Advanced Vaccine Research : Methods for the Decade of Vaccines [1 ed.] 9781910190043, 9781910190036

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Edited by Fabio Bagnoli and Rino Rappuoli

Advanced Vaccine Research Methods for the Decade of Vaccines

Edited by Fabio Bagnoli and Rino Rappuoli Novartis Vaccines Research Center Siena Italy

Caister Academic Press

Copyright © 2015 Caister Academic Press Norfolk, UK www.caister.com British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-1-910190-03-6 (hardback) ISBN: 978-1-910190-04-3 (ebook) Description or mention of instrumentation, software, or other products in this book does not imply endorsement by the author or publisher. The author and publisher do not assume responsibility for the validity of any products or procedures mentioned or described in this book or for the consequences of their use. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the publisher. No claim to original U.S. Government works. Cover design adapted from Figures 3.1 and 3.2

Contents

Contributorsv Prefacexi Part I Innovative Technologies and Approaches in Vaccine Research

1

1

Deep Sequencing in Vaccine Research, Development and Surveillance

3

2

New Bioinformatics Algorithms Applied to Deep Sequencing Projects

33

3

Comparative Genomics Approaches for Tracking the Emergence and Spread of Disease-associated Bacteria

65

4

Quantitative Proteomics in Vaccine Research

75

5

Structural Biology in Vaccine Research

103

6

Cellular Screens to Interrogate the Human T- and B-cell Repertoires and Design Better Vaccines

133

Novel Strategies of Vaccine Administration: The Science Behind Epidermal and Dermal Immunization

157

8

Toll-like Receptors as Targets to Develop Novel Adjuvants

187

9

The Importance of Cell-mediated Immunity for Bacterial Vaccines

219

T-cell-inducing Vaccines

251

Stefano Censini, Silvia Guidotti, Giulia Torricelli, Rino Rappuoli and Fabio Bagnoli Mina Rho

Tracy H. Hazen and David A. Rasko

Massimiliano Biagini and Nathalie Norais

Danilo Donnarumma, Matthew J. Bottomley, Enrico Malito, Ethan Settembre, Ilaria Ferlenghi and Roberta Cozzi

Jens Wrammert and Kaja Murali-Krishna

7

Béhazine Combadière and Hélène Perrin

10

Şefik Şanal Alkan

Alison G. Murphy and Rachel M. McLoughlin Sarah Gilbert

iv  | Contents

11

Exploiting the Mutanome for Personalized Cancer Immunotherapy

Ugur Sahin, Sebastian Kreiter, John C. Castle, Martin Löwer, Cedrik M. Britten and Özlem Türeci

263

Part II Challenges for the Decade of Vaccines

271

12

273

Malaria Vaccine Development: Progress to Date

Philip Bejon, Ally Olotu and Kevin Marsh

13

Tuberculosis305

14

HIV-1 Vaccine Development

335

15

Cancer Immunotherapy: The Road to Rejection

359

16

Global Health Vaccines Against the Invasive Salmonelloses: Enteric Fever and Invasive Non-typhoidal Salmonella Disease

387

17

The Path to a Respiratory Syncytial Virus Vaccine

411

18

Staphylococcus aureus 425

Else Marie Agger and Peter Andersen

Barton F. Haynes, Georgia D. Tomaras, Hua-Xin Liao and Andrew J. McMichael Peter E. Fecci, Christina Chen, Susanne Baumeister and Glenn Dranoff

Calman A. MacLennan

Christine A. Shaw, Max Ciarlet, Brian W. Cooper, Lamberto Dionigi, Paula Keith, Karen B. O’Brien, Maryam Rafie-Kolpin and Philip R. Dormitzer Linhui Wang and Jean C. Lee

Index449

Contributors

Else Marie Agger Department of Infectious Disease Immunology Statens Serum Institut Copenhagen Denmark

Philip Bejon Department of Pathogen, Vector, and Host Biology KEMRI Wellcome Trust Research Programme Kilifi Kenya

[email protected]

[email protected]

Şefik Şanal Alkan Alkan Consulting LLC Basel Switzerland

Massimiliano Biagini Research Centre – Protein Biochemistry Structural Mass Spectrometry & Proteomics Unit Novartis Vaccines Siena Italy

[email protected] Peter Andersen Department of Infectious Disease Immunology Statens Serum Institut Copenhagen Denmark [email protected] Fabio Bagnoli Novartis Vaccines Research Center Siena Italy [email protected] Susanne Baumeister Department of Pediatrics Boston Children’s Hospital Harvard Medical School; and Department of Pediatric Oncology Dana-Farber Cancer Institute Boston, MA USA [email protected]

[email protected] Matthew J. Bottomley Novartis Vaccines Research Center Siena Italy [email protected] Cedrik M. Britten Translational Oncology (TRON) Translational Research Centre Johannes Gutenberg-University Mainz Mainz Germany [email protected]

vi  | Contributors

John C. Castle Translational Oncology (TRON) Translational Research Centre Johannes Gutenberg-University Mainz Mainz Germany

Lamberto Dionigi Dompé Farmaceutici Milan Italy

[email protected]

Danilo Donnarumma Novartis Vaccines Research Center Siena Italy

Stefano Censini Novartis Vaccines Research Center Siena Italy [email protected] Christina Chen Department of Neurosurgery Massachusetts General Hospital Harvard Medical School Boston, MA USA [email protected] Max Ciarlet Novartis Vaccines Cambridge, MA USA [email protected] Béhazine Combadière Centre d’Immunologie et des Maladies infectieuses Institut National de Santé et de Recherche Médicale Paris France [email protected] Brian W. Cooper University of Connecticut School of Medicine Farmington, CT USA [email protected] Roberta Cozzi Novartis Vaccines Research Center Siena Italy [email protected]

[email protected]

[email protected] Philip R. Dormitzer Novartis Vaccines Cambridge, MA USA [email protected] Glenn Dranoff Department of Medicine Brigham and Women’s Hospital Harvard Medical School; and Department of Medical Oncology Cancer Vaccine Center Dana-Farber Cancer Institute Boston, MA USA [email protected] Peter E. Fecci Department of Neurosurgery Massachusetts General Hospital Harvard Medical School Boston, MA USA [email protected] Ilaria Ferlenghi Novartis Vaccines Research Center Siena Italy [email protected]

Contributors |  vii

Sarah Gilbert The Jenner Institute Nuffield Department of Medicine University of Oxford Oxford UK [email protected] Silvia Guidotti Novartis Vaccines Research Center Siena Italy [email protected] Barton F. Haynes Duke Human Vaccine Institute School of Medicine Duke University Durham, NC USA [email protected] Tracy H. Hazen Department of Microbiology and Immunology Institute for Genome Sciences School of Medicine University of Maryland Baltimore, MD USA [email protected] Paula Keith Novartis Vaccines Cambridge, MA USA [email protected] Sebastian Kreiter Translational Oncology (TRON) Translational Research Centre Johannes Gutenberg-University Mainz Mainz Germany [email protected]

Jean C. Lee Division of Infectious Diseases Department of Medicine Brigham and Women’s Hospital Harvard Medical School Boston, MA USA [email protected] Hua-Xin Liao Duke Human Vaccine Institute School of Medicine Duke University Durham, NC USA [email protected] Martin Löwer Translational Oncology (TRON) Translational Research Centre Johannes Gutenberg-University Mainz Mainz Germany [email protected] Calman A. MacLennan Wellcome Trust Sanger Institute Cambridge UK [email protected] Rachel M. McLoughlin Host Pathogen Interactions Group School of Biochemistry and Immunology Trinity College Dublin Ireland [email protected] Andrew J. McMichael The Jenner Institute Nuffield Department of Medicine University of Oxford Oxford UK [email protected]

viii  | Contributors

Enrico Malito Novartis Vaccines Research Center Siena Italy

Ally Olotu Department of Pathogen, Vector, and Host Biology KEMRI Wellcome Trust Research Programme Kilifi Kenya

[email protected]

aolotu@ kemri-wellcome.org

Kevin Marsh Department of Pathogen, Vector, and Host Biology KEMRI Wellcome Trust Research Programme Kilifi Kenya

Hélène Perrin Centre d’Immunologie et des Maladies infectieuses Institut National de Santé et de Recherche Médicale Paris France

[email protected]

[email protected]

Kaja Murali-Krishna Department of Pediatrics School of Medicine; and Emory Vaccine Center Emory University Atlanta, GA USA; and ICGEB – Emory Vaccine Center International Center for Genetic Engineering and Biotechnology (ICGEB) New Delhi India

Maryam Rafie-Kolpin AstraZeneca Drug Safety and Metabolism Waltham, MA USA

[email protected] Alison G. Murphy Mucosal Immunology Group/Maloy Lab Sir William Dunn School of Pathology University of Oxford Oxford UK [email protected] Nathalie Norais Research Centre – Protein Biochemistry Structural Mass Spectrometry & Proteomics Unit Novartis Vaccines Siena Italy [email protected] Karen B. O’Brien Novartis Institutes of Biomedical Research Cambridge, MA USA [email protected]

[email protected] Rino Rappuoli Novartis Vaccines Research Center Siena Italy [email protected] David A. Rasko Department of Microbiology and Immunology Institute for Genome Sciences School of Medicine University of Maryland Baltimore, MD USA [email protected] Mina Rho Division of Computer Science and Engineering Hanyang University Seoul Korea [email protected]

Contributors |  ix

Ugur Sahin Translational Oncology (TRON) Translational Research Centre Johannes Gutenberg-University Mainz Mainz Germany [email protected] Ethan Settembre Novartis Vaccines Cambridge, MA USA [email protected] Christine A. Shaw Novartis Vaccines Cambridge, MA USA [email protected] Georgia D. Tomaras Duke Human Vaccine Institute School of Medicine Duke University Durham, NC USA [email protected]

Giulia Torricelli Novartis Vaccines Research Center Siena Italy [email protected] Özlem Türeci Translational Oncology (TRON) Translational Research Centre Johannes Gutenberg-University Mainz Mainz Germany [email protected] Linhui Wang Division of Infectious Diseases Department of Medicine Brigham and Women’s Hospital Harvard Medical School Boston, MA USA [email protected] Jens Wrammert Department of Pediatrics School of Medicine; and Emory Vaccine Center Emory University Atlanta, GA USA [email protected]

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Preface

Only clean water has contributed to improving global health more than vaccines (Andre et al., 2008). Vaccines have completely, or nearly, eradicated some of the most deadly viral and bacterial infections (e.g. smallpox, poliomyelitis, diphtheria, tetanus, pertussis, measles, mumps and rubella) (Rappuoli et al., 2011). On top of direct effects, by preventing infections in vaccinated subjects, vaccines also have a number of indirect benefits for the individual and society (Andre et al., 2008). Indeed, vaccines can generate herd immunity, which plays a key role in protecting individuals at higher risk of infection including the immunocompromised, elderly and cancer patients, those in which the use of the vaccines is contraindicated, and those with limited or no access to resources to buy them. Vaccination has also been shown to reduce the incidence of certain cancers (Chang, 2003; Harper et al., 2006). Indeed, some infective agents are associated with cancer, such as HBV with liver cancer and HPV with cervical cancer. Furthermore, vaccines are a key component in the fight against antibiotic resistance both directly and indirectly. By targeting bacterial pathogens, vaccines directly reduce the need for the use of antibiotics. Antiviral vaccines, such as the ones against influenza, can also have an indirect effect on reducing the emergence of antibiotic resistant strains by decreasing complications associated with super-infections, which routinely require antibiotic use. Most of the vaccines currently available for human use were developed on the basis of Louis Pasteur’s principle of inactivating or killing the infectious agent and then using it to induce protective immunity into the host (Rappuoli et

al., 2011). However, scientists have recently realized that for several pathogens (e.g. serogroup B Neisseria meningitidis (MenB), HIV, malaria), conventional vaccinology methods are not sufficient or adequate. After the publication of the first bacterial genome in 1995 (Fleishmann et al., 1995), it became clear that availability of the genomic sequence of pathogens was an invaluable source of information for vaccine research. In fact, only five years later, a new antigen identification approach, named reverse vaccinology, was applied to MenB (Pizza et al., 2000). The approach was termed reverse vaccinology because antigens were selected prior to experimental testing (Rappuoli, 2000). Later, with the explosion of the omics era, vaccine discovery could benefit from techniques that generate data complementary to reverse vaccinology. With the advent of high-throughput sequencing technologies, the availability of multiple genomes of the same species allowed comparative genomics studies to be performed, critical to determine the level of conservation of vaccine candidates (He et al., 2010). However, none of the genomic approaches can provide all the information required for vaccine design and characterization. Techniques based on immunomics, such as the so-called antigenomics, can identify candidates expected to be immunogenic in humans (Meinke et al., 2005; Rinaudo et al., 2009; Vytvytska et al., 2002). Approaches based on transcriptomics or proteomics are able to identify candidates expressed by pathogens under different growth conditions. Studies done to date using the different approaches have generally shown a significant degree of overlap and have

xii  | Preface

identified subsets of the surface and secreted antigens predicted by reverse vaccinology (Bagnoli et al., 2011; Bensi et al., 2012; Doro et al., 2009; Etz et al., 2002; Grifantini et al., 2002; RodriguezOrtega et al., 2006; Stranger-Jones et al., 2006). However, each approach supplies different information that altogether can be used to select the best candidates. Despite the recent progress made by omics science and high-throughput technologies we should not assume that vaccine research can be performed without the tight support of basic research. Indeed, it is still highly dependent on experimental studies and empirical observations. It is of critical importance to determine the role played by antigens in virulence, and interactions with the host, as well as their function and biochemical properties such as the structure. Structural biology represents a powerful means to identify protective epitopes, especially in highly variable antigens. Available vaccines are against pathogens whose antigens are relatively stable. Microbes that have rapid and extensive antigenic variability, remain a major challenge for vaccine researchers (Rappuoli and Aderem, 2011). Structural studies on the antigens can be performed to understand the degree of surface exposure of the epitopes and to design peptides optimized to generate neutralizing antibodies (Dormitzer et al., 2008). Another important aspect that requires a basic research approach is the discovery of mechanisms of protection. Pathogens against which successful vaccines have been developed, have known protective mechanisms and in all cases humoral response appears to be the driving mechanism (Moriel et al., 2010). On the contrary, when protective mechanisms and correlate of protection are not clear (e.g. Staphylococcus aureus, malaria, HIV, Candida albicans, tuberculosis), successful vaccines could not be developed (Bagnoli et al., 2012; Dubensky et al., 2012; He et al., 2010). Therefore, basic immunology studies to shed light on their mechanisms of protection are needed to support vaccine development against these pathogens. Accumulating literature indicate that innate and cell mediated immunity are important against several pathogens, such as Mycobacterium

tuberculosis (Doherty and Andersen, 2005; Hoft, 2008), Candida albicans and S. aureus. In this regard, adjuvant formulations stimulating T-cell-mediated immunity are certainly another important area of investigation for next generation vaccines. Traditionally, adjuvants have been used to increase antibodymediated responses. However, the important role of adjuvants in stimulating T-cell responses is also becoming clear. Recently, the role of Tolllike receptors as adjuvant targets is emerging as a promising area of investigation. Usually, prior to clinical trials, most of the information available on protective efficacy of candidate vaccines is obtained in animal models and in in vitro studies. However, this approach has several limitations in predicting human immune response to vaccines. This is particularly true for those pathogens mentioned earlier for which correlate of protection in humans are unknown. Indeed, several failures in phase III clinical trials on HIV, malaria, and S. aureus have been recorded (Proctor, 2012; Shinefield et al., 2002; Spellberg and Daum, 2010, 2012). The possibility to use different high-throughput technologies (e.g. next generation sequencing) to monitor the host response to vaccination and disease as well as to interrogate T- and B-cell repertoires in a large collection of individuals will allow the discovery of signatures of protection in humans. By integrating as many biological measurements as possible, systems biology will provide a powerful tool to analyse and interpret host responses to vaccines in clinical trials. The aim of this book is therefore to illustrate the impressive technological advance that is increasing the quality standards of vaccines and is paving the way to develop vaccines against diseases for which efficacious medical treatments are still lacking. The examples that we have used comprise very different diseases; we include not only infectious diseases, but also cancer. We believe that these will be the vaccines of the future, the ‘vaccines for 2020’. References Andre, F.E., Booy, R., Bock, H.L., Clemens, J., Datta, S.K., John, T.J., Lee, B.W., Lolekha, S., Peltola, H., Ruff, T.A., et al. (2008). Vaccination greatly reduces disease,

Preface |  xiii

disability, death and inequity worldwide. Bull. World Health Organ. 86, 140–146. Bagnoli, F., Baudner, B., Mishra, R.P., Bartolini, E., Fiaschi, L., Mariotti, P., Nardi-Dei, V., Boucher, P., and Rappuoli, R. (2011). Designing the next generation of vaccines for global public health. OMICS 15, 545–566. Bagnoli, F., Bertholet, S., and Grandi, G. (2012). Inferring reasons for the failure of Staphylococcus aureus vaccines in clinical trials. Front. Cell. Infect. Microbiol. 2, 16. Bensi, G., Mora, M., Tuscano, G., Biagini, M., Chiarot, E., Bombaci, M., Capo, S., Falugi, F., Manetti, A.G., Donato, P., et al. (2012). Multi high-throughput approach for highly selective identification of vaccine candidates: the group a streptococcus case. Mol. Cell. Proteomics 11, M111.015693. Chang, M.H. (2003). Decreasing incidence of hepatocellular carcinoma among children following universal hepatitis B immunization. Liver Int. 23, 309–314. Doherty, T.M., and Andersen, P. (2005). Vaccines for tuberculosis: novel concepts and recent progress. Clin. Microbiol. Rev. 18, 687–702. Dormitzer, P.R., Ulmer, J.B., and Rappuoli, R. (2008). Structure-based antigen design: a strategy for next generation vaccines. Trends Biotechnol. 26, 659–667. Doro, F., Liberatori, S., Rodriguez-Ortega, M.J., Rinaudo, C.D., Rosini, R., Mora, M., Scarselli, M., Altindis, E., D’Aurizio, R., Stella, M., et al. (2009). Surfome analysis as a fast track to vaccine discovery: identification of a novel protective antigen for Group B Streptococcus hypervirulent strain COH1. Mol. Cell. Proteomics 8, 1728–1737. Dubensky, T.W. Jr., Skoble, J., Lauer, P., and Brockstedt, D.G. (2012). Killed but metabolically active vaccines. Curr. Opin. Biotechnol. 23, 917–923. Etz, H., Minh, D.B., Henics, T., Dryla, A., Winkler, B., Triska, C., Boyd, A.P., Sollner, J., Schmidt, W., von Ahsen, U., et al. (2002). Identification of in vivo expressed vaccine candidate antigens from Staphylococcus aureus. Proc. Natl. Acad. Sci. U.S.A. 99, 6573–6578. Fleishmann, J.A., Mor, V., and Laliberte, L.L. (1995). Longitudinal patterns of medical service use and costs among people with AIDS. Health Serv. Res. 30, 403–424. Grifantini, R., Bartolini, E., Muzzi, A., Draghi, M., Frigimelica, E., Berger, J., Ratti, G., Petracca, R., Galli, G., Agnusdei, M., et al. (2002). Previously unrecognized vaccine candidates against group B meningococcus identified by DNA microarrays. Nat. Biotechnol. 20, 914–921. Harper, D.M., Franco, E.L., Wheeler, C.M., Moscicki, A.B., Romanowski, B., Roteli-Martins, C.M., Jenkins, D., Schuind, A., Costa Clemens, S.A., and Dubin, G. (2006). Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomised control trial. Lancet 367, 1247–1255. He, Y., Rappuoli, R., De Groot, A.S., and Chen, R.T. (2010). Emerging vaccine informatics. J. Biomed. Biotechnol. 2010, 218590.

Hoft, D.F. (2008). Tuberculosis vaccine development: goals, immunological design, and evaluation. Lancet 372, 164–175. Meinke, A., Henics, T., Hanner, M., Minh, D.B., and Nagy, E. (2005). Antigenome technology: a novel approach for the selection of bacterial vaccine candidate antigens. Vaccine 23, 2035–2041. Moriel, D.G., Bertoldi, I., Spagnuolo, A., Marchi, S., Rosini, R., Nesta, B., Pastorello, I., Corea, V.A., Torricelli, G., Cartocci, E., et al. (2010). Identification of protective and broadly conserved vaccine antigens from the genome of extraintestinal pathogenic Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 107, 9072–9077. Pizza, M., Scarlato, V., Masignani, V., Giuliani, M.M., Arico, B., Comanducci, M., Jennings, G.T., Baldi, L., Bartolini, E., Capecchi, B., et al. (2000). Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 287, 1816–1820. Proctor, R.A. (2012). Is there a future for a Staphylococcus aureus vaccine? Vaccine 30, 2921–2927. Rappuoli, R. (2000). Reverse vaccinology. Curr. Opin. Microbiol. 3, 445–450. Rappuoli, R., and Aderem, A. (2011). A 2020 vision for vaccines against HIV, tuberculosis and malaria. Nature 473, 463–469. Rappuoli, R., Mandl, C.W., Black, S., and De Gregorio, E. (2011). Vaccines for the twenty-first century society. Nat. Rev. Immunol. 11, 865–872. Rinaudo, C.D., Telford, J.L., Rappuoli, R., and Seib, K.L. (2009). Vaccinology in the genome era. J. Clin. Invest. 119, 2515–2525. Rodriguez-Ortega, M.J., Norais, N., Bensi, G., Liberatori, S., Capo, S., Mora, M., Scarselli, M., Doro, F., Ferrari, G., Garaguso, I., et al. (2006). Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome. Nat. Biotechnol. 24, 191–197. Shinefield, H., Black, S., Fattom, A., Horwith, G., Rasgon, S., Ordonez, J., Yeoh, H., Law, D., Robbins, J.B., Schneerson, R., et al. (2002). Use of a Staphylococcus aureus conjugate vaccine in patients receiving hemodialysis. New Engl. J. Med. 346, 491–496. Spellberg, B., and Daum, R. (2010). A new view on development of a Staphylococcus aureus vaccine: insights from mice and men. Human Vaccin. 6, 857–859. Spellberg, B., and Daum, R. (2012). Development of a vaccine against Staphylococcus aureus. Semin. Immunopathol. 34, 335–348. Stranger-Jones, Y.K., Bae, T., and Schneewind, O. (2006). Vaccine assembly from surface proteins of Staphylococcus aureus. Proc. Natl. Acad. Sci. U.S.A. 103, 16942–16947. Vytvytska, O., Nagy, E., Bluggel, M., Meyer, H.E., Kurzbauer, R., Huber, L.A., and Klade, C.S. (2002). Identification of vaccine candidate antigens of Staphylococcus aureus by serological proteome analysis. Proteomics 2, 580–590.

Part I Innovative Technologies and Approaches in Vaccine Research

Deep Sequencing in Vaccine Research, Development and Surveillance

1

Stefano Censini, Silvia Guidotti, Giulia Torricelli, Rino Rappuoli and Fabio Bagnoli

Abstract Since it was established in the 1970s by Frederick Sanger, DNA sequencing has brought enormous contribution to virtually all areas of life and medical sciences. More recently, next-generation sequencing technologies (NGS) have gradually flanked and then replaced the Sanger method in most sequencing projects. The major aim of this chapter is to describe the extraordinary technological advances from the first to the thirdgeneration sequencing platforms as well as the consequent expanding range of their applications. NGS is enabling large-scale genomics, metagenomics, transcriptomics and epigenomics studies at extremely deep resolution. Another key area of NGS is the analysis of human B- and T-lymphocyte repertoires. The explosion of new sequencing technologies was paralleled by a similar advance in computational science and synthetic biology leading to the era of ‘teleporting life’. Altogether, these new technologies and their applications are significantly increasing chances to discover and develop efficacious vaccines. Impact of DNA sequencing on vaccines research and development The value of vaccines Along with improved nutrition and hygiene practices, vaccines with either killed, live attenuated or purified subunits of microorganisms have drastically improved human health, leading to the complete or nearly complete eradication of many infectious diseases that historically plagued the

mankind (Plotkin, 2005, 2009). Immunization is actually recognized as the most successful, costeffective and cost-beneficial health intervention to save human lives and improve life expectancy and quality worldwide, that led to an increased lifespan in developed countries by greater than 30 years during the last century (Centers for Disease Control and Prevention, 1999; Bunker et al., 1994). However, the value of vaccines goes far beyond the individual benefit (Rappuoli et al., 2002), being further enriched by ‘herd immunity’ in unvaccinated people (Kim et al., 2011) as well as the reduction of super-infections and of antibiotic resistance phenomena (Andre et al., 2008; Bloom, 2011; Bloom et al., 2004, 2005; Ehreth, 2003; Mishra et al., 2012). While vaccines are actually estimated to prevent 2.5 million child deaths per year, two million lives could be saved in addition if existing vaccines would be more universally available. In addition, an expanded and timely access to key life-saving vaccines is thought to be pivotal to fulfil WHO’s global Millennium Development Goals 1 and 4 by 2015, in reducing poverty and improve human equity, life and development (Maurice and Davey, 2009; World Health Organization, 2005). In other words, the relevance of mass vaccination is tied to the value that our society attributes to the condition of ‘global health’ and how this translates into choices and investments of social and political economy (Ozawa et al., 2012; Postma and Standaert, 2013). From variolation to today’s vaccines The history of vaccination stems from the empirical observation that people can only get sick

4  | Censini et al.

once from a certain disease. Earliest attempts to deliberately induce human protection against plagues and pestilences were conducted somewhere in Asia during the tenth century, with the administration to unexposed individuals of crude material from active cases of smallpox (variola virus). Apparently, that was enough to induce a milder disease and a lifelong resistance to natural smallpox reinfection (Levine, 2009; Plotkin et al., 2012). When the variolation practice was introduced in Europe during the early eighteenth century thanks to Lady Wortley Montagu, smallpox was still killing over 400,000 Europeans and as many as one-third of city children per year, leaving severe and painful consequences among scarred survivors. Despite some initial distrust on such risky procedure (approximately 2% of variolated people could contract the disease and die) the variolation approach became soon quite popular and widely adopted since the chance of dying from variolation was 10 times lower than that related to the naturally acquired smallpox during a raging epidemic (Baxby, 1999; Brunham and Coombs, 1998). A safer approach came at the end of the same century, when Edward Jenner noticed that milkmaids with mild cowpox disease (variolae vaccinae) were ‘resistant’ to more contagious and deadly smallpox infections, encouraging him to accomplish in 1796 the first systematic ‘vaccination’ effort consisting of a ‘smallpox challenge’ following a ‘cowpox administration’ ( Jenner, 1798). The Jennerian ‘vaccination’ practice (from ‘vacca’, Latin for ‘cow’) was proven to be more effective and safer than variolation, and its broad adoption allowed a significant decrease in smallpox mortality throughout the nineteenth century. However, the explanation behind the vaccination effectiveness remained unknown until Pasteur’s ‘germ theory’ unambiguously established the cause-effect link between pathogens and diseases (Pasteur, 1881). Such fundamental discovery progressively encouraged the wide adoption of improved hygiene practices to minimize the microbial spread and led the take-off of vaccination practices with either live-attenuated or inactivated microorganisms from that moment onwards.

Reversing Pasteur’s paradigm The empirical Pasteur’s approach to ‘isolate, inactivate, and inject’ the causative microorganism of the infectious disease, dominated the scene of mass vaccination during the twentieth century. Even if vaccines were developed at that time with limited knowledge about the mechanisms underlying the protective immunity, the strategy was based on the unifying rationale that microbial virulence could be attenuated while preserving the original immunogenicity. During the last four decades, remarkable progresses were made in the vaccine field due to the introduction of recombinant DNA technologies, and chemical conjugation of proteins to polysaccharides, as well as advances on adjuvant molecules capable to increase the protective immunity. The combination of those technological breakthroughs with the accumulation of knowledge about the mechanisms of microbial pathogenesis enabled the identification of virulence determinants, and expanded our ability to prepare vaccines according to the Pasteur’s principle by gradually moving away from killed/live attenuated microorganisms in favour of safer protein subunit vaccines. By the end of last century, great efficacy was achieved in controlling or eradicating many pathogens with relatively stable antigenic profiles by means of antibody-mediated immunity (De Gregorio and Rappuoli, 2012; Delany et al., 2013; Lipsitch and O’Hagan, 2007; Rappuoli, 2004, 2007; Serruto and Rappuoli, 2006; Telford, 2008). While smallpox disease was declared to be officially eradicated by 1980, vaccines against diphtheria, tetanus, polio, measles, mumps, rubella, pneumococcus, hepatitis B and meningitis (caused by type B Haemophilus influenzae and serogroup C meningococci) has led to the reduction of the incidence of those infections by 97–99% worldwide (Rappuoli et al., 2002). However, since even most improved approaches failed to provide an effective solution against new or reemerging pathogens more complex to study (like the uncultivable or hyper variable ones), faster and more effective approaches were required to meet such emerging needs (Delany et al., 2013; Lipsitch and O’Hagan, 2007; Rappuoli, 2007; Telford, 2008). The first major innovation in vaccine design came with the advent of ‘microbial

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genomics’ during the 1990s, when the genome sequencing became a prerequisite to understand the complete biology of microorganisms and an irreplaceable tool for vaccine development. As a result, a new vaccine design approach called Reverse Vaccinology (RV) was established since the early 2000s, based on whole-genome sequencing and bioinformatic data mining of worldwide circulating pathogens (Capecchi et al., 2004; Delany et al., 2013; Donati and Rappuoli, 2013; Fraser and Rappuoli, 2005; He et al., 2010, 2013; Masignani et al., 2002; Rappuoli, 2000; Rappuoli et al., 2011; Rinaudo et al., 2009; Seib et al., 2009, 2012; Serino et al., 2012; Serruto and Rappuoli, 2006; Serruto et al., 2009; Sette and Rappuoli, 2010). The RV method was first applied to the Neisseria meningitidis serogroup B (MenB), a Gram-negative capsulated bacterium causing up to 90% of invasive meningococcal disease worldwide, with a mortality rate of up to 15% (Kaaijk et al., 2013; Pizza et al., 2000). Although the use of vaccines based on polysaccharide antigens was found appropriate for most species causing bacterial meningitis (type-B Haemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitidis serogroups A, C, Y and W135), such approach was not feasible in the case of MenB, since its capsular polysialic acid shares significant similarity to a mammalian glycoprotein on neural tissues, and it is therefore poorly immunogenic. After in silico screening of a MenB genome sequence (Tettelin et al., 2000) it was possible to identify genes encoding proteins with attributes of good vaccine targets, as predicted to be surface exposed and conserved across multiple circulating strains (Muzzi et al., 2013; Pizza et al., 2000). Selected antigens were expressed in E. coli and used to immunize mice in order to evaluate both immunogenicity and protective efficacy by means of in vitro bactericidal properties of related antisera as correlate of immunity. Starting with a set of 570 in-silico predicted vaccine candidates, this approach led to the ultimate selection and combination of five antigens [factor H binding protein (f HBP), neisserial adhesin A (NadA), neisserial heparinbinding antigen (NHBA), GNA1030, and GNA2091] into a multicomponent vaccine to prevent MenB. The purified protein antigens were

formulated together with detergent-extracted outer membrane vesicles (dOMV) from the New Zealand epidemic strain NZ98/254 to increase vaccine coverage and avoid the selection of escape mutants (Gorringe and Pajon, 2012). This vaccine (Bexsero), was shown to be safe and capable of eliciting high protective immunity in infants, adolescents, and adults against a wide panel of heterologous MenB isolates, covering up to 90% of circulating strains in Europe (Carter, 2013; Giuliani et al., 2006; Gorringe and Pajon, 2012; Lucidarme et al., 2009, 2010; Riedmann, 2012; Toneatto et al., 2011a,b; Vogel et al., 2013). Bexsero recently received the European license for distribution (European Public Assessment Report EMA/755874/2012), providing the ultimate validation of RV strategy for vaccine development. Since the original in silico analysis of a MenB genome sequence (Pizza et al., 2000; Tettelin et al., 2000) the RV approach has been applied to a wide range of bacterial pathogens (Barocchi et al., 2007; Delany et al., 2013; Mora et al., 2005). Meanwhile, the rapid advancement of DNA sequencing technology and the availability of multiple genome sequences from different isolates of a single species suggested that the genome sequence of a single isolate might not be enough to understand the complex pathogenesis of a bacterial species and to identify broadly protective vaccine candidates. The comparative analysis of genomes from multiple strains and/or closely related pathogenic and non-pathogenic microorganisms, allowed the definition of the ‘pan-genome’ as global gene repertoire of a species (Donati et al., 2010; Medini et al., 2005, 2008; Muzzi et al., 2007; Tettelin et al., 2005, 2008). As the number of sequences across genera increases, so does the power to estimate genomic heterogeneity and the chance to develop more universal vaccines. Various refinements have been applied to the RV approach to overcome its original limitations, especially when dealing with microorganisms with high intra-species diversity or species consisting of both commensal and pathogenic strains (Bambini and Rappuoli, 2009; Delany et al., 2013; Donati and Rappuoli, 2013; Mora et al., 2006; Seib et al., 2012; Serino et al., 2012; Serruto et al., 2009). Just to list few examples, a pan-genome approach was

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applied to identify antigens from the extended gene repertoire of Group-B streptococci (Maione et al., 2005; Tettelin et al., 2002, 2005, 2006) and a subtractive strategy has been implemented to identify antigens restricted to pathogenic but not commensal strains of E. coli (Brzuszkiewicz et al., 2006; Moriel et al., 2010; Rasko et al., 2008). Pioneering DNA sequencing efforts Since the establishment of DNA as the hereditary material (Avery et al., 1944; Griffith, 1928; Hershey and Chase, 1952) and the elucidation of its double-helix structure (Watson and Crick, 1953), much effort has been devoted to decode the DNA sequence in its linear order. Among the pioneering approaches, the ‘dideoxy chain termination method’ proposed by Frederick Sanger and collaborators during the 1970s (Sanger et al., 1977a) was based on the ability of a DNA polymerase to incorporate nucleotide analogues while synthesizing template driven DNA, and quickly became the most popular one to accomplish DNA sequencing (Sanger et al., 1977b, 1982). However, the Sanger procedure was too costly, time consuming, and labour intensive (1 Mb throughput per day at 500 amino acids sequence variants have been reported,) ( Jolley and Maiden, 2010), which has generated much interest in identifying the key protective epitopes, to aid design of a broadly protective vaccine antigen (see the section on ‘Examples of structural vaccinology for bacterial and viral pathogens’, below). A clear advantage of this simple approach is that it only requires knowledge of the antigen sequence, thus removing the need to produce and purify the native antigen. Moreover, peptide synthesis, the experiment itself and the data analysis are rapid. All peptide fragments are analysed in parallel, and the experimental surfaces can usually be reused, making this both a quick and cost-effective approach overall. A disadvantage of the approach is that it can only determine interactions of the antibody with linear peptide epitopes. This shortcoming was revealed in the same study described above, where PepScan was unable to identify conformational functional epitopes in the C-terminal domain of f Hbp, requiring the use of purified recombinant folded proteins in order to more thoroughly perform the epitope mapping (Giuliani et al., 2005). Similar issues were observed recently in a multi-technique epitope

mapping study of the interaction between f Hbp and a monoclonal antibody, mAb 12C1, in which the Pepscan approach reliably revealed only a single linear peptide ultimately found to be part of a much bigger conformational epitope (Malito et al., 2013). Fragment-display approaches Epitope mapping can be performed by studying the binding of antibodies to bacteriophage displaying libraries of peptides on their surface. Such libraries can be pre-made to include millions of different, randomly generated sequences, or can be smaller custom-built targeted libraries displaying only peptide fragments of a specific antigen of interest. The former typically results in the identification of several linear peptides, ideally revealing a consensus target motif that can subsequently be matched to an epitope on the corresponding target antigen, as described elsewhere (Bottger and Bottger, 2009). The latter approach is more similar to other linear epitope mapping techniques, and allows identification of linear epitopes within a given target. For example, in epitope mapping studies of f Hbp, the antigen-specific phage display approach was used in parallel with Pepscan performed on relatively short synthetic peptides. In theory, the phage display approach can be tailored to allow the display and subsequent recognition of antigen fragments produced in vivo and displayed on the phage surface, and the fragment size-range can be approximately defined by the user. Compared to Pepscan, this phagedisplay approach may have the advantage of being able to present some conformational epitopes. However, in the study described above, when analysing an f Hbp fragment library of average size 55 amino acids, the linear epitope identified was essentially the same as that found by Pepscan, and additional conformational epitopes were not revealed (Malito et al., 2013). The possibility that surface-display approaches can rapidly identify linear peptide epitopes – and may even allow identification of larger, folded antigen fragments bearing conformational epitopes – has prompted the development of several different methods in addition to phage display system. For example, antigen fragment display has been performed on E. coli (Christmann et al., 2001),

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yeast (Chao et al., 2004; Levy et al., 2007) and Staphylococcus carnosus (Rockberg et al., 2008). These approaches take advantage of normalized fragment expression, fluorescence-activated cell sorting (FACS) and high-throughput sequencing for rapid selection and characterization of the cloned epitope inserts. Moreover, in a recent adaptation of the staphylococcal display system, a library containing fragments from 60 diseaserelated human antigens was constructed, for simultaneous epitope mapping of mono- and polyclonal antibodies via a multiplex detection method (Hudson et al., 2012). Interestingly, using a subset of this library specific for vascular endothelial growth factor A (VEGF), with an average DNA fragment size of 100 bp, epitope mapping was performed for the therapeutic monoclonal antibody, Avastin, which is known to target a large conformational epitope (Chen et al., 1999). In agreement with the Avastin Fab/ VEGF 3-D structure determined by X-ray crystallography, the staphylococcal library revealed the relevant VEGF fragment spanning residues 34–121 as the high-affinity epitope, suggesting that this display library can also detect conformational epitopes. However, the wider applicability of such libraries to reliably detect conformational epitopes is not guaranteed, because the presence of correctly folded conformational epitopes within the fragment library may be relatively rare, and for each new antigen being studied will depend on the distinct 3-D structure of the protein and its subdomains. To summarize, while Pepscan is a simple and rapid approach, and can readily identify epitopes targeted both by mAbs and polyclonal antibodies, it is likely to present only a partial picture when attempting to detect and fully define a conformational epitope. Similarly, display technologies have the advantage of eliminating the need for native antigen purification and have been successfully used to identify new vaccine candidates (Etz et al., 2002). Linear epitope mapping has also allowed the creation of a database of linear B-cell epitopes that was compared with regions of human proteins capable of generating antibodies, revealing that pathogen-derived antigens eliciting antibodies do not share epitopes with host proteins, an important consideration when selecting candidate

vaccine antigens (Amela et al., 2007). However, although fast and economical, epitope mapping based on displaying short fragments (20–150 amino acids) seems unlikely to systematically identify all conformational epitopes. These limitations have led to considerable efforts to enable the mapping of conformational epitopes, principally in studies of pairwise antigen–antibody combinations, since the application to polyclonal antibody populations is much more complicated. The following section aims to illustrate the pros and cons of several of the more powerful epitope mapping techniques. Many of these have recently been applied to studies of the meningococcal f Hbp, which will therefore be used in several cases to facilitate the comparisons. Mass spectrometry Mass spectrometry (MS) is nowadays the stateof-the-art method for the characterization of peptides and proteins. This analytical technique allows the detection of peptides and proteins of increasing molecular weight (up to MDa) with very high sensitivity (sub-femtomole concentration for peptides) as well as sub-isotopic resolution and high mass accuracy. Moreover, the time required for the sample preparation and the analysis is much lower than that for several other techniques. For all these reasons, in the past years researchers have developed various MS strategies in order to map epitopes of monoclonal antibodies and some of the main approaches are discussed below. The epitope extraction approach is suitable only for the identification of linear epitopes and consists of three main steps (Zhao and Chalt, 1994). In the first step the antigen is enzymatically digested in order to produce an appropriate set of peptides. The use of proteases with known specificity allows the prediction of the cleavage sites and the rapid identification of the peptides by mass analysis. Moreover, the parallel digestion using two or more proteolytic enzymes with different specificity allows the production of a set of overlapping peptides spanning the entire sequence of the protein, resulting in increased resolution. In the second step the peptides bearing the linear epitope are enriched by immune-affinity selection using a column containing immobilized

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antibodies. The column is thoroughly washed to avoid any unspecific binding. The last step consists of the elution of the bound peptides and their identification by MS. The epitope is deduced by comparing the sequences of the affinity-selected peptides and selecting only the common regions. The epitope excision approach consists of the proteolytic digestion of the antigen non-covalently bound to the antibody (Suckau et al., 1990). It is based on the observation that mAbs show very high resistance to proteases, that the region of the antigen bound to the antibody is protected from digestion, and that the proteolysis does not dissociate the immune complex. This method is very similar to the epitope extraction approach discussed above, with the main difference being that the antigen is first bound to the immobilized antibodies, and then digested with one or more proteolytic enzymes. Also in this case the use of proteases with different specificity can increase the resolution of the technique. After the washes to avoid unspecific binding, the peptides containing the epitope are eluted and identified by MS. A combination of the epitope excision and extraction methods has been used to map the epitope of the mAb 1331A raised against the HIV envelope protein gp120, where a combination of different proteases was used to demonstrate the conformational nature of the epitope (Hochleitner et al., 2000b). Since this approach is relatively easy and does not require a high level of technical sophistication, it can perform high-throughput analyses. However, it also presents a major drawback in the lack of reliability for detecting conformational epitopes. Epitope mapping by differential chemical modification relies on the different solvent accessibility of surface-exposed residues of an antigen in the presence and absence of the antibody, which results in a different relative rate of chemical modification of the amino acids involved in the binding in presence of a labelling reagent (Fiedler et al., 1998). In order to successfully apply this method some key factors need to be considered during the choice of the derivatizing agent, including (i) it needs to be specific for a set of amino acids, (ii) it does not modify the conformation of the protein, (iii) it reacts only with surface-exposed residues, and (iv) it needs to be

compatible with mass spectrometry. Most of the MS-compatible reactive reagents available on the market are specific for lysine, arginine, tyrosine, tryptophan, aspartic acid and glutamic acid. After the labelling, the excess reagent is quenched, the antigen is enzymatically digested and the peptides obtained are identified by MS. The position of the chemical modification can then be determined by tandem MS. The comparison of the degree of labelling in the peptides generated from the antigen alone against the peptides derived from the antigen bound to the antibody allows the identification of the region of the protein protected from the chemical modification. Residues showing a reduced reactivity in presence of the mAb are assumed to be part of the epitope. This approach has been successfully applied to identify the epitope of the HIV core protein p24 (Hochleitner et al., 2000a). In this case the primary amines of the antigen, with and without the antibody, were acetylated under mild conditions in order to modify only the surface-exposed residues. After the purification of the antigens from both conditions, the acetylated proteins were derivatized with hexadeuteroacetic anhydride in denaturing conditions, resulting in the trideuteroacetylation of all the primary amines not previously acetylated. The extent of acetylation/trideuteroacetylation of the peptides derived from the antigen alone and the antigen bound to the antibody was determined and compared in order to characterize the shielding effect of the antibody and identify the epitope. This approach provides more information about the structure of a conformational epitope compared to the epitope excision, but the difficulties in controlling the reaction and the necessary presence of the specific amino acid recognized by the labelling agent within the epitope represent some limitations. Epitope mapping by Hydrogen-Deuterium exchange MS (H/DX-MS) is based on the differential rate of deuterium incorporation by the antigen when it is bound or not by an antibody (Baerga-Ortiz et al., 2002). This technique takes advantage of a natural process occurring when a protein is in solution: hydrogen located on polar side chains or at the N/C termini and bonded to heteroatoms such as –N, –O or –S exchange quite easily with hydrogens in the surrounding

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solvent. This H/H conversion cannot be detected by mass analysis. In contrast, exposing a protein to a D2O-containing environment leads to H/D replacements that increase the mass of the protein by one unit per exchange event (Konermann et al., 2011). The reaction is then quenched using low-temperature and pH conditions, the protein is digested using acidic proteases, usually pepsin, and the generated peptides are analysed by MS. All these steps are performed in an aqueous solution, which means that exchange will continue at a slow rate leading to a partial reversion of deuterated positions after the quench step; this process is referred to as back-exchange. Although H/DX also takes place at side chains, the back exchange of these hydrogens is so fast that is not compatible with the timescale of the experiment, thus only the backbone amide hydrogens, that require a longer time both for the exchange and the back exchange (typically on a timescale spanning milliseconds to hours), are taken into account for the analysis. Every residue (with the exception of prolines and the N-terminal amino acid) possesses an amide N–H group, and therefore H/DX can probe features affecting the entire protein. Isotope exchange is fastest for completely solvent-exposed amides that are not involved in hydrogen bonding and are located on the surface of the protein. Therefore, regions embedded within the structure or occluded by the presence of a ligand, such as an antibody, will exchange more slowly than regions fully exposed to the solvent. H/DX-MS was used to map the epitope of the mAb 12C1 raised against f Hbp in the study partially described above (Malito et al., 2013), clearly showing that the mAb 12C1 binding region involves both N- and C-terminal domains, defining a conformational epitope that includes the region identified by Pepscan and phage display library screening. In the same study, these H/ DX-MS results were confirmed to be in full agreement with the structure of the antigen-binding fragment (Fab) 12C1 complexed with f Hbp determined by X-ray crystallography. Moreover, H/DX-MS has been recently used for the first time to map the epitope of a polyclonal antibody preparation directed against a subunit of the food allergen protein, cashew Ana o 2 (Zhang et al., 2013). Although the analysis was restricted only

to the large subunit (31 kDa) this work clearly shows that Ab sample heterogeneity is no more an insuperable obstacle to H/DX analysis of protein/ protein interactions. Some limitations of H/DX-MS include the analysis of heavily glycosylated proteins in which it may be difficult to identify glycosylated peptides after digestion, and heavily disulfidebonded proteins, which are resistant to digestion and make it difficult to identify peptic fragments. Also, proteins larger than ~100 kDa may have reduced sequence coverage. In this case strategies to reduce the complexity of the antigen without modifying its conformation can be applied, like mutagenesis and mild enzymatic pre-treatment, but this will significantly increase the timeframe of the analysis. Additionally, when using collision induced dissociation (CID) to perform tandem mass spectrometry on the H/D-exchanged proteolytic peptides, gas-phase ‘hydrogen-deuterium scrambling’ (intra-molecular migration of peptide amide hydrogens or deuterons) can occur and may hamper the detailed analysis of amino acid specific exchange rates. Non-ergodic fragmentation techniques with minimal vibrational excitation such as electron transfer dissociation (ETD) or electron capture dissociation (ECD) can be used in HDX-MS experiments to minimize hydrogen scrambling increasing the spatial resolution of the technique, although this approach is still far from routine. X-ray crystallography Since the first crystal structure of an antibody– hapten complex determined almost four decades ago (Segal et al., 1974), and the earliest co-crystal structures of antigens complexed with antibody Fab fragments (e.g. the Fab-lysozyme (Amit et al., 1986; Sheriff et al., 1987) and Fab-neuraminidase structures (Colman et al., 1987)), it has been clear that X-ray crystallography can provide the most unambiguous mapping of epitopes, by providing an immediate and visual definition of the epitope and paratope atoms forming the interface. A cocrystal structure of an antigen–antibody complex can give an atomic-level description of the interface and thus for many this approach represents the ‘gold standard’ for mapping of conformational epitopes (Gershoni et al., 2007). To date, this is

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one of the most powerful and important applications of structural biology in the field of vaccines research. Taking a recent example, and continuing the epitope mapping analyses of f Hbp, the first co-crystal structure of f Hbp bound to a cognate antibody (the Fab fragment of mAb 12C1) was reported recently at 1.8Å resolution (Malito et al., 2013). The structure revealed that both the N- and C-terminal domains of f Hbp engage in extensive interactions with the variable heavy (VH) and variable light (VL) domains of mAb 12C1, and the antigen does not undergo notable conformational changes upon binding. Analysis of the structure showed that 23 f Hbp residues and 33 Fab residues create a large interface, burying surface patches of ~1000 Å on f Hbp and ~880 Å on Fab 12C1. The crystallographically determined epitope on f Hbp includes atoms from the linear peptide identified by Pepscan and phage-display approaches described above, but also includes atoms from many other regions – thus defining a discontinuous, conformational epitope. This structure also confirmed the power of the HDX-MS approach, and showed that linear epitope mapping techniques may provide useful but incomplete descriptions of B-cell epitopes. To date there are over 100 non-similar/nonredundant antibody-antigen (i.e. Fab-protein) complex structures deposited in the protein data bank (PDB), providing a wealth of information about molecular recognition by the immune system (Kringelum et al., 2013). Moreover, on a few rare but notable occasions, X-ray crystallography has also been used for epitope mapping studies of antibody complexes with carbohydrate (as opposed to protein) antigens. For example, the co-crystal structure of Fab 2G12 bound to di- and oligosaccharide mannose moieties from HIV-1 (Calarese et al., 2003). The structure revealed a remarkable VH domain-swapped interlocked dimeric organization of two Fab molecules and a consequently unexpected interaction with the carbohydrate, mediated by an extended antibody–antigen interface. However, X-ray crystallography has a few limitations, such that it cannot be considered a universal solution to reliably answer all questions of epitope mapping in a timely manner. For instance:

(i) the generation of crystals typically requires large amounts of sample material (≥0.1 ml of antigen–antibody complex typically at 5–10 mg/ ml concentration); and (ii) even when sufficient sample is available, there is no guarantee that any protein, or antigen–antibody complex, will actually produce high-quality crystals. Despite this, the use of Fabs or nanobodies (single-domain antibodies) is actually an emerging tool to facilitate the crystallization of recalcitrant proteins, and so it could be anticipated that the probabilityof-success when crystallizing antigen–antibody complexes may in fact be higher than that when crystallizing proteins alone, largely due to the stabilizing effect of the antibody component and the generation of new regions that can provide crystal packing interfaces. While the timelines for the crystallization and structure determination processes can sometimes be very short (days-weeks), they are nevertheless notoriously unpredictable and may even never be achieved. However, the growing ability to produce recombinant Fabs for co-crystallization studies will help to mitigate this issue – providing more shots on goal than possible when using only Fabs derived from standard hybridoma techniques. NMR spectroscopy Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to study biological macromolecules in solution. In particular, heteronuclear NMR studies of proteins can provide information about molecular structure and dynamics, yielding insights into folding, function and associated conformational changes. Moreover, NMR is a very powerful way to study ligand binding events, allowing calculation of binding affinities and the definition of atoms or residues involved in binding. In mapping of linear epitopes, NMR can be used to determine the boundaries of the peptide region recognized by an antibody. This approach is based on the difference in mobility between the bound peptide residues and those adjacent residues that are outside the bound epitope, as described extensively elsewhere (Rosen and Anglister, 2009). More interestingly, if the 3-D structure of the protein is known, NMR can be used to map protein–ligand interfaces, i.e. for the mapping of conformational epitopes. The

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latter is achieved by chemical shift perturbation studies, in which NMR spectra of the protein antigen (usually uniformly labelled with NMR-active isotopes, 15N and 13C) are first recorded and allow chemical shift assignment, such that each peak in the spectrum is associated with a specific atom of the protein. In a second step, the putative ligand or antibody (usually unlabelled) is introduced and, if binding occurs, the ligand induces changes in a subset of chemical shifts corresponding to those atoms involved in the interaction (Gao et al., 2004). For example, the binding site of the bactericidal mAb 502 on f Hbp was determined by heteronuclear NMR spectroscopy, revealing a conformational epitope within a well-defined area of the immunodominant C-terminal domain (Scarselli et al., 2009). NMR spectroscopy can rapidly provide detailed epitope mapping information. However, there are a number of important limitations to the approach that have probably discouraged a more widespread implementation, including: (i) the prerequisite for 3-D structure determination of the antigen (although a good homology model could be a reasonable substitute), (ii) the relatively large amount of material required (≥0.25 ml isotope-labelled antigen in low millimolar concentrations per sample); (iii) the relatively long time, and high-degree of expertise, required to collect the NMR spectra and complete the chemical shift assignment; (iv) the overall size of the antigen–antibody complex. NMR spectroscopy for chemical shift assignment and structure determination can be readily applied to proteins of up to 40–50 kDa. However, the reduced quality of NMR spectra for larger macromolecular complexes, e.g. greater than 60–100  kDa, often renders NMR-based approaches impractical. One partial solution to this issue is to use Fab fragments, rather than whole mAbs, when performing titrations with antigens for epitope mapping. However, even the Fab fragment is ~50 kDa, such that studies of antigen–Fab complexes using antigens greater than ~50 kDa (and thus complexes of >100 kDa) is challenging. A typical strategy employed by NMR spectroscopists in order to overcome this issue is to ‘divide and conquer’ – i.e. instead of using the fulllength antigen, a recombinant protein construct

spanning the smallest relevant domain(s) is used to recapitulate the antigen–antibody complex. This approach provides initial knowledge of the epitope region, and requires the successful production of the domain, either by rational-design or, if such a domain is not readily identified based on amino acid sequence analyses or by inspection of known structures of homologous proteins, by application of emerging ‘domain-hunting’ techniques (Reich et al., 2006; Yumerefendi et al., 2010), which apply high-throughput screening of randomly generated gene constructs in order to reveal highly expressing, stably folded protein subdomains. Cryo-electron microscopy Colloidal gold immunohistochemistry, first introduced in the early 1970s by Faulk and Taylor (1971), is a useful methodology to highlight very specific labelling of both monoclonal and polyclonal antibodies designed to bind specific proteins or cell components. Typically, a secondary mAb is labelled with colloidal gold and is used to localize the primary mAb thus allowing localization of the epitope with respect to larger structures. This approach has been named ‘electron microscopy mapping’ (Claviez et al., 1982). Since Faulk and Taylor the use of antibodies has been extended to negative stain single particle EM firstly to label domains within a complex (Aebi et al., 1977; Nakagawa et al., 2005) and secondly to increase the size of a target protein for better visualization in cryoEM and negative stain EM ( Jiang et al., 2004; Wu et al., 2012). In recent years functionally active monoclonal antibodies (mAbs) and in particular of Fabs have been extensively used in negative stain single particle electron microscopy to determine the location of epitopes in large structures like viruses, cellular organelles and bacterial appendages determined by electron microscopy (Aebi et al., 1977; Clark et al., 2009; Dormitzer et al., 2008) and even for the identification of different conformational states of single proteins forming the complexes. Furthermore, although the resolution of the 3-D structure obtained by negative stain single particle methods cannot reach that generated by X-ray crystallography, single domains of the protein can be identified in the whole structure

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by labelling the protein with relevant monoclonal antibodies and Fabs. The single domain, unless larger than 100 kDa, is not usually distinguishable from rest of the density map, but the differences in the spatial arrangement between the bound and unbound form of the protein can be clearly observed (Nakagawa et al., 2003). Thus, mAbs and Fabs emerge as powerful tools to gain insights into the structure and thus into the physiological mechanisms of large biological complexes. Very recently an important application of cryo-EM in epitope mapping has been developed: single-particle electron microscopy combined with 3-D reconstruction (3-D-EM) (Austin et al., 2012; Cardone et al., 2013; Earl et al., 2013). As discussed previously, an atomic-resolution crystal structure of the antibody/antigen (Ab/Ag) complex is the gold standard for epitope mapping as it allows direct visualization of the interaction between the antigen and antibody and can be regarded as the ultimate description of the epitope and the paratope. However, this method requires large amounts of purified samples and crystallization. In parallel, although NMR does not require crystallization, it requires intensive data collection, data analysis and calculations that can be challenging when spectral quality is low. Both X-ray and NMR become more difficult for larger proteins. The new method of mapping epitopes by single-particle electron microscopy combined with three-dimensional reconstruction (3-DEM), overcomes the limitations present in the classical methods of structural analysis. In spite of its many recent achievements, this method has not been widely used to study proteins smaller than 100 kDa, because it is generally difficult to accurately align images of small particles embedded in vitreous ice. For large macromolecular assemblies like the eukaryotic ribosome, viruses, flagella, each single particle image contains sufficient information for accurate alignment to near-atomic resolution (Armache et al., 2010; Chen et al., 2009; Wolf et al., 2010; Zhang et al., 2010). However, small particles often do not have easily recognizable structural features, thus not allowing accurate image analysis and becoming a major obstacle for high-resolution structural analysis. Many efforts have been invested in developing novel approaches to enable structure

determination of small proteins by single particle cryoEM (Henderson, 1995; Kratz et al., 1999). A valid strategy to overcome this limitation is the selection of a Fab to form a stable and rigid complex with a target protein, thus providing a defined feature for accurate image alignment. By using this approach, Wu and co-workers (Wu et al., 2012) determined a 3-D structure of a ~65 kDa protein. In this study, the authors introduced a new approach using a monoclonal Fab to enable single particle cryoEM studies of small proteins. Moreover, they showed that in absence of a previously known structure the bound Fab provides a fiducial marker with specific features to facilitate image alignment and to validate the final 3-D reconstruction. In the past, Fabs were not used as references for image alignment in single particle cryoEM because of the potential flexibility between the Fab and its target ( Jiang et al., 2004). However, Fabs bound to conformational epitopes forming rigid complexes displayed specific Fab densities in the 3-D reconstruction (Stewart et al., 1997). Wu and co-workers also demonstrated that once a Fab is able to form a rigid complex with its target protein it could be used as marker, although the use of Fabs recognizing a genetically introduced protein tag should be avoided because a linker between a tag and a target protein is too flexible (Sotriffer et al., 2000). Wu and co-workers also suggested a possible solution to an important concern: the potential internal flexibility within a Fab between its variable and constant domains. Fabs are thought to be flexible because there is a wide range of variation in angles between variable and constant domains. These angles, referred as the elbow angles, and corresponding to the angle between the pseudo-2-fold axes relating the VL to VH and CL to CH1 are able to assume a wide range of values, even very large (>195°). These structural differences due to an additional residue present in the λ chain of the switch region could generate more flexibility in the Fab structure, thus enhancing the capability of an antibody to bind to the pathogen surface (Stanfield et al., 2006). It was found, however, that the angle variation of the same Fab crystallized in different conditions is in general less than 15° (Sotriffer et al., 2000) and this small angle fluctuation may not be detectable at the resolutions currently achieved by cryoEM.

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Nevertheless, a Fab forming a rigid complex with its target protein can serve as a point of reference to facilitate image alignment and as an internal control to validate the correctness of the final 3-D reconstruction. In the future this method in conjunction with other developments in the field of cryoEM has the potential of determining correct 3-D reconstructions of small proteins, including integral membrane proteins. Finally, an important application of cryo-EM and of cryo-electron tomography in particular, is the analysis of flexibility of the antibodies (Conway et al., 2003; Jiang et al., 2004). Antibodies, like most of the proteins are not rigid and their flexibility is fundamental to promote their ability to link antigens to immunological effectors. In particular, since the antigen binding sites, also named paratopes, are the most flexible regions of the antibodies, they show an intrinsic freedom to bend and to twist in order to fit with a wide variety of antigens and link them to a relatively reduced number of effectors (Bongini et al., 2005). By comparing the models from a population of flexible molecules, such as IgG, each in a slightly different shape, it is possible to analyse the statistical distribution of shapes (Bongini et al., 2005). Bongini and co-authors developed a model for the potential energy of IgG. By using cryo-electron tomography they noted that the average angle between the Fab arms (110°) differs significantly from the value determined by X-ray (172°) and suggested that the difference is due to the packing environment of the crystal. Such distortion may be generally present in X-ray structures and that the more flexible the protein, the larger and more likely the errors. Closely related to its ability to study molecular dynamics, is the potential for cryoEM to investigate protein interactions. The ability to visualize the binding site and binding dynamics of an antibody with its target antigen has been demonstrated previously (Savage, 2003) using a small (30 kDa) single chain antibody (ScFv) that binds to human complement factor C5. The authors were able to visualize the epitope and confirm that the shape of the bound complex agreed with their modelling results. In a drug development programme, protein imaging studies such as this one could allow the visual selection of the most appropriate antibody from an available

library subset based on the ability to recognize a specific region of a particular antigen. Computational prediction of epitopes for antigen design As reviewed elsewhere (Ponomarenko and Van Regenmortel, 2009; Van Regenmortel, 2009), several epitope prediction methods have been developed so far. However, these did not yet result in finding effective antigenic or immunogenic regions, and it is believed that the main reasons are our somewhat weak or wrong hypotheses on what truly makes a B-cell epitope, or what are the molecular bases for protective epitopes (Van Regenmortel, 2009). However, the constant improvements in structure-based computational methods, which clearly benefit from and depend upon the availability of an increasing number of macromolecular structures, now promise to address these same issues (Gourlay et al., 2009). Specifically, the dramatic growth of available protein structures, as well as the constant evolution and sophistication of computational methods for protein folding and design, can help elucidating the molecular bases of protein–protein and (specifically antigen–antibody) interactions, and these in turn can improve the automation of protective epitope prediction discovery and design. Importantly, automated screenings currently employed for the characterization of antigen properties include studies of their expression level, solubility, stability, aggregation, protease resistance, and epitope presentation (Dormitzer et al., 2008). Instead, methods that reliably allow an automatic and rapid discovery of epitopes or protective domains still lag behind, and as shown above mapping epitopes of an antigen requires extensive experimental work (Ladner, 2007; Malito et al., 2013). Therefore, epitope discovery or mapping is believed to be one of the major areas where structural biology and structure-based computational methods can play a crucial role for accelerating the antigen selection processes (Gourlay et al., 2009; Scarselli et al., 2011). Several examples of the combination of structural biology and computational protein design have been previously described for HIV-1 proteins, where the goal was to engineer scaffolds that could host and show previously known epitopes in a

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more stable and reliable fashion than in their native context. Schief and co-workers recently described computational methods for the design of optimal epitope scaffolds that show high conformational stability and are therefore good candidates for presentation of known structural epitopes to the immune system (Correia et al., 2010). In a similar study, the same group performed the grafting of a discontinuous gp120 epitope onto a scaffold protein not related to gp120, showing binding to an antibody with similar high affinity and specificity to those of gp120 (Azoitei et al., 2011). Also, Kwong and co-workers recently described computer-aided structure-based antigen design to graft neutralizing epitopes of HIV-1 gp41 onto a protein backbone scaffold that is more stable than gp41 (Ofek et al., 2010). Here we review a few examples of recent studies that by combining structural biology and computational methods focus on the identification of protective B-cell epitopes. The aim of this line of research is to elucidate the physicochemical determinants of antibody recognition by an antigen, presenting therefore the prospect of ultimately providing a full understanding of the molecular bases of what makes protective epitopes able to induce the production of functional antibodies, thus enabling an effective immune response. Recently, Bolognesi and co-workers applied computational epitope prediction to the OppA protein from Burkholdeira pseudomallei, a promising target antigen for the development of a vaccine against melioidosis (Lassaux et al., 2013). Starting from the 3-D structure of OppA, the authors discovered antigenic substructures, or epitopes that were later confirmed experimentally to be recognized by antibodies. Once structural information on OppA became available, its surface properties were analysed in silico using two computational methods: (i) electrostatic desolvation profiles (EDP) (Fiorucci and Zacharias, 2010) and (ii) matrix of local coupling energies (MLCE) (Scarabelli et al., 2010). Both these methods are based on analyses of protein surfaces in order to understand the molecular bases of optimal interface formation for protein–protein complexes in general (EDP), and for antigen–antibody complexes (MLCE). EDP exploits the solvation properties of

protein surface regions, and the free energy penalty associated with removal of water molecules from regions that will form the interface in a protein–protein complex (Fiorucci and Zacharias, 2010). First, a protein surface is explored using neutral low-dielectric probes that will perturb the electric field and give rise to an energetic penalty. Electrostatic free energies are then calculated using the finite-difference Poisson-Boltzmann approach. The main advantage of EDP is that the calculated energies are influenced by several factors such as the locally buried area and charge distribution, the shape of dielectric boundary, and most importantly long-range electrostatic interactions. As shown by Zacharias and co-workers (Fiorucci and Zacharias, 2010), most known binding interfaces involved in protein–protein complexes correspond or overlap with regions of low electrostatic desolvation penalty, suggesting that electrostatic or hydrophobic interactions involved in protein–protein interactions generally compensate for desolvation penalties measured in favourable interfaces. Importantly, when EDP was tested for the prediction of antigenic epitopes, most of the known binding regions of antibody– antigen complexes analysed overlapped with regions of low desolvation. While EDP was designed for the identification of general protein–protein interactions, MLCE has been developed specifically to perform computational epitope mapping (Scarabelli et al., 2010). MLCE takes into account both structuraldynamic and energetic properties of a protein surface, in order to find sites that because of their intrinsic low-intensity energetic couplings with the rest of the protein will likely undergo conformational changes, as well as mutations with minimal energetic expense. These are both desirable properties for antigenic epitopes, and will determine the way an antigen–antibody complex forms. For example, conformational changes in the antigen loop regions often influence the formation of such complexes, while mutations in epitope regions with no major perturbation form the basis of the mechanisms of immune evasion, with pathogens that use these to evade recognition by the immune defence system of the host without impairing the stability and original function of the cognate protein. Starting from molecular dynamics (MD)

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simulations to select representative trajectories or optimal ensembles of the antigen of interest, the MLCE algorithm looks for contiguous regions in the 3-D structure that are not significantly coupled to the rest of the protein. This method does not require previous knowledge on antibody binding or of related structures of antibody complexes, and can be applied to any isolated protein antigen and can also be benchmarked with real (experimentally determined) antigen–antibody complexes. Once MLCE discovers regions that being minimally coupled to the rest of the antigen are likely to be involved in protein-protein recognition, two possible scenarios are possible: (i) the introduction of mutations that will not influence the overall stability of the antigen in order to select new sequences with maximum affinity for antibodies; (ii) the engineering of the stable region of the antigen that would allow to use an optimized, likely more stable or dominant conformation of the scaffold. A combination of the two scenarios can ultimately allow the re-engineering of the original antigen, specifically modified for an optimal interaction with the antibody. The crystal structure of OppA at 2.1 Å resolution was first solved by Bolognesi and co-workers, and then used for epitope prediction and design of antigenic peptides. A consensus model of epitopes found by EDP and MLCE included three different minimal epitopes, while experimental epitope mapping found a different set of three more epitopes. All the initial six epitopes found were synthesized as peptides and shown to be recognized by human antibodies from plasma samples of patients recovering from melioidosis, confirming that antigens may contain multiple epitopes and suggesting that the computational methods favoured the identification of different epitope regions. To find a consensus between the computationally and experimentally detected epitopes, a further computational step was introduced, which consisted of domain decomposition to separate a folded protein in small fragments (Genoni et al., 2012). The underlying hypothesis of this method is that the difference in epitopes found might be due to the processing of the antigen after injection in mice, as performed for the experimental epitope mapping experiments. This might have exposed regions of OppA that were otherwise

inaccessible in the fully folded state used for EDP and MLCE. Thus, the decomposition of the antigen in subdomains was utilized to overcome the limitation of EDP and MLCE that only probe the antigen surface, or its solvent-accessible residues. This allowed discovering clusters of subdomains of OppA that mapped to three distinct fragments of the antigen. A further cycle of epitope prediction by EDP and MLCE was then performed on these new fragments, returning new epitopes overlapping with those found by experimental epitope mapping. The main implication of this work is methodological, as it shows that epitope discovery by computational methods can now be reliably performed. Another study that employed and validated the MLCE method has also been recently published by Colombo and co-workers (Peri et al., 2012). Here, epitope prediction was performed on two proteins, FABP and S100B, previously proposed as markers for neurological disorders and damage. Computationally predicted epitopes were first synthesized as free peptides, and once coupled to a carrier protein were used to elicit antibodies in rabbits. Importantly, these antibodies were able to bind the original full-length proteins used for the epitope discovery, as shown by microarray-based experiments. This result suggests that the antigen fragments used for immunization reproduced the key properties of the antigen surface required for antibody recognition. In another similar study, Grandi and co-workers employed computational analysis coupled to experimental epitope mapping methods to find immunogenic domains of two protein orthologues from Chlamydia trachomatis and Chlamydia pneumoniae (CT ArtJ and CPn ArtJ, respectively) (Soriani et al., 2010). Intriguingly, although these proteins share ~60% of sequence identity, they possess different immunogenic properties (Finco et al., 2005). In order to elucidate whether these differences could be explained by structural or dynamic properties, the structure of the two proteins were first solved and then utilized for epitope prediction by the EDP and MLCE methods. Distinct conformational epitope regions for the two ArtJ proteins were predicted, with main differences for possible antigenic regions clustered on one specific domain (the N-C terminal

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domain called D1). This in turn suggested that CPn ArtJ, which overall possesses a larger number of predicted epitopes, could likely be more immunogenic than CT ArtJ. Importantly, the predicted epitope regions overlapped with experimentally determined linear epitope sequences, and this computational analysis suggested qualitative differences in the distribution of antigenic regions between the two domains of ArtJ thus supporting the initial hypothesis that the different immunogenic behaviour that could be associated with distinct physicochemical properties. Consensus models built from the intersection of sets of predicted epitopes, confirmed local differences of the two protein surfaces, such as number of epitopes and surface extension. Additional experimental linear epitope mapping using both polyclonal and monoclonal antibodies were performed, confirming the results of the computational epitope mapping. The main implication of this study is the possibility of better understanding how pathogens deploy antigenic variability to escape immune recognition and at the same time preserve functional properties. As the authors of this study speculate, it is likely that ArtJ remodels its surface in order to diversify the profile of immunodominant epitopes, and this in turn supports the idea of epitope selection as a necessary step in bacterial adaptation. Future directions for epitope mapping in structural vaccinology The sections above have provided an overview of past and present activities in the field of epitope mapping, and the different approaches are also summarized in Table 5.1. However, a small number of recent reports suggest that the field may be undergoing a transition, largely due to the emerging ability to produce large quantities of recombinant mAbs, or Fabs, obtained from humans, via B-cell cloning and sequencing efforts. For example, following immunization, B-cell sorting for antigen specificity, cloning and sequencing can yield a set of antibody sequences specific for the vaccine antigen. In addition, such studies can be performed under different conditions – e.g. following immunization with the antigen formulated with or without adjuvants, thus providing information that can guide vaccine optimization

in order to elicit the desired immune response. Intriguingly, analysis of antibody sequences at various time points after immunization also provides insights into the process of affinity maturation. This has raised the interesting possibility of trying to design antigens that target specific germline B-cell receptors ( Jardine et al., 2013). Remarkably, a recent report has described not only the sequence repertoire of polyclonal antibodies in response to immunization of rabbits with haemocyanin, but has also provided the first quantitative proteomic analysis of the entire antibody set (Wine et al., 2013). This landmark study revealed an oligoclonal response of approximately 30 different circulating antibodies with different sequences, but which cluster into related groups. Such a study would be well-complemented by epitope mapping analyses, thus providing a full quantitative understanding of the diversity of antibodies produced and the range of epitopes recognized by the subset of most immunodominant regions. If epitope mapping is combined with functional studies of these antibody repertoires, these approaches will yield detailed molecular insights into the immune response, aiding identification of the most potently immunogenic and protective epitopes of an antigen. In an iterative process, such insights will provide a solid platform for future design of more effective vaccine antigens. Examples of structural vaccinology for bacterial and viral pathogens Structure-based strategies to improve the protective and immunogenic properties of Group-B Streptococcus pilus 2a backbone protein Group-B Streptococcus [GBS] is the most common cause of sepsis and meningitis in neonates and a vaccine is an unmet medical need (Baker, 1997; Baker and Kasper, 1985; Dillon et al., 1982). GBS pilus proteins were discovered as vaccine candidates while searching for protective antigens using the reverse vaccinology approach (Maione et al., 2005; Margarit et al., 2009). GBS

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Table 5.1  Different epitope mapping methods and approaches Approach

Advantages

No need of purify the antigen/s Synthetic peptide libraries Fast and economical (PepScan) Can be used with polyclonal Abs Fragmentdisplay Approaches

No need to purify the antigen/s Can identify consensus motifs Fast and economical

Drawbacks Identify only linear epitopes

Not reliable for the identification of conformational epitopes (nevertheless can be done)

Can be used with polyclonal Abs MS epitope extraction

High sensitivity (low amount required)

Identify only linear epitopes

MS epitope excision

High sensitivity (low amount required)

MS differential chemical modification

High sensitivity (low amount required)

Difficulties in controlling the reaction

Reliably identify conformational epitopes

Limited resolution (need to have the aa targeted by the used reagent in the epitope region)

H/DX-MS

High sensitivity (low amount required)

Analysis of heavy glycosylated/disulfide bonded proteins

Reliably identify conformational epitopes

Limited resolution due to ‘hydrogendeuterium scrambling’ in CID (can be solved using ETD/ECD but not routine)

Fast and economical Fast and economical

Quite fast

Give information about conformational changes Give information about dynamics of the antigen

Not reliable for the identification of conformational epitopes (nevertheless can be done)

Quite fast (can be automated) Can be used with polyclonal Abs (need some development) X-ray crystallography

Atomic resolution

Low sensitivity (high antigen amount required)

Reliably identify conformational epitopes and paratopes

No guarantee of obtaining high quality crystals (although the use of Fabs can facilitate the crystal formation)

Give information about conformational changes Time consuming

NMR

In rare cases can identify epitopes on carbohydrates

No information about dynamic

Atomic resolution

3-D structure or model of the antigen needed

Reliably identify conformational epitopes

Low sensitivity (high antigen amount required)

Give information about conformational changes Time consuming Give information about dynamics of the antigen Size of the antigen–antibody complex (can be reduced using Fabs complexed to antigen domains) CryoEM

Reliably identify conformational epitopes

3-D structure or model of the antigen needed

Medium-high resolution

Difficulties in the alignment of images from protein smaller than 100 kDa (can be reduced using Fabs to create rigid complexes to facilitate imagine alignment)

Quite fast Can give information of antigens in their biological context High sensitivity (low amount required) Give information about dynamics of the antigen

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pili are constituted by three protein subunits, one forming the shaft of the pilus, the backbone protein (BP), and two ancillary proteins (AP1 and AP2 respectively). All three proteins are covalently linked to each other through pilus related sortase C enzymes (Dramsi et al., 2006; Oh et al., 2008; Sauer et al., 2000; Telford et al., 2006). As all GBS isolates carry pili conferring protection in mice, the combination of the backbone proteins (BP) of pilus 1 and 2b and the 6 BP variants of pilus 2a (BP-2a) can potentially provide a universal-coverage solution for the prevention of GBS infections (Madzivhandila et al., 2013; Margarit et al., 2009). However, the existence of six different variants of the pilus 2a backbone protein, all highly protective in mice, represents a real challenge in vaccine manufacturing. Nuccitelli et al. (2011) using a structural-vaccinology approach, overcame this obstacle, through the generation of a chimeric fusion of the minimal protein region carrying the key protective epitopes of each of the 6 variants of pilus 2a backbone proteins. The chimeric protein was generated by combining structural information with immunological assays, resulting in an antigen able to confer protection in mice challenged with any of the GBS strains carrying pilus 2a variants (Nuccitelli et al., 2011) (Fig. 5.1).

Recently, Nuccitelli et al. (2013) also elucidated the mechanisms by which antibodies raised against each BP-2a variant can neutralize only GBS strains expressing the homologous variant, by combining computational strategies for the modelling of antibodies and functional assays (Nuccitelli et al., 2013). Mouse monoclonal antibodies targeting epitopes of the pilin protein BP-2a 515 variant were generated and three functional mAbs were selected by Flow Cytometry (FACS) analysis and by in vitro opsonophagocytosis assay. The functional mAbs (27F2/H2/H9, 17C4/ A3 and 4H11/B7) were further used for epitope mapping experiments to identify the neutralizing epitope on BP-2a 515 variant. MS-based epitope extraction and epitope excision approaches were used, to target both linear and conformational epitopes (Nuccitelli et al., 2013). The results showed that all the three different monoclonal antibodies recognize the same region of BP-2a515 in domain D3, the same protein region previously characterized as the domain carrying most of the epitopes inducing protective antibody responses, and thus contained in the chimeric fusion protein. Information derived from the mapping of the functional epitope and the crystal structure of BP-2a 515 was then used to perform mAb-antigen molecular docking and molecular

Figure 5.1  Chimeric fusion protein GBS 6×D3 BP-2a. A model of the D3 domains of GBS BP-2a protein from six variants (labelled on top and bottom) is shown as a single fusion protein and with GSGS linkers depicted as thick and darker cartoons. PDB code 2XTL (residues 333–444).

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dynamics simulations, with the aim of predicting the binding of two of the monoclonal antibodies and to characterize the mode of action of neutralizing mAbs on the BP-2a 515 antigen (Nuccitelli et al., 2011). The computational analysis shows that the functional mAbs target the same key residues, Val429 and Asn430, in binding BP-2a 515. These two residues are able to reach the deepest cavity formed by the antibody binding interface, through specific hydrophobic interactions for residue Val429 and hydrogen bonds and polar contacts for Asn430. Moreover, mutagenesis studies of these two residues showed that mutations introducing a drastic perturbation of the shape and electrostatics of the antigen surface inhibit mAb binding (Nuccitelli et al., 2013). Finally, as GBS BP-2a exists as six non-cross-protective variants, the variability of the epitope region identified in BP-2a was investigated, showing how the epitope targeted by the functional mAbs is subjected to sequence variation and selective pressure, highlighting the molecular correlation between BP-2a variability and its variant-specific immunological properties (Nuccitelli et al., 2013). In conclusion, epitope mapping and computational docking analysis of the antibodies in complex with the target antigen and the identification of specific residues in the domain 3 of BP-2a, confirmed and strengthened the importance of D3 domain for immunogenicity and protection capacity of GBS pilus 2a backbone protein. The knowledge of the native molecular architecture of protective determinants might be useful to engineer the antigen for an improved vaccine against GBS infections. In this context, recombinant polymerized backbone pilus proteins, equivalent in structure to a naturally occurring pilus, could contain additional epitopes with respect to monomeric proteins, therefore enhancing immunogenicity. Recently, Cozzi et al. (2013) demonstrated that an efficient polymerization of GBS pilin proteins in high molecular weight (HMW) complexes can be achieved in vitro by using a recombinant sortase C lid mutant, expressed in soluble form and purified from E. coli. This mutant was generated based on structural analysis of the crystal structure of SrtC1 from GBS pilus island 2a (SrtC1-2a) (Cozzi et al., 2011). The SrtC1-2a crystal structure shows

that the aromatic ring of Tyr86 in the lid region interacts with the catalytic Cys219 of the enzyme active site, blocking the enzyme in a closed, auto-inhibited conformation. Starting from these observations, in vitro experiments using recombinant GBS PI-2a SrtC1WT or the engineered lid mutant SrtC1Y86A, mixed with the purified recombinant backbone protein from pilus 2a (BP-2a), have been performed. While the wild type enzyme was totally inactive, the lid mutant SrtC1Y86A was able to efficiently assembly the backbone subunit in high molecular weight polymers, clearly detectable by SDS-PAGE. In agreement with the crystal structure, the mutation of this crucial residue in sortases might break the interaction between lid residues and the catalytic cysteine, making the active site available for substrate binding (Cozzi et al., 2011, 2013). This newly generated active sortase C mutant now opens a broad perspective of applications for characterizing the GBS pilus assembly mechanism and for studying the pilus proteins as vaccines candidates in their native polymerized structure, with the final purpose of further improving their immunological properties. The application of structural vaccinology in the development of a meningococcal antigen inducing broad protection Neisseria meningitidis is a major cause of bacterial septicaemia and meningitis, diseases that can kill children and young adults in hours. Five main serogroups of meningococcal bacteria – A, B, C, W-135 and Y cause the majority of all meningococcal disease cases worldwide (Harrison et al., 2009). Preventative vaccines using a carrier protein conjugated to capsular polysaccharides from all these serogroups are now available, except for serogroup B (Dull and Rosenstein, 2001; Feavers and Pizza, 2009; Sow et al., 2011). A vaccine against serogroup B has been more difficult to develop, due to the low immunogenicity, and potential human cross-reactivity, of the B capsular polysaccharide (Serruto et al., 2012; Sow et al., 2011). Recently, the first broad-coverage proteinbased vaccine for MenB, developed by Novartis Vaccines has been approved by European and Australian authorities (Bioscience, 2013; Carter,

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2013; Gorringe and Pajon, 2012; Riedmann, 2012). This vaccine, called Bexsero®, contains four main antigenic components: factor H binding protein (f Hbp), neisserial adhesin A (NadA), Neisseria heparin binding antigen (NHBA) and outer membrane vesicles from a New Zealand epidemic strain (which provides PorA). Factor H binding protein (f Hbp) is a surface-exposed outer membrane lipoprotein of meningococcus B that is essential for pathogenicity, as it is essential for survival of the organism in vivo (Seib et al., 2009). Around 500 sequence variants of the antigen f Hbp have been identified ( Jolley and Maiden, 2010), which can be divided into three major groups (known as antigenic variants 1, 2 and 3) or two families (Family A, including all variants 2 and 3 and Family B, including variant 1). The cross-reactivity is high within a variant group but unfortunately the 3 variant classes are not cross-protective (Fletcher et al., 2004; Marsh et al., 2011; Masignani et al., 2003). In order to overcome this problem, Scarselli and colleagues used a structure-based approach to design a single antigenic f Hbp molecule conferring protection against all possible MenB strains from the three different classes (Scarselli et al., 2011). The available three-dimensional structure of f Hbp was

analysed using variant-specific monoclonal antibodies, enabling the identification of the amino acidic residues within several epitopes crucial for immunogenicity (Giuliani et al., 2005; Mascioni et al., 2009; Scarselli et al., 2009). Importantly, epitope-mapping analyses revealed that residues contributing to the immunogenicity of variant 1 or variants 2 and 3 were located in non-overlapping areas, leading to the hypothesis that residues from f Hbp variants 2 and 3 could be substituted onto the scaffold of f Hbp variant 1, in order to obtain a cross-protective chimeric antigen (Scarselli et al., 2009, 2011) (Fig. 5.2). Following structural analysis, 54 different chimeric variant 1 proteins were designed, engineered to display epitopes from all three MenB variants on its surface. Each chimeric protein was expressed, purified and their structural integrity was verified by biophysical methods (size-exclusion chromatography, CD and NMR spectroscopy). The inserted groups of point mutations did not disrupt protein architecture, indicating that the designed conformational epitopes were successfully grafted onto the molecular scaffold of f Hbp variant 1. The 54 mutant proteins were used to immunize mice and the collected sera were tested in vitro for the ability to kill seven meningococcal strains expressing

Figure 5.2  Three-dimensional structure of MenB fHbp and mAbs specificities. Dark- and light-grey cartoons depict the N- and C-terminal domains of fHbp, while light- and dark-spheres are used to show the relative position of residues recognized by mAbs elicited by variant 1, or variants 2 and 3, respectively. PDB code 2W80 (chain H).

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different f Hbp subvariants. The most promising chimeric proteins were further tested for their ability to induce protective immunity in mice against an enlarged panel of meningococcus B strains. Three chimaeras were bactericidal against all of the tested strains and the ‘G1’ chimera was identified as the best candidate, inducing high titres against most of the MenB strains. Finally, the f Hbp G1 mutant protein was crystallized and the X-ray structure solved, showing that the mutations in the G1 chimera had indeed generated a new surface displaying broadly protective epitopes without causing major alterations in protein folding (Scarselli et al., 2011). Structure-guided approach for detoxification of vaccine targets There are several other examples in which structural knowledge has been used for formulating effective strategies leading to antigen optimization (Dormitzer et al., 2012; Schneewind and Missiakas, 2011). Bacterial toxins are essential and well known virulence factors, and chemically or genetically detoxified bacterial toxins have been used extensively as vaccines (Halperin et al., 2000; Rappuoli et al., 1995; Robbins et al., 2005). A systematic structureguided approach has the potential to identify suitable mutation sites for eliminating toxin activity while maintaining the structural features necessary for inducing neutralizing antibodies. The structure-based detoxification approach has been successfully applied to the pneumococcal pneumolysin (PLY) and staphylococcal alphatoxin, allowing promising progress on difficult but extremely important antigen targets (Adhikari et al., 2012; Oloo et al., 2011). Streptococcus pneumoniae is one of the most common causes of bacterial meningitis in adults and young adults, and infections by S. pneumoniae cause around 1 million deaths worldwide every year (O’Brien et al., 2009). Conjugate vaccines against invasive disease are effective, but the extent of vaccine coverage is limited to the number of polysaccharides included in the formulations (Munoz-Almagro et al., 2008; Singleton et al., 2007). The development of a universal vaccine against pneumococcal disease is a global health priority and several efforts have been made to

discover and develop vaccines based on conserved surface protein antigens. Several recombinant antigens have been investigated as potential vaccine candidates in different animal models, with promising results (Barocchi et al., 2006; Briles et al., 2003; Gianfaldoni et al., 2007; Goulart et al., 2013; Ogunniyi et al., 2007). Pneumolysin (PLY), a cholesterol-dependent cytolysin, is a major virulence factor of S. pneumoniae (Paton, 1996; Rubins et al., 1996). PLY binds to cholesterol in the membranes of host cells and forms pores, thus causing host cell lysis (Gilbert, 2002; Kelly and Jedrzejas, 2000; Marriott et al., 2008). Purified wild-type toxin injection is sufficient to reproduce many aspects of pneumococcal pneumonia in the immunized animals (Feldman et al., 1991). Given that PLY plays a key role in the pathogenesis of S. pneumoniae infections, this toxin represents a prime candidate for a protein-based vaccine (Malley et al., 2003; Marriott and Dockrell, 2006; Paton, 1996; Rubins et al., 1996). However, since PLY is toxic in its native form, it is not possible to use the wild-type toxin as a human vaccine due to safety concerns. Several less-toxic mutant forms of PLY, named pneumolysin derivatives (Pds), were generated by site-directed mutagenesis or chemical detoxification, and evaluated for their protective effect in different animal models (Alexander et al., 1994; Kirkham et al., 2006). Of those detoxified forms, the most studied are PdB, carrying a Trp-Phe substitution at position 433 (Paton et al., 1991), and PdT, containing three mutations, D385N, C428G and W433F (Berry et al., 1995). Recently, Oloo and colleagues combined bioinformatics, prediction of unknown threedimensional structures, and molecular dynamics simulations to engineer the Pneumolysin into a detoxified antigen (Oloo et al., 2011). They identified and introduced amino acid mutations in PLY, cause the abrogation of its transitioning to the pore-forming state without leading to a completely detoxified form of PLY that retains its ability to elicit neutralizing antibodies against the wild type toxin (Oloo et al., 2011). Firstly, different conformations of PLY were modelled, using as templates the low-resolution structures of PLY and the high-resolution coordinates of its homolog, perfringolysin O (PFO) of Clostridium perfringens (Hotze et al., 2001; Oloo et al., 2011).

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PLY is formed by four domains; inter- and intradomain rearrangements, inducing the switch from the water-soluble, pre-pore, and pore-forming states, have been analysed (Walz, 2005). Rigorous structure-driven approaches have been applied to identify suitable mutation sites. The analysis of contact maps of the soluble and pore-forming conformations of PLY, led to the hypothesis that the substitution of Gly-293 and Gly-294, critical for haemolytic activity, with residues containing side chains larger than Ala, introduce a steric interference that obstruct the progressive transformation of PLY to the pore-forming state (Oloo et al., 2011). Moreover, the authors introduced disulfide linkages to induce conformational trapping, as this approach was successful for abrogating the pre-pore complex conversion into the pore-forming state of the PFO toxin (Hotze et al., 2001). Disulfide bridges were introduced between cysteine-substituted residues resulting in disulfide-constrained mutants. Nine in silico designed mutants were expressed as recombinant proteins, seven trapped-mutants were completely inactive, and their haemolytic activity was recovered only in presence of reducing conditions, when the disulfides were reduced (Oloo et al., 2011). The detoxified mutants were successively tested for their ability to elicit antibodies that neutralized the wild-type toxin, to be sure that the mutations inserted to eliminate toxicity, do not interfere with the correct presentation of functional epitopes. The PLY(T65C,G293C,C428A) mutant, which is detoxified by both mechanisms, disulfide constraints and steric interference, was the most promising in terms of antibody response, and it is currently in clinical trials (Oloo et al., 2011). A second example of detoxification of an antigen through structural knowledge, is the staphylococcal alpha-toxin Hla, which represents a prime candidate as a component of a Staphylococcus aureus vaccine (Bubeck Wardenburg and Schneewind, 2008; Kennedy et al., 2010; Ragle and Bubeck Wardenburg, 2009). Mutations that attenuate or completely abrogate the lytic activity of Hla are absolutely necessary, as the wild type antigen is not considered safe for use in a human vaccine (Bubeck Wardenburg and Schneewind, 2008; Kennedy et al., 2010; Menzies and

Kernodle, 1996; Ragle and Bubeck Wardenburg, 2009). Although the non-cytolytic mutant form of Hla (HlaH35L) can be used as protective antigen against S. aureus infections (Kennedy et al., 2010; Menzies and Kernodle, 1996), an alternative strategy is the identification of subdomain mutants of Hla, capable of inducing protective immunity (Adhikari et al., 2012). The high resolution crystal structure of S. aureus alpha-haemolysin showed that Hla is a heptameric trans-membrane pore with the N-terminal domain located on the top of the pore (Song et al., 1996). Adhikari et al. (2012) applied a rational, structure-based approach, to design engineered recombinant α-haemolysin vaccine candidates. The N-terminal domain of Hla is composed of four anti-parallel β-strands (Ragle and Bubeck Wardenburg, 2009), the linear sequence of residues 1–62 contains three of the β-strands, while the fourth β-strand is composed by residues 228–234; this domain is considered a potential vaccine candidate itself, as it contains protective epitopes (Ragle and Bubeck Wardenburg, 2009). In theory, antigens designed to contain the N-terminal region in a stabilized structure could be effective and safe vaccine candidates. Structural information was used to analyse the Hla N-terminal domain, focusing on the residues 1–50, 1–62 or the fusion of residues 1–62 to 228–236, containing all the four anti-parallel β-strands (Adhikari et al., 2012). The in silico analysis suggested that the new 1–62 construct is predicted to be more stable than the previously reported 1–50 construct (Ragle and Bubeck Wardenburg, 2009). In order to design a chimeric Hla N-terminal domain containing all the four anti-parallel β-strands, residues 1–62 were fused to the residues 223–236. Six fusion proteins with different linkers were generated by molecular modelling (Adhikari et al., 2012). From the simulation, the linkers composed of more than three glycines resulted to confer the lowest energy to the (1–62)-(223–236) fusion protein structure, thus improving the folding and stability of the protein. The results of the modelling predicted that the 1–62 construct (named AT-62aa), the fusion of 1–62-(GGG)-223–236 (named AT-79aa), as well as fusions containing more than three glycines would fold into functional domains (Adhikari et al., 2012). The constructs AT-50aa

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(residues 1–50 construct) and AT-62aa, and the fusion AT-79aa were produced as recombinant proteins in E. coli. Moreover, several recombinant fusion proteins containing more than three glycine residues in the linker region were also expressed and the fusion containing 5 glycine residues in the linker resulted to be the best one, in terms of protein folding. Finally the AT50aa and AT-62aa truncated constructs and the new fusion AT-79aa (1–62-G5-223–236), which replaced the construct with 3 Gly, were tested as vaccine candidates in two infection models of S. aureus, with the 1–62 and AT-79 constructs, showing total protection and higher immunogenicity with respect to the AT50aa construct. In particular, the AT-62aa construct represents a reduced toxicity version of the Hla antigen that could be used for the future development of a safe and efficient vaccine against S. aureus infection (Adhikari et al., 2012). The recombinant AT-50aa protein was unfolded, as no β-sheets could be detected by circular dichroism analysis, while the fusion protein AT-79aa is partially folded, as evidences of both β-sheets and α-helices were detected. The β-sheet content of these recombinant proteins is strictly connected to the ability of the two proteins to induce neutralizing antibodies (Adhikari et al., 2012). The hypothesis put forward by Adhikari and co-workers is that the partial misfolding observed in the AT-79aa is due to the linker region connecting the third beta sheet of the N-terminal domain to the distal fourth sheet, portion that is not included in the AT-62aa construct. Optimization of the linker region could improve the folding of the AT-79aa protein, with the hope of obtaining the complete four β-strand structure with the potential of higher immunogenicity with respect to the AT-62aa protein. In addition to alpha-haemolysin, S. aureus also produces other pore-forming toxins including leukotoxins such as Panton–Valentine leukocidin (PVL) and LukED (Lina et al., 1999). Recently, Karauzum and co-workers followed the same approach used for structure-detoxification of alpha-haemolysin described above, to generate highly attenuated mutants of PVL subunits LukS-PV and LukF-PV (Karauzum et al., 2013). Based on an octameric structural model of the toxin (Aman et al., 2010), point mutations were

designed to disrupt the oligomerization of the S and F subunits, which is a pre-requisite for pore formation, while maintaining the overall structural integrity and immunogenicity of the wild type subunits (Karauzum et al., 2013). Consequently, the authors proposed that attenuated forms of LukS-PV and/or LukF-PV could potentially be used to develop a vaccine against S. aureus infection without risks associated to active pore-forming toxins. Structural vaccinology for viral pathogens Viral pathogens pose a particularly challenging target for vaccination. Generally, viruses contain a small set of viral proteins whose functions relate to cell entry, replication, immune evasion, and viral structure. A virus is dependent on cellular machinery for a number of functions so viral entry is a key step that viral vaccines aim to block. Depending on the virus, humoral and cellular immunity can play differing roles in the generation of an effective immune response. Owing to the small number of viral proteins, it may be assumed that the generation of a viral vaccine would be straightforward since there are only very few relevant immunogenic targets. However, viruses have multiple immune evasion mechanisms some of which have been outlined in great detail for HIV and Influenza (Kwong and Wilson, 2009). To highlight the role of structural vaccinology in antiviral vaccine design we will focus on recent approaches to address the generation of a universal influenza vaccine and a respiratory syncytial virus (RSV) vaccine. Influenza is a constantly evolving viral pathogen and, despite the availability of safe and effective vaccines, is a major cause of morbidity and mortality especially in the young and elderly populations. Beyond the burden of seasonal infections, the importance of influenza is underscored by the fear that a new pandemic will arise with results similar to that of the 1918 pandemic that claimed more than two million lives (Wright et al., 2007). Influenza evolution is marked by antigenic drifts (where particular dominant epitopes on a circulating strain vary) and shifts (where gene segments transferred between strains results in a new variant) resulting in a constantly changing target

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for the immune system. Further, zoonoses from animal reservoirs (e.g. birds, pigs) as represented by H5N1, the so called ‘bird flu’, or the recent H7N9 outbreak shows that the threat of a new pandemic is very real. Structural biology had been applied for many years to influenza resulting in key discoveries relating to the functional role of the main viral antigen and surface-exposed, entry protein, haemagglutinin (HA) (Wilson et al., 1981). In 2009, the first structures of a broadly neutralizing antibody (CR6261) to HA were discovered giving hope that a universal influenza vaccine based on antibody responses could be generated (Ekiert et al., 2009). The epitope is in the conserved stem region of HA rather than the traditionally immunodominant and variable head region (Fig. 5.3). Using the structure of the CR6261 Fab/ HA complex as a guide, several research groups have attempted to generate immunogens aimed at focusing the immune response on the broadly neutralizing stem epitope. One set of approaches rely on the generation of constructs that lacked the variable head region, preserving only residues

in the stem. Animals immunized with these ‘stem only’ constructs showed some immune response but lacked substantially improved breadth. The stem region is a complicated folded domain whose role in entry is to significantly change conformation, thus a properly folded ‘stem only’ construct may be challenging to obtain. The elucidation of the broadly neutralizing antibodies was paralleled temporally with two other events that suggested other structural approaches to universal influenza vaccine design: the appearance of the 2009 pandemic H1N1 virus and improvements in B-cell repertoire analysis. The 2009 H1N1 pandemic virus was a zoonosis from swine introducing an H1 variant of HA that shows very few conserved residues in the head region, but high conservation in the stem region (Wei et al., 2010). Researchers analysed the B-cell repertoire of patients infected with H1N1 pandemic virus and discovered that it was easier to find B-cells expressing broadly neutralizing antibodies than when looking in individuals that had not been exposed to the virus (Wrammert et al., 2011). Furthermore, many of these antibodies

Figure 5.3  Crystal structure of the complex between influenza haemagglutinin and the Fab of the human antibody CR6261. The complex is shown with dark cartoons for the Fab CR6261 and light grey for HA. Head and proximal-membrane stem regions of HA are labelled, to highlight how CR6261 binds to the base of the stem. PDB code 3GBN.

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were directed to the broadly neutralizing stem epitope. This led to the hypothesis that a vaccination strategy involving sequential immunization using immunogens that only shared broadly neutralizing epitopes could have the effect of boosting a pre-existing B-cell response to these conserved regions (Settembre et al., 2011). This concept has been demonstrated through sequential immunization of mice using haemagglutinins containing heads from exotic influenza viruses fused to identical stem regions. Mice immunized with these constructs showed both increased anti-stem antibody titres and expanded breadth of protection in challenge models (Krammer et al., 2013). A recent example of a structural vaccinology success is the generation of a stable pre-fusion F protein antigen from RSV, the leading cause of infant hospitalization in developed countries. The F protein has multiple relevant conformational states as required for viral entry and, when expressed recombinantly, favours states other than the pre-fusion state. Much like HA, a prefusion form is functionally required for viral entry and hence the form on the virus to which it would be valuable for a vaccine to elicit a strong neutralizing response. McClellan and colleagues (McLellan et al., 2013b), determined the structure of prefusion RSV F bound to a neutralizing antibody (D25) and used it as a starting point for antigen design. The structure revealed the presence of a metastable neutralizing site (0) not present in the

post fusion antigen. They used an iterative cycle of antigen design, protein expression, small scale analyses of key characteristics, and immunization to generate improved antigens (McLellan et al., 2013a). They discovered that those antigens that were more stable resulted in higher neutralizing titres post vaccination in animal models. This effort demonstrates a practical methodology, given the appropriate reagents, to generate improved antigens in a direct manner. Traditional vaccine design has failed for many of the remaining viral pathogens. The strategies described above are examples of how structural knowledge can be used, combined with techniques to analyse the immune system, to generate novel immunogens not only for influenza and RSV but, more broadly, to any virus that evolves quickly. Perhaps structural vaccinology, with its root in understanding viral protein structure and function, may be a way to finally generate vaccines for these viral pathogens. Concluding remarks and future directions In this chapter we have provided an overview of how structural biology can be applied to drive the design of vaccine antigens (Fig. 5.4). One particular area that can yield important insights is that of epitope mapping, and here we have outlined several different practical approaches to achieve that

Figure 5.4  Flow chart of the structural vaccinology concepts.

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goal. Our comparison of the various techniques underlines the power of X-ray crystallography and clearly highlights the growing importance of EM and H/DX-MS as emerging techniques with strong advantages, both bringing a great reduction in the amount of sample required for analysis (approximately three orders of magnitude less than for crystallographic studies) and also promising significantly increased throughput (summarized in Table 5.1). In addition to these technological advances in epitope mapping made in recent years, the increasing number of antigen and of antigen– antibody complex structures present in the PDB has provided a platform for the development of computational pipelines that exploit antigen structures to quickly screen their structural and dynamic properties to predict putative epitopes. Outcomes of these analyses will ideally allow the formulation of large libraries of B-cell epitopes that could then be formulated as antigenic peptides, and these could in turn be used for immunizations. However, the presentation of isolated antigenic peptides has met with limited success to date, likely due to the difficulty of faithfully recreating an immunogenic conformational epitope outside the context of the fully folded native protein. Nevertheless, as described above, by combining structural and computational modelling approaches, there appears to be the growing possibility that immunogenic peptides can be mounted on selected scaffold proteins with desirable properties. In addition to the presentation of protective peptide epitopes grafted onto a single ‘scaffold’ protein, there are also some interesting possibilities raised by the presentation of full antigens (or folded antigen domains) in multi-valent formats. Recent examples include the structure-guided design of an influenza haemagglutinin HA–ferritin fusion protein that allowed presentation of HA on a self-assembling ferritin nanoparticle, resulting in increased immunogenicity, neutralization potency and breadth of protection compared to trimeric HA or a traditional trivalent influenza vaccine (TIV) formulation containing equimolar HA (Kanekiyo et al., 2013). Following the same principle of increasing antigen performance through greater valency in a large particle format,

an HIV gp120 outer-domain immunogen presented on a lumazine synthase nanoparticle of 60 subunits also showed enhanced performance ( Jardine et al., 2013). It is therefore possible to envisage how the well-established structural techniques, and the emerging ones, described here could be combined with upraising computational advances and innovative antigen presentation strategies, in order to dramatically simplify the processes of antigen optimization in future vaccine design and development projects. We anticipate that the identification of epitopes based on antigen structures and their physicochemical properties, as well as a detailed molecular knowledge of epitopes, will eventually allow the development of tailor-made neutralizing antibodies able to provide protection against many challenging pathogens. References Adhikari, R.P., Karauzum, H., Sarwar, J., Abaandou, L., Mahmoudieh, M., Boroun, A.R., Vu, H., Nguyen, T., Devi, V.S., Shulenin, S., et al. (2012). Novel structurally designed vaccine for S. aureus alpha-hemolysin: protection against bacteremia and pneumonia. PLoS One 7, e38567. Aebi, U., ten Heggeler, B., Onorato, L., Kistler, J., and Showe, M.K. (1977). New method for localizing proteins in periodic structures: Fab fragment labeling combined with image processing of electron micrographs. Proc. Natl. Acad. Sci. U.S.A. 74, 5514–5518. Alexander, J.E., Lock, R.A., Peeters, C.C., Poolman, J.T., Andrew, P.W., Mitchell, T.J., Hansman, D., and Paton, J.C. (1994). Immunization of mice with pneumolysin toxoid confers a significant degree of protection against at least nine serotypes of Streptococcus pneumoniae. Infect. Immun. 62, 5683–5688. Aman, M.J., Karauzum, H., Bowden, M.G., and Nguyen, T.L. (2010). Structural model of the pre-pore ring-like structure of Panton–Valentine leukocidin: providing dimensionality to biophysical and mutational data. J. Biomol. Struct. Dyn. 28, 1–12. Amela, I., Cedano, J., and Querol, E. (2007). Pathogen proteins eliciting antibodies do not share epitopes with host proteins: a bioinformatics approach. PLoS One 2, e512. Amit, A.G., Mariuzza, R.A., Phillips, S.E., and Poljak, R.J. (1986). Three-dimensional structure of an antigen– antibody complex at 2.8 A resolution. Science 233, 747–753. Anderson, A.S., Jansen, K.U., and Eiden, J. (2011). New frontiers in meningococcal vaccines. Expert Rev. Vaccines 10, 617–634. Armache, J.P., Jarasch, A., Anger, A.M., Villa, E., Becker, T., Bhushan, S., Jossinet, F., Habeck, M., Dindar, G., Franckenberg, S., et al. (2010). Localization of

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Cellular Screens to Interrogate the Human T- and B-cell Repertoires and Design Better Vaccines

6

Jens Wrammert and Kaja Murali-Krishna

Abstract The success of vaccines depends upon their ability to induce memory T-cells, B-cells and long-lasting antibody-secreting plasma cells. These memory cells differ in quantity, quality, homing characteristics and persistence depending upon vaccine antigen, adjuvants and delivery systems. A thorough understanding of the receptor repertoire associated with protective immunity, and in some cases infection-driven pathology, or in situations such as autoimmunity, cancer, allergy or transplant rejection, is important for rational design of vaccines and therapeutics. In this chapter we will first briefly introduce general processes involved in the generation of naive T- and B-cell receptor repertoire and the complex functional changes that can occur in these cells when they encounter antigen. Following this, we will discuss recent progress in the development of novel tools for Band T-cell receptor repertoire analysis combined with cellular states and functions. The concurrent development of genomics and detailed functional analyses, both on a global as well as at a single cell level, also allows us to gain additional insight into the mechanisms of immunological memory. Together, these cellular screens allow defining optimal antigens and adjuvants and ultimately enable the development of more efficacious vaccines. Introduction Vaccine development relies on understanding the complex interplay between pathogen and their mammalian host. Most successful vaccines in use today, have been developed by immunizing the

host with attenuated pathogens capable of inducing protective immunity against future infections without causing disease. The success of these vaccines depends upon their ability to induce both memory B- and T-cells (Ahmed and Gray, 1996). The memory cells, and their products, contribute to sterilizing immunity, or elicit rapid recall responses, if there were to be a re-infection. It is interesting to note that historically, some of the most successful vaccines developed, such as the small pox vaccine, were successful even though the scientists had had little knowledge at that time about the disease-causing organism or of the host immune system (Plotkin and Orenstein, 2013). In spite of the overall success of vaccination versus many pathogens, developing vaccines against several of emerging and remerging infectious agents with a complex set of immune evasive strategies have proved to be challenging. We still do not have efficacious vaccines against several infectious agents including, but not limited to, HIV, tuberculosis, rotaviruses, many respiratory viruses, malaria, several flaviviruses and arboviruses such as dengue, Japanese encephalitis and Chikungunya virus (Plotkin and Orenstein, 2013). Fortunately, the past few years have seen great scientific advances, revealing the complexity of microbial structures, the host immune system and their interplay. One of the critical aspects of these recent advances is the detailed knowledge about receptor repertoire usage of T- and B-cells, which is the focus of the discussion in this chapter. An average human produces millions of naive T- and B-cells each day, each of these cells equipped with a unique receptor. The individual receptors expressed at the population level are referred as

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the receptor repertoire (Pannetier et al., 1995). A majority of the cells bearing receptors capable of recognizing self-antigens are deleted soon after formation, through central tolerance mechanisms (Hogquist et al., 2005; Kyewski and Klein, 2006; LeBien and Tedder, 2008). Those cells that do not recognize self-antigens are maintained for a substantial time. These cells circulate through secondary lymphoid organs surveying for foreign antigens. Although these circulating naive T- and B-cells have a diverse receptor repertoire, the actual frequency of naive cells bearing a specific receptor to a given antigen is extremely low ( Jenkins and Moon, 2012). When these cells encounter foreign antigens presented along with clues of infection, they undergo clonal expansion, convert into effector cells and contribute to eliminating the infection. Once the infection subsides, some of these antigen-stimulated cells differentiate into memory cells (Pulendran and Ahmed, 2011). The memory cells persist for longer periods at heighted frequencies and are poised to elicit recall responses much more rapidly than their naive counterparts. Because of their qualities, the memory cells can confer life long protection against re-infection. Vaccines, in which a pathogen or pathogen-derived foreign antigen is used along with cues of infection (e.g. adjuvants) similarly activate naive T- and B-cells, some of which can potentially differentiate into long lasting memory B- and T-cells. A thorough understanding of the receptor repertoire associated with protective immunity, and in some cases infection-driven pathology, or in situations such as autoimmunity, cancer, allergy or transplant rejection, is important for rational design of vaccines and therapeutics. In this chapter we will first briefly introduce general processes involved in the generation of naive T- and B-cell receptor repertoire and the complex functional changes that can occur in these cells when they encounter antigen. Following this, we will discuss recent progress in the development of novel tools for Band T-cell receptor repertoire analysis combined with cellular states and functions. The concurrent development of genomics and detailed functional analyses, both on a global as well as at a single cell level, also allows us to gain additional insight into the mechanisms of immunological memory.

Together, this will allow us to define optimal antigens and adjuvants and ultimately enable the development of more efficacious vaccines. Additionally, some of these new tools provide a large selection of human monoclonal antibodies, some of which can be directly used for both therapeutic and diagnostic purposes in humans. Generation of naive B- and T-cell repertoires B-cells develop in the bone marrow in a series of carefully orchestrated steps that balance the almost infinite amounts of possible rearrangements and modifications, with stringent central tolerance mechanisms (Pieper et al., 2013). Naive B-cells are derived from haematopoietic stem cells (HSC) expressing VDJ recombinases RAG-1 and RAG-2 in bone marrow. Although all the nucleated cells in our body carry identical germline BCR loci, these germline genes cannot be transcribed into mRNAs that encode functional antigen receptor proteins. Functional B-cell receptor (BCR) genes are only created by somatic rearrangement with the help of RAG-1/2 expressed in developing cells in the bone marrow (LeBien and Tedder, 2008). These recombinases initially help rearranging immunoglobulin heavy chain (IgH) loci. In the pre B-cell stage functional IgM heavy chain polypeptides associate with a surrogate light chain leading to the formation of a pre-BCR, which is essential for cell survival and proliferation. Exiting the cell cycle leads to their transition into pre-BII-cells that initiate rearrangement of the κ or λ light chain loci. Functional rearrangement of these loci and successful immunoglobulin assembly results in B-cells with a mature BCR that is capable of binding to antigen. The infinite combinations created through V-D-J recombinationandassociationofIgheavychainwith or λ light chains results in a highly diverse B-cell receptor repertoire ( Janeway et al., 2001), a majority of which is directed against self-antigens. A majority of these auto-reactive immature B-cells are either physically deleted or inactivated by immunological tolerance mechanisms in the bone marrow itself. The remaining B-cells passes through transitional B-cell phases characterized by phenotypic changes and surface IgM/IgD

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expression and exit into the periphery wherein they undergo a second level of peripheral immune tolerance processes before entering the mature naive B-cell pool (Meffre and Wardemann, 2008). As important as these mechanisms are to maintain tolerance and prohibit auto immunity, both the central and peripheral tolerance mechanisms are not absolute. In fact, both the naive and memory B-cell compartments contain a surprisingly large amount of auto-reactive specificities (Shlomchik, 2008; Tiller et al., 2007; Yurasov and Nussenzweig, 2007). An understanding of the dynamic changes in B-cell receptor repertoire associated with these developmental processes, the escape of the autoimmune repertoire that can potentially cause autoimmune diseases, and the ability of humans to maintain a broad and efficient repertoire able to respond to infectious challenge is only beginning to be understood. Similar to B-cell development, the human immune system produces millions of T-cells in the thymus each day. Each of these cells is equipped with a unique T-cell receptor (TCR), composed of a unique combination of α and β chains (Koch and Radtke, 2011). The recombinase mediated TCR germline rearrangements allow bringing randomly chosen TCR germline V, (D), and J gene segments into contiguity, forming functional genes encoding TCR a and b chains. Such germline recombination machinery generates huge number of permutations and combinations leading to expression of functional TCR genes, whose diversity is expected to exceed 20 million combinations in an individual. After successfully rearranging a β-chain gene, a developing T-cell immediately switches off the recombination machinery, with the result that there is allelic exclusion at the β-chain locus to ensure that the TCR expressed by each developing T-cell is equipped with a single rearranged TCR b-chain and an a chain. These developing T-cells, each equipped with a complete TCR, then encounter several checkpoints in the thymus. Their fate is largely determined by the strength of signal perceived by the antigen receptor. CD4+CD8+ double-positive (DP) thymocytes with low affinity for self-peptide MHC (pMHC) ligands undergo positive selection, whereas those with high affinity undergo negative selection (Wakim and Bevan, 2010).

Because the TCR α-chain gene is not subject to allelic exclusion in the same way as the β-chain gene, rearrangements can occur at both copies of the α-chain locus, and double-positive thymocytes can potentially express two different α chains thereby leading to the generation of some T-cells with two different types of TCRs. However, the proportion of T-cells bearing two different types of TCR with each receptor succeeding positive selection is expected to be very small. Thus, the majority of mature T-cells exhibit one receptor, and among those that express two receptors, one receptor is expected to be practically non-functional (Brady et al., 2010). Those naive T-cells that successfully withstand the stringent thymic selection processes then exit into lymphoid circulation and continue to survey for foreign antigens. The enormous diversity in the T-cell receptor repertoire is instrumental in generating an immune response to virtually any foreign antigen that can be processed into peptides that bind to MHC molecules. But the frequency of naive T-cells specific to a given antigen is expected to be extremely low, usually in the orders of one in millions ( Jenkins and Moon, 2012). An understanding of which of these naive T-cells, each with a unique TCR, are actually involved in a productive immune response is important for rational design of vaccines and immunotherapeutic strategies. Hence there is large amount of interest focused on probing the human TCR repertoire associated with protective immunity. Shaping of the receptor repertoire of B- and T-cells during immune responses Once past the central and peripheral tolerance checkpoints, a naive B-cell is able to respond when it encounters an antigen that fits to its receptor specificity along with appropriate costimulatory signals and T-cell help (Packard and Cambier, 2013). These responses occur primarily in peripheral lymphoid organs, where B-cells can differentiate along several different pathways, leading to the generation of either short-lived plasmablasts, long-lived plasma cells or memory B-cells (Kalia et al., 2006; Victora and Nussenzweig, 2012) (Fig. 6.1). The plasmablasts and plasma cells actively secrete their immunoglobulin

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Short-lived plasmablasts

Memory B cells long-lived circulate in blood

Antigen re-exposure

Germinal Center Reaction Antigen And T-cell help

Naïve B cells

Activation and proliferation

Memory plasma cells isotype switched, somatic hypermutation & affinity maturation long-lived ; home to Bone Marrow

Figure 6.1 General schematic outline of the BCR repertoire shaping during an immune response. The naive B-cell pool with a highly diverse receptor repertoire circulate through secondary lymphoid organs. When a particular B-cell receptor is engaged in the context of appropriate co-stimulation, that B-cell clone undergoes activation, expands, and differentiate into antibody secreting plasmablasts. The plasmablasts are generally short-lived and circulate through blood compartment. Some of the activated B-cells also undergo germinal centre reaction with appropriate T-cell help. During the germinal centre reaction the B-cells and their receptors are subjected to several changes. The receptors can change their isotype, or improve/alter specificity through somatic hypermutation or affinity maturation processes. Consequently, the receptor repertoire of the B-cells emerging from the germinal centre reaction need not be identical to that of the initial B-cell clone from which they originate. Thus, it is important to consider cellular states and functions while assessing the receptor repertoire. Some of the B-cells emerging from germinal centre reaction differentiate into long-lasting plasma cells that home to bone marrow and constitutively secrete antibody. Some of them differentiate into memory B-cells that do not secrete antibody, but circulate through secondary lymphoid organs at heightened frequencies and poised to elicit faster and efficient recall responses by rapidly differentiating into plasmablasts or plasma cells upon encounter with antigen.

receptors in the form of soluble antibody, which can migrate to other compartments of the body away from the B-cell from which it originated; and are thus capable of mediating different effector functions (e.g. complement fixation, opsonization, neutralization) depending upon the type of antibody made (e.g. IgG, IgA, IgM) and the location and the configuration of the antigen it encounters (e.g. systemic antigens vs. mucosal antigens, surface antigens versus soluble antigens). Long lived plasma cells primarily take up residence in the bone marrow and continually secrete soluble immunoglobulin receptor (antibody), and are, thus, responsible for maintaining

humoral immunity over long periods of time, in some cases likely life long (Slifka and Ahmed, 1996; Slifka et al., 1998; Wrammert and Ahmed, 2008). Memory B-cells, unlike plasma cells, do not secrete their immunoglobulin receptor. Instead, these cells circulate through secondary lymphoid organs so as to rapidly respond with greater vigour in case of a re-exposure to the pathogen, using its preselected BCR repertoire. The receptor repertoire of those B-cells selected to respond against a given antigen/pathogen also dynamically changes with each exposure to antigen through germinal centre reaction, somatic hypermutation, affinity maturation and isotype switching processes

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(Victora and Nussenzweig, 2012). Together, all of these mechanisms contribute to the conversion of the selected BCR repertoires to an improved configuration that better suits the pathogen. Such improvement in the receptor repertoire of those B-cells selected to respond against a given antigen/pathogen allow our immune system to respond with greater vigour against the pathogen or prepare better in anticipation of a changing pathogen. Thus, an understanding of the key differences in the repertoire of long-lived memory B-cells and plasmacells, and the dynamic changes in these compartments are important for a better understanding of protective humoral immunity. Similar to naive B-cells, mature naive CD4 and CD8 T-cells, upon exit from the thymus, continue surveillance of the peripheral immune system for encounter with foreign antigens. The enormous diversity in the T-cell receptor repertoire of these circulating naive T-cells allows an immune response to virtually any foreign peptide antigen that can be processed and presented by the MHC molecules to which the T-cell is restricted. It is estimated that the frequency of naive T-cells capable of recognizing a given peptide ranges anywhere between 1 and 200 per million cells ( Jenkins and Moon, 2012). But even among the pool of naive cells specific to a given peptide, each cell may not necessarily bear identical receptor. Indeed, within a population of T-cells recognizing a given peptide, a highly variable TCR repertoire can be found (Blattman et al., 2000). Thus, the T-cell arm of the immune system can respond to an infinite variety of antigens, including the possibility of self-antigens, foreign antigens and altered antigens produced during developmental changes. Immune responses by the potentially self-reactive T-cells that escape thymic negative selection are generally hampered by several peripheral tolerance mechanisms including the ones mediated by Fox-p3+ regulatory (Treg) cells (Mueller, 2010). When a foreign antigen is presented in the context of appropriate co-stimulation and inflammatory pathways, naive cells become activated, clonally expand, alter their circulatory properties and exert effector functions (Fig. 6.2). Some of the clonally expanded effector T-cells are short-lived, whereas others differentiate into long-lasting memory cells that can elicit rapid

recall responses if there were to be a secondary exposure to the same antigen/pathogen (MuraliKrishna et al., 1998a,b). If the initial response is insufficient to eliminate the infection, as seen in the case of persistent/chronic infections, the effector cells continue fighting the infection – but in the process they may become functionality exhausted or physically deleted (Blattman et al., 2000), leading to further changes in the repertoire. As a consequence of these processes, it would become important to understand the dynamic changes in the receptor repertoire of the T-cells during the course of an immune response. Because the functionality of a TCR is linked to the effector functions of the cells bearing that receptor, it is important to understand what TCR specificity is associated with what cellular functions; and what changes in cellular functions occur depending upon the stage/status of the immune response (Seder et al., 2008). Which of the effector functions are exerted by a T-cell bearing a particular receptor is not simply dependent upon the antigen or the receptor, but highly dependent upon the context of pathogen, the context of the MHC through which the antigen is presented and the status of the T-cell recruited into the response. CD8 T-cells generally recognize MHC class-I presented antigens and differentiate into cytotoxic effector T-cells (CTL) capable of killing the cells that they recognize. CD4 T-cells, which recognize antigens presented by MHC-class-II and result in the generation of different types of effectors depending upon the context: Th1 effector cells, which are generally protective against intracellular bacteria and viruses, Th2 cells which are protective against nematodes but also responsible for allergic reactions, and Th17 cells which appear to be evolved for conferring protection against extracellular bacteria (Zhu et al., 2010). Each of these different effector cells produce and secrete their own signature cytokines upon engaging their receptors, thus leading to a different effector response. Thus, a mere understanding of the receptors selected during an immune response is unlikely to provide sufficient information for rational vaccine design. What is really needed is information on which cells bearing which receptors are selected to expand and how the functional qualities of those

138  | Wrammert and Murali-Krishna Effector activation, clonal expansion, acquisition of effector functions & altered circulation

Peptide presentation by appropriate MHC + costimulation Naïve CD4/ CD8 T cell pool

Infection Persists Functional exhaustion, physical deletion or

Infection cleared Memory differentiation

Figure 6.2  General schematic outline of TCR repertoire shaping during an immune response. Naive CD8 or CD4 T-cells when stimulated with appropriate peptide along with co-stimulation in the context of the MHC to which they are restricted, undergo activation, clonal expansion and acquire effector functions. The effector functions that they acquire largely depend upon the context of stimulation rather than the receptor per se. The fate of the effector cells is largely influenced by the dynamic nature of the host–pathogen interaction. In most conditions, where the infection is cleared, a majority of the effectors dies, but a significant number of them differentiate into long lasting memory cells. In situations wherein the infection persists, the effectors cells can undergo functional exhaustion or physical deletion, thereby altering the repertoire. In some situations of latent infections, such as the one seen in the case of cytomegalo virus, the memory cells specific to some of the epitopes can undergo inflation thereby changing the repertoire. Note that the receptor repertoire of B-cells continue to change during the evolution of an immune response, whereas, in the case of T-cells, the functional properties of the cells, rather than the receptor, changes during the course of the immune response.

cells are affected during a protective immune response. Thus, cellular screens for interrogation of the receptor repertoire acquires significance. Several key differences in the biology of B-cells versus T-cells must be considered when making efforts to understand their receptor repertoire. Unlike B-cells, which tend to improve their BCR structure and specificity during progression through immune response, T-cells do not change their receptors. A T-cell born with a given receptor retains that particular receptor throughout its life. But the functions of the T-cell expressing a given receptor can change depending upon the antigen exposure status of the T-cell (Kim and Ahmed, 2010). Activated B-cells secrete their immunoglobulin receptors, which exerts effector functions away from the cell from which they were produced. Activated T-cells, however, do not

secrete their receptor. Instead, the effector functions of the T-cell are mediated by the cell itself upon engaging the receptor. Thus, in the case of T-cells, an understanding of the functionality of the TCR in question is stringently dependent upon understanding the functions of the cell expressing that particular receptor. Unlike B-cells, T-cells do not recognize free-floating antigens. T-cells recognize antigens displayed on the surface of the other cells (e.g. pathogen-derived antigens on an infected cell, altered antigens expressed by cancer cells or allo-antigens displayed by transplanted cells) only if these antigens are also presented in the context of the MHC molecule to which the T-cell is restricted. Thus, TCR functionality studies need to take into account the correct antigen-presenting cells with the appropriate MHC restriction element. Considering these

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factors, an understanding of the TCR repertoire of an individual with a given HLA haplotype may not necessarily reflect the repertoire in a different individual with a different HLA haplotype (Rodriguez-Caballero et al., 2007). Thus, meaningful human studies with reference to TCR repertoire must account for diversity of the HLA haplotypes at the population level. Cellular screens for T- and B-cell repertoire analysis Historically, the origins of the analysis of receptor usage during an immune response relied on laborious methods involving cloning of single cells by limiting dilution followed by production of clones through immortalization (e.g. fusing to tumour cells), analysis of their receptor by cloning, crystallization or functional characterization. Indeed, the elegant work by Cesar Milstein and co-workers in the 1970s (Kohler and Milstein, 1975), through establishment of hybridoma technology for producing monoclonal antibodies, revolutionized our ability to generate and characterize mouse monoclonal antibodies and has proven essential to the development of almost all aspects of modern biotechnology (Margulies, 2005). Other tools to generate monoclonal antibodies took advantage of powerful genetic library approaches (Bradbury et al., 2011; Marasco and Sui, 2007; Marks et al., 1992) to identify particular specificities or functional properties needed, although these approaches did not maintain the natural pairing of the immunoglobulin heavy and light chains. Similarly, elegant studies generated T-cell clones for isolation and characterization of TCRs that led to great advances in basic science and transgenic animal models expressing those defined TCRs (Barnden et al., 1998); and also paved way for potential use of TCR genes to modify haematopoietic stem cells for gene therapy (Clay et al., 1999). But none of these studies provides an idea of the complexity of the receptor repertoire and the cellular functions associated with a particular receptor during an immune response. Over the last half a decade or so, there has been a renewed interest in understanding the repertoire breadth of human immune responses at a cellular level. This has led to a rapid development of several novel technologies for repertoire

analysis of both B- and T-cells in humans, both on a population level as well as on a single cell level. More over, these methods also paved the way for characterization of the antigen epitopes against which protective immune responses are elicited – thus leading to efforts for rational vaccine design. In the following sections we will first outline cellular screens currently used for interrogation of human T- and B-cell repertoires and then discuss their implications for designing better vaccines and therapeutics. Although such ongoing efforts are focused on many different disease conditions, such as infection, autoimmunity and cancer, for the purpose of the discussion in this chapter, we will primarily focus on cellular screens to understand human protective immunity in the situation of two prominent pathogens that have been at the forefront of this development, namely influenza and HIV. Cellular screens for interrogation of the BCR repertoire Recent years have seen a rapid development and expansion of analytical tools to understand pathogen specific B-cell responses, after either infection or vaccination, often at a single cell resolution. Defining human B-cell repertoires responding to ongoing infection is critical for design of vaccines that induce broadly protective antibodies. But historically this has not been an easy task. Characterization of the breadth of the functional repertoire of serum circulating antibodies can be analysed by methods such as peptide mapping (Khurana et al., 2011, 2012), but this approach misses conformational epitopes as it only analyses linear peptide epitopes, it does not allow distinction of the antibodies responding to ongoing infection versus those merely elicited by previous exposures, and does not provide any functional characteristics of the antibodies. Furthermore, it is only recently that tools to identify rearrangements directly from serum antibodies have been available (Cheung et al., 2012). Instead, the rapid progress of B-cell repertoire studies in the last decade relies on several novel tools to analyse the cells making these antibodies. This primarily includes high-throughput sequencing, improved immortalization strategies, single cell isolation of Ig rearrangements as well as the identification and

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isolation of single cell, antigen-specific plasmablasts or memory B-cell populations. In order to fully understand the immunoglobulin repertoire usage and the dynamics of those repertoires, one needs information both from different cellular differentiation stages and from different tissues. Such comprehensive analyses would require using all of these novel tools. Plasmablasts: Until recently, technologies to characterize the receptor repertoire of the human B-cells specifically responding to a given antigen, during the ongoing immune response, was lacking, with available tools focused on analysing memory B-cells from convalescent patients. Recent work from the laboratories of Drs Ahmed and Wilson (Smith et al., 2009; Wrammert et al., 2011; Wrammert et al., 2008), for the first time, allowed overcoming these technological difficulties. They devised exciting novel tools and technologies for identification and enumeration of blood circulating antibody secreting B-cells (also called plasmablasts) that were specifically responding to vaccination or infection (Li et al., 2012; Wrammert et al., 2011, 2012). The plasmablasts are short-lived antibody secreting cells (ASC) originating from rare populations of B-cells responding to the specific infection/ vaccination. They enter blood circulation shortly after infection or vaccination and the timing of acute plasmablast appearance in the blood is strikingly consistent after immunization or infection (Halliley et al., 2010). Flow cytometry analysis or ELISPOT performed ex vivo with human peripheral blood mononuclear cells (PBMCs) sampled after vaccination with inactivated influenza vaccine (Cox et al., 1994; Halliley et al., 2010; He et al., 2011; Moldoveanu et al., 1995; Wrammert et al., 2008) or tetanus vaccine (Odendahl et al., 2005; Qian et al., 2010), and after infection with respiratory syncytial virus (RSV) (Lee et al., 2010) or dengue virus (Balakrishnan et al., 2011; Wrammert et al., 2012) showed that antigen-specific plasmablast numbers during a recall response peak consistently at day 6 or 7. As the vast majority of these cells are induced by the infection or vaccination, they represent a unique population of almost entirely antigen-specific cells. The magnitude of these responses correlate well with the serum antibody titres at a later time point and can

in fact be used as a predictor for vaccine-induced antibody titres (Nakaya et al., 2011) at later time points. The functional repertoire of the antibodies produced by these cells can be analysed using ELISPOT assays (Li et al., 2012; Wrammert et al., 2011), through bulk cultures of the plasmablast cells (He et al., 2011) as well as peptide library screens (Khurana et al., 2011). In addition to providing information about the magnitude and quality of the immune response at a population level, the plasmablasts are also an excellent source for generating panels of antigen-specific monoclonal antibodies through single cell RT-PCR expression cloning (Smith et al., 2009; Wrammert et al., 2008), to further characterize both the repertoire of the responding cells as well as provide large panels of antibodies that will provide insight into the functional characteristics of the responding B-cells and the antibodies that they produce. This approach is dependent on the efficient identification of Ig rearrangements from single human B-cells by RT-PCR. Initially designed by Wardeman et al. (2003) for use in understanding human B-cell development and tolerance, this technology represents a major step forward in our ability to analyse subpopulations of human B-cells, and has now found broad use for understanding human B-cells in both healthy and immuno-compromised patients. In addition to these analyses of B-cell development and function, this technology has also been key for developing single cell expression cloning of pathogen specific monoclonal antibodies, both from antigen induced plasmablasts as described here, as well as from antigen-specific memory B-cells stained with fluorescently labelled antigen (see below). Single cell expression cloning from plasmablasts allows for a detailed, and unbiased, functional characterization of the ongoing immune responses at a single cell level, as well as provides information regarding the repertoire breadth of the responding cells. This technology has been used successfully to generate panels of monoclonal antibodies against influenza (Li et al., 2012; Wrammert et al., 2008, 2011), rotavirus (Di Niro et al., 2010), dengue virus (Xu et al., 2012) and tetanus (Poulsen et al., 2011) and is also being brought to bear on autoimmune diseases such as rheumatic arthritis, coeliac disease (Di Niro et al., 2012) and

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Pemphigus Vulgaris. One of the strengths of this approach is that it provides an unbiased picture of the ongoing immune response, i.e. the repertoire and fine specificities of the cells activated in the current immune response. This is in contrast to the memory B-cell-based approaches below that provides a summary of all the antigenic exposures that a particular individual has been exposed to in a lifetime. Furthermore, experiments from mice suggests that the compartment of long lived plasma cells in bone marrow and memory B-cells have quite distinct repertoires, with the former primarily selected for high affinity antibodies while the memory pool is selected more for breadth (Purtha et al., 2011; Smith et al., 1997). The drawbacks of this approach is that these cells are only present in circulation very transiently, and thus samples have to be obtained at the optimal time point. Furthermore, although these cells can be isolated from frozen PBMCs, this approach really works best if performed on fresh cells. Finally, as the rearrangement of each individual cell is first cloned and expressed, before the specificity can be analysed, this approach does not easily lend itself to large-scale functional screening efforts for very rare types of antibodies, such as in the case of identifying rare broadly HIV neutralizing antibodies. However, recent progress with high throughput, automated systems for generating large panels of antibodies using this method, initial success with single cell cultures for micro scale screening prior to single cell cloning (Corti et al., 2011), as well as the use of barcoding technologies allow the combination of this approach with highthroughput sequencing, with retained heavy and light chain pairing, allowing more comprehensive repertoire analyses to be performed (Reddy and Georgiou, 2011). Memory B-cells While long-lived plasma cells primarily reside in the bone marrow and are responsible for maintaining serum antibody titres, and thus provides immediate protection against re-exposure to a particular pathogen, memory B-cells on the other hand do not produce soluble antibody, but can respond vigorously upon encounter with it’s cognate antigen, rapidly expand to large numbers and subsequently differentiate into antibody

secreting cells, thus providing a marked, albeit transient, increase in serum antibody titres. The memory B-cell compartment represents a summary of all the different antigen exposures that a person has been through in a lifetime. This difference is of importance for analysis of diseases such as dengue, influenza and HIV, where clearly sampling the memory B-cell compartment or the plasmablast compartment would yield different outcomes. A number of different approaches for analysing the memory B-cell compartment have been developed, both for bulk analyses as well as at a single cell level, and includes immortalization of memory B-cell clones, expression cloning from single cell cultures without immortalization, as well as direct isolation of antigen-specific memory B-cells by staining with fluorophore labelled antigen (Wilson and Andrews, 2012). While hybridoma technology has been an absolutely essential tool for studying B-cell repertoires and generating monoclonal antibodies in mice, it has never become a very useful tool working with human cells. An alternative option was immortalization of B-cells using EBV transformation. While this certainly was a feasible route, as evidenced by numerous early publications (Atlaw et al., 1985; Buchacher et al., 1994; Burioni et al., 2010; Cole et al., 1984; Habersetzer et al., 1998; Purtscher et al., 1994; Steinitz et al., 1977), it remained a very inefficient method. Work pioneered by Lanzavecchia and his team (Traggiai et al., 2004) in the early 2000s demonstrated that the introduction of a memory B-cell specific TLR signal (TLR9; stimulated through use of CpG DNA) to the in vitro cultures significantly improved the rate of immortalization. Typically this approach involves the isolation of IgG positive memory B-cells from either fresh or frozen PBMCs, followed by an in vitro stimulation in the presence of a TLR9 ligand and allogeneic irradiated feeder cells for about two weeks at semi-limiting dilutions. At this point the supernatants can be screened for binding, neutralization or any other functional readout required. Positive wells are subsequently subcloned prior to sequencing of the Ig heavy and light chain genes. The Lanzavecchia group and many others have since used this approach to understand the repertoire of pathogen-specific memory B-cells and to generate panels of monoclonal antibodies

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to a large range of different pathogens including influenza virus (Corti et al., 2010; Krause et al., 2011, 2012), SARS virus (Traggiai et al., 2004), dengue (Beltramello et al., 2010; Dejnirattisai et al., 2010; Schieffelin et al., 2010; Smith et al., 2012), CMV (Macagno et al., 2010), RSV (Collarini et al., 2009) and Chikungunya virus (Warter et al., 2011), as well as antibodies involved in autoimmune diseases such as pemphigus (Di Zenzo et al., 2012). One of the major advantages of this approach is that one can obtain antibody upfront, before genetic cloning, and the approach is thus amiable to efficient functional screening for rare types of antibodies. Another benefit is that this approach can be readily performed on cryopreserved cells allowing analysis even many years after the exposure or vaccination took place. It does require handling large numbers of clones for initial screening and subsequent sub cloning as the frequency of the specific cells of interest is much lower then for the plasmablast approach above, where the vast majority of the cells are antigen specific. This approach will also only provide information based on the screening technique used and as such might not be the best choice to address the overall breadth of the immune response. Recent publications have shown that a similar approach with clonal expansion of single memory B-cells, but without the need for immortalization is also feasible (Walker et al., 2009). In this case memory B-cells are cultured in a single cell format, followed by binding or functional screening of the antibody secreted into the culture supernatant. Once a well of interest is identified, RT-PCR cloning of that particular clone can be performed and the corresponding antibody expressed. Finally, isolation of antigen-specific memory B-cells through surface staining of the BCR with fluorescent labelled antigen probes, antigen baiting, followed by single cell RT-PCR expression cloning has also found a lot of traction in the last several years (Scheid et al., 2009a,b). One particular strength with this approach is that by using multiple protein mutants, antibodies specific for a particular epitope on a protein antigen can readily be identified and cloned (Scheid et al., 2011; Sundling et al., 2012). This approach has been used successfully in the HIV field (Mouquet

et al., 2011; Scheid et al., 2009a, 2011; Wu et al., 2010), but also for identification of mucosal plasmacells specific for rotavirus (Di Niro et al., 2010) or for transglutaminase-2 (Di Niro et al., 2012) in patients with coeliac disease. The latter IgA positive mucosal plasmacells are unique compared to other plasmacells in that they express immunoglobulin on their cell surface, allowing antigen-specific plasmablasts to be identified through antigen baiting. High-throughput sequencing The significant developments in generating and analysing monoclonal antibodies against many different pathogens using either memory B-cells or plasmablasts, as described above, have certainly not only provided a wealth of antibodies with possible therapeutic potential, but also provided insight into the repertoire breadth of human B-cell responses. However, most of these approaches are single cell based and only allows for the analysis of relatively small numbers of cells. However, in addition to this, various high-throughput sequencing technologies are being developed that allows for a more comprehensive and exhaustive analysis (Reddy and Georgiou, 2011). In its basic form, these high through-put sequencing approaches only provide a very large number of sequence reads from a particular sample. As such, it is certainly very informative for specific questions, for example following a particular rearrangement and its evolution over time as was recently reported in an elegant study describing the evolution of a broadly neutralizing HIV specific antibody (Liao et al., 2013). These approaches also allow for the direct comparison of repertoire breadths in various tissues and cellular compartments, and tracking of the distribution of certain rearrangements within various tissues or compartments. However, a major obstacle for these high throughput sequencing approaches of antigen-specific plasmablasts or memory B-cells has been that they do not maintain the cognate heavy and light chain pairing, preventing downstream functional analysis of the importance of the repertoire findings. The more recent use of barcoding amplified material from individual single cells prior to the high through-put sequencing now promise to allow the re-assembly of the cognate pairing and

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thus preserving the potential for downstream functional analysis of the antibody sequences derived from this methodology. In concert with improvements to single cell genomics analysis this technology is likely to provide a wealth of information connecting individual rearrangements and their functional properties, with the phenotype of the cells expressing them (Reddy and Georgiou, 2011). Cellular screens for interrogation of the TCR repertoire As mentioned above, T-cells exert effector functions depending upon context of the stimulation, pathogen, and the stage of the immune response. Moreover, their recognition of the antigen is dependent upon MHC restriction. Thus, with reference to TCR repertoire, it is not only important to understand which TCR repertoire is expanded during an immune response, but which phenotypic and functional qualities of the particular T-cells bearing a particular receptor are affected, and how they change depending upon the pathogen, its persistence and the stage of the immune response (i.e. naive to effector and memory stage conversion) (Fig. 6.3). There are several different approaches currently in practice for TCR repertoire analysis, many of them are complementary and offer

Naive

Effector

different levels of advantages, but when combined together provide a much more comprehensive picture on the TCR repertoire. These range from identification of T-cell frequencies using stimulation with overlapping peptide pools followed by scoring the responding cells using cytokine ELISPOT, identification of specific T-cells by MHC tetramers, and a single cell analysis of function, phenotype and specificity using flow cytometry. Moreover, these methods can be combined with cell sorting methods to gain a comprehensive understanding of how a given receptor specificity in a specific condition affects the gene expression profiles and or the receptor repertoire. Although the power of cellular screens for interrogation of T-cell repertoire is largely dependent upon our ability to combine these multiple assays, each of them is individually outlined below. Sequence-based screening of the TCR repertoire One of the widely used methods for interrogating TCR repertoire in the past, was through profiling of TCR β-chains. Conventional methods to analyse TCR Vβ repertoire relied on flow cytometry-based assays employing a panel of monoclonal antibodies recognizing individual members of the TCR Vβ family (Pilch et al., 2002). Such assays can be combined with staining for V-alpha chains

Memory/ Exhausted cells

1. What is the epitope specificity? 2. What are the cellular functions? 3. What are the phenotypes? 4.  How are these properties affected by the cell type (CD4/CD8), their specificity, cellular location (e.g., mucosal/ tissue compartments, lymphoid organs, blood), pathogen, disease outcome (e.g., latent versus active infection)?

Figure 6.3  Questions revolved around cellular screens for interrogation of TCR repertoire during the course of the immune response.

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to analyse the over all frequency of cells exhibiting individual V-α and V-β-chains. While such monoclonal antibody-based assays allow quantitation, they do not allow understanding the qualitative diversities because each V-beta family is further comprised of diverse sequence variations generated by V-D-J recombination that leads to unique CDR3 regions, which are the principle site for determining antigen recognition. Another widely used methods for identifying sequence variability in the CDR3 regions is spectratyping (Ciupe et al., 2013). TCR spectratyping uses multiplex RT-PCR methods to compare relative frequencies of different clonal length products within the CDR3 region of a particular TCR Vβ family. Although in humans the TCR α/β receptor diversity is estimated of the order of 1015 to 1018 rearrangements, the size of the β-chain diversity is much more limited (~106 rearrangements). Thus, spectratyping is generally useful only for analysis of the relative hierarchy of T-cell populations within a given V β population. More recent approaches use deep sequencing of the TCR-beta genes using next-generation sequencing (NGS)-based platforms (Clemente et al., 2013). These methods provide a higher capacity to differentiate and monitor many unique DNA rearrangements in parallel at the population level in a given pool of cells. More recent methods attempt to generate tags that are specific for one in more than a million clones. These new strategies should eventually allow single-clone analysis of the complete TCRβ repertoire, and thus may provide much more detailed information. Although it is possible to spectratype following sorting different populations (Estorninho et al., 2013; Warren et al., 2011), the information gained from spectratyping is generally limited to relative diversity of the TCRs at population level (Woodsworth et al., 2013) rather than providing functional insight on the linked cellular phenotypes or functions. Linking the TCR repertoire with specificity, cellular phenotype and function In the case of the B-cells, receptor specificity can be assessed in a simple soluble antigen-binding assay. But determining the antigen specificity of the T-cell receptor is much more complex. Instead

of recognizing and binding the antigen directly, TCR recognizes short peptide fragments, which are bound to MHC molecules on the surfaces of other cells. Which peptides are presented by MHC class-I or MHC class-II is dependent upon whether the peptides are derived from endogenous processing or exogenous uptake by the presenting cells. T-cell co-receptors expressed by the CD4 and CD8 T-cells facilitate this interaction by binding to MHC class-I or class-II respectively. Unlike immunoglobulin receptors, which can dramatically change in their structure and effector functions by processes such as affinity maturation, isotype switching or somatic hypermutation, the structural properties of the TCR remain constant throughout the life of a given T-cell. What changes are the properties of the cells depending upon the levels of antigen, persistence and intensity of the signals transmitted through the receptor. Unlike immunoglobulin, which bind soluble antigens and mediate effector function through neutralization, opsonization or complement fixation, TCRs exert their effector functions by transmitting intracellular signals into the T-cell, which then exert effector functions. The effector functions are highly complex, and depend upon several parameters. These include the type of the T-cell under investigation (i.e. whether they are CD4 T-cells, CD8 T-cells, regulatory cells), the stage of the T-cell (i.e. whether they are naive cells, recently antigen experienced effector cell, memory cell, or those cells trying to fight persistent antigenic exposure such as the ones expected in chronic infections, autoimmunity or cancer, or those cells receiving anergic signals as expected in situations of peripheral tolerance), or even the circulatory properties of the T-cells (i.e. depending upon their location in lymphoid circulation, nonlymphoid tissues) (Mueller et al., 2013; Wherry and Ahmed, 2004). Many of these functions are also dependent upon disease manifestations and bystander inflammatory events in the host. Thus, approaches that allow linking the receptor specificity with cellular phenotypes and functions offer many advantages. The ability to count the antigen-specific T-cells and characterize their phenotypic and functional characteristics using cellular screens in different situations led to great advances in immunology

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in the past decade. Until a decade ago, the most common method used to enumerate antigenspecific CD8 T-cells against a given specificity relied on limiting dilution analysis (LDA). LDA uses the laborious methods of in vitro expansion of T-cells using peptide pulsed antigen-presenting cells at graded numbers followed by their functional enumeration using radio-active chromium release assays. These methods, although considered ‘gold standard’ at that time- are usually subjected to a variety of errors, arising due to differences in the expandability of different T-cell subsets in the in vitro culture conditions. There are several advances that arouse in the past decade that now allow comprehensive understanding of the TCR repertoire by linking their specificity, phenotype, and function (Murali-Krishna et al., 1998a,b). These include (1) methods that score TCR specificity, frequencies, phenotypes, and functions using (a) stimulation with overlapping peptide libraries, (b) MHC tetramer staining, and (c) multiparametric flow cytometric analysis. Overlapping peptide stimulation methods Mapping of which antigenic peptide sequences of the pathogen are recognized by CD8 or CD4 T-cells is crucial first step in repertoire analysis and vaccine development. But, epitope mapping is a challenging task. Most pathogens carry several proteins, and each protein will have several peptide epitopes that can be recognized by human T-cells. The peptide epitopes recognized by CD4 and CD8 T-cells are usually different in length and amino acid sequences. Moreover, because of the MHC restriction differences, the peptide epitopes recognized by T-cells of an individual may not necessarily be epitopes in a different individual. One of the methodologies commonly used for T-cell epitope mapping is by using stimulation with synthetic peptides (Li Pira et al., 2010). A complete panel of peptides, with a given length of 9–15 amino acids and a predetermined overlapping sequence, that encompass the entire protein length are synthesized and then used to stimulate T-cells in vitro. The frequency of cells responding to each of these peptides can then be assessed by scoring the frequency of cytokine (for example IFN-γ) producing cells, using functional readouts

such as ELISPOT technique or by intracellular cytokine staining followed by flow cytometry. The flow cytometry is generally suitable for differentiating the cytokine-producing cell population as CD4 or CD8 subsets, whereas the ELISPOT method does not offer such advantage. However, ELISPOT method is generally used in most of these assays because of the low frequency of epitope specific cells, which may be difficult to differentiate from back ground in flow cytometry. These methods do not require prior knowledge about the HLA haplotype of the individual or the MHC restriction of the peptide. If T-cells of an individual score positive for stimulation with a given peptide, then one can refine the precise epitope by stimulation with truncated peptides around that sequence, and understand whether the identified epitope stimulates CD4 or CD8 T-cells. Using this information, and depending on the HLA type of the individual, one can then generate MHC class I or class II tetramers that allow physical, functional and phenotypic characterization of T-cells specific to that particular peptide (Altman and Davis, 2003). But peptide scanning approaches often poses challenges because it is not easy to obtain blood cells from subjects in sufficient quantities that allow for stimulating with large numbers of overlapping peptides. In such cases, an alternative approach is to first stimulate with pools of peptides in such a way that any given peptide pool shares only one or more peptide from a different pool (Precopio et al., 2008). If a given peptide pool turns out to be positive, one can then deconvolute the positive peptide by deducing from the information on which of the peptides are shared between two pools that gave positive result. Although this approach offers several advantages, the mapping of peptide epitopes using these approaches is impossible for large pathogens that carry multiple proteins. For example, a large pathogen such as Mycobacterium tuberculosis carry over 500 proteins. Producing the peptide libraries scanning the entire genome of such a large pathogen and mapping them using the limited volumes of blood that can be obtained from patients is a daunting task. To overcome these limitations, algorithms have been developed to test the potential capacity of a given peptide to bind a predetermined MHC allele (Kim and

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Ahmed, 2010). From this information it can be assumed that a T-cell with such a specificity can be present in the T-cell repertoire. A large-scale validation study for CTL epitope prediction has been recently published by comparing five different web-based tools, and these algorithms have now successfully tested to map human T-cell epitopes to large pathogens such as M. tuberculosis (Lindestam Arlehamn et al., 2013). Other variations include enumeration of individual protein specific T-cells, instead of peptide epitope specific T-cells. This can be done by stimulating human PBMCs with individual proteins followed by enumeration of antigen-specific CD4 T-cells or by stimulating with replication deficient recombinant viral vectors (e.g. vesicular stomatitis vectors) expressing the pathogen derived individual proteins followed by enumeration of CD8 and CD4 T-cells (Moseley et al., 2012). MHC tetramer staining Tetramers consist of four biotinylated HLA–peptide epitope complexes bound to streptavidin conjugated with fluorescent dye (Davis et al., 2011). The MHC class-I tetramers, or classII tetramers, when bound to correct peptide, identify the T-cells bearing receptor specific to that particular epitope with great sensitivity. We published the first tetramer experiments that provided an accurate picture of response kinetics and the extent of CD8+ T-cell proliferation following virus challenge in LCMV models in mice in 1998 (Murali-Krishna et al., 1998b). The technology rapidly revolutionized the field of immunology and is now an important aspect of human immunology for understanding immune responses against vaccines, infections, autoimmunity, allergy and cancer. The combination of MHC tetramer technology with flow cytometry and intracellular cytokine staining methods opened up significantly better ways of studying T-cell receptor repertoire in conjunction with phenotypic and functional characteristics (Akondy et al., 2009; Miller et al., 2008). Such techniques allow interrogating how the TCR specificity and the state of the immune response affects the cellular functions such as cell cycle, activation, inhibitory receptor expression, life and death issues, migratory properties, cytotoxic and cytokine functions. Additionally,

one can flow cytometrically sort T-cells specific to a given peptide epitope and further interrogate their gene expression, signalling profiles or receptor usage. Multiparametric flow cytometric analysis Intracellular cytokine staining is a flow cytometric technique consisting of culturing stimulated cytokine-producing cells in the presence of a protein secretion inhibitor, followed by fixation, permeabilization and staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies (O’Donnell et al., 2013). Up to 18 different colours can be detected by modern flow cytometers, making it the only immunological technique allowing simultaneous determination of antigen-specific T-cell function and phenotype. In addition, cell proliferation and viability can be also measured. For this reason, it is probably the most popular method to measure antigenicity during vaccine trials and in the study of infectious diseases, along with ELISPOT. Single cell mass cytometry (CytOF) is a recent advance in this field, with a variation of conventional flow cytometry using metal tagged antibodies instead of fluorochromes and detection by time of flight of discrete masses of the metal tags (Newell et al., 2012). The lack of any significant mass spectral overlap allows analysis up to 100 parameters simultaneously on single cells using the CyTOF, with 33 stable isotope tags currently commercially available. Repertoire analysis and implications for vaccine development As outlined above the tools available for comprehensive BCR repertoire analysis, and the use of the information that it provides, to better understand protective antibody responses, to generate therapeutically potent monoclonal antibodies, and to improve vaccine design have literally exploded over the last several years, focusing on a large number of pathogens. Two pathogens that have been at the forefront of these developments are the influenza and HIV viruses. In this section we will briefly go over the progress made using these two pathogens as models, and discuss the implications that these findings have for understanding

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protective immunity, what the implications are for vaccine development, and how this knowledge can be applied to other pathogens. Influenza virus Influenza remains one of the most important respiratory infections in humans, and even though there is ample availability of efficacious vaccines, this disease remains one of the foremost causes of morbidity and mortality worldwide, especially in children and the elderly. As influenza vaccination essentially relies on the induction of strain specific neutralizing antibodies, it requires annual re-vaccination to retain protective antibody titres against the most current strains, and this protection is highly susceptible to be overcome by either drifted escape mutants or reassortant virus strains. This fact has made it difficult to generate a vaccine with broad and more long lasting protective capacity. Recent events such as the rapid worldwide spread of the 2009 pandemic H1N1 influenza strain, along with the very high mortality of avian strains such as the H5N1, has led to renewed concerns about the possibility of a more lethal future pandemic, such as what was seen in the beginning of the century, with the 1918 Spanish Influenza, which is thought to have killed as many as 100 million people. These concerns has led to large-scale efforts to identify conserved epitopes in the influenza virus that can be used to generate broadly protective vaccines, as well as to generate monoclonal antibodies that can be used for passive immunotherapy. Traditionally, this would not be feasible as the influenza virus would rapidly mutate and become non-responsive to such therapies. However, recent work by us and by others suggests that such broadly neutralizing antibody responses can be induced targeting highly conserved regions in the stem of the HA molecule (Chiu et al., 2013; Pica and Palese, 2013). Neutralizing antibodies directed against influenza typically react with either the HA or the NA proteins of the influenza virus, inhibiting viral entry or spread, respectively. There are 16 different influenza strains based on the HA molecule, which can be loosely grouped into two main phylogenetic subgroups, named groups 1 and 2. The HA trimeric molecule itself is composed of two subunits,

HA1 and HA2. The HA1 forms the globular head of the molecule and contains the sialic acid binding site, while the HA2 subunit forms the stalk of the molecule and also the membrane attachment portion. Historically, essentially all neutralizing antibodies described against influenza up until 2008, bound to the hyper-variable regions of the globular head of the HA molecule. In 2008 and 2009, several papers showed that broadly neutralizing antibodies targeting the HA2 stem portion of the HA molecule could be identified by phage display libraries (Ekiert et al., 2009; Sui et al., 2009; Throsby et al., 2008). As this region of the HA2 subunit is highly conserved these antibodies were able to neutralize most strains of group 1 HAs, including the H1, H5 and H9 clades. Using the EBV immortalization approach described above, Lanzavecchia and co-workers could show that this type of antibody is actually present in the human memory B-cell compartment, although at very low frequencies (Corti et al., 2010). This was done by screening PBMC samples from patients vaccinated with the seasonal influenza vaccine, for neutralization of a non-related H5 influenza strain. Out of these experiments about 20 antibodies were identified that were able to neutralize both H5 and H1 containing viruses. Almost all of these were shown to bind to the stem region of the HA2 molecule, as described above. Subsequent work by the same group then used a combination of EBV transformed memory B-cells and single cell plasmablast cultures to identify an additional antibody able to neutralize both group 1 and group 2 viruses (Corti et al., 2011). However, the frequency of this type of antibody is exceedingly rare, as only one was identified after screening more then 100,000 plasmablasts from eight preselected donors. Also, they were unable to identify this particular type of antibody screening memory B-cells from the same donors, illustrating how rare this particular type of antibody is in the context of seasonal influenza vaccination. The emergence of the 2009 pandemic H1N1 presented an interesting opportunity to analyse the acute plasmablast responses induced by this re-assorted virus in infected patients. This virus was a re-assortant of human and swine strains, with the HA and the NA proteins deriving from swine (Xu et al., 2010). In fact, compared

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Table 6.1  Different approaches for the generation and analysis of monoclonal antibodies Approach

Examples

PlClassical EBV transformation

Atlaw et al. (1985), Buchacher et Functional screening possible al. (1994), Burioni et al. (2010), Cole et al. (1984), Habersetzer et al. (1998), Purtscher et al. (1994), Steinitz et al. (1977)

Phage display

Bradbury et al. (2011), Marasco and Sui (2007), Marks et al. (1992)

Plasma blast expression cloning

Plasmablast antigen baiting

Li et al. (2012), Poulsen et al. (2011), Smith et al., (2009), Wrammert et al. (2008, 2011), Xu et al. (2012)

Di Niro et al. (2010, 2012)

Benefits

Drawbacks Poor cloning efficiency

High-throughput functional screening possible

Heavy and light chain cognate pairs not retained

Multiple cell types can be analysed

No frequency information retained

No functional screening prior Unbiased repertoire of activated cells responding to cloning to ongoing infection or vaccination Rapidly generate large numbers of antigenspecific monoclonals

Only possible at the peak of the immune response

As above but allows for identification of rare events

Only possible using intestinal plasmacells that are surface immunoglobulin A positive

Also allows analysis of certain mucosal tissues

Only possible when good baiting reagents are available

Works best on fresh PBMCs

No functional screening prior to cloning Optimized EBV immortalization

Beltramello et al. (2010), Collarini et al. (2009), Corti et al. (2010), Dejnirattisai et al. (2010), Di Zenzo et al. (2012), Krause et al. (2011, 2012), Macagno et al. (2010), Schieffelin et al. (2010), Smith et al. (2012), Traggiai et al. (2004), Warter et al. (2011)

High-throughput screening for specificity/ function

Not possible for plasmablasts /plasmacells

Allows for identification of memory B-cells generated decades ago

Thousands of cells screened to identify antibody of interest

Works well on banked PBMC samples

Represents immunological history, does not characterize an ongoing immune response

Memory B-cell expression cloning without immortalization

Walker et al. (2009)

Reduces the amount of clonal lines to be maintained

Small amounts of antibody limits functional screening options compared to immortalization

Memory B-cell antigen baiting

Mouquet et al. (2011), Scheid et al. (2009a, 2011), Sundling et al. (2012), Wu et al. (2010)

Highly efficient for obtaining antibodies with a defined specificity

Only possible when good baiting reagents are available No functional screening prior to cloning

EBV, Epstein–Barr virus; PBMC, peripheral blood mononuclear cell.

to the previously circulating H1N1 strain (A/ Brisbane/10/2007) this novel strain only shared about 50% homology. In contrast, the HA of this new virus did show extensive homology with the

1918 influenza. Interestingly, it became apparent that a large proportion of the responding cells produced antibodies that were cross-reactive against both H1N1 and H5N1 strains. In contrast to

Cellular Screens to Interrogate the Human T- and B-cell Repertoires and Design Better Vaccines |  149

what we had observed earlier analysing seasonal influenza vaccine responses, the majority of the neutralizing antibodies identified in this study were broadly neutralizing and targeted the stem of the HA molecule (Wrammert et al., 2011), not the globular head. The high levels of mutation observed in these antibodies along with their cross reactivity patterns, suggests that they are derived from rare memory B-cells, present in the normal repertoire at low frequencies, but heavily expanded during the current infection. In contrast, the normally immuno-dominant memory B-cells directed against the globular head did not get activated as they were unable to recognize the structures in the HA head that was not conserved between the pandemic H1N1 and the seasonal H1N1 (A/Brisbane/10/2007). These findings illustrate that exposure to a novel influenza strain with little homology to recently circulating strains, can induce potent responses against non-dominant, conserved epitopes in the stem of the HA molecule. Subsequently, it has also been shown that these types of stem-reactive, broadly neutralizing antibody responses can be induced also after vaccination with the pandemic H1N1 vaccine (Li et al., 2012). What makes this epitope even more interesting is that in addition to it’s conservation across a large number of clades, the virus cannot readily incorporate mutations in this region, without loosing viability. These findings provide important proof of concept that broadly neutralizing immune responses can be induced in humans given the correct immunogen. This remains a very active field of research, both with the aim of optimizing the induction of this type of broadly neutralizing antibody responses with novel vaccines, as well the use of this type of antibodies for therapeutic purposes (Pica and Palese, 2013). Similar approaches for repertoire analysis, identification of novel potent epitopes for vaccine development and monoclonal antibodies for therapeutic use are currently being employed to dissect B-cell responses against many other pathogens, such as for example dengue infection and HIV infection. The detailed analysis of the development of broadly neutralizing antibodies in a subset of HIV-infected patients is especially worth mentioning. While early HIV vaccine trials

generally gave rise to serum antibodies that were unable to neutralize divergent primary isolates (Burton et al., 2012a; Pantophlet and Burton, 2006), and largely refocused most HIV research onto T-cell responses and T-cell-based vaccines for the next few decades, more recent analyses of HIV infected patients have shown that some donors generate broadly cross-reactive neutralizing antibody responses, that interestingly does not develop until several years after infection (Binley, 2009; Binley et al., 2008; Doria-Rose et al., 2009; Gray et al., 2009, 2011; Sather et al., 2009). Mapping of these broadly neutralizing antibody reactivities using serum revealed targeting of a relatively small number of epitopes on the envelope molecule, including the CD4 binding site, the membrane proximal external region (MPER), quaternary structure dependent epitopes as well as glycan dependent epitopes (Gray et al., 2009; Stamatatos et al., 2009; Walker et al., 2010). By focusing on this subset of patients and taking full advantage of novel technologies for generating monoclonal antibodies in large scale, allowing for screening for rare events, either through memory B-cell cultures, through antigen baiting methods, or by screening plasmablasts, starting around 2009 several novel broadly cross-reactive monoclonal antibodies targeting these sites have been identified (Burton et al., 2012a,b; Mouquet et al., 2011; Scheid et al., 2009a, 2011; Walker et al., 2011; Wu et al., 2010). Even though there are likely additional broadly neutralizing epitopes remaining to be discovered, these existing monoclonals are valuable tools for vaccine discovery and the generation of novel immunogens in this field. In addition, these antibodies also show promise in a therapeutic setting, where a cocktail of broadly neutralizing antibodies led to a rapid and precipitous decline in plasma viraemia. In addition to this, a subset of animals did not show viral rebound, even after the antibody infusions had ended suggesting that there was also an impact in the host immune response (Barouch et al., 2013). As mentioned above, an interesting fact is that most of these broadly neutralizing antibodies do not develop until relatively late after infection, i.e. often more then two years later. These antibodies as a whole also share some unusual properties, in that they for example carry highly mutated

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V genes, display very long CD3 loops and often show some degree of poly-reactivity. This illustrates that designing a vaccine able to induce these particular characteristics is no small task and will require intimate knowledge of not only the initial immune response but also how these immune responses develop over time, to generate this type of antibodies. Elegant studies from the laboratories of Dr Haynes and co-workers recently published in Nature (Liao et al., 2013) used a combination of analysis of monoclonal antibodies and high-throughput sequencing to analyse the co-evolution of the HIV virus and the antibody response over time in a particular donor. Notably, they found that the un-mutated version of the CH103 antibody isolated from this patient, could bind the founder virus, but that the neutralizing breadth was only observed later in the response, after both extensive viral and antibody diversification. While additional broadly neutralizing antibodies directed at other epitopes is likely to be identified, the currently described antibodies provides a lot of insight into broadly neutralizing HIV antibodies, their potential role as a therapeutic agent and are teaching us more about modelling novel vaccine antigens, designed to elicit this type of antibodies (Barouch, 2013; Burton et al., 2012a). The cellular screens for T-cell repertoire analysis have also provided great strides in our understanding of mechanisms of immunity and protection in the recent past. An understanding of the breadth of the epitopes recognized by host immune system is important for designing vaccines that are widely protective and allow combating viral escape mutants. Human studies suggest that targeting of a large number of T-cell epitopes in HIV Gag rather than Env is advantageous for better vaccine efficacy (Rolland et al., 2008). The ability to characterize systemic and mucosal responses and an understanding of the T-cell phenotypes and the associated virological outcomes in mucosally transmitted infections such as HIV led to renewed interest in designing vaccines that induce better mucosal responses (Barnett et al., 2008). CD4 T-cells are at centre stage of regulating both B-cell-mediated immunity, controlling extracellular pathogens as well as regulating CD8 T-cell

immunity. CD8 T-cells are important in conferring protection against several intracellular pathogens including viruses, bacteria and parasites. The success of vaccines against intracellular pathogens such as tuberculosis, malaria, HIV and other viral infections, thus, require designing vaccines that induce durable T-cell responses with sufficient quantity and quality. The recent advances in T-cell peptide epitope mapping, MHC tetramer analysis, functional and phenotypic studies and their combinations are now opening novel insight into the quality and quantity of immune response associated with protection; which will have enormous implications in designing rational vaccines down the line. For example, it is now well established that differences in proliferative capacity or functional qualities of HIV-specific CD4 T-cells, rather than their magnitude alone, are associated with improved control of HIV. Poly-functionality of T-cells, rather than simple numbers, appear to be important determinants in conferring protection in several situations, including HIV and several other situations (Albareda et al., 2010; Betts et al., 2006; Lepone et al., 2010; Sutherland et al., 2010). The comparison of T-cell quality between disease susceptible and resistant groups (for e.g. HIV long-term non-progressors vs. those that succumbed to disease and put on anti-retroviral regimen) opens up critical T-cell-mediated functional differences that can potentially determine disease outcome. Similar approaches can be used on different situations, for example, by comparing T-cell responses in patients exposed to a given pathogen but exhibit with differential disease outcomes. Examples include, but not limited to, dengue haemorrhage fever patients versus those with mild dengue; chikunguyna arthritis patients versus those with no arthritis; M. tuberculosis exposed subjects with active versus latent TB, etc. Our ability to analyse a combination of T-cell antigen specificity with phenotypes and functions have revolutionized conceptual frameworks on the type of immune responses that need to be elicited by successful vaccines. These combination assays now reveal the ability of the immune system to induce different cell subsets (for example central and effector memory cells) that are geared to provide immediate protection against invading pathogens by homing to tissues and producing

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effector cytokines (e.g. effector memory cells) versus those that circulate through secondary lymphoid organs and elicit rapid recall responses (e.g. central memory cells). This knowledge now allows testing which vaccine formulations are capable of inducing both of these cell subsets. More importantly, the comprehensive knowledge on dynamic changes in T-cell receptor repertoire, by cellular screens that allow combining epitope specificity along with cellular phenotypes, function and cellular location, has now revolutionized our basic understanding of mechanism of immunity and memory differentiation, which have huge implications for vaccine design. In conclusion, the last several years have seen great developments of novel tools and techniques to analyse both the B- and the T-cell compartments of immune responses. These methods have provided, and continue to promise, profound steps forward with regards to our basic understanding of vaccine or infection induced immune responses, to epitope discovery, immunogen design and vaccine development, as well as providing an ever-increasing number of monoclonal antibodies with therapeutic potential, against many different pathogens. References

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Novel Strategies of Vaccine Administration: The Science Behind Epidermal and Dermal Immunization

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Béhazine Combadière and Hélène Perrin

Summary Following the era of vaccinia virus intradermal vaccination, the past two decades have seen interest focusing once again on the cutaneous routes of vaccination. The definition of the skin antigen-presenting cell (APC) phenotype and its function, gene regulation and interaction with the skin microenvironment and inflammatory reactions has added further complexity to the skin immune system. Indeed, activating the appropriate arm of the immune system, that which induces protection, is now one of the major challenges to be faced in the development of vaccines against infectious diseases. Numerous concepts for vaccine delivery to the skin have been developed. However, they have not yet met expectations: this challenge remains to be met by using innovative approaches in immunology and vaccine design. Therefore, understanding the tight skin molecular and cellular network might refresh our knowledge on skin immunization procedures and vaccine development and have important impact on future vaccination strategies. Introduction As the primary protective barrier of the body, the skin plays a crucial role in immunosurveillance through a dense and complex immune network. Our understanding of the high immune potential of skin tissue grows together with the urgency of making innovative vaccines have motivated the development of alternative routes of immunization. Novel approaches have been developed over the past two decades, both facilitating vaccination and improving targeting of superficial skin layers.

These approaches have already brought new insights into vaccination regimens and doses but they might also improve the quality and effectiveness of the immune response and thus open new paths for vaccination. In addition, nanotechnology has enabled the development of vaccines based on solid or biodegradable nanoparticles, viruslike particles (VLPs), virosomes, or liposomes – good alternatives to recombinant proteins for immunization. These vaccine formulations aimed to increase antigen uptake by APCs and thus improve the intensity of immune responses. A combination of innovative devices for skin immunization and inventive vaccine formulations could provide fresh approaches to vaccination strategies by improving immunity to infectious diseases. However, progress is still needed in the area of clinical applications, especially in terms of their tolerance, safety, acceptability, feasibility, immune response mechanism and induction of long-term protection in humans. The skin layers The skin is the largest organ of the human body and provides the first line of protection against opportunistic pathogens and physical and chemical injuries from the environment (Bos and Kapsenberg, 1993; Di Meglio et al., 2011; Kupper and Fuhlbrigge, 2004; Proksch et al., 2008). Therefore, it is a major immunological organ with a dense network of professional APCs consisting mainly of Langerhans cells (LCs) in the epidermis and various subpopulations of dermal dendritic cells (dDCs) and dermal macrophages (Fig. 7.1) (Romani et al., 2010a; Teunissen et al., 2012).

158  | Combadière and Perrin HUMAN MARKERS

CD207+ CD1aHi CD1c+

CCR6 +

CD11c lo

« en route » LC

Hair duct

Infundibulum

CD1aDim CD1c+ CCR7+ CD207Di m

Neutrophils

pDC

LCs

CD1a dDC CD1aInt CD208+ CD11c Hi/lo CD141 dDC CD141+ CD1a+/CD11clo

Monocytes

Blood vessel Macrophages

Hair duct

CD14 dDCs CD14+ CD163+ CD209+ CD1c+/CD11cHi/lo

LCs

CD207Hi CD11bint CCR6+

Stratum corneum Epidermis

CD11bHi dDC CD11c Hi CD11bHi

Papillary Dermis

CD103 dDC

Precursors (CCR6) Precursors (CCR2)

MOUSE MARKERS

CD103+ CD207+ Chemokines

CD11blo dDC CD11cHi CD11blo F4/80+ Lymphatic vessel

Capillary Dermis

Figure 7.1  Antigen-presenting cells in human and mouse skin. A dense network of professional antigenpresenting cells is present in the skin. Human and mice markers are indicated in the figure for each cell population. Langerhans cells (LCs) can be found at the superficial layers of the skin, at more basal layer of the epidermis, at the epidermal–dermal junction during inflammation as ‘en route LCs’, and in the epidermal sheet surrounding the hair follicle, the dendritic extension of LCs can be seen scanning the lumen of the hair follicle. Inflammation induces up-regulation of activation markers and costimulatory molecules at the surface of the skin dendritic cell subset, with increases in HLA-DR, CD86, CD80, CD83, CD40, and CCR7, associated with their migration into the lymph node.

Structurally, human skin is made up of three superimposed tissues, each of which can be vaccination target tissue. From the most external to the most internal, we find the: epidermis, dermis, and hypodermis. The overall skin thickness, although averaging 2 mm, is highly variable over the body, between individuals, and as a function of age (Lambert and Laurent, 2008) (Skin Vaccine Summit, Washingto,n DC, USA, 2011). The skin contains appendages (Kanitakis, 2002): local sweat glands in the deep dermis, which move sweat from the plasma to the skin surface through excretory ducts, and sebaceous glands in the intermediate dermis, located between hair follicle ducts and epidermal sheaths. Epidermis The epidermis is composed of stratified layers of squamous and keratinized cells that protect

the integrity of the skin (Callard and Harper, 2007; Proksch et al., 2008). It has an internal part made up of three layers of living cells: the stratum basale or germinative layer, the spinous layer (stratum spinosum), and the granular layer (stratum granulosum). The thicker superficial portion of the epidermis comprises multiple layers of dead keratinocytes, also called corneocytes (between 4 and 20 cell layers, according to body site). This portion is a horny, cornified, keratin layer known as the stratum corneum. Its principal function is to protect the organism from external aggression, by ensuring the cohesion of the epithelial cells, attached to one other by intercellular desmosomes (that link microfilaments and microtubules from the cytoskeletons of adjacent keratinocytes), and by producing keratin, a resistant fibrous protein (Proksch et al., 2008). The stratum corneum is pierced by

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several appendages, including sweat glands and hair follicles (HFs). Keratinocytes (more than 80% of all epidermal cells) are found at the basal layer. They undergo substantial morphologic and biochemical changes as they migrate centrifugally towards the surface. Dead keratinocytes die by a highly specialized apoptotic mechanism and are then eliminated by desquamation. Melanocytes, Merkel cells, and some nerve endings are also included among the epidermal cells (Kanitakis, 2002). Blood vessels are absent from the epidermis, the integrity of which depends on exchanges through the epidermal–dermal junction. LCs, a particular subset of dendritic cells (DCs), account for 1–5% of the epidermal cells. LCs establish a dendritic network between keratinocytes and ensure essential immunosurveillance of cutaneous. They are considered the first line of the immune barrier against invading pathogens but also essential for the induction of tolerance (Callard and Harper, 2007; Romani et al., 2010a). Hence, LCs present a key target for the cutaneous vaccination route (Combadiere and Liard, 2011; Sparber et al., 2010). Moreover, antigen-specific CD8+ cytotoxic memory T-cells have been found in the stratum basale and the stratum spinosum (Clark, 2010; Lopez-Bravo and Ardavin, 2008; Mueller et al., 2012) Dermis The dermis is a fibrous and elastic tissue containing collagen and elastic fibres, rich in lymphatic and blood vessels, that serves as the solid support for the skin (Kanitakis, 2002). They provide the structural framework for many tissues and play a critical role in wound healing. The epidermal–dermal junction is made of a basement membrane composed of collagen IV, structural glycoproteins and proteoglycans. Fibroblasts are the main cell population of the dermis; they are mesenchymal cells that synthesize the extracellular matrix components. Several subpopulations of immune cells are present in the dermis: subsets of dDCs described below, resident macrophages (Dupasquier et al., 2004), mast cells, CD8+ and CD4+ memory T-cells, γδ T-cells and B-cells (Clark, 2010; Geherin et al., 2012).

Hypodermis Finally, one of the skin layers most often used for drug and vaccine delivery is the hypodermis, or the subcutaneous tissue, a layer of white fat, also called adipose tissue, connected to the lower part of the dermis by extensive collagen fibres. It is composed largely of fibroblasts and adipocytes (Kanitakis, 2002). The presence of these lipids enables injected vaccine preparations to be maintained there for a period of time, by a depot mechanism (Lambert and Laurent, 2008). The specific anatomical site determines the extent of hypodermal vascularization and thus the recruitment of immune cells. Unlike the epidermis and the dermis, the hypodermis is naturally devoid of resident immune cells. Vaccine injected subcutaneously is thus more apt to remain in the fatty layers, to be taken up by monocytes and transported at a low rate to peripheral lymphoid tissues. Skin antigen-presenting cells and other players in vaccination Epidermal immune cells Streilein first described the expression of major histocompatibility complex (MHC) molecules on LCs, enabling the processing and presentation of endogenous and exogenous antigens (Streilein et al., 1982). LCs (1–5% of all epidermal cells) form a network around keratinocytes in the suprabasal layer of the epidermis (Mulholland et al., 2006; Tang et al., 1993) (Fig. 7.1). Human and mice LCs are characterized by the expression of the E-cadherin molecule, an adhesion marker in epithelial cells (Mayumi et al., 2013; Tang et al., 1993; Van den Bossche et al., 2012; Van den Bossche and Van Ginderachter, 2013), and by the presence of unique intracytoplasmic organelles, Birbeck granules, a hallmark of LCs (Romani et al., 2010a). LCs express CD1c, MHCClass II and DEC-205 (CD205) and a high level of the human CD1a marker (Klechevsky et al., 2008; Ochoa et al., 2008). Langerin is a C-lectin type II receptor internalized in Birbeck granules when LCs mature (McDermott et al., 2004). The discovery of langerin (CD207) in humans and

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then in mice led to changes in the characterization of LCs (Valladeau et al., 2000, 2002). The expression of CD207 in mice is not confined to LCs, but extends to a subpopulation of dDCs (Bursch et al., 2007; Poulin et al., 2007; Romani et al., 2010a). Also, in humans, dDCs express a low level of CD1a. LCs can be found at the superficial level of the skin or at the more basal layer of the epidermis or, during inflammation, at the epidermal–dermal junction and the epidermal sheet surrounding the hair follicle (Fig. 7.1). Sometimes, the dendritic extension of LCs can be seen scanning the lumen of the hair follicle (Kubo et al., 2009). LCs are a relatively independent population, replenished by local long-lived precursors of haematopoietic origin that seed the skin during embryonic development and self-renew under steady-state conditions (Chorro et al., 2009; Merad et al., 2002; Schuster et al., 2009). It has been recently shown that two type of LCs – longterm and short-term – develop through different pathways, one in steady state and the other in inflammation (Seré et al., 2012). Indeed, experiments in mice have showed that Gr-1hi monocytes replenish the epidermal LC population, under inflammatory conditions, with the progressive acquisition of MHC-II and langerin markers (Ginhoux et al., 2006). These precursors are recruited from circulating blood by the MIP-3α chemokine (CCL20), which binds to the CCR6 that they express strongly (Dieu-Nosjean et al., 2000; Jakob et al., 2001), while CCR8 ligands somehow negatively regulate their recruitment. Nagao et al. (2012) further characterized this pathway by identifying an intermediate population of precursor LCs and showing that they are recruited to the epidermis via hair follicles. Using mice deficient in the transcription factor Id-2, which lack LCs and langerin, Seré et al. (2012) also elegantly demonstrated that two distinct types of LCs repopulate the epidermis in two waves to reconstitute the epidermal LC network after UV light exposure. First to arrive are short-term langerin–/low LCs, developed from circulating Gr-1hi monocytes and independent of Id-2. They are progressively replaced by long-term langerin+ LCs that continuously arise, Id-2 dependently, from bone marrow precursors in steady state. Recent studies in mice also identified IL-34 (ligand of the colony

stimulating receptor-1, Csf-1) as a non-redundant cytokine for the control of the development of LCs during embryogenesis and LC homeostasis in adult (Greter et al., 2012; Wang et al., 2012). Whereas IL-34 is not required for inflammationinduced repopulation of LCs from monocytes, LC survival depends again on keratinocyte-derived IL-34 once the inflammation is resolved (Greter et al., 2012). The development of transgenic mice expressing langerin combined with enhanced green fluorescent protein (EGFP) (langerin-EGFP) (Kissenpfennig and Malissen, 2006; Merad et al., 2008) answered some questions about the in situ behaviour of LCs during migration and differentiation. Overall, 2 to 3% of LCs circulate naturally from the skin to the lymph nodes, even in the absence of inflammatory signals (Kissenpfennig et al., 2005). Their homeostatic migration from the epidermis to the lymphoid tissues is regulated by the expression of several chemokine receptors: CCR7 allows LCs to migrate to the lymph nodes under the influence of chemokines constitutively expressed by lymphatic vessel endothelial cells, such as CCL20. This circulation allows continuous monitoring of the cutaneous environment and contributes to making the skin such a potentially valuable target for vaccination. Indeed, LCs can practice what has been nicknamed dSEARCH: dendrite monitoring extension and retraction cycling habits (Nishibu et al., 2006; Udey, 2006), interacting with keratinocytes and searching for surrounding antigens. With these two mechanisms, LCs also contribute to the tolerance of self-antigens as well as tolerance to the cutaneous bacterial flora (Geissmann et al., 2002). Recent studies have examined the relative contributions of these subsets to the generation of immunity or tolerance. Resting epidermal LCs selectively and specifically induce activation and proliferation of skin resident Treg cells (Seneschal et al., 2012). In the presence of foreign pathogen, however, LCs activated and induced proliferation of the skin T effector/ memory TEM cells (Nestle et al., 2009) that contribute to the rapid antigen-specific cell responses in the skin (Clark, 2010). Interactions between intra-epithelial T-cells and LCs generate a particularly effective local anti-infectious defence

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system (Lopez-Bravo and Ardavin, 2008; Paus et al., 1999, 2003; Ruckert et al., 1998; Seneschal et al., 2012). Moreover, LCs initiate epicutaneous sensitization with protein antigens in allergy and induce Th2-type immune responses (Nakajima et al., 2012). They might thus play a role in both the maintenance of tolerance in normal skin and the activation of protective skin-resident TEM cells on infectious challenge. LCs are called the gatekeeper of the skin, monitoring constantly for foreign antigens that they capture, transport to the draining lymph nodes and then present to the adaptive immune cells (Heath and Mueller, 2012; Igyártó and Kaplan, 2012). Hair follicles and the chemokine microenvironment may also play a role in regulating LC migration between the dermis and epidermis, according to Nagao et al. (2012). Under inflammatory conditions, the morphology of LCs changes rapidly, their dendrites become smaller, and they begin migrating from the skin to the dermis (Liard et al., 2011b) giving rise to the ‘en route’ LCs. The main subsets – LCs, CD1a+ dDCs and CD206+ dDCs (CD14 is likely to be lost during migration) – are found in skin draining lymph node. Another subtype of epidermal DCs, known as inflammatory dendritic epidermal cells (IDECs), are distinguished from LCs by their expression of the macrophage mannose receptor CD206. This cell subset has been found in the inflamed epidermis of patients with atopic dermatitis (Wollenberg et al., 2002), however its role and origin remain to be elucidated. Keratinocytes represent the major cell population in the epidermis. They are considered to be sensors of danger through alert systems such as the inflammasome complex (Di Meglio et al., 2011; Nestle et al., 2009; Singh and Morris, 2012). A recent study reports that an autocrine effect enables the IL-36 members of the IL-1 family to induce keratinocytes to secrete more IL-36 (Tortola et al., 2012); at the same time, the keratinocytes also produce antibacterial peptides When pathogens start to invade the upper layers, keratinocytes can react by producing proinflammatory mediators and chemokines such as CXCL10 and CCL2, which attract LCs. These two epidermal populations are closely intertwined, with E-cadherin mediating the adhesion of the

epidermal LCs to keratinocytes (Mayumi et al., 2013; Tang et al., 1993; Van den Bossche and Van Ginderachter, 2013). Dermal immune cells The principal cellular components of the dermis are fibroblasts, which are mesenchymal cells that synthesize components of the extracellular matrix. They secrete cytokines that play an essential role in the mechanisms of cutaneous immunity; these include IL-6, which is involved in DC activation (Saalbach et al., 2010; Yellin et al., 1995), and TGF-β, which participates in the maturation of LC precursors (Bauer et al., 2012; Geissmann et al., 1999). Fibroblast proliferation is regulated by inflammatory factors such as the myeloid-derived CCL2 chemokine (Wong et al., 2012). The dermis contains several APCs, including dDCs, dermal macrophages and plasmacytoid DCs (Teunissen et al., 2012). In normal healthy human skin, γδ CD3 T-cells, and both natural killer (NK) and NK T-cells comprise the innate immune compartments. The rich vascularization of the dermis by blood and lymphatic vessels facilitates recruitment of other inflammatory immune cells (neutrophils, monocytes, monocyte-derived DCs, and memory T-cells) and their recirculation through the activity of multiple locally produced inflammatory chemokines and cytokines (Kupper and Fuhlbrigge, 2004; Nestle et al., 2009). Dermal DCs and macrophages have common morphological features (Ochoa et al., 2008; Zaba et al., 2009), but can nonetheless be distinguished by a well-defined panel of cell markers and by their distinct localization in the dermis (Klechevsky et al., 2008). Factor XIIIa, long used in dermatology, is no longer considered to characterize dDCs but rather CD209+ macrophages (Zaba, 2007). Dermal macrophages are localized around the capillaries and in the upper part of the deep (reticular) dermis. Besides the CD14 marker, these cells express other monocyte/macrophage markers, including CD68, CD163 and CD209 (DC-SIGN). The strong expression of CD209 by macrophages differentiates them quite clearly from dDCs in histologic sections. Macrophages are DC-like CD209+CD1c–, while dDCs are CD209–CD1c+ (Ochoa et al., 2008). In flow cytometric analyses of cells extracted from human

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dermis, these cells are CD1a–CD14+HLA–DR+. This phenotype is also found in vitro in cultures of differentiated CD34+ cells (Klechevsky et al., 2008). In mice, four populations of dermal DCs can be identified according to their level of expression of CD103, CD207 and CD11b (Bursch et al., 2007; Guilliams et al., 2010; Poulin et al., 2007; Romani et al., 2010a), as Fig. 7.1 illustrates. Three populations of dDCs are found in humans: CD1a+ dDCs, CD14+ dDCs, and CD141+ dDCs (Angel et al., 2007b; Chu et al., 2012; Haniffa et al., 2012; Nestle et al., 2009; Teunissen et al., 2012). The CD1a+ dDC subset, in the papillary zone of the dermis, is the largest of the three, strongly expressing the CD1c (BDCA-1) marker and low levels of CD1a (Larregina et al., 2001; Nestle et al., 1993; Ochoa et al., 2008). According to these reports, CD1a-CD14+ dDCs show increased phagocytic activity and weak T-cell stimulatory potential. The CD141+ dDCs described recently in human skin, as the equivalent of the cross-presenting CD103+ mouse dDCs, are also a heterogeneous population containing mainly CD1c+ CD1a+ cells but also CD1a- cells in capillary locations of the dermis (Chu et al., 2012; Haniffa et al., 2012). Inflammation induces up-regulation of activation markers and costimulatory molecules at the surface of the skin DC subset, with increases in HLA-DR, CD86, CD80, CD83, CD40, and CCR7, associated with their migration into the lymph node (Angel et al., 2007a; Nestle et al., 2009; Segura et al., 2012). Upon activation, the shape of LCs changes to rounded as they become « en route » LCs which acquire CCR7 and can then migrate through the dermis into the lymph (Nestle et al., 2009; Tal et al., 2011) (Fig. 7.1). Although human skin LCs and dDCs have been extensively studied, most of our knowledge about them comes from in vitro studies of cultured cells or skin explants that do not fully reflect the complexity of the skin immune system at steady state or in inflammatory conditions in humans. The relative paucity of the highly diverse DC subsets and their phenotypic similarity to one another and to cells of the mononuclear phagocytic lineage are obstacles to deciphering their precise functions and interrelations. Recent advances in biotechnical methods and gene-expression profile

analyses offer access to rare cell populations in the tissues and have thus increased our knowledge of the complex skin DC network (Pandey et al., 2013; Satpathy et al., 2012). Such recent studies have revealed the particularity of LCs within the skin DC populations (Harman et al., 2013; Miller et al., 2012). Miller et al. (2012) have showed that skin-resident LCs display lineage markers more a macrophage-like related lineage rather than other DC subsets, while migratory LCs acquire the hallmarks of the conventional DC signature. Skin sensors and local microenvironment While protecting against pathogenic bacteria, fungi or viruses, the skin sustains a highly diverse symbiotic microbiome that influences health and disease (Grice et al., 2009; Naik et al., 2012). Skin barrier disruption due to injury or chemical or dermatological disorders such as psoriasis and dermatitis can lead to invasion of the cutaneous tissue by microorganisms (Di Meglio et al., 2011). However, the cutaneous immune system generally stops potential infections rapidly. Phagocytic and cytotoxic activities by macrophages, granulocytes, mast cells, NK cells and γ/δ T-cells eradicate most of the potential pathogens that invade the dermis. A dense, efficient innate immune system allows rapid responses in the skin through the recognition of non-specific danger signals: surface pattern recognition receptors (PRRs) sense pathogenassociated molecular pattern molecules (PAMPs) from bacteria, viruses, fungi and parasites and damage-associated molecular pattern molecules (DAMPs) from wounded skin cells (Blander and Sander, 2012; Lai and Gallo, 2008; Nestle et al., 2009). In human skin, many immune cell types as well as keratinocytes, endothelial cells, fibroblasts and other stromal cells express PRRs (Lai et al., 2009; Nestle et al., 2009). Distinct TLR expression patterns and related innate receptors (C-type lectin receptors, NOD-like receptors, RIG-1 like receptors) most likely contribute to cutaneous immune responses and tolerance (Figdor et al., 2002; Kang et al., 2006; Klechevsky et al., 2010). Innate immunity begins at the first contact with a vaccine preparation and contributes to differing extents (depending on the type of vaccine component) to an inflammatory response

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that maintain an environment propitious to APC activation in the cutaneous tissue (Kupper and Fuhlbrigge, 2004; Teunissen et al., 2012). Moreover, pro-inflammatory molecules and chemokines secreted by LCs, dDCs, keratinocytes, mast cells and activated macrophages (IL-1, TNFα, IL-6, IL-12, CCL2 (MCP-1) and CXCL8 (IL-8)) help to increase the expression of endothelial adhesion molecules, such as the E-selectins and P-selectins (ICAM-1, principally by NF-κB transcription) on lymphatic and blood vessel cells (Blumberg et al., 2007; Cumberbatch et al., 1996; Nakamura et al., 2012). Thus, It also contributes to the rapid recruitment of monocytes and neutrophils from the blood (Abadie et al., 2005, 2009; Le Borgne et al., 2006; Leon and Ardavin, 2008). Plasmacytoid DCs are also recruited upon inflammation (Sisirak et al., 2011). All these supplementary sources of APCs flowing in the skin strongly contribute to the primary events of the immune response: activation of cutaneous APCs and antigen capture. Epidermal keratinocytes play a primordial role in the early immune events in the skin (Di Meglio et al., 2011; Nestle et al., 2009; Singh and Morris, 2012). In particular, they express numerous TLRs on their surface (TLR-1, TLR-2, TLR-4, TLR-5 and TLR-6) or in endosomes (TLR-3, TLR-7, and TLR-9) NOD-like receptors that allow the keratinocytes to recognize microbial components (Chen et al., 2013; Kuo et al., 2012; Lai et al., 2009; Martinon et al., 2009). Thus, recognition of PAMPS activate the inflammasome complex and promotes the production of pro-inflammatory cytokines and chemokines. TNFα, which is a multifunctional cytokine also produced by inflammatory and Th1 cells, induces increased IL-6 production and ICAM-1 expression by keratinocytes, as well as the production of antimicrobial peptides such as beta-defensins (Harder et al., 2001; Kondo and Sauder, 1997). TNFα and IL-1 give the signals necessary both for LCs and DCs to migrate through the lymphatic vessels and accumulate in draining lymph nodes and for the development of specific immune responses (Cumberbatch, 1997; Bhushan, 2002). LCs and immature DCs, which are key sensors of danger, constantly sample the environment and self-antigens in the skin and also express several TLRs. By inducing the massive production of

pro-inflammatory cytokines and chemokines, these receptors play an essential role in the activation of cutaneous DCs (Lai and Gallo, 2008). For this reason some vaccine preparations use TLR agonists as adjuvants, to recreate the activation of DCs by PAMPs artificially at the injection site. Interestingly, dDCs present a broad profile of TLR expression, but LCs do not express receptors for TLR-2, TLR-4, or TLR-5 – all involved in the recognition of bacterial motifs (Flacher et al., 2006; Harman et al., 2013; Matthews et al., 2012; van der Aar et al., 2007). Accordingly, LCs are less sensitive than dDCs to the ligands of these TLRs, and they therefore produce fewer cytokines when they encounter bacteria. They are nonetheless able to recognize viral motifs as effectively as dDCs, via specific TLRs, such as TLR-3 (dsRNA). Recognition of exogenous or endogenous nucleic acids through TLR-7 and -9 also induced type I IFN production by plasmacytoid DCs that are recruited upon inflammation and have an important role in antiviral response. However, excessive stimulation of plasmacytoid and skin resident DCs through TLRs are associated to chronic inflammation and skin diseases (de Koning et al., 2012; Guiducci et al., 2010). In non-inflammatory conditions, cutaneous dermal and epidermal DCs are, for the most part, immature (that is, they have a strong endocytotic capacity and weak expression of MHC class II proteins). Thus, only 5% of dDCs express the CD208 marker (DC-Lamp) that is characteristic of mature DCs in non-inflamed skin (Ochoa et al., 2008). Skin DCs must be activated before they can migrate towards the draining lymph nodes (Fig. 7.2), and this activation depends highly on the cutaneous environment and especially on the balance between proinflammatory (TNFα, IL-1, and IL-6) and anti-inflammatory (IL-10) signals. DCs that have been activated become mature, with greater capacities for processing and presenting antigens. Specifically, as DCs mature, their morphology changes substantially (dendrite retraction), their capacity for endocytosis/phagocytosis diminishes, and a variety of modifications occur in their expression of some co-stimulation and adhesion molecules (increased levels of CD80, CD86, CD40, and DC-Lamp (CD208), decreased CD68, and internalization of MHC

164  | Combadière and Perrin DRAINING LYMPH NODE

SKIN

MOUSE MARKERS

HUMAN MARKERS Vaccines

Neutrophils Vaccine 1-4 h compounds

« en route » LC CD1a dDC 2-4 h

dDC

CD103 dDC

CD14 dDC

Macrophage s

1-16 h Lymphatic vessel

BONE MARROW CCR1 dependent neutrophils migration to the bone marrow : induction of LN-independent source of memory CD8+ cells

CD4+ TFH BCL-6, ICOS

Naive CD4+ T LC Skin DC

0-1 h

5-7 Days

Macrophages

TFH CCR7CXCR5 up-regulation PD-1+

LN resident DC

8-24 h

IL-21 & TGFβ Follicular DC

CD11blo dDC

2-8 h

Monocytes

Blood vessel 1-2 h

CD11bHi

CD141 dDC

Neutrophils CCR1+

30 μm), the stronger the immune response induced (Singh et al., 2004). This led to the hypothesis that smaller and more readily engineered nanoparticles would preferentially target APCs. DNA injection followed by electroporation dramatically increases the amount of antigen at the immunization site and thus increases both humoral and cellular immune responses in mice (Abdulhaqq and Weiner, 2008) and in humans (Murakami and Sunada, 2011). Although many electroporation methods have been used for muscle tissue, both electroporation and bioinjector methods have been applied to skin tissue and shown to induce direct presentation of the antigen by skin APCs (Lee et al., 2010) or cross-presentation after uptake by keratinocytes (Kim et al., 2007; Mkrtichyan et al., 2008; Rice et al., 2008).

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Immunological advantage of epidermal and dermal immunization Intradermal immunization More than 100 clinical trials of intradermal delivery of vaccines against 11 diseases, including rabies, hepatitis B and seasonal influenza, have been conducted (Hickling et al., 2011). For influenza and rabies vaccines, intradermal delivery of reduced doses resulted in immune responses equivalent to the standard dose delivered by the standard route (Lambert and Laurent, 2008; Laurent et al., 2007). One licensed vaccine is the microinjection system developed for intradermal immunization by inactivated seasonal influenza vaccine (Intanza, Sanofi-Pasteur-MSD, France). In addition, the high dosage level (15 µg HA per strain) is approved for persons 60 years of age and older (Atmar et al., 2010). The study by Young and Marra (2011) suggested that the efficacy of intradermal administration of seasonal split-virus influenza vaccines does not differ from that of intramuscular administration in the population aged 18–60 years and is similar or superior in those older than 60 years. Adverse events during the first 3 days after vaccination were also similar for both routes of administration, but the rates of local adverse events such as erythema, swelling, induration and pruritus were consistently higher in the intradermal group. The immunogenicity of the Intanza® 15-μg intradermal vaccine tended to be higher than that of Inflexal® V intramuscular vaccine in healthy elderly subjects, but injection site reactions appeared to be more frequent (Ansaldi et al., 2013). Similar results have been obtained for comparisons of the intramuscular (15  µg HA) and intradermal microinjection routes (3.6 µg HA) of the inactivated split virion influenza vaccine (alpha-Rix®, GSK Biologicals) (Van Damme et al., 2009). Dose-sparing intradermal trivalent influenza vaccine can overcome reduced immunogenicity of the H1N1 strain, and according to some measures, for the H3N2 strain. This vaccine should thus be considered for intradermal immunization of at-risk subjects, to compensate for reduced immunogenicity (Hung et al., 2012).

Despite the difficulties associated with the need for skilled medical practitioners to use the Mantoux technique, intradermal vaccination by this method has been tested in humans in numerous clinical trials of more than 12 different vaccines. The vaccines studied most frequently were those against rabies, HBV, and seasonal influenza (Nicolas and Guy, 2008; Roukens et al., 2010). Many of these protocols sought to assess the potential of the intradermal route compared with the standard intramuscular and subcutaneous routes, in terms of vaccination efficacy and antigenic dose. Unsurprisingly, the outcome of these clinical trials varied significantly according to the vaccines tested. Nonetheless, it is now generally agreed that the intradermal route can induce immune responses (neutralizing antibodies and long-term protection) similar in intensity to those of the other routes with a lower antigen dose (generally one-fifth). These studies nonetheless have weaknesses. First, they often consider only the humoral response. Assessment of the potential cellmediated response (CD4+ and CD8+) induced intradermally would have been useful, in view of the fundamental role of CD4+ T-cells in building the immune memory and the importance of inducing CD8+ T-cells capable of recognizing several viral variants for influenza vaccination (Combadiere et al., 2010). The variation in clinical protocols and the lack of standardized methods for analysing results sometimes prevent an accurate assessment of the potential of the intradermal route. It is also difficult to compare the efficacy of two immunization routes without knowing the optimal antigen dose. These trials have only rarely tested dose–response relations. Nonetheless, these vaccines can be classified in two groups: (1) vaccines for which intradermal injection has induced better humoral response with a lower antigen dose, i.e. the rabies vaccine (Vien et al., 2008), HBV vaccine in patients on dialysis (Micozkadioglu et al., 2007), and seasonal influenza vaccine in the elderly (Fiore et al., 2010), and (2) vaccines for which results conflict, i.e. the trivalent seasonal influenza vaccine. But we know that the intensity of influenza immune response depends strongly on the patient’s infection, vaccine history and age.

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Mouse studies with inactivated influenza vaccines showed that intradermal injection with microneedles greatly improved vaccine efficacy (measured by the level of neutralizing antibodies), compared with intramuscular injection (Kim et al., 2009), and intradermal injection of a VLP influenza vaccine with microneedles induced better protection during a lethal viral challenge in mice than did an intramuscular injection (Quan et al., 2010; Zhu et al., 2009). Although these results are very encouraging, the feasibility of these techniques must be tested in humans, in view of the limitations of animal models. For this reason, vaccine manufacturers try to integrate these new techniques into their clinical studies very early on, to optimize the efficacy and cost of the combined product. Needle-free epidermal immunization Numerous studies have used diverse techniques to disrupt the horny layer and showed that it is possible to induce humoral, CTL, and mucosal responses by the transcutaneous route in mice and in humans. The numerous techniques available for epidermal immunization today include (1) patch systems, (2) topical application of viral vectors in vesicular systems, such as liposomes, niosomes, transfersomes and vesosomes, (3) low-frequency ultrasound, (4) iontophoresis or electroporation, (5) tape-stripping, and (6) nanoor micro-needles (Bal et al., 2010). The potential for needle-free vaccination with devices, patches or tape-stripping has been validated with several vaccine models in animal studies and human clinical trials. G. Glenn’s group has pioneered in the domain of transcutaneous vaccination and the use of patch vaccine delivery (Glenn et al., 2000). It was the first to show that transcutaneous vaccination combining a patch and a cholera toxin in mice protected the animals against a lethal mucosal challenge (Glenn et al., 1998). Clinical trials with E. coli lymphotoxin applied in a patch (Glenn et al., 2000) showed that patch application promotes a high rate of anti-lymphotoxin antibodies. The first patch vaccine against traveller’s diarrhoea (turista) that uses lymphotoxin as an adjuvant has reached phase III clinical testing in the United States (Frech et al., 2008). Other

studies of seasonal influenza vaccine in the elderly found better seroconversion with a patch than with intramuscular injection (Frech et al., 2005). Transcutaneous routes have been studied in various diseases including diphtheria (Matsuo et al., 2011), cholera (Glenn et al., 1998, 2000, 2007), tetanus (Shi et al., 2001), yersiniosis (Eyles et al., 2004), herpes virus simplex 1 (El-Ghorr et al., 2000), HIV (Belyakov and Ahlers, 2011), influenza (Combadiere et al., 2010; Vogt et al., 2008), vaccinia (Mahe et al., 2009), anthrax (Kenney et al., 2004) and some types of tumours (Takigawa et al., 2001; Yagi et al., 2006). Rechtsteiner and colleagues have even suggested increasing the intensity of the CTL response induced by transcutaneous vaccination by adding to the vaccination preparation a cream containing TLR-7 ligands (imiquimod), which stimulate LC maturation (Rechtsteiner et al., 2005). Finally, Belyakov et al. (2004) applied Rechtsteiner and Glenn’s ideas and used adjuvants to induce a good CTL response to transcutaneous vaccination. They immunized mice by the transcutaneous/patch route with HIV peptides administered simultaneously with cholera toxin or lymphotoxin and thereby induced very good specific CTL response in the spleen but also in the intestinal, vaginal and nasal mucosa and the Peyer patches. They also demonstrated that transcutaneous vaccination protected animals against a mucosal challenge with a recombinant MVA that expressed HIV gp160 (Belyakov et al., 2004). This study showed for the first time that transcutaneous vaccination (with lymphotoxin or cholera toxin adjuvants) could induce specific systemic and mucosal CTL responses. Indeed, one of the most notable benefits of transcutaneous immunization is the induction of immune responses in both systemic and mucosal compartments (Lawson et al., 2012). However, the immune mechanism for this is still unknown. Finally, transcutaneous vaccination using CSSS and cutaneous application of trivalent inactivated influenza vaccine induced both CD4+ and CD8+ T-cell response, whereas intramuscular injection of vaccine induced a strong effector CD4+ T-cell response but no influenza-specific CD8+ cell response (Combadiere et al., 2010; Vogt et al., 2008). A phase I/II clinical trial with a live

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attenuated measles vaccine (Rouvax®) patch produced IgA in saliva and a measles-specific CD8+ T-cell immune response (Etchart et al., 2007). Moreover, in a clinical trial with seven patients with advanced melanoma, five CSSS applications of a mixture of melanoma and HIV peptides induced both melanoma and HIV-specific CTLs in vivo (Yagi et al., 2006). Numerous studies suggest that targeting mostly epidermal DCs, as by vaccine delivery through hair follicles, induces a CD8+ T-cell immune response whereas the intramuscular route does not. Therefore, any attempt to redirect the immune response by using specific skin routes of vaccination requires paying special attention to the choice of vaccination technique, especially for intradermal and transcutaneous vaccination methods (e.g. patch versus hair follicle targeting, or microneedle versus conventional intradermal immunization). Conclusion Numerous concepts for vaccine delivery to the skin have been developed. However, they have not yet met expectations: this challenge remains to be met by immunologists and vaccine designers. Further comparative studies are necessary. Other key components of vaccination remain to be elucidated in more detail: (i) the nature of the antigens against which adaptive immune responses are elicited, (ii) the nature of the vaccine components that can favour the initiation of the immune response via certain intrinsic adjuvant effects, and (iii) the delivery system that will ensure optimal presentation to both the innate and the adaptive immune systems. Moreover, these routes of immunization must be considered for each of these factors. Intramuscular and subcutaneous routes, widely used for vaccination, have proven to be successful in inducing systemic humoral immunity towards several pathogens but generally failed to induce efficient and long-term protection. The challenge today involves especially manipulating the immune system so as to produce predictable vaccine efficacy. The nature and intensity of acquired immune protection vary depending on the targeted pathogens and host responses. Progress is needed to understand the immune pathways and networks induced after vaccination by novel skin routes of immunization. It

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Toll-like Receptors as Targets to Develop Novel Adjuvants Şefik Şanal Alkan

Abstract This chapter starts with a summary of our current understanding of innate immunity, which is the rapid responding arm of the immune system. Innate cells recognize microorganisms via molecular sensors collectively called pattern-recognition receptors (PRR). These evolutionarily conserved sensors consist of four families based on their molecular structures: C-type lectin receptors, NOD-like receptor, and RIG-I-like receptors and Toll-like receptors (TLRs). We will focus on the latter, as TLR agonists are molecules most advanced as vaccine adjuvants. Various degrees of information are available about TLR (TLR2– TLR9) agonists that are under development for the treatment of infectious diseases and cancer: while some are at very early stage, many are in clinical trials and a few are approved as vaccine adjuvants. Decades of TLR research have taught us several lessons. Just to name a few: There is variation in the cellular distribution of TLRs in different species, which makes the translation of animal data into the clinic difficult; simultaneous delivery of TLR agonist and the antigen is more efficacious; multiple TLR activation produces much better adjuvant effects. Equipped with these lessons, we are not far from utilizing multiple TLR agonists as adjuvants in 21st century vaccines against infectious diseases and cancer. Introduction Empirical vaccines were among the first ‘medicines’ humanity discovered in the distant past. Now, the time has arrived to ‘design’ vaccines rationally, thanks to the enormous progress made

8

in four related areas. First, we understand better how the immune system works, notably, antigen recognition, generation of diversity, specificity and memory ( Janeway et al., 2001; Pulendran and Ahmed, 2006; Sallusto et al., 2010). Second, by molecular engineering, we can dissect the structures of antigens and synthesize small subunits (Sirskyj et al., 2011; Quakkelaar and Melief, 2012; Moyle and Toth, 2013). Third, through modern methods, we can deliver vaccines more efficiently (Giudice and Campbell, 2006; O’Hagan and Rappuoli, 2006). Finally, we have begun to utilize novel synthetic adjuvants that are needed to potentiate the desired, protective immune responses (Bhardwaj and Gnjatic 2010; Coffman et al., 2010; Duthie et al., 2011; Levitz et al., 2012). For historical reasons, and perhaps for convenience, immunologists divide the immune system into two, namely the innate and adaptive immunities. Fig. 8.1 depicts this artificial division by showing the most relevant cells and molecules of both systems, which in reality, work intimately together. Knowledge on adaptive immunity has developed earlier and went deeper, while the significance of innate immunity was not fully realized for decades. The 2011 Nobel Prize for medicine rewarded J. Hoffmann, B. Beutler and R. Steinman for their revolutionary findings concerning the activation of the immune system, and showing the significance of understanding the mechanisms of activation of innate immunity. Thanks to the recent discovery of pattern recognition receptors (PRR), we are now on in the middle of a renaissance of studies in this field. Owing to its relevance to vaccinology, let’s briefly recall, some of the basic features of innate immunity.

188  | Alkan Innate immunity

Macrophage

Adaptive immunity

T cell

T cell

CD4

CD8 CD8

Dendritic cell δλ T cells

Th2

Th1 Neutrophil

Basophile Natural killer cell Natural killer T cell

Complement proteins

Mast cell Eosinophil

• Fast • Germ-line encoded • Lacks fine specificity • No memory

B cell

Th9 Th22

Th17

Treg

• Slow • Gene rearrangement • Highly specific • Lasting memory

Figure 8.1 Cells of the innate and adaptive immune system. The innate immune response functions as the first line of defence against infection. It consists of soluble factors, such as complement proteins, and diverse cellular components. The adaptive immune response is slower to develop, but manifests as increased antigenic specificity and memory. It consists of B-cells, and several subsets of CD4+ cells and CD8+ T-lymphocytes. Natural killer T-cells and γδ T-cells are cytotoxic lymphocytes at the interface of innate and adaptive immunity. Modified from Dranoff (2004).

Innate immunity This ancient system operates under the direct control of germ-line genes that co-evolved during our long co-existence with microorganisms. The system uses evolutionarily ancient mechanisms like the complement protein cascade, mannose recognition receptors, phagocytosis, programmed cell death (apoptosis), etc. The field of innate immunity has enjoyed tremendous progress in the past decade. We now know that cells of this system provide rapid response to invading pathogens, by recognizing evolutionarily conserved microbial signature structures named pathogen-associated molecular patterns (PAMPs). Detection of PAMPs by the host occurs via germline-encoded pattern-recognition receptors (PRRs). Many PRRs are expressed on many cells of the innate immunity, where they can be located on the cell surface, or in endocytic compartments or the cytoplasm. Upon recognition of PAMPs, the cardinal cells of innate immunity, dendritic cells (DCs), for example, can initiate an adaptive immune response, i.e. T- and B-cell responses (Fig. 8.1). Owing to requirement of somatic gene

rearrangements, adaptive responses take days to weeks to develop, but can leave behind a lasting memory. Since the goal of most vaccines is the generation of memory B- and T-cells, we need to know which specific PRRs to stimulate in order to develop effective vaccines (Iwasaki and Medzhitov, 2010; Hajishengallis and Lambris, 2011; Levitz and Douglas, 2012) Pattern recognition receptors (PRRs, sensor molecules) Pattern-recognition molecules can be divided into multiple families based on their molecular structure. Fig. 8.2 depicts four groups in the ever-growing list of PRRs, all of which might be relevant for vaccine adjuvant development. These include C-type lectin receptors (CLRs), NODlike receptor (NLRs), and RIG-I-like receptors (RLRs) and Toll-like receptors (TLRs) (Levitz and Golenbock, 2012; O’Neill and Bowie, 2010). The CLRs are cell surface receptors, whereas the NLRs and RLRs reside in cytoplasm. The interaction of a PRR with its ligand results in a signalling cascade ultimately leading to an inflammatory response.

TLR Agonists as Novel Adjuvants |  189 CLRs

TLRs

NLRs

TLR2/1

NLRP3 NLRC4 NOD2

DEC-205

Dectin-1

TLR7 TLR9

TLR2/6

TLR8

TLR3

Mincle

TLR4-

MD2

RLRs Mannose R

RIG-I

TLR5

MDA5

Figure 8.2  Pathogen recognition receptors (PRR) relevant for vaccination. TLR1, TLR2, TLR4, TLR5, and TLR6 are located on the cell surface. TLR2 forms a heterodimer with TLR1 or TLR6. TLR3, TLR7, TLR8 and TLR9 are endolysosomal. The C-type lectin receptors (CLRs) are cell surface receptors, whereas the NLRs (NOD-like receptors) and RLRs (RIG-I-like receptors) reside in cytoplasm. Modified from Levitz and Golenbock (2012).

CLRs (C-type lectin receptors) are transmembrane proteins, many of which are expressed on DCs and recognize pathogen-associated glycans such as β-glucans and mannoses (Osorio and Reis e Sousa, 2011). Since some CLRs recognize endogenous (self) ligands as well, they may not be ideal receptors for vaccine development. NLRs (NOD-like receptors and RLRs (RIGI-like receptors) are all intra cytoplasmic sensor molecules. More than 20 NLRs are predicted based upon the human genome (Franchi et al., 2010; Elinav et al., 2011). All NLRs contain a nucleotide-binding oligomerization domain and a leucine-rich repeat (LRR). LRRs are also present on TLRs and are responsible for ligand recognition (Fig. 8.3). Sensing of PAMPs by many NLRs leads to formation of a proteolytically active multiprotein complex termed the inflammasome, which causes maturation of the cytokines IL-1β and IL-18. RLRs, RIG-I and MDA5, recognize dsRNA and stimulate nuclear factor kappa-light-chainenhancer of activated B-cells (NF-κB) and interferon regulatory factor 3 (IRF3/7) signal transduction pathways (O’Neill and Bowie, 2010). Ligand recognition by RLR/MDA5 leads to inflammasome activation. Recently, several

cytoplasmic DNA sensors have been described, each utilizing a distinct signalling complex (Hornung et al., 2009; Hornung and Latz, 2010; Sharma et al., 2011). TLRs (toll-like receptors) The discovery of these phylogenetically ancient, evolutionarily conserved molecules, and their role in sensing infections, represents one of the most important advances in immunology. We now know that TLRs play a fundamental role in recognition of microbes, and stimulate and tune the quality of the ensuing adaptive immune responses. To date, 13 TLRs have been reported in human and mouse. A full account of PAMPs and TLRs is given in recent reviews (Akira and Takeda, 2004; Gay and Gangloff, 2007; Iwasaki and Medzhitov, 2010; Kawai and Akira, 2011). Fig. 8.3 describes TLRs’ locations, their ligands and their common signalling pathway. TLRs recognize a remarkably diverse number of PAMPs expressed by viral, bacterial, fungal, and parasitic pathogens (Akira et al., 2006). TLR1, TLR2, TLR4, TLR5 and TLR6 are primarily expressed on the plasma membrane, where they recognize specific molecules on the surface of microbes. On the other hand, TLR3, TLR7,

190  | Alkan Lipoprotein, PGN (bacteria)

Peptidoglycan, Zymozan (bacteria, fungi)

TLR2/2

TLR1/2

Lipoproteins, MALP-2, LTA, PGN

LPS (bacteria)

TLR4

TLR2/6

Flagellin (bacteria)

TLR5/?

Profilin (protozoa)

TLR11

LLR domain

TIR domain

Endopllasmic Virus ds RNA TLR3

Virus ssRNA TLR7/8

Bacteria DNA TLR9

MyD88 IRAK TRAF6

NF-kB

Inflammatory responses

Figure 8.3  Toll-like receptors and their ligands. There are about a dozen TLRs in humans. Surface TLRs (TLR1, TLR2, TLR4, TLR5, TLR6, TLR10, TLR11) usually recognize bacterial products. Endosomal TLRs (TLR3, TLR7, TLR8, TLR9) recognize usually viral RNA and DNA. All TLR signalling utilizes MyD8 except TLR3. LRR: Leucine-rich repeats; TIR: Toll-IL-1 receptor. Modified from Kaufmann (2007) and Takeda and Akira (2004).

TLR8 and TLR9 are located in endolysosomal compartments, where they recognize single- or double-stranded RNA and DNA molecules (Latz et al., 2004a; Brinkmann et al., 2007; Iwasaki and Medzhitov, 2010). TLRs activate the NF-κB pathway, which regulates cytokine expression, through several adaptor molecules including MyD88. Activation of the NF-κB pathway initiates production of inflammatory cytokines such as IL-1, IL-6, IL-8, IL-12, TNF and chemokines, and induction of co-stimulatory molecules such as CD80, CD86, and CD40. Each immune cell expresses a different set of TLRs (Alkan, 2012; Coffman et al., 2010; Duthie et al., 2011; Hornung et al., 2002; Iwasaki et al., 2004; Kaufmann 2007). Cellular distribution of human TLRs is depicted in Fig. 8.4. Distribution of other sensors as shown in Fig. 8.2, is summarized elsewhere (Duthie et al., 2011). After engagement with a PAMP, TLRs initiate signalling pathways through interactions with several adaptor proteins, which include MyD88, IRAK, TRAP and TRAF6 (TRIF for TLR3) (Akira and

Takeda, 2004; Gay and Gangloff, 2007). This results in activation of nuclear factor (NF-κB), and interferon regulatory factor (IRF)-responsive genes (Fig. 8.3). TLRs activate multiple steps in the inflammatory reactions that help to eliminate the invading pathogens by linking innate and adaptive immune responses, and hence coordinate systemic defences. TLRs control multiple DC functions and activate signals that are critically involved in the initiation of adaptive immune responses. Recent studies have provided important clues about the mechanisms of TLR-mediated control of adaptive immunity orchestrated by DC populations in distinct anatomical locations (Iwasaki and Medzhitov, 2004; Pulendran et al., 2010). After the realization that innate immunity directs the ensuing adaptive immunity, it became the basis of vaccine adjuvant research. Currently, there is an intense focus on molecules that trigger a given TLR. The search of agonists of NLRs and RLRs is also expected to intensify in the near future.

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Mono

mDC

pDC

B cell

1,2,4,5,6

1,2,3,4,5,6,10

1,6,10

1,2,4,5,6,10

RLR NLR

Mφ 1,2,3?,4,5,6

RLR NLR

PMN 1,2,4,5,6,10

NK cell 1,2,3,4?,5?,6

RLR NLR

NKT cell 2,3,4,6

Figure 8.4 TLR expression pattern of human cells. Despite overlaps, each cell appears to be unique in its TLR expression. mDC: myeloid dendritic cell; pDC: plasmacytoid DC; Mφ: Macrophage; PMN: polymorphonuclear cell. NK: Natural killer cell; NKT: Natural killer T-cell. Data collected from Alkan (2012), Coffman et al. (2010), Duthie et al. (2011), Hornung et al. (2002) and Iwasaki and Medzhitov (2004).

Adjuvants While immunogens contained in a vaccine provide the antigen-specific stimulus, vaccine adjuvants direct the quantity and quality of the ensuing immune response (Pulendran and Ahmed, 2011). For most live vaccines, such as bacillus Calmette– Guérin (BCG), measles mumps and rubella (MMR), and for some inactivated vaccines, antigen and adjuvant activity reside in the same vaccine ingredient. However, modern vaccines, which increasingly consist of purified microbial subunits, often lack the adjuvant activity necessary to induce an adequate immune response. Inclusion of adjuvants has been key to the efficacy of these subunit vaccine formulations, especially for vaccination of children (Levy et al., 2012). Non-TLR adjuvants Before going into novel and mostly experimental TLR agonist-based vaccine adjuvants, it is pertinent that we touch briefly upon several other adjuvants, some of which have been used for decades without knowing the mode of action. Adjuvants that trigger these non-TLRs, most likely will be used in combination with TLR agonists. We know that these adjuvants trigger other sensor molecules that we mentioned earlier (Figs. 8.2 and 8.3). The topic has been reviewed recently (Ofer et al., 2012; Levitz and Golenbock, 2012).

Aluminium salts Before the discovery of TLRs the most widely used adjuvants were based on aluminium salts. The mode of action of these empirically discovered adjuvants has been clarified only recently, although the final mechanism is not yet well defined. Crystalline alum binds lipid moieties on DCs, which promotes lipid sorting in the DC plasma membrane (Flach et al., 2011). Upon phagocytosis by antigen-presenting cells (APCs), alum particles trigger lysosomal membrane damage and cathepsin B-dependent activation of the NALP3 (NLR family) inflammasome and release of IL-1, IL-18 and IL-33 (Kool et al., 2012). Emulsions MF59 (Novartis) and AS03 (GSK) are both oil-in-water emulsions based on squalene, an oil that is more readily metabolized than the paraffin oil used in Freund’s adjuvants. MF59 and AS03 are licensed adjuvants for influenza vaccination. (Mosca, 2008; O’Hagan et al., 2012; Vesikari et al., 2011). Such emulsions trigger local recruitment of innate cells at the injection site and draining lymph node and enhance subsequent induction of antibody responses. The adjuvant effect of MF59 on antibody production appears to be independent of the NALP3 inflammasome, but MyD88-dependent (O’Hagan et al., 2012, 2013).

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Cationic adjuvant formulations They consist of a delivery vehicle (liposomes) plus synthetic mycobacterial cord factor (CAF01) as the immunomodulator (Christensen, 2009). CAF01 has shown promising results as adjuvant for a wide range of diseases. Virus-like particles Delivery systems comprised of phospholipid membrane vesicles containing viral proteins, virosomes have been effectively used as adjuvants in mice (Kamphuis, 2012) and humans (Herzog et al., 2009). Before we close the topic of non-TLR adjuvants, here a word of caution might be appropriate: Most of these molecules exert their effects through inflammasomes. Inflammasomes are multiprotein complexes that activate caspase-1, which leads to maturation of the proinflammatory cytokines interleukin 1β (IL-1β) and IL-18. Members of the NLR family (NLRP1, NLRP3 and NLRC4, and the cytosolic receptor AIM2) are critical components of inflammasomes and link microbial and endogenous danger signals to the activation of caspase-1. However, this pathway, which is protective against invaders, when deregulated, causes auto/inflammatory diseases and other pathologies (Franchi et al., 2012). It should be mentioned, however, that alum, which has been extensively used in humans in several different vaccines, has never been associated with such adverse reactions. TLR-based adjuvants Bacterial pathogens impose a heavy burden of disease on human populations worldwide. Highly virulent respiratory pathogens, enteric pathogens, malaria and HIV-associated infections are still enormous treats to humanity. Tuberculosis alone is responsible for the deaths of 1.5–2 million people annually. And among bacterial pathogens drug resistance is rapidly spreading. While waiting for population-level whole-genome sequencing studies to yield new treatment modalities (Wilson, 2012) our best choice is vaccinations with better adjuvants. Along with our experience with first generation vaccines, recent progress in TLR research suggests that an increased number of effective vaccines containing TLR agonists will be available in

the future. Below, I summarized the current status of TLR agonists in vaccine development. The possibilities of using TLR agonists either alone, or in combination with non-TLRs, to provide prophylactic or therapeutic protection against infectious diseases and cancer have been reviewed recently (Bhardwaj and Gnjatic 2010; Coffman et al., 2010; Duthie et al., 2011; Levitz et al., 2012). TLR2 agonists TLR2 agonists stimulate robust IL-12 production in DCs in vitro and thus would be predicted to promote Th1-type responses. Also, TLR2 binds lipoproteins and the lipidated cysteine. (Brightbill et al., 1999). In vivo, animals vaccinated with a peptide from Mycobacterium tuberculosis conjugated to the TLR2 ligand Pam2Cys mounted Th1-type responses and were protected against mycobacterial challenge (Gowthaman et al., 2011). The neisserial outer membrane protein (OMP) complex has been used as a vaccine adjuvant in some Haemophilus influenzae type b (‘Hib’) vaccines. Only years after widespread use of OMP-adjuvant in Hib vaccines in infants it was discovered that OMP is in fact a TLR2 agonist (Latz et al., 2004b). The neisserial outer membrane protein PorB was found to have TLR2-dependent effects and induced a Th2-type profile (Burke et al., 2007). The outer surface lipoprotein (OspA) of Borellia burgdorferi, the causative agent of Lyme disease, is recognized by TLR2/TLR1. Low antibody titres to recombinant OspA, the major component of a withdrawn vaccine against Lyme disease, were associated with defects in the TLR2/1 signalling pathway (Alexopoulou et al., 2002). In a recent study, it was demonstrated that intranasally administered Pam2Cys triggers a cascade of inflammatory signals, by attracting neutrophils and macrophages and inducing secretion of IL-2, IL-6, IL-10, IFN-γ, MCP-1 and TNF-α. (Tan et al., 2012). It provided increased resistance against influenza A virus challenge and also reduced the potential for transmission of infection, reduced weight loss and lethality associated with virulent influenza virus infection in a TLR 2-dependent manner. Treatment did not affect the animals’ ability to generate an adaptive immune response, measured by the induction of functional influenza A virus-specific CD8+ T-cells following exposure

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to virus. Because this compound demonstrated efficacy against distinct strains of influenza, it could be a candidate for development as an agent against influenza and possibly other respiratory pathogens. TLR3 agonists The discovery that double-stranded viral RNA (dsRNA) is a potent activator of innate immunity was an important finding for understanding host immunity against viral infection (Alexopoulou et al., 2001). Synthetic analogues of dsRNA such as Poly IC, have been used as adjuvants (StahlHennig et al., 2009; Longhi et al., 2009). Viral or synthetic dsRNA activates TLR3 in endosomes (Alexopoulou et al., 2001) or through cytosolic RLRs, and melanoma differentiation-associated gene 5 (MDA5) (Kato et al., 2006). TLR3 mediates its effects through the adaptor TRIF (Alexopoulou et al., 2001), whereas RLRs signal through the adaptor IFN-β promoter stimulatory-1 (Kato et al., 2006). TLR3 activation in DCs induces IL-12 and type I IFN and improves MHC class II expression and cross-presentation (Coffman et al., 2010; Kadowaki et al., 2001). Stimulation of MDA-5 strongly enhances production of type I IFNs, which play a critical role in enhancing T- and B-cell immunity with dsRNA through several mechanisms that include activation of DCs, NK cells, and direct effects on T-cells. Several synthetic analogues of dsRNA (Poly IC, Poly ICLC, and Poly IC12U) have been used as adjuvants with soluble proteins, DC-targeting constructs, or inactivated viral vaccines (Gowen et al., 2007; Stahl-Hennig et al., 2009; Trumpfheller et al., 2008). Poly IC activates both TLR3 and MDA, whereas Poly IU signals through TLR3 only. The formulation of Poly IC is critical for its potency. While long dsRNA is required to activate MDA-5 (Kato et al., 2008), Poly IC + poly-l-lysine and carboxymethylcellose (poly ICLC) complex prolongs the adjuvant effect in vivo (Stahl-Hennig et al., 2009; Levy et al., 1975). Poly (I):poly (C12U), which is structurally similar to dsRNA, promotes protective mucosal and systemic antibody responses in mice following intranasal administration with influenza antigens (Ichinohe et al., 2007). Studies on the ontogeny of the TLR3-mediated responses

indicate that newborns and infants up to 1 year of age produce these three cytokines at much lower levels than in adults (Corbett, 2010; De Wit, 2003). In a recent clinical study, the efficacy and safety of a TLR-3 agonist, rintatolimod (Poly I: C(12) U), were examined in patients with debilitating chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) (Strayer et al., 2012). CFS/ ME is a disease with unknown pathogenesis and shows a variety of symptoms including severe fatigue. In this phase III prospective, doubleblind, randomized, placebo-controlled trial, the effect of rintatolimod in 234 subjects with severe CFS/ME was studied. Several improvements were observed. Rintatolimod produced objective improvement in exercise tolerance and a reduction in CFS/ME-related concomitant medication usage as well as other secondary outcomes (ClinicalTrials.gov NCT00215800). In summary, an optimally formulated Poly IC is an effective adjuvant for inducing adaptive immunity through both TLR and RLR signalling pathways. TLR4 agonists Although the adjuvant effect of bacterial lipopolysaccharides (LPS) has long been recognized, due to their pyrogenic activity they could not be used in man. Later, less toxic preparations of LPS, and ultimately the substantially detoxified derivative monophosphoryl lipid A (MPL), have been prepared (Qureshi et al., 1982). Since then, several adjuvants have been licensed including adjuvant system 03 (AS03) and AS04. AS04, which consists of 3-O-desacyl-42-monophosphoryl lipid A (MPL) adsorbed onto aluminium hydroxide, is used as an adjuvant in licensed vaccines against human papillomavirus (HPV, Cervarix), and hepatitis B virus (FENDrix). MPL is a derivative of LPS from Salmonella minnesota, which lacks some acyl chains, polysaccharide side groups, and phosphates. MPL has less toxicity than its parent molecule, lipid A and has proven to be both safe and effective (Casella and Mitchell, 2008). Both LPS and MPL stimulate TLR4, but MPL leads to signalling only through the TRIF adaptor, whereas LPS leads to TLR4 activation through both the TRIF and MyD88 pathways (Mata-Haro et al., 2007), the latter pathway resulting in high

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levels of many inflammatory cytokines, prominently TNF-α. MPL formulated on alum (AS04) stimulates a polarized Th1 cell response in contrast to the mixed Th1-Th2 cell response of alum alone (Casella and Mitchell, 2008; Didierlaurent et al., 2009). Much of the adjuvant activity of this mixture can be attributed to the MPL component, although alum helps prolong stimulation by MPL (Didierlaurent et al., 2009). In addition to the MPL, other synthetic TLR4 ligands are in development for use as adjuvants (Coler et al., 2011). In a recent study, the effect of TLR4 agonist E6020 was evaluated with recombinant Men B antigens delivered in MF59 submicron adjuvant emulsion (Singh et al., 2012). The co-delivery of E6020 within MF59 enhanced both the serum and bactericidal titres for Men B antigens and for Men ACWY–CRM conjugate vaccine. The delivery of the TLR4 agonist within MF59 emulsion oil droplets led to a more potent response than when it was simply mixed with the emulsion. MPL’s mechanism of action appears to decrease inflammasome priming and IL-1 production, while maintaining overall adjuvanticity (Embry et al., 2011). Interestingly, immune responses to MPL-containing vaccines would likely differ if given at birth versus later in life (Levy et al., 2012). MPL-containing vaccines given near birth are more likely to support a Th17- or Th2-polarized response, instead of a Th1 type response. However, the issue of age-dependency of the immune response has not yet been fully analysed. TLR5 agonists Flagellin, the major structural protein of flagella on Gram-negative bacteria, which was known as a potent T-cell-independent antigen, has received much attention as an adjuvant. It was found that extracellular flagellin is sensed via TLR5, whereas intracellular flagellin stimulates the NLRC4 inflammasome by a process requiring direct recognition by the NOD-like receptor protein NAIP5 (Mizel and Bates, 2010; Zhao et al., 2011). TLR5 signalling appears to be more important for adjuvanticity because most flagellin-formulated vaccines are designed to stimulate DCs extracellularly. However, a Listeria monocytogenes strain engineered to express flagellin in the host cell cytoplasm was attenuated because of its ability

to hyperactivate the NLRC4 inflammasome, yet was able to confer protective immunity against virulent L. monocytogenes (Warren et al., 2011). An advantage of flagellin as an adjuvant is that it can be incorporated as a fusion protein in recombinant vaccines (Huleatt et al., 2007; Mizel and Bates, 2010). In general, flagellin-adjuvanted vaccines stimulate robust antibody and CD4+ T-cell responses. Unlike many other TLR agonists, flagellin tends to produce mixed Th1 and Th2 cell responses rather than strongly polarized Th1 cells (Huleatt et al., 2007). In phase I and phase II trials, a recombinant haemagglutinin influenza–flagellin fusion vaccine was safe and induced seroprotective titres, even in the elderly (Taylor et al., 2011). TLR7 agonists (e.g. imiquimod) ssRNAs rich in guanosine and uridine were first identified as natural agonists for TLR7 and 8 (Diebold et al., 2004; Heil et al., 2004; Lund et al., 2004). Interestingly, a number of small synthetic compounds originally developed as type I IFN inducers were found to be TLR7/8 agonists. Thus, 3M’s imidazoquinolines (Imiquimod, TLR7 and Resiquimod, TLR7-TLR8) and some guanosine and adenosine analogues have been shown to activate TLR7, TLR8, or both (Gorden et al., 2005; Heil et al., 2003; Hemmi et al., 2002). TLR7 and TLR8 both mediate their effects through MyD88dependent signalling (Hemmi et al., 2002). Mainly because of its small molecular size (less than 500 Da) and ease of applications to skin, the therapeutic potential of TLR7 agonist imiquimod, was recognized in the clinical setting more than a decade ago. Beginning with an approved indication for the treatment of external genital warts caused by human papillomavirus (HPV) in 1997, imiquimod 5% topical cream (Aldara) has received further approval for treating actinic keratosis and superficial basal cell carcinoma. Currently, imiquimod 5% topical cream is the most widely studied and characterized TLR agonist available in the clinical setting (Miller et al., 2008). This drug, which is the first approved TLR7 agonist, activates innate immune cells to produce interferon-α and other cytokines. The induced cytokine cascade, in combination with effects in enhancing antigen presentation,

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promotes an antigen-specific T-helper type 1 cellmediated immune response. This immunological mechanism provides activity against a number of viruses and other intracellular pathogens. Activity of imiquimod against several other viruses has been described in case reports of ‘off-label’ usage (Miller et al., 2008). In early clinical trials topical administration of imiquimod as adjuvant followed by injection of vaccine antigen through the treated skin enhanced recruitment of mononuclear cells, activation of dendritic cells and enhanced both humoral and cellular adaptive immunity in vivo (Adams et al., 2008). Recombinant, full-length NY-ESO-1 protein was administered intradermally into imiquimod-preconditioned sites followed by additional topical applications of imiquimod. The formulation elicited both humoral and cellular responses in a significant fraction of patients. Skin biopsies were assessed for imiquimod’s in situ immunomodulator effects. Compared with untreated skin, topical imiquimod induced dermal mononuclear cell infiltrates in all patients composed primarily of T-cells, monocytes, macrophages, myeloid DCs, NK cells, and, to a lesser extent, plasmacytoid DCs. Thus, feasibility and excellent safety profile of a topically applied TLR7 agonist as a vaccine adjuvant in cancer patients has been demonstrated (Adams, 2008). As expected, the anti-tumour effect of imiquimod is multifactorial. In vitro studies suggested a role for plasmacytoid DCs (pDCs) (Inglefield et al., 2008). However, a direct contribution of pDCs to tumour killing in vivo has been lacking. In a recent study, using a mouse model of melanoma, it was demonstrated that pDCs could directly clear tumours without the need for the adaptive immune system (Drobits et al., 2012). Topical imiquimod treatment led to TLR7-dependent and IFN-α/β receptor 1-dependent (IFNAR1dependent) up-regulation of expression of the chemokine CCL2 in mast cells. This was essential to induce skin inflammation and for the recruitment of pDCs to the skin. The recruited pDCs were CD8α+ and induced tumour regression in a TLR7/MyD88- and IFNAR1-dependent manner. Lack of TLR7 and IFNAR1 or depletion of pDCs or CD8α+ cells from tumour-bearing mice completely abolished the effect of imiquimod.

TLR7 was essential for imiquimod-stimulated pDCs to produce IFN-α/β, which led to TRAIL and granzyme B secretion by pDCs via IFNAR1 signalling. Blocking these cytolytic molecules impaired pDC-mediated tumour killing. These mechanistic studies demonstrate that imiquimod treatment leads to CCL2-dependent recruitment of pDCs and their transformation into a subset of killer DCs able to directly eliminate tumour cells (Drobits et al., 2012; Holcmann et al., 2012). As the skin metastases of breast cancer remain a therapeutic challenge in the clinic, Adams et al. (2012) tested the ability of topically applied imiquimod to stimulate local anti-tumour immunity and induction of regression of breast cancer skin metastases. Although only 10 patients completed the study, imiquimod treatment was well tolerated and responders showed histological tumour regression with evidence of an immunemediated response. This study showed that imiquimod could promote a pro-immunogenic tumour microenvironment in breast cancer. Additional preclinical data generated suggest even superior results with a combination of imiquimod and ionizing radiation (Adams et al., 2012). The mechanisms of action of TLR7 agonists, approved indications, exploratory indications and the role of combination therapy, add-on molecules, and new formulations, etc. are discussed recently (Gaspari et al., 2009; Schön and Schön, 2008; Meyer et al., 2008). The predominant antitumoral mode of action of these agents appears to be activation of the central nuclear transcription factor nuclear factor NF-κB, which leads to induction of proinflammatory cytokines and other mediators. Cutaneous dendritic cells are the primary responsive cell type and initiate a strong Th1-biased antitumoral cellular immune response. Research has shown that dendritic cells themselves acquire direct antitumoral activity upon stimulation by imiquimod (Schön and Schön, 2008). It should be noted that the proinflammatory activity of imiquimod, but not resiquimod, appears to be augmented by suppression of a regulatory mechanism, which normally limits inflammatory responses. This is achieved independently of TLR7/8 through interference with adenosine receptor signalling pathways. Finally, at higher concentrations imiquimod exerts

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Bcl-2- and caspase-dependent proapoptotic activity against tumour cells (Schön and Schön, 2008). Anti-tumour activity of prolonged subcutaneous dosing of systemic 852A, a TLR-7 agonist was tested in recurrent breast, ovarian and cervix cancers (Geller et al., 2010). Patients failing multiple therapies received 0.6 mg/m2 of 852A subcutaneously twice weekly for 12 weeks. In this first human experience of a TLR-7 agonist delivered subcutaneously using a prolonged dosing schedule, 852A demonstrated sustained tolerability in some patients but not others. Clinical benefit was modest, but immune activation was observed suggesting further study of anti-tumour applications is warranted (Geller et al., 2010). TLR7/8 agonists (e.g. resiquimod) A dual TLR7/8 agonist, resiquimod, was also evaluated in clinical studies. It was applied topically for treatment of HSV and systemically for hepatitis C virus, with mixed success (Miller et al., 2008). Important differences between mice and humans with regard to tissue expression and function of TLR7 and TLR8 were noticed. In both species, TLR7 is expressed in B-cells, neutrophils, and plasmacytoid DCs (pDCs); however, in mice TLR7 is expressed by macrophages and CD8-, but not CD8+, DC subsets (Iwasaki and Medzhitov, 2004). In contrast, monocyte lineage cells and myeloid DCs in man express TLR8, whereas it was thought to be a non-functional receptor in mice ( Jurk et al., 2002). Although these endosomal TLRs are close relatives, TLR7-deficient mice are unresponsive to TLR8 agonists. Similarly, natural ssRNA cannot activate murine TLR8, leading to the belief that murine TLR8 is non-functional. However, a combination of polyT oligodeoxynucleotides (ODN) plus the TLR8 agonist activated an ILR8-NF-kappa B reporter gene, whereas polyT ODN plus the TLR7 agonist did not (Gorden et al., 2006). Also, primary mouse cells responded to the TLR8/polyT ODN by secreting TNF. Cells from TLR7(–/–) and TLR9(–/–) mice responded to the TLR8 agonist/polyT ODN combination, whereas MyD88(–/–) cells did not respond. Thus, it was concluded that the mouse TLR8 is functional (Gorden et al., 2006). The wider cellular distribution in humans of TLR7/8, compared to TLR9, makes these

compounds attractive as adjuvants. Activation of TLR7 and TLR8 in human pDCs and mDCs, respectively, increases the expression of costimulatory molecules and production of type I IFN and IL-12. A dual TLR7- TLR8 agonist may be more effective than a monospecific agonist by activating multiple DC subsets and B-cells to induce cytokines optimal for Th1 cell immunity, cross-presentation, and antibody production. Small TLR7 or 8 agonists are not very effective as adjuvants when simply mixed with antigens, but their activity can be substantially improved by formulation with (Smirnov et al., 2011) or conjugation to the antigen (Wille-Reece et al., 2005, 2006; Wu et al., 2007). Recently, a dendrimeric molecule bearing six units of TLR7/TLR8 dual-agonistic imidazoquinoline were synthesized and effect of multimerization of TLR7/8 studied (Shukla et al., 2012). A complete loss of TLR8-stimulatory activity with selective retention of the TLR7-agonistic activity was observed in the dendrimer. The dendrimer was superior to the imidazoquinoline monomer in inducing high titres of high-affinity antibodies to bovine α-lactalbumin. Additionally, epitope mapping experiments showed that the dendrimer induced immunoreactivity to more contiguous peptide epitopes along the amino acid sequence of the model antigen. Outcomes of clinical studies with the TLR7/8 agonist resiquimod on patients with genital herpes, hepatitis C or actinic keratosis (AK) were reviewed recently (Meyer and Stockfleth, 2008; Meyer et al., 2013). Although resiquimod is effective against genital HSV-2 in animal models, its development for the topical treatment of recurrent genital herpes in humans was stopped due to inconsistent results in clinical trials. Reduction of HCV viral load was achieved by oral application but was associated with unacceptable side effects. Topical treatment of AK was well tolerated and effective, giving higher clearance rates than the TLR7 agonist imiquimod. The molecular mode of action underlying the clinical efficacy depends primarily on cytokine induction in TLR7/8-expressing dendritic cells in the skin. In summary, topical resiquimod was shown to be a safe and effective treatment option for AK, and appears to have potential as a treatment modality

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for patients with extended skin areas affected with AK. Overall findings regarding the development of TLR7/8 response indicate that this pathway is one of the most active around birth and in the first year of life, and thus provide a rationale for adjuvant development for neonatal and early life vaccination (Levy et al., 2012). However, to date, no clinical data are available to determine if the theoretical benefits of selective TLR7/8 molecular agonists as a neonatal and early life adjuvant can materialize. Chronic lymphocytic leukaemia (CLL) may be especially amenable to treatment by TLR agonists, because it is an immunologically susceptible tumour with strong expression of several TLRs, particularly TLR-7 and TLR-9. TLR-7 agonists delivered locally showed strong activity against CLL, and thus, phase I/II trials utilizing systemically administered imidazoquinolines (and also TLR-9 agonists) are currently ongoing at different centres (Spaner and Masellis, 2007; Spaner et al., 2010). Success is expected in these trials because TLR agonists can sensitize tumour cells to respond to cytotoxic agents. Probably, a more successful therapy of CLL will depend on combinations involving radiotherapies, chemotherapies, monoclonal antibodies and cancer vaccines TLR-8 agonists (e.g. 3M-002) The contribution of TLR8 to innate immunity remained poorly understood. As mentioned earlier, despite phylogenetic and locational similarities between TLR7-8-9, there are important differences between mice and humans with regard to tissue expression and function of these endosomal TLRs (Gorden et al., 2005, 2006; Gorski et al., 2006; Iwasaki and Medzhitov, 2004). The true TLR8 agonist in the mouse has not yet been identified. Most of 3M’s imidazoquinoline compounds stimulate both TLR7 and TLR8. However, there exist TLR selective compounds a well (Gorden et al., 2005; Dzopalic et al., 2010; Hackstein et al., 2011; Lan et al., 2009). In a recent study, the role of TLR8 signalling in immunity in mice was studied (Demaria, 2010). It was found that TLR8–/– DCs overexpressed TLR7, and were hyper-responsive to various TLR7 agonists. Tlr8–/– mice showed splenomegaly, defective development of marginal

zone and B1 B-cells, and increased serum levels of IgM and IgG2a: they also exhibited increased serum levels of autoantibodies against small nuclear ribonucleoproteins, ribonucleoprotein and dsDNA. and developed glomerulonephritis, whereas neither TLR7–/– nor TLR8–/–/TLR7–/– mice showed any of the phenotypes observed in TLR8–/–) mice. These data provided evidence for a pivotal role for mouse TLR8 in the regulation of mouse TLR7 expression and prevention of spontaneous autoimmunity (Alexopoulou, 2012). For rational vaccine design it is imperative to know which DC should be preferentially activated. A recent study directly addressed this question by employing selective TLR7 ligands and resiquimod-co-culture experiments with inhibitory oligonucleotides (iODN) suppressing TLR7, TLR7 + 8 or TLR7 + 8 + 9 (Hackstein et al., 2011). Selective TLR7 ligands did not affect conventional monocyte derived DC (moDC) differentiation as analysed by CD14/CD1a expression. iODN experiments confirmed that resiquimod’s effects during DC differentiation were antagonized only with TLR8 iODNs. Direct comparison of resiquimod DC with TLR7- and control-DC revealed significantly higher T-cell costimulatory molecule and MHC class II expression. Resiquimod DC promoted significantly stronger allogeneic T-cell proliferation and stronger naive CD4+ T-cell proliferation. These results indicate the relevance of TLR8 for human monocyte-derived DC differentiation and maturation and may be relevant for clinical trials as well as vaccine adjuvants employing resiquimod. Newborns have frequent infections and manifest impaired vaccine responses, necessitating a search for neonatal vaccine adjuvants. TLR agonists are candidate adjuvants, but human neonatal cord blood monocytes demonstrate impaired Th1 polarizing responses to many TLR agonists caused by plasma adenosine. A recent study characterized activation of neonatal monocytes and moDCs induced by the TLR8 agonist imidazoquinoline 3M-002 (Philbin et al., 2012). Imidazoquinolines were more potent and effective than alum at inducing TNF and IL-1β from monocytes. 3M-002 induced robust TLR pathway transcriptome activation and Th1-polarizing

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cytokine production in neonatal and adult monocytes and MoDCs, signalling through TLR8 in an adenosine/cyclic AMP-refractory manner. Newborn MoDCs displayed impaired LPS/ATPinduced caspase-1-mediated IL-1β production but robust 3M-002-induced caspase-1-mediated inflammasome activation independent of exogenous ATP. TLR8 agonists induced robust TNF and IL-1β in whole blood of rhesus macaques at birth and infancy. It was concluded that the TLR8 agonists induce robust monocyte and MoDC activation and therefore represent promising neonatal vaccine adjuvants. In summary, while designing vaccines it is critical to remember the differences in cellular distribution between TLR7 and 8 agonists: In man, TLR7 agonists mainly activate plasmacytoid DC, while TLR8 agonists activate mainly macrophages, monocytic and myeloid-DCs. The TLR7/8 dual analogue (resiquimod, R-848) activates both cell types. In mice the situation is different. It appears that TLR8 is functional and its role may be regulation of TLR7. However, its natural ligand and its role are not well understood. TLR9 agonists (e.g. CpG-ODN) TLR9 is an endosomal PRR that specifically recognizes bacterial and viral DNA. (Blasius and Beutler, 2010). Unmethylated CpG dinucleotides are prevalent in bacterial and viral DNA, but not in vertebrate genomes. Oligodeoxynucleotides (ODN), which may contain 18–25 bases, have been optimized for CpG motifs (CpG-ODN) and used as adjuvants, in various formulations, such as nanoparticles (Marshall et al., 2004) or virus-like particles ( Jennings and Bachmann, 2009). Just like TLR7 agonists as discussed above, CpG-ODNs enhance antibody responses and induce Th1 cell responses (Kobayashi et al., 1999). However, the target cell distribution is restricted to pDCs and B-cells in man (Campbell et al., 2009; Hornung et al., 2002; Iwasaki and Medzhitov, 2004). Thus, it is a better adjuvant for mice. In studies of newborn mice, CpG-ODN can circumvent Th2 polarization of neonatal responses to vaccines, but does not fully redirect Th2 responses after neonatal priming (Kovarik et al., 1999). In humans, the immunogenicity of pneumococcal conjugate vaccine was improved by CpG-based adjuvants in

HIV-infected adults (Sogaard et al., 2010). Thus, under certain conditions TLR9-based adjuvants may hold some promise. A number of immunostimulatory DNA sequences (ISS), designed to stimulate TLR9 have been developed as vaccine adjuvants. These exert strong antibody and Th1-biased T-cell responses. However, as mentioned earlier, the value of these preclinical studies using ISS is questionable because the cellular distribution of TLR9 is much more limited in the human compared to the mouse. Despite this limitation, Heplisav, a vaccine combining a CpG-rich TLR9 agonist (1018 ISS) with rHBsAg, has been tested in phase III clinical trials (Barry and Cooper, 2007). It is expected that this formulation may increase immunogenicity and reduce the number of required doses (Cooper and Mackie 2011). CpG-rich TLR9 agonists have been tested in multiple phase II and phase III human clinical trials, as adjuvants to cancer vaccines and in combination with conventional chemotherapy and other therapies (Krieg, 2007; Vollmer and Krieg 2009; Holtick et al., 2011). In a recent study, a therapeutic vaccination strategy that combines local radiation with the injection into the tumour of a TLR9 agonist has been tested in the clinic (www.clinicaltrials.gov as NCT00226993) (Kim et al., 2012). Fifteen patients with mycosis fungoides, which is a common form of cutaneous T-cell lymphoma, have been treated. Clinical responses were assessed at the distant, untreated sites as a measure. Five clinically meaningful responses were observed. The procedure was well tolerated. The immunized sites showed a significant reduction of CD25+, Foxp3+ T-cells and a similar reduction in S100+, CD1a+ dendritic cells. There was a trend towards greater reduction of CD25+ T-cells and skin dendritic cells in clinical responders versus non-responders. This in situ vaccination strategy warrants further study with modifications to augment these therapeutic effects (Kim et al., 2012). Research and development on nucleic acid vaccines started about two decades ago. However, there is still no commercial product for human use. A recent study developed a self-amplifying RNA vaccine using systemic delivery of short interfering RNA (siRNA) using lipid nanoparticles

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(Geall et al., 2012). They showed that non-viral delivery of a 9 kb self-amplifying RNA encapsulated within nanoparticles substantially increases immunogenicity. These results suggest that nonviral technologies for delivering self-amplifying RNA vaccines may hold promise. Enthusiasm about triggering of innate immunity against intracellular pathogens by the nucleic acid sensing machinery still remains high. A recent review explains how these mechanisms could be harnessed for the design of new vaccines (Desmet and Ishii, 2012). However, it should be stressed that synthetic TLR9 agonist CpG-ODN, despite great promise as immunotherapeutic agents and vaccine adjuvants in laboratory animal models of infectious disease, allergy and cancer have not yet fulfilled these expectations. It is highly possible that the disparity in immune responses between rodents and mammals is mainly due to differences in cellular expression of TLR9 in the various species (Mutwiri, 2012). On the other hand, as we shall see later, this is not the only lesson we have learned from decades of TLR experience. The myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs), express Toll-like receptors TLR7 and TLR9 and produce large amounts of type I interferons (IFNs) in response to pathogenic agents. pDCs play a central role not only in viral infections, but also in allergy and autoimmune diseases. Thus, using TLR agonists in individuals with these conditions (e.g. lupus or psoriasis) might exacerbate their disease (Gilliet et al., 2004; Sun et al., 2007; Morizane et al., 2012). TLR-based adjuvants in highpriority vaccines HIV, tuberculosis, and malaria are among the most devastating infectious diseases of our time, but despite heroic efforts, there are no widely effective protective vaccines against these diseases. Also, currently there is extensive work on cancer vaccines. Therefore, these four topics warrant a separate section in which we will briefly mention ongoing vaccine work that involves TLR agonists. TLR agonists in HIV Resiquimod is a modest adjuvant for HIV-1 gagbased genetic immunization in a mouse model

(Otero et al., 2004). Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specific Th1 and CD8+ T-cell responses and improves the magnitude and quality of responses in non-human primates (Wille-Reece et al., 2005; Wille-Reece et al., 2006). Also, adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus (Kwissa et al., 2007). It has been found that endocytosis of HIV-1 activates plasmacytoid dendritic cells via TLR7viral RNA interactions (Beignon et al., 2005). Recently, another TLR7 agonist gardiquimod, showed anti-HIV effects in activated PBMC and macrophages in vitro (Buitendijk et al., 2013). The HIV-1 matrix protein p17 can be efficiently delivered by intranasal route in mice, using the TLR 2/6 agonist MALP-2 as mucosal adjuvant (Becker et al., 2006). Poly-IC induces durable and protective CD4+ T-cell immunity together with a DC- targeted vaccine in mice (Trumpfheller et al., 2008). The T-cells proliferate and continue to secrete IFN-gamma in response to HIV gag p24. The adjuvant role of poly IC requires Toll-like receptor TLR3 and MDA5 receptors. The study suggests that poly IC be tested as an adjuvant with DC-targeted vaccines to induce numerous multifunctional CD4+ Th1 cells. CpG-ODNs on the other hand, enhance proliferative and effector responses of B-cells in HIV-infected individuals (Malaspina et al., 2008). Despite abnormalities in naive and memory B-cells of HIV-infected individuals, irrespective of disease status, they can respond to TLR9, thus the incorporation of such agents in vaccine formulations may enhance their Ab responses to vaccination. HIV-1 lentiviral vector immunogenicity is mediated by TLR3 and TLR7 (Breckpot et al., 2010). Utilizing ligands for three TLRs (TLR2/6, TLR3, and TLR9) together greatly increased the protective efficacy of vaccination with an HIV envelope peptide in mice, when compared with using ligands for only any two of these TLRs (Zhu et al., 2010). Surprisingly, the combination of these 3 TLR ligands augmented the quality but not the number of the responding T-cell. The triple combination increased production of DC IL-15 along with its receptor, IL-15Ralpha, which

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contributed to high avidity, and decreased induction of Tregs. Therefore, selective TLR ligand combinations can increase protective efficacy by increasing the quality rather than the quantity of T-cell responses (Zhu et al., 2010). Another combination study shows that TLR agonists and/or IL-15- adjuvanted mucosal SIV vaccine reduces gut CD4+ memory T-cell loss in SIVmac251challenged rhesus macaques (Sui et al., 2011). Recent vaccine trials utilizing adenovirusbased vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. In a recent study a novel vaccine strategy utilizing an Adeno-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine (Appledorn et al., 2010). The data showed that the quantity and quality of HIV-Gag specific CD8+ and CD8– T-cell responses were significantly improved when coupled with (rEA) expression. The data presented in this study illustrate the potential utility of Adbased vectors expressing TLR agonists to improve immune responses in vivo. Sublingual administration of an adenovirus serotype 5 (Ad5)-based vaccine confirms Toll-like receptor agonist activity in the oral cavity and elicits improved mucosal and systemic cell-mediated responses against HIV antigens despite pre-existing Ad5 immunity (Appledorn et al., 2011). In a mouse study, it was shown that intranasal immunization with HIV-gp140-adsorbed nanoparticles greatly enhances serum and vaginal IgG and IgA responses (Arias et al., 2011). HIV-gp140-carrying wax nanoparticles induce no inflammation and thus may be of potential use as effective mucosal adjuvants for HIV vaccine candidates. Successful vaccine development against HIV will likely require the induction of strong, long lasting humoral and cellular immune responses in both the systemic and mucosal compartments. Based on the known immunological linkage between the upper-respiratory and urogenital tracts, investigators explored the potential of nasal adjuvants to boost immunization for the induction of vaginal and systemic immune responses to gp140 (Arias et al., 2012). Mice were

immunized intranasally with HIV gp140 together with micellar and emulsion formulations of a synthetic TLR4 agonist, Glucopyranosyl Lipid Adjuvant (GLA), and responses were compared to R848, a TLR7/8 agonist, or chitosan, a nonTLR adjuvant. GLA and chitosan but not R848 greatly enhanced serum immunoglobulin levels when compared to antigen alone. Both GLA and chitosan induced high IgG and IgA titres in nasal and vaginal lavage and faeces. Whilst both GLA and chitosan induced T-cell responses to immunization, GLA induced a stronger Th17 response and chitosan induced a more Th2skewed response. These results show that GLA is a highly potent intranasal adjuvant (Arias et al., 2012). In a recent study, several TLR agonists (TLR3, TLR4, TLR9) as potential mucosal adjuvants for HIV gp140 and tetanus toxoid have been evaluated in a mouse model (Buffa et al., 2012). Some TLRs displayed differential activity dependent upon the route of administration. As mentioned earlier, the data demonstrate the importance of route, antigen and adjuvant effects that need to be considered in the design of mucosal vaccine strategies. Based on the partial efficacy of the HIV/ AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Thus, a recent study describes, using systems analysis, the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C) (Gómez et al., 2012). Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T-cell and B-cell responses. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T-cell memory responses that are polyfunctional and with effector memory phenotype. This systems analysis of immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the

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prevalent subtype virus in the most affected areas of the world (Gómez et al., 2012). In summary, in HIV, it has been shown that polymorphism of the TLR4, 7 and 8 and 9 plays a role in disease progression and viral load (Stevceva, 2011). TLR3 has shown good results if used with vaccine proteins selectively delivered to DCs by antibodies to DEC-205/CD205. TLR7/8 and TLR9 agonists enhanced immune responses if conjugated to the vaccine protein. A triple combination of TLR2/6, -3, and -9 agonists and IL-15 synergistically up-regulated immune responses to vaccine formulated as recombinant MVA viruses expressing SIVmac239 Gag, Pol, Env and Rev, Tat, Nef (Stevceva, 2011). These and other studies are just beginning to unravel the potential of TLR agonists and much more and broader research is needed in order to revitalize the field of HIV vaccines. Synthetic peptide vaccines are attractive, because they allow rational improvement of vaccine design and detailed pharmacokinetic and pharmacodynamic studies not possible with conventional vaccines. Although no major success has been achieved in preventative vaccine development against HIV, the possibilities are by no means exhausted (Quakkelaar and Melief, 2012). TLR agonists in malaria Recently, it was shown that the TLR9 agonist CpG-ODN promotes the acquisition of Plasmodium falciparum-specific memory B-cells in malaria-naive individuals. Despite the central role of memory B-cells (mBC) in protective immune responses, little is understood about how they are acquired in naive individuals in response to Ag exposure, and how this process is influenced by concurrent activation of the innate immune system’s TLR. In a longitudinal study of malaria-naive individuals, investigators examined the mBC response to twocandidate malaria vaccines administered with or without CpG-ODN (Crompton et al., 2009). They show that the acquisition of mBC is a dynamic process in which the vaccine-specific mBC pool rapidly expands and then contracts, and that CpG-ODN enhances the kinetics, magnitude, and longevity of this response. The best malaria vaccine results yet are with CS protein-based RTS,

S and AS02 adjuvant, which contains MPL (and QS-21) (Abdulla et al., 2013). Malaria subunit vaccines require potent adjuvants, as they lack known pathogen-associated molecular patterns that act as TLR agonists to stimulate dendritic cells and initiate strong adaptive immune responses. In a study, the topical application of TLR7 agonist imiquimod at the site of subcutaneously injected P. falciparum circumsporozoite (CS) peptides elicited strong parasite-specific humoral immunity that protected against challenge with transgenic rodent parasites that express P. falciparum CS repeats (Othoro et al., 2009). In addition, injection of a simple linear peptide followed by topical imiquimod elicited strong Th1 CD4+ T-cell responses, as well as high antibody titres. The correlation of high anti-repeat antibody titres with resistance to sporozoite challenge in vivo and in vitro supports use of this topical TLR7 agonist adjuvant to elicit protective humoral immunity. Later, it was found that immunization of mice with a crude blood stage extract of the malaria parasite P. falciparum elicits parasite antigen-specific immune responses via TLR 9 and that the malarial haem-detoxification by-product, haemozoin (HZ), but not malarial DNA, produces a potent adjuvant effect (Coban et al., 2010). Malarial and synthetic (s) HZ bound TLR9 directly to induce conformational changes in the receptor. Although natural HZ acts as a TLR9 ligand, the adjuvant effects of synthetic HZ are independent of TLR9 or the NLRP3-inflammasome but are dependent on MyD88. Thus, HZ can influence adaptive immune responses to malaria infection and may have therapeutic value in vaccine adjuvant development. Using the Plasmodium vivax antigen PvRII, Wiley et al. (2011) showed that the use of a TLR agonist in the vaccine formulation increased the diversity of the variable region sequences in comparison to the use of an oil-in-water emulsion adjuvant alone. Moreover, increased variable domain diversity in response to the use of TLR agonist-based adjuvants correlated with improved antigen neutralization. The use of TLR agonists also broadened the range of polymorphic variants against which these antibodies could be effective. In addition, a peptide microarray demonstrated

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that inclusion of adjuvants changed the profile of linear epitopes from PvRII that were recognized by serum from immunized animals. Thus, during vaccine design, tailored adjuvants can be used to obtain a broad spectrum of antibodies to neutralize drifted and polymorphic pathogenic strains (Wiley et al., 2011). TLR agonists against tuberculosis Vaccine efficacy largely depends upon DC targeting and activation. Using a promising candidate vaccine against tuberculosis antigen, Ag85BESAT-6, formulated in the TLR9 agonist IC31 adjuvant, DC targeting and activation was studied in mice (Kamath et al., 2008). Thus, potent protective IFN-γ-producing responses may be elicited by the very selective activation of a minute number of in vivo targeted DC. Both in mice and in men, the importance of Th1 cells producing IFN-γ in controlling Mycobacterium tuberculosis is well known. Also, IL17-producing T-cells (Th17) have been observed during mycobacterial infections. Recently, it was demonstrated that TLR2 has a major impact on the regulation of p19 (IL-23) expression in response to M. tuberculosis and therefore on the establishment of Th17 cell responses to M. tuberculosis infection (Teixeira-Coelho et al., 2011). TLR2-deficient animals showed reduced chemokine production and diminished Th17 responses in the lung. One of the newest adjuvants in approved products is AS04, which combines monophosphoryl lipid A, a TLR-4 agonist, with alum. Baldwin et al. (2012) compared two adjuvants: a stable oilin-water emulsion (SE) and a stable oil-in-water emulsion incorporating glucopyranosyl lipid adjuvant, a synthetic TLR-4 agonist (GLA-SE), each together with a recombinant protein, ID93. Both the emulsion SE and GLA-SE adjuvants elicited potent cellular responses in combination with ID93 in mice. ID93/SE induced Th2-biased immune responses, whereas ID93/GLA-SE induced multifunctional Th1 cell responses (IFNγ, TNF-α, and IL-2). The ID93/GLA-SE vaccine candidate generated significant protection in mice and guinea pigs, whereas no protection was observed with ID93/SE. These results demonstrate the importance of adjuvant and the

formulation in the development of a tuberculosis vaccine. TLR agonists against cancer The immune surveillance theory, which found widespread acceptance (Burnet, 1970), stimulated numerous studies on immune-based cancer therapies. During many years, it stirred much discussion because in practice, the results were disappointing with sporadic successes. Today we have a few examples of success: the approvals of cancer vaccine sipuleucel‑T for advanced prostate cancer (Madan and Gulley, 2011) and ipilimumab (antiCTLA-4; CD152 mAb) for metastatic melanoma (Shapira-Frommer and Schachter, 2012). After the discovery of TLRs hopes were again raised again in favour of immune surveillance theory. However, compared to other adjuvants, TLR agonists suffered the most. In a recent example, TLR9 agonist IMO‑2055, CPG 7909 (ProMune) KGaA (Murad et al., 2007), failed to demonstrate efficacy when used in combination with chemotherapy in Phase III trials. Despite several disappointments it is possible that these agents still offer promise if they are better combined with anticancer drugs or if they are used as therapeutic cancer vaccine adjuvants (Guha, 2012). Also, one should consider the potential effect of TLR agonists on the tumour cells themselves as well as on the entire organism. Recent data demonstrate that TLR activation in tumour cells could trigger both pro- or anti-tumoral effect depending on the context (Goutagny et al., 2012). In any case, therapeutic cancer vaccination still faces significant challenges. This may be because the activation of T-cells and B-cells is compromised in most cancer patients due to immune suppression, loss of tumour antigen expression, and dysfunction of antigen-presenting cells (APC). Recent studies focus on novel adjuvants that enhance APCs, in particular dendritic cells. TLR agonists are being used clinically either alone or in combination with tumour antigens and showing initial success in eliciting anti-tumour activity (Bhardwaj and Gnjatic, 2012). From early on there were concerns that the neoplastic process may subvert TLR signalling pathways to advance cancer progression (Killeen et al., 2006). Thus, the TLR system is thought

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to be a double-edged sword, which is needed to be carefully handled in the setting of neoplastic disease. These concerns may still be valid and we still know very little about the TLR expression by tumour cells (Goutagny et al., 2012). Despite these apprehensions, one of the first studies examined the effects of the systemic administration of the TLR7 agonist 852A, in patients with advanced cancer (Dudek et al., 2007). Eligible adult patients with refractory solid organ tumours received i.v. 852A thrice weekly for 2 weeks. Significant correlations were found between pharmacodynamic biomarkers and pharmacokinetic variables, and an objective clinical response was seen. 852A was safely administered i.v. at doses up to 1.2 mg/ m2 thrice weekly for 2 weeks with transient or reversible adverse effects. They concluded that a TLR7 agonist holds promise for stimulating innate immune responses. Evidence was provided for a multifaceted anti-tumour function of the imiquimod, which rapidly recruits plasmacytoid dendritic cells and possibly other immune cells into tumours by inducing the secretion of CCL2 by dermal cells (Holcmann et al., 2012). In fact, imiquimod induces pDC maturation and their conversion into cytolytic killer cells, which are capable of eliminating tumours independently of the adaptive immune system. In summary, today, only three TLR agonists are approved by FDA for use in humans: TLR2 and TLR4 agonist bacillus Calmette–Guérin (BCG) (Tsuji et al., 2000), TLR4 agonist monophosphoryl lipid A (MPL) and TLR7 agonist imiquimod. BCG (an attenuated strain of Mycobacterium bovis) is mainly used as a vaccine against tuberculosis, but also for the immunotherapy of in situ bladder carcinoma. MPL is included in the formulation of Cervarix®, a vaccine against human papillomavirus-16 and -18. Imiquimod (a synthetic imidazoquinoline) is routinely employed for actinic keratosis, superficial basal cell carcinoma, and external genital warts. In a recent Trial Watch the results of recently completed clinical trials were reviewed and progress on ongoing FDA-approved TLR agonists as off-label medications for cancer therapy was evaluated (Vacchelli et al., 2012). Clinical trials with TLR agonists either alone or in combination with other therapeutic agents are summarized in

Table 8.1. (http://clinicaltrials.gov/ct2/results?t erm=tlr+agonists&Search=Search) Lessons learned from studies of TLR agonists as adjuvants Simultaneous delivery of antigens and TLR agonists enhances effectiveness During evolution, the immune system codeveloped with microorganisms and hence has been selected to respond rapidly and forcefully to certain microbial antigens that carry PRR ligands. For this reason, APC respond optimally to ‘naturally adjuvanted’ viral and microbial infections and live attenuated vaccines. Thus, to imitate this natural situation, attempts have been made to co-deliver purified antigens covalently coupled to TLR9 agonists (Tighe et al., 2000) or TLR7-TLR8 agonists to purified proteins (WilleReece et al., 2005, 2006; Wu et al., 2007). In the case of TLR agonists, a construction was made using recombinant fusion proteins consisting of antigen and the TLR5 agonist flagellin (Huleatt et al., 2007). In these examples, the potency of the linked vaccine is 10–100 times that of a comparable mix of separate components. In the case of CpG–ODN conjugates, it was found that TLR 9 expression was not required for CpG DNA-aided cross-presentation of DNA-conjugated antigens but was essential for cross-priming of CD8 T-cells (Heit et al., 2003). The physical association of antigen and agonist may be important mainly because DCs have been shown to preferentially process and present antigen from compartments that also contain TLR agonists (Blander and Medzhitov, 2006). The qualitative nature of the adaptive immune response may depend upon which TLR is stimulated (Querec et al., 2006). Moreover, combinations of TLR agonists can have synergistic effects on cytokine production as shown in vitro (Ghosh et al., 2006, 2007) and when used as adjuvants in vivo (Kasturi et al., 2011; Zhu et al., 2008). In fact, antigen-TLR agonist conjugates induce greater and more immune durable responses with lower doses of the antigen. A recent review summarized studies on individual TLR ligand–antigen

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Table 8.1  List of adjuvants targeting TLRs currently being studied clinically TLR target

Candidate adjuvant

Disease

Statutes of clinical trial

TLR2 and TLR5

Entolimod

Locally advanced or metastatic solid tumours that cannot be removed by surgery

Recruiting

TLR2 and TLR5

Entolimod

Stage III–IV squamous cell head and neck cancer

Not yet recruiting

TLR3

Poly I:Poly C12U

Influenza

Recruiting

TLR3

Poly-ICLC

Melanoma

Active, not recruited

TLR3/TLR7/8 Poly ICLC /resiquimod

Dendritic cell vaccine for brain tumours

Recruiting

TLR4

Lipopolysaccharide

Endotoxaemia; sepsis;

Completed

TLR4

R848 gel

Melanoma

Recruiting

TLR4

Glucopyranosylphospholipid A

Hookworm infection

Recruiting

TLR5

CPG 7909

Malaria

Completed

TLR7

Imiquimod

Metastatic breast cancer

Recruiting

TLR7

Imiquimod

Breast cancer

Has results

TLR7

Imiquimod

Melanoma (skin); metastatic cancer

Unknown

TLR7

GSK2245035

Asthma and rhinitis

Recruiting

TLR7

GS-9620

Chronic hepatitis B

Active, not recruiting

TLR7

Imiquimod

Human papillomavirus infection

Unknown

TLR7/8

Resiquimod

Influenza vaccination in seniors

Completed

TLR7/8

Resiquimod

Tumours

Active, not recruiting

TLR8

VTX-2337

Fallopian tube cancer; ovarian cancer;

Active not recruiting

TLR8

VTX-2337

Low grade B-cell lymphoma

Terminated

TLR8

VTX-2337

Carcinoma, squamous cell of head and neck Recruiting

TLR8

VTX2337

Epithelial ovarian cancer; fallopian tube cancer; primary peritoneal cancer

Recruiting

TLR9

EMD 1201081

Squamous cell carcinoma of the head and neck

Terminated

TLR9

URLC10-177, TTK-567, CpG-7909

Oesophageal cancer

Unknown

TLR9

CYT003

Moderate to severe allergic asthma

Recruiting

TLR9

CPG 7909

Pneumococcal vaccines in HIV infected

Completed

TLR9

GSK2445053

Rhinitis, allergic, seasonal

Completed

TLR9

SD-101

Chronic hepatitis C

Completed

TLR9

CPG 7909

Lymphoma, non-Hodgkin

Completed

TLR9

EMD 1201081

Squamous cell carcinoma of the head and neck cancer

Active, not recruiting

TLR9

IMO-2125

Hepatitis C infection

Completed

TLR9

IMO-2055

Colorectal cancer

Terminated

This table is constructed from NIH Clinical website: http://clinicaltrials.gov/ct2/results?term=tlr+agonists&Searc h=Search. Almost all the treatments include combination of drugs, however, here for clarity; only the TLR targets are given.

conjugates using Pam3/2Cys, lipid A analogues, flagellin, imidazoquinoline analogues and unmethylated CpG motifs to activate immune

systems through TLR2, TLR4, TLR5, TLR7/8 and TLR9, respectively (Fujita and Taguchi, 2012). Thus, several studies have demonstrated

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that TLR ligands covalently coupled to the antigens have several benefits over non-conjugated antigens. Also, physical properties of TLR agonists were found to be critical for adjuvanticity. For example, it was noticed that TLR7 and TLR8 agonists sometimes could not induce the desired immune potentiation in some models (Kwissa et al., 2007). Highly soluble, small molecular weight TLR7 and TLR8 agonists may reach the draining lymph node faster and ‘alarm’ the system before arrival of the antigen. This idea is supported by the observation that ‘slowing down’ a TLR7/8 agonist with chemical modification induces a better local cytokine response (Alkan et al., 2006; Smirnov et al., 2011). Glycoconjugate vaccines have provided great health benefits globally, but they have been less successful in some populations. To identify new approaches to enhancing glycoconjugate effectiveness, Avci et al. (2011) investigated molecular and cellular mechanisms governing the immune response to glycoconjugate vaccines. They found that when group B streptococcal type III polysaccharide is coupled to a carrier protein it can induce carbohydrate-specific CD4+ T-cell responses (Avci et al., 2011). This novel glycoconjugate vaccine enhanced the presentation of carbohydrate-specific T-cell epitopes 50–100 times more potently and was substantially more protective in a neonatal mouse model. It is tempting to speculate that incorporation of some TLR agonists into this conjugate could enhance the vaccine efficacy. TLR agonists can work through both direct and indirect ways Several studies have shown that TLR activation in the same DC that is presenting the antigen is critical for CD4+ T-cell activation and Th1 cell differentiation (Blander and Medzhitov, 2006; Joffre et al., 2009; Sporri and Reis e Sousa, 2005; Coffman et al., 2010). However, more recent evidence also shows that production of type I IFNs from DCs or non-haematopoietic stromal cells not presenting the antigen can profoundly influence Th1 and CD8+ T-cell immunity (Hou et al., 2008; Longhi et al., 2009; Wang et al., 2010). If we think of the totality of cells in a given environment the picture becomes even more

complex. Cells of haematopoietic and nonhaematopoietic origin, despite overlaps, each express a unique combination of TLRs (Duthie et al., 2011). As one example, purified NK cells do not express TLR7 and thus should not be able to respond to a TLR7 agonist. Indeed, highly purified NK cells could not respond to TLR7 agonists (Gorski et al., 2006). However, in the presence of other cells (in a PBMC mixture), they respond to a TLR7 agonist as assessed by IFN-gamma release and tumour killing. It was demonstrated that NK cell activation by TLR7 agonists occurs indirectly, via activation of TLR7-positive neighbour cells, and this cross-activation is dependent on IL-12 and IL-18 release (Gorski et al., 2006). Multiple TLR activation can be advantageous Presumably, immune systems increase the likelihood of dealing successfully with an infection if they would utilize redundant innate pathways instead of a single PRR. Indeed, studies of live attenuated vaccines showed that activation of multiple innate receptors may be more effective than activation of a single pathway (Querec et al., 2006). The live attenuated yellow fever vaccine 17D (YF-17D) is one of the most effective vaccines available, with a 65-year history of use in more than 400 million people globally. Investigations of the mechanisms of this ‘super’ immunogenicity for the 17D strain have revealed that YF-17D activates multiple TLRs on dendritic cells and elicits a broad spectrum of innate and adaptive immune responses. Specifically, YF-17D activates multiple DC subsets via TLRs (TLR2, 7, 8, and 9) to elicit the proinflammatory cytokines, such as IL-12p40, IL-6 and interferon-alpha. Interestingly, the resulting adaptive immune responses are characterized by a mixed Th1/Th2 cytokine profile. These data highlight the potential of vaccination strategies that use combinations of different TLR agonists to stimulate polyvalent immune responses. Also, in vitro studies with combinations of TLR agonists support this idea and suggest combinations that may be especially useful for adjuvants (Ghosh et al., 2006, 2007; Trinchieri and Sher, 2007). Targeting of protein antigens to DC via the DEC205 receptor enhances presentation of antigen-derived peptides on MHC-I and MHC-II

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molecules. In the absence of adjuvants, targeting of DNA-encoded ovalbumin to DCs suppressed CD8+ T-cell responses after an adenoviral booster immunization (Grossmann et al., 2009). CD8+ T-cell responses to the DEC205-targeted DNA vaccines were increased only slightly by adding either the CpG-ODN, the poly-IC, or CD40 ligand expression plasmids. However, the combination of both TLR agonists led to a strong enhancement of CD8+ T-cell responses compared to a non-targeted DNA vaccine. This finding was confirmed using HIV Gag as antigen. Thus, the CD8+ T-cell responses can be further improved by targeting the DNA- encoded antigen to DEC205 in the presence of synergistic TLR agonists CpGODN and Poly I:C. In summary, effective adjuvant systems developed by companies already utilize combinatorial approaches, for example, by combining MPL and alum (AS04) or MPL, QS-21, and either oil-inwater emulsion (AS02) or liposomes (AS01). Many more combinations and formulations are in late preclinical or early clinical stages of development. Clinical trials with single or multiple TLR agonists either alone or in combination with other therapeutic agents are summarized in Table 8.1. TLR cross talks may effect the adjuvanticity Stimulation with multiple TLR agonists may result in synergistic, complementary or inhibitory effects on innate immune responses (Ghosh et al., 2006, 2007; Klinman et al., 2003). When human PBM were stimulated with tandem TLR2–9 agonists, most combinations of TLR agonists were found to be either neutral, additive, or synergistic while a few were antagonistic with respect to cytokine production. The TLR7/8 agonists in combination with TLR2 or TLR4 agonists always gave synergistic responses. Also, if T-cell receptors (TCR) are activated in the presence of a TLR7/8 agonist, the cytokine response is synergistically enhanced (Ghosh et al., 2006). However, combinations of TLR7 agonists with poly-T ODNs (thymidine homopolymer oligodeoxynucleotides), but not with poly-A or G were suppressive. Surprisingly, poly-T ODN enhanced the cytokine production when combined with a TLR8 agonist. (Ghosh et al., 2006, 2007; Jurk et al., 2006). The

functional effects of one TLR on another, among TLR7, TLR8, and TLR9, have been studied using HEK293 cells transfected with TLRs in pairwise combinations (Wang et al., 2006). The results indicated that TLR8 inhibits TLR7 and TLR9, and TLR9 inhibits TLR7 but not vice versa. In another study, Booth et al. (2010) observed that co-stimulation of PBMC with CpG ODN + ORN (RNA oligoribonucleotides) or CpG ODN + imiquimod (another TLR7/8 agonist) resulted in significant reduction in CpG ODN-induced IFNalpha production, B-cell proliferation and IgM responses. In addition, ODNs can be used as microbicides that inhibit human immunodeficiency virus type 1 (HIV-1) infection and block TLR7 and TLR9 triggering by HIV-1 (Fraietta et al., 2010). Finally, due to the above-mentioned unexpected plasticity in the ligand specificities of TLR7 and TLR8, the TLR8 agonist + poly-T ODN combination was tested in the mouse in which TLR8 is believed to be inactive, or non-functional. This combination activated the TLR8 (Gorden et al., 2006). Apart from TLR–TLR cross-talk mentioned above, there exists cross-talk between TLRs and other elements of innate immune system such as the complements system (Hajishengallis and Lambris, 2010; Song, 2012). The complementTLR interplay reinforces innate immunity or regulates excessive inflammation, through synergistic or antagonistic interactions, respectively. As the innate immune system relies on a variety of PRRs to sense microbial structures that are present in pathogens, various levels of cross-talk between the TLR and other sensors such as NLR pathways would be expected (Becker and O’Neill, 2007). Indeed, cross talk between NOD1/NOD2 and TLRs has been established (Petterson et al., 2011). NOD1 and NOD2 have the ability to augment the B-cell receptor-induced activation independently of physical T-cell help. Hence, NLRs represent a new pathway for B-cell activation and a potentially important host defence system against bacterial infections. TLR expression by non-immune cells may be critical for some vaccines The routes of vaccination in humans involve various epithelial cells, thus a brief survey of the role of

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TLRs in epithelial cells located at different sites is appropriate. Epithelial cells express several TLRs and respond to microbial products (Coffman et al., 2010; Duthie et al., 2011; Rakoff-Nahoum et al., 2004). Gut epithelium: The intestine is continuously exposed to dietary and microbial antigens. The host has to maintain intestinal homeostasis to keep the commensal and pathogenic bacteria under control (Abt et al., 2012). Commensal bacteria are recognized by epithelial TLRs under normal steady-state conditions, and this interaction plays a crucial role in the maintenance of intestinal epithelial homeostasis. Furthermore, activation of TLRs by commensal microflora is critical for the protection against gut injury. These findings reveal a novel function of TLRs-control of intestinal epithelial homeostasis and protection from injury- and provide a new perspective on the evolution of host–microbial interactions. As described in (Abreu, 2010; Santaolalla and Abreu, 2012), dysregulation of this microorganisminduced program of epithelial cell homeostasis and repair in the intestine can result in chronic inflammatory diseases. The TLR adaptor TIR domain is necessary to activate innate immunity and MyD88 is required to keep the intestinal microbiota under control (Santaolalla and Abreu, 2012). Toll-like receptors (TLRs) are involved in B-cell homing to the intestine and recruitment of mast cells. Lung epithelium: Allergic airway inflammation develops in the context of innate immune cells that express TLRs. Therefore, understanding the regulatory role of TLRs in the pathogenesis of allergic airway inflammation may shed light on improving inflammation control in asthmatic patients (Iwamura and Nakayama 2008). Skin epithelium: TLRs have emerged as a major class of PRRs that are involved in detecting invading pathogens in the skin and initiating cutaneous immune responses. TLRs are expressed on many different cell types in the skin, including keratinocytes and Langerhans cells in the epidermis (Miller, 2008; Duthie et al., 2011). Certain TLRs have been implicated in the pathogenesis of skin diseases, such as atopic dermatitis, psoriasis, and acne vulgaris. Since the discovery that topical TLR agonists (TLR7 agonist imiquimod, the

TLR7/8 agonist resiquimod) it has been shown that they promote antiviral and anti-tumour immune responses (Miller et al., 2008; Schön and Schön, 2008). On the other hand, overstimulation of TLRs and induction of excess IFNs may cause or exacerbate skin lesions in psoriasis (Farkas et al., 2012). Also, it should be noted that there is an age dependent expression and function of Toll-like receptors in human skin (Iram et al., 2012). A recent study compared the impact of a range of TLR agonists and chitosan as potential adjuvants for different routes of mucosal immunization (sublingual (SL), intranasal (IN), intravaginal (IVag) and a parenteral route (subcutaneous (SC) in the murine model (Buffa et al., 2012). Antibody responses to HIV-1 CN54gp140 (gp140) and Tetanus toxoid (TT) in systemic and vaginal compartments were measured. A number of trends were observed by route of administration. In general, co-administration with adjuvants increased specific systemic IgG responses where IN = SC > SL, while in the vaginal compartment IN>SL>SC for specific IgA. In contrast, for systemic and mucosal IgA responses to antigen alone SL > IN = SC. A number of adjuvants increased specific systemic IgA responses where in general IN > SL > SC immunization, while for mucosal responses IN = SL > SC. In contrast, direct intravaginal immunization failed to induce any detectable systemic or mucosal responses to gp140 even in the presence of adjuvant. However, significant systemic IgG responses to TT were induced by intravaginal immunization with or without adjuvant, and detectable mucosal responses (IgG and IgA) were observed when TT was administered with FSL-1 or poly-IC. Interestingly some TLRs displayed differential activity dependent upon the route of administration. MPLA (TLR4) suppressed systemic responses to SL immunization while enhancing responses to IN or SC immunization. CpG B enhanced SL and IN responses, while having little or no impact on SC immunization. These data demonstrate importance of the route, antigen and adjuvant mixture in the design of mucosal vaccine strategies. In summary, studying the expression of all TLRs by cells that are located at ports of microbial entry such as skin, lung, and

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intestine is likely to provide novel vaccine strategies for vaccines that are administered by dermal, inhalation and oral routes. In vitro systems and animal models are of limited value For obvious reasons, investigators still use human PBMC for in vitro evaluation, but rely on animal (mainly rodent models) for preclinical in vivo studies. Because the distribution of TLRs is not identical in different species, there can be substantial differences in the responses of rodents and humans to TLR agonists or to complex adjuvant formulations. For example, studying endosomal TLRs, similarities and differences between TLR7, -8 and -9 agonists have been found (Alkan 2012; Campbell et al., 2009; Hanten et al 2008). Both TLR7 and -9 are found in plasmacytoid DCs (pDCs) and B-cells and they induce high IFN-α levels in man. However, more mouse cells express TLR9 (Edwards et al., 2003). Thus, mouse studies indicated that TLR9 agonists CpG-ODN are superior to TLR7 agonists such as imiquimod (Krieg, 2007). In this sense, rodents provided limited value for clinical studies (Krieg, 2012). The use of non-human primates is much more appropriate (Pulendran, 2010). In addition, we should put more emphasis on thorough analysis of human innate immune responses in clinical vaccine trials (Kwissa et al., 2012). Studies of the immune response in humans, when analysed by systems biology approaches can generate new biomarkers, new signatures of protection, and new questions that can be addressed by in vitro, in silico, and animal studies (Bernstein, 2011). Safety considerations of TLR agonists Safety is of primary concern in vaccine development. TLR agonists as powerful adjuvants may increase the risk of inflammatory/autoimmune diseases. This is of more concern for newborns and infants. A study in mice, utilizing systemic, repetitive, and high dose exposure to TLR2 agonists, resulted in perinatal brain injury (Du et al., 2011). Systemic administration of TLR agonists is expected to induce a ‘cytokine storm’ with unwanted side effects. Therefore, it is important

that TLR agonists, especially small molecular weight agonists should preferentially stay at the site of administration, and their effects should be transient. Thus, recently an imidazoquinoline designed for local activity without systemic cytokine induction has been constructed (Alkan et al., 2006). For example, 3M-052, a TLR7/8 agonist with a lipid tail and increased hydrophobicity, given subcutaneously, drives a strong Th1 response to haemagglutinin and serum neutralization of viable H1N1 virus in the absence of circulating TNFα or the induction of Th1 cytokines (Smirnov et al., 2011). From the few approved human vaccine adjuvants (alum and the oil-in-water-based emulsions MF59 (Novartis), AS03 and AS04 (GSK), AF03 (Sanofi), and liposomes (Crucell) we learned the importance of properly formulating subunit vaccines (Baldwin et al 2012). Adjuvants contained in our current vaccines seem to be safe even if given early in life (Demirjian and Levy, 2009; Levy et al., 2012). However, optimization of the dose of TLR agonists and antigen-TLR agonist conjugates require more attention. Challenges for the next decade The discovery of a remarkable number of PRRs has prompted the rational development of adjuvants based upon their ability to stimulate the innate immune sensors. We have a fairly good idea of how existing empirically designed adjuvants and live attenuated vaccines work (Levitz and Golenbock, 2012). Major challenges for the next decades will be to translate these findings into protective human vaccines. These challenges include: 1

In more challenging areas such as HIV, malaria, tuberculosis and tumours, adjuvants may need to induce ‘multidimensional’ immune responses (Coffman et al., 2010). Here, the systems biology approach may provide vital information. Understanding of the multiple dynamic molecular and cellular networks operating in the immune system requires a more integrative or systems approach to immunology (Gottschalk et al.,

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2

3

4

2012). Systems biology is the comprehensive and quantitative analysis of the interactions between all of the components of biological systems over time and can be used for rational design of vaccines and to accelerate the pace of clinical vaccine trials (Diercks and Aderem, 2013). The so-called systems vaccinology approach employs high-throughput technologies (e.g. microarrays, RNA-sequencing and mass spectrometry-based proteomics and metabolomics) and computational modelling to describe the complex interactions between all the parts of immune system, with a view to elucidating new biological rules capable of predicting the behaviour of the system (Nakaya and Pulendran, 2012). The changing demographics of the world’s population increases the need for vaccines that target and protect specific groups (Poland, et al., 2011; Rappuoli et al., 2011) such as very young, elderly and immunocompromised, who may have poor or different responses to traditional vaccines. In this century, sophisticated variants of vaccines will be developed for different segments of society such as pregnant, infant, adolescent, adult as well as elderly and immunocompromised individuals. Unique problems exist in developing regions of the world where the healthcare infrastructure is often underfunded and malnutrition may decrease vaccination responses. The possibility of using TLR agonists, either in combination or with non-TLR agonists such as NLRs, CLRs, and RLRs, to synergistically potentiate vaccine-induced responses needs to be thoroughly investigated. Such combinatorial vaccines may provide not only prophylactic, but therapeutic protection against infectious diseases and cancer (Duthie et al., 2011)

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The Importance of Cell-mediated Immunity for Bacterial Vaccines Alison G. Murphy and Rachel M. McLoughlin

Abstract Despite the fact that vaccines have been pivotal in controlling many serious diseases, traditional vaccine strategies are not without limitations. Current challenges include the development of novel strategies to confer protection against antigenically hypervariable pathogens, opportunistic pathogens, rapidly evolving anti-microbial resistant pathogens and non-cultivatable pathogens. In addition, some recently licensed vaccines offer only limited coverage (e.g. pneumococcal vaccines) and well-established vaccines have displayed decreasing immunogenicity (e.g. pertussis vaccine). Continued development of effective anti-bacterial vaccines will rely upon a clear understanding of the correlates of immunity against distinct pathogens. During the course of natural infection both cellular and humoral immune responses are utilized, therefore vaccines should induce antigen-specific responses by both arms of immunity. Novel anti-bacterial vaccines should strive to activate adaptive cellular immunity, i.e. antigen-specific T-cells, due to their well documented roles in induction of antibody isotype switching, cytolytic functions and regulation of phagocyte responses. The development of such vaccines will require a more lucid understanding of the contribution played by specific T-cell subsets in mediating immunity to natural infection. Additionally, inducing T-cell responses via immunization will require further investigation into the use of appropriate adjuvants, which have the capacity to direct specific T-cell responses. New vaccines, which specifically target cellular immunity in addition to humoral immunity, maybe key to providing protection against bacterial infections

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for which antibiotics are currently the only available treatment. Furthermore, the incorporation of elements targeting cellular immunity in currently licensed vaccines could potentially improve their efficacy. Introduction Core functions of the immune system The immune system comprises a myriad of cells and molecules which fulfil a series of core functions. Initially, the immune system must distinguish infectious non-self from self. This recognition then triggers the immune system to mount an appropriate effector immune response against the invading pathogen. This complex multi-component process translates initial recognition into a response that specifically eliminates or neutralizes the invading pathogen. Concurrently, the immune response is under tight control, to limit collateral damage to the host. Immune regulation, or the ability of the immune response to self-regulate, is an essential feature of immunity, the loss of which can lead to the development of allergic and autoimmune diseases. Finally, the immune system strives to protect against the reoccurrence of the same infection by generating immunological memory. Immunological memory is the hallmark of the adaptive immune response and the fundamental basis of vaccination. Innate and adaptive immunity Though referred to as a single entity the immune response can be broadly divided into two systems,

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the innate and the adaptive immune system. The innate and adaptive immune responses can be further subdivided into humoral and cellular components. The humoral immune response comprises of secreted molecules, present in the plasma, lymph and tissue fluids which are particularly important in protection against extracellular pathogens. As the name suggests the cellular immune response comprises immune cells which protect the host against infection. Innate immune responses are rapid, non-specific and incapable of generating immunological memory. This effective first line of defence comprises both cellular (e.g. phagocytes) and humoral (e.g. complement, antimicrobial peptides) components that are often sufficient to prevent or eliminate infections at the outset. During a bacterial infection the primary functions of the innate immune system are to recognize invading bacteria, actively clear the bacteria and facilitate initiation of adaptive anti-bacterial immunity. Innate immune recognition is executed via a series of constitutively expressed receptors, known as pathogen recognition receptors (PRRs), which efficiently recognize pathogen-associated molecular patterns (PAMPs) conserved amongst different microbial species. However, they are not equipped to distinguish between subtle differences in molecules expressed by individual species. Professional phagocytes including neutrophils, macrophages and dendritic cells (DCs), are the most important effector cells of the innate immune system. These cells have specifically evolved to recognize, engulf and ultimately kill bacteria. In addition, these cells can function as antigen-presenting cells (APCs) processing and presenting bacterial antigens to activate antigen-specific T-cells and are thus instrumental for initiating adaptive immune responses. The second arm of immunity, known as the adaptive immune response, develops and adapts to recognize and eliminate pathogens in a specific manner and is capable of distinguishing antigens from different bacterial species. Once activated it provides a comprehensive line of defence to eliminate pathogens that have overcome innate immunity. The adaptive response also involves both cellular (e.g. T-cells, B-cells) and humoral components (e.g. antibodies), which are specific

for particular pathogens. A critical aspect of the adaptive immune response is the generation of immunological memory. Memory cells formed during the adaptive immune response to primary bacterial infection are capable of persisting in the absence of antigenic stimulation. Memory T-cells have a lower activation threshold and enhanced effector functions compared to naive T-cells and somatic hypermutation enables memory B-cells to secrete antibodies with increased affinity for their target antigen, thus upon re-infection the invading bacteria is rapidly targeted and eliminated. It is important to note that innate and adaptive immune responses do not function independently of one another but rather act in concert to clear pathogenic threats and protect the host. Ligation of PRRs on innate APCs initiates and directs the subsequent adaptive immune response via production of pro-inflammatory cytokines that polarize specific T-cell responses. Activation of these innate signals is an important aspect of immunity for consideration in vaccine design. Adjuvants (molecules included in vaccine preparations to enhance the immunogenicity of an antigen) initiate signalling pathways in innate cells (Li et al., 2007), the downstream effects of which can direct or shape the adaptive immune response that is induced against the particular candidate antigen, leading to the generation of immunological memory that is appropriate to protect against the infection. Vaccine induced immunity Vaccination is the process by which an individual is intentionally exposed to a modified version of a pathogen or part thereof, which results in the establishment of long-term immunological memory to the specific pathogen. Most successful anti-bacterial vaccination strategies rely on the induction of memory B-cells, or effector plasma B-cells, which produce antigen-specific antibodies as the primary correlate of immune protection. Upon subsequent exposure to the bacteria, preexisting antibodies rapidly exert their effector functions; blocking attachment of the bacteria to host tissues, opsonization of bacteria for efficient uptake and killing by phagocytes and/or neutralization of the invading bacteria and its associated toxins. These effects ensure swift resolution of the

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infection (Manz et al., 2005). To date, licensed anti-bacterial vaccines that strive to generate antibody-mediated protection have been incredibly successful in eradicating or controlling a number of serious infectious diseases (Table 9.1). So, why are vaccines not available against all pathogenic bacteria? Current vaccine strategies are not without limitations, given the propensity of bacteria to display increased antigen variability, express immune evading factors and employ intracellular lifecycles culminating in the evasion of humoral responses. Therefore, antibody responses alone may not be sufficient to control infection with many bacterial species. Vaccine strategies targeting T-cells Development of effective vaccination strategies will rely upon a clear understanding of the correlates of immune protection. In other words: what constitutes a protective immune response against specific pathogens? The nature of the infecting organism and the associated disease pathology dictates the nature of the protective immune response that needs to be elicited. During the course of a natural infection all aspects of the immune response are utilized from innate to adaptive and cellular to humoral. It is therefore logical that the most efficient vaccines would induce antigen-specific responses in both the cellular and humoral arms of immunity. Novel anti-bacterial vaccines should therefore strive to activate antigen-specific T-cells. In addition

to their well documented role in providing help for the induction and maintenance of antibody responses (Mahon et al., 1995; Poulsen and Hummelshoj, 2007; Savelkoul and van Ommen, 1996), certain T-cell subsets possess direct cytolytic functions and are therefore capable of recognizing and destroying host cells infected with traditional intracellular pathogens or cells in which bacteria are surviving intracellularly as an immune evasion strategy. Furthermore, T-cells play an instrumental role in regulating downstream phagocyte responses (McLoughlin et al., 2008). Given that phagocytic cells represent the most important effector cells required for clearance of bacterial infection it stands to reason that manipulating T-cell responses which control or regulate the effector functions of phagocytes represents an attractive approach for enhancing immune protection against infection. Advancing the development of novel antibacterial vaccines to elicit antigen-specific T-cell responses first requires a more lucid understanding of the role played by specific T-cell subsets in mediating protective immunity to natural infection against the invading pathogens. The development of vaccines to protect against infections caused by opportunistic pathogens such as commensal species e.g. Staphylococcus aureus, Escherichia coli as well as environmental bacteria e.g. Pseudomonas aeruginosa represent a major challenge, in part because immunization strategies must elicit protective immunity against

Table 9.1 Examples of past and present licensed vaccines adapted from (Plotkin, 2005) Vaccine type

Infecting organism

Immunogenic component(s)

Approximate time of availability

Live, attenuated

Rabies virus

Chemical attenuated whole organism

1885 1881

Bacillus anthracis Killed

B. pertussis

Inactivated whole organism

Polio vaccines

1926 1938

Toxoids

Corynebacterium diphtheriae

Diphtheria toxin

1923

Clostridium tetani

Tetanus toxin

1927

Purified recombinant proteins/subunits

Hepatitis B virus

Hepatitis B surface antigen

1986

B. pertussis

Pertactin, pertussis toxin, fimbriae, filamentous haemagglutinin

1996

Conjugate vaccines

S. pneumoniae

Capsular polysaccharide conjugated to a protein carrier

2002

N. meningitidis C

2002

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organisms to which we are not immunologically naive. Using the bacterium S. aureus as an example we know that due to its propensity to colonize the anterior nares (Miller et al., 2012; Peacock et al., 2001; van Belkum et al., 2009) the majority of the population possess high serum antibody titres against S. aureus antigens (Verkaik et al., 2009), yet the presence of anti-S. aureus antibodies in the sera does not translate to protection against invasive infection (David et al., 2008; Huang and Platt, 2003; Wertheim et al., 2004). S. aureus has evolved numerous strategies for evading the immune response, several of which, such as protein A, specifically target antibody-mediated immunity (reviewed in Foster, 2005). These issues may explain why active and passive immunization strategies against S. aureus which primarily target protective immunity provided by antibodies, have thus far failed to show efficacy in humans. Strategies designed to elicit robust cellular mediated responses, in addition to humoral immunity, may be key for the development of effective vaccines against these types of bacterial infections. T-cell responses to bacterial infection Antigen specific T-lymphocytes are at the core of the adaptive cellular immune response. Owing to the expression of highly variable antigen receptors on their surface, these cells enable the body to respond specifically to a wide range of foreign antigens. Upon antigen recognition, naive T-cells become activated and differentiate into effector lymphocytes which mediate a range of protective functions culminating in efficient clearance of the invading pathogen and importantly provide long lasting memory against subsequent re-exposure. Haematopoietic stem cells differentiate in the bone marrow into putative common lymphocyte progenitors, from which Natural Killer (NK), T, and B-lymphocytes originate (Fig. 9.1). When T-cell precursors egress to the thymus from the bone marrow these progenitor cells lack most of the surface receptors characteristic of mature T-cells. Interaction with the thymic stroma initiates sequential phases of differentiation and maturation involving ordered, rearrangement of antigen receptor genes leading to the eventual

formation of the complete heterodimeric antigen receptor. Two distinct lineages of T-cells are produced during thymic development and can be distinguished based on their distinct TCRs; α:β and γ:δ. αβT-cells further differentiate into CD4+ and CD8+ T-cells. In addition, another lineage of cells, known as NKT cells, bearing an αβTCR of limited diversity that also express the NK1.1 receptor, develop. In the periphery the effector functions of the mature T-cell relies upon their interaction with a target cell (usually an APC) displaying a specific antigen. However, the effector actions initiated by this antigen recognition can be distinct depending on the nature of the T-cell. While the classic antigen-specific T-cell lineages of CD4+ and CD8+ T-cells are fundamental components of the adaptive immune system other lineages such as γδT-cells and NKT cells possess both innate and adaptive characteristics. These cells are capable of rapid innate-like effector functions particularly at mucosal sites and are also capable of generating antigen-specific responses. In addition there are subsets of innate lymphoid cells (ILCs), which produce many adaptive T-cell cytokines but do not express the cell surface markers associated with classical lineages. These cells which include NK cells and lymphoid tissue inducer cells (LTis) develop in the bone marrow like classical T-cells, but do not necessarily require localization to the thymus for maturation. NK cells are large, granular, non-specific lymphocytes that play an important role in immunity against tumours and virally infected cells. NK cells are capable of spontaneously killing infected cells and producing high levels of cytokines particularly interferon γ (IFNγ) (Stetson et al., 2003). LTicells are involved in the development of lymph nodes (Eberl et al., 2004; Mebius et al., 1997; Yoshida et al., 1999) and have also been linked to the maintenance of T-cell memory (Withers et al., 2012). LTi-cells were shown to play an important role in the immune response to enteric infections due to their propensity for rapid interleukin (IL)17A and IL-22 production (Sonnenberg et al., 2011; Takatori et al., 2009). Knowledge gained from studying the role played by T-cells in natural immunity to bacterial infection will facilitate the development of novel vaccine strategies capable of harnessing the

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Haematopoietic stem cell

Common Myleoid progenitor

Common Lymphoid progenitor

Myeloblast Lymphoblast

Basophil

B-cell

T-cell

NK-cell

Monocyte

Eosinophil Neutrophil

αβT-cell Macrophage

γδ T-cell

Myeloid Dendritic cell

NKT-cell Lymphoid Dendritic cell

CD4+ T-cell

CD8+ T-cell

Figure 9.1  Lymphoid cell lineage. Haematopoietic stem cells (HSCs) are the blood cells from which all other blood cells are derived, namely the myeloid (phagocytes and APCs) and lymphoid lineages. Lymphocytes can be broadly categorized into B-cells, which mature in the bone marrow, T-cells, which mature in the thymus and NK-cells which can mature in the bone marrow, lymph nodes, spleen or thymus. Once stimulated, B-cells differentiate into effector cells known as plasma cells, responsible for antibody production. T-cells can be further differentiated into αβT-cells, γδT-cells and NKT cells, all of which carry out a diverse array of effector functions within the immune system. NK cells are large granular lymphocytes important in antitumour and anti-viral immunity.

protective effector functions of these individual T-cell subsets. Conventional antigen-specific T-cells αβT-cells αβT-cells express a TCR composed of disulfide linked heterodimer between a TCRα chain and a TCRβ chain. The TCR is encoded by homologous genes and its enormous diversity is generated by somatic recombination of variable (V), diversity (D) and joining ( J) gene segments (Chien et al., 1984; Petrie et al., 1995). The αβ TCR facilitates recognition of antigenic peptides displayed by

MHC molecules on APCs. The variable regions of the peptide binding groove in TCRs are specific for a wide array of possible antigens, thus facilitating recognition of the diversity of antigens expressed by potential pathogens. During an infection bacteria are taken up by APCs which migrate to the peripheral lymphoid organs (lymph nodes, spleen and mucosal lymphoid tissues) where they present antigen to naive T-cells. When a naive T-cell encounters an antigen it requires three signalling events that are provided by the APC for complete activation (Fig. 9.2). This interaction exhibits the important interplay that exists between innate and adaptive arms of the immune response for the generation

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1.

Antigen MHC I/II

TCR

APC

T-cell 2.

CD80/ CD28 CD86

PRR

3.

PAMP

Cytokine receptor

Cytokine

Figure 9.2  APC: T-cell interactions. The interaction of peptide loaded MHC molecules with specific TCRs, referred to signal 1, is the first activating stimulus received by the T-cell. Activation of the APC by the pathogen interactions through PRR ligation also induces the expression of co-stimulatory molecules such as CD80 and CD86. These molecules engage their counter receptors on the T-cell, particularly CD28, transmitting the signals required for T-cell proliferation and survival. This constitutes as signal 2. Finally, signal 3 comprises of cytokine secretion by the APCs, which is needed for effector T-cell differentiation and inhibition of T-cell anergy.

of antigen-specific T-cell responses. These controlled activation steps prevent unnecessary T-cell activation which could lead to tissue damage and autoimmunity, but also allows for specific immune responses directed towards particular pathogens. Upon activation, antigen-specific naive T-cells undergo clonal expansion, which involves repeated T-cell proliferation of differentiated effector cells bearing a TCR identical to the specificity of the parental cell. As part of their differentiation these effector cells up-regulate specific adhesion molecules and chemoattractant receptors that allow for interactions with the vascular endothelia, directing migration of the antigen-specific T-cells to particular tissues where they can execute their effector functions (Bromley et al., 2008). Resolution of infection is followed by a contraction phase where approximately 90% of the effector T-cells die, leaving behind a population of long lived memory T-cells. Under steady-state conditions, memory T-cells slowly turn over in the absence of antigen. Upon antigen re-exposure memory T-cells have a higher sensitivity to antigenic stimulation and are less dependent on co-stimulation allowing for rapid activation and amplification of the antigen-specific T-cell effector

response. Long term immunological protection depends on both the quantity and quality of the memory T-cell population. αβT-cells are further classified as either CD4+ helper T-cells or CD8+ cytotoxic T-cells. Both CD molecules function as co-receptors aiding in the process of antigen presentation by APCs. During antigen presentation CD4 or CD8 molecules associate with the TCR on the T-cell surface and bind to invariant sites on Major histocompatibility class (MHC) I or MHC II molecules respectively, expressed by APCs. Binding of CD4 and CD8 molecules increases the antigen sensitivity of the TCR and the TCR signal ( Janeway, 1992). CD4+ T-cells CD4+ T-helper cells function by activating and directing or ‘helping’ the function of other effector immune cells primarily via cytokine release. The downstream effects of these cytokines enhance phagocytic and bactericidal activity of innate effector cells, induce antibody class switching, and promote activation and expansion of CD8 T-cells. CD4+ T-cells can be divided into different effector subsets that are characterized by their expression of specific transcription factors, signal

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IL-1β, IL-23

TGFβ, IL-6

Bacteria

Naive CD4+ T cell

Naive CD4+ T cell

Naive CD4+ T cell

Naive CD4+ T cell

Naive CD4+ T cell

Th1

Th2

Th17

Th22

iTreg

T-bet STAT4

GATA3 STAT6

RORγT STAT3

Ahr

FoxP3

IFNγ IL-2

IL-4 IL-5 IL-13

IL-17A IL-17F IL-22

IL-22 AMPs

IL-10 TGFβ

Macrophage Activation Neutrophil recruitment Humoral Immunity Immunity against Immunity against Anti-helminth immunity intracellular bacteria extracellular bacteria

Mucosal Immunity Epithelial barrier protection

Regulation

Figure 9.3  CD4+ helper T-cell differentiation depends on the cytokine milieu. Following antigen presentation by the APC naive CD4+ T-cell precursor cells can differentiate into subsets of effector T-cells (Th1, Th2 and Th17, and in humans Th22 cells) and a subset of inducible T-regulatory cells. Subset differentiation is driven by selective cytokines and transcription factors. Each subset carries out specialised functions within the immune response to pathogens and the eventual resolution of inflammation.

transduction pathways and cytokine secretion profiles (Fig. 9.3). T helper 1 (Th1) cells Th1 cells are characterized by their secretion of IFN-γ and are vital for the cellular immune response to infections with intracellular pathogens such as Listeria monocytogenes (Hsieh et al., 1993) and Mycobacterium spp. (Cooper et al., 1993; Dalton et al., 1993). IL-12 produced by innate immune cells upon PRR activation combined with IFN-γ produced by NK cells polarize CD4+ T-cells towards Th1 differentiation through the activation of signal transducer and activator of transcription (STAT) 4 and STAT 1 and T-box transcription factor (T-bet) (Aaronson and Horvath, 2002; Hsieh et al., 1993).

In the context of a bacterial infection antigenspecific Th1 responses are vital for enhancing the effector functions of macrophages and transforming them into potent anti-bacterial effector cells. IFN-γ has been shown to augment macrophage activation in response to bacterial products and pro-inflammatory cytokines (Stout and Bottomly, 1989). In addition, IFN-γ enhances the production of reactive nitric oxide (NO) (Erwig et al., 1998) by up-regulation of nitric oxide synthases (NOS) which is involved in the formation of microbicidal NO by macrophages (MacMicking et al., 1997). IFN-γ signalling also enhances respiratory burst and the formation of reactive oxygen species (ROS) by up-regulating gp91phox, a subunit of NADPH oxidase in both macrophages and neutrophils (Cassatella

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et al., 1990). Macrophages also up-regulate their expression of FcRγ1, a receptor involved in binding of opsonized bacteria or their components, in response to IFN-γ signalling (Erbe et al., 1990). In addition, IFN-γ promotes expression of co-stimulatory molecules and MHC complex expression, enabling more efficient antigen presentation by the macrophage (Dalton et al., 1993). IFN-γ can also regulate B-cell functions, namely immunoglobulin production and isotype switching to IgG2a thus promoting opsonophagocytic killing (Finkelman et al., 1988a). Th1 cells have also been shown to promote the recruitment of leucocytes to infection sites. IFN-γ signalling enhances the production of chemokines such as IP-10/CXCL-10, which is involved in T-cell trafficking, MCP-1, a chemoattractant for monocytes/macrophages and MIP-1α and MIP-1β, chemoattractants for CD4+ and CD8+ cells (Gil et al., 2001). Furthermore, it can increase ICAM-1 expression by endothelial cells thus facilitating immune cell migration (Hou et al., 1994). Taken together, we can conclude that Th1 cells have the potential to exert powerful pro-inflammatory effects which promote the clearance of infection. The critical role played by Th1 cells in protective immunity to intracellular pathogens is evidenced by the fact that patients with defects in IFN-γ signalling, including defects in IFN-γ receptor expression, IL-12 signalling pathways or mutations in STAT1, suffer from severe intracellular bacterial infections (de Jong et al., 1998; Dorman and Holland, 1998; Dupuis et al., 2001; Newport et al., 1996). In murine models, infection with L. monocytogenes induces a strong Th1 response (Hsieh et al., 1993) and these Th1 cells were found to enhance CD8 T-cell-mediated protection against L. monocytogenes (Geginat et al., 1998). Neutralization of IFN-γ production in mice increased susceptibility to infection and impaired the generation of protective T-cells (Yang et al., 1997). Mice deficient in IFN-γ are also highly susceptible to infections with Mycobacterium spp. due to impaired macrophage activation and decreased MHC II expression by macrophages (Cooper et al., 1993; Dalton et al., 1993). Th1 cells have also been shown to a play a protective role during extracellular infections, as mice deficient in IL-12 display increased susceptibility to infections with

Klebsiella pneumoniae (Happel et al., 2005). This effect is probably due to inefficient macrophage activation. These results demonstrate the importance of Th1 protective immunity against both intracellular and extracellular bacterial infections through their ability to enhance phagocytic (particularly macrophage) responses. Vaccine strategies have employed the use of adjuvants to induce Th1 cell responses. Most Th1 inducing adjuvants consist of ligands which when ligated in vivo induce Th1 polarizing cytokines, namely IL-12. Examples of Th1 inducing adjuvants include complete Freunds adjuvant (CFA) which enhances inflammasome activation (Coffman et al., 2010; Su et al., 2005), Poly I:C a TLR3 and RLR ligand (Longhi et al., 2009) and Monophosphoryl lipid a (MPL), a TLR4 ligand (Didierlaurent et al., 2009). CpG is a TLR9 ligand that promotes DC maturation, antigen presentation, IFN-γ production by T-cells and production of IgG2a (Chu et al., 1997; Kobayashi et al., 1999). The combination of chitosan and CpG together was shown further enhance the secretion of Th1 polarizing cytokines by DCs (Mori et al., 2012). The selection of adjuvants that target the Th1 response will be extremely beneficial in the design of vaccines against both intracellular and extracellular bacterial infections and have already showed promise in improving anti-cancer (Dredge et al., 2002), anti-fungal (de Oliveira et al., 2008) and anti-parasite vaccine efficacy (Corral and Petray, 2000). T helper (Th) 2 cells Th2 cells are characterized by their secretion of IL-4, IL-5 and IL-13 and are primarily involved in the induction of humoral immunity and antihelminth immunity through the activation of basophils and mast cells (Urban et al., 1991). IL-4 produced by innate cells upon PRR ligation polarizes naive CD4+ T-cells towards Th2 differentiation through the activation of signal transduction pathways involving GATA3, a master gene regulator, and STAT6 (Shinkai et al., 2002; Sokol et al., 2008). Downstream effects of Th2 cells involve stimulation of B-cell proliferation, induction of B-cell antibody class switching to IgE (which binds to FCεR1 on the surface of basophils and mast cells) and augmentation of

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neutralizing antibody (e.g. IgG1) production (Finkelman et al., 1988b). IL-4 signalling also induces the production of thymic stromal lymphopoietin by epithelial or stromal cells (Kato et al., 2007), which enhances the maturation of DCs (Reche et al., 2001). Alum, one of the few adjuvants licensed for human use, predominantly induces Th2 driven humoral immunity which then provides protective humoral immunity against infection (Gavin et al., 2006). Alum has also been shown to activate the inflammasome leading to the production of mature IL-1β and IL-18, two cytokine capable of polarizing the T-helper cell response (Li et al., 2007). An acellular pertussis vaccine containing alum was shown to induce Th2 and Th17 cells in vivo (Ross et al., 2013b). In the context of bacterial infection it is possible that the expansion of Th2 cells by vaccination may not be optimal as Th2 cells favour the development of IgE, an antibody isotype important in helminth infections, but not necessarily bacterial infections. In fact, vaccine induced Th2 immunity against B. pertussis infection appears to be dispensable as vaccinated IL-4 deficient mice had comparable bacterial burden to their wild-type counterparts (Ross et al., 2013a,b). Therefore we can conclude that vaccine induced Th2 cells, whilst useful for the generation of humoral immunity, may have limitations. In fact enhanced vaccine induced protection against hepatitis B virus was achieved when alum was switched to a more effective adjuvant AS04, a combination of alum and MPL. AS04 was found to facilitate macrophage interactions, leading to a T-cell response alongside the induction of humoral immunity (Beran, 2008; Halperin et al., 2006). T helper (Th) 17 cells Th17 cells are characterized by their secretion of IL-17A, IL-17F and IL-22 and are vital for protection against extracellular bacterial infection, particularly at mucosal surfaces through their abilities to regulate CXC chemokine induced neutrophil recruitment to sites of infection. More recently Th17 cells have been associated with the induction of IgA, an antibody important in mucosal defences (Hirota et al., 2013). Production of TGF-β, IL-6, IL-21 and IL-23 by activated

innate immune cells polarizes naive CD4+ T-cells towards Th17 differentiation through activation of STAT3 signalling and the transcription factor retinoid-related orphan receptor (ROR)γt (Bettelli et al., 2006; Harrington et al., 2005; Wei et al., 2007; Zhou et al., 2007). Th17 cells are fundamentally important for controlling neutrophil responses. IL-17A promotes granulopoiesis through the induction of SCF and G-CSF by epithelial cells which in turn signal to bone marrow stimulating the proliferation of haemopoietic stem cells. IL-17A is also a potent activator of CXC chemokine secretion by endothelial cells, epithelial cells and keratinocytes which attracts neutrophils to sites of infection (Andreasen et al., 2009; Fossiez et al., 1996; Schwarzenberger et al., 2000; Ye et al., 2001a). Leucocyte migration is further facilitated by IL-17A induced up-regulation of ICAM-1 expression by keratinocytes (Albanesi et al., 1999). Not only does IL-17A aid in the recruitment of neutrophils, it also enhances direct bacterial killing by neutrophils (Lu et al., 2008). In addition to their role in mobilizing and activating neutrophils, Th17 cells are capable of stimulating other innate immune responses including the production of AMP production, such as β-defensin, by epithelial cells (Kao et al., 2004; Liang et al., 2006). In humans, patients with hyper-IgE syndrome (HIES) have genetic mutations in their STAT3 genes leading to impaired Th17 cell function and decreased IL-17 levels. As a result these patients suffer from recurrent and often severe Candida albicans and S. aureus infections (Ma et al., 2008; Milner et al., 2008). The importance of IL-17 in protection against extracellular bacterial infection was first described in murine models of K. pneumoniae infection. IL-17R deficient mice had impaired clearance of the infection compared with wild-type mice and this correlated with defective G-CSF and CXC chemokine production and consequently reduced neutrophil recruitment to the infection site (Ye et al., 2001a,b). IL-23-deficient mice also demonstrated substantial mortality following a sublethal dose of K. pneumoniae which was directly associated with decreased IL-17 production by T-cells. Administration of IL-17 restored bacterial control in the IL-23 deficient mice (Happel et al., 2005). Immunity to S. pneumoniae

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also appears to be mediated by Th17 cells, as adoptive transfer of CD4+ T-cells isolated from mice immunized with killed or live pneumococci, protected T-cell-deficient mice against pulmonary infection via IL-17A production and neutrophil recruitment (Lu et al., 2008). It has also been shown that IL-17A could increase pneumococcal killing by human neutrophils suggesting that in this model of S. pneumoniae infection IL-17 may contribute to bactericidal effects of neutrophils (Lu et al., 2008). IL-17A- and IL-17F-deficient mice also demonstrated increased susceptibility to S. aureus cutaneous infections; however, the same mice were not significantly impaired in their abilities to clear systemic S. aureus infection, suggesting a site-specific role for IL-17 in anti-S. aureus immunity (Ishigame et al., 2009). During B. pertussis infection, pertussis toxin has been shown to induce a Th17 response, the inhibition of which leads to reduced chemokine expression locally in the lung, corresponding to impaired neutrophil recruitment and consequently reduced bacterial clearance (Andreasen et al., 2009). Given their defined role in protective immunity to bacterial infections vaccine strategies targeting Th17 cells are being considered for a number of extracellular bacterial infections. Immunization with whole cell pertussis vaccine has been shown to evoke protective Th17 responses in a murine model. In this model the authors noted the novel effect of IL-17 in promoting macrophage killing of B. pertussis (Higgins et al., 2006). Th17 responses have also been proposed to play a role in vaccine induced immunity to S. aureus. In murine model studies a vaccine based on clumping factor A (ClfA), a staphylococcal surface protein (Narita et al., 2010) or a molecule structurally analogous to ClfA (Lin et al., 2009) mediated protection in wild-type mice but were ineffective in IL-17A deficient mice. Vaccine induced Th17 cells were shown to mediate protection through their downstream effects on phagocytes (Lin et al., 2009). A large-scale proteomic screen of an expression library of S. pneumoniae antigens that primarily induce Th17 cell cytokines has also been proposed as a strategy to identify appropriate antigens for future vaccines against S. pneumoniae that harness Th17-mediated immunity (Moffitt et al., 2011).

It appears that the selection of specific adjuvant molecules maybe the key to expanding protective Th17 cells. As previously mentioned chitosan administered with CpG was shown to drive not only enhanced secretion of Th1 polarizing cytokines but also the induction of Th17 polarizing cytokines (Mori et al., 2012). Complete Freund’s adjuvant also induces strong Th17 responses through enhancing inflammasome processing of the Th17 polarizing cytokine IL-1β (Coffman et al., 2010). The adjuvant, heat-liable enterotoxin (LT) promotes antigen-specific Th17 responses, via enhanced IL-1β and IL-23 secretion by DCs, which were protective in a murine model of B. pertussis infection (Brereton et al., 2011). Emerging evidence also suggests that efficient protection against invading bacteria, particularly at mucosal sites, may require synergy between the Th1 and Th17 cell lineages. In a model of B. pertussis infection extended induction of IL-17 preceding the Th1 response was seen, prior to the peak of infection, with the production of IL-17 persisting until clearance (Andreasen et al., 2009). Furthermore, adoptive transfer of Th1 and Th17 cells purified from mice previously infected with B. pertussis into naive mice provided enhanced protection against B. pertussis challenge compared with transfer of either cell type alone (Ross et al., 2013b). In IL-12- and IL-23-deficient mice, defects in Th1 and Th17 lineages respectively are observed; however both types of mice experience increased but comparable susceptibility to K. pneumoniae suggesting that both Th1 and Th17 cells contribute to immunity in this model (Happel et al., 2005). In addition, vaccination against M. tuberculosis expanded a population of IL-23-dependent IL-17 producing antigen-specific T-cells in the lung, which preceded the Th1 response. In the absence of IL-23, impaired IFN-γ recall responses were observed (Khader et al., 2007). The authors postulated that the Th17 response was indirectly required for anti-mycobacterial immunity mediated by Th1 cells. Taken together these studies support the notion that an anti-bacterial vaccine inducing IL-17 responses could provide surveillance in the periphery which when triggered by infection would initially induce a rapid Th17 response

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which in turn may be capable of facilitating a Th1 response, ultimately leading to complete protective immunity against both intracellular and extracellular bacteria. T helper (Th) 22 cells Th22 cells are a recently described lineage of T-helper cells identified in humans. They are characterized by their propensity to express the skin homing chemokine receptors CCR4 and CCR10 in conjunction with CCR6 (characteristic of Th17 cells) and to selectively produce large amounts of IL-22 but little to no IL-17 or IFN-γ (Duhen et al., 2009; Trifari et al., 2009). IL-22 production by Th22 cells is associated with the expression of the transcription factor for the aryl hydrocarbon receptor (AhR), which interacts with environmental toxins and metabolites. In vitro studies revealed that the differentiation of Th22 cells can occur in the presence of IL-6, TNF, AhR agonists, vitamin D3 metabolites and that interaction with plasmacytoid DCs favoured Th22 development compared to interaction with conventional DCs (Duhen et al., 2009; Trifari et al., 2009). Given the propensity of Th22 cells to home to the skin, IL-22 has been shown to be important for protection against infections at barrier sites (e.g. skin, respiratory tract) where it promotes AMP production in the epithelium (Aujla et al., 2008; Zheng et al., 2008). In the skin, IL-22 induces AMPs, such as defensins but also promotes keratinocyte proliferation and inhibits differentiation demonstrating IL-22’s role in wound healing and tissue remodelling (Boniface et al., 2005; Eyerich et al., 2009). IL-22 was shown to play an important role in protection against cutaneous infection with C. albicans through AMP production and maintenance of the epithelia (Eyerich et al., 2011). IL-22 also plays a role in immunity to pulmonary K. pnuemoniae infection (Aujla et al., 2008) and enteric Citrobacter rodentium infection (Zheng et al., 2008). During murine enteric C. rodentium infection, which is analogous to human enterohaemorrhagic E. coli infection, researchers demonstrated that IL-22 deficient animals were more susceptible to the infection than their wild-type counterparts and that adoptive transfer of Th22 cells differentiated in vitro conferred complete protection against the

infection in IL-22-deficient mice whereas transfer of Th17 cells did not (Basu et al., 2012). Vaccine targeting of Th22 cells which would induce AMP production and possibly improve epithelial barriers could be potentially beneficial for protection against enteric and also cutaneous pathogens. Similar to other helper T-cell subsets, it may be possible to induce Th22 cells through the use of adjuvants which target the pathways involved in driving a Th22 phenotype. Targeting these cells would require identification of the particular PRRs which when ligated specifically induce APCs to produce Th22 polarizing cytokines. Further characterization of this novel subset is therefore required in order to fully elucidate the mechanisms which give rise to the Th22 phenotype. T regulatory cells (Treg) Homeostasis within the immune system depends on a balance between pro-inflammatory and regulatory responses. Regulatory T-cells are a diverse population of lymphocytes with the crucial role of maintaining this balance. They are required to suppress immune responses to self antigens and critically important in protection against autoimmunity. Tregs are also required to regulate the pro-inflammatory environment initiated during an infection thus limiting collateral host tissue damage. Mice deficient in the immunosuppressive cytokine IL-10 demonstrated multiple organ failure and increased mortality during E. coli infection. This was observed despite an accelerated bacterial clearance compared to wild-type mice (Sewnath et al., 2001). Similar observations have been made using IL-10 deficient mice infected with L. monocytogenes (Deckert et al., 2001), Helicobacter pylori (Chen et al., 2001) and Helicobacter hepaticus (Kullberg et al., 2001). Therefore in the absence of Tregs an exaggerated immune response takes place which initially deals with the bacterial infection, but then the lack of inflammatory control damages host tissues leading to increased mortality. Efficient clearance of infection will probably depend upon an appropriate balance of effector and regulatory T-cells present during the infection, i.e. sufficiently low levels of Treg activation early in infection enabling a robust

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effector T-cell response followed by sufficient regulation of these effector responses to control inflammation induced pathology (Mills, 2004). Immunotherapies targeting Tregs have been shown to be beneficial for non-infectious diseases. For example co-immunization of OVA epitope peptide (a substance used to induce asthma in murine models) with a DNA vaccine encoding the same epitope prevented experimental asthma by inducing a population of OVA specific Tregs. Treg induction correlated with a decrease in eosinophil and lymphocyte migration to the site of challenge ( Jin et al., 2008). In the context of an anti-bacterial vaccine it is important to consider the fact that induction of suppressive Treg cells is not likely to be beneficial. For example, a vaccine striving to induce protective Th1 or Th17 effector cells would not simultaneously want to induce antigen-specific Treg cells. Novel vaccine strategies targeting tumours aim to induce polyfunctional T-cells that can produce multiple cytokines such as IFNγ, IL-17 and IL-2, while at the same time inhibiting the secretion of suppressive cytokines. This was achieved by inhibiting phosphoinositide-3-kinase signalling in DCs leading to decreased production of IL-10 and TGF-β (which drive Treg cell differentiation) but did not affect the production of the pro-inflammatory cytokines IL-12 and IL-1β (which drive effector T-cell differentiation) (Marshall et al., 2012). CD8+ T-cells The primary function of CD8+ T-cells known as cytotoxic T-lymphocytes (CTLs) is to kill intracellularly infected, cancerous or abnormal host cells. These cells interact with MHC I molecules on the surface of nucleated cells which can present cytosolic antigens (i.e. antigens derived from viruses and intracellular bacteria). To minimize damage to the surrounding tissues CD8+ T-cells kill in a specific manner by stimulating target cells to undergo apoptosis (programmed cell death). Upon the detection of an infected cell the CTLs release special lytic granules which contain perforins and granzymes amongst other proteins. The perforin released by the CTL polymerizes the target cell membrane, creating a pore through which granzyme can gain entry to the intracellular space (Nakajima et al., 1995; Shi et al., 1992a;

Shi et al., 1992b; Shi et al., 1997). Once inside granzymes A and B cleave and activate multiple protein substrates, primarily pro-apoptotic caspases, which eventually results in the initiation of apoptosis (Adrain et al., 2005; Rousalova and Krepela, 2010; Talanian et al., 1997). CTLs can also induce killing of target cells by the engagement and aggregation of target cell death receptors (e.g. Fas) with their cognate ligands (e.g. Fas ligand) on the CTL membrane. These interactions result in classical caspase dependent apoptosis of the target cell (Nagata and Golstein, 1995; Trapani and Smyth, 2002). CD8+ T-cells are also capable of modulating other effectors of the immune response through cytokine secretion (Hausmann et al., 2005; Liu et al., 2001; Patterson et al., 2002). Secretion of these cytokines can modulate the effector responses elicited by CD4+ T-cells. In turn, CD4+ cells have also been shown to be important in modulating CD8+ responses via the maintenance of memory CD8+ T-cells (Shedlock and Shen, 2003; Sun and Bevan, 2003). CD8+ T-cells can be alternatively activated via cross presentation. This process involves the ability of APCs to load peptides derived from exogenous antigens normally destined for MHC II presentation onto MHC I molecules. This process is atypical as MHC I molecules normally present cytosolic or endogenous antigens. These MHC I molecules are then capable of presenting non-cytosolic antigen to CD8+ T-cells and inducing immunogenic CD8+ T-cells to atypical or exogenous antigens. Granzymes secreted by the CD8+ T-cell can act as regulators of cross-presentation. They achieve this through induction of ‘eat me’ signals on transformed or infected target cells which aid in the endocytosis of these dying cells by DCs. Endocytosis allows DCs to present exogenous antigens derived from the dying cells on their MHC I molecules thus enabling them to cross prime CD8+ T-cells (Hoves et al., 2011; Hoves et al., 2012). Cross priming has been shown to be required for defence against many viruses and tumours (Yewdell and Haeryfar, 2005). In the context of bacterial infections, CD8+ T-cells play well-defined roles in protection against mycobacterium infections (Weerdenburg et al., 2010; Woodworth and Behar, 2006).

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During M. tuberculosis infection, mice deficient in TAP (an essential protein for loading antigen onto MHC I molecules and thus presentation to CD8+ T-cells) rapidly die and display severe tissue pathology and increased bacterial burden compared to their wild-type counterparts (Behar et al., 1999). Additionally, mice lacking CD8 expression on the T-cell surface and mice deficient for MHC I molecules also show increased susceptibility to M. tuberculosis infection (Sousa et al., 2000). Cross priming of CD8+ T-cells plays an important role in protection against M. tuberculosis infection, as apoptotic vesicles derived from mycobacteria infected macrophages contained antigens which were cross-presented to CD8+ T-cells. This cross presentation resulted in CD8+ T-cell stimulation and protection against tuberculosis (Winau et al., 2006). CD8+ T-cells have also been shown to play a protective role during L. monocytogenes infections (Kaufmann and Ladel, 1994; Ladel et al., 1994). Highly specific CD8+ T-cells contributed more substantially to long-term protective immunity against L. monocytogenes in comparison to CD4+ T-cells, as mice with impaired MHC I presentation succumbed to both primary and secondary infection more readily than mice deficient in MHC II presentation ( Jiang et al., 2003; Ladel et al., 1994). Adoptive transfer studies using L. monocytogenes specific CD4+ or CD8+ T-cells demonstrated that CD8+ T-cells were capable of mediating protection independently of IFNγ, unlike CD4+ T-cells which required IFN-γ to mediate protective immunity (Harty and Bevan, 1995; Harty et al., 1992) suggesting that direct killing of intracellularly infected cells by the CD8 T-cells contributes to clearance of L. monocytogenes infection. Evidence also exists for the importance of CD8+ T-cells in immunity against extracellular bacterial infections. Mice deficient in CD8 T-cells were found to be more susceptible to infection with Yersinia pseudotuberculosis. During infection Y. pseudotuberculosis binds to host cells, releasing proteins into the cytoplasm resulting in the inhibition of phagocytosis. CD8+ T-cell-derived perforin was shown to mediate cytolytic killing of Y. pseudotuberculosis-associated cells, resulting in phagocytosis of both bacteria and cells by neighbouring phagocytes (Bergman et al., 2009). CD8+

T-cells were also shown to play a protective role during infection with S. pneumoniae serotype 3, a serotype not covered by the pneumococcal vaccine. This study demonstrated that CD8 deficient mice succumbed to infection more readily than their immunocompetent counterparts. This effect was associated with increased bacterial burden and inflammatory infiltrate to infected lungs. Using perforin-deficient and IFN-γ deficient mice the authors concluded that CD8+ T-cell protection was mediated by the effects of these molecules (Weber et al., 2011). These studies demonstrate a previously unexpected role for CD8+ T-cells in protection against extracellular bacteria. The ability of CD8+ cells to be cross-primed by DCs may be utilized for vaccine design against pathogens with both intracellular and extracellular phases in their lifecycles (Kurts et al., 2010). Enhancing presentation of bacterial antigens to CD8+ T-cells has been shown to increase vaccine efficacy, as immunization with a M. tuberculosis BCG vaccine expressing a mutant gene which inhibited phagolysosome escape improved antigen presentation resulting in better CD8+ T-cell stimulation and protection against tuberculosis infection compared to vaccination with unmodified BCG (Grode et al., 2005). Thus, CD8 T-cells make attractive targets for vaccine design, especially for inducing protection against viruses and intracellular bacteria, and are being successfully targeted in trials for influenza vaccines (Berthoud et al., 2011). In order to successfully target these cells in vaccines against extracellular bacteria, further understanding of the mechanisms involved in targeting antigens for cross presentation will need to be elucidated. Innate-like lymphocytes Gamma delta (γδ) T-cells γδT-cells are a distinct subset of T-cells that mature in the thymus like classical αβT-cells, however unlike classical T-cells they express a TCR comprised of a γ and a δ chain. Like αβ TCRs, γδ TCRs are generated through somatic rearrangement of V, D and J gene segments. Individual γδT-cell subsets resident in particular locations, are biased towards the expression of particular TCR V gene segments and in some

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cases express invariant TCRs. Expression of particular Vγ chains and/or Vδ chains can be used to categorize γδ subsets with specific Vγ subsets associated with particular locations and cytokine profiles (O’Brien and Born, 2010). γδT-cells constitute 1–5% of circulating lymphocytes but are present in greater numbers at epithelial surfaces such as the skin or the intestines. However, upon microbial infection in humans γδT-cell numbers have been shown to increase dramatically within the circulation, in some cases reaching more than 50% of peripheral T-cells within days (Morita et al., 2007). γδT-cell expansion was observed in infection with bacteria such as M. tuberculosis (Ito et al., 1992), S. pneumoniae (Raziuddin et al., 1994) and L. monocytogenes ( Jouen-Beades et al., 1997). This physiological expansion is one of the most impressive of any immune cell and indicates that we maybe potentially underestimating the significant role played by γδT-cells in protection against bacterial infection. Like classical adaptive T-cells, human γδTcells are capable of antigen recall whereby they recognize a bacterial antigen upon re-exposure to the organism allowing for a more rapid and efficient immune response (Ryan-Payseur et al., 2012; Shen et al., 2002; Zeng et al., 2012). However, unlike classical T-cells they are capable of recognizing conserved self and foreign non-peptide antigens, such as phosphoantigens (Morita et al., 2007). Phosphoantigens can directly activate γδT-cells but activation is enhanced in the presence of monocytes which most likely help present the phosphoantigen to the γδ TCR (Eberl and Moser, 2009; Wei et al., 2008). γδT-cells can also respond rapidly to cytokine activation, for example in the absence of antigen γδT-cells were shown to secrete IL-17 in response to IL-1 and IL-23 stimulation (Sutton et al., 2009). Recently, antigen activation of γδT-cells in conjunction with inflammatory cytokine stimulation was shown to stimulate increased γδT-cell activation compared to either stimulus alone, suggesting that γδT-cells are capable of synergising both activation mechanisms enabling increased effector function (Zeng et al., 2012). γδT-cells are also capable of responding to pathogen products directly via expression of their PRRs and environmental signals via their Ahr (Martin et al., 2009). Additionally, γδT-cells

differ from classical T-cells in that they exhibit a pre-activated phenotype, with high expression of the activation marker CD44 and the chemokine receptors CCR5 and CCR6 (Haas et al., 2009; Kabelitz and Wesch, 2003). This expression profile allows for rapid recruitment and induction of effector functions upon stimulation. Vγ2Vδ2 are the predominant γδ subset in human blood and these cells recognize phosphoantigen metabolites from isoprenoid synthesis pathways. The most potent γδT-cell activating phosphoantigen documented thus far is hydroxymethyl-but-2-enyl-pryophosphate (HMBPP) which is an intermediate metabolite of the nonmelavonate pathway of cholesterol synthesis (Morita et al., 2007). HMBPP molecules are produced exclusively by prokaryotes e.g. E. coli, L. monocytogenes and M. tuberculosis (Begley et al., 2004; Hintz et al., 2001). Vγ2Vδ2 cells are also activated by isopentenyl pyrophosphate (IPP) an intermediate of the melavonate pathway conserved in both prokaryotes and eukaryotes. Thus, IPP represents a self phosphoantigen induced under conditions of stress (Morita et al., 1995). In vitro phosphoantigen activated γδT-cells are capable of producing IFNγ, TNFα and of lysing infected cells via the secretion of perforin (Ali et al., 2007; Dieli et al., 2001; Eberl et al., 2009; Martino et al., 2007; Shao et al., 2009; Shen et al., 2002; Troye-Blomberg et al., 1999). In vivo Vγ2Vδ2 cells specifically undergo major expansion during bacterial infections, e.g. M. tuberculosis (Ito et al., 1992), S. pneumoniae (Raziuddin et al., 1994) and L. monocytogenes ( Jouen-Beades et al., 1997). In murine models, γδT-cells have been shown to display an innate-like phenotype which is associated with their abilities to rapidly produce IL-17 upon stimulation, or an antigen experienced adaptive-like phenotype associated with IFN-γ production (Ribot et al., 2010). These individual phenotypes can be distinguished by expression of the surface marker CD27. IFN-γ producing cells express CD27 whereas IL-17 producing γδT-cells do not (Ribot et al., 2009). In mice, systemic γδTcells are generally thought to be biased towards an IFNγ-producing phenotype and mucosal or peripheral γδT-cells predisposed to IL-17 production potentially due to interactions with

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commensal bacteria at these locations (Duan et al., 2010). IL-17 producing γδT-cell are particularly important for host infection against both extracellular bacterial infections such as S. aureus (Cheng et al., 2012; Cho et al., 2010) and E. coli (Mokuno et al., 2000; Shibata et al., 2007; Tagawa et al., 2004), as well as intracellular infections with M. tuberculosis and L. Monocytogenes (Dalton et al., 2003; Hamada et al., 2008). Mice deficient in total γδT-cells, or particular subsets of γδT-cells were shown to succumb to bacterial infections more rapidly than their wild-type counterparts (Cheng et al., 2012; Cho et al., 2010; Tagawa et al., 2004). This decrease in bacterial clearance correlated with a decrease in neutrophil influx to the infection site. γδT-cell derived IFN-γ has been shown to have an important role in anti-tumour immunity (Gao et al., 2003), malarial infection (Ribot et al., 2009) and fungal infection in immune-compromised individuals (Fenoglio et al., 2009). IFN-γ producing γδT-cells also play a protective role against viruses, such as coxsackeivirus B3 (Huber et al., 2002; Huber et al., 2000), west nile virus (Wang et al., 2003), influenza (Qin et al., 2011) and human cytomegalovirus (Couzi et al., 2012). Thus, it has been proposed that IL-17 producing γδT-cells are Th17-like, representing essential components of mucosal immunity against extracellular bacterial infection, whereas IFN-γ producing γδT-cells are Th1-like playing important roles in protection against intracellular infection and tumours. Both these subsets of γδT-cells represent novel targets for vaccine design as a consequence of their T-helper celllike functions early on in the immune response. Not only do γδT-cells have T-helper cell-like characteristics (in terms of cytokine production) but they also exhibit some CTL-like functions in that they are capable of directly killing infected or transformed cells through engagement of death inducing receptors such as FAS and the release of cytotoxic effector molecules such as perforin and granzymes (Dieli et al., 2001; Qin et al., 2009). In addition, γδT-cells can produce bacteriostatic or lytic molecules such as granulysin and defensins directly contributing to bacterial clearance (Dieli et al., 2001; Dudal et al., 2006). The cytotoxic capacity of γδT-cells could potentially be

harnessed in vaccine strategies against intracellular bacteria and are currently being explored as an immunotherapy for cancer treatment (reviewed by Fournié et al., 2013). γδT-cells encompass a unique niche within the immune system due to their potent multi-effector functions, their capacity to recognize distinct phosphoantigens and their prime location at mucosal sites, all of which highlight their critical role in anti-bacterial immunity. As previously mentioned γδT-cells are currently being targeted in novel anti-cancer immunotherapies in which γδT-cells are activated in vivo by administration of phosphoantigens and IL-2 or autologous γδTcells are harvested and activated in vitro using the same molecules prior to being adoptively transferred back into the patient (reviewed by Fournié et al., 2013). The drugs zoledronate or pamidronate are also being used to initiate the accumulation of intracellular IPP resulting in potent activation of Vγ2Vδ2 cells. Kobayashi and colleagues demonstrated that activation and expansion of reactive Vγ2Vδ2 cells ex vivo followed by adoptive transfer into a patient with metastatic renal cell carcinoma, who then received doses of IL-2 and zoledronate resulted in complete remission of the tumour. This effect was associated with a sharp increase in IFN-γ production by the patient’s Vγ2Vδ2 cells (Kobayashi et al., 2010). Vaccines targeting γδT-cells have also shown to be efficacious during West Nile virus infection. In murine models, administration of active hexose correlated compound, a fungal extract rich in α-glucans which promotes γδT-cell expansion, attenuated viraemia and mortality following lethal infection due to induction of West Nile virus-specific IgG and IgM and the specific expansion of Vγ1.1+ cells (Wang et al., 2009). Furthermore, treatment with HMBPP and IL-2 resulted in prolonged accumulation of γδT-cells in the lungs of macaques and correlated with ameliorated lung lesion formation upon infection with Yersinia pestis (Huang et al., 2009). In order to fully harness the powerful effects of γδT-cells for anti-bacterial vaccines, novel phosphoantigens appropriate for use in vaccines will need to be identified as well as establishing how polarizing cytokines contribute to activation of specific γδTcell subsets.

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Natural killer T (NKT) cells NKT cells are a heterogeneous group of T-cells that share properties of both T-cells and NK cells. NKT cells were originally named because of their co-expression of a αβTCR along with typical NK cell surface receptors, such as NK1.1 (Godfrey et al., 2004). This co-expression reflects the hybrid nature of NKT cells which like γδT-cells are attributed with bridging innate and adaptive immunity. Like innate cells NKT cells exist in a poised effector state (Stetson et al., 2003) and can swiftly respond to infection through rapid cytokine secretion and cell lysis, without the need for extensive cell division and differentiation. However, like conventional T-cells they are also capable of responding to antigen presentation. NKT cells in particular recognize lipid antigens, such as glycolipids, presented by CD1d molecules on APCs. CD1d molecules have a deeper, narrower and more hydrophobic antigen binding groove than their MHC-encoded counterparts and thus are tailored for lipid presentation (Blomqvist et al., 2009; Brigl and Brenner, 2004). The best known subset of CD1d-dependent NKT cells expresses an invariant TCR-α chain and are referred to as type I or invariant NKT cells (iNKT) cells (Godfrey et al., 2004). Mature iNKT cells are widely distributed and can be found in the blood, gastrointestinal tract, liver, lymph nodes and spleen (Gumperz et al., 2002; Matsuda et al., 2000). These cells are conserved between humans and mice and are implicated in many immunological processes. In the absence of microbial glycolipid iNKT cells can be indirectly activated via cytokines (Nagarajan and Kronenberg, 2007), endogenous stress ligands (Mallevaey et al., 2006) or a combination of the two (Brigl et al., 2003; Mattner et al., 2005), initiating a rapid response to a wide range of pathogens. iNKT cells can also be activated directly through the interactions of their TCR with lipid antigen presented by CD1d molecules. A number of lipid antigens expressed by bacterial species such as S. pneumoniae (Kinjo et al., 2011), and Mycobacteria spp. (Fischer et al., 2004) have already been identified as potent NKT cells antigens. It also important to note that ligation of the NK cell-like receptors expressed by iNKT cells, such as NK1.1 (a receptor with no known ligand)

and the stress receptor NKG2-D, may also contribute to iNKT cell activation (Arase et al., 1996; Kuylenstierna et al., 2011). Rapid cytokine production is a major outcome of NKT cell activation. The eventual functionality of an iNKT cell is likely to be determined by the same master regulators that control Th cell differentiation, although unlike Th cells iNKT cells appear to acquire their effector identities during thymic development rather than in the periphery (Watarai et al., 2012). Th1-like iNKT cells which produce IFNγ, are predominantly NK1.1+ and require IL-15 for development. Th2-like iNKT cells are identified by their expression of IL-17RB and CD4 and once activated can produce IL-4, IL-9, IL-10 and IL-13. These cells can be activated by IL-25 and are enriched in the lungs (Terashima et al., 2008; Watarai et al., 2012). Th17-like iNKT cells produce IL-17A and are mainly contained within the CD4−NK1.1− subpopulation of iNKT cells. Like Th17 cells these iNKT cells express CCR6, IL-23R and require RORγt for development. These iNKT cells are enriched in the peripheral lymph nodes, lungs and skin (Coquet et al., 2008; Michel et al., 2007, 2008; Rachitskaya et al., 2008; Watarai et al., 2012). In the lungs these Th17-like iNKT cells are capable of responding to microbial infection (Michel et al., 2007). iNKT cells play a critical protective role during numerous bacterial infections. During pulmonary S. pneumoniae infection, activated iNKT cells, recruit neutrophils to the lung through secretion of CXCL2 and IL-17A (Kawakami et al., 2003; Watarai et al., 2012) and activate macrophages through IFN-γ secretion leading to enhanced microbial clearance (Nieuwenhuis et al., 2002). Furthermore, mice deficient in iNKT cells displayed increased streptococcal burden and impaired neutrophil responses compared to their wild-type counterparts (Kawakami et al., 2003). During pulmonary infection with P. aeruginosa Cd1d–/– mice or mice treated with anti-CD1d antibodies had decreased levels of bacterial clearance, this was again associated with decreased neutrophil recruitment to the lungs (Nieuwenhuis et al., 2002). iNKT cells were also shown to be important during M. tuberculosis infection where they recognized infected macrophages, produced IFN-γ and in turn facilitated the killing

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of the intracellular bacteria. Furthermore, adoptive transfer of iNKT cells protected mice against subsequent M. tuberculosis infection (Sada-Ovalle et al., 2008). The above studies highlight the critical role played by iNKT cells during bacterial infections through their ability to recognize novel lipid antigens, resulting in rapid cytokine production leading to swift recruitment and activation of effector phagocytes. To date relatively little is known about how human iNKT cells contribute to bacterial clearance. If iNKT cells are important, however, it could be possible to incorporate glycolipids that activate iNKT cells into vaccines, which was shown to enhance the efficacy of a vaccine against tuberculosis. The authors demonstrated that the incorporation of glycolipids into the BCG vaccine (licensed vaccine for M. tuberculosis) mediated enhanced protection against infection compared to unmodified BCG. This effect was associated with robust NKT cell responses, enhanced DC maturation and augmented priming of CD8+ T-cells (Venkataswamy et al., 2009). Further investigation into the types of microbes that produce CD1d restricted antigens is required to inform vaccine development. As CD1d molecules are not polymorphic, and iNKT cells usually respond similarly in different individuals, iNKT cell activation represents an attractive target for vaccine design as the potential variability of responses between individuals may be limited. In addition, activated iNKT cells are capable of modulating DC functions promoting adaptive MHC restricted T-cell responses (Fujii et al., 2003; Hermans et al., 2003), this represents a mechanism whereby activated iNKT cells could also feedback into classical adaptive immunity further amplifying anti-bacterial immune responses. Targeting cellular immunity for novel anti-bacterial vaccine design Vaccines are one of the most effective and sustainable ways of preventing infectious disease. However, whilst traditional vaccination strategies have been pivotal in controlling or eradicating many serious diseases (Table 9.1), these conventional methods are not without limitations. These

limitations include failure to provide protection against; antigenically hypervariable pathogens, opportunistic pathogens, rapidly evolving antimicrobial resistant pathogens and non-cultivatable pathogens. The design of new vaccines specifically targeting cellular immunity in addition to humoral immunity could theoretically solve these issues. Furthermore the incorporation of elements targeting cellular immunity in currently licensed vaccines could potentially improve their efficacy. New vaccines with limitations Advances in technology has allowed for the development of new vaccines against bacterial pathogens for which traditional immunization strategies were ineffective. Conjugate vaccines have played a major role in the last decade in providing protection against a number of bacterial infections e.g. H. influenzae type b, N. meningitidis and S. pneumoniae that are particularly critical in children. S. pneumoniae is a Gram-positive coccal bacterium that can cause bacterial pneumonia as well as bacteraemia, sepsis and meningitis. Pneumococci colonize the nasopharynx of approximately 50% of children and 2.5% of adults (De Lencastre et al., 1999). In developing countries 10–20% of all childhood deaths are caused by pneumoncocci, with up to 1 million children dying worldwide of pneumococcal disease each year. S. pneumoniae is an encapsulated bacterium and the capsule contributes to overall virulence by protecting the bacterium from phagocytosis. Ninety-three different capsular types (i.e. serotypes) have been identified so far. Conjugate vaccines are currently licensed against S. pnuemoniae for use in children under 2 years of age which induce serotype specific antibodies against the polysaccharide capsules expressed by epidemiologically important serotypes of S. pneumoniae. The first conjugate vaccine was a heptavalent vaccine consisting of 7 different S. pneumoniae capsular polysaccharides conjugated to the diphtheria toxoid protein CRM197. Polysaccharides normally have low immunogenicity therefore conjugation of these antigens to highly immunogenic cross reactive material enhances their capacity to induce immunity. The vaccine has now been improved by adding a

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further 6 capsular polysaccharide serotypes thus providing expanded protection (Gruber et al., 2012), Despite the successes of the pneumococcal conjugate vaccines, serotypes not covered by the vaccine are slowly beginning to colonize the vaccinated population through selective pressure, replacing the prevalent pneumococci strains (Kyaw et al., 2006). In the long term, it is therefore likely that protection against existing serotypes provided by the current vaccines may have to be replaced with new vaccines that contain polysaccharide antigens from new serotypes emerging in the target population. Numerous studies have been published demonstrating the importance of cellular responses in protection against S. pneumoniae infections. NKT cells (Kawakami et al., 2003), CD8 T-cells (Weber et al., 2011) and Th17 cells (Moffitt et al., 2011) have all been identified to play protective roles during natural S. pneumoniae infection. Murine studies have demonstrated that acquired immunity to pneumococcal colonization, a risk factor of infection, is antibody independent and CD4+ T-cell dependent (Malley et al., 2005; Trzcinski et al., 2005). These studies highlight that CD4+ T-cells are important for preventing colonization suggesting they play an important role in promoting anti-pneumococcal immune responses resulting in bacterial clearance. In fact, clinical data has shown that patients with HIES who have impaired Th17 responses, are also highly susceptible to S. pneumoniae infections clearly highlighting the protective role of Th17 cells in this disease (Milner et al., 2008). Current efforts are under way to develop an anti-S. pneumoniae vaccine based on non-capsular antigens, thus increasing the chances of providing cross-serotype protection, through the induction/activation of Th17 cells. Moffitt et al. (2011) identified antigens recognized by Th17 cells derived from mice immune to pneumococcal colonization using a gene expression library. A selection of antigens identified by the screen, were then used to intranasally immunize mice. Upon challenge with live pneumococci, vaccinated mice displayed significantly lower levels of nasal colonization than control groups. Both depletion of CD4+ T-cells or neutralization of IL-17A was found to eliminate this protection (Moffitt et al.,

2011). Induction of adaptive cellular responses (to complement induction of opsonizing antibodies) which recognize novel conserved antigens that are serotype independent could potentially generate broad-spectrum protection against multiple serotypes, thereby circumventing the problem of serotype replacement in the vaccinated population. Established vaccines with decreasing immunogenicity Some established vaccines have recently been associated with decreasing efficacy. A clear example of this is the acellular pertussis vaccine. B. pertussis is a Gram-negative bacterium that causes whooping cough, a severe respiratory tract infection. Almost 200,000 children die annually from this disease, mainly in developing countries (Black et al., 2010). Despite worldwide vaccination programmes, epidemiological evidence suggests that the incidence of pertussis is increasing in many developed countries (Black et al., 2010; Cherry, 2010). A whole-cell vaccine against the bacterium was introduced in the 1950s resulting in a significant reduction in the incidences of pertussis. Concerns over the safety of this vaccine (side effects included febrile seizures, high fevers, and even fainting) led to the development of an acellular vaccine composed of purified antigens (pertussis toxin, filamentous haemagglutinin, pertactin, fimbrial-2 and fimbrial-3) administered in combination with the adjuvant alum. This acellular vaccine had a reduced efficacy compared to the whole cell vaccine (Greco et al., 1996; Simondon et al., 1997), however due to the reduced safety concerns associated with this vaccine it was licensed for widespread use. Further studies revealed that the two vaccines were eliciting two different types of cellular immunity with the cellular vaccine inducing more protective Th1 responses whereas the acellular vaccine induced primarily Th2 responses (Ausiello et al., 1997; Redhead et al., 1993; Ryan et al., 1998). Recently, evidence emerged that protection mediated by the whole-cell vaccine is actually as a result of synergy between both Th1 and Th17 cellular responses, whereas the acellular vaccine in contrast elicits Th2 and Th17 cellular responses.

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In fact these studies revealed that Th2 responses were dispensable during whole-cell induced protection against pertussis infection. The authors postulated that the decreased efficacy of the acellular vaccine directly resulted from a lack of a Th1 response. Alterations to the adjuvant used was sufficient to correct this, with mice receiving acellular vaccine adjuvanted with CpG (which promoted a Th1 response) generating protective immunity akin to mice vaccinated with the wholecell vaccine (Ross et al., 2013b). Thus, it appears that during B. pertussis infection, inducing synergy between Th1 and Th17 cellular responses generates more efficient prophylactic protection than driving Th2 and Th17 responses. This highlights not only the importance of both cellular and humoral immunity for vaccine induced protection against B. pertussis, but also the importance of driving the ‘correct type’ of cellular immunity to induce full prophylactic protection against the target bacterium. Antibiotic resistant bacteria with no available vaccines Owing to the reliance on antibiotics for the treatment of infections caused by bacteria with no available vaccines, antibiotic resistance among these bacterial strains has become rampant over the last number of years. Initially in hospitals and healthcare-associated settings, but now more worryingly in the community (Deleo et al., 2010). S. aureus is one of the leading causes of nosocomial infections worldwide and the treatment of staphylococcal infections is becomingly increasingly difficult due to the prevalence of strains that are resistant to methicillin (antibiotic of choice), known as MRSA. The MRSA epidemic has become an increasing problem in Western Europe and North America. In the USA, a staggering statistic has been reported that invasive hospital acquired (HA)-MRSA infections result in more deaths annually than HIV/AIDS, viral hepatitis and influenza combined (Boucher and Corey, 2008; Klevens et al., 2007). Of growing concern is the emergence of MRSA infections in the community (community acquired (CA)-MRSA)) in young, immunocompetent individuals who have no healthcare-associated risk factors (Deleo et al., 2010). Recently, S. aureus strains resistant

to vancomycin (Hiramatsu, 2001; Weigel et al., 2003), linezolid (Sánchez García et al., 2010) and daptomycin (Marty et al., 2006), the last viable treatment options for severe MRSA infections, have been reported, revealing S. aureus’s rapid ability to become resistant to even the newest forms of antibiotics. Alternative approaches to standard antibiotic therapies are therefore urgently required. It is likely that future success in the treatment of staphylococcal infections will rely upon immunomodulatory therapies and/or the development of anti-S. aureus vaccines which would prevent the development of infections in the first place. Recently, substantial efforts have been employed to develop anti-S. aureus vaccines (Spellberg and Daum, 2012). Thus, far however, all passive and active immunization strategies have failed to show efficacy in humans. There are several potential reasons for this, not least of which is a lack of understanding of the correlates of protective immunity against S. aureus infections in humans, which likely relies upon the induction of both cellular and humoral responses. The memory immune response induced as a consequence of invasive S. aureus infection is typified by an elevation of S. aureus specific antibody titres in the serum post infection (Hermos et al., 2010; Verkaik et al., 2010). It is not clear however if this memory response is sufficient to protect against subsequent re-infection as recurrence is often a hallmark of S. aureus pathologies (Daum, 2007; Stryjewski and Chambers, 2008). Furthermore, it appears that the majority of the population already possess antibodies against S. aureus given the prevalence of this organism as a commensal colonizer (Miller et al., 2012; Toshkova et al., 2001; van Belkum et al., 2009; Verkaik et al., 2009). Consequently most people who succumb to invasive S. aureus infection will likely already possess antibodies against this organism, supporting the notion that antibodies alone are not sufficient to protect against subsequent infection. In a recent study it was revealed that anti-S. aureus antibodies may actually neutralize one another’s opsonophagocytic activity when mixed together (Skurnik et al., 2010). The authors postulated that these antibodies could bind together impeding one another’s effector functions. These observations may in part explain the lack of

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effective protection induced by humoral immunity during S. aureus infections in humans. The absolute requirement of humoral immunity for protection against S. aureus infections is further brought into question as murine models demonstrated that T-lymphocyte deficient mice were more susceptible to S. aureus infection that B-lymphocyte deficient mice. These experiments revealed that the adoptive transfer of antigenspecific CD3+ T-cells but not B-cells or serum containing antibodies from mice vaccinated with a model S. aureus vaccine, mediated protection against subsequent S. aureus infection in naive recipient mice (Spellberg et al., 2008). Further studies demonstrated that T-cells, specifically Th1 and Th17 cells, were capable of mediating vaccine induced protection through enhanced macrophage and neutrophil killing (Lin et al., 2009). These studies categorically identify the importance of specific T-cell subsets in conferring protection against systemic S. aureus infection. However, it is important to stipulate the differences in murine studies and humans, a major contrast being the lack of natural staphylococcal colonization in mice, suggesting that mice are naturally more resistant to S. aureus than humans. Human clinical data has revealed however that B-cell or antibody deficiency in patients does not correlate with increased susceptibility to severe S. aureus infections (Rich et al., 2001). In contrast, patients with defects in T-cells (Popovich et al., 2010), particularly Th17 cells (Ma et al., 2008; Milner et al., 2008; Popovich et al., 2010), or those with neutrophil disorders (Song et al., 2011) are at increased risk of developing staphylococcal infections. These studies highlight the importance of effective cellular mucosal immunity in protection against S. aureus infections which likely acts in concert with induced antibody responses, leading to efficient clearance of the bacterium via opsonophagocytic killing. An effective vaccine against S. aureus would ideally target both the cellular and humoral arms of immunity to induce full prophylactic immunity. Improving anti-bacterial vaccination strategies Immunology will play a vital role in the design of next generation vaccines (Fig. 9.4). It is essential

that the immune correlates of protection against natural infection are established for particular diseases before any advances in vaccine development can be made. The development of strategies targeting T-cells should certainly be employed given the diverse roles played by T-cells in protective immunity, i.e. phagocyte control, antibody isotype switching and direct cytotoxic activities. Individual T-cell subsets have powerful abilities to modulate phagocytic cell responses. Phagocytes in turn work in concert with humoral responses to mediate opsonophagocytosis. In fact generating antibody responses alone severely limits their capacity to protect if sufficient phagocytes are not recruited/activated to complement them and facilitate optimal opsonophagocytic killing of the invading bacterium. Therefore it is still unclear if antibody-mediated responses should be the primary goal of vaccination against all bacteria. Rather, the identification of antigenic targets which induce potent T-cell immune responses (that in turn regulate phagocyte responses) capable of responding to a broad array of bacterial strains will likely be advantageous. The selection of antigens which synergise both humoral and cellular immunity in order to induce comprehensive prophylactic protection against bacterial infections is the ultimate goal and will rely upon the identification of novel bacterial antigens or more likely a combination of antigens to achieve this. Vaccination strategies should also strive to target specific T-cell subsets thus tailoring the induced immune response to the specific pathogen, e.g. activation of Th1 and CD8 T-cells for protection against intracellular bacteria or the Th1/Th17 axis for protection against extracellular bacteria. In addition to enhancing the immunogenicity of the antigen, the selection of appropriate adjuvants and identification of novel adjuvants which activate specific innate signalling pathways, has the potential to dictate or direct the nature of the antigen-specific T-cell response. Finally, the development of novel strategies to target innate like lymphocytes, such γδT-cells and iNKT cells may be possible due to these cells’ abilities to recognize distinctive antigens, such as phosphoantigens and lipid antigens. Targeting these cell types could be particularly useful in the design of vaccines to protect against mucosal

Cellular Immunity |  239 VaccineAntigens + Adjuvants Antigens MHC I/II, CD1d

PRR

APC

Peptide Antigen

Cytokines

Antibodies

NKT

Lipid Antigen Phosphoantigen

Y

Y

γδ

Inhibition of virulence factors & toxins

Y

Adjuvant

Infection Site

Invading bacterium

Opsonophagocytic Killing

αβ

PMN

Mϕ

ROS

Phagocyte Activation

IFNγ, IL-17, IL-22 CXC Chemokines, Growth Factors

PMN

Mϕ

Y

RNS

Phagocyte Recruitment

Blood Vessel

Figure 9.4  An ideal vaccine would induce both cellular and humoral immunity. Induction of cellular immunity would allow for the generation of both classic MHC restricted T-cells (i.e. αβT-cells) and non-MHC restricted T-cells (i.e. γδT-cells and NKT cells). Activated T-cells produce proinflammatory cytokines (such as IFNγ, IL-17 and IL-22) the downstream effects of which would promote phagocyte recruitment to the infection site. Upon phagocyte egression to the infection site these cytokines are also capable of enhancing the phagocyte’s bactericidal properties. IL-17 and IL-22 are also capable of inducing AMP production. Induction of humoral immunity would allow for antibody generation, which alone could neutralize virulence factors and toxins, and in the presence of activated phagocytes mediate opsonophagocytic killing of the invading bacterium. Induction of both arms of immunity would therefore facilitate more efficient clearance of bacterial threats.

infections, as γδT-cells and iNKT cells preferentially reside at mucosal sites, where they detect and respond to invading pathogens at the port of entry for many bacteria. Future development of novel anti-bacterial vaccines absolutely relies upon knowledge of the fundamental correlates of natural immune protection against the specific organisms. Precise understanding of what drives ‘appropriate’ cellular and humoral immunity to a specific infection will provide the immunological framework for the design of these vaccines (Kaufmann, 2007; Rappuoli, 2007). It is imperative that the basic biological functions underlying natural immunity to bacterial infections are fully elucidated in order to propel vaccine design into the future and improve global health.

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10

T-cell-inducing Vaccines Sarah Gilbert

Abstract For some infectious pathogens a protective T-cell response as well as or instead of a functional antibody response is necessary to protect against disease. The development of vaccines designed to induce T-cell responses has been hampered by difficulties in finding appropriate technologies for measuring and characterizing

the immune response as well as defining safe and highly immunogenic methods of vaccination. This chapter will review progress in both of these areas, and report on advances in development of vaccines for malaria, HIV, influenza, tuberculosis and cancer that rely on inducing T-cell responses. Key publications are listed in Table 10.1.

Table 10.1 Some key developments in the field of T-cell-inducing vaccines. This list is not intended to be exhaustive, but rather to provide a basis for further reading Technology

Disease

Pre-clinical or Clinical Reference

Lipopeptides

Influenza

Pre-clinical

Deres et al. (1989)

Peptides on VLPs

Influenza (also covers gonadotropin releasing hormone, angiotensin II, S. typhi outer membrane protein D2, CXCR4 receptor, HIV1 Nef)

Pre-clinical

Tissot et al. (2010)

Virosomes carrying peptides

Hepatitis C

Pre-clinical

Amacker et al. (2005)

Ty virus-like particles

HIV

Pre-clinical

Layton et al. (1993)

Recombinant VP2

Porcine circovirus

Pre-clinical

Pan et al. (2008)

DNA vaccine

Influenza

Pre-clinical

Ulmer et al. (1993)

DNA vaccine

Malaria

Clinical

Moorthy et al. (2003b)

DNA vaccine

Influenza

Clinical

Jones et al. (2009)

DNA vaccine with electroporation

HIV-1

Clinical

Vasan et al. (2011)

Recombinant MVA

Influenza

Pre-clinical

Sutter et al. (1994)

Recombinant MVA

Malaria

Clinical

McConkey et al. (2003)

Recombinant adenovirus

Herpes simplex virus

Pre-clinical

Johnson (1991)

Recombinant adenovirus

HIV-1

Clinical

Catanzaro et al. (2006)

Recombinant BCG

HIV-1

Pre-clinical

Chapman et al. (2010)

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Introduction The assessment of immunity to infectious pathogens and of the immune response to vaccination typically relies on the measurement of antibody responses to the pathogen or the vaccine antigens. In some cases, humoral correlates of protection have become the accepted indirect measure of vaccine efficacy, providing easily assessed information on response to vaccination, or of maintenance of that response. For example, a serum titre greater than 10 milli-International Units per millilitre against hepatitis B virus surface antigen is deemed to be protective, and indicative of protective vaccination, and a haemagglutination titre greater than 1:40 has long been accepted as a correlate of protection for seasonal influenza vaccines. This approach to defining protective immunity against viral infections in particular led to an emphasis on designing vaccines that induced antibody responses to particular antigens of the pathogen, and the role of T-cell immunity was largely ignored. In more recent times there has been a greater appreciation of the requirement for T-cell help for the induction and maintenance of antibody responses, as discussed in the preceding chapter, but in addition, it is the aim of some vaccine development programmes to induce cytolytic T-cells capable of recognizing host cells that are infected with an intracellular pathogen and destroying both the cell and the pathogen inside it, thereby stopping the pathogen from spreading any further and aiding recovery from infection. This chapter will focus on the induction of T-cell responses against intracellular pathogens, covering methods for achieving this, approaches to measuring the induced responses, and progress that has been made in both pre-clinical and clinical vaccine research. How can we measure T-cell responses? As for measurements of antibodies, T-cell responses are most frequently measured in blood samples, but may also be detected in solid tissues such as the spleen, liver, or a tumour, or in samples obtained by the lavage of mucosal surfaces. The most frequently used assay to quantitate T-cell responses is the interferon gamma ELISpot assay.

This determines the number of cells within a sample that produce interferon gamma in response to stimulation with a particular antigen, with the results typically presented as spot-forming cells per million peripheral blood mononuclear cells (PBMC). To perform the assay, a 96-well filter plate is first coated with anti-interferon gamma antibodies, then counted PBMCs are added to the wells along with the test antigen, and the plate is incubated, typically for 16–18 hours. During this time, any interferon gamma secreted by the cells in response to the antigen will be captured by antibodies bound to the plate, leaving behind a ‘footprint’ of each cytokine-secreting cell. The cells are then washed off, and the interferon gamma detected with a second antibody, which is conjugated to a reagent to allow colour development in the manner of a sandwich ELISA. Once developed, spots are visible in the well for each interferon gamma secreting cell, and after washing and drying, these may be counted using an automated counter. This assay is relatively simple to perform, and has been successfully used in many different laboratories all over the world. It is therefore frequently used as the main assay for quantitating T-cell response in clinical trials of vaccines. The ‘test antigen’ usually consists of pools of overlapping synthetic peptides spanning the entire vaccine antigen, although single peptides may also be used. Both CD4+ and CD8+ T-cells may secrete interferon gamma, and by depleting either from the PBMCs prior to setting up the ELISpot assay it is possible to determine the source of the cytokine secretion. Dual colour assays may be used to test for production of two different cytokines within the same assay. An alternative approach to assessing T-cell responses (with or without antigen stimulation) is to use flow cytometry, in which cells are stained with multiple fluorescently labelled antibodies, which may then be detected using a flow cytometer. The antibodies to be used are a matter of choice for the investigator, but may include cell surface markers such as CD3, CD4, CD8, or those used to define memory status, such as CD62L and CCR7. If the cells are fixed and permeabilized prior to staining, antibodies may also be used to

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detect intracellular cytokines. The technique has recently been extended by the use of antibodies labelled with transition element isotopes which are then detected by mass spectrometry time of flight (single cell mass cytometry, or CyTOF), increasing the number of markers that can be analysed simultaneously to more than 30, and potentially up to 100 in the future. Technologies for inducing T-cell responses by vaccination For those researchers engaged in developing vaccines designed to induce a humoral response, the words ‘vaccine’ and ‘antigen’ are often used interchangeably, and the simplest approach to producing a new vaccine is to use a recombinant protein or polysaccharides which are injected with or without an adjuvant to stimulate production of antibodies to that antigen. When a cytolytic T-cell response is desired, it is necessary to consider the means of antigen delivery, since in order to induce a T-cell response, epitopes from the antigen must be presented to T-cells on the surface of an antigen-presenting cell in the context of major histocompatibility (MHC class I molecules. This can either be achieved by vaccinating with peptides encoding minimal T-cell epitopes which may complex with empty MHC class I molecules on the surface of antigenpresenting cells, or by using a vaccine delivery system designed to result in expression of the antigen within the cells of the vaccinee, thereby gaining entry to the host’s antigen processing and presentation pathway. The following sections will cover the most widely used approaches to solving this problem. Peptide vaccines It is possible to define the minimal 8 to 12 amino acid residue T-cell epitopes presented by different MHC class I molecules within any given antigen, produce these peptides synthetically and vaccinate with them. The first hurdle to be overcome is that since humans express many different MHC molecules it is necessary to include many different peptides within the vaccine in order to have a high probability that any individual will respond to at least one of the peptides. If the antigens from

which the epitopes are derived are polymorphic, some epitopes may exist as several variants, which may have little effect on the T-cell response or may entirely abrogate peptide binding and T-cell immune response. In this latter case, this could then lead to the selection of pathogens able to evade the immune response, and so the selection of peptide sequence to use must be made with this in mind. It is also necessary to ensure that the peptides are not homologous to any human protein sequences, since immunization could potentially result in the induction of auto-immunity. Synthetic peptides are themselves weakly immunogenic, and a number of different approaches have therefore been employed to increase immunogenicity. The use of lipopeptides, in which a lipid molecule, such as tripalmitoylS-glycerylcysteinyl-seryl-serine (P3CSS), is conjugated to each peptide, was one of the first approaches to be tested (Deres et al., 1989). Another approach is to incorporate the peptides into a synthetic virus-like particle. Particulate formulations, which present ordered arrays of epitopes to the immune system are inherently more immunogenic than individual peptides or soluble proteins. Conjugating synthetic peptides to a virus-like particle is one way of improving the immunogenicity of peptides ( Jennings and Bachmann, 2008), and peptides may also be incorporated into virosomes, which can be produced using proteins purified from influenza A virus and assembled by an in vitro process, without the incorporation of any genetic material (Moser et al., 2011). A virosomal vaccine against influenza, without the addition of any peptides, was licensed in 1997, and induces humoral responses against the influenza antigens in the vaccines (Herzog et al., 2009). The long history of safe use of this product encourages the use of virosomes carrying peptides, although a different intranasally administered influenza vaccine consisting of a virosome containing heat labile toxin of Escherichia coli as an additional mucosal adjuvant was withdrawn from use following the increased incidence of Bell’s palsy in vaccinees. Virosomes carrying peptides derived from hepatitis C virus, administered by intramuscular injection, have been used to induce cytotoxic and IFN-γ-secreting T-cells (Amacker et al., 2005).

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Virus-like particles Virus-like particles consist of a protein derived from a viral capsid, that when expressed as a recombinant protein will spontaneously assemble into a particle. These particles are taken up by antigen-presenting cells after immunization and processed into peptides by the proteasome, leading to peptide presentation to T-cells. Examples of proteins that have been employed to carry additional antigenic sequences are the TyA protein from the Ty retrotransposon found within Saccharomyces cerevisae (Layton et al., 1996), and the VP2 capsid protein of porcine or canine parvovirus (Feng et al., 2011; Pan et al., 2008). Ty VLPs expressing a minimal T-cell epitope derived from Plasmodium berghei are immunogenic, resulting in a stronger T-cell response than a DNA vaccine expressing the same epitope (Gilbert et al., 1999). A string of 15 defined T-cell epitopes fused to the Ty protein did not affect particle assembly, and immunogenicity of all the epitopes was demonstrated (Gilbert et al., 1997). A recombinant porcine parvovirus VP2 protein expressing a defined CD8+ T-cell epitope from lymphocytic choriomeningitis virus (LCMV) nucleoprotein induced a protective T-cell response against LCMV challenge in mice (Rueda et al., 1999). DNA vaccines DNA vaccines consist of plasmids produced in Escherichia coli from which the expression of the vaccine antigen is controlled by a strong mammalian promoter. They can be taken up by cells around the vaccination site after immunization, resulting in the expression of the vaccine antigen inside the cell, thus leading to the generation of an immune response directed against the vaccine antigen. Intracellular expression of the antigen within antigen-presenting cells leads to antigen processing via the proteasome, and presentation of peptides derived from the antigen on MHC Class I molecules on the cell surface, and thus to T-cell stimulation. Early experiments with DNA vaccines expressing influenza A haemagglutinin in chickens demonstrated that protective antibodies could be induced by DNA vaccination (Robinson et al., 1993), and that in mice, T-cell responses to influenza A nucleoprotein were induced by

DNA vaccination, resulting in partial protection against influenza virus challenge (Ulmer et al., 1993). DNA vaccines are easily produced in a research laboratory, but although they are capable of generating protective immune responses in mice, they are less immunogenic in non-human primates and immunogenicity in clinical trials has been disappointing. Although T-cell responses to the antigens encoded by DNA vaccines can be detected in clinical trials, they are low level responses, and often only a subset of vaccinees make any detectable response (McConkey et al., 2003; Mwau et al., 2004), although vaccine safety was good (Moorthy et al., 2003a). Much research effort has therefore focused on improving the immunogenicity of DNA vaccines. Since they consist of DNA produced in bacteria, the plasmid itself, which is unmethylated, is recognized as non-self by the mammalian innate immune system, resulting in the production of IL6, IL12 and IFN-γ, which has a small adjuvant effect on the response to the encoded antigen (Sato et al., 1996). Optimization of these immunostimulatory sequences had some effect in pre-clinical studies, but has not resulted in vectors with greater immunogenicity in humans. Expression of cytokines from plasmids given with the DNA vaccine has also been tested, without notable improvements in immunogenicity. To date, a veterinary DNA vaccine to protect horses against West Nile disease has been licensed, but only one clinical trial of a DNA vaccine has demonstrated efficacy, which was mediated by humoral, rather than cellular responses ( Jones et al., 2009). This trial used a helium-powered delivery device (the so-called gene-gun) to vaccinate with DNA precipitated onto gold beads. This approach to DNA vaccine delivery has been found to greatly reduce the amount of DNA vaccine required to induce an immune response. A different approach which also allows for considerable reduction of the amount of DNA vaccine required is to base the DNA vaccine on an alphavirus, which results in the amplification of the nucleic acid encoding the vaccine antigen following entry of the DNA vaccine into a cell (Atkins et al., 2008). However, as with the ‘genegun’ approach, although the dose may be reduced, immune responses are not enhanced over those

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obtained by using larger amounts of DNA vaccine delivered by intramuscular injection. Similar results have been obtained by using virosomes to deliver DNA vaccines ( Jamali et al., 2012). More recently, in vivo electroporation has been tested as a means of enhancing DNA vaccine immunogenicity. The DNA vaccine is injected intramuscularly, and electrical stimulation is then applied to the site of injection, increasing the uptake of DNA into cells and the recruitment of immune cells to the site of immunization. In a trial of a DNA vaccine against HIV, responses to the vaccine antigen were increased 70-fold when electroporation was used (Kopycinski et al., 2012; Vasan et al., 2011), as well as improving the breadth and durability or responses. The effects of electroporation demonstrated in this study were dramatic, but the requirement for the use of an electrical stimulation device following vaccination may limit the deployment of this type of vaccination. However, promising results have also been demonstrated in trials of therapeutic cancer vaccines (Low et al., 2009), and research will continue in this area. Poxvirus-vectored vaccines The first vaccine to be used was a live, replicationcompetent poxvirus, capable of causing mild cutaneous infection, and inducing an immune response that could protect against subsequent infection with Variola, the causative agent of smallpox. The virus that we now know as Vaccinia (with more genetic similarity to horsepox than cowpox) was extensively used, eventually resulting in the eradication of smallpox. Adverse events following vaccination were not trivial, and could be fatal in immunocompromised individuals. The immune response to vaccination consisted of production of antibodies that protected against smallpox infection and a T-cell response that was required to limit the Vaccinia infection following vaccination (Gordon et al., 2011). Despite the severity of adverse events following vaccination, the widespread use of Vaccinia provides an extensive amount of safety data, confirming that the virus is not oncogenic, and since the viral DNA replicates in the cytoplasm of the host cell, integration of viral sequences into the host genome does not occur (Verardi et al., 2012).

Recombinant Vaccinia virus may be used to express the antigens of different pathogens, and the large size of the viral genome allows for multiple complete antigen sequences to be incorporated without deleterious effects on vaccine production. Originally produced in the skin of animals, modern cell culture techniques allow for controlled production, and a recombinant Vaccinia rabies vaccine has been used to control rabies in wildlife in Europe and North America. For that application, the ability of the vaccine vector to replicate after oral administration (via bait capsules) has been advantageous (Verardi et al., 2012). However, for prophylactic human vaccination, a reduction in the severity of adverse events is required, and most vaccine development employing poxvirus vectors has therefore focused on replication-deficient derivatives. Modified vaccinia virus Ankara was produced by multiple passages of the replication competent Ankara strain of Vaccinia in primary chicken embryo fibroblasts (Mayr and Munz, 1964). This resulted in the loss of many host range genes, and those involved in modifying host immune responses. MVA is replication-deficient in mammalian cells, with the puzzling exception of BHK cells, a fibroblast cell line originally derived from the kidneys of baby hamsters. However, the virus is still capable of infecting many different mammalian cell types, and the block in viral replication occurs late in the life cycle of the virus after viral gene expression has taken place. This results in a viral vector that can be produced in avian primary cells or cell lines, will infect human cells and express viral and recombinant genes after immunization, but is not capable of causing a disseminated infection and is therefore safe to use even in immunocompromised individuals (Stittelaar et al., 2001). Recombinant MVA vaccines have been used in many clinical trials (Gilbert, 2013). A different approach to attenuation of the vector was taken to produce NYVAC. This replication-deficient poxvirus vector is based on the Copenhagen strain of vaccinia, from which 18 viral genes were deleted (Tartaglia et al., 1992). A number of wild type poxviruses infect avian species, but these viruses are replication-deficient in mammals, and have also been used as vaccine vectors. FP9 is an attenuated fowlpox virus which

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has been used to vaccinate chickens against fowlpox (Laidlaw and Skinner, 2004), TROVAC is also derived from fowlpox (Karaca et al., 2005) and ALVAC is derived from canarypox (Cadoz et al., 1992). With any live viral vector, even if replicationdeficient, immunity to the vector itself may reduce the immunogenicity of recombinant vaccines based on that vector. For MVA vectored vaccines this has not presented a barrier to immunogenicity. Prior smallpox immunization had no effect in trials of either a malaria or cancer vaccine (Cottingham and Carroll, 2013). Repeated administration of an MVA-vectored therapeutic cancer vaccine, used up to 12 times, did result in anti-vector immunity, but with no apparent effect on immunogenicity (Harrop et al., 2010). This may be because poxviruses are capable of infecting many different cell types and do so very rapidly after immunization. Anti-vector antibodies have a very short time in which to exert their effect, and anti-vector T-cells will arrive too late to prevent the expression of recombinant antigen from an early viral promoter inside the infected cell. For poxviruses, it appears that anti-vector immunity is only likely to be important if a replication-competent vector is used, and in most cases a replication-deficient vector is preferred for reasons of safety. Adenovirus-vectored vaccines Adenoviruses can also be genetically manipulated to express recombinant antigens. The viral genome is smaller than that of poxviruses (around 40 kb compared to 200 kb for vaccinia) and only recombinant genomes of up to 42 kb can be packaged into infectious viral particles, but the removal of the E1 and E3 genes from the viral genome not only prevents viral replication following immunization but allow for the insertion of a recombinant antigen coding sequence under the control of a strong promoter, such as the cytomegalovirus (CMV) immediate early promoter. The vaccine is produced in cells which express the E1 gene, and high level expression of the recombinant antigen immediately after immunization, plus persistent very low level expression of the transgene results in a vaccine vector capable of inducing very strong T-cell responses against the recombinant antigen (Bassett et al., 2011). Originally developed as

gene therapy vectors, for which application the immunogenicity of the vector was disadvantageous, replication-deficient adenoviruses have now been employed in many vaccine development programmes. Unlike poxvirus-vectored vaccines, the presence of pre-existing antibodies to adenoviral vectors does appear to have a deleterious effect on the immunogenicity of the vector. Adenovirus serotype 5 has been used in many pre-clinical experiments, but a high proportion of humans have neutralizing antibodies to this common adenovirus serotype, which causes mild respiratory infections in humans. To reduce this problem, one approach has been to use rarer human serotypes such as Ad26 or Ad35, modifying the Ad5 capsid to remove neutralizing antibody epitopes, producing chimeras of different serotypes, or use adenoviruses isolated from non-human primates, to which humans do not have prior immunity (Seregin and Amalfitano, 2009). The use of simian adenoviruses has been particularly successful, and T-cell-inducing vaccines against malaria, influenza and hepatitis C, based on replication-deficient simian adenovirus are now in clinical trials. Bacterial vaccines One of the earliest vaccines to be used, bacillus Calmette–Guérin (BCG) is a live bacterial vaccine that induces T-cell responses against mycobacterial antigens. An attenuated, but replication-competent strain of Mycobacterium bovis, BCG is the most widely used vaccine in the world, typically given very early in life to prevent tuberculosis. As with Vaccinia, in a small number of cases disseminated infections can result from vaccination. It induces a T-cell response, chiefly CD4+, against mycobacterial antigens, and has long been the main weapon available to prevent disease caused by Mycobacterium tuberculosis. However, its efficacy is limited, does not extend beyond childhood, and cannot be improved to any great extent by a second vaccination given in adolescence (Barreto et al., 2011). Recombinant BCG expressing increased amounts of mycobacterial antigens has been produced, with improved protection against M. tuberculosis challenge in guinea pigs (Horwitz and Harth, 2003). Recombinant BCG expressing

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human immunodeficiency virus (HIV antigens) has also been developed (Chapman et al., 2010). For further information about Mycobacterium tuberculosis vaccines, see Chapter 13. Heterologous prime-boost immunization Clearly there are many approaches that can be taken to inducing T-cell responses to epitopes or whole antigens, which have been successfully used at least in pre-clinical studies. However, once it has been demonstrated that it is possible to induce a T-cell response by vaccination, it is necessary to consider the magnitude of the response that is required to protect against the pathogen, and to test whether a vaccination is protective after deliberate infection. The mouse/ Plasmodium berghei model is one example that facilitates this. P. berghei is a pathogen of Grammomys surdaster tree rats, but will also infect laboratory mice, and the complete life cycle from mouse to mosquito and back can be maintained in the laboratory. The pb9 epitope from the circumsporozoite (CSP) antigen is known to be a protective epitope in mice, and passive transfer of a T-cell clone recognizing this epitope was shown to be protective against P. berghei sporozoite challenge (Romero et al., 1989). A variety of vaccines expressing either the minimal epitope or the whole CSP antigen were then tested for induction of T-cell responses in mice, with varying results, but when cytotoxic T-cells were generated they were capable of recognizing the naturally processed epitope as well as the minimal peptide (Allsopp et al., 1996). Some vaccines were then taken forward into P. berghei sporozoite challenge studies of immunized mice. Although DNA vaccines and MVA-vectored vaccines were both clearly immunogenic, neither were protective. Only when responses were primed with DNA and boosted with MVA were the mice protected against infection, and protection correlated with the highest level of T-cell response (Schneider et al., 1998). Priming with Ty virus like particles, as described above, and boosting with MVA (Plebanski et al., 1998), or priming with adenovirus and boosting with MVA (Gilbert et al., 2002) also achieved protection, and similar vaccination

regimens have now been tested with many different antigens. Heterologous prime-boost immunization is far more immunogenic than homologous prime boost immunization. Repeated doses of vaccines, which are clearly immunogenic at a low level after a single dose may result in a small increase in immunogenicity, but DNA prime/MVA boost results in T-cell responses an order of magnitude higher than DNA/DNA or MVA/MVA. However, MVA/DNA is not more immunogenic than MVA alone. The reasons for this are complex. Firstly, at any immunization, any pre-existing memory T-cells will be boosted in preference to priming naive T-cells against any given epitope. A DNA vaccine typically expresses a single antigen, and although may be only weakly immunogenic will result in the generation of memory T-cells specific for that antigen. These memory T-cells will expand when boosted with a viral vector expressing the same antigen, such that even if the response was undetectable after DNA priming, there is a greater response after viral vector boosting than if the viral vector was used alone. However, not all immunogens are efficient at boosting, and this property appears to be confined to the live immunogens such as viral vectors. If a response is primed by a viral vector, memory T-cells to viral antigens as well as the recombinant antigen will be primed, and all of these memory T-cells will then be boosted if the same viral vector is used to boost. However, if a different, unrelated viral vector is used to boost, only the recombinant antigen is present in both the priming and boosting immunizations, with the result that memory T-cells are first primed to this antigen and then expanded, whereas each viral antigen is encountered only once and responses to these remain at low levels. Thus, a heterologous prime/boost regimen may employ DNA/MVA, or TyVLP/MVA, in which only the boosting vector is live, or avipox/ MVA, MVA/adenovirus, in which two distinct live vectors are used, and either may be used to prime or to boost. Clearly this requires the use of two different vaccines to achieve protective levels of T-cell following vaccination, and presents a challenge for the manufacturing of novel vaccines, but in order to achieve the level of T-cell response

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required for protective efficacy it may be necessary to overcome this challenge. Successful clinical development of T-cell vaccines Irradiated sporozoite vaccination is protective against malaria infection in mice and humans. To achieve protection by this method, mosquitoes infected with the relevant Plasmodium species are irradiated prior to allowing the mosquitoes to feed on a human volunteer, or sporozoites are dissected from mosquito salivary glands and used to immunize a mouse. Repeated rounds of immunization are required, but sterile protection against subsequent live sporozoite infection can be achieved, and is mediated by low level T-cell responses against many different antigens. This means of immunization is not one that can be employed on a large scale, but does provide evidence that successful vaccination against P. falciparum infection in humans can be achieved, and the P. berghei mouse model can be employed for pre-clinical testing of T-cell-inducing vaccines. As stated above, heterologous prime/boost immunization was required to achieve protection in the mouse model, and this has now been tested in clinical trials. In the first clinical trials, DNA and MVAvectored vaccines were found to be well tolerated (Moorthy et al., 2003a), immunogenic (McConkey et al., 2003) and partially protective when one antigen was used, but not with a different antigen (Dunachie et al., 2006). Following further pre-clinical development, FP9 prime MVA boost vaccination was shown to be safe, immunogenic and capable of achieving sterile protection in some volunteers after controlled human malaria infection (Webster et al., 2005) but in an efficacy study in a malaria endemic area, no significant protection was demonstrated (Bejon et al., 2006). Simian adenovirus-vectored vaccines have now entered clinical development, demonstrating very high levels of immunogenicity (Sheehy et al., 2012), and are likely to be the focus of ongoing clinical development. A number of clinical trials employing the same approaches to inducing T-cell responses to HIV antigens have also been conducted. In a trial of an

Ad5 vectored vaccine, low level immunogenicity was demonstrated, but protective efficacy was not (Buchbinder et al., 2008). Vaccination with an MVA-vectored vaccine was also immunogenic in both HIV seronegative and seropositive individuals (Howles et al., 2010). More clinical studies employing simian adenovirus-vectored vaccines both alone and in prime/boost combinations are now in progress. For influenza, the problem is not how to prime T-cell responses (since natural exposure to the virus results in memory T-cell responses in all but the youngest children) but how to boost them and maintain the protective effect of T-cells recognizing conserved antigens of influenza over time. Naturally acquired T-cell responses are known to have a protective effect for a relatively short time after recovery from infection (McMichael et al., 1983). Clinical studies of MVA expressing two influenza antigens, NP and M1, have demonstrated an impressive level of boosting of T-cell responses in adults of different ages (Antrobus et al., 2012; Berthoud et al., 2011), and preliminary evidence of vaccine efficacy was demonstrated in a small influenza challenge study (Lillie et al., 2012). Similarly, BCG given early in life is known to prime T-cell response to mycobacterial antigens, but much attention has been paid to boosting responses to at least one of these antigens in an attempt to improve the protective efficacy of BCG. MVA has again proved highly effective in boosting responses, but in an efficacy study there was no significant improvement of BCG prime MVA boost over BCG alone (Tameris et al., 2013). A therapeutic vaccine has now been licensed for the treatment of prostate cancer. Sipuleucel-T (also known as Provenge) is an autologous cellular therapy produced from the patient’s white blood cells collected by leukapheresis and cultured with a recombinant fusion protein of human prostatic acid phosphatase with granulocyte–macrophage colony-stimulating factor prior to returning those cells to the patient (Sims, 2011). Despite the very low level of T-cell response that was induced in vivo, median survival time of the treatment group was increased by 4 months. A key factor in the development of therapeutic vaccines against prostate cancer is that there is a serum biomarker

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which allows patients to be stratified and followed up, thus greatly facilitating the conduct of efficacy studies. Thus, it is likely that further development of prostate cancer vaccines will lead the field in the testing of novel therapeutic cancer vaccines, resulting in innovative strategies that will then be transferred to other types of cancer, in the same way that the relative ease by which malaria vaccines may be tested for efficacy has led to successful approaches to vaccination being applied against other infectious diseases. Thus, although the first vaccine to be used exerted its protective effect partly through a T-cell response in the vaccinees, in the twenty-first century there is still much for us to learn about how to vaccinate to induce protective T-cell responses. What we have learned is that only a limited amount of what works well in animal models can be translated into clinical trials with any degree of success. In order to make progress, we will need a continued focus on clinical research, and must seek to transfer information between the fields of infectious disease research and cancer therapy. References

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focus on recombinant adenovirus vectors. Expert Rev. Vaccines 10, 1307–1319. Bejon, P., Mwacharo, J., Kai, O., Mwangi, T., Milligan, P., Todryk, S., Keating, S., Lang, T., Lowe, B., Gikonyo, C., et al. (2006). A phase 2b randomised trial of the candidate malaria vaccines FP9 ME-TRAP and MVA ME-TRAP among children in Kenya. PLoS Clin. Trials 1, e29. Berthoud, T.K., Hamill, M., Lillie, P.J., Hwenda, L., Collins, K.A., Ewer, K.J., Milicic, A., Poyntz, H.C., Lambe, T., Fletcher, H.A., et al. (2011). Potent CD8+ T-cell immunogenicity in humans of a novel heterosubtypic influenza A vaccine, MVA-NP+M1. Clin. Infect. Dis. 52, 1–7. Buchbinder, S.P., Mehrotra, D.V., Duerr, A., Fitzgerald, D.W., Mogg, R., Li, D., Gilbert, P.B., Lama, J.R., Marmor, M., Del Rio, C., et al. (2008). Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial. Lancet 372, 1881–1893. Cadoz, M., Strady, A., Meignier, B., Taylor, J., Tartaglia, J., Paoletti, E., and Plotkin, S. (1992). Immunisation with canarypox virus expressing rabies glycoprotein. Lancet 339, 1429–1432. Catanzaro, A.T., Koup, R.A., Roederer, M., Bailer, R.T., Enama, M.E., Moodie, Z., Gu, L., Martin, J.E., Novik, L., Chakrabarti, B.K., et al. (2006). Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 candidate vaccine delivered by a replication-defective recombinant adenovirus vector. J. Infect. Dis. 194, 1638–1649. Chapman, R., Chege, G., Shephard, E., Stutz, H., and Williamson, A.L. (2010). Recombinant Mycobacterium bovis BCG as an HIV vaccine vector. Curr. HIV Res. 8, 282–298. Cottingham, M.G., and Carroll, M.W. (2013). Recombinant MVA vaccines: dispelling the myths. Vaccine 31, 4247–4251. Deres, K., Schild, H., Wiesmuller, K.H., Jung, G., and Rammensee, H.G. (1989). In vivo priming of virusspecific cytotoxic T lymphocytes with synthetic lipopeptide vaccine. Nature 342, 561–564. Dunachie, S.J., Walther, M., Epstein, J.E., Keating, S., Berthoud, T., Andrews, L., Andersen, R.F., Bejon, P., Goonetilleke, N., Poulton, I., et al. (2006). A DNA prime-modified vaccinia virus ankara boost vaccine encoding thrombospondin-related adhesion protein but not circumsporozoite protein partially protects healthy malaria-naive adults against Plasmodium falciparum sporozoite challenge. Infect. Immun. 74, 5933–5942. Feng, H., Liang, M., Wang, H.L., Zhang, T., Zhao, P.S., Shen, X.J., Zhang, R.Z., Hu, G.Q., Gao, Y.Q., Wang, C.Y., et al. (2011). Recombinant canine parvoviruslike particles express foreign epitopes in silkworm pupae. Vet. Microbiol. 154, 49–57. Gilbert, S.C. (2013). Clinical development of Modified Vaccinia virus Ankara vaccines. Vaccine 31, 4241–4246. Gilbert, S.C., Plebanski, M., Harris, S.J., Allsopp, C.E., Thomas, R., Layton, G.T., and Hill, A.V. (1997). A

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influenza virus H5 gene (TROVAC AIV-H5) in cats. Clin. Diagn. Lab. Immunol. 12, 1340–1342. Kopycinski, J., Cheeseman, H., Ashraf, A., Gill, D., Hayes, P., Hannaman, D., Gilmour, J., Cox, J.H., and Vasan, S. (2012). A DNA-based candidate HIV vaccine delivered via in vivo electroporation induces CD4 responses towards the α4β7-binding V2 loop of HIV gp120 in healthy volunteers. Clin. Vaccine Immunol. 19, 1557–1559. Laidlaw, S.M., and Skinner, M.A. (2004). Comparison of the genome sequence of FP9, an attenuated, tissue culture-adapted European strain of Fowlpox virus, with those of virulent American and European viruses. J. Gen. Virol. 85, 305–322. Layton, G.T., Harris, S.J., Gearing, A.J., Hill Perkins, M., Cole, J.S., Griffiths, J.C., Burns, N.R., Kingsman, A.J., and Adams, S.E. (1993). Induction of HIVspecific cytotoxic T lymphocytes in vivo with hybrid HIV-1 V3:Ty-virus-like particles. J. Immunol. 151, 1097–1107. Layton, G.T., Harris, S.J., Myhan, J., West, D., Gotch, F., Hill Perkins, M., Cole, J.S., Meyers, N., Woodrow, S., French, T.J., et al. (1996). Induction of single and dual cytotoxic T-lymphocyte responses to viral proteins in mice using recombinant hybrid Ty-virus-like particles. Immunology 87, 171–178. Lillie, P.J., Berthoud, T.K., Powell, T.J., Lambe, T., Mullarkey, C., Spencer, A.J., Hamill, M., Peng, Y., Blais, M.E., Duncan, C.J., et al. (2012). Preliminary assessment of the efficacy of a T-cell-based influenza vaccine, MVA-NP+M1, in humans. Clin. Infect. Dis. 55, 19–25. Low, L., Mander, A., McCann, K., Dearnaley, D., Tjelle, T., Mathiesen, I., Stevenson, F., and Ottensmeier, C.H. (2009). DNA vaccination with electroporation induces increased antibody responses in patients with prostate cancer. Hum. Gene Ther. 20, 1269–1278. Mayr, A., and Munz, E. (1964). [Changes in the vaccinia virus through continuing passages in chick embryo fibroblast cultures]. Zentralbl. Bakteriol. Orig. 195, 24–35. McConkey, S.J., Reece, W.H., Moorthy, V.S., Webster, D., Dunachie, S., Butcher, G., Vuola, J.M., Blanchard, T.J., Gothard, P., Watkins, K., et al. (2003). Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat. Med. 9, 729–735. McMichael, A.J., Gotch, F.M., Dongworth, D.W., Clark, A., and Potter, C.W. (1983). Declining T-cell immunity to influenza, 1977–82. Lancet 2, 762–764. Moorthy, V.S., McConkey, S., Roberts, M., Gothard, P., Arulanantham, N., Degano, P., Schneider, J., Hannan, C., Roy, M., Gilbert, S.C., et al. (2003a). Safety of DNA and modified vaccinia virus Ankara vaccines against liver-stage P. falciparum malaria in non-immune volunteers. Vaccine 21, 2004–2011. Moorthy, V.S., Pinder, M., Reece, W.H., Watkins, K., Atabani, S., Hannan, C., Bojang, K., McAdam, K.P., Schneider, J., Gilbert, S., et al. (2003b). Safety and immunogenicity of DNA/modified vaccinia virus Ankara malaria vaccination in African adults. J. Infect. Dis. 188, 1239–1244.

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Exploiting the Mutanome for Personalized Cancer Immunotherapy Ugur Sahin, Sebastian Kreiter, John C. Castle, Martin Löwer, Cedrik M. Britten and Özlem Türeci

Abstract Cancer mutations conceptually represent ideal targets for cancer immunotherapy as they combine a favourable safety due to the lack of their expression in healthy tissues and capability of supreme immunogenicity as they are not affected by central tolerance mechanisms. However, the systematic immunotherapeutic targeting of cancer mutations is hampered so far as 95% of the mutations in a tumour are unique to that single patient and only a small number of mutations are shared between patients. We have recently introduced a personalized immunotherapy approach targeting the spectrum of individual mutations by joining innovations from different technology fields. Next-generation sequencing (NGS) is applied to enable the rapid identification of somatic mutations in individual tumours (the mutanome). Immunoinformatic tools are applied for prioritization of mutated epitopes that are predicted to be highly immunogenic and presented on MHC molecules. RNA-based vaccines encoding multiple mutations can be rapidly and affordably synthesized as custom GMP drug products. Integration of these cutting edge technologies into a clinically applicable process holds the promise of successful targeting of cancer heterogeneity by the multi-epitope approach. Cancer mutations as therapeutic targets Cancer is a primary cause of mortality in industrialized countries, accounting for one in four of all deaths. The high medical need, the limited efficacy of available treatments and increasing costs

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pose a socio-economical challenge and mandate expedited development of innovative therapeutics. The treatment of illness has traditionally been based on the law of averages – what works best for the largest number of patients. However, owing to the molecular heterogeneity in cancer, often less than 25% of treated individuals profit from the approved therapies. Personalized medicine, enabling physicians to tailor patient treatment based upon defined characteristics, combined with knowledge-based targeted therapeutics, is regarded as a potential solution to low efficacies and high costs for innovation in drug development and health systems (Chan and Ginsburg, 2011; Hodgson et al., 2012). An increasing number of compounds targeting cancer mutations such as imatinib (Gleevec) for treatment of BCR/ABL, vemurafenib (Zelboraf) for BRAF V600E and crizotinib (Xalkori) for EML4-ALK mutations show unequivocal clinical benefit. Based on the success of these drugs, the identification of causative ‘driver’ mutations shared by a subpopulation of patients and the subsequent design of small molecule inhibitors against them emerged as a novel blueprint in cancer drug development (Hait and Hambley, 2009). The introduction of next-generation sequencing technologies has revealed that human cancers carry dozens to hundreds of non-synonymous mutations (Shah et al., 2009). Multicentre genome-wide mutation profiling and documentation initiatives have been launched, including The Cancer Genome Association (TCGA) and the International Cancer Genome Consortium (ICGC). As in general the number of mutations varies (Alexandrov et al., 2013), also the number

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of non-synonymous mutations varies between 10s and 100s in different tumour entities (Greenman et al., 2007; Stratton, 2011a; Ding et al., 2010; Parsons et al., 2008; Jones et al., 2008; Totoki et al., 2011; Sjoblom et al., 2006; Wood et al., 2007; Pleasance et al., 2010a,b; Wei et al., 2011; Lee et al., 2010). However, for most cancers only a small number of mutations are shared between two patients with the same tumour type. More 95% of the mutations in a patient`s tumour appear to be unique to that tumour (Stratton, 2011). Moreover, only a fraction of the individual mutations are of biological relevance such that their functional inhibition is detrimental for the tumour cell and thus of therapeutic benefit. Owing to these limitations, only a small fraction of patients are eligible for a classical driver mutation targeting drug approach. Mutanome vaccines – concept and preclinical proof Cancer mutations can lead to changes in the protein sequence (e.g. non-synonymous single nucleotide variations (SNV), indels, fusions, splice site mutations) thereby forming novel immunogenic epitopes recognized by T-cells. From the immunologic perspective, mutations may be particularly potent vaccination targets as they can create neo-antigens that are not subject to central immune tolerance. Indeed, a number of immunogenic cancer mutations have been described in mice and patients. Some mutations are capable of inducing rejection of tumours in mice (Dunn et al., 2004; Matsushita et al., 2012; Vesely and Schreiber, 2013) and others are targets of spontaneously occurring dominant immune responses in patients with malignant melanoma (Lennerz et al., 2005; Wolfel et al., 1995). Importantly, at least in some patients T-cell responses against neo-antigens might become dominant. Also in adoptive T-cell transfer therapies, it was shown that patient derived T-cell lines from clinically responding patients recognized mutated antigens (Tran et al., 2014; Robbins et al., 2013). The recent observation that a melanoma patient clinically responding to anti-CTLA4 treatment developed a strong neo-antigen-specific T-cell response further supports the hypothesis that

those T-cells can be of therapeutic value (van Rooij et al., 2013). Major advances in immunotherapy, specifically in the cancer vaccine field, enable us exploit the individualized mutanome data. Most adult epithelial cancers arise by accumulation of mutations. Only a small fraction of mutations are driver mutations and thus essential for tumour-growth or survival. Within the driver mutations only some are druggable and useful for function-inhibiting targeted approaches. The fraction of immunogenic mutations, in contrast, might be considerably higher. Computational predictions suggest that tumours contain many antigenic mutations (Segal et al., 2008; Srivastava and Srivastava, 2009). Mutated tumour antigens tested have been assessed on the single epitope level and induction of immune responses against mutations by tumour cell vaccines has been confirmed in patients (Lennerz et al., 2005) and as rejection antigens in mouse models (Matsushita et al., 2012). Moreover, recent studies indicate that polyepitopic tumour-specific immune responses might be predictive for therapeutic efficacy and are correlated with complete remissions in autologous patient specific vaccines in advanced melanoma (Andersen et al., 2012). As tumours typically have tens to hundreds of non-synonymous mutations, the mutanome may provide many targets for poly-epitope vaccines for each individual patient. Furthermore, novel drug platforms suitable for the on-demand production of GMP-quality individualized vaccines are emerging, including synthetic peptides (Rammensee and Singh-Jasuja, 2013; Yajima et al., 2005) and RNA (Kreiter et al., 2011b; Schlake et al., 2012). Among the different antigen formats for vaccination, coding messenger RNA (mRNA) is a particularly attractive option (Kreiter et al., 2010). Synthesized mRNA can be engineered to encode multiple types of transcripts, including synthetic poly-epitopic nucleotide sequences and has a favourable safety profile (Kuhn et al., 2011). Further, RNA production is established, rapid and cost efficient. Thus, a personalized therapy concept that combines individualized tumour genome sequencing and on-demand RNA vaccine manufacture to

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create customized drug products is both an exciting new therapeutic vision and is within our grasp (Kreiter et al., 2011b). We tested the feasibility of such an individualized vaccine using the B16F10 murine melanoma model and the C57BL/6 background for (i) establishing a sensitive and accurate method for identifying tumour mutations, (ii) testing the immunogenicity of a representative list of tumour mutations confirmed by Sanger sequencing and (iii) studying the efficacy and antitumoral efficacy of a cancer vaccine stimulating T-cells specific for selected mutations. B16F10 is a highly metastatic clone of a melanoma cell line which spontaneously originated in a C57BL/6 mouse (Fidler, 1973). The B16F10 cell line is one of the most widely used model for experimental cancer therapies. In part due to low MHC expression (Boegel et al., 2013), B16 cells are poorly immunogenic, making B16F10 a model ‘in which treatment is notoriously difficult’ (Overwijk and Restifo, 2001). In contrast to its frequent preclinical application, there was almost no knowledge of the mutations underlying the malignant phenotype of B16F10. To identify the cancer mutanome, covering tumour-specific protein coding mutations, we sequenced and compared the exomes of B16F10 cells and of C57BL/6 wild type mouse cells. We captured and sequenced each exome in triplicate. Moreover, we sequenced the B16F10 transcriptome using RNA-Seq to determine tumour gene expression. We applied existing algorithms for NGS data processing (Li and Durbin, 2009), mutation discovery (Castle et al., 2010; Larson et al., 2012; Li et al., 2009), and gene expression (Langmead et al., 2009). However, the available tools to identify tumour-specific single nucleotide point variations (SNVs) had a poor congruence. A poor analytical performance is not acceptable if data for a diagnostic procedure defining a therapeutic intervention. Therefore, we developed a novel statistical approach incorporating exome sequencing using replicate DNA to accurately identify and prioritize true somatic mutations (Lower et al., 2012). Mutations were discovered in crucial signalling pathways (e.g. RAS/MAPK/ERK and PIEK/AKT), in the DNA repair machinery and

in genes of relevance to oncogenesis (e.g. Alk, Flt1 and Fat1). Within the study we designed a rational approach to select mutations qualifying for immunogenicity testing based on the following criteria: (i) the mutation is present in all B16F10 triplicates and is not detected in all C57BL/6 triplicates, (ii) the detected sequence variation has a high statistical confidence, (iii) the mutation occurs in a protein coding gene that is expressed in B16F10, (iv) the mutation causes a non-synonymous change in the protein sequence (v) the mutated epitope has a high-score for presentation on B16F10 MHC molecules (Lower et al., 2012; Lundegaard et al., 2008). We identified 3570 somatic mutations, including 1392 in transcripts, of which 1266 were in protein coding regions, of which 962 cause non-synonymous protein changes and, of these, 563 occur in an expressed gene based on the RNA-Seq sequencing. Of the 563 expressed non-synonymous mutations, we selected 50 and confirmed all 50 by PCR and Sanger sequencing. The immunogenicity and specificity of these 50 validated mutations were assessed by immunizing mice with long synthesized peptides encoding the mutated epitope. The long peptides were 27 amino acids in length with the mutated or wild-type amino acid positioned centrally. Approximately one-third (16) of the 50 mutant peptides were found to be immunogenic, with 60% (11) of this immunogenic group eliciting immune responses directed preferentially against the mutated sequence as compared to the wild-type sequence. For peptides inducing a strong mutation-specific T-cell response, immune recognition was confirmed by an independent approach that showed that induced, mutationspecific T-cells recognize endogenously processed epitopes. Significantly, the study demonstrated the immunogenicity of novel somatic mutations that are not known to promote a tumour phenotype, thus indicating that, regardless of their function, immunity against these mutations, as long as they are stably expressed in a relevant malignant cell population, may mediate tumour control. Finally, we assessed whether immune responses elicited by immunization with mutations translate into anti-tumoural effects. Prophylactic immunization with 27mer peptides encoding selected mutations

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achieved tumour protection and survival in 40% of the mice, whereas all mice in the control group died within the observation period. In the immunized mice that developed tumours, tumour growth was delayed and median survival increased. In the therapeutic setting, the immunization significantly delayed tumour growth. Clinical translation The B16F10 mutanome vaccination study and similar studies applying the CT26 colon cancer model provided a preclinical proof-of-concept demonstrating that mutation targeting vaccines can elicit potent immunogenicity and confer tumour growth inhibition and control. For clinical translation of this approach we addressed several challenges. The entire process, from patient sample through individualized drug product back to the patient, has to be performed in a controlled fashion. The process has to be rapid and robust and must comply with the regulatory requirements of a controlled drug development process defined by drug development guidelines. Here we discuss the key requirements and challenges for the discovery and selection of the set of mutations to be used for vaccine design in the individual patient, the on-demand manufacturing of the poly-epitopic mutation-based vaccine and the appropriate clinical trial concepts and regulatory approval. Mutanome analyses of clinical samples Identification of cancer mutations is performed by NGS-based exome analyses using DNA extracted from primary and metastatic tumour samples. Genomic analysis of clinical samples within studies requires an ethics approval and patient informed consent explicitly allowing analysis of patient genomes. Auditable standard operating procedures have to be in place for the acquisition, handling, transport and documentation of patient sample material. Once in the biobank, the reception, labelling, processing, storing and tracking of each sample and its derivatives must be done correctly and correctly documented. Ideally, this process is coupled to a laboratory information management system (LIMS) for the collection and documentation of all operational steps and the resultant data (Scholtalbers et al., 2013). In

addition, relevant clinical annotation and outcome information must be securely and privately stored. For the clinical grade NGS process we define input quality criteria for incoming samples, for example amount and quality of DNA and used quality controlled reagents. For analytical steps calibration processes must be defined for each step. The nucleic acid extraction protocols vary depending on the sample and must be optimized for each type of input material. We have established processes for fresh-frozen tissue and blood, and derivatives (RNA and DNA). RNA and DNA extraction and NGS library preparation from formalin-fixed and paraffin-embedded (FFPE) tissues is much more challenging. In many cases the use of FFPE material is required as FFPE is the most commonly used method of tissue preservation. Exome re-sequencing kits are available from different vendors: the protocols, the library preparation steps, the target regions and the data generated differ for each kit. Thus, also for the use of vendor supplied kits, standard operating procedures, effective calibration for each sample type, defined workflows, and reagent and lot number tracking are mandatory. We found that replicate DNA sequencing reduces the number of false positive mutation calls (Lower et al., 2012). Thus, for the clinical protocol we are using replicate DNA tumour and germline sequencing followed by independent mutation confirmation. Genomic positions with predicted mutations can be amplified by PCR using specific primer assays for Sanger sequencing of the tumour and PBMC (germline) samples. As DNA upstream and downstream of the predicted mutation is also determined by the Sanger sequencing, the resultant sequence also allows for verification of assay specificity. Selection of mutations for vaccine design Based on the confirmed mutanome map of a patient’s tumour, mutations are selected for the vaccines which have a higher likelihood of inducing immunotherapeutic tumour control. A mutation to be incorporated into a poly-epitope vaccine should (i) be confirmed by

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an independent sequencing method, (ii) be specific to the tumour sample and not in the patient germline genome, (iii) occur in a protein coding transcript, (iv) cause a change in protein sequence and (v) be expressed in the tumour cells. We determine the tumour gene expression by RNASeq of mRNA obtained from the patient’s tumour. A new field of research is the accurate prediction and the selection of mutations that are capable of inducing a robust T-cell response recognizing the respective mutated epitope. For an immunotherapeutic activity this requires that the mutation-containing epitope is presented on the patient´s MHC molecules. We have developed a bioinformatic approach for determination of HLA haplotypes directly from RNA-Seq data, called seq2HLA (Boegel et al., 2013). In addition, seq2HLA provides information about expression levels of the respective HLA-alleles in the tumour sample. By obtaining the patient HLA haplotype information, MHC binding epitopes can be predicted with algorithms such as those from the Immune Epitope Database (IEDB) (Lundegaard et al., 2008). We analyse the immunogenicity of the computationally predicted epitopes using short-time in vitro T-cell stimulation approaches (Kreiter et al., 2007). The use of the patient´s own lymphocytes in combination with dendritic cells transfected with mutation encoding mRNA in such assays allows identification of mutations that have already induced a spontaneous T-cell response in the tumour-bearing patient. Production of personalized vaccines Once the mutated epitopes for the patient’s individualized vaccine has been selected, a tailored vaccine must be designed and manufactured in a GMP-compliant manner as a small-scale batch specific for the single-patient. In vitro transcribed (IVT) antigen encoding RNA is an attractive format for rapid GMP-compliant manufacturing of vaccines that are tailored to the mutation signature of an individual patient’s tumour (Kreiter et al., 2011b). IVT RNA is easy and fast to synthesize in a GMP-compliant manner. RNA vaccines have an excellent safety profile and do not integrate into the host genome, facilitating regulatory hurdles. RNA-based vaccines have a number of additional

advantages. RNA vaccines (i) exhibit an intrinsic adjuvant activity by stimulating TLR7, TLR8 and TRL3, (ii) can be used to encode multiple epitopes in a single molecule, (iii) are translated in cytoplasm and effectively enter the endogenous antigen processing and presentation machinery, and (iv) are able to elicit potent anti-tumoural immunity (Diken et al., 2011, 2013; Kreiter et al., 2010, 2011a,b; Kuhn et al., 2011). The administration can be done using autologous dendritic cells that are transfected with RNA or by direct in vivo RNA administration. Both intranodal as well as the intradermal injections of RNA are feasible, have demonstrated delivery to DCs and efficacious inducement of T-cells (Diken et al., 2011) and are currently under clinical investigation (Kreiter et al., 2010; Weide et al., 2008). Clinical evaluation of mutanome vaccines Safety is one key aspect requiring particular attention in the clinical translation of novel therapeutic drugs. As the sequence composition of the individualized drug product will be patient-specific, formal pre-clinical toxicity studies are not feasible. Thus, pre-clinical toxicity studies must be designed in accordance with FDA (U.S. Food and Drug Administration), ICH (International Conference on Harmonisation) and CHMP (Committee for Medicinal Products for Human Use) guidelines to test the safety of the approach itself based on a representative process and drug products. For clinical testing of the individualized vaccine approach the entire process from sample acquisition, mutation discovery and target selection, design and production of the vaccine, the vaccine administration and clinical monitoring must be run in the framework of a regulatory-approved clinical trial. Regulatory challenges for the clinical development of individualized mutanome vaccines arise because each patient will receive a tailored vaccine that will be unique and therefore vary between patients. This is different to classical molecularly defined drug products that are invariant in their molecular compositions. This paradigm shift creates a series of challenges for drug developers, physicians and regulators that have been intensively discussed over a period of several years. The CIMT

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regulatory research group (RRG) proposed regulatory principles for clinical development of individualized vaccines. Following a meeting with the Innovation Task Force at the European Medicine Agency, the regulatory agency endorsed the proposed blueprint for clinical development of such innovative new therapies. It was clarified that the development of individualized vaccines differs from the development of existing cancer therapeutics and that the existing EU regulatory framework does not directly address individualized vaccines. On the other side several regulatory principles already in place for the development of cellular and autologous therapeutics may be applicable to the development and testing of recombinant individualized vaccines (Britten et al., 2013). Thus, suitable regulatory principles were identified and are being actively addressed in conjunction with the regulatory authorities, resulting in blueprints that are already published. From clinical safety perspective, a vaccine encoding a mutation–containing peptide might induce a T-cell reaction against the wild-type analogue peptide that is expressed by healthy tissues. Even though cancer vaccines have been demonstrated to be very safe and we have not seen evidence of autoimmunity-associated toxicities in mouse models, toxicity by recognition of the wild type epitope in normal tissues cannot be fully excluded. Further, the target antigens used for the individualized vaccine will differ for every patient, resulting in a variation of potential adverse effects mediated by this toxicity. Therefore, diligent safety monitoring of patients is mandatory and the investigators and clinicians should be provided with information listing in which organs each wild-type gene is highly expressed. The activity of individualized mutanome vaccines can be assessed by clinical, molecular and immunological endpoint assessments throughout the clinical development. Quantifying the frequency, function and phenotype of immune cell subsets following therapy in the peripheral blood, in the skin following the induction of delayed type hypersensitivity (DTH) reactions, and most importantly in the tumour may identify additional biomarkers. Measuring the induction of functional mutation-specific T-cell responses as well as their trafficking to the tumour as determined

by functional immunological readouts and TCR repertoire profiling are a straightforward strategy to achieve the clinical proof of concept in first in human trials. Longitudinal studies with sequential analyses of antigen-specific T-cell frequency and phenotype in blood samples and in tumour biopsies at multiple time points before and after vaccination will be instructive to understand the immunogenicity of each individual mutation. Immunogenicity data generated from such studies can be used to further optimize and refine the target selection for the individualized vaccination approach. Combining the individualized mutanome vaccine approach with potent immunomodulatory therapies such as anti-checkpoint inhibitor antibodies opens additional avenues for further clinical development (Garber, 2013). Whereas the immunomodulatory therapy would release the brakes of the immune system, the individualized vaccine approach would instruct the immune system by providing a tailored target map to maximize anti-tumoural effects. In addition to the regulatory and clinical challenges, the costs of individualized treatments pose a significant development risk. In their early development phase the cost per patient of individualized vaccines will be high, mainly due to costs for genome sequencing and manufacture of small, patient-specific GMP drug product batches. Commercialization of individualized vaccines to reach larger number of patients requires not only evidence for superior clinical efficacy but also affordability of a personalized treatment. We believe that cost-effective delivery of vaccines can be achieved in the future, not only due to decreasing genome sequencing costs but also by full automation and optimization of the manufacturing process and increase of the scale. References Andersen, R.S., Thrue, C.A., Junker, N., Lyngaa, R., Donia, M., Ellebaek, E., Svane, I.M., Schumacher, T.N., Thor Straten, P., and Hadrup, S.R. (2012). Dissection of T-cell antigen specificity in human melanoma. Cancer Res. 72, 1642–1650. Boegel, S., Lower, M., Schafer, M., Bukur, T., de Graaf, J., Boisguerin, V., Tureci, O., Diken, M., Castle, J.C., and Sahin, U. (2013). HLA typing from RNA-Seq sequence reads. Genome Med. 4, 102. Britten, C.M., Singh-Jasuja, H., Flamion, B., Hoos, A., Huber, C., Kallen, K.J., Khleif, S.N., Kreiter, S.,

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Nielsen, M., Rammensee, H.G., et al. (2013). The regulatory landscape for actively personalized cancer immunotherapies. Nat. Biotechnol. 31, 880–882. Castle, J.C., Biery, M., Bouzek, H., Xie, T., Chen, R., Misura, K., Jackson, S., Armour, C.D., Johnson, J.M., Rohl, C.A., and Raymond, C.K. (2010). DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing. BMC Genomics 11, 244. Chan, I.S., and Ginsburg, G.S. (2011). Personalized medicine: progress and promise. Annu. Rev. Genomics Hum. Genet. 12, 217–244. Diken, M., Kreiter, S., Selmi, A., Britten, C.M., Huber, C., Tureci, O., and Sahin, U. (2011). Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation. Gene Ther. 18, 702–708. Diken, M., Kreiter, S., Selmi, A., Tureci, O., and Sahin, U. (2013). Antitumor vaccination with synthetic mRNA: strategies for in vitro and in vivo preclinical studies. Methods Mol. Biol. 969, 235–246. Dunn, G.P., Old, L.J., and Schreiber, R.D. (2004). The three Es of cancer immunoediting. Annu. Rev. Immunol. 22, 329–360. Fidler, I.J. (1973). Selection of successive tumour lines for metastasis. Nat. New Biol. 242, 148–149. Garber, K. (2013). Melanoma combination therapies ward off tumor resistance. Nat. Biotechnol. 31, 666–668. Hait, W.N., and Hambley, T.W. (2009). Targeted cancer therapeutics. Cancer Res. 69, 1263–1267. Hodgson, D.R., Wellings, R., and Harbron, C. (2012). Practical perspectives of personalized healthcare in oncology. N. Biotechnol. 29, 656–664. Kreiter, S., Diken, M., Selmi, A., Diekmann, J., Attig, S., Husemann, Y., Koslowski, M., Huber, C., Tureci, O., and Sahin, U. (2011a). FLT3 ligand enhances the cancer therapeutic potency of naked RNA vaccines. Cancer Res. 71, 6132–6142. Kreiter, S., Diken, M., Selmi, A., Tureci, O., and Sahin, U. (2011b). Tumor vaccination using messenger RNA: prospects of a future therapy. Curr. Opin. Immunol. 23, 399–406. Kreiter, S., Konrad, T., Sester, M., Huber, C., Tureci, O., and Sahin, U. (2007). Simultaneous ex vivo quantification of antigen-specific CD4+ and CD8+ T cell responses using in vitro transcribed RNA. Cancer Immunol. Immunother. 56, 1577–1587. Kreiter, S., Selmi, A., Diken, M., Koslowski, M., Britten, C.M., Huber, C., Tureci, O., and Sahin, U. (2010). Intranodal vaccination with naked antigen-encoding RNA elicits potent prophylactic and therapeutic antitumoral immunity. Cancer Res. 70, 9031–9040. Kuhn, A.N., Diken, M., Kreiter, S., Vallazza, B., Tureci, O., and Sahin, U. (2011). Determinants of intracellular RNA pharmacokinetics: Implications for RNA-based immunotherapeutics. RNA Biol. 8, 35–43. Langmead, B., Trapnell, C., Pop, M., and Salzberg, S.L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25. Larson, D.E., Harris, C.C., Chen, K., Koboldt, D.C., Abbott, T.E., Dooling, D.J., Ley, T.J., Mardis, E.R., Wilson, R.K., and Ding, L. (2012). SomaticSniper:

identification of somatic point mutations in whole genome sequencing data. Bioinformatics 28, 311–317. Lennerz, V., Fatho, M., Gentilini, C., Frye, R.A., Lifke, A., Ferel, D., Wolfel, C., Huber, C., and Wolfel, T. (2005). The response of autologous T cells to a human melanoma is dominated by mutated neoantigens. Proc. Natl. Acad. Sci. U.S.A 102, 16013–16018. Li, H., and Durbin, R. (2009). Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., and Durbin, R. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079. Lower, M., Renard, B.Y., de Graaf, J., Wagner, M., Paret, C., Kneip, C., Tureci, O., Diken, M., Britten, C., Kreiter, S., et al. (2012). Confidence-based somatic mutation evaluation and prioritization. PLoS Comput. Biol. 8, e1002714. Lundegaard, C., Lamberth, K., Harndahl, M., Buus, S., Lund, O., and Nielsen, M. (2008). NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8–11. Nucleic Acids Res. 36, W509–512. Matsushita, H., Vesely, M.D., Koboldt, D.C., Rickert, C.G., Uppaluri, R., Magrini, V.J., Arthur, C.D., White, J.M., Chen, Y.S., Shea, L.K., et al. (2012). Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting. Nature 482, 400–404. Overwijk, W.W., and Restifo, N.P. (2001). B16 as a mouse model for human melanoma. Curr. Protoc. Immunol., Chapter 20, Unit 20.1. Rammensee, H.G., and Singh-Jasuja, H. (2013). HLA ligandome tumor antigen discovery for personalized vaccine approach. Expert Rev. Vaccines 12, 1211–1217. Robbins, P.F., Lu, Y.C., El-Gamil, M., Li, Y.F., Gross, C., Gartner, J., Lin, J.C., Teer, J.K., Cliften, P., Tycksen, E., et al. (2013). Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nat. Med. 19, 747–752. Schlake, T., Thess, A., Fotin-Mleczek, M., and Kallen, K.J. (2012). Developing mRNA-vaccine technologies. RNA Biol. 9, 1319–1330. Scholtalbers, J., Rossler, J., Sorn, P., de Graaf, J., Boisguerin, V., Castle, J., and Sahin, U. (2013). Galaxy LIMS for Next Generation Sequencing. Bioinformatics 29, 1233–1234. Segal, N.H., Parsons, D.W., Peggs, K.S., Velculescu, V., Kinzler, K.W., Vogelstein, B., and Allison, J.P. (2008). Epitope landscape in breast and colorectal cancer. Cancer Res. 68, 889–892. Shah, S.P., Morin, R.D., Khattra, J., Prentice, L., Pugh, T., Burleigh, A., Delaney, A., Gelmon, K., Guliany, R., Senz, J., et al. (2009). Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution. Nature 461, 809–813. Srivastava, N., and Srivastava, P.K. (2009). Modeling the repertoire of true tumor-specific MHC I epitopes in a human tumor. PLoS One 4, e6094. Stratton, M.R. (2011). Exploring the genomes of cancer cells: progress and promise. Science 331, 1553–1558.

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Part II Challenges for the Decade of Vaccines

Malaria Vaccine Development: Progress to Date Philip Bejon, Ally Olotu and Kevin Marsh

Abstract Malaria is a major global health problem with substantial morbidity and mortality. Some progress in reducing this burden has been made with existing malaria control measures and a vaccine would be an important addition. The malaria parasite is complex, presenting several thousand possible antigens expressed in various lifecycle stages. We examine the difficulties in translating the findings from immuno-epidemiology to vaccination strategies, and the difficulties in selecting an appropriate animal model. Despite the apparent complexity of the problem a partially successful vaccination approach has been progressed through to phase III trials and a license application is expected. Furthermore whole parasite vaccination approaches appear highly effective in experimental challenge studies. Further improvements are expected as antigen selection and antigen delivery are optimized with rapid down-selection of approaches using in vitro functional studies, global genetic parasite diversity, appropriately designed and interpreted animal models before proceeding to Controlled Human Malaria Infection studies and field trials. A highly effective malaria vaccine based on a multi-stage, multi-component approach is technically feasible. Introduction Historically malaria has been a major killer throughout the tropics and sub tropics and even into temperate regions. The evidence of its wide ranging historical impact on human mortality is reflected in the prevalence of many human genetic polymorphisms which confer partial protection

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against the disease. Today, despite some successes in control, malaria remains a major cause of ill health and death on a global scale with 2.5 billion exposed, an estimated 500 million cases and up to a million deaths annually (Snow et al., 1999, 2005; Hay et al., 2010; Murray et al., 2012). Malaria is a protozoan parasite with a complex life cycle involving obligatory phases in both vertebrate hosts as well as in insect vectors. The complexity of the organism and the life cycle means that developing vaccines against malaria presents a challenge more akin to developing vaccines against cancer than against ‘simpler’ organisms such as viruses and bacteria which have been the target of the majority of successful human vaccines to date. None the less, there are several reasons to have confidence that it is technically feasible to develop effective vaccines which could play a critical role in the control and eventual elimination of malaria: (a) humans do become immune to malaria, (b) this immunity can be passively transferred in the immunoglobulin fraction of plasma and, (c) prototype vaccines are protective in a range of model systems including human challenge studies and field trials (albeit with only partial efficacy in the latter). In this chapter we first briefly outline salient features of the epidemiology of malaria which have a bearing on the need for and potential role of malaria vaccines. We then go on to consider the points in the malaria life cycle that can potentially be targeted by vaccines and discuss the desired characteristics of an effective vaccine. We briefly outline the various model systems including challenge in humans used to assess potential vaccine candidates before turning to the question

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of assessment of efficacy in the field. We give an overview of vaccine development to date including RTSS (or ‘Mosquirix’), the lead candidate and the only vaccine to have gone into phase III trials. Finally we discuss the way forward and argue that recent developments in high-throughput techniques in biology will revolutionize the field and allow much more rapid development of second generation vaccines. Malaria parasites causing disease in humans Although there are numerous species of malaria parasites infecting a wide range of vertebrates from primates through to birds and lizards, only five species are regularly transmitted to humans. Plasmodium falciparum is by far the major cause of severe disease and death due to malaria. It is endemic in 85 countries throughout the tropics and subtropics but in terms of population burden the greatest number of cases occur in south Asia and Africa, with 90% of the deaths occurring in Africa. Plasmodium vivax is the second most important cause of significant illness and is widely distributed throughout the tropics and subtropics, though absent in much of Africa. It is estimated that there are around 2.5 billion at risk globally (Gething et al., 2012). Two other species, Plasmodium malariae and Plasmodium ovale, are found patchily worldwide overlapping with P. falciparum and P. vivax. Their epidemiology is imperfectly understood and while both can cause clinical disease, this forms only a very small proportion of total cases in any country and is rarely severe (though P. malariae is associated with a poorly understood nephropathy which can be life-threatening (Abdurrahman et al., 1990; Neri et al., 2008). Recently a fifth species, Plasmodium knowlesi, has been described as a significant cause of ill health and death in some parts of south East Asia, particularly Malaysia (William et al., 2013; Rajahram et al., 2013; Cox-Singh et al., 2008). Currently this infection appears to occur as a zoonosis without human to human transmission (Roughton and Green, 2012). The majority of work on vaccine development has focussed on P. falciparum, though there is increasing interest in

both the possibility and the importance of developing vaccines against P. vivax. Approaches to the control of malaria Following the discovery of the malaria parasite in 1897 and of its transmission by mosquitos, early attempts at control focussed on environmental modification, such as drainage schemes in order to achieve breeding site control. The development of insecticides targeted at adult mosquitos, particularly DDT in the 1940s gave great impetus to these efforts. In 1955 WHO declared a policy of global malaria eradication. This is often criticized as a failure because it never incorporated or dealt with malaria transmission in Africa, the heartland of P. falciparum. None the less, the forty years following the second world war saw remarkable successes in eliminating malaria from many countries and these gains have been maintained. Even in Africa over the period of the much criticized global eradication programme there is evidence to suggest that in many parts there were considerable gains in controlling the ill-effects of malaria (Snow et al., 2012, 2013). However, from the late 1980s malaria morbidity and mortality rose considerably, probably mostly related to the development of resistance to chloroquine, a widely used, cheap safe and effective drug (Trape, 2001). By the nineteen nineties it was recognized that malaria in Africa was a disaster and this finally spurred concerted international action reflected in the Abuja declaration by African heads of states (Global Partnership to Roll Back Malaria, 2000), the formation of the Roll Back malaria partnership (Global Partnership to Roll Back Malaria, 2000) and the initiation of the Global Fund for HIV, TB and HIV (Global Fund, 2013b, 2002). Increased international advocacy and collaboration had a remarkable effect on funding for global malaria control, which rose from a few hundred million dollars per annum to over 2 billion dollars per annum in 2011. Control efforts have depended on four key strategies: prompt treatment of disease with effective antimalarials, the use of insecticide impregnated bed nets, protection of pregnant women against malaria and the control of epidemics (WHO, 1993). New

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approaches have also been developed including the use of intermittent presumptive treatment of infants and children (WHO, 2011b) and, more recently, seasonal malaria chemoprophylaxis for all children has been shown to be remarkably effective in areas of highly seasonal malaria like the Sahel (Meremikwu et al., 2012; Wilson and IPTc Taskforce, 2011). There is increasing evidence from many parts of Africa of reductions in both malaria transmission and of malarial disease over recent years. Measuring malaria cases and deaths in Africa is remarkably difficult but the WHO estimates that there has been a fall of between 25 and 47 million cases of malaria from 2000 to 2010 (WHO, 2011a). However, it is important to recognize that this is not the case everywhere: malaria cases are stable or even rising in some parts of Africa (Okiro et al., 2010, 2011; Roca-Feltrer et al., 2012; Jagannathan et al., 2012). Furthermore, although control efforts are critical, in some areas falls in transmission began well before major control efforts for reasons that we do not understand (Okiro et al., 2007). We therefore cannot be sure that these falls will be maintained, and ought not to be complacent. The need for vaccines in malaria control It is sometimes said that we already have the tools to control malaria and the key issue is to ensure that they are used. There is considerable truth in this and morbidity and mortality due to malaria could almost certainly be substantially reduced in all areas by maximal and sustained application of all the currently available effective tools. However, there is a marked difference between reducing malaria to a relatively minor cause of death and eliminating its transmission. Furthermore, gains in malaria control are continually under threat, not simply from potential difficulties in maintaining international funding, but also by the enormous potential of parasites to develop resistance to antimalarials and of vectors to develop resistance to insecticides. Most experts believe that accelerating the elimination of malaria in areas of historically high transmission will require the development of novel interventions.

These could include a range of new approaches including genetic modification of vectors or mass population treatment with safe and long lasting drugs. Historically vaccines are one of the most successful public health interventions and the development of a highly effective vaccine would not only offer a new means of malaria control but would also be the critical tool in allowing us to move to elimination in many areas and eventually global eradication. Vaccine development is a long process which can take between 10 and 20 years, requiring substantial financial investment with no guarantee of a viable product at the end. Diseases of poverty such as malaria carry a limited incentive of future profits. Public–private initiatives play a central role in creating push mechanisms (i.e. ‘up front’ funding of pre-clinical and/or clinical research) and pull mechanisms (e.g. advance market commitments where donor countries have committed funds to purchase vaccines for developing world consumption). Naturally acquired immunity to malaria Humans in populations exposed to stable transmission of malaria do develop significant acquired immunity (Marsh and Kinyanjui, 2006). This is responsible for shaping the epidemiology, including determining who is most at risk of disease and death and determining the clinical picture disease under different levels of transmission (Marsh and Snow, 1999). In endemic areas the burden of severe disease and death disease falls on young children, with older children and adults progressively having less disease. In essence any situation where older individuals show evidence of acquired immunity can be considered ‘stably endemic’. Such conditions can be maintained over a several-log order range of transmission intensity, from around an average of one infected bite per individual per year to thousands of infected bites per individual per year. Thus, naturally acquired immunity for a population is bought at the cost of a high death rate in young children. Furthermore it is never complete in that even adults remain susceptible to infection and occasional symptomatic illness.

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An exception to the idea that adults in endemic areas are relatively protected against malaria occurs in pregnant women, who have higher prevalence of parasitaemia and incidence of clinical episodes and suffer a number of ill effects, most notably severe anaemia (Steketee et al., 2001). This does not appear to be due to a reduction in immunity per se but rather to the presence of a new in vivo ecological niche for the parasite (i.e. the placenta), which may become heavily parasitized leading to the spill over of parasites into the peripheral circulation (Rogerson et al., 2007). Placental infection also has important effects on the fetus and is a major cause of low birthweight (Guyatt and Snow, 2004). The ability of parasites to colonize this new niche is due to the expression of a specific subgroup of variant antigens on the infected red cell surface with the ability to adhere to the syncytio-trophoblast (Fried and Duffy, 1996). Women develop partly effective protective antibody responses to this subgroup of receptors and placental parasite load is progressively less with increasing parity (Duffy and Fried, 2003). These observations provide the basis for attempts to develop specific vaccines to prevent pregnancy related malaria (Staalsoe et al., 2004a). Does understanding naturally acquired immunity help vaccine development? The fact that humans develop significant immunity to malaria under conditions of natural exposure provides support to the effort to develop vaccines. It is clearly the case that natural exposure-induced immunity is slowly acquired and incomplete, and is not what one would ideally want from a vaccine. However, it is arguable that one could identify key immunological responses by measuring the diversity of immunological responses between individuals acquired in the field, and then identifying individuals in the field who most rapidly acquire immunity. Children acquiring immunity in the field do so at very different rates, and after a clinical malaria episode may fail to seroconvert to well characterized malaria antigens, or generate only short-lasting antibody responses. Studies of the heterogeneous development of immunity would be particularly important, in order to

understand the regulation and dysregulation of the immune response to malaria antigens. An alternative and attractive approach is to look for targets and mechanisms to which immunity does not occur naturally, but for which a critical function in the parasite life-cycle can be inferred. A prime example would be targeting sexual stage antigens primarily expressed in the mosquito and therefore never subject to human immune pressure, but where an antibody response may prevent the sexual stage parasites from completing their life-cycle. It could be argued that the RTS,S vaccine is also an example of this ‘Achilles heel’ approach in that although humans in endemic areas do make low-titre anti-circumsporozoite antigen (CS) responses, there is to date no evidence that these are sufficiently well developed to provide any significant protection. Potential targets of immunity Identification of potential immune targets in the vertebrate host can be considered in relation to three phases of the life cycle: the pre-erythrocyte, erythrocytic asexual stages and the pre-erythrocytic stages. At each stage there is a series of obvious potential targets for naturally acquired or vaccine-induced immune responses. Malaria life cycle The malaria life cycle starts with the injection of sporozoite by an infected mosquito into the vertebrate host skin, subcutaneous tissue or blood capillary The sporozoites which find the blood vessel travel to the liver and infect hepatocytes. Each single sporozoite is capable of developing into thousands of merozoites. This early stage is termed the ‘pre-erythrocytic stage’ and it is clinically silent. The rupture of infected hepatocytes and release of merozoites into the blood circulation marks the beginning of the erythrocytic stage of malaria infection. Merozoites invade erythrocytes and through asexual multiplication develop into thousands of merozoites. Upon release, these merozoites invade more erythrocytes. This stage is responsible for the clinical manifestations of malaria and if left unabated can lead to fatal consequence. A small proportion of merozoites develop into male and female gametocytes which

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when imbibed by the female mosquito mate and develop into oocysts within the mosquito midgut. The oocyst further divides asexually to generate thousands of sporozoites which migrate from the gut to the salivary gland of the mosquito where they can be injected into the host during mosquito feeding and restart the cycle all over again. Pre-erythrocytic immunity Infection is initiated by the injection of sporozoites through the bites of female anopheline mosquitoes while feeding. The sporozoites move quickly (over minutes or hours) through both lymphatic and blood vessels to the liver where they enter hepatocytes, in which the parasite undergoes a period of rapid multiplication over a period of around six days to form the hepatic schizont. The sporozoite is one of the few points in the life cycle where the parasite is not hidden within another host cell and present in few numbers, making it a good potential target for host immune responses. Given its extracellular location, relatively small numbers and rapid movement from the periphery to the hepatocyte, the most likely effective mechanisms would involve antibodies directed at surface antigens leading to either antibody-dependent cell-mediated clearance (for instance opsonization leading to phagocytosis) or to blocking of receptor mediated contact and invasion of the hepatocyte. Although humans in endemic areas clearly make humoral and cellular responses to sporozoites antigens (Webster et al., 1988; Khusmith et al., 1999; Doolan et al., 1994) there is limited evidence that these are effective in protecting against infection although it should be noted that few studies have looked in detail at the anti-sporozoite response as a whole (as opposed to response to specific antigens). Whatever the case for the involvement of natural immunity, aborting this earliest stage in the parasites human cycle would prevent infection, disease and transmission and is therefore an attractive target for vaccine induced responses. The leading vaccine candidate, RTSS, is based on a subunit repeat from the circumsporozoite protein. Although the relative importance of antibody-mediated response to RTSS vaccination versus cell-mediated responses (targeted at CS peptides exported to the infected hepatocyte surface) is not clear, the balance of

evidence is towards a dominant role for anti-CS antibodies (Olotu et al., 2010, 2011). The parasite within the hepatocyte Once the sporozoite has invaded the hepatocyte it undergoes a process of rapid division over around six days to form the hepatic schizont comprising up to 30,000 daughter merozoites. At this stage the parasite is ensconced within a host cell and assumed not to be directly susceptible to antibody-mediated responses. However, the infected hepatocyte could clearly be the target for a range of cell-mediated responses directed at parasite antigens presented at the hepatocyte surface and a range of effector mechanisms involving CD4, CD8 lymphocytes as well as other cells have been demonstrated in animal models and in vitro models as well as in naturally infected humans and humans given a range of experimental vaccines (Sedegah et al., 1992; Calvo-Calle et al., 2005; Hoffman et al., 1989). The potential for pre-erythrocytic vaccines The pre-erythrocytic stages have been the focus of considerable effort, and some success, in vaccine development. The strongest direct evidence for the feasibility of vaccination comes from classic experiments in which Clyde showed that inoculation with irradiated sporozoites could lead to sterile immunity sustained over 6 months (Clyde, 1990). These observations provided the basis for much of the work to develop malaria vaccines over the subsequent forty years, attempting to reproduce this kind of immunity through the use of sub unit vaccines. The assumption was that it would not be practical to deliver irradiated sporozoites as a vaccine in the field (an assumption which has recently been challenged by the commercial company Sanaria who have demonstrated the feasibility of producing GMP quality irradiated sporozoites at a scale which would allow large numbers to be vaccinated (Hoffman et al., 2010). The search for a subunit vaccination approach formed the basis of the development of RTSS, (based on a subunit of the circumsporozoite protein which forms the major surface protein of the sporozoite) the leading candidate currently in phase III trials. The only other pre-erythrocytic

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vaccine to reach phase IIb trials has been ME TRAP (Bejon et al., 2007), which is based on another pre-erythrocytic antigen together with B-cell epitopes from a range of other liver stage antigens. Immunity to the erythrocytic stages The erythrocytic cycle comprises two obvious immune targets: the free merozoite and the infected erythrocyte. The most likely direct mechanisms for immune attack on either stage would be antibody mediated (the merozoite being free and the red cell lacking machinery for presentation of antigens in association with HLA molecules). A classical set of studies 50 years ago using passive transfer of immunoglobulin established that immune individuals in endemic areas have circulating antibodies that are effective in rapidly controlling parasitaemia in children with clinical episodes of malaria (Cohen et al., 1961). However, cellular responses could also play key roles, either secondary to antibody through Fc mediated interactions, or through induction of cytokine release in response to blood stage antigens processed by cells of the reticulo-endothelial system. Merozoites are released when the infected red cell containing a mature schizont bursts. The merozoite has to attach to and invade a new red cell. Although only present briefly in its extra-cellular state, the merozoite offers an obvious target for antibody-mediated attack, either through cell assisted clearance of antibody sensitized or damaged parasites, or by blocking receptor ligand interactions involved in the attachment and invasion process. Humans make specific antibodies to a wide range of merozoite antigens (Marsh and Kinyanjui, 2006). Early studies of natural immune responses tended to be focussed on a very limited set of merozoite antigens that were being considered as vaccine candidates. More recently the number of characterized antigens has expanded enormously as a result of having the full genome sequence of the parasite (Gardner et al., 2002). The picture that is emerging is that one can define an association between antibody-mediated responses to an increasing number of antigens with a degree of protection from clinical disease

in the field (Osier et al., 2008) and with a range of mechanisms modelled in vitro, including prevention of invasion of merozoites into red cells as well as phagocytosis of merozoites and a range of antibody dependent cellular killing mechanisms (Wipasa et al., 2002). Although it is tempting to think in terms of identifying a key target and mechanism, the picture emerging does not readily identify any single response as being either necessary or sufficient to mediate protection. The exact components responsible for mediating protection may be different in different individuals and even in the same individuals over time (Quakyi et al., 1989; Garcia et al., 1998). Once inside the red cell the parasite is, on the face of it, protected from immune attack. However, rather than quietly developing in this potentially hidden niche, the parasite modifies the host red cell surface in multiple ways, involving both modification of host molecules and the insertion of parasite molecules on to the red cell membrane (Moxon et al., 2011). This seemingly unnecessary exposure of antigen results in the parasite adhering to the walls of blood vessels. It is assumed that adherence plays an important role for the parasite, possibly in avoiding passage through the spleen and that antigenic variation represents a way of evading consequent immune responses to the parasite molecules exposed to the host immune system. Parasite molecules exposed at the infected red cell surface include the products of several multi gene families, the best characterized of which are the var genes coding for Pfemp1. Each parasite genome has around 60 different var genes which are expressed in a mutually exclusive manner leading to the phenomenon of clonal antigenic variation (i.e. the ability of even clonal line to sequentially express distinct versions of the molecule (Scherf et al., 2008). The var gene products are involved in mediating adherence of infected red cells to a range of host cells and play a direct role in the pathogenesis of severe disease. Although Pfemp 1 is the best characterized set of variant antigens expressed on the host infected red cell surface, there are several other large families including stevor and Rifins (Sherman et al., 2003) where both the function and the role of host immune responses are not yet well characterized. As with the merozoite, antibodies directed

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to antigens on the infected red cell surface could lead either to cellular assisted killing or clearance, or interfere directly with receptor–ligand interactions, for instance by blocking cytoadherence, and leading to clearance on passage through the spleen. Parasite molecules expressed on the infected red cell surface present an obvious potential target for the natural immune response. Infected individuals make responses to the set of antigens expressed at the time of presenting with a clinical episode of malaria (i.e. homologous responses) (Bull et al., 1998) and these responses are associated with strong protection against subsequent episodes of clinical malaria by parasites expressing the same set of antigens. This leads to the idea that immunity may be acquired by building up a repertoire of such responses. However, the potential repertoire of variant antigens is so large that it would not be possible for immunity to rely on this (especially immunity to severe disease which is acquired relatively rapidly) and it is increasingly clear that it is also possible to develop responses that are cross-reactive to other red cell surface antigens (i.e. heterologous responses) (Mackintosh et al., 2008; Elliott et al., 2007) and to antigens showing varying degrees of conservation. These heterologous responses may play a role in naturally acquired immunity (Kheliouen et al., 2010; Staalsoe et al., 2004b; Dodoo et al., 2001). The potential for blood stage vaccines The starting point for efforts to develop blood stage vaccines is that naturally acquired humoral responses to these stages are effective in protecting large numbers from disease and death. As with the pre-erythrocytic stages efforts to date have largely focussed on developing subunit vaccines based on a limited set of specific merozoite antigens supported by evidence from their location, function and results of vaccination in a range of animal models. Three full scale phase 2b field trials have been reported to date (AMA1, MSP1 and combination B) and several others are under way (for instance MSP3 in combination with glutamate-rich protein (GLURP)). However, none have shown significant overall efficacy though in

several cases there has been evidence of possible allele specific protection (Genton et al., 2002; Thera et al., 2011). There are a number of challenges to be faced. In contrast to pre-erythrocytic vaccine development, where several antigens can be distinguished by key biological roles and expression patterns, there are many more well characterized bloodstage antigens with apparently critical roles in red cell invasion or cytoadherence. Many of the antigens considered as targets show marked polymorphism, presumably as an evolutionary adaptation to evade host responses. However, it is becoming clear that some functionally important antigens show minimal polymorphism and going forward these will likely be a major focus. Some have expressed doubts whether the blood stages are a suitable target at all because the level of immunity achieved by natural responses, whilst important, is far from complete. While there is no doubt that a 100% effective pre-erythrocytic vaccine would be ideal in preventing any manifestations of malarial infection, at a time when this is still elusive it would seem premature to ignore a critical part of the life cycle which if successfully targeted would have enormous benefits. Immunity to the sexual stages of the parasite A proportion of parasites leave the 48-hour erythrocytic cycle to develop as sexual stages (male and female gametocytes). These are subsequently taken up by feeding mosquitos, where they undergo mating in the mid gut, forming oocysts which produce progeny sporozoites, which in turn eventually migrate to the salivary glands to complete the cycle by being injected when the mosquito next bites a human host. Clearly immune responses which affected the viability of gametocytes or any subsequent stage in the mosquito could limit transmission of the parasite. As with the blood stages, responses to gametocytes (which are themselves contained within the host red cell) would likely be depend on antibody-mediated responses. A number of antigens specific to the sexual stages have been identified (Carter, 2001). Humans in endemic areas make specific antibody responses to some of these antigens (Mendis et al., 1990; Targett,

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1990). It has also been demonstrated that these antibodies are capable of blocking to varying degrees the transmission of gametocytes when examined in bioassays in which mosquitoes are fed on infected blood containing the antibodies and then monitored for the development of infection (Carter, 2001; Kaslow, 2002). However, the role of such responses in limiting transmission remains to be established. One of the most promising sexual stage targets are antigens found on ookinetes (i.e. Pf25 and Pf28), a stage found only in the mosquito but which can be targeted by human antibodies carried in a blood meal (Wu et al., 2008). Because this stage is never seen by the human immune system, there has been no pressure on it to drive antigenic polymorphism. An extension of this idea is work examining the possibility of using key mosquito proteins involved in the parasite life cycle (Dinglasan and Jacobs-Lorena, 2008). The potential for sexual stage vaccines To date vaccines targeted against the sexual stages have received less investment of effort than those to other stages. To some degree this may reflect concerns as to how one would deploy a vaccine that, whilst producing benefit to the population as a whole, would confer no individual benefit to the recipient, and perhaps concern that large cluster randomized trials would be required to demonstrate efficacy in the field. The ideal might be to combine them with effective vaccines against other stages in the life cycle. The increasing emphasis on elimination and even eradication as a driver for research in recent years has brought transmission blocking vaccines to the fore again. Animal models The need for laboratory challenge models As described above, the ‘natural experiment’ of observing naturally acquired immunity in the field does not provide an unequivocal basis for choosing one particular antigen of the 5000 falciparum

genes available for vaccine development, and nor does it provide a basis for defining the magnitude or functional characteristics of a protective response. Laboratory challenge models allow mechanistic studies of a complex immune response, and are often used to provide ‘proof-of-concept’ regarding a protective mechanism, and then to select the most promising vaccination approaches, before field trials are undertaken. The challenge models available include mice, non-human primates, and controlled human malaria infections. Murine models Mice are convenient laboratory models, and their immune system has been extensively studied. Unfortunately Plasmodium falciparum does not infect mice (except in ‘humanized’ mice, as described below). During field surveys in central Africa in the 1950s, a tree rat, Thamnomys rutilans, was found to be naturally infected with Plasmodium berghei (Vanderberg, 2009). Further field studies identified Plasmodium yoelii, Plasmodium chabaudi and Plasmodium vinckei as naturally infecting strains of the tree rat. Although these parasites can be used to successfully infect mice in the laboratory, they are not natural parasites of mice, and therefore do not all produce chronic infections or transmissible forms, which would be required to sustain transmission in the wild. Nevertheless, various parasite and mouse strain combinations have been used to model aspects of human infection and pathology. The utility of these models in examining the pathophysiological consequences of malaria has been reviewed elsewhere (White et al., 2010; Craig et al., 2012). Here, we consider how these models have been used to examine (a) pre-erythrocytic immunity (b) erythrocytic immunity and (c) transmission blocking immunity. Models of pre-erythrocytic immunity Sporozoites for P. berghei and P. yoelii are readily generated in the laboratory, but it has been more difficult to optimize the production of P. chabaudi sporozoites (Spence et al., 2012). Hence groups studying pre-erythrocytic immunity have tended to focus on P. berghei and P. yoelii models.

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Irradiated sporozoites It had been known since the early 40s that killed sporozoites could protect birds against subsequent challenge with malaria (Richards, 1966). After several attempts, this effect was eventually reproduced in mice using X-ray irradiation (Nussenzweig et al., 1967), an approach which generalized to human studies (Clyde et al., 1973). The requirement for attenuated rather than killed sporozoites in mice and humans suggests that successful hepatocyte invasion is required, therefore implying that CD8 T-cell induction is an important protective mechanism. Although there is some diversity of findings according to the mouse model used, in most models protection requires CD8 T-cell responses acting via IFNg production (Doolan and Hoffman, 2000). There is evidence for both cellular and antibody-mediated protection in humans studies (Epstein et al., 2011). Pre-erythrocytic antibody-mediated immunity Circumsporozoite proteins (CS) comprise a dense coat around invading malaria sporozoites (Pancake et al., 1992; Gwadz et al., 1979). Humans exposed to malaria in the field acquire low-titre antibody responses to the CS, which is not consistently associated with protection (Hoffman et al., 1987). On the other hand, monoclonal antibodies against the CS were found to be protective in the Plasmodium yoelii mouse model (Charoenvit et al., 1991), and mice could also be protected by vaccination with DNA encoding CS (Sedegah et al., 1994). These studies led to human challenge studies (Stoute et al., 1997) and field trials (Bojang et al., 2001). These human studies confirm the prediction from the mouse model that anti-CS antibodies would be protective. However, substantially lower levels of protection have been observed in humans than were observed in mice. This reduction in efficacy probably reflects the lower antibody responses raised in humans compared with mice. Indeed the first synthetic CS peptides examined in humans were very poorly protective, and higher immunogenicity was required by engineering virus-like-particles and using potent adjuvants before significant protection was seen in human challenge studies (see below). Studies of immunity to CS in mice may

be poorly analogous to human malaria, since the immunologically dominant epitope falciparum has a unique NANP epitope, but later became possible with transgenic parasites (see below). Pre-erythrocytic T-cell immunity Models of pre-erythrocytic immunity have been developed using both P. berghei and P. yoelii. The incubation period in the liver is only 2 days for both of these parasites (compared with 6.5 days for P. falciparum in humans), which in theory might give less opportunity for host immunity to clear parasites from the liver before they give rise to a blood-stage infection. However, in practice, several protective vaccinations inducing T-cell immunity have been described. A number of different platforms for delivering pre-erythrocytic antigens have been assessed in mice, including; DNA (Doolan et al., 1996), recombinant viral vectors and proteins (Gilbert et al., 1999). Mice of the strain ‘BALBc’ readily produce high frequency CD8+ T-cell responses to a single H2 class I epitope of the CS antigen in P. berghei (pb9) (Plebanski et al., 1998) and this model has become very widely used. However, T-cell responses to the CS antigen, which have been more difficult to induce in humans, are of much lower frequency than BALBc mice (Walther et al., 2006; Ndungu et al., 2012), tend to be CD4+ rather than CD8+ (Ansong et al., 2011), broadly distributed rather than to a single epitope (Lalvani et al., 1999), and associated with much lower levels of protection in humans than in BALBc mice (Kester et al., 2009). Thus, clinical development of T-cell-inducing vaccines in humans has focused on prime-boost approaches to enhance immunogenicity, and on the antigen thrombospondin-related adhesion protein (TRAP) rather than CS. This latter decision was based on in vitro data suggesting more durable transcription of TRAP than CS during the intra-hepatic phase (Bodescot et al., 2004) and data suggesting limited polymorphism in the field (Robson et al., 1990). That said, mouse models were used to demonstrate that vaccination with TRAP was protective in more than one strain of mouse (Schneider et al., 1998), and also to establish improved immunogenicity and protection of varying prime-boost approaches

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with different viral vectors (Anderson et al., 2004; Reyes-Sandoval et al., 2010). Qualitatively, the conclusions reached in mouse studies have held true in human studies. T-cells responding to TRAP are protective, and the hierarchy of immunogenicity and protective efficacy of a range of single-vaccination and prime-boost regimens determined in mice has been similar in human studies (Moore and Hill, 2004; Anderson et al., 2004; Reyes-Sandoval et al., 2010). However, absolute immunogenicity and protective efficacy has been substantially lower in humans than in mice when considering equivalent vaccinations. The protective threshold for CD8+ T-cell responses was defined in a mouse model where varying experimental conditions resulted in mice with wide variation in reactive CD8+ T-cell frequencies. In this study, mice are only protected when CD8+ T-cell responses exceed a high threshold, considerably above that required to protect against other pathogens (Schmidt et al., 2008). This may explain the reduced protective efficacy seen in humans, since the T-cell responses raised to date in humans are substantially below an equivalent threshold. Erythrocytic-stage antibodymediated immunity Following blood-stage infection with P. berghei or P. yoelii, the outcome of infection is either uncontrolled expansion of parasite numbers leading to death of the mouse, or rapid clearance of the parasites by host immunity, dependent on the mouse and parasite strain (Greenberg and Kendrick, 1957; Weiss, 1989). Combinations of mouse and parasite strains are therefore described as ‘lethal’ or ‘non-lethal’ models. In human chronic parasitaemia is a more common outcome of malaria infection than either rapid progression to death or spontaneous clearance. Plasmodium chabaudi has not been favoured by some investigators because of the difficulty of successfully culturing parasites through the mosquito stages of its development in order to generate infective mosquitoes (although protocols for achieving this are now available). However, P. chabaudi chronically infects mice and therefore strikes a closer likeness to human

infection (Stephens and Langhorne, 2010; Spence et al., 2012). The P. chabaudi model has therefore been studied extensively as a model of chronic blood-stage infection. In the P. chabaudi model, the cytokine response in the very early phases determines initial parasite growth (in particular a balance between pro-inflammatory cytokines such as IFNγ to control growth and regulatory cytokines such as TGFβ to prevent immunopathology). Beyond the early phase, antibody-mediated responses appear to be critical in controlling parasitaemia (Artavanis-Tsakonas et al., 2003). The published record shows a preponderance of P. yoelii use in models assessing vaccine-induced antibody-mediated protection against erythrocytic stage parasites (Alaro et al., 2010; Shi et al., 2007). In these P. yoelii models, antibody appears sufficient to account for protection. At the time of challenge there is no requirement for T-cells (not withstanding a need for T-cell help to generate antibody in the first place), or for natural killer cells, monocytes or Fc receptors (Rotman et al., 1998; Hirunpetcharat et al., 1997; Murphy and Lefford, 1979). Hence it may be argued that this model is a murine in vivo growth inhibition assay, analogous to the growth inhibition assays commonly conducted in vitro, except for the requirement to generate antibodies by vaccinating the mouse. It is not explicit in many reports why investigators favour P. yoelii over P. berghei for models of blood-stage immunity. However, it is more difficult to protect mice against blood-stage challenge in models using P. berghei rather than with P. yoelii (Playfair et al., 1977; Holder and Freeman, 1981; Narum et al., 2000; Cox, 1970; Longley et al., 2011; Yoshida et al., 2010). One study demonstrated failure to protect against P. berghei using a series of vaccinations that had been protective in P. yoelii (Goodman et al., 2013). Curiously, the situation is reversed in models of pre-erythrocytic immunity where P. berghei parasites are more susceptible to T-cell killing than P. yoelii parasites (Schmidt et al., 2011). Hence in both circumstances investigators have tended to work with mouse models in which their particular vaccines were more likely to be protective.

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Erythrocytic-stage T-cell Immunity Despite the importance of antibody responses in controlling natural infection in the models of naturally acquired immunity and some models of vaccine-induced immunity described above, this does not exclude the possibility that one might generate T-cell effectors capable of mediating immunity in other models. For instance, T-cell responses to specific vaccination protocols using whole parasites may account for protection in mice in some P. chabaudi (Taylor-Robinson et al., 1993) and P. yoelii (Amante and Good, 1997) models, perhaps due to responses to the antigen HGXPRT (Makobongo et al., 2003). However, in human studies T-cell-inducing vaccination with whole parasites (Sauerwein et al., 2012) or with blood-stage antigens (Sheehy et al., 2012) did not demonstrate any convincing protection against blood-stage multiplication. Outbred mice The use of outbred mice has been proposed as a method for avoiding the selectivity inherent in choosing a particular mouse strain as a model for testing vaccines (Doolan and Hoffman, 2000). A diversity of genetic background implies a risk of diversity of immunological responses. This may avoid the very restricted cellular responses seen in inbred mouse strains, but it does not necessarily follow that the more diverse response will be similar to the immunological responses in humans. It does, however, avoid the danger that the outcome in any given model will be determined in an ‘all or none’ fashion by the presence or absence of responses to a particular epitope, and remove the need for an arbitrary (or not so arbitrary) choice of mouse strain to be made by the investigator. Transgenic parasites It is possible to generate transgenic parasites that allow testing of vaccines based on P. falciparum proteins rather than the mouse malaria orthologues (Mlambo and Kumar, 2008). This is convenient, and arguably allows an epitopespecific conclusion that could not be drawn from vaccination with the mouse malaria orthologue. For instance, the efficacy of particulate protein delivering the NANP repeats present in falciparum CS could not have been tested in a mouse

model without the use of transgenic parasites (Calvo-Calle et al., 2006). On the other hand, the transgenic parasites are altered in one of 5000 genes only, and therefore retain many points of dissimilarity with P. falciparum parasites. Furthermore, transgenic parasites may be less fit and show altered growth characteristics as a result of the foreign protein. The transgenic parasite approach may be particularly beneficial when testing transmission blocking vaccines, where antibody binding and functional outcomes occur in the mosquito midgut rather than in the host. Hence the points of dissimilarity between mice and humans are rather fewer with regard to transmission blocking than they would be for a host in vivo process. Humanized mice In order to allow infection with P. falciparum (rather than using a murine malaria species with a single falciparum epitope), mice have been generated with engrafted human red blood cells (Angulo-Barturen et al., 2008) or engrafted human hepatocytes (Morosan et al., 2006). These approaches have not yet been widely used in vaccine testing. This may partly reflect the technical difficulty of producing these mice as well as the perception that the advantages of these models may be limited where the human parasite is tested against a mouse immunological response. This may be partly addressed by further ‘humanizing’ various aspects of the mouse immunological response such as engrafting human T- and B-cells (Danner et al., 2011), but this carries the expense of further technical complexity. The utility of the murine models It is easy to be critical of the many ways in which murine models do not resemble human malaria, ranging from parasite life cycles, immune responses and pathophysiology. On the other hand, the need for laboratory models remains pressing given the limited experimentation that can be carried out in humans, and our lack of a clear understanding of the acquisition of immunity to malaria in the field. As we have seen, some conclusions gained from mouse models have led to promising lines of investigation, for instance; the protective

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efficacy of anti-circumsporozoite antibodies (a conclusion that could not have been gained from field immuno-epidemiology given the low titres induced by natural exposure) and the protective efficacy of irradiated and other variously attenuated sporozoites. On the other hand, enthusiasm for a range of apparently protective vaccinations has proved to be premature. There may be a tendency for investigators to use a ‘good model’ for particular immunological outcomes, which then creates a misleading impression that protection can be induced relatively easily. Although the models provide quantitative over-estimates of vaccine efficacy, a qualitative conclusion that, in principle, a T-cell or antibody response to a particular antigen (or attenuated whole parasite) is capable of mediating protection appears more likely to be supported by subsequent trials in humans. Furthermore, hierarchies of protective efficacy may be predictive, and therefore studies comparing multiple vaccination modalities or antigens may be preferable to ‘stand-alone’ tests of a single vaccination approach. Non-human primate malaria models Non-human primates are phylogenetically more related to humans than other animals and therefore may be more relevant as an animal model. Several non-human primates such as New World monkeys (Aotus or Saimiri), Rhesus macaques, Cercopithecus monkeys and chimpanzees have been used in preclinical studies to assess the immunogenicity and efficacy of candidate malaria vaccines. P. falciparum MSP-1 was the first to be tested in owl monkeys and shown to be protective against intravenous challenge with blood stage parasites (Siddiqui, 1977). Other blood stage antigens including EBA175 ( Jones et al., 2001), AMA-1 ( Jones et al., 2002) and EMP1 (Baruch et al., 2002) have been tested in Aotus monkeys and shown to be highly immunogenic with functional antibody production. Pre-erythrocytic candidate malaria vaccines such as RTS,S (Pichyangkul et al., 2008, 2009) and live attenuated sporozoite vaccine (Epstein et al., 2011) have all been tested in non-human primates for safety and immunogenicity assessment before proceeding to human clinical trials, but not

for efficacy. The development of T-cell-inducing vaccines have also included sporozoite challenge studies in macaques (Capone et al., 2010a). However, there are several challenges facing non-human primate models. Human malaria parasites do not immediately grow in non-human primates and this necessitates the use of adapted parasite lines. For example whereas Santa Lucia strains of P. falciparum and FVO grows in Aotus genus, the NF54 and 3-D7 strains do not (Herrera et al., 2002; Heppner et al., 2001). There also are species-specific differences in terms of immune response to various malaria antigens (Kumar et al., 1995), susceptibility to different malaria parasite strains (Gramzinski et al., 1999), dose of infectious challenge required, progression of parasitaemia and severity of disease (Thomas et al., 1994; Heppner et al., 2001). The data reported to date do not immediately indicate that studies in non-human primates closely reflect what would be expected in human trials. Controlled human malaria infection (CHMI) Although a number of insights into malaria vaccine development have depended on the use of animal models there are a number of limitations to this approach as indicated above, and in many ways the ideal approach is to study human subjects, using controlled human malaria infection (CHMI) studies. Practical considerations Mosquito-bite CHMI is undertaken using infected mosquitoes. Two clonally related strains of parasite (3-D7 and NF54) have been used most widely, a few studies have used the 7G8 isolate (Sauerwein et al., 2011), and recently a new parasite clone has been developed for use in challenge studies (Teirlinck et al., 2013). A single mosquito bite does not reliably infect challenged volunteers, and current protocols use five infected bites. Mosquitoes that have been applied to the volunteer are confirmed to have delivered an infection by dissection to identify (a) sporozoites in the salivary glands and (b) a blood meal. It is time consuming to deliver individual potentially infectious bites, and so batches of mosquitoes are applied to each

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volunteer. The intensity of the challenge depends on the number of sporozoites injected by each mosquito. This has been estimated at a mean of 11 sporozoites per bite in one study (Beier et al., 1992), but the skewed distribution around this mean (i.e. occasionally several hundred are injected in a bite) implies that challenge can be very variable between individuals. Furthermore, the NF54 challenge model appears to lead to blood-film patent infection two days earlier than the 3-D7 model (Roestenberg et al., 2009, 2012; Bejon et al., 2005), suggesting variation in inoculum between challenge models. Once challenged, pre-erythrocytic incubation of 3-D7 parasites lasts for 6.5 days, after which careful monitoring for parasitaemia should begin. This is usually done with twice-per-day blood films for the first 7 days, and then daily blood films for the next 7 days. Anti-malarials are given on first identification of blood-stage parasitaemia by microscopy. Volunteers who remain uninfected at 21 days are considered to be protected, but usually treated with curative anti-malarials as a precaution. It is not unusual for some degree of objective fever, febrile symptoms and malaise to be reported prior to blood film diagnosis and/or for a few days after, but CHMI appears to be very safe (Church et al., 1997). More than a thousand challenges have been undertaken worldwide, and very few linked serious adverse events have been reported. Myocardial events have been reported in two volunteers, one with prior cardiovascular risk factors and evidence of atherosclerotic infarction (Verhage et al., 2005), and one without risk factors and a less clear clinical picture (Nieman et al., 2009). These isolated events were not clearly linked to CHMI and are not widely recognized consequences of natural malaria infection. Furthermore, no volunteer has thus far developed an illness meeting criteria for severe malaria. Efficacy endpoints The most obvious endpoint is sterilizing immunity. CHMI studies are necessarily small, given the expense of conducting them. Thus, formal statistical analysis of a single study is limited. In order for the study to be informative, the intensity of the challenge must be sufficient to reliably infect all unvaccinated control volunteers.

Instead of complete or sterilizing protection, vaccines may provide partial protection, manifest as a delay to the detection of parasitaemia (McConkey et al., 2003). This is particularly valuable in assessing the significance of a single protected vaccinated volunteer, since isolated sterilizing protection in the absence of any partial protection among other volunteers is less likely to be evidence of vaccine-mediated protection, and more likely to represent a failure in technical aspects of the challenge experiment. Quantitative PCR monitoring A delay to blood film patent infection may be the result of a reduced liver to blood inoculum (thus requiring more rounds of multiplication before parasitaemia reaches the threshold required for diagnosis), or the result of a reduced rate of multiplication. These effects can be distinguished by PCR monitoring (Bejon et al., 2005; Hermsen et al., 2004). PCR can identify parasites well before they reach the threshold required for microscopic detection by blood film (i.e. at 20 parasites per ml vs. 10,000 parasites per ml), and can also quantify the parasitaemia. By using the longitudinal readings for an individual, the growth rate and liver-to-blood inoculum can be calculated. Sophisticated models have been applied to fit the effect of asynchronous sequestration of the parasite to blood vessel walls, which causes periodic variability in parasitaemia, but in fact these models may offer no advantage over a simple log-linear growth model (Douglas et al., in press). Blood stage challenge In order to be completely clear that protection is mediated by antibodies to blood stage parasites rather than pre-erythrocytic immunity, it is possible to challenge with infected human blood cells (Sanderson et al., 2008; Pombo et al., 2002). This is essentially a blood product, and must be subject to stringent checks to avoid transmitting blood-borne pathogens (aside from malaria). A further advantage of this approach is that the blood stage inoculum can be precisely controlled, thus allowing the growth rate of parasites to be modelled with less uncertainty than when the liver-to-blood inoculum is variable. The method also avoids a reliance on producing sufficient

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infective mosquitoes in the laboratory at the right time to conduct challenge. Cryopreserved sporozoites The company Sanaria Inc has been formed to develop the whole-sporozoite vaccination approach as a viable option for routine vaccination. There are various technical challenges that have been addressed in developing this technology, including the need to prepare large numbers of dissected, viable sporozoites which can be cryopreserved and injected by syringe when required. Aside from the possibility of using cryopreserved attenuated sporozoites as a means of generating immunity, cryopreserved un-attenuated sporozoites may simplify CHMI studies substantially. Mosquito delivery of sporozoites relies on timing the incubation of blood-stage cultures, feeding and infection of laboratory reared mosquitoes and subsequent successful incubation to infective mosquitoes in sufficient numbers to conduct a challenge study, ready in time for the appropriate post-vaccination window. Cryopreserved sporozoites can be secured in advance of the clinical trials, and can be transported to and used in centres which lack the specialized Level 3 containment facilities necessary for mosquitobite challenge (Roestenberg et al., 2013). Pre-erythrocytic vaccines tested by CHMI The first subunit vaccines tested by CHMI delivered the circumsporozoite antigen (CS). The early trials of synthetic CS peptides demonstrated occasional protection (Herrington et al., 1987). The formulation of CS contained in ‘RTS,S’ improved on immunogenicity by the addition of a carrier antigen to assemble virus-like-particles and additional adjuvant systems, leading to greater protection in CHMI (Stoute et al., 1997). The results from CHMI were replicated in the field, with partial protection being observed in both settings and, for the two formulations tested in the field, a similar hierarchy of protection (Polhemus et al., 2009). T-cell-inducing vaccines against the antigens TRAP and CS have also been tested in CHMI (Vuola et al., 2005; Dunachie et al., 2006). It appears that viral vectors induce a more substantial

response to TRAP than to CS, and significant protection has been observed in CHMI for the former but not the latter. Protection has not been observed in field studies to date, perhaps owing to lower immunogenicity when tested in the field (Bejon et al., 2006; Moorthy et al., 2004). Several other attempts to deliver CS antigen have been assessed in CHMI, and all found to be non-protective, probably due to lower immunogenicity than required for protection (Audran et al., 2009; Walther et al., 2005). A variety of DNAbased vaccines have been tested in an attempt to induce protective T-cells, but have been poorly immunogenic and non-protective (Moore and Hill, 2004). The efficacy of whole-parasite methods have also been established using CHMI. Irradiated sporozoites (Clyde, 1975), and large doses of sporozoites delivered under chloroquine cover (Roestenberg et al., 2009) generate high levels of protection against subsequent CHMI, but have not yet been tested in the field. Clinical development decisions based on CHMI It has become standard practice to use CHMI data to make a critical go/no-go decision in the development plan for pre-erythrocytic vaccines. In a CHMI study the following factors are in favour of a positive result; that the challenge strain is known (and usually related to the vaccine strain), the challenge is timed for peak immune responses, the experiment is well controlled for extraneous factors and healthy volunteers are recruited. It is therefore hard to justify proceeding to field trials in the absence of protection on CHMI, where none of these advantages apply. Empirically, RTS,S delivers lower efficacy in the target population than it did in CHMI studies (Agnandji et al., 2011, 2012), and other vaccinations with efficacy in CHMI did not show efficacy in field studies (Bejon et al., 2006; Moorthy et al., 2004). Perhaps surprisingly, the same consensus has not emerged for testing blood-stage vaccines. This has been justified on the basis that one might see protection in the field at higher parasitaemias than could be allowed in CHMI, or that there may be a synergistic interaction between naturally acquired immunity and vaccine-induced immunity. In

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support of this contention, observations in the field do indeed suggest that natural immunity is able to limit parasite growth but not lead to sterilizing immunity, and this leads to chronic parasitaemia in the absence of febrile illness at a level of parasitaemia well above that allowed in CHMI (Langhorne et al., 2008). However, it seems unlikely that a clinically relevant limitation of growth at higher parasitaemias can be achieved without even the slightest effect on the rate of growth at lower parasitaemias, and indeed studies of natural challenge following curative anti-malarials strongly suggest that lower parasite multiplication rates are observed in semi-immune adults (Douglas et al., 2011). Furthermore, several blood-stage vaccines have now been tested in the field, all without detectable overall efficacy. As the number of blood-stage vaccine candidates that could be tested proliferate (Schwartz et al., 2012), it seems essential to apply more rigorous selection criteria on candidates taken forward to field studies. .

What kinds of vaccines should we be aiming at? The ideal malaria vaccine would be safe, cheap to produce and give 100% lifelong protection against infection following a single injection. However, recognizing that this is a high bar and that less effective vaccines may have great utility, attempts have been made to set consensus targets through wide consultation. This resulted in the development in 2006 of the Malaria Vaccine Technology Roadmap (WHO, 2006), which set as a goal developing and licensing a malaria vaccine that has a protective efficacy of more than 80% against clinical disease and lasts longer than four years’ by 2025. A general assumption around vaccine development for many years was that the target population would be young children and that it would have to be capable of being delivered through the extended programme of immunization (EPI) schedule. More recently, an increasing focus on elimination and eradication has led to a change in emphasis by some funders and researchers to focus on prevention of transmission as the primary target of vaccine development. The

circumstances in which such a vaccine would be used mean that consideration would have to be given to delivering it to all age groups. Clearly a totally effective pre-erythrocytic vaccine would achieve both aims. However, it needs to be borne in mind that a highly but not completely effective (either by virtue of individual variability or failure to cover the entire population) pre-erythrocytic or transmission blocking vaccine would lead to a reduction in population immunity against blood stages. If protection were to wane in the face of sustained malaria transmission there would be the potential for massive epidemics of severe malaria, as has occurred many times in the past, for instance in Sri Lanka in the 1960s (Nájera et al., 1998) and in Madagascar in the 1980s (Mouchet et al., 1997). Under such circumstances a vaccine which included an element of protection against disease would be very important. Clinical development plans for malaria vaccines Vaccines go through numerous trials during the four clinical development phases (Friedman et al., 2010). Distinctive features of malaria vaccine development are; (a) we have a limited understanding of the correlates of natural immunity (see above); (b) a human challenge model is available (see above) and (c) in many field settings malaria transmission is sufficiently high for phase II trials to be powered to detect efficacy against infection in adults or clinical disease and/or infection in children (thus termed ‘Phase IIb’). Primary endpoints in malaria vaccine evaluation There are four main endpoints which could potentially be used in the evaluation of protective efficacy of a malaria vaccine (a) malaria infection, (b) uncomplicated clinical malaria (i.e. the occurrence of fever in association with malaria parasitaemia), (c) severe (complicated) clinical malaria and (d) death. When choosing an endpoint, consideration needs to be given to the following issues; (a) the relevance of the endpoint to the stage of development (i.e. whether proof-of-principle in a

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modestly sized Phase II study or pre-licensure in Phase III) (b) the rate of occurrence of the endpoints in the population under consideration (c) reliability in measuring the endpoint and (d) the public health relevance of an endpoint. The choice of endpoint in proof-of-concept studies in the field may vary according to the mode of action of the vaccine. For instance, a pre-erythrocytic malaria vaccine is intended to prevent blood-stage infection. Any form of blood-stage infection resulting (whether associated with fever or not) may be regarded as failure of protection. On the other hand, a blood stage malaria vaccine may be intended to reduce the exponential increase in blood stage parasitaemia. Hence asymptomatic, low-level parasitaemia may still result despite the individual being protected against higher parasitaemia and the development of fever. Transmission blocking vaccines work at the interface between human host and mosquitoes to reduce infection of mosquitoes, therefore blocking infectivity in mosquito feeding assays is an appropriate endpoint in phase II. More prevalent endpoints are easier to study than rare endpoints. For instance in order to investigate the vaccine efficacy against severe malaria in a typical area on the coast of east Africa with a severe malaria incidence rate of 4.2 per 1000 per year, a sample size of 24,220 children (12,110 in each arm) would be required in order to have 80% power to detect a 50% efficacy against severe malaria given the alpha error of 0.05. In such areas uncomplicated malaria might be the only practical endpoint (provided that an argument is accepted that this is a worthwhile public health measure). Areas where severe malaria is prevalent offer an opportunity to study the effect on severe malaria. For instance in Bandiagara, Mali, where annual incidence of severe malaria was estimated to be around 2.3%, a sample size of 4580 would provide 80% power to detect 50% efficacy against severe malaria given alpha of 0.05 (Lyke et al., 2004). Investigating the efficacy against malariaattributable deaths is difficult even in areas with high prevalence of severe malaria. This is because, fortunately, only a small proportion of children with severe malaria die (around 10%) implying a much larger sample size than would be required for severe malaria is needed. With the enhanced

care that results when infrastructure is improved to support the clinical monitoring of a trial, the mortality is likely to fall further (Maitland et al., 2011; Dondorp et al., 2010). Phase III trials to evaluate the efficacy of candidate malaria vaccines may therefore include both uncomplicated and complicated malaria as co-primary endpoints (Agnandji et al., 2012). This avoids the ethical dilemma of having to do sequential trials on each endpoint when proof-of-concept studies have confirmed the beneficial effect of the vaccine on uncomplicated malaria. Field evaluation of candidate malaria vaccines Randomized controlled trials are regarded as the gold standard in providing evidence for protective efficacy of a new vaccine. There has been considerable debate regarding the appropriate measure of protective efficacy against malaria in field trials (Moorthy et al., 2007). To provide biologically generalizable estimates of individual vaccine efficacy, the majority of phase II malaria vaccine trials have used time to first event as the primary analysis (Alonso et al., 2004; Bojang et al., 2001; D’Alessandro et al., 1995). However, because this kind of analysis precludes including episodes beyond the first episode in estimating efficacy, adaptions of the survival analysis which take into account all episodes have been recommended (Moorthy et al., 2007). The later provides greater efficiency in estimating the efficacy estimates compared to time to first episode only analysis (Ghosh, 2000). On the other hand, a public health perspective may be better informed by the overall reduction in cumulative episodes once vaccine is introduced in the population. This overall reduction in number of episodes takes into account baseline population risk. Therefore even for a leaky vaccine with partial efficacy one might see a significant number of malaria cases averted in a population with a high malaria burden. There have been few studies on the views of parents and guardians of children in malaria endemic areas to determine the value that would be placed on various measures of vaccine efficacy such as the overall reduction in the number of malaria episodes versus the chance of

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being completely protected from malaria in a given period of time. Two qualitative studies in Mozambique and Kenya have explored some community perceptions (Ojakaa et al., 2011; Bingham et al., 2012), indicating a diversity of views regarding whether partially effective vaccines with limited durations of efficacy would be acceptable. Heterogeneity in malaria exposure and vaccine efficacy Heterogeneity in exposure has been previously described as a complicating factor in an analysis of vaccine efficacy (Halloran et al., 1992; Valim et al., 2008). This may be especially acute in malaria studies. Spatial heterogeneity in malaria exposure has been described at a micro-epidemiological level at varying transmission settings (Drakeley et al., 2003; Kreuels et al., 2008) and accounts for substantial variation in the degree of exposure to malaria from person to person. It is responsible for variations in disease risk within a small area and is evidenced by geographical clustering of malaria infections. It has been attributed to factors such as varying local density of malaria vectors (Thompson et al., 1997), the pattern of contact between human host and vectors and intrinsic human host factors (Knols et al., 1995; Takken and Knols, 1999). Heterogeneity in malaria exposure can affect the efficacy estimates due to (a) a true biological effect and (b) as an artefact of statistical analysis, particularly when relying on time to first event analysis. Higher malaria exposure can result in lower efficacy because (i) the intensity of challenge overwhelms a limited protective efficacy, (ii) reduced exposure to malaria parasite leads to slower acquisition of natural immunity in vaccinated children relative to the control group, an effect which is more marked at higher transmission intensity or (iii) because enhanced transmission intensity reduces vaccine immunogenicity (Olotu et al., 2013). In any cohort consisting of individuals with variable levels of susceptibility to clinical malaria, highly susceptible individuals will experience clinical malaria episodes earlier in the follow-up. In time to first event analysis, individuals are removed from the ‘at risk’ set once they suffer

from infection. Subsequently, the malaria incidence will decline over time as less susceptible individuals remain. In a randomized trial, the distribution of malaria exposure is balanced between the two groups at the start of the trial but if the candidate vaccine is protective, the vaccine group will be compared with a control group consisting of progressively less susceptible individuals as more time since vaccination elapses. The vaccine efficacy against first event will therefore appear to wane overtime despite sustained biological protection of the vaccine. This effect is more marked as time since randomization increases and more events occur. Updates in malaria vaccine development Development of malaria vaccines has made significant progress in the last 10 years. This has been a result of increases in funding from private– public partnership ventures as well as advances in malaria vaccine science and technologies which have facilitated production and testing of new vaccine candidates. The WHO rainbow table contains the list of current candidate malaria vaccine candidates under clinical evaluation (WHO, 2013). There are currently 40 registered vaccine trials evaluating candidate vaccine in different stages. Pre-erythrocytic and blood stage candidate vaccine form the large proportion of vaccines under development. Only one candidate vaccine RTS,S manufactured by GlaxoSmithKline Biologicals is in phase III trials, a milestone as far malaria vaccine development is concerned. Below we review the development of different types of malaria vaccines and highlight salient milestones, challenges and current status. Development of preerythrocytic malaria vaccines The field was originally dominated by trials of SPf66 in the 1990s, but following little or no efficacy being found in several phase II trials clinical development was discontinued (Valero et al., 1993; Alonso et al., 1994; D’Alessandro et al., 1995, Graves and Gelband, 2006).

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RTS,S candidate malaria vaccine The most clinically advanced candidate vaccine is RTS,S which is based on the circumsporozoite protein (CSP). RTS,S is a recombinant protein produced in the yeast cells and consists of central repeats (NANP) and C-terminal flanking regions of the circumsporozoite protein (CSP) from P. falciparum fused with the Hepatitis B virus surface antigen. The antigen is co-expressed with unfused native Hepatitis B virus surface antigen. When produced, the complex spontaneously assembles into virus-like particles. History of RTS,S development Its development started in early 1980s using CS protein constructs containing central repeat regions alone. Although the antigen induced antigen-specific antibody and cellular immune responses, there was little or no protection against experimental challenge (Fries et al., 1992; Herrington et al., 1987; Rickman et al., 1991). Inclusion of some T-cell epitopes in the C terminus part of CS protein and improved presentation of the antigen by using Hepatitis B virus as a carrier matrix improved both antibody levels and cell-mediated immune responses in immunized malaria naive adults but efficacy remained disappointingly low (Gordon et al., 1995). Further efforts to improve the immunogenicity included the use of more potent adjuvant systems, developed by GSK Biologicals. In a series of studies in non-human primates three adjuvants emerged as promising candidates and were later compared in a phase 2a trial in malaria naive adults (Garcon et al., 2003; Stewart et al., 2006). They include AS02 (RTS,S in an oil-in-water emulsion plus the immune stimulants MPL and QS21), AS03 (RTS,S in an oil-in-water emulsion) and AS04 (RTS,S with alum and MPL). The results of the phase 2a trial were remarkable with 85% efficacy (albeit with wide confidence intervals) against homologous challenge in individuals who received RTS,S with AS02 adjuvant (Stoute et al., 1997). This study formed the initial proof of concept for RTS,S and prompted its clinical evaluation in the field.

RTS,S evaluation in malaria endemic countries In 1998, a phase 2b trial of RTS,S given with AS02 adjuvant was conducted among semi-immune adults in Gambia. The vaccine induced high titres of CSP-specific antibodies and T-cell responses (Doherty et al., 1999) and had an efficacy of 34% (95% CI 8% to 53%; P = 0.014) against malaria infection over 15 months of follow-up (Bojang et al., 2001). However, the efficacy waned over time, from 71% (95% CI 46% to 85%) during the first nine weeks to 0% (–52 to 34) in the last six weeks of follow-up (Bojang et al., 2001). A booster dose of RTS,S given one year later boosted immune responses and provided additional protection (47%) against malaria infection during the ensuing malaria transmission season. The first proof of concept study of RTS,S formulated with AS02 in children was conducted in Mozambican children following a series of age de-escalation and dose finding studies (Bojang et al., 2005; Macete et al., 2007). The study consisted of two cohorts. In the first larger cohort older children (1–4 years) were recruited to assess efficacy against uncomplicated and severe malaria. In the second smaller cohort, young infants (6–12 weeks) were recruited to assess safety, immunogenicity and efficacy of RTS,S against infection. In the smaller cohort the adjusted efficacy against infection was 35.4% (95% CI 4.5% to 56.3%; P = 0.029) over 6 months and 9.0% (CI 30.6% to 36.6%; P = 0.609) over the subsequent 12 months showing evidence of waning over time. In the larger cohort adjusted efficacy was at 27.4% (95% CI 6.2% to 43.8%; P = 0.014). and 57.7% (95% CI 16.2% to 80.6%; P = 0.019) against all clinical episodes and severe malaria, respectively, over eight months of surveillance (Alonso et al., 2004). Over four years of follow-up adjusted efficacy against clinical malaria was 25.6% (95% CI 13.4% to 36.0%; P 20% between clades (Fig. 14.2). But even within clades, where variability is around 10%, there is a real risk that vaccine and virus will fail to match (Li et al., 2011). For T-cell vaccines the problem is worsened by the fact that even the best vaccine-induced T-cell responses have failed to prevent infection; they can only contain or, rarely, eliminate infection after transmission (Hansen et al., 2011; Picker et al., 2012). Therefore, there is the high probability that T-cell responses even if well matched initially, will simply select escape mutants. There is evidence that this happened in the failed Merck STEP vaccine trial that utilized rAd5 (Rolland et al., 2011).

A series of related approaches to this problem have been taken. An early approach was to design consensus sequence vaccines that differed minimally from all common circulating strains (reviewed by Korber et al., 2009). Consensus genes were shown to be superior to wild-type HIV-1 genes for induction of breadth in rhesus macaques (Santra et al., 2008). A similar approach was to design ancestral vaccines that lie at the base of major branches of the virus phylogenetic trees (Korber et al., 2009). But even this approach still could not reduce variation to a level that was immunologically irrelevant. A more promising approach has been to make mosaic immunogens, combining two or more such constructs and so cover the most common virus variants and reduce virus variability (Barouch et al., 2010; Fischer et al., 2007; Korber et al., 2009; Santra et al., 2010). Mosaic constructs have been tested for cell immunogenicity in macaques, and have shown encouraging results (Barouch et al., 2010; Santra et al., 2010). In each of two studies, a mosaic immunogen of two or three mosaic sequences, induced T-cell responses that were greater in magnitude, broader in terms of the number of epitopes recognized and deeper in the sense that each response could cross recognize a number of natural variants of the epitope. The approach is scheduled to go into human trials, and the indications are that the approach could deal effectively with infection by common variant viruses. It is less clear that the mosaic approach could deal effectively with post-infection escape mutations. A study of virus deep sequencing in acute HIV-1 infection by Fischer et al (Fischer et al., 2010) showed that each escape mutation arose from a large number (>50) of possible mutations in each epitope, with one variant epitope usually dominating eventually, but new variants emerge at later times. A mosaic vaccine-induced T-cell response might make such escape harder but would be unlikely to contain total viral replication thus limiting long-term virus control, if infection was not prevented. An alternative approach that is attracting interest is to focus T-cell responses on the most conserved regions of the virus by making artificial constructs based only on these regions (Letourneau et al., 2007; Mothe et al., 2011;

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Figure 14.2  Maps showing the subtype designation of all HIV-1 sequences in the Los Alamos database as of 1 January 2009. The maps reflect what is known about global distributions of HIV-1 subtypes. The figures were made using the HIV geography tool at the Los Alamos database (http://www.hiv.lanl.gov/components/ sequence/HIV/geo/geo.comp). Used with permission from Korber (2009).

Rolland et al., 2007). One such conserved vaccine, the conserved construct expressed by chimpanzee adenovirus CH63 and Modified Vaccinia Virus Ankara (MVA) in a prime-boost regimen, has entered a phase I clinical trial. These vaccines have induced high levels of T-cell responses in humans measured in the conventional Elispot assay and the vaccine induced CD8+ T-cells suppress replication

of several virus strains in vitro, though not as effectively as by CD8 T-cells from infected patients who control virus well (Borthwick, 2014). This study has proved the principle that an artificial construct comprising several (fourteen) short regions of the HIV-1 genome is immunogenic, and that at least a proportion of the induced T-cells can recognize virus-infected cells. There was a risk

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that the construct was so artificial that induced T-cells might not see naturally processed virus antigens. There is discussion ongoing whether this construct or one of the alternatives, designed on similar principles, offers the best coverage of the range of potential infecting viruses. In addition, one report suggests that conserved epitopes are better recognized in the context of full proteins (Stephenson et al., 2012). Nonetheless, targeting conserved HIV-1 epitopes holds considerable promise for HIV-1 vaccine development. The potential advantage of the conserved immunogen approach is that, while not preventing infection, it could make virus escape less likely, because virus could only change these conserved regions with a viral fitness cost. While a mosaic vaccine might also generate responses to these same regions, they would at the same time stimulate responses to variable regions; the latter, if immunodominant, might interfere with the antiviral potency of CD4 or CD8 T-cells specific for the conserved epitopes (Liu et al., 2013). Ultimately a direct comparison between these related vaccine approaches should be made. Probably the best in vitro test for monitoring conserved or mosaic vaccine-induced effector functions, will be measure how T-cells suppress the replication of viruses in vitro (Freel et al., 2012b; Yang et al., 2012). Similar to the design of good Env constructs for antibodies, only those vaccines that induce in vivo potent antiviral activity as measured in vitro should be considered for large scale efficacy trials. Protective innate immunity The use of adjuvants to stimulate innate immune responses to enhance primary adaptive immune responses is well known, and the adjuvant mechanisms have been elegantly revealed over the last 15 years (Medzhitov and Janeway, 1999). However, induction of innate immune memory is a new concept to vaccinology (Gillard et al., 2011; Paust and von Andrian, 2011; Sun et al., 2009, 2012). Besides contributing to adaptive responses, a key question recurs, is there evidence that innate immune responses impact directly on the control of HIV-1? In the very first stages of SIV infection of the genital mucosa, it has been argued that induction

of type I interferon and beta chemokines by plasmacytoid dendritic cells has potential antiviral effects, but also recruits new T-cells to the site of infection which are targets for the virus, promoting early spread (Bosinger et al., 2009). Type I interferon, produced in the very earliest stages of infection by plasmacytoid dendritic cells (PDC) ought to have a significant antiviral effect. However, two recent studies have shown that the primary T/F viruses are significantly resistant to type I interferon (Parrish et al., 2013; Fenton-May et al., 2013). This may be part of the process that limits the founder virus to a single species in most infections (Keele et al., 2008). If this is the case, vaccines that enhance type I interferon production on initial HIV-1 contact might be effective in further limiting acquisition of infection. Once early HIV-1 infection is established and spreads beyond local genital or rectal mucosa, there is a dramatic cytokine storm with induction of different cytokines in waves as the virus reaches peak viraemia. Interferon type I is central in this storm but other major contributors are IL-15, IL-10, IL-18 and IP10 (Stacey et al., 2009). Studies on leucocyte transcriptomes during acute infection reveal the multiple downstream consequences of this cytokine storm (Chang et al., 2012). What is not clear is whether these cytokines are beneficial or harmful; likely they are both. If the beneficial effects could be teased out, their activity might be enhanced by a vaccine, but again, this would need a vaccine that could prepare the innate system to make an enhanced response to HIV-1 at a later date. There is increasing evidence of immunological memory in the innate immune system (Chang et al., 2012; Gillard et al., 2011; Paust and von Andrian, 2011; Sun et al., 2009). This has been shown in mouse and macaque models where transfer of innate cells, with knock down of adaptive immunity, has given protection against reinfection or infection of the recipient animals. The mechanistic details are not known, but if it can be shown empirically that certain vaccine regimens have this property, then it could be harnessed for protection from HIV-1. In ongoing HIV-1 infection there is good genetic evidence that innate immune responses contribute to protection (Carrington and Walker,

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2012). The most striking is the association between the killer inhibitory receptor (KIR)3DL1 and KIR3-DS1 receptors on natural killer (NK) and T-cells, and HLA Bw4 molecules with isoleucine at position 80, with control of virus load and delayed progression (Martin et al., 2007). KIR3-DL1 is known to bind to Bw4 HLA molecules (a group of mostly B HLA allotypes that have a particular amino acid sequence motif between positions 77 and 81 on the alpha-1 helix). The binding is peptide-dependent, and the KIR3-DL1 delivers a negative signal back to the NK or T-cell. KIR3-DS1 is an activating receptor, also on NK and T-cells, which is an allelic form of KIR3-DL1 that has been postulated to bind to HLA Bw4 molecules. However, KIR3-DS1 does appear to mediate NK cell recognition of HIV infected cells that carry the HLA Bw4 epitope (Alter et al., 2007). It is curious that an inhibitory receptor and an activating receptor show the same properties, and it is possible that the protective effects in HIV-1 infection reflect another shared function of these receptors. Binding of KIR3-DL1 is peptidedependent, and one might therefore expect to see escape mutations selected in ligand peptides within the virus (Brackenridge et al., 2011). If KIR3-DS1 has a similar specificity, such escapes might be more prevalent, but so far, none have been shown convincingly. However, a repeatedly selected mutation in the VPU gene has been shown to enhance NK binding and inhibition through the KIR2-DL2 receptor, thereby downregulating NK cell attack and possibly enabling virus escape (Alter et al., 2011). These findings raise the intriguing possibility that better understanding of the peptides involved could lead to peptide-based vaccines that enhance NK cells activity in acute HIV-1 infection and this induce HIV-1-specific NK cell memory. Other ways that NK cell activity could be favourably enhanced involve the use of adjuvants. These effects are thought to be short-lived in nature, but ways might be devised to prolong such activation (i.e. memory) and thereby induce a strong barrier to subsequent HIV-1 infection. Clearly, this is an area of HIV-1 vaccinology that needs further investigation. While NK-mediated effects are unlikely to be fully protective on their own, they

could play and important accessory role in protecting from HIV-1 infection. Protective B-cell responses to HIV-1 Currently the field is working to learn to induce two types of antibody responses as possible mediators of protection from HIV infection: those antibodies that neutralize HIV-1, and those antibodies that do not neutralize HIV-1, but rather bind to the surface of virus-infected CD4 T-cells and mediate anti-HIV-1 activity by cell killing (antibody-dependent cellular cytotoxicity ADCC) or otherwise inhibiting virus-infected cells. Neutralizing antibodies The field of HIV-1 vaccine development originally worked to induce antibodies against the envelope third variable (V3) loop that was shown to be the principle neutralizing determinant of laboratoryadapted HIV-1 strains (Goudsmit et al., 1988; Javaherian et al., 1989; Palker et al., 1988). However, it was soon realized that laboratory-adapted, easy-to-neutralize or ‘tier 1’ HIV-1 strains were not representative of those strains in the field involved in transmission (Matthews, 1994). Keele et al. (2008) demonstrated that in ~80% of heterosexual HIV-1 transmissions, only 1 T/F virion is responsible for infection. Characterization of neutralization sensitivity of T/F viruses demonstrated that they were resistant to neutralizing monoclonal antibodies (mAbs) against the V3 loop, and were only sensitive to the rare broadly neutralizing antibodies (BnAbs). T/F viruses are generally more difficult to neutralize, and like many chronic primary virus strains isolated from infected patients, are classified as ‘tier 2’ viruses that are difficult-to-neutralize HIV-1 strains (Keele et al., 2008). Thus, a successful vaccine will need to neutralize and contain tier 2 HIV-1 T/F strains. Passive protection studies in rhesus macaques with neutralizing antibodies have consistently demonstrated protection from transmission of chimeric simian-human immunodeficiency viruses (SHIVs) (Hessell et al., 2009a; Hessell et al., 2009b; Mascola et al., 1999, 2000). Recently

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newer more potent BnAbs have been isolated that protect at remarkably low plasma antibody levels (2 µg/ml) – a level that is obtainable by a vaccine should a vaccine be available that could induce BnAbs (Moldt et al., 2012; Walker et al., 2011). Only ~15% of HIV-1 chronically infected patients develop high levels of plasma BnAbs (reviewed in Mascola and Haynes, 2013). Those HIV-1 patients who make BnAbs do not develop them soon after transmission; rather it takes 2–3 years on average to develop BnAbs (Doria-Rose et al., 2010; Gray et al., 2011; Mikell et al., 2011). Recent breakthroughs in recombinant antibody technology and clonal memory B-cell cultures have allowed for a number of new BnAbs to be isolated over the last two years and have revealed 4 general Env conserved areas, the gp41 membrane proximal external region (MPER), the gp120 CD4 binding site (CD4bs), the gp120 variable 1 and variable 2 (V1V2) N156, N160 site, and the base of the third variable (V3) loop and other glycan sites (reviewed in Burton et al., 2012; Mascola and Haynes, 2013) (Fig. 14.3 and Table 14.1). Interestingly, each of the BnAbs isolated thus far have one or more of the following unusual traits: reactivity with non-HIV-1 antigens, either host (autoreactivity, reactivity with host molecules) or non-human (polyreactivity, reactivity with environmental antigens), high levels of somatic mutations, and long and frequently

hydrophobic third heavy chain complementaritydetermining regions (HCDR3s) (Table 14.1) (Haynes et al., 2012b; Verkoczy et al., 2011b). All of these unusual traits predispose antibodies to forms of immune regulation, most commonly due to immune tolerance mechanisms (reviewed in Haynes et al., 2012b; Verkoczy et al., 2011b). For antibodies with extremely unusual germline ancestor antibodies (i.e. those with long HCDR3s), another factor is rarity of accessible germline precursors available to respond to the particular epitope. Finally, each of the BnAb epitopes is, by definition, subdominant, and competition with other dominant Env epitopes, even when BnAb precursors are not deleted, can lead to non-neutralizing antibodies dominating in the Env-driven germinal centre response. Specificities of broad neutralizing antibodies Gp41 MPER Antibodies against the membrane proximal region of the gp41 component of the HIV-1 envelope were among the first BnAbs reported (Fig. 14.3). One, 2F5, binds to the ELDKWA sequence in the MPER while another mAb, 4E10, binds to the NWFDIT in the same membrane proximal area (Muster et al., 1993; Stiegler et al., 2001). Both of these antibodies are extremely 2F5, 4E10, 10E8 Gp41 membrane proximal region CD4 binding site 1b12, VRC01, HJ16, NIH45-46

V3 glycan PGT121–PGT123, PGT125–PGT128 gp41 Transmembrane

V1V2 region PG9, PG16, CH01-05, PGT141–PGT145

gp41 Ectodomain

gp120 Outer domain

gp120 Trimer association domain

Figure 14.3  Subunit organization of the membrane bound HIV-1 trimeric envelope with general locations of the broadly neutralizing antibody binding sites. Representative names are shown associated with antibody binding site locations. Adapted with permission from Mao (2012).

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Table 14.1  Specificity, VH and VL gene rearrangements and unusual traits of select HIV-1 broadly neutralizing antibodies

Antibody name

Env specificity

Isotype VH

HCDR3 % VH mutation length Polyreactivity Vκ/Vλ frequency (aa)

2F5

gp41 membrane proximal

IgG3

2–5

k1–13 15

24

Yes

4E10

gp41 membrane proximal

IgG3

1–69 k3–20 20

20

Yes

CAP206-CH12

gp41 membrane proximal

IgG1

1–69 k3–20 14

17

Yes

10e8. 7H6

gp41 membrane proximal

IgG3

3–5

λ3–19 26

22

No

2G12

gp120 carbohydrate

IgG1

3–21 k1–5

16

No

PGT121–PGT123

V3 epitope involving carbohydrate

IgG1

4–59 λ3–21 21–27

24

No

PGT125–PGT128, PGT130, PGT131

V3 epitope involving carbohydrate

IgG1

4–39 λ2–8

25–33

19

No

PGT135–PGT137

V3 epitope involving carbohydrate

IgG1

4–39 κ3–15 25–29

18

No

PGT141–PGT145

V1/V2 conformational epitope IgG1

1–8

k2–28 21–29

31/32

No

1b12

gp120 CD4 binding site

IgG1

1–3

k3–20 20

20

Yes

VRC01, VRC02

gp120 CD4 binding site

IgG1

1–2

k3–11 40–48

14

No

VRC-PG4, VRCPG4b

gp120 CD4 binding site

IgG1

1–2

k3–11 38–39

16

No

CH30-CH34

gp120 CD4 binding site

IgG1

1–2

k1–33 13

15

No

NIH45–46

gp120 CD4 binding site

IgG1

1–2

k3–20 41

18

Yes

CH03–CH106

gp120 CD4 binding site

IgG1

4–59 λ3–1

22

13

Yes

HJ16

gp120 CD4 core

IgG1

3–3

37

21

ND

PG9, PG16

V1/V2 conformational epitope IgG1

3–33 l2–14

17–20

30

No

CH01–CH04

V1/V2 conformational epitope IgG1

3–20 k3–20 23–29

24

Yes, CH03

polyreactive with 2F5 reactive with anionic lipids and the tryptophan pathway enzyme kynureninase (KYNU) and 4E10 reactive with lipids and splicing factor 3b subunit 3 (SF3B3) (Alam et al., 2007, 2009; Dennison et al., 2009; Haynes et al., 2005a; Yang et al., 2013). The auto-reactivity of these mAbs raised the possibility that induction of these types of BnAbs were controlled by tolerance mechanisms (Haynes et al., 2005a,b), and through the use of homologous recombinant BnAb VH(D)JH + VLJL knock-in (KI) mice, it has now been shown that indeed this is the case (Verkoczy et al., 2010, 2011a,b). In the case of both 25F and 4E10 KI mouse strains, >95% of BnAb precursor B-cells expressing the BnAb B-cell receptors are deleted in the bone marrow, while the 5% of remaining BnAb B-cells reside in peripheral tissues in an anergic state. Immunization with gp41 constructs that bind the 2F5 BnAb

k4–1

32

can ‘wake up’ tolerized anergic peripheral BnAb B-cells and in the absence of competition from non-BnAb B-cells, can make clinically significant levels of plasma BnAbs (Verkoczy et al., 2013). Thus, strategies are being developed to induce rare clones of anergic BnAb B-cells to respond and clonally expand. Recently, a new MPER mAb 10e8 was described that binds to an epitope central to the MPER regions bounded by the 2F5 and 4E10 epitopes, that does not have obvious polyreactivity, but does have remarkably high levels of somatic hypermutations (~21%) (Huang et al., 2012). Other members of this lineage do have polyreactivity and the hypothesis is that 10e8 was a rare member of the lineage with sufficient mutations to achieve BnAb activity and yet not have polyreactivity – its other clonal lineage members being reduced in number due to polyreactivity Haynes BF et al. (unpublished).

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Gp120 CD4bs The prototype gp120 BnAb, 1b12, was derived from a phage displayed library with a randomly paired light chain, and showed moderate neutralization breadth (Saphire et al., 2001). More recently, using single memory B-cell sorting and recombinant antibody technology, a number of new specificities of CD4bs BnAbs have been isolated with much more potent and broader neutralization magnitude and breadth than 1b12 (Fig. 14.3). These include the VRC 01 and a host of VRC01-like BnAbs that all share VH1 VH(D) JH chains (Chen et al., 2009; Diskin et al., 2011; Kwong and Mascola, 2012; Scheid et al., 2011; Zhou et al., 2010). While of different sequences, these antibody Fab regions all bind to gp120 in a manner homologous to CD4, and indeed resemble CD4 in structure (Zhou et al., 2010). Other CD4bs BnAbs recently isolated include CD4bs/DMR/Core specificities that bind near the CD4bs and depend on gp120 amino acids D474, M475 and R476 for reactivity (Pietzsch et al., 2010). Finally, Liao et al. (2013b) have described a CD4bs BnAb lineage, the CH103 lineage, with moderate breadth as well as moderate levels of somatic mutations (12–17%), that bind to the CD4 binding site loop and V5 loop in a novel loop-based mechanism. The importance of this latter antibody for vaccine design is that the lineage arose within 14 weeks of transmission, and it was relatively uncomplicated with fewer mutations than other CD4bs BnAbs, and thus may be easier to induce (Liao et al., 2013b). Gp120 quaternary V1V2 The first V1V2 quaternary neutralizing antibody (quaternary refers to mAb that preferentially bind HIV-1 trimers vs. monomers) was mAb 2909 that potently neutralize the Tier 1 HIV-1 strain, SF162 (Spurrier et al., 2011). The first BnAbs against this epitope were mAbs PG9 and PG16 (Walker et al., 2009) followed by MAbs Ch01-CH04 (Bonsignori et al., 2011, 2012a) (Fig. 14.3). This class of broad neutralizing antibodies recognizes the V1V2 region glycans at N156 and N160 as well as the V2 region around K169. In contrast, the restricted neutralizing antibody 2909 only recognizes the glycan at N156 and requires a lysine at 160 to bind. All of these antibodies are

characterized by an extraordinarily long HCDR3 region with 2909 21 aa, PG9 and PG16 30aa, and Ch01–04 24 aa (Bonsignori et al., 2011, 2012a; Walker et al., 2009). The long HCDR3 of this BnAb class is significant for two reasons. First, antibodies with long HCDR3 regions are commonly deleted from the repertoire in the bone marrow and thus are rare in the germline repertoire (reviewed in Haynes et al., 2012b). Second, structural studies have demonstrated those rare V1V2 quaternary BnAbs that are made require the long HCDR3 to form a projection that can engulf glycans at N160 and N156 and project to the V2 core (McLellan et al., 2011). Thus, the hypothesis why these BnAbs are rare is that the requirement for long HCDR3s for neutralization predisposes these BnAb precursors to be rare. Moreover, like other BnAbs, the mutations accumulated during V1V12 BnAb maturation are high at ~15–20% (Haynes et al., 2012b). V3 and other glycan sites The first prototypic anti-glycan BnAb was mAb 2G12 that binds to a conformational cluster of glycans on Env gp120 (Trkola et al., 1995). This is a dimeric BnAb with a unique heavy and light chain domain swap structure that would be expected improbable to be induced with a vaccine (Calarese et al., 2003). More recently, new, more traditional structured anti-glycan BnAbs have been isolated that bind glycans 301 and 332 at the base of the V3 loop (Pejchal et al., 2011; Walker et al., 2011; Mao et al., 2012) (Fig. 14.3). The prototypes of these antibodies, PGT121 and PGT128, are more potent than previously described antibodies and can mediate complete protection from the R5 SHIV SF162P3 at ~2 µg/ml, a plasma amount achievable with a vaccine (Moldt et al., 2012). However, the glycans found on HIV-1 virions are derived from host human cells and therefore are poorly immunogenic in humans. Hence, induction of glycan antibodies is complicated by gp120 glycan similarity with host glycans, and as well, again, by the BnAb requirement for long tortuous pathways of maturation when they do occur – manifested by extraordinary SHM levels in the PGT antibodies of ~20% (Walker et al., 2011). Thus, all of the BnAbs reported to date are unusual in a number of respects and their induction

348  | Haynes et al.

will require circumventing aspects of peripheral tolerance and anergy. Equally important is the need to find germline BnAb precursors that are sufficiently common to be able to target with a vaccine prime immunization, and then drive to BnAb development with immunogen boosts. Such a strategy is known as B-cell lineage vaccine design, and is a strategy that is currently being pursued for practical vaccine development (Haynes et al., 2012b). Non-neutralizing antibodies In 2009 the ALVAC-HIV/AIDSVAX gp120 B/E vaccine was tested in Thailand in a low-risk heterosexual population, and unlike prior gp120 and recombinant Adenovirus 5 vaccines that showed no vaccine efficacy, the Thai RV144 vaccine showed an estimated vaccine efficacy of 31.2% (Fig. 14.1) (Rerks-Ngarm et al., 2009). An extensive immune correlate analysis of immune responses present in vaccinees to a greater extent compared to placebo recipients demonstrated two immune responses that correlated with transmission risk. IgG antibodies to the gp120 V1V2 loop inversely correlated with transmission risk (i.e. the greater the V1V2 antibody, the lower the infection risk), and plasma IgA antibodies to Env directly correlated with infection risk (i.e. the greater the Env IgA level, the higher the level of infection risk) (Haynes et al., 2012a). A viral genetic analysis studying virus envelope V2 sequences of viruses that occurred in vaccinees vs. placebos demonstrated that the vaccine-exerted immune pressure at amino acid 169 in viruses that matched the vaccine in V2 sequences (Rolland et al., 2012). To study the V1V2 correlate of infection risk, four V2 mAbs were isolated from RV144 vaccinees and studied for their effector functions in order to further develop hypotheses how protection might have occurred in the RV144 trial (Liao et al., 2013a). Each of these V2 antibodies contained V2 K169 in their footprint of binding, and crystal structure of two of these mAbs complexed with V2 peptides confirmed the mAb contacts with K169 (Liao et al., 2013a). Importantly, the isolation of these 169-targeted mAbs allowed the profiling of their immune effector functions. The RV144 V2 antibodies all targeted K169, neutralized tier 1 but not tier 2 viruses, and did not

capture tier 2 infectious virions. Since most of the plasma neutralizing activity induced in RV144 was only against tier 1 and not tier 2 virions, these data suggested that either any protective effect of V2 antibodies induced by RV144 were targeted for neutralization against tier 1 transmitted/founder viruses in the study (which are likely to be few), or more likely, were mediated by non-neutralizing antibody effector functions. Thus, the RV144 V2 antibodies were studied for their ability to bind to tier 2 virus-infected CD4 T-cells and to mediate ADCC. Indeed, all 4 V2 antibodies from RV144 vaccinees could bind to the surface of tier 2 virusinfected CD4 T-cells and mediate ADCC (Liao et al., 2013a). Thus, a prime hypothesis that is as yet unproven, is that protection in the RV144 trial was mediated by ADCC (Bonsignori et al., 2012b; Liao et al., 2013a). The second correlate of risk that was found in the RV144 immune correlates analysis was the surprising finding that the higher the plasma Env IgA levels, the higher the risk of transmission (Haynes et al., 2012a). There was no enhancement of infection risk with vaccination in the trial. Rather, the findings gave rise to the hypothesis that IgA binding to sites of ADCC-antibody binding could mitigate the effect of potentially protective ADCC-mediating antibodies (Haynes et al., 2012a). The ADCC response in RV144 was indeed targeted to the V2 region, but the dominant epitope induced in the trial was to a conformational ADCC epitope in the first constant (CI) gp120 region as defined by MAb A32 (Bonsignori et al., 2012b). Evidence for this hypothesis that IgA could block ADCC also came from an association analysis that demonstrated that ADCC antibodies in the presence of low plasma Env IgA, correlated with decreased transmission, whereas ADCC in the presence of high plasma Env IgA did not. Further studies have demonstrated that IgA MAbs isolated from RV144 vaccinees indeed can block RV144 vaccinee IgG MAbs in mediation of ADCC (Tomaras, 2013). Thus, in-depth dissection of the RV144 efficacy trial immune responses has uncovered multiple layers of interacting antibodies that appear to have modulated the outcome of the trial. This manner of analysis of clinical trials has set the standard for monitoring of future HIV-1 vaccine efficacy trials, and as

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well, allowed the field to focus on the V1V2 region and other gp120 regions such as the C1 region, for evaluation as targets of protective antibodies. Thus, the field is approaching the protective antibody problem from two directions. First attempts are being made to improve on what was achieved in RV144 by increasing the quantity of what was induced by use of a more powerful adjuvant, and by improving the quality of nonneutralizing antibodies that were induced by using more antigenic and immunogenic Env gp120s. Second, progress has been made in understanding both the targets and the nature of the developmental pathways of broad neutralizing antibodies, and the strategy of use of B-cell lineage immunogen design is a promising strategy for understanding how to induce and drive BnAbs (Haynes et al., 2012b). Examples of current vaccine strategies A number of vaccine strategies that are currently being used or considered for future clinical efficacy trials. A multivalent clade A, B and C DNA prime, recombinant Adenovirus type 5 boost is currently in the HVTN 505 efficacy trial that is scheduled to be completed in 2014 (Churchyard et al., 2011). In addition, recombinant Adenovirus 26 (rAd26) prime, modified vaccinia Ankara (MVA) boost has shown robust induction of T-cell responses in rhesus macaques with limited protection in the SIV challenge model (Barouch et al., 2012). DNA prime, NYVAC (attenuated New York strain vaccinia) has been immunogenic in humans and is being considered for an efficacy trial in South Africa, with NYVAC as a prime for a protein boost (McCormack et al., 2008; Perreau et al., 2011; Precopio et al., 2007). Both rAd26/ MVA and DNA prime, NYVAC boost will include mosaic inserts discussed above for clinical trial testing. The conserved vaccine discussed above is in human clinical trials and is being tested in a DNA prime chimpanzee adenovirus 63 (chAd63) regimen (O’Hara et al., 2012). The hope is that chAd63 immunogenicity will not be affected by any pre-existing immunity in humans as are human rAds. The ALVAC prime, rgp120 boost regimen is

being considered for a follow-up efficacy trial in Thailand with a more potent adjuvant than alum that was used in RV144, as well as two or more immunogenic Envs (Haynes BF, Korber, BT, Kim J, Michael, N, unpublished). Finally, a number of strategies are being considered based on preclinical studies that include DNA and pox vector prime with a protein boost (Goepfert et al., 2011), replicating vectors such as replicating recombinant Ad4 (Gurwith et al., 2013), and attenuated cytomegalovirus (CMV) (Hansen et al., 2011). As mentioned above, the latter strategy has been of interest because of the remarkable protection induced in 50% of rhesus macaques via vaccine induction of effector memory T-cells (Hansen et al., 2011). Summary The results of the RV144 vaccine efficacy trial has given the HIV vaccine development field hope that a vaccine for HIV-1 can indeed be made. Similarly, the identification of new targets of BnAbs, and the realization that a number of these specificities can be made in chronically infected patients has provided new hope for inducing these types of antibodies. Finally, the design of conserved and mosaic vaccine constructs to overcome T-cell epitope diversity and the use of novel replicating vectors has provided a degree of efficacy in pre-clinical studies. Nonetheless, major hurdles remain such as induction of BnAbs by vaccination, and either inducing a truncated pathway of BnAb or learning how to elicit the high degree of SHM needed for the most potent BnAbs to develop. A final successful vaccine will require not only multiple specificities of protective B-cell responses, but multiple specificities of protective T-cell responses to provide complete and lasting protection from most HIV-1 strains. Recent insights have suggested this daunting goal is achievable, but will require quite novel vaccine approaches. Acknowledgements The authors acknowledge the expert secretarial assistance of Kim McClammy. This work supported by the Center for HIV-1/AIDS Vaccine Immunology-Immunogen Discovery grant

350  | Haynes et al.

from the NIH, NIAID, Division of AIDS, UM1AI00645 and a Collaboration for AIDS Vaccine Discovery grant to BFH from the Bill & Melinda Gates Foundation. References

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Haynes, B.F. (2010). Autoreactivity in an HIV-1 broadly reactive neutralizing antibody variable region heavy chain induces immunologic tolerance. Proc. Natl. Acad. Sci. U.S.A. 107, 181–186. Verkoczy, L., Kelsoe, G., Moody, M.A., and Haynes, B.F. (2011b). Role of immune mechanisms in induction of HIV-1 broadly neutralizing antibodies. Curr. Opin. Immunol. 23, 383–390. Verkoczy, L., Chen, Y., Bouton-Verville, H., Zhang, J., Diaz, M., Hutchinson, J., Ouyang, Y.B., Alam, S.M., Holl, T.M., Hwang, K.K., et al. (2011a). Rescue of HIV-1 broad neutralizing antibody-expressing B cells in 2F5 VH x VL knockin mice reveals multiple tolerance controls. J. Immunol. 187, 3785–3797. Verkoczy, L., Chen, Y., Zhang, J., Bouton-Verville, H., Newman, A., Lockwood, B., Scearce, R.M., Montefiori, D.C., Dennison, S.M., Xia, S.M., et al. (2013). Induction of HIV-1 broad neutralizing antibodies in 2F5 knock-in mice: selection against membrane proximal external region-associated autoreactivity limits T-dependent responses. J. Immunol. 191, 2538–2550. Walker, L.M., Phogat, S.K., Chan-Hui, P.Y., Wagner, D., Phung, P., Goss, J.L., Wrin, T., Simek, M.D., Fling, S., Mitcham, J.L., et al. (2009). Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326, 285–289. Walker, L.M., Huber, M., Doores, K.J., Falkowska, E., Pejchal, R., Julien, J.P., Wang, S.K., Ramos, A., ChanHui, P.Y., Moyle, M., et al. (2011). Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 477, 466–470.. Wiktor, S.Z., Ekpini, E., Karon, J.M., Nkengasong, J., Maurice, C., Severin, S.T., Roels, T.H., Kouassi, M.K., Lackritz, E.M., Coulibaly, I.M., et al. (1999). Short-course oral zidovudine for prevention of motherto-child transmission of HIV-1 in Abidjan, Cote d’Ivoire: a randomised trial. Lancet 353, 781–785. Wilkinson, T.M., Li, C.K., Chui, C.S., Huang, A.K., Perkins, M., Liebner, J.C., Lambkin-Williams, R., Gilbert, A., Oxford, J., Nicholas, B., et al. (2012). Preexisting influenza-specific CD4+ T cells correlate with disease protection against influenza challenge in humans. Nat. Med. 18, 274–280. Williams, L.D., Bansal, A., Sabbaj, S., Heath, S.L., Song, W., Tang, J., Zajac, A.J., and Goepfert, P.A. (2011). Interleukin-21-producing HIV-1-specific CD8 T cells are preferentially seen in elite controllers. J. Virol. 85, 2316–2324. Wong, J.K., Strain, M.C., Porrata, R., Reay, E., SankaranWalters, S., Ignacio, C.C., Russell, T., Pillai, S.K., Looney, D.J., and Dandekar, S. (2010). In vivo CD8+ T-cell suppression of siv viremia is not mediated by CTL clearance of productively infected cells. PLoS Pathog. 6, e1000748. Yamane, H., and Paul, W.E. (2012). Cytokines of the gamma(c) family control CD4+ T cell differentiation and function. Nat. Immunol. 13, 1037–1044. Yang, G., Holl, T.M., Liu, Y., Li, Y., Lu, X., Nicely, N.I., Kepler, T.B., Alam, S.M., Liao, H.X., Cain, D.W., et al. (2013). Identification of autoantigens recognized

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Cancer Immunotherapy: The Road to Rejection Peter E. Fecci, Christina Chen, Susanne Baumeister and Glenn Dranoff

Abstract Immunotherapy continues to gain both momentum and legitimacy as a rational mode of cancer therapy. The idea that the immune system, if correctly manipulated, might be capable of surveying, identifying, and eradicating, in precise fashion, those cells that have mutant or newly expressed proteins en route to neoplastic growth patterns has long proved intriguing. This idea has been gradually shaped into a variety of immunotherapy approaches ranging from antibodies to cell transfers to vaccines. A large number of tumour types are also under study, including melanoma, glioma, lung, prostate, breast, head/neck, cervical, ovarian, pancreas, renal cell, colorectal cancers, and lymphoma. This chapter will review the current state, applications, adjuncts, and challenges to the successful immune-based treatment of cancer, emphasizing clinical trials. Introduction In 2010, the FDA approved sipuleucel-T (PROVENGE, Dendreon Corp.) for the treatment of metastatic hormone-refractory prostate cancer (Kantoff et al., 2010), marking the first endorsement of a cancer vaccine in the U.S. and heralding the evolving trend towards legitimacy for tumour immunotherapy. This endorsement mounted the shoulders of the first FDA approval for immunotherapy more generally (traztuzumab) in 1998 (Slamon et al., 2001) and was further underscored by the 2011 approval of ipilimumab (anti-CTLA-4 monoclonal antibody) for metastatic melanoma.

15

Significantly, these FDA approvals span multiple immunotherapeutic modalities, which are most classically divided into ‘passive’ and ‘active’ forms. Here, ‘passive’ denotes the transfer of a non-native mode of immunity, whose lifespan is directly related to the half-life of the modality so transferred (such as antibodies directed against tumour antigens). By contrast, ‘active’ immunotherapies aim to stimulate nascent immunity and ultimately constitute resident long-term memory. They, in turn, are distinguished primarily by vaccine-based approaches, which typically aspire to solicit activation of anti-tumour T-cells. As immunotherapies advance in number and complexity, however, these traditional lines demarcating active and passive become more blurred, and therapies such as adoptive transfer of ex vivo-activated autologous lymphocytes or antibodies endeavouring towards immune checkpoint blockade fit less neatly into these historical boxes. The features of a particular cancer likely make it suited to treatment with one modality of immunotherapy over another. The presence of an over-expressed receptor on an immunologically accessible tumour might proffer the ideal scenario for a monoclonal antibody-based therapy, for instance, while a hematologic malignancy requiring bone marrow transplantation might present a favourable opportunity for transferring lymphocytes into a myeloablated host, where they might homeostatically expand ( Jameson, 2002; Surh and Sprent, 2008). To the contrary, weakly over-expressing tumours sheltered behind the blood–brain barrier are likely to prove challenging for an antibody-based therapy, and promiscuous

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regulatory T-cells may suggest utility for immune checkpoint blockade. Ultimately, understanding the nuances to the various immunotherapeutic modalities, as well as to the immune contexts in which various cancers reside, will be critical to realizing the potential of immune-based tumour therapies. This review will focus on the rationale and developments characterizing anti-tumour vaccines, immune checkpoint blockade, and adoptive lymphocyte transfer (ALT) (Table 15.1). Vaccines Vaccination against most tumours represents an effort to stimulate host immunity to established disease, where this immunity has already failed in its cancer surveillance task. The challenge then is to ‘reprogram’ the immune response by providing it some combination of new targets, new contexts, or new resistance to tumour-elaborated counter-measures. Differences among strategies relate to the vehicle used to deliver the antigenic target, the numbers of targets provided, and the immune-stimulant (adjuvant) that is often co-administered. Taken in aggregate, vaccination strategies offer the capacity for potentiated adaptive immune-activation, antigen(s)/targetspecificity, multi-arm immune engagement, and the realization of immunological memory. Cell-based strategies Tumour cell vaccines Early generation cancer vaccines took a traditional and familiar form, simply employing ‘killed or inactivated’ tumour cells as fodder for the immune system. After some ill-fated early attempts combining irradiated tumour cells with adjuvant (Finke et al., 2007), various groups in the 1980s and early 1990s engineered a variety of cytokine-secreting tumour cell vaccines, the most notable of which was designed to elaborate GM-CSF (Dranoff et al., 1993). While GM-CSF exercises an array of immune effects, its influence in the context of vaccination appears to be the local recruitment of multiple immune cells, particularly dendritic cells. These, in turn, contribute to vaccine efficacy by phagocytosing, processing, and presenting the tumour cells co-provided. The ultimate effectors

are CD4+ and CD8+ T-cells, CD1d restricted NKT cells, and antibodies, which collectively contribute to tumour destruction. Following an initial phase I study in metastatic renal cell carcinoma (RCC) (Simons et al., 1997), GM-CSF vaccines were trialled in melanoma and prostate cancer (Simons et al., 1999; Soiffer et al., 1998; Luiten et al., 2005) and eventually nonsmall cell lung cancer (NSCLC), breast cancer, and pancreatic cancer (Salgia et al., 2003; Nemunaitis et al., 2006; Emens et al., 2009; Jaffee et al., 2001; Laheru et al., 2008). The vaccine format evolved from retroviral-mediated gene transfer to an adenoviral vector (Salgia et al., 2003), subsequently progressing to GM-CSF secreting allogeneic cell lines employed alone ( Jaffee et al., 2001) or in combination with autologous irradiated tumour cells (Nemunaitis et al., 2006). While employment of an allogeneic GM-CSFproducing line permits the standardization of cell manufacture, the antigenic relationship to autologous tumours remains uncertain, and the lines might trigger a response to irrelevant histocompatibility antigens. Allogeneic GM-CSF secreting prostate carcinoma cell vaccines (termed GVAX, Cell Genesys) have advanced to two phase III trials for hormone-refractory metastatic prostate cancer. The first trial, VITAL-1, randomized 626 patients to prostate-GVAX alone versus a standard combination of docetaxel + prednisone and targeted improvement in overall survival as the primary endpoint (Higano et al., 2009a). Meanwhile, VITAL-2 evaluated GVAX and docetaxel in conjunction versus a docetaxel and prednisone combination (Small et al., 2009). Cell Genesys terminated the VITAL-2 early when the GVAX/ docetaxel group revealed more deaths than the docetaxel/prednisone arm (67 >47), although this difference did not persist with longer followup. VITAL-1 was also terminated early when it became clear that the goal of improved overall survival (OS) was unlikely to be met. Failures of the allogeneic GM-CSF prostate vaccines may be revealed in the complex biology of GM-CSF. Despite its use as adjuvant in the trials highlighted above, in some systems, tonic GM-CSF production by tumours facilitates immune-evasion and progression (Sotomayor et

Cancer Immunotherapy: The Road to Rejection |  361

al., 1991), and some peptide vaccine trials have highlighted a potentially tolerogenic role for the cytokine, inhibiting vaccine efficacy (Slingluff et al., 2009). GM-CSF’s split personality, soliciting immunity in some instances and tolerance at others, may prove to be context-dependent. A deciding factor appears to be the molecular ‘switch’ milk fat globule EGF-8 (MFG-E8), a phosphatidylserine binding protein that facilitates the integrin-mediated and tolerizing phagocytosis of apoptotic cells by APC (Hanayama et al., 2002). In heralded moments of immunologic ‘danger,’ MFG-E8 becomes down-regulated, and GM-CSF preferentially triggers cytotoxic immunity via MFG-E8-independent mechanisms ( Jinushi et al., 2007). This protective pathway can be encouraged via the application of a dominant negative MFG-E8 mutant (RGD to RGE), which retains the ability to bind phosphatidylserine but cannot engage tolerogenic integrin signalling on APCs. Indeed, GM-CSF-secreting tumour cell vaccines incorporating RGE have their in vivo effects potentiated in mouse models ( Jinushi et al., 2007), and in vitro systems recapitulate the ability of a human MFG-E8 mutant to block the tolerizing effects of GM-CSF in patient samples (N Souders and G Dranoff, unpublished observations). These findings suggest a new strategy to maximize the activity of GM-CSF secreting tumour cell vaccines, which is currently being translated to phase I clinical testing. Dendritic cell vaccines The concept of professional antigen presentation was conceived following the discovery of the dendritic cell (DC) by Steinman and Cohn in 1973. By the early 1990s, a firm role for the DC system in immunogenicity was established, this encompassing most notably the functions of peripheral antigen capture and presentation; migration to the T-dependent areas of lymphoid organs; and presentation to and activation of naive T-cells (Steinman, 1991). Following the realization that antigens presented by DC could include tumour antigens (Huang et al., 1994), DC were similarly ascribed a role in physiologic anti-tumour immunity (Grabbe et al., 1995). Accordingly, the expansion, loading, and activation of DC with tumour antigens ex vivo were soon advocated as

a rational vaccination strategy for triggering antitumour immunity (Fig. 15.1). The first published trial of DC-based cancer vaccination enrolled patients with B-cell lymphoma in 1996 (63), and with regard to solid tumours, melanoma soon followed suit (64–66), as did prostate cancer (Murphy et al., 1999; Fong et al., 2001a), renal cell carcinoma (Holtl et al., 1999; Kugler et al., 2000), non-small cell lung carcinoma (Fong et al., 2001b), and colon cancer (Morse et al., 2000). Early trials (as above) most typically pulsed dendritic cells with either tumour cell lysate or specific peptide antigens, and pursuant trials experimented with further modes of antigen loading; immature versus mature DC; methods of maturation; and routes of administration; none of which remain standardized to this day. DCs continue as one of the most tried methods of tumour vaccination, however, with additional cancers such as breast (Peethambaram et al., 2009; Koski et al., 2012) and glioma (Okada et al., 2011; Prins et al., 2011; Sampson et al., 2009; Wheeler et al., 2008; Yu et al., 2001, 2004) having served also now as frequent subjects of study. Most prominently, however, DCs form a presumed mainstay of sipuleucel-T, the first FDA approved anti-tumour vaccination strategy (Kantoff et al., 2010). Approval of sipuleucel-T was predicated on results from a collection of phase III trials (Small et al., 2006; Higano et al., 2009b) whose culmination was the IMPACT study, published by Dendreon in 2010 (Kantoff et al., 2010). IMPACT was designed to reflect an increasingly observed immunotherapeutic trend. Prior trials had noted survival benefits despite no change in time to disease progression, a phenomenon thought to represent initial ‘inflammatory expansion’ of lesions on imaging prior to eventual disease regression. IMPACT was thus designed differently, with ‘overall survival’ as its primary endpoint. IMPACT enrolled 512 men with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer. Patients were randomized to receive either sipuleucel-T (n = 341) or placebo (n = 171). Peripheral blood mononuclear cells (PBMC) were harvested by leukapheresis and cultured for 36–44 hours in the

Glioma

Melanoma

MART-1 encoded plasmid DNA (80) CMV-specific (97)

Anti-PD1/PDL-1 (133-135)

Tyrosinase Gp100/ Pmel17-like epitope encoded antigen (57) plasmid DNA (79)

Immune Anti-idiotype checkpoint vaccine blockade

Anti-CTLA4 (109,110,112,116)

DNA Vaccine

Viral-based vaccine

Synthetic Gp100 encoded peptide (56) plasmid DNA (78)

EGFRviii Alpha-type 1 peptide polarized dendritic variant (42) cells (33) Glioma lysate-pulsed DCs (34, 36-38) EGFRviii targeted (35)

Gp100 peptide (66)

GM-CSF-transduced (10)

Self-derived differentiation antigens (147)

Retrovirus encoded T-cell receptor transduced (148)

Synthetic long peptides (64)

GM-CSF-secreting (9)

Tumour cell vaccine

Antigenic Dendritic cell vaccine vaccine

Autologous TIL (144)

CD19- specific chimera coupled with CD137, CD3-zeta (171)

Leukaemia Autologous CD19targeted T-cells (170)

Autologous CD19 targeted T-cells (171)

Lymphoma CD20-specific chimera (168, 173)

Hematological

Adoptive lymphocyte transfer

Clinical trials

Table 15.1  A summary of immunotherapy clinical trials highlighted in this chapter, organized by cancer type and modality.

GM-CSF secreting (14) GM-CSF vs. GM-CSF + cyclophosphamide (15)

Pancreas

ERBB2 transduced T-cells (174)

Autologous TIL vs. TIL + cisplatin (145) Autologous TIL (146) Anti-folate receptor chimeric T-cells (166)

Cervical

Ovarian

Colon

GM-CSF secreting + cyclophosphamide, doxorubicin (13)

Breast

Sipuleucel-T vs. placebo(39, 40)

GVAX + docetaxel vs. docetaxel + prednisone (17)

Flt3 expanded dendritic cells (30)

DC1-polarized dendritic cells (32)

Lapuleucel- T (31)

Flt3 expanded dendritic cells (29)

PAP pulsed DC (26)

GVAX vs. docetaxel + prednisone (16)

GM-CSF Transduced (8) Dendritic cells + PSM-P1, -P2 (25)

GM-CSF-secreting (11) GVAX + autologous tumour (12)

Anhydrase IX T-cells (165)

Prostate

Tumour cell-dendritic cell hybrids (28)

GM-CSF Transduced (7) CD83+ dendritic cells (27)

NSCLC/ SCLC

Anti-Carbonic

RCC

Carcinoma

E6, E7 synthetic long peptide (69)

Modified vaccinia virus expressing MUC1 (91, 92)

Poxviral-based (89, 93)

Cervarix (94, 96) Cervarix vs. Gardasil (95)

ONYX015 + gemcitabine

HER2 encoded TRICOM vector plasmid DNA (83) based (88)

PAP encoded (81, 82)

1E10 antiidiotype (99, 100)

Anti-PD-1/PDL-1 (135)

Anti-PD-1/PDL-1 (135)

Anti-PD-1/PDL-1 (133-135)

Anti-PD1/PDL-1 (133, 134)

Anti-CTLA4 (113115)

Anti-PD1/PDL-1 (133-135)

Anti-CTLA4 antibody (111)

364  | Fecci et al.

Y

Y

Y

Tumor antigen linked to cytokine

Mature DC infused back into patient

Y

Y

Antigen/cytokine complex binds to dendritic cell (DC)

Complex taken in by DC

T cell

Y

Activated T cells attack cancer cells

Y

Y

Y

Y

DC displaying tumor antigen activates T cells

Cancer cells

Y

Figure 15.1  Dendritic cells (DC) are isolated or manufactured from patient’s blood and loaded with tumour antigen(s) via one of a variety of mechanisms. Typically, the DCs are then triggered into a maturation program to enhance co-stimulatory and migratory capacities. The mature DCs are injected back into the patient as a vaccine, with the goals of migration to lymph nodes and presentation of tumour antigens to T-lymphocytes.

presence of PA2024, a fusion of prostate cancer antigen prostatic acid phosphatase (PAP) and GM-CSF. The activated cells, which potentially include dendritic cell ‘precursors,’ were then delivered intravenously every two weeks for a total of three treatments. Placebo consisted of a one-third dose of PBMC cultured in the absence of PA2024; the remainder was cryopreserved, and could be thawed subsequently and administered as a crossover option for patients on the control arm. The proposed mechanism of therapeutic action was ex vivo uptake of antigen (PA2024) by the activated APC precursors, which subsequently elicit either vaccine-inclusive or nascent T-cell priming. The specific cellular content and properties of the vaccine remain under active investigation. IMPACT met its primary overall survival endpoint, as patients treated with sipuleucel-T demonstrated an increased median survival of 25.8 months compared with 21.7 months in placebo-treated patients, resulting in a 22% relative reduction in risk of death (HR, 0.78;

95% confidence interval, 0.61–0.98; P = 0.03) (overall, 63.7% of placebo patients eventually received APC8015F – these patients had a 23.8 month median survival compared to 11.6 months for those who received placebo only; non-randomized, no statistics available). As with prior studies, no differences were seen between sipuleucel-T and placebo with regard to time to ‘objective’ or ‘clinical’ disease progression, despite the benefit to overall survival. Development of either antibody or T-cell immunity to the immunizing PAP–GM-CSF fusion (PA2024) far exceeded development of similar immunity to the nascent PAP antigen (i.e. T-cell responses to PA2024 arose in 73% of patients in the sipuleucel arm, whereas responses to PAP developed in only 27.3%). Furthermore, the appearance of such T-cell responses to either the proprietary fusion or native antigen proved of unclear significance, as no correlate between T-cell responses and survival was observed (a correlation was observed however for the emergence

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of high antibody titres to either antigen). Adverse events were prevalent (98%) but mild, represented by mostly flu-like symptoms, presumably due to cytokine release. Ultimately, the significant extensions observed for median and 3-year survival and the reasonable safety profile precipitated what has been a landmark FDA approval. The experience with sipuleucel-T has not yet resolved many of the uncertainties with DC vaccines, which range from methods of production, to delivery mode, to means of cell activation. One major question remains the optimal mode of DC antigen loading. While manufacturing of sipuleucel-T consists of co-culture (pulsing) of APC with a tumour-associated peptide (fused to ‘adjuvant’ cytokine), little consensus on such methodology exists to date, and DC have been additionally pulsed with tumour extract or lysate; apoptotic bodies; RNA; acid-eluted peptides; or even fused to tumour cells prior to enlistment (Fecci et al., 2003). As antigenic sources, tumour lysates and eluted peptides offer the incentive of a broadened target repertoire, but with the concordant risk for autoimmunity to contaminating self-antigens. Additionally, as with tumour cell-based vaccines, the requirement for large amounts of available tumour can be prohibitive. Tumour-associated (or -specific when available) antigens (TAA or TSA) obviate the need for tumour tissue and offer the potential for a tumour-specific immune response, but tumour heterogeneity makes escape a likelihood when only one or a few antigens are targeted (Sampson et al., 2010). Furthermore, if the antigens are peptides, MHC-restrictions and the availability of satisfactory MHC I- and MHC II-helper epitopes within the antigenic target become restrictive. Whole protein antigen can bypass concerns over MHC restrictions, but the costs and efforts involved in synthesizing protein are limiting factors. Currently, reliable and prevalent TAAs and TSAs are few in number but are being increasingly identified through a variety of techniques. A recent meta-analysis attempted to examine and prioritize some of the unresolved issues regarding DC vaccines, assessing the factors that influence clinical response rates (clinical benefit rate (CBR) defined as summed rates of partial,

complete, and mixed responses, as well as stable disease). Beginning with manufacturing methodology, comparisons of monocyte-derivation and density-enrichment revealed an advantage to the latter, lending support to the methods employed in IMPACT for sipuleucel-T production. With regard to DC activation, Chi-square tests revealed a significant positive influence of DC maturation status on the CBR in prostate cancer patients. Route of administration was also examined, with analysis yielding better clinical response rates for vaccination routes with presumed access to draining lymph nodes (intradermal (id); subcutaneous (sc); intranodal (in); intralymphatic (il)) than for the intravenous (iv) route (Draube et al., 2011). Despite limitations to the study, the efforts to address what remain unresolved issues for a promising therapeutic modality are laudable. Other cell-based vaccine approaches Although most technically a ‘cell-free’ vaccination modality, it is relevant here to mention an additional strategy that employs not entire dendritic cells, but rather somewhat poorly understood cell-derived ‘components’ termed exosomes. Exosomes are nanovesicles originating from late endosomal compartments and are secreted by most living cells under ex vivo culture conditions. Exosomes were initially described among reticulocytes, where they were ascribed functions permitting the elimination of such molecular vestiges as the transferrin receptor. Ultimately, however, DC-derived exosomes, or Dex, were shown capable of modulating immune responses (Zitvogel et al., 1998; Thery et al., 1999), either directly by exposing MHC and costimulatory molecules, or indirectly by conveying internal components, even genetic material, to surrounding cells. Dex have proven to be fairly potent immunogens in murine models (Zitvogel et al., 1998) and confer singly an advantage over DC that they may be more efficiently manufactured from cell lines ex vivo. A phase II clinical trial employing peptide-pulsed autologous DEX as maintenance immunotherapy for inoperable nonsmall cell lung cancer (NSCLC) was launched in November 2009 and continues currently at the Gustave Roussy and Curie Institutes. A relatively unique tumour cell-derived

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approach has been to administer the equivalent of multiple non-identified peptides in the form of tumour-derived heat shock protein–peptide complexes (HSPPC). Heat shock proteins (HSP) are highly conserved, stress-induced proteins that possess chaperone function within a cell and may associate with intracellular peptides (including tumour antigens), shuttling them from the proteasome to the endoplasmic reticulum, and potentially mediating transfer to MHC I. One such HSP is glycoprotein-96 (gp-96), which when isolated from autologous tumour homogenate as a complex with peptides (HSPPC-96, VItespen, formerly Oncophage), has served as a vaccination platform in phase III trials for metastatic melanoma and renal cell carcinoma. In both cases, no differences in survival were observed between treatment and control arms (Wood et al., 2008; Testori et al., 2008). Antigenic vaccines T-cells demonstrate the capacity to recognize a variety of proteins whose expression may be limited (specific) to or increased (associated) among tumours. Many tumour-specific antigens (TSA) represent mutational products (i.e. EGFRvIII in malignant glioma (Sugawa et al., 1990)) or viral proteins, the latter as the result of a viral superinfection (i.e. questionably CMV in malignant glioma (Cobbs et al., 2002; Mitchell et al., 2008)) or even aetiology (i.e. HPV-16 in cervical cancer (Walboomers et al., 1999)). Tumour-associated antigens (TAA), being more so simply overexpressed by tumours, are not surprisingly often developmental, differentiation, or growth-promoting proteins. The first TAA uncovered was MAGE-1 for melanoma in 1991, and the capacity to generate CTL recognizing the protein was simultaneously demonstrated (van der Bruggen et al., 1991). Following its discovery, vaccines specifically targeting MAGE-1, as well as other promptly identified antigenic targets, rapidly emerged (Cormier et al., 1997; Marchand et al., 1999) (the first clinical trial actually targeted gp100 (Rosenberg et al., 1998a)). Popular melanoma targets have since then included gp100 (Kawakami et al., 1994b), MART-1 (Kawakami et al., 1994a), MAGE-1 (van der Bruggen et al., 1991), MAGE-3 (Chaux et al.,

1999), tyrosinase (Brichard et al., 1993), and NY-ESO-1 (Zeng et al., 2000). ‘Antigenic’ vaccines comprise the delivery of a protein or peptide antigen itself, often in conjunction with an immune-stimulating adjuvant. Much as peptides are used to pulse DC vaccines ex vivo, this is, in effect, an attempt at in vivo pulsing of nascent DC. Thus, the goal remains antigen uptake by native APC and priming of similarly resident T-cells, while adjuvant [e.g. incomplete Freund’s adjuvant (IFA), poly-I:C] provides the necessary contextual ‘danger.’ Decision points mostly relate to format (i.e. whole protein versus peptide), route of delivery (much as for DC vaccination) and choice of adjuvant (a thorough discussion of which is beyond the scope of this chapter). Regarding the former, factors include cost (advantage: peptide), in vivo stability (advantage: peptide), and breadth of antigenic selection (advantage: protein). In dissecting proteins into antigenic components, computer modelling can be used to predict the MHC class I and class II epitopes within proteins that are most likely to instigate CTL-mediated immunity and CD4 help, respectively. This, in turn, permits the synthesis of the relevant epitope-spanning peptides, which is often a more cost-effective means of vaccine production than manufacturing complete recombinant protein sequences, for instance. The price, however, of focusing on specific peptide epitopes versus whole protein is likely the same as focusing on a single antigen versus whole cell lysates – the door is left increasingly ajar for immune escape. A salient demonstration of this proclivity, even in the face of successful immunological targeting, is found in the reports of Sampson et al. regarding their phase II experience with peptide vaccination for malignant glioma, during which 82% of recurrent tumours proved to be antigen-loss variants (Sampson et al., 2010). In essence, this is a ‘real life’ depiction of Schreiber’s immunoediting process, whereby the immune system controls tumour outgrowth while simultaneously shaping tumour immunogenicity (Dunn et al., 2002; Matsushita et al., 2012). Peptide vaccines are also subject to HLArestrictions that limit their patient pool, as any employed epitope possesses a specific, constrained HLA-binding capacity. Patients’ HLA haplotypes

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(and thus their range of recognizable epitopes for any antigenic protein) are exceedingly varied, although HLA-A2 still appears frequently amidst the multitude in the US Caucasian population. Classically then, U.S. peptide vaccine trials have employed HLA-A2-restricted peptides, making HLA-typing and positive ‘A2 status’ a frequent inclusion criterion in the respective trials. This restrictiveness can at times be partially abrogated by the administration of longer or additional peptides harbouring multiple epitopes. The employment of synthetic long peptides (SLP) likely proffers a number of advantages for vaccine efficacy over short peptide administration, beyond simply a broadened epitope repertoire (reviewed in (Melief and van der Burg, 2008)). It is increasingly suggested that minimum length peptides (i.e. 8–10 amino acid peptides that mimic a single predicted MHC I epitope) prove tolerogenic when administered in vaccine form (Bijker et al., 2007). Most salient is the proposition that such short peptides are directly capable of binding the empty MHC I of non-professional APC, including B- and T- cells, an event that can prove to be an immunologic dead end of sorts. Nonetheless, the co-administration of cytokine adjuvants such as IL-2 or GM-CSF may overcome some of these limitations (Schwartzentruber et al., 2011; Walter et al., 2012). By contrast, SLPs uniquely require uptake and processing by the desired professional APC (DC), which will more selectively present antigen in the intended inflammatory contexts/locales. Efficacy is further enhanced, it appears, by co-joining helper epitopes and adjuvant (i.e. TLR agonists) in tandem within the SLP, a notion substantiated early in the HIV vaccine literature (Shirai et al., 1994). Regarding cancer therapies, a noteworthy SLP clinical trial produced complete responses in 9 of 19 women with vulvar intra-epithelial neoplasia vaccinated against the HPV-16 oncoproteins E6 and E7 (Kenter et al., 2009). Newer generation antigenic vaccines look to improve on the immunogenicity of the peptide or proteins employed. Such strategies include furnishing peptides with leader amino acids sequences that improve affinity for class I MHC, increasing the durability of presentation and theoretically improving the likelihood of CTL priming

(Bei and Scardino, 2010). Additionally, the central deletion of self-reactive T-cells during thymic development can limit the availability of a reliable effector arm with specificity for immunodominant epitopes; this limitation can be circumvented via the provision of subdominant or cryptic epitopes, which may solicit immunity either directly or indirectly through epitope spreading. Additional strategies involve the creation of ‘vectors’ to improve antigenic delivery to immune cells, the most salient examples of which are likely liposomal delivery systems, meant to increase APC-uptake. Phase III trials with L-BLP25, a liposomal vaccine targeting the extracellular core peptide of MUC1 are under way for NSCLC (Stimuvax, Merck) (Gridelli et al., 2009). A similar approach involves targeting antigenic vaccines for in vivo DC-mediated uptake by adjoining a moiety that binds the DEC-205 receptor on DC. Employed in pre-clinical models to date, targeting the breast cancer antigen HER2 to DEC-205 substantially improves long-term mouse survival to tumour challenge when compared to nontargeted HER2 vaccine (Wang et al., 2012). Given the central role for DC in mediating the efficacy of antigenic vaccines, such strategies represent a rational move along the path to next-generation approaches. DNA vaccines DNA vaccines, in their simplest form, are characterized by the administration of plasmids encoding single epitopes, multi-epitopes, or whole protein versions of selected TAA(s). Most typically, they are delivered intramuscularly with aspirations for myocyte plasmid uptake and TAA expression or, alternatively, they can be delivered into the skin and keratinocytes, respectively. TAA-expressing myocytes may subsequently serve as immune targets, whose cytotoxic destruction releases antigen and inflammatory mediators into the milieu, compelling local APC uptake and cross-presentation. Alternatively, it may simply be that local APC within the muscle serve as targets for transfection. In either event, the vaccine then proffers an in vivo ‘antigen depot’ of sorts, usurping host cellular machinery (much as would a virus) to produce the vaccine target. The resultant human ‘expression system’ ensures appropriate folding and

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post-translational modification when relevant, and therefore fewer errors en route to presentation (Bei and Scardino, 2010; Rice et al., 2008; Stevenson et al., 2010). Furthermore, plasmid DNA, being double-stranded and rich in unmethylated CG sequences, serves as a relatively potent danger signal for the innate immune system, and therefore as an adjuvant in and of itself. DNA vaccines additionally afford the capacity to more pro-actively mould immune context and garner anti-tumour favour, for instance by ‘plugging’ in ‘extra’ cDNAs for pro-inflammatory molecules, such as cytokines or chemokines (Zhu and Stevenson, 2002). More clever vaccine adaptations include fused gene sequences for Fc receptors and antibodies that can ‘lead’ the TAA to APCs, or even sequences that alter the intracellular destinies of the TAA and optimize its processing and presentation. Regarding the latter, targeting an antigen to the cytosol and/or incorporating a ubiquitin sequence can facilitate class I presentation, for instance, while encoding a marker for endoplasmic reticulum shuttling can likewise enhance class II presentation and antibody production (Leifert et al., 2004; Rice et al., 2008). It is therefore a not untoward expectation to ‘manufacture’ the desirable combination of innate, adaptive, and humoral immunity. Clinical experience with DNA vaccines has disproportionately involved melanoma (Rosenberg et al., 2003; Tagawa et al., 2003; Triozzi et al., 2005), prostate (McNeel et al., 2009; Becker et al., 2010), and breast cancers (Norell et al., 2010). Interestingly, DNA vaccines have sustained their greatest advancement in the veterinary arena (Bergman et al., 2003, 2006). However, with regard to human cancers and DNA vaccines, it is a small minority that has demonstrated evidence of the desired immune reactivity, with exceedingly little suggestion of objective clinical responses. Some modicum of success has been elicited by boosting the vaccines, either with the same plasmid or with viral vectors. This is demonstrated in a recent prostate cancer trial, where such boosters indeed proved requisite for soliciting PAP-specific IFN-γ responses (Becker et al., 2010). Continued proponents of DNA vaccination cite difficulties translating dosing schedules and poor in vivo transfection rates as some of the leading

reparable culprits. One means of enhancing transfection efficiency has been via electroporation. Electroporation involves the application of electrical current to the skin or muscle at the site of vaccination, concomitant with plasmid injection (or immediately thereafter). Pre-clinically, it has overtly demonstrated the capacity to improve transfection rates and elevate antigen expression 10- to 100-fold, as well as to instigate danger and inflammatory cell recruitment (Liu et al., 2008). It has similarly been employed as a means of loading RNA or DNA into DC’s in vitro prior to DC vaccination. Electroporation may help to abrogate requirements for large volumes or plasmid loads, although its clinical efficacy for anti-tumour DNA-based immunization still awaits appropriate examination. Viral-based vaccines A variety of viral-based anti-cancer approaches are being explored today, ranging from immune-targeting antigen-delivery systems to tumour-targeting suicide gene delivery vectors to directly oncolytic viruses. A noteworthy example of the latter is the employment of oncolytic herpes virus encoding GM-CSF (OncovexGM–CSF) in patients with metastatic melanoma (Kaufman et al., 2010). Most commonly, however, virus-based vaccination strategies involve utilizing virus as a vector for conveyance of antigen to the immune system. If DNA vaccines are defined by the administration of ‘naked’ DNA, then viral vaccines are analogously the ‘clothed’ equivalent, with the viral coat enhancing delivery into APC. It has not been uncommon, in fact, for viral delivery of the same DNAs to be used as a ‘boost’ strategy following initial priming with a naked DNA vaccine. Trials have been most prevalent in breast (Garnett et al., 2006), prostate (Gulley et al., 2010), pancreatic (Hecht et al., 2003), and lung cancers (Rochlitz et al., 2003; Ramlau et al., 2008), with CEA, MUC-1, and PSA serving as common antigenic targets conveyed by virus. PROSTVAC-VF/ TRICOM (BN ImmunoTherapeutics Inc.), was currently in phase III trials for castration-resistant prostate cancer as of 2011. In a preceding phase II study, therapy began with a vaccinia virus carrying a modified immunogenic PSA gene, as well as genes for three immune-stimulatory molecules

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meant to enhance T-cell priming: CD80, ICAM1, and LFA-3 (the TRICOM component). This priming immunization was followed by a series of fowlpox boosters containing the same genes (to limit neutralizing anti-viral antibodies), and adjuvant GM-CSF was delivered subcutaneously in proximity. Once again, there was no statistically significant difference in PFS, but patients in the treatment arm (n = 82) experienced an 8.5-month OS advantage compared with patients in the control (empty virus + saline, n = 40) arm (25.1 months vs. 16.6 months, HR 0.56; 95% CI 0.37– 0.85, P = 0.0061). PSA-specific T-cell responses were not assessed, although have been observed in other phase II studies with the therapy. Antibodies to PSA were measured but not detected, while all patients demonstrated antibodies to the viral vectors, a phenomenon that did not correlate with survival (Gulley et al., 2010). As noted, viruses have also served as the antigenic target of interest, in both preventative and therapeutic vaccine strategies. Preventative cancer vaccines targeting viral antigens are most relevant when a viral aetiology to the cancer of interest is understood. In this regard, cervical cancer and vaccines targeting HPV have clearly earned the discussional forefront. Two vaccines, Cervarix and Gardasil, targeting HPV 16/18 and HPV 6/11/16/18 respectively, are now widely available commercially and being instituted in international vaccination programmes for at-risk women and even men (McKeage and Romanowski, 2011). In phase III trials of Cervarix, which appears to be the more effective vaccine in head to head studies (Einstein et al., 2009), preventative rates for cervical intraepithelial neoplasia (CIN) are greater than 80–90% depending on grade, and approach 100% in certain cohorts (Paavonen et al., 2009). Regarding therapeutic vaccines, the use of viral peptides as antigenic targets has primarily been employed in glioma, where recent studies (albeit not entirely non-controversial) have uncovered the selective re-expression of seemingly latent CMV proteins within tumour cells (Cobbs et al., 2002; Mitchell et al., 2008). The debatable origin of this CMV expression aside, tumour immunologists may be afforded a potent, non-self antigen for targeting, a highly coveted scenario. As a result, three clinical trials combining DC-pulsed

with CMV peptides and CMV-specific T-cells are active at Duke University Medical Center for patients with malignant glioma (Hickey et al., 2010). Anti-idiotype antibody vaccines: a brief word Although this mode of immunotherapy is characterized by delivery of an antibody, it is more accurately an antigenic mimic designed to instigate the development of a host immune response, and thus qualifies for mention in a tumour vaccine review. Anti-idiotype antibodies are produced as xenogeneic antibodies with specificity for the antigen-binding groove of antibodies to the tumour antigens of interest. This ‘antibody to an antibody’ thus actually more closely resembles the antigen of interest, but its xenogeneic nature makes it quite highly immunogenic, and its mimicry permits cross recognition of the resident protein antigen. The effector arm of this vaccine mode primarily enlists tumour-specific antibody induction, and this predilection for humoral immune responses makes it perhaps best suited to ‘liquid’ tumours. It has however, been employed with some success in models of melanoma and breast cancer (Vazquez et al., 2000), and in patients with NSCLC (Alfonso et al., 2007). Of particular note, it has been one of the most widely employed modes of immunotherapy for small cell lung carcinoma (SCLC), which is often non-operative at presentation and treated with chemotherapy/radiation and observation alone (Manjili, 2007). Unfortunately, a phase III trial of an anti-GD3 ganglioside antibody (BEC) plus BCG conducted by Merck for SCLC revealed no significant survival benefit (Giaccone et al., 2005). Continued proponents of this vaccine mode, however, point to more immunogenic gangliosides, such as N-glycolyl GM3 and its anti-idiotype counterpart 1E10, as potential harbingers of future success with this approach. Vaccines: addressing limitations Tumour vaccines share the common goal of active, adaptive immunity, the realization of which is necessarily dependent on an intact T-cell effector arm. Unfortunately, the state of nascent ‘immune affairs’ is frequently more troublesome.

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Specific tumours exhibit varying capacities for immune-escape, which provide increasingly recognized challenges to successful vaccine therapy. Such capacities range in their proclivities from ‘simple’ down-regulation of MHC I and co-stimulatory molecules (tumour invisibility) to outright ‘attacks’ on the immune response with accompanying global systemic immunosuppression (tumour countermeasures/immune subterfuge). Some of the latter include the induction or expansion of regulatory T-cells (Tregs) and the shuttling of activated T-cells into ‘dead end’ immune checkpoints, typified by binding of surface inhibitory molecules that solicit inactivation (CTLA-4) or even apoptosis (PD-1). The next waves of tumour vaccine/immunotherapy trials, then, must incorporate means of side-stepping, or even meeting head-on, tumour mechanisms for immune-subterfuge. Traditionally, strong adjuvants have provided limited means for overcoming what are at the very least raised thresholds for productive immune activity. More tailored and ‘intelligent’ approaches have included depletion of Tregs with e.g. anti-CD25 monoclonal antibodies, although these have the caveat of collaterally removing recently activated T-cells that express intermediate levels of the same IL-2 receptor α-chain ( Jacobs et al., 2010). Increasingly popular strategies for releasing the brakes on immune responses address the aboveintroduced notion of immune-checkpoints. Immune checkpoints Following the fruitful commencement of an adaptive immune response, routine modes for switching off that same response must exist, lest the potential for unwanted inflammation or autoimmunity become realized. The physiologic provisions for this mechanism are ‘immune checkpoints,’ typified by molecules on activated T-cells, signalling via which can lead to inactivation (CTLA-4) or even apoptosis (PD-1). Conversely, blockade or antagonism of these same molecules and their intracellular signalling pathways can potentiate T-cell responses and even render them insensitive to some of the tumours’ evolved mechanisms for inhibition (Pardoll, 2012). CTLA-4 has additionally

been implicated in mediating regulatory T-cell function or sensitivity, and PD-1 has been characterized as an inducible marker for peri-tumoural T-cell exhaustion, particularly in melanoma and prostate cancer to date. Strategies targeting both molecules represent viable and promising modalities, either alone, or perhaps more appropriately, in combination with current vaccine platforms to synergistically amplify or potentiate vaccinestimulated responses (Fig. 15.2). CTLA-4 The most extensively studied immune check point receptor is CTLA-4. It is expressed exclusively on T-cells and acts to regulate the amplitude of early stages of T-cell activation. It dampens the activation of T-cells by competing with the costimulatory CD28 for binding of B7 on APC. Conversely, the blockade of CTLA-4 can increase the availability of CD28 co-stimulation and amplify/perpetuate T-cell activation. Such effects on T-cells are global and antigen non-specific, a fact, which, when combined with the lethal autoimmune phenotypes of CTLA-4 knockout mice, spurred initial fears for the safety of anti-CTLA-4 as an anti-tumour modality. A landmark study led by Leach et al. (1996) helped pave the path to anti-CTLA-4 utilization. This study demonstrated, in mouse models, the ability of transient CTLA-4 blockade to achieve anti-tumour responses in partially immunogenic tumours without overt immune toxicities. Pursuant studies highlighted the rejection of several types of established transplantable tumours in treated mice, including colon carcinoma, fibrosarcoma, prostatic carcinoma, and renal carcinoma (van Elsas et al., 1999; Kwon et al., 1997; Shrikant et al., 1999; Korman et al., 2006). Tumour-suppressive activity, however, proved dependent on the immunogenicity of the tumour. In poorly immunogenic tumours such as B16 melanoma, anti-CTLA-4 monotherapy was ineffective, but a combination with GM-CSF-secreting tumour cell vaccines achieved effective responses (van Elsas et al., 1999). Analogous combinations with other vaccines or chemoradiation likewise proved efficacious (Korman et al., 2006). These preclinical results led to the testing of the fully humanized anti-CTLA-4 monoclonal antibodies (mAb)

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CTLA-4

T-cell

Proliferation of Effector T cell

Anti-CTLA-4 mAb

Effector T-cells

B7.1 PD-1

PDL-1

TCR

Tumor infiltration and destruction

CD28

MHC

B7.1/7.2

APC IDO

-4

CTLA

Treg

Cancer Cells Inhibitory cytokines

CTLA-4

Figure 15.2  Antigen presenting cells (APC) present tumour antigens and elicit activation of tumour-specific T-cells. The amplitude of the T-cell response may be limited by signalling via the immune checkpoints CTLA-4 and PD-1. Antibody blockade of CTLA-4 and PD-1 in turn perpetuates T-cell activation, in part by increasing permissiveness for CD28-mediated co-stimulation.

ipilimumab (MDX-010) and tremelimumab (CP675,206) in 2000. The first clinical demonstration of anti-CTLA-4 was carried out by Hodi et al. (2003) when they administered ipilimumab (anti-CTLA-4, Bristol-Myers-Squibb) to nine previously vaccinated metastatic melanoma or advanced ovarian cancer patients (Hodi et al., 2003). Ipilimumab was administered as a one-time dose (3 mg/kg) over 1.5 hours. Results indicated safety for the single-dose profile, and raised the possibility of combination-specific synergy with other immune modalities (five of five patients previously immunized with G-VAX demonstrated responses, but minimal effects were seen in four patients previously immunized with defined melanoma antigens). Autoimmune effects in these patients were minimal (rash, but no evidence of vitiligo). Combined data from three subsequent monotherapy studies administering various doses and schedules of ipilimumab indicated a total response rate of 5%, and a 10% incidence of severe adverse

events, mainly grade III–IV gastrointestinal toxicities (Weber, 2008). Other data on ipilimumab as monotherapy come from studies comparing single agent ipilimumab with combination therapies. Response rates remain 5–15%, with typically better rates seen in combination groups. Parallel study of ipilimumab monotherapy was conducted in renal cell carcinoma, where the antibody was given as a 3 mg/kg loading dose followed by either 1 mg/kg or 3 mg/kg every three weeks. Treatment had a response rate of 5% in the lower dose group and 13% in the higher dose group. Response duration was segregated based on prior IL-2 therapy. Patients who had previous IL-2 therapy had shorter duration responses, while longer responses were seen among patients who had not had prior IL-2. Adverse events were also correlated with tumour regression: all six responders were in the group of 20 patients with significant adverse immune events (Yang et al., 2007). The profile of adverse events was similar to that reported in melanoma patients (enteritis,

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colitis, hypophysitis, and rash), suggesting that such events hold no correlation with the type of cancer being treated. Similarly suggested is the notion that autoimmunity may be a ‘welcome’ and even necessary surrogate for the breaking of self-tolerance that can mark the achievement of proper anti-tumour immunity, although this remains controversial. One of the most notable trials with ipilimumab was a phase III randomized clinical trial comparing patients with advanced melanoma receiving either: (1) a gp100 specific peptide vaccine alone; (2) gp100 vaccine plus ipilimumab; or (3) ipilimumab alone. This study demonstrated a 3.5month survival benefit among patients in either of the groups receiving ipilimumab compared with the group receiving gp100 peptide vaccine alone. The gp100 vaccine itself appeared relatively inert, conferring no benefit as monotherapy, and drawing no survival distinction between the two groups receiving ipilimumab (± gp100) (Hodi et al., 2010). Impressively, 18% of ipilimumabtreated patients survived beyond two years. Once again, the achieved results were accompanied by a 15–20% immune-related grade 3–4 adverse event profile (including a proclivity for hypophysitis), although this was not correlated with improved survival. The overall clinical activity, however, prompted FDA approval of ipilimumab for patients with metastatic melanoma in 2011. Since then, ipilimumab has increasingly been employed alongside vaccination therapy, with prominent recent examples including combinations with either Prostvac or GVAX for prostate cancer (Dranoff, 2012; Madan et al., 2012; van den Eertwegh et al., 2012). Numerous other studies with ipilimumab have been initiated including combination trials with existing chemotherapy in melanoma and monotherapy in pancreatic and cervical cancers. Similar to ipilimumab, clinical experience with tremelimumab came mainly from melanoma patients. Studies have been more geared towards tremelimumab as monotherapy and the optimal dose/scheduling. The first phase III randomized clinical trial completed with tremelimumab was conducted by Ribas et al. Patients with advanced melanoma either received 15  mg/kg tremelimumab monotherapy every three months or

standard dacarbazine chemotherapy. Survival benefit was not seen in the tremelimumab-treated group, however, perhaps because of insufficient dosing (Ribas et al., 2007). The pattern of often delayed responses observed with ipilimumab in clinical trials also did much to highlight the weaknesses associated with our current means of judging our successes and failures in immunotherapy trials. Classically, researchers have applied WHO criteria (Miller et al., 1981), and more recently, RECIST criteria (Therasse et al., 2000) for evaluating responses to cancer therapies. Terms such as complete response (CR), partial response (PR), mixed response (MR), stable disease (SD), and progressive disease (PD) are strictly defined and used to evaluate therapies, which have most typically comprised chemotherapy. As mentioned above, a notable pattern seen in immunotherapy trials was an unaltered time to progression, yet improved overall survival, a discordance that belied the common findings of disease regression following an initial progression, or, perhaps, of reductions in overall tumour burden, despite the appearance of new lesions. These patterns are not frequently observed with cytotoxic therapies, and WHO/ RECIST criteria mandate that such patients be included in the PD group when results are reported, necessarily hindering statistical analyses and diluting observed therapeutic effects. Furthermore, PD might require ceasing an experimental therapy. As a result, groups such as the Cancer Immunotherapy Consortium of the Cancer Research Institute have since attempted to design more appropriate ‘immune-response criteria’ to be applied during immunotherapy trials (Hoos et al., 2010; O’Regan et al., 2011; Wolchok et al., 2009). These are based more on tumour burden and broaden the types of observed patterns (including SD) that might qualify as response to therapy, in an effort to permit reasonable alignment between clinical outcome (overall survival) and reported responses to therapy. These have not been universally accepted, but continue to expand in recognition and use. PD-1 A second immune checkpoint under active study is programmed death-1 (PD-1, CD279),

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a member of the CD28 family that is expressed on activated T-cells, B-cells, dendritic cells, and macrophages (Sharpe et al., 2007; Pardoll, 2012). PD-1 engages two ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), which are both members of the B7 family. PD-L1 is expressed on a variety of immune and non-haematopoietic cells, while PD-L2 is restricted to myeloid cells. The PD-1 pathway functions to tune down inflammatory responses under normal physiological conditions, but may be exploited in cancer to accomplish immune escape. PD-1 is highly expressed on Tregs, and may signal to enhance proliferation and suppressive function upon ligand engagement. PD-1 is detected on a large proportion of tumour infiltrating lymphocytes, and PD-1 ligands (especially PD-L1) may be up-regulated on the surface of diverse tumour cells, including melanoma, ovarian cancer, lung cancer, renal cell cancer and glioblastoma (Pardoll, 2012). The enforced expression of PD-L1 on tumour cells diminishes susceptibility to cytotoxic lymphocytes and fosters tumour growth (Iwai et al., 2002). Moreover, high tumour PD-L1 expression is linked to inferior clinical outcomes in patients with cancers of the bladder, breast, kidney, stomach, and pancreas (Thompson et al., 2004; Hamanishi et al., 2007; Inman et al., 2007). While PD-1 expression on tumour infiltrating lymphocytes cells is associated with immune escape, antibody blockade of the receptor increases T-cell cytokine production and attenuates Treg mediated suppression in vitro (Pardoll, 2012; Sharpe et al., 2007). Consistent with these findings, aged PD-1 deficient mice manifest moderate autoimmune pathology, with high titres of autoantibodies and activated effector T-cells (Nishimura et al., 1999, 2001). The administration of blocking anti-PD-1 antibodies thereby reduces metastatic spread of B16 melanoma and CT26 colon carcinoma cells (Iwai et al., 2005) and decreases the growth of murine pancreatic adenocarcinoma cells (Nomi et al., 2007). Similar results may be obtained with anti-PD-L1 antibodies, whereupon tumour growth inhibition or rejection was observed in breast carcinoma, myeloma, and squamous cell carcinoma murine models (Strome et al., 2003; Hirano et al., 2005).

Based upon these encouraging pre-clinical experiments, human clinical trials with anti-PD-1 or PD-L1 monoclonal antibodies were initiated. The first study of a fully human anti-PD1 IgG4 antibody (MDX-1106) was undertaken in 39 patients with various solid tumours including metastatic melanoma, colorectal cancer, castrateresistant prostate cancer, non-small cell lung carcinoma (NSCLC), and renal cell carcinoma (RCC) (Brahmer et al., 2010). Cohorts of six patients received a single intravenous infusion of anti-PD-1 mAb at 0.3, 1, 3, or 10 mg/kg, with a 15 patient expansion cohort at the highest dose level. Treatment was generally well tolerated, and tumour regressions were observed in melanoma and lung, colon, and renal carcinomas. These exciting findings stimulated a second trial involving repetitive antibody dosing, with administration at 1, 3, or 10 mg/kg at 20week intervals for up to 12 cycles. Results of the first 126 patients revealed that these schedules were tolerable and biologically active (Pardoll, 2012). A follow-up report on 296 patients documented objective responses in a substantial proportion of NSCLC (14/122), melanoma (26/104), and RCC (9/34) patients, whereas high PD-L1 expression on tumours appeared to predict for higher response rates (Topalian et al., 2012). Phase I testing of a fully human anti-PD-L1 mAb (MDX-1105) has also been launched in patients with advanced melanoma, lung, ovarian, kidney, colon, stomach, pancreas, and breast cancer (Flies et al., 2011). The therapeutic benefits of PD-1 blockade appear to be durable in some cases, even after therapy is discontinued (136). Moreover, retreatment after tumour relapse may result in additional clinical responses (Lipson et al., 2013). Thus, phase III studies of PD-1 blockade are being developed for non-small-cell lung cancer, melanoma, and renal cell carcinoma. Because PD-1 blockade releases the ‘brakes’ on the immune system in a non-specific fashion, concerns have been expressed about potential autoimmune adverse effects. However, in contrast to the results with anti-CTLA-4 antibodies, anti-PD1 mAbs appear, at least thus far, to be better tolerated, although potentially lethal pneumonitis has been observed (Pardoll, 2012). The most common side effects of PD-1 blockade include fatigue,

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rash, diarrhoea, pruritus, decreased appetite, and nausea. In some animal models, antibodies blocking PD-L1 show greater anti-tumour effects than antibodies blocking PD-1 (Butte et al., 2007). This might reflect the ability of PD-L1 to signal in tumour cells, conferring enhanced resistance to apoptosis (Azuma et al., 2008). Some evidence also suggests that engagement of B7–1 and PD-L1 may inhibit T-cell activation and proliferation (Park et al., 2010). The effects of PD-L1/B7-1 interactions in the suppression of tumour immunity are not fully understood, however, and will require further investigation. A comparison of the clinical results with anti-PD-1 versus anti-PDL-1 mAbs will indeed be of great interest. Adoptive T-cell therapy The potential therapeutic role of lymphocytes directly was first identified through studies in metastatic melanoma, where the T-cell cytokine interleukin 2 (IL-2) was shown to mediate measurable responses in 15% of patients, leading to the drug’s FDA approval for this indication (Atkins et al., 1999, 2000; Rosenberg et al., 1998b). Melanoma also provided the first platform in which tumour infiltrating lymphocytes were isolated from tumour specimens, expanded and adoptively transferred back into the patient (Dudley et al., 2003). Depending on the preparative regimen, this resulted in clinical response rates of up to 72% in selected patients (Rosenberg et al., 2011). Adoptive transfer of autologous TILs has also shown promising results in pilot clinical trials for ovarian cancer (Aoki et al., 1991; Fujita et al., 1995). A major limitation of this approach, however, is that isolation of TILs is a labour intensive and technically difficult approach, and TILs cannot be obtained in all patients. To bypass this problem, Morgan et al. (2006) cloned the alpha and beta chains for a T-cell receptor (TCR) recognizing the tumour-associated antigen (TAA) MART-1 from a patient with metastatic melanoma who demonstrated near complete regression after adoptive transfer of TILs (Dudley et al., 2002). This allowed for adoptive T-cell therapy of HLA-matched melanoma patients after their autologous T-cells were engineered to express the

anti-MART1 TCR via retroviral gene transfer. In selected patients who were treated in this manner and sustained high levels of circulating engineered T-cells, objective regressions of metastatic melanoma lesions were seen (Morgan et al., 2006). Eshhar and colleagues pioneered an alternative approach for the rapid generation of TAA-specific T-cells through genetic modification of non-reactive T-cells to express a chimeric antigen receptor (CAR), which specifically binds to tumour antigens in an MHC-unrestricted fashion (Gross et al., 1989; Gross and Eshhar, 1992). CARs are fusion genes comprised of a single-chain variable fragment (scFv) antibody or other extracellular domain recognizing the TAA of interest, linked to intracellular signalling modules that mediate T-cell activation upon ligation of the CAR’s extracellular domain. Upon gene transfer of the CAR into T-cells, which can be performed using γ-retroviral or lentiviral vectors or electroporation ( June et al., 2009), the transduced T-cell acquires specificity for the targeted TAA, while retaining its endogenous TCR. One of the major advantages is that CARs recognize TAA in HLA–independent fashion and do not require presentation of the TAA via the MHC complex. This allows CAR+ T-cells (CARTs) to attack tumour cells in the immunosuppressive tumour microenvironment, where MHC-down-regulation is a major immune evasion mechanism. Furthermore, this obviates the need for HLA-matching and allows universal use of the respective CAR-construct for the transduction of patient T-cells and large-scale production of the viral vector for clinical use. However, CARTs still have to be manufactured individually, either from a patient’s autologous T-cells or from matched allogeneic donor-derived T-cells in the setting of hematopoietic stem cell transplantation. Additionally, CARTs can be targeted to any cell surface antigen to which a mAb can be generated, including targets such as glycolipids, which cannot be recognized by endogenous TCRs. However, CARs utilizing scFvs derived from murine antibodies can elicit a humoral and cellular immune response to the murine fraction and lead to CART-elimination (Lamers et al., 1995), thus prompting the development of humanized scFvs (Hombach et al., 2000).

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CARTs have become increasingly sophisticated, and several generations of CARs have been developed. First generation CARs typically consist of an extracellular single-chain variable fragment (scFv) of a monoclonal antibody, linked via a hinge and transmembrane region to an intracellular immunoreceptor tyrosine-based activation motif (ITAM) such as the CD3ζ-chain or less commonly the FcεRIγ. These CARs deliver ‘signal 1’ resulting in T-cell activation, target cell lysis, modest IL-2 secretion and in vivo anti-tumour function. However, they lack a costimulatory ‘signal 2’, which may result in T-cell anergy under physiological conditions. Therefore, second generation CARs have been designed to include an intracellular costimulatory domain such as CD28, ICOS or a TNFR family member such as 4-1BB or OX40 to mimic physiologic T-cell activation. The optimal combination of costimulatory signals within a CAR is still subject to debate and may vary depending on the clinical scenario. Comparative studies are complicated by other variables such as use of different scFvs, methods of transduction, tumour models and culture conditions. Unifying features of second-generation CARs include expression of anti-apoptotic proteins in resting T-cells and enhanced proliferation in response to antigen when compared to first-generation CARs. Several studies have demonstrated that inclusion of CD28 in the CAR construct enhances T-cell proliferation, cytokine production, up-regulation of anti-apoptotic proteins such as Bcl-xL and delayed activation induced cell death (Maher et al., 2002; Hombach et al., 2001; Kowolik et al., 2006). It further enhances resistance to the immunosuppressive effects of T-regulatory cells (Loskog et al., 2006) and transforming growth factor (TGF)-β (Koehler et al., 2007) in the tumour microenvironment. In contrast to first-generation CARs, 4-1BB co-stimulation results in improved T-cell survival and sustained proliferation, activation-induced death resistance and expression of anti-apoptotic proteins, enhanced cytokine production and notably increased antigen-specific tumour lysis (Zhao et al., 2009; Wang et al., 2007). Compared to TNFR containing CARs, CD28 costimulation leads to higher levels of IL-2, IFN-γ, TNF-α and GM-CSF secretion, whereas ICOS best mediates antigen-specific tumour cell lysis

(Finney et al., 2004). 4-1BB appeared superior in a xenograft model of acute lymphoblastic leukaemia (ALL) (Carpenito et al., 2009), but in a mesothelioma tumour model, CD28 and 4-1BB containing CARs were equivalent (Carpenito et al., 2009). Although 4-1BB signalling domains show a reduced propensity to trigger IL-2 and TNF-α (Milone et al., 2009) compared to CD28 domains, lower levels of these cytokines may be beneficial as they carry a reduced risk for cytokine storm and may lead to sustained clinical activity. Furthermore, lower IL-2 levels may be beneficial given that substantial amounts of IL-2 can lead to Treg-mediated suppression of CARTs (Lee et al., 2011). Finally, third generation CARs encompassing two costimulatory molecules have been designed. More stringent comparison of both second and third generation CARs in clinical trials should provide better insight as to the ideal combination in different clinical settings. CARs recognizing a variety of different targets have been developed. Some of the early proof-of-principle trials were directed against carboxy-anhydrase-IX (CAIX) for the treatment of renal cell carcinoma (Lamers et al., 2006), folate-receptor for metastatic ovarian cancer (Kershaw et al., 2006), CD171 for neuroblastoma (Park et al., 2007), and CD20 for non-Hodgkin’s lymphoma (Till et al., 2008). These trials showed minimal or manageable toxicities, but no clear efficacy and poor persistence of T-cells. To address persistence, the concept of transducing EBV+ or CMV+ T-cells has been explored, hypothesizing improved persistence due to antigenic TCR-stimulation and avoiding theoretical off-target toxicity against unknown antigens via the endogenous TCR. An anti-GD2-CAR trial in paediatric patients with neuroblastoma utilized this approach by infusing both EBV specific CARTs and polyclonal CARTs. While this trial showed encouraging clinical results in several patients, CART persistence was concordant with the percentage of CD4+ T-cells and central memory T-cells, rather than EBV + CARTs (Louis et al., 2011). Studies have further identified an inverse correlation between disease burden and persistence of CARTs and patients may thus benefit from conditioning chemotherapy (Brentjens et al., 2011; Park et

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al., 2007). Other factors that may contribute to CART persistence include CAR-costimulatory domains, exogenous administration of IL-2 and absence of an immune response against the CAR transgene. Overall, the degree of persistence of CARTs clearly appears to correlate with superior clinical responses (Louis et al., 2011; Porter et al., 2011). The target that has been best characterized in clinical trials so far is CD19. Expressed on B-cell malignancies as well as on healthy B-cells, it is absent on hematopoietic stem cells and early B-precursor cells, thus limiting undesired on-target toxicity to B-cell aplasia, which can be supported with intravenous immunoglobulin replacement. Several phase I/II clinical trials have evaluated the safety and efficacy of CD19 CARs in patients with chronic lymphocytic leukaemia (CLL), acute lymphoblastic leukaemia (ALL) and non-Hodgkin’s lymphoma. While the CD19–CAR constructs used in the respective trials differ in the scFvs against CD19, type of costimulatory domain(s) used as well as lentiviral versus γ-retroviral transduction regimens, the therapeutic promise of CD19-CARTs has been demonstrated by several independent groups (Brentjens et al., 2011; Kochenderfer et al., 2010). Porter et al. (2011) reported on three patients with CLL treated with chemotherapy followed by split infusion of a CD19–41BB-zetaCAR, whereby two patients achieved a complete and one patient a partial remission. Whereas in vivo persistence of CARTs beyond a few weeks was problematic in many of the early CART trials, this group demonstrated not only persistence of CARTs in the blood and bone marrow exceeding 6 months, but also in vivo expansion of infused CARTs by >3 log steps. Some patients were observed to develop a non-fatal cytokine release syndrome, but remarkable anti-tumour efficacy was demonstrated in patients who had been refractory to chemotherapeutic modalities. A pilot clinical trial using a third generation CD20-CAR containing CD28 and 4-1BB in patients with relapsed indolent B-cell and mantle cell lymphomas following cyclophosphamide lymphodepletion, showed continued freedom of disease in two and a partial remission lasting 12 months in the third patient (Till et al., 2012).

Clinical trials which are currently ongoing or have not reported clinical results are targeting CEA in breast cancer, IL-13Rα2 in glioblastoma, κ-light chain in non-Hodgkin’s lymphoma and B-CLL, CD30 in CD30+ lymphomas as well as Her2 in advanced Her2+ sarcomas and glioblastoma multiforme. Further, infusion of allogeneic CD19 CARs is now being evaluated in patients who relapsed after stem cell transplantation. One adverse event was reported for a patient with metastatic colon cancer who received a large dose of a third generation Her-2/CD28/41BB-zeta-CARTs and developed an ARDS-like clinical picture followed by multi-organ failure, likely due to low level Her-2 expression in healthy lung tissue (Morgan et al., 2010). A CLL patient treated with a CD19-CAR following chemotherapy died within days of the T-cell infusion, but this was attributed to an infectious etiology after data-review and autopsy (Brentjens et al., 2010). With careful dose-escalation regimens, CARTs have otherwise been infused safely and with manageable toxicity profiles at many different centres and even Her-2 CARs are now being employed again in clinical trials with cautious dose-escalation regimens. To improve safety and specificity of CARTs, several preclinical strategies have been investigated. These include the integration of inducible suicide genes such as HSV-tk or iCaspase 9 which can be pharmacologically activated and have been explored for the treatment and prevention of GVHD (Bonini et al., 1997; Tey et al., 2007; Di Stasi et al., 2011). Another strategy which may render T-cells specific for a tumour in the absence of a truly tumour-restricted antigen, involves combinatorial antigen recognition in which T-cells are transduced both with a CAR that provides suboptimal activation upon binding of one antigen, and a chimeric costimulatory receptor recognizing a second antigen. In this scenario, full T-cell activation is only achieved if both CARs bind to their respective antigen (Kloss et al., 2013). Contrary to gene transfer into hematopoietic stem cells, gene transfer into differentiated T-cells has not been associated with insertional mutagenesis or leukaemogenesis. Over 540 patient-years of follow-up from three clinical trials evaluating a CD4ζ-CAR for HIV infection, showed no evidence of persistent clonal

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expansion or enrichment for integration sites near genes implicated in growth control or transformation (Scholler et al., 2012). CAR-technology will no doubt be optimized by systematically comparing the role of different costimulatory domains, scFVs and transduction regimens. Additionally, several interesting CAR constructs have been explored in preclinical models and hold promise for clinical application. Examples include a CAR containing the NKG2D-receptor which targets the natural ligands of NKG2-D that are overexpressed on a multitude of tumours (Zhang et al., 2005), a polypeptide against VEGFR-2 (Niederman et al., 2002), IL-12 secreting CARTs (Pegram et al., 2012), and an anti-FITC CAR which could be used more universally to eradicate cells marked by a FITC-labelled antibody against various desired tumour-associated antigens (Curran et al., 2012). Other strategies of interest include expression of chemokine receptors such as CXCR2 and CCR4 to enhance trafficking of CARTs to the site(s) of tumour (Kershaw et al., 2002; Di Stasi et al., 2009), and coupling of negative T-cell regulators such as PD-1 to an activating intracellular signal allowing the T-cell to prevail over PD-L1 signals in the tumour microenvironment. Lastly, design of CARs requiring several conditional signals for full T-cell activation may allow for safer CARtherapy against targets that are not exclusively expressed on the tumour. Immunotherapy: a final word The lines connecting the currently emerged immunotherapeutic modalities represent a manner of divergent evolution, with multiple solutions sprouting as adaptations to a common challenge. Each modality has garnered validation, with successive renditions growing in complexity and applicability, expanding our understanding of the complexity that similarly characterizes the problem and its interface with the proposed solutions. Thus, we have reached new appreciation for the unique difficulties afforded by each tumour type; the variety of tumour-employed mechanisms for immune escape; the limitations of our attempts to produce and monitor responses; the considerations in choosing a particular modality alone or in

combination; the reasons for failure; and, most importantly, the opportunities for success. Maintaining objective views of our progress, flexibility in our approach, and recognition of the complexity of the task will be essential ingredients to the derivation of improved immunotherapy platforms and continued stepwise progress towards an ultimate goal. Web resources Cancer vaccine study for unresectable stage III non-small cell lung cancer. Available at: http://www.clinicaltrials. gov/ct2/show/NCT00409188 A Phase 3 efficacy study of a recombinant vaccinia virus vaccine to treat metastatic prostate cancer. Available at: http://www.clinicaltrials.gov/ct2/ results?term=NCT01322490 Trial of a vaccination with tumor antigen-loaded dendritic cell-derived exosomes (CSET 1437). Available: http:// www.clinicaltrials.gov/ct2/show/NCT01159288

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Global Health Vaccines Against the Invasive Salmonelloses: Enteric Fever and Invasive Non-typhoidal Salmonella Disease

16

Calman A. MacLennan

Abstract There is a growing awareness of the significance of Salmonella disease as a major public health concern, particularly in the developing world. This encompasses both enteric fever, caused by Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A), and gastrointestinal and invasive disease caused by Salmonella serovars collectively known as non-typhoidal Salmonellae (NTS). While responsible for gastroenteritis in high-income countries, NTS are a common cause of fatal invasive disease in low-income countries, particularly in Africa. Currently available licensed vaccines for use in man are Vi capsular polysaccharide (Vi CPS) and the Ty21a live attenuated vaccine. Both vaccines are targeted against S. Typhi and offer limited or no crossprotection against other serovars of Salmonella. Even against S. Typhi, these vaccines have some significant draw-backs, the main one being their lack of efficacy in children under two years of age. There is increased recognition that new vaccines are required to deal effectively with the global problem of Salmonella. A proper understanding of the modalities of protective immunity against Salmonella is required for the rational development of such vaccines, along with an appreciation of the targets of protective acquired immunity. At an epidemiological level, it is critical to know which population groups most need vaccination against salmonellosis. This chapter deals with the challenges posed by Salmonella to vaccine development. It considers the optimal requirements for new vaccines, particularly those with broad specificity that can be used across a spectrum of

ages. The chapter also reviews vaccines against Salmonella that are currently in development and discusses innovations and prospects for vaccines of the future. Introduction Salmonella is a genus of the Enterobacteriaceae family consisting of two species, Salmonella bongori and Salmonella enterica. All types of Salmonella that cause disease in man belong to S. enterica, which can be subdivided into over 2000 serovars based on the characteristics of their surface antigens. Additionally, clinically significant diversity is emerging through genome analysis (Holt et al., 2008; Kingsley et al., 2009; Okoro et al., 2012a). The most important disease-causing serovars in man are Typhi, Paratyphi A, Typhimurium and Enteritidis. S. Typhi and Paratyphi A are host-restricted to man and are the causative agents of enteric fever. This clinical condition has a very variable clinical presentation ranging from fever alone, to abdominal symptoms with altered bowel habit, to toxaemia involving many organ systems with intestinal bleeding and perforation, and typhoid encephalopathy (Bhan et al., 2005). Transmission is directly from human to human and through contaminated water supplies (Baker et al., 2011). S. Typhimurium and Enteritidis are able to infect a range of animals and are commonly associated with gastroenteritis in humans in high-income countries. These NTS infections are usually transmitted through contaminated foodproducts and contact with animals. In marked contrast, NTS in low-income countries, especially

388  | MacLennan

in sub-Saharan Africa, are a major cause of fatal invasive disease. The most common presentation of invasive NTS (iNTS) disease is bacteraemia (Feasey et al., 2012; MacLennan and Levine, 2013), but iNTS can also manifest as meningitis (Molyneux et al., 2009) and septic arthritis (Lavy, 2007). As will be discussed below, there is strong evidence that these differences between high- and low-income country NTS disease are associated with genetic diversity, which is not reflected by serological differences. The transmission of invasive forms of NTS in Africa is not well understood, but there is evidence from Kenya to support human-to-human, rather than zoonotic transmission (Kariuki et al., 2002, 2006). The antigens used to type Salmonella serovars comprise the O (somatic) antigen, H (flagellin) antigen and the Vi (virulence) antigen. The Vi antigen is only found in S. Typhi, Dublin and Paratyphi C, and forms a polysaccharide capsule. Given their surface location, each of the three antigens has been proposed as a vaccine candidate. O-antigen is the variable polysaccharide moiety of lipopolysaccharide, which extends from the bacterial membrane and consists of multiple repeats of a monomer consisting of four sugar units. Flagellin consists of polymerized protein units, which form the flagella responsible for Salmonella motility. As a protein, flagellin, unlike the other two antigens, is recognized by T-cells in its native form. Salmonellae are facultative intracellular bacteria that are able to survive within monocytes and macrophages. As NTS are also capable of extracellular survival, it is important clinically, as well as for vaccine development, to understand immunity against both free infection and infection within phagocytes. Chronic carrier states can occur in Salmonella infections, particularly with S. Typhi, where survival in the gall bladder is found in around 2 to 5% of adults after acute infection (Bhan et al., 2005; Levine et al., 1982). The standard clinical management of Salmonella infections is with antibiotics. This approach is increasingly hampered by multi-drug resistance (Gordon et al., 2008; Holt et al., 2011; Zaki and Karande, 2011). Effective control of Salmonella is also made difficult by the absence of a clear clinical presentation in many instances and a lack of rapid affordable diagnostics. Blood culturing facilities

are required to make a diagnosis of invasive Salmonella disease and these are rarely available in low-income countries (MacLennan and Levine, 2013). Global burden of Salmonella disease In relation to the most serious forms of Salmonella disease, typhoid fever is commonest in South and South-East Asia while iNTS disease mostly affects sub-Saharan Africa (Fig. 16.1). The global burden of enteric fever due to S. Typhi was estimated for the year 2000 as 21.7 million cases, mostly in pre-school and school-aged children, with a casefatality rate of 1% giving 217,000 deaths (Crump et al., 2004). A recent review of communityacquired bacteraemia in South and South-East Asia confirmed that S. Typhi is the most common blood culture isolate in the region, accounting for 30% of pathogenic isolates in adults and 25% in children (Deen et al., 2012). S. Paratyphi A also causes enteric fever with a similar geographical distribution. The clinical presentation is indistinguishable from that caused by S. Typhi and is of equal severity (Maskey et al., 2006). In 2000, the global burden of enteric fever due to S. Paratyphi A was estimated at 5.4 million cases, approximately a quarter of the number due to S. Typhi (Crump et al., 2004). However, the relative contribution of S. Paratyphi A to enteric fever varies and, in some regions, notably South-East China, exceeds that of S. Typhi (Ochiai et al., 2005). There are currently no published figures for the global burden of iNTS disease, though at 20–25%, this has a much higher case-fatality rate that that of enteric fever and is likely to account for over 100,000 deaths per year. These values indicate that iNTS disease is responsible for at least as many deaths as enteric fever. The vast majority of cases (over 2 million) occur in sub-Saharan Africa. A meta-analysis of community-acquired bacteraemia in Africa found that Salmonella is the commonest pathogenic blood culture isolate overall (adult and children combined) and among adults, and the second commonest isolate in children (Reddy et al., 2010). Salmonella accounts for 29% of pathogenic isolates in the continent, 58% of which are from non-typhoidal serovars,

Vaccines Against Invasive Salmonellosis |  389

Sub-Saharan Africa Typhimurium/ Enteritidis

South Asia Typhi/ Paratyphi A

Figure 16.1  Geographical distribution of enteric fever and invasive non-typhoidal Salmonella (iNTS) disease indicating global regions bearing the highest burden of these two principles form of invasive salmonellosis. Enteric fever (caused by S. Typhi and S. Paratyphi A) is commonest in South and South-East Asia, while iNTS disease (caused by S. Typhimurium and S. Enteritidis) is mostly a problem in sub-Saharan Africa. Medium incidence levels of enteric fever, mainly due to S. Typhi, are found throughout the rest of Asia, in South America and increasingly in Africa (based on findings from Crump et al., 2004; Deen et al., 2012; Reddy et al., 2010).

although this increases to over 90% in sub-Saharan Africa. Recent data from the RTS,S/AS01 malaria vaccine phase 3 trials across eleven sites in sub-Saharan Africa give a combined incidence for invasive Salmonella disease in children under 2 years of 478 and 530/100,000/year among the two cohorts recruited into the study (Agnandji et al., 2011). While S. Typhi is well-recognized as a problem in North Africa, where it accounts for 99% of Salmonella blood culture isolates (Reddy et al., 2010), typhoid fever has become an increasing problem in recent years in sub-Saharan Africa. The reasons for this are not clear, though typhoid fever is a particular problem in urban slums (Breiman et al., 2012; Tabu et al., 2012) rather than rural settings. Target populations for vaccination Understanding the populations at greatest risk of disease and death from Salmonella infections, and therefore those groups that need to be targeted for health-care interventions, is an important

consideration for vaccine development. Typhoid fever typically affects pre-school and school-aged children (Crump et al., 2004) (Fig. 16.2), although in Africa the distribution of cases encompasses older children and young adults (Feasey et al., 2010), while rates of chronic carriage of S. Typhi are more common in women and those over 50 years (Levine et al., 1982). In contrast to enteric fever, the prevalence of iNTS disease with age typically follows a bi-modal distribution (Feasey et al., 2010), affecting young children under two years of age and neonates, but with a relative sparing of those between 1 and 6 months of age (MacLennan et al., 2008). One study from Africa found it to be the second commonest cause of neonatal bacteraemia after Group-B Streptococcus (Milledge et al., 2005). The incidence of iNTS disease among adults mirrors the prevalence of HIV infection (Feasey et al., 2010) with which iNTS disease is strongly associated. Surprisingly, studies from sites in Africa where iNTS disease and enteric fever both occur, have found a lack of association between HIV infection and S. Typhi (Crump et al., 2011).

390  | MacLennan A Enteric fever – S. Typhi Ty21a ViCPS

Proportion of cases

future vaccine

0

5

10

15

20

25

Age (years)

B iNTS – S. Typhimurium/ S. Enteritidis future vaccine

Proportion of cases

future vaccine

0

5

10

15

20

25

Age (years)

Figure 16.2  Age distribution of global cases of invasive salmonellosis indicating age groups most in need of protection from vaccines. (A) Enteric fever affects pre-school and school-aged children with incidence gradually declining into adulthood. Currently available vaccines, Ty21a (for children over five years) and Vi capsular polysaccharide (ViCPS for children over two years), cannot protect children in the second year of life where incidence of typhoid fever peaks (Saha et al., 2001). A vaccine is required that can be administered to children under one year of age. (B) Invasive non-typhoidal Salmonella (iNTS) disease has a striking bimodal distribution affecting children under two years and HIV-infected adults. No vaccine is currently available, but one is required that can be given in infancy and protect through the susceptible period of early childhood, as well as in the teenage years to protect against iNTS disease in the event of infection with HIV (based on incidence findings from Crump et al., 2004; Feasey et al., 2010; MacLennan et al., 2008).

While a similar chronic gall bladder carriage state to that seen with S. Typhi has not been described for iNTS, recurrence of iNTS disease in HIVinfected African adults is common (Gordon et al., 2002) and can result from either reinfection or recrudescence (Okoro et al., 2012b). Immunity to Salmonella Rational, rather than purely empirical vaccine design, requires an understanding of the

underlying basis of protective immunity, particularly the acquired aspects of such immunity, against a pathogen. As mentioned above, Salmonella, is a facultative intracellular pathogen that can survive both in the cell-free environment and intracellular niche within macrophages. The basis for immunological protection against both these phases of infection must be considered. Salmonellae have a collection of conserved genes that permit survival within monocyte and macrophage (De Groote et al., 1995; Vazquez-Torres

Vaccines Against Invasive Salmonellosis |  391

et al., 2000b). Mutations in these genes render Salmonellae incapable of survival within phagocytes and cause loss of virulence (Fields et al., 1986). There is also evidence for several extracellular stages during prolonged Salmonella infection and transmission (Mastroeni et al., 2009). It has been shown that cell-mediated immunity has a key role in limiting intracellular infection, while humoral immunity is required to protect against the extracellular spread of Salmonella. The relative importance of both these modes of immunity has in the past been a controversial issue (Blanden et al., 1966; Mackaness et al., 1966; Robbins et al., 1992, 1995), but the balance of evidence suggests that vaccine development should consider both cellular and humoral immunity. Support for the importance of cellular immunity against Salmonella in man is provided by clinical findings from rare patients with primary immunodeficiencies. Individuals with chronic granulomatous disease have defects in the genes controlling the oxidative burst mechanism which is required to kill intracellular bacteria within neutrophils, as well as monocyte and macrophages. Prior to the use of prophylactic antibiotics, Salmonella was one of the three commonest causes of invasive disease in patients with these immunodeficiencies (Lazarus and Neu, 1975; Mouy et al., 1989). Further support for the importance of cellular immunity comes from patients with deficiencies of the IFNγ–IL12/23 axis, which is required for proper activation of macrophages (Bustamante et al., 2008; MacLennan et al., 2004; Prando et al., 2013). Disseminated Salmonella and mycobacterial disease is the most common infectious problem in such patients, who go on to develop Salmonella-associated granulomata (Lammas et al., 2002). Such infections often require the administration of subcutaneous IFN-γ (Altare et al., 1998). Recent evidence to support the importance of these cellular mechanisms comes from mechanistic studies of malaria and Salmonella co-infection where an increase in haem oxygenase-1 following malaria infection leads to defective oxidative burst (Cunnington et al., 2012). Nevertheless, in relation to acquired immunity, these cellular effector mechanisms can be delivered by the innate immune system. Although Salmonella-specific T-cells can deliver

IFNγ, this can also be produced by innate signals, particularly those acting through NK cells, γδT cells and NKT cells (Nyirenda et al., 2010). In a mouse model of salmonellosis, the importance of the oxidative burst has been confirmed in studies using animals with similar genetic defects to people with chronic granulomatous disease (Mastroeni et al., 2000; Vazquez-Torres et al., 2000a). In relation to T-cells and antibodies, there is evidence from multiple studies to support the importance of each in protection against Salmonella. Mice without T-cells have great difficulty in controlling Salmonella infections (Mittrucker et al., 1999; Sinha et al., 1997), and in some studies, antibodies appear superfluous. However, passive transfer studies have indicated a role for antibodies (Mastroeni et al., 1993; McSorley and Jenkins, 2000). Part of the explanation for these apparently conflicting results can be attributed to the relative virulence of the Salmonella strain used and the susceptibility of mice to Salmonella (Eisenstein et al., 1984; Simon et al., 2011a), in particular whether they have the SLC11A (formally Nramp1) resistance or susceptibility polymorphism to intracellular infection (Blackwell et al., 2001; Vidal et al., 1995). It is now generally accepted that both T-cells and antibodies are important for immunity to Salmonella in mice (Mastroeni et al., 1993) with T-cells required later rather than earlier in infection (Hormaeche et al., 1990; O’Brien and Metcalf, 1982). Antibodies have a role in enhancing the uptake and killing of Salmonella by neutrophils and monocyte/macrophages, as well as preventing the subsequent spread of diseases from phagocyte to phagocyte (Mastroeni et al., 2009). By contrast, T-cells have an important role in eliminating persistent infection within phagocytic cells (Blanden et al., 1966; Mackaness et al., 1966). The relative importance of humoral immunity and antibodies in protection against Salmonella in man has become clear from epidemiological studies of iNTS infection in Africa. Invasive Salmonella disease has not been a particular problem in European and North American patients with primary antibody deficiencies, such as common variable immunodeficiency (CVID), although increased episodes of Salmonella-related gastroenteritis have been reported (Leen et al.,

392  | MacLennan

1986). Similarly, problems with Salmonella have only rarely been found among individuals with complement deficiencies (Morgan and Walport, 1991; Ross and Densen, 1984). The work in subSaharan Africa has shown an inverse relationship between the acquisition of Salmonella-specific antibodies in early childhood and the incidence of invasive Salmonella disease. Few cases are recorded in children older than two to three years of age. There is a relative sparing of children under six months of age who have the protection of antibody transferred from the mother during the last trimester of pregnancy (MacLennan et al., 2008). These anti-Salmonella antibodies are able to kill Salmonella both through the complementmediated cell-free mechanism (MacLennan et al., 2008) and through opsonization for uptake and killing by blood phagocytes (Gondwe et al., 2010). There is close association between human immunodeficiency virus (HIV) infection and iNTS disease. At face value, this would seem to indicate an overwhelming importance of cellular immunity, particularly CD4+ T-cells, for defence against Salmonella, but this may not be a valid interpretation of the susceptibility of these individuals. iNTS disease is a WHO stage III-defining illness and HIV-infected patients with iNTS often have CD4+ T-cell counts less than 200 cells/µl. There is evidence for dysregulated cytokine responses in such patients, although these responses are characterized by elevated levels of the cytokines normally associated with protection against Salmonella (Gordon et al., 2007). The situation is further complicated by the occurrence of dysregulated humoral immunity to NTS in HIV-infected African adults. In particular, some individuals have high levels of anti-Salmonella LPS antibodies that are counter-intuitively associated with iNTS disease (MacLennan et al., 2010). Also, HIV interferes with the integrity of the gastrointestinal mucosa and work in macaques has shown that simian immunodeficiency virus (SIV) infection leads to increased translocation of Salmonella across the gastrointestinal wall (Raffatellu et al., 2008). Finally, although HIV-infected individuals are highly susceptible to iNTS, they do not appear to be at increased risk of developing typhoid, and epidemiological studies from Africa have

suggested a potentially protective effect against typhoid (Crump et al., 2011). In summary, work in both man and mice indicates roles for both antibodies and cell-mediated immunity for optimal protection against Salmonella (summarized in Fig. 16.3). Although there is uncertainty about the relative importance of each, these two principal forms of immunity are critical at different points in the infection cycle. Further insights into the modalities of protective immunity come from the deployment of existing vaccines against Salmonella. Targets of protective immunity Salmonellae are immunogenic and well known to induce antibody responses to the three main groups of surface components: polysaccharides – Vi and O-antigen of LPS, flagellin, and outer membrane proteins (Mastroeni, 2002) (Table 16.1). As previously mentioned, antibodies to LPS and flagellin (and Vi for S. Typhi) are key to the identification of the Salmonella serovars according to the Kauffman–White classification. However, it has proved more difficult to ascribe protective efficacy to the antibodies targeting these different antigens. Polysaccharides Owing to the host restriction of S. Typhi to man, in relation to polysaccharides, more work has been performed on antibodies to LPS in mice, particularly the O-antigen of LPS rather than the Vi capsular polysaccharide. Passive transfer of monoclonal antibodies against LPS O-antigen can confer protection to subsequent challenge with Salmonella in mice (Carlin et al., 1987; Singh et al., 1996). Protection can also be afforded by active immunization of mice with O-antigen glycoconjugate vaccines (Simon et al., 2011b; Watson et al., 1992). A recent study immunizing mice with heatkilled African invasive S. Typhimurium D23580 found that most of the induced bactericidal response was directed against O-antigen (Rondini et al., 2013). A protective role for antibodies to Salmonella O-antigen in humans has been less clear. High levels of IgG antibodies to O-antigen of S. Typhimurium have been shown to impair killing induced in vitro by antibodies to outer

Vaccines Against Invasive Salmonellosis |  393

Salmonellae

neutrophil

Location

Blood/Lymphatics

Gastrointestinal Tract/Gut-Associated Lymphoid Tissues

(intracellular – neutrophil/ monocyte)

(extracellular)

(extracellular/ intracellular)

Key Immune Components

B cells Mucosal antibodies IgG/IgA Neutrophils CD4+ Th1/Th17 cells Anti-microbial peptides

Clinical Consequence of Failure to Control Infection

macrophage

B cells Systemic bactericidal Antibodies - IgG/IgM Complement

Systemic opsonising antibodies – IgG Neutrophils/Monocytes Oxidative burst

Bacteraemia Enteric Fever

Diarrhoea

Liver/Spleen/Lymph Nodes/ Bone Marrow/ Gall bladder (intracellular – macrophage)

CD4+ Th1 cells/ CD8+ T cells NK cells/γδ T cells/NKT cells IL12/IL18/IFNγ/TNFα

Chronic carriage Latent/recurrent clinical infection Abscess/lymphadenitis

Figure 16.3 Key modalities of protective immunity against invasive salmonellosis. Invasive Salmonella disease normally begins in the gastrointestinal tract and spreads through the blood and lymphatics, to the liver, spleen, lymph nodes, bone marrow, and, in the case of S. Typhi, to the gall bladder. Different components of the immune system are required at different points in the infection cycle and there are roles for both B-cell and T-cell arms of the acquired immune response in effecting protective humoral and cellmediated immunity against Salmonella. Table 16.1 Potential vaccine coverage of main invasive Salmonella enterica serovars by candidate antigens Clinical presentation Enteric fever

iNTS diseasea

S. enterica serovar Typhi

S. enterica serovar Paratyphi A

S. enterica serovar Typhimurium

S. enterica serovar Enteritidis

1. Vi

+

–

–

–

2. O:2

–

+

–

–

3. O:4,5

–

–

+

–

4. O:9

+

–

–

+

1. Omp F/Omp C

+

+

+

+

2. Omp D

–

+

+

+

3. Other

+/–d

+/–d

+/-d

+/–d

+/–e

+/–e

+/-e

+/–e

+/–e

+/–e

+/-e

+/–e

Antigen (A) Polysaccharide

(B) Protein

(C) Mixed 1. LAVb 2.

GMMAc

iNTS disease = invasive non-typhoidal Salmonella disease. attenuated vaccine. cGMMA = Generalized modules for membrane antigens. d+/- for ‘Other’ protein antigens indicates dependency on identity of antigen. e+/– for ‘LAV’ and ‘GMMA’ indicates dependency on choice of production strain and presence/expression levels of key antigens in production strain and target serovar. a

bLAV = live

394  | MacLennan

membrane proteins of Salmonella (MacLennan et al., 2010). Nevertheless, a number of studies have shown that some anti-O-antigen antibodies do have bactericidal potential (Trebicka et al., 2013; MacLennan and Tennant, 2013). Flagellin Flagellin, the principal ligand of TLR5, can provide innate signals to the immune system through this receptor. It has been associated with immunomodulatory effects in mice, for example, in influencing T-cell maturation into different functional CD4+ T-helper cell subsets (Cunningham et al., 2004), inducing IgA antibody production following systemic administration (Flores-Langarica et al., 2012) and promoting increased IgG2a production by a live attenuated S. Paratyphi A vaccine (Gat et al., 2011). Notably these effects are dependent on whether flagellin is polymerized and present in cell-associated form or administered in unpolymerised soluble subunits. Studies with candidate O-antigen-based glycoconjugate vaccines incorporating flagellin have demonstrated a protective effect in mice (Simon et al., 2011b, 2013), which can also be achieved through immunization with flagellin alone (Simon et al., 2011b). Outer membrane proteins An increasing area of interest has been the potential for various outer membrane proteins to induce immunity to Salmonella. Outer membrane protein antibodies have the added attraction that they could potentially provide broadly protective immunity where outer membrane protein targets are conserved across a range of serovars. As with LPS, monoclonal antibodies to outer membrane proteins can provide passive protection in mice (Singh et al., 1996). Protective immunity in mice can be elicited through immunization with porins OmpC and OmpF (Cunningham et al., 2007) and, more recently, by OmpD, which is expressed by almost all Salmonella serovars, but not S. Typhi (Gil-Cruz et al., 2009). An array consisting of 2700 Salmonella proteins has been used to probe immune and control sera in order to try to identify new targets of protective immunity. Using this method, overlap has been demonstrated between the antibody profiles elicited by active

immunization with the live attenuated vaccine strain S. Typhimurium BRD509 in mice and clinical iNTS disease in African children. This provides support for the use of mice in the study of immunity to Salmonella (Lee et al., 2012). SseB, a Salmonella protein identified through the analysis of sera from bacteraemic Malawian children, was able to protect against Salmonella infection when used to immunize mice (Lee et al., 2012). T-cell antigens Owing to the accessibility of antibodies and ease of characterization compared with T-cell receptors and associated antigen, more is known about antibody than T-cell responses to Salmonella. Interestingly, a recent study of Salmonella T-cell antigens found that the most promising candidate antigens are associated with the Salmonella surface. The authors hypothesized that this is due to the release of these molecules by live Salmonellae within the intracellular vacuole of macrophages (Barat et al., 2012). Whatever the exact mechanism, these findings suggest that Salmonella surface protein antigens may serve both as B-cell and T-cell antigens. Existing vaccines Vaccines against Salmonella have been in use for over a hundred years and insights gained from the experience of vaccination against Salmonella can help in the design and development of a new and innovative generation of vaccines. Of the three types of Salmonella vaccines that have been widely licensed for use in man, all have been developed to prevent S. Typhi and no vaccine is currently available against NTS. All have their drawbacks, particularly the lack of effectiveness in young children, which has led to the absence of widespread implementation of any of these vaccines (Table 16.2). The oldest vaccines against Salmonella are inactivated whole cell formulations which were first introduced in 1896 (Pfeiffer, 1896) to prevent typhoid and were subsequently used extensively among British (Wright and Leishman, 1900) and US army personnel (Hawley and Simmons, 1934). The introduction of such vaccines was accompanied by a dramatic fall in cases of typhoid among

Vaccines Against Invasive Salmonellosis |  395

Table 16.2 Advantages and disadvantages of past, present and future vaccines against Salmonella enterica Vaccine

Advantages

Disadvantages

73% 3-year efficacy

Reactogenicity

Single dose

Not licensed for infants

Low reactogenicity

Lack of memory response

(A) Vaccines of the past Whole cell inactivated (B) Vaccines of the present 1. Vi CPS

Lack of affinity maturation Only protects against S. Typhi 2. Ty21a

Oral administration

Not licensed for infants

Some cross-protection against S. Paratyphi B

Requires multiple doses

(C) Vaccines of the future 1. Vi glycoconjugate vaccines Higher efficacy than current vaccines

Only protects against S. Typhi

T-dependent antibody response Memory induction Affinity maturation Low reactogenicity 2. O-antigen glycoconjugate vaccines

As for Vi glycoconjugates

Only protects against serovars with same O-antigen specificity

3. New live attenuated vaccines

Salmonella-specific B- and T-cell immunity

Attenuating for optimal balance of immunity and reactogenicity

Clearance of residual infection

Breadth of coverage may be limited by insufficient expression of key antigens Possibility of disease in immunocompromised subjects

4. Recombinant proteins

Salmonella-specific B- and T-cell immunity

Issues with antigen conformation may limit ability to induce effective B-cell response

Potential for pan-specific immunity Low reactogenicity 5. Proteins purified from whole Salmonellae

Salmonella-specific B- and T-cell immunity

Difficulties with purification of integral membrane proteins

Potential for pan-specific immunity Low reactogenicity 6. GMMA

Salmonella-specific B- and T-cell immunity

Balance of reactogenicity and immunogenicity in man not currently known

Potential for pan-specific immunity Enrichment of membrane antigens Ease of manufacture

the soldiers in these armies. A meta-analysis of clinical trials performed with the various forms of Salmonella vaccines indicates that the inactivated whole cell vaccines have been the most effective, with a three year cumulative efficacy of 73%

following two doses (Engels et al., 1998). However, high levels of adverse reactions to such whole cell vaccines have proved unacceptable (Ivanoff et al., 1994; Wahdan et al., 1975) and consequently they are no longer used (Engels et al., 1998).

396  | MacLennan

A live attenuated oral vaccine, Ty21a, was developed by non-specific chemical mutagenesis of the wild type Ty2 S. Typhi strain (Germanier and Fuer, 1975). This has a cumulative three-year efficacy of 51% for three doses (Engels et al., 1998; Fraser et al., 2007) and is tolerated in children as young as two years of age (Cryz, Jr. et al., 1993). Unfortunately, the use of Ty21a in young children is associated with a decreased rate of seroconversion (Cryz, Jr. et al., 1993) and the vaccine is not immunogenic in infants (Murphy et al., 1991). It is commonly available as an enteric-coated capsule which is licensed only for children over 5 years. Other problems associated with this vaccine are its thermal instability, requiring a cold chain, and need for multiple dosing. There is evidence that Ty21a can induce herd protection (Levine et al., 1989) and offers some cross-protection against S. Paratyphi B (Levine et al., 2007). There were insufficient cases in these clinical studies to make conclusions about an effect on S. Paratyphi A or iNTS, although ex vivo studies on blood from Ty21a vaccinees has demonstrated the presence of antibody-secreting cells which are cross-reactive with S. Paratyphi A and S. Paratyphi B, as well as S. Typhi (Pakkanen et al., 2012; Wahid et al., 2012). The final licensed Salmonella vaccine is a subunit vaccine consisting of purified Vi polysaccharide (ViCPS). Although it has similar efficacy to Ty21a (55% at 3 years) (Engels et al., 1998; Fraser et al., 2007), these results were achieved with a single vaccine dose. Vi polysaccharide is a TI2 (T-independent type 2) antigen, making it non-immunogenic in infants. Although licensed for children down to 2 years of age, two cluster randomized controlled trials in children aged 2–5 years, one in Kolkata (Sur et al., 2009) and one in Karachi (Khan et al., 2012), gave conflicting results. Protective efficacy in this age group was only demonstrated in the Kolkata study. In common with other pure polysaccharide vaccines, ViCPS does not produce memory responses. It can also lead to hyporesponsiveness to subsequent vaccination with polysaccharide–protein conjugates (Overbosch et al., 2005; Poolman and Borrow, 2011). As for Ty21a, a cold chain is required for its distribution. Despite these reservations, where implemented, vaccination with Vi polysaccharide

has led to a marked reduction in the number of typhoid cases (Acharya et al., 1987; Khan et al., 2010). Anti-Vi IgG antibodies induced by ViCPS appear to relate to vaccine efficacy (Klugman et al., 1996; Robbins and Robbins, 1984). From a design perspective, this vaccine provides important evidence that protection against typhoid in man can be mediated by antibodies, since pure polysaccharide antigens are unable to induce T-cell immunity. Vaccines of the future As discussed above, currently there are only two forms of licensed vaccines against Salmonella still in use and these do not cover non-typhoidal serovars. These vaccines lack effectiveness in young children. Despite the recommendation of the immunizations Strategic Advisory Group of Experts (SAGE) to the WHO for programmatic used of existing typhoid vaccines in endemic countries (WHO, 2008), their implementation has been limited (WHO, 2011). Clearly an opportunity exists for the development and implementation of new and more effective vaccines against Salmonella (Fig. 16.4). Perhaps the biggest obstacle to developing such vaccines is the absence of a clear commercial driver, making them unattractive to large commercial vaccine companies (Maclennan, 2013). Salmonella infections, particularly iNTS disease and enteric fever, which have high associated case fatality rates, are primarily problems of low- and some middleincome countries and not North America and Europe. However, substantial interest in the development of new Salmonella vaccines has recently emerged among vaccine companies based in emerging economies, particularly India (BioMed, Shantha Biotech and Bharat Biotech) and China (Lanzhou Institute), new global health vaccines institutes such as the International Vaccines Institute (IVI; http://www.ivi.int/web/www/home), and Novartis Vaccines Institute for Global Health (NVGH; http://www.nibr.com/research/developing_world/NVGH/about.shtml), as well as academic institutes, particularly the Center for Vaccine Development at the University of Maryland (CVD; http://medschool.umaryland.edu/

Vaccines Against Invasive Salmonellosis |  397 Current Vaccines

Polysaccharide

Live Attenuated

Whole Cell Inactivated

Glycoconjugate polysaccharideprotein

New Live Attenuated

GMMA

Vi CPS

Vaccines of the Future

Ty21a

Vaccines of the Past

Recombinant/ Purified Protein

Figure 16.4  Salmonella vaccines of the past, present and future and how they relate to each other.

cvd/). Such efforts have been encouraged by the recently formed Coalition against Typhoid (CaT) (http://www.coalitionagainsttyphoid.org/), an advocacy group for Salmonella vaccination overseen by the Sabin Vaccine Institute and funded by the Bill and Melinda Gates Foundation. Requirements for new Salmonella vaccines General The general requirements for new vaccines against Salmonella are largely determined by the populations who need them most. As for all vaccines, they need to be safe and effective in reducing burden of disease. Their use to prevent both enteric fever and iNTS disease is most likely to involve immunization in early childhood. Ideally these vaccines should induce life-long immunity with a high level of efficacy after a minimum of doses (ideally one) and without the need for subsequent boosting. Modelling of the implementation of vaccines which induce short-lived immunity, indicates that such vaccines may merely shift the age at which disease occurs without affecting the overall burden of disease (Saul et al., 2013). Vaccines that lead to herd immunity by preventing transmission of infection will be more effective at the population level than those that have no impact on transmission. Such an effect will most likely rely

on the induction of sterilizing immunity (which prevents infection), rather than clinical immunity (where infection can occur, but without clinical symptoms), and should prevent asymptomatic carriage and shedding, particularly of S. Typhi. Affordability Since new vaccines are most likely to be used in a widespread manner in low-income countries, their affordability will be key to deciding whether their use will be supported by WHO and the GAVI Alliance. Low cost of goods is determined partly by the cost of the materials needed for the vaccine and partly by the production methods employed. Hence, complex vaccines requiring multiple steps in the production pathway, or relying on the use of hazardous biological or chemical agents are best avoided. A second reason to favour relatively simple vaccine design and production methods is that these vaccines are likely to be produced by emerging economy manufacturers which may lack the same access to sophisticated vaccine technologies and handling conditions for hazardous agents present in high-income countries. Cost per dose can be partly defrayed by multi-dose vial formulations, although this can lead to many wasted doses. Another key impact on the final cost of implementing a vaccine, particularly in widespread vaccination programmes, is the cost of vaccine delivery. This can be offset by implementing new vaccines into the Expanded

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Programme on Immunisations (EPI). Since typhoid fever incidence peaks in the second year of life in endemic countries (Saha et al., 2001), co-administration of new typhoid vaccines with the measles EPI vaccine at nine months could be a preferable approach (Podda et al., 2010). An iNTS vaccine would ideally be given to young infants from two to four months, together with their first EPI immunizations, due to the peak of iNTS disease at one year of age (Feasey et al., 2010; MacLennan et al., 2008). Mode of immunity The effectiveness of the ViCPS vaccine, while by no means complete, is a strong indication for the role of antibodies in protection against Salmonella disease. While there is growing immune-epidemiological and laboratory evidence for the role of systemic antibodies in preventing fatal bacteraemia (Gondwe et al., 2010; MacLennan et al., 2008), mucosal antibodies may be important for reducing carriage and breaking transmission cycles, thereby promoting herd immunity. Along with the importance of antibodies, T-cells are likely to be required for the clearance of Salmonella from the intracellular niche and prevention of recurrent infection seen in patients with HIV/ AIDS in Africa (Gordon et al., 2002) and those with impaired cellular immunity due to deficiencies of the IFNγ–IL12 axis (Altare et al., 1998; Prando et al., 2013). Therefore vaccines that can recruit protective responses from Salmonellaspecific B-cells as well as T-cells are particularly attractive. Breadth of coverage A major drawback of the two current Salmonella vaccines is their restriction to S. Typhi. Since it is increasingly clear that the range of serovars contributing to the burden of invasive life-threatening salmonellosis extends well beyond serovar Typhi, vaccines with greater breadth of coverage need to be developed (Table 16.1). The main need for vaccines against Salmonella divides between at-risk populations in South Asia (where enteric fever is widespread) and sub-Saharan Africa (where iNTS disease dominates) (Fig. 16.1). A vaccine that can protect against the two main aetiological agents of enteric fever, S. Typhi and S. Paratyphi A,

could prevent the majority of Salmonella disease in South and South-East Asia. Conversely, a vaccine against the two main causes of iNTS disease, S. Typhimurium and S. Enteritidis, could do the same in Africa. Using subunit vaccines (e.g. new glycoconjugate vaccines), such a strategy could be achieved by separate Asian and African bivalent Salmonella vaccines. In the time that it takes to develop a new vaccine (ten years minimum), the current epidemiological paradigm of Salmonella disease could markedly change and new disease-causing serovars emerge which the potential bivalent subunit vaccines mentioned above would offer no protection against. To an extent, this is already occurring with the emergence of enteric fever caused by S. Typhi in several sub-Saharan African countries, particularly in urban settings. Alternative vaccine strategies need to be explored that can offer panspecific protection against Salmonella. Such an approach is facilitated by the increased availability of whole genome sequences of Salmonella from a range of endemic countries (Holt et al., 2008; Kingsley et al., 2009; Okoro et al., 2012a). Glycoconjugate vaccines Glycoconjugate vaccine technology, whereby polysaccharide antigens are covalently linked to carrier proteins such as tetanus toxoid and the non-toxic recombinant form of diphtheria toxin, CRM197, effectively converting the polysaccharide from a T-independent to a T-dependent antigen, is by no means a new vaccine technology. The advantages of this strategy over pure polysaccharide are well-recognized and include induction of memory, affinity maturation, improved class switching and, of key importance for Salmonella vaccines, ability to induce immune responses in infants (MacLennan, 2013; Pollard et al., 2009) (Table 16.2). Glycoconjugates against all four main serovars responsible for invasive Salmonella have clear potential as effective vaccines (Simon and Levine, 2012). Their development has lagged a long way behind the development and implementation of glycoconjugate vaccines against the main encapsulated bacteria, pneumococcus, meningococcus and Haemophilus influenzae b. As mentioned above, this is probably because

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Salmonella does not represent a major health problem in high-income countries. In 2001, a phase III clinical trial in Vietnamese children aged two to five years with a NIH (National Institutes of Health, US) vaccine consisting of Vi conjugated to Pseudomonas aeruginosa recombinant exoprotein A (Vi-rEPA), showed a 91% efficacy over 27 months (Mai et al., 2003). This has subsequently been shown to be compatible with co-administration with EPI vaccines (Thiem et al., 2011). Despite the gap of 12 years since results from the first phase III study using this Vi conjugate vaccine were published (Lin et al., 2001), conjugate vaccines against typhoid have only just become available, and so far only for in-country use in India and China. These include Vi-tetanus toxoid vaccines, Peda Typh (BioMed; http://biomed.co.in/typh_vaccine.html) and Typbar-TCV (Bharat Biotech; http://www. bharatbiotech.com/) in India, and a Vi-rEPA vaccine (Lanzhou Institute) in China. Other Vi-based conjugate vaccines are in various stages of development (Martin, 2012; McGregor et al., 2013). NVGH has completed phase II studies in India, Pakistan and the Philippines with a Vi–CRM197 conjugate vaccine (Bhutta et al., 2013; van Damme et al., 2011). An innovation in the method of production for the NVGH vaccine serves to increase safety and decrease production costs. Instead of using S. Typhi to produce Vi polysaccharide, an organism which requires manipulations, such as fermentation, to be carried out under BSL level 3 containment conditions, a Vi-expressing strain of Citrobacter is used (Micoli et al., 2011). Citrobacter is non-pathogenic and requires no special laboratory containment conditions. Although there is good potential for efficacy of all the above vaccines against S. Typhi, none of them will protect against enteric fever caused by S. Paratyphi A or strains of S. Typhi that do not express Vi. Since S. Paratyphi A does not express Vi, an alternative antigen is required to generate a subunit vaccine against this serovar. The antigen of choice to date has been the O:2 antigen of the S. Paratyphi A LPS molecule. Results from phase I and phase II studies with an O:2-tetanus toxoid vaccine conducted in Vietnamese children, teenagers and adults, were published in 2000 (Konadu et al., 2000). These indicate good tolerability and

immunogenicity of the vaccine with induction of anti-LPS antibodies and serum bactericidal activity. Both IVI and NVGH are developing bivalent enteric fever vaccines with Vi and O:2 polysaccharide antigens conjugated separately to either diphtheria toxoid or CRM197 (Martin, 2012; McGregor et al., 2013). These vaccines have the potential to give broader coverage against enteric fever than the monovalent Vi-based conjugate vaccines. Like S. Paratyphi A, NTS serovars lack Vi, but have O-antigens. Diversity of the O-antigen structure means that separate O-antigen-based vaccines are required to induce antibodies against S. Typhimurium (O:4,5) and Enteritidis (O:9). Development of such vaccines is a long way behind glycoconjugates for typhoid and paratyphoid, probably due to the lack of recognition of iNTS as a major cause of morbidity and mortality in low-income countries and a lack of demand for such vaccines. There is increasing awareness of the problem of iNTS disease, particularly in Africa (Feasey et al., 2012; MacLennan and Levine, 2013; Okoro et al., 2012a), and groups are actively developing O-antigen-based bivalent vaccines against S. Typhimurium and Enteritidis. NVGH is developing such a vaccine using CRM197 as the carrier protein, while University of Maryland is using flagellin (Simon et al., 2011b, 2013). The latter approach has the advantage of being able to induce Salmonella-specific T-cell immunity, which may be important for the clearance of infection as well as induction of antibodies against flagellin and a T-dependent antibody response to the O-antigen. This vaccine has been shown to protect against challenge in mice (Simon et al., 2011b). An innovation by NVGH in NTS vaccine design is the use of acetic acid hydrolysis to cleave O-antigen directly from the bacteria. This reduces the number of production steps and avoids the use of hot phenol, a hazardous chemical, for extraction of LPS (Micoli et al., 2013). Again, such innovations serve to improve the safety of the manufacturing process and reduce costs. Currently bivalent glycoconjugate vaccine strategies are targeting either enteric fever or iNTS disease. Experience with multivalent pneumococcal and meningococcal vaccines indicate that it should be feasible to extend these to trivalent or

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quadrivalent vaccines in order to cover three or four of the principal disease-causing serotypes. The most pressing extension of vaccine coverage is likely to be the need for a vaccine in Africa that protects against S. Typhi as well as iNTS disease. Fortuitously, the bivalent iNTS vaccines described above may have activity against S. Typhi, since this serovar shares the O:9 antigen with S. Enteritidis. There is proof of concept that these new glycoconjugate vaccines will be effective and have advantages over the ViCPS vaccine. Nevertheless the impact of not inducing Salmonella-specific T-cell immunity may lead to some loss of effectiveness, especially in relation to transmission in view of the adaptation of Salmonellae to survive within macrophages. Apart from the CVD vaccine, where flagellin is used as the carrier protein, all other Salmonella glycoconjugates in development employ non-Salmonella carrier proteins, most of which have been used extensively in previous vaccines. Second and third generation glycoconjugate vaccines against Salmonella may switch to using Salmonella proteins, such as Salmonella outer membrane proteins. Proteins that contain T-cell epitopes shared by multiple serovars would be particularly attractive. New live-attenuated vaccines Whereas Ty21a was generated by random mutagenesis, our increased understanding of molecular biology and genomics, combined with access to the whole genome sequences of many Salmonella isolates (Holt et al., 2008; Okoro et al., 2012a), allows a targeted mutagenesis approach for the generation of new live-attenuated vaccines against Salmonella. These contain the majority of antigens of the particular Salmonella strain used. Consequently, there is enhanced potential for developing a vaccine that can protect across multiple serovars. T-cell immunity, as well as antibodies, is induced by live-attenuated vaccines and the mouse salmonellosis model suggests greater ability of live attenuated vaccines to clear residual infection than either glycoconjugate vaccines or killed whole cell vaccines. A further advantage is oral administration, which permits needle-free delivery, and potentially a greater likelihood of inducing mucosal as well as systemic immunity.

The challenge with this type of vaccine is to select the right mutation or combination of mutations needed to provide acceptable tolerability without compromising immunogenicity and ultimately protection. Safety is also an important issue for live vaccines, particularly in regions, such as sub-Saharan Africa, where many recipients could be immunocompromised secondary to HIV/AIDS. A final challenge is to develop a vaccine requiring fewer doses than Ty21a, ideally a single dose, and that will induce lasting protection. Three new live attenuated vaccines, all based on the Ty2 parent strain, have been developed and tested in phase II clinical trials (Martin, 2012). CVD 909 from the University of Maryland has mutations in the aroC, aroD and htrA genes, and the PtviA promoter has been replaced by the strong constitutive Ptac promoter to ensure constitutive Vi expression (Tacket and Levine, 2007; Wahid et al., 2007, 2008; Wang et al., 2000), which has been lacking from many live attenuated S. Typhi vaccines. Ty800, developed by Avant Immunotherapeutics, also has a disrupted aroC gene, and mutated ssaV gene (Hohmann et al., 1996a,b), while M01ZH09 of Emergent Biosolutions has mutations in the Pho/PhoQ regulator genes ( Jain, 2009; Kirkpatrick et al., 2005a,b, 2006). All have good safety, tolerability and immunogenicity profiles. As expected, all induce mucosal as well as systemic antibodies. It remains to be seen what protection they can deliver in man. Less progress has been made to date with the development of live attenuated oral vaccines against NTS. A phase I study with a S. Typhimurium live attenuated vaccine with same aroC and ssaV attenuations as the S. Typhi Ty800, found prolonged stool shedding in volunteers up to 23 days (Hindle et al., 2002). More recently, preclinical studies have been conducted with candidate live attenuated S. Typhimurium and S. Enteritidis vaccines with deleted guaBA and clpP genes, at the CVD. These were found to be immunogenic and protected against lethal challenge with NTS infections using the homologous serovar (Tennant et al., 2011). Protein-based subunit vaccines Salmonella proteins in recombinant or purified form are alternative forms of subunit vaccines

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to the glycoconjugates. These have the potential advantage of being able to induce immunity that covers a broad range of serotypes. Careful bioinformatic analysis of a range of Salmonella genomes allows comparison of the nucleotide and predicted amino acid sequences of protein antigens. This should enable candidates to be identified that are likely to include conserved B-cell and T-cell epitopes. Analyses searching for common B-cell antigens focus on predicted outer membrane proteins, periplasmic proteins, lipoproteins and extracellular proteins, as these are most likely to harbour epitopes accessible to antibodies. As mentioned earlier, a recent study suggests that many Salmonella T-cell antigens are located at the Salmonella surface (Barat et al., 2012). Such an approach can form the basis of a reverse vaccinology project for candidate vaccine antigen discovery. This would have the objective of identifying conserved and universally expressed surface-exposed proteins that induce similar broadly cross-protective immunity against Salmonellae as factor H binding protein (f Hbp) does against meningococcus (Pajon et al., 2011). The most studied potential Salmonella subunit vaccine proteins are the porin outer membrane proteins, OmpC, OmpF and OmpD. Early studies in mice suggested that immunization with Salmonella porins can protect against subsequent challenge and that this protection is antibodymediated, since it can be passively transferred (Kuusi et al., 1979). More recent animal studies have confirmed the protective potential of immunization with OmpC and OmpF (Cunningham et al., 2007) and OmpD alone (Gil-Cruz et al., 2009). Although these antigens are widely conserved across Salmonella enterica, OmpD is not present in S. Typhi, limiting the potential of this as a stand-alone candidate vaccine antigen. An S. Typhi porin preparation containing OmpC and OmpF induced long-lasting antibody responses in mice in which OmpC was immunodominant (Secundino et al., 2006) and has been used in a phase I study in Mexico where it was found to be safe and immunogenic eliciting antibodies and cell-mediated responses (Salazar-Gonzalez et al., 2004). Interestingly, serum cross-reactivity did not extend to S. Typhimurium, suggesting that S. Typhi and S. Typhimurium porins may need to

be used in combination to give protection against typhoid and iNTS. A potential drawback of these promising vaccine antigens is their multiple membrane spanning domains, which requires them to be purified from whole Salmonella, rather than synthesized as recombinant proteins. A recent study found that recombinant Salmonella porins did not protect mice against subsequent challenge (Toobak et al., 2013). GMMA bacterial particle vaccines An innovative vaccine strategy, enabling expression of outer membrane proteins in their native conformation, takes advantage of the natural ability of Gram-negative bacteria to shed outer membrane in the form of blebs containing periplasm. This process can be up-regulated by the insertion of targeted mutations of genes encoding components of the Tol-Pal system which maintains the integrity of the inner and outer membrane (Berlanda et al., 2008; MacLennan, 2013). The particles, known as generalized modules for membrane antigens (GMMAs) or native outer membrane vesicles (NOMVs), are highly enriched for outer membrane components including proteins which, unlike recombinant proteins, are maintained in their native conformation and orientation (Fig. 16.5). The approach has so far been applied to Neisseria meningitidis with mutation of the gna33 gene (Koeberling et al., 2014; Ferrari et al., 2006), and Salmonella and Shigella where mutation of tolR up-regulates GMMA production (Berlanda et al., 2008; MacLennan, 2013). NVGH has developed a simple and affordable two-step tangential flow filtration process for the rapid purification of GMMA following fermentation of parent bacterial strains (Berlanda et al., 2008). Early indications are that 0.5 to 5 million vaccine doses could be generated from a single 50 litre fermentation. In addition to being enriched in surface proteins, GMMA contain other surface components, including LPS and O-antigen. Consequently, they could potentially be used as a delivery vehicle for O-antigen in place of glycoconjugates, with the benefit of co-delivery of multiple Salmonellaspecific proteins and innate signals. Advantages

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outer membrane protein periplasm

lipopolysaccharide inner membrane protein

GMMA

periplasm

cytoplasm

Gram-negative bacterial cell wall outer membrane proteoglycan inner membrane

Figure 16.5 The GMMA (generalized modules of membrane antigens) platform technology for the production of novel Salmonella vaccines. GMMA consist of blebs of outer membrane and are enriched in surface components including O-antigen and outer membrane proteins. Mutagenesis of genes encoding components of the Tol-Pal system of Gram-negative bacteria up-regulates the generation and release of these particles. GMMAs are highly immunogenic and constitute a particularly affordable vaccine strategy (adapted from MacLennan, 2013).

of this approach compared with glycoconjugates include simplicity and low-cost of production, equivalent or greater levels of immunogenicity, and a potential self-adjuvanting effect due to the presence of innate signalling molecules such as TLR ligands (e.g. LPS for TLR4). A disadvantage is that modifications may be required to reduce reactogenicity, for example, by detoxifying the lipid A moiety of LPS, through the deletion of genes, such as those encoding the late acyltransferases HtrB (Clementz et al., 1996) and MsbB (Clementz et al., 1997). The approach also permits the overexpression of key target antigens and could also be used as a vehicle for expression of increased levels of porins in their native conformation. Use of adjuvants in new Salmonella vaccines Salmonellae contain a number of molecules that stimulate innate immune responses and are incorporated in the new vaccine approaches. Notably

these include the TLR agonists, LPS, which stimulates both TLR4 and CD14, and flagellin, the principal known ligand for TLR5. Careful manipulation of these components in candidate vaccines may serve to potentiate the protective immune response without leading to unacceptable reactogenicity. There is evidence that some components, notably flagellin, can modulate the T-cell response to vaccination (Cunningham et al., 2004) and impact on mucosal immunity. As just described, vaccines incorporating GMMA are likely to provide self-adjuvanting signals. Conclusions We are currently in exciting times for the development and implementation of new vaccines against the Salmonellae responsible for invasive disease presenting as enteric fever and iNTS disease. With an enhanced understanding of the impact of these pathogens on global health and a growing appreciation of the need for new vaccines, there are a range of Salmonella vaccines in the development

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pipeline (Fig. 16.4; Table 16.2). Some of these are in early preclinical studies, while others have passed through clinical studies and are near licensure. While the majority of the more advanced vaccines employ glycoconjugate technology, new live attenuated vaccines with targeting mutations have been used in phase II clinical studies and promising novel technologies, particularly GMMA, could enter phase I studies in the next couple of years. The progress of these vaccines has been facilitated by the initiatives of the new global health vaccine institutes and manufacturers in emerging economies, together with support and advocacy from CaT. Innovations in Salmonella vaccine development have been aided by the availability of whole genome sequences of an increasing number of Salmonella isolates allowing both the identification of conserved potential candidate vaccine antigens and the targeting of specific genes for disruption in live attenuated strategies. With much investigation already undertaken to understand immunity to this facultative intracellular pathogen, the progress of these new vaccines, informed by such knowledge, is likely to provide answers to some of the outstanding questions related to what is really required for protection against this pathogen in man. As the first new vaccines against Salmonella become available, the challenge will remain for the development of second and third generation vaccines that can offer the increased effectiveness, duration of action and breadth of protection needed in the face of the evolving global epidemiology of invasive Salmonella disease. Acknowledgements I am grateful to Ian MacLennan, Colette O’Shaughnessy and Allan Saul for helpful comments on the manuscript. References

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Salazar-Gonzalez, R.M., Maldonado-Bernal, C., Ramirez-Cruz, N.E., Rios-Sarabia, N., Beltran-Nava, J., Castanon-Gonzalez, J., Castillo-Torres, N., PalmaAguirre, J.A., Carrera-Camargo, M., Lopez-Macias, C., et al. (2004). Induction of cellular immune response and anti-Salmonella enterica serovar typhi bactericidal antibodies in healthy volunteers by immunization with a vaccine candidate against typhoid fever. Immunol. Lett. 93, 115–122. Saul, A., Smith, T., and Maire, N. (2013). Stochastic simulation of endemic Salmonella enterica serovar Typhi: the importance of long lasting immunity and the carrier state. PLoS One 8, e74097. Secundino, I., Lopez-Macias, C., Cervantes-Barragan, L., Gil-Cruz, C., Rios-Sarabia, N., Pastelin-Palacios, R., Villasis-Keever, M.A., Becker, I., Puente, J.L., Calva, E., et al. (2006). Salmonella porins induce a sustained, lifelong specific bactericidal antibody memory response. Immunology 117, 59–70. Simon, R., and Levine, M.M. (2012). Glycoconjugate vaccine strategies for protection against invasive Salmonella infections. Hum. Vaccin. Immunother. 8, 494–498. Simon, R., Tennant, S.M., Galen, J.E., and Levine, M.M. (2011a). Mouse models to assess the efficacy of nontyphoidal Salmonella vaccines: revisiting the role of host innate susceptibility and routes of challenge. Vaccine 29, 5094–5106. Simon, R., Tennant, S.M., Wang, J.Y., Schmidlein, P.J., Lees, A., Ernst, R.K., Pasetti, M.F., Galen, J.E., and Levine, M.M. (2011b). Salmonella enterica serovar enteritidis core O polysaccharide conjugated to H:g,m flagellin as a candidate vaccine for protection against invasive infection with S. enteritidis. Infect. Immun. 79, 4240–4249. Simon, R., Wang, J.Y., Boyd, M.A., Tulapurkar, M.E., Ramachandran, G., Tennant, S.M., Pasetti, M., Galen, J.E., and Levine, M.M. (2013). Sustained protection in mice immunized with fractional doses of salmonella enteritidis core and o polysaccharide-flagellin glycoconjugates. PLoS One 8, e64680. Singh, S.P., Williams, Y.U., Benjamin, W.H., Klebba, P.E., and Boyd, D. (1996). Immunoprotection by monoclonal antibodies to the porins and lipopolysaccharide of Salmonella typhimurium. Microb. Pathog. 21, 249–263. Sinha, K., Mastroeni, P., Harrison, J., de Hormaeche, R.D., and Hormaeche, C.E. (1997). Salmonella typhimurium aroA, htrA, and aroD htrA mutants cause progressive infections in athymic (nu/nu) BALB/c mice. Infect. Immun. 65, 1566–1569. Sur, D., Ochiai, R.L., Bhattacharya, S.K., Ganguly, N.K., Ali, M., Manna, B., Dutta, S., Donner, A., Kanungo, S., Park, J.K., et al. (2009). A cluster-randomized effectiveness trial of Vi typhoid vaccine in India. N. Engl. J. Med. 361, 335–344. Tabu, C., Breiman, R.F., Ochieng, B., Aura, B., Cosmas, L., Audi, A., Olack, B., Bigogo, G., Ongus, J.R., Fields, P., et al. (2012). Differing burden and epidemiology of non-Typhi Salmonella bacteremia in rural and urban Kenya, 2006–2009. PLoS One 7, e31237.

Tacket, C.O., and Levine, M.M. (2007). CVD 908, CVD 908-htrA, and CVD 909 live oral typhoid vaccines: a logical progression. Clin. Infect. Dis. 45 (Suppl. 1), S20-S23. Tennant, S.M., Wang, J.Y., Galen, J.E., Simon, R., Pasetti, M.F., Gat, O., and Levine, M.M. (2011). Engineering and preclinical evaluation of attenuated nontyphoidal Salmonella strains serving as live oral vaccines and as reagent strains. Infect. Immun. 79, 4175–4185. Thiem, V.D., Lin, F.Y., Canh, d.G., Son, N.H., Anh, D.D., Mao, N.D., Chu, C., Hunt, S.W., Robbins, J.B., Schneerson, R., et al. (2011). The Vi conjugate typhoid vaccine is safe, elicits protective levels of IgG anti-Vi, and is compatible with routine infant vaccines. Clin. Vaccine Immunol. 18, 730–735. Toobak, H., Rasooli, I., Talei, D., Jahangiri, A., Owlia, P., and Darvish Alipour, A.S. (2013). Immune response variations to Salmonella enterica serovar Typhi recombinant porin proteins in mice. Biologicals 41, 224–230. Trebicka, E., Jacob, S., Pirzai, W., Hurley, B.P., and Cherayil, B.J. (2013). Role of anti-lipopolysaccharide antibodies in serum bactericidal activity against Salmonella enterica serovar Typhimurium in healthy adults and children in the United States. Clin. Vaccine Immunol. 20, 1491–1498. Vazquez-Torres, A., Jones-Carson, J., Mastroeni, P., Ischiropoulos, H., and Fang, F.C. (2000a). Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro. J. Exp. Med. 192, 227–236. Vazquez-Torres, A., Xu, Y., Jones-Carson, J., Holden, D.W., Lucia, S.M., Dinauer, M.C., Mastroeni, P., and Fang, F.C. (2000b). Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase. Science 287, 1655–1658. Vidal, S., Tremblay, M.L., Govoni, G., Gauthier, S., Sebastiani, G., Malo, D., Skamene, E., Olivier, M., Jothy, S., and Gros, P. (1995). The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene. J. Exp. Med. 182, 655–666. Wahdan, M.H., Sippel, J.E., Mikhail, I.A., Rahka, A.E., Anderson, E.S., Sparks, H.A., and Cvjetanovic, B. (1975). Controlled field trial of a typhoid vaccine prepared with a nonmotile mutant of Salmonella typhi Ty2. Bull. World Health Organ. 52, 69–73. Wahid, R., Salerno-Goncalves, R., Tacket, C.O., Levine, M.M., and Sztein, M.B. (2007). Cell-mediated immune responses in humans after immunization with one or two doses of oral live attenuated typhoid vaccine CVD 909. Vaccine 25, 1416–1425. Wahid, R., Salerno-Goncalves, R., Tacket, C.O., Levine, M.M., and Sztein, M.B. (2008). Generation of specific effector and memory T cells with gut- and secondary lymphoid tissue- homing potential by oral attenuated CVD 909 typhoid vaccine in humans. Mucosal Immunol. 1, 389–398. Wahid, R., Simon, R., Zafar, S.J., Levine, M.M., and Sztein, M.B. (2012). Live oral typhoid vaccine Ty21a

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of Experts, November 2007—conclusions and recommendations. Wkly Epidemiol. Rec. 83, 1–16. World Health Organization (WHO) (2011). Meeting of the Strategic Advisory Group of Experts on Immunization, November 2010—summary, conclusions and recommendations. Wkly Epidemiol. Rec. 86, 1–16. Wright, A.E., and Leishman, W.B. (1900). Remarks on the results which have been obtained by the antityphoid inoculations and on the methods which have been employed in the preparation of the vaccine. BMJ 1, 122–129. Zaki, S.A., and Karande, S. (2011). Multidrug-resistant typhoid fever: a review. J. Infect. Dev. Ctries 5, 324–337.

The Path to a Respiratory Syncytial Virus Vaccine Christine A. Shaw, Max Ciarlet, Brian W. Cooper, Lamberto Dionigi, Paula Keith, Karen B. O’Brien, Maryam Rafie-Kolpin and Philip R. Dormitzer

Abstract Respiratory syncytial virus (RSV) is the greatest remaining unmet infant vaccine need in developed countries and an important unmet infant vaccine need worldwide. More than 40 years of effort have yet to result in a licensed RSV vaccine for humans. Key challenges to RSV vaccine development include a peak of severe disease at 2–3 months of age, problematic biochemical behaviour of key vaccine antigens, a history of vaccine-mediated disease enhancement, and reliance on animal models that may not accurately reflect human disease processes. Potential paths to overcome these challenges include maternal immunization, structure-based engineering of vaccine antigens, the design of a novel platform for safe infant immunization, and the development of improved animal models for vaccine-enhanced disease. RSV background RSV belongs to the genus Pneumovirus in the family Paramyxoviridae. It is a large (120–300 nm diameter), pleomorphic enveloped virus with a non-segmented, negative sense, single-stranded RNA genome (~15–16 Kb), which encodes 11 proteins. Two surface glycoproteins are the major neutralization antigens: the heavily glycosylated attachment protein (G) has moderate-to-high sequence diversity and defines antigenic groups A and B; the fusion protein (F) is highly conserved between strains of both groups, recognized by broadly cross-neutralizing antibodies, and the preferred RSV vaccine antigen.

17

RSV disease in infants classically presents as bronchiolitis, a respiratory disease marked by obstruction of airflow through small airways. RSV also causes pneumonia, rhinitis, and otitis media. Severe RSV infection in infancy is associated with higher rates of asthma later in childhood (Escobar et al., 2010; Wu and Hartert, 2011). The global medical and economic impact of RSV is very high. It is the most important cause of acute lower respiratory tract infections (ALRIs) that result in hospital visits during infancy and early childhood. For example, in the United States, more than 60% of infants are infected by RSV during their first RSV season, and nearly all have been infected by 2–3 years of age (Glezen et al., 1986). Approximately 2.1 million US children less than 5 years of age are treated for RSV disease each year: 3% as inpatients, 25% in emergency departments, and 73% in paediatric practices (Hall et al., 2009). Globally, among children less than 5 years of age, RSV causes an estimated 33.8 million ALRIs each year (more than 22% of all ALRIs), resulting in 66,000–199,000 deaths, 99% of which occur in developing countries (Nair et al., 2010). RSV is also a common cause of respiratory disease among the elderly, resulting in as many hospitalizations as influenza in a heavily influenza-immunized population (Falsey et al., 2005). RSV spreads by respiratory droplets and close contact with infected persons or contaminated objects. In temperate climates, there is an annual winter epidemic. Infants are at highest risk for severe RSV disease in their first 6 months, and hospitalization peaks at 2–3 months of age (Hall

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et al., 2009; Parrott et al., 1973). Preterm birth and cardiopulmonary disease are risk factors for severe RSV disease. RSV infection of infants elicits partially protective immunity, which appears to wane more rapidly than immunity against most other respiratory viruses. Most children infected with RSV during their first year are reinfected the next year, generally with less severe disease (Glezen et al., 1986). Re-infections continue throughout life, often with upper respiratory tract symptoms, and sometimes with lower respiratory tract or sinus involvement (Hall et al., 2001). Recommended treatment of RSV bronchiolitis consists primarily of respiratory support and hydration (American Academy of Pediatrics Subcommittee on Diagnosis and Management of Bronchiolitis, 2006). No specific anti-viral therapy is recommended. The neutralizing monoclonal antibody Palivizumab® is used for prophylaxis of infants at highest risk for severe infection but is too expensive and impractical for universal use (Prescott et al., 2010). Elicitation of neutralizing antibodies should be the primary goal of vaccination because the severity of RSV disease is largely determined by the extent of viral replication (Graham, 2011). There is no licensed RSV vaccine, and developing a safe and effective RSV vaccine is a global public health priority (Anderson et al., 2013). History of RSV vaccinemediated enhanced respiratory disease (ERD) The history of RSV vaccine-mediated ERD raises safety concerns for vaccine studies in RSV-naive infants. Understanding ERD is difficult because the phenomenon has only been observed in humans in studies that took place almost 50 years ago. In a vaccine trial in the 1960s, infants and young children were immunized with a formalin-inactivated whole virion RSV preparation (FI-RSV) or an equivalent paramyxovirus preparation (FI-PIV). Five per cent of the subjects who were immunized with FI-PIV and then naturally infected by RSV during the next RSV season were hospitalized; 80% of those who were immunized with FI-RSV and then infected by RSV were

hospitalized, and two children died (Kim et al., 1969). Given this history, how can infant RSV immunization trials be conducted safely? As detailed below, three conclusions about vaccine-mediated ERD, based on the published literature, can guide strategies for safe testing of RSV vaccines: (a) individuals who have been infected with RSV in the past are not at risk for ERD after immunization; (b) ERD has not occurred after immunization of RSV-naive infants with replicating RSV vaccines; and (c) human and animal studies have defined a desired, safe immune profile to be elicited by an RSV vaccine. The conclusion that individuals who have been infected with RSV in the past are not at risk for ERD after immunization is supported by a commonplace clinical observation: an initial RSV infection does not predispose to enhanced disease upon re-infection. Instead, subsequent childhood RSV infections are typically less severe than the first infection (Glezen et al., 1986; Henderson et al., 1979). FI-RSV immunization of older children, likely to have been infected with RSV before immunization, did not predispose to ERD (Chin et al., 1969; Fulginiti et al., 1969; Kapikian et al., 1969). In BALB/c mice, RSV infection before FI-RSV immunization abrogates FI-RSVenhanced pulmonary inflammation upon RSV challenge (Waris et al., 1997). To date, there have been 15 published clinical trials of RSV subunit vaccine candidates in previously RSV-infected children and adults, and these trials have provided no evidence of ERD upon subsequent RSV infection. Many of these trials are reviewed in Hurwitz (2011). In addition, live attenuated RSV vaccine candidates have been tested in multiple trials in RSV-naive infants with no evidence of ERD upon subsequent RSV infection. Many of these trials are reviewed in Wright et al. (2007). No vectored RSV vaccine has been tested in RSV-naive infants, but one RSV vaccine candidate that expresses the RSV F glycoprotein from a bovine parainfluenza virus vector has been tested in previously RSVinfected children and adults, with no evidence of ERD upon subsequent RSV infection (Gomez et al., 2009). Other RSV vaccine candidates that express RSV F from adenovirus vectors, Sendai

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virus vectors, poxvirus vectors, or alphavirus replicons have been tested in RSV-naive experimental animals without significantly enhancing pulmonary inflammation upon RSV challenge (Hurwitz, 2011). Studies in humans and experimental animals have defined a desirable, safe immune profile to be elicited by immunization. The immune response elicited by FI-RSV is distinct from the response elicited by RSV infection or replicating RSV vaccines (Graham, 2011). In infants, FI-RSV immunization elicited little, if any, neutralizing antibody but did elicit RSV-specific cellular and non-neutralizing antibody responses (Kim et al., 1969, 1976). Autopsies on the two FI-RSVimmunized children who died upon subsequent RSV infection revealed a peri-bronchiolar monocytic infiltrate with excess eosinophils, suggesting a T-helper type 2 (Th2)-biased cellular response. Th2 CD4+ T-cells produce IL-4, IL-5, and/or IL-13, which enhance B-cell activation and antibody production, particularly IgE, to promote allergic reactions and eosinophilic inflammation and to control extracellular pathogens. In contrast, Th helper type 1 (Th1) CD4+ T-cells produce IFNγ, which stimulates the production of complement-fixing and opsonizing antibodies and directs cell-mediated inflammatory reactions to control intracellular pathogens. Experiments in several animal models corroborate that FI-RSV-elicited Th2-biased immune responses are associated with ERD upon subsequent RSV infection (reviewed in Graham, 2011). In contrast, the prophylactic efficacy of Palivizumab® and its polyclonal predecessor Respigam® demonstrate that neutralizing antibodies prevent rather than enhance RSV disease (The IMpact-RSV Study Group, 1998; Groothuis et al., 1993). Based on these findings, FI-RSV-mediated ERD has been attributed to FI-RSV’s failure to elicit neutralizing antibodies combined with its priming for an exaggerated, Th2-predominant immune response to subsequent RSV infection. The considerations detailed in this section define a desirable, safe immune profile to be elicited by RSV immunization: an antibody response that neutralizes the virus and a cellular response that is not Th2-biased.

Need for an improved animal model for RSV vaccine-mediated disease enhancement In practice, elicitation of the desired immune profile by a non-replicating RSV vaccine candidate in small animal studies appears insufficient to quell fears that a non-replicating RSV vaccine candidate might elicit ERD in RSV-naive infants. Although the preponderance of evidence in small animal models strongly supports the role of Th2 responses in causing ERD, the literature also includes small animal studies that support roles for immune complexes between RSV antigens and non-neutralizing antibodies and for cytotoxic T-cells in the pathogenesis of ERD (Cannon et al., 1988; Polack et al., 2002). Some of the inconsistency may result from deficiencies in the primary small animal models that have been used to study ERD. Superficially, these models, mice and cotton rats, do mimic the ERD that occurred in the FI-RSV trials in the 1960s. For example, FI-RSV primes for enhanced pulmonary lesions in RSV-challenged cotton rats, and the lesions resemble those in the fatal childhood cases (Prince et al., 2001). However, in rodent studies of ERD, the FI-RSV immunogen and RSV challenge stocks often contain large quantities of non-viral, cell culture-derived proteins, such as cellular constituents or bovine serum albumin (BSA) from the culture medium. In the human cases of ERD in the 1960s, the FI-RSV vaccine candidate was produced in cell culture and may have contained some quantities of cell culture-derived impurities; however, the subsequent natural exposures of the clinical trial subjects to RSV presumably resulted from droplet- or fomite-mediated transmission of relatively small inoculae of virus from other infected people, free of cell culture-derived impurities (Kim et al., 1969). Observations during pre-clinical RSV vaccine development suggest that the difference between the natural person-to-person spread of RSV that occurred during the FI-RSV trial and experimental inoculation of cell culture-produced RSV in small animal studies may limit the predictive value of the small animal experimental work for ERD in humans (Shaw et al., 2013a). Specifically, we observed that the purity of the challenge virus has

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a large impact on the severity of ERD in FI-RSVimmunized cotton rats: those animals challenged with crude medium from RSV-infected cell cultures developed robust alveolitis, a characteristic hallmark of ERD, whereas those challenged with semi-purified virus developed markedly reduced alveolitis. Additionally, the non-viral components of vaccine and challenge preparations that were devoid of infectious RSV or RSV antigens were sufficient to produce a low level of alveolitis, supporting previous reports that non-viral antigens contribute to ERD in cotton rats and mice (Boelen et al., 2000; Piedra et al., 1989). Further investigations demonstrated that FI-RSV induced robust T-cell responses to the non-viral antigen BSA, that the degree of alveolitis upon challenge paralleled BSA-specific T-cell responses, and that the cellular responses to BSA were enhanced by RSV (Shaw et al., 2013a). These findings suggest that the cotton rat model of ERD is primarily one of T-cell-driven hypersensitivity pneumonitis to non-RSV antigens (for which BSA is a convenient experimental marker), with RSV antigens having a potentiating role. This is strikingly demonstrated by experiments in which cotton rats immunized with a highly purified RSV F subunit vaccine candidate, which does not elicit T-cell responses against BSA, were protected from a crude RSV nasal challenge with no significant alveolitis (Shaw et al., 2013a). Although the F subunit vaccine elicited neutralizing antibodies capable of blocking viral replication, this did not explain the lack of alveolitis. Immunization with experimentally denatured RSV F subunit antigen did not elicit neutralizing antibodies; however, no alveolitis resulted from the RSV challenge, even though RSV-specific T-cells were present. Addition of virus-free cell-culture medium to the intact RSV F subunit preparation resulted in alveolitis upon crude RSV challenge despite the presence of high neutralizing antibody titres that protected against RSV replication, and this alveolitis was accompanied by BSA-specific T-cell responses (Shaw et al., 2013a). Given that the infants who experienced enhanced RSV disease after FI-RSV immunization were not re-exposed to cell culture-derived non-viral antigens during their natural RSV challenge, it appears that the cotton rat model of ERD

does not accurately model the illness that the FIRSV-immunized children experienced. Despite these shortcomings, the cotton rat model does have a role as a negative gatekeeper, analogous to the role of the desired immune profile. A vaccine candidate that exacerbates RSV pathology in the cotton rat is unlikely ever to be tested in human infants. However, the converse is not true: failure of a non-replicating vaccine to elicit ERD in the cotton rat model is considered insufficient evidence that the vaccine does not pose a risk of ERD in RSV-naive infants. In fact, subsequent to the FI-RSV tragedy, no non-replicating RSV vaccine has been intentionally tested in RSV-naive infants. An animal model of RSV ERD sufficiently robust to ensure safety for testing in infants would greatly advance the field. The key characteristics of such a model would include: 1

2

3

Sufficient susceptibility to RSV that a small viral inoculum amplifies many-fold in the host, as it does in humans. In contrast, cotton rats and mice require large viral inocula, with their accompanying large volume (resulting in chemical pneumonitis) and antigenic load (potentially resulting in hypersensitivity). The production of a respiratory disease endpoint that mimics human disease. In the cotton rat and mouse models, protection from virus shedding, not from RSV disease, is the endpoint. The absence of cell culture-derived impurities in the RSV challenge preparation, either because it is highly purified or because it has been passaged from animal to animal of the species used in the challenge experiment, with no intervening propagation in cell culture.

The bovine model may provide such characteristics. Bovine RSV (BRSV) causes infection of calves in the field, spreading through herds. A small infectious inoculum of BRSV amplifies, with infection sometimes involving the lower respiratory tract to cause bronchiolitis and a broncho-interstitial pneumonia (Valarcher and Taylor, 2007). Signs of BRSV disease in cows resemble those of RSV disease in humans, including wheezing, fever, and respiratory distress (Valarcher

The Path to an RSV Vaccine |  415

and Taylor, 2007). Experimentally, BRSV can be passaged from cow to cow to produce viral challenge stocks that contain no cell culturederived impurities. BRSV is distinct from RSV, but the F glycoproteins are closely related with approximately 80% amino acid sequence identity and some cross-reactive neutralizing epitopes (Orvell et al., 1987). Although data on ERD in cows are much more limited than data from small animal models, controlled experiments indicate that immunization with cell culture-produced FI-BRSV accelerates clinical disease after challenge with non-cell culture-produced live BRSV, although no enhancement of lung pathology has been observed after such challenges, and the effect of FI-BRSV on the overall severity of disease has varied between studies (Antonis et al., 2003; West et al., 1999). Although the bovine model of ERD poses significant practical challenges, it has potential for studying vaccine-enhanced clinical disease without confounding by a superimposed hypersensitivity pneumonitis directed against the non-viral antigens shared by the vaccine and challenge virus preparations. If fully developed, such a model could better reflect the pathophysiology of ERD in the FI-RSV clinical trial and provide a path towards the safe testing of an expanded range of RSV vaccine candidates in RSV-naive infants. The maternal immunization solution to ERD Given the evidence that those previously infected with RSV are not at risk for ERD and that Th2biased cellular responses are the predominant contributor to ERD, immunizing pregnant women is an attractive strategy to circumvent the challenges of direct neonatal immunization and provide short-term protection to young infants. Protective antibodies can pass from mother to fetus via the placenta; lymphocytes do not. Maternal immunization can also address the challenge of protecting infants by the peak of severe RSV disease at 2 to 3 months of age. It is difficult to protect so early in life through active immunization of infants because there is not enough time to develop a sufficiently matured immune response, which may require more than one dose of vaccine. Direct neonatal immunization

is further complicated by the immaturity of immune system, in which antigen-presenting cell responses are inefficient, T-helper responses are type 2-biased, and antibody affinity maturation is impaired (Philbin and Levy, 2009; Wood and Siegrist, 2011). In addition, in neonates, maternal antibody can inhibit the humoral response to immunization (Crowe, 2001). The inverse correlation between maternal, cord blood, or infant serum RSV neutralizing titres and early RSV disease in infants demonstrates that maternal antibody can protect infants (Glezen et al., 1981; Piedra et al., 2003). Levels of passive RSV-specific antibodies in infants mirror those in their mothers, with transplacentally transferred RSV-specific antibody titres reaching term levels after 29–33 weeks of gestation (de Sierra et al., 1993; Simister, 2003). By approximately 3 months after birth, RSV-specific antibody titres drop below protective levels, and the incidence of RSV bronchiolitis rises. Although no consensus has been reached on a precise correlate of protection, in part because of inter-laboratory variability in assays, studies of passive protection by maternal antibody or administered immune globulin and studies of RSV infection of cotton rats suggest that infant serum neutralizing titres of approximately 1:200–1:400 prevent severe RSV disease (Groothuis et al., 1993; Piedra et al., 2003; Prince et al., 1985; Siber et al., 1994). Immunization during pregnancy is an established approach to increase passive antibody titres in infants and protect from disease, as demonstrated by dramatic reductions in neonatal tetanus through maternal immunization (Blencowe et al., 2010). Immunization against influenza during pregnancy is increasingly common and likely to increase, as is supported by recent data showing improvement in pregnancy outcomes and infant protection from febrile respiratory illness (Steinhoff et al., 2012). Infecting pregnant guinea pigs with RSV increases anti-RSV titres in the pups and reduces viral replication upon challenge (Buraphacheep and Sullender, 1997). In a small clinical trial, infants born to women immunized in the third trimester with an RSV subunit vaccine had higher RSV-specific IgG than controls up to 6 months of age (Munoz et al., 2003). The vaccine was well tolerated with no associated systemic

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al., 2009). If immunization in the 3rd trimester increases antibody titres 8-fold, the median peak of RSV disease in infants would be delayed until approximately 5 to 6 months of age (Fig. 17.1). Given the early peak of infant mortality and hospitalization from RSV, a maternal vaccine could be a highly cost-effective means to decrease the burden of disease, making it especially appealing in resource-limited settings. Nevertheless, a significant paediatric RSV disease burden would remain at 6–24 months, beyond the likely duration of maternally transferred, passive immunity.

reactions, fever, or serious adverse events in the pregnant women. All infants (20 born to vaccine recipients and 15 to placebo recipients) were born healthy, and no significant differences between the groups were observed with respect to perinatal or neonatal outcome, growth and development in the first year of life, and frequency or morbidity of respiratory illness. RSV infection was documented in two infants of vaccine recipients and four infants of placebo recipients. There was no evidence of greater T-cell or cytokine activity at 2 and 6 months of age in infants of vaccine recipients than in infants of placebo recipients. Thus, immunization during pregnancy is supported by ongoing mass immunization of pregnant women to prevent a neonatal disease (tetanus) and the precedent of a safe clinical trial of a subunit RSV vaccine in pregnancy. The maternal immunization approach to protect young infants without risking ERD promises an elegant but partial solution to the burden of paediatric RSV disease. The 1 to 2 month halflife of maternal antibody in infants predicts that each doubling of neutralizing titre should extend passive infant protection by approximately one month (Brandenburg et al., 1997; Ochola et

Replicating vaccine approaches to active immunization of infants against RSV disease Given that maternal immunization against RSV is likely to leave an unmet medical need for protection against RSV in later infancy, active immunization of infants is likely to be necessary as well. Because of concerns over ERD, the vaccine candidates now advancing towards trials in seronegative infants are replicating vaccines. Intranasal live attenuated RSV vaccine candidates have the longest history of development. However, this class of vaccines has repeatedly failed to achieve an

Passive neutralizing Ab titer with maternal immunization Passive neutralizing Ab titer without maternal immunization Disease peak without maternal immunization Disease peak with maternal immunization Active neutralizing Ab titer after infant immunization with a replicating vaccine, possibly with boosting

Protective Ab titer in infants Combined maternal and infant immunization – continuously protective Ab titers, no disease peak

Pregnancy (3rd trimester) 0

3

6

9

12

24

Age (months) Maternal immunization with a non-replicating vaccine

Infant immunization with a replicating vaccine

Figure 17.1 The dual immunization approach to protect infants from RSV disease. Passively acquired and actively elicited antibody from dual immunization could provide combined titres above the protective threshold from birth until 24 months. Curve dimensions are illustrative only.

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acceptable balance between immunogenicity and tolerability (Wright et al., 2007). In large part, this is due to inherent tolerability challenges of infecting an infant’s airway. Even nasal congestion can be sufficient to interfere with a newborn’s breastfeeding, and airway-related safety concerns have prevented the use of live attenuated intranasal influenza vaccine in infants, despite their efficacy and safety in older children (Belshe et al., 2008). Some vectored vaccine candidates, such as Sendai virus-vectored or bovine paramyxovirus-vectored candidates that express the RSV F glycoprotein are also administered intranasally (Bernstein et al., 2012; Jones et al., 2012). It remains to be determined if expressing RSV F in the infant airway from these vectors with inherently less pathogenicity for humans will result in an acceptable balance of immunogenicity and tolerability. Vectored candidates that express RSV F after intramuscular injection, such as adenovirus-vectored and alphavirus replicon-vectored vaccine candidates, avoid compromising the infant airway and can elicit immune profiles that mimic natural infection (Elliott et al., 2007; Krause et al., 2011). Challenges with vectored approaches include anti-vector immunity that interferes with vaccine efficacy and safety concerns related to the vectors. Anti-vector immunity may be passively transferred from the mother, actively acquired from previous infection by an infectious agent similar to the vector, or actively acquired from a previous dose of the vaccine, inhibiting the take of subsequent doses. Nucleic acid-based vaccines that express RSV antigens in the immunized hosts’ cells could elicit the immune profile of RSV infection without the airway risks of live attenuated or some vectored RSV vaccine candidates, with no possibility of generating infectious virus during vaccine production, and with a lower likelihood of interference from anti-vector immunity. Because the formulations that deliver nucleic acid vaccines into host cells, such as liposomal preparations, are not encoded by the vaccines, no nucleic acid delivery apparatus will be produced in the transfected host cells, and there is no risk that such vaccines will cause spreading infection in the host. A new generation of nucleic acid vaccines, based on RNA, has potential to provide a

platform that elicits non-Th2-biased immune profile without the risk of chromosomal integration. The SAM® vaccine against RSV encodes an RNA-dependent RNA polymerase to provide a burst of self-amplification of the message encoding the RSV F glycoprotein and is in preclinical development (Geall et al., 2012). The RSV SAM vaccine is substantially more potent than a DNAbased vaccine in BALB/c mice. The pre-clinical potency of the RSV SAM vaccine may reflect the ability of an RNA-based vaccine to express RSV F once the nucleic acid payload reaches the cytoplasm; a DNA payload must reach the nucleus to express. Triggering of innate immunity by cytoplasmic RNA replication may also increase immunogenicity. To date, DNA-based vaccines have demonstrated disappointingly low potency in humans relative to their potency in small animals (Kutzler and Weiner, 2008). Clinical trials will determine whether SAM RNA-based immunization is as potent in humans as it is in experimental animals. Combined modality immunization against RSV The challenge of immunization of infants against RSV may require a dual immunization strategy for complete protection from birth to 2 years of age. Maternal immunization, potentially with a subunit vaccine that maximally increases neutralizing antibody titres in pregnant women, would protect the newborns passively; immunization of infants with a replicating or nucleic acid-based vaccine during the disease-free interval opened by maternal immunization would elicit active protection that extends after the waning of transplacentally transferred immunity, without risking ERD. For the most premature infants, who are born with little transplacentally transferred antibody, the current regimen of passive prophylaxis with an intramuscularly administered monoclonal antibody is likely to continue. Because passively transferred antibody appears to interfere with humoral immunity elicited by RSV vaccine candidates, not only by inhibition of the replication of live attenuated or vectored RSV vaccine candidates, but also by antigen-specific mechanisms, multiple doses of a vaccine for infants are likely to be necessary (Kim et al., 2011). The first doses

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administered to an infant could provide priming, with subsequent doses raising high neutralizing antibody titres. Antigen-specific suppression of immunity by pre-existing antibody has relatively little effect on cell-mediated responses (Siegrist et al., 1998). The effect of a priming dose in setting the immune phenotype in response to RSV antigens also raises the potential of priming of infants with a replicating or nucleic acid vaccine followed by boosting with a subunit or VLP vaccine to elicit high neutralizing titres (Waris et al., 1997). Because implementing such a regimen in clinical practices carries the risk of inadvertent initial immunization with the non-replicating vaccine, demonstration of the safety of unprimed infant immunization with the non-replicating vaccines would likely be necessary. Can a non-replicating RSV vaccine be safe for testing in infants? It is unfortunate for RSV vaccine development that the first candidate to be tested in humans, FI-RSV, contained a damaged and atypical RSV antigen. Formalin treatment of RSV virions greatly reduces their ability to elicit neutralizing antibodies (Murphy and Walsh, 1988). The physical basis for this damage remains obscure. Formalin-treated RSV is extensively carbonylated, and reversal of this carbonylation by reduction diminishes the Th2-bias of the elicited immune response in mice but does not restore the ability of the antigen to elicit neutralizing antibodies (Moghaddam et al., 2006). The antigenic deficiencies of FI-RSV are not representative of non-replicating RSV vaccine candidates now in development. For example, some subunit vaccine candidates that have not been treated with formalin or denaturing agents elicit neutralizing antibodies in cotton rats and mice and boost neutralizing antibody titres in humans (Glenn et al., 2013; Hurwitz, 2011; Shaw et al., 2013a; Smith et al., 2012; Swanson et al., 2011). Immunization with native or denatured, pure subunit F does not cause alveolitis in cotton rats upon RSV challenge (Shaw et al., 2013a). However, intramuscular coimmunization with purified, recombinant RSV F is not sufficient to prevent the hypersensitivity pneumonitis elicited by intramuscular immunization with cell-culture-derived, non-viral antigens

followed by pulmonary challenge with the same non-viral antigens, mixed with infectious RSV, in the cotton rat model (Shaw et al., 2013a). The margin of safety from ERD provided by vaccineelicited neutralizing antibodies could be greater in humans than in small animal models. In humans, the RSV antigenic challenge after droplet or fomite introduction of small volumes of infectious RSV-containing matter requires multiple viral replication cycles, which could be blocked by neutralizing antibodies; in small animal models, because of the inherently limited replication of RSV in a non-native host, large viral challenges are required, providing a substantial immediate antigenic challenge, even if the limited viral replication is further reduced by neutralizing antibodies. Despite uncertainty over how to extrapolate small animal experimental results to human ERD, the demonstrable role of cell culture-derived impurities in experimental ERD makes high purity a desirable characteristic for any nonreplicating RSV vaccine candidate that might be considered for testing in infants. Without protein engineering to improve the biochemical characteristics of F, the major neutralization antigen of RSV, preparations of this antigen tend to have suboptimal purity, homogeneity, and reproducibility. This is in part due to the inherent ‘stickiness’ of the exposed hydrophobic transmembrane and fusion peptide regions of full length, wild type RSV F glycoprotein that is extracted from viral or cell membranes. Exposed hydrophobic regions can bind protein and non-protein components of initial viral or cell lysates and complicate purification. Structural engineering of the RSV F glycoprotein can eliminate exposed hydrophobic regions and improve the purity and homogeneity of RSV F to the point where the expressed antigen crystallizes (Swanson et al., 2011). Next generation non-replicating vaccines could further address ERD concerns by eliciting an antibody repertoire that more closely resembles that elicited by RSV infection. RSV F, like other paramyxovirus fusion proteins, is a dynamic molecular machine, which functions during cell entry to fuse the viral and cell membranes (Magro et al., 2012; Yin et al., 2005, 2006). Before it ‘fires’ to fuse membranes, F has a ‘prefusion’ conformation, with a C-terminal coiled-coil anchoring it in the

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viral membrane, a roughly spherical globular head (giving it a ‘lollipop’ shape), and a fusion peptide compactly packed into the top of the globular head together with a folded extension apparatus (Magro et al., 2012; Yin et al., 2006). During cell entry, the fusion peptide is thrust out of head at the tip of a newly extended coiled-coil to insert into a cellular membrane, and the C-terminal coiled-coil of the pre-fusion form dissociates and wraps back around the head to pack against the new coiled-coil, form a 6-helix bundle, and bring the viral membrane-anchored transmembrane region and cellular membrane–anchored fusion peptide together. This rearrangement yields a much more stable molecule, which has a crutch-shaped contour (Magro et al., 2012; McLellan et al., 2011b; Swanson et al., 2011; Yin et al., 2005). When extracted from a membrane, or expressed as a recombinant ectodomain (containing the parts of the native molecule external to the membrane) F flips to the post-fusion state (Calder et al., 2000). Therefore, RSV F subunit vaccines tested to date have probably contained F predominantly or entirely in the post-fusion conformation. A greater proportion of F on RSV virions and virus-like particle vaccine candidates may be in the pre-fusion conformation, based on the necessity of pre-fusion F for virion infectivity and the restraining effect of membrane anchoring on rearrangement. However, electron micrographs of parainfluenza virus 5 virions show a predominance of post-fusion F, demonstrating that predominantly pre-fusion conformation of the F glycoprotein in membrane-anchored vaccine antigen preparations cannot be assumed (Ludwig et al., 2008). Detailed structural analysis has demonstrated that the well-characterized neutralization epitopes of RSV F, those characterized by single monoclonal antibody (mAb) escape mutations and co-crystal structures, are shared by pre-fusion and post-fusion F (McLellan et al., 2011b; Swanson et al., 2011). The presence of these shared epitopes is likely the basis for the elicitation of neutralizing antibodies by immunization with post-fusion RSV F. However, a recent study using antibody depletion techniques to probe the specificities of neutralizing antibodies in polyclonal human and experimental animal antisera indicates that a

significant proportion of the neutralizing activity of post-infection serum may be directed against epitopes present only on pre-fusion RSV F (Magro et al., 2012). The contrast between this finding and the specificities of known RSV neutralizing mAbs suggests that the screening techniques used to identify RSV neutralizing mAbs may be biased towards those recognizing post-fusion as well as pre-fusion F and that mAbs directed against neutralizing epitopes found only on pre-fusion F have yet to be discovered. Although there is now experimental evidence that the pre-fusion form of RSV F can be stabilized off the virion through structure-based engineering (Magro et al., 2012), to date there is no preparation of RSV F that has been verified to be homogenously pre-fusion, stable, and purifiable in sufficient quantity to be used in the immunization and other studies needed to test definitively the match between the antibody repertoire and protective responses elicited by human RSV infection and by pre-fusion RSV F immunization. Modern techniques of human antibody repertoire analysis, which have yielded important insights into immunity against HIV and influenza (Bonsignori et al., 2012; Jiang et al., 2013; Wrammert et al., 2008), have yet to be applied to RSV vaccine research. In coming years, the development of homogenous, stabilized pre-fusion F preparations and human antibody repertoire cloning studies of RSV infection and immunization may provide translational medicine information that could complement evidence from improved animal models to assess more reliably the safety of testing modern nonreplicating RSV vaccine candidates in previously RSV-naive infants. The complexity of a multi-functional and multi-epitope antigen, like RSV F, contributes to the challenge of safety assessment. F mediates membrane fusion, contributes to cell attachment by binding heparan sulfate, and may even modulate the host immune response by signalling through toll-like receptor 4 (although an effect of contaminants in RSV F preparations is difficult to exclude) (Feldman et al., 2000; Kurt-Jones et al., 2000). F has a mixture of epitopes recognized by neutralizing and non-neutralizing antibodies (Beeler and van Wyke Coelingh, 1989). The clinically well-established finding that prophylaxis by

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intramuscular injection of the neutralizing mAb, Palivizumab, substantially protects high risk newborns from RSV disease suggests a potential means to immunize newborns safely against RSV with a non-replicating vaccine (The IMpact-RSV Study Group, 1998). A vaccine that elicited only antibodies with the specificity of Palivizumab without eliciting T-cell immunity should protect without risk of disease enhancement. Of course, such a vaccine is not possible. Cellular immunity inevitably accompanies humoral immunity, and protein antigens do not elicit antibody responses limited to a single specificity. However, it may be possible to produce, through scaffolding or other molecular mimicry, an antigen that elicits Palivizumab-like antibodies with a substantially lower background of other antigen-specific cellular and humoral specificities. To date, attempts to do so have not been successful. Even scaffolded antigens that appear substantially similar to the Palivizumab epitope (known from crystal structures of RSV F and of RSV F peptides in complex with a related antibody, Motavizumab) fail to elicit a high proportion of antibodies with the desired specificity and functional activity (McLellan et al., 2011a). Although a scaffolding approach has limitations, such as the generation of neo-epitopes in a scaffolded construct and limited extent of the surface presented by the scaffold, it is possible that a mimetic that more closely models a desired RSV neutralization epitope could elicit a sufficiently directed immune response to reduce ERD concerns. Addition of the RSV G glycoprotein to nonreplicating RSV vaccine candidates could either increase or decrease ERD concerns, depending on the interpretation of the experimental literature. Although G is more diverse than F and responsible for the serological differences between the A and B groups of RSV, it is also an important target of neutralizing antibodies. Because higher neutralizing titres correlate with protection from disease (Glezen et al., 1981; Piedra et al., 2003), including G in an RSV vaccine could plausibly reduce the risk of ERD in RSV-naive vaccinees. However, immunization of BALB/c mice with a vaccinia virus that expresses RSV G leads to pulmonary eosinophilia upon experimental RSV challenge; immunization with a vaccinia virus

that expresses RSV F does not (Openshaw et al., 1992). On the other hand, the BALB/c T-helper response to vaccinia virus-expressed G is dominated by a highly restricted set of T-cell receptors and G peptide specificities, and immunization of BALB/c mice with formalin-inactivated RSV that lacks G also causes pulmonary eosinophilia upon RSV challenge, making it difficult to extrapolate the small animal experimental findings implicating G in ERD to human immunization ( Johnson et al., 2004; Varga et al., 2001). RSV G appears to be an immunomodulatory glycoprotein. It is mucin-like in the extent of its glycosylation, and a soluble form of the G glycoprotein is secreted in large quantities from RSV-infected cells, possibly as an immune decoy (Bukreyev et al., 2012). The conserved G central domain contains a CX3C chemokine motif that acts as a functional mimic of the chemokine fractalkine, and passive administration of antibodies that recognize this motif quell the inflammation associated with RSV infection in BALB/c mice (Caidi et al., 2012; Tripp et al., 2001). In theory, a vaccine based on this G motif could elicit antibodies that quell pulmonary inflammation upon RSV infection. However, with the current state of knowledge, trials of such a vaccine in RSV-naive infants would be precluded by safety concerns. The formulation of non-replicating vaccine candidates also can reduce the risk of ERD. Immunomodulators, such as toll-like receptor agonists, when added to non-replicating vaccines before immunization of experimental animals can shift the T-helper phenotype away from Th2 and can promote antibody affinity maturation, increasing neutralizing titres (Delgado et al., 2009). Such adjuvants can influence the immune profile elicited by non-replicating vaccine to more closely resemble the desired profile elicited by replicating vaccines. This approach is effective in a laboratory setting and, although levels of caution are high around the licensing of novel adjuvants (particularly for newborn immunization), the licensure of a human papillomavirus vaccine containing a toll-like receptor 4 agonist for adolescent immunization suggests that such barriers may be surmountable (Paavonen et al., 2009). For immunization of the elderly against RSV, barriers to the use of novel adjuvants should be much lower. In

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the elderly, whose immune response profile to RSV antigens has been set by previous infection, the T-helper phenotype elicited is not a primary concern. An ability of immunomodulators to overcome immuno-senescence could make them useful vaccine components of an RSV vaccine to protect the elderly. Conclusion RSV is one of the most important remaining unmet infant vaccine needs. The need remains unmet because of the multiple challenges of newborn immunization against this pathogen. Improved animal models, structure-based antigen design, innovative nucleic acid and vectored vaccines, and novel immunization regimens targeting pregnant women may all contribute to meeting these challenges. Acknowledgements The authors are employees of Novartis Vaccines and Diagnostics and Novartis shareholders. We thank Fred Porter and Jeffrey Ulmer for scientific discussions. A substantially similar manuscript has been published in Current Opinion in Virology (Shaw et al., 2013b). References

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Paavonen, J., Naud, P., Salmeron, J., Wheeler, C.M., Chow, S.N., Apter, D., Kitchener, H., Castellsague, X., Teixeira, J.C., Skinner, S.R., et al. (2009). Efficacy of human papillomavirus (HPV)–16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women. Lancet 374, 301–314. Parrott, R.H., Kim, H.W., Arrobio, J.O., Hodes, D.S., Murphy, B.R., Brandt, C.D., Camargo, E., and Chanock, R.M. (1973). Epidemiology of respiratory syncytial virus infection in Washington, D.C. II. Infection and disease with respect to age, immunologic status, race and sex. Am. J. Epidemiol. 98, 289–300. Philbin, V.J., and Levy, O. (2009). Developmental biology of the innate immune response: implications for neonatal and infant vaccine development. Pediatric research 65, 98R–105R. Piedra, P.A., Faden, H.S., Camussi, G., Wong, D.T., and Ogra, P.L. (1989). Mechanism of lung injury in cotton rats immunized with formalin-inactivated respiratory syncytial virus. Vaccine 7, 34–38. Piedra, P.A., Jewell, A.M., Cron, S.G., Atmar, R.L., and Glezen, W.P. (2003). Correlates of immunity to respiratory syncytial virus (RSV) associatedhospitalization: establishment of minimum protective threshold levels of serum neutralizing antibodies. Vaccine 21, 3479–3482. Polack, F.P., Teng, M.N., Collins, P.L., Prince, G.A., Exner, M., Regele, H., Lirman, D.D., Rabold, R., Hoffman, S.J., Karp, C.L., et al. (2002). A role for immune complexes in enhanced respiratory syncytial virus disease. J. Exp. Med. 196, 859–865. Prescott, W.A., Jr., Doloresco, F., Brown, J., and Paladino, J.A. (2010). Cost-effectiveness of respiratory syncytial virus prophylaxis: a critical and systematic review. PharmacoEconomics 28, 279–293. Prince, G.A., Horswood, R.L., and Chanock, R.M. (1985). Quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats. J. Virol. 55, 517–520. Prince, G.A., Curtis, S.J., Yim, K.C., and Porter, D.D. (2001). Vaccine-enhanced respiratory syncytial virus disease in cotton rats following immunization with Lot 100 or a newly prepared reference vaccine. J. Gen. Virol. 82, 2881–2888. Shaw, C.A., Galarneau, J.R., Bowenkamp, K.E., Swanson, K.A., Palmer, G.A., Palladino, G., Markovits, J.E., Valiante, N.M., Dormitzer, P.R., and Otten, G.R. (2013a). The role of non-viral antigens in the cotton rat model of respiratory syncytial virus vaccine-enhanced disease. Vaccine 31, 306–312. Shaw, C.A., Ciarlet, M., Cooper, B.W., Dionigi, L., Keith, P., O’Brien, K.B., Rafie-Kolpin, M., and Dormitzer, P.R. (2013b). The path to an RSV vaccine. Curr. Opin. Virol. 3, 332–342. Siber, G.R., Leombruno, D., Leszczynski, J., McIver, J., Bodkin, D., Gonin, R., Thompson, C.M., Walsh, E.E., Piedra, P.A., Hemming, V.G., et al. (1994). Comparison of antibody concentrations and protective activity of respiratory syncytial virus immune globulin and

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Staphylococcus aureus Linhui Wang and Jean C. Lee

Abstract Development of an effective vaccine to prevent Staphylococcus aureus disease in humans continues to be a major challenge for the research community. There have been four phase III clinical trials aimed at proving efficacy with a single component vaccine or immunotherapeutic, but each has failed at different stages of development. Vaccines comprising multiple staphylococcal proteins and polysaccharides and vaccines aimed at eliciting a T-cell response are currently being explored, but there is little agreement on the ideal formulation. Major limitations include our failure to identify convincing correlates of protective immunity to S. aureus, as well as less than optimal models of staphylococcal infection in rodents. Despite these challenges, there are new approaches and ongoing efforts by both industry and academic labs to design an effective vaccine to contain this formidable pathogen. Introduction Staphylococcus aureus is a major cause of invasive human infections, including bacteraemia, endocarditis, pneumonia, septic arthritis, osteomyelitis, and infections associated with wounds and prosthetic devices. Methicillin-resistant S. aureus (MRSA) have become endemic in hospitals, and community-associated MRSA strains are spreading worldwide, posing a major global challenge (Bassetti et al., 2009; Chambers, 2005; Dantes et al., 2013). MRSA strains account for more than half of all community and hospital infections (Klevens et al., 2007). The effectiveness of vancomycin against MRSA strains has diminished

18 with the rise of strains with decreased susceptibility to this ‘antibiotic of last resort’ (de Lassence et al., 2006; Holmes et al., 2011). Moreover, the recognition of reduced vancomycin susceptibility among S. aureus isolates (heteroresistance and the MIC creep) over the past decade has further diminished the value of therapy (Dhand and Sakoulas, 2012). A vaccine to reduce the morbidity, mortality, and economic impact associated with staphylococcal disease is urgently needed. Preclinical trials in rodents have identified a variety of protective S. aureus antigens (Anderson et al., 2012; Dhand and Sakoulas, 2012; Fattom et al., 1990, 1993; Josefsson et al., 2001; Kim et al., 2010; Kuklin et al., 2006; Maira-Litran et al., 2005; Mishra et al., 2012; Schluepen et al., 2013; Shinefield et al., 2002; Stranger-Jones et al., 2006; Tuchscherr et al., 2008; Wacker et al., 2014). However, these individual preclinical studies are limited in scope, evaluated against a handful of laboratory and clinical isolates, and have not translated into an effective vaccine for humans. Several candidate S. aureus vaccines have advanced to clinical vaccine trials (Table 18.1), but capsular polysaccharide (CP) conjugates and vaccines targeting individual protein antigens have failed at various developmental stages, underscoring the need for novel vaccines with broader efficacy (Broughan et al., 2011; Fowler et al., 2013; Shinefield et al., 2002). Multicomponent vaccines are now being developed and tested by numerous research groups. Some are a combination of CP and protein antigens, but the appropriate mix of antigens to elicit a protective immune response in humans remains controversial. Critical parameters

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Table 18.1  S. aureus vaccines in active immunization clinical trials Product

Sponsor

Composition

StaphVAX

Nabi

CP5- and CP8-conjugates Phase III failed

Status

V710

Merck

IsdB

Phase IIb/III terminated

Toxoids

Nabi (GSK)

Alpha toxoid and PVL LukS

Phase I-II completed

Tetravalent vaccine

GlaxoSmithKline

????

Phase I completed

SA4Ag

Pfizer

CP5, CP8, ClfA, MntC

Phase I/II completed

Tetravalent protein vaccine

Novartis

Two surface proteins Two secreted proteins

Phase I completed

STEBVax

NIH (NIAID) and IBT

Non-toxic SEB

Phase I in progress

NDV-3

NovaDigm Therapeutics

rAls3p-N

Phase Ib completed

of vaccine antigens for inclusion in a multivalent vaccine include the percentage of clinical S. aureus isolates producing the antigen, the extent of antigenic diversity among those isolates, the level of antigen expression in vivo, and accessibility of the target antigen on the bacterial surface. Many of the early attempts towards S. aureus vaccine development targeted antigens that would elicit a brisk and long-lasting humoral immune response. However, with the failure of multiple vaccine trials in humans, there is now scepticism about the importance of antibodies and a new interest in the notion that T-cells may play a critical role in immunity to S. aureus infections. Multicomponent S. aureus vaccines that elicit both humoral and cell-mediated immune responses are likely the most desirable, and several of these are currently under evaluation (Anderson et al., 2012; Proctor, 2012). Moreover, new information on mechanisms of immune escape by S. aureus may provide critical clues towards development of an effective vaccine. Nonetheless, many questions remain concerning the choice of vaccine antigens and the types and measures of immune effectors that an S. aureus vaccine should elicit. Active immunization strategies to prevent S. aureus – clinical trials StaphVAX The first staphylococcal vaccine to reach phase III clinical trials was based on the two capsular

polysaccharides (CPs) that are most prevalent among clinical strains of S. aureus. Fattom et al. (1990) at Nabi Biopharmaceuticals conjugated the serotype 5 (CP5) and serotype 8 (CP8) polysaccharides to non-toxic recombinant Pseudomonas aeruginosa exoprotein A (rEPA). CP5- and CP8-EPA were immunogenic in mice and humans, and they induced opsonic antibodies that showed efficacy in protecting rodents from lethality and reducing the bacterial burden in non-lethal staphylococcal infections (Fattom et al., 1990, 1993; Fattom et al., 1996; Lee et al., 1997). Passive immunization studies indicated that CP-specific antibodies reduced staphylococcal endocarditis in rats (Lee et al., 1997) and experimental mastitis in mice (Tuchscherr et al., 2008). Nabi Biopharmaceuticals combined the CP5- and CP8-conjugate vaccines into a bivalent vaccine called StaphVAX for immunization of humans at elevated risk for S. aureus infection. A phase III clinical trial of the vaccine, conducted between April 1998 and April 2000, enrolled 1804 patients with end-stage renal disease who were receiving haemodialysis (Shinefield et al., 2002). Subjects were randomized to receive either a single intramuscular injection of the vaccine or a placebo injection. The primary hypothesis of the study was that the vaccine would prevent S. aureus bacteraemia during the period from week 3 to week 54 after immunization. The vaccine significantly reduced the incidence of S. aureus bacteraemia between weeks 3 and 40 after immunization. During this period, S. aureus bacteraemia occurred in 11 of the

S. aureus Vaccine Challenges |  427

892 patients who received the vaccine, compared with 26 of the 906 control patients. The vaccine efficacy up to 40 weeks after immunization was 57% (P = 0.02). However, at the study endpoint (week 54) the vaccine efficacy was only 26%, which was not statistically significant (Shinefield et al., 2002). The reduction in vaccine efficacy after week 40 correlated with a decline in CP5 and CP8 antibody levels among the vaccine recipients. A confirmatory phase III clinical trial enrolled 3600 haemodialysis patients who were evaluated for bacteraemia from 3 to 35 weeks after receiving StaphVAX (NCT00071214). To boost their antibody levels, a second dose of StaphVAX was administered, and the patients were followed for an additional six months. Results from the second trial, announced in November 2005 showed that StaphVAX offered no significant protection against bacteraemia over the placebo control [3–35 weeks, −23% efficacy; 3–60 weeks, −8% efficacy (Matalon et al., 2012)]. These results led Nabi to halt further development of StaphVAX. The company attributed the clinical failure of the vaccine to the immunocompromised status of the patients in the trial. In addition, a manufacturing problem was confirmed since the functional characteristics of the antibodies generated by the vaccine used in the second clinical study were inferior to antibodies generated by previous vaccine lots. The conjugate used for the confirmatory phase III study was produced by a different contract manufacturing organization, and the functionality of the antibodies was only tested after completion of the second phase III trial. The failed Nabi clinical trials suggest that a conjugate vaccine that targets the S. aureus CPs alone is insufficient to protect against staphylococcal bacteraemia. Because of the complexity of this pathogen and its myriad of virulence factors, the inclusion of multiple staphylococcal antigens would likely result in a more effective vaccine. The iron-regulated surface determinant (IsdB) IsdB is an S. aureus cell wall-anchored protein that plays a role in staphylococcal haem iron acquisition (Mazmanian et al., 2002). It was first identified as a vaccine target by its reactivity with convalescent-phase serum from S. aureus-infected

patients (Etz et al., 2002). As a vaccine candidate, IsdB (designated V710 by Merck) enhanced survival in mice after a lethal S. aureus challenge ( Joshi et al., 2012), and it showed good immunogenicity in humans (Harro et al., 2012; Moustafa et al., 2012). Merck conducted a phase II/III clinical trial between December 2007 and August 2011 among ~8000 adult patients undergoing cardiothoracic surgery involving a median sternotomy (NCT00518687). The subjects received either placebo or a single 60-µg dose of V710 (no adjuvant) for the prevention of S. aureus bacteraemia and deep sternal wound infection. The trial was terminated in June 2011 after an interim data analysis revealed safety concerns and low efficacy (Fowler et al., 2013). The use of V710 elicited a robust antibody response in the recipients, but it did not reduce the rate of serious postoperative S. aureus infections compared with placebo. The mortality rate in patients with staphylococcal infections was significantly higher among V710 than placebo recipients. Among patients who developed S. aureus infection, those in the vaccine group were ~5 times more likely to die, and to die of multiorgan system failure, than those in the placebo group. The mechanism by which receipt of V710 worsened the outcome of post-operative staphylococcal infection is not known, but it is of grave concern to investigators in the field of S. aureus vaccine development. rAls3p-N The Candida albicans agglutinin-like sequence (Als)3p protein belongs to a family of adhesins responsible for colonization of the pathogen on host mucosal surfaces (Hoyer et al., 2008). Through molecular modelling, scientists predicted that candidal Als1p and Als3p adhesins have similar three-dimensional structures to the surface adhesin S. aureus clumping factor A (ClfA) (Sheppard et al., 2004). Two very high doses (300 µg each) of a vaccine derived from the recombinant N terminus of Als3p reduced lethality in mice challenged by the intraperitoneal (IP) route with S. aureus (Spellberg et al., 2008). Neither adoptive transfer of sera elicited by rAls3p-N vaccination nor primed B220+ B-cells protected mice against lethal challenge with S. aureus. In contrast, primed CD4+ or CD8+ T-cells

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transferred protection. rAls3p-N vaccination induced a Th1/Th17 response in the mice, resulting in recruitment and activation of phagocytes at the infection site, resulting in effective S. aureus clearance (Lin et al., 2009). In a phase I clinical trial among 40 subjects, an intramuscular (IM) dose of either 30 or 300 µg of rAls3p-N (designated NDV-3 by Novadigm Therapeutics, Inc.) was immunogenic in humans. NDV-3-primed peripheral blood mononuclear cells produced both IFN-gamma and IL-17A after stimulation with rAls3p-N in vitro. After a booster dose, a memory T-cell response was noted in the majority of subjects (Schmidt et al., 2012). In a recently completed phase Ib trial (NCT01447407) in 160 healthy humans, one dose of NDV-3 formulated with or without alum was given IM. In addition, a lower dose of NDV-3 without alum was given intradermally. Results from the trial have not yet been made public. STEBVax S. aureus superantigens (SAgs) are important virulence factors, and they represent a potential target for therapeutic and prophylactic intervention. At least 19 SAgs can be produced by S. aureus, and ~80% of clinical isolates harbour ≥1 SAg gene (Becker et al., 2003; Ferry et al., 2005). The major SAgs include staphylococcal enterotoxin A (SEA), SEB, SEC, SED, SEE, and TSST-1, and the proteins range in size from 23 to 29 kDa. SEB is a category B bioterrorism agent, and non-toxic mutant SEB protein (STEBVax) carries three point mutations that abrogate the binding of SEB to human MHC II molecules. Consequently, SEB is rendered nontoxic while retaining most of its immunogenicity (Boles et al., 2003). The staphylococcal SAg toxins share some amino acid homology, as well as secondary and tertiary structures. Thus, immunization with a single SAg can induce cross-protective antibodies to heterologous SAgs (Bavari et al., 1999; Ulrich, 1998). Neutralizing antibodies play a key role in protection from SAg-induced toxicity (Bonventre et al., 1984; Notermans et al., 1983; Strandberg et al., 2010). A phase I clinical trial of STEBVax administered to 28 healthy adults (age 18–40) was begun in February 2011. The primary objective is to develop a hyperimmune human IgG product for

treatment of toxic shock syndrome originating from nosocomial, environmental, or potential bioterrorist events. A secondary goal is to develop a vaccine that will protect against a broad range of staphylococcal SAgs. The vaccine recipients receive a single injection of STEBVax in escalating doses (0.01 µg to 20 µg). A subset of adults will receive two injections of the 20-µg vaccine dose. The trial is scheduled to be completed in 2014. Multicomponent S. aureus vaccines under evaluation by Big Pharma Neither CP-conjugates nor IsdB showed protective efficacy in human clinical vaccine trials, underscoring the need for novel vaccines with broader efficacy (Broughan et al., 2011; Shinefield et al., 2002). Multi-component S. aureus vaccines that elicit both humoral and cell-mediated immune responses are currently under study by several large pharmaceutical companies (Anderson et al., 2012; Proctor, 2012). Preclinical studies have indicated that combination vaccines are in general more effective than single component vaccines (Lattar et al., 2014; Stranger-Jones et al., 2006; Tuchscherr et al., 2008). Pfizer Inc. Pfizer initially described a trivalent S. aureus vaccine comprised of CP5, CP8, and clumping factor A (ClfA). Antibodies to CP5-CRM197 and CP8CRM197 have documented opsonic activity for encapsulated S. aureus in in vitro opsonophagocytic killing assays (Nanra et al., 2009, 2012). ClfA is a surface protein adhesin that binds to fibrinogen (McDevitt et al., 1994) and promotes the attachment of S. aureus to biomaterial surfaces (Vaudaux et al., 1995), blood clots (Moreillon et al., 1995), and damaged endothelial surfaces (Moreillon et al., 1995). ClfA also plays an important role in S. aureus binding to platelets, an interaction that is critical in animal models of catheter-induced staphylococcal endocarditis (Sullam et al., 1996). Preclinical studies showed that mice immunized with the fibrinogen-binding domain of ClfA showed reduced staphylococcal mastitis (Gong et al., 2010), as well as protection from lethality following intravenous (IV) challenge with S.

S. aureus Vaccine Challenges |  429

aureus (Narita et al., 2010). Other investigators documented reductions in arthritis and lethality induced by S. aureus, although ClfA-mediated protection was strain-dependent ( Josefsson et al., 2001). Therapy with ClfA antibodies and vancomycin resulted in better bacterial clearance from the blood of rabbits with catheter-induced S. aureus endocarditis than did vancomycin treatment alone (Vernachio et al., 2003). However, the bacterial burdens in the tissues of the infected animals were not significantly reduced. A fibrinogen binding-deficient mutant of the ClfA protein was subsequently constructed and used as an immunogen in mice ( Josefsson et al., 2008). Compared to animals given BSA, immunization with the recombinant A domain of the ClfAP336SY338A mutant formulated with Freund’s adjuvant resulted in a reduction in lethality (but not arthritis) following IV challenge with strain Newman. Pfizer scientists have reported that vaccine-induced human antibodies to ClfA block the binding of S. aureus cells to immobilized fibrinogen (Hawkins et al., 2012). Furthermore, the antibodies were able to displace S. aureus bound to Fg, suggesting that the ligand-binding activity of ClfA may be effectively neutralized through vaccination. Pfizer conducted a phase I clinical trial of their trivalent vaccine called SA3Ag in 2010. The subjects included ~400 adults in Australia that were either 18–24 years or 50–85 years old, and they received one of three ascending doses of CP5CRM, CP8-CRM, and the recombinant fibrinogen binding deficient ClfA protein (rClfAm). A single dose of the 3-component S. aureus candidate vaccine was well tolerated and induced a rapid and persistent functional antibody response in adults for 12 months (Richmond et al., 2012). Subsequently, Pfizer developed a tetravalent vaccine candidate SA4Ag, containing CP5-CRM, CP8-CRM, rClfAm, and MntC, a manganese transport protein. MntC is an in vivo expressed and conserved surface metal binding subunit of MntABC, a heterotrimeric manganese transporter embedded in the bacterial cell membrane (Anderson et al., 2012). Manganese acquisition plays important roles in bacterial metabolism, cell wall synthesis and virulence (Horsburgh et al., 2002a,b; Papp-Wallace and Maguire, 2006). Preclinical studies (Anderson et al., 2012) showed

that mice given a three-dose vaccination regimen with 10 μg of MntC showed reduced bacteraemia following challenge by a single strain each of S. aureus and S. epidermidis. Likewise, passive immunization with 0.4 mg of a MntC-specific monoclonal antibody (mAb) significantly reduced S. epidermidis bacteraemia levels in infant rats. More recently, Pfizer evaluated a single dose of SA4Ag given IM to 456 subjects (18–64 years old) in a phase I/II clinical trial. In a separate trial, they are vaccinating older adults (65–85 years old) with a similar vaccine formulation, and for each population they are evaluating vaccine immunogenicity (with escalating doses) and effects on S. aureus colonization. The results of these studies are not yet available. GlaxoSmithKline (GSK) biologicals GSK evaluated a four-component vaccine in a phase I trial (NCT01160172), which was completed in September 2012. The trial evaluated 88 healthy adult subjects in Belgium who received one of several formulations of the multicomponent vaccine given IM. The investigators monitored the safely and immune response to the vaccine components, as well as S. aureus colonization in the subjects. The data from this trial and the identity of the vaccine antigens tested have not been made public. However, the vaccine antigens tested were not PentaStaph, which GSK acquired from Nabi in 2009. Novartis vaccines and diagnostics Novartis is currently developing a tetravalent protein-based vaccine against S. aureus. Although details about the composition of the vaccine have not been publicly disclosed, the vaccine includes two surface proteins and two secreted proteins. The company embarked on a phase I clinical trial in healthy adults in 2012 but the details of its clinical development are still confidential. One of the staphylococcal surface-associated vaccine components in the Novartis vaccine is FhuD2 (ferric hydroxamate uptake), a novel lipoprotein responsible for the uptake of siderophore iron into the bacterial cell via a dedicated ABC transport system (Sebulsky and Heinrichs, 2001). FhuD2 is encoded within the core genome of S. aureus and conserved among clinical isolates. In

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active vaccination experiments, mice were immunized IP with FhuD2 formulated with alum on days 0 and 14 (Mishra et al., 2012). In an IP sepsis model, mice vaccinated with FhuD2 showed reduced lethality compared with mice given alum alone. In the renal abscess model, FhuD2 immunization resulted in a 1- to 2-log reduction in the bacterial load from kidneys of immunized mice. A similar level of protection was observed when mice were passively immunized with rabbit sera against FhuD2. In opsonophagocytic in vitro killing assays, FhuD2 antibodies mediated killing of ~34% of wild type S. aureus but not the fhuD2 mutant. Another cell surface components in the Novartis vaccine is a member of a previously uncharacterized family of conserved staphylococcal antigens (Csa) that are taxonomically restricted to S. aureus. The Csa family of proteins is organized into four loci with a variable number of paralogues per locus. There are 11 Csa paralogues in strain NCTC8325 and 18 paralogues in strain Newman. The extent of amino acid similarity within the Csa family ranges from 54% to 91% (Schluepen et al., 2013). The Csa paralogues are mostly annotated as tandem lipoproteins based on the presence of a characteristic lipoprotein signature sequence. The Csa1A protein was shown to be primarily associated with the cell membrane, expressed in all bacterial growth phases, and secreted into the supernatant during the stationary growth phase. However, the function of the Csa proteins in staphylococcal virulence remains largely unknown. To evaluate the Csa proteins as vaccine candidates, the four Csa proteins within locus 1 were formulated with alum, and 20 µg of each protein was used to immunize separate groups of mice on days 0 and 14. Ten days later, the animals were challenged IV with a sublethal dose of S. aureus. Compared with control mice, immunization with Csa1A resulted in a ~1.5 log reduction in the kidney bacterial burden. Syntiron LLC Syntiron is a vaccine development company that is investigating multi-protein subunit vaccines comprising surface-embedded iron transport proteins that are produced under low iron conditions. In December of 2009, Syntiron granted

Sanofi Pasteur an exclusive worldwide licence to its experimental human vaccine against S. aureus. Further details about vaccine development at Syntiron and Sanofi Pasteur have not been made public, as the project is still in the preclinical stages of development. Passive immunization with S. aureus antibodies – clinical trials Several candidate S. aureus passive immunotherapies have also advanced to clinical vaccine trials (Table 18.2); these are described below. AltaStaph AltaStaph, produced by Nabi, is a hyperimmune polyclonal antibody preparation derived from healthy volunteers immunized with the bivalent StaphVAX preparation. The product contains high levels of vaccine-induced antibodies to the S. aureus serotype 5 and 8 CPs. In a phase II study, 206 neonates were given an initial 1000 mg/kg IV dose of AltaStaph or placebo (Benjamin et al., 2006). A second dose was given 14 days later. No significant difference was seen in the rate of adverse events between the two arms, and the rates of S. aureus bacteraemia were nearly identical in both groups, at about 3%. Another phase II trial looking at AltaStaph enrolled 40 patients with S. aureus bacteraemia and persistent fever (Rupp et al., 2007). Five of 21 patients (24%) who received AltaStaph died, compared with 2 of 18 patients (11%) in the placebo group (P = 0.42). These results underscore the premise that vaccine-induced antibodies to CP5 and CP8 are insufficient to significantly reduce S. aureus bacteraemia in at-risk populations. Veronate Veronate was a pooled human immunoglobulin preparation from donors with high antibody titres against staphylococcal adhesins that bind fibrinogen and fibrin (S. aureus ClfA and S. epidermidis SdrG). A phase II trial of INH-A21 (sponsored by Inhibitex) showed a trend towards fewer episodes of S. aureus sepsis, candidaemia, and lower mortality among infants receiving 750 mg/kg of INH-A21, as compared with placebo (Bloom et

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Table 18.2 Target populations for an S. aureus vaccine Active immunization

Passive immunization

Haemodialysis patients

Low-birthweight neonates

Patients undergoing elective surgery

Patients in intensive care units

Residents of long-term care facilities

Trauma victims

Diabetics

Immunocompromised individuals

Intravenous drug users

Patients undergoing emergency surgery

Patients with HIV

Patients with intravascular or prosthetic devices

Military personnel Prisoners Athletes School children Men who have sex with men Patients with health care-associated community onset S. aureus infections

al., 2005). A follow-up phase III double-blinded, placebo-controlled study of INH-A21 was conducted in 1983 infants that received either placebo or 750 mg/kg of INH-A21 (DeJonge et al., 2007). There was no significant difference between the late onset-sepsis rate or mortality in the INHA21 group vs. the placebo group. The failure of INH-A21 was striking since the infants that were given INH-A21 were compared with infants that received placebo, rather than an IVIG preparation lacking elevated levels of antibodies to ClfA and SdrG. The INH-A21 product, although selected for its antibodies to ClfA and SdrG, likely contained antibodies to many other staphylococcal antigens, and so could be considered ‘multicomponent’ passive immunotherapy. However, the INH-A21 product was not elicited by immunization but by natural exposure to staphylococci, and so the antibodies may have been of low affinity or avidity or non-functional, as suggested by scientists at Pfizer (Hawkins et al., 2012). Another Inhibitex product was the murine mAb 12–9 that binds ClfA and inhibits fibrinogen binding to ClfA. A humanized version of mAb 12–9 is known as Tefibazumab (or Aurexis). In a rabbit model of S. aureus endocarditis, two doses of Tefibazumab (30 mg/kg) in combination with vancomycin resulted in fewer animals with bacteraemia and a significant reduction in the bacterial

load recovered from the spleens and kidneys of challenged rabbits, compared to animals given vancomycin alone (Patti, 2004). A phase II study of Tefibazumab enrolled hospitalized patients with documented S. aureus bacteraemia (Weems et al., 2006). Subjects were randomized to receive either a single Tefibazumab dose of 20 mg/kg plus standard therapy or standard therapy alone. To evaluate efficacy, a composite clinical endpoint was used, consisting of a relapse of S. aureus bacteraemia, a complication related to the S. aureus bacteraemia (such as endocarditis), or death. In the Tefibazumab group, 2 of 30 (6.7%) patients reached the composite clinical endpoint, compared with 4 of 30 (13.3%) patients in the placebo group (P = 0.455). Although preliminary clinical trials of Tefibazumab in haemodialysis patients (Hetherington et al., 2006) and cystic fibrosis patients were performed, follow-up clinical trials of Tefibazumab never occurred. Pagibaximab Biosynexus Inc. developed a humanized mAb (Pagibaximab) against Gram-positive lipoteichoic acid, a conserved poly-1,3-(glycerolphosphate) polymer with a glycolipid membrane anchor. Although preclinical studies were never published, scientists at the company sought to evaluate Pagibaximab for the prevention of

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staphylococcal sepsis in the very low birth weight neonates. Several phase I and II trials preceded a phase IIb/III clinical trial that was carried out between 2009 and 2011. Low birth weight neonates (600–1200 g; n = 1550) were the target population for this study. Pagibaximab (100 mg/ kg) was delivered IV on days 0, 1, 2, 9, 16 and 23. The product was ineffective, however, since the number of participants with staphylococcal sepsis from days 0–35 was 85 in the treatment group and 79 in the placebo group (http://clinicaltrials.gov/ ct2/show?term=biosynexus&rank=1). Although this clinical trial failed, interest remains in utilizing lipoteichoic acid (either purified from a bacterial source or synthetic) as a conserved antigen in a multicomponent vaccine formulation. This antigen, like ClfA (Nanra et al., 2009; Risley et al., 2007), may be useful in inducing opsonic antibodies against unencapsulated strains of S. aureus (Chen et al., 2013; Theilacker et al., 2012). Alpha toxin as a vaccine antigen and target for protective immunotherapy Alpha toxin (also known as alpha haemolysin; Hla) is a pore-forming cytotoxin secreted by S. aureus to which a variety of cells, including platelets, endothelial cells, lymphocytes, type 1 alveolar cells, erythrocytes, and monocytes are sensitive (Bhakdi and Tranum-Jensen, 1991; McElroy et al., 1999). In mouse models of dermonecrosis and lethal pneumonia, the severity of disease was shown to correlate with the production of Hla by different S. aureus strains (Bubeck Wardenburg and Schneewind, 2008; Kennedy et al., 2010). Adlam et al. (1977) first reported that vaccination with a glutaraldehyde treated Hla protected against the lethal gangrenous form of S. aureus mastitis in rabbits but did not prevent the abscess form of the disease. Similarly, in an experimental model of S. aureus keratitis, rabbits immunized with an Hla toxoid showed less corneal pathology and epithelial erosion than rabbits immunized with adjuvant alone (Hume et al., 2000). However, there was no difference in the number of bacteria recovered from the infected corneas of immunized or control rabbits. Menzies and Kernodle created a non-toxic and non-haemolytic Hla mutant toxin (H35L) by site-directed mutagenesis (Menzies

and Kernodle, 1994). Mice were passive immunized in the thigh muscle with 200 µl of rabbit anti-H35L serum or pre-immune serum. The H35L antiserum protected mice from lethal IP challenge with native Hla and against acute lethal challenge with a high Hla-producing S. aureus strain (Menzies and Kernodle, 1996). Active immunization with the Hla (H35L) protein (in complete Freund’s adjuvant) protected mice against lethal pneumonia (Bubeck Wardenburg and Schneewind, 2008), dermonecrotic skin lesions (Kennedy et al., 2010), and lethal peritonitis (Rauch et al., 2012). Moreover, passive immunization with neutralizing antibodies to the H35L protein protected mice against staphylococcal disease in the same three infection models. Immunization with Hla (H35L) did not prevent S. aureus peritoneal abscess formation, nor did it have much effect on the bacterial burden in the pneumonia model. In a multiple infection model study, Brady et al. (2013b) reported that mice immunized with HlaH35L formulated with alum and CpG showed a less severe infection and decreased S. aureus burden in a skin and soft tissue (SSTI) model compared to controls. In contrast, vaccination with HlaH35L resulted in a modest 1 log reduction in the kidney bacterial burden in an IV challenge model. In a prosthetic implant model of osteomyelitis, immunnization with HlaH35L resulted in no significant reduction in the bacterial burden when compared to controls. Adhikari et al. (2012b) described a detoxified Hla mutant protein called AT-62aa, which is a peptide containing the N-terminal 62 amino acids from native Hla. The authors use a modified sepsis model wherein 5 × 104 CFU of S. aureus USA300 were suspended in 3% mucin and injected IP into mice. Animals given AT-62aa survived the challenge, which was lethal for mice given other AT peptides or adjuvant alone. Similar protection elicited by AT-62aa was seen in the murine pneumonia model. In passive protection studies, mice given 4 mg of AT-62aa rabbit antibodies IP were protected from lethality following challenge IP with a low dose of S. aureus mixed with 3% hog mucin. Mice euthanized 12 h after bacterial challenge showed a lower bacterial burden than controls in the blood, kidneys, spleen and liver.

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Hla is an antigen that is included in several multicomponent vaccines tested preclinically and in phase I trials. Lattar et al. (2014) immunized rats with S. aureus CP5- or CP8-conjugate vaccines alone or together with rClfA or a genetically detoxified Hla (dHla). The vaccines were delivered according to a preventative or a therapeutic regimen, and their efficacy in an experimental model of osteomyelitis was evaluated (Lattar et al., 2014). Addition of dHla (but not ClfA) to the CP5 or CP8 vaccine induced reductions in bacterial load and bone morphologic changes compared to immunization with either conjugate vaccine alone. Both the prophylactic and therapeutic regimens were protective. The S. aureus CP components of the bivalent vaccine were crucial for protective efficacy since immunization with dHla together with a pneumococcal conjugate vaccine used as a control did not reduce staphylococcal osteomyelitis. A novel glycoengineering technology for creation of a multicomponent bioconjugate staphylococcal vaccine was recently described (Wacker et al., 2014). Genes encoding S. aureus CP5 or CP8 biosynthesis, PglB (a Campylobacter oligosaccharyl transferase), and a protein carrier (detoxified Pseudomonas aeruginosa Epa or S. aureus Hla) were co-expressed in Escherichia coli. Recombinant proteins N-glycosylated with S. aureus serotype 5 or 8 CPs were purified from E. coli. Rabbits and mice immunized with the glycoprotein vaccines produced functional antibodies that mediated in vitro opsonophagocytic killing of several S. aureus strains and neutralized Hla in an in vitro haemolytic assay. Active and passive immunization strategies targeting the CPs (CP5-Epa, CP8-Epa, or CP5-Hla) protected mice against staphylococcal bacteraemia, and the CP-Hla bioconjugate vaccine protected against both bacteraemia and lethal pneumonia. Glycoengineering technology, wherein polysaccharide and protein antigens are enzymatically linked in an E. coli production system, is a novel approach for use in vaccine development against encapsulated microbial pathogens. A phase I/II clinical trial (NCT01011335) was performed by Uniformed Services University of the Health Sciences in collaboration with Nabi Pharmaceuticals to evaluate the safety and

immunogenicity of an Hla toxoid (rAT), the native S component of Panton Valentine leukocidin (rLukS-PV) (see below), or a combination of the two proteins in military recruits (Lalani et al., 2013). The 25 µg and 50 µg monovalent and bivalent rAT/rLukS-PV vaccines were welltolerated and resulted in significantly higher anti-rAT and anti-rLukS-PV antibody levels compared to placebo. Neutralizing activity of rAT IgG and rLukS-PV IgG was measured by its ability to inhibit alpha-toxin induced haemolyses and inhibition of PVL-mediated neutrophil lysis, respectively. A two-fold or greater increase in anti-rAT and anti-rLukS-PV neutralizing antibodies at day 84 compared to baseline was noted in 83% (10/12) of subjects in each of the 50 µg monovalent and 50 µg bivalent cohorts. There was no benefit observed with a booster dose of the vaccine. Preclinical data in murine models have also shown that passive immunization strategies that target Hla afford protection against dermonecrosis, sepsis, and pneumonia (Bubeck Wardenburg and Schneewind, 2008; Kennedy et al., 2010; Rauch et al., 2012), validating the potential of Hla as a target for immunoprophylaxis against these infections. Kenta Biotech developed a humanized mAb 243-4 (KBSA301) to Hla that is under evaluation in a phase I/II clinical trial. The trial is a double blinded, placebo-controlled study assessing the safety and therapeutic potential of the mAb as an adjunctive therapy to standard of care antibiotics in patients with hospital-acquired pneumonia or ventilator-associated pneumonia. Patients are given a single IV dose of KBSA301 along with standard antibiotic treatment. Enrolment was set at 44 patients in four European countries. Four ascending doses of the mAb are being evaluated for adverse events, patient survival, bacterial burden, and clinical outcome. The trial was set to extend from June 2012 to October 2013, but it was suspended in January 2013, apparently due to funding issues. In May 2013, under a strategic agreement with Kenta Biotech, Aridis Pharmaceuticals in the US acquired the entire Kenta mAb portfolio. The product has been renamed AR-301, and the study has resumed (http://www.aridispharma.com/ar301.html). Similarly, MedImmune developed a fully

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humanized mAb against Hla, designated 2A3.1. A neutralization assay showed that this antibody protected against Hla-induced lysis of A549 cells, human monocytic cells, and rabbit erythrocytes by preventing assembly of the heptameric form of the toxin (Tkaczyk et al., 2013; Tkaczyk et al., 2012). Furthermore, mice given 5 mg/kg of 2A3.1 showed smaller skin lesions in a dermonecrosis model following challenge with S. aureus strains USA100, USA300 or CC30 (Tkaczyk et al., 2013). Subsequently, the investigators evaluated the activity of an affinity-optimized variant mAb (LC10) in a mouse S. aureus pneumonia model. Passive immunization with LC10 increased survival following intranasal challenge with diverse clinical isolates (Hua et al., 2014). mAb LC10 also reduced the bacterial load in the lungs of mice challenged with USA300 strain SF8300 and blunted an Hla-mediated inflammatory cytokine response in the lung, resulting in reduced immune cell infiltration and pathology. MedImmune is currently testing the safety, tolerability, and pharmacokinetics of IV administration of a single dose of 2A3.1 (designated MEDI4893) in a phase I human trial (NCT01769417). The mAb, which purportedly has an extended half-life, is being tested in a dose escalation trial in 33 healthy adults. Scientists at Rinat Laboratories, Pfizer Inc., isolated a single-chain variable fragment (scFv) against Hla (Foletti et al., 2013) from a library built directly from the complete germline diversity of 654 human donor IgM repertoires (Glanville et al., 2009) and converted to a full IgG (designated LTM14). In preclinical studies, mice that received LTM14 24 h before (1, 3, 10, 30 mg/kg) or 12 h after (30 mg/kg) challenge with S. aureus showed significantly improved survival in a lethal pneumonia model compared with mice given PBS. In a skin abscess model, mice dosed with 50 mg/kg of LTM14 24 h before challenge with USA300 showed reductions in abscess size and bacterial burden. Furthermore, administration of LTM14 showed partial protection in an IV lethal challenge model. Antibodies to poly-Nacetylglucosamine (PNAG) PNAG, also known as polysaccharide intercellular adhesin, is a surface polysaccharide produced by

many microbes, including S. aureus and S. epidermidis (Cywes-Bentley et al., 2013; Maira-Litran et al., 2004). PNAG promotes staphylococcal biofilm formation and enhances staphylococcal virulence in a mouse model of systemic infection (Kropec et al., 2005). Of note, antibodies to a deacetylated (backbone) form of PNAG (dPNAG), but not the native acetylated form of PNAG, are opsonic, i.e. promote antibodydependent opsonophagocytic killing of S. aureus by human neutrophils. Maira-Litran et al. (2005) immunized mice, rabbits, and goats with either native PNAG or dPNAG conjugated to diphtheria toxoid and transferred immune or non-immune serum IP to mice 48 h prior to IV challenge with S. aureus. Quantitative blood cultures performed 2 h after bacterial challenge revealed that mice given dPNAG antibodies had 54–91% fewer S. aureus in their blood than mice given normal rabbit serum. Antibodies to the native (acetylated) PNAG conjugate were ineffective in clearing the bacteraemia in mice. A human mAb (F598) that bound to the backbone epitopes of dPNAG showed in vitro opsonic killing activity and protected mice against a lethal IP challenge with S. aureus (KellyQuintos et al., 2006). Alopexx Pharmaceueticals initiated a phase I trial of the F598 mAb in 20 human volunteers in May 2010. Administration of F598 (1–20 mg/kg) was well tolerated and no serious side effects were noted. Subsequently, Sanofi Aventis licensed the mAb, which they dubbed SAR279356. In 2011 they embarked on a phase II clinical trial (NCT01389700) to evaluate the safety and efficacy of IV administration of SAR279356 to mechanically ventilated patients in intensive care units. However, the trial was terminated in January 2013 because of the insufficiency of enrolment (seven patients). Further development of the mAb by Sanofi Aventis is pending. S. aureus vaccine antigens in preclinical development Panton–Valentine leukocidin (PVL) PVL is a bi-component, pore-forming toxin that lyses leucocytes (Kaneko and Kamio, 2004). PVL production among hospital-associated S. aureus strains is rare. Nonetheless, the major community

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acquired (CA)-MRSA lineages have the lukPV operon that encodes PVL (Diep and Otto, 2008), which is carried by a number of related lysogenic phages. CA-MRSA isolates often affect children and young adults that lack typical predisposing risk factors (Gillet et al., 2002; Lina et al., 1999), attesting to their virulence. There have been conflicting views on the role of PVL in the pathogenesis of staphylococcal infections. Epidemiological data and certain experimental animal infection studies suggested that PVL toxin contributes to the severity of life-threatening necrotizing pneumonia and was associated with SSTIs (Labandeira-Rey et al., 2007; Lina et al., 1999; Rasigade et al., 2011). However, other investigations indicated that PVL was not essential for disease pathogenesis provoked by MRSA clones (Bubeck Wardenburg et al., 2007, 2008; Cremieux et al., 2009; Diep et al., 2010; Kobayashi et al., 2011; Voyich et al., 2006). The inconsistent in vivo data on the role of PVL in CA-MRSA pathogenesis prompted an evaluation of the lytic effect of PVL on neutrophils from different species including humans, mice, rabbits and monkeys (Loffler et al., 2010). These investigations demonstrated that PVL confers strong and rapid cytotoxic activity against neutrophils from humans and rabbits, but not on neutrophils from mice or monkeys. More recently, identification of the C5a receptor as the ligand for the PVL S subunit (Spaan et al., 2013), its expression and interspecies variations dictate the species specificity of PVL. Together, these differences indicate that murine models of staphylococcal infection are not appropriate for the study of the role of PVL in S. aureus pathogenesis or its usefulness as a vaccine target. Ongoing studies are evaluating PVL antigens in rabbit models of staphylococcal disease. There has been some interest in PVL as an S. aureus vaccine antigen. Karauzum et al. (2013) described highly attenuated recombinant mutants of PVL subunits LukS-PV and LukF-PV. Mice immunized with the non-toxic mutant proteins were protected against a lethal challenge of S. aureus CA-MRSA strain USA300 (5 × 104 CFU) mixed with 3% mucin. Because murine neutrophils are resistant to the lytic effects of PVL, more important than the protective efficacy observed in that study was the fact that antibodies elicited

against the PVL toxin were cross-reactive serologically and neutralized the lytic activity associated with certain other leukocidins produced by S. aureus, such as gamma haemolysin (Karauzum et al., 2013). Because S. aureus produces a number of leukocidin proteins and these proteins share some homology at the amino acid level, a vaccine that elicited neutralizing antibodies to a number of these lytic proteins would be desirable. This study lends some support to the inclusion of attenuated toxins in a multivalent vaccine for prophylaxis against S. aureus infection. Candidate toxoids include Hla, leukocidins, and the SAgs. To determine whether human serum antibody to PVL might modulate the course of infection, Hermos et al. (2010) measured antibody levels to PVL in children with SSTI or invasive staphylococcal infection, as well as uninfected control patients. Children with a primary PVL-positive MRSA infection mounted a robust antibody response to PVL. Individuals with prior MRSA infection or SSTI had high levels of antibody to PVL after the onset of PVL-positive MRSA infection, and no increase in antibody to PVL occurred after the onset of infection. Convalescent-phase serum samples from children with PVL-positive MRSAassociated SSTIs, and particularly those with invasive PVL-positive MRSA infection, potently inhibited PVL-induced lysis of polymorphonuclear cells. Data from this study indicate that neutralizing antibodies to PVL in children with prior infection do not provide protection against recurrent PVL-positive MRSA-associated SSTI. Functional antibodies to Hla or Panton– Valentine leukocidins can neutralize the detrimental effects induced by these toxins. Similarly, antibodies to S. aureus SAgs can neutralize these toxins. In an effort to define a relationship between the serological response of humans to S. aureus toxins and protective immunity, Fritz et al. (2013) performed a 12-month follow-up study in a cohort of 235 children with S. aureus colonization, primary or recurrent SSTI, or invasive disease. Serum binding antibody levels in the children to two major staphylococcal exotoxins, Hla and PVL, were measured by ELISA and correlated with the incidence of recurrent infection. Patients with invasive infections had lower preexisting antibody levels against Hla and LukF than

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patients with SSTI. Despite a transient elevation in acute antitoxin antibody levels in the recurrent SSTI cohort, the incidence of subsequent infection was similar in both primary and recurrent SSTI groups. Consistent with the data published by Hermos et al. (2010), antibody levels against LukF and LukS did not correlate with sustained protection against infection. In contrast, anti-Hla levels were associated with a lower incidence of subsequent S. aureus infection for the 12-month follow-up period. However, in this study the antibody levels were measured by ELISA readings taken at a single serum dilution (1:100), and considerable overlap between antibody levels was observed among the groups and between acute and convalescent sera. Moreover, a measurement of neutralizing antibody levels was not performed. To determine whether pre-existing antibodies to S. aureus exotoxins was associated with a lower risk of sepsis in adults with S. aureus bacteraemia, Adhikari et al. (2012a) measured serum IgG levels to 11 staphylococcal exotoxins. Twenty-seven of 100 patients in the study developed sepsis during the study period. Compared to patients without sepsis, patients with sepsis had significantly lower antibody levels to S. aureus Hla, delta haemolysin, PVL, staphylococcal enterotoxin C-1, and phenol soluble modulin-α3. Higher pre-existing antibody levels to PVL and SEB were protective against sepsis only when PVL or SEB genes were present in the infecting isolate. The investigators concluded that the presence of pre-existing antibody against these toxins could serve as a serological signature and may protect against sepsis in patients with invasive S. aureus infections (Adhikari et al., 2012a). Protein A Protein A (Spa) is a cell wall-anchored surface protein of S. aureus that binds to the Fcγ of immunoglobulin (Ig) and the Fab portion of VH3-type B-cell receptors. As a result, Spa interferes with opsonic antibody binding and modulates the host adaptive immune response to S. aureus (Kim et al., 2010). Interactions of Spa with B-cell receptors (IgM) result in clonal expansion and subsequent apoptosis of B-cell populations. Hence, Spa modulates the innate and adaptive immune response to staphylococcal infection (Kim et al.,

2010), leading to a net immunosuppressive effect. Immunization with native Spa did not result in antibodies that protected against systemic staphylococcal disease (Greenberg et al., 1989). Kim et al. generated a non-toxigenic Spa vaccine (SpaKKAA) by mutating glutamine 9 and 10 and aspartate 36 and 37 in each of the five Spa Ig binding domains (Kim et al., 2010). In contrast to the native protein, this construct no longer bound human IgG, Fc or Fab2, IgM, or von Willebrand factor. SpaKKAA also lacked B-cell SAg activity and failed to result in B-cell apoptosis. Immunization of mice with the non-toxigenic Spa mutant protein formulated with Freund’s adjuvant resulted in decreased lethality and a reduction in renal abscess formation in mice challenged systemically with S. aureus Newman or USA300 LAC. Furthermore, vaccination with SpaKKAA neutralized the immunosuppressive effects of Spa, enabling infected mice to mount an antibody response to many different staphylococcal antigens. This elegant study supports the use of SpaKKAA as a vaccine component. The limitations of this study were the use of Freund’s adjuvant in the immunization scheme and demonstration of efficacy only in the renal abscess infection model, a clinical syndrome rarely observed in humans. Furthermore, the potential toxicity of the protein for humans due to its binding to the TNF alpha receptor protein (Gomez et al., 2004) has not yet been addressed. Leukocidins S. aureus leukotoxins consist of two subunits (F and S) and include PVL, the gamma haemolysins (HlgAB and HlgCB), Leukocidin ED, and leukocidin AB (also known as LukHG). These leukotoxins exert pore formation on the membranes of human neutrophils (Finck-Barbancon et al., 1993), as well as monocytes and macrophages (Miles et al., 2002; Pedelacq et al., 2000) with various degrees of functional overlap and specificity. Although PVL is phage encoded (Diep et al., 2006; Kaneko et al., 1998; Narita et al., 2001), most leukotoxin genes are chromosomal and expressed by the majority of S. aureus strains. There is strong sequence identity within each of the subunit classes (F and S), suggesting that a broadly neutralizing antibody response to these toxins may be possible. Antibodies to the PVL

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S subunit have been shown to block the activity of HlgCB on host cells (Karauzum et al., 2013; Laventie et al., 2011). Coagulase (Coa) and von Willebrand protein (vWbp) Coa and vWbp are clotting factors secreted by S. aureus that promote the non-proteolytic activation of prothrombin, resulting in the conversion of fibrinogen to fibrin. The majority of S. aureus strains expressed Coa and vWbp, but there is considerable sequence diversity among strains. Active immunization of mice with recombinant Coa and/or vWbp (emulsified with Freund’s adjuvant) or passive transfer (5 mg/kg) of rabbit antibodies to Coa and/or vWbp to recipient mice delayed death and conferred limited protection against renal abscess formation in an S. aureus IV challenge infection model (Cheng et al., 2010; McAdow et al., 2012). Because of the antigenic variability among Coa and vWbp from different S. aureus strains, McAdow et al. (2012) subsequently combined the variable portions of the prothrombin binding portion of Coa and vWbp from different North American isolates into a hybrid Coa and vWbp subunit vaccine. The recombinant vaccine (composed of four Coa-type D12 domains and two vWbp-type D12 domains) was administered with Freund’s adjuvant to rabbits and mice. Active immunization or passive administration to mice of affinity-purified antibodies (5 mg/kg) only delayed death following challenge with five different S. aureus strains (McAdow et al., 2012). Moreover, reductions in the renal bacterial burden of infected mice were minimal, suggesting that these proteins are less than ideal vaccine candidates. PNAG As noted above, PNAG is a surface polysaccharide produced by many microbes, including S. aureus and S. epidermidis (Cywes-Bentley et al., 2013; Maira-Litran et al., 2004). PNAG promotes staphylococcal biofilm formation and enhances staphylococcal virulence in a mouse model of systemic infection (Kropec et al., 2005). Immunization with a synthetic conjugate vaccine containing dPNAG-TT reduced skin abscess induced by S. aureus (Gening et al., 2010), and

antibody to dPNAG-TT protected mice against lethal peritonitis caused by USA300 (Skurnik et al., 2012). A bivalent vaccine conjugate containing dPNAG and ClfA protected mice against bacteraemia induced by four S. aureus strains (Maira-Litran et al., 2012). Despite the positive data regarding PNAG as an immunogen, Skurnik et al. (2010, 2012) observed that both immunization-induced antibodies in experimental animals and natural antibodies to PNAG in some human sera may interfere with the protective efficacy of immunization-induced antibody to S. aureus CP5 and CP8 antigens, representing potential barriers to successful use of PNAG-specific vaccines. A vaccine to prevent nasal colonization Nasal carriage of S. aureus is a documented risk factor for staphylococcal infection. The source of ~80% of S. aureus bacteraemias is endogenous since infecting bacteria have been shown by genotypic analysis to be identical to organisms recovered from the nasal mucosa (von Eiff et al., 2001; Wertheim et al., 2004). Moreover, individuals with immune dysfunction have an increased carriage rate (Geerlings and Hoepelman, 1999; Padoveze et al., 2008; Smith and O’Connor, 1966). These observations support an approach in which systemic S. aureus infections are prevented by eliminating or reducing nasal carriage. Although topical treatment with mupirocin is usually effective in decolonizing nasal carriers, the emergence of mupirocin resistance in S. aureus abrogates the effectiveness of this approach. Moreover, recolonization of mupirocin-sensitive strains often occurs from extranasal carriage sites, which include skin, throat, perineum, gastrointestinal tract, and vagina. The feasibility of reducing nasal colonization is based on clinical studies that have documented decreased nasopharyngeal carriage of Haemophilus influenzae and Streptococcus pneumoniae vaccine serotypes following immunization with relevant capsule conjugate vaccines (Barbour et al., 1995; Ghaffar et al., 2004; Kauppi et al., 1993; Pelton et al., 2004). Secretory IgA responses are best achieved by mucosal immunization (Brandtzaeg, 2003), and mucosal immunization often results in a more Th1-like antibody subclass

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pattern of response than immunization by the systemic route (Hanniffy et al., 2007). Nonetheless, the relative importance of IgG versus IgA abs in preventing S. aureus colonization is not known. The mucosal immunization route has been shown to stimulate both mucosal and systemic immunity and thus might offer immune protection against both invasive disease and nasal carriage. S. aureus mutants deficient in sortase A or the surface-associated protein clumping factor B (Clf B) show reduced nasal colonization in an experimental mouse colonization model (Schaffer et al., 2006). Mice immunized intranasally with killed exponential-phase S. aureus cells showed reduced nasal colonization compared with control animals. Likewise, mice that were immunized systemically or intranasally with a recombinant vaccine composed of domain A of Clf B demonstrated lower levels of colonization compared with control animals (Pozzi et al., 2012; Schaffer et al., 2006). Passive immunization of mice with a Clf B specific mAb resulted in reduced nasal colonization compared with an isotype-matched control antibody. Thus, Clf B is an attractive component for inclusion in a vaccine to reduce S. aureus nasal colonization in humans, which in turn may diminish the risk of staphylococcal infection. Using a different approach to identifying candidate antigens for a vaccine against nasal carriage, Clarke et al. (2006) probed S. aureus expression libraries with sera from infected and uninfected patients to identify immunogenic proteins. Eleven proteins identified in their screen were further investigated by measuring antibodies reactive with the recombinant proteins in serum from healthy individuals (S. aureus nasal carriers and non-carriers), as well as patient sera. Antibodies to IsdA and IsdH were higher among non-carriers than nasal carriers of S. aureus, and so the investigators immunized cotton rats with the recombinant proteins. Rats immunized with IsdA or IsdH and inoculated with S. aureus showed reduced nasal colonization compared with control rats. Another antigen that may be worthy of inclusion in an S. aureus vaccine to prevent nasal colonization is wall teichoic acid. These ribitol phosphate polymers are produced by all S. aureus strains, they are essential for nasal colonization (Weidenmaier et al., 2004), and they play a role

in adherence to human endothelial cells under high flow conditions (Weidenmaier et al., 2005). However, studies to prevent nasal colonization by targeting S. aureus wall teichoic acid have not yet been reported. Target populations Considering the disappointing results of the clinical vaccine trials to date, the choice of a target population for evaluation of S. aureus vaccine efficacy in humans has become exceedingly difficult. There are target populations, listed in Table 18.2, that are obvious candidates for vaccination against staphylococcal disease. Haemodialysis patients have a relatively high incidence of S. aureus infection because their vascular compartment is frequently accessed, they spend many hours a week in healthcare facilities, they have numerous comorbidities, and the uraemia accompanying their renal failure may suppress their immune system (Minnaganti and Cunha, 2001). However, after the failure of the Nabi’s StaphVAX trial in haemodialysis patients, it is unlikely that another phase III trial will occur in this population that is associated with so many underlying co-morbidities. Similarly, peritoneal dialysis patients and individuals undergoing elective major surgery are at risk for staphylococcal infection and are prime targets for an S. aureus vaccine. However, Merck’s failed IsdB clinical trial has diminished enthusiasm for the latter population, since the incidence of staphylococcal infection in surgical patients continues to decrease, and the cardiac surgery patients in both arms of the vaccine trial experienced many serious adverse events. Moreover, the multi-organ failure associated with staphylococcal infection in the V710 vaccine recipients who developed S. aureus infection still has no explanation. Other groups, such as individuals (especially children) with recurrent infections, athletes, prisoners, men who have sex with men could also benefit from an S. aureus vaccine. However, most of the staphylococcal infections experienced by these groups are skin and soft tissue infections, rather than invasive S. aureus disease. Other populations that could be tested in an S. aureus vaccine clinical trial include military personnel, firefighters, policemen, intravenous drug abusers, or the

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elderly. A newly described population with a high risk of S. aureus invasive disease is the subset of individuals with health care-associated community onset staphylococcal infections (Dantes et al., 2013; Lenz et al., 2012). These patients were hospitalized during the previous year, and many of them were culture positive for MRSA at some point. This population with a high rate of staphylococcal infection may be suitable for evaluation of protective efficacy of the next generation S. aureus vaccine. Passive immunoprophylaxis against staphylococcal infections is indicated for persons who are unable to respond to active immunization because they are not immunocompetent (Table 18.3). This group includes patients undergoing chemotherapy and patients with immune disorders. Passive immunotherapy would also be appropriate for individuals at immediate risk of infection and for whom time constraints prohibit an active immunization approach. Patients undergoing emergency surgeries and premature neonates would benefit from passive immunotherapy. Because Inhibitex and Biosynexus failed to prevent infection among very-low-birth-weight infants with Veronate and Pagibaximab, respectively, there is diminished enthusiasm for testing additional S. aureus vaccine candidates in this patient group. Factors predisposing premature neonates to sepsis include hypogammaglobulinaemia (given that transplacental transfer of maternal antibodies occurs after 32 to 35 weeks gestation), an immature oxidative burst from neutrophils, and indwelling catheters (Benjamin et al., 2006; Kaufman and Fairchild, 2004). Passive immunotherapy with polyclonal or mAbs may provide a benefit, however, when used

in combination with antibiotics to treat bacterial infections (Foletti et al., 2013) Challenges in the development of a vaccine for S. aureus There are substantial limitations in the design of a universal S. aureus vaccine, i.e. one that can protect against the different manifestations of staphylococcal disease, such as bacteraemia, endocarditis, pneumonia, septic arthritis, osteomyelitis, skin and soft tissue infections, and staphylococcal infections associated with prosthetic devices. Importantly, our understanding of host defence elements critical in protective immunity against S. aureus is minimal, at best. Prior infection with S. aureus is not associated with immunity to reinfection, and as such the correlates of protection are not known. Single component vaccines that have shown protective efficacy in preclinical studies have failed to meet their clinical endpoints in human clinical trials. New data from studies designed to systematically evaluate how well research from murine clinical models mimics human disease indicate that the transcriptional response to inflammatory stresses in mouse models is a poor reflection of the human response to similar stresses (Seok et al., 2013). These findings question the usefulness of efforts spent on preclinical vaccine studies, which generally rely on rodent models of staphylococcal colonization and infection. Preclinical studies of potential vaccine candidates in rabbits have been initiated, since these animals are more susceptible to S. aureus toxins than rodents (Lipinska et al., 2011; Wilson et al., 2011; Diep et al., 2008). Nonetheless,

Table 18.3  S. aureus passive immunotherapies in clinical trials Product

Sponsor

Target

Status

Pagibaximab

Biosynexus

Lipoteichoic acid

Phase II/III failed

INH-A21 (Veronate)

Inhibitex

ClfA (selected IVIG)

Phase III failed

Tefibazumab (Aurexis)

Inhibitex

ClfA (mAb)

Phase II completed

AltaStaph

Nabi

CP5 and CP8 (IgG)

Phase II completed

SAR279356

Sanofi Aventis

Poly-N-Acetyl Glucosamine (mAb)

Phase II terminated

AR-301

Aridis Pharmaceuticals

Hla (mAb)

Phase I/II

MEDI4893

MedImmune LLC

Hla (mAb)

Phase I

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humans are not immunologically naive to S. aureus, and so their response to staphylococcal infection or an S. aureus vaccine is likely to be different from that of either rodents or rabbits. Another significant challenge in vaccine development is the fact that S. aureus clinical isolates are heterogeneous in their repertoire of virulence factors, such as secreted proteins (Ziebandt et al., 2010), including those implicated in immune evasion and surface proteins implicated in adherence (McCarthy and Lindsay, 2010). Although vaccine development focuses on antigens conserved among clinical isolates, another important issue is how much the primary amino acid sequence of the proteins vary among strains. For example, the amino acid sequence of S. aureus ClfA is 86% conserved overall among diverse isolates (Murphy et al., 2011), but the majority of diversity resides in exposed protein epitopes that appear to be immunodominant (Brady et al., 2013a). This could potentially impact the protective efficacy of vaccine proteins that are variable in expression as well as serologically. A multivalent S. aureus vaccine comprised of multiple purified proteins or polysaccharides will most likely need to be formulated with an adjuvant to enhance or modulate the host immune response. Adjuvants such as aluminium salts enhance the antibody titre towards a vaccine, as well as antibody affinity and functionality (Reed et al., 2013). Alum modulates antigen uptake by antigen-presenting cells, recruits a variety of immune cells, and elicits a Th2 response. Because aluminium-based adjuvants do not elicit a T-cell response, an effective S. aureus vaccine may need to be formulated with an alternate adjuvant, such as monophosphoryl lipid A, oil-in-water, or the TLR9 ligand CpG that induce Th1 and Th17 responses (Didierlaurent et al., 2009; Reed et al., 2013). Conclusions and considerations Many questions remain in the quest for an effective S. aureus vaccine for humans. Which antigens are best suited for inclusion in a multivalent staphylococcal vaccine? Can we reach an understanding of the correlates of protective immunity to S. aureus

infection? Will we overcome the limitations of preclinical models of S. aureus infection? If efficacy can at last be demonstrated in a clinical trial, what will be the duration of immunity? If efficacy is achieved in a relatively healthy population (e.g. military recruits), how will the vaccine perform in high-risk groups, such as the elderly, diabetics, and haemodialysis patients? If skin and soft tissue infections can be prevented, will protection be afforded against other manifestations of staphylococcal disease, such as bacteria, pneumonia, wound infection? Will S. aureus colonization be affected by vaccination? Can we expect a vaccine to prevent against staphylococcal infections involving a prosthetic device? Many questions remain in the search for a vaccine to prevent S. aureus disease. Acknowledgements This work was supported by the National Institutes of Health (grant number R01 AI088754 to J.C.L.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. L.W. was partially supported by the Chinese Scholarship Council. We thank Drs Gerald Pier and Ravi Mishra for many useful discussions about vaccine development. References Adhikari, R.P., Ajao, A.O., Aman, M.J., Karauzum, H., Sarwar, J., Lydecker, A.D., Johnson, J.K., Nguyen, C., Chen, W.H., and Roghmann, M.C. (2012a). Lower antibody levels to Staphylococcus aureus exotoxins are associated with sepsis in hospitalized adults with invasive S. aureus infections. J. Infect. Dis. 206, 915–923. Adhikari, R.P., Karauzum, H., Sarwar, J., Abaandou, L., Mahmoudieh, M., Boroun, A.R., Vu, H., Nguyen, T., Devi, V.S., Shulenin, S., et al. (2012b). Novel structurally designed vaccine for S. aureus alphahemolysin: protection against bacteremia and pneumonia. PLoS One 7, e38567. Adlam, C., Ward, P.D., McCartney, A.C., Arbuthnott, J.P., and Thorley, C.M. (1977). Effect of immunization with highly purified alpha- and beta-toxins on staphylococcal mastitis in rabbits. Infect. Immun. 17, 250–256. Anderson, A.S., Miller, A.A., Donald, R.G., Scully, I.L., Nanra, J.S., Cooper, D., and Jansen, K.U. (2012). Development of a multicomponent Staphylococcus aureus vaccine designed to counter multiple bacterial virulence factors. Hum. Vaccin. Immunother. 8, 1585–1594.

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Index

1000 Genomes Project  34 16S rRNA  53, 65

A Acetic acid hydrolysis  399 Acinetobacter baumannii 72 Activation-induced cytidine deaminase (AID)  338 Acute lower respiratory infection  411 Adaptive immunity  219, 220 ADCC 348 Adenovirus  251, 256 AdH5 291 Adjuvants  134, 191, 317, 321, 324, 402, 420 Adoptive T-cell therapy  374–377 chimeric antigen receptor (CAR)  374–377 Adults  388–390, 392, 399 Africa  387–392, 394, 398–399, 400 Kenya 388 Malawi 394 AIDS vaccine  335 Alignment 36 αβT-cells  223, 224, 239 Alpha toxin  432–434 AltaStaph  430, 439 Aluminum salts  191 ALVAC-HIV/AIDSVAX gp120 B/E vaccine  348 Alveolitis 414 AMA-1  293, 279 Animal models  280 Antibiotic resistance  69, 70 Antibiotics  388, 391 Antibodies  220, 222, 239, 391–394, 396, 398–401 affinity maturation  398 class switching  395, 398 IgA  393, 394 IgG  392–394, 396 IgG2a 394 IgM 393 monoclonal antibodies  392, 394 mucosal antibodies  393, 398 Antigen–antibody complex  103–119, 122, 124–126 Antigen baiting  142 Antigen binding region  43 Antigen design  103, 105, 113–114, 116, 120, 122, 125–126

Antigen-presenting cells (APC)  157, 161, 163–164, 166–168, 171–173, 220, 223, 229 Antigens (Salmonella)  387, 388, 392–396, 398–403 flagellin  388, 392, 394, 399, 400, 402 O:2  393, 399 O:4,5  393, 399 O:9  393, 399, 400 O-antigen  388, 392, 394, 395, 399, 401, 402 OmpC  393, 394, 401 OmpD  393, 394, 401 OmpF  393, 394, 401 SseB 394 Vi  387, 388, 390, 392, 393, 396, 399, 400 Animal model  413 Anti-retroviral therapy (ART)  335 Apical merozoite antigen 1  293 see also AMA-1 AS01 291 AS02 290 AS04 290 Asia  388–389, 398 China  388, 396, 399 India  396, 399 Pakistan 399 Philippines 399 Vietnam 399 ASO3 191 Assembly  37, 47 Asthma 411

B B16F10 265 Bacillus Calmette–Guerín see BCG Bacteraemia  388, 389, 393, 398 BAM format  41 Basophil 223 B-cells  220, 222, 223, 226, 336, 393–395, 398, 401 B-cell germinal centres  337 B-cell memory  133 B-cell receptor (BCR)  17, 335 BCG  251, 256, 258, 306–312 BEAST analysis  70 β-chemokines 337 Bexsero 4 Bill and Melinda Gates Foundation  397

450  | Index

BLAST 36 Blood culture  388, 389 Blood stage challenge  285 Blood stage vaccines  293 BnAbs 344–350 Boosting 397 Bordetella pertussis  227, 228, 236, 237 Bovine RSV  414 Bovine serum albumin  414 Broadly neutralizing antibodies  335 Bronchiolitis 411–412 BRSV 414–415 BW transform  36

C C57/B6 265 C type lectin receptors CLR  188–189 Cancer immunotherapy  359–377 Cancer Immunotherapy Consortium (CIMT)  267 Cancer mutations  263-264 discovery 263 non-synonymous 266 selection 266 Cancer vaccines  360–370 antigenic  362–363, 366–367 see also peptide under Cancer vaccines anti-idiotype  362–363, 369 dendritic cell  361–365 DNA  362–363, 367–368 exosomes 365 heat shock proteins  366 peptide  362–363, 366–367 tumor cell  360–363 viral  362–363, 368–369 Candida albicans 229 Carrier proteins  398, 399, 400 CRM197  398, 399 Pseudomonas aeruginosa recombinant exoprotein A (rEPA) 399 tetanus toxoid  398, 399 Case fatality  388, 396 CD107a 340 CD14 402 CD4 T-cell  222, 224, 226, 236, 311, 313–314, 325, 335–339, 348 TCM  311, 325 TEM  311, 325 Th1 313 Th17 313–314 CD8 T-cell  222–224, 226, 230, 231, 238, 314 CD8 T cytolytic  337, 338 cDNA 13 Cellular immunity  219, 220, 235, 420 Cellular phenotype and function  144 Cellular screens  139 Clonal expansion  134 CH63 342 Challenge (with Salmonella)  392, 399–401 Challenges for next decade  208 Chemokines  160, 161, 163, 165 Children  395, 396, 399

ChIP-seq  15, 35, 51–52 CHMI  284, 286, 287 Choice of endpoints  288 Chronic carriage  389, 393 Circumsporozoite protein  281 see also CS Circumsporozoite protein  290 Citrobacter 399 Class II HLA  337 Clinical management  388 Clinical presentation  387, 388 Clinical trials  395, 399, 400 Clostridium difficile  65, 66, 70 Clumping factor A (ClfA)  428–429 Clustering 53 CMV vaccine  341 Coalition against Typhoid (CaT)  397, 403 Cold chain  396 Colonization 437–438 Comparative genomics  12, 65–74 Complement  392, 393 Controlled human malaria infection  284 see also CHMI Correlate of protection  415 Cost of goods  397 Cotton rats  413–414, 418 CP5-CRM197 428–429 CP5-Epa 426–427 CP8-CRM197 428–429 CP8-EPA 426–427 CpG ODN  198 Cre-Lox recombination  18 Cross-protection  395, 396 Cross-reactivity 401 Cryo-EM  111–113, 117 Cryopreserved sporozoites  285 CS  281, 286 Csa1A 430 CSP 290 see also CS CXCR5 338 Cytokines 392 IFNγ  391, 393, 398 IL-12 393 IL-18 393 TNFα 393 Cytotoxic lymphocyte  229

D De Bruijn graph  38 Deep sequencing  7 Dendritic cells  157, 220, 223, 226, 230, 235 see also Dermal dendritic cells Dermal dendritic cells (dDC)  157–176 Dermis  158, 159, 161, 162, 165–167, 169–172 Diabetes 305 Diagnosis 388 Diarrheagenic E. coli  67, 68, 70 Dideoxy chain termination method  6 DNA  3, 337, 349 DNA methylation  14

Index |  451

DNA sequencing  3 DNA vaccine  251, 254, 257, 258 Driver mutation  264 Drug-resistant TB  306, 326 MDR-TB 306 XDR-TB 306

E EAEC  71, 72, 73 EBA-175 293 EBV transformation  141 ECGC 263 Effectiveness  394, 398, 400, 403 Effector functions  137 Efficacy  387, 392, 395–397, 399 Efficacy endpoint  285 EHEC  71, 72 Electron microscopy  103–104, 111–112 ELISPOT assay  140, 252 EM 54 Emulsions 191 Enhanced respiratory disease  412 Enteric fever  387–390, 393, 396–399, 402 Environmental mycobacteria  308–310 Eosinophil 223 EPI 287 Epidemiological  387, 391, 392, 398, 403 Epidemiology 70 Epidermal immunization  169, 175 Epidermis 157–176 Epigenomics  10, 14 Epitope mapping  103–111, 113–126 Epitopes  44–46, 419 ERD  412–416, 418, 420 Erythrocyte-binding antigen-175  293 Erythrocytic-stage antibody mediated immunity  282 Erythrocytic-stage T-cell immunity  283 Escherichia coli  65–68, 221, 233 ETEC  66, 71, 72 Expanded Programme on Immunizations (EPI) 397–399 Expectation Maximization see EM Extracellular  388, 391, 393

F FhuD2 429–430 Field evaluation of malaria vaccine  288 First-generation sequencing (FGS)  8 FI-RSV  412–413, 418 FM index  36 Formalin-inactivated 412 Food and Drug Administration (FDA)  267 Fragment-display  106–107, 110, 117 Fusion protein  411, 418

G Gall bladder  388, 390 GAPs 292 Gastroenteritis  387, 391 GAVI Alliance  397 GBS 116–119

Global burden  388 Global health  387, 402 Gene prediction  39 Genes  390, 391, 400–403 aroC 400 aroD 400 clpP 400 gna33 401 guaBA 400 htrA 400 htrB 402 msbB 402 PhoP/PhoQ 400 ssaV 400 tolR 401 Genetic diversity  388 Genetically attenuated parasites  292 see also GAPs Genome  387, 398, 400, 401, 403 Genome-wide association studies (GWAS)  12 Genomic viewer  40–41 Genomics  10, 12, 133 Gd T-cell  222, 223, 231–233, 238, 239 GLURP  279, 293 Glutamate-rich protein  293 gp120 349 gp120 CD4 binding site  345 Granuloma 312

H Haemophilus influenzae B  398 Hair follicle  160, 169–172, 176 Haitian cholera outbreak  69 HapMap 34 Hash table  36 HCV 35 H/DX-MS  108–109, 117 Helicobacter 229 Heterogeneity in malaria exposure  289 Hidden Markov model see HMM High-income countries  387, 397, 399 High-throughput sequencing  142 HIV  149, 305–306, 389, 390, 392, 398, 400 HIV-1 335–352 HIV vaccine  334, 349 HLA  34, 278 HMM 39 HMP 34 Human adenovirus  291 see also AdH5 Human Genome Project (HGP)  6 Humanized mice  283 Humoral immunity  219, 220, 227 Hybridoma technology  141 Hyporesponsiveness 396

I ICOS 337 ICTV 41 IEDB Immune Epitope database  267 Ig 42

452  | Index

Ig-seq 17 IMG 41 Imiquimod 194 Immune checkpoint blockade  370–374 CTLA-4 370–372 PD-1 371–374 Immune recognition  265 Immune system  219, 220 Immunity  387, 388, 390–403 acquired immunity  387, 391 cellular immunity  391–393, 396, 398–400 humoral immunity  391–393 herd immunity  397, 398 innate immunity  391, 394, 402 mucosal immunity  400, 402 pan-specific immunity  395 systemic immunity  400 Immunity to erythrocytic stages  278 Immunity to sexual stages  279 Immunodeficiency 391 chronic granulomatous disease  391 common variable immunodeficiency (CVID)  391 Immunogenicity  395, 399, 400, 402 Immunogenicity testing  265 Immunoglobulin see Ig Immunological memory  133 Immunome 16 Immunomodulatory 394 Immunotherapy, cancer  359–377 Incidence  389, 390, 392, 398 Infants  395, 396, 398 Infection under treatment vaccination  292 see also ITV Influenza virus  147 Innate immunity  187, 219, 220 Innate like-lymphocyte  222, 231, 238 Innovation  387, 394, 399, 401, 403 Interferon-γ  222, 225, 226, 231–233 Interleukin-10 (IL-10)  229 Interleukin-17 (IL-17)  222, 227, 232, 234, 239 Interleukin-22 (IL-22)  222, 229, 239 International Conference on Harmonization (ICH)  267 Intracellular  388, 390, 391, 393, 394, 398, 403 Intradermal  166–167, 169, 172, 174–176 iNTS disease  387–394, 396–402 Invasive non-typhoidal Salmonella 70 In vitro transcribed (IVT) RNA  267 Irradiated sporozoites  280 IsdB  427, 428 ITV 292

K Killer inhibitory receptor (KIR)  344 Klebsiella pneumoniae  65, 66, 69, 227, 229 KPC 69 Kynureninase (KYNU)  346

L Langerhans cells (LC)  157–175 Lessons learned from TLR agonists  203 Licensed  387, 394–396

Limited values of animal work  208 Lipopeptides 251 Lipopolysaccharide (LPS)  388, 392, 394, 399, 401, 402 Live attenuated  412, 416 Long lived plasma cell  136 Low-income countries  387, 388, 397, 399 Lujo virus  34 Lymphoid tissue inducer cells (iLT)  222

M Macrophage  223, 225, 226, 239 Major histocompatibility complex  223, 224, 226, 230, 231 Malaria  273, 389, 391 Malaria control approaches  274 Malaria life cycle  276 Malaria parasites  274 Malaria vaccine development  289 Manufacturers  397, 403 Mapping 35–36 Markov model  39 Mass spectrometry  103, 107–109 Massive parallel sequencing  7 Maternal immunization  415–417 Maximum likelihood  41 Mechanism  391, 392, 394 Membrane proximal external region (MPER)  345 Memory  395, 396, 398 MenB 119–121 Meningitis 388 Meningococcus  398, 399, 401 Merozoite surface protein  1 293 see also MSP-1 Merozoite surface protein 2  293 see also MSP-2 Merozoite surface protein 3  293 see also MSP-3 Merozoites 276–278 Meta-analysis 395 Metagenomic analysis  19 Metagenomics  10, 52 MetaHIT 34 Methicillin-resistant Staphylococcus aureus (MRSA)  237 ME-TRAP  277, 291 MF59 191 MHC 44-45 MHC tetramers  146 Microbiome 12 Middle-income countries  396 MIP-1β 340 miRNA  13, 48–49 MLEE 65 MLST 65 MLVA 65 MntC 429 Modified Vaccinia virus Ankara see MVA Molecular 72 Monoclonal antibodies  140 Monocyte 223 Mouse  391, 392, 394, 399–401 MRSA  65, 66, 69

Index |  453

MSP-1  293, 279, 283 MSP-2 293 MSP-3  279, 293 Mucosa 392 Multiparametric flow cytometry  146 Multiple TLR activation  205 Murine model  280, 283 Mutagenesis  396, 400, 402 Mutanome 263 Mutanome vaccines  264 Mutation  391, 400, 401, 403 MVA  251, 255, 257, 258, 291, 337, 342 Mycobacterium tuberculosis  231, 234

N NANP 290 National Institutes of Health (NIH), US  399 Native outer membrane vesicles (NOMV)  401 Natural killer T-cells (NKT-cells)  222, 223, 234, 235, 238, 239 NDV-3 426 see also rAls3p-N Needle-free 169 Neisseria meningitidis  4 Neo-antigen 264 Neutrophil  223, 225, 227, 239 Next-generation sequencing see NGS NGS  7, 41, 263 clinical grade  266 exome analyses  266 non-synonymous 266 NK cells  391 NMR  103–104, 110–112, 117, 120 NOD like receptors NLR  188–189 Non TLR adjuvants  191 Non-coding regulatory elements (ncRNAs)  13 Non-human primate malaria models  283 NYVAC 349

O O104:H4  65, 66, 71 O157:H7  65, 66, 71, 72 O55:H7 72 Opsonization  220, 392 OUT 53 Outbreak  66, 67 Out-bred mice  283 Outer membrane vesicles (OMV)  5 Overlap extension PCR  18 Overlap-layout consensus  37 Oxidative burst  391, 393

P P52 292 Pagibaximab  431, 438 Palivizumab  412, 420 Panton–Valentine leukocidin  434–436 Paratyphoid 399 Passive transfer  391, 392 Pathogen-associated molecular patterns (PAMPs)  188, 220

Pathogen recognition receptors (PRR)  220, 229 Pattern recognition receptors (PRR)  188 PCR 285 PD1 338 Peptide stimulation  145 Personalized cancer immunotherapy  263 Personalized medicine  263 Personalized vaccines  267 Pf EMP-1 278 PFGE 69 Pfs230 294 Pfs25 294 Pfs28 294 Pfs45/48 294 Phagocytes  388, 391, 392 macrophages  388, 390, 391, 393, 394, 400 neutrophils  391, 393 Phylogeny  41, 66 piRNAs 13 Plasma cell  133 Plasmablasts  18, 136 Plasmacytoid dendritic cells (PDC)  343 Plasmodium falciparum 274 Plasmodium knowlesi 274 Plasmodium malariae 274 Plasmodium ovale 274 Plasmodium vivax 274 Pneumococcus  398, 399 Pneumovirus 411 Poisson model  50 Polymorphism 391 Poly-N-acetylglucosamine (PNAG)  434, 436–437 Polysaccharide  387, 388, 390, 392, 393, 396–399 Population  387–389, 397 Post-fusion 419 Post-licensure surveillance  19 Pre-erythrocytic immunity  277 Pre-erythrocytic malaria vaccine  289 Pre-erythrocytic vaccines  277, 286 Pre-fusion 418–419 Primary malaria endpoints  287 Primer-/ bead barcoding  18 Prophage 54 Protection  390–392, 394, 396, 398, 400, 401, 403 Protective immunity  133 Protein A  435–436 Proteins  388, 392–402 outer membrane proteins  392, 394, 400–402 porins  394, 401, 402 purified proteins  395, 397, 400, 402 recombinant proteins  395, 397, 400, 401 Pseudomonas aeruginosa 221 Public health  387

Q Quasispecies 54

R Radiation attenuated sporozoites  292 see also RAS RAS 292

454  | Index

rAls3p-N 426 see also NDV-3 RD1 316 Reactogenicity  395, 402 Receptor repertoire  133 REP-PCR 65 Resiquimod 196 Respiratory syncytial virus  411 Reverse vaccinology  4, 401 Ribo-seq 14 RIG-I like receptors RLR  188–189 RNA  13, 417 RNA-seq  13, 35, 46 RPKM 51 rRNAs 13 RSV 411–421 RSV F  412–420 RSV G  420 RTS,S  277, 290 RTS,S development  290 AS01 291 AS02 290 AS04 290 RV144  335–336, 348

S Safety  399, 400 Safety considerations of TLR agonists  208 Salmonella vaccines  387–403 Generalized modules for membrane antigens (GMMA)  393, 395, 397, 401–403 glycoconjugate  392, 394–403 inactivated whole cell  394, 395, 397, 400 live-attenuated  387, 393–397, 400, 403 subunit  396, 398–401 Ty21a  387, 390, 395–397, 400 Vi capsular polysaccharide vaccine (Vi CPS)  387, 390, 395–398, 400 SAM vaccine  417 SAP1 292 Scaffolded antigen  420 Second-generation systems (SGS)  7 SERA-5 293 Serine repeat antigen 5  293 see also SERA-5 Serovars (of Salmonella enterica)  387, 388, 392–394, 396, 398–401 Dublin 388 Enteritidis  397, 389, 390, 393, 398, 399, 400 Paratyphi A  387–389, 393, 394, 396, 398, 399 Paratyphi B  395, 396 Paratyphi C  388 Typhi  387–390, 392–401 Typhimurium  387, 389, 390, 392–394, 398–401 Shigella  67, 68 Shigella sonnei 68 Simian immunodeficiency virus (SIV)  392 Single cell expression cloning  140 Single nucleotide polymorphisms (SNPs)  12, 65, 66, 69, 70 Single nucleotide variations (SNV)  37, 264

siRNAs 13 Skin 157–176 Skin resident T cells (TRM)  165, 169 SLC11A 391 snoRNAs 13 SNP phylogeny  66, 67 SNP typing  68 snRNAs 13 Somatic hypermutation (SHIM)  42–43, 337 Somatic mutation  42 spa typing  69 SPf66 289 Splice junction  47 Sporozoites 276 ST239 69 Staphylococcus aureus  18, 221, 222, 228, 237, 238, 425–440 StaphVAX 426–427 Statistical confidence  265 STEBVax  426, 428 Streptococcus pneumoniae  232, 234–236 Structural 418–419 Structural genomics  15 Structural vaccinology  103–106, 116, 118–119, 123, 125, 128 Structure-based detoxification  121–123 Subunit vaccine approach  293 Suffix tree  36 Synthetic biology  18 Synthetic libraries  105–106, 117 Synthetic seed  19 Synthetic virus  19 Systems biology  16 Systems vaccinology  17

T T helper 1 cell (Th1)  225, 226, 228, 236–238 T helper 17 cell (Th17)  225, 227, 228, 236–238 T helper 2 cell (Th2)  225–227 T helper 22 cell (Th22)  225, 229 T regulatory cell (Treg)  225, 229, 230, 337 TBV 293 T-cell memory  133 T-cell receptor see TCR T-cells  220, 221, 222, 224, 238, 223, 232, 336, 388, 391–396, 398–402 CD4+ T-cells  392–394 CD4+ T-cells  393 γδT cells  391 NKT cells  391 Th17 cells  393 TCGA (The Cancer Genome Atlas)  263 TCR  17, 42 T-dependent  395, 398, 399 Tefibazumab  431, 439 Teleporting life  18 T-follicular helper (Tfh)  337 Th1 337 Th17 337 Th2  337, 413 Third-generation systems (TGS)  7

Index |  455

Third heavy chain complementarity-determining region (HCDR3s) 345 Third variable (V3) loop  345 Thrombospondin-related adhesion protein  281 see also TRAP T-independent  396, 398 TLR agonists against cancer  202 TLR agonists against tuberculosis  202 TLR agonists direct and indirect  205 TLR agonists in HIV  199 TLR agonists in malaria  201 TLR based adjuvants  192 TLR cross talk  206 TLR2 192 TLR3-4 193 TLR4 402 TLR5  394, 402 TLR5,7 194 TLR7/8 196 TLR8 197 TLR9 198 TLRs in non-immune cells  206 TMM 51 Tn21-like element  70 Tn6192 70 Tolerability  399, 400 Toll-like receptor (TLR)  162, 163, 173, 175, 189–206 Toll-like receptor agonists  420 Tol-Pal system  401, 402 Transcriptome 46 Transcriptomics  10, 13 Transgenic parasites  283 Transmission  387, 388, 391, 397, 398, 400 Transmission blocking vaccines  293 see also TBV Transmitted/founder (T/F) viron  336 TRAP  281, 282, 286 Treatment 412 tRNAs 13 Tumour immunotherapy  359–377 Typhoid  388–390, 392, 394, 396, 398, 399, 401 Ty VLPs  251, 254, 257

U UIS3 292 UIS4 292

Up-regulated infectious sporozoite gene-3  292 see also UIS3 Up-regulated infectious sporozoite gene-4  292 see also UIS4

V Vaccination see Vaccines Vaccine companies  396 Bharat Biotech  396, 399 BioMed  396, 399 Emergent Biosolutions  400 Lanzhou Institute  396, 399 Shantha Biotech  396 Vaccine coverage  393, 395, 398–400 Vaccine design  390, 394, 396, 397, 399 Vaccine institutes  396, 403 Center for Vaccine Development (CVD)  396, 400 International Vaccines Institute (IVI)  396, 399 Novartis Vaccines Institute for Global Health (NVGH)  396, 399, 401 Sabin Vaccine Institute  397 Vaccines  133, 220, 221, 235, 237, 238, 412–421 anti-tumor see Cancer vaccines Vaccinomics 17 Variants calling  37 VDJ recombination  42, 134 Vectored 417 Veronate  430–431, 439 Vibro cholerae  66, 69 Viral pathogen  105–106, 116, 123, 125 Viral vectors  317, 321, 322–323 adenovirus  321, 323 fowl pox (FP)  317–318 modified vaccinia ankara (MVA)  317 Virulence  388, 391 Virus-like particle (VLP)  173, 172, 175, 192 VLPs with peptides  251, 254

W WHO  392, 396, 397 Whole genome alignment  68–71 Whole-genome sequencing (WGS)  12 Whole-shotgun sequencing  6

X X-ray crystallography  103–104, 109–111, 117, 126

Advanced Vaccine Research Methods for the Decade of Vaccines

Since the publication of our first book Vaccine Design: Innovative Approaches and Novel Strategies in 2011, the field of vaccinology has advanced significantly. This has prompted the need for this new volume, which aims to distil the most important new findings to provide a timely overview of the field. As before the book has been divided into two main parts. The first explores in considerable depth the key innovations that we think are dramatically changing the field; for both preclinical and clinical vaccine research fields. Some of the topics covered include: applications of deep sequencing, cellular screens to interrogate the human T and B cell repertoires, microbial comparative genomics, quantitative proteomics, structural biology, novel strategies for vaccine administration, T-cell inducing vaccines, etc. The second part focuses on diseases for which current medical treatment is not sufficiently effective and that could be either prevented or treated by vaccination. The examples that we have used comprise very different diseases including infectious diseases (e.g. malaria, tuberculosis, HIV, and Staphylococcus aureus), as well as cancer. We believe that these will be the vaccines of the future, the “vaccines for 2020”. This books is essential reading for everyone working in vaccine R&D in academia, biotechnology companies, and the pharmaceutical industry and a recommended volume for all microbiology libraries.

www.caister.com