Measles: Pathogenesis and Control [1 ed.] 354070616X

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Current Topics in Microbiology and Immunology Volume 330

Series Editors Richard W. Compans Emory University School of Medicine, Department of Microbiology and Immunology, 3001 Rollins Research Center, Atlanta, GA 30322, USA Max D. Cooper Department of Pathology and Laboratory Medicine, Georgia Research Alliance, Emory University, 1462 Clifton Road, Atlanta, GA 30322, USA Tasuku Honjo Department of Medical Chemistry, Kyoto University, Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan Hilary Koprowski Thomas Jefferson University, Department of Cancer Biology, Biotechnology Foundation Laboratories, 1020 Locust Street, Suite M85 JAH, Philadelphia, PA 19107-6799, USA Fritz Melchers Biozentrum, Department of Cell Biology, University of Basel, Klingelbergstr. 50–70, 4056 Basel Switzerland Michael B.A. Oldstone The Scripps Research Institute, Department of Immunology and Microbial Science, 10550 N. Torrey Pines, La Jolla, CA 92037, USA Sjur Olsnes Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello 0310 Oslo, Norway Peter K. Vogt The Scripps Research Institute, Dept. of Molecular & Exp. Medicine, Division of Oncovirology, 10550 N. Torrey Pines. BCC-239, La Jolla, CA 92037, USA

Diane E. Griffin • Michael B.A. Oldstone Editors

Measles Pathogenesis and Control

Editors: Diane E. Griffin Johns Hopkins University School of Hygiene and Public Health Department of Molecular Microbiology 615 N. Wolfe Street Baltimore, MD 21205 USA [email protected] [email protected]

ISBN 978-3-540-70616-8

Michael B.A. Oldstone Scripps Research Institute Department of Immunology and Microbial Science 10550 N. Torrey Pines Road La Jolla, CA 92037 USA [email protected]

e-ISBN 978-3-540-70617-5

DOI 10.1007/978-3-540-70617-5 Current Topics in Microbiology and Immunology ISSN 0070-217X Library of Congress Catalog Number: 2008931704 © 2009 Springer-Verlag Berlin Heidelberg This work is subject to copyright. All rights reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September, 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publisher cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Cover design: WMXDesign GmbH, Heidelberg, Germany Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com

Contents

Introduction ......................................................................................................

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Making It to the Synapse: Measles Virus Spread in and Among Neurons ............................................................................ V.A. Young and G.F. Rall Modeling Subacute Sclerosing Panencephalitis in a Transgenic Mouse System: Uncoding Pathogenesis of Disease and Illuminating Components of Immune Control ............ M.B.A. Oldstone

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Measles Studies in the Macaque Model ................................................. R.L. de Swart

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Ferrets as a Model for Morbillivirus Pathogenesis, Complications, and Vaccines ................................................................... S. Pillet, N. Svitek, and V. von Messling

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Current Animal Models: Cotton Rat Animal Model ........................................................................................... S. Niewiesk

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Current Animal Models: Transgenic Animal Models for the Study of Measles Pathogenesis ................................................... 111 C.I. Sellin and B. Horvat

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Molecular Epidemiology of Measles Virus ............................................ 129 P.A. Rota, D.A. Featherstone, and W. J. Bellini

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Human Immunology of Measles Virus Infection .................................. 151 D. Naniche

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Measles Control and the Prospect of Eradication................................. 173 W.J. Moss

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Measles: Old Vaccines, New Vaccines .................................................... 191 D.E. Griffin and C.-H. Pan

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Measles Virus for Cancer Therapy ........................................................ 213 S.J. Russell and K.W. Peng

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Measles Virus-Induced Immunosuppression ........................................ 243 S. Schneider-Schaulies and J. Schneider-Schaulies

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Hostile Communication of Measles Virus with Host Innate Immunity and Dendritic Cells ................................................................ 271 B. Hahm

Index .................................................................................................................. 289

Contributors

W.J. Bellini Measles, Mumps, Rubella and Herpesvirus Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA R.L. de Swart Department of Virology, Erasmus MC, University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands, [email protected] D.A. Featherstone Immunization, Vaccines and Biologicals, World Health Organization, Geneva, Switzerland D.E. Griffin Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe St. Rm E5132, Baltimore, MD 21205, USA, [email protected] B. Hahm Departments of Surgery and Molecular Microbiology and Immunology, Center for Cellular and Molecular Immunology, University of Missouri-Columbia School of Medicine, One Hospital Dr., Columbia, MO 65212, USA, [email protected]. edu B. Horvat U758-ENS Lyon, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France, branka. [email protected] W.J. Moss Department of Epidemiology and the W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore MD, USA, [email protected]

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Contributors

D. Naniche Barcelona Center for International Health Research (CRESIB), Hospital Clinic, Institut d’Investigacions Biomedicas August Pi i Sunyer (IDIBAPS), C/Rossello 132, 4 08036, Barcelona, Spain, [email protected] S. Niewiesk College of Veterinary Medicine, Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210, USA, niewiesk.1@osu. edu M.B.A. Oldstone The Scripps Research Institute, Department of Immunology and Microbial Science, La Jolla CA, USA, [email protected] C.-H. Pan Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe St. Rm E5132, Baltimore, MD 21205, USA K.W. Peng Mayo Clinic, Department of Molecular Medicine, 200 1st Street SW, Rochester, MN 55905, USA S. Pillet INRS-Institut Armand-Frappier, University of Quebec, 531, boul. des Prairies, Laval, QC, H7V 1B7, Canada P.A. Rota Measles, Mumps, Rubella and Herpesvirus Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA, USA, [email protected] S.J. Russell Mayo Clinic, Department of Molecular Medicine, 200 1st Street SW, Rochester, MN 55905, USA, [email protected] J. Schneider-Schaulies Institute for Virology and Immunobiology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany S. Schneider-Schaulies Institute for Virology and Immunobiology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany, [email protected]

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S. Svitek INRS-Institut Armand-Frappier, University of Quebec, 531, boul. des Prairies, Laval, QC, H7V 1B7, Canada C.I. Sellin U758-ENS Lyon, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France V. von Messling INRS-Institut Armand-Frappier, University of Quebec, 531, boul. des Prairies, Laval, QC, H7V 1B7, Canada, [email protected] V.A. Young Division of Basic Science, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA

Introduction

Measles virus, one of the most contagious of all human viruses, has been largely contained by the development and use of a vaccine that was introduced 50 years ago. These two volumes were timed to honor the introduction of the vaccine and to record the enormous advancements made in understanding the molecular and cell biology, pathogenesis, and control of this infectious disease. Where vaccine has been effectively delivered, endemic measles virus transmission has been eliminated. However, difficulties in vaccine delivery, lack of health care support and objection to vaccination in some communities continue to result in nearly 40 million cases and over 300,000 deaths per year from measles. By itself measles virus infection has and still provides some of the most interesting phenomena in biology. Following infection of dendritic cells, measles virus causes a profound suppression of the host’s immune response that lasts a number of months after apparent recovery from infection. Indeed, measles virus was the first virus to be associated with immunosuppression with many of the manifestations to be observed one hundred years later with HIV infection. Measles is also associated with development of both post-infectious encephalomyelitis, an autoimmune demyelinating disease, and subacute sclerosing panencephalitis, a slowly progressive neurodegenerative disorder. How measles virus infects cells, spreads to various tissues and causes disease, as well as the role of the immune response, generation of new vaccines, and use as a vector for gene delivery are topics covered in these two volumes. A unique highlight for readers of this series and those interested in the history of a major and profound biomedical research accomplishment is the chapter written by one of the participants who worked on the initial discovery and use of the vaccine who records the events that occurred at that time. Baltimore, MD La Jolla, CA

Diane E. Griffin Michael B.A. Oldstone

D.E. Griffin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. © Springer-Verlag Berlin Heidelberg 2009

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Making It to the Synapse: Measles Virus Spread in and Among Neurons V.A. Young and G.F. Rall(*)

Contents Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measles Virus: Genome and Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measles Virus Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Understanding MV CNS Complications: Results from Brains of Infected Individuals and Persistently Infected Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Big Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tools. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transgenic Mouse Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vitro Culture Systems and Techniques in Cellular Transport Studies. . . . . . . . . . . . . . . Reverse Genetics for MV. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Access to the CNS: Lessons from CDV, PV, and WNV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MV Spread and Transport in Non-neuronal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MV Spread in Non-neuronal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Use of Microtubules and Their Associated Motor Proteins to Achieve Viral Transport Within an Infected Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MV Spread in Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MV Movement Within and Among Neurons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Immune Responses on MV Spread in Neurons. . . . . . . . . . . . . . . . . . . . . . . . . . Remaining Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Measles virus (MV) is one of the most transmissible microorganisms known, continuing to result in extensive morbidity and mortality worldwide. While rare, MV can infect the human central nervous system, triggering fatal CNS diseases weeks to years after exposure. The advent of crucial laboratory tools to dissect MV neuropathogenesis, including permissive transgenic mouse models, the capacity to manipulate the viral genome using reverse genetics, and cell biology advances in understanding the processes that govern intracellular

G.F. Rall Division of Basic Science, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA, e-mail: [email protected] D.E. Griffin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. © Springer-Verlag Berlin Heidelberg 2009

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trafficking of viral components, have substantially clarified how MV infects, spreads, and persists in this unique cell population. This review highlights some of these technical advances, followed by a discussion of our present understanding of MV neuronal infection and transport. Because some of these processes may be shared among diverse viruses, comparisons are made to parallel studies with other neurotropic viruses. While a crystallized view of how the unique environment of the neuron affects MV replication, spread, and, ultimately, neuropathogenesis is not fully realized, the tools and ideas are in place for exciting advances in the coming years.

Abbreviations BBB CNS CSF EGFP F FIP H HSV IC IFNAR IL IN IV KO L M MAP-2 MIBE MV N NK P PIE PV PrV RAG RNP SLAM SSPE WNV YAC

Blood–brain barrier Central nervous system Cerebrospinal fluid Enhanced green fluorescence protein MV fusion Fusion inhibitory peptide MV hemagglutinin Herpes simplex virus Intracerebral Interferon alpha receptor Interleukin Intranasal Intravenous Knockout MV large (viral polymerase) MV matrix Microtubule associated protein 2 Myelin inclusion body encephalitis Measles virus MV nucleoprotein Neurokinin MV phosphoprotein Postinfectious encephalomyelitis Poliovirus Pseudorabies virus Recombinase activating gene Ribonucleoprotein Signaling lymphocyte activation molecule Subacute sclerosing panencephalitis West Nile virus Yeast artificial chromosome

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Introduction The basic steps of the infectious cycle of any pathogen’s entry into, spread within, and exit from the host offer crucial insights into how pathogens cause disease. As a result, a full understanding of the stages of a microorganism’s life cycle may inform the rational design of therapeutics to prevent or ameliorate consequent disease. Measles virus (MV) is one of the most transmissible microorganisms known; it continues to result in hundreds of thousands of infections annually throughout the world, many of which have serious pathogenic consequences that can result in death. Despite the tremendous progress that has been made in deciphering the basis of MV pathogenesis and in the creation of effective attenuated vaccines, how MV causes disease, including rare, but serious, central nervous system (CNS) complications, remains poorly understood. It is our view that defining the pathogenesis of MV in the CNS necessitates an understanding of the interaction of the virus with the host in trafficking to and spreading within the CNS. This is the focus of this review.

Measles Virus: Genome and Proteins MV is a member of the Paramyxoviridae, within the Morbillivirus genus. Its genome consists of approximately 16,000 bases of nonsegmented, single-stranded negativesense RNA, meaning that the viral genome is transcribed immediately upon entry into the cell. Virions are spherical and enveloped, and the envelope is derived from the host cell as the viral particle buds from the plasma membrane. The viral genome encodes eight proteins, the function of which are briefly noted here. Inserted into the envelope of the virion are the two MV glycoproteins, hemagglutinin (H) and fusion (F). These proteins mediate attachment to cellular receptors and fusion of the virion with the target cell or fusion of an infected cell with an adjacent uninfected cell. The matrix protein (M) lies immediately underneath the virion envelope and serves in virus assembly and budding. The two polymerase proteins, large (L) and phosphoprotein (P), are closely associated with the genome, which is encapsidated by the nucleocapsid (N) protein. The nonstructural proteins V and C, encoded within the P cistron, are also packaged within the virion; these proteins have recently been shown to play a role in counteracting host antiviral immune responses (reviewed in Griffin 2001).

Measles Virus Pathogenesis MV is a human-restricted pathogen that spreads among individuals by release of aerosol droplets. An infected individual will undergo a latent period of 10–14 days followed by a few days of fever, cough, coryza, and rash. Primary infection occurs in the upper respiratory tract, but MV will secondarily infect lymphoid cells (reviewed in Griffin 2001; Rall 2003; Schneider-Schaulies et al. 2003; Sips et al.

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2007), though a contrasting hypothesis has recently been proposed that lymphoid cells are a primary traget for MV replication, followed then by engagement of an as yet unidentified receptor on the basolateral membrance of lung epithelia (de Swart et al., 2007; Leonard et al., 2008). Although the immunological response made by most infected, immunocompetent individuals is sufficient to clear the virus and provide life-long protection, a period of transient immunosuppression is a notorious characteristic of MV infection and is likely the basis of most of the complications and the subsequent fatalities following acute infection (reviewed in Dhib-Jalbut and Johnson 1994; Griffin et al. 2008; Rall 2003). Additional consequences of acute infection include diarrhea pneumonia, and encephalitis.

Immunosuppression Immunosuppression following MV infection can last for weeks, extending beyond the classical MV symptoms. The chief risk of immunosuppression is that it renders infected individuals more susceptible to secondary infections, though precisely how this occurs is not fully understood. In humans, MV-induced immunosuppression is characterized by a loss of delayed type hypersensitivity responses to recall antigens (e.g., tuberculin; Tamashiro et al. 1987), a limited response of lymphocytes to mitogens when cultured ex vivo (Hirsch et al. 1984), and impaired responses to new antigens (Coovadia et al. 1978). To date, a number of mechanisms have been proposed based on animal and cell culture studies, all of which could be pertinent in the natural infection. For example, Griffin and colleagues showed that MV infection of antigen-presenting cells suppresses interleukin-12 (IL-12) production, which then in turn skews the CD4+ T cell response toward a Th2 profile (Karp et al. 1996). This altered CD4+ T cell response leads to inappropriate priming of T cells and a failure of T cells to proliferate following interaction with MV-infected dendritic cells (Fugier-Vivier et al. 1997; Servet-Delprat et al. 2000). These studies correlate well with serum profiles in MV-infected macaques and humans that also show a skewing toward Th2-like cytokines (Atabani et al. 2001; Moss et al. 2002; Polack et al. 2000). In addition to influencing the Th1/Th2 balance, acute MV infection may precipitate immunosuppression by causing an overall lymphopenia, due to effects on T cell proliferation and progression through the cell cycle (Naniche et al. 1999; Niewiesk et al. 1999, 2000; Schnorr et al. 1997), as well as specifically inhibiting immune function via the production of unidentified, immunosuppressive molecules from infected T cells (Sun et al. 1998). A detailed discussion of MV-induced immunosuppression can be found in other chapters within this volume.

CNS Complications Following Acute MV Infection Approximately 1 in 100,000 acutely infected individuals later go on to develop CNS complications. These diseases differ in terms of immune status of the affected

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host, onset of symptoms, presence of MV within the CNS, host survival rate, and neuropathological findings. These are briefly discussed below. Subacute Sclerosing Panencephalitis Subacute sclerosing panencephalitis (SSPE) is a slow, progressive disease that is invariably fatal, and can occur from 1 to 15 years following acute MV infection (Dubois-Dalcq et al. 1974). Children are far more likely to develop this complication than adults (reviewed in Johnson 1998). The disease initially manifests as subtle cognitive losses, progressing to more overt cognitive dysfunction, followed by motor loss, seizures, and eventual organ failure in virtually all affected individuals. The rate of SSPE occurrence ranges from 1 in 10,000–300,000 acute MV infections (reviewed in Rima and Duprex 2005; Takasu et al. 2003). Neurons are predominantly infected, though at late times of infection, oligodendrocytes, astrocytes, and endothelial cells may also be involved (reviewed in Rima and Duprex 2005). SSPE affects both gray and white matter and is histologically characterized by the presence of cellular inclusion bodies, inflammation, glial activation, loss of blood–brain barrier (BBB) integrity, and neuronal loss (reviewed in Dhib-Jalbut and Johnson 1994; Rall 2003). A serologic hallmark of SSPE, as compared to the other CNS complications, is the elevation of measlesspecific antibodies in the blood and cerebrospinal fluid (CSF) (Dubois-Dalcq et al. 1974). Importantly, evidence from brain biopsies of SSPE patients indicates that infected neurons do not release budding virus (Paula-Barbosa and Cruz 1981). Based on extensive sequencing studies of MV from these specimens and from cells persistently infected with MV isolates from SSPE patients, it has been proposed that the failure of infected neurons to produce complete extracellular virus may be due to defects in protein expression caused by extensive point mutations in the envelope-associated genes, H, F, and M (Cattaneo et al. 1988; reviewed in DhibJalbut and Johnson 1994; Rima and Duprex 2005), though what role these viral proteins play in neuronal spread of MV and how mutations may affect MV biology in infected neurons are not known. Postinfectious Encephalomyelitis Postinfectious encephalomyelitis (PIE) occurs more frequently than SSPE, affecting approximately 1 in 1,000 infected individuals. Symptoms of PIE normally appear 5–14 days after the characteristic MV rash but can predate the rash (reviewed in Johnson 1998). This complication is thought to be an autoimmune reaction, perhaps to myelin basic protein (Johnson et al. 1984). MV antigen and nucleic acids have not been detected in PIE brain biopsies by immunohistochemistry or in situ hybridization (Johnson et al. 1984; reviewed in Dhib-Jalbut and Johnson 1994; Norrby and Kristensson 1997), supporting the notion that this

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is an autoimmune disease. Additional hallmarks of PIE include perivascular inflammation and demyelination (reviewed in Norrby and Kristensson 1997). Unlike SSPE, intrathecal production of MV antibodies has only been found in a few cases of PIE. Affected individuals present with seizures, deafness, ataxia, and movement disorders. There is an approximate 25% mortality rate associated with PIE, and survivors are likely to suffer from frequent neurologic sequelae. Measles Inclusion Body Encephalitis Measles inclusion body encephalitis (MIBE), a rare CNS complication following acute MV infection, has been described in children and adults receiving immunosuppressive drugs and therefore is thought to chiefly affect immunocompromised hosts. The neurologic disease appears 3–6 months after the acute MV rash (reviewed in Dhib-Jalbut and Johnson 1994; Johnson 1998). As the name suggests, MIBE is characterized by inclusion bodies in both neurons and glia, with accompanying neuronal loss but an overall lack of inflammation (reviewed in Dhib-Jalbut and Johnson 1994; Johnson 1998; Norrby and Kristensson 1997; Rall 2003). Measles antigen is present in the brain, and virus has been isolated directly from the brains of affected individuals (Johnson 1998). MIBE differs from SSPE in the absence of elevated serum and cerebrospinal fluid neutralizing antibodies (reviewed in Dhib-Jalbut and Johnson 1994; Rima and Duprex 2005). The disease course is relatively short, lasting from days to weeks, causing seizures, motor deficits, and stupor, often leading to coma and death (reviewed in Johnson 1998). Importantly, even though only a small percentage of acute MV infections will go on to develop CNS complications, a few studies have detected MV RNA in various organs, including brain, upon autopsy of elderly individuals who died of nonviral and non-CNS causes (Katayama et al. 1995, 1998). These findings suggest that MV may persist in the brains (and other organs) of healthy individuals, and that the frequency with which MV invades the CNS cannot be determined by summing the occurrence of the above-described CNS complications.

Understanding MV CNS Complications: Results from Brains of Infected Individuals and Persistently Infected Cell Lines The development of a robust live attenuated vaccine has profoundly decreased the infection rate in vaccinated populations. Vaccination has not been associated with SSPE, and only wild-type virus sequences have been isolated from SSPE tissues. However, vaccine strains have been isolated from the CNS of immunocompromised patients with MIBE (Bitnun et al. 1999; reviewed in Rima and Duprex 2005), implying that the attenuated vaccine strain can traffic to the brain under conditions of poor immune surveillance.

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Both SSPE and MIBE are characterized by mutations that apparently render the virus defective, though spread in neurons may still occur. As noted above, it has long been hypothesized that these mutations are the reason why infectious virus is not released from infected brain cells, especially since the mutations map to the envelope-associated proteins M, F, and H (Baczko et al. 1988; Billeter et al. 1994; reviewed in Johnson 1998). In SSPE, M protein expression is reduced, likely through one of two mechanisms: either a steeper gradient of transcription (Cattaneo et al. 1987a, 1987b) or increased read-through across the P–M junction of the MV genome (reviewed in Rima and Duprex 2005). In support of these data, hyperimmune antibody responses in SSPE are directed to all MV proteins with the key exception of M (reviewed in Rima and Duprex 2005). Mutations in F have been described for both SSPE and MIBE cases (Baczko et al. 1988; Billeter et al. 1994; reviewed in Johnson 1998). Interestingly, in all SSPE cases, mutations in F characteristically consist of the loss of the C-terminal pentadecapeptide, a sequence that is strictly conserved among morbilliviruses and is therefore thought to play an essential role in F protein function. This region contains the basolateral sorting signal of F (Maisner et al. 1998; reviewed in Rima and Duprex 2005), suggesting that missorting of F may contribute to altered MV spread and lack of complete MV assembly in neurons of SSPE patients. Interestingly, a study of ten SSPE cases by immunohistochemistry and in situ hybridization suggests that MV likely spreads transneuronally, an observation subsequently validated by in vitro model studies (Lawrence et al. 2000; Oldstone et al. 1999). The analysis of these infected brains revealed that MV infection in neuronal processes was predominantly dendritic, though there were signs of occasional axonal involvement as well (Allen et al. 1996). Whether mutations in viral proteins influence how MV is transported within neurons is currently under investigation.

The Big Questions Years of research on MV CNS complications have provided key insights into MV neuronal spread and have identified future areas of study concerning the relationship between viral spread and pathogenesis. For example: how does MV move from the site of entry to the perinuclear space and then again to the site of viral egress? Once present at the synaptic membrane, what cellular and viral proteins mediate MV transsynaptic spread? Is the mechanism of interneuronal spread related to the pathogenesis of MV within the brain? The establishment of permissive animal and cell culture models, coupled with advances in manipulating the viral genome, and the advent of cell biology resources to explore intracellular trafficking have been essential for key developments over the past few years. A discussion of these recent observations serves as the basis for the remainder of this review. We first describe some of these important technical advances and then discuss how they have been applied to MV neuronal spread.

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Tools Transgenic Mouse Models To date, two human receptors for MV have been identified: CD46 (Dorig et al. 1993; Naniche et al. 1993) and signaling lymphocyte activation molecule (SLAM; Erlenhoefer et al. 2001; Hsu et al. 2001; Tatsuo et al. 2000). While this is still an evolving field, and many believe that other receptors will be identified in the future, the general consensus is that CD46 is principally a receptor for vaccine strains, such as Edmonston, whereas SLAM, though restricted to hematogenous cells, permits entry of wild-type MV. As the mouse and rat homologs of CD46 and SLAM do not confer susceptibility to MV infection (Dorig et al. 1993; Manchester et al. 1994; Naniche et al. 1993; Ono et al. 2001b), transgenic mice expressing human CD46 or SLAM were established, with the hypothesis that expression of these human proteins would overcome the initial barrier to viral entry (Mrkic et al. 1998; Oldstone et al. 1999; Rall et al. 1997; Sellin et al. 2006; reviewed in Manchester and Rall 2001). Importantly for neuron-focused studies, these transgenic mice also provide a source of primary neurons for ex vivo experiments to complement observations made in vivo (Lawrence et al. 2000; Makhortova et al. 2007). A brief introduction to some of the transgenic model systems follows. NSE-CD46 mice, developed in the laboratory of Michael Oldstone, were engineered to express the BC1 isoform of human CD46 under the control of the neuronspecific enolase (NSE) promoter, restricting CD46 expression to CNS neurons. These mice can be infected intranasally (IN) or intracranially (IC) with a vaccine strain of MV (e.g., Edmonston); as predicted, only CD46-expressing neurons are initially permissive for infection. Adult immunocompetent mice mount an aggressive T cell response and survive infection, whereas NSE-CD46 neonatal mice, or adults on an immunodeficient background succumb to CNS disease. CD46+ primary hippocampal neurons can be cultured from transgenic embryos, providing a parallel in vitro culture system to corroborate and extend the in vivo observations (Lawrence et al. 1999, 2000; Makhortova et al. 2007; Patterson et al. 2002, 2003; Rall et al. 1997). Another CD46 transgenic model that was developed in Oldstone’s lab is the YAC-CD46 mouse, in which CD46 is expressed from its own promoter, more closely mirroring expression in humans. Indeed, CD46 distribution is found throughout the mouse, and this model has been used for both CNS and immunosuppression studies. Importantly, in this model, all four major isoforms of CD46 are expressed to levels and in locations similar to those seen in humans. Furthermore, overall expression of CD46 was shown to be greater than that previously reported for other transgenic mouse models, including the NSE-CD46 mice (Oldstone et al. 1999; Patterson et al. 2001). Mrkic et al. engineered mice to express CD46 on a mouse background lacking the interferon-α receptor [IFNAR–/– (Duprex et al. 2000; Ludlow et al. 2007; Mrkic et al. 1998)]. IFNAR–/– mice can be infected intranasally with MV, though

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replication of MV is limited. Engineering mice to express the MV receptor CD46 on the IFNAR–/– background resulted in higher levels of MV infection in the respiratory tract with subsequent inflammation, providing a more relevant model for the acute MV infection seen in humans. A perplexing observation from the establishment of a number of other CD46 transgenic mice was that, despite CD46 expression and ability to infect such cells when cultured ex vivo, little if any infection was observed in vivo. For example, Blixenkrone-Moller et al. generated a CD46 transgenic mouse that expressed a genomic copy of CD46 on a Bl/6 × SJL mouse background (Blixenkrone-Moller et al. 1998). Despite apparently robust CD46 expression, MV replication could not be detected following intraperitoneal (IP) or IN inoculation, though kidney and lung cell cultures from these mice were permissive. An important observation made by these authors was that, in these cultures, MV did not reach full replication levels as compared to MV-infected Vero cells. Furthermore, while IC challenge of CD46 transgenic mice did result in limited CNS infection, infection was also observed in nontransgenic mice, suggesting that factors other than CD46 play a role in mediating MV uptake into murine cells, and that key intracellular factors are needed to achieve optimal levels of MV replication. This work was supported by the findings of Horvat et al. and Evlashev et al. who found that cells from CD46 transgenic mice showed cell-type specific susceptibility to MV infection, indicating a role for host factors other than CD46 in mediating productive MV infection (Evlashev et al. 2001; Horvat et al. 1996). Thus far, four different SLAM-expressing transgenic mouse models have been created (Ohno et al. 2007; Sellin et al. 2006; Shingai et al. 2005; Welstead et al. 2005). However, to date, only one group has used these mice to look at MV CNS infections. Sellin et al. engineered mice to ubiquitously express SLAM. Wild-type and vaccine strains of MV could infect transgenic mice, either by IC or IN routes, but vaccine strains were less virulent (Sellin et al. 2006).

Other Animal Models Other investigators have focused on rodent-adapted MVs to infect nontransgenic (i.e., wild-type) mice, rats, or hamsters (Castro et al. 1972; Duprex et al. 1999a; Griffin et al. 1974; Moeller-Ehrlich et al. 2007; Schubert et al. 2006; Johnson and Swoveland 1977; Johnson and Norrby 1974; Roos et al. 1978). Such studies have been ongoing since the 1970s and have allowed researchers to study MV in a small animal model prior to the identification of the human MV receptors. Importantly, despite the mutations that occurred in adapting MV to rodents, the pathogenesis of such strains is remarkably similar to that seen following MV infection of humans or transgenic mouse models. Efforts to create animal models to study MV infection and pathogenesis have not been limited only to mice. For example, MV-infected cotton rats recapitulate the immunosuppression seen in MV-infected humans (Niewiesk 1999; Niewiesk et al.

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1999; Wyde et al. 1992), even with wild-type (clinical) isolates (Wyde et al. 1999). Furthermore, studies on rat brain slices grown in culture and infected with MV have revealed important clues about how MV spreads through an infected brain (Ehrengruber et al. 2002). Interestingly, the generation of a transgenic CD46expressing rat demonstrated that although MV was taken up by CD46-expressing cells, subsequent intracellular blocks in MV replication prevented robust infection of the animal (Niewiesk et al. 1997). Ferret models have also been used by some researchers as an SSPE model of MV infection in the CNS (Brown et al. 1985, 1987; Mehta and Thormar 1979; Thormar et al. 1983). Finally, while rhesus macaques have been chiefly used to study immune responses to MV, immune suppression induced by MV and efficacy of possible vaccines against MV (de Swart et al. 2007; Pan et al. 2005; Pasetti et al. 2007; Polack et al. 2000), some investigators have used tissues collected from infected monkeys to model human CNS diseases (Albrecht et al. 1977; Steele et al. 1982). How results from all of these model systems have been used to advance our understanding of MV interneuronal spread and neuropathogenesis will be discussed in the final section of the chapter.

In Vitro Culture Systems and Techniques in Cellular Transport Studies Primary Neuron Cultures and Neuron-Like Cell Lines The ability to culture primary neurons from various CNS substructures (e.g., hippocampus, cortex, dorsal root ganglia, cerebellum) of transgenic mice has been a powerful tool in dissecting aspects of intra- and interneuronal MV transport. These cultures are validated as neurons in their expression of characteristic neuronal markers such as MAP-2 and NeuN, their ability to form synapses in culture, and the fact that, once plated, these cells are mitotically inactive. While in general quite pure neuron cultures can be obtained (90%–95%), contaminating glial cells, culture-to-culture variability, difficulty in establishing these cultures and a fairly short lifespan in culture (10–14 days, typically), are some of the disadvantages. Cells lines such as NT2 (human teratocarcinoma cells that can be terminally differentiated into neuronal cells by retinoic acid treatment), human astrocytoma cells, and mouse neuroblastoma cells offer an alternative that is free of the complexities and challenges of primary neuron cultures (Duprex et al. 1999b, 2000; Lawrence et al. 2000; Ludlow et al. 2005; McQuaid et al. 1998). The ease of using these established lines of CNS cells is balanced by concerns about whether these cells accurately reflect primary CNS cell biology.

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Slice Cultures The introduction of organotypic monolayer cultures of nervous tissue has added an intermediate technique to the neurologist’s toolbox, falling between the complexity of a whole animal model and the culturing of cell lines or pure primary cultures that do not recapitulate the cellular heterogeneity of the CNS. Advantages to organotypic brain slice cultures are the ability to take cells from a defined region of the brain, the ability to use phase microscopy on the monolayer of resulting brain cells, the relevance of culturing primary cells without performing single cell dissociation, and the power of studying heterogeneous cell populations and how such diverse cells impact viral spread (Gahwiler 1981). This approach has been used to assess the spread of rMV-EGFP through neurons of cultured rat brain slices (Ehrengruber et al. 2002) as well as the spread of SSPE isolates through hamster cerebellum slices (Sheppard et al. 1975).

Reagents to Study the Role of Motor Proteins in Viral Spread Progress in our understanding of the cellular cytoskeleton and how organelles, signaling complexes, and proteins are transported within cells has greatly advanced our understanding of how viruses are transported within cells (Jouvenet et al. 2004; reviewed in Leopold and Pfister 2006; Mackenzie et al. 2006; reviewed in Ploubidou and Way 2001; Rietdorf et al. 2001; Ward and Moss 2004). For example, the development of dominant negatives to genetically manipulate the cellular transport system, as well as the increased availability of genetically altered mice with defined motor protein deficiencies, have enabled a dissection of which cellular proteins are utilized by viruses to enable transport. Although pharmaceutical approaches to ablate specific microtubule motors or microtubules themselves are broadly used (e.g., vanadate, colchicine, cytochalasin, nocodazole, etc.), the harsh and somewhat nonspecific consequences of using these cytotoxic reagents was always a limitation that can now be obviated with more precise and less caustic genetic strategies. For example, the generation and characterization of mice via ENU-mutagenesis containing a single point mutation in dynein heavy chain has helped to define the role of dynein in the spread of mouse-adapted scrapie prions from the periphery to the CNS of infected mice (Hafezparast et al. 2004). These loa (legs at odd angles) mice are viable as heterozygotes, and are currently being used by a number of neurovirology labs to assess the role of dynein in virus transport (Ahmad-Annuar et al. 2003; Hafezparast et al. 2003, 2004). Another tool to define viral–cytoskeletal interactions are dominant negative constructs. For example, overexpression of p50 dynamitin, a member of the multiprotein complex dynactin, which acts as an accessory factor to dynein, specifically disrupts dynein function (Ahmad et al. 1998; Burkhardt et al. 1997; Echeverri et al. 1996). This approach was used by Dohner et al. to show that herpes simplex virus type 1 (HSV-1) utilizes dynein following its entry into a cell to travel to the nucleus (Dohner et al. 2002). Similar reagents have been established for kinesins,

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motors that govern intracellular retrograde transport (Verhey et al. 1998; Verhey et al. 2001).

Reverse Genetics for MV The establishment of a reverse genetics system for vaccine strains of MV has also furthered our understanding of MV replication in neurons, and in MV intra- and interneuronal spread (Devaux et al. 2007; Radecke et al. 1995; Schneider et al. 1997). Future studies using this approach will be key to identifying what roles each of the viral proteins play in neuronal infection, and will define whether some viral proteins may be dispensable for neuronal infection. Moreover, given the speculation that point mutations found in SSPE isolates might make these viruses more neuropathogenic, reverse genetics offers an opportunity to directly test these hypotheses in a controlled setting. Already, MV reverse genetics has been used to address the role of the H protein of rodent brain-adapted MV in conferring neurovirulence to MV Edmonston in nontransgenic mice (Duprex et al. 1999a). Moreover, Maisner’s group engineered viruses with altered basolateral sorting signals in F and H and showed that these domains are important for MV propagation through lymphoid cells, in addition to their previously described role in MV spread through epithelial cells (Runkler et al. 2008). Furthermore, the ability of SSPE-associated viral genes to confer certain phenotypes on wild-type or vaccine strains of MV has been tested through reverse genetics approaches. For example, a recombinant MV expressing the M gene of an SSPE isolate was found to replicate less efficiently and led to a CNS infection in YAC-CD46 transgenic mice with a longer course than that seen following infection with MV Edmonston. This MVexpressing SSPE M also showed defects in viral assembly and subsequent budding of progeny virus from infected cells (Patterson et al. 2001). Finally, the power of reverse genetics has provided some experimental convenience in that we can now molecularly tag MV with marker proteins such as GFP to more easily follow MV infections in vivo, in brain slices, and in cell culture (de Swart et al. 2007; Duprex et al. 1999b, 2000; Ehrengruber et al. 2002; Ludlow et al. 2007; Plumb et al. 2002; Schubert et al. 2006).

Access to the CNS: Lessons from CDV, PV, and WNV Despite this broad palette of tools that are available to study MV–neuron interactions, many of the reagents and strategies have been developed only recently, and as a result we have more questions than answers about how MV gets to the CNS, spreads within CNS cells, and mediates neurological disease. How MV gains access to the CNS from the periphery is not known, though it has been previously proposed that MV spreads into the brain by infecting endothelial cells during secondary viremia (Esolen et al. 1995). Alternatively, there is speculation that MV may infect

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the CNS by infiltration of infected lymphoid cells, e.g., Alternatively there is speculation that MV may infect the CNS by infiltration of infected lymphoid cells, e.g., infiltrating macrophages. Despite a lack of certainty regarding MV neuroinvasion, clues about how MV spreads to the CNS can be gained by looking at the spread of other neurotropic viruses from the periphery to the CNS. Two routes of CNS infection have been proposed for a related morbillivirus (CDV): the hematogenous route and anterograde trafficking via the olfactory nerve. Like MV, CDV can infect lymphocytes during the systemic phase of infection, thus providing an opportunity for infected lymphocytes to traffic across the BBB and release infectious virus to initiate infection within the brain parenchyma. Importantly, the work by Rudd et al. showed that entry of CDV into the CNS can also occur via the olfactory bulb and suggested that this may be a common mechanism of entry for neurotropic paramyxoviruses (Rudd et al. 2006). Similar portals of entry have been proposed and tested for both polio virus (PV) and West Nile virus (WNV). One hypothesis is that these viruses cross the BBB; the second is that PV or WNV is transmitted via peripheral nerves (Ohka et al. 1998; Samuel et al. 2007). Yang et al. demonstrated that circulating PV crosses the BBB at a high rate that is independent of polio virus receptor (PVR) expression (Yang et al. 1997). More recently, studies in transgenic PVR-expressing mice have shown that PV is transported through the sciatic nerve by fast retrograde axonal transport. Furthermore, PV accesses the sciatic nerve from its intramuscular inoculation site by a process dependent on PVR (Ohka et al. 1998), and the mechanism by which PV is transported retrogradely along the sciatic nerve is thought to be an interaction of the PVR cytoplasmic domain with dynein light chain Tctex1 (Ohka et al. 2004). As with polio, axonal transport of WNV can also occur, mediating entry into the CNS. However, WNV can spread to the CNS even when the sciatic nerve has been transected, suggesting that WNV likely uses multiple routes to access the brain (Samuel et al. 2007). One of these routes of WNV entry has been shown to be dependent on Toll-like receptor 3 (TLR3)-mediated inflammation and subsequent opening of the BBB (Wang et al. 2004). Thus, as for CDV (Rudd et al. 2006), multiple non-mutually exclusive routes of spread are available for PV and WNV entry into the CNS. How MV penetrates into the CNS, and perhaps more importantly, how often this occurs in a human population, remain to be determined.

MV Spread and Transport in Non-neuronal Cells MV Spread in Non-neuronal Cells MV infection is initiated when H binds to one of its cellular receptors, CD46 or SLAM (Dorig et al. 1993; Naniche et al. 1993; Tatsuo et al. 2000). The receptor usage correlates with the source of the MV H protein: wild-type isolates typically use SLAM (Erlenhoefer et al. 2001; Ono et al. 2001a, 2001b; Tatsuo et al. 2000), whereas attenuated vaccine strains, such as Edmonston and the strains derived from

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it, preferentially use CD46. Receptor selection is not exclusive, however: SLAM has been shown to be an effective receptor for MV entry for vaccine strains and conversely a few wild-type strains have the ability to bind to CD46 (Erlenhofer et al. 2002). It has been suggested that the receptor usage of a given MV isolate may depend on the cell line used to isolate and amplify the virus rather than the wildtype or vaccine strain status of the MV isolate (Manchester et al. 2000). Furthermore, genetic and structural studies of MV H proteins have illustrated that the H binding domains to CD46 and SLAM are spatially distinct (Colf et al. 2007; Erlenhofer et al. 2002), providing support for the idea that H can interact with both CD46 and SLAM, albeit with different affinities for each. In any case, given that SLAM expression is limited to lymphoid cells (McQuaid and Cosby 2002), it is likely that other MV receptors await discovery. In non-neuronal cells, MV buds from the apical surface, resulting in the release of free virus or in cell fusion (reviewed in Griffin 2001; Fig. 1.1A). Transport of the viral nucleocapsid to the plasma membrane is dependent on levels of M protein and its accumulation at the cell surface (Runkler et al. 2007). Apical budding occurs despite the preferential sorting of MV glycoproteins F and H to the basolateral membrane through a tyrosine-based sorting signal (Blau and Compans 1995; Maisner et al. 1998; Moll et al. 2001). Appropriate budding is achieved by restricted expression of M at the apical surface, which retargets some of F and H in polarized cells (Naim et al. 2000; Riedl et al. 2002). The interaction of the cytoplasmic tails of F and H with M mediates virus assembly (Cathomen et al. 1998a, 1998b). However, the predominant presence of the glycoproteins F and H at the basolateral membrane has been hypothesized to play a key role in MV spread through an infected individual. The tyrosine-based sorting signals in F and H are not only required for basolateral sorting, but are also required for the fusogenicity of the F/H complex in polarized epithelial cells (Maisner et al. 1998; Moll et al. 2001). Consequently, it has been proposed that the basolaterally expressed F/H complex promotes cell–cell fusion, thus allowing the virus to spread to underlying tissues

Fig. 1.1 A, B Immunohistochemistry for MV antigen with hematoxylin counterstain. A MVinfected Vero cells form syncytia, or multinucleated giant cells. B MV-infected primary hippocampal neurons from NSE-CD46 mice do not form syncytia

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and facilitating systemic MV dissemination (Moll et al. 2004). This hypothesis emphasizes that apically released budding virus and basolaterally mediated cell– cell fusion are both important components of MV systemic dissemination. It should be noted that the classical MV spread in non-neuronal cells (extracellular progeny virus budding and cell–cell fusion) is dependent on MV glycoproteins. Thus, MV H interaction with its receptor likely triggers F protein fusogenic activity, initiating viral infection (Griffin 2001; Lamb and Kolakofsky 2001). As we point out later, these events may be substantially altered upon MV infection of neurons.

Use of Microtubules and Their Associated Motor Proteins to Achieve Viral Transport Within an Infected Cell Introduction to Microtubules How are the glycoproteins and RNP complexes shuttled through a cell to achieve appropriate assembly and egress? Microtubules are part of the cellular cytoskeleton and play a critical role in mediating movement of cellular proteins and organelles to their proper destinations. In neurons, their role is even more critical, as neurons rely on microtubules for neurite extension, synaptic vesicle trafficking, and synapse formation (Zhai and Bellen 2004). Microtubules are long, hollow cylindrical polymers of tubulin that assemble with a head-to-tail orientation (reviewed in Henry et al. 2006). The plus-end of microtubules typically extends to the plasma membrane and away from the nucleus, or toward the axon or dendrite and away from the cell body in neurons. In non-neuronal cells, microtubule minus-ends are bound and stabilized at the microtubule organizing center (MTOC) near the nucleus, whereas in neurons the minus-ends are oriented to the cell body, but lack an MTOC. The protein motors that move cargo along these microtubules are grouped into two families: the predominately plus-end-directed kinesin family of motors, which move cargo away from the cell body (anterograde transport), and the minus-end directed dynein family of motors, which move cargo to the cell body (retrograde transport), often as part of the lysosomal pathway. Cytoplasmic kinesin (or conventional kinesin) consists of two heavy chains and two light chains. The heavy chains contain the motor domains, which allow for the generation of force and subsequent movement along the microtubule. Furthermore, the heavy chains facilitate dimerization and cargo binding. The kinesin light chains may also facilitate binding to cargo (reviewed in Mandelkow and Mandelkow 2002). Cytoplasmic dynein binds to microtubules via its two heavy chains, but other components of this complex (including six light chains, two light intermediate chains, and two intermediate chains) mediate cargo binding and contribute to the specificity of the cargo that is transported. Furthermore, cytoplasmic dynein associates with another protein complex dynactin, which acts as a co-factor to facilitate cargo binding and processivity along the microtubule (reviewed in Leopold and Pfister 2006; Radtke et al. 2006).

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Viruses of many families utilize the microtubule pathway during infection. Some viruses associate with microtubules and retrograde motors to move to the nucleus, including murine polyomavirus, adeno-associated virus, adenovirus, herpes simplex virus 1, and human immunodeficiency virus (reviewed in Greber and Way 2006; Radtke et al. 2006). Other viruses are associated with anterograde transport, as has been shown for both Vaccinia virus and African swine fever virus transport to the plasma membrane through an interaction of viral cargo with kinesin-1 (Jouvenet et al. 2004; Rietdorf et al. 2001). Presumably, for these viruses, interaction with anterograde motors facilitates viral egress. Moreover, recent work with the alphaherpesvirus pseudorabies, a large DNA virus, illustrated fast axonal transport through neurons, a process mediated by microtubule motors (Smith et al. 2001). However, little is known about how neurotropic RNA viruses, including MV, interact with molecular motors in neurons. MV and the Cytoskeleton It has been known for some time that actin plays a key role in MV trafficking within cells. Actin is packaged within MV virions (Tyrrell and Norrby 1978), and treatment of infected cells with the actin-disrupting agent cytochalasin B resulted in disruption of MV virion formation (Stallcup et al. 1983). Electron microscopy studies of cytoskeletal preparations of MV-infected cells showed that the growing end of actin filaments protruded into budding virions. The authors suggested that the vectorial action of actin promotes MV budding and may also be involved in the transport of nucleocapsids to the cell surface (Bohn et al. 1986). Furthermore, actin was associated with transcriptionally silent MV nucleocapsids in vitro, supporting a role of actin in the budding of mature (i.e., not transcriptionally active) MV nucleocapsids. The same study showed that tubulin promoted MV RNA synthesis in in vitro RNA synthesis assays and could be co-immunoprecipitated with MV L, implying a further requirement for tubulin in MV replication (Moyer et al. 1990). These points, in addition to the large body of work illustrating the essential role of microtubules in facilitating longdistance transport within cells (reviewed in Greber and Way 2006), strongly support the notion that MV engages the microtubule network for transport within infected neurons. Ongoing work in a number of MV-focused laboratories is addressing this hypothesis, recognizing that it is hard to imagine a neurotropic virus achieving transport from one end of a neuron to the other without hijacking the cell’s railroad, the microtubule system. Transport of Other Viruses in Neurons The best-studied viruses, with regard to neuronal transport and spread, are the alphaherpesviruses, including herpes simplex virus 1 (HSV1) and pseudorabies virus (PRV). Cell imaging studies with both viruses have revealed by time-lapse

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fluorescence microscopy that these viruses travel retrogradely down the dendrite to the cell body (Bearer et al. 2000; Feierbach et al. 2007). This intracellular trafficking of the virus along microtubules can be disrupted by inhibitors of microtubule assembly, such as colchicine, vinblastine, or nocodazole (Sodeik et al. 1997; Topp et al. 1994). HSV-1 capsids entering a cell associate with the dynein complex, and overexpression of the dynactin component dynamitin (p50) blocks the transport of HSV1 to the nucleus (Dohner et al. 2002). Two somewhat contradictory models have emerged to explain virus transport away from the nucleus and subsequent viral egress. In the first model, herpesviral capsids and glycoproteins are transported separately, and assembly takes place somewhere along the axon shaft or at the axon terminus. The second model proposes that fully assembled enveloped virions are transported down the axon in vesicles (reviewed in Diefenbach et al. 2008; Lyman et al. 2007). As of last count, five different laboratories, three for HSV, two for PRV, have demonstrated contradictory findings as to which model is correct, and the controversy continues as the differences between variables, viruses, types of neurons, kinetics, and technical differences are sorted out (reviewed in Diefenbach et al. 2008). In either case, these two models provide the RNA virologist with an impetus for thought as to how smaller enveloped RNA viruses achieve egress from an infected neuron.

MV Spread in Neurons MV Movement Within and Among Neurons MV-infected CD46-expressing mice illustrated that, in vivo, neurons are the primary cell of the CNS infected by MV, even in animals where the expression of CD46 was not neuronally restricted (Oldstone et al. 1999). Similarly, neurons are the main target of MV infection in the brain of infected SLAM transgenic mice (Sellin et al. 2006), as is the case for infected human CNS tissues (Allen et al. 1996). The Edmonston strain of MV engineered to express EGFP (rMV-EGFP) spread through brains of infected CD46+/IFNAR–/– neonatal mice by neuronal processes. Furthermore, the authors detected EGFP-positive cells whose morphology was not neuronal and that the authors believed to be ependymal cells and neuroblasts (Duprex et al. 2000). This finding is of significance since it widens the population of cells susceptible to MV in the CNS of this animal model, and the result differs from the predominant neuronal infection described in other CD46-expressing animal models. Interestingly, the first report describing the generation and characterization of CD46+/IFNAR–/– mice indicated that IC infection with MV Edmonston also resulted in infection of ependymal cells, oligodendrocytes, and neurons (Mrkic et al. 1998). One key difference among these studies and those of other CD46 transgenic mouse models and of infected humans is the absence of an intact type I interferon system, which may allow the virus the opportunity to access other cell populations.

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MV does not bud from infected neurons and MV-infected primary neurons do not form syncytia (Fig. 1.1B). The presence of nucleocapsids in the axons and at the presynaptic membranes of infected neurons suggests a contact-dependent, trans-synaptic spread of MV (Lawrence et al. 2000; Oldstone et al. 1999). This finding is consistent with data from autopsy specimens of SSPE cases, where MV budding is not apparent (Paula-Barbosa and Cruz 1981). Moreover, spread of MV-Edmonston in a neuronal population is independent of the receptor CD46 (Lawrence et al. 2000); thus, in this model, while CD46 was needed for viral entry, it was dispensable for neuron-to-neuron transmission. MV spread via neuron–neuron contact has been confirmed in studies using rat organotypic hippocampal slices, as well as in tissue culture studies using differentiated human NT2 neurons (Lawrence et al. 2000; Ludlow et al. 2005; McQuaid et al. 1998). The spread of rMV-EGFP through hippocampal slices followed neuronal tracts, and no release of extracellular virus particles was observed. In this study, the interneuronal spread of rMV-EGFP was determined to be retrograde based on the spread of MV-EGFP from CA1 to CA3 pyramidal cells to granule cells within the slices and the known synaptic connectivity of these CNS substructures. Furthermore, the MV envelope proteins (F, H, and M) and P proteins were detected in dendrites of infected neurons (Ehrengruber et al. 2002). The mutations that accumulate upon MV adaptation to rodents (Rima et al. 1997; Vanchiere et al. 1995) do not seem to affect neuronal spread, as these rodent-adapted MVs have yielded data consistent with observations in humans and data from MV-infected CD46-expressing mice and primary neurons cultured from such mice (Duprex et al. 1999a; Mrkic et al. 1998; Schubert et al. 2006). For example, the hamster-neurotropic strain (HNT) of MV was used to infect Balb/c mice, and the resulting infection was limited to neurons and was not cytolytic. No virus assembly was detected (Van et al. 1979). Other work with HNT supports the lack of virus assembly following infection of weanling or adult mice (Griffin et al. 1974). The infection of weanling hamsters with HBS, another rodent-adapted MV isolated from an SSPE case, also revealed no budding virions in infected brains (Johnson and Swoveland 1977). As mentioned earlier, MV neuronal spread can occur independent of the expression of CD46, suggesting that H may not be required for MV trans-synaptic spread. Recently, Makhortova et al. indirectly investigated the role of MV F in trans-synaptic spread by using FIP, fusion inhibitory peptide (Makhortova et al. 2007). FIP, a synthetic tripeptide (z-D-Phe-L-Phe-Gly), prevents fusion by multiple viruses, though its strongest activity is against MV (Norrby 1971; Richardson et al. 1980; Richardson and Choppin 1983). FIP reduced MV infection and subsequent spread through primary CD46-expressing neuron cultures, implicating a role for F in trans-neuronal transport. Furthermore, the neurotransmitter substance P, whose active site is identical to FIP, also blocked MV neuronal spread. Genetic deletion of the substance P receptor, neurokinin-1 (NK-1), or pharmacological inhibition of NK-1 decreased MV replication and subsequent disease in MV-infected mice. Overall, these data suggest that MV F may interact with NK-1 at the synapse to mediate trans-synaptic spread of MV (Makhortova et al. 2007). The idea that MV engages a cellular receptor other than SLAM or CD46 is not

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new (Andres et al. 2003; Blixenkrone-Moller et al. 1998; Takeda et al. 2007), though the potential that such a receptor may be bound by MV F, rather than H, broadens this original hypothesis. As a result of this work, the current model for MV trans-synaptic spread is a microfusion event at the synapse, illustrated in Fig. 1.2. In this model, at least the MV RNP and F glycoprotein are actively transported to the synapse. F engagement of NK-1 may then allow a membrane fusion event to permit movement of the RNP from one neuron to the next. The neurotransmitter receptors neurokinin-2 and -3 (NK-2 and NK-3) bind to neurotransmitters of the same family as substance P and therefore may also play a role in MV trans-synaptic spread.

Effects of Immune Responses on MV Spread in Neurons It has long been known that multiple host factors contribute to the ability of a virus to replicate in a given cell. The literature contains several examples in which the elicitation or removal of a host immune response can alter MV replication and subsequent spread. While most of these studies have measured the consequences of ablation of a particular immune cell type (e.g., recombinase-activating gene (RAG) knockout (KO) mice that lack functional T and B cells), intrinsic responses of neurons can also influence the outcome of infection. For example, expression of the heat shock protein hsp72, which would be induced during a febrile illness, increased MV gene expression following infection of neonatal hsp72 transgenic mice. The increased MV RNA levels correlated with a higher mortality rate (Carsillo et al. 2006), providing an example wherein the cellular response to viral infection increases MV pathogenesis. Conversely, the removal of the adaptive immune response protein TAP1 (the transporter associated with antigen presentation) led to enhanced spread of MV into the brains of infected mice. The HNT strain of MV trafficked from the olfactory bulb into the limbic

Fig. 2.2 Model for MV-trans-synaptic spread. RNPs and F are transported to the synapse. The interaction of F with neurokinin-1 triggers a microfusion event, allowing the RNP to cross the synapse and infect the synaptically connected neuron. Neurokinin-2 and -3 may act as receptors for MV F, given their homology to neurokinin-1

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system of wild-type and TAP1–/– mice but showed a greater dissemination into the brains of TAP1–/– mice, as compared to that seen in wild-type mice (Urbanska et al. 1997). Finally, an association was found between promoter polymorphisms of the innate immunity protein MxA and the occurrence of SSPE in Japan. The most frequent polymorphisms caused the MxA promoter to be more responsive to type I interferon than the wild-type promoter sequence. Therefore, the increased activity of MxA in response to type I interferon positively correlated with SSPE occurrence, suggesting that MxA may play a role in the establishment of persistent MV infections in neurons (Torisu et al. 2004), though these studies have been somewhat controversial (Pipo-Deveza et al. 2006). The analysis of SSPE lesions of affected individuals revealed that the cells staining positive for MxA were mainly astrocytes located in a belt around the region of MV antigen-positive cells in affected areas of the brain. Similar to the conclusions drawn from the Japanese cases of SSPE, the authors suggested that MxA plays an important role in slowing down viral spread in SSPE and therefore may contribute to the persistent nature of this MV CNS infection (Ogata et al. 2004; Torisu et al. 2004).

Remaining Questions As this review has highlighted, the molecular mechanisms that govern MV transport in neurons are slowly coming into focus. Ehrengruber’s study on the spread of rMV-EGFP in cultured rat hippocampal slices found that MV spreads through the rat hippocampus in a retrograde direction, with the MV F, H, M, and P proteins localizing to the dendrite of infected neurons (Ehrengruber et al. 2002). Lawrence et al. demonstrated by electron microscopy the presence of MV nucleocapsids at the presynaptic membrane of infected primary hippocampal neurons, suggesting that MV may, in fact, spread in an anterograde direction, or from the axon of an infected neuron to the dendrite of a neighboring neuron (Lawrence et al. 2000). However, these studies could not distinguish between nucleocapsids that were exiting or nucleocapsids that were entering the neuron. Furthermore, there have been reports in support of both anterograde and retrograde trafficking of MV in neurons (McQuaid et al. 1998; Urbanska et al. 1997), which may not be unexpected as the virus must travel to the cell body to replicate and then back to the periphery of the neuron for egress, regardless of which neuronal process it uses (the dendrite or the axon) for entry and for exit. While a comprehensive view of how the unique environment of the neuron affects MV replication, spread and, ultimately, neuropathogenesis awaits further study, the tools and ideas are in place for exciting advances in the coming years. Acknowledgements We gratefully acknowledge the assistance of Christine Matullo and Lauren O’Donnell in the preparation of this review. V.A. Young is supported by T32 NS007180-25 from the University of Pennsylvania. G. R. is supported by NIH grants NS40500, MH56951, and CA006927, as well as generous support from the F.M. Kirby Foundation.

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Takasu T, Mgone JM, Mgone CS, Miki K, Komase K, Namae H, Saito Y, Kokubun Y, Nishimura T, Kawanishi R, Mizutani T, Markus TJ, Kono J, Asuo PG, Alpers MP (2003) A continuing high incidence of subacute sclerosing panencephalitis (SSPE) in the Eastern Highlands of Papua New Guinea. Epidemiol Infect 131:887–898 Takeda M, Tahara M, Hashiguchi T, Sato TA, Jinnouchi F, Ueki S, Ohno S, Yanagi Y (2007) A human lung carcinoma cell line supports efficient measles virus growth and syncytium formation via a SLAM- and CD46-independent mechanism. J Virol 81:12091–12096 Tamashiro VG, Perez HH, Griffin DE (1987) Prospective study of the magnitude and duration of changes in tuberculin reactivity during uncomplicated and complicated measles. Pediatr Infect Dis J 6:451–454 Tatsuo H, Ono N, Tanaka K, Yanagi Y (2000) SLAM (CDw150) is a cellular receptor for measles virus. Nature 406:893–897 Thormar H, Mehta PD, Lin FH, Brown HR, Wisniewski HM (1983) Presence of oligoclonal immunoglobulin G bands and lack of matrix protein antibodies in cerebrospinal fluids and sera of ferrets with measles virus encephalitis. Infect Immun 41:1205–1211 Topp KS, Meade LB, LaVail JH (1994) Microtubule polarity in the peripheral processes of trigeminal ganglion cells: relevance for the retrograde transport of herpes simplex virus. J Neurosci 14:318–325 Torisu H, Kusuhara K, Kira R, Bassuny WM, Sakai Y, Sanefuji M, Takemoto M, Hara T (2004) Functional MxA promoter polymorphism associated with subacute sclerosing panencephalitis. Neurology 62:457–460 Tyrrell DLJ, Norrby E (1978) Structural polypeptides of measles virus. J Gen Virol 39:219–229 Urbanska EM, Chambers BJ, Ljunggren HG, Norrby E, Kristensson K (1997) Spread of measles virus through axonal pathways into limbic structures in the brain of TAP1–/– mice. J Med Virol 52:362–369 Van PC, Rammohan KW, McFarland HF, Dubois-Dalcq M (1979) Selective neuronal, dendritic, and postsynaptic localization of viral antigen in measles-infected mice. Lab Invest 40:99–108 Vanchiere JA, Bellini WJ, Moyer SA (1995) Hypermutation of the phosphoprotein and altered mRNA editing in the hamster neurotropic strain of measles virus. Virology 207:555–561 Verhey KJ, Lizotte DL, Abramson T, Barenboim L, Schnapp BJ, Rapoport TA (1998) Light chaindependent regulation of kinesin’s interaction with microtubules. J Cell Biol 143:1053–1066 Verhey KJ, Meyer D, Deehan R, Blenis J, Schnapp BJ, Rapoport TA, Margolis B (2001) Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules. J Cell Biol 152:959–970 Wang T, Town T, Alexopoulou L, Anderson JF, Fikrig E, Flavell RA (2004) Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis. Nat Med 10:1366–1373 Ward BM, Moss B (2004) Vaccinia virus A36R membrane protein provides a direct link between intracellular enveloped virions and the microtubule motor kinesin. J Virol 78:2486–2493 Welstead GG, Iorio C, Draker R, Bayani J, Squire J, Vongpunsawad S, Cattaneo R, Richardson CD (2005) Measles virus replication in lymphatic cells and organs of CD150 (SLAM) transgenic mice. Proc Natl Acad Sci U S A 102:16415–16420 Wyde PR, Ambrose MW, Voss TG, Meyer HL, Gilbert BE (1992) Measles virus replication in lungs of hispid cotton rats after intranasal inoculation. Proc Soc Exp Biol Med 201:80–87 Wyde PR, Moore-Poveda DK, Daley NJ, Oshitani H (1999) Replication of clinical measles virus strains in hispid cotton rats. Proc Soc Exp Biol Med 221:53–62 Yang WX, Terasaki T, Shiroki K, Ohka S, Aoki J, Tanabe S, Nomura T, Terada E, Sugiyama Y, Nomoto A (1997) Efficient delivery of circulating poliovirus to the central nervous system independently of poliovirus receptor. Virology 229:421–428 Zhai RG, Bellen HJ (2004) Hauling t-SNAREs on the microtubule highway. Nat Cell Biol 6:918–919

Chapter 2

Modeling Subacute Sclerosing Panencephalitis in a Transgenic Mouse System: Uncoding Pathogenesis of Disease and Illuminating Components of Immune Control M.B.A. Oldstone

Contents Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction: Measles Virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Historical Background: Subacute Sclerosing Panencephalitis. . . . . . . . . . . . . . . . . . . . . . . . . Transgenic Mouse Model of SSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contribution of the Hypermutated M Protein to the Chronic Progressive CNS Disease . . . . Hypothesis to Explain the Initiation and Pathogenesis of SSPE . . . . . . . . . . . . . . . . . . . . . . . Contribution of Biased Hypermutation Predominantly in the M Gene of Measles Virus to the Pathogenesis of SSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contribution of Antibody-Induced Modulation of Measles Virus Antigens to the Pathogenesis of SSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dual-Hit Hypothesis: Acute Immunosuppressive Event Preceding Measles Virus Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of the Components of Measles Virus-Specific Immune Response Required for Clearing Measles Virus from the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future: Studies to Further Dissect the Molecular Pathogenesis of SSPE . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Subacute sclerosing panencephalitis (SSPE) is a chronic neurodegenerative disease of the central nervous system (CNS) that afflicts eight to 20 individuals per one million of those who become infected with measles virus (MV). The six cardinal elements of SSPE are: (1) progressive fatal CNS disease developing several years after MV infection begins; (2) replication of MV in neurons; (3) defective nonreplicating MV in the CNS that is recoverable by co-cultivation with permissive tissue culture cells; (4) biased hypermutation of the MV recovered from the CNS with massive A to G (U to C) base changes primarily in the M gene of the virus; (5) high titers of antibody to MV; and (6) infiltration of B and T cells into the CNS. All these parameters can be mimicked in a transgenic (tg) mouse model that expresses the MV receptor, thus enabling infection of a usually uninfectable mouse in which the immune system is or is not manipulated. Utilization and analysis of

M.B.A. Oldstone Viral-Immunobiology Laboratory, Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla CA, USA, e-mail: [email protected] D.E. Griffin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. © Springer-Verlag Berlin Heidelberg 2009

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such mice have illuminated how chronic measles virus infection of neurons can be initiated and maintained, leading to the SSPE phenotype. Further, an active role in prolonging MV replication while inhibiting its spread in the CNS can be mapped to a direct affect of the biased hypermutations (A to G changes) of the MV M gene in vivo.

Abbreviations SSPE CNS MV

Subacute sclerosing panencephalitis Central nervous system Measles virus

Introduction: Measles Virus Although diseases caused by viruses, including measles, were known and described in antiquity, viruses per se were not recognized as separate infectious agents until the late 1890s, over 110 years ago. Their recognition followed the pioneering work in bacteriology by Louis Pasteur and Robert Koch and their associates in the mid1800s. During that interval, the laboratory culturing process was developed, so it became possible to grow bacteria in culture for eventual placement on glass slides, staining, and observation under the microscope. Importantly, bacteria placed on Pasteur-Chamberland filters of varying pore sizes were retained upon vacuum pressure enabling the identification of specific bacteria, which when re-introduced into the appropriate host recaused the specific illness, thus linking the infectious agent with a particular disease state. It was on this framework that the first viruses were uncovered in 1898 (Loeffler and Frosch 1898; Beijerinck 1899; Ivanovski 1899). In contrast to the retention of bacteria on the Pasteur-Chamberland filters, viruses were characterized by their passage through the filters, their invisibility under light microscopy and their inability to grow in bacterial (cell-free) cultures. Thirteen years later, in 1911, Goldberger and Anderson (1911) showed that respiratory tract secretions from measles virus-infected patients were not retained but passed through the Pasteur-Chamberland-like filter, were invisible to light microscopy, and were unable to replicate in cell-free bacterial cultures. Moreover, macaque monkeys inoculated with material that passed through the filter developed the features of a measles virus-like disease. Another 43 years passed before MV was isolated and adapted to grow in cell cultures (Enders and Peebles 1954). This feat was accomplished by John Enders, to whom this volume is dedicated. His work formed the basis of subsequent extensive biological research on the pathogenesis of MV, methods for diagnosing MV, and eventually the development of the measles virus vaccine, again the achievement of Enders and his laboratory associates (Katz and Enders 1959; Enders et al. 1960; Katz 1965). Sam Katz, a protégé of Enders who was instrumental in the development and testing of the vaccine has recorded some of his personal observations with Enders in chapter 1 of volume 329.

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In addition to the usual transient CNS symptoms and signs that accompany acute measles virus infection, two serious and debilitating manifestations of CNS injury, although uncommon, are consequences of acute MV infection: postinfectious encephalitis and subacute sclerosing panencephalitis (SSPE). Measles virus infection can occasionally be complicated by sudden onset of encephalitis. The incidence of MV postinfection encephalitis is 1 per 1000 cases (Miller 1964; Scott 1967) and the clinical picture includes high fever, headache, vomiting, and convulsions, often leading to coma in an individual who has begun to recover from the initial acute infection. This manifestation usually occurs at 5 days, but in some instances 25 days after the measles virus rash appears. Mortality is approximately 10% with one-quarter of those who survive showing a permanent neurologic defect. Rarely is MV recovered from the patient’s cerebral spinal fluid or brain, and the pathogenesis of this disease is immunologic in nature. Infection by other viruses such as mumps, varicella, rubella, smallpox, and vaccinia may cause a similar postinfection encephalitis. In contrast to post-MV encephalitis, SSPE does not appear for several years after the primary MV infection, and no fever or other signs of acute CNS injury are present. SSPE presents with behavioral changes and mental deterioration followed by neurodegeneration of the pyramidal, extrapyramidal, and cerebellar systems, invariably leading to death. In this situation, MV genetic material is easily identified in the CNS. Despite its rarity, the lethal sequence of SSPE and its long period of latency have motivated the author’s investigation of this disease. This chapter discusses SSPE and recent observations from the author’s laboratory detailing the molecular pathogenesis of the disease.

Historical Background: Subacute Sclerosing Panencephalitis As early as the 1930s, the clinical picture of SSPE was suggestive of a potential infectious etiology. Neuropathologic evaluation (Dawson 1933; van Bogaert 1945) showed acidophilic intranuclear and cytoplasmic inclusion bodies in neurons and glia cells of the CNS. However, although infection was the suspected cause, attempts to implicate any microorganism failed. Indeed, evidence for a viral etiology required an additional 30 years. Not until the mid-1960s did Bouteille and his associates (1965) view by electron microscopy structures resembling nucleocapsids of a paramyxovirus within neurons from the brain material of a patient with SSPE. Two years later, Connolly and colleagues (1967) demonstrated that patients with SSPE had excessively high titers of antibodies to measles virus in their sera and cerebral spinal fluid. Further, by immunohistochemistry measles virus antigens were found in neurons and glia. The last and essential piece of evidence establishing an association with MV came independently from two laboratories, one at the NIH from Horta-Barbosa and colleagues (1969) and the second from a team at the University of Michigan lead by Francis Payne (Payne et al. 1969). Their work verified that the MV was defective in replication and that the recovery of virus required the assistance of a co-cultivation assay. Thus, viable brain tissue from SSPE

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patients when co-cultivated in tissue culture with cells permissive for MV lead to recovery of the virus. These results have now been confirmed multiple times and extended by direct cloning of measles virus from the brains of SSPE patients, thus firmly establishing the relationship between SSPE chronic progressive neurologic disease and MV infection. Thereafter, a plethora of papers appeared concerning measles virus and SSPE and occasionally also implicating mumps virus with SSPE (see PubMed). The fact that SSPE cases have decreased in parallel with the implementation of vaccination against MV clearly indicates that MV vaccination does not cause SSPE (Campbell et al. 2007). Noteworthy and seminal was the report concerning SSPE from the laboratories of Cattaneo, ter Meulen, and Billeter (Cattaneo et al. 1988), who performed direct cloning and molecular analysis of the MV genome recovered from brains of SSPE patients. These investigators noted biased hypermutation in the MV genome, most often affecting the matrix (M) gene, with conversion of U to C and A to G bases. The debate that followed centered on the question could such a defective MV cause or result from the persistent infection? The evidence on hand given later in this review indicates that the defective virus does not initiate infection but is a consequence of it. Further, the defective virus contributes to the ongoing pathogenesis of the disease. Currently, there is no reliable treatment that delays or aborts this chronic fatal neurodegenerative disease. Thus, investigation has centered first on establishing animal model(s) to mimic this disorder and second, on utilizing such models to understand how measles virus can establish, under rare conditions, a persistent infection and why the host cannot clear the infection. With the availability of such models, the pathogenesis of this disease can be decoded and therapeutic approaches designed and tested. An animal model that mimics SSPE must exhibit six cardinal elements: 1. Progressive fatal CNS disease; 2. Replication of measles virus in neurons; 3. Defective (nonreplicating) virus that can be recovered by co-cultivation with permissive tissue culture cells; 4. Dramatic biased hypermutation in the measles virus genome primarily involving one of the eight measles virus genes, the M gene, with conversion of U to C and A to G bases; 5. High titers of antibodies to measles virus; 6. Infiltration of B cells and T cells into the CNS. Several animal models discussed in this current volume of CTMI or the CTMI volume 191 on this subject satisfy one or more of these demands. However, the one described below fulfills all six criteria. The tg mouse model we use in which the MV receptor CD46 is transcriptionally expressed by its authentic CD46 promoter (YAC-CD46 tgs), placed on the C57Bl/6 background and then crossed onto the Rag1 genetically deleted (Rag1−/−) background provides all of the six necessary fingerprints cited above. First, such tg mice express CD46 on neurons as well as all nucleated cells. Crossing these tg mice on to the Rag1−/− background results in offspring whose B and T lymphocytes have been deleted. This maneuver removes the adaptive immune response and allows

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MV infection to persist. This approach was first successfully employed in the Rall laboratory where Rag−/− mice (Lawrence et al. 1999) were crossed with previously constructed tg mice that expressed the CD46 molecule exclusively on neurons by use of the neuron-specific enolase (NSE) promoter (Rall et al. 1997). A further benefit of using either the YAC-CD46 or NSE-CD46 mice is that reconstitution of purified and specific components of the host’s immune system can be adaptively transferred to the measles virus-infected host deleted of immune cells (YAC-CD46 × Rag−/− or NSE-CD46 × Rag−/− tg mice), thereby allowing insight into which the missing component(s) of the immune system is essential for the control and purging of measles virus from the CNS.

Transgenic Mouse Model of SSPE The primary host for measles virus is humans. Measles virus can infect monkeys but as far as we know, that occurs by transmission from humans to monkeys either by experimental inoculation or by air droplet spread to susceptible monkeys from humans infected with the virus. Earlier investigators utilized forced adaptation of measles virus by serial passage through brains of a variety of animals. Eventually, use of this method produced rodent adapted MV capable of infecting in the rodent host in which the virus had been passed. Although useful data were obtained from such experimental systems, the models were limited primarily by the disease phenotype produced, the age of the host required for infection, and the forced passage production of a mutated measles virus genome that was very distant from the sequence of the measles viruses isolated from humans and used for adaptation. Once the receptors (CD46, SLAM) for measles virus in humans were discovered, this limitation was overcome by expressing the authentic human virus receptor in mice using tg protocols. The transcriptional expression of CD46 or SLAM in mice, especially in C57Bl/6 (B6) mice, provided an easily manipulatable small-animal model in which the genetics were known and abundant markers were present to dissect the immunologic response to the virus by deletion and reconstitution of unique cells of the immune system. However, a limitation remained. Although the mouse and human genomes are over 98% identical, not all the host factors required for optimal measles virus replication and control of infection may be present or optimally functional in the rodent. Nevertheless, either tissue-specific promoters to transcriptionally express MV in cells of the immune system (e.g., CD11c for dendritic cells, LCK for T cells) in neurons in NSE for neurons or promoters to express receptors more generally have been used by a number of laboratories (Rall et al. 1997; Thorley et al. 1997; Blixenkrone-Moller et al. 1998; Mrkic et al. 1998; Oldstone et al. 1999; Hahm et al. 2003, 2004; Carsillo et al. 2006; Kurihara et al. 2006; Sellin et al. 2006; Ohno et al. 2007). Our tack and the tg model we utilized were the CD46 molecule expressed under control of its own CD46 promoter. This strategy allowed us to infect cells of the immune system and neurons in the brain. Since the CD46 genome was obtained from a yeast artificial chromosome (YAC) library (Yannoutsos et al. 1996), the

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resultant tg mice were termed YAC-CD46. Infection of YAC-CD46 mice led to virus replication in and recovery from cells of the animal immune system. This process was associated with suppression of primary and secondary humoral and cell-mediated immune responses (Oldstone et al. 1999). Further, the use of YACCD46 tg mice, measles virus infection, and challenge with a secondary bacterial infection, Listeria monocytogenes, demonstrated that measles virus suppressed both the innate and adoptive immune responses required to control the Listeria infection. Further, the molecules and immune cells involved in the process were mapped (Slifka et al. 2003). Pertinent for this chapter, infectious virus also replicated in and was recovered from neurons in the CNS. However, replication of MV in the CNS was markedly enhanced and prolonged when YAC-CD46 mice were placed on a Rag1−/− background. Rag1−/− mice have a deletion in B and T immune cells and thus cannot generate an immune response when challenged with an antigen. Thus, by avoiding a host-generated immune response to MV, adult YAC-CD46 × Rag1−/− mice, replicated and spread MV infections in their neurons. This allowed the infection to persist in the CNS for months. Figure 2.1 shows neurons of YAC-CD46 × Rag1−/− tg mice 60 days after the initiation of MV infection in adult mice. However, no MV could be

Fig. 2.1 Infection of neurons and accumulative mortality of YAC-CD46 × Rag1−/− tg mice infected with MV (Edmonston strain)

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recovered from brains of infected YAC-CD46 × Rag1−/− tg mice unless viable brain tissue was co-cultured with viable permissive tissue culture cells such as Vero cells. Electron microscopy performed by Samuel Dales, currently in Gunter Blobel’s laboratory at the Rockefeller University in New York, showed a picture in which structures of measles virus nucleocapsids were clearly present in the cytoplasm and nucleus of hippocampal neurons in MV-infected YAC-CD46 × Rag1−/− tg mice (Fig. 2.2). Further, direct cloning of the MV genome from brains of such tg mice by Lee Martin, when a postdoctoral fellow in my laboratory, revealed biased hypermutations in the M gene. Figure 2.3 displays the geography of A to G hypermutation in the M protein from one such mouse, #1919, in which a remarkable 35%–40% of the gene is mutated. Also listed in Fig. 2.3 is the A to G hypermutations in four other YAC-CD46 × Rag1−/− mice. Their clinical state at the time of sacrifice and the number of A to G mutations found are displayed. Hypermutation of the M gene commonly knocked out a unique Alu1 restriction site. This allowed Martin to devise a scheme for enrichment of additional hyperimmune M gene sequences (see Oldstone et al. 2005 for details). Cattaneo and Billeter (1988, 1992) had previously reported similar mutations and hypermutations in MV persisting in brain material from patients with SSPE. Thus the YAC-CD46 × Rag1−/− tg mouse infected with measles virus (Edmonston strain) mimicked the SSPE disease in humans by the following criteria. The first is a progressive fatal CNS disease with measles virus replication in neurons. Second, measles virus in the CNS was defective in that it was recoverable only by co-cultivation on permissive cells in culture. Third, analysis of the measles virus genome revealed biased hypermutation primarily in the M gene

Fig. 2.2 Electron micrographs of brains from YAC-CD46 × Rag1−/− tg mice infected with MV. Note presence of viral nucleocapsids in neurons. EM courtesy of Sam Dales

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Fig. 2.3 Biased hypermutations (A to G) in the M gene of MV from brains of YAC-CD46 × Rag1−/− tg mice persistently infected with MV. Each dot records A to G mutation

with A to G (U to C) changes. Further, classic SSPE-like nucleocapsid structures were seen by electron microscopic examination of infected neurons. In addition, when such YAC-CD46 × Rag1−/− tg mice were reconstituted with splenic lymphocytes and infected with measles virus, high titers of measles virus antibodies were recorded in the sera, presumably in response to repetitive stimulation of the immune system by measles virus (Oldstone et al. 2005; Tishon et al. 2006). The heightened anti-measles virus antibody response was accompanied by a modest infiltration of B and T cells in the CNS (Oldstone et al. 2005; Tishon et al. 2006).

Contribution of the Hypermutated M Protein to the Chronic Progressive CNS Disease Now that a reproducible small-animal model of SSPE was available, we could answer the question of whether or not the hypermutated A to G bases in the M gene of measles virus directly contributed to the chronic progressive CNS disease. This was accomplished by John Patterson, a former postdoctoral fellow in my laboratory, who used the reverse genetics system for MV developed by Martin Billeter and his colleagues in Zurich (Radecke et al. 1995). Patterson replaced the M gene

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of Edmonston strain measles virus with the M gene taken from Biken strain SSPE MV (Patterson et al. 2001a). The newly constructed recombinant virus was then injected into adult YAC-CD46 × Rag1−/− tg mice. Comparison of Edmonston measles virus (contains non-A to G mutated M gene) with recombinant Edmonston/M genome Biken (A to G mutated M gene) indicated that the Edmonston/M gene Biken recombinant was infectious and produced a protracted CNS disease in the range of 1–3 months longer than Edmonston alone. Both viruses caused death. Further, the recombinant virus appeared predominantly in clusters so that the infection of neurons was limited. That is, samples of multiple levels of brain tissues from tg mice infected with the recombinant Edmonston/Biken SSPE virus when compared to those infected with Edmonston virus showed less spread of the Edmonston/ Biken SSPE virus in neurons. The best interpretation of these results is that the biased hypermutations of the M gene most likely slowed the migration of the virus and thereby prolonged the infection. These data noted in vivo supported the earlier observations in vitro of Cathomen et al. (1998) that a functioning M gene is not required for measles virus replication or transcription. The other conclusion from this work is that mutations in the M gene do not harm the virus, as do some mutations to other MV genes required for replication, transcription, or viral assembly. Those detrimental mutations in other MV genes likely result in the virus’ elimination, whereas mutations in M are well tolerated and have little if any affect on virus survival.

Fig. 2.4 Evidence of a pathogenic role for the biased hypermutated M gene in the pathogenesis of SSPE. The M gene of Edmonston strain of MV was replaced by the hypermutated M gene from human SSPE MV (Biken isolate). The resultant recombinant MV when inoculated into YACCD46 × Rag1−/− tg mice significantly prolonged the time of the progressive CNS degenerative disease (left panel). Further, MV-infected neurons appeared as clumps, suggesting impairment of viral spread in vivo (compare infected neurons in Fig. 1 and Fig. 5, Edmonston MV infection) with neurons in right panels (recombinant Edmonston/Biken M gene MV). For details see Cattaneo et al. 1988; Wong et al. 1989; Patterson et al. 2001a

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Figure 2.4 illustrates the accumulated mortality curves of both groups and reveals the enhanced survival time of YAC-CD46 × Rag1−/− mice infected with the recombinant measles virus bearing the A to G hypermutated M gene. The insert shows clumping of MV-infected neurons as detected by immunochemistry.

Hypothesis to Explain the Initiation and Pathogenesis of SSPE Hypotheses regarding the initiation and pathogenesis of SSPE revolve around three clinical/pathological findings. The first concerns the issue of whether biased hypermutations in the M gene of measles virus contribute to the development of SSPE or if such mutations per se are not involved in either the initiation or maintenance of pathogenesis, but simply accumulate because the M gene is not necessary for the virus’s life cycle during CNS infection. For instance, it has been suggested that the measles virus causing SSPE may be a unique strain or recombinant virus. However, this explanation is unlikely because epidemiologic analysis of identical twins infected simultaneously, i.e., with the same measles virus, show discordance in that only one twin developed SSPE (Houff et al. 1979; Cianchetti et al. 1983; DhibJalbut and Haddad 1984). Thus, although it is unlikely that a unique strain of measles virus (carrying the biased hypermutation primarily of the M gene) is responsible for initiation of measles virus infection leading to SSPE, it is possible that such biased hypermutated viruses in the CNS, once formed, contribute to ongoing SSPE disease. Further, a suspected enzyme may be responsible for the U to C (A to G) M hypermutations and could be triggered by virus-induced cytokines in the CNS. Such cytokines would enhance the interferon-inducible double-stranded RNA adenosine deaminase (ADAR1). ADAR1 is presumed to propagate the biased hypermutations by adenosine to inosine conversion in double-stranded RNA intermediates (Cattaneo et al. 1988; Bass and Weintraub 1988; Bass et al. 1989; Baczko et al. 1993; Patterson and Samuel 1995; Patterson et al. 2001a, 2001b). Further, MVs, as they continue their infection of neurons, may alter these cells’ expression of selected genes and function without killing the infected cell. ADAR is associated with Ca++ flux and glutamate receptor editing. The second hypothesis focuses on the role of excessively high titers of antibody to measles virus in the blood and cerebral spinal fluid of patients with SSPE. This theory questions whether such antibody could participate in modulating MV gene products and thus alter the virus’s life cycle, favoring its ability to persist in infected cells. The basic idea here is that MV per se is not cytotoxic for cells but causes their injury by cell-to-cell fusions and syncytial formation, which depends on the viral glycoproteins (Joseph and Oldstone 1975; Oldstone and Tishon 1978; Fujinami and Oldstone 1979, 1980, 1983; Oldstone et al. 1980). Fusion/syncytial formation is the sine qua non of measles virus infection in tissue culture and during acute infection, i.e., syncytia of lymphoid cells in lymphoid organs, but is surprisingly absent or minimally present in neurons of the CNS in SSPE patients (Fenner 1974; Adams et al. 1984). The third hypothesis centers on the profound suppression of the immune system associated with measles virus infection. Indeed, among viruses, measles was the

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first to be recognized as causing a profound immunosuppression (Osler 1904; von Pirquet 1908; Wagner 1968), which was discovered several years before the virus was isolated (Goldberger and Anderson 1911). Measles virus-induced immunosuppression led to the activation of a latent tuberculosis, changing it to a miliary or systemic spread of tuberculin disease (von Pirquet 1908). Further, the strength of immune system suppression by MV is profound and, in fact, measles was used by physicians therapeutically before the discovery of corticosteroids to treat aggressive autoimmune nephritis (Blumberg and Cassady 1947). The activation of secondary microbial infections and other infectious agents was linked with MV well before MV was isolated and parallels the later observations with the human immunodeficiency (HIV, AIDS) virus (reviewed in McChesney and Oldstone 1989). The foundation of this hypothesis rests on three observations. The first is the study of identical twins infected simultaneously with MV (presumably the same strain) show a discordance because SSPE develops in only one twin (Houff et al. 1979; Cianchetti et al. 1983; Dhib-Jalbut and Haddad 1984). This observation suggests that an environmental effect, not a genetic predisposition, is important in initiating SSPE. This observation also disputes a component of the first hypothesis that a unique measles virus strain or altered measles virus is responsible for initiation of SSPE. The second arm of the immunosuppressive hypothesis is the clinical documentation that unimmunized patients who receive intensive immunosuppressive therapy are more vulnerable to SSPE than immunocompetent individuals infected with measles virus (Coulter et al. 1979; Fukuya et al. 1992). The third arm is that the majority of SSPE cases occur in children infected before the age of 2 years (Cattaneo et al. 1988; Griffin 2007) when the immune system is still immature. In the next section follows the experimental support for these various hypotheses, along with the possibility or realization that components of each and not necessarily only one may be involved in the initiation and pathogenesis of SSPE.

Contribution of Biased Hypermutation Predominantly in the M Gene of Measles Virus to the Pathogenesis of SSPE To determine whether a biased hypermutation of the M gene directly participates in SSPE pathogenesis we used reverse genetics to construct and rescue a recombinant measles virus containing the hypermutated M gene. The Edmonston strain of MV backbone was modified to contain the biased hypermutated A to G M gene of a human SSPE isolate Biken strain (Cattaneo et al. 1988; Wong et al. 1989). Once CNS infection was initiated in CD46 MV receptor expressing tg mice, this recombinant virus was found to be defective in budding from the plasma membranes of neurons and was recoverable only by co-cultivation of infected brains with tissue culture cells permissive for measles virus. The recombinant virus was expressed in neurons and the time course for the persistent infection was dramatically prolonged by months when compared to similar tg mice persistently infected with Edmonston virus alone. Further, recombinant Edmonston/Biken SSPE biased hypermutated

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M gene measles virus appeared predominantly in clusters and did not spread as widely through the CNS as did the Edmonston measles virus. Together these observations indicated that the biased hypermutations in the M gene likely slow the migration of the virus through the CNS and prolong the infectious cycle in the host. This outcome in an experimental animal model documented clearly and for the first time that the biased hypermutated M gene of SSPE virus per se contributes to the chronicity of SSPE disease. Probably, however, the M protein is not necessary for MV replication and transcription (Cathomen et al. 1998). Hence, mutations in the M gene are not lethal for the virus. Yet, the hypermutated M gene still allows the virus to replicate and spread. This conclusion amplifies that of Baczko et al. (1993) who recorded clonal expansion of a different hypermutated MV variant. Similarly, our use of Edmonston measles virus infection in YAC-CD46 × Rag1−/− tg mice resulted in long stretches of A to G hypermutations in the M gene and the COOH−-terminal end of the F gene (Oldstone et al. 2005). The preference for A to G and U to C mutations was dramatic accounting for over 95% of mutations in M and 73% for F genes, respectively. Further, all five mice studied showed clonal expansion of M genes with biased hypermutations. Each of the mice had Alu1 hypermutated M genes, thus mimicking the end stage found in brains of humans with SSPE (Cattaneo and Billeter 1992; Baczko et al. 1993; Radecke et al. 1995). Further, the high percentage of A to G and U to C mutations suggests the possible superimposition of mutations induced by a host enzyme such as adenosine deaminase (ADAR) (Cattaneo et al. 1988; Bass et al. 1989; Patterson and Samuel 1995). Accordingly, the hypothetical scenario is that cytokines made in the CNS during persistent MV infection enhances the interferon-inducible ADAR1 enzyme. ADAR1 would then propagate the biased hypermutation by adenosine to inosine conversion in viral double-stranded intermediates. Since the measles virus M gene is not essential to virus replication or transcription, it could absorb multiple mutations, whereas, for example, the virus polymerase–replication complex could not, resulting in the virus’s survival. Further, by bearing an increase of mutations in M, the infecting virus survives longer, thereby establishing a near continuous feedback loop cycle over a number of years in SSPE patients to favor persistence and continuous stimulation for the production of antibody to MV.

Contribution of Antibody-Induced Modulation of Measles Virus Antigens to the Pathogenesis of SSPE Patients with SSPE possess logarithmically higher titers of antibodies to measles virus than found in individuals following either acute measles virus infection or vaccination against measles. Presumably the cause is continuous immunogenic stimulation from the persisting MV. Over three decades ago, investigators recognized that MV-infected cells in culture bathed in antibodies to MV resulted in a redistribution of MV antigens on the cell surface and the suppression of both

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immune mediate injury and, more importantly, the infected cells continued to survive and switch into a state of persistent infection (Joseph and Oldstone 1974, 1975; Oldstone et al. 1980). Impressively, by removing (capping off) the MV glycoproteins the hemagglutinin (HA) and the fusion (F) protein, antibody to MV prevented cell-to-cell fusion and giant cell syncytial formation, essential components for MV-induced death. In addition, by antibody modulation therapy a time occurred when restoration of HA and F proteins (by removal of anti-measles virus antibody) no longer occurred on the surface of infected cells and, uniquely, measles virus nucleocapsids similar to those in SSPE patients appeared in the nucleus and cytoplasm of infected cells. Of interest here is that the prevention of cell-to-cell fusion by another means recapitulates these events. When MV-infected cells are actively maintained in suspension to prevent cell–cell contact, the cell lysis does not occur and persistent infection follows. Thus the abnormal nucleocapsid formation seen in patients with SSPE is also observed in either antibody-treated cells or in tg mice expressing the CD46 measles virus receptor and whose neurons are persistently infected with measles. At the molecular level, MV antibody-induced modulation occurs after using monoclonal antibodies to specific epitopes on the measles virus HA and results in a signal delivered to the plasma membrane of infected cells that alters the measles virus transcriptional complex, especially the measles virus phosphoprotein inside the cell (Fujinami and Oldstone 1979, 1980; Oldstone et al. 1980; Fujinami et al. 1984). These results from our experiments with antibodies to MV used to treat measles-infected HeLa cells were subsequently confirmed in ter Meulen’s laboratory in mouse neuroblastoma cells or rat glioma cells infected with MV (Schneider-Schaulies et al. 1992). Further, we noted that this antibody induced modulation of measles-infected cells was specific since antibodies to HeLa cellsurface antigens as well as antibodies to other viruses, i.e., influenza, failed to modulate MV-infected cells. Conversely, the antibodies to MV failed to modulate off-surface antigens of influenza or lymphocytic choriomeningitis virus on infected cells (Fujinami and Oldstone 1979, 1980; Oldstone et al. 1980; Fujinami et al. 1984; Go et al. 2006). Recently, we obtained RNA profiles and performed mass spectrometry on the antibody-modulated cells in order to identify the host genes or proteins either up- or downregulated. These gene products are still being characterized (Go et al. 2006). One gene product of interest is ADAR1. The role played by MV antibody-induced antigen modulation in vivo has been more difficult to define, although two observations are noteworthy. Wear and Rapp (1971) demonstrated that only MV (adapted to the hamster brain) infected newborn hamster pups suckled to mothers having antibodies to measles developed a persistent infection in their CNS. In contrast, similarly infected pups suckled to nonimmune mothers died of acute encephalitis. Later, working with rats and rat-adapted measles virus, ter Meulen’s laboratory (Liebert et al. 1990) found that passive transfer of neutralizing antibodies directed against selected MV HA determinants were able to modulate MV gene expression at the level of transcription and switched an expected acute necrotizing encephalitis in either Lewis rats or brown Norway rats into a persistent CNS infection.

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Dual-Hit Hypothesis: Acute Immunosuppressive Event Preceding Measles Virus Infection Clinical reports that SSPE was associated with measles virus infection occurring after drug-induced immunosuppression (Coulter et al. 1979; Fukuya et al. 1992) usually used in treatment of acute childhood leukemia, coupled with the discordance of SSPE in identical twins infected at the same time with measles virus, raised the possibility that an immunosuppressive event narrowly preceding acute MV infection might allow the virus to initially escape the host’s immune response that would otherwise terminate the acute virus infection. Persistent virus infection would then follow. Further, the timing of both events would be critical and thus account for the low frequency of SSPE. To test this hypothesis, we initially infected tg mice expressing the MV receptor with a virus, lymphocytic choriomeningitis virus (LCMV) Clone 13 (Cl 13), known to transiently suppress the mouse immune system yet itself be noncytopathic (Oldstone 2002). Infection with measles virus 10 days later, a time of maximal immunosuppression by LCMV Cl 13 in its natural murine host (Sevilla et al. 2000), resulted in persistent measles virus infection of neurons. Such mice then developed a chronic CNS disease leading to death. Analysis of their brains showed the biased A to G (U to C) hypermutations in the measles virus M gene. Further, the other cardinal manifestations of SSPE occurred in this dual-hit model: (1) generation of a defective measles virus in the CNS that could be recovered by co-cultivation with tissue culture cells permissive for measles virus; (2) high titers of antibodies to measles, and (3) infiltration of B and T cells into the CNS. The timing between initiation of the immunosuppressive event and contracting measles virus infection was crucial. LCMV Cl 13 infection of mice causes a generalized suppression of both T (cell-mediated) and B cell (humoral antibody) arms of the adoptive immune system (Sevilla et al. 2000; Oldstone 2002). Thus, antigenspecific T cell responses to several RNA and DNA viruses as well as antibody responses to soluble and particulate antigens are significantly dampened. The decreased immune response peaks at day 10–14 following initiation of infection, wanes after day 20, and by 60–90 days after LCMV Cl 13 infection, the virus is usually cleared from the mouse (except from neurons and glomeruli in which viral clearance is delayed until days 110–120) (see Oldstone et al. 1986; Tishon et al. 1993). Further, LCMV Cl 13, by virtue of its tropism for dendritic cells, also interferes with or suppresses the early innate immune response (Zuniga et al. 2008) as well as the later adoptive immune response (Sevilla et al. 2000). Thus, when MV infection occurred 3 days after LCMV Cl 13 infection, a time when there is no LCMV-induced immunosuppression, none of the inoculated mice developed persistent infection of neurons. When MV was administered 30 days after the initiation of LCMV Cl 13 infection, a time when immunosuppression has begun to wane, only a minority of mice had evidence of persistent MV infection in their neurons. In contrast, when LCMV Cl 13 was given initially followed by MV 10 days later, the time of maximal LCMV-induced immunosuppression, all mice developed

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a persistent measles virus infection of neurons (see Fig. 2.5). Cloning of virus from their brains revealed A to G (U to C) biased hypermutations prevalent in the M gene and electron microscopy revealed viral nucleocapsids in the cytoplasm and nuclei of infected neurons (Oldstone et al. 2005). Sera harvested from these mice had antibody titers that were 20- to 30-fold higher than observed in immunocompetent mice immunized with measles virus. Further, like human patients with SSPE, such mice displayed infiltration of predominantly CD4 T cells and B cells as well as CD8 T cells in their brains.

Fig. 2.5 Experimental evidence for dual-hit strategy to explain the initiation of persistent MV infection in CNS neurons. Lymphocytic choriomeningitis virus (LCMV) Clone (Cl) 13 induces a generalized immunosuppression in mice due to its replication in dendritic cells (DCs) and interferes with DC activation of T and B cells (see Sevilla et al. 2000 and Oldstone 2002 for details). Judged by results of mixed leukocyte reaction (MLR), there is negligible inhibition on MLR by day 3 but maximal inhibition at day 10 with modest inhibition at day 30 post-LCMV Cl 13 infection. The SSPE phenotype in YAC-CD46 mice infected first with LCMV and then with measles refers to: (1) persistent MV infection of neurons with chronic progressive neurologic disease; (2) presence of nucleocapsids in disarray in the cytoplasm and nuclei of infected neurons; (3) biased hypermutation in MV M genome cloned from CNS; (4) recovery of defective MV from the CNS only by co-cultivation of viable brains with MV permissive cultured cells; (5) markedly elevated amounts of antibodies to MV in the sera of infected mice (significantly higher than antibody titers in acutely infected or vaccinated mice); and (6) infiltration of T and B cells into the brains of infected mice. See Oldstone et al. 2005 for details

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Patients with clinical manifestations of SSPE when their disease emerges, several years after the initial MV infection, are not severely immunosuppressed. Our early studies with Rag1−/−- mice in which T and B cells are deleted, crossed to YACCD46 recapitulated all the findings of the LCMV Cl 13/measles virus dual-hit model (10-day interval before the second infection). The longer-lasting persistent MV infection observed with the dual-hit model then likely results from two separate factors. One is the ability of antibody to MV to prolong the lifespan of MV-infected cells (Joseph and Oldstone 1975; Oldstone et al. 1980). This observation and its consequence are discussed above in Sect. 5.2. The second factor is the enhanced A to G (U to C) mutation in the M gene in patients with SSPE (Cattaneo et al. 1988) and in the murine model outlined here (Patterson et al. 2001a; Oldstone et al. 2005). Indeed, mice given recombinant Edmonston/Biken M SSPE measles virus in which the M gene of the Biken strain from an SSPE patient replaced the normal nonmutated M gene in Edmonston measles virus (Patterson et al. 2001a) have a persistent infection lasting several months longer than mice given the Edmonston virus alone. Two essential points emerge from this model. First, a system is available to further dissect the molecular pathogenesis of SSPE and, second, an animal model of SSPE is available for testing and evaluating various protocols and therapies for treatment of this chronic neurologic disease. At present, therapy for SSPE remains controversial and overall has not worked. Drugs such as steroids, ribavirin, type I and II interferons, have been tried singularly or in combination but with either conflicting or negative results (reviewed in Gascon 2003).

Identification of the Components of Measles Virus-Specific Immune Response Required for Clearing Measles Virus from the CNS Measles virus infection is most often an acute self-limiting disease. Both humoral (antibody) and cell-mediated (T cell) immune responses act to restrict infection, although T cells appear to be the major players (Burnet 1968; Whitton and Oldstone 2001; Griffin 2007). However, clearance of persisting virus from the CNS may require a unique combination of immune cells. For example, CD8 T cells alone can overcome an acute LCMV infection, but the mix of CD4 T cells along with CD8 T cells is an absolute requirement to purge this virus from persistently infected neurons (Tishon et al. 1995). To determine the essential prerequisite for clearance of MV from neurons during a persistent MV infection, we took advantage of the YACCD46 tg mouse model and the technical ability to purify or delete individual cell constituents of the immune response and adaptively transfer them into the YACCD46 × Rag1−/− tg mice so infected. Measles virus fails to establish a persistent infection of neurons in adult YAC-CD46 tg mice because such mice have a competent and mature immune system. However, when the immune system is voided by crossing YAC-CD46 mice onto the Rag1−/− background, infection with MV then causes a persistent neuronal infection. Adaptive transfer of 5 × 107 or 1 × 107 splenic

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colleagues, we have received NOD-SCID mice implanted when 1-day-old with human neuronal stem cells. Accordingly, 1 × 105 stem cells were inoculated into six different CNS locations. These cells express the CD46 molecule and are permissive to MV infection in vivo. When the mice were 8 weeks old, they were inoculated with MV, which subsequently induced a persistent infection in the animals’ acquired human neurons. Since the MV was engineered to express GFP, infected neurons were easily identified. This technique allowed analysis of the MV genome biased mutations of A to G (U to C) and use of gene display to profile RNA expression of human neuronal genes during persistent MV infection. The MV-infected neurons in this stem cell model, as well as those in the YAC-CD46 × Rag1−/− model, mirror the situation of most infected neurons in brains of SSPE patients in that none of these neurons engage in fusion. Additionally, since MV per se is not cytotoxic for cells, neuronal apoptosis was not an issue. The hypothesis being tested is that, in SSPE, persistent MV infection of neurons affects the differentiation (luxury) function of neurons without killing them. As a result, the altered homeostasis of neuronal function leads to disease. This mechanism of disease, e.g., disturbed cell function in the absence of cell cytotoxicity, was first uncovered during study of persistent LCMV infection of several differentiated cell types in vivo – e.g., neurons, endocrine cells, immune cells – and has been extended to a variety of RNA and DNA viruses (reviewed in Oldstone 2002). A second ongoing study tests the hypothesis that cytokines released in the brain during persistent MV infection induce the dsRNA adenosine deaminase enzyme responsible for A to G (U to C) changes in the virus’s M gene (see Fig. 2.8). The resultant behavioral/neurodegenerative phenotype may likely be caused by faulty glutamate receptor editing in neurons.

Fig. 2.8 Illustrated hypothesis for the induction of cytokines inducing ADAR1 enzyme and its conversion of A to G base changes in the MV genome

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ADAR is an interesting candidate to evaluate for four reasons. First, the presence of ADAR could account for the molecular hallmark of prolonged MV infection of the CNS, the biased conversion of A to G (U to C) found in humans with SSPE (Cattaneo et al. 1988; Bass and Weintraub 1988; Bass et al. 1989; Baczko et al. 1993; Patterson and Samuel 1995; Patterson et al. 2001a, 2001b) and in the tg animal model of SSPE (Patterson et al. 2001a; Oldstone et al. 2005). Second, we found elevations of ADAR in brains of some measles virus-infected YAC-CD46 mice compared to uninfected YAC-CD46 controls. Third, ADAR is involved in neuronal glutamate receptor editing, defects that can cause neuronal dysfunction due to excess Ca++ flux (Seeburg et al. 1998; Liu and Samuel 1999). Fourth, ADAR is an interferon-inducible protein (Patterson and Samuel 1995; Patterson et al. 1995) and amounts of this cytokine are elevated in MV-infected brains. To explore the role of ADAR in the pathogenesis of SSPE disease, Bumsuk Hahm and Lee Martin, former postdoctoral fellows in my laboratory, in collaboration with Charles Samuel and Cyril George at the University of California, Santa Barbara, constructed an ADAR1a plasmid with a neo-insert for the purpose of producing ADAR1a knockout mice. At present, several potential ADAR1a knockout founders have been identified and are currently being bred to determine germline transmission of the gene. Once ADAR1a knockout lines have been confirmed, these mice will be used to generate triple tg mice: ADAR1a−/− × YAC-CD46 × Rag1−/−. Such mice, after infection with MV, will provide a valuable resource to determine the roles of ADAR in the generation of biased hypermutations A to G (U to C) and the role, if any, of glutamate receptor editing in the pathogenesis of chronic measles virus infection of neurons. Acknowledgements This is Pub. No. 19363 from the Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA. This work was supported in part by NIH grant AI036222 and NIH postdoctoral training fellowships to Glenn Rall, Lee Martin, and John Patterson. Bumsuk Hahm was supported by NIH grants AI036222 and AI074564.

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Oldstone MB, Tishon A (1978) Immunologic injury in measles virus infection. IV. Antigenic modulation and abrogation of lymphocyte lysis of virus-infected cells. Clin Immunol Immunopathol 9:55–62 Oldstone MB, Fujinami RS, Lampert PW (1980) Membrane and cytoplasmic changes in virusinfected cells induced by interactions of antiviral antibody with surface viral antigen. Prog Med Virol 26:45–93 Oldstone MBA, Blount P, Southern PJ, Lampert PW (1986) Cytoimmunotherapy for persistent virus infection: unique clearance pattern from the central nervous system. Nature 321:239–243 Oldstone MBA, Lewicki H, Thomas D, Tishon A, Dales S, Patterson J, Manchester M, Homann D, Naniche D, Holz A (1999) Measles virus infection in a transgenic model: virus-induced central nervous system disease and immunosuppression. Cell 98:629–640 Oldstone MBA, Dales S, Tishon A, Lewicki H, Martin L (2005) A role for dual viral hits in causation of subacute sclerosing panencephalitis. J Exp Med 202:1185–1190 Osler W (1904) The principles and practice of medicine. New York Patterson JB, Samuel CE (1995) Expression and regulation by interferon of a double-stranded RNA-specific adenosine deaminase from human cells: evidence for two forms of the deaminase. Mol Cell Biol 15:5376–5388 Patterson JB, Cornu TI, Redwine J, Dales S, Lewicki H, Holz A, Thomas D, Billeter MA, Oldstone MB (2001a) Evidence that the hypermutated M protein of a subacute sclerosing panencephalitis measles virus actively contributes to the chronic progressive CNS disease. Virology 291:215–225 Patterson JB, Manchester M, Oldstone MBA (2001b) Disease model: dissecting the pathogenesis of the measles virus. Trends Mol Med 7:85–88 Patterson JB, Thomis DC, Hans SL, Samuel CE (1995) Mechanism of interferon action: doublestranded RNA-specific adenosine deaminase from human cells is inducible by alpha and gamma interferons. Virology 210:508–511 Payne FE, Baublis JV, Habashi HH (1969) Isolation of measles virus from cell cultures of brain from a patient with subacute sclerosing panencephalitis. N Engl J Med 281:585–589 Radecke F, Spielhofer P, Schneider H, Kaelin K, Huber M, Dotsch C, Christiansen G, Billeter MA (1995) Rescue of measles viruses from cloned DNA. EMBO J 14:5773–5784 Rall GF, Manchester M, Daniels LR, Callahan EM, Belman AR, Oldstone MB (1997) A transgenic mouse model for measles virus infection of the brain. Proc Natl Acad Sci U S A 94:4659–4663 Schneider-Schaulies S, Liebert UG, Segev Y, Rager-Zisman B, Wolfson M, ter Meulen V (1992) Antibody-dependent transcriptional regulation of measles virus in persistently infected neural cells. J Virol 66:5534–5541 Scott T (1967) Postinfectious and vaccinial encephalitis. Med Clinics North Am 51:701–707 Seeburg PH, Higuchi M, Sprengel R (1998) RNA editing of brain glutamate receptor channels: mechanism and physiology. Brain Res Rev 26:217–229 Sellin CI, Davoust N, Guillaume V, Baas D, Belin MF, Buckland R, Wild TF, Horvat B (2006) High pathogenicity of wild-type measles virus infection in CD150 (SLAM) transgenic mice. J Virol 80:6420–6429 Sevilla N, Kunz S, Holz A, Lewicki H, Homann D, Yamada H, Campbell KP, de la Torre JC, Oldstone MBA (2000) Immunosuppression and resultant viral persistence by specific viral targeting of dendritic cells. J Exp Med 192:1249–1260 Slifka MK, Homann D, Tishon A, Pagarigan R, Oldstone MB (2003) Measles virus infection results in suppression of both innate and adaptive immune responses to secondary bacterial infection. J Clin Invest 111:805–810 Thorley BR, Milland J, Christiansen D, Lanteri MB, McInnes B, Moeller I, Rivailler P, Horvat B, Rabourdin-Combe C, Gerlier D, McKenzie IF, Loveland BE (1997) Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection. Eur J Immunol 27:726–734

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Tishon A, Eddleston M, de la Torre JC, Oldstone MB (1993) Cytotoxic T lymphocytes cleanse viral gene products from individually infected neurons and lymphocytes in mice persistently infected with lymphocytic choriomeningitis virus. Virology 197:463–467 Tishon A, Lewicki H, Rall G, von Herrath M, Oldstone MB (1995) An essential role for type 1 interferon-gamma in terminating persistent viral infection. Virology 212:244–250 Tishon A, Lewicki H, Andaya A, McGavern D, Martin L, Oldstone MBA (2006) CD4 T cell control primary measles virus infection of the CNS: regulation is dependent on combined activity with either CD8 T cells or with B cells: CD4, CD8 or B cells alone are ineffective. Virology 347:234–245 van Bogaert L (1945) Une leucoencéphalite sclérosante subaigüe. J Neurol, Neurosurg, Psych 8:101–120 von Pirquet C (1908) Das Verhalten del kutanen Tuberkulin-Reaktion Wahrend der Masern. Dtsch Med Wochenschr 34:1297 Wagner R (1968) Clements von Pirquet: his life and work. Baltimore, MD Wear DJ, Rapp F (1971) Latent measles virus infection of the hamster central nervous system. J Immunol 107:1593–1598 Whitton JL, Oldstone MBA (2001) The immune response to viruses. In: Knipe DM, Howley PM (eds) Fields virology, 4th edn. Lippincott Williams Wilkins, Philadelphia, pp 285–320 Wong TC, Ayata M, Hirano A, Yoshikawa Y, Tsuruoka H, Yamanouchi K (1989) Generalized and localized biased hypermutation affecting the matrix gene of a measles virus strain that causes subacute sclerosing panencephalitis. J Virol 63:5464–5468 Yannoutsos N, Ijzermans JN, Harkes C, Bonthuis F, Zhou CY, White D, Marquet RL, Grosveld F (1996) A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection. Genes Cells 1:409–419 Zuniga EI, Liou LY, Mack L, Oldstone MBA (2008) In vivo virus infection inhibits type 1 interferon production by plasmacytoid dendritic cells thereby facilitating opportunistic infections. Cell Host Microbe (in press)

Chapter 3

Measles Studies in the Macaque Model R.L. deSwart

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vaccination Studies in Macaques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Atypical Measles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Live-Attenuated Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New-Generation Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative Routes of Administration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis Studies in Macaques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Lines and Virus Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differences Between Macaque Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunity, Protection, and Immunosuppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Infections with MV Expressing EGFP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Much of our current understanding of measles has come from experiments in non-human primates. In 1911, Goldberger and Anderson showed that macaques inoculated with filtered secretions from measles patients developed measles, thus demonstrating that the causative agent of this disease was a virus. Since then, different monkey species have been used for experimental measles virus infections. Moreover, infection studies in macaques demonstrated that serial passage of the virus in vivo and in vitro resulted in virus attenuation, providing the basis for all current live-attenuated measles vaccines. This chapter will review the macaque model for measles, with a focus on vaccination and immunopathogenesis studies conducted over the last 15 years. In addition, recent data are highlighted demonstrating that the application of a recombinant measles virus strain expressing enhanced green fluorescent protein dramatically increased the sensitivity of virus

R.L. deSwart Department of Virology, Erasmus MC, University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands, e-mail: [email protected] D.E. Griffin and M.B.A. Oldstone (eds.) Measles – Pathogenesis and Control. © Springer-Verlag Berlin Heidelberg 2009

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detection, both in living and sacrificed animals, allowing new approaches to old questions on measles vaccination and pathogenesis.

Introduction At the beginning of the twentieth century, it was demonstrated that measles virus (MV) could be transmitted from humans to non-human primates. One to 2 weeks after inoculation with blood collected from measles patients, macaques developed fever and skin rash (Anderson and Goldberger 1911). Disease transmission could also be achieved using filtered respiratory secretions, demonstrating that the causative agent of measles was a virus (Goldberger and Anderson 1911). A decade later, Blake and Trask followed up on these studies by experimentally infecting macaques with nasopharyngeal washings of measles patients, thereby successfully inducing measles in 16 animals (Blake and Trask 1921a). The virus could be passaged from monkey to monkey by intratracheal inoculation of tissue homogenates or by intravenous injection of citrated blood (Blake and Trask 1921a). The authors carefully documented incubation time, fever, leukopenia and pathology of enanthem and exanthem (Blake and Trask 1921b). In addition, they demonstrated that experimental MV infection resulted in complete immunity against reinfection (Blake and Trask 1921c). In the 1950s, it was demonstrated that experimental infections with MV isolated in cell culture also caused measles in macaques (Peebles et al. 1957). The authors were able to reisolate the virus and detect MV-specific serum antibody responses, thus bringing the model close to its current state. Although experimental MV infection of macaques had been demonstrated successfully, a number of other studies yielded negative results. Retrospectively, it can be concluded that in many of these cases animals were immune to measles due to prior exposure to the virus. Peebles et al. detected MV-specific serum antibodies in 22 out of 24 macaques tested from US laboratories, whereas animals screened immediately after capture all proved to be serum antibody-negative (Peebles et al. 1957). This illustrates that measles is a disease of humans and normally does not affect non-human primates unless they are brought in close contact with humans. Monkeys used as experimental animals were at that time in most cases wild-caught animals. Before and during transport they were housed under crowed conditions, and contacts with MV-infected humans could result in virus transmission (Meyer et al. 1962). On several occasions, measles outbreaks occurred at animal facilities after arrival of new animals, in some cases associated with significant morbidity (Potkay et al. 1966; Hime and Keymer 1975; Scott and Keymer 1975; Remfry 1976; MacArthur et al. 1979; Welshman 1989). Thus monkeys intended to be used for experimental MV infections needed to be transported using special precautions (Tauraso 1973). At present, all macaques used for experimental measles virus infections are purpose-bred animals, but prescreening for the absence of MV-specific antibodies remains imperative. The high susceptibility of non-human primates to MV infection provided the first animal model for measles. The two species used most often are the rhesus

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macaque (Macaca mulatta) and the cynomolgus macaque (Macaca fascicularis) (El Mubarak et al. 2007). As a result of the close similarity of clinical, virological, immunological and pathological parameters to those associated with measles in humans, the model continues to be used almost a century later. Although several rodent species and other small laboratory animals have been inoculated with MV, most of these do not or poorly replicate the virus (Stittelaar et al. 2002a; Pütz et al. 2003). Studies in cotton rats or transgenic animal species do reproduce certain aspects of measles, but none of these show the same level of similarity with the pathogenesis of measles in humans as the macaque model (see chapters 5 and 6). New world monkeys proved to be even more susceptible to MV infection than old world monkeys, but developed a disease with a different pathogenesis than that of measles in humans, which was associated with high mortality (Levy and Mirkovic 1971; Albrecht et al. 1980). Due to their high susceptibility, marmosets (Saguinus mystax) were later used for encephalogenicity studies, as reviewed elsewhere (Van Binnendijk et al. 1995). The current review will address macaque models of measles, with a focus on studies published after 1995.

Vaccination Studies in Macaques The first isolation of MV in cell culture (Enders and Peebles 1954) was immediately followed by attempts to develop a vaccine against measles. Two different strategies were pursued in parallel: inactivated vaccines and live-attenuated vaccines. Whereas the first category of vaccines proved to predispose for enhanced disease, the second was highly successful. In the 1990s studies were initiated to develop new-generation vaccines as potential successors of the current live-attenuated vaccines, some of which showed promise in preclinical studies in macaques. However, none of these candidate new measles vaccines have been pushed forward towards licensing for human use. Studies in the 1980s had already demonstrated that administration of the current MV vaccine by aerosol held great promise (Sabin et al. 1982). However, regulatory authorities consider a vaccine and its route of administration as one entity, which means that before the aerosol route for measles vaccination can be implemented licensure will be required, for which preclinical studies were conducted in macaques.

Atypical Measles In the 1960s, classical inactivated measles vaccines were manufactured by formalin inactivation of whole MV preparations, which were subsequently precipitated with aluminum salts (referred to as FI-MV). Initially, vaccination with FI-MV was shown to induce MV-specific serum antibody responses (Hilleman et al. 1962;

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Norrby et al. 1964; Foege et al. 1965), and the vaccine was used in large-scale clinical trials in humans. However, after a number of years it turned out that vaccination predisposed for enhanced disease upon natural MV infection, which was referred to as atypical measles (Fulginiti et al. 1967) (see also chapter 10). During the same period, vaccination trials with formalin-inactivated respiratory syncytial virus (FI-RSV), another member of the family Paramyxoviridae, also proved to predispose for enhanced disease upon natural infection (Fulginiti et al. 1969; Kapikian et al. 1969; Kim et al. 1969). Whereas availability of small animal models for RSV allowed extensive studies on the pathogenesis of this vaccinemediated enhanced disease, this remained difficult for measles. Because the pathogenesis remained subject to speculation, the risk of inducing atypical measles continued to form a major stumbling block for the development of new generation nonreplicating MV vaccines. In 1999, Polack and Griffin were successful in reproducing atypical measles in macaques (Polack et al. 1999), thus providing an opportunity to study the pathogenesis of the disease as well as a model to test candidate new MV vaccines for predisposition of similar aberrant responses upon challenge infection. They immunized macaques with the original FI-MV preparation that had been manufactured in the 1960s. When challenged with pathogenic wild-type MV, two out of five rhesus macaques vaccinated with FI-MV developed atypical measles, characterized by a petechial rash and pneumonitis associated with the presence of eosinophils and immune complexes in their lungs. The authors concluded that atypical measles “results from previous priming for a nonprotective type 2 CD4 T cell response rather than from lack of functional antibody against the fusion protein” (Polack et al. 1999). In follow-up studies, they further characterized immune responses in these animals, demonstrating in vivo impairment of interleukin (IL)-12 production and increased production of IL-4 by peripheral blood mononuclear cells (Polack et al. 2002). When characterizing the antibody responses, they found that these were not only transient but also lacked avidity maturation. Challenge infection resulted in anamnestic production of low avidity antibodies, which could explain the immune complex deposition in FI-MV-primed animals (Polack et al. 2003a). Before reproduction of atypical measles in the macaque model, it was speculated that inactivation with formalin had resulted in destruction of critical B cell epitopes on the fusion protein (Norrby et al. 1975). However, this hypothesis could be rejected on the basis of two macaque experiments: animals primed with FI-MV developed atypical measles in the presence of fusion-inhibiting antibodies (Polack et al. 1999), and macaques primed with a DNA vaccine encoding for the haemagglutinin gene that developed H- but not F-specific antibodies did not develop any of the clinical signs associated with atypical measles (Polack et al. 2000). Interestingly, vaccination of macaques with formalin-inactivated respiratory syncytial virus (RSV) or human metapneumovirus (hMPV) preparations also predisposed for hypersensitivity to challenge infection with the respective viruses, suggesting that these phenomena are characteristic for all members of the family Paramyxoviridae (De Swart et al. 2002, 2007c).

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Live-Attenuated Vaccines As mentioned above, the MV strain Edmonston isolated by Enders and colleagues in 1954 induced measles-like clinical signs in macaques (Peebles et al. 1957). Serial passage of this virus in vivo (in chicken embryos) and in vitro (in chicken embryo fibroblast [CEF] cells) resulted in virus attenuation: upon experimental infection of macaques, the virus still induced MV-specific immune responses but virtually no clinical signs (Enders et al. 1960). More than 30 years later, direct comparison of MV isolated in lymphoid cells (strain Bilthoven) with MV passaged in human and monkey kidney cells (strain Edmonston wild type) or live-attenuated MV passaged in CEF cells (strain Schwarz) demonstrated that these strains displayed high, intermediate and low pathogenicity in macaques, respectively (Van Binnendijk et al. 1994). Virus loads in peripheral blood mononuclear cells and in bronchoalveolar lavage cells were several log values lower in animals infected with MV strains of reduced pathogenicity. Whereas levels of MV-specific serum IgM antibodies seemed directly related to the magnitude of virus loads, levels of specific serum IgG and virus neutralizing (VN) antibodies induced by the three virus strains were on the same order of magnitude (Van Binnendijk et al. 1994). Macaques and other non-human primate species have been used to assess the levels of attenuation of different candidate vaccine virus strains. In most cases, this was done by studying pathological lesions induced by intracerebral MV infection (Buynak et al. 1962; Nii et al. 1964b; Albrecht et al. 1981; Sharova et al. 1984). At present, vaccine manufacturers still use this method to assess appropriate attenuation of new MV vaccine virus seed stocks.

New-Generation Vaccines Despite its documented safety and efficacy, live-attenuated MV vaccines also have drawbacks, most importantly their dependence on cold chain maintenance and their ineffectiveness in the presence of maternal antibodies (Stittelaar et al. 2002a; Pütz et al. 2003). Live-attenuated MV vaccines are ineffective when used before the age of 9 months, resulting in a window of susceptibility in young infants between waning of maternal immunity and acquisition of vaccine-induced immunity. Since the 1980s, new-generation candidate MV vaccines have been developed to address these issues, including subunit vaccines, vectored vaccines and nucleic acid vaccines (see chapter 10). In the macaque model, passive transfer of MV-specific VN antibodies inhibited effectiveness of the live-attenuated MV vaccine, thus providing a model to evaluate the potential of candidate new vaccines in the presence of maternal antibodies (Van Binnendijk et al. 1997). The scientific steering committee of the World Health Organization (WHO) adopted the macaque model for preclinical comparison of the different candidates. A strategy was developed in which vaccine candidates were first used to immunize juvenile animals, either in the absence or presence of passively transferred MV-specific antibodies. This would

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allow assessment of immunogenicity and longevity of the induced specific immune responses. Approximately 1 year after vaccination, animals were challenged with a pathogenic wild-type MV strain to assess levels of protection. Vaccine candidates that performed well in this evaluation were subsequently tested in infant macaques, to assess their potential effectiveness at an early age in the presence of true maternally derived VN antibodies. This strategy resulted in the identification of a number of promising candidate MV vaccines. However, further preclinical and clinical evaluation of a new MV vaccine will require huge financial investments. Moreover, improved coverage of the live-attenuated MV vaccine in recent years in combination with the implementation of a two-dose strategy has been highly successful in reducing measles mortality. As a result, it remains uncertain if any of the candidate new-generation MV vaccines will ever be licensed for human use.

Alternative Routes of Administration For regulatory purposes, a vaccine and the device used for administration form one integral entity: the current live-attenuated MV vaccines are licensed for injection only. However, alternative routes of administration have extensively been studied in humans. Whereas intradermal, conjunctival, oral or intranasal administration were not particularly successful, aerosol administration of nebulized MV vaccine proved highly effective (Cutts et al. 1997). The aerosol route closely mimics the natural route of MV infection and may result in both mucosal and systemic immunity (Valdespino-Gomez et al. 2006). The WHO, in partnership with the American Red Cross and the Centers for Disease Control and Prevention and with funding from the Bill and Melinda Gates Foundation, has initiated a Product Development Group (PDG) for measles aerosol vaccination. The ultimate objective of the PDG is to achieve licensure of a combination of measles vaccine and nebulizer, which is equally safe, effective and cheap as the currently licensed measles vaccines administered by injection, but is easier to administer, less invasive, and could be administered by nonmedical personnel. The PDG evaluated two alternative strategies for measles aerosol vaccination: nebulization of a reconstituted vaccine (Dilraj et al. 2000) or inhalation of a dry powder aerosol (LiCalsi et al. 2001). Both vaccination strategies were evaluated in immunocompetent and immunocompromised macaques and proved equally safe. However, vaccination with a nebulized aerosol proved more effective than inhalation of a dry powder vaccine (De Swart et al. 2006, 2007a). The animals used in these studies had body weights of 1.8–4.5 kg and consequently had much smaller tidal volumes than those of children. To mimic vaccination using a similar dose to that inhaled by a child in 30 s, the exposure time therefore had to be prolonged (De Swart et al. 2006). Before proceeding to clinical trials, a toxicology study was conducted using larger study groups than those used in the exploratory studies. These studies were also

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conducted in macaques, to allow MV vaccine replication in all exposed tissues. This study revealed no adverse events, and measles aerosol vaccination is currently under investigation in clinical trials. The PDG aims to achieve licensure for this vaccination route in 2009.

Pathogenesis Studies in Macaques Experimental MV infections of macaques have been crucial for our understanding of the pathogenesis of measles. Infections with MV isolated in cell culture demonstrated the importance of passage history of the virus: MV strains exclusively cultured in lymphoid cells retained pathogenicity in macaques, whereas passage in other cell lines often resulted in virus attenuation. However, nonattenuated MV strains were in some cases also associated with subclinical infections, which seemed to be related to species differences between rhesus and cynomolgus macaques rather than to virus differences. A large number of studies have focused on the development of MV-specific immune responses, and the role of different arms of the immune system in protection from measles. Measles is usually not recognized before onset of rash, approximately 2 weeks after MV infection, making studies on the early events following virus transmission in humans difficult to perform. Pathological studies of experimentally infected macaques have provided important insights in the tissue distribution and cell tropism of the virus. Recently, infections with recombinant MV expressing enhanced green fluorescent protein (EGFP) resulted in improved sensitivity of virus detection, and provided new insights in the role of different target cells. This new approach to an old animal model provides alternative possibilities to further unravel the pathogenesis of measles and measles-associated immunosuppression.

Cell Lines and Virus Strains The introduction of Epstein-Barr virus (EBV)-transformed marmoset B cell line B95a as a substrate for isolation of MV (Kobune et al. 1990) made it possible to isolate non-culture-adapted wild-type MV strains. Whereas isolation of wild-type MV from patient samples in Vero cells usually takes at least 2 weeks (and in many cases requires serial blind passage), virus isolation in B95a cells can be as rapid as 2–4 days (WHO 2007). Experimental infections with the Edmonston wild-type strain did not always result in detectable clinical signs (Enders et al. 1960; Hicks et al. 1977; Van Binnendijk et al. 1994), whereas non-cell culture-passaged virus usually induced more fulminant infections (Yamanouchi et al. 1973; Sakaguchi et al. 1986; McChesney et al. 1989). Kobune and colleagues demonstrated that MV isolated in B95a cells retained its pathogenicity in macaques, resulting in development of viremia, lymphopenia and rash (Kobune et al. 1996).

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Van Binnendijk et al. isolated wild-type MV in human EBV-transformed Blymphoblastic cell lines (BLCL) and demonstrated that this virus also retained pathogenicity (Van Binnendijk et al. 1994). These authors applied the infectious center assay to the model, thus quantifying the frequency of MV-infected cells during viremia. Intratracheal inoculation of macaques with a single infectious unit of wild-type MV strain Bilthoven could spark infection associated with similar viral load kinetics as infection with 104 infectious units, with the only difference that the peak of viremia shifted slightly backwards in time with decreasing infectious dosage (Van Binnendijk et al. 1994). McChesney and colleagues developed a measles model in macaques using a MV strain isolated during an outbreak of measles in a primate facility (McChesney et al. 1997). The virus was isolated in Raji cells, a human BLCL isolated from a patient with Burkitt’s lymphoma, a disease resulting from in vivo transformation of B lymphocytes by EBV infection. Subsequently, the virus was passaged in vivo in macaques, after which a challenge stock was produced in macaque mononuclear cells (McChesney et al. 1997). This virus was also fully pathogenic in macaques, as demonstrated by induction of clinical and pathological changes typical for measles (McChesney et al. 1997) and MV-specific immune responses (Zhu et al. 1997). Auwaerter and colleagues compared the pathogenicity in macaques of six different MV strains, demonstrating that the nonadapted Bilthoven strain was fully pathogenic while the other cell culture-adapted strains were not (Auwaerter et al. 1999). Interestingly, a recent study addressing genetic changes that affect the virulence of MV in macaques demonstrated that wild-type MV isolated in Vero cells expressing CD150 also retained pathogenicity in macaques and induced skin rash (Bankamp et al. 2008). In contrast, the same MV isolated and passaged in normal Vero cells or in CEF cells did not induce rash. This suggests that expression of CD150 on the cells used for virus isolation is crucial, and that the cells are not required to be of lymphoid origin. Cell culture adaptation does not necessarily result in adaptation to the use of CD46 as a receptor, as the adapted MV isolates in the above-mentioned study did not infect Chinese Hamster Ovary cells expressing CD46 (Bankamp et al. 2008).

Differences Between Macaque Species Although experimental MV infections have been conducted both in rhesus and cynomolgus macaques, clinical signs such as rash and conjunctivitis were especially reported in rhesus macaques (Blake and Trask 1921b; McChesney et al. 1997; Auwaerter et al. 1999). Although skin rash has also been reported in cynomolgus macaques (Kobune et al. 1996), this symptom seemed to be less prominent in this species. The first direct comparison of MV infection in these two macaque species by experimental infection with two different non-culture-adapted wild-type MV strains indeed seemed to confirm this assumption: although animals of both species displayed similar virus replication curves in peripheral blood and broncho-alveolar

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lavage cells as well as similar MV-specific immune responses, the appearance of skin rash was more prominent in rhesus macaques (El Mubarak et al. 2007). Infection studies with a recombinant MV strain expressing EGFP conducted in parallel in rhesus and cynomolgus macaques showed that in both animal species MV-infected cells were detected in many different tissues, including the skin (De Swart et al. 2007b). Also with respect to other virological and immunological parameters, including infection of specific lymphocyte subsets, MV infection followed a virtually identical course in both animal species, suggesting that both macaque species can be used for measles pathogenesis studies.

Immunity, Protection, and Immunosuppression Both in humans and macaques recovery from measles is accompanied by lifelong immunity (Blake and Trask 1921c). MV infection induces strong specific humoral and cellular immune responses, and many different assays have been developed to characterize these ex vivo in macaques. The most important serological parameter is the detection of VN antibodies in serum, of which levels above 0.1–0.2 IU/ml have been identified as a correlate of protection from measles in infants (Chen et al. 1990; Samb et al. 1995). In macaques, similar levels of passively transferred VN antibodies were shown to interfere with MV vaccination (Van Binnendijk et al. 1997). Measurement of specific cell-mediated immunity (CMI) is less well standardized, but several techniques including assessment of lymphoproliferation (Van Binnendijk et al. 1997; Pan et al. 2005), cytotoxicity (Van Binnendijk et al. 1997; Zhu et al. 1997, 2000), cytokine production (Auwaerter et al. 1999; Polack et al. 2002, 2003b; Stittelaar et al. 2002b; Premenko-Lanier et al. 2003; Pan et al. 2005; Pasetti et al. 2007) or flow cytometry-based stimulation assays (Stittelaar et al. 2000; Pahar et al. 2005; De Swart et al. 2006) have been employed as correlates of CMI. VN antibodies can confer complete protection from measles, as also illustrated by the fact that infants born of a mother with adequate MV-specific antibody titers are protected from measles during their first months of life. In contrast, clearance of an established MV infection is largely dependent on CMI. Agammaglobulinemic patients recover normally from measles, while individuals with impaired CMI may succumb to MV infection (Burnet 1968). The role of specific lymphocyte populations in clearance of MV was addressed in the macaque model by depleting single or multiple populations using monoclonal antibodies to CD20 and/or CD8. Macaques depleted of all T lymphocytes or of CD8+ T lymphocytes only at the moment of MV infection exhibited a more extensive rash, increased viral loads and delayed viral clearance (Hicks et al. 1977; Permar et al. 2003). In contrast, depletion of CD20+ B lymphocytes did not result in alterations of clinical signs or kinetics of MV clearance (Permar et al. 2004). These studies demonstrate that CMI (and more specifically CD8+ T lymphocyte responses), but not humoral immunity, plays a crucial role in MV clearance. These data also highlight a major strength of the

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macaque model, correlating specific immune responses to protection from or clearance of experimental MV infection. Measles is not only associated with the induction of strong MV-specific immune responses but also with a transient immunosuppression, leading to enhanced susceptibility to opportunistic infections (see also chapter 12). Many putative mechanisms have been suggested, but in vivo assessment of the relative importance of these hypotheses has remained difficult. MV infects lymphocytes in vitro and in vivo (Yamanouchi et al. 1973; McChesney et al. 1989), but may also alter functionality of noninfected lymphocytes (Okada et al. 2000) or antigen-presenting cells (Schneider-Schaulies et al. 2003). Furthermore, MV infection interferes with production of specific cytokines, thus changing the host response to invading pathogens (Griffin et al. 1994; Moss et al. 2002). Paradoxically, MV inhibits proliferation of lymphocytes in vitro (Hirsch et al. 1984), but recovery from measles is associated with extensive lymphocyte expansion in vivo (Mongkolsapaya et al. 1999). Due to difficulties in standardization of CMI assays, it has been difficult to evaluate mechanisms of immunosuppression in macaques. Lymphopenia and reduced responses to mitogen stimulation have been described extensively (McChesney et al. 1989; Kobune et al. 1996; Zhu et al. 1997; Auwaerter et al. 1999). MV infection of macaques specifically alters the production of IL-10 and IL-12, thus affecting the balance between phenotypically different T lymphocyte populations (Polack et al. 2002; Hoffman et al. 2003a, 2003b). Perhaps the most functional assessment of immunocompetence of macaques during measles is the assessment of responses to immunization with other antigens, e.g., tetanus toxoid, which has been applied both to assess recall of previously primed immune responses (Bankamp et al. 2008) or as primary immunization during measles (Premenko-Lanier et al. 2004). Flow cytometry studies on lymphocytes of macaques infected with MV-EGFP suggest that infection of specific memory T lymphocyte subsets may also play an important role in measles-associated immunosuppression.

Pathology Early studies on the pathology of measles in macaques demonstrated the remarkable similarity to measles in humans (Blake and Trask 1921b). However, experimental infections in an animal model offer the possibility to evaluate pathological changes during different phases of the pathogenesis, whereas human tissue samples collected during the prodromal phase of measles are relatively rare. The classical Warthin-Finkeldey-type syncytial cells observed in human lymphoid tissues during the prodromal phase (Warthin 1931; Finkeldey 1931) were also observed in macaque tissues (Nii et al. 1964a; Yamanouchi et al. 1970, 1973; Hall et al. 1971; Sakaguchi et al. 1986; McChesney et al. 1997). The major tissues affected by MV infection of macaques are the upper and lower respiratory tract, the gastrointestinal tract, the lymphoid system and the skin (Blake

3 Measles Studies in the Macaque Model

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and Trask 1921b; Sergiev et al. 1960; Nii et al. 1964a; Hall et al. 1971; McChesney et al. 1997). However, the cellular origin of the lesions has been debated for decades (Nii et al. 1964a; De Swart et al. 2007b). Both in human and macaque tissues, MV antigen is commonly detected in association with epithelial tissues. In addition, culture-adapted MV strains grow very well in epithelial cells, which has led to the assumption that respiratory epithelial cells were a primary target for MV infection. However, epithelial cells do not express CD150 and are not easily infected with non-culture-adapted wild-type MV strains in vitro (Takeuchi et al. 2003). These observations warrant a reevaluation of the pathogenesis of measles, which may be facilitated by the combination of modern immunohistochemistry and flow cytometry techniques for characterizing cell subsets with experimental infections of macaques with MV strains expressing EGFP.

Infections with MV Expressing EGFP The development of reverse genetics techniques for non-culture-adapted MV strains resulted in the rescue of a MV strain from cloned cDNA that retained pathogenicity in macaques (Takeda et al. 2000). This molecular clone was used as a backbone for insertion of the gene encoding EGFP as an additional transcription unit upstream of the MV N gene. The resulting recombinant virus displayed similar in vitro replication characteristics as its parental strain, and infected cells produced high amounts of EGFP (Hashimoto et al. 2002). The MV-EGFP strain still proved to be virulent in macaques, and EGFP could be visualized macroscopically in both living and sacrificed animals, and microscopically by confocal microscopy and flow cytometry (De Swart et al. 2007b). As illustrated in Fig. 3.1, EGFP fluorescence was detected in skin, respiratory tract and digestive tract, but most intensely in lymphoid tissues. B and T lymphocytes expressing CD150 were the major target cells for MV infection. Highest percentages (up to more than 30%) of infected lymphocytes were detected in lymphoid tissues. In peripheral tissues, large numbers of MV-infected CD11c+ MHC class-II+ myeloid dendritic cells (DCs) were detected in conjunction with infected T lymphocytes, suggesting transmission of MV between these cell types. In the respiratory tract, the majority of MV-infected cells was detected in subepithelial tissues, but infected ciliated epithelial cells were also observed. In the T lymphocyte compartment, MV preferentially infected CD45RA− cells, which have a memory phenotype. This observation, which is in good accordance with recent data from MV infections in a human tonsillar explant model (Condack et al. 2007), pointed to a possible role for depletion of specific lymphocyte subsets in the transient disappearance of recall responses, as described in the early twentieth century (Von Pirquet 1908). By using cell-sorting techniques, the model allows both identification and functional assessment of MV-infected cell populations. The macaque model using the pathogenic autofluorescent wild-type MV strain IC323/EGFP clearly opens new possibilities for measles pathogenesis

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