Challenges and Opportunities of mRNA Vaccines Against SARS-CoV-2: A Multidisciplinary Perspective 3031189027, 9783031189029

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Siguna Mueller

Challenges and Opportunities of mRNA Vaccines Against SARS-CoV-2 A Multidisciplinary Perspective

Challenges and Opportunities of mRNA Vaccines Against SARS-CoV-2

Siguna Mueller

Challenges and Opportunities of mRNA Vaccines Against SARS-CoV-2 A Multidisciplinary Perspective

Siguna Mueller Kaernten, Austria

ISBN 978-3-031-18902-9 ISBN 978-3-031-18903-6 https://doi.org/10.1007/978-3-031-18903-6

(eBook)

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, 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. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

To my parents, who have demonstrated love, faith, and authenticity.

Foreword

It all began on March 11, 2020, when WHO director general in his opening remarks at the media briefing on COVID-19.1 stated the following: “In the past two weeks, the number of cases of COVID-19 outside China has increased 13-fold, and the number of affected countries has tripled. There are now more than 118,000 cases in 114 countries, and 4291 people have lost their lives. Thousands more are fighting for their lives in hospitals. In the days and weeks ahead, we expect to see the number of cases, the number of deaths, and the number of affected countries climb even higher. WHO has been assessing this outbreak of a new virus around the clock and we are deeply concerned both by the alarming levels of spread and severity, and by the alarming levels of inaction. We have therefore made the assessment that COVID19 can be characterized as a ‘Pandemic.”’ It was the very first step in the journey sailing into uncharted waters both for scientists and policy makers performing in this complicated and sensitive medical area. The area became even more demanding when an entire novel type of vaccine was introduced to fight the disease: mRNA vaccines. The idea of writing this book came as an imperative to convey basic and applied scientific knowledge in a clear and organized form contributing to the understanding of the many open issues concerning this new medical initiative intended to save millions of people’s lives. Therefore, it is not a conventional book to be read only. It is rather a book that would act as a compass for the journey undertaken by anybody concerned or interested to understand the novel groundbreaking technology. However, even though this book is written in a defining moment of human history while the whole globe is grappling with the worst threats to humankind (not only the Pandemic but also other erupting issues evolving around the world such as wars, poverty, lack of ecojustice, and climate change, just to name a few), there was a light at the end of the tunnel that brought hope and relief: vaccines.

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Vaccines that we all were expecting to see effectively fight the virus (SARS-CoV2 as the name of the new virus named by WHO on 11 February 2020) promised to save lives and restore our daily life. But these vaccines are made with a novel technology that even most of the medical doctors who are asked to administer them do not know much about. These COVID-19 mRNA gene-based vaccines that were asked to be implemented in fast-track due to the urgency of the rising death toll of the Pandemic victims have raised questions in the scientific community and beyond. And here starts the end of the thread that was picked by Dr. Siguna Mueller. Dr. Siguna Mueller, early with the onset of the Pandemic, picked up the thread of the difficult task, to unveil and explain the mode of action with all the possible effects, implementing her qualified knowledge, while she sensed the complexity of the issue. With her unique scientific background in mathematics, cryptography, and biomedical sciences, she has the capacity to understand and carefully uncover the challenges of these novel technologies and to depict them with fully referenced documentation in a quite friendly way that can easily be understood. Despite the difficulties she faced and the long hours she devoted to aligning the scientific content of this book with the state of the art to reflect on the underlying complexity of the new technology, she persisted with loyalty and managed to accomplish her task that was not only to finish it but also to construct a ladder of knowledge that can be climbed step by step leading to unbiased personal decisions of the reader going beyond the limited concept that RNA is only the traditional transporter of signals from the nucleus to the cytoplasm. This book provides a clear guide to understand the basics and the applications of this new vaccine establishment and production technology. It offers necessary complementary reading material for a wide array of (semi-)professional and interested people involved in these aspects of vaccinology. As a medical scholar and lifelong learner, I wish to thank Dr. Siguna Mueller sincerely for undertaking a Herculean task to walk the difficult path of loads of numerous published information, showing the way out through the challenges and opportunities of these mRNA vaccines in a clear and organized form. Prof. Dr. P. Nicolopoulou Stamati, MD, PhD

Preface

This book offers an objective treatment of findings in genetics, molecular biology, biochemistry, epidemiology, statistics, public health, and biosafety into the risks and opportunities of mRNA vaccines. While many have been, and are, trying to illuminate this extremely complex subject, this book essentially found me. It begged to be born. My training and experience in different disciplines have equipped me with a unique approach to analyze connections and relationships—based on logical, rational, and dialectic science. So, how did this book find me? When the SARS-CoV-2 virus first emerged, I began to go through a series of denial programs such as “It is never going to get from China to where I live” or “It does not even exist—nobody has seen it.” Very much in the same way, I opposed the first reports of vaccine development and dismissed everything as being orchestrated by pharma companies in their “crooked way to just make money.” There were times when I even thought the virus had intentionally been made in the lab by those companies and their compliances. I have gone through all sorts of crazy ideas in my mind—only to continually feel a great level of unease with all those grave accusations. The harder I blamed someone out there, the greater was my inner agony. Slowly, very slowly, another thought began to cross my mind: “What if there is nobody to accuse? What if everyone is doing the best they can, and always has been?” While the year 2020 had already divided people in their opinion over the origin of the virus, different attitudes related to the different vaccines have led to an even bigger divide—worse than anything else I have ever seen in my life. Close friends and family members became divided over the issue, with individual beliefs ranging from vaccines being our only hope to them being secret biological weapons carefully orchestrated to control the mind of people in an apocalyptic manner. But once again, “What if everyone is just doing their best, just as I am?” This book is about my investigative journey into the world of RNA vaccines, based on this very premise that they came from the best intentions of researchers and vaccine developers alike. Arguably, I have identified both challenges and opportunities. But this does not mean I want to blame anyone or judge them for intentionally doing something wrong. I am not an anti-vaxxer, nor do I want to be a stubborn antiix

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anything. When I am suggesting in the book some opportunities and areas for further research, whenever I say it would be important to do something, or that this and that “should” be done, this is not meant as a “shooting” enterprise. I aim to highlight what I have found in intense research as I have been eager to contribute to the one thing that truly unites us: we all want to be healthy and well. Nothing would excite me more if my insights turn out to be instrumental to support vaccine development programs so that future RNA vaccines can work most effectively with absolutely minimal adverse effects. Even if the information provided, or any measures prompted, by my book just helps one person, I consider my work a great success. This one person could be you or me, or my mother, father, or anyone else most near to us. Kaernten, Austria

Siguna Mueller

Acknowledgments

I would like to thank Jack Heinemann and Sarah Agapito for reading an earlier version of this book, and for their helpful feedback and encouragement. Jack, your insightful comments have been valuable pointers on how to proceed. I also very much appreciate your directing me to ENSSER (European Network of Scientists for Social and Environmental Responsibility). ENSSER has become a true home for me. Not only do I enjoy the open, critical, and yet heartfelt discussions which continue to challenge me regarding what true science really is, the small group within ENSSER that meets regularly has become my family. First and foremost, I would like to thank P. Nicolopoulou-Stamati, who first introduced the idea of writing a book. I have come to appreciate your authenticity, depth, wisdom, and skill, but more than that, I consider you a dear friend. Thank you! When during the many 80+ hour weeks I often felt like giving up, you so often pointed me to a deep stirring inside, as if this book seemingly wanted to be written. On the one hand, the realization that it concerns a sensitive topic that profoundly touches us all has been utterly humbling, paradoxically, at the same time, these pointers have helped me draw upon some deeper power and strength. I am also most grateful to Diederick Sprangers, arguably the heart of ENSSER, for his uniquely warm, skilled, and supportive way. Through your very presence, you are demonstrating again and again that it is not only about what we discuss but also how we do it. You are such a great example of how our being drastically shapes what we say and do—even in science! Thanks are also due to the other members of ENSSER that I have come to know: Ignacio Chapela—you always keep asking the most profound questions, and always encourage us to look beyond our own ways of thinking and conceptualization. Through your genuine and deep presence, you are also such a great example of how to “do science.” Giuseppe Longo—I have come to know you as one of my favorite colleagues indeed. We can communicate without needing a lot of explanations and words. It’s perhaps our common background in mathematics that allows us to have this unique common approach to the life sciences. Your way of seeing and interpreting things xi

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is always so near and dear to my heart. It just takes someone in math—and with an open and curious mind and heart like yours! Angelika Hilbeck—what a joy it always is to exchange with you! I love your wits and your humor, and yet, at the same time, your incredible gift to see through things and to cut through the most difficult situations. Ulrich Loening—in many regards, you have become a father to me. I am so thankful for your presence. You are always willing to share your vast experience and wisdom, and yet, you are such a great team player who allows us to think out loud, even if some of it may be crazy. Thomas Bøhn, you do not say too much in our group, but when you do, it is important and deep. Thank you for challenging us all, and always asking such great questions. I have come to appreciate each and every one of you, more than you may believe. For years, I never had anyone to talk to, save for my horse, until I needed to sell him. Albeit, I have to say, while I still miss “Ben’s” big ears (who were always so willing to listen), he was not so great at asking questions or providing feedback. Again, thank you for many interesting discussions, and for providing a platform for me to bounce off my ideas and concerns, in a way where I can just be myself. Without you, I would not be doing what I am doing. Many thanks to Lucas Wirl, for not only noticing that my old laptop has been causing troubles. What a surprise it was that you donated one of your laptops and even went through the trouble to install a different keyboard! Thank you for your generosity and for always being willing to help! I am also indebted to Ole Faergeman, Ricarda Steinbrecher, Amela Skiljan, Sanjay Kumar, June Rebekka Bresson, and Brian Wynne for your kind words and encouragement these last 2 years. I would also like to thank M.M. for your very conscientious proofreading and getting the job done so quickly. The specific topics covered in the book have to a large part been guided by the insightful feedback received by Springer. The many skillful and relevant pointers are much appreciated. Many thanks to the reviewers for challenging me with important questions and for pointing me to relevant literature and topics that still needed to be covered. The book has been greatly enriched by this. I also very much appreciate the great work done by Springer in terms of type-setting and book-cover design. Countless people across the globe have sent good thoughts and prayers. Many of you are from different religious and spiritual orientations, and yet, I felt the united support from you all. How can I express my gratitude to the inner promptings that I received so often as if I was guided by some higher power to the right question, material, and source. Last but not least, thanks are due to God and all Heavenly Beings, the Universe, the Source of all creativity, wisdom, power, generosity, and love. I have often been humbled by the task, knowing the division this topic has brought, along with so much agony, strife, hostility, and pain. In this sense, to powers and forces higher than myself, I return this work, so that it may be a blessing to all!

About the Author

Brief Bio The author spent the first 20 years of her academic career in mathematics (MS and Ph.D., University of Klagenfurt, Austria, both summa cum laude). She was among the first women in Austria to receive habilitation in discrete mathematics and cryptography (2002). The following 15 years she spent as a research associate at the Centre for Information Security and Cryptography Research, University of Calgary, and as an assistant professor at the University of Wyoming. A lifelong passion for biology and medicine eventually triggered her decision to radically expand her field of expertise. Consequently, she earned her second Ph.D. (biomedical sciences) in 2014 at the University of Wyoming (with a GPA of 4.0). Since then, she has been utilizing her experience in the theoretical fields to help advance a broader appreciation and comprehension of phenomena and challenges arising in the biological and life science domains. For almost 10 years, she has been warning of new biosafety and public health dangers, but for years was ignored. Only recently, her concerns about new safety and new security challenges in synthetic biology have been supported by others, and she has become one of the leaders in the new field of cyberbiosecurity. Her interests and curiosities are not limited to those fields as she finds great excitement in physics, theology, philosophy, ethics, spirituality, and the complexity and fascination of nature altogether. As such, it has been natural for her, employing her multi-disciplinary experience, to appraise the new Covid-19 vaccines, through rigorous, balanced, and scientific logic.

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Great hope is being placed in mass vaccination efforts to end once and for all the ongoing Covid-19 pandemic. Due to the scale and urgency of the situation, difficult decisions needed to be made regarding the choice of the most promising vaccines. While under normal circumstances, development and safety trials of vaccines take 10–15 years, Operation Warp Speed policies have enabled the incredibly fast approval of new vaccines to target the pandemic. Two of the first candidates that received emergency approval are mRNA vaccines which rely upon different technologies than those based on known vaccine formats. mRNA vaccines are believed to offer unique benefits as follows: • They are faster and cheaper to produce than traditional ones. • Given they are not produced using infectious elements, they are believed to be safer for the patient. • RNA is not expected to integrate itself into the host genome. • The RNA strand in the vaccine is expected to be degraded once the protein is made. • The synthesized complexes are believed to act as adjuvants themselves, triggering desirable immune responses. The first part of the book is especially devoted to these foundational cornerstones, via a clear and detailed appraisal of their rationale and scientific underpinnings, based on evidence from post-inoculation experience. Later parts of the book take a deeper dive into the vast pool of information that has been accumulating since the mass vaccine rollout, all the way up to the Omicron variants. Public acceptance of Covid-19 vaccines is rather low. Fear about unknown or undesirable effects of those vaccines which lack large-scale human trials increased as soon as the first recipients of the jab experienced severe allergic reactions and other severe adverse events. Data released by the Centers for Disease Control and Prevention (CDC) on the number of injuries and deaths reported to the Vaccine Adverse Event Reporting System (VAERS) following Covid-19 vaccines show a total of 1,287,595 injuries, including 28,532 deaths and 235,041 serious injuries, between December 14, 2020, and May 27, 2022. xv

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Thus, by May 2022, the total number of Covid-19 vaccine adverse events reported to VAERS far exceeds the overall total number (930,952) of adverse events reported in the 32-year history of this database, overwhelming various vaccine injury compensation programs. By way of comparison, in the USA, a total of 21 deaths were reported during the 2020–2021 flu season after more than 180 million flu vaccines [1]. On May 18, 2021, the official estimate for Covid-19 vaccines given by US health officials [2] was 1.7 deaths per 100,000. This is about 1.7 times that of the fatality rate that Pfizer reported during the first 3 months following authorization of its Covid-19 vaccine. In a series of court-released documents (Chap. 8), Pfizer observed 42,086 reports of adverse events containing 158,893 adverse events, including 1223 fatal outcomes, among the 126,212,580 doses of BNT162b2 which had been shipped worldwide. This means a rate of reported adverse events of 1 in 1000 doses distributed (with many of them graded as serious and only a small fraction of events reported as resolved), and a fatality rate of almost 1 in every 100,000 doses, for the time between December 1, 2020, and February 28, 2021. Reporting of vaccine injuries and deaths is a controversial issue, not just because cases are difficult to interpret (Chap. 5). There is a huge contrast between surveillance and reporting itself. For example, in their weekly report covering adverse reactions to approved Covid-19 vaccines, the UK listed a total of 374 fatal outcomes for Pfizer, 786 for AstraZeneca, and an additional 4 for Moderna, until May 13, 2021 [3]. As of May 13, 2021, about 36 million people in the UK had received at least one inoculation with either of the Covid-19 vaccines [4]; therefore, the post-vaccine death estimate, in this case, would be 1 in 31,000. A clear attribution to the individual Covid-19 vaccines in this case is difficult, as vaccination status in the UK does not automatically link the type of vaccines used. Obviously, estimating the risks and benefits of a vaccine is difficult, amounting to more than just estimating fatality rates. Although every “case” or “fatal outcome” is a person—someone’s parent, child, or spouse—clear attribution to vaccines can be difficult to make. Numbers assigning associations of long-term sequelae or even death are ordinarily incomplete. Unexpectedly, the same applies even to Covid-19 death tolls. In May 2022, scientists working with the World Health Organization (WHO) corrected some errors in its estimates of how many deaths the pandemic has caused.1 Surprisingly, this revealed that, e.g., Germany’s pandemic-related deaths estimate had been over-counted by 37%; on the other hand, the WHO argues that Sweden should raise their estimate by 19%. Rather than fighting over numbers, blaming, and accusation, this book carefully analyzes the relevant models and predictions used for assessing the safety and efficacy of the new vaccines, but it also highlights the challenges of scientific

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https://www.nature.com/articles/d41586-022-01526-0?utm_source=Nature+Briefing& utm_campaign=1436525283-briefing-dy-20220601&utm_medium=email&utm_term= 0_c9dfd39373-1436525283-45026129.

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reporting and surveillance and the problem of computing risk-benefit ratios at both the individual and population level. In its appraisal of the risks and opportunities of mRNA vaccines, this book develops and offers a critical, objective, rational, and independent approach, enabled by findings and insights in genetics, molecular biology, epidemiology, statistics, public health, biosafety, modeling in the life sciences, and ethics, supported by an extensive review of published literature. The approach taken is based on inquiry, analysis, interpretation, scientific logic, and deduction. This way, the many individual topics are taken up by this book, converging from different disciplines, increasing our ability to target the pandemic with its unprecedented scale and complexity. Despite the urgency and complexity of the topic, the chain of causation of the individual findings and evidence gathered as fostered by a multidisciplinary approach attains a high level of confidence in the whole, of the fascinating interrelationships between humans, pathogens, technical innovation, and health. Several previously unassessed and non-obvious assumptions regarding RNA vaccine safety, which are implicitly relied upon, and which lead to questions that previously do not seem to have been clearly asked, are identified and analyzed. Focusing on what can be gleaned from biological modeling, in-vitro experiments, various RNA technologies, and their biochemistries, and recent findings related to regulatory RNAs, retro-integration, (epi)genetic inheritance, and the dichotomous nature of the self-adjuvant effect, several opportunities and areas of improvement are identified. Much focus is placed on the postulated modus operandi of mRNA vaccines, in terms of its level of predictability and completeness. This includes the anticipated fate of the generated complexes, from their synthetic processing to their fate in human cells. This leads to various questions regarding the interplay between humoral and cellular immunity, the influence of the cellular environment such as inflammation, and interrelationships with various innate immune processes. Numerous previously unexplored processes and interrelationships are considered, including additional players such as vaccine RNAs and their potential to interfere with human micro RNA regulation. Much focus is also placed on immunity beyond mere antibody responses, as well as pharmaceutical aspects of the mRNA-LNP complex. In particular, Part II of the book is devoted to, among others, analyzing the most central question: where, when, and for how much time vaccine-induced production of the spike protein occurs, and to what extent it may affect anticipated and unanticipated (adverse) vaccine effects. In terms of individual patient responses, a huge range of potential side effects is critically analyzed, ranging from those triggered by the dichotomous nature of the self-adjuvant response of mRNA vaccines and those evoked by excessive Type-1 IFN signaling to various possible forms of disease enhancements. An entire chapter is devoted to the spike protein itself and its postulated role, and potential gaps in its anticipated action, both in Covid-19 and induced by the injections. Many of the relevant epidemiological issues go beyond mRNA vaccines alone and are included for their importance. The difficulty of testing, modeling, statis-

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tical evaluation, relevant efficacy measures, and predictability of viral escape are described in detail. Overall, this book offers a comprehensive overview of mRNA vaccines that addresses the most important issues known so far but also highlights additional challenges and questions that to date have not received a lot of attention. While some of the content may be technical, the most difficult to comprehend material is usually summarized in specially designed “Boxes.” In addition to the text section itself, 60 figures have been added to summarize and depict the most important findings in an even less technical way. Again, it needs to be stressed that while the topic is complex, it is treated in a rational and unbiased way via the most rigorous scientific logic. Much emphasis is also placed on how risks and benefits are evaluated, and the most critical component that is missing—the use of technology, and how it scales both benefits and harm. Inspired by the very promising approach by Heinemann et al. [5] for risk assessment in the context of agricultural gene technologies, this book extends this new methodology to mRNA vaccines. As in [5], it is recognized that any potential harmful outcomes of a new technology may increase when it is used more. This not only applies to agricultural gene technologies but mRNA vaccines as well. Therefore, what can make new technologies useful is also what makes them risky. The regulation of agricultural gene technology and mRNA technology have both led to numerous discussions on how their risk/benefit assessment ought to be done best. As for the former, mRNA technologies are not any safer when used more. Surprisingly, there are many implicit scaling characteristics that shape both technologies. Albeit “scale” is not merely a measure of time, cost, and the like. Indeed, “scaling” the discussion of safety assessment provides a strong basis to identify where risks and benefits scale differentially. Identifying those transitions not only helps distill where and how the risk of harm grows. These critical control points also help inform both risk assessment and risk mitigation. The technology risk equation is more complex than counting deaths or opinions of lives saved. As will be clearly laid out, bringing the human factor to the center of the discussion is most urgently needed, even in terms of how the risks and benefits of the vaccines are evaluated. This will impact not only each patient but humanity at large. The biochemical and epidemiological aspects of mRNA vaccines developed in the first part of the book are critically important for their scientific understanding. But these alone cannot curb the pandemic. It is suggested, however, that the framework of the critical control points developed in the second part can help bridge the gap between science and the actual use of the vaccines in order to target the most effective, precautionary outcomes. Also included in the book are challenging issues involving attribution, diagnostics, reporting, transience of vaccine immunity, and causality assessment. An entire chapter deals with the WHO-AEFI criteria, which are a detailed set of rules to assess when a vaccine caused an adverse event, how (and if) these criteria apply to mRNA vaccines, and how this played out during the pandemic.

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Later chapters in particular analyze which outcomes have been formally measured in trials and studies (and over how much time), which outcomes have been inferred, and what is known so far about the (in)effectiveness of vaccines to prevent SARS-CoV-2 infection, severe disease, and death. The book concludes with questions that point beyond common models rooted in the Central Dogma and amino acid–based determinants to assess viral evolution and postulates what this means in the context of mass vaccination and induction of escape mutants. It is hoped that the findings described here will help guide the safe and responsible use of Covid-19 vaccines and future vaccines built on the same mRNA technology. Overall, the ultimate hope is to demonstrate how curiosity, openmindedness, and critical thinking are the most powerful tools to most competently advance science, technology, medicine, and health.

References 1. Centers for Disease Control and Prevention (n.d.) The vaccine adverse event reporting system (vaers). https://vaers.hhs.gov/data.html 2. The Centers for Disease Control and Prevention (CDC) (2021) Selected adverse events reported after covid-19 vaccination. https://www.cdc.gov/coronavirus/ 2019-ncov/vaccines/safety/adverse-events.html 3. Medicines and Healthcare products Regulatory Agency (n.d.) Coronavirus (covid-19) vaccine adverse reactions - a weekly report covering adverse reactions to approved covid-19 vaccines. https://www.gov.uk/government/publications/ coronavirus-covid-19-vaccine-adverse-reactions 4. Ritchie H, Ortiz-Ospina E, Beltekian D, Mathieu E, Hasell J, Macdonald B, Giattino C, Appel C, Rodes-Guirao L, Roser M (2020) Coronavirus pandemic (covid-19). Our World in Data. https://ourworldindata.org/coronavirus 5. Heinemann JA, Paull DJ, Walker S, Kurenbach B (2021) Differentiated impacts of human interventions on nature: Scaling the conversation on regulation of gene technologies. Elementa Sci Anthropocene 9(1):00086

Contents

1

Introduction .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .

Part I

2

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Scientific Underpinnings, Early Expectations, and Emerging Challenges from the Global Inoculation Experience

Appraisal of Some of the Key Postulates Underlying mRNA Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 2.1 Potential Uncertainties of the Model . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 2.1.1 The Synthesized RNA Molecules May Not Play the Role of Messenger RNAs . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 2.1.2 Maximizing the Expression of the Immunogenic Gene from a Network Perspective . . . . .. . . . . . . . . . . . . . . . . . . . 2.1.3 The Role of Metabolism.. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 2.1.4 The Spike Is a Functional Protein Whose Immunogenicity Is Determined and Targeted by Various Non-orthodox Modification Strategies . . . . . . . . . . . 2.2 The Assumption “The IVT mRNA’s self-adjuvant property is beneficial” .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 2.2.1 Harmful dsRNAs, as First Known from the in Vitro Synthesis of the mRNAs . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 2.2.2 DsRNAs Play a Key Role in the Dichotomous Self-Adjuvant Effect of mRNA . . . . . . . .. . . . . . . . . . . . . . . . . . . . 2.2.3 The mRNA Vaccine Effect of Type I IFNs Modulation on CD8+ T Cell Immunity Is a Double-Edged Sword .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 2.3 The Assumption “Vaccine mRNAs do not integrate into the human genome” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 2.3.1 Implicit Assumptions Regarding Their Non-integrative and Non-mutagenic Nature . . . . . . . . . . . . . .

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2.3.2 Reverse-Transcriptase Activity in Human Cells . . . . . . . . . . The Assumption “IVT mRNA is not mutagenic”.. . . . . . . . . . . . . . . . . . 2.4.1 DsRNAs Are Mediators of RNAi Pathways and Potential Mutagens . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 2.5 The Assumption That “vaccine mRNAs immediately get degraded” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 2.6 The Assumption “Any excessive immune-stimulatory activity can be eliminated from IVT processing” .. . . . . . . . . . . . . . . . . . 2.6.1 The Concern That Undesirable Immune Processes Get Triggered in Vivo . . . . . . .. . . . . . . . . . . . . . . . . . . . 2.6.2 dsRNA Production in Vivo from Synthetic mRNAs . . . . . . 2.6.3 Vaccines with Self- and Trans-Amplifying mRNAs . . . . . . References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .

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Relevance for mRNA Vaccine Safety . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 3.1 Detrimental Consequences of Type I IFN Stimulation and New Disease Patterns .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 3.1.1 Dichotomous Immunogenicity and Cytotoxicity of Vaccine RNAs and Byproducts . . . . .. . . . . . . . . . . . . . . . . . . . 3.1.2 Autoimmune Conditions and New Pathologies . . . . . . . . . . . 3.2 Cross-Reactivities, Vaccine Self-Adjuvancy, and Adverse Immune Responses .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 3.2.1 mRNA Vaccines as Potential Agents in the Initiation of AI, or as Triggers of AI Flares . . . . . . . . . . . . . . . 3.2.2 Immune-Mediated Hepatitis Following mRNA Inoculation and Type III Hypersensitivity Reactions . . . . . 3.2.3 The Potential of Immunopathologic Th2 Responses.. . . . . 3.3 Shooting the Messenger of Critical Human Proteins . . . . . . . . . . . . . . . 3.3.1 Impairment of the Adaptive Immune System . . . . . . . . . . . . . 3.3.2 Shooting the Messenger of Tumor-Suppressing Proteins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 3.4 Contaminants of IVT Processing . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 3.5 Dichotomous Immune Response and Attribution of Adverse Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 3.6 The Real Potential of Creating Genetically Modified Humans . . . . 3.6.1 DsRNAs and Their Role in Mutagenesis .. . . . . . . . . . . . . . . . . 3.6.2 Pathogenic Endogenous RT Activity in Human Cells via Transposable Elements.. . . . . .. . . . . . . . . . . . . . . . . . . . 3.6.3 Retro-Integration of IVT RNAs May Be Triggered by Several Mechanisms .. . . .. . . . . . . . . . . . . . . . . . . . 3.6.4 Genomic Integration and False PCR Tests Following Vaccination .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 3.6.5 Relevance for Clinical Trials and Antiviral Therapies .. . . 3.6.6 Ribonucleotides as Harmful DNA Lesions .. . . . . . . . . . . . . . .

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Impact of Truncated IVT mRNA Species and Other Short RNAs Derived from RNA Vaccines . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 3.7.1 Interfering with Endogenous RNAi Processes and miRNA-Regulated Gene Expression .. . . . . . . . . . . . . . . . . 3.7.2 Interfering with the Balance Between IFN and RNAi-Based Antiviral Defense Mechanisms in Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 3.7.3 Disrupting Other Activities of Human miRNAs at the Cellular Level . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 3.7.4 Disrupting Circulating/Extracellular miRNAs . . . . . . . . . . . . 3.8 External RNAs as miRNA Activity Modulators .. . . . . . . . . . . . . . . . . . . References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .

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4

From Challenges to Opportunities and Open Questions .. . . . . . . . . . . . . . 4.1 Differentiating Whether Adverse Reactions Are Geared Against the Lipid Nanoparticles or Against Vaccine RNAs and Their Byproducts .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 4.2 The Need for Clear Attribution .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 4.3 Guarding Against Cross-Reactivities and Aberrant Immune Responses .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 4.4 Guarding Against Other Medium and Longer-Term Side Effects.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 4.5 The Opposing Role of mRNA Vaccine-Induced Type I IFN Signaling in the Regulation of T Cell Immunity.. . . . . . . . . . . . . . 4.6 Discerning the Function and Impact of Vaccine-Derived Regulatory RNAs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 4.6.1 The Interplay Between IFN Responses and RNAi Mechanisms in Self- and Non-self Recognition . . . 4.6.2 Mechanisms and Effects of Externally Derived Regulatory RNAs . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 4.6.3 Off-Target Effects, Effects on the Human Microbiota, and the Larger Environment .. . . . . . . . . . . . . . . . . 4.6.4 Small Vaccine-Derived RNAs in the Extracellular Space . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 4.7 Can Vaccine RNAs Act as Micro RNA Activity Modulators? .. . . . 4.8 The Spike During Infection, as Opposed to the Spike Expressed via a Vaccine.. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 4.8.1 The Spike Itself as a Driver of Severe Disease . . . . . . . . . . . . 4.8.2 Fate of the Vaccine Induced Spike Unclear Even at the Beginning of the Global Vaccination Campaign . . . 4.8.3 The Spike and Potential Analogs of Virally Mediated AI. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 4.9 When the Vaccine Just “Does Not Work” . . . . . . .. . . . . . . . . . . . . . . . . . . . 4.10 The Need for a Clear Understanding of How mRNA Vaccines Affect Reproductive Health . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .

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87 89 91 93 94 95 95 98 99 101 102 105 105 106 110 111 114

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4.10.1 Pregnancy Safety Studies of mRNA Covid-19 Vaccines .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 4.10.2 Incompletely Understood Mechanisms and Impact of Antibody Transfer to Infants .. . . . . . . . . . . . . . . . . . . 4.10.3 Sperm Parameters Before and After mRNA Inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 4.11 Mutagenic Risks Impacting Future Generations .. . . . . . . . . . . . . . . . . . . 4.12 RNA Vaccines Need to Be Classified as Gene Therapies, with Corresponding Testing, Surveillance, and Long-Term Follow-up Practices . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 4.12.1 RNA Vaccines Do Satisfy the Criteria of the FDA to Be Classified as GT Products .. . . . . . . . . . . . . . . . . . . . 4.12.2 Persistence, Resistance, and Viral Escape .. . . . . . . . . . . . . . . . 4.12.3 All GT Products, Including mRNA Vaccines, Require Long-Term Follow-up Studies .. . . . . . . . . . . . . . . . . . . References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 5

6

The Challenge of Evaluating Vaccine Safety and Effectiveness . . . . . . . 5.1 The Problem of Adequate and Unified Testing and Surveillance, and the Utilization of Statistics in the Biological Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 5.2 The Challenge of Unified and Transparent Reporting of Adverse Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 5.3 The Need for Unbiased Scientific Reporting and Interpretation of Vaccine Safety and Effectiveness .. . . . . . . . . . . . . . . . 5.3.1 Variations Among the Reported Case Numbers.. . . . . . . . . . 5.3.2 Global Patterns, as Opposed to Those at a Smaller Scale . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 5.4 The Problem of Computing Vaccine Effectiveness . . . . . . . . . . . . . . . . . 5.4.1 Dying from or with . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 5.4.2 Lack of a Single Cause in Population-Wide Infection Dynamics .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 5.4.3 The Problem of Assessing Covid-19 Vaccine Effectiveness, During the Delta Time and Beyond.. . . . . . . 5.4.4 Statistical Models and their Interpretations . . . . . . . . . . . . . . . 5.4.5 Other Statistical Measures and Complications .. . . . . . . . . . . References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . Safeguarding Against the Analog of Antimicrobial-Resistance Development .. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 6.1 Resistance Development to SARS-CoV-2 Vaccines .. . . . . . . . . . . . . . . 6.1.1 Breakthroughs Involving the SARS-CoV-2 Variants B.1.1.7 and B.1.351 .. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 6.1.2 Breakthroughs Involving the SARS-CoV-2 Variant B.1.617 . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 6.1.3 Further Lab Experiments .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .

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6.1.4 Breakthroughs After First and Second Doses . . . . . . . . . . . . . Risks of Common Public Antibody Responses .. . . . . . . . . . . . . . . . . . . . 6.2.1 Non-traditional Antibodies .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 6.2.2 Selection by Neutralizing Antibodies May be a Key Driver to Induce a Viral Escape Mutant .. . . . . . . . . . . . . References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .

169 171 171

Scales, Pseudoscales, the Human Factor, and a Way Forward . . . . . . . . 7.1 Scale Relationships and Human Activities . . . . . .. . . . . . . . . . . . . . . . . . . . 7.2 Pseudoscales as Problems.. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 7.2.1 The Pseudoscale PCR Positiveness .. . .. . . . . . . . . . . . . . . . . . . . 7.2.2 “Naturalness” of Vaccine-Induced Immune Response.. . . 7.2.3 The Pseudoscale Preventiveness of Disease and Death .. . 7.2.4 The Pseudoscale “Predictiveness of Sequence Differences and Determinants” . . . . . . . .. . . . . . . . . . . . . . . . . . . . 7.2.5 The Pseudoscale “Antibody-Titer” . . . .. . . . . . . . . . . . . . . . . . . . 7.2.6 Natural Immunity Versus Vaccine-Induced Immunity .. . . 7.2.7 Similar Viral Load Among the Vaccinated and Unvaccinated .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 7.3 Critical Control Points via Genuine Scaling Features . . . . . . . . . . . . . . 7.3.1 Contamination and Quality Control Are Distinctive Scaling Concerns . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 7.3.2 Negative Scaling of Antibody Diversity and Flexibility Exerted by Vaccination .. . . .. . . . . . . . . . . . . . . . . . . . 7.3.3 “Sky-High” Antibody Levels: A Vaccine Induced Scaling Feature with Potentials for Adverse Effects . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 7.3.4 Scaling of Antibody-Driven Selection Pressure Leading to the Emergence of Viral Variants in Immune-Compromised Individuals .. . .. . . . . . . . . . . . . . . . . . . . 7.3.5 Scaling of Antibody-Driven Selection Pressure Leading to the Emergence of New VOCs In Vitro .. . . . . . . 7.3.6 Epidemiological Findings: Increased Dominance of VOCs Scales with the Increased Rate of Mass Vaccination Across Nations .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 7.3.7 Boosters and Scaling of Disease Severity and Adverse Events . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 7.3.8 “Vaccines” Themselves, as Newly Defined by the CDC, as the Main Scale Driver . . . .. . . . . . . . . . . . . . . . . . . . 7.3.9 Scaling of Human Intervention: When Apparent Benefits Escalate Risks Instead . . . . . . . .. . . . . . . . . . . . . . . . . . . . 7.4 Ethical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 7.5 Critical Control Points Allow for Optimal Risk Mitigation .. . . . . . . References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .

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Part II 8

A Deeper Dive into mRNA Vaccine Safety and Security, and Developments Until Delta

mRNA Vaccine Safety and Efficacy—Official Criteria When AEs Are Caused by the Injection . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.1 Death and Other Adverse Events Following Immunization: The Three Basic Levels of the WHO Manual.. . . . . 8.1.1 Evaluation of AEFIs in the Context of mRNA Vaccines—The Population Level . . . . . .. . . . . . . . . . . . . . . . . . . . 8.1.2 AEFIs in the Context of mRNA Vaccines—The Individual Level . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.2 Individual Criteria in the WHO-AEFI Manual in Regard to mRNA Vaccines—A Closer Look .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.2.1 The Concept of Biological Plausibility Does Not Extend to mRNA Vaccines .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.2.2 Is the Event Classifiable? . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.2.3 Causality in the WHO Manual Requires No Other Factors Involved . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.3 The Traditional vs. the Modified Definition of a Vaccine: From the Perspective of Causation . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.3.1 “Covid-19 Vaccines Prevent Infection and Transmission” as a Causal Relationship .. . . . . . . . . . . . . . . . . . 8.3.2 A Moving Endpoint—Goal of the Vaccine .. . . . . . . . . . . . . . . 8.3.3 The Moving Target as to Who Counts as Vaccinated . . . . . 8.4 Lessons Learned from the Pandemic—The WHO-AEFI Criteria Revisited .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.4.1 Covid-19 Vaccines as Single Causative Agents .. . . . . . . . . . 8.4.2 Impact on WHO-AEFI Criteria to Determine AEFIs .. . . . 8.4.3 How Signals Are Being Identified, Measured, and Interpreted .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.4.4 Optimism, Bias, and Disparate Interpretations .. . . . . . . . . . . 8.4.5 Comparing a Subtle Outcome and Computer Decisions .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.5 The Investigation of Signals—Assessing Causation Without Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.5.1 Data Not Getting Published . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.5.2 Another Aspect of Underreporting . . . .. . . . . . . . . . . . . . . . . . . . 8.6 Criteria for Assessing Causation—Too Ambiguous vs. Too Strict . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.6.1 Notions and Criteria to Assess Vaccine Safety Are Too Ambiguous .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8.6.2 Criteria Are Too Strict—“No causation shown” Does Not Mean There Is No Causation . . . . . . . . . . . . . . . . . . . References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .

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9

mRNA Covid-19 Vaccines Best Reflect Effective Pharmaceuticals—Basic Considerations and LNPs . . . . . . . . . . . . . . . . . . . . 9.1 Concerns About mRNA “Vaccines,” Regarding Their Potentials as Vaccines .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 9.1.1 Do mRNA Injections Resemble Actual “Vaccines”? . . . . . 9.1.2 Do mRNA Inoculations Act Like Therapeutics? .. . . . . . . . . 9.1.3 No Clearly Specified end of mRNA-LNP Activity, Amplifying Cell Damage and Adverse Reactions After Each Inoculation .. . . . .. . . . . . . . . . . . . . . . . . . . 9.2 Tissue Tropism of the LNP “Delivery System” .. . . . . . . . . . . . . . . . . . . . 9.2.1 Selective Versus Unselective Tissue Tropism . . . . . . . . . . . . . 9.2.2 The Leaked Japanese Study .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 9.2.3 Potential Consequences . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 9.3 Findings from Related Vaccines .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 9.3.1 Biodistribution of Moderna’s 2017 Flu “Vaccine” Candidate.. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 9.3.2 Moderna’s 2022 Flu Vaccine Candidate .. . . . . . . . . . . . . . . . . . 9.3.3 A Common Denominator . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 9.4 Lipid NPs as Active Compounds . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 9.4.1 LNPs Used for Preclinical Studies Are Highly Inflammatory and May Be Key Drivers of the Antibody Response .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 9.4.2 Mechanism of the LNPs as Active Compounds Are Poorly Understood .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 9.4.3 Polyethylene Glycol (PEG) and More .. . . . . . . . . . . . . . . . . . . . References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .

10 mRNA Covid-19 Pharmaceuticals and the Spike Antigen . . . . . . . . . . . . . 10.1 Design Criteria of the Spike . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 10.1.1 Basic Assumptions and Open Questions . . . . . . . . . . . . . . . . . . 10.1.2 Toxicity of the Spike in Covid-19 Disease and When Produced Upon Vaccination . . . .. . . . . . . . . . . . . . . . . . . . 10.2 Biodistribution and Persistence of the Vaccine-Induced Spike . . . . 10.2.1 Spike Protein Fragments and Entire Spike Protein Detected in the Plasma of mRNA-1273 Vaccine Recipients. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 10.2.2 Spike Protein Induced by BNT162b2 Found on Exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 10.2.3 Vaccine Spike and mRNA Found to Persist in Lymph Node Germinal Centers and Confirmed in the Blood of Vaccinated Individuals . . . . . . . . . . . . . . . . . . . . 10.2.4 More Evidence That the Vaccine mRNA Is Not Degrading But Continues to Produce Protein . . . . . . . . . . . . .

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10.3 Vaccine-Derived Products Are Not Limited to the Cytosol But Found in the Nucleus. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 10.3.1 Reverse-Transcriptase Activity Following SARS-CoV-2 Infection or Injection . . .. . . . . . . . . . . . . . . . . . . . 10.3.2 In vitro, Pfizer Vaccine mRNA Becomes DNA in Liver Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 10.3.3 Spike Protein Goes to the Nucleus and Impairs DNA Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 10.3.4 Controversies or Incomplete Model? . .. . . . . . . . . . . . . . . . . . . . References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 11 Other Facets of SARS-CoV-2 Immunity, the Risk of Immune Tolerance and T Cell Exhaustion . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 11.1 Sars-CoV-2 Immunity Beyond B Cell Protection .. . . . . . . . . . . . . . . . . . 11.1.1 Beyond the Notion of Antibodies as Necessary and Sufficient Agents .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 11.1.2 Beyond Antibodies: The Impact of Vaccination on Innate and Adaptive Immunity . . . . .. . . . . . . . . . . . . . . . . . . . 11.2 Covid-19 as a Mucosal Disease and the Risk of Vaccine-Induced Tolerance . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 11.2.1 Mucosal Immune Tolerance, a Foundational Pillar of the Mucosal Immune System.. . . . . . . . . . . . . . . . . . . . 11.2.2 Covid-19 as a Two-Part Disease and mRNA Injections from a Mucosal Perspective . . . . . . . . . . . . . . . . . . . . 11.2.3 mRNA Injections May Be Subject to Suppressor Functions of the Mucosal Immune System .. . . . . . . . . . . . . . . 11.2.4 Is the Suppressive Effect of Mucosal Immunity Responsible for the Decline of mRNA Vaccine Efficacy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 11.2.5 The Opposite: Boosters Evoking Hyperinflammatory Immune Responses.. . . . . . . . . . . . . . . . . . 11.3 T Cell Exhaustion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 11.3.1 The EMA Concern of Boosters Potentially Weakening the Immune System . . . . . . .. . . . . . . . . . . . . . . . . . . . 11.3.2 Plausibility of the Concern .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 11.4 Signals of Adverse Immunological Reactions . .. . . . . . . . . . . . . . . . . . . . 11.4.1 Large-Scale Study to Assess the Fourth Dose of BNT162b2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 11.4.2 A Deeper Analysis of the Study Outcome: A Signal of an Adverse Immune Effect? .. . . . . . . . . . . . . . . . . . . . 11.5 The Impact of Vaccination During the Pandemic: Declining VE, No VE, or Negative Effects? . . . .. . . . . . . . . . . . . . . . . . . . 11.5.1 Immune System Habituation and More . . . . . . . . . . . . . . . . . . . 11.5.2 No Effect or a Negative Effect? .. . . . . . .. . . . . . . . . . . . . . . . . . . . 11.5.3 Possible Scenarios of Vaccine Protection and Decline . . .

311 311 312 317 321 322 325 325 326 327 331 331 334 336

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11.5.4 The Main Surrogates of Vaccine Protection Proved Inappropriate . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 358 References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 361 Part III

The Omicron Variants

12 Omicron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.1 Indicators of an Essentially New Virus.. . . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.1.1 High Degree of Escape from Previous Protection . . . . . . . . 12.1.2 A Drastic Increase in Infectivity . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.1.3 Overall, Omicron is Causing Less Severe Disease Compared to Previous SARS-CoV-2 Variants . . . 12.2 Why Is Omicron so Much More Infectious? .. . .. . . . . . . . . . . . . . . . . . . . 12.3 Decline in Vaccine-Induced Immunity Intensifies During the Omicron Wave(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.3.1 Overall Declining VE Data . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.3.2 For Children, VE Shows an Immediate and Radical Decline .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.3.3 Recognition that Next-Generation Vaccines are Needed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.3.4 Accumulating Evidence of Negative VE Against Omicron .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.4 Evidence of Increased Vaccine Mismatch. . . . . . .. . . . . . . . . . . . . . . . . . . . 12.4.1 Neutralization of Omicron Compared to Other VOCs, up to 3 Doses . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.4.2 Neutralization of Omicron Compared to Other VOCs: 3 and 4 Doses . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.4.3 A Synopsis of Factors that Seem to Drive Viral Neutralization Resistance . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.5 “Number of Doses Administered” as a Scaling Feature .. . . . . . . . . . . 12.5.1 Secondary Attack Rate (SAR) Differences Between Delta and Omicron in Denmark Point to the Number of Doses as an Important Scale Driver .. . . 12.5.2 A Vaccine-Dose-Dependent Rise in Omicron Infections Also Seen from a Large Study from California . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.5.3 The Number of Shots: Likely the Main Scale Driver on Both the Viral and the Host Side . . . . . . . . . . . . . . . 12.6 Omicron, as an Escape Mutant, Can Use a New Way to Enter Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.7 Emerging Trends and Open Questions . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 12.7.1 IgG Bias, High Ab Levels, and Immune Imprinting .. . . . . 12.7.2 Omicron-Specific Boosters and Immune Priming . . . . . . . . 12.7.3 The Origin of Omicron, and Why It Matters .. . . . . . . . . . . . .

367 367 367 369 370 374 376 376 377 379 381 384 385 386 387 387

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12.7.4 Future Variants: A Guaranteed Trajectory of Common-Cold CVs? . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 408 References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 410 13 Conclusion .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 415 References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 437

Abbreviations

Ab Ag ACE2 AI ADE AEFI APC ARR BNT162b2 CDC CI Covid-19, aka COVID-19 DAE dNTPs dsRNA EMA EC endo exo FDA GT HIV IFN-1 IgA IgG ISG IVT LINEs LTFU LNP mAb

Antibody Antigen Angiotensin converting enzyme 2 Autoimmunity Antibody-dependent enhancement Adverse events following immunization Antigen-presenting cell Absolute risk reduction The Pfizer-BioNTech COVID-19 Vaccines Centers for Disease Control and Prevention Confidence interval Coronavirus disease 2019 Delayed adverse event Deoxynucleoside triphosphates, Double-stranded RNA European Medicines Agency Eextra-cellular Endogenous Exogenous U.S. Food and Drug Administration Gene-therapy Human immunodeficiency viruses Type I interferons Immunoglobulin A Immunoglobulin G Cytotoxic antiviral IFN-stimulated genes In vitro transcription Long interspersed nuclear elements Long-term follow-up Lipid nanoparticles Monoclonal antibody xxxi

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MDA5 mRNA-1273 miRNA MRE NELF NNH NNT NNV ORF ONS PCR PRNT50 RBD RRR RIG-1 RISC RdRP RNAi rNTPs RT RT-PCR SARS-CoV-2, aka Sars-CoV-2 siRNA SIgA SLE SNP Th cells T7 pol UKHSA URT VAERS VE VOC VSV WHO

Abbreviations

Melanoma differentiation-associated protein 5 The Moderna COVID-19 (mRNA-1273) vaccine Micro RNA miRNA response elements Nasal epithelial lining fluid Number needed to harm Number needed to treat Number needed to vaccinate Open reading frame The UK’s Office for National Statistics Polymerase chain reaction Plaque reduction half-maximal neutralization Receptor binding domain Relative risk reduction Retinoic acid-inducible gene I RNA-induced silencing complex RNA-dependent RNA polymerase RNA interference Ribonucleoside triphosphates Reverse transcriptase Reverse transcription polymerase chain reaction Severe acute respiratory syndrome coronavirus 2 Short-inhibitory RNA Secretory IgA Systemic lupus erythematosus Single nucleotide polymorphisms T helper cells, are a type of T cell that play an important role in the immune system T7 RNA Polymerase The UK Health Security Agency Upper respiratory tract Vaccine Adverse Event Reporting System Vaccine efficacy (Viral) variant of concern Vesicular stomatitis virus World Health Organization

Chapter 1

Introduction

Continued improvements in numerous vaccine technologies have led to their increased potential to help target various public health challenges related to many different conditions and socioeconomic environments. Ever since the beginning of the unprecedented Covid-19 pandemic, there has been great hope in RNA vaccines. The realization of this new vaccine platform has been made possible by the fact that mRNA can now be synthetically produced through a cell-free enzymatic transcription reaction. Yet, just because it is possible to produce RNAs, this alone is not the same as having a complete vaccine. This short chapter is meant as an introduction to the basic model of mRNA vaccines and their believed mechanisms of action. Later chapters will take a deeper dive into implicit assumptions, analyzing why, and the extent to which, mRNA technologies in actuality resemble that of traditional vaccines. The chapter concludes with a detailed outline of the remainder of this book. The Basic Model of mRNA Vaccines As their name implies, the central player of mRNA vaccines is the synthetic RNA. In a nutshell, the idea is that if the vaccine RNA in a human cell acts the same as natural RNA, then it will get translated into a specific protein. And if that protein is an immunogen, then this process will evoke an adaptive immune response, all the while without ever having the body exposed to the actual pathogen itself. Therefore, the basic premise of mRNA technologies appears to be simple. One only needs to deliver an mRNA transcript encoding one or more immunogen(s) into the host cell cytoplasm (see Table 1.1 for the design of the Pfizer-BioNTech COVID19 vaccine BNT162b2 as an example), and the body will do the rest. In the case of Covid-19 mRNA vaccines, the antigen is the viral spike protein, a protein on the surface of the SARS-CoV-2 virus which the virus needs to enter the body’s cells. It is generally regarded as the main immunogenic part of this new virus and as such expected to trigger an immune response. The above summarizes only the main functionality of mRNA vaccines. It does not describe how the synthetic RNA, as a foreign material, evades detection—and © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Mueller, Challenges and Opportunities of mRNA Vaccines Against SARS-CoV-2, https://doi.org/10.1007/978-3-031-18903-6_1

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The lipid nanoparticle (LNP) part

Component The mRNA part

The vaccine mRNA is adapted by modified nucleosides and coding sequence optimization (esp., avoiding rare codons with low utilization) The vaccine mRNA is encapsulated in LPNs. The lipid components include an ionizable lipid, cholesterol, a polyethylene glycol conjugated lipid, and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) [3]

The ORF is flanked by 5 and 3 untranslated regions (UTRs), a 5 cap and a poly(A) tail. These structures are enzymatically added to the transcriptional product at the end of the reaction or as a synthetic analog in a single-step procedure [19]

The open reading frame (ORF) of the mRNA encodes the viral full length spike S of SARS-CoV-2 The spike protein RNA is modified by the introduction of 2 proline mutations

Description Synthetic production of the single mRNA is realized in the form of in vitro transcription (IVT) processes from DNA templates

• LPNs protect the naked mRNAs from RNases after injection and enhance their uptake into cells by endocytosis. • They have their own adjuvant activity and elicit innate immune sensors that are believed to augment antigen expression (see also Chaps. 2 and 9).

These modifications aim to enhance mRNA abundance and in vivo translation, to maximize protein production and stability (see also Chaps. 2 and 10)

• The 5 UTR is involved in translation initiation. • The 3 UTR facilitates mRNA stability and durability of protein expression. • The poly(A) tail helps initiate translation and delay degradation.

The aim of this modification is to stabilize the S protein in a conformation that is better suited for antibody-mediated neutralization These additions are deemed necessary to resemble mature, fully processed mRNA molecules as naturally found in the cytoplasm of eukaryotic cells and to protect the mRNA from intracellular nuclease digestion:

Design Principle [4, 17] Once the synthesized molecules reach the cytoplasm of “transiently transfected” cells, the ribosomal translation machinery facilitates the expression of the mRNA into the target antigen (here, the spike (S) glycoprotein) S is the target of virus-neutralizing antibodies

Table 1.1 Basics of mRNA Vaccines exemplified by the BioNTechPfizer BNT162b2 vaccine

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destruction—by the body’s immune system, nor does it say where the injected material will be distributed, for how long the synthesized mRNA will survive inside the human body, how it will be cleared, and how it will be taken up by cells; it also does not explain where, for how much time, and in which quantity vaccine-induced production of spike protein occurs, and to what extent it may affect intended—and unintended—vaccine effects. The first part of the book examines and appraises the basic model of mRNA vaccines as described above—to act as a shortcut for adaptive immunity without requiring some live or attenuated virus (as had been the case with traditional vaccines). The issues related to target tissue, biodistribution, and related important pharmaceutical questions will be analyzed in Chaps. 9 and 10 below. Basic Believed Differences Between mRNA Vaccines and Traditional Vaccines Once again, the idea of mRNA vaccines is that once the mRNA is inside the body’s cells, they will read the mRNA instructions and “temporarily produce the spike protein” [16]. The person’s immune system will then recognize this protein as foreign and produce antibodies and activate T cells (white blood cells) to attack it. This mechanism is unlike that of the types of vaccines that previously have been utilized, which consist of specific components such as inactivated diseasecausing organisms or proteins made by the pathogen. With mRNA technologies, it is believed that this step can be replaced by genetic instructions resembling an intended activity and progress—to get the body to produce the spike antigen itself. The hope is that later on, in case of infection with SARS-CoV-2, the immune system will recognize the spike on the real virus; already being primed, the body will remember the invader and be ready to quickly defend the body against the virus [16]. Some Known Technical Hurdles mRNA vaccines have been in development for almost three decades [18]. Before the Covid-19 pandemic, there were only a few clinical studies that assessed infectious disease mRNA vaccines (e.g., [2, 7]). Major technical hurdles had hampered their practical use for many years. As it is believed that RNA vaccines have several advantages relative to more traditional vaccines, two mRNA vaccine candidates (by Pfizer and Moderna) were among the first to receive emergency approval as Covid vaccines. This, despite the fact they were tested via limited clinical trials which lasted a few months only and included merely a very small fraction of the global population [1, 12]. Adverse effects of a new vaccine are not new, nor are they unexpected. They are nevertheless difficult or impossible to anticipate when developers are unable to include all the relevant facts in their formulation plan. For many years, the safety and effectiveness of mRNA vaccines were based on foundational key principles, with several of them rooted in assumptions that are difficult to fully appreciate. One of the main challenges with these platforms has been the appropriate level of the spike antigen to be produced from the synthetic transcript; it should not be too much to cause immune overreactions, but it also ought not to be too low either.

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This requires that an appropriate amount of the mRNA is taken into the right type and number of cells, evoking the anticipated antigen expression for the appropriate length of time. With mRNA Covid-19 vaccines, as they are injected into the deltoid, one of the basic assumptions is that the material will be taken up by nearby cells only, which will then produce the spike antigen and expose fragments of it on their surface, triggering adaptive immune responses. All this relies on several assumptions that will be analyzed in a very detailed way in Part II of the book. Chapter 9 will talk more about the lipid nanoparticles (LNP) that are generally merely regarded as carrier vehicles to ferry the mRNA payload into appropriate tissues and cells. Issues regarding the spike, such as where and for long it is produced, where it goes, and its anticipated and unanticipated effects, will be critically analyzed in Chap. 10. Another challenge is to prevent recognition of the mRNA-LNP complex by immune cells, as these would otherwise destroy the injected material before it could evoke the desired immune memory response. To achieve this, the entire mRNA-LNP complex is usually hidden inside Polyethylene Glycol (PEG), a process that is done also for many other PEGylated drugs. Problems with this step are well established, evoking severe immune reactions in many people, and will be briefly summarized in Part II. Nonetheless, even for people who are not allergic to PEG, once the substances are released, these, too, are not invisible to the immune system. Normally, RNAs are not just floating around freely in high amounts. They are very transient in nature and for the vaccines needed to be modified to be more stable. Problems about this unnaturally enhanced stability will be analyzed in Chap. 10 below. Without going into detail about any of the above, we are now left with the main player of mRNA vaccines, the mRNA. Now, the innate arm of the immune system is specially equipped to recognize foreign RNAs as these are usually a sign of invaders and pathogens. It is clear then that exogenous RNA in general will be targeted by the host immune system. For therapeutic mRNA in particular, produced by in vitro transcription (IVT) from a plasmid backbone, the strong inflammatory response to the synthesized RNA is known to trigger many endosomal and cytoplasmic innate immune receptors. This recognition of the mRNA by the innate immune system thereby establishes an intrinsic adjuvant activity of mRNA vaccines (for an overview, see, e.g., Figure 1 in [13]). This was long believed to be an added benefit, with the mRNA being both the messenger and its own adjuvant. In the context of immunotherapy, this inherent immune-activating activity of RNA is seen as highly desirable in that it can increase the potency of the vaccine [5, 13]. Very new findings, however, challenge the overall correctness of this view and point out that the intrinsic immune-activating property of IVT mRNA may be beneficial in some applications such as “RNA cancer vaccines,” or when very strong T and B cell immune responses are needed, but not in others [9, 14]. For decades, the question of how to protect the foreign RNA from destruction by the innate immune system remained unresolved. Specifically, it became apparent that innate immune sensing of mRNA can lead to the inhibition of antigen

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expression and may negatively affect the immune response [9]. In particular, it was found that contaminants during IVT production, most notably related to doublestranded RNAs (dsRNAs), could have detrimental effects on the mRNAs themselves (this has become known as the host immune response “shooting the messenger” [5]). The above has prompted intense efforts to modulate the immunostimulatory profile via different formats of mRNA vaccines, e.g., how purification of IVT mRNA is achieved, the incorporation of modified nucleotides, sequence optimization, and other approaches (e.g., [6, 8, 9, 15]). The undesirable effects of innate immune sensing of synthetic RNA have led to extensive efforts during the last ten years [15], to increase the translation capacity and stability of mRNA [11, 14]. During the years leading up to the pandemic, several studies have revealed conflicting results and discrepancies related to the various modifications of different mRNA vaccine platforms; this is in part due to the intrinsic microheterogeneity of RNA related to cell type, the physiochemical properties of the mRNA complexes [9], and related to their unique impacts on humans as opposed to animals, as seen from various animal trials [10]. Nonetheless, before the mass-vaccination rollout, there have only been limited considerations about undesirable adverse effects of the IVT mRNAs. It has only been recently that the beneficial self-adjuvant nature of IVT mRNA production has been challenged, and in some cases, a clear debilitating effect has been shown. Yet, there is no clear understanding of the adverse effects evoked by the inherent stimulation of the innate immune system. While some clinical findings point to the concern of triggering autoimmune conditions, the realization that the immune-stimulatory effect of the IVT mRNAs is not necessarily beneficial radically overturns the unproven paradigm of this being an added benefit. As such, there are only a few hypothetical, let alone clinical, studies that investigate this. Believed Advantages Compared to DNA Vaccines Parallel to RNA vaccines, DNA vaccines have been considered to directly take DNA encoding a certain protein into host cells. Yet, a disadvantage of DNA technologies is that the DNA needs to be taken into the cell nucleus, potentially endangering various serious genotoxicity issues. In this regard, a major believed advantage of mRNA vaccines is their assumed difference to DNA vaccines. In contrast to DNA (and viral vectors), mRNA is believed to: (1) not be able to integrate into the host genome, (2) be expressed in a dose-dependent manner, (3) be transient, and (4) not disrupt genes unless mRNAs encoding DNA-modifying enzymes are delivered. For these reasons, regulators determined already years ago that resolving the legal regulatory framework [13] of mRNA vaccines is “a simple decision.” In fact, as RNA is not believed to result in “modification of genetic material of living cells,” mRNA vaccines are not classified as gene therapy in the United States. Moreover, in the European Union, they do not fall into the category of a gene-therapy medicinal product as the latter, by very definition, do not include vaccines against infectious diseases. This decision was based on the understanding that mRNA is degraded within cells at the time of their adoptive transfer to the patient [13].

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Fig. 1.1 Underlying safety assumptions of mRNA vaccines and how these influence testing policies. The notion of nucleic-acid-based vaccines such as viral vectors, plasmid DNA, and mRNA has been around for decades. Because they do not generate infectious particles and are not believed to get integrated into the genome of the host cells, mRNA vaccines are regarded as the most promising ones. These and other key postulates (as depicted) have become the basis of why they are not classified as genetic therapies. This decision has important and far-reaching consequences, most notably related to safety and testing. The arguments leading to each of the underlying assumptions are carefully analyzed in this book. The resulting questions and arguments are corroborated by findings unique to SARS-CoV-2 and the pandemic in general

Based on the above assumptions (summarized in Fig. 1.1), the conclusion was made that “there is no scientifically sound rationale to test for genome integration, germline transmission, genotoxicity or carcinogenicity of IMPs (investigational medicinal products), or carry out long-term observation of patients in clinical studies” [13]. Nonetheless, the question arises of how these critical assumptions were obtained, to what degree they have been assessed in various animal models or clinical trials, and whether or not their validity deserves to be taken for granted. Given the pivotal nature of these assumptions, they will here be critically discussed below. In fact, the decision for granting emergency approval of the first two mRNA vaccines by Pfizer-BioNTech and Moderna was based on rather short clinical trials that excluded certain high-risk people such as those with allergies. It will be argued here that some of the rationales used to establish the regulatory framework, and therefore, how R&D efforts and safety trials have been conducted, is based on interpretations of experiments and mechanisms (or often the lack thereof) of transfection, exogenous RNAs, the regulatory nature of RNAs, specific biochemical mechanisms in human cells, pathological presentations consequent to the stimulation of RNA

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sensors, or the potential of RNA integration into the human genome, which have not been fully appreciated. Not all of the most well-intended programs and efforts achieve their goals when time is an impeding factor. This book offers several areas of concern relative to hidden underlying assumptions of core principles of mRNA vaccines and potentials for further improvement. The hypotheses developed here are not conspiracy theories. They are rooted in a series of questions and arguments that during the emergency approval do not seem to have been rigorously investigated, and which are developed here based on extensive research of relevant literature and critical analysis. Outline of the Book mRNA vaccines trigger questions and pose us with situations that we have never seen before. This book offers a comprehensive appraisal of mRNA vaccines, Covid-19 vaccines in general, and issues of public health altogether, covering relevant areas in genetics, molecular biology, and public health. Now, because of the large deployment of these vaccines, it is imperative for researchers, scientists, health officials, and the public, to scrutinize every aspect of the new vaccines. In this sense, great emphasis will be placed, first, on the underlying assumptions (Fig. 1.1) that shape the current paradigm mRNA vaccine safety and testing policies, namely the belief that: • The IVT mRNA’s self-adjuvant property is beneficial. • Any excessive immune-stimulatory activity can be eliminated from IVT processing. • mRNA does not integrate into the host genome. • mRNA is not mutagenic. • mRNA is quickly degraded. In addition to these, new challenges will be discussed that have arisen since the large-scale rollout of the first Covid-19 vaccines. The book is laid out in three parts, roughly, corresponding with the timeline of the pandemic: • Part I (Chaps. 2–7): Scientific underpinnings, early expectations, and emerging challenges from the global inoculation experience: This part critically appraises key assumptions underlying mRNA vaccines and raises key questions that previously do not seem to have been fully appreciated. This part not only analyzes biological plausibility but also examines early challenges regarding the evaluation of vaccine safety and effectiveness. The limits and challenges identified invite a different approach to capture the tradeoff between risks and benefits. It is suggested that the notion of scaling can be a valuable tool in this regard, especially in the context of a global intervention. • Part II (Chaps. 8–11): A deeper dive into mRNA vaccine safety and security, and developments until Delta. This part first focuses on adverse events postinoculation and the WHO policy to determine when these are indeed caused by the jabs. This framework will be critically discussed for the mRNA vaccines, analyzing how this played out during the pandemic (until roughly the Delta era). Numerous challenges and difficulties will be identified, also for related causality

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1 Introduction

questions that have shaped the course of the pandemic. The second focus of Part II is mRNA vaccines as pharmaceutical agents rather than traditional vaccines, based on increasing evidence of their mechanisms of action, both related to mRNA-LNP complex, the active components of these products, and immunity beyond antibody responses. • Part III (Chap. 12): The Omicron variants. Granted, Omicron is radically different than previous SARS-CoV-2 VOCs. Yet, in many regards, this variant amplifies the challenges that arose with previous variants already, highlighting some expected trends, with many unknowns, nonetheless. Omicron answers some of the questions raised previously and raises a host of new ones. The book concludes with a consideration of the most relevant open questions and opportunities to tackle some of the concerns that have been identified. Overall, rooted in an analysis of published literature and logical analysis, the consequences of possible gaps or misconceptions that led to the current view of mRNA technologies will be illuminated, and potentials and opportunities for improvement presented.

References 1. Baden LR, El Sahly HM, Essink B, Kotloff K, Frey S, Novak R, Diemert D, Spector SA, Rouphael N, Creech CB, et al (2020) Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N Engl J Med 384:403–416 2. Bloom K, van den Berg F, Arbuthnot P (2020) Self-amplifying RNA vaccines for infectious diseases. Gene Therapy, 1–13 3. Bowman CJ, Bouressam M, Campion SN, Cappon GD, Catlin NR, Cutler MW, Diekmann J, Rohde CM, Sellers RS, Lindemann C (2021) Lack of effects on female fertility and prenatal and postnatal offspring development in rats with bnt162b2, a mRNA-based Covid-19 vaccine. Reproductive Toxicology 103:28–35 4. Buschmann MD, Carrasco MJ, Alishetty S, Paige M, Alameh MG, Weissman D (2021) Nanomaterial delivery systems for mRNA vaccines. Vaccines 9(1):65 5. Devoldere J, Dewitte H, De Smedt SC, Remaut K (2016) Evading innate immunity in nonviral mRNA delivery: don’t shoot the messenger. Drug Discov Today 21(1):11–25 6. Foster JB, Choudhari N, Perazzelli J, Storm J, Hofmann TJ, Jain P, Storm PB, Pardi N, Weissman D, Waanders AJ, et al (2019) Purification of mRNA encoding chimeric antigen receptor is critical for generation of a robust t-cell response. Hum Gene Ther 30(2):168–178 7. Jackson NA, Kester KE, Casimiro D, Gurunathan S, DeRosa F (2020) The promise of mRNA vaccines: A biotech and industrial perspective. NPJ Vaccines 5(1):1–6 8. Karikó K, Buckstein M, Ni H, Weissman D (2005) Suppression of RNA recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23(2):165–175 9. Pardi N, Hogan MJ, Porter FW, Weissman D (2018) mRNA vaccines-a new era in vaccinology. Nat Rev Drug Discov 17(4):261 10. Pardi N, Hogan MJ, Weissman D (2020) Recent advances in mRNA vaccine technology. Curr Opin Immunol 65:14–20 11. Pepini T, Pulichino AM, Carsillo T, Carlson AL, Sari-Sarraf F, Ramsauer K, Debasitis JC, Maruggi G, Otten GR, Geall AJ, et al (2017) Induction of an ifn-mediated antiviral response by a self-amplifying RNA vaccine: implications for vaccine design. J Immunol 198(10):4012– 4024

References

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12. Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, Perez JL, Pérez Marc G, Moreira ED, Zerbini C, et al (2020) Safety and efficacy of the bnt162b2 mRNA Covid-19 vaccine. N Engl J Med 383:2603–2615 13. Sahin U, Karikó K, Türeci Ö (2014) mRNA-based therapeutics—developing a new class of drugs. Nat Rev Drug Discov 13(10):759–780 14. Verbeke R, Lentacker I, De Smedt SC, Dewitte H (2019) Three decades of messenger RNA vaccine development. Nano Today 28:100766 15. Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, et al (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7(5):618–630 16. WHO (2021) The moderna COVID-19 (mRNA-1273) vaccine: what you need to know. https:// www.who.int/news-room/feature-stories/detail/the-moderna-covid-19-mrna-1273-vaccinewhat-you-need-to-know 17. World Health Organization et al (2020) mRNA vaccines against Covid-19: Pfizer-biontech Covid-19 vaccine bnt162b2: prepared by the strategic advisory group of experts (sage) on immunization working group on Covid-19 vaccines, 22 december 2020. Tech. rep., World Health Organization 18. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL (1990) Direct gene transfer into mouse muscle in vivo. Science 247(4949):1465–1468 19. Zhang C, Maruggi G, Shan H, Li J (2019) Advances in mRNA vaccines for infectious diseases. Front Immunol 10:594. https://doi.org/10.3389/fimmu.2019.00594, https://www.frontiersin. org/article/10.3389/fimmu.2019.00594

Part I

Scientific Underpinnings, Early Expectations, and Emerging Challenges from the Global Inoculation Experience

Chapter 2

Appraisal of Some of the Key Postulates Underlying mRNA Vaccines

As described in the introduction, key foundational assumptions (Fig. 1.1) have guided the approval and large-scale deployment of mRNA vaccines. Arguably, they were most influential in their fast approval, and they shaped decision making and public understanding. Many of them have been widely broadcasted by mainstream media and give the impression of being rooted in clear and detailed modeling, a sound comprehension of underlying biological mechanisms, and supported by clinical experience. These assumptions come across as long-established facts. This section critically analyzes each one of them, asking how they were developed and established. Doing so raises several uncertainties, questions, and problems, as can be seen from previous experience with these technologies, insights from previous studies, biological plausibility, and basic issues related to modeling itself. Rooted in a rational and logic-based approach, the section discusses a number of foundational questions that have not been fully appreciated, highlighting the urgency for further investigation. Understanding the (in)validity of foundational assumptions is essential for everything that follows. Therefore, the beginning of the book pays close attention to the general underpinnings and assumptions guiding mRNA platforms. Later parts will dig deeper into the points raised here, further corroborated by ongoing experience from the global vaccination regime all the way to the Omicron variants.

2.1 Potential Uncertainties of the Model Before analyzing the central pillars that shape safety assumptions of mRNA vaccines, it is worthwhile to explore some relevant factors at a yet more fundamental level. This section considers questions related to the model itself and the paradigm of “messenger RNAs.” © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Mueller, Challenges and Opportunities of mRNA Vaccines Against SARS-CoV-2, https://doi.org/10.1007/978-3-031-18903-6_2

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2.1.1 The Synthesized RNA Molecules May Not Play the Role of Messenger RNAs Arguably, the synthesized mRNA is the most important component of mRNA vaccines. Thanks to various manufacturing techniques (see Table 1.1 for the BioNTechPfizer BNT162b2 vaccine as an example), the mRNA is designed to reach cells and evoke the translation machinery for the production of the desired proteins. This is one, if not the single, most critical step of the entire mRNA vaccine paradigm. Nonetheless, intending the synthesized molecules to play the role of “messenger” RNAs does not mean that they will. Even if those components are, as designed, not rejected as foreign and indeed recognized as RNA molecules, there is no guarantee they will be translated into the desired protein. First, the production process may lead to truncated species, introduce transcription errors, or lead to other modifications that may evade various purification strategies. Thanks to the ubiquity and variety of mRNA splicing and various post-transcriptional modification events, even those species that have been correctly synthesized in vitro may not lead to the intended final product in vivo. A recognized problem with IVT processes is that the RNA molecules may form double-stranded byproducts that tend to trigger various undesirable immune recognition pathways (Sect. 2.2); alas, even if they escape those (as intended), some of their byproducts may be interpreted as regulatory RNAs (Sect. 3.7) and lead to unanticipated downstream processes. RNAs in general (and not only dsRNAs) have effects on organisms that range from gene regulation to intergenerational inheritance (for more, see below), and so the synthesized molecules may enter different pathways than intended.

2.1.2 Maximizing the Expression of the Immunogenic Gene from a Network Perspective One of the key goals of mRNA vaccines is “to efficiently utilize the translational machinery of the host cell to generate a sufficient quantity of the encoded immunogen” [41]. The view is that expressing the highest quantity of the gene of interest leads to the most optimal immune response, as this is fostered by the largest quantity of the desired immunogen possible. This notion is rooted in the understanding first shaped by Francis Crick in 1957 that places genes at the center and proteins at the end of biological information transfer [17, 18]. However, recent years have revealed an operational role of the proteome, as an active agent of biological function [28]; perturbations in both protein level and function contribute to the pathogenesis of human diseases, despite their molecular, genetic, and physiological origins [4]. Indeed, protein homeostasis has become one of the key objectives for post-genomic therapeutic strategies [4]. Targeting their

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precise function and interrelationships has proven difficult as many proteins can perform two or more biochemical functions (moonlighting). This phenomenon may explain the collateral effects of some drugs and is of great biological relevance: about 80% of the proteins involved in human diseases present alternative functions, usually related to cellular localization, cell type, oligomeric state, the concentration of cellular ligands, substrates, cofactors, or post-translational modifications; in some cases, the different functions have been clearly associated with different diseases and it is also possible that moonlighting protein may participate in other as yet unassociated pathologies [27]. During the last two decades, rather than relying on individual molecules and reactions, a completely different picture has been emerging. This has revealed that to understand both homeostasis and disease development, it is necessary to analyze the distinct tiers of information in an integrated (systems) approach [28]. Systems biology recognizes the complex dynamics of networks [26], with interactions as key determinants of cellular function [69], and where stimuli are integrated and responses coordinated often according to nonlinear principles [3]. Both proteins [28] and the metabolome [21] act as functional units of cellular phenotype. Accordingly, disease is caused by perturbations within those networks, rather than genomic underpinnings alone, as these cannot account for differential modification of the genomic structure in different cell types, or within the cell under varied transcriptional and metabolic states [21]. Therefore, even if vaccine mRNAs lead to the translation of the desired proteins, expecting their sole function as clearly predictable immune triggers is problematic, especially since they share close homologies (see below) to many human proteins [46, 66]. Overall, intending a unique and singular activity (antigen production in response to the antigens produced) does not mean that it will, especially when there is so much focus on the quantity of the output alone.

2.1.3 The Role of Metabolism In the area of individualized medicine, genetic information as the key determinant of a certain anticipated function (e.g., to trigger a desired immune response) is likely not going to do justice to the range of effects experienced by different individuals. Despite being absent from the standard representation of the Central Dogma of Molecular Biology, metabolism rears its head in virtually all living systems. As made clear in [21], fundamental parameters of metabolites including the availability of their precursors, cofactors and signals, their chemical structure, or their fluxes, cannot be directly predicted from the genetic information involved. As such, the production of antigens in human cells is implicitly—albeit critically—checked by their environmental conditions; in particular, stress or pre-existing pathologies are known to alter translation and may even lead to errors. In fact, studies involving genetic alterations demonstrate that physiological imbalances have a greater impact on the output of biological networks than changing (or adding) genetic instructions.

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“Very few things can happen [as anticipated] if instructions from one or other gene antagonize the kinetic and thermodynamic network of the living carrier” [21]. This does not mean, however, that metabolism merely constrains the production of the spike antigen, as predicted. It may also be important from the perspective of viral evolution and how pathogens adapt to their host. Metabolism plays a huge role in adaptive virulence programs. For instance, the presence of reactive oxygen species may not only damage genes and increase mutagenesis; it may also act as a driver for pathogens to explore new metabolic pathways: most importantly, the gain of new metabolic activity could establish a fitness advantage over the host and enable them to become more pathogenic [21]. All of these may be important contributors in processes that enable SARS-CoV-2 to become resistant to infection or treatment (Box 8 below).

2.1.4 The Spike Is a Functional Protein Whose Immunogenicity Is Determined and Targeted by Various Non-orthodox Modification Strategies One of the key assumptions with synthetic vaccines is that the mRNAs related to the immunogenic protein(s) in question closely resemble their viral counterparts. The mRNA of Covid-19 vaccines encodes the SARS-CoV-2 spike (S) protein that is believed to be the key component necessary to trigger an immune reaction. However, the characterization of epitopes by their amino acid sequence alone misses the fact that coronavirus S proteins are heavily glycosylated. The human immune system has evolved a range of non-traditional antibody refinement strategies to target such beyond-sequence-based viral evasion tricks (reviewed in [42]). Some studies have even shown that such non-orthodox antibodies target the carbohydrates on glycoproteins rather than the peptide part itself [42, 56]. Little is known about how glycosylation affects the immunogenicity of the SARS-CoV-2 S protein [49]. The fact that glycosylation can mask peptide epitopes (such as S) has long been a major problem that has hampered vaccine design efforts [39, 52]. Although glycosylated peptides were suggested years ago as an alternative to more traditional peptide epitopes [39], exploiting this fact for vaccine design has proven challenging. First, the glycan shield may be composed of familiar host glycans. This may not only shield it from detection of the innate immune system but at the same time also makes it difficult to use these components for adaptive immunity without evoking unwanted systemic responses or autoimmune (AI) conditions. Second, detailed experiments have revealed a strong dependence of both the composition and the glycan microheterogeneity on the cell type used for glycoprotein production [35]. Furthermore, both the glycoprotein composition and the non-traditional adaptive immune response have shown to be strongly influenced by pathophysiological condition of the host [42]. Finally, glycosylation patterns can also change rapidly as the virus mutates [39].

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All these reasons attenuate the ability of the host immune system to raise antibodies in an a priori determined fashion, as predicted by traditional epitopes via their amino acid sequence alone. In fact, both healthy and disease responses to SARS-CoV-2 infection seem to critically rely on glycosylation and other nonorthodox antibody diversification strategies [49]. It is also known that SARS-CoV-2 uses viral entry methods beyond that of the spike S protein binding to the ACE2 receptor [14, 66]. Thus, for prophylactic treatment, relying on the genetic information of the spike alone is certainly not going to be enough. The possibility for co-/post-translational modifications of viral proteins is manifold [29] and not determined by linear, explicit sequence information alone. Indeed, many of these modification processes may be consequent to viral–host interactions beyond what is encoded in the viral genome, and unique to the host and the infected cell [29, 42]. Non-orthodox antibody diversification [42] to counteract glycosylation or other viral masquerading tricks is often mounted as a last-resort mechanism and may actually be detrimental to the host itself [42]. In fact, in [49], the author suggests that a successful disease response to SARS-CoV-2 heavily relies on such mechanisms; conversely, depending on the overall pathophysiological state of the patient, these could easily go awry and in part be responsible for severe disease evolution or even death.

2.2 The Assumption “The IVT mRNA’s self-adjuvant property is beneficial” Despite being one of the celebrated features of RNA vaccine safety, the issue of selfadjuvancy is one of the least understood. In fact, modulating the inherent immunestimulatory activity of IVT RNA has been one of the major hurdles to RNA drug therapies for decades. This section describes some of the challenges and why it has been so difficult to reach a solution.

2.2.1 Harmful dsRNAs, as First Known from the in Vitro Synthesis of the mRNAs While IVT using the bacteriophage T7 RNA polymerase (T7 pol) is one of the most popular methods to produce mRNA, it is also known to generate various kinds of aberrant byproducts (reviewed in [32, 48, 61]). In particular, double-stranded RNA (dsRNA) contaminants of the IVT process display an immune-stimulatory activity that is incompletely understood, often undesirable, and uncontrollable. Yet, the precise mechanisms and the source of immunogenicity have proven difficult to elucidate [38, 58, 64].

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It is well established that viral infections are detected by sensor molecules that initiate innate antiviral responses, including the activation of type I interferons (IFNs) and proinflammatory cytokines. Viral dsRNAs are recognized by the major cytosolic sensors RIG-I and MDA5 [74], inter alia, and the same is true for the dsRNA byproducts of the IVT processes. Moreover, sequence and secondary structures formed by vaccine mRNAs are recognized by a number of innate immune receptors. In particular, mRNA vaccines have been shown to stimulate innate immunity through TLRs 3,7, and 8, RIG-1, and MDA5 [76]. The immunogenic nature of synthetic RNAs, and dsRNA contaminants created during IVT in particular, has long been an obstacle to the practical and safe application of mRNA technologies (see, e.g., [48, 60, 61] and references therein). Comprehending the exact nature and mechanisms leading to these contaminants remained an open question for almost a decade. Years of dedicated research finally revealed that the aberrant dsRNA byproducts are the result of unexpected mechanisms of T7 pol, such as self-complementary 3 extension and other nontraditional mechanisms [2, 41]. Other unique aspects of T7 pol to enable the formation of the unwanted byproducts were revealed only in 2018 [48]. While this study offers a “previously unappreciated mechanism by which T7 transcripts stimulate the innate immune system,” it also demonstrates the lack of a comprehensive understanding of all the immunostimulatory activities in this context. Many questions related to these “undesirable and uncontrollable” [48] mechanisms remain unanswered. Furthermore, Mu et al. [48] used very specific cell types able to transiently express RIG-1 or MDA5 and stimulated these cells by transfecting RNAs of interest. They demonstrated yet different pathways to generate erroneous dsRNA products. Notably, their findings revealed that T7 pol can initiate transcription from a promoter-less DNA end. Moreover, this promoter-independent transcriptional activity of T7 pol was observed for a wide range of DNA sequences and lengths, although structural mechanisms by which this enzyme can do this are still unclear. It seems as if the logical follow-up question, as to whether IVT RNAs could analogously lead to dsRNAs formation in vivo, has not been regarded a real concern and never been critically assessed. This may, in part, be rooted in an outdated comprehension of T7 pol—as an RNA-based RNA polymerase found in several viruses, and limited in their action to viral replication (only). This view also seems to have shaped the notion that “RNAs of the host cell are generally not replicated” [50]. In fact, basic principles of such enzymes were postulated many years ago, such as that they require RNA as a template, that they cannot function with DNA, and that they are specific for the RNA of the virus [50]. Unfortunately, the mechanisms leading to the hazardous dsRNAs in vitro heavily rely on different (“aberrant”) activities of T7 pol—which are in radical contrast to the orthodox mechanisms just stated.

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2.2.2 DsRNAs Play a Key Role in the Dichotomous Self-Adjuvant Effect of mRNA The intrinsic immunogenicity of IVT RNA is believed to happen via several distinct pathways. Immune activation is thought to be evoked either by the formation of secondary RNA structures or through the introduction of contaminants during the production process. As previously described, dsRNA contaminants play a key role as their binding to the cell danger sensors RIG-1 and MDA5 triggers various downstream signaling molecules that lead to the production of type I IFN and other proinflammatory cytokines. In turn, type I IFNs initiate processes leading to the expression of hundreds of proteins involved in antiviral immunity and the activation of distinct innate and adaptive immune responses, to activate neighboring cells and recruit immune cells, all of which have come to be known as the “self-adjuvant effect” of RNA [70]. While this had long been assumed to be beneficial, it has only been recently that the self-adjuvant effect of mRNA has been recognized as a double-edged sword for vaccine effectiveness [70]. Contradictory results have been reported about the role of type I IFN responses on the immunogenicity of mRNA vaccines. For instance, experiments on mice with a certain mRNA vaccine platform [57] revealed two opposing findings: on the one hand, an early and robust induction of type I IFN responses has the potential to provide an adjuvant effect on vaccine potency, but on the other hand, it could also establish a temporary state that inhibits the vaccineencoded antigen expression. This inflammatory response to synthetic RNA in terms of its inhibitory ramification on translation has been confirmed by others (now known as “shooting the messenger” [62]). These detrimental effects on the efficiency and strength of the targeted immune response [57, 70] have spurred efforts to develop different mRNA platforms to improve RNA vaccine potency [41, 57]. For instance, it was found that robust and sustained antigen expression of nucleoside-modified mRNA was associated with more optimal antibody responses compared to unmodified mRNA [45, 54]. In addition to the introduction of modified nucleosides, various efforts such as complexing the mRNA with various carrier molecules (see [55] and references) have been trying to determine how to best modulate the immunostimulatory profile of mRNA. Purification of IVT mRNA seems to be necessary—but not sufficient—for maximizing immunogen production and for minimizing unwanted innate immune activation. Numerous experiments (see, e.g., [43, 44, 55]) demonstrated that while the incorporation of modified nucleosides indeed increases the translation of mRNA, residual induction of type I IFNs and proinflammatory cytokines still remains. Crucially, Kariko et al. [43] showed that contaminants from nucleoside-modified IVT RNA, most notably dsRNAs, are responsible for the activation of these unwanted innate immune responses. Their experiments also revealed that the removal of these contaminants by various chromatographic methods increases RNA translation and that, as a result, this does not induce IFNs and inflammatory cytokines.

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However, as pointed out in [55], there are several inconsistencies, as other studies revealed seemingly contradictory results. The discrepancies between the different experiments may arise from variations in RNA sequence optimization, the level of innate immune sensing in the targeted cell types, the stringency of purification methods to remove dsRNA contaminants, or processes and contaminants that activate in the presence of nucleoside modifications [44, 55]. The inconsistency of these approaches (see also Sect. 2.6) is reflected in the different Covid-19 mRNA vaccines (those already approved and those currently undergoing human trials), which individually utilize all of these distinct—and often mutually opposing— categories to minimize immunity [10]. Type I IFNs can affect every step of the immune response to mRNA vaccination, from early modulation of antigen expression to dendritic cell (DC) function, and T cell differentiation [20]. Yet, the process of vaccine mRNA recognition by cellular sensors, the mechanisms of sensor activation, and the paradoxical effects of innate immune sensing on different formats of mRNA vaccines remain poorly understood [76]. While it has become clear that purity and delivery systems play central roles in instigating the immune response, there is no consensus on how different administration routes and vaccine formulations induce a correct balance of type 1 IFN induction [20, 76] (not too much and not too little). Moreover, as there has been so much focus on modulating the intrinsic immunity of mRNA to increase the production of the target protein(s), only a few studies have considered related safety problems arising from the self-adjuvant nature of the different IVT mRNA formats [55, 70].

2.2.3 The mRNA Vaccine Effect of Type I IFNs Modulation on CD8+ T Cell Immunity Is a Double-Edged Sword The negative impact of excessive IFN activation by mRNA vaccines can also manifest at the level of T cells. T cells are one of the important white blood cells of the immune system and play a central role in modulating immune responses in health and diseases. It has been known for well over 50 years that the thymus is the maturation site for T cells that provide cell-mediated immunity against offending pathogens. Nonetheless, the multifaceted role of T cell immunity is far from fully understood. T cell differentiation is one the key events that are absolutely essential, not only to eliminate intra- and extracellular pathogens but, upon dysregulation, could also lead to the onset of inflammation that causes pathogenesis of disease. A key component of T cells is CD8+ “killer” T cells, named after their capability to directly kill virus-infected cells, as well as cancer cells. On the one hand, CD8+ T cells are cytotoxic. On the other, CD8+ T cells might be an important factor in longevity, and tumor-specific CD8+ T cells have been investigated for their potential to attack cancer cells [34]. In recent years, several conflicting studies have revealed opposing roles for type I IFNs in modulating CD8+ T cell functions to mRNA vaccines; these range

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from profoundly stimulatory to strongly inhibitory, with either a beneficial or a detrimental impact on CD8+ T cell immunity [20]. As has been well established, the effectiveness of any vaccine to instigate T cell immunity is governed by the early interplay between the vaccine and innate immune cells, and the resulting inflammatory environment. mRNA vaccines, by virtue of their antigen-encoding nature, exhibit a high capacity to elicit cytolytic CD8+ T cells, with a strong capacity to destroy infected cells [20]. The opposing role of type I IFNs on CD8+ T cell responses is reminiscent of what has been reported in the context of viral infections [19], which led De Beuckelaer and collaborators [20] to postulate that similar mechanisms govern the dual effects of type I IFNs of mRNA vaccines as well. In particular, they suggest that whether type I IFN inhibits or stimulates the CD8 T cell response to mRNA vaccines depends on the timing and intensity of the type I IFN induced, as follows: • In the case that T cell receptor signaling (TCR) precedes type I IFN signaling, this is believed to promote the expansion and differentiation of antigen-primed CD8+ T cells into cytotoxic effectors, which leads to antigen presentation via upregulation of major histocompatibility complex (MHC)-I and -II (1) and of costimulatory ligands. • On the other hand, as mentioned above, type I IFN exposure is known to stimulate the expression of proinflammatory cytokines, and this can lead to a positive feedback mechanism. Along these lines, strong type I IFN responses elicit apoptosis by enhancing the expression of proapoptotic genes. Therefore, De Beuckelaer et al. [20] suggest that signaling of type I IFNs before TCR activation imposes an anti-proliferative status and activates a proapoptotic program. Evidently, clear comprehension of the mechanisms underlying the apparently opposing role of type I IFNs in the context of mRNA vaccines is urgently needed. The last few years have seen conflicting results regarding the inhibitory as opposed to the stimulatory role of type I IFN signaling depending on systemic versus topical immunization with mRNA vaccines. In 2017, De Beuckelaer et al. [20] suggested that i.d. versus i.v. administration will not only strongly affect the type of cells that encounter the vaccine mRNA, but also heavily impact the relative kinetics of antigen presentation versus type I IFN secretion, and thereby be a key component to instigate one as opposed to the other process described above. The first evidence for this hypothesis was obtained via a mouse study in 2020 [68]. Van Hoecke and coauthors were able to demonstrate the opposing role of type I IFN on T cell immunity upon i.v. versus s.c. administration. Nonetheless, as this was done in the context of nonmodified mRNA-Lipoplex vaccines that are currently explored in Phase II clinical trials for the treatment of patients with advanced solid tumors, these findings do not automatically translate to current Covid-19 mRNA vaccines which rely on other platforms [53, 71], and also target different clinical outcomes. The safe and effective utilization of prophylactic vaccines in the context of infectious diseases likely involves different processes than that for cancer treatment— where high-magnitude T cell responses and prominent antitumor efficacy following i.v. administration are desired. As such, [68] highlights the double-edged sword

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character of type I IFN induction upon mRNA-based vaccine treatment: Type I IFNs can promote CD8+ T cells responses, but their action on DC cells can also be pernicious to T cell immunity. For instance, it is known that bacterial sepsis causes a profound release of IFN α/β that can block antigen presentation and weaken cytolytic CD8+ T cells responses. Of note in this context, as reviewed in [68], sustained exposure to type I IFNs, associated with a chronic viral infection, has been demonstrated to cause the differentiation of monocyte-derived DCs into T cell inhibitory DCs and to suppress the generation of T-cell-stimulatory conventional DCs. Moreover, Type I IFNs also regulate DC turnover, and it has been speculated that "transient" DC loss might be a means to minimize excessive immune activation. The downsides of this do not seem to have been investigated. Translating the opposing findings of Type I IFN signaling on T cell immunity to mRNA vaccines was recognized as a major outstanding question just a few years ago [20]. Unfortunately, to my knowledge, there have been no clinical studies with Covid-19 mRNA vaccines that demonstrate how to optimally balance the potency of Covid-19 vaccines with inflammatory toxicity, appropriate immune activation, and inherent safety risks.

2.3 The Assumption “Vaccine mRNAs do not integrate into the human genome” A celebrated feature of mRNA vaccines, which is believed to create a major advantage compared to DNA vaccines, is that while the latter have to reach the nucleus, the former only need to reach the cytosol. This makes mRNA vaccines easier to deliver because they do not require crossing the nuclear membrane. At the same time, however, this quality is generally extended to mean that these molecules stay in the cytosol and never get into the nucleus. As a result, mRNA vaccines are believed to be non-integrative and non-mutagenic and, hence, much safer than DNA technologies. Nonetheless, in this regard, mRNA technologies have no advantage over DNA technologies, as detailed below.

2.3.1 Implicit Assumptions Regarding Their Non-integrative and Non-mutagenic Nature Unfortunately, it does not seem that the foundational premises to establish the nonintegrative nature of mRNA vaccines have been clearly assessed. Just as laid out in [37] in the context of gene-edited plants, for these important characteristics to be true, a number of assumptions are implicitly relied upon. The same hidden propositions are obviously applied to mRNA vaccines as well (Fig. 2.1) and will be analyzed in more detail below.

2.3 The Assumption “Vaccine mRNAs do not integrate into the human genome”

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Fig. 2.1 Breakdown of some of the main points believed to establish the safety of mRNA vaccines, with a special focus on their difference from DNA vaccines. When the idea of DNA vaccine technologies was first developed about 30 years ago [72], concerns were soon raised about whether they could act as potent mutagens. RNA vaccines, with their believed biochemistries happening in the cytosol only, are thought to eliminate these serious concerns. Unfortunately, it seems that Operation Warp Speed policies and the impetus to quickly develop a vaccine for Covid-19 have caused an incomplete analysis of some of these most fundamental postulates underlying mRNA vaccine safety. For the celebrated key principles (left-hand side) to hold true, additional (albeit less recognized, right-hand side) principles need to be fulfilled also. It seems that this elucidation into the unrecognized underlying assumptions has previously not been perceived (a-mRNA, artificial messenger RNA; SGM, synthesized genetic molecules; SRTs, synthetic RNA technologies)

2.3.2 Reverse-Transcriptase Activity in Human Cells The unproven assertion that vaccine RNA cannot be integrated into the human genome is also likely fostered by the conception of the Central Dogma as commonly known as “from DNA to RNA to protein.” If one excepts the premise of this linear logic as a general rule, then the only exceptions are retroviruses that transcribe RNA into DNA through the use of their own special enzyme called reverse transcriptase (RT). Alas, this reversal of the flow of genetic information applies not only to those special viruses alone, and indeed, also exists in human cells. In particular, (1) endogenous RT activity has been observed in human cells, and (2) the product of such reverse-transcription events has been shown to become integrated into the

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genome. Potential sources of RT to catalyze an RNA-dependent polymerization of DNA and related mechanisms are: (a) Various known human DNA polymerases—with often unexpected functionality. Surprisingly, human DNA polymerase η (hpol η) has very versatile functions and is capable of using both RNA and DNA templates as primers; in addition, hpol η can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) with a reverse-transcription efficiency almost as high as DNA polymerase [67]. (b) A specific RNA polymerase ribozyme. This has recently been shown to catalyze reverse-transcriptase activity and can incorporate all four dNTPs [63]. (c) LINE-1 elements. These self-replicating stretches of the genome use RT for DNA replication, employing RNA as an intermediate. The DNA of these retrotransposons is copied into RNA, which is then copied back into DNA (with the latter being the “retro” step facilitated by an RT enzyme encoded by the retrotransposon). Interestingly, the LINE-1 elements, which are autonomous retrotransposons, are capable of retro-transposing themselves and other nonautonomous elements including cellular RNA (see also Sects. 3.6.2 and 3.6.3 below). In a different context, LINE-1 proteins function as nucleic acid chaperones with high RNA-binding affinity. From this view, as noted by [77], it is not so surprising they can also integrate exogenous viral RNAs; one may even wonder if other chaperones could have a similar functionality. (d) Human polymerase θ (Pol θ ). A 2021 publication in Science [12] describes that human Pol θ reverse transcribes RNA, similar to retroviral RTs, and indeed acts like retroviral RTs in many regards: Pol θ exhibits a similar rate of RT activity as HIV-RT under identical conditions. Also, just as HIV RT and other retrovirus RTs are highly error-prone, Pol θ s RNA-dependent DNA synthesis activity is prone to nucleotide misincorporations and indels. Importantly, Pol θ RNA-dependent DNA synthesis activity has been observed under various conditions and on different template constructs and sequences. (e) RT activity in human cells facilitated by human retroviruses such as HIV-1. As for items (c) and (e), a preprint in 2020 [77] (later published as [78] in the Proceedings of the National Academy of Sciences) showed that these two mechanisms enable the reverse transcription of SARS-CoV-2 RNAs and their integration into the human genome. This activity was experimentally corroborated in cells overexpressing human LINE-1 or HIV-1 RT and can provide an explanation for the paradoxical finding of viral RNA shedding and recurrence of PCR-positive tests with patients recovered from Covid-19. These experiments also suggest that the integrated SARS-CoV-2 sequences could be transcribed, as demonstrated by RNA-Seq and smRNA-FISH data. Integration into the host chromosome has also been demonstrated for a variety of entire viruses, not only viral RNAs. Although this step is part of the life cycle of retroviruses, it may also occur incidentally for other DNA and RNA viruses as well [22]. The integration of various RNA viruses is believed to be facilitated by retrotransposons encoding RT and may lead to drastic consequences for the

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host cells, including gene disruption, insertional mutagenesis, and cell death, and has been associated with numerous forms of disease in numerous hosts, including humans (reviewed in [22]). Altogether then, based on the established literature as well as recent experiments, it must be concluded that the key assertion regarding the non-integrative nature of mRNA vaccines is unsubstantiated.

2.4 The Assumption “IVT mRNA is not mutagenic” Clearly, any potential for mutagenesis triggered by the products of Covid injections needs to be carefully scrutinized. This section does so in most general terms, analyzing how mRNA vaccines may create issues that are still incompletely assessed. Nonetheless, for Covid-19 vaccines, it is not only about any mRNAs coding for an arbitrary antigen. As will be discussed more fully in Part III of this book, the spike protein plays a rather unique role in and of itself. Genotoxic concerns and others specific to the spike antigen will be discussed more fully in later chapters. In general, RT processes are not the only way to facilitate the integration of RNAs into the genome. Other ways how this may be realized have been carefully detailed by Heinemann [37] in the context of gene-edited plants and externally derived dsRNAs. The latter have received a lot of attention in recent years as regulatory elements, but it does not seem that their relationship with RNA vaccines has been investigated.

2.4.1 DsRNAs Are Mediators of RNAi Pathways and Potential Mutagens Before diving into the issue of mutagenesis, this section considers the potential of vaccine RNAs, or their byproducts, to act as regulatory RNAs. RNA interference (RNAi) is a form of gene regulation in eukaryotes that, due to its many potential biotechnological applications, has received a lot of attention in recent years. RNAi results in what is called post-transcriptional and transcriptional gene silencing but is also known to sometimes cause an increase in the expression of genes. The most well-known mechanisms for the former are based on dsRNAmediated endonucleolytic cleavage or exonucleolytic destruction of the transcript, or inhibition of translation of the transcript. In recent years, several techniques have been developed to introduce dsRNA into the cells of organisms to target and alter their gene expression and lead to new traits. Research on dsRNA-mediated gene regulation has advanced rapidly. It has been guided by the view that such treatments do not create new or genetically modified organisms. Correspondingly, there is the belief that exogenously derived

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(exo) RNAs do not modify genes or other genetic materials. In the year 2018, these views became the basis for the New Zealand Environmental Protection Agency (EPA) to deregulate the use of dsRNA technologies. Nevertheless, as carefully detailed by Heinemann [37], “each of the major scientific justifications relied upon by the EPA was based on either an inaccurate interpretation of evidence or failure to consult the research literature.” Although [37] centered on the decisions made by the EPA, some of the same arguments apply to dsRNAs in much broader contexts. Unfortunately, however, the important findings in [37] do not seem to have been integrated into mRNA vaccine development efforts. This is probably due to the unfortunate assumption that there is no relationship between synthesized mRNAs and dsRNAs altogether (as the latter are believed to be eliminable from IVT processes), let alone those dsRNAs in vivo with regulatory capabilities. The nomenclature regarding RNAi-instigating dsRNA reagents is extensive. The main classes include microRNA (miRNA), short-interfering RNA (siRNA), and piwi-interacting RNA (piRNA). These types are foundational substrates in biochemical pathways involving Argonaute proteins that eventually determine the mechanisms of RNAi. However, as pointed out in [37], nomenclature should be used as an indicative guide to the biogenesis of the dsRNA only, but not to distinguish activity of the active form. The implications of this are far-reaching: regardless of their source, dsRNAs share the same pathways in the cell. More explicitly, their biochemistries overlap, no clear distinction can be made in the kinds of silencing they cause, and much of this applies to externally derived fragments of RNA vaccines in human cells as well (Box 1). Here, and as used in [37], this term is therefore used more generically (and often simply referred to as micro RNA or si/miRNA), rather than as used in biotechnological applications involving specific exo-dsRNAs meant to trigger specific processes. Although vaccine RNAs are designed to resemble full-length human mRNAs, this is not always the case. Figure 2.2 gives a short synopsis of how vaccine RNAs could lead to unexpected byproducts with si/miRNA-like features (for more, see Sect. 3.7.1). As a result, this means that dsRNAs from RNA vaccines will share many of the same pathways and issues as described in [37] regarding their potential to create unintended genetic changes (Sect. 3.6). It is also likely that vaccinederived byproducts enter the RNAi pathway and interfere with small endo RNA regulation involved in a variety of biological processes essential for normal animal development (Sect. 3.7).

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Fig. 2.2 How vaccine RNAs could lead to unexpected byproducts with si/miRNA-like features. Two main categories of small regulatory RNAs are short-interfering RNAs (siRNAs) and microRNAs (miRNAs). Canonically, the former are derived from exogenous RNAs and the latter from endogenous ones. However, despite differences, the distinct pathways of these short RNAs are intertwined. Moreover, while these regulatory RNAs may be born differently, they are not readily distinguished by their biochemistries once in the cytoplasm [11, 23, 37, 51]. Both derive from a variety of dsRNAs that are picked up by Dicer and further processed to regulate endogenous genes and defend the genome from foreign and invasive nucleic acids. Vaccine RNAs are based on the idea they perfectly resemble human transcripts and are as such not recognized as foreign. As detailed in the main text, this does not always need to be the case. This figure describes several factors in how vaccine RNAs could end up as precursors of novel short regulatory RNAs and thereby have si/miRNA-like functions. As their potential presence is unexpected, they could mimic and perturb many of the regulatory functions of endogenous small RNAs

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Box 1 Small RNAs Over the last two decades, 20–30 nucleotide RNA molecules have emerged as critical regulators in the expression and function of eukaryotic genomes. These small RNAs serve as factors that direct bound effector proteins to target nucleic acid molecules via base-pairing interactions [11]. As the effect of these interactions is generally inhibitory (although it can also lead to an increase in the expression of genes), the collective mechanisms have become known as RNAi (RNA interference). Three primary categories of these small RNAs are short siRNAs, miRNAs, and piRNAs. While the latter exert their function primarily in the germline (and will not be considered here) and derive from single-stranded precursors, the other two derive from double-stranded RNAs. During the years, our understanding of the origins and mechanisms of these dsRNAs has changed considerably. In the canonical view: • siRNAs are defenders of genome integrity in response to foreign or invasive nucleic acids, including viruses, transposons, and transgenes, whereas miRNAs are regulators of endogenous genes [11]. • Correspondingly, siRNAs were viewed as primarily exogenous in origin (exo-siRNA) and excised from long, fully complementary dsRNAs, whereas miRNAs were thought to be endogenous and purposefully expressed products of an organism’s own genome [11]. • siRNAs operate in a sequence-specific manner, in that they mediate silencing by inducing mRNA cleavage and subsequent mRNA degradation of target transcripts with full complementarity, whereas miRNAs repress target mRNAs with partially complementary binding sites [23, 75]. • siRNAs usually silence the same locus from which they were derived (although sometimes they can silence other loci as well), whereas miRNAs mostly do not silence their own loci but other genes [11]. During the last two decades, there has been a growing realization that small RNA pathways are all intertwined. Many of the believed differences turned out to be more fluid than originally thought. Accordingly, core features of both miRNA and siRNA silencing include the following: • They both depend on the same two families of proteins: Dicer enzymes excise them from their precursors, and AGO proteins support their silencing effector function [11]. • Several different categories of dsRNAs, including viral RNAs, transposons, or other exo-dsRNAs, can be sources of precursors for both siRNAs and miRNAs [11]. These long dsRNAs (which may be transcribed from cellular genes or introduced into the cells by infecting pathogens, or artificially via transfection or transduction by a viral-derived vector [4]) are processed by Dicer into ≈20–30 nt fragments. One strand (the guide (continued)

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Box 1 (continued) strand) of the processed duplex is loaded unto an AGO protein, and the other strand is discarded. The remaining strand (the guide) directs target recognition by Watson–Crick base-pairing. • Despite differences in the biogenesis of miRNAs and siRNAs, this separation does not extend to the activity of the active form. In fact, regardless of their source, siRNA and miRNA share a piece of overlapping machinery in plants and animals, including humans [23, 51]. In fact, siRNAs can function as miRNAs and vice versa. The fact that siRNAs with only partial complementarity to mRNAs can have miRNA-like undefined regulatory mechanisms leads to various “off-target effects.”

2.5 The Assumption That “vaccine mRNAs immediately get degraded” The fact that natural mRNA molecules synthesized in the cell nucleus are relatively unstable is well established. In the cytoplasm, RNAs are protected from the attack of exonucleases by the 5’-cap structure and the 3’-poly(A) tail. Therefore, to enhance the stability of the synthesized RNAs, various modifications needed to be done to the vaccine mRNAs. In general, it is believed that these modifications do not affect the very action of the vaccines. This is based on the overall—and oversimplified—view of how these platforms work. As such, the focus is mainly on the RNA, as it is meant to evoke the production of the spike antigen, and no more. Part III below will examine the pharmaceutical aspects of both the vaccine mRNA, along with the modifications done for these vaccines. Chapters 9 and 10 will explicitly discuss some of the emerging evidence from clinical experience post-vaccine rollout, affecting both their distribution, degradation, and clearance. Nonetheless, to lay the basis, it is relevant to examine more foundational questions related to the basic model itself, which will be covered in this, and the following, section. More specifically, while the previous sections looked at unexpected byproducts of mRNA vaccines, this section considers the opposite where the synthesized entities in fact do assume the role of mRNAs. However, even in this case, the fate of the (IVT) mRNAs is not straightforward. The traditional notion of gene expression first began to change some 15 years ago when it became obvious that the large-scale contribution made by mRNA decay could no longer be ignored [30]. Thus, the notion that vaccine RNAs immediately get degraded after translation needs to be re-evaluated. Conventionally, the “mRNA life-cycle” has been separated into two phases: the first one being nuclear and transcriptional and the other one being cytoplasmic and post-transcriptional. However, this traditional model has been overthrown in recent

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years and is not supported by recent molecular findings. The notion that is now emerging is that factors thought to specialize in a single step of gene regulation in one cellular compartment can contribute to the regulation of mRNAs at multiple steps along the mRNA life cycle; by regulating the expression of large subsets of messenger RNAs [8, 15, 16], this allows cells to quickly respond to various stimuli such as temperature, oxidative stress, or even pathological conditions [16, 36]. The last ten years or so have seen a rise in publications demonstrating that mRNA decay is linked to the act of transcription itself, but also to some additional factors that have not been fully elucidated. It is now confirmed that although the stability of mRNAs depends on their innate features, it is also predetermined by the nucleotide sequence, and more critically, by functions of the protein it encodes [65]. As a result, the process of mRNA decay and surveillance is itself one of the main posttranscriptional gene expression regulation platforms in eukaryotes [65]. The current notion of RNA vaccines is that the artificial messenger RNA will be degraded after translation by the DNA polymerase. This breakdown (and also that of the lipid carrier) is believed to happen within a matter of hours, thereby assuaging concerns about long-term risks [1]. However, several mechanisms that could lead to a prolonged—and undesirable (see Sect. 3.1)—expression of the IVT mRNAs include the following (Fig. 2.3): • Coupling between transcription and mRNA decay (which can be realized by multiple mechanisms). Although the most studied mechanisms affecting decay involve the direct imprinting or association of different mRNAs with transactivating factors that are recruited onto the mRNA during transcription, some decay-regulatory mechanisms also happen in the cytoplasm. In some cases, cisacting elements directly regulate the stability of the mRNA, either by attracting cytoplasmic RNA-binding factors that regulate decay or by interacting with the decay factors themselves. Furthermore, transcription and decay can also regulate each other kinetically, or through the involvement of unconventional RNA– protein interactions and/or protein–protein interactions [16, 36]. • Coupling via specific promoters as well as transcription factors: Basics of these mechanisms were first elucidated almost a decade ago [9] when it was demonstrated that yeast promoters can regulate mRNA decay even after the mRNA leaves the nucleus. Extensions of this study suggest that such a promotermediated coordination between transcription and mRNA degradation is an evolutionarily conserved phenomenon in gene regulation and can also occur in humans [24]. This mechanism is analogous to swapping promoters of the globin gene reported almost 30 years ago [25, 36]. It was shown that in HeLa cells replacement of this promoter with that obtained from the Herpes simplex virus 1 stabilizes a certain nonsense mutation of the β-globin mRNA and thereby reveals that mammalian and viral promoters can affect the stability of mRNA. Coupling transcription and decay via a promoter is a unique regulatory mechanism because the specificity of mRNA turnover is encoded entirely in the promoter sequence itself [36]. Notably, this same principle is utilized in trans-amplifying RNA vaccines via the incorporation of specific subgenomic promoters. These types of

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Fig. 2.3 Examples of how short abortive RNA fragments or stray RNAs from full-length products introduced through vaccination can lead to hazardous dsRNA products, in analogous ways as described for in vitro experiments. IVT processing of vaccine mRNA (which relies on T7 pol or similar enzymes) is known to generate harmful byproducts fostered by unexpected biochemistries of T7 pol. While in human cells it may not be the case that one enzyme alone harbors the exact full scope of these undesirable mechanisms seen in vitro, it is unlikely that none will. Neither the presence of similar enzymatic activities in human cells (possibly enabled by the human microbiome) nor the presence of residual human or nonhuman RNAs with homologies to the synthesized RNAs can be ruled out. Both of these factors suggest the real potential for harmful dsRNA generation in vivo also. What is highly concerning is that the core feature of T7 pol that supports those undesirable processes—to behave like viral RNA-dependent RNA polymerases that self-replicate specific RNA sequences [32]—are now intentionally introduced into human cells via recent self- (and trans-) amplifying RNA vaccine platforms [5–7]

mRNA vaccine platforms are still in development—see Chap. 3 for their unique characteristics and opportunities. One concern is that this added feature of RNA amplification in turn favors the development of harmful dsRNAs. Alas, while these novel technologies intentionally utilize promoters of certain RNA viruses, the same concern likely applies to all mRNA vaccines via promoters stemming from acute, latent, or chronic viral infections as well. • Various cis-elements in the nucleotide sequences of mRNAs that can be recognized by RNA-binding proteins (RBPs) and that lead to associations in the form of ribonucleoprotein particles (RNPs): In the human genome, 1542 RBP genes have so far been identified with about half of them involved in the post-

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transcriptional regulation of gene expression and, therefore, on the intracellular fate of mRNAs [31]. • Reversal of deadenylation and ongoing translation: In eukaryotes, the main mRNA degradation pathway starts with the shortening of the poly(A) tail (also known as deadenylation [13]), which is therefore often the rate-limiting step of mRNA decay as well. However, this critical step is reversible to some extent: it is known that transcripts can be readenylated and translated into functional proteins [30]. Additional mechanisms are known to initiate several cycles of translation [33]. The function of regulatory RNAs, with many of them taking place in the cytosol, is still incompletely understood. Many of the involved mechanisms not only employ physical but also functional linkage [15, 16], and some may even re-translocate to the nucleus. All these processes (Fig. 2.4) may determine the fate of the synthesized mRNAs in a highly dynamic spatiotemporal (and likely, undesirable) way. Thus, not only will they not always get degraded after translation; the lack of comprehensive knowledge of their interconnectedness and regulation may also trigger a host of unintended consequences (most notably, dsRNA formation in vivo, see below).

2.6 The Assumption “Any excessive immune-stimulatory activity can be eliminated from IVT processing” Generally, for IVT processes from linear DNA, RNA polymerases are predicted to synthesize a defined full-length (runoff) product. However, it is not uncommon to observe products longer or shorter than the expected runoff transcript [32], or, as discussed, dsRNA contaminants with undesirable immunostimulatory activity such as via type I IFN and inflammatory cytokines. In general, the understanding is that these can be eliminated during the synthetic production process (see also Sect. 3.5). This seems to have shaped the understanding that the problem of dsRNA contaminants has been taken care of. This section analyzes why this notion needs to be re-evaluated.

2.6.1 The Concern That Undesirable Immune Processes Get Triggered in Vivo Even though dsRNA formation during IVT processing is a known concern, it does not seem that the possibility of these harmful products forming in vivo has been thoroughly assessed. This concern is made even more plausible when inspecting the various attempts made to minimize the in vitro formation of such hazardous byproducts. For example, co-translational incorporation of modified nucleotides seems to a certain degree decrease excessive immunogenicity of IVT mRNA (albeit,

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Fig. 2.4 mRNA degradation as determined by the mRNA life cycle. Recent findings related to the “mRNA life-cycle” reveal an intricate degradation process of the mRNAs, far more involved than previously appreciated. Some of the mRNA degradation pathways have only recently been discovered and remain poorly understood [65]. They vary among different organisms, tissues, cells, cell-differentiation types, and pathophysiological conditions. As a result, the key assertion that all mRNAs quickly get degraded after they are translated is not substantiated. Thus, we cannot say for sure that human cells will only get “transiently” transfected [53] upon inoculation with RNA vaccines. What is more likely is that some vaccine mRNAs will not immediately get degraded but lead to prolonged production of antigenic proteins and probably influence other processes as well. This critical concern is increased through retro-integration and transcription of some of the RNAs (Sect. 3.6.3). Being that this has been demonstrated for SARS-CoV-2 RNAs [77], the same concern is relevant for vaccine RNAs encoding the spike. Just as in the study by Zhang et al. [77] (see also Chap. 10), this could mean that antigenic protein(s) may be expressed in an ongoing manner. The on- and off-target effects of these sustained processes and the evoked sustained inflammatory immune responses were not determined prior to the global vaccine rollout

unexpected consequences of this step do not seem to have been fully envisioned, see Chap. 10). In addition, [73] showed that this does not affect their MDA5stimulatory activity. Moreover, [48] also showed that the transcription reaction conditions can affect the formation of these unintended dsRNAs. While this led to the suggestion that lowering MgCl2 concentration can “suppress the aberrant dsRNA byproduct formation” in vitro, these types of conditions can obviously not be regulated in vivo. The reason for this is because MDA5 primarily recognizes dsRNA backbone structure with little specific contact with RNA bases. Therefore, for mRNA vaccine manufacturing, the various steps known for dsRNA purification are likely not appropriate for clinical applications.

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It is still possible that hazardous dsRNAs formed from stray vaccine RNAs can trigger excessive and pathogenic processes in vivo as well. The following section suggests specific mechanisms of how these dsRNAs could be formed in human cells.

2.6.2 dsRNA Production in Vivo from Synthetic mRNAs At present, harmful dsRNAs in the context of RNA vaccines are believed to be problematic mainly because of IVT production challenges. For this view to be complete, however, at least the following two conditions need to be fulfilled: • Vaccine RNAs immediately get degraded once the immunogenic protein is made. • The underlying biochemistries that lead to dsRNA formation in vitro are not supported in vivo. Alas, as discussed, there is no guarantee that artificial RNAs in human cells immediately get degraded (see also Chaps. 9 and 10). Neither can the existence of these biochemical processes be ruled out. In general, single-stranded RNA molecules tend to trigger various mechanisms that will lead to the formation of dsRNA products. The same applies to vaccine-derived stray RNAs, whether they are truncated or full-length species. Several mechanisms have been characterized that lead to the formation of harmful dsRNAs during IVT processing. These include the 3 extension of RNA, RNA-dependent RNA polymerization, DNA-primed RNA synthesis, as well as hybridization of the intended sense transcript and its fully complementary antisense transcript produced by promoter-independent transcriptional initiation from a DNA end [48]. These could also be realized in human cells as summarized in Box 2. In particular, past (latent) or future infection with RNA viruses is a critical factor to support dsRNA formation in vivo, as their enzymes can act as an RNA-dependent RNA polymerase (RdRP) to facilitate RNA-dependent RNA polymerization. On account of the different processes described in Box 2, it can therefore not correctly be concluded that all human cells are devoid of RdRPs or their ability to mimic the undesirable effects of T7 pol seen in vitro experiments. Crucially, dsRNA production is expected to demonstrate a strong correlation between RNA accumulation and formation of the spurious RNA [61], and this likely pertains to vaccine RNAs as well. This suggests that booster vaccines, but also mRNA platforms based on self-amplifying mRNAs (Sect. 2.6.3) may increase the risk of excessive immune responses.

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Box 2 Harmful dsRNA Production in Human Cells Several of the processes identified that enable dsRNA formation in vitro exist in vivo as well: • RNA-dependent RNA polymerase present in living cells One of the key mechanisms leading to the formation of the dangerous dsRNA products from synthetic RNAs in vitro [61] is facilitated by the ability of T7 pol to act as an RNA-dependent RNA polymerase (RdRP) to catalyze the replication of RNA from an RNA template. Although this mechanism was first identified for the specific bacteriophage RNA polymerase T7 pol, RdRP activities are rather common. In fact, RdRP is an essential protein encoded in the genomes of all RNA viruses. Viral RdRPs were first discovered in the early 1960s in the context of mengovirus and polio virus and are key components of all RNA viruses (except for retroviruses). Some eukaryotes also have RdRPs involved in RNA interference. They have been shown to amplify microRNAs and small temporal RNAs and produce dsRNAs using small interfering RNAs as primers [40]. • Potential of RNA vaccines to form dsRNAs from short abortive RNA fragments or full-length vaccine runoff transcripts Another way of how dsRNAs could form in vivo is based on the fact that other short RNA fragments can anneal to complementary sequences of any such stray RNAs (in trans). Given that the spike S protein exhibits considerable homologies to human proteins [46, 66], these free fragments may come from residual human RNA molecules. Even in the absence of T7 pol, other polymerases can mimic its features and therefore can support some of the in vitro observed mechanisms that result in the formation of dsRNA products. For instance, free RNAs from the vaccine can serve as a template for any RdRP to enable the extension of its 3 end. If the 3 end has sufficient complementarity (in cis), it will fold back and result in the extension of the free RNA strand. In addition to free RNAs binding to such a polymerase to initiate extension, RdRPs may also generate RNA using another RNA as a template. In general then, dsRNA formation can occur if the 3 end of the primary transcript has sufficient complementarity to sit down in trans on a second RNA or fold back on itself in cis to form extendible inter- or intramolecular duplexes, respectively [32]. • dsRNA formation from stray RNAs in the presence of viral infection T7 pol is markedly versatile in its activity. In addition to the above, many of these processes may also be realized through viral infections. Viruses bring their own enzymes and activities similar to T7 pol. In many ways, this enzyme behaves like viral PdRPs to self-replicate various different RNA sequences (see [32] and references therein), including the synthesis (continued)

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Box 2 (continued) of RNA from single- and double-stranded RNA, in the presence of a double-stranded DNA (dsDNA) promoter sequence, but also in the absence of promoter DNA. This applies to RNA viruses in particular, with their replication machinery completely taking place in the cytoplasm. However, infection with certain pathogens often leads to a marked instability of the genome [59]. Thus, because of the overall collapse of the strict separation and compartmentalization (Sect. 2.5), and/or the general genomic instability that is triggered upon infection, the same concern applies to DNA viruses as well. All these features are exactly the same as those “aberrant” features of T7 pol observed in vitro and can therefore analogously support the formation of dsRNA molecules in vivo from abortive products from RNA vaccines.

2.6.3 Vaccines with Self- and Trans-Amplifying mRNAs The above mechanisms of hazardous dsRNA products forming in living cells are relevant for the simplest type of RNA vaccine—non-replicating mRNA constructs encoding the coding sequences and being flanked by the 3 and 5 UTRs, a 5 -cap structure and a 3 -poly-(A)-tail [41]. A different RNA vaccine technology is actively being pursued to obtain greater quantities of antigen made from a smaller amount of vaccine: an in vivo self-amplifying mRNA (SAM) construct encodes additional replicase components able to direct intracellular mRNA amplification to express high levels of the antigen of interest [41, 47]. SAM mRNA vaccines are commonly based on the engineered RNA genome of positive-sense single-stranded RNA viruses such as alphaviruses. Their genomes are translated directly from the genomic RNA. Importantly, the replicase genes of these viruses encode an RdRP complex to synthesize RNA from an RNA template without a DNA intermediate. For self-amplifying vaccines, structural genes of these RNA viruses are replaced with the antigen of interest. In contrast to vaccines based on attenuated viruses, for these new RNA vaccine platforms, the full-length self-amplifying mRNA can be produced by IVT processes from a plasmid DNA (pDNA) template that has the required structural genes, including a subgenomic promoter, either provided in trans, or as synthetically formulated RNA. Following in situ translation of the vaccine immunogen, the viral proteins form an RdRP complex that interacts with host factors to form “replication factories.” In this way, self-/trans-amplifying RNA vaccines amplify the vaccine-encoded synthetic transcripts in situ that therefore results in the accumulation of the antigen in the transfected cells [47]. The critical issue, however, is the delivery of the pDNA expressing the alphavirus-derived RdRP complex. It is not clear to what extent this could

References

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exacerbate in vivo creation of hazardous dsRNAs. It seems unlikely this will not be the case, simply because RdRP activities are some of the key components to enable dsRNA formation processes (Box 2). In fact, Ref. [7] admits that, for selfamplifying RNA vaccines, “the formation of new dsRNA intermediates is inevitable during self-amplification.” Nonetheless, while there have been numerous attempts to eliminate dsRNA contaminants, these target IVT processes and mechanisms only. Detailed studies to better understand their formation in vivo may help mitigate the harmful induction of excessive inflammatory and systemic responses (see also Sect. 3.1).

References 1. Abbasi J (2020) Covid-19 and mRNA vaccines-first large test for a new approach. JAMA 324(12):1125–1127 2. Baiersdörfer M, Boros G, Muramatsu H, Mahiny A, Vlatkovic I, Sahin U, Karikó K (2019) A facile method for the removal of dsRNA contaminant from in vitro-transcribed mRNA. Mol Therapy Nucl Acids 15:26–35 3. Barabasi AL, Oltvai ZN (2004) Network biology: understanding the cell’s functional organization. Nat Rev Genet 5(2):101–113 4. Bartoszewski R, Sikorski AF (2019) Editorial focus: understanding off-target effects as the key to successful RNAi therapy. Cell Mol Biol Lett 24(1):1–23 5. Beissert T, Perkovic M, Vogel A, Erbar S, Walzer KC, Hempel T, Brill S, Haefner E, Becker R, Türeci Ö, et al (2020) A trans-amplifying RNA vaccine strategy for induction of potent protective immunity. Molecular Therapy 28(1):119–128 6. Blakney AK, McKay PF, Shattock RJ (2018) Structural components for amplification of positive and negative strand veev splitzicons. Front Mol Biosci 5:71 7. Bloom K, van den Berg F, Arbuthnot P (2020) Self-amplifying RNA vaccines for infectious diseases. Gene Therapy, 1–13 8. Braun KA, Young ET (2014) Coupling mRNA synthesis and decay. Mol Cell Biol 34(22):4078–4087 9. Bregman A, Avraham-Kelbert M, Barkai O, Duek L, Guterman A, Choder M (2011) Promoter elements regulate cytoplasmic mRNA decay. Cell 147(7):1473–1483. https://doi.org/10.1016/ j.cell.2011.12.005, http://www.sciencedirect.com/science/article/pii/S0092867411015030 10. Buschmann MD, Carrasco MJ, Alishetty S, Paige M, Alameh MG, Weissman D (2021) Nanomaterial delivery systems for mRNA vaccines. Vaccines 9(1):65 11. Carthew RW, Sontheimer EJ (2009) Origins and mechanisms of miRNAs and siRNAs. Cell 136(4):642–655 12. Chandramouly G, Zhao J, McDevitt S, Rusanov T, Hoang T, Borisonnik N, Treddinick T, Lopezcolorado FW, Kent T, Siddique LA, et al (2021) Polθ reverse transcribes RNA and promotes RNA-templated DNA repair. Science Advances 7(24):eabf1771 13. Chen CYA, Shyu AB (2011) Mechanisms of deadenylation-dependent decay. Wiley Interdisciplinary Reviews: RNA 2(2):167–183 14. Cheng MH, Zhang S, Porritt RA, Rivas MN, Paschold L, Willscher E, Binder M, Arditi M, Bahar I (2020) Superantigenic character of an insert unique to SARS-CoV-2 spike supported by skewed tcr repertoire in patients with hyperinflammation. Proc Natl Acad Sci 15. Collart MA, Reese JC (2014) Gene expression as a circular process: cross-talk between transcription and mRNA degradation in eukaryotes; International University of Andalusia (unia) Baeza, Spain

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16. Coppin L, Leclerc J, Vincent A, Porchet N, Pigny P (2018) Messenger RNA life-cycle in cancer cells: emerging role of conventional and non-conventional RNA-binding proteins? Int J Mol Sci 19(3):650 17. Crick F (1970) Central dogma of molecular biology. Nature 227(5258):561–563 18. Crick FH (1958) On protein synthesis. In: Symp Soc Exp Biol, vol 12, p 8 19. Crouse J, Kalinke U, Oxenius A (2015) Regulation of antiviral t cell responses by type i interferons. Nat Rev Immunol 15(4):231–242 20. De Beuckelaer A, Grooten J, De Koker S (2017) Type i interferons modulate cd8+ t cell immunity to mRNA vaccines. Trends Mol Med 23(3):216–226 21. de Lorenzo V (2014) From the selfish gene to selfish metabolism: revisiting the central dogma. Bioessays 36(3):226–235 22. Desfarges S, Ciuffi A (2012) Viral integration and consequences on host gene expression. In: Viruses: essential agents of life. Springer, pp 147–175 23. Doench JG, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. Genes Dev 17(4):438–442 24. Dori-Bachash M, Shalem O, Manor YS, Pilpel Y, Tirosh I (2012) Widespread promotermediated coordination of transcription and mRNA degradation. Genome Biology 13(12):R114 25. Enssle J, Kugler W, Hentze MW, Kulozik AE (1993) Determination of mRNA fate by different RNA polymerase ii promoters. Proc Natl Acad Sci 90(21):10091–10095 26. Ferrell Jr JE (2002) Self-perpetuating states in signal transduction: positive feedback, doublenegative feedback and bistability. Curr Opin Cell Biol 14(2):140–148 27. Franco-Serrano L, Huerta M, Hernández S, Cedano J, Perez-Pons J, Piñol J, Mozo-Villarias A, Amela I, Querol E (2018) Multifunctional proteins: involvement in human diseases and targets of current drugs. Protein J 37(5):444–453 28. Franklin S, Vondriska TM (2011) Genomes, proteomes, and the central dogma. Circ Cardiovasc Genet 4(5):576–576 29. Fung TS, Liu DX (2018) Post-translational modifications of coronavirus proteins: roles and function. Future Virology 13(6):405–430 30. Garneau NL, Wilusz J, Wilusz CJ (2007) The highways and byways of mRNA decay. Nat Rev Mol Cell Biol 8(2):113–126 31. Gerstberger S, Hafner M, Tuschl T (2014) A census of human RNA-binding proteins. Nat Rev Genet 15(12):829–845 32. Gholamalipour Y, Karunanayake MA, Martin CT (2018) 3’ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character-RNA-Seq analyses. Nucl Acids Res 46(18):9253–9263. https://doi.org/10.1093/nar/gky796, https://academic.oup. com/nar/article-pdf/46/18/9253/26001463/gky796.pdf 33. Gorgoni B, Gray NK (2004) The roles of cytoplasmic poly (a)-binding proteins in regulating gene expression: a developmental perspective. Brief Funct Genomics 3(2):125–141 34. Goswami R, Awasthi A (2020) Editorial: T cell differentiation and function in tissue inflammation. Front Immunol 11:289. https://doi.org/10.3389/fimmu.2020.00289, https:// www.frontiersin.org/article/10.3389/fimmu.2020.00289 35. Grant OC, Montgomery D, Ito K, Woods RJ (2020) Analysis of the SARS-CoV-2 spike protein glycan shield: implications for immune recognition. bioRxiv 36. Haimovich G, Choder M, Singer RH, Trcek T (2013) The fate of the messenger is predetermined: a new model for regulation of gene expression. Biochim Biophys Acta (BBA)Gene Regul Mech 1829(6-7):643–653 37. Heinemann JA (2019) Should dsRNA treatments applied in outdoor environments be regulated? Environment International 132:104856 38. Hornung V, Ellegast J, Kim S, Brzózka K, Jung A, Kato H, Poeck H, Akira S, Conzelmann KK, Schlee M, et al (2006) 5’-triphosphate RNA is the ligand for RIG-I. Science 314(5801):994– 997 39. Hütter J, Rödig JV, Höper D, Seeberger PH, Reichl U, Rapp E, Lepenies B (2013) Toward animal cell culture-based influenza vaccine design: viral hemagglutinin n-glycosylation markedly impacts immunogenicity. J Immunol 190(1):220–230

References

39

40. Iyer LM, Koonin EV, Aravind L (2003) Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases. BMC Struct Biol 3(1):1–23 41. Jackson NA, Kester KE, Casimiro D, Gurunathan S, DeRosa F (2020) The promise of mRNA vaccines: A biotech and industrial perspective. NPJ Vaccines 5(1):1–6 42. Kanyavuz A, Marey-Jarossay A, Lacroix-Desmazes S, Dimitrov JD (2019) Breaking the law: unconventional strategies for antibody diversification. Nat Rev Immunol 19(6):355–368 43. Karikó K, Buckstein M, Ni H, Weissman D (2005) Suppression of RNA recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23(2):165–175 44. Kariko K, Muramatsu H, Ludwig J, Weissman D (2011) Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucl Acids Res 39(21):e142–e142 45. Lindgren G, Ols S, Liang F, Thompson EA, Lin A, Hellgren F, Bahl K, John S, Yuzhakov O, Hassett KJ, Brito LA, Salter H, Ciaramella G, Loré K (2017) Induction of robust b cell responses after influenza mRNA vaccination is accompanied by circulating hemagglutininspecific ICOS+ PD-1+ CXCR3+ t follicular helper cells. Front Immunol 8:1539. https:// doi.org/10.3389/fimmu.2017.01539, https://www.frontiersin.org/article/10.3389/fimmu.2017. 01539 46. Lyons-Weiler J (2020) Pathogenic priming likely contributes to serious and critical illness and mortality in covid-19 via autoimmunity. J Translat Autoimmunity 3:100051 47. Maruggi G, Zhang C, Li J, Ulmer JB, Yu D (2019) mRNA as a transformative technology for vaccine development to control infectious diseases. Molecular Therapy 27(4):757–772 48. Mu X, Greenwald E, Ahmad S, Hur S (2018) An origin of the immunogenicity of in vitro transcribed RNA. Nucl Acids Res 46(10):5239–5249. https://doi.org/10.1093/nar/gky177, https://academic.oup.com/nar/article-pdf/46/10/5239/24962288/gky177.pdf 49. Mueller S (2021) Rarely recognized antibody diversification in covid-19 evolution to counteract advanced SARS-CoV-2 evasion strategies, and implications for prophylactic treatment. Front Physiol 12:1186. https://doi.org/10.3389/fphys.2021.624675, https://www.frontiersin. org/article/10.3389/fphys.2021.624675 50. Nelson DL, Lehninger AL, Cox MM (2008) Lehninger principles of biochemistry. Macmillan 51. Neumeier J, Meister G (2021) siRNA specificity: Rnai mechanisms and strategies to reduce off-target effects. Front Plant Sci 11:2196. https://doi.org/10.3389/fpls.2020.526455, https:// www.frontiersin.org/article/10.3389/fpls.2020.526455 52. Oostra M, De Haan C, De Groot R, Rottier P (2006) Glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and m. J Virol 80(5):2326–2336 53. Organization WH, et al (2020) mRNA vaccines against covid-19: Pfizer-biontech covid-19 vaccine bnt162b2: prepared by the strategic advisory group of experts (sage) on immunization working group on covid-19 vaccines, 22 december 2020. Tech. rep., World Health Organization 54. Pardi N, Hogan MJ, Naradikian MS, Parkhouse K, Cain DW, Jones L, Moody MA, Verkerke HP, Myles A, Willis E, et al (2018) Nucleoside-modified mRNA vaccines induce potent t follicular helper and germinal center b cell responses. J Exp Med 215(6):1571–1588 55. Pardi N, Hogan MJ, Porter FW, Weissman D (2018) mRNA vaccines-a new era in vaccinology. Nat Rev Drug Discov 17(4):261 56. Peng W, de Vries RP, Grant OC, Thompson AJ, McBride R, Tsogtbaatar B, Lee PS, Razi N, Wilson IA, Woods RJ, et al (2017) Recent h3n2 viruses have evolved specificity for extended, branched human-type receptors, conferring potential for increased avidity. Cell Host Microbe 21(1):23–34 57. Pepini T, Pulichino AM, Carsillo T, Carlson AL, Sari-Sarraf F, Ramsauer K, Debasitis JC, Maruggi G, Otten GR, Geall AJ, et al (2017) Induction of an ifn-mediated antiviral response by a self-amplifying RNA vaccine: implications for vaccine design. J Immunol 198(10):4012– 4024

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58. Pichlmair A, Schulz O, Tan CP, Näslund TI, Liljeström P, Weber F, e Sousa CR (2006) Rig-i-mediated antiviral responses to single-stranded RNA bearing 5’-phosphates. Science 314(5801):997–1001 59. Robbiani DF, Deroubaix S, Feldhahn N, Oliveira TY, Callen E, Wang Q, Jankovic M, Silva IT, Rommel PC, Bosque D, et al (2015) Plasmodium infection promotes genomic instability and aid-dependent b cell lymphoma. Cell 162(4):727–737 60. Roy B, Robb G (2018) Use of thermostable RNA polymerases to produce RNAs having reduced immunogenicity. US Patent 10,034,951 61. Roy B, Wu MZ (2019) Understanding and overcoming the immune response from synthetic mRNAs: New england biolabs focuses on formation and detection of dsRNA byproducts during in vitro transcription. Genet Eng Biotechnol News 39(12):56–58 62. Sahin U, Karikó K, Türeci Ö (2014) mRNA-based therapeutics—developing a new class of drugs. Nat Revi Drug Discov 13(10):759–780 63. Samanta B, Joyce GF (2017) A reverse transcriptase ribozyme. Elife 6:e31153 64. Schlee M, Roth A, Hornung V, Hagmann CA, Wimmenauer V, Barchet W, Coch C, Janke M, Mihailovic A, Wardle G, et al (2009) Recognition of 5’ triphosphate by rig-i helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus. Immunity 31(1):25–34 65. Siwaszek A, Ukleja M, Dziembowski A (2014) Proteins involved in the degradation of cytoplasmic mRNA in the major eukaryotic model systems. RNA Biology 11(9):1122–1136 66. Sørensen B, Susrud A, Dalgleish A (2020) Biovacc-19: A candidate vaccine for covid-19 (sarscov-2) developed from analysis of its general method of action for infectivity. QRB Discovery, 1–17 67. Su Y, Ghodke PP, Egli M, Li L, Wang Y, Guengerich FP (2019) Human dna polymerase η has reverse transcriptase activity in cellular environments. J Biol Chem 294(15):6073–6081 68. Van Hoecke L, Roose K, Ballegeer M, Zhong Z, Sanders NN, De Koker S, Saelens X, Van Lint S (2020) The opposing effect of type i ifn on the t cell response by non-modified mRNA-lipoplex vaccines is determined by the route of administration. Mol Ther-Nucleic Acids 22:373–381 69. Venkatesan K, Rual JF, Vazquez A, Stelzl U, Lemmens I, Hirozane-Kishikawa T, Hao T, Zenkner M, Xin X, Goh KI, et al (2009) An empirical framework for binary interactome mapping. Nature Methods 6(1):83–90 70. Verbeke R, Lentacker I, De Smedt SC, Dewitte H (2019) Three decades of messenger RNA vaccine development. Nano Today 28:100766 71. WHO (2021) The moderna COVID-19 (mRNA-1273) vaccine: what you need to know. https:// www.who.int/news-room/feature-stories/detail/the-moderna-covid-19-mrna-1273-vaccinewhat-you-need-to-know 72. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL (1990) Direct gene transfer into mouse muscle in vivo. Science 247(4949):1465–1468 73. Wu MZ, Asahara H, Tzertzinis G, Roy B (2020) Synthesis of low immunogenicity RNA with high-temperature in vitro transcription. RNA 26(3):345–360 74. Yoneyama M, Fujita T (2010) Recognition of viral nucleic acids in innate immunity. Rev Med Virol 20(1):4–22 75. Zamore PD, Haley B (2005) Ribo-gnome: the big world of small RNAs. Science 309(5740):1519–1524 76. Zhang C, Maruggi G, Shan H, Li J (2019) Advances in mRNA vaccines for infectious diseases. Front Immunol 10:594. https://doi.org/10.3389/fimmu.2019.00594, https://www.frontiersin. org/article/10.3389/fimmu.2019.00594 77. Zhang L, Richards A, Khalil A, Wogram E, Ma H, Young RA, Jaenisch R (2020) SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome. bioRxiv 78. Zhang L, Richards A, Barrasa MI, Hughes SH, Young RA, Jaenisch R (2021) Reversetranscribed sars-cov-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues. Proc Natl Acad Sci 118(21)

Chapter 3

Relevance for mRNA Vaccine Safety

This chapter describes how the above unsubstantiated assumptions underlying mRNA vaccines may impact vaccine safety. Again, the analysis here is done in a more general way, aiming to uncover mechanisms and consequences which previously do not seem to have been anticipated. Previously, the broad reliance on the underlying assumptions never necessitated a deeper analysis of the relevant biological mechanisms necessary. Apparently, as the above pillars were taken for granted, there seemed to be no need to consider “what-if” scenarios. On the other hand, now that these assumptions are unjustified, it is imperative to envision clinical and epidemiological implications. While some of these are described in general terms, these will be corroborated by publications and findings related to Covid-19 disease pathology and early responses to vaccination. As before, this chapter aims to highlight the general plausibility of previously unexpected mechanisms. During the unfolding of the pandemic, some of these have been accentuated by specific experiences from global vaccinations and will be revisited in Parts II and III below.

3.1 Detrimental Consequences of Type I IFN Stimulation and New Disease Patterns If dsRNAs can form in vivo, then this can evoke the same immunogenicity reactions and result in the same safety concerns as identified for dsRNA contaminants from IVT processing (Sect. 2.2.2), such as the release of proinflammatory cytokines, cytokine storm, and adverse systemic effects [74].

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Mueller, Challenges and Opportunities of mRNA Vaccines Against SARS-CoV-2, https://doi.org/10.1007/978-3-031-18903-6_3

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3.1.1 Dichotomous Immunogenicity and Cytotoxicity of Vaccine RNAs and Byproducts In terms of inflammatory cytokines, it is interesting to note that some mRNA vaccine platforms not only induced potent type I IFN responses in clinical trials [25, 79]; these also pointed to a clear dichotomous nature of this response which is most evident when contrasting acute versus chronic activation of the type I IFN pathways. In regards to the former, the activation of acute type I IFN responses can trigger several beneficial processes in both cellular and humoral immunity, as reviewed by Verbeke et al. [98]. On the other hand, however, these authors describe the detrimental role of type I IFNs via insights gleaned from persistent viral infections. They argue that chronic activation of the type I IFN pathway leads to, among others, the deletion of virus-specific B cells and strongly impairs T cell response. Chronic activation may also lead to undesirable modulation of CD8+ T cell immunity (see also Sect. 2.2.3). As previously described (Sect. 2.2.3), refs. [17, 97] suggest that in specific situations which depend on the duration and relative timing between type I IFN signaling and T cell priming, rather than stimulating T cells, type I IFNs have completely opposite effects and exert deleterious effects. That short-term (“transient,” Table 1.1) immune activation can be radically different than that of longer applications was also proposed by Sahin et al. [85]. These authors warn that long-term repetitive systemic application of mRNAs may result in anti-RNA antibody formation and mediate immune pathology. They propose that for transient applications this should not be a concern and that transient immune activation could in fact be desirable. Unfortunately, for mRNA vaccines, the line between transient and chronic is difficult to draw. As discussed, this is not only because the mRNAs may not be degraded as quickly as anticipated (see also Chaps. 9 and 10). Of greater concern are self- and trans-amplifying mRNA platforms for which the ongoing production of dsRNAs is an automatic side effect (Sect. 2.6.3). A related issue in this context is the integration of IVT RNA into the genome (Sect. 3.6), which could mimic a chronic viral infection, and in turn, ongoing dsRNA formation in vivo.

3.1.2 Autoimmune Conditions and New Pathologies It seems plausible that some adverse consequences of mRNA vaccines can be triggered by chronic or long-term immune stipulation. The greatest detrimental effect of vaccine RNAs or their byproducts may be caused by the prolonged presence of the same antigen(s). While this can lead to immune-tolerance and immune-non-responsiveness on the one hand (which will be further explored in Chap. 11), the opposite can happen as well. The development of autoreactive B and T cells (reviewed in [85]) is known to even lead to severe autoimmune

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consequences, such as systemic lupus erythematosus and diabetes. While this has not received a lot of attention during the vaccine trials, evidence of AI disease associated with RNA vaccines meanwhile has been accumulating.

3.1.2.1 Bell’s Palsy and Guillain-Barre Syndrome Prior to the pandemic, in a small clinical trial (n = 101) of mRNA vaccines against Rabies [3], 78% of the participants reported systemic adverse events (including Grade III). Immunogenicity across the different groups was clearly affected by the route of administration. More specifically, from the 24 young and healthy individuals in a specific trial group (where the vaccine was administered intramuscularly by needle-syringe), one moderate case of Bell’s palsy occurred one week after administration of the second dose. Furthermore, phase 3 studies of both the vaccines of Pfizer-BioNTech and Moderna already demonstrate an imbalance of cases of Bell’s palsy in the vaccine groups compared with the placebo groups. This trend has continued during the global Covid vaccinations [13]. A publication in The Lancet estimates that this increased incidence in the vaccine groups is between 3.5times and 7-times higher than would be expected in the general population [73]. Specifically, through May 15, 2021, the number of post-vaccination cases of Bell’s Palsy or related disorders (Facial discomfort or Facial dysmorphism or Facial nerve disorder or Facial neuralgia or Facial palsy or Facial paralysis or Facial paresis or Facial spasm) reported to VAERS are: those attributed to Moderna: 826, and those to Pfizer/BioNTech: 969 [13]. The number of Bell’s palsy alone, reported to EudraVigilance are, for TOZINAMERAN: 702, and for Moderna: 264 [29]. Although the exact etiology of Bell’s palsy is unknown, it is associated with both viral infection and autoimmune disease. It has often been observed with children after vaccination but can also occur in adults. Ref. [39] postulates that cell-mediated autoimmune mechanisms prompted by viral infections may be important factors in its pathogenesis. This is in line with studies that prove a clear association between Bell’s palsy and Guillain-Barre syndrome (GBS), a condition that is known to be a cell-mediated autoimmune neuritis [39]. Indeed, the first case of GBS after receiving the first dose of the Pfizer—COVID-19 vaccine was investigated in a case study published in [99]; the study authors suggest that the immune response evoked by the vaccine can “trigger autoimmune processes that lead to the production of antibodies against the myelin and cause GBS.” Of note, by May 15, 2021, other forms of myelitis were reported to VAERS, including acute disseminated encephalomyelitis (Pfizer-BioNTech: 2, Moderna: 9) which is characterized by widespread inflammation in the brain and spinal cord that damages myelin. The immunotoxic response induced by vaccine RNAs or their byproducts may be an additional driver of the disease, as corroborated by the fact that serum samples of patients with Bell’s palsy show elevated concentrations of inflammatory cytokines. The inflammatory response and AI processes may lead to a cascade, however, as a cross-regulation between the different cytokines, most notably involving type I IFN, has been proposed as a driver of different autoimmune diseases [8].

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3.1.2.2 Transverse Myelitis and Myelitis A mysterious form of post-vaccine paralysis was first reported in April 2021, when a healthy 33-year-old woman experienced no feeling in her arms or legs 12 h after getting the first dose of the Pfizer vaccine. “An MRI and spinal tap were clear, and her blood work all came back negative” WPXI-TV reported [42]. While Pfizer responded that “At this time, our ongoing review has not identified any safety signals with paralysis and the Pfizer-BioNTech Covid-19 vaccine” [42], cases of paralysis (transverse myelitis (TM), myelitis, paralysis, and GBS) were reported to VAERS soon after Covid-19 vaccine rollout. Until May 15, 2021, the count attributed to Moderna was 173, and that to Pfizer/BioNTech was 174. Myelitis, which refers to an inflammatory disease process affecting the spinal cord, is a component of TM. One may postulate that the overarching component of these may indeed be linked to an AI response. In fact, while TM can have many causes, the Mayo Clinic notes that these can include infections and immune system disorders that attack the body’s tissues [65]. The NIH explicitly describes a potential cause of TM as a “post-vaccine autoimmune phenomenon, in which the body’s immune system mistakenly attacks the body’s own tissue” [71].

3.1.2.3 Blood Clots and Bleeding Disorders The discovery of a mysterious blood clotting disorder among some recipients of the Oxford-AstraZeneca COVID-19 vaccine (not an mRNA vaccine) has led to quite some uproar soon after their rollout and caused several countries to stop using this vaccine altogether. This particular disorder manifests as a strange combination of blood clots, which can be dangerous, and potentially fatal, if they block blood flow to the brain or lungs. As detailed in [58], the disease manifests as both blood clots blocking blood vessels (thrombosis) and “a counter-intuitive deficiency of cell fragments called platelets that promote clotting” (thrombocytopenia). Given that the clots also appear in unusual parts of the body, such as the brain and abdomen, rather than in the legs, where most deep-vein blood clots form, several health authorities have acknowledged a possible link to the Oxford-AstraZeneca coronavirus vaccine. Although the syndrome seems to be seen more often in women than in men (particularly, in women aged under 60 [58]), “vaccine-induced thrombotic thrombocytopenia syndrome” has been reported in men as well and analyzed in a case study published in the Journal of Neurology, Neurosurgery & Psychiatry [2]. Determining whether an adverse event post-inoculation has actually been caused by the injection is a difficult and controversial task and will be more fully explored in relation to mRNA vaccines in Chap. 8. When blood clots following the injections first emerged, this quickly stirred questions about a possible link to the vaccine, or if these events had just occurred by chance: “Somebody who gets the vaccine could have a stroke or a heart attack a week later because they were already going to have a stroke or a heart attack” cardiologist Behnood Bikdeli is quoted in [58].

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To identify the underlying cause, attention was first directed to factors unique to the vaccine, “maybe it’s something with the vector, maybe it’s an additive in the vaccine, maybe it’s something in the production process ... ” [58]. Alas, cases for these mysterious blood clots have been mounting for all currently approved Covid19 mRNA vaccines. In April 2021, the CDC temporarily paused the J&J vaccine after confirming they were investigating six reported cases of blood clots in the USA [34]. In turn, EU regulators decided that while they had identified a possible link between the blood clots and the AstraZeneca vaccine, they thought that the benefits of the vaccine outweighed the risks [30]. Denmark’s health officials, on the other hand, decided to remove the J&J vaccine from the country’s vaccine program after concluding that the benefits of the vaccine did not outweigh the risk of blood clots [81]. Soon after that, the CDC recommends the continued use of the J&J vaccine based on the same arguments as given by the EU [56]. Nonetheless, as reports of cases of blood clots and cerebral vein thrombosis after vaccination have been increasing, both the AstraZeneca and the J&J vaccine have been suspended or recalled in Europe and the USA for that very reason. This strange type of thrombosis with thrombocytopenia syndrome, or TTS, was initially believed to be a very rare event. “If 1 million people were vaccinated with the J&J, there would be 2000 fewer deaths and 6000 fewer hospitalizations with two additional TTS cases,” one of Johnson & Johnson’s chief medical officers predicted [56]. However, already during the spring of 2021 it became clear that reports to EudraVigilance do not reflect these (and other blood clotting disorders) to be so rare at all: By May 1, 2021, there were: • For the COVID-19 VACCINE JANSSEN (AD26.COV2.S): 50 cases and 3 fatalities for the “Reaction Groups & Reported Suspected Reaction” Thrombocytopenia and Thrombotic thrombocytopenic purpura; 3 cases and 1 fatality for Embolism; and 273 cases and 25 fatalities for various forms of thrombosis. • For ASTRAZENECA (CHADOX1 NCOV-19), 5947 cases, including 71 fatalities, for the “Reaction Groups & Reported Suspected Reaction” Immune thrombocytopenia, Thrombocytopenia, Thrombocytopenic purpura, Thrombocytosis, Thrombocytosis, Thrombotic thrombocytopenic purpura; (these are the same numbers as attributed to (Autoimmune) Heparin-induced thrombocytopenia). There were also 9949 cases, including 112 deaths, associated with various forms of embolism (which is the same count as for various forms of thrombosis). • For the COVID-19 MRNA VACCINE PFIZER-BIONTECH: there were 11,931 cases and 50 fatalities for the “Reaction Groups & Reported Suspected Reaction” Thrombocytopenia, Thrombocytopenic purpura, Thrombocytosis, Thrombotic microangiopathy, Thrombocytopenic purpura; 9099 cases and 186 fatalities for the groups Embolism, Embolism arterial, Embolism venous (which is the same count as for various forms of thrombosis). • For COVID-19 MRNA VACCINE MODERNA (CX-024414): 64 cases of Thrombocytopenia, including 7 deaths; 1337 cases and 91 fatalities for the

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groups Embolism, Embolism arterial, Embolism venous (which is the same count as for various forms of thrombosis). A population-based cohort study in Denmark and Norway, published 05 May 2021 in the BMJ, confirms increased rates of venous thromboembolic events, including cerebral venous thrombosis, after vaccination with Oxford-AstraZeneca ChAdOx1-S [80]. As reports of blood clots and deaths continued to rise, in May 2021 more countries decided to restrict AstraZeneca’s COVID vaccine, including Brazil, Norway, Slovakia, and the two Canadian provinces Alberta and Ontario [28, 70, 82, 84]. Likewise, the CDC, at that time counting 28 cases (including 3 deaths) of blood clotting among people who received the J&J COVID-19 vaccine, suspected a “plausible causal association” but still maintained that the percentage of such cases was so rare that the benefits outweigh the risks [26]. The common occurrence (in significantly high numbers) of the clots implies that this is not a phenomenon unique to either of the vaccine platforms but likely relates to a common mechanism which may be linked to the massive SARS-CoV-2 viral peptide sharing with humans (Sect. 3.2), or the same vaccine-induced antigens (the spike, in particular). It is relevant to point out that in COVID-19, blood clotting disorders may be caused by the virus’s unique spike protein. In fact, it has been found that the virus triggers the production of an autoimmune antibody that is circulating in the blood, leading to a relentless, self-amplifying cycle of inflammation and clotting throughout the body [55, 106]. Given that mRNA vaccines instruct human cells to make that very spike protein, it seems plausible that the vaccine will have similar effects as seen during viral infection (see also Box 6 and Chap. 10 for more on the vaccine-induced spike). Several studies (reviewed in [102]) have corroborated the above concerns. In a particular case study, Greinacher et al. [40] analyzed 11 patients in Austria and Germany, nine of them women, with a median age of 36. They all developed thrombosis or thrombocytopenia between five and 16 days after receiving the AstraZeneca vaccine, and six died. All 11 patients, as well as 17 others who were included in the study, tested positive for antibodies against platelet factor 4 (PF4)—a protein that is seemingly unrelated to the vaccine reaction. As noted by the authors, clinically this mimics autoimmune heparin-induced thrombocytopenia. Intriguingly, none of the patients had received heparin before their symptoms started. HIT in the absence of heparin, while it is known to have occurred, is believed to be “extremely rare” [58]. The study concluded that “Whether these antibodies are autoantibodies against PF4 induced by the strong inflammatory stimulus of vaccination or antibodies induced by the vaccine that cross-react with PF4 and platelets requires further study.” Clues for the involvement of an AI reaction were already present in January 2021 in a related situation involving a healthy 56-year-old Florida doctor who died after receiving the Pfizer vaccine and developing immune thrombocytopenic purpura (ITP) [96].

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Immune thrombocytopenic purpura (ITP) is an autoimmune bleeding disorder. In the case of ITP, the immune system destroys platelets, which are necessary for normal blood clotting. As explained by the American Autoimmune Related Diseases Association,1 persons with the disease have too few platelets in the blood. Of note, ITP affects women more often than men. With girls, it is known to manifest as abnormally heavy menstruation and others. In this context, it is relevant to point out that reports to EudraVigilance following mRNA vaccine include, as of May 18, 2021, Menstrual discomfort/disorder or Menstruation delayed/irregular (1397 cases, including 3 fatalities), for the Covid-19 vaccine by Pfizer/BioNtech.

3.2 Cross-Reactivities, Vaccine Self-Adjuvancy, and Adverse Immune Responses Molecular mimicry between the virus and human proteins may be a key factor associated with the severity and multitude of the diseases encompassed within Covid-19. Several studies have suggested that the immune response raised against the virus can cross-react with those human proteins which share sequence or structure similarities with the pathogen. Significantly, this process is known to lead to harmful AI conditions [50, 63]. It seems plausible that analogous detrimental cross-reactivities could be triggered by the vaccine-induced spike antigens—which also have significant similarities with human proteins. In fact, already in 2020, Sørensen et al. [89] discovered that the spike protein of SARS-CoV-2 has 78.4% similarity with human-like epitopes. This number was obtained via a 6-amino-acid rolling window search for antibody epitopes (on the grounds that antibodies can only recognize 5-6 amino acids). Specifically, these authors found that “if all epitopes on the 1255-amino acid long SARS-CoV-2 spike protein can be used by antibodies then there will be 983 antibody binding sites which also could bind to epitopes on human proteins”—which amounts to the 78.4% human similarity to the SARS-CoV-2 spike protein. These substantial homologies between human proteins and the spike were confirmed by two other studies as well: Kanduc and Shoenfeld [50] analyzed the peptide overlap between SARS-CoV-2 spike glycoprotein and mammalian proteomes at the 6-mer and 7-mer level and discovered a massive peptide commonality with humans and mice but not with the other animals tested. Finally, a study by Lyons-Weiler concluded that “all SARS-CoV-2 immunogenic epitopes have similarity to human proteins except one” [63]. Apropos of cross-reactivities, several phenomena and notions are related, and all are known to evoke severe pathological consequences (Box 3), also in the context of vaccines.

1

https://www.aarda.org/.

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Box 3 Cross-Reactivities and Immune Responses Several phenomena have been identified that are implied with severe immuneoverreactions, including AI development, in response to vaccination: • Pathogenic priming: Peptide commonalities have long been suggested to explain some problems linked to vaccines. They have the potential to evoke dangerous cross-reactivities and have been confirmed in the context of a number of human pathogens [16, 49]. As for coronaviruses, in SARS-CoV, a type of “priming” of the immune system has been described in animal studies where vaccination against SARS led to increased morbidity and mortality in vaccinated animals when they were subsequently exposed to the wild SARS virus. Strikingly, the respective vaccines not only failed to be protective against the virus, but the animals (mice, ferrets, rhesus monkeys) which had been vaccinated against SARS-CoV developed strong immunopathologic responses and experienced markedly enhanced SARSCoV infection as opposed to their vaccinated controls [18, 100, 101]. • Antibody-dependent enhancement (ADE). The above types of unfortunate outcomes are often also referred to as (antibody-dependent) immune enhancement. It is now well known that ADE can exacerbate disease not only of coronavirus infections but those with other viruses as well. Wang et al. [100] first elucidated a mechanism enabling ADE of SARS-CoV infection in vitro and rhesus macaques. They were also able to identify antigenic peptides from the spike protein of SARS-CoV by high-cross-reactivity with a large number of antisera from convalescent SARS patients; several antibodies generated against those significantly enhanced SARS-CoV infection in vitro and in those experimental monkeys. Although the basis of ADE remains to be further elucidated, “enhancing” antibodies seem to have dual specificity and bind to virus virions and/or a crossreactive protein on the host’s cell surface [46]. This way, they may expose specific conformations that catalyze viral attachment to and/or membrane fusion with target cells that are more advantageous for the virus than for “non-enhancing” antibodies. Not much is known as to what causes the generation of enhancing antibodies, although cross-reactivities arguably play a critical role. • Original antigenic sin: In addition to the notion of ADE acting via peptide commonalities, a distinct feature of pathogen priming focuses on the increased risk of illness and death due to prior exposure [16, 49, 63] of the immunogenic protein. When triggered again, the immune system will react with an intense antibody response that involves the very specific antibodies remembered from the very first (priming) encounter with that specific immunogen (infection or injection). The dilemma that ensues is that as viruses adapt and evolve, those antibodies may be rather ineffective (continued)

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Box 3 (continued) in neutralizing a mutated virus, leading to viral escape. A particular area of concern is when vaccines do not stop all infections, something that does seem to be the case with Covid-19 vaccines as well [36]. A situation like this often renders established vaccines ineffective against new strains. This first became a concern for the 501Y.V2 SARS-CoV-2 variant identified in South Africa for which all current vaccines show reduced efficiency [83]. Serious concerns have been raised for the B.1.617 variant which first emerged in India, and which a preliminary study found to be resistant to Bamlanivimab (an antibody used for COVID-19 treatment) and which evades antibodies induced by infection and vaccination [44]. Related and more recent developments are discussed in more detail in Chaps. 6 and 10.

In the context of adverse reactions to vaccines, the self-adjuvant property of mRNA vaccines is likely playing a critical role as well. This is because of their intended functionality, to enhance the immune system’s response to the presence of an antigen. As explained by the CDC,2 “Adjuvants help the body to produce an immune response strong enough to protect the person from the disease he or she is being vaccinated against.” Albeit, as discussed, this “strong” immune response triggered by mRNA vaccines may lead to excessive immune reactions which could engender the analogous situation seen in the etiology of severe and fatal Covid19—which also resembles characteristics of an autoimmune response [10, 22, 106]. Thus, the immune response would not be raised against the virus but the vaccine RNA and its carrier molecules. Given they are not immediately degraded (Sect. 2.5 and Chaps. 9 and 10), the ongoing immune-stimulatory process could be directed at homologous human peptides as well (see also Box 6 below).

3.2.1 mRNA Vaccines as Potential Agents in the Initiation of AI, or as Triggers of AI Flares As is well known, type I IFNs have pronounced immunostimulatory effects which can promote loss of B cell self-tolerance and autoantibody production [41]. The latter can include autoantibodies against nuclear antigens, including nucleic acids. The mechanism of misguided innate immune recognition of nucleic acids has been well-studied in the context of Systemic lupus erythematosus (SLE) [41], a complex autoimmune disease characterized by glomerulonephritis, arthritis, general vasculitis, and others [67]. Triggers of SLE include viral infection, circulating

2

https://www.cdc.gov/vaccinesafety/concerns/adjuvants.html.

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nucleic acids released from apoptotic cells, or UV light. All these factors could enhance and prolong IFN production in susceptible individuals to initiate autoimmunity [41]. The key observation here is that nucleic acids can trigger type I IFN production in some contexts, provoking AI. Therefore, it is plausible that AI initiation or flares could not only be initiated by vaccine mRNAs (or their byproducts) as described above via the prolonged presence of the antigen (Sect. 3.2) but also via this intrinsic feature of nucleic acids (including synthesized ones), as promoters of systemic autoimmunity [41]. Overall, the attribution of AI flares is difficult, due to the multifaceted nature of these disorders. The coordination between pathways that regulate both degradation and sensing of intracellular nucleic acids has been recognized as pivotal to preventing inappropriate innate immune responses to self-nucleic acids [41]. Albeit, the extent to which vaccine mRNAs could disrupt this regulation is unknown. Furthermore, the impact of mRNA vaccines on immunocompromised patients is uncertain, given that these were excluded from clinical trials altogether [14]. Similarly, as older people with comorbidities and frailty as well as those with multiple chronic diseases were either largely excluded or underrepresented in the vaccine trials, little is known about safety and efficacy in these groups as well [47, 88]. Importantly, as highlighted in [47], “Pfizer/BioNTech included healthy participants, but chronic diseases could lead to the proband’s exclusion based on the investigator’s judgment in the absence of any structured selection process.”

3.2.2 Immune-Mediated Hepatitis Following mRNA Inoculation and Type III Hypersensitivity Reactions In October 2021, a case report from the UK provided conclusive evidence of immune-mediated liver damage triggered by the Moderna vaccine, published in the Journal of Hepatology [95]. The case involved a 47-year-old previously healthy man. Three days after his first Moderna Covid-19 injection he developed malaise and jaundice. Liver tests showed concerning results with markedly elevated levels, including serum bilirubin 190 µmol/L (normal 0–20), alanine aminotransferase (ALT) 1048 U/L (normal 10–49), and alkaline phosphatase (ALP) 229 U/L (normal 30–130). Also, as reported in the article [95], serum IgG was raised at 25.1 g/L (normal 6–16), IgM was at 2.2 g/L (0.5–2), and the serum was positive for antinuclear antibody, suggesting some AI or immune-mediated component. During the next couple of months, the man’s jaundice and liver function tests improved. But a few days following his second dose of the Moderna shot, jaundice returned, serum bilirubin was 355 µmol/L, and ALT 1084 U/L. The study concludes: “The pattern of injury on histology was consistent with acute hepatitis, with features of autoimmune hepatitis or possible drug-induced liver injury (DILI), triggering an autoimmune-like hepatitis.”

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The case in [95] is not an isolated one. The Journal of Hepatology [95] describes other severe forms of autoimmune hepatitis following either Moderna or Pfizer inoculation as well. In all these cases, liver histology revealed similar findings, indicating acute hepatitis with interface hepatitis. In a fascinating lecture on this,3 Dr Mobeen Syed gave an interesting explanation of how the blood vascular system may have become inflamed and damaged. He reasons that (vaccine-induced) antibodies may be circulating in the blood. Consecutively then, when they come out of the blood, they attack whatever cells are nearby. In this case, it would be the cells at the boundary between the liver and the blood system. More generally, Mobeen believes that the antibodies could form complexes with the spike from vaccination. When these immune complexes incorrectly sit down on the surface of blood vessels, the kidney, or other barriers (e.g., the BBB), macrophages will attack that surface, leading to local inflammatory reactions.4 The potential that products from the injections could lead to type III hypersensitivity reactions has not received much attention, certainly because of the prevailing view that the injected material does not get into circulation, at least in large enough quantities. However, as detailed in Chap. 10 below, vaccine-induced spike has been confirmed in the blood and also carried on exosomes for weeks following inoculation, and in quantities higher than after severe Covid-19.

3.2.3 The Potential of Immunopathologic Th2 Responses The puzzling and often disparate function of T cell immunity is also evident in the context of another type of T helper cells (Th cells), known as CD4+ cells. These also play important roles during an adaptive immune response. They help to polarize the immune response into the appropriate kind, depending on the nature of the immunological insult. Differentiated CD4+ T cells play a crucial role in providing beneficial immune responses against invading pathogens, but conversely, they are also implicated in the pathology of autoimmune inflammation. All T helper lymphocytes start out as naive Th0 cells, which, after being activated, are capable of differentiating into either Th1 or Th2 effector cells. For most infections, type 1 immunity is protective, whereas type 2 responses assist with the resolution of cell-mediated inflammation [90]. Albeit, these responses can also have significant pathologic consequences. Interestingly, a study on Th polarization in acute COVID-19 published in Frontiers in Cellular and Infection Microbiology [37] found a higher proportion of activated Th2 cells in the COVID-19 patients compared with the reference population. Moreover, a higher percentage of senescent Th2 cells was found in the

3 4

https://www.youtube.com/watch?v=Tfjopb-2kKs. https://www.youtube.com/watch?v=-Y7dTMzn9B8&t=4s.

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patients who died than in those who survived. On the other hand, less severe cases of SARS were associated with accelerated induction of a Th1 cell response [105]. Accordingly, the T helper cell phenotype of vaccine-induced T cells has been recognized as important to the protection they mediate. While it has been recognized that Covid-19 vaccines should evoke a Th1 response [48], there is limited knowledge as to the exact type of the Th-immune response that is actually triggered in different clinical contexts. Importantly, differentiation and regulation of CD4+ T cells depend on a plethora of factors including the strength of antigen-antibody interaction, amount of costimulation, cytokines present in the milieu, and others [38]. Notably, all these are factors that are poorly understood in the context of mRNA-based vaccine immunity. Immunopathologic Th2 responses have long been a major problem in the design of coronavirus vaccines as they are associated with disease enhancement. This is a syndrome which has been reported in the past for a few viral vaccines. Not only were those who had been immunized found to have an increased frequency of infection; tragically, many of them suffered increased disease severity when they later encountered the virus, and some children even died (reviewed in [57]). Given that some Middle East respiratory syndrome (MERS) and SARS-CoV-1 vaccines have shown evidence of disease enhancement in vaccinated animals after viral challenge, the same is a particular concern for SARS-CoV-2 vaccines [57]. Of note, this syndrome of disease enhancement is distinct from ADE (Sect. 3.2) which enhances uptake of the virus (e.g. when the back-end of the antibody binds to macrophages and thereby helps the virus enter those cells [77]). Although the pathogenesis of disease enhancement is still unclear, animal models with the Respiratory syncytial virus (RSV) have shed some light on the underlying mechanisms. Trials with inactivated RSV vaccine have suggested that life-threatening disease enhancement is characterized by immunopathology via Th2 biased antibody responses, wherein the pathology was associated with a high ratio of non-neutralizing antibodies to neutralizing antibodies (reviewed in [57]). To mitigate these types of adverse events, focus has been placed on specific adjuvants which are expected to drive the immune response toward Th1 [57]. However, as pointed out in [89], most adjuvants have a strong Th2 bias. Indeed, this has long been deemed necessary to achieve a good neutralizing antibody response. In this regard, there is no consensus among vaccine developers, and this is reflected in vaccine design as well. For mRNA vaccines, T cell immunity was assessed in some animal models, which did not show any evidence of a Th-2-directed CD4+ T cell response in those animals [31, 32]. It is unclear if the same applies in a variety of clinical contexts, especially since the self-adjuvant nature of mRNA vaccines has been recognized as a “double-edged sword.” Overall, then, it cannot be concluded that mRNA vaccines cannot evoke any life-threatening Th2 responses. At particular risk may be immunocompromised individuals and the still largely unknown group of people who do not mount a sufficient neutralizing antibody response (Sect. 4.9). The problem may get further accentuated by viral immune escape mutants (Sect. 6.2).

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The sobering result could be a vicious cycle: people may end up being less protected and instead experience increased symptoms or even death. Yet, this would not be because they had not been inoculated. In fact, it would be the result of vaccine-induced disease enhancement—of the type that is fostered by vaccines with poor neutralization activity as postulated in ref. [57].

3.3 Shooting the Messenger of Critical Human Proteins A known concern with RNA technologies is that the synthesized mRNA is itself often targeted and destroyed by the innate immune system [20]. In particular, this unwanted immune response is known to be evoked by dsRNA contaminants, derived from aberrant RNA polymerase activities, which leads to the inhibition of translation and degradation of cellular mRNA and ribosomal RNA [98, 103]. The same could be initiated by dsRNA byproducts formed in vivo, however. Thus, due to the massive SARS-CoV-2 epitope peptide sharing with humans, it is likely that this dsRNAmediated destructive process of “shooting the messenger” [20] could not only demolish the vaccine mRNAs but also those of essential human proteins. Clearly, the pathological consequences of this could be manifold. Two specific situations are worth mentioning.

3.3.1 Impairment of the Adaptive Immune System Due to the above-mentioned commonalities between SARS-CoV-2 immunogenic epitopes and human proteins, it is plausible that a dsRNA-mediated anti-mRNA response may even be mounted against the adaptive immune system itself. In fact, ref. [63] found that over 1/3 of the human proteins that potentially may be targeted (putative autoantigens) are key to the adaptive immune system. As discussed, these same epitopes might also be responsible for autoimmunological pathogenic priming due to prior infection or following SARS-CoV-2 vaccination. Overall, then, vaccination could result in a misguided immune response that involves both arms (innate and acquired) of the immune system. As with other AI issues, this could be further worsened by chronic exposure to the antigen(s) (infection or injection).

3.3.2 Shooting the Messenger of Tumor-Suppressing Proteins Another problem with dsRNA-instigated inhibition of translation and degradation of mRNA [20] is in the context of genes with tumor-suppressing function. Notably, changes at the mRNA level could be drivers of leukemia and other cancers, as only discovered in recent years. Surprisingly, a study [59] published in Nature

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found that it is indeed specific mRNA events, and not DNA mutations, that have the same ultimate effect as known drivers in chronic lymphocytic leukemia (CLL), and likely other cancers as well. Precisely, these authors found that a substantially greater number of CLL patients had an inactivation of a tumor-suppressor gene (TSG) at the mRNA level than those who had it at the DNA level. It is believed that due to changes at the mRNA level, the protein made from the mRNA can be truncated and not function properly. In addition to the inactivation of known TSGs through aberrant mRNA processing, this study also identified previously unrecognized TSGs that are inactivated in leukemia. This leads to the question if the inactivation of tumor suppressors could be realized through other mechanisms than those identified—which occurred through intronic polyadenylation (IPA) or more general alterations in mRNA processing [23]. Critically, via the above-mentioned aberrant dsRNA-mediated mechanisms to destroy not only vaccine mRNAs but also human mRNAs with sufficient homologies, then, if the latter involve TSGs, this could likely promote tumorigenesis as well.

3.4 Contaminants of IVT Processing During the last few years, the unwanted immunogenicity of RNA vaccines triggered by RNA sensor activation was thought to be resolved by a number of critical innovations; these include the incorporation of modified nucleosides, optimization of coding sequences, and stringent purification of IVT mRNA from dsRNA and other manufacturing contaminants [75]. However, there are several reasons to believe that this problem has not been adequately resolved. • There is no scientific consensus on how these modifications and purification strategies ought to be realized, as is obvious by the (often mutually opposing) approaches pursued by different vaccine manufacturers (see, e.g., [7]). • While under stringent laboratory research conditions, it may be possible to eliminate a large proportion of contaminants, this may change under the pressure of mass-production processing. Indeed, the EMA Public Assessment Report of the Pfizer/BioNTech vaccine admits that “Truncated RNA species are regarded as product-related impurities and can be expected due to the principle of the in-vitro transcription reaction” [31]. • Neither of these strategies can fully prevent harmful dsRNA formation in human cells, and neither can they prevent the generation of other undesirable products such as truncated species derived from IVT processing. Although the EMA is aware of this, in their assessment they state that “the overall characterization of the truncated species is still limited,” and “[t]he company [BioNTech Manufacturing] does not expect truncated transcripts formulated in the finished product to pose a safety or efficacy concern, as in their view no protein expression is expected from truncated transcripts” [31]. However, the potential of those

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contaminants to engender dsRNA generation in vivo, to act as RNA activity modulators, or otherwise interfere with endogenous RNA regulation does not seem to have been considered (for more, see below).

3.5 Dichotomous Immune Response and Attribution of Adverse Events The induced expression of type I IFNs (IFN-α and IFN-β), accompanied by proinflammatory cytokines such as TNF-α, IL-6, and IL-12 in the same cell and adjacent cells, activates more than 300 IFN-stimulated genes, including the protein kinase PKR [7]. This, and the systemic distribution and expression of immunogens, likely evokes dichotomous immune response mechanisms, which over time can result in deleterious systemic effects including AI pathologies. The difference between the acute and prolonged immune response discussed above has far-reaching consequences of how adverse events after vaccination are captured and interpreted. Databases such as VAERS cannot and do not determine whether a vaccine caused a certain adverse event. By mid-May 2021, the US Centers for Disease Control and Prevention (CDC), which monitors these events, maintains that they have no reason to believe that COVID-19 vaccines cause death [68]. Yet, it is unclear how a conscientious investigation of, at that point, over 4 thousand deaths, and still counting, was possible in such a short time (see also Sect. 5.4). The CDC seems to have a very limited view of when adverse events could be linked to a vaccine. While details of their policy are unknown in general, certain criteria have been made public. For instance, “Anaphylaxis and nonanaphylaxis allergic reaction cases with symptom onset occurring later than the day after vaccination (i.e., outside of the 0–1-day risk window) [are] excluded because of the difficulty in clearly attributing allergic reactions with onset outside this risk window to vaccination” [12]. This perspective does not align with the dichotomous self-adjuvant nature of the vaccine mRNA. This short time frame is also contrary to previously established immunological testing routines. Before the pandemic, specific molecular signatures in the blood induced a few days after vaccination were used to predict the magnitude of later innate and adaptive responses to vaccination [25]. This problem is even more pronounced from the perspective of genetic alterations fostered by vaccination (see below), for which delayed adverse events are a major problem. In fact, according to the FDA, one of the greatest concerns with gene-therapy products is delayed adverse events (Sect. 4.12). As a result, the short risk window suggested by the CDC does not do justice to the full spectrum of adverse events which can be triggered by mRNA vaccines. The topic of attribution of adverse events to the vaccine and related causality associations will be further analyzed in Chap. 8 based on experience that emerged post-vaccination rollout.

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3.6 The Real Potential of Creating Genetically Modified Humans Humans are excluded from the very definition of genetically modified organisms (GMO). This exclusion was probably based on the assumption that the intended act of turning humans into GMOs would be much too unethical, controversial, and risky. This section considers the danger of this happening via RNA vaccines.

3.6.1 DsRNAs and Their Role in Mutagenesis It does not seem to be the case that the issue of dsRNA byproducts from the vaccines and their potential for mutagenesis has been assessed before mRNA vaccine rollout. This section aims to raise concerns in this regard.

3.6.1.1 From Synthetic mRNAs to dsRNAs to Genetic Modifications As succinctly spelled out by Heinemann, there are numerous mechanisms how exodsRNAs could act as mutagens [43]. Although ref. [43] considered the general framework of eukaryotic cells or organisms, many of the means of modifying genes or genetic material described in [43] are also relevant to humans. Firstly, the processes and mechanisms of [43] may be fostered by the human microbiome (the many micro-“organisms,” including viruses, which collectively contribute to what has become known as the human metagenome); arguably, this can play a critical role, e.g., through RNA-RNA recombination, to enable dsRNA formation from vaccine RNAs, but then also to enable transport to the nucleus and retro-integration. Indeed, as reviewed in [43], various forms of recombinations via microorganisms are known, involving different dsRNAs which later have even been identified in the chromosomes. The second group of mechanisms studied in [43], which leads to dsRNAs mediated mutagenesis, relies on special activities such as via RT and regulatory RNAs. Albeit, these processes are present in human cells as well. Specifically, in terms of dsRNAs enabling mutagenesis, Table 3.1 summarizes the most relevant arguments from [43] which also pertain to vaccine RNAs and their dsRNA byproducts. It demonstrates that synthetically derived mRNA, via dsRNA intermediates, can cause modification of genes or other genetic material that is capable of being inherited by the progeny of the organism, or capable of causing a characteristic or trait that can be inherited. In the following, these critical issues will be further investigated from different contexts.

Key findings Exo-dsRNAs and their activities are not confined to the cytoplasm

Facts to support these findings—as cited from [43]a “However, the [EPA] Committee incorrectly concluded that exo-dsRNA always would be confined to the cytoplasm of exposed cells of organisms. This error undermines the conclusion that dsRNA is not heritable or cannot act as a modifier of genes or other genetic material...” “...exo-dsRNAs may be conducted to the nucleus in association with a variety of proteins including Dicer and NRDE-3 (Mao et al., 2015; Various, n.d.)” “Further evidence of transport is provided by Djupedal and Ekwall (2009) writing about heterochromatin formation. They said: ‘Exogenous siRNAs are thus capable of stable and specific epigenetic regulation of target genes.’ Those genes are located in the nucleus and the epigenetic regulation comes from chemical modifications made to DNA nucleotides and/or histones” “...in many eukaryotes cytoplasmic and nuclear compartments regularly mix. The nuclear envelope breaks down every cell cycle in eukaryotes with open mitosis, resulting in mixing with the cytoplasm (Gorlich and Kutay 1999; Smoyer and Jaspersen 2014). This cyclic breakdown provides, for example, the Argonaute protein-associated RNA access to the chromosomes (Li 2008). In animals at least, the nuclear envelope can also rupture, resulting in mixing of content (Hatch and Hetzer 2014)”

(continued)

• Obviously, this is true in general, regardless of the type and source of the RNAs in question • In addition, infection with certain parasites or chronic infections can induce widespread genomic instability, as has been shown with Plasmodium infection [51], for example

The same is true for vaccine-derived dsRNA fragments or their byproducts (Box 1, Fig. 2.2); see also Sects. 3.6.2, 3.6.3 Again, based on the observation that dsRNA byproducts from vaccine RNAs can be transported to the nucleus via several mechanisms (Sect. 2.3), the same conclusion as in [43] can be made here

Comment Given the real potential for aberrant dsRNA production from mRNA inoculations (Sect. 3.1), the same argument of [43] applies to these vaccines as well

Table 3.1 Insights and lessons learned from environmental exo-RNA treatments regarding their potential for mutagenesis, applied to mRNA vaccines

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Exo-RNAs can be replicated by reverse transcription

Key findings Genes are not confined to the nucleus

Table 3.1 (continued)

Facts to support these findings—as cited from [43] “Even if it were the case that exo-dsRNA was confined to the cytoplasm, eukaryotes have genes there too. Cytoplasmic organelles called mitochondria and chloroplasts have DNA genomes. Separate from them, some eukaryotes have self-replicating DNA and RNA elements in the cytoplasm” (cont.) “Moreover, these [self-replicating] elements have acquired genes from other organisms and other dsRNA elements through RNA-RNA recombination, making it possible for them to acquire sequences directly from exo-dsRNAs (Ramirez et al. 2017)” “Presumptive exclusion of dsRNAs from the nucleus does not prevent interaction with these cytoplasmic genes and therefore possible ongoing replication of the exo-dsRNA through linkage” “Another potential barrier to inheritance would exist if exo-dsRNAs could not be reverse transcribed” “...there is substantial evidence indicating that RNAs can be, and have been, reverse transcribed and incorporated into eukaryotic genomes. A variety of enzymes commonly found in eukaryotes have reverse transcriptase activity (Goic et al. 2013). By some estimates, as much as 30% of the mammalian genome, and 10% of the human, was created by the action of reverse transcriptase activity originating from retroviruses (de Parseval et al. 2003)” “We do not know whether all exo-dsRNA molecules could serve as substrates for reverse transcriptase, but it is unlikely that none could”

The same can be said for IVT mRNAs and their dsRNA byproducts

The analogous erroneous presumptive exclusion is applied to IVT RNAs—where those and similar mechanisms do not seem to have been considered by vaccine manufacturers The analogous flawed assumption is applied to externally derived RNAs from vaccines Reverse transcription is a broad mechanism, not limited to specific types of RNAs. It is very versatile and can proceed in the presence of specific but also non-particular primer molecules, even in human cells (Sect. 2.3.2 and Chap. 10)

The analogous applies to vaccine-derived dsRNA products via self-replicating elements known for humans (Sects. 3.6.2, 3.6.3)

Comment Obviously, the same applies to externally derived mRNAs from vaccines and their unassessed impact on mitochondrial genes

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a For

“chemical modifications of DNA and chromatin, for instance, affecting the degree of chromatin compaction or the accessibility of regulatory sequences to transcription factors... is a kind of modification that can result from a treatment with exo-dsRNAs...” “RNA-directed DNA methylation caused by dsRNA can result in heritable effects... Once methylation has occurred, it can be propagated independently of further stimulation by exo-dsRNA... The use of exo-dsRNA could result in targeted mutations in the eukaryotes that have RNA-directed DNA methylation pathways” “...exo-dsRNA may replicate independently of the DNA genome using RdRP-based amplification” “exo-dsRNAs can modify DNA in chromosomes in some cell types or species. Modifications include heritable methylation of nucleotides and histones, DNA deletions and rearrangements, and changes in chromosome copy number”

“dsRNA can cause the same range of modifications that can be caused by mutagens,” including DNA deletions, changes in chromosome copy number, modifications of nucleotides, or other heritable DNA rearrangements

the exact details of the literature quoted in the table, please see ref. [43]

Exo-RNAs are heritable

Exo-RNAs can act as mutagens

This range of modifications is not limited to specific RNA species; it is therefore highly likely these are also relevant for IVT mRNAs, or products thereof

The same concerns also RNAs from vaccines (Sect. 2.6.2)

As above

Heinemann [43] did not analyze this question for IVT RNAs. However, given that byproducts from RNA vaccines can enter the same pathways as other short RNAs (Box 1), these types of effects on humans or their microbiome cannot be ignored (see also Sects. 2.3, 3.6.2, 3.6.3) Again, the same likely also applies to dsRNAs formed from IVT RNAs (Box 1)

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3.6.1.2 DsRNAs, Even in Small Amounts Can Regulate Gene Expression RNAi pathways are commonly triggered upon the recognition of structural differences between viral and cellular RNAs. Notably, dsRNAs can activate such innate immune triggers, even when present in small amounts only. For instance, ref. [66] details how RNAi represents a potent response to infection with vesicular stomatitis virus (VSV), which is a negative-strand RNA virus. More generally, RNAi is known to represent a major host-defense pathway against other RNA viruses, irrespective of their genome. However, surprisingly, VSV does not produce readily detectable amounts of dsRNA in infected mammalian and Drosophila cells. Yet, these authors were able to show that the small RNAs triggering RNAi were derived from dsRNA. As they were unable to detect dsRNA, they posit that very small amounts of dsRNAs are sufficient to trigger this pathway, or that dsRNAs undergo some unexpected modifications that do not allow their identification via the detection methods used. In the context of mRNA vaccines, this shows the difficulty of identifying dsRNAs in living cells, and of predicting their effects. Even if RNA vaccines may only lead to low levels of dsRNAs in vivo, this may be enough to trigger unwanted and harmful RNAi processes, including those described in [43] leading to mutagenesis.

3.6.1.3 Repeated Vaccination May Increase the Risk of Unintended Heritable Changes As detailed by Heinemann [43], dsRNAs acting as regulatory molecules may evoke ongoing RNAi processes and may even be heritable. Albeit, as pointed out in [43], the issue of transgenerational effects of exo-RNAs has not been extensively studied. Two of these less-appreciated features are particularly relevant to RNA vaccines: (1) while RNAi can be self-limiting, it does not in all cases self-extinguish, and moreover, (2) the limiting response can be reduced by repeat exposures to the exo-dsRNA [43, 45]. Because mRNAs from vaccines may form dsRNAs, this can become a serious issue with booster vaccines. Notably, exogenous and endogenous dsRNAs compete for biochemical components of the RNAi pathway. It is therefore likely that traits made stable and heritable by critical endo-dsRNA become destabilized through competition with exo-dsRNA. Heinemann [43] concluded that any attempt to determine the longevity of exo-dsRNA-mediated RNAi must define how often an organism will be exposed to exo-dsRNA. This is because the “ ‘transgenerational timer’ is being reset by initiation of new RNAi responses, and therefore ‘second triggers’ extend the inheritance of ancestral silencing” (Houri-Ze’evi et al. 2016). Exposure frequencies will determine the duration of the effect both in time and number of generations.

Due to the potential of dsRNAs forming from vaccine RNA, the same critical observation also applies to mRNA vaccines. It is important to realize that this type of inheritable effect is not specific to the sequence of the genes controlled by particular RNAs and can affect the production

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of proteins necessary for intergenerational transmission of RNAi caused by specific dsRNAs [43]. So, overall, there are various ways how the innate immunogenicity and resulting activation of RNA sensors can lead not only to deleterious inflammatory responses but simultaneously promote the integration of some of the foreign RNA into the human genome (see Fig. 3.1 for a summary).

Fig. 3.1 DsRNAs in human cells and through mRNA vaccines. The nuclei of human cells contain dsRNA that are involved in many biological processes. By contrast, when dsRNA enters the cells from the extracellular milieu into their endosome or cytoplasm, it is sensed as a viral invader. DsRNAs trigger various innate immune receptors, which prompt the secretion of inflammatory cytokines, general viral defense mechanisms, and an anti-mRNA translation response, independently of whether its source is from viral or other exogenous sources (including IVT processes). This has led to quite some effort to reduce dsRNA contaminants from IVT processing, which in part can be achieved, and also leads to increased translatability of the synthetic mRNAs (but see also Chap. 10). Until recently, the self-adjuvant feature of vaccine RNA was regarded as beneficial. However, triggering of the innate immune processes can result in excessive and harmful inflammatory processes (Sect. 3.1), or evoke other unknown components of the innate immune system [75]. As described in the text, the same mechanisms that result in harmful dsRNA formation in vitro may happen in living cells as well (see also Fig. 2.3)

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3.6.2 Pathogenic Endogenous RT Activity in Human Cells via Transposable Elements As outlined in Sect. 2.3.2, human cells harbor a variety of mechanisms that support reverse transcription and integration of vaccine RNAs into the genome. The majority of these are incompletely understood, including those that derive from mobile pieces of DNA (even though this comprises more than half of the human genome). Although the bulk of these transposable elements cannot transpose, some of them have the ability to mobilize [53]. A major class of mobile DNA includes the retrotransposons which incorporate both an RT activity and facilitate reverse transcription of DNA and integration into the human genome. More specifically, the retrotranscription of human LINE-1 elements is mediated by a LINE-1 encoded reverse-transcriptase (ORF2p) and supporting proteins. Interestingly, these are known to retro-transpose not only LINE-1 transcripts (in Cis) but also other transcripts (in Trans) including Alu elements and cellular mRNAs [52]. Mobile elements have shaped, over evolutionary time, a transcriptional network, affecting, e.g., interferon response of innate immunity, and are also responsible for a great deal of interindividual variation in the population [53]. Actually, among all the LINE-1 poly(A) retrotransposons (of which we have about 500,000) and other “jumping genes,” a small number is still active, can disrupt genes, and even mediate disease. Owing to a lack of evolutionary adaptation, the obtained mutations are believed to always be deleterious, and the same likely applies to the retro-integration of vaccine RNAs. In fact, in humans, LINEs are the major source of insertional mutagenesis and are involved in both germinal and somatic mutant phenotypes [27]. Pathogenic retrotransposition events mediated by LINE-1 alone account for approximately 1 in every 250 pathogenic human mutations. In addition to somatic mutations in exons, introns, or regulatory regions, these types of events can generate copy-number variations and other genomic rearrangements and may contribute to cancer and neuropsychiatric disease [53].

3.6.3 Retro-Integration of IVT RNAs May Be Triggered by Several Mechanisms Section 2.3.1 mentioned briefly how RT activity in human cells even involves SARS-CoV-2 RNAs. The study by Zhang et al. [104] rests on two mechanisms. The ability of SARS-CoV-2 to reverse-transcribe and become integrated into the human genome was demonstrated in cells overexpressing a specific RT (LINE-1 or HIV-1 RT). Surprisingly, however, it was also revealed that LINE-1 expression could be mediated either upon SARS-CoV-2 infection or by cytokine exposure in cultured cells.

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With vaccine RNAs, we are encountering both of these situations. On the one hand, they mimic actual infection, and, on the other, they (or their dsRNA byproducts) activate the secretion of interferons and inflammatory cytokines. Alas, if cytokines alone can induce LINE-activation—as demonstrated in [104]—then the same may happen also upon vaccination and lead to the same effects as described in [104]. Altogether, the integration of vaccine RNAs into the human genome cannot be ruled out. The consequences of such events are likely going to disrupt cellular homeostasis and elicit severe forms of disease. An overview of the main consequences is given in Fig. 3.2. The above issues are heavily debated and will be further explored in Chap. 10.

Fig. 3.2 Potential consequences of vaccine RNA integration events. Viral integration is a phenomenon that remains incompletely understood. While obligatory for some viruses (such as retroviruses), it is also known to occur for other viruses, including RNA viruses as well [19]. Viral integration may promote long-term persistence of the virus and can have drastic consequences for the host, such as gene disruption, oncogenesis, and cell death, and even lead to lasting consequences for the host by ultimately shaping their genome [19]. The figure predicts the possibility of specific integration events of vaccine RNAs, in analogy to those known for viruses. Vaccine RNA integration may not engender the full spectrum of effects as known for viruses. For instance, virus-induced cellular transformation can trigger rapid tumorigenesis simply because specific viruses such as onco-viruses can directly code for oncogenes [19]. Nonetheless, vaccine-induced integration events may, as known for different viruses, induce slow tumorigenesis processes and other events which, depending on the type and site of integration may have multiple adverse clinical consequences

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3.6.4 Genomic Integration and False PCR Tests Following Vaccination The above-mentioned study by Zhang and coauthors also demonstrates that in addition to SARS-CoV-2 being reverse-transcribed and genome integrated, it is also possible that the integrated sequences can be transcribed. This may help explain the puzzling clinical situation of the presence of viral sequences in the absence of detectable infectious viruses. Importantly, this may explain why patients, already recovered from disease symptoms, may become positive again for viral sequences as detected by PCR. However, as pointed out in [104], this also leads to a rather critical issue: The reliance on the PCR test may not reliably reflect infection status. This is because the PCR assay may pick up the viral transcripts from those sequences stably integrated into the genome as opposed to those from the infectious virus. The same concern applies to RNA vaccines. Since it seems feasible that their RNAs could also get integrated into the genome (Fig. 3.3) via the action of various RTs, then it is similarly possible they could get expressed as well. Given that the expressed antigens resemble unique epitopes of the spike, then, in addition to potential adverse effects, these proteins could impair the sensitivity of PCR assays to correctly identify those patients who actually have Covid-19.

3.6.5 Relevance for Clinical Trials and Antiviral Therapies Ref. [104] raises an important issue that is equally relevant to RNA vaccines. Zhang et al. suggest that the route via retro-integration constitutes a novel aspect of infection by itself, and that it may even affect the course and treatment of the disease. Although it is unclear if, or to what degree, the integrated sequences can express viral antigens, the retro-integration of the RNAs could be detrimental nonetheless. The clinical consequences may depend on unique factors, such as the integration site, epigenetic regulation, and individual’s underlying conditions. Zhang et al. posit that this could cause a more severe immune response such as cytokine storm or autoimmune reactions. Given that the same mechanisms can be evoked by vaccineRNA integration and expression, these same concerns apply in this situation as well; it is important to note that these harmful consequences are not by virtue of the (detrimental) self-adjuvant effect discussed above (Sects. 3.2 and 3.2.1), but now owing to retro-integration of vaccine RNAs.

3.6.6 Ribonucleotides as Harmful DNA Lesions The presence of ribonucleotides in DNA has been recognized as a critical driver of severe pathologies. Ribonucleotides are the most common DNA “lesions.”

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Fig. 3.3 The interrelationship between the inherent immunogenicity properties of vaccine RNAs and their detrimental consequences on safety and mutagenesis. Traditionally, RNAs were regarded as having a clearly defined role that is easily predictable, in particular as this relates to mRNAs—to carry the genetic information from DNA in the nucleus (“the message”) to the ribosomes so these can make the appropriate protein. However, intending a particular RNA to be mRNA does not mean that it always will be. The role of RNAs in terms of their entire life cycle, as regulatory elements, as key players to trigger immune responses, and their potential to act as mutagens, are far beyond what is predicted (and possibly, predictable), from their envisioned role as vaccine mRNAs. This figure summarizes the potential for undesirable effects of vaccine RNAs in terms of their inherent immunogenicity and, moreover, how this could support mutagenesis

Embedded ribonucleotides in DNA interfere with DNA repair, disturb normal biological processes such as transcription, and are also responsible for chromosomal instability [91]. In particular, ref. [41] provides strong evidence that embedded ribonucleotides can cause UV-induced cyclobutane pyrimidine dimer (CPD) formation and enhanced type I IFN signaling which can promote autoimmunity. It was found that certain mutations that impair the function of important ribonucleases result in the accumulation of ribonucleotides in genomic DNA. It is believed that the ensuing chronic low level of DNA damage triggers a DNA damage response, and in turn, enhanced type I IFN response processes to the immunostimulatory nucleic acid. It seems plausible that the same process could also be triggered by the retro-integration of vaccine RNAs, leading to either AI flares, or engendering the development of new AI diseases.

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A range of different AI conditions seems to be induced by the defective removal of ribonucleotides from DNA [41]. The etiology of all these has been linked to a misappropriate coordination of pathways that regulate the degradation and sensing of intracellular nucleic acids. Any resulting imbalance triggered by the unexpected accumulation of vaccine RNAs (which would be further intensified by self- or transamplifying vaccine platforms), may critically impair these processes. As in [41], this could lead to an inappropriate innate immune response to self-RNA or -DNA and thereby promote systemic autoimmunity. It seems likely that the mutagenic nature of vaccine RNAs could initiate the same deleterious processes. Albeit, under the assumption that mRNA vaccines cannot get integrated into the genome (Fig. 1.1), the potential effects of vaccine RNA lesions have apparently not been elucidated.

3.7 Impact of Truncated IVT mRNA Species and Other Short RNAs Derived from RNA Vaccines Above, the focus of dsRNAs has been, firstly, related to their potential to provoke excessive and undesirable immune responses, and secondly, as RNAi-instigating agents enabling mutagenic processes. Short RNAs play many different roles, however. Indeed, RNA biology has changed significantly over the last decades as more and more surprising activities of short regulatory RNAs have been identified. Regulatory RNAs such as miRNAs play critical roles in modulating gene expression and are involved in various physiological and pathological events [72]. They help control mRNA stability, translation, and even transcription and act as extracellular/circulating messengers, and others [9, 72]. A critical question that does not seem to have received a lot of attention concerns the extent to which vaccine RNA byproducts can act as, and/or interfere with, human short RNA regulation.

3.7.1 Interfering with Endogenous RNAi Processes and miRNA-Regulated Gene Expression The RNAi interference pathway was first recognized in Caenorhabditis elegans [35] as a defensive response to exogenously introduced long dsRNAs and has meanwhile been recognized as a ubiquitous form of gene regulation with many biotechnological applications. From the core process of RNAi itself, several reasons can be inferred as to why RNA byproducts may turn into RNAs with active si/miRNA-like regulatory functions (see Box 1 above).

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3.7.1.1 Vaccine Byproducts May Become Precursors of Regulatory RNAs The canonical inducers of RNAi are various long dsRNA molecules introduced into the cytoplasm or taken up from the environment [9]. Such RNA is further processed to siRNAs of about 20–30 nucleotide (nt) length by cytoplasmic Dicer (although Dicer-independent biogenesis pathways have also been reported [15]); then, by interfering with the expression of genes with complementary nucleotide sequences, they form the heart of the RNAi machinery. Importantly, the RNAi pathway shares protein components of the miRNA pathway [93]. Thus, while the former employs siRNAs derived from long dsRNAs, in animals, endogenous miRNAs function similarly to RNAi processes (Box 1); however, they allow only partial complementarity between the miRNA and the target RNA, and instead of cleavage of the target RNA, lead to translational inhibition [54]. Thus, owing to the vast range of precursors to the miRNA/RNAi pathways (including foreign or invasive nucleic acids and viruses [9]), it is likely that truncated species and other vaccine-derived RNAs (Fig. 2.2) will enter these as well.

3.7.1.2 The Initiation of RNAi Processing Does Not Hinge Upon Specific Genetic Features of the Instigating dsRNAs Regardless of the different forms of biogenesis, all of the si/miRNAs derive from longer dsRNAs and are then converted into their active form. That is, RNAi action is not dictated by specific genetic determinants of their precursors. Instead, both cell type and cellular environment dictate the directionality of the two strands, and which of these get loaded into the Argonaute (AGO) family of proteins to form an RNA-induced silencing complex (RISC). Moreover, the target specificity of the single-stranded siRNA (part of the RISC complex) is determined by complementary sequences on target mRNA, called micro RNA response elements (MREs). And finally, it is the degree of MRE complementarity which dictates the type of RNAi response (silencing or cleavage of the target mRNA) [72]. Overall, it is impossible to say that mRNA vaccines or their byproducts are incapable of initiating RNAi processes as well.

3.7.1.3 Overlapping Activities of si/miRNA Precursors Can Be Induced by a Range of Different dsRNAs The pathways of miRNA- and siRNA- mediated gene silencing were long thought to be completely different. Arguably, miRNA-guided gene silencing is distinct from siRNA-guided targeted protein knockdown. Nonetheless, as mentioned, despite unique characteristics according to their biogenesis and related to different organisms and cell types, distinctions between small RNA regulation are difficult to obtain. MiRNAs can function as siRNAs and vice versa (Box 1 above), and this

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also applies to mammals. Once a long dsRNA is encountered by Dicer, the pathway induced by the siRNAs mechanistically essentially merges with that induced by miRNAs [93]. In this regard, siRNAs cannot readily be distinguished from miRNAs, and no clear distinction can be made in the kinds of silencing that these dsRNAs cause. In fact, this mechanistic overlap, once in the cytoplasm, is the core of all geneediting technologies. As such, the experimental gene-knockdown in mammalian cells is either instigated by short RNAs which are synthetically generated siRNAs or by expressed miRNA-like molecules [93]. All this may have important consequences for unexpected vaccine byproducts (of various lengths), namely to become substrates in the pathways leading to RNAi. There is one feature, however, which could limit the ability of vaccine RNAs to enter the RNAi pathway: Long dsRNAs are often sensed as foreign by animals with a more sophisticated immune system and consequently targeted by various innate immune responses. However, this is not always the case as will be analyzed more fully in Sect. 3.7.2. Overall then, it seems plausible that both short and long dsRNA vaccine byproducts (Fig. 2.2) can assume si/miRNA-like functions and thereby exhibit unanticipated regulatory activities with rather undesirable implications.

3.7.2 Interfering with the Balance Between IFN and RNAi-Based Antiviral Defense Mechanisms in Mammalian Cells As is well established, innate immune responses are essential for protection against viral invasion, and inadequate responses are associated with severe disease. Most non-vertebrates, such as insects, plants, and nematodes, rely mainly on RNAi for antiviral defense. By contrast, in vertebrates, while RNAi is regarded as an ancient defense mechanism against viruses and mobile elements [93], the exact role of RNAi as a defense mechanism is less clear [86]. It is believed that in mammals, endogenous RNAi was largely outstripped during evolution by the current innate and acquired immunity [93]. Animals with more advanced immune systems possess large-scale, protein-based innate, and adaptive immune sensory systems to discriminate self from non-self nucleic acids. In particular, long dsRNAs in the cytosol are a hallmark of viral replication as they are absent from uninfected host cells. Notably, higher eukaryotes have host cell pattern recognition receptors (PRRs) which sense viral dsRNAs as pathogenic. DsRNA sensing by cytosolic PRRs can be divided into several pathways. As key components of the immediate antiviral response, type I IFNs are crucial for restricting viral replication and spread. Type I IFNs trigger the expression of hundreds of genes called IFN-stimulated genes (ISGs) with antiviral activities. These and parallel processes activated by the dsRNAs lead to the degradation of

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viral and host RNA, resulting in protein synthesis shutdown, restriction of viral replication, and stalling of the cell cycle [62, 87] (see also Sect. 3.3). Thanks to these potent processes, it was long assumed that dsRNA-induced innate immune responses block an efficient RNAi response to infection or other foreign nucleic acids. Nonetheless, as Box 4 describes, the RNAi machinery can be active as an antiviral mechanism in human cells as well.

Box 4 Intracellular Antiviral Signaling and the RNAi Response in Mammalian Cells Artificial induction of RNAi in mammalian somatic cells with long (as opposed to short) dsRNA does not readily induce the extent of robust silencing as expected. This has led to the belief that in mammalians long dsRNA is recognized as toxic and that, in turn, RNAi is readily masked by a robust IFN response. However, this is not always the case. For instance, coronaviruses, in general, are adept at evading all antiviral pathways induced by dsRNAs. (In particular, SARS-CoV-2 has been shown to induce minimal levels of interferon only [62]; albeit, ref. [61] reveals that SARS-CoV-2 induces substantial but delayed IFN-β production). Moreover, already twenty years ago, experiments with mice revealed the strange situation where dsRNA-induced RNAi, but not an IFN response. Notably, infection with encephalomyocarditis virus yielded siRNAs generated from long dsRNA in a Dicer-dependent manner [5, 64]. Importantly, then RNAi also has an innate antiviral function in mammals [64], and “A picture of hidden and inefficient RNAi in somatic cells may be oversimplified because there are several reports of long dsRNAinduced RNAi in transformed and primary somatic cells” [93]. While many aspects of these RNAi mechanisms remain unknown [86, 93], a few principles have been emerging: • Svoboda [93] suggested that RNAi may become fully functional in somatic cells in scenarios where dsRNAs do not provoke the IFN response. • A more general scenario is shown by Seo et al. [87] who suggest a close interplay between the RNAi pathway and the IFN response. This finding is striking as it not only points to the involvement of RNAi, but also that it is in fact a critical regulator to either enhance or quench the antiviral response. This interplay is likely highly beneficial to cells, as follows: – Given that many ISGs are toxic, the IFN antiviral defense is expected to be controlled by multiple layers of negative regulation. In particular, ISGs associated with cell proliferation and cell death are expected to be significantly more regulated by miRNAs. This is indeed what was seen in [87]. This study observed a negative regulation of many of the cytotoxic effectors of the IFN response which likely serves as a part of (continued)

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Box 4 (continued) a negative feedback loop to quench the antiviral response. That is, in the absence of infection, miRNAs suppress ISGs associated with cell death and proliferation. On the other hand, infection results in suppression of the RISC activity which contributes to an increased expression of ISGs [87]. – These findings point not only to a pivotal role of RNAi as a mammalian defense mechanism but also to a close interplay between the RNAi machinery and the IFN response. Thus, on the one hand, RNAi may act directly, for instance, in the degradation of genomic RNA of some viruses [93]. On the other hand, RNAi processes and intracellular antiviral signaling can influence each other in a competitive rather than cooperative way [86] to ensure pathogenic defense and normal cell physiology. Therefore, the traditional view that the RNAi machinery, due to its capacity of evading this immune recognition, readily gets masked by a robust IFN-based immune response needs to be drastically updated. Further research is needed to comprehend all possible scenarios involving negative or positive feedback mechanisms between RNAi pathways and the IFN antiviral defense (Fig. 3.4).

Actually, since the mechanisms of action of mammalian si-/miRNAs overlap, it is possible that defense processes against external nucleic acids utilize modification of cellular gene expression by suppressing the activity of miRNAs [87, 93]. Specifically, then, the interplay between the IFN response and the miRNA pathway (Fig. 3.4) may establish a fundamental balancing mechanism to ward off defenders most optimally without causing excessive toxicity to the host. The extent to which RNAs byproducts from vaccines activate or antagonize these interactions is unknown. Dysregulation or imbalanced IFN response is associated with severe coronavirus infection and linked to processes that promote inflammation and cellular stress [62]. In particular, this includes those of coronavirus infections and Covid-19 as well [62], where deregulated miRNA expression is associated with severe forms of the disease. Notably, SARS-CoV-2 can replicate rapidly during the incubation phase without detectably triggering IFNs, leading to high viral loads [6]. The potential of IFN dysregulation as a key determinant of COVID-19 pathogenesis has been gaining increased attention [1]. On the other hand, miRNAs have been associated with an inflammatory response due to their role in the regulation of cytokines, as well as with gene targets that regulate endothelial function. Of note, Centa et al. [11] identified a correlation between specific miRNA expression with fatal outcomes in patients with severe forms of SARS-CoV-2 infection. Specifically, they demonstrated the involvement of miR-26a-5p, miR-29b-3p, and miR-34a5p in endothelial dysfunction and inflammatory response and the occurrence of severe lung injury and immunothrombosis. To the best of my knowledge, the

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Fig. 3.4 Different scenarios of the interplay between mammalian intracellular antiviral signaling and RNAi processes in response to foreign nucleic acids. In many eukaryotes, RNAi (light yellow) serves as the primary antiviral defense by directing the RISC machinery to cleave viral RNAs, thereby inhibiting viral replication. However, unlike plants and invertebrates, mammals have evolved an elaborate protein-based antiviral system (light blue) which may represent an alternative arm to the RNAi system for combating viral infection. The IFN response is a key player in this antiviral defense and leads to the expression of hundreds of cytotoxic antiviral genes called IFNstimulated genes (ISGs). In contrast to the widely held belief that RNAi mechanisms in mammalian cells are inactivated by the IFN response, it may instead be that there is a sophisticated interplay (central blue and yellow arrows) between those processes. In fact, [93] demonstrated that the RNAi machinery may have evolved to negatively regulate the toxic host antiviral effectors of the IFN response. In non-infected cells (central part - yellow), miRNAs suppress IFN-stimulated genes (ISGs), probably because of their cytotoxic effects. On the other hand, the detection of foreign invaders (central part - blue) results in the suppression of RISC activity and thereby increases the expression of ISGs. Thus, miRNAs may promote an antiviral state in some contexts, or engender a negative feedback loop to promote infection in others, which overall leads to a reciprocal feedback (red line) between intracellular antiviral signaling and the RNAi machinery in mammalian cells [87]. Nonetheless, depending on parameters that are poorly understood (incl. the immunological context of the host), positive/negative (green) or minimal (brown) feedback mechanisms between those two main defense systems are possible as well

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extent to which analogous processes could be triggered by vaccine RNAs and their byproducts has not received any attention (see also Box 6 below).

3.7.2.1 Unexpected Silencing Effects of RNA Vaccines Although siRNAs are known to sequence-specifically cleave complementary RNAs—a characteristic that all RNAi applications want to exploit—siRNAs may also bind to an undefined number of target sites in an miRNA-like manner [21, 69], leading to their silencing and unwanted off-target effects. As pointed out in [69], these off-target sequences are only 6–7 nt long. Because of their high frequency of occurrence, this makes these unspecific target sites almost impossible to predict and difficult to avoid. This and the fact that not all such off-target matches will immediately lead to detectable knockdown effects, are a critical concern with all RNAi silencing therapies. Despite numerous efforts to minimize these off-target activities for RNAi applications, for instance, via specific modifications of the siRNA guide strand [69], they continue to be a major problem. This is even more so the case with RNA vaccines where this dilemma has not been fully appreciated. In contrast to RNAi therapies, the siRNAs cannot even be designed on purpose but emerge as unexpected vaccine byproducts. As a result, they could evoke the translational inhibition and repression of similar human mRNAs (Sect. 3.3). The latter is particularly concerning in light of the considerable overlap between immunogenic epitopes of SARS-CoV-2 proteins and human proteins [50, 63].

3.7.2.2 Vaccine RNAs Interfering with miRNA Regulations The differential expression of miRNA is fundamental for most biological processes, and impairment or changes in the level of miRNAs have been shown to lead to numerous animal and human diseases. This leads to the concern that the same could be initiated by the synthetic RNAs, as they assume unanticipated regulatory functions. As miRNAs are known to regulate 50% of protein-coding genes, the unintended involvement of vaccine RNAs or byproducts (Fig. 2.2) could have many undesirable consequences. In the context of gene therapy, it is known that exogenous siRNAs compete with endogenous miRNAs for RISC (see also Sect. 4.7), leading to the saturation of the RNAi machinery, which is, in addition to the regulation of other transcripts and their innate immune stimulation, one of the major challenges in RNAi therapy [94]. In this sense, vaccine-derived RNA byproducts, when they enter the RNAi pathway, could lead to numerous deleterious effects, for instance, when they distort the balance of natural miRNA:MRE interactions, compete for miRNA targets of endogenous mRNAs, or otherwise antagonize human micro RNA regulation. This may support the development of a myriad of diseases and metabolic disorders associated with aberrant expression of miRNAs, including diabetes,

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cardiovascular disease, kidney disease, and cancer [76, 78]. Most notably, in cancer cells, miRNAs have been found to be heavily dysregulated, and cancer cells with abnormal miRNA expression exhibit key hallmarks of all cancers: they evolve the capacity to sustain proliferative signaling, evade growth suppressors, resist cell death, activate invasion and metastasis, and induce angiogenesis (reviewed in [78]). Ref. [78] gives a careful analysis of mechanisms by which miRNA expression can become dysregulated in human cancer. Many of these share similarities to the vulnerabilities described in Fig. 3.5, how byproducts of mRNA vaccines could interfere with endogenous miRNA regulation. A synopsis of parallels of the mechanisms of [78] via vaccine-derived RNAs is given in Table 3.2. As is well known, miRNAs may function as either oncogenes or tumor suppressors under certain conditions. Thus, byproducts of mRNA vaccines, once they enter the RNAi pathway and resemble si/miRNAs, could disrupt normal anti-proliferative, pro-apoptotic, and tumor-suppressor effects of human miRNAs or trigger oncogenic mechanisms in some cellular environments. By May 3, 2021, EudraVigilance [29] reported 9 fatalities in the category “Neoplasms benign, malignant and unspecified (incl cysts and polyps)” attributed to the BioNTech/Pfizer Covid-19 vaccine. At that point, it appeared it was too soon after the start of Covid-19 vaccination campaigns for their oncogenic potential to become evident. However, within the first year of Covid-19 vaccinations, concerns about cancers, metabolic disorders, heart conditions, and other unexpected forms of disease have rapidly increased and will be analyzed more fully in Chaps. 8 and 10 below. While the vaccine-induced spike may be responsible for many of these adverse clinical implications, it does not seem that the possibility of micro RNA deregulation caused by vaccination has been considered in this regard.

3.7.3 Disrupting Other Activities of Human miRNAs at the Cellular Level MiRNAs play critical roles at the cellular level where they can regulate gene networks both transiently and dynamically [72]. Figure 3.5 depicts some of the core features of this regulation, and mechanisms of how byproducts of RNA vaccines resembling micro RNAs (Fig. 2.2) could perturb these processes. Despite much progress in micro RNA research to better comprehend their involvement in numerous biological processes (Fig. 3.6), many questions regarding their functioning and dysregulation have not been fully appreciated. As vaccinederived micro RNAs were not a guiding factor in vaccine safety testing, their unanticipated involvement could disrupt many of the regulatory aspects at the cellular level (Fig. 3.5) and result in pathological consequences. It seems possible that the introduction of exo-miRNAs could • Distort the dynamic—and yet highly regulated—ability of miRNAs to promote stable gene expression and to buffer out small fluctuations in transcription.

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Fig. 3.5 Factors influencing coupling of miRNA dynamics and cellular gene expression, and points of possible disruption via vaccine-derived micro RNAs. Many factors contribute to the activity of miRNAs, including cell type/state, functionalized compartmentalization, and shuttling of miRISC within cells (ref. [72] and the references therein). Several of these seem to be highly sensitive to influences from externally derived RNAs. The seven sectors in the circle depict these types of modulators of miRNA activities and how they could be adversely influenced by micro RNAs derived from vaccine RNA byproducts (Fig. 2.2), especially since the pathways of siRNAs and miRNAs are intertwined (Box 1). A—The addition of new micro RNAs: short fragments of RNA vaccines or their byproducts may resemble novel micro RNAs able to bind to proteins or vesicles associated with endo si/miRNA processes and networks. B—Byproducts of the vaccines acting as si/miRNAs may have a higher affinity than endogenous miRNAs. C—The exo-RNAs derived from vaccines may mimic and possibly outcompete endogenous miRNAs and could sequester miRISC from target mRNAs (see also Sect. 3.8). D—Impact of the nanoparticle components, interactions with drugs, and other factors that influence the distribution and bioavailability of the vaccine mRNA complexes

Issues with transcriptional control of miRNAs

Overarching mechanism Alterations involving miRNA genes

• Dysregulation of functional c-Myc-miRNA feedback loops, • Disruption of p-53-regulated expression of miRNA genes.

Association with human cancer Abnormal miRNA expression in malignant cells compared to normal cells are often attributed to changes in genomic miRNA copy numbers Many miRNA genes are located in cancer-associated genomic regions. Abnormal expression of miRNAs compared with normal cells is often attributed to miRNA gene or location change (amplification, deletion, or translocation) miRNA expression is finely tuned by multiple factors. Disturbances can lead to tumorigenesis by dysregulation of some key transcription factors such as c-Myc and p53, via

(continued)

• The dysregulation of key transcription factors may analogously be triggered by vaccine-derived micro RNAs that mimic, outcompete, or otherwise interfere with, endomiRNAs • Arguably, p53 is one of the major tumor suppressors. It is one of the most commonly mutated genes in human cancers As vaccine RNAs could lead to mutagenesis through various mechanisms (Sect. 2.3.2 and Chap. 10), this could involve such critical genes associated with tumorigenesis as well

Possible analogs via byproducts from mRNA vaccines It is plausible that such copy-number changes can also be triggered by RNA vaccine byproducts that share sufficient similarities with human miRNAs These types of changes could similarly result from vaccine RNAs or their byproducts if they get retro-integrated (Sect. 3.6.3 and Chap. 10), disrupting cancer-associated genes

Table 3.2 Known mechanism of endo-miRNA dysregulation [78] in human cancer cells and potential analogs involving vaccine-derived RNA byproducts

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Other abnormal expression of micro RNAs

Overarching mechanism Dysregulated epigenetics change

Table 3.2 (continued)

• Aberrant expression of any component of the miRNA biogenesis machinery could lead to abnormal expression of miRNAs • Some of the key enzymes involved with the biogenesis of miRNAs, including Drosha, Dicer, and AGO, have been shown to be mutated/dysregulated in certain tumors, leading to absent or defective miRNA activities

• miRNAs, similar to protein-coding genes, are susceptible to epigenetic modulation • In particular, DNA demethylation and histone deacetylase inhibition can lead to a significant upregulation of expression of certain miRNAs in some cancer cells and also affect the regulation of associated proto-oncogenes

Association with human cancer

• On the one hand, their binding to these enzymes with increased affinity, or in increased number, can disrupt required network dynamics and presence of critical endomiRNAs • On the other hand, the vaccine-derived byproducts may be similar to, but yet different enough, from endo-miRNAs, to directly perturb the activity of the enzymes involved in endogenous miRNA biogenesis

Possible analogs via byproducts from mRNA vaccines Some vaccine-derived RNA fragments, by mimicking endo RNAs, may be susceptible to the same modulation, and thereby have analogous oncogenic properties. In addition, depending on different vaccine platforms or possibly following retro-integration and genome uptake, some may be perpetually expressed; this ongoing expression itself may become an indirect disruptor of endogenous epigenetic micro RNA modulation Vaccine-derived micro RNAs may compete with endo-miRNAs for binding to the same components of the miRNA processing system

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Fig. 3.6 Micro RNAs play regulatory roles in a variety of biological processes. In addition to specific siRNA/miRNA-regulated processes (Box 1) involved in gene silencing or activation, miRNAs act more globally at the cellular level. Thus, miRNAs may not solely, or even predominantly, function as target-specific regulators, but act via cellular gene expression networks that involve all targetable RNA molecules within a cell (the “MRE load,” Fig. 3.5). This leads to a sophisticated interplay to facilitate specific miRNA activities in response to changes in cellular environments, to transiently regulate gene expression, and do so dynamically by buffering out small fluctuations in transcription. In addition, miRNAs can be secreted into extracellular fluids and transported to target cells via special vesicles (e.g., exosomes) or by binding to proteins (esp. AGOs). This way, they mediate cell-cell communication. They are also secreted and transferred to recipient cells where they may act as autocrine, paracrine, and/or endocrine regulators (see [72] and the references therein)

• Disrupt cellular gene expression networks by perturbing endogenous “MRE loads” (Fig. 3.5) of miRNAs: In effect, through the contribution of vaccinederived micro RNAs, the total collection of cellular miRNAs may not be adequately diluted amongst their potential targets. The subcellular localization of the different RNAs could further be disturbed by the NP component of the vaccines, vaccine interactions with drugs, and the overall pathophysiological context of the cells. It is relevant to point out that during mRNA Covid-19 vaccine development, no pharmacodynamics drug interaction studies and no safety pharmacology studies were conducted (see e.g. [31]). The involvement of vaccine RNAs or byproducts could disrupt the proportion of human target mRNAs bound to miRNAs at any given time, and disturb the balance required [72] for homeostasis and for achieving specifically targeted effects.

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• Interfere with the synergistic nature of miRNA regulation of target mRNA, a situation which has been implied with diseases such as oncogenesis [72].

3.7.4 Disrupting Circulating/Extracellular miRNAs One of the least understood areas of miRNAs may relate to their activities and functions as chemical messengers to mediate intercellular communication. Of note, miRNAs can be secreted into extracellular (EC) fluids, including plasma and serum, saliva, breast milk, urine, tears, and others. They can be delivered to target cells via vesicles such as exosomes, microvesicles, and apoptotic bodies, but also by associating with proteins, especially those of the AGO family [72]. Many miRNAs are functionally active in recipient cells and are shown to regulate them via hormone-like activities. Although it is known that such miRNAs are associated with various forms of disease, and are also explored for therapeutic and medicinal purposes (see, e.g., [94]), our understanding of when and how these RNAs exert their functions is very limited. It is more and more evident that the release of human EC miRNAs is itself a highly regulated process [72]. Obviously, this could be interrupted by the introduction of new miRNAs from vaccines acting as miRNAs. This may lead to the same deleterious effects as with other aberrant functioning of circulating/EC miRNAs. This is important, as they are, inter alia, known to bind to Toll-like receptors and activate downstream signaling events that eventually lead to tumor growth, metastasis, and neurodegeneration [33, 60].

3.8 External RNAs as miRNA Activity Modulators Interestingly, a study published in Frontiers in Cellular and Infection Microbiology adds pivotal insights into the potential of micro-RNA-mediated interactions as important drivers of severe Covid-19 disease [4]. In contrast to the above, where the focus was on SARS-CoV-2 infection leading to processes which interfere with endogenous RNA regulation [11], this study investigated if the virus itself (e.g., acting as a competitive RNA) could become a modulator of endogenous miRNA activity. The starting point of this investigation was the observation that viral sequences, once expressed, can interact with the host’s miRNA regulatory machinery in that they sequestrate the selected miRNAs. In this sense, viral RNAs can indeed act as competing RNA modulators [92] for endogenous miRNAs: importantly, this means that when expressed at a high level, sequestration of the miRNAs rescues the expression of their endogenous target mRNAs leading to their over-expression. Details of this interaction, and novel mechanisms that likely contribute to the

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modulation of mRNA levels associated with specific inflammatory processes, are summarized in Box 5.

Box 5 The Role of miRNAs in SARS-CoV-2 Infection An extensive analysis of human miRNA binding sites on the viral genome led to the discovery that human miRNA:viral RNA interactions may contribute to disease pathology of severe COVID-19. This important finding has been published in Frontiers in Cellular and Infection Microbiology [4]. As is well established, the transcriptional and post-transcriptional control of mRNA constitutes a key regulatory step during infection. Although ref. [4] did not discuss this, the observation that viral RNAs can act as important mediators in inflammatory processes may be associated with the fact that SARS-CoV-2 is able to escape a potent IFN response (Box 4). The main points of the virally mediated inflammatory responses are: • An extensive analysis of feasible human miRNA targets led to the identification of a small number of miRNA:viral RNA interacting pairs. • In particular, specific human lung-specific miRNAs may bind SARS-CoV2 RNAs. • The human miRNA miR-1207-5p was identified as a potential regulator of the viral spike protein. • In a healthy cell, miRNA-1207-5p plays an important role in shaping the inflammatory milieu, with CSF1 (colony-stimulating factor 1, also known as macrophage colony-stimulating factor, M-CSF) as one of its direct targets. • Based on the fact that exogenous RNA can compete for miRNA targets on endogenous mRNAs, it was hypothesized that the virus itself can act as a competing RNA [92]. In particular, it was predicted that viral RNA will sequestrate endogenous miRNAs (here, miRNA-1207-5p), rescuing the expression of their endogenous targets. Thus, it was hypothesized that, following SARS-CoV-2 infection, endogenous targets (here, CSF1) will increase their expression level (Fig. 4.3 in Chap. 4). • Analysis of datasets derived from lung epithelial cells infected with SARSCoV-2 revealed significant upregulation of CSF1 and therefore confirmed this hypothesis. These results are striking, for two reasons, as also explained in [4]. • The interaction between the SARS-CoV-2 genome and human miRNAs may establish a potent mechanism to modulate mRNA levels of specific inflammatory mediators. In particular, the CSF1 gene, a known target of miRNA-1207-5p, plays a key role in the innate immune system in that it regulates the survival, proliferation, differentiation, and chemotaxis (continued)

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Box 5 (continued) of tissue macrophages and dendritic cells (DC). Its over-expression may contribute to the acute inflammatory response observed in severe COVID19. • Exogenous RNA can compete with human RNA for shared miRNA targets: While competing RNAs were previously known in the context of endogenous RNA [92] and in experimental transgenic settings [24], the findings by Bertolazzi et al. [4] demonstrate that viral RNA, too, can compete for miRNA targets of endogenous mRNAs and lead to their deregulated expression. Importantly, this means that SARS-CoV-2 itself can act as an exogenous competing RNA and facilitate the over-expression of its endogenous targets.

Importantly, however, if viral RNAs may act as competing RNA for certain host miRNAs, leading to an increased expression level of miRNA targets of endogenous mRNAs, then similar types of unpredictable and dangerous dysregulations of mRNA expression can likely be instigated by vaccine RNAs as well. It seems plausible that this problem is further exacerbated with self- and trans-amplifying vaccine platforms. It is unclear why the possibility of vaccine RNAs themselves, as mediators and disruptors of human RNA regulation, was not studied during Covid-19 vaccine trials. This notable omission may be due to the misunderstanding that the fate of all vaccine RNAs will always be exactly as expected (see also Box 6 below).

References 1. Acharya D, Liu G, Gack MU (2020) Dysregulation of type I interferon responses in covid-19. Nat Rev Immunol 20(7):397–398 2. Al-Mayhani T, Saber S, Stubbs MJ, Losseff NA, Perry RJ, Simister RJ, Gull D, Jäger HR, Scully MA, Werring DJ (2021) Ischaemic stroke as a presenting feature of ChAdOx1 nCoV19 vaccine-induced immune thrombotic thrombocytopaenia. J Neurol Neurosurg Psychiatry 92(11):1247–1248 3. Alberer M, Gnad-Vogt U, Hong HS, Mehr KT, Backert L, Finak G, Gottardo R, Bica MA, Garofano A, Koch SD et al. (2017) Safety and immunogenicity of a mRNA rabies vaccine in healthy adults: an open-label, non-randomised, prospective, first-in-human phase 1 clinical trial. Lancet 390(10101):1511–1520 4. Bertolazzi G, Cipollina C, Benos PV, Tumminello M, Coronnello C (2020) mir-1207-5p can contribute to dysregulation of inflammatory response in covid-19 via targeting SARS-CoV-2 RNA. Front Cell Infect Microbiol 10:673 5. Billy E, Brondani V, Zhang H, Müller U, Filipowicz W (2001) Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl Acad Sci 98(25):14428–14433

References

81

6. Blanco-Melo D, Nilsson-Payant BE, Liu WC, Uhl S, Hoagland D, Møller R, Jordan TX, Oishi K, Panis M, Sachs D et al. (2020) Imbalanced host response to SARS-CoV-2 drives development of covid-19. Cell 181(5):1036–1045 7. Buschmann MD, Carrasco MJ, Alishetty S, Paige M, Alameh MG, Weissman D (2021) Nanomaterial delivery systems for mRNA vaccines. Vaccines 9(1):65 8. Cantaert T, Baeten D, Tak PP, van Baarsen LG (2010) Type i ifn and tnfα cross-regulation in immune-mediated inflammatory disease: basic concepts and clinical relevance. Arthritis Res Therapy 12(5):1–10 9. Carthew RW, Sontheimer EJ (2009) Origins and mechanisms of miRNAs and siRNAs. Cell 136(4):642–655 10. Casadevall A, Pirofski La (2020) In fatal covid-19, the immune response can control the virus but kill the patient. Proc Natl Acad Sci 117(48):30009–30011 11. Centa A, Fonseca AS, da Silva Ferreira SG, Azevedo MLV, De Paula CBV, Nagashima S, Machado-Souza C, dos Santos Miggiolaro AFR, Pellegrino Baena C, De Noronha L et al. (2021) Deregulated miRNA expression is associated with endothelial dysfunction in post-mortem lung biopsies of covid-19 patients. Am J Physiol-Lung Cell Mol Physiol 320(3):L405–L412 12. Centers for Disease Control and Prevention (2021) Allergic reactions including anaphylaxis after receipt of the first dose of Pfizer-BioNTech covid-19 vaccine—United States (2020). https://www.cdc.gov/mmwr/volumes/70/wr/mm7002e1.htm 13. Centers for Disease Control and Prevention. The vaccine adverse event reporting system (vaers). https://vaers.hhs.gov/data.html 14. Cha AE (2021) Coronavirus vaccines may not work in some people. It’s because of their underlying conditions. https://www.washingtonpost.com/health/2021/05/18/ immunocompromised-coronavirus-vaccines-response/ 15. Cheloufi S, Dos Santos CO, Chong MM, Hannon GJ (2010) A dicer-independent miRNA biogenesis pathway that requires ago catalysis. Nature 465(7298):584–589 16. Davenport FM, Hennessy AV, Francis J Thomas, With the Technical Assistance of Phyllis Fabisch (1953) Epidemiologic and immunologic significance of age distribution of antibody to antigenic variants of influenza virus . J Exp Med 98(6):641–656 17. De Beuckelaer A, Grooten J, De Koker S (2017) Type i interferons modulate CD8+ t cell immunity to mRNA vaccines. Trends Mol Med 23(3):216–226 18. Deming D, Sheahan T, Heise M, Yount B, Davis N, Sims A, Suthar M, Harkema J, Whitmore A, Pickles R et al. (2006) Vaccine efficacy in senescent mice challenged with recombinant sars-cov bearing epidemic and zoonotic spike variants. PLoS Med 3(12):e525 19. Desfarges S, Ciuffi A (2012) Viral integration and consequences on host gene expression. In: Viruses: essential agents of life. Springer, Berlin, pp 147–175 20. Devoldere J, Dewitte H, De Smedt SC, Remaut K (2016) Evading innate immunity in nonviral mRNA delivery: don’t shoot the messenger. Drug Discovery Today 21(1):11–25 21. Doench JG, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. Genes Dev 17(4):438–442 22. Dorward DA, Russell CD, Um IH, Elshani M, Armstrong SD, Penrice-Randal R, Millar T, Lerpiniere CE, Tagliavini G, Hartley CS, Randall NP, Gachanja NN, Potey PM, Anderson AM, Campbell VL, Duguid AJ, Al Qsous W, BouHaidar R, Baillie JK, Dhaliwal K, Wallace WA, Bellamy CO, Prost S, Smith C, Hiscox JA, Harrison DJ, Lucas CD, (2020) Tissuespecific tolerance in fatal covid-19. medRxiv 23. Dvinge H, Kim E, Abdel-Wahab O, Bradley RK (2016) Rna splicing factors as oncoproteins and tumour suppressors. Nat Rev Cancer 16(7):413 24. Ebert MS, Sharp PA (2010) MicroRNA sponges: progress and possibilities. RNA 16(11):2043–2050 25. Edwards DK, Jasny E, Yoon H, Horscroft N, Schanen B, Geter T, Fotin-Mleczek M, Petsch B, Wittman V (2017) Adjuvant effects of a sequence-engineered mRNA vaccine: translational profiling demonstrates similar human and murine innate response. J Transl Med 15(1):1–18

82

3 Relevance for mRNA Vaccine Safety

26. Erman M, Steenhuysen J. U.S. CDC finds more clotting cases after j&j vaccine, sees causal link. https://www.reuters.com/business/healthcare-pharmaceuticals/us-cdc-findsmore-clotting-cases-after-jj-vaccine-sees-causal-link-2021-05-12/ 27. Esnault C, Maestre J, Heidmann T (2000) Human line retrotransposons generate processed pseudogenes. Nat Genet 24(4):363–367 28. Euronews. Slovakia suspends use of the AstraZeneca jab for first-time vaccinations. https://www.euronews.com/2021/05/11/slovakia-suspends-use-of-the-astrazenecajab-for-first-time-vaccinations 29. European Medicines Agency. Eudravigilance—European database of suspected adverse drug reaction reports. http://www.adrreports.eu/en/search_subst.html 30. European Medicines Agency. https://www.ema.europa.eu/en/news/astrazenecas-covid-19vaccine-ema-finds-possible-link-very-rare-cases-unusual-blood-clots-low-blood 31. European Medicines Agency. Assessment report–comirnaty—common name: Covid19 mRNA vaccine (nucleoside-modified). https://www.ema.europa.eu/en/documents/ assessment-report/comirnaty-epar-public-assessment-report_en.pdf 32. European Medicines Agency. Covid-19 mRNA vaccine risk management plan (RMP). https://www.ema.europa.eu/en/documents/rmp-summary/covid-19-vaccine-moderna-eparrisk-management-plan_en.pdf 33. Fabbri M, Paone A, Calore F, Galli R, Gaudio E, Santhanam R, Lovat F, Fadda P, Mao C, Nuovo GJ et al. (2012) MicroRNAs bind to toll-like receptors to induce prometastatic inflammatory response. Proc Natl Acad Sci 109(31):E2110–E2116 34. FDA Office of Media Affairs (2021) Joint CDC and FDA statement on Johnson https://apnews.com/press-release/pr-newswire/ & Johnson covid-19 vaccine. business-diseases-and-conditions-health-care-industry-anne-schuchat-health561bd901bd02aeb7fbf7935b7db3e481 35. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans. Nature 391(6669):806–811 36. Getty Micah Green, Bloomberg. Don’t be surprised when vaccinated people get infected. https://www.theatlantic.com/science/archive/2021/03/vaccine-breakthrough-cases/618330/ 37. Gil-Etayo FJ, Suàrez-Fernández P, Cabrera-Marante O, Arroyo D, Garcinuño S, Naranjo L, Pleguezuelo DE, Allende LM, Mancebo E, Lalueza A, et al. (2021) T-helper cell subset response is a determining factor in covid-19 progression. Front Cell Infect Microbiol 11:79 38. Goswami R, Awasthi A (2020) Editorial: T cell differentiation and function in tissue inflammation. Front Immunol 11:289 39. Greco A, Gallo A, Fusconi M, Marinelli C, Macri G, De Vincentiis M (2012) Bell’s palsy and autoimmunity. Autoimmun Rev 12(2):323–328 40. Greinacher A, Thiele T, Warkentin TE, Weisser K, Kyrle PA, Eichinger S (2021) Thrombotic thrombocytopenia after chadox1 ncov-19 vaccination. N Engl J Med 384(22):2092–2101 41. Günther C, Kind B, Reijns MA, Berndt N, Martinez-Bueno M, Wolf C, Tüngler V, Chara O, Lee YA, Hübner N et al. (2015) Defective removal of ribonucleotides from DNA promotes systemic autoimmunity. J Clin Invest 125(1):413–424 42. Hartmann J (2021) Bethel park woman paralyzed 12 hours after getting first dose of pfizer vaccine, doctors searching for answers. https://www.wpxi.com/news/top-stories/bethelpark-woman-paralyzed-12-hours-after-getting-first-dose-pfizer-vaccine-doctors-searchinganswers/ZSYTEX4H4FHKDFVSPGS3ILJZUI/ 43. Heinemann JA (2019) Should dsRNA treatments applied in outdoor environments be regulated? Environ Int 132:104856 44. Hoffmann M, Hofmann-Winkler H, Krüger N, Kempf A, Nehlmeier I, Graichen L, Sidarovich A, Moldenhauer AS, Winkler MS, Schulz S, Jäck HM, Stankov MV, Behrens GMN, Pöhlmann S (2021) Sars-cov-2 variant b.1.617 is resistant to bamlanivimab and evades antibodies induced by infection and vaccination. bioRxiv 45. Houri-Zeevi L, Rechavi O (2017) A matter of time: small RNAs regulate the duration of epigenetic inheritance. Trends Genet 33(1):46–57

References

83

46. Huang KJ, Yang YC, Lin YS, Huang JH, Liu HS, Yeh TM, Chen SH, Liu CC, Lei HY (2006) The dual-specific binding of dengue virus and target cells for the antibody-dependent enhancement of dengue virus infection. J Immunol 176(5):2825–2832 47. Incalzi RA, Trevisan C, Del Signore S, Volpato S, Fumagalli S, Monzani F, Bellelli G, Gareri P, Mossello E, Malara A et al. (2021) Are vaccines against covid-19 tailored to the most vulnerable people? Vaccine 39(17):2325–2327 48. Jeyanathan M, Afkhami S, Smaill F, Miller MS, Lichty BD, Xing Z (2020) Immunological considerations for covid-19 vaccine strategies. Nat Rev Immunol 20(10):615–632 49. Kanduc D, Shoenfeld Y (2018) Inter-pathogen peptide sharing and the original antigenic sin: solving a paradox. Open Immunol J 8(1) 50. Kanduc D, Shoenfeld Y (2020) Molecular mimicry between SARS-CoV-2 spike glycoprotein and mammalian proteomes: implications for the vaccine. Immunol Res 68(5):310–313 51. Kanyavuz A, Marey-Jarossay A, Lacroix-Desmazes S, Dimitrov JD (2019) Breaking the law: unconventional strategies for antibody diversification. Nat Rev Immunol 19(6):355–368 52. Kazazian HH, Moran JV (1998) The impact of l1 retrotransposons on the human genome. Nat Genet 19(1):19–24 53. Kazazian Jr HH, Moran JV (2017) Mobile DNA in health and disease. N Engl J Med 377(4):361–370 54. Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 10(2):126–139 55. King S (2020) New cause of covid-19 blood clots identified. https://labblog.uofmhealth.org/ lab-report/new-cause-of-covid-19-blood-clots-identified 56. Kutner B (2021) CDC panel recommends lifting pause of j&j vaccine. https://www. courthousenews.com/cdc-panel-recommends-end-to-pause-of-jj-vaccine/ 57. Lambert PH, Ambrosino DM, Andersen SR, Baric RS, Black SB, Chen RT, Dekker CL, Didierlaurent AM, Graham BS, Martin SD et al. (2020) Consensus summary report for CEPI/BC March 12–13, 2020 meeting: assessment of risk of disease enhancement with covid19 vaccines. Vaccine 38(31):4783–4791 58. Ledford H (2021) How could a covid vaccine cause blood clots? Scientists race to investigate. Nature 592(7854):334–335 59. Lee SH, Singh I, Tisdale S, Abdel-Wahab O, Leslie CS, Mayr C (2018) Widespread intronic polyadenylation inactivates tumour suppressor genes in leukaemia. Nature 561(7721):127– 131 60. Lehmann SM, Krüger C, Park B, Derkow K, Rosenberger K, Baumgart J, Trimbuch T, Eom G, Hinz M, Kaul D et al. (2012) An unconventional role for miRNA: let-7 activates toll-like receptor 7 and causes neurodegeneration. Nat Neurosci 15(6):827–835 61. Lei X, Dong X, Ma R, Wang W, Xiao X, Tian Z, Wang C, Wang Y, Li L, Ren L, et al. (2020) Activation and evasion of type I interferon responses by SARS-CoV-2. Nat Commun 11(1):1–12 62. Li Y, Renner DM, Comar CE, Whelan JN, Reyes HM, Cardenas-Diaz FL, Truitt R, Tan LH, Dong B, Alysandratos KD et al. (2021) SARS-COV-2 induces double-stranded RNAmediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes. Proc Natl Acad Sci 118(16):e2022643118 63. Lyons-Weiler J (2020) Pathogenic priming likely contributes to serious and critical illness and mortality in covid-19 via autoimmunity. J Transl Autoimmun 3:100051 64. Maillard P, Ciaudo C, Marchais A, Li Y, Jay F, Ding S, Voinnet O (2013) Antiviral RNA interference in mammalian cells. Science 342(6155):235–238 65. Mayo Clinic. Transverse myelitis. https://www.mayoclinic.org/diseases-conditions/ transverse-myelitis/symptoms-causes/syc-20354726#:~:text=Transverse%20myelitis%20is %20an%20inflammation,nerves%20send%20throughout%20the%20body 66. Mueller S, Gausson V, Vodovar N, Deddouche S, Troxler L, Perot J, Pfeffer S, Hoffmann JA, Saleh MC, Imler JL (2010) Rnai-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in drosophila. Proc Natl Acad Sci 107(45):19390–19395

84

3 Relevance for mRNA Vaccine Safety

67. Napirei M, Karsunky H, Zevnik B, Stephan H, Mannherz HG, Möröy T (2000) Features of systemic lupus erythematosus in DNAse1-deficient mice. Nat Genet 25(2):177–181 68. Nebraska Medicine (2021) You asked, we answered: does VAERS list deaths caused by covid-19 vaccines? https://www.nebraskamed.com/COVID/does-vaers-list-deaths-causedby-covid-19-vaccines 69. Neumeier J, Meister G (2021) siRNA specificity: RNAi mechanisms and strategies to reduce off-target effects. Front Plant Sci 11:2196 70. Nia Williams AM (2021) Two Canadian provinces stop offering Astrazeneca’s covid vaccine. https://www.reuters.com/business/healthcare-pharmaceuticals/canadas-alberta-provincestops-giving-first-doses-astrazeneca-vaccine-2021-05-11/ 71. NIH Neurological Institute. Transverse myelitis fact sheet. https://www.ninds.nih.gov/ Disorders/Patient-Caregiver-Education/Fact-Sheets/Transverse-myelitis-fact-sheet#causes 72. O’Brien J, Hayder H, Zayed Y, Peng C (2018) Overview of microRNA biogenesis, mechanisms of actions, and circulation. Front Endocrinol 9:402 73. Ozonoff A, Nanishi E, Levy O (2021) Bell’s palsy and SARS-CoV-2 vaccines. Lancet Infect Diseases 21(4):450–452 74. Pardi N, Hogan MJ, Porter FW, Weissman D (2018) mRNA vaccines-a new era in vaccinology. Nat Rev Drug Discovery 17(4):261 75. Pardi N, Hogan MJ, Weissman D (2020) Recent advances in mRNA vaccine technology. Curr Opin Immunol 65:14–20 76. Paul P, Chakraborty A, Sarkar D, Langthasa M, Rahman M, Bari M, Singha RS, Malakar AK, Chakraborty S (2018) Interplay between miRNAs and human diseases. J Cell Physiol 233(3):2007–2018 77. Peeples L (2020) News feature: Avoiding pitfalls in the pursuit of a covid-19 vaccine. Proc Natl Acad Sci 117(15):8218–8221 78. Peng Y, Croce CM (2016) The role of microRNAs in human cancer. Signal Transduction Targeted Therapy 1(1):1–9 79. Pepini T, Pulichino AM, Carsillo T, Carlson AL, Sari-Sarraf F, Ramsauer K, Debasitis JC, Maruggi G, Otten GR, Geall AJ et al. (2017) Induction of an IFN-mediated antiviral response by a self-amplifying RNA vaccine: implications for vaccine design. J Immunol 198(10):4012– 4024 80. Pottegård A, Lund LC, Karlstad Ø, Dahl J, Andersen M, Hallas J, Lidegaard Ø, Tapia G, Gulseth HL, Ruiz PLD, Watle SV, Mikkelsen AP, Pedersen L, Sørensen HT, Thomsen RW, Hviid A (2021) Arterial events, venous thromboembolism, thrombocytopenia, and bleeding after vaccination with Oxford-AstraZeneca ChAdOx1-S in Denmark and Norway: population based cohort study. BMJ 373:n1114 81. Reuters. Denmark ditches J&J covid-19 shots from vaccination programme. https://www. reuters.com/world/europe/denmark-excludes-jj-shot-vaccine-programme-local-mediareports-2021-05-03/ 82. Reuters. Norway drops AstraZeneca vaccine, J&J remains on hold. https://www.usnews. com/news/world/articles/2021-05-12/norway-will-not-use-astrazeneca-covid-19-vaccinesays-daily-vg 83. Reuters Staff. New covid-19 variant defeats plasma treatment, may reduce vaccine efficacy. https://www.reuters.com/article/us-health-coronavirus-safrica-variant-idUKKBN29P2HJ 84. Ricardo Brito Pedro Fonseca. Brazil suspends use of AstraZeneca vaccine in pregnant women nationally after death. https://www.reuters.com/business/healthcare-pharmaceuticals/brazilhealth-agency-calls-halt-astrazeneca-vaccine-pregnant-women-2021-05-11/ 85. Sahin U, Karikó K, Türeci Ö (2014) mRNA-based therapeutics—developing a new class of drugs. Nat Rev Drug Discovery 13(10):759–780 86. Schlee M, Hartmann G (2016) Discriminating self from non-self in nucleic acid sensing. Nat Rev Immunol 16(9):566 87. Seo GJ, Kincaid RP, Phanaksri T, Burke JM, Pare JM, Cox JE, Hsiang TY, Krug RM, Sullivan CS (2013) Reciprocal inhibition between intracellular antiviral signaling and the RNAi machinery in mammalian cells. Cell Host Microbe 14(4):435–445

References

85

88. Soiza RL, Scicluna C, Thomson EC (2020) Efficacy and safety of COVID-19 vaccines in older people. Age Ageing 50(2):279–283 89. Sørensen B, Susrud A, Dalgleish A (2020) Biovacc-19: a candidate vaccine for covid-19 (SARS-CoV-2) developed from analysis of its general method of action for infectivity. QRB Discovery 1:e6. 90. Spellberg B, Edwards Jr. JE (2001) Type 1/type 2 immunity in infectious diseases. Clin Infect Diseases 32(1):76–102 91. Su Y, Ghodke PP, Egli M, Li L, Wang Y, Guengerich FP (2019) Human DNA polymerase η has reverse transcriptase activity in cellular environments. J Biol Chem 294(15):6073–6081 92. Sumazin P, Yang X, Chiu HS, Chung WJ, Iyer A, Llobet-Navas D, Rajbhandari P, Bansal M, Guarnieri P, Silva J et al. (2011) An extensive microRNA-mediated network of RNA-RNA interactions regulates established oncogenic pathways in glioblastoma. Cell 147(2):370–381 93. Svoboda P (2014) Renaissance of mammalian endogenous RNAi. FEBS Lett 588(15):2550– 2556 94. Tian Z, Liang G, Cui K, Liang Y, Wang Q, Lv S, Cheng X, Zhang L (2021) Insight into the prospects for RNAi therapy of cancer. Front Pharmacol 12:308 95. Tun GSZ, Gleeson D, Al-Joudeh A, Dube A (2022) Immune-mediated hepatitis with the modeRNA vaccine, no longer a coincidence but confirmed. J Hepatol 76(3):747–749 96. USA Today. Death of florida doctor after receiving covid-19 vaccine under investigation. https://eu.usatoday.com/story/news/health/2021/01/06/death-florida-doctor-followingpfizer-covid-19-vaccine-under-investigation-gregory-michael/6574414002/ 97. Van Hoecke L, Roose K, Ballegeer M, Zhong Z, Sanders NN, De Koker S, Saelens X, Van Lint S (2020) The opposing effect of type I IFN on the t cell response by non-modified mRNA-lipoplex vaccines is determined by the route of administration. Mol Therapy-Nucleic Acids 22:373–381 98. Verbeke R, Lentacker I, De Smedt SC, Dewitte H (2019) Three decades of messenger RNA vaccine development. Nano Today 28:100766 99. Waheed S, Bayas A, Hindi F, Rizvi Z, Espinosa PS (2021) Neurological complications of covid-19: Guillain-barre syndrome following pfizer covid-19 vaccine. Cureus 13(2) 100. Wang Q, Zhang L, Kuwahara K, Li L, Liu Z, Li T, Zhu H, Liu J, Xu Y, Xie J, et al. (2016) Immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates. ACS Infect Diseases 2(5):361–376 101. Weingartl H, Czub M, Czub S, Neufeld J, Marszal P, Gren J, Smith G, Jones S, Proulx R, Deschambault Y et al. (2004) Immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets. J Virol 78(22):12672–12676 102. Wise J (2021) Covid-19: Rare immune response may cause clots after AstraZeneca vaccine, say researchers. BMJ 373:n954 103. Zhang C, Maruggi G, Shan H, Li J (2019) Advances in mRNA vaccines for infectious diseases. Front Immunol 10:594 104. Zhang L, Richards A, Khalil A, Wogram E, Ma H, Young RA, Jaenisch R (2020) SARSCOV-2 RNA reverse-transcribed and integrated into the human genome. bioRxiv 105. Zhao J, Zhao J, Mangalam A, Channappanavar R, Fett C, Meyerholz D, Agnihothram S, Baric R, David C, Perlman S (2016) Airway memory CD4+ t cells mediate protective immunity against emerging respiratory coronaviruses. Immunity 44(6):1379–1391 106. Zuo Y, Estes SK, Ali RA, Gandhi AA, Yalavarthi S, Shi H, Sule G, Gockman K, Madison JA, Zuo M, Yadav V, Wang J, Woodard W, Lezak SP, Lugogo NL, Smith SA, Morrissey JH, Kanthi Y, Knight JS (2020) Prothrombotic autoantibodies in serum from patients hospitalized with covid-19. Sci Transl Med 12(570):eabd3876

Chapter 4

From Challenges to Opportunities and Open Questions

An unprecedented pandemic requires unprecedented approaches, including how vaccines are developed and tested. Typically, the development of a new vaccine takes about 10–15 years [45]. To combat the pandemic, all current Covid vaccines were developed and tested in record-breaking time and approved for “emergency use authorization.” It is important to keep this in mind, with the understanding that (a) their rapid rollout was deemed necessary to protect those most vulnerable, (b) that they are acknowledged as products whose underpinnings and biological actions remain incompletely known, and (c) that it is critical to continually assess their weaknesses and strengths and work toward updates and improvements. Above, a number of opportunities for RNA vaccines have been identified. These, and additional ones which have been emerging from increased clinical experience with mRNA vaccines, are highlighted here in the following.

4.1 Differentiating Whether Adverse Reactions Are Geared Against the Lipid Nanoparticles or Against Vaccine RNAs and Their Byproducts Soon after their first release, some healthy individuals who received the first dose of Pfizer-BioNTech COVID-19 Vaccine experienced severe allergic reactions, including anaphylaxis. According to the CDC [17], “Anaphylaxis is a severe, life-threatening allergic reaction that occurs rarely after vaccination.” A host of other severe adverse events following vaccination with mRNA vaccines have been reported, including thousands of cases of death during the first few months postauthorization [19]. When these first emerged, most of them were believed to be attributed to an unfortunate combination of events and circumstances, such as old age, pre-existing medical conditions, or others. To help explain these tragic reactions, vaccine developers have focused on pre-existing sensitization to © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Mueller, Challenges and Opportunities of mRNA Vaccines Against SARS-CoV-2, https://doi.org/10.1007/978-3-031-18903-6_4

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a component of the vaccine such as the lipid-based nanoparticle carrier system and its polyethylene glycol (PEG) 2000 lipid conjugate [16, 25]. Developers and regulators maintain that the vaccines are not responsible for these (or other) adverse events. However, as discussed, several pivotal principles have incorrectly been excluded from the conceptual framework of the underlying mechanisms and action. One of them is the intrinsic immunogenicity of mRNA vaccines which has not been fully appreciated. Therefore, it will be important to better differentiate between the mRNA part itself as opposed to its carrier system, to identify potential drivers of adverse reactions. The approaches taken by the different producers to eliminate immunotoxicity are inconsistent and likely insufficient. While it is believed that harmful immunestimulatory byproducts can be eliminated from IVT processing, there is no clear scientific consensus for this, and the steps needed for purification are rather demanding and known to be highly problematic for large-scale production. In fact, seemingly overwhelmed with mass-production demands, a Johnson & Johnson (J&J) COVID vaccine manufacturing plant admitted an ingredient mix-up which resulted in 15 million doses being contaminated and needing to be discarded. While this particular event did not include any mRNA vaccines, an investigation by the US Food and Drug Administration (FDA) reported multiple problems related to clean and sanitary conditions, and also that this plant had “failed to adequately train personnel involved in manufacturing operations, quality control sampling, weigh and dispense, and engineering operations to prevent cross-contamination of bulk drug substances” [78]. Analogous problems involving incomplete purification of mRNA vaccines likely will have negative impacts on large groups of the population. Thus, there is an urgent need for • Optimal IVT purification methods that are suitable for mass-applications. • A better understanding of the potential of vaccine dsRNA contaminants as drivers of disease. • New approaches that eliminate harmful dsRNA formation in vivo, even when these technologies are applied at scale. According to some leaked documents, the European Medicines Agency (EMA) had concerns about early batches of the Pfizer Covid-19 vaccine having lower than expected levels of intact mRNA. Published in the BMJ [86], this reveals, among others that “EMA scientists tasked with ensuring manufacturing quality—the chemistry, manufacturing, and control aspects of Pfizer’s submission to the EMA— worried about ‘truncated and modified mRNA species present in the finished product.”’ Among the many files leaked to The BMJ, one email dated 23 November, 2020 identified “‘a significant difference in % RNA integrity/truncated species’ between the clinical batches and proposed commercial batches—from around 78% to 55%. The root cause was unknown and the impact of this loss of RNA integrity on safety and efficacy of the vaccine was ‘yet to be defined.”’ While there are no further details about the truncated species, it does sound as if they could have been dsRNA contaminants. Pfizer’s vaccine received EMA authorization on 21 December 2020, based on the assessment that the quality of this medicinal product was sufficiently

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consistent and acceptable. “It’s unclear how the agency’s concerns were satisfied,” the BMJ article notes. The question raised by the BMJ is what this data leak tells us about mRNA instability and product consistency. Regardless of the exact type and extent of the contamination, the real question is how known quality issues can effectively be resolved when millions of doses need to be produced in a short time.

4.2 The Need for Clear Attribution When Covid-19 vaccines were first rolled out, longer-term adverse effects (e.g., outside “the 24 h window” for anaphylaxis [17]) were not believed to be attributable to mRNA vaccines. Based on their believed mechanism of action, they were not expected to lead to adverse events. With hindsight, when unexpected side effects following vaccination first appeared, it seems understandable this came across as unbelievable: they just did not fit the model. It took quite some time until unexpected side effects have been more critically assessed (see Part II below for more detail). Unfortunately, initial signals have led to not only quite some controversies but also to substantial polarities and divisiveness among researchers, scientists, and physicians.1 In early 2021, a relatively high number of reported deaths in Norwegian nursing homes attracted attention both in Norway and internationally. In the period between 27 December 2020 and 15 February 2021, the Norwegian Medicines Agency received 100 reports of suspected fatal adverse reactions to the Pfizer/BioNTech vaccine BNT162b2. Consequently, The Norwegian Medicines Agency and the Norwegian Institute of Public Health asked an expert group to assess the extent of a causal link between vaccination and death and came to the official conclusion, “Of the 100 reported deaths, the expert group classified 10 (10%) as most likely related to the vaccine, and considered that there could be a possible link for 26 (26%)” [98]. The assessment had been done according to the World Health Organization’s classification system for monitoring adverse drug reactions [85]. Nonetheless, for many cases classified as “possible,” important information was not available to arrive at a definite conclusion [98]. Given that no autopsies were reported, it is possible that the “confirmed” cases may be considerably higher (see Sect. 5.4 and Chap. 8 below). Nonetheless, at that point, the CDC maintained that “VAERS has not detected patterns in cause of death that would indicate a safety problem with COVID19 vaccines” and that “[s]erious side effects that would cause a long-term health problem are extremely unlikely following COVID-19 vaccination” [88]. It also stated that “Vaccine monitoring has historically shown that if side effects are going to happen, they generally happen within six weeks of receiving a vaccine dose.”

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See, e.g., Senator Ron Johnson Roundtable Discussion: COVID 19—A Second Opinion, https:// rumble.com/vt62y6-covid-19-a-second-opinion.html.

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Based on the understanding that RNA vaccines act like any other (traditional) vaccine, at that point, longer-term follow-up was not deemed necessary. The reason for this decision was based on their legal classification (Fig. 1.1), which gives rise to the following devastating circular argument. • Firstly, because RNA vaccines are not regarded as gene therapies, patient studies do not assess long-term or delayed adverse events, and clinical tests, including those for genome integration, germline transmission, genotoxicity or carcinogenicity, have never been conducted [32, 33]. • Secondly, by the same token, after vaccine rollout, when severe adverse effects following vaccination occur, then the same unsubstantiated premise that these consequences cannot occur is used to determine that there is no link between those events and vaccination. So, both during R&D and mass clinical application, unexplainable, delayed, and long-term adverse events—which are not anticipated according to the expected framework (Fig. 1.1)—are ruled out based on unsupported and often hidden assumptions which, in turn, engender unwarranted follow-up and attribution decisions via a circular argument (see Fig. 4.1 for a summary). With gene therapies, delayed long-term effects are a known issue of concern. A well-studied example that illustrates this point occurred in a retrovirus-mediated gene-therapy trial that aimed to correct severe combined immunodeficiency-X1 disease (SCID-X1). Although this trial was successful with regards to the goal of restoring an immune function, 4 out of 9 patients developed leukemia in the 5 years following viral transduction (which later was linked to integration events close to an oncogene [42]). Thus, to clearly understand the implications of the “transient” transfection mechanisms underlying all of the mRNA vaccines, detailed investigations should involve suitable animal models with sufficient follow-up time and limited clinical studies that specifically screen for delayed adverse events. To get a better understanding between injurious events that are, or are not, related to vaccination, it therefore seems warranted to • Appreciate the factor of time, in particular with booster vaccines, related to dichotomous immune responses and the development of adverse reactions with undesirable mid- or long-term effects. • Develop clear transcriptional and proteomic profiling techniques to assess the dissemination and impact of vaccine RNAs throughout the body. • Comprehend the ongoing systemic effects of transfection, in different situations and pathophysiological states such as pregnancy, poor nutritional or immune status, comorbidities, age, and genetic predisposition.

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Fig. 4.1 Possible adverse events of mRNA vaccines. The most obvious adverse events are immediate and short-term (top of the iceberg) and are the basis for evaluating the safety of these vaccines [17]. The main arguments for their limited follow-up are based on their expected modus operandi. Albeit, there is no guarantee that mRNA vaccines will only reach professional antigenpresenting cells (APCs) in the injection site, nor that they will only evoke the anticipated antigen presentation with the desired immune response. Critically, the products may reach the bloodstream and be distributed throughout the body. Moreover, the synthetic mRNA may lead to truncated, fragmented or otherwise harmful mRNA species such as dsRNAs, and their associated proteins may have numerous adverse effects. In fact, the delivered products are actually processes but are not assessed as such. Another argument for their limited follow-up is the mistaken view that RNA vaccines are not gene-therapy products (Fig. 1.1). This flawed premise severely impacts both R&D efforts, as well as ongoing surveillance and safety evaluations. On the one hand, thanks to their current legal status, they bypass much more stringent guidelines as would be necessary for GT products [18]. On the other hand, this unsubstantiated premise has shaped the understanding that other effects, including delayed long-term adverse events, cannot take place, and hence, cannot be associated with vaccination. Rationally speaking, this is a classic circular argument, built on a false logical assumption. As laid out here, numerous factors that critically undermine the prevailing improper framework of mRNA vaccines have not been adequately considered (the hidden part of the iceberg)

4.3 Guarding Against Cross-Reactivities and Aberrant Immune Responses As discussed, homology between human and the SARS-CoV-2 spike protein is considered a key factor in the development of serious illness and mortality in Covid19. Strikingly, ref. [77] identified a 78.4% human similarity to the SARS-CoV-2 spike protein to all human known proteins. An extensive expression of the spike is

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believed to trigger harmful antibodies leading to either ADE or AI conditions, as has been alerted to by several authors [56, 73, 77, 90, 93]. This concern is made even more plausible by the observation that a considerable percentage of patients with severe disease have autoantibodies [15]. In this light, several studies suggest that severe or fatal Covid-19 may actually be the response of an aberrant and/or over-reactive immune reaction—which may be able to control the virus but thereby enhance disease [15, 21, 29, 48, 56, 95]. Intriguingly, transcriptional and proteomic insights of some patients who had died during the first wave of the pandemic in Wuhan, China revealed that they “had a low viral burden, a finding that suggests the patients’ deaths may be due to uncontrolled host inflammatory processes rather than an active viral infection” [97]. Thus, if an aberrant immune response drives severe disease, then it will be critical to assess the extent to which this could happen via vaccination as well. The factor of time is also critically important in regard to sensitization and development of serious disease. Indeed, ref. [56] posits that the lengthy asymptomatic period in Covid-19 may reflect the immune system’s response to SARS-CoV-2, as it could lead to an impaired immune response first, and then, over time, to the development of autoimmunity. They argue that the risk of illness and death is likely increased by prior exposure to the spike protein, and it is plausible the same applies to repeat exposure (infection or injection). To help guard against these types of adverse events, it will be helpful to • Obtain a better understanding of the immunotoxic effects triggered over time, in response to the synthesized molecules, due to their similarities to human proteins, and how this maps to the general population as opposed to those with pre-existing hyperinflammatory, AI, or other conditions. • Determine if priming via prior infection or injection—especially related to coronaviruses and other RNA viruses—has the potential to evoke vaccine-induced immune responses in form of cross-reaction with human proteins (Sect. 3.2). • Clearly assess the effect of utilizing novel epitopes and determine the possibility of (a) cross-reactivities and pathogenic effects via peptide commonalities, and (b) the potential of an increased number of epitopes to enhance undesirable vaccinederived micro RNA formation, and their potential to interfere with endogenous small RNA regulation. • Assess cross-regulatory actions of interferons and inflammatory cytokines triggered by vaccination, to help identify individuals with an increased risk of AI or other severe adverse events. • Analyze the intrinsic self-adjuvant nature of RNA vaccines in form of their antimRNA translation response, if this destructive process extends to human mRNAs with significant homologies to the spike, and how this may impair critical cellular processes, disrupt proteins, or enable tumorigenesis.

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4.4 Guarding Against Other Medium and Longer-Term Side Effects During a healthy disease response, at some point, the body terminates defensive immune responses. In contrast, with RNA “vaccines” the goal is the opposite, namely the ongoing and sustained generation of antibodies, on the argument that otherwise immunity may not be maintained. This not only means that key components of the disease response (mimicking active infection) are artificially sustained in an ongoing manner. A consequence of the unsubstantiated assumption that products of the injections will immediately get degraded is that these can get disseminated in the body. Albeit, the fate of these products was not clearly ascertained prior to vaccine rollout. As summarized in Chaps. 9 and 10 below, the first year of post-vaccine experience has brought preliminary but convincing evidence that the vaccine-induced spike antigen reaches the bloodstream and is detectable in high amounts in circulation for much longer than previously believed possible. The long persistence of vaccine-induced antigens could be further exacerbated if vaccine RNAs may get integrated into the genome and expressed. Thus, to ensure patient safety in light of prolonged execution of non-natural processes, it is important to • Clearly determine the distribution and persistence of vaccine RNAs or their byproducts throughout the body, including unexpected and non-targeted tissue and organs (e.g., the brain). Any broad localization or distribution of these products must effectively be ruled out in ongoing and future vaccination programs. • Obtain a detailed understanding of the potential of (a) vaccine RNAs, (b) dsRNA byproducts, to trigger inflammatory cytokines, complement activation, or otherwise impair immune response generation. • Determine if repeated transfection through boosted injection or self-/transamplifying vaccine formats results in a dose-dependent manner when it comes to dissemination and clearance. Potentials for inflammatory immune responses to create a hazardous positive-feedback loop as previously known for some cellular antiviral defenses involving type I IFNs [69] must be ruled out. • Analyze the propensity of vaccine RNAs to generate DNA lesions and to engender the development of known associated diseases such as cancer, neuropsychiatric disease, and others. • Perform single-cell sequencing to assess the frequency and fate of genetic modifications due to retro-integration events stemming from vaccine products and how this could disrupt critical genes or trigger unwanted events such as tumorigenesis.

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4.5 The Opposing Role of mRNA Vaccine-Induced Type I IFN Signaling in the Regulation of T Cell Immunity One of the key observations with mRNA vaccines is that IVT mRNA triggers the same innate RNA sensors that have evolved to recognize microbial RNAs, hence evoking the type I IFN signature that is typically associated with viral infections. Yet, as previously described (Sect. 2.2.2, Chap. 3), Type I IFNs are pleiotropic antiviral cytokines that can affect nearly every step of the immune response. In particular, they can act as stimulatory cytokines that evoke cytolytic CD8+ T cells, as well as antibody responses, but, conversely, can also have profoundly inhibitory and strongly detrimental effects. Yet, the complex and opposing role of type I IFN in the regulation of T cell immunity to mRNA vaccines remains to be elucidated. A study in mice for specific mRNA cancer vaccines [91] suggests that the two different mechanisms can be attributed to the route of administration—with (a) systemic application promoting T cell proliferation, differentiation, and survival, but (b) local application evoking a pro-apoptotic and anti-proliferative transcription program. This same study warns that systemic administration of mRNA-lipoplexes comes with an increased safety risk and the concern for potential infusion reactions, liver toxicity, and systemic inflammatory responses. Translating these apparently opposing mechanisms into safe and effective mRNA vaccines constitutes a major challenge. As previously described, the last couple of years have seen many efforts to decrease IFN responses through the use of highly purified mRNA, the incorporation of modified nucleosides, and others, as reflected by the different Covid-19 mRNA vaccines (under emergency approval or in development). However, as noted in [24], such strategies may coincide with a general decrease in PRR triggering, DC activation, and cytokine release, which could hamper rather than enable T cell activation. Therefore, the critical open question remains: when is the response enough, and when is it too much? To date, there is no clear understanding of how to disentangle the various mRNA vaccine and/or administration routes, and how they influence the cellular networks and critical host factors which determine potency and inflammatory toxicity. The mechanisms behind the differential outcome of IFN signaling on T cell immunity to mRNA vaccines are still unresolved and warrant further investigation. While findings of studies in mice can provide valuable insights, the very nature of the assumed key modulators of these processes points to the immediate limits of such animal models. If the outcome of type I IFNs on CD8+ cell immunity, for example is largely determined by the relative kinetics, timing, and intensity of the corresponding innate signaling processes [24, 91], then such results may be radically different in a clinical context. The Covid-19 mRNA vaccines by both Pfizer and Moderna are administered intramuscularly [34, 83]. Yet, in this context of CD8+ cell immunity, they exhibit safety patterns as would be expected from systemic administration routes as suggested in [24, 91]. Post-mRNA-vaccine systemic reactions include anaphylaxis, flu-like illness, breathlessness, multiple organ dysfunction syndrome, and many

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others. Related signals were already visible on May 22, 2021: at that point, VAERS reports of Covid-19 post-vaccine fatalities included the following (including number of cases): for Moderna: unresponsive to stimuli (174), sudden death (40), sudden cardiac death (7), seizures (21 cases), sepsis or septic shock (41); and for Pfizer/BioNTech: loss of consciousness (33), seizures (17), sepsis or septic shock (63), shock (11), sudden death (25), and unresponsive to stimuli (153). Detailed clinical studies are warranted to disentangle how coinfections and comorbidities (esp. those involving inflammatory processes), previous infection and inoculation status, or unexpected vaccine-drug interactions could impair the assumed IFN-evoked timing processes in innate signaling. Moreover, it is important to assess how the latter could be disrupted by vaccine RNAs in the—rather likely— event they are not immediately degraded, when they purposefully get translated in an ongoing manner via self-/trans-amplifying vaccine platforms, or when vaccine RNAs get retro-integrated and expressed.

4.6 Discerning the Function and Impact of Vaccine-Derived Regulatory RNAs Regulatory RNAs are most critical in many aspects of homeostasis, disease, and pathogen defense. Despite evoking a great deal of interest during the last few years, many questions about the regulatory nature of RNAs are still unknown. The same concerns the fate of RNAs from vaccines. Therefore, it will be important to clearly assess the potential of unexpected vaccine RNA byproducts to influence endogenous small RNA regulation, and how this could impact vaccine safety and effectiveness.

4.6.1 The Interplay Between IFN Responses and RNAi Mechanisms in Self- and Non-self Recognition That homology between human and the SARS-CoV-2 spike protein could lead to pathologies via autoimmunity is a great concern (see above). Nonetheless, the problem of how vaccine RNAs influence the balance between self- and non-self recognition is likely much more extensive, in particular also because mRNA vaccine developers did not seem to regard the RNAi antiviral defense in human cells. As mentioned, the reciprocal inhibition between intracellular antiviral signaling and the RNAi machinery described above (Box 4) is likely beneficial to mammalian cells in several ways [75]. The interplay of, and balance between, the expression of miRNAs and that of cytotoxic ISGs allow for responsive control of timing, degree, and resolution [75]. For instance, dysregulation of ISG expression susceptible to miRNA-mediated regulation has been associated with cancer [75].

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Nevertheless, the reciprocal regulation does not apply to all viruses and cell lines [75, 80]. Which factors determine the directionality and strength (if any) of various feedback mechanisms between the IFN and RNAi responses (Fig. 3.4) are major understudied areas of research. The challenge of balancing both of these arms of the immune response can easily lead to paradoxical situations, as is especially evident in the context of IVT RNAs: (a) On the one hand, as IVT RNA is capable of both triggering immune recognition and destruction of the mRNA, the latter may attenuate RNAi, whereas the former may engender adverse and toxic immune responses. However, the attempts of vaccine manufacturers to minimize dsRNA formation and to prevent those undesirable IVT responses seem to be rooted in the view that RNAi antiviral defense mechanisms are absent in human cells. Thus, (b) on the other hand, then, if IVT RNA does not trigger the IFN arm—as intended by the manufacturers—then unexpected byproducts (Fig. 2.2) can become substrates in the pathways leading to RNAi and may evoke undesirable gene silencing and related mechanisms. Above, the two main mechanisms were separately discussed, ignoring their interplay. Moreover, the focus of the IFN pathway was on warding off invaders or RNAi, as understood in its broad context as a gene-regulatory mechanism triggered by long dsRNAs. Adverse consequences that the intended blunting of the IFN arm may have in terms of immune responsiveness in general, do not seem to have been investigated. It is unclear to what extent mRNA vaccines may disrupt the interplay and balance between various immune response pathways (see Fig. 4.2 for problematic issues). Given that the interrelationship between the RNAi system and the protein-based antiviral response has received little attention (if any), it will be important to assess if vaccine-derived byproducts can perturb balanced ISG expression or otherwise interfere with various feedback processes between these two main antiviral response systems. Studies are warranted to assess • The extent to which antiviral signaling and RNA sensor activation via dsRNA byproducts from vaccines can alter endogenous RNAi responses. • Whether vaccine-derived byproducts disrupt the antiviral defense mechanism and the recognition of foreign nucleic acids. • Whether those byproducts perturb the balance between IFN responses, RNAi mechanisms, and other immune reactions. • To what degree vaccine-derived byproducts can hamper the interplay between promoting and quenching an antiviral state, including those of secondary infections. • Whether modifications done to vaccine RNAs compromise immunity against infections, cancer, and AI conditions. In this context, it may be relevant to point to a publication in Rheumatology that confirms herpes zoster (HZ) reactivation following BNT162b2 mRNA Covid19 vaccination in patients with some AI conditions [37]. This reactivation may be triggered by suppressed innate immune signaling, given that the modifications done to the vaccine RNAs aim to quench the (excessive) induction of type I IFNs –

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Fig. 4.2 The fate of vaccine RNAs. Ideally, the mRNAs from the vaccines are full-length species that resemble mature human transcripts and will not provoke any undesirable effects. Unanticipated byproducts will have different lengths. Left triangle: Long dsRNAs (these may include truncated vaccine RNA species) are toxic for animals (including humans) as their sophisticated immune systems are capable of recognizing these as foreign. This undesired immunogenic feature has long hampered the practical use of mRNA vaccines and has also been observed independently of vaccine research [72]. Elimination of dsRNAs from IVT processes continues to be challenging, as is confirmed by the many—and often mutually opposing—approaches utilized by the different vaccine platforms. Short dsRNAs (right triangle) may, however, bypass immune sensing [31]. This observation is exploited in form of biotechnological RNAi applications such as siRNAmediated targeted gene knockout for therapy and in form of exo-dsRNA-mediated gene therapies for environmental applications. Alas, if dsRNA byproducts of RNA vaccines do not trigger immune recognition, they may be processed by Dicer into short fragments and trigger unanticipated RNAi pathways. As with other exo-siRNAs (e.g., from viruses or other invasive nucleic acids), these short dsRNAs may evoke the destruction of their own RNAs; even so, owing to the intertwined nature of siRNA and miRNA silencing processes (Box 1), it is likely that this process simultaneously promotes the repression of partially complementary sequences of human RNAs as well: they may bind to an undefined number of target sites in a miRNA-like manner and as such interfere with various endogenous processes of regulatory RNAs that are essential for normal health and development. Finally (top triangle), even if vaccine RNAs resemble full-length species, the modifications done to suppress type I IFN signaling and excessive immune responses, may compromise immune function overall

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which, however, may disrupt downstream B and T cell immune responses as well. In [37], it is suggested that this may “negatively affect antigen expression potentially contributing to HZ reactivation.” Thus, the goal to limit (excessive) IFN responses may in reality perturb appropriate immune function overall. As both, imbalanced IFN responses and deregulated miRNA expression are associated with severe forms of Covid-19 (Sect. 3.7.2), studies are warranted to assess if analogous disturbances can be triggered by vaccination as well. To this end, it will be important to determine (Fig. 3.4), • The extent to which siRNAs derived from RNA vaccines activate or antagonize endogenous RNAi system that regulates the expression of ISGs, and in particular, those associated with toxicity. • If IFN triggered by RNA vaccines can result in a feedback loop that represses other essential human genes. • To what extent those byproducts could disrupt regulatory feedback mechanisms and lead to an excessive toxic ISG response.

4.6.2 Mechanisms and Effects of Externally Derived Regulatory RNAs Despite their notable involvement in nucleic acid sensing and antiviral defense, arguably the most critical function of mammalian micro RNAs is in regulating endogenous gene expression [80]. Short siRNAs specifically suppress the expression of endogenous and heterogenous genes in various mammalian cell lines [31], and miRNAs play important roles in most biological processes [67]. Therefore, the effect of vaccine-derived byproducts with si/miRNA-like functions on human micro RNA pathways merits further investigation, especially since this cannot necessarily be inferred from lab experiments with limited animal models—whose RNAi system is often quite different from humans. As is well known, si/miRNA-based gene regulation (Box 1) exhibits major differences between plants and animals, both related to sequence complementarity requirements for target recognition and the extent of the regulation. Critically, mammalian miRNAs are not perfectly complementary to their target mRNA sequence and also have multiple targets [5]. The imperfect nature of this pairing and the small size of the miRNAs are major hurdles toward their identification, as is the observation that miRNA:RNA pairing does not happen in a one-toone manner; indeed, miRNA-mediated repression can include multiple targets and involve miRNA complexes leading to cooperative rather than additive effects [27]. These considerations make it even more compelling to get a detailed understanding of the range of mechanisms triggered by vaccine byproducts when they enter RNAi processes. Toward this end, it will be important to

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• Get a clear picture of the proportion of truncated species during IVT production (Sect. 3.5) and of all vaccine RNA species which may become substrates of RNAi processes (Fig. 2.2). • Include small vaccine RNA byproducts as additional factors during surveillance and analysis of adverse events. • Expand on previous miRNA profiling and deep sequencing studies2 to determine the extent to which vaccine-derived RNA byproducts share similarities with known human miRNA sequences. • Determine if vaccine-derived si/miRNAs could impair signature patterns of endo miRNA expression [67], as they are known for (a) healthy individuals, and (b) implicated with the formation of cancer or other diseases. • Perform genome-wide profiling of vaccinated individuals to compare their miRNA expression signatures with those that are unvaccinated. • In addition to the targeted products of the injections, obtain quantitative and qualitative information about the fate of vaccine byproducts (e.g., truncated species), their subcellular locations, and spatiotemporal distribution, in a variety of real clinical settings. • Assess the potential of small regulatory RNAs derived from vaccines to be taken up into the nucleus, and whether they—just as endogenous miRNAs [2]—may interfere in the regulation of chromatin state and genomic remodeling, disrupt alternative splicing profiles, or other related processes implicated with certain (hereditary) diseases, premature aging, and cancer [14].

4.6.3 Off-Target Effects, Effects on the Human Microbiota, and the Larger Environment Despite the rapid increase in the number and type of biotechnological RNAi applications in recent years, many challenges have not been fully appreciated, even in those areas where those technologies have been intensely studied. In the context of gene therapy, issues such as insufficient therapeutic efficiency, on the one hand, and immune-related toxicities and undesirable off-target activity, on the other, have prevented the use of RNAi drugs for most of the last decade (see, e.g., [5] and the references therein). Even though several RNAi drug candidates are currently progressing through clinical trials, considerable technical barriers and hazards still need to be overcome: most notably, as summarized in [5], “we are very far from understanding the long-term effects of siRNA and miRNA therapy in vivo in human subjects.” Both for RNAi drugs and gene-edited crops, the risk of off-target effects has been considered perhaps the most challenging risk as it could result in off-target degradation and disturb miRNA biogenesis. Due to various nucleotide changes in the human genome such as single nucleotide polymorphisms (SNPs), targeting the 2

e.g., http://wwwmirbase.org.

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specific RNA sequences remains one of the main challenges for the drug candidates as it could critically interfere with recognition of the target mRNA [5]. For gene-editing and gene-silencing in plants, the challenge of precisely predicting the dynamic and highly stochastic nature of sequence interaction is especially problematic as plants are engaged in a myriad of interactions with their environment. Therefore, off-target effects could not only have adverse consequences on the plant itself; plants expressing specific siRNAs could be taken up by animals, could prove to be toxic to some species, and even end up in the food chain. Non-target animals, including their microbiota and soil-bacteria, might incorporate these RNAs, and even though they may not have RNAs with full complementarity, the expression of partially complementary sites could be adversely affected through their endogenous miRNA systems [44, 64]. Furthermore, as highlighted in [43], genetic changes may even be inherited and lead to a permanent alteration of plants or their associated microbiota. It is unclear if siRNAs derived from vaccines could adversely affect the human microbiota and furthermore, via numerous cross-reactivities extend to the larger environment. Therefore, in this regard, it will be critical to: • Determine the interrelationship of the synthesized products on the human microbiome, to influence their propensity to act as mutagens, transition to more serious pathogenic forms (e.g., biofilms, see [43] and the references therein) or enhance the colonization of human organs or tissue by pathogens. • Clearly assess the potential of vaccine (by)products, whether they can be transferred between species and act as regulatory RNAs in off-target organisms as well. It seems relevant to point out in this context that some of the reported adverse events seem to point to some associations between vaccination and the human microbiome. For instance, as of May 13, 2021, the UK database [61] lists for COVID-19 mRNA Pfizer- BioNTech a total of 17,170 “Gastrointestinal disorders,” including 16 deaths. These cases are still higher than for “Respiratory disorders” (total of 7039, including 35 deaths), and next to “General disorders” (total of 47,111 cases, including 146 deaths), “Nervous system disorders” (30,778 cases, including 37 deaths), “Muscle & tissue disorders” (total of 21,628), are one of the leading categories with adverse events. Similarly, the categories of “Skin disorders” (total of 12,406, including one death) and “Infections” (including Influenza, Lower and upper respiratory tract and lung infections) have a rather high count (4167 cases, including 64 deaths). This compares to “Blood disorders” (5795 cases, including 2 fatalities), “Psychiatric disorders” (2846 cases, 1 death), “Eye disorders” (2687 cases), “Vascular disorders” (2416 cases, including 10 deaths), “Cardiac disorders” (2140 cases, including 51 fatalities), “Ear disorders” (2103 cases), and others. It is also relevant to note that in a healthy human body, microbes live in a dynamic equilibrium with the host and that each new invading microbe tries to reset this balance in an attempt to create favorable conditions for its own existence [53]. Thus, if mRNA vaccines can facilitate the reactivation of persistent viruses (as already demonstrated [37]), or further the invasion by new viruses, this could

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result in numerous downhill consequences, simply because it could help distort the equilibrium of those microbes with the host. This could favor the uncontrolled replication of some of these microbes, a known factor leading to the imbalance of the immune system [53]. In this imbalanced system, both symbiotic viruses and new invaders can cause different pathological conditions. For instance, it has been shown that HIV-1 interacts with coinfecting viruses (Herpes viruses in particular), thereby accelerating the disease [53]. Strikingly, a study published in Vaccines [11] suggests that SARS-CoV-2 may be able to act as a bacteriophage or induce the activity of other bacteriophages. In this study, the authors analyzed stool samples of Covid-infected patients and uninfected controls and found strong evidence of interaction of this virus not only with human but also non-mammalian cells. The authors suggest it is not only able to infect eukaryotic cells as previously elucidated but also the human bacterial flora, in an interaction that could be both lytic and lysogenic. The clinical consequences remain to be further elucidated, but it seems likely this could lead to a disruption of the gut microbiome and possibly also cause other issues, such as AI.

4.6.4 Small Vaccine-Derived RNAs in the Extracellular Space Circulating/EC miRNA species are highly stable and resist degradation at various deleterious conditions [65]. In addition to their manifold functions mentioned above (Fig. 3.6), evidence has been accumulating demonstrating a cross-kingdom action of miRNAs. Notably, it was shown that plant miRNAs present in the human circulating system through dietary intake can target human genes and regulate human gene expression [54]; some of these findings have even been explored for medical intervention [22, 50]. Nonetheless, their detection, including their appropriate levels, has proven challenging, in part due to the lack of standardized protocols and intraand inter- laboratory variations [67]. To achieve desired therapeutic effects, in vivo mRNA delivery of RNA vaccines has long been a critical challenge as exogenous mRNA needs to get through the barrier of the lipid membrane and into the cytoplasm. mRNA uptake, delivery, and organ distribution are dependent on many parameters such as cell type, physicochemical properties of the mRNA complexes, and others [13, 66]. The last few years have seen many advances, via NP delivery systems and others [13], to achieve this purpose. For therapy, there has been an intense focus on how uptake can most efficiently be realized. Yet, there is no guarantee that all IVT RNA molecules will indeed reach the cytosol of targeted cells. Thus, the amount of EC species and the extent of vaccinederived byproducts involved in cell-cell or even cross-kingdom communication and their possible consequences are unknown. In fact, during mRNA vaccine trials and development, there were no toxicokinetic studies to clearly assess what happens to these chemicals once they are in human bodies [32]. Moreover, there were no studies to determine how the injection reacts with other drugs [32]. Thus, detailed

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theoretical and clinical studies are warranted to help shed light on critical questions such as: • The fate, under various pathophysiological conditions, of vaccine-derived byproducts in the EC milieu. • The extent to which RNA byproducts from vaccines could mimic or perturb activities of endogenous circulatory/EC miRNAs. • Whether these byproducts could be transferred to other humans or the extended environment, via breast milk, saliva, urine, tears, and others.

4.7 Can Vaccine RNAs Act as Micro RNA Activity Modulators? Traditionally, miRNAs are believed to induce repression or translational inhibition of target genes through partial complementarities with various miRNA response elements (MREs) of the target mRNAs. Accordingly, miRNAs are mainly regarded as active regulatory elements, whereas the target mRNAs are viewed as passive targets of repression. During the last decade, this notion has begun to change. Already in 2009, Seitz hypothesized that computationally identified miRNA binding sites can titrate miRNAs and thereby regulate micro RNA availability [74]. Along those lines, it was then determined that pseudogenes, due to their high sequence homology with their associated genes, can act as genuine miRNA competitors, actively competing with their ancestral protein-coding genes for the same pool of miRNAs through common sets of MREs [68]. Consequently, a number of theoretical and experimental studies have demonstrated that large classes of RNAs can influence each other’s levels by competing for a limited pool of miRNAs. In fact, there is now mounting evidence that all types of coding and non-coding RNA transcripts may cross-talk with, and actively influence each other via miRNAmediated interactions. Two main types of miRNA activity modulators have been experimentally verified [79]. Sponge modulators include both mRNAs and non-coding RNAs which share miRNA binding sites with other RNAs. Actually, these miRNA sponges include long non-coding RNAs, pseudogenes, mRNAs, and circular RNAs [82], which, by competing with each other, can release parental target mRNAs from miRNA’s control (Fig. 4.3). Nonsponge modulators, on the other hand, do not necessarily share miRNA binding sites with their modulated targets. They are implemented by proteins and RNAs acting via a variety of alternative mechanisms, including activation or suppression of miRISC-mediated regulation of target RNAs, and can affect target degradation and transport [79]. Remarkably, as discussed above for the special case of SARS-CoV-2, viruses, too, can act as competitive RNAs [7]. Notably, as previously described, it seems that it is this very function of this viruses’ RNA which contributes to the acute

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Fig. 4.3 Micro RNA modulators. This last decade has revealed that the modulation of miRNA activity leads to an extensive post-transcriptional regulation layer of target genes of surprising magnitude. miRNA activity modulation is known to be implemented by several mechanisms [79]. Firstly, RNAs modulate each other through their common miRNA regulatory program (depicted). Such “miRNA sponges” include a wide variety of coding and non-coding RNAs which share miRNA response elements with other RNAs. (a) These miRNA sponges, or competitive RNAs, can decrease the number of free miRNA molecules available to repress other functional targets. Thus, while in the absence of miRNA sponges (top right), target mRNAs for some particular miRNAs are repressed: when sponge RNAs are expressed at a high level, they sequester the miRNA complexes with common binding sites, rescuing the expression of the endogenous targets (bottom right). (b) Importantly, sponge interactions are symmetric: if one RNA regulates another via competition for specific common miRNA binding sites (left: here, depicted as same color rectangles), then the latter RNA also regulates the former via competition for other common miRNA programs. As a result, RNAs in such a network both regulate and are regulated by their neighbors, leading to a complex dynamic cross-talk and competition. Remarkably, it was recently shown that SARSCoV-2 itself can act as a miRNA sponge [7]. Due to the extensive similarity between the spike protein and the human proteins (Sect. 3.2), this suggests the same can be true for mRNA vaccines: hence, vaccine-derived RNAs could compete with endo RNAs for miRNA binding. The prospect that mRNA vaccines may act as sponge- or also non-sponge- modulators could have important implications for disease development such as cancer.

inflammatory response observed in severe COVID-19. This raises the question if vaccine RNAs could similarly function as sponge modulators, or even nonsponges. If so, this could have far-reaching harmful impacts on post-transcriptional regulation, including disease initiation and progression, for several reasons. Firstly, although miRNAs regulate tens or hundreds of targets, the repression of a few targets alone can have a physiological role [74]. Moreover, even though individual miRNA-mediated interactions may be weak, the combined regulatory effect of the combined interaction layer is substantial. Moreover, their ability to

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affect cellular phenotype is significant [71]. Thus, it has been suggested that this extensive miRNA layer, which allows gene regulation without direct transcriptional or even post-transcriptional interactions, plays a significant role in disease initiation and progression when dysregulated, as has been demonstrated for cancer in particular [41, 71, 79]. Although several studies shed light on the intricate interplay among diverse RNA species, many questions regarding their co-regulatory functions are still unanswered. The potential of vaccine RNAs as key players in this network is supported by the following observations, whose details merit further investigation. • Vaccine RNAs may share a sufficient number of miRNA bindings sites with endogenous RNAs: As it is well established, there is extensive sharing of common MREs between human RNAs. Therefore, due to the significant homology between spike and human sequences (Sect. 3.2), it seems plausible that this inherent feature of endogenous RNAs extends to vaccine RNAs as well. • In the case of such shared MREs, vaccine RNAs may establish the same symmetric type of interactions as known for endogenous sponge interactions: That is, (a) vaccine RNAs and, (b) endogenous RNAs, may modulate each other through their common miRNA regulatory program. In this sense, up/down changes to the expression of either one of these RNAs may perturb the relative abundance of functioning miRNAs which target both of these RNAs, leading to a corresponding down/up- regulation of the other RNA (Fig. 4.3). In addition, nonsponge modulators (not shown in Fig. 4.3) have been identified, which act via a variety of alternative mechanisms. For instance, they assist or inhibit components of the miRNA-mediated post-translational regulatory apparatus, may help or prevent recruitment of miRISC to the target RNA or affect target degradation and transport [79]). • Depending on their expression levels and on the total number of functional miRNA binding sites that vaccine RNAs share with endogenous RNAs, it is possible that all the RNAs in this network both regulate and get regulated by each other, establishing a complex functional network. • It is possible then that vaccine RNAs—as part of this regulatory layer—act as significant drivers of (epi)genetic alterations leading to disease. While all of this is purely hypothetical, studies and experiments are warranted, given the substantial influence that these types of networks have on the regulation of normal cell physiology, and in the development of pathogenesis when dysregulated. In fact, during the last decade, numerous studies have documented the pervasive transcription across 70–90% of the human genome [82]. This is in sharp contrast to the less than 2% of the total genome which encodes protein-coding genes and implies that non-coding RNAs represent most of the human transcriptome. Indeed, it has been found that aside from around 21,000 protein-coding genes, the human transcriptome includes about 9000 small RNAs, about 10,000–32,000 long noncoding RNAs (lncRNAs) and around 11,000 pseudogenes [30, 92]. The importance of lncRNAs has become increasingly clear in recent years and is now often referred to as the Dark Matter of the genome [63]. They can vary in

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length from 200 nucleotides to 100 kilobases and have been implicated in a diverse range of biological processes from pluripotency to immune responses [63]. They are also important and powerful cis- and trans-regulators of gene activity that can function as scaffolds for chromatin-modifying complexes and nuclear bodies, and as enhancers and mediators of long-range chromatin interactions [63]. In analogy to SARS-CoV-2 RNAs—acting as competing RNAs—implied in severe forms of Covid-19 [7], it is therefore suggested here that the same is possible for vaccine RNAs; this would mean that they, too, play a potential role in this intricate interplay between protein-coding messenger RNAs and non-coding RNAs. How many of them will be able to interact with endogenous mRNAs, and also the largely unexplored pool of non-coding transcripts, is difficult to say; but it is unlikely they will not be involved as active regulators at all. Understanding how RNA vaccines are part of this huge RNA regulatory network can have significant implications for the development of disease and help increase our knowledge of previously unexplored etiologies of some of the adverse events.

4.8 The Spike During Infection, as Opposed to the Spike Expressed via a Vaccine To date, more than 100 AI diseases are known. This includes some of the common diseases which previously were not thought to belong to this category. Because of the significant homology between spike and human proteins (Sect. 3.2), it would be prudent to re-evaluate many of the known post-vaccine injuries reported in this regard. It seems plausible that some of the mysterious new side effects of mRNA vaccines could actually be linked to AI mechanisms fostered by peptide commonalities involving the spike. However, with SARS-CoV-2, the spike itself seems to play a much more critical role, than just via homologies to human proteins. In the first two years of the pandemic, evidence of various toxic aspects of the spike has been mounting (Chap. 10). This raises the question if the spike, as expressed by the vaccine, could itself be an important driver of serious adverse events. This section describes the vaccine-induced spike, even long after the global vaccine rollout, was not believed to cause any clinical concerns and difficulties with this view. Specific aspects related to the biodistribution and persistence of the vaccine spike and newer insights regarding the spike’s toxic potential will be more fully analyzed in Chap. 10.

4.8.1 The Spike Itself as a Driver of Severe Disease As is well-established, the spike protein of coronaviruses is important during the receptor recognition and cell membrane fusion process. In addition to its function in

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infection, several pivotal studies show that the spike also plays critical roles in the pathogenesis of the Covid-19 disease itself. For instance, in [52], Lei and coauthors demonstrate how the SARS-CoV-2 virus damages and attacks the vascular system on a cellular level. Although this study used a noninfectious pseudovirus, their findings reveal that it is the spike protein alone which attacks the vascular system, damaging the endothelium. Similarly, exposure to the spike protein on the SARSCoV-2 virus alone seems to be enough to induce symptoms analogous to COVID-19 [36], including severe inflammation, an influx of white blood cells into their lungs, and evidence of a cytokine storm.

4.8.2 Fate of the Vaccine Induced Spike Unclear Even at the Beginning of the Global Vaccination Campaign The striking result that the SARS-CoV-2 spike protein causes injury to the lungs or endothelium even without the presence of the intact virus has led to the vital question of how this affects vaccines that purposefully express (almost) the same protein (Chap. 10). More explicitly, the question is: if we are causing people to express the spike protein through inoculation, are we damaging them similarly as if they had been infected with the virus? Even during the beginning of the global vaccination campaign, this serious matter did not receive much attention. When the question first arose, some suggested that the answer was definitely “no,” and that there was no concern at all. This view was based on the following arguments [55]. • The injection of mRNA vaccines is intramuscular (IM), and not into the bloodstream. According to this view, a thick muscle like the deltoid is a good target without any easily hit veins or arteries at the site of injection; therefore, even if vaccine components end up in the intercellular fluid, they quickly drain through the lymphatic system, and not the bloodstream. • Once the host cell expresses the spike protein from the vaccine, it is recognized as foreign and presented to the immune system, as an abnormal intruding protein on a cell surface [55]. As the spike protein has a transmembrane anchor region, it is believed to remain sitting on the surface of muscle and lymphatic cells, close to the injection site. • Even if some of the injected material makes it into the bloodstream, it is believed that is only a small fraction. In fact, some animal models (see, e.g., [32]) suggest that the injected material only hits cells at the site of injection and outside the liver. Again, when they express the spike, it is expected that pieces of this antigen will get anchored on their surface so that the spike will never enter circulation. It seems, however, that all these views are based on implicit assumptions, with some of them not justified.

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• There is no guarantee the vaccine mRNA, if expressed, will evoke an immune response and get recognized as foreign. This may be due to the significant SARS-CoV-2 epitope peptide sharing with humans, apply to individuals with compromised immune systems, or for other reasons. • Even if the immune system mounts a response, there is no guarantee this will lead to the anticipated T cell differentiation and expression of the expected antigen. As previously described (Sect. 2.2.3), there is a lot of controversy about this believed mechanism, as it could easily be impaired by the timing of the relative kinetics of antigen presentation as opposed to type I IFN response (see also the following item). • Thus, the vaccine-induced protein may not be recognized as foreign, may not trigger an adaptive immune response and effectively get cleared. Rather, it may assume a completely different role—the synthetic mRNAs may result in truncated/fragmented or otherwise aberrant species whose associated proteins can have numerous adverse effects that have not been assessed. • As mRNA vaccines do not seem to involve “new formulations” or “novel excipients,” the evaluation of the Covid-19 mRNA vaccines by the European Medicines Agency (EMA) [32, 33] did not rely on clinical pharmacokinetic studies. Some distribution studies of mRNA vaccines were performed in animals. Yet, Box 6 summarizes several gaps and concerns with the completeness of these models and nonclinical findings. Indeed, the hypothesized fate of the spike, when expressed by the vaccine, may be radically different in real clinical settings than inferred from these animal models—which had been developed for a different situation altogether. Consequently, there is no scientific or clinically proven reason as to why the spike, when expressed after inoculation, “cannot wander around to cause trouble” [55] nor why it always has to trigger the desired immune response. Of concern are firstly people with a compromised immune system (see also Sect. 4.9). For example, in contrast to immunocompetent participants, a study published in JAMA demonstrates that only a low proportion (17%) of solid organ transplant recipients mounted a positive antibody response to the first dose of the two mRNA Covid-19 vaccines mRNA—1273 (Moderna) and BNT162b2 (PfizerBioNTech) [9]. Furthermore, in this same study, the poor humoral response was persistently associated with the use of antimetabolite immunosuppression. The spike protein behavior from the vaccine is unknown for immunocompromised individuals, as these were excluded from trials [20, 94]. However, it cannot be adequately predicted for the general population either. Clinical trials did not assess where, and for how long vaccine-induced production of the spike antigen occurs and to what extent it may affect vaccine effects (intended and unintended). Moreover, no pharmacodynamics drug interaction studies were conducted to determine how vaccines react with other drugs; startlingly, the use of immunosuppressants appears to diminish the vaccine response; the same is true for some steroids (e.g., prednisone) which lead to a marked reduction in antibody production, regardless of the dose given, if administered around the time of the vaccine [20]. Furthermore,

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toxicokinetic studies were only done in limited animal models [32]. As described in Sect. 4.5, it is all these unique clinical parameters that dictate the fate of mRNA vaccines, and ergo, that of the vaccine-based spike. It is also important to note that the very limited, animal-based biodistribution studies of both of the current Covid19 mRNA vaccines showed areas of concern, even though these were performed on different mRNAs acting as proxies, rather than that encoding the spike [32, 33]).

Box 6 The Spike Protein During Infection and Expressed Through Vaccines One of the most urgent questions which is insufficiently resolved is: can the spike protein, when expressed via Covid-19 vaccines, evoke similar harmful responses as during natural infection? At present, a “no” answer to this pivotal issue almost seems to be taken for granted. This is based on believed differences regarding the fate of the spike in these two different situations [55], as follows. In terms of natural infection, a key component of this process is lysis of the host cell and subsequent release of a load of new viral particles into the cellular neighborhood and the bloodstream. It is mainly in the bloodstream where the virus causes greater damage. By contrast, it is not believed that the vaccine-induced spike can reach the bloodstream nor that it can disseminate throughout the body. According to vaccine developers, this is because of the postulated biodistribution characteristics of the vaccine, believed to be the same as for other IM drug products. Yet, as described (Sect. 4.5), many details of the underlying mechanisms have only recently become known; many of them are critically determined by patient-specific parameters. According to the EMA, “nonclinical pharmacokinetic studies such as biodistribution studies are not usually required to support the development and authorization of vaccines for infectious diseases (only in the case of new formulations or novel excipients used)” [35]. Nonetheless, some analyses were conducted without “traditional pharmacokinetic or biodistribution studies” [32], and in a nonclinical setting. 1. Assessment of Moderna included a biodistribution study via a proxy— mRNA-1647, an mRNA-based vaccine against human cytomegalovirus formulated in SM-102 (the same novel lipid nanoparticle as used for the Covid-19 vaccine mRNA-1273)—in “a male rat” (singular?). Although the highest mRNA concentrations were observed at the injection site, a “relatively small fraction of the administered mRNA-1647 dose distributed to distant tissues” [33]. Furthermore, “the mRNA constructs did not persist past 1–3 days in tissues other than the injection site, lymph nodes, and spleen where it persisted in general 5 days” [33]. (continued)

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Box 6 (continued) 2. For the case of BNT162b2, unique animal experiments were used based on “LNP formulated luciferase-encoding RNA” (as a proxy for the Covid19 mRNA). Notably, they revealed a long half-life of the nanolipid carrier. Moreover, while the liver and the injection site were the major sites of distribution, extensive biodistribution in most tissues in rats was identified as well. Even these very limited animal models, utilizing only a proxy mRNA (rather than that encoding the spike), raise several critical concerns. • Why are mRNA vaccines, along with their LNPs, not considered new formulations/novel excipients? • How can a few animals (or one male rat, in the case of Moderna) represent all the different clinical situations of kinetics and timing parameters of human immune signaling processes which, inter alia, determine the fate of IM-administered drugs (Sect. 4.5)? • Why is the observed spatial and temporal distribution as seen from the animal experiments not regarded as problematic? Even for rats, 3 days is a long time. This time interval is also relevant for post-Covid-19-vaccination injuries, especially since almost a quarter of the deaths after vaccination reported to VAERS (as of May 14, 2021) occurred within 48 h [19]. Therefore, the view that the vaccine-induced spike cannot evoke similar pathogenesis programs as when expressed during natural infection, is unsubstantiated. This topic will be discussed in greater detail in Chaps. 9 and 10.

Overall then, many of the mechanisms that are believed to establish the above “no” answer have never been assessed in clinical trials. Consequently, there is no guarantee of anticipated spike protein behavior in real clinical settings. Importantly, there is no reason to see why mRNA vaccines cannot reach the circulatory system, be taken up by endothelial cells, and possibly be expressed throughout the body. Intending antigen-expressing injections to only reach antigen-expressing cells at the injection site does not mean they will. Likewise, intending that the generated molecules will be recognized as antigens and engender the anticipated immune response does not mean any of this will actually happen. Therefore, as the postulated fate of mRNA vaccines may not pan out in real clinical settings, there is no guarantee it will not evoke similarly dangerous pathogenesis programs as the viral spike from natural infection. To summarize the above, two of the believed main assumptions underlying mRNA vaccines is their transient nature and that the injected material will rapidly be degraded. Moreover, initially, it was assumed that only one booster would be required. The fact that additional boosters have become necessary has led to suggestions that

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• At each round, the duration of the vaccine-induced antibody production is transient, and the amount produced is low. • A sufficient number of boosters are required for the immune system to even begin to recognize the new spike antigen. • Without sufficiently many boosters, the immune system will not be able to respond to the new antigen which is only present for a short time and in small amounts in cells close to the injection site. However, as has become apparent from post-vaccine experience (Parts II and III below), the 2-dose regimen for mRNA vaccines as initially authorized did not prove to be enough to provide adequate protection. This shows that the above underlying assumptions regarding the spike are not substantiated and rather lead to new questions (which will be revisited in Chap. 10). • The official requirement for more boosters is no proof that the amount of the injected material is low. Neither does it say anything about the distribution and persistence of the products of the injections. • The observation that more boosters are required does not mean that the level of vaccine-induced spike was too low to establish adaptive immunity. • A diminishing antibody titer post-vaccination, while meant as a proxy for immunity (but see Chap. 11), does not inform about biodistribution and persistence of the vaccine spike. It has only been after the global vaccine rollout that complex features of the LNP and the spike as active compounds have been emerging. These will be further discussed in Chaps. 9 and 10.

4.8.3 The Spike and Potential Analogs of Virally Mediated AI Some serious forms of COVID-19 display a very unusual pattern of organ damage that is not easily explained by prior knowledge of virally induced pathogenesis. On the other hand, it is well-established that the majority of people with COVID19 are asymptomatic. These disparities have raised the question of whether the symptoms are primarily caused by the viral infection or whether they are the result of an AI response instead. Some hypotheses appeared already in 2020 which link a significant proportion of SARS-CoV-2—induced organ damage to an AI response [59, 87]. Given the high affinity between ACE-2 and the Receptor Binding Domain of the spike protein, ref. [87] postulates that the association of ACE-2 with the spike protein is likely long-lived. According to Townsend, it can therefore be expected that the spike will remain associated with ACE-2 even when it enters the cells. Further extending on this, McMillan et al. [60] focus, in addition to the cellular attached form of the ACE-2 enzyme, on its soluble form. They suggest that in people with high levels of soluble ACE-2, the SARS-CoV-2 spike protein, binding tightly

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with ACE-2, could form complexes that could end up in circulation. When these get endocytosed by macrophages (which could function as antigen-presenting cells), the combined entity will be regarded as part of a new pathogen. This may lead to antibody production against ACE-2 as well. As ACE-2 has a 42% homology to the angiotensin-converting enzyme (ACE), this could mean that autoantibodies formed against ACE-2 might cross-react with ACE also. Given that the AI hypothesis gives a possible explanation for the unusual pattern of organ damage in Covid-19, it would be important to investigate the clinical implications of these same mechanisms post-inoculation. Notably, [60] argues that for Covid-19, the AI theory can explain the entire spectrum of symptoms, from mild to severe. Of great interest to a possible vaccine-induced AI response are the following types of hypersensitivity response implicated with Covid: • Type 2 Hypersensitivity (the production of IgM or IgG antibodies to cellular or extracellular matrix proteins). As a concrete example of an immune response in SARS-CoV-2, McMillan and coauthors [60] describe immune thrombocytopenia as a cellular immune response to platelets with subsequent platelet destruction by this antibody response. They suspect that this consists of IgM produced against ACE and ACE-2, primarily targeting the endothelial blood vessels in the lung and small intestines. • Type 3 Hypersensitivity (antigen and antibody complexes are formed in the blood and become deposited in tissues, resulting in inflammation). This is in particular implied with a systemic vasculitic response with endothelial swelling, inflammatory infiltrates, and fibrinoid necrosis of the arterial wall. Again, McMillan et al. argue that this type of hypersensitivity is associated with COVID19 as well, as confirmed by autopsy studies. • Type 4 Hypersensitivity (the antigen triggers the activation of CD8 lymphocytes which target cells that are infected with a virus). Several aspects of this type of immune hypersensitivity responses are also observed in COVID-19, including perivascular T cell infiltration with evidence of distal organ involvement in the adrenals and giant cell pathology [60]. This high combination of immune hypersensitivity—involving Types 2, 3, and 4—has not previously been described in any other disease. As spike-based vaccines are also rooted in the production of the spike, it would be important to assess vaccine-induced autoimmunity as presented in [60], and to what degree this may explain the vast spectrum of side effects following vaccination. This may be particularly relevant to people with elevated levels of serum ACE-2—the elderly and obese, and those with hypertension, heart disease, kidney disease, and diabetes.

4.9 When the Vaccine Just “Does Not Work” Soon after the global vaccine rollout, research showed that 15–80% of people with certain medical conditions are generating few antibodies, if any, after receiving

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mRNA-based coronavirus vaccines [84]. For those, the shots may not work fully, or not at all. Obviously, this has been creating considerable frustration and fear, as many do not know whether they are affected or not. In the USA alone, it is now recognized that “millions of immunocompromised Americans may not be fully protected” by the two current mRNA Covid-19 vaccines [84]. Although studies are still ongoing, so far several groups of individuals have already been identified who do not have the desired antibody response after receiving either the vaccine by Moderna or the one by Pfizer/BioNTech [9, 26, 84]: • Immunocompromised individuals (e.g., people with HIV). • Patients with AI conditions, including inflammatory bowel disease, systemic lupus, rheumatoid arthritis, and another wide range of illnesses. In this group, mRNA-based vaccine immunogenicity is greatly varied. While many have a healthy antibody response, others have a very blunted or undetectable antibody response. • Individuals with certain blood cancers. Surprisingly, patients with chronic lymphocytic leukemia had a very weak response even if they were not undergoing treatment. • Those who have had organ transplants/those who take immunosuppressants. • Patients on treatments that impact B cell function. • Those who take commonly prescribed steroids. These troubling findings beg the question: is this just the tip of the iceberg? Given the urgent rush to produce Covid-19 vaccines as quickly as possible, vaccine trials excluded people with compromised immune systems and those who take immunosuppressants [84]. Most phase-3 trials also excluded those with chronic and serious conditions such as tuberculosis, hepatitis C, autoimmunity, and cancer, as well as pregnant and lactating volunteers. Additionally, with few exceptions, SARS-CoV-2 vaccine trials excluded the elderly, making it impossible to identify, inter alia, the occurrence of post-vaccination eosinophilia and enhanced inflammation in elderly people. Yet, experiments with mice showed that Covid19 immunized elderly mice were at particularly high risk of life-threatening Th2 immunopathology (see ref. [12] and references). Moreover, as stated above, there were no studies that assessed how SARS-CoV-2 vaccines react with other drugs, and neither were there toxicokinetic studies to determine the action of the vaccine once inside of a human body. While the above results demonstrate the ineffectiveness of the vaccine relative to the specific underlying conditions and exact prescription drugs which have been studied so far, not much is known about the effect of the vaccines in the large context of all possible pathophysiological conditions, or how they interact with common over-the-counter drugs. A related question is to what degree breakthrough infections are associated with an inadequate antibody response, and if so, the extent to which those individuals fall into the above categories. At present, it is suggested that individuals who do not mount a sufficient antibody response either receive boosters or a high-dose version of the vaccine. However, there are grave concerns with this approach:

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• The limited trials done for mRNA vaccines were based on a specific dosage, which, however, was not even tested for patients with a dysregulated immune system (including those already over-sensitized). Even for healthy individuals, the immune system can be a capricious thing, serving as a defensive shield one minute, and then shifting into overdrive the next, attacking itself [84]. Thus, simply ramping up the vaccine may result in significant safety issues that have not been investigated. • There is no reason to believe that boosters or high-dose shots will always be able to enhance antibody production. In this book, numerous mechanisms have been described that demonstrate that the anticipated action of mRNA vaccines cannot be taken for granted. Indeed, specific situations or circumstances may obliterate a desired neutralizing antibody response altogether. This may be due to the dichotomous nature of the immune response, the timing of, and interaction with, specific innate defense mechanisms, and others. Unless the exact mechanisms leading to reduced or absent antibody production have been clearly elucidated, higher doses or increased boosters may still not lead to actual protection and have hazardous effects on the patient instead (Fig. 4.4). • One of the greatest worries with mRNA vaccines is that no one knows what levels of antibodies are effective against the virus [84]. Albeit, with SARS-CoV2 one may question the role of neutralizing antibodies altogether, as necessary and sufficient actors to foster immunity (see also Sect. 6.2 and Chap. 11). The interaction of the virus in patients who do not mount a strong antibody response has become one of the pandemic’s “most fraught questions” [84]. One of the concerns is that, as discussed, this may in fact support the development of more virulent or contagious variants. Crucially, emerging research seems to confirm just that: variants of concern may emerge in a single host when they are unable to mount a sufficient neutralizing antibody response [23]. However, as analyzed in more detail below (Sect. 6.2), this urgently important concern also applies to all those with a large number of commonly shared antibodies [96]. Consequently, it is not merely a few isolated immunocompromised patients who could act as incubators of new viral variants. This very same trouble may actually involve the large group of those who have been mass-vaccinated via the same spike-based approach [96], especially since those vaccines are leaky (Chap. 12). One of the main problems is that Covid-19 vaccine design and efficacy studies tend to focus on the ability to prevent severe disease and death. The trouble with this is that it is not a clear-cut measure, but rather encompasses a large range of estimates. This leads to the predicament that vaccinated people could catch the virus without getting ill, pass it on [1], and, under the selective pressure of common antibodies, could induce viral escape mutants. All this will be examined in greater detail below.

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Fig. 4.4 Incomplete antibody response means much more than just no protection. For many, mRNA vaccines simply do not lead to the desired neutralizing antibody response. The immediate concern about this lack of mRNA vaccine immunity is that those individuals do not have the anticipated protection. While this is in itself a tragic situation that may endanger both those vaccinated and their contacts, the consequences may be even worse than that. Rather than not experiencing a protective effect of the vaccine, those individuals may experience rather serious adverse events. Whereas most human antiviral vaccines depend largely on the induction of antibody responses, emerging evidence suggests that protection against SARS-CoV-2 requires both antibody-mediated and T-cell-mediated immunity [46]. The induction of immune memory by mRNA vaccines [32, 33] rests on the generation of a specific immunogen. Nonetheless, if this is not the case, the synthesized proteins could trigger several hazardous events (dark and blue arrows), simply because they do not assume the anticipated role – precisely, as an antigen that provokes a neutralizing antibody response. On the other hand, T cell immunity is usually targeted via the addition of adjuvants [77]. Albeit, thanks to the dichotomous nature of the self-adjuvant response of mRNA vaccines, there is no guarantee that harmful predominant Th2 responses can be avoided. Immunopathologic Th2 responses may result in more serious Covid-19 breakthrough infection (red arrows), including death, the same way as they have been observed in studies with vaccines against coronaviruses and others, wherein pathology was associated with an antibody response with poor neutralizing activity (reviewed in [51])

4.10 The Need for a Clear Understanding of How mRNA Vaccines Affect Reproductive Health Because mRNA vaccines contain RNA and not live viruses, it is commonly believed that they do not create problems for reproductive health either. However, as has become increasingly clear only after the massive vaccine rollout, these products

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actually resemble pharmaceuticals, with both the RNA and the lipid NPs being biologically active (Chap. 9), but their effects poorly understood.

4.10.1 Pregnancy Safety Studies of mRNA Covid-19 Vaccines Pregnant women (and their fetuses) are at high risk for many medical interventions and are often excluded from safety trials of new drugs. Because of the unknown risks, vaccination of pregnant women remains poorly understood and hotly debated (see, e.g., [28]). For mRNA Covid-19 vaccines, the issue of vaccinating pregnant women has been concerning in several regards, and remains largely underappreciated.

4.10.1.1 Rigorous Exclusion During the Trials As they are regarded as high-risk individuals, pregnant women were excluded from all the Covid-19 vaccine trials. Actually, the FDA admitted:3 “Available data on SPIKEVAX administered to pregnant women are insufficient to inform vaccineassociated risks in pregnancy.” Moreover, prior to emergency approval of the jabs, both Pfizer and Moderna promised future studies on pregnant women [32, 33]. Peculiarly, Pfizer’s trial description4 of “A PHASE 1/2/3, PLACEBOCONTROLLED, RANDOMIZED, OBSERVER-BLIND, DOSE-FINDING STUDY TO EVALUATE THE SAFETY, TOLERABILITY, IMMUNOGENICITY, AND EFFICACY OF SARS-COV-2 RNA VACCINE CANDIDATES AGAINST COVID-19 IN HEALTHY INDIVIDUALS” adds yet another level of seriousness to their exclusion criteria. Not only are pregnant women to be excluded. Additionally, Sect. 8.3.5 in this document defines “Exposure During Pregnancy or Breastfeeding, and Occupational Exposure” and how this ought to be handled. Exposure During Pregnancy (EOP) occurs, if • “A female participant is found to be pregnant while receiving or after discontinuing study intervention.” • “A male participant who is receiving or has discontinued study intervention exposes a female partner prior to or around the time of conception.” • “A female is found to be pregnant while being exposed or having been exposed to study intervention due to environmental exposure. Below are examples of environmental exposure during pregnancy”:

3

https://dailymed.nlm.nih.gov/dailymed/fda/fdaDrugXsl.cfm?setid=23e93ef9-c51c-4e13-9cb3e65b6a0f1968. 4 https://cdn.pfizer.com/pfizercom/2020-11/C4591001_Clinical_Protocol_Nov2020.pdf.

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– “A female family member or healthcare provider reports that she is pregnant after having been exposed to the study intervention by inhalation or skin contact.” – “A male family member or healthcare provider who has been exposed to the study intervention by inhalation or skin contact then exposes his female partner prior to or around the time of conception.” The criteria involving inhalation or skin contact are perplexing. No reason is given why an intramuscularly administered product should trigger such a high level of alarm. Such a high level of caution would make sense from the perspective of micro RNAs and extracellular vesicles (Sect. 3.7.4). However, as described, these issues have not received much attention even after global vaccination. Astonishingly, Pfizer also details the steps that must be taken after EDP, among others, “in a participant or a participant’s partner:” “The investigator must report EDP to Pfizer Safety within 24 h of the investigator’s awareness, irrespective of whether an SAE has occurred.” The question that arises if this type of reporting during the trials was even possible. After all, the sheer number of reports of such EDPs would have been insurmountable. And moreover, did investigators even know of this reporting mandate?

4.10.1.2 How Has the Safety of mRNA Injections During Pregnancy Been Established? Despite the above rigorous exclusion policy during the trials, the global vaccination campaign has been accompanied by numerous statements that the shots are safe and effective for pregnant women. Indeed, in many countries, the latter are regarded as individuals who should get priority for (ongoing) booster injections.5 Given pregnant women were excluded from the trials, the believed safety of mRNA injections during pregnancy has been determined via the following. Limited Animal Studies Studies with rats were the basis of both Pfizer’s and Moderna’s reports that led to the approval of those vaccines. • Moderna’s study involving rats: In their EU Risk Management Plan for Spikevax, Moderna stated [33] that their developmental and reproductive study with rats had revealed no adverse findings. However, they went on to warn that “Animal studies do not indicate direct or indirect harmful effects with respect to pregnancy, embryo/foetal development, parturition or postnatal development. Administration of Spikevax in pregnancy should only be considered when the potential benefits outweigh any potential risks for the mother and foetus.” 5

e.g., in May 2022, Sweden specifically recommended the 5th COVID-19 shot to people over 65 and pregnant women, https://abcnews.go.com/Health/wireStory/sweden-5th-covid-19-shotpeople-65-pregnant-84929370.

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• The Pfizer-BioNTech DART study on female Wistar rats [32]. This rat study, too, reported no signs of adverse effects on the exposed animals or their pups. Notwithstanding this, the report specifically stated [32, p. 50]: – “There was an increase (∼2×) of pre-implantation loss (9.77%, compared to control 4.09%).” – “Among foetuses (from a total of n = 21 dams/l), there was a very low incidence of gastroschisis, mouth/jaw malformations, right sided aortic arch, and cervical vertebrae abnormalities.” – “Regarding skeletal findings, the exposed group had comparable to control group levels of presacral vertebral arches supernumerary lumbar ribs, supernumerary lumbar short ribs, caudal vertebrae number 180 days prior, 91 − 180 days prior, and ≤90 days prior). For each of these, the OR for Omicron, as opposed to Delta infection, remains in the range of approximately 2 − 2.5—that is, higher than for the 1x jabbed and less than the 3x jabbed. Therefore, the clearly increasing trend of the ORs relative to the number of injections is maintained for those without prior infection across different time points, implying that it is not waning immunity, but the number of doses, which is the key factor of Omicron’s increase relative to Delta. Hybrid Immunity Interestingly, the small number of people (1% of cases) who were infected before study begin, when later vaccinated, essentially exhibited the opposite trend to the above. As seen from Table S13 in [23], for both of the mRNA vaccines, the adjusted (unadjusted) ORs for Omicron vs Delta infection were after 1 dose: 6.0 (5.51), after 2 doses: 3.66–4.36 (3.21–4.28), according to time since vaccination, and after 3 doses: 2.43 (1.8). Nonetheless, the adjusted OR for the unvaccinated with prior infection was 3.32, that is, lower than that for the 1 or 2x RNA vaccinated with prior infection. Again, this shows that the odds for Omicron infection were greater among the vaccinated than the unvaccinated, even for those with documented prior infection. For the people who had a history of prior infection, the OR values were substantial. Overall, one sees that among the 1x and 2x mRNA vaccinated people, Omicron infection as opposed to Delta infection was most common among those previously infected. For example, the adjusted odds of prior infection and 1x inoculation were sixfold higher among cases with Omicron compared to Delta variant infection, while the adjusted odds for the non-previously infected and 1x inoculated were only 1.73. That is, other than in the 3x mRNA group, previous infections were more common among the Omicron compared to the Delta cases. However, the severity, length, and number of previous infections are unknown. It is possible that the group with prior infections also included repeat infections—likely indicating some form of immune compromise. Above, the percentage of those with previous infections was minimal (1% of cases only). However, the ongoing Omicron surges will likely shift measures. The concern now is that repeated inoculations may be associated with some form of immune dysregulation (Chap. 11) and repeat breakthrough infections—which will further advance viral escape.

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12.5.3 The Number of Shots: Likely the Main Scale Driver on Both the Viral and the Host Side The results from the two studies from Denmark and California reveal population characteristics that are most favorable to the evolution of Omicron as a new dominant variant. Specifically, the group of people with higher OR values are indicative of conditions that are more conducive to the Omicron than the Delta variant, allowing it to take over at the population level. Escape mutants clearly have an advantage in immunocompromised people. They may not only succumb to (repeated) infections but do so despite vaccination as well. This is in line with the observation in [23], where those with prior infection plus vaccination were associated with some of the highest OR values in their study. Nonetheless, even if some escape mutants emerge in some individuals with poor immunization capabilities, this does not automatically mean that they become dominant in a population or even globally. At the population level, both the studies from Denmark and California agree on one key scale driver—the number of Covid vaccines administered in the population. The evolution of Omicron as a new variant that quickly has become dominant worldwide has been enabled by a certain environment in which Omicron can do very well. An increased number of shots seems to enable Omicron to thrive epidemiologically, as corroborated by the larger OR values among those more frequently jabbed. Omicron’s increased advantage—in line with the number of vaccine doses administered at the population level—is likely not only fostered Omicron’s escape to offset targeted immune pressure; the number of injections is also a key scale driver on the host’s side to potentially provoke immune dysfunction (Chap. 11). Thus, via this critical scaling influence, viral escape mutants may not only have a better chance to evade vaccine-induced antibody pressure, but they may be able to exploit possible vaccine-induced weakening of immune response in some individuals as well (summarized in Fig. 12.5). Overall, Omicron seems to have confirmed the notion developed in Chap. 7, with the number of injections being one of the most significant scaling features to support the development and persistence of dominant escape mutants.

12.6 Omicron, as an Escape Mutant, Can Use a New Way to Enter Cells Strong evidence is now emerging to resolve the paradox (Fig. 12.1) of how Omicron can simultaneously be more infectious and less pathogenic. In Ref. [27], Gupta and collaborators not only confirmed Omicron’s significant evasion to therapeutic monoclonal and vaccine-elicited polyclonal NAbs. Perhaps even more importantly, they showed that Omicron’s spike-mediated immune escape is accompanied by a

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Fig. 12.5 The number of injections as the main scale driver. There is increasing evidence that the number of inoculations acts as a strong scale driver (Chap. 7), both at the viral and at the host’s side. Regarding the former, an increased number of boosters both aggravates the risk of immune dysfunction and also raises the prospect of increased injury overall, further minimizing the ability of the host to successfully neutralize the virus. On the viral side, pressure on the virus increases both via specific host immune responses and their frequency as well. Thus, an increased number of injections likely engenders a multiplicative effect to foster the emergence and dominance of immune-escape variants, as seems to have occurred for Omicron

new mechanism for how this VOC enters a cell. Notably, the new way, causing a significant cellular tropism shift, also makes it less likely to cause severe Covid-19 illness in the lungs (as already previously suggested, see section “First Evidence that Omicron Causes Less Severe Disease”). Their work also extends the previous fascinating study [46], which identified Omicron’s new pathway to enter cells. In a nutshell: • SARS-CoV-2’s cell entry before Omicron: SARS-CoV-2 efficiently uses several human host factors for viral attachment and entry. Central to this process is the viral spike attaching to the host cell receptor ACE2 via the use of additional attachment factors. Prior to Omicron, it was already well established that SARSCoV-2 utilizes the serine protease TMPRSS2 and endosomal proteases for the activation of the spike protein to facilitate membrane fusion [20]. Specifically, viral and cellular membrane fusion relies on conformational changes of the spike, which is achieved via cleavage of the S1/S2 junction by furin, after which the S2’ site is cleaved by either the cell surface TMPRSS2 or

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the endosomal cathepsin [20]. For Delta, efficient utilization of TMPRSS2 has proven essential for optimal cell entry/cell–cell fusion and is believed to be associated with its enhanced viral replication [27, 46]. • Altered TMPRSS2 usage observed for Omicron, which affects replication and fusion activity: Analyzing differences in viral replication kinetics and entry pathways, Ref. [46] surprisingly found that replication and fusion activity of the Omicron variant is much less dependent on TMPRSS2 than seen in previous variants. When specifically comparing differences in entry pathways in various cell lines, it was found that Omicron infection mainly depends on endocytosis rather than the TMPRSS2 pathway. That is, in contrast to Delta, Omicron is not as effective in using TMPRSS2 for viral replication. In addition, the Omicron variant also shows much weaker fusion activity than Delta. • The altered TMPRSS2 usage affects Omicron’s tropism: Omicron’s less efficient utilization of TMPRSS2 for spike cleavage at S1/S2 is also associated with a shift in cellular tropism away from TMPRSS2 expressing cells. Notably, TMPRSS2 is highly expressed in alveolar type I and type II cells of the lung. Just as the enhancement of viral replication of the Delta variant is in line with its ability to effectively utilize TMPRSS2, the opposite is now the case for Omicron. This means that Omicron has poorer replication in the lungs when compared with the Delta variant. The shift in cellular tropism has profound implications for its altered pathogenesis. • The altered TMPRSS2 usage affects Omicron’s pathogenesis: The final observation to deduce the differential pathogenesis between Delta and Omicron was obtained in [27]. Consistent with both suboptimal S1/S2 cleavage and inability to utilize TMPRSS2, the Omicron spike shows low fusogenic potential and is much less able than Delta to cause fusion between cells (syncytia formation). This is likely one of the key reasons why Omicron is intrinsically less dangerous. Syncytia are mainly formed in lung tissue and result in extensive immune reaction, which marks the excessive inflammatory response of previous SARS-CoV-2 variants during severe disease [27]. Synctia formation has also been found in postmortem lung specimens, in line with the notion that fusion activity is associated with disease severity [46]. Altogether then, the novel cell-entry mechanism is one of the most substantial changes of Omicron relative to the previous VOCs. It explains why Omicron is both more infectious and still less dangerous overall. As Omicron is not so well equipped to enter lung cells and, once entered the cells, is less able than Delta to cause cell fusion, this variant causes less lower respiratory infection and less lung damage. At the same time, Omicron’s reliance on endocytosis rather than the TMPRSS2 pathway makes Omicron more efficient to infect cells as well.

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12.7 Emerging Trends and Open Questions Omicron has raised a number of questions that are rather intriguing. This section introduces some of them.

12.7.1 IgG Bias, High Ab Levels, and Immune Imprinting In addition to the seminal finding of vaccine mRNA and spike antigen persisting in lymph node GCs (Chap. 10), the paper by Röltgen and collaborators published in Cell [41] also reports significant differences in immune responses stimulated by vaccination and natural infection. Let us backtrack. A special aim of their study was to compare differences in variant binding RBD by vaccinee and Covid-19 patient plasma IgG, of early SARS-CoV-2 variants, compared to Wuhan-Hu-1. The variants considered were Epsilon, Kappa, B.1.526.2, B.1.214.2, Alpha, Eta/Iota, Gamma, P.3, and Beta. To ascertain these differences, the study authors calculated the ratios of anti-RBD IgG concentrations for Wuhan-Hu-1 compared with the other viral variants. Remarkably, this revealed that vaccination evokes a greater breadth of IgG binding to viral variants compared to natural infection, at least at earlier time points [41]. This applied to all four COVID-19 vaccines, BTN162b2, ChAdOx1-S, GamCOVID-Vac, and BBIBP-CorV, that were tested. While this seems like a great advantage over natural infection, it must be noted that SARS-CoV-2 is a respiratory virus and more effectively neutralized by mucosal antibodies. The differences in immune responses between SARS-CoV-2 infection and vaccination are significant: indeed, Ref. [41] further reports the following: • Natural infection stimulates robust but short-lived IgM and IgA responses. • The BNT162b2 vaccine induced a highly IgG-polarized serological response with minimal IgM- and IgA-binding spike and RBD. • While IgG concentrations decreased rapidly following vaccination, boosting with a third dose resulted in concentrations after 7 to 8 days actually exceeded prior peak concentrations (Chap. 11). Systemic Versus Mucosal Immunity The potent effect of vaccines to drive early and extensive IgG class-switching, and the further increase in IgG concentration beyond previous levels, demonstrates a significant immune bias. One may wonder if this is at the expense of IgM and IgA responses. As argued above (Chap. 11), IgA and IgM antibodies produce the strong mucosal immune response needed for respiratory diseases. In particular, secretory IgA, effectively produced by natural infection, confers strong immunity in our mouth and the upper respiratory pathways. Thus, because of the pronounced bias for IgG production with a relative absence of IgM and IgA responses, vaccinated people do not have the protective mucosal immunity seen in natural infection.

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Now, since they cannot neutralize the virus in the location of the body where the virus comes in, this may help explain why vaccinated people carry a high volume of virus and also effectively pass the virus to others. Ab Breadth Versus Actual Immune Memory: Strong Immune Imprinting Effects After Vaccination In Ref. [41], the greater breadth of variant RBD binding by vaccinee IgG compared with COVID-19 patient IgG was also seen in a different cohort of predominantly mild patients (this also included the Delta variant). Nonetheless, a comparative analysis of variant binding assessed in the lab is not the same as the actual immune response triggered by real infection. Notably, even the greatest antibody breath against viral variants cannot account for immune memory effects such as imprinting (Box 3), whereby primary exposure to an antigen affects B cell and antibody responses against variant epitopes in future infection. In fact, in contrast to variant-specific serological responses following Alpha and Delta SARS-CoV-2 infection, vaccination was actually shown to evoke strong imprinting of the serological response to variant RBD. Specifically, in [41], analysis of plasma from individuals vaccinated with WuhanHu-1-like antigens and subsequently infected with Alpha or Delta variants revealed that imprinting from the initial antigens altered IgG responses to the other variants. Despite breakthrough infection with Alpha or Delta variants, BNT162b2 inoculated individuals showed patterns of IgG binding to viral variant RBDs similar to those of individuals exposed to only Wuhan-Hu-1. In addition to the deleterious effects on individuals, it begs the question if vaccine-caused imprinting could support the development of escape mutants as well. High IgG Levels Versus Ongoing Evolution It is interesting to note that despite initially lower variant binding, natural infection in Ref. [41] demonstrated an ongoing evolution of antibody response. Indeed, the antibody breath against viral variants after infection was shown to improve through at least 7 weeks post-onset of symptoms, supporting the notion that fluidity and evolution may be more important than a sustained high level of virus-specific antibodies—which we now know is not a reliable surrogate of immune protection (Sect. 11.5.4). Differences in immune responses between natural infection and injections are significant. Yet, the above confirms that the actual mechanisms, underpinnings, and sequelae remain insufficiently appreciated. While it is technically easier to do studies in the lab, e.g., to predict which variants will effectively be targeted by the injections, the interplay between systemic and mucosal immunity is poorly understood, as are immune memory effects such as imprinting. Both of these factors not only affect those vaccinated but the entire population at large as well, via their potential to influence viral escape.

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12.7.2 Omicron-Specific Boosters and Immune Priming The recognition of Omicron’s enhanced immune escape has prompted vaccine makers to work on Omicron-specific jabs. While it is essential that vaccines are updated, the question is what strain actually will be circulating by the time they are released and how they compare to existing vaccines in terms of safety and real-world effectiveness. The question to what degree Omicron-specific shots will offer greater immunity or protection has led to a somewhat surprising result. In a work by the National Institutes of Allergy and Infectious Disease’s Vaccine Research Center [16], mRNA1273 was compared to an Omicron-specific formulation in nonhuman primates. Immune responses were evaluated via several means, including serum antibody responses, neutralization titers, B cell cross reactivity, and virus replication in lower airways. Yet, surprisingly, the findings revealed that animals boosted with the original vaccine had similar levels of protection compared to those boosted with the updated version, suggesting that products specifically designed for Omicron do not improve the efficacy of existing boosters. According to the study authors, this surprising finding can likely be explained by the principle of original antigenic sin (aka, imprinting), whereby prior immune memory is recalled by a related antigenic encounter (Sect. 3.2): the imprinting of the original vaccine seems to have generated B cells which, after the booster, ended up cross-reactive to the previous viral strains. This same phenomenon had been observed by the same group when comparing an mRNA-1273 booster with a boost specially matched to the Beta-VOC spike protein. Likewise, both of these boosters provided similar levels of protection, despite one of them being specifically designed for Beta. A Potential Deadlock Situation? According to Ref. [16], both the booster with the original vaccine and the one specific for Omicron led to a significant increase in neutralizing antibodies against the different VOCs. Now, if this is enough to neutralize the virus, then neutralization activity could potentially extend to closely related variants as well. However, this does not seem to have happened during the pandemic, as demonstrated by the rising vaccine mismatch and decline in VE— which is in line with the recognition that NAb titer is not a suitable proxy of actual immunity. This also leads to the concern that vaccine-induced imprinting may aid the development of viral escape, as the immune system may be locked in a detrimental memory state, unable to update to future variants. Imprinting Effects of Covid-19 Vaccines Concerns about the effects of immune imprinting were already articulated early during the pandemic (Box 3). The lack of improved protection via specific boosters raises the critical question if current boosters have exhausted the protective potential against SARS-CoV-2. By contrast, because of the influential involvement of mucosal immunity, it seems unlikely that imprinting affects natural infections the same deleterious way.

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Further evidence of immune imprinting as a result of Covid-19 vaccination has been documented elsewhere: • Several studies involving Beta-specific vaccines suggest immune imprinting effects, reflected in the antibody response developed against newer viral variants: for example, vaccination with mRNA-1273 followed by an mRNA booster that specifically expresses Beta-spike resulted in higher neutralization titers to Wuhan-Hu-1-like variants compared to Beta (see [41] and references therein). • Vaccination with Wuhan-Hu-1 spike antigen: a strong imprinting effect, confirmed by antibody specificities, can be seen in numerous breakthrough infections [41]. • Vaccination causing imprinting of humoral immunity, altering IgG responses to viral variants: as described in Sect. 12.7.1, BNT162b2 vaccinees who experienced breakthrough infections with Alpha or Delta showed patterns of IgG binding to viral variant RBDs similar to those who had only been exposed to Wuhan-Hu-1. This is in contrast to variant-specific serological responses following natural Alpha and Delta SARS-CoV-2 infection [41]. A Lower Anti-N Antibody Conversion Rate Among the Vaccinated in Moderna’s Trials In April 2022, a study [15] from Moderna and collaborators announced a new diagnostic tool to identify recent and remote infections with SARS-CoV-2. However, the actual finding that is based on may even be more interesting in its own right. The analysis uses data from Moderna’s adult trial, where trial participants later exposed to the virus were compared to those in the placebo group. The hypothesis was that since vaccine recipients will have anti-spike antibodies, they will therefore have different anti-N antibody seroconversion and/or seroconversion profiles after SARS-CoV-2 infection, compared to placebo recipients. It was indeed found that there was a significant difference in the two groups when they later became infected. At a median follow-up of 53 days post-diagnosis, among participants with PCR-confirmed Covid-19 illness, seroconversion to antiN antibodies occurred only in 40% of the mRNA-1273 vaccine recipients—as opposed to 93% of the placebo recipients. Interestingly, the study also showed that the 2x Moderna vaccinated have a lower anti-N Ab conversion rate than the unvaccinated. Notably, the discrepancy was not an effect of different viral loads, as it was maintained across all the different levels (Figure 2 (B) in [15]). The finding that those who had received the vaccine did not generate antibodies to a key epitope of the virus the same way as the unvaccinated do is sobering indeed: • The data of Ref. [15] imply that vaccinated people have a reduced ability to mount an Ab response to other components of the virus. • The duration of the diminished seroconversions is unknown. One may even wonder that this sobering finding is a type of immune imprinting to the vaccineinduced spike protein, in which case the effect could go on indefinitely. • Given that vaccinated people are less able to mount a complete immune response compared to natural infection, this increases the concern for viral escape mutants.

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12.7.3 The Origin of Omicron, and Why It Matters Despite normally being linked to South Africa, the origin of the Omicron VOC is still unclear. According to the Dutch health agency,27 Omicron was identified in retests of samples in Europe nearly a week before it was discovered by South African scientists. Actually, since Johannesburg is home to the largest airport on the African continent, the variant could have originated anywhere in the world. Still, its emergence is puzzling, mostly not only because of its vast number of mutations but also because it does not branch off the SARS-CoV-2 family tree in any obvious way.

12.7.3.1 Several Theories About Omicron’s Origin Three possible scenarios of Omicron’s emergence are now regarded as most likely. As described in Nature:28 (1) It may have silently been spreading through a simple process of gradual evolution: this option is not regarded as very likely, as personto-person spread is not believed to be conducive to the accumulation of the many mutations seen in Omicron. (2) It may have developed over weeks or months in a person with chronic infection: as described in Sect. 7.3.4, similar developments have been observed in people with compromised immune systems and have been shown to lead to mutations, especially under immune pressure (e.g., monoclonal Ab treatment). (3) It may have come from an animal intermediate: the idea is intriguing—it could have given the virus enough time and also enabled it to come up with some of its rare mutations. However, no such animal has been identified.

12.7.3.2 Could Omicron Have Arisen as a Vaccine-Escape Mutant? There is no concrete evidence for either of the above possibilities. On the other hand, previous sections suggest that option (2) could also have taken place in vaccinated people, on the basis of Covid-19 vaccines being leaky and possibly evoking some form of immune dysfunction, especially with boosters. In addition, breakthrough infections may not be serious enough for the host to seek and require other ways to eliminate the virus. As seen from the large number of asymptomatic Covid cases, many are not even aware they are infected, especially if they think they have immune protection.

27 https://www.npr.org/2021/11/30/1060025081/omicron-variant-netherlands-europe-southafrica, https://www.fox6now.com/news/omicron-variant-was-in-europe-before-it-was-detectedin-south-africa-dutch-health-agency-says. 28 https://www.nature.com/articles/d41586-022-00215-2?utmsource=Nature+Briefing& utm_campaign=b124caafb6-briefing-dy-2022028&utm_medium=email&utm_term= 0c9dfd39373-b124caafb6-45026129.

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Although the possibility of Omicron’s origin in the context of breakthrough infections has not been widely acknowledged, it may be worth considering, for several reasons: Plausibility of Omicron’s Emergence via Vaccine Breakthrough Infections Evidence that antibody-resistant SARS-CoV-2 lineages comprise a higher percentage of cases in fully vaccinated, as compared with unvaccinated (or all) cases, was first presented in a paper published in Nature Microbiology [43]. This study, based on SARS-CoV-2 whole-genome sequences and viral loads from 1373 persons with COVID-19 from the San Francisco Bay Area from 1 February to 30 June 2021, shows that vaccine breakthrough infections are more commonly associated with circulating antibody-resistant SARS-CoV-2 variants than infections in unvaccinated individuals. Evidence that Omicron specifically could have emerged via vaccine breakthrough infections from antibody-resistant VOCs can also be seen at the genetic level. Notably, Ref. [14] stresses that Omicron has mutations in both the NTD and the RBD which go beyond the protective threshold provided by vaccines and antibodies. The authors caution: “Playing catchup to SARS-CoV-2 selects for more resistant and transmissible variants and may not be successful in the long run.” As noted, a clear discord in percentages of patients infected with Omicron compared to Delta was also seen in [11], where this number was substantially higher for the fully vaccinated than those who were not (Table 12.1). The Predicament of Immune Tolerance and Immune Imprinting The potential of vaccines leading to tolerance, imprinting or other weakened immune responses (Chap. 11) is a reason for serious concern, and not only for those vaccinated. While they may thereby be prone to repeated infections, they may also be less able to fully neutralize the virus, effectively providing the virus a niche where it can thrive. Omicron Found to Emerge in Household Settings At the epidemiological level, as analyzed in Sect. 12.5, independent studies show a clear increase in the odds of being infected with Omicron compared to Delta, in a dose-dependent manner relative to the number of vaccine doses administered, beginning with those never inoculated (lowest odds) to those 3x infected (highest odds). The study from Denmark [25] specifically describes the emergence of Omicron in a household setting. In fact, Table 7 in [25] summarizes the intra-household correlation of variants, i.e., the probability that the primary case infects household members with the same viral variant. Notably, not all the household infections were of the same variant though. It is suggested here that this remarkable finding can be explained as follows: • A spectrum of variants: Altogether, Lyngse et al. [25] identified 6397 secondary infections during their 1–7 day follow-up period. Among these, 5077 positive secondary cases also had a Variant PCR result. No further information is given about the remaining positive secondary cases. • Apparently, not all secondary infections were identified by Variant PCR, indicating they were infected with other variants—a finding interesting in its own

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right. Yet, one should remember that designating something a certain variant is a technical decision based on some snap-shot information alone. Even if confirmed by whole-genome sequencing (WGS), such an assessment cannot account for the ongoing viral mutations, both within one infected person, and more so, at large. Furthermore, for most samples, due to practical reasons, differentiation between variants is realized with the aid of specific target sequences alone (such as the SGTF). In this case, of course, mutations outside this target are completely unaccounted for [29, 30]. These technical constraints make it impossible to fully capture what is going on in nature where there is a large spectrum of variants, rather than just one or two exact same variants only. • Different variants between primary and secondary cases: The spectrum of variants may explain the somewhat unexpected observation in [25] regarding the different variants seen in the same household. Specifically, of the 1137 cases where the primary case was Omicron, there were 23 secondary cases designated as Delta, and conversely, of the 3917 primary Delta cases, 90 of the secondary infections were labeled as Omicron. Given that Lyngse and collaborators [25] carefully corrected for various confounders, e.g., previous undetected infection, their data strongly point to the plausibility of new variants arising very quickly in a household setting. A Common Factor Driving the Emergence and Persistence of Escape Mutants? The distribution of different SARS-CoV-2 variants29 since the beginning of the pandemic reveals an interesting trend. During 2020, several major variants coexisted globally, in line with the natural flux and dynamics of viral mutations. However, since the rollout of the vaccines, the many different variants have more and more been replaced by fewer dominant ones. While the classification of the sub-lineages remains in constant flux, the radical decline in variant diversity concomitantly happening with mass vaccination is in line with survival advantage of fewer variants optimized to escape targeted selective pressure. This type of development was clearly documented in the above-mentioned publication in [43], which during the first part of 2021 identified multiple variant lineages in their study population. By contrast, the distribution of study lineages among vaccinated cases was found to be skewed. As already mentioned above, antibody-resistant lineages were shown to comprise a higher percentage of cases in fully vaccinated, compared with unvaccinated or all cases. The authors find that in contrast to previous studies, “vaccine breakthrough infections are overrepresented by immunity-evading variants as compared with unvaccinated infections” [43]. The above trend is fascinating as during 2020 and 2021 SARS-CoV-2, apart from some cross-immunity via common-cold CVs, still largely encountered an immunologically naive population, and immunity was to a large part afforded by common vaccination strategies. Meanwhile, however, pretty much everyone has been infected with Omicron. As vaccine-immunity has been waning off but natural

29 e.g.,

https://nextstrain.org/,https://covariants.org/.

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immunity increasing, there has been less of a common selection pressure, as is also seen in the many different Omicron subvariants that have been emerging since 2022.

12.7.3.3 Beyond the Current Models Limits of prevalent models in predicting the safety and efficacy of new vaccines have been described in previous chapters. Many of these models also limit our comprehension of a new variant, as particularly obvious with Omicron. Problems Associated with Over-Emphasis of Sequence-Only Determinants As noted, Omicron is extremely different from earlier variants, with its closest genetic ancestor estimated to date back more than a year. ago30 Yet, this concept of evolutionary distance is based on complex genetic modeling—a problem that likewise arose in the search of the SARS-CoV-2’s origin and species susceptibility. In the context of the ancestral strain, an article in Scientific Reports [36] posits that the prevalent distance measures, as e.g. centered on amino acid differences, may not be the only, or even best, choice (see also Sect. 2.1). A surprising finding based on in silico comparisons revealed that the binding affinity of the S protein to various animal ACE2 receptors was radically inconsistent with what would be expected from ACE2 sequence analysis. Despite high similarities in protein sequences, different animals often exhibited drastically different binding energies to the spike protein. For instance, after humans, the pangolin ACE2 showed the next highest binding affinity despite having a relatively low sequence homology. Conversely, the affinity of monkey ACE2 was much lower despite its high sequence similarity to human ACE2. These and related surprising discrepancies suggest that structural rather than sequence-based parameters would be more suited for crossspecies analysis [36]. The study authors note the significant role of spatial and physiochemical surface features such as hydrogen bonding, electrostatic, hydrophobic, and lipophilic interactions. Albeit, how these are best assessed and combined to yield a comprehensive metric is a hot topic of discussion. Similarly then, via an alternative, structure-based distance measures, it seems feasible that Omicron could be more closely related to the other variants; accordingly, it may also have arisen sooner than previously suggested. Mutations as Bursts? The notion of the genetic “code” conveys the idea of biological information being transmitted from cell to cell, in a manner not unlike that of man-made information being transmitted across the internet. Via the digitization of biology—most literally, the conversion of nucleotide codes of DNA to machine-

30 https://www.nature.com/articles/d41586-022-00215-2?utmsource=Nature+Briefing& utmcampaign=b124caafb6-briefing-dy-2022028&utm_medium=email&utmterm=0c9dfd39373b124caafb6-45026129.

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readable formats—the linear representation of the genetic material is often seen as analogous to computer words sent across space. Although there are major limitations of such a simplified conception [32], this allegory invites a broader view about the rate of natural mutations. A phenomenon well known in information theory is that errors happening to codewords during transmission do not necessarily happen bit by bit. This may be best visualized by codewords sent across space, where electromagnetic or other influences can corrupt the word that was sent. This often manifests as bursts of errors affecting several consecutive bits, rather than at independent locations and happening in a piecemeal fashion only. On the other hand, errors (aka, mutations) in genetics are typically assumed to happen little by little, in contrast to error bursts known from information theory. This poses the question if in some situations genetic changes could happen as bursts rather than mere single-nucleotide changes happening slowly over time. A similar phenomenon has been described by Denise Noble,31 who posits that organisms can hypermutate and shuffle their genome when under stress. Clearly, this fascinating concept overthrows the idea that mutations always happen minimally and slowly. These and other surprising epigenomic features have been described in an editorial in Future Medicine [33]. As these override deep-seated notions in genomics, they have not been widely appreciated. Nonetheless, these considerations might serve an important role in, e.g., explaining how Omicron could have gained its significant number of mutations in such a short time, and others.

12.7.4 Future Variants: A Guaranteed Trajectory of Common-Cold CVs? The problem of vaccine-induced pressure on viral evolution is succinctly summarized in the Nature Microbiology publication [43] mentioned above. It posits that the predominance of immune-evading variants among post-vaccination cases in their study population seems to indicate “selective pressure for immune-resistant variants locally over time in the vaccinated population concurrent with ongoing viral circulation in the community” [43]. While this study was conducted during the Delta era, Omicron’s combination of (a) increased infectivity and (b) decreased disease severity, may even further exaggerate the issue. The believed beneficial feature of the injections to protect from more serious disease drastically likely amplifies the problem, as mild or asymptomatic breakthrough infections become a significant factor at the epidemiological level: they give the virus time to further evolve, especially in this context of nonsterilizing vaccines.

31 https://www.youtube.com/watch?v=F-QF7QOax4&list=PLkFGg6nuHI8qMg4MRAbe5rSvw XxlGdqT&index=5.

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Unfortunately, from what is known now, Omicron effectively escapes immunity from the first-generation Covid-19 vaccines. Moreover, it seems that Omicronspecific boosters may not be any better in neutralizing the virus either (Sect. 12.7.2). Regardless of the exact immune protection that Omicron-specific vaccines may bring, they will likely not evoke sterilizing immunity, for the same reasons as detailed in Chap. 11. As mRNA vaccines continue to pursue the goal of “minimizing severe disease and death,” the incomplete neutralization of the virus will continue to provide a significant advantage to escape mutants. Nonetheless, as seen from Omicron, escape variants can emerge with features that previously never had been expected. Will we be as lucky as before, so that future variants will also be less pathogenic? Or will the pendulum swing in the other direction, causing more serious disease again? Key questions remain unresolved. Before the Covid-19 pandemic, we have not seen such an intense mass vaccination regimen ever before. We really do not know how the virus is going to respond. It is tempting to think that the Omicron VOC has mutated into a form of virus that has changed its phenotypic profile in a way that it just has to remain less virulent. Some may say SARS-CoV-2’s evolution into a much milder form of the virus is essentially guaranteed, in line with the trajectory of other coronaviruses and previous pandemics ending in endemicity. However, some worrying aspects related to this belief are as follows: Omicron’s Evolution Difficult to Comprehend A key feature of transmissibility and disease severity of pre-Omicron variants is the polybasic cleavage site (PBCS) between S1 and S2, with cleavage efficiency at this site correlating with TMPRSS2 dependency. In terms of SARS-CoV-2’s evolution, it is interesting to note that Delta seems to have optimized the TMPRSS2 pathway, exhibiting the highest fusion activity of the pre-Omicron variants [46]. Surprisingly, however, while Omicron also has mutations in the furin cleavage site, it took a turn in the previous optimization development, switching to the endosomal entry route instead. This was not really predictable with previous models. Incompleteness of Sequence-Based Methods Despite the presence of three mutations that individually were predicted to favor spike S1/S2 cleavage, Omicron exhibits S1/S2 cleavage deficiency and an inability to effectively utilize TMPRSS2. The reduced cleavage efficiency of spike and the impaired TMPRSS2-mediated entry are favoring a TMPRSS2 independent mechanism for cell entry via endocytosis which does not seem to directly follow from sequence-based determinants. That Omicron favors the endocytic pathway seems to be heavily influenced by mutations which lead to an increase in the overall charge of the S protein. In fact, Gupta et al. [27] suggest that this may have made S more sensitive to low-pH induced conformational changes, which could facilitate the low-pH endosomal entry route and/or entry in the lower pH environment in the upper airway. As described before, structure- rather than sequence-based modeling has proven challenging, given that it is not clearly resolved how structural differences are obtained. Parameters such as polarity, charge, molecular volume, size, hydrogen

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bonding ability, salt bridge formation, and others are crucial contributors to structure and function; yet, computer simulations reveal how difficult it is to align them with sequence-based analyzes [36]. Consequently, it does not seem straightforward at all to model characteristics of future escape mutants. Under ongoing immune pressure, who can tell if future variants will take another surprise turn, just as they did for Omicron? New Pathways? Considering the extraordinarily high degree of infectivity of Omicron, both of the original variant, and even more so of more recent subvariants, one may wonder if future variants will maintain its current mechanism of how it infects cells. It seems difficult to believe that the evolution of yet more infectious escape mutants will go on indefinitely, unless these take a different turn again, further expanding alternative ways to enter cells. Doing so may also make them more pathogenic again, possibly whilst maintaining a high degree of infectivity. Viral evolution may be radically different whether or not it is in the absence or presence of large-scale immune pressure (as exerted by vaccination). This has been the main fear of Geert Vanden Bossche who has been speaking out for years now against vaccinating into a pandemic32 worried about the development of SARSCoV-2 superstrains and the “dramatic consequence on humanity” this could entail.

References 1. Altarawneh HN, Chemaitelly H, Ayoub HH, Tang P, Hasan MR, Yassine HM, Al-Khatib HA, Smatti MK, Coyle P, Al-Kanaani Z, Al-Kuwari E, Jeremijenko A, Kaleeckal AH, Latif AN, Shaik RM, Abdul-Rahim HF, Nasrallah GK, Al-Kuwari MG, Butt AA, Al-Romaihi HE, AlThani MH, Al-Khal A, Bertollini R, Abu-Raddad LJ (2022) Effects of previous infection and vaccination on symptomatic omicron infections. N Engl J Med 387(1):21–34 2. Altarawneh HN, Chemaitelly H, Hasan MR, Ayoub HH, Qassim S, AlMukdad S, Coyle P, Yassine HM, Al-Khatib HA, Benslimane FM, Al-Kanaani Z, Al-Kuwari E, Jeremijenko A, Kaleeckal AH, Latif AN, Shaik RM, Abdul-Rahim HF, Nasrallah GK, Al-Kuwari MG, Butt AA, Al-Romaihi HE, Al-Thani MH, Al-Khal A, Bertollini R, Tang P, Abu-Raddad LJ (2022) Protection against the omicron variant from previous SARS-Cov-2 infection. N Engl J Med 386(13):1288–1290 3. Bar-On YM, Goldberg Y, Mandel M, Bodenheimer O, Amir O, Freedman L, Alroy-Preis S, Ash N, Huppert A, Milo R. (2022) Protection by a fourth dose of bnt162b2 against omicron in Israel. N Engl J Med 386(18):1712–1720 4. Beekman M, Latty T (2015) Brainless but multi-headed: decision making by the acellular slime mould physarum polycephalum. J Mol Biol 427(23):3734–3743 5. Boisseau RP, Vogel D, Dussutour A (2016) Habituation in non-neural organisms: evidence from slime moulds. Proc R Soc B Biol Sci 283(1829):20160446 6. Buchan SA, Chung H, Brown KA, Austin PC, Fell DB, Gubbay JB, Nasreen S, Schwartz KL, Sundaram ME, Tadrous M, Wilson K, Wilson SE, Kwong JC, on behalf of the Canadian

32 https://www.voiceforscienceandsolidarity.org/.

References

7.

8.

9. 10.

11.

12. 13.

14. 15.

16.

17.

18.

19.

411

Immunization Research Network (CIRN) Provincial Collaborative Network (PCN) Investigators (2022) Effectiveness of covid-19 vaccines against omicron or delta infection. medRxiv Buchan SA, Chung H, Brown KA, Austin PC, Fell DB, Gubbay JB, Nasreen S, Schwartz KL, Sundaram ME, Tadrous M, Wilson K, Wilson SE, Kwong JC, the Canadian Immunization Research Network (CIRN) Provincial Collaborative Network (PCN) Investigators (2022) Effectiveness of covid-19 vaccines against omicron or delta symptomatic infection and severe outcomes. medRxiv Cameroni E, Bowen JE, Rosen LE, Saliba C, Zepeda SK, Culap K, Pinto D, VanBlargan LA, De Marco A, di Iulio J, et al (2021) Broadly neutralizing antibodies overcome SARS-Cov-2 omicron antigenic shift. Nature 602:664–670 Chen J, Wang R, Gilby NB, Wei G-W (2022) Omicron variant (b. 1.1. 529): Infectivity, vaccine breakthrough, and antibody resistance. J Chem Inf Model 62(2):412–422 Chia PY, Xiang Ong SW, Chiew CJ, Ang LW, Chavatte J-M, Mak T-M, Cui L, Kalimuddin S, Chia WN, Tan CW, et al (2021) Virological and serological kinetics of SARS-Cov-2 delta variant vaccine-breakthrough infections: a multi-center cohort study. Clin Microbiol Infect 28(4):612-e1. Christensen PA, Olsen RJ, Long SW, Snehal R, Davis JJ, Saavedra MO, Reppond K, Shyer MN, Cambric J, Gadd R, et al. (2022) Signals of significantly increased vaccine breakthrough, decreased hospitalization rates, and less severe disease in patients with covid-19 caused by the omicron variant of SARS-Cov-2 in Houston, Texas. Am J Pathol 192(4):642–652 Cohn BA, Cirillo PM, Murphy CC, Krigbaum NY, Wallace AW (2021) SARS-Cov-2 vaccine protection and deaths among us veterans during 2021. Science 375(6578):331–336 Dorabawila V, Hoefer D, Bauer UE, Bassett MT, Lutterloh E, Rosenberg ES (2022) Effectiveness of the bnt162b2 vaccine among children 5–11 and 12–17 years in New York after the emergence of the Omicron variant. medRxiv Fang FF, Shi PY (2022) Omicron: a drug developer’s perspective. Emerging Microbes and Infections 11(1):208–211 Follmann D, Janes HE, Buhule OD, Zhou H, Girard B, Marks K, Kotloff K, Desjardins M, Corey L, Neuzil KM, Miller JM, El Sahly HM, Baden LR (2022) Anti-nucleocapsid antibodies following SARS-Cov-2 infection in the blinded phase of the mRNA-1273 Covid-19 vaccine efficacy clinical trial. medRxiv Gagne M, Moliva JI, Foulds KE, Andrew SF, Flynn BJ, Werner AP, Wagner DA, Teng IT, Lin BC, Moore C, Jean-Baptiste N, Carroll R, Foster SL, Patel M, Ellis M, Edara V-V, Maldonado NV, Minai M, McCormick L, Honeycutt CC, Nagata BM, Bock KW, Dulan CNM, Cordon J, Todd J-PM, McCarthy E, Pessaint L, Van Ry A, Narvaez B, Valentin D, Cook A, Dodson A, Steingrebe K, Flebbe DR, Nurmukhambetova ST, Godbole S, Henry AR, Laboune F, Roberts-Torres J, Lorang CG, Amin S, Trost J, Naisan M, Basappa M, Willis J, Wang L, Shi W, Doria-Rose NA, Olia AS, Liu C, Harris DR, Carfi A, Mascola JR, Kwong PD, Edwards DK, Andersen H, Lewis MG, Corbett KS, Nason MC, McDermott AB, Suthar MS, Moore IN, Roederer M, Sullivan NJ, Douek DC, Seder RA (2022) mRNA-1273 or mRNA-omicron boost in vaccinated macaques elicits comparable B cell expansion, neutralizing antibodies and protection against omicron. Cell 185(9):1556–1571 Hansen CH, Schelde AB, Moustsen-Helm IR, Emborg HD, Krause TG, Mølbak K, ValentinerBranth P, on behalf of the Infectious Disease Preparedness Group at Statens Serum Institut (2021) Vaccine effectiveness against SARS-CoV-2 infection with the Omicron or Delta variants following a two-dose or booster BNT162b2 or mRNA-1273 vaccination series: A danish cohort study. medRxiv Hay JA, Kennedy-Shaffer L, Kanjilal S, Lennon NJ, Gabriel SB, Lipsitch M, Mina MJ (2021) Estimating epidemiologic dynamics from cross-sectional viral load distributions. Science 373(6552):eabh0635 Hay JA, Kissler SM, Fauver JR, Mack C, Tai CG, Samant RM, Connelly S, Anderson DJ, Khullar G, MacKay M, Patel M, Kelly S, Manhertz A, Eiter I, Salgado D, Baker T, Howard B, Dudley JT, Mason CE, Ho DD, Grubaugh ND, Grad YH (2022) Viral dynamics and duration of pcr positivity of the SARS-CoV-2 Omicron variant. medRxiv

412

12 Omicron

20. Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, et al (2020) SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. cell 181(2):271–280 21. Kissler SM, Fauver JR, Mack C, Tai CG, Breban MI, Watkins AE, Samant RM, Anderson DJ, Metti J, Khullar G, et al (2021) Viral dynamics of SARS-CoV-2 variants in vaccinated and unvaccinated persons. N Engl J Med 385(26):2489–2491 22. Lewnard JA, Hong VX, Patel MM, Kahn R, Lipsitch M, Tartof SY (2022a) Clinical outcomes among patients infected with omicron (b.1.1.529) SARS-CoV-2 variant in southern california. medRxiv 23. Lewnard JA, Hong VX, Patel MM, Kahn R, Lipsitch M, Tartof SY (2022b) Clinical outcomes among patients infected with Omicron (b.1.1.529) SARS-CoV-2 variant in Southern California. medRxiv 24. Li B, Deng A, Li K, Hu Y, Li Z, Xiong Q, Liu Z, Guo Q, Zou L, Zhang H, Zhang M, Ouyang F, Su J, Su W, Xu J, Lin H, Sun J, Peng J, Jiang H, Zhou P, Hu T, Luo M, Zhang Y, Zheng H, Xiao J, Liu T, Che R, Zeng H, Zheng Z, Huang Y, Yu J, Yi L, Wu J, Chen J, Zhong H, Deng X, Kang M, Pybus OG, Hall M, Lythgoe KA, Li Y, Yuan J, He J, Lu J (2021) Viral infection and transmission in a large, well-traced outbreak caused by the SARS-CoV-2 delta variant. medRxiv 25. Lyngse FP, Mortensen LH, Denwood MJ, Christiansen LE, Møller CH, Skov RL, Spiess K, Fomsgaard A, Lassaunière MM, Rasmussen M, Stegger M, Nielsen C, Sieber RN, Cohen AS, Møller FT, Overvad M, Mølbak K, Krause TG, Kirkeby CT (2021) SARS-CoV-2 Omicron voc transmission in Danish households. medRxiv 26. Mahase E (2022) Covid-19: Second boosters may benefit at-risk groups but have “minimal” impact for others, says WHO. BMJ 377:o1259 27. Meng B, Abdullahi A, Ferreira I, et al (2022) Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts tropism and fusogenicity. Nature 603:706–714 28. Morens DM, Folkers GK, Fauci AS (2022) The Concept of Classical Herd Immunity May Not Apply to COVID-19. J Infect Dis 226(2):195–198 29. Mueller S (2019a) Are market GM plants an unrecognized platform for bioterrorism and biocrime? Front Bioeng Biotechnol 7:121 30. Mueller S (2019b) On DNA signatures, their dual-use potential for GMO counterfeiting, and a cyber-based security solution. Front Bioeng Biotechnol 7:189 31. Mueller S (2020a) Are paradoxes of amoeboid cognition, memristors, and memory mandating a re-conceptualization of actions and behaviors? EXPLORE 16(4):250–256 32. Mueller S (2020b) Facing the 2020 pandemic: What does cyberbiosecurity want us to know to safeguard the future? Biosafety and Health 3(01):11–21 33. Noble D, Hunter P (2020) How to link genomics to physiology through epigenomics. Epigenomics 12(4):285–287 34. Nordström P, Ballin M, Nordström A (2021) Effectiveness of Covid-19 vaccination against risk of symptomatic infection, hospitalization, and death up to 9 months: a Swedish total-population cohort study. Hospitalization, and Death Up to 9 35. Pérez-Then E, Lucas C, Monteiro VS, Miric M, Brache V, Cochon L, Vogels CBF, De la Cruz E, Jorge A, De los Santos M, Leon P, Breban MI, Billig K, Yildirim I, Pearson C, Downing R, Gagnon E, Muyombwe A, Razeq J, Campbell M, Ko A, Omer SB, Grubaugh ND, Vermund SH, Iwasaki A (2021) Immunogenicity of heterologous BNT162b2 booster in fully vaccinated individuals with coronavac against SARS-CoV-2 variants delta and Omicron: the dominican republic experience. medRxiv 36. Piplani S, Singh PK, Winkler DA, Petrovsky N (2021) In silico comparison of SARS-CoV-2 spike protein-ACE2 binding affinities across species and implications for virus origin. Sci Rep 11(1):1–13 37. Puhach O, Adea K, Hulo N, Sattonnet P, Genecand C, Iten A, Bausch FJ, Kaiser L, Vetter P, Eckerle I, Meyer B (2022) Infectious viral load in unvaccinated and vaccinated patients infected with SARS-CoV-2 WT, Delta and Omicron. medRxiv

References

413

38. Regev-Yochay G, Gonen T, Gilboa M, Mandelboim M, Indenbaum V, Amit S, Meltzer L, Asraf K, Cohen C, Fluss R, Biber A, Nemet I, Kliker L, Joseph G, Doolman R, Mendelson E, Freedman LS, Harats D, Kreiss Y, Lustig Y (2022a) Efficacy of a fourth dose of Covid-19 mRNA vaccine against omicron. N Engl J Med 386(14):1377–1380 39. Regev-Yochay G, Gonen T, Gilboa M, Mandelboim M, Indenbaum V, Amit S, Meltzer L, Asraf K, Cohen C, Fluss R, Biber A, Nemet I, Kliker L, Joseph G, Doolman R, Mendelson E, Freedman LS, Harats D, Kreiss Y, Lustig Y (2022b) 4th dose COVID mRNA vaccines’ immunogenicity and efficacy against Omicron VOC. medRxiv 40. Riemersma KK, Grogan BE, Kita-Yarbro A, Halfmann PJ, Segaloff HE, Kocharian A, Florek KR, Westergaard R, Bateman A, Jeppson GE, Kawaoka Y, O’Connor DH, Friedrich TC, Grande KM (2021) Shedding of infectious SARS-CoV-2 despite vaccination. PLoS Pathog 18(9):e1010876 41. Röltgen K, Nielsen SC, Silva O, Younes SF, Zaslavsky M, Costales C, Yang F, Wirz OF, Solis D, Hoh RA, et al (2022) Immune imprinting, breadth of variant recognition and germinal center response in human SARS-CoV-2 infection and vaccination. Cell 185(6):1025–1040 42. Saigusa T, Tero A, Nakagaki T, Kuramoto Y (2008) Amoebae anticipate periodic events. Phys Rev Lett 100(1):018101 43. Servellita V, Morris MK, Sotomayor-Gonzalez A, Gliwa AS, Torres E, Brazer N, Zhou A, Hernandez KT, Sankaran M, Wang B, et al (2022) Predominance of antibody-resistant SARSCoV-2 variants in vaccine breakthrough cases from the San Francisco Bay Area, California. Nat Microbiol 7(2):277–288 44. Shitrit P, Zuckerman NS, Mor O, Gottesman BS, Chowers M (2021) Nosocomial outbreak caused by the SARS-CoV-2 Delta variant in a highly vaccinated population, Israel, July 2021. Eurosurveillance 26(39):2100822 45. Wilhelm A, Widera M, Grikscheit K, Toptan T, Schenk B, Pallas C, Metzler M, Kohmer N, Hoehl S, Helfritz FA, et al (2021) Reduced neutralization of SARS-CoV-2 Omicron variant by vaccine sera and monoclonal antibodies. MedRxiv 46. Zhao H, Lu L, Peng Z, Chen LL, Meng X, Zhang C, Ip JD, Chan WM, Chu AWH, Chan KH, et al (2021) SARS-CoV-2 Omicron variant shows less efficient replication and fusion activity when compared with Delta variant in TMPRSS2-expressed cells: Omicron variant replication kinetics. Emerging Microbes and Infections 11(1):277–283

Chapter 13

Conclusion

In face of the ongoing pandemic, heroic efforts have been made to develop Covid19 vaccines, including those that utilize new technology with no previous clinical experience. Given the urgency of the situation, it was simply not possible to conduct long-term and comprehensive clinical studies. Everyone knows that. Despite the unprecedented seriousness of the situation, this book has gathered significant evidence that suggests that the emergency approval of the first Covid-19 vaccines was premature.

Modeling and Predictions Vs. Clinical and Human Reality Although numerous areas of concern were identified, the list of safety risks of mRNA vaccines considered in this book is not intended to be comprehensive. Arguably, the most significant problem identified concerns the gap between the theorized modus operandi of those vaccines and their possible, or even observed, mode of action. Despite being claimed otherwise, there is no guarantee that the manufactured entities will be as desired, that the generated molecules will assume the role of mRNAs as intended, only be taken up by professional antigen-presenting cells, trigger the anticipated immune response, and no more. Even if those vaccines engender the desired generation of the spike protein, this may have the opposite effect than intended and result in the same serious pathologies as seen during SARS-CoV-2 infection or lead to new pathologies altogether. Indeed, the spike alone is a critical driver of serious Covid-19 disease and arguably the main culprit of fatalities. Analogous pathogenic processes can be triggered by injection, despite several modifications done to the vaccine mRNAs which may, in fact, make the situation even worse. The injected material reaches the circulation and disseminates throughout the body—a situation is believed to be excluded by its very postulated modus operandi, which during the trials was © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Mueller, Challenges and Opportunities of mRNA Vaccines Against SARS-CoV-2, https://doi.org/10.1007/978-3-031-18903-6_13

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only assessed in a limited nonclinical context. Prior to the mass vaccinations, there were no detailed experiments and tests that determined the biodistribution and biodynamics of the synthesized molecules in human cells and in varied clinical contexts, such as those fostered by drug interactions or in specific pathophysiologic situations. It has been only after millions of people had been injected that postvaccine experience has begun to reveal a host of previously overlooked, unassessed, and deleterious clinical implications these may entail, including harmful immune stimulatory processes, Th-2 immunopathology, heart disease, autoimmunity, cancer, neurologic disease, death, and many other chronic debilitating conditions. The spike, other proteins expressed from unintended vaccine mRNAs (e.g., truncated species), or the RNA molecules themselves, can interfere with endogenous microRNA regulation or act as hazardous RNA activity modulators the same way as it is known for the SARS-CoV-2 virus itself, via a recently discovered mechanism [2], which tends to cause severe disease or even death. Importantly, the significant peptide commonalities between the spike and human proteins in itself are expected to provoke various adverse cross-reactive responses, possibly leading to excessive immune responses or autoimmune development. Also emerged have numerous insights confirming that mRNA “vaccines” rather act as drugs than vaccines. Part II of this book, in particular, focuses on pharmaceutical aspects of these interventions, based on numerous new pieces of evidence that bring a deeper understanding of waning immunity and the immune response triggered beyond antibodies, and what this means for beneficial but also adverse effects. Based on what has been considered in this book, there is strong evidence that the notion mRNA “vaccine” is unjustified and, in fact, highly misleading. Following an extensive literature review and independent analysis, it must be concluded they are gene-therapy (GT) products instead. In fact, as carefully examined in this book, the basic arguments leading to the widely held belief as to why they are not GTs (Fig. 1.1) are counterfactual, with a complete synopsis of the unsubstantiated arguments given in Fig. 4.5. During the time of writing this book, it surprisingly became known that the manufacturers are indeed aware of this. This, however, is not widely known. Ref. [17] states that “Referring to the ‘mRNA technology’ in its Vaccine, Moderna admits the ‘novel and unprecedented nature of this new class of medicines’ in its Securities and Exchange Commission filings18. Further, it admits that the FDA classes its Vaccine as a form of ‘gene therapy.”’ I believe producers were overly optimistic about what this new class of medicines can do. It is obvious that the term “gene therapy” would instill fear and rejection by the consumers of these products and would also mandate a different level of evaluation and decision making. Nonetheless, it is also clear that developers of mRNA technologies have long believed in the benefit of this approach. It is worthwhile to look into the history of mRNA therapies (now called “vaccines”). Doing so reveals that these are rooted in a comprehension of biology that is

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meanwhile largely found to be rather incomplete (what Giuseppe Longo1 has termed the geocentric view). The origin of mRNA “vaccines” dates back several decades. Following the early work by Malone et al. [12, 24], mRNA has been extensively investigated for the transient modulation of immune cells. For example, IVT mRNA has been studied to encode tumor–antigen-specific T cell receptors. Transfected cells carrying such mRNA-encoded receptors have been investigated for their potential to recognize and kill tumor cells that express the target antigen [19]. While mRNA vaccines utilize the same underlying mechanism of “transfection,” this term is hardly ever used in this context, although this is exactly what is happening with mRNA “vaccines” as well. Transfection is the process of introducing nucleic acid into eukaryotic cells using various chemical or physical methods.2 According to the US National Library of Medicine Medical, “it is analogous to bacterial transformation” as it is “routinely employed in GENE TRANSFER TECHNIQUES.3 ” From this perspective alone, it is inconceivable then, why mRNA “vaccines” are not classified as gene therapy, the same way as this notion is used in the context of IVT mRNAs for cancer treatment. Currently, it is believed that the transient nature of mRNA reduces the risk of unwanted side effects; in this sense, mRNA “vaccines” are thought to transfect human cells merely in a “transient” way [23]. However, it was shown above that this assertion regarding the transient nature is not true in general. In January 2020, the FDA developed a detailed guidance on how to conduct trials involving human GT products. They also gave a clear description of potential risks of delayed adverse events following exposure to human GT products. As detailed, most of these criteria and characteristics utilized by the FDA actually do apply to RNA vaccines as well (Table 4.1). This is in sharp contrast to the decision made by the FDA: they explicitly state that their “guidance does not apply to vaccines for infectious disease indications” [21]. The reason why the FDA believed that RNA vaccines do not fall into the category of GT products is based on their view that there is “no” propensity of RNA vaccines to modify genomes. The criteria given by the FDA to detail this propensity are “[b]ased on product design (i.e., lack of any known mechanism to facilitate integration or genome editing), as well as cumulative preclinical and clinical evidence suggesting that a GT product does not integrate into or edit the genome or integrates into/modifies the genome at very low frequencies” [21] (emphasis mine).

1

https://ensser.org/from-our-members/book-review-programming-evolution-a-crack-in-science/. https://www.sciencedirect.com/topics/medicine-and-dentistry/genetic-transfection. 3 https://meshb.nlm.nih.gov/record/ui?name=Transfection. 2

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These “facts” as relied upon by the FDA and the many stakeholders globally that utilize RNA “vaccines” have been shown to be unjustified based on the following arguments presented: • Integration of vaccine RNAs or their byproducts: The key assertion that IVT RNAs are confined to the cytoplasm was shown to be false. Even in human cells, the existence of reverse-transcriptase activities via different mechanisms has clearly been demonstrated. • Genome editing: There is no guarantee that unexpected byproducts cannot enter RNAi pathways in human cells. In fact, different methods for purification of IVT contaminants utilize conflicting approaches and cannot eliminate all harmful dsRNA byproducts and other contaminants; furthermore, the generation of dsRNAs in vivo cannot be ruled out and is even a mandatory step of some mRNA vaccine platforms. In addition to evoking inflammatory responses, it was shown above that these byproducts may even become substrates of RNAi processes. Therefore, as siRNA and miRNA pathways also overlap in human cells, mRNA vaccines may have significant gene-editing propensities. Furthermore, as they could themselves act as competitive RNAs, they may disrupt endogenous microRNA regulation in other austere ways as well. • Other modifications of the genome: additional modifications were shown to be plausible, such as via interactions of vaccine-derived products with cytoplasmic genetic material including that of the human microbiome, and chemical modification of DNA and chromatin. Preclinical and clinical evidence has been accumulating, of both RNA “vaccines” and various RNA viruses, which seriously undermine the views relied upon by all decision makers: • Viral integration into human chromosomes is a fact for numerous RNA viruses, not only those for whom this step is obligatory (retroviruses). • The integration of RNAs from SARS-CoV-2 into the human genome has been experimentally verified in cell culture. • A lack of LTFOs: clinical experience has been seriously hampered by the following circular argument. For mRNA vaccines, all R&D developments, safety trials, and ongoing clinical evaluations are based on the understanding they are not GT products. Ever since this viewpoint was first established years ago already, there has been “no rationale” to test for unique off-target effects (as for other gene therapies), or to carry out long-term observation of patients in clinical trials [19]. As LTFOs have not been deemed necessary, none have been conducted, and adverse events that may be associated with the action of a GT are by definition excluded. As a sobering consequence of the limiting conceptual framework that RNA vaccines are like traditional vaccines, a vast majority of (delayed) severe adverse events and risk possibilities are not included in safety analyses (Fig. 4.1). Yet, as with any other GT, the longer it persists, “the greater the duration and degree of risk of delayed adverse events” [21].

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Arguably, delayed adverse events are much more difficult to link to vaccination. This is certainly the case when possible relationships are excluded by the way they are classified. In this regard, short-term safety analyses under the assumption these jabs work like traditional vaccines amount to looking for the key under the light post. Initially, both the BNT162b2 and the mRNA-1273 SARS-CoV-2 vaccine boasted a 90+ % efficacy in preventing infection. However, this number is broadly misinterpreted as it represents one statistical measure of risk reduction alone that does not apply to the general public. By contrast, using the absolute risk reduction measure— which before the pandemic was required by the FDA as well—those numbers radically change to 1% or even less. As with other statistical applications, additional statistical metrics (such as ARR, NNT, and NNH) are needed to assess the risks and benefits to the overall population. Shockingly, these estimates have rarely ever been done for Covid vaccines—and those rare papers that have been highlighting the devastating results in this regard have often been censored or retracted. Who would have thought at the beginning of the pandemic, that by the summer of 2021 people would be forced to get Covid-vaccinated? But how do you truly measure the promise, success, or failure of a vaccine? Families have become divided over this issue, as have close friends, businesses, companies, and, in fact, the entire globe. Proponents argue that the vaccine has saved thousands of lives. People on the other side counter that the vaccine has cost multiple times more lives than all the other vaccines combined. Others posit that the unvaccinated are responsible for causing the ongoing spread of the virus, and in some places, a “war” has been declared on the unvaccinated. I wonder how scientists and doctors will judge these policies, some 5, 10, or 20 years from now, when they will better understand the many unknown aspects of these new medicines and gene therapy in general. Just these last few years since their rollout have revealed shocking results about their disappointing intended and unintended effects alike. And whatever happened to the notion of individualized medicine based on proven modalities? During the time of writing the book, it has become next impossible to keep track of the numbers of post-vaccine adverse events. By September 2021, the USA alone had hundreds of thousands of reported injuries and 15 thousand deaths following vaccination, while it is possible that the real number may be ten times as high, as even said under sworn testimony [17]. By June 10, 2022, these numbers reached dimensions that were even less comprehensible: data released by the CDC showed 1,301,356 reports of adverse events from all age groups following COVID-19 vaccines, including 28,859 deaths and 238,412 serious injuries. Tragically, lives have been lost to both the disease and the vaccines. But what is the balance? Clearly, the largest burden of Covid-19 is carried by the elderly. However, 94% of the global population is younger than 70 years. That Covid-19 is much less deadly than previously thought, at least in the non-elderly population, was shown by a major retrospective study [15] that assessed the infection fatality rate (IFN) until the end of 2020. Seroprevalence data revealed that at the global level, prevaccination IFR may have been as low as 0.03% and 0.07% for 0–59 and 0–69 year

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old people, respectively. Ioannidis et al. conclude that “it is reassuring that even in the wild strains that dominated the first year of the pandemic, the IFR in non-elderly individuals was much lower than previously thought.” Can we now say that because of Covid-19 vaccines lives have been saved? Even if we were to measure this in raw numbers only, how big is that success? Paradoxically, many nations are not reporting any increased all-cause mortality numbers at all since the beginning of the pandemic. And ironically, for others, fatalities significantly increased only after rollout of the injections. And even more paradoxically, even these foundational and final estimates about fatalities are off, and often by a very large factor (Sect. 5.3.2). From this perspective, it is even less understandable how publications that have raised questions about the vaccines have often quickly been debunked. For example, a publication in Toxicology Reports [10] which had raised some controversies, argued that based on a very conservative analysis and involving only the most susceptible individuals, the deaths attributable to each Covid inoculation are at least five times that of the disease it aims to prevent. The paper ended up being retracted by the editorial office, as it seemingly demonstrated inappropriate bias, and the findings were deemed unreliable. The question arises, how easy is it to fully represent the total “truth” related to new medical interventions, especially when public health agencies choose to withhold raw data and publish the results of some modeling instead? Doesn’t the success of science and medicine rely on independent scrutiny and ongoing process? Isn’t the essence of science that of inquiry, questioning, debate, and learning, as has been demonstrated time and again by generations of researchers? The more we have been learning about mRNA vaccines, the more it is clear how little we still do not know about the underlying mechanisms, nor how to track, collect, and identify the enormous amount of data, including those that do not fit the expectations of those products. The first 1.5 years of the mass-vaccination campaign have revealed major gaps in how adverse events post-injections are assessed and reported, as well as related to other basic causality questions, such as the degree to which these products prevent infection and transmission. Chapter 8 describes this struggle, revealing why the very WHO-AEFI manual is not able to determine a causal association. This means that when examining adverse events, this global policy will almost certainly determine a non-causal or indeterminate association. Albeit, such a negative decision is a technical artifact, caused by the re-definition of what a vaccine is, numerous issues, flaws, and problems with various efficacy estimates and reporting, the discrepancy between models and clinical experience, and a momentous knowledge gap about these interventions altogether. By what rules does mRNA vaccine-imposed immunity play, and what is the true risk–benefit analysis of those vaccines? Is it really the life of one person— potentially lost to Covid—versus the life of another potentially lost to the vaccine? What about severe and chronic and debilitating health issues? How can we measure the value of friendship, love, trust, openness, ease and playfulness, curiosity and creativity, independent thinking and free speech, and a flourishing economy?

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Of people being able to be human beings, living and being treated in dignity, appreciating the gift of their own life, as well as that of their loved ones, children, and all future generations? How about the value of community and society, of people trusting their governments and cheering their leaders? On the one side, the pandemic has cost more lives via indirect consequences, such as businesses going under, people losing their jobs, and young children growing up in fear, isolation, and trauma. And then, on the other side, lives are lost to the vaccine too, and in terms of numbers only, who knows how these two fatality values relate! Is it really lives saved by the vaccine? Do the benefits still outweigh the risks as we hear constantly in the news? Despite the entire world being polarized and in fear, heroic critics have dared to speak up, arguing that the numbers do not work out. For instance, grounded on the publication [5], Oregon State Senators filed a petition for a Grand Jury investigation into the alleged violations of Federal Law and subsequent acts of Willful Misconduct by the CDC and the FDA. The allegations involve their policy actions taken to hyperinflated Covid cases, hospitalization and fatality data, their refusal to collaborate with independent subject matter experts “to address the severe flaws surrounding PCR testing,” and others [20]. Likewise, several whistleblowers have given testimonies under penalty of perjury [6, 9, 11], and countless brave organizations and individuals have been sharing the painful experiences of vaccine injuries. Soberingly, the last 1.5 years have often required legal interventions for critical information to be released. For example, in June 2022, the CDC admitted it had never even assessed the Vaccine Adverse Event Reporting System (VAERS) for safety signals for the COVID-19 vaccines. The admission was revealed in response to a Freedom of Information Act (FOIA) request submitted by Children’s Health Defense (CHD). As pointed out by Dr Mobeen Syed,4 while some may not like CHD, what matters is the issue admitted by the CDC.5 Likewise, an unprecedented huge number of documents obtained by Pfizer during its trials and post-authorization analysis are also only being made available to the public because of some court decision. Yet, these documents contain vitally important information about adverse reactions experienced by trial participants, details regarding animal studies, and information about changes made to the study protocol. Clearly, dead numbers alone are a very poor measure. A number is neither able to adequately assess the present, nor can it predict future trends. What has been missing in all the risk–benefit estimates is the only “thing” that actually matters in this context—human involvement. It is because of human involvement that the virus further evolves—how else could it, if it did not have a host? It is also with humans, wherein the disease lies, where it could get treated—or where disease prevention

4

https://www.youtube.com/watch?v=cBT87_egYa8. https://childrenshealthdefense.org/defender/cdc-vaers-covid-vaccine-safety/?utm_source= substack&utm_medium=email.

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rests on the vaccine and so many other insufficiently appreciated factors such as nutrition status and availability of supplements and well-established medications. Perhaps, after all, the pandemic has shown us that each person truly matters. We still do not know how the virus originated. It could have been just a few people initially infected in the form of a spill-over event from some still unknown natural source. It could also have been one or a few lab workers who got infected during one of the numerous lab experiments, which in recent years raised alarm regarding their biosafety and biosecurity concerns fostered by automation, cyber-overlaps, and the digitization of biology [14]. While everyone is trying to find out which of these it had been, the most important factor is overlooked: it is about humans. Had we known the index patient, or the group first infected, would we not have acted differently? Now, the question about the vaccine takes this even further. The virus, once it has infected some individuals, can indeed evolve further. This problem is highly amplified by Covid vaccines—which, according to the new definition of vaccines, do not even need to prevent infection. That such vaccines can drive the evolution of more transmissible or virulent pathogens is a well-established fact [13, 16]. This has clearly been shown for leaky vaccines before, which are vaccines that “let the hosts survive but do not prevent the spread of the pathogen” [16]. Previously, the danger of such vaccines was clearly recognized, and they were never used at scale. Alarmingly, soon after the beginning of the massvaccination campaign, studies first began to demonstrate a clear association between breakthrough cases and increased virulence of SARS-CoV-2. Meanwhile, further evidence related to these associations has been mounting, raising the prospect of viral escape and the concern that the common global approach could result in the analogous development as seen with antimicrobial resistance. Now, knowing that SARS-CoV-2 could evolve into more dangerous variants, don’t we want to learn how exactly it can do so? It, again, requires human beings. We are at the center—not risk–benefit estimates absent of human involvement. The most critical question we need to face is: where are we going as humanity? Will we provide an environment for the virus to further evolve?

With mRNA Vaccines, Risks and Benefits Do Scale Differently The incomplete and even flawed interpretations of data surrounding mRNA vaccine development and deployment have discouraged legislators and regulators around the world from contesting the embedded uncertainty in scientific discourse by ignoring one of the most significant issues: vaccines in their particular patient and societal contexts differ in their potential to channel and amplify harm. Chapter 7 presents two ideas, inspired by the work of Heinemann and collaborators [8], that are equally valuable in the context of mRNA vaccines to move beyond the present limited arguments and risk–benefit assessments. The first is that mRNA

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vaccines are distinctively scalable. And second, their feature of scalability may be used to inform how we may best control policy for the most effective, precautionary outcomes. In contrast to the many incomplete, controversial, and contestable risk–benefit estimates that have been, and still are being conducted to assess how Covid-19 vaccines fare when applied at large, placing human activity at the center of such an analysis provides a more consistent, verifiable, and practical approach; it succinctly identifies where the scale of effects of a human activity diverges between risk and safety. These so-called critical control points were first introduced in [8] to clearly lay out where technical experts can collaborate with public officials with different expertise to manage the regulations specific to genetically engineered/modified organisms. Implicitly, scale has been at the foundation of vaccine risk–benefit estimates before. However, it has not explicitly been understood in this way, and misunderstandings of pseudoscales have, as in the case of legislation of agricultural gene technologies, led to “muddy thinking in the semantic (re)interpretation of legislation” [8]. Pseudoscales are normative judgments that have features that resemble relevant scales “but have no or only a limited perceptual basis” [8]. Just as in the context of agricultural gene technologies, pseudoscales are inappropriate surrogates for measuring vaccine safety and effectiveness as well. Therefore, mRNA vaccines can scale risks one way, while vaccine mandates go another way. There is no experience with the manipulation of the scales of time, side effects, or viral evolution in the context of mRNA vaccines. I agree with Heinemann et al. who described analogous properties for gene-silencing technologies and posit that with mRNA gene technologies too, this lack of experience is the main feature that demands strict regulation and oversight—in the context of mRNA vaccines, in the forms of lab and animal experiments and, based on informed consent, strictly voluntary use, rather than a global deployment of one-size-fits-all medicine. Chapter 7 shows that it is because of a lack of involvement of human activity in the risk–benefit estimates of vaccines, that these cannot predict true scaling features, nor address the most urgent questions of how to make correct decisions. As shown above, all of the most celebrated metrics used by decision makers are in fact pseudoscales. These include: • PCR positiveness to diagnose Covid • Pseudoscales of “naturalness” as a quantity, as in how much natural the immune response triggered by mRNA vaccine is, or how much better it is • Neutralizing antibody titer, as a predictor of immunity • Predictiveness of amino acid sequence differences and determinants While these seem like measurable characteristics, this book demonstrated major limits when applied in the biological realm. The expected genetic action of the vaccine RNAs is not the only source of risk—the actual clinical context adds an entirely different world with a much greater number of interrelationships and unknowns.

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Scale is not merely a notion of length, weight or time. Importantly, with safety– risk estimates, scale can also include factors that are not readily appreciated, including the individuality of vaccinees, host–viral interactions, or population dynamics. It can even include those that previously did not even exist in this context, but which may turn out to be most critical, such as viral escape instilled by antibodydriven selection pressure from the vaccines. In fact, Chap. 7 shows that mRNA vaccines have clearly defined inherent scaling characteristics, as dictated by the outcomes of their increased use (i.e., with scale), including: • • • • • • • •

Contamination and quality control problems Vaccine-induced antibody diversity and flexibility Artificially elevated antibody levels in relation to overall clinical outcome Biodistribution, persistence, and accumulation of the (expected and unexpected) products of the injections A lack of capacity of the injections to neutralize the virus Antibody-driven pressure selection and the development of viral VOCs Reverse transcription of vaccine mRNAs and integration into the human genome, including expression of viral or neoantigens The very essence of gene therapies themselves, as well as booster shots, leading to (delayed) adverse effects

Following the advice of [8], this work has identified how the potential to cause harm or improve benefits can vary in how mRNA vaccines are used. Importantly, risks and benefits scale differentially. Overall, they do not scale together, meaning that the potential for adverse events, but not that of benefits, increases with use (e.g., mass vaccinations, boosters). For these reasons, this framework suggests a critical control point structure for the governance of these technologies (Fig. 13.1). Critical control points are at the transitions of use where adverse effects are amplified. These identify where regulation and policy can optimally target effective outcomes (Fig. 13.1), with examples of such simple measures given in Fig. 13.2. Despite their apparent simplicity, the risk scaling method [8] assures that such interventions will effectively interrupt the escalation of risk/hazardous outcomes.

The Necessity of Mass Vaccination, Revisited When the virus first emerged, initial shelter-in-place and lockdown orders were put in place based on announcements of a “new, contagious virus with 3.4% fatality rate and no asymptomatic infections.6” Thankfully, the years 2020 and 2022 (to date) have proved that the frightening estimate did not pan out. The disease is hardest on the elderly, with an estimated case-fatality rate of 2.5 times the rate provided by the

6

https://www.hsgac.senate.gov/imo/media/doc/Testimony-Ioannidis-2020-05-06.pdf.

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Fig. 13.1 The critical control point framework applied to mRNA vaccines. Critical control points identify where risk and safety scale differently. In the figure, each arrow is a critical control point. Such points interrupt the connection between the use of mRNA vaccines and the generation of harm and therefore show where regulation and policy can target the most effective, precautionary outcomes. The first line (representing the first control point) is not explicitly included in the figure as mRNA vaccines have already been deployed. Examples of beneficial intervention at the other critical control points (black circles), i.e., where the process of risk escalation can be halted, are depicted in Fig. 13.2

CDC for seasonal influenza [3]. For the vast majority of the population, infection with SARS-CoV-2 only causes either mild or even asymptomatic disease. For example, on April 17, 2021, Worldometer data revealed that of the active cases, 99.4% were in mild condition, and only 0.6% in serious or critical. Now, with the Omicron variants, the disease is even more mild, overall. For instance, by May 2022, the ZOE study identified the top ten symptoms as runny nose, fatigue, sore throat, sneezing, headache, persistent cough, hoarseness, chills or shivers, joint pains, and dizziness. The marked decrease in disease severity compared to Delta has been corroborated by numerous studies and practical experience with this variant

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Fig. 13.2 A way forward. Placing human activity at the center is an alternative to the less fruitful risk–benefit evaluations that are rooted in biochemical, genetic, or other scientific notions and often resemble inappropriate “pseudoscales” (Chap. 7). Doing so also clearly identifies critical control points where risks and benefits scale differently with the use of Covid-19 vaccines (the central bullet points). These control points (here, depicted as blue circles) provide opportunities for effective intervention: they oppose and halt the scaling of risk with increased use (center red)

(Chap. 12). Further, on May 9, 2022, Bill Gates suggested7 that Covid is “a disease mainly of the elderly, kind of like the flu.” The observation that the virus is causing no symptoms for the vast majority of people begs the question of why response measures are needed on a large scale. John Campbell, discussing the stringent Zero-Covid policy in China,8 suggested there are indeed some lessons we can learn from China. In their case, where during the spring of 2022 over 90% of the cases were asymptomatic, many wonder why they are worried about the virus, to the extent even of locking down millions? More generally, what about the mass-vaccination campaigns? Are these justified as well, from the perspective of disease severity alone?

7

https://www.dailywire.com/news/bill-gates-covid-disease-of-elderly-low-fatality-rate-kind-oflike-the-flu. 8 https://www.youtube.com/watch?v=TryFMOekX8w.

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Sadly, by the spring of 2022, it has become clear that those most susceptible to the virus, the elderly and the comorbid, are not sufficiently protected by the vaccines either. On April 29, 2022, The Washington Post9 wrote: “The pandemic’s toll is no longer falling almost exclusively on those who chose not to or could not get shots,” going on to explain that Covid fatalities are again concentrated among the elderly. At that point, “nearly two-thirds of the people who died during the Omicron surge were 75 and older, according to a Post analysis, compared with a third during the Delta wave. Seniors are overwhelmingly immunized, but vaccines are less effective and their potency wanes over time in older age groups.” The incomplete immune protection from the vaccines is now undisputed and corroborated by numerous studies (Chaps. 8, 11, and 12). Surprisingly, at the end of May 2022, the CEO of Pfizer, responding to concerns about the politicization of the pandemic, diminishing immunity from vaccines and natural infections, and others, predicted that “antiviral drugs would replace vaccines as the key weapon in fighting the coronavirus, at least until shots providing a longer period of immunity are developed.10” More generally, it is now also clear that vaccines fail to confer good protection to the general population, neither can they prevent long-COVID and organ damage after breakthrough infection [1]. At the same time, while unable to prevent infection, evidence of serious adverse events following the injections is mounting, as can be seen from numerous studies and reports. Independent estimates (Chap. 8) now suggest serious adverse events in 1 out of every 125 vaccinated, i.e., a sobering eight per 1000 serious-event-rate overall. Paradoxically, the high incidence rate of adverse events had already been known to the manufacturers and regulators, not been disclosed to the public, and obviously not been regarded as notable. In fact, a document released April 1, 2022 by the FDA and made public as part of a court-ordered disclosure schedule stemming from a FOIA request,11 reveals: the rate of reported adverse events per dose during Pfizer’s initial post-marketing analysis was over 1:1000, with many of them graded as serious. And the more we learn about the underlying mechanisms of the genetic inoculations, without being able to account for long-term sequelae, it is clear that the incidence of adverse events increases with each shot. As fourth boosters are being rolled out, fifth are being discussed, and as postvaccine experience has begun to unravel previously underappreciated insights in terms of biochemistry, genetics, epidemiologic estimates, and others, it is imperative

9

https://www.washingtonpost.com/health/2022/04/29/covid-deaths-unvaccinated-boosters/.

10 https://www.businessinsider.com/pfizer-ceo-constant-waves-covid-19-coronavirus-

complacency-albert-bourla-2022-5. 11 The document had been previously released in November 2021 but had been partially redacted. The unredacted April version also revealed that Pfizer-BioNTech had shipped 126,212,580 doses of its vaccine worldwide between December 2020 and February 2021. This new piece of information now exposes the unprecedented incidence of adverse events reported previously (Chap. 8); the court-released document is available at https://phmpt.org/wp-content/uploads/2022/04/reissue5.3. 6-postmarketing-experience.pdf.

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to look for true scaling properties to avoid being bogged down by pseudoscale risk– benefit estimates, and instead discern where risk truly scales with use. We are at a place in humanity where fear and competitiveness to get more (in this case, Covid vaccines) may finally prove to result in the opposite, driving the virus to evolve— and possibly escape all treatment options altogether. In the context of regulation of agricultural gene technologies and their risk– safety evaluations, Heinemann and collaborators conclude: “We have presented evidence that regulators, science advisors, research societies, and governments have been to varying degrees nudged further toward a form of risk discourse that is unreflective on questions of why to use gene technology to one that is protective of it, legitimizing adoption because it satisfies safety criteria disproportionately influenced by those who develop the technology” [8]. I contest that the same is true for the gene technology applied to humans (although the true nature is hidden under the concept of mRNA “vaccines”). Even though that these technologies have been rolled out, and at an unprecedented scale, the risk–benefit discussions have been bogged down by the same shortcomings as just described for their agricultural gene-technology counterparts that we are much more experienced with. Any deep reflections as to why these technologies should be employed to begin with have been relinquished by pseudoscale-based arguments enforcing their large-scale rollout.

Lessons (to Be) Learned from Omicron Overall, while Part I of the book was to a large degree shaped by rational thinking, logical reasoning, predictions, comparisons, and conclusions based on early evidence of mRNA vaccines, later parts were written from the perspective of the increased post-injection experience. During the first 1.5 years since their rollout, numerous insights have emerged. These have not only drastically overturned the very first underlying assumptions of mRNA vaccines but also provide insights into previously unrecognized mechanisms and relationships. Already toward the end of the Delta time, the sobering reality was that neither of the early hopes and promises in the mRNA Covid injections played out in real life. These included: • Those who are vaccinated will not get infected and will not be able to pass on the virus to others. • mRNA Covid-19 vaccines are safe and highly effective, with VE values in the 90+ range. • Protection from vaccination is much better than natural immunity. • The virus can only get transmitted by unvaccinated people. • It is mostly unvaccinated people who get infected, who end up in hospital, and who end up dying from Covid.

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• Herd immunity can only be achieved if sufficiently many in the population are fully, i.e., doubly, vaccinated. When Omicron emerged, it accentuated the trend seen before, offering some additional surprises too. At the beginning of the Omicron era, it was still believed that: • Even when in rare cases the 2-dose (3-dose) mRNA regime can result in breakthrough infections, 3 (4) doses will provide lasting protection from infection. • Protection from mRNA vaccines against infection lasts at least 6 months. • mRNA boosters protect against severe disease and death. • mRNA boosters furnish high (neutralizing) antibody levels—which are a proxy of immune protection afforded by the injections. • Frequent boosters will enable us to get ahead of the virus. • Herd immunity can only be achieved if sufficiently many people get boosted. • Ignoring the theoretical potential of animals as a potential reservoir, Omicron can only have arisen in unvaccinated or immune-compromised individuals. That the above beliefs, too, turn out to be incorrect can be seen from the following observations that have become irrefutable during the various Omicron waves: • Vaccine immunity is short-lived; some individuals do not benefit from their anticipated protection and may instead experience a number of adverse events whose clinical and biological mechanisms are increasingly being elucidated. • A high antibody titer is no measure of protection. In fact, repeat boosters evoke antibody levels at least as high as first seen during the basic vaccine regime, but they still offer no durable protection. • Repeat boosters may lead to tolerance development or T cell exhaustion. In addition to the potentially deleterious effects on the vaccinee, this gives an additional advantage to the virus. • A targeted antibody response is a strong scale driver to foster viral escape, as suggested by a distinctive dose–response relationship: 3-dose inoculated people have been linked to a higher secondary attack rate (SAR) than the 2-dose inoculated; likewise, the SAR of 2-dose vaccinated people is greater than that of unvaccinated individuals. • The number of injections is a potent scale driver on both sides of the virus– host relationship. As the virus is repeatedly pressured with the same antigens, escape mutants from the leaky vaccines further benefit from a potential weakened/downregulated immune response of the host, driven by the number of injections received. • Vaccinated people effectively pass on the virus to others. Even those 4x inoculated, when infected, have viral loads high enough to be infective. • The virus, when infecting vaccinated people, is able to mutate into a different VOC. In particular, vaccinated people infected with the Delta VOC have triggered Omicron VOC infections among household members. • Omicron has learned a new way to infect cells. This mechanism was unpredictable via previous models.

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Some of these assumptions are overlapping, but all point to foundational issues with the underlying model, and the anticipated modus operandi of the shots. Basic Underpinnings are Still Largely Unknown Some have said that the global vaccinations are a mass experiment on all of humanity. Indeed, the more we learn about mRNA injections, the more we know they are not the type of vaccine as promised. Somewhat surprisingly, on May 20, 2022, Fauci admitted12 that “he’s not sure why the immune response triggered by mRNA vaccines may not be longer lasting. He has some theories, though.” Incomplete Immune Surrogates It is now known that the most celebrated player of vaccine immunity, the antibody response, turned out to be insufficient and incomplete (Fig. 13.3). More specifically, the main proxies of immune protection relied upon during the pandemic have proven to no longer be valid with Omicron (Chap. 11). The sobering realization that even the neutralization antibody titer is rather useless when it comes to Omicron is more than just a testimony to waning immunity and viral neutralization escape. It also indicates that the very believed model of trained immune induction apparently does not work out in clinical practice. mRNA Vaccines Offer Poor Immune Protection at Both the B Cell, T Cell, Antibody Binding, and Neutralizing Antibody Level Now that the previous correlates of immune protection are no longer substantiated, the focus has started to shift. The immune response triggered by mRNA has recently been described as follows:13 “The mRNA vaccines, both Moderna and Pfizer, generated these four categories: antibodies, memory B cells, helper T cells, and killer T cells. Overall, the mRNA vaccines generated the best of all four of those.” One may ask how we can know for sure this to be the case, and that said immunity is indeed protective? Concerns and limitations in this regard include: • Several mechanisms are now known (Chaps. 11 and 10) that provide the basis for how these injections dysregulate, impair, or reprogram, both adaptive and innate immunity altogether. • That the injections lead to some (“transient”) immune suppression has been observed in numerous studies, including those by the manufacturers themselves. On the other hand, that repeated exposure to the same antigen (or one exposure with a very high dose) can lead to immune-non-responsiveness is a wellestablished fact in immunology in general. This concern has been articulated in the context of ongoing boosters but has not been sufficiently appreciated (Chap. 11). Particular worrying in this regard are the COVID shots for kids as young as 6 months, i.e., the two-dose series of Moderna’s and that of Pfizer’s that requires 3 shots to begin with.14 It is inconceivable that a young immune

12 https://edition.cnn.com/2022/05/20/health/mrna-vaccine-technology-covid-19-durability/

index.html. footnote 12. 14 https://www.axios.com/2022/06/15/fda-moderna-covid-vaccine-children. 13 See

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Fig. 13.3 Estimated impact of vaccination on immune evasiveness of the Omicron VOC. The pandemic has centered on the action of vaccines, whose anticipated protection has been assessed by the antibody count they evoke. Nonetheless, as the pandemic has been unfolding, more and more questions and challenges have been arising. It is increasingly obvious that antibody titers do not serve as suitable correlates of protection. Against the many underappreciated and unresolved mechanisms and relationships, a limited focus on antibodies may be regarded as looking for the key under the lamp post. More concerted efforts are urgently needed to tackle those issues that previously had been in the dark

system will not be distinctively primed by such repeated genetic injections whose long-term implications are rooted in but hopes and wishful expectations. • Overall, the effectiveness of any vaccine to instigate T cell immunity is dictated by the early interplay between the vaccine, innate immune cells, and the inflammatory environment. Albeit, for mRNA vaccines, these parameters have not been analyzed in clinical studies. In fact, it was only in December 2020 that a study in mice demonstrated the opposing role of Type I IFN signaling in this context [22]. As described above, these findings point to critical issues involving the timing and kinetics of innate signaling mechanisms. These cannot be resolved by the one-size-fits-all approach of untried mRNA modifications to abrogate innate immune signaling but depend on pharmacokinetic and biodistribution dynamics, drug interactions of the vaccine, and other factors that have not been determined in clinical contexts. Likewise, while it has been recognized that antigen-encoding mRNA vaccines have a high capacity to elicit cytolytic CD8+

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T cells, knowledge of the effect of vaccine-based innate sensor activation and their impact on the immunogenicity of a given mRNA vaccine is rather limited. • Other than the many poorly appreciated risks, a wide range of primary data and recent publications indicate poor mRNA protection not only in terms of neutralizing antibody responses (Chap. 11). Remarkably, a June 2022 publication in Science [18] that investigated T and B cell immunity against B.1.1.529 (Omicron) in triple mRNA vaccinated healthcare workers (HCW) found: – An increased immunological escape from B cell-mediated (antibody) control and T cell immunity after three Pfizer vaccine doses – A significantly reduced T cell cross-recognition of the S1 antigen and related peptides – Evidence that Omicron has evolved to completely evade antibodies from (a) injection and (b) infection with some of the earlier SARS-CoV-2 strains The reason for these devastating developments seems to lie in differentiated imprinting responses (Chap. 11). Those who were least protected were the HCWs who were infected with the original Wuhan strains, later vaccinated, and then reinfected with Omicron. Moreover, according to the study authors, “[b]y three vaccine doses antibody responses had plateaued, regardless of infection history.” This raises the urgent question of vaccinating those who had previously been infected, notably our children who generally have cleared the virus with little problem. As described above (Chap. 11), the concern of immune imprinting has been present during the entire pandemic. Yet, this paper in Science highlights just how severe the problem is, depicting a potentially large-scale deadlock situation. Reynold et al. conclude that “mRNA vaccination carrying the B.1.1.529 (Omicron) spike sequence (Omicron third dose after ancestral sequence prime/boosting) offers no protective advantage.” mRNA Vaccines Cannot Prevent Infection and Yet They are Believed to Prevent Severe Disease and Death A new conundrum related to the action of the vaccines is now emerging. It is now beyond doubt that they cannot prevent infection. Yet, many still believe their protective advantage lies in their ability to prevent severe disease and death. Alas, how is this possible, seeing they cannot prevent disease to begin with? Although an incomplete allegory, this reminds me of a leaking dam—where the vaccines would mimic the steps taken to stop this leak. If it turns out that these cannot keep more water from running out, then handling the excess runoff water at the many places it goes to is a different problem than stopping the leakage altogether. From this perspective, if mRNA vaccines help prevent more severe forms of infection and death, then, in part, this may be attributable to them acting as medicines rather than (traditional) vaccines. Nonetheless, it is important to appreciate the enormous knowledge gap in this regard. These products have not been developed for, or assessed as, drugs to treat Covid infection. Although one such case was described above (Chap. 11), the immune response to those pharmaceuticals varies substantially between individuals. This is evidenced by the large spectrum of

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responses to these injections, as well as the unprecedented large confidence intervals seen from various vaccine efficacy estimates. Albeit, in terms of their action of vaccines, it is also not true in general that those who have been vaccinated always have sufficiently many antibodies (even if nonneutralizing) to keep the virus from spreading too far, from reaching different organs and from getting into the bloostream. As experience has shown, their believed benefits to prevent severe disease and death are also no longer substantiated. In fact, the Washington Post15 reported data from the CDC showing that this notion is no longer true. By the fall of 2022, various health agencies reported higher Covid19 numbers for the vaccinated versus the unvaccinated, even in the severe/fatal category. For instance, according to an analysis conducted by the Kaiser Family Foundation, in August 2022, a majority of Covid-19 deaths in the U.S. were people who were vaccinated or boosted. Sadly, this extends the analogous trends described above in terms of the inability of the shots to prevent infection or mild disease. And even more sadly, the share of deaths of people who were vaccinated has been increasing over the past year. Whereas in September 2021, those who were vaccinated made up 23% of Covid-19 fatalities, by the beginning of 2022, it was up to 42%. There is now ample evidence that Covid VE numbers are plummeting globally, even after several boosters. The rapid decline of these estimates confirms one major issue: we are not going to vaccinate ourselves out of the pandemic. Notably, a VE of zero means that an infinite number of people would need to be inoculated to prevent one additional case of an infection (that for most is non-severe)—depicting a situation that is beyond comprehension. However, that a VE estimate of zero is not the result of some technical glitches can be seen in many studies that even report negative VE numbers, reflecting a common trend. It is astonishing why most negative VE estimates are not clearly reported as such and why this has not raised alarm across the board. Some even argue that as soon as VE estimates cross unity, that this just means that the corresponding statistics do not say anything meaningful anymore. However, the definition of VE (Chap. 5) clearly shows when VE metrics become negative—it is precisely when the risk of infection is greater among the vaccinated than the unvaccinated. Previously, risk–benefit comparisons revealing negative protection were unheard of for approved medicines. For the Covid vaccines, the observation that they actually increase the risk of Covid has been heavily disputed. Albeit, negative VE estimates are more and more showing up in independent statistics and situations (Chap. 11). In fact, it is not only about statistics. Several biological factors have come to light that can explain an actual negative effect of the shots, including immune suppression by means of the modified RNAs, T cell exhaustion and immune non-responsiveness, immune imprinting, their highly inflammatory actions as systemic vaccines, the

15 https://www.washingtonpost.com/politics/2022/11/23/vaccinated-people-now-make-upmajority-covid-deaths/

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potential of the injections to evoke genetic alterations, and others (Fig. 11.6). Neither of these has been adequately appreciated. Viral Immune Escape Evidence has been emerging (Chaps. 7 and 12) that suggests that vaccinated people, when later infected, may provide a niche for viral escape mutants. Specifically, they may also have fostered the development of the Omicron VOC. This leads to the concern they likewise become a reservoir for other escape mutants too. Thus, the epidemiological role of vaccinated people in potentially inducing viral escape mutants cannot be ignored and must be taken seriously. While each of the SARS-CoV-2’s VOCs has had characteristics of escape mutants, their potential for immune escape has been radically increasing. Even though the original Omicron variant was believed to have reached the highest degree of transmissibility of such types of viruses, subsequent subvariants have proven to be more transmissible yet. Often this has been linked to waning immunity, including that afforded by the “vaccines.” What has been overlooked is the enormous number of mutations the virus has been accumulating during the pandemic. In this regard, how could it be expected then that a vaccine, based on the original variant, would still match the 2021 and 2022 circulating strains? With this, the urgency has shifted. It is no longer about protection of individuals against infection—mRNA vaccines cannot do this. Yet, their leaky nature, combined with the substantial vaccine mismatch, create an environment that is most conducive for further escape mutants. During the writing of this book, the new bivalent boosters by Pfizer and Moderna have been rolled out, albeit without human studies. The limited trial information based on a few mice16 does not give much reason for optimism. Moreover, while the payload of the bivalent boosters consists of mRNAs targeting different viral variants, the underlying mRNA vaccine platform has not changed. Explicitly, then, the very same foundational problems as with the previous mRNA Covid vaccines remain. The notion that vaccines could possibly induce an escape mutant has been vehemently denied by many public health authorities. Nonetheless, that mutants strive in an environment for which they are better suited should not be a surprise. In this regard, the CDC has made a perplexing statement: they argue that there are “no documents supporting claim vaccines don’t cause variants.17” Astonishingly, the way this has been interpreted means the opposite of this statement. The latter, however, is a clear double negation and may thereby lead to the same confusion as detailed in Box 9 above, which involved the very same issue of variant induction.

16 https://www.science.org/content/article/omicron-booster-shots-are-coming-lots-questions 17 https://www.theepochtimes.com/cdc-no-documents-supporting-claim-vaccines-dont-causevariants4464871.html.

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In addition to the above, two other recent publications in Nature and the New England Journal of Medicine [4, 7] have raised alarm: these papers also show that the radical immune escape of the virus has accelerated to the extent that some lineages of Omicron have now escaped neutralization of both vaccine- and natural immunity altogether. The sobering news is that this does not merely mean escape, but neutralization escape—i.e., related to neutralizing antibodies. And while these studies examined a limited number of people, we now know from various other studies that current vaccines indeed provide very transient immune protection at best, with VE numbers that can be negative. Cao et al., in their paper in Nature [4], also raise alarm about the new variants’ strong neutralization evasion against the plasma from 3x vaccination, pointing out that “post-vaccination BA.1 infection mainly recalls wildtype-induced humoral memory.” As above, the potential of immune imprinting raises the urgent question to what degree mass vaccination has led to an epidemiological immune response that is substantially non-responsive to new variants.

Looking Ahead The next urgent question we are now facing, as humanity, is where the virus is going. Is it going to become yet more transmissible? More pathogenic? In this regard, one cannot but pause, and note that Omicron, despite its toll overall, has been doing humanity a favor. This variant could have continued the trajectory seen in previous variants, which seemingly culminated with the Delta VOC that optimized some of the most dangerous ways how coronaviruses can infect cells. Somehow or another, Omicron has taken a turn. In this book, details have been provided on how the development of the new VOCs can be linked to mass vaccinations. Nonetheless, we truly do not understand the ins and outs of viral virulence development in general (Chap. 12). And what do we know about the future at all? If anything, the pandemic has shown how fragile many of our human systems have been. We have seen enormous loss and suffering at all levels, to a degree that we never could have predicted. Many researchers and scientists have devoted their careers to modeling and predicting. And yet, can it be that the virus has taught us another lesson? Isn’t it that we now find out that so many notions, models, concepts, and dogmas in science and medicine just

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turned out to be incorrect? Omicron, in particular, has shown that we were wrong in so many regards. This is not new. Science and all forms of human understanding have always and will always be like this: an ongoing form of learning, based on our recognition of the shortcomings and mistakes we previously made. Clearly, this book has raised issues that many may not agree with. The future will show that this work, too, is incomplete. And there will be a time when it will also need to be corrected and updated. If anything else, the ultimate goal of this book is not to bring further division, to condemn past flaws in the underlying model, or how health regulators and others have responded during the pandemic. What I have hoped to do with this book is to show the very complexity of the problem, the infeasibility of a one-size-fits-all solution, and the mystery of life. At this stage, this prompts a question that is usually not articulated in academia or science. But I believe it is the type of question that the pandemic itself urges us to ask. If Omicron could speak, what would it say? Why did it take the other turn? Is it really against us, out to kill, harm, and destroy? The most important question, I believe, the virus might be asking is: Are the laws of nature that of hopelessness, scarcity, and polarization? Or is the very essence of life that of value, creativity, integrity, and love? Seeing an increase in both breakthroughs and emergence of new VOCs, it is imperative to consider the issues raised above—before assuming that global vaccination will be a sufficient and safe solution (and the only one for that matter). A failure to consider basic medical, ethical, and humanitarian principles may not only endanger vaccinees but lead to an unnatural balance between a one-size-fits-all experimentation vs. the flourishing of viral escape mutants. In confronting the overwhelmingly harsh and protective governance of pandemic research, the radical censorship, injudicious fact checking, and other forms of polarizing supervision, I hope to have provided valuable insights into many of the fundamental underpinnings of mRNA inoculations and have offered a more comprehensive approach to assessing the many knowns and unknowns of science, epidemiology, public, and personal health. Nothing would be more welcome than to see these resolved, to help shift the risk–benefit balance (Fig. 13.4), to turn Covid-19 vaccines into a proven and safe technology. And beyond this technology, I hope that the points raised above also help trigger in each of us the type of questions that shape how we think and feel not only toward the virus, but about our fellow human beings, and nature at large.

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Fig. 13.4 Summary of advantages and disadvantages of mRNA vaccines. The safe and effective use of vaccines has been a long-held promise for addressing infectious diseases and other persistent health challenges in public health. However, with Covid-19 mRNA vaccines, their rushed approval, limited clinical experience, and lack of reliance on the precautionary principle have led to an unprecedented number of deleterious adverse events; as more data and insights are emerging, for many who have been inoculated, this may defeat their trust in those technologies and public acceptance of vaccines altogether. Based on an independent review of the published literature, it is found that the main scientific assertions regarding the safety and efficacy of RNA vaccines are based on a failure to: (1) perform clear and independent preclinical and clinical studies and experiments, (2) make all the data related to studies before and after the global rollout of these new technologies publicly available, and (3) adequately interpret and scrutinize existing information and relevant literature. Although mRNA vaccines have some advantages (left-hand side), these do not outweigh their known and unknown risks (right-hand side), demonstrating that a shift (black arrow) is required before these platforms can be used on humans and classified as safe and effective

References 1. Al-Aly Z, Bowe B, Xie Y (2022) Long Covid after breakthrough SARS-CoV-2 infection. Nat Med: 28:1461–1467. https://doi.org/10.1038/s41591-022-01840-0 2. Bertolazzi G, Cipollina C, Benos PV, Tumminello M, Coronnello C (2020) miR-1207-5p can contribute to dysregulation of inflammatory response in Covid-19 via targeting SARS-CoV-2 RNA. Front Cell Infect Microbiol 10:673 3. Blackburn J, Yiannoutsos CT, Carroll AE, Halverson PK, Menachemi N (2021) Infection fatality ratios for covid-19 among noninstitutionalized persons 12 and older: results of a random-sample prevalence study. Ann Intern Med 174(1):135–136

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4. Cao Y, Yisimayi A, Jian F, Song W, Xiao T, Wang L, Du S, Wang J, Li Q, Chen X, et al. (2022) BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection. Nature 608:593–602 5. Ealy H, McEvoy M, Chong D, Nowicki J, Sava M, Gupta S, White D, Jordan J, Simon D, Anderson P (2020) Covid-19 data collection, comorbidity and federal law: A historical retrospective. ETHICS 2:4–22 6. FOX6 News Milwaukee (2021) Sen. Ron Johnson with families on adverse reactions to Covid vaccine. https://youtu.be/6mxqC9SiRh8 7. Hachmann NP, Miller J, Collier AY, Ventura JD, Yu J, Rowe M, Bondzie EA, Powers O, Surve N, Hall K, Barouch DH (2022) Neutralization escape by SARS-CoV-2 Omicron subvariants ba.2.12.1, ba.4, and ba.5. N Engl J Med 387(1):86–88 8. Heinemann JA, Paull DJ, Walker S, Kurenbach B (2021) Differentiated impacts of human interventions on nature: Scaling the conversation on regulation of gene technologies. Elementa: Science of the Anthropocene 9(1):03 00086 9. Johnson R (2022) Covid-19: A second opinion. https://rumble.com/vt62y6-covid-19-asecond-opinion.html 10. Kostoff RN, Calina D, Kanduc D, Briggs MB, Vlachoyiannopoulos P, Svistunov AA, Tsatsakis A (2021) Why are we vaccinating children against Covid-19? Toxicol Rep 8:1665–1684 11. Law R (2021) Whistleblower Jane Doe declaration—45k death count from Covid shot lawsuit. https://renz-law.com/45k-whistleblower-suit 12. Malone RW, Felgner PL, Verma IM (1989). Cationic liposome-mediated RNA transfection. Proceedings of the National Academy of Sciences 86(16):6077–6081 13. Moyer MW (2018) Vaccines are pushing pathogens to evolve. https://www.quantamagazine. org/how-vaccines-can-drive-pathogens-to-evolve-20180510/ 14. Mueller S (2020) Facing the 2020 pandemic: What does cyberbiosecurity want us to know to safeguard the future? Biosafety and Health 3(01):11–21 15. Pezzullo AM, Axfors C, Contopoulos-Ioannidis DG, Apostolatos A, Ioannidis JPA (2022) Age-stratified infection fatality rate of COVID-19 in the non-elderly population. Environ. Res. 114655 16. Read AF, Baigent SJ, Powers C, Kgosana LB, Blackwell L, Smith LP, Kennedy DA, WalkdenBrown SW, Nair VK (2015) Imperfect vaccination can enhance the transmission of highly virulent pathogens. PLoS Biol 13(7):e1002198 17. Renz Law (2021) US District Court of Alabama civil action no. 2:21-cv-00702-clm (pdf). https://img1.wsimg.com/blobby/go/3c6a0774-cfad-46fa-aa97-af5aa5e74f00/M%20for %20PI%20file%20stamped.pdf 18. Reynolds CJ, Pade C, Gibbons JM, Otter AD, Lin K-M, Muñoz Sandoval D, Pieper FP, Butler DK, Liu S, Joy G, et al (2022) Immune boosting by B.1.1.529 (Omicron) depends on previous SARS-CoV-2 exposure. Science 377(6603):eabq1841 19. Sahin U, Karikó K, Türeci Ö (2014) mRNA-based therapeutics–developing a new class of drugs. Nat Rev Drug Discov 13(10):759–780 20. Stand for Health Freedom (2021) Breaking: OR senators filed grand jury petition into CDC’s willful misconduct to hyperinflate Covid-19 data. https://standforhealthfreedom. com/press-release/or-senators-grand-jury-petition/?utm_campaign=Solo%20Newsletter %3A%20Oregon%20Senators%20Filed%20a%20Grand%20Jury%20Petition%20for %20Investigation%20into%20CDC%20and%20FDA.%20Read%20the%20Press%20Release %20%28TKw2cE%29&utm_medium=email&utm_source=Daily%20Newsletter&kx= im3Aw7g3cZmueeIEhGV5PfhuY7jK1YdygFZg5DW4UJwqRfmO-Jhj34yOtgu2VqRG. K2vXAy 21. US FDA (2020) Long term follow-up after administration of human gene therapy products. Guidance for Industry. https://www.fda.gov/regulatory-information/search-fdaguidance-documents/long-term-follow-after-administration-human-gene-therapy-products

References

439

22. Van Hoecke L, Roose K, Ballegeer M, Zhong Z, Sanders NN, De Koker S, Saelens X, Van Lint S (2020) The opposing effect of type I IFN on the t cell response by non-modified mRNAlipoplex vaccines is determined by the route of administration. Mol. Ther.-Nucleic Acids 22:373–381 23. World Health Organization et al. (2020) mRNA vaccines against Covid-19: Pfizer-BioNTech Covid-19 vaccine BNT162b2: prepared by the Strategic Advisory Group of Experts (SAGE) on Immunization working group on Covid-19 vaccines, 22 December 2020. Technical report, World Health Organization 24. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL (1990) Direct gene transfer into mouse muscle in vivo. Science 247(4949):1465–1468