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The COVID-19 TEXTBOOK Science, Medicine, and Public Health

William A. Haseltine, PhD Chair and CEO ACCESS Health International Ridgefield, Connecticut

Roberto Patarca, MD, PhD Chief Medical Officer, ACCESS Health International Ridgefield, Connecticut

Acquisitions Editor: Chris Teja Development Editor: Ariel S. Winter Editorial Coordinator: Venugopal Loganathan Marketing Manager: Kirsten Watrud Production Project Manager: Bridgett Dougherty Manager, Graphic Arts & Design: Stephen Druding Manufacturing Coordinator: Lisa Bowling Prepress Vendor: S4Carlisle Publishing Services Copyright © 2025 Wolters Kluwer.

All rights reserved. This book is protected by copyright. No part of this book may be reproduced or transmitted in any form or by any means, including as photocopies or scanned-in or other electronic copies, or utilized by any information storage and retrieval system without written permission from the copyright owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals as part of their official duties as U.S. government employees are not covered by the above-mentioned copyright. To request permission, please contact Wolters Kluwer at Two Commerce Square, 2001 Market Street, Philadelphia, PA 19103, via email at permissions@lww.com, or via our website at shop.lww.com (products and services). 9 8 7 6 5 4 3 2 1 Printed in Mexico Library of Congress Cataloging-in-Publication Data

Names: Haseltine, William A., editor. | Patarca-Montero, Roberto, editor. Title: The COVID-19 textbook: science, medicine, and public health / [edited by] William A. Haseltine, Roberto Patarca. Description: Philadelphia: Wolters Kluwer Health, [2025] | Includes bibliographical references and index. Identifiers: LCCN 2023036538 | ISBN 9781975202330 (paperback) | ISBN 9781975202354 (ebook) Subjects: MESH: COVID-19 | SARS-CoV-2—pathogenicity | COVID-19 Drug Treatment—methods | COVID-19 Vaccines | BISAC: MEDICAL / Infectious Diseases Classification: LCC RA644.C67 | NLM WC 506 | DDC 362.1962/4144—dc23/eng/20230908 LC record available at https://lccn.loc.gov/2023036538

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Preface

COVID-19 took the world by storm beginning in the closing months of 2019. The world was simply not prepared for a natural disaster of such magnitude. The true toll in terms of lives lost, long-term social and economic impacts, and opportunities to live full and productive lives taken from many hundreds of millions may never be known. The devastation has been profound enough that the COVID-19 pandemic will affect our lives for many years to come. If there is a bright spot, it is the collective effort of our medical, scientific, and public health efforts to respond to the pandemic, to understand the disease, to save the lives of those infected, to develop new means to prevent and treat infection, to understand the causal virus, and to prepare for future pandemics to come. This textbook summarizes those valiant efforts. This text is written not as a historical account but instead as a guide for all those working to pre vent this and similar disasters from occurring. In that respect, it is intended for advanced university students, graduate students, postdoctoral fellows, and young professionals preparing for life in public health, research, medicine, and public policy. I am pleased that my coeditor, Roberto Patarca, and I have been able to draw upon world-leading experts in each aspect of the pandemic, including natural history, epidemiology, molecular biology, immune response, drug and vaccine development, and medical treatment. We are grateful for the participation of all our contributors and thank the publisher, Wolters Kluwer, for their help in the preparation of the text.

Acknowledgments

Our sincere thanks to all of our contributors. Each one brought dedicated expertise and a rigorous analysis of current and emerging research to their chapters. We would like to thank ACCESS Health International for their support of this project, in particular Courtney Biggs, communications direc tor, who assisted in the editorial coordination of this book. In addition, we would like to extend our gratitude to the Massachusetts Consortium on Patho gen Readiness for being such an informative and inspiring brainstorming venue that allowed the fruition, implementation, and sharing of ideas, initiatives, and resources. We would also like to thank the patients who contributed to acquiring knowledge and running clinical trials globally. Finally, we would like to thank Wolters Kluwer and the editorial team who made this important book possible, including Acquisition Editor Chris Teja, Development Editor Ariel Winter, and Edi torial Coordinator Venugopal Loganathan. Dr. William Haseltine would like to thank his wife Maria Eugenia, children Mara, Alexander, Karina, Manuela, and Camila, and his three grandchildren, Pedro, Enrique, and Carlos, for their ongoing support. Dr. Roberto Patarca dedicates this work to his wife Carole Ann, children Alexander, David, and Emily, and grandchildren Willow and Bodie.

vii

Contributors

Galit Alter, PhD Vice President of Immunology Research Department of Infectious Disease Research Moderna Therapeutics Cambridge, Massachusetts Dan H. Barouch, MD, PhD Professor of Medicine Director, Center for Virology and Vaccine Research Beth Israel Deaconess Medical Center

Akshamal Gamage, PhD, BSc (Hons) Research Fellow Program in Emerging Infectious Diseases Duke-NUS Medical School Singapore, Singapore Rajesh T. Gandhi, MD Director, HIV Clinical Services and Education Infectious Diseases Division Massachusetts General Hospital Boston, Massachusetts David E. Golan, MD, PhD Dean for Research Operations and Global Programs George R. Minot Professor of Medicine Professor of Biological Chemistry and Molecular Pharmacology Department of Biological Chemistry and Molecular Pharmacology

Harvard Medical School Boston, Massachusetts Nagme Bilgehan Medical Student Faculty of Medicine Imperial College London London, UK

Mary Carrington, MS, PhD Director, Basic Science Program

Harvard Medical School Boston, Massachusetts William A. Haseltine, PhD Chair and CEO ACCESS Health International Ridgefield, Connecticut Howard M. Heller, MD, MPH Assistant Professor of Medicine Infectious Diseases Division Massachusetts General Hospital

Laboratory of Integrative Cancer Immunology Frederick National Laboratory for Cancer Research National Cancer Institute Bethesda, Maryland Bing Chen, PhD Rosalind Franklin PhD Professor of Pediatrics Division of Molecular Medicine Boston Children’s Hospital/Harvard Medical School Boston, Massachusetts Gabriel Dagotto, BSc PhD Candidate

Boston, Massachusetts Carole Henry, PhD Director, Immunology Department of Infectious Disease Research Moderna Therapeutics Cambridge, Massachusetts Catherine Jacob-Dolan, BSc PhD Candidate, Immunology

Virology Program Harvard University Boston, Massachusetts

George Q. Daley, MD, PhD Dean of the Faculty of Medicine Department of Biological Chemistry and Molecular Pharmacology

Harvard Medical School Boston, Massachusetts

Contributors

Michael J. Mina, MD, PhD Epidemiologist, Immunologist, and Clinical Pathologist Previously a Professor of Epidemiology, Immunology and Clinical Pathology Harvard T.H. Chan School of Public Health and Harvard Medical School Boston, Massachusetts Mark Namchuk, PhD Professor of the Practice Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School Boston, Massachusetts Christopher Newton-Cheh, MD, MPH

Nikolaus Jilg, MD, PhD Instructor in Medicine Massachusetts General Hospital

Boston, Massachusetts Mahima Kaur, MSc Research Assistant Yale School of Public Health Yale University New Haven, Connecticut Mehak Zahoor Khan, PhD Senior Scientist Department of Infectious Disease Research Moderna Therapeutics Cambridge, Massachusetts Rachel Leeson, MSciComm Consultant, Medical Writer Immunology Moderna Therapeutics Cambridge, Massachusetts Jacob E. Lemieux, MD, DPhil Assistant Professor of Medicine Division of Infectious Diseases Department of Medicine Massachusetts General Hospital and Harvard Medical School Boston, Massachusetts M. William Lensch, PhD Associate Provost for Research Office of the Vice Provost for Research Harvard University Cambridge, Massachusetts Jeremy Luban, MD Professor Program in Molecular Medicine University of Massachusetts Chan Medical School Worcester, Massachusetts Anna Martens, MD Resident Physician Department of Pediatrics Massachusetts General for Children Boston, Massachusetts Maureen P. Martin, MD Senior Scientist Laboratory of Integrative Cancer Immunology Frederick National Laboratory for Cancer Research National Cancer Institute Bethesda, Maryland

Physician, Cardiology Division Massachusetts General Hospital Boston, Massachusetts Roberto Patarca, MD, PhD Chief Medical Officer ACCESS Health International Ridgefield, Connecticut Shiv Pillai, MD, PhD Professor of Medicine Ragon Institute Massachusetts General Hospital Harvard Medical School Cambridge, Massachusetts Arlene H. Sharpe, MD, PhD Chair and Professor, Immunology

Harvard Medical School Boston, Massachusetts Galatea Stavraka Medical Student Faculty of Medicine Imperial College London London, UK

Justin Stebbing, MD, PhD Professor of Cancer Medicine Department of Surgery and Cancer Imperial College London London, UK Sten H. Vermund, MD, PhD

Anna M.R. Lauder Professor of Public Health Department of Epidemiology of Microbial Diseases Yale School of Public Health New Haven, Connecticut

Contributors

Lael Yonker, MD Assistant Professor Department of Pediatrics Massachusetts General Hospital Boston, Massachusetts Adil Yunis, MD Physician, Cardiology Massachusetts General Hospital Boston, Massachusetts Feng Zhu, PhD Senior Research Fellow Program in Emerging Infectious Diseases Duke-NUS Medical School Singapore, Singapore

Chee Wah Tan, PhD Senior Research Fellow Program in Emerging Infectious Diseases Duke-NUS Medical School Singapore, Singapore Bruce D. Walker, MD Director Ragon Institute of MGH, MIT, and Harvard Mass General, MIT and Harvard Nahant, Massachusetts Lin-Fa Wang, PhD Professor Program in Emerging Infectious Diseases Duke-NUS Medical School Singapore, Singapore

Contents

Preface v Acknowledgments vii Contributors ix

Section 1: Introduction

Epidemiology of COVID-19

Sten H. Vermund • Mahima Kaur

Molecular Biology of SARS-CoV-2

Roberto Patarca • William A. Haseltine

Section 2: Virology

115

Bat Coronaviruses

117

Chee Wah Tan • Feng Zhu • Akshamal Gamage • Lin-Fa Wang

4 Structure and Function of SARS-CoV-2 Spike Protein

136

Bing Chen • Jeremy Luban

SARS-CoV-2 Variants Jacob E. Lemieux • Jeremy Luban

156

Section 3: Immunology

173

6 Genetic Determinants of Susceptibility and Resistance to SARS-CoV-2

175

Maureen P. Martin • Mary Carrington

Roberto Patarca • William A. Haseltine

xiii

xiv

Contents

Antibodies in COVID-19

247

Mehak Zahoor Khan • Rachel Leeson • Carole Henry • Galit Alter

T Cells and COVID-19

264

Shiv Pillai

Section 4: Pathogenesis

277

10 Organ Pathogenesis: Lung, Heart, Kidney, Liver, Pancreas, Brain

279

Nagme Bilgehan • Galatea Stavraka • Justin Stebbing

11 Pediatric COVID

303

Anna Martens • Lael Yonker

12 Long COVID

326

Roberto Patarca

Section 5: Medical Response

369

13 COVID-19 Testing for Medicine and Public Health

371

Michael J. Mina

14 A Brief History and Future Directions for Small Molecule Antivirals for COVID-19 and the Next Pandemic 409 Mark Namchuk 15 Treatment of COVID-19 in Adults 434 Nikolaus Jilg • Adil Yunis • Christopher Newton-Cheh • Rajesh T. Gandhi 16 Potential Treatments for SARS-CoV-2 Beyond Current and Related New-Generation Antivirals and Monoclonal Antibodies 444 Roberto Patarca • William A. Haseltine 17 COVID-19 Vaccines 470 Gabriel Dagotto • Catherine Jacob-Dolan • Dan H. Barouch 18 MassCPR: Research Collaboration to Confront a Pandemic 492 M. William Lensch • Arlene H. Sharpe • Bruce D. Walker • Howard M. Heller • George Q. Daley • David E. Golan Index 523

CHAPTER

Antibodies in COVID-19

Mehak Zahoor Khan • Rachel Leeson • Carole Henry • Galit Alter

Introduction Antibodies Fc-Receptors Antibody Effector Functions Fc-Mediated Effector Functions Importance of Antibodies in Control of COVID-19 Role of Antibodies Following Vaccination Next-Generation Vaccine Challenges for COVID and Beyond Variants of Concern Preventing Transmission Optimized Boosting Monoclonal Antibodies as Therapeutics mAb Targets Next-Generation Monoclonal Antibodies Newer Gene Delivery Strategies Conclusion

INTRODUCTION Antibodies, or immunoglobulins (Igs), were first mentioned in the literature in 1890, when Emil von Behring and Shibasaburo Kitasato published diphtheria serum transfer studies, referring to neu tralizing agents, or “antikörper,” in the blood of immunized animals that conferred pathogen-specific immunity. 1,2 A decade later, Paul Ehrlich proposed the side-chain theory, describing antibodies as branched molecules containing multiple “lock-and-key” binding sites. Although it was incorrectly described as “chains” that grew out from the cell, the lock-and-key mechanism was confirmed in the 1940s by Linus Pauling, 3 who demonstrated the exquisite specificity conferred by these serum pro teins. Molecular structural work on antibodies was published in 1959, in independent studies from Gerald Edelman and Rodney Porter. 4,5 In 1948, Astrid Fagraeus deepened the understanding of the source of these molecules, demonstrating the highly specialized role of plasma cells that produce an tibodies. This was followed by the publication by Frank Burnet and David Talmage describing clonal selection theory in 1957, 6 a theory that was experimentally confirmed in a 1958 publication by Gustav Nossal and Joshua Lederberg. 7 However, the major explosion in antibody research occurred after Georges Köhler and César Milstein developed a method to produce monoclonal antibodies (mAbs) in vitro, 8 in 1975, greatly expanding our ability to study antibodies and explore their ther apeutic and research applications. mAbs now represent the fastest growing class of drugs, 9,10 with

247

248

SECTION 3 • Immunology

hundreds of therapeutics in clinical development and testing across disease indications from autoim munity to infectious disease. The coronavirus disease 2019 (COVID-19) pandemic illustrated the critical role of antibodies in the prevention of disease, in the setting of prophylactic vaccination, and as therapeutics for the treatment of disease. Here, we will review the anatomy of antibodies, their origin, their role in vaccination, and the promise of next-generation strategies through the lens of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ANTIBODIES Antibodies are Y- or T-shaped polypeptide proteins, comprised of four peptide chains: two heavy chains and two light chains (Figure 8.1). Together, these four chains form two identical antigen-binding fragments (Fabs) and a crystallizable fragment (Fc), linked by a hinge domain. 11 They are expressed in B cells, acting as B-cell receptors (BCRs), and produced by antibody-secreting cells, including short-lived plasmablasts and long-lived plasma cells. 12 Each B cell has a unique BCR, formed via a random recombination event during B-cell maturation in the bone marrow, resulting in the gener ation of up to 10 12 unique BCRs with distinct antigen recognition capabilities. 13 At the start of the maturation process, one of two light chains ( κ or λ ) is selected to form the antibody Fab. The light chains pair with the heavy chains to form a polypeptide of two light chains and two heavy chains, forming two identical antigen-binding sites. Variation in the antigen-binding sites arises from the recombination of VJ (light chain) and VDJ (heavy chain) segments found in the Ig locus, and the ensuant random repair following end-joining creates random variation that collectively forms the complementarity determining regions (CDRs), 14 which are supported by framework regions found on the end of each of the polypeptide chains. 14,15 The CDRs are responsible for antibody specificity and variation. Typically, B cells encounter and sample antigens with their BCR in dedicated regions of the lymph node called germinal centers (GCs), 16-18 where antigens are arrayed on specialized set of fibroblasts known as follicular dendritic cells (fDCs). 19 When the BCRs productively engage an antigen, B cells rapidly become activated, acquiring signals to proliferate, travel to a newly devel oped dark zone of the GC, and begin hypermutating their BCR. 20 Hypermutation involves the specialized incorporation of additional mutations in the CDRs of the antibody, aimed at randomly allowing for the creation of almost unlimited variability in antigen specificity and affinities. 14 Hy permutated daughter cells then circulate back to the light zone of the GC, resampling the arrayed antigen. Cells that bind the antigen more tightly gain survival and proliferative signals, present the processed antigen to follicular T-helper cells (Tfh), and return to the dark zone for further hyper mutation. Cells that do not gain affinity, and therefore do not receive additional signals, die off. Following several mutational rounds, a subset of B cells differentiates into short-lived plasmablasts primed to populate the system with antibodies. A second subset of high-affinity GC B cells dif ferentiate into plasma cells that take up long-term residence in the bone marrow. Finally, a third subset become memory B cells that circulate widely through the body, providing memory upon pathogen/antigen reencounter. The Fc-domain, which is composed of two polypeptide chains, can also change during the life span of a B cell. In humans, the Ig locus contains nine different potential Fc-domains that can be attached to any Fab. These include the five antibody isotypes, IgD, IgM, IgG, IgA, and IgE, in which IgG and IgA are composed of the subclasses IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively 21,22 (Table 8.1 23 ). Each Fc-domain can bind to isotype-specific Fc-receptors (FcRs) found in different combinations and at different levels across cells of the immune system. 24,25 Specific inflammatory cues and T-helper signals, present at the time of B-cell programming, lead to the selection of a particular Fc-domain able to recruit and drive the most appropriate immunologic functions to clear the pathogen the antibody recognizes, that is, mucosal pathogens tend to drive IgA Fc-selection, known to play a critical role at mucosal barriers, whereas systemic viral infections tend to elicit IgG1/IgG3 responses that have high affinity for FcRs present on systemic immune cells. 22,24 Together, the pairing of the optimal Fab and Fc results in the generation of bifunctional molecules able to fully leverage the immune system to fight disease.

V(D)J recombination Somatic hypermutation ( )

D 2 D 1 V n V 3 V 2 V 1

FR2 FR1

FR3

FR4 FR3 CDR3 CDR2 CDR1

C C J n J 2 J 1 D n Immunoglobulin heavy chain

CDR3 CDR2 CDR1

J D

FR4

site

Light chain

FcRn binding site

Complement binding

Heavy chain

Fc Glycosylation

Disulfide bridges

CH2

CH3

~25 kDa

CH1

FIGURE 8.1 The structure of an antibody . The inset shows the CDR loops and framework (FR) of the variable region. CDR, complementarity determining region. 249

Hinge

region

~50 kDa

Fab

~50 kDa

250

SECTION 3 • Immunology

TABLE 8.1 Summary of major classes of antibodies in humans

IgM

IgG

IgA

IgE

IgD

γ 1, γ 2, γ 3, γ 4

α 1, α 2

Heavy chain Light chain

κ , λ

Structure

Monomer, pentamer

Monomer

Monomer, dimer Monomer

Mono mer

Percentage of total antibody in serum Half-life (days) Fixes complement

10%

75%

15%

< 1%

6-8 No

1-5 No

2-8 No

Yes

Fc γ RI/CD64, Fc γ RII/ CD32, Fc γ RIII/CD16 Secondary anti body responses; neutralization, opsonization, fixes complement

Fc α RI/CD89

Fc δ R

Fc receptor

FcμR

Fc e RI, Fc e RII

Major function Monomer form of IgM serves as

Predominant an tibody in mucosa and secretions

Allergy and antiparasitic activity

BCR

BCR; fixes comple ment; primary anti body responses

BCR, B-cell receptor; Ig, immunoglobulin. From Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the Cell . 4th ed. Garland Science; 2002. ©1983, 1989, 1994. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. Used by permission of W. W. Norton & Company, Inc.

F c -RECEPTORS The Fc-domain of an antibody plays two critical roles. First, it provides instructions to the im mune system on how the antigen to which the antibody is bound should be destroyed. Second, the Fc-domain dictates the half-life of the antibody. Interaction of the antibody Fc-domain with a range of FcRs, found across immune and nonimmune cells, dictates these two functions. Individual FcRs, expressed on a variety of innate immune cell types, exist for nearly all antibody isotypes and subclasses, allowing each to deploy specific immunologic functions (Table 8.2). For example, the Fcμ-receptor (FcµR) has been implicated in opsonophagocytosis and T-cell activation. 26,27 Fc α R expression is more restricted, but it can activate a large number of functions. For example, Fc α RI activation can result in phagocytosis, degranulation, superoxide generation, release of neutrophil extracellular traps (NETs), antibody-dependent cellular cytotoxicity (ADCC) , release of cytokines and chemokines, or antigen presentation. 28 A diverse set of activating and inhibitory Fc γ Rs, expressed across distinct immune cell types, can deploy a range of immunologic functions that may be essential to the elimi nation of pathogens or aberrant cells/materials in the body. All FcRs except Fc γ RIIIB possess or are associated with signaling receptors that provide activating or inhibitory signals. Fc γ RIIIB is a Glyco sylphosphatidylinositol (GPI)-anchored protein, which captures antibodies largely on neutrophils. Although FcRs for IgE and IgA exhibit high affinity for their antibodies, Fc γ RI is a high-affinity receptor and the remaining four Fc γ Rs—Fc γ RIIA, Fc γ RIIB, Fc γ RIIIA, and Fc γ RIIIB—are all low-affinity receptors, 29 requiring multimerization to activate cells. 30 This affinity threshold across the Fc γ Rs acts as a quality control and ensures the appropriate activation threshold is met on innate im mune cells prior to cellular activation, preventing pathologic activation of the innate immune system. Antibody half-life is dictated by the ability of the antibody Fc-domain to interact with the neonatal FcR (FcRn). 31,32 FcRn is expressed across both endothelial cells and a subset of immune cells. In nonimmune cells, antibody binding results in antibody capture and recycling back into the circulation. 33 FcRn is also involved in transferring antibodies across barriers, such as the placental or blood-brain barrier, to ensure the presence of antibodies across tissues and compartments. 34,35 IgG

F γ Rn

ADCC, antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cellular phagocytosis; CDC, complement-dependent cytotoxicity; DC, dendritic cell; Ig, immunoglobulin. From Gillis C, Gouel-Cheron A, Jonsson F, Bruhns P. Contribution of human Fc γ Rs to disease with evidence from human polymorphisms and transgenic animal studies. Front Immunol . 2014;5:254. 251

Recycling, transport, Ag uptake

Major role Activation Activation Inhibition Activation Activation Decoy; activation IgG recycling; transport Major expression Monocytes/mac B cells, DCs, NK cells, mono NK cells, monocytes/ Monocytes/

macrophages,

neutrophils, DCs,

endothelium, and

syncytiotrophoblasts

Fc γ RIIIB

Neutrophils. Induc ible expression in basophils

ADCC/ADCP, CDC, degranulation

CD CD64 CD32A CD32B CD32C CD16A CD16B

F γ RIIIA

macrophages

ADCC/ADCP, CDC, degranulation

Fc γ RIIC

Antigen presenta

tion, ADCC, ADCP

cytes/macrophages, neutrophils

Fc γ RIIB

Antigen presenta

tion, ADCC, ADCP

basophils. induc ible expression in neutrophils

and monocytes/ macrophages

Fc γ RIIA

Antigen presenta

tion, ADCC, ADCP

Monocytes/macro

phages, neutrophils, DCs, basophils,

eosinophils and mast cells

Fc γ RI

TABLE 8.2 Summary of IgG Fc-receptors in humans Name

Main

functions

252

SECTION 3 • Immunology

antibodies bind to FcRn with a higher affinity and thus they both persist for longer periods of time and can be transferred more efficiently to tissues. In immune cells, FcRn is involved in driving anti gen processing and presentation for the potent induction of T-cell immune responses. Finally, FcRn also contributes to the delivery of antibody-antigen/pathogen complexes to antigen-presenting cells (APCs) that are involved in T-cell education. 36 ANTIBODY EFFECTOR FUNCTIONS Antibodies act through synergy, typically binding a target as a polyclonal pool of molecules, meaning that antibodies with different affinities, geometries, and Fc-domains bind simultaneously to a target, forming a large complex, also known as an immune complex (IC). Thus, given the multitude of different Fc-domains that may be present within a given IC, antibodies can drive a range of different functional responses. In viral infection, these effector functions include either direct antiviral activity through neutralization or indirect antiviral activity through the recruitment of immune functions via Fc:FcR interactions. Direct neutralization occurs when neutralizing antibodies (nAbs) bind to a pathogen and phys ically prevent the pathogen from infecting a new cell. nAbs often bind to the receptor or close to the receptor used by the pathogen to attach to the host cell, such as the receptor-binding domain (RBD) on SARS-CoV-2, preventing viral binding to the host cell, and thereby preventing entry and thus infec tion. As this is largely driven by the Fab-domain, all isotypes and subclasses of antibodies can be nAbs. However, viruses often evolve rapidly to evade direct nAbs. Thus, the Fab must perpetually evolve to catch-up to pathogen evolution. For example, over the COVID-19 pandemic, several variants emerged that were able to evade the nAbs induced by various vaccines. 37 Although the vaccine-induced immune responses, programmed with the original SARS-CoV-2 Spike antigen, were insufficient to provide pro tection against the new variants, the ensuing humoral immune responses observed in breakthrough vaccine cases evolved to cover the new variants, illustrating the plasticity of the memory B-cell response able to rapidly adapt to an evolving pathogen (see Figure 8.2). F c -MEDIATED EFFECTOR FUNCTIONS ADCC is the antibody-driven killing of target cells by natural killer (NK) cells, lymphocytes that belong to the innate arm of the immune system, and represent the first line of defense against incom ing pathogens. NK cells recognize their targets via an array of receptors that detect transformed or infected cells. 38 Mature NK cells express copious amounts of FcRs, specifically the Fc γ RIIIA recep tor, enabling a pathogen-specific response to antibody-opsonized targets. Upon activation by IgG/ Fc γ RIIIA, NK cells can release cytotoxic granules containing perforin, which creates channels in the cell membrane, and granzyme, which drives apoptosis in the cell. Although NK cells are primarily responsible for ADCC in vivo, multiple other cytotoxic innate effector cells, including neutrophils, monocytes, macrophages, and NK T cells, have been shown to also induce ADCC in vitro. ADCC has been implicated in the control of influenza 39,40 and human immunodeficiency virus (HIV), 41-43 and has also been observed in the setting of SARS-CoV-2 infection. 44-46 Antibody-dependent cellular phagocytosis (ADCP) , or opsonophagocytosis, is the antibody-mediated mechanism whereby antibodies drive pathogen-antibody complex uptake into phagocytic cells, where the complexes are degraded in the lysosomes. Phagocytosis can be induced by a wide range of receptors, including high- and low-affinity receptors, as well as by activating and inhibitory receptors. 47 Monocytes, macrophages, neutrophils, B cells, and dendritic cells (DCs) can all mediate opsonophagocytic uptake of ICs. Depending on the ligated cell type, ADCP can induce cytokine release, neutrophil NETosis (a process in which neutrophils release extracellular webs of chromatin, antimicrobicidal proteins, and oxidant enzymes that kill extracellular pathogens 48,49 ), antigen presentation by DCs to promote more effective T-cell immunity, and/or antiviral mecha nisms. ADCP may play a role in the immune response to HIV, 50,51 adenovirus, 52 West Nile virus, 53 and coxsackieviruses. 54,55 Antibody-mediated complement deposition , also known as complement-dependent cyto toxicity (CDC) , occurs when antibodies activate the classical complement pathway, enabling the

Fc α R

IgA

Fc γ RIIA

Fc γ R

IgG

NK cell

Neutrophil

• Fc γ Rs on innate effector cells are engaged by the Fc domain of antibodies that are bound to viral proteins on the surface of virus-infected cells

• IgG or IgA mediated neutrophil activation opsonophagocytosis and stimulation of NETogis • Infected cells cleared by ADCP and ADCC

• Viral particles are trapped and inactivated in NETS resulting in release of proteases, antimicrobial peptides and ROS that can result in aberrant inflammation

ADCC

• Release of cytotoxic granules contributes to pathology

Perforins

& granzymes

B cell

Infected cell

• Crosslinking of BCR activates B cells • Uptake of immune complexes by DCs and antigen presentation to T cells SARS-CoV-2 spike protein

IgG BCR TCR

Membrane attack complex (MAC)

• IgG or light binds to viral antigen on the surface of infected cell • Clq binds to these antibodies resulting in formation of MAC and lysis of target cell

T cell DC

CDC

Complement

SARS-CoV-2

MHC

FIGURE 8.2 Antibody effector functions . ADCC, antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cellular phagocytosis; BCR, B-cell receptor; CDC, complement-dependent cytotoxicity; DC, dendritic cell; Ig, immunoglobulin; MHC, major histocompatibility complex; SARS-CoV, severe acute respiratory syndrome coronavirus; TCR, T-cell receptor. (Adapted from Zhang A, Stacey HD, D’Agostino MR, Tugg Y, Marzok A, Miller MS. Beyond neutralization: Fc-dependent antibody effec tor functions in SARS-CoV-2 infection. Nat Rev Immunol . 2022. doi:10.1038/s41577-022-00813-1) 253

Costimulatory signals

• Antibody-opsonized target cells activate the Fc γ Rs on the surface of effector cells to induce phagocytosis • This results in internalization and degradation of the target cell through phagosome acidification

Immunomodulation

Fc γ R

Phagocytic cell

Fc γ RIIA

• Viral degradation results in enhanced MHCI and MHCII antigen presentation

lysed target cell

Cytokines

254

SECTION 3 • Immunology

first member of the cascade, C1q, to deposit on the surface of the pathogen. 56 C1q, a pentamer of C1 molecules, can interact with IgM (also a pentamer) or multimerized IgG3 and IgG1 antibodies via their Fc-domains, once they have bound to the surface of a pathogen. 57,58 C1q binding then leads to a cascade of enzymatic reactions and catalytic events, wherein additional members of the comple ment cascade deposit, forming the membrane attack complex (MAC) that ruptures the membrane of the pathogen and death. The deposition of complement components, midway in the formation of the MAC, such as C3b or C5b, can lead to rapid opsonophagocytosis by phagocytic cells. Thus, antibody-mediated complement may have an effect via direct destruction or a pathogen clearance. CDC is a protective mechanism in mAbs against influenza virus, 59,60 vaccinia virus, 61 cytomegalo virus, 62 and HIV. 50,51 However, complement activation may contribute to disease severity in both dengue virus (DV) 63,64 and HIV infection. 65,66 Antibody-mediated adaptive cell activation occurs when T cells and B cells, which both ex press complement receptors and FcRs, 67,68 are modulated by the presence of antibody-pathogen complexes. For example, IC interactions with Fc γ RIIB on B cells temper B-cell activation, helping to control inflammatory immune responses. 69,70 Complement receptors on T cells play a critical role in the initiation of the T-cell response, allowing ICs that may form immediately after infection to drive rapid activation and maturation of T cells. 71 These processes are known as antibody-mediated immunomodulation and have important therapeutic implications. Antibody-dependent enhancement (ADE) is the process by which antibodies either facilitate the uptake of pathogens into cells or contribute to the pathogenesis associated with the infection. ADE is principally mediated by IgG antibodies; it has also been shown to be mediated by IgM and complement or IgA antibodies. 72-75 For example, in the setting of DV infection, preexisting non-neutralizing or sub-neutralizing concentrations of DV-specific antibodies have been shown to facilitate DV uptake upon reinfection into monocytes/macrophages, providing the virus with a direct conduit to their target cells thereby promoting infection and disease. 76-78 Specifically, cross-reactive antibodies from the first infection form ICs upon reinfection with a different serotype of DV; these stabilize the virus and promote viral uptake by macrophages, leading to dengue hemorrhagic fever and shock syndrome. 79-82 ADE has been observed in other flaviviruses such as Zika virus, where in vitro studies show both convalescent plasmid therapy and mAbs enhance infection. 83 In an animal model, SARS-1 pox viral vector–based vaccination of nonhuman primates induced antibodies that enhanced inflammation and pathology in the lungs, rather than enhance infection, 84,85 pointing to a second mechanism by which antibodies could contribute to enhanced disease. Thus, understanding both the immune-protective and also potential pathologic activities of antibodies is key to harnessing their full vaccine and therapeutic potential. 86 IMPORTANCE OF ANTIBODIES IN CONTROL OF COVID-19 Following SARS-CoV-2 infection, SARS-CoV-2-specific antibody development occurs in a predict able pattern (Figure 8.3). First, IgM antibodies appear rapidly within the first weeks of infection, fol lowed closely by IgA antibodies in the mucosal membranes. Then, IgG antibodies appear, peaking at 3 to 7 weeks postsymptom onset. 87 Neutralizing Abs, 90% of which target the RBD, 88 appear within 7 to 15 days following symptom onset, increasing over time, then level off and decay, 89 similar to natural decay patterns observed for nAbs to other common coronaviruses. 90 This decay process ren ders previously infected individuals newly susceptible to infection every 2 to 4 years. 91 SARS-CoV 2-specific antibodies primarily target the immunogenic S and nucleocapsid (N) proteins, particularly the highly immunogenic RBD. Antibody production correlates with severity of disease, with the highest number of antibod ies observed in individuals with severe COVID-19 and the lowest levels detected in asymptomatic cases. 92,93 Thus, antibody levels are linked to the level of viral replication, or antigen levels, suggesting that individuals with more severe disease likely harbor higher levels of virus in their lungs, triggering stronger B-cell responses and thus eliciting higher levels of antibodies. 94,95 Individuals with milder disease also show enhanced FcR binding, Fc-effector function, and neutralization, along with an increased number of antibody subclasses present. 96-98 Individuals with mild disease also have lower viral loads and lower antibodies. This suggests that qualitative differences in the antiviral functions of

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CHAPTER 8 • Antibodies in COVID-19

Longitudinal profile of COVID-19 antibody response

Incubation period

Disease

Convalescence

Live virus

IgG

Viral RNA via PCR

IgA

IgM

Serum antibody titer

Week 1

Week 2

Week 3

Week 4

Week 5

Weeks postinfection

FIGURE 8.3 Profile of COVID-19 antibody response. Ig, immunoglobulin; PCR, polymerase chain reaction.

antibodies, rather than quantitative levels, are likely key to control and clear infection. Dysfunctional Fc-effector functions have been linked to poor clinical outcome and have been associated with altered Fc-glycosylation. 99,100 Specifically, decreased Fc fucosylation and increased titers of proinflammatory but poorly functional IgG2 antibodies are observed in severely ill COVID-19 patients, especially in male patients that typically suffer from enhanced morbidity and mortality in COVID-19. 99 Collec tively, these data suggest that in natural infection, Fc-effector functions may play a critical role in the control and clearance of infection, resulting in milder disease. The majority of nAbs against SARS-CoV-2 target the RBD and work through steric hindrance, physically preventing the RBD from binding to its host angiotensin-converting enzyme 2 (ACE2) receptor, effectively preventing viral entry into the cell. Antibodies against the N-terminal domain (NTD) of the S protein can also have neutralizing activity, 101 either by preventing the structural rearrangement of S protein required for ACE2 binding or through other, poorly defined mecha nisms. 102,103 When induced by natural infection, the evolution of nAbs plays a critical role in con ferring transient immunity against that same viral strain, 104 but confer limited protection against additional strains. 105-110 Emerging data suggest that individuals that survive severe disease more rap idly evolve a higher proportion of nAbs to non-nAbs than those who ultimately pass away. 111 Sim ilarly, nAb potency, which is measured by the nAbs’ ability to outcompete ACE2 in binding to the RBD, is a predictor of survival in COVID-19 patients. 112 Thus, the production of nAbs, linked to Fc-effector function, likely enables antibodies to simultaneously block further rounds of viral repli cation while clearing the infection. ROLE OF ANTIBODIES FOLLOWING VACCINATION All current licensed vaccines demonstrated robust immunogenicity, inducing robust binding of the virus and nAbs, often at levels far superior to those induced by natural infection. 113-117 Antibodies induced by vector and messenger RNA (mRNA) vaccines are more durable than infection-induced antibodies, 118 and approved vaccines conferred 60% to 95% protection against severe disease and death, 114-116 providing the protection that was urgently required to curb the pandemic. Early phase 3

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correlates of protection analyses pointed to peak immune nAb titers as a key predictor of protection against severe disease. 112,119-121 As mentioned previously, new variants of concern (VOCs) able to evade nAbs arose and continue to arise regularly, with the virus perpetually evolving to evade both infection- and vaccine-induced nAbs. 105-110 Against these VOCs, however, vaccinated individuals still show reduced morbidity and mor tality, 122-124 suggesting that additional immunologic mechanisms, beyond nAbs, contribute to protection against disease and death. Analyses from both preclinical and clinical studies point to a role for T-cell im munity and non-neutralizing, Fc-effector functions in protection against nAb-resistant VOCs. 125-128 T-cell responses are more resistant to mutations in the S protein, 129,130 suggesting that these cells could contribute to durable control of VOCs. Moreover, patients lacking antibodies, because of natural or chemothera peutically induced agammaglobulinemia, did not experience more severe COVID-19 131,132 , suggesting that T cells likely play a compensatory role in viral control after infection. However, T-cell depletions in animal models did not lead to loss of control of virus (measured by viral replication rates) in the lung, 133 suggesting that other, antibody-mediated mechanisms likely control and clear the virus after transmission. Binding antibodies, able to drive antibody effector functions, are also more resilient to viral evolution and recognize VOCs, 134,135 and the passive delivery of sub-nAbs with robust Fc-effector function significantly attenuated viral replication in the lung and led to faster clearance of infection. 136 This suggests that both non-nAb functions and T cells likely collaborate to attenuate disease if transmission occurs. Vaccine boosting has been shown to confer transient protection from infection and provides enhanced protection against mortality in vulnerable populations, 137,138 although breakthrough in fections can occur after boosting. 139 Beyond increased nAb titers, boosting can expand the breadth of variants covered by a vaccine-induced nAb response. 140 Emerging data suggest that boosting with combinations of antigens or with heterologous antigens can lead to a potent diversification of neu tralization and potentially provide protection against a broader array of variants. 141 Boosting also increases T-cell response and Fc-effector-inducing antibodies that may also contribute to longer-term protection against severe disease and death. Thus, beyond the impact of simply raising more antibod ies, boosting can also play a critical part in driving enhanced breadth and T-cell quality, which could ultimately have an important impact on response to future evolving VOCs. Although overall approved vaccines confer robust protection against disease, with boosting help ing to increase that protection, breakthrough disease continues to occur. Next-generation vaccines that can induce high titers of persistent, broadly reactive nAb, protecting against both disease and infection, are required. Despite major successes in vaccine and therapeutic development, the transition of SARS-CoV-2 to endemicity points to a need to evolve new therapeutics and vaccines to tackle this evolving pathogen. However, the pandemic has taught us that nAb activity is partly but perhaps not the sole mechanism by which antibodies confer protection against SARS-CoV-2. Both vaccine and therapeutic development must look beyond traditional design strategies to leverage the full spectrum of antiviral functionality held within the humoral immune response. Critically, the COVID-19 pandemic led to the collection of terabytes upon terabytes of antibody evolution, viral evolution, clinical, and genomic data, offering a unique opportunity to apply machine learning and artificial intelligence to define the rules by which antibodies may be fully used to fight COVID-19 and beyond. Current and future researchers will need to address a variety of issues, outlined later, in order to continue combating this and future pandemics. VARIANTS OF CONCERN The rapid evolution of VOCs across different parts of the globe, durability concerns, and immune-deficient or aging immunity represent major challenges in the transition of vaccine design from one intended to control an ongoing pandemic to one able to provide population-level immu nity against an evolving, endemic virus. 142 NEXT-GENERATION VACCINE CHALLENGES FOR COVID AND BEYOND

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As VOCs can systematically escape vaccine-induced nAbs, research efforts have shifted to focus on the development of pancoronavirus vaccines. 143,144 Significant progress has already emerged in the generation of pan-sarbecovirus vaccines, able to provide protection against all SARS-CoV-2 VOCs and SARS1. 144 Specifically, nanoparticle vaccines, using RBD domains from an array of variants, have already demonstrated striking progress toward the induction of broad responses able to limit VOC-mediated infections. 145,146 Novel designs focused on more conserved regions of the S pro tein, 147 or even other proteins, such as N proteins harboring many conserved T-cell epitopes, have also emerged, 148 aimed at leveraging both the T-cell and antibody response able to target current and potentially future VOCs while limiting disease. Both approaches are also being used to develop pan-betacoronavirus vaccines targeting sarbecoviruses and many common endemic coronaviruses that cause seasonal infections. 145,146 These strategies aim to significantly enhance the breadth of the immune response by targeting conserved elements and inducing broad antibody responses. PREVENTING TRANSMISSION Key to next-generation vaccines will be the development of strategies to drive immunity able to confer long-term protection against transmission. 142 Tissue-resident memory B and T cells form in response to mucosal infection 149 and help prevent reinfection, 150 with mucosal IgA known to be protective against coronaviruses. 151 High titers of S protein–specific IgA in the nasal passage are associated with milder COVID-19 in both children and adults. 152 Vaccine strategies aimed at driving sustained IgA responses at the virus’ portal of entry, the upper respiratory tract, could have a profound impact on preventing infection. However, most approved vaccines are administered intramuscularly and induce very limited tissue-resident antibodies. 153-156 Intranasal vaccination induces more robust and durable immune re sponses at the mucosal barrier, 157 potentially affording enhanced protection at the time of exposure. Intramuscular immunization with a mucosal boost has been shown to populate the mucosa with robust T and antibody responses. 158 Additionally, vectored vaccine administration to the mucosa has also shown robust nasal IgA responses. 159 Providing long-term protection via mucosal immunity may be challenging, as evidenced by some COVID-19 breakthrough cases occurring just weeks after an in fection, despite individuals being consistently exposed at the mucosal barrier. 160 Defining the precise immunologic mechanisms to drive broad and persistent nAbs in the upper respiratory tract is a key goal for next-generation vaccine development for SARS-CoV-2 and many other respiratory pathogens. OPTIMIZED BOOSTING Boosting is a common strategy to increase immunity, particularly in vulnerable populations such as im munocompromised individuals who require more doses to reach robust nAb titers or aging populations with partially anergized immunity. 137,138,161 mRNA vaccine–mediated boosting, for instance, reduces the risk of infection by 10- to 12-fold compared to the original (two-dose) vaccine in both the general population and in those more vulnerable to severe disease. 162 Cross-platform boosting may further increase effectiveness, by delivering the S antigen in the setting of different innate immune platform- specific stimuli that lead to higher quality T-cell activation and enhanced antibody breadth. 141 This is of particular concern in older populations, who have limited numbers of naive T cells, 163 where stronger responses using heterologous prime boosting strategies may be of particular advantage. Boosting with mRNA vaccines leads to higher levels of protection, 164 suggesting that specific platforms may induce superior activation of nAbs in older individuals. Further research is needed to define optimal boosting strategies to confer the highest level of immunity to vulnerable populations. MONOCLONAL ANTIBODIES AS THERAPEUTICS Early in the pandemic, convalescent plasma therapy (CPT) was trialed as a potentially high-impact, easily accessible therapeutic 165 from recovered patients. 166 Although several trials demonstrated lim ited benefit from CPT, 167-170 trials that used CPT early in the disease, in vulnerable populations,

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