Monoclonal Antibodies: The Once and Future Cure for Covid-19

Monoclonal Antibodies: The Once and Future Cure for Covid - 19

William A. Haseltine PhD and Griffin McCombs

Copyright © 2023 by William A.

Haseltine, PhD

Cover art by Kim Hazel

All rights reserved. No part of this book may be used or reproduced by any means, graphic, electronic, or mechanical, including photocopying, recording, taping, or by any information storage retrieval system, without the written permission of the publisher except in the case of brief quotations embodied in critical articles and reviews.

All author proceeds from the sale of this book will be donated to the nonprofit global think tank ACCESS Health International.

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Welcome to Monoclonal Antibodies: The Once and Future Cure forCovid-19!

Here, we investigate many of the questions raised by the discovery, fall, and resurrection of monoclonal antibodies for Covid-19. These questions are of vital importance to understanding the future of the pandemic. The stories included here were written in the heat of the moment as the news of antibody treatments emerged throughout the pandemic. As such, these stories should be considered to be snapshots in time of what we knew and when we knew it. Each story is followed by a link to the original publication, which may include more detailed figures. For the latest information about Covid-19 and new variants, please visit www.accessh.org/covid-19/

The format of this book is something that I have dubbed a living ebook a format suitable for a rapidly evolving pandemic such as COVID-19. I will continue to update Monoclonal Antibodies: The Once and Future Cure for Covid-19 as we learn more. You may download these updates at no additional cost by visiting www.williamhaseltine.com/antibody Password: antibody

Thank you for your interest.

Foreword

It was late 2020. The President was deathly ill. For a few days, amid a pandemic, he had severe Covid symptoms and refused to admit them. At long last, it became clear that he would shortly expire unless he was treated immediately. He was helicoptered with haste to Walter Reed Hospital for emergency treatment.

Within days, he had returned to the White House, spotted inside the presidential limousine waving to the crowds. What happened to cause such a remarkable turnaround? He was treated with a combination of monoclonal antibodies directed against a virus and another antiviral to quell virus production.

What were these drugs responsible for such rapid improvement? Where did they come from? Why did they work? More importantly, why have they recently stopped working? Will a time come when drugs of this sort work again?

This book answers these questions. Following the introduction, this book chronicles the rise, implementation, fall, and rebirth of monoclonal antibodies for treating Covid-19 in real-time. Each entry is accompanied by the original publication date, providing a unique lens through which to observe monoclonal antibodies throughout the Covid pandemic.

As the book approaches the present day, we outline why we believe the future for monoclonal antibodies is bright, not only for Covid19 but also for other infectious diseases.

The following introduction briefly describes monoclonal antibodies: how they work, why they failed, and how they could make their resurgence. Then we will embark on our journey through time.

Introduction

What isanAntibody?

Antibodies are small proteins crucial to our immune system’s defense against invading pathogens. Their primary role is identifying and neutralizing foreign objections and protecting the host’s cells and vital organs from infection and disease. Each antibody features a hand and glove fit, a definite shape of atoms to lock onto the pathogen it is designed to counteract, down to the resolution of one-tenth of an atomic radius. Billions of antibodies are continuously produced within human and nonhuman hosts, protecting us from common diseases such as the cold or deadly pathogens such as Covid-19.

NaturalImmunity

Most vaccines work by inducing long-lived antibodies and memory. Upon infection, there is an immediate reaction to the virus, the innate immune response, which mobilizes intracellular defenses before the development of antibodies. These defenses hold off the virus as well as they can until the immune system can initiate antibody development.

Source:ACCESSHealthInternational

Upon contact with a pathogen, the immune system sends macrophages and dendritic cells to capture and deconstruct the pathogen, presenting the invader to B cell lymphocytes. The B cell lymphocyte then carves a unique key on a blank antibody that locks solely to the captured pathogen. Therefore, every antibody is specifically tailored to the captured pathogen for which it was created. This is known as somatic hypermutation and is the bedrock of the adaptive immune response. The encoded antibody is cloned

Figure 1. Timelineshowingtherise andfalloftheinnateandadaptive immuneresponses.

millions of times and released into the bloodstream to defend against a repeat invader. However, these antibodies fade after about a year, but lasting memory allows for long-term protection against the worst severe progression, explaining why those who have received the vaccine are more protected long-term than those who do not. Were that same pathogen to return, the memory B cells with the imprinted key would produce a wave of protective antibodies that would lock onto the pathogen's surface and either disable it themselves or signal other immune cells to come to neutralize the invader.

Monoclonal antibody treatments are a way of loading up the body system with the most effective antibodies that exist, in the most ideal case, to prevent and treat the viral disease.

Structure

Roughly 10nm long, an antibody presents in a ‘Y’ shape, consisting of four polypeptide chains. The four chains consist of two identical light chains and two identical heavy chains.

Source:ACCESSHealthInternational

The two light chains consist of a variable and constant domain, whereas the heavy chains consist of one variable and three constant domains. The chains are held together by a series of disulfide bonds.

The key region of the antibody that locks to the pathogen is known as an antigen-binding fragment. These are the two arms of the ‘Y’ shape consisting of the light chains and the variable and one constant domain of the heavy chains. The B cell lymphocyte encodes the variable domains with the antigen-binding site to the pathogen.

Figure 2. SchematicdesignofanImmunoglobulin(IgG)

The trunk of the ‘Y’ shape is the Fc region, comprised of the other two constant domains of the heavy chains. This region enables effector molecule binding and half-life modification, which we will discuss later. While most antibodies we discuss in the ‘Y’ configuration are immunoglobulin G (IgG), there are also IgE and IgD antibodies in a similar configuration. Additionally, IgM and IgA are present in their unique shapes.

Source:ACCESSHealthInternational

Figure 3.Thefivetypes ofantibodiesareIgA,IgD,IgE,IgG,andIgM.

The germline characteristics of antibodies are inherited, selected over many millions of years to approximate pathogens we are likely to encounter. Before being matched to a specific pathogen, antibodies are close but imperfect matches to an invader, much like an off-the-rack suit compared to a tailored one. An antibody is tailored to that specific target only once a pathogen is introduced. Both off-the-rack and tailored antibodies are derived from B cells, which begin in the body’s lymph nodes. The lymphatic system involves the spleen, tonsils, etc. They may become inflamed during viral infection due to B cell activation. The outer portion of lymph nodes consists of groups of inactivated B cells called follicles. Once activated, these cells produce tailored antibodies to a specific antigen.

The off-the-rack antibodies are extrafollicular and can recognize pathogens, but to a lesser extent, creating the first line of defense. Aggressive symptoms such as cytokine storms or thyroid complications may arise due to extrafollicular activation upon pathogen entry. Microorganisms will not activate all extrafollicular antibodies, only the ones that approximate, akin to a smaller man only pulling smaller suits off the rack. However, it seems some microorganisms use the approximation to their advantage, only activating extrafollicular antibodies that are least harmful to themselves. The attacker is choosing which defense our body employs.

Our aim is to find antibodies that have been activated that cause the most damage to an invading pathogen, countering its effort to activate the least damaging. Different antibody types may be activated in some cases, such as IgM or IgA. In others, there may be

broadly neutralizing antibodies with a very low affinity that need scientific modification to optimize.

AntibodyVariability

From the off-the-rack antibodies, how does our body produce antibodies with such incredible specificity? Some estimates suggest a human produces roughly 10 billion distinct antibodies; all copied countless times. From the ancestral germ line cells that adapted over many years, the immune system uses several tricks and methods to re-engineer antibodies in various ways to create an army of pathogen-specific antibody soldiers.

Our immune systems have overcome this hurdle by evolving B cell antibody generation to include a swathe of complex processes such as V(D)J recombination.

V(D)J recombination is the removal and recombination of gene segments in antibodies to yield vast arrays of antibody combinations from a limited building supply. The variable domains of antibodies are comprised of gene segments categorized as variable (V), diversity (D), and joining (J). These gene segments can be rearranged significantly without altering the antigen-binding site. Take the alphabet, for example. In the English language, there are 26 letters from ‘A’ to ‘Z.’

ABCDEFGHIJKLMNOPQRSTUVWXYZ

V(D)J combination removes unnecessary clutter from an antibody blueprint. For instance, we may not need the letters ‘C’ through ‘F’ and ‘J’ through ‘P’ AB-GHI-QRSTUVWXYZ

This process takes a set number of inputs, the letters A to Z, and yields a countless number of outputs depending on the blueprint drawn from the pathogen in question.

The result is a broad range of antibodies that bind and neutralize a wide swathe of pathogens.

How to Make Monoclonal Antibodies

Monoclonal antibodies are tailor-fitted antibodies that can be harvested, cloned, and produced in bulk by pharmaceutical companies for the purpose of antiviral treatment and prevention. We can take the tailor-fitted antibody response our bodies create against varying pathogens, identify the most effective antibodies at neutralizing those pathogens, and create a drug based on that antibody.

Monoclonal antibodies are among our greatest assets in preventing and treating moderate to severe cases of Covid-19. As new variants of SARS-CoV-2 arise, new monoclonal antibody candidates must be discovered and examined to overcome the more immune-evasive virus versions. Here we dive deeper into the discovery process and describe how our antibody drugs are isolated and examined.

InfectedHumanSeraIsolation

Throughout the pandemic, most monoclonal antibodies have been discovered by analyzing the blood samples of previously infected Covid-19 patients. When we are infected, our immune systems naturally develop antibodies against an invading pathogen to prevent the same pathogen from infecting us in the future. However, the antibodies we develop are variable, slightly differing in binding epitope, neutralization effectiveness, etc.

Sera samples are extracted from previously infected patients, and the variable SARS-CoV-2 antibodies are located and isolated for further examination. This allows for precise data on individual antibodies’ neutralization capacity, binding affinity, etc.

The hundreds, if not thousands, of antibodies are typically sorted by binding affinity, meaning the strength to which antibodies latch onto the virus target. Those that bind poorly or not at all are discarded from contention.

Next, antibody candidates are analyzed for neutralization capacity. In the case of SARS-CoV-2, antibodies are often tested against the wild-type virus and the latest emerging variants of SARS-CoV-2. An antibody that neutralizes the Wuhan virus of 2020 may not neutralize the newest Omicron versions. Those with the most robust neutralizing capacity are then closely examined by cryo-electron microscopy to determine the exact makeup of the antibody and whether it should advance to further trial.Many of the FDAapproved antibodies in the pandemic have been found using this discovery method, though most lose neutralization of later SARSCoV-2 variants.

VaccinatedHumanSeraIsolation

Since Pfizer/BioNTech and Moderna's wide release of mRNA vaccines in the Spring and Summer of 2021, roughly 68% of the US population has been fully vaccinated with two doses. When a pathogen invades a patient, and the immune system reacts by creating antibodies tailored to the pathogen, a similar process occurs with vaccination. The Covid-19 mRNA vaccines are essentially a weakened form of the SARS-CoV-2 spike protein, the portion of the virus that binds to host cells. The vaccine teaches our immune

system to fight against the virus in a lower-stakes setting than an infection.

Vaccinated human sera isolation is the same process as infected human sera isolation, though the target patient is those vaccinated instead of infected. While less common, this method yields similar results to the first method. However, antibodies isolated from vaccinated sera may be less effective against later SARS-CoV-2 variants, as the vaccines were designed using the wild-type spike protein.

AnimalSeraIsolation

The third method for antibody isolation comes not from human patients, but from animals. Animal sera isolation follows the same steps as infected human sera isolation but in nonhuman hosts such as mice, hamsters, camelids, and monkeys.

There are two main benefits of animal sera isolation for antibody discovery. The first, more practical benefit is the increased flexibility of animal subjects in laboratory settings. Humans have variable schedules, lifestyles, etc.; relying on human subjects for experimentation and scientific pursuits can be more taxing than necessary. Animal subjects, however, are highly controlled, both in their availability for experimentation and lifestyles outside of the lab, as their health is closely and easily monitored. Many animals, such as mice, hamsters, camelids, and monkeys, have similar immune systems to humans and can be substituted for human subjects.

Modified germline sequencing is the second, more theoretical benefit of animal sera isolation. Throughout the pandemic, the SARS-CoV-2 virus has developed mutations to evade and overcome our immune defenses, fueling the search for new monoclonal

treatments to which the virus may evolve to overcome once again. This game of cat and mouse may be avoided using animal sera isolation. The viruses circulating today have been selected against human immune responses, not animals.

For instance, a recently isolated antibody from macaque monkeys displays an inherited germline sequence not found in antibodies developed from human hosts. The core sequential structure of the antibody is different than human antibodies. This may be the key to enabling isolated antibodies to overcome rapidly mutating SARSCoV-2 variants.

PhageDisplay

The final popular method of antibody discovery is phage display. This technique involves encoding proteins on a phage coat gene, which displays the protein outside the gene. The protein can then be introduced to various other molecules to detect interactions. In simpler terms, phage display can show which proteins interact with which molecules in vitro in a very concise and organized manner.

Figure 4.Phagedisplaycycle.1)fusionproteinsfora viralcoatprotein + thegene to beevolved(typicallyanantibodyfragment)areexpressedin bacteriophage.2)thelibraryofphageis washedover an immobilized target.3)theremaininghigh-affinitybindersare usedto infectbacteria.

4)thegenesencodingthehigh-affinitybindersare isolated.5)those genesmay haverandommutations introducedandusedtoperform anotherroundofevolution.Theselectionandamplificationsteps can be performedmultipletimes atgreaterstringency to isolatehigher-affinity binders.

Source:WikipediaPublicDomain

In the early 1990s, researchers at the Scripps Research Institute reported the first use of phage display to detect and isolate human antibodies which bound tetanus toxin. Since then, laboratories and pharmaceutical companies worldwide have used phage libraries containing millions of antibodies to isolate monoclonal antibodies for human use.

CamelidNanobodies

One particular antibody-deriving animal group of note is the camelid. Camelids consist of camels, llamas, alpacas, and other members of the Camelidae family. Single-domain antibodies, or nanobodies, are fragments of a sole variable antibody domain. The first nanobodies were engineered from camelid heavy chain antibodies. They contain a single variable domain in the arms of the ‘Y’ shape and no light chain.

The main therapeutic advantage of camelid antibodies is that they are more easily administered in drug form via inhalation. Most antibody therapies require at least an intramuscular shot or, more likely, an intravenous administration, which mandates a professional to administer. They are also often as, or more specific

than, regular antibodies, creating an opportunity for a highly effective treatment.

Researchers Koenig et al. isolated a panel of nanobodies from alpacas immunized with wild-type SARS-CoV-2 spike protein. Four nanobodies effectively bound and neutralized live SARS-CoV-2: E, U, V, and W. Two of these, E and V, worked well together to lock the receptor-binding domain. This region of the spike shifts between the down and up positions based on the stage of infection. By locking the receptor-binding domain in the “up” position, the virus cannot release from the host cell upon ACE2 binding, neutralizing it by preventing further spread.

In conjunction, the V and E nanobodies display a 62-fold increase in neutralization and a 22-fold increase in binding compared to the nanobodies in isolation. This trend could also be duplicated in other pathogens, as nanobodies are more effective than their IgG counterparts. Their smaller stature could lead to a more densely packed drug in the latter stages of development. More nanobodies per mL could lead to more effective moderate to severe disease treatment.

AntibodyModification

BispecificMonoclonalAntibodies

Bispecific antibodies have two binding sites directed at two antigens or two different epitopes on the same antigen. In baseball, most pitchers are only pitchers, but some are two-way players, capable of pitching and hitting, making them incredibly valuable. Bispecific antibodies are equally as valuable but uncommon. They are more commonly produced in a lab than isolated naturally. Despite their

difficulties, the clinical therapeutic effects of bispecific antibodies are superior to those of monoclonal antibodies.

Immunotoxins

Immunotoxins are cytolytic fusion proteins developed for cancer therapy, composed of an antibody fragment that binds to a cancer cell and a protein toxin fragment that kills the cell. Immunotoxins are a relatively novel but effective treatment in the oncology field, potentially filling a large void for accessible drugs. Two such drugs approved or in development are Lumoxiti for hairy cell leukemia, approved in the United States in 2018, and Oportuzumab monatox for bladder cancer, pending review for approval in the US.

Intrabodies

An intrabody is an antibody that works within the cell to bind to an intracellular protein. Due to the lack of a reliable mechanism for bringing antibodies into a living cell from the extracellular environment, these antibodies are modified for intracellular localization. Since 2007, intrabodies have been developed for several therapeutic applications, including hepatitis B, bird flu, prion disease, inflammation, Parkinson’s, and Huntington’s.

ArtificialIntelligence

Antibodies, while highly effective and relatively inexpensive compared to some treatments, require a time-intensive development process. Much of the time spent developing antibodies is finding ones that work. Our bodies produce seemingly countless antibodies in reaction to infection with a pathogen. When infected with something new, our immune systems go into overdrive, producing waves of new antibodies from B cells.

The antibody researcher is tasked with isolating highly effective antibodies against a given pathogen from the sera of an infected or vaccinated patient. Many of the antibodies analyzed will have little to no effect, but some could be potently neutralizing. The trick is isolating the winners. While human researchers could take days, weeks, or even months to test a large panel of antibody candidates, what if there were an artificially intelligent program to do the work in a fraction of the time?

In recent months, new and exciting advances in artificial intelligence biotechnology may pave the way for antibody discovery in the near future. Designed by researchers Parkinson etal.from the University of California - San Diego, the new AI mechanism, RESP, finds and tests antibody binding at a highly accelerated rate.

Antibody discovery typically means isolating antibodies from the convalescent sera of a patient, testing a panel of the isolated antibodies against the pathogen, then focusing on those that produce the most robust binding and neutralization. RESP does this process at a much faster rate and more.

A particularly compelling aspect of RESP is its testing of acutely mutated antibodies. It takes the antibodies isolated from sera, tests those antibodies against the pathogen, then tests the same antibodies with slightly mutated structures to see if improvements can be made to the isolated antibodies. The antibody is fine-tuned by slightly modifying the structure to be as highly binding and neutralizing as possible.

For instance, RESP found a slightly mutated version of the approved cancer immunotherapy Atezolizumab that displayed a 17fold increase in binding affinity. RESP can be used for all sorts of

pathogens, Covid-19 notwithstanding. There is only one approved monoclonal antibody drug for Covid-19: Tocilizumab. This drug yields a modest 4-8% reduction in death compared to a placebo control in clinical studies.

If RESP can increase the effectiveness of the cancer therapy

Atezolizumab by 17-fold, it is not out of the question to assume similar results can be seen with Covid-19 drugs. An anti-Covid monoclonal treatment with a 68+% reduction in death rate sounds much more appealing than 4-8%. That is what makes RESP and AI antibody identification all the more special. It can improve the drugs we already produce.

If we can harness the full potential of RESP or an AI mechanism similar, it could lead to a new wave of highly effective antiviral treatments and prophylactics.

AntibodyOptimization

A critical issue with monoclonal antibodies is that they fade. Similar to how protection from severe disease fades over time with most vaccines, monoclonal antibody treatments are not permanent shields against their given pathogen. Researchers have recently developed a few methods to extend and optimize monoclonal antibodies.

FcRegions

As described in a previous section, the Fc region is the trunk of an antibody's “Y” shape. The Fc binds the Fc receptor on an effector cell. When an antibody binds a pathogen, it either neutralizes it directly or signals effector cells to neutralize it. Effector cells could be B lymphocytes, macrophages, killer cells, etc. Modifying the Fc

interaction of monoclonal antibodies may yield an increased halflife for therapeutic and prophylactic use.

Source:WikipediaPublicDomain

Extended Half-Life

Using structure-guided design, researchers from Visterra Inc. extended the half-life of monoclonal antibodies over nine-fold in transgenic mice models. They observed Fc binding interactions between the Fc region and effector cell Fc receptor that mutational alterations could enhance.

Figure 5. Schematicrepresentation ofEffectorcellFcreception.

They engineered a panel of unique Fc variants that could enhance Fc binding while maintaining a threshold of binding affinity and overall structural integrity. Their panel yielded several mutational sets in the Fc region that increased the half-life of monoclonal antibodies in a mouse mode. The most prominent were YTE (M252Y/S254T/T256E) and LS (M428L/N434S).

Including these mutations in monoclonal antibodies used for therapeutic use may extend their half-life by weeks, which could be crucial in critical cases of Covid-19 and Long Covid patients.

FcClassSwitching

Antibody class is determined by the heavy chain constant region. Antibodies may switch classes if the variable region is maintained and the constant region is modified. In an irreversible Fc class switching process, various enzymes create nicks in the DNA sequence at switch regions, allowing the constants to be excised. A repair enzyme joins the remaining variable segment onto a new constant region.

The differing antibody classes, IgG, IgM, and IgA, often have variable affinities and neutralizing capabilities. In many cases, IgG antibodies neutralize invading pathogens, though IgM antibodies may yield a stronger response in some cases, such as with Zika Virus.

Recent studies even indicate that IgG3 antibodies, which have a longer Fc fragment than IgG1, more efficiently activates Fc-related immune functions, leading to stronger recovery and protection for those infected with SARS-CoV-2.

EffectorFunctions

An antibody has four main methods by which antibodies exert their effects, known as effector functions: neutralization, opsonization, complement activation, and antibody-dependent cellular cytotoxicity (ADCC).

Neutralization, as we have discussed, is the process of stopping a pathogen from infecting healthy host cells.

Opsonization is a process by which the Fc region of antibodies is recognized by phagocytic cells. Instead of the antibody directly neutralizing a pathogen, it latches onto the pathogen and signals phagocytic cells to neutralize and clear the invader.

ADCC occurs when the Fc portion of an antibody bound to a cell's surface interacts with the Fc receptor on immune cells such as macrophages, NK cells, and neutrophils. This triggers the immune cell to target the antibody-coated cell for lysis.

Complement is a system of plasma proteins activated by antibodies bound on a cell. The complement cascade leads to the formation of a protein complex in bacteria that can kill the bacteria. The complement system can also activate phagocytes to destroy bacteria that would otherwise not be recognized by the immune system.

FcsthatBindComplement

In addition to binding Fc receptors on effector cells, Fc regions also bind complement proteins. The complement system is a segment of the innate immune system that enhances antibody clearance of microbes and damaged cells from the host. Complement activation promotes inflammation and pathogen cell membrane targeting.

The system is a series of fragments and subunits signaled by a bound antibody. These fragments and subunits are also called the complement cascade. Once signaled, the initial C1 complex fragments and fragments until small subunits of the initial complex recombine into the cylindrical membrane attack complex, which infiltrates and destroys the infected cell.

Source:WikipediaPublicDomain

Figure 6. Schematicrepresentation ofcomplement cascadesystem.

Complement activation is not nearly as common as Fc receptor binding, as the complement system can damage host tissue and present additional negative health effects. However, its availability is a valuable tool for the antibody.

Sugar(Glycosylation)Modifications

Another antibody optimization method involves glycosylation. In the molecules of most microorganisms, there are chain-like structures composed of single sugar molecules linked together by chemical bonds. These are glycans, which are involved in the structural integrity of a molecule, as well as energy storage and systemic regulatory processes. Glycosylation is the process of covalent attachment between the glycan and the targeted macromolecule.

One macromolecule that attracts these glycans is antibodies, specifically the Fc portion of the antibody. All antibodies, including IgG, IgM, IgD, IgE, and IgA, bear N-linked glycosylation sites in their Fc heavy chain, each with varying levels of complexity due to differing Fc structures.

Recent advances in microbiological technologies have enabled us to look closely at antibody glycosylation. Firstly, it seems antibody glycosylation acts as a biomarker for disease severity. Antibody glycosylation is directly correlated to autoimmune and chronic disease severity, most notably high inflammation.

Secondly, and more directly, antibody glycosylation improves the immune response to pathogen infection. In both HIV and tuberculosis cases, patients with higher levels of glycosylated antibodies in their immune responses yielded stronger neutralizing

reactions. Harnessing glycosylation in monoclonal antibody treatments could prove fruitful.

SARS-CoV-2 Monoclonal Antibodies

ApprovedAntibodyTreatments

While antibodies represent our strongest tools against the virus, we lack approved and effective antibodies against the latest variants. Because the virus constantly mutates to evade antibody neutralization, treatments that may have worked against earlier versions of the virus are greatly reduced against more recent variants. As of May 2023, there are only two FDA-authorized monoclonal antibody treatments Evusheld and Tocilizumab, which is fully approved for use in hospitalized adults.

Evusheld is the combination therapy of the Tixagevimab and Cilgavimab antibodies. It was first isolated from Covid-19 patients infected in 2020, likely with the D614G SARS-CoV-2 virus. While the emergency use authorization still stands, it was revised in early 2023 to limit its use to when the “combined frequency of nonsusceptible SARS-CoV-2 variants nationally is less than or equal to 90%.” If later virus variants comprise more than 90% of infections, Evusheld cannot be used, rendering the treatment obsolete.

Tocilizumab, while approved, only modestly improves severe Covid outcomes. In the clinical studies that led to the drug’s FDA approval, cohorts who received Tocilizumab had reduced mortality rates of 4-8%.

Drugsagainst

Source:ACCESSHealthInternational

BroadlyNeutralizingMonoclonalAntibodies

While currently available treatments are somewhat limited, several monoclonal antibody candidate drugs are in development, many of which are effective against Omicron. Those in development are focused on broad neutralization by targeting conserved regions of the spike protein. Many older monoclonals target the virus at positions mutated in later strains, whereas newer antibody candidates specifically target spike residues that are conserved across many viral variants.

For instance, a number of broadly neutralizing monoclonal antibodies currently in development target portions of the S2 subunit of the spike. Mutations that occur in later variants of SARSCoV-2 are far more common in the receptor-binding and Nterminal domains of the S1 subunit. By targeting S2, antibodies are more likely to neutralize a wider range of variants. A notable example target is the fusion peptide, which plays a crucial role in membrane fusion post-contact with the host ACE2 receptor. An antibody may impede the fusion peptide, therefore impeding fusion and halting infection via a different avenue.

Figure 7.Efficacy(ng/mL)ofMonoclonalAntibodiesandAntiviral

OmicronSubvariantsin Vitro.

A particularly exciting antibody in development is AZD3152, a combination antibody therapy by AstraZeneca. A press release by the company claims the cocktail neutralizes all variants of SARSCoV-2, combining a new antibody candidate with cilgavimab, a crucial component of the successful Evusheld treatment. We eagerly await new details about this exciting drug.

These antibodies will be discussed in great detail in later sections of this book. Broadly neutralizing antibodies represent one of the better tools we could forge against the virus. Combining two or three broadly neutralizing monoclonal antibodies into a single treatment could be a particularly effective and perhaps even post-exposure prophylactic. We need only to continue their development in the coming months and years.

AlternateCovidAntibodies

Another effective strategy for Covid neutralization is cross-trimer binding. The SARS-CoV-2 spike protein attacks a host cell in sets of three, or a trimer. While most antibodies bind the epitope of a single spike monomer, a select few bind across the spike trimer, locking the entire mechanism and preventing infection. A notable example are the camelid nanobodies described by Koenig et al. early in the pandemic. This class of antibody often demonstrates a strong neutralization capacity thanks, in large part, to the cross-trimer strategy.

Further removed from traditional antibodies, a number of recent antibody candidates have targeted aspects of the human immune system. One such example is the anti-ACE2 described by scientists at Rockefeller and Stanford Universities. The antibody, h11b11, targets the host cell ACE2 receptor, blocking the virus from binding

and infecting the cell. Another example is the T cell antibody

Foralumab developed by Tiziana Life Sciences. Foralumab modulates immune reactions brought about by infection, reducing disease severity. The concern with antibodies that bind host cells is the possibility of autoimmune reactions, but those have not occurred in these cases.

MonoclonalAntibodiesforNon-CovidPathogens

A positive dividend from the emergence of SARS-CoV-2 monoclonal antibody discovery is the reinvigorated effort to develop antibodies for other problematic pathogens as well. In just the past few years, highly effective antibody candidates have been developed for some of the world’s deadliest diseases, including Ebola, Lassa Virus, Malaria, Zika, and Yellow Fever, among others.

Many of these pathogens are disproportionately active in low income nations in tropical regions and Sub-Saharan Africa. As they become available for public use, it will be critical to ensure the populations that need these interventions receive them, regardless of potential costs.

EconomicsofAntibodies

One of the major barriers to the widespread use of monoclonal antibody treatments is the cost. Throughout the pandemic, the United States government footed the bill for millions of vaccine doses and some treatments for specfic populations, shielding the patient from that out-of-pocket expense. With monoclonal antibody treatments, however, the cost to use the drug would fall on the patient and their insurer, particularly with the end of public health emergency declarations globally. Bebtelovimab, an monoclonal

antibody that was previously used to treat Covid-19 but is now ineffective against Omicron, costs an eye-watering $2,100 per dose. Almost six in ten Americans do not have $1,000 to spare in case of an unexpected medical bill, much less $2,100 per dose. If a family of four sought treatment with Bebtelovimab, they would need $8,400, pending insurance contributions. That is over 10% of the median household income. Meanwhile, most antibodies only cost between $100-200 to produce per gram. Granted, this does not include the cost of research and development, but the profit margin per dose of monoclonal antibody treatment is significant. The cost could be brought down spectacularly with sufficient government intervention and regulation. Whether that occurs remains to be seen.

AZD3152:ANewHopeOnTheHorizon

A new Covid-19 antibody may be the renewed hope for SARS-CoV2 monoclonal therapies we desperately need in the ongoing Omicron era. For the past three years, emerging variants of SARSCoV-2 mutated to overcome neutralization by monoclonal antibodies. As new treatments were authorized by the FDA, new variants would circulate that evaded these treatments. In due course, antibody treatments lost the luster they once demonstrated.

At the time of writing, only Evusheld (tixagevimab + cilgavimab) and Actemra (tocilizumab) and authorized for use against Covid19. Over the past several months, we have written about the potential of many antibody candidates in development. Many of these antibodies target conserved regions of the SARS-CoV-2 spike protein that are less likely to mutate, while others may have different

targets than the virus altogether, such as the anti-ACE2 monoclonal antibody.

Recently, a new antibody candidate has entered the ring: AstraZeneca’s AZD3152. While data on the antibody is limited and efficacy trials are ongoing, AZD3152 shows as much promise as any potential treatment we have discussed.

Structurally, AZD3152 is similar to many of the antibodies that have come and gone over the past three years. It is an IgG1 antibody, the most common of the four IgG subtypes. AZD3152 even has a duplicate constant region of that found in Evusheld, with only the Fab fragment antigen-binding domain replaced.

AZD3152 also does not bind an uncommon epitope. Whereas some recent antibodies we described target the S2 subunit or the fusion peptide, AZD3152 binds the often-targeted receptor-binding domain. The vast majority of the epitope is highly conserved, more than 99.9% of the time in observed sequences, but other binding residues mutate as much as 50% of the time.

However, what AZD3152 lacks in creativity, it makes up for in neutralizing capacity. Unlike any antibody we have observed, AZD3152 potently neutralizes all known variants of SARS-CoV-2, both dormant and current. At 100 ng/mL, every variant from D614G at the start of the pandemic to the many iterations of Omicron is neutralized by greater than 80%. At 1,000 ng/mL, that percentage jumps to over 95%.

We would have liked to analyze this antibody in far greater detail, but preprint or published research on AZD3152 is unavailable, as of yet. The above data was derived from a poster first displayed at

the 33rd European Congress of Clinical Microbiology & Infectious Diseases in Copenhagen in mid-April.

AstraZeneca has communicated that efficacy trials are expected to be completed later this year, and early indications are that the drug will be highly successful. We eagerly anticipate any further news on this exciting treatment, as it could be the silver bullet we so desperately need to protect and treat at-risk populations from a disease that is very much still thriving.

MovingForward

There is a genuine opportunity with monoclonal antibodies to create tools that will prevent Covid-19 from further devastating communities worldwide without disrupting our daily lives. Many thousands continue to die weekly as a direct result of and due to complications from Covid-19. Only by accelerating and consistently funding research into these monoclonal antibodies will we find our way out of the Covid-19 crisis once and for all. We shall see how the United States and other countries worldwide choose to proceed with monoclonal treatments. We recommend they be pursued with great haste.

PART ONE

April 23, 2020: Monoclonal Antibodies Could Help Fight Against Coronavirus

Agroup of Chinese scientists report the isolation of two human monoclonal antibodies with the potential to treat and to prevent SARS-CoV-2 infections, the causative agent of COVID-19. The work is described in a manuscript made available by Nature CellularandMolecularImmunology.

The two monoclonal antibodies block binding of the virus to the receptor preventing entry.

Such antibodies hold great promise for treatment as they are expected to prevent the virus spreading from cell to cell. The antibodies are also likely to protect those exposed, especially healthcare workers, from infection at least for a limited time. In both cases, the drugs would be administered intravenously.

No results demonstrating the activity of these potential drugs in animals or humans are reported. Nonetheless, previous experience with antivirus monoclonal antibodies of this type have been shown to be effective in treatment of both human and animal infections by viruses and other microorganisms. Monoclonal antibodies are among the most successful class of new drugs. Methods for manufacture and testing are well established. The FDA has approved more than 70 monoclonal antibodies for the treatment of human disease, including one for the treatment of respiratory syncytial virus a major cause of lower respiratory disease in children.

Several key questions remain open:

How soon will drugs be available? My guess, within three to four months for the first approvals.

 It may take several weeks to demonstrate effectiveness in animals.

 Human safety studies can be done with no more than 30 people in about a month. These drugs are generally known to be safe though more testing for safety will be required.

 Tests of the drugs’ effect in reducing the viral load in infected patients should take no longer than a month.

 Most time consuming will be large scale manufacturing of the drugs, once approved.

Some of these steps are undoubtedly already in process at this moment. China has the necessary skills and capacity to complete the research and testing and to manufacture at scale, as do many other countries. I know whereof I speak as I developed a similar drug for the treatment of and protection from anthrax infection and a monoclonal antibody for the treatment of Lupus.

Will the drugs work once patients become critically ill? The damage may already be too great to reverse. It is highly likely that the earlier the drug is administered post infection, the better.

If given to healthcare workers to protect them from infection, how long will the protection last? Best estimates for first generation antibody drugs is from 3 to 6 weeks. Follow on prophylactic treatments will be needed. Subsequent preparations may provide protection for up to four months.

There are several caveats.

 Coronaviruses, specifically those closely related to the SARS virus, are known to mutate to escape monoclonal antibody neutralization. The drugs may work for a while before the virus develops immunity. One way to counteract virus escape is to treat with two or more antibodies simultaneously.

 The drugs are relatively expensive to manufacture. The first generation must be grown in cell culture. It should be possible to develop future generations of drugs that act similarly but are far less expensive to produce.

 The antibodies were isolated from COVID-19 convalescent blood. Of the 26 convalescent patients studied, only three had the potential to block binding of the virus to its receptor, the ACE2 surface protein. Cells that produce antibodies were isolated from these three patients.

 The genes that produce the antibodies were isolated from these cells and used to create the two new drug candidates.

The work was completed by a team of scientists working in four different cities from across the length and breadth of China. While still in its early phases, the discovery is significant. There has been much talk in recent weeks about the use of convalescent serum and hyperimmune globulins essentially, collecting blood from patients who have recovered from COVID-19 and giving the plasma to people who are sick. This discovery offers us the hope of a purer and potentially safer form of this type of treatment.

This article originally appeared in Forbes and is available online here: Monoclonal Antibodies Could Help Fight Against Coronavirus

June 23, 2020: Progress In Mo noclonal Antibodies For The Treatment And Prevent - Of - COVID - 19

Ateam of scientists recently announced progress in the discovery of a pair of monoclonal antibodies that may be useful for the prevention and treatment of COVID-19. The work was described in the June 12th issue of Science Magazine.

The scientists began the process by isolating live antibodyproducing cells from the blood of convalescent patients. They used a key fragment of the virus spike protein as bait to fish for cells that make the antibodies of interest. The bait fragment is the precise region of the spike protein that attaches to the ACE2 surface structure to begin the process of infection. The idea behind their choice was that antibodies that block the attachment of the virus to the cell will prevent infection altogether. Their fishing expedition was successful. They settled on four antibody-producing cells that met their criteria.

The next step was to isolate the genes (the bits of DNA in the cell) that specify each of the antibodies. Again, they were successful.

The next challenge was to develop each as a potential drug. To do so, they needed to insert the genes into a cell suitable for large scale production of the antibodies. This they also did.

The first test of the four antibodies was to determine whether the antibodies attached to the SARS-CoV-2 spike protein as expected.

All passed this test. The virus that causes SARS-CoV-1 also uses the ACE2 protein as a receptor but none of the four antibodies attached to the SARS-1 spike protein.

The next question the researchers needed to answer was whether the antibodies blocked binding of the spike protein to the ACE2 receptor. Two of the four antibodies did and the other two did not. The team then focused their attention on the two ACE2-blocking antibodies.

Did both blocking antibodies bind to the same part of the spike protein? If they did, binding of one should interfere with binding by the other. What they found was the two antibodies did interfere with one another but only weakly, suggesting that they attach to overlapping but not identical regions of the spike.

To understand exactly how the antibodies block binding to the ACE2 receptor, the atom-to-atom contacts of one of the antibodies to the spike protein was determined by X-ray crystallography of the antibody-spike protein complex. The result: the antibody studied binds to 18 of the 21 ACE2 attachment sites. In other words, when the antibody binds to the spike protein it almost completely covers the ACE2 attachment site.

The next obvious question is, do the antibodies prevent infection? The hope is that if they do, two together should be more powerful than one alone. The experiments fulfilled that hope. Each of the antibodies on its own can neutralize the virus. The two together are more effective than either alone.

The final experiment was to determine if the antibody acted against SARS-CoV-2 in an infected animal. They chose mice for convenience even though SARS-CoV-2 does not usually kill those

animals. The antibodies were introduced in the mice 12 hours after the viral challenge. The experiment showed that the antibody treatment reduced the amount of virus in the animals by 32.8% for one of the antibodies and 26% for the other, when compared to a control group three days post-infection. The animals treated with each antibody also had fewer lung lesions than the placebo control animals.

A next logical question, do the antibodies prevent infection of naive animals if not reported? It is likely that they will. Monoclonal antibodies used to treat respiratory syncytial virus protect very young children from infection. A monoclonal antibody that treats anthrax infections also prevents infection.

The work is a tour de force not only for the clarity and completeness of the results but also for the speed with which all the many steps were accomplished. The work reveals the stepwise question and answer process of successful scientific experiments each performed in a clear logical progression. The work also attests to the power of modern bioscience as each of the steps is, in itself, demanding technical exercise.

Apart from elegant science, the work offers hope that combinations of monoclonal antibodies can be used to treat and to prevent COVID-19. As an aside, combinations of antiviral drugs often work better than drugs used alone as coronaviruses, like many other viruses, are known to develop resistance to single drugs.

As a treatment, it is likely that combinations of monoclonal antibodies can prevent those infected from becoming ill, and if used early enough, may prevent those who are seriously ill from dying.

Used as prophylactics, such antibodies may protect healthcare workers. They may also be used to prevent those exposed at home or in the workplace, and perhaps even those identified as exposed by contract tracing. One happy way to describe such a success is that monoclonal antibodies may be the equivalent of a short-acting vaccine.

For now, all such good news must await human trials. The experiments described here demonstrate that success is in reach, not only for the effort of this group, but for many others who are on the same trail.

This article originally appeared in Forbes and is available online here:Progress In Monoclonal Antibodies For The Treatment And Prevention OfCOVID-19

October 28, 2020: Eli Lilly Stops

Antibody Trial In Hospitalized Covid - 19 Patients

How do we understand the decision of the National Institutes of Health (NIH) to halt the Eli Lilly antibody treatment trial? Is it a sign that monoclonal antibodies to SARS-CoV-2, the virus that causes Covid-19, are ineffective?

The Lilly trial combined two drugs designed to interfere with SARSCoV-2 infection: remdesivir, a drug that is intended to inhibit the viral RNA polymerase, and the Lilly drug, a monoclonal antibody that is meant to prevent the spread of the virus within an infected person. The trial participants were all hospitalized volunteers with serious complications from Covid-19. The intent of the trial was to speed up recovery and prevent further progression of the disease. I do not have information regarding the exact endpoints and whether or not they include prevention of progression, the need for intensive care, and death.

Why two drugs? The presumptive benefits of remdesivir make it a drug of choice for most patients with Covid-19. A recent WHO study of remdesivir conducted in many countries found that remdesivir had no effect on hospitalized patients neither with respect to progression nor more serious disease and death. This finding did not surprise me, as the effects of the drug even at their best are described as weak to marginal. Nonetheless, most

volunteers in a study of a new drug would rather receive remdesivir along with the new drug.

The NIH halted the trial because no effect on the hoped-for endpoints was observed. Patients on remdesivir fared as well (or as badly) as patients on the two-drug combination. In other words, the addition of the Lilly drug had no measurable effect. The preliminary report mentions no adverse events that occurred as a result of the two-drug combination.

Is this the end for the Lilly drug? Not at all. Drugs designed to stop virus replication should work, if they work at all, during the phase when the virus is most active. There is a phase of a few days to a week following infection when virus growth is very slow. This is followed by a second phase when virus growth is rapid and the concentration of virus particles in nasal fluids, the lung, and the intestine is very high. That is followed by a third phase in which the growth of the virus is typically contained and reduced to nothing or near nothing. Antiviral drugs should work during the incubation phase and the phase of rapid growth, but not after the virus growth is controlled.

Most people infected with SARS-CoV-2 do not experience symptoms until after the peak of virus replication. Even then the symptoms may be mild and not require hospitalization. It is only later, after the virus is no longer replicating, that the most serious symptoms appear those that require hospitalization. Treating hospitalized Covid-19 patients with antiviral drugs designed to impede a virus that is no longer replicating is the equivalent of the proverbial closing the barn door after the horse is gone.

When, then, should antiviral drugs be used? The answer is very early on in infection, or even before infection occurs. The obstacles that prevent us from using drugs early on in infection are related to how and who we test for infection. The people tested most frequently are those who have symptoms. By then the virus is already on the way out, and much of the damage the virus can do is already in progress. (There may be an exception for people who fail to make good interferon responses.)

There is a potential solution: frequent universal testing, whereby most people are tested every two to three days using tests that yield answers in 5 to 10 minutes. Such a testing regime, which I believe to be the surest way forward for contagion control, will identify those in the earliest stage of infection. Identification of an infected person should be followed by immediate treatment. Then and only then might antiviral drugs work, be they chemicals like remdesivir or monoclonal antibodies.

Antiviral drugs may also prevent infection, as is currently the case with HIV. Monoclonal antibodies are used to prevent respiratory syncytial virus (RSV) infection in infants. Antimalarial drugs are used to prevent malaria infections. The caveat is that such drugs must undergo rigorous evaluations for safety, for they are intended to be used on healthy people who are not yet infected. The safety profiles of treatments for the healthy are much more stringent than those required to treat the ill.

I suspect that Lilly will go on to test their drug in those reporting mild symptoms. The first issue of that trial design is that only some of those with mild symptoms will be detected early enough for the drug to have an effect. An even greater issue is that the great majority of those with early cold-like symptoms perhaps 80 percent or

more will recover from the cold and not progress to serious disease whether given a drug or not. Therefore, any possible therapeutic benefit will be diluted by the great majority of those that will not progress in any event. Such is the life of a drug developer.

The only way I see such trials as possible is the advent of very low cost, universally available rapid virus tests to identify those recently infected. Even then, the signal to noise ratio will be low.

In conclusion, this is not the end of the line for the Lilly drug, but it is the beginning of a very long and rough road for not just their drug, but any other Covid-19 antiviral.

This article originally appeared in Forbes and is available online here: Eli Lilly Stops Antibody Trial In Hospitalized Covid-19 Patients

November 3,

2020: What Are Autoantibodies? The Latest Risk Factor For Severe Covid - 19

We know that people who are older, obese, immunocompromised, pregnant, or diabetic are at greater risk of developing severe Covid-19. Now evidence has emerged that establishes another determinant of risk something that isn’t inherited, but acquired over a lifetime. That is the inability to mount a robust interferon response.

Interferons play a critical role in defending our bodies against invading pathogens like SARS-CoV-2, the coronavirus that causes Covid-19. They function as warning signals, alerting the immune system whenever an intruder is on sight. Some people, however, develop what are called autoantibodies against their own interferons and research shows that they’re more susceptible than most to the more devastating effects of Covid-19, including death.

If antibodies are the defenders our B cells produce to battle oncoming infection, autoantibodies are defectors that do precisely the opposite. Rather than detecting and debilitating viral genetic material, autoantibodies target us A new study, made available on medRxiv but currently undergoing peer review, examined 52 patients with severe Covid-19 and found that nearly half had autoantibodies of some kind. In those most critically ill specifically the top 50 percent that number exceeds 70 percent.

None of the patients who participated in the study reported a history of autoimmune disease, but it may be the case that survivors continue to be prone to severe Covid-19 symptoms upon future encounters with the virus potentially even worse than the first time around. The flipside is that they might benefit from drug therapies for conditions like lupus. Autoantibodies are more prevalent in older adults than younger, which might explain, at least in part, why Covid-19 is, too.

If the results of this study aren’t momentous enough to impact how we treat Covid-19, at the very least they have bearing on how we diagnose and prevent it. First thing’s first, people who develop serious Covid-19 symptoms, whether they’re hospitalized or not, should be tested for autoantibodies against interferons and other relevant targets. Those who test positive, now that we know they’re more vulnerable to critical illness or death, must receive priority status for vaccinations and be cared for accordingly.

More generally, people aged 65 and older should be routinely tested for anti-interferon antibodies as part of their annual checkup. Again, those who test positive will know to be especially cautious to protect themselves from infection. After all, regardless of whether the tide of the current pandemic is turned by widespread vaccination or any other deterrent, there’s no telling how long we’ll be dealing with Covid-19 in some form particularly those most vulnerable. Any precautions we can take to prevent further spread, we should, and with haste.

The last two articles in this series on fading natural immunity to Covid-19 and fading vaccine-mediated immunity to influenza feature research that puts into perspective the timeframe of infection, disease, and potential protection. For many, all of the

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

above will tend towards the short-term, which has manifold implications for our methods of preventing and vaccinating against Covid-19.

But in the months to come, more studies will be conducted that give us a clearer understanding of the long-term effects of this disease. These in turn will force us to reconsider our methods once more. The issue of autoantibodies is one to watch, as well as one we should act to address sooner rather than later. If you have autoantibodies against interferon, you may be in it for the long haul.

This article originally appeared in Forbes and is available online here: What Are Autoantibodies? The Latest Risk Factor For Severe Covid-19

Janua ry 26, 2021: Autoantibodies May Be A Driver Of Severe Covid - 19 Reactions

While vaccine distribution and President Biden’s inauguration occupied most of the media’s attention, January marked the peaks of hospitalization and deaths among Covid-19 patients in the United States. Over 130,000 were in the hospital with severe Covid19 symptoms in the past two weeks. Emerging evidence suggests one cause of severe Covid-19 reaction may be autoantibodies or antibodies that attack the body and not an invading pathogen. What do we know about autoantibodies, and how can our understanding of them inform Covid-19 research moving forward?

A recent study out of NYU analyzed the sera of 86 hospitalized Covid-19 patients. Researchers were on the hunt for autoantibodies, self-attacking proteins mistakenly turn on the body in their attempt to root out pathogens. It is unknown how many people have these antibodies present in their immune systems, though ongoing research is looking into the matter. The NYU researchers hoped their presence could provide some clarity on why some patients get so sick from SARS-CoV-2 infection.

They indeed found autoantibodies geared against the annexin A2 protein, which stabilizes cell membranes and blood vessels in the lungs. On average, hospitalized Covid-19 patients had a significantly higher level of these autoantibodies than non-severe patients, and their targeting of the lungs may be one of the causes for Covid-19 related respiratory issues.

Autoantibodies targeting interferon responses are noted by several studies as a significant factor in severe Covid-19. The interferons are warning signals that alert the immune system whenever a pathogen invades, which I’ve written about here. A study out of Science showed autoantibodies reducing interferon circulation in the blood to extremely low levels, meaning the pathogen could easily spread throughout the body without having to worry about the substantial immune response. Autoantibodies also seem more prevalent in older adults, which falls in line with who is more likely to have a severe Covid-19 reaction.

A major question remains unresolved. Are annexin A2 and interferon autoantibodies activated by Covid-19 infection, preexisting in the patient, and therefore predisposing them to infection? Or are they actually induced by the infection itself? That is an interesting question for these specific autoantibodies, but also for autoantibodies in general found in patients ill with Covid-19. To this point, the existence of autoantibodies in patients prior to arriving at the hospital is unreported.

Why do autoantibodies arise? Some are created when the immune system recognizes similar structures in the invading pathogens and human proteins. Some antibodies may be produced by a flood of cellular debris released upon pathogen destruction of cells. In the case of Covid-19, we don’t know the answer.

A leading theory for autoantibody development in reference to Covid-19 relates to disfunction in the follicular node when a patient becomes infected. The follicular node is the B cell’s learning center; it is where an antibody learns to neutralize a pathogen. In hospitalized patients, this node is often damaged. This may lead to a B cell less equipped to create virus-fighting antibodies properly

and may produce harmful autoantibodies that prompt severe symptoms.

While research doesn’t seem to lean one way or the other on why we have autoantibodies waiting to pounce, studies are in the midst of production to find the answers. Rockefeller University in New York was one of the first to observe autoantibodies in Covid-19 patients back in September and are currently screening as many as 40,000 patients to see how many had preexisting autoantibodies.

If autoantibodies are found to be a significant driver of severe Covid19 symptoms, pharmaceutical developers should be inclined to research further Covid-19 therapies geared towards them. Treatments that boost specific proteins in weakened immune systems may just be creating more targets for autoantibodies to attack. Testing for these autoantibodies could even be a strong indicator to identify at-risk people for severe Covid-19.

Furthering our understanding of how Covid-19 works and why some experience severe Covid-19 symptoms may lead to more effective treatments for patients. Even if an autoantibody therapy or test could save a few lives, the research is worth doing. Ultimately, autoantibody research may open doors for future understanding of this virus and how it affects us.

This article originally appeared in Forbes and is available online here: Autoantibodies May Be A Driver Of Severe Covid-19 Reactions

January 29, 2021: Eli Lilly’s Latest

C ombination Antibody Therapy Yields Strong Effectiveness Against Covid - 19

While Covid-19 infection rates have taken a downturn in recent days, hospitalizations and deaths are still at all-time highs. The need for effective therapies to treat severe Covid-19 symptoms has never been greater. Eli Lilly is one of the pharmaceutical companies taking on the task of developing these therapies, and a new antibody therapy trial released last week proved one combination therapy to be quite effective at reducing viral load in Covid-19 patients.

The study aimed to determine the effect of bamlanivimab (BAM) as an antibody monotherapy and bamlanivimab paired with etesevimab (ETE) as an antibody combination therapy to reduce the viral load for severe SARS-CoV-2 patients. 613 patients were given BAM monotherapy, BAM and ETE combination therapy, or a placebo treatment.

The BAM monotherapy yielded results only slightly better than the placebo group. The change in log viral load for the placebo group after 11 days was -3.80, whereas the change for a 700mg dose of BAM was -3.72 for 700 mg, -4.08 for 2800 mg, and -3.49 for 7000 mg. Across all dosages, it seems that the BAM monotherapy yielded around the same reduction in viral load as the placebo group did naturally over the time period.

Researchers additionally tested BAM in conjunction with ETE to see if the combination therapy would yield a greater result. It did just that. After 11 days, while again the placebo reduction in viral load was -3.80, the combination therapy reduction was -4.37. According to the researchers, this was a statistically significant result, whereas the BAM monotherapy did not yield statistical significance.

The secondary outcomes of this study also note that none of the therapies completely clear the virus from the patient. Neither the combination therapy nor the BAM monotherapy prompted two consecutive PCR negative test results for SARS-CoV-2 within the 29 days of the start of the study. This means that none of the therapies completely cleared the virus from the patient. The remaining bits of the virus may not be enough to transmit to another host, but it’s noteworthy nonetheless.

Another noteworthy piece of data from the secondary endpoints, and arguably a primary endpoint to many, is that all doses of the BAM monotherapy and the BAM/ETE combination reduced hospitalizations in patients with severe Covid-19. Among the placebo group, 5.8% of patients were hospitalized during the course of the trial. That percentage dropped to 1% for the 700 mg group, 1.9% for the 2800 mg group, 2% for the 7000 mg group, and .9% for the combination therapy.

This is a positive result. It seems that some therapies in conjunction can yield greater results than monotherapies. This may speak to the strength of the virus to evade neutralizing antibodies or perhaps therapies in conjunction simply prompt a stronger immune response. As more data becomes available about the BAM/ETE combination therapy, greater confidence can be taken in

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

distributing this therapy to those in overcrowded ICUs and hospitals across the country.

Though a limiting hesitation remains. We don’t know the exact strain the patients involved in this study were infected with. Emerging data is indicating that mutant strains, like those originating in the UK and South Africa, may be immune resistant to neutralizing antibodies. Meaning the variants may resist antibodies from previous infection, antibodies delivered via vaccine, or even antibodies used in severe Covid-19 therapies.

While we await new studies working to specifically target new variants with Covid-19 combination therapies, these results are encouraging. Bamlanivimab-etesevimab combination therapy could save countless lives that remain at risk. We eagerly await the results of its next stage of trial.

This article originally appeared in Forbes and is available online here:SARS-CoV-2Immunity: AMovingTarget

April 26, 2021: New Antibody Therapy And Prophylactic Shows Promise In Defending Against SARS - CoV - 2 Variants Of Concern

In a search for an Achilles’ heel for the virus, the scientific world is monitoring potential targets for virus neutralization. One of these targets is a highly conserved region of the receptor-binding domain in the spike protein and two new monoclonal antibodies targeting this region are rapidly advancing in development and trial. The first of Vir Biotechnology and GSK’s two experimental antibodies, VIR 7831, aims to be used as a monotherapy for the early treatment of Covid-19 in adults at high risk of hospitalization. Early trial returns showed an 85% reduction in hospitalization or death, indicating the potential success of the antibody. The second, VIR 7832, is still in the preliminary stages of study as a dual-action prophylactic that potentially blocks viral entry to healthy cells and enhances clearance of infected cells. These antibodies are highly engineered and merit deeper analyses, as they could have wideranging therapeutic and prophylactic success against Covid-19, including SARS-CoV-2 variants, in the near future.

Work began on these antibodies as early as April and May 2020 as researchers searched for neutralizing monoclonal antibodies for SARS-CoV that may carry over effectiveness to SARS-CoV-2. One SARS-CoV antibody extracted from B-cells in a SARS-CoV patient, S309, potently neutralized both SARS-CoV and SARS-CoV-2,

prompting a deeper study of the antibody and the epitope to which it binds.

The S309 antibody recognizes a proteoglycan epitope on the receptor-binding domain of SARS-CoV-2. The antibody is composed of 6 complementarity-determining regions (CDR) loops which come in contact with amino acids 337-344, 356-361, and 440-444 in the spike protein. Two of these CDRs, CDRH3 and CDRL2 sandwich the glycan of the SARS-CoV-2 spike protein at position N343, making it a significant point of contact for the antibody.

The reason S309 carries over its neutralizing effects from SARSCoV is the significant conservation of contact amino acids between viruses. Of the 22 residues involved in antibody binding, 17 are strictly conserved, four are conservatively conserved, and one is semi-conserved between viruses. This research concludes by noting the potential of S309 as a Covid-19 countermeasure and that variants of S309 that may have boosted half-lives and effector functions have already entered accelerated development.

Early in 2021, GSK and Vir Biotechnology began work on the S309 antibody to optimize it for SARS-CoV-2 prevention and treatment, resulting in VIR-7831 and VIR-7832. Both antibodies were mutated to contain an “LS” mutation in the Fc region to prolong antibody half-life and potentially enhance distribution to the respiratory mucosa. This would block viral entry to greater effect and would provide protection against infection for longer.

The VIR-7832 antibody additionally contains an Fc GAALIE mutation shown to boost T-cell immunity. GAALIE is an acronym for the amino acid mutations in the Fc region, specifically G236A,

A330L, and I332E. These mutations would enable antibodydependent complement-mediated cytotoxicity without inducing antibody-dependent enhancement. In other words, it would neutralize the virus and clear out infected cells faster. Early trials indicate that these antibodies effectively neutralized wild-type SARS-CoV-2 at highly effective levels. Additionally, and perhaps more notably, both antibodies additionally neutralize the United Kingdom B.1.1.7, South Africa B.1.351, and Brazilian B.1.1.28.1 variants at high levels. The Brazilian and South African variants are neutralized as well as the wild-type and the United Kingdom variant neutralized about 25% less effectively for both VIR-7831 and VIR-7832.

As a word of caution, there are now variants with mutations in the highly conserved target region that these antibodies attack. While B.1.1.7, B.1.351, and B.1.1.28.1 are all mostly conserved in the regions 337-344, 356-361, and 440-444, there are a number of sequenced viruses in the GISAID database which carry mutations at some of these locations. The most frequent of these are at position 440 where there are 1,689 mutants, position 357 where there are 699 mutants, and position 344 where there are 307 mutants. While these numbers are relatively low in comparison to the 1.2 million sequences in GISAID and the millions of infections worldwide, India has taught us that a strong and resistant virus can spread rapidly. Some of these mutations may have a large effect on antibody resistance, for instance, some mutations to P337 and E340 reduce the neutralizing capability of the antibodies up to 200-fold, showing that this potential Achilles’ heel may have an Achilles’ heel of its own.

These antibodies are ultimately a promising step towards furthering Covid-19 therapeutics and prophylactics. They show strong neutralization of the wild-type virus and some of the major variants of concern. However, there are mutants out there that may be resistant to VIR-7831 and VIR-7832. We suggest more research on these antibodies could clear up concerns about mutations in the conserved region. While this targeted region is not the ultimate Achilles’ heel, it may turn out to be a good starting point as we explore more possibilities in future additions to this series.

This article originally appeared in Forbes and is available online here: New Antibody Therapy And Prophylactic Shows Promise InDefendingAgainst SARS-CoV-2 Variants Of Concern

April 29, 2021: An Antibody Cocktail To Lay Low A Mighty Foe

Viruses like SARS-CoV-2, influenza, and respiratory syncytial virus, are escape artists. These viruses evade immune recognition by mutations that reduce immune recognition yet preserve the ability to infect and cause disease.

SARS-CoV-2 changes many sites of the exterior spike protein to avoid neutralization, all while maintaining replication competency and in some cases, boosting competency and receptor binding avidity. However, there are some amino acids of the spike protein that are not often changed. These conserved areas may be an Achilles’ heel of the virus. Understanding these conserved regions is vital to both therapeutic and prophylactic antibody design, as well as design for future generation coronavirus vaccines.

In a recent paper, Starr et al. describe SARS-CoV-2 antibodies that neutralize not only the B.1 virus but also all variants tested as well as SARS-CoV, several bat coronaviruses and even a bat coronavirus that binds to a different receptor.

Most therapeutic antibodies in use today were isolated based on avidity for the SARS-CoV2 receptor, the cell surface protein ACE2, and for neutralization potency. In contrast, the Starr etal.antibodies were selected for both neutralization and their ability to neutralize a diverse set of coronaviruses, most of which bind to the ACE2 receptor. The breadth of neutralization rather than potency was a primary selection criterion.

In a series of experiments, they determined the binding sites of each of twelve antibodies on the SARS-CoV-2 receptor-binding domain. They also developed a detailed map of possible escape variants of each amino of the entire domain. Finally, they measured the ability of each antibody to neutralize a broad set of SARS-CoV-2 variants of concern as well as an ACE2 binding, including SARS-CoV and one non-ACE2 binding coronaviruses of diverse origin.

The twelve antibodies studied naturally sort themselves into three classes, those that bind to the region of the receptor-binding domain that interacts directly with the ACE2 receptor, those that bind near the ACE2 binding (the receptor-binding motif), and a third category that binds to a distal region named the core region.

Antibodies targeting the receptor-binding motif and ACE2 contact epitopes neutralize SARS-CoV-2 effectively but fail to effectively neutralize many of the variants and all of the non-SARS-CoV-2 viruses. There is one exception, S2E12, which both neutralizes a broad range of viruses and which is unaffected by almost all amino acid substitutions in the receptor-binding domain. The S2E12 exception is attributed to the recognition of contacts between the subunits of the trimeric receptor that are constrained to maintain consistent protein interactions even though individual amino acids at the contact points may vary.

The core receptor-binding domain antibodies bind to sites distant from ACE2 binding have lower potency than those which bind to the receptor-binding motif. However, these antibodies typically neutralize most variants and non-SARS-CoV-2 viruses. The binding and neutralization of these antibodies are impervious to most amino acid substitutions in the receptor-binding domain.

As an analogy, think of the receptor-binding domain having a mushroom shape, with a cap and a stem. In this analogy, the ACE2 binding site occupies the top of the cap. Some of the conserved sites occur on the stem and to some extent the edges of the cap. Specifically, S309 and S2X35 bind high up on the stem and the underside of the cap. Others including S304 and S2H97, bind on the lower parts of the stem.

The four most effective core receptor-binding domain antibodies were S2H97, S304, S309, and S2X35. The part of the ACE2 receptor to which the spike binds is colored pink. The intensity of the color blue of the receptor-binding domain denotes how well the region tolerates amino acid substitutions. Notice that the base of the receptor domain is highly conserved.

The four antibodies neutralize SARS-CoV-2, most of the variants of concern tested, as wells many non-SARS-CoV-2 coronaviruses. All, but S309, effectively neutralize the Kenyan bat coronavirus BtKY72.

S2H97 and S304 effectively neutralize European bat coronavirus BM48-31. S2H97 takes it one step further than the rest and also neutralizes a number of Asian coronaviruses that use a different receptor altogether.

Amino acids in the spike protein receptor-binding domain were changed to every other possible amino acid. The majority which remain functioning were then tested to examine whether or not the spike, with these mutations, could be neutralized. In every case, some laboratory-generated mutations are capable of eliminating antibody binding. The letters denote the amino acid substitutions at that site and the height represents the reduction in the binding capability of the antibody. For example, S2H97 has reduced

binding when E516 and L518 mutations are introduced, whereas those mutations can be sustained by S309. Each antibody recognizes epitopes that, if mutated, render the virus resistant to neutralization. For S309, mutations to P337 and E340 confer 12-fold increase in resistance. For S2H97, mutations to S514, E516, and L518 can reduce neutralization three-fold. For S304, mutations to S383, T385, and K386 result in 15-fold or more reduction. Finally, for S2X35, mutations to G504 can induce neutralization 18-fold or more. resistance. While these mutations have not appeared in variants of concern and are relatively rare due to the region in natural isolates, they do still appear in GISAID database sequences, ranging between 9 and 759 viruses.

This research provides a blueprint for future anti-SARS-CoV-2 antibody drug development. Some of these antibodies are impressive on their own, such as S309, the basis for VIR-7831 and VIR-7832 drug candidates currently in clinical development. Cocktails containing two or more of these antibodies may be even more effective in countering resistance and neutralization of variants than will any one alone. I suggest an ideal cocktail would contain two bi-specific antibodies covering all four conserved core footprints. It might help to add the modifications in the Fc portion of these antibodies that increase the half-life of the antibodies, mucous membrane transport, and antibody-mediated cytotoxicity as was done to create the in the VIR-7831 and VIR-7832 drug candidates.

Conservation of the “mushroom stem” binding sites amongst distantly relates coronaviruses does necessarily not mean they are immutable. There are two potential interpretations of such conservation. The first is that there is no selective pressure to change

because the virus has been successful in diverting immune attention away from these conserved regions, specifically to the ACE2 binding site and to epitopes of the N-terminal domain of the S1 protein. The second is that the conserved areas are functionally relevant and cannot be changed. The first interpretation may have merit, as one report noted that the cold-causing coronavirus’s receptor-binding domain naturally varies in amino acid composition by as much as 17% and yet remains functional.

An additional caution: The Starr et al. studies are restricted to the receptor-binding domain, a small but critically important region of the entire S protein. The development of antibodies to the receptorbinding domain alone may not be the only winning strategy. Mutations in the spike protein within and outside of the receptorbinding domain as well as in the extra-spike proteins of the virus may have compensatory effects. Decreases in viral infectivity and transmission lost by changes in the conserved regions may be made up for by increases in the activity of the replicative complex, the stability of the virus particle, and increases in the activity of the accessory proteins. Indeed, all variants characterized to date have more mutations in proteins other than the spike than they do in the spike protein itself. Infectivity, pathogenesis, and neutralization are composite properties of all viral functions.

These and other studies may have found at least one Achilles’ heel of SARS-CoV-2. I doubt that will be the end of the story for this wily escape artist. In time I believe we will finally throttle this manyheaded hydra with a combination of antiviral drugs, each targeted to a different essential viral protein, combined with a cocktail of antibodies such as those so elegantly described by Starr et al.

This article originally appeared in Forbes and is available online here:AnAntibody Cocktail ToLay Low AMighty Foe

May 5, 2021: New Antiviral Drug Cocktail

Could Hel p India Control Brutal Covid19 Surge

As a brutal second wave of Covid-19 infections rages on in India, more and more states are reporting critical shortages of ventilators and vaccines. The relative scarcity of these provisions means they’ll be difficult to wrangle in time to staunch further suffering and death. But there are drug therapies that could in fact, they might be sitting on the shelves of physicians and pharmacists across the country.

Experts have known for some time that no single therapy will effectively prevent and treat Covid-19. What we need, especially now that highly infectious variants of SARS-CoV-2 are on the loose, is a veritable cocktail of drugs that overwhelms the virus through multiple lines of attack, giving it little opportunity to resist, evolve, or escape. The results of a new study, published in Cell magazine last month, suggest that a cocktail of one polymerase inhibitor in this case remdesivir and two hepatitis C drugs might do the trick. The RNA-dependent RNA polymerase makes a good drug target because it is so essential to the replication strategy of SARS-CoV-2. Viewed in terms of the entire viral genome, this enzyme consists of three nonstructural proteins: nsp7, nsp8, and nsp12. Another exemplary target what the seven different hepatitis C virus (HCV) drugs used in this study inhibit are two proteases, enzymes that break down proteins into pieces a virus can use to copy itself.

The researchers behind the Cell study predicted that certain protease-inhibiting HCV drugs might inhibit also the main protease of SARS-CoV-2, known as nsp5. When they used a supercomputer to model and test their theory, all seven HCV drugs did indeed dock at nsp5. Interestingly enough, however, it was blockage of another protease, nsp3, that significantly boosted antiviral activity. Given that nsp3 precedes nsp5 in the virus’s replication strategy, it makes sense that blocking one has synergistic effects while the other is additive. But when combined with remdesivir, each of the four HCV drugs that inhibited nsp3 increased the potency of the treatment in cell cultures tenfold.

The tenfold increase in potency means the adverse effects of each drug can be mitigated using lower doses. If successful, these treatments should be effective not only at treating the disease, but very importantly preventing it in people who are likely to be exposed. This strategy is used routinely in malaria zones and even for HIV. No vaccine was ever invented for HIV, but high-risk individuals can still protect themselves from infection through a regimen known as pre-exposure prophylaxis (PrEP). India has the pharmaceutical research, development, and manufacturing capacity to produce large quantities of such a treatment and, more importantly, sell it to consumers at low cost, which is how generic drug prices in India are among the cheapest in the world. But for Covid-19, the choice of which treatment to scale up for mass consumption wasn’t easy or obvious until now.

Of course, discovering a breakthrough product in the laboratory is one thing. Rolling it out with great haste is another. Before rush orders of the HCV drug and polymerase inhibitor combo can be placed, the therapy must be tested in animal models and clinical

studies. At this stage, more convenient substitutes for remdesivir which at present can only be administered intravenously in hospitals should be considered and tested alongside it. One polymerase inhibitor, developed by Ridgeback Biotherapeutics LP and Merck & Co, that comes in pill form and has promising preliminary data behind it is molnupiravir. Pfizer is also developing an oral drug that should inhibit nsp5 as well, which would make it another viable addition to the cocktail.

Then comes the question of how to scale up production and distribution using existing infrastructure. Egypt did this with tremendous success with its 100 Million Healthy Lives program, which eliminated hepatitis C from the country in a matter of months by screening more than 60 million adults and providing free treatment to all who tested positive. The India-based multinational Cipla already manufactures HCV drugs en masse at low prices. In fact, they may have the capacity to produce the entire combination at scale. India has the added benefit of being able to supply its own active pharmaceutical ingredients (APIs). Hepatitis C treatments that cost US customers thousands of dollars cost only $25 to $35 in India. There is no good reason why Covid-19 combination therapy shouldn’t cost less than $5.

Were India to succeed at leveraging existing HCV drugs and pharmaceutical infrastructure alongside Covid-19-specific therapies, the strategy could have considerable import in countries facing similarly dire straits. It is impossible to know when the vaccine shortages plaguing not just India, but many countries around the world, will resolve. We also can’t expect new variants of SARS-CoV-2, many of which have learned to evade immune detection, to cease appearing in the meantime. Government and

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

public health officials should take advantage of the opportunity to pump out combination drug therapies while they still can. Otherwise, many more countries may soon find themselves in circumstances not unlike India’s overwhelmed to the point of paralysis. These drugs may be their only way out. This article originally appeared in Forbes and is available online here: New Antiviral Drug Cocktail Could Help India Control Brutal Covid-19Surge

May 11, 2021: Newly Discovered

Antibody Neutralizes Covid Variants By Locking Receptor - Binding Domain In A Closed Position

Cocktails of monoclonal antibodies are valuable therapies for the prevention and treatment of Covid-19. However, their utility has been compromised by variants of SARS-CoV-2 that evade one or more of the antibodies in the mix.

A new paper by Scheid et al . describes a new monoclonal antibody BG10-19 that has a valuable set of characteristics. It is highly potent and active against a broad range of variants. BG10-19 and other antibodies analyzed in the study bind to the receptorbinding domain of the spike protein. The receptor-binding domain can be either open or closed. It must be in the open configuration to bind the ACE2 receptor to initiate infection .

The BG10-19 antibody acts by bonding tightly to the top of the trimeric receptor and locking all three receptor-binding domains in the closed configuration.

The special properties of BG10-19 derive, in part, its ability to bind to two adjacent receptor-binding domains simultaneously. The majority of the interactions are pictured with the receptor-binding site “RBD-A,” while binding simultaneously to the adjacent receptor-binding domain of the trimer designated as “RBD-B.” BG10-19 binds to two N-linked glycans on RBD-A as well.

This versatile antibody was discovered through a selective process of elimination. B cells from 14 patients who had recovered from mild cases of Covid-19 were isolated. The researchers selected 6,113 B cells that either bound to the SARS-CoV-2 S-protein or to the receptor-binding domain. They narrowed the search to B cells originating from four of the 14 patients who had the highest concentration of neutralizing antibodies against SARS-CoV-2 lentivirus pseudotyped with the B.1 spike protein, derived from the wild-type Wuhan strain carrying the D614G spike mutation.

The FAB fragments of 92 monoclonal antibodies isolated from the four patients were added to an IgG1 framework. They then screened the 92 antibodies for polyreactivity, nonspecific binding to unrelated antigens, testing for binding to single-stranded DNA, double-stranded DNA, insulin, bacterial lipopolysaccharide, and streptavidin-APC. They found that 11 of the 92 monoclonal antibodies showed reactivity against two or more of the listed antigens. These were eliminated from future consideration. The search narrowed further by selecting those that bound the SARS-CoV-2 spike protein and the receptor-binding domain. Fortytwo of the antibodies bound to both, nine bound exclusively to the spike protein, and five bound exclusively to the receptor-binding domain. Next, they screened the antibodies for neutralization capability against both SARS-CoV-2 pseudotypes and live virus (the B.1 variant of SARS-CoV-2). Twenty-seven of the 92 showed neutralizing activity with IC50 values ranging from 3 ng/ml to 25 µg/ml.

They narrowed their search to six potential antibody candidates based on neutralization potency. BG10-19 was selected for further study as it was one of the most potent neutralizing antibodies and

capable of neutralizing two variants of concern, the B.1.1.7 variant first isolated in the UK and the B.1.351 variant that was discovered in South Africa.

The set of antibodies was also tested for its ability to bind to spike proteins, each of which carried a single mutation on the spike found in many variants of concern. Two of the tested antibodies, BG10-19 and BG1-28, are largely unaffected by any single mutation and bind to both SARS-1 and the WIV1 spike proteins. Sequence conservation at the BG10-19 epitope explains the crossneutralization against SARS-1, as 23 of 29 residues are conserved between SARS-1 and SARS-CoV-2 RBDs. Notably, despite strong binding to the WIV1 spike protein, there is little neutralizing activity, despite a highly parallel amino acid sequence to SARS-1. Again, BG10-19 was selected for further study as it binds and neutralizes SARS-CoV-2 much with a much lower IC50 than does BG-1-28.

This result is somewhat surprising as at least five of the spike proteins tested alter amino acids well within the binding footprint of BG1019, specifically the mutations R433S, V367F, N439K, N440K, and V445E. Two of the naturally occurring mutations, N439K and V445E, do reduce binding affinity modestly between 1 and 5 fold.

It is notable that the binding site of BG10-19 does not overlap with the residues of the receptor-binding domain that interact directly with the ACE2 receptor. Additionally, the binding site of another broadly neutralizing monoclonal antibody selected for clinical development S309 overlaps that of BG10-19.

This is not to say there are no escape variants that reduce binding and neutralization to the BG10-19 antibody. In fact, they found two

mutations that reduce BG10-19’s neutralizing capability: G339R and L441P. The G339R mutation reduces neutralization as much as ten-fold and the L441P mutation reduces neutralization about one hundred-fold. While these mutations have not yet been sequenced and deposited into the GISAID mutation database, there have been other mutations at both positions. There have been 238 sequenced mutations at position 339, including changes to aspartic acid, serine, valine, as well as some deletions. Additionally, there have been 101 sequenced mutations at position 441, including changes to isoleucine, phenylalanine, arginine, as well as some deletions. While these are not the mutations that inhibit neutralization, their existence indicates that mutations to these positions are tolerated.

All of the antibodies discussed in this paper are very interesting in terms of their potential use. They could be used in various combinations. We suggest the BG10-19 antibody be combined with the S309 and S2H97 antibodies described by Starr et al.to create a cocktail that recognizes conserved epitopes and that is both potent and resistant to naturally arising variants.

This article originally appeared in Forbes and is available online here:Newly DiscoveredAntibody Neutralizes CovidVariantsBy LockingReceptor-Binding Domain In A Closed Position

May 13, 2021: A New Twist To Antibody

Cocktails To Prevent And Treat Covid - 19

Monoclonal antibodies have proved effective in the prevention and treatment of Covid-19. Their effectiveness depends on the recognition of specific structures on the surface of the viral spike protein. Over the past six months, we have learned that many of these shape-specific determinants change in ways that abrogate the effectiveness of individual antibodies and even of antibody cocktails. This is the fifth in a series that describes a search for monoclonal antibodies that may successfully address the problem of antigenic variation.

Monoclonal antibodies have been successful in both prevention and early treatment of Covid-19. The monoclonal antibodies that have been studied to date bind to and interact with the spike protein, and more specifically, the receptor-binding domain. Over the past several months, as reviewed in this series, new monoclonal antibodies have been discovered that have broad neutralizing capabilities. All of these antibodies bind to the receptor-binding domain, either to the receptor-binding motif, which interacts directly with the ACE2 receptor, or to other parts of the receptorbinding domain to inhibit function.

Despite the broad activity of these antibodies or camelid nanobodies, none are entirely neutralizing. SARS-CoV-2 is capable of mutating, leading to viral variants which inhibit antibody function that escape neutralization, triggering a search for effective neutralizing antibodies that recognize epitopes in regions outside

the receptor-binding domain which might be constrained functionally, and therefore, less likely to mutate.

In a new study by Garrett et al., a complete set of overlapping linear peptides on a phage display backbone was developed, which spanned the entire spike protein. For reference, the receptorbinding domain is only about 15% of the full spike protein, stretching from amino acid 332 to 523.

To examine naturally occurring antibodies that recognize these epitopes, they identified those linear regions which are recognized by convalescent sera. It is important to note that these are very different from what is seen when the entire spike protein or receptorbinding domain is used as a target. When the spike protein or receptor-binding domain are targeted, the recognized structures are three-dimensional and yield different binding actions. Whereas in this study, linear epitopes are recognized and the receptor-binding domain is nearly ignored. This is notable as the receptor-binding domain is the principal source for neutralizing monoclonal antibodies in most previous studies. Additionally, antibody responses vary from patient to patient, as no two immune responses are identical.

The antibodies were next exposed to the linear epitopes and examined their ability to neutralize a pseudotype virus. Their samples indicated that 0% to 59% of the neutralization activity present in patient plasma is directed at non-receptor-binding domain epitopes. After depleting the neutralizing antibodies targeting the receptor-binding domain, the researchers found that non-receptor-binding domain antibodies, while not completely responsible for virus neutralization in patients, are at least partially

responsible for residual neutralization, and are therefore important to the neutralization process.

The linear non-receptor-binding domain epitopes of interest map into four specific regions. These are the carboxy-terminal, the nterminal domain, the fusion peptide, and the carboxy-terminus of the S2 subunit. To grasp whether these antibodies could tolerate amino acid mutations in these regions, the researchers developed a heat map of every mutation to individual positions. For example, in the figure below for the fusion peptide, the red indicates a reduction in binding affinity, and the blue indicates a boost. The intensity of the color shows the magnitude of reductions or promotions.

Using this technique, they were able to divide these regions into two classes: those in which amino acid mutations in the region affect binding and those that are relatively resistant to amino acid mutations. It is this second class that provides the most interest. These results may have identified linear regions outside the receptor-binding domain that are constant regardless of variation, most likely because they are required for a conserved function.

In summary, these results suggest that it would be valuable to add to the current cocktails that recognize the receptor-binding domain. One or two additional antibodies that recognize highly conserved epitopes, perhaps in the vicinity of the fusion peptide, may be most useful, as this region was conserved across many variants and viruses. Additionally, we have commented that highly effective cocktails for treatment and prevention might be combined with combination chemoprophylaxis with alternate drugs for maximum effect, creating multiple targets on the spike protein for neutralization. It is unlikely that variants can quickly arise that are resistant to antibody and drug cocktails simultaneously. As new variants spread through

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

populations, this combination strategy may be successful in the prevention of new variant-associated outbreaks. This article originally appeared in Forbes and is available online here: A New Twist ToAntibody Cocktails To Prevent And Treat Covid-19

May 19, 2021 : Discovery Of A Novel Monoclonal Antibody That Neutralizes A Broad Range Of Coronaviruses

The Covid-19 pandemic is no isolated incident. Coronaviruses have been coming after us for years. In the past 60 years, there have been as many as five seasonal cold-causing coronaviruses. In the past 20 years, we have dealt with three lethal coronaviruses: SARS-CoV, MERS-CoV, and SARS-CoV-2, the last of which has conservatively killed 3.5 million people and counting.

The SARS-CoV-2 virus belongs to the B sarbecovirus clade, along with SARS-CoV. There are also C clade viruses, including MERSCoV, and A clade viruses, which include many cold-causing coronaviruses. As we search for treatments for SARS-CoV-2, the question is raised: can we find measures to treat all broad classes of coronavirus unilaterally?

This search has already begun and one approach of note is monoclonal antibodies. These antibodies have been found to effectively treat and prevent early infection when used as a therapy. Within monoclonal antibodies, researchers aim to find those which potently neutralize not only SARS-CoV-2 but also SARS-CoV and viruses from the A and C clades as well.

In a study by Sauer et al., researchers aimed to find a broadly neutralizing monoclonal antibody not in human convalescent sera, but in that of mice. To do this, they inoculated the mice twice with a stabilized MERS-CoV spike trimer and once with a stabilized

SARS-CoV-2 spike trimer. Their aim was to locate antibodies that broadly combated both viruses.

The researchers gathered Fab fragments from the vaccinated mice and connected them to human Fc receptors. After introducing the antibodies to the different coronaviruses, they selected one antibody, B6, which strongly bound to A and C clade viruses, but not for B clade viruses. Additionally, B6 was only able to potently neutralize A and C clade viruses, namely MERS-CoV and coldcausing coronaviruses. SARS-CoV and SARS-CoV-2 were left unneutralized.

It seems, however, that the B6 antibody is capable of binding in SARS-CoV and SARS-CoV-2 to what the researchers label a cryptic epitope. Why, then, does the binding and neutralization fall off? To examine this, the researchers used cryo-electron microscopy to see exactly where the antibody was binding. In A and C clade viruses, the cryo visual of the region to which the antibody bound was clear.

In SARS-CoV and SARS-CoV-2, however, the binding was disordered. This is surprising as this is usually a highly ordered area. They investigated the hidden binding by taking higher resolution pictures of the unusual binding between B6 and SARS-CoV-2.

Testing individual peptide fragments across the general region of binding noted in other viruses, the researchers found their clearer picture. It seems that in A and C clade viruses, the trimer is less tightly bound than in B clade viruses. The looser trimer stem helix bundle in MERS-CoV and cold-causing coronaviruses was unraveled during binding, allowing a clearer picture during cryoelectron microscopy. In SARS-CoV and SARS-CoV-2, the trimer bundle is much tighter and does not unravel during antibody binding. This is consistent with the distortion during cryo-electron

microscopy and likely explains the lack of neutralization by the B6 antibody. Another study by Garrett et al. , which located a number of linear binding epitopes in this region was unable to locate the cryptic epitope Sauer etal.describe, most likely because it is hidden by the stem helix bundle.

What good is an antibody that does not neutralize SARS-CoV-2, as we are in the midst of a pandemic? We may have a way around this shortcoming. In a study by Koenig et al. , a nanobody derived from camelid mammals was able to neutralize SARS-CoV-2 by binding tightly to the receptor-binding domain. The nanobody locked the receptor-binding domain in the open configuration and nullified its binding to ACE2 receptors. Why is this relevant? Perhaps when combined with this camelid nanobody, they will open the structure, exposing the cryptic epitope hidden by the tightly bound stem helix bundle in SARS-CoV and SARS-CoV-2. Then, B6 could be able to enter SARS viruses and neutralize them.

This combination therapy would, however, be dependent on the conservation of the targeted region: positions 1147 to 1161.. In the GISAID database, there are some mutations in these areas, but many are likely dead viruses as they include long strings of deletions or insertions. The most common mutations include H1159Y, which has been sequenced 329 times, S1147L, which has been sequenced 385 times, and D1153Y, which has been sequenced 689 times. While these are no insignificant numbers of sequencing, none of these mutations appear in major variants of interest or concern, which suggests that this region is significantly conserved, not only cryogenically, but also in over 1.5 million GISAID entries.

This reminds me very much of the first effective vaccine against the feline leukemia retrovirus that I made in the early 1980s. The

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

peptide in use was a membrane-spanning peptide that covered the carboxy terminus of the exterior glycoprotein, which deleted the membrane-spanning region, and included a small portion of the cytoplasmic domain. This vaccine provided then and still provides broad protection against leukemia in cats. That shows that peptide vaccines to conserved regions of spike proteins can be very important vaccines. Therefore, as the authors imply, peptides spanning this region may play a role in neutralizing many different variants. We echo their calls for increased research on this and other antibodies in the name of pandemic preparedness, as well as to find combination therapies that may control the SARS-CoV-2 virus today.

This article originally appeared in Forbes and is available online here:DiscoveryOfANovelMonoclonalAntibodyThatNeutralizes ABroadRangeOfCoronaviruses

November 17, 2021: Detailed Description

Of A Highly Potent SARS - CoV - 2

Neutralizing Antibody: Bamlanivimab

Monoclonal antibodies have been shown to be effective in both the prevention and early treatment of SARS-CoV-2 infections. One of the antibodies currently approved for clinical use by emergency use authorization is a combination antibody treatment of bamlanivimab and etesevimab. A recent paper by Jones et al.describes bamlanivimab and its properties in detail, as well as details how bamlanivimab was discovered, providing a potential blueprint for antibody discovery in the future.

Monoclonal antibodies are typically laboratory-produced molecules that mimic the adaptive immune system’s response to invading pathogens. They neutralize a virus by blocking the Spike (S) protein from attaching to the host ACE2 receptor, which reduces viral reproduction and immune reactions.

SelectingBamlanivimab

To find potential SARS-CoV-2 antibodies, Jones et al. extracted peripheral blood mononuclear cells (PBMCs) from a convalescent patient 20 days after initial symptoms. They extracted and screened about 5.8 million PBMCs, which contained 2,238 single antibodysecreting cells. Using custom machine-learning-based sequencing, Jones et al.narrowed their search to 440 high-confidence heavy and light chain antibodies.

From these 440 candidates, Jones et al.selected antibodies capable of rapid cloning, recombinant expression, and were observed in high frequency, of which there were 175. They further selected for more specific requirements, including SARS-CoV-2 and SARSCoV-1 binding, binding to the S protein receptor-binding domain, and binding to certain receptor-binding domain mutations commonly found in SARS-CoV-2 variants, including V378F and V483A. This led to a lead panel of antibodies for further testing.

Jones et al. tested the neutralizing capacity of the panel antibodies against a SARS-CoV-2 pseudovirus and a live SARS-CoV-2 isolate. In both instances, one antibody, bamlanivimab, was by far the most potent neutralizing antibody, with a half-maximal concentration of about 0.05 micrograms per milliliter neutralizing at roughly 75%.

BamlanivimabBinding

Bamlanivimab had a 10-fold greater neutralization capacity than any other identified antibody candidate. This is despite binding in a similar place and pattern to two other tested antibodies: Ab128 and Ab133, which is notably a similar configuration to advanced antibody candidate etesevimab.

To investigate the increased neutralization, Jones et al. performed x-ray crystallography on bamlanivimab and found that in addition to binding the receptor-binding domain, it also was found to bind to an epitope overlapping ACE2 binding sites.

The researchers then conclude that this allows bamlanivimab to bind the receptor-binding domain in both the “up” and “down” configurations, whereas the other antibodies, including etesevimab, only bind in the “up” configuration.

BamlanivimabProtection

To test for protection by bamlanivimab against Covid-19 related symptoms, Jones et al. used nonhuman primates as a simulation of how the antibody may be accepted by the human immune system. Testing for RNA copies per milliliter in bronchoalveolar lavage fluid, lung tissue, nasal swabs, and throat swabs, the researchers noticed distinct drops in RNA content across the board within days of monoclonal antibody inoculation.

In other words, in all areas of the respiratory tract, the monoclonal antibody bamlanivimab reduces SARS-CoV-2 replication and viral load, which in theory results in weakening and waning of symptoms sooner in the infection cycle. This is great news for general and atrisk populations alike. If you were to come in contact with a known SARS-CoV-2 infection or were early on in the symptom onset, taking bamlanivimab could greatly reduce the risk for at-least respiratory-related illness.

While bamlanivimab on its own neutralized SARS-CoV-2 more effectively etesevimab in the Jones et al. study, further research indicated that together they neutralize live virus even more effectively.

The combination therapy, now produced by Eli Lilly, is now approved by the Food and Drug Administration via emergency use authorization for post-exposure prophylaxis and treatment. In addition to immediate treatment, the antibody can be used in immunocompetent infected patients up to five to seven days postinfection and it remains effective. Additionally, the antibody is successful in preventing infection and disease in those known to be exposed to SARS-CoV-2 infected hosts.

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

However, we note that as SARS-CoV-2 continues to evolve into new and more mutated variants, the virus can and will become more resistant to treatments like bamlanivimab + etesevimab. For example, a recent study of three SARS-CoV-2 variants all originating in Africa indicates some resistance already occurs. The Beta variant from South Africa is completely resistant to the combination cocktail. Additionally, the Eta variant from Nigeria and the A.30 variant from Tanzania are substantially resistant to the cocktail in comparison to the Triad variant which drove infections in the Summer of 2020. In other words, these variants already contain mutations that render major approved monoclonal antibodies less effective or completely ineffective.

Despite this, we implore more such wide studies to be undertaken as hidden gems like bamlanivimab await discovery. More treatments and prophylactics of this nature could greatly reduce the risk for hospitalization and death, especially among at-risk individuals. Using bamlanivimab’s discovery as a blueprint may lead to numerous more to be discovered in the critical months ahead.

This articleoriginallyappeared on Forbes, and can be read online here: Detailed Description Of A Highly Potent SARS-CoV-2

NeutralizingAntibody:Bamlanivimab

November 19, 2021:

Intramuscular Injection Of Monoclonal Antibodies Simplifies Covid Treatment

Until effective and accessible SARS-CoV-2 antivirals are available, monoclonal antibodies remain our strongest treatment and prophylactic against Covid-19. These antibodies are typically administered by a drip, which often requires medical assistance. One antibody, however, sotrovimab, could be administered intramuscularly (by shot), making delivery of the treatment far simpler. Here we review a press release by sotrovimab producers GSK and VIR that details the intramuscular administration of sotrovimab.

The trial to test sotrovimab was a phase 3, randomized, doubleblind, placebo-controlled study, which is the standard for an efficacy test of this nature. The aim of the trial was to determine whether intramuscular administration of the antibody was similarly effective to intravenous (IV) administration for the early treatment of mildto-moderate Covid-19 in high-risk populations.

Primary Outcomes

A detailed review of the study is yet to be released, but according to the GSK press release, the results for intravenous and intramuscular administration of sotrovimab were roughly equal. There was a 2.7% rate of progression to hospitalization for more than 24 hours or death among high-risk patients with mild-to-moderate Covid-19 in the

intramuscular group. In contrast, that rate in the intravenous cohort was 1.3%.

The adjusted difference between the two administration methods was 1.07% with a 95% confidence interval of -1.25% to 3.39%, which falls below the upper bound set by the Food and Drug Administration for non-inferiority. In other words, the receiving sotromivab from a shot in the arm is of the same clinical effectiveness as from an IV drip.

SotrovimabStructure

We have previously described the origins and activities of sotrovimab in greater detail. To recount, the antibody is a derivative of another antibody, S309, which was first isolated from memory B cells from the sera of a recovered SARS-CoV-1 patient. S309, like many other antibodies, targets the Spike protein of the viral genome, which modulates viral entry into host cells and carries several antibody binding sites.

S309 targets a specific residue on the Spike protein, N343, which was later determined to be a consistently conserved glycan in the Sarbecovirus subgenus. As SARS-CoV-2 belongs to this subgenus and maintains many similarities to SARS-CoV-1, S309 was a promising neutralizing antibody candidate for inhibition of SARSCoV-2.

Sotrovimab differs from S309 in a particular way. It is purposefully engineered to possess two mutations in the Fc region. These are M428L and N434S, colloquially referred to as the LS mutation. These two mutations confer enhanced antibody binding to the Fc receptor, a critical binding agent for monoclonal neutralization. Studies describe that the two mutations result in an extended half-

life of roughly 50 days and enhanced drug distribution to the respiratory system, protecting from significant lung damage or other Covid-related respiratory complications.

CaveatsandConclusions

While the finding that intramuscular and intravenous administration of sotrovimab is significant, there are several further endpoints that should be considered with this monoclonal antibody and with all antibody candidates.

The trial described follows patients through 29 days of trial. As the half-life of sotrovimab is detailed by some studies as roughly 50 days, it would be noteworthy to follow these patients past the 29-day marker to investigate the ongoing protection from SARS-CoV-2 reinfection that sotrovimab may grant.

While the two administration methods were statistically equivalent, more patients were hospitalized or died in the intramuscular cohort than the intravenous cohort. It would be interesting to analyze the health context and complications of those in the intramuscular group versus the intravenous group to potentially find some correlation in symptom protection.

It is worth commenting on the relative merits and disadvantages of a single intramuscular injection of long-acting monoclonal antibodies versus orally-administered pills both for the treatment and prevention of COVID-19.

The advantage of pill formulation is that they do not require medical personnel for administration. However, the patient must determine that they are infected, see a doctor, obtain a prescription, and take the pills as recommended, at present a five-day course

involving a dose every 12 hours. Moreover, the treatment must begin shortly after infection and usually within three to four days of symptom onset, which translates to between six and 12 days postinfection. Additionally, once oral treatment ceases, the patient is only protected by existing convalescent antibodies.

A single muscular injection, however, requires no further treatment or medication past the moment of treatment, though this requires medical personnel for administration. Moreover, the half-life of sotrovimab is roughly 50 days, which means the protection from subsequent infections following exposure could last three months or longer. In the case of one antibody cocktail developed by Regeneron, protection is estimated to last eight months, though that treatment is an IV drip.

Perhaps the best use for antiviral drugs occurs prior to infection, either as pre-exposure or post-exposure prophylaxis. In other words, the drug is administered as a preventative either in general or immediately following exposure to a known positive infection. In this context, a single intramuscular injection of an antibody is far superior to orally-administered pills. The single intramuscular injection may provide protection for up to four months, whereas ingestible pills only protect as long as they are taken. Additionally, long-term use of antiviral pills could lead to dangerous side effects, including carcinogenesis. This is the case for aspiring drug candidate Molnupiravir, which is toxic and mutagenic in extended use.

One major advantage of small-molecule ingestible antivirals over intramuscular injections is the price. Prices may vary and both Merck and Pfizer have stated that costs will be lower in low-income countries. For Molnupiravir, the cost of production for a full course

is roughly $5. Most small-molecule drugs will be of similar production cost: around $20 or less. In higher-income countries, the five-day regimen of antiviral pills is closer to $500. A full 8-milliliter dose of sotrovimab will cost roughly $2000, which is far from a competitive price point compared to other existing and emerging treatments for Covid-19. Were the sotrovimab treatment closer to the $500 to $1000 range, it may be more competitive in the treatment and prophylaxis market.

For congregate living centers, particularly eldercare facilities where the residents are at high risk due to age and comorbidities, we first recommend triple vaccination. Then, if a Covid-19 case is detected, we would recommend all residents and employees receive an intramuscular injection of monoclonal antibodies. We call this the “belt and suspenders” strategy, conferring two layers of significant protection to high-risk populations. This strategy is universally beneficial, as it boosts all recipients' antibody levels tremendously. Were “belt and suspenders” a routine procedure in every potential host, we would all be better protected if exposed to the virus.

Orally-administered pills, on the other hand, only provide additional protection so long as the treatment is ingested. We also note that antiviral pills are mostly used in the context of postexposure and post-symptom-onset patients. Their benefits for preexposure prophylaxis have yet to be significantly evaluated.

It is welcome news that a variety of approaches for both treatment and prevention of SARS-CoV-2 infectious disease are either here or will soon be available. It will be up to both governments and individuals to decide which treatment is appropriate in what setting to join vaccines in medical control of the COVID-19 pandemic.

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

This articleoriginallyappeared on Forbes, and can be read online here:IntramuscularInjectionOfMonoclonalAntibodiesSimplifies CovidTreatment

November 24, 2021:

A New Monoclonal Antibody That Has The Potential To Neutralize All Viral Variants

Monoclonal antibodies offer what is now a proven way to prevent and treat Covid-19 infections. Until recently, antibodies were administered by intravenous (IV) injection, which limited their widespread use. Newer technologies allow antibodies to be administered intramuscularly or subcutaneously. Additionally, new data indicates that with minor modifications, monoclonal antibodies can provide protection for many months. These advances add to the ability of monoclonal antibodies to treat and prevent Covid-19.

One limitation of Covid-19 monoclonal antibodies is that virus variants can escape neutralization. Single point mutations or deletions in certain areas of the SARS-CoV-2 genome can eliminate or gravely reduce antibody potency. Here we describe a novel antibody: DH1047. The distinguishing characteristic of this antibody is that it is capable of neutralizing all known variants of interest and concern by targeting highly conserved epitopes.

Martinez etal at the University of North Carolina aimed to identify, isolate, and analyze an RBD-targeting monoclonal antibody with the potential to neutralize beyond just the SARS-CoV-2 wild type.

Building on the research by Rappazzo et al . that noted that an engineered RBD-directed antibody, ADG-2, could neutralize and protect against multiple SARS-related viruses, Martinez et al.

hypothesized that conserved RBD glycans could create potent targets for cross-reactive antibodies.

IdentifyingDH1047

Thousands of antibodies develop within us upon infection with an invading pathogen. To uncover their winning antibody, Martinez et al. built on the foundations of previous research. Beginning with a pool of 1737 monoclonal antibodies isolated from SARS-CoV and SARS-CoV-2 convalescent sera described by Li et al. , Martinez et al. then focused on 50 which bound to both SARS-CoV, SARSCoV-2, and other animal sarbecoviruses such as bat virus WIV-1.

To further restrict their candidates, Martinez et al. tested the 50 antibodies for neutralizing activity on a mouse-adapted SARS-CoV2 virus, a SARS-CoV-1 virus, bat coronavirus WIV-1, and bat coronavirus RsSHC014. These tests resulted in four primary candidates that displayed broadly neutralizing capability: DH1235, DH1073, DH1046, and DH1047.

Martinez et al. further analyzed their refined group of four against several more sarbecoviruses, including horseshoe bat viruses RaTG13-CoV and RsSHC014, pangolin virus GXP4L-CoV, camel virus MERS-CoV, human cold virus HuCoV OC43, and others. Notably, the four antibodies bound to some viruses but not others. Specifically, they only bound to Group 2B betacoronaviruses, leaving out viruses like MERS-CoV. Martinez et al. conclude that this is likely due to the targeted epitope of the four antibodies existing in Group 2B betacoronaviruses, but not others.

Martinez et al.then aimed to analyze the protective activities of the four antibodies to determine whether their binding and neutralization carried over to animal models. Neither DH1235,

DH1073, nor DH1046 protected infected mice from SARS-CoV lung viral replication, demonstrating a contrast to the potent neutralization displayed invitro DH1047, however, fully protected mice subjects from lung virus replication. Further tests indicated that DH1047 as a prophylactic prevented illness-related weight loss and pulmonary complications as well. Martinez et al. had found their winner.

DH1047Binding

Using cryo-electron microscopy, Martinez et al . observed the techniques DH1047 and its related antibody use to bind to a broad range of sarbecoviruses so effectively. They note three antibody fragments bound to three SARS-CoV RBDs in the “up” position. This is the position required for virus interaction with the host cell. The residues making antibody contact are 356-372, 390-404, and 488-492. Two of these sets, 356-372 and 390-404, are located in the receptor-binding core, whereas 488-492 is located in the receptorbinding motif, which is the part of the receptor-binding domain that makes contact with the host ACE2 receptor. Martinez et al.further note that these sets of residues closely resemble that of the SARSCoV-2 spike and others, defining the cross-vulnerability of sarbecoviruses.

Source:ACCESSHealthInternational

DH1047SARS-CoV-2Variant Neutralization

Perhaps the most immediate use for a broadly neutralizing crosssarbecovirus antibody is the neutralization of pervasive mutated SARS-CoV-2 variants. Using pseudovirus neutralization assays, Martinez et al introduced DH1047 to several variants, including the Triad (or D614G), Alpha, Beta, Gamma, B.1.429, B.1.526, Kappa, and Delta. They found that in the case of all SARS-CoV-2 variants, the 50% neutralization assay values ranged between 0.1214 and 0.1609 micrograms per milliliter. In other words, DH1047 potently neutralized all SARS-CoV-2 variant pseudoviruses. They also tested the antibody against live virus samples of the Triad, Alpha, and Beta variants. Again, 50% neutralization assay values were low, running from 0.059 to 0.111 micrograms per milliliter, confirming that DH1047 broadly neutralizes SARS-CoV-2 variants.

Figure 8. Linearrepresentation oftheSARS-CoV-2receptor-binding domainwithDH1047bindingregions notedinblue.

Conclusion

We look forward to the rapid development of this antibody and others like it. Cross-neutralizing antibodies such as DH1047 would make an important contribution to the control of the Covid-19 pandemic.

This articleoriginallyappeared on Forbes, and can be read online here: A New Monoclonal Antibody That Has The Potential To NeutralizeAllViralVariants

December 10, 2021: FDA Approves Anti -

SARS - CoV - 2 Monoclonal Antibodies For The Vaccine Insensitive Immune Suppressed Population

Immunocompromised Americans who are not adequately protected by their Covid-19 vaccination series will now have an additional option for long-term protection in the form of an antibody cocktail drug developed by AstraZeneca.

Antibody drugs have been a standard treatment for Covid-19 infections for over a year, but the AstraZeneca drug is the first intended for long-term prevention against COVID-19 infection, rather than short-term treatment. This development demonstrates the potential for monoclonal antibodies to be used in tandem with vaccines for the strongest possible long-term protection against this ever-evolving virus.

The FDA has issued an emergency use authorization for AstraZeneca’s drug which will be sold as Evusheld. The drug is a cocktail of tixagevimab co-packaged with cilgavimab and administered together and is authorized for the pre-exposure prophylaxis of Covid-19 in adults and children 12 and older whose immune systems haven't responded adequately to COVID-19 vaccines or have a history of severe allergic reactions to the shots. The drug is given via an intramuscular injection.

Specific groups who could benefit from the antibody drug include cancer patients, organ transplant recipients, elderly populations, and people taking immune-suppressing drugs for conditions like rheumatoid arthritis. A study published earlier this year found that out of 3 million insured Americans, 2.8% were taking immunosuppressant drugs.

The US has a contract with AstraZeneca to purchase up to 700,000 doses of the treatment which will be allocated proportionally to states and distributed at no cost within the next few weeks. Yet the decision by AstraZeneca to price the drug commercially while negotiating contracts with governments around the world during the pandemic will broadly constrain the effectiveness of the drug and create issues of equity.

Monoclonal antibodies are laboratory-made proteins that mimic the immune system’s ability to fight off harmful pathogens such as viruses. Tixagevimab and cilgavimab are long-acting monoclonal antibodies that are specifically directed against the spike protein of SARS-CoV-2, designed to block the virus’ attachment and entry into human cells. Tixagevimab and cilgavimab bind to different, nonoverlapping sites on the spike protein of the virus.

AstraZeneca tested the Evusheld drug in approximately 5150 adults who were older than 59, had a prespecified chronic medical condition or were at increased risk of Covid infection in a randomized, double-blind, placebo-controlled trial. In the primary analysis, those who received the drug saw a 77% reduced risk of developing Covid-19 compared to those who received a placebo. In further analyses, the reduction in risk of developing Covid-19 was maintained for drug recipients through six months.

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

Evusheld has not presently been authorized for the treatment of post-exposure prevention of Covid-19. But it is important to ask why and continue to collect data on how monoclonal antibodies can be used prophylactically in high-risk congregate settings such as nursing homes, prisons, and military ships. We also need greater research into how antibodies can match the variant in circulation. Although widespread vaccination against Covid-19 remains the top priority, the emergence of the Omicron variant highlights that additional lines of defense are needed.

This articleoriginallyappeared on Forbes, and can be read online here:FDAApprovesAnti-SARS-CoV-2MonoclonalAntibodiesFor TheVaccine Insensitive ImmuneSuppressedPopulation

December 23, 2021: Omicron Evades

Most But Fortunately Not All Monoclonal Antibodies

The Omicron variant of SARS-CoV-2 has rapidly ricocheted around the world. Reported cases are at their highest since May 2021. There is a growing need for effective monoclonal antibody treatments to prevent and treat Omicron infections. Unfortunately, most approved anti-SARS-CoV-2 antibodies have a diminished potency or are ineffective against Omicron. The Omicron mutations which render the virus resistant to antibodies induced either by natural infection or vaccine also render them resistant to most monoclonal antibody treatments. Most does not mean all. At present, there are at least five well-characterized antibodies that retain potency against most variants of SARS-CoV-2 including Omicron.

S2K146

S2K146 is a monoclonal antibody developed jointly by Vir and GSK. Isolated from the memory B cells of a symptomatic Covid-19 patient, S2K146 bind the SARS-CoV-2 S protein, as well those of SARS-CoV-1, bat coronavirus WIV-1. Additionally, the antibodybound and potently neutralized a number of variants of concern or interest, including Alpha, Beta, Gamma, Delta, Epsilon, and Lambda. Structurally, the antibody locks two of the trimer’s three receptor-binding domains in the open configuration while the remaining binding domains remain closed.

As outlined by Park et al. , the S2K146 antibody footprint mimics that of the ACE2 binding sites. As many as 21 of the amino acids of the receptor binding site of the virus’s spike protein also bind the S2K146 antibody. These amino acids include heavily mutated sites in variants of concern, including N501, E484, and K417. Mutations of these mutations normally reduce antibody binding. The research speculates that neutralization is preserved as the interaction between the antibody and the receptor occurs over so many contacts. To me, it makes sense that an antibody has a structured surface that closely mimics that of the ACE2 neutralizers most variants. However much it may mutate, the spike protein must bind to ACE2 itself to initiate infection. I expect to see many more such ACE2 antibodies in the future, some with binding affinities that exceed that of S2K146.

CV3-1

The CV3-1 monoclonal antibody also neutralizes several variants of concern and may also retain activity against Omicron. The antibody binds to a structure designated the 485-GFN-487 loop in the receptor binding domain. The three amino acids are well conserved in Omicron and most other variants.

The antibody recognizes the receptor binding domain in the open configuration. Evidently binding of the antibody triggers a conformational change that mimics that of binding to the natural receptor. Upon binding, the S1 protein dissociates from the membrane-bound S2, laying bare the structures on the S2 protein that drives viral to cell membrane fusion required for virus entry. According to the researchers binding, by CV3-1 triggers premature release of the S1 protein from the virus surface, rending it incapable

of infection. In other words, the antibody imitates ACE2 binding and springs a trap before the virus enters the cell. Rather than blocking binding as does S2K146, CV3-1 inactivates the virus prematurely. Wenwei et al. report that this loop structure is critical for S1 activation.

CV3-25

A third antibody CV3-25 binds to the S2 protein of the Spike. This region is highly conserved. among betacoronaviruses. Specifically, CV3-25 bindings the stem helix near the membrane attachment site.

Wenwei et al.findthat the antibody binds to one or two units of the S protein trimer. The main site of interaction between CV3-25 and S2 lies between residues 1153 to 1165. More detailed analysis, including binding to an isolated peptide corresponding to this region, reveals that the antibody recognizes a linear epitope. A threedimensional conformation is not required. The ability of the antibody to bind and neutralize the virus via a linear epitope raises the exciting possibility that it may be possible to develop a vaccine using the linear peptide that directs the immune response to this conserved neutralizing site, a possibility suggested by the authors.

Both CV3-1 and CV3-25 efficiently neutralize the Wuhan wildtype virus, as well as variants of concern Alpha, Beta, Gamma, Delta.

We have previously discussed other monoclonal antibodies for SARS-CoV-2 that could be used in conjunction with those described here to create a potently effective treatment. For example, a SARS-CoV-2 camelid nanobody binds across the receptor-binding domain in such a way that the trimer’s receptor-binding domains remain in the closed configuration.

Sotrovimab

Sotrovimab antibody was developed by Vir and is marketed by VIR/GSK. I have previously described this antibody here. The potency of Sotrovimab is only moderately diminished by Omicron as compared to its activity against other variants of interest. Sotrovimab is approved for emergency use in the United States and elsewhere. The epitopes to which Sotrovimab are moderately conserved in existing variants of concern. Whether Sotrovimab will retain activity against variants to come is uncertain.

Evusheld

Evusheld, developed by Astra-Zeneca, is a combination of two antibodies tixagevimab and cilgavimab, both of which were isolated from Covid-19 patients. The two antibodies bind to the receptor binding domain of the S1 protein albeit at the different and complementary sites. The antibodies were engineered to enhance their half-life. Evusheld is designed to be administered as an intramuscular injection, as opposed to an intravenous infusion.

Early data indicated that the combination antibody effectively neutralized variants of concern including the Delta variant. Recent studies show that Evusheld is substantially less effective against Omicron as compared to earlier variants. Nonetheless, Omicron retains what appears to be sufficient activity to provide at least some protection against Omicron.

Conclusions

As potentially dangerous as Omicron may be more transmissible than delta and resistant to most vaccines and monoclonal antibodies at least two approved monoclonal antibody treatments

remain in our arsenal; Sotrovimab, and Evusheld. More are in active development. The recent emergency use authorization of Paxlovid, a small-molecule antiviral protease inhibitor for the treatment of the infected vulnerable, is what we hope will be the first of many new drugs that in combination with broadly neutralizing antibodies may effectively treat and eventually prevent SARS-CoV-2 infections.

This articleoriginallyappeared on Forbes, and can be read online here: Omicron Evades Most ButFortunately Not All Monoclonal Antibodies

December 23, 2021: Pfizer’s New Antiviral Drug Could Transform The Pandemic, But Challenges Still Lie Ahead

The approval of Pfizer’s Paxlovid antiviral pill for Covid by the FDA is a transformative development at this critical stage of the pandemic. Paxlovid has been authorized for use in adults and pediatric patients with mild-to-moderate COVID-19 who are at high risk of progressing to severe illness. The pill could reduce the burden on our overwhelmed hospitals during this current surge of Omicron-driven cases that has already exceeded the peak of the recent Delta wave.

Final clinical trial data, conducted during the Delta surge, showed that the drug reduced the risk of hospitalization or death by 88 percent when given to high-risk unvaccinated adults within five days of the start of their symptoms. Pfizer’s laboratory studies indicate that its pills are likely to work against the Omicron variant, but this has not yet been confirmed.

Paxlovid consists of nirmatrelvir, which inhibits a SARS-CoV-2 protein to stop the virus from replicating, and ritonavir, which slows down nirmatrelvir’s breakdown to help it remain in the body for a longer period at higher concentrations. Paxlovid is administered as three tablets (two tablets of nirmatrelvir and one tablet of ritonavir) taken together orally twice daily for five days, for a total of 30 tablets.

Presently, the pill has been authorized for Covid patients age 12 and over who are vulnerable to becoming severely ill because they are

older or have medical conditions such as obesity or diabetes. However, once manufacturing increases and supplies are more plentiful there is potential for the pill to be used prophylactically for those potentially exposed in high-risk, congregate settings such as nursing homes, prisons, and military ships. However, there are several challenges that will prevent us from utilizing the full power of this tool. The first is insufficient supplies of the drug. In the coming week, the US is contracted to receive enough pills to cover 65,000 Americans. With a current daily average of 242,794 cases nationwide that wouldn’t even cover half of the people testing positive for the virus daily. Pfizer needs to be working with other manufacturers to significantly increase production.

The US is purchasing the drug at a cost of $530 per patient, a cost significantly out of reach for poorer countries. Based on comparable drugs manufactured in India, I estimate that the true cost should be more like $25 - $30 per patient. By pricing these countries out of the market, we risk making the same mistakes we have with global vaccine inequity. As we have learned from bitter experience, in our highly globalized world, no population is safe until we all are.

The second major issue is our woefully inadequate testing infrastructure. Currently, Americans are facing long wait times to access a Covid test and even longer wait times to receive the results. Rapid tests are nearly impossible to find during the holiday period. Paxlovid is only effective if taken within the first few days of symptoms, without an efficient testing infrastructure that cannot be achieved. We need to be dramatically increasing testing sites across the country.

I have long been an advocate of free, mass rapid testing to control the spread of Covid-19 since March 2020. Yet well over a year since the first at-home rapid antigen test was approved, despite desperate pleas from many epidemiologists and public health experts, the US is yet to embrace the power of mass rapid testing, focusing instead on vaccines. The Biden administration recently announced they would ship as many as 500 million at-home test kits to Americans in January, but the amount is still insufficient.

Widespread accessibility to rapid tests in the US is presently hampered by a cumbersome F.D.A. process intended for high-tech medical devices. To be approved, the rapid tests must demonstrate that they are nearly as sensitive as the gold standard PCR tests. But rapid tests are the “public health gold standard”, they deliver the most important public health metric by detecting when someone is infectious and is likely to transmit the virus. Therefore they should be regulated as a Public Health Good. President Biden could accomplish this with a simple Executive Order, increasing competition among manufacturers and flooding the market with inexpensive, high-quality rapid tests.

Finally, we should be aware that Paxlovid does carry a number of drug contradictions including blood thinners, (warfarin), hormonal contraceptives (ethinyl estradiol), steroids (prednisone), cholesterol drugs (statins), the full list can be viewed here. The drug may not be right for everyone and those who are prescribed Paxlovid should always give their doctor a full medical history and medication list, including any vitamin or herbal supplements.

Despite the transformative impact that antiviral drugs could have on the pandemic, we must still proceed with caution. The FDA has unfortunately approved a second antiviral drug Molnupiravir from

Merck today which has the potential to supercharge SARS-CoV-2 mutations and unleash a more virulent variant. A risk not worth taking when the drug only reduces the risk of hospitalization and death among high-risk Covid patients by 30 percent compared to Paxlovid’s 88 percent. We also have two viable treatment alternatives in the form of two monoclonal antibodies treatments Sotrovimab and Evusheld which are effective against the Omicron variant.

This articleoriginallyappeared on Forbes, and can be read online here:Pfizer’sNewAntiviralDrugCouldTransformThePandemic, ButChallengesStillLieAhead

January 4, 2022: Difficulties Of Single Monoclonal Antibody Treatment Of SARS - CoV - 2: The Sotrovimab Experience In Australia

The SARS-CoV-2 Omicron variant has spread around the globe at unprecedented speed. Neither prior infection nor multiple vaccinations impede transmission. One hope was that early treatment with monoclonal antibodies for those most susceptible to serious disease would reduce hospitalization and death. Unfortunately, Omicron has proved to be resistant to most FDAapproved monoclonal antibody treatments. Laboratory experiments suggested that at least one monoclonal antibody, sotrovimab, retained significant activity. Unfortunately, recent data from Australia raises serious issues of sotrovimab as a stand-alone treatment. Rockett et al. demonstrate that resistance to sotrovimab as a monotherapy rises rapidly in treated patients. Moreover, they worry that such viable variants may also be highly resistant to existing vaccines.

Rockett et al. determined the sequence of virus isolated from patients treated with sotrovimab. A patient cohort of 100 received sotrovimab at a treatment center in New South Wales, Australia. The treatment was a single 500mg dose targeted at patients within five days of symptom onset thought to be at risk for severe disease progression. Of the initial 100 patients, 23 tested positive for SARSCoV-2 infection at least 10 days post-infusion, and of these, the pre-

and post-infusion respiratory tract samples of eight were collected. Of these eight, seven were hospitalized, six were partially vaccinated or unvaccinated, and four were given additional antibody treatment (Table 1).

Viable virus samples obtained from four of the patients contained a mutation at amino acid E340 of the spike protein, either E340K, E340A, or E340V (Table 1). The virus in one patient contained an additional mutation: the P337L mutation.

These mutations are predicted to yield virus resistance to sotrovimab. Analyses of mutations in this region of the Spike, the receptor-binding domain, indicate that the observed mutations decrease sotrovimab neutralization by almost 300 fold.

Sotrovimab, like many other SARS-CoV-2 monoclonal antibodies in development, targets highly conserved regions of the Spike protein. This strategy is an attempt to create pan-variant neutralizing treatments. Residues 337 and 340 are both among the most highly conserved epitopes in the SARS-CoV-2 Spike protein. Each is only found mutated in a few hundred of the millions of sequences in the GISAID sequence database.

Rockett et al. note that in addition to monoclonal antibody resistance, similar resistance may be found in vaccine-elicited neutralizing antibodies. The experimental evidence showing sotrovimab treatment induces dangerous mutations suggests we should conduct similar experiments with current generations of vaccines as the subject.

This study provides several sobering lessons:

Resistance to singular antibody treatment can develop rapidly in high-risk patients.

Mutant viruses that persist for ten days or more may transmit further antibody and vaccine-resistant infections. Invitroneutralization activity provides little assurance for treatment efficacy.

Effective therapy will require the use of two or more monoclonal antibodies each targeting different epitopes.

The rapid appearance of antibody resistance should come as no surprise for those who follow the literature on the use of convalescent sera to treat persistently infected patients. Variants resistant to the neutralizing activity of such polyclonal sera develop in a matter of days to weeks. Some speculate that Omicron arose from such a patient.

The data of Rockett et al. also serve as a warning for the use of monotherapy low molecular weight antiviral drugs such as paxlovid and molnupiravir. Laboratory experiments demonstrate that resistance to these drugs does arise. We should not be surprised if such resistance develops rapidly.

We conclude by noting that molnupiravir may create even more vaccine and monoclonal antibody drug-resistant variants than sotrovimab. The drug is a polymerase inhibitor that induces a destructive number of mutations to the virus. The possibility of molnupiravir introducing new mutations to the virus, without neutralizing the virus, seems a significant risk. In vivo human trials akin to the one examined here could shed light on this dangerous possibility.

This articleoriginallyappeared on Forbes, and can be read online here: Difficulties Of Single Monoclonal Antibody Treatment Of SARS-CoV-2:TheSotrovimabExperience In Australia

March 23, 2022: Antibodies Team Up Against Omicron

Omicron was an unpleasant surprise in many respects. First, the sheer number of mutations in the Spike and throughout the genome yield other significant effects, such as resistance to many vaccines and modified cellular entry. Second, Omicron is now known to be not one, but a family of variants BA.1, BA.2, BA.3, and the recombinant BA.4. Third, the Omicron family is more infectious than any variant preceding it. BA.1 is more infectious than Delta and BA.2 is more infectious than BA.1.

Source:ACCESSHealthInternational

Here we address another unpleasant surprise of Omicron. The family of variants, in addition to being highly resistant to many vaccines, is also resistant to currently available monoclonal antibodies, which are often the first line of defense for treating people infected, but not yet seriously ill, in hospital settings. Antibodies developed to treat infections resulting from the original Wuhan virus or initial B.1 variant have reduced effectiveness against Omicron.

Figure 9.IncreasinginfectivityofSARS-CoV-2variants.

Evushield and bebtelovimab are two examples of antibodies with reduced Omicron neutralization. Both antibody treatments target binding sites that are altered in the Omicron receptor-binding domain. Among Evushield’s targeted residues, S477, T478, E484, and Q493 are all altered in BA.1, BA.1.1, and BA.2. Bebtelovimab’s binding map is also altered at positions N440, G446, Q498, and N501

Thankfully, there are a few antibodies that may still have significant neutralization of the Omicron family. These may become the next great tools in the fight against severe Covid-19. Here we describe a study by Fenwicket al.that describes these antibody candidates and how they are capable of neutralizing BA.1, BA.2, and all their subvariants.

Screening for the presence of anti-Spike antibodies in over 100 samples of sera donors, Fenwick et al. identified two monoclonal antibody candidates that showed promising results against previous variants of concern, including Alpha, Beta, Gamma, and Delta. They compared these two antibodies against currently available monoclonal treatments, such as the AstraZeneca combination cocktail, Regeneron cocktail, and sotrovimab.

The two antibodies, P2G3 and P5C3, strongly neutralized not only the Wuhan strain of SARS-CoV-2, but also Alpha, Beta, Gamma, Delta, and, most notably, BA.1, BA.1.1, and BA.2 remarkably effectively. P2G3 was between 5 and 907-fold more potent at neutralizing the Omicron family Spike proteins as compared to other monoclonal therapies. P5C3 was slightly less potent than P2G3, but still more neutralizing than the other tested therapies.

Notably, Fenwick et al. identified upon cryo-electron microscopy that P5C3 bound the virus noncompetitively with P2G3, meaning they could be combined in a single treatment. This combination performed roughly the same as P2G3 on its own, though we note that a combination would be more difficult for the virus to mutate to overcome.

The two are non-competitive because they attach to the Spike receptor-binding domain at different angles. The authors believe that P5C3 takes a common approach as previous monoclonal antibodies, latching to the up-configuration of the receptor-binding domain. P2G3 attacks at an unusual angle, targeting the side of the protein rather than the top, meaning the antibody can bind in the up or down configurations. Together, the antibodies effectively lock the Spike protein in place, limiting its transmission.

Throughout the pandemic, variants have mutated to evade our latest defenses against severe illness. Just as Omicron mutated a number of Spike and genome-wide mutations leading to reduced effectiveness of vaccines and monoclonal treatments, future variants may mutate to overcome P2G3 and P5C3. When administered together, we are essentially delaying that process by giving the virus more targets to mutate against.

Recent studies of the sotrovimab antibody indicate that resistance to the treatment, when used as a monotherapy, rises rapidly in treated patients. Particularly in those that are immunosuppressed, Rockett et al. note that over time, infected hosts with persisting infections may develop mutant viruses that reduce sotrovimab neutralization by as much as 300-fold.

We suggest avoiding the monotherapy approach of antibodies altogether. There are a number of monoclonal antibodies that attach to relatively conserved sites across SARS-CoV-2 variants. Among these are Vir/GSK’s S2K146 antibody, which uses a wide footprint to overcome heavily mutated sites like N501 and E484. Another is the CV3-1 monoclonal antibody that binds the 485GFN-487 loop in the receptor-binding domain, which is a highly conserved site in Omicron and other variants.

A third is CV3-25, which avoids the receptor-binding domain altogether and inhibits the Spike protein via the S2 region. This one is of particular interest as it binds a different region altogether than the receptor-binding domain.

Now that there are many antibodies available, and more coming soon, we should be combining at least three or more of the described antibodies in one treatment, making the task of overcoming neutralization that much more difficult for the virus. One of the included antibodies should definitely be CV3-25, as it differs from most circulating antibody candidates to date. Ultimately, SARS-CoV-2 will continue to mutate and overcome our tools for the months and perhaps years to come. During such time, we must continue to innovate and develop strategies to fight back against severe illness, and combination monoclonal therapies are one such avenue.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Antibodies TeamUpAgainst Omicron

April 29, 2022: 35B5: A Potent, Broadly -

Neutralizing Monoclonal Antibody

Effective Against All Known Variants

Monoclonal antibodies have proven to be a potent tool in our ability to prevent and treat SARS-CoV-2 infections. The current epidemic is driven in part by naturally occurring variants that elude vaccine-induced and monoclonal antibodies. The search is on broadly neutralizing antibodies. Here we describe one such antibody recently reported by Wang et al.in Japan.

Discovering35B5

In a previous study from earlier this year, Wang et al. attempted to discover broadly neutralizing monoclonal antibodies that would be effective against both existing and future variants. Their experiments took place in the latter stages of the Delta surge of infections. Sorting the B cells for immune memory of the SARSCoV-2 receptor-binding domain, they found a match.

Wang et al. cloned the monoclonal antibody 35B5. They then analyzed the neutralizing capacity, finding that 35B5 potently neutralized not only the SARS-CoV-2 wild-type but also a broad spectrum of variants, including Beta and Delta, leading the researchers to investigate the structural-functional mechanisms of 35B5 in greater detail.

35B5AlsoNeutralizesOmicron

After the rise of the Omicron variant, Wang et al . repeated their experiments in the context of the new variant. They observed 35B5 exhibiting binding efficiency to Omicron comparable to Delta and the SARS-CoV-2 wild-type, as well as trimer dissociation. The authors note that Omicron’s binding efficiency with 35B5 is slightly weaker than the wild-type likely because of a hydrogen bond between the antibody and N481 in the receptor-binding domain, which is impaired in the Omicron Fab interface.

In a neutralization assay, they found that both pseudotype and authentic Omicron viruses were potently neutralized by 35B5. While Omicron was not neutralized as effectively as Delta or the wild-type, it was still efficient enough to disable the virus.

35B5'sResidueTarget

Despite dozens of mutations in the Omicron Spike receptorbinding domain and N-terminal domain, 35B5 still binds tightly over a footprint of 29 interacting residues. These residues interact via salt bridges and hydrogen bonds to allow for interaction between the antibody and the Spike.

One crucial glycan that 35B5 interacts with is N165. One of the functions of glycans is to shield the virus particle beneath, but N165, along with N234, also possesses a unique responsibility. These two act together as a molecular switch to control the Spike conformational transition. The Spike alternates between up and down conformation based on whether it is currently infecting a host cell. The two glycans clamp the two sides of the receptor-binding domain and enable the trimer to alter between the up and down configurations, acting as a switch. The 35B5 antibody displaces

N165 from its binding pocket, disabling the switch mechanism, like breaking a light switch lever.

Were the Spike in the up configuration, Wang et al.found that the 35B5 antibody would continue to take a toll on the virus. When superimposed into the up configuration, the researchers observed the N165 and N234 glycans are displaced from their native binding pocket. This displacement results in significant dysfunction of the glycan switch necessary for transition between up and down configurations, suggesting that the 35B5 antibody forces the trimer in the up configuration, thereby destabilizing the Spike.

What makes this antibody so exciting is the conservation of the N glycans across SARS-CoV-2 genomes. Of the 10.5 million SARSCoV-2 genomes in the GISAID database, only 3,129 contain a mutation at position N165 or less than .03%. The same can be said about N234, which only displays a mutation in 1,723 viruses or less than .02%. This suggests that 35B5 could have neutralized over 99% of the viruses circulated since the start of the pandemic, meaning they could be a great tool for variants yet to come. Additionally, the mutations in Omicron are far from the 35B5 epitope, suggesting an additional advantage to 35B5. Because the 35B5 epitope residues are highly conserved, it is unlikely that Omicron RBD mutations interfere with 35B5’s virus neutralization.

This is not the only antibody that recognizes highly conserved sequences in the Spike. We note that Zhou et al. describe two antibodies that target conserved sequences in the Omicron Spike: Ly-CoV1404 and S2E12. Additionally, Li et al. describe the CV325 antibody, which binds a linear epitope in the S2. We suggest that a combination of 35B5 and CV3-25 might be ideal for the prevention and treatment of Covid-19. Similar combination

antibodies that bind both the membrane-associated protein and the receptor-binding protein display broad neutralizing activity against most of the existing strains of Ebola. Such a combination antibody therapy for Covid-19 could be a successful path for treatment and prophylaxis against current and future strains of the virus.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: 35B5: A Potent, Broadly-Neutralizing Monoclonal Antibody

EffectiveAgainst All Known Variants

July 13, 2022: Pasteur Institute Scientists

Discover SARS - CoV - 2 BroadlyNeutralizing Antibody

From the early days of the Covid pandemic, monoclonal antibodies have been billed as an effective means to treat and possibly prevent SARS-CoV-2 infections. The originally sparkling promise has been substantially tarnished as one monoclonal drug after another has succumbed to the rapid variation of the SARSCoV-2 virus, rendering them essentially useless for treatment.

Now a global search for new antibodies that hone in on regions of the virus Spike protein that are required for effects and, therefore, resist change is in effect. We have already discussed one such antibody, 35B5, as described by a group of virologists from China. Here we discuss another such antibody as described by scientists at the Pasteur Institute in Paris, France.

IsolationoftheVirus

As described by Planchais et al in the Journal of Experimental Medicine last month, the two antibodies Cv2.1169 and Cv2.3194 carry a notable headline: they potently neutralize both Omicron BA.1 and BA.2. Planchais et al. observed and cloned 102 human SARS-CoV-2 Spike monoclonal antibodies from the memory B cells of 10 convalescent Covid-19 patients. Most of the 102 mAbs are bound to the S2-region of the Spike protein, meaning nonbinding to the receptor-binding or N-terminal domain. None of

these S2 mAbs were neutralizing against the Wuhan strain in vitro. Roughly one-third of the remaining receptor-binding and Nterminal domain mAbs neutralized SARS-CoV-2 in vitro. The most potent of these were Cv2.1169 and Cv2.3194. Further analyzing both antibodies, the researchers found that both were fully active against previous variants of concern Alpha, Beta, Gamma, and Delta, as well as the earliest strains of Omicron BA.1 and BA.2. Between the two, 1169 most potently neutralized all variants from the Wuhan strain up through BA.2.

The researchers examined 1169 in greater detail as it neutralized recent variants more effectively than 3194, including Delta+, BA.1, and BA.2. Planchais et al. further enhanced 1169 with a purified Jchain containing IgA dimers as 1169 as a monomeric IgA antibody displayed muted effects against SARS-CoV-2. In other words, they greatly enhanced its neutralization against early and later variants by modifying the structure of the cloned antibody.

Cv2.1169Protection in AnimalModels

The Cv2.1169 antibody showed positive therapeutic effects during in vivo mouse and hamster SARS-CoV-2 infection models small rodents infected with SARS-CoV-2 mirror the disease course of a mild-to-moderate case of Covid-19 in humans.

A single injection of Cv2.1169 significantly reduced pulmonary viral infectivity and RNA levels. Intra-lung viral infectivity and RNA loads also reduced dramatically after using the 1169 antibody. These results were repeated with mice infected with Beta, among the most pathogenetically severe variants to circulate. The mice made a full recovery after treatment with 1169.

StructureofCv2.1169SpikeBinding

The Cv2.1169 antibody binds in a set of three to the trimer of the SARS-CoV-2 Spike protein receptor-binding domain in the up configuration.

Via cryo-electron microscopy, we can see the exact contact points of 1169 to the SARS-CoV-2 Spike protein, illuminating targets for current and future Covid-19 therapies. These contact points include Y473, S477, T478, F486, N487, and Q493.

PotentialEscapefromCv2.1169

Inspection of the GISAID database shows that mutations involving the binding sites for Cv2.1169 occur but in relatively low frequency. However, there is more immediate cause for concern as some binding sites are altered in the most recent iterations of Omicron BA.4, BA.5, and BA.2.75.

Y473 is unmutated in all Omicron strains, S477 is mutated to S477N in all Omicron strains, T478 is mutated to T478K in all Omicron strains, and N487 is unmutated in all Omicron strains. Because these are consistent, they should not have an altering effect in later Omicron variants. F486 is unmutated in BA.1 and BA.2, but is mutated to F486V in BA.4 and BA.5. Q493 is mutated to Q493R in all strains but BA.2.75, in which the position remains Q493.

Source:ACCESSHealthInternational

Whether these changes are significant enough to cause later Omicron variants to escape Cv2.1169 is unknown, so we implore further research to be conducted on Cv2.1169 to analyze the neutralization of newer strains.

This is but one of many potential broadly neutralizing antibodies that could work against currently circulating and future variants. We will continue to cover these antibodies as they are released because rather than playing catch up with SARS-CoV-2, we must start future-proofing our treatment arsenal as variants continue to arise.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere:

Pasteur Institute Scientists Discover SARS-CoV-2 BroadlyNeutralizingAntibody

Figure 10. TableofMutationaldifferencesin 1169contactpoints betweendifferentOmicronstrains.

July 20, 2022: New SARS - CoV - 2 Variant

BA.2.75 Evades All Approved Monoclonal Antibody Therapies

Viral variation has proved to be a critical weak point in our approach to medical solutions for controlling Covid-19. Over the last two and a half years, we've seen successive waves of reinfection by new variants of those who've been previously infected, those who have been vaccinated and boosted, and those who have been infected, vaccinated, and boosted as well. Behind this unfortunate dynamic is the dramatic variation in the structure of the virus exterior, specifically the Spike protein, which plays a critical role early in infection by binding to the cell surface and forcing entry.

Antibodies that recognize this structure can block infection. However, changes in the exterior structure negate antibody collections in convalescent sera and monoclonal antibodies from binding and neutralizing the virus. A recent study by Yamasoba et al. summarizes the effectiveness of existing monoclonal antibodies against a successive set of virus variants, namely the BA.2 variant, which first emerged in late 2021 and quickly spread around the world, driving the most infectious wave of the virus to date, BA 4/5, which are the predominant strains circulating at the time of writing, and BA.2.75, a new sublineage of BA.2 which is likely more infectious and immune evasive than its predecessors, suggesting it may be the predominant variant in the coming weeks and months.

Immediately, we note that five antibodies: adintrevimab, bamlanivimab, casirivimab, etesevimab, and imdevimab failed to neutralize any of the three Omicron sublineages. Casirivimab and imdevimab, as well as etesevimab and bamlanivimab, are designed to be used in tandem in an antibody cocktail, yet their combination antibodies were just as ineffective. Adintrevimab is intended for individual use, meaning its neutralization potency for the strains circulating today is nonexistent. These were among the first monoclonal antibodies developed, rationalizing why they are so ineffective against recent strains.

This leaves five individual monoclonal antibodies. Regdanvimab, sotrovimab, and tixagevimab did not neutralize the previously circulating BA.2 and the currently circulating BA.4/5. However, the three effectively neutralized the BA.2.75 pseudovirus. This suggests that if BA.2.75 became the dominant strain in the coming weeks and months, these three monoclonal antibodies could be effective treatments for those suffering from Covid due to this strain.

Of the two remaining antibodies, cilgavimab poorly neutralized BA.2 and BA.4/5 but was 24.4-fold worse against BA.2.75. Although bebtelovimab effectively neutralized BA.2 and BA.4/5, it again was much worse against BA.2.75, this time 21.2 to 25.6-fold. Despite poorly neutralizing BA.2.75 compared to BA.4/5, bebtelovimab still neutralized the strain better than any other antibody. Even newer generations of viruses recently detected in South Africa with more extensively mutated Spike proteins, against which bebtelovimab and others may perform even more poorly.

New variants evading monoclonal antibodies should come as no surprise. After infection, the convalescent sera of a recovered patient

contains many antibodies designed to inhibit the virus the host just overcame. For the virus to reinfect, it must mutate considerably to evade the convalescent antibodies. Monoclonal antibodies are effectively the same as convalescent antibodies on an individual scale. They are designed to overcome a virus by binding to specific amino acids on the Spike. If the virus mutates enough, the monoclonal antibody can no longer bind. This is how the cat and mouse game of developing antibodies and the virus mutating has continued for two and a half years.

What then can be done? The search is on for monoclonal antibodies that recognize regions of the virus that are critical to the virus life cycle and therefore are resistant to most mutations. In other words, scientists worldwide are rushing to identify and develop antibodies with broadly neutralizing capabilities, i.e., antibodies that recognize highly conserved sequences of the Spike protein that may overcome all viral variants.

The good news is that many such antibodies have already been identified. We recently described the Cv2.1169 antibody discovered by scientists at the Pasteur Institute and will continue to detail others as data is released. Whether these antibodies recognize and neutralize the latest variants such as BA.2.75 remains an open question.

A second potential solution is to use extensive combinations of functional monoclonal antibodies. While many fail to neutralize, some retain neutralizing capability against the latest variants, and new monoclonal antibodies are constantly advancing. Combining two, three, or four antibodies into a single treatment may suppress infection. Our hope remains high for monoclonal antibodies as a

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

short-term relief for those infected and, in the long run, as a prophylactic against infection in the first place.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: New SARS-CoV-2 Variant BA.2.75 Evades All Approved Monoclonal AntibodyTherapies

July 26, 2022: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies III

Alongside vaccines, monoclonal antibodies have been billed as the most effective means of treating and preventing Covid-19. This rang true for the first several months of the pandemic, as antibodies designed to neutralize the SARS-CoV-2 Spike protein were significantly effective. However, as the virus mutated, the potency of antibodies declined while virus immune evasion rose. To counter this trend, there is now an ongoing search for broadly neutralizing antibodies that can overcome not just a single strain but all strains of SARS-CoV-2, current and future.

In this series, we have discussed several pan-variant monoclonal antibodies, all of which show promise against current Omicron strains and previous variants of concern such as Alpha, Beta, and Delta. Here we analyze another described in a study by Luo et al.: the SP1-77 antibody.

SP1-77AntibodyOrigin

The typical search for monoclonal antibodies includes the collection of sera from Covid-19 patients and isolating the often dozens or hundreds of distinct antibodies found, testing them individually on pseudoviruses with the SARS-CoV-2 Spike for binding capacity, then sorting for the best neutralizers.

A team of researchers from the Massachusetts Institute of Technology took a different approach. Rather than a blind treasure hunt in the convalescent sera of a Covid patient, the researchers developed a specialized mouse model modified with human gene segments VH1-2 and Vκ1-33. These genes are associated with complementarity-determining-region-3 (CDR3) sequences. In essence, this modification results in a far more diverse B cell response when exposed to pathogens like SARS-CoV-2, leading to the identification of more unique and distinct monoclonal antibodies to test.

Luo et al. immunized the mouse models by exposing them to the Wuhan Spike twice over four weeks. All mice involved developed strong resistance to the SARS-CoV-2 Spike, yielding 96 antibodies for the researchers to test.

SP1-77AntibodyNeutralization

Of the 96 candidates, the researchers narrowed the field down to nine that targeted the Spike protein specifically and then down to three that potently neutralized the Wuhan strain of the virus: VHH7-5-82, VHH7-7-53, and SP1-77. These three were tested for neutralization against current and previous variants of concern, both pseudotype and live viruses.

Figure one demonstrates the neutralizing prowess of SP1-77. In the pseudotyped virus assays, SP1-77 neutralized all variants of concern tested to varying degrees, including Alpha, Beta, Gamma, Delta, and both previous and current Omicron strains. Alternatively, the other two antibodies tested failed to neutralize any Omicron strains and struggled against many other previous variants of concern.

The live virus neutralization paints a similar picture. Omicron was omitted from the live virus tests for safety, but SP1-77 again neutralizes Alpha, Beta, Gamma, and Delta, whereas the others again struggle.

SP1-77AntibodyTarget

A closer examination of the SP1-77 antibody via cryo-electron microscopy reveals an alternate solution to neutralization aside from inhibiting ACE2 binding. SP1-77 targets sites on the opposite side of the receptor-binding domain from the ACE2 binding site. In particular, the antibody epitope targeted positions ranging from 339346, 440-450, and 499. In most variants of concern, these amino acids are unmutated, with the lone exception being G339 and G446 in some Omicron strains being changed.

Further analysis found that the antibody binding results in either the blockage of S2’ cleavage and/or inhibition of S1 dissociation. What does this mean? After the virus binds a human ACE2 receptor, the S1 and S2 portions of the Spike must disengage for membrane fusion to occur. If the antibody is blocking either S2’ cleavage or S1 dissociation, it is inertly preventing the virus from fusing with the host cell, preventing that virus from replicating within the host cell and moving on to another.

We have previously described two other antibodies that bind to different sites. The first, 35B5, targets the N-terminal domain positions N165 and N234, which act together as a molecular switch for the Spike’s changing up and down conformations. The second, Cv2.1169, targets a different region of the receptor-binding domain than SP1-77, namely contact points including Y473, S477, T478,

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

F486, N487, and Q493. We will describe more in the coming days and weeks.

Echoing Luo et al., SP1-77 represents an opportunity for highly potent monoclonal treatments. In addition to being an effective antibody on its own, due to its distinct residue targets, SP1-77 could be easily paired with one or two other antibodies to create a highly potent antibody cocktail for the treatment and prevention of Covid19. Such cocktails should be immediately investigated.

Additionally, the novel humanized mouse model is a triumph of antibody discovery technology. The methods by Luo et al. should be further explored in all antibody discovery trials, potentially leading to dozens of antibodies to aid the millions continuing to get sick in this extended pandemic.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies III

July 28, 2022: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies

IV

Monoclonal antibody treatments remain our greatest asset in the fight against Covid-19, though in recent months, the effectiveness of these treatments has waned. New variants with more mutations have evolved to overcome antibodies from monoclonal therapies, vaccines, and prior infections. The search for monoclonal antibodies that neutralize not one but all strains is underway to counter these new variants.

In this series, we have discussed several pan-variant monoclonal antibodies, all of which promise against current Omicron strains and previous variants of concern such as Alpha, Beta, and Delta. Here we analyze another described in a study by Zhou et al.: the ZCB11 antibody.

ZBC11AntibodyOrigin

Hong Kong researchers used an uncommon antibody identification method in their study. Antibodies described previously in this series were identified either from the sera of those naturally infected with Covid-19 or from a specially-engineered mouse model. Here researchers use the sera of mRNA vaccine inoculated patients as the basis for antibody collection.

Collecting the sera of 34 Pfizer vaccinated subjects, Zhou et al. found that only two of the 34 samples had neutralizing activities

against all variants of concern included in the study, notably Alpha, Beta, Gamma, Delta, and Omicron BA.1. Subject #26 displayed the highest neutralization titers against Beta and Delta. They showed above-average neutralization of Alpha, Gamma, and Omicron. Zhou et al., therefore, took a closer look at the sera of this vaccine for broadly neutralizing antibodies.

The researchers collected another blood sample 130 days after the second vaccination. Fourteen antibodies were identified, while only seven (ZCB3, ZCB8, ZCB9, ZCB11, ZCC10, ZCD3, ZCD4) showed positive responses to WT spike. They narrowed these seven to four by limiting their search to receptor-binding domain-specific antibodies that displayed neutralization against the wildtype Wuhan strain, namely ZCB3, ZCB11, ZCC10, and ZCD3. Along with a control antibody, ZB8, these four moved onto VOC neutralization assays.

ZCB11AntibodyNeutralization

Zhou et al. conducted both pseudovirus and live virus assays for the four antibody candidates. Against the Alpha, Beta, Gamma, Delta, and Omicron BA.1 pseudotypes, ZCB11 was the only candidate to neutralize all variants and consistently outperformed other antibodies when they did neutralize.

Against authentic viruses, this result was replicated to an even greater degree. ZCB11 consistently and effectively neutralized all variants in the lineup, including Omicron BA.1, BA.1.1, and BA.2. We note that the currently circulating BA.4 and BA.5 were not included in the neutralization assays. Still, the potent neutralization of BA.2 and all earlier strains strongly indicates ZCB11’s broad neutralization.

ZCB11AntibodyTarget

Alongside many other monoclonal antibodies, The ZCB11 antibody targets the receptor-binding motif. Cyro-electron microscopy analysis of the antibody revealed the antibody binding to the Spike protein in the “up” conformation, wherein the Spike is preparing to bind the ACE2 receptor of the host cell.

Why does this antibody neutralize much more effectively than previous monoclonal treatments when they all bind the receptorbinding domain? We can attribute this to two things. One reason for the broad activity found with ZCB11 is that most of the amino acid contacts are highly conserved among all known coronavirus sequences.

The antibody footprint binds the Spike amino acids D420, L455, F456, N460, A475, S477, T478, F486, and N487. Three of these, S477, T478, and F486 are mutated in the latest Omicron strains to S477N, T478K, and F486V. In the GISAID SARS-CoV-2 sequence database, all positions aside from the three Omicron exceptions are mutated less than 5,000 times in over 12 million sequences, meaning the footprint of this antibody is highly conserved.

Notably, Zhou et al. found that S477N and T478K increase the binding affinity between the antibody and Spike, rather than interfering with neutralizing capability. They postulate that this is at least a partial explanation for the antibody's substantial neutralization of the variant.

The second reason is that most of the amino acid positions in ZCB11 are relatively unmutated in natural strains. Previous antibodies have contact points at major residues of mutation like

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

N501 and E484, but ZCB11 mostly lacks contact points at major mutational residues.

As we recommend with all broadly neutralizing antibodies, there is no reason to limit treatment to just one. An antibody cocktail of two or three monoclonal antibodies covering a broad footprint of conserved residues could be a powerful weapon against current and future strains, which are sure to continue mutating to evade immunity. We must prioritize and expedite these antibodies' production as the pandemic continues to rage.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies IV

August 01, 2022: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies V

As novel SARS-CoV-2 variants develop new mutations, their evasion from existing treatments and vaccines continues to increase. Antibodies from vaccines wane after a few months, antibodies from previous infections are often ineffective, and monoclonal therapies that worked against earlier variants struggle against current strains. The current state of the pandemic has sparked a search for monoclonal antibodies that neutralize not one but all strains to counter these new variants.

In this series, we have discussed several pan-variant monoclonal antibodies, all of which promise against current Omicron strains and previous variants of concern such as Alpha, Beta, and Delta. Here we analyze another described in a study by Dacon et al.: the COV44-62 and COV44-79 antibodies.

COV44-62andCOV44-79AntibodyOrigin

Researchers from the National Institute of Health and Scripps Research Institute began their search for monoclonal antibodies by a traditional method: examining the plasma samples of 142 previously infected donors. They scanned these samples not only for SARS-CoV-2 recognition but also for six other human coronaviruses, including SARS-1 and MERS. Of the 142 samples, 19 recognized SARS-CoV-2 and at least two other

betacoronaviruses. By finding antibodies that recognize multiple betacoronaviruses, the likelihood increases of finding antibodies that recognize various strains of SARS-CoV-2 as well.

From these 19 samples, they identified a staggering 673,671 IgG B cells. They tested for reactivity and binding specificity to a panel of coronavirus Spike proteins to narrow this pool. They eventually found a set of six monoclonal antibodies that bound all seven human coronaviruses tested.

COV44-62andCOV44-79AntibodyNeutralization

Introducing the set of six monoclonal antibodies to a neutralization assay of pseudotyped betacoronaviruses, Dacon et al. found that two antibodies, COV44-62 and COV44-79, showed the broadest functional neutralization, disabling SARS-CoV-2, SARS-CoV-1, and HCoV-OC43, as well as the alphacoronavirus HCoV-NL63 and HCoV-229E.

Perhaps more notably for our search, both COV44-62 and COV4479 strongly neutralized a host of SARS-CoV-2 variants. In more pseudotype assay neutralization tests, COV44-62 and COV44-79 effectively neutralized Alpha, Beta, Gamma, Delta, Mu, Omicron BA.1, BA.2, and BA.4/5. Neutralization of Omicron BA.4/5 is significant as they are still the majority sequence fueling infections worldwide as this is written in late July. In general, COV44-62 achieved neutralization at lower concentrations, indicating a more efficient antibody, but we note that COV44-79 neutralizes BA.4/5 more efficiently than COV44-62.

COV44-62andCOV44-79AntibodyTarget

While the rest of the antibodies in this series have also neutralized many variants of concern, what makes these two antibodies particularly special is their variant neutralization and Spike residue target. Most monoclonal antibodies target amino acids on either the receptor-binding domain, the N-terminal domain, or a combination of the two. These two domains facilitate ACE2 binding to the host cell, and blocking these connections is often an effective way to prevent host cell infection.

The COV44-62 and COV44-79, as well as the other four antibodies from the broadly recognizing set, all prefer to bind in the S2 portion of the Spike, specifically the fusion peptide. The two antibodies bind from amino acid positions 812-830 in the SARS-CoV-2 Spike protein.

Dacon et al. note that critical binding residues include R815, E819, D820, L822, and F823. These five residues are amongst the most conserved in the coronavirus genera Spike protein, all of which are conserved in at least 34 of 35 coronavirus species. This also holds true for SARS-CoV-2 variants of concern; none carry mutations at these five amino acids. In fact, in the GISAID SARS-CoV-2 sequence database, all five mutations are identified less than 5,000 times among 12 million sequences, many of which are likely dead viruses.

Early in vivo trials on Syrian hamster models found that those treated with COV44-79 recovered from moderate to severe symptoms within 3-7 days, and those treated with COV44-62 recovered in 5-7 days.

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

As we recommend with all broadly neutralizing antibodies, there is no reason to limit treatment to just one. An antibody cocktail of two or three monoclonal antibodies covering a broad footprint of conserved residues could be a powerful weapon against current and future strains, which are sure to continue mutating to evade immunity. We must prioritize and expedite these antibodies' production as the pandemic continues to rage.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies V

September 01, 2022: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VI

SARS-CoV-2 variants that evade the immune system are characteristic of the Covid-19 pandemic. As novel SARS-CoV2 variants develop new mutations, their evasion from existing treatments and vaccines continues to increase. Antibodies from vaccines wane after a few months, antibodies from previous infections are often ineffective, and monoclonal antibody therapies that worked against earlier variants struggle against current variants. The current state of the pandemic has sparked a search for monoclonal antibodies that neutralize not one but all variants to counter these new variants.

In this series, we have discussed several pan-variant monoclonal antibodies, all of which promise against current Omicron variants and previous variants of concern, such as Alpha, Beta, and Delta. Here we analyze two new antibodies described in a study by Low et al. that target a conserved site not only in SARS-CoV-2 variants, but in many similar coronaviruses as well.

AntibodyDiscovery

Low et al. began their search in a familiar fashion: isolating memory B cells from convalescent and vaccinated SARS-CoV-2 patients. This yielded many thousand samples from 43 individuals.

Next, the researchers introduced the samples to an ELISA panel of many viruses, testing for cross-reactivity to a broad range of coronaviruses. The tested viruses included SARS-CoV, MERSCoV, human coronaviruses OC43, HKU1, NL63, and 229E. A group of seven antibodies emerged cross-reacted against all Spike proteins tested, including alphacoronaviruses NL63 and 229E. The antibodies were sorted into three groups depending on their broad binding capacity, with Group 3 designated for those that bound all tested coronaviruses.

AntibodyNeutralization

The next step was to examine those that broadly bound for neutralizing capability. Testing all seven Group 3 antibodies for neutralization capabilities, Low and colleagues narrowed their search to two: C77G12 and VN01H1. The VN01H1 antibody neutralized all viruses examined, specifically SARS-CoV-2, SARSCoV, MERS-CoV, NL63, and 229E from the binding assay and bat virus WIV-1 and merbecovirus PDF-2180. C77G12 neutralized the binding assay betacoronaviruses but to the highest degree of all Group 3 antibodies.

HamsterStudy

Extending their studies to in vivo examination, they found that the Gamma variant SARS-CoV-2 virus in infected hamsters was potently neutralized by both antibodies, as were authentic Omicron BA.1 and BA.2 viruses. The researchers found that prophylactic administration of either C77G12 or VN01H1 reduced viral RNA copies and lung titers and ameliorated lung pathology at statistically significant levels.

Notably, earlier SARS-CoV-2 variants such as Alpha, Beta, and Delta were not examined in the study, nor were later Omicron variants, including the currently dominant BA.5 or BA.2.75 lineages.

AntibodyBindingSite

Low et al. examined the exact binding of the antibodies by cryoelectron microscopy, a method of freezing small proteins and enzymes to enable a high-definition image of very small biological masses. Such broad cross-reactivity with a wide variety of SARSCoV-2 relative viruses would require a highly conserved region of the Spike to bind. C77G12 and VN01H1 bind amino acids 811 through 825, which are highly conserved among all genera of the Orthocoronavirinae subfamily.

This conservation holds with all circulating SARS-CoV-2 variants to date. In the GISAID SARS-CoV-2 database containing 12.8 million sequences, the antibody binding epitope amino acids are only mutated a few thousand times.

Source:ACCESSHealthInternational

The Low antibodies share a very similar epitope to the Dacon antibodies, described in a previous entry in this series. Both target the highly conserved fusion peptide and many of the amino acid contact points overlap between the two, and as such, both are viable candidates for monoclonal treatment in the months to come.

Figure 11.Antibodyepitope residuemutations in theGISAIDdatabase as of8/31/2022.

Source:ACCESSHealthInternational

Notably, the epitope target is buried toward the core of the Spike trimer and is highly inaccessible.

How, then, do these antibodies bind a hidden target? Low and colleagues identified a crack in the fusion peptide’s defenses using transfected human embryonic kidney cells. During ACE2 binding, conformational shifting of the Spike trimer exposes the cryptic epitope. The coronavirus Spike can be in one of two conformations based on its infecting host stage. The “down” conformation is before ACE2 binding occurs, and the “up” conformation is as ACE2 binding occurs. The receptor-binding domain attaches to the ACE2

Figure 12. Venndiagramcomparing theamino acidfootprintofthe LowantibodiesandtheDaconantibodies.

receptor, revealing the fusion peptide, at which point C77G12 and VN01H1 can bind.

Antibody-MediatedPhagocytosis

Based on personal communications with the authors, we also note preliminary evidence that the antibodies may induce antibodydependent cellular phagocytosis. ADCP is a mechanism of elimination by which the monoclonal antibodies target cells to promote clearance from the body by phagocytic immune cells. The phagocyte engages with the antibody and engulfs the infected cell, leading to quicker, more favorable outcomes for the patient.

Summary

The Low study adds another useful pair of broadly neutralizing monoclonal antibodies to our growing armamentarium of SARSCoV-2 antibody treatments and prophylactics. In tandem with the Low antibody, additional antibodies could be used in combination to target other binding epitopes or trigger conformational changes to maximize antibody potency. Combination antibody therapies present the best weapon to overcome the virus and clear infected cells in Covid-19 patients. We recommend immediate high scale production of such combinations as the pandemic continues to rage in its third year.

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Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VI

September 06, 2022: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VII

SARS-CoV-2 variants that evade the immune system are characteristic of the Covid-19 pandemic. As novel SARS-CoV2 variants develop new mutations, their evasion from existing treatments and vaccines continues to increase. Antibodies from vaccines wane after a few months, antibodies from previous infections are often ineffective, and monoclonal antibody therapies that worked against earlier variants struggle against current variants. The current state of the pandemic has sparked a search for monoclonal antibodies that neutralize not one but all variants to counter these new variants.

In this series, we have discussed several pan-variant monoclonal antibodies, all of which promise against current Omicron variants and previous variants of concern, such as Alpha, Beta, and Delta. Here we analyze two new antibodies described in a study by Fenwick et al.that target a conserved site in SARS-CoV-2 variants.

AntibodyDiscovery

Collecting sera samples from over 100 donors of varying vaccine and infection status, Fenwick and colleagues focused their search on the sera of a post-infected donor who had received a full dose of Moderna mRNA vaccine, yielding six monoclonal antibodies for cloning and testing.

The six clones were introduced to a binding assay against a panel of SARS-CoV-2 variants to whittle further the field, including the original Wuhan Spike, Alpha, Beta, Gamma, and Delta. Two antibodies, P2G3 and P5C3, outperformed the five others and became the focal point of examination for Fenwick and colleagues moving forward.

NeutralizationComparisontoExistingmAbTherapies

Currently available monoclonal antibody treatments, such as the AstraZeneca cocktail (cilgavimab + tixagevimab), the Regeneron cocktail (casirivimab + imdevimab), and Sotrovimab, are not effective against the Omicron family of variants, including BA.1, BA.2, BA.5, and BA.2.75, with the possible exception of bebtelovimab and sotrovimab against BA.2.75.

Not only does the P2G3 antibody bind and neutralize the Wuhan, Alpha, Beta, and Delta variants to a greater degree than any of the above-listed monoclonal treatments. It also wholly neutralizes live Omicron BA.1 and BA.2 virus. Both alone and in combination with P5C3, the P2G3 antibody’s IC50 values consistently remain under .025 µg/ml.

Notably, Fenwick and colleagues did not include neutralization data for the currently circulating BA.5 or BA.2.75 variants. However, they suggest that the novel mutations in those viruses would likely not impact neutralizing activity. Regardless, a further examination should be taken to confirm that hypothesis.

Antibody-DependentCellularCytotoxicityandAntibodyDependentCellularPhagocytosis

The researchers next examined the antibodies for antibodydependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Both function to clear infected cells from the host. ADCC is the enabling of host immune cells to kill virus-infected cells, whereas ADCP is the enabling of host immune macrophages to absorb and kill infected cells.

They found that P2G3-mediated ADCC was more robust than any other monoclonal antibody tested, including the other cloned antibodies and the AstraZeneca and Regeneron cocktails. For ADCP, Fenwick and colleagues found greater activity in the P2G3 + P5C3 combination rather than in isolation, adding a significant data point in favor of the tandem.

LiveAnimalStudies

To examine the use of P2G3 as both a prophylactic and therapeutic in live hosts, Fenwick and colleagues administered the antibody to both hamster and macaque models. In hamsters, the antibody was administered two days before infection with the Wuhan virus. After four days of infection, only one of six infected hamsters had detectable infectious virus in the lungs, which was a massive fourlog reduction from the control group.

In the macaques, the P2G3 antibody was administered 72 hours before infection with the BA.1 virus. The researchers found that peak viral load in the treated macaques was roughly half that of the control group, suggesting P2G3 maintains therapeutic efficacy even against later Omicron viruses.

BindingEpitope

P2G3 and P5C3 fall into the antibody group that binds the receptorbinding domain, many of which we have described in this series.

Using the widely adopted method of cryo-electron microscopy, Fenwick and co. found the exact binding epitope of the antibodies. The two antibodies in conjunction interact with residues 344-347 and 440-451 for a total of 16 receptor-binding domain contact points. According to the GISAID sequence database, these residues are highly conserved, aside from three notable exceptions, all commonly found in the major versions of the Omicron family. However, as the neutralization assays demonstrate, these mutations clearly do not impede Omicron neutralization.

Source:ACCESSHealthInternational

The Fenwick antibodies share many overlapping residues with antibodies we have previously described in this series. Most notably, the Luo and Wang antibodies. All three are viable receptor-binding domain targeting antibodies that would prove vital to combination therapy in tandem with fusion domain and S2 targeting antibodies.

Source:ACCESSHealthInternational

The researchers also make a note of P2G3’s angle of attack. Many monoclonal antibodies are structured such that they can only bind at certain angles to the receptor-binding domain when it is in a specific conformation, whether up or down. This includes the Regeneron cocktail REGN10987, which can only bind to the upRBD. In contrast, P2G3 can bind in either conformation from a

Figure 13.Antibodyepitope residuemutations in theGISAIDdatabase as of9/2/2022.

Figure14. Venndiagramcomparingtheamino acidfootprintofthe Fenwickantibodies,theWangantibody,andtheLuoantibody.

different angle, giving the antibody more flexibility and potentially one catalyst behind its broad neutralization.

Conclusion

While this antibody lacks data on the latest versions of Omicron sweeping the world, its binding and neutralization speak for themselves. It shares many binding residues with antibodies that we know are effective against the latest variants and outcompetes many commercially available monoclonal therapies. Add this antibody to the growing list of weapons we can use to combat the everpresent SARS-CoV-2 virus as Omicron continues to infect, mutate, and spread.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VII

September 12, 2022: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VIII

SARS-CoV-2 variants that evade the immune system are characteristic of the Covid-19 pandemic. As novel SARS-CoV2 variants develop new mutations, their evasion from existing treatments and vaccines continues to increase. Antibodies from vaccines wane after a few months, antibodies from previous infections are often ineffective, and monoclonal antibody therapies that worked against earlier variants struggle against current variants. The current state of the pandemic has sparked a search for monoclonal antibodies that neutralize not one but all variants to counter these new variants.

In this series, we have discussed several pan-variant monoclonal antibodies, all of which show promise against current Omicron variants and previous variants of concern, such as Alpha, Beta, and Delta. Here we describe one promising new antibody described in a study by Ishimaru et al. that targets a conserved site in SARS-CoV2 variants.

AntibodyDiscovery

Ishimaru and colleagues began searching for antibodies in previously infected, two-dose-vaccinated patients with the Pfizer mRNA vaccine, examining the sera of three patients led to ten antibodies cloned for further testing.

Three of the ten stood out in ELISA binding assays: MO1, MO2, and MO3. All three efficiently bound the D614G variant of SARSCoV-2 and Omicron BA.1 and BA.2. MO1 and MO2 also bound to the Delta variant. Most notably, MO1 effectively bound the pervasive BA.5 variant driving the majority of infections worldwide at the time of writing.

AntibodyBroadNeutralization

Ishimaru and colleagues next performed neutralization examinations on the three antibody candidates to determine if they killed virus particles to the same efficiency as they bound. MO3 was the worst of the three, failing to neutralize even D614G strongly. In second place was MO2, which eliminated Omicron BA.1 and BA.1.1 with excellent efficiency, and D614G, Delta, and BA.2 to a lesser degree.

The true standout of the group was the MO1 antibody. It strongly neutralized the D614G, Delta, BA.1, BA.1.1, BA.2 variants, and BA.5 easily in the 10-100 ng/mL range. Most of the monoclonal antibodies described in this series strongly neutralize BA.1 and BA.2. However, they drop off significantly against BA.5. Furthermore, Ishimaru and colleagues note that MO1 also binds and neutralizes BA.2.75 with significant efficiency, another feat often lost among other monoclonal antibody candidates.

BindingEpitope

Unfortunately, the exact binding map of MO1 is unavailable at the time of writing. Cryo-electron microscopy analyses are underway as MO1 is further investigated as a vital anti-Covid monoclonal antibody treatment or prophylactic. The researchers note that the

antibody binds the receptor-binding domain and likely the receptorbinding motif, the contact region between the virus and the human host cell.

As the exact amino acids are unavailable, we cannot compare MO1 to other similar monoclonal antibodies described previously. However, given the antibody's substantial neutralization of BA.5 and BA.2.75, MO1 likely avoids major mutation sites in the latest Omicron variants, such as L452R and F486V.

Conclusion

When the binding map becomes available, it would be worth investigating a combination antibody that includes MO1. While the receptor-binding domain is one of the most highly active regions of the virus in a mutational context, MO1’s neutralization of the latest Omicron variants suggests an evasion of these mutations. Pairing MO1 with one or two others could yield a powerful treatment for Covid-19 as of yet unseen in the ongoing pandemic.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Progress In The Search For Broadly Neutralizing Monoclonal Antibodies VII

October 05, 2022: Broadly Neutralizing

Monoclonal Antibodies For Covid - 19 Treatment, Prevention, And Vaccine Design

Recent progress on monoclonal antibodies raises the possibility that the means to prevent and treat SARS-CoV-2 infections may soon be at hand. The hope arises from discoveries of antibodies that have the potential to neutralize all known SARS-CoV-2 variants and other related coronaviruses, including SARS-1 and MERS. A strategy for Covid control is possible using combinations of these antibodies for the treatment and pre-and post-exposure prophylaxis. Such treatments may eventually be combined with highly active antiviral drugs to end the pandemic.

There is an acute need for drugs to treat and prevent SARS-CoV-2 infection regardless of variants. Current vaccines dramatically reduce hospitalization and death from multiple variants. However, protection from infection and transmission diminishes with time and as the virus mutates. Recurrent viral variants are common despite multiple vaccine boosts and prior infection. The infection of hundreds of millions of people dramatically increases the risk that even more transmissible and virulent variants may arise, not to mention the ever-increasing burden of Long Covid. The possibility of increased virulence is real. Recall that SARS-1 and MERS kill 10% and 30% of those infected, respectively. The recently discovered broadly neutralizing monoclonal antibodies provide

near-term hope effective for variant-independent prevention and treatment as we await the discovery of highly active antiviral drugs.

MonoclonalAntibodiesthatNeutralizeSARS-CoV-2

Monoclonal antibodies are one of the most powerful tools for treating viruses and other infectious diseases. They target specific surface structures and either eliminate the virus from the host bloodstream or destroy the infected cell in which it resides. An advantage and disadvantage of monoclonal antibodies reside in their specificity. Monoclonal antibodies target specific structures on the surfaces structures leading to their destruction and clearance. Mutations that alter the structure of the binding site render monoclonal antibodies useless.

Pharmaceutical and biotechnology companies have developed monoclonal antibodies that neutralize SARS-CoV-2. These antibodies have been directed at the Spike protein on the virus's exterior. This is a favored target, as studies show that greater than ninety percent of naturally-occurring antibodies that neutralize SARS-CoV-2 are directed against the Spike protein. The targets in the Spike protein are the primary receptor-binding domain (RBD) and the secondary N-terminal domain (NTD).

The Spike is an intertwined trimer of three S proteins. Each of the three is composed of two subunits: S1, the membrane distal region, including the receptor-binding and N-terminal domains, and S2, the membrane-proximal protein. The receptor-binding domain atop S1 can assume two configurations: up, capable of binding the ACE 2 receptor, or down, non-binding.

The original monoclonal antibodies approved for clinical use were met with great success, potentially neutralizing the virus both

singularly or in combination. Unfortunately, the utility of these antibodies waned quickly. The virus has mutated significantly over the last two and a half years, leading to evasion of the immune response to infection. These mutations also abrogate neutralization by many of the FDA-approved monoclonal antibodies. This seems to be a never-ending game of catch-up. First, the virus changes, then new antibodies are created to recognize the new variants. Next, variants mutate to evade natural and monoclonal antibody immunity. This endless cycle resulted in the rapid deterioration of antibody potency against themes' recent variants (Table 1). Note that even the most broadly neutralizing antibody available today, bebtelovimab, has reduced activity against the BA.2.75 variant currently circulating in Asia and Europe.

Figure 15. Neutralizationbyavailableantibodytreatmentsagainstthe latestOmicronvariants as definedbytheir50%inhibitoryconcentration (IC50;ng/mL)values.+++ indicates<20ng/mL.++ indicates20-50 ng/mL.+ indicates50-200ng/mL. - indicates>200ng/mL.

Source:ACCESSHealthInternational

Discovery ofa SetofBroadlyNeutralizingAntibodies

To circumvent this cycle, researchers worldwide sought and found monoclonal antibodies that recognize highly conserved regions of the virus to neutralize most, if not all, variants. Some also neutralize SARS-1, MERS, and related Bat betacoronaviruses, and some even neutralize human betacoronaviruses that use the ACE2 protein as the receptor (Table 2). These antibodies have multiple origins, from convalescent and vaccinated volunteers to mice, alpacas, and macaques.

(+)indicatesthatthegivenvirus islikelyneutralizeddueto the conservation oftheaminoacidsequencetargettedbythecorrelating SP1-77antibody.+++ indicates<20ng/mL.++ indicates20-50ng/mL. + indicates50-200ng/mL. - indicates>200ng/mL.

Source:ACCESSHealthInternational

Despite differing binding sites on the Spike protein, the broadly neutralizing antibodies share one common property. Most amino acid contacts between the antibody and the spike are highly conserved, not only amongst all SARS-CoV-2 variants but also with the closely-related human and bat coronaviruses. Specifically,

Figure 16. NeutralizationofearlySARS-CoV-2variants, Omicron familyvariants, otherbetacoronaviruses,andhumanbetacoronaviruses.

mutations in the amino acid binding sites are exceedingly rare in the over 12 million sequences deposited in international databanks. These amino acids likely perform a critical function. For example, the monoclonal antibody COV44-62/79 targets highly conserved the fusion peptide. The 35B5 antibody binds a crucial region that serves as a hinge for the up-down configuration father receptor binding domain. Wang et al. report that binding to the region dissociates the trimer. The Camelid antibodies lock the receptor binding domain in the down, inactive configuration. The Li antibody binds the very highly conserved linear epitope in the S2 region near the virus membrane.

Source:ACCESSHealthInternational

Figure 17.Broadlyneutralizingantibodiesandtheiraminoacidepitope footprints.

At the time of writing, there is another preliminary antibody candidate by Cao et al., SA55+SA58, which shows strong Omicron neutralization. This set binds the receptor-binding domain in a similar configuration to the Fenwick antibodies P2G3/P5C3. We will do additional in-depth analysis of this new set at a later time. We have reached out to the researchers behind each antibody to discern their current status. While none of those described here are clinically approved or are currently undergoing human trials, many of the authors indicated that after further preliminary data collection, they anticipate clinical trials in the coming months.

New MonoclonalTriadsforTreatment andPrevention

The protean nature of SARS-CoV-2 to sustain viable mutations in the Spike protein merits caution. A strategy of combining three of the broadly neutralizing antibodies rewires an escape variant that alters three highly conserved bindings sites simultaneously, an unlikely possibility..

ofthebebtelovimab,Li,and Dacon/Lowantibodies.Bebtelovimabingreen bindsthereceptorbindingdomain,inhibitingACE2contact. TheLiantibodyin blue

Figure 18.(A)Bindingepitopes

bindstheS2region near theviralmembrane,inhibitingfusion.The DaconandLowantibodiesin redbindthefusionpeptide,inhibiting post-contact fusion.(B)Venndiagramcomparingtheepitopes ofthe bebtelovimab,Dacon,andLi antibodies.(C)Venndiagramcomparing thebebtelovimab,Low,andLi antibodies.

Source:ACCESSHealthInternational

We note there are a number of mutations common to binding sites in bebtelovimab. Specifically, key residues such as N440, Q498, and N501 are commonly mutated in the latest Omicron family variants. In the future, more mutations in these residues may occur in emerging variants, potentially rendering bebtelovimab less effective.

Furthermore, cryptic sewershed variants described by Marc Johnson and colleagues display similar mutations throughout the receptorbinding domain. Were these variants to gain traction, bebtelovimab’s neutralization capacity would likely be severely hampered due to mutations in key binding sites.

Treatment andPrevention

For treatment, monoclonal antibodies must be used in the first five or six days of infection, as the virus concentration decreases sharply in most people after five days.

Post-exposure prophylaxis is the prevention of infection and disease in people known to be exposed to the virus. The duration of such treatment with antiviral drugs and monoclonal antibodies should be no longer than ten days, the maximum incubation period for signs and symptoms of Covid-19.

The third use, pre-exposure prophylaxis, is the use of long-acting monoclonal antibodies and highly-active antiviral drugs for those under conditions of high risk of infection. This may include people

living in close quarters, for example, military bases, ships, sea cruisers, schools and hospitals, and nursing homes. High-risk situations may also pertain to entire communities where the infection rate exceeds five percent of the local population.

Source:ACCESSHealthInternational

Delivery

At present, most monoclonal antibodies require intravenous infusion. However, some monoclonal antibodies, such as Evusheld, are administered via an intramuscular injection. Engineering the proposed combination antibody to be administered by intramuscular or subcutaneous injection would significantly increase widespread public access and acceptance of the drug.

Caveats

One caveat is that each of the broadly neutralizing antibodies described (and there will be more) was developed by different

Figure 19. Covid-19infection/symptomdurationaftermonoclonal antibodytreatment.

independent labs and may be licensed to diverse biotechnology and pharmaceutical companies. Development of the ideal antibody cocktails may require that the pharmaceutical industry and the NIH work in tandem. Solutions to such issues have been successfully addressed to develop effective combination therapies for cancer and HIV using the global National Institutes of Health office network.

A second caveat is potency. Some of the antibodies are active in the IC50 in the low nanogram range. Others are significantly less potent. All the antibodies described here may require additional engineering for potency, extended half-life, and antibodydependent cellular cytotoxicity and phagocytosis all possible with extant technology.

The cost to manufacture is another caveat. At present, the price of monoclonal antibody treatments is loosely tied to manufacturing costs. Antibodies can be produced at a cost of about $250 a gram. At this price, the manufacturing cost of a potent triad dose might well be under $100.

Summary

Combinations of broadly neutralizing monoclonal antibodies administered in tandem with long-awaited highly effective small molecule antiviral drugs are our best chance to deliver the final knockout blow to Covid-19 by preventing infection, transmission, and disease.

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Broadly Neutralizing Monoclonal Antibodies For Covid-19 Treatment, Prevention, And VaccineDesign

October 21, 2022: Broadly Neutralizing

SARS - CoV - 2 Antibodies From Immunized Macaque Monkeys

Monoclonal antibodies have been effective in both preventing and treating SARS-CoV-2 infections. However, just as SARSCoV-2 evolves to evade immune responses by vaccines and natural infection, so too do variants arise that evade neutralization by specific monoclonal antibodies, hence the search for antibodies that broadly neutralize regardless of the source. We have previously described eight such antibodies derived from infected humans, mice, and camelids. Here we describe a different approach in which antibodies are isolated from monkey subjects.

In the search for such antibodies, He et al. discovered a series of antibody candidates induced in macaque monkeys. The researchers immunized macaques over a ten-week period and drew the antibodies from sera post-inoculation. They used a recombinant prefusion-stabilized soluble S-protein plus a saponin (SMNP) adjuvant as their vaccine, yielding strong antibody responses in the animals.

NeutralizationofSARS-CoV-1andSARS-CoV-2

Not only did the enhanced adjuvant immune serum grant the macaques with significant virus neutralization of SARS-CoV-2 but the treatment also neutralized macaques infected with SARS-CoV1, indicating broad neutralization capability. This response is in

stark contrast to human natural infection, which rarely induces cross-neutralizing activity against SARS-CoV-1. Inoculation with mRNA SARS-CoV-2 did not yield SARS-CoV-1 neutralization in monkeys or humans, and inoculation with the enhanced adjuvant S protein only yielded a significant SARS-CoV-1 response in monkeys. The sera of adjuvant vaccinated monkeys bound and neutralized both viruses with great consistency.

IsolationofMonoclonalAntibodies

The intention behind using enhanced adjuvant S protein as a vaccine in monkeys is to enable enhanced memory B cell responses, from which strong antibody candidates can be isolated. These differ from most monoclonal antibody candidates, which derive from plasma cells.

The researchers isolated many monoclonal antibody candidates from the macaque sera, but two stood out for binding and neutralization: K288.2 and K398.22. Both bind and neutralize a wide variety of viruses, including both SARS viruses and bat viruses WIV1 and RaTG13. Additionally, both neutralize variants of SARSCoV-2 from the wildtype through Delta effectively. However, K288.2 loses neutralization capability against BA.1, whereas K398.22 maintains it.

Cyro-ElectronMicroscopy:TheAngleMay MakeAllThe Difference

The researchers note that differences in binding capacity between different viruses may be attributed to inherited differences between macaque and human sera from the immune system. Each species uses a different immunodominant germline gene segment in its

antibody development, leading to different approach angles and binding modes when introduced to the receptor-binding domain of SARS-CoV-2 and others.

High-ResolutionX-RayCrystallography

X-ray crystallography allows researchers to observe specific amino acid binding residues as the antibodies come in contact with the virus. Not only do K288.2 and K398.22 bind conserved regions in the receptor-binding domain that are less often targeted in other monoclonal candidates, such as 373-375, 404-408, and 502-508, but there is additional evidence suggesting that the macaque germline introduces a specifically unique binding motif not found in humanderived antibodies, E33 in CDRH1, which perhaps is another cause for the bolstered SARS-CoV-1 neutralization in the macaque antibodies.

Caveats

The first caveats come from the isolation of the antibodies themselves. The sera was developed from an irregular and powerful adjuvant that was administered over a much longer 10-week period, meaning comparison to mRNA-derived sera in a more consistent four-week period is not directly apples to apples.

These antibodies would be of much greater significance if they demonstrated binding and neutralization of later Omicron variants such as BA.5, BA.2.75, or BA.4.6, though there have been no published results discussing these variants. Through personal communications with the authors, we have confirmed that K398.22 neutralization drops significantly against BA.2, which does not bode well for later Omicron variants.

Speculation

The macaque isolation process may be a favorable system for isolating new monoclonal antibodies to be used as drugs. The Omicron viruses circulating today have been selected against the human immune responses, not macaque. This study shows that the inherited germline sequences in macaque antibodies may give them a leg up against emerging variants tailored to evade human antibodies. That is not to say that K288.2 and K398.22 evade currently circulating strains. Rather, the method used to discover these two may yield others yet to come and the macaque isolation process is one that should be thoroughly investigated.

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BroadlyNeutralizingSARS-CoV-2AntibodiesFromImmunized MacaqueMonkeys

October 26, 2022: As Protection From Current Covid - 19 Monoclonal Antibody Treatments Fades, The Discovery Of

A New Class Of Antibodies Brings Hope

The latest Omicron subvariants are evolving to become more immune evasive, rendering monoclonal antibody treatments such as Evusheld and the forthcoming bebtelovimab ineffective against new strains. With surges predicted in the winter and few mitigation measures left in place, over 17,000 immunocompromised Americans and older populations in nursing homes who rely on these treatments could be left vulnerable to severe disease and high mortality rates. As reported in STAT news, Biden administration officials are “racing to game out other antibody options [to protect these populations].”

However, there is hope in the discovery of at least two new classes of antibodies, which appear to be impervious to new virus mutations. They could provide a long-term solution to the issue of SARS-CoV-2 drug resistance fueled by evolving variants. Unlike previous waves driven by a single ominous variant, current and future waves will be driven by multiple subvariants of Omicron. Each new subvariant accumulates mutations in similar parts of the receptor binding domain, a critical spot in the spike protein where virus-blocking antibodies dock, continuing to enhance the virus’s immune dodging capabilities.

The most concerning of the emerging immune-evading variants are BQ.1.1 and XBB. The two variants currently represent under 5% of cases in the United States and worldwide, but that figure is likely to rise. In addition to the various mutations established in their ancestral strains, BQ.1.1 and XBB carry several new mutations in the spike protein that enable their further evasion of approved monoclonal antibodies, including, but not limited to V213G/E, G339D/H, and F486V/S.

among themost immuneevasivemutationsto date.

Source:ACCESSHealthInternational

All previously approved monoclonal antibodies work by binding the receptor-binding domain at positions mutated in these new strains,

Figure 20.Comparativespikemutations ofBQ.1.1,XBB,andDelta variants ofSARS-CoV-2.Thosehighlightedare

suggesting that they will lose most, if not all, neutralization against new subvariants.

However, two new groups of monoclonal antibodies in development may circumvent these and future Omicron variants by binding at alternative conserved sites to the receptor-binding domain that rarely mutate.

The first set is antibodies that bind the fusion peptide and surrounding region. Two examples are COV44-62/79 by Dacon et al. , and VN01H1/C77G12 by Low et al.These two antibodies have binding epitopes between positions 812 and 824. As with most variants throughout the pandemic, BQ.1.1 and XBB lack mutations in this range, meaning the antibodies likely maintain neutralization capabilities despite the highly mutated spike protein.

The second type of antibody binds in the lower S2 region proximal to the membrane. An example in this set CV3-25 by Li et al. CV325 binds from positions 1149 to 1165, which is unmutated in both BQ.1.1 and XBB.

Not only could these antibodies neutralize all SARS-CoV-2 variants, but other related coronaviruses such as MERS and SARS-CoV-1.

These antibodies are not approved, nor have they been approved for human trial, but ongoing experiments in mice and hamsters show great promise in their Omicron neutralization. Most significantly, COV44-62/79 demonstrated substantial neutralization even against BA.5.

Using combinations of antibodies could also further protect against SARS-CoV-2 drug resistance. We can create an insurance policy against future mutated variants by pairing conserved targets.

Another issue is that the strength of binding of non-receptor-binding domain antibodies is lower than those that bind the receptorbinding domain. The binding strength can be boosted through modification of the variable end of the antibody. They can be further optimized by increasing half-life by up to three months or improving cell toxicity by modifying the stem portion of the antibody known as the Fc receptor. All such techniques are wellestablished and reasonable methods to be explored.

Here we have discussed the use of monoclonal antibody drugs for the prevention and pre and post-exposure and treatment of Covid19. The alternative approach, which has been successful for HIV, is the use of small-molecule drugs to prevent and treat AIDS and other HIV-related complications. Currently, more than 30 drugs are approved for the prevention and treatment of HIV infection, some of which provide durable protection between two and six months with a single injection. It is clear that while we continue to pursue the development of broadly active monoclonal antibodies, we should accelerate our programs to discover and deploy highly effective, long-acting small molecule drugs targeted to Covid-19.

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As Protection From Current Covid-19 Monoclonal Antibody

TreatmentsFades,TheDiscovery OfANew ClassOfAntibodies

BringsHope

December 22, 2022: New Monoclonal Antibody Fully Approved For The Treatment Of Covid - 19

The Food and Drug Administration has approved an early gift for those suffering from Covid-19 this holiday season.

Tocilizumab, branded Actemra by pharmaceutical company Genentech, is a monoclonal antibody now approved for treating Covid-19 in hospitalized adult patients in moderate to severe conditions. All treatments to this point of the pandemic were only temporarily approved via emergency use authorization, speaking to the value of Tocilizumab. Here we discuss how the antibody was discovered and how it works.

Unlike many monoclonal antibody treatments we discuss, Tocilizumab has a long history dating back 25 years. Clinical development of the antibody began in 1997 by Chugai Pharmaceuticals in Japan to treat rheumatoid arthritis. Chugai Pharmaceuticals and Genentech are now under the Roche Group umbrella, a Swiss multinational healthcare company.

The researchers at Chugai Pharmaceuticals found that specific mouse antibodies contained anti-human IL-6 receptors. Interleukin-6, or IL-6, is a secreted cytokine protein expressed by white blood cells. In terms of disease, IL-6 stimulates inflammatory processes when the body is exposed to various pathogens and clinical conditions such as diabetes, multiple sclerosis, and rheumatoid arthritis.

Upon identifying the anti-human IL-6 receptors in mice, the researchers grafted the receptor onto a human IgG constant Fc region using recombinant DNA technology, effectively humanizing the receptor.

Later research by Kishimoto et al. confirmed that the engineered mouse antibody receptor grafted to the human IgG constant Fc exclusively blocked IL-6 trans-signaling without affecting other significant pathways.

IL-6 inhibition sufficiently blocks inflammation without inhibiting other immune defenses against infection. Contrary to popular belief, many of the classic symptoms we experience when we are sick are not directly caused by an invading pathogen but rather consequences of our immune system fighting off the pathogen.

Inflammation is one of the most prominent causes of body pain, fatigue, fever, rash, and other significant symptoms most endure during sickness. In severe cases, inflammation may cause severe pain, lack of appetite, and severe headaches and chills.

To produce the drug en masse, Chugai Pharmaceutical used genetic manipulation of host cells. They encoded the genes that code for tocilizumab production onto a human host T-cell. The encoded T-cell is then transfected into Chinese hamster ovary cells. These cells are then manipulated to express countless tocilizumab genes, creating a master that can be copied several times.

In 2005, Tocilizumab was approved in Japan to treat Castleman’s disease, a rare disorder involving hyperactive immune systems and chronic hyper-inflammation. By 2010, the drug was approved in the United States and the European Union for the treatment of rheumatoid arthritis.

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

More recently, the drug was approved via emergency use authorization by the FDA in June 2021 for the treatment of Covid19 in hospitalized patients receiving systemic corticosteroids and require supplemental oxygen, non-invasive or invasive mechanical ventilation, or extracorporeal membrane oxygenation (ECMO). Its full approval by the FDA is a testament to the drug's effectiveness in hospitalized patients. While much of the world is content to declare the pandemic over, case numbers and hospitalizations beg to differ. We must continue to search for every available treatment and pursue their distribution with the same urgency we had in the early months of the pandemic. In the next article, will discuss tocilizumab’s efficacy for the treatment of Covid-19, leading to its approval.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: New MonoclonalAntibodyFullyApproved For The Treatment Of Covid-19

De cember 28, 2022: How Recently

Approved Tocilizumab Treats Covid - 19

The treatment of severe Covid-19 has proven difficult over the past three years, illustrated most clearly by the lack of approved drugs to treat the disease. Recently, the Food and Drug Administration has just its third fully approved treatment, joining Baricitinib and Remdesivir. Baricitinib is approved for the treatment of COVID-19 in hospitalized adults requiring supplemental oxygen, non-invasive or invasive mechanical ventilation, or extracorporeal membrane oxygenation (ECMO). In contrast, Remdesivir is approved for all patients with mild-tomoderate COVID-19 who are at high risk for progression to severe COVID-19, including hospitalization or death.

Tocilizumab, branded Actemra by pharmaceutical company Genentech, is a monoclonal antibody now approved for treating Covid-19 in hospitalized adult patients who receive systemic corticosteroids and require supplemental oxygen, non-invasive or invasive mechanical ventilation or extracorporeal membrane oxygenation (ECMO), akin to Barcitinib. All treatments to this point of the pandemic were only temporarily approved via emergency use authorization. In a previous article, we discussed the antibody’s discovery and mechanism of action. Here we discuss how Tocilizumab treats those suffering from moderate to severe Covid19.

Chief among Covid-19 symptoms are inflammatory conditions such as body pains, headaches, fatigue, and others. Recent studies even

link long-term Covid-19 inflammation to permanently impacting the lungs, kidneys, and brain. Treating inflammation in moderate to severe Covid-19 patients could quell both short and long-term consequences of infection.

As a highly active anti-inflammatory monoclonal antibody treatment used for over ten years to treat Castleman’s disease, rheumatoid arthritis, and other forms of chronic inflammation, Tocilizumab was a prime candidate for treating Covid-19 inflammation. Tocilizumab is an IL-6 inhibitor, a secreted cytokine protein expressed by white blood cells. In terms of disease, IL-6 stimulates inflammatory processes when the body is exposed to various pathogens and clinical conditions such as diabetes, multiple sclerosis, and rheumatoid arthritis.

Both the emergency use authorization issued for Tocilizumab in June 2021 and the recent full approval by the Food and Drug Administration were based on the University of Oxford-led trial RECOVERY and supported by Genentech-sponsored trial EMPACTA.

In the RECOVERY trial, 4,116 adults with progressive Covid-19 (defined as oxygen saturation <92% on room air or receiving oxygen therapy, and CRP ≥75 mg/L) received either the current standard of care or Tocilizumab treatment in addition to the current standard of care. Most patients also received systemic corticosteroids at the start of their disease progression.

The cohort who received Tocilizumab had a mortality rate of 30.7%, whereas those who only received the standard of care had a mortality rate of 34.9%. Therefore the reduced risk of death from Covid-19 for those who received the drug was -4.1%.

Notably, those who received systemic corticosteroids in addition to Tocilizumab saw even more robust results. Their reduced risk of death was -5.9%, suggesting the drug should be administered alongside corticosteroids in Covid-19 patients. We note that these are somewhat marginally positive reductions in the risk of death for a drug to receive full approval.

The EMPACTA study delivered similarly modest results in a smaller 389-patient study. 12% of those receiving the drug required ventilation or died, whereas 19.3% of those receiving the placebo required ventilation or died.

On Wednesday, December 21st, Genentech announced that Acterma, the clinical name for Tocilizumab, was fully approved for use in hospitalized adults. It joins Baricitinib as an approved treatment solely for hospitalized adults and Remdesivir, which remains the only approved drug for all ages.

The emergency use authorization for Tocilizumab remains, allowing hospitalized patients as young as two years old to receive the drug.

Currently, there are three fully approved drugs and 13 treatments approved via emergency use authorization. The marginal effect of Tocilizumab and the recent withdrawal of some antibody treatments from the Emergency Use Authorization list outlines the research difficulty for Covid-19 antivirals. This underlines the necessity to continue expanding our search for highly effective, safe, long-acting antiviral drugs that can be used singularly or combined to prevent and treat Covid-19. Thisarticleis featuredon

January 13, 2023: Emergence Of I gG4 In Long - Term Vaccines: Winning Or Losing The Race?

Entering the fourth year of Covid-19, vaccination has become the frontline of protective measures to control the disease, as global nations have all but given up on mass mitigation strategies such as masks and regular testing.

The initial vaccines released in 2021, both adenovirus and mRNA versions, displayed a remarkable efficacy in preventing infection and symptoms peaking at roughly 90%.

Two years later, our enthusiasm for the Covid vaccines has been chastened for two reasons. Firstly, the protection against infection for the version of Covid-19 the vaccines were designed for has waned heavily, and many people were subject to reinfection only months later. Secondly, virus variations occurred much more rapidly than most anticipated, resulting in little to no protection against later strains such as the current XBB.1.5.

Vaccines do protect against hospitalization and death, though to varying degrees. Those under 65 are much less likely to be hospitalized due to Covid-19 if they are fully vaccinated and boosted with an additional bivalent dose, however, those over 65, and especially those over 80, are far less protected.

For this reason, it is important to understand exactly what these vaccines may be doing. Here we summarize the results of a study on

the nature of antibodies produced after multiple rounds of vaccination. The study by Irrgang et al. describes how repeated exposure to vaccines produces a rare form of antibody, IgG4, which has some unique properties.

UnderstandingIgG4

There are five types of antibodies: IgG, IgM, IgA, IgD, and IgE. Each antibody performs a different function and is characterized by a different heavy chain. The study by Irrgang and colleagues focuses on IgG, which is among the most stable and critical to neutralizing functions.

Within IgG, there are four subclasses: IgG1, IgG2, IgG3, and IgG4. The two most apparent distinctions between the subclasses of IgG antibodies are their structural properties and their activation of complement effector functions. In terms of structure, IgG antibodies all have a distinct ‘Y’ shape, with a constant Fc region at the stalk and a variable Fab region at the arms, with a hinge combining them. Regarding structural differences, the subclasses share a nearly identical Fc region, but the hinge region varies considerably.

The constant regions for all four of these subclasses are derived from combinations of germline-inherited DNA sequences, which are serially constructed. The rarest among these are IgG4 antibodies.

All four antibody subclasses may bind to the same epitope, but they differ in the consequence of that binding. The Fc portion (the trunk of the ‘Y’ shape) determines how the body reacts to an antigen once it is bound to the antibody. These reactions are effector functions, and each subclass activates effector functions to varying degrees.

The three main functions are 1) the activation of inflammation; (2) the opsonization (labeling) of pathogens and cells for clearance/destruction; (3) the direct killing of target cells/microbes by lysis.

IgG1, IgG2, and IgG3 provide protection not only by blocking the virus from entering cells but also by their Fc regions activating effector functions and signaling the immune system to kill infected cells. IgG4, however, does not activate the effector functions, meaning their presence may impact how the body responds to Covid-19.

IgGSubclassesin VaccineSera

To measure the relative frequency of IgG subclasses in the sera of vaccine recipients, Irrgang et al. followed a cohort of 29 healthcare workers, analyzing their sera ten days after a first, second, and third dose, as well as 210 days after the second, and 180 days after the third.

In line with initial efficacy reports for the vaccine, antibody levels were robust throughout the cohort post-first and second doses. The researchers also found that 210 days after the second dose, antibody levels had fallen significantly, reaffirming the loss of antibody protection over time. Again, following the third dose, antibodies rose significantly, only to fall 180 days after the booster.

The most interesting data regards the growing concentration of IgG4 in the cohort’s sera. On average, only 0.04% of the antibody response post-second vaccination was IgG4. 210 days after the second dose, that percentage rose to 4.82%. Following the third dose, IgG4 comprised 13.91%, rising to 19.27% 180 days after.

Why is this happening? IgG4 antibodies have distinct characteristics. They are highly matured, highly variable, and have a high affinity for the receptor for the target. Therefore, they should be exceedingly effective at stopping the virus via neutralization activity and ACE2 direct blocking. Because it is not triggering the complement effector functions, IgG4’s sole and primary activity is blocking and neutralizing the virus.

WhatThisCouldMean

The exact impacts of an increased frequency of IgG4 are unclear and could be argued either way as a positive or negative. IgG4 occurrence correlates with increased avidity but decreased antibody effector function. Avidity measures the overall or accumulated strength of a protein-protein complex, which may serve as a marker for increased virus neutralization. However, IgG4’s decreased effector functions could signal less robust neutralization and clearance of virus and infected cells.

The addition of IgG4 may explain the increased ability of sera from multiple vaccinated or infected patients to recognize a broader range of strains because it binds more tightly. While it lacks the effector functions, that may not be as serious a disadvantage as one may think. More than 80% of antibodies in analyzed sera were still IgG1, IgG2, and IgG3, meaning effector functions are still activated, creating a healthy mix. However, it remains to be seen how IgG4 concentrations impact disease outcomes such as hospitalization and death.

We echo the closing thoughts of the researchers, noting that these results should be further examined to understand the exact impacts of IgG4 concentration in antibody titers. Not only could this

information impact the future of Covid-19 vaccination, but of mRNA vaccine technology in general.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Emergence Of IgG4 In Long-Term Vaccines: Winning Or Losing The Race?

January 19, 2023: Bivalent Antibodies For Covid -

19: Two Hands Are Better Than One

The current generation of monoclonal antibodies is ineffective against the predominant variants of SARS-CoV-2 in circulation. Recent studies by Callaway et al. open the possibility that a new type of monoclonal antibody may regain some of the lost activity. These are bivalent antibodies that recognize two sites on the virus simultaneously.

From the early days of the pandemic, the SARS-CoV-2 virus began to mutate. Over the following years, entire new lineages of variants emerged, such as Alpha, Beta, Gamma, Delta, and now Omicron. Each lineage introduced new mutations that altered the virus’s structure and enhanced its ability to infect host cells and evade neutralizing antibodies.

Omicron and its sublineages are the current exemplars of antibody evasion. Vaccines and antibody treatments used against earlier variants had greatly reduced effect on the Omicron family. There are some antibodies, however, that maintain some, if not all, neutralizing capacity against the new viruses, namely bivalent antibodies.

Antibodies that recognize the Omicron spike bivalently attach to two receptor binding domains on the spike trimer, as opposed to one. Most monovalent antibodies fall short against Omicron, but bivalent antibodies show promise. Researchers Callaway et al.

examined the neutralizing capacities of nearly 400 antibody therapeutics to further understand the supremacy of bivalent antibodies. Here we discuss their results and the further implications on vaccine and treatment design.

The397-AntibodyPanel

The researchers introduced a massive panel of nearly 400 antibodies to a wide variety of viruses, including D614G, Beta, Delta, Mu, and three sublineages of Omicron. At a low concentration of 250 ng/mL, only 66 potently neutralized BA.1, the base of the Omicron tree. At the higher 25 μg/mL, an additional 67 antibodies neutralized BA.1.

The researchers further tested 20 antibodies that potently neutralized the full panel against BA.4/5 and BA.2.12.1, later strains of Omicron known for their additional mutations and antibody resistance. The 20 maintained potent neutralization of Ba.2.12.1, but 14 had reduced or complete loss of neutralization against BA.4/5. The authors attributed this to the F486V receptor-binding domain mutation unique to BA.4/5. Most notably, the remaining antibodies neutralized the Omicron sublineages as well or better than the D614G parent lineage.

BivalentBinding

Upon further examination using electron microscopy, Callaway et al. found that of the Omicron-neutralizing antibodies, 13 bound bivalently, with each arm of the antibody engaging a neighboring receptor-binding domain in the spike. An IgG antibody is ‘Y’ shaped, with two arms and a base. The arms are those that bind the spike protein.

The researchers confirmed the connection between bivalency and antibody-spike affinity by conducting the same electron microscopy with nine antibodies that failed to neutralize Omicron. Eight of the nine bound partially or entirely monovalent, suggesting a link between bivalency and Omicron neutralization.

Impact ofBivalencyon Neutralization

Callaway et al. further investigated the supremacy of bivalency by isolating the Fab fragments of the bivalent Omicron-neutralizing antibodies. Essentially, they tested one arm of the ‘Y’ as opposed to the entirety of the ‘Y’ to deduce the importance of the arms working in tandem.

Using eight bivalent potent antibody fragments, they found that all had lower neutralization than intact IgG antibodies against D614G. All eight had an even more precipitous drop against Omicron BA.1 pseudovirus.

Discussion

The disproportionate effectiveness of bivalency in Omicron neutralization may be a marker for antibody and vaccine design in the near future. For over a year, Omicron sublineages have dominated SARS-CoV-2 virus frequencies. Given the widespread nature of the Omicron family, this may be unlikely to change anytime soon. If bivalent antibodies are those prone to bind and neutralize Omicron viruses, perhaps we should focus efforts on the discovery and isolation of bivalent antibodies for treatment and vaccines.

Bivalent antibodies are far from a new concept. For instance, the discovery of bivalent virus-like particles geared towards Ebola has

led to recent advancements in the search for additional effective Ebola vaccine candidates. A bivalent vaccine for Respiratory Syncytial Virus also takes a similar approach.

With bivalency as a prerequisite for Covid-19 antibody treatments, we may conduct a more thorough search of broadly neutralizing antibodies in development, bringing forward those with bivalent tendencies. Omicron and its many descendants will likely remain in the public sphere for some time. Bivalent antibody treatments would be another arrow in the quiver to keep the Omicron family at bay.

This article is featured on Forbes.org, and can be read online here: Bivalent Antibodies For Covid-19: Two Hands Are Better Than One

February 07, 2023: Artificial Intelligence

Opens The Door To More Effective Antibodies - Potential Applications For Covid - 19

One of the major time hindrances to the development of monoclonal antibodies is one of the first steps: identification. Advances in artificial intelligence antibody modeling may pave the way to reduce time spent during the identification process. Rather than sorting through millions of B-cell receptor sequences manually or with the assistance of some software, what if that work could be expedited by artificial intelligence?

There's an urgent need for drugs to prevent and treat Covid-19, particularly for immunosuppressed people. Unfortunately, the current Covid-19 variants have mutated to escape our currently approved antibodies. There are, however, a series of broadly neutralizing antibodies in research and development, though these typically suffer with lower affinity than would be ideal. Fortunately, a recent paper outlines a solution to this conundrum: using artificial intelligence to dramatically increase the binding affinity of antibodies.

Researchers Parkinson et al. present an artificial intelligence pipeline, RESP, which efficiently and independently identifies high-affinity antibody candidates. RESP selects the best fit to improve it for existing antibodies, given a particular antibody target. Using RESP, the researchers demonstrate an increased affinity to an

existing drug by almost 20-fold. Here we examine the function of RESP and how it could improve antibody development in the near future.

RESPComponents

The researchers describe the artificial intelligence system in four components. The first is an encoding scheme to pick out human Bcell receptor sequences from a larger panel. B-cells carry the genetic sequence used to encode and produce antibodies. By immediately recognizing human B-cell receptor sequences, RESP can immediately filter out innumerable sequences the researchers are not interested in.

The second component is a yeast surface display library to demonstrate the relative impact of mutations in an identified antibody’s sequence. Modest mutations of just one amino acid could radically impact a given antibody's binding and neutralization capabilities. The sorted library can then be easily analyzed for different factors such as off-rate, half-life, binding affinity, and so on.

The third component is a sorting model for understanding the potential affinity of an antibody. RESP analyzes the off-rate of predicted antibody candidates and selects the highest affinity. Offrate is essentially a ligand-protein binding component that negatively correlates with affinity. The lower the off-rate, the higher the affinity. They note this model can be retrained for different antigens, expanding the usefulness of RESP even further.

The fourth component aggregates the data from the preceding three to predict B-cell sequences with significantly lower off-rates, yielding antibodies with significantly higher affinity. Altogether, RESP seems impressive, but does the data back it up?

RESPTesting

The first test the researchers gave to RESP was for reconstructing sequences and discarding decoy sequences. The researchers introduced over 2.7 million human B-cell receptor sequences to RESP, as well as another 2.7 million decoys that had been modified in several positions. The system reconstructed the 5.4 million sequences with >99.99% accuracy, while its identification of decoys was a more modest but still strong 97.4%.

They next tested the validity of the yeast surface display library. Using approved monoclonal antibody treatment Atezolizumab, a cancer immunotherapy treatment for non-small cell lung cancer, they scanned for potential modifications in the antibody coding sequence that could improve the off-rate. Upon examining the heavy chains of the antibodies for positive mutations, the yeast library revealed over 92,000 unique sequences that were at least equal to the off-rate of the original Atezolizumab, many of which were lower, which we will speak to in a moment.

Using the reconstruction and yeast library functions, RESP identified 21 final candidates with higher affinity than the original Atezolizumab. The 21 sequences carry mutations at residues A40, K43, T58, I70, N77, A79, S85, A97, and R98 in various combinations. None of these mutations appear in the original Atezolizumab and likely account for the increased binding affinity of the new candidates.

One of the 21, mutant 4, carried mutations I70A, A79T, and A97V. Mutant four displayed significantly slower off-rates as compared to the unaltered Atezolizumab. And finally, this selected mutant bound the antigen 17-fold tighter than the approved drug.

Discussion

As with many aspects of our lives, artificial intelligence presents an opportunity to reduce manual input significantly and significantly improve productive output in antibody discovery. The most important element of RESP is its adaptability. This is not a Covid19 antibody discovery mechanism or a mechanism for this specific version of cancer. RESP can be modestly altered to discover new or improved antibody candidates for all antigens, potentially even those without treatment.

If the trial run of RESP can identify a mutated alternative to an approved drug with a 17-fold increase in binding affinity, it could do the same for existing Covid-19 medicines and drug candidates. While further testing should always be pursued, the advancement of RESP should be pursued with haste.

Artificial intelligence mechanisms like RESP should be applied to the in-development panel of monoclonal antibodies. While previously and currently approved antibodies typically bind the receptor-binding domain of SARS-CoV-2, that is not the only target. For instance, the CV3-25 antibody described by Li et al. binds the S2 region of the virus on the complete other end of the spike. Another is the COV44-62/79 antibodies described by Dacon et al. These bind the fusion peptide.

Source:ACCESSHealthInternational

Again, these antibodies are broadly neutralizing across many variants but fall short of binding affinities of a bebtelovimab or Evusheld. With the assistance of RESP, perhaps slight modifications to in-development antibodies could yield a powerful monoclonal antibody candidate primed to save thousands of lives. Thisarticleis featuredon Forbes.org,andcan be readonlinehere:

Artificial Intelligence Opens The Door To More Effective Antibodies - PotentialApplications For Covid-19

Figure 21.Bindingepitopes ofthebebtelovimab,Li,andDacon antibodies.

February 10, 2023: Inherited Differences

In Antibody Genes May Explain Variable Responses To Covid - 19 Infection

Three years into the Covid-19 pandemic, it is clear that some of us produce stronger immune reactions to infection than others. Some may only experience mild flu-like symptoms, while others could be hospitalized or worse. Recent research suggests there is more to humans’ variable ability to control infection than pure chance. Common variations in immune-related genes heavily influence our immune reactions to invading pathogens, SARSCoV-2 included.

Antibodies inherit their structure from the genetic Ig germline locus. The exact physical structure of the antibody is determined by the genetic predispositions of the patient, which is why one patient may develop much more effective antibodies than another, even if infected with the same virus at the same time.

Following exposure to Covid-19, our immune systems develop antibodies specifically tailored against the virus. These antibodies are highly variable in their efficacy. Pushparaj etal.shine some light on this variation. They find that minor differences in our genes and in the antibody genes that we inherit may also influence the structure and efficiency of the antibodies we develop post infections. Here we discuss the implications of their findings and how we may use this data to inform antibody discovery efforts better moving forward.

MeasuredVariation inResponseto Covid-19Infection

The researchers extracted the sera of 14 healthcare workers seven months after their infections in May 2020. Every patient had developed antibodies towards SARS-CoV-2 but in a wide array of genetic diversity. Antibodies are encoded by B-cell receptor sequences, which often vary significantly, leading to the significant genetic diversity of produced antibodies.

From the sera, they analyzed individual antibodies. From these antibody structures, they could determine the exact inherited germline, meaning every individual genetic detail of the sequence that would encode the antibody. By analyzing each allele individually, they could compare for relative presence in neutralizing antibodies.

They found that the number of encoded alleles tallied between 44 and 61 per patient. One of the most critical alleles to antibody potency, IGHV1-69, was found to have six variants among the 14 patients: IGHV1-69∗01, ∗02, ∗04, ∗06, ∗09, and ∗20. This will be the focus of Pushparaj et al.’s study moving forward, as they aim to understand the impact of gene polymorphisms on antibody efficacy.

AntibodiesEfficacyis Relatedto GeneticVariation

Using the sera of SP14, a patient who displayed the IGHV1-69∗01, IGHV1-69∗02, and IGHV1-69∗20 alleles, the researchers generated several heavy and light chains. These are the building blocks of antibodies. From this sole patient, they generated 29 spike-specific monoclonal antibodies, 15 of which were neutralizing against the wildtype SARS-CoV-2. All those that neutralized bound the receptor-binding domain of the virus.

Notably, most neutralizing antibodies used a variation of IGHV169, specifically IGHV1-69∗02 or IGHV1-69∗20. To reiterate, these patients had alleles between 44 and 61 each. Still, a variation of IGHV1-69 was found in a large majority of neutralizing antibodies in this particular patient, reinforcing the notion that this specific allele is critical to developing potently neutralizing antibodies in the immune system.

However, IGHV1-69 has allelic variation. Each variant allele may contain one or more somatic hypermutations that characterize it differently from others. These minor genetic variations in the alleles once encoded onto a monoclonal antibody, may account for some or all of the neutralizing capacity of the antibody, or lack thereof.

IGHV1-69AlleleUsageInfluencesNeutralizingAntibodyActivity

They next tested the value of somatic hypermutations (SHMs) in the allele to the final neutralization of the produced antibody. In other terms, do mutations in the important alleles make a difference? Pushparaj et al.found that by removing SHMs from the IGHV1-69∗20 allele found in patient SP14, the resultant antibody lost neutralizing capacity against SARS-CoV-2.

They repeated this process with all other variations of IGHV1-69 and found similar results. In essence, the SHMs are critical to neutralizing capability.

StructuralAnalysisRevealstheBasisforIGHVAlleleRequirement

To understand further why certain somatic hypermutational differences in specific alleles are so important, the researchers conducted a cryo-electron microscopy analysis of the previous antibody in question: CAB-I47. They wanted up close the binding

differences between the antibody produced from the SHM and nonSHM alleles.

As with many monoclonal antibodies, CAB-I47 makes contact with the receptor-binding domain to block binding to the human ACE2 receptor. There are a few critical contacts between the antibody and the receptor-binding domain, most notably the interaction between R50 of the antibody and E484 and G482 of the receptor-binding domain.

One amino acid can make a big difference. A single amino acid variation in the inherited structure may yield a significant difference in antibody binding and neutralization. R50 is variable among the different versions of IGHV1-69. Some mutate the arginine (R) to glycine (G), while others leave it unmutated. The R50G mutation seems to be the culprit for the loss of neutralizing activity, as the unmutated version forms hydrogen bonds and salt bridges between the virus and antibody.

Discussion

This is but a single example of one minor variation in the antibody germline completely reducing the neutralizing capacity of an antibody candidate. There are likely thousands, if not millions, of similarly crucial allele modifications that can make or break a potential monoclonal antibody. Not only can minor variations negatively impact an antibody, but it would follow that such minor variations could also drastically improve neutralizing efficacy.

How can we identify and maximize these minor differences to our advantage? We recently detailed an artificial intelligence mechanism designed to do just that; it identifies and tests minor antibody variations to identify the most potent germline sequence

possible. It is conceivable that genetic allele variation could be incorporated into that mechanism to optimize the system further. We can only hope such a process will be implemented shortly as hundreds of people succumb to Covid-19, desperately needing effective monoclonal drugs to fight this disease.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Inherited Differences InAntibody GenesMayExplain Variable Responses To Covid-19 Infection

March 07, 2023: Prophylactic Antibodies

Alter Vaccine Responses To Covid - 19

We are now in the third year of the Covid-19 pandemic. It is likely that Covid and the virus that causes it will be with us for many years. Most of us already have a complex history with Covid-19, including infection by the virus and exposure to a mix of vaccines and antiviral drugs. It is time to examine how the interplay of infections, antiviral drugs, and vaccines condition our response to new infections. Such a study is critical, as we now know that longterm protection from disease depends on the efficacy of memory response, not on initial neutralizing responses.

A recent study by Schaefer-Babajew et al. in the journal Nature begins to do just that by examining the influence of prophylactic antibody treatment on our antibody and memory B cells' response to vaccines. Surprisingly, they find that protective antibody treatments significantly affect our B cell memory response and may diminish the ability of vaccines to protect us from serious diseases.

StudyDesign

Schaefer-Babajew et al. examined a cohort of 18 patients who received a combination antibody treatment of C144-LS and C135LS, a combination yet to be approved or authorized for public use in the United States. Both of these antibodies bind to different regions of the receptor-binding domain and together have significant neutralizing potency against authentic SARS-CoV-2 wild-type virus obtained from human patients.

These antibodies were first described by Robbiani et al . in June 2020. C144 and C135 were isolated along with 50 other neutralizing antibodies from the sera of over 150 patients infected with SARS-CoV-2 in the early months of the pandemic. C144 and C135 were later modified with the ‘LS mutations,’ which is a method of improving the half-life of an antibody by enhancing the binding affinity between the Fc of an antibody and the human Fc receptor.

The cohort examined by Schaefer-Babajew et al. participated in a preliminary study to test the efficacy of these antibodies for submission to the Food and Drug Administration for emergency use authorization or approval. However, neither antibody is approved or authorized today. No patients in this study, whether experimental or control, were infected by SARS-CoV-2 before this study.

The 18 patients then received a two-dose mRNA vaccine regimen after a median of 82 days for the first dose and 103 days for the second. The cohort was compared to another group of patients vaccinated with two mRNA doses but with no previous history of infection or monoclonal antibody treatment. Patients received either the Moderna or Pfizer-BioNTech mRNA vaccine. The researchers examined the antibody binding, antibody neutralization, memory B cells, and antibodies produced by memory B cells.

TheEffectofPriorTreatment on AntibodiesthatBindtheSpike Protein

The first query was whether previous antibody treatment impacted antibody binding in patient sera. They examined the binding levels of two antibody types: IgM, which arises earlier in infection, and

IgG, which is the most common type of antibody and is typically the type used for antibody treatments.

The IgM antibody binding efficiency between treatment and nontreatment patients was relatively stable. For IgG antibodies, binding in the treatment group rose above the non-treatment after the first dose, but stabilized after the second. These results, however, were solely when tested against the wild-type Wuhan virus. When tested against a virus coded with receptor-binding domain mutations R346S/E484K or N440K/E484K, directly interfering with the epitope for both C144 and C135 binding, efficiency drops below the non-treatment group, though only marginally. The pseudovirus was also encoded with mutation R683G, far from the epitope involved with C144 and C135. The mutation disrupts the function of the furin-cleavage site, increasing particle infectivity without compromising the binding affinity between the receptorbinding domain and the antibodies.

Ultimately, the results show that monoclonal antibody infusions have little to no impact on IgM and IgG binding responses.

TheEffectofPriorTreatment on VirusNeutralization

They next examined the effect of infused antibody treatment on post-vaccine neutralization. They again introduced C144 and C135 to a wild-type spike protein pseudovirus and a mutated version.

Against the wild-type, those who received the antibodies beforehand had significantly higher neutralizing titers after both the first and second doses. Their past monoclonal treatment enhanced their post-vaccine defense against the virus.

For the following mutated pseudoviruses, the antibodies made a negative impact rather than a positive one. The researchers introduced patient sera to pseudoviruses with R346S/Q493K and R346S/N440K/E484K mutations. Once again, R346S, N440K, Q493K, and E484K directly interfere with the binding epitopes of C144 and C135. After the first dose, those in the antibody group saw neutralizing titers fall 2.7-fold and 3.5-fold against the mutant viruses compared to the control group.

Neutralization rebounds to just a 0.6-fold to 0.85-fold drop from the control group after the second vaccination, which is less statistically significant, though still relevant. Against highly mutated spike proteins, previous antibody treatments may lower your immune defenses post-vaccination. This is notable as all viruses circulating today are heavily mutated in the receptor-binding domain, potentially causing concern for those with many previous antibody treatments.

TheEffectofPriorTreatment on Memory BCells

Perhaps the researchers’ most crucial finding relates to memory B cell responses. One critical aspect of memory is the persistence of memory B cells. These cells have full antibody maturation and are stable for months or even years. Reinfection by a similar virus causes rapid proliferation and production of protective B cell antibodies.

Schaefer-Babajew et al. found that mRNA vaccination elicited memory B cell responses approximately three-fold higher in the antibody group than in the control.

TheEffectofPriorTreatment on TypeandNeutralizationof AntibodiesProducedbyMemory BCells

Notably, the composition of the memory B cells was heavily altered in the antibody group. While the absolute number of IgG antibodies rose in the antibody group compared to the control, the relative percentage fell from a vast majority to just 45%. After the second dose, IgM antibodies catapulted from marginal levels in the control to 49% in the antibody group. The researchers attribute this to the pre-exposure to anti-RBD antibodies. IgM antibodies carry far fewer mutations than IgG, meaning the antibody treatment designed to neutralize the wild-type virus was potentially predisposed to favor IgM memory B cell expression.

Upon taking a closer look at the isolated antibodies from the antibody treatment group versus the control, some major concerns are revealed. To reiterate, IgM antibodies are much more heavily concentrated in the test group than in the control group.

First, examining the binding capacity of isolated antibodies, 62% of isolated antibodies bound the wild-type receptor binding domain poorly, if at all, compared to just 5% in the control group.

The results are worse when it comes to neutralization, as only 1 of the 45 IgG antibodies and none of the 32 IgM antibodies from the test group were neutralizing, as compared to 63% of the control IgG antibodies and 17% of the IgM antibodies.

These results are attributed to shifting the target epitope in the antibody group isolated memory B cell antibodies. Whereas half of the antibodies in the control group target epitope classes one, two, or three, just 20% do so in the treatment group, favoring the class four epitope.

Discussion

This study shows that preexisting treatment with anti-SARS-CoV-2 monoclonal antibodies significantly impacts the development of memory B cell responses in post-vaccinated patients. While the initial antibody levels were not harshly impacted, in some cases even increasing, memory B cell development suffered. Affinity thresholds for memory B cell development were lowered, leading to weaker antibodies that bound and neutralized relatively poorly compared to the control.

The increase in IgM memory also aligns with previous observations of rising IgM levels post-third and fourth doses of the mRNA vaccine. The shifting memory was accelerated in the patients that previously received monoclonal treatment. However, the increasing breadth of the memory antibody responses is countered by lacking neutralization and affinity.

This is not to say stay away from monoclonal antibody treatments. They can be life-saving in many instances and should be pursued early in infection, especially for those at high risk of severe disease progression. However, once you receive antibodies, your memory responses will be altered moving forward, and you may be at risk. Continue receiving mRNA boosters every three to six months to maximize immediate protection and lasting memory against SARSCoV-2.

These studies are the first of what we hope will be many in examining the complex interaction between infection and both prophylactic and therapeutic interventions for Covid-19. The results show significant effects, particularly on memory B cells and the antibodies they produce. The consequence of these perturbations

regarding protection from new variants remains to be seen, as new variants will inevitably arise. Nonetheless, we hope that these are the first of many studies investigating what is now a critical question three years into the pandemic.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Prophylactic Antibodies Alter VaccineResponses To Covid-19

March 16, 2023 : New T Cell Antibody

Treatmen t Improves Outcomes For Covid Patients

The need for effective Covid-19 drugs is ever-expanding. Hundreds of millions, if not billions, have been infected by SARS-CoV-2 throughout the pandemic. Even today, thousands continue to be infected in what many claim to be a post-pandemic world. Antiviral drugs can prevent infection in those recently exposed to the virus and help those infected stave off the worst effects of Covid-19.

A new study by Moreira et al. suggests that the drug Foralumab, designed to treat multiple sclerosis and other neurodegenerative diseases, may do just that.

One of the most vital types of anti-Covid drugs is the monoclonal antibody treatment. Typically targeting the SARS-CoV-2 spike protein, these drugs neutralize the virus by binding to the portion of the virus that infects our cells, preventing further spread. However, these drugs are susceptible to virus mutation and may lose potency against more recent variants.

Foralumab does not bind the virus spike protein but rather the T cells of the immune system. T cells are immune modulators involved early and often during Covid-19 infection. They are heavily involved in immune memory and lasting immunity against the virus post-infection. However, they also cause symptomatic

responses during infection through effector functions and cytokine reactions.

Developed by Tiziana Life Sciences during the pandemic, Foralumab was designed to treat patients suffering from multiple sclerosis, reducing inflammatory responses brought on by the immune system. It is one of the only T cell targeting monoclonal antibodies in clinical development, and Moreira et al. aimed to deduce whether its anti-inflammatory mechanisms worked against Covid-19 as well.

This study is derived from a previous pilot study of Foralumab. Patients were split into three cohorts: control, Foralumab, and Foralumab, in addition to dexamethasone. Over the two-week observation period, patients who received Foralumab or Foralumab/dexamethasone displayed more rapid clearance of lung infiltrates.

Foralumab patients showed a more substantial reduction of serum IL-6 and C-reactive protein, meaning the monoclonal antibody was well tolerated and may effectively treat immune hyperactivity.

How, then, does Foralumab work to impact T cells and improve the overall condition of a Covid patient? Moreira et al. followed their pilot study with a mechanism of action analysis for Foralumab.

Foralumab begins down and upregulating specific genes and proteins upon binding with activated host T cells. Many of these have little effect, if any, on Covid disease outcomes. Some, however, are crucial to the immune responses.

One such upregulated gene was GIMAP7. While little is known about this specific gene, as with many expressed genes in the human immune response, it is reportedly linked to T cell regulatory

efficiency, meaning Foralumab upregulating this gene leads to a stronger immune response from the host, yielding more promising disease outcomes.

Numerous genes, proteins, and cytokine pathways are impacted by Foralumab; too many to mention in this article. Still, the culmination leads patients treated with Foralumab to more efficient lung clearance of the virus and higher levels of immune protection.

In all, Foralumab induces many factors that impact improved tissue remodeling, induction of immune cells, and restriction of effector function, improving disease outcomes while fighting the virus to full strength. These benefits are not limited to Covid-19 patients, as similar results were observed in patients with multiple sclerosis.

Discussion

Foralumab represents a novel approach to treating Covid-19 illness. While the drug does not prevent infection, it can lessen the impact of mild to moderate symptoms by regulating the T cell immune response. In conjunction with a spike-targeting monoclonal antibody, Foralumab could be a welcome addition to the anti-Covid toolkit.

The anti-CD3 monoclonal treatment effectively modulates the T cell inflammatory response by up or downregulating specific gene and protein expressions, resulting in less severe disease. This novel avenue should be further explored, as more drugs with similar mechanisms could prevent the deaths of thousands.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: New T CellAntibodyTreatmentImproves Outcomes For Covid Patients

March 18, 2023: Progress Toward

Effective Monoclonal Antibodies

Treatments Against Covid - 19 And Other Coronavirus Diseases

The greatest difficulty for vaccine and drug design three years into the Covid-19 pandemic is the proliferation of viral variants. Most antibodies target the SARS-CoV-2 receptor-binding domain, but this region is highly variable, leading to variants of concern such as Beta, Delta, and now Omicron.

A potential solution to this problem is to find antibodies that target conserved sites unlikely to mutate. Many have attempted to locate these antibodies that target sites like this in the receptor-binding domain, though they are often still overwhelmed by the latest iterations of Omicron.

Researchers Zhou et al. from the Scripps Research Institute in La Jolla, California, examine a different approach. Their study analyzes a panel of broadly neutralizing antibodies that target not the receptor-binding domain but the S2 stem-helix fusion region of the spike. Zhou and his team previously isolated the CC40.8 antibody, which targets this conserved region of SARS-CoV-2, binding and neutralizing in mouse models. Notably, the antibody also protected against sister viruses SARS-CoV-1 and MERS-CoV, speaking to the broad capabilities of S2 binding antibodies. Here we examine their results and the impact on antibody development moving forward.

StudyDesign

Zhou et al. isolated antibodies from the sera of a wild variety of donors. 15 patients were Covid-19 recoverees, 10 had received two doses of mRNA vaccine, nine had received three doses, and 15 had recovered from Covid-19 and received one dose of mRNA vaccine. Notably, none of the sera from the recoveree, two-dose, or threedose groups bound to spike S2 stem-helix peptides. However, 80% (12/15) exhibited strong binding to these epitopes in the recoveredvaccinated group. Accordingly, Zhou et al. focused on this group from here, aiming to isolate antibodies for a larger panel examination.

From ten of the 12 recovered-vaccinated patients, Zhou et al. isolated 40 stem-helix monoclonal antibodies. 38 of the 40 bound a range of coronaviruses, including SARS-CoV-1, MERS-CoV, and SARS-CoV-2. Some antibodies were duplicates, reducing their pool to 32 broadly binding antibody candidates.

BroadNeutralizationofBetacoronaviruses

They next introduced the panel of 32 antibodies to another host of viruses to determine neutralization efficiency. All 32 antibodies neutralize viruses such as SARS-1, MERS, and SARS-CoV-2 to varying degrees. When including MERS-CoV, only 72% (23/32) efficiently neutralized across the board. Further narrowing their group, they selected the top ten highly potent antibodies against the previous group of viruses to introduce to a range of SARS-CoV-2 variants of concern. Consistent with their previous findings, all ten demonstrated highly effective neutralization against some of the most mutated strains of Omicron.

Recognition ofa CommonHydrophobicCoreEpitope

Upon further examination of the 32 broadly neutralizing antibodies, Zhou et al. found that many exhibited dependence on a range of residues on the Spike S2 stem-helix, specifically from positions 1146 to 1156 in SARS-CoV-2.

All 32 antibodies competed strongly for similar S2 stem-helix epitopes, targeting a similar region containing the hydrophobic core residues of the spike fusion machinery. This machinery is highly conserved across betacoronaviruses and may explain why this panel is highly broad in binding and neutralization capabilities.

MechanismofNeutralization

To understand the structural basis behind the antibody panels’ broad neutralization, Zhou et al. employed cryo-electron microscopy on four antibodies to determine the complex interactions between the antibody and S2 stem-helix.

The epitope previously described is highly conserved across betacoronaviruses. It is located at the interface within a helix bundle at the base of the prefusion spike. The virus spike undergoes a significant conformational change upon binding with a host cell. Many antibodies aim to block this conformational change to prevent membrane fusion, which is precisely the aim of this antibody panel.

Stem-HelixAntibodiesin LiveAnimalExperiments

To determine the in vivo efficacy of these antibodies matched the invitroobservations, they introduced three of the broadest and most potent antibodies, CC25.106, CC68.109, and CC99.103, to a group of aged mice.

12 groups of ten mice each were given either CC25.106, CC68.109, CC99.103, or a placebo. Each group was then administered mouseadapted SARS-CoV-2, SARS-CoV-1, or MERS-CoV.

The animals were monitored for daily weight changes, pulmonary function, and viral load in the lungs upon euthanization. Mice that received the antibodies displayed significantly lower weight loss, more normal pulmonary function, and fewer viral titers in lung tissue than in the control group.

Among the three antibodies, CC25.106 provided superior protection, but all were significantly protective against the three betacoronaviruses.

Discussion

Three years into the pandemic, we desperately need new effective prophylactics and treatments for SARS-CoV-2. Hundreds die from Covid-related symptoms every week in the United States alone.

Many of these deaths are in susceptible populations such as the elderly, children, and the immunocompromised, and they could be prevented with an effective and accessible monoclonal antibody treatment.

Unfortunately, many monoclonal antibodies are ineffective against Omicron's latest variants, as the receptor-binding domain has wildly mutated past recognition by previously promising antibodies.

Enter this panel of antibodies by Zhou et al. Their binding to the S2 stem-helix bypasses the mutated receptor-binding domain for a more conserved virus region. They do this so efficiently that the panel effectively neutralizes even the most mutated versions of Omicron.

We caution that previous attempts at stem-helix antibodies have been made, and they typically lack significant affinity or never made it past initial experiments. That being said, the panel of antibodies by Zhou etal.displays significant promise, and those with the power to do so should pursue them as a potential antibody treatment for the future with haste.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Progress Toward Effective Monoclonal Antibodies Treatments Against Covid-19 And Other Coronavirus Diseases

March 30, 2023: Anti - ACE2 Monoclonal Antibodies To Prevent And Treat COVID19

Early in the pandemic, monoclonal antibodies proved to be one of the most effective ways to prevent and treat Covid-related disease. Neutralizing antibodies are directed toward specific structures on the spike protein, specifically to the receptor binding domain.

Unfortunately, the virus mutates under selective pressure over time, altering the structure of the receptor-binding domain. The mutated virus retains binding to the surface of lung and other cells, but then they avoid recognition by antibodies. Over time, each of the antibodies, whether singly or in combination, fails to protect against these virus variants.

Researchers Zhang et al. from the Rockefeller and Stanford Universities investigated a different approach. Rather than directing antibodies to the receptor binding domain, their strategy was to develop antibodies that recognize ACE2 on the host cell, to which the virus spike binds.

Here we analyze their findings, which may have the potential to offer an alternative therapeutic pathway for antibody development moving forward.

GenerationofhACE2-bindinghumanmonoclonalantibodies

To find ACE2 binding monoclonal antibody candidates, Zhang et al. immunized humanized mice models that produce chimeric antibodies consisting of human Fab domains and a murine Fc domain. They were immunized with ACE2 extracellular domains.

Following a 35-day observation period, hybrid B cells, or hybridomas, were isolated from mice sera. Ten candidates were selected that secreted antibodies that impeded SARS-CoV-2 pseudotype infection.

Human anti-hACE2mAbsbroadlyinhibitsarbecovirusinfection

Six of the 10 chimeric human-mouse antibodies were chosen to be adapted to a human IgG expression for their genetic diversity and binding capacity. All six generated human antibodies blocked a pseudotype SARS-CoV-2 wildtype virus from infecting target cells. Notably, two antibodies were more than 10-fold more potently neutralizing than a previously investigated anti-ACE2 antibody, h11b11.

The group of six also inhibited infection by pseudotype variants, including Beta, Delta, and Omicron BA.1.

The researchers found that the antibodies also inhibited a SARSCoV-1 pseudovirus, as well as two pangolin and two bat viruses with similar potencies.

Vero E6 cells were incubated with the 05B04 antibody candidate and later introduced to the wild-type live virus. 05B04 also potently inhibited the live BA.1 virus, demonstrating its capability to neutralize a range of SARS-CoV-2 variants.

Structuralanalysisofanti-hACE2mAbs

The ACE2 site on human cells has natural functions and it is crucial the introduction of antibodies does not impact these functions. The anti-ACE2 antibodies must bind an epitope that solely impacts the binding capacity of the SARS-CoV-2 receptor-binding domain and not other cellular enzymatic activity. With rare exception, human genes remain unmutated in regards to ACE2 design, and therefore, it is crucial that the antibody does not overlap the receptor-binding domain and substrate-binding site.

Source:ACCESSHealthInternational

Figure 22. ACE2schematic.TheACE2(green)is boundbythespike receptor-bindingdomain(blue),withthesubstratebindingsite(red) denoted.

With the aid of cryo-electron microscopy, they found that antibody interactions mimicked favorable binding between SARS-CoV-2 receptor-binding domains and an N-terminal helix of ACE2, a critical juncture for SARS-CoV-2 infection. This molar mimicry enables high binding affinity, despite a smaller binding epitope than the virus.

hACE2mAbsprotect hACE2knock-in miceagainst SARS-CoV-2 infection

In animal models, the anti-ACE2 antibodies continue their positive data points. In mice adapted with a human ACE2, subjects were subcutaneously injected with 250 micrograms of anti-ACE2 monoclonal antibody. There were no observed pharmacokinetic issues in the mice during a 14-day observation period.

Two days later, the mice were introduced to live wild-type SARSCoV-2. Pre-treatment with the anti-ACE2 antibody reduced lung virus replication to near undetectable levels as compared to the control. Therefore, the anti-ACCE2 antibodies were successful as a prophylactic for SARS-CoV-2 infection, albeit against the wild-type virus.

Discussion

This is a promising new approach to antibody development, though some concerns must be addressed. Firstly, can a high enough concentration of these antibodies be delivered to block enough receptors to be effective against the invading pathogen? Secondly, would blocked receptors interfere with normal cellular physiology? Until extensive human studies are conducted, we cannot know the answer.

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

Moreover, we have continually underestimated the genetic and structural flexibility of the coronavirus family. For instance, a close relative of the MERS virus binds not to the typical MERS receptor CD26, but rather it uses a different domain to bind to bat and even human ACE2 receptors, albeit using an alternative face.

Given this flexibility, it is conceivable that variants of SARS-CoV-2 may evolve to use not the typical front face of the receptor-binding domain but rather other receptor-binding domain epitopes.

Nonetheless, this is an interesting and promising approach. Another approach that we often describe is the use of broadly neutralizing monoclonal targeting conserved epitopes on the spike protein, a number of which have been discovered and some of which are in clinical development. These strategies may be complementary and antibody cocktails that include ACE2 binding antibodies may be part of the answer following phase one safety and phase two efficacy trials.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere:

Anti-ACE2 Monoclonal Antibodies To Prevent And Treat

COVID-19

April 12, 2023: FDA Authorizes New Antibody Treatment For Severe Covid - 19

Anew antibody drug by pharmaceutical company inflaRx received FDA emergency use authorization to treat critically ill Covid-19 patients. One of the greatest hurdles for monoclonal antibody development during the Covid-19 pandemic is the ongoing mutation of the SARS-CoV-2 virus. Most antibodies introduced throughout the pandemic targeted the virus spike protein, which mutates significantly with every emerging variant. The monoclonal antibody, vilobelimab, does not target the spike protein, nor the virus. Rather, it targets a specific product of the complement system; an immune response that triggers most of the severe symptoms we experience when battling a dangerous pathogen, including inflammation, pain, fatigue, etc.

The antibody recently underwent phase one and two safety and efficacy trials in human volunteers, demonstrating a strong response in controlling severe symptoms. Here we discuss the antibody and its trial results, discussing the potential impacts it may have now that it has been authorized.

MechanismofAction

The targeted complement molecule for vilobelimab is C5a. The complement system comprises a cascade of molecules C1-C9 activated upon pathogen contact with an antibody. The cascade initiates a series of reactions that destroy infected cells, but in the process, the host must deal with side effects that manifest as

symptoms. Less severe cases may include moderate tiredness or physical pain, whereas severe symptoms include inflammation and significant fatigue.

The inflaRx team focused on one such molecule fragment, C5a, which acts as a chemoattractant for other immune system mechanisms. Once activated, C5a signals other cells, such as macrophages, to attack invaders and infected cells.

They found that a specific epitope within the C5a molecule could be exploited for a potent blocking monoclonal antibody, which could temper the C5a signaling and reduce the severity of complement-related symptoms in severe disease cases.

Their research led to vilobelimab. Originally, the monoclonal antibody was developed to treat hidradenitis suppruativa, or HS; a common skin condition that causes painful lumps deep in the skin. These lumps are a result of hyperinflammation and infection in the sweat glands. An antibody that could reduce inflammation by impeding the complement system could relieve those living with HS. Such reduction in inflammation could also be applied to Covid patients.

The antibody delivered a complete immunological block of C5ainduced effects, resulting in strong therapeutic responses. The drug was also fully selective and has high binding affinity, meaning it did not interfere with related cellular activities such as membrane attack complex, reducing autoimmune complications.

EfficacyTrials

A trial conducted by Vlaar et al. aimed to determine survival outcomes impacted by vilobelimab as a therapeutic. Their phase

three trial included 368 patients in nine countries worldwide, all suffering from severe Covid-19 and requiring ventilation.

In the treatment group, 54 patients of 177 (31%) died due to Covidrelated health complications within 28 days of treatment, whereas 77 of 191 (40%) died in the control placebo group in the same timeframe.

While modest, the drug did improve survival outcomes for patients by nine percentage points, which considers the drug a suitable therapy for patients with severe Covid-19.

Discussion

Ultimately, vilobelimab is not a miracle cure for Covid-19. The modest reduction in Covid-related death in severe patients is notable but not groundbreaking. Combining vilobelimab with other effective monoclonal antibody treatments may improve the disease outcomes even further, and additional research into this possibility should be explored. Notably, monoclonal antibodies that target highly conserved regions of the spike, such as the S2 region, or anti-ACE2 antibodies we have previously discussed could be possible combination options.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere:

FDA Authorizes NewAntibody Treatment For Severe Covid-19

April 18, 2023: Novel Broadly

Neutralizing SARS - C oV - 2 Monoclonal Antibodies That Bind Across The Subunits Of The Spike Protein

Anew antibody pair neutralizes all current variants of concern of SARS-CoV-2. There is an effective need for monoclonal antibody treatments for those suffering from moderate to severe Covid-19. While a number of treatments have been developed throughout the pandemic, the virus is continuously mutating, rendering many of these treatments obsolete. The key is to find antibodies that target conserved regions of the virus, allowing the antibody to neutralize a broad range of variants.

Two such broadly neutralizing antibodies were recently identified by Liu et al.Many monoclonal antibodies target the spike receptorbinding domain, the region that makes contact with the host cell's ACE2 receptor, triggering infection. The two antibodies defined by Liu et al.target different regions, the N-terminal domain, SD1, and the S2 region. Here we analyze these antibodies and discuss how their addition impacts the current monoclonal antibody treatments pool.

Isolationandcharacterizationofbroadlyneutralizingmonoclonal antibodiesagainst SARS-CoV-2variants

The researchers evaluated a panel of sera samples against a range of 11 SARS-CoV-2 variants in search of neutralizing activity. Sera from

patient 12 demonstrated the highest levels of neutralization. From patient 12’s sera, they isolated 27 monoclonal antibodies, of which five neutralized Omicron BA.1. From these five, antibody candidates 12-16 and 12-19 demonstrated the strongest neutralization.

Both 12-16 and 12-19 further neutralized some of the latest and most prevalent variants of SARS-CoV-2, including BQ.1.1, XBB.1.5, and CH.1.1, all of which have highly mutated spike proteins.

In hamster models, 12-16 and 12-19 significantly reduced virus titers in subjects infected with Omicron BA.1, reducing the virus to undetectable levels within days.

Antibodies12-16and12-19target a quaternaryepitope between NTD andSD1

Liu et al. used cryo-electron microscopy to determine the binding epitope of 12-16 and 12-19 to the SARS-CoV-2 spike protein. Rather than binding the typical receptor-binding domain, the antibodies bound the N-terminal domain, subdomain 1, and a small section of the S2 region.

Most antibodies throughout the pandemic bound the receptorbinding domain from residues 333 to 527. The N-terminal domain runs from residues 14 to 305, subdomains 1 and 2 run from residues 541 to 685, and S2 is the latter half of the virus from residues 685 to 1273.

12-16 and 12-19 bind the spike at slightly different angles. 12-16 binds at a higher angle, favoring the N-terminal domain, whereas 12-19 binds at a lower angle, binding more sites on SD1. From here,

we will focus on 12-19 as it binds more completely invitro than 1216, likely due to its slightly more cross-region epitope.

12-19neutralizeSARS-CoV-2bylockingRBD in thedown conformation

Akin to some antibodies seen throughout the pandemic, 12-19 blocks infection by locking the receptor-binding domain in the down conformation. Before contact with the host cell, the spike shifts from a down to an up conformation, allowing binding and enabling infection of the cell. Infection is prevented if the receptorbinding domain is locked in the down conformation.

But how can an antibody that binds the N-terminal domain, subdomain 1, and S2 impact the receptor-binding domain? Part of the 12-19 epitope binds residues that are shifted during conformation change, namely in subdomain one and a linker region between the N-terminal and receptor-binding domains. Imagine two gears in the same link. They may not necessarily be consecutive, but block the movement of one, and the whole line of gears is stalled.

By blocking the conformational shift, 12-19 impedes ACE2 binding between the receptor-binding domain and the host cell.

Another set of antibodies that uses conformation locking is the camelid nanobodies described in 2021. These bind across receptorbinding domains of a spike trimer, preventing the spring-loaded conformation switch from engaging.

Theepitope ofantibody12-19ishighlyconserved

As with any monoclonal antibody candidate, we must consider the potential escape mutations enabling a viral variant to escape

neutralization from 12-19. Using deep mutational scanning libraries, Liu et al.found a few potential problem residues.

The areas most likely to produce escape mutations were the N4 loop of the N-terminal domain from residues 172-176, deletions throughout the N-terminal domain loops, namely residues 103 and 121, as well as residues 522, 561, and 577 in the subdomain 1. However, these residues are rarely mutated in currently circulating variants. The most frequent is L176F, found in only 0.2% of sequenced variants. This indicates that the epitope of 12-19 is largely conserved, at least so far in the pandemic.

Discussion

On first inspection, 12-19 would serve as a valuable antibody treatment, given its strong neutralization of the latest SARS-CoV-2 variants. An advantage it may have over other treatments is its unique binding epitope. The figure below compares the binding epitope of 12-19 to other neutralizing antibody epitopes from throughout the pandemic.

Figure 23. Schematicrepresentations ofbindingepitopes fordiffering classesofmonoclonalantibodytreatmentsthroughoutthepandemic.

Source:ACCESSHealthInternational

Compounding the unique binding epitope of 12-19 is it's reaching across spike domains to lock conformation. We believe conformation locking is among the most potent mechanisms by which an antibody can neutralize a virus.

The 12-19 antibody is not our first encounter with conformationlocking antibodies. In fact, some of our most antibody candidates to other dangerous pathogens employ a similar method. Lassa virus antibody 8.9F binds across the three faces of the Lassa trimer, locking the glycoproteins into place. Ebola antibodies 1C3 and 1C11 use a similar mechanism, locking the whole of the Ebola structure in place. Even parasites such as Malaria can be overcome with conformation-locking antibodies, such as CIS43, which prevents a cleavage function required for malaria infection.

The need for effective monoclonal antibody treatments is urgent. We should constantly look for antibodies that employ this conformation-locking tactic against SARS-CoV-2 and other major pathogens. These treatments consistently provide strong neutralization and protection. Their addition to the antibody arsenal would be well worth the effort to find them.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Novel Broadly Neutralizing SARS-CoV-2 Monoclonal AntibodiesThatBindAcrossTheSubunitsOfTheSpikeProtein

April 25, 2023: Are We Leaving Useful Antibodies Behind? The Value Of NonNeutralizing Protective Monoclonal Antibodies

While common sense may suggest that antibodies that do not neutralize the SARS-CoV-2 virus are of little value, recent studies show they can still confer protection against infection. Monoclonal antibodies have been a very effective method for preventing and treating Covid-19. Unfortunately, the current generation of virus variants has mutated to a degree that currently approved monoclonal antibodies that were previously successful are no longer effective.

There is a clear need for new approaches to antibody therapies. One method we have described is the use of combination antibodies that target highly conserved sites. Here we outline a different approach: using antibodies that bind, but are non-neutralizing, yet still protective in high doses. Part one will describe how this method was uncovered, and part two will describe more recent progress in this approach.

A recent study from Swedish scientists Bahnan et al. was the first to show the potential of non-neutralizing antibodies. What is a protective non-neutralizing antibody? These antibodies bind the virus spike, but do not prevent infection in vitro . However, they do protect from infection in live animal models.

How can that be? The virus still enters the cells as the antibody does not block infection, but the infected cells are killed before the virus spreads throughout the body. Bahnan et al.found that high levels of non-neutralizing monoclonal antibodies mediate the interactions between infected cells and immune system monocytes. The following study describes this discovery.

IsolationofSpike-ReactiveHuman MonoclonalAntibodies

They isolated the antibodies found in the sera of 20 Covid-19 patients. Ten of the 96 they isolated were reactive when exposed to the in vitro spike protein. Those most notably reactive were Ab11, 57, 59, 66, 77, 81, 94, and 95.

They identified the binding epitopes using cryo-electron microscopy to narrow down those that may be neutralizing antibodies. Of those most strongly reactive, they identified six antibodies that bound the spike receptor-binding domain (Ab11, 57, 59, 66, 77, 81) and one that binds both the receptor-binding and Nterminal domains (Ab94). However, they note that only Ab59’s epitope allows for virus neutralization. The rest were nonneutralizing.

The most important factor in our typical search for monoclonal antibody treatments is their neutralizing capacity. As suggested by their binding epitopes, only Ab59 showed a reduction of virus titers.

HighLevelsofNon-NeutralizingAntibodiesCanProtectAgainst SARS-CoV-2Infection

Given the varying levels of neutralization among the 96 isolated antibodies, Bahnan et al. introduced each antibody individually to

spike-monocyte interactions to determine the role of antibodies as mediators of immune interactions.

They found that modulated interactions were correlated with the dosage of monoclonal antibodies involved in the interaction. The highest dosage yielded a reduction in spike-monocyte interaction. Ab59, the only neutralizing antibody among the set of 96, showed the same trend of modulating monocyte interaction as other nonneutralizing antibodies, suggesting that this phenomenon is independent of neutralizing capacity.

Using humanized ACE2 mice subjects, they found that subjects treated with a large dose (250 µg) of non-neutralizing Ab94 were better protected from Covid-19 using weight loss as the measure as compared to the calculated protective dose (100 µg) of neutralizing Ab59.

Discussion

While neutralizing monoclonal antibodies will continue to be the focus of Covid-19 treatment, this discovery unlocks a new world of potential for antibody treatment. There have been dozens, if not hundreds, of antibody candidates in the past three years that either were not completely neutralizing initially, or lost neutralization as emerging variants developed. At the right dosage, some of these antibodies may prove to be valuable protective treatments.

In part two, we will describe another study that explores the use of monoclonal antibodies outside of their traditional role as neutralizing agents against the SARS-CoV-2 spike. With any luck, this new avenue may yield new treatments for those continuing to suffer from Covid-19 sooner rather than later.

April 27, 2023: Resurrection Of Covid

Antibody Therapy: IgG3 Fc Fusion To The Rescue

Recent studies indicate the previously unknown protective value of non-neutralizing monoclonal antibodies against SARS-CoV2. Throughout the pandemic, the search for monoclonal antibody treatments centered around neutralizing capacity. How well can an antibody neutralize the latest variants? As new variants arose, previously neutralizing antibodies lost their potency.

In a previous description, we analyzed the discovery of nonneutralizing antibodies protecting humanized mice models from Covid-19. In fact at proper dosages, some non-neutralizing antibodies protected better than their neutralizing counterparts. The key was opsonization: the tagging of pathogens for elimination by phagocytes. Non-neutralizing antibodies efficiently opsonized, leading to rapid clearance of the virus from infected subjects.

Here we discuss a study by Izadi et al. , which expands on the promise of non-neutralizing antibodies, as well as the potential of antibody subclass switches to enhance immune functions.

The two main components of an antibody are the Fab fragment and the Fc fragment. The Fab is the section that binds the pathogen and modulates neutralization. The Fc fragment binds host cell Fc receptors and modulates immune activities. Most human monoclonal antibodies isolated from sera are IgG1 antibodies. Neutralizing capacity aside, IgG1 is the joint-most potent activator

of Fc gamma receptors on immune cells, alongside IgG3. However, IgG3 is the single most potent activator of the classical complement pathway, which mediates immune functions such as inflammation, opsonization, and pathogen clearance.

Izadi et al. reengineered eight high affinity and non-neutralizing SARS-CoV-2 antibodies to use an IgG3 structure as opposed to an IgG1.

For six of the eight, the altered Fc fragment did not impact the binding affinity of the antibodies to the SARS-CoV-2 virus, meaning the enhanced immune functions of IgG3 did not come at the cost of binding capacity. For the remaining two antibodies, altering the Fc fragment substantially altered avidity. In one case, Ab11, the binding affinity for IgG3 was far worse than IgG1, yet for Ab57, IgG3 far outperformed IgG1, suggesting that Fc class switching can have an impact on avidity, but to exactly what degree remains unknown.

IgG3-switched antibodies also induced a stronger phagocytosis response than their IgG1 counterparts. Phagocytosis is the ingestion and elimination of pathogens by immune system phagocytes. Opsonization is the flagging of these cells to destroy an invader. All antibodies, aside from Ab11, saw phagocytosis efficiency increase up to threefold.

Furthermore, cocktails of the antibodies improved phagocytosis even further. A cocktail of all eight IgG3 antibodies was 12-fold more potent at phagocytosis induction than a cocktail of their IgG1 counterparts. A dual cocktail of the top two IgG3 antibodies was 8fold more potent than its IgG1 counterpart.

As for complement activation, IgG3 completely outperforms IgG1, which failed to even activate the system. Ab94, the best performing

singular IgG3, was the only single antibody that could induce complement deposition. The dual cocktail and octo-cocktail each deposited roughly 2.5-fold more than Ab94. Taken together, IgG3, and more specifically IgG3 antibody cocktails more effectively trigger the complement system, which would lead to more rapid clearance of the virus in a patient.

Finally, Izadi et al.aimed to validate their findings in live models to determine the relevancy of Fc mediated functions against true SARS-CoV-2 infection. Recall from our previous description that these antibodies do not have neutralizing capacity, so any and all protective capability is dependent upon Fc mediation. They prophylactically injected mice with Ab94 IgG1, Ab94 IgG3, Ab81 IgG3, DuomAb IgG3 or a vehicle control, followed by infection with the Wuhan strain of SARS-CoV-2.

All mice inoculated with some form of antibody outperformed those in the control group, in terms of body weight, mortality, and lung viral load. Notably, the Ab94 IgG1 performed the best in all areas, but the IgG3 antibodies and IgG3 cocktail were not far behind. This data adds to the growing evidence that both Fc-mediated immune functions are crucial to antibody development, as well as the value of non-neutralizing antibodies as treatment avenues.

Discussion

The use of these highly engineered antibodies for increased opsonization may be the recipe that's needed to revive antibody therapy to revive antibodies as effective treatments for a broad range.

One caveat of note is that of resistance. These antibodies mostly bind the receptor-binding domain, aside from Ab94, which binds both receptor-binding and N-terminal domains. This opens up the

possibility of virus mutations impacting the binding affinity of these non-neutralizing antibodies, which could make them less effective.

The search for more such antibodies not only that S1 subdomains, but also antibodies such as the camelid antibodies and others bind mind multiple faces of the trimeric structure to lock the spike from fusion. This style of antibody worked to improve the neutralizing activity for other viruses and we suggest coupling that with the major advance here of increasing opsonization by IgG3 FC fusion. A combination in that model offers a way forward for resurrecting monoclonal antibodies as effective treatments for a wide range of variants both past and future.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Resurrection Of Covid Antibody Therapy: IgG3 Fc Fusion To The Rescue

PART TWO

Antibodies and Other Major Pathogens

March 29, 2022: New And Effective Monoclonal Antibody Treatment For Ebola On The Horizon

Scientists at the Center for Infectious Disease and Vaccine

Discovery in La Jolla, California have developed a remarkable pair of monoclonal antibodies that may be the long-sought answer for effective treatments of multiple ebolavirus strains. Ebola poses a threat to human health not only in Africa, where it originates but with the possibility that it may spark another pandemic. We can all recall the fears of Ebola as it spread from the African continent to international shores. Fortunately, these cases were isolated and a pandemic did not ensue. There are no guarantees that ebolaviruses could not be the cause of the next global pandemic.

At present, our tools to confront Ebola are lacking and not fully effective. Current vaccines only treat one strain of ebolavirus, EBOV, and only with partial protection that fades over time. There are mRNA vaccines in development to treat ebolavirus, but it remains to be seen if those will have lasting durability against disease and death.

Although many small molecule drugs have been tested for efficacy, none have yet been approved. The most effective drug against Ebola is Regeneron’s monoclonal antibody cocktail: REGN-EB3. The drawback is that it only protects against one of the major strains of ebolavirus, leaving strains like Sudan virus (SUDV) untreated.

IsolationOfBroadlyNeutralizingAntibodies

The target for neutralizing antibodies in ebolaviruses, much like SARS-CoV-2, is the surface protein that regulates binding cell surface and cell entry. The protein binds receptors on the cell, triggering fusion of the viral and cell membrane, providing an opening for the virus particle to enter the cell. Entry is mediated by the trimeric surface protein of Ebola. There are three glycoproteins to a trimer and each is divided into two separate proteins: GP1 and GP2.

GP1 binds to the receptor, forming a binding pocket while GP2 mediates the fusion of the virus and cell membranes. GP1 is noncovalently attached to GP2, which oversees fusion to the cell surface once receptor-binding is complete.

SearchForBroadlyNeutralizingAntibodies

Milligan et al.isolated B-cell samples from patients within one year of their Ebola infection. They identified 36 mAb candidates. From these, they screened for binding strength to Ebola (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), all members of the ebolavirus genus. They also screened for antibodies that bound outside the ectodomain, meaning they were not restricted to the head and core regions.

This screening process narrowed their search to nine, which they examined individually for their neutralization of the diverse ebolavirus strains. A subset of those that bound these viruses neutralized the SUDV and BDBV strains. They separated the nine into two groups: those that bound to GP1 and those that bound to GP2.

Their search resulted in the discovery of two new antibodies: 1C11 and 1C3. Both of these strongly neutralized all of EBOV, SUDV, and BDBV, with the exception of BDBV for 1C3. 1C11 targeted the base region of the ebolaviruses glycoprotein, whereas 1C3 targeted the head region. Milligan etal.note that 1C3 also induces antibodydependent cytotoxicity, not only neutralizing the virus but also targeting and killing the infected cell.

1C3Binding

Please note that the antibody makes asymmetric contacts, binding overlapping but distinctly different sets of amino acids of the three receptor binding interfaces. 1C3, therefore, has a dual function. The three-face asymmetric binding not only inhibits binding to the cell but also effectively locks GP1 into position preventing fusion. Please note the simultaneous asymmetric binding residues of 1C3 to the trimer below.

1C11Binding

1C11 also has very unusual properties. Not only does it bind across two of the trimer surfaces, but it also binds an entirely different site down below 1C3 in the base of the glycoprotein, GP2, where the fusion domain lies. 1C11 contacts GP1 residues 34, 88–90, and 155 and the GP2 residues 523–524, 527–532, 534–536, and 563–566. The net result is the locking of the entire structure in place, without the ability to release glycoproteins after infection.

CombinationAntibody

The resultant combination therapy was more effective than both antibodies in isolation. In comparison to 1C3, the combination was

2.64-fold more effective and in comparison to 1C11, the combination was 3.93-fold more effective in vitro.

There are two reasons this combination therapy is so broadly binding. First, the base region that is bound by 1C11 varies little across ebolavirus strains. Second, while the amino acids of the chalice differ, the 1C3 footprint touches so many residues that the commonalities may compensate for the variation.

Protection FromDisease AndDeathIn RodentsAndNon-Human Primates

They also tested the combination therapy in vivo on both rodents and nonhuman primates. They first used a mouse-adapted virus. In pretreated EBOV-infected mice, 1C3 and 1C11 provided 90-100% protection from death as compared to the 10% survival rate of the control group. Similar results were found in BDBV-infected mice, in which the combination yielded 100% protection from death. In guinea pigs, neither antibody was effective individually, but the combination protected 80% of subjects for both EBOV and SUDV.

In nonhuman primate models, SUDV and EBOV-infected primates treated with the combination therapy had a 100% survival rate with the viruses being completely cleared according to PCR test between two and three weeks post-infection. The viruses were undetectable only days after inoculation with the treatment.

Milligan et al. also emphasize the restriction of low dosages throughout their testing process as these antibodies can be expensive and would primarily be used in low-income countries.

In a piece of truly excellent work involving antibody isolation, characterization, and animal model work, Milligan et al. present a

tour de force . This work provides a key not only for effective treatment for ebolaviruses, but we believe for many other viruses as well. The analog for SARS-CoV-2 would be antibodies that bind across the S1/S2 subunits in a similar fashion, locking the trimer into place. An equivalent may be the camelid antibodies that lock the SARS-CoV-2 Spike protein in place in a similar way, which may be a class of antibodies worth exploring for future treatments.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: New And Effective MonoclonalAntibody Treatment For Ebola On The Horizon

April 11, 2022: Two Emerging Viral Adversaries Nipah And Hendra Virus May Soon Meet Their Match

Two emerging viral adversaries Nipah and Hendra Virus may soon meet their match. Our recent experience with Covid-19 has taught us to be aware of new and emerging viruses that have pandemic potential. Amongst these viruses, we draw attention to two members of the zoonotic henipavirus genus: Nipah virus and Hendra virus.

These two viruses are responsible for outbreaks of encephalitis, or the inflammation of brain tissue, and respiratory illness, with a staggering 50-100% fatality rate. To date, Nipah Virus and Hendra Virus outbreaks have been localized in poorer communities within Bangladesh, India, the Philippines, and Africa. Exciting new work underway may pave a path for preventing these viruses from growing out of hand in the coming years.

People have long noted the potential of these viruses to cause much wider outbreaks. The viruses are transmitted both from infected animals to humans via direct contact or contaminated food supplies, as well as from infected human-to-human via direct contact. Personto-person transmission is most commonly noted in families and healthcare settings. The virus is not airborne but can spread via droplets, such as sneezing or coughing.

NipahVirusGlycoproteinStructure

Monoclonal antibodies have proven to be a mainstay for postexposure prophylaxis and treatment of Covid-19. It is therefore of great interest to understand how monoclonal antibodies may work for other viruses and in turn, such knowledge may inform antiCovid monoclonal strategies as well.

The following paper by Veesler et al. is a welcome insight. It elucidates both the structure of the Nipah virus external protein, as well as describes a neutralizing antibody combination that could overcome both Nipah Virus and Hendra Virus. This information may provide a pathway for future treatments and vaccines to follow.

The Nipah Virus binds and fuses to a host cell via the glycoprotein. This protein comes in sets of four, or as a tetramer, like a claw in an arcade crane game with four arms. It is an asymmetrical tetramer consisting of a stalk, neck, linker, and four heads. The Nipah tetramer binds ephrin-B2 or ephrin-B3, which are transmembrane protein tyrosine kinases on the host cell surface.

The figure above is a beautiful depiction of the tetramer. The amino terminus of the glycoprotein is a stalk, which descends from the protein as an alpha helix to the membrane. Next is the neck region which connects the stalk to the large head region comprising over two-thirds of the glycoproteins amino acids. We emphasize the asymmetric nature of the tetramer. Many viruses, including HIV and coronaviruses, form symmetric trimers or sets of three, but Nipah Virus is an asymmetric tetramer, wherein only one head binds. A detailed understanding of virus Spike proteins allows for a more intricate analysis of potential antibody treatments, such as the following.

ThreeMonoclonalAntibodiesForNipahVirusAndHendraVirus

After modeling the Nipah Virus glycoprotein in clear detail using cryo-electron microscopy, Veesler et al. then began testing a number of potential antibody candidates in hopes of finding a treatment for the disease.

BlockingTheReceptor-BindingSite:m102.4

One antibody, m102.4 targets the receptor-binding domains of the tetramer’s individual monomers, essentially mimicking the binding surface of the host cell and preventing transmission. It directly competes with ephrin binding sites, blocking any chance of the virus binding to the cell surface. This antibody recently completed a phase 1 clinical trial in Australia against Hendra Virus or Nipah Virus infection. With IC50 values ranging between 17-58 ng/mL, the neutralization potency was relatively solid against both Nipah Virus and Hendra Virus.

InterferingWithFusion-TriggeringMechanism:nAH1.3

The second antibody, nAH1.3, was isolated from a mouse-adapted Nipah Virus infection back in 2016. The nAH1.3 antibody targets each Nipah Virus head domain, interacting with antigenic sites away from the receptor-binding domain. Unlike m102.4, nAH1.3 does not impact binding but rather inhibits HNV entry into cells by interfering with fusion triggering mechanisms. If the virus is unable to fuse, it cannot transmit its RNA into the host cell and continue to reproduce within the host. The IC50 values range between 33 and 32 ng/mL for the Nipah Virus and Hendra Virus viruses, which the authors note is a similar neutralizing capability to that of m102.4.

AntibodySynergy

Against SARS-CoV-2 and other viruses, combination antibody treatments have been used to give antibodies an edge against escape viruses by targeting multiple nonconflicting sites. Both m102.3 and nAH1.3 binding to the Nipah Virus and Hendra Virus tetrameric glycoproteins in a combination treatment resulted in a highly synergistic neutralization. Synergy is determined by a ZIP score, of which greater than ten indicates a stronger combination together than separate. For Nipah Virus, the ZIP score of the two antibodies was 10.628 and for Hendra Virus, the score was 17.248.

Yet AnotherArrow In TheQuiver:HENV-32

They also note a third antibody, HENV-32, which binds the neck and stalk regions of the viruses. These regions are typically more conserved among the henipavirus genus, indicating potential crossreactivity. Based on its target region, HENV-32 would be very unlikely to negatively interfere with nAH1.3, m102.3, or their synergistic effects. A triple antibody combination of the three could potentially be our most effective strategy for treating Nipah Virus and Hendra Virus in the near future.

This paper is a dramatic understanding of the structure and function of the Nipah Virus that should lead to a strong step forward in attempts to both prevent and control infections. We urge continued work by researchers and pharmaceutical companies to develop vaccines, small molecule drugs, and monoclonal antibodies. These medical advances could treat current epidemics of viruses such as these and prevent the potential for future pandemic spread of a deadly pathogen.

Two Emerging Viral Adversaries Nipah And Hendra Virus

May Soon Meet Their Match

November 03, 2022: New Monoclonal Antibody Cocktail Neutralizes Lassa Virus

Monoclonal antibodies are among our greatest assets in treating and preventing virus-induced disease. While the spotlight has focused squarely on Covid-19 monoclonal antibodies throughout the pandemic, antibody candidates for other severe pathogens have also made strides forward. Here we describe a new antibody cocktail that neutralizes a lesser known but nonetheless dangerous virus circulating West Africa: Lassa Virus.

Whatis LassaVirus?

Lassa Virus is a pathogen responsible for Lassa hemorrhagic fever, which afflicts between 300,000 and 500,000 people per year, commonly in West African countries, including Nigeria, Liberia, Sierra Leone, Guinea, and Ghana. Lassa fever has a mortality rate of roughly one percent. Pregnant women are at the greatest risk of death, with up to a 90% fatality rate. The symptomatic cases present issues such as fever, headaches, vomiting, and muscle pain. Spread typically occurs via contact with infected mice excrement or urine, though direct contact spread from person to person is also common. Unfortunately, a vaccine is not yet available for the virus, and antivirals are weak at best. Lassa Virus, like SARS-CoV-2, is an RNA virus that constantly mutates. A therapy or prophylactic to fill the void of Lassa Virus drugs is needed.

LassaVirusMonoclonalAntibodyTherapy

Fortunately, a new candidate may be just around the corner. A group of researchers from the La Jolla Insitute for Immunology in California led by Dr. Erica Ollmann Saphire have identified a cocktail of three antibodies that bind and neutralize the Lassa Virus. This same group discovered a broadly accepted cocktail of monoclonal antibodies for Ebolavirus, which we have previously described. This cocktail arrived just in time as the current to treat the current epidemic caused by the Sudan strain of Ebolavirus in Nigeria.

The new Lassa Virus therapy, denoted Arevirumab-3, is comprised of three distinct antibodies, each one binding distinct regions of the Lassa Virus glycoprotein.

The Lassa Virus spike protein comes in a set of three identical subunits embedded into the membrane. The virus is synthesized as a single long polypeptide and is cleaved into three parts, a leader sequence, GP1 which encodes the receptor-binding function, and GP2 embedded into the membrane.

8.9Finhibitsvirion-cellattachmentbyblockingtheGPC/α -DG interaction

The first antibody, 8.9F, binds the top of the spike trimer, stretching across all three portions of the glycoprotein. 8.9F is an unusual antibody as the single antibody binds all three faces of the trimer. The antibody also binds the three cleavage sites of the GP1 subunits. It binds the portion of the glycoprotein that attaches to the host cell, directly inhibiting virion-cell attachment by mimicking the cell alpha-dystroglycan receptor.

The 8.9F antibody also specifically binds a glycan, N119, which is required for neutralization activity. This glycan is required for alpha-dystroglycan receptor-binding, so 8.9F’s recognition is surprising.

Anative N89glycanplaysa centralrolein 12.1F/LASVGPC recognition

The second antibody, 12.1F, binds to a separate site on GP1, directly interacting with N-glycans for neutralization activity. The antibody binds more membrane-proximal and has direct interaction with six crucial amino acid residues and critical glycans N89 and N109.

To infect a cell, the Lassa Virus must bind the cell membrane and be engulfed by a cellular endosome. Then the protein undergoes a structural change to bind the LAMP-1 protein in the endosome interior. Binding to LAMP-1 is essential for fusion of viral and cell membranes and entry into the cytoplasm where replication occurs.

37.2DneutralizesLASVbylockingtheGPCtrimer in an inactive configuration.

The third antibody, 37.2D, binds two adjacent subunits of GP2. The antibody stretches across subunits, locking the trimer in place by binding conserved peptides and conserved glycans N390 and N395.

37.2D is special in that it attacks GP2 at a unique angle, stabilizing the pre-fusion trimer, preventing fusion activation and, therefore, infection.

These three mechanisms work together to yield a highly effective antibody cocktail.

One critical question for monoclonal antibody treatment, is can the virus escape via mutation in the binding sites? Dr. Ollmann Saphire's team shows that such is the case for treatment with a single 8.9 F antibody. However, no resistance mutations arose when using the cocktail of all three antibodies, nor was the virus capable of escaping.

The Arevirumab-3 antibody may provide a long-awaited answer to the lack of treatment and prevention of Lassa infections. This is particularly important for cases involving pregnant women and their fetuses.

It will be important to reduce the costs of this antibody cocktail as much as possible so that it is available when needed in West Africa. Current technologies allow monoclonal antibodies to be produced at $200 and $250 per gram.

The next step in the development of the control of Lassa Fever is the development of a vaccine effective against all Lassa Virus variants. This work may serve as a guide for the creation of such a vaccine.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: New MonoclonalAntibody Cocktail Neutralizes Lassa Virus

November

2022: New Monoclonal Antibody For Treatment Of Malaria

14,

Monoclonal antibodies are among our greatest assets in treating and preventing virus-induced disease. While the spotlight has focused squarely on Covid-19 monoclonal antibodies throughout the pandemic, antibody candidates for other severe pathogens have also made strides forward. Here we describe a new antibody candidate that neutralizes a parasitic foe that has circulated much longer than SARS-CoV-2 and is responsible for hundreds of thousands of deaths annually: Malaria.

While the incidence of Malaria is much lower than 20 years ago, poverty-stricken countries, particularly in Africa, continue to struggle with the disease. Progress has halted in recent years due to the lack of effective treatments for the disease. Existing monoclonal antibody treatments are few and far between, and vaccines are relatively ineffective. While newly emerging vaccines backed by the WHO show more promise, Malaria expert Dr. Umberto D’Alessandro notes we should use all the tools we can gather, specifically in reference to monoclonal treatments.

A new study by Kayentao et al. from the Mali Malaria mAb Trial Team discusses the phase two clinical trials of the CIS43LS antibody, which is already demonstrating protection against controlled malarial infection in phase one.

The new antibody was discovered back in 2018 by Kisalu et al. of the National Institutes of Health. It was isolated with other antibody

candidates from humans vaccinated with the malarial vaccine RTS,S/ASO1. Protection from this vaccine wanes over time, but the researchers discovered that the isolated antibody CIS43 demonstrated extended protection against malaria in mice. They found that the antibody preferentially binds a junction between two critical domains of the malaria parasite, resulting in remarkably high binding affinity and the inhibition of cleavage, rendering the pathogen inert. The antibody targets the sporozoite form of malaria, which is the infective stage. The parasite cannot bind and spread within the human liver due to the lack of cleavage. This antibody target is notable as it's the same target used in the new GSK malarial vaccine: Mosquirix.

The antibody was also modified with an LS mutation to increase its half-life. First discovered by Zalevsky et al. in 2010, LS mutations refer to a set of two mutations in an antibody, M428L and N434S. These two mutations decrease the antibody dissociation rate and increase Fc binding affinity to human receptors 11-fold. In various experiments, LS mutations to the Fc receptor yield a 2-to-3-fold increase in antibody half-life, which has led to LS mutations becoming common practice among monoclonal antibody developers to extend the efficacy of their treatments.

CIS43LS then underwent phase one trials, described in an August 2021 paper by Gaudinski et al.While the trial was relatively limited with 25 participants, Gaudinski and colleagues found that none of the participants who received CIS43LS displayed malarial parasitemia 21 days after controlled human malarial infection. These results were encouraging, as the RTS,S/ASO1 malarial vaccine’s efficacy ranges from only 30-60%.

The Mali Malaria mAb Trial Team expands Gaudinski’s controlled phase one trial to a more uncontrolled setting in phase two. 330 adults across Mali received either 10 mg of CIS43LS per kilogram of body weight, 40 mg, or a placebo over a six-month period. Of the 110 participants randomly assigned to the 40 mg group, 20 (18.2%) were infected within the six-month period. In the 10 mg group, 39 (35.5%) were infected. In the placebo group, 86 (78.2%) were infected.

The final efficacy of the 40 mg treatment, as compared to the placebo, was 88.2% within a 95% confidence interval, while the 10 mg treatment was slightly lower at 75%.

The only notable side effect was the risk of moderate headache in the 40 mg group, which was reported 3.3 times as often as the placebo group. Aside from headaches, no significant side effects were noted, and no safety concerns were observed.

Both 75% and 88.2% efficacies for CIS43LS are far higher than the 30-60% efficacy of existing malarial vaccines.

Upon approval, the greatest challenge for the antibody will be keeping the production cost low, as those needing the treatment most reside in lower-income nations. Current technologies allow monoclonal antibodies to be produced at $200 and $250 per gram. Ensuring the treatment reaches those that need it most without a high financial barrier to entry is crucial if the drug is to be successful.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: New MonoclonalAntibody For Treatment Of Malaria

November 23, 2022: Hope For A New Treatmen t On The Horizon For Zika Virus

Researchers from Duke University, UC Berkeley, Purdue University, and elsewhere have made what will likely be a significant breakthrough in drug development to treat Zika Virus infections. They discovered an unusual monoclonal antibody, not of the common immunoglobulin G family, but rather immunoglobulin M. This antibody is extraordinarily potent in neutralizing the Zika Virus in tissue cultures, as well as in live animal experiments.

Monoclonal antibodies are among our greatest assets in treating and preventing virus-induced disease. While the spotlight has focused squarely on Covid-19 monoclonal antibodies throughout the pandemic, antibody candidates for other severe pathogens have also progressed. Here we describe a new antibody candidate that neutralizes the Zika Virus, which is responsible for thousands of infections annually.

While not nearly as prevalent as its peak in 2016, documented Zika Virus infections still occur in over 80 countries, with roughly 18,000 cases per year. Infections most often occur in areas closer to the equator, as mosquitoes are the primary mode of transmission for the virus.

The incubation period for Zika Virus disease is between three and 14 days, at which point symptomatic patients yield fever, rash,

conjunctivitis, joint pain, and headache for up to a week. One of the more severe Zika Virus complications occurs in pregnant hosts. Roughly 14% of host fetuses develop severe brain and eye defects. Many cases result in stillbirth, premature birth, or miscarriage. Researchers Singh et al. aimed to discover a monoclonal antibody treatment to placate the virus that still rages in tropical regions. The scientists were surprised to find that a specific type of antibody, immunoglobulin M (IgM), was particularly active concerning Zika Virus immunity for the fetus during pregnancy. The vast majority of antibodies are immunoglobulin G (IgG). While most antibodies are a single ‘Y’ shaped monomer, the IgM antibody presents in a set of five, or a pentamer. IgM antibodies are the largest produced antibody and are the first to respond to initial exposure to an antigen. The monomers are bound to their adjacent monomer by a disulfide bond, and a joining chain keeps the large antibody intact.

Singh et al. extracted plasma IgM to test for binding and neutralization from a cohort of 10 pregnant Brazilian women during the 2015-2016 Zika Virus outbreak. They eventually isolated one Zika Virus monoclonal antibody candidate, DH1017.IgM, which demonstrated the most robust Zika-neutralizing capacity in early testing.

In addition to neutralizing the parental Zika Virus, DH1017.IgM also neutralized ZIKV PRVABC59 and ZIKV H/PF/2013, two prominent variants of the parental virus. DH1017.IgM also had a lower mutation rate than other antibody candidates in early testing, meaning neutralization capacity is unlikely to change due to chance mutations in the antibody.

Notably, Singh et al. found that the pentameric form of the IgM is crucial to DH1017 binding. As a Fab fragment, meaning the arms of the ‘Y’ shape solely, the antibody bound poorly. DH1017 bound roughly 20-fold stronger as an IgG monomer, but as an IgM pentamer, the antibody bound more than five-fold stronger than the monomer. In parallel, neutralization for IgM was 40-fold stronger than the IgG monomer.

The researchers conducted further tests on mice models to determine in vitro neutralization for DH1017. They found that the IgM antibody protects against severe and lethal cases of Zika Virus infection at lower 50 or 100 microgram doses but protects against viremia much more efficiently at higher doses. Again, the researchers found that the IgG monomer version of DH1017 fails to achieve the marks set by the pentameric IgM.

Singh et al.note that the five-armed IgM antibody “may contact up to five epitope pairs compared to a single epitope pair for the bivalent DH1017.IgG.” This would explain the vastly increased binding and neutralization. Picture a chest with a single lock and a chest with five locks. Which is more secure?

The researchers also found that the IgM may bind epitope pairs across different virus particles, creating a cross-linked virion epitope. Now picture multiple chests chained and locked together. The IgM antibody form presents fascinating advantages over its IgG counterpart.

The DH1017 antibody, like many antibodies treating viruses primarily impacting lower-income nations, would need to be produced at a low cost to ensure those who need the treatment most could afford it. Current technologies allow monoclonal antibodies

to be produced at $200 and $250 per gram. DH1017 could be a godsend for those still impacted by Zika Virus, particularly pregnant women in low-income countries.

In a broader sense, this study opens a new avenue for monoclonal antibody development. It is clear that pentameric IgM antibodies bind and neutralize much more effectively than monomeric IgG antibodies. It would be relatively straightforward to convert potent IgG antibodies to IgM by replacing the Fc portion of the antibody. If researchers could harness that advantage and engineer antibodies to other global antigens, such as SARS-CoV-2, the reward would be well worth the effort.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere:

Hope For A New Treatment On The Horizon For Zika Virus

December 01, 2022: Crimean - Congo

Hemorrhagic Fever Virus Monoclonal Antibodies: A Work In Progress

Crimean-Congo Hemorrhagic Fever is among the deadliest diseases in the world; a tick-born disease touting a mortality rate of up to 40%. First discovered almost eight decades ago, there is no effective treatment at present. Here we describe a work in progress of attempts to develop monoclonal antibodies to prevent and treat this deadly disease.

CCHFVBackground

The virus was first identified in Crimea in 1944 and later in the then-Belgian Congo in 1967. More recent reports date the virus as far back as ancient Celtic settlements between 1500 and 1100 BCE. Since its discovery, outbreaks have plagued dozens of countries in Eastern Europe, the Middle East, Africa, and South Asia. The most substantial was an outbreak in Turkey from 2002 to 2008, in which 3,128 cases were reported.

CCHFV is most commonly transmitted by ticks. In southeast Iran, 31 different species of tick carry the pathogen. The pathogen has also been found in hares, hedgehogs, rats, birds, and domesticated animals such as sheep, goats, and cattle.

The virus is a member of the genus Orthonairovirus , family Nairoviridaeof RNA viruses. Other viruses in this genus include the Dugbe virus, the Nairobi sheep disease virus, and the Kasokero

virus. Throughout the decades, attempts have been made at vaccines and treatments for the virus and its subsequent disease, but all were tabled due to low efficacy or associated toxicity. A new antibody treatment could be helpful for hundreds impacted by the disease annually and thousands that could be spared from infection altogether.

CC5Human MonoclonalAntibodies

The first objective for researchers Durie et al. was to find a worthwhile target for monoclonal antibody treatment. All previous treatments had failed, so a novel site would be a quality starting point.

Their strategy was to use a previously discovered antibody that prevented severe disease in mice against CCHFV, but failed to do so in humans. They analyzed the antibody, 13G8, and its primary binding site: CCHFV glycoprotein GP38.

Using this site as a template, the researchers searched for human monoclonal antibodies targeting this same site. They pulled sera from six verified survivors of CCHFV infection and isolated a panel of antibodies for further testing from one patient: CC5.

Of the seven antibodies isolated from CC5, all matched or exceeded the binding affinity of the mouse antibody 13G8. The three most potent binders overall were CC5-6, CC5-16, and CC5-17. Three of the seven human monoclonal antibodies directly competed with the binding epitope of 13G8: CC5-6, CC5-12, and CC5-17. This overlap would suggest that GP38 is a prime target for antibody binding moving forward for CCHFV treatments.

Upon further cryo-electron microscopy experiments, Durie et al. found that CC5-17, one of the stronger binding human monoclonals, attacks GP38 at a differing angle of 22 degrees, which may account for the more thorough binding affinity. The researchers also note the binding capability of CC5-17 to another closely related virus, the Aigai Virus. The GP38 binding site is shared between the two viruses, and minor mutational differences do not affect binding affinity.

Unfortunately and unsurprisingly, the human antibodies were again non-neutralizing in human in vitro models. The antibody may still protect against severe disease and death in human CCHFV patients, but it seems the GP38 antibody binding site is exclusively nonbinding. This suggests another receptor could yield a more substantial neutralizing capacity when bound by an as-of-yetundiscovered antibody. The CC5 antibodies may have some diagnostic value but fall short as a tool for protection.

ConcludingThoughts

While it is disappointing that CC5-17 and its CC5 alternatives were not innately neutralizing, their shortcoming could enable another treatment's success in the future. The GP38 binding site is a prime target for broad binding affinity. GP38 likely has a significant role in virus maturation and localization of viral particles to the host cell's surface. A viable antibody candidate could disrupt this process and effectively neutralize the virus. GP38 could also serve as a starting platform for vaccine treatment soon. Otherwise, different binding sites should be investigated throughout the virus genome in the search for a treatment.

MonoclonalAntibodies:TheOnceandFutureCureforCovid-19

This study confirms the possibility of highly effective anti-CCHFV treatments; we need only find them.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere:

Crimean-Congo Hemorrhagic Fever Virus Monoclonal Antibodies: A Work InProgress

March 29, 2023: Opening The Door To The Use Of Antibodies To Protect Plants From Pathogens

New technologies are currently being explored to protect our agriculturally important plants from invading pathogens. The advent of mass monoculture, that is to say, enormous numbers of plants with a similar genetic background, has provided an almost perfect environment for a wide variety of invading organisms, including viruses, bacteria, fungi, and parasites. Although plants are well defended by their innate natural immunity against invading organisms, they lack the fundamental adaptive immune system present in animals. There is an urgent need to develop new ways to allow plants to protect themselves. Recent advances raise the exciting possibility that at least some components of the adaptive immune systems of animals, through genetic engineering, can be added plant defenses against specific pathogens. Here we describe a recent paper by Kourelis et al. , researchers from the University of East Anglia in the United Kingdom that details this exciting development.

The researchers found the camelid nanobodies more convenient to work with firstly, because of their single chain. Secondly, nanobodies lack an Fc region, so they are unlikely to engage other cellular activities such as cytotoxicity.

They inserted the nanobody into a gene usually induced by plants to counter a pathogen as part of the innate immune system. This

gene is activated when a plant is infected by one of a broad array of pathogens. When the plant was infected, this re-engineered antibody structure would be produced for the specific pathogen. They refer to the re-engineered antibody as a pikobody. Kourelis et al. used Potato virus X to demonstrate the binding and neutralization specificity of the pikobody, demonstrating the efficacy of this new plant antibody technology.

Six of the 11 fusions they developed did not exhibit autoimmune activities, meaning the engineered immune response did not turn on itself. From those six, four demonstrated hypersensitive cell death responses.

The researchers next tested their four candidates against live pathogens. Against a recombinant Potato virus X expressing fluorescent proteins, the top two performing pikobodies substantially reduced the accumulation of virus expressed compared to a control.

Further testing against Potato virus X revealed that stacking pikobodies allowed for enhanced reduction of virus accumulation.

While the pikobodies demonstrated a strong reaction to the Potato virus X sample, a different pikobody would need to be developed for every pathogen that could impact the plant, as the innate immune system is insufficient to ward off all pathogens.

Researchers emphasize that “nanobodies can be readily generated to bind virtually any antigen.” Plant pikobodies can be created against a broad range of plant pathogens, including but not limited to fungi, bacteria, protozoa, and viruses.

Pikobodies represent a vital step forward and a new pathway to create disease-specific plants capable of defending themselves

against specific diseases, both with their native immune system and now with human-like defenses against specific pathogens.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere:

Opening TheDoor ToTheUse OfAntibodiesTo Protect Plants

FromPathogens

April 04, 2023: Promising Monoclonal Antibodies For The Treatment Of Yellow Fever Virus

Yellow Fever virus is among the most dangerous pathogens circulating today. As many as half of hospitalized patients succumb to disease complications. The disease typically impacts tropical climates, such as central Africa and South America, where mosquitos are heavily present. However, the Yellow Fever virus has historically been a serious problem for the United States. Throughout the 18th and 19th centuries, Yellow Fever ravaged port cities as far north as Boston as trade in the post-revolution United States reached its full potential. Cities such as New Orleans, Savannah, and Charleston were hit the hardest, accounting for over 100,000 Yellow Fever deaths. Many thousands of Americans were also infected during the building of the Panama Canal. While recent vaccine advances have limited new infections, effective treatments for those with a Yellow Fever virus infection are nonexistent.

Yellow Fever virus is estimated to cause 30,000 deaths annually, with 90% occurring in central Africa. The primary transmission method is by the bite of the Aedes or Haemagogus mosquito, with symptoms coming on roughly five days after the initial infection.

The only presently available treatment or prophylactic is the YFV17D vaccine. The US Department of Health and Human Services recommends a YFV-17D booster every ten years for those traveling to areas affected by the Yellow Fever virus. The vaccine was developed in 1938 at Rockefeller University. Since that time, no other live attenuated vaccine has been created, despite ongoing efforts.

In a recent study by Ricciardi et al. , human monoclonal antibodies were isolated from the sera of volunteers vaccinated with the Yellow Fever virus vaccine YFV-17D. The researchers analyzed the isolated antibodies against both in vitro and in vivo viruses, finding that several antibodies sufficiently neutralized virus samples. Here we analyze their findings and discuss the implications on global Yellow Fever virus outbreaks.

AntibodyScreening

Ricciardi et al. isolated 489 antibodies from memory B-cells extracted from the sera of over 1,200 Yellow Fever virus vaccinees. They selected 38 candidates based on their high neutralizing capacity.

The antibodies were further selected based on the neutralization of Yellow Fever virus alternate strains, specifically the common isolate, YFV-ES504, associated with the 2016 outbreak in Brazil. Just 16 of the 38 neutralized the Brazilian isolate with a sufficient IC50 value. The final antibodies selected were two that do not compete in binding, suggesting they bind different epitopes. These maintained the strongest Brazilian isolate neutralization at low concentrations.

The two antibodies, MBL-YFV-01 and MBL-YFV-02, also effectively neutralized three other varying Brazilian isolates, YFV-

4408-1E, YFV-RJ155, and YFV-GO09, all associated with different Yellow Fever virus outbreaks. Again, the two independently neutralized all three isolates.

TherapeuticmAbadministrationprotects hamstersandmacaques frompathogenicYFV infection

Ricciardi et al. next infected Syrian golden hamsters with adapted Yellow Fever virus to analyze the in vivo efficacy of MBL-YFV-01 and MBL-YFV-02. Three days after infection, the hamsters were administered a single antibody dose of either MBL-YFV-01 or MBLYFV-02.

Both antibodies significantly improved hamsters’ survival rate, with 100% of those treated with MBL-YFV-01 overcoming the disease. In contrast, only four of 15 in the control group survived their disease course.

Additionally, health markers such as weight fluctuation and liver disease found that those treated with monoclonal antibody candidates survived their symptoms and experienced a wholly reversed disease course, indicating strong protection from death and severe illness.

In further invivotesting, Ricciardi etal.infected macaques with the adapted Yellow Fever virus. Following the same pattern as the Syrian golden hamsters, macaques treated with MBL-YFV-01 or MBL-YFV-02 on day two survived three weeks following infection, compared to control macaques succumbing to the disease on day five of infection. All but one macaque had no detectable Yellow Fever virus in their sera 21 days post-infection., suggesting MBLYFV-01 and MBL-YFV-02 are strong candidates as anti-Yellow Fever virus treatments.

Discussion

Recent advances in antibody technologies have enabled new and exciting treatments for pathogens previously untreatable. Highmortality diseases such as Yellow Fever, Dengue, Zika, and Ebola have ravaged poorer communities without access to high-quality healthcare. While vaccines effectively prevent some of these diseases, treatment methods are often limited to general medical support, which is often limited in these communities.

MBL-YFV-01 and MBL-YFV-02 represent a step forward in treatment for the Yellow Fever virus and high mortality diseases in general. To understand potential protection against current and future variants of the Yellow Fever virus, future studies must include structural analyses of the antibodies to understand the exact binding epitope to the virus. While further human safety and efficacy are necessary to consider these two antibody candidates a success, their initial in vitro and in vivo data show great promise. We can only hope these antibodies soon advance to clinical trials before helping those in desperate need of an effective treatment.

Thisarticleis featuredon Forbes.org,andcan be readonlinehere: Promising Monoclonal Antibodies For The Treatment Of Yellow Fever Virus

Acknowledgments

Iwould like to thank Griffin McCombs for his tireless work and good cheer. I could not have attempted this book without the dedicated assistance of the ACCESS Health US team: Courtney Biggs, Koloman Rath, Amara Thomas, Roberto Patarca, and Kim Hazel.

I would also like to thank my Boston area colleagues at Mass CPR; the Variants Group; and the Vaccine Group.

This work is supported by ACCESS Health International (www.accessh.org).

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