Isolation and characterization of the functional breadth of neutralizing antibodies that cross-react with diverse sarbecovirus strains

Abstract

Coronaviruses, specifically viruses in the Betacoronavirus genus, recurrently spillover into humans from other animal reservoirs and are a cause of substantial morbidity and mortality globally. Severe acute respiratory syndrome coronavirus (SARS-CoV-1) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have caused significant outbreaks, and there are numerous diverse SARS-like coronavirus strains (sarbecoviruses) circulating in animal reservoirs with human spillover potential. In the context of SARS-CoV-2, the viral entry protein, spike (which is the major target of neutralizing antibodies), continues to accumulate mutations leading to reduced efficacy of therapeutic monoclonal antibodies (mAbs) and the elicited plasma response against variants of concern. Cross-reactive mAbs, defined here as antibodies capable of recognizing SARS-CoV-2 as well as diverse sarbecovirus strains, have been isolated from SARS-CoV-2 convalescent and vaccinated individuals. This indicates there are conserved sarbecovirus spike epitopes recognized by cross-reactive mAbs, though many are not durable against SARS-CoV-2 evolution. Harnessing what has been learned from studying the immune response to SARS-CoV-2, could inform what a protective immune response may look like and minimize the impact of future outbreaks. To improve our understanding of the development of broad mAbs that can neutralize across sarbecoviruses and gauge their durability against evolving SARS-CoV-2, here we conducted two studies to comprehensively characterize a collection of cross-neutralizing antibodies elicited in different SARS-CoV-2 contexts. We previously identified a cross-reactive mAb in one individual, C68, that experienced a vaccine breakthrough infection with the Delta SARS-CoV-2 variant, which led us to hypothesize the existence of additional broad neutralizing antibodies in this individual. In chapter II, I explore the functional activities of multiple cross-reactive mAbs isolated from this individual that target epitopes within the receptor binding domain (RBD) of the spike protein. Not all cross-reactive mAbs are equal and I describe some mAbs displaying broad and potent binding and neutralizing activity against recently circulating SARS-CoV-2 variants and animal sarbecoviruses with spillover potential. Given this finding, I collaborated on studies to identify the escape pathways of these antibodies in a SARS-CoV-2 deep mutational scanning assay. In most instances, these identified sites of escape would negatively impact viral functions (like proper RBD folding), and thus SARS-CoV-2 spike would be less tolerant to variation at these sites. For cross-reactive mAbs with limited breadth, these escape maps largely predicted loss of neutralization activity against SARS-CoV-2 variants or explained loss of activity against sarbecovirus strains (due to variation at these sites). Since the identified broadly neutralizing antibodies have complementary neutralization profiles and minimal to no overlapping escape maps, they could form the basis for a pan-sarbecovirus antibody cocktail. In the studies of chapter III, we sought to determine whether mAbs with even greater breadth or potency might be present in C68 after subsequent exposures to SARS-CoV-2 based on studies showing that multiple antigen exposures and antigenic diversity may drive the development of enhanced neutralizing antibody responses. To address this, we examined the affinity maturation of clonal lineage members using a longitudinal sample from C68 after additional vaccination and breakthrough infection (Omicron strain). We applied an antibody isolation approach using both SARS-CoV-1 and SARS-CoV-2 spike protein to select for broadly active memory B cells and did identify a collection of cross-reactive mAbs from multiple expanded clonal lineages, some of which exhibited enhanced neutralizing activity relative to clonal lineage members from the earlier time point. We also identified a broadly reactive mAb that exhibited pan-sarbecovirus binding and robust neutralization activity against every single sarbecovirus tested in a multi-clade virus panel. Overall, the major targets for mAbs with broad sarbecovirus activity were epitopes within conserved regions of the RBD. This includes RBD class 5 (explored in chapter II) that rarely mutates in nature for SARS-CoV-2 variants and may be functionally constrained from evolving. In addition, it includes the RBD class 4 epitope (explored in chapter III) that is highly conserved across all sarbecoviruses assessed though targeting an interface that is susceptible to evolving SARS-CoV-2. Thus, these studies describe a diversity of broad-spectrum, and in some cases potent, mAbs targeting different epitopes that could be valuable medical countermeasures against sarbecoviruses with spillover potential. Additionally, these studies demonstrate that exposure to SARS-CoV-2 can lead to cross-reactive mAbs that target conserved epitopes and additional exposure to diverse SARS-CoV-2 spike can lead to enhancement of neutralizing activity for clonal lineage members. Future studies should focus on characterizing cross-reactive mAbs from individuals with multiple diverse exposures to SARS-CoV-2 antigen to inform how to elicit broadly neutralizing antibodies by vaccination.