The S2 subunit of spike encodes diverse targets for functional antibody responses to SARS-CoV-2 (preprint)

The SARS-CoV-2 virus responsible for the COVID-19 global pandemic has exhibited a striking capacity for viral evolution that drives continued evasion from vaccine and infection-induced immune responses. Mutations in the receptor binding domain of the S1 subunit of the spike glycoprotein have led to considerable escape from antibody responses, reducing the efficacy of vaccines and monoclonal antibody (mAb) therapies. Therefore, there is a need to interrogate more constrained regions of Spike, such as the S2 subdomain. Here, we describe a collection of S2 mAbs from two SARS-CoV-2 convalescent individuals that target multiple regions in the S2 subdomain and can be grouped into at least five epitope classes. Most did not neutralize SARS-CoV-2 with the exception of C20.119, which bound to a highly conserved epitope in the fusion peptide and showed broad binding and neutralization activity across SARS-CoV-2, SARS-CoV-1, and closely related zoonotic sarbecoviruses. Several of the S2 mAbs tested mediated antibody-dependent cellular cytotoxicity (ADCC) at levels similar to the S1 mAb S309 that was previously authorized for treatment of SARS-CoV-2 infections. Three of the mAbs with ADCC function also bound to spike trimers from HCoVs, such as MERS-CoV and HCoV-HKU1. Our findings suggest there are diverse epitopes in S2, including functional S2 mAbs with HCoV and sarbecovirus breadth that likely target functionally constrained regions of spike. These mAbs could be developed for potential future pandemics, while also providing insight into ideal epitopes for eliciting a broad HCoV response.

Jointly modeling deep mutational scans identifies shifted mutational effects among SARS-CoV-2 spike homologs (preprint)

Deep mutational scanning (DMS) is a high-throughput experimental technique that measures the effects of thousands of mutations to a protein. These experiments can be performed on multiple homologs of a protein or on the same protein selected under multiple conditions. It is often of biological interest to identify mutations with shifted effects across homologs or conditions. However, it is challenging to determine if observed shifts arise from biological signal or experimental noise. Here, we describe a method for jointly inferring mutational effects across multiple DMS experiments while also identifying mutations that have shifted in their effects among experiments. A key aspect of our method is to regularize the inferred shifts, so that they are nonzero only when strongly supported by the data. We apply this method to DMS experiments that measure how mutations to spike proteins from SARS-CoV-2 variants (Delta, Omicron BA.1, and Omicron BA.2) affect cell entry. Most mutational effects are conserved between these spike homologs, but a fraction have markedly shifted. We experimentally validate a subset of the mutations inferred to have shifted effects, and confirm differences of >1,000-fold in the impact of the same mutation on spike-mediated viral infection across spikes from different SARS-CoV-2 variants. Overall, our work establishes a general approach for comparing sets of DMS experiments to identify biologically important shifts in mutational effects.

Elevated binding and functional antibody responses to SARS-CoV-2 in infants versus mothers (preprint)

Infant antibody responses to viral infection can differ from those in adults. However, data on the specificity and function of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in infants, and direct comparisons between infants and adults are limited. We characterized antibody binding and functionality in convalescent plasma from postpartum women and their infants infected with SARS-CoV-2 from a vaccine-naive prospective cohort in Nairobi, Kenya. Antibody titers against SARS-CoV-2 Spike, receptor binding domain and N-terminal domain, and Spike-expressing cell-surface staining levels were significantly higher in infants than in mothers. Plasma antibodies from mothers and infants bound to similar regions of the Spike S2 subunit, including the fusion peptide (FP) and stem helix-heptad repeat 2. However, infants displayed higher antibody levels and more consistent antibody escape pathways in the FP region compared to mothers. Finally, infants had significantly higher levels of antibody-dependent cellular cytotoxicity (ADCC), though, surprisingly, neutralization titers between infants and mothers were similar. These results suggest infants develop distinct SARS-CoV-2 binding and functional antibody repertoires and reveal age-related differences in humoral immunity to SARS-CoV-2 infection that could be relevant to protection and COVID-19 disease outcomes.

Distinct antibody responses to endemic coronaviruses pre- and post-SARS-CoV-2 infection in Kenyan infants and mothers (preprint)

Pre-existing antibodies that bind endemic human coronaviruses (eHCoVs) can cross-react with SARS-CoV-2, the betacoronavirus that causes COVID-19, but whether these responses influence SARS-CoV-2 infection is still under investigation and is particularly understudied in infants. In this study, we measured eHCoV and SARS-CoV-1 IgG antibody titers before and after SARS-CoV-2 seroconversion in a cohort of Kenyan women and their infants. Pre-existing eHCoV antibody binding titers were not consistently associated with SARS-CoV-2 seroconversion in infants or mothers, though we observed a very modest association between pre-existing HCoV-229E antibody levels and lack of SARS-CoV-2 seroconversion in infants. After seroconversion to SARS-CoV-2, antibody binding titers to endemic betacoronaviruses HCoV-OC43 and HCoV-HKU1, and the highly pathogenic betacoronavirus SARS-CoV-1, but not endemic alphacoronaviruses HCoV-229E and HCoV-NL63, increased in mothers. However, eHCoV antibody levels did not increase following SARS-CoV-2 seroconversion in infants, suggesting the increase seen in mothers was not simply due to cross-reactivity to naively generated SARS-CoV-2 antibodies. In contrast, the levels of antibodies that could bind SARS-CoV-1 increased after SARS-CoV-2 seroconversion in both mothers and infants, both of whom are unlikely to have had a prior SARS-CoV-1 infection, supporting prior findings that SARS-CoV-2 responses cross-react with SARS-CoV-1. In summary, we find evidence for increased eHCoV antibody levels following SARS-CoV-2 seroconversion in mothers but not infants, suggesting eHCoV responses can be boosted by SARS-CoV-2 infection when a prior memory response has been established, and that pre-existing cross-reactive antibodies are not strongly associated with SARS-CoV-2 infection risk in mothers or infants.

Macaque-human differences in SARS-CoV-2 Spike antibody response elicited by vaccination or infection (preprint)

Macaques are a commonly used model for studying immunity to human viruses, including for studies of SARS-CoV-2 infection and vaccination. However, it is unknown whether macaque antibody responses recapitulate, and thus appropriately model, the response in humans. To answer this question, we employed a phage-based deep mutational scanning approach (Phage-DMS) to compare which linear epitopes are targeted on the SARS-CoV-2 Spike protein in humans and macaques following either vaccination or infection. We also used Phage-DMS to determine antibody escape pathways within each epitope, enabling a granular comparison of antibody binding specificities at the locus level. Overall, we identified some common epitope targets in both macaques and humans, including in the fusion peptide (FP) and stem helix-heptad repeat 2 (SH-H) regions. Differences between groups included a response to epitopes in the N-terminal domain (NTD) and C-terminal domain (CTD) in vaccinated humans but not vaccinated macaques, as well as recognition of a CTD epitope and epitopes flanking the FP in convalescent macaques but not convalescent humans. There was also considerable variability in the escape pathways among individuals within each group. Sera from convalescent macaques showed the least variability in escape overall and converged on a common response with vaccinated humans in the SH-H epitope region, suggesting highly similar antibodies were elicited. Collectively, these findings suggest that the antibody response to SARS-CoV-2 in macaques shares many features with humans, but with substantial differences in the recognition of certain epitopes and considerable individual variability in antibody escape profiles, suggesting a diverse repertoire of antibodies that can respond to major epitopes in both humans and macaques.

SARS-CoV-2 antibody binding and neutralization in dried blood spot eluates and paired plasma (preprint)

Widescale assessment of SARS-CoV-2-specific antibodies is critical to understanding population seroprevalence, correlates of protection, and the longevity of vaccine-elicited responses. Most SARS-CoV-2 studies characterize antibody responses in plasma/sera. While reliable and broadly used, these samples pose several logistical restrictions such as requiring venipuncture for collection and cold chain for transportation and storage. Dried blood spots (DBS) overcome these barriers as they can be self-collected by fingerstick and mailed and stored at ambient temperature. Here, we evaluate the suitability of DBS for SARS-CoV-2 antibody assays by comparing several antibody responses between paired plasma and DBS from SARS-CoV-2 convalescent and vaccinated individuals. We found that DBS not only reflected plasma antibody binding by ELISA and epitope profiles using phage-display, but also yielded SARS-CoV-2 neutralization titers that highly correlated with paired plasma. Neutralization measurement was further streamlined by adapting assays to a high-throughput 384-well format. This study supports the adoption of DBS for numerous SARS-CoV-2 binding and neutralization assays.

High resolution profiling of pathways of escape for SARS-CoV-2 spike-binding antibodies (preprint)

Defining long-term protective immunity to SARS-CoV-2 is one of the most pressing questions of our time and will require a detailed understanding of potential ways this virus can evolve to escape immune protection. Immune protection will most likely be mediated by antibodies that bind to the viral entry protein, Spike (S). Here we used Phage-DMS, an approach that comprehensively interrogates the effect of all possible mutations on binding to a protein of interest, to define the profile of antibody escape to the SARS-CoV-2 S protein using COVID-19 convalescent plasma. Antibody binding was common in two regions: the fusion peptide and linker region upstream of the heptad repeat region 2. However, escape mutations were variable within these immunodominant regions. There was also individual variation in less commonly targeted epitopes. This study provides a granular view of potential antibody escape pathways and suggests there will be individual variation in antibody-mediated virus evolution.

Epitope profiling reveals binding signatures of SARS-CoV-2 immune response and cross-reactivity with endemic HCoVs (preprint)

A major goal of current SARS-CoV-2 vaccine efforts is to elicit antibody responses that confer protection. Mapping the epitope targets of the SARS-CoV-2 antibody response is critical for innovative vaccine design, diagnostics, and development of therapeutics. Here, we developed a phage display library to map antibody binding sites at high resolution within the complete viral proteomes of all human-infecting coronaviruses in patients with mild or moderate/severe COVID-19. The dominant immune responses to SARS-CoV-2 were targeted to regions spanning the Spike protein, Nucleocapsid, and ORF1ab. Some epitopes were identified in the majority of samples while others were rare, and we found variation in the number of epitopes targeted by different individuals. We also identified a set of cross-reactive sequences that were bound by antibodies in SARS-CoV-2 unexposed individuals. Finally, we uncovered a subset of enriched epitopes from commonly circulating human coronaviruses with significant homology to highly reactive SARS-CoV-2 sequences.