Antibodies to the SARS-CoV-2 receptor-binding domain that maximize breadth and resistance to viral escape

An ideal anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse SARS-related coronaviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid development of antibody therapeutics and guide vaccine design. Here, we comprehensively characterize escape, breadth, and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD), including S3094, the parental antibody of the late-stage clinical antibody VIR-7831. We observe a tradeoff between SARS-CoV-2 in vitro neutralization potency and breadth of binding across SARS-related coronaviruses. Nevertheless, we identify several neutralizing antibodies with exceptional breadth and resistance to escape, including a new antibody (S2H97) that binds with high affinity to all SARS-related coronavirus clades via a unique RBD epitope centered on residue E516. S2H97 and other escape-resistant antibodies have high binding affinity and target functionally constrained RBD residues. We find that antibodies targeting the ACE2 receptor binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency, but we identify one potent RBM antibody (S2E12) with breadth across sarbecoviruses closely related to SARS-CoV-2 and with a high barrier to viral escape. These data highlight functional diversity among antibodies targeting the RBD and identify epitopes and features to prioritize for antibody and vaccine development against the current and potential future pandemics.

Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016

Monoclonal antibodies and antibody cocktails are a promising therapeutic and prophylaxis for COVID-19. However, ongoing evolution of SARS-CoV-2 can render monoclonal antibodies ineffective. Here we completely map all mutations to the SARS-CoV-2 spike receptor binding domain (RBD) that escape binding by a leading monoclonal antibody, LY-CoV555, and its cocktail combination with LY-CoV016. Individual mutations that escape binding by each antibody are combined in the circulating B.1.351 and P.1 SARS-CoV-2 lineages (E484K escapes LY-CoV555, K417N/T escape LY-CoV016). Additionally, the L452R mutation in the B.1.429 lineage escapes LY-CoV555. Furthermore, we identify single amino acid changes that escape the combined LY-CoV555+LY-CoV016 cocktail. We suggest that future efforts should diversify the epitopes targeted by antibodies and antibody cocktails to make them more resilient to antigenic evolution of SARS-CoV-2.

Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail

The SARS-CoV-2 pandemic has led to an urgent need to understand the molecular basis for immune recognition of SARS-CoV-2 spike (S) glycoprotein antigenic sites. To define the genetic and structural basis for SARS-CoV-2 neutralization, we determined the structures of two human monoclonal antibodies COV2-2196 and COV2-21301, which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor binding domain (RBD) of SARS-CoV-2. COV2-2196 forms an "aromatic cage" at the heavy/light chain interface using germline-encoded residues in complementarity determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals1-4. The structure of COV2-2130 reveals that an unusually long LCDR1 and HCDR3 make interactions with the opposite face of the RBD from that of COV2-2196. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the critical residues of both antibodies and identified positions of concern for possible viral escape. Nonetheless, both COV2-2196 and COV2-2130 showed strong neutralizing activity against SARS-CoV-2 strain with recent variations of concern including E484K, N501Y, and D614G substitutions. These studies reveal germline-encoded antibody features enabling recognition of the RBD and demonstrate the activity of a cocktail like AZD7442 in preventing escape from emerging variant viruses.

Prospective mapping of viral mutations that escape antibodies used to treat COVID-19

Antibodies are becoming a frontline therapy for SARS-CoV-2, but the risk of viral evolutionary escape remains unclear. Here we map how all mutations to SARS-CoV-2s receptor-binding domain (RBD) affect binding by the antibodies in Regenerons REGN-COV2 cocktail and Eli Lillys LY-CoV016. These complete maps uncover a single amino-acid mutation that fully escapes the REGN-COV2 cocktail, which consists of two antibodies targeting distinct structural epitopes. The maps also identify viral mutations that are selected in a persistently infected patient treated with REGN-COV2, as well as in lab viral escape selections. Finally, the maps reveal that mutations escaping each individual antibody are already present in circulating SARS-CoV-2 strains. Overall, these complete escape maps enable immediate interpretation of the consequences of mutations observed during viral surveillance.

Complete mapping of mutations to the SARS-CoV-2 spike receptor-binding domain that escape antibody recognition

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and make a major contribution to the neutralizing antibody response elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding, and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same RBD surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies, and enable us to design escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same surface of the RBD but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.

Dynamics of neutralizing antibody titers in the months after SARS-CoV-2 infection

Most individuals infected with SARS-CoV-2 develop neutralizing antibodies that target the viral spike protein. Here we quantify how levels of these antibodies change in the months following SARS-CoV-2 infection by examining longitudinal samples collected between ~30 and 152 days post-symptom onset from a prospective cohort of 34 recovered individuals with asymptomatic, mild, or moderate-severe disease. Neutralizing antibody titers declined an average of about four-fold from one to four months post-symptom onset. This decline in neutralizing antibody titers was accompanied by a decline in total antibodies capable of binding the viral spike or its receptor-binding domain. Importantly, our data are consistent with the expected early immune response to viral infection, where an initial peak in antibody levels is followed by a decline to a lower plateau. Additional studies of long-lived B-cells and antibody titers over longer time frames are necessary to determine the durability of immunity to SARS-CoV-2.

Seroprevalence of SARS-CoV-2 among children visiting a hospital during the initial Seattle outbreak

Children are strikingly underrepresented in COVID-19 case counts1-3. In the United States, children represent 22% of the population but only 1.7% of confirmed SARS-CoV-2 cases1. One possibility is that symptom-based viral testing is less likely to identify infected children, since they often experience milder disease than adults1,4-7. To better assess the frequency of pediatric SARS-CoV-2 infection, we serologically screened 1,775 residual samples from Seattle Children's Hospital collected from 1,076 children seeking medical care during March and April of 2020. Only one child was seropositive in March, but seven were seropositive in April for a period seroprevalence of {approx} 1%. Most seropositive children (6/8) were not suspected of having had COVID-19. The sera of seropositive children had neutralizing activity, including one that neutralized at a dilution >1:18,000. Therefore, an increasing number of children seeking medical care were infected by SARS-CoV-2 during the early Seattle outbreak despite few positive viral tests.

Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral and VSV particles, but the reagents and protocols are not widely available. Here we detail how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.