Adam S Dingens
Profile Url: adam-s-dingens
Researcher at Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
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. ### Competing Interest Statement H.Y.C. is a consultant for Merck and Glaxo Smith Kline and receives research funding from Sanofi Pasteur. The other authors declare no conflicts of interest.
Mapping the epitope specificities of polyclonal serum is critical to rational vaccine design. However, most high-resolution mapping approaches involve isolating and characterizing individual monoclonal antibodies, which incompletely defines the full polyclonal response. Here we use two complementary approaches to directly map the specificities of the neutralizing and binding antibodies of polyclonal anti-HIV-1 sera from rabbits immunized with BG505 Env SOSIP trimers. To map the neutralizing specificity, we used mutational antigenic profiling to determine how all amino-acid mutations in Env affected viral neutralization. To map the binding specificity, we used electron microscopy polyclonal epitope mapping (EMPEM) to directly visualize the Fabs in serum bound to Env trimers. Mutational antigenic profiling showed that the dominant neutralizing specificities were the C3/V5 and/or 241/289 glycan hole epitopes, which were generally only a subset of the more diverse binding specificities mapped with EMPEM. Additional differences between binding and neutralization reflected antigenicity differences between virus and soluble Env trimer. Further, mutational antigenic profiling was able to refine epitope specificity in residue-level detail directly from sera, revealing subtle differences across rabbits. Together, mutational antigenic profiling and EMPEM allow for a holistic view of the binding and neutralizing specificity of polyclonal sera and could be used to finely evaluate and guide vaccine design. ### Competing Interest Statement The authors have declared no competing interest.
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.
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-2's receptor-binding domain (RBD) affect binding by the antibodies in Regeneron's REGN-COV2 cocktail and Eli Lilly's 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.
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.
An ideal anti-SARS-CoV-2 antibody would resist viral escape, have activity against diverse SARS-related coronaviruses, and be highly protective through viral neutralization and effector functions. 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 S309, 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.