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Comprehensive mapping of mutations to the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human serum antibodies

Video Abstract (AI generated) Paper Preprint (github,"") (jbloomlab,github)

The evolution of SARS-CoV-2 could impair recognition of the virus by human antibody-mediated immunity. To facilitate prospective surveillance for such evolution, we map how convalescent serum antibodies are impacted by all mutations to the spikes receptor-binding domain (RBD), the main target of serum neutralizing activity. Binding by polyclonal serum antibodies is affected by mutations in three main epitopes in the RBD, but there is substantial variation in the impact of mutations both among individuals and within the same individual over time. Despite this inter- and intra-person heterogeneity, the mutations that most reduce antibody binding usually occur at just a few sites in the RBDs receptor binding motif. The most important site is E484, where neutralization by some sera is reduced >10-fold by several mutations, including one in emerging viral lineages in South Africa and Brazil. Going forward, these serum escape maps can inform surveillance of SARS-CoV-2 evolution.

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

Viruses, 2020-05-06

Video Abstract (AI generated) Paper Preprint File S1 (plasmid maps in Genbank format) Viruses

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.

High-resolution mapping of the neutralizing and binding specificities of polyclonal rabbit serum elicited by HIV Env trimer immunization

Video Abstract (AI generated) Paper Preprint Figure 2 - Figure supplements 1 to 6 Figure 2 - source data 1

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 mRNA-1273 vaccine elicits more RBD-focused neutralization, but with broader antibody binding within the RBD

Video Abstract (AI generated) Paper Preprint (jbloomlab,github) (github,"")

The emergence of SARS-CoV-2 variants with mutations in key antibody epitopes has raised concerns that antigenic evolution will erode immunity. The susceptibility of immunity to viral evolution is shaped in part by the breadth of epitopes targeted. Here we compare the specificity of antibodies elicited by the mRNA-1273 vaccine versus natural infection. The neutralizing activity of vaccine-elicited antibodies is even more focused on the spike receptor-binding domain (RBD) than for infection-elicited antibodies. However, within the RBD, binding of vaccine-elicited antibodies is more broadly distributed across epitopes than for infection-elicited antibodies. This greater binding breadth means single RBD mutations have less impact on neutralization by vaccine sera than convalescent sera. Therefore, antibody immunity acquired by different means may have differing susceptibility to erosion by viral evolution.

Identification of antibodies targeting the H3N2 hemagglutinin receptor binding site following vaccination of humans

Cell Reports, 2019-12-24

Video Abstract (AI generated) Paper Preprint Cell Reports

Antibodies targeting the receptor binding site (RBS) of the influenza virus hemagglutinin (HA) protein are usually not broadly-reactive because their footprints are typically large and extend to nearby variable HA residues. Here, we identified several human H3N2 HA RBS-targeting monoclonal antibodies (mAbs) that were sensitive to substitutions in conventional antigenic sites and were not broadly-reactive. However, we also identified one H3N2 HA RBS-targeting mAb that was exceptionally broadly reactive despite being sensitive to substitutions in residues outside of the RBS. We determined that similar antibodies are present at measurable levels in the sera of some individuals but that they are inefficiently elicited by conventional vaccines. Our data indicate that some HA RBS-targeting antibodies can be surprisingly effective against variable viral strains even if they are somewhat sensitive to substitutions in HA residues adjacent to the RBS.

Mapping person-to-person variation in viral mutations that escape polyclonal serum targeting influenza hemagglutinin

eLife, 2019-08-27

Video Abstract (AI generated) Paper Preprint eLife

A longstanding question is how influenza evolves to escape human immunity, which is polyclonal and can target many distinct epitopes on the virus. Here we map how all amino-acid mutations to influenza's major surface protein affect viral neutralization by polyclonal human sera. The serum of some individuals is so focused that it selects single mutations that reduce viral neutralization by over an order of magnitude. However, different viral mutations escape the sera of different individuals. This individual-to-individual variation in viral escape mutations is not present among ferrets, which are frequently used as a model in influenza studies. Our results show how different single mutations help influenza escape the immunity of different members of the human population, a phenomenon that could shape viral evolution and disease susceptibility.