Structural basis for continued antibody evasion by the SARS-CoV-2 receptor binding domain

Many studies have examined the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on neutralizing antibody activity after they have become dominant strains. Here, we evaluate the consequences of further viral evolution. We demonstrate mechanisms through which the SARS-CoV-2 receptor binding domain (RBD) can tolerate large numbers of simultaneous antibody escape mutations and show that pseudotypes containing up to seven mutations, as opposed to the one to three found in previously studied variants of concern, are more resistant to neutralization by therapeutic antibodies and serum from vaccine recipients. We identify an antibody that binds the RBD core to neutralize pseudotypes for all tested variants but show that the RBD can acquire an N-linked glycan to escape neutralization. Our findings portend continued emergence of escape variants as SARS-CoV-2 adapts to humans. Description Evasive maneuvers by the spike protein Throughout the course of the COVID-19 pandemic, variants have arisen in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus that increase infectivity or reduce its susceptibility to existing antibodies. Nabel et al. focus on mutations in the spike protein, which is found on the viral surface and is responsible for binding and entering host cells, and show that the structure exhibits plasticity in resisting neutralizing antibodies. Working with pseudoviruses, the authors identify combinatorial mutations that might lead to resistance. They identify an antibody that neutralizes a pseudotype with a highly mutated spike, but also show that SARS-CoV-2 can acquire a glycan to escape this neutralization. Understanding the consequences of changes in the antigenic landscape of the spike protein is important if we are to rapidly respond to new variants of concern. —VV As it evades antibodies, the SARS-CoV-2 spike protein receptor-binding domain can acquire multiple mutations and a glycan. INTRODUCTION As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates under selective pressure from natural and vaccine-induced immunity, variants of concern (VOCs) continue to emerge. Through adaptative evolution, these variants acquire mutations in the spike protein receptor binding domain (RBD) that binds the cellular receptor angiotensin-converting enzyme 2 (ACE2). The effects of spike protein mutations on immune responses make it important to monitor viral variants. While previously studied VOCs contain one to three RBD mutations that at times overlap, the potential for composite variants that contain larger numbers of mutations is being closely monitored. RATIONALE As parts of the world continue to face waves of infection and mitigation strategies are relaxed, viral replication in human hosts under antibody selective pressure continues to shape the antigenic landscape of the SARS-CoV-2 spike protein. As variants containing composite mutations begin to emerge, proactive approaches examining the impact of variants before they become dominant strains are critical. RESULTS We determined the x-ray crystal structure of human ACE2 in complex with a SARS-CoV-2 RBD that contains six substitutions that arose during persistent infection of an immunocompromised individual. We found that structural plasticity at the RBD–ACE2 interface allowed the RBD to tolerate a large number of mutations while retaining ACE2 affinity. We generated a panel of pseudotypes bearing composite RBD mutations (up to seven) from immunocompromised host-derived sequences and VOCs. Composite variants more adeptly evaded therapeutic antibody neutralization than did previously studied VOCs. After first immunization but before the second dose of an mRNA vaccine, we observed a loss in vaccine recipient serum neutralizing activity for all variants tested, although the severity differed depending on the variant. However, sampling after the second immunization revealed detectable neutralizing activity against all variants in the serum of vaccine recipients, including against a pseudotype that contains seven composite RBD mutations [denoted receptor binding mutant-2 (RBM-2)]. To identify evolutionary barriers that restrict neutralization breadth, we used the SARS-CoV spike protein to isolate a neutralizing antibody from a COVID-19 convalescent donor. Through structural analysis and functional assays, we show that N-linked glycan acquisition by the SARS-CoV-2 RBD confers pseudotype resistance to neutralization by the isolated cross-reactive antibody and at least one other antibody that binds a similar, otherwise highly conserved epitope. Therefore, acquisition of an N-linked glycan on the SARS-CoV-2 RBD is an additional means through which the virus could continue to evade immune responses. CONCLUSION We find that accumulation of large numbers of RBD mutations is facilitated by structural plasticity at the RBD–ACE2 interface and further erodes the activity of therapeutic antibodies and serum from vaccine recipients. Furthermore, acquisition of an N-linked glycan on the SARS-CoV-2 RBD is an additional neutralization escape pathway that should be closely monitored during viral antigenic drift. Immune escape at the SARS-CoV-2 spike protein RBD. Structural plasticity accommodates the accumulation of composite substitutions in the RBD ACE2 binding site and allows the RBD to adeptly escape therapeutic antibodies. Cross-neutralizing antibodies bind the RBD core, but acquisition of an N-linked glycan at RBD residue Asn370 (N370) drives further neutralization escape. Single-letter abbreviations for the amino acid residues are as follows: D, Asp; E, Glu; F, Phe; H, His; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; and Y, Tyr. LC, light chain; HC, heavy chain.