Tyler N. Starr

The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor and is a major determinant of host range and a dominant target of neutralizing antibodies. Here, we experimentally measure how all amino acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD's surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity-enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open analysis pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations observed during viral surveillance.

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and are a major contributor to neutralizing antibody responses 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 surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies. They further enable the design of escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same RBD surface 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.

Antibodies are a potential therapy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the risk of the virus evolving to escape them remains unclear. Here we map how all mutations to the receptor binding domain (RBD) of SARS-CoV-2 affect binding by the antibodies in the REGN-COV2 cocktail and the antibody LY-CoV016. These complete maps uncover a single amino acid mutation that fully escapes the REGN-COV2 cocktail, which consists of two antibodies, REGN10933 and REGN10987, targeting distinct structural epitopes. The maps also identify viral mutations that are selected in a persistently infected patient treated with REGN-COV2 and during in vitro viral escape selections. Finally, the maps reveal that mutations escaping the individual antibodies are already present in circulating SARS-CoV-2 strains. These complete escape maps enable interpretation of the consequences of mutations observed during viral surveillance.

The structure, function, and evolution of proteins depend on physical and genetic interactions among amino acids. Recent studies have used new strategies to explore the prevalence, biochemical mechanisms, and evolutionary implications of these interactions-called epistasis-within proteins. Here we describe an emerging picture of pervasive epistasis in which the physical and biological effects of mutations change over the course of evolution in a lineage-specific fashion. Epistasis can restrict the trajectories available to an evolving protein or open new paths to sequences and functions that would otherwise have been inaccessible. We describe two broad classes of epistatic interactions, which arise from different physical mechanisms and have different effects on evolutionary processes. Specific epistasis-in which one mutation influences the phenotypic effect of few other mutations-is caused by direct and indirect physical interactions between mutations, which nonadditively change the protein's physical properties, such as conformation, stability, or affinity for ligands. In contrast, nonspecific epistasis describes mutations that modify the effect of many others; these typically behave additively with respect to the physical properties of a protein but exhibit epistasis because of a nonlinear relationship between the physical properties and their biological effects, such as function or fitness. Both types of interaction are rampant, but specific epistasis has stronger effects on the rate and outcomes of evolution, because it imposes stricter constraints and modulates evolutionary potential more dramatically; it therefore makes evolution more contingent on low-probability historical events and leaves stronger marks on the sequences, structures, and functions of protein families.