Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition

Abstract

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.

Keywords: SARS-CoV-2; antibody escape; antigenic evolution; deep mutational scanning.

Graphical abstract

Figure 1

A Yeast-Display System to Completely Map SARS-CoV-2 RBD Antibody-Escape Mutations (A) Yeast display RBD on their surface. The RBD contains a c-Myc tag, enabling dual-fluorescent labeling to quantify RBD expression and antibody binding by flow cytometry. (B) RBD expression and antibody binding as measured by flow cytometry for yeast expressing unmutated RBD and an RBD mutant library. (C) Yeast expressing RBD mutant libraries are sorted to purge mutations that abolish ACE2 binding or RBD folding. These libraries are labeled with antibody, and cells expressing RBD mutants with decreased antibody binding are enriched by using FACS (the “antibody-escape” bin; see Figure S1 for gating). Deep-sequencing counts are used to compute the “escape fraction” for each mutation. Escape fractions are represented in logo plots, with tall letters indicating mutations that strongly escape antibody binding.

Figure 2

Complete Maps of Escape Mutations from 10 Human Monoclonal Antibodies (A) Properties of the antibodies as reported by Zost et al. (2020a). SARS-CoV-2 neutralization potency is represented as a gradient from black (most potent) to white (non-neutralizing). Antibodies that bind SARS-CoV-1 spike or compete with RBD binding to ACE2 or rCR3022 are indicated in black. (B) Structure of the SARS-CoV-2 RBD (PDB: 6M0J; Lan et al., 2020), with residues colored by whether they are in the core RBD distal from ACE2 (orange), in the receptor-binding motif (RBM, light blue), or in direct contact with ACE2 (dark blue). ACE2 is in gray. RBD sites where mutations escape antibodies are indicated with spheres. (C) Maps of escape mutations from each antibody. The line plots show the total escape at each RBD site (sum of escape fractions of all mutations at that site). Sites with strong escape mutations (indicated by purple at bottom of the line plots) are shown in the logo plots. Logo plots are colored by RBD region as in (B). Different sites are shown for the rCR3022-competing antibodies (top four) and all other antibodies (bottom six). For interactive escape maps, see https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_Crowe_antibodies. (D) Multidimensional scaling projection of the escape mutant maps, with antibodies having similar escape mutations drawn close together. Each antibody is shown with a pie chart that uses the color scale in (B) to indicate the RBD regions where it selects escape mutations. See also Figures S1 and S2 and Table S1.

Figure 3

Neutralization Assays Validate Antibody-Escape Maps (A) For four antibodies, we validated two mutations that our maps indicated should escape antibody binding and one or two that should not. Logo plots show the escape maps for all tested sites, with the tested mutations expected to escape antibody binding in red. Dot plots show the fold change in neutralization (inhibitory concentration 50%, IC₅₀) relative to wildtype measured using spike-pseudotyped lentiviral particles. Fold changes greater than one (dashed gray line) mean a mutation escapes antibody neutralization. Points in red and blue correspond to mutations expected to mediate or not mediate escape, respectively. Blue letters are not visible in the logo plots due to small escape fractions. Dotted pink lines indicate the upper limit to the dynamic range; points on the line indicate a fold change greater than or equal to this value. See Figure S3A for the raw neutralization curves and Figures S3B and S3C for similar validation for the non-neutralizing antibody rCR3022. Mutations were chosen that had among the largest effects for each of the four antibodies, escaped from multiple antibodies, are present in circulating strains (A475V) or other sarbecoviruses (F490K, E484V) or were surprising in their lack of escape. (B) RBD structure colored as in Figure 2B, with labeled spheres indicating sites where mutation effects on neutralization were validated.

Figure 4

Structural Mapping of Antibody Binding and Escape (A–D) For each antibody, the structure shows the RBD surface colored by the largest-effect escape mutation at each site, with white indicating no escape and red indicating the strongest escape mutation for that antibody. ACE2 contact residues are outlined in black. Antibodies are arranged by structural epitope: (A) core RBD, (B) ACE2-binding ridge, (C) the opposite edge of the RBM, or (D) the saddle of the RBM surface. (E) Crystal structure of the rCR3022-bound RBD (PDB: 6W41; Yuan et al., 2020), with Fab in purple, ACE2 contact sites outlined, and RBD colored according to sites of escape as in (A). (F) For 5 antibodies, Fab bound to SARS-CoV-2 spike ectodomain trimer was visualized by negative-stain EM. The modeled RBD is colored according to sites of escape as in (A). Fab chains are modeled in gold. Antibody names are colored according to Figure 2B: core-binding, orange; RBM-binding, cyan; ACE2 contact site-binding, dark blue. See https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_Crowe_antibodies/ for interactive versions of the escape-colored structures in (A)–(D). See also Table S2.

Figure 5

Functional and Evolutionary Constraint on Antibody-Escape Mutations (A) Variation at sites of antibody escape among currently circulating SARS-CoV-2 viruses. For each site of escape from at least one antibody, we counted sequences in GISAID with an amino-acid change. Sites with at least 5 GISAID variants (of 93,858 sequences at the time of analysis) are shown ordered by count; black cells indicate antibodies with escape mutations at that site. Sites are colored by RBD region. Antibodies are colored according to where the majority of their sites of escape fall. See also Figure S4. (B) Escape maps (as in Figure 2C), with letters colored according to how deleterious mutations are for ACE2 binding or RBD expression (Starr et al., 2020). Only sites of escape mutations for each antibody are depicted. See Figure S5 for similar logo plots for all antibodies. (C) Mutational constraint on sites of escape. For each antibody, the mean effects of all possible amino acid mutations at sites of escape on ACE2 binding and RBD expression are shown. (D) Top: effective number of amino acids (N_eff) in the sarbecovirus RBD alignment at sites of escape for each antibody. N_eff is a measure of the variability of a site (the exponentiated Shannon entropy), and ranges from 1 for a position that is completely conserved to 20 for a site where all amino acids are present at equal frequency. Bottom: escape fraction for each sarbecovirus RBD homolog from the yeast display selections; 1 means complete escape (no binding), and 0 means no escape (complete binding).

Figure 6

Viral Escape Mutant Selections with Individual Antibodies and Antibody Cocktails (A) Results of viral selections with five individual monoclonal antibodies. The number of replicates where escape variants were selected are indicated, color-coded according to whether escape was selected frequently (red) or rarely (white). Mutations present in the RBD of the selected escape variants are indicated. (B) Each point represents a different amino-acid mutation to the RBD, with the x axis indicating how strongly the mutation ablates antibody binding in our escape maps and the y axis indicating how the mutation affects ACE2 binding (negative values indicate impaired ACE2 binding). All selected mutations were accessible by single-nucleotide changes. The only accessible escape mutation from COV2-2165 that is not deleterious to ACE2 binding is D420Y, but this mutation is highly deleterious for RBD expression (Figure 5B; Figure S5). (C) Results of viral selections with antibody cocktails, indicating the number of replicates with escape out of the total tested. The data for the single antibodies are repeated from (A). In all panels, antibody names are colored according to where in the RBD the majority of their sites of escape fall: orange for the core RBD, light blue for the RBM, and dark blue for ACE2 contact residues. See also Figure S6.