Mutational escape from the polyclonal antibody response to SARS-CoV-2 infection is largely shaped by a single class of antibodies

Abstract

Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasma, including plasma from individuals from whom some of the antibodies were isolated. The plasma-escape maps most closely resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is dominated by a single class of antibodies targeting an epitope that is already undergoing rapid evolution.

Figure 1.. Maps of mutations to the RBD that escape binding by three classes of monoclonal antibodies that target the receptor-binding motif.

(A) Epitopes for each of the three antibody classes. ACE2 is shown as a gray cartoon. Some sites fall under both class 1 and class 2 and are shown as an intermediate pink-purple. (B) Escape maps for monoclonal antibodies from each of the three classes. The line plots at left indicate the summed effect of all mutations at each site in the RBD, with larger values indicating a greater reduction in antibody binding. The logo plots at right show the effects of individual mutations at key sites (indicated by purple highlighting on the x-axis of the line plots). In these logo plots, the height of each letter is that mutation’s escape fraction, so larger letters indicate mutations that cause a greater reduction in antibody binding. Sites in the logo plots are colored by RBD epitope. Sites that contact antibody (non-hydrogen atoms within 4Å in high-resolution structures) are highlighted with gray backgrounds. For C110, sites 444, 446, and 447 are unresolved in the structure but are likely in close contact with the antibody, and so are highlighted in gray. The data for LY-CoV016 were previously reported and are replotted here. (C) Multidimensional scaling projection of the escape maps, such that antibodies with similar escape mutations are drawn close together. Each antibody is represented as a pie chart colored according to the amount of escape in each RBD epitope. Antibodies for which escape maps are shown in panel (B) have black outlines and colored names. The other antibodies were profiled previously^,,,. Escape maps for all class 1, 2, and 3 antibodies in the plot are shown in Figure S3; for the class 4 antibody-escape maps, see Greaney et al. (2021). Colors used in all panels: sites are colored according to epitopes, as defined in Greaney et al. (2021). Bright pink for class 1, dark purple for class 2, medium pink-purple for class 1/2 overlap sites (455, 456, 486, 487, 489), cyan for class 3, and gray for all other RBD sites. Escape maps colored according to mutation effects on RBD expression and ACE2 binding are in Figure S4. The ACE2-bound RBD structure in (A) is from PDB 6M0J.

Figure 2.. Mutations that escape antibody binding are usually in the direct structural footprint.

(A) The total escape at each site is mapped onto the surface of the Fab-bound RBD, with white indicating no escape and red indicating the site with the most escape from that antibody. Sites where no mutations are tolerated for RBD folding or ACE2 binding are indicated in dark gray. For C105 and LY-CoV016, gray labels with dashed lines indicate example contact sites with no tolerated mutations. For C110, the general area where site 444 (unresolved in structure) would be located is indicated. (B) Total escape at each site in the RBD, with sites classified according to whether they are an antibody contact (within 4Å), antibody-proximal (4 to 8Å), antibody-distal (>8Å), or unresolved in the Fab-trimer structure. Text indicates the number of sites in each structural category that are sites of “strong escape,” (n / total) shown in orange. See Methods for details and PDB accessions.

Figure 3.. The mutations that reduce binding of polyclonal plasma often differ from those that reduce binding by monoclonal antibodies isolated from the same individual.

(A) Table indicating which plasma and antibodies were derived from the same individual. (B) Escape maps for the polyclonal plasma antibodies, as in Figure 1B. The y-axis is scaled separately for each plasma (see Methods). When there are monoclonal antibodies isolated from the same individual, the total monoclonal antibody escape at each site is shown using the heat maps above the escape maps, with white indicating no effect and black indicating strong escape. (C) Correlation of plasma and monoclonal antibody escape for each plasma / antibody pair from the same individual. Each point in the scatter plots is a site, with the x-axis indicating the total escape at that site for the antibody and the y-axis indicating the total escape at that site for the plasma. Key sites are labeled. Pearson’s R shown above each plot. Colors in B, C reflect antibody classes as in Figure 1.

Figure 4.. The escape maps of convalescent polyclonal plasma most resemble class 2 antibodies.

(A) Multidimensional scaling projection of the escape maps of polyclonal plasma and monoclonal antibodies of each class. Antibodies or plasma that are nearby in the plot have their binding affected by similar RBD mutations. The antibodies are those in Figure 1C, colored according to antibody class. The five plasma newly mapped in this study are shown in green, and the previously mapped 23 plasma are shown in white. (B) Structural projection of sites where mutations reduce binding by each class of monoclonal antibodies (left) or polyclonal plasma (right). The RBD surface coloring is scaled from white to red, with white indicating no escape, and red indicating the site with the greatest average site-total escape for all antibodies or plasma in that group. Mutations to sites such as E484, F456, and F486 have some of the largest effects on binding by polyclonal plasma and class 2 antibodies.

Figure 5.. Escape maps predict mutations that are selected during viral growth in the presence of monoclonal antibodies.

(A) Mutations selected when chimeric VSV encoding the SARS-CoV-2 spike was grown in the presence of each of the three indicated antibodies by Weisblum et al. (2020). Each point represents a different amino-acid mutation, with the x-axis indicating how strongly the mutation escapes antibody binding (measured in the current study) and the y-axis indicating how well the mutant binds to ACE2 (measured in Starr et al. (2020)). The red diamonds indicate the mutations selected in VSV-spike by Weisblum et al., the gray circles indicate all other amino-acid mutations accessible by a single-nucleotide change, and the brown x’s indicate amino-acid mutations that require multiple nucleotide changes to the codon. (B) Logo plots showing the effects of only single-nucleotide accessible amino-acid mutations on antibody binding. Mutations selected in VSV-spike virus by Weisblum et al. are colored red. (C) The correlation of the effects of mutations on antibody binding measured in the current study and effects on viral neutralization previously measured by Weisblum et al. 7 using chimeric VSV (top) or lentiviral particles (bottom). The x-axis shows the escape fraction measured in the current study, and the y-axis shows the fold-change in IC50 for viral neutralization caused by that mutation, such that larger numbers correspond to greater reductions in neutralization sensitivity. For effects of all antibody- and plasma-binding-escape mutations on ACE2 binding and RBD expression, see Figure S4. For each mutation’s escape fraction compared to fold-change IC50 against each monoclonal antibody or polyclonal plasma tested in Weisblum et al., see Figure S6.

Figure 6.. Mutations that escape binding by antibodies and plasma among sequenced SARS-CoV-2 isolates.

(A) The total escape at each site averaged across antibodies in each class versus frequency of mutations at each site in GISAID sequences as of March 15, 2021. (B) The total escape at each site averaged across the polyclonal plasma versus frequency of mutations at each site in GISAID sequences. Left: plasma samples profiled in this study, right: plasma samples profiled previously. (C) Antibody-escape mutations found in emerging viral lineages. RBD mutations in each lineage are assigned to the class of antibody they most strongly escape (e.g., E484K most strongly escapes class 2 antibodies but may also affect some class 1 antibodies). Other RBD mutations present in each viral lineage with negligible effects on binding of these antibodies are listed at right. For numbers of antibodies in each class, see n indicated in each panel in A and B. In each plot, key sites are labeled and colored according to RBD epitope using the same color scheme as in Figure 1.