Epistasis lowers the genetic barrier to SARS-CoV-2 neutralizing antibody escape

Figures

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  Fig. 1. Selection of SARS-CoV-2 spike variants with antibodies. a Receptor-Binding Domain (RBD) structure (PDB ID: 7C8J [https://www.rcsb.org/structure/7c8j]) illustrating salient features: yellow = ACE2-binding site; blue = residues changed in BA.1 relative to Wuhan-Hu-1; red = residues changed in BA.2. Highlighted in green are all positions where substitutions were selected by neutralizing antibodies during replication of rVSV/SARS-CoV-2 encoding Wuhan-Hu-1, BA.1, or BA.2 spike proteins. b Substitution frequency at residues along the length of the RBD following passage of rVSV/SARS-CoV-2 encoding Wuhan-Hu-1, BA.1, and BA.2 spike proteins with antibodies.

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  Fig. 2. Effects of substitutions in Wuhan-Hu-1, BA.1, and BA.2 backgrounds on neutralizing antibody sensitivity. a Inhibition of Wuhan-Hu-1, BA.1, and BA.2 receptor-binding domain (RBD) point mutant pseudotypes by broadly neutralizing antibodies. Relative infection is defined as the decimal fraction of infection measured (with 1 μg/ml antibody), relative to an uninhibited virus control (without antibody). Median value of two experiments.

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  Fig. 3. Resistance to broadly neutralizing class 4 and 1/4 antibodies. Receptor-binding domain (RBD) structure (PDB ID: 7C8J [https://www.rcsb.org/structure/7c8j]) illustrating epitopes of two prototype class 4 antibodies (C022 and C118, yellow), substitutions that confer resistance to class 4 and 1/4 antibodies (green), and pre-existing substitutions in BA.2 (red).

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  Fig. 4. Epistatic effects of RBD substitutions on neutralizing antibody sensitivity. a Neutralization of the D420N, N460K, and F486S RBD point mutant pseudotypes in Wuhan-Hu-1, BA.1, and BA.2 backgrounds by antibody C3095. b Neutralization of the K444N, L452R, and F490S RBD point mutant pseudotypes indicated in Wuhan-Hu-1, BA.1, and BA.2 backgrounds by antibody B1034. The median value is plotted and the error bars indicate the range of 1–3 independent titrations as detailed in the accompanying source data files.

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  Fig. 5. Pre-existing and escape substitutions in broadly neutralizing class 1 and class 3 antibody epitopes. a Substitutions that confer context-dependent escape from the class I antibody C099, depicted on the C099:RBD complex structure (PDB ID 7R8L[https://pdbj.org/mine/summary/7r8l]). Green indicates C099 escape substitutions. Blue and red indicate BA.1 and BA.2 substitutions, respectively. C099 heavy and light chains are magenta and yellow respectively. b Substitutions that confer context-dependent escape from C080 class 3 antibody, depicted on the Receptor-Binding Domain (RBD) complex structure (PDB ID 7C8J [https://www.rcsb.org/structure/7c8j]). Yellow indicates the C032 (clonal ancestor of C080) epitope. Green indicates escape substitutions. Blue and red indicate BA.1 and BA.2 substitutions, respectively.

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  Fig. 6. Context-dependent effect of BA.5, BA.2.75, and BA.2.75.2 substitutions on broadly neutralizing antibody sensitivity. a Naturally occurring Receptor-Binding Domain (RBD) substitutions in a selection of BA.1, BA.2, and BA.5 derived SARS-CoV-2 variants. Positions marked with asterisks indicate those at which substitutions arose during broadly neutralizing antibody selection experiments. b Inhibition of Wuhan-Hu-1 and BA.2 RBD pseudotypes with L452R or F486S substitutions by broadly neutralizing antibodies, in comparison with BA.5. c Inhibition of Wuhan-Hu-1 and BA.2 RBD pseudotypes with R346W, G446N, or N460K substitutions by broadly neutralizing antibodies, in comparison with BA.2.75 and BA.2.75.2. b, c Relative infection is defined as the decimal fraction of infection measured (with 1 μg/ml antibody), relative to an uninhibited virus control (without antibody) and median values from two independent experiments are displayed.

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  Fig. 7. Resistance of synthetic SARS-CoV-2 variants to broadly neutralizing antibodies. a BA.5 and synthetic SARS-CoV-2 receptor-binding domain (RBD) variants. Red: substitutions in BA.2 RBD relative to Wuhan-Hu-1; magenta: substitutions BA.5 RBD relative to BA2. Highlighted in green are substitutions in the synthetic variants, chosen based on antibody selection experiments. b Inhibition of synthetic variants by broadly neutralizing antibodies. Relative infection was calculated as a decimal fraction of infection measured (with 1 µg/ml antibody) relative to an uninhibited virus control (without antibody). A median of two independent experiments is shown. c Fold inhibition of natural and synthetic variant infection by individual broadly neutralizing antibodies. Individual data points represent the median of two independent experiments for n = 40 individual antibodies. Red line = group median fold inhibition for all antibodies tested.

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  Fig. 8. Reduced potency of polyclonal plasma antibodies against synthetic SARS-CoV-2 variants. The 50% neutralization titers (NT50) for plasmas from various volunteer cohorts (see methods). Fifteen randomly selected plasma samples from each cohort were tested against natural variants; Wuhan-Hu-1, BA.1, BA.2, BA.5 (black), and synthetic variants; BA.5(+3), BA.5(+7b) BA.5(+8) (red). Individual points represent individual plasma samples (n = 15 for each cohort); median of two independent experiments is plotted. Group median NT50 is indicated by red/black lines and numbers above each group.