Extended Data Fig. 7: Identification by deep mutational scanning of mutations affecting antibody binding and method of selection of antibody resistant mutants with VSV-SARS-CoV-2 virus.
From: Genetic and structural basis for SARS-CoV-2 variant neutralization by a two-antibody cocktail
a. Top: Plots showing gating strategy for selection of single yeast cells using forward- and side-scatter (first three panels) or RBD expression (right panel). Each plot is derived from the preceding gate. Bottom: Plots showing gating for RBD + , antibody- yeast cells. Selection experiments are shown for AZD8895 or AZD1061 - two independent libraries each. b. Correlation of sites of escape between yeast library selection experiments. The x-axes show cumulative escape fraction for each site for library 1, and the y-axes show cumulative escape fraction for each site for library 2. Correlation coefficient and n are denoted for each graph. c. Correlation of observed mutations that escape antibody binding between yeast library selection experiments. The x-axes show each amino acid mutation’s escape fraction for library 1, and the y-axes show each amino acid mutation’s escape fraction for library 2. Correlation coefficient and n are denoted for each graph. d-f. DMS results for AZD8895 (d), AZD1061 (e), or AZD7442 (f). Left panels: sites of escape across the entire RBD indicated by peaks that correspond to the logo plots in the middle/right panels. Middle panel: logo plot of cumulative escape mutation fractions of all RBD sites with strong escape mutations. Mutations are colored based on degree to which they abrogate RBD binding to hACE2. Right: logo plots show cumulative escape fractions, but colored based on degree to which mutations affect RBD expression. g. RTCA sensograms showing neutralization escape. Cytopathic effect was monitored kinetically in cells inoculated with virus in the presence of 5 μg/mL AZD1061. Representative escape (magenta) or lack of escape (blue) are shown. Green - uninfected cells; red -cells inoculated with virus without antibody. Magenta/blue curves - a single representative well; red/green controls - mean of technical duplicates. h. Representative RTCA sensograms validating that a virus selected by AZD1061 in (g) escaped AZD1061 (magenta) but not AZD8895 (light blue). i. Example sensograms from wells of 96-well E-plate analysis for escape selection. Instances of escape from AZD1061 are noted, while escape was not detected in the presence of AZD8895 or AZD7442. Positive and negative controls are denoted on the first plate.
