Fig. 6: A combination of MAbs 8C4 and 9B5 prevents CVA16 virus escape mutations.

a Overlay of the cryo-EM maps of CVA16–9B5 C1 and CVA16–8C4 C1. The color bars indicate the corresponding radius from the center of the particle (unit in Å). The densities for 8C4 and 9B5 Fabs were colored in violet red and blue, respectively. b Neutralization of single MAbs and the antibody cocktail against CVA16/SZ05 was measured by the cell viability assay. 8C4 + 9B5, MAbs 8C4 and 9B5 were combined at a ratio of 1:1. For MAb cocktail, the concentration on the x axis is that of the 9B5 antibody and only IC50 value for 9B5 is shown. IgG-ctr, SARS-CoV-2 MAb 2H2. Data are mean ± SEM of five replicate wells. c–e Screening of neutralization escape mutants. 100 TCID₅₀ of wild-type CVA16/SZ05 was incubated with various concentrations of MAb 8C4 (c), 9B5 (d), or 8C4 + 9B5 cocktail (e) for 1 h before infection of RD cells. After 4 days, the culture supernatants and cell lysates (passage 1 [P1] virus) were incubated with the indicated concentrations of the MAbs prior to infection of fresh RD cells. After 3 days, the resultant P2 virus was subjected to another round of selection with higher concentrations of the MAbs, yielding P3 virus. During these passages, cells were observed for CPE. Green circle indicates no CPE, orange circle indicates partial CPE, and red complete CPE. Antibody concentrations used are shown in the circles. For the cocktail, only the concentrations of MAb 9B5 are shown. The mutants used for sequencing were blue boxed. f Information of neutralization escape mutants selected by MAb 8C4 and 9B5. g Location of escape mutations on the CVA16 protomer (PDB: 5C4W) surface. The residues replaced in 8C4-selected and 9B5-selected mutants are colored in red and blue, respectively.