Figure 5.
A combination of AX290 and AX677 prevents rapid mutational escape of authentic live SARS-CoV-2 virus.
(a) A schematic diagram of microplate well allocations in the SARS-CoV-2 escape mutants experiment. MAbs were serially five-fold diluted starting with 50 µg/mL (left panels), pre-incubated with live SARS-CoV-2 (Slovakia/SK-BMC5/2020) virus (diluted to MOI=0·5) and added to Vero E6 cells (1st passage, middle panels). Culture medium from the first wells of the mAb dilution series where CPE appeared (indicated with the yellow circles) were used for the 2nd passage (right panels). White squares highlight wells with CPE where virus genomes were sequenced. C - control wells with virus without mAbs were sequenced to monitor possible tissue culture adaptations.
(b) The table shows amino acid changes resulting from nonsynonymous mutations in the virus genome that appeared under selection pressure of mAbs. Combination of the two antibodies prevented appearance of escape mutations in the Spike protein.
(c) The positions of the amino acids that were mutated in the viruses that escaped from the neutralization by the antibodies are highlighted in pink in the structure of SARS-CoV-2 Spike receptor-binding domain bound to ACE2 (reproduced from PDB 6M0J, see also Figure 2c, d). T345 is located close to the AX677 binding site (orange and yellow, the structure on the left), S477 is located in the binding site of AX290 (blue, structure on the right). (d) Changes in the proportion of the mutant viruses in the mixture with the parental virus before and after three days of co-cultivation with Vero E6 cells in the absence of the monoclonal antibodies determined by nanopore sequencing. The mutant viruses were arbitrarily named A1, B11 and C25 for the escape viruses from AX290, AX677 and AX290+AX677 mix, respectively. Mean and SD are shown from three separate passages of the viral mixtures.