Fig. 4

Recombinant SARS-CoV-2 S:E484 mutants show distinct replicative fitness in competition assays in the presence of neutralizing antibodies. Competition assays were performed in A and E in the absence of SARS-CoV-2 antiserum and in B and F in the presence of SARS-CoV-2 antiserum. In C and D, competition assays were performed as described above in the presence of hamster serum raised against WT (1:3000) or rSARS-CoV-2 E484K (1:2000). In B, virus inocula (total 10,000 PFU in three defined ratios) were pre-incubated in a 1:200 dilution of a 3x vaccinated serum for 1 h prior to infection in Calu-3 cells, or left untreated (no serum control) in A. Prior to infection (p0), every 24 hpi, 200 µl of virus containing supernatant were collected and incubated with a 1:200 final serum concentration, before being transferred to naïve Calu-3 cells for a total of three consecutive passages. Viral RNA was isolated from the initial inoculum (p0) and from culture supernatants at p1 and p3 (exceptionally p5) and subjected to RT-PCR yielding an 800-bp PCR fragment for Sanger-sequencing. Peak heights in sequencing chromatograms were analyzed using the ChromatQuantitator online tool. WT = wildtype.