Generation of escape mutants against neutralizing antibodies

Escape mutants were generated by serial passage of the virus in increasing amounts of CR6261, 1G01, or BiAb by following a previously described protocol⁷⁰ with several modifications. First, 2.3 × 10² PFU/mL of PR8 was incubated with 0.001 μg/μL of CR6261 or 0.002 μg/μL of 1G01 or 0.002 μg/μL of BiAb in serum-free DMEM with 1 μg/mL TPCK-treated trypsin (Sigma) and for 30 min at 37 °C. The viruses were then separately incubated with MDCK cells seeded on a six-well plate after washing with 1× PBS. The plate was kept for 1-h incubation at 37°C with repeated shaking of the plate every 10–15 min. After the incubation, the wells were washed and supplemented with complete DMEM and kept for 48-h incubation at 37°C, 5% CO₂. Then, 48 h following the infection, the infected cells were checked for cytopathic effect (CPE) and the supernatants were collected for further passages in MDCK cells. In the next passage, if the infected cells showed gross 70%–90% CPEs, the antibody concentration was increased 2-fold. However, if CPE was moderate to mild, the antibody concentration was maintained as the previous passage. Moreover, the rate of infections was also evaluated by flow cytometry every 48 h by evaluating the percentage of HA-positive cells. After 10 rounds of passages and similar antibody concentrations between the different groups, supernatants were collected, and viruses were sequenced by SGS for escape mutants.