Converting non-neutralizing SARS-CoV-2 antibodies into broad-spectrum inhibitors

All proteins were expressed in Expi293F cells. Expi293F cells were cultured in media containing 66% Freestyle/33% Expi media (Thermo Fisher Scientific) and grown in TriForest polycarbonate shaking flasks at 37 °C in 8% CO2. The day before transfection, cells were spun down and resuspended to a density of 3 × 106 cells per ml in fresh media. The next day, cells were diluted and transfected at a density of approximately 3–4 × 106 cells per ml. Transfection mixtures were made by adding the following components: mirA-prepped or maxi-prepped DNA, culture media and FectoPro (Polyplus) would be added to cells to a ratio of 0.5–0.8 µg:100 µl:1.3 µl:900 µl. For example, for a 100-ml transfection, 50–80 µg of DNA would be added to 10 ml of culture media, and then 130 µl of FectoPro would be added to this. After mixing and a 10-minute incubation, the resultant transfection cocktail would be added to 90 ml of cells. The cells were harvested 3–5 days after transfection by spinning the cultures at >7,000g for 15 minutes. Supernatants were filtered using a 0.22-µm filter. To determine hCoV protein expression, spun-down Expi293F supernatant was used without further purification. For proteins containing a biotinylation tag (Avi-Tag), Expi293F cells containing a stable BirA enzyme insertion were used, resulting in spontaneous biotinylation during protein expression.