Investigating SARS-CoV-2 Spike Glycoprotein Variants

ExpiCHO-S cells were seeded at 6 × 10⁶ cells/mL in a volume of 5 mL in a 50 mL bioreactor. The following day, cells were transfected with SARS-CoV-2 spike glycoprotein-encoding pcDNA3.1(+) plasmids (BetaCoV/Wuhan-Hu-1/2019, accession number MN908947, Wuhan D614; Omicron BA.1 and BA.2 generated by overlap PCR mutagenesis of the Wuhan D614 plasmid) harboring the Δ19 C-terminal truncation^{27 (link)}. Spike encoding plasmids were diluted in cold OptiPRO SFM (Life Technologies, 12309-050), mixed with ExpiFectamine CHO Reagent (Life Technologies, A29130) and added to cells. Transfected cells were then incubated at 37˚C with 8% CO₂ with an orbital shaking speed of 250 RPM (orbital diameter of 25 mm) for 24 to 48 h. Transiently transfected ExpiCHO-S cells were harvested and washed twice in wash buffer (PBS 2% FBS, 2 mM EDTA). Cells were counted and distributed into round bottom 96-well plates (Corning, 3799) and incubated with serial dilutions of mAb starting at 10 μg/mL. Alexa Fluor647-labelled Goat Anti-human IgG secondary Ab (Jackson ImmunoResearch, 109–606–098) was prepared at 2 μg/mL and added onto cells after two washing steps. Cells were then washed twice and resuspended in wash buffer for data acquisition at ZE5 cytometer (BioRad).