Antibody-induced cell-cell fusion requires spike proteolysis at the S1/S2 junction.

(A) Immunoblots showing shedded S1 subunits and human IgG heavy chain (IgG Hc), full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions of antibody-treated HEK293T cells expressing WT full-length spike for 16 hours. CB6, FD20, REGN10933 and REGN10987 were diluted in PBS and used at 12.5 nM, blots are representative of at least three independent experiments; (B) Immunoblots showing Proteinase K-resistant S2’ cleavage product, obtained from cell lysates described in (A) and were then treated in the absence or presence of 10 μg/mL proteinase K for 30 min at 37°C. blots are representative of two individual repeats; (C) Luciferase activity (RLU) measured from cell-cell fusion assay and immunoblots showing shedded S1 subunits, IgG Hc, full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions of CB6-stimulated HEK293T cells. CB6 doses used were 0.1, 0.5, 2.5 and 12.5 nM respectively, human IgG isotype was used at 12.5 nM for the negative control. Data and blots are representative of four individual repeats; (D) Immunoblots showing shedded S1 subunits, full-length spike and S1 collected from supernatant and cell lysate fractions of lentivirus-transduced A549 cells expressing WT full-length spike, stimulated without or with 12.5 nM CB6 antibody. Blots are representative of three individual repeats; (E) Luciferase activity (RLU) measured from cell-cell fusion assay and immunoblots showing shedded S1 subunits, IgG Hc, full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions of CB6-stimulated HEK293T cells expressing Beta spike VOCs carrying N417 or K417 revert mutation, blots and data are representative of four individual repeats; (F) Schematics of the R685A (furin-cleavage site deficient) spike mutant, and representative fluorescent images captured at 594 nm and 405 nm from HEK293T cells expressing WT and R685A spike mutant, stimulated without or with 12.5 nM CB6; scale bars are representative of 50 μm, images were representative of two individual repeats; (G) Luciferase activity (RLU) measured from antibody-induced cell-cell fusion assay, where co-cultured HEK293T cells expressing WT or R685A spike mutant were stimulated without or with 12.5 nM CB6 for 16 hours (Top). Supernatants and cell lysates were used for immunoblots of shedded S1 subunits, IgG Hc, full-length spike, S1, S2 and cleaved S2’ (Bottom). Blots are representative of three independent experiments.