Dose-dependent neutralization of cell-cell fusion by CB6 in the R685A spike mutant.

(A) Fluorescent images of REGN10933-mediated neutralization of cell-cell fusion in HEK293T cells co-expressing ZsGreen reporter with WT or R685A spike mutant, preincubated without or with 100 nM REGN10933 for 1 hour, then co-cultured with HEK293T cells expressing ACE2 for 16 hours. Scale bars are indicative of 50 μm and images are representative of two independent experiments; (B) Luciferase activity (RLU) measured from HEK293T cells co-expressing Cre, siControl or siFURIN with WT spike pretreated with 100 nM CB6 for 1 hour, before mixing with Stop-Luc-expressing cells carrying ACE2 for further 6 hours (top); and immunoblots showing shedded S1, hIgG Hc, full-length spike, S2 and cleaved S2’ collected from stimulated cell supernatants and lysates (bottom). Data are representative of four individual repeats, blots are representative of two independent experiments; (C) Immunoblots showing shedded S1 subunit and hIgG IgG Hc, full-length spike, S1, S2 and cleaved S2’ collected from supernatants and lysates of HEK293T cells co-expressing ZsGreen reporter with WT and Delta spike VOCs, preincubated without or with 100 nM CB6 and 25 μM RVKR for 1 hour, then co-cultured with HEK293T cells expressing ACE2 for 16 hours. Blots are representative of two individual repeats; (D) Zsgreen fluorescent images of CB6-mediated neutralization of cell-cell fusion described in (C). Scale bars are indicative of 50 μm, images are representative of two independent experiments. (TIF)