Fig. 3: S2-targeting VHHs inhibit spike-mediated membrane fusion.
From: Ultrapotent SARS coronavirus-neutralizing single-domain antibodies that clamp the spike at its base
a Binding of hACE2-muFc to coated recombinant 2P-stabilized spike proteins in the presence of a dilution series of R3DC23, GBP (negative control), VHH72_S56A and CB6 (positive controls), measured in ELISA (mean (N = 2) OD450 signal). b Anti-S1 Western blot analysis of the growth medium (SN) and cell lysates (LYS) of HEK293T cells expressing the SARS-CoV-2 D614G spike protein (HEK S (D614G)) or not (HEK EV) incubated for 30 min with the indicated VHHs, CB6 (positive control), S309 and GBP (negative controls). Arrowheads at the right indicate cell-associated uncleaved spike and the S1 spike subunit. c Binding of hACE2-muFc to cell surface-expressed spike in the presence of R3DC23, VHH72_S56A and GBP (N = 2 samples). Lower dotted line: ACE2-muFc binding to cells that do not express spike, upper dotted line: ACE2-muFc binding to spike-expressing cells in the absence of antibody. d GFP expression of Vero E6 cells that were treated with PBS, GBP, palivizumab (negative controls), R3DC23 or huR3DC23-Fc 4 h after co-transfection of a GFP and a spike expression vector. Scale bar: 250 μm. e Syncytia formation by spike-expressing cells in the presence of R3DC23, GBP or VHH55 (negative control) with live cell imaging. Mean ± SD (N = 3 wells) GFP-positive area of wells is shown. f ACE2-independent spike-mediated syncytia formation, as measured by GFP reconstitution of split GFP. HEK293T cells, co-transfected with a spike and a GFP1-10 expression plasmid, were stimulated with CB6 (20 µg/mL) or PBS, followed by treatment with R3DC23, GBP or S309 and added to HEK293T cells that were transfected with a GFP11 expression plasmid in a 96 well plate. Median (line) and individual (dots) fluorescence intensity (N = 4 wells) is shown. g Syncytia formation of VeroE6/TMPRSS2 cells infected with GFP-expressing replication-competent VSV pseudotyped with SARS-CoV-2 spikes, in the presence of a dilution series of R3DC23, huR3DC23-Fc, GBP or S309. Mean (N = 2) GFP fluorescence of infected wells is shown, images: GFP fluorescence of the indicated samples at 40 h post-infection. Source data are provided as a source data file.
