Antibody-mediated spike activation during SARS-CoV-2 infection.

(A) Schematic representation of antibody-treatment on SARS-CoV-2 post-infected cells. Wildtype Vero E6 cells or Vero E6-TMPRSS2 cells were inoculated with 2 MOI live SARS-CoV-2 (isolate Wuhan-Hu-1) virus for 4 hours to ensure efficient viral entry; culture growth media were then replaced without or with antibody to investigate the possible role of antibody on post-infected cells. Supernatants and cell lysates were harvested 48 hours post infection (hpi) for viral titer and spike cleavage; (B) Brightfield images of 2 MOI SARS-CoV-2 infected wildtype Vero E6 cells, treated without or with 12.5 or 25 nM CB6 antibody for 24 and 48 hours. White arrows indicate patched cytopathic effect under the effect of antibody; scale bars are indicative of 50 μm and images are representative of two independent experiments; (C) SARS-CoV-2 viral titer measured on fresh Vero E6 cells by 50% tissue culture infectious dose (TCID50), inoculated with serially diluted supernatants collected from (B). TCID50 was measured with the Reed–Muench method, data were representative of two individual repeats; (D) Immunoblots of SARS-CoV-2 S, S2, S2’ and N proteins, collected from wildtype Vero E6 cell lysates24 and 48 hpi as described in (B). Blots are representative of three individual experiments; (E) Immunofluorescent images showing morphology of 2 MOI SARS-CoV-2 infected VeroE6 and VeroE6-TMPRSS2 cells, treated without or with 12.5 nM CB6 antibody 48 hours post infection (hpi). Anti-SARS-CoV-2 N and Anti-human IgG (H+L chains) were stained with Alexa fluor 488 and 555 respectively. Scale bars are indicative of 50 μm; (F) Schematic representation of anti-ACE2 blocking antibody and CB6 treatment on SARS-CoV-2 post-infected cells, wildtype Vero E6 cells were inoculated with 2 MOI live SARS-CoV-2 (isolate Wuhan-Hu-1) virus for 4 hours to before medium was replaced without or with 1 μg/mL anti-ACE2 blocking antibody and 12.5 nM CB6, cells were further incubated for 48 hours; (G) Immunofluorescent images showing morphology of 2 MOI SARS-CoV-2 infected wildtype Vero E6 cells, treated without or with 12.5 nM CB6 antibody in the absence or presence of 1 μg/mL anti-ACE2 blocking antibody for48 hpi. Anti-SARS-CoV-2 N and Anti-human IgG (H+L chains) were stained with Alexa fluor 488 and 555 respectively. Scale bars are indicative of 50 μm; (H) Immunoblots of SARS-CoV-2 full-length S, S2, S2’ and N, collected from wildtype Vero E6 lysates described in (G), blots are representative of two individual repeats.