FIG 5.

The combination of cobicistat and remdesivir synergistically inhibits SARS-CoV-2 activity. (A to F) Synergistic activity of cobicistat and remdesivir in inhibiting replication and cytopathic effects of SARS-CoV-2 in Vero E6 cells. Cells were infected at 0.5 MOI and left untreated or treated with the drugs at the indicated concentrations 2 h postinfection. Forty-eight hours posttreatment, cells were fixed for immunofluorescence (IF) staining (A and B), supernatants were collected for qPCR (C to E), or cellular viability was analyzed (F). For IF detection, cells were stained with sera of SARS-CoV-2 patients and with the J2 antibody, which binds to double-stranded RNA (36). The percentage of infected cells was determined by automatic acquisition of nine images per well (A), as described in Materials and Methods. Scale bar = 100 μm. Viral RNA in supernatants was detected by qPCR using an in vitro-transcribed standard curve for absolute quantification (C to E). Data, expressed as mean ± SD, were transformed as log₁₀ to restore normality and analyzed by one-way ANOVA, followed by the Holm-Sidak posttest (C). Cellular viability was measured by MTT assay (F). Isobologram analysis of synergism (D) (82) was performed using the IC₉₀ values for SARS-CoV-2 replication of cobicistat, remdesivir, or their combination, calculated by nonlinear regression. Synergism analyses of the inhibition of viral replication (E) or cytopathic effects (F) were performed with the SynergyFinder web tool (79) using the Zero Interaction Potency (ZIP) model based on inhibition values calculated as described in Materials and Methods. (G) Effect of the combination of cobicistat and remdesivir on SARS-CoV-2 RNA expression in supernatants of a primary human colon organoid. Treatment with cobicistat/remdesivir was performed 2 h postinfection, and supernatants were collected 48 h posttreatment. Viral RNA was quantified as described for panel C. For all panels, n equals 3 independent experiments, except for panel E (n = 2 independent experiments) and panel G (n = 2 replicates from one colon organoid donor). *, P < 0.05; **, P < 0.01; ***, P < 0.001.