Figure 8.

ORF8 resultsin decreased infectivity of Spike-pseudotyped virus (S-VSV) but limits the reactivity of anti-SARS-CoV-2 human sera towards the infected cells.A, experimental workflow and summary of results. B, HEK293T cells cotransfected with plasmids encoding Spike or ORF8-Strep genotypes (ORF8-Strep or ORF8-Strep S84L) were infected with replication-incompetent GFP-encoding VSV (VSVΔG-GFP), which resulted in production of Spike incorporated ΔG-GFP (S-VSV). The culture medium containing S-VSV was collected, and Spike levels in S-VSV were evaluated by immunoblot analysis with antibodies against S2 (detects both uncleaved and S2 fragment) and VSV-M (loading control, a VSV structural protein). C–F, HEK293T cells stably expressing ACE2/TMPRSS2 were infected with S-VSV produced in the absence or presence of ORF8-Strep or ORF8-Strep S84L. C, the infectivity of S-VSVs was evaluated by flow cytometry analysis for the percentage of GFP-expressing cells. The cells were further incubated with antibodies against S2 (detects both uncleaved and S2 fragment) (D) or anti-SARS-CoV-2 human sera collected from three COVID-19 convalescent (E) or six vaccinated (F) (three Pfizer and three Moderna, before first shot (prevaccination) and after second shot (postvaccination)) donors. The levels of cell-surface Spike in S-VSV–infected cells (D) and the reactivity of anti-SARS-CoV-2 human sera (E and F) towards S-VSV–infected cells were evaluated by flow cytometry in S-VSV–infected (GFP-positive) but yet viable (LIVE/DEAD-negative) cells. The data represent or are combined from three independent experiments and are presented as mean ± s.d. Statistical significance was analyzed using one-way (B–E; Dunnett’s) or two-way (F; Tukey’s) ANOVA. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; VSV, vesicular stomatitis virus; VSV-M, VSV membrane protein.