Fig. 7: EEEV-373 inhibits SINV/EEEV entry through receptor blockade and inhibition of early fusion events.

Competition-binding ELISA to assess the ability of bivalent EEEV-373 (green) or isotype-matched control (black) IgG1 molecules to block ApoER2 (A) or VLDLR (B) protein receptors from binding to EEEV VLPs at the concentrations indicated (67 to 3 nM; x-axis). The average optical density at 450 nm of the isotype-matched control mAb at each concentration was calculated and used to normalize the percent binding of EEEV-373 to EEEV VLPs (y-axis). Data represent mean ± SD of technical triplicates of two independent experiments. Data were analyzed at the specified concentrations using multiple unpaired t-tests (***ρ < 0.000001, **ρ < 0.0001, *ρ < 0.001). The exact ρ values are noted for each comparison. C Dynamic light scattering (DLS) of EEEV-373 (green) as bivalent IgG1 (top), F(ab′)2 (middle), or monovalent Fab (bottom) molecules at different Ab:VLP molar ratios (x-axis). The peak hydrodynamic diameter (nm) is shown on the y-axis. Data represent mean ± SD of technical duplicates and are representative of two independent experiments. D Representative neutralization curves of EEEV-373 (green) as bivalent IgG1 (top), F(ab′)2 (middle), or monovalent Fab (bottom) molecules assessed by a post-attachment assay (open circles) or focus reduction neutralization test (FRNT; closed circles). Ab concentration (nM) is on the x-axis and percent relative infectivity is shown on the y-axis. Data represent mean ± SD of technical duplicates and are representative of two independent experiments. E Liposomal fusion assay of DiD-labeled SINV/EEEV particles incubated with liposomes in the presence of EEEV-373 (closed green circles), EEEV-94 (closed cyan circles; positive control), or an isotype-matched control (open black squares; negative control) as bivalent IgG1 (top left), F(ab′)2 (top right), or monovalent Fab (bottom left) molecules. Assay controls in which no antibody (closed black circles) or liposomes (closed grey circles) were also included. Data are representative of two independent experiments. F An egress inhibition assay was performed to determine the relative RNA copies/µL present in supernatant harvested at either 1 h (left) or 6 h (right) after addition of EEEV-373 (green) or the previously published⁴⁰ irrelevant isotype control mAb, rDENV-2D22 (black), as bivalent IgG1 molecules. RNA copies/µL were determined using a standard curve with quantitative EEEV RNA (ATCC). SINV/EEEV RNA levels were compared to the previously published⁴⁰ SINV/EEEV only control group (purple) using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test (ns = not significant). The previously published negative control mAb, rDENV-2D22, and SINV/EEEV only control groups were included for comparative purposes⁴⁰. Data represent mean ± SD of technical quadruplicates and are representative of two independent experiments.