Fig. 1: EEEV−373 potently neutralizes and protects against wild-type EEEV.

A Neutralization curves of EEEV-373 (hybridoma-derived; green squares) as bivalent IgG1 molecules against wild-type EEEV strain FL93-939 in a plaque reduction neutralization assay. The previously published irrelevant isotype mAb (rDENV-2D22; black squares) or mouse anti-EEEV ascites fluid (ATCC (+)ve; purple squares) were included as negative or positive controls, respectively, for comparative purposes³⁹. Each curve displays mAb concentration (nM) or ascites fluid dilution on the x-axis and percent relative viral infectivity on the y-axis. Data represent mean ± SD of technical triplicates of one biological replicate. B Percent of EEEV strain FL93-939 residual fraction (y-axis) present at the highest concentration tested (75 nM) of mAb (EEEV-373 [2%], rDENV-2D22 [99%], or ascites fluid [0%]). Data represent the median values of technical triplicates of one biological replicate. C–E EEEV (strain FL93-939; 10^3.3 CCID₅₀) was inoculated subcutaneously (s.c.) into C57BL/6 mice. After 24 h, EEEV-373 (green) as bivalent IgG1 molecules was administered intraperitoneally (i.p.) at 10 mg/kg (200 µg/mouse; n = 10). The previously published negative control treatment group, rDENV-2D22 (black; n = 10), or the mock-inoculated normal controls (grey; n = 5)^40,42 also were included for comparative purposes. C EEEV-373 (green circles) or mock-inoculated normal control (grey circles) groups mediated 100% survival compared to the negative control mAb (rDENV-2D22; black circles) treatment group. Survival curves were compared using the log-rank test with Bonferroni multiple comparison correction (*ρadj < 0.002). D Percent body weight change of each mouse from the EEEV-373 (green circles), rDENV-2D22 (black circles), or mock-inoculated normal control (grey circles) groups over the course of 18 days post-inoculation. E Serum was collected 3 days post-inoculation from each mouse of the treatment groups (EEEV-373 [green circles], rDENV-2D22 [black circles], or mock-inoculated normal controls [grey circles]) for quantification of virus titer (log₁₀ CCID₅₀/mL; y-axis) in an infectious cell culture assay. Data represent mean ± SD. The EEEV-373 and mock-inoculated normal control groups were compared to the rDENV-2D22 negative control treatment group using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test (*ρ < 0.05).