Fig. 4. ZE4B-36 induces potent B and T cell responses.

a–j Four- to 6-week old AB6 mice (a–i) or WT B6 (j) mice were infected with ZE4B-36, WT ZIKV FSS13025ic, or PBS (mock). On day 28 pi, splenocytes and blood were harvested. a Splenocytes were stimulated in vitro for 7 d with R848 plus rIL-2 and seeded onto ELISpot plates coated with Ig capture Ab or ZIKV- VLP. Images of total IgG-secreting, ZIKV-specific MBCs, and control wells are shown. b Frequencies of ZIKV antibody-secreting cells per 10⁷ input cells. c Sera ZIKV IgG antibody detected by ELISA. n = 4 to 6. d Sera NAb titers of mice vaccinated with 2.2 × 10⁵ FFU (high dose) or 1 × 10⁴ FFU ZE4B-36 (low dose). n = 4 to 5. Splenocytes of AB6 mice (e, f) or WT B6 mice (j) were cultured ex vivo with WT ZIKV for 24 h and stained for IFN-γ, CD3, and CD4 or CD8. Percentage (e) and the total number of IFN-γ⁺ (f, j) T-cell subsets is shown. n = 4 to 7. g–i Cytokine production in the ex vivo culture following stimulation with WT-ZIKV for 48 h. n = 4 to 10. k, l Four-week-old WT B6 mice were infected with ZE4B-36 or PBS (mock). On day 60 pi, splenocytes were cultured with WT ZIKV for 24 h and stained for IFN-γ, CD3, and CD4 or CD8. Cell number (k) and percent positive (l) of IFN-γ⁺ T-cell subsets is shown. n = 4. Data are presented as means ± standard error of the mean (s.e.m). ***P < 0.001, **P < 0.01, or *P < 0.05 compared to mock group.