Fig. 4.

Safety evaluation of ZIKV-3′UTR-∆20-LAV (∆20) vaccine candidate. a Viral loads in organs of infected A129 mice. Three-week-old A129 mice (n = 7) were subcutaneously immunized with 10³ FFU of WT ZIKV FSS13025 (left panel) and its derivative ∆20 vaccine candidate (right panel). Organs from infected mice were collected and homogenized at days 6 and 10 post infection. The amounts of viruses were quantified on Vero cells using a focus-forming assay. The mean results from seven animals are presented. Bars denote standard errors. The dashed lines indicate the limit of detection (LOD) of the assay. b–f Effect of ∆20 vaccination on the testis. Three-week-old A129 mice (n = 5) were subcutaneously infected with 1 × 10³ FFU of WT ZIKV FSS13025 or ∆20 vaccine candidate. At day 28 post infection, animals from each group were analyzed for testis weight (b), testis size (c), total sperm counts (d), motile sperm counts (e), and viral RNA load (f). Scale bar, 1 mm. g Comparison of neurovirulence of WT ZIKV FSS13025 and ∆20 vaccine candidate in outbred CD-1 mice. One-day-old CD-1 mice (n = 7–8/group) were injected intracranially with 10–10⁴ FFU of WT ZIKV or 10³–10⁴ FFU of ∆20 vaccine candidate. Surviving mice and total infected animals are indicated. h Analysis of vector competency. Aedes aegypti were fed on artificial blood-meals spiked with 10⁶ FFU/ml of WT ZIKV FSS13025 or ∆20 vaccine virus. At day 7 post feeding, individual engorged mosquitoes were assayed for infection by immunostaining of viral protein expression on inoculated Vero cells. The number of infected mosquitos and total number of engorged mosquitoes are indicated. Asterisks indicate significant differences (one-way ANOVA: ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05). Nonsignificant (n.s.) with P-value > 0.05. All negative samples are plotted at the half value of LOD. Error bars represent SDs