FIG 6.

Anti-ZIKV immune response in mice immunized with prM-E/C-prM-E DNA or VLPs. (A) Serum samples collected from different groups of immunized mice were used in the RVP-based microneutralization assay. A serum sample from each mouse was serially diluted in DMEM and incubated with a predetermined amount of ZIKV RVPs for 1 h at room temperature. All samples were assayed in technical duplicates. Subsequently, the virus-serum mixtures were added to Vero cells in 96-well plates. The cells were incubated for 72 h, after which the plates were fixed and images acquired as described in the legend to Fig. 2. Curves were fit by using GraphPad Prism software, and neutralizing antibody EC₅₀ (B) and EC₉₀ (C) values were calculated. Statistical analysis was performed using the unpaired t test. Significant differences in EC₅₀ (P = 0.0083) and EC₉₀ (P = 0.0006) values were observed between prM-E and C-prM-E VLP-immunized mice. The dotted line denotes the limit of detection for the RVP assay (defined as the highest concentration of serum used in the neutralization experiments). Samples with titers of <20 are reported at half the limit of confidence (1:10). Neutralization data from one of two independent repeats are shown. (D) Neutralization of the clinical ZIKV isolate PRVABC59 with immune serum samples from mice. Pooled serum samples from each immunized group were serially diluted in serum-free medium as for panel A and then incubated with a predetermined amount of ZIKV for 2 h at 37°C. All samples were assayed in technical duplicates. Subsequently, the virus-serum mixtures were added to Vero cells in 96-well plates. The cells were incubated for 48 h, after which the plates were stained using MAB10216. Images were acquired as described in the legend to Fig. 2, antibody-positive cells were quantitated, and curves were fit using GraphPad Prism software. (E) Protein A beads were coated with 3 μl or 0.6 μl of pooled serum samples from each group of immunized mice. The antibody-coated beads were then incubated with radiolabeled cell lysates derived from C-prM-E-expressing cells. Cell lysates were resolved in an SDS-PAGE gel, followed by PhosphorImager analysis. The photo-stimulated luminescence (PSL) values for the bands are depicted in the graphs at bottom. (F) Pooled serum samples from each group of immunized mice were used in technical duplicates to determine the inhibition of WNV RVPs as described for panel A.