Fig. 5: pNS3 and pNS4 DNA vaccines elicit robust CD8⁺ T cell responses against ZIKV non-structural proteins.

A Schematic representation of the codon-optimized DNA vaccine constructs encoding full-length ZIKV NS3 (pNS3) and NS4 (pNS4), cloned into the pVAX1 vector under a CMV promoter. B Vaccination schedule. Female BALB/c mice (6–8 weeks old, n = 7/group) were immunized with 50 µg of pNS3, pNS4, or pVAX control, on days 0, 14, and 28. T cell responses were assessed 2 weeks after the final dose. C IFN-γ ELISpot quantification of total NS3-specific T cell responses following stimulation with five peptide pools spanning NS3. D Functional assessment of NS3-specific cytotoxic (CD8⁺) and helper (CD4⁺) T cell responses by in vivo fluorescent target array (FTA). Peptide pools P1–P5 (5 μg/mL) were used for stimulation. E IFN-γ ELISpot quantification of total NS4-specific T cell responses following stimulation with two peptide pools covering NS4A/B. F CTL and Th responses in pNS4-vaccinated mice as determined by in vivo FTA using NS4 peptide pools P1 and P2. Bars represent mean ± SEM; comparisons between groups were performed using Mann–Whitney U tests. To account for multiple testing, p-values were adjusted using the Benjamini–Hochberg false discovery rate (FDR) method. **P ≤ 0.01.