Fig 4. POWV-SEV vaccine elicits antigen-specific CD4⁺ and CD8⁺ T cell responses in mice.

(A-B) ELISpot analysis of splenocytes secreting IFN-γ in response to POWV-SEV immunizations measured in spot-forming units (SFUs) per 10⁶ splenocytes. Harvested splenocytes of POWV-SEV vaccinated C57BL/6 mice (n = 5) were stimulated ex vivo with POWV envelope peptides spanning the entire length of the protein. (A) IFN-γ-secreting SFU per 10⁶ stimulated with linear peptide pools or (B) matrix peptide pools. The immunodominant epitope matched with the H2-Db-restricted prediction of dominant epitope is highlighted in arrows. (C) Percent intracellular cytokine population for antigen-specific CD4⁺ and CD8⁺ T cells. Splenocytes that were stimulated with POWV-envelope peptides spanning the entire length of the protein were evaluated for CD4⁺ and CD8⁺ T cells producing IFN-γ, IL-2, and TNF-α via flow cytometry. (D) Polyfunctionality of antigen-specific CD4⁺ and CD8⁺ T cells. Frequency of total CD4⁺ and CD8⁺ T cells expressing double- or triple- positive cytokines (IFN-γ, IL-2, and/or TNF-α) using Boolean gating strategy.