Two-component, DNA-prime/protein-boost (D/P) vaccine elicits poly-functional T cell response in mice.

<p>C57BL/6 mice were immunized with an empty vector, cytokines only, or D/P vaccine (V1 dose: TcG2- and TcG4-encoding plasmids ± IL-12- and GMCSF-expression plasmids; and V2 dose: recombinant TcG2 and TcG4 proteins), as detailed in Materials and Methods. <b>(A&B)</b> Splenocytes were obtained at 14 days post-vaccination (dpv) and <i>ex vivo</i> labeled with PE-conjugated anti-CD4, FITC-conjugated anti-CD8, PerCPCy5.5-conjugated anti-CD62L, and APC-conjugated anti-CD44 antibodies. Shown are the PE⁺CD4⁺<b><i>(A)</i></b> and FITC⁺CD8⁺<b><i>(B)</i></b> T cell subsets that were of effector/effector memory (T_EM: CD44⁺CD62L^-) and central memory (T_CM, CD44⁺CD62L⁺) phenotype, determined by flow cytometry. <b>(C-E)</b> Splenocytes were <i>in vitro</i> stimulated for 48 h with TcG2 and TcG4 recombinant antigens and labeled with fluorescent-conjugated antibodies. The mean percentage of PE⁺CD4⁺<b><i>(C)</i></b> and FITC⁺CD8⁺<b><i>(D)</i></b> T cells that were IFN<i>γ</i>⁺ (e-Fluor) and/or TNFα⁺ (Cy5) with or without Ki67⁺ (PerCPCy 5.5) phenotype are shown. <b>(E)</b> The percentage of antigen-specific CD8⁺IFNγ⁺ T cells that were CD107a⁺ (Alexa-Fluor 488) and/or perforin⁺ (APC) was acquired by flow cytometry analysis. In all figures, data are presented as mean ± SD (n = 8/group, triplicate observations per experiment). Significance is presented as *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 (vaccinated versus non-vaccinated or vaccinated/infected versus non-vaccinated/infected).