Figure 5. DNA vaccination generated E7-specific CD8⁺ T-cell responses against spontaneous HPV oral tumors.

(A) Schematic diagram of treatment regimens, and the overall time course. C57BL/6NCr mice (n=5) were CD3-depleted, as described, for three consecutive days, followed by submucosal injection and electroporation in the buccal area with plasmids containing HPV16-E6/E7, luciferase, NRas^G12V, and SB100. Ten days after plasmid injection, mice were vaccinated with pNGVL4a-CRT/E7 DNA vaccine or empty pNGVL4a plasmid vector control for a total of four times at four-day intervals. (B) Kaplan-Meier survival analysis of mice. (C) Peripheral blood from tail arteries were examined by flow cytometry for E7-specific CD8⁺ T cells. Percentages were checked over time from two weeks after plasmid injection at one-week intervals. (D) Representative flow cytometry images of the percentage of E7-specific CD8⁺ T cells in PBMCs of tumor-bearing mice treated with pNGVL4a control (top) and pNGVL4a-CRT/E7(detox) (bottom) on day 21 after plasmid injection. A total number of 120,000 cells were acquired. (E) Real-time bioluminescence image of tumor-bearing mice. (F) Line graph depicting the change in tumor volume of tumor-bearing mice after plasmid transfection. Three independent experiments were performed. P values were calculated by log-rank test (B) or two-tailed Student t-test (C). P values < 0.05 were considered significant. Data are presented as mean ± SD.