(A) hHuDEC205-Tg mice were immunized with different combinations of vaccines for the prime and the boost, which were scheduled 10 days apart. Mice were s.c. challenged with 2 × 10⁵ EBNA1-expressing EL4 cells (EL4-E1) either 14 days after the boost in a prophylactic setting (B, C, and D) or 1 to 7 days before the prime vaccination in a therapeutic setting (E, F, and G). Mice were monitored every second day, weight was measured, and tumor size was analyzed by caliper. Mice were sacrificed when the tumor reached 15 mm or more in diameter. (B and E) The tumor volume was calculated using the formula A² × B × 0.52, where A is the shortest diameter perpendicular to the longest diameter, and B is the longest diameter. The mean tumor volume plus the SD of 3 independent experiments with at least 3 mice per group is shown. *P < 0.05 versus PBS-treated mice; 2-way ANOVA with Tukey’s multiple comparisons test. (C and F) The percentage survival from 3 independent experiments with at least 3 mice per group is shown. *P < 0.05 and ***P < 0.001; Mantel-Cox test. (D and G) At sacrifice, bulk single-cell suspensions of LN cells were harvested and analyzed by EBNA1 qPCR from representative prophylactic (D) and therapeutic (G) EL4-E1 tumor challenges. Abundance of the EBNA1 gene was normalized to the UBC gene as the tumor load. A tumor-load cutoff of 0.005 or greater was set. The percentage of mice per condition with and without tumor burden in the LNs is depicted. Statistical analysis was done by 2-way ANOVA with Tukey’s multiple comparisons test using the quantitation cycle (c_q) value of the qPCR.