(A) Splenocytes from immunized mice with LO DNA, AZ DNA, and multi-epitope polypeptides alone or combined were harvested for 10–14 days after immunization and exposed to LO or AZ polypeptide for ex vivo IFN-γ expression. Quantitation of IFN-γ from mouse splenocytes was evaluated in 2 separate experiments. Each experiment (n = 4 mice) was evaluated for comparison for vaccination with AZ protein compared with control and for LO protein compared with control; significance evaluated with Student’s t test. (B) HLA-A*11:01 transgenic mice survival curve after challenge with Type II parasites. Two weeks after last immunization, the transgenic mice immunized with empty vector, LO DNA + LO polypeptide, or LO polypeptide, or were injected with PBS were infected with 2,000 T. gondii ME49-Fluc (Type II) parasites. The survival rates of the 2 groups were recorded. Mice vaccinated with either LO protein alone or LO DNA + LO protein were compared with control mice (PBS or empty vector). Kaplan-Meier curves were generated and survival compared across groups using the log-rank test, P < 0.05. (n = 8 mice per group in 2 replicate experiments with 4 mice, shown pooled). (C and D) CD8⁺ memory T cells. Flow cytometry gating for CD8⁺ memory T cells. Spleen cells are gated on CD3⁺CD8⁺ T cells. Memory T cells were defined as CD44^hiCD45RB^lo. For each group, a representative FACS plot is shown with the percent of CD8⁺ memory T cells shown. All mouse experiments were repeated at least twice (n = 2–4 mice in each group). * P < 0.001, ** P < 0.001. In A and D; one-way ANOVA was performed before the Student’s t test to determine whether there was an overall difference between the groups.