Figure 6.

Antigen-decorated AAV vector-based vaccine-induced Ova-specific immune responses potently inhibit the growth of EG7 tumor cells injected 80 days post-vaccination

Experimental design as depicted (A). C57BL/6j mice (7 to 8 weeks old; n = 8 per group) received via intramuscular injection AAV2::sOva or the negative control AAV2::eGFP at a dose of 3 × 10⁹ vector genome-containing particles (vg) per animal, or AAV-Vac_Ova4+8⁵⁸⁷ at two different doses—either a total dose of 3 × 10⁹ vg per animal (AAV-Vac_Ova4+8⁵⁸⁷-low) or a total dose of 6 × 10⁹ vg per animal (AAV-Vac_Ova4+8⁵⁸⁷-high). Eighty days after immunization, 1 × 10⁶ Ova-expressing EG7 syngeneic thymoma cells were injected subcutaneously into the flank of each mouse. Blood samples were collected on days (D) 14, 21, 28, 73, 82, and 88, and serum samples on D17, 24, 31, and 95. Blood samples were evaluated by flow cytometry after staining with H-2K^b/Ova_257-264 dextramer for the presence of Ova-specific CD8⁺ T lymphocytes (B). Splenocytes were isolated at D95 post-vaccination and evaluated by ELISpot assays to determine the number of anti-Ova CD8⁺- (C), (left graph) and anti-Ova CD4⁺- (C) (right graph) specific T cells secreting IFN-γ upon in vitro re-stimulation. Serum samples were evaluated by ELISA for the presence of Ova-specific IgG antibodies (D). Percentage and number of mice that remained tumor-free until day 10 post-tumor challenge are depicted (E). Statistical analysis: Kruskal-Wallis test with Dunns post test; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. Data are represented as mean with SEM.