Humoral and cellular immune responses elicited by vaccination with <i>Salmonella</i> expressing the VapA protein.

<p>(A) Fecal IgA antibody response. Groups of BALB/c mice were orally immunized on days 0 and 14 with <i>S. enterica</i> Typhimurium χ3987-pYA3137<i>vapA</i> (closed triangles/dashed line), or inoculated with either <i>S. enterica</i> Typhimurium χ3987-pYA3137 (closed squares) or PBS (open circles). Fecal extracts were obtained on days 7, 14, 21, 28, 35, and 42 after the second immunization, for measurement of specific IgA by ELISA. The <i>R. equi</i> surface antigen (APTX) was used as the coating antigen (1 µg/mL). Results are expressed as the mean of OD values of five mice per group and are a representative experiment of three assays. ***p<0.001 compared to control groups. (B) Lymphocyte proliferative response. Spleen cells were harvested from immunized (pYA3137<i>vapA</i>) and control mice (pYA3137 or PBS) on day 14 after the last immunization and cultured in the presence of medium alone, APTX (5 µg/mL), or Concanavalin A (2 µg/mL) for 72 h. Cell proliferation was measured by [³H]-thymidine incorporation assay. Each bar represents the average of five mice per group ± SD and is representative of four independent experiments. ***p<0.001 compared to control groups. (C–F) Cytokines production. Whole spleen cells were collected from immunized (pYA3137<i>vapA</i>) and control mice (pYA3137 or PBS) on day 14 after the last immunization and cultured in the presence of medium alone, APTX (5 µg/mL), or LPS (1 µg/mL) plus IFN-γ (1 ng/mL) for 48 h. The concentration of IL-12 (C), IFN-γ (D), TNF-α (E), IL-10 (F), and IL-4 (not shown) cytokines in the supernatants of the cell cultures was measured by ELISA. Each bar represents the mean ± SD of triplicate samples and is a representative experiment of three assays. ***p<0.001 compared to the two control groups; ⁺⁺⁺p<0.001 compared to PBS-inoculated mice.