Fig 6. GRA24 induces protective immunity in the absence of MyD88.
(A) MyD88+/+ and MyD88-/- animals (n = 5–6 mice per group) were i. p. inoculated at Day 0 and Day 14 with 106 cps1-1 or cps1-1:Δgra24 tachyzoites, then at day 28 splenocytes were isolated and subjected to in vitro stimulation with soluble tachyzoite antigen (STAg; 50 μg/ml). Supernatants were collected at 72 hr for cytokine ELISA. Each point represents a single mouse. (B) MyD88-/- mice (n = 3 mice per group) were administered IL-12 neutralizing antibody or isotype control antibody during the course of infection initiated by inoculation with 5 x 106 cps1-1 tachyzoites. At Day 10 post-infection, splenocytes were isolated and stimulated with STAg at the indicated concentrations (μg/ml), then 72 hr later supernatants were collected for IFN-γ assay. NI, splenocytes from noninfected mice. M1, M2, M3 are individual MyD88-/- mice treated with the isotype control and M4, M5, M6 are individual MyD88-/- mice treated with the IL-12 neutralizing antibody. (C) Mesenteric lymph node cells were pooled from vaccinated mice, and subjected to STAg re-stimulation and cytokine assay as in (A). C, control cells cultured in medium alone. (D) MyD88-/-IL-12p40eYFP/eYFP mice (n = 7 per group) were vaccinated by i. p. inoculation with cps1-1 or cps1-1:Δgra24 tachyzoites. Two weeks after the final inoculation, mice were challenged with 103 RH tachyzoites. Nine days post challenge tissues were harvested from each individual mouse and processed independently for parasite quantitation by qPCR. In E and F, MyD88+/+ and MyD88-/- animals were vaccinated by i. p. inoculation with cps1-1 or cps1-1:Δgra24 tachyzoites as in (D). Two weeks after the final inoculation, mice were challenged by subcutaneous injection with 103 RH strain tachyzoites, and survival was monitored. Data shown are the means ± SD of cells cultured in triplicate (A, B and C). Statistical significance was evaluated using Student’s t test (A, C, D). A Mann-Whitney test was used to measure statistical significance between isotype controls and the corresponding anti-IL-12 concentrations (B). Log rank Mantel-Cox test was used to measure the statistical significance between the survival curves. (E and F). The asterisks in F indicate MyD88-/- mice vaccinated with the cps1-1:Δgra24 strain were significantly more susceptible than cps1-1 vaccinated mice, and that animals vaccinated with the cps1-1:Δgra24 strain were significantly more resistant than the nonvaccinated group. For the results shown, * p < 0.05, ** p < 0.01, and *** p < 0.001. These collective experiments were repeated with the same result two to three times.