Figure 4. Immune responses associated with IrrPb immunization in C57BL/6 mice.

C57BL/6 mice were immunized once with 10³ or 10⁷ IrrPb, challenged with virulent pRBC, and tissues were harvested on day 6 when ECM appeared in naïve mice. A and B) Serum IgG antibodies (subclasses IgG1, IgG2a, IgG2b, and IgG3) to blood-stage Pb-A antigen were measured by endpoint ELISA. Values are averaged from two independent experiments. Parasite-specific IgG antibodies were not detected (n.d.) in sera from naïve mice or 10³ IrrPb immunized mice before (A) or after challenge (B) with virulent pRBC. Parasite-specific IgG antibodies were detected in sera from 10⁷ IrrPb immunized mice before challenge (A), and the antibody titers increased between 1 to 2 log-fold in the sera of these mice after challenge (B). C and D) Cellular responses: IFN-γ responses in the spleen during a virulent infection are reduced in immunized mice. Spleen cells from unchallenged (C) or challenged (D) mice were cultured with blood-stage Pb-A antigen (Pb Ag), uninfected RBC (RBC), or media control (Unstim). IFN-γ protein was measured in cell culture supernatants by sandwich ELISA (mean +/− SEM) C) Among unchallenged mice, IFN-γ was not detected (n.d.) in samples from naïve mice or 10³ IrrPb immunized mice. A low level of IFN-γ was detected in samples from 10⁷ IrrPb immunized mice after stimulation with parasite antigen (Pb Ag, black bar). D) During a virulent infection, the highest levels of IFN-γ were detected in samples from naïve and 10³ IrrPb immunized mice. 10⁷ IrrPb immunized mice had lower levels of antigen-stimulated IFN-γ in comparison to antigen-stimulated samples from naïve mice (black bars, mean difference −596 (411, 781) pg/ml, p<.0001, Two Way ANOVA).