Cellular and humoral response induced in BALB/c mice by immunization with rLiHyp1 plus saponin.

<p>Single cells suspensions were obtained from the spleens of mice, four weeks after vaccination. Cells were non-stimulated (medium; background control) or stimulated with rLiHyp1 (20 µg mL⁻¹) for 48 h at 37°C, 5% CO₂. IFN-γ, IL-12, GM-CSF, IL-4, and IL-10 levels were measured in culture supernatants by capture ELISA (<b>A</b>). Each bar represents the mean ± standard deviation (SD) of data from four individual mice per group. Statistically significant differences in the IFN-γ, IL-12 and GM-CSF levels between the rLiHyp1 plus saponin group and control mice (saline and saponin groups) were observed (*** <i>P</i><0.0001). The ratio between IFN-γ/IL-10 and IFN-γ/IL-4 levels (<b>B</b>); and between IL-12/IL-10 and IL-12/IL-4 levels (<b>C</b>) are also showed. Statistically significant differences in the ratios between the rLiHyp1 plus saponin group and control groups were observed (*** <i>P</i><0.0001). The ratio between rLiHyp1-specific IgG1 and IgG2a antibodies was obtained for sera of each individual mouse within their respective vaccination group and statistically significant difference between the rLiHyp1 plus saponin group and control groups was also observed (* <i>P</i><0.005) (<b>D</b>).</p

Analysis of the cellular and humoral response and of the involvement of IL-12, CD4 and CD8 T cells in the IFN-γ production after <i>L. infantum</i> challenge.

<p>Single cells suspensions were obtained from the spleens of mice, 10 weeks after infection. Cells were non-stimulated (medium; background control) or stimulated with <i>L. infantum</i> SLA (25 µg mL⁻¹) for 48 h at 37°C, 5% CO₂. Levels of IFN-γ, IL-12, GM-CSF, IL-4 and IL-10 were measured in culture supernatants by capture ELISA. Mean ± standard deviation (SD) of the cytokines levels determined in four individual mice per group is shown (<b>A</b>). Statistically significant differences between the rLiHyp1 plus saponin group and the control mice (saline and saponin groups) were observed (*** <i>P<0.0001</i>). The analysis of the involvement of IL-12 and CD4 and CD8 T cells in the IFN-γ production is showed (<b>B</b>). Levels of IFN-γ in the supernatants of spleen cells cultures stimulated with SLA, as explained above, in the absence (positive control) or in the presence of anti-IL-12, anti-CD4, or anti-CD8 monoclonal antibodies were measured. Statistically significant differences between non-treated control cells and cultures incubated with anti-CD4 and anti-IL-12 monoclonal antibodies were observed (*** <i>P</i><0.0001). The ratio between IFN-γ/IL-10 and IFN-γ/IL-4 levels (<b>C</b>), and between IL-12/IL-10 and IL-12/IL-4 levels (<b>D</b>), are also showed. Statistically significant differences between the rLiHyp1 plus saponin group and the control groups were observed (***<i>P</i><0.0001). The ratio between SLA-specific IgG1 and IgG2a antibodies levels were calculated for sera of each individual mouse within their respective vaccination group and statistically significant difference between the rLiHyp1 plus saponin group and the control groups was also observed (* <i>P</i><0.005) (<b>E</b>).</p

Protection of BALB/c mice vaccinated with rLiHyp1 plus saponin against <i>L. infantum</i>.

<p>Mice inoculated with saline, saponin, or rLiHyp1 plus saponin were subcutaneously infected with virulent 1×10⁷ stationary-phase promastigotes of <i>L. infantum</i>. The number of parasites in the liver (<b>A</b>), spleen (<b>B</b>), bone marrow (<b>C</b>), and paws' draining lymph nodes (<b>D</b>) was measured, 10 weeks after challenge by a limiting-dilution technique. Mean ± standard deviation (SD) of four mice in each group is shown. Statistically significant differences in the parasite load in all evaluated organs between the rLiHyp1 plus saponin group and control mice (saline and saponin groups) are showed (in numbers). Data shown in this study are representative of two independent experiments, which presented similar results.</p