Fig. 5. Cytokine responses before L. infantum infection.

BALB/c mice (n = 8 per group) were immunized with ChimeraT, rPHB, rEIF5a, rLiHyp1 or rLiHyp2 (15 μg each per mouse) mixed with saponin. Controls received saline or saponin alone. Thirty days after the last vaccine dose, their spleen cells were collected and cultured (5 × 10⁶ cells per mL) in DMEM (Medium) or stimulated with each homologous protein used for immunization (10 µg/mL each) or L. infantum SLA (50 µg/mL) for 48 h at 37 °C with 5% (v/v) CO₂. a Measurement of IFN-γ, IL-12, GM-CSF, IL-4, and IL-10 cytokine levels in cell supernatants. b Ratios between mean IFN-γ and IL-10 levels. c Evaluation of IFN-γ, IL-12, GM-CSF, IL-4, and IL-10 production in spleen cells of the Chimera T/saponin-immunized mice stimulated in vitro with synthetic peptide epitopes. The bars indicate the mean and the error bars denote the standard deviation of the groups. (*) indicates significant difference in relation to the groups of mice immunized with the recombinant proteins plus adjuvant (P < 0.005). (**) indicates significant difference in relation to using the individual peptides as stimuli (P < 0.01). Statistics were done using one-way analysis of variance (ANOVA), followed by Bonferroni´s post-test, which was used for multiple comparisons, and P values < 0.05 were considered significant.