4). Although the same trend described in Fig. 3A was observed, the predominance of the CA4 IDR against the Leishmania lysate was in this experiment even more pronounced (mean = 0.416 mm and 0.430 at 24 h, before and after challenge, respectively) ( Fig. 4A and C). The CA3 vaccine, on the other hand, showed means = 0.202 and 0.217 at 24 h, before

and after challenge, respectively ( Fig. 4A and C). In this experiment, the predominance of the CA4 saponin vaccine see more was sustained even after challenge. IDR reactions after injection with either FML or NH36 antigens were higher in mice vaccinated with CA4 than with CA3 saponin. While all reactions to promastigote lysate were sustained after challenge, the IDR to FML or NH36 antigens showed to be reduced ( Fig. 4C and D). Following the analysis of the cellular immune response, the increase of the percents of spleen

Leishmania-specific T cells after challenge was evaluated by fluorescent cytometry analysis ( Fig. 5). We observed that only the CA4 vaccine increased both the CD4+ and the CD8+ Leishmania-specific T cell proportions over the saline controls while the CA3 vaccine increased only the CD8+ specific T cell proportions ( Fig. 5). There was no difference between the CA3 and CA4 vaccines to the gold standard R. Finally, the splenocytes were also labeled through the ICS JAK inhibitor method and the results are shown as double positive cells ( Fig. 6). We observed that others the CA4 vaccine induced enhancements of the TNF-α-producing CD4+ T cells and of the IFN-γ-producing CD8+-T cells while the CA3 vaccine induced the increase of the IFN-γ-producing CD4+-T cell proportions. No significant variations among treatments were observed in the proportions regarding the TNF-α or the IL-10 production by the CD8+ T cells. The analysis of the parasite load in livers showed that all vaccines induced protection when compared to saline controls (p < 0.0001) ( Fig. 7). Besides the QS21 containing saponin positive control which induced a 89% significant reduction, in agreement with the above described results of the analysis of the immune response, the C. alba CA4 induced

the highest protection (78%, p < 0.0001) that was followed by the CA3 saponin with 57% (p < 0.0001) of parasite load reduction. The difference between CA4 and CA3 was significant (p < 0.0125) hence confirming the superiority of the CA4 saponin in protection against visceral leishmaniasis ( Fig. 7). The gain in body weight along the experiment induced by R saponin was superior to that of the saline controls (p = 0.0407) but not significantly different from the increases in the CA3 and CA4 saponin vaccinated mice (not shown). The increases in IDR after vaccination and infection were strong correlates of protection and were significantly correlated to the decrease of parasite load (p = −0.007) and to the gain in corporal weight (p = 0.0001). The increases in CD4–TNF-α (p < −0.001), CD8–IFN-γ (p < −0.002) and CD8–TNF-α (p < −0.