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Abstract: The Nucleoside Hydrolase (NH36) is the main marker of the FML complex of Leishmania donovani, antigen of the licensed Leishmune® vaccine for prophylaxis of canine visceral leishmaniasis. As a DNA vaccine in mice, the NH36 induces a TH1 immune response. Aiming at the identification of the main epitopes of the NH36 recognized by MHC class I and II restricted T cells, the sequences of three fragments composed by the aminoacids 1-103 (F1), 104-198 (F2) and 199-314 (F3) were cloned in the pET28b plasmid system, expressed them in E. coli Bl21DE3 cells and purified in a Ni-NTA column. Balb/c mice were vaccinated with NH36 recombinant protein and Riedel de Haen saponin and challenged with Leishmania chagasi amastigotes. The DTH response to leishmanial antigen was significantly increased (70%) in vaccines over controls (p=0.000) remaining unchanged after infection and the parasite load in liver suffered a 67.8% significant reduction (p<0.05). Fragments F1 and F3 were capable of inducing a significantly higher secretion of IFN gamma and TNF alpha by cultured splçeen cells. Both are pro-inflammatory TH1 cytokines expected in a protective response against visceral leishmaniasis. The IFN gamma/IL-10 ratios following stimulation with F1 or with F3 were, repectively, 6.73 (138.57/20.57 pg/ml) and 2.8 (181.42/64.71 pg/ml), indicating the induction of a cellular protective immune response, probably related to a TH1 expansion. Taken together, our results of DTH response, liver parasitemia and cytokine secretion disclosed that the protective potential of the saponin-NH36 vaccine is probably related to the relevant contribution of fragments F1 and F3 stimulating the identification of their major involved epitopes. [Copyright &y& Elsevier]