Vaccination, challenge and development of NH36-specific humoral immune response.

<p>(<b>A</b>) Study design: Balb/c mice were vaccinated with NH36sap, F1sap, F2sap or F3sap at the indicated time intervals, through the sc route, followed by intravenous challenge with <i>L. chagasi</i> amastigotes<b>.</b> Bars represent the mean ± SE of the absorbance values of anti-NH36 antibodies from 1/100 diluted serum of two independent experiments (n = 11–12 mice per treatment) after immunization (<b>B</b>) and after challenge (<b>C</b>). <b>*</b> p<0.05 different from the saline control. p<0.05 different from F1sap vaccine; <b>○</b> p<0.05 different from the F2sap vaccine; ◆ p<0.05 different from NH36sap vaccine; p<0.05 different from all other vaccines.</p

Superiority of the F3 over the NH36 vaccine.

<p>Calculation was performed according the following equation  =  (F3-NH36/F3) values x 100 =  protective effect increment.</p

Development of NH36-specific cellular immune response as disclosed by intracellular staining analysis of splenocytes <i>in vitro</i> cultured with NH36 before and after <i>L. chagasi</i> infection.

<p>Anti-CD4-FITC and anti-CD8-FITC antibodies were used for labeling the cell surfaces and anti-IFN-γ-APC, anti-TNF-α-PE and anti-IL-10-PE for intracellular staining. In order characterize the TH1 response bars represent the ratio of IFN-γ/IL-10 and TNF-α/IL-10 producing cells. Results represent mean + SE of two independent experiments (n = 7–8 mice per treatment). * p<0.05 indicate significant differences from the saline treated controls, from F1sap, ○ from F2sap, and ◆ from the NH36sap vaccine.</p

Nucleoside hydrolase NH36 T cell and antibody epitope mapping.

<p>The peptide sequence of MHC class II-IA^d and IE^d, haplotype H2^d CD4+ T cell epitopes (bold), of MHC class I L^d-CD8+ T cell predicted epitopes (underlined) and of epitopes for antibodies (grey background) in the F1, F2 and F3 fragments of the NH36 Nucleoside hydrolase of <i>Leishmania donovani</i>.</p

NH36 antibody epitope mapping.

<p>Percent of the antibody binding inhibition from sera of Leishmune vaccinated dogs by each of the predicted synthetic peptides in a competitive ELISA assay.</p

Long-term protection generated by the F3sap vaccine.

<p>Balb/c mice were vaccinated with NH36sap, F1sap, F2sap or F3sap at the indicated time intervals, through the sc route, followed by the intravenous challenge with <i>L. chagasi</i> amastigotes 28 days after the last immunization<b>.</b> Bars represent the mean ± SD of the individual parasite load in liver measured by LDU (one experiment, n = 3–4 mice). <b>*</b>p<0.05 significant differences from the saline controls and ○ from the F2sap vaccine.</p

Development of cell-mediated immune response as disclosed by <i>in vivo</i> depletion with anti-CD4+ and anti-CD8+ monoclonal antibodies.

<p><i>Leishmania chagasi</i> parasite-load (<b>A</b> and <b>B</b>) and percent of liver/corporal relative weight (<b>C</b> and <b>D</b>) in mice vaccinated with NH36sap and F3 sap vaccine and treated with rat serum, anti-CD4+ or anti-CD8+ or the combination of anti-CD4+ and anti-CD8+ MAbs. Maximal parasite load reduction was achieved in mice that received either the NH36sap or the F3sap vaccines and rat serum (rat IgG) as controls for antibody treatment. Bars represent the mean + SD (5 mice per each treatment). The parasite load is expressed in LDU values (number of amastigotes per 600 liver cell nuclei/mg of liver weight) (<i>A</i> and <i>B</i>). Hepatomegaly was assessed by the individual increment in liver relative weight expressed as percent of the body weight. <i>+</i> p<0.05 significant differences between treatments.</p

Development of NH36-specific cellular immune response. ELISA of cytokines in supernatants of mice splenocytes.

<p>The mean of one group of data of vaccinated mice (results of one experiment with n = 7–8 mice per treatment) is or not included in the 95% CI of control groups.</p

Protective efficacy of vaccinated mice against <i>L. chagasi</i> infection.

<p>The individual <i>L. chagasi</i> liver parasite load of vaccinated and control groups is expressed in LDU values (number of amastigotes per 600 liver cell nuclei/mg of liver weight) of 2 independent experiments, each with 4–8 mice per vaccine group. <b>*</b>p<0.05 significant differences from the saline controls, from the F1sap and ○ from the F2sap vaccines. The mean averages of LDU values are: 1632.64 (sal); 1027.50 (NH36sap); 1806.49 (F1sap); 1469.91 (F2sap) and 192.14 (F3sap<i>)</i>.</p

Protective efficacy of vaccinated mice against <i>L. amazonensis</i> infection.

<p>(<b>A</b>) Evolution of the size of footpad lesions of mice challenged with 10⁵ metacyclic promastigotes of <i>L.amazonensis</i> one week after completing vaccinations. Results are from 2 independent experiments with 5 animals per treatment. Lesions development was followed by measuring the increment in the thickness of the infected footpad compared to the thickness of the contra-lateral non-infected footpad. Results represent the mean + SE of the footpad measurements. (<b>B</b>) The number of promastigotes of <i>Leishmania amazonensis</i> in the footpad lesions as disclosed by Real Time PCR assay. The horizontal lines represent the mean averages. <b>*</b>p<0.05 significant differences from the saline controls and ○ from the F2sap vaccines.</p