Adaptive immunity against Leishmania nucleoside hydrolase maps its c-terminal domain as the target of the CD4+ T cell-driven protective response

Abstract

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

Figure 1. Vaccination, challenge and development of NH36-specific humoral immune response.

(A) 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 L. chagasi amastigotes. 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) and after challenge (C). * p<0.05 different from the saline control. p<0.05 different from F1sap vaccine; ○ p<0.05 different from the F2sap vaccine; ◆ p<0.05 different from NH36sap vaccine; p<0.05 different from all other vaccines.

Figure 2. Nucleoside hydrolase NH36 T cell and antibody epitope mapping.

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 Leishmania donovani.

Figure 3. Intradermal response to the leishmanial antigen.

(A) 24 h and 48 h after complete immunization and (B) after challenge with 3×10⁷ amastigotes of L. chagasi obtained from hamster spleens. Results of 3 independent experiments with 20–24 mice per treatment (A) and 8–11 mice (B) per vaccine group are shown as mean + SE. * p<0.05 significantly different from the saline treated controls, the F1, ○ the F2 or from all the other vaccines.

Figure 4. Development of NH36-specific cellular immune response as disclosed by flow cytometry analysis.

Splenocytes stained with anti-CD4 (A) or and anti-CD8 (B) antibodies in vaccinated mice challenged with L. chagasi. Results are shown as mean + SE of two independent experiments (n = 14–16 mice per treatment). ** Significant increase in the CD8+ T cell proportions after challenge, * p<0.05 significant differences from the saline treated controls, ○ from the F2sap and ◆ from the NH36sap vaccine.

Figure 5. Development of NH36-specific cellular immune response as disclosed by intracellular staining analysis of splenocytes in vitro cultured with NH36 before and after L. chagasi infection.

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.

Figure 6. Development of cell-mediated immune response as disclosed by in vivo depletion with anti-CD4+ and anti-CD8+ monoclonal antibodies.

Leishmania chagasi parasite-load (A and B) and percent of liver/corporal relative weight (C and D) 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) (A and B). Hepatomegaly was assessed by the individual increment in liver relative weight expressed as percent of the body weight. + p<0.05 significant differences between treatments.

Figure 7. Protective efficacy of vaccinated mice against L. chagasi infection.

The individual L. chagasi 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. *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).

Figure 8. Protective efficacy of vaccinated mice against L. amazonensis infection.

(A) Evolution of the size of footpad lesions of mice challenged with 10⁵ metacyclic promastigotes of L.amazonensis 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) The number of promastigotes of Leishmania amazonensis in the footpad lesions as disclosed by Real Time PCR assay. The horizontal lines represent the mean averages. *p<0.05 significant differences from the saline controls and ○ from the F2sap vaccines.

Figure 9. Long-term protection generated by the F3sap vaccine.

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 L. chagasi amastigotes 28 days after the last immunization. Bars represent the mean ± SD of the individual parasite load in liver measured by LDU (one experiment, n = 3–4 mice). *p<0.05 significant differences from the saline controls and ○ from the F2sap vaccine.