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.
Highlights
In recent years, Nucleoside hydrolases (NHs) of trypanosomatid protozoa have emerged as strong phylogenetic markers of the Leishmania genus [1,2] and vital protagonists of pathways for parasite replication and establishment of infection
Since all Leishmanias species share high identity in their Nucleoside hydrolases amino acid sequences, our study represents a major step forward in the development of a bivalent synthetic vaccine against leishmaniasis and a potential future multivalent vaccine against pathogens that are dependent on NHs for replication
Mice were immunized with NH36, F1, F2 or F3 proteins and saponin (NH36sap; F1sap, F2sap and F3sap vaccines, respectively), challenged with amastigotes of Leishmania chagasi on week 4 and euthanized on week 6 (Figure 1A)
Summary
Nucleoside hydrolases (NHs) of trypanosomatid protozoa have emerged as strong phylogenetic markers of the Leishmania genus [1,2] and vital protagonists of pathways for parasite replication and establishment of infection. NHs cleave the Nglycosidic linkage of imported nucleosides making the purines available for further parasite DNA synthesis. NHs of Leishmania have been described in the parasite stages which infect the mammal host [1,2,6,7,8] and in the exosporium membrane of Bacillus anthracis being important for anthrax transmission [10]. Vaccines against NHs would prevent the replication of many different pathogens at the very first stage of their life-cycle and prevent infection, mild disease, severe disease and death while vaccine with antigens present in later stages of the parasite cycle would only protect from severe disease and death [12]
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