Abstract
BackgroundThe development of a vaccine conferring long-lasting immunity remains a challenge against visceral leishmaniasis (VL). Immunoproteomic characterization of Leishmania donovani proteins led to the identification of a novel protein NAD+-dependent Silent Information regulatory-2 (SIR2 family or sirtuin) protein (LdSir2RP) as one of the potent immunostimulatory proteins. Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity. In the present study, an immunobiochemical characterization of LdSir2RP and further evaluation of its immunogenicity and prophylactic potential was done to assess for its possible involvement as a vaccine candidate against leishmaniasis.Methodology/Principal FindingsLdSir2RP was successfully cloned, expressed and purified. The gene was present as a monomeric protein of ~45 kDa and further established by the crosslinking experiment. rLdSir2RP shown cytosolic localization in L. donovani and demonstrating NAD+-dependent deacetylase activity. Bioinformatic analysis also confirmed that LdSir2RP protein has NAD binding domain. The rLdSir2RP was further assessed for its cellular response by lymphoproliferative assay and cytokine ELISA in cured Leishmania patients and hamsters (Mesocricetus auratus) in comparison to soluble Leishmania antigen and it was observed to stimulate the production of IFN-γ, IL-12 and TNF-α significantly but not the IL-4 and IL-10. The naïve hamsters when vaccinated with rLdSir2RP alongwith BCG resisted the L. donovani challenge to the tune of ~75% and generated strong IL-12 and IFN-γ mediated Th1 type immune response thereof. The efficacy was further supported by remarkable increase in IgG2 antibody level which is indicative of Th1 type of protective response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of rLdSir2RP was done using computational approach.Conclusion/SignificanceThe immunobiochemical characterization strongly suggest the potential of rLdSir2RP as vaccine candidate against VL and supports the concept of its being effective T-cell stimulatory antigen.
Highlights
Leishmaniases is caused by an intracellular protozoan parasite complex by the invasion of the reticuloendothelial system
LdSir2RP was immunodetected in whole cell lysate of L. donovani and further it immunolocalized in cytoplasm of the Leishmania parasite
Recombinant protein rLdSir2RP shown immunogenicity in Peripheral blood mononuclear cells (PBMCs) of cured Leishmania patients and hamsters (Mesocricetus auratus). rLdSir2RP stimulated the production of IFN-γ, IL-12 and TNF-α but not IL-4 and IL-10
Summary
Leishmaniases (cutaneous, mucocutaneous, and visceral) is caused by an intracellular protozoan parasite complex by the invasion of the reticuloendothelial system. The available antileishmanial drugs are very costly and having long-term course of treatment with adverse side effects Apart from this in the various region of endemicity the increasing drug resistance has worsened the scenario. In active VL cases cell-mediated immune responses are absent [5,6,7] and in the patients that are cured, the Th1 type immune response is increased [8,9,10] leading to long time immunity This provides a rationale that Th1 immune response play a major role in cure and prevention of VL [7] the antigenic proteins that modulate Th1 type arm of the immune response could be exploited as vaccine candidates. An immunobiochemical characterization of LdSir2RP and further evaluation of its immunogenicity and prophylactic potential was done to assess for its possible involvement as a vaccine candidate against leishmaniasis
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