Characterization of Glycolytic Enzymes - rAldolase and rEnolase of Leishmania donovani, Identified as Th1 Stimulatory Proteins, for Their Immunogenicity and Immunoprophylactic Efficacies against Experimental Visceral Leishmaniasis

Th1 immune responses play an important role in controlling Visceral Leishmaniasis (VL) hence, Leishmania proteins stimulating T-cell responses in host, are thought to be good vaccine targets. Search of such antigens eliciting cellular responses in Peripheral blood mononuclear cells (PBMCs) from cured/exposed/Leishmania patients and hamsters led to the identification of two enzymes of glycolytic pathway in the soluble lysate of a clinical isolate of Leishmania donovani--Enolase (LdEno) and aldolase (LdAld) as potential Th1 stimulatory proteins. The present study deals with the molecular and immunological characterizations of LdEno and LdAld. The successfully cloned and purified recombinant proteins displayed strong ability to proliferate lymphocytes of cured hamsters' along with significant nitric-oxide production and generation of Th1-type cytokines (IFN-γ and IL-12) from stimulated PBMCs of cured/endemic VL patients. Assessment of their prophylactic potentials revealed ∼ 90% decrease in parasitic burden in rLdEno vaccinated hamsters against Leishmania challenge, strongly supported by an increase in mRNA expression levels of iNOS, IFN-γ, TNF-α and IL-12 transcripts along with extreme down-regulation of TGF-β, IL-4 and IL-10. However, animals vaccinated with rLdAld showed comparatively lesser prophylactic efficacy (∼ 65%) with inferior immunological response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of both the proteins was done using computational approach. Additionally, in-silico 3-D modelling of the proteins was done in order to explore the possibility of exploiting them as potential drug targets. The comparative molecular and immunological characterizations strongly suggest rLdEno as potential vaccine candidate against VL and supports the notion of its being effective T-cell stimulatory protein.