Abstract
The present study includes cloning and expression of recombinant Leishmania donovani histone proteins (rLdH2B, rLdH3, rLdH2A and rLdH4), assessment of their immunogenicity in Leishmania infected cured patients/endemic contacts as well as in cured hamsters and finally evaluation of their prophylactic efficacy in hamsters against L. donovani challenge. All recombinant proteins were expressed and purified from the heterologous bacterial host system. Leishmania infected cured patients/endemic contacts as well as cured hamsters exhibited significantly higher proliferative responses to individual recombinant histones and their pooled combination (rLdH2B+rLdH3+rLdH2A+rLdH4) than those of L.donovani infected hosts. The L.donovani soluble antigens (SLD) stimulated PBMCs of cured/exposed and Leishmania patients to produce a mixed Thl/Th2-type cytokine profile, whereas rLdH2B, rLdH3, rLdH2A, rLdH4 and pooled combination (rLdH2-4) stimulated the production of Th1 cytokines IFN-γ, IL-12 and TNF-α but not Th2 cytokines IL-4 or IL-10. The immunogenicity of these histone proteins along with their combination was also checked in cured hamsters where they stimulated higher lymphoproliferation and Nitric oxide production in lymphocytes of cured hamsters than that of infected controls. Moreover, significantly increased IgG2 response, an indicative of cell mediated immunity, was observed in cured hamsters against these individual proteins and their combination as compared to infected hamsters. Further, it was demonstrated that rLdH2B, rLdH3, rLdH2A and rLdH4 and pooled combination were able to provide considerable protection for hamsters against L. donovani challenge. The efficacy was supported by the increased inducible Nitric Oxide Synthase (iNOS) mRNA transcripts and Th1-type cytokines - IFN-γ, IL-12 and TNF-α and down-regulation of IL-4, IL-10 and TGF-β. Hence, it is inferred that pooled rLdH2-4 elicits Thl-type of immune responses exclusively and confer considerable protection against experimental Visceral Leishmaniasis.
Highlights
Visceral leishmaniasis (VL) is one of the most severe forms of Leishmanases caused by an intracellular protozoan parasite of the Leishmania donovani/L. infantum/L. chagasi complex and transmitted through the bites of the sand fly Phlebotomus argentipes
Immunoblots of lysates from L. donovani promastigote was performed with the polyclonal antibodies raised against each of rLdH2B, rLdH3, rLdH2A and rLdH4 respectively, which detected one dominant protein band of,17kDa,21kDa,17kDa and,21kDa respectively (Fig1. i, j, k & l)
Histones are structural proteins and building units of nucleosomes which play an important role in DNA packaging, transcription and regulation of gene expression as well as the organization and function of DNA within the eukaryotic nucleus
Summary
Visceral leishmaniasis (VL) is one of the most severe forms of Leishmanases caused by an intracellular protozoan parasite of the Leishmania donovani/L. infantum/L. chagasi complex and transmitted through the bites of the sand fly Phlebotomus argentipes. The disease is known to cause progressive fatal disease in poor people belonging to North-eastern part of Indian subcontinent. An average of more than 90% of VL cases in India is reported from Bihar alone [1,2]. Recent epidemics of VL in Sudan and India have resulted in over 100,000 deaths [3]. Due to the nonavailability of an ideal antileishmanial drug 350 million people are globally at the risk of acquiring infection with Leishmania parasites worldwide [5]. The disease is emerging as an important opportunistic infection in immunocompromised patients, especially those co-infected with HIV [6,7,8]
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