Recombinant NAD-dependent SIR-2 protein of Leishmania donovani: immunobiochemical characterization as a potential vaccine against visceral leishmaniasis.
BACKGROUND
The 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.


RESULTS
LdSir2RP 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.


CONCLUSIONS
The 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.
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, India.
Rajendra K
RK
Baharia
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, India.
Rati
R
Tandon
Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India.
Tanuj
T
Sharma
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow, India.
Manish K
MK
Suthar
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, India.
Sanchita
S
Das
Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India.
Mohammad Imran
MI
Siddiqi
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow, India.
Jitendra Kumar
JK
Saxena
Shaym
S
Sundar
Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
Shyam
S
Sunder
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, India.
Anuradha
A
Dube
D000328:Adult; D000818:Animals; D019295:Computational Biology; D006224:Cricetinae; D016207:Cytokines / Q000276:immunology; D006801:Humans; D007114:Immunization; D007893:Leishmania donovani / Q000276:immunology*; D054332:Leishmaniasis Vaccines / Q000276:immunology*; D007898:Leishmaniasis, Visceral / Q000517:prevention & control*; D008213:Lymphocyte Activation; D008297:Male; D008647:Mesocricetus; D009243:NAD / Q000502:physiology*; D009569:Nitric Oxide / Q000096:biosynthesis; D015800:Protozoan Proteins / Q000276:immunology*; D056565:Sirtuin 2 / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000276:immunology
D016207:Cytokines; D054332:Leishmaniasis Vaccines; D015800:Protozoan Proteins; D014614:Vaccines, Synthetic; D009243:NAD; D009569:Nitric Oxide; D056565:Sirtuin 2