Thus, there remains considerable scope for improvement of antileishmanial vaccine design to maximize the chances of clinical benefit. as a triple antigen cocktail for antileishmanial vaccination in hamsters. We found the antigens to be highly immunoreactive Mouse monoclonal to CD152(FITC) and persistent anti-CPA, anti-CPB and Toreforant anti-CPC antibodies were detected in VL Toreforant patients even after cure. The liposome-entrapped CPs with monophosphoryl lipid A-Trehalose dicorynomycolate (MPL-TDM) induced significantly high nitric oxide (up to 4 fold higher than controls) mediated antileishmanial activity in vitro, and resulted in strong in vivo protection. Among the three CPs, CPC emerged as the most potent vaccine candidate in combating the disease. Interestingly, a synergistic increase in protection was observed with liposomal CPA, CPB and CPC antigenic cocktail which reduced the organ parasite burden by 1013C1016 folds, and increased the disease-free survival of 80% animals at least up to 6 months post infection. Robust secretion of IFN- and IL-12, along with concomitant downregulation of Th2 cytokines, was observed in cocktail vaccinates, even after 3 months post infection. Conclusion/Significance The present study is the first report of a comparative efficacy of leishmanial CPs and their cocktail using liposomal formulation with MPL-TDM Toreforant against in a hamster model. The three CPs acted synergistically in the cocktail to induce almost complete protection against form an attractive group of vaccine candidates for future studies in human VL. Introduction Visceral leishmaniasis (VL) caused by is a fatal disease with an estimated 360,000 new cases all over the world with almost 10% annual case fatality in the Indian subcontinent alone . It is a neglected tropical disease inevitably associated with poverty and immunosuppression. High toxicity of available drugs (amphotericin B, miltefosine and paromomycin), HIV co-infection, and resistant parasites pose a global threat against leishmaniases. Despite recent advances in pharmaceutics and molecular immunology, there is no licensed vaccine available against the disease till date . Encapsulation of antigens within nanocarriers promises stable and customized vaccine delivery to related immune cells against various intracellular pathogens including induced immunosuppression , . Thus, there remains considerable scope for improvement of antileishmanial vaccine design to maximize the chances of clinical benefit. Outcome of prophylactic vaccination largely depends on the choice of right immunopotentiating adjuvants and/or delivery systems coupled to right antigen(s). Cationic liposomes protect the labile antigens from lysosomal degradation and take the advantage of electrostatic interactions with the cells’ negative charge which makes them a natural target for antigen presenting cells (APCs), Toreforant crucial for immune stimulation , . Monophosphoryl lipid A (MPLA) is a Toll-like receptor 4 (TLR4) agonist with more than 100,000 human doses safely administered as a part of licensed hepatitis B and Human papillomavirus vaccines . Mycobacterial glycolipid trehalose-6,6-dimycolate (TDM; cord factor) is a potent immunostimulant known for its macrophage activation properties and induction of proinflammatory cytokines, and anti-tumor activity . Recently, TDM has been shown to act via macrophage receptor with collagenous structure (MARCO), TLR2, CD14 and also macrophage-inducible C-type lectin (Mincle) receptors to exert its immunomodifying effects , . When used together, both the adjuvants i.e. MPL and TDM non-specifically activate the immune system, allowing a better response to the associated immunogen . Recently, we have developed a cationic liposome and MPL-TDM (monophosphoryl lipid-trehalose dicorynomycolate) delivery platform that is suitable for subcutaneous delivery of leishmanial antigens in mice model . Compared to an array of antigens that have been tested, very few are sufficiently promising to be carried out to Phase I clinical trials or advanced preclinical work against VL . Lysosomal cysteine proteases (CP) of (MHOM/IN/83/AG83) originally isolated from an Indian kala-azar patient was maintained by serial passage in Syrian golden hamsters as described earlier . Parasites from stationary-phase culture were sub-cultured to maintain an average density of 2106 cells/ml. Cloning, expression and purification of cysteine proteases and (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”KF018070″,”term_id”:”532164762″,”term_text”:”KF018070″KF018070), (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”KC609324″,”term_id”:”478212837″,”term_text”:”KC609324″KC609324) and (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”JX968801.1″,”term_id”:”409905639″,”term_text”:”JX968801.1″JX968801.1) from (pET28a-and pET28a-promastigotes was subjected to polymerase chain reaction (PCR) with sets of gene specific primers corresponding to and genes based on and gene sequences (Table S1, supporting information). PCR conditions for rCPA and rCPB were one cycle of 5 min at 94C, 35 cycles of 1 1 min at 94C, 1 min at 59C, and 1 min 10 s at 72C, followed by a final cycle of 7 min at 72C. PCR conditions for rCPC were one cycle of.