Purified Splenic amastigotes of Leishmania donovani-Immunoproteomic approach for exploring Th1 stimulatory polyproteins.

Visceral leishmaniasis (VL) represents one of the most challenging infectious diseases worldwide. The reason that once infected, patient develops immunity against Leishmania parasite has paved way to develop prophylactic vaccines against disease, but only some of these have moved ahead for clinical trials. Herein, the study to explore novel and potential vaccine candidates was extended to pathogenic form of parasite, that is, amastigote form which is less explored due to complexity of its purification process. Methods and results. Classical protocol of purification of splenic amastigotes was modified to obtain highly pure amastigotes which was confirmed by Western blotting in support with proteomics studies. Fractionation and sub-fractionation of purified splenic amastigotes revealed four sub-fractions, belonging to 97 to 68 kDa and 68 to 43 kDa ranges, which showed long-lasting protection with remarkable Th1-type cellular responses in hamsters vaccinated with these sub-fractions (LTT, NO, QRT-PCR). Further proteomics analysis, to identify and understand the precise nature and function of these protective protein sub-fractions, identified a total of 47 proteins including twenty-five hypothetical proteins/unknowns. Amastigote stage has potential Th1-stimulatory vaccine candidates, notably, among identified proteins, major were uncharacterized proteins/hypothetical proteins, which once characterized may serve as novel and potential vaccine candidates/drug targets.