Isolation of integral membrane proteins of Leishmania promastigotes and evaluation of their prophylactic potential in hamsters against experimental visceral leishmaniasis

The integral membrane proteins (IMP's) of promastigotes of two virulent strain of Leishmania (L.) donovani Dd8 and Leishmania (L.) infantum LV9 and one avirulent viscerotropic strain Leishmania tropica UR6 were extracted by phase separation technique using a non-ionic detergent “Triton X-114”. This detergent is homogeneous at 0 °C but divides in an aqueous phase and a detergent phase at above 20 °C. The phase partition resulted in solubilisation of hydrophilic proteins in aqueous phase, and IMP's with an amphiphilic nature were recovered in the detergent phase. The strain wise quantitative recovery rates of IMP's were estimated. These proteins were purified by chill methanol centrifugation and used as vaccinogen, alone or in combination with adjuvant against L. donovani challenge in hamster model. Among all the combinations, hamsters immunised with IMP of L. donovani (Dd8) in combination with CFA resulted in 75% parasite inhibition in spleen (P < 0.001), however, 61.14% (P < 0.001) and 77.60% (P < 0.001) parasitic inhibition was achieved in liver and bone marrow respectively as compared to their unvaccinated counter part. Similar combinations with UR6 and LV9 strain inhibited the parasite establishment up to 65.12% (P < 0.001) and 66.87% (P < 0.001), respectively on splenic site. The specific IgG level against (Dd8 strain) soluble leishmania promastigote antigen was monitored at different stages by enzyme linked immunosorbent assay (ELISA) corresponds to the level of parasitic establishment. Similar observations were made in cases of LV9 and UR6 strains. However, significant lymphoproliferative response to IMPs of Dd8 strain (SI 3.5-4.9, P < 0.001) was noticed in all IMP + CFA vaccinated animals. Thus, this study will provide a lead for more manipulative trials to develop a subunit vaccine against the fatal disease.