Figure 1. Silver-stained SDS-PAGE gel of Leishmanial antigens (LAg) and purified antigens by electroelution. Lane 1, molecular mass markers; lane 2, LAg ; lane 3, 91-kDa (LD91); lane 4, 72-kDa (LD72); lane 5, 51-kDa (LD51); lane 6, 31-kDa (LD31). The positions of molecular mass standards are shown on the left in kilodaltons. doi:10.1371/journal.pone.0005820.g001 Novel Antigens of L. donovani the highest degree of both DTH (Figure 3A, p,0.0001) and proliferative response of splenocytes (Figure 3B, p,0.0001) compared to controls. Liposomal LD72 and LD91 also induced considerably higher responses than controls (p,0.0001). Controls exhibited almost similar level of splenocyte proliferation after stimulation with different antigens. Stimulation with total LAg also enhanced the proliferative response of the vaccinated groups but lower in magnitude as observed with specific antigens (data not shown). The responses were further measured after 3 months of L. donovani infection. At this stage, all the groups of mice presented increased DTH and proliferative responses, compared to their respective levels before infection (p,0.001). Similar to prechal- lenge, mice immunized with all four liposomal LD31, LD51, LD72 and LD91 displayed enhanced but varying magnitudes of both DTH and antigen-induced splenocytes proliferative respons- es (p,0.0001 compared to controls) at 3 months post infection. Cytokine responses in vaccinated mice before and after challenge with L. donovani Next, we analyzed different cytokine production by splenocyte following restimulation with specific antigens in vaccinated mice. Following immunization, different vaccinated groups produced substantial amounts of IFN-c (Figure 4A), IL-12 (Figure 4B) and IL-4 (Figure 4C) compared to controls (p,0.0001). There was no difference in the cytokine production in control animals when stimulated in vitro with different antigens. Both liposomal LD31 and LD51 secreted highest level of IFN-c and IL-12 compared to controls (p,0.0001). Interestingly, these groups also secreted IL-4 in high magnitude (p,0.0001 compared to controls). Before infection there was no stimulation of IL-10 production in any of the vaccinated groups in response to antigens (Figure 4D). Splenocytes of different vaccinated mice also secreted increased amounts of IFN-c, IL-12 and IL-4 when stimulated with total LAg but response was lower than with the specific antigens (data not shown). Taken together, the quality of cytokine responses in Figure 2. Parasite burdens in BALB/c mice vaccinated with cationic liposomal antigens after L. donovani challenge infection. Mice were vaccinated intraperitoneally 3 times with 2.5 mg of each antigen entrapped in cationic liposomes at 2-week intervals. Control groups received PBS or empty liposomes (Lip). At 10 days after the last immunization, the mice were challenged intravenously with 2.56107 promastigotes of L. donovani. Liver (A) and spleen (B) parasite burden were measured 3 months after challenge as Leishman Donovan Units (LDU). The results are mean LDU6S.E. of five individual mice per group, representative of two independent experiments with similar results. * p,0.001 in comparison to control groups as assessed by one-way ANOVA and Tukey’s multiple comparison test. doi:10.1371/journal.pone.0005820.g002 PLoS ONE | www.plosone.org 4 June 2009 | Volume 4 | Issue 6 | e5820