Figure 5. Prechallenge antigen-specific IgG isotype responses in BALB/c mice vaccinated with cationic liposomal antigens. Ten days after last vaccination serum samples were collected and assayed for antigen-specific IgG1 and IgG2a antibodies by ELISA. Each sample was examined in duplicate. The results are shown as the mean absorbance values6S.E. of five individual mice per group, representa- tive 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.g005 required. Additionally, the immune response that mediate susceptibility or resistant to L. donovani infection differ significantly from other species of Leshmania, especially L. major. In CL a Novel Antigens of L. donovani polarized Th1 response is sufficient for protection and a concomitant Th2 abrogates even a strong Th1 function to favor the disease progression. However, this Th1/Th2 dichotomy is lacking in VL and a mixed response is essential for protection [6,13,16,33]. But the immune parameters, which were required to predict protection in VL, are still unresolved. Although cell mediated immune responses are thought to play important role in controlling leishmaniasis, ability to induce Th1 response might not be the only criteria to select vaccine targets against VL [10,34]. Screening of expression libraries with sera from infected animals and humans have been widely used to identify most immunogenic novel vaccine antigens against leishmaniasis [35]. As individuals with acute VL develop a polyclonal B-cell response generating antibodies to a large number of parasite antigens, a two-step library screening procedure first by reactivity with patients’ sera followed by their ability to induce T cell proliferation and IFN-c responses in immune mice might be more useful [36]. Subsequently, antigens such as HASPB1 and A2 that induce strong protection against VL also showed serologic reactivity with patients’ sera [37,38]. In our attempt to design a vaccine against VL, we used a different approach and selected four antigens that exhibited humoral reactivity with not only sera of infected VL patients but also demonstrated persistence recognition with cured sera. Moreover, PBMC from VL patients showed proliferative response with an elevation in IFN-c and IL-12 production when stimulated with these antigens (unpublished observation). Thus, in the absence of an efficient method to predict protective antigens, we then critically evaluated the vaccine efficacy of these antigens individually using an in vivo experimen- tal mice model against L. donovani challenge infection. Figure 6. Correlates of immune responses and reduced parasite burdens in BALB/c mice vaccinated with cationic liposomal antigens. Liver and spleen parasite burden after 3 months challenge infection with L. donovani promastigotes was tested for correlation with antigen-specific prechallenge IFN-c, IL-12 and IL-4 responses. Magnitudes of the antigen-specific IFN-c, IL-12 and IL-4 production were inversely correlated with the liver (A to C) and spleen (D to F) parasite burdens. Each symbol represents an individual animal from a vaccinated group. The p- values were calculated for correlation efficiency and were considered significant at p,0.05. Spearman’s rank correlation coefficient values are represented (r). Diagonal lines represent linear regression. doi:10.1371/journal.pone.0005820.g006 PLoS ONE | www.plosone.org 7 June 2009 | Volume 4 | Issue 6 | e5820