Novel Antigens of L. donovani Figure 3. DTH and splenocyte proliferation in BALB/c mice vaccinated with cationic liposomal antigens before and after challenge infection. Ten days after the last vaccination and 3 months after challenge infection DTH (A) and splenocyte proliferative (B) responses were measured. DTH response is expressed as the difference (in millimeters) between the thickness of the test (LAg-injected) and control (PBS-injected) footpads at 24 h. Spleens were collected and splenocytes were restimulated in vitro for 72 h with 2.5 mg/ml of specific antigens. Antigen-specific splenocyte proliferation was determined by thymidine incorporation and expressed as counts per minute. Each sample was examined in triplicate. The results are shown as the mean6S.E. for five individual mice per group, representative of two independent experiments with similar results. # p,0.0001 in comparison to control groups and * p,0.001 in comparison to data before infection as assessed by two-way ANOVA and Bonferroni’s correction. doi:10.1371/journal.pone.0005820.g003 different vaccine groups represented production of IFN-c, IL-12 and IL-4 before infection which correlated with the decreased parasite level in post infection. In addition, these results suggested a link between the magnitude of these cytokine responses and the extent of reduction in parasite burden. With progressive infection, levels of IFN-c and IL-12 remained low although pronounced stimulation of IL-4 and IL-10 (p,0.001) compared to the level before infection, were observed in the supernatants from splenocytes of control infected mice stimulated with different antigens. Splenocytes from mice immunized with liposomal LD31, LD51 and LD72 secreted high levels of IFN-c and IL-12 (p,0.0001 compared to controls) significantly enhanced in comparison to prechallenge (p,0.001). Levels of IL-4 and IL-10 remaind low (p,0.0001 compared to controls) in these vaccinated groups with decreased parasite load. In contrast, IFN-c and IL-12 levels were comparatively lower in LD91 immunized mice with higher levels of IL-4 and IL-10 production. An increase, however was observed in the levels of all the cytokines when compared to prechallenge response (p,0.001). Production of IgG2a and IgG1 before infection is predictive of successful vaccination Because cytokines such as IFN-c and IL-4 direct immunoglobin class switching for IgG2a and IgG1, respectively [31] we measured antigen specific production of these antibody isotypes before infection to provide an indirect but physiologic in vivo assessment of the pattern of cytokine production and consequently reduction of parasite load after challenge infection. Sera from mice immunized with liposomal LD31 and LD51 contained elevated and highest levels of both the IgG2a and IgG1 isotypes (Figure 5, p,0.001 compared to controls), although the level of IgG2a was higher than that of IgG1. Significant enhancement for both the isotypes with a dominance of IgG2a were also observed in liposomal LD72 immunized groups with minimum production of the isotypes in liposomal LD91 group (p,0.001 compared to controls) These results further substantiate the magnitude of the production of both the cytokines, IFN-c and IL-4, before infection is predictive of success of vaccination. Assesment of immune correlates of protective immunity Differences in the IFN-c, IL-12 and IL-4 production between vaccine groups prompted us to evaluate the potential correlations between the cytokine responses after immunization and parasite burden in the vaccinated animals of the representative groups. Importantly there was a strong inverse correlation between the liver parasite burden and magnitude of the IFN-c production before infection (Figure 6A). Also, the differential production of IL-12 (Figure 6B) and IL-4 (Figure 6C) correlated with control of infection, suggesting the identification of associates of reduced parasite burden after immunization. A statistical correlation between the cytokine responses and pathogenesis was then determined in spleen. There was also a striking inverse correlation between the IFN-c, IL-12 and IL-4 (Figure 6D to F) productions and the parasite burden in spleen, signifying a direct correlation of parasite burden with the magnitude of the cytokine responses after immunization. Identification of antigens which conferred protection against challenge infection Immunization with liposomal LD31, LD51 and LD72 exhibited enhanced reduction in parasite burden with a preferable immune response against L. donovani challenge. To identify these candidate vaccine antigens, the bands were excised, subjected to in-gel trypsin digestion. The digested proteins were analyzed by MALDI-TOF/TOF mass spectrometry, and the MS and MS/MS spectrum were searched against all the known Leishmania spp. sequences in MSDB and NCBInr databases using MASCOT search engine. LD31 was identified as ATP synthase a chain of L. major (Table 1). The band of LD51 was identified as b-tubulin of L. major. Again, the N-terminal sequence of LD51 found to be PLoS ONE | www.plosone.org 5 June 2009 | Volume 4 | Issue 6 | e5820