Novel Antigens of L. donovani Figure 4. Cytokine responses 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 spleens were collected and splenocytes were restimulated in vitro with 2.5 mg/ ml of specific antigens. After 72 h supernatants were collected and assayed for IFN-c (A), IL-12 (B), IL-4 (C) and IL-10 (D) levels by ELISA. Each sample was examined in duplicate. 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.g004 MREIVGCQAGQCGNQ, which corresponds to the b-tubulin of L. major and L. donovani. Mass spectrometry of trypsin digested LD72 was also identified as heat shock 70-related protein 1 mitochondrial precursor of L. major. Interestingly, the tryptic digests of LD91 demonstrated the presence of b-tubulin of L. major with 6 matched peptides. Discussion Over the past years major efforts have been dedicated to develop novel vaccine candidates against leishmaniasis and evaluate them in a variety of animal models. These efforts were fueled by the publication of the L. major and L. infantum genome and renewed interest in the field of leishmaniasis prophylaxis led to the identification of a plethora of putative candidates for vaccine development [2]. The vaccine components identified from genome however, requires critical in vivo experimental evaluation to establish their utility as candidate antigens. Although recombinant proteins fulfill the key criteria, like consistent product quality and cost-effectiveness, studies involving native proteins rather than recombinant ones are needed, as the latter may lack similar immunogenic characteristics [32]. Several vaccine candidates have been identified and evaluated against different Leishmania spp. However, in spite of homology between the different species and a certain degree of cross protection, the need for identification of new immunostimulatory molecules from individual species is still PLoS ONE | www.plosone.org 6 June 2009 | Volume 4 | Issue 6 | e5820