Novel Antigens of L. donovani Table 1. Proteins identified by MALDI-TOF/TOF mass spectrometry. Band LD31 LD51 LD72 LD91 Protein name ATP synthase a chain (L. major) b-tubulin (L. major) Heat shock 70-related protein 1 mitochondrial precursor ( L. major) b-tubulin (L. major) aAccession number in NCBInr database. doi:10.1371/journal.pone.0005820.t001 Accession Numbera 68223738 68224035 57015345 68224035 Matching Peptides 18 18 11 6 Probability-based Mowse score % Sequence coverage 324 223 71 57 20 27 18 14 The findings in this report demonstrated that vaccination with these polypeptides entrapped in cationic liposomes conferred reduction in parasite burden in BALB/c mice against visceral infection with L. donovani. Experiments performed herein also demonstrated that stimulation of three cytokines, IFN-c, IL-12 and IL-4, before infection correlated with decreased parasite level. All the vaccinated groups produced these three prechallenge cytokines suggesting this quality of response is required for successful vaccination against VL. We also observed a direct correlation between the magnitude of the response and the level of reduction in parasite load. Liposomal LD31 and LD51 immuni- zation exihibited highest level of these three cytokines, reduced parasite level in mice to the greatest degree. Immunization with LD72 with moderate response led to a lower level of reduction whereas liposomal LD91 immunization induced lowest level of these cytokines and reduction of parasite load. Thus, not only the quality but also the magnitudes of the prechallenge cytokine responses were critical correlates of protection in VL. The findings in this report with subunit vaccines also substantiate our earlier observation with leishmanial antigens that an early mixed Th1/ Th2 response was required for success against VL [27,29]. Significantly elevated levels of IgG2a antibody along with increased levels of IgG1 in vaccinated groups before infection observed herein suggested a mixed Th1/Th2 cytokine profile. Again the magnitude of the antibody response correlated with the degree of reduction of parasite burden further supporting the implication of this immune correlate. The quality and magnitude of the immune response may be regulated by the synergistic effect of these three cytokines. An IL-12 driven IFN-c-dependent Th1 response associated with protective immunity, has a direct effect on the macrophage microbicidal activity and other effector killing mechanisms [13]. IL-12 is known to be involved not only in activation of NK cell but also development of Th1 response and granuloma formation. Moreover, it may have important role to establish cell-mediated immunity by promoting the differentiation of Th1 cells from naive T cells and also to maintain that immunity [39]. The essential role of IL-12 has been confirmed in vivo by the treatment of mice with recombinant IL-12 at the time of infection that shifted the immune response to Th1 and prevented susceptibility to infection [40]. Additionally, immunization with a combination of leishmanial antigens and IL-12 led to the development of a Th1 response and conferred protection in BALB/c mice [41]. Another important candidate is IL-4, a known Th2 cytokine. Previous reports have shown that IL-4 is needed to drive Th1 differentiation [42], to maintain IFN-c production [43] and to prime IL-12 production [44]. This suggests that an early IL-4 may have a role in an initial induction of protective immunity. The finding that IL-42/2 knockout mice were more susceptible to L. donovani [18] and the reported beneficial role of IL-4 in effective chemotherapy [45], suggest the protective ability of this cytokine in VL. Thus, combinatorial effects of these cytokines cause an additional paramount effect on magnitude of the protective immune response. None of the protected groups produced IL-10 after immunization, suggesting that IL-10 has no role in protection. It is hypothesized that production of IL-10 may be a prechallenge indicator for vaccine failure in CL [19], and thus absence of IL-10 after immunization may also have beneficial role in VL for protection. As shown in this study LD31, LD51 and LD72 are protective vaccine antigens, we had identified the proteins by MALDI-TOF and N-terminal sequencing. The success of this approach was made possible due to the availability of the entire sequence of L. major and L. infantum genome. 31-kDa polypeptide was identified as ATP synthase a chain of L. major. 51-kDa band was identified as b- tubulin and 72-kDa was identified as heat shock 70-related protein 1 precursor of L. major. To our knowledge vaccine potentiality of two of these antigens ATP synthase and b-tubulin have never been evaluated against any form of leishmaniasis. L. major HSP70 was evaluated as recombinant protein but failed to induce protection against L. major [46]. ATP synthase complex subunits are functionally associated with the membrane and thus can be potential targets for drugs, diagnostic probes or vaccine components against Leishmania. ATP synthase a subunit was already identified as immunogenic proteins in Mycobacterium bovis [47] and intracellular pathogens like Brucella [48] when probed with antisera from bovine and human patient. The discrepancy in the observed and predicted molecular weight, which seems to be 62-kDa, suggests a probable truncated form of this protein. This phenomenon has been widely reported in the proteomes of several Leishmania spp. as well as in Trypanosoma [49,50]. Moreover, for membrane proteins often the gel molecular weights do not correspond to the actual molecular weights based on amino acid composition and bands may migrate anomalously in SDS-PAGE [51]. Additionally, the difference in observed mass from that predicted by the leishmanial genome, a common feature reported in most proteomic analyses, may be the effect of protein ‘maturation’ events including co- or post-translational modifica- tions (PTM). The observations from other studies of Leishmania suggest that PTM is widely prevalent in this organism [49,50]. 51- kDa band, identified as b-tubulin has previously been character- ized as a T-cell stimulating antigen from Leishmania by CD4+ T cell expression cloning [52]. Moreover, tubulin, has also been identified as a vaccine candidate from a phage expression library using sera of VL patients [53]. The 91-kDa polypeptide with a homology with b-tubulin did not show immunogenicity and protective immunity to that level as observed with 51-kDa antigen. This 91-kDa band suggested that PTMs are the most likely cause of variant forms of tubulin [49,50]. There are several different PLoS ONE | www.plosone.org 8 June 2009 | Volume 4 | Issue 6 | e5820