We further assessed whether the vaccination with LdTPI-DNA was able to generate immune response in hamsters since, a major factor of the immune mechanism is the development of strong CMI Cinoxacin responses like T-cell responses, NO production and DTH responses which are responsible for protection and are also supposed to contribute to healing in VL. In the present study, it was evident that all LdTPI-DNA vaccinated hamsters challenged with L. donovani have a specific active T-cell response because they displayed significant LTT response after challenge; on the other hand, this response was severely impeded in non-immunized infected and healthy control hamsters. Further, the supernatant of SLD-stimulated lymphocytes from LdTPI-DNA vaccinated hamsters produced a remarkable level of NO in the macrophages of naive hamsters which also supported the view regarding the up-regulation of iNOS by Th1 cell-associated cytokines and confirms that the NO-mediated macrophage effector mechanism is critical in the control of parasite replication in the animal model. In addition, successful vaccination of Tulathromycin B humans and animals is often related to antigen-induced DTH responses in vivo and T-cell stimulation with antigen in vitro, suggesting a correlation between CMI responses and immunity to infection in this model. There was a low level of parasite-specific DTH responses observed in infected and vector control animals which, on the other hand, was strongly expressed in hamsters immunized with DNA vaccine. Apart from diminished cellular responses, active VL is also associated with the production of high levels of the Leishmania specific antibody particularly IgG and IgG1, which are observed before detection of parasite-specific T cell response. On the other hand, as a measure of CMI, the elevation of IgG2 is consistent with the development of effective immune responses. The transcript of IFN-c, a signature cytokine of the Th1-type response that has a dominant effect on macrophage microbicidal responses and other effector killing mechanisms, along with TNF-a��, often reported to act in concert to activate iNOS for the production of NO, were found to be down-regulated in infected hamsters, whereas their expression was observed to be increased many fold in the immunized hamsters. We have also found an extreme down-regulation in the of IL-10, reported to be down regulate IL-12 for disease progression and IL-4, considered to be a marker for Th2 response, in LdTPI-DNA vaccinated hamsters compared with infected control hamsters. The level of other Th1 cytokine-IL-12, an immune-regulatory cytokine for initiation and maintenance of the Th1 response and plays an important role in the induction of IFN-a? production by T and NK cells, was completely down-regulated in infected 
hamster, where as high levels of IL-12 mRNA transcripts were observed in vaccinated hamsters. However, TGF-a?-a pleiotropic cytokine, having immunosuppressive properties, is also documented in leishmania disease progression and known to be expressed at a moderate level even in normal hamsters, was apparently down-regulated in all the immunized hamsters throughout the experiment. Further, it has been well established that level of IgG and IgG1 antibody increases with the L. donovani loads, which however, was present at very low level in the vaccinated group and thus consistent with the decreasing parasite loads. The significant increase in the IgG2 levels in the vaccinated animals only is a phenotypic marker of enhanced CMI. In a nutshell, our studies have for the first time indicated that LdTPI, a glycolytic enzyme, is also capable of inducing a robust cellular immune response in vitro against both L. donovani-primed lymphocytes from cured kala-azar patients and hamsters and also eliciting a strong protective response against experimental VL.