Evaluation of Immunopharmacological efficacy of live Leishmania donovani overexpressing Ld_ζ1domain for protection against experimental human visceral Leishmaniasis

Objective: To evaluate the efficacy and immunogenicity of the zeta domain over-expressing Leishmania donovani (Ld_zeta 1domain) as a vaccination candidate against visceral leishmaniasis (VL). Methods: In this study, Leishmania overexpressor Ld_zeta 1domain (OE) were transformed by electroporation using a GFP-tagged Ld_zeta 1domain recombinant plasmid. The resulting overexpressing cells were analysed in vitro to assess their growth dynamics and infectivity. We also investigated the immune-protective potential of these overexpressor in a mouse model challenged with Leishmania donovani. The immune response, including Th1 and Th2 pathways, was thoroughly characterized using RT-PCR and ELISA assays. In addition, the study conducted a thorough evaluation of the mouse's spleen and liver parasites, as well as quantitative evaluation of tissue pathological changes. Results: Ld_ zeta 1 domain (OE) parasites exhibited significantly lower viability and replication rates than WT parasites, and in vivo studies showed that mice immunized with the Ld_zeta 1(OE) domain had lower parasite numbers than mice infected with LdWT. Spleen and liver showed significant histological changes suggestive of protection. Parasite load in the spleen and liver of vaccinated mice were significantly reduced. The immune response showed increased IFN-gamma levels and lower IL-10 production, resulting in a greater IFN-gamma/IL-10 ratio, indicating parasite elimination. The vaccination also caused a significant IgG humoral response and increased nitric oxide production in immunized mice. Conclusion: Our findings demonstrated that overexpressing the zeta toxin resulted in controlled parasite attenuation, lowering pathogenicity while retaining immunogenic features. Our work established the zeta overexpressor's protective efficacy, immunogenicity, and proliferation in response to a Leishmania challenge in vitro and in vivo. This preliminary prototype study suggested that Ld_zeta 1domain (OE) parasites may be suitable for developing an attenuated vaccine against leishmaniasis.