Subcutaneous immunization with kharon1-deficient leishmania infantum induces immunogenicity and protects hamsters...

Leishmanization, the deliberate inoculation of live parasites into the skin, was historically the only effective immunization strategy against leishmaniasis. Advances in molecular biology have since transformed this concept, particularly through gene-editing technologies targeted attenuation of virulence-associated genes, leading development of a new generation of live attenuated parasites. These next-generation leishmanization approaches preserved broad antigenic diversity while offering improved safety profiles inducing durable protective immunity. Deletion of the kharon1 gene in Leishmania infantum generated an attenuated strain (Likh1-/-) capable of infecting mice while eliciting a robust protective immune response. In the present study, we evaluated the immunogenicity and vaccine efficacy of Likh1-/- in Golden Syrian Hamsters (Mesocricetus auratus), a well-established and representative experimental model of visceral leishmaniasis (VL). Animals received one or two subcutaneous doses of Likh1-/- and were evaluated one month after immunization, as well as one and eight months following challenge with virulent L. infantum promastigotes. Likh1-/- immunization induced a mixed Th1/Th2 cellular immune response, as evidenced by flow cytometry and RT-qPCR analyses. Immunized animals exhibited higher IFN-γ-producing cells compared with non-immunized challenged controls, accompanied by a reduction in IL-10-producing lymphocytes. Gene-expression analysis of key immunological markers (IL-1β, TNF, IFN-γ, IL-10, and TGF-β) revealed marked upregulation post-immunization, followed by modulation toward a more balanced response after challenge. Immunization with Likh1-/- also triggered a strong humoral immune response, with increased serum levels of total IgG and IgG2, with IgG2 predominating across all immunized groups and remaining elevated after challenge. Importantly, animals that received two subcutaneous Likh1-/- vaccine doses exhibited a significant reduction in both hepatic and splenic parasite burdens compared with control animals at both post-challenge time points. Collectively, these findings demonstrate that Likh1-/- is immunogenic in hamsters, eliciting a balanced Th1/Th2 immune response associated with effective parasite control, and support its further development as a promising immunoprophylactic strategy against VL.