Heterologous adenovirus A2/Leish-Tec® vaccination strategy in a murine model of visceral leishmaniasis: Potential canine vaccine with enhanced protection and potent T-cell immunity.

Zoonotic visceral leishmaniasis caused by Leishmania infantum is the most severe clinical form of leishmaniasis and remains endemic in multiple regions, where it can be fatal if untreated. Domestic dogs act as the main urban reservoirs, sustaining zoonotic transmission. This study evaluated the immunogenicity and ability to reduce parasite burden of homologous and heterologous vaccination regimens based on the Leishmania amastigote A2 antigen administered as a recombinant adenovirus vector (AdA2) or a purified recombinant protein (LeishTec vaccine - LT) in a murine model. A total of 120 female BALB/c mice were allocated into two experimental arms: Parasite Burden (PB) and Immune Response (IR), each comprising six protocols: Ad (adenovirus-β-galactosidase), AdA2 (adenovirus-A2), LT (LeishTec), AdA2+LT, LT+AdA2, and PBS. Anti-A2 antibodies were quantified by indirect ELISA. In the PB arm, mice were challenged with Leishmania infantum 30 days after immunization, and parasite burden in spleen and bone marrow was determined by quantitative PCR. In the IR arm, splenocytes were analyzed by flow cytometry to assess cellular immune responses. The homologous LT regimen induced the highest anti-A2 antibody titers. Notably, the heterologous AdA2+LT protocol was associated with a marked reduction in bone marrow parasite burden, with undetectable parasite DNA in most animals in this organ. This regimen also induced robust cellular immunity, characterized by increased TNF-α- and IFN-γ-producing CD4+ T cells, expansion of central and effector memory subsets, and enhanced TNF-α and IFN-γ production by CD8+ T lymphocytes after antigen stimulation. Overall, the AdA2+LT heterologous strategy outperformed the other regimens and represents a promising vaccination approach against visceral leishmaniasis.