Adenovírus expressando proteína A2 em protocolos vacinais homólogos e heterólogos: uma nova abordagem para a imunoprofilaxia da leishmaniose visceral

Abstract

Visceral leishmaniasis is the most severe form of leishmaniasis, endemic in various regions, and potentially fatal. Domestic dogs are the primary urban reservoirs of the disease due to their proximity to humans, which facilitates transmission. In this study, we evaluated the efficacy of homologous and heterologous immunization protocols composed of adenoviruses expressing the recombinant rA2 protein in a murine experimental model. A total of 120 female BALB/C mice, aged 6 to 8 weeks, were divided into two groups: Parasitic Burden (PB) and Immune Response (IR), each consisting of six distinct immunization protocols: Ad (adenovirus expressing galactosidase), AdA2 (adenovirus expressing the A2 protein), LT (LeishTec, CEVA Animal Health), AdA2 + LT (adenovirus expressing the A2 protein + LeishTec), LT + AdA2 (LeishTec + adenovirus expressing the A2 protein), and PBS (1X phosphate- buffered saline). Blood was collected at predetermined times for complete blood count, differential leukocyte count, and indirect ELISA for the detection of specific anti-A2 antibodies. To assess parasitic burden reduction, mice from the PB group were challenged with Leishmania infantum (MHOM/67/ITMAP-263) 30 days after immunization. Thirty days post-challenge, the animals were euthanized, and the spleen and bone marrow were removed for DNA extraction, followed by quantitative PCR analysis of parasitic burden. In the IR group, 30 days after the final immunization dose, the mice were euthanized, and flow cytometry was performed on spleen cells to evaluate the cellular immune profile. No significant hematological changes were observed among the protocols, and the homologous LT protocol yielded the highest antibody titers. The heterologous AdA2 + LT protocol demonstrated 90% protection in the spleen and 97.6% in the bone marrow compared to the PBS protocol. Additionally, the AdA2 + LT protocol induced a robust cellular response, with activation of CD4+ T cells expressing TNF-α and IFN-γ, and activation of central memory and effector memory CD4+ T cell subsets. An increase in TNF-α expression in CD8+ T lymphocytes and IFN-γ production in cytotoxic lymphocytes was also observed following stimulation with soluble Leishmania antigen. In conclusion, the heterologous AdA2 + LT protocol proved superior to others in controlling visceral leishmaniasis, and the development of a vaccine based on this protocol may represent an important strategy for controlling the infection.