“Desenvolvimento de uma composição vacinal contendo multiepitopos de LTCD4+ E TCD8+ contra a leishmaniose visceral em camundongos BALB/C”

Abstract

Diseases caused by parasites of the Leishmania genus are currently classified as neglected infections worldwide, despite their significant medical relevance. Visceral leishmaniasis (VL) is the most severe form of the disease, with a high annual incidence globally. In Brazil, VL is a zoonotic disease, with dogs serving as important reservoirs, exhibiting high levels of skin parasitism and often remaining asymptomatic, which facilitates the persistence of the protozoan's biological cycle. Given the urgent need for an effective strategy to reduce the incidence of the disease, this study aimed to develop a gene encoding a chimeric protein containing multiple epitopes for CD4+ and CD8+ T lymphocytes, intended for use in a vaccine formulation against canine visceral leishmaniasis (CVL) in combination with the MPLA adjuvant. Using bioinformatics analysis tools, specific CD4+ and CD8+ T lymphocyte epitopes were predicted from hypothetical protein sequences previously identified in the immunoproteome of Leishmania infantum promastigotes to design the chimeric protein. The pET-28a(+) plasmid containing the gene encoding the protein was used to obtain Escherichia coli BL21 clones. To evaluate the vaccine formulation in vivo, Balb/c mice were used and divided into four experimental groups: saline control; adjuvant-only (MPLA); chimeric protein-only (rQui); and chimeric protein combined with adjuvant (rQui+MPLA). Following immunization, half of the animals were euthanized to assess the immune response. The remaining animals were subjected to a challenge infection and, after 60 days, were also euthanized for comparative analysis of immunogenicity and infection outcomes. The results demonstrated that the chimeric protein formulation, in association with monophosphoryl lipid A (MPLA), induced a protective immune response with a TH1-polarized profile against the Leishmania parasite. This response was characterized by the activation of CD4+ and CD8+ T lymphocytes, along with an elevated production of pro-inflammatory cytokines, such as IFN-γ and TNF-α. Additionally, a strong induction of specific antibodies against both the chimeric protein and the parasite was observed, as well as significant protection against challenge infection, evidenced by a reduced parasite load in analyzed organs and a substantial increase in nitric oxide production. These findings underscore the potential of this antigen as a promising candidate for the development of an effective vaccine against visceral leishmaniasis, which may contribute in the future to the prevention and control of the disease in endemic areas of Brazil.