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•Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniasis, which can be fatal.•Visceral leishmaniasis treatment may be toxic, and resistance has been reported.•There are currently no vaccines available for humans.•Transgenic L. infantum could be an excellent vaccine candidate.•Prime-boosted mice have shown maximized protection. The etiologic agents of visceral leishmaniasis are Leishmania infantum and Leishmania donovani. Despite the variety of drugs available to treat leishmaniasis, most lead to serious adverse effects, and resistance to these drugs has been reported. Currently, no leishmaniasis vaccine is available for humans. That is why the group developed transgenic L. infantum promastigote lines, which express toxic proteins after differentiation into amastigotes. That is why group developed the pFL-AMA plasmid and transfected it into L. Infantum promastigotes. This plasmid was expressed only in the amastigote form of the parasite. Sequences encoding toxic proteins (active bovine trypsin and egg avidin) were inserted in this plasmid, and the transfected parasites died after the differentiation process. In this study, two immunization protocols were performed in BALB/c mice: prime and prime-boost immunization prior to challenge with the wild-type L. infantum (WT). The parasite burdens in the spleen, liver, and bone marrow were evaluated to verify immunological protection. Histopathological analysis of the spleen and liver and the humoral immune response were also performed. The data showed that the parasite burden was reduced in prime-boosted mice in the spleen, liver, and bone marrow, indicating that mice immunized with two doses of the transfected parasites were satisfactorily protected. High levels of IgG, IgG1, and IgG2a antibodies were observed, as well as the presence of anti-inflammatory cytokine Interleukine-10 and pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α) and Interferon-γ (IFN – γ) suggesting a Th1/Th2 mix response, in addition to the presence of multinucleated giant cells in the spleen and lymphocyte infiltration in the liver. Therefore, L. infantum transfected with a toxic plasmid is an excellent vaccine candidate against visceral leishmaniasis and the application of a boost before the challenge promotes greater protection against WT L. infantum infection.

Leishmania amazonensis is an intracellular protozoan parasite and the main cause of Localized Cutaneous Leishmaniasis (LCL). A practical problem that can worsen the condition of infected individuals is secondary co-infection caused by opportunistic bacteria. This study evaluated the influence of Bacterial Membrane Vesicles (BMVs) from Pseudomonas aeruginosa and Staphylococcus aureus—both commonly associated with LCL—on macrophages previously infected with L. amazonensis. The diameter and concentration of BMVs were consistent with previous findings. We assessed infection dynamics, macrophage viability, and cytokine production. Results showed significant reductions in these parameters when L. amazonensis-infected macrophages were subsequently treated with BMVs, compared to the control group (infected but not treated with BMVs). Quantification of IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α revealed elevated levels of the last cytokine, suggesting an inflammatory response. Co-cultures treated with BMVs from P. aeruginosa (POMVs) exhibited a more pronounced inflammatory profile—marked by higher IL-1β and TNF-α concentrations—compared to those treated with BMVs from S. aureus (SEVs). IL-4 and IL-6 levels remained low relative to IL-1β and TNF-α. In conclusion, our data suggest that macrophage infection with L. amazonensis followed by exposure to bacterial MVs simulates a host-parasite-bacterial interaction, inducing strong immunogenic and inflammatory responses, which may represent potential targets for future vaccine strategies.