Chagas disease, caused by the protozoan Trypanosoma cruzi, remains a major public health challenge affecting millions in Latin America and worldwide. Although significant progress has been made in vector control, no vaccine exists to prevent infection or mitigate disease pathogenesis. We developed a rationally designed chimeric protein vaccine, N-Tc52/TSkb20, incorporating immunodominant epitopes from two T. cruzi antigens, the amino-terminal portion of Tc52 and the TSkb20 epitope derived from trans-sialidase. The objectives of this study were to construct and characterize the antigen and evaluate its protective potential in an immunoprophylactic murine model of T. cruzi infection. The N-Tc52/TSkb20 protein was recombinantly expressed in E. coli and its identity was confirmed using mass spectrometry and Western blotting. Immunization with the chimeric protein significantly controlled parasitemia and reduced the heart, colon, and skeletal muscle parasite burdens compared to non-vaccinated mice. Protection was superior to vaccination with the individual parental antigen components. Mechanistically, the vaccine induced potent CD8+ T-cell and IFNγ responses against the incorporated epitopes and a protective IgG antibody profile. A relatively low IL-10 response favored early parasite control. These results validate the promising multi-epitope approach and support the continued development of this type of rational vaccine design strategy against Chagas disease.

Chagas disease, caused by Trypanosoma cruzi, remains a major health concern in Latin America, particularly affecting low-income and rural communities. Among the many vaccine strategies explored, live attenuated parasites have shown the strongest ability to trigger protective immune responses. In this study, we investigated whether adding saponin-based adjuvants—Immunostimulant Particle Adjuvant (ISPA) and Quil-A—could improve the effectiveness and safety of a live parasite attenuated T. cruzi vaccine. Mice were vaccinated with a T. cruzi attenuated strain (TCC) alone or in combination with each adjuvant, and immunoglobulin G (IgG) subtypes in the serum of vaccinated mice, and interferon gamma (IFN-γ) and interleukin-10 (IL-10) in the supernatants of stimulated cells were measured at two weeks and twelve months post-vaccination. While protection levels were similar across all groups, the adjuvants assist in modulating the immune response over time: ISPA and Quil-A initially shifted antibody production toward IgG1 but later favored a balanced T_H1/T_H2 profile. ISPA also promoted long-term regulation through increased IL-10. Both adjuvants reduced tissue inflammation and enhanced clearance of vaccine-derived parasites. These findings suggest that while adjuvants may not boost protection directly, they significantly improve vaccine safety and immune quality, reinforcing their value in developing better vaccines for Chagas disease.

Background: Chagas disease, caused by Trypanosoma cruzi, remains a major public health problem in Latin America and an emerging concern worldwide. Current chemotherapies show limited efficacy during chronic infection, and no licensed vaccine is currently available. We previously developed the chimeric antigen N-Tc52/TSKb20 as a vaccine candidate against T. cruzi infection. In a murine model, this vaccine induced robust antigen-specific immune response associated with protection shortly after vaccination. Objectives: Here, we investigated the long-term persistence and effector functions of the immune responses elicited by this vaccine candidate. Methods: Both female and male C57BL/6 mice were immunized with three doses of N-Tc52/TSKb20 formulated with QuilA adjuvant. Serum samples collected 170 days post-immunization were analyzed for antigen-specific antibodies by ELISA and for trypanolytic activity against cell-derived trypomastigotes using an in vitro functional assay. Cellular immune responses were evaluated by measuring cytokine production, T cell activation, and memory T cell responses following in vitro re-stimulation with the vaccine antigen or T. cruzi antigens. Results: N-Tc52/TSKb20 vaccination induced a sustained antigen-specific humoral response, characterized by long-lasting IgG2c antibodies and functional activity persisting for up to 170 days post-immunization. In parallel, vaccination promoted long-term activation of antigen-specific CD8⁺ T cells and production of TNF-α and IFN-γ upon antigen re-encounter. A sex-dependent tendency was observed for IL-10, with increased production in vaccinated female mice. Moreover, vaccinated animals exhibited increased frequencies of central and effector memory CD4⁺ and CD8⁺ T cells in response to T. cruzi antigens, with a predominant contribution of CD8⁺ T cells, indicating the establishment of parasite-specific T cell memory. Conclusions: Together, these findings demonstrate that vaccination with N-Tc52/TSKb20 induces a long-lasting Th1-biased immune response characterized by trypanolytic antibodies, functional and durable T cell responses, and parasite-specific memory T cells. This immunological profile supports the potential of N-Tc52/TSKb20 as a promising vaccine candidate for Chagas disease and highlights its capacity to elicit immune mechanisms that have been associated with protection against T. cruzi infection.

American tegumentary leishmaniasis (ATL) remains a therapeutic challenge due to the toxicity and limited effectiveness of conventional agents. This study evaluated Enterococcus mundtii CRL35, a food-grade lactic acid bacterium, as a dual-mechanism therapeutic platform against Leishmania (L.) amazonensis. Initial screening identified E. mundtii CRL35 as the most active strain among four enterococci tested, demonstrating high selectivity against promastigotes (SI = 95) and intracellular amastigotes (SI = 25). In a BALB/c murine model of cutaneous leishmaniasis, three treatment modalities were evaluated: topical application of cell-free supernatant (CFS-T), oral viable bacteria (LB-O), and their combination (CFS-T+LB-O). All three treatments achieved significant reductions in lesion parasite burden (approximately 2–3 orders of magnitude versus untreated controls), substantially exceeding the parasitological efficacy of amphotericin B under identical experimental conditions. CFS-T and CFS-T+LB-O additionally produced significant macroscopic lesion improvement. Importantly, no treatment was associated with adverse changes in organ indices or serum biochemical parameters. E. mundtii CRL35-based treatments promoted higher IgG2a/IgG1 antibody ratios compared to untreated controls and amphotericin B, generating an immune profile more favorable for intracellular parasite control. Taken together, these findings support the therapeutic potential of these modalities as safe, effective, and affordable alternatives for ATL, particularly in resource-limited endemic regions where toxicity and logistical barriers limit access to conventional therapy.