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.