Targeting Amastigote and Trypomastigote Phases: Multi‐Epitope Vaccine Strategy Against <i>Trypanosoma cruzi</i>

Effective vaccines against Trypanosoma cruzi, the causative agent of Chagas disease, are urgently needed. Here, we report the design and in silico validation of a novel multiepitope vaccine construct targeting the key surface proteins ASP-2 and gp82. Using a comprehensive immunoinformatics pipeline, we identified and selected 38 potent T-cell (CTL/HTL) and B-cell epitopes, ensuring high antigenicity, immunogenicity, and safety. The final chimeric protein, integrated with adjuvants and stabilizing linkers, demonstrated favorable physicochemical properties, high solubility, and was predicted to be non-allergenic and non-toxic, with a significant population coverage of approximately 62% in Latin America. Structural modeling and refinement confirmed a stable tertiary structure. Crucially, molecular docking predicted high-affinity interactions with the immune receptors TLR2 and TLR4 (docking scores: -1360.4 and -1278.7, respectively). The stability and flexibility of these vaccine-receptor complexes were further validated by 300 ns molecular dynamics simulations. Finally, codon optimization and in silico cloning projected high expression potential in an Escherichia coli system. Immune simulations predicted robust responses: Innate (elevated cytokines, dendritic cell activation), humoral (IgG/IgM production), and cellular (CD4+/CD8+/memory T-cell activation) across simulated doses. These findings strongly support the potential of this vaccine candidate and provide a solid foundation for its further preclinical development against T. cruzi.