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Here, we report the design and in silico validation of a novel multiepitope vaccine construct targeting the key surface proteins ASP\u20102 and gp82. Using a comprehensive immunoinformatics pipeline, we identified and selected 38 potent T\u2010cell (CTL\/HTL) and B\u2010cell 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\u2010allergenic and non\u2010toxic, 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\u2010affinity interactions with the immune receptors TLR2 and TLR4 (docking scores: \u22121360.4 and \u22121278.7, respectively). The stability and flexibility of these vaccine\u2013receptor complexes were further validated by 300\u00a0ns molecular dynamics simulations. Finally, codon optimization and in silico cloning projected high expression potential in an\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    system. Immune simulations predicted robust responses: Innate (elevated cytokines, dendritic cell activation), humoral (IgG\/IgM production), and cellular (CD4+\/CD8+\/memory T\u2010cell 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\n                    <jats:italic>T. cruzi<\/jats:italic>\n                    .\n                  <\/jats:p>","DOI":"10.1002\/bab.70185","type":"journal-article","created":{"date-parts":[[2026,5,30]],"date-time":"2026-05-30T05:07:07Z","timestamp":1780117627000},"page":"2457-2481","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Targeting Amastigote and Trypomastigote Phases: Multi\u2010Epitope Vaccine Strategy Against\n                    <i>Trypanosoma cruzi<\/i>"],"prefix":"10.1002","volume":"73","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9497-5164","authenticated-orcid":false,"given":"Maria Karolaynne da","family":"Silva","sequence":"first","affiliation":[{"name":"Department of Biophysics and Pharmacology, Bioscience Center Federal University of Rio Grande Do Norte  Natal Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caio Patr\u00edcio de Souza","family":"Sena","sequence":"additional","affiliation":[{"name":"Chemistry Institute Federal University of Rio Grande Do Norte  Natal Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Davi Serradella","family":"Vieira","sequence":"additional","affiliation":[{"name":"Chemistry Institute Federal University of Rio Grande Do Norte  Natal Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Magdi E. 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