Rational <i>in silico</i> discovery and serological validation of <i>Trypanosoma cruzi</i> -specific B-cell epitopes for high-precision Chagas disease diagnosis

Abstract Chagas disease is caused by the parasite Trypanosoma cruzi and remains a neglected tropical disease presenting a substantial global health burden. Crude antigen-based assays have historically been limited in specificity; however, even contemporary recombinant-antigen tests may exhibit residual cross-reactivity, depending on antigen composition and geographic context. To overcome this limitation, this study developed a novel diagnostic strategy that integrates computational and experimental approaches to identify specific linear B-cell epitopes within the T. cruzi proteome. The strategy was developed to exclude sequences homologous to H. sapiens and Leishmania spp. proteins, thereby minimizing potential cross-reactivity. Using a consensus approach across five prediction algorithms, B-cell epitopes were identified and subsequently clustered to reveal conserved, immunoreactive consensus sequences. The peptide sequences were characterized for optimal physicochemical properties and subsequently modeled to interact with a human antibody using protein-peptide docking and molecular dynamics simulations to assess complex stability. The most promising candidates were chemically synthesized and validated using ELISA against a cohort comprising Chagas disease patients (chronic indeterminate and cardiac forms), healthy donors, and a cross-reactive control group (visceral and tegumentary leishmaniasis and leprosy). From the initial set of 19,245 proteins, the multi-tiered bioinformatic analysis identified 4,431 unique, non-homologous sequences. Consensus prediction yielded 401 high-confidence epitopes, which were refined to 179 structurally stable candidates. Computational analyses identified five top-ranking epitopes capable of forming high-affinity, stable complexes with a human antibody. Experimental validation confirmed the high diagnostic accuracy of two epitopes, which demonstrated exceptional diagnostic performance: Epitope 4 and Epitope 5 achieved 100% sensitivity. Notably, Epitope 5 exhibited superior specificity, reaching 96.67% against healthy controls and 90.91% against the cross-reactive group. This study establishes a basis for the development of an improved immunoassay for Chagas disease and provides a reproducible framework for targeted epitope discovery. Consequently, this study validates a high-precision computational pipeline capable of discovering T. cruzi -specific antigens that effectively circumvent cross-reactivity with Leishmania spp., proposing Epitope 5 as a qualified candidate for reliable serological diagnosis in co-endemic regions.