Deep serological profiling of the Trypanosoma cruzi TSSA antigen reveals different epitopes and modes of recognition by Chagas disease patients

BackgroundTrypanosoma cruzi, the agent of Chagas disease, displays a highly structured population, with multiple strains that can be grouped into 6-7 evolutionary lineages showing variable eco-epidemiological traits and likely also distinct disease-associated features. Previous works have shown that antibody responses to 'isoforms' of the polymorphic parasite antigen TSSA enable robust and sensitive identification of the infecting strain with near lineage-level resolution. To optimize the serotyping performance of this molecule, we herein used a combination of immunosignaturing approaches based on peptide microarrays and serum samples from Chagas disease patients to establish a deep linear B-cell epitope profiling of TSSA. Methods/Principle findingsOur assays revealed variations in the seroprevalence of TSSA isoforms among Chagas disease populations from different settings, hence strongly supporting the differential distribution of parasite lineages in domestic cycles across the Americas. Alanine scanning mutagenesis and the use of peptides of different lengths allowed us to identify key residues involved in antibody pairing and the presence of three discrete B-cell linear epitopes in TSSAII, the isoform with highest seroprevalence in human infections. Comprehensive screening of parasite genomic repositories led to the discovery of 9 novel T. cruzi TSSA variants and one TSSA sequence from the phylogenetically related bat parasite T. cruzi marinkellei. Further residue permutation analyses enabled the identification of diagnostically relevant or non-relevant substitutions among TSSA natural polymorphisms. Interestingly, T. cruzi marinkellei TSSA displayed specific serorecognition by one chronic Chagas disease patient from Colombia, which warrant further investigations on the diagnostic impact of such atypical TSSA. Conclusions/SignificanceOverall, our findings shed new light into TSSA evolution, epitope landscape and modes of recognition by Chagas disease patients; and have practical implications for the design and/or evaluation of T. cruzi serotyping strategies. Author summaryChagas disease, caused by the protozoan Trypanosoma cruzi, is a chronic, debilitating illness of major significance in Latin America. Due to the extensive genetic and phenotypic variability of this parasite, methods able to reliably assign the infecting strain type are expected to have a positive effect on epidemiologic surveillance, prevention of transmission, and clinical management of Chagas disease. Herein we describe the diversity of human antibody specificities directed to a polymorphic T. cruzi antigen, TSSA, using a combination of bioinformatics and state of the art immunoassays based on peptide arrays. Our results led to the identification of novel variants of the antigen, distinct epitopes, and their key residues for antibody binding across diverse human populations and have practical implications for the design and/or evaluation of T. cruzi serotyping strategies.