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

BACKGROUND: Trypanosoma 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 FINDINGS: Our 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/SIGNIFICANCE: Overall, 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.

The Trypanosoma cruzi Antigen and Epitope Atlas: antibody specificities in Chagas disease patients across the Americas.

During an infection the immune system produces pathogen-specific antibodies. These antibody repertoires become specific to the history of infections and represent a rich source of diagnostic markers. However, the specificities of these antibodies are mostly unknown. Here, using high-density peptide arrays we examined the human antibody repertoires of Chagas disease patients. Chagas disease is a neglected disease caused by Trypanosoma cruzi, a protozoan parasite that evades immune mediated elimination and mounts long-lasting chronic infections. We describe a proteome-wide search for antigens, characterised their linear epitopes, and show their reactivity on 71 individuals from diverse human populations. Using single-residue mutagenesis we revealed the core functional residues for 232 of these epitopes. Finally, we show the diagnostic performance of identified antigens on challenging samples. These datasets enable the study of the Chagas antibody repertoire at an unprecedented depth and granularity, while also providing a rich source of serological biomarkers.

APRANK: Computational Prioritization of Antigenic Proteins and Peptides From Complete Pathogen Proteomes.

Availability of highly parallelized immunoassays has renewed interest in the discovery of serology biomarkers for infectious diseases. Protein and peptide microarrays now provide a rapid, high-throughput platform for immunological testing and validation of potential antigens and B-cell epitopes. However, there is still a need for tools to prioritize and select relevant probes when designing these arrays. In this work we describe a computational method called APRANK (Antigenic Protein and Peptide Ranker) which integrates multiple molecular features to prioritize potentially antigenic proteins and peptides in a given pathogen proteome. These features include subcellular localization, presence of repetitive motifs, natively disordered regions, secondary structure, transmembrane spans and predicted interaction with the immune system. We trained and tested this method with a number of bacteria and protozoa causing human diseases: Borrelia burgdorferi (Lyme disease), Brucella melitensis (Brucellosis), Coxiella burnetii (Q fever), Escherichia coli (Gastroenteritis), Francisella tularensis (Tularemia), Leishmania braziliensis (Leishmaniasis), Leptospira interrogans (Leptospirosis), Mycobacterium leprae (Leprae), Mycobacterium tuberculosis (Tuberculosis), Plasmodium falciparum (Malaria), Porphyromonas gingivalis (Periodontal disease), Staphylococcus aureus (Bacteremia), Streptococcus pyogenes (Group A Streptococcal infections), Toxoplasma gondii (Toxoplasmosis) and Trypanosoma cruzi (Chagas Disease). We have evaluated this integrative method using non-parametric ROC-curves and made an unbiased validation using Onchocerca volvulus as an independent data set. We found that APRANK is successful in predicting antigenicity for all pathogen species tested, facilitating the production of antigen-enriched protein subsets. We make APRANK available to facilitate the identification of novel diagnostic antigens in infectious diseases.

Serological Approaches for Trypanosoma cruzi Strain Typing.

Trypanosoma cruzi, the protozoan agent of Chagas' disease, displays a complex population structure made up of multiple strains showing a diverse ecoepidemiological distribution. Parasite genetic variability may be associated with disease outcome, hence stressing the need to develop methods for T. cruzi typing in vivo. Serological typing methods that exploit the presence of host antibodies raised against polymorphic parasite antigens emerge as an appealing approach to address this issue. These techniques are robust, simple, cost-effective, and are not curtailed by methodological/biological limitations intrinsic to available genotyping methods. Here, we critically assess the progress towards T. cruzi serotyping and discuss the opportunity provided by high-throughput immunomics to improve this field.