Identificação de epitopos lineares de célula B conservados e polimórficos em diferentes cepas de Trypanosoma cruzi com potencial aplicação para sorotipagem

Abstract

During chronic phase of Chagas disease, because of the low parasitemia and high anti-T.cruzi antibody titers, the diagnosis is made preferably by serological tests. However, contradictory or inconclusive results mainly related to the characteristics of the antigens used are observed. The factors influencing the variation in the clinical manifestation of Chagas disease have not been elucidated, but it is likely that genetic of the host and parasite is involved. Several studies trying to correlate the parasite strain and clinical manifestation have used hemoculture or PCR-based genotyping. However, both techniques have limitations. The goal of this work is to identify T. cruzi conserved and polymorphic B-cell linear epitopes that could be used for serodiagnostic and serotyping using ELISA. We have performed B-cell epitope prediction on proteins derived from single copy genes represented by pair of alleles in the CL Brener genome. The rationale behind this strategy is that because CL Brener is a recent hybrid between TcII and TcIII lineages, it is likely that polymorphic epitopes in its pair of alleles could also be polymorphic in the parental genotypes. The reactivity of 150 peptides has been tested using sera from C57BL/6 mice chronically infected with Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain. A total of 36 peptides were reactive and the cross-reactivity between the strains is in agreement with the evolutionary origin of the different T. cruzi lineages. Two peptides similarly reactive with different strains and three with reactivity specific for each strain were synthesized in solution and tested in ELISA and ELISA affinity experiments. One of the peptides with similar reactivity showed no statistical difference between the strains and was recognized by intermediate affinity antibodies with 93% of specificity and sensitivity of 87%. This peptide was also differentially recognized by sera from chagasic patients with the cardiac form compared with serum from patients with the indeterminate form of the disease. In addition to the serodiagnosis, it could be used to prognosis or to aid in the identification of clinical manifestations. The other peptide had low specificity (35%) and sensitivity (58%) values . The peptide reactive with the Y strain was also reactive with sera from patients infected with TcII and the amino acid critical for recognition by antibodies were identified only in the allele of haplotype Esmo. Conversely, the peptide reactive only with Colombia was not recognized by sera from patients infected with TcII and mapping of the amino acids showed that only the allele of Non-esmo haplotype has the pattern of antibodies binding. The last peptide correctly discriminated strain CL Brener. The three peptides were derived from polymorphic repetitive regions and together had a high potential use for serotyping of the parasite.