Immunogenomic screening approach to identify new antigens for the serological diagnosis of chronic Chagas’ disease

Abstract

Serological tests are preferentially used for the diagnosis of Chagas' disease (CD) during the chronic phase because of the low parasitemia and high anti-Trypanosoma cruzi antibody titers. However, the current methods showed several disadvantages, as contradictory or inconclusive results, mainly related to the characteristics of the antigens used, in general, crude or whole parasites, but also due to antigen production protocol and the experimental conditions used in serological tests. Thus, better-quality serological assays are urgently needed. Here, we performed a wide immunogenomic screen strategy to identify conserved linear B-cell epitopes in the predicted proteome based on genome sequence from T. cruzi strains to will be applied as synthetic peptides in the serodiagnosis of the chronic CD. Three B-cell epitopes derived from mucin-associated surface protein (MASP) family, expressed in both infective parasite stages, trypomastigote and amastigotes, conserved in T. cruzi strains, and highly divergent as compared with Leishmania spp. proteome, were selected for this study. The results demonstrated that synthetic peptide 2 and a mixture of peptides (Mix II: peptides 2 and 3) were able to identify all chronic CD cases, indeterminate or Chagas cardiomyopathy clinical presentation, and simultaneously able to discriminate infections caused by Leishmania parasites, with high accuracy (98.37 and 100.00%, respectively) and agreement (kappa index = 0.967 and 1.000, respectively) with direct methods as compared to current diagnostic pipeline performed by reference laboratories in Brazil. This study represents an interesting strategy for the discovery of new antigens applied to serologic diagnosis of infectious diseases and for the technological development of platforms for large-scale production of diagnostic tests.

Antibody epitope repertoire analysis enables rapid antigen discovery and multiplex serology

“…Here, Chagas-specific motifs mapped to distinct candidate antigens. Several of these antigens have been reported 25 , while others have not been described previously. Approaches to antigen discovery from biospecimens often require laborious antigen cloning, expression, purification, and reactivity testing 26 .…”

New antigens for the serological diagnosis of human visceral leishmaniasis identified by immunogenomic screening

“…In order to solve the problems of sensitivity and/or specificity, as described above, studies has been carried out to identify new antigens candidates based on experimental approaches evaluating in silico prediction data by immunoinformatics and published genomes [27, 45]. This strategy allows the rational search for antigenic proteins, as well as to determine the regions of these targets that determine the antigenicity and specificity for the recognition of the targets by sera from patients [27, 48].…”

“…In fact, evaluating peptides ELISA assays, high sensitivity values were observed, that ranging from 82.86–100.00%, included peptide mixtures (Mix I, II, III and IV), and can be explained by high expression in intracellular stages, subcellular localization (secreted/excreted) and high antigenicity demonstrated by B-cell prediction. In according, previous studies have demonstrated that proteins expressed in stages found in vertebrate host are possibly associated with with the infection process and/or the intracellular survival of the parasite within the host, and may be attractive diagnostic targets [30, 45, 49]. Serological tests have also presented limitations regarding the absence of correlation between the antibody levels and the current stage of the disease [21, 50].…”

“…infantum and H . sapiens proteins, which avoid the possibility of the targets being similar to the host's self-antigens, guaranteeing a higher production of antibodies and, consequently, greater sensitivity [45].…”

“…A mixture containing all peptides in the same proportion (Mix II: 1+2+3+4+5+6; 1.66 μg for each one/well) was evaluated. The ELISA assay was carried out as described previously [45]. To eliminates the need of combining three or more peptides at a time, we also evaluated the combination between peptides 2 and 6 (Mix III: 2+6; 5.0 μg for each one/well) and peptides 3 and 6 (Mix IV: 3+6; 5.0 μg for each one/well).…”