Genome-wide screening and identification of new Trypanosoma cruzi antigens with potential application for chronic Chagas disease diagnosis.

The protozoan Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that afflicts approximately 8 million people in Latin America. Diagnosis of chronic Chagas disease is currently based on serological tests because this condition is usually characterized by high anti-T. cruzi IgG titers and low parasitemia. The antigens used in these assays may have low specificity due to cross reactivity with antigens from related parasite infections, such as leishmaniasis, and low sensitivity caused by the high polymorphism among T. cruzi strains. Therefore, the identification of new T. cruzi-specific antigens that are conserved among the various parasite discrete typing units (DTUs) is still required. In the present study, we have explored the hybrid nature of the T. cruzi CL Brener strain using a broad genome screening approach to select new T. cruzi antigens that are conserved among the different parasite DTUs and that are absent in other trypanosomatid species. Peptide arrays containing the conserved antigens with the highest epitope prediction scores were synthesized, and the reactivity of the peptides were tested by immunoblot using sera from C57BL/6 mice chronically infected with T. cruzi strains from the TcI, TcII or TcVI DTU. The two T. cruzi proteins that contained the most promising peptides were expressed as recombinant proteins and tested in ELISA experiments with sera from chagasic patients with distinct clinical manifestations: those infected with T. cruzi from different DTUs and those with cutaneous or visceral leishmaniasis. These proteins, named rTc_11623.20 and rTc_N_10421.310, exhibited 94.83 and 89.66% sensitivity, 98.2 and 94.6% specificity, respectively, and a pool of these 2 proteins exhibited 96.55% sensitivity and 98.18% specificity. This work led to the identification of two new antigens with great potential application in the diagnosis of chronic Chagas disease

Immunoblot of a peptide array with sera from C57BL/6 mice chronically infected with different <i>T. cruzi</i> strains.

<p>(A) Sera from mice infected with Colombiana (TcI); (B), Y (TcII) or (C), CL Brener (TcVI); or (D) uninfected mice.</p

Recognition of <i>r</i>Tc_11623.20 and <i>r</i>Tc_N_10421.310 by the sera from chronic chagasic patients or <i>Leishmania</i>-infected individuals.

<p>Sera from chronic chagasic patients and sera from patients with cutaneous and visceral leishmaniasis were assayed by ELISA using <i>r</i>Tc_11623.20 (A, D), <i>r</i>Tc_N_10421.310 (B, E) and pooled recombinant proteins (C, F). Tc-non typed, sera from chronic chagasic patients; TcII, sera from patients infected with <i>T. cruzi</i> TcII DTU; TcVI, sera from patients infected with <i>T. cruzi</i> TcVI DTU, Tc-Ind, sera from chagasic patients in the indeterminate form of Chagas disease; Tc-Card, sera from chagasic patients in the cardiac stage of Chagas disease; CL, sera from patients with cutaneous leishmaniasis; VL, sera from patients with visceral leishmaniasis; C-, uninfected individuals. Squares represent sera from chagasic patient in the initial cardiac stage. The dotted line represents the cutoff value that was obtained based on the ROC curve. The solid line corresponds to the mean values.</p

Recognition of <i>r</i>Tc_11623.20 and <i>r</i>Tc_N_10421.310 by sera from C57BL/6 mice chronically infected with different <i>T. cruzi</i> strains.

<p>Sera from mice chronically infected with CL Brener (CL-B), Colombiana (Col) or Y (Y) <i>T. cruzi</i> strains, mice infected with <i>T. rangeli</i> (T.rang) or uninfected mice (C-) were screened by ELISA with <i>r</i>Tc_11623.20 (A), <i>r</i>Tc_N_10421.310 (B) or with a pool of these two recombinant proteins (C). The dotted line represents the cutoff value that was obtained based on the ROC curve. The solid line corresponds to the mean values. Col, mice infected with Colombiana; Y, mice infected with the Y strain; CL-B, mice infected with the CL Brener; T. rang, <i>T. rangeli</i>-infected mice. C-, uninfected mice.</p

Predictions of B-cell linear epitopes and intrinsically unstructured/disordered regions in <i>r</i>Tc_11623.20 (A) and <i>r</i>Tc_N_10421.310 (B).

<p>The complete sequences of the recombinant proteins were submitted to the BepiPred and IUPred algorithms. The dashed arrow corresponds to the complete amino acid sequence of the recombinant proteins. The orange boxes indicate linear B-cell epitopes as predicted by BepiPred, and the gray boxes indicate unfolded regions that were predicted by IUPred. The epitope region that contains the peptide screened in the immunoblotting for each one of the proteins is highlighted by a blue box.</p