Characterization of Novel Trypanosoma cruzi-Specific Antigen with Potential Use in the Diagnosis of Chagas Disease.

Chagas disease is caused by the parasite Trypanosoma cruzi. In humans, it evolves into a chronic disease, eventually resulting in cardiac, digestive, and/or neurological disorders. In the present study, we characterized a novel T. cruzi antigen named Tc323 (TcCLB.504087.20), recognized by a single-chain monoclonal antibody (scFv 6B6) isolated from the B cells of patients with cardiomyopathy related to chronic Chagas disease. Tc323, a ~323 kDa protein, is an uncharacterized protein showing putative quinoprotein alcohol dehydrogenase-like domains. A computational molecular docking study revealed that the scFv 6B6 binds to an internal domain of Tc323. Immunofluorescence microscopy and Western Blot showed that Tc323 is expressed in the main developmental forms of T. cruzi, localized intracellularly and exhibiting a membrane-associated pattern. According to phylogenetic analysis, Tc323 is highly conserved throughout evolution in all the lineages of T. cruzi so far identified, but it is absent in Leishmania spp. and Trypanosoma brucei. Most interestingly, only plasma samples from patients infected with T. cruzi and those with mixed infection with Leishmania spp. reacted against Tc323. Collectively, our findings demonstrate that Tc323 is a promising candidate for the differential serodiagnosis of chronic Chagas disease in areas where T. cruzi and Leishmania spp. infections coexist.

Expression of Inhibitory Receptors TIGIT, TIM-3, and LAG-3 on CD4+ T Cells from Patients with Different Clinical Forms of Chronic Chagas Disease.

T cells are central to the adaptive immune response against Trypanosoma cruzi infection. In chronic Chagas disease (CCD), circulating parasite-specific memory T cells show reduced functionality and increased expression of inhibitory receptors as a result of persistent antigenic stimulation. This phenotype has been linked to progression of cardiac pathology, whereas the presence of polyfunctional T cells shows association with therapeutic success. In this study, we demonstrate that T. cruzi-specific human CD4+ T cells can be identified by their expression of OX40 and CD25 upon in vitro stimulation. We characterized the expression of the inhibitory receptors T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell Ig and mucin-domain containing-3 (TIM-3), and lymphocyte activation gene 3 (LAG-3) in CD4+ T cells from CCD patients with and without cardiac alterations. Our results show that, independently of their clinical stage, CCD patients present an increased frequency of CD4+ T cells expressing TIGIT in comparison with non-T. cruzi-infected donors. Exposure to parasite Ags increases the expression of TIM-3 in CD4+ T cells from CCD patients, especially in those with cardiac compromise. Upregulation of LAG-3 was also detected in CCD individuals without cardiac manifestations, predominantly within the subpopulation of cells that did not become activated upon stimulation. Further differences were found between groups in the coexpression of these receptors. Blockade of each individual receptor did not affect activation or the production of IFN-γ and IL-10 by CD4+ T cells in response to parasite Ags. Our results suggest a role for TIGIT, TIM-3, and LAG-3 in the modulation of inflammatory phenomena thought to ultimately lead to tissue damage and cardiac pathology.

Trypanosoma cruzi Induces Regulatory B Cell Alterations in Patients With Chronic Chagas Disease.

The clinical evolution of patients with chronic Chagas disease (CCD) is mainly associated with an excessive inflammation and a defective immunomodulatory profile caused by the interaction between T. cruzi and the host. Regulatory B (Breg) cells exert immune suppression mostly through IL-10 production (B10 cells), but also through IL-10-independent mechanisms. Previously, we demonstrated that CCD patients with cardiomyopathy show changes in the ex vivo Breg cell phenotypic distribution although maintain IL-10 production capacity. Here, we sought to identify potential alterations on Breg cells upon in vitro stimulation. Isolated B cells from CCD patients with or without cardiomyopathy and non-infected (NI) donors were stimulated with T. cruzi lysate or CpG + CD40L, and characterized by flow cytometry based on the expression of CD24, CD27, CD38, and the regulatory molecules IL-10 and PD-L1. IL-10 and IL-17 secretion in the supernatant of B cells was evaluated by ELISA. Data showed that T. cruzi stimulation diminished the expression of CD24 and CD38 on CD27- B cells while reducing the percentage of CD24high inside CD27+ B cells. Furthermore, T. cruzi induced a regulatory B cell phenotype by increasing B10 cells and IL-10 secretion in all the groups. The innate-like B10 cells expansion observed in patients with cardiomyopathy would be associated with CD27- B10 cell subsets, while no predominant phenotype was found in the other groups. Patients with cardiomyopathy also displayed higher IL-17 secretion levels in T. cruzi-activated B cells. CpG + CD40L stimulation revealed that B cells from CCD patients and NI donors had the same ability to differentiate into B10 cells and secrete IL-10 in vitro. Additionally, CCD patients showed an increased frequency of CD24-CD27- B cells and a reduction in the percentage of CD24highCD27+ Breg cells, which appeared to be inversely correlated with the presence of T. cruzi DNA in blood. Finally, CCD patients exhibited a higher frequency of PD-L1+ B cells in T. cruzi-stimulated samples, suggesting that IL-10-independent mechanisms could also be tangled in the control of inflammation. Altogether, our results provide evidence about the potential role of Breg cells in the immune response developed against T. cruzi and its contribution to chronic Chagas cardiomyopathy.

Ex vivo characterization of Breg cells in patients with chronic Chagas disease.

Despite the growing importance of the regulatory function of B cells in many infectious diseases, their immunosuppressive role remains elusive in chronic Chagas disease (CCD). Here, we studied the proportion of different B cell subsets and their capacity to secrete IL-10 ex vivo in peripheral blood from patients with or without CCD cardiomyopathy. First, we immunophenotyped peripheral blood mononuclear cells from patients according to the expression of markers CD19, CD24, CD38 and CD27 and we showed an expansion of total B cell and transitional CD24highCD38high B cell subsets in CCD patients with cardiac involvement compared to non-infected donors. Although no differences were observed in the frequency of total IL-10 producing B cells (B10) among the groups, CCD patients with cardiac involvement showed an increased proportion of naïve B10 cells and a tendency to a higher frequency of transitional B10 cells compared to non-infected donors. Our research demonstrates that transitional B cells are greatly expanded in patients with the cardiac form of CCD and these cells retain the ability to secrete IL-10. These findings provide insight into the phenotypic distribution of regulatory B cells in CCD, an important step towards new strategies to prevent cardiomyopathy associated with T. cruzi infection.

Recombinant antibody against Trypanosoma cruzi from patients with chronic Chagas heart disease recognizes mammalian nervous system.

BACKGROUND: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients. METHODS: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target´s orthologues, focusing mainly on post-translational modifications. FINDINGS: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system. INTERPRETATION: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection.

In Silico Guided Discovery of Novel Class I and II Trypanosoma cruzi Epitopes Recognized by T Cells from Chagas' Disease Patients.

T cell-mediated immune response plays a crucial role in controlling Trypanosoma cruzi infection and parasite burden, but it is also involved in the clinical onset and progression of chronic Chagas' disease. Therefore, the study of T cells is central to the understanding of the immune response against the parasite and its implications for the infected organism. The complexity of the parasite-host interactions hampers the identification and characterization of T cell-activating epitopes. We approached this issue by combining in silico and in vitro methods to interrogate patients' T cells specificity. Fifty T. cruzi peptides predicted to bind a broad range of class I and II HLA molecules were selected for in vitro screening against PBMC samples from a cohort of chronic Chagas' disease patients, using IFN-γ secretion as a readout. Seven of these peptides were shown to activate this type of T cell response, and four out of these contain class I and II epitopes that, to our knowledge, are first described in this study. The remaining three contain sequences that had been previously demonstrated to induce CD8+ T cell response in Chagas' disease patients, or bind HLA-A*02:01, but are, in this study, demonstrated to engage CD4+ T cells. We also assessed the degree of differentiation of activated T cells and looked into the HLA variants that might restrict the recognition of these peptides in the context of human T. cruzi infection.

Evaluation of the immune response against Trypanosoma cruzi cytosolic tryparedoxin peroxidase in human natural infection.

Trypanosoma cruzi, the aetiological agent of Chagas disease, has a highly efficient detoxification system to deal with the oxidative burst imposed by its host. One of the antioxidant enzymes involved is the cytosolic tryparedoxin peroxidase (c-TXNPx), which catalyses the reduction to hydrogen peroxide, small-chain organic hydroperoxides and peroxynitrite. This enzyme is present in all parasite stages, and its overexpression renders parasites more resistant to the oxidative defences of macrophages, favouring parasite survival. This work addressed the study of the specific humoral and cellular immune response triggered by c-TXNPx in human natural infection. Thus, sera and peripheral blood mononuclear cells (PBMC) were collected from chronically infected asymptomatic and cardiac patients, and non-infected individuals. Results showed that levels of IgG antibodies against c-TXNPx were low in sera from individuals across all groups. B-cell epitope prediction limited immunogenicity to a few, small regions on the c-TXNPx sequence. At a cellular level, PBMC from asymptomatic and cardiac patients proliferated and secreted interferon-γ after c-TXNPx stimulation, compared with mock control. However, only proliferation was higher in asymptomatic patients compared with cardiac and non-infected individuals. Furthermore, asymptomatic patients showed an enhanced frequency of CD19+ CD69+ cells upon exposure to c-TXNPx. Overall, our results show that c-TXNPx fails to induce a strong immune response in natural infection, being measurable only in those patients without any clinical symptoms. The low impact of c-TXNPx in the human immune response could be strategic for parasite survival, as it keeps this crucial antioxidant enzyme activity safe from the mechanisms of adaptive immune response.