Plasma-Derived Extracellular Vesicles as Potential Biomarkers in Heart Transplant Patient with Chronic Chagas Disease

Chagas disease is emerging in countries to which it is not endemic. Biomarkers for earlier therapeutic response assessment in patients with chronic Chagas disease are needed. We profiled plasma-derived extracellular vesicles from a heart transplant patient with chronic Chagas disease and showed the potential of this approach for discovering such biomarkers.

(Biokit, https://www.biokit.com). Together with clinical management of dysphagia and constipation, a pacemaker in the context of third-degree atrioventricular block was implanted. In July 2015, an echocardiogram revealed iterative cardiac failure and severe ventricular dysfunction (ejection fraction 15%-20%). On November 28, 2015, the patient underwent heart transplantation without incident, and results of followup endomyocardial biopsies showed no early signs of transplant rejection.
After transplantation and in the context of immunosuppression therapy (Table 1), quantitative PCR (qPCR) was performed weekly to detect T. cruzi in the blood (Tc-qPCR) (7). First benznidazole treatment was started when several consecutive and positive Tc-qPCRs confirmed Chagas disease reactivation. Three weeks after benznidazole treatment, the Tc-qPCR became negative. After completion of 80% of the treatment, bronchopulmonary aspergillosis developed, and the benznidazole course was interrupted. The Tc-qPCR became positive and a second benznidazole course was initiated; this time the patient completed the initial prescribed dose without evidence of therapeutic failure based on Tc-qPCR results. Plasma samples for purification and characterization of EVs were collected before the first benznidazole treatment and just after the second course (Table 1). Unexpectedly, the patient died in August 2016 because of a late organ rejection. Therefore, samples at 6 and 12 months posttreatment, already included in the approved protocol, were not collected.
To determine whether circulating EVs from this patient could have been used as predictive biomarkers to evaluate therapeutic response and disease outcome in the Chagas disease context, we collected pretreatment and posttreatment plasma samples, and EVs were enriched by size-exclusion chromatography (SEC) and characterized as described (8) ( Figure  1, panel A). As negative controls, plasma samples from 2 healthy donors were also subjected to SEC. We characterized eluting EVs by bead-based assay and Nanoparticle Tracking Analysis ( Figure 1, panels B, C). We pooled aliquots (100 µL) from SEC fractions 7-10 and determined protein composition using 2Dliquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). In brief, samples were digested with trypsin and resulting peptides were resolved by high-pH reversed-phase peptide fractionation (9), followed by C18 reversed-phase nanoflow ultrahighperformance liquid chromatography coupled to a Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (QE Plus MS; Thermo Fisher Scientific, https://www.thermofisher.com), as described (10). Raw 2D-LC-MS/MS data were analyzed using Proteome Discoverer version 2.1.1.21 software (Thermo Fisher Scientific), followed by Scaffold perSPECtives version 4.8.7 (Proteome Software; http://www.proteomesoftware.com). A protein database with combined human, T. cruzi, and potential contaminants was generated from UniProt (https://www.uniprot. org). Using a false-discovery rate <1% and 1 unique peptide per protein, we identified 12 T. cruzi proteins and 338 human proteins (Appendix, https:// wwwnc.cdc.gov/EID/article/26/8/19-1042-App1. xlsx). However, when we applied the more stringent criterion of >2 unique peptides per protein, we detected only 1 T. cruzi protein (i.e., pyruvate phosphate dikinase [PPDK]), and 288 human proteins, of which we identified 19 only in pretreatment samples ( Table 2). PPDK has been identified by proteomic  (6), and/or the combined use of posaconazole and benznidazole because a second round of the latter was started in April 2016.
analysis of T. cruzi total secretome and EVs (10)(11)(12). This protein plays a central role in the metabolism of T. cruzi glycosomes and has been shown to be upregulated when trypomastigote forms are incubated with the extracellular matrix, an obligatory step before host-cell invasion and differentiation of trypomastigote into amastigote forms (13). The specific role of PPDK in EVs secreted by this patient remains to be determined. Among the 19 human proteins uniquely identified in EVs from the patient with chronic Chagas disease before treatment, the mannan binding lectin serine protease 2 (MASP2) is worth highlighting. A recent study with human samples showed that MASP2 gene polymorphisms and MASP2 levels are associated with high risk for chronic Chagas disease cardiomyopathy (14). Furthermore, mannose-binding lectin, which activates complement on T. cruzi through MASP2, has been related to a decrease in blood and tissue parasite load and in myocarditis and cardiac fibrosis in experimental T. cruzi infection (15). In this study, mRNA levels of collagen-1 and -6 increased in the infected animals' hearts (15). These results could support our findings because collagen α-1 is one of the proteins identified exclusively in EVs before patient treatment ( Table 2).
Another important observation is the identification of a higher number of human proteins in patient-derived EVs than in the 2 healthy donor-derived EV samples (Figure 2; Appendix). Of the total proteins identified, in which statistical analysis was feasible, 4 were significantly upregulated in patientderived EVs before treatment, particularly for the proteins complement C1s subcomponent, isoform CRA_b, FLJ00385 protein, and cDNA FLJ75416 (Appendix). Complement C1s subcomponent recently was identified among the 6 upregulated EV biomarkers with potential for clinical applications in myocardial infarction (3).

Conclusions
Proteins associated with EVs secreted by T. cruzi have been identified in the conditioned medium of different parasite stages (11-13) but not in biofluids from Chagas disease patients. We described the proteomic profiling of plasma-derived EVs purified directly from a heart transplant patient with chronic Chagas disease who exhibited reactivation after immunosuppression. We identified human and parasite proteins present or upregulated in plasmaderived EVs from a chronic Chagas disease patient before chemotherapy and that are absent or downregulated after treatment. We thus hypothesize that EV proteins released by the host or parasite during infection might be potential biomarker candidates for evaluating therapeutic response and disease outcome in chronic Chagas disease, independently of the immunologic status of patients. Table 2. Trypanosoma cruzi and human proteins identified in plasma-derived EVs from a heart transplant patient with chronic Chagas disease before benznidazole chemotherapy but absent after treatment and in healthy donors However, our results should be interpreted with caution because they represent a single clinical case. Further research is needed to validate and provide stronger evidence that circulating EVs in patients with chronic Chagas disease can serve as biomarkers in disease progression and early assessment of therapeutic outcomes. Moreover, the future incorporation of such validated biomarkers in a point-of-care device could help in the detection of very low parasites in circulation, particularly when concentrations are below the PCR detection level (2).

Acknowledgments
We thank the patient and healthy donors who participated in this study. We are particularly grateful to Hernando A. del Portillo for helpful discussions and support throughout this project and to Marc Nicolau for technical assistance. Human proteomic profile of plasma-derived EVs from a heart transplant patient with chronic Chagas disease before and after BZN treatment and from 2 healthy donors. Heatmap of the identified human EV-derived proteins. Heatmap was generated from proteomic data by the Scaffold perSPECtives software (Proteome Software; http://www.proteomesoftware.com). Hierarchical clustering (on the left), using single-linkage clustering with a Euclidean distance metric, was performed based on the normalized weighted spectrum counts of the identified proteins. Color gradient scale bar (Y-axis) indicates normalized spectrum counts (numerical matrix) translated into a color image, as described in the Scaffold perSPECtives version 3.0 User's Manual (http://www.proteomesoftware.com/ pdf/scaffold_perspectives_users_guide.pdf?v=232de555). More human proteins were found in EVs isolated from the patient, before and after BZN treatment than from EVs derived from the 2 healthy donors (H1, H2). BZN, benznidazole; EV, extracellular vesicle. Program), Generalitat de Catalunya. Work in the laboratory of C.F.B. is funded by Fundació La Marató de TV3 (reference 566/U/2018) and Fundación Mundo Sano. This project was co-financed by the European Union through the European Regional Development Fund with the support of Secretaria d'Universitats i Recerca del Departament d'Empresa i Coneixement de la Generalitat de Catalunya. N.C., M.G., J.G., and M.J.P. receive funds from the Redes temáticas de investigación cooperativa en salud (RETICS), Spanish Tropical Diseases Network "RD12/0018/0010" and from the Agencia de Gestió d'Ajuts Universitaris i de Recerca, Generalitat de Catalunya; grant "2017 SGR 00924." M.G., C.B., J.G., M.J.P., and I.C.A. belong to the Ibero-American Nuevas Herramientas para el Diagnóstico y la Evaluación del Paciente con Enfermedad de Chagas network. I.C.A. is partially supported by grants no. 2G12MD007592 and 5U54MD007592 from the National Institute on Minority Health and Health Disparities of the US National Institutes of Health. We are grateful to the Biomolecule Analysis Core Facility at University of Texas at El Paso, Border Biomedical Research Center, funded by National Institute on Minority Health and Health Disparities grants 2G12MD007592 and 5U54MD007592. M.T.M. received a PhD fellowship from the Science Without Borders Program, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil.