Field of the Art

The present invention relates to a method for predicting the therapeutic response and/or disease prognosis in a subject suffering Chagas disease. The invention also relates to the use of a protein expression product obtained from a biological sample of a chronic Chagas disease subject as potential biomarker in the context of said therapeutic response and prognosis.

Background of the Invention

Parasitic infections, which affect millions of people worldwide and are considered a serious public health concern, are the most common among infectious diseases. Furthermore, worldwide economic growth, climate changes, and migratory movements in recent years have contributed to the global resurgence of some parasitic infections.

Chagas disease, caused by Trypanosoma cruzi, is one of the most prevalent parasitic infections in Latin America and responsible for millions of clinical cases. However, mainly due to migratory movements, the epidemiology of Chagas disease has changed in recent decades with a significant increase of cases in nonendemic countries of North America, Europe, and Asia [1 ]. Thus, raising awareness of this debilitating or deadly neglected tropical disease and promoting the creation of global strategies for its accurate diagnosis, treatment, and control are of paramount importance. The detection of T. cruzi- specific antibodies in serological assays is the current gold standard technique for diagnosing chronic Chagas disease. However, this so-called conventional serology is not a valid indicator of chemotherapeutic outcomes because most posttreatment patients remain seropositive for 10-20 years [2] Therefore, there is a lack of validated biomarkers for early assessment of therapeutic responses for testing current and new drugs or treatment regimens.

Extracellular vesicles are cell-derived membranous nanoparticles present in most biological fluids. Biofluid-derived extracellular vesicles are minimally invasive molecular tools for the diagnosis and screening of diseases [3]. They can be released by various mammalian cells and pathogens, and its use as predictive biomarkers for disease progression and treatment outcomes has been reported for different pathologies, including parasitic diseases [4, 5]. Patent application US-A1 -201231621 1 discloses methods for detecting evidence of Chagas disease in a biological sample, comprising the step of measuring the presence of at least one protein selected from the group consisting of gelsolin, myosin light chain 2, vimentin, myosin heavy chain 1 1 , vinculin, and plasminogen in said sample, wherein the significantly elevated levels of the protein is informative of the presence or severity of Chagas disease.

Biomarkers for earlier therapeutic response assessment in chronic Chagas disease patients are therefore needed.

Brief Description of the Invention

To that end, the present invention provides, according to a first aspect, a method for predicting the therapeutic response and/or disease prognosis in a subject suffering Chagas.

The method comprises the analysis of an isolated biological sample, wherein it is detected at least one protein expression product comprising human and/or T. cruzi parasite proteins, said human proteins including at least one of: the group of mannose binding lectin-associated serine proteins 2, MASP2, the group of angiopoietin-related proteins, the group of collagen alpha-2 chain, VI, and the group of myosin regulatory light chain 12A, and said T. cruzi parasite protein including pyruvate phosphate dikinase.

Therefore, the detected protein expression product can serve as potential biomarker in the context of the therapeutic response and prognosis of the disease during chronic infection

According to the proposed method, the biological sample can be selected from plasma, for example obtained from peripheral blood of the subject/patient, urine or saliva from the patient.

In a particular embodiment, the analysis comprises isolating and characterizing extracellular vesicles present in said biological sample by using size-exclusion chromatography (SEC), for example, by:

- packaging a specific volume, preferably from 1 to 10 ml, of sepharose in a plastic instrument, for example a syringe, and equilibrating said volume with a phosphate buffered saline solution, producing a chromatography column; and

- loading 0.1 - 1 ml aliquot of the biological sample in said chromatography column and collecting a specific series of fractions, preferably 12 fractions, of 0.1 - 0.5 ml each only with a phosphate buffered saline.

A microsphere-based flow cytometry assay using specific extracellular vesicles markers can be performed for the characterization. In one embodiment, the mentioned markers include the detection of the CD5L and CD71 molecules on the surface of the extracellular vesicles. In other embodiments, the markers include the detection of CD5L, CD9, CD63 and CD71 molecules.

In another embodiment, the protein expression product is detected by performing a proteomic analysis. The mentioned proteomic analysis can be performed by means of liquid chromatography-mass spectrometry, after digesting the proteins with trypsin.

The proposed method can further comprise analyzing the detected protein expression product by means of a computational analysis, for example using commercial software such as“Proteome Discoverer” and“Scaffold”, and determining which of the detected proteins are absent in the sample after treatment for Chagas disease and in healthy controls.

Another aspect of the present invention also relates to the use of a protein expression product obtained from a biological sample of a chronic Chagas disease subject as potential biomarker in the context of therapeutic response and prognosis of the disease during chronic infection. In particular, an analysis of said biological sample is performed and at least one protein expression product comprising human and/or T. cruzi parasite proteins is detected, said human proteins including at least one of: the group of mannose-binding lectin-associated serine proteins 2, MASP2, the group of angiopoietin-related proteins, the group of collagen alpha-2 chain, VI, and the group of myosin regulatory light chain 12A, and said T. cruzi parasite protein including pyruvate phosphate dikinase.

Brief description of the Drawings

The foregoing and other features and advantages will be more fully understood based on the following detailed description of several merely illustrative, non-limiting embodiments in reference to the attached drawings, in which:

Fig. 1 illustrates the isolation and characterization of plasma-derived extracellular vesicles, according to a preferred embodiment of the present invention. Fig. 1A is a schematic diagram of the isolation and characterization of the extracellular vesicles derived from plasma samples. SEC, size-exclusion chromatography; NTA, nanoparticle tracking analysis; BBA, bead-based assay; LC-MS/MS, liquid chromatography-tandem mass spectrometry. Fig. 1 B extracellular vesicles characterization was done by BBA using the classical EVs markers CD5L, CD9 and CD63; MFI, Median Fluorescence Intensity. Fig. 1 C illustrates the NTA of SEC fractions F7-10. Left panel: pretreatment; right panel: posttreatment. Fig. 2 illustrates the human proteomic profile of plasma-derived extracellular vesicles from a heart-transplanted patient with chronic Chagas disease, before and after benznidazole treatment, and from two healthy donors. Heatmap of the identified human extracellular vesicles-derived proteins. Heatmap was generated from proteomic data by the Scaffold perSPECtives software. Hierarchical clustering was performed based on the normalized weighted spectrum count. Scale indicates intensity. Higher numbers of human proteins were found in extracellular vesicles isolated from the patient, pre- and post-BZN treatment, when compared with extracellular vesicles-derived from the two healthy donors (H1 , H2).

Detailed Description of Several Embodiments

Present invention provides an innovative approach for predicting the therapeutic response and/or prognosis in a subject/patient suffering Chagas disease. The invention comprises the analysis of an isolated biological sample, for example a plasma sample obtained from the peripheral blood of the patient, not limitative as in other embodiments the biological sample can be urine or saliva from the patient. Moreover, the invention comprises detecting at least one protein expression product comprising human and/or T. cruzi parasite proteins. The human proteins can include at least one of: the group of mannose-binding lectin-associated serine proteins 2, MASP2, the group of angiopoietin- related proteins, the group of collagen alpha-2 chain, VI, and the group of myosin regulatory light chain 12A, and the T. cruzi parasite protein can include pyruvate phosphate dikinase. Therefore, the detected protein expression product can serve as potential biomarker in the context of the therapeutic response and prognosis of the Chagas disease during chronic infection.

In an embodiment, a group of patients with cardiomyopathy not related with Chagas disease (for example, N=10) and a group of healthy individuals (for example, N=10) can be included as control. The patients of the different groups will be offered treatment with benznidazole at standard doses. A biological sample preferably plasma, will be taken before treatment (day 0) and 6 and 12 months after treatment.

In a particular embodiment, the human and/or parasite (T. cruzi) proteins are included in extracellular vesicles present in the biological sample. The protein composition of extracellular vesicles can be identified by performing a proteomic analysis which detects at least 2 unique peptides for each of the proteins identified in the extracellular vesicles.

In one embodiment, the method described by (Menezes-Neto et al., J. Extracell. Vesicles, 2015) is used for purifying and characterizing the extracellular vesicles originating from the mentioned biological samples. To that end, 10 ml of sepharose, a polysaccharide polymer, are packaged in a syringe and equilibrated with a phosphate buffered saline solution, preferably 0.32% PBS citrate, producing a chromatography column. Next, 1 ml_ of plasma aliquot is loaded in the column and 12 0.5 ml_ fractions are collected, each with a citrate-phosphate buffered saline. The protein concentrations of the fractions are determined using BCA ( Thermo Scientific).

Likewise, the concentration and particle size distribution can be determined by means of analysis with a nanoparticle tracking instrument such as NTANanoSight LM10.

Furthermore, the extracellular vesicles can be characterized by a microsphere- based flow cytometry assay using specific markers, such as CD5L, CD63, CD9 and CD71 . Briefly, the SEC fraction samples are incubated with 0.5 mI of microspheres of 4 pm-aldehyde/sulfate latex (4% w/v, Invitrogen) for 15 minutes at room temperature. The microspheres are resuspended in 1 mL of microsphere coupling buffer and incubated for at least 8 hours (for example overnight) at room temperature under rotation. The exosome coated microspheres are centrifuged at a specific speed, for example 2000 x g, for 10 minutes at room temperature, and washed with phosphate buffer before incubation with the specific primary antibodies, CD5L, CD63, CD9 and also CD71 , for a specific period of time, preferably 30 minutes, at 4 ⁵ C. After washing, the microspheres are incubated with the secondary antibody for 30 minutes at 4 ⁵ C, producing exosome microspheres. The exosome microspheres are preferably washed twice with phosphate buffered saline with bovine serum albumin before finally being resuspended only in phosphate buffered saline and subjected to flow cytometry, where about 10,000 samples will be required. Preferably, Flow Jo software will be used for comparing the mean fluorescence intensity (MFI) of the microsphere populations between the extracellular vesicle preparations.

The cited proteomic analysis is preferably performed using liquid chromatography-mass spectrometry after digesting the proteins (100-1000 ng) with trypsin. For the proteomic analysis of the extracellular vesicles, tryptic peptides are dissolved in 0.1% formic acid (FA) (1 pg/mI) and 1 pL (equivalent to 10-100 ng of the original protein content) is loaded in a reversed-phase (RP) column at a speed of about 300 nl/min, for 120 minutes. The elution of the peptides is performed in a multi-step gradient (5% to 40% in 95 min; 40% to 95% in 5 min; 95% in 9 min; and 95% to 5% in 1 1 min) of solvent B. The mass spectrometry spectra are collected at a mass resolution of 70,000, in a scan range of 400 to 1600 m/z and with an AGO target value of 1 E6. The ten most intense ions are subjected to fragmentation at a mass resolution of 17,500 (AGO target 2e5, NCE 28%, isolation width 4 m/z).

To increase peptide coverage and therefore protein identification, the tryptic peptides are first fractionated using a high pH reversed-phase peptide fractionation kit. The resulting fractions are analyzed by means of mass spectrograph-liquid chromatography as indicated above.

In one embodiment, the identified proteins are analyzed by a computational analysis and the proteins identified in the extracellular vesicles which are absent in samples after treatment for Chagas disease and in healthy controls are determined.

A particular study will be now detailed. In 2009, a 51 -year-old patient from Bolivia, with past history of chronic Chagas disease, exhibiting severe organ involvement (chronic cardiomyopathy Kuschnir III, and megacolon and megaesophagus degree IV) [6], was admitted at the International Health Department (Hospital Clinic, Barcelona). Serological diagnosis for chronic Chagas disease was performed using two commercial ELISA kits (Ortho-Clinical Diagnostics and BioELISA Chagas). 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 (EF 15-20%). In September 2015, after being included in the transplantation program, a positive IgG serology for cytomegalovirus infection and toxoplasmosis, and a subclinical hypothyroidism were diagnosed. On November 28, 2015, the patient had a heart transplant without incidences in the short follow-up period. The patient started immunosuppressive therapy with tacrolimus, azathioprine, and prednisone and no early transplant rejection signs were found in the follow-up endomyocardial biopsies.

After transplantation and in the context of immunosuppression treatment, qRT- PCR was performed weekly to detect T. cruzi in the blood (Tc-qPCR) [7] Benznidazole (BZN) treatment (2.5 mg/kg, bis in die, 60 days) was started when a Chagas disease reactivation was confirmed by several consecutive, positive Tc-qPCR assays, without clinical criteria. Three weeks following BZN treatment, the Tc-qPCR became negative. After having completed 80% of the treatment, the patient presented bronchopulmonary aspergillosis and BZN course was interrupted. The Tc-qRT-PCR became positive and a new BZN course was initiated, completing this time the total dose, without evidence of therapeutic failure based on Tc-qPCR results. Plasma samples for purification and characterization of extracellular vesicles were collected before first treatment and just after the last treatment. Unexpectedly, the patient died in August 2016, due to a late organ rejection.

To determine whether circulating extracellular vesicles from this patient could have been used as predictive biomarkers for the evaluation of therapeutic response and disease outcome in the Chagas disease context, pre- and posttreatment plasma samples were collected and extracellular vesicles enriched by size-exclusion chromatography (SEC), as described [8] (Fig. 1A). As negative controls, plasma samples from two healthy donors (HDs) were also subjected to SEC. Eluting extracellular vesicles were characterized by bead-based assay (BBA) and Nanoparticle Tracking Analysis (NTA) (Fig. 1 B, C). Aliquots (100 mI_) from SEC fractions 7-10 were pooled and protein composition was determined by 2D-liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). Briefly, samples were digested with trypsin and resulting peptides were resolved by high-pH reversed-phase peptide fractionation [9], followed by C18-reversed phase UHPLC coupled to a Thermo QE Plus Orbitrap MS, as described [10]. Raw MS data were analyzed by Proteome Discoverer™ (v.2.1.1.21 ) software (Thermo), followed by Scaffold perSPECtives (v.4.8.7) (Proteome Software). Using a false-discovery rate (FDR) <1 % and one unique peptide per protein, 12 T. cruzi proteins and 338 human proteins were identified. However, when the more stringent criterium of >2 unique peptides per protein was applied, only one T. cruzi protein (i.e., pyruvate phosphate dikinase (PPDK)) was detected, and 288 human proteins, of which 19 were identified only in pretreatment samples (Table 1 ). PPDK has been identified by proteomic analysis of T. cruzi total secretome and extracellular vesicles [10-12] This protein has 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 extracellular vesicles secreted by this chronic Chagas disease patient remains to be determined.

Among the 19 human proteins uniquely identified in extracellular vesicles from the chronic Chagas disease patient before treatment, the mannan-binding lectin serine protease 2 (MASP2) is worth highlighting. A recent study with human samples has shown that MASP2 polymorphisms and MASP-2 levels are associated with high risk of chronic Chagas disease cardiomyopathy [4] Furthermore, mannose-binding lectin (MBL), 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]. Noteworthily, an increase of mRNA levels of collagen-1 and -6 in the heart of the infected animals was observed in this study [15]. These results could support present invention findings, as collagen alpha-1 is one of the proteins identified exclusively in extracellular vesicles before patient treatment (Table 1 ).

Another important observation here is the identification of a higher number of human proteins in patient-derived extracellular vesicles, when compared with the two HD-derived extracellular vesicles samples (Fig. 2). Of the total proteins identified, in which statistical analysis was feasible, four were significantly upregulated in patient- derived extracellular vesicles before treatment. This was particularly the case for the proteins complement C1 s subcomponent, isoform CRAJo, FLJ00385 protein, and cDNA FLJ75416. Notably, complement C1 s subcomponent has been recently identified among the six up-regulated extracellular vesicles biomarkers with potential for clinical applications in myocardial infarction [4]

Hence, proteins associated with extracellular vesicles secreted by T. cruzi have been identified in the conditioned medium of different parasite stages [10-12], but never in biofluids from Chagas disease patients. This is the first proteomic profiling of plasma- derived extracellular vesicles purified directly from a heart-transplanted chronic Chagas disease patient who exhibited reactivation following immunosuppression. Here, it has been identified human and parasite proteins present or upregulated in plasma-derived extracellular vesicles from a chronic Chagas disease patient before chemotherapy and that are absent or downregulated following treatment. It can be thus hypothesized that extracellular vesicles proteins released by the host or parasite during infection might serve as potential biomarker candidates for the evaluation of therapeutic response and disease outcome in chronic Chagas disease.

T. cruzi Protein Name Accession Number ChD ChD Healthy 1 Healthy 2

Pre- Post-BZN

BZN

Pyruvate, phosphate dikinase OS=Trypanosoma cruzi K2MVM1 TRYCR 2 0 0 0 marinkellei GN=MOQ_000480 PE=3 SV=1 (0.96)

Human Protein Name Accession Number ChD ChD Healthy 1 Healthy

Pre- Post-BZN 2

BZN

Collagen alpha-l(VI) chain OS=Homo sapiens GN=COL6Al C06A1 HUMAN 3 0 0 0 PE=1 SV=3 (1.44)

Group of Angiopoietin-related protein 6 OS=Homo sapiens ANGL6 HUMAN 3 0 0 0 GN=ANGPTL6 PE=1 SV=1+1 (+1) (1.44) Myosin regulatory light chain 12B OS=Homo sapiens ML12B HUMAN 2 0 0 0

GN=MYL12B PE=1 SV=2 (1.92)

Collagen alpha-2(VI) chain OS=Homo sapiens GN=COL6A2 C06A2 HUMAN 2 0 0 0

PE=1 SV=4 (1.44)

Collectin sub-family member 10 (C-type lectin), isoform tr| A0A024R9J3 | A0A 2 0 0 0

CRA_a OS=Homo sapiens GN=COLEC10 PE=4 SV=1 024R9J3_HUMAN (1.44)

Group of Coagulation factor XIII A chain OS=Homo sapiens F13A HUMAN (+2) 2 0 0 0

GN=F13A1 PE=1 SV=4+2 (1.44)

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase tr| A0A024R1K7 | A0 2 0 0 0 activation protein, eta polypeptide, isoform CRA_b A024R1K7 HUMAN (1.44)

OS=Homo sapiens GN=YWHAH PE=3 SV=1

Fibrinogen-like protein 1 OS=Homo sapiens GN=FGL1 PE=1 FGL1 HUMAN 2 0 0 0

SV=3 (0.96)

Group of L-lactate dehydrogenase A chain OS=Homo sapiens LDHA HUMAN (+1) 2 0 0 0

GN=LDHA PE=1 SV=2+1 (0.96)

Group of Laminin subunit alpha-2 OS=Homo sapiens A0A087WX80 HUM 2 0 0 0

GN=LAMA2 PE=1 SV=1+1 AN (+1) (0.96)

Group of Serum amyloid A protein OS=Homo sapiens E9PQD6 HUMAN 2 0 0 0

GN=SAA1 PE=1 SV=l+2 (+2) (0.96)

Group of Transforming growth factor beta-induced 68kDa tr| A0A0S2Z4K6 | A0 2 0 0 0 isoform 2 (Fragment) OS=Homo sapiens GN=TGFBI PE=2 A0S2Z4K6_HUMAN (0.96)

SV=1+1 (+1)

Heparan sulfate proteoglycan 2 (Perlecan). isoform CRA_b tr| A0A024RAB6 | A0 2 0 0 0

OS=Homo sapiens GN=HSPG2 PE=4 SV=1 A024RAB6_HUMAN (0.96)

Neurogenic locus notch homolog protein 3 OS=Homo NOTC3 HUMAN 2 0 0 0 sapiens GN=NOTCH3 PE=1 SV=2 (0.96)

Table 1. Parasite and human proteins identified in plasma-derived extracellular vesicles from a chronic Chagas disease patient prior to benznidazole chemotherapy, but absent following treatment, and in healthy donors. All proteins were identified by at least two unique peptides. Exclusive unique peptides for the patient before treatment (ChD-Pre- BZN) are shown. Normalized total spectrum count values are indicated in parenthesis.

The present invention contemplates developing a kit for the analysis of biomarkers identified in extracellular vesicles of subjects with different stages of Chagas disease. The proteins of the extracellular vesicles with antigenic domains are analyzed by means of a computing algorithm to predict the lineal epitopes of B cells using a database containing data relating to antibodies and T-cell epitopes for humans, non human primates, rodents, and other animal species. The genes of interest are amplified by means of PCR or RT-PCR using specific primers. The amplified fragments are cloned in the already modified pIVEX vector to express recombinant proteins fused to the glutathione-S-transferase (GST) in the N-terminal region, using the wheat germ system (Roche). All the plasmid constructs are verified by DNA sequencing. The free wheat germ protein is expressed according to the manufacturer’s instructions. All the recombinant proteins are purified by affinity using GST columns (Health Care) and the protein concentration is determined by the Bradford assay (Bio-Rad). The soluble proteins are anchored to carboxylated magnetic particles to determine those reacting with the sera of the patients, through Luminex suspension assays.

Additionally, a statistical analysis can be carried out to determine if the new antigens identified in the extracellular vesicles are immunogenic in natural infections and the possible associations thereof with clinical cardiac manifestations in patients with Chagas disease, using sera from the different groups. At the same time, (parasite and/or human) proteins specifically identified in the extracellular vesicles originating from infection, which are absent in samples after treatment and in healthy controls, can also be determined.

The scope of the present invention is defined in the attached claims.

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