WO2010139826A1 - Use of the masp-52 protein for the diagnosis, treatment and prevention of chagas disease - Google Patents

Abstract

The invention relates to the use of the MASP-52 protein, from the mucin-associated surface protein (MASP) family, for the diagnosis, treatment and prevention of Chagas disease, including a method for detecting the presence of the T. cruzi parasite in individuals, a method for obtaining data that can be used for diagnosing Chagas disease and a diagnostic kit containing the elements necessary for analysing the quantity of MASP-52 protein in a biological sample.

Description

 Use of the Masp 52 protein for diagnosis, treatment and prevention of Chagas disease.

The present invention is within the field of molecular biology and medicine, and specifically refers to the use of a protein of the MASP family (Mucin associated Surface proteins) for the diagnosis, treatment and prevention of Chagas disease .

STATE OF THE PREVIOUS TECHNIQUE

Chagas disease (Chagas-Mazza disease, Chagas disease or American trypanosomiasis), is a tropical parasitic disease mainly from Central and South America, generally chronic, and whose etiologic agent is Trypanosoma cruzi. It is estimated to result in about 21,000 deaths each year (WHO, 2002, 2005), with approximately 50,000-200,000 new cases diagnosed per year (Tarieton RL, 2007. PLoS Med 4 (12): e332). Although the disease has traditionally been confined to Latin America, it is currently expanding as a result of migratory processes, so it has been necessary to implement diagnostic tests in blood banks and health centers in those countries with a high rate of immigrant population from endemic areas. Thus, the incidence of the disease in said immigrant population is 16 per 1000 in Australia, 9 per 1000 in Canada, 25 per 1000 in Spain and 8 to 50 per 1000 in the USA. (Schmunis GA et al, 2007. Mem Inst Oswaldo Cruz. 102 Suppl 1: 75-85).

T. cruzi is a flagellated protozoan, belonging to the Kinetoplastid Order, being the only one of the trypanosomes that presents an obligatory phase of intracellular multiplication in the vertebrate host, the metacyclic Trypomastigote forms the infective phase in the life cycle of the protozoan responsible for said cell invasion The parasite transmitted to the vertebrate host in the feces of the insect is called at this stage, therefore, metacyclic trypomastigote. In the blood, the parasite is observed as a fusiform trypomastigote, in the form of "C" or "S" 20 μm long and 1 μm wide. During this stage, the trypomastigote does not multiply in the host's blood. When the parasite infects striated heart muscle fibers or to phagocytes, the scourge is shortened and transformed into a round amastigote of 2 to 5 μm in diameter and with a very short or non-existent external scourge, this is multiplied by binary fission forming “clusters” or “nests” accumulate in the host cell until it breaks. The parasites released from the cell become blood trypomastigotes, which are released into the circulating blood, are of a total size that varies between 15 and 20 μm, have a free flagellum, a bulky, terminal or underground cinetoplast, and an oval nucleus. These trypomastigotes can infect other cells, but they are not able to multiply in the blood since the only replicative form in the vertebrate is the intracellular amastigote form, invading other cells to repeat the cycle.

Invertebrate hosts acquire the parasite by feeding on man or infected domestic or wild animals. The trypomastigotes migrate to the midgut of the insect where they become epimastigotes, wide flagellates, very mobile, with the kinetoplast between the nucleus and the free scourge. There they divide a large number of times, leaving the insect infected for life. The epimastigotes become metacyclic trypomastigotes and migrate to the posterior intestine from where they are excreted with feces at the time of the bite.

Any biological infectious process can be divided into varying degrees according to its severity and depending on the treatments necessary to relieve its symptoms. In the case of infections caused by Trypanosoma cruzi in man, the disease has two severe states: the acute phase, shortly after the infection, and the chronic phase that can develop even after ten years. In the case of infections caused by Trypanosoma cruzi, some acute cases (10 to 20%) resolve in a period of two to three months giving rise to an asymptomatic chronic phase now called an undetermined phase, which is characterized by the persistence of The infection without presenting clinical problems, to reappear only several years later.

The diagnostic methods mainly include serological techniques, and these can be direct or indirect hemagglutination, IFA (indirect immunofluorescence), complement fixation reaction and ELISA, as well as the microscopic examination of the cell interface after centrifuging the blood (Stront and microStront), and blood culture. These and other methods show different sensitivity and specificity. During the acute phase, the xenodiagnosis is the most sensitive test (92%), which can also be used for the study of the susceptibility of laboratory animals to different strains of T. cruzi. While the xenodiagnosis has been negative after treatment with effective trypanocides, conventional serological tests such as immunofluorescence and complement fixation remain positive. Consequently, the evaluation of the cure is still controversial. By means of the complement-mediated lysis test (CML), lytic antibodies (LA) of T. cruzi were detected that are attached to the epitopes of live trypomastigotes and are related to the host's resistance to infection (Krettli et al. ., 1982. Trans R Soc Trop Med Hyg 76 (3): 334-340). Conventional antibody serology (CSA) detects immunoglobulins in sera of patients with chagasic infections, but unlike LA, it does not recognize live trypomastigotes. The presence of LA has been used as an important criterion for the evaluation of Chagas disease.

Using the flow cytometry, a sensitive immunometry was introduced for the detection of live membrane-bound anti-trypomastigote antibodies (Martins-Filho et al. 1995. Clin Diagn Lab Immunol 2 (5): 569-573). Based on serological tests (detection of LA-lytic antibodies and CSA-conventional serology antibodies) and parasitological evaluations as blood cultures (HE), it is possible to classify patients into: a) chronically infected untreated patients (NT) and uncured treated patients (TNC), with positive HE and LA and CSA antibodies in their serum; b) "dissociated" patients (DIS); that is, with HE-negative, in which LAs are not detected while CSAs are present; c) cured patients (CUR), with HE-negative, who do not have LA or CSA antibodies, and d) as a control, a group of non-chagasic people (NC).

The sera of the patients are examined by incubation of live trypomastigotes from the bloodstream, which have been exposed to fluorescein isothiocyanate conjugated with antihuman immunoglobulin G. The parasites are fixed, run on the cytometer and are identified based on their size and granulations. With the experience in the CLM test a level of 20% is used of parasites positive to the fluorescence of the conjugate as a cut-off line between effective and non-effective treatments.

The differential diagnosis is essential to distinguish the stage of development of the disease, as well as the monitoring of the course of the disease, especially in the chronic phase, to eliminate or minimize autoimmune mechanisms, which cause negative effects and even lethal, in patients. In addition, the only two medications available for the treatment of Chagas disease are Nifurtimox, developed in 1960 by Bayer and Benzinidazole, developed in 1974 by

Roche. Both have a low cure rate and side effects.

The family of MASPs (Mucin associated Surface Proteins) proteins was described for the first time following the sequencing of the entire genome of the Trypanosoma cruzi parasite, and seems exclusive of it. As described in El Sayed et al., 2005. (Science, 309, 409), there are more than 1300 copies of MASP genes, (1377 genes identified) and have been named for being downstream of TcMUC Il mucins (subfamily of 884-member mucins), and structurally resemble them (although not at the sequence level). Of the 1377 MASP genes identified, 771 appear to encode well-preserved N-terminal and C-terminal regions, and 433 are pseudogenes. Approaches with proteomic techniques have not been able to detect a large number of these proteins, so it is thought that they may show post-translational modifications similar to those of mucins, described as N-linked glycoproteins (Atwood Ml et al, 2006. J Protect Res. 3376-3384), or alternatively, these genes are expressed in a manner similar to the surface variant glycoproteins (VSGs) of T. brucei (Atwood III et al., 2005. Science 309, 473-476)

Regarding immunization against T. cruzi, there are several approaches in the state of the art, ranging from attenuated parasites in their virulence to gene immunization. Thus, Segura et al., 1976 (J. Parasitol., 62, 131-133) used subcellular fractions of epimastigotes obtained from differential centrifugation in sucrose density gradients. Snary et al., 1981 (Mol. Biochem. Parasitol., 3, 1-14) employed 72 kDa molecular weight surface glycoproteins, which protected animals against a challenge with metacyclic tripmastigotes but not against a challenge with blood trypomastigotes Scott et al., 1982 (Trans. R. Soc. Trop. Med. Hyg. 76, 698-700) used a 90 kDa molecular weight glycoprotein, which does not cross with mammalian tissues and induced protection and increased reaction levels of delayed hypersensitivity when using saponin as an adjuvant (Scott et al., 1984. Int. Archs. Allergy Appl. Immun., 74, 373-377), but did not cause negativity of parasitemia, after a challenge with parasites in mice and monkeys A glycoprotein of similar molecular weight but present only in metacyclic tipomastigotes was mentioned as inducing resistance against acute infection generating a satisfactory protective immune response (Yoshida et al., 1990. Mol. Biochem. Parasitol., 39, 39-46). Purified monoclonal antibody antigens have also been used (Gómez et al., 1995 .Appl. Biochem. Biotechnol., 50, 57-69), proteins purified through affinity columns (Plumas-Marty et al, 1993. Res. Immunol., 144, 553-563), excretion products - parasite secretion (Ouassi et al., 1990. Parasitol., 100, 115-124) as an immunogen to induce a response capable of nullifying the development of parasite evasive mechanisms to avoid the immune response (Taibi et al., 1993. J. Immunol., 151, 2676-2689).

The use of defined defined antigens from T. cruzi presents the disadvantage of its difficult obtaining in adequate volumes. Obtaining antigens by molecular biology and recombinant DNA techniques allowed cloning, expressing and producing T. cruzi antigens in sufficient volumes.

Thus, proteins known as Amastigote Surface Protein-2 (ASP-2) (Silveira et al., 2008. Clin Vaccine Immunol., 15, 1292-300), and transialidases (TS) linked to CpG motifs (Hoft et al., 2007. J. Immunol., 10, 6889-900) or recombinant adenoviruses with TS or ASP-2 (Machado et al., 2006. Hum Gene Ther., 17, 898-908) have been used with high levels of protection against to the challenge with T. cruzi. However, the large expansion of the Transialidase family throughout the genome and its ease of mutation due to immune pressure, is a difficulty in treating the disease due to the large variety of parasite strains distributed throughout Latin America .

In addition, the development of vaccines has been dramatically limited due to the debate of the mechanisms involved in Chagas disease. So, Some studies seem to indicate that tissue damage is due to the replication of intracellular amastigotes, while others suggest that it is due to autoimmunity induced by parasite antigens that mimic host proteins. This is one of the causes for which there are no effective vaccines available for the prevention of this infection, and the current perspective on their eventual development is uncertain. In addition, currently available treatments have low efficacy (≥ 80% of therapeutic failures) in the established chronic phase of the disease, and also the antiparasitic efficacy of the compounds varies according to the geographical region, probably as a result of the different intrinsic susceptibility to drugs of the T. cruzi strains that circulate in different endemic areas. Finally, these compounds frequently have side effects, which include anorexia, vomiting, peripheral polyneuropathy and allergic dermopathy, and which can lead to interruption of treatment.

There is, therefore, the need to develop a method of diagnosis of Chagas disease, of high sensitivity and specificity, and that allows patients to be classified according to the stage of their disease, as well as a method of treatment and prevention adequate and effective against this disease.

DESCRIPTION OF THE INVENTION

The authors of the present invention have purified and characterized a 52 kDa protein secreted to the medium during the T. cruzi-host cell interaction, belonging to this family of proteins and which they have designated as Masp52.

The detection of this protein allows to differentiate the different forms of Trypanosoma cruzi, both quantitatively (detection of a single band with different degree of expression in the different stages of the parasite by SDS PAGE electrophoresis and its subsequent analysis by Western blot using the anti-center IgG catalytic of Masp52 (CR), or by studying the mRNA by RTqPCR as qualitatively (using the anti-signal peptide IgG (SP), to observe the pattern of protein expression of the MASP family by Western blot). In this way, reference values could be established that would allow the classification of Chagasic patients in different groups depending on the stage of the disease, as well as the severity of the disease and the post-treatment prognosis.

Thus, a first aspect of the invention relates to a method of detecting the presence of the T. cruzi parasite in an individual, hereinafter the first method of the invention, comprising: a) obtaining an isolated biological sample of said individual, b) detect the Masp52 protein in the biological sample isolated from (a).

The organisms of the species Trypanosoma cruzi belong to the Superukine Eukaryota, Order Kinetoplastida, Family Trypanosomatidae, Genus Trypanosoma and subgenus Schizotrypanum.

An isolated biological sample includes, but is not limited to, cells, tissues and / or biological fluids of an organism, obtained by any method known to a person skilled in the art. Preferably, the isolated biological sample is a biological fluid. More preferably, the biological fluid is blood or plasma or blood serum. The term "individual", as used in the description, refers to animals, preferably mammals, and more preferably, humans. The detection of this protein in an isolated biological sample of a subject is indicative of the presence of the T. cruzi parasite. The first method of the invention thus allows the detection of T. cruzi in a biological sample any organism capable of being parasitized by T. cruzi, therefore including vectors and reservoirs. In a preferred embodiment, the organism where the presence or absence of the T. cruzi parasite is detected is a mammal. In another even more preferred embodiment is a human mammal (man).

The Masp52 protein belongs to the family of MASPs proteins {Mucin associated Surface proteins).

In the context of the present invention, the term "Masp52 protein" is defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 1, and which can comprise various variants from: a) nucleic acid molecules encoding a polypeptide that It comprises the amino acid sequence of SEQ ID NO: 1, b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to degeneration of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 1. In which the polypeptide encoded by said nucleic acids has the activity and structural characteristics of the Masp52 protein. It includes, therefore, various variants of the Masp52 protein, that is, proteins resulting from posttranslational modifications such as, but not limited to, glycosylation, phosphorylation or methylation. The term "variant" is defined later in this report.

The detection of the Masp52 protein can be carried out by any means known in the state of the art. The authors of the present invention have shown that the detection of the amount or concentration of this protein semi-quantitatively or quantitatively allows differentiating between the different stages of the parasite. In this way, a differential diagnosis could be established in individuals affected by Chagas disease, which would allow them to subclassify them. This subclassification, in turn, would allow the establishment and adaptation of the treatment individually. For example, the expression of the Masp52 in the different stages of T. cruzi can be performed by SDS PAGE electrophoresis, and its subsequent analysis by Western blot using the anti-CR IgG, shows us how this protein is recognized as a single band with different degree of expression at different stages of the parasite. Thus, the expression in metacyclic trypomastigotes could be of the order of 12 times higher than that found in epimastigotes, five times higher than that of amastigotes and twice greater than that found in trypomastigotes derived from cell cultures. When the recognition in the four phases of T. cruzi is carried out using the anti-SP IgGs, to observe the expression pattern of proteins of the MASP family by Western blot, the expression of 6 bands in the metacyclic trypomastigotes, 4 bands, was obtained in trypomastigote forms, 3 bands in amastigotes and 3 bands in epimastigotes, although in the latter the level of expression was very low. The result of the study of mRNA using RTqPCR showed that all stages had synthesis of the specific mRNA of the Masp 52 protein, but with different levels of expression. In the metacyclic trypomastigote forms the synthesis of specific mRNA could be of the order of three times greater than that of tissue-derived trypomastigote, eleven times greater than that of amastigotes and fifty times greater than that of the epimastigotes.

Thus, another aspect of the invention relates to a method of obtaining useful data for the diagnosis of Chagas disease, hereinafter the second method of the invention, comprising: a) obtaining an isolated biological sample from an individual , b) detect the amount of Masp52 protein present in the biological sample isolated from (a).

In a preferred embodiment, the second method of the invention further comprises: c) comparing the amount detected in step (b) with a reference amount.

The steps (b) and / or (c) of the methods described above can be totally or partially automated, for example, by means of a robotic sensor device for the detection of the amount in step (b) or the computerized comparison in step (c). In addition to the steps specified above, it may include other additional steps, for example related to the pre-treatment of the sample or the evaluation of the results obtained by these methods.

The term "diagnosis", as used in the present invention, refers to the ability to detect the presence of Trypanosoma cruzi parasitizing an individual, preferably in an animal, more preferably in a mammal and even more preferably in a human. It also refers, in a more preferred embodiment, to the ability to discriminate between samples from patients presenting with different states of Chagas disease: the acute phase, shortly after the infection, the undetermined phase and the chronic phase. In turn, according to the second method of the present invention, other subclassifications could be established within this principal, thus allowing the choice and establishment of regimes adequate therapeutic. This detection, as understood by an expert in the field, is not intended to be correct in 100% of the samples analyzed. However, it requires that a statistically significant amount of the analyzed samples be classified correctly. The amount that is significantly statistical can be established by an expert in the field through the use of different statistical tools, for example, but not limited, through the determination of confidence intervals, determination of the p-value, Student's test or discriminant functions of Fisher Preferably, the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. Preferably, the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001. Preferably, the present invention makes it possible to correctly detect the disease differentially by at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a certain group or population analyzed.

The measurement of the amount or concentration, preferably semi-quantitatively or quantitatively, can be carried out directly or indirectly. The direct measurement refers to the measurement of the amount or concentration of the protein, based on a signal that is obtained directly from the protein, and that is directly correlated with the number of molecules of the protein, present in the sample. Said signal - to which we can also refer to as an intensity signal - can be obtained, for example, by measuring an intensity value of a chemical or physical property of the protein. The indirect measurement includes the measurement obtained from a secondary component (for example, a component other than the product of the gene expression) or a biological measurement system (for example the measurement of cellular responses, ligands, "labels" or enzymatic reaction products ).

The term "quantity", as used in the description, refers to but is not limited to the absolute or relative amount of the protein, as well as any other value or parameter related to them or that may be derived from them. . Said values or parameters comprise intensity values of the signal obtained from any of the physical or chemical properties of the protein obtained by direct measurement, for example, intensity values of mass spectroscopy or nuclear magnetic resonance. Additionally, said values or parameters include all those obtained by indirect measurement, for example, any of the measurement systems described elsewhere in this document.

The term "comparison", as used in the description, refers to, but is not limited to, the comparison of the amount of Masp52 protein of the biological sample to be analyzed, also called the problem biological sample, with an amount of Masp52 protein. of a desirable reference sample described elsewhere in this description. The reference sample can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. The comparison described in section (c) of the methods of the present invention can be performed manually or assisted by a computer.

The term "reference amount", as used in the description, refers to the amount of the absolute or relative amount of the protein forms

Masp52 that allows to discriminate a stage of T. cruzi from other stages. Suitable reference amounts can be determined by the method of the present invention from a reference sample that can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample.

The reference sample can be, for example, a protein extract obtained from the serum of a patient with Chagas disease in a certain clinical phase. In another preferred embodiment of this aspect of the present invention, the reference amount is obtained from a reference sample. The reference amount can also be obtained, for example, from the normal distribution limits of an amount found in samples obtained from a population of patients with Chagas disease in different phases, by means of well-known statistical techniques.

In a preferred embodiment, the detection of the amount of Masp52 protein is performed by incubation with a specific antibody in an immunoassay. The term "immunoassay", as used in the present description refers to any analytical technique that is based on the reaction of the conjugation of an antibody with the sample obtained. Examples of immunoassays known in the state of the art are, for example, but without limited: immunoblot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry or protein chips.

The term "antibody" as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts) with the protein Masp52. Examples of portions of immunologically active immunoglobulin molecules include F (ab) and F (ab ') 2 fragments that can be generated by treating the antibody with an enzyme such as pepsin. The antibodies can be polyclonal (typically include different antibodies directed against different determinants or epitopes) or monoclonal (directed against a single determinant in the antigen). The monoclonal antibody can be biochemically altered, by genetic manipulation, or it can be synthetic, possibly lacking the antibody in whole or in parts, of portions that are not necessary for the recognition of the Masp52 protein and being substituted by others that communicate to the antibody additional advantageous properties. The antibody can also be recombinant, chimeric, humanized, synthetic or a combination of any of the foregoing. A "recombinant antibody or polypeptide" (rAC) is an antibody that has been produced in a host cell that has been transformed or transfected with the nucleic acid encoding the polypeptide, or produces the polypeptide as a result of homologous recombination. There are five major isotypes or classes of immunoglobulins: immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin E (IgE).

Antibodies that recognize the Masp52 protein or certain fragments or variants thereof are described, but not limited, in the examples herein. These antibodies can be used to carry out the methods of the present invention, for example, but not limited, by immunoblot, ELISA or immunhistochemistry. In a preferred embodiment, the immunoassay is an immunoblot or Western blot. To carry out a Western blot, a protein extract is obtained from an isolated biological sample of a subject and the proteins are separated in a support medium capable of retaining them by electrophoresis. Once separated the proteins are transferred to a different support where they can be detected by use of specific antibodies that recognize the Masp52 protein. In a more preferred embodiment of this aspect of the invention, the Masp52 protein is detected by Western blotting using anti-CR IgG. In another preferred embodiment, the Masp52 protein is detected by Western blotting using anti-SP IgG.

The separation of proteins is usually done by electrophoresis. Electrophoresis is an analytical technique of separation of kinetic foundation based on the movement or migration of macromolecules dissolved in a certain medium (electrophoresis buffer solution), through a matrix or cross-linked support as a result of the action of an electric field. The behavior of the molecule is given by its electrophoretic mobility and this by the charge, size and shape thereof. The higher the charge / size ratio, the faster an ion migrates within the electric field. There are numerous variations of this technique depending on the equipment used, support and physical-chemical conditions in which the separation will be carried out. Some variations of this technique are, for example, but not limited to, capillary electrophoresis, paper electrophoresis, agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), isoelectric focusing or two-dimensional electrophoresis. PAGE electrophoresis can be carried out under denaturing or non-denaturing conditions. Preferably, to carry out the detection of the amount of Masp52 protein by Western blotting, the proteins obtained from an isolated biological sample of a subject are separated by monodimensional PAGE electrophoresis under denaturing conditions (SDS-PAGE).

Alternatively, proteins obtained from an isolated biological sample of a subject can be separated by two-dimensional electrophoresis or 2D-PAGE. This technique is based on the separation of proteins based on two characteristics: first, proteins are separated according to their isoelectric point (pl) by isoelectric focusing (IEF) and, secondly, the separation is carried out according to their mass molecular, by electrophoresis under denaturing conditions (SDS-PAGE). The DIGE (Differential In GeI Electrophoresis) technique is based on the protein mapping of the study samples with one of the three fluorochromes (Cy2, Cy3 or Cy5) before separation. The samples are then mixed and separated in a single two-dimensional gel, minimizing the experimental variability. Due to the tide specific to each sample can be observed individually and perform a comparative analysis of the differential expression of proteins, allowing precise quantification.

Once the proteins are separated by electrophoresis, and before detection, the proteins are transferred to a support or to a membrane, for example, but not limited to, PDVF, nitrocellulose or cellulose acetate. This membrane is hybridized with a specific antibody (also called primary antibody) that recognizes the Masp52 protein. Then, the membrane is hybridized with an antibody (also called secondary antibody) capable of specifically recognizing the primary antibody and which is conjugated or bound with a marker compound. In another preferred embodiment, it is the antibody that recognizes the Masp52 protein that is conjugated or bound to a marker compound, and the use of a secondary antibody is not necessary. Once the protein is detected, its relative molecular size can be determined, comparing its migration with the migration of a control protein that is detected simultaneously, preferably on the same support, which has a known size.

In another preferred embodiment, the immunoassay is an enzyme-linked immunosorbent assay or ELISA (Enzyme-Linked ImmunoSorbent Assay). The ELISA is based on the premise that an immunoreactive (biological sample antigen or antibody) can be immobilized on a solid support, then putting that system in contact with a fluid phase containing the complementary reagent that can bind to a marker compound .

There are different types of ELISA. In the direct ELISA or simple two-layer ELISA assay, the solid support is coated with the biological sample and incubated with an antibody that recognizes the Masp52 protein, conjugated or bound to a marker compound. In the indirect ELISA, the solid support is coated with the biological sample and incubated with a primary antibody, which recognizes the Masp52 protein and then a secondary antibody, which recognizes the primary antibody, conjugated or bound to a marker compound. . In the sandwich ELISA or antigen capture and immunocomplete detection assay, the well is coated with a first antibody that recognizes the Masp52 protein, the problem biological sample is applied, so that the Masp52 protein will be retained in the well when it is recognized for the first antibody, and then a second antibody is applied that recognizes the Masp52 protein, conjugated or bound to a marker compound.

The term "marker compound", as used in the present description, refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive and / or chemiluminescent signal that allows the detection and quantification of the amount of the Masp52 protein. . The marker compound is selected from the list comprising radioisotopes, enzymes, fluorophores or any molecule capable of being conjugated with another molecule or detected and / or quantified directly. This marker compound can bind to the antibody directly, or through another compound. Some examples of directly binding marker compounds are, but are not limited to, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as ³³ P or ³⁵ S, fluorochromes such as fluorescein or metal particles, for direct detection by colorimetry, auto-radiography. , fluorimetry, or metallography respectively.

The quantitative detection of the Masp52 protein can also be performed by real-time PCR (RT-PCR or RTqPCR). The real-time detection of the amplified products can be carried out by the use of fluorescent molecules that are intercalated in the double-stranded DNA or by hybridization with different types of probes. Thus, in another preferred embodiment, the Masp52 protein is detected by RTqPCR using primers SEQ ID NO: 2 (primer MASP.220F) and SEQ ID NO: 3 (primer MASP.220R).

Another aspect of the present invention is a diagnostic kit or device, hereinafter kit of the invention, which comprises the elements necessary to analyze the amount of Masp52 protein in a biological sample. In a preferred embodiment, the kit of the invention also comprises the elements necessary to compare the amount detected in (a) with a reference amount. In another more preferred embodiment, it comprises the elements suitable for carrying out any of the methods of the present invention.

Said kit may contain all those reagents necessary to analyze the amount of Masp52 protein by means of any of the methods described previously in this document, such as, but not limited to, antibodies specific to the Masp52 protein, secondary antibodies or positive and / or negative controls. The kit can also include, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc. In the case of detection by RTqPCR, it may contain, but not limited to, primers, probes and all those reagents necessary to determine the expression of the Masp52 protein. The kit can also include, without any limitation, the use of buffers, polymerases, cofactors to obtain optimum activity of these, agents to prevent contamination, etc. On the other hand, the kit can include all the supports and containers necessary for commissioning and optimization. Preferably, the kit also includes the instructions for carrying out the methods of the invention.

The authors of the present invention have also demonstrated, as shown in the examples, that the use of specific humoral antibodies (IgG) against the catalytic region (hereinafter referred to as anti-CR) at dilution 1/50, 1 / 100 and 1/200, are capable of inhibiting cell invasion by metacyclic Trypomastigotes.

The Masp52 protein or any of its variants can be formulated in compositions for use as an immunogen (hereinafter, immunogens of the invention). These immunogens can also be used as vaccines in animals, and more particularly in mammals, including humans, or produce a response in the production of antibodies in animals. For the formulation of such compositions, an immunologically effective amount of the Masp52 protein or any of its variants is mixed with a suitable physiologically acceptable transporter for administration to mammals including humans. The immunogens may be covalently linked to each other, to other peptides, to a transporter protein or to other carriers, incorporated into liposomes or other similar vesicles, and / or mixed with an adjuvant or absorbent as is known in the field of vaccines. For example, they can be mixed with other immunostimulatory complexes. Alternatively, the immunogens are not coupled and merely mixed with a physiologically acceptable transporter such as a normal buffer or saline compound suitable for administration to mammals including humans. Therefore, another aspect of the invention relates to the use of the isolated Masp52 protein, or any of its variants, for the preparation of a medicament, or to the Masp52 protein, or any of its variants, for use as a medicament. A preferred embodiment of this aspect of the invention relates to the use of the isolated Masp52 protein, or any of its variants, for the preparation of a medicament for the treatment and / or the prevention of Chagas disease, or the Masp52 protein, or Any of its variants, for use in the treatment and / or prevention of Chagas disease.

The term "isolated" refers to a material (nucleic acid, peptide or protein) that is: (1) substantially or completely free of the components that normally accompany or interact with it in its natural form. The isolated material may optionally comprise another material that is not associated with the isolate in its natural form; or (2) in case the material is in its natural environment, said material has been synthetically altered by a deliberate human intervention that modifies its composition. The alteration that gives rise to the synthetic form of the material can be directed to the material (nucleic acid and / or protein) or to the natural environment.

In the sense used in this description, the term "variant" refers to a protein substantially homologous to the Masp52 protein. In general, a variant includes additions, deletions or substitutions of amino acids. The term "variant" also includes proteins resulting from posttranslational modifications such as, but not limited to, glycosylation, phosphorylation or methylation.

As used herein, a protein is "substantially homologous to the Masp52 protein" when its amino acid sequence has a good alignment with the amino acid sequence SEQ ID NO: 1, that is, when its amino acid sequence has a degree of identity regarding the amino acid sequence SEQ ID NO: 1, of at least 50%, typically of at least 80%, advantageously of at least 85%, preferably of at least 90%, more preferably of at least 95%, and even more preferably of at least 99%. The term "identity", as used herein, refers to the proportion of identical amino acids between two amino acid sequences that are compared. The percentage of identity existing between two sequences can be easily identified by one skilled in the art, for example, with the help of an appropriate computer program to compare sequences, which includes, but is not limited to, the BLASTP or BLASTN program , and FASTA (Altschul et al., J. Mol. Biol. 215: 403-410 (1999).

The term "fragment", as used in the present description refers to a portion of the Masp52 protein or one of its variants.

The term "functionally equivalent", as used herein, means that the protein or the fragment of the protein in question essentially maintains the immunological properties described herein. Such immunological properties can be determined by conventional methods such as those described in the examples that accompany this description.

As described above, the immunogens of the invention have protective antigenic sequences. These protective antigens are capable of generating a protective (immunogenic) host immune response, that is, a host response that leads to the generation of immune effector molecules, antibodies or cells that damage, inhibit or kill the invading biological entity, " thus protecting "the host from a clinical or sub-clinical disease and a loss of productivity. Such protective immune response can commonly be manifested by the generation of antibodies.

Therefore, another aspect of the invention refers to the antibodies generated by the immunization, with the Masp52 protein or any of its variants, of the animal, preferably a mammal, and more preferably a human mammal. In another aspect of the invention, the antibodies produced after the immunization of the animal, preferably a mammal, and more preferably human, hereinafter referred to as antibodies of the invention, are used as a medicine, that is, this aspect refers to the use of the antibodies of the invention in the preparation of a medicament. A preferred embodiment of this aspect of the invention refers to the use of antibodies of the invention for the preparation of a medicament for the treatment of Chagas disease, or to the antibodies of the invention for use in the treatment of Chagas disease.

The antibodies of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways. Thus, the antibodies may be in sterile aqueous solution or in biological fluids, such as serum. Aqueous solutions may be buffered or unbuffered and have additional active or inactive components. Additional components include salts to modulate the ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelants, and the like, and nutrients including glucose, dextrose, vitamins and minerals. Alternatively, the antibodies can be prepared for solid administration. Antibodies can be combined with various inert carriers or excipients, including but not limited to: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or corn starch; lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint or methyl salicylate.

Antibodies or their formulations can be administered to an animal, including a mammal and, therefore, to man, in a variety of ways. Such means include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intracecal, intraventricular, oral, enteral, parenteral, intranasal or dermal.

The dosage of antibodies to obtain a pharmaceutically effective amount depends on a variety of factors, such as, for example, age, weight, sex, tolerance, etc. of the animal, preferably mammal, and more preferably human.

Another aspect of the invention relates to a composition, hereinafter the composition of the invention, comprising the isolated Masp52 protein or any of its variants, an antibody of the invention, or any combination thereof, for use as a medicine. In a preferred embodiment In this aspect of the invention, the composition of the invention is used for the treatment and / or prevention of Chagas disease.

More preferably, the isolated Masp52 protein, or any of its variants, are found, or translated, in a therapeutically effective amount, capable of generating antibodies for use in the preparation of vaccines.

In the context of the present invention the term "vaccine" refers to an antigen preparation used to establish the immune system response to a disease. They are prepared of antigens that once inside the organism cause the response of the immune system, through the production of antibodies, and generate immunological memory producing permanent or transient immunity.

The term "antigen" herein refers to a molecule (generally a protein or a polysaccharide), which can induce the formation of antibodies. There are many different types of molecules that can act as antigens, such as proteins or peptides, polysaccharides and, more rarely, other molecules such as nucleic acids. Specifically, in this report, it refers to the Masp52 protein or any of its variants.

The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals. It includes, therefore, anti-CR antibodies that prevent the entry of metacyclic tripomastigotes into the cell. In the context of the present invention, it also refers to the Masp52 protein or any of its variants, which are capable of generating an immune response against a given organism, which is causing said disease in man or animals. It includes, therefore, what is known as a vaccine, as previously defined herein.

Therefore, in another preferred embodiment, the composition of the invention also comprises pharmacologically acceptable excipients. In another more preferred embodiment, the composition of the invention additionally comprises another active principle. In a more preferred embodiment, the composition of the invention is a vaccine, henceforth a vaccine of the invention. In other Even more preferred embodiment, the vaccine comprises an adjuvant.

In this specification, "active substance", "active substance", pharmaceutically active substance "," active ingredient "or" pharmaceutically active ingredient "means any substance or mixture of substances that is endowed with pharmacological, metabolic or immunological activity, or other direct effect on the diagnosis, cure, mitigation, treatment, or prevention of a disease or medical condition, or that affects the structure or function of the body.

In this report, the term "adjuvant" refers to an agent, as long as it does not have an antigenic effect in itself, which can stimulate the immune system by increasing its response to the vaccine. Although not limited to them, aluminum salts "aluminum phosphate" and "aluminum hydroxide" are the two adjuvants most commonly used in vaccines. Other substances, such as squalene, can also be used as adjuvants.

An alternative method of vaccine production is the use of molecular biology techniques to produce a fusion protein that contains one or more of the amino acid sequences of the present invention and a highly immunogenic peptide or protein, against a certain infection. . Therefore, in another preferred embodiment of this aspect of the invention, the vaccine of the invention has a recombinant origin.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Shows the 12.5% SDS-PAGE and silver nitrate staining of the proteins purified by WGL-Agarose from the Excretion Secretion Product (ESP) and of the fraction purified by Wheat Germ Lectin.

Fig. 2. It shows the MALDI-TOF obtained after the digestion with trypsin of the Masp52 and matches obtained with the Masp52 after the search for homologous sequences with the MASCOT 2.0 software (Matrix Science) integrated with the Biotool 2.2 software.

Fig. 3. Shows the structural and compositional motifs found through Expasy's proteomic tools server (http://expasy.org). There are two transmembrane regions in N- and C-terminal, a signal peptide (SEQ ID NO: 4) of amino acid 1 to 25, an ATP / GTP-binding site motif A (P-loop) motif (SEQ ID NO: 5) from 159 to 166 and an N-glycosylation site (SEQ ID NO: 6) from 465 to 473 B) Hydrophobicity plot for the Masp52 according to the Kyte-Doolitte hydrophobicity scale.

Fig. 4. Shows the Western blot of the trypomastigote metacyclic (M), trypomastigote from cell cultures (T), amastigotes (A), epimastigote (E) and Excretion-secretion product (ESP) forms of Trypanosoma cruzi using the Anti-CR B IgG) Western blot against anti-SP IgG in the forms trycycomastigotes metacyclic (M), trypomastigote from cell cultures (T), amastigotes (A) and epimastigote (E).

Fig. 5. Shows the RTqPCR to quantify the relative amount of transcribed mRNA of the Masp52 by comparing it against the expression of ribosomal 18S according to the method of ΔCT. The relative quantity for the trypomastigote metacyclic, trypomastigote derived from cell culture, amastigote phases was measured. and epimastigote.

Fig. 6. Shows the Focal Laser Microscopy of Ia Masp52 using anti-CR IgG. A) Mareaje for the different stages of the parasite, Trypomastigote derived from stumpy forms cell culture (A) Metacyclic trypomastigote (C),

Epimastigote (D) and Amastigote (E). Bar = 5 μm B) Marking of metacytic trypomastigotes after 2 hours of interaction with the host cell. The moment of cell-parasite interaction is observed. Bar = 5 μm (Fig. 6 B1), Bar = 10 μm (Fig. 6 B2) C) Tide for cells infected by T. cruzi containing amastigotes inside. Bar = 5 μm Fig. 7. It shows the immunocytochemistry of the Masp52 using the anti-CR IgG A) Flagellar Pocket of Trypomastigote derived from cell culture B) Flagellar Pocket of Trypomastigote Metacyclic C) Vacuoles of Trypoamstigote metacyclic D) Trypomastigote derived from cell culture, locator! in membrane, cytosol and exterior cell E) Negative control. Bar = 0.25 μm

Fig. 8. It shows the effect after 4 hours of interaction with Vero cells of bentonite particles adsorbed to Ia Masp52 and BSA as a control by locating them using anti CR IgG and anti BSA polyclonal serum A) Masp52 Mareaje We see a distribution by the cellular cytosol (arrows) of the particles B) Marking of the BSA. We do not observe the presence of proteins in the cells.

Fig. 9. It shows the effect of antibodies against Masp 52 on the invasiveness of metacyclic trypomastigotes. We tested with dilution anti-CR IgG

1/50, 1/100 and 1/200, incubating it for half an hour with the metacyclic Trypomastigotes and after washing the serum parasites, leaving them to interact for 2 hours in front of Vero cells. The bars represent the mean are the mean ± the standard deviation of triplicates for each dilution. To find if there are significant differences between the data groups, the Bonferroni test was used.

EXAMPLES

Next, the invention will be illustrated by tests carried out by the inventors, which shows the effectiveness of the detection of the Masp52 protein in obtaining useful data for the differential diagnosis of Chagas disease.

Material and methods.

Parasite strains, cultures and cells used.

The strain of Trypanosoma cruzi used in these experiments was strain PAN2, (isolated from a 32-year-old male patient residing in the District of Arraiján, community of Burunga, Panama), donated in 2006 by Dr. A. Ying de Ia

University of Panama and maintained by cryopreservation from that moment. The epimastigote forms were grown in culture at 28 ⁰ C in MTT medium supplemented with 10% Inactivated Fetal Bovine Serum (SBFI) (Ruiz-Perez et al., 1986. Arzneimittel Forschung 36: 13-16). Infectious metacyclic trypomastigote forms were obtained "in vitro" in modified Grace Medium, according to Osuna et al. 1979. Rev Iber Parasitol 39: 129-133, and Osuna et al., 1990 International Journal of Parasitology. 20 (5): 673-676.

Cells Vero host (ECACC 84113001) were grown at 37 ⁰ C 5% CO ² in plastic bottles 75 cm2 (Costar) containing Dulbecco ^'s modified ^Eagle' s medium (DMEM, Gibco) supplemented with 10% v / v SBFI (Gibco). The procedure to infect host cells was developed according to Osuna A et al, 1984 (inter. J. for Parasitol. 14 (3): 253-257). The metacyclic trypomastigote forms obtained "in vitro" were resuspended in DMEM medium without serum and added to a cell culture. The parasite-cell ratio was adjusted to 5: 1 and the infection was performed for 12h 37 ⁰ C. Cultures were washed with culture medium to eliminate metacyclic trypomastigotes that had not penetrated. Infected cells were cultured in DMEM 10% SBFI medium (pH: 7.2).

The trypomastigote forms were obtained from cultures of Vero cells previously infected with the metacyclic forms of T. cruzi. After 96h of infection, the supernatant containing the trypomastigotes was centrifuged at 30Og for 10 min. The button with the trypomastigote forms was resuspended in DMEM medium and centrifuged twice to obtain the parasitic forms.

Infected cultures were maintained for 8 days, then amastigote forms were purified by discontinuous gradient (1,100, 1,090, 1,080, 1,070 g / ml) and prepared as described below. The purified amastigote forms were collected from the interface 1,070 / 1,080 g / ml.

All forms of the biological cycle of T. cruzi used in the development of the work were purified in a discontinuous gradient of percoll as described in Gil et al., 2003 {Parasitol Res 90: 268-272) and had at least 95% of proven purity both under staining and by Neubauer chamber counting. Purification of Ia Masp52 and sequencing

The obtaining and purification of the Masp52, was made from the excretion-secreetion product (ESP) during the cell-parasite interaction. For this, semiconfluent cultures of Vero cells were removed the medium and after washing them repeatedly in DMEM without serum, they were infected with a suspension of trypomastigote forms of the parasite in DMEM without serum with a ratio, conditions and time of infection equal to how It has been cited above.

After the interaction period, and the medium was removed, it was centrifuged at 150Og 10 minutes at 4 ⁰ C in order to eliminate forms of the parasite that had not penetrated and filtered the supernatant through a 0.22 μm diameter filter (Sartorius, Sartolab twenty).

ESP proteins were concentrated with 5 kDa molecular exclusion filters (Amicon, ultra ™, Millipore) and subsequently chromatographed through the mono column P 5 / 200GL (GE) with polybuffer 96 (GE) with liquid phase, collecting the fractions between the pl of 5.2 to 4.6. These fractions were chromatographed again through a column of Wheat Germ Lectin-Agarose (WGL) (Sigma) in order to purify the N-glycosylated proteins. As a wash buffer, a 0.1 M pH: 9 Carbonate Buffer was used, and as eluent of the lectin bound fraction, 2 volumes of 0.5 M N-Acetylglucosamine (Sigma) in Carbonate Buffer. All chromatographs were performed on an AKTA ™ Purifier (GE) device.

The result of the chromatography was evaluated by electrophoresis in 12.5% SDS_PAGE polyacrylamide gels. as described below.

The sequencing and identification of the Masp52 was carried out by the Proteomics Service of the Severo Ochoa Molecular Biology Center in Madrid. The band of interest was trimmed manually minimizing the amount of gel. Digiting "in-situ" in automatic mode (using a Bruker robot digester) by trypsin using a protocol based on that described by Shevchenko et al., 1996. Anal. Chem. 68: 850-858. The digestion supernatant (containing the peptides) was acidified with TFA (0.1% final concentration) drying in a Speedvac to resuspend it in 5 ml of TA (Trifluoroacetic acid 0.1% Acetonitrile 33%). A small aliquot (0.5 ml) was deposited on an "Anchor-chip" (Bruker) plate using DHB (2,5- Dihydroxybenzoic acid) as a matrix at a concentration of 5 g / l using the "fast evaporation" method. The plate was measured in a mass spectrometer of the MALDI-TOF type (matrix-assisted laser desorption ionisation / time-of-flight), autoflex model (Broker) equipped with reflector. The mass spectra obtained were used as "peptide fingerprint" for the identification of proteins in the databases using search engines accessible in the network (Mascot, Profound).

Search for homologies and structural reasons.

The homology of the amino acid and nucleotide sequence was studied using the T. cruzi genome database in T. cruziDB (http://tcruzidb.org/tcruzidb/) using the BLASTP and BLASTN algorithms in GenBank, of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The search for structural motifs was carried out through the Expasy proteomic tools server (http://expasv.org) with the programs: Motif Sean for the search for structural motifs; SignalP 3.0 for the search of the signal sequence; Tm Pred for the transmembrane sequence and Protscale to predict the hydrophobicity of our sequence using the Kyte-Doolitte hydrophobicity scale.

Synthesis of synthetic peptides and obtaining antibodies.

The synthesis of the designed peptides was carried out in the proteomics service of the CBM. Two peptides were designed corresponding to the N-proximal zones (signal peptide) of residues 1-25 and of the catalytic zone of Ia Masp52 (ATP / GTP binding motif A) of residues 159 to 181.

Each peptide was used to immunize 5 female Balb / c mice by intraperitoneal inoculation of 50 μg of the peptide, according to the protocol described by Espino et al., 2007. (Exp Parasite !. 2007 Sep; 117 (1): 65- 73. Epub

2007 Mar 27). Three weeks after the first immunization, the antibody titer by indirect ELISA test. Subsequently, we purified the IgG from both the polyclonal serum against the synthetic peptides, and from a preimmune mouse serum using the HP Spin Trap Protein A Kit (GE health care). Subsequently, the specific IgGs obtained were purified through an affinity column using the immobilized antigen on a Sepharose BrCN (GE) column. The concentration of the purified IgGs was adjusted to the original one containing the serum sample. The specific IgGs against the catalytic region will be referred to as anti-CR and against the signal peptide as anti-SP.

Cell Invasion Assay

Semiconfluent Vero cells (3 x IO ⁵ cells / well) were infected with metacyclic trypomastigote forms. The parasite / cell ratio was 5: 1, developed Ia invasion 2h at 37 ⁰ C in serum free DMEM with dilutions 1/50, 1/100 and 1/200 anti-Ig CR. Immunoglobulins of a mouse preimmune serum were used as a control. At the end of the interaction period, the cultures were washed, fixed in methanol and stained with the Preimmunized Diff-Quick (Medion Diagnostics, GMBH, CH-3186 Düdingen). After being stained, the cells were studied under a microscope to determine the percentage of parasitization, adhesion and number of intracellular parasites. A minimum of 500 cells per well were examined, and each experiment was repeated at least 3 times, calculating the number of parasitized cells and calculating the cell-parasite ratio.

SDS-PAGE and Western Blot analysis

The expression studies of the Masp52 in the different forms of the life cycle of the parasite was evaluated by immunoblot in the different stages, 5 x 10 ⁷ organisms of each of the phases of the parasite were resuspended in

2 ml of lysis buffer; 2OmM PBS, 0.25mM Sucrose, 1mM EDTA,

0.145mM KCI, 1 mM DTT at pH: 7.4 plus a protease inhibitor cocktail

Complete Mini (Roche Molecular Biochemicals) After treatment for 10 min of the protozoa in said buffer, they were sonicated at 0 ^o C for three cycles of 30 seconds and electrophoresis in 12.5% of SDS-PAGE (Laemmli,

1970) with the PhastSystem (GE). The protein sample was adjusted to Ia same concentration, so that all the wells of the electrophoresis contained the same amount of proteins and prepared in the same buffer (10 mM Tris / HCI; 1 mM EDTA, pH 8.0; 2.5% SDS and 17% glycerin and bromophenol blue 0.01%) and heated to 98 ° C 5 min.

Western blots were developed with hydrophobic membranes of polyvinyl difluoride (PVDF) (Hybond-P. Amersham) according to the method developed by Towbin and Staehelin 1979. (Proc Nati Acad Sci USA 1979; 76: 4350-4354). The spots were exposed to anti-CR or anti-SP (tested at 1: 50) for 2h at 37 ⁰ C followed by a peroxidase conjugate (Polyclonal Goat Anti Mouse Immunoglobulins HRP (DakoCytomation) for 2 h at 37 ⁰ C. The reaction it was revealed with 3-3 ^' Diaminobenzidine Tetrahydrochlorhydric in Tris HCI buffer at pH 7.2 The gels were digitized and processed by QuantiScan software (Biosoft, Cambridge, UK) in order to quantify the bands. I perform using the Bradford method (Bradford, 1976. Analytical Biochemistry 72: 248-254).

MRNA isolation and RTqPCR development

The total mRNA of the different stages was isolated using the SV Total RNA Isolation kit (Promega), the quality of the samples was verified by agarose gels.

For the reverse transcription, the iScript ™ cDNA Synthesis Kit (Biorad), which contains oligo (dT) and random primers, was used to obtain the most representative RNA template possible.

The quantification of the expression of the Masp52 was evaluated by performing a RTqPCR using the Sensimix dT Kit (Quantace). Quantification was performed in all stages of the life cycle studied.

The primers used were: MASP.220F (SEQ ID NO: 2) and MASP.220R (SEQ ID NO: 3) which give rise to a 198 bp amplicon (SEQ ID NO: 7). To normalize the amount of Masp52 cDNA present in each sample, the primers were used:

V1 (SEQ ID NO: 8) and V2 (SEQ ID NO: 9)

for the 18S rRNA ribosomal gene (Clark and Pung, 1994. Mol Biochem Parasite / 66: 175-179), from T. cruzi obtaining an amplicon of 179 bp (SEQ ID NO. 10). The bands obtained by PCR were confirmed by sequencing using the BigDye® Terminator v 1.1 cyclesequencing kits (Applied Biosystems, CA, USA). The relative quantification of the samples was carried out, according to the ΔC _T method _, where the Ratio 18S / Masp52 = 2 ^{CT 18S "cτ Masp52} . All tests were performed in triplicate.

Immunolocation and immunofluorescence.

The protein was located in the different phases of the T. cruzi life cycle, obtained as previously described. The different buttons of the four forms were washed three times with 5 ml of PBS 0.125 M and fixed for 12 h at 4 ⁰ C in 1% glutaraldehyde and 2% formaldehyde in Cacodylate Buffer with 0.1 M sucrose (pH: 7), being then embedded in LRWhite resin.

Samples were incubated with anti-CR 1 h at 37 ⁰ C and treated Ia to visualize antigen-antibody reaction marking the primary antibody with a secondary antibody anti - mouse IgG labeled with gold (Sigma). Finally, the sample was stained with uranyl acetate under a Zeiss EM-10C transmission microscope.

In those experiments in which it was intended to locate the protein inside the parasite forms or its location during the infection process, the cells or the parasite forms were fixed in acetone at -20 ° C, at 2 hours of interaction parasite / cell, or after 60 hours of having performed the infection in order to observe the presence and location of the protein in amastigotes or in parasitized cells. Once fixed and washed with PBS, we incubated the preparations 1 h with the anti-CR IgG at a dilution to 1/100.

As a secondary antibody, an anti mouse labeled with

Fluorescein isothiocyanate (FITC) (Sigma) in Blocking Buffer for 1 ha room temperature. Finally, the preparations were treated for 15 minutes with a 10μg / ml DAPI solution. The preparations were mounted and preserved in medium mounting (Prolong Antifade Kit, Molecular Probes), being observed in a Leica DMI6000 confocal laser microscope that incorporates a filter system for FITC (average wavelength 530 nm and maximum 490nm).

Adsorption of Ia Masp52 to Bentonite particles

For the adsorption to bentonite particles, the technique of Kagan &Norman's (1970. Manual of Clinical Microbiology 453-486) was used to select the bentonite particles for the adsorption of the Masp52. A suspension of 100ul of particles with a diameter of 1 to 2 μm was incubated 12 h at 4 ⁰ C with 50 ul of a solution of 20 μg / ml protein. The particles were adsorbed to Bovine Serum Albumin (BSA) as a control at the same concentration and conditions as those used as described below. Masp52 and BSA adsorbed on bentonite particles were incubated with Vero cells (3 x 10 ⁵ ) at 37 ° 4 h. The cells were washed with PBS and fixed in acetone to study them by immunofluorescence, being treated with a 1/100 dilution of the anti-CR antibody and with a secondary anti-mouse antibody labeled with fluorescein as described above. A 1/100 dilution of a polyclonal serum in anti-BSA mouse (Sigma) and the same secondary antibody used previously was used in the control cells. A 0.01% solution of Evans Blue was used as a bleach, studying the preparations under confocal fluorescence microscopy as described in the previous section.

RESULTS

1. Purification, sequencing and search for structural reasons of the masp52.

From the process of purification of proteins from the interaction medium, two bands, one of 66 kDa and another of 52 kDa, were obtained by electrophoresis (Fig. 1) which, analyzed by MALDI-TOF MS and MS / MS, corresponded to bovine bovine albumin - Bovine Serum Albumin (BSA) - from one of the culture media and the 52 kDA band that through a Peptide mass fingerprinting (PMF) or peptide fingerprint found 7 matches, obtaining a value of 47.9% and 17% coverage for the sequence shown in Figure N ⁰ 2. This sequence was in the databases with the name of mucin-associated surface protein (MASP), putative and with an EMBL access number: XP_820015.1, calling it here as Masp52.

A search of possible structural motifs present in the Masp 52 was performed through the use of Expasy's proteomic tools server (http://expasy.org), a signal peptide (amino acids 1-25) was obtained, two transmembrane regions at the ends N-Terminal (amino acids 9-28) and C-terminal (amino acids 493-510), an ATP / GTP-binding site motif A (P-loop) that we can name as a catalytic center, (amino acids 159-166) and a site for N-glycosylation (465-470) (Fig. 3).

2. Protein expression, Western blot and RTqPCR.

The analysis of the expression of the Masp52 in the different stages of T. cruzi by SDS PAGE electrophoresis and its subsequent analysis by Western blot using the anti-CR IgG, shows us how this protein is recognized as a single band with different degree of expression in the different stages of the parasite (Fig. 4A). Thus, the expression in metacyclic trypomastigotes is 12 times higher than that found in epimastigotes, five times higher than that of amastigotes and twice higher than that found in trypomastigotes derived from cell cultures.

The result of the study of mRNA by RTqPCR showed similar results (Fig. 5), all stages had synthesis of the specific mRNA of Ia Masp 52, but with different levels of expression. In the metacyclic trypomastigote forms the synthesis of specific mRNA is three times greater than that of tissue-derived trypomastigote, eleven times greater than that of amastigotes and fifty times greater than that of epimastigotes.

On the other hand, when the recognition in the four phases of T. cruzi was carried out using the anti-SP IgG, to observe the expression pattern of proteins of the MASP family by western blot, the 6-band expression was obtained in the metacyclic trypomastigotes, 4 bands in the trypomastigote forms, 3 bands in the amastigotes and 3 bands in the epimastigotes, although in the latter the level of expression was very low (Fig. 4B).

Using the same technique, using antibodies against the catalytic domain, it is shown that Ia Masp52 is a secreted protein, being in the ESP of the metacyclic trypomastigotes in their interaction with Vero cells, their membranes or fixed Vero cells, which seems to indicate given of the absence of the protein in the cell-free media that said protein is secreted in an inducible way to the environment after contact of the parasite with the cell membranes.

3. Location of the Masp52 in the different stages of T. cruzi and adsorbed to bentonite particles.

The studies carried out by confocal laser microscopy using the Immunoglobulins in front of the catalytic center showed how the presence of the Masp 52 is observed both in the cytosol and linked to the plasma membrane in the different stages of the parasite, as shown in Fig. 6 A.

In those preparations where the study of the immunolocation of the protein was carried out during the metacyclic tripomastigote-cell interaction process, the presence of the protein in the cytosol of the trypomastigotes was also observed, detecting the protein at the point of contact between the parasite and the cell. The fluorescence was also dispersed in the cytoplasm of the host cell together with the presence of the parasite (Fig. 6B).

In those cells where the parasite forms had already been transformed and multiplied as amastigote forms, it was observed how the Masp52 appears surrounding the amastigotes in the space between the parasite and the cytoplasm of the host cell (Fig. 6C).

The studies of ultrastructural location in the different stages (trypomastigote, metacyclic trypomastigote derived from cell culture and epimastigote), using the IgG against the catalytic domain of Ia Masp52, are Observe how the gold marks are located both in the parasite membrane, and in the cytosol, the number of particles being higher in the stages with high infective capacity, that is trypomastigotas (Fig. 7A, 7C). The aggregates of marks are located inside vacuoles (Fig. 7B), close to the Flagelar Pocket and the root of the scourge in the infecting forms (Fig. 7A and C), while the Masp52 appears in the amastigotes in the cytosol .

Confocal laser optical microscopy studies using anti CR or anti BSA antibodies as a control in the interaction between Masp52 absorbed in inert bentonite particles and non-phagocytic Vero cell cultures and BSA coated bentonite particles, revealed that the cells endocyted the particles of Masp52 coated bentonite, while BSA coated control particles were removed in washes after the interaction process (Fig. 8). Masp 52 particles are located within vacuoles in the cytoplasm of the cells that have interacted.

5. Effects of the antiserum against Masp52 in the cell invasion by metacyclic trypomastigotes

Fig. 1 summarizes the results obtained by treating cell-parasite interactions with anti-CR antibodies in different dilutions (1: 50; 1: 100; 1: 200) of specific immunoglobulins. The percentage of inhibition versus control ranged in a significant range from 17.14% to 1/200, 61.9% to 1/100 and 77.14% to 1/50 for intracellular parasites in Vero cells. Penetration levels were reduced by 30.7% for 1/200, 74.38% for 1/100 and 78.9% for 1/50, and the percentage of inhibition of metacyclic trypomastigotes adhered to cells of 49.92% for 1/200, 74.35% for 1/100 and 74.48% for 1/50 dilution.

Claims

1. Method of detecting the presence of the T. cruzi parasite in an individual, comprising: a. obtain an isolated biological sample from said individual, b. Detect the Masp52 protein in the biological sample isolated from (a).

2. Method of detecting the presence of the T. cruzi parasite in an individual according to the preceding claim, wherein the individual of step (a) is a mammal.

3. Method of detecting the presence of the T. cruzi parasite in an individual according to any of claims 1 or 2, wherein the individual of step (a) is a human mammal.

4. Method of obtaining useful data for the diagnosis of Chagas disease, which comprises: a. obtain an isolated biological sample from an individual, b. Detect the amount of Masp52 protein present in the biological sample isolated from (a).

5. Method of obtaining useful data for the diagnosis of Chagas disease according to the preceding claim, which further comprises: c. compare the amount detected in step (b) with a reference amount.

6. Method of obtaining useful data for the diagnosis of Chagas disease according to any of claims 4 or 5, wherein the detection of the amount of Masp52 protein is carried out by incubation with a specific antibody in an immunoassay.

7. Method of obtaining useful data for the diagnosis of Chagas disease according to any of claims 4 to 6, wherein the immunoassay is a Western blot.

8. Method of obtaining useful data for the differential diagnosis of Chagas disease according to any of claims 4 to 7, wherein the Western blot is carried out using anti-CR IgG.

9. Method of obtaining useful data for the differential diagnosis of Chagas disease according to any of claims 4 to 7, wherein the Western blot is carried out using anti-SP IgG.

10. Method of obtaining useful data for the differential diagnosis of Chagas disease according to any of claims 4 or 5, wherein the detection of the amount of Masp52 protein is performed by RTqPCR.

11. Method of obtaining useful data for the differential diagnosis of Chagas disease according to the preceding claim, wherein the RTqPCR is carried out using the primers whose sequence is included in SEQ ID NO: 2 and SEQ ID NO: 3.

12. Use of the isolated Masp52 protein, or any of its variants, for the preparation of a medicine.

13. Use of isolated Masp52 protein, or any of its variants, for the preparation of a medicament for the treatment and prevention of Chagas disease.

14. Antibodies generated by the immunization of an animal with the Masp52 protein, or any of its variants.

15. Use of the antibodies generated by the immunization of an animal with the Masp52 protein for the preparation of a medicament.

16. Use of the antibodies generated by the immunization of an animal with the Masp52 protein for the preparation of a medicament for the treatment and prevention of Chagas disease.

17. Composition comprising the Masp52 protein according to any of claims 12 or 13, or an antibody according to any of claims 14 to 16.

18. Composition according to claim 17, which further comprises pharmacologically acceptable excipients.

19. Composition according to any of claims 17 or 18, which is a vaccine.

20. Composition according to the preceding claim, which further comprises an adjuvant.

21. Composition according to any of claims 19 or 20, wherein the vaccine has a recombinant origin.

22. Composition according to any of claims 19 or 20, which additionally comprises another active ingredient.