ES2343236B2 - USE OF MASP 52 PROTEIN FOR DIAGNOSIS, TREATMENT AND PREVENTION OF CHAGAS DISEASE. - Google Patents

Abstract

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

The use of the Masp52 protein, of the MASP family (Mucin associated Surface proteins) is proposed for the diagnosis, treatment and prevention of Chagas disease, including a method of detecting the presence of the T. cruzi parasite in an individual , a method of obtaining useful data for the diagnosis of Chagas disease and a diagnostic kit comprising the elements necessary to analyze the amount of Masp52 protein in a biological sample.

Description

Use of the Masp 52 protein for diagnosis, the 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 MASP family protein ( Mucin associated Surface proteins ) for the diagnosis, treatment and prevention of Chagas disease. .

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Prior art

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 (Tarleton 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 Kinetoplástida Order, being the only one of the trypanosomes that presents an obligatory phase of intracellular multiplication in the vertebrate host, the metacyclic Tripomastigote forms being 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 by 1 µm wide. During this stage, the trypomastigote does not multiply in the host's blood. When the parasite infects the striated cardiac muscle fibers or phagocytes, the flagellum is shortened and transformed into a round amastigote of 2 to 5 µm in diameter and with a very short or nonexistent external flagellum, this is multiplied by fission binary forming "clusters" or "nests" that 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 when feeding on man or domestic animals or Infected wild Trypomastigotes migrate to the middle intestine of the insect where they become epimastigotes, flagellated wide, very mobile, with the cinetoplast between the nucleus and the scourge free. There they divide a large number of times, leaving the Infected insect for life. The epimastigotes are transformed into metacyclic trypomastigotes and migrate to the posterior intestine of 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 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 within a period of two to three months resulting in 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.

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 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 xenodiagnostic is the most sensitive test (92%), which can also be used to study 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 healing is still controversial. By means of the complement-mediated lysis test (CML), lytic antibodies (LA) of T. cruzi were detected that are attached to epitopes of live trypomastigotes and are related to host 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 evaluation criterion for Chagas disease.

Using 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 the serological tests (detection of LA - lytic antibodies - and CSA - conventional serology antibodies -) and parasitological evaluations as a blood culture (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 to say, with HE-negative, in which LAs are not detected while the CSAs are present;

c) cured patients (CUR), with HE-negative, which have no LA or CSA antibodies, Y

d) as a control, a group of people do not Chagasic (NC).

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

The differential diagnosis that allow to distinguish in which stage of development the disease, as well as monitoring the course of it, 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 the Nifurtimox, developed in 1960 by Bayer and Benzinidazole, developed in 1974 by Roche. Both have a low rate of Healing and side effects.

The family of MASPs ( Mucin associated Surface Proteins ) proteins was described for the first time following sequencing of the entire genome of the Trypanosoma cruzi parasite, and seems exclusive to 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 the TcMUC II 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 III et al , 2006. J .Proteome 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 various 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 surface glycoproteins of 72 kDa molecular weight, 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) employed a 90 kDa molecular weight glycoprotein, which does not cross with mammalian tissues and induced protection and increased reaction levels of delayed hypersensitivity when saponin is used as an adjuvant (Scott et al ., 1984. Int. Archs. Allergy Appl. Immun. , 74, 373-377), but did not cause parasltemia negativization, after a challenge with parasites in mice and monkeys A glycoprotein of similar molecular weight but present only in metacyclic tipomastigotes was mentioned as a resistance inducer 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), parasite excretion-secretion products (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 circumvent the immune response (Taibi et al ., 1993. J. Immunol. , 151, 2676-2689).

The use of purified 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 great expansion of the Transialidase family throughout the genome and its ease to mutate 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, vaccine development has been dramatically limited due to the debate on the mechanisms involved in Chagas disease. Thus, 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 reasons why 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 in addition the antiparasitic efficacy of the compounds varies according to the geographical region, probably as a result of the different intrinsic susceptibility to the 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 discontinuation of treatment.

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

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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 termed 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 the Masp52 (CR), or by studying the mRNA by RTqPCR as qualitatively (using the IgG anti-peptide signal (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 into 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:

to)

obtain an isolated biological sample of said individual,

b)

detect Masp52 protein in the biological sample isolated from (a).

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Organisms of the Trypanosoma cruzi species belong to Superuk 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 , thus 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 ( Mucin associated Surface proteins ).

In the context of the present invention, the term "Masp52 protein" is defined by a sequence of nucleotides or polynucleotide, which constitutes the sequence protein encoder collected in SEQ ID NO: 1, and which may comprise various variants from:

a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: one,

b) nucleic acid molecules whose chain complementary hybrid with the polynucleotide sequence of a),

c) nucleic acid molecules whose sequence differs from a) and / or b) due to code degeneration genetic,

d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with a identity of at least 80%, 90%, 95%, 98% or 99% with the SEQ ID NO: 1. In which the polypeptide encoded by said acids Nucleic has the activity and structural characteristics of Masp52 protein. It includes, therefore, various variants of the Masp52 protein, that is, proteins resulting from modifications posttranslational, such as, but not limited to, glycosylation, phosphorylation or methylation. The term "variant" is defined later in this report.

Masp52 protein can be detected 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 Masp52 in the different stages of T. cruzi can be performed by SDS PAGE electrophoresis, and its subsequent analysis by Western blot using 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 higher than that found in trypomastigotes derived from cell cultures. When recognition in the four phases of T. cruzi is performed using anti-SP IgGs, to observe the expression pattern of MASP family proteins by Western blotting , 6-band expression was obtained in the 4-band metacyclic trypomastigotes. 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 by 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 higher than that of tissue-derived trypomastigote, eleven times greater than that of amastigotes and fifty times greater than that of epimastigotes.

Thus, another aspect of the invention is refers to a method of obtaining useful data for diagnosis of Chagas disease, hereafter second method of the invention, comprising:

a) obtain an isolated biological sample from a individual,

b) detect the amount of Masp52 protein present in the biological sample isolated from (a).

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In a preferred embodiment, the second method of the invention further comprises:

c) compare the amount detected in step (b) With a reference amount.

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Steps (b) and / or (c) of the methods described previously they can be totally or partially automated, by 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 you can understand other additional steps, for example related to the pre-treatment of the sample or evaluation of Results obtained through 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 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 appropriate therapeutic regimens. This detection, as understood by one skilled in the art, 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 a person skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining 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 measure of quantity or concentration, preferably semi-quantitatively or quantitative, can be carried out directly or hint. The direct measure refers to the measure of the quantity or protein concentration, based on a signal that is obtained directly from protein, and that is directly correlated with the number of protein molecules, present in the sample. This signal - which we can also refer to as a signal of intensity - can be obtained, for example, by measuring a value of intensity of a chemical or physical property of the protein. The indirect measure includes the measure obtained from a component secondary (for example, a different component of the product of the gene expression) or a biological measurement system (for example the measurement of cellular responses, ligands, "tags" or enzymatic reaction products).

The term "quantity" as it is used in the description, refers but is not limited to the absolute or relative amount of protein, as well as any another value or parameter related to them or that may derive from these. These values or parameters include values of signal strength obtained from any of the physical or chemical properties of the protein obtained by direct measurement, for example, spectroscopy intensity values of masses 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 but is not limited to the comparison of the amount of Masp52 protein in the sample biological to analyze, also called biological sample problem, with an amount of Masp52 protein from a reference sample Desirable described elsewhere in this description. The reference sample can be analyzed, for example, simultaneously or consecutively, together with the biological sample problem. The comparison described in section (c) of the methods of the The present invention can be performed manually or assisted by computer.

The term "reference amount", as used in the description, refers to the amount of the absolute or relative amount of the Masp52 protein forms that makes it possible 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 a patient's serum with Chagas disease in a certain clinical phase. In other preferred embodiment of this aspect of the present invention, the reference quantity is obtained from a sample of reference. The reference amount can also be obtained, by example, of the normal distribution limits of an amount found in samples obtained from a population of patients with Chagas disease in different phases, using techniques well known statistics.

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 herein, refers to any analytical technique that is based on the reaction of conjugation of an antibody with the sample obtained. Examples of immunoassays known in the state of the art are, for example, but not limited to: immunoblot , enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry or protein chips .

The term "antibody" as used herein, it refers to immunoglobulin molecules and immunologically active portions of molecules of immunoglobulins, that is, molecules that contain a site of antigen binding that specifically binds (immunoreacts) with the Masp52 protein. Examples of portions of molecules of immunologically active immunoglobulins, include fragments F (ab) and F (ab ') 2 that can be generated by treating the antibody with an enzyme such as pepsin. Antibodies they can be polyclonal (typically include different antibodies directed against different determinants or epitopes) or monoclonal (directed against a single determinant in the antigen). He monoclonal antibody can be altered biochemically, by genetic manipulation, or it can be synthetic, lacking, possibly the antibody in whole or in part, of portions that are not necessary for the recognition of the Masp52 protein and being replaced by others that communicate to antibody additional advantageous properties. The antibody can be also recombinant, chimeric, humanized, synthetic or a combination of any of the above. An "antibody or recombinant 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 isotypes or main 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 through the 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 Gel Electrophoresis ) technique is based on the protein mapping of the study samples with one of three fluorochromes (Cy2, Cy3 or Cy5) before separation. The samples are then mixed and separated in a single two-dimensional gel, minimizing experimental variability. Due to the specific marking of each sample they 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, proteins are transfer to a support or to a membrane, for example, but without limited, PDVF, nitrocellulose or cellulose acetate. This membrane hybridizes with a specific antibody (also called antibody primary) that recognizes the Masp52 protein. Then the membrane hybridizes with an antibody (also called antibody secondary) capable of specifically recognizing the antibody primary and that is conjugated or bound with a compound marker. 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 an antibody is not necessary secondary. Once the protein is detected, it can be determined relative molecular size, comparing its migration with migration of a control protein that is detected simultaneously, preferably on the same support, which has a size known.

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

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 upon recognition. by 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 signal and / or chemiluminescent that allows the detection and quantification of the amount 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 it can bind to the antibody directly, or through another compound. Some examples of binding compounds that bind directly are, but not limited to, enzymes such as phosphatase alkaline 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.

Quantitative detection of Masp52 protein can also be done by real-time PCR (RT-PCR or RTqPCR). The real-time detection of amplified products can be carried out by means of use of fluorescent molecules that intercalate in the DNA double chain 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 kit or diagnostic device, hereafter 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 further comprises the necessary elements to compare the amount detected in (a) with A reference amount. In another more preferred embodiment, it comprises the appropriate elements to carry out any of The methods of the present invention.

This kit can contain all those Reagents needed to analyze the amount of Masp52 protein by any of the methods described above in this document as, for example, but not limited to antibodies specific for Masp52 protein, secondary antibodies or positive and / or negative controls. The kit can also include, without No type of limitation, buffers, extraction solutions proteins, agents to prevent contamination, inhibitors of protein degradation, etc. In the case of detection by RTqPCR may contain, but is 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 type of limitation, the use of buffers, polymerases, cofactors to obtain an optimal activity of these, agents to prevent pollution, etc. On the other hand, the kit can include all supports and containers necessary for commissioning and optimization Preferably, the kit further comprises the instructions for carrying out the methods of the invention.

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

The Masp52 protein or any of its variants it can be formulated in compositions for use as an immunogen (from hereinafter, immunogens of the invention). These immunogens they 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 effective amount immunologically of the Masp52 protein or any of its variants is mixed with a suitable acceptable conveyor physiologically for administration to mammals including humans. Immunogens may be covalently linked between them, to other peptides, to a transporter protein or to others transporters, incorporated into liposomes or other vesicles similar, 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 carrier such as a normal buffer or saline compound suitable for administration to mammals including humans.

Therefore, another aspect of the invention is refers to the use of isolated Masp52 protein, or any of its variants, for the preparation of a medicine, or to the protein Masp52, or any of its variants, for use as a medicine. A preferred embodiment of this aspect of the invention relates to when using isolated Masp52 protein, or any of its variants, for the preparation of a medication for the treatment and / or the prevention of Chagas disease, or Masp52 protein, or any of its variants, for use in the treatment and / or the Chagas disease prevention.

The term "isolated" refers to a material (nucleic acid, peptide or a protein) found: (1) substantially or completely free of the components that they usually accompany him or interact with him in his natural way. The insulated material may optionally comprise another material that it is not associated with the isolate in its natural form; O well (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 results in the synthetic form of the material can be directed to the material (nucleic acid and / or protein) or to the environment natural.

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, for example, but without limited, glycosylation, phosphorylation or methylation.

As used here, a protein is "substantially homologous to the Masp52 protein" when its amino acid sequence presents a good alignment with the amino acid sequence SEQ ID NO: 1, that is, when its sequence of amino acids has a degree of identity with respect to the sequence of amino acids SEQ ID NO: 1, of at least 50%, typically of, at least 80%, advantageously at least 85%, preferably of at least 90%, more preferably of, at less, 95%, and, even more preferably, 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 easily be 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. Blol. 215: 403-410 (1999).

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

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

As described above, the immunogens of the invention have antigenic sequences protective These protective antigens are capable of generating a host protective (immunogenic) 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, "protecting" so to the host of a clinical illness or sub-clinical and a loss of productivity. Such Protective immune response can be commonly manifested by the Antibody generation

Therefore, another aspect of the invention is refers to the antibodies generated by immunization, with the Masp52 protein or any of its variants, of the animal, preferably a mammal, and more preferably a mammal human. In another aspect of the invention, the antibodies produced after immunization of the animal, preferably a mammal, and more preferably human, hereinafter referred to as antibodies of the invention, they are used as a medicine, that is, this aspect refers to the use of the antibodies of the invention in the Preparation of a medicine. A preferred embodiment of this aspect of the invention relates to the use of the antibodies of the invention for the preparation of a medicament for the treatment of Chagas disease, or to the antibodies of the invention to its 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 ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, 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 salicylate
methyl.

Antibodies or their formulations can administered to an animal, including a mammal and, therefore, to Man, in a variety of ways. Such means include, but without 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 age, weight, sex, tolerance, etc. of the animal, preferably mammal, and more preferably human.

Another aspect of the invention relates to a composition, hereinafter composition of the invention, which comprises 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 of 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 vaccine production.

In the context of the present invention the term "vaccine" refers to an antigenic preparation used to establish the immune system response to a disease. They are antigen preparations that once inside the organism provoke the response of the immune system, by means of antibody production, and generate immune memory producing permanent or transient immunity.

The term "antigen" herein is refers to a molecule (usually a protein or a polysaccharide), which can induce the formation of antibodies. There is 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 memory, refers to the Masp52 protein or any of its variants.

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

Therefore, in another preferred embodiment, the composition of the invention further comprises excipients pharmacologically acceptable. In another more preferred embodiment, the composition of the invention additionally comprises another principle active. In a more preferred embodiment, the composition of the Invention is a vaccine, hereinafter vaccine of the invention. In another even more preferred embodiment, the vaccine comprises a adjuvant

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

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

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

Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be partly detached of the description and in part of 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 SDS-PAGE 12.5% and silver nitrate staining of purified proteins via WGL-Agarosa from the Excretion Secretion Product (ESP) and the fraction purified by Wheat Germ Lectin.

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

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

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

Fig. 6. Shows the Focal Laser Microscopy of 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 [mu] 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) Marking for cells infected with T. cruzi containing amastigotes inside. Bar = 5 \ mum

Fig. 7. Shows the immunocytochemistry of the Masp52 using the anti-CR IgG A) Flagellar Pocket of Trypomastigote derived from cell culture B) Flagellar Pocket de Metacyclic Trypomastigote C) Metacyclic Trypoamstigote Vacuoles D) Trypomastigote derived from cell culture, location in membrane, cytosol and outer cell E) Negative control. Bar = 0.25 \ mum

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

Fig. 9. Shows the effect of antibodies against to Masp 52 in the invasiveness of metacyclic trypomastigotes. We tested with anti-CR IgG at dilution 1/50, 1/100 and 1/200, incubating it for half an hour with the Trypomastigotes metacyclic and after washing the parasites of the serum, leaving them interact for 2 hours against Vero cells. The bars represent the mean are the mean ± the standard deviation of triplicates for each dilution. To find if they exist significant differences between the data groups the Bonferroni test.

Examples

The invention will be illustrated below through tests carried out by the inventors, which puts manifested 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 Arraiján District, community of Burunga, Panama), donated in 2006 by Dr. A. Ying of the University of Panama and maintained by cryopreservation from that moment.

The epimastigote forms were grown in culture at 28 ° 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.

Vero Hosting Cells (ECACC 84113001) were grown at 37 ° C 5% CO 2 in 75 cm 2 plastic bottles (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 12 h 37 ° C. Cultures were washed with culture medium to remove 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 96 h of infection, the supernatant containing the trypomastigotes was centrifuged at 300 g 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 by 8 days, then amastigote forms were purified by gradient discontinuous (1,100, 1,090, 1,080, 1,070 g / ml) and prepared as outlined below. The purified amastigote forms are They collected 1,070 / 1,080 g / ml from the interface.

All forms of the T. cruzi biological cycle 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 possessed at least 95% of proven purity both under staining and by Neubauer chamber counting.

Masp52 purification and sequencing

The obtaining and purification of the Masp52, was made from excretion-secreetion product (ESP) during cell-parasite interaction. For this, Vero cell semiconfluent cultures were removed on medium and after washing them repeatedly in DMEM without serum, they were infected with a suspension of metacyclic trypomastigotes of the parasite in DMEM without serum with a relationship, conditions and Infection time as mentioned above.

After the interaction period, and the medium removed, it was centrifuged at 1500 g 10 minutes at 4 ° C in order to eliminate parasite forms that had not penetrated and filtered the supernatant through a 0.22 µm diameter filter (Sartorius, Sartolab 20).

ESP proteins were concentrated with 5 kDa molecular exclusion filters (Amicon, ™ ultra, Millipore) and subsequently chromatographed through the mono P column 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 Wheat Germ Lectin-Agarosa (WGL) column (Sigma) a in order to purify N-glycosylated proteins. How Wash buffer was used a 0.1M Carbonate Buffer pH: 9, and as eluent of the lectin bound fraction, 2 volumes of 0.5 M N-Acetylglucosamine (Sigma) in Buffer Carbonate. All chromatographs were performed in a team AKTA ™ Purifier (GE).

The result of the chromatography was evaluated by electrophoresis in polyacrylamide gels SDS_PAGE at 12.5% .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) by drying in a Speedvac to resuspend 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 a "peptide fingerprint" for the identification of proteins in the databases using search engines accessible on the web (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. cruzi DB (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 using the Expasy proteomic tools server ( http://expasy.org ) with the programs: Motif Sean for the search for structural motifs; SignalP 3.0 for searching 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. They were designed two peptides corresponding to the N-proximal zones (signal peptide) of residues 1-25 and the zone Catalytic Masp52 (ATP / GTP binding motif A) of waste 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 Parasitol. 2007 Sep; 117 (1): 65- 73. Epub 2007 Mar 27). Three weeks after the first immunization the antibody titer was evaluated 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 were infected (3 x 10 5 cells / well) with metacyclic trypomastigote forms. The parasite / cell ratio was 5: 1, the invasion developing 2 h at 37 ° C in DMEM without serum with dilutions 1/50, 1/100 and 1/200 of anti-CR Ig. Serum immunoglobulins Preimmune mice were used as control. At the end of the period of interaction, the cultures were washed, fixed in methanol and Dyed 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 parasites intracellular 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 calculated the ratio parasite cell

SDS-PAGE and Western Blot analysis

The expression studies of the Masp52 in the different forms of the parasite's life cycle was evaluated by immunoblot at different stages, 5 x 10 7 organisms of each phase of the parasite were resuspended in 2 ml of tisis buffer; 20 mM PBS, 0.25 mM Sucrose, 1 mM EDTA, 0.145 mM KCl, 1 mM DTT at pH: 7.4 plus a cocktail inhibitor of Complete Mini protease (Roche Molecular Biochemicals). Behind the treatment for 10 min of the protozoa in said buffer, were sonicated at 0 ° 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 it. concentration, so that all the wells of the electrophoresis contained the same amount of protein and prepared in the same buffer (10 mM Tris / HCl; 1 mM EDTA, pH 8.0; 2.5% SDS and 17% glycerin and 0.01% bromophenol blue) and heated at 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 Natl Acad Sci USA 1979; 76 : 4350-4354). The spots were exposed to anti-CR or anti-SP (tested at 1:50) for 2 h at 37 ° C followed by a peroxidase conjugate (Polyclonal Goat Anti Mouse Immunoglobulins HRP (DakoCytomation) for 2 h at 37 ° C. The reaction was developed with 3 -3'Diaminobenzidine Tetrahydrochlorhydric in Tris HCl buffer at pH 7.2 The gels were digitized and processed by QuantiScan software (Biosoft, Cambridge, UK) in order to quantify the bands In all cases the protein determination was carried out by the method Bradford (Bradford, 1976. Analytical Biochemistry 72: 248-254).

MRNA isolation and RTqPCR development

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

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

The quantification of the expression of 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)

resulting in 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 Parasitol 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 ΔCT method, where the Ratio 18S / Masp52 = 2 CT \ 18S-CT \ Masp52}. All trials 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 0.125 M PBS and fixed for 12 h at 4 ° C in 1% glutaraldehyde and 2% formaldehyde in Cacodylate Buffer with 0.1 M sucrose (pH: 7), then being embedded in LRWhite resin.

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

In those experiments in which it was intended locate the protein inside the parasite forms or their location during the infection process, the cells or the parasite forms were fixed in acetone at -20 ° C, at 2 hours of parasite / cell interaction, or after 60 hours of having performed the infection in order to observe the presence and location of the protein in amastigotes or cells parasitized Once fixed and washed with PBS, we incubate the 1 h preparations with anti-CR IgG at dilution to 1/100. As a secondary antibody, a labeled anti mouse was used with Fluorescein isothiocyanate (FITC) (Sigma) in Blocking Buffer for 1 h at room temperature. Finally, the preparations were treated for 15 minutes with a solution of DAPI 10 \ mug / ml. The preparations were assembled and preserved in mounting medium (Prolong Antifade Kit, Molecular Probes), observed in a Leica DMI6000 confocal laser microscope that incorporates a filter system for FITC (average wavelength 530 nm and maximum of 490 nm).

Adsorption of Masp52 to Bentonite particles

For the adsorption to bentonite particles, the Kagan &Norman's technique (1970. Manual of Clinical Microbiology 453-486) was used to select the bentonite particles for the adsorption of the Masp52. A suspension of 100 ul of particles with a diameter of 1 to 2 µm was incubated 12 h at 4 ° 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.

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

From the purification process 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 serum albumin - Bovine Serum Albumin (BSA) - from one of the culture media and the 52 kDA band that by means of a Peptide mass fingerprinting (PMF) or peptide fingerprint 7 matches were found, obtaining a value of 47.9% and a 17% coverage for the sequence shown in Figure No. 2. This sequence was found in the databases with the name of mucin-associated surface protein (MASP), putative and with an EMBL access number: XP_820015.1, naming it here as Masp52 .

A search of possible structural motifs present in the Masp 52 was carried out through the use of Expasy's proteomic tools server ( http://expasy.org ). a signal peptide (amino acids 1-25), two transmembrane regions at the N-Terminal (amino acids 9-28) and C-terminal (amino acids 493-510), an ATP / GTP- binding site motif A (P-) were obtained loop) that we can name as a catalytic center, (amino acids 159-166) and a site for N-glycosylation (465 to 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 through RTqPCR showed similar results (Fig. 5), all stages they had synthesis of the specific mRNA of Masp 52, but with Different levels of expression. In the trypomastigotas forms metacyclic synthesis of specific mRNA is three times higher than that of tissue-derived trypomastigote, eleven times greater than that of amastigotes and fifty times higher 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 MASP family proteins by western blot, the expression of 6 bands in the trypomastigotes was obtained metacyclic, 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).

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

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

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

In those preparations where the study of Protein immunolocation was performed during the process of Metacyclic trypomastigote-cell interaction, it also observed the presence of the protein in the cytosol of the trypomastigotes, detecting the protein at the point of contact between the parasite and the cell. Fluorescence was observed equally dispersed in the cytoplasm of the host cell next to the presence of the parasite (Fig. 6B).

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

Ultrastructural location studies in the different stages (trypomastigote, metacyclic trypomastigote derived from cell culture and epimastigote), using IgG against the catalytic domain of Masp52, it is observed as the Gold marks are located both on the parasite's membrane, and in the cytosol, the number of particles in the stadiums being greater with high infective capacity, that is trypomastigotas (Fig. 7A, 7C). Brand aggregates are located inside vacuoles (Fig. 7B), close to the Flagellar Pocket and the root of the scourge in the infecting forms (Fig. 7A and C), while the Masp52 appears in amastigotes in the cytosol.

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

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

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

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<210> 3

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

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<213> Artificial

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         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> primer MASP.220R

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 3

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip

tgcagatgct tcaactgctg

\hfill

20

 \ hskip-.1em \ dddseqskip 

tgcagatgct tcaactgctg

 \ hfill 

twenty

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<210> 4

         \ vskip0.400000 \ baselineskip
      

<211> 25

         \ vskip0.400000 \ baselineskip
      

<212> PRT

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<213> Trypanosoma cruzi

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<400> 4

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<210> 5

         \ vskip0.400000 \ baselineskip
      

<211> 8

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Trypanosoma cruzi

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         \ vskip0.400000 \ baselineskip
      

<400> 5

\ hskip1cm 5

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<210> 6

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<211> 9

         \ vskip0.400000 \ baselineskip
      

<212> PRT

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<213> Trypanosoma cruzi

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<400> 6

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<210> 7

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<211> 1524

         \ vskip0.400000 \ baselineskip
      

<212> DNA

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<213> Trypanosoma cruzi

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         \ vskip0.400000 \ baselineskip
      

<400> 7

7

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<210> 8

         \ vskip0.400000 \ baselineskip
      

<211> 23

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

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         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> primer v1

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         \ vskip0.400000 \ baselineskip
      

<400> 8

         \ vskip1.000000 \ baselineskip
      

\ hskip1cm 8

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         \ vskip0.400000 \ baselineskip
      

<210> 9

         \ vskip0.400000 \ baselineskip
      

<211> 23

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<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> primer v2

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         \ vskip0.400000 \ baselineskip
      

<400> 9

         \ vskip1.000000 \ baselineskip
      

\ hskip1cm 9

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         \ vskip0.400000 \ baselineskip
      

<210> 10

         \ vskip0.400000 \ baselineskip
      

<211> 2240

         \ vskip0.400000 \ baselineskip
      

<212> PRT

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<213> Trypanosoma cruzi

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 10

10

eleven

12

13

14

fifteen

16

17

18

Claims (22)

1. Method of detecting the presence of the T. cruzi parasite in an individual, comprising:

to.

obtain an isolated biological sample of said individual,

b.

detect Masp52 protein in the biological sample isolated from (a).

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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 one of claims 1 or 2, wherein the individual in step (a) is a human mammal. 4. Method of obtaining useful data for diagnosis of Chagas disease, which includes:

to.

obtain an isolated biological sample of an individual,

b.

detect the amount of Masp52 protein present in the biological sample isolated from (a).

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5. Method of obtaining useful data for the diagnosis of Chagas disease according to claim previous, which also includes:

C.

compare the amount detected in the step (b) with a reference amount.

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6. Method of obtaining useful data for Chagas disease diagnosis according to any of the claims 4 or 5, wherein the detection of the amount of Masp52 protein is performed by incubation with an antibody specific 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 differential diagnosis of Chagas disease according to anyone of claims 4 or 5, wherein the detection of the amount Masp52 protein is performed using RTqPCR. 11. Method of obtaining useful data for the differential diagnosis of Chagas disease according to previous claim, wherein the RTqPCR is carried out using the primers whose sequence is collected in SEQ ID NO: 2 and SEQ ID NO: 3. 12. Use of isolated Masp52 protein, or any of its variants, for the elaboration of a medicine. 13. Use of isolated Masp52 protein, or any of its variants, for the preparation of a medicine 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 antibodies generated by the immunization of an animal with the Masp52 protein for processing of a medicine. 16. Use of antibodies generated by the immunization of an animal with the Masp52 protein for processing of a medication 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 It also comprises pharmacologically acceptable excipients. 19. Composition according to any of the claims 17 or 18, which is a vaccine. 20. Composition according to claim above, which also comprises an adjuvant. 21. Composition according to any of the claims 19 or 20, wherein the vaccine has an origin recombinant 22. Composition according to any of the claims 19 or 20, further comprising another active principle.