MXPA00005473A - Trypanosoma cruzi - Google Patents

Abstract

The nucleotide sequence of Tc40, a gene encoding a new antigenic protein from Trypanosoma cruzi called PTc40 is disclosed. The amino acid sequence of the PTc40 protein is also disclosed, along with the amino acid sequence of the dominant antigenic epitope of the PTc40 protein. The PTc40 protein and Tc40 gene, or a fragment thereof, modified or otherwise, can be used directly or indirectly for the detection of Trypanosoma cruzi, or for the monitoring of an infection generated by T. cruzi in man or animals.

Description

NEW ANTIGEN OF CRYZI TRIPANOSOMA, THIS CODING GENE, AND METHOD OF DETECTION AND TREATMENT OF THE CHANGAS DISEASE This application is in part a continuation of the application Serial Number 08 / 480,917, filed on June 1, 1995. The entire disclosure of the prior application is incorporated herein in its entirety for reference.

FIELD OF THE INVENTION The subject of the present invention is a new genetic material that encodes a new protein and its fragments and their antigenic determinants recognized by an anti-Trypanosoma cruzi antiserum, and is related to a promoter sequence and to the use of said gene and protein and / or determinant ... antigenic, especially for diagnosis, therapeutic and pharmaceutical purposes.

BACKGROUND OF THE INVENTION Trianapanosoma cruzi is a flagellated protozoan parasite, a member of the order Kinetoplastida and of the family Tripanosomatidae, which is responsible for Chagas disease which naturally affects millions of people, mainly in Latin America.

REF .: 120557 In vertebrate hosts, Trypanosoma cruzi is present in two forms: one which is mobile by means of its flagellum or trypomastigote and which is not divided; the other is aflagelated, or intracellular amastigote, which is multiplied by means of binary division.

The transmission of the protozoan in man occurs through hematophagous insects of the family Reduviidae, during a blood supply followed by debris at the picket site. Thus the insect vector releases the infectious forms of metacyclic trypomastigote which colonize many types of cells through the blood circulation. Trypanosoma cruzi infects skeletal and cardiac muscle cells, glia cells, and cells of the mononuclear phagocytic system. After passive penetration of the host cell, the trypomastigote form of the parasite differs in the amastigote form, actively dividing and then this is followed by a release of the trypomastigote forms, thereby causing a new cell invasion.

The insect vectors complete the parasitic cycle by ingestion, during a blood supply, the forms of trypomastigote in the host. It then differs in the forms of epimastigote in the midgut of the vector and finally in the infectious forms of metacyclic trypomastigote in the hindgut.

Two phases can be distinguished in Chagas disease: the acute phase and the chronic phase. The acute phase occurs after a type of transfusion, congenital, or vector contamination and remains for a few weeks. This is characterized by a large number of parasites circulating in the blood and corresponds to an exponential division of the protozoan. The acute phase is usually more symptomless. However, in infants contaminated by their mother, the acute phase, which is marked by acute cardiac disease, can be critical. The chronic phase can extend for many years. In some individuals, this phase is asymptomatic. On the other hand, other patients have tissue lesions in the heart or digestive-type manifestations. In any case, the clinical diagnosis must always be confirmed by tests for the detection of both antibodies directed against the antigens of the parasite, as well as the parasite itself.

This disease has become a worldwide problem due to contamination through blood transfusion. This has therefore made it essential to have available diagnostic tests which make it possible to determine the presence of the parasite in individuals. Several serological tests are available, such as direct agglutination, immunofluorescence (IIF), complement fixation tests (CFR), and ELISA tests (Enzyme Linked Immunosorbent Assay). The Trypanosoma cruzi antigens currently used for serological tests are obtained from a total lysate or. of protein fractions partially purified from non-infectious phases of the parasite. Nevertheless, these fractions do not allow the antigens to be obtained in sufficient quantity and quality for the production of a reliable serological diagnostic test. In addition, the complexity of the parasite and the chain-to-chain antigenic polymorphism introduce an additional difficulty in the reproducibility of the different preparations. Finally, there are many risks of cross-reactivity with other protozoa, more particularly with the family Leishmania and Trypanosoma rangeli, a non-pathogenic parasite.

In order to solve these several problems, it was contemplated to produce a serological diagnostic equipment composed of recombinant proteins which would be highly sensitive and specific for Trypanosoma cruzi.

Several groups of researchers have projected libraries for the expression of genomic DNA or DNA complementary to Trypanosoma cruzi in the vector 9gtll, using serum from patients suffering from Chagas disease. The 9gtll phage allows the insertion of a foreign DNA of a maximum size of 7kB into the EcoRI site located in a recombinant protein fused with beta-galactosidase, which is inducible by IPTG (isopropyl beta-D-thiogalactosidase).

Several genes of Trypanosoma cruzi, which encode proteins recognized by the Chagasic serum, were thus characterized (Moncayo and Luquetti, 1990 and Levin et al. (1991), FEMS Microbiol, Immunol., 89: 11-20). Among the written recombinant antigens, the H49 antigen can be mentioned (Paranhos et al., 1994 (1)). However, this antigen does not allow a sensitivity of serological detection of 100% of patients in the chronic or acute phase. This was therefore contemplated to combine the H49 antigen with the CRA antigen (Cytoplasmic Repetitive Antigen) (Lafaille et al., (1989) (2)) but this problem has not yet been resolved.

BRIEF DESCRIPTION OF THE INVENTION The present inventors have identified and obtained a new genetic material that encodes a new protein, its fragments, and antigenic determinants recognized by anti-Trypanosoma cruzi antiserum, which make this possible to overcome the aforementioned disadvantages. The genetic material can be used to produce proteins or polypeptides for the production of diagnostic tests or for the preparation of pharmaceutical or vaccination compositions, or they can themselves be used both as a test, and for the determination of specific tests which can be used. to be used in nucleic acid hybridization tests for the detection of Trypanosoma cruzi infections. Likewise, the protein or any corresponding polypeptide can be used for the production of antibodies specific for the parasite, for diagnostic or passive protection purposes.

DETAILED DESCRIPTION OF THE INVENTION The new genetic material is designated Tc40 and it encodes a protein designated PTc40 by the applicant.

Accordingly, the subject matter of the present invention is a DNA or RNA molecule consisting of at least one chain comprising a nucleotide sequence represented by the identification means SEQ ID No. 1, or a complementary or counter-sense sequence or equivalent for said sequence identified by the identification means SEQ ID No. 1, and specifically a sequence having, for any succession of 100 contiguous monomers, at least 50%, preferably at least 60%, or more preferably at least 85% homology with said sequence.

The invention further relates to fragments of DNA or RNA whose nucleotide sequence is identical, complementary, contradictory, or equivalent to any of the following sequences: -those starting at nucleotide 1232 and ending at nucleotide 2207 of SEQ ID NO. 1, -those starting at nucleotide 1232 and ending at nucleotide 1825 of SEQ ID NO. 1, -and that starting at nucleotide 1266 and ending at nucleotide 1207, and specifically the DNA or RNA fragments whose sequence has, for any succession of 30 contiguous monomers, at least 50%, preferably at least 60%, or more preferably at minus 85% homology with any one of said sequences.

The preferred fragment of DNA or RNA which is fragment 1232-1825 of SEQ ID NO. 1, the invention relates to fragments of DNA or RNA which hybridize with these fragments knowing that up to 10% error is tolerated in the bases they copy.

The DNA or RNA fragments could be of any length and be comprised in the sequence of 266 to 3010 of SEQ ID NO. 1, followed by knowing that this sequence is highly specific for Trypanosoma cruzi. Any fragment which hybridizes with the DNA or RNA of this sequence is considered to be a specific equivalent nucleotide or deoxynucleotide sequence.

The nucleotide sequence is understood to refer to either a DNA strand as its complementary strand, or an RNA strand or its opposite strand or its corresponding complementary DNAs. The DNA sequence as represented in the identification means SEQ ID NO. 1 corresponds to a sequence of messenger RNA, it being understood that thymine (T) in the DNA is replaced by a uracil (U) in the RNA.

A more preferred DNA or RNA fragment is still the nucleotide sequence of 1471 to 1543 of SEQ ID NO. 1.

According to the invention, two nucleotide sequences are mentioned to be equivalent in relation to each other, or in relation to a reference sequence if, functionally, the corresponding biopolymers can play essentially the same role, without being identical, with respect to the application or use considered, or in the technique in which they are involved; two sequences obtained because of natural variability, especially spontaneous mutation, of the species of which they were identified, or because of induced variability, as well as homologous sequences, homology that is defined below, are especially equivalent.

Variability is understood to refer to any modification of a spontaneous or induced sequence, especially by substitution and / or insertion and / or deletion of nucleotides and / or nucleotide fragment, and / or extension and / or shortening of the sequence to at least one from the ends; unnatural variability may result from the genetic engineering techniques used; this variability may result in modifications of any initiating sequence, considered as a reference, and capable of being expressed by a degree of relative homology for the aforementioned reference sequence.

Homology characterizes the degree of identity of two fragments of nucleotides (or peptides) compared; this is measured by the percent identity which is especially determined by direct comparison of the nucleotide (or peptide) sequences, related to the reference nucleotide (or peptide) sequences.

Any nucleotide fragment is mentioned to be equivalent for a reference fragment if it has a nucleotide sequence which is equivalent for the reference sequence; according to the preceding definition; the following are especially equivalent for a reference nucleotide fragment: a) any fragment capable of at least partially hybridizing with the complementary strand of the reference fragment, knowing that up to 10% of error in the copy bases is tolerated. b) any fragment whose alignment with the reference fragment leads to the detection of identical contiguous bases, in a larger number than with any other fragment obtained from another taxonomic group, c) any fragment resulting or capable of resulting from the natural variability of the species, from which it is obtained, d) any fragment capable of resulting from the genetic engineering techniques applied for the reference fragment, any fragment, containing at least 30 contiguous nucleotides, encoding a peptide homologous or identical to the peptide encoded for the reference fragment, and / or f) any different fragment of the reference fragment by insertion, deletion or substitution of at least one monomer, extension or shortening at least one of its ends; for example any fragment corresponding to the reference fragment flanked at least one of its ends by a nucleotide sequence that does not encode a pslipptide.

The subject of the invention is also a protein, called PTc40 by the Applicant, which has an apparent molecular mass of about 100 kDa, which is recognized by anti-Trypanosoma cruzi antiserum, an immunological equivalent of this protein, and fragments thereof. . The amino acid sequence of this protein is represented by the identification means sequence SEQ ID NO. 2, the sequence that starts at amino acid 1 and ends at amino acid 915 of the sequence defined in the identification means SEQ ID NO. 2.

Immunological equivalent is understood to refer to any polypeptide or peptide capable of being immunologically recognized by antibodies directed against said PTc40 protein.

The invention also relates specifically to a fragment of this protein represented by the sequence identification means SEQ ID NO. 2, the sequence that starts at amino acid 323 and ends at amino acid 520 of the sequence defined in the identification means SEQ ID NO. 2.

The invention also relates specifically to the antigenic determinant or epitope of the PTc40 protein, a fragment of 25 amino acids, which start at amino acid 403 and end at amino acid 426 of SEQ ID NO. 2, called S23G.

The PTc40 protein and said PTc40 fragment, as well as the antigenic determinant S23G can contain modifications, especially chemical modifications, which do not alter their antigenicity, such as for example N-terminal binding by oxidation of serine or N-terminal binding by function of cysteine or hydrazine (see Keith Rose et al., Natural Peptides as Building Blocks for the Synthesis of Large Protein-like Molecules with Hydrazone and Oxime Linkages, Department of Medical Biochemistry, (1996)).

The PTc40 protein fragment is understood to refer to any fragment of the PTc40 protein which preferably has at least the sequence from amino acid 323 to 520 of SEQ ID NO. 2 and / or the S23G sequence, together with some other amino acids. It is clear that the SG23 fragment contains an antigenic epitope of the PTc40 protein that reacts with antibodies generated during Chagas disease.

Moreover, the subject matter of the present invention is also an expression cartridge which is especially functional in a cell derived from a prokaryotic or eukaryotic organism, and which allows the expression of DNA encoding the complete PTc40 protein or a fragment thereof. , in particular of a DNA fragment as defined above, placed under the control of elements necessary for its expression; said protein and said protein fragments being recognized by anti-Trypanosoma cruzi antiserum.

Generally, any cell derived from a prokaryotic or eukaryotic organism can be used within the structure of the invention. Such cells are known to persons skilled in the state of the art. By way of example, cells derived from eukaryotic organisms, such as cells derived from a mammal, especially CHO cells (Chinese Hamster Ovary); insect cells; cells derived from a fungus, especially a unicellular fungus or from a yeast, especially from the strain Pichia, Saccharomyces, Schizosaccharomyces and more particularly selected from the group consisting of Saccharomyces cerévisiae, Schizosaccharomyces pombe, Schizosaccharomyces malidevorans, Schizosaccharomyces sloofiae, Schizosaccharomyces octosporus, or other systems such as the Semliki virus of the forest and the vaccinia virus. Likewise, among the cells derived from a prokaryotic organism, cells of a strain of Escherichia coli (E. coli) other enterobacterial cells can be used, without constituting a limitation. A large number of these cells are commercially available in collections, such as ATCC (Rockville, MD, USA) and AFRC (Agriculture &Food Research Concil, Norfolk, UK). The cell can also be wild type or mutant type. Mutations are described in accessible literature for persons qualified in the state of the art.

For the purposes of the present invention, an E. coli DH5a cell (marketed by the company CLONTECH under the reference: C2007-1) is used; however, other cells can be used in the same way.

The expression cartridge of the invention is proposed for the production of the PTc40 protein or for fragments of said protein, such as the PTc40 fragment and / or the S23G epitope, which are produced by the aforementioned E. coli cell, and which are recognized by human antiserum. Such antisera are obtained from patients who have recently or previously been infected with Trypanosoma cruzi, and contain immunoglobulins that specifically recognize the PTc40 protein and / or a PTc40 protein fragment. Of course, the PTc40 protein can also be recognized by other antibodies, such as for example monoclonal or polyclonal antibodies obtained by immunization of several species with the aforementioned natural protein, the recombinant protein, or peptide fragments thereof.

The PTc40 protein is understood to refer to the native cytoplasmic antigen of Trypanosoma cruzi, or the antigen produced especially by the genetic recombination techniques described in the present application, or any fragment or mutant of this antigen, for example the recombinant protein GST-Tc40 and the peptide BIO-S23G (Tc40), provided that it is immunologically reactive with antibodies directed against the PTc40 protein of this parasite.

Advantageously, such a protein has an amino acid sequence that has a degree of homology of at least 70%, preferably at least 85%, and more preferably at least 95% related to the sequence identified by the identification means SEQ ID. NO 2. In practice, such an equivalent can be obtained by deletion, substitution, and / or addition of one or more amino acids of the recombinant protein sequence. Such homology means that the amino acid sequence of the S23G peptide, contained in the PTc40 protein and its PTc40 protein fragment, is at least preserved in order to potentially maintain the antigenicity. It is within the ability of the people qualified in the state of the art to execute, using known techniques, these modifications without affecting the immunological recognition.

Within the structure of the present invention, the PTc40 protein and the PTc40 protein fragment can be modified in vi tro, especially by deletion or addition of chemical groups, such as phosphates, sugars or ironic acids, in order to improve stability antigenic or the presentation of one of several epitopes, such as the S23G epitope.

The expression cartridge according to the invention allows the production of a PTc40 protein (having an amino acid sequence as specified above) and fragments of said protein, fused with an exogenous element which can help its stability, its purification, its production, its presentation for its recognition. The choice of such an exogenous element is within the capacity of the persons qualified in the state of the art. This can be especially a hapten, a hexogen peptide or a protein.

The expression cartridge according to the invention comprises the necessary elements for the expression of said DNA fragment in the considered cell. "Elements necessary for expression" is understood to refer to the elements as a whole which allow the transcription of the DNA fragment in a messenger RNA (mRNA) and the translation of the second into protein.

The present invention also relates to a putative promoter specifically for T. cruzi, which can be used in an expression cartridge for the expression of eukaryotic genes. The tests could also be defined in this region to detect the parasite.

The present invention also extends to a vector comprising an expression cartridge according to the invention. This can be a viral vector and especially a vector derived from a baculovirus, more particularly proposed for expression in insect cells, or an adenovirus-derived vector for expression in mammalian cells. This can also be a plasmid vector that replicates autonomously and in particular a multiplicative vector.

The present invention also relates to a cell derived from a prokaryotic or eukaryotic organism, comprising an expression cartridge, both in an integrated form in the cellular genome, and inserted into a vector which can be maintained as an extra-genomic entity . Such a cell was previously defined.

The subject matter of the present invention is also a process for the preparation of a PTc40 protein, or fragments of said protein, according to which: (i) a cell derived from a prokaryotic or eukaryotic organism, comprising the expression cartridge according to the invention, is cultured under appropriate conditions; Y (ii) The expressed protein derived from the aforementioned organisms is recovered.

The present invention also relates to one or more peptides whose amino acid sequence corresponds to a portion of the PTc40 protein sequence and exhibits, either alone or as a mixture, a reactivity with the whole serum of individuals or animals infected with Trypanosoma cruzi. . The preferred portion is the PTc40 protein fragment or the peptide defined above and the S23G peptide. The peptides can be obtained by chemical synthesis, lysis of PTc40 protein or by genetic recombination techniques.

The invention also relates to monoclonal or polyclonal antibodies obtained by immunological reaction of an animal or human organism to an immunogenic agent consisting of a natural or recombinant PTc40 protein and fragments thereof, or of a peptide as defined above.

The present invention also relates to a reagent for the detection and / or monitoring of an infection by Trypanosoma cruzi, which comprises, as a reactive substance, a PTc40 protein as defined above, or fragments thereof, a peptide or a mixture of peptides as defined above, or at least one monoclonal or polyclonal antibody as described above. The above reagent can be linked directly or indirectly to an appropriate solid support. The solid support can be especially in the form of a cone, a tube, a receptacle, a molding, and others.

The term "solid support" as used herein, includes all materials in which a reagent can be immobilized for use in diagnostic tests. Natural or synthetic materials, chemically modified or otherwise, can be used as solid supports, especially polysaccharides such as cellulose-based materials, for example paper, cellulose derivatives, such as cellulose acetates and nitrocellulose; polymers such as vinyl chloride, polyethylene, polystyrenes, polyacrylate or copolymers such as polymers of vinyl chloride and propylene, polymers of vinyl chloride and vinyl acetate; styrene-based copolymers, natural fibers such as cotton and synthetic fibers such as nylon; and magnetic particles. Preferably the solid support is a polystyrene polymer or a butadiene / styrene copolymer. Advantageously, the support is a polystyrene or a styrene-based copolymer comprising between about 10 and 90% by weight of styrene units.

The binding of the reagent within the solid support can be carried out in a direct or indirect manner. Using the direct way, two approaches are possible: either by absorption of a reagent within the solid support, ie by non-covalent bonds (mainly hydrogen, Van der Waals or ionic type), or by formation of covalent bonds between the reactant and the support. Using the indirect manner, an "anti-active" compound capable of interacting with the reagent in order to immobilize the total within the solid support can be pre-bound (by covalent binding or absorption) into the solid support. By way of example, an anti-PTc40 antibody can be mentioned, provided that it is immunologically reactive with a portion of the protein different from that involved in the reaction to recognize the antibodies in the serum; a ligand receptor system, for example by grafting into the PTc40 protein a molecule such as a vitamin, and by immobilization within the solid phase of the corresponding receptor (for example the biotin-streptavidin system). The indirect manner is also understood to refer to the preliminary graft or fusion by genetic recombination - of a protein, or a fragment of this protein, towards one of the ends of the PTc40 protein, and the immobilization of the second within a solid support by passive absorption or covalent binding of the grafted or fused protein or polypeptide.

The invention also relates to a process for the detection and / or monitoring of a Trypanosoma cruzi infection in a biological sample, such as blood or plasma serum, urine, saliva, or tear samples of an individual or an animal suspected of having been infected with Trypanosoma cruzi, characterized in that said sample and a reagent as defined above are contacted, under conditions that allow a possible immunological reaction, and then the presence of an immune complex with said reagent is detected.

By way of example without limitation, sandwich-type detection processes can be mentioned in one or more stages, as described in particular in the patents FR2,481,318 and FR2,487,983, which consist of reacting a first specific monoclonal or polyclonal antibody for a desired antigen, bound within a solid support, with the sample, and revealing within it the possible presence of an immune complex thus formed, using a second antibody indicated by any appropriate marker known to persons skilled in the art, especially by a radioactive isotope, an enzyme, for example peroxidase or alkaline phosphatase and others, using the so-called competition techniques well known to persons skilled in the state of the art.

The subject of the invention is also an active immunotherapeutic composition, especially a vaccine preparation, which comprises as an active ingredient, a natural or recombinant PTc40 protein or fragments thereof, or the peptides identified above, the active ingredient being optionally conjugated to a pharmaceutically acceptable carrier, and optionally an excipient and / or an appropriate adjuvant.

The present invention also covers a pharmaceutical composition proposed for the treatment or prevention of a Trypanosoma cruzi infection in man or an animal, comprising a therapeutically effective amount of an expression cartridge, a vector, a cell derived from an prokaryotic or eukaryotic organism as defined above, a PTc40 protein according to the invention, or fragments thereof, or the peptide identified above, or an antibody of the invention. The subject matter of the present invention is also specific tests and primers for T. cruzi, and their uses in diagnostic tests.

The term "test" or "probe" as used in the present invention refers to a DNA or RNA containing at least one strand having a nucleotide sequence which allows hybridization to nucleic acids having a nucleotide sequence as represented by the means of identification SEC ID NO. 1, or a sequence complementary or in contradiction, or a sequence equivalent to said sequence, and especially a sequence having, for any sequence of 5 to 100 contiguous monomers, at least 50%, preferably at least 60%, or more preferably at minus 85% homology with SEQ ID NO. 1, with fragments thereof, or with a synthetic oligonucleotide allowing such hybridization, unmodified or comprising one or more modified bases such as inosine, 5-methyl deoxycytidine, deoxyuridine, 5-dimethylaminodeoxyuridine, 2,6-diaminopurine, -bromodeoxyuridine or any other modified base. Likewise, these tests can be modified at the sugar level, specifically the replacement of at least one deoxyribose with a polyamide (PE NIELSEN et al. (1991) (13)), or at the level of the phosphate group, for example its replacement with esters, especially chosen from diphosphate, alkyl or arylphosphonate and phosphorothionate esters.

The tests can be much shorter than the sequence identified in the identification means SEC ID. DO NOT. 1. In practice, such tests comprise at least 5 monomers, advantageously 8 to 50 monomers, which have a specificity of hybridization, under defined conditions, to form a hybridization complex with DNA or RNA having a nucleotide sequence as defined before. The conditions are well known and / or easily determined by those of skills in the state of the art.

A test according to the invention can be used for diagnostic purposes, such as starter by amplification of biological material, as capture and / or detection test, or for therapeutic purposes. The capture test can be immobilized on a solid support by any appropriate means, i.e. directly or indirectly, for example by "covalent binding or passive absorption." The detection test is signaled by means of a chosen radioactive isotope marker, especially enzymes. chosen from peroxidase and alkaline phosphatase, and those capable of hydrolyzing a chromogenic, fluorigenic, or luminescent substrate, chromophoric, chromogenic, fluorigenic, or luminescent compounds, nucleotide base analogues, and biotin.The tests of the present invention which are used for diagnostic purposes can be used in any known hybridization technique, and especially the so-called "Dot-Blot" technique.

(Maniatis et al. (1982) (14)), Southern technique Blatting (Southern EM (1975) (15)), Northern blot technique, which is a technique identical to the Southern blot technique but which uses RNA as a target, sandwich technique (Dunn AR et al. (1977) ( 16)). Advantageously, the sandwich technique is used which comprises a specific capture test and / or a specific detection test, it being understood that the capture test and the detection test must have a nucleotide sequence which is at least partially different .

Another application of the invention is a therapeutic test for treatment of infections due to Trypanosoma cruzi, said test being able to hybridize in vivo with the DNA or RNA of the parasite to block the translation and / or transcription and / or replication phenomenon.

An initiator is a test comprising from 5 to 30 monomers, having a specificity of hybridization, under predefined conditions, for the initiation of an enzymatic polymerization, for example in an amplification technique such as PCR (Polymerase Chain Reaction), in an elongation process such as sequencing, in a reverse transcription method and so on. Such predefined conditions are well known and / or easily determined by those of skill in the state of the art.

A preferred test or primer will contain a selected nucleotide sequence of the sequences SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO. 7 The invention also relates to a reagent for the detection and / or identification of Trypanosoma cruzi in a biological sample, comprising at least one test as defined above, and in particular a capture test and a detection test, one or both of which correspond to the previous definition.

The invention therefore provides a process for selectively detecting and / or identifying Trypanosoma cruzi in a biological sample, according to which the RNA, extracted from the parasite and optionally denatured, or the DNA, denatured extract, or the DNA obtained from the reverse transcription of the RNA is exposed to at least one test as defined above and the hybridization of said test is detected.

BRIEF DESCRIPTION OF THE FIGURES The invention will be understood more clearly with the reading of the detailed description below which is made with reference to the accompanying figures in which: Figure 1 represents the restriction map of the Tc40 gene, whose map is deduced by Southern blotting of different fragments obtained after digestion of Trypanosoma cruzi DNA with restriction endonucleases.

Figure 2 is a schematic representation of the 3 superimposed regions corresponding to the 5 ', central, and 3' regions of ccTN of Tc40. The arrows marked SL, 1, 2, 3, 4, AD indicate the position of the PCR primers used for the amplification. The resulting full-length cDNA is represented by the black box.

Figure 3 (A) represents the characterization of the Tc40 antigen with respect to the identification of the native T. cruzi antigen related to the Tc40 fusion protein. Western blots of T. cruzi epimastigote (G chain) were incubated with the following guinea-pig antiserum: anti-GST serum (l), pre-immune serum (2) and anti-GST-Tc40 serum (3). The arrows indicate the polypeptides specifically recognized by anti-GST-Tc40 serum. The sizes of the markers are shown on the left.

Figure 3 (B) represents analysis by Southern hybridization showing the location of the Tc40 gene in T. cruzi G and CL chains. The chromosomal bands of the G and CL epimastigotes were separated by pulsed-field gel electrophoresis (PFGE) and analyzed by Southern blot hybridization with 594-bp of Tc40 inserted as a test. The molecular weights of the indicated chromosomes, in megabase pairs, corresponding to G and CL chains are indicated to the left and to the right of the gel, respectively.

Figure 4 represents the Southern blot analysis of the Tc40 gene. 5 μg of G (1), Y (2), CL (3), Dm30 (4) DNA of T. cruzi and DNA of Leishmania mexicana amazónica (5) were digested with the corresponding restriction enzymes and analyzed by Southern blot with 594-bp radiolabelled from Tc40 inserted as a test. The sizes of the markers are shown on the left.

Figure 5 represents: (A) Southern blot analysis of the Tc40 gene in the G chain. 5 μg of Trypanosoma cruzi DNA were restricted with (1) HaelII, (2) EcóRI / HaelII, (3) £ coRI (4) EcoRl / Pstl, ( 5) Pstl, (6) PvuII, (7) Sacl, and (8) Pvull / Sacl, and subjected to Southern blot analysis using the 597-bp test of Tc40.

(B) The restriction map of the Tc40 locus gene, deduced from the combination of many Southern blot analysis. (Ps): PstI, (E): EcoRI; (S): Sacl; (D): Oral; (H): HindIII; (B): BamHl; (Ha): HaelII; (P): PvuII; (A): Accl. The striped box represents the 594 bp insert of the clone? Gtll-Tc40 isolated.

Figures 6 (A) and 6 (B) represent the nucleotide and deduced amino acid sequences of the Tc40 cDNA the asterisk at the end of the adenine segments and the "T-and GT-rich" regions are doubly underlined. The general consensus SL is underlined in dotted line.

Figure 7 represents: (A) Schematic representation of the genomic clones? Gtll and? GtlO. The 594-bp and 3.7-kb .EcoRI clones of Tc40 are represented by a striped box and a bold line, respectively.

(B) The nucleotide sequence of the 5 'region of the clone? GtlO-Tv40 of 3.7 kb (Gen Bank accession number TCU96914). The sequence of the protein corresponding to the reading opening formed is indicated. The promoter sequence is underlined and the binding site of the transcription factors are indicated in bold letters; the initial site of the transcript is signaled (+1). The undetermined nucleotide bases are designated N.

Figure 8 depicts the location of the human antigenic determinant in the amino acid sequence of the? Gtll-Tc40. The number of amino acids is the only one of the Tc40 protein deduced from the translation of Tc40 cDNA. The sequence of the antigenic determinant is underlined.

Figure 9 (A) represents the transcription in vi tro and the cDNA translation of Tc40. The translation in vi tro of products marked from the Tc40 cDNA, cloned in the vector pSP64 poly (A) downstream (3 '-5') of the SP6 promoter were analyzed by SDS-PAGE (l), by Western blotting, and tested with a guinea pig anti-GST antiserum (2) and guinea pig anti-GST-Tc40 serum (3). The size of the markers, shown on the left, is in kilodaltons (kDa).

Figure 9 (B). represents the reactivity of the recombinant protein GST-Tc40 with Chagasic human serum. The purified proteins related to GST after the induction of IPTG of recombinant pGEX-Tc40 (4) and non-recombinant pGEX (2) were separated by 12% SDS-PAGE, Western blot, and tested with a combination of Chagas' human serum chronic. The sizes of the markers are shown on the left.

Figure 10 represents the serological evaluation of the peptide BIO-S23G (Tc40) in a direct ELISA test. The distribution of the DO values was obtained in relation to the serum populations evaluated. The trace of the box indicates for each population of serum evaluated the distribution and the average value of OD492nm. The five horizontal lines of the boxes correspond to the percentiles 10.25.50 (medium), 75.90. The values on both sides of the 10th and 90th percentiles are experimental points (circles).

A corresponds to patients suffering from chronic Chagas disease (n = 184). B corresponds to normal serum (n = 63). C corresponds to patients suffering from toxoplasmosis (n = 7). D corresponds to patients suffering from visceral leishmaniasis (n = 26). E corresponds to patients suffering from mononucleosis (n = 5). F corresponds to patients who present antinuclear antibodies (n = 5) G corresponds to patients suffering from filariasis (n = 8).

MATERIALS AND METHODS USED IN THE PREVIOUS EXAMPLES The metacyclic epimastigotes and tryomastigotes of Trypanosoma cruzi were grown in liquid medium infusion of triptose liver (LIT) supplemented with 10% fetal calf serum inactivated at 28 ° without stirring. The trypomastigote and amastigote cell cultures were obtained from infected monolayers of Vero and HeLa cells. The E. coli DH5K chain (Gibco BRL, France) was used to clone and also for the expression of the fusion protein in the pGEX plasmid (Pharmacia Biotech, France) although the Y1090 chain was used for the growth and expression of the phage 9gtll. The 9gtl0 phage (Amersham France) was grown in the Y1089 chain of E. coli.

The DNAs and RNAs were isolated from axenic cultures of T. cruzi by conventional protocols (see Maniatis T et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). The recombinant clone Tc40 was isolated from a genomic expression library of T. cruzi (G chain) projected with a combination of chronic Chagasic serum (see Ozaki et al, Plaque antibody selection: rapid immunological anaysis of a large number of recombinant phage clones positive to sera against Plasmodium falciparum antigens J. Immunol Methods 1986; 89: 213-9). The insert of the purified original DNA clone was subcloned into the Pucl9 plasmid (Gibco BRL) for sequence analysis, and into the pGEX expression plasmid to produce a fusion protein with the Schistosoma japonicum glutathione S-transferase (GST-Tc40) induced by isopropyl-2-D-thiogalactopyranoside.

Serum samples were collected from patients with chronic Chagas disease diagnosed by serological methods and clinical symptoms. Serological analyzes were performed by direct immunofluorescence (ImmunoCruzi, Biolab-Mérieux, Brazil) and ELISA (BioELISAcruzi, Biolab-Mérieux, Brazil). Antibodies against the GST protein and the GST-Tc40 fusion protein were cultured in Guinea pigs by injection of partially purified recombinant and non-recombinant antigens extracted from polyacrylamide gels.

The GST and GST-Tc40 proteins were induced and purified within Sepharose Glutathione 4B (Pharmacia LKB Biotechnology, France) in the presence of one mM phenylmethylsulfonyl fluoride. The parasites were used in the presence of a solution containing 150 mM NaCl / 10 mM Tris-Hcl, pH 7.5 / 1 mM EDTA / 1% Nonidet-ketone / 2 U mL-1 aprotinin / 25: g ml-1 leupeptin / 25: g ml-1 antipain (Boehrinfer Mannheim, France). The parasite lysate was incubated at 4 ° C for 30 minutes, centrifuged for 10 minutes at 18,000 x g at 4 ° C. This supernatant was used directly in the immunoblot study. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with parasites and bacteria in 10 or 12% gels in the Laemmli buffer system (see Laemmli UK, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 1970; 227: 680-5). The immunoblot was carried out by the method of Towbin et al. (See Towbin et al., J. Electrophoretic Proc Nati Acad Sci USA 1979; 76: 4350-4). The bound antibodies were revealed with both goat anti-human IgG paired to alkaline phosphatase (Jackson Immuno Research Laboratories) and protein A [1251] (Amersham, France).

For the intracellular staining of the parasites, the Vero cells grown on strips of glass covers were infected with T. cruzi. The immunofluorescence reaction was carried out at 72-96 hours post-infection, as previously described using the GST-Tc40 specific antibodies cultured in Guinea pigs (see Cotrim PC et al., J. Expression in Escherichia coli of a dominant immunogen of 'Trypanosoma cruzi recognized by human chagasic sera J Clin Microbiol 1990; 28: 519-24).

To clone the Tc40 cDNA, the T. cruzi mRNA, the G chain, was first reverse transcribed using random hexanucleotide primers, and then amplified by the polymerase chain reaction (PCR). The 5 'portion was generated using an SL-sense primer (5'-AACGCTATTATTAGAACAGTT-3') (SEQ ID NO: 3) deduced from the spliced leader sequence (SL) of T. cruzi (See Parsons M er al., Trypanosoma mRNAs share a common 5 'spliced leader seq., Cell 1984; 38: 309-16) and an inverse-1 primer (5'-TGCAGCAGCAGCGGCAGAAGT-3') (SEQ ID NO: 4) of the original Tc40 sequence corresponding to nucleotides 1442-1459 of the Tc40 cDNA). The central region was obtained using an initiator-2 in the direction (5'-CAGCCGACGGTAGCTGCGTCCT-3 ') (SEQ ID NO: 6) corresponding to nucleotides 2187-2207 of the Tc40 cDNA. To clone the 3 'ends of the Tc40 cDNA, the polyadenylated RNA of T. cruzí, the G chain, was converted into a single-stranded cDNA according to the rapid amplification protocol for the 3' ends of cDNAs (RACE) (see Frohman MA et al., Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotid primer Proc Nati Acad Sci USA 1988; 85: 8998-9002), using an adapter-17 primer (dT) hybrid [(dT) 17-AD]. The cDNA was amplified by PCR using a gene-specific sense primer (5'-CGAAGAGACCATGAACAACTT-3 ') 8SEC ID NO. 7) corresponding to the positions of the 1997- 2017 nucleotide of the Tc40 cDNA and the AD adapter-initiator. The sequences of primers numbered 3 and 4 were obtained from an enpecific clone isolated from a 3.7 kb genomic library of T. cruzi EcolRl 9gtl0 which hybridized with the original insert Tc40. PCR experiments were performed for 35 cycles of 1 minute at 94 ° C, 1 minute at 50 ° C, 1 minute at 72 ° C followed by extension at 72 ° C for 7 minutes, using 50 pmol of each primer and 100 ng of T. cruzi simple chain cDNA. The Taq polymerase used was obtained from Perkin Elmer Cetus, France. The PCR products that hybridized to the original Tc40 clones were cloned and sequenced within the PCR II vector using the TA cloning kit for PCR products (in Vitrogen, San Diego, CA).

The complete open reading frame of Tc40 (ORF) was obtained after assembling the 5 ', central, and 3' regions obtained by PCR, as described above. The sense-initiating sequence included the consensus Tc40"kozak" and the initiator AUG, while the initiator in contradictory contained the three consecutive stop codons of Tc40. The PCR products were cloned into the pSP64 Poly (A) vector (Promega, Madison, Wl), downstream (3 '-5') of the SP6 RNA polymerase promoter and transcribed and translated in vi tro using the Transcription system. - Matched Translation TNT (Reticulocytes used rabbit, Promega). The 35S-labeled products translated were separated by SDS-PAGE as described above.

The nucleotide sequence of the insert plasmid was determined by double-linked sequencing according to the dideoxynucleotide chain termination method (see Sanger et al, DNA sequencing with chain-terminating inhibitors, Proc Nati Acad Sci USA 1977; 74: 5463-7), using Sequence (Amersham, US Biochemichal, France). The analysis of the Tc40 sequence was carried out using the Mac Vector 4.5 software (Kodak). The sequence was also compared, at the level of the nucleotide and at the level of the protein. To all the major sequence databases (BLASTN, BALSTP, TBALSTN). The databases were provided by the NCBI GENINFO (R) Experimental BLAST Network Service.

DNA restriction fragments were radiolabelled with (K '-32 PjdATP using a randomizing DNA marker kit (Boehringer Mannheim) .The northern and southern blots were prepared using standard methods.Hybridizations for both DNA analysis and of RNA were performed overnight at 42 ° C in 6 x SSC (1 x SSC is 0.15 M NaCl / 0.015 M sodium citrate, pH 7.5) / 5 x Denhardt's solution (1 x Denhardt's solution: Ficoll 0.2 mg ml-1 / polyvinylpyrrolidone 0.2 mg ml-l / bovine serum albumin 0.2 mg ml-1) 50% formamide / 0.5% SDS / 100: g ml-1 of sonicated herring sperm DNA. After hybridization with the Tc40 test, the filters were washed in 2 x SSC / 01% SDS at room temperature for 15 minutes, then in 0.1 x SSC / 0.5% SDS at 37 ° C, for 30 minutes and, finally at 65 ° C, for 30 minutes.

Pulsed-field gel electrophoresis (PFGE) samples were obtained as described in Cano MI et al., J. Molecular karyotype of clone CL. brener chosen for the * Trypanosoma cruzi genome project. Mol Biochem Parasitol 1995; 71: 273-8. The agarose blocks containing 108 chains of epimastigotes CL and G were prepared and stored in 0.5 M EDTA, pH 9.0. The equivalent of 107 parasites were electrophoresed at 80 V for 132 hours at 13 ° C, with pulse times varying from 90 to 800 seconds. The chromosomal DNA bands were transferred to nylon filters and the spot hybridized and washed as described above.

The evaluation of the BIO-S23G peptide by the indirect ELISA process was provided in 96 wells contained in plates impregnated with 100 μl streptavidin in 10 μg / ml PBS overnight at 4 ° C. Plates were washed three times with PBS-Tween 20 at 0.05%. Loo μl of the BIO-S23G peptides in 10 μg / ml of PBS were absorbed in the plates for 2 hours at 37 ° C. Then the plates were washed. The serum was diluted 1/100 in a total volume of 100 μl and incubated for 2 hours at 37 ° C. After three washes, the plates were incubated for 90 minutes at 37 ° C with 100 μl of goat anti-IgG IgG coupled with PA and diluted to 1/30000. OD was measured at 492 nm. Each serum was double tested.

The library of the Tc40 peptide domains was constructed in the vector pTOPE-T, according to the protocol provided by the manufacturer (Novatope Library Construction System, Novagen).

The peptides were synthesized using an Applied Biosystems 413a synthesizer, with the Emoc / tBu strategy.

Example 1: Isolation and characterization of the recombinant antigen Tc40.

An expression library in the 9gtll vector was made directly from randomly generated fragments of T. cruzi nuclear DNA. Approximately 50000 recombinant phages were projected with a combination of chronic Chagasic serum and forty phages expressing the T. cruzi antigens were purified. Based on the signal intensity, the clone Tc40 (594-bp) was chosen for further characterization. The tc40 insert was subcloned into the expression vector pGEX in order to produce high amounts of the GST fusion protein in bacterial cultures induced with isopropyl thiogalactosidase. The reactivity of the fusion protein was analyzed by immunoblotting assays using a combination of chronic human Chagasic serum. The Tc40 clone encodes a GST fusion protein of approximately 48 kDa which reacts strongly with the antibodies of chronic Chagasic patients. Some proteolysis products could be observed in this antigen preparation even in the presence of a serine protease inhibitor. In the same experiment, the non-recombinant GST protein failed to react with human Chagasic serum showing the specificity of the European protein GST-Tc40 (Figure 9B).

To identify the native Cruzx protein that shares common antigenic determinants with the Te 40 clone, immunoblots were tested by using the T. cruzi epimastigote with a monospecific guinea pig antiserum against the recombinant protein Tc40. The monospecific antiserum reacted with the three polypeptides of molecular masses of 100.41 and 38 kDa (Figure 3A, line 3). These polypeptides were also detected at comparable levels in all stages of parasite development. In contrast, the anti-GST control serum, such as antibodies from a pre-immunized animal and serum from an animal immunized with GST, failed to react with T. cruzi polypeptides (Figure 3A, lanes 1 and 2). This result shows that the antigens known for anti-GST-Tc40 serum are more related to Tc40.

All three polypeptides were detected by anti-GST-Tc40 antibodies even when the parasites were used in the presence of a mixture of protease inhibitors (serine, cysteine and metalloproteinases), which is normally used to protect the T. cruzi peptides against the activity of endogenous proteases. It is noteworthy that the anti-GST-Tc40 antibodies reacted with almost the same intensity with the 100 and 41 kDa peptides (Figure 3A, line 3), suggesting that they are present in equivalent amounts in the cell. The available data suggest that peptides of molecular masses of 100.41, and 38 kDa represent different molecular entities that share common epitopes. The 41 and 38 kDa species appear to be products of the proteolysis of the 100 kDa protein.

The cellular localization of the Tc40 antigen was investigated by indirect immunofluorescence using parasites fixed in formaldehyde. The anti-Tc40 antibodies discolored the cytoplasm of amastigotes and trypomastigotes. A reaction with the nucleus or the kinetoplast was not obtained. The human Chagasic antibodies immunopurified in the recombinant antigen also discolored the cytoplasm of the T. cruzi cells giving a fluorescence model similar to that obtained with the monospecific guinea pig antiserum. As expected, the anti-GST control serum does not react with intact T. cruzi cells (amastigotes and trypomastigotes) in indirect immunofluorescence assays. The living parasites were not labeled with anti-Tc40 antibodies, suggesting that the proteins recognized by these antibodies are located in intracellular structures.

Example 2: Cloning and sequencing of the Tc40 gene.

Northern blots were hybridized by carrying mRNAs from the epimastigote stage of G and CL chains with the insert of the Tc40 genomic clone. The test or probe hybridized with a transcription of around 3.9 kb in both chains. No additional bands were observed even after a longer exposure (10 days). These results indicate that the gene length for Tc40 was at least 3.9 kb.

In an attempt to define the entire transcribed region of the Tc40 gene, three overlapping cDNA fragments corresponding to the 5 'and 3' regions of the Tc40 gene have been cloned. The cloning strategy was based on PCR amplification using as primers a combination of specific sequences of 9gtll-Tc40 (594-bp) and T. cruzi (Figure 2). The 5 'region of the Tc40 gene was amplified using a pair of primers derived from the SL sequence (sense) and the genomic sequence 594-bp (contradictory) (Figure 2). The amplified cDNA fragment was 1459-bp long carrying the entire 5 'region of the Tc40 gene including the SL sequence. The central region of the Tc40 gene including the genomic sequence of 594-bp was amplified using a pair of Tc40-specific primers.

To obtain more information on the organization of the Tc40 gene, the 594-bp fragment was hybridized with a genomic Southern blot carrying T. cruzi DNA digested with several restriction enzymes (see Figure 4 and 5A and 5B). a conserved 3.7-kb EcoRI fragment was detected in all the chains tested. For this reason, a genomic library of T. cruzi was constructed in 9gtl0 using fragments of approximately 3.7 kb obtained by complete digestion with EcoRI of T. cruzi DNA (G chain) and a recombinant phage carrying the expected genomic fragment of 3.7 kb. EcoRI was isolated after hybridization with clone 594-bp. The region within the 9gtl0 recombinant clone homologous to the 594 bp sequence was identified and sequenced using specific primers. The 3 'end of the 3.7 kb EcoRI fragment was also sequenced and used in the isolation of the 3' end of the transcribed region (Figure 4, 5B and 7A). The 3 'end of the Tc40 gene was amplified by the 3' RACE method with a hybrid adapter initiator d (T) 17 and an initiator in the sense of an internal sequence of the genomic fragment - 3.7 kb EcoRI. The product generated was 1405 bp long and presented a tail of 18 adenine residues which could correspond to the poly (A) tail. Since the Northern blot suggests that the Tc40 mRNA is 3.9 kb and the cDNA compound includes 3.4 kb (Figure 6), it is possible that the RACE 3 'has identified an A-rich region in the 3' untranslated region (UTR) and unidentified the true poly (A) tail at the end of the mRNA that could currently have another 0.5 kb in its 3 'UTR.

The complete nucleotide sequence and predicted amino acids of the Tc40 cDNA are shown in Figure 6. The Tc40 cDNA is 3402 bp long with an ORF of 2745 bp encoding a polypeptide of 915 amino acids with a predicted molecular mass of approximately 100 kDa These ORFs joined with the original insert of Tc40 of 594 bp. It is interesting to note that no internal repeats were found in this gene, in contrast to more cloned genes encoding T. cruzi antigens. The first primer codon, located 266 bp downstream (3 '-5') of the SL sequence, met the nucleoside sequence frequency flanking the protozoan start codons, particularly finding the purine at position -3. The upstream sequence (5 '-3') of the initiator site contains stop codons in all three of its structures. At the end of 3 ', three consecutive stop codons were detected 390-bp upstream (5' -3 ') from the tail of the poly- (A) putative.

As shown above, the genomic fragment 3.7 kb- £ coRI cloned in 9gtl0 carries the region flanking 5 'of the Tc40 gene (Figure 1 and Figure 7). To see the putative promoter and the regulatory sequences, we sequenced the 5 'region immediately downstream (3' -5 ') of the Tc40 ORF. The 5'-flanking sequence of 1006 bp was analyzed with the Prosean program (version 1.7) for the search for potential promoter RNA polymerase II sequences (Prestidge, DS (1995) Predicting Pol II promoter sequences using transcription factor binding sites. Biol. 245 (5): 923-32). This program identified a potential TATA box containing a promoter, with a putative transcription initiator site at position 609. The promoter region was predicted between position 350 and 600 (positions- in the GenBank accession number U96914).

The Tc40 polypeptide shows no significant similarities with other published sequences as indicated by the search conducted in all databases of larger sequences. Of the predicted amino acid sequence, in addition to thirteen sites for N-glycosylation, there are 17 potential sites for myristylation.

Example 3: Transcription and genomic organization of the Tc40 gene.

Northern blot analysis showed that the insert of the Tc40 genomic clone was strongly hybridized to a 3.9 kb copy which is long enough to encode a 100 kDa peptide. In addition, to confirm the size of the protein, a full-length Tc40 mRNA was transcribed and translated in vi tro. Two polypeptides labeled ~ 35S of 100 and 80 kDa were detected (Figure 9, line 1), but only the 100 kDa peptide was recognized by the anti-GST-Tc40 antiserum (Figure 9, line 3). Thus, the size of the protein translated in vi tro correlated with that of the native protein found in the extracts of T. cruzi and the protein encoded by the ORF was shown in Figure 6 and 8. Clearly, this indicates that the peptides of 41 and 38 kDa, also found in extracts of T. cruzi, share common epitopes with the 100 kDa protein.

The insert of the Tc40 genomic clone was hybridized with Southern blotting carrying genomic T. cruzi DNAs (G, CL, Y and DM30 chains) Leishmania mexicana amazonensis (Figure 4). The test hybridized with all T. cruzi DNAs tested but not with Leishmania DNA, suggesting that it carries specific sequences of T. cruzi species. When the T. cruzi DNA, G chain was digested with several restriction enzymes and probed with the Tc40 genomic clone (Fig. 5A), a pattern was observed which is consistent with the presence of a few copies of Tc40 genes in the parasite genome.

The chromosomal mapping of the Tc40 genes was carried out by hybridizing the genomic fragment 594-bp with the chromosomes separated by pulsed-field gel electrophoresis. Fig. 3B shows the occurrence of only one hybridization band of 1.1 Mbp in the G chain, according to the results above, while the same probe hybridized with two chromosomal bands of 0.80 and 0.70 Mbp in the CL chain. These results suggest the existence of two allelic forms of the Tc40 gene, on chromosomes III and IV, in the CL Brener chain (see Cano MI et al., J. Molecular karyotype of the CL Brener clone chosen for the Trypanosoma cruzi genome project. Mol Biochem Parasitol 1995; 71: 273-8).

Example 4: antigenic relevance of the recombinant antigen Tc40.

The antigenic relevance of the recombinant antigen GST-Tc40 was tested by the immunoblot assay with a large panel of human serum samples from chronic chagasic patients (n = 201), non-chagasic patients (leishmaniasis, toxoplasmosis, filariasis, leprosy, mononucleosis, arthritis rheumatoid, autoimmune diseases) (n = 67) and healthy individuals (n = 36). The Tc40 fusion protein reacted with 92% of the serum samples from chronic chagasic patients. From 103 non-chagasic sera tested, only one serum sample showed a reaction with the recombinant antigen GST-Tc40 (Table 1). This result suggests that the presence of serum antibodies to the Tc40 antigen could be associated specifically with Chagas disease.

Table 1 This demonstrates that the structure of the Tc40 protein gene does not possess a repetitive amino acid pattern found in the majority of recombinant antigens isolated by protection of the T. cruzi expression libraries with human chagasic serum. In this context it is interesting to note that other T. cruzi proteins (ribosomal proteins P), which do not have repetitive motifs, are also antigenic in Chagas disease (see Levin MJ et al., The family of ribosomal proteins P of Trypanosoma cruzi: classification and antigenicity, Parasitol Today 1993; 9: 381-4).

The natural humoral immune responses to several T. cruzi antigens appear to be broadly directed to epitopes encoded by the repeat units. The strength of the signals given by chronic chagasic serum in Western blot immunoassays of recombinant Tc40 proteins indicates that antibodies against non-repetitive antigens are also present in the chronic phase of Chagas disease. High antibody titers against repetitive amino acid motifs in the vast majority of individuals living in endemic areas appear inconsistent with repetitive epitopes being the target of host immune responses.

Recently, several studies have demonstrated that T. cruzi recombinant antigens can potentially be used in the serological diagnosis of Chagas disease (see Moncayo A et al., Multicenter double blind study for evaluation of Trypanosoma cruzi defined antigens as diagnostic reagents. Ment Inst Oswaldo Cruz, 1990; 85: 489-95). The recombinant peptide Tc40 was used in immunoblotting assays to protect the standard serum classified as chagasic and non-chagasic based on conventional serological tests. Sensitivity (92%) and specificity (99%) of the Tc40 antigen are comparable with the other serodiagnostic tests of T. cruzi based on recombinant antigens.

Example 5: Identification of an immunodominant domain of PTc40 To more accurately determine the antigenic domain (s) of the PTc40 protein, a Tc40 fragment expression library was made from the genomic clone? Gtll-Tc40 DNA. The 594 bp insert of clone? Gtll-Tc40 was digested with deoxyribonuclease I and populations of fragments of various sizes from 50 to 150 bp were cloned into the vector pTOPE-T. The immunological protection of the library obtained was carried out with the chronic phase human chagasic serum well. The protection of about 3000 bacterial clones with - the chagasic human serum well led to the isolation of an identified clone.

The total protein extracts of the isolated clone and the non-recombinant control were analyzed by immunoblotting. The fusion proteins with Protein 10 of phage T7 (36 kDa) have been identified with the anti- "T7. Tag". The recombinant proteins expressed by the human "tc40 epitope" clone are recognized with the human chagasic serum well of the chronic phase.

The nucleotide sequence of the clone "human Tc40 epitope" was located at position 1472-1543 in the Tc40 DNA (amino acids 403 to 426 of the PTc40 protein) (Fig. 8).

The human chagasic serum antibodies are directed against the antigenic domain located at position 403-426 of the PTc40 protein.

The antigenic domain determined with the antibodies of the human chagasic serum present some peculiarities in its sequence. It has 6 prolines, which can introduce a constriction in the polypeptide chain and thus induce a secondary or tertiary structure. Two motifs of three consecutive alanine amino acids are also observed.

The use of peptides corresponding to the immunodominant human domain sequence of the PTc40 protein can eliminate problems related to the purification of the GST-Tc40 antigen as well as problems of reactivity of some sera with the GST protein.

To determine if the sequence of the "human Tc40 epitope" represents the major antigenic determinant of the Tc40 protein of T. cruzi, a peptide referred to as S23G (Tc40) presenting that sequence was synthesized. The serine at position 402 of the PTc40 sequence was integrated into the peptide sequence [S23G (Tc40) and not P22G (Tc40)] to allow binding of this peptide to an amine via the intermediary of a free alcohol function at N-terminal end of the peptide.

The peptide S23G (Tc40) corresponding to the human antigenic domain Te has been validated in indirect standard ELISA with a well of human chagasic serum of chronic phase and two characterized non-chagasic sera: No reactivity was observed with the negative serum and with the serum well human chagasic chronic phase.

The reactivity of the S23G peptide (Tc40) was checked with an inhibition test against the recombinant protein GST-Tc40 adsorbed under standard conditions in the ELISA plate. Added to 20 μg / ml of the human chagasic serum well, the inhibitory peptide caused the inhibition of about 80% of the binding of anti-GST-Tc40 antibodies from the human chagasic well. A similar result was obtained when a GST-Tc40 control recombinant antigen was added to the human chagasic serum well. On the other hand, no reactivity was observed with the serum tested negative. These results clearly demonstrate the reactivity of the S23G peptide (Tc40) towards the anti-Tc40 antibodies.

To explain the absence of reactivity of the S23G peptide (Tc40) in the solid phase, the secondary structure of the S23G peptide was verified, and it shows that the S23G peptide (Tc40) has no secondary structure, and that the human Tc40 epitope is sequential.

To maintain the accessibility of the human Tc40 epitope, a biotin has been linked to the N-terminal end of the S23G (Tc40) peptide (BIO-S23G (Tc40)). The BIO-S23G peptide was tested with indirect ELISA. Comparison of the results obtained in an ELISA using the BIO-S23G peptide and in a Western blot using the GST-Tc40 antigen, as demonstrated in Table II, suggests that the 'S23G peptide contains the immunodominant epitope of the recombinant Tc40 antigen in the natural infection of T. cruzi.

Table II at the crosses indicate the relative degree of reactivity of the serum with the antigen GST-Tc40.

The reactivity of the biotinylated peptide S23G (Tc40) was analyzed by an ELISA test using an important panel of chagasic and non-chagasic serum. 184 sera from chronic phase chagasic patients (indeterminate form, cardiac and digestive forms), including more than 95% of the serum tested in Western blot on the GST-Tc40 antigen. recombinant, were tested. To better evaluate the specificity of the test, the number of sera from healthy individuals was increased to 63, including 40 samples from French donors. The reactivity of BIO S23G (Tc40) was analyzed in serum of patients suffering from toxoplasmosis (n = 7), mucosal leishmaniasis (n = 26), mononucleosis (n = 5), filariasis (n = 8) and serum of patients who present antinuclear antibodies (n = 5). All these sera have been tested in Western blotting of the recombinant GST-Tc40 antigen. Each plate includes two negative control sera, two anti-Tc40"threshold" control sera, previously identified in a Western blot, as well as a strongly positive control anti-Tc40 serum. The distribution of the values obtained from OD492 is presented in Fig. 10 according to the different populations tested. The discrimination between chagasic and non-chagasic serum is satisfactory. In fact, the average of OD492 obtained in the entire Chagas population is 1.6 +/- 0.86, while the average of OD492 obtained in the entire non-Chagasic population is 0.17 +/- 0.24. No interference was observed with the serum of patients suffering from toxoplasmosis, mononucleosis, filariasis, or patients suffering from autoimmune diseases. However, among the population of sera from normal individuals, five sera from French blood donors show a weak reactivity with the BIO peptide S23G (Tc40). Serum from patients suffering from visceral leishmaniasis has a higher reactivity than other populations (OD + 0.39 +/- 0.38). The cut was determined as the means of all the non-chagasic populations + 3 standard deviations. It was evaluated at OD = 0.vity has been determined at 81%, and the specificity at 96% in the tested population.

Accuracy can also be increased by combining two or more antigens to construct a multi-antigenic immunoassay. The repertoire of B cell epitopes in a non-repeating antigen is greater than that found in repetitive antigens. To improve the specificity of serodiagnostic tests, non-repetitive antigens, such as Tc40 and / or S23G and 1F8 (see Godsel LM et al., Utility of recombinant flegellar calcium-binding protein for serodiagnosis of Trypanosoma cruzi infection J Clin Microbilo 1995; 33: 2082-5 and Enge an DM et al., J Biol. Chem 1989; 264: 18627-31,) should be included in the multi-antigen immunoassays.

Example 6: Location of the Ptc40 epitope To determine more precisely the major epitope of the Ptc40 protein, nine overlapping peptides of 9 to 12 amino acids, corresponding to the S23G sequence, were synthesized: Pl (1-10) SPPVSAPAKA P2 (3-12) PVSAPAKAAA P3 (5-16) SAPAKAAAPPAA P4 (7-16) PAKAAAPPAA P5 (9-18) KAAAPPAAAR P6 (11-20) AAPPAAARSA P7 (13-24) PPAAARSAEPHV P8 (15-24) AAARSAEPHV P9 (17-25) ARSAEPHVG The reactivity of each peptide was plotted with an inhibition test against the recombinant GST-Tc40 protein adsorbed under standard conditions in an ELISA plate. Added to 10 micrograms / ml of the human chagasic serum well, two peptides (P3 (5-16) and P4 (7-16)) showed 83 and 80% inhibition, respectively, of the binding of anti-GST-Tc40 antibodies of the chagasic human well. No inhibition was observed with the other peptides. These results indicate that the major epitope of PTc40 is located more precisely at positions 406-417 of the PTc40 protein.

SEQUENCE LIST (1. GENERAL INFORMATION APPLICANT: BIO MERIEÜX (i) TITLE OF THE INVENTION: NEW ANTIGEN OF TRYPANOSOMA CRUZI, CODING GENE OF THIS, AND METHODS OF DETECTION AND TREATMENT OF CHAGAS DISEASE (ii) NUMBER OF SEQUENCES: (iii) CORRESPONDENCE TO THE ASSIGNER: (A) CONSIGNEE: Cabmet GERMAIN & MAÜREAU (B) STREET: 12 RUÉ Boileau (OR CITY: Lyon (D) COUNTRY: France (E) ZIP: 69006 (iv) LEGIBLE FORMAT IN COMPUTING: (A) TYPE OF MEDIA: DISKETTE 1.44 Mb, 3.5 PÜLG (B) COMPUTER: IBM COMPATIBLE PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patent In Relay # 1.0, version # 1.30 (v) CURRENT APPLICATION REFERENCE: (A) APPLICATION NUMBER: (B) REGISTRATION DATE: (C) CLASSIFICATION: (viii) INFORMATION FROM THE LAWYER / AGENT (A) NAME: Dommique GÜERRE (B) REGISTRATION NUMBER: (C) REFERENCE / CELLULAR NUMBER: AD / MG / BO5B2082 (ix) TELECOMMUNICATION INFORMATION (A) TELEPHONE: 472-69-84-30 (B) TELEFAX: 472-69-84-31 (2) INFORMATION FOR SEC ID NO: l (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3 402 base pairs (B) TYPE: nucleic acid (C) CHAIN: double (D) TOPOLOGY: linear TYPE OF MCLÉCUI cALN (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: I AACGCTATTA TTAGAACAGT TTCTGTACTA TATTGTCATT TGGGGAGGGG GGAAAGGGGG 60 GAAGTACTTG CCGTTTTGTG TGGGTGACGA GACAACACAC ATCGAGCGGG AAGAAAAAAA 120 AAAAGGAAAT AAATTAAATT AAATTATTTG TTCTTTGAAT AGGCAAAGAA GAAGAAGAAG 180 AAAAGGTGCG GGGGAGGGAG GAGAAAGCGA CACACACACA AAAAAAAAAA AAGGAATTGC 240 GGAAATAACA ACGCAAGGCG CGGACATGAC CGTGACGGTG GATTTGTTCA ATCATGCGAA 300 GCCGAGCAAC AATGAGGGCC GCGTGTGGTC TGTGGACGCC GCGACATTTA ACGAGGTGCC 360 TGAGGCGCAG CGTGTGCTGG CGGATTCGCA GTTTTATCTT GCCTACACCA TGAAGCGGCG 420 TCACGTGCTG CGTGTGGTGA AGCGCTCGAA CCTTTTGAAG GGCACCGTGC GGGCACACTC 480 AAAGCCCATT CATGCGGTGA AGTTTGTGAA TTACCGCAGT AACGTCGCAG CATCGGCTGG 540 GAAGGGGGAG TTCTTCGTGT GGGTTGTGAC GGATGAAACG GAGGCGAGCA ACGGCAAGCC 600 • GGATCTCGCA GCCCGCCTCA CAGTGAAGGT GTACTTTAAG CTTCAGGATC CTGTCACAAT 660 TCCATGCTTT TCTTTCTTTA TCAACGCCGA GAGTCAGCGG CCTGATCTGC TTGTCCTTTA 720 CGAAACGCAG GCGGCAATTC TTGACAGCTC CTCCCTCATT GAGCGCTTTG ACGTGGAATC 780 10 ACTGGAGGCA ACACTACAGC GGAATTGCAC AACCCTGCGA ACCCTGACTC AACCGGTTAG_840_T TTATGCTCCG TTGGCTCTGG CGGATGGTTC ACCTTTACCA CGGAACCA? C 900 AATGGTAGCG GCATGCACAT TACGAAACCG CAGCACTCCA TCATGGGCGT GTTGCGAGGG 960 15 TGAGCCAGTG AAGGCATTGC ATCTCCTTGA CGCAACCGTT GAGGAAAATG TCAGTGTTCT 1020 CGTGGCCGCA TCTACAAAAG GGGTGTACCA ATGGCTCCTT ACGGGTGTAG CAGAACCAAA 1080 CTTGTTGCGC AAGTTTGTCA TTGATGGATC TATTGTCGCG ATGGAAAGCT CACGAGAAAC 1140 20 GTTTGCCGTG TTTGACGACA GGAAGCAGCT GGCGCTGGTC • AACATGCATT CCCCTCATAA 1200 CTTTACCTGC ACACACTACA TGATGCCTTG TCAGGTACAG CGTAACGGCT TTTGCTTCAA 1260 TCGTACAGCC GACGGTAGCT GCGTCCTGGC TGACATGTCG ATTCGATTGA CGATCTTCCA 1320 25 TCTCCGGTCC TCCCGCAGGG AAGAACAGCA GCCAGGCCAA AAAACATCGG TAGTGGCGAC 1380 GGCGAAACCG GGGTGTGTGT CCTCGGGCAC TGACGCGGCG AGTAGCAGTC ATACCAATAC 1440 GACTTCTGCC GCTGCTGCAT CCCCTGCATC ACCCCCTGTT TCAGCOCCAG CCAAGGCAGC 1500 CGCGCCTCCT GCCGCGGCGC GATCGGCTGA GCCGCACGTG GGGAGCAAGA TCATTGCTAA 1560 TCTAGTGAAT CAGCTGGGGA TTAATGTCAC CCAAAGGAGC GTCGTCAGCA CTGGAGCGCC 1620 GGCCACGACG AGGTCTACGG CGGTGACGTC CACGACTACC GCCCCGCAGC GAACAAGTCC 1680 ATACGGGCAC AATGGCCGAC CTGTGACGGC TGGATTGGTG GCAGCTAATA GTGGTGCCAG 1740 CGCGGCCTCG TCTCCCACAG CCGCGGCGAA ACCAACAGGA GAAGAAAAGG CCTCCGCGGC 1800 ATGTGAAACG AGCTCCGTGG CGATAAATGC GACACGCCCG GCGCTTCACA ACGCCTCTCT 1860 CCCGCAGGCG CCAACGGATG GCGTTTTGGC GGCAGCAGTA TACCAGTCGG AGGGCGAGGT 1920 TCATCAGTCG CTGGAGCGGC TGGAGTCCGT CATAACCAAC ACGTCTCGGG TTCTGAAGTT 1980 GCTCCCTGAC ACCATTCGAA GAGACCATGA ACAACTTCTG AATCTGGGTT TAGAGGCACA 2040 GATGACAGAG CTGCAGCAGA GCCGTCCAAC ACCGCAAACA CAGCCGAGAG ACACAAGCTC 2100 CGCGAAATCA TCCGTGTTTG AGACGTACAC CCTTGTTCTC ATTGCGGATT CCCTCTCTCG 2160 CAACATCACG AAGGGGGTGA AGCGTGGTGT GAACGAGGCC ATTATGTTGC ATCTCGACCA 2220 TGAGGTGCGG CACGCCATAG GGAACCGGCT TCGGCAAACA CAAAAGAACA TCATCAAGAG 2280 CCGCCTCGAT GAAGCGTTGA AGGAAAGCAC TACACAGTTT ACGGCTCAAT TGACGCAAAC 2340 GGTGGAGAAT CTGGTGAAGC GCGAGCTTGC CGAGGTGCTT GGTAGCATCA ACGGCTCCCT 2400 CACTTCTCTC GTGAAGGAAA ATGCCTCATT ACAGAAAGAG TTGAATTCCA TAATGTCTAG_2460_TGGGGTGTTG GATGAAATGC GTCGTATGCG GG AAGAGCTG TGCACATTGC GAGAGTCCGT 2520 10 TGCGAAGCGG AAGGCAACAA TGCCAGATTC TTCTCTTCAC GCCACGAGCT CCTTTCAAGG 2580 • AAGAAGGTCT GCGCCCGAGA CAATTCTTGC AACCGCGTTA TCGATGGTGC GAGAGCAGCA 2640 ATACCGTCAG GGACTGGAAT ACATGTTGAT GGCTCAGCAG CCCTCTCTCC TCCTGCGGTT 2700 15 CCTCAGCATA CTTACAAGGG AAAACGAAAA CGCCTACTCG GAACTTATTG AAAATGTAGA 2760 GACGCCGAAT GACGTGTGGT GTTCGGTTCT GTTGCAACTC ATAGAGGCCG CGGCGACCGA 2820 GGCTGAGAAG GAGGTGGTTG TTGGCGTCGC CATTGATATT CTCTCCGAGC GCGATCAAAT 2880 20 TGCTCAGAAC GGCGCACTCG GCTCGAAACT CACCACCGCC ATGCGAGCCT TTGAGCGACA 2940 GGCAAGGTCG GAGACAACGA GCAGGTCATT CTTGCAATGC CTGAAGAACC TGGAAAAGCT 3000 TCTGCAATCA TGATAATAAA AAGAACTCAA CGAATACAGT TGTTGATTAT TAAGGAAGGG 3060 25 GAGAGAGAGA AAAAGAGAGA AAGAGAGAGA AATGTAATGG GCOTTTAGTT ACGGTAGAAA 3120 GAAAACGTGT GGATAAGAAG GAGGGGTTTT GTGTGCGACC AGGAATTACT GGGGAACGCT 3180 GCTACACGGC GGAATCGACC ATTTTATTAT TATTATTATT GTCTTTAGTA TTATGTTTTT 3240 TCTTGTGTGT OTGTGTGTGT GTTTGTGTGT GTGCGGTTAT TTTGTATCCG TTTGCTCCCG 3300 CCCCTGCCCC CCATCACCCG AGGAGAAAGT AGAATAAGAC ACATACGATT GTTGTTTTTG 3360 TTATCCTTAA AAGGAAGAGA GACCAAAAAA AAAAAAAAAAA AA 3402 (2) INFORMATION FOR SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 915 amino acids (B) TYPE: amino acids (C) CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "protein". , x ?: DESCRIPTION OF THE SEQUENCE: SEQ ID NO: Met Thr Val Thr Val Asp Leu Phe Asn His Wing Lys Pro Ser Asn Asn 1 5 10 15 Glu Gly Arg Val Trp Ser Val Asp Wing Wing Thr Phe Asn Glu Val Pro 20 25 30 Glu Wing Gln Arg Val Leu Wing Asp Ser Gln Phe Tyr Leu Wing Tyr Thr 35 40 45 Met Lys Arg Arg His Val Leu Arg Val Val Lys Arg Ser Asn Leu Leu 50 55 60 Lys Gly Thr Val Arg Wing His Ser Lys Pro lie His Wing Vai Lys Phe 65 70 75 80 Val Asn Tyr Arg Ser Asn Val Wing Wing Wing Gly Lys Gly Glu Phe 85 90 95 Phe Val Trp Val Val Thr Asp Glu Thr Asp Ala Ser Asn Gly Lys Pro 100 105 1 10 Asp Leu Ala Ala Arg Leu Thr Val Lys Val Tyr Phe Lys Leu Gln Asp 1 15 120 125 Pro Val Thr He Pro Cys Phe Ser Phe Phe Lie Asn Wing Glu Ser Gln 130 135 140 Arg Pro Asp Leu Leu Val Leu Tyr Glu Thr Gln Ala Wing He Leu Asp 145 150 155 160 Being Ser Leu lie Glu Arg Phe Asp Val Glu Be Leu Giu Wing Thr 165 170 175 Leu Gln Arg Asn Cys Thr Thr Leu Arg Thr Leu Thr Gln Pro Val Ser 180 185 190 Glu Asn Ser Leu Cys Ser Vai Gly Ser Gly Giy Trp Phe Thr Phe Thr 195 200 205 Thr Glu Pro Thr Met Val Wing Wing Cys Thr Leu Arg Asn Arg Ser Thr 210 215 220 Pro Ser Trp Wing Cys Cys Glu Glu Glu Pro Val Lys Wing Leu His Leu 225 230 235 240 Leu Asp Wing Thr Val GI.u Glu Asn Val Ser Val Leu Val Ala Wing Ser 245 250 255 Thr Lys Gly Val Tyr Gln Trp Leu Leu Thr Gly Vai Wing Glu Pro Asn 260 265 270 Leu Leu Arg Lys Phe Val lie Asp Gly Ser lie Val Wing Met Glu Ser 275 280 - 285 Ser Arg Glu Thr Phe Wing Vai Phe Asp Asp Arg Lys Gln Leu Wing Leu 290 295 300 Val Asn Met His Ser Pro His Asn Phe Thr Cys Thr His Tyr Met Met 305 310 315 320 Pro Cys Gln Val Gln Arg Asn Gly Phe Cys Phe Asn Arg Thr Wing Asp 325 330 335 Gly Ser Cys Val Leu Wing Asp Met Ser Asn Arg Leu Thr He Phe His 340 345 350 Leu Arg Cys Ser Arg Glu Glu Gln Gln Pro Gly Gln Lvs Thr Ser 355 360 365 Val Val Wing Thr Wing Lys Pro Gly Cys Val Being Ser Gly Thr Asp Wing 370 375 380 Wing Being Ser His Thr Asn Thr Thr Ser Wing Wing Wing Wing Pro 385 390 395 400 Wing Pro Pro Pro Val Wing Wing Pro Wing Lys Wing Wing Wing Pro Pro Wing 405 410 415 Wing Wing Arg Wing Wing Glu Pro His Val Gly Ser Lys lie Wing Asn 420 '425 430 Leu Val Asn GIn Leu Gly He Asn Goes! Thr Gln Arg Ser Val Val Ser 435 440 445 Thr Gly Ala Pro Aia Thr Thr Arg Ser Thr Wing Val Thr Ser Thr Thr 450 455 460 Thr Wing Pro Gln Arg Thr Pro Pro Tyr Gly His Asn Gly Arg Pro Val 465 470 475 480 Thr Ala Gly Leu Val Ala Ala Asn Ser Gly Ala Be Ala Ala Be Ser 485 490 495 Pro Thr Wing Wing Wing Lys Pro Thr Giy Glu Giu Lys Wing Wing Ala Wing 500 505 510 Cvs Glu Thr Ser Ser Val Wing He Asn Wing Thr Arg Pro Ala Leu His 5 15 520 525 Asn Ala Be Leu Pro Gln Ala Pro Thr Asp Giy Val Leu Ala Ala Ala 530 535 540 Val Tyr Gln Ser Glu Gly Glu Val His Gln Ser Leu Glu Arg Leu Glu 545 550 555 560 Ser Vai He Thr Asn Thr Ser Arg Val Leu Lys Leu Leu Pro Asp Thr 565 570 575 He Arg Arg Asp His Glu Gln Leu Leu Asn Leu Giy Leu Glu Aia Gln 580 585 590 Met Thr Glu Leu Gln Gln Ser Arg Pro Thr Pro Gln Thr Gln Pro Arg 595 600 605 Asp Thr Ser Be Ala Lys Ser Ser Val Phe Glu Thr Tyr Thr Leu Val 610 615 620 Leu He Wing Asp Ser Leu Ser Arg Asn He Thr Lys Gly Val Lys Arg 625 630. 635 640 Gly Val Asn Glu Ala He Met Leu His Leu Asp His Glu Val Arg His 645 650 655 Wing He Gly Asn Arg Leu Arg Gln Thr Gln Lys Asn He He Lys Ser 660 665 670 Arg Leu Asp Glu Ala Leu Lys Glu Ser Thr Thr Gln Phe Thr Aia Gln 675 680 685 Leu Thr Gln Thr Val Glu Asn Leu Val Lys Arg Glu Leu Wing Glu Vai 690 695 700 Leu Gly Ser He Asn Gly Ser Leu Thr Ser Leu Val Lys Glu Asn Ala 705 710 715 720 Ser Leu Lys Lys Glu Leu Asn Being He Met Being Ser Gly Val Leu Asp 725 730 735 Glu Met Arg Arg Met Arg Glu Glu Leu Cys Thr Leu Arg Glu Ser Val 740 745 750 Wing Lys Arg Lys Wing Thr Met Pro Asp Being Ser Leu His Wing Thr Ser 755 760 765 Ser Phe Gin Gly Arg Arg Ser Aia Pro Glu Thr He Leu Wing Thr Aia 770 775 780 Leu Ser Met Val Arg Glu Gln Gln Tyr Arg Gln Gly Leu Glu Val Met 785 790 795 800 Leu Met Wing Gln Gln Pro Be Leu Leu Leu Arg Phe Leu Ser lie Leu 805 810 815 Thr Arg Glu Asn Glu Asn Wing Tyr Ser Glu Leu lie Glu Asn Val Glu 820 825 830 Thr Pro Asn Asp Val Trp Cys Ser Val Leu Leu Gln Leu He Glu A 835 840 845 Wing wing Thr Glu Wing Glu Lys Glu Val Val Val Gly Val Wing Asp 850 855 860 He Leu Ser Glu Arg Asp GIn He Wing GIn Asn Gly Aia Leu Gly Ser 865 870 875 880 Lys Leu Thr Thr Wing Met Arg Wing Phe Glu Arg Gln Wing Arg Ser Glu 885 890 895 Thr Thr Ser Arg Ser Phe Leu Gln Cys Leu Lys Asn Leu He Lys Leu 900 905 910 Leu GIn Ser 915 (2) INFORMATION FOR SEQ ID NO: 3: (i) ' CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs 5 (B) TYPE: nucleic acids (C) CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (i) SEQUENCE DESCRITION: SEQ ID NO: 3 10 AACGCTATTA TTAGAACAGT T 21 (2) INFORMATION FOR SEQ ID NO: 4 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid 15 (C) CADEN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF 'THE SEQUENCE: SEQ ID NO: 4 TGCAGCAGCG GCAGAAGT 18 (2) INFORMATION FOR SEQ ID NO: 5; i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid 25 (C) CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5 CAGCCGACGG TAGCTGCGTC CT? "> (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 460 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear } (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: ACATAATGGC CTCGTTCACA C 21 (2: PAPA INFORMATION S? C ID NO: 7: : Í) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs 0 (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear : Ü) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7 CGAAGAGACC ATGAACAACT T 21 Declaration including process It is noted in relation to this date, that the best method known to the applicant to carry out the said invention / is that which is clear from the present description of the invention.

Having described the invention as above, the contents of the following are declared as property:

Claims (6)

1. The DNA or RNA sequence characterized in that it consists of the sequence of nucleotides 1472 to 1543 of SEQ. ID NO. 1 or an equivalent of this.
2. The expression cartridge for eukaryotic expression > ao prokaryotic, characterized in that it comprises a putative promoter comprising the sequence between position 350 and 600 of the sequence described in figure 7 for prokaryotic and eukaryotic expression of the sequence of claim 1, together with the DNA sequence according to the claim 1.
3. The vector characterized in that it comprises an expression cartridge according to claim 2.
4. The cell derived from a prokaryotic or eukaryotic organism, characterized in that it comprises an expression cartridge according to claim 2, both in a form integrated in the cellular genome, and inserted in a vector according to claim 3.
5. An epitope determinant of the PTc40 protein characterized in that it comprises a fragment of 24 amino acids, starting at amino acid 403 and terminating at amino acid 426 of SEQ ID NO. NO 2, or an immunological equivalent of this.
6. 6. The process for the preparation of an antigenic determinant according to claim 5, characterized in that: a cell derived from a prokaryotic or eukaryotic organism, comprising the expression cartridge according to claim 3, is cultured under appropriate conditions; Y the expressed antigenic determinant derived from the aforementioned organisms is recovered. Monoclonal or polyclonal antibodies obtained by immunological reaction of a human organism or. animal with a characterized immunogenic agent, because it consists of the antigenic determinant according to claim 5. The reagents for the detection and / or monitoring of Trypanosoma cruzi infection, characterized in that it comprises, as a reactive substance, an antigenic determinant according to claim 5. The process for the detection and / or monitoring of Trypanosoma cruzi infection in a biological sample, such as blood serum or plasma, urine, saliva, or tear samples from an individual or animal likely to have been infected with Trypanosoma cruzi, characterized in that said sample and a reagent according to claim 9 are brought into contact, under conditions that allow a possible immunological reaction, and the presence of an immune complex with said reagent is then detected. The pharmaceutical composition characterized in that it comprises a therapeutically effective amount of an expression cartridge according to claim 2, a vector according to claim 3, or a cell according to claim 4, or an antigenic determinant according to claim 5, or a antibody according to claim 7, optionally conjugated to a pharmaceutically acceptable carrier, and an excipient and / or an appropriate adjuvant. The use of a pharmaceutical composition according to claim 10 characterized in that it serves for the manufacture of a medicament, for the treatment or for the prevention of a Trypanosoma cruzi infection in man or in an animal. RESUb &N The nucleotide sequence of Tc40, a gene encoding a new antigenic protein of Trypanosoma cruzi called PTc40, is revealed. The amino acid sequence of the PTc40 protein is also revealed, together with the amino acid sequence of the dominan antigenic epitope of the PTc40 protein. The PTc40 protein and the Tc40 gene, or a fragment thereof, modified or different, can be used directly or indirectly for the detection of Trypanosoma cruzi, or for the monitoring of an infection generated by T. cruzi in humans or animals.