MX2008005541A - Compositions and methods for the detection of trypanosoma cruzi infection - Google Patents

Abstract

Compositions comprising fusion polypeptides ofT. cruziepitopes are provided, together with methods for the use of such compositions in the diagnosis ofT. cruziinfection and in screening blood supplies. Diagnostic kits comprising such compositions are also provided.

Description

COMPOSITIONS AND METHODS OF POPULATION THE DETECTION OF TRYPANOSOMA CRUZI INFECTION DESCRIPTION FIELD OF THE INVENTION The present invention is generally related to the diagnosis of the infection of Trypanosoma cruzi (T. cruzi). More specifically, the invention relates to the use of antigenic polypeptides and T. cruzi fusion polypeptides in methods for selecting individuals and blood supplies for T. cruzi infection.

BACKGROUND OF THE INVENTION Protozoan parasites are a serious threat to health in many areas of the world. Trypanosoma cruzi (T. cruzi) is a parasite that infects millions of individuals. Ten to thirty percent of individuals infected with T. cruzi develop symptomatic and chronic Chagas disease, which can in turn lead to heart disease and a variety of immune system disorders. T. cruzi infection has been a major public health problem in Central and South America. It is estimated that 18 Millions of people around the world are chronically infected with G. cruzi, but the available drug treatments lack effectiveness and often cause serious side effects. The most significant route of transmission in areas where the disease is endemic is through contact with an infected triatomid insect. However, in other areas blood transfusions are the dominant means of transmission. Therefore, to inhibit the transmission of T. cruzi, it is necessary to develop exact methods to diagnose G. cruzi infection in individuals and to select blood sources. Blood bank selection is particularly important in South America, where 0.1% -62% of blood samples may be infected and where the parasite is often transmitted by blood transfusion. Due to the high flow of immigrants to the US. from many countries of Central and South America where G. cruzi infection is endemic, the source of blood in the United States. It is becoming a high risk of contamination from blood donors infected with T. cruzi. Although there is some evidence available to diagnose the infection in individuals, there is currently no FDA approved test available in the US. for screening or selection of the blood donor for G. cruzi infection.

The diagnosis of G. cruzi infection has been problematic, since exact methods to detect the parasite that are convenient for routine use have been unaffordable. During the acute phase of the infection, which may last for decades, the infection may remain quiet and the host may be asymptomatic. As a result, serological tests for G. cruzi infection are the most reliable and the most commonly used forms of diagnosis. Such diagnoses are complicated; however, due to the complex life cycle of the parasite and the various immune responses of the host. The parasite goes through an epimastigote stage in the insect vector and two main stages in the mammalian host. A stage of the host occurs in the blood (the trypomastigote stage), while a second stage is intracellular (the amastigote stage). The different stages give rise to a diversity of antigens that are presented by the parasite during the infection. In addition, immune responses to protozoan infection are complex, involving humoral responses and transmitted by cells to the arsenal of parasite antigens. Although detection of antibodies against parasite antigens is the most common and reliable method to diagnose clinical and subclinical infections, current tests for T. cruzi infection are usually insensitive, lack specificity, and are not convenient for filtering or selecting blood sources. Most serological tests use T. cruzi complete or lysate and require positive results in two of three tests, including complement fixation, indirect immunofluorescence, passive agglutination or ELISA, to detect T. cruzi infection with veracity. The cost and difficulty of such tests have impeded the selection of blood or sera in many endemic areas. US Patents 5,876,734 and 6,228,601 disclose compositions useful for diagnosing Chagas disease comprising a non-repetitive region of the TCR27 protein of T. cruzi and fusion polypeptides including such regions. Patent US 6,419,933 describes a fusion polypeptide designated TcF containing the four antigenic peptides G. cruzi PEP-2, TcD, TcE and TcLol.2, together with methods for the use of the fusion polypeptide for the detection of infection T. cruzi. Although TcF is highly reactive with sera infected with T. cruzi from South America, it shows low activity and is sometimes negative with sera from Central America. Patent US 6,458,922 describes a test for T. cruzi infection using compositions comprising at least six T. cruzi antigenic peptides, selected from the group consisting of: SAPA, CRA, FRA, TcD, Tc24, Ag39 and MAP. Patent application number US2004 / 0132077-A1 discloses recombinant polypeptides and fusion polypeptides (designated FP3, FP4, FP5, FP6, FP7, FP8, FP9 and FP10) useful for diagnosing G. cruzi infection. The disclosed fusion polypeptides comprise modified versions of previously identified T. cruzi epitopes, including TCR27, TCR39, SAPA and MAP.

SUMMARY OF THE INVENTION The present invention provides compositions and methods for detecting G. cruzi infection in individuals and in biological samples, including blood sources. The inventive compositions can be used to detect G. cruzi infection in all geographic areas where Chagas disease is present and with greater sensitivity compared to the analyzes currently in use. In one embodiment, the inventive compositions comprise the fusion polypeptide of T. cruzi TcF (SEQ ID No. 1), or a variant thereof, and at least one peptide selected from the group consisting of: SAPA (SEQ ID NO. : 2); PEP30 (SEQ ID NO: 3); PEP36 (SEQ ID NO: 4); KMP-11 (SEQ ID NO: 5); Peptide 1 (SEQ ID NO: 6; also designated FRA; Lafaille et al., Mol. Biochem. Parasitol. 35: 127-136, 1989); a modified version of peptide 1 (SEQ ID NO: 7); and its variants. The TcF fusion polypeptide and the at least one peptide can be present as individual components within the composition or can be ligated to form a fusion polypeptide. In certain embodiments, the inventive compositions comprise the TcF fusion polypeptide in conjunction with the Pep30, Pep36 and SAPA peptides. Such compositions may, for example, include the fusion polypeptide of SEQ. ID NO: 8 (designated here as ITC-6). In alternate embodiments, the inventive compositions include a fusion polypeptide comprising ITC-6 in conjunction with at least one repeat of the KMP-11 peptide, and / or the sequences of SEQ peptide 1. ID NO: 6 or 7. The amino acid sequence of a representative fusion polypeptide comprising ITC-6 plus a repeat of KMP-11 (designated ITC7.1) is provided in SEQ. ID NO: 15, with the amino acid sequence of a representative fusion polypeptide comprising ITC-6 plus two repeats of KMP-11 (designated as ITC7.2) which is provided in SEQ. ID NO: 17. SEQ. ID NO: 19 is the amino acid sequence of a representative fusion polypeptide comprising ITC7.2 plus peptide 1 (designated ITC8.2), with the corresponding DNA sequence provided in SEQ. ID NO: 18. The amino acid sequence of a shortened version of ITC8.2, designated ITC8.1, is provided in SEQ. ID NO: 20. SEQ. ID NO: 21 and 22 are the DNA and amino acid sequences, respectively, for a shortened version of ITC8.1. Polynucleotides encoding the inventive fusion polypeptides and host cells transformed or transfected with such expression vectors are also provided by means of the current invention. As described in detail below, the inventors have determined that the inventive compositions can be used to effectively detect G. cruzi infection in a biological sample. Accordingly, in one aspect, the present invention provides methods for detecting T. cruzi infection in a biological sample, comprising: (a) contacting the biological sample with a composition of the present invention; and (b) detecting in the biological sample the presence of antibodies that bind to an epitope present within the inventive composition, thereby detecting T. cruzi infection in the biological sample. In a further aspect, diagnostic kits are provided for detecting G. cruzi infection in a biological sample, such kits comprising: (a) a composition of the present invention; and (b) a detection reagent. The inventive compositions may also comprise at least one component selected from the group consisting of: carriers and immunostimulants physiologically acceptable. Also included in the present invention are methods for inducing protective immunity against Chagas disease in a patient by administering such compositions. The above and additional features of the present invention and the manner of obtaining them will become apparent, and the invention will be better understood by reference to the following more detailed description. All references herein disclosed are incorporated in their totality as a reference as if each were incorporated individually.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 shows the reactivity of the TcF and ITC-6 fusion polypeptides with sera infected with T. cruzi and control sera from uninfected individuals, as determined by ELISA. Figs. 2A-C show the reactivity of TcF and ITC-6 with several sera as determined by ELISA. The RIPA status of the individual sera is shown in Table 1. FIG. 3 shows the reactivity of the fusion polypeptides TcF, ITC-6, ITC7.1 and ITC 7.2 in a panel of sera as determined by ELISA.

Fig. 4 shows the reactivity of the ITC7.2 fusion polypeptides and the shortened version of ITC8.2 provided in SEQ. ID NO: 22 in a panel of sera as determined by ELISA. Fig. 5 shows the activity of non-absorbed and absorbed sera of 1-positive peptide with ITC7.2, ITC8.1 and shortened ITC8.2 (SEQ ID NO: 22). Figs. 6A and 6B show the isolated DNA sequence and the corresponding amino acid sequence, respectively, for TC5. The insert is shown in bold type and the complementary sequence in non-bold font. Figs. 7A and 7B show the isolated DNA sequence and the corresponding amino acid sequence, respectively, for TC48. The insert is shown in bold type and the complementary sequence in bold type. Figures 8A and 8B show the isolated sequence of DNA and the corresponding amino acid sequence, respectively, for TC60. The insert is shown in bold type and the complementary sequence in bold type. Figs. 9A and 9B show the isolated sequence of DNA and the corresponding amino acid sequence, respectively, for TC70. The insert is shown in bold type and the complementary sequence in bold type.

DETAILED DESCRIPTION OF THE INVENTION As noted above, the present invention generally relates to compositions and methods for detecting T. cruzi infection in individuals and for selecting blood sources for T. cruzi infection. The compositions of this invention generally comprise the known T. cruzi TcF fusion polypeptide (patent 6, 419, 933; SEQ ID NO: 1), or one of its variants and at least one antigenic epitope or polypeptide of T. cruzi selected from the group consisting of: SAPA (SEQ ID NO: 2); Pep30 (SEQ ID NO: 3); Pep36 (SEQ ID NO: 4); KMP-11 (SEQ ID NO: 5); peptide 1 (SEQ ID NO: 6); modified peptide 1 (SEQ ID NO: 7); and its variants. The use of one or more additional epitopes of G. cruzi polypeptides, either before or in conjunction with one or more of the specific G. cruzi polypeptides disclosed herein, to increase the sensitivity and specificity of the detection are also included herein and they are comprised within the current invention. The TcF fusion polypeptide and the at least one peptide can be present as individual components within the inventive composition or can be ligated to form a fusion polypeptide. Fusion polypeptides comprising more than one repeat of the polypeptides or the antigenic epitopes of G. cruzi are also included and are included in the present invention, as well as fusion polypeptides to which the peptides are linked in an order which differs from those shown in the specific fusion polypeptide sequences provided herein. In one embodiment, the inventive compositions include a fusion polypeptide comprising TcF, Pep30, Pep36 and SAPA, here referred to as ITC-6 (SEQ ID NO: 8). In alternate embodiments, the inventive compositions include a fusion protein selected from the group consisting of SEQ. ID NO: 15 (designated ITC7.1), SEQ. ID NO: 17 (designated ITC7.2), SEQ. ID NO: 19 (designated ITC8.2), and SEQ. ID NO: 20 (designated ITC8.1). The DNA sequences for ITC-6, ITC7.1, ITC7.2 and ITC8.2 are provided in SEQ. ID NO: 11, 14, 16 and 18, respectively. As described in US Pat. No. 6,419,933, the disclosure of which is hereby incorporated by reference, the TcF fusion polypeptide includes four antigenic epitopes, or peptides, designated PEP2, TcD, TcE and TcLol.2. In an alternative embodiment, the inventive compositions may include these four individual peptides in place of the fusion TcF protein. In accordance with what is used herein, the term "polypeptide" comprises chains of amino acids of any length, including integral or full-length proteins, wherein the amino acid residues are bound by covalent peptide bonds. The polypeptides described herein can be naturally purified products or can be produced partially or totally with recombinant techniques. Such polypeptides may be glycosylated with mammalian or other eukaryotic carbohydrates or may not be glycosylated. A polypeptide comprising an epitope may consist entirely of the epitope or may contain additional sequences. Additional sequences can be derived from the native antigen or can be heterologous and such sequences can be (but not necessarily) antigenic. As used herein, a "fusion polypeptide" is a polypeptide in which the epitopes of various antigens, or their variants, are linked, for example through a peptide coupling, to a single chain of amino acids. The chain of amino acids thus formed may be linear or branched. The epitopes can be linked directly (i.e., without amino acids intervening) or can be assembled by a linking sequence that does not significantly alter the antigenic characteristics of the epitopes. Peptide epitopes can also be linked via non-peptide couplings, such as hetero- or homo-bifunctional agents that chemically or photochemically couple between specific functional groups on peptide epitopes such as amino, carboxyl, or direct sulfhydryl. The bifunctional agents that can be usefully employed in the combination polypeptides of the present invention are known to those of ordinary skill in the art. The epitopes can also be linked via a complementary ligand / antiligand pair, such as avidin / biotin, with one or more epitopes that bind to a first member of the ligand / antiligand pair and then bind to the complementary member of the ligand / pair. antiligand either in solution or in solid phase. A fusion polypeptide may contain epitopes of one or more different antigens of T. cruzi, bound or linked to an epitope described herein. A polynucleotide encoding a fusion protein of the present invention is constructed using known recombinant DNA techniques to assemble separate polynucleotides that encode the first and second polypeptides into an appropriate expression vector. The 3 'end of a polynucleotide encoding a first polypeptide is linked, with or without a binding peptide, to the 5' end of a polynucleotide encoding a second polypeptide so that the reading frames of the sequences are in phase to allow the translation of the mRNA of the two polynucleotides into a single fusion protein that retains the biological activity of both the first and the second polypeptides.

As indicated above, a peptide linking sequence can be used to separate the first and second polypeptides by a sufficient distance to ensure that each polypeptide double in its secondary and tertiary structures. Such a peptide linking sequence is incorporated into the fusion protein using standard techniques known in the art. Suitable peptide linking sequences can be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) its inability to adopt a secondary structure that could interact with functional epitopes in the first and second polypeptides; and (3) the lack of hydrophobic or charged resi that could react with the functional polypeptide epitopes. The preferred linker sequences of the peptide contain resi of Gly, Asn and Ser. Other nearby neutral amino acids, such as Thr and Ala can also be used in the linking sequence. The amino acid sequences that can be usefully employed as bonds include those disclosed in Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. National. Acad. Sci. USA 83: 8258-8262, 1986; Patents No. US 4,935,233 and 4,751,180. The linker sequence can be from 1 to about 50 amino acids in length. Peptide binding sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and to prevent steric interference. The ligated polynucleotides encoding the fusion proteins are cloned into convenient expression systems using techniques known to those of ordinary skill in the art. The present invention also provides polynucleotides that encode a fusion polypeptide or polypeptide of the present invention. Also provided are polynucleotides comprising complements of such polynucleotide sequences, inverse complements of such polynucleotide sequences or reverse sequences of such polynucleotide sequences, together with variants of such sequences. The definition of the terms "complement (s)," "inverse complement (s)," and "inverse sequence (s)," as used herein, are best illustrated by the following example. For the 5 'AGGACC 3' sequence, the complement, inverse complement, and reverse sequence are as follows: complement 31 TCCTGG 5 'reverse complement 3' GGTCCT 5 'reverse sequence 5' CCAGGA 3 '.

Preferably, the sequences that are complements of a specifically described polynucleotide sequence are complementary for the entire length of the specific sequence of the polynucleotide. The term "polynucleotide (s)," as used herein, means a single or double-stranded polymer of deoxyribonucleotide bases or ribonucleotides and includes DNA and corresponding RNA molecules, including hRNA and mRNA molecules, both in sense and in chains. in contrast, and comprising cDNA, genomic DNA and recombinant DNA, as well as polynucleotides synthesized totally or partially. An RNA molecule contains introns and corresponds to a DNA molecule in a generally one-to-one manner. An mRNA molecule corresponds to an RNA molecule and DNA from which the introns have been deleted. A polynucleotide can consist of a whole gene or any portion thereof. The operable antisense polynucleotides may comprise a fragment of the corresponding polynucleotide and the definition of "polynucleotide" therefore includes all of these operable antisense fragments. All polypeptides, fusion polypeptides and polynucleotides described herein are isolated and purified, as these terms are commonly used in the medium. Preferably, the polypeptides, the polypeptides of fusion and the polynucleotides are at least about 80% pure, preferably at least about 90% pure and preferably at least about 99% pure. The compositions and methods of the present invention also comprise variants of the polypeptides, the fusion polypeptides and the above-mentioned polynucleotides. In accordance with what is mentioned herein, the term "variant" comprises nucleotide or amino acid sequences different from the specifically identified sequences, wherein one or more nucleotide or amino acid residues are deleted, substituted or added. The variants can be allelic variants that occur naturally or variants that do not occur naturally. The variable sequences (polynucleotide or polypeptide) preferably exhibit at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, and even more preferably, at least minus 98% identity for a sequence of the current invention. The percent identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the sequence (requested) inventive and multiplying the result by 100. In addition to displaying the described level of sequence identity, the variable sequences of the present invention preferably exhibit a functionality that is substantially similar to the functionality of the specific sequences disclosed herein. The variable sequences of fusion polypeptides thus preferably preserve the antigenic and diagnostic characteristics of the fusion polypeptides disclosed herein. Preferably, a variable sequence of polypeptide or fusion polypeptide will generate at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably 100% of the response generated by the polypeptide or fusion polypeptide sequence specifically identified in an antibody binding assay, such as an ELISA assay. Such variants can generally be identified by modifying one of the polypeptide or fusion polypeptide sequences described herein and evaluating the antigenic and / or diagnostic characteristics of the modified polypeptide or fusion polypeptide using, for example, the representative procedures described herein. Suitable assays to evaluate reactivity with sera infected with T. cruzi, such as an enzyme-linked immunosorbent assay (ELISA), are described in more detail below, and in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Variable sequences generally differ from the sequence specifically identified only by substitutions, deletions or conservative modifications. As used herein, a "conservative or conservative substitution" is one in which an amino acid is replaced by another amino acid having similar characteristics, such that a person with average knowledge in the field of peptide chemistry would expect the secondary structure and the hydropathic nature of the polypeptide will remain substantially unchanged. In general, the following amino acid groups represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. The variants may also, or alternatively, contain other modifications, including the cancellation or addition of amino acids that have minimal influence on the antigenic characteristics, secondary structure and hydropathic nature of the polypeptide. For example, a polypeptide can be conjugated to a signal sequence (or leader) at the N-terminus of the protein that co-translationally or post-translationally directs the transfer of the protein. The polypeptide can also be conjugating the binder or the other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to improve the binding of the polypeptide to a solid support. For example, a polypeptide can be conjugated to an immunoglobulin Fe region. The polypeptide and polynucleotide sequences can be aligned and percentages of identical nucleotides can be determined in a specified region against another polynucleotide, using computer algorithms that are available to the public. Two exemplary algorithms for aligning and identifying the identity of the polynucleotide sequences are the algorithms of BLASTN and FASTA. The alignment and identity of the polypeptide sequences can be examined using the BLASTP algorithm. The BLASTX and FASTX algorithms compare the translated nucleotide query sequences in all reading frames against polypeptide sequences. The algorithms of FASTA and FASTX are described in Pearson and Lipman, Proc. Nati Acad. Sci. USA 85: 2444-2448, 1988; and in Pearson, Methods in Enzymol, 183: 63-98, 1990. The FASTA software package is available from the University of Virginia, Charlottesville, VA 22906-9025. The FASTA algorithm, established for the defect parameters described in the documentation and distributed with the algorithm, can be used in the determination of the polynucleotide variants. The files read me for FASTA and FASTX the version 2.0x that is distributed with the algorithms describe the use of the algorithms and describe the defect parameters. The BLASTN software is available on the anonymous FTP server of NCBI and is available from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894. The BLASTN algorithm version 2.0.6 [Sep -10-1998] and version 2.0.11 [January-20-2000] established for the defect parameters described in the documentation and distributed with the algorithm, are preferred for use in the determination of variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN, is described on the NCBI website and in the publication of Altschul, et al., "Gapped BLAST and PSI-BLAST: a new generation of protein datbase search programs," Nucleic Acids Res. 25: 3389-3402, 1997. "Successes" for one or more database sequences by a query sequence produced by BLASTN, BLASTP, FASTA or a similar algorithm, align and identify similar portions of sequences. The successes are arranged in order of the degree of similarity and the length of the overlap of the sequence. The successes for a database sequence generally represent an overlap over only a fraction of the length of the sequence of the sequence asked.

The percent identity of a polynucleotide or polypeptide sequence is determined by aligning polynucleotide and polypeptide sequences using appropriate algorithms, such as BLASTN or BLASTP, respectively, set for defect parameters; identifying the number of identical amino acids or nucleic acids on the aligned portions; dividing the number of identical amino acids or nucleic acids by the total number of amino acids or nucleic acids of the polynucleotide or polypeptide of the present invention; and then multiplying by 100 to determine the percentage of identity. In general, T. cruzi polypeptides and fusion polypeptides and polynucleotide sequences encoding such polypeptides and fusion polypeptides, can be prepared using any of a variety of methods. For example, a G. cruzi DNA or a genomic DNA expression library can be selected with wells from sera from individuals infected with T. cruzi. Such filtrations can generally be performed using techniques known to those of ordinary skill in the art, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989. Briefly , the bacteriophage library can be plated and transferred to filters. The filters can be Then incubate with serum and a detection reagent. In the context of this invention, a "detection reagent" is any compound capable of binding to the antibody-antigen complex, which can then be detected by any of a variety of means known to those of ordinary skill in the art. Typical detection reagents for screening purposes contain a "binding agent," for example protein A, protein G, IgG or a lectin, coupled to an indicator group. Preferred indicator groups include, but are not limited to, enzymes, substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. More preferably, the indicator group is horseradish peroxidase (HRP), which can be detected by incubation with a substrate such as tetramethylbenzidine (TMB) or 2, 2'-azino-di-3-ethylbenzthiazoline sulphonic acid. Plates containing ssDNAs that express a protein that binds to an antibody in the serum can be isolated and purified by techniques known to those of ordinary skill in the art. Appropriate methods can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989. Alternatively, the polynucleotides encoding the fusion polypeptides described herein can be amplify from genomic DNA or cDNA of G. cruzi via the polymerase chain reaction (PCR). For this approach, specific sequence primers can be designed based on the polynucleotide sequence and can be purchased or synthesized. An amplified portion of the DNA sequences can then be used to isolate the entire genomic full length or cDNA clones using well known techniques, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories. , Cold Spring Harbor, NY (1989). Polypeptides having less than about 100 amino acids and generally less than about 50 amino acids, can be synthesized using, for example, the Merrifield solid-phase synthesis method, where the amino acids are sequentially added to an increasing chain of amino acids. See Merrifield, J. Am. Chem. Soc. 85: 2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from such providers COITK > Perkin Elmer / Applied Biosystems Division, Foster City, CA. Polypeptides and fusion polypeptides can also be produced recombinantly by inserting a polynucleotide encoding the fusion polypeptide into an expression vector and expressing the antigen in an appropriate host. You can use any of a variety of Expression vectors known to people with average knowledge in the field. Expression can be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule encoding a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, mycobacteria, insect, yeast or a mammalian cell line such as COS or CHO. The polynucleotides expressed in this way can encode naturally occurring antigens, portions of naturally occurring antigens or other variants of them. The expressed polypeptides and fusion polypeptides are generally isolated in substantially pure form. Preferably, the polypeptides and fusion polypeptides are isolated at a purity of at least 80% by weight, preferably, at a purity of at least 95% by weight and more preferably at a purity of at least 99% by weight . In general, such purification can be achieved using, for example, standard ammonium sulfate fractionation techniques, SDS-PAGE electrophoresis, and affinity chromatography. The present invention also provides methods for detecting T. cruzi infection in individuals and sources of blood. G. cruzi infection can be detected in any biological sample that contains antibodies. Preferably, the sample is blood, serum, plasma, saliva, cerebrospinal fluid or urine. Preferably, the sample is a blood or serum sample obtained from a patient or from a blood source. Briefly, G. cruzi infection can be detected using any one or more of the fusion polypeptides or polypeptides described above, or variants thereof, to determine the presence or absence of antibodies to the polypeptide or fusion polypeptide in the sample, in relation to a predetermined cutoff value. There are a variety of assay formats known to those of ordinary skill in the art to use the purified antigen to detect antibodies in a sample. See, eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In a preferred embodiment, the assay involves the use of immobilized fusion polypeptide or polypeptide on a support to bind and remove the antibody from the sample. The binding antibody can then be detected using a detection reagent that binds to the antibody / fusion polypeptide complex and contains a detectable reporter group. Suitable detection reagents include antibodies that bind to the antibody / fusion polypeptide complex and the polypeptide free labeling with an indicator group (e.g., in a semi-competitive analysis). Alternatively, a competitive assay can be used, wherein an antibody that binds to the fusion polypeptide is labeled with a reporter group and allowed to bind to the immobilized antigen after incubation of the antigen with the sample. The degree to which the components of the sample inhibit the binding of the labeled antibody to the fusion polypeptide is indicative of the reactivity of the sample with the immobilized fusion polypeptide. The solid support may be any solid material known to those of ordinary skill in the art to which the fusion polypeptide binds. For example, the solid support can be a test well in a microtiter plate or a nitrocellulose or other convenient membrane. Alternatively, the support can be a grain or disc, formed of glass, fiberglass, latex or plastic material such as polystyrene or polyvinyl chloride. The support can also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in US Pat. No. 5,359,681. The fusion polypeptide can be bound to the solid support using a variety of techniques known in the art, which are widely described in the patent and scientific literature. In the context of the current invention, the term "linkage" refers both to the non-covalent association, such as adsorption, as well as to the covalent coupling (which may be a direct link between the antigen and functional groups on the support or may be a coupling by means of an agent of crosslinking). Adsorption to a sample in a microtiter plate or to a membrane is preferred. In such cases, adsorption can be achieved by contacting the polypeptide, in a convenient buffer solution, with the solid support for a convenient amount of time. Contact time varies with temperature, but is typically between approximately 1 hour and 1 day. In general, contacting a well of a microtiter plastic plate (such as polystyrene or polyvinyl chloride) with an amount of fusion polypeptide ranging from about 10 ng to about g, and preferably about 100 ng, is sufficient. to bind an adequate amount of antigen. The nitrocellulose will bind approximately 100 g of protein per cm. The covalent linkage of the fusion polypeptide or polypeptide to a solid support can generally be achieved first by reacting the support with a bifunctional reagent that will react both with the support and with a functional group, such as a hydroxyl or an amino group, in the polypeptide of fusion. For example, him Fusion polypeptide can be linked to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen in the fusion polypeptide (see, eg, Pierce Immunotechnology Catalog and Handbook ( 1991) at A12-A13). In certain embodiments, the assay is an enzyme-linked immunosorbent assay (ELISA). This analysis may be performed first by contacting a polypeptide or fusion polypeptide that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that antibodies to the polypeptide or polypeptide are allowed. of fusion within the sample bind to the immobilized fusion polypeptide or polypeptide. The unbound sample is then removed from the immobilized fusion polypeptide or polypeptide and a detection reagent capable of binding to the immobilized antibody-polypeptide complex is added. The amount of detection reagent remaining bound to the solid support is then determined using a method appropriate for the specific detection reagent. Once the polypeptide or fusion polypeptide is immobilized on the support, the remaining protein binding sites on the support are typically blocked using any convenient blocking agent known for an ' person with average skill in the art, such as bovine serum albumin or Tween 20MR (Sigma Chemical Co., St. Louis, O). The immobilized fusion polypeptide or polypeptide is then incubated with the sample and the antibody (if present in the sample) is allowed to bind to the antigen. The sample can be diluted with a convenient diluent, such as phosphate buffered saline (PBS) before incubation. In general, an appropriate contact time (i.e., incubation time) is that period sufficient to detect the presence of the T. cruzi antibody within a sample infected with T. cruzi. Preferably, the contact time is sufficient to reach a binding level that is at least 95% of that achieved in the balance between the bound and unbound antibody. People with average knowledge in the field will recognize that the time necessary to reach equilibrium can be easily determined by testing the level of linkage that occurs over a period. At room temperature, an incubation time of approximately 30 minutes is generally sufficient. The unbound sample can then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20MR. The detection reagent can then be added to the solid support. An appropriate detection reagent is any compound that binds to the antibody-polypeptide immobilized complex and that can be detected by any of a variety of means known to those of ordinary skill in the art. Preferably, the detection reagent contains a binding agent (such as, for example, protein A, protein G, immunoglobulin, lectin or free antigen) conjugated to a reporter group. Preferred indicator groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. The conjugation of the binding agent to the indicator group can be achieved using standard methods known to those of ordinary skill in the art. Common binding agents can also be purchased conjugates for a variety of indicator groups from many sources (e.g., Zymed Laboratories, San Francisco, CA and Pierce, Rockford, IL). The detection reagent is then incubated with the immobilized antibody-polypeptide complex for a sufficient amount of time to detect the bound antibody. An appropriate amount of time can usually be determined from the manufacturer's instructions or by analyzing the link level that occurs over a period. The unbound detection reagent is then removed and the detection reagent is detected without binding using the indicator group. The method used to detect the indicator group depends on the nature of the indicator group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. The spectroscopic methods can be used to detect dyes, luminescent groups and fluorescent groups. Biotin can be detected using avidin, coupled to a different indicator group (commonly a radioactive or fluorescent group or an enzyme). Enzyme indicator groups can generally be detected by the addition of substrate (generally for a specific period), followed by spectroscopic analysis or other analysis of the reaction products. To determine the presence or absence of T. cruzi antibodies in the sample, the detected signal of the reporter group that remains limited to the solid support is generally compared to a signal corresponding to a predetermined cutoff value. This cutoff value is preferably the average average signal obtained when the immobilized antigen is incubated with samples from an uninfected patient. In general, a sample that generates a signal that is three standard deviations above the mean is considered as positive for G. cruzi antibodies and for T. cruzi infection. In an alternate preferred embodiment, the cut value is determined using a curve of Receptor-Operator, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, p. 106-7 (Little Brown and Co., 1985). Briefly, in this modality, the cut-off value can be determined from a diagram of pairs of true positive values (ie, sensitivity) and false positive values (100% -specificity) that correspond to each possible cutoff value for the result of diagnostic test. The cutoff value in the diagram that is closest to the upper left corner (that is, the value that includes the largest area) is the most accurate cutoff value, and a sample that generates a signal that is higher than The cut-off value determined by this method can be considered positive. Alternatively, the cut-off value can be changed to the left along the diagram, to minimize the false positive value, or to the right, to minimize the false negative value. In general, a sample that generates a signal that is higher than the cut-off value determined with this method is considered positive for T. cruzi infection. In a related embodiment, the analysis is performed in a strip or through flow test format, wherein the polypeptide or fusion polypeptide is immobilized on a membrane such as nitrocellulose. In the through-flow test, the antibodies within the sample are bind to the immobilized fusion polypeptide or polypeptide while the sample passes through the membrane. A detection reagent (e.g., colloidal gold of protein A) then binds to the antibody-polypeptide complex while the solution containing the detection reagent traverses the membrane. The detection of the link detection reagent can then be performed as described above. In the strip test format, one end of the membrane to which the polypeptide or fusion polypeptide is bound is immersed in a solution containing the sample. The sample migrates along the membrane with a region containing the detection reagent and the area of the immobilized fusion polypeptide or polypeptide. The concentration of the detection reagent in the fusion polypeptide or polypeptide indicates the presence of G. cruzi antibodies in the sample. Such tests can typically be performed with a very small amount (e.g., one drop) of the patient's serum or blood. In yet another aspect of this invention, the methods are provided for detecting T. cruzi in a biological sample using monospecific antibodies (which may be polyclonal or monoclonal) for one or more T. cruzi fusion polypeptides or polypeptides. Antibodies to purified or synthesized polypeptides can be prepared by any of a variety of techniques known to the people with average knowledge in the matter. See, eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one such technique, an immunogen comprising the antigenic polypeptides is initially injected into any of a wide variety of mammals (eg, mice, rats, rabbits, sheep and goats). In this step, the polypeptides of this invention can serve as the immunogen without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response can be extracted if the polypeptide is linked to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably in accordance with a predetermined schedule incorporating one or more reinforcing immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide can then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a convenient solid support. Monoclonal antibodies specific for the antigenic polypeptide of interest can be prepared, for example, by the technique of Kohler and Milstein, Eur. J. Immunol. 6: 511-519, 1976, and its improvements. Briefly, these methods involve the preparation of cell lines immortals capable of producing antibodies with the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines can be produced, for example, from spleen cells obtained from an animal immunized as described above. Spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques can be used. For example, spleen cells and myeloma cells can be combined with a non-ionic detergent for a few minutes and then plated at low density in a selective medium that supports the growth of hybrid cells, but not of myeloma cells. A preferred screening technique uses HAT detection (hypoxanthine, aminopterin, thymidine). After a sufficient time, generally around 1 to 2 weeks, colonies of hybrids are observed. The single colonies are selected and tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred. The monoclonal antibodies can be isolated from the supernatants of the growing hybridoma colonies. In addition, various techniques can be employed to improve production, such as injection of the cell line of the Hybridoma in the peritoneal cavity of a convenient vertebrate host, such as a mouse. The monoclonal antibodies can then be collected from the ascites or blood fluid. The contaminants can be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Monospecific antibodies to epitopes of one or more of the fusion polypeptides or polypeptides described herein can be used to detect T. cruzi infection in a biological sample using any of a variety of immunoassays, which can be direct or competitive. Suitable biological samples for use in this aspect of the current invention are as described above. Briefly, in a direct analysis format, a monospecific antibody can be immobilized on a solid support (as described above) and come into contact with the sample to be tested. After removal of the unbound sample, a second monospecific antibody, which has been labeled with a reporter group, can be added and used to detect the bound antigen. In an exemplary competitive analysis, the sample can be combined with the monoclonal or polyclonal antibody, which has been labeled with a convenient reporter group. The mixture of the sample and the antibody can then be combined with the antigen of the immobilized polypeptide on a convenient solid support. The antibody that has not been bound to an antigen in the sample is allowed to bind to an immobilized antigen, and the remainder of the sample and the antibody are removed. The level of the antibody bound to the solid support is inversely proportional to the level of the antigen in the sample. Thus, a low level of the antibody bound to the solid support indicates the presence of G. cruzi in the sample. To determine the presence or absence of the G. cruzi infection, the detected signal from the indicator group remaining bound to the solid support is generally compared to a signal corresponding to a predetermined cut-off value. Such cut-off values can generally be determined as described above. Any of the indicator groups discussed above can be used in the context of ELISAs to label the monospecific antibodies, and the linkage can be detected by any of a variety of techniques appropriate for the indicator group employed. Other formats for using monospecific antibodies to detect T. cruzi in a sample will be apparent to those with average knowledge in the field, and the above formats are provided for exemplary purposes only. In another aspect of this invention, compositions are provided for the prevention or treatment of G. cruzi infection, and its complications, in a mammal. Such compositions generally comprise one or more fusion polypeptides disclosed herein, together with at least one component selected from the group consisting of: carriers and physiologically acceptable immunostimulants. The routes and frequency of administration and dose of the fusion polypeptide will vary from individual to individual and may be similar to those currently used in immunization against other protozoan infections. In general, the compositions can be administered by injection (eg, intracutaneous, intramuscular, intravenous or subcutaneous), intranasal (eg, by aspiration), transdermally, orally or by transcutaneous patch as described, for example, in patents 5,910,306 and 5,980,898 of the United States of America, whose descriptions are incorporated herein by reference. You can administer between 1 and 4 doses for a period of 2-6 weeks. Preferably, two doses are administered, with the second dose 2-4 weeks after the first. A convenient dose is an amount of fusion polypeptide that is effective to raise the antibodies in a treated mammal so that they are sufficient to protect the mammal against G. cruzi infection for a period. In general, the amount of fusion polypeptide present in a dose ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 μ ?, and preferably from about 100 pg to about 1 μg. Suitable dose sizes will vary with the size of the animal, but will typically range from about 0.01 ml to about 5 ml per 10-60 kg of the animal. Although any convenient carrier known to those of ordinary skill in the art may be used in the compositions of the present invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer solution. For oral administration, any of the above carriers or a solid carrier can be used, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose and magnesium carbonate. Biodegradable microspheres (e.g., polylactic galactide) can also be used as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in US Pat. Nos. 4, 897, 268 and 5, 075, 109. Any of a variety of immunostimulants can be employed in the compositions of this invention for not specifically improve the immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bortadella pertussis or mycobacterium tuberculosis. Suitable adjuvants are commercially available as, for example, incomplete Freund's adjuvant and Freund's complete adjuvant (Difco Laboratories) and Merck's adjuvant 65 (Merck and Company, Inc., Rahway, NJ). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and quil A. The following examples are offered by way of illustration and not limitation.

Example 1 Design and preparation of a multiepitope fusion polypeptide Serological expression studies using serum infected with G. cruzi from Mexico and Central America that show little or no reactivity with the TcF fusion protein (SEQ ID NO: 1) were performed to identify known T. cruzi antigens that will complement TcF in order to achieve high sensitivity and specificity.

TcF (SEQ ID NO: 1), SAPA (SEQ ID NO: 2), Pep30 (SEQ ID NO: 3) and Pep36 (SEQ ID NO: 4) were found to have very good specificity and complement each other in samples where the TcF reactivity is low or zero. After these studies, a fusion polypeptide containing the sequences TcF, Pep30, Pep36 and SAPA was prepared in two steps. In step one, SAPA, Pep30 and Pep36 were fused together with the EcoRI and XhoI restriction sites to provide the SEQ polypeptide sequence. ID NO: 9 (designated SAPA3036; DNA sequence for the expression of E. coli provided in SEQ ID NO: 10) and cloned into a related pUC vector using standard techniques. In step two, SAPA3036 was digested with EcoRI and XhoI, and subcloned into the 3 'end of TcF (SEQ ID NO: 1). The amino acid sequence of the resulting fusion polypeptide (designated ITC-6) is provided in SEQ. ID NO: 8, with the corresponding DNA sequence that is provided in SEQ. ID NO: 11. After sequence verification, expression in the E. coli chain of pLysS from Rosetta ™ (Novagen, Madison, Wisconsin) was performed. The ITC-6 fusion polypeptide has sufficient flexibility at the DNA level for insertion of other antigenic epitopes, such as the T. cruzi specific sequence KMP-llc (SEQ ID NO: 5, Thomas et al., Clin. Exp. I munol, 123: 465-471, 2001), the peptide sequence 1 (SEQ ID NO: 6) and the sequence of modified peptide 1 SEQ. ID NO: 7) Oligonucleotides (SEQ ID NO: 12 and 13) were designed to PCR the SAPA3036 fusion protein to be cloned directly into the pET28 vector to check expression and viability without the TcF component. The resulting PCR product was digested with Ndel and Xhol before cloning into pET28. There was no problem with the expression of SAPA3036 without TcF. It is anticipated that the order of the peptides in the ITC-6 recombinant fusion polypeptide could be altered without significantly changing the activity of the polypeptide. As well, the inclusion of a Gly-Cys-Gly coupling between the peptides can improve the solid phase binding without significantly affecting the activity of the polypeptide.

Example 2 Detection of T. cruzi infection in sera using ITC7.1 and ITC 7.2 The reactivity of sera from individuals infected with G. cruzi and control sera from uninfected individuals against the TcF and ITC-6 fusion polypeptides was determined by ELISA. procedure described above. The results of this study are shown in fig. 1. Sera infected with T. cruzi RR39, 40218, 63221, PMT201-13, RR66, 40219, 74043 and PMT201-9 all had been shown to be positive for T. cruzi by radioimmunoprecipitation (RIPA) analysis. Serum 63225 is consensus positive, with the status of RIPA being equivocal. The NHS86, NHS92 and PMT201-15 sera were all normal sera. Sera 197001, 312001, 38001 and 505001 were all from individuals infected with visceral leishmaniasis (VL). The sera were tested at a final dilution of 1/100. Goat anti-human IgG-HRP was used for detection followed by the TMB substrate. As can be seen from fig. 1, ITC-6 is able to recognize sera that are negative or low with TcF. In subsequent studies, the reactivities of 56 sera (American Red Cross, 42, BBI 14) from the United States of America and Central and South America were determined for positive by RIPA, and four endemic control sera from Mexico and from Central American countries were tested against the TcF and ITC-6 recombinants by ELISA. The RIPA status of the individual sera is shown in Table 1 below. The coating concentration was 100 ng per well. All sera were tested at 1/100 final dilution. After the incubation, the analyzes were developed using anti-human substrate IgG-HRP and TMB. The results of these studies are shown in Figs. 2A-C. As previously stated, ITC-6 detected the sera infected with G. cruzi much better than TcF. All the endemic control sera were negative with TcF and with ITC-6. Due to the very low background and cross reaction with TcF and ITC-6, these can be used even at 200 ng per well. Although there was 100% agreement with RIPA, with some sera (mixture of RR and Teragenix mixture), the OD was low. These are mixtures of several low-order sera and therefore represent dilutions of individual sera that were subsequently used in filtering or serological expression selection for additional antigens. In these cases, it is likely that the use of other peptides, such as KMP-11 and peptide 1, together with ITC-6 will produce higher OD.

Table 1: RIPA status of various sera from North, South and Central America In fig. 3, the reactivities of TcF and ITC-6 are compared with those of ITC-7.1 and 7.2 in a panel of sera. ITC-7.1 and 7.2 contain one and two repeats of the K-P-11 specific sequence of T. cruzi. The analyzes were performed as described above. The reactivity of these sera with TcF, ITC-6, ITC-7.1 and 7.2 are compared in fig. 3. Constructions ITC-7.1 and 7.2 showed activity comparable to ITC-6 with possibly higher signal indications.

Example 3 Detection of G. cruzi infection in sera using ITC 7.2 and ITC 8.2 Recombinant protein for ITC8.2 (SEQ ID NO: 19) was expressed as described in Example 6 below. This protein was then used as the solid phase antigen in an ELISA analysis and compared to ITC7.2 (SEQ ID NO: 17) which differs from ITC8.2 by not incorporating peptide 1 (SEQ ID NO: 6) ). A serum sample (sample No. 7190014) which was known to react with peptide 1 as well as with other components of the recombinant multiepitope proteins was included in the group. The results of this study are shown in fig. 4. As shown, activity comparable to ITC8.2 and ITC7.2 was seen. To ensure that peptide 1 is functional in ITC8.1 (containing the short form of peptide 1 provided in SEQ ID NO: 7) and ITC8.2 (containing the full length peptide 1 provided in SEQ ID NO. : 6), absorption studies were carried out with a reactive serum of peptide 1 (sample No. 7190014, University of Chile) absorbed with beads covered with ITC7.2. The preparation of ITC7.2 coated beads treated with serum # 7190014 was performed as follows: 1) 100 beets of ITC-7.2 beads were centrifuged at 2000 RPM for 1 min. 2) The floating buffer solution was carefully removed completely and added to the covered beads ITC7.2 a serum sample 7190014 diluted 1/10 in lOmM Tris, pH 8. 3) The mixture was gently oscillated for about 1 hour at RT. 4) The tube of the mixture was centrifuged again at 2000 rpm for 1 min. 5) The float was further diluted to a final dilution of 1/20 and 1/100 with Sample Dilution Buffer for IgG, 15mM EDTA. The untreated serum # 7190014 also it was diluted directly to 1/20 and 1/100 in serum dilution buffer. All samples were then tested in an ELISA with ??? μ? per well of diluted sera that were added to coated ITC7.2, shortened ITC8.1 (SEQ ID NO: 22) and ITC8.2 (SEQ ID NO: 19) microtiter strips (3.12 ng / well). ELISA of T. cruzi was performed as indicated above in example 1. FIG. 5 shows the activity of the non-absorbed and absorbed positive sera of peptide 1. Residual reactivity of peptide 1 was observed only in ITC8.2, indicating that the shortened form of peptide 1 in shortened ITC8.1 was unreacted or was less active than the largest peptide 1 sequence present in ITC8.2.

Example 4 Immunoblotting with ITC recombinant proteins Immunoblots of colloidal gold were performed to determine if the recombinant proteins of ITC-6 and ITC7.2 detected positive T. cruzi sera that were of low reactivity or negative by the recombinant father TcF protein. We also tested 12 samples of visceral leishmaniasis (VL) sera and sera from four normal donors. The data is shown in table 2 below. Briefly, the recombinant proteins were sprayed on nitrocellulose, blocked with nonfat dry milk powder, washed with PBS Tween and cut into strips. The strips were incubated in serum at a dilution of 1/50 for 15 minutes at 37 ° C. The strips were then washed in PBS Tween and further incubated for 6 minutes at room temperature with PA-gold (OD5) with 10% ECL. The spots were washed with PBS tween 20 and observed for the presence or absence of a colored line. The data (expressed as line intensity) indicate that both ITC6 and ITC7.2 markedly improve the detection capacity of positive T. cruzi sera relative to TcF without damaging specificity as indicated by any change in the reactivity profile with both VL serums and normal donor sera.

Table 2 Example 5 Rapid test data with ITC8.2 Rapid lateral flow immunoassays were performed using graduated rods prepared with ITC8.2 on the membrane and were compared with radioimmunoprecipitation, immunofluorescence, lysate and ITC8.2 ELISA data. The comparative data are shown in table 3, below. Good correlation is observed among all the analysis formats. Table 3 Example 6 Expression of ITC8.2 in SUMO expression system and improvement of specificity Two expression systems were compared to determine if it was possible to improve the expression of ITC8.2 and also to improve with respect to specificity. Positive and negative T. cruzi sera, and an antibody dilution panel were run in an analysis. Negative sera included problematic normal sera. The compared systems were the expression of an ITC8.2 labeled with Hexahistidine tag in a pET17 vector against an expression system using the SUMO expression system (without His tag, Life Sensors, Inc., Malvern, PA) where the SUMO fragment It was deleted with the appropriate protease. The data, shown in Table 4, show the potential for improvement in the specificity achieved by using the recombinant SUMO derivative against the preparation of hexahistidine ITC8.2 in a pET vector system.

Table 4 Example 7 Sequences derived from cloning of serological expression Preparation of T.cruzi bank: A bank was built of genomic randomized expression by sonicating genomic DNA from the CL chain of Trypanosoma cruzi. The sonicate produced fragment sizes of 0.5-2.0 KB. Fifteen micrograms of sonicated DNA were treated with T4 polymerase (NEB) for 15 minutes at 12 ° C followed by incubation for 20 minutes at 75 ° C to produce terminal diamond fragments. The EcoRI adapters were then ligated to the fragments and the adapters were then ligated to the fragments and adapters where they were phosphorylated with E. coli polynucleotide kinase. The fragments were then fractionated with a Sephacryl S400 column and finally ligated to a lambda ZAP Express vector (Stratagene). He bound vector was packed with packaging extract from Gigapack III Gold (Stratagene).

Filtration: The amplified library was plated on agarose plates at a concentration of 20,000 unit forming plates (PFU) per 35 plates. After incubation at 42 ° C for 4 hours, nitrocellulose filters soaked in 10m of IPTG were soaked and the plates were incubated at 37 ° C overnight. The filters were removed and washed 3x with PBS containing 0.1% Tween 20 (PBST), blocked for 1 hour with 1% BSA in PBST, washed 3x (washed 3 times) with PBST, blocked for another 1 hour with 1% Tween 20 in PBS, 3x wash with PBST and then incubation overnight at 4 ° C in serum, patient well # 1 (RR mixture) and / or well # 2 (Teragenix mixture). Both patient serum wells were obtained from low reactivity T. cruzi sera confirmed by RIPA. The next day, after washing 3x with PBST, the filters were incubated in an alkaline phosphatase secondary antibody goat anti-human Ig (IgG, IgA, IgM) IgM for 1 hr at room temperature. Finally the filters were washed 3x with PBST, 2x with buffer solution of AP and developed with BCIP / NBT. Positive clones were purified using the same technique. The phagemid was deleted and the resulting plasmid DNA was sequenced and searched against the T.cruzi databases.

Characteristics and result of the library filtration: Vector lambda: Lambda Zap Express (Stratagene) Plasmid vector: pBK-C V (kanamycin) DNA: Geni T. cruzi. Library title: 2.5xl08 / ml (amplified) (total of 30 ml) Insert size: 0.5-2.0 Kbp (average = 1.1) Filtering: 20,000 pfu per 35 plates Serum: patient well # 1 and well # 2 ( from normal donors) 1: 200 dilution Primary collections: 31 (human Ig) from well # 1 of patient (from 15 plates) 47 (human Ig) from well # 2 of patient (from 20 plates) Secondary purified: 38 (strong-weak signal) Sent to sequencing: 12 Table 5: Results of the filtration of the T.cruzi genomic library with serum collected from infected patients Additional sequences, designated Tc48, Tc60 and Tc70, were also identified. These sequences, plus Tc5, were of great interest for further evaluation. The DNA sequences for Tc5, Tc48, Tc60 and Tc70 are provided in SEQ. ID NO: 23, 25, 28 and 30, respectively, with the amino acid sequences for Tc5, Tc60 and Tc70 being provided in SEQ. ID NO: 24, 29 and 31. The identified partial amino acid sequence of Tc48 is provided in SEQ. ID NO: 26, with the corresponding total sequence obtained from the public database provided in SEQ. ID NO: 27. From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described for the purpose of illustration, various modifications may be made without departing from the essence and scope of the invention. I KNOW THAT. ID NO: 1-31 are established in the attached sequence listing. The codes for the polynucleotide and polypeptide sequences used in the attached sequence listing confirm WIPO Standard ST.25 (1988), Appendix 2. All references described herein, including patent references and non-patent references, are incorporated into way of reference in its entirety as if each were incorporated individually.

Claims (17)

1. A fusion polypeptide comprising an amino acid sequence of SEQ. ID NO: 1 and at least one amino acid sequence selected from the group consisting of: SEQ. ID NO: 2-7.

2. The fusion polypeptide according to claim 1, characterized in that the fusion polypeptide comprises the amino acid sequences SEQ. ID NO: 1, 2, 3 and 4.

3. The fusion polypeptide according to claim 1, characterized in that the fusion polypeptide comprises the amino acid sequences SEQ. ID NO: 1. 2, 3, 4 and 5.

4. The fusion polypeptide according to claim 1, characterized in that the fusion polypeptide comprises the amino acid sequences SEQ. ID NO: 1. 2, 3, 4, 5 and 6.

5. The fusion polypeptide according to claim 1, characterized in that the fusion polypeptide comprises the amino acid sequences SEQ. ID NO: 1. 2, 3, 4, 5 and 7.

6. The fusion polypeptide according to claim 1, characterized in that the polypeptide of The fusion comprises an amino acid sequence selected from the group consisting of: SEQ. ID NO: 8, 15, 17, 19 and 20.

7. A fusion polypeptide comprising an amino acid sequence selected from the group consisting of: (a) sequences having at least 85% identity to SEQ. ID NO: 8, 15, 17, 19 or 20; (b) sequences having at least 90% identity to SEQ. ID NO: 8, 15, 17, 19 or 20; and (c) sequences having at least 95% identity to SEQ. ID NO: 8, 15, 17, 19 or 20, wherein the fusion polypeptide is capable of generating at least 80% of the response generated by a fusion polypeptide of SEQ. ID NO: 8, 15, 17, 19 or 20 for sera infected with G. cruzi in an antibody binding assay.

8. An isolated polynucleotide sequence encoding a fusion polypeptide according to any of claims 1-7.

9. A recombinant expression vector comprising a polynucleotide sequence according to claim 8.

10. A host cell transformed or transfected with an expression vector according to claim

11. A method for detecting T. cruzi infection in a biological sample, comprising: (a) contacting the biological sample with a fusion polypeptide of any of claims 1-7; and (b) detecting in the biological sample the presence of antibodies that bind to the fusion polypeptide, thereby detecting T. cruzi infection in the biological sample. The method according to claim 11, characterized in that the biological sample is selected from the group consisting of: blood, serum, plasma, saliva, cerebrospinal fluid and urine. 13. A diagnostic kit for detecting G. cruzi infection in a biological sample, comprising: (a) a fusion polypeptide of any of claims 1-7; and (b) a detection reagent. 14. The kit in accordance with the claim 13, characterized in that the detection reagent comprises a reporter group. 15. The kit in accordance with the claim 14, characterized in that the indicator group is selected from the group consisting of: enzymes, substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. 16. A composition comprising a fusion protein according to any of claims 1-7 and at least one component selected from the group consisting of: carriers and physiologically acceptable immunostimulants. 17. A method for the prevention or treatment of T. cruzi infection in a patient, comprising administering to the patient a composition according to claim 16.