MXPA97007020A - Compounds and methods for the detection of infection by t. cr - Google Patents

Abstract

The present invention relates to compounds and methods for diagnosing infection by Trypanosoma cruzi, or for screening infection by T. cruzi or Leishmania. The disclosed compounds are polypeptides, or antibodies thereto, that contain one or more antigenic epitopes of T. cruzi proteins. The compounds are useful in a variety of immunoassays to detect T. cruzi infection. The polypeptide compounds are also useful for vaccines and pharmaceutical compositions to prevent Chagas disease in individuals exposed to T. cru

Description

COMPOSITIONS AND METHODS FOR THE DETECTION OF INFECTION BY T. CRUZI TECHNICAL FIELD The present invention relates generally to the diagnosis of T. cruzi infection and leishmaniasis. The invention is more particularly related to the use of one or more T cruzi antigenic peptides, or antibodies to the same, in diagnostic methods and equipment for sifting individuals and supplying blood for the presence of antibodies to T cruzi. The invention is also directed to vaccine compositions for immunizing an individual to prevent C agase disease. Background of the Invention Protozoa parasites They are a serious threat to health in many areas of the world Trypanosoma cruzi. { T cross? is one of these parasites that infects millions of individuals, mainly in Central and South America. Infections with this parasite can cause Chaguas disease, which can result in a chronic heart disease and a variety of immune system disorders. It is estimated that 18 million people in Latin America are infected with T cruzi in these regions, it is necessary to develop precise methods to diagnose T Cruzi infection in individuals and to screen blood supplies Blood bank screening is particularly important in Sudamépca, wherein 0 1% to 62% of the samples can be infected and where the parasite is frequently transmitted by blood transfusion. There is also a growing interest in the blood supply in certain cities of the United States. it may be contaminated with T. cruzi parasites. The diagnosis of T. cruzi infection has been problematic, given that the exact methods to detect the parasite that are suitable for routine use, have not been available. During the acute phase of infection, which can last for decades, the infection can remain immobile and the host can be asymptomatic. As a result, serological tests for T. cruzi infection are the most reliable and the most commonly used. However, such diagnoses are complicated due to the complex life cycle of the parasite and the various immune responses of the host. The parasite passes through an epimastigote state in the insect vector and two main stages in the mammalian host. A stage of the host is present in the blood (the trypomastigote stage) and a second stage is intracellular (the amastigote stage). The multiple steps result in a variety of antigens presented by the parasite during infection. In addition, the immune responses to protozoan infection are complex, involving both humoral and cell-mediated responses for the arrangement of parasite antigens. While antibodies against parasite antigens are detected, it is the most common and reliable method of clinical and subclinical diagnostic infections, the current tests are expensive and difficult. Most serological tests use 7. complete cruzi or lysate and require positive results in two of three tests, including complement fixation, indirect immunofluorescence, passive aglutination or E LISA, to accurately detect T. cruzi infection. The cost and difficulty of these tests has prevented the screening of blood or serum in many endemic areas. An improved method for detecting infection with T. cruzi was described in a patent of E. U.A. Do not . 5, 304, 371, which is incorporated herein by reference. In this patent, an antigenic epitope of T. cruzi was described that detected antibodies to T. cruzi and therefore infection with the parasite, in the majority of infected patients. However, while this method is an improvement over previous methods, the sensitivity of the technique is only about 93% (ie, only about 93% of infections could be diagnosed). Similar difficulties arise in the diagnosis of Leishmania infections. A variety of Leishmania species infect humans, causing diseases of humans characterized by visceral, cutaneous or mucosal lesions. Millions of cases of leishmaniasis exist throughout the world and at least 400,000 new cases of visceral leishmaniasis (IV) have been diagnosed annually. Leishmania species are transmitted to humans and other mammals by the bite of a blackfly or by blood transfusions with contaminated blood.

LV is usually caused by L. donovani in Africa and India, L. infantium in Southern Europe, or L. chagasi in Latin America. In LV, high levels of parasite-specific antibodies were observed prior to the detection of antigen-specific T cell responses (Ghose et al., Clin.Exp.Imunol.40: 318-326, 1980). This antibody response has been used for serodiagnostics (commonly by immunofluorescence techniques) of infection with L. chagasi and L. donovani. Those serodiagnostic methods currently available to diagnose VL usually use complete parasites or lysates. Such methods tend to inaccurate and cross-react with a variety of other diseases and fail to detect some cases of potentially fatal disease early enough to allow effective treatment. Consequently, there is a need in the art for more specific and sensitive methods of detecting T. cruzi and Leishmania infections in blood supplies of individuals. The present invention meets these needs and also provides other related advantages. SUMMARY OF THE INVENTION In summary, this invention provides compounds and methods for detecting and preventing T. cruzi infection in individuals and in blood supplies and for screening T. cruzi and Leishmania infections in biological samples. In one aspect, the present invention provides method for detecting T. cruzi infection in a biological sample, comprising (a) contacting the biological sample with a first polypeptide comprising at least 7 consecutive residues of the portion of SEQ ID NO. : 1 between the lysine at residue 137 and alanine at residue 247, or an antigenic variant thereof that differs only in substitutions or conservative modifications, with the proviso that the first polypeptide contains no more than five consecutive residues of the of SEQ ID NO: between amino acid 1 and amino acid 136; and (b) detecting in the biological sample the presence of antibodies that bind to the polypeptide, thereby detecting T. cruzi infection in the biological sample. In a related aspect, the present invention provides methods for detecting T. cruzi infection in a biological sample, comprising (a) contacting the biological sample with a first polypeptide comprising the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys, or an antigenic variant thereof that differs only in substitutions or conservative modifications: (b) contacting the biological sample with a second polypeptide comprising an amino acid sequence selected from the group consisting of Ala Glu Pro Lys Ser Wing Gly Pro Lys Pro Wing Glu Pro Lys Ser, or an antigenic variant thereof that differs only in substitutions or modifications or conservative and Ala Gly Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro, or an antigenic variant of the which differs only in substitutions or conservative modifications; and (c) detecting in the biological sample the presence of antibodies that bind to the first or second polypeptide, thereby detecting T. cruzi infection in the biological sample. In another related aspect of this invention, methods are provided for detecting T. cruzi infection in a biological sample, comprising (a) contacting a biological sample with a polypeptide comprising the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys, or an antigenic variant thereof that differs only in substitutions or conservative modifications; and further comprising an amino acid sequence selected from the group consisting of Ala Glu Pro Lys Ser Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser, or an antigenic variant thereof which differs only in conservative substitutions or modifications and Ala Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Gl u Pro Lys Pro, or an antigenic variant of the same that differs only in substitutions or conservative modifications; and (b) detecting in the biological sample the presence of antibodies that bind to the polypeptide, thereby detecting T. cruzi infection in the biological sample. In another aspect of this invention, polypeptides comprising at least 7 consecutive residues of the portion of SEQ ID NO: 1 are provided between the lysine at residue 137 and the alanine at residue 247 or an antigenic variant thereof which differ only in substitutions or conservative modifications.

In a related aspect of the present invention, polypeptides are provided comprising (a) the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala Ala Gly Asp Lys, or an antigenic variant thereof which differs only in substitutions or conservative modifications; and (b) an amino acid sequence selected from the group consisting of Ala Gly Pro Lys Ser Ala Glu Pro Lys Pro Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser, or an antigenic variant thereof that differs only in substitutions or conservative modifications , and Ala Gly Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro, or an antigenic variant thereof that differs only in substitutions or conservative modifications. Within the related aspects, the DNA sequences encoding the above polypeptides, expression vectors comprising these DNA sequences and host cells transformed or transfected with said expression vectors are provided as well. In another aspect, the present invention provides diagnostic kits for detecting T. cruzi infection in a biological sample, comprising (a) a first polypeptide consisting essentially of at least 7 consecutive residues of the portion of SEQ ID NO: 1 between the lysine at residue 137 and the alanine at residue 247, or a variant of antigen thereof that differs only in substitutions or conservative modifications; and (b) a detection reagent.

In a related aspect, diagnostic kits are provided to detect infection by 7 cruzi in a biological sample, comprising (a) a first polypeptide comprising the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys, or an antigenic variant thereof, which differs only in conservative substitutions or modifications and a second polypeptide comprising an amino acid sequence selected from the group consisting of Ala Glu Pro Lys Ser Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser, or an antigenic variant thereof it differs only in substitutions or conservative modifications thereof and Ala Glu by Lys Pro Ala Glu Pro Lys Ser Ala Glu Pro Lys Pro or an antigenic variant thereof that differs only in substitutions or conservative modifications and (b) a detection reagent. In another aspect, the present invention provides a method for screening infection of Leishmania or T. cruzi in a biological sample, comprising (a) contacting the biological sample with a T. cruzi antigen comprising the portion of SEQ ID NO: 1 between the arginine in residue 1 and the alanine in position 143, or an antigenic variant thereof that differs only in substitutions or conservative modifications; and (b) detecting the biological sample the presence of antibodies that bind to the antigen, thus detecting Leishmania infection or 7. cruzi in the biological sample. In yet another aspect, this invention provides diagnostic equipment for detecting leishmaniasis or 7. cruzi infection, comprising (a) an antigen of 7. cruzi comprising amino acids 1 to 143 of SEQ ID NO.:1, or an antigenic variant of the same ones that differ only in substitutions or conservative modifications; and (b) a detection reagent. Within related aspects, the pharmaceutical compositions, comprising the above polypeptides and also a physiologically acceptable carrier, and vaccines, comprising the above polypeptides and an auxiliary are provided. In another aspect of the invention, method is provided for detecting the presence of 7. cruzi infection in a biological sample, comprising (a) contacting a biological sample with a monoclonal antibody that binds to a polypeptide consisting essentially of at least 7 consecutive residues of the SEC portion. ID. NO .: 1 between the lysine at residue 137 and the alanine at residue 247, or an antigenic variant thereof which differs only in substitutions or conservative modifications; and (b) detect in the biological sample the presence of parasites of 7. cruzi that bind to the monoclonal antibody. In a related aspect, this invention provides method for detecting the presence of 7. cruzi infection in a biological sample, comprising (a) contacting a biological sample with a monoclonal antibody that binds to a polypeptide comprising an amino acid sequence selected from the group consists of Ala Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser, or an antigenic variant thereof that differs only in substitutions or conservative modifications and Ala Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro, or an antigenic variant thereof that differs only in substitutions or conservative modifications; and (b) detect in the biological sample the presence of parasites of 7. cruzi that bind to the monoclonal antibody. These and other aspects of the present invention will be apparent by reference to the following detailed description and accompanying drawings. Brief Description of the Drawings Figure 1 shows the DNA sequence of the TcE cDNA. Figure 2 depicts the deduced amino acid sequence of the TcE cDNA, with copies arranged in tandem of the seven-amino acid repeat unit underlined. Figure 3 shows the amino acid sequence of TcEr, a polypeptide containing the three degenerate seven amino acid repeat units present in TcE. Figure 4 describes the amino acid sequence of TcD, with the sequence of an antigenic TcD polypeptide underlined. Figure 5 shows the amino acid sequence of PEP2. Figure 6 illustrates the results of an ELISA comparing the reactivities of infection serum of 7. cruzi (Te) with lysate (Lys), TcD and TcE. Serum reactivities of patients with visceral leishmaniasis (AVL) and normal serum (N) not infected with TcE are also shown.

Figure 7 illustrates the results of a competition ELISA of 7. cruzi infection serum in TcE in the absence (-) or presence of 5 μg of synthetic control (CON) or the TcEr peptide. Figure 8 illustrates the results of an ELISA that evaluates the reactivities of infection serum of 7. cruzi (Te) in the lysate (Lys) TcEr. Serum reactivities of patients with visceral leishmaniasis (LAV), cutaneous leishmaniasis (LC) and normal non-infected control serum (N) in TcEr are also shown. Detailed Description of the Invention As noted above, the present invention is generally directed to compounds and methods for detecting and protecting against 7. cruzi infection in individuals and in blood supplies. The compounds of this invention generally comprise one more antigenic epitopes of 7. cruzi proteins. In particular, polypeptides comprising an antigenic epitope of a homolog of 7. cruzi of 35 kD to the eukaryotic ribosomal protein L19E are described. The homolog sequence of 7. cruzi of 35 kD (referred to herein as TcE) is shown in Figure 2, as well as in SEQ ID NO: 1. As used herein, the term "polypeptide" encompasses chains of amino acids of any length, where the amino acid residues are bound by covalent peptide ligations. The use of antigenic epitopes of additional 7. cruzi proteins, in combination with a TcE epitope, is also described to improve the sensitivity and specificity of the diagnosis.

The compounds and methods of this invention also encompass antigenic variants of the antigenic polypeptides. As used herein, an "antigenic variant" is a polypeptide that differs only from the polypeptide recited in conservative substitutions or modifications, so that it retains the antigenic properties of the recited polypeptide. A "conservative substitution" is one in which the amino acid is replaced by another amino acid having similar properties, so that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to remain substantially unchanged. . In general, the following amino acid groups present conservative changes: ala, pro, gly, glu, asp, gln, asn, ser, thr, cys, ser, tyr, thr, val ile, leu, met, ala, phe, lys, arg, his; and phe tyr, trp, his. Preferred substitutions include changes in valine, threonine and alanine and changes between serine and proline. The variants also, or alternatively, may contain other conservative modifications, including the deletion addition of amino acids that has minimal influence on the antigenic properties, secondary structure and hydropathic nature of the polypeptide. For example, the polypeptide can be conjugated to a linker or other sequence for ease of synthesis or to increase the binding of the polypeptide to a solid support. In one aspect of the invention, polypeptides comprising an antigenic epitope of the L19E homologue of 7. cruzi are provided. L19E homologs of 39 kD can be isolated by screening an expression bank of 7. cruzi for clones expressing antigens that have the following properties: (1) strong reactivity with serum from patients infected with 7. cruzi, (2) reactivity with serum of patients infected with 7. cruzi whose infections can not be detected using an antigenic epitope of the TcD antigen and (3) lacks reactivity with serum from normal patients and heterolols (ie, serum from patients with other pathologies, such as leishmaniasis, leprosy and tuberculosis). Consequently, it is first possible to sift a 7. cruzi amastigote cDNA expression bank with mixed serum from individuals infected with 7. cruzi. Clones expressing proteins that react with the mixed serum can then be subjected to a second sieve using sera from 7. cruzi infected individuals whose infection can be detected with antigenic polypeptides derived from an antigenic epitope of the TcD antigen of 7. cruzi. Finally, the clones expressing proteins which react with the serum in the first sieves can be subjected to a third screen using serum from normal or heterologous patients. All of the above sieves can generally be made using methods known to those skilled in the art or as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. 1989, which is incorporated herein by reference. In summary, the bacteriophage bank can be plated and transferred to filters. The filters can then be incubated 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. Normal detection reagents for screening purposes contain a "binding agent", such as Protein A, Protein g, IgG or a lectin, coupled to a reporter group. Preferred reporter groups include, but are not limited to, enzymes, substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. More preferably, the reporter group is horseradish peroxidase, which can be detected by incubation with a substrate such as tetramethyl Ibenzidine or 2,2'-azino-di- 3-ethylbenzthiazolin-sulphonic acid. Plates containing cDNAs 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. A cDNA encoding the L19E homologue of 7. cruzi (ie, TcE) that was isolated using the above sieves is shown in Figure 1, and the deduced amino acid sequence of the TcE cDNA is shown in Figure 2. The N-terminal portion of TcE (the region not underlined in Figure 2) is homologous to the eukaryotic ribosomal protein L19E. Following the homology region of L19E there are sixteen copies of a repeat of seven amino acids arranged in tandem, which are underlined in Figure 2. The antigenic regions of TcE can generally be determined by generating polypeptides containing portions of the TcE sequence and evaluating the reactivity of the polypeptides with serum from individuals infected with 7. cruzi using, for example, an enzyme-bound, absorbent, immunoassay (ELISA). Appropriate assays to evaluate reactivity with serum 7. cruzi infected are described in more detail below. Portions of TcE containing at least 7 amino acids from the tandem repeat region (i.e., residues 137-247 in Figure 2) have generally been found to be antigenic. Accordingly, polypeptides comprising at least a 7 amino acid portion of the sequence between residues 137 and 247 of TcE and antigenic variants thereof, are within the scope of this invention preferably, the antigenic polypeptides contain at least a portion of 14 amino acids and more preferably at least a 21 amino acid portion of the TcE sequence between residues 137 and 247. In certain embodiments, the N-terminal region of TcE that is homologous to L19E (i.e., residues 1-136) it is substantially excluded from the antigenic polypeptide to avoid cross-reactivity with anli-Lesihmania antibodies. In these embodiments, the polypeptide generally contains no more than about 5 consecutive amino acids from the N-terminal region. More preferably, the antigenic polypeptide is TcEr, a 21 amino acid peptide comprising three degenerate 7 amino acid repeat units. The sequence of TcEr is provided in Figure 3. In a related aspect, combination polypeptides comprising antigenic epitopes of multiple peptides of 7. cruzi are described. A "combination polypeptide" is a polypeptide in which the antigenic epitopes of different 7 cruzi peptides or antigen variants thereof, are joined by a peptide linkage in a single chain of amino acids. The epitopes can be linked directly (ie, without amino acids intervening) or can be linked in the manner of a binding sequence (eg, Gly, Cys, Gly) that does not significantly alter the antigenic properties of the epitopes. In preferred embodiments, the combination polypeptide comprises an antigenic TcE epitope together with an antigenic epitope derived from TcD antigen of 7. cruzi (described, in U.S. Patent No. 5,304,371) and / or the antigenic epitope PEP2 (treated, for example, in Peralta et al., J. Clin. Microbiol. 32971-74, 1994). The preferred TcE epitopes for use in combination peptides are as described below. The antigenic epitope of TcD preferably has the amino acid sequence Ala Glu Pro Lys Ser Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser or the amino acid sequence Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser Ala Glu Pro Lys Pro and the epitope of PEP2 preferably has the amino acid sequence Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala Wing Wing Gly Asp Lys Pro Ser Pro Phe Gly Gln Wing (provided in Figure 5. The combination polypeptides of this invention may also contain an antigenic epitope of TcD in combination with PEP2, with or without an antigenic TcE epitope It has been found that the location of the TcE epitope at one end of the combination polypeptide proceeds superiorly to a solid support.Subsequently, for polypeptides containing a TcE epitope, that epitope is preferably located at either N-terminal or C-terminal end of the combination polypeptide The polypeptides of this invention can be generated I know techniques well known by those of ordinary experience in the field. Polypeptides having less than about 100 amino acids and generally less than about 50 amino acids, they can be synthesized using, for example, the Merrifield solid phase synthesis method, wherein the amino acids are added sequentially to an increasing amino acid chain. See Merrifield, J. Am Chem. Soc. 85: 2149-2146, 1963. Equipment for automatic polypeptide synthesis is commercially available from suppliers such as Applied Biosystems, Inc., Foster Cityu, CA. Thus, for example, the PEP2 polypeptide of 22 amino acids, or portions thereof, can be synthesized by this method. Similarly, the antigenic epitopes of TcE or TcD, which preferably contain 1 to 3 repeating units of those proteins, can be prepared using an automated synthesizer.

Alternatively, the polypeptides of this invention can be prepared by expressing recombinant DNA encoding the polypeptide in cultured host cells. Preferably, the host cells are E. coli, yeast, an insect cell line (such as Spodoptera or Trichoplusia) or a mammalian cell line, including (but not limited to) CHO, COS and NS-1. The DNA sequences expressed in this form can encode proteins that occur in nature, such as TcE and TcD, portions of proteins present in nature, or antigenic variants of said proteins. The expressed polypeptides of this invention are generally isolated in substantially pure form. Preferably, the polypeptides are isolated at a purity of at least 80% by weight, more preferably at a purity of at least 95% by weight and even more preferably at a purity of at least 99% by weight. In general, such purification can be achieved using, for example, normal ammonium sulfate fractionation techniques, SDS-PAGE electrophoresis, and affinity chromatography. In another aspect of this invention, methods for detecting infection with 7. cruzi can be detected in individuals and blood supplies. In general, 7. cruzi infection can be detected in any biological sample that contains antibodies. Preferably, the sample is blood, serum, plasma, saliva, cerebrospinal fluid or urine. More preferably, the sample is a sample of blood or serum obtained from a patient or a blood supply. In summary, infection with 7. cruzi can be detected using any of the polypeptides or combination polypeptides discussed above, or antigenic variants thereof. More specifically, the polypeptide or polypeptides are used to determine the presence or absence of antibodies to the polypeptide polypeptides in the sample, relative to a predetermined reduction value. There is a variety of assay formats known to those of ordinary skill in the art to use purified antigen in order to detect antibodies in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, which is incorporated herein by reference. In a preferred embodiment, the assay involves the use of immobilized polypeptide on a solid support to bind to remove the antibody from the sample. The bound antibody can then be detected using a detection reagent that binds to the antibody / peptide complex and contains a detectable reporter group. Suitable detection reagents include antibodies that bind to the antibody / polypeptide complex and free polypeptide labeled with a reporter group (e.g., in a semi-competitive analysis). Alternatively, a competitive assay may be used, in which an antibody that binds to the polypeptide is labeled with a reporter group and allowed to bind 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 polypeptide indicates the reactivity of the sample with the immobilized polypeptide. The solid support can be any solid material known per os of ordinary experience in the material to which the antigen can be bound. For example, the solid support can be a microtiter plate, a nitrocellulose and another suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinyl chloride. The support can also be a magnetic particle or a fiber optic sensor, such as that described, for example, in US Pat. No. 5,359,681. The polypeptide can be attached to a solid support using a variety of techniques known to those skilled in the art, which are described extensively in the patent and scientific literature. In the context of the present invention, the term "attached" refers to both the non-covalent association, such as adsorption and the covalent bond (which may be a direct binding between the antigen and functional groups on the support or may be a binding agent as an agent). interlacing). Bonding by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, the adsorption can be achieved by contacting the polypeptide, in a buffer solution of suitable pH, with the solid support for a suitable amount of time. The contact time varies with the temperature, but normally it is between approximately 1 hour and 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinyl chloride) with an amount of polypeptide ranging from about 10 ng to about 1 μg of protein per cm 3. The covalent attachment of polypeptide to a solid support can generally be achieved by first reacting the support with a bifunctional reagent which will react with the support with a functional group, such as a hydroxyl or amino group, on the polypeptide. For example, the polypeptide can be attached to supports having an appropriate polymer coating using benzoquinone or by condensing an aldehyde group on the support with an amine and an active hydrogen in the polypeptide (see, e.g., Pierce Immunotechnology Catalog and Handbook (1991) in A12-A13; Jerry March Advanced Organic Chemistry (2nd Ed. 1977) at 820-823). In certain embodiments, the assay is an enzyme-linked immunosorbent assay (ELISA). This analysis can be performed by first contacting a polypeptide antigen that has been immobilized on a solid support, commonly the well of a microtitre plateau, with the sample, so that antibodies to the polypeptide within the sample are allowed to bind to the polypeptide immobilized. The unbound sample is then removed from the immobilized polypeptide by a detection reagent capable of binding to the immobilized polypeptide. The unbound sample is then removed from the immobilized polypeptide and a detection reagent capable of binding to the immobilized antibody-polypeptide complex is added. The amount of detection reagent remaining attached to the solid support is then determined using a method appropriate for the specific detection reagent. Once the polypeptide is immobilized in the support, the binding sites of the remaining protein in the support are normally blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20 ™ (Sigma Chemical Co.). The immobilized polypeptide is then incubated with the sample and the antibody (if present in the sample) is allowed to bind the antigen. The sample can be diluted with a suitable diluent, such as phosphate buffered saline (PBS) before incubation. In general, an appropriate contact time (i.e., incubation time) is the time that is sufficient to allow detection of the presence of 7. cruzi antibody with a sample infected with 7. cruzi. Preferably, the contact time is sufficient to achieve a binding level that is at least 95% of that achieved in equilibrium between the binding and non-binding antibody. Those skilled in the art will recognize that the time necessary to achieve equilibrium can easily be determined by analyzing the level of binding that occurs over time. At room temperature, an incubation time of approximately 30 minutes is usually sufficient. The unbound sample can then be removed by washing the solid support with an appropriate buffer solution, such as PBS containing 0.1% Tween 20. The detection reagent can then be added to the solid support. An appropriate detection reagent is any compound that binds to the immobilized antibody-polypeptide complex and that which can be detected by any of a variety of means known to those skilled in the art. Preferably, the detection reagent contains a binding agent (such as, for example, Protein α, G protein, immunoglobulin, lectin or free antigen) conjugated to a reporter group. Preferred reporter groups include enzymes (such as horseradish peroxidase), substrate, cofactors, inhibitors, dye, radionuclides, luminescent groups, fluorescein biotin groups. The conjugation of the agent linked to the reporter group can be achieved using normal methods known to those skilled in the art. Common binding agents can also be purchased conjugated to a variety of reporter groups from many sources (e.g., Zuymed Laboratiries, San Francisco, CA and Pierce, Rockford, IL). The detection reagent is then incubated with the immobilized antibody-polypeptide complex for a sufficient time to detect the bound antibody. An appropriate amount of time can usually be determined by the manufacturer's instructions and by analysis of the level of bonding that occurs over time. The unbound detection reaction is then removed and the bound detection reagent is detected using the reporter group. The method used to detect the reporter group depends on the nature of the reporter group. For radioactive groups, scintillation or autoradiographic counting methods are generally appropriate. Spectroscopic methods can be used to detect dyes, luminescent groups and fluorescent groups. Biotin can be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme report groups can usually be detected by the addition of substrate (usually over a specific period), followed by spectroscopic analysis or another of the reaction products. To determine the presence or absence of 7. cruzi antibodies in the sample, the signal detected from the reporter group remaining attached to the solid support is generally compared to a signal corresponding to a predetermined reduction value. This reduction value is preferably the average average signal obtained when the immobilized antigen was incubated with samples from an uninfected patient. In general, a sample that generates a signal that is three normal deviations above the predetermined reduction value is considered positive for 7. cruzi antibodies and 7. cruzi infection. In a related embodiment, the analysis is carried out in a test format through flow or strip, where the antigen is immobilized or a membrane such as nitrocellulose. In the flow-through test, antibodies within the sample bind to the immobilized polypeptide since the sample passes through the membrane. A detection reagent (e.g., A-colloidal protein gold) is then bound to the antibody-polypeptide complex since the solution containing the detection reagent flows through the membrane. The detection of the attached detection reagent can then be performed as described above. In the strip test format, one end of the membrane to which the polypeptide binds is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing detection reagent and to the immobilized polypeptide area. The concentration of the detection reagent in the polypeptide indicates the presence of 7. cruzi antibodies in the sample. Such tests can usually be performed with a very small amount (eg, one drop) of patient's serum or blood. Of course, there are numerous other assay protocols that are suitable for use with the polypeptides of the present invention. The above descriptions are intended to be illustrative only. In one embodiment of the analyzes discussed above, the antibodies are detected using a polypeptide comprising at least a portion of 7 amino acids, preferably a portion of 14 amino acids and more preferably at least a portion of 21 amino acids of the sequence between the residues. 137 and 247 of TcE, or an antigenic variant thereof. In general, the N-terminal region of TcE that is homologous to L19E (i.e., residues 1-136) is substantially excluded from the antigenic polypeptide to avoid cross-reactivity with the an-Leishmania antibodies. More preferably, the antigenic polypeptide is TcEr. In further embodiments, methods for improving the sensitivity of the analyzes described above are described. In general, the sensitivity can be significantly improved by using one or more additional 7. cruzi antigens in combination with the TcE epitope. In particular, the antigenic epitopes of TcD and / or PEP2 (or antigenic variants thereof) that are provided above, can be mixed with the TcE polypeptides. Alternatively, an antigenic epitope of TcD can be combined with PEP2, in the absence of the TcE antigen. These analyzes can be performed using different polypeptide groups. In a two polypeptide embodiment, one of the polypeptides contains an antigenic epitope of TcE and the others contain an antigenic epitope of TcD. In another embodiment, one of the polypeptides contains an antigenic epitope of TcE and the other contains PEP2 or an antigenic portion thereof. In a third of such embodiments, one of the polypeptides contains an antigenic epitope of PEP2. In a third of these modalities, one of the polypeptides contains an antigenic epitope of PEP2 and the other contains an epitope of TcD. The analysis can also be performed using three polypeptides, one containing a TcE epitope, one containing a TcD epitope and a third containing an epitope of PEP2.

Preferably, the antigenic polypeptides are immobilized by adsorption to a solid support such as a well of a microtiter plate or membrane, as described above, so that an almost similar amount of each polypeptide is contacted with the support and so that the total amount of polypeptide in contact with the support varies from about 10 ng to about 100 μg. The rest of the steps can generally be carried out as described above. In an alternative embodiment, the combination polypeptides are employed. As discussed above, a combination polypeptide is a polypeptide in which the antigenic epitopes of different 7. cruzi peptides are linked through one or more peptide linkages in a single chain of amino acids. Any of the above antigenic epitopes or antigenic variants thereof can be incorporated into a combination of polypeptides. Therefore, a combination of polypeptides may contain a TcE epitope linked to a TcD epitope, a TcE epitope attached to a PEP2; an epitope of TcD bound to PEP2, or a TcE epitope, an epitope of TcD and PEP2 joined together within the same polypeptide. In another aspect of this invention, methods are provided for screening a biological sample for 7. cruzi and / or Leishmania species. In these methods, the biological sample is analyzed for antibodies to TcE, or certain portions thereof. In general, the assays can be carried out as described above, except that the polypeptide employed comprises amino acids 1 to 143 of TcE, as depicted in Figure 2. It has been found that the N-terminal portion of this antigen (amino acids 1 -136) reacts with antibodies to Leishmania. Any Leishmania species can be detected using this sequence, including L. major, L. tropic, L. chagasi, L. donovani, L. infantum, L. guyanesis, L. braziliensis, L. amazonensis and L. panamensisl. The inclusion of amino acid sequence of the repeated portion in tamc of TcE results in the detection of specific antibodies also for 7. cruzi. Additional amino acids of the TcE portion between amino acid 145 and the carboxy terminus can also be included. In a preferred embodiment, the antigen used in the sieve for both 7. cruzi and Leishmania is the full-length TcE protein, shown in Figure 2. Antigenic variants of TcE, or a portion thereof, may also be used comprising minus amino acids 1-136 and a repeating unit. Following the previous sieve for 7. cruzi and / or Leshmania, the parasite can be identified using specific methods for 7. cruzi or Leishmania. For example, any of the methods described above can be used to detect the presence of 7. cruzi in the sample. Any of the methods known to those skilled in the art can be used to detect Leshmania. In yet another aspect of this invention, a method is provided for detecting 7. cruzi in a biological sample using monospecific antibodies (which may be polyclonal or monoclonal) to polypeptides comprising epitopes of one or more of TcE, TcD and PEP2. Preferred epitopes are those recited before the antigenic variants thereof. Antibodies to these purified or synthesized polypeptides can be prepared by a variety of techniques known to those of ordinary skill in the art. See. V. gr. , Harlow and Land, Antibodies: A Laboratory Manual, Cold Spring Harbor The Boratory, 1988. In one of these techniques, an immunogen that comprises the antigenic polypeptide is initially injected into any of a wide variety of mammals (Fig. gr., 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 may be produced if the polypeptide binds to a carrier protein., such as bovine serum albumin or orifice limpet hemocyanin. The immunogen is injected into the host at least preferably according to a predetermined schedule incorporating one or more booster immunizations and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide can be purified from said antisera, for example, by affinity chromatography using the polypeptide coupled to a suitable solid support. Monoclonal antibodies specific for the antigenic polypeptide of interest can be prepared, for example, using the technique of Kohier and Milstein, Eur. J. Immunol. 6.51 1 -519, 1976, and improve the same. In summary, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Said cell lines can be produced, for example, from spleen cells obtained from an immunized animal as described above. Spleen cells are then immortalized, for example, by fusion with a myeloma cell fusion partner, preferably one that is syngenic with the immunized animal. A variety of fusion techniques can be employed. 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 melanoma cells. A preferred selection technique uses HAT selection (hypoxanthine, aminopterin, thymidine). After a sufficient time, usually about 1 to 2 weeks, the hybrid colonies are observed. The colonies alone are selected and tested to bind activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred. Monoclonal antibodies can be isolated from the supernatants of growth hybridoma colonies. In addition, various techniques can be employed to increase performance, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. The monoclonal antibodies can then be cultured from the ascites fluid or the blood. The contaminants can be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation and extraction. Monospecific antibodies for polypeptides comprising epitopes of one or more of TcE, TcD PEP2 can be used to detect infection with 7. cruzi in a biological sample using one of a variety of immunoanalysis, which can be direct or competitive. In summary, in a direct analysis format, a monospecific antibody can be immobilized on a solid support (as described above) and contacted with the sample to be tested. After the removal of the unbound sample, a second monospecific antibody can be added, which has been labeled with a reporter group, and can be used to detect the bound antigen. In a competitive illustrative analysis, the sample can be combined with the monoclonal or polyclonal antibody, which has been labeled with a suitable reporter group. The mixture of the sample and antibody can then be combined with immobilized polypeptide antigen on a suitable solid support. The antibody that has not bound to an antigen in the sample is allowed to bind to the immobilized antigen and the rest of the sample and antibody is removed. The level of antibody bound to the solid support is inversely related to the level of antigen in the sample. Therefore, a lower level of antibody bound to the solid support indicates the presence of 7. cruzi in the sample. Any of the reporter groups treated earlier in the ELISA context can be used to label monospecific antibodies and the binding can be detected by any of a variety of techniques appropriate to the reporter group employed. Other formats for using monospecific antibodies to detect 7. cruzi in a sample will be apparent to those skilled in the art and the above formats are provided for illustrative purposes only. In another aspect of this invention, vaccines and pharmaceutical compositions are provided for the prevention of 7. cruzi infection and the complications thereof, in a mammal. The pharmaceutical compositions generally comprise one or more polypeptides, containing one or more antigenic epitopes of 7. cruzi proteins and a physiologically acceptable carrier. The vaccines comprise one or more of the above polypeptides and an auxiliary, to increase the immune response. The routes and frequency of administration and dose of polypeptides will vary from individual to individual and may be parallel to those that are currently being used in immunization against other protozoan infections. In general, the pharmaceutical compositions and vaccines can be administered by injection (e.g., intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. They can be given from 1 to 4 doses for a period of 2-6 weeks. Preferably, two doses of polypeptide are administered which is effective to raise antibodies in a treated mammal that are sufficient to protect the mammal from 7. cruzi infection for a time. In general, the amount of polypeptide present in a dose ranges from about 1 pg to about 100 mg per host kg, usually from about 10 pg to about 1 mg and preferably from about 100 pg to about 1 pg. The appropriate dose sizes will vary with the size of the animal, but will usually vary from about 0.01 to about 5 ml for an animal of 10-60 kg. While using any suitable vehicle known to those of ordinary skill in the art can be employed in the pharmaceutical compositions of this invention, the type of vehicle will vary depending on the mode of administration. For parenteral administration, such as by subcutaneous injection, the vehicle preferably comprises water, saline, alcohol, a fat, a wax or a pH regulator. For oral administration, any of the above vehicles or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) can also be employed as carriers for the pharmaceutical compositions of this invention. Any of a variety of auxiliaries can be employed in the vaccines of this invention to not specifically enhance the immune response. Most auxiliaries contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil and a non-specific stimulated immune response, such as lipid A, Bordella pertussis or Mycobacterum tuberculosis. Such auxiliaries are commercially available as, for example, Incomplete Freund's Assistant and Full Assistant (Difco Laboratories, Detroit, Ml) and Merk Adjuvant 65 (Merck and Company, Inc., Rahway, NJ). The following Examples are offered by way of illustration and not by way of limitation. EXAMPLES Example 1 Preparation of TcE This Example illustrates the isolation of cDNA encoding TcE and the preparation of TcE using the cDNA. Total RNA was isolated from the amastigote stage of the strain of 7. cruzi MHOM / CH / 00 / Tulahuen using the guanidino acid isothiocyanate method. An unamplified cDNA expression library was prepared from this RNA using the unidirectional ZAP / cDNA cloning kit (Stratagene, Inc., La Jolla, CA). In summary, the first cDNA was constructed by curing an oligo dT primer with Xho I adapters. Following the synthesis of the second thread, the Eco R1 adapters were added and the double stranded cDNA was digested with Xho 1 and ligated into the phage. unizap lambda predigested with Eco Rl and Xho 1. To immunize a bank, serum samples from five infected individuals were mixed with 7. cruzi. Anti-E reactivity was removed. serum coli before sifting by adsorption. 60,000 pfu of the unamplified bank was screened with the mixture of sera and plaques expressing that the proteins that react with the serum were detected using radish protein peroxidase (with the ABTS substrate) and isolated. The pBSK (-) phagemid excision (Stratagene, Inc., La Jolla, CA) was carried out according to the manufacturer's protocol. Clone overlap was generated by digestion of exonuclease III and the single-stranded primers were isolated after infection with helper phage VSCM 13. The DNA was sequenced by the dideoxy chain termination method or by the Taq terminator system. , using an Applied Biosystem automatic sequencer, Model 373A. Forty-two clones that expressed proteins that were reacted with serum were isolated after this screen. Of the isolated clones, 33 which reacted strongly or very strongly with the patient's serum were purified and sequenced. Twelve of these clones (approximately 35%) were members of a highly immunogenic P. cruzi protein family. One clone corresponded to a clone shock antigen gene. Two clones showed sequence identity for ubiquitin genes of 7. cruzi. The remaining 18 clones represented new genes of 7. cruzi. Six of these had sequence similarity with eukaryotic ribosomal proteins (L19E, S8 and S-phase specific) and 12 genes represented that were not homologous to sequences in GenBank.

The isolated clones were further screened by the above procedure with serum from heterologous patients of individuals infected with Leishmania. The members of the P protein family showed cross-reactivity with the heterologous serum and were not followed further. The remaining clones were then screened with serum from individuals who were infected with 7. cruzi, but whose infections could not be detected using the antigenic epitopes of TcD. Clones that had sequence similarity with eukaryotic ribosomal proteins were strongly reactive with the negative serum of TcD. Of these clones, the L19E homolog was unique in that its homology to the eukaryotic ribosomal protein was confined to the N-terminal portion of the protein. This homologue (TcE) was exceptionally reactive with the test serum. The sequence of the cDNA encoding TcE is shown in Figure 1 and the predicted amino acid sequence is provided in Figure 2. Full-length TcE was produced and purified from transformed E. coli with an expression vector containing the cDNA insert coding TcE. A transformed bacterial colony was used to inoculate 10 ml of LB broth developed at 37 ° C during the night. A 500 ml culture was then inoculated with the non-induced culture overnight at a 50: 1 dilution. This culture was grown at 37 ° C until the A560 was approximately 0.4 to 0.5. I PTG was then added to a final concentration of 2 mM the culture was allowed to develop for 4 hours. The cells were cultured by centrifugation, smoothed and fractionated into a pellet and soluble components. The TcE that remained in the soluble supernatant was fractionated by sequential ammonium sulfate precipitations. Purification for homogeneity was achieved by preparative SDS-PAGE electrophoresis, followed by excision and electroelution of the recombinant antigen. Example 2 Preparation of TcEr This Example illustrates the preparation of a polypeptide comprising an antigenic epitope of TcE. While the minimal sequence representing the antigenic epitope of TcE is a repeat of 7 amino acids, a sequence of peptides having three degenerate repeats was selected to be studied in order to maximize reactivity. The TcEr polypeptide was synthesized on an ABI 430A peptide synthesizer using FMOC chemistry with HPTU activation. (O-Benzotriazole hexafluorophosphate-N, N, N'-N'-tetramethyluronium). A Gly-Cys-Bly sequence was attached to the amino terminus of the peptide to provide a method of conjugation or labeling of the peptide. The separation of the peptides from the solid support was carried out using the following separation mixture, trifluoroacetic acid: ethanedithiol: thioanisole: water: phenol (40: 1: 2: 2: 3). After separating for 2 hours, the peptides were precipitated in methyl t-butyl ether. The peptide pellets were then dissolved in water containing 0.1% trifluoroacetic acid (TFA) and lyophilized before purification with C18 reverse phase HPLC. A gradient of 0% -60% acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) was used to elute the peptides. The following lyophilization of the pure fractions, the peptides were characterized using mass spectrometry of the sprayed spectrum and by amino acid analysis. The synthesized peptide (TcEr) has the sequence shown in Figure 3. Example 3 Detection of 7. cruzi infection in serum This Example illustrates the detection of 7. cruzi infection using the compounds and methods of this invention. The analyzes described below were performed in the ELISA format. A. TcE The TcE antigen was purified from extracts of E. coli induced as described above for the serological evaluation of the serum of patients by ELISA. The ELISA analysis was performed as follows. The microtitre plates were coated overnight at 4 ° C with 25 ng per well of recombinant TcE in 50 μl of pH buffering solution. After washing with PBS / 0.1% Tween ™ 20 (PBS-T). 50 μl of serum (diluted 1: 100) were added and incubated for 30 minutes at room temperature. An additional washing step was performed with PBS-T. Protein A-horseradish peroxidase conjugate was diluted 1: 10,000 in PBS-T and 50 μl of the diluted conjugate was added to each well and incubated for minutes at room temperature. The washing step with PBS-T was repeated again and 100 μl of substrate (ABTS / H203, Zymed Kit, Catalog No. 0-2011) was added per well and incubated for 30 minutes at room temperature under dim light. The colorimetric reaction was terminated with 100 μl of 5% sodium dodecyl sulfate (SDS) and the absorbance was read at 405 nm. Of 36 sera with 7. cruzi infection that were initially tested, 35 (97.2%) were tested positive using TcE, with absorbance values ranging from 0.25 to over 2.0. The average absorbance value is shown in Figure 6, which also compares the reactivity of TcE with that of TcD and lysate. Of particular importance, the serum of 8 patients who were negative or had low titers of antibodies to TcD reacted relatively strongly with TcE. These results are shown in the following Taba 1. Table 1 Serum Reactivities of TcD-Negative with TcE It was also found that some of the serum from patients who had low absorbance values with TcE were reactive with TcD. Therefore TcE has the ability to complement TcD, thus increasing diagnostic sensitivity. The specificity of TcE was further evaluated using serum from individuals infected with Leishmania as well as normal serum. It was observed that the cross-reactivity with serum of individuals infected with Leishmania (LAV), as shown in Figure 6. Consequently, the full-length TcE antigen can be used to screen patients for the presence of 7. cruzi infection or Leishmania After a positive result using TcE, however, additional tests specific for 7. cruzi or Leishmania will need to be performed in order to distinguish it between two parasites. B. TcEr To evaluate the reactivity of TcEr, the peptide was used in an inhibition study, where its capacity to complete the binding of serum infected with 7. cruzi was measured. Competition ELISA was performed in the following manner. The microtitre plates were coated overnight at 4 ° C with 25 ng per well of recombinant TcE in 50 μl of pH buffering solution. After washing with PBS-T, 50 μl of serum obtained from an individual infected with 7. cruzi (diluted to 1: 100 and pre-incubated with 5 μg of peptide for 1 hour at room temperature) was added and incubated for 30 minutes at room temperature. room temperature. Bound antibody was detected using protein A-horseradish peroxidase with ABTS substrate and the absorbance was measured at 405 nm. From serum tested infected with 7. cruzi from seven individuals, TcEr was efficient to compete with serum binding in TcE with inhibition values varying from 62% -90%. A control peptide with amino acid residues derived from the reading frame without coding had minimal effect in the same competition analysis. These results are described in Figure 7. Consequently, TcEr represents the epitope of immunodominant B cells of TcE. The specificity of TcEr reactivity was also evaluated, together with the serum-reactivity compared with lysate and TcD. In these experiments, ELISA were performed in which the microtiter plates were coated overnight at 4 ° C with 100 ng per well of 7. cruzi lysate, 250 ng per well of recombinant TcD peptide (i.e., the polypeptide having the 15 amino acid sequence underlined in Figure 4, with a Gly-Cys-Gly sequence attached to the amino terminus) or 25 ng per synthetic TcEr peptide well in 50 μl of coating pH buffer. After washing with PBS-T, 50 μl of serum from an individual infected by 7. cruzi (diluted 1: 100) was added and incubated for 30 minutes at room temperature. The bound antibody was detected using the protein A-horseradish peroxidase and the absorbance was measured at 405 nm. The results of this experiment are described in Figure 8. Using three normal deviations above the average normal serum average as a criterion to classify a patient's serum as positive, 66 of 69 (95.6%) of serum samples infected with 7. cruzi were positive when tested with TcEr and had an average absorbance value of 1.16. Figure 8 also shows the reactivity of TcEr with serum from patients with visceral leishmaniasis (LAV), cutaneous leishmaniasis (LC) and normal uninfected control serum (N). The 16 sera with LAV infection that were positive in the full length TcE antigen were negative when the analysis was performed with TcEr. Therefore, the cross-reactivity of heterologous Leishamania infection serum with TcE was directed against the homology domain of L19E without repetition. We also tested the patient's serum reactivities of individuals with cutaneous leishmaniasis (LC) with TcEr. All serum from 39 patients with LC was negative when the analysis was performed with TcEr. These results indicate that TcEr is as reactive as TcD with serum from individuals infected by 7. cruzi and that TcEr is highly specific for the detection of 7. cruzi. Therefore, TcEr met the requirements as a sensitive and specific diagnostic antigen for 7. cruzi infection. C. Multiple Antigenic Polypeptides In order to increase the sensitivity of the analysis described above, the analyzes were repeated using multiple polypeptides, each of which contained an epitope of a different 7. cruzi antigen. In particular, the TcD and TcE polypeptides were combined, as the TcD and PEP2 polypeptides. The PEP2 polypeptide in all these experiments consisted of the 22 amino acid sequence shown in Figure 5, with a Gly-Cys-Gly sequence attached to the amyloid terminus and the TcD peptide was as described above. The reactivity of these combinations was evaluated using the ELIS format and compared with the reactivities of each of the polypeptides individually. The ELISA analyzes were carried out in the following manner. 96-well plastic plates (Cornígn Easy Wash High Binding, Corning Laboratories, Corning, NY) were coated with 50 μl of the peptide or peptide mixture. The TcD peptide used in these analyzes has the sequence Ala Gly Pro Lys Ser Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser and was present in the 50 μl at a concentration of 10 μg / ml. The sequence of PEP2 was the sequence of 22 amino acids shown in "Figure 5, and this peptide was present in the 50 μl at a concentration of 2.5 μg / ml. The TcEr polypeptide had the sequence shown in Figure 3 and 25 ng were present in the 50 μl peptides were diluted in 0.05 M carbonate solution (pH 9.6) The plates were incubated for 1 hour at 37 ° C and kept at 4 ° C until used for 1 month. used, the sensitized wells were washed with 0.01M phosphate buffered saline (pH 7.2) containing 0.3% Tween 20 (PBS / T), and positive control, negative control and unknown serum samples were diluted 1:20 in PBS / T containing 0.5% bovine serum albumin and 50 μl each well was added.After 30 minutes of incubation at room temperature, the wells were washed six times with PBS / T. Fifty μl of 1.5% H2O2, 2.5 ml were added. of 0.1M citrate buffer (pH 4.1), Zymed Laboratorie s, San Francisco, CA). After 15 minutes at room temperature, the enzymatic reaction was stopped by adding 100 μl of 10% sodium dodecylsulfate. A 05 values were determined with ELISA reader (Titertek Multiskan, Flow Laboratories, McLean, V.A.). For each test, 5 negative control samples and 2 serum samples from patients with positive Chagas were included. The test results were considered acceptable only when the negative control serum had absorbance values above 0.2 and the positive control serum had absorbances between 0.6 and 0.8 (low positive), or between 1.2 and 1.4 (high positive). was determined for each test by calculating the negative serum mean plus three normal deviations. In an initial experiment, serum from 26 individuals living in an endemic area of Chagas' disease were analyzed for 7. cruzi infections described above. One hundred and seventy-nine positive serum samples and 81 negative serum samples, characterized according to clinical findings and conventional serological tests (indirect immunofluorescent analysis, indirect hemagglutination and ELISA) were analyzed. In this analysis, the TcD peptide was found to be 95% sensitive and the PEP 2 peptide was 91% sensitive. However, only one positive serum sample did not react with any peptide. These results are shown in the following Table 2. Table 2 Reactivity of Serum Samples with Peptides TcD and PEP2 Consequently, the ELISA test that employed a mixture of PEP2 and the TcD peptide had a sensitivity greater than 99%. The specificity of the TcD / PEP2 test was evaluated using sera from individuals living in an endemic area of Chagas disease, who had serology of 7. negative cruzi, as well as serum from patients with other pathologies. In these samples, 2 of 81 analyzes were positive, but no false positive results were found among the 37 serum samples of individuals against pathologies. The other pathologies represented in this study were cutaneous leishmaniasis, visceral leishmaniasis, leprosy, and tuberculosis. All serum samples of cutaneous and visceral leishmaniasis were negative in the analysis of mixed peptides.

In a similar experiment, the reactivity of TcEr, alone and in combination with the TcD peptide, was evaluated and compared with the reactivity of 7. cruzi lysate, the TcD peptide, PEP2 and the TcD / PEP2 mixture. Sixty-nine serum samples obtained from individuals with chronic Chagas disease were analyzed as described above using each of the above antigens. For comparison, similar analyzes were performed using 16 serum samples from individuals with acute visceral leishmaniasis and 33 serum samples from uninfected individuals. The average mean absorbance for the uninfected samples was determined for each of the different antigens and is shown in Table 3, below, along with the normal deviation. Table 3 Absorbances at 405 nm for Human Serum with Peptides of T cruzi and Plaster of Pest Consequently, the mixtures containing the TcD polypeptide and any PEP2 or TcEr were more sensitive and specific in these analyzes than any of the individual peptides. This was due to the fact that these polypeptides exhibit complementary reactivities. As shown in Table 4, below, many of the patient sera that were negative or had low titers of antibodies to TcD reacted relatively strongly with PEP2 and / or TcE. Table 4 Reactivity of Serum Samples with Antigens of 7. cruzi These results demonstrate that combinations of PEP2 and / or TcE significantly increase the sensitivity of the analysis beyond that obtained with TcD alone.

In the third experiment, TcD was mixed with PEP2 a fragment of PEP2. Specifically, the PEP2 fragments containing residues 2 to 13 or residues 2 to 15 were used. In each case, the portions of PEP2 were reactive, either alone or when mixed with TcD, but the PEP2 sequence of 22 amino acids showed reactivity higher. Therefore, mixtures employing the PEP2 sequence of 22 amino acids are more sensitive for 7. cruzi infection than mixtures using the shorter sequences. D. Combination polypeptides The experiments described above were repeated using combination polypeptides. First, serum from 12 patients infected with 7. cruzi was analyzed using D / 2 combination polypeptide, which consisted of the TcD peptide linked to the pEP2 sequence by means of the Gly-Cys-Gly linker. In addition, the serum of 15 negative individuals for 7. cruzi was analyzed with the D / 2 combination polypeptide. In this experiment, the absorbance was measured at 450 nm since the substrate was tetramethylbenzidine (TMB), instead of ABTS. All 12 serum analyzes of individuals infected with 7. cruzi were positive (100%) and none of the sera of individuals negative for 7. cruzi produced a positive result. As a result, the D / 2 polypeptide is highly specific and sensitive for infection with 7. cruzi. In another experiment, a D / E polypeptide combination containing the TcD peptide and the TcEr sequence, bound by the Gly-Cys-Gly linker, was evaluated alone and in combination with the D / 2 peptide. Forty-four samples Serum samples from individuals infected with 7 cruzi were analyzed along with 24 samples from clinically normal individuals in the endemic regions of Brazil and 24 samples from clinically normal individuals in the United States. The results of each analysis performed on the above serum samples are shown in Table 5 Table 5 Reactivity of Serum Samples with Combination Polypeptides Consequently, peptide D / E detected 41 of 44 positive samples (93%) and the mixture of peptides D / E and D / 2 detected all 44 positive samples (100%). None of the peptides produced a positive result in the analysis. of clinically normal serum samples from the United States Because "normal endemic" samples were only clinically normal (ie serodiagnosis analysis was performed), the positive result produced by the mixture of D / E and D / 2 peptides may indicate an undiagnosed infection E- Tripeptide mixture The above analyzes were prepared using a mixture of the TcD peptide TcEr, and PEP2. The 44 samples from individuals infected with 7. cruzi, together with the 48 samples from clinically normal individuals (24 from the United States and 24 from endemic regions), which were described in Section D above, were analyzed using a mixture of three polypeptides separated, each one containing one of the previous epitopes. In this experiment, the 4 positive serum samples for 7. cruzi resulted in absorbances at 450 nm that were greater than three standard deviations above the average mean and none of the normal serum samples from the United States produced a positive result. Two of the negative samples from Brazil's endemic regions produced a positive result but, again, this may be the result of undiagnosed infections. Consequently, the tripeptide mixture detected 100% of the positive serum samples and showed high specificity. From the foregoing, it will be appreciated that, although the specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention.

LIST OF SEQUENCES (I) GENERAL INFORMATION: (i) APPLICANT: Reed Steven G. (ii) TITLE OF THE INVENTION: COMPOUNDS AND METHODS FOR THE DETECTION OF INFECTION BY T. CRUZI (iii NUMBER OF SEQUENCES: 6 (iv ADDRESS OF CORRESPONDENCE : (TO RECIPIENT: SEED and BERRY (B STREET: 6300 Columbia Center, 701 Fifth Avenue (C CITY: Seattle (D STATE: Washington (E COUNTRY: USA (F ZP: 98104-7092 (v READY ON THE COMPUTER : (A TYPE OF MEDIUM: Soft disk (B COMPUTER: IBM compatible PC (C OPERATING SYSTEM: PC-DOS / MS-DOS (D SOFTWARE: Patentln Reléase # 1.0, Version # 1.30 (vi CURRENT REQUEST DATA: (A APPLICATION NUMBER OF TCP: US (B DATE OF SUBMISSION: March 14, 1996 (C CLASSIFICATION: (vi) EMPLOYEE / AGENT INFORMATION: (A NAME: Kadlecek, Ann T. (B REGISTRATION NUMBER: P-29,244 ( C) REFERENCE NUMBER / CASE: 210121.406 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (206) 622-4900 (B) TELEFAX: (206) 682-6031 (C) TELEX: 3723836 SEEDANDBERRY (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 262 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULES: protein (x) ) DESCRIPTION OF SEQUENCES: SEQ ID NO: 1 Glu Gly Thr Arg Glu Wing Arg Met Pro Ser Lys Glu _eu Trp Met Arg 1 5 10 15 Arg Leu Arg He Leu Arg Arg Leu Leu Arg Lys Tyr Arg Glu Glu Lys 20 25 30 Lys He Asp Arg His He Tyr Arg Glu Leu Tyr Val Lys Wing Lys Gly 35 40 45 Asn Val Phe Arg Asn Lys Arg Asn Leu Met Glu His He His Lys Val 50 55 60 Lys Asn Glu Lys Lys L s Giu Arg Gln Leu Wing Glu Glp Leu Wing Wing 65 70 75 80 Lys Arg Leu Lys Asp Glu Gln His Arg His Lys Wing Arg Lys Gln Glu 85 90 95 Leu Arg Lys Arg Glu Lys Asp Arg Glu Arg Wing Arg Arg Glu Asp Wing 100 105 110 Ala Ala Ala Ala Ala Ala Lys Gln Lys Ala Ala Ala Lys Ala Ala Ala 115 120 125 Wing Pro Ser Gly Lys Lys Ser Wing Lys Wing Wing He Wing Pro Wing Lys 130 135 140 Ala Ala Ala Ala Ala Pro Ala Ala Ala Ala Ala Pro Ala Ala Ala Ala 145 150 155 160 Wing Wing Pro Wing Lys Wing-Wing Wing Pro Wing Wing Wing Wing? '. d ICO 17G 1"5 Pro Ala Lys Ala Ala Thr A-a Pro Ala Ala Ala Ala Ala Ala Ala Ala 180 185 185 Lys Thr Wing Wing Pro Wing Lys Wing Wing Pro Wing Wing Lys Wing Wing 195 200 205 Ala Ala Pro Ala Ala Ala Ala Thr Ala Pro Ala Ala Ala Ala Ala Ala 210 215 220 Pro Wing Lys Wing Wing Thr Wing Pro Wing Lys Wing Wing Thr Wing Pro Wing 225 230 235 240 Lys Ala Ala Ala Ala Pro Ala Lys Ala Ala Thr Ala Pro Val Gly Lys 245 250 255 Lys Wing Gly Gly Lys Lys 260 (2) SEQUENCE INFORMATION ID NO: 2 (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 786 base pairs (B) TYPE: nucleic acid (C) THREAD: single (D) TOPOLOGY : linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CTCCGCCGCC TGCTGCGCA.A GTACCGCGAG 3AGAAGAAGA TTGA.CC3CCA CATCTACCGC 1 0 GAGCTGTAC3 TGAAGGCGA.A GGGGAACGTG TTTCGCAACA AGCGTAACCT CATGGAGCAC 180 ATCCACAAGG TGAAGAACGA GAAGAAGAAG GAAAGGCAGC TGGCTGAGCA GCTCGCGGCG 240 AA.GCGCCTGA AGGATGAGC GCACCGTCAC AAGGCCCGCA AGCAGGAGCT GCGTAAGCGC 300 GAGAA.GGACC GCGAGCGTGC GCGTCGCGAA GATGCTGCCG CTGCCGCCGC CGCGAAGCAG 360 AAAGCTGCTG CGAAGAAGGC CGCTGCTCCC TCTGGCAAGA AGTCCGCGAA GGCTGCTATT 420 GCACCTGCGA AGGCCGCTGC TGCACCTGCG AAGGCCGCTG CTGCACCTGC GAAGGCTGCT 480 GCTGCACCTG CGAAGGCCGC TGCTGCACCT GC3AAGGCTG CTGCTGCACC TGCGAAGGCT 540 GCTACTGCAC CTGCGAAG3C TGCTGCTGCA CCTGCCAAGA CCGCTGCTGC ACCTGCGAAG 600 GCTGCTGCAC CTGC3AAG3C CGCTGCTGCA CCTGC3AAGG CCGCTACTGC ACCTGCGAAG 660 (2) SEQUENCE INFORMATION ID NO: 3: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) THREAD: (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEC ID NO: 3: Lys Wing Wing Wing Wing Pro Wing Lys Wing Wing Wing Wing Pro Wing Lys Wing 1 5 10 15 Wing Thr Wing Pro Wing 20 (2) SEQUENCE INFORMATION ID NO: 4 (1) SEQUENCE CHARACTERISTICS: (A ) LENGTH: 22 amino acids (B) TYPE: amino acids (C) THREAD: (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Gly Asp Lys Pro Ser Pro Phe Gly Gln Wing Wing Wing Gly Asp Lys Pro 1 5 10 15 Ser Pro Phe Gly Gln Wing 20 20 (2) SEQUENCE INFORMATION ID NO: 5: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 836 base pairs (B) TYPE: nucleic acid (C) THREAD: simple (D) TOPOLOGY: linear (ix) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 8..628 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GAATTCA GAG GAG CCC AAA CCA GCG GAG CCG AAG TCA GCA GAG CCT AAA 49 Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys 1 5 10 CCA GCG GAG CCG AAA TCG GCA GAG CCC AAA CCA GCG GAG CCG AAA TCG 97 Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser 15 20 25 30 GCA GAG CCC AAA CCA GCG GAG CCG AAA TCA GCG GGG CCT AAA CCA GCG 145 Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Gly Pro Lys Pro Wing 35 40 45 GAG CCG AAG TCA GCG GAG CCT AAA CCA GCG GA.G CCG AAA TCA GCA GAG 193 Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu 50 55 60 CCC AAA CCA GCG GA.G CCG AAA TCG GCA GAG CCC AAA CCA GCG GAG CCG 241 Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu Pro 65 70 75 AAG TCA. GCA GAG CCC AAA CCA GCG G.-G TCG A.-G TCA GCA GAG CCT AA-25? Lys Ser Ala Glu Pro Lys Pro Ala G'.u Ser Lys Ser Ala Glu Fro Lys 80 85 90 CCA GCG GAG CCG AAA TCA GCA GA.G CCC AAA CCA GCG GAG TCG AAG TCA 337 Pro Wing Glu Pro Lys S r Wing Glu Pro Lys Pro Wing Glu Ser Lys Ser 95 100 105 110 GCA GAG CCC AAA CCA Í3CG GAG CCG AAG TCA GCA. GAG CCC AAA CCA GCG 385 Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing 115 120 125 GAG CCG AAG TCA GCA GAG CCC AAA CCA GCG GAG CCG AAA TCA. GCG GAG 433 Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu 130 • 135 140 CCC AAA 6CA GCG GAG CCG AAA TC.A GCA GAG CCC AAA CC.A GCG GAG TCG 481 Pro Lys Pro Wing Glu Prc Lys Se Wing Glu Pro Lys Pro Wing Glu Ser 145 150 155 AAA TCA GCG GGG CCT AAA CC.A GCG GAG CCG AAG TCA GCG GAG CCA AAA 529 Lys Ser Wing Gly Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys 160 165 170 CCA GCG GAG CCG AAA TCA GCG GAG CCA AAA CCA 0CG GAG CCG AAA TCG 577 Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser 175 180 185 190 GCA GAG CCC AAA CCA GCG GAG CCG AAG TCA GCA GAG CCA AAA CCA GCG 625 Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing 195 200 205 GA.G CCGAATTC 636 Glu (2) SEQUENCE INFORMATION ID NO: 6: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 207 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 : Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing 1 5? O 15 Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu 20 25 30 Protys Pro Wing Glu Pro Lys Ser Wing Gly Pro Lys Pro Wing Glu Pro 35 40 45 Lys Ser Wing Glu Pro Lys Pro Wing Glu Pro Lys Se- Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser 65 70 75 80 Wing Glu Pro Lys Pro Wing Glu Ser Lys Ser Wing Glu Pro Lys Pro Wing 85 90 95 Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu Ser Lys Ser Wing Glu 100 105 110 Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu Pro 115 120 125 Lys Ser Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys 130 135 140 Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu Ser Lys Ser 145 150 155 160 Wing Gly Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing 165 170 175 Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu 180 185 190 Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu 195 200 205

Claims (64)

1. CLAIMS 1. A method for detecting T. cruzi infection in a biological sample, comprising: (a) contacting a biological sample with a polypeptide in at least 7 consecutive residues of the portion of SEQ ID NO: 1 between lysine at residue 137 and alanine at residue 247, or an antigenic variant thereof that differs only in substitutions or conservative modifications, as long as the first polypeptide contains no more than five consecutive residues of the portion of SEQ ID NO: 1 enters amino acid 1 and? amino acid 136; and (b) detecting in the biological sample the presence of antibodies that bind to the polypeptide, thereby detecting T cruzi infection in the biological sample. The method of claim 1, wherein the first polypeptide comprises the amino acid sequence Lys Ala Ala lie Ala Pro Ala Lys Ala Ala Ala Ala Pro Ala Lys Ala Ala Thr Ala Pro Ala or an antigenic variant thereof which differs only in substitutions or conservative modifications. The method of claim 1, further comprising contacting the sample with a second polypeptide comprising an amino acid sequence selected from the group consisting of Ala Glu Pro Lys Ser Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser, or a variant antigenic of the same that differs only in substitutions or conservative modifications, and Ala Gly Pro Lys Pro Ala glu Pro Lys Ser Ala Glu Pro Lys Pro, or an antigenic variant thereof that differs only in substitutions or conservative modifications. 4. The method of any of claims 1 or 3, further comprising contacting the sample with a third polypeptide comprising the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys,, or an antigenic variant of the same ones that differ only in substitutions or conservative modifications. The method of claim 4, wherein the third polypeptide comprises the amino acid sequence Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala. The method of claim 1, wherein the first polypeptide further comprises an amino acid sequence selected from the group consisting of Ala Gly Pro Lys Ser Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser, or an antigenic variant thereof it differs only in substitutions or conservative modifications and Ala Gly Pro Lys Pro Ala Glu Pro Lys Ser Ala Gl or Pro Lys Pro, or an antigenic variant thereof that differs only in substitutions or conservative modifications. The method of any of claims 1 or 6 wherein the first polypeptide further comprises the amino acid sequence Pro Ser Pro Phe gly Gln Ala Ala Ala Ala Gly Asp Lys, or an antigenic variant thereof which differs only in substitutions or conservative modifications. The method of claim 7, wherein the first polypeptide further comprises the amino acid sequence Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala, or an antigenic variant thereof which differs only in substitutions or conservative modifications. The method of claim 1, wherein the biological sample is selected from the group consisting of blood, serum, plasma, saliva, cerebrospinal fluid and urine. The method of claim 1, wherein the first polypeptide is attached to a solid support. The method of claim 10, wherein the detection step comprises: (a) removing the unbound sample from the solid support, (b) adding a detection reagent to the solid support; and (c) determining the level of detection reagent bound to the solid support, relative to a predetermined reduction value, by detecting the same T. cruzi infection in the biological sample. The method of claim 11, wherein the detection reagent comprises a reporter group conjugated to a binding agent. 13. A polypeptide comprising at least 7 consecutive residues of the portion of SEQ ID. NO: 1 between lysine at residue 137 and alanine at residue 247, or an antigenic variant thereof that differs only in substitutions or conservative modifications, as long as the polypeptide contains no more than five consecutive residues of the portion of SEQ ID. NO: 1 between amino acid 1 and amino acid 136. 14. The polypeptide of claim 13 further comprising the amino acid sequence Lys Ala Ala lie Ala Pro Ala Lys Ala Ala Ala Ala Ala Pro Ala Lys Ala Ala Thr Ala Pro Ala, or a antigenic variant of the same that differs only in substitutions or conservative modifications. 15. A polypeptide comprising at least 7 consecutive residues of the portion of SEQ ID NO: 1 between the lysine at residue 137 and the alanine in residue 247 or an antigenic variant thereof which differs only in substitutions or conservative modifications , wherein the polypeptide further comprises an amino acid sequence selected from the group consisting of Ala Glu Pro Lys Ser Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser, or an antigenic variant thereof that differs only in conservative substitutions or modifications and the amino acid sequence Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser Ala Glu Pro Lys Pro or a ntigenic variant thereof that differs only in substitutions or conservative modifications. 16. A polypeptide comprising at least 7 consecutive residues of the portion of SEQ ID NO: 1, between the lysine at residue 137 and the alanine in residue 247. or an antigenic variant thereof which differs only in substitutions or conservative modifications wherein the polypeptide further comprises the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys or an antigenic variant thereof which differs only in substitutions or conservative modifications. 17. The polypeptide of claim 16, further comprising the amino acid sequence Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala or an antigenic variant thereof which differs only in substitutions or conservative modifications. The polypeptide of claim 15, further comprising the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys, or an antigenic variant thereof which differs only in substitutions or conservative modifications. 19. A diagnostic kit for detecting T. cruzi infection in a biological sample, comprising: (a) a first polypeptide comprising at least 7 consecutive residues of the portion of SEQ ID NO: 1 between the lysine at residue 137 and the alanine at residue 247, or an antigenic variant thereof that differs only in substitutions or conservative modifications, as long as the first polypeptide contains no more than five consecutive residues of the portion SEQ ID NO: 1 between amino acid 1 and amino acid 136: and (b) a detection reagent. 20. The kit of claim 19, wherein the first polypeptide comprises the amino acid sequence Lys Ala Ala lie Ala Pro Ala Lys Ala Ala A Ala Pro A Lys Ala A Thr Ala Pro A la, or antigenic variant thereof It differs only in substitutions or conservative modifications. twenty-one . The kit of claim 19, further comprising a second polypeptide comprising an amino acid sequence selected from the group consisting of Ala Gly Pro Lys Ser Ala Gly Pro Lys Pro Ala Glu Pro Lys Ser, or an antigenic variant thereof which differs only in substitutions or conservative modifications, and Ala Gly Pro Lys Pro Ala Glu Pro Lys Ser Ala Glu Pro Lys Pro, or an antigenic variant thereof that differs only in substitutions or conservative modifications 22. The kit of claim 21, further comprising a third polypeptide comprising the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys or an antigenic variant thereof that differs only in substitutions or conservative modifications. 23. The kit of claim 22, wherein the third polypeptide comprises the amino acid sequence Gly Ala Lys Pro Be Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala or an antigenic variant thereof that differs only in substitutions or conservative modifications. The kit of claim 1, wherein the first polypeptide further comprises an amino acid sequence selected from the group consisting of Ala Gly Pro Lys Ser Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser or an antigenic variant thereof differs only in conservative substitutions or modifications and Ala Gly Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro or an antigenic variant thereof that differs only in substitutions or conservative modifications. 25. The kit of any of claims 19 or 24 wherein the first polypeptide comprises the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys or an antigenic variant thereof which differs only in substitutions or conservative modifications. 26. The kit of claim 19, wherein the first polypeptide is attached to a solid support. 27. A method for detecting T. cruzi infection in a biological sample, comprising: (a) contacting a biological sample with a polypeptide comprising the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys or a antigenic variant thereof that differs only in conservative substitutions or modifications, and further comprising an amino acid sequence selected from the group consisting of an amino acid sequence selected from the group consisting of Ala glu Pro Lys Ser Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser, or an antigenic variant thereof that differs only in substitutions or conservative modifications and Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser Ala Glu Pro Lys Pro, or an antigenic variant thereof that differs only in substitutions or conservative modifications; and (b) detecting in the biological sample the presence of antibodies that bind to the polypeptides, thereby detecting infection by t. cruzi in the biological sample. The method of claim 27, wherein the polypeptide further comprises the amino acid sequence Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala or an antigenic variant thereof which differs only in substitutions or conservative modifications. 29. The method of claim 27 wherein the polypeptide is attached to a solid support. The method of claim 29, wherein the detection step comprises: (a) removing the unbound sample from the solid support: (b) adding a detection reagent to the solid support; and (c) determining the level of detection reagent bound to the solid support relative to a predetermined reduction value, by detecting the same T. cruzi infection in the biological sample. 31. The method of claim 30. wherein the detection reagent comprises a reporter group conjugated to a binding agent. 32. The method of claim 27, wherein the biological sample is selected from the group consisting of blood, serum, plasma, saliva, cerebrospinal fluid and urine. 33. A polypeptide comprising: (a) the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys or an antigenic variant thereof which differs only in substitutions or conservative modifications; and (b) an amino acid sequence selected from the group consisting of Ala Glu Pro Lys Ser Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser, or an antigenic variant thereof that differs only in conservative substitutions or modifications and Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser Ala Glu Pro Lys Pro or an antigenic variant thereof that differs only in substitutions or conservative modifications. 34. The polypeptide of claim 33, further comprising the amino acid sequence Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala, or an antigenic variant thereof which differs only in substitutions or conservative modifications. 35. A diagnostic kit for detecting T. cruzi infection in a biological sample comprising: (a) the recombinant polypeptide of claim 33; and (b) a detection reagent. 36. The kit of claim 35 wherein the recombinant polypeptide is attached to a solid support. 37. A method for screening infection by Leishmania or T. cruzi in a biological sample, comprising: (a) contacting a biological sample with a polypeptide comprising the portion of SEQ ID NO: 1 between the arginine at residue 1 and the alanine at position 143, or an antigenic variant thereof which differs only in substitutions or conservative modifications; and (b) detecting in the biological sample the presence of antibodies that bind to the polypeptide thereby detecting Leishmania or T. cruzi infection in the biological sample. 38. The method of claim 37, wherein the polypeptide is attached to a solid support. 39. The method of claim 38, wherein the detection step comprises; (a) removing an unbound sample from the solid support; (b) adding a detection reagent to the solid support; and (c) determining the level of detection reagent bound to the solid support, relative to a predetermined reduction value, thereby screening for infection by Leishmania and T. cruzi in the biological sample. 40. The method of claim 39, wherein the detection reagent comprises a reporter group conjugated to a binding agent. 41. The method of claim 39, wherein the biological sample is selected from the group consisting of blood, serum, plasma, saliva, cerebrospinal fluid and urine. 42. A diagnostic kit for detecting Leishmania or T. cruzi infection comprising: (a) a polypeptide comprising amino acids 1 to 143 of SEQ ID NO: 1, or an antigenic variant thereof that differs only in substitutions or conservative modifications; and (b) a detection reagent. 43. The kit of claim 42, wherein the detection reagent comprises a reporter group conjugated to a binding agent. 44. An isolated DNA sequence encoding the polypeptide of any of claims 13-18, 33 or 34. 45. A recombinant expression vector comprising the DNA sequence of claim 44. 46. A host cell transformed with the vector of expression of claim 45. 47. The host cell of claim 46, wherein the host cell is selected from the group consisting of E. coli, yeast, insect cell lines and mammalian cell lines. 48. A pharmaceutical composition comprising the recombinant polypeptide of any of claims 13-18, 33 or 34 and a physiologically acceptable carrier. 49. A pharmaceutical composition comprising: (a) a first polypeptide comprising at least 7 consecutive residues of the portion of SEQ ID NO: 1 between the lysine at residue 137 and the alanine in residue 14, or an antigenic variant of the which differs only in substitutions or conservative modifications; (b) a second polypeptide comprising an amino acid sequence selected from the group consisting of Ala Glu Pro Lys Be Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser, or an antigenic variant thereof that differs only in substitutions or conservative modifications and Ala Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro, or an antigenic variant of the which differs only in substitutions or conservative modifications; Y (c) a physiologically acceptable vehicle. 50. The pharmaceutical composition of claim 49, further comprising a third polypeptide comprising the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys or an antigenic variant thereof which differs only in substitutions or conservative modifications. 51. The pharmaceutical composition of claim 50, wherein the third polypeptide further comprises the amino acid sequence Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala or an antigenic variant thereof which differs only in substitutions or conservative modifications. 52. A pharmaceutical composition comprising: (a) a first polypeptide comprising at least 7 consecutive residues of the portion of SEQ ID NO: 1, between the lysine at residue 137 and the alanine in residue 143, or a variant antigenic of the same that differs only in substitutions or conservative modifications; (b) a second polypeptide comprising the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys, or an antigenic variant thereof which differs only in substitutions or conservative modifications; and (c) a physiologically acceptable vehicle. 53. The pharmaceutical composition of claim 52, wherein the second polypeptide further comprises the amino acid sequence Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala or an antigenic variant thereof which differs only in substitutions or conservative modifications. 54. A pharmaceutical composition comprising: (a) a first polypeptide comprising the amino acid sequence Pro Ser Pro Phe Gly Gln Ala Ala Ala gly Asp Lys, or an antigenic variant thereof which differs only in substitutions or conservative modifications; (b) a second polypeptide comprising an amino acid sequence selected from the group consisting of Ala Glu Pro Lys Ser Ala Glu Pro Lys Pro Ala Glu Pro Lys Ser, or an antigenic variant thereof which differs only in substitutions or conservative modifications, and Ala Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro or an antigenic variant thereof that differs only in substitutions or conservative modifications, and (c) a physiologically acceptable vehicle. 55. The pharmaceutical composition of claim 54, wherein the first polypeptide further comprises the amino acid sequence Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala or an antigenic variant thereof which differs only in substitutions or conservative modifications. 56. A vaccine for stimulating the production of antibodies that bind to T. cruzi comprising the recombinant polypeptide of any of claims 13-18, 33 or 34 and an auxiliary. 57. A vaccine for stimulating the production of antibodies that bind to T. cruzi comprising a pharmaceutical composition according to any of claims 49-55 and an auxiliary. 58. A method for detecting T. cruzi infection in a biological sample, comprising: (a) contacting a biological sample with a first polypeptide consisting essentially of at least 7 consecutive residues of the portion of SEQ ID NO: 1 between the lysine at residue 137 and the alanine at residue 247 or an antigenic variant thereof which differ only in substitutions or conservative modifications; and (b) detecting in the biological sample the presence of antibodies that bind to the polypeptide, thereby detecting infection by t. cruzi in the biological sample. 59. A method to detect the presence of infection by t. cruzi in a biological sample, comprising: (a) contacting a biological sample with a monoclonal antibody that binds to a polypeptide consisting essentially of at least 7 consecutive residues of the portion of SEQ ID NO: 1 between the lysine in residue 137 and alanine in residue 247 or an antigenic variant thereof that differs only in substitutions or conservative modifications; and (b) detect in the biological sample the presence of T. cruzi parasites that bind to the monoclonal antibody. 60. a method for detecting the presence of T. cruzi infection in a biological sample, comprising: (a) contacting a biological sample in a monoclonal antibody that binds to a polypeptide comprising an amino acid sequence selected from the group consisting of of Ala Glu Pro Lys Ser Wing Glu Pro Lys Pro Wing Glu Pro Lys Ser, or an antigenic variant thereof that differs only in substitutions or conservative modifications and Ala Glu Pro Lys Pro Wing Glu Pro Lys Ser Wing Glu Pro Lys Pro or a antigenic variant thereof that differs only in substitutions or conservative modifications; Y (b) detect in the biological sample the presence of T. cruzi parasites that bind to the monoclonal antibody. 61. A method for detecting the presence of T. cruzi infection in a biological sample comprising: (a) contacting a biological sample with a monoclonal antibody that binds to a polypeptide comprising the amino acid Gly Asp Lys Pro Ser Pro Phe Gly Gln Ala Ala Ala Gly Asp Lys Pro Ser Pro Phe Gly Glu Ala; and (b) detect in the biological sample the presence of T. cruzi parasites that bind to the monoclonal antibody. 62. The method of any of claims 59-61 wherein the monoclonal antibody binds to a solid support. 63. The method of claim 62, wherein the detection step comprises: (a) removing the unbound sample from the solid support; (b) adding a solid support detection reagent; and (c) determining the level of detection reagent bound to the solid support, relative to a predetermined reduction value, thereby detecting T. cruzi infection in the biological sample. 64. The method of claim 63, wherein the detection reagent comprises a reporter group coupled to an antibody.