Patent Publication Number: US-2015072354-A1

Title: Compounds and methods for diagnosis and treatment of leishmaniasis

Description:
PRIORITY CLAIM 
     This is a continuation of U.S. application Ser. No. 11/733,440 filed on Apr. 10, 2007 (pending), which application claims the benefit of priority U.S. Provisional Application No. 60/791,226 filed on Apr. 10, 2006, and Provisional Application No. 60/744,798 filed on Apr. 13, 2006, all of which applications are incorporated herein by reference in their entireties for all purposes. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates generally to the serodiagnosis of  Leishmania  infection. The invention is more particularly directed to the use of one or more  Leishmania  polypeptides in methods and diagnostic kits to screen organisms and blood supplies for  Leishmania,  and to identify those individuals that are likely to progress to acute visceral leishmaniasis. The invention is also directed to vaccines and pharmaceutical compositions for treating and immunizing an organism against leishmaniasis. 
     BACKGROUND OF THE INVENTION 
       Leishmania  organisms are intracellular protozoan parasites of macrophages that cause a wide range of clinical diseases in humans and domestic animals, primarily dogs. In some infections, the parasite may lie dormant for many years. In other cases, the host may develop one of a variety of forms of leishmaniasis. For example, the disease may be asymptomatic or may be manifested as sub-clinical visceral leishmaniasis, which is characterized by mild symptoms of malaise, diarrhea and intermittent hepatomegaly. Patients with sub-clinical or asymptomatic disease usually have low antibody titers, making the disease difficult to detect with standard techniques. Alternatively, leishmaniasis may be manifested as a cutaneous disease, which is a severe medical problem but is generally self-limiting, or as a highly destructive mucosal disease, which is not self-limiting. Finally, and most seriously, the disease may be manifested as an acute visceral infection involving the spleen, liver and lymph nodes, which, untreated, is generally a fatal disease. Symptoms of acute visceral leishmaniasis include hepatosplenomegaly, fever, leukopenia, anemia and hypergammaglobulinemia. 
     Leishmaniasis is a serious problem in much of the world, including Brazil, China, East Africa, India and areas of the Middle East. The disease is also endemic in the Mediterranean region, including southern France, Italy, Greece, Spain, Portugal and North Africa. The number of cases of leishmaniasis has increased dramatically in the last 20 years, and millions of cases of this disease now exist worldwide. About 2 million new cases are diagnosed each year, 25% of which are visceral leishmaniasis. There are, however, no vaccines or effective treatments currently available. 
     Diagnosis of Visceral leishmaniasis can not always be made only on the basis of clinical symptoms because visceral leishmaniasis shares its clinical features with other diseases such as malaria, typhoid fever and tuberculosis occurring commonly in the same endemic areas. Thus, the diagnosis of visceral leishmaniasis largely relies on parasitological or serological methods. The former is microscopic detection of amastigotes in aspirates of spleen and bone marrow or detection of promastigotes through cultivation of the aspirates. Unfortunately, this method requires the biopsy of bone marrow, liver, spleen, or lymph nodes may be required, which may lead to secondary infection or further disfigurement of the patient. Additionally, along with being an invasive diagnosis, it takes a long period of time to diagnose and results are commonly non-conclusive. Therefore, this method is invasive, time-consuming, and not sufficiently sensitive, thereby rendering it inefficient. 
     Less invasive and time consuming laboratory procedures do exist, however, these procedures suffer from either lack of sensitivity or cumbersome implementation. For example the Liquid Direct Agglutination Test (LQ DAT: Ahfad University, Khartoum and IPB, Addis Abbeba) and the Freeze Dried DAT (FD DAT: Meredith et al. 1995) have good sensitivity, but require multiple pipetting steps and incubation, which makes implementation of this test difficult in developing countries. Similarly, the Latex Antigen Agglutination Test in Urine (KATEX®: Kalon Biological Ltd-UK) has good sensitivity and specificity, but requires a cumbersome urine boiling step and further suffers from low reproducibility. Finally, the rK39 and rK26 dipstick tests (Inbios®, Seattle, Wash.) can rapidly give results within a matter of minutes, but these tests lack sensitivity in certain geographic regions such as Sudan, Ethiopia and Kenya. Because the dipstick tests can be easily, quickly, and affordably implemented, yet suffer from lack of antigen sensitivity, discovery of new antigens is necessary for more accurate diagnosis of leishmaniasis. 
     Among defined leishmanial antigens reported previously, rK39 appears to be the best antigen for serodiagnosis of visceral leishmaniasis in terms of both sensitivity and specificity. rK39 is sensitive and reliable even on a strip format, which is feasible for field use, and the rK39 strip test has high sensitivity in India, Nepal and Brazil. In Sudan, Ethiopia and Kenya, however, the sensitivity of the strip test falls to 67% and the negative responses on the strip test appear to correlate with lower reactivity by ELISA. Thus, new diagnostic antigens are needed to complement rK39 to contribute to the development of a more accurate diagnosis of leishmaniasis. The present invention fulfills these needs and many other related needs. 
     SUMMARY OF THE INVENTION 
     Briefly stated, this invention relates to compounds and methods for detecting and treating leishmaniasis in individuals and in blood supplies. 
     More specifically, compounds and methods are provided for diagnosing, preventing, treating and detecting leishmaniasis infection and stimulating immune responses in patients are disclosed. The compounds disclosed include polypeptides and fusion proteins that contain at least one immunogenic portion of one or more  Leishmania  antigens, or a variant thereof. Additionally, methods of screening a screening library for tandem repeat proteins that have immunogenic properties are disclosed. Vaccines and pharmaceutical compositions comprising polynucleotides, polypeptides, fusion proteins and variants thereof that may be used for the prevention and therapy of leishmaniasis, as well as for the detection of Leishmaniasis infection are described. 
     In one embodiment a method for detecting  Leishmania  infection is disclosed that comprises the steps of first contacting a biological sample with polypeptides comprising at least one tandem repeat unit, wherein the tandem repeat unit comprises an amino acid sequence having homology to, an amino acid sequence selected from the group consisting of SEQ ID NO: 1-59; and second detecting the presence of antibodies in the biological sample to detect Leishmaniasis infection. Related embodiments include similar methods of using tandem repeat proteins and fusion proteins that comprise one or more of the amino acid sequences of SEQ ID NO: 1-59. In still further embodiments, diagnostic kits for detecting  Leishmania  infection are disclosed that comprise the polypeptides described above. 
     In still further embodiments of the present invention, DNA sequences encoding the polypeptides described above are disclosed in addition to expression vectors and host cells comprising the same. 
     Additional embodiments are disclosed wherein the polypeptides disclosed are used in pharmacological compositions and related methods of using these pharmacological compositions to treat, detect, and immunizing against  Leishmania  infection are disclosed. 
     In other embodiments, methods for screening for immunogenic polypeptides are disclosed, which comprise the steps of constructing a screening library, screening the library with a biological sample and analyzing identified sequences to select for tandem repeat genes. Still further methods are disclosed for embodiments of the present invention where one or more polypeptide or polynucleotide sequences are selected and then screened for tandem repeat domains so as to screen for immunogenic polynucleotides or immunogenic polypeptides. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Preferred and alternate embodiments of the present invention are described in detail below with reference to the following drawings: 
         FIG. 1  shows a representative PCR analysis of tandem repeat genes. PCR reactions performed with primer sets specific for the tandem repeat regions of LinJ16.1750, LinJ22.1590 and LinJ33.2870 or for a  T. cruzi  gene using total DNA of  L. infantum  (Li),  L. donovani  (Ld),  L. major  (Lm),  L. amazonensis  (La) and  T. cruzi  (Tc) as templates. Sizes are shown in base pairs. 
         FIG. 2  illustrates an exemplary expression and purification of  L. infantum  recombinant proteins. Shown are coomassie blue-stained SDS/4-20% polyacrylamide gradient gels of uninduced  E. coli  lysates (lane 1), induced lysates (lane 2) and purified proteins (lane 3). Sizes are shown in kDa. 
         FIG. 3  presents a demonstrative enzyme-linked immunosorbent assay evaluation of patient seroreactivity to  L. infantum  recombinant proteins. Patient sera from patients of visceral leishmaniasis (VL, n=10), cutaneous leishmaniasis (CL, n=10), tuberculosis (TB, n=10), malaria (n=6), or sera from healthy controls in United States (n=10) were used. Mean and SEM of OD values in each group are shown. 
         FIG. 4  shows representative reactivity of human visceral leishmaniasis patient sera to tandem repeat proteins. Sera from visceral leishmaniasis patients (n=35) and healthy controls (n=20) were tested for reactivity to rLinJ16.1750r2 or rK39 by ELISA. Mean in each group is shown as a solid line. Dotted lines represent cutoff values calculated as mean+3SD of OD values from healthy controls. 
         FIG. 5  illustrates exemplary recognition of recombinant proteins by sera from eight visceral leishmaniasis patients, which showed low reactivity to rK39 (OD&lt;1.0, circled in A). 
         FIG. 6  shows demonstrative antibody responses of visceral leishmaniasis patient sera to tandem repeat proteins. Sera from visceral leishmaniasis patients (closed circles: n=16) and healthy controls (open circles: n=8) were tested for their reactivity to tandem repeat proteins by ELISA and OD values of each individuals are shown. Bars represent means of each group. 
     
    
    
     DETAILED DESCRIPTION 
     As stated above the present invention relates to compositions and methods for detecting and protecting against  Leishmania  infection in a biological sample or organism, in addition to methods for screening and identifying compounds that have efficacy in detecting and protecting against  Leishmania  infection in a biological sample or organism. 
     In one embodiment, the invention provides a method compounds for detecting  Leishmania  infection in a biological sample or organism, comprising: (a) contacting a biological sample with one or more polypeptides at least one of which comprises one or more tandem repeat units or a variant thereof that only differs in conservative substitutions or modifications, which tandem repeat unit in certain embodiments comprises an amino acid sequence having at least 8 consecutive amino acids of, and at least 70% sequence homology to, an amino acid sequence selected from SEQ ID NOS:1-59, under conditions and for a time sufficient for binding to the polypeptide(s) by an antibody in the sample to take place; and (b) detecting in the biological sample the presence of one or a plurality of antibodies that specifically bind to the polypeptide, thereby detecting  Leishmania  infection in the biological sample. 
     In another embodiment, the invention provides methods and compositions for detecting  Leishmania  infection in a biological sample or organism, comprising: (a) contacting a biological sample with a composition comprising a polypeptide that comprises a tandem repeat unit, or a variant thereof that only differs in conservative substitutions or modifications under conditions and for a time sufficient for binding to the polypeptide(s) by an antibody in the sample to take place; and (b) detecting in the biological sample the presence of antibodies that bind to the polypeptide, thereby detecting  Leishmania  infection in the biological sample. In these and other related embodiments the composition may include a single tandem repeat as provided herein, or may include two or more tandem repeat units which may be the same or different. This composition may thus comprise one or more species of tandem repeat unit, and/or may also include fusion proteins comprising one or more species of tandem repeat unit. 
     In a still further embodiment, the invention provides methods for screening and selecting tandem repeat proteins that have efficacy in detecting  Leishmania  infection in a biological sample or organism comprising: (a) constructing a  Leishmania  screening library; (b) screening the  Leishmania  screening library; and (c) analyzing the genes identified from the screening the  Leishmania  screening library to select genes that are tandem repeat genes. 
     In yet another embodiment, the invention provides systems and methods for treating and detecting leishmaniasis in patients with clinical or sub-clinical leishmaniasis infection. 
     Polypeptides that are contemplated according to certain embodiments of the present invention include, but are not limited to, polypeptides comprising immunogenic portions of  Leishmania  antigens comprising the sequences recited in SEQ ID NO:1-59. As used herein, the term “polypeptide” encompasses amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are covalently linked as linear polymers by peptide bonds. Thus, a polypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences. The additional sequences may be naturally occurring sequences such as sequences derived from the native  Leishmania  antigen, or may be heterologous (e.g., derived from other sources including exogenous naturally occurring sequences and/or artificial sequences), and such sequences may (but need not) be immunogenic. An antigen “having” a particular recited sequence is an antigen that comprises a recited sequence, e.g., that contains, within its full length sequence, the recited sequence. The native antigen may, or may not, contain one or more additional amino acid sequences. A material, molecule, preparation or the like which is “isolated” refers to its having been removed from the environment or source in which it naturally occurs. For example, a polynucleotide sequence which is part of a gene present on a chromosome in a subject or biological source such as an intact, living animal is not isolated, while DNA extracted from a biological sample that has been obtained from such a subject or biological source would be considered isolated. In like fashion, “isolating” may refer to steps taken in the processes or methods for removing such a material from the natural environment in which it occurs. 
     As used herein, the term “tandem repeat” refers to a region of a polynucleotide sequence (e.g., a sequence of DNA, RNA, recombinantly engineered or synthetic oligonucleotides including linear polymers of non-naturally occurring nucleotides or nucleotide analogs or the like, including nucleotide mimetics) or to a region of a polypeptide or protein comprising a sequence, respectively, of about 6 to 1200 nucleotides or 2 to 400 amino acids, that is repeated in tandem such that the sequence occurs at least two times. As used herein the term “tandem repeat unit” refers to a single unit of the sequence that is repeated in tandem. Additionally, the term “tandem repeat” also encompasses a region of DNA wherein more than a single 2- to 400-amino acid or 6- to 1200-nucleotide tandem repeat unit is repeated in tandem or with intervening bases or amino acids, provided that at least one of the sequences is repeated at least two times in tandem. Moreover, the term “tandem repeat” also encompasses regions of DNA or a protein wherein the tandem repeat units are not identical. Where two or more sequences are at least 70% homologous to each other or are reasonable variants of each other, these sequences will be considered tandem repeat units for the purpose of comprising and constituting a tandem repeat. 
     Also, where a sequence is at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more amino acids of a tandem repeat unit, this sequence will be considered a tandem repeat unit for the purpose of comprising and constituting a tandem repeat. 
     Also, where a sequence is at least about 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51 or any intervening integer of nucleotides, the sequence will be considered a tandem repeat unit for the purpose of comprising and constituting a tandem repeat. 
     Additionally, the term “tandem repeat” also encompasses tandem repeats where one or more tandem repeat unit of a tandem repeat is the reverse sequence of the other tandem repeat units. Reverse tandem repeat units and non-reverse tandem repeats can be configured in any way and with or without intervening nucleotide bases or amino acids. Configurations of reverse and non-reverse sequences include, but are not limited to, those where a non-reverse sequence is followed by reverse sequence; where a reverse sequence is followed by non-reverse sequence; and where a reverse sequence is followed by a reverse sequence. In the case of double-stranded polynucleotides having tandem repeats two or more such repeats may be present on the same strand or may occur on opposite strands. 
     In certain preferred embodiments a tandem repeat may comprise an immunogenic portion of a  Leishmania  antigen. An immunogenic portion of a  Leishmania  antigen is a portion that is capable of eliciting an immune response (i.e., cellular and/or humoral) in a presently or previously  Leishmania -infected patient (such as a human or a dog) and/or in cultures of lymph node cells or peripheral blood mononuclear cells (PBMC) isolated from presently or previously  Leishmania -infected individuals. Those skilled in the art will be familiar with any of a wide variety of methodologies and criteria for determining whether an immune response has been elicited. (See, e.g., Current Protocols in Immunology, John Wiley &amp; Sons Publishers, NY 2000, Chapter 2, Units 2.1-2.3) The cells in which a response is elicited may comprise a mixture of cell types or may contain isolated component cells (including, but not limited to, T-cells, NK cells, macrophages, monocytes and/or B cells). In particular, immunogenic portions are capable of inducing T-cell proliferation and/or a dominantly Th1-type cytokine response (e.g., IL-2, IFN-.gamma., and/or TNF-.alpha. production by T-cells and/or NK cells; and/or IL-12 production by monocytes, macrophages and/or B cells). Immunogenic portions of the antigens described herein may generally be identified using techniques known to those of ordinary skill in the art, including the representative methods provided herein. 
     The compositions and methods of the present invention also encompass variants of the above polypeptides. A polypeptide “variant,” or a polypeptide that is “homologous” to another protein as used herein, is a polypeptide that differs from a native (e.g., naturally occurring) protein in one or more substitutions, deletions, additions and/or insertions, such that the immunogenicity of the polypeptide is not substantially diminished. For instance, the ability of a variant to react with an antigen-specific antibody, antiserum or T cell may be enhanced or unchanged, relative to the native protein, or may be diminished by less than 50%, and preferably less than 20%, relative to the native protein. Such variants may generally be identified by modifying one of the herein described polypeptide sequences and evaluating the reactivity of the modified polypeptide with antigen-specific antibodies or antisera as described herein. Preferred variants include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed. Other preferred variants include variants in which a small portion (e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removed from the N- and/or C-terminal of the mature protein. 
     Polypeptide variants encompassed by the present invention include those exhibiting at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity (determined as described below) to the polypeptides disclosed herein. 
     Preferably, a variant contains conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide. 
     The polypeptides of the present invention can be prepared in any suitable manner known in the art. Such polypeptides include naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Similarly certain embodiments disclosed herein contemplate polynucleotides comprised of the naturally occurring polynucleotides having sugar—(e.g., ribose or deoxyribose) phosphate backbones in 5′-to-3′ linkage, but the invention is not so limited and also contemplates polynucleotides comprised of any of a number of natural and/or artificial polynucleotide analogs and/or mimetics, for example those designed to resist degradation or having other desirably physicochemical properties such as synthetic polynucleotides having a phosphorothioate backbone, or the like. 
     Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a protein or a portion thereof) or may comprise a variant, or a biological or antigenic functional equivalent of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, as further described below, preferably such that the immunogenicity of the encoded polypeptide is not diminished, relative to a native tumor protein. The effect on the immunogenicity of the encoded polypeptide may generally be assessed as described herein. The term “variants” also encompasses homologous genes of xenogenic origin. 
     When comparing polynucleotide or polypeptide sequences, two sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. 
     Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730. 
     Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection. 
     One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always &gt;0) and N (penalty score for mismatching residues; always &lt;0). For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=-4 and a comparison of both strands. 
     Preferably, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 160 to yield the percentage of sequence identity. 
     Therefore, the present invention encompasses polynucleotide and polypeptide sequences having substantial identity to the sequences disclosed herein, for example those comprising at least 50% sequence identity, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a polynucleotide or polypeptide sequence of this invention using the methods described herein, (e.g., BLAST analysis using standard parameters, as described below). One skilled in this art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. 
     In additional embodiments, the present invention provides isolated polynucleotides and polypeptides comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences disclosed herein. For example, polynucleotides are provided by this invention that comprise at least about 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths there between. It will be readily understood that “intermediate lengths”, in this context, means any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like. 
     The polynucleotides of the present invention, or fragments thereof, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, illustrative DNA segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful in many implementations of this invention. 
     In other embodiments, the present invention is directed to polynucleotides that are capable of hybridizing under moderately stringent conditions to a polynucleotide sequence provided herein, or a fragment thereof, or a complementary sequence thereof. Hybridization techniques are well known in the art of molecular biology. For purposes of illustration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50.degree. C.−65.degree. C., 5×SSC, overnight; followed by washing twice at 65.degree. C. for 20 minutes with each of 2.times., 0.5.times. and 0.2.times.SSC containing 0.1% SDS. 
     Moreover, it will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison). 
     “Polypeptides” as described herein also include combination polypeptides, also referred to as fusion proteins. A “combination polypeptide” or “fusion protein” is a polypeptide comprising at least one of the above immunogenic portions and one or more additional immunogenic  Leishmania  sequences, which are joined via a peptide linkage into a single amino acid chain. The sequences may be joined directly (i.e., with no intervening amino acids) or may be joined by way of a linker sequence (e.g., Gly-Cys-Gly) that does not significantly diminish the immunogenic properties of the component polypeptides. 
     Fusion proteins may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3′ end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5′ end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in frame. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides. 
     A peptide linker sequence may be employed to separate the first and the second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference. 
     The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5′ to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3′ to the DNA sequence encoding the second polypeptide. 
     In on embodiment of the present invention fusion proteins comprise one or more tandem repeat units. In further embodiments the one or more tandem repeat units are selected from a group consisting of SEQ ID NO. 1-59. In still further embodiments, fusion proteins further comprise the antigenic portions of proteins such as, but not limited to, rK26, rK39 and rLiA2, which are specifically disclosed in SEQ ID NO.&#39;s 119-121. 
     Methods of Screening for Immunogenic Polypeptides 
     In one embodiment, the invention provides methods for screening and selecting tandem repeat proteins that may be useful in diagnosis, treatment and/or immunization of a subject or biological source such as a human patient, for example, in detecting  Leishmania  infection in a biological sample or in an organism, comprising: (a) constructing a  Leishmania  screening library; (b) screening the  Leishmania  screening library; and (c) analyzing the genes identified from the screening the  Leishmania  screening library to select genes that are tandem repeat genes. In further embodiments, a screening library can be constructed from the genomic DNA of any organism, for example by way of illustration and not limitation, an infectious or non-infectious organism, or an infectious organism that may be pathogenic or non-pathogenic, such as a bacterium, a virus, a protozoan, a fungus, a yeast, a diplomonoadid and a kinetoplastid. In certain embodiments the screening library may be constructed from genetic material (i.e., nucleic acid) or an organism that causes or is capable of causing leishmaniasis, such as  Leishmania infantum, Leishmania donovani, Leishmania major, Leishmania amazonensis, Trypanosoma cruzi,  or a natural or unnatural bacterial strain that causes leishmaniasis In a still further embodiment, libraries can be constructed with nucleotide species, including but not limited to DNA and cDNA. 
     For instance, one such embodiment contemplates a method of identifying an immunogenic polypeptide for diagnosis, treatment or immunization in a patient, comprising (a) expressing, in one or a plurality of host cells, expression products of a polynucleotide expression library which comprises one or a plurality of candidate immunogenic polypeptide-encoding polynucleotides at least one of which is capable of expressing a candidate immunogenic polypeptide that comprises a tandem repeat, to obtain a host cell population comprising expression products (b) contacting, under conditions and for a time sufficient for specific binding of at least one antibody in the biological material to at least one expression product, (i) the host cell population comprising expression products of (a) with (ii) a biological material that is obtained from a subject or biological source that has been infected with an infectious or non-infectious organism, or with a pathogenic or non-pathogenic infectious organism such as a bacterium, a virus, a protozoan, a fungus, a yeast, a diplomonoadid or a kinetoplastid, or with a  Leishmania  organism or other organism that is capable of causing leishmaniasis, wherein the biological material comprises at least one antibody and is selected from blood, serum and urine (c) detecting at least one host cell that comprises the at least one expression product to which the at least one antibody specifically binds; (d) isolating from the host cell detected in (c) a polynucleotide that encodes the expression product to which the at least one antibody specifically binds to obtain an isolated polynucleotide; and (e) analyzing a nucleotide sequence of the isolated polynucleotide of (d) for presence or absence of a tandem repeat, wherein the presence of a tandem repeat indicates the isolated polynucleotide encodes an immunogenic polypeptide, and therefrom identifying the immunogenic polypeptide. 
     Constructing a polynucleotide screening library such as a polynucleotide expression library as described herein can be achieved by cleaving or shearing DNA with methods such as sonication or enzyme restriction, which are well known in the art. Resulting nucleotide fragments can be of any size, however, preferably averaging 1, 2, or 3 kb. Resulting nucleotide fragments are then amplified through methods that are well known in the art such as ligation into recombinant polynucleotide vectors including amplification and/or expression vectors and expression with expression vectors such as the λ phage vector or the ZAP Express© vector (Stratagene, La Jolla, Calif.), to generate a polynucleotide screening library, for instance, a polynucleotide expression library. The screening library is then screened by exposing biological material to host cells comprising the expressed expression library products where the biological material may be sera, blood, urine, saliva, or any biological material that has been obtained from a subject or biological source that has been, or is, infected by  Leishmania infantum, Leishmania donovani, Leishmania major, Leishmania amazonensis, Trypanosoma cruzi,  or other natural or unnatural strains of bacteria that causes leishmaniasis. The host cell can be a higher eukaryotic cell, such as a mammalian cell (including a tumor cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Representative examples of appropriate host cells according to the present invention include, but need not be limited to, bacterial cells, such as  E. coli, Streptomyces, Salmonella typhimurium;  fungal cells, such as yeast; insect cells, such as  Drosophila  S2 and  Spodoptera  Sf9; animal cells, such as CHO, COS or 293 cells; adenoviruses; plant cells, or any suitable cell already adapted to in vitro propagation or so established de novo. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa, and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Introduction of the construct into the host cell can be effected by a variety of methods with which those skilled in the art will be familiar, including but not limited to, for example, calcium phosphate transfection, DEAE-Dextran mediated transfection, electroporation (e.g., Davis et al., 1986 Basic Methods in Molecular Biology) or other techniques known to the art. 
     Host cells expressing expression products that comprise an immunogenic polypeptide (e.g., a polypeptide that reacts with an antibody present in a test biological material via a specific binding interaction) may be identified according to any of a number of known methodologies. Readily observed host cell expression of an expression library product, for example, resulting plaques within the expression library, can then be detected, and the original antigenic epitope-encoding polynucleotide fragments may be excised from the expression vector and recovered. In alternative embodiments, the screening library is derived from a subject or biological source that has been exposed to biological material infected by an organism (e.g., an infectious organism) such as a pathogenic or non-pathogenic bacteria, protozoan, fungus, yeast, virus, a diplomonadid, a kinetoplastid or other infectious organism. The excised nucleotide fragments can then be sequenced using methods that are well known in the art. In an additional embodiment these sequences are compared to known genes, which can be found in databases such as the  Leishmania infantum  data gene database (GeneDB: The Wellcome Trust Sanger Institute, www.genedb.org), or the GenBank gene database (National Center for Biotechnology Information (NCBI) www.ncbi.nih.gov). 
     The nucleotide sequences obtained from sequencing the nucleotide fragments are then analyzed to determine if these sequences comprise tandem repeats. Tandem repeats can be identified, for example, using Tandem Repeats Finder (http://tandem.bu.edu/trf/trfhtm) or other similar programs or methods that identify tandem repeats. Typically, these programs or methods identify the period size of the tandem repeats and the number of copies aligned with the consensus pattern. In one embodiment of the present invention, screening may exclude small tandem repeat motifs that have a period size (e.g., periodicity) of about 24, 21, 18, 15, 12 or 10 base pairs or less. 
     In yet another embodiment, sequence libraries or genomes of any organism can be screened for tandem repeats to find epitopes that can be used for the serodiagnosis or treatment of diseases including, but not limited to leishmaniasis, tuberculosis, HIV, and cancer. Where one or more sequences of DNA, cDNA, RNA, or amino acids of any organism are known, these sequences can be screened for tandem repeats. As described above, programs such a Tandem Repeats Finder can be used to analyze and identify sequences that comprise tandem repeats, which are likely to comprise epitopes that are useful in the serodiagnois or treatment of a disease. In one embodiment, the genome of  L. infantum  is screened for tandem repeats. 
     In a further embodiment, tandem repeat sequences that are identified from a sequence library are subsequently screened by exposure to a biological sample from an infected individual or blood supply to identify tandem repeat sequences that have the greatest efficacy in the serodiagnosis or treatment of a disease. 
     In a still further embodiment, the tandem repeat unit or units from the identified sequences are isolated and used to construct new proteins with one or more tandem repeat units of one or more sequence of tandem repeat unit. For example, among other possibilities, constructed proteins can comprise a single tandem repeat unit, multiple tandem repeat units, or be a fusion protein with one or more tandem repeat units, with the tandem repeat units being of different or homologous sequences. 
     Examples of tandem repeat units discovered through these and other methods are disclosed in SEQ ID NO&#39;s 1-59. 
     In a yet further embodiment of the present invention, the tandem repeat units or tandem repeat sequences identified by the aforementioned methods are subsequently screened for homology to other known or unknown proteins. As used herein the term “least homology” refers to a subset of one or more sequences that have less homology to one or more reference sequence compared to at least one sequence within the set. Reference sequences may be, for example, the sequences of polypeptides of organisms that potentially infect organisms that are potentially infected by leishmaniasis, or organisms that are potentially infected by leishmaniasis. Homology screening can be achieved by the methods of homology screening described above, or by the many methods that are well known in the art. For example, tandem repeat sequences or tandem repeat units can be screened for homology to known parasites such as  Trpanosoma Cruzi,  which are potentially present in patients who are being tested for Leishmaniasis. By screening for proteins that are not homologous to proteins in such parasites, proteins can be selected for pharmaceutical or diagnostic purposes that will have more specificity to the disease being treated or tested for, which in this example is Leishmaniasis. By screening for proteins with high specificity to Leishmaniasis, false positives and misdiagnosis can be avoided. 
     In a still further embodiment, the tandem repeat units or tandem repeat sequences identified by the aforementioned methods are subsequently screened for homology to other known or unknown proteins in mammals such as mouse, dog or human, which are potential hosts or test hosts for Leishmaniasis. Screening for low homology to proteins in these mammals again allows pharmaceutical and diagnostic applications to have more specificity to Leishmaniasis and reduces or eliminates false positives or misdiagnosis. 
     In yet further embodiments, the screening methods described above can be applied to any disease. 
     Compounds and Methods for Detecting  Leishmania  Infection 
     As described above, the present invention discloses methods of screening and selecting tandem repeat proteins that have efficacy in detecting  Leishmania  infection in a biological sample or organism comprising the steps: (a) constructing a  Leishmania  screening library; (b) screening the  Leishmania  screening library; and (c) analyzing the genes identified from the screening the  Leishmania  screening library to select genes that are tandem repeat genes. Polypeptides screened and selected by this and other methods of the present invention can be used for various applications, including but not limited to systems and methods for detecting, treating, preventing, monitoring and immunizing against leishmaniasis infection in organisms or blood supplies. 
     Accordingly, in another embodiment of this invention, methods are disclosed for detecting and monitoring  Leishmania  infection, in individuals and blood supplies. In general,  Leishmania  infection may 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 blood or serum sample obtained from a patient or a blood supply. Briefly,  Leishmania  infection may be detected using one or more tandem repeat polypeptides, fusion proteins or other polypeptides as discussed above, or variants thereof. The one or more tandem repeat polypeptides, fusion proteins or other polypeptides are then used to determine the presence or absence of antibodies that are capable of specifically binding to the polypeptide or polypeptides in the sample. 
     Polypeptides within the scope of the present invention include, but are not limited to, polypeptides comprising immunogenic portions of  Leishmania  antigens comprising the sequences recited in SEQ ID NO:1-59. As used herein, the term “tandem repeat” refers to a region of DNA or a protein comprising a sequence of 4 to forty 400 nucleotides or amino acids repeated in tandem at least two times. As used herein the term “tandem repeat unit” refers to a single unit of the sequence that is repeated in tandem. 
     As used herein, references to “binding” interactions between two molecules, such as between an antibody and its cognate antigen, may include binding that may according to non-limiting theory be the result of one or more of electrostatic interactions, hydrophobic interactions, steric interactions, van der Waals forces, hydrogen bonding or the like, or other types of interactions influencing such binding events, such as binding of an antibody to a polypeptide, binding of a detection reagent to an antibody/peptide complex, or any other binding interaction of molecules, including in preferred embodiments specific binding interactions wherein in “specific” binding the affinity constant, Ka, may typically be less than about 10-9 M, less than about 10-8 M, less than about 10-7 M, less than about 10-6 M, less than about 10-5 M or less than 10-4 M. 
     There are a variety of assay formats known to those of ordinary skill in the art for using a polypeptide to detect antibodies in a sample. See, e.g., Current Protocols in Immunology (Coligan et al., eds., John Wiley &amp; Sons, publishers), and Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, which are incorporated herein by reference. In a preferred embodiment, the assay involves the use of a polypeptide (e.g., a polypeptide antigen comprising one or more tandem repeat units as described herein) immobilized on a solid support to bind to and remove the antibody from the sample. The bound antibody may then be detected using a detection reagent that specifically binds to the antibody/polypeptide complex, and that comprises a readily detectable moiety such as 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 assay). Alternatively, a competitive assay may be utilized, in which an antibody that binds to the polypeptide is labeled with a reporter group and allowed to bind to the immobilized polypeptide after incubation of the polypeptide with the sample. The extent to which components of the sample inhibit the binding of the labeled antibody to the polypeptide is indicative of the reactivity of the sample with the immobilized polypeptide. 
     The solid support may be any material known to those of ordinary skill in the art to which the polypeptide may be attached. For example, the support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. 
     The polypeptide may be bound to the solid support using a variety of techniques known to those in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term “bound” refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the antigen and functional groups on the support or may be a linkage by way of a cross-linking agent). Binding by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the polypeptide, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of polypeptide ranging from about 10 ng to about 1 .mu.g, and preferably about 100 ng, is sufficient to bind an adequate amount of antigen. Nitrocellulose will bind approximately 100 .mu.g of protein per cm.sup.3. 
     Covalent attachment of polypeptide to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the polypeptide. For example, the polypeptide may be bound to a support having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the polypeptide (see, e.g., Pierce Immunotechnology Catalog and Handbook (1991) at A12-A13). 
     In certain embodiments, the assay is an enzyme linked immunosorbent assay (ELISA). This assay may be performed by first contacting a polypeptide antigen that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that antibodies to the polypeptide within the sample are allowed to bind to the immobilized polypeptide. 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 that remains bound to the solid support is then determined using a method appropriate for the specific detection reagent. 
     Once the polypeptide is immobilized on the support, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin (BSA) or Tween 20™ (Sigma Chemical Co., St. Louis, Mo.) may be employed. The immobilized polypeptide is then incubated with the sample, and antibody (if present in the sample) is allowed to bind to the antigen. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is that period of time that is sufficient to permit detection of the presence of antibody within a  Leishmania -infected sample. Preferably, the contact time is sufficient to achieve a level of binding that is at least 95% of that achieved at equilibrium between bound and unbound antibody. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient. 
     Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20™. Detection reagent may then be added to the solid support. An appropriate detection reagent is any compound that binds to the immobilized antibody-polypeptide complex and that can be detected by any of a variety of means known to those in the art. Preferably, the detection reagent contains a binding agent (such as, for example, Protein A, Protein G, immunoglobulin, lectin or free antigen) conjugated to a reporter group. Preferred reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. The conjugation of binding agent to reporter group may be achieved using standard methods known to those of ordinary skill in the art. Common binding agents may also be purchased conjugated to a variety of reporter groups from many sources (e.g, Zymed Laboratories, San Francisco, Calif. and Pierce, Rockford, Ill.). 
     The detection reagent is then incubated with the immobilized antibodypolypeptide complex for an amount of time sufficient to detect the bound antibody. An appropriate amount of time may generally be determined from the manufacturer&#39;s instructions or by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products. 
     To determine the presence or absence of anti- Leishmania  antibodies in the sample, the signal detected from the reporter group that remains specifically bound to the solid support is generally compared to a signal that corresponds to an appropriate control according to art-accepted methodologies, for example, a predetermined cut-off value. In one preferred embodiment, the cut-off value may be the average mean signal obtained when the immobilized polypeptide is incubated with samples from an uninfected patient. In general, a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive (i.e., reactive with the polypeptide). In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, p. 106-7 (Little Brown and Co., 1985). Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper lefthand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. 
     In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the antigen (e.g., one or ore polypeptides, each comprising at least one tandem repeat unit) is immobilized on a solid support, for instance, a membrane such as nitrocellulose. In the flow-through test, the fluid sample is contacted with the solid support under conditions and for a time sufficient to permit antibodies, if present within the sample, to bind specifically to the immobilized polypeptide as the sample passes through the membrane. A detection reagent (e.g., protein A-colloidal gold) that may be present in the solid support, or that may alternatively be applied, then binds to the antibody-polypeptide complex as the solution containing the detection reagent flows through the membrane. Determination of bound detection reagent may then be performed as described above. In certain related embodiments of the strip test format, one end of a solid support membrane to which the polypeptide antigen is bound, is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing detection reagent and to the area of immobilized polypeptide antigen. Concentration of detection reagent at the area of the immobilized polypeptide antigen indicates the presence of  Leishmania  antibodies in the sample. Typically, the concentration of detection reagent at that site generates a pattern, such as a line or a series of two or more lines, which can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of polypeptide immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of antibodies that would be sufficient to generate a positive signal in an ELISA, as discussed above. Preferably, the amount of polypeptide immobilized on the membrane ranges from about 25 ng to about 1 .mu.g, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount (e.g., one drop) of patient serum or blood. 
     Of course, numerous other assay protocols exist that are suitable for use with the polypeptides of the present invention, and these will be known to those familiar with the art for detecting the presence of an antibody that is capable of specifically binding to a particular polypeptide antigen. The above descriptions are intended to be exemplary only. 
     Systems and Methods of Treating, Preventing, and Immunizing Against Leishmaniasis 
     As described above, the present invention discloses methods of screening and selecting tandem repeat proteins that have efficacy in detecting  Leishmania  infection in a biological sample or organism comprising the steps: (a) constructing a  Leishmania  screening library; (b) screening the  Leishmania  screening library; and (c) analyzing the genes identified from the screening the  Leishmania  screening library to select genes that are tandem repeat genes. As described herein, polypeptides screened and selected by this and other methods of the present invention can be used for various applications, including but not limited to systems and methods for detecting, treating, preventing, monitoring and immunizing against leishmaniasis infection in organisms or blood supplies. 
     Accordingly, in certain aspects of the present invention, described in detail below, the polypeptides, antigenic epitopes, tandem repeat units, immunogentic sequences, fusion proteins and/or soluble  Leishmania  antigens of the present invention may be incorporated into pharmaceutical compositions or vaccines. For clarity, the term “polypeptide” will be used when describing specific embodiments of the inventive therapeutic compositions and diagnostic methods; however, it will be clear to one of skill in the art that the antigenic epitopes, polypeptides, tandem repeat units and fusion proteins of the present invention may also be employed in such compositions and methods. 
     Pharmaceutical compositions comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable carrier. Vaccines, also referred to as immunogenic compositions, comprise one or more of the above polypeptides and an immunostimulant, such as an adjuvant (e.g., LbeIF4A, interleukin-12 or other cytokines) or a liposome (into which the polypeptide is incorporated). Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A,  Bordetella pertussis  or  Mycobacterium tuberculosis  derived proteins. Certain adjuvants are commercially available as, for example, Freund&#39;s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2,-7,-12, and other like growth factors, may also be used as adjuvants. 
     Within certain embodiments of the invention, the adjuvant composition is preferably one that induces an immune response predominantly of the Th1 type. By virtue of its ability to induce an exclusive Th1 immune response, the use of LbeIF4A, and variants thereof, as an adjuvant in the vaccines of the present invention is particularly preferred. Certain other preferred adjuvants for eliciting a predominantly Th1-type response include, for example, Imiquimod, Res-Imiquimod, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt. MPL™ adjuvants are available from Corixa Corporation (Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996. Another preferred adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; or Gypsophila or  Chenopodium quinoa  saponins. Other preferred formulations include more than one saponin in the adjuvant combinations of the present invention, for example combinations of at least two of the following group comprising QS21, QS7, Quil A, .beta.-escin, or digitonin. 
     Alternatively the saponin formulations may be combined with vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc. The saponins may also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOMs. Furthermore, the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM. The saponins may also be formulated with excipients such as Carbopol.sup.R to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose. 
     In one preferred embodiment, the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and 3D-MPL™ adjuvant, as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. Another particularly preferred adjuvant formulation employing QS21, 3D-MPL™ adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95/17210. 
     Another enhanced adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 is disclosed in WO 00/09159. Preferably the formulation additionally comprises an oil in water emulsion and tocopherol. 
     Additional illustrative adjuvants for use in the compositions of the invention include Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), EnhanZyn™ (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in U.S. Pat. No. 6,113,918 and pending U.S. patent application Ser. No. 09/074,720, the disclosures of which are incorporated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549A1. 
     Other preferred adjuvants include adjuvant molecules of the general formula (I): HO(CH.sub.2CH.sub.2).sub.n-A-R, wherein, n is 1-50, A is a bond or —C(O)—, R is C.sub.1-50 alkyl or Phenyl C.sub.1-50 alkyl. One embodiment of the present invention consists of a vaccine formulation comprising a polyoxyethylene ether of general formula (1), wherein n is between 1 and 50, preferably 4-24, most preferably 9; the R component is C.sub.1-50, preferably C.sub.4-C.sub.20 alkyl and most preferably C.sub.12 alkyl, and A is a bond. The concentration of the polyoxyethylene ethers should be in the range 0.1-20%, preferably from 0.1-10%, and most preferably in the range 0.1-1%. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12.sup.th edition: entry 7717). These adjuvant molecules are described in WO 99/52549. The polyoxyethylene ether according to the general formula (I) above may, if desired, be combined with another adjuvant. For example, a preferred adjuvant combination is preferably with CpG as described in the pending UK patent application GB 9820956.2. 
     Vaccines may additionally contain a delivery vehicle, such as a biodegradable microsphere (disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109). Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other  Leishmania  antigens, either incorporated into a combination polypeptide or present within one or more separate polypeptides. 
     Alternatively, a pharmaceutical or immunogenic composition may contain an immunostimulant, such as an adjuvant (e.g., LbeIF4A, interleukin-12 or other cytokines, or DNA coding for such enhancers), and a polynucleotide (e.g., DNA) encoding one or more of the polypeptides or fusion proteins described above, such that the polypeptide is generated in situ. In such compositions, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as  Bacillus -Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be “naked,” as described, for example, in Ulmer et al., Science 259:1745-1749 (1993) and reviewed by Cohen, Science 259:1691-1692 (1993). The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. 
     While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109. 
     In one preferred embodiment, compositions of the present invention include multiple polypeptides selected so as to provide enhanced protection against a variety of  Leishmania  species. Such polypeptides may be selected based on the species of origin of the native antigen or based on a high degree of conservation of amino acid sequence among different species of  Leishmania.  A combination of individual polypeptides may be particularly effective as a prophylactic and/or therapeutic vaccine because (1) stimulation of proliferation and/or cytokine production by a combination of individual polypeptides may be additive, (2) stimulation of proliferation and/or cytokine production by a combination of individual polypeptides may be synergistic, (3) a combination of individual polypeptides may stimulate cytokine profiles in such a way as to be complementary to each other and/or (4) individual polypeptides may be complementary to one another when certain of them are expressed more abundantly on the individual species or strain of  Leishmania  responsible for infection. A preferred combination contains polypeptides that comprise immunogenic portions of M15, Ldp23, Lbhsp83, Lt-1 and LbeIF4A. Alternatively, or in addition, the combination may include one or more polypeptides comprising immunogenic portions of other  Leishmania  antigens disclosed herein, and/or soluble  Leishmania  antigens. 
     In another preferred embodiment, compositions of the present invention include single polypeptides selected so as to provide enhanced protection against a variety of  Leishmania  species. A single individual polypeptide may be particularly effective as a prophylactic and/or therapeutic vaccine for those reasons stated above for combinations of individual polypeptides. 
     In another embodiment, compositions of the present invention include individual polypeptides and combinations of the above described polypeptides employed with a variety of adjuvants, such as IL-12 (protein or DNA) to confer a protective response against a variety of  Leishmania  species. 
     In yet another embodiment, compositions of the present invention include DNA constructs of the various  Leishmania  species employed alone or in combination with variety of adjuvants, such as IL-12 (protein or DNA) to confer a protective response against a variety of  Leishmania  species. 
     The above pharmaceutical compositions and vaccines may be used, for example, to induce protective immunity against  Leishmania  in a patient, such as a human or a dog, to prevent leishmaniasis. Appropriate doses and methods of administration for this purposes are described in detail below. 
     The pharmaceutical and immunogenic compositions described herein may also be used to stimulate an immune response, which may be cellular and/or humoral, in a patient. For  Leishmania -infected patients, the immune responses that may be generated include a preferential Th1 immune response (i.e., a response characterized by the production of the cytokines interleukin-1, interleukin-2, interleukin-12 and/or interferon-.gamma., as well as tumor necrosis factor-.alpha.). For uninfected patients, the immune response may be the production of interleukin-12 and/or interleukin-2, or the stimulation of gamma delta T-cells. In either category of patient, the response stimulated may include IL-12 production. Such responses may also be elicited in biological samples of PBMC or components thereof derived from  Leishmania -infected or uninfected individuals. As noted above, assays for any of the above cytokines may generally be performed using methods known to those of ordinary skill in the art, such as an enzyme-linked immunosorbent assay (ELISA). 
     Suitable pharmaceutical compositions and vaccines for use in this aspect of the present invention are those that contain at least one polypeptide comprising an immunogenic portion of a  Leishmania  antigen disclosed herein (or a variant thereof). Preferably, the polypeptides employed in the pharmaceutical compositions and vaccines are complementary, as described above. Soluble  Leishmania  antigens, with or without additional polypeptides, may also be employed. 
     The pharmaceutical compositions and vaccines described herein may also be used to treat a patient afflicted with a disease responsive to IL-12 stimulation. The patient may be any warm-blooded animal, such as a human or a dog. Such diseases include infections (which may be, for example, bacterial, viral or protozoan) or diseases such as cancer. In one embodiment, the disease is leishmaniasis, and the patient may display clinical symptoms or may be asymptomatic. In general, the responsiveness of a particular disease to IL-12 stimulation may be determined by evaluating the effect of treatment with a pharmaceutical composition or vaccine of the present invention on clinical correlates of immunity. For example, if treatment results in a heightened Th1 response or the conversion of a Th2 to a Th1 profile, with accompanying clinical improvement in the treated patient, the disease is responsive to IL-12 stimulation. Polypeptide administration may be as described below, or may extend for a longer period of time, depending on the indication. Preferably, the polypeptides employed in the pharmaceutical compositions and vaccines are complementary, as described above. A particularly preferred combination contains polypeptides that comprise immunogenic portions of LmSTI1, Ldp23, Lbhsp83, Lt-1 and LbeIF4A, Lmsp1a, Lmsp9a, and TSA. Soluble  Leishmania  antigens, with or without additional polypeptides, may also be employed. 
     Routes and frequency of administration, as well as dosage, for the above aspects of the present invention will vary from individual to individual and may parallel those currently being used in immunization against other infections, including protozoan, viral and bacterial infections. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Between 1 and 12 doses may be administered over a 1 year period. For therapeutic vaccination (i.e., treatment of an infected individual), 12 doses are preferably administered, at one month intervals. For prophylactic use, 3 doses are preferably administered, at 3 month intervals. In either case, booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of polypeptide or DNA that, when administered as described above, is capable of raising an immune response in an immunized patient sufficient to protect the patient from leishmaniasis for at least 1-2 years. In general, the amount of polypeptide present in a dose (or produced in situ by the DNA in a dose) ranges from about 100 ng to about 1 mg per kg of host, typically from about 10 .mu.g to about 100 .mu.g. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL. 
     In another aspect, this invention provides methods for using one or more of the polypeptides described above to diagnose  Leishmania  infection in a patient using a skin test. As used herein, a “skin test” is any assay performed directly on a patient in which a delayed-type hypersensitivity (DTH) reaction (such as induration and accompanying redness) is measured following intradermal injection of one or more polypeptides as described above. Such injection may be achieved using any suitable device sufficient to contact the polypeptide or polypeptides with dermal cells of the patient, such as a tuberculin syringe or 1 mL syringe. Preferably, the reaction is measured at least 48 hours after injection, more preferably 72 hours after injection. 
     The DTH reaction is a cell-mediated immune response, which is greater in patients that have been exposed previously to a test antigen (i.e., an immunogenic portion of a polypeptide employed, or a variant thereof). The response may be measured visually, using a ruler. In general, induration that is greater than about 0.5 cm in diameter, preferably greater than about 1.0 cm in diameter, is a positive response, indicative of  Leishmania  infection, which may or may not be manifested as an active disease. 
     The polypeptides of this invention are preferably formulated, for use in a skin test, as pharmaceutical compositions containing at least one polypeptide and a physiologically acceptable carrier, as described above. Such compositions typically contain one or more of the above polypeptides in an amount ranging from about 1 .mu.g to 100 .mu.g, preferably from about 10 .mu.g to 50 .mu.g in a volume of 0.1 mL. Preferably, the carrier employed in such pharmaceutical compositions is a saline solution with appropriate preservatives, such as phenol and/or Tween .sub.80T. 
     The inventive polypeptides may also be employed in combination with one or more known  Leishmania  antigens in the diagnosis of leishmaniasis, using, for example, the skin test described above. Preferably, individual polypeptides are chosen in such a way as to be complementary to each other. Examples of known  Leishmania  antigens which may be usefully employed in conjunction with the inventive polypeptides include K39 (Bums et al., Proc. Natl. Acad. Sci. USA, 1993 90:775-779). 
     The following examples are offered by way of illustration, and not by way of limitation. 
     EXAMPLES 
     Example 1 
     Parasite and Infection of Hamsters 
       Leishmania infantum, Leishmania donovani, Leishmania major, Leishmania amazonensis  and  Trypanosoma cruzi  were used to infect Hamsters and thereby produce infected sera. Hamsters were infected intracardiacally with 1×10 7    Leishmania infantum, Leishmania donovani, Leishmania major, Leishmania amazonensis  or  Trypanosoma cruzi  promastigotes in a stationary phase. After eight to twelve weeks the infected hamsters were sacrificed and the sera were collected. 
     Example 2 
     Patient Sera 
     Sudanese visceral leishmaniasis patient sera were collected from patients with active disease diagnosed clinically and proven parasitologically. Patient sera of cutaneous leishmaniasis (Brazil), tuberculosis (USA) and malaria (Brazil) were collected from well-characterized patients, including parasitological diagnosis (cutaneous leishmaniasis, malaria) or culture positive diagnosis (tuberculosis). Normal sera were obtained from healthy individuals in the United States. 
     Example 3 
     Serological Screening of  L. infantium  Expression Library 
     Construction and screening of library was performed. In brief, total DNA from  L. infantum  was randomly sheared by sonication to an average size of ˜2 kb, blunt ended with T4 DNA polymerase, and followed by the addition of EcoRI adaptors. The insert was subsequently ligated into the ZAP Express© vector predigested with EcoRI (Stratagene, La Jolla, Calif.) and packaged using Gigapack III© Gold Packaging Extract (Stratagene). The phage library was amplified and then screened according to the manufacturer&#39;s instruction with pooled  L. infantum -infected hamster sera or pooled Sudanese visceral leishmaniasis patient sera described above. Approximately 5×10 5  plaques were screened using the sera at a dilution of 1:100 and immunoreactive plaques were detected with the alkaline phosphatase-conjugated goat anti-hamster IgG or goat anti-human IgG (KPL, Gaithersburg, Md.) and the substrate, BCIP/NBT (KPL). PBK-CMV phagemid vectors were excised from the immunoreactive phage clones according to the manufacturer&#39;s protocol. The inserts were sequenced and analyzed using  Leishmania infantum  gene database (GeneDB: The Wellcome Trust Sanger Institute, www.genedb.org). 
     Example 4 
     Tandem Repeat Gene Analysis 
     The genes identified by the screening were analyzed to determine whether they are tandem repeat genes. Tandem Repeats Finder, a program to locate and display tandem repeats in DNA sequences, was used for this analysis (. As a control for the screened genes, 108 genes which were randomly picked from the  L. infantum  gene database were also analyzed for the presence of tandem repeat motifs. The program calculates the score according to the nature of the tandem repeat genes such as the period size of the repeat, the number of copies aligned with the consensus pattern and the percent of matches between adjacent copies overall. In this study the genes were regarded as tandem repeat genes if the scores from the Tandem Repeats Finder analysis were higher than 500, thus excluding genes containing small tandem repeat motifs such as LinJ01.0470 and LinJ13.0810 (Table I). 
     
       
         
           
               
             
               
                 TABLE I 
               
             
            
               
                   
               
               
                   L. infantum  proteins identified by serological screening 
               
            
           
           
               
               
            
               
                 GENE DB 
                 TR analysis 
               
            
           
           
               
               
               
               
               
               
            
               
                 ID 
                 Gene 
                 Size 
                 PS 
                 CN 
                 Score 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 LinJ01.0470 
                 hypothetical protein 
                 151 
                 3 
                 11 
                 66 
               
               
                 LINJ03.0120 
                 hypothetical protein 
                 237 
                 117 
                 31.8 
                 7033 
               
               
                 LinJ05.0380 
                 microtubule-associated 
                 165 
                 114 
                 28.5 
                 6336 
               
               
                   
                 protein 
               
               
                 LinJ05.0590 
                 hypothetical protein 
                 86 
               
               
                 LinJ08.0860 
                 mitochondrial DNA poly- 
                 154 
               
               
                   
                 merase beta-PAK 
               
               
                 LinJ08.1010 
                 stress-induced protein sti1 
                 62 
               
               
                 LinJ08.1130 
                 hypothetical protein 
                 50 
               
               
                 LinJ10.1460 
                 hypothetical protein 
                 56 
               
               
                 LinJ13.0810 
                 hypothetical protein 
                 76 
                 15 
                 3.7 
                 74 
               
               
                 LinJ14.1160 
                 kinesin K39 (rK39) 
                 241 
                 117 
                 27.9 
                 5237 
               
               
                 LinJ14.1190 
                 kinesin K39 
                 95 
                 105 
                 6.2 
                 1198 
               
               
                 LinJ14.1540 
                 hypothetical protein 
                 112 
                 72 
                 6.1 
                 806 
               
               
                 LinJ15.0490 
                 tb-292 membrane 
                 164 
                 105 
                 31.6 
                 6027 
               
               
                   
                 associated protein-like 
               
               
                 LinJ16.1540 
                 kinesin 
                 230 
                 42 
                 138.5 
                 10588 
               
               
                 LinJ16.1560 
                 kinesin 
                 88 
                 42 
                 2.5 
                 172 
               
               
                 LinJ16.1750 
                 hypothetical protein 
                 346 
                 219 
                 8.7 
                 3691 
               
               
                 LinJ17.0100 
                 elongation factor 1-alpha 
                 49 
               
               
                 LinJ17.0610 
                 hypothetical protein 
                 86 
               
               
                 LinJ18.0610 
                 hypothetical protein 
                 68 
               
               
                 LinJ18.1150 
                 hypothetical protein 
                 107 
               
               
                 LinJ21.0440 
                 1a RNA binding protein 
                 37 
               
               
                 LinJ22.0680 
                 hypothetical protein 
                 45 
                 30 
                 34.2 
                 1137 
               
               
                 LinJ22.1590 
                 hypothetical protein 
                 234 
                 84 
                 29.2 
                 3993 
               
               
                 LinJ24.1570 
                 basal body component 
                 164 
               
               
                 LinJ26.0980 
                 dynein heavy chain 
                 458 
               
               
                 LinJ26.1200 
                 Hsp70.4 heat shock protein 
                 70 
               
               
                   
                 70 
               
               
                 LinJ27.0290 
                 nucleoporin 
                 159 
                 27 
                 2.4 
                 74 
               
               
                 LinJ27.0410 
                 calpain-like cysteine 
                 702 
                 12 
                 2.6 
                 53 
               
               
                   
                 peptidase 
               
               
                 LinJ27.1480 
                 hypothetical protein 
                 247 
               
               
                 LinJ28.2310 
                 glycoprotein 96-92 
                 61 
                 315 
                 2.2 
                 1398 
               
               
                 LinJ28.3170 
                 hypothetical protein 
                 75 
                 60 
                 23.4 
                 2546 
               
               
                 LinJ31.0530 
                 amastin 
                 21 
               
               
                 LinJ31.1430 
                 hypothetical protein 
                 95 
               
               
                 LinJ32.2730 
                 hypothetical protein 
                 173 
                 150 
                 10.3 
                 2916 
               
               
                 LinJ32.2780 
                 membrane associated 
                 131 
                 30 
                 60.9 
                 3125 
               
               
                   
                 protein 
               
               
                 LinJ33.2870 
                 hypothetical protein 
                 413 
                 444 
                 7 
                 6041 
               
               
                 LinJ34.0710 
                 hypothetical protein 
                 306 
                 168 
                 16.3 
                 4487 
               
               
                 LinJ34.2140 
                 hypothetical protein 
                 296 
                 249 
                 7.4 
                 3604 
               
               
                 LinJ35.0590 
                 proteophosphoglycan ppg4 
                 536 
                 45 
                 246.1 
                 10667 
               
               
                 LinJ35.0600 
                 proteophosphoglycan ppg3 
                 — 
                 135 
                 37.8 
                 8773 
               
               
                 LinJ35.4250 
                 poly(A) binding protein 
                 65 
                 36 
                 3.6 
                 111 
               
               
                 LinJ36.4560 
                 chaperonin Hsp60, 
                 59 
               
               
                   
                 mitochondrial 
               
               
                 LinJ36.4930 
                 sterol 
                 40 
               
               
                   
                 24-c-methyltransferase 
               
               
                   
               
            
           
         
       
     
     ID numbers in GeneDB are temporary and may vary. Sizes are shown in kDa. Data of PS, CN and score in TR analysis are from a program analysis using Tandem Repeats Finder. Tandem repeat genes are shown in bold letters. Blanks in PS, CN and Score columns represent no repeat found in the genes. PS: period size (bp) of the repeat, CN: number of copies aligned with the consensus pattern. 
     Example 5 
     PCR Analysis and Cloning of Tandem Repeat Genes 
     Tandem repeat sequences of LinJ16.1750, LinJ22.1590 or LinJ33.2870 were amplified by PCR using primers corresponding to both ends of a single copy of the tandem repeat (primer sequences are shown in Table II). The entire gene sequence of LinJ28.2310 was amplified by PCR and primers used for this reaction are also shown in Table II. To analyze whether or not those genes are conserved among  Leishmania  species, total DNA of  L. infantum, L. donovani, L. major, L. amazonensis  and  Trypanosoma cruzi  were used as templates. As a control, a  T. cruzi  gene (GenBank: XM 810936) was amplified by PCR using  L. infantum  and  Trypanosoma cruzi  DNA as templates (primer sequence in Table II). For the cloning of the genes for producing recombinant proteins,  L. infantum  total DNA was used as a template for the PCR reactions. 
     
       
         
           
               
             
               
                 TABLE II 
               
             
            
               
                   
               
               
                 Primers used in this study 
               
            
           
           
               
               
            
               
                 Gene 
                 Primer sequence 
               
               
                   
               
               
                 LinJ16.1750/SEQ ID 
                 CAA TTA CAT ATG TAC CCG TTC CTA CGG CTG 
               
               
                   
                 CAA TTA GGA TCC CTA GCG CGA CGC CAG CTC GTC 
               
               
                   
               
               
                 LinJ22.1590/SEQ ID 
                 CAA TTA CAT ATG GCT GAC CTG AGG GAG CAG 
               
               
                   
                 CAA TTA GGA TCC CTA CAC CTC GGC GTC CCT GTC 
               
               
                   
               
               
                 LinJ28.2310/SEQ ID 
                 CAA TTA CAT ATG AGC GCT GCA CCG TCC 
               
               
                   
                 CAA TTA GAA TTC CTA CGC AAG TCC GAG GGC 
               
               
                   
               
               
                 LinJ33.2870/SEQ ID 
                 CAA TTA CAT ATG CAG CGG CTG GTG CTC 
               
               
                   
                 CAA TTA GGA TCC CTA CGA CGT CCG CGG CAG CGC 
               
               
                   
               
               
                   T. cruzi  XM_810936 
                 CAA TTA CAT ATG TGC ATT GCT CTT GGC ATCGTC 
               
               
                   
                 CAA TTA AAG CTT CTG GGG CGT GAA GCG TAT GTA 
               
               
                   
                 CTC 
               
               
                   
               
            
           
         
       
     
     Example 6 
     Expression of Recombinant Proteins 
     The PCR product corresponding to two copies of LinJ16.1750 repeat (LinJ16.1750r2), three copies of LinJ22.1590 repeat (LinJ22.1590r3), a copy of LinJ33.2870 repeat (LinJ33.2870r1) or an entire gene of LinJ28.2310 was inserted into NdeI/BamHI or NdeI/EcoRI site of pET28 vector. The sequence of the inserts was then analyzed. These pET28 vectors were transformed into  E. coli  Rosetta for expression of the recombinant proteins. Expression of the recombinant proteins was induced by cultivation with 1M isopropyl-β-D-thiogalactoside. The recombinant proteins were then purified as 6× His-tagged proteins using Ni-NTA agarose (Qiagen Inc., Valencia, Calif.). Purity of the proteins was assessed by Coomassie blue-staining following SDS-PAGE. The concentrations of these proteins were measured by BCA protein assay (Pierce Biotechnology Inc., Rockford, Ill.). 
     Example 7 
     Enzyme-Linked Immunosorbent Assay (ELISA) 
     rLinJ16.1750r2, rLinJ22.1590r3, rLinJ28.2310, rLinJ33.2870, rK39 or  L. infantum  promastigote soluble lysate antigen (LiSLA) were diluted in ELISA coating buffer, and 96-well plates were coated with rLinJ16.1750r2, rLinJ22.1590r3, rLinJ28.2310, rLinJ33.2870 (200 ng), rK39 (50 ng) or LiSLA (1 μg) followed by blocking with phosphate-buffered saline containing 0.05% Tween-20 and 1% bovine serum albumin. Next, the plates were incubated with patient sera (diluted 1:100) as well as healthy controls and then with horseradish peroxidase-conjugated protein G (Zymed laboratories, South San Francisco, Calif.). The plates were developed with TMB peroxidase substrate (KPL) and read by a microplate reader at a 450 nm wavelength. 
     Example 8 
     Detection of Tandem Repeat Genes in Serologically Screened Genes 
     Pooled sera from  L. infantum -infected hamsters or Sudanese visceral leishmaniasis patients were used to screen a  L. infantum  expression library. A half million plaques covering about eight times  L. infantum  genomic equivalents were screened. PBK-CMV phagemids were excised from positive clones; the inserts were sequenced and analyzed using GeneDB. A total of 43 genes were identified as genes encoding B-cell antigens (Table I). Some of the genes encoded previously identified antigens such as HSP70 and K39 , but most of them encoded previously unidentified antigens. 
     The genes identified by the screening were then analyzed by Tandem Repeats Finder. In this study, a gene was regarded as a tandem repeat gene if a score from the analysis was 500 or higher thereby excluding genes containing small tandem repeat domains. Of the 43 genes identified by the serological screening, 19 genes were identified as tandem repeat genes (Table I). For example, LinJ16.1750 contained 8.7 copies of a 219 by sequence and LinJ28.3170 contained 23.4 copies of a 60 by sequence. With the exception of LinJ14.1160 (known as rK39), these genes were newly identified as genes encoding B-cell antigens. One hundred and eight genes, which were picked randomly from the database (3 genes from each chromosome), were also analyzed with Tandem Repeats Finder to compare the prevalence of tandem repeat genes with that in the genes identified by screening. In contrast to the screened genes, no tandem repeat genes were found in the 108 randomly picked genes. 
     Example 9 
     PCR Analysis of Tandem Repeat Genes 
     PCR amplifications were performed with total DNA from  Leishmania  species and  T. cruzi  to analyze whether or not tandem repeat genes were conserved among these parasites. Using sets of primers specific for tandem repeat domains of LinJ16.1750 and LinJ22.1590, the PCR products showed ladder bands corresponding to one or multiple copies of the repeat ( FIG. 1 ). These genes were conserved among  Leishmania  species because similar band patterns were observed through all four  Leishmania  species tested. When primers for tandem repeat domains of LinJ33.2870 were used, the PCR product also showed bands which sizes corresponded to one or two copies of the repeat in  Leishmania  species, but not in  L. amazonensis  ( FIG. 1 ). When primers for the whole gene of LinJ33.2870 were used, a 1.5 kb band was found in  L. infantum  and  L. donovani,  and bands of 2.1 kb and 1.8 kb were found in  L. major  and  L. amazonensis,  respectively. In all cases, no bands were found in PCR reactions using  T. cruzi  DNA. In contrast, a single band with an expected size was found in  T. cruzi  but not in  L. infantum  when primers for a  T. cruzi  gene were used for PCR. Thus, while the tandem repeat genes are not conserved between  Leishmania  and  T. cruzi,  they are well conserved between  Leishmania  spp. 
     Example 10 
     Recognition of Recombinant Tandem Repeat Proteins by Visceral Leishmaniasis Patient Sera 
     To formally test the identified tandem repeat proteins as potential diagnostic candidates, LinJ16.1750r2, LinJ22.1590r3, LinJ28.2310 and LinJ33.2870r1 were expressed as recombinant proteins ( FIG. 2 ). 
     The prevalence of antibodies to the recombinant proteins in a preliminary screen of ten Sudanese visceral leishmaniasis patient sera by ELISA. The visceral leishmaniasis patient sera showed significantly stronger reactivity to all the recombinant proteins than sera from the groups without visceral leishmaniasis, i.e. tuberculosis, malaria and cutaneous leishmaniasis patients or healthy individuals, despite the finding that cutaneous leishmaniasis patients showed antibody responses to LiSLA ( FIG. 3 ). Among the tested proteins, rLinJ16.1750r2 was the best antigen recognized strongly by visceral leishmaniasis patient sera with a high degree of specificity. Next, rLinJ16.1750r2 was tested with additional Sudanese visceral leishmaniasis patient sera. Among 35 sera tested, 34 showed antibody responses to rLinJ16.1750r2 (the cutoff value was the mean+3 SD of healthy controls:  FIG. 4 ). The sensitivity using rLinJ16.1750r2 was 97%, comparable to that of rK39 (35/35, 100%:  FIG. 4 ). Mean O.D. values to rLinJ16.1750r2 and rK39 were 1.159 and 1.771, respectively. 
     Although these 35 sera were 100% positive using rK39, eight sera showed low reactivity to rK39 (OD values were &lt;1.0:  FIG. 4 ). These eight samples were tested for reactivity to rLinJ16.1750r2, rLinJ22.1590r3, rLinJ28.2310 or rLinJ33.2870r1 to examine whether or not those antigens could complement rK39 for more sensitive antibody detection. Five of the eight sera showed better responses to the new antigens than to rK39 (No. 3-7 in  FIG. 5 ). Five patients (No. 3-7) showed better responses to rLinJ22.1590r3, four (No. 4-7) to rLinJ28.2310, and three (No. 3, 5 and 6) to rLinJ33.2870r1. rLinJ22.1590r3 was recognized weakly by all the eight sera compared to rK39. 
     Example 11 
     Bioinformatic Computer Analysis for Finding Tandem Repeat Genes 
     DNA sequences of 8,191  L. infantum  genes were obtained from  L. infantum  GeneDB 0. Tandem Repeats Finder, a program used to locate and display tandem repeats in DNA sequences, was used for this analysis ( ). The program calculates the score according to the nature of the tandem repeat genes such as the period size of the repeat, the number of copies aligned with the consensus pattern and the percent of matches between adjacent copies overall. Thus, an output of a high score in gene analysis means the gene possesses a large tandem repeat sequence and the repeat is highly conserved among the copies. A low score means the opposite; for example, a score of a gene with 3.6 copies of a 36 by repeat was 111. In this study, the genes were regarded as tandem repeat genes if the scores from the Tandem Repeats Finder analysis were higher than 500, thus excluding genes containing small tandem repeat motifs. Using these screening parameters, the following genes were identified from the  L. infantum  GeneDB. (Table 3) 
     
       
         
           
               
             
               
                 TABLE III 
               
             
            
               
                   
               
               
                 L. infantum TR genes identified by Tandem Repeats Finder 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Size 
                 PS3 
                   
                   
                   
               
               
                 Gene ID1 
                 C/I2 
                 Product 
                 (kDa) 
                 (bp) 
                 CN3 
                 Score3 
                 Ref4 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 LinJ03.0120 
                 C 
                 hypothetical protein 
                 237 
                 117 
                 31.8 
                 7033 
                 1 
               
               
                 LinJ05.0340 
                 C 
                 viscerotropic leishmaniasis antigen 
                 95 
                 99 
                 13.8 
                 2545 
                 2 
               
               
                 LinJ05.0380 
                 C 
                 microtubule-associated protein 
                 165 
                 114 
                 28.5 
                 6336 
                 1 
               
               
                 LinJ09.0950 
                 C 
                 polyubiquitin 
                 74 
                 228 
                 8 
                 3621 
                   
               
               
                 LinJ11.0070 
                 C 
                 hypothetical protein 
                 147 
                 138 
                 12.9 
                 2435 
                   
               
               
                 LinJ13.0780 
                 C 
                 hypothetical protein 
                 107 
                 63 
                 14.2 
                 1637 
                   
               
               
                 LinJ14.0370 
                 C 
                 hypothetical protein 
                 302 
                 84 
                 10.9 
                 1475 
                   
               
               
                 LinJ14.1160 
                 C 
                 kinesin K39 
                 242 
                 117 
                 27.9 
                 5237 
                 1.3 
               
               
                 LinJ14.1180 
                 I 
                 kinesin K39 
                 278 
                 168 
                 8.2 
                 2671 
                   
               
               
                 LinJ14.1190 
                 I 
                 kinesin K39 
                 95 
                 315 
                 6.1 
                 2828 
                 1 
               
               
                 LinJ14.1200 
                 C 
                 kinesin K39 
                 79 
                 468 
                 3.4 
                 1971 
                 3 
               
               
                 LinJ14.1210 
                 I 
                 kinesin K39 
                 337 
                 483 
                 10.9 
                 3676 
                   
               
               
                 LinJ14.1540 
                 C 
                 hypothetical protein 
                 112 
                 72 
                 6.1 
                 806 
                 1 
               
               
                 LinJ15.0490 
                 I 
                 tb-292 membrane associated 
                   
                 105 
                 31.6 
                 6027 
                 1 
               
               
                 LinJ15.1570 
                 I 
                   
                 112 
                 105 
                 29.9 
                 5588 
                   
               
               
                 LinJ16.1540 
                 C 
                 kinesin 
                 230 
                 42 
                 138.5 
                 10588 
                 1 
               
               
                 LinJ16.1750 
                 C 
                 hypothetical protein 
                 346 
                 219 
                 8.7 
                 3691 
                 1 
               
               
                 LinJI8.1030 
                 C 
                 Hypothetical repeat protein 
                 46 
                 21 
                 30.4 
                 1036 
                   
               
               
                 LinJ19.0940 
                 C 
                   
                 24 
                 6 
                 95 
                 1076 
                   
               
               
                 LinJ19.1560 
                 I 
                   
                 166 
                 81 
                 21.1 
                 3094 
                   
               
               
                 LinJ20.1220 
                 C 
                 calpain-like cysteine peptidase 
                 112 
                 39 
                 11.3 
                 826 
                   
               
               
                 LinJ21.2010 
                 C 
                 hypothetical protein 
                 306 
                 192 
                 5.3 
                 2003 
                   
               
               
                 LinJ22.0410 
                 C 
                 hypothetical protein 
                 130 
                 183 
                 15.9 
                 5779 
                   
               
               
                 LinJ22.0680 
                 C 
                 hypothetical protein 
                 45 
                 216 
                 5.9 
                 1240 
                 1.4 
               
               
                 LinJ22.1510 
                 C 
                 hypothetical protein 
                 179 
                 81 
                 13.5 
                 1984 
                   
               
               
                 LinJ22.1520 
                 C 
                   
                 72 
                 39 
                 42.9 
                 3197 
                   
               
               
                 LinJ22.1550 
                 C 
                   
                 126 
                 81 
                 10.4 
                 1504 
                   
               
               
                 LinJ22.1560 
                 I 
                   
                 172 
                 267 
                 16.9 
                 8614 
                   
               
               
                 LinJ22.1570 
                 C 
                   
                 210 
                 81 
                 23.5 
                 3230 
                   
               
               
                 LinJ22.1580 
                 C 
                   
                 175 
                 267 
                 17.1 
                 8591 
                   
               
               
                 LinJ22.1590 
                 C 
                 hypothetical protein 
                 234 
                 84 
                 29.2 
                 3993 
                 1 
               
               
                 LinJ23.1180 
                 C 
                 hydrophilic surface protein (HASPB) 
                 26 
                 42 
                 11.2 
                 832 
                 5 
               
               
                 LinJ25.1100 
                 C 
                 hypothetical protein 
                 91 
                 66 
                 9.5 
                 1142 
                   
               
               
                 LinJ25.1910 
                 C 
                 hypothetical protein 
                 91 
                 369 
                 2 
                 1443 
                   
               
               
                 LinJ26.2140 
                 C 
                 hypothetical protein 
                 215 
                 48 
                 63.4 
                 5289 
                   
               
               
                 LinJ27.0140 
                 I 
                 kinetoplast-associated protein-like 
                 33 
                 30 
                 19.9 
                 1086 
                   
               
               
                 LinJ27.0170 
                 C 
                 kinetoplast-associated protein-like 
                 95 
                 30 
                 62.1 
                 3283 
                   
               
               
                 LinJ27.0400 
                 C 
                 calpain-like cysteine peptidase 
                 687 
                 204 
                 43.8 
                 17362 
                   
               
               
                 LinJ28.2310 
                 C 
                 glycoprotein 96-92 
                 61 
                 315 
                 2.2 
                 1398 
                 1 
               
               
                 LinJ28.3170 
                 C 
                 hypothetical protein 
                 75 
                 60 
                 23.4 
                 2546 
                 1 
               
               
                 LinJ29.0110 
                 C 
                 hypothetical protein 
                 278 
                 24 
                 28.6 
                 967 
                   
               
               
                 LinJ30.0400 
                 C 
                 hypothetical protein 
                 56 
                 117 
                 7.4 
                 1716 
                   
               
               
                 LinJ31.1820 
                 C 
                 hypothetical protein 
                 49 
                 75 
                 4.1 
                 581 
                   
               
               
                 LinJ31.1840 
                 C 
                 hypothetical protein 
                 52 
                 24 
                 18.1 
                 814 
                   
               
               
                 LinJ31.2660 
                 C 
                 hypothetical protein 
                 247 
                 456 
                 2.2 
                 1973 
                   
               
               
                 LinJ31.3360 
                 C 
                 hypothetical protein 
                 71 
                 30 
                 11.1 
                 556 
                   
               
               
                 LinJ32.2730 
                 C 
                 hypothetical protein 
                 173 
                 150 
                 10.3 
                 2916 
                 1 
               
               
                 LinJ32.2780 
                 C 
                 membrane associated protein-like 
                 132 
                 30 
                 60.9 
                 3125 
                 1 
               
               
                 LinJ32.3710 
                 C 
                 hypothetical protein 
                 292 
                 99 
                 3.9 
                 730 
                   
               
               
                 LinJ33.2870 
                 C 
                 hypothetical protein 
                 413 
                 444 
                 7 
                 6041 
                 1 
               
               
                 LinJ34.0710 
                 I 
                 hypothetical protein 
                   
                 336 
                 9.5 
                 4517 
                 1 
               
               
                 LinJ34.2140 
                 C 
                 hypothetical protein 
                 296 
                 249 
                 7.4 
                 3604 
                 1 
               
               
                 LinJ34.4250 
                 C 
                 hypothetical protein 
                 168 
                 168 
                 6.1 
                 1960 
                   
               
               
                 LinJ35.0590 
                 C 
                 proteophosphoglycan ppg4 
                 536 
                 45 
                 246.1 
                 10667 
                 1 
               
               
                 LinJ35.0600 
                 I 
                 proteophosphoglycan ppg3 
                   
                 135 
                 37.8 
                 8773 
                 1 
               
               
                 LinJ35.0610 
                 C 
                 proteophosphoglycan ppg4 
                 291 
                 45 
                 183.2 
                 13275 
                   
               
               
                 LinJ35.0620 
                 I 
                 proteophosphoglycan 5 
                 495 
                 90 
                 152.5 
                 15050 
                   
               
               
                 LinJ35.0630 
                 I 
                 proteophosphoglycan ppg4 
                 281 
                 45 
                 176.6 
                 10813 
                   
               
               
                 LinJ35.0640 
                 I 
                 hypothetical protein 
                 95 
                 45 
                 58.4 
                 4766 
                   
               
               
                 LinJ35.1530 
                 C 
                 hypothetical protein 
                 328 
                 141 
                 2.4 
                 661 
                   
               
               
                 LinJ35.1620 
                 I 
                 hypothetical protein 
                   
                 126 
                 8.7 
                 1855 
                   
               
               
                 LinJ35.4500 
                 C 
                 hypothetical protein 
                 60 
                 165 
                 4.5 
                 1438 
                   
               
               
                 LinJ36.0320 
                 C 
                 histidine secretory acid phosphatase 
                 71 
                 72 
                 6.5 
                 861 
                   
               
               
                 LinJ36.5810 
                 C 
                 hypothetical protein 
                 365 
                 276 
                 4.3 
                 2341 
               
               
                   
               
            
           
         
       
     
     The following notes correspond to the superscript notes in Table III: (1) ID numbers in GeneDB are temporary and may vary; (2) C or I mean the gene is a complete or incomplete gene, respectively; (3) Data of PS, CN and score in TR analysis are from a program analysis using Tandem Repeats Finder. TR genes are shown in bold letters. Blanks in PS, CN and Score columns represent no repeat found in the genes. PS: period size (bp) of the repeat, CN: number of copies aligned with the consensus pattern; and (4) antigenicity of the protein has been reported in the referenced paper. 
     Example 12 
     Analysis of Amino Acid Compositions of Tandem Repeat Proteins 
     Tandem repeat proteins, which encoded by the tandem repeat genes identified by the bioinformatic analysis, were analyzed their isometric points and amino acid compositions using a software, EditSeq (DNASTAR Inc., Madison, Wis.). Lysine and Arginine were classified as strongly basic amino acids, Aspartic Acid and Glutamic Acid as strongly acidic amino acids, Asparagine, Cysteine, Glutamine, Sering, Threonine and Tyrosine as other polar amino acids, and Alanine, Isoleucine, Leucine, Phenylalanine, Tryptophan and Valine as hydrophobic amino acids. Tandem repeat domains of the tandem repeat proteins were analyzed in a same way. As a control, 108 genes, which were randomly selected from the  L. infantum  gene database, were also analyzed isometric points and amino acid compositions of their deduced amino acid sequences. 
     Example 13 
     Expression of Recombinant Proteins 
     Sequences encoding whole or partial tandem repeat domains of LinJ20.1220, LinJ25.1100, LinJ32.3710 were amplified by PCR. The amplified PCR products were inserted into multi cloning sites of pET28a and the inserts were confirmed their sequences matching with the ones on database. The vectors with the inserts were transformed into expression host  E. coli  and the recombinant proteins were purified as 6× His-tagged proteins using Ni-NTA agarose (Qiagen Inc., Valencia, Calif.). The concentrations of these proteins were measured by BCA protein assay (Pierce Biotechnology Inc., Rockford, Ill.). 
     Example 14 
     Recognition of Tandem Repeat Proteins by Sera From Leishmaniasis Patients 
     To evaluate antigenicity of the tandem repeat proteins, ELISA was performed using 200 ng antigen per well in 96-well plates. Sera from visceral Leishmaniasis patients (Sudan), cutaneous leishmaniasis patients (Brazil) and healthy individuals (United States) were used at 1:100 dilutions for the ELISA. 
     The visceral Leishmaniasis patient sera showed significantly stronger reactivity to all the recombinant proteins than sera from non-visceral Leishmaniasis groups, i.e. tuberculosis, malaria and cutaneous leishmaniasis patients or healthy individuals, despite the finding that cutaneous leishmaniasis patients showed antibody responses to LiSLA ( FIG. 6 ). Among the tested proteins, rLinJ16.1750r2 was the best antigen, and was recognized strongly by visceral Leishmaniasis patient sera with a high degree of specificity. Next, rLinJ16.1750r2 was tested with additional Sudanese visceral Leishmaniasis patient sera. Among 35 sera tested, 34 showed antibody responses to rLinJ16.1750r2 (the cutoff value was the mean+3 SD of healthy controls:  FIG. 6 ). The sensitivity using rLinJ16.1750r2 was 97%, comparable to that of rK39 (35/35, 100%:  FIG. 6 ). Mean O.D. values to rLinJ16.1750r2 and rK39 were 1.159 and 1.771, respectively. 
     Although these 35 sera were 100% positive using rK39, eight sera showed low reactivity to rK39 (OD values were &lt;1.0:  FIG. 6 ). These eight samples were tested for reactivity to rLinJ16.1750r2, rLinJ22.1590r3, rLinJ28.2310 or rLinJ33.2870r1 to examine whether or not those antigens could complement rK39 for more sensitive antibody detection. Five of the eight sera showed better responses to the new antigens than to rK39 (No. 3-7 in  FIG. 6 ). Five patients (No. 3-7) showed better responses to rLinJ22.1590r3, four (No. 4-7) to rLinJ28.2310, and three (No. 3, 5 and 6) to rLinJ33.2870r1. Finally, rLinJ22.1590r3 was recognized weakly by all the eight sera compared to rK39. 
     Example 15 
     ELISA Analysis of Tandem Repeat Protiens Identified by Serological Screening and Bioinformatic Analysis 
     Recombinant tandem repeat proteins or  L. infantum  promastigote soluble lysate antigen (LiSLA) were diluted in ELISA coating buffer, and 96-well plates were coated with rK39 (50 ng), other recombinant proteins (200 ng) or LiSLA (1 μg) followed by blocking with phosphate-buffered saline containing 0.05% Tween-20 and 1% bovine serum albumin. Next, the plates were incubated with patient sera (n=16) as well as healthy controls (n=8) at 1:100 dilution and then with horseradish peroxidase-conjugated anti-human IgG (Rockland Immunochemicals, Inc., Gilbertsville, Pa.). The plates were developed with TMB peroxidase substrate (KPL) and read by a microplate reader at a 450 nm wavelength (570 nm as a reference). 
     Compared to healthy controls, higher levels of antibody were detected being bound to tandem repeat proteins not only from serological screening but also solely from the bioinformatic analysis in VL patients (Table IV) ( FIG. 6 ). This demonstrates that tandem repeat proteins not only from serological screening but also identified solely by the bioinformatic analysis are antigenic. 
     
       
         
           
               
             
               
                 TABLE IV 
               
             
            
               
                   
               
               
                 Reactivity of L. infantum Tandem Repeat proteins in ELISA 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 PSa 
                   
                 Size 
                 OD: 
                 OD: 
                 OD: 
               
               
                   
                 (aa) 
                 CNa 
                 (kDa) 
                 VL 
                 HC 
                 P value 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Recombinant 
                   
                   
                   
                   
                   
                   
               
               
                 Identified by 
                   
                   
                   
                   
                   
                   
               
               
                 serology 
                   
                   
                   
                   
                   
                   
               
               
                 rLinJ05.0380r6 
                 38 
                 6 
                 28 
                 0.786 
                 0.142 
                 ** 
               
               
                 rLinJ16.1750r2 
                 73 
                 2 
                 18 
                 0.847 
                 0.058 
                 *** 
               
               
                 rLinJ22.1590r3 
                 28 
                 3 
                 12 
                 0.474 
                 0.042 
                 *** 
               
               
                 rLinJ28.2310TR 
                 105 
                 2.2 
                 35 
                 1.326 
                 0.123 
                 *** 
               
               
                 rLinJ33.2870r1 
                 148 
                 1 
                 18 
                 0.476 
                 0.137 
                 ** 
               
               
                 rLinJ34.2140r2 
                 83 
                 2 
                 20 
                 0.327 
                 0.126 
                 ** 
               
               
                 Identified 
                   
                   
                   
                   
                   
                   
               
               
                 solely from 
                   
                   
                   
                   
                   
                   
               
               
                 bioinformatics 
                   
                   
                   
                   
                   
                   
               
               
                 rLinJ11.0070r2 
                 46 
                 2 
                 12 
                 0.359 
                 0.065 
                 *** 
               
               
                 rLinJ21.2010TR 
                 64 
                 5.3 
                 38 
                 0.274 
                 0.048 
                 *** 
               
               
                 rLinJ25.1100TR 
                 22 
                 9.6 
                 27 
                 0.520 
                 0.032 
                 *** 
               
               
                 rLinJ27.0400r2 
                 68 
                 2 
                 18 
                 0.748 
                 0.117 
                 *** 
               
               
                 rLinJ29.0110TR 
                 8 
                 28.8 
                 31 
                 0.724 
                 0.185 
                 ** 
               
               
                 rLinJ32.3710TR 
                 33 
                 3.8 
                 17 
                 0.192 
                 0.124 
                 * 
               
               
                 CRUDE 
                   
                   
                   
                   
                   
                   
               
               
                 LiSLA 
                   
                   
                   
                 0.885 
                 0.082 
                 *** 
               
               
                   
               
            
           
         
       
     
     Notes: aPS: length of one copy of the repeat in units of amino acids, CN: copy number. bMean OD values of VL patients (VL: n=16) and healthy controls (HC: n=8) are shown. P values are from statistical analyses using Mann-Whitney test. *P&lt;0.05, **P&lt;0.01. ***P&lt;0.001. 
     From the foregoing, it will be appreciated that, although specific embodiments of this invention have been described herein for the purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims.