Chagas disease serology antigen

Abstract

T, cruzi antigens that are useful for detection infection are provided. Methods of detection and various assay formats are also provided.

Description

PATENT Attorney Docket No.110221-1519890-011910WO Client Ref. No. CZB-315F-PC / SF2025-004 CHAGAS DISEASE SEROLOGY ANTIGEN CROSS-REFERENCE OF RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 692,888 filed September 10, 2024, the content of which is hereby incorporated by reference in its entirety for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0001] This invention was made with government support under K38 HL154203 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0002] Chagas disease is caused by infection of the protozoan parasite, Trypanosoma cruzi. This disease is endemic to the Americas, with the highest prevalence in Latin America. In the United States, the major burden of disease is in Latinx immigrant populations, though autochthonous transmission has been documented throughout the southern half of the country. Chronic infection is assumed lifelong in the absence of treatment, where the parasite persists intracellularly within the cardiac and gastrointestinal systems leading to end-organ damage after decades. Given the mechanism of disease, detection of anti-T. cruzi antibodies is the most sensitive method for parasite detection in the chronic phase. However, imperfect performance of currently available commercial serological assays combined with the low overall prevalence of disease have necessitated detection by at least two separate tests for confirming the diagnosis. The medical implications of Chagas disease detection are numerous, including clinical diagnosis, blood donor screening, and solid organ or hematopoietic stem cell transplant donor and recipient testing. As such, improvements in T. cruzi serological diagnostics could have great implications across many specialties of healthcare.1 KILPATRICK TOWNSEND 800161941BRIEF SUMMARY OF THE INVENTION

[0003] In some embodiments, methods of detecting the presence or absence of T. cruzi in a sample from a human subject are provided. In some embodiments, the method comprises contacting a sample from the human subject to a polypeptide having an epitope comprising DILLEFR(D / E)LA (SEQ ID NO:1); and detecting the presence or absence of an antibody in the sample that binds to the epitope, thereby detecting the presence or absence of Trypanosoma cruzi in the sample.

[0004] In some embodiments, the sample is a blood sample, bodily fluid or a tissue sample. In some embodiments, the sample is a blood sample for blood transfusion or a separate sample from the individual who has donated the blood sample, and the method further comprises: (i) adding the blood sample to blood supply for blood transfusion if the antibody is not present in the sample, or (ii) discarding or quarantining the sample if the antibody is present in the sample.

[0005] In some embodiments, the detecting detects the presence of the antibody, and the method further comprises treating the human to kill or inhibit Trypanosoma cruzi in the human. In some embodiments, the treating comprises administering nifurtimox or benznidazole to the human subject.

[0006] In some embodiments, the polypeptide comprises IPSDHDILLEFR(D / E)LA (SEQ ID NO:2). In some embodiments, the polypeptide comprises GGADAAPTPSSAAPGETKVPSELNATIPSDHDILLEFRDLAAMALIG (SEQ ID NO:3) or TAPGETKIPSELNATIPSDHDILLEFRELAAMALIGDSTVHVCVSRV (SEQ ID NO:4).

[0007] In some embodiments, the method further comprises detecting an antibody in the sample that binds to: RAQELAREKKLADRAFLDQKPEGVPLRELPLDDDSDFVAMEQERRQQLEKDPRRNAKE IA (SEQ ID NO:5); PSPFGQAAAGDKPSPFGQAAAGDK (SEQ ID NO:6); KSAEPKSAEPKSAEP(SEQ ID NO:7); EKQKAAEATKVAEA(SEQ ID NO:8); ALPQEEQEDVGPRHVDPDHFRSTTQDAYRPVDPSAYKR(SEQ ID NO:9);2 KILPATRICK TOWNSEND 800161941DSTAHGTPSTPADSSAHSTPSTPA(SEQ ID NO:10); QKAAENERLADELE(SEQ ID NO:11); QKAAEATKVAEAEKQRAREATKVAEAEK(SEQ ID NO:12); PFGQAAAGDKPS(SEQ ID NO:13); AAPAKAA(SEQ ID NO:14); AEPKSAEPKP(SEQ ID NO:15); KAAIAPAKAAAAPAKAATAPA(SEQ ID NO:16); KTAAPPAKTAAPPAKTAAPPA(SEQ ID NO:17); GTSEEGSRGGSSMPSGTSEEGSRGGSSMPA(SEQ ID NO:18);or KFAELLEQQKNAQFPGK(SEQ ID NO:19).

[0008] In some embodiments, the detecting is performed as part of a chemiluminescent immunoassay, lateral flow assay, immunohistochemical staining, immunoprecipitation, microarray analysis, enzyme-linked immunosorbent assay (ELISA), or Western blot analysis.

[0009] In some embodiments, the polypeptide is immobilized on a solid support and the method comprises incubating the sample with the polypeptide under conditions that antibodies specific for the epitope bind to the polypeptide; washing unbound components of the sample from the polypeptide immobilized on solid surface; and detecting the presence, absence or amount of antibody bound the polypeptide immobilized on the solid surface.

[0010] In some embodiments, a polypeptide is provided comprising an epitope fused to a heterologous amino acid sequence, wherein the epitope comprises DILLEFR(D / E)LA (SEQ ID NO:1). In some embodiments, the polypeptide comprises IPSDHDILLEFR(D / E)LA (SEQ ID NO:2). In some embodiments, the polypeptide comprises GGADAAPTPSSAAPGETKVPSELNATIPSDHDILLEFRDLAAMALIG (SEQ ID NO:3) or TAPGETKIPSELNATIPSDHDILLEFRELAAMALIGDSTVHVCVSRV (SEQ ID NO:4).

[0011] In some embodiments, a polypeptide is provided immobilized to a solid support, wherein the polypeptide comprises an epitope comprising DILLEFR(D / E)LA (SEQ ID NO:1).3 KILPATRICK TOWNSEND 800161941In some embodiments, the polypeptide is covalently linked to the solid support.In some embodiments, the solid support is part of a lateral flow. In some embodiments, the solid support is a bead, membrane, well, or plate. In some embodiments, the polypeptide comprises IPSDHDILLEFR(D / E)LA (SEQ ID NO:2). In some embodiments, the polypeptide comprises GGADAAPTPSSAAPGETKVPSELNATIPSDHDILLEFRDLAAMALIG (SEQ ID NO:3) or TAPGETKIPSELNATIPSDHDILLEFRELAAMALIGDSTVHVCVSRV (SEQ ID NO:4).

[0012] In some embodiments, the solid support further comprises at least a second polypeptide immobilized on the solid support, wherein the second polypeptide comprises: RAQELAREKKLADRAFLDQKPEGVPLRELPLDDDSDFVAMEQERRQQLEKDPRRNAKE IA (SEQ ID NO:5); PSPFGQAAAGDKPSPFGQAAAGDK (SEQ ID NO:6); KSAEPKSAEPKSAEP (SEQ ID NO:7); EKQKAAEATKVAEA (SEQ ID NO:8); ALPQEEQEDVGPRHVDPDHFRSTTQDAYRPVDPSAYKR (SEQ ID NO:9); DSTAHGTPSTPADSSAHSTPSTPA(SEQ ID NO:10); QKAAENERLADELE (SEQ ID NO:11); QKAAEATKVAEAEKQRAREATKVAEAEK (SEQ ID NO:12); PFGQAAAGDKPS (SEQ ID NO:13); AAPAKAA (SEQ ID NO:14); AEPKSAEPKP (SEQ ID NO:15); KAAIAPAKAAAAPAKAATAPA (SEQ ID NO:16); KTAAPPAKTAAPPAKTAAPPA (SEQ ID NO:17); GTSEEGSRGGSSMPSGTSEEGSRGGSSMPA (SEQ ID NO:18); or KFAELLEQQKNAQFPGK (SEQ ID NO:19). KILPATRICK TOWNSEND 800161941

[0013] In some embodiments, a polypeptide is provided linked to a heterologous label, wherein the polypeptide comprises an epitope comprising DILLEFR(D / E)LA (SEQ ID NO:1). In some embodiments, the polypeptide comprises IPSDHDILLEFR(D / E)LA (SEQ ID NO:2). In some embodiments, the polypeptide comprises GGADAAPTPSSAAPGETKVPSELNATIPSDHDILLEFRDLAAMALIG (SEQ ID NO:3) or TAPGETKIPSELNATIPSDHDILLEFRELAAMALIGDSTVHVCVSRV (SEQ ID NO:4). In some embodiments, the label comprises a fluorescent or chemiluminescent moiety.

[0014] Also provided are kits comprising the polypeptide as described above or elsewhere herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG.1 depicts results of measurement of antibody reactivity from patients having and not having Chagas disease. The figure shows trans-sialidase peptide fragment 23 is a Chagas disease antigen that cross-reacts with antibodies specifically in Chagas- disease individuals across multiple patient cohorts. Bolivian cohort: patients with Chagas cardiomyopathy, patients with T. cruzi infection but no heart disease, and seronegative patients. Blood donor cohort: remnant plasma from United States blood donors with confirmed seropositive or seronegative screening for anti-T. cruzi serology testing. The data depicts is PhIP-seq data, plotting reads per 100,000 (RPK) for a given trans-sialidase peptide fragment. Each dot represents the RPK for a given patient’s serum sample run on the assay. RPK is a read-out of general reactivity to a given peptide. For example, if a patient’s RPK for a given peptide is 0, they had no reactivity to that peptide, whereas if it is above zero, they have some degree of reactivity. RPK is generally proportional to how reactive a patient’s antibodies are to that peptide (antibody affinity).

[0016] FIG.2 depicts exemplary trans-sialidase antigenic peptide sequences.

[0017] FIG.3 depicts results of an orthogonal validation (split-luciferase binding assay) that confirmed Chagas disease-specific trans-sialidase antigen reactivity in the depicted trans- sialidase peptides.

[0018] FIG.4 summarizes results from a split-luciferase binding assay used to identify the reactive epitope from trans-sialidase fragment 2. Using an alanine-scanning approach, the reactive epitope was mapped. Each bar represents the average normalized antibody index across5 KILPATRICK TOWNSEND 800161941four patients for each sequentially mutated peptide. Each patient’s plasma was reacted against each sequentially mutated peptide. Loss of binding (decreased antibody index) with a specific peptide indicates that the antigenic epitope of the peptide is contained within the alanine-mutated sequence. In this case, the shared antigenic epitope across all patients is IPSDHDILLEFRDLA. DEFINITIONS

[0019] Unless otherwise dictated by context, singular terms shall include pluralities, and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry are those well-known and commonly used. Known methods and techniques are generally performed according to conventional methods well-known and as described in various general and more specific references, unless otherwise indicated. The nomenclatures used in connection with the laboratory procedures and techniques described in the present disclosure are those well-known and commonly used.

[0020] As used herein, the terms “a”, “an”, and “the” can refer to one or more unless specifically noted otherwise.

[0021] The use of the term “or” is used to mean “and / or,” unless explicitly indicated to refer to alternatives only, or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” can mean at least a second or more.

[0022] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20% (%); preferably, within 10%; and more preferably, within 5% of a given value or range of values. Any reference to “about X” or “approximately X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions “about X” or “approximately X” are intended to teach and provide written support for a claim limitation of, for example, “0.98X.” Alternatively, in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated6 KILPATRICK TOWNSEND 800161941otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated. When “about” is applied to the beginning of a numerical range, it applies to both ends of the range.

[0023] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers and non-natural amino acid polymers, as well as to amino acid polymers in which one (or more) amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid. The terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0024] An “isolated” or “purified” polypeptide or protein, or biologically active portion a polypeptide or a protein, is substantially or essentially free from components that normally accompany or interact with the polypeptide or protein as found in its naturally occurring environment. Thus, an isolated or purified polypeptide or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% (total protein) of contaminating protein. When the protein of the invention or its biologically active portion is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% (by concentration) of chemical precursors or non-protein-of-interest chemicals.

[0025] The term “amino acid” refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally-occurring -amino acids and their stereoisomers, as well as unnatural (non-naturally occurring) amino acids and their stereoisomers. “Stereoisomers” of a given amino acid refer to isomers having the same molecular formula and intramolecular bonds but different three-dimensional arrangements of bonds and atoms (e.g., an L-amino acid and the corresponding D-amino acid).

[0026] Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, -carboxyglutamate, and O- phosphoserine. Naturally-occurring -amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), KILPATRICK TOWNSEND 800161941histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and their combinations. Stereoisomers of a naturally-occurring -amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D- isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D- Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D- threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and their combinations. In some embodiments, a polypeptide comprising a trans-sialidase epitope sequence as described herein can comprise all natural amino acids or one or more amino acid can be unnatural amino acid.

[0027] Unnatural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in either the L- or D-configuration that function in a manner similar to the naturally- occurring amino acids. For example, “amino acid analogs” can be unnatural amino acids that have the same basic chemical structure as naturally-occurring amino acids (i.e., a carbon that is bonded to a hydrogen, a carboxyl group, an amino group) but have modified side-chain groups or modified peptide backbones, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. “Amino acid mimetics” refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally-occurring amino acid. Amino acids may be referred to by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0028] The expression “conservatively modified variant” and related expression may apply to amino acid sequences, as well to nucleic acid sequences encoding amino acid sequence, for example, comprising or encoding a trans-sialidase epitope sequence as described herein. Substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively8 KILPATRICK TOWNSEND 800161941modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).

[0029] The terms “identity,” “substantial identity,” “similarity,” “substantial similarity,” “homology” and the related terms and expressions used in the context of describing nucleic acid or amino acid sequences refer to a sequence that has at least 60% sequence identity to a reference sequence e.g., a trans-sialidase epitope sequence as described herein). Examples include at least: 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity, as compared to a reference sequence using the programs for comparison of nucleic acid or amino acid sequences, such as BLAST using standard parameters. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default (standard) program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. A “comparison window” includes reference to a segment of any one of the number of contiguous positions (from 20 to 600, usually about 50 to about 200, more commonly about 100 to about 150), 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. Methods of alignment of sequences for comparison are well-known. Optimal alignment of sequences for comparison may be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, by the homology9 KILPATRICK TOWNSEND 800161941alignment algorithm of Needleman and Wunsch, 1970, by the search for similarity method of Pearson and Lipman, 1988, by computerized implementations of these algorithms (for example, BLAST), or by manual alignment and visual inspection.

[0030] Algorithms that are suitable for determining percent sequence identity and sequence similarity include BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, and Altschul et al., 1977, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is 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 word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1989). The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (Karlin and Altschul, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic10 KILPATRICK TOWNSEND 800161941acid is less than about 0.01, more preferably less than about 10-5, and most preferably less than about 10-20.

[0031] The term “antibody” and the related terms refer to an immunoglobulin or its fragment that binds to a particular spatial and polar organization of another molecule. Immunoglobulins include various classes and isotypes, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgG4, IgM, etc. Naturally occurring antibodies are encoded by immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are typically classified as either kappa or lambda. Heavy chains are typically classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes. A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms “variable light chain” (VL) and “variable heavy chain” (VH) refer to these light and heavy chains respectively. An antibody can be specific for a particular antigen. The antibody or its antigen can be either an analyte or a binding partner. The term “antibody,” as used herein, also includes antibody fragments that retain binding specificity, including but not limited to Fab, F(ab’)2, Fv, and scFv.

[0032] An exemplary immunoglobulin (antibody) structural unit comprises two identical pairs of polypeptide chains, each pair having one “light” chain (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. Thus, the terms “variable heavy chain” or “VH” refer to the variable region of an immunoglobulin heavy chain, including an Fv, scFv, dsFv or Fab; while the terms “variable light chain” or “VL” refer to the variable region of an immunoglobulin light chain, including an Fv, scFv, dsFv or Fab.

[0033] The term “antigen” refers to a molecule, such as a polypeptide, containing one or more epitopes (either linear, conformational or both) that can stimulate a subject’s immune system to produce antigen-specific immune response. A polypeptide epitope may be for example, between11 KILPATRICK TOWNSEND 800161941about 7 and 17 amino acids, such as, 9, 10, 12 or 15 amino acids, which in some embodiments, are contiguous amino acids.

[0034] The terms “individual”, “subject”, and “patient” can be used interchangeably in the present disclosure to refer to a non-human animal or a human. Examples of subjects include, but are not limited to: humans and other primates, including non-human primates, such as chimpanzees and other apes and monkey species; farm animals, such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs; birds, including domestic, wild and game birds, such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The terms individual, subject, and patient, by themselves, do not denote a particular age, sex, race, or clinical status. Thus, subjects of any age, whether male or female, are intended to be covered by the present disclosure and include, but are not limited to the elderly, adults, children, babies, infants, and toddlers. Likewise, the methods of the present invention can be applied to any human race, including, for example, Caucasian (white), African-American (black), Native American, Native Hawaiian, Hispanic, Latino, Asian, and European.

[0035] The terms “administering” or “administration,” when using in the context of the present disclosure (and the related terms and expression), refer to the act of physically delivering a substance as it exists outside the body (for example, an immunogenic composition described in the present disclosure) into a subject. Administration can be by mucosal, intradermal, intravenous, intramuscular, subcutaneous delivery and / or by any other known methods of physical delivery. Administration encompasses direct administration, such as administration to a subject by a medical professional or self-administration, or indirect administration, which may be the act of prescribing a composition described in the present disclosure.

[0036] As used herein, the term “biological sample” encompasses any sample obtained from a biological source. A biological sample can, by way of non-limiting example, include blood, serum, plasma, tissue biopsy, cell-free DNA, amniotic fluid, cerebrospinal fluid, sera, urine, feces, epidermal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample and / or chorionic villi. Convenient biological samples may be obtained by, for example, scraping cells from the surface of the buccal cavity. The term biological sample encompasses samples which have been processed to release or otherwise make available a12 KILPATRICK TOWNSEND 800161941nucleic acid or protein for detection as described herein. The biological sample may be obtained from a stage of life such as a fetus, young adult, adult, and the like. Fixed or frozen tissues also may be used. DETAILED DESCRIPTION OF THE INVENTION

[0037] From a peptide library from Trypanosoma cruzi, the inventors have discovered a peptide epitope that generates antibodies in humans infected with T. cruzi. Accordingly, the presence of human antibodies that bind to the epitope can be used to detect humans carrying T. cruzi with a high specificity and sensitivity. The present disclosure provides methods of detecting T. cruzi as well as reagents and kits useful for detection of T. cruzi.

[0038] As discussed in the examples in detail, antibodies that bind to a polypeptide comprising DILLEFR(D / E)LA (SEQ ID NO:1) indicate the presence of T. cruzi with high specificity and sensitivity. Accordingly, methods of detecting T. cruzi comprising detection of antibodies that specifically bind to SEQ ID NO:1 in a sample from a human are provided. In general, methods of detection can comprise providing a polypeptide comprising or consisting of SEQ ID NO:1, contacting a sample from a human to the polypeptide, and detecting antibodies from the sample that bind to the polypeptide. As shown in the examples, both GGADAAPTPSSAAPGETKVPSELNATIPSDHDILLEFRDLAAMALIG (SEQ ID NO:3) and TAPGETKIPSELNATIPSDHDILLEFRELAAMALIGDSTVHVCVSRV (SEQ ID NO:4) bind to antibodies from T. cruzi individuals and thus in some embodiments, the polypeptides used to detect antibodies in a sample from a human can comprise or consist of SEQ ID NO:3 or SEQ ID NO:4. The bolded portions of the sequences above completely overlap except for one amino acid underlined and represented by “(D / E)” in SEQ ID NO:1. As shown in the examples, alanine scanning indicates that all polypeptides comprising SEQ ID NO:1 react with infected patient serum, indicating that antibody-binding to SEQ ID NO:1 is indicative of infection with T. cruzi. In some embodiments, the polypeptide comprises or consists of IPSDHDILLEFR(D / E)LA (SEQ ID NO:2). In some embodiments, the polypeptide comprises or consists of IPSDHDILLEFRDLA (SEQ ID NO:20) or IPSDHDILLEFRELA (SEQ ID NO:21) or DILLEFRDLA (SEQ ID NO:22) or DILLEFRELA (SEQ ID NO: 23).13 KILPATRICK TOWNSEND 800161941

[0039] In some embodiments, the polypeptide comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 but comprises no additional (or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 contiguous) amino acids from T. cruzi trans-sialidase. In some embodiments, the polypeptide can however comprise other additional heterologous amino acid sequences. Heterologous amino acid sequences can be included in the polypeptide for a variety of reasons, for example as spacer between a solid support and the epitope trans-sialidase sequence. In other embodiments, the polypeptide can comprise one or more additional T. cruzi epitopes from a polypeptide other than can T. cruzi trans-sialidase. In some embodiments, the one or more additional epitopes are selected from Table 1. Table 1: Other T. cruzi epitopes14 KILPATRICK TOWNSEND 800161941

[0040] The polypeptide comprising the epitope can be any length so that the polypeptide allows for presentation of the epitope to sample antibodies and optionally short enough to avoid presentation of epitopes unrelated to T. cruzi, or for example in some cases the polypeptide does not comprise other epitopes for which no more than 1 or 2 people in 100 carry antibodies.

[0041] Any sample from an individual that contains antibodies can be assayed with the T. cruzi trans-sialidase epitope described herein. Exemplary samples can be, but are not limited to, blood or tissue samples. Exemplary blood samples can include but are not limited to blood of any volume for testing from the individual. In some embodiments, the blood sample is for use in blood transfusion or otherwise for entry into the blood donation and transfusion system. In some embodiments, the blood sample is an umbilical cord sample.

[0042] In some embodiments, the sample is from a potential organ or blood donor. In these embodiments, if T. cruzi is detected, then the blood transfusion does not take part and if already collected, the blood product from the infected individual is discarded. If T. cruzi is not detected, then the organ or blood donation can take place, i.e., introduced into a second individual. Likewise, in some embodiments, the subject is a potential heart donor. If T. cruzi is detected, then heart transplant does not take part and if already collected, the heart from the infected individual is discarded. If T. cruzi is not detected, then the heart transplant can take place, i.e., introduced into a second individual. For other organs, in some embodiments, the same criteria can apply or a different clinical management decision can be applied.15 KILPATRICK TOWNSEND 800161941

[0043] In some embodiments, the individual is determined to have antibodies that bind to the T. cruzi trans-sialidase epitope described herein and is subsequently treated to kill T. cruzi in the individual. Any effective treatment for killing T. cruzi or Chagas disease can be used. In some embodiments, nifurtimox or benznidazole are administered to the individual.

[0044] Any detection assay format can be used that detects antibodies in a sample that bind to the T. cruzi trans-sialidase epitope (e.g., comprising SEQ ID NO:1) described herein. In some embodiments, an isolated, purified polypeptide comprising a T. cruzi trans-sialidase epitope may be used in the provided methods. In some embodiments, the polypeptides can be recombinantly produced and / or purified. Modified polypeptides comprising one or more T. cruzi trans-sialidase epitopes are also contemplated, such as those in which one or more amino acid residues are substituted or modified (such as with glutaraldehyde or an unnatural amino acid).

[0045] In some embodiments, methods of detecting the presence of an antibody that specifically binds to a T. cruzi trans-sialidase epitope as described herein in a biological sample from a subject are provided, comprising the steps of contacting the biological sample with a polypeptide comprising a T. cruzi trans-sialidase epitope according to the present disclosure (e.g., comprising SEQ ID NO:1) and detecting binding of the polypeptide or the fragment or the variant thereof to the antibody in the biological sample. In some embodiments according to the present disclosure, a liquid sample comprising antibodies from a subject that bind to the T. cruzi trans-sialidase epitope may be used to practice the methods. Exemplary liquid samples include cell lysate, blood, serum, cerebrospinal fluid (CSF), and urine. In some embodiments, a liquid sample, such as a cell lysate, can be produced from a non-liquid sample, such as a biopsy sample or a sample of brain tissue. A step of contacting a liquid sample comprising antibodies that bind to the T. cruzi trans-sialidase epitope with a polypeptide comprising a T. cruzi trans-sialidase epitope may be carried out by incubating an immobilized form of said polypeptide in the presence of the liquid sample under conditions that are compatible with the formation of a complex comprising said polypeptides and the antibodies that bind to the T. cruzi trans-sialidase epitope. Optionally, the liquid sample, then at least partially depleted of antibodies that bind to the T. cruzi trans-sialidase epitope may subsequently be removed to facilitate detection of a complex between the antibodies that bind to the T. cruzi trans-sialidase epitope and the16 KILPATRICK TOWNSEND 800161941polypeptide comprising a T. cruzi trans-sialidase epitope. Optionally, one or more washing steps may be contemplated.

[0046] In some embodiments, the biological sample is contacted with a polypeptide comprising a T. cruzi trans-sialidase epitope and a secondary antibody. In some embodiments, the secondary antibody is an antibody raised against the IgG of the animal species in which the primary antibody originated (e.g., an “anti-human antibody” that binds to the Fc portion of human IgGs). Secondary antibodies bind to the primary antibody to assist in detection, sorting, and purification of target antigens to which a specific primary antibody is first bound. The secondary antibody can have specificity both for the antibody species as well as the isotype of the primary antibody being used. If a antibody that binds to the T. cruzi trans-sialidase epitope is present in the biological sample, under appropriate conditions, a complex is formed between the polypeptide comprising a T. cruzi trans-sialidase epitope, the antibody (that binds to the T. cruzi trans-sialidase epitope) in the biological sample, and the secondary antibody.

[0047] A complex comprising the the polypeptide comprising a T. cruzi trans-sialidase epitope and the antibody that binds to the T. cruzi trans-sialidase epitope may be detected using a variety of methods known to the person skilled in the art, for example immunofluorescence microscopy or spectroscopy, luminescence, NMR spectroscopy, immunodiffusion, radioactivity, chemical crosslinking, surface plasmon resonance, native gel electrophoresis, or enzymatic activity. Depending on the nature of the sample, either or both immunoassays and immunocytochemical staining techniques may be used. Enzyme-linked immunosorbent assays (ELISA), Western blot, and radioimmunoassays are methods used in the art, and can be used as described herein to detect the presence of antibodies that binds to the T. cruzi trans-sialidase epitope in a biological sample. While some of these methods allow for the direct detection of the complex, in some embodiments, the second antibody is labeled such that the complex may be detected specifically owing to intrinsic properties of the label such as, for example, fluorescence, radioactivity, enzymatic activity, visibility in NMR, or MRI spectra or the like. In some embodiments, the detection method may include any of Western blot, dot blot, protein microarray, ELISA, line blot radioimmune assay, immunoprecipitation, indirect immunofluorescence microscopy, radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistostaining, complement fixation assay, FACS, and protein17 KILPATRICK TOWNSEND 800161941chip, but is not limited thereto. Methods and compositions are described herein that can be used for detecting, by immunohistochemistry, the presence of antibodies specific for the epitopes described herein in a biological sample. Immunohistochemical methods are well known in the art, and non-limiting exemplary methods are described in U.S. Pat. Nos. 5,073,504; 5,225,325; and 6,855,552. See also Dabbs, Diagnostic Immunohistochemistry, 2ndEd., 2006, Churchill Livingstone; and Chu & Weiss, Modern Immunohistochemistry, 2009, Cambridge University Press. It would be understood by those skilled in the art that immunohistochemistry routinely includes steps that are not necessarily discussed herein in detail such as washing the tissue samples to remove unbound secondary antibodies and the parallel staining experiments with proper controls.

[0048] Assays to detect epitope-antibody interactions can include direct to indirect assays. In some embodiments, for example, the assay is a competitive assay or a non-competitive assay. Exemplary competitive assays can involve, for example, contacting the sample with a reagent that binds to the target molecules, in this case an antibody that binds to the target epitope, and then contacting the resulting mixture with a detection antibody that also binds the epitope. The detection antibody in some embodiments is immobilized on a surface the epitope is part of a polypeptide comprising a detectable label. In competitive assays, in general, the amount of signal (e.g., the amount of epitope that binds to the detection antibody) is inversely proportional to the signal generated.

[0049] Exemplary assay formats can include, but are not limited to, a chemiluminescent immunoassay, lateral flow assay, immunohistochemical staining, immunoprecipitation, microarray analysis, enzyme-linked immunosorbent assay (ELISA), or Western blot analysis.

[0050] Exemplary chemiluminescent immunoassay formats can comprise copies the T. cruzi trans-sialidase epitope described herein immobilized on magnetic or paramagnetic beads. Accordingly the disclosure provides one or more polypeptides comprising a T. cruzi trans- sialidase epitope described herein immobilized on magnetic or paramagnetic beads. Exemplary chemiluminescent immunoassay formats are reviewed in Cinquanta, et al., Auto Immun Highlights.2017 Dec; 8(1): 9. In a chemiluminescent immunoassay, the beads are mixed with a sample from an individual under conditions to allow for any reactive antibodies to bind to the epitope. Non-binding components of the assay can be removed by washing, for example while18 KILPATRICK TOWNSEND 800161941the beads are held in place by a magnetic force. A secondary antibody can then be bound to antibodies from the sample bound to the T. cruzi trans-sialidase epitope immobilized on the beads. The secondary antibody can then be detected as desired. In some embodiments, the secondary antibody is linked to one or more chemiluminescent moiety. Exemplary chemiluminescent moieties can include but are not limited to isoluminol or derivatives thereof. See, e.g., Messeri et al, J Biolumin Chemilumin.1989 Jul;4(1):154-8. The quantity of signal will be proportional to the amount of antibodies in the sample that react with the epitope immobilized on the beads.

[0051] Exemplary lateral flow assays can involve a flow path caused by capillary flow of a liquid. A lateral flow device can include a location on the flow path to which the sample is loaded and downstream can be located one or more capture reagents (e.g., the sample comprising antibodies can flow through the lateral flow device, encountering immobilized polypeptides comprising an epitope as described herein). Binding can be detected as described herein, for example via inclusion of detectable labels in the solution added to the later flow device such that bound antibodies are detected for example at the location of the capture reagent. A variety of later flow configurations have been described and can be used in the assays described herein.

[0052] In some instances, a primary or secondary antibody, or a polypeptide comprising the epitope (e.g., SEQ ID NO:1) is conjugated to a detectable label. Detectable labels are well known in the art and include, without limitation, a fluorescent label, an enzymatic label, a radioactive label, a luminescent label, or an affinity tag such as biotin or streptavidin. Exemplary fluorescent dyes include water-soluble rhodamine dyes, fluoresceins, 2’,7’-dichlorofluoresceins, fluorescein isothiocyanate (FITC), DyLight™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor®700, Cy5, allophycocyanin, Cy7, benzoxanthene dyes, and energy transfer dyes, as disclosed in the following references: Handbook of Molecular Probes and Research Reagents, 8thed. (2002), Molecular Probes, Eugene, OR; U.S. Patent Nos.6,191,278, 6,372,907, 6,096,723, 5,945,526, 4,997,928, and 4,318,846; and Lee et al.1997. Exemplary enzymatic labels include but are not limited to alkaline phosphatase (AP) and horseradish peroxidase (HP)). Luminescent labels include, e.g., any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the europium chelate of diethylene triamine pentaacetic acid (DTPA) or tetraazacyclododecane-19 KILPATRICK TOWNSEND 8001619411,4,7,10-tetraacetic acid (DOTA). Suitable radioactive labels include, for example,32P,33P,14C,125I,131I,35S, and3H. In some instances, the detectable label can be a heterologous polypeptide such as an antigenic tag such as, for example, FLAG, polyhistidine, hemagglutinin (HA), glutathione-S-transferase (GST), or maltose-binding protein (MBP)) for use in detection of the secondary antibody. In some instances, the detectable label can be a heterologous polypeptide that is useful as diagnostic or detectable marker such as, for example, luciferase, a fluorescent protein (such as a green fluorescent protein (GFP)), or chloramphenicol acetyl transferase (CAT). Another labeling technique which may result in greater sensitivity is the coupling the antibodies to low molecular weight haptens. These haptens can then be specifically altered by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific anti- hapten antibodies.

[0053] In some embodiments, the method comprises contacting a polypeptide comprising the T. cruzi trans-sialidase epitope with a biological sample from a subject and a secondary antibody having a suitable label thereon under conditions in which a complex is formed between the polypeptide comprising the T. cruzi trans-sialidase epitope , a corresponding antibody in the biological sample, if present, that binds to the T. cruzi trans-sialidase epitope and the secondary antibody; and detecting the complex formed, if formed, by detecting the label of the secondary antibody, wherein the presence of the secondary antibody is indicative of the presence of a T. cruzi in the biological sample, and wherein the absence of the secondary antibody is indicative of the absence of a T. cruzi in the biological sample. In some instances, the secondary antibody is detectably-labeled. Immobilization of the polypeptide comprising the T. cruzi trans-sialidase epitope on a solid carrier can facilitate detection. In some instances, the method comprises contacting a polypeptide comprising the T. cruzi trans-sialidase epitope having a suitable label thereon with a biological sample from a subject, and immunoprecipitating any complex formed between the polypeptide and a corresponding antibody in the biological sample that binds to the T. cruzi trans-sialidase epitope, and monitoring for said label on any of said complexes, wherein the presence of said label is indicative of the presence of antibodies in the biological sample that bind to that binds to the T. cruzi trans-sialidase epitope and the absence of said label is indicative of the absence of such an antibody in the biological sample. Exemplary labels include any of the20 KILPATRICK TOWNSEND 800161941detectable labels described in this disclosure including, for example, fluorescent dyes and radioactive labels.

[0054] Compositions, systems, and kits containing one or more polypeptides comprising a T. cruzi trans-sialidase epitope described in the present disclosure are included among the embodiments of the disclosure. An exemplary kit may include a polypeptide comprising a T. cruzi trans-sialidase epitope according to the present disclosure, and one or more components for detecting binding of the polypeptide to an antibody that binds to the epitope in the biological sample. In some embodiments, a kit may include a polypeptide comprising a T. cruzi trans- sialidase epitope immobilized on a solid support. A composition comprising a polypeptide comprising a T. cruzi trans-sialidase epitope may be a solution, such as an aqueous solution, a suspension, such as an aqueous suspension, or may be in dry form, such as in lyophilized form.

[0055] In some embodiments, one or more polypeptides comprising a T. cruzi trans-sialidase epitope described in the present disclosure can be immobilized on a solid carrier insoluble in an aqueous solution, such as via a covalent bond, electrostatic interactions, encapsulation or entrapment, for example by denaturing a globular polypeptide in a gel, or via hydrophobic interactions such as via one or more covalent bonds. Various suitable carriers, for example paper, metal, silicon or glass surfaces, microfluidic channels, membranes, beads such as magnetic beads, column chromatography media, biochips, polyacrylamide gels and the like have been described in the literature, for example in Kim et al.2013. This way, the immobilized molecule, together with the insoluble carrier, may be separated from an aqueous solution in a straightforward manner, for example by filtration, centrifugation or decanting. An immobilized molecule may be immobilized in a reversible or irreversible manner. For example, the immobilization is reversible if the molecule interacts with the carrier via ionic interactions that can be masked by addition of a high concentration of salt or if the molecule is bound via a cleavable covalent bond such as a disulfide bridge which may be cleaved by addition of thiol- containing reagents. By contrast, the immobilization is irreversible if the molecule is tethered to the carrier via a covalent bond that cannot be cleaved in aqueous solution, for example a bond formed by reaction of an epoxide group and an amine group as frequently used to couple lysine side chains to affinity columns. The protein may be indirectly immobilized, for example by immobilizing an antibody or other entity having affinity to the molecule, followed by formation21 KILPATRICK TOWNSEND 800161941of a complex to the effect that the molecule-antibody complex is immobilized. Various ways to immobilize molecules are described in the literature such as, for example, in Kim et al.2013. In addition, various reagents and kits for immobilization reactions are commercially available such as, for example, from Pierce Biotechnology. EXAMPLE

[0056] PhIP-Seq was performed using the T. cruzi peptide phage display library on less than 1 L plasma samples with confirmed blood donor testing (n = 90, 64 seropositive and 26 seronegative). Two rounds of enrichment were performed. To identify Chagas disease-specific enriched peptides and avoid false positives, a conservative analysis pipeline was used as follows. Peptide-level enrichment across seronegative samples was calculated and used to generate z- scores for the seropositive and seronegative samples. Peptides were considered Chagas disease- specific if they had a z-score 3 in at least eight seropositive samples and zero seronegative samples. Among the significantly enriched peptides, the trans-sialidase peptide fragment was reactive in 95% of seropositive patients and 0 seronegative patients (95% sensitivity, 100% specificity). A validation cohort was also immunoprecipitated against the T. cruzi peptide display library using the same experimental and analytical approach. This cohort consisted of plasma from 121 seropositive Bolivian patients and 22 seronegative Bolivian patients. Serum from 96 healthy blood donors from the New York Blood Center (US controls) were also run for background correction, based on the assumption that none of these patients had ever been exposed to T. cruzi antigens. Within this cohort, 90% of seropositive patients and 0 seronegative patients demonstrated reactivity above the z-score threshold to the trans-sialidase peptide fragment (90% sensitivity, 100% specificity).

[0057] Seroreactivity to the trans-sialidase peptide was orthogonally validated using a split- luciferase binding assay (SLBA). In short, oligos with the trans-sialidase sequence linked to a HiBiT tag were used to synthesize the trans-sialidase peptide with a wheat germ extract in vitro transcription / translation (IVTT) assay (Promega Cat #L4140). Peptides were then immunoprecipitated with seropositive and seronegative patient plasma and quantified using the Promega Nano-Glo® HiBiT Lytic Detection System (Cat #N3030). An antibody index for each KILPATRICK TOWNSEND 800161941plasma sample was calculated as (RLU sample – mean RLU blank wells) / (mean RLU anti- HiBiT antibody – mean RLU blank wells). Reactivity was considered positive if the antibody index was positive, and negative if the normalized antibody index was less than 0.1, implying a similar level of immunoprecipitation as a blank well in which no antibody was added at all. All seropositive patient samples tested (n = 4) showed positive reactivity to both trans-sialidase peptides (fragment 2 and fragment 23), and no seronegative patient samples (n = 5) demonstrated reactivity to either peptide fragment.

[0058] The precise reactive epitope was mapped using an alanine-scanning approach and further SLBAs. Oligonucleotides encoding trans-sialidase fragment 2 were designed with tiled windows of 10 alanines that sequentially covered the entire fragment with 5 amino acid overlaps in alanine blocks per peptide. These oligonucleotides were used to generate trans-sialidase peptides using the IVTT system described above. The synthesized peptides were normalized and immunoprecipitated with patient plasma as above. The resulting antibody index for each peptide was normalized to the full, wild type peptide fragment, and then normalized across each patient’s reactivity to all the peptides. Loss of binding to a given mutant peptide indicated that that mutant sequence covered the key antigenic epitope of the trans-sialidase peptide. All seropositive patient samples tested (n=4) demonstrated a loss of reactivity to peptides in which the sequence IPSDHDILLEFRELA was mutated.

[0059] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this specification should be considered as limiting the scope of the present invention. All examples presented are representative and non-limiting. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.23 KILPATRICK TOWNSEND 800161941

Claims

WHAT IS CLAIMED IS:

1. A method of detecting the presence or absence of Trypanosoma cruzi in a sample from a human subject, the method comprising, contacting a sample from the human subject to a polypeptide having an epitope comprising DILLEFR(D / E)LA (SEQ ID NO:1); and detecting the presence or absence of an antibody in the sample that binds to the epitope, thereby detecting the presence or absence of Trypanosoma cruzi in the sample.

2. The method of claim 1, wherein the sample is a blood sample, bodily fluid or a tissue sample.

3. The method of claim 1, wherein the sample is a blood sample for blood transfusion or a separate sample from the individual who has donated the blood sample and the method further comprises: (i) adding the blood sample to blood supply for blood transfusion if the antibody is not present in the sample, or (ii) discarding or quarantining the sample if the antibody is present in the sample.

4. The method of claim 1 or 2, wherein the detecting detects the presence of the antibody, and the method further comprises treating the human to kill or inhibit Trypanosoma cruzi in the human.

5. The method of claim 4, wherein the treating comprises administering nifurtimox or benznidazole to the human subject.

6. The method of any one of claims 1-5, wherein the polypeptide comprises IPSDHDILLEFR(D / E)LA (SEQ ID NO:2).

7. The method of any one of claims 1-5, wherein the polypeptide comprises GGADAAPTPSSAAPGETKVPSELNATIPSDHDILLEFRDLAAMALIG (SEQ ID NO:3) or TAPGETKIPSELNATIPSDHDILLEFRELAAMALIGDSTVHVCVSRV (SEQ ID NO:4).

8. The method of any one of claims 1-7, wherein the method further comprises detecting an antibody in the sample that binds to:24 KILPATRICK TOWNSEND 800161941RAQELAREKKLADRAFLDQKPEGVPLRELPLDDDSDFVAMEQERRQQLE KDPRRNAKEIA (SEQ ID NO:5); PSPFGQAAAGDKPSPFGQAAAGDK (SEQ ID NO:6); KSAEPKSAEPKSAEP(SEQ ID NO:7); EKQKAAEATKVAEA(SEQ ID NO:8); ALPQEEQEDVGPRHVDPDHFRSTTQDAYRPVDPSAYKR(SEQ ID NO:9); DSTAHGTPSTPADSSAHSTPSTPA(SEQ ID NO:10); QKAAENERLADELE(SEQ ID NO:11); QKAAEATKVAEAEKQRAREATKVAEAEK(SEQ ID NO:12); PFGQAAAGDKPS(SEQ ID NO:13); AAPAKAA(SEQ ID NO:14); AEPKSAEPKP(SEQ ID NO:15); KAAIAPAKAAAAPAKAATAPA(SEQ ID NO:16); KTAAPPAKTAAPPAKTAAPPA(SEQ ID NO:17); GTSEEGSRGGSSMPSGTSEEGSRGGSSMPA(SEQ ID NO:18);or KFAELLEQQKNAQFPGK(SEQ ID NO:19).

9. The method of any one of claims 1-8, wherein the detecting is performed as part of a chemiluminescent immunoassay, lateral flow assay, immunohistochemical staining, immunoprecipitation, microarray analysis, enzyme-linked immunosorbent assay (ELISA), or Western blot analysis.

10. The method of any one of claims 1-9, wherein the polypeptide is immobilized on a solid support and the method comprises incubating the sample with the polypeptide under conditions that antibodies specific for the epitope bind to the polypeptide; washing unbound components of the sample from the polypeptide immobilized on solid surface; and detecting the presence, absence or amount of antibody bound the polypeptide immobilized on the solid surface.25 KILPATRICK TOWNSEND 80016194111. A polypeptide comprising an epitope fused to a heterologous amino acid sequence, wherein the epitope comprises DILLEFR(D / E)LA (SEQ ID NO:1).

12. The polypeptide of claim 11, wherein the polypeptide comprises IPSDHDILLEFR(D / E)LA (SEQ ID NO:2).

13. The polypeptide of claim 11, wherein the polypeptide comprises GGADAAPTPSSAAPGETKVPSELNATIPSDHDILLEFRDLAAMALIG (SEQ ID NO:3) or TAPGETKIPSELNATIPSDHDILLEFRELAAMALIGDSTVHVCVSRV (SEQ ID NO:4).

14. A polypeptide immobilized to a solid support, wherein the polypeptide comprises an epitope comprising DILLEFR(D / E)LA (SEQ ID NO:1).

15. The polypeptide of claim 14, wherein the polypeptide is covalently linked to the solid support.

16. The polypeptide of claim 14 or 15, wherein the solid support is part of a lateral flow.

17. The polypeptide of claim 14 or 15, wherein the solid support is a bead, membrane, well, or plate.

18. The polypeptide of any one of claims 14-17, wherein the polypeptide comprises IPSDHDILLEFR(D / E)LA (SEQ ID NO:2).

19. The polypeptide of any one of claims 14-17, wherein the polypeptide comprises GGADAAPTPSSAAPGETKVPSELNATIPSDHDILLEFRDLAAMALIG (SEQ ID NO:3) or TAPGETKIPSELNATIPSDHDILLEFRELAAMALIGDSTVHVCVSRV (SEQ ID NO:4).

20. The polypeptide of any one of claims 14-19, wherein the solid support further comprises at least a second polypeptide immobilized on the solid support, wherein the second polypeptide comprises: RAQELAREKKLADRAFLDQKPEGVPLRELPLDDDSDFVAMEQERRQQLE KDPRRNAKEIA (SEQ ID NO:5); PSPFGQAAAGDKPSPFGQAAAGDK (SEQ ID NO:6);26 KILPATRICK TOWNSEND 800161941KSAEPKSAEPKSAEP (SEQ ID NO:7); EKQKAAEATKVAEA (SEQ ID NO:8); ALPQEEQEDVGPRHVDPDHFRSTTQDAYRPVDPSAYKR (SEQ ID NO:9); DSTAHGTPSTPADSSAHSTPSTPA(SEQ ID NO:10); QKAAENERLADELE (SEQ ID NO:11); QKAAEATKVAEAEKQRAREATKVAEAEK (SEQ ID NO:12); PFGQAAAGDKPS (SEQ ID NO:13); AAPAKAA (SEQ ID NO:14); AEPKSAEPKP (SEQ ID NO:15); KAAIAPAKAAAAPAKAATAPA (SEQ ID NO:16); KTAAPPAKTAAPPAKTAAPPA (SEQ ID NO:17); GTSEEGSRGGSSMPSGTSEEGSRGGSSMPA (SEQ ID NO:18); or KFAELLEQQKNAQFPGK (SEQ ID NO:19).

21. A polypeptide linked to a heterologous label, wherein the polypeptide comprises an epitope comprising DILLEFR(D / E)LA (SEQ ID NO:1).

22. The polypeptide of claim 21, wherein the polypeptide comprises IPSDHDILLEFR(D / E)LA (SEQ ID NO:2).

23. The polypeptide of claim 21, wherein the polypeptide comprises GGADAAPTPSSAAPGETKVPSELNATIPSDHDILLEFRDLAAMALIG (SEQ ID NO:3) or TAPGETKIPSELNATIPSDHDILLEFRELAAMALIGDSTVHVCVSRV (SEQ ID NO:4).

24. The polypeptide of any one of claims 21-23, wherein the label comprises a fluorescent or chemiluminescent moiety.

25. A kit comprising the polypeptide of any one of claims 11-24.27 KILPATRICK TOWNSEND 800161941

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