Protein for the serological detection of streptococcus EQUI subspecies EQUI exposure or infection

Abstract

The current disclosure provides polypeptides and peptides comprising SEQ ID NO: 1. Antibodies or antigen binding fragments thereof specifically bind to a polypeptide comprising SEQ ID NO: 1 or peptide fragments thereof (e.g., SEQ ID NOs: 2 and / or 3) are also provided. Accordingly, the current disclosure provides polypeptides and peptides comprising ant one of SEQ ID NOs: 1-6 or fragments thereof. The antibodies or antigen binding fragments thereof can be monoclonal antibodies (Mab), chimeric antibodies, single chain antibodies, single chain fragment variable (scFv) antibodies, or fragment antigen-binding (Fab fragment). The antibodies or antigen binding fragments thereof can be labeled with an enzyme, a radioisotope, a fluorescent label, or a bioluminescent label and / or used in diagnostic assays. Diagnostic assays and vaccines using the disclosed polypeptides or fragments thereof are also provided.

Description

[0001] PROTEIN FOR THE SEROLOGICAL DETECTION OF STREPTOCOCCUS EQUI SUBSPECIES EQUI EXPOSURE OR INFECTION

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 581,391, filed September 8, 2023, the disclosure of which is hereby incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences.

[0004] STATEMENT REGARDING SEQUENCE LISTING

[0005] The Sequence Listing for this application is labeled “Seq-List.xml” which was created on September 4, 2024 and is 10,548 bytes. The entire content of the sequence listing is incorporated herein by reference in its entirety.

[0006] BACKGROUND

[0007] The disease known as strangles remains one of the most common infectious disorders of horses in the United States (US) and beyond: outbreaks were reported to the Equine Disease Communication Center from 38 of the 50 US states during the last 12-24 months, and many other US outbreaks were unreported.

[0008] Strangles has plagued horses worldwide for centuries because the bacterium causing this disease (viz., Streptococcus equi subspecies equi [SEE]) can survive long-term in carrier horses that do not show clinical signs. Control and prevention of strangles is greatly hampered by the limited accuracy of testing to identify infected horses, particularly inapparent carrier horses. These inapparent carriers can test negative for long periods of time by standard diagnostic methods of either microbiological culture or polymerase chain-reaction (PCR). Other important limitations for controlling strangles include absence of a point-of-care test for diagnosis and a vaccine that is highly effective and safe.

[0009] An accurate test to detect antibodies in blood indicative of SEE infection would be useful to detect carrier horses, to identify horses exposed to strangles during outbreaks, and confirm diagnosis of horses that have overt signs of strangles. However, the only available blood test for detecting exposure to SEE available in the US lacks diagnostic accuracy in part because it detects antibodies to SEE that cross-react with Streptococcus equi subspecies zooepidemicus (SEZ). SEZ, from which SEE evolved, is a common commensal inhabitant of the upper airway of horses. Unlike SEZ which has a wide range of mammalian hosts, SEE is a host restricted pathogen for equine members of the genus Equus.

[0010] We have identified a novel protein for which antibodies in blood (serum) can differentiate horses infected with SEE from either uninfected or SEZ-infected horses. Moreover, we have identified portions (peptide fragments) of this protein that are identical in strains of SEE but which are highly variable in SEZ. Targeting these peptides will improve the accuracy of a diagnostic test for SEE infection and solve a long-felt need for the identification of animals infected with SEE. The polypeptide and peptide fragments disclosed in this application can be adapted for testing either in veterinary diagnostic laboratories using a standard technology known as enzyme-linked immunosorbent assay (ELISA) or for point-of- care (i.e., stall-side) test platforms to improve control of strangles. The protein and peptide fragments can also be used to develop reagents for stall-side antigen capture test for rapid detection of SEE and to develop a more effective strangles vaccine.

[0011] BRIEF SUMMARY

[0012] The current disclosure provides polypeptides, peptides, and / or fragments thereof comprising any one of SEQ ID NOs: 1-6. Isolated antibodies or antigen binding fragments thereof that specifically bind to the polypeptide of SEQ ID NO: 1, wherein the antibodies or antigen binding fragments thereof specifically bind to a polypeptide comprising SEQ ID NO: 1 or peptide fragments thereof (e.g., SEQ ID NOs: 2 and / or 3). Accordingly, the current disclosure provides polypeptides and peptides comprising any one of SEQ ID NOs: 1-6 or fragments thereof. The antibodies or antigen binding fragments thereof can be monoclonal antibodies (Mab), chimeric antibodies, single chain antibodies, single chain fragment variable (scFv) antibodies, or fragment antigen-binding (Fab fragment). The antibodies or antigen binding fragments thereof can be labeled with an enzyme, a radioisotope, a fluorescent label, or a bioluminescent label.

[0013] The current disclosure also provides methods of detecting SEE infections in animals, the method comprising performing an assay using the polypeptides or peptides disclosed herein or antibodies or antigen binding fragments thereof that bind the disclosed polypeptides in a biological sample obtained from the animal. The assay can be an enzyme-linked immunosorbent assay (ELISA), for example, sandwich ELISA or competitive ELISA. An infected animal may be infected naturally (e.g., animals at commercial farms, etc.) or experimentally (e.g., in laboratories or experimental facility). The current disclosure also provides kits, for example ELISA kits, comprising the antibody or antigen binding fragment thereof and / or antigens disposed upon a substrate (for example, a microtiter plate or a lateral flow immunoassay substrate). The antibodies or antigen binding fragments thereof can be labeled with an enzyme in the ELISA kits or labeled antibodies can be used to detect the presence of a polypeptide comprising SEQ ID NO: 1 or fragments thereof, that have been captured by a first antibody that binds the polypeptide (the labeled antibodies binding to an epitope separate and distinct from the antibody that captures the polypeptide). Alternately, the antibodies or antigen binding fragments thereof can be coated onto immunoassay plates or other solid substrates and used to capture an antigen (e.g., a polypeptide comprising SEQ ID NO: 1 or fragments thereof). The kits can further comprise an immunoassay plate or lateral flow immunoassay upon which the polypeptide of SEQ ID NO: 1 or peptide fragments thereof (e.g., SEQ ID NOs: 2 and / or 3) can be disposed.

[0014] Further, the current disclosure provides antibodies or antigen binding fragments thereof obtained from an animal that has been immunized with a polypeptide or peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-6. Methods of immunizing animals with the disclosed polypeptide and / or peptides are also provided as are immunogenic conjugates comprising the disclosed polypeptides and peptides. Additionally, nucleic acid sequences (polynucleotides), nucleic acid constructs, vectors, host cells expressing the disclosed polypeptides and peptides are also provided.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication, with color drawing(s), will be provided by the Office upon request and payment of the necessary fee.

[0017] FIG. 1. Heatmap of 92 proteins (rows) comparing individual horses (columns): 48 horses with strangles or recovered from strangles (left, salmon-colored band), horses without history of strangles (n=30; green band), and horses infected with SEZ (blue bands); the magenta band represents the SEZ-infected and healthy groups. The top 4 rows of the heat map (maroon arrow) represent the 4 proteins that best discriminated horses with strangles from those without. The bottom of the heatmap shows the 8 homologous (i.e., shared by SEE and SEZ) control proteins.

[0018] FIG. 2. Receiver operator characteristic (ROC) curves from microarray data: Protein 92 (green line) had significantly (P<0.05) greater area under the ROC curve than Antigen A (orange), Antigen C (N terminus of SeM, light blue), or Protein 37 (pink line; another novel antigen). Combining Antigens A+C with Protein 92 (dark blue line) did not significantly improve the accuracy of results based on Protein 92 alone.

[0019] FIG. 3. Receiver-operator characteristic (ROC) curves comparing ELISA results (OD ratios) for Protein 92 (solid line) versus SeM (hatched line). The area under the ROC curve was significantly (P = 0.0003) greater than that for SeM. The optimal cut-point selected for the Protein 92 curve (OD ratio of 0.1) yielded significantly better sensitivity (92.2%) and specificity (90.2%) than the optimal cut-point (OD ratio of 0.2) for the SeM ELISA.

[0020] FIG. 4. Boxplot of activity level of IgG (ratio of sample to positive control) recognizing Protein / Antigen 92 (P92) among 103 healthy horses without known history of strangles, 102 horses with clinical signs of strangles caused by Streptococcus equi subspecies equi (SEE), and 24 horses infected with Streptococcus equi subspecies zooepidemicus (SEZ). The top and bottom of a box represent the 75thand 25thpercentiles of the data, respectively; the horizontal line bisecting a box is the median (50thpercentile); thin vertical lines extend to a multiple of 1.75 of the respective interquartile distance.

[0021] FIG. 5. Boxplot of activity level of IgG (ratio of sample to positive control) recognizing the M protein (SeM) of Streptococcus equi subspecies equi (SEE) among 103 healthy horses without known history of strangles, 102 horses with clinical signs of strangles caused by SEE, and 24 horses infected with Streptococcus equi subspecies zooepidemicus (SEZ). The top and bottom of a box represent the 75thand 25thpercentiles of the data, respectively; the horizontal line bisecting a box is the median (50thpercentile); thin vertical lines extend to a multiple of 1.75 of the respective interquartile distance.

[0022] BRIEF DESCRIPTION OF THE SEQUENCES

[0023] SEQ ID NO: 1: Sequence of protein 92

[0024] MAKKEMKYYLRKSAFGLASVFCTLLVGTASVSAQVTMRGQKYATKAEATARLTTL KQQHPELASTIAGFESKINDSNLIQVDDINKILAEAEQTIAVKAEAERSKRAEEEAANE ALRKEKSTVIYIFGSKQGMTLPQELLDVAKMSELQLVSGDLLVGHVTQMMVKLNLQ RAIQHQVMEILL. Amino acids 1-34 constitute a signal peptide and the mature protein comprises amino acids 35-182.

[0025] SEQ ID NO: 2: Amino acid sequence for peptide 1

[0026] TMRGQKYATKAEATARLTTLKQQHPELASTIAGFESKINDSNLIQVDDINKILAEAEQ TIAV. SEQ ID NO: 3: Amino acid sequence of peptide 2

[0027] VAKMSELQLVSGDLLVGHVTQMMVKLNLQRAIQHQVMEILL

[0028] SEQ ID NOs: 4-6: peptide fragments: STIAGFESKINDSNL (SEQ ID NO: 4), LNLQRAIQHQVMEIT (SEQ ID NO: 5), and LNLQRAIQHQVMEIL (SEQ ID NO: 6).

[0029] SEQ ID NO: 7 : His-His-His-His-His-His peptide (a histidine tag).

[0030] SEQ ID NO: 8: Trp- Ser-Hi s-Pro-Gln-Phe-Glu-Lys (STREP-TAG II, a peptide that exhibits intrinsic affinity toward streptavidin, STREP-TACTIN, or STREP-TACTIN XT; IB A Lifesciences GmbH, Gottingen, Germany).

[0031] SEQ ID NO: 9: WSHPQFEK-(GGGS)2-GGSA-WSHPQFEK (TWIN-STREP-TAG, a peptide that exhibits intrinsic affinity toward streptavidin, STREP-TACTIN, or STREP- TACTIN XT; IBA Lifesciences GmbH, Gottingen, Germany).

[0032] DETAILED DISCLOSURE

[0033] The term “prokaryotic system” refers to prokaryotic cells such as bacterial cells (e.g., bacterial cells having the genus Escherichia or Salmonella) or prokaryotic phages or bacterial spores. The term “eukaryotic system” refers to eukaryotic cells including cells of animal, plants, fungi and protists, and eukaryotic viruses such as retrovirus, adenovirus, baculovirus. Prokaryotic and eukaryotic systems may be, collectively, referred to as “expression systems”.

[0034] The term “expression cassette” is used here to refer to a functional unit that is built in a vector for the purpose of expressing recombinant polypeptides in the periplasm. An expression cassette includes a promoter or promoters, a transcription terminator sequence, a ribosome binding site or sites, and the DNA encoding the fusion proteins. Other genetic components can be added to an expression cassette, depending on the expression system (e.g., enhancers and polyadenylation signals for eukaryotic expression systems).

[0035] As used herein the term “vector” refers to a nucleic acid molecule, preferably selfreplicating within a cell, which transfers an inserted nucleic acid molecule into and / or between host cells. Typically, vectors are circular DNA comprising a replication origin, a selection marker, and / or viral package signal, and other regulatory elements. Vector, vector DNA, plasmid DNA, phagemid DNA are interchangeable terms in description of this invention. The term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and / or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. As used herein the term “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, leads under appropriate conditions to the transcription and translation of one or more polypeptides. The term “expression vector”, refers to vectors that direct the expression of polypeptides of interest fused in frame with a binding motif.

[0036] As used herein the terms “nucleic acids” and “polynucleotides” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the nucleotide polymer.

[0037] As used herein the terms “polypeptide”, “peptide”, and “protein,” are used interchangeably herein to refer to polymers of amino acids of any length. A “fragment” of a polypeptide, peptide, or protein refers to a part of the polypeptide, peptide, or protein and contains fewer amino acids than the intact or complete polypeptide, peptide, or protein.

[0038] As used herein the term “host cell” includes an individual cell or cell culture which can be, or has been, a recipient for the disclosed expression constructs. Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical to the original parent cell due to natural, accidental, or deliberate mutation. A host cell may be prokaryotic or eukaryotic.

[0039] Two nucleic acid sequences or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4: 11-17 (1988) e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, Calif., USA).

[0040] Sequences are “substantially identical” to each other if they have a specified percentage of nucleotides or amino acid residues that are the same (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a specified region or the entire designated sequence if a region is not specified), when compared and aligned for maximum correspondence over a comparison window.

[0041] 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 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.

[0042] A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 10 to 600, about 10 to about 300, about 10 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. The comparison window can also be the entire length of either the reference or the test sequence.

[0043] Percent sequence identity and sequence similarity can be determined using the BLAST 2.0 algorithm, which is described in Altschul et al. (J. Mol. Biol. 215:403-10, 1990). Software for performing BLAST 2.0 analyses is publicly available through the National Center for Biotechnology Information (Worldwide Website: ncbi.nlm.nih.gov / ). This 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 (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. 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 BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0044] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (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 nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0045] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or). As an example, where a combination of elements (listed items) is provided (for example, “... a, b, and / or c”), the combinations include “a” alone, “b” alone, “c” alone, “a” and “b” together, “a” and “c” together, “b” and “c” together, and “a”, “b” and “c” together.

[0046] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Further, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”.

[0047] The transitional terms “comprising,” or “comprises” or grammatical variations thereof, which are synonymous with “including”, “includes”, “containing”, or “contains”, is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Use of the term “comprising” contemplates other embodiments that “consist” or “consist essentially of’ the recited component(s). The phrases “consisting essentially of’ or “consists essentially of’ indicate that the claim encompasses embodiments containing the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim.

[0048] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 0-20%, 0 to 10%, 0 to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. In the context of compositions containing amounts of ingredients where the terms “about” or “approximately” are used, these compositions contain the stated amount of the ingredient with a variation (error range) of 0-10% around the value (X±10%).

[0049] In the present disclosure, ranges are stated in shorthand, so as to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. Where a range is disclosed, the range can be specified as being between any two of the listed values for the range. The current disclosure provides a novel polypeptide and peptide fragments thereof from SEE and antibodies and antigen binding fragments thereof that specifically bind to the disclosed polypeptide and peptide fragments. This disclosure also describes methods, techniques, approaches, kits for use in conjunction with the present disclosure.

[0050] The current disclosure provides methods of using the antibodies or antigen binding fragments thereof and the disclosed polypeptides and / or peptides for detection for strangles (caused by SEE) infection in animals) and for distinguishing SEE infected animals from uninfected animals or animals infected by SEZ. The methods of the current disclosure provide improved sensitivity and specificity over the existing methods of detection of SEE in animals and reduces the time necessary to identify and / or distinguish animals positive for SEE infection from animals infected with SEZ.

[0051] Accordingly, the current disclosure provides antibodies and antigen binding fragments thereof that specifically binds to the polypeptide of SEQ ID NO: 1 or fragments thereof (e.g., SEQ ID NOs: 2 and 3). In one embodiment, the antibody is a monoclonal antibody. Additionally, the antibodies can be polyclonal antibodies. In further embodiments, the antibody or antigen binding fragments thereof can be chimeric antibodies, single chain antibodies, scFv antibodies, or Fab fragments. The antibodies and antigen binding fragments thereof can be labeled with a detectable label. The detectable label, which in another embodiment, is fluorescent, or in another embodiment, luminescent, or in another embodiment, radioactive, or in another embodiment, electron dense. In other embodiments, the detectable label comprises, for example, green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), beta-galactosidase, luciferase,32P,1251,3H and14C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone, luciferin or any number of other such labels known to one skilled in the art. The particular label used will depend upon the type of immunoassay used or the intended use of the polypeptide or fragment thereof. In certain embodiments, the polyclonal antibodies are non-equine antibodies, for example, polyclonal antibodies raised in mice, rats, sheep, goats, pigs or other animals not infected by SEE (which is a host restricted pathogen of equine animals).

[0052] A further embodiment provides antibodies or antigen binding fragments thereof obtained from an animal that has been immunized with a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1, 2, 3 or fragments of said polypeptides (e.g., SEQ ID NOs: 4-6). These antibodies can also be monoclonal antibodies or polyclonal antibodies. In one aspect, this disclosure provides a polypeptide comprising SEQ ID NOs: 1, 2 or 3 or fragments of such polypeptides. Fragments of the disclosed polypeptides can be referred to as “peptide fragments”, “polypeptide fragments” and / or “fragments”. The phrase “polypeptide and fragments thereof’, as used herein, is intended to designate the polypeptide of SEQ ID NO: 1 and fragments thereof, such as SEQ ID NO: 2, SEQ ID NO: 3, or fragments of SEQ ID NOs: 1, 2 or 3 as defined herein.

[0053] Fragments of SEQ ID NO: 1 can comprise or consist of a span of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

[0054] 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,

[0055] 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,

[0056] 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,

[0057] 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 122, 123, 124, 125, 126, 127,

[0058] 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,

[0059] 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,

[0060] 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, or 181 consecutive amino acids of SEQ ID NO: 1 or a span between any two of the listed values that is at least 5 amino acid in length.

[0061] In preferred embodiments, the fragment begin at position 35 of SEQ ID NO: 1. Thus, the fragments can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23,

[0062] 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,

[0063] 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,

[0064] 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,

[0065] 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, or 147 consecutive amino acids of the mature protein of SEQ ID NO: 1 (amino acids 35-182 of SEQ ID NO: 1) or a span between any two of the listed values that is at least 5 amino acid in length (amino acids 35-182 of SEQ ID NO: 1).

[0066] For SEQ ID NO: 2, fragments can comprise or consist of a span of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 consecutive amino acids of SEQ ID NO: 2 or a span between any two of the listed values that is at least 5 amino acid in length. For SEQ ID NO: 3, fragments of SEQ ID NO: 1 can comprise or consist of a span of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive amino acids of SEQ ID NO: 3 or a span between any two of the listed values that is at least 5 amino acid in length. Other exemplary polypeptides / peptides are provided in SEQ ID NOs: 4-6 and fragments of those peptides that are 5, 6, 7, 8, 9, 10, 11, 12,13, or 14 consecutive amino acids in length (for SEQ ID NOs: 4-6) are also expressly contemplated herein.

[0067] It should be understood that the phrase “fragments of SEQ ID NO: X comprising consecutive amino acids of SEQ ID NO: X” (where X is 1, 2, 3, 4, 5 or 6) indicate a polypeptide fragment that has fewer amino acids than the full length of the corresponding sequence (i.e., SEQ ID NOs: 1, 2, 3, 4, 5 or 6). The disclosed polypeptide fragments can be used in the diagnostic and therapeutic protocols disclosed herein.

[0068] The disclosed polypeptides and fragments thereof can also be modified such that the polypeptide and fragments further comprise a detectable label, tag, or additional amino acid (such as a N-terminal or C-terminal Cys to facilitate attachment of the polypeptide or fragment to a substrate or a label or tag).

[0069] Thus, the disclosed polypeptide and fragments thereof can further comprise a detectable label, which in another embodiment, is fluorescent, or in another embodiment, luminescent, or in another embodiment, radioactive, or in another embodiment, electron dense. In other embodiments, the detectable label comprises, for example, green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), beta-galactosidase, luciferase,32P,1251,3H and14C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone, luciferin or any number of other such labels known to one skilled in the art. The particular label used will depend upon the type of immunoassay used or the intended use of the polypeptide or fragment thereof.

[0070] In another embodiment, the polypeptide or a fragment thereof can be linked to a substance to increase an elicited an immune response. In such embodiments, a polypeptide or fragment is linked to a carrier. In another embodiment, the carrier is KLH. In other embodiments, the carrier is any other carrier known in the art, including, for example, thyroglobulin, albumins such as human serum albumin, polyamino acids such as poly (lysine:glutamic acid), influenza, tetanus toxoid, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like. Each possibility represents a separate embodiment. In another embodiment, peptides of this invention are linked to other molecules, such as a cross-linking agent (for example, carbodimides). Examples of carbodimides are 1- cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), l-ethyl-3-(3- dimethyaminopropyl) carbodiimide (EDC) and l-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide. In other embodiments, the cross-linking agents comprise cyanogen bromide, glutaraldehyde and succinic anhydride. In general, any of a number of homo-bifunctional agents including a homo-bifunctional aldehyde, a homo-bifunctional epoxide, a homo- bifunctional imido-ester, a homo-bifunctional N-hydroxysuccinimide ester, a homo- bifunctional maleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyl disulfide, a homo-bifunctional aryl halide, a homo-bifunctional hydrazide, a homo-bifunctional diazonium derivative and a homo-bifunctional photoreactive compound can be used. Also envisioned, in other embodiments, are hetero-bifunctional compounds, for example, compounds having an amine-reactive and a sulfhydryl-reactive group, compounds with an amine-reactive and a photoreactive group and compounds with a carbonyl-reactive and a sulfhydryl-reactive group.

[0071] In certain embodiments, the homo-bifunctional cross-linking agents include the bifunctional N-hydroxysuccinimide esters dithiobi s(succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional imido-esters dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the bifunctional sulfhydryl-reactive crosslinkers l,4-di-[3'-(2'-pyridyldithio)propionamido]butane, bismaleimidohexane, and bis- N-maleimido-l,8-octane; the bifunctional aryl halides l,5-difluoro-2,4-dinitrobenzene and 4,4'-difluoro-3,3'-dinitrophenylsulfone; bifunctional photoreactive agents such as bis-[b-(4- azidosalicylamido)ethyl]disulfide; the bifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde, glutaraldehyde, and adipaldehyde; a bifunctional epoxide such as 1,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipic acid dihydrazide, carbohydrazide, and succinic acid dihydrazide; the bifunctional diazoniums o-tolidine, diazotized and bis-diazotized benzidine; the bifunctional alkylhalides NlN'-ethylene- bis(iodoacetamide), N IN'-hexamethylene-bis(iodoacetamide), N IN' -undecamethyl ene- bis(iodoacetamide), as well as benzylhalides and halomustards, such as ala'-diiodo-p-xylene sulfonic acid and tri(2-chloroethyl)amine, respectively.

[0072] In other embodiments, hetero-bifunctional cross-linking agents used to link the disclosed polypeptide and fragments thereof to other molecules, as described herein, include, but are not limited to, SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-l- carboxylate), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N- succinimidyl(4-iodoacteyl)aminobenzoate), SMPB (succinimidyl-4-(p- maleimidophenyl)butyrate), GMBS (N-(gamma-maleimidobutyryloxy)succinimide ester), MPBH (4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H (4-(N-maleimidomethyl) cyclohexane- 1-carboxyl-hydrazide), SMPT (succinimidyloxycarbonyl-a-methyl-a-(2- pyridyldithio)toluene), and SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate).

[0073] In yet other embodiments, the disclosed polypeptide and fragments thereof of the invention are formulated as non-covalent attachment of monomers through ionic, adsorptive, or biospecific interactions. Complexes of peptides with highly positively or negatively charged molecules can be accomplished, in another embodiment, through salt bridge formation under low ionic strength environments, such as in deionized water. Large complexes can be created, in another embodiment, using charged polymers such as poly-(L-glutamic acid) or poly-(L- lysine), which contain numerous negative and positive charges, respectively. In another embodiment, peptides are adsorbed to surfaces such as microparticle latex beads or to other hydrophobic polymers, forming non-covalently associated peptide-superantigen complexes effectively mimicking cross-linked or chemically polymerized protein, in other embodiments. In another embodiment, peptides are non-covalently linked through the use of biospecific interactions between other molecules. For instance, utilization of the strong affinity of biotin for proteins such as avidin or streptavidin or their derivatives could be used to form peptide complexes. The disclosed polypeptide and fragments thereof, according to this aspect, and in another embodiment, can be modified to possess biotin groups using common biotinylation reagents such as the N-hydroxysuccinimidyl ester of D-biotin (NHS-biotin), which reacts with available amine groups.

[0074] In another embodiment, a peptide of this invention is linked or adsorbed to a solid support or substrate, which, in another embodiment, serves as a carrier or a surface upon which an immunoassay can be performed. The term “solid support” or “substrate” (and grammatical equivalents of these terms) are used to denote a solid inert surface or body to which an agent, such as an antibody or a peptide or protein can be immobilized. These terms (“solid support” or “substrate” (and grammatical equivalents of these terms)) may be used interchangeably. Non-limiting examples of a solid support or substrate include plastic, polystyrenes, nitrocellulose, membranes, chips, and particles. If solid supports other than particles are used, for instance, glass, polymeric or silica chips (such as microchips), plates, slides, etc., the disclosed polypeptide and fragments thereof(target analytes) disclosed herein can be immobilized on the surface of the support at specific locations (e.g., in specific wells of a plate (e.g., microtiter plate) or at specific locations on a chip, microchip, plate or slide). Thus, it is possible to differentiate antibodies within a sample by the location at which specific binding between antibodies in a sample and the disclosed polypeptide and fragments thereof disclosed herein occurs on the surface of the support.

[0075] Alternatively, lateral flow immunoassays can be performed in a manner analogous to those disclosed in U.S. Pat. Nos. 5,851,776 and 6,777,190 (each of which is hereby incorporated by reference in their entireties and which relate to lateral flow chromatographic assays on a membrane or other porous or non-porous materials, such as a particle). The polypeptide and / or fragments thereof or antibodies (or antigen binding fragments thereof), disclosed herein are immobilized at discrete locations on the membrane or other porous or non- porous material (each location being specific for a particular antigen / target analyte / antibody / antigen binding fragment) and then contacted with a biological sample containing antibodies that specifically bind to the disclosed polypeptide or fragments thereof or a biological sample comprising a polypeptide comprising SEQ ID NO: 1 or a fragment thereof. The specific binding of antibodies to the disclosed polypeptide and fragments thereof that are immobilized at discrete locations on the membrane or other porous or non-porous material is then detected using conventional methods. Similarly, binding of the disclosed polypeptide and fragments thereof to antibodies or antigen binding fragments thereof that are specific to SEQ ID NO: 1 or fragments thereof that are immobilized at discrete locations on the membrane or other porous or non-porous material is then detected using conventional methods.

[0076] The term “particle” is used herein to refer to a solid or semisolid body, often with linear dimensions on the micron scale (i.e., less than about 100 microns), of any shape or surface texture. Except as noted, the term is used interchangeably with “particle,” which refers to a micron scale particle, and “bead,” which refers to particles that are spherical or near-spherical in shape, often polymeric in composition. Where used in this application, the terms “particle” and “bead” (and grammatical equivalents of these terms) can be interchanged without altering the context of the passages within this application.

[0077] The term “immobilized” as used herein denotes a molecular-based coupling that is not significantly de-coupled under the conditions imposed during the steps of the assays described herein. Such immobilization can be achieved through a covalent bond, a non-covalent bond, an ionic bond, an affinity interaction (e.g., avidin-biotin or polyhistidine-Ni++), or any other chemical bond.

[0078] Immobilization of the various combinations of the polypeptide and fragments thereof disclosed in this application can be performed by covalent or non-covalent immobilization on a substrate. For example, non-covalent immobilization can be non-specific (e.g., non-specific binding of a combination of one or more disclosed polypeptide and fragments thereof to a polystyrene surface). Specific or semi-specific binding to a substrate can be achieved by the polypeptide and / or fragments thereof having a moiety that enables covalent or non-covalent binding of the peptide and / or protein to the substrate that is coated with a ligand that binds to the moiety. For example, the moiety can be a biotin or biotinyl group or an analogue thereof bound to an amino acid group of the peptide, such as 6-aminohexanoic acid, and the ligand is then avidin, streptavidin or an analogue thereof. Alternatively, the moiety can be a His-His- His-His-His-His peptide (SEQ ID NO: 7, a histidine tag) and the substrate can be derivatized with a nitrilotriacetic acid derivative (NTA) charged with Ni++ions. In other embodiments, the moiety can be Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8, STREP -TAG II, a peptide that exhibits intrinsic affinity toward streptavidin, STREP-TACTIN, or STREP-TACTIN XT; IB A Lifesciences GmbH, Gottingen, Germany) or WSHPQFEK-(GGGS)2-GGS A- WSHPQFEK (SEQ ID NO: 9, TWIN-STREP-TAG, a peptide that exhibits intrinsic affinity toward streptavidin, STREP-TACTIN, or STREP-TACTIN XT; IBA Lifesciences GmbH, Gottingen, Germany) and the substrate can be derivatized with streptavidin, avidin or an analogue thereof).

[0079] Various substrates suitable for use in the disclosed methods include, and are not limited to, magnetic beads, polystyrene beads, latex beads, beads comprising co-polymers such as styrene-divinyl benzene; hydroxylated styrene-divinyl benzene; polystyrene; carboxylated polystyrene; carbon black; non-activated, polystyrene or polyvinyl chloride activated glass; epoxy-activated porous magnetic glass; gelatin or polysaccharide particles; protein particles or red blood cells.

[0080] In other embodiments, the substrate can be the floor or wall of a microtiter well; a filter surface or membrane (e.g., a nitrocellulose membrane or a PVDF (polyvinylidene fluoride) membrane, such as an Immobilon membrane); a hollow fiber; a beaded chromatographic medium (e.g., an agarose or polyacrylamide gel); a magnetic bead; a fibrous cellulose matrix; an HPLC matrix; an FPLC matrix; or any other suitable carrier, support or surface. In one embodiment of the invention, one or more disclosed polypeptide and / or fragments thereof disclosed herein are immobilized onto polystyrene beads (microspheres), wherein each peptide or protein is immobilized onto a bead with a unique detectable physical parameter, and are analyzed by the xMAP® technology developed by Luminex Technology (Austin, Tex.) and described in their world wide web site luminexcorp.com. Such assays may be referred to as “multiplex immunoassays”.

[0081] In other embodiments, antibodies or antigen binding fragments thereof can be immobilized on the floor or wall of a microtiter well; a filter surface or membrane (e.g., a nitrocellulose membrane or a PVDF (polyvinylidene fluoride) membrane, such as an Immobilon membrane); a hollow fiber; a beaded chromatographic medium (e.g., an agarose or polyacrylamide gel); a magnetic bead; a fibrous cellulose matrix; an HPLC matrix; an FPLC matrix; or any other suitable carrier, support or surface. In one embodiment of the invention, one or more disclosed antibodies or antigen binding fragments thereof are immobilized onto polystyrene beads (microspheres), wherein each peptide or protein is immobilized onto a bead with a unique detectable physical parameter, and are analyzed by the xMAP® technology developed by Luminex Technology (Austin, Tex.) and described in their world wide web site luminexcorp.com. Such assays may be referred to as “multiplex immunoassays”.

[0082] Devices for performing specific binding assays, especially immunoassays, are known and can be readily adapted for use in the present methods. Solid phase assays, in general, are easier to perform than heterogeneous assay methods which require a separation step, such as precipitation, centrifugation, filtration, chromatography, or magnetism, because separation of reagents is faster and simpler. Solid-phase assay devices include microtiter plates, flow-through assay devices, chips, microchips, lateral flow substrates dipsticks and immunocapillary or immunochromatographic immunoassay devices.

[0083] The terms “receptacle,” “vessel,” “tube,” “well,” etc. refer to a container that can hold reagents or an assay. If the receptacle is in a kit and holds reagents, it will typically be closed or sealed. If the receptacle is being used for an assay, it will typically be open or accessible during steps of the assay.

[0084] Various assays for detection of the polypeptide of SEQ ID NO: 1 or a fragment thereof (such as one or more of SEQ ID NOs: 2, 3, 4, 5, or 6) can be used to detect SEE infection in an animal or an animal exposed to SEE using the antibodies or antigen binding fragments thereof of the current disclosure. Non-limiting examples of protein detection assays include Western blot analysis, lateral flow immunoassays, ELISA, antigen capture immunoassays, immunohistochemistry, or immunoprecipitation. Assays disclosed herein can be performed on a biological sample obtained from an animal. In certain embodiments, the biological sample obtained from the animal contains antibodies that bind to the polypeptide of SEQ ID NO: 1 or a fragment thereof (such as SEQ ID NOs: 2 and / or 3). In certain embodiments the biological sample is a fluid sample from an animal. It should be appreciated that the assays may be used as part of an approach to determine the presence or absence of the polypeptide of SEQ ID NO: 1 or fragments thereof, such as SEQ ID NOs: 2 or 3 in some instances. In certain embodiments, the animal is an equine animal.

[0085] This disclosure also provides methods of screening a population of animals for SEE infection or exposed to SEE. An exemplary method includes: a) obtaining a biological sample from each animal from the population of animals, b) conducting an assay comprising contacting said biological sample with an polypeptide comprising SEQ ID NOs: 1, 2, and / or 3 (or fragments thereof (e.g., any one of SEQ ID NOs: 4-6), said polypeptide being optionally bound to a substrate, c) detecting the presence of antibodies to said polypeptide, and d) identifying individual animals from the population of animals as SEE infected animals or exposed to SEE based on the presence of antibodies to said polypeptide(s).

[0086] Alternatively, the subject disclosure provides for detecting a polypeptide comprising SEQ ID NO: 1 or fragments thereof in a biological sample obtained from an animal (e.g., an antigen-capture immunoassay). In various embodiments, a solid substrate comprising antibodies that specifically bind to a first epitope of a polypeptide comprising SEQ ID NO: 1 or fragments thereof (e.g., one or more of SEQ ID NOs: 2-6) are contacted with a biological sample from an animal such that the polypeptide is captured by the antibody bound to the substrate. A labeled antibody that binds to a second epitope of the polypeptide comprising SEQ ID NO: 1 or fragments thereof (e.g., one or more of SEQ ID NOs: 2-6) is then contacted with the captured polypeptide to detect its presence.

[0087] Non-limiting examples of biological samples (e.g., body fluid samples) include nasopharyngeal lavage, nasal lavage, guttural pouch lavage, nasal swabs, nasopharyngeal swabs, aqueous humor, vitreous humor, blood serum, blood plasma, cerebrospinal fluid, endolymph, perilymph, exudates (e.g., nasal discharge), lymph, mucus, pericardial fluid, pleural fluid, synovial fluid, milk, or oral fluids.

[0088] The current disclosure further provides kits containing the disclosed polypeptide and / or fragments thereof. A kit, for example, is used to detect the presence of antibodies in a sample obtained from an infected animal. The kits in accordance with this disclosure can comprise reagents for conducting various assays. The reagents provided in a kit depend on the purpose and design of the assay to be performed. For example, a kit designed for a western blot analysis contains a labeled secondary antibody and additional reagents for visualizing the label. Various embodiments of western blot analyses are contemplated.

[0089] Kits for performing ELISA are also disclosed. The kits can be designed to perform different types of ELISA, for example, a sandwich ELISA, an antigen capture ELISA, or a competitive ELISA. A person of ordinary skill in the art knows various reagents and tools required for conducting each of the different types of ELISAs and accordingly, the current disclosure provides kits containing appropriate reagents and tools for performing these ELISAs. For example, a kit for conducting a sandwich ELISA can contain an immunoassay plate coated with the disclosed polypeptide and / or fragments thereof.

[0090] In one embodiments, an immunizing agent is constructed by covalently conjugating a polypeptide selected from the disclosed polypeptide and / or fragments thereof to an immunogenic carrier protein, preferably by means of a crosslinker, such as a glutaraldehyde moiety or other known linkers. Immunogenic carriers include compounds to which any one of the disclosed polypeptide and fragments thereof disclosed herein attached so as to render the peptide immunogenic. Non-limiting examples of such carriers include proteins such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), tetanus toxoid and human thyroglobulin. One function of the crosslinker is to introduce into the immunizing agent a spacer of sufficient size to prevent the carrier protein from masking the polypeptide. Suitable crosslinking agents are known in the art and are commercially available. The immunizing agent is then administered to a subject, such as a goat, horse, donkey, sheep, rabbit, mouse, rat, guinea pig, pig, cow, buffalo or other appropriate non-human mammal in an amount sufficient to raise an immune response to the immunizing agent. The immunizing agent can be administered in combination with an adjuvant, such as Freund’s Complete adjuvant, Freund’s Incomplete adjuvant, aluminum salts, oil based adjuvants, saponins, chemically synthesized adjuvants, or other adjuvants. The vaccines (immunizing agents) can be administered by intramuscular, subcutaneous or intranasal routes.

[0091] In one embodiment, an immunizing agent is constructed by covalently conjugating a polypeptide selected from the disclosed polypeptide and / or fragments thereof (e g., any one of SEQ ID Ns: 1-6 or fragments thereof) to an immunogenic carrier protein, preferably by means of a crosslinker, such as a glutaraldehyde moiety or other known linkers. Immunogenic carriers include compounds to which any one of the disclosed polypeptide and fragments thereof disclosed herein atached so as to render the peptide immunogenic. Non-limiting examples of such carriers include proteins such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), tetanus toxoid and human thyroglobulin. One function of the crosslinker is to introduce into the immunizing agent a spacer of sufficient size to prevent the carrier protein from masking the polypeptide. Suitable crosslinking agents are known in the art and are commercially available. The immunizing agent is then administered to a subj ect, such as a goat, horse, donkeys, asses, mules, zebras, sheep, rabbit, mouse, rat, guinea pig, pig, cow, buffalo or other appropriate non-human mammal in an amount sufficient to raise an immune response to the immunizing agent. The immunizing agent can be administered in combination with an adjuvant, such as Freund’s Complete adjuvant, Freund’s Incomplete adjuvant, aluminum salts, oil based adjuvants, saponins, chemically synthesized adjuvants, or other adjuvants. The immunizing agent(s) can be administered by intramuscular, subcutaneous or intranasal routes.

[0092] In another embodiment, an immunizing agent can comprise the disclosed polypeptide and / or fragments thereof (e g., any one of SEQ ID NOs: 1-6 or fragments thereof) thereof to immunize a subject. The immunizing agent is then administered to a subject, such as a goat, horse, donkey, ass, mule, zebra, sheep, rabbit, mouse, rat, guinea pig, pig, cow, buffalo or other appropriate non-human mammal in an amount sufficient to raise an immune response to the immunizing agent. The immunizing agent can be administered in combination with an adjuvant, such as Freund’s Complete adjuvant, Freund’s Incomplete adjuvant, aluminum salts, oil based adjuvants, saponins, chemically synthesized adjuvants, or other adjuvants. The immunizing agent(s) can be administered by intramuscular, subcutaneous or intranasal routes.

[0093] Yet another aspect of the invention provides a microorganism comprising: a) a polynucleotide sequence encoding a polypeptide comprising SEQ ID NOs: 1, 2, and / or 3 or fragments thereof; b) a genetic construct comprising a polynucleotide sequence as set forth in (a); or c) a vector comprising a polynucleotide or genetic construct as set forth in (a) or (b). Genetic constructs of the subject invention can also contain additional regulatory elements such as promoters and enhancers and, optionally, selectable markers. Also within the scope of the subject instant invention are vectors or expression cassettes containing genetic constructs as set forth herein or polynucleotides encoding the polypeptides operably linked to regulatory elements (e.g., promoters or enhancers). The vectors and expression cassettes may contain additional transcriptional control sequences as well. The vectors and expression cassetes may further comprise selectable markers. The expression cassete may contain at least one additional gene, operably linked to control elements, to be co-transformed into the organism. Alternatively, the additional gene(s) and control element(s) can be provided on multiple expression cassettes. Such expression cassettes are provided with a plurality of restriction sites for insertion of the sequences of the invention to be under the transcriptional regulation of the regulatory regions. The expression cassette(s) may additionally contain selectable marker genes operably linked to control elements. Promoters that are operably linked to nucleic acid sequence encoding the disclosed polypeptides or fragments thereof are heterologous promoters. The term “heterologous” is used to designate an element that not associated with, for example, the nucleic acid sequence encoding the disclosed polypeptide in its native environment (in this case a SEE microorganism). Thus, a genetic construct, as disclosed herein, comprises a heterologous promoter that is operably linked to a nucleic acid sequence that encodes a polypeptide, or fragment thereof, as is disclosed herein.

[0094] As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter, or another transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Such expression control sequences are known in the art, such as promoters, enhancers (such as cis-regulatory modules (CRM)), introns, polyA signals, etc.

[0095] Another aspect of the invention provides vectors for the cloning and / or the expression of a polynucleotide sequence taught herein. Vectors of this invention can also comprise elements necessary to allow the expression and / or the secretion of the said nucleotide sequences in a given host cell. The vector can contain a promoter, signals for initiation and for termination of translation, as well as appropriate regions for regulation of transcription. In certain embodiments, the vectors can be stably maintained in the host cell and can, optionally, contain signal sequences directing the secretion of translated protein. These different elements are chosen according to the host cell used. Vectors can integrate into the host genome or, optionally, be autonomously-replicating vectors.

[0096] The subject invention also provides for the expression of a polypeptide or peptide fragment encoded by a polynucleotide sequence disclosed herein comprising the culture of a host cell transformed with a polynucleotide of the subject invention under conditions that allow for the expression of the polypeptide and, optionally, recovering the expressed polypeptide. The host cell / microorganism, as disclosed herein, may be chosen from eukaryotic or prokaryotic systems, such as for example bacterial cells, (Gram negative or Gram positive), yeast cells (for example, Saccharomyces cereviseae or Pichia pastoris), animal cells (such as Chinese hamster ovary (CHO) cells), plant cells, and / or insect cells using baculovirus vectors. In some embodiments, the host cells for expression of the polypeptides include, and are not limited to, those taught in U.S. Patent Nos. 6,319,691, 6,277,375, 5,643,570, or 5,565,335, each of which is incorporated by reference in its entirety, including all references cited within each respective patent. Host cells / microorganisms can be selected from Escherichia coll, Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricatum, Brevibacterium lactof er mentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusilium, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Pianococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhimurium, Salmonella schottmulleri, Xanthomonas citri, Sacchromyces spp. or algae. In certain embodiments, the genetically modified host cell is an E. coir yeast or algae. Non limiting examples of algae that can be used in this aspect of the invention include: Botryococcus braunii; Neochloris oleoabundans; Scenedesmus dimorphus; Euglena gracilis; Nannochloropsis salina; Dunaliella tertiolecta; Tetraselmis chui; Isochrysis galbana; Phaeodactylum tricornutum; Pleurochrysis carterae; Prymnesium parvum; Tetraselmis suecica; or Spirulina species.

[0097] Furthermore, a host cell may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post- translational processing and modification (e.g., glycosylation, phosphorylation) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an unglycosylated core protein product. Expression in yeast will produce a glycosylated product. Expression in plant cells can be used to ensure “native” glycosylation of a plant-derived protein. Furthermore, different vector / host expression systems may effect processing reactions to different extents.

[0098] The disclosure also provides mRNA-based vaccines useful for the immunization of horses or other equine animals (such as donkeys, asses, mules, and zebras). In various embodiments, mRNA encoding a polypeptide comprising any one of SEQ ID NOs: 1-6 or fragments thereof that are at least 5 consecutive amino acids in length. These mRNA-based vaccines can be encapsulated within various delivery systems. For example, in one embodiment mRNA vaccines can be encapsulated in species-agnostic polymeric formulations for inhalable delivery to the lung (see, for example, Rotolo et al., Nature Materials, 2023, 22:369-379 and International Publication No. W02023 / 108111A2 (PCT / US2022 / 081249), the disclosures of which are hereby incorporated by reference in their entireties). In other embodiments, the mRNA vaccines can be incorporated into liposomal formulations for administration intramuscularly (see, for example, U.S. Patent Nos. 11,246,933, 10,369,226, and 9,061,063 which are hereby incorporated by reference in its entireties). These formulations can also be used to administer polypeptides comprising any one of SEQ ID NOs: 1-6 or fragments thereof comprising at least 5 consecutive amino acids to horses or other equine animals.

[0099] Another aspect of the invention provides for methods of vaccinating equine animals (e.g., horses, donkeys, asses, mules, and zebras) with the mRNA-based vaccines disclosed herein. Such methods comprise administering an inhalable formulation comprising a mRNA vaccine to an equine animal into the airway of an equine animal (e g., as an aerosol or via nebulization) or the intramuscular administration of a mRNA vaccine incorporated into a liposomal formulation to an equine animal.

[0100] In certain embodiments, the disclosure also provides for the treatment of animals identified as having an SEE infection. These treatments include, and are not limited to, lancing or incising abscessed lymph nodes to promote drainage, lavage of the treatment of guttural pouch empyema using repeated lavages of pus-filled pouches trans-endoscopically or via rigid or indwelling catheters using isotonic saline or polyionic fluid accompanied by subsequent lowering of the head to allow drainage or use of a suction pump attached to the endoscope, aid the removal of pus. This treatment can be augmented by both topical and prolonged systemic benzylpenicillin or beta-lactam antibiotics (such as penicillins, cephalosporins, and / or cehamycins. The treatments may also comprise, or further comprise topical installation of an acetylcysteine solution (e.g., a 20% (w / v) solution).

[0101] The current disclosure also provides the following non-limiting embodiments:

[0102] 1. A polypeptide comprising SEQ ID NO: 1, 2, 3, 4, 5 or 6 or a fragment thereof.

[0103] 2. The polypeptide of embodiment 1, wherein the polypeptide is a fragment of SEQ ID NO: 1, 2, 3, 4, 5 or 6.

[0104] 3. The polypeptide of embodiment 2, wherein said fragment comprises: a) a span of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,

[0105] 121 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,

[0106] 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,

[0107] 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,

[0108] 178, 179, 180, or 181 consecutive amino acids of SEQ ID NO: 1 or a span between any two of the listed values that is at least 5 amino acid in length. In preferred embodiments, the fragment begin at position 35 of SEQ ID NO: 1. Thus, the fragments can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

[0109] 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,

[0110] 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,

[0111] 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,

[0112] 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, or 147 consecutive amino acids of the mature protein of SEQ ID NO: 1 or a span between any two of the listed values that is at least 5 amino acid in length (amino acids 35-182 of SEQ ID NO: 1); b) a span of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 consecutive amino acids of SEQ ID NO: 2 or a span between any two of the listed values that is at least 5 amino acid in length; c) a span of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive amino acids of SEQ ID NO: 3 or a span between any two of the listed values that is at least 5 amino acid in length. Other exemplary polypeptides / peptides are provided in SEQ ID NOs: 4-6 and fragments of those peptides that are 5, 6, 7, 8, 9, 10, 11, 12,13, or 14 consecutive amino acids in length (for SEQ ID NOs: 4-6) are also expressly contemplated herein; or d) a span of 5, 6, 7, 8, 9, 10, 11, 12,13, or 14 consecutive amino acids of any one of SEQ ID NOs: 4, 5, or 6.

[0113] 4. The polypeptide of any one of embodiments 1-3, said polypeptide further comprising a detectable label, tag, additional amino acid (such as a N-terminal or C-terminal Cys to facilitate attachment of the polypeptide or fragment to a substrate or a label or tag), carrier, Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, Hise, or a cross-linking agent.

[0114] 5. The polypeptide of embodiment 4, wherein the detectable label is fluorescent, luminescent, radioactive, or electron dense. In various embodiments, the detectable label comprises, for example, green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), beta-galactosidase, luciferase,32P,125I,3H and14C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone, luciferin or any number of other such labels known to one skilled in the art. 6. The polypeptide of embodiment 4, wherein the polypeptide further comprises a carrier. In one embodiment, the carrier is KLH. In other embodiments, the carrier is any other carrier known in the art, including, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly (lysine:glutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like. Each possibility represents a separate embodiment.

[0115] 7. The polypeptide according to embodiment 4, wherein the polypeptide further comprises a cross-linking agent (for example, carbodimides). Examples of carbodimides are 1- cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), l-ethyl-3-(3- dimethyaminopropyl) carbodiimide (EDC) and l-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide. In other embodiments, the cross-linking agents comprise cyanogen bromide, glutaraldehyde and succinic anhydride. In general, any of a number of homo-bifunctional agents including a homo-bifunctional aldehyde, a homo-bifunctional epoxide, a homo- bifunctional imido-ester, a homo-bifunctional N-hydroxysuccinimide ester, a homo- bifunctional maleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyl disulfide, a homo-bifunctional aryl halide, a homo-bifunctional hydrazide, a homo-bifunctional diazonium derivative and a homo-bifunctional photoreactive compound can be used. Also envisioned, in other embodiments, are hetero-bifunctional compounds, for example, compounds having an amine-reactive and a sulfhydryl-reactive group, compounds with an amine-reactive and a photoreactive group and compounds with a carbonyl-reactive and a sulfhydryl-reactive group.

[0116] In certain embodiments, the homo-bifunctional cross-linking agents include the bifunctional N-hydroxysuccinimide esters dithiobi s(succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional imido-esters dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the bifunctional sulfhydryl-reactive crosslinkers l,4-di-[3'-(2'-pyridyldithio)propionamido]butane, bismaleimidohexane, and bis- N-maleimido-l,8-octane; the bifunctional aryl halides l,5-difluoro-2,4-dinitrobenzene and 4,4'-difluoro-3,3'-dinitrophenylsulfone; bifunctional photoreactive agents such as bis-[b-(4- azidosalicylamido)ethyl]disulfide; the bifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde, glutaraldehyde, and adipaldehyde; a bifunctional epoxide such as 1,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipic acid dihydrazide, carbohydrazide, and succinic acid dihydrazide; the bifunctional diazoniums o-tolidine, diazotized and bis-diazotized benzidine; the bifunctional alkylhalides NIN'-ethylene- bis(iodoacetamide), N IN'-hexamethylene-bis(iodoacetamide), N IN' -undecamethyl ene- bis(iodoacetamide), as well as benzylhalides and halomustards, such as ala'-diiodo-p-xylene sulfonic acid and tri(2-chloroethyl)amine, respectively.

[0117] In other embodiments, hetero-bifunctional cross-linking agents used to link the disclosed polypeptide and fragments thereof to other molecules, as described herein, include, but are not limited to, SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-l- carboxylate), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N- succinimidyl(4-iodoacteyl)aminobenzoate), SMPB (succinimidyl-4-(p- maleimidophenyl)butyrate), GMBS (N-(gamma-maleimidobutyryloxy)succinimide ester), MPBH (4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H (4-(N-maleimidomethyl) cyclohexane- 1-carboxyl-hydrazide), SMPT (succinimidyloxycarbonyl-a-methyl-a-(2- pyridyldithio)toluene), and SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate).

[0118] 8. A solid support comprising a polypeptide according to any one of embodiments 1-3 immobilized on said solid support. Non-limiting examples of a solid support or substrate include plastic, polystyrenes, nitrocellulose, membranes, chips, and particles. If solid supports other than particles are used, for instance, glass, polymeric or silica chips (such as microchips), plates, slides, etc., the disclosed polypeptide and fragments thereof (target analytes) disclosed herein can be immobilized on the surface of the support at specific locations (e.g., in specific wells of a plate (e.g., microtiter plate) or at specific locations on a chip, microchip, plate or slide). The term “particle” is used herein to refer to a solid or semisolid body, often with linear dimensions on the micron scale (i.e., less than about 100 microns), of any shape or surface texture. Except as noted, the term is used interchangeably with “particle,” which refers to a micron scale particle, and “bead,” which refers to particles that are spherical or near-spherical in shape, often polymeric in composition. Where used in this application, the terms “particle” and “bead” (and grammatical equivalents of these terms) can be interchanged without altering the context of the passages within this application).

[0119] 9. A vector comprising a nucleic acid sequence encoding a polypeptide according to any one of embodiments 1-3 or a nucleic acid sequence encoding a polypeptide according to any one of embodiments 1-3 operably linked to a heterologous promoter.

[0120] 10. A host cell comprising a nucleic acid sequence or vector according to embodiments 9.

[0121] 11. An immunogenic carrier protein covalently attached to a polypeptide comprising any one of SEQ ID NOs: 1, 2, 3, 4, 5 or 6 or a fragment thereof. 12. An immunogenic composition comprising: a) an adjuvant and a immunogenic carrier protein covalently attached to a polypeptide comprising any one of SEQ ID NOs: 1, 2, 3, 4, 5, or 6 or a fragment thereof; or b) an adjuvant and a polypeptide comprising any one of SEQ ID NOs: 1, 2, 3, 4, 5, or 6 or a fragment thereof.

[0122] 13. A kit comprising a polypeptide according to embodiment 7 or a polypeptide according to any one of embodiments 1-3 linked or adsorbed to a solid substrate.

[0123] 14. The kit of embodiment 13, wherein said polypeptide is biotinylated and is linked to the solid substrate with avidin or streptavidin.

[0124] 15. A method of vaccinating an animal comprising administering an immunogenic composition according to embodiment 12 or an immunogenic composition according to embodiment 11 to an animal, preferably an equine animal. The immunogenic composition can be administered by any route, such as intramuscular, subcutaneous or intranasal routes of administration.

[0125] 16. A method of detecting an SEE infection in an equine animal, the method comprising contacting a sample from an equine animal with an substate comprising a polypeptide, or a fragment thereof, according to any one of embodiments 1-3 and detecting the binding of antibodies in said sample to said polypeptide, or a fragment thereof, the method optionally distinguishing animals infected with SEE from animals infected with SEZ and optionally further comprising the treatment of an infected animal.

[0126] 17. The method of embodiment 16, wherein the sample is a serum sample, nasopharyngeal lavage, nasal lavage, guttural pouch lavage, mucous, or an exudate.

[0127] 18. The method of embodiment 16 or 17, wherein the assay is an ELISA or a lateral flow immunoassay.

[0128] 19. The method of embodiments 16-18, wherein the assay is an ELISA.

[0129] 20. The method of embodiments 16-18, wherein the assay is a lateral flow immunoassay.

[0130] 21. A method of detecting an SEE infection in an equine animal or an equine animal exposed to SEE, the method comprising: contacting a sample from an animal with an substate comprising a first antibody that binds to a first epitope of a polypeptide, or a fragment thereof according to any one of embodiments 1-3 and capturing said polypeptide or a fragment thereof; contacting the captured polypeptide or fragment thereof with a second antibody that binds to a second epitope of said polypeptide or a fragment thereof and detecting the binding of said second antibody to said polypeptide, or a fragment thereof, the presence of a label indicating that the animal is infected with SEE or has been exposed to SEE.

[0131] 22. The method of embodiment 21, wherein the sample is a serum sample, nasopharyngeal lavage, nasal lavage, guttural pouch lavage, mucous, nasal or nasopharyngeal swabs, or an exudate.

[0132] 23. The method of embodiment 21 or 22, wherein the assay is an ELISA or a lateral flow immunoassay and the first antibody is, optionally, immobilized on said substrate.

[0133] 24. The method of any one of embodiments 21-23, said method distinguishing an animal infected with SEE from an animal infected with SEZ and, optionally, further comprising the treatment of an animal identified as being infected by SEE.

[0134] 25. An isolated antibody or antigen binding fragment thereof that specifically binds to a polypeptide according to any one of embodiments 1-3.

[0135] 26. The antibody or antigen binding fragment thereof of embodiment 25, said antibody specifically binding SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0136] 27. The antibody or antigen binding fragment of any one of embodiments 25-26, wherein the antibody is a monoclonal antibody.

[0137] 28. The antibody or antigen binding fragment of embodiments 25-26, wherein the antibody is a polyclonal antibody, preferably a non-equine polyclonal antibody.

[0138] 29. The antibody or antigen binding fragment of any one of embodiments 25-28, wherein said antibody is labeled.

[0139] 30. A solid support comprising an antibody or antigen binding fragment according to any one of embodiments 25-28 immobilized on said solid support.

[0140] 31. The solid support according to embodiment 30, wherein the solid support is selected from the group consisting of plastics, polystyrenes, nitrocellulose, membranes, chips, beads, and particles.

[0141] 32. A mRNA vaccine comprising a mRNA molecule encoding a polypeptide, or a fragment thereof, according to any one of embodiments 1-3 formulated in a liposomal carrier or a composition comprising beta-amino-ester and beta-thio-ester polymers.

[0142] 33. A method of vaccinating an equine animal comprising the administration of an mRNA vaccine according to embodiment 32 to said equine animal. 34. The method according to embodiment 33, wherein the mRNA vaccine is formulated in a liposomal carrier and is administered to said equine animal intramuscularly.

[0143] 35. The method according to embodiment 33, wherein the mRNA vaccine is formulated in a composition comprising beta-amino-ester and beta-thio-ester polymers and is administered into the airway of said equine animal.

[0144] 36. The method according to embodiment 35, wherein the mRNA vaccine is administered as an aerosol or is nebulized for administration.

[0145] EXAMPLE MATERIALS AND METHODS

[0146] Serological testing offers a convenient method to detect horses exposed to SEE that are not showing clinical signs, including inapparent carriers. Currently, two serological tests for SEE have been commercialized. The first test developed detects antibodies targeting the SeM protein of SEE. Currently, this is the only test licensed and available in the US, but has limited sensitivity and specificity, in large part because of cross-reactivity with a homologous protein found in SEZ (Robinson et al., 2013; Kelly et al., 2006; Davidson et al. 2008). As a result, another test was developed in England based on comparative genomics of a single strain each of SEE and SEZ (Holden et al., 2009, Robinson et al., 2013). This test simultaneously recognizes antibodies against 2 antigens: antigen A and antigen C (a fragment of the SeM protein). Although this newer ELISA has improved specificity (Robinson et al., 2013; Tscheschlok etal., 2018), reports of limited accuracy have been described (Pringle etal., 2020; Sebire etal., 2019; Riihimaki etal, 2019; Durham etal, 2020 and it is not available in the US. Thus, great need exists for an improved serological test for strangles in the US allowing for identification, isolation, and - when appropriate - treatment of infected horses that lack clinical signs.

[0147] We have identified 84 proteins expressed on the surface of SEE that differed substantially (>75%) from SEZ. The genes encoding these proteins (plus 8 genes with homologous sequences in SEE and SEZ as positive controls) were cloned and proteins expressed using a cell-free system. These proteins were printed onto a microarray and probed with serum from 30 horses infected with SEE, 30 healthy horses, and 18 SEZ-infected horses with respiratory disease (Figure 1). This array identified 4 proteins that appeared promising for discriminating between SEE-infected horses and either healthy or SEZ-infected horses. As expected, proteins A and C (SeM) were identified using the microarray; however, we identified a novel protein (SEQ ID NO: 1) that outperformed the other proteins (Figure 2). Further analysis of SEQ ID NO: 1 (also referred to as Protein 92) revealed two subsequences (SEQ ID NOs: 2 and 3) that were highly homologous among the genomes of several hundred strains of SEE but dissimilar to sequences of several hundred strains of SEZ (data not shown). We mapped one epitope (SEQ ID NOs: 4, 5 and 6) expected to be readily recognized by the immune system of horses for each of these two polypeptide regions.

[0148] Example 2

[0149] Sera were collected from a) 102 horses with recent or active clinical signs of strangles including fever, nasal discharge, abscesses of the submandibular or retropharyngeal lymph nodes, and positive results for SEE from microbiologic culture, PCR, or both, b) 104 apparently healthy horses without clinical signs of SEE, and c) 24 horses with respiratory tract disease attributed to SEZ based on microbiologic culture. This sample size was based on 80% power to detect a difference in area under the receiver operator characteristic (ROC) curve of Protein 92 of 96% and SeM of 88% (based on findings of the microarray study), with significance level of 5% and variance parameters derived from the microarray study using power. roc. test command in the pROC package of R.

[0150] Protein 92 and SeM genes were cloned, expressed in a bacterial vector, and highly purified. Immulon 4 HBX 96-well immunoassay plates were coated overnight with 2 pg / mL of either Protein 92 or SeM. After blocking of excess protein binding sites with 5% skim milk in PBS, the plates were incubated with the sera collected from study horses diluted 1:10,000; a positive control consisting of serum from a donor horse hyperimmunized against SEE was included on each plate. After rinsing the plates, the primary antibodies were visualized using a horse-radish peroxidase-conjugated isotype-specific (z.e., IgG) secondary antibody and a substrate system containing tetramethylbenzidine (TMB) and peroxide. The ELISA data were analyzed as the ratio of the optical density (OD) of the sample (minus blank) to the positive control sample (minus blank), and referred to as the OD ratio.

[0151] The areas under the ROC curves were determined and compared between the 2 ELISA tests using the pROC program in R to identify the test with highest accuracy. Additionally, the sensitivity and specificity of the ELISA at the optimal cut-point of the data were estimated and compared between groups using the pROC program. Significance was set at P < 0.05 for all analyses.

[0152] The mean area under the ROC curve for Protein 92 ELISA was 95% (95% CI = 92 to 98%), and this was significantly (P < 0.05) greater than that for the SeM ELISA (88%; 95% CI = 83% to 92%) (Figure 3). Moreover, the estimated sensitivity (92.2%) and specificity (90.2%) for the Protein 92 ELISA at the optimal threshold from the ROC curve data were significantly (P < 0.05) higher than those for the SeM ELISA (82.2% sensitivity and 78.4% specificity). The distribution of the 3 groups of horses (healthy controls, horses with strangles, and 24 horses infected with SEZ (Figures 4 and 5). The 2 control horses with high P92 OD ratios were older horses that had previously been vaccinated for strangles.

[0153] The ELISA based on the novel Protein 92 ELISA was significantly more accurate than an ELISA based on the SeM protein. Limitations in the diagnostic accuracy of the SeM ELISA have been recognized, leading to the development of a newer ELISA test based on 2 antigens of SEE. Unfortunately, this test - known as the A+C ELISA - is not available in the US. Thus, results of this study indicating a single-protein ELISA can provide significantly greater accuracy for detecting horses infected with SEE than an ELISA based on SeM is important.

[0154] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and / or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

[0155] REFERENCES

[0156] Robinson C, Steward KF, Potts N, Barker C, Hammond TA, Pierce K, Gunnarsson E, Svansson V, Slater J, Newton JR, Waller AS. Combining two serological assays optimises sensitivity and specificity for the identification of Streptococcus equi subsp. exposure. Vet J 2013;197:188-191.

[0157] Kelly C, Bugg M, Robinson C, Mitchell Z, Davis-Poynter N, Newton JR, Jolley KA, Maiden MC, Waller AS. Sequence variation of the SeM gene of Streptococcus equi allows discrimination of the source of strangles outbreaks. J Clin Microbiol 2006; 44:480- 486.

[0158] Davidson A, Traub-Dargatz JL, Magnuson R, Hill A, Irwin V, Newton R, Waller A, Smith K, Callan RJ, Meehan M, Owen P, Salman M. Lack of correlation between antibody titers to fibrinogen-binding protein of Streptococcus equi and persistent carriers of strangles. J Vet Diagnost Invest 2008;20:457-462.

[0159] Holden MTG, Heather Z, Paillot R, et al. Genomic evidence for the evolution of Streptococcus equi: host restriction, increased virulence, and genetic exchange with human pathogens. PLoS Pathogens 2009; 5(3):el000346.

[0160] Tscheschlok L, Venner M, Steward, Bose R, Riihimaki M, Pringle J. Decreased clinical severity of strangles in weanlings associated with restricted seroconversion to optimized Streptococcus equi ssp equi assays. J Vet Intern Med 2018;32:459-464.

[0161] Pringle J, Storm E, Waller A, Riihimaki. Influence of penicillin treatment of horses with strangles on seropositivity to Streptococcus equi ssp. equi-specific antibodies. J Vet Intern Med 2020;34:294-299.

[0162] Sebire S, Lagy C, Castagnet S, Pradier S, Paillot R, Giard JC, Leon-Seck. Comparison of diagnostic tests to improve detect of Streptococcus equi ssp. equi and management of strangles epizooties in France. Paper presented at: Dorothy Havemeyer Foundation Workshop: Getting to grips with strangles and other streptococcal diseases; May 2-4, 2019. Reykjavik, Iceland.

[0163] Riihimaki M, Steward K, Pringle. Persistence of Streptococcus equi ssp. equi antibodies in a prolonged strangles outbreak, assessed by an enhanced indirect enzyme-linked immunosorbent assay. Paper presented at: Dorothy Havemeyer Foundation Workshop: Getting to grips with strangles and other streptococcal diseases; May 2-4, 2019. Reykjavik, Iceland. Durham AE, Kemp-Symonds J. Failure of serological testing for antigens A and C of Streptococcus equi subspecies equi to identify guttural pouch carriers. Equine Vet J 2020; DOI:10.1111. evj.13276.

Claims

CLAIMSWe claim:

1. A polypeptide comprising SEQ ID NO: 1, 2, 3, 4, 5, 6 or a fragment thereof.

2. The polypeptide according to claim 1, wherein the polypeptide is a fragment of SEQ ID NO: 1, 2, 3, 4, 5, or 6.

3. The polypeptide according to claim 2, wherein said fragment comprises: a) a span of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, or 181 consecutive amino acids of SEQ ID NO: 1 or a span between any two of the listed values that is at least 5 amino acid in length. In preferred embodiments, the fragment begin at position 35 of SEQ ID NO: 1; b) a span of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, or 147 consecutive amino acids of the mature protein of SEQ ID NO: 1 (amino acids 35-182) or a span between any two of the listed values that is at least 5 amino acid in length; c) a span of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 consecutive amino acids of SEQ ID NO: 2 or a span between any two of the listed values that is at least 5 amino acid in length;d) a span of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive amino acids of SEQ ID NO: 3 or a span between any two of the listed values that is at least 5 amino acid in length;; or e) a span of 5, 6, 7, 8, 9, 10, 11, 12,13, or 14 consecutive amino acids of any one of SEQ ID NOs: 4, 5, or 6.

4. The polypeptide according to any one of claims 1-3, said polypeptide further comprising biotin, a detectable label, tag, additional amino acid, carrier, Trp-Ser-His-Pro-Gln- Phe-Glu-Lys, Hise, or a cross-linking agent.

5. The polypeptide according to claim 4, wherein the detectable label is fluorescent, luminescent, radioactive, or electron dense.

6. The polypeptide according to claim 5, wherein the detectable label comprises green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), beta-galactosidase, luciferase,32P,125I,3H and14C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl or umbelliferone.

7. The polypeptide according to claim 4, wherein the polypeptide further comprises a carrier.

8. The polypeptide according to claim 7, wherein the carrier is KLH, thyroglobulin, an albumin, tetanus toxoid, a polyamino acids, poly (lysine:glutamic acid), an influenza protein, or hepatitis B virus core protein.

9. The polypeptide according to claim 4, wherein the polypeptide further comprises a cross-linking agent, a homo-bifunctional cross-linking agent, or a heterobifunctional cross-linking agent.

10. The polypeptide according to claim 9, wherein the crosslinking agent is a carbodimide, a homo-bifunctional aldehyde, a homo-bifunctional epoxide, a homobifunctional imido-ester, a homo-bifunctional N-hydroxysuccinimide ester, a homobifunctional maleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyldisulfide, a homo-bifunctional aryl halide, a homo-bifunctional hydrazide, a homo-bifunctional diazonium derivative, a homo-bifunctional photoreactive compound, a hetero-bifunctional compounds having an amine-reactive and a sulfhydryl-reactive group, an amine-reactive and a photoreactive group or a carbonyl -reactive and a sulfhydryl-reactive group.

11. The polypeptide according to claim 10, wherein the crosslinking agent is 1- cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), l-ethyl-3-(3- dimethyaminopropyl) carbodiimide (EDC), l-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide, a bifunctional N-hydroxysuccinimide ester, dithiobis(succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional imido-esters dimethyl adipimidate, dimethyl pimelimidate, dimethyl suberimidate, a bifunctional sulfhydryl-reactive crosslinkers, l,4-di-[3'-(2'-pyridyldithio)propionamido]butane, bismaleimidohexane, bis-N- maleimido-l,8-octane, a bifunctional aryl halide, l,5-difluoro-2,4-dinitrobenzene, 4,4'- difhroro-3,3'-dinitrophenylsulfone, a bifunctional photoreactive agent, bis-[b-(4- azidosalicylamido)ethyl]disulfide, a bifunctional aldehyde, formaldehyde, malondialdehyde, succinaldehyde, glutaraldehyde, adipaldehyde, a bifunctional epoxide, 1,4-butaneodiol diglycidyl ether, a bifunctional hydrazide, adipic acid dihydrazide, carbohydrazide, succinic acid dihydrazide, a bifunctional diazonium, o-tolidine, diazotized or bis-diazotized benzidine, a bifunctional alkylhalide, NlN'-ethylene-bis(iodoacetamide), NlN'-hexamethylene- bis(iodoacetamide), NlN'-undecamethylene-bis(iodoacetamide), a benzylhalide, a halomustard, SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl(4- iodoacteyl)aminobenzoate), SMPB (succinimidyl-4-(p-maleimidophenyl)butyrate), GMBS (N-(gamma-maleimidobutyryloxy)succinimide ester), MPBH (4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H (4-(N-maleimidom ethyl) cyclohexane- 1 -carboxylhydrazide), SMPT (succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene), or SPDP (N-succinimi dy 1 3 -(2-pyri dy 1 dithi o)propi onate) .

12. A solid support comprising a polypeptide according to any one of claims 1-3 immobilized on said solid support.

13. The solid support according to claim 12, wherein the solid support is selected from the group consisting of plastics, polystyrenes, nitrocellulose, membranes, chips, beads, and particles.

14. A vector comprising a nucleic acid sequence encoding a polypeptide according to any one of claims 1-3 or a nucleic acid sequence encoding a polypeptide according to any one of claims 1-3 operably linked to a heterologous promoter.

15. A host cell comprising a nucleic acid sequence or vector according to claims 14.

16. An immunogenic carrier protein covalently attached to a polypeptide according to any one of claims 1-3.

17. An immunogenic composition comprising: a) an adjuvant and a immunogenic carrier protein covalently attached to a polypeptide according to any one of claims 1-3; or b) an adjuvant and a polypeptide according to any one of claims 1-3.

18. A kit comprising a polypeptide according to claim 7 or a polypeptide according to any one of claims 1-3 immobilized on, linked, or adsorbed to a solid substrate.

19. The kit according to claim 18, wherein said polypeptide is biotinylated and is immobilized on or linked to the solid substrate with avidin or streptavidin.

20. A method of vaccinating an animal comprising administering an immunogenic composition according to claim 17 or an immunogenic carrier protein according to claim 16 to an animal, preferably an equine animal.

21. A method of detecting an SEE infection in an equine animal or an equine animal exposed to SEE, the method comprising contacting a sample from an equine animal with an substate comprising a polypeptide, or a fragment thereof, according to any one of claims 1-3and detecting the binding of antibodies in said sample to said polypeptide, or a fragment thereof.

22. The method according to claim 21, wherein the sample is a serum sample, mucous, nasopharyngeal lavage, nasal lavage, guttural pouch lavage, nasal or nasopharyngeal swab, or an exudate.

23. The method according to claim 21 or 22, wherein the assay is an ELISA or a lateral flow immunoassay.

24. The method according to any one of claims 21-23, said method distinguishing an animal infected with SEE from an animal infected with SEZ or identifying an animal as having been exposed to SEE and, optionally, further comprising the treatment of an animal infected with SEE.

25. A method of detecting an SEE infection in an equine animal or an equine animal exposed to SEE, the method comprising: contacting a sample from an equine animal with an substate comprising a first antibody that binds to a first epitope of a polypeptide, or a fragment thereof according to any one of claims 1-3 and capturing said polypeptide or a fragment thereof; contacting the captured polypeptide or fragment thereof with a second antibody that binds to a second epitope of said polypeptide or a fragment thereof and detecting the binding of said second antibody to said polypeptide, or a fragment thereof, the presence of a label indicating that the animal is infected with SEE or has been exposed to SEE.

26. The method according to claim 25, wherein the sample is a serum sample, mucous, nasopharyngeal lavage, nasal lavage, guttural pouch lavage, nasal or nasopharyngeal swab, or an exudate.

27. The method according to claim 25 or 26, wherein the assay is an ELISA or a lateral flow immunoassay and the first antibody is, optionally, immobilized on said substrate.

28. The method according to any one of claims 25-27, said method distinguishing an animal infected with SEE from an animal infected with SEZ and, optionally, further comprising the treatment of an animal infected with SEE.

29. An isolated antibody or antigen binding fragment thereof that specifically binds to a polypeptide according to any one of claims 1-3.

30. The antibody or antigen binding fragment thereof according to claim 29, said antibody or antigen binding fragment specifically binding SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

31. The antibody or antigen binding fragment according to any one of claims 29- 30, wherein the antibody is a monoclonal antibody.

32. The antibody or antigen binding fragment according to claim 29 or 30, wherein the antibody is a polyclonal antibody, preferably a non-equine polyclonal antibody.

33. The antibody according to any one of claims 29-32, wherein said antibody is labeled.

34. A solid support comprising an antibody according to any one of claims 29-32 immobilized on said solid support.

35. The solid support according to claim 34, wherein the solid support is selected from the group consisting of plastics, polystyrenes, nitrocellulose, membranes, chips, beads, and particles.

36. A mRNA vaccine comprising a mRNA molecule encoding a polypeptide, or a fragment thereof, according to any one of claims 1-3 formulated in a liposomal carrier or a composition comprising beta-amino-ester and beta-thio-ester polymers.

37. A method of vaccinating an equine animal comprising the administration of an mRNA vaccine according to claim 36 to said equine animal.

38. The method according to claim 37, wherein the mRNA vaccine is formulated in a liposomal carrier and is administered to said equine animal intramuscularly.

39. The method according to claim 37, wherein the mRNA vaccine is formulated in a composition comprising beta-amino-ester and beta-thio-ester polymers and is administered into the airway of said equine animal.

40. The method according to claim 39, wherein the mRNA vaccine is administered as an aerosol or is nebulized for administration.

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