Feline leukemia virus antigens and epitopes and proteins that bind thereto

Abstract

Provided are highly conserved antigens and epitopes of Feline Leukemia Virus that can be used in vaccines and to produce binding gp70 or pl5E proteins (e.g., antibodies) for treating, preventing, or reducing the risks of infections caused by Feline Leukemia Virus, and as targets for detecting Feline Leukemia Virus infection.

Description

FELINE LEUKEMIA VIRUS ANTIGENS AND EPITOPES AND PROTEINS THAT BIND THERETO Cross-Reference To Related Applications[0001 This application claims priority under 35 USC § 119 (e) to U.S. Provisional Application No. 63 / 561590, filed March 5, 2024, the entire contents of which are incorporated herein by reference.Sequence Listing

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 28, 2025, is named 116540-0128_SL.xml and is 23,443 bytes in size.Field

[0003] The present disclosure relates to the field of viral vaccines, therapeutics, and diagnostics, and in particular, discloses antigens and epitopes of Feline Leukemia Virus (FeLV) that can be used in vaccines and to produce binding proteins (e.g., antibodies) for treating, preventing, or reducing the risks of infections caused by Feline Leukemia Virus, which antigens and binding proteins also can be used in assays and kits for detecting infection by Feline Leukemia Virus.Background

[0004] The following discussion is merely provided to aid the reader in understanding the disclosure and is not admitted to describe or constitute prior art thereto.[0005} Feline leukemia virus, (FeLV) is an RNA virus of Gamma-retrovirus (y-retrovirus) genus, Retroviridae family.

[0006] Approximately 2 to 3% of all cats are infected with this rare disease. However, rates rise significantly depending on whether the cats live outdoors, their immune system is deficient or compromised, their age, and their contact with other cats that already have the virus.(0097) Thus, there is a need for prophylactic, therapeutic, and diagnostic agents useful against various variant strains of Feline Leukemia Virus, including variants that have not yet been identified and / or that have not yet emerged.Summary

[0008] Described herein are highly conserved antigens and epitopes of Feline Leukemia Virus (FeLV), binding proteins (e.g., antibodies) that bind to the disclosed antigens and epitopes, vaccines based on the antigens, methods of treating, preventing, or reducing the risks of infection with the antigens or binding proteins, and methods and kits for detecting or diagnosing infection by Feline Leukemia Virus using the antigens or binding proteins.

[0009] In one aspect, the present disclosure provides an isolated peptide antigen comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 1-26.

[0010] The peptide can be a recombinant peptide.

[0011] The amino acid sequence of the peptide can be selected from any one of SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25.

[0012] In another aspect, the present disclosure provides a vaccine composition comprising a peptide antigen described herein (e g., a peptide antigen of the foregoing aspect) and a pharmaceutically acceptable carrier.

[0013] In another aspect, the present disclosure provides an isolated binding protein that binds to a conserved epitope of a Feline Leukemia Virus (FeLV) wherein the conserved epitope comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1-26.

[0014] The amino acid sequence of the conserved epitope can be selected from any one of SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25. The amino acid sequence of the conserved epitope can be SEQ IDN0:2. The amino acid sequence of the conserved epitope can be SEQ ID N0:3. The amino acid sequence of the conserved epitope can be SEQ ID NON. The amino acid sequence of the conserved epitope can be SEQ ID NO:11. The amino acid sequence of the conserved epitope can be SEQ ID NO: 13. The amino acid sequence of the conserved epitope can be SEQ ID NO: 16. Theamino acid sequence of the conserved epitope can be SEQ ID NO:22. The amino acid sequence of the conserved epitope can be SEQ ID NO:25.

[0015] The conserved epitope can be a FeLV GP70 epitope that comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1-16. The conserved epitope can be a FeLV pl5E epitope that comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 17-26.

[8016] The binding protein can be an antibody or an antibody fragment, such as a monoclonal antibody.

[0017] In another aspect, the present disclosure provides a method of reducing the risk of Feline Leukemia Virus infection in a subject, comprising administering to the subject an effective amount of an isolated peptide antigen described herein (e.g., the first aspect above) or a vaccine described herein (e.g., the second aspect above).

[8018] In another aspect, the present disclosure provides a method of treating, preventing, or reducing the risk of Feline Leukemia Virus infection in a subject, comprising administering to the subject a therapeutically effective amount of an isolated binding protein described herein (e.g., the third aspect above).

[8019] For the purposes of the disclosed methods, the isolated peptide antigen or isolated binding protein can be administered by subcutaneous or intramuscular injection.

[8020] For the purposes of the disclosed methods, the subject may be a feline.

[8021] In another aspect, the present disclosure provides an isolated peptide antigen described herein (e.g., the first aspect above) or a vaccine described herein (e.g., the second aspect above) for inducing an immune response to Feline Leukemia Virus.

[8022] In another aspect, the present disclosure provides an isolated binding protein described herein (e.g., the third aspect above), for treating, preventing, or reducing the risk of Feline Leukemia Virus infection in a subject in need thereof.(0023) In another aspect, the present disclosure provides uses of an isolated peptide antigen described herein (e.g., the first aspect above) or a vaccine described herein (e.g., the second aspect above) for inducing an immune response to Feline Leukemia Virus.

[0024] In another aspect, the present disclosure provides uses of an isolated binding protein described herein (e.g., the third aspect above) in the preparation of a medicament for treating, preventing, or reducing the risk of Feline Leukemia Virus in a subject in need thereof.

[0025] In another aspect, the present disclosure provides a method of preparing an antibody that binds to a peptide antigen described herein, comprising:(a) identifying an asymptomatic patient that has been infected with Feline Leukemia Virus as a donor for obtaining immune B-lymphocytes that produce high titers of Feline Leukemia Virusneutralizing antibodies;(b) collecting the B-lymphocytes from the patient;(c) immortalizing the B-lymphocytes;(d) collecting antibodies produced by the immortalized B-lymphocytes; and(e) screening the antibodies for binding to a peptide antigen disclosed herein.

[0026] The method may further comprise testing the antibodies for binding to Feline Leukemia Virus. Additionally or alternatively, the method may further comprise epitope mapping the antibodies that tested positive for binding to Feline Leukemia Virus. For the purposes of this method, immortalizing the B-lymphocytes may comprise fusing a B-lymphocyte with a heteromyeloma cell in order to produce a heterohybridoma cell.

[0027] In another aspect, the present disclosure provides an in vitro method of analyzing a biological sample obtained from a subject, comprising contacting the sample with a binding protein that specifically binds to a Feline Leukemia Virus peptide antigen selected from SEQ ID NOs: 1-26, and detecting binding between the binding protein and any Feline Leukemia Virus antigen present in the sample.

[0028] The method may comprise contacting the sample with a panel of from 2 to 26 binding proteins that each specifically binds to a different Feline Leukemia Virus peptide antigen selectedfrom SEQ ID NOs: 1-26, and detecting binding between the binding proteins and any Feline Leukemia Virus antigen present in the sample.

[0029] In another aspect, the present disclosure provides an in vitro method of analyzing a biological sample obtained from a subject, comprising contacting the sample with a Feline Leukemia Virus peptide antigen comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1-26, and detecting binding between the peptide antigen and any anti-Feline Leukemia Virus antibodies present in the sample.

[0030] The method may comprise contacting the sample with a panel of from 2 to 26 peptide antigens each comprising or consisting of a different an amino acid sequence selected from SEQ ID NOs: 1-26, and detecting binding between the peptide antigen and any anti-Feline Leukemia Virus antibodies present in the sample.

[0031] In another aspect, the present disclosure provides an in vitro method of analyzing a biological sample obtained from a subject, comprising extracting nucleic acids from the biological sample, contacting the extracted nucleic acids with a pair of primers that specifically amplify a nucleic acid sequence encoding a peptide of any one of SEQ ID NOs: 1-26, and detecting the presence of the amplified nucleic acid sequence if present in the sample.

[0032] The method may comprise contacting the sample with a panel of from 2 to 26 primer pairs that each specifically amplify a nucleic acid sequence that encodes a different amino acid sequence selected from SEQ ID NOs: 1-26, and detecting amplification of each nucleic acid sequence if present in the sample. For the purposes of this method, the sample can be selected from saliva, nasal fluid, nasal cells, throat cells, blood, plasma, serum, urine, and feces. For the purposes of this method, the subject may be a feline. For the purposes of this method, the subject may be suspected of having a Feline Leukemia Virus infection, has been exposed to Feline Leukemia Virus, or is suspected of having been exposed to Feline Leukemia Virus. The method may further comprise determining that the subject is infected with Feline Leukemia Virus when binding or amplification is detected.

[0033] In another aspect, the present disclosure provides a kit comprising at least one binding protein that specifically binds to a peptide comprising or consisting of any one of SEQ ID NOs: 1-26, a solid substrate to which the at least one binding protein is attached, and a detectably labeled antibody that specifically binds to the peptide to which the at least one binding protein specifically binds.

[0034] In another aspect, the present disclosure provides a kit comprising at least one peptide comprising or consisting of any one of SEQ ID NOs: 1-26, a solid substrate to which the at least one peptide is attached, and a detectably labeled antibody that specifically binds to IgE or IgD, wherein the IgE or IgD are optionally feline; optionally, wherein the at least one peptide comprises or consists of any one of SEQ ID NOs: 1-26.

[0035] In another aspect, the present disclosure provides a kit comprising at least one primer pair capable of specifically amplifying a nucleic acid sequence that encodes a peptide selected from any one of SEQ ID NOs: 1-26, wherein:(a) at least one primer of the primer pair is detectably labeled; or(b) the kit further comprises a detectably labeled probe that hybridizes to the nucleic acid sequence amplified by the primer pair.

[0036] The foregoing general description and following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure.Brief Description of the Drawings

[0037] FIG. 1 shows the structure of Feline Leukemia Virus.

[0038] FIG. 2 shows a scheme of the genomic structure of Feline Leukemia Virus.

[0039] FIG. 3 shows an alternative scheme of the genomic structure of Feline Leukemia Virus.Detailed Description

[0040] The present disclosure provides highly conserved antigens and epitopes of the Feline Leukemia Virus envelope, specifically epitopes of the gp70 glycoprotein and the transmembrane protein pl5E. The disclosed antigens and epitopes are highly conserved across all viral isolates, indicating that there is strong selective pressure to maintain these sequences, even as variants ofFeline Leukemia Virus develop. As a result, proteins (e g. antibodies) that bind to the disclosed antigens and epitopes may provide pan-protection, both in terms of treatment and prevention, against variant strains of Feline Leukemia Virus. Similarly, vaccines based on the disclosed highly conserved antigens may provide broadly universal and temporally durable protection against a range of Feline Leukemia Virus variants and retrovirus strains. Also, diagnostic methods and kits based on the disclosed antigens may permit detection of infection by known and future strains of Feline Leukemia Virus.I. Definitions[00411 It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.[0042J Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined. Unless otherwise specified, materials and / or methodologies known to those of ordinary skill in the art can be utilized in carrying out the methods described herein, based on the guidance provided herein.

[0043] As used herein, the singular terms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Reference to an object in the singular is not intended to mean “one and only one” unless explicitly so stated, but rather “one or more.”

[0044] As used herein, “about” when used with a numerical value means the numerical value stated as well as plus or minus 10% of the numerical value. For example, “about 10” should be understood as both “10” and “9-11

[0045] As used herein, a phrase in the form “A / B” or in the form “A and / or B” means (A), (B), or (A and B); a phrase in the form “at least one of A, B, and C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

[0046] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but does not exclude others.

[0047] An used herein, the term “isolated” when used in the context of referring to a peptide antigen or binding protein or antibody as discussed herein refers to one which has been separatedfrom at least some of the components with which it existed in nature (for those isolated from nature) or with which it was produced (for those produced, e.g., in a laboratory setting).

[0048] As used herein, a “variant” when used in the context of referring to a peptide means a peptide sequence that is derived from a parent sequence by incorporating one or more amino acid changes, which can include substitutions, deletions, or insertions. For the purposes of this disclosure, a variant may comprise an amino acid sequence that shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to about 100% sequence identity or homology with the reference (or “parent”) sequence. For purposes of this disclosure, the terms “variant” and “derivative” when used in the context of referring to a peptide are used interchangeably.

[0049] As used herein, the phrases “effective amount,” “therapeutically effective amount,” and “therapeutic level” mean the dosage or concentration of an antigen, antibody or binding protein that provides the specific pharmacological effect for which the antigen, antibody or binding protein is administered in a subject in need of such treatment, e.g., to induce a protective immune response against FeLV or to treat or prevent FeLV infection (e.g., an infection with Feline Leukemia Virus). It is emphasized that a therapeutically effective amount or therapeutic level of an antigen, antibody or binding protein will not always be effective in inducing a protective immune response or treating or preventing the infections described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art. The therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and / or the subject’s condition, including the type and severity of the FeLV infection.[0050J The terms “treat,” “treatment” or “treating” as used herein with reference to FeLV infection refer to reducing or eliminating viral load.

[0051] The terms “prevent,” “preventing” or “prevention” as used herein with reference to FeLV infection refer to precluding or reducing the risk of an infection from developing in a subject exposed to FeLV, or to precluding or reducing the risk of developing a high viral load of FeLV. Prevention may also refer to the prevention of a subsequent infection once an initial infection has been treated or cured.(0052) The terms “individual,” “subject,” and “patient” are used interchangeably herein, and refer to any individual mammalian subject, e.g., bovine, canine, feline, equine, or human. In specific embodiments, the subject, individual, or patient is a feline. In a specific embodiment, the subject, individual, or patient is a domesticated cat, e.g., a house cat.II. Antigens and Epitopes of gp70 and pl5E Proteins of FeLV

[0953] The present disclosure provides highly conserved peptide sequences of the Feline Leukemia Virus (FeLV) gp70 and pl 5E proteins that can be used as antigens or epitopes of binding proteins (e.g., antibodies) for vaccines, targets for drugs and / or for treating and / or preventing and / or diagnosing Feline Leukemia Virus infection.[0054} The structure of feline leukemia virus as shown in FIG.1. Gamma-retroviruses contain two identical strands of RNA and associated enzymes, which include reverse transcriptase, integrase, and protease, packaged into a core composed of the capsid protein (p27) with a surrounding matrix, all enclosed by a phospholipid membrane envelope derived from the host cell. The envelope contains a gp70 glycoprotein and a transmembrane protein pl5E.

[0055] The FeLV genome is a linear single stranded 8.3 kb RNA molecule. It has major structural and nonstructural genes gag, pol, env, flanked by long terminal repeats (LTR) (FIG. 2 and FIG. 3). Transcription is regulated by 5'-LTR. The FeLV genome contains two ORFs: one for gag and pol, and another for env genes.

[0956] The disclosed epitopes of SEQ ID NOs: 1-26 were discovered using computational prediction and machine learning approaches to assess and compare 401 viral isolates to identify highly conserved regions.

[0057] The pronounced conservation of these regions across this many isolates indicates that these regions are unlikely to mutate as Feline Leukemia Virus evolves and spawns new variants. The conserved nature of these epitopes also indicates that antibodies or other binding proteins that bind to these regions and vaccines that contain an antigen that comprises or consists of a peptide having one of the amino acid sequences may provide broad treatment and / or protection against a wide FeLV variants, and that these epitopes and antibodies or other binding proteins that bind to theseregions can be used in diagnostic assays useful for detecting or diagnosing Feline Leukemia Virus infection.

[0058] Specific antigens / epitopes of the gp70 glycoprotein of the Feline Leukemia Virus disclosed herein are shown in Table 1 below.Table 1 - FeLV gp70 Antigens / Epitopes

[0059] Specific antigens / epi topes of the pl5E transmembrane protein of the Feline Leukemia Virus spike protein disclosed herein are shown in Table 2 below.Table 2 - FeLV pl5E Antigens / Epitopes[0060 Three-dimensional artificial intelligence analysis of the Feline Leukemia Virus gp70 and pl5E proteins was undertaken to identify accessible regions for antibody binding, and it was determined that all of the disclosed epitopes are linear epitopes on the proteins. In particular, starting with Feline Leukemia Virus gp70 and pl5E structures (see GenBank: AAA43048.1, GenBank: AAA4305L1, GenBank: AAC31802.1, GenBank: AAA43050.1; PIR: VCMVFP; see also Nicolaisen-Strouss et al., J. Virol., 1987, 61(11):3410-5; Donahue et al., J. Virol., 1988, 62(3):722-31; Kumar et al., J. Virol., 1989, 63(5):2379-84; and Overbaugh et al., Virology, 1992, 188(2): 558-69), a three-dimensional model for each protein was constructed. Using these models, individual models were built for each of the 26 epitopes disclosed herein. These models indicate the epitopes are conserved, accessible, and linear on the respective proteins.

[0061] The peptide sequences disclosed in Tables 1 and 2 can be used as antigens in a vaccine or can be used to develop a binding protein, such as an antibody, to be used in passive immunization or therapy. Any one or more of the peptide sequences in Tables 1 or 2 (i.e., SEQ ID NOs: 1-26) can be formulated in a vaccine to immunize a subject and / or to induce an immune response (e.g., to induce antibodies) in the subject, and can be used singly or in combination (e.g., in multivalent vaccines or multivalent diagnostics as discussed below). Alternatively, any one or more of the peptide sequences in Tables 1 or 2 (i.e., SEQ ID NOs: 1-26) can be used to produce (raise) antibodies, such as by immunizing an animal, such as a mouse or rat or human, to produce (raise)antibodies that specifically bind to the immunizing peptide sequence, including to produce monoclonal antibodies. The antibodies induced or produced by such methods will bind to the corresponding sequences on the gp70 protein or the pl5E protein, e.g., will bind to that epitope on the gp70 protein or the pl5E protein. Such antibodies can be used singly or in combination for analytical purposes or in passive immunotherapy as discussed below.[0062) Studies indicate that the antigens / epitopes comprising a cysteine residue may be the most immunogenic and likely to give rise to neutralizing antibodies. Thus, peptides comprising or consisting of any of SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25 may be particularly useful as vaccine antigens, and antibodies raised against peptides comprising or consisting of any of SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25 may be particularly useful for passive immunization or passive immunotherapy therapy. Thus, the amino acid sequence of the antigen / epitope can be SEQ ID NO:2. The amino acid sequence of the antigen / epitope can be SEQ ID NO:3. The amino acid sequence of the antigen / epitope can be SEQ ID NON. The amino acid sequence of the antigen / epitope can be SEQ ID NO:11. The amino acid sequence of the antigen / epitope can be SEQ ID NO: 13. The amino acid sequence of the antigen / epitope can be SEQ ID NO: 16. The amino acid sequence of the antigen / epitope can be SEQ ID NO:22. The amino acid sequence of the conserved epitope can be SEQ ID NO:25.III. FeLV Vaccines[0063J The present disclosure provides vaccines comprising one or more antigen(s) that comprise or consist of a peptide having one of the amino acid sequences in Tables 1 or 2 (i.e., SEQ ID NOs: 1-26). In some embodiments, the vaccines comprise one or more antigen(s) that comprise or consist of a peptide having an amino acid sequence of any of SEQ ID NOs: 1-26. In some embodiments, the vaccines comprise one or more antigen(s) that comprise or consist of a peptide having an amino acid sequence of any of SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25. The antigens can be prepared by methods known in the art, such as chemical synthesis, or by recombinant methods. Techniques for making peptides are known in the art, and can be used to obtain antigens as disclosed herein. The vaccines may comprise one or more antigen(s) formulated in a pharmaceutically acceptable carrier for the intended route of administration, as discussed in more detail below.[0064J The immune response elicited by immunization with a vaccine as disclosed herein (e.g., comprising one or more antigen(s) that comprise or consist of a peptide having one of the sequences in Tables 1 or 2 (SEQ ID NOs: 1-26, including SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25) is expected to induce production of antibodies that bind highly conserved epitopes of Feline Leukemia Virus and provide broad spectrum immune protection against Feline Leukemia Virus and variants thereof.[0065} A vaccine as disclosed herein, comprising one or more antigen(s) that comprise or consist of one or more of the peptide sequences in Tables 1 or 2 (i.e., each comprising or consisting of one of SEQ ID NOs: 1-26, including SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25), can be used for treating or preventing Feline Leukemia Virus infection (e.g., FeLV). Optimal doses and routes of administration may vary, such as based on the route of administration and dosage form, the age and weight of the subject, and / or the subject’s condition, and can be determined by the skilled practitioner. The vaccine may be formulated for injection and administered parenterally, such as intramuscularly, subcutaneously, or intradermally. The vaccine may be formulated for intravenous injection or infusion. The disclosed vaccines may be formulated to be administered alone or concurrently with another therapeutic agent for treating FeLV infection. The vaccines may be formulated to be administered in sequence with another therapeutic agent. For example, the vaccine may be administered either before or after the subject has received a regimen of an antiviral therapy. The vaccines may be administered as a single dose or an initial dose followed by one or more booster doses.

[0066] A vaccine as disclosed herein, comprising one or more antigen(s) that comprise or consist of one or more of the peptide sequences in Tables 1 or 2 (i.e., each comprising or consisting of one of SEQ ID NOs: 1-26, including SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25), can be formulated or administered with an adjuvant to improve immune responses and promote protective responses. An adjuvant is an ingredient used in some vaccines that helps create a stronger immune response in people receiving the vaccine. Adjuvants help the body to produce an immune response strong enough to protect the person from the disease he or she is being vaccinated against. Those skilled in the art are aware of pharmaceutically acceptable adjuvants that may be combined with one or more of the disclosed antigens to prepare a vaccine.IV. FeLV Binding Proteins

[0067] The present disclosure also provides binding proteins, such as antibodies, including monoclonal antibodies that specifically bind to the disclosed antigen / epitope sequences (i.e., to one of SEQ ID NOs: 1-26, including SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25). In some embodiments, the binding proteins may selectively bind to an epitope that comprises or consists of a peptide having an amino acid sequence of any of SEQ ID NOs: 1-26. In some embodiments, the binding proteins may selectively bind to an epitope that comprises or consists of a peptide having an amino acid sequence of any of SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25. The disclosed binding proteins can be used for passive immunization or as therapeutics or diagnostics. The disclosed binding proteins can be used in methods for treating, preventing, or reducing the risk of infection by Feline Leukemia Virus or the development of FeLV infection in an individual in need thereof. For example, the disclosed binding proteins can be administered in a therapeutically effective amount to a subject in need thereof to reduce circulating levels of Feline Leukemia Virus, reduce viral load, and / or reduce, ameliorate, or eliminate one or more signs or symptoms of FeLV infection.

[0068] The disclosed binding proteins include antibodies and antibody fragments, monomers, dimers, single-domain antibodies, and other immunoglobulin fragments, variants, or derivatives. The binding proteins disclosed herein can be obtained by any means, including from in vitro sources (e.g., a hybridoma or a cell line producing the peptide recombinantly) and in vivo sources (e.g., rodents, rabbits, humans, etc.). In some embodiments, the binding proteins may be produced by a heterohybridoma, as discussed in more detail below. In some embodiments, the binding proteins may be monoclonal antibodies.

[8069] The binding proteins disclosed herein specifically bind to an epitope on the Feline Leukemia Virus gp70 or pl5E protein disclosed herein (i.e., to one of SEQ ID NOs: 1-16 (gp70) or SEQ ID NOs: 17-26 (pl 5E)). In some embodiments, one of SEQ ID NOs: 1-26 may represent the minimal epitope to which the disclosed binding proteins specifically bind, i.e., the minimal essential core epitope(s).

[0070] In general, the disclosed binding proteins comprise at least a least a portion of an immunoglobulin heavy chain. For instance, in some embodiments, the binding protein maycomprise a heavy chain monomer, a heavy chain dimer, or may be a single-domain antibody (i.e., a VHH fragment, a “nanobody,” or a “camelid-like” antibody). A single-domain antibody may comprise or consist of a VH domain, a Cm domain, and a Cm domain, but not a VK domain or a CHI domain.

[0071] The disclosed binding proteins can comprise, but do not require, an immunoglobulin light chain in order to bind a FeLV gp70 or pl5E protein epitope disclosed herein. In some embodiments, the disclosed binding proteins comprise both a heavy and light chain. In some embodiments, the disclosed binding proteins are full antibodies (e.g., complete IgGs). Human, partially humanized, fully humanized, and chimeric versions of the binding protein disclosed can be made by methods known in the art, such as using a transgenic animal (e.g., a mouse) wherein one or more endogenous immunoglobulin gene sequences are replaced with one or more human immunoglobulin gene sequences. Examples of transgenic mice wherein endogenous antibody genes are effectively replaced with human antibody genes include, but are not limited to, the HUMAB-MOUSE™ , the Kirin TC MOUSE™, and the KM-MOUSE™ (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181 : 69-97 (2008)).

[0072] The disclosed binding proteins may be an antibody. Typically, an antibody consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C- terminal constant (CHI, CH2 and CH3) regions, and each light chain contains one N- terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody, however, some of the disclosed peptides may comprise a heavy chain without a light chain. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs.” The extent of the framework region and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, and framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.[0073 The disclosed binding proteins may be an “antibody fragment,” which refers to one or more portions of an antibody that exhibits the ability to bind to an epitope on the Feline Leukemia Virus gp70 and / or pl5E protein defined by any one of SEQ ID NOs: 1-26 (i.e., to one of SEQ ID NOs: 1-16 (gp70) or SEQ ID NOs: 17-26 (pl5E)). Examples of binding fragments include (i) Fab fragments (monovalent fragments consisting of the VL, VH, CL and CHI domains); (ii) F(ab')2 fragments (bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region); (iii) Fd fragments (comprising the VH and CHI domains); (iv) Fv fragments (comprising the VL and VH domains of a single arm of an antibody), (v) dAb fragments (comprising a VH domain); and (vi) isolated complementarity determining regions (CDRs), e.g., VH CDR3. Other examples include single chain Fv (scFv) constructs. See e.g., Bird et al., Science, 242:423- 26 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-83 (1988). Other examples of types of antibody fragments include binding domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide (such as a heavy chain variable region, a light chain variable region, or a heavy chain variable region fused to a light chain variable region via a linker peptide) fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the Cm constant region, where the hinge region may be modified by replacing one or more cysteine residues with, for example, serine residues, to prevent dimerization.

[0074] As noted above, the disclosed binding proteins may or may not comprise a light chain. Similarly, he disclosed binding proteins may or may not comprise a CHI region. For instance, in some embodiments, a binding protein may comprise or consist of a VH domain, a CH2 domain, and a CH3 domain. In some embodiments, a binding protein may comprise or consist of a VH domain, a CHI domain, a CH2 domain, and a CH3 domain. In some embodiments, the constant domains may comprise one or more modifications, such as an amino acid substitution.

[0075] The disclosed binding proteins include monoclonal antibodies (mAbs) and fragments thereof, which may be obtained by methods known in the art, for example, by fusing antibodyproducing cells with immortalized cells to obtain a hybridoma, and / or by generating mAbs from mRNA extracted from bone marrow, B cells, and / or spleen cells of immunized animals using combinatorial antibody library technology and / or by isolating monoclonal antibodies from serum from subjects immunized with a peptide antigen, such as a peptide antigen comprising orconsisting of any one of SEQ ID NOs: 1-26, or a peptide antigen comprising or consisting of any of SEQ ID NOs: 2, 3, 9, 11, 12, or 18; or created from “immune B-cells” obtained from peripheral mononuclear cells of a cat that has convalesced after having FeLV.

[0076] Recombinant versions of the disclosed binding proteins may be obtained by methods known in the art, for example, using phage display technologies, yeast surface display technologies (Chao et al., Nat. Protoc., 1(2): 755-68 (2006)), mammalian cell surface display technologies (Beerli et al., PNAS, 105(38): 14336-41 (2008), and / or by expressing or co-expressing component polypeptides, such as heavy and light chain polypeptides. Other techniques for making peptides and antibodies are known in the art and can be used to obtain binding proteins as well.

[0077] The disclosed binding proteins may be or be derived from a human IgGl antibody, a human IgG2 antibody, a human IgG3 antibody, or a human IgG4 antibody. In some embodiments, the binding protein may be or be derived from a class of antibody selected from IgG, IgM, IgA, IgE, and IgD. That is, the disclosed binding proteins may comprise all or part of the constant regions, framework regions, or a combination thereof of an IgG, IgM, IgA, IgE, or IgD antibody. For instance, a disclosed binding protein comprising an IgGl immunoglobulin structure may be modified to replace (or “switch”) the IgGl structure with the corresponding structure of another IgG-class immunoglobulin or an IgM, IgA, IgE, or IgD immunoglobulin. This type of modification or switching may be performed in order to augment the neutralization functions of the peptide, such as antibody dependent cell cytotoxicity (ADCC) and complement fixation (CDC). A person of ordinary skill in the art will understand that, for example, a recombinant IgGl immunoglobulin structure can be “switched” to the corresponding regions of immunoglobulin structures from other immunoglobulin classes, such as recombinant secretory IgAl or recombinant secretory IgA2, such as may be useful for topical application onto mucosal surfaces. For example, immunoglobulin IgA structures are known to have applications in protective immune surveillance directed against invasion of infectious diseases, which makes such structures suitable for methods of using the disclosed binding proteins in such contexts, e.g., treating or preventing FeLV infection or the spread of FeLV from one individual to another.

[0078] In some embodiments, a disclosed binding protein may comprise one or more mutations, alterations, or modifications that improve one or more properties or functions of the bindingprotein. Such mutations, alterations, or modifications may comprise, for example, changes to the Fc region to increase the ability of the peptide to mediate cellular cytotoxicity functions like antibody dependent cell cytotoxicity (ADCC), antibody dependent cell mediated phagocytosis (ADCP), and / or complement fixation (CDC). A wide number of mutations to the Fc domain that enhance binding to Fc receptors have been reported, for example, S239D / A330L / I332E, F243L, and G236A. Additionally or alternatively, mutations to the Fc region that increase the circulating half-life of a disclosed FeLV-binding peptide may be incorporated into the structure. For example, mutations to engineer the pH-dependent interaction of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4, can increase half-life and improve efficacy under physiological conditions. Exemplary mutations that may be incorporated in order to enhance Clq receptor or Fc receptor binding are shown in the table below.Table 3 - Potential Fc Mutations{0079J In some embodiments, the disclosed binding proteins may be conjugated to polyethylene glycol (PEG) and / or albumin, which may increase the half-life and decrease the potential immunogenicity of the peptide.[0O8O The disclosed binding proteins may bind to a conserved FeLV epitope as disclosed herein (e g., one of SEQ ID NOs: 1-26) with a high affinity. For example, the disclosed binding proteins and antibodies can have a KD of at least 3.0x1 O'8, at least 2.5x1 O'8, at least 2.0xl0'8, at least 1.5x10'8, at least l .OxlO'8, at least 0.5xl0'8, at least 9.95xl0'9, at least 9.90xl0'9, at least 9.85xl0'9, at least 9.80xl0'9, at least 9.75xl0'9, at least 9.70xl0'9, at least 9.65xl0'9, at least 9.60xl0'9, at least 9.55xl0'9, at least 9.5xl0'9, at least 9.45x1 O'9, at least 9.40x1 O'9, at least 9.35xl0'9, at least 9.30x10'9, at least 9.25xl0'9, at least 9.20xl0'9, at least 9.15xl0'9, at least 9.10xl0'9, at least 9.05xl0'9, at least 9.0xl0'9, at least 8.95xl0'9, at least 8.90xl0'9, at least 8.85xl0'9, at least 8.80xl0'9, at least 8.75xl0'9, at least 8.70xl0'9, at least 8.65xl0'9, at least 8.60xl0'9, at least 8.55xl0'9, at least 8.5x10'9, at least 8.45xl0'9, at least 8.40xl0'9, at least 8.35xl0'9, at least 8.30xl0'9, at least 8.25xl0'9, at least 8.20xl0'9, at least 8.15xl0'9, at least 8.10xl0'9, at least 8.05xl0'9, at least 8.0xl0'9, at least 7.95xl0'9, at least 7.90xl0'9, at least 7.85xl0'9, at least 7.80xl0'9, at least 7 ,75xl0'9, at least 7.70xl0'9, at least 7.65xl0'9, at least 7.60xl0'9, at least 7.55xl0'9, at least 7.5xl0'9, at least 7.45x10'9, at least 7.40xl0'9, at least 7.35xl0'9, at least 7.30xl0'9, at least 7.25xl0'9, at least 7.20xl0'9, at least 7.15xl0'9, at least 7.10xl0'9, at least 7.05xl0'9, at least 7.0xl0'9, at least 6.95xl0'9, at least 6.90xl0'9, at least 6.85xl0'9, at least 6.80xl0'9, at least 6.75xl0'9, at least 6.70xl0'9, at least 6.65xl0'9, at least 6.60xl0'9, at least 6.55xl0'9, at least 6.5xl0'9, at least 6.45xl0'9, at least 6.40x10'9, at least 6.35xl0'9, at least 6.30xl0'9, at least 6.25xl0'9, at least 6.20xl0'9, at least 6.15xl0'9, at least 6.10xl0'9, at least 6.05xl0'9, at least 6.0xl0'9, at least 5.95xl0'9, at least 5.90xl0'9, at least 5.85xl0'9, at least 5.80xl0'9, at least 5.75xl0'9, at least 5.70xl0'9, at least 5.65xl0'9, at least 5.60xl0'9, at least 5.55x1 O'9, at least 5.5xl0'9, at least 5.45xl0'9, at least 5.40xl0'9, at least 5.35x10'9, at least 5.30xl0'9, at least 5.25xl0'9, at least 5.20xl0'9, at least 5.15xl0'9, at least 5.10xl0'9, at least 5.05xl0'9, at least 5.0xl0'9, at least 4.95xl0'9, at least 4.90xl0'9, at least 4.85xl0'9, at least 4.80xl0'9, at least 4.75xl0'9, at least 4.70xl0'9, at least 4.65xl0'9, at least 4.60xl0'9, at least 4.55xl0'9, at least 4.5xl0'9, at least 4.45x1 O'9, at least 4.40x1 O'9, at least 4.35xl0'9, at least 4.30x10'9, at least 4.25xl0'9, at least 4.20xl0'9, at least 4.15xl0'9, at least 4.10xl0'9, at least 4.05xl0'9, at least 4.0xl0'9, at least 3.95xl0'9, at least 3.90xl0'9, at least 3.85xl0'9, at least 3.80xl0'9, at least 3.75xl0'9, at least 3.70xl0'9, at least 3.65xl0'9, at least 3.60xl0'9, at least 3.55x1 O'9, at least 3.5x10'9, at least 3.45xl0'9, at least 3.40xl0'9, at least 3.35xl0'9, at least 3.30xl0'9, at least 3.25xl0'9, at least 3.20xl0'9, at least 3.15xl0'9, at least 3.10xl0'9, at least 3.05xl0'9, at least 3.0xl0'9, at least 2.95xl0'9, at least 2.90xl0'9, at least 2.85xl0'9, at least 2.80xl0'9, at least 2.75xl0'9, at least2.70x1 O'9, at least 2.65x1 O'9, at least 2.60x1 O'9, at least 2.55x1 O'9, at least 2.5x1 O'9, at least 2.45x1 O'9, at least 2.40xl0'9, at least 2.35xl0'9, at least 2.30xl0'9, at least 2.25xl0'9, at least 2.20xl0'9, at least 2.15xl0'9, at least 2.10xl0'9, at least 2.05xl0'9, at least 2.0xl0'9, at least 1.95xl0'9, at least 1.90xl0'9, at least 1.85xl0'9, at least 1.80xl0'9, at least 1.75xl0'9, at least 1.70xl0'9, at least 1.65xl0'9, at least 1.60xl0'9, at least 1.55xl0'9, at least 1.5xl0'9, at least 1.45xl0'9, at least 1.40x10"9, at least 1.35xl0'9, at least 1.30xl0'9, at least 1.25xl0'9, at least 1.20xl0'9, at least 1.15xl0'9, at least l . lOxlO'9, at least 1.05xl0'9, at least l .OxlO'9, at least 0.95xl0'9, at least 0.90xl0'9, at least 0.85xl0'9, at least 0.80xl0'9, at least 0.75xl0'9, at least 0.70xl0'9, at least 0.65xl0'9, at least 0.60xl0'9, at least 0.55xl0'9, at least 0.5xl0'9, at least 0.45x1 O'9, at least 0.40x1 O'9, at least 0.35x10'9, at least 0.30xl0'9, at least 0.25xl0'9, at least 0.20xl0'9, at least 0.15xl0'9, at least O. lOxlO'9, at least 0.05xl0'9, at least 9.5xlO10, at least 9.OxlO10, at least 8.5xlO10, at least 8.OxlO10, or any value in between.10081] Any of the binding proteins or antibodies disclosed herein can be used for treating and / or preventing FeLV infection. Optimal doses and routes of administration may vary, such as based on the route of administration and dosage form, the age and weight of the subject, and / or the subject’s condition, including the type and severity of the FeLV infection, and can be determined by the skilled practitioner. The disclosed binding proteins can be formulated in a pharmaceutical composition suitable for administration to a subject by any intended route of administration, as discussed in more detail below.V. Methods of Making Binding Proteins

[0082] While the disclosed binding proteins may be prepared using any known method of protein or antibody production, they also can be prepared using the methodologies disclosed herein. In particular, human neutralizing monoclonal antibodies or binding protein can be produced according to the following processes, rather than “humanizing” mouse or rat antibodies / peptides. In general, this process allows for the development of an effective, strong, and robust library of biologies (e.g., binding proteins) that have pharmaceutical applications with significant benefits to patients or animals in the global marketplace.

[0083] Using a parent hybridoma cell line, any one or more of four distinct and effective products can be produced: (1) a fully human neutralizing monoclonal antibody — directed against anypathogen (e.g., virus or bacteria) — through use in passive immunotherapy; (2) an effective humoral active vaccine that is safe and effective; (3) an oral mini-antibody peptide-based medication with an efficacy that is equivalent to the immunologic capacity of the monoclonal antibody produced by a parent hybridoma cell; and (4) an entry-fusion inhibitor that is immunologic in character and scope. The applications for these products are broad, effective and beneficial for therapeutic use. For example, monoclonal antibodies for therapeutic use may be made to treat FeLV infection.[0084J In some embodiments, the disclosed method of producing a binding protein or antibody against any one of the disclosed antigens or epitopes (i.e., any one of SEQ ID NOs: 1-26) may comprises the steps of: (a) identifying an asymptomatic patient after natural infection by a target infectious agent (e.g., Feline Leukemia Virus) as a donor for obtaining immune B-lymphocytes that produce high titers of plasma neutralizing antibodies directed against the target infectious agent; (b) collecting B-lymphocytes from the patient; (c) immortalizing the human B-lymphocytes to obtain immortalized cell lines; and (d) collecting antibodies produced by the immortalized cell lines. This process may optionally include the steps of (e) stabilizing and augmenting neutralizing antibody production by the immortalized cells lines; (f) screening supernatants from the immortalized cell lines for antibody production; and (g) testing the antibodies for binding against protein components of the infectious agent. The method may further comprise one or more of epitope mapping the antibodies that tested positive for binding to the infectious agent to screen for antibodies / binding proteins that specifically bind to any one of SEQ ID NOs: 1-26; purifying the antibodies by affinity chromatographic techniques; and in vitro testing of the antibodies to confirm neutralization reactivity against the target infectious agent at physiologic concentrations.VI. Pharmaceutical Compositions

[0085] Also provided herein are pharmaceutical compositions comprising one or more disclosed peptide antigen(s) (comprising or consisting of one or more of SEQ ID NOs: 1-26, optionally including one or more of SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25) (e.g., for a vaccine composition) or one or more disclosed binding protein(s) (each specifically binding to any of SEQ ID NOs: 1-26, optionally including one or more of SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25) (e.g., for a passive immunization or therapeutic composition) and a pharmaceutically acceptable carrier or diluent. A vaccine composition as disclosed herein may include one or a plurality of peptide antigens, each comprising or consisting of a different one of SEQ ID NOs: 1 -26, includingone, two, three, four, five, six, seven, eight, nine, ten, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 26 of SEQ ID NOs: 1-26, optionally including one or more of SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25. A passive immunization composition as disclosed herein may include one or a plurality of binding proteins, each binding to the same or different one of SEQ ID NOs: 1-26, including binding proteins binding to one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 26 of SEQ ID NOs: 1-26; or binding proteins binding to one or more or all of SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25. Additionally or alternatively, a passive immunization composition may comprise a plurality of binding proteins (i.e., antibodies) that each bind to the same one of SEQ ID NOs: 1-26. As noted above, in any embodiments, the binding proteins may be monoclonal antibodies.[0086 The disclosed pharmaceutical compositions may be formulated for any suitable route of administration, including intravenous, subcutaneous, intraperitoneal, intramuscular, or oral administration. In typical embodiments, the binding proteins are formulated for intravenous, subcutaneous, intraperitoneal, or intramuscular administration, such as in a solution, suspension, emulsion, liposome formulation, etc. More specifically, the disclosed binding proteins can be formulated for intravenous, subcutaneous, or intramuscular administration. The pharmaceutical composition can be formulated to be an immediate-release composition, sustained-release composition, delayed-release composition, etc., using techniques and excipients that are known in the art.[00871 Pharmaceutically acceptable carriers for various dosage forms and routes of administration are known in the art. For example, solvents, solubilizing agents, suspending agents, isotonicity agents, buffers, and soothing agents for liquid preparations are known. In some embodiments, the pharmaceutical compositions include one or more additional components, such as one or more preservatives, antioxidants, colorants, sweetening / flavoring agents, adsorbing agents, wetting agents and the like.

[0088] Pharmaceutical compositions of the disclosed antigens or binding proteins can be prepared as formulations according to standard methods (see, for example, Remington's Pharmaceutical Science, Mark Publishing Company, Easton, USA). The pharmaceutical compositions generally comprise a carrier and / or additive in addition to the antigen or binding protein (e.g., antibody). Forexample, the pharmaceutical composition may comprise one or more surfactants (for example, PEG and Tween), excipients, antioxidants (for example, ascorbic acid), preservatives, stabilizers, buffering agents (for example, phosphoric acid, citric acid, and other organic acids), chelating agents (for example, EDTA), suspending agents, isotonizing agents, binders, disintegrators, lubricants, fluidity promoters, corrigents, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmelose calcium, carmelose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetaldi ethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, and inorganic salt. The pharmaceutical composition may comprise one or more other low-molecular-weight polypeptides or proteins, such as serum albumin, gelatin, immunoglobulin, or amino acids such as glycine, glutamine, asparagine, arginine, and lysine.

[0089] When the antigens or binding proteins are prepared as an aqueous solution for injection, the antigen or binding protein may be formulated in an isotonic solution containing, for example, physiological saline, dextrose, or other excipients or tonifiers. The tonifier may include, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride. In addition, appropriate solubilizing agents, for example, alcohols (for example, ethanol), polyalcohols (for example, propylene glycols and PEGs), and non-ionic detergents (polysorbate 80 and HCO-50) may be used concomitantly.

[0090] The disclosed antigens and binding proteins may be formulated for administration by injection or infusion, such as an intravenous injection or infusion, an intramuscular injection, or a subcutaneous injection. Alternatively, the disclosed antigens or binding proteins may be formulated for oral administration.

[0091] Any of the pharmaceutical compositions disclosed herein can be used for inducing an immune response to or treating and / or preventing FeLV infection. Optimal doses and routes of administration may vary, such as based on the route of administration and dosage form, the age and weight of the subject, and / or the subject’s condition, including the type and severity of the infection, and can be determined by the skilled practitioner.VII. Treatment and Prevention of FeLV Infection

[0092] The present disclosure provides methods of inducing an immune response against Feline Leukemia Virus, by administering an antigen as disclosed herein, i.e., an antigen that comprises or consists of a peptide having one of the sequences in Tables 1 or 2 (i.e., selected from SEQ ID NOs: 1-26, optionally selected from SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25), in the form of a vaccine as discussed above. The present disclosure also provides uses of the disclosed antigens and pharmaceutical compositions (vaccines) for inducing an immune response to Feline Leukemia Virus. The immune response may be effective to reduce the risk of infection by Feline Leukemia Virus. The immune response may be effective to partially or fully protect against infection, such as by preventing infection or reducing the viral load if the subject does get infected.

[0093] The present disclosure also provides methods of treatment and prevention of Feline Leukemia Virus infections (e g., FeLV) by administering a binding protein that specifically binds to at least one of the epitopes disclosed herein, i.e., having one of the sequences in Tables 1 or 2 (i.e., selected from SEQ ID NOs: 1-26, optionally selected from SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25). The present disclosure also provides uses of the disclosed binding proteins and pharmaceutical compositions for treating or preventing Feline Leukemia Virus infections (e.g., FeLV), or in the preparation of a medicament for treating or preventing Feline Leukemia Virus infections (e.g., FeLV). As noted above, in any embodiments, the binding proteins may be monoclonal antibodies.

[0094] The disclosed methods comprise administering to a subject an effective amount of one or more of the antigens or binding proteins or pharmaceutical compositions disclosed herein. Administration may be performed via intravenous, intra-arterial, intramuscular, subcutaneous, or intradermal injection. In some embodiments, the subject may be at risk of exposure to Feline Leukemia Virus. In some embodiments, the administration of the antigen prevents the subject from developing a FeLV infection. In some embodiments, the administration of the antigen reduces the risk the subject will develop a severe FeLV infection, such as reducing the risk of infection requiring hospitalization. In some embodiments, the subject may have previously been exposed to Feline Leukemia Virus. In some embodiments, particularly embodiments using binding proteins, the subject may have an active infection (e.g., a FeLV infection) which may be treated as a result of the administration. In some embodiments, the administration of the binding protein prevents thesubject from developing a a FeLV infection. In some embodiments, the effective amount of a binding protein is sufficient to reduce circulating viral load and / or to reduce, ameliorate, or eliminate one or more symptoms or effects of a FeLV infection. In some embodiments, the effective amount of a binding protein is effective to prevent binding of a Feline Leukemia Virus Gp70 or p!5E protein to an ACE2 receptor. The specific amount of antigen or binding protein administered may depend on one or more of the age and / or weight of the subject and / or the stage or severity of the disease and / or the dosage form and route of administration, and can be determined by the skilled practitioner.

[0095] For the purposes of the disclosed methods and uses, treatment and / or prevention of all strains and variants of FeLV are specifically contemplated.

[0096] Dosage regimens can be adjusted to provide the optimum desired response. For example, in some embodiments, a single bolus of an antigen or binding protein may be administered, while in some embodiments, several doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the situation. In some embodiments, a subject may be administered more than one distinct antigen or binding protein, such as two or three or more distinct antigens or antibodies that each bind to different epitopes disclosed herein.VIH. Detection of FeLV Infection

[0997] As noted above the peptide antigens and binding proteins thereto described herein also are useful for detecting Feline Leukemia Virus infection.

[0098] Tests for Feline Leukemia Virus infection can detect a component of the virus in a sample taken from the subject, either by detecting a viral component in the sample or by detecting antibodies against the virus in the sample. For detecting viral components, the test format can be a molecular test that detects viral RNA or an antigen test that detects viral protein. Molecular tests also are called Nucleic Acid Amplification Tests (NAATs), and involve amplifying nucleic acids present in the sample until they are detectable. The polymerase chain reaction (PCR) is viewed as the “gold standard” for diagnostic tests, but can show false-negative results at early stages of infection. RT-PCR is expensive, requires expert handling, and takes about four hours to complete the assay. Antigen tests are less expensive, and offer nearly instant results without requiring skilledpersonnel, but may be less sensitive, so the rate of false negatives is high, and false positives also may occur.

[0099] The evolution of Feline Leukemia Virus variants having mutations in their genomic and protein sequences poses another problem in the development of effective methods for detecting infection. Mutations can impact test performance if the mutation impairs or prevents the test reagent from being able to detect the virus. The impact of mutations on a test's performance may be influenced by several factors, including the sequence of the variant (including the number, identity and location of mutations), the design of the test, and the prevalence of the variant in the population. For example, tests with single targets are more likely to fail to detect new variants. On the other hand, tests with multiple targets (e.g., a PCR test designed to detect more than one section of the Feline Leukemia Virus genome or an antigen test designed to detect more than one region of the gp70 or pl5E protein) are more likely to be able to detect new variants.

[0100] The peptide antigens and binding proteins described herein offer significant advantages in this context due to the highly conserved nature of the corresponding epitopes. As discussed above, the epitopes of SEQ ID NOs: 1-26 have been confirmed to be conserved across variants. This indicates that detection tests targeting these epitopes (or antibodies that bind these epitopes) will be able to detect infection by all currently known variants, as well as future variants. Testing can involve detecting one or more of SEQ ID NOs: 1 -26 using a binding protein or, alternatively, can involve detecting antibodies that bind to one or more SEQ ID NOs: 1-26, as described herein.

[0101] Thus, provided herein are an “antigen test” and kit for detecting Feline Leukemia Virus infection, comprising detecting any one or more or all of SEQ ID NOs: 1-26 in a sample obtained from a subject, including detecting any one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 26 of SEQ ID NOs: 1-26 in a sample. For the purposes of such antigen tests, an ELISA (i.e., enzyme-linked immunoassay) is a particularly useful format, but other known methods in the art may be used, such as Western blotting, dot blotting, immunohistochemistry, immunofluorescence, immunoprecipitation, immunoelectrophoresis, or mass-spectrometry, and any other assay format that can detect the presence of any one or more or all of SEQ ID NOs: 1-26 in a sample.(0192) For example, an ELISA-based method of detecting Feline Leukemia Virus infection may comprise contacting a sample obtained from a subject with one or more probe binding proteins or antibodies that each specifically bind at least one of SEQ ID NOs: 1-26, and detecting binding between the probe binding proteins / antibodies and any Feline Leukemia Virus antigen present in the sample. In some embodiments, such a method may comprise using a single monoclonal antibody that is specific for any one of SEQ ID NOs: 1-26 or a panel of binding proteins / antibodies that each specifically binds one of SEQ ID NOs: 1-26, typically where each specifically binds a different one of SEQ ID NOs: 1-26, such as a panel of 2-26 binding proteins / antibodies where each specifically binds a different one of SEQ ID NOs: 1-26, although binding proteins / antibodies that specifically bind the same one of SEQ ID NOs: 1-26 can be included to potentially increase sensitivity. In such embodiments, the probe antibodies or binding proteins that bind to the Feline Leukemia Virus antigens may be bound to a solid substrate (e.g., a plate, well, slide, bead, strip, etc.) and a biological sample obtained from a subject (such as an individual suspected of having or having been exposed to Feline Leukemia Virus) may be applied to the substrate. If a target antigen (i.e., antigens comprising or consisting of SEQ ID NOs: 1-26) is present in the sample and a probe antibody / binding protein that is specific for the target antigen is bound to the substrate, then the target antigen will be bound. The biological sample can then be removed and the substrate may be washed to remove any unbound protein or debris. Next, a detection antibody that also binds to the target antigen may be contacted to the substrate. The detection antibody typically is detectably labeled, such that it can be detected as evidence of the presence of the target antigen in the sample. Detectable labels that can be used for this purpose are known in the art and can include, but are not limited to, a fluorophore (e.g., FTIC, rhodamine, GFP, lanthanide, etc.), a chromogen, a chemiluminescent agent, an enzymatic label (e.g., luciferase, horseradish peroxidase, alkaline phosphatase), an acridinium moiety, a radiolabel, a colorometric label, a magnetic agent, or a metal (e.g., a gold particle).

[9193] Also provided herein is an antibody test for detecting Feline Leukemia Virus infection, comprising detecting antibodies to any one or more or all of SEQ ID NOs: 1-26 in a sample obtained from a subject, including antibodies to any one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 26 of SEQ ID NOs: 1-26 in a sample. Antibodies to each of SEQ ID NOs: 1-26 can be detected using a respective peptide antigen as described herein as a probe. Thus, a method of detecting Feline Leukemia Virus infection maycomprise contacting a sample obtained from a subject with one or more peptide antigens, each comprising or consisting of one of SEQ ID NOs: 1-26. In some embodiments, such a method comprises using a panel of peptide antigens, each comprising or consisting of one of SEQ ID NOs: 1-26, typically where each comprises or consists of a different one of SEQ ID NOs: 1-26, such as a panel of 2-26 peptide antigens, typically where each comprises or consists of a different one of SEQ ID NOs: 1-26, although peptide antigens the comprise or consist of the same one of SEQ ID NOs: 1-26 can be included to potentially increase sensitivity. For the purposes of such embodiments, the probe antigen(s) may be bound to a substrate (e.g., a plate, well, slide, bead, strip, etc.) either directly or indirectly (e.g., via a polymer, such as polyethylene glycol (PEG), a peptide linker, or a protein, such as an antibody). The substrate may be contacted with a biological sample obtained from a subject (such as an individual suspected of having or having been exposed to Feline Leukemia Virus). If there are antibodies present in the sample that bind to any one of the probe antigens, then the presence of such antibodies can be detected, such as by removing the sample and washing the substrate to remove any unbound protein or debris, and then contacting the substrate with a detection antibody that binds to antibodies from the sample (e.g., human IgE or IgD). The detection antibody typically is detectably labeled, as discussed above.

[0104] The present disclosure also provides methods for detection of Feline Leukemia Virus, by detecting the presence of nucleic acid sequence(s) that encode any one or more or all of SEQ ID NOs: 1-26. Such methods include, but are not limited to, RT-qPCR, RT-PCR, RNA-seq, Northern blotting, Serial Analysis of Gene Expression (SAGE), or DNA or RNA microarrays. The starting material for detection of polynucleotides encoding the disclosed biomarkers may be genomic DNA, cDNA, RNA or mRNA. In such embodiments, nucleic acid primers and, optionally, nucleic acid probes, may be designed to specifically amplify and detect the nucleic acid sequences that encode any one or more or all of SEQ ID NOs: 1-26. Primers and probes may comprise a detectable label or a plurality of detectable labels. The detectable label associated with the primer or probe can generate a detectable signal directly. Additionally, the detectable label associated with the primer or probe can be detected indirectly using a reagent, wherein the reagent includes a detectable label, and binds to the label associated with the probe.

[0105] Detectably labeled nucleic acid primers and probes can be used to monitor the amplification of a target nucleic acid sequence (e.g., nucleic acid sequences that encode any oneor more or all of SEQ ID NOs: 1-26). In some embodiments, detectably labeled primers or probes present in an amplification reaction are suitable for monitoring the amount of amplicon(s) produced as a function of time. Examples of such probes include, but are not limited to, the 5'- exonuclease assay (TAQMAN® probes described herein (see also U.S. Pat. No. 5,538,848) various stem-loop molecular beacons (see for example, U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, 1996, Nature Biotechnology 14:303 - 308), stemless or linear beacons (see, e.g., WO 99 / 21881), PNA Molecular Beacons™ (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, for example, Kubista et al., 2001, SPIE 4264:53-58), non- FRET probes (see, for example, U.S. Pat. No. 6,150,097), Sunrise® / Amplifluor™ probes (U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion probes (Solinas et al., 2001, Nucleic Acids Research 29:E96 and U.S. Pat. No. 6,589,743), bulge loop probes (U.S. Pat. No. 6,590,091), pseudo knot probes (U.S. Pat. No. 6,589,250), cyclicons (U.S. Pat. No. 6,383,752), MGB Eclipse™ probe (Epoch Biosciences), hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA) light-up probes, self-assembled nanoparticle probes, and ferrocene-modified probes described, for example, in U.S. Pat. No. 6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et al., 1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000, Molecular Cell Probes. 14:321-328; Svanvik et al., 2000, Anal Biochem. 281 :26-35; Wolffs et al., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002, Nucleic Acids Research. 30:4208-4215; Riccelli et al., 2002, Nucleic Acids Research 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329- 332; Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et al., 2002, Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem. Res. Toxicol. 15:118-126; and Yu et al., 2001, J. Am. Chem. Soc 14: 11155-11161. In some embodiments, the detectable label is a fluorophore. Suitable fluorescent moieties include but are not limited to the following fluorophores working individually or in combination: 4-acetamido-4'-isothiocyanatostilbene- 2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes); 5-(2- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4- anilino-1- naphthyl)maleimide; anthranilamide; Black Hole Quencher™ (BHQ™) dyes (biosearch Technologies); BODIPY dyes: BODIPY® R-6G, BOPIPY® 530 / 550, BODIPY® FL; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5 "-dibrom opyrogallol- sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4- methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'- disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5- [dimethylamino]naphthalene-l -sulfonyl chloride (DNS, dansyl chloride); 4-(4'- dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'- isothiocyanate (DABITC); Eclipse™ (Epoch Biosciences Inc.); eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)amino fluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), hexachloro-6-carboxyfluorescein (HEX), QFITC (XRITC), tetrachlorofluorescem (TET); fiuorescamine; IR144; IR1446; lanthamide phosphors; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B -phycoerythrin, R-phycoerythrin; allophycocyanin; o-phthaldialdehyde; Oregon Green®; propidium iodide; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 - pyrene butyrate; QSY® 7; QSY® 9; QSY® 21; QSY® 35 (Molecular Probes); Reactive Red 4 (Cibacron®Brilliant Red 3B-A); rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6- carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, riboflavin, rosolic acid, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); terbium chelate derivatives; N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); and VIC®. Detector probes can also comprise sulfonate derivatives of fluorescenin dyes with S03 instead of the carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of CY 5 (commercially available for example from Amersham).

[0106] Nucleic acid primers or probes may be designed to selectively hybridize to any portion of a nucleic acid sequence encoding any one or more or all of SEQ ID NOs: 1-26. Methods for preparing nucleic acid primers or probes are well known in the art.(0197) Accordingly, the present disclosure provides in vitro methods of analyzing a biological sample obtained from a subject, comprising contacting the sample with a binding protein that specifically binds to a Feline Leukemia Virus peptide antigen selected from SEQ ID NOs: 1-26, and detecting binding between the binding protein and any Feline Leukemia Virus antigen present in the sample. The methods may comprise contacting the sample with one binding protein or a panel of from 2 to 26 binding proteins that each specifically binds to a different Feline Leukemia Virus peptide antigen selected from SEQ ID NOs: 1-26, and detecting binding between the binding proteins and any Feline Leukemia Virus antigen present in the sample. The methods also may comprise contacting the sample with a plurality of binding proteins that each binds to the same Feline Leukemia Virus peptide antigen selected from SEQ ID NOs: 1-26.[0198} The present disclosure also provides in vitro methods of analyzing a biological sample obtained from a subject, comprising contacting the sample with a Feline Leukemia Virus peptide antigen comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1-26, and detecting binding between the peptide antigen and any anti-Feline Leukemia Virus antibodies present in the sample. The methods may comprise contacting the sample with one peptide antigen or a panel of from 2 to 26 peptide antigens each comprising or consisting of a different an amino acid sequence selected from SEQ ID NOs: 1-26, and detecting binding between the peptide antigen and any anti-Feline Leukemia Virus antibodies present in the sample. The methods also may comprise contacting the sample with a plurality of peptide antigens that comprise or consist of the same one of SEQ ID NOs: 1-26,[0199} The present disclosure also provides in vitro methods of analyzing a biological sample obtained from a subject, comprising extracting nucleic acids from the biological sample, contacting the extracted nucleic acids with a pair of primers that specifically amplify a nucleic acid sequence encoding any one of SEQ ID NOs: 1-26, and detecting the presence of the amplified nucleic acid sequence if present in the sample. The methods may comprise contacting the sample with one primer pair or a panel of from 2 to 26 primer pairs each specific for a nucleic acid sequence that encodes a different amino acid sequence selected from SEQ ID NOs: 1-26, and detecting amplification of each nucleic acid sequence if present in the sample. The methods also may comprise contacting the sample with a plurality of primer pairs specific for a nucleic acid sequence that encodes the same amino acid sequence selected from SEQ ID NOs: 1-26.[£ 110 As discussed above, the epitopes of SEQ ID NOs: 1-26 to be targeted for detection can be selected depending on the aim of the analysis.

[0111] In some embodiments of the disclosed methods, the sample may be selected from saliva, nasal fluid, nasal cells, throat cells, blood, plasma, serum, urine, and feces. However, the sample is not necessarily limited to these sample types. In some instances, a blood sample, a plasma sample, a serum sample, or a tissue sample may be appropriate. In some instances, the sample may comprise urine or feces, which may be useful for epidemiological studies and public health tracking that relies on wastewater. Thus, in some embodiments the sample comprises biological samples obtained from a plurality of subjects.

[0112] In some embodiments of the disclosed methods, the subject is suspected of having a Feline Leukemia Virus infection, has been exposed to Feline Leukemia Virus, or is suspected of having been exposed to Feline Leukemia Virus. In some embodiments of the disclosed methods, the method may further comprise determining that the subject is infected with Feline Leukemia Virus when binding or amplification is detected.[011.3] As noted above, in some embodiments, the sample comprises biological samples obtained from a plurality of subjects. Some such embodiments further comprise determining a level of infection in the plurality of subjects.

[0114] The present disclosure additionally provides kits for implementing any of the foregoing methods of detection.

[0115] For example, the present disclosure provides kits comprising at least one binding protein that specifically binds to a peptide comprising or consisting of any one of SEQ ID NOs: 1-26, a solid substrate to which the at least one binding protein is attached, and a second detectably labeled antibody that specifically binds to the peptide to which the at least one binding protein specifically binds. Similarly, the present disclosure provides kits comprising at least one peptide comprising or consisting of any one of SEQ ID NOs: 1-26, a solid substrate to which the at least one peptide is attached, and a detectably labeled antibody that specifically binds to IgE or IgD, wherein the IgE or IgD are optionally human. The solid substrate can be selected from a bead, a plate, a well, a dish, a slide, or a strip.[£ 116 The present disclosure also provides kits comprising at least one primer pair capable of specifically amplifying a nucleic acid sequence that encodes a peptide selected from any one of SEQ ID NOs: 1-26, wherein: (a) at least one primer of the primer pair is detectably labeled; or (b) the kit further comprises a detectably labeled probe that hybridizes to the nucleic acid sequence amplified by the primer pair. In some embodiments, the kit may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 26 primer pairs, each primer pair being capable of specifically amplifying a different peptide selected from SEQ ID NOs: 1-26, wherein: (a) at least one primer of each primer pair is detectably labeled; or (b) the kit further comprises a detectably labeled probe that hybridizes to each nucleic acid sequence amplified by primer pairs included therein.[0117} The following examples are given to illustrate the present disclosure. It should be understood that the invention is not to be limited to the specific conditions or details described in these examples.ExamplesExample 1 - Identification of Highly Conserved Antigens and Epitopes[0118} This example describes the analytical methods used to identify the disclosed putative epitopes and antigens of SEQ ID NOs: 1-26. The identification process used computational prediction and machine learning (ML) approaches to identify optimal targets and assess antigenicity.

[0119] Data Collection

[0012] A protein antigen dataset was created to train an artificial intelligence model to predict and map other linear, sequential, conserved and neutralizable B-cell epitopes within the Feline Leukemia Virus Gp70 or p!5E protein. FELV Gp70 or pl5E protein sequences were collected and multiple sequence alignments were performed to identify conserved sequences. Additional epitope databases (e.g., IEDB and AntiJen, BciPep, Epitome, SDAP, FLAVIdB, and Influenza Sequence and Epitope Database) were used to obtain independent and relevant data points to ensure unbiased training.

[0121] Curating Dataset[0122) All identical epitopes (sequence and structure based) and non-immunogenic peptides were removed, so as to achieve unique experimentally proved epitopes. Generally, majority of B-cells epitope have length less than or equal to 20 amino acid, hence in this analysis all the epitopes having length more than 20 residues were removed. For training of machine learning technique, it was necessary to have fixed length patters whereas B-cell epitopes have varying length, hence if the epitope length were less than 20 amino acids, then the length was increased by introducing equal number of residues at both terminals derived from its original antigenic sequence. To generate a negative dataset, non-epitopes were created using random peptides of length 20 residues from the proteins in Swiss-Prot. All the random peptides that are identical to B-cells epitopes were excluded. Next, the unique epitope data was scored based on biophysical and biochemical metrics such as computed volume, polarity, hydrophobicity, linearity, activity and immunogenicity. These metrics served as dimensions across which regression, classification methods were performed. After a cleaned and labelled dataset was generated a portion of the dataset was used for training purposes. In order to achieve five-fold cross-validation approach. Multilayer perceptron method was implemented. The dataset was randomly divided into five subsets each containing an equal number of epitopes data. The three of the subsets were used for training purpose and from remaining two subsets each subset was used for validation and testing. This process was repeated five times so that each set was used once for testing. The final prediction results gave the average of five testing sets.(0123) ANN Model Training

[0124] To predict the probability that a given antigen residue is part of an epitope, artificial neural network (Jordon Network) was implemented using Keras and TensorFlow. The networks were trained using back-propagation algorithm and with various window lengths from 10 to 20 residues. The target output consists of a single binary number with one or zero (B-cell epitopes or nonepitopes). At the beginning of each simulation, the weights were initialized with random values and the training was carried out by using error back- propagation, with a sum of square error function. In each cycle of the training, the magnitude of the error sum in the test and training set were monitored and the ultimate number of cycles were determined when the network converges.Also a cut off value for each network was set up, which was used to compare the output produced by the network. Thus, when the output value was greater than the threshold value, then that peptide was predicted as B-cell epitope, otherwise as a non-epitope. Additionally, each amino acid composition, along with other parameters such as computed volume, polarity, hydrophobicity, linearity, neutralizing activity and immunogenicity were used for prediction purpose. We accurately predict the likelihood of each residue being an epitope candidate. To ensure the capability of the model in determining the correct epitope for a given antibody, these models with two hidden layers were implemented. Parameters of prediction include amino acid composition, exposed donors / receptors, hydrophobicity, aromatic / positive / negative residues, size, antigen patch density and structural conjoint triads to represent the specified protein sequences by considering not only the composition of amino acids but also the neighbor relationships in that sequence. Once the system was trained on the curated database, the blind dataset evaluation was performed using 1) clinically validated immunogenic proteins, 2) IgE epitopes of allergenic proteins (SDAP), and none of these datasets were used in the training or testing.[0125} Statistical Evaluation of Linear Epitope Dataset

[0126] FASTA sequences of the FeLV virus peptides were inputted into the system and epitope predictions with percentage of conservedness, neutralizing activity and immunogenicity were established. Each model was tuned by comparing prediction accuracy from the test set to predict the accuracy on the validation set. Models that perform the best were scored on the test set and evaluated using metrics such as precision, recall, true positive rate, false positive rate, and ROC- area under the curve using sci-kitleam, Keras, and TensorFlow. Once potential candidates were identified, the performance for each antigen in terms of the area under the receiver operation curve, the positive predictive rate and the true positive rate of the top predictions was evaluated for immune recognition.

[0127] Thus, SEQ ID NOs: 1-26 (Tables 1 and 2) were identified as highly conserved epitopes having the conservation across isolates.Example 2 -Confirmation of Disclosed Antigens and Epitopes[0128) Utilizing artificial intelligence, over 400 isolates of Feline Leukemia Virus were analyzed, and the 26 conserved immutable sites on Feline Leukemia Virus (of SEQ ID NOs: 1-26) were confirmed. The conserved sites remained conserved regardless of the mutations, thus making these region ideal for vaccine development, antibody targeting, and diagnostic development.This extensive artificial intelligence analysis has been done in three phases. Phase I analyzed Feline Leukemia Virus isolates and identified the 26 conserved sites of SEQ ID NOs: 1- 26. The conserved sites were confirmed to be conserved in other variants. In Phase-2, the 26 identified conserved sites of SEQ ID NOs: 1-26 were screened against further isolates to confirm their presence these variants. Phase 3 analyzed more Feline Leukemia Virus isolates and confirmed the 26 conserved sites of SEQ ID NOs: 1-26.

Claims

What is claimed is:

1. An isolated peptide antigen consisting of an amino acid sequence of any one of SEQ ID NOs.: 2, 3, 4, 11, 13, 16, 22, 25, 1, 5-10, 12, 14, 15, 17-21, 23, 24, or 26.

2. The isolated peptide antigen of claim 1, wherein the peptide is a recombinant peptide.

3. The isolated peptide antigen of claim 1 or claim 2, wherein the amino acid sequence is selected from any one of SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25.

4. A vaccine composition comprising a peptide antigen of any one of claims 1-3 and a pharmaceutically acceptable carrier.

5. An isolated binding protein that binds to a conserved epitope of a Feline Leukemia Virus (FeLV) wherein the conserved epitope consists of an amino acid sequence of any one of SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, 25, 1, 5-10, 12, 14, 15, 17-21, 23, 24, or 26.

6. The isolated binding protein of claim 5, wherein the amino acid sequence is selected from any one of SEQ ID NOs: 2, 3, 4, 11, 13, 16, 22, or 25.

7. The isolated binding protein of claim 5, wherein the conserved epitope is a FeLV GP70 epitope that consists of an amino acid sequence of any one of SEQ ID NOs: 1-16.

8. The isolated binding protein of claim 5, wherein the conserved epitope is a FeLV pl5E epitope that consists of an amino acid sequence of any one of SEQ ID NOs: 17-26.

9. The isolated binding protein of any one of claims 5-8, wherein the binding protein is an antibody or an antibody fragment, optionally wherein the binding protein is a monoclonal antibody.

10. A method of reducing the risk of Feline Leukemia Virus infection in a subject, comprising administering to the subject an effective amount of an isolated peptide antigen according to any one of claims 1-3 or a vaccine according to claim 4.

11. A method of treating, preventing, or reducing the risk of Feline Leukemia Virus infection in a subject, comprising administering to the subject a therapeutically effective amount of an isolated binding protein according to any one of claims 5-9.

12. The method of claim 10 or 11 wherein the isolated peptide antigen or isolated binding protein is administered by subcutaneous or intramuscular injection.

13. The method of any one of claims 10-12, wherein the subject is a feline.

14. An isolated peptide antigen according to any one of claims 1-3 or a vaccine according to claim 4, for inducing an immune response to Feline Leukemia Virus.

15. An isolated binding protein according to any one of claims 5-9, for treating, preventing, or reducing the risk of Feline Leukemia Virus infection in a subject in need thereof.

16. Use of an isolated peptide antigen according to any one of claims 1-3 in the preparation of a vaccine for inducing an immune response to Feline Leukemia Virus.

17. Use of an isolated binding protein according to any one of claims 5-9 in the preparation of a medicament for treating, preventing, or reducing the risk of Feline Leukemia Virus in a subject in need thereof.

18. A method of preparing an antibody that binds to a peptide antigen of any one of claims 1- 3, comprising:(a) identifying an asymptomatic patient that has been infected with Feline Leukemia Virus as a donor for obtaining immune B-lymphocytes that produce high titers of Feline Leukemia Virus-neutralizing antibodies;(b) collecting the B-lymphocytes from the patient;(c) immortalizing the B-lymphocytes;(d) collecting antibodies produced by the immortalized B-lymphocytes; and(e) screening the antibodies for binding to the peptide antigen of any one of claims 1-2.

19. The method of claim 18, further comprising testing the antibodies for binding to FelineLeukemia Virus.

20. The method of claim 19, further comprising epitope mapping the antibodies that tested positive for binding to Feline Leukemia Virus.

21. The method of any one of claims 18-20, wherein immortalizing the B-lymphocytes comprises fusing a B-lymphocyte with a heteromyeloma cell in order to produce a heterohybridoma cell.

22. An in vitro method of analyzing a biological sample obtained from a subject, comprising contacting the sample with a binding protein that specifically binds to a Feline Leukemia Virus peptide antigen selected from SEQ ID NOs: 1-26, and detecting binding between the binding protein and any Feline Leukemia Virus antigen present in the sample.

23. The method of claim 22, comprising contacting the sample with a panel of from 2 to 26 binding proteins that each specifically binds to a different Feline Leukemia Virus peptide antigen selected from SEQ ID NOs: 1-26, and detecting binding between the binding proteins and any Feline Leukemia Virus antigen present in the sample.

24. An in vitro method of analyzing a biological sample obtained from a subject, comprising contacting the sample with a Feline Leukemia Virus peptide antigen consisting of an amino acid sequence selected from SEQ ID NOs: 1-26, and detecting binding between the peptide antigen and any anti -Feline Leukemia Virus antibodies present in the sample.

25. The method of claim 24, comprising contacting the sample with a panel of from 2 to 26 peptide antigens each consisting of a different an amino acid sequence selected from SEQ ID NOs: 1-26, and detecting binding between the peptide antigen and any anti-Feline Leukemia Virus antibodies present in the sample.

26. An in vitro method of analyzing a biological sample obtained from a subject, comprising extracting nucleic acids from the biological sample, contacting the extracted nucleic acids with a pair of primers that specifically amplify a nucleic acid sequence encoding a peptide of any one of SEQ ID NOs: 1-26, and detecting the presence of the amplified nucleic acid sequence if present in the sample.

27. The method of claim 26, comprising contacting the sample with a panel of from 2 to 26 primer pairs that each specifically amplify a nucleic acid sequence that encodes a different amino acid sequence selected from SEQ ID NOs: 1-26, and detecting amplification of each nucleic acid sequence if present in the sample.

28. The method of any one of claims 26-27, wherein the sample is selected from saliva, nasal fluid, nasal cells, throat cells, blood, plasma, serum, urine, and feces.

29. The method of any one of claims 26-28, where the subject is a feline.

30. The method of any one of claims 26-29, where the subject is suspected of having a Feline Leukemia Virus infection, has been exposed to Feline Leukemia Virus, or is suspected of having been exposed to Feline Leukemia Virus.

31. The method of any one of claims 26-30, further comprising determining that the subject is infected with Feline Leukemia Virus when binding or amplification is detected.

32. A kit comprising at least one binding protein that specifically binds to a peptide consisting of any one of SEQ ID NOs: 1-26, a solid substrate to which the at least one binding protein is attached, and a detectably labeled antibody that specifically binds to the peptide to which the at least one binding protein specifically binds.

33. A kit comprising at least one peptide consisting of any one of SEQ ID NOs: 1-26, a solid substrate to which the at least one peptide is attached, and a detectably labeled antibody that specifically binds to IgE or IgD, wherein the IgE or IgD are optionally feline; optionally, wherein the at least one peptide consists of any one of SEQ ID NOs: 1-26.

34. A kit comprising at least one primer pair capable of specifically amplifying a nucleic acid sequence that encodes a peptide selected from any one of SEQ ID NOs: 1-26, wherein:(a) at least one primer of the primer pair is detectably labeled; or(b) the kit further comprises a detectably labeled probe that hybridizes to the nucleic acid sequence amplified by the primer pair.

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