Cow milk allergen epitopes, megapools and uses thereof

Abstract

The present invention includes compositions, including epitope megapools, and methods for detecting the presence of cow milk allergen(s), specifically T cell epitopes for T cells responsive to one or more cow milk peptides or proteins comprising, consisting of, or consisting essentially of: one or more peptides or proteins selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). The invention further provides vaccines, diagnostics, therapies, and kits, comprising such protein(s) or peptide(s).

Description

COW MILK ALLERGEN EPITOPES, MEGAPOOLS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 610,662, filed December 15, 2023, the entire contents of which is incorporated herein by reference.TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates in general to the field of proteins and peptides that are T cell epitopes and / or antigens for cow milk allergens, including epitopes and antigens from cow milk, and more particularly, to compositions and methods for the prevention, treatment, diagnosis, kits, and uses of such T cell epitopes and antigens, including megapools, for use in detecting and characterizing cow milk specific allergic responses.STATEMENT OF GOVERNMENT SUPPORT

[0003] The inventions described in the present disclosure were made with government support under Contract No. U19AI135731, awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

[0004] The 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 December 11, 2024, is named “LJII2032WO.xml” and is 1,344,582 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTION

[0005] Without limiting the scope of the invention, its background is described in connection with cow milk allergens.

[0006] Food allergies affect 6-8% of children in the United States1, 2and prevalence is increasing3. Of these food allergies, cow milk allergy (CMA) is the most common in infants and young children affecting 2-3%4. Avoidance is currently the best treatment for this food allergy, but due to the widespread use of milk and milk products, accidental ingestion is common. This can be dangerous as evidenced by 13% of fatal allergy-induced anaphylaxis being due to milk allergy5. Current methods of diagnosing CMA based on IgE titers or skin prick tests do not reliably detect all forms of the disease, and the gold standard of using double -blinded food challenges is resource-intensive and not a pleasant experience for the patient.

[0007] Allergen-specific CD4 T cells are known to play key roles in both IgE and non-IgE-mediated food allergies6, 7. but significant challenges to studying them exist. The first is rooted in the ability to identify them accurately and distinguish them from bystanders. A critical step for this is the identification of T cell antigen and epitope targets in cow milk (CM). While several studies have characterized T cell epitopes for milk allergens8 11, none of these has comprehensively considered all proteins found in milk, leaving potentially novel antigens yet to be identified. The second challenge is the ability to isolate and characterizethese rare cell populations. Advancements in activation marker assays coupled with single-cell technologies have led to recent discoveries in the heterogeneity of antigen-specific T cell subsets in allergy and other diseases12 14. Specific to food allergy, subsets of disease-associated Th2 cells have been recently identified, which are associated with the production of cytokines that lead to downstream allergic symptoms ’ ’ .

[0008] Further, CMA, unlike the more widely studied peanut allergy, is typically outgrown with age. The heterogeneity of disease manifestations and progressions of CMA coupled with individual variability necessitates more targeted approaches to diagnosing and treating this disease. Additionally, both allergic and non-allergic individuals have T cells specific to milk proteins, and the ability to use them as diagnostics relies on finding phenotypic differences between them that are associated with the disease. Identifying the phenotypes and functions of milk-specific T cells in CMA is critical for understanding disease pathology and identifying new diagnostic and treatment methods14.

[0009] Thus, a need remains for identifying antigens and T cell epitopes for use in diagnostics, treatments, kits, etc., for cow milk-related allergic reactions. There is additionally a specific need in the art for optimized megapools for use in detecting and characterizing cow milk-related allergic reactions.SUMMARY OF THE INVENTION

[0010] As embodied and broadly described herein, an aspect of the present disclosure relates to a composition comprising: one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more or more peptides comprising, consisting of, or consisting essentially of amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof. In one aspect, one or more peptides or proteins comprises, or wherein the fusion protein comprises two or more or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof. In another aspect, the amino acid sequence is selected from a cow milk T cell epitope selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In another aspect, the composition comprises one or more cow milk peptides amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes oneor more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof. In another aspect, the peptide or protein comprises a cow milk T cell epitope. In another aspect, the one or more peptides or proteins comprises a cow milk CD8+ or CD4+ T cell epitope. In another aspect, the one or more peptides or proteins has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids. In another aspect, the one or more peptides or proteins elicits, stimulates, induces, promotes, increases, or enhances a regulatory T cell response to a cow milk protein or peptide. In another aspect, the one or more peptides or proteins that elicits, stimulates, induces, promotes, increases or enhances the regulatory T cell response to the cow milk protein or peptide, or a variant, homologue, derivative or subsequence thereof. In another aspect, the composition further comprises the one or more peptides or proteins formulated into an immunoregulatory formulation with an adjuvant. In another aspect, the adjuvant is selected from the group consisting of aluminum hydroxide, calcium phosphate, microcrystalline tyrosine (MCT), and monophosphoryl lipid A (MPL). In another aspect, the composition further comprises a downregulator of immune response. In another aspect, the pool or to or more peptides can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, ,100, 1050, or 1081 peptides.

[0011] As embodied and broadly described herein, an aspect of the present disclosure relates to a composition comprising monomers or multimers of: peptides or proteins comprising, consisting of, or consisting essentially of: one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3,concatemers, subsequences, portions, homologues, variants or derivatives thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

[0012] As embodied and broadly described herein, an aspect of the present disclosure relates to a composition comprising one or more peptide-major histocompatibility complex (MHC) monomers or multimers, wherein the peptide-MHC monomer or multimer comprises a peptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), in a groove of the MHC monomer or multimer.

[0013] As embodied and broadly described herein, an aspect of the present disclosure relates to a composition comprising: one or more peptides or proteins comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); a pool of 2 or more peptides selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide thatencodes one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof. In one aspect, the one or more peptides or proteins comprises, or wherein the fusion protein comprises, 2 or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof. In another aspect, the protein or peptide comprises cow milk T cell epitope. In another aspect, the one or more peptides or proteins comprises a cow milk CD8+ or CD4+ T cell epitope. In another aspect, the one or more peptides or proteins has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids. In another aspect, the one or more peptides or proteins elicits, stimulates, induces, promotes, increases or enhances a regulatory T cell response to comprises cow milk peptides or proteins. In another aspect, the one or more peptides or proteins that elicits, stimulates, induces, promotes, increases or enhances the regulatory T cell response to cow milk protein(s) or peptide(s), or a variant, homologue, derivative or subsequence thereof. In another aspect, the composition further comprises formulating the one or more peptides or proteins into an immunogenic formulation with an adjuvant. In another aspect, the adjuvant is selected from the group consisting of adjuvant is selected from the group consisting of aluminum hydroxide, calcium phosphate, microcrystalline tyrosine (MCT), and monophosphoryl lipid A (MPL). In another aspect, the composition further comprises an immunoregulator of immune response. In another aspect, the pool or to or more peptides can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, ,100, 1050, or 1081 peptides.

[0014] As embodied and broadly described herein, an aspect of the present disclosure relates to a composition comprising monomers or multimers of: one or more peptides or proteins comprising, consisting of, or consisting essentially of: one or more cow milk amino acid sequences selected from any one ofthose sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: I to 1081), concatemers, subsequences, portions, homologues, variants or derivatives thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof. In another aspect, the pool or to or more peptides can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, ,100, 1050, or 1081 peptides.

[0015] As embodied and broadly described herein, an aspect of the present disclosure relates to a composition comprising one or more peptide-major histocompatibility complex (MHC) monomers or multimers, wherein the peptide-MHC monomer or multimer comprises a peptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), in a groove of the (MHC) monomer or multimer.

[0016] As embodied and broadly described herein, an aspect of the present disclosure relates to a method for detecting the presence of one or more cow milk antigens, comprising: providing one or more proteins or peptides for detection of an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells; contacting a biological sample suspected of having cow milk antigen-specific T- cells to one or more proteins or peptides for detection; and detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample, wherein the one or more proteins or peptides for detection comprise one or more amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or comprise a pool of 2 or more or more amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In one aspect, detecting the amount or a relative amount of, and / or activity of antigen-specific T-cells comprises one or more steps of identification or detection of the antigen-specific T-cells and measuring the amount of the antigen-specific T-cells. In another aspect, the one or more peptides or proteins comprises 2 or more amino acid sequences selected from those set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In another aspect, the detecting the amount or a relative amount of, and / or activity of antigen-specific T-cells comprises indirect detection and / or direct detection. In another aspect, the method of detecting an immune response to the one or more cow milk antigens comprises the following steps: providing an MHC monomer or an MHC multimer; contacting a population T-cells to the MHC monomer or MHC multimer; and measuring the number, activity or state of T-cells specific for the MHC monomer or MHC multimer. In another aspect, the MHC monomer or MHC multimer comprises a protein or peptide of cow milk. In another aspect, the protein or peptide comprises a CD8+ or CD4+ T cell epitope. In another aspect, the protein or peptide has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids. In another aspect, the proteins or peptides comprise 2 or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof. In another aspect, the method further comprises detecting the presence or amount of the one or more peptides in a biological sample, or a response thereto, which is diagnostic of a cow milk allergy. In another aspect, the detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample comprises measuring one or more of a cytokine or lymphokine secretion assay, T cell proliferation, immunoprecipitation, immunoassay, ELISA, radioimmunoassay, immunofluorescence assay, Western Blot, FACS analysis, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay, a reporter assay, a luciferase assay, a microarray, a surface plasmon resonance detector, a florescence resonance energy transfer, immunocytochemistry, or a cell mediated assay, or a cytokine proliferation assay. In another aspect, the method further comprises administering a treatment comprising the composition described hereinabove to the subject from which the biological sample was drawn that increases the amount or relative amount of, and / or activity of the antigen-specific T-cells. In another aspect, the pool or to or more peptides can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, ,100, 1050, or 1081 peptides.

[0017] As embodied and broadly described herein, an aspect of the present disclosure relates to a method for detecting the presence of an allergy to cow milk antigens, comprising: providing one or more proteins or peptides for detection of an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells; contacting a biological sample suspected of having cow milk-specific T-cells to one or more proteins or peptides for detection; and detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample, wherein the one or more proteins or peptides for detection comprise one or more amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or comprise a pool of 2 or more amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In one aspect, the detecting the amount or a relative amount of, and / or activity of antigen-specific T-cells comprises one or more steps of identification or detection of the antigen-specific T-cells and measuring the amount of the antigen-specific T-cells. In another aspect, the one or more peptides or proteins comprises 2 or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In another aspect, the detecting the amount or a relative amount of, and / or activity of antigen-specific T-cells comprises indirect detection and / or direct detection. In another aspect, the method of detecting an immune response to one or more cow milk antigens comprises the following steps: providing an MHC monomer or an MHC multimer; contacting a population T-cells to the MHC monomer or MHC multimer; and measuring the number, activity or state of T-cells specific for the MHC monomer or MHC multimer. In another aspect, the MHC monomer or MHC multimer comprises a protein or peptide of cow milk. In another aspect, the protein or peptide comprises a B. pertussis CD8+ or CD4+ T cell epitope. In another aspect, the protein or peptide has a length from about 9-15, 15-20, 20- 25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids. In another aspect, the proteins or peptides comprise 2 or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant, or derivative thereof. In another aspect, the method further comprises detecting the presence or amount of the one or more peptides in a biological sample, or a response thereto, which is diagnostic of an allergy to cow milk. In another aspect, the detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample comprises measuring one or more of a cytokine or lymphokine secretion assay, T cell proliferation, immunoprecipitation, immunoassay, ELISA, radioimmunoassay, immunofluorescence assay, Western Blot, FACS analysis, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay, a reporter assay, a luciferase assay, a microarray, a surface plasmon resonance detector, a florescence resonance energy transfer, immunocytochemistry, or a cell mediated assay, or a cytokine proliferation assay. In another aspect, the method further comprises administering a treatment comprising the composition described hereinabove to the subject from which the biological sample was drawn that increases the amount or relative amount of, and / or activity of the antigen-specific T-cells. In another aspect, the pool or to or more peptides can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125,150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, ,100, 1050, or 1081 peptides.

[0018] As embodied and broadly described herein, an aspect of the present disclosure relates to a method detecting an allergy to cow milk antigens in a subject, the method comprising, consisting of, or consisting essentially of: contacting a biological sample from a subject with a composition described hereinabove; and determining if the composition elicits an immune response from the contacted cells, wherein the presence of an immune response indicates that the subject has an allergy to cow milk antigens. In one aspect, the sample comprises T cells. In another aspect, the response comprises inducing, increasing, promoting, or stimulating a regulatory activity of T cells. In another aspect, the T cells are CD8+ or CD4+ T cells. In another aspect, the method comprises determining whether the subject has been exposed to the cow milk antigens more than once by determining if the subject elicits a secondary T cell immune response profde that is different from a primary T cell immune response profde. In another aspect, the method further comprises diagnosing an allergy to cow milk in a subject, the method comprising contacting a biological sample from a subject with a composition described hereinabove, and determining if the composition elicits a T or B cell immune response, wherein the T cell immune response identifies that the subject is allergic to cow milk. In another aspect, the method is conducted three or more days following the date of exposure to cow milk.

[0019] As embodied and broadly described herein, an aspect of the present disclosure relates to a method detecting a cow milk allergy in a subject, the method comprising, consisting of, or consisting essentially of: contacting a biological sample from a subject with a composition described hereinabove; and determining if the composition elicits an immune response from the contacted cells, wherein the presence of an immune response indicates that the subject has an allergy to cow milk. In one aspect, the sample comprises T cells. In another aspect, the response comprises inducing, increasing, promoting or stimulating a regulatory activity of T cells. In another aspect, the T cells are CD8+ or CD4+ T cells. In another aspect, the method comprises determining whether the subject has been exposed to cow milk more than once by determining if the subject elicits a secondary T cell immune response profde that is different from a primary T or B cell immune response profde. In another aspect, the method further comprises diagnosing an allergy to cow milk in a subject, the method comprising contacting a biological sample from a subject with a composition described hereinabove; and determining if the composition elicits a T or B cell immune response, wherein the T or B cell immune response identifies that the subject has an allergy to cow milk. In another aspect, the method is conducted three or more days following the date of suspected exposure to cow milk.

[0020] As embodied and broadly described herein, an aspect of the present disclosure relates to a kit for the detection of an allergy to cow milk in a subject comprising, consisting of or consisting essentially of: one or more T cells that specifically detect the presence of: one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; or a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to1081); or a pool of 2 or more or more peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In one aspect, the one or more amino acid sequences are selected from a cow milk T cell epitope set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In another aspect, the composition comprises: one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In another aspect, the fusion protein has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids. In another aspect, the kit includes instruction for a diagnostic method, a process, a composition, a product, a service or component part thereof for the detection of an allergy to cow milk. In another aspect, the kit includes reagents for detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T- cells in the biological sample comprises measuring one or more of a cytokine or lymphokine secretion assay, T cell proliferation, immunoprecipitation, immunoassay, ELISA, radioimmunoassay, immunofluorescence assay, Western Blot, FACS analysis, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay, a reporter assay, a luciferase assay, a microarray, a surface plasmon resonance detector, a florescence resonance energy transfer, immunocytochemistry, or a cell mediated assay, or a cytokine proliferation assay. In another aspect, the kit includes reagents for determining a Human Leukocyte Antigen (HLA) profile of a subject, and selecting peptides that are presented by the HLA profile of the subject for detecting an immune response to cow milk. In another aspect, the pool or to or more peptides can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, ,100, 1050, or 1081 peptides.

[0021] As embodied and broadly described herein, an aspect of the present disclosure relates to a kit for the detection of an allergy to cow milk in a subject comprising, consisting of or consisting essentially of: one or more T cells that specifically detect the presence of: one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In one aspect, the one or more amino acid sequences is selected from one or more cow milk CD4 T cell epitopes selected from any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or both. In another aspect, the amino acid sequence comprises one or more cow milk CD8+ or CD4+ T cell epitopes. In another aspect, the fusion protein has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids. In another aspect, the kit includes instruction for a diagnostic method, a process, a composition, a product, a service or component part thereof for the detection of an allergy to cow milk. In another aspect, the kit includes reagents fordetecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T- cells in the biological sample comprises measuring one or more of a cytokine or lymphokine secretion assay, T cell proliferation, immunoprecipitation, immunoassay, ELISA, radioimmunoassay, immunofluorescence assay, Western Blot, FACS analysis, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay, a reporter assay, a luciferase assay, a microarray, a surface plasmon resonance detector, a florescence resonance energy transfer, immunocytochemistry, or a cell mediated assay, or a cytokine proliferation assay. In another aspect, the kit includes reagents for determining a Human Leukocyte Antigen (HLA) profile of a subject, and selecting peptides that are presented by the HLA profile of the subject for detecting an immune response to cow milk.

[0022] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of stimulating, inducing, promoting, increasing, or enhancing an immunoregulatory response against one or more cow milk antigens in a subject, comprising: administering a composition described hereinabove, in an amount sufficient to stimulate, induce, promote, increase, or enhance an immune response against the cow milk in the subject. In one aspect, the immunoregulatory immune response provides the subject with protection against cow milk allergies, or one or more physiological conditions, disorders, illnesses, diseases or symptoms caused by or associated with cow milk allergies. In another aspect, the immune response is specific to: one or more cow milk peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof. In another aspect, the pool or to or more peptides can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, ,100, 1050, or 1081 peptides.

[0023] A method of stimulating, inducing, promoting, increasing, or enhancing an immunoregulatory response against cow milk in a subject, comprising: administering a composition described hereinabove, in an amount sufficient to stimulate, induce, promote, increase, or enhance an immune response against cow milk in the subject. In one aspect, the immunoregulatory response provides the subject with protection against cow milk, or one or more physiological conditions, disorders, illnesses, diseases or symptoms caused by or associated with cow milk allergies. In another aspect, the immunoregulatory response is specific to: one or more cow milk peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

[0024] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of stimulating, inducing, promoting, increasing, or enhancing a regulatory T cell response in a subject with an allergy to one or more cow milk antigens, comprising: administering to a subject an amount of a protein or peptide or a polynucleotide that expresses the protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence of two or more cow milk protein or peptide, or a variant, homologue, derivative or subsequence thereof, wherein the protein or peptide comprises at least two peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081)or asubsequence, portion, homologue, variant or derivative thereof, in an amount sufficient to stimulate, induce, promote, increase, or enhance an immunoregulatory immune response to one or more cow milk antigens in the subject. In one aspect, the immune response provides the subject with protection against an allergy to cow milk, or one or more physiological conditions, disorders, illnesses, diseases or symptoms caused by an allergy to cow milk.

[0025] A method of treating, preventing, or immunizing a subject against cow milk allergies, comprising administering to a subject an amount of a protein, peptide or a polynucleotide that expresses the protein or peptide comprising, consisting of, or consisting essentially of an amino acid sequence of a cow milk protein or peptide, or a variant, homologue, derivative or subsequence thereof, wherein the protein or peptide comprises at least one amino acid sequence selected from any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081)or a subsequence, portion, homologue, variant or derivative thereof, in an amount sufficient to treat, prevent, or immunize the subject to prevent the allergy to cow milk, wherein the protein or peptide comprises or consists of cow milk T cell epitope that elicits, stimulates, induces, promotes, increases, or enhances an anti- cow milk regulatory T cell immune response. In one aspect, the one or more amino acid sequences are selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In another aspect, the cow milk T cell response is a CD8+, a CD4+ T cell response, or both. In another aspect, the subject is a mammal or a human. In another aspect, the method reduces one or more adverse physiological conditions, disorders, illness, diseases, symptoms or complications caused by or associated with cow milk allergies. In another aspect, the method improves one or more adverse physiological conditions, disorders, illness, diseases, symptoms or complications caused by or associated with cow milk allergies. In another aspect, the symptom is fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea or vomiting, or diarrhea. In another aspect, the method reduces or inhibits susceptibility to cow milk allergies. In another aspect, the protein or peptide, or a subsequence, portion, homologue, variant or derivative thereof, is administered prior to, substantially contemporaneously with or following exposure to cow milk. In another aspect, a plurality of cow milk T cell epitopes are administered prior to, substantially contemporaneously with or following exposure to cow milk. In another aspect, the protein or peptide, or a subsequence, portion, homologue, variant or derivative thereof is administered within 2-72 hours, 2-48 hours, 4-24 hours, 4-18 hours, or 6-12 hours after a symptom of cow milk allergy. In another aspect, the protein or peptide, or a subsequence, portion, homologue, variant or derivative thereof is administered prior to exposure to cow milk. In another aspect, the method further comprises administering a modulator of immune response prior to, substantially contemporaneously with or following the administration to the subject of an amount of a protein or peptide.

[0026] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of treating, preventing, or immunizing a subject against cow milk allergies, comprising administering to a subject the composition described hereinabove in an amount sufficient to treat or prevent the cow milk allergy. In another aspect, the method reduces one or more adverse physiological conditions, disorders, illness, diseases, symptoms or complications caused by cow milk allergy. In another aspect, the method improves one or more adverse physiological conditions, disorders, illness, diseases, symptoms or complications caused by or associated with cow milk allergies. In another aspect, the method reduces or inhibits susceptibility to cow milk allergies. In another aspect, the composition is administered prior to, substantially contemporaneously with or following exposure to cow milk allergens. In another aspect, the composition is administered prior to, substantially contemporaneously with or following exposure to cow milk allergens. In another aspect, the composition is administered within 2-72 hours, 2-48 hours, 4-24 hours, 4-18 hours, or 6-12 hours after a symptom of cow milk allergies. In another aspect, the composition is administered prior to exposure to cow milk. In another aspect, the pool or to or more peptides can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, ,100, 1050, or 1081 peptides.

[0027] As embodied and broadly described herein, an aspect of the present disclosure relates to a peptide or peptides that are immunoprevalent or immunodominant in cow milk obtained by a method comprising, consisting of, or consisting essentially of: obtaining an amino acid sequence of one or more cow milk proteins; determining one or more sets of overlapping peptides spanning one or more cow milk antigen using unbiased selection; synthesizing one or more pools of cow milk peptides comprising the one or more sets of overlapping peptides; combining the one or more pools of cow milk peptides with Class I major histocompatibility proteins (MHC), Class II MHC, or both Class I and Class II MHC to form peptide-MHC complexes; contacting the peptide-MHC complexes with T cells from subjects exposed to the cow milk; determining which pools triggered cytokine release by the T cells; and deconvoluting from the pool of peptides that elicited cytokine release by the T cells, which peptide or peptides are immunoprevalent or immunodominant in the pool. In one aspect, the immunodominant peptides are selected from 1, 2 or more peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081) . In another aspect, the immunodominant peptides are selected from 1 , 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In another aspect, the pool or to or more peptides can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, ,100, 1050, or 1081 peptides.

[0028] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of selecting an immunoprevalent or immunodominant peptide or protein of cow milk comprising, consisting of, or consisting essentially of: obtaining an amino acid sequence of the cow milk; determining one or more sets of overlapping peptides spanning one or more cow milk antigen using unbiased selection; synthesizing one or more pools of cow milk peptides comprising the one or more sets of overlapping peptides; combining the one or more pools of cow milk peptides with Class I major histocompatibilityproteins (MHC), Class II MHC, or both Class I and Class II MHC to form peptide-MHC complexes; contacting the peptide-MHC complexes with T cells from subjects exposed to cow milk; determining which pools triggered cytokine release by the T cells; and deconvoluting from the pool of peptides that elicited cytokine release by the T cells, which peptide or peptides are immunoprevalent or immunodominant in the pool. In one aspect, the immunodominant peptides are selected from 1, 2 or more peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In another aspect, the immunodominant peptides are selected from 1, 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In another aspect, the pool or to or more peptides can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, ,100, 1050, or 1081 peptides.

[0029] As embodied and broadly described herein, an aspect of the present disclosure relates to a polynucleotide that expresses one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more or more peptides comprising, consisting of, or consisting essentially of amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

[0030] A vector that comprises the polynucleotide that expresses one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more or more peptides comprising, consisting of, or consisting essentially of amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In one aspect, the vector is a bacterial vector.

[0031] A host cell that comprises the vector that expresses one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more or more peptides comprising, consisting of, or consisting essentially of amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

[0032] A polynucleotide that expresses: one or more peptides or proteins comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of thosesequences set forth in Tables l, 2, and 3 (SEQ ID NOS: I to 1081); or apool of2 or more peptides selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

[0033] As embodied and broadly described herein, an aspect of the present disclosure relates to a vector that comprises a polynucleotide that expresses: one or more peptides or proteins comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables l, 2, and 3 (SEQ ID NOS: I to 1081); or apool of2 or more peptides selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In one aspect, the vector is a bacterial vector.

[0034] As embodied and broadly described herein, an aspect of the present disclosure relates to a host cell that comprises the vector that comprises a polynucleotide that expresses: one or more peptides or proteins comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: I to 1081).BRIEF DESCRIPTION OF THE DRAWINGS

[0035] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

[0036] FIGS. 1A to 1G show the discovery and assessment of a T cell epitope megapool from cow milk extract. FIG. 1A) 2D immunoblot of cow milk extract showing protein spots (green) reactive with IgG(pink), IgE(blue) or both(white). 96 circled spots were selected for mass spectrometry analysis. FIG. IB) Table showing the known allergen or novel antigen associated with each spot on the gel as determined by mass spectrometry. FIG. 1C) Peptide pools were evaluated by 14-day culture followed by Fluorospot assay. Bar graph showing the percentage of IL-5 responders across all pools for each antigen. FIG. ID) Screened peptides were assessed for homology to human peptides using PepMatch. The pie charts show percentage of indicated reactive or non-reactive peptides where the top human homolog hit had the indicated number of mis-matched amino acids. FIG. IE) Table describing the contents and peptide inclusion criteria for each of the three megapools. FIG. IF, FIG. 1G) Bar graphs showing percentage of responders in (FIG. IF) allergic (CMA-S and CMA-M) versus non-allergic controls or (FIG. 1G) CMA-S versus CMA-M donors expressing IL-5, IFNy, and IL-10 (n= 11 controls, 3 CMA-M, 5 CMA-S).

[0037] FIGS. 2A to 2F show the scRNA-Sequencing of Ml 11 epitope megapool reactive memory CD4 T cells. FIG. 2A) Experimental design for the study. PBMC from Control, CMA-M, and CMA-S donors were stimulated with the Ml 11 megapool for 6 hours and antigen specific cells (CM+) were sorted using flow markers CD137 and CD154 for 10X scRNA-Seq. FIG. 2B) Dot plot showing percentage CM+ cells in eachdonor. FIG. 2C) UMAP of 98,883 cells colored by CM+ and CM- sort fractions. FIG. 2D) UMAP colored by identified Seurat cluster. FIG. 2E) Dot plot showing expression of TNFRSF9 (CD 137) and CD40LG (CD 154) in each cluster where size represents percent of cells expressing the marker and color represents expression level. FIG. 2F) Stacked bar graphs representing the distribution of clusters within CM+ and CM- sort fractions to determine true antigen specific cells. Statistics were performed using a Yates’ continuity corrected chi-square test where *=p<0.05, ***=p<0.001, and ****=p<0.0001.

[0038] FIGS. 3A to 31 shows that detection of increased CM+ FOXP3+ cells with Severe CMA by scRNA-Seq and flow cytometry. CM+ cells from clusters 3, 7, and 15 were subset from the total dataset. FIG. 3 A) Heatmap showing expression of genes differentiating clusters 3, 15, and 7 where each row is a gene and each column a cell. Selected genes with high expression in each cluster are boxed to the left and clusters are identified as FOXP3+ or FOXP3-. FIG. 3B) Dot plots showing the proportions of cells in each of the three clusters per donor and separated by allergic group. FIG. 3C) Plot showing the ratio of CM+ FOXP3- / FOXP3+ cells per donor where the CMA group is shown both as a total CMA and split into severity groups. FIG. 3D) Dot plot showing expression of genes differentiating the FOXP3+ and FOXP3- clusters where size represents percent of cells expressing the marker and color represents expression level. FIG. 3E) Example flow plots showing CD127(IL7R) / CCR7 expression in CM+ cells. FIG. 3F, FIG. 3G) Quantification of the gates drawn in (FIG. 3E) compared across allergy groups. FIG. 3H) Example flow plots showing CD127(IL7R) / CD25(IL2RA) expression in CM+ cells. FIG. 31) Quantification of the gate drawn in (FIG. 3E). Statistics were performed by Mann-Whitney test with correction for multiple comparisons where appropriate. *=p<0.05, **=p<0.01. Trending values are included.

[0039] FIGS. 4A to 4F shows the population of CM+ FOXP3+ cells present with CMA shows strong interferon signature. CM+ FOXP3+ cells were subset and re-clustered. FIG. 4A) UMAP colored by Seurat cluster and annotated by distinguishing gene expression signatures. FIG. 4B) Pie charts showing representation of cells from each allergy group within each cluster. Number in paratheses is the number of cells in that cluster. FIG. 4C) Volcano plot showing differentially expressed genes in total CM+ FOXP3+ cells between CMA-S and Control groups. Colored dots are statistically significant (adjusted p value < 0.05; control=blue, CMA-S=red) and genes involved in indicated GO processes are labeled. FIG. 4D) Dot plot showing expression of selected genes in each allergy group where size represents percent of cells expressing the marker and color represents expression level. FIG. 4E) CM+ FOXP3+ cells were merged with CM- FOXP3+ cells and re-clustered. UMAP is colored by indicated cluster. FIG. 4F) Violin plots showing expression levels of interferon responsive genes associated with CM+ FOXP3+ cluster C2.

[0040] FIGS. 5A to 5E shows that CMA-S donors have small population of pathogenic Th2 cells. FIG. 5A) UMAP of 6,568 re-clustered CM+ FOXP3- cells. FIG. 5B) Heatmap showing cluster marker identification genes for CM+ FOXP3- cells. FIG. 5C) Feature plots showing expression of Th2 cytokines and pathogenic Th2 marker genes. FIG. 5D) Dot plot showing expression of selected genes in Th2 and Activated cluster 3 where size represents percent of cells expressing the marker and color represents expression level. FIG. 5E) Bar plot showing top functional enrichment pathways of upregulated genes in CMA-S donors determined from total CM+ FOXP3- cells.DETAILED DESCRIPTION OF THE INVENTION

[0041] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0042] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive.

[0043] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.Definitions:

[0044] Definitions.

[0045] As used herein, the term “expression” or “expressed” as used herein in reference to a gene means the transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell. The level of expression of non-coding nucleic acid molecules (e.g., sgRNA) may be detected by standard PCR or Northern blot methods well known in the art. See, Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1- 18.88.

[0046] As used herein, the term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g. , norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functionsin a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0047] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0048] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may, in embodiments, be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0049] Proteins and peptides include isolated and purified forms. Proteins and peptides also include those immobilized on a substrate, as well as amino acid sequences, subsequences, portions, homologues, variants, and derivatives immobilized on a substrate.

[0050] Proteins and peptides can be included in compositions, for example, a pharmaceutical composition. In particular embodiments, a pharmaceutical composition is suitable for specific or non-specific immunotherapy, or is a vaccine composition.

[0051] As used herein, the term “gene” means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a “protein gene product” is a protein expressed from a particular gene.

[0052] Isolated nucleic acid (including isolated nucleic acid) encoding the proteins and peptides are also provided. Cells expressing a protein or peptide are further provided. Such cells include eukaryotic and prokaryotic cells, such as mammalian, insect, fungal, and bacterial cells.

[0053] Methods, uses, and medicaments of proteins and peptides of the invention are included. Such methods, uses and medicaments include modulating the immune activity of the immune system to cow milk allergen(s).

[0054] As used herein, the term “peptide mimetic” or “peptidomimetic” refers to protein-like chain designed to mimic a peptide or protein. Peptide mimetics may be generated by modifying an existing peptide or by designing a compound that mimics peptides, including peptoids and [3-peptides.

[0055] As used herein, the term “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refer to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every positionwhere an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0056] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well-known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure. The following eight groups each contain amino acids that are conservative substitutions for one another: (1) Alanine (A), Glycine (G); (2) Aspartic acid (D), Glutamic acid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); (6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (7) Serine (S), Threonine (T); and (8) Cysteine (C), Methionine (M) (see, e.g.. Creighton, Proteins (1984)).

[0057] A “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e. , gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0058] As used herein, the terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like.Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0059] An amino acid or nucleotide base “position” is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N- terminus (or 5 ’-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0060] As used herein, the terms “numbered with reference to” or “corresponding to,” when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.

[0061] As used herein, the term “multimer” refers to a complex comprising multiple monomers (e.g., a protein complex) associated by noncovalent bonds. The monomers be substantially identical monomers, or the monomers may be different. In embodiments, the multimer is a dimer, a trimer, a tetramer, or a pentamer.

[0062] As used herein, the term “Major Histocompatibility Complex” (MHC) is a generic designation meant to encompass the histocompatibility antigen systems described in different species including the human leucocyte antigens (HLA). Typically, MHC Class I or Class II multimers are well known in the art and include but are not limited to dimers, tetramers, pentamers, hexamers, heptamers and octamers.

[0063] As used herein, the term “MHC / peptide multimer” refers to a stable multimeric complex composed of MHC protein(s) subunits loaded with a peptide of the present invention. For example, an MHC / peptide multimer (also called herein MHC / peptide complex) include, but are not limited to, an MHC / peptide dimer, trimer, tetramer, pentamer, dextramer, or higher / other valency multimer. Several examples of dextramers are those of IMMUDEX®. In humans there are three major different genetic loci that encode MHC class I molecules (the MHC molecules of the human are also designated human leukocyte antigens (HLA)): HLA- A, HLA-B, HLA-C, e.g., HLA-A*01, HLA-A*02, and HLA-A* 11 are examples of different MHC class I alleles that can be expressed from these loci. Non-classical human MHC class I molecules such as HLA-E (homolog of mice Qa-lb) and MICA / B molecules are also encompassed by the present invention. In some embodiments, the MHC / peptide multimer is an HLA / peptide multimer selected from the group consisting of HLA-A / peptide multimer, HLA-B / peptide multimer, HLA-C / peptide multimer, HLA-E / peptide multimer, MICA / peptide multimer and MICB / peptide multimer. Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

[0064] In humans there are three major different genetic loci that encode MHC class II molecules: HLA- DR, HLA-DP, and HLA-DQ, each formed of two polypeptides, alpha and beta chains (A and B genes). For example, HLA-DQAl*01, HLA-DRBl*01, and HLA-DRBl*03 are different MHC class II alleles that can be expressed from these loci. It should be further noted that non-classical human MHC class II molecules such as HLA-DM and HL-DOA (homolog in mice is H2-DM and H2-O) are also encompassed by the present invention. In some embodiments, the MHC / peptide multimer is an HLA / peptide multimer selected from the group consisting of HLA-DP / peptide multimer, HLA-DQ / peptide multimer, HLA- DR / peptide multimer, HLA-DM / peptide multimer and HLA-DO / peptide multimer.

[0065] An MHC / peptide multimer may be a multimer where the heavy chain of the MHC is biotinylated, which allows combination as a tetramer with streptavidin. MHC -peptide tetramers have increased avidity for the appropriate T cell receptor (TCR) on T lymphocytes. The multimers can also be attached to paramagnetic particles or magnetic beads to facilitate removal of non-specifically bound reporter and cell sorting. Multimer staining does not kill the labelled cells, thus, cell integrity is maintained for further analysis. In some embodiments, the MHC / peptide multimer of the present invention is particularly suitable for isolating and / or identifying a population of CD8+ T cells having specificity for the peptide of the present invention (in a flow cytometry assay).

[0066] The peptides or MHC class I or class II multimer as described herein is particularly suitable for detecting T cells specific for one or more peptides of the present invention. The peptide(s) and / or the MHC / multimer complex of the present invention is particularly suitable for diagnosing cow milk allergies in a subject. For example, the method comprises obtaining a blood or PBMC sample obtained from the subject with an amount of a least peptide of the present invention and detecting at least one T cell displaying a specificity for the peptide. Another diagnostic method of the present invention involves the use of a peptide of the present invention that is loaded on multimers as described above, so that the isolated CD8+ or CD4+ T cells from the subject are brought into contact with the multimers, at which the binding, activation and / or expansion of the T cells is measured. For example, following the binding to antigen presenting cells, e.g., those having the MHC class I or class II multimer, the number of CD8+ and / or CD4+ cells binding specifically to the HLA-peptide multimer may be quantified by measuring the secretion of lymphokines / cytokines, division of the T cells, or standard flow cytometry methods, such as, for example, using fluorescence activated cell sorting (FACS). The multimers can also be attached to paramagnetic ferrous or magnetic beads to facilitate removal of non-specifically bound reporter and cell sorting. The MHC class I or class II peptide multimers as described herein can also be used as therapeutic agents. The peptide and / or the MHC class I or class II peptide multimers of the present invention are suitable for treating or preventing cow milk allergies in a subject. The MHC Class I or Class II multimers can be administered in soluble form or loaded on nanoparticles.

[0067] As used herein, the term “antibody” refers to a polypeptide encoded by an immunoglobulin gene or functional fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as eitherkappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

[0068] As used herein, the phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein or peptide, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0069] Antibodies are large, complex molecules (molecular weight of -150,000 or about 1320 amino acids) with intricate internal structure. A natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable (“V”) region involved in binding the target antigen, and a constant (“C”) region that interacts with other components of the immune system. The light and heavy chain variable regions come together in 3 -dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions (“CDRs”). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in 3 -dimensional space to form the actual antibody binding site which docks onto the target antigen. The position and length of the CDRs have been precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The part of a variable region not contained in the CDRs is called the framework (“FR”), which forms the environment for the CDRs.

[0070] Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)52, a dimer of Fab which itself is a light chain joined to VH-CHI by a disulfide bond. The F(ab)’2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)52 dimer into a Fab’ monomer. The Fab’ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinantDNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) orthose identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0071] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. The Fc (i.e., fragment crystallizable region) is the “base” or “tail” of an immunoglobulin and is typically composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

[0072] As used herein, the term “antigen” and the term “epitope” refer to a molecule or substance capable of stimulating an immune response. In one example, epitopes include but are not limited to a polypeptide and a nucleic acid encoding a polypeptide, wherein expression of the nucleic acid into a polypeptide is capable of stimulating an immune response when the polypeptide is processed and presented on a Major Histocompatibility Complex (MHC) molecule. Generally, epitopes include peptides presented on the surface of cells non-covalently bound to the binding groove of Class I or Class II MHC, such that they can interact with T cell receptors and the respective T cell accessory molecules. However, antigens and epitopes also apply when discussing the antigen-binding portion of an antibody, wherein the antibody binds to a specific structure of the antigen.

[0073] Proteolytic Processing of Antigens. Epitopes that are displayed by MHC on antigen-presenting cells are cleavage peptides or products of larger peptide or protein antigen precursors. For MHC I epitopes, protein antigens are often digested by proteasomes resident in the cell. Intracellular proteasomal digestion produces peptide fragments of about 3 to 23 amino acids in length that are then loaded onto the MHC protein. Additional proteolytic activities within the cell, or in the extracellular milieu, can trim and process these fragments further. Processing of MHC Class II epitopes generally occurs via intracellular proteases from the lysosomal / endosomal compartment. The present invention includes, in one embodiment, pre- processed peptides that are attached to the anti-CD40 antibody (or fragment thereof) that directs the peptides against which an enhanced immune response is sought directly to antigen-presenting cells.

[0074] The present invention includes methods for specifically identifying the epitopes within antigens most likely to lead to the immune response sought for the specific sources of antigen presenting cells and responder T cells.

[0075] As used herein, the term “T cell epitope” refers to a specific amino acid that when present in the context of a Major or Minor Histocompatibility Complex provides a reactive site for a T cell receptor. The T-cell epitopes or peptides that stimulate the cellular arm of a subject’s immune system are short peptidesof about 8-25 amino acids. T-cell epitopes are recognized by T cells from animals that are immune to the antigen of interest. These T-cell epitopes or peptides can be used in assays such as the stimulation of cytokine release or secretion or evaluated by constructing major histocompatibility (MHC) proteins containing or “presenting” the peptide. Such immunogenically active fragments are often identified based on their ability to stimulate lymphocyte proliferation in response to stimulation by various fragments from the antigen of interest.

[0076] As used herein, the term “immunological response” refers to an antigen or composition is the development in a subject of a humoral and / or a cellular immune response to an antigen present in the composition of interest. For purposes of the present disclosure, a “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and / or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells (“CTL”s). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A “cellular immune response” also refers to the production of cytokines, chemokines, and other such molecules produced by activated T-cells and / or other white blood cells, including those derived from CD4+ and CD8+ T-cells. Hence, an immunological response may include one or more of the following effects: the production of antibodies by B-cells; and / or the activation of effector and / or suppressor T-cells and / or gamma-delta T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest. These responses may serve to neutralize an allergic response. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art.

[0077] As used herein, the terms “immunogenic composition” and “vaccine” refer to a composition that comprises an antigenic molecule where administration of the composition to a subject or patient results in the development in the subject of a humoral and / or a cellular immune response to the antigenic molecule of interest. “Vaccine” refers to a composition that can provide active acquired immunity to and / or therapeutic effect (e.g., treatment) of a particular disease or a pathogen. A vaccine typically contains one or more agents that can induce an immune response in a subject against a pathogen or disease, i.e., a target pathogen or disease. The immunogenic agent stimulates the body’s immune system to recognize the agent as a threat or indication of the presence of the target pathogen or disease, thereby inducing immunological memory so that the immune system can more easily recognize and destroy any of the pathogen on subsequent exposure. Vaccines can be prophylactic (e.g., preventing or ameliorating the effects of a future cow milk allergies) or therapeutic (e.g., reducing symptoms or aberrant conditions associated with cow milk allergies). The administration of vaccines is referred to as vaccination or alternatively an immunization.

[0078] In some examples, a vaccine composition can provide nucleic acid, e.g., mRNA that encodes antigenic molecules (e.g., peptides) to a subject. The nucleic acid that is delivered via the vaccine composition in the subject can be expressed into antigenic molecules and allow the subject to acquire immunity against the antigenic molecules. In the context of the vaccination against cow milk allergen(s), the vaccine composition can provide mRNA encoding antigenic molecules that are associated with a certain pathogen, e.g., one or more peptides that are known to be found in cow milk.

[0079] The present invention provides nucleic acid molecules, specifically polynucleotides, primary constructs, and / or mRNA that encode one or more polynucleotides that express one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in the tables herein, or a subsequence, portion, homolog, variant or derivative thereof for use in immune modulation. The term “nucleic acid” refers to any compound and / or substance that comprises a polymer of nucleotides, referred to herein as polynucleotides. Exemplary nucleic acids or polynucleotides of the invention include but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs), including diastereomers of LNAs, functionalized LNAs, or hybrids thereof.

[0080] One method of immune modulation of the present invention includes direct or indirect gene transfer, i.e., local application of a preparation containing the one or more polynucleotides (DNA, RNA, mRNA, etc.) that expresses the one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homolog, variant or derivative thereof. A variety of well-known vectors can be used to deliver to cells the one or more polynucleotides or the peptides or proteins expressed by the polynucleotides, including but not limited to adenoviral vectors and adeno- associated vectors. In addition, naked DNA, liposome delivery methods, or other novel vectors developed to deliver the polynucleotides to cells can also be beneficial. Any of a variety of promoters can be used to drive peptide or protein expression, including but not limited to endogenous promoters, constitutive promoters (e.g., cytomegalobacteria, adenoviral, or SV40), inducible promoters (e.g., a cytokine promoter such as the interleukin- 1, tumor necrosis factor-alpha, or interleukin-6 promoter), and tissue-specific promoters to express the immunogenic peptides or proteins of the present invention.

[0081] The immunization may also include inserting the one or more polynucleotides (DNA, RNA, mRNA, etc.) that express the one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homolog, variant or derivative thereof into the bacterial vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, such that the vector is now target specific. Bacterial vectors can be made target-specific by attaching, for example, a sugar, a glycolipid, or a protein. Targeting can also be accomplished by using an antibody to target the bacterial vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences that can be inserted into the bacterial genomeor atached to a bacterial envelope to allow target-specific delivery of the bacterial vector containing the gene.

[0082] Another example of immunization includes colloidal dispersion systems that include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes and one or more polynucleotides that express the one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables l, 2, and 3 (SEQ ID NOS: I to 1081), or a subsequence, portion, homolog, variant or derivative thereof. One non-limiting example of a colloidal system for use with the present invention is a liposome. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 micrometers can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA, and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (Zakut and Givol, supra) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (Feamhead, et al., supra) preferential and substantial binding to a target cell in comparison to non-target cells; (Korsmeyer, S. J., supra) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (Kinoshita, et al., supra) accurate and effective expression of genetic information (Mannino, et al., Bio Techniques, 6:682, 1988).

[0083] The composition for immunizing the subject or patient may, in certain embodiments comprise a combination of phospholipid, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based on whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) in organs that contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

[0084] For any of the above approaches, the immune-modulating polynucleotide construct, composition, or formulation is preferably applied to a site that will enhance the immune response. For example, the immunization may be intramuscular, intraperitoneal, enteral, parenteral, intranasal, intrapulmonary, or subcutaneous. In the gene delivery constructs of the instant invention, polynucleotide expression is directed from any suitable promoter (e.g., the human cytomegalovirus, simian bacteria 40, actin or adenoviralconstitutive promoters; or the cytokine or metalloprotease promoters for activated synoviocyte specific expression).

[0085] In one example of the immune modifying peptide(s) or protein(s) includes polynucleotides, constructs, and / or mRNAs that express the one or more polynucleotides that express the one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in tables herein, or a subsequence, portion, homolog, variant or derivative thereof, that are designed to improve one or more of the stability and / or clearance in tissues, uptake and / or kinetics, cellular access by the peptide(s) or protein(s), translational, mRNA half-life, translation efficiency, immune evasion, protein production capacity, accessibility to circulation, peptide(s) or protein(s) half-life and / or presentation in the context of MHC on antigen-presenting cells.

[0086] The present invention contemplates immunization for use in both active and passive immunization embodiments. Immunogenic compositions, proposed to be suitable for use as a vaccine, may be prepared most readily directly from immunogenic peptides, proteins, monomers, multimers, and / or peptide-MHC complexes prepared in a manner disclosed herein. The antigenic material is generally processed to remove undesired contaminants, such as small molecular weight molecules, and incomplete proteins, or when manufactured in plant cells, plant components such as cell walls, plant proteins, and the like. Often, these immunizations are lyophilized for ease of transport and / or to increase shelf-life and can then be more readily dissolved in a desired vehicle, such as saline.

[0087] The preparation of immunizations (also referred to as vaccines) that contain the immunogenic proteins of the present invention as active ingredients is generally well understood in the art, as exemplified by United States Letters Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4.578,770, all incorporated herein by reference. Typically, such immunizations are prepared as injectables. The immunizations can be a liquid solution or suspension but may also be provided in a solid form suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, buffers, or the like and combinations thereof. In addition, if desired, the immunization may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.

[0088] The immunization is / are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual’s immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.

[0089] The manner of application of the immunization may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to also include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size of the host.

[0090] Various methods of achieving adjuvant effect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° to 101°C for 30 second to 2-minute periods respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.

[0091] In many instances, it will be desirable to have multiple administrations of the vaccine, usually not exceeding six to ten immunizations, more usually not exceeding four immunizations and preferably one or more, usually at least about three immunizations. The immunizations will normally be at from two to twelve-week intervals, more usually from three to five-week intervals. Periodic boosters at intervals of 1- 5 years, usually three years, will be desirable to maintain protective levels of the antibodies. The course of the immunization may be followed by assays for antibodies for the supernatant antigens. The assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescent agents, and the like. These techniques are well known and may be found in a wide variety of patents, such as Hudson and Cranage, Vaccine Protocols, 2003 Humana Press, relevant portions incorporated herein by reference.

[0092] Techniques and compositions for making useful dosage forms using the present invention are described in one or more of the following references: Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, New York, 1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition, McGraw Hill, 2007; Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001; Remington’s Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000, and updates thereto; Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (The Pharmaceutical Press, London, 1999); all of which are incorporated by reference, and the like, relevant portions incorporated herein by reference.

[0093] Many suitable expression systems are commercially available, including, for example, the following: baculoviral expression (Reilly, P. R., et al., BACULOVIRAL EXPRESSION VECTORS: A LABORATORY MANUAL (1992); Beames, et al., Biotechniques 11:378 (1991); Pharmingen; Clontech, Palo Alto, Calif.)), vaccinia expression systems (Earl, P. L., et al., “Expression of proteins in mammalian cells using vaccinia” In Current Protocols in Molecular Biology (F. M. Ausubel, et al. Eds.), Greene Publishing Associates & Wiley Interscience, New York (1991); Moss, B., et al., U.S. Pat. No. 5,135,855,issued Aug. 4, 1992), expression in bacteria (Ausubel, F. M., et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, Inc., Media Pa.; Clontech), expression in yeast (Rosenberg, S. and Tekamp-Olson, P., U.S. Pat. No. RE35,749, issued, Mar. 17, 1998, herein incorporated by reference; Shuster, J. R., U.S. Pat. No. 5,629,203, issued May 13, 1997, herein incorporated by reference; Gellissen, G., et al., Antonie Van Leeuwenhoek, 62(l-2):79-93 (1992); Romanos, M. A., et al., Yeast 8(6):423-488 (1992); Goeddel, D. V., Methods in Enzymology 185 (1990); Guthrie, C., and G. R. Fink, Methods in Enzymology 194 (1991)), expression in mammalian cells (Clontech; Gibco-BRL, Ground Island, N.Y.; e.g., Chinese hamster ovary (CHO) cell lines (Haynes, J., et al., Nuc. Acid. Res. 11:687-706 (1983); 1983, Lau, Y. F„ et al., Mol. Cell. Biol. 4: 1469-1475 (1984); Kaufinan, R. J., “Selection and coamplification of heterologous genes in mammalian cells,” in Methods in Enzymology, vol. 185, pp 537- 566. Academic Press, Inc., San Diego Calif. (1991)), and expression in plant cells (plant cloning vectors, Clontech Laboratories, Inc., Palo-Alto, Calif., and Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.; Hood, E„ et al., J. Bacteriol. 168: 1291-1301 (1986); Nagel, R„ et al., FEMS Microbiol. Lett. 67:325 (1990); An, et al., “Binary Vectors”, and others in Plant Molecular Biology Manual A3: l, 2, and 3 (1988); Miki, B. L. A., et al., pp. 249-265, and others in Plant DNA Infectious Agents (Hohn, T., et al., eds.) Springer- Verlag, Wien, Austria, (1987); Plant Molecular Biology: Essential Techniques, P. G. Jones and J. M. Sutton, New York, J. Wiley, 1997; Miglani, Gurbachan Dictionary of Plant Genetics and Molecular Biology, New York, Food Products Press, 1998; Henry, R. J., Practical Applications of Plant Molecular Biology, New York, Chapman & Hall, 1997), relevant portion incorporated herein by reference.

[0094] As used herein, the term “effective amount” or “effective dose” refers to that amount of the peptide or protein T cell epitopes of the invention sufficient to induce immunity, to prevent and / or ameliorate a cow milk allergy or to reduce at least one symptom of cow milk allergies and / or to enhance the efficacy of another dose of peptide or protein T cell epitopes. An effective dose may refer to the amount of peptide or protein T cell epitopes sufficient to delay or minimize the onset of cow milk allergies. An effective dose may also refer to the amount of peptide or protein T cell epitopes that provides a therapeutic benefit in the treatment or management of cow milk allergies. Further, an effective dose is the amount with respect to peptide or protein T cell epitopes of the invention alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of cow milk allergies. An effective dose may also be the amount sufficient to enhance a subject’s (e.g., a human’s) own immune response against a subsequent exposure to cow milk allergens. Levels of immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and / or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay. In the case of a vaccine, an “effective dose” is one that prevents disease and / or reduces the severity of symptoms. A “reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) refers to a decrease in the severity or frequency of the symptom(s), or elimination of the symptom(s) of cow milk allergies. A “prophylactically effective amount” of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, pathology, or condition,or their symptoms, in this case, an allergy to cow milk allergen(s). The full prophylactic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, for the given parameter, an effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. The exact amounts will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins), relevant portions incorporated herein by reference.

[0095] As used herein, the term “immune stimulator” refers to a compound that enhances an immune response via the body’s own chemical messengers (cytokines). These molecules comprise various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro- inflammatory activities, such as interferons, interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony-stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc. The immune stimulator molecules can be administered in the same formulation as peptide or protein T cell epitopes of the invention, or can be administered separately. Either the protein or an expression vector encoding the protein can be administered to produce an immunostimulatory effect.

[0096] As used herein, in certain embodiments, the phrase “immune response” refers to an immune response mediated by B cell or T cells against an allergen, which is exhibited by a vertebrate (e.g., a human), which causes or triggers cow milk allergies. Peptide and protein T cell epitopes of the invention can stimulate the production of antibodies that, for example, neutralize cow milk allergens or that stimulate suppressor T cells.

[0097] The terms “biological sample” or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage -like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

[0098] As used herein, a “cell” refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including,for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells. Cells may be useful when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization.

[0099] As used herein, the term “contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species to become sufficiently proximal to react, interact or physically touch. It should be appreciated, however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, an amino acid sequence, protein, or peptide as provided herein and an immune cell, such as a T cell.

[0100] As used herein, a “control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a test condition, e.g., in the presence of a test compound, and compared to samples from known conditions, e.g., in the absence of the test compound (negative control), or in the presence of a known compound (positive control). A control can also represent an average value gathered from a number of tests or results. One of skill in the art will recognize that controls can be designed for assessment of any number of parameters. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.

[0101] As used herein, the term “modulator” refers to a composition that increases or decreases the level of a target molecule or the function of a target molecule or the physical state of the target of the molecule relative to the absence of the modulator.

[0102] As used herein, the term “modulate” is used in accordance with its plain ordinary meaning and refers to the act of changing or varying one or more properties. “Modulation” refers to the process of changing or varying one or more properties. For example, as applied to the effects of a modulator on a target protein, to modulate means to change by increasing or decreasing a property or function of the target molecule or the amount of the target molecule.

[0103] As used herein, the terms “associated” or “associated with” in the context of a substance or substance activity or function associated with a disease that is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function, specifically, cow milk allergen(s). As used herein, what is described as being associated with a disease, if a causative agent, could be a target for treatment of the disease.

[0104] As used herein, the term “aberrant” as used herein refers to different from normal. When used to describe enzymatic activity or protein function, aberrant refers to activity or function that is greater or less than a normal control or the average of normal non-diseased control samples. Aberrant activity may refer to an amount of activity that results in a disease, wherein returning the aberrant activity to a normal or nondisease-associated amount (e.g., by administering a compound or using a method as described herein), results in a reduction of the disease or one or more disease symptoms.

[0105] As used herein, the terms “subject” or “subject in need thereof’ refer to a living organism that is at risk of or prone to having a disease or condition, or that is suffering from a disease or condition that can be treated by administration of a composition or pharmaceutical composition as provided herein. Nonlimiting examples include humans and other primates, but also include non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered. The system described above is intended for use in any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly.

[0106] As used herein, the terms “disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with a compound, pharmaceutical composition, or method provided herein. In embodiments, a patient or subject is human. In embodiments, the disease is cow milk allergy. In certain alternative embodiments, the disease is anaphylaxis caused by cow milk allergen(s).

[0107] As used herein, the terms “treatment” or “treating,” or “palliating” or “ameliorating” are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated or the disorder resulting from cow milk allergens. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with cow milk allergies or the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder or may still be allergic. For prophylactic benefit, the compositions may be administered to a patient at risk of cow milk allergies, of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made. Treatment includes preventing or reducing cow milk allergies, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition prior to the induction of the cow milk allergies; suppressing the cow milk allergies, that is, causing the clinical symptoms of the cow milk allergies not to develop by administration of a protective composition after the inductive event of cow milk allergies but prior to the clinical appearance or reappearance of the disease; inhibiting the disease, that is, arresting the development of clinical symptoms by administration of a protective composition after their initial appearance; preventing re-occurring of the disease and / or relieving the disease, that is, causing the regression of clinical symptoms by administration of a protectivecomposition after their initial appearance. “Treatment” can also refer to any of (i) the prevention of cow milk allergies, as in a traditional vaccine or immunization, (ii) the reduction or elimination of symptoms, and (iii) the substantial or complete elimination of the cow milk allergies. Treatment may be affected prophylactically (prior to exposure to cow milk allergen(s)) or therapeutically (following an allergic reaction to cow milk allergen(s)).

[0108] In addition, in certain embodiments, “treatment,” “treat,” or “treating” refers to a method of reducing the effects of one or more symptoms of cow milk allergies. Thus, in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of the cow milk allergies. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition and / or complete prevention of cow milk allergies. Further, as used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level and such terms can include but do not necessarily include complete elimination.

[0109] As used herein, the terms “diagnose” or “diagnosing” refer to recognition of a cow milk allergy, by signs and symptoms. Diagnosing can refer to the determination of whether a subject has an allergy to cow milk allergen(s). Diagnosis may refer to the determination of the type of cow milk allergen(s) to which a subject is allergic or the type of allergy to cow milk allergens.

[0110] Diagnostic agents provided herein include any such agent, which are well-known in the relevant art. Among imaging agents are fluorescent and luminescent substances, including, but not limited to, a variety of organic or inorganic small molecules commonly referred to as “dyes,” “labels,” or “indicators.” Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes, and cyanine dyes. Enzymes that may be used as imaging agents in accordance with the embodiments of the disclosure include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, [3-galactosidase, [3- glucuronidase or [3-lactamase. Such enzymes may be used in combination with a chromogen, a Anorogenic compound, or a luminogenic compound to generate a detectable signal.

[0111] The peptide(s) or protein(s) of the present invention can also be used in binding assays including, but are not limited to, immunoassays such as competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, Meso Scale Discovery (MSD, Gaithersburg, Md.), immunoprecipitation assays, ELISPOT, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, relevant portions incorporated herein by reference).

[0112] Radioactive substances that may be used as imaging agents in accordance with the embodiments of the disclosure include, but are not limited to,18F,32P,33P,45Ti,47Sc,52Fe,59Fe,62Cu,64Cu,67Cu,67Ga,68Ga,77As,86Y,90Y.89Sr,89Zr,94Tc,94Tc,99mTc, "Mo,105Pd,105Rh,i nAg,i nIn,123I,124I,125I,131I,142Pr,143Pr,149Pm,153Sm,154'1581Gd,161Tb,166Dy,166Ho,169Er,175Lu,177Lu,186Re,188Re,189Re,194Ir,198Au,199Au,211At,211Pb,212Bi,212Pb,213Bi,223Ra and225Ac. Paramagnetic ions that may be used as additional imaging agents in accordance with the embodiments of the disclosure include, but are not limited to, ions of transition and lanthanide metals (e.g., metals having atomic numbers of 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

[0113] When the imaging agent is a radioactive metal or paramagnetic ion, the agent may be reacted with another long-tailed reagent having a long tail with one or more chelating groups attached to the long tail for binding to these ions. The long tail may be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which the metals or ions may be added for binding. Examples of chelating groups that may be used according to the disclosure include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), DOTA, NOTA, NETA, TETA, porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups.

[0114] As used herein, the terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on a number of factors, including the range of normal doses for a given therapy, frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; and the route of administration. One of skill will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical or pharmaceutical composition, and depends on the route of administration. For example, a dosage form can be in a liquid form for nebulization, e.g., for inhalants, in a tablet or liquid, e.g., for oral delivery, or a saline solution, e.g., for injection.

[0115] As used herein, the term “administering” means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini- osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. The compounds of the invention can be administered alone or can be co-administered to the patient. Co-administration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation). The compositions of the present invention can be delivered orally,transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0116] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the antibodies provided herein suspended in diluents, such as water, saline, or PEG 400; (b) capsules, sachets, or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, com starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.

[0117] Pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized sepharose (TM), agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants).

[0118] As used herein, the term “adjuvant” refers to a compound that when administered in conjunction with the compositions provided herein including embodiments thereof, augments the composition’s immune response. Generally, adjuvants are non-toxic, have high purity, are degradable, and are stable.

[0119] Adjuvants can augment an immune response by several mechanisms including lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. The adjuvant increases the titer of induced antibodies and / or the binding affinity of induced antibodies relative to the situation if the immunogen were used alone. A variety of adjuvants can be used in combination with the agents provided herein including embodiments thereof, to elicit an immune response. Preferred adjuvants augment the intrinsic response to an immunogen without causing conformational changes in the immunogen that affect the qualitative form of the response. Preferred adjuvants include aluminum hydroxide and aluminum phosphate, 3 De-O-acylated monophosphoryl lipid A (MPL™) see GB 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Montana, now part of Corixa). Stimulon™ QS-21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree found in South America (see Kensil el al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); US Patent No. 5,057,540), (Aquila BioPharmaceuticals, Framingham, MA). Other adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute etal.,N. Engl. J. Med. 336, 86-91 (1997)), pluronic polymers, and killed mycobacteria. Another adjuvant is CpG (WO 98 / 40100). Adjuvants can be administered as a component of a therapeutic composition with an active agent or can be administered separately, before, concurrently with, or after administration of the therapeutic agent.

[0120] Other adjuvants contemplated for the invention are saponin adjuvants, such as Stimulon™ (QS-21, Aquila, Framingham, MA) or particles generated therefrom such as ISCOMs (immunostimulating complexes) and ISCOMATRIX. Other adjuvants include RC-529, GM-CSF and Complete Freund’s Adjuvant (CFA) and Incomplete Freund’s Adjuvant (IF A). Other adjuvants include cytokines, such as interleukins (e.g., IL-1 a and P peptides, IL-2, IL-4, IL-6, IL-12, IL-13, and IL-15), macrophage colony stimulating factor (M-CSF), granulocyte -macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), chemokines, such as MIPla and and RANTES. Another class of adjuvants is glycolipid analogues including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted in the sugar residue by an amino acid, as immuno-modulators or adjuvants (see US Pat. No. 4,855,283). Heat shock proteins, e.g., HSP70 and HSP90, may also be used as adjuvants.

[0121] Suitable formulations for rectal administration include, for example, suppositories, which consist of the packaged peptides or nucleic acids with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the compound of choice with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.

[0122] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally. Parenteral administration, oral administration, and intravenous administration are the preferred methods of administration. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

[0123] Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced by nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above.

[0124] The pharmaceutical preparation is preferably in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The composition can, if desired, also contain other compatible therapeutic agents.

[0125] The combined administration contemplates co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.

[0126] Effective doses of the compositions provided herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. However, a person of ordinary skill in the art would immediately recognize appropriate and / or equivalent doses looking at dosages of approved compositions for treating and preventing cancer for guidance.

[0127] As used herein, the term “pharmaceutically acceptable” is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration. As used herein, the terms “pharmaceutically acceptable” or “pharmacologically acceptable” refer to a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual in a formulation or composition without causing any unacceptable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0128] As used herein, the terms “pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer’s solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances, and the like., that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0129] As used herein, the term “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

[0130] As used herein, the term “preparation” is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0131] The pharmaceutical preparation is optionally in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packetedtablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The unit dosage form can be of a frozen dispersion.

[0132] The compositions of the present invention may additionally include components to provide sustained release and / or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides, and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). In embodiments, the formulations of the compositions of the present invention can be delivered by the use of liposomes that fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46: 1576-1587, 1989). The compositions of the present invention can also be delivered as nanoparticles.

[0133] Example 1. Identification of cow milk epitopes to characterize and quantify disease-specific T cells in allergic children.

[0134] Cow milk allergy (CMA) is the most prevalent food allergy in young children in the US. Current diagnostic tests are either unreliable (IgE, skin prick test), or resource-intensive (food challenges). The inventors determined if allergen-specific T cells in CMA patients have a distinct quality and or quantity that could potentially be used as a diagnostic marker. Starting from cow milk extract, T cell responses were mapped to a set of reactive epitopes that were compiled in a peptide pool. This pool induced cytokine responses in in vitro cultured cells between allergic and non-allergic individuals. Using single-cell RNA- Seq significant changes were detected in the transcriptional program of cow milk antigen-specific (CM+) T cells elicited by the pool in allergic vs. non-allergic individuals ex vivo. CM+ T cells from allergic individuals had increased percentages of FOXP3+ over FOXP3- cells. FOXP3+ cells are often equated with regulatory T cells (Tregs) that have suppressive activity, but CM+ FOXP3+ cells from allergic individuals showed significant expression of interferon-responsive genes and dysregulated chemokine receptor expression compared to non-allergic individuals, suggesting that these are not conventional Tregs. It was found that surface markers (CD25, CD 127, CCR7) can be used to quantify these CM+ FOXP3+ cells by a simple flow cytometry assay that showed increased percentages of these cells from CMA donorsin an independent cohort. Looking specifically for Th2 cells normally associated with allergic diseases, the inventors found a small population of CM+ Th2 cells that were only present in CMA donors and that had high expression of Th2 cytokines and pathogenic Th2 markers. Overall, these findings demonstrate that there are several differences in the phenotype of CM+ T cells with CMA and that the increase in CM+ FOXP3+ cells is a diagnostic marker of an allergic state. Such markers can be used to monitor natural disease outgrowth and / or the efficacy of immunotherapy.

[0135] Study population. Blood samples from 157 pediatric study participants were acquired from three participating centers; Johns Hopkins Hospital Allergy Clinic (Baltimore, MD) Rady Children’s Hospital (San Diego, CA) and University Hospital Southampton (Southampton, UK). The clinical phenotype of each study participant was assigned at each center based on multiple parameters including reaction history, dietary history, and food challenge information when available. Briefly, Cow milk Allergy (CMA) diagnosis was determined by symptomatic reactivity to raw and / or baked milk. Clinical signs included eczema, urticaria, upper or lower respiratory tract symptoms, gastrointestinal disturbances, rash, or oral symptoms. CMA severity diagnosis was determined by reactivity to raw milk and / or baked milk-containing food, where CMA-Mild refers to patients allergic only to raw milk and CMA-Severe refers to allergy to baked milk as well. Finally, the non-allergic participants were defined as consuming milk with no symptoms and no prior history of milk allergy.

[0136] PBMC isolation. Each study participant provided a sample between 1 mE and 10 mEs, depending on age at sampling time. PBMCs were isolated by density gradient centrifugation (Ficoll-Hypaque, Amerhsam Biosiences, Uppsala, Sweden) from the obtained blood, cryopreserved at a concentration between IxlO7and 2X107cells / mE in FBS (GeminiBio, Sacramento, CA) + 10% DMSO (Sigma-Aldrich, St. Eouis, MO), and shipped to Ea Jolla Institute for Immunology as previously described20.

[0137] Identification of IgG and / or IgE reactive antigens in cow milk using Two-Dimensional Difference Gel Electrophoresis (DIGE) and Immunoblotting. In order to identify antigens recognized by allergic individuals, commercial cow milk extract (Cat#XPF395D3A2.5, Greer Laboratories, Lenoir, NC, USA) was run using a two-dimensional (2D) difference gel electrophoresis (DIGE) by Applied Biomics (San Francisco, California, USA). A pool of plasma from 7 CM allergic pediatric donors was then used to probe the resulting gel for the identification of IgG and IgE antibody-binding proteins. Selected spots were cut out from the gel, and proteins contained in each spot were identified by mass spectrometry. Proteins containing amino acid sequences with >90% similarity were then assigned a specific protein ID as previously described19, resulting in the identification of 27 distinct proteins.

[0138] Peptide prediction and generation of peptide pools. Sequences of the 27 milk proteins identified were split into clusters of highly homologous proteins, and each cluster was separately aligned using the MEGA software tool with ClustalW. Stretches of fifteen amino acid residues overlapping by 10 amino acids were generated overlapping the sequence alignment. HLA class II binding predictions using the 7- allele method were performed as previously described using the standalone version of the IEDB class II binding prediction tool21and redundancy analysis to remove peptides with >70% of sequence identity was performed22. The final list of peptides was composed by 677 peptides. Peptides were ordered and purchasedfrom A&A (San Diego, CA, USA) and resuspended in DMSO after at 40mg / ml upon arrival. In order to generate the pools, lOul (400ug) of each peptide was pooled in a tube and lyophilized if more than 20 peptides are required. Finally, the lyophilized pools were resuspended to a final concentration per peptide of 2ug / ml. The 677 peptides were split into 32 peptide pools of ~20 peptides each, with peptides from the same protein put into the same pool as much as possible.

[0139] In Vitro Expansion of milk-specific T Cells. In order to screen the 32 pools, PBMCs were cultured with RPMI 1640 (Omega Scientific, Tarzana, CA, USA) supplemented with 5% human AB serum (Gemini Bio-Products, West Sacramento, CA, USA), 1% penicillin / streptomyscin (Omega Scientific), and 1% Glutamax (Gemini Bio-Products, West Sacramento, CA, USA) at a density of 2 x 106cells per mE in 24- well plates (Gennesee scientific, San Diego, CA, USA), in the presence of CM extract Cat#XPF395D3A2.5 (10 pg / mL)(Greer, Eenoir, NC). Cells were incubated at 37C in 5% CO2, IL-2 (10 U / mL; Prospec, Ness Ziona, Israel) was added every three days after initial antigenic stimulation. After 14 days, cells were harvested, washed, and screened for reactivity against the CM extract and CM peptide pools by FluoroSPOT. For the individual peptide deconvolution, the same procedure was conducted, however, on day 14, cells were tested for reactivity against the mesopools. The remaining cells were put back into culture, fed with IL-2, harvested at day 17, and screened for reactivity against the CM extract and the individual peptides from positive mesopools using FluoroSPOT. This procedure allowed the inventors to identify the individual reactive epitopes.

[0140] FluoroSpot. IL-5, IL-10, and IFNy production after CM extract, pool, or peptide stimulation was measured by Fluorospot assay as described in23. Briefly, in vitro expanded cells (1 x 105cells / well) were restimulated with the same extract as the original culture at 250 pg / mL, CM peptide pools (lOug / mL), peptides (lOug / mL), medium alone and PHA (10 pg / mL) as negative and positive controls, respectively. After 24 h at 37°C, cells were removed and the plates were incubated at room temperature with detection antibodies for IL-5 diluted at 1:200 (mAb 5A10-WASP; Mabtech), IFNy diluted at 1:200 (mAb 7-B6-1- BAM; Mabtech), and biotinylated IL-10 Ab at 2 pg / mL (Clone 12G8; Mabtech). After 2 h, plates were developed by adding fluorophore conjugates for IL-5 (anti-WASP-640; Mabtech), IFNy (anti-BAM-490; Mabtech), and IL-10 (SA-500; Mabtech) diluted at 1:200. Plates were then treated with Fluorescence enhancer-II (Mabtech) after 1 h for 15 min. Spot forming cells (SFC) were counted by computer-assisted image analysis (AID iSpot ELR07IFL reader; Strasberg, Germany).

[0141] Antigen Reactive T Cell Enrichment Assay (ARTE assay) and sorting of memory CD4 T cells for scRNA-Seq. In order to identify and isolate CM+ T cells based on Ml 11 peptide pool stimulation, FACS sorting of CD154+ and / or CD137+ memory CD4+ T cells from PBMC was conducted as previously described13, 24. Briefly, PBMC of 12 study participants were thawed and plated overnight at 5% CO2, 37° C using 24-well culture plates at a concentration of 10 x 106cells / mL in 1 mL of serum-free media for each experiment. Blocking CD40 antibody (1 pg / mL; Miltenyi Biotec) was added and cells were stimulated with DMSO (negative control) PHA (Positive control), and Mi l l peptide pool at a concentration of 2 pg / mL for 6 hours. The cells were then washed with PBS and incubated with a viability dye (eFluor506, eBioscience) and incubated for 10 minutes at room temperature. The viability dye was then inactivated bythe addition of PBS +20% FBS (GeminiBio, Sacramento, CA), washed with PBS and cells were surface incubated for 30 minutes with a cocktail containing Fcr blocking TruStain FX (Biolegend 422302), anti- CD4 (APCef780, clone RPA-T4, Thermo- Fisher), anti-CD3 (AF700, clone UCHT1, Thermo- Fisher), anti- CD8 / CD14 / CD19 / CD56 (V500, clones RPA-T8, M5E2, H1B19, NCAM16.2, all from BD), anti- CD45RA (eFluor450, clone HI1000, BD), anti- CCR7 (PerCy5.5, clone G043H7, BD), anti-CD154 (PE, 24-31, Biolegend), and anti-CD137 (APC, clone 4B4-1, Thermo- Fisher). During surface staining, each donor was hashtagged adding 2 ul of TotalSeq™-anti-human Hashtags 1 to 10, TotalSeq™-C0048 antihuman CD45, and TotalSeq™-C0391 anti-human CD45 Antibody (all from BioLegend). After surface staining, cells were resuspended at a concentration of 20 x 106cells / ml in MACS buffer and kept in ice until sorting. Memory CD4 T cells (TCM and TEM) expressing CD154 and / or CD137 (CM+) were FACS- sorted using a FACS Symphony S6 (BD) and to avoid cell losses, CM- CD154-CD137- cells were also sorted. In each tube, the CM- fraction of 3 donors were combined with the CM+ fraction of 3 different donors. All flow cytometry data were analyzed using FlowJo software (BD).

[0142] scRNA-Seq Library Preparation and Sequencing. For single cell RNA-seq assays (lOx Genomics), the maximum amount of CM+ memory T cells and 15,000 CM- cells was collected by sorting in low retention and sterile ice-cold 1.5 mL collection tubes with 500 pL of PBS and FBS in equal volume containing RNase inhibitor (1: 100). -50,000 sorted cells per tube were resuspended and loaded on the 10X Chromium Controller (10X Genomics). cDNA amplification and library preparation (10X v2 chemistry) were performed according to manufacturer's protocol and sequenced on a NovaSeq 6000 (Illumuina) to a depth of >25,000 reads / cell for GEX and >5,000 reads / cell for hashtag oligo libraries.

[0143] scRNA-Seq Analysis- Hashtag Demultiplexing, QC filtering, Clustering. Sequencing reads were aligned to the GRCh38 human reference genome using the multi pipeline in Cellranger (v5.0). Downstream analysis was performed using the package Seurat (v4.0.3; satijalab.org / seurat / index.html) in R (v4.1.0). To overcome donor-specific expression of individual TCR genes, TCRA / B / D / G genes were pulled from the gene expression matrix and counts were aggregated into a single gene feature for each. Due to the complex sorting scheme and unequal mix of CM+ / - cells in each sample, samples containing matching barcodes were merged prior to hashtag oligo demultiplexing to overcome barcode number imbalances. Demultiplexing was performed using MULTIseqDEMUX25. Cells called as doublets or negatives and those with high mitochondrial content (>8%) and / or low feature number (<200) were removed for downstream analysis. Samples were merged and normalized using SCTransform with parameters to regress out mitochondrial percentage and donor sex, to remove lowly expressed genes from normalization using “v2” regularization, and using 2000 variable features. Principal component analysis was performed using the RunPCA function with the top 50 PCs. RunUMAP and FindNeighbors were performed with 30 dimensions and a k.param of 30. Finally, FindClusters was applied and cluster specific markers were obtained using the FindAllMarkers function using default parameters. All visualization plots were produced in Seurat, ggplot2, or pheatmap.

[0144] scRNA-Seq Analysis- Differential Expression and Functional Enrichment. Differential gene expression analysis between groups was performed using the MAST algorithm26on the normalized RNAexpression matrix. A gene was considered to be significantly differentially expressed if it had an adjusted p-value < 0.05 and a logFC > 0.25. All functional enrichment analyses were performed using Metascape27with GO Biological Processes selected.

[0145] Proteomic and serologic analysis identified 27 protein antigens in cow milk (CM). A proteomic and serologic analysis method to systematically identify protein antigens in cow milk8, 18> 19> 28-32. A 2D electrophoresis gel was used to separate CM extract into its components based on pH and molecular weight. This was followed by immunoblotting with a pool of sera from 7 CM allergic donors for detection of IgE and IgG reactive proteins (FIG. 1A). The inventors identified spots reactive to either IgE, IgG or both. These spots were cut out and evaluated by mass spectrometry resulting in the identification of 27 proteins based on their sequence identified with proteins encoded in the cow genome (FIG. IB). Of those proteins, 8 were well-known CM allergens (Bos d 4, Bos d 5, Bos d 6, Bos d 7, Bos d 9, Bos d 10, Bos d l l, and Bos d 12), while 19 proteins were not previously described as CM antigens (FIG. IB).

[0146] Bioinformatics and T cell screening approach identified 9 proteins recognized by T cells in CM. To identify targets of T cell responses in the 27 antigens, 15-mer peptide sequences were generated overlapping by 10 residues that span these protein sequences, and filtered them for peptides predicted to frequently bind MHC class II molecules present in the general population21> 22. After redundancy removal, this resulted in 677 candidate peptides that were synthesized and assembled into 32 pools. To identify T cell reactive peptides, PBMCs from milk allergic pediatric donors were cultured for 14 days with CM extract and restimulated separately with the 32 peptide pools. Due to restricted sample volumes from pediatric patients, the screen was performed in a staggered fashion, with different sets of pools being screened in each patient. Reactivity was assessed using Fluorospot assays measuring the secretion of IL-5, IFN-g, and IL-10 from antigen-specific T cells. In an initial screen of 29 donors (17 non-allergic controls and 12 CM allergic (CMA)), 7 of the starting 32 pools resulted in production of IL-5, IFNg, and / or IL-10 in allergic individuals. The reactivity pattern between CMA and non-allergic donors was highly similar, but non-allergic donors gave overall weaker responses. The 7 reactive pools were then subdivided into 14 “mesopools” each of which contained peptides from single proteins. These 14 mesopools were tested for reactivity in an additional 24 non-allergic controls and 36 CMA donors, which showed reactivity for 10 mesopools containing 111 peptides from 9 proteins. The combined screening results for IL-5 production are summarized in FIG. 1C, which shows the percent of allergic individuals tested for a given protein that gave a positive response. Notably, seven of the nine reactive proteins were known IgE reactive allergens (7 / 8 tested) while only two were not (2 / 19 tested), suggesting that for CM there is a strong association between IgE and T cell antigen reactivity (p=0.00029, Fisher Exact test).

[0147] T cell reactivity to specific CM peptides is associated with dissimilarity to human milk homologs. The 10 peptide mesopools covering the 9 reactive antigens were deconvoluted to identify specific reactive peptides in individual donors. Donor samples that showed reactivity for a given peptide pool after 14-day culture were subsequently tested for reactivity to each individual peptide contained in that pool. Due to sample volume limitations, pools were deconvoluted in a limited number of individuals ranging from 2 to 5. This screen identified a total of 43 reactive peptides in individual donors.

[0148] The sequence similarity of the 43 reactive peptides was compared to their closest match in the human proteome. It was found that for a majority (93%) of the 43 reactive peptides, the closest human homolog had 3 or more mis-matches (FIG. ID). This was significantly different from the 634 non-reactive peptides from the screens where only 26% had 3 or more mis-matches (p = 2.6 x 10'19, Fisher exact test). Interestingly, the 68 non-reactive peptides from the 111 total peptides considered in the deconvolution also had a majority (76%) of 3 or more mis-matches (FIG. ID), which makes them more similar to the reactive peptides identified from the same antigens than the other non-reactive peptides. Overall, this strongly suggests that dissimilarity to human milk proteins is a driving factor of allergic T cell reactivity for the specific CM epitopes that were identified and the 9 reactive antigens from which they were derived.

[0149] Table 1 - Epitopes and Pools

[0150] Table 1 - Legend:

[0151] Table 2. Mesopool and Proteins.

[0152] Table 2. Legend:

[0153] Table 3. Megapools.

[0154] Table 3. Legend.

[0155] Comparison of T cell reactivity identified Mi l l pool as the most sensitive and specific stimulus for CMA patients. The inventors compiled 3 different “megapools” of peptides: 1) M43, containing the 43 peptides that elicited individual peptide responses, 2) Ml 11, containing all peptides from the 9 reactive antigens in the screen, and 3) M388, containing all 15-mer peptides overlapping the 7 reactive antigens having >15% responders (FIG. IE). The M388 pool was designed to evaluate if the exclusion of peptides not predicted to broadly bind HLA class II molecules in the screen may have led to lower reactivity. To assess the reactivity of the three pools, PBMCs were stimulated from an independent cohort (8 CMA, 11 non-allergic controls) with milk extract for 14 days, followed by a 24-hour stimulation with the defined megapools. The inventors found that the Ml 11 and M388 megapools resulted in increased reactivity across IL-5, IFNg, and IL-10 as compared to the M43 pool (FIG. IF). As the inventors did not see a substantial increase in reactivity in the more than three times larger M388 pool compared to Ml 11, it was concluded that the peptides predicted to promiscuously bind HLA class II molecules in Mi l l accounted for the majority of reactivity (FIG. IF). The Mi l l pool was additionally able to discriminate CM allergic disease severity, with mildly allergic donors tolerating baked milk products (CMA-M) vs. severely allergic donorswho do not (CMA-S), especially in terms of IFNg production (FIG. IF). This made the Mi l l pool the choice for subsequent studies.

[0156] scRNA-Seq of cow milk epitope specific CD4 T cells in pediatric milk allergy. To characterize milk allergen-specific CD4 T cells were used from the Ml 11 megapool to perform an antigen reactive T cell enrichment assay (ARTE) (FIG. 2A). PBMC from a total of 36 pediatric participants (12 non-milk allergic controls, 9 CMA-M, and 15 CMA-S) were stimulated with the Mi l l megapool for 6 hours followed by a sort for memory CD4+ T cells that were milk allergen specific, defined as memory CD4 T cells (CD3+CD4+CD45RA-) expressing CD154 and / or CD137 (Cow milk(CM)+). Mi l l stimulation resulted in significant induction of CM+ cells by flow cytometry. However, the inventors did not see significant differences in the frequencies of CM+ cells between individuals with different milk allergic disease status (FIG. 2B).

[0157] Given that T cells from CMA individuals differed from non-allergic individuals after a 14-day culture with Mi l l stimulus, it was hypothesized that there is a difference in phenotype of the CM+ cells immediately after stimulation. To assess this, the inventors determined the transcriptional profile of CM+ T cells using 10X scRNA-Seq from the same 36 participants following the ARTE assay (FIG. 2A). Next, 98,883 sequenced cells were retrieved from the CM+ and CM- (as a control) sorting fractions, which were clustered together using uniform manifold approximation (UMAP) (FIGS. 2C, 2D). The CM+ sort fraction clustered closely together with a vast majority of cells falling in clusters 3, 7 and 15 (FIGS. 2C, 2D). These clusters also showed the highest RNA expression of the sort markers TNFRSF9 (CD 137) and CD40LG (CD154) (FIG. 2E). Additionally, clusters 3, 7, and 15 were significantly enriched within the CM+ sort fraction compared to CM- (FIG. 2F). The remaining 13 clusters were composed of cells from the CM- fraction (FIGS. 2D, 2F). Re-clustering the CM- fraction showed that these cells were primarily made up of naive, central- and effector-memory, Tregs, and small clusters of gd T and activated T cells. No significant differences were observed within the CM-cell clusters between individuals with different allergic status. This demonstrated that the cell surface marker sorting approach separated activated antigen-specific cells (CM+) from non-activated cells into clusters with distinct transcriptional profiles, and that allergy- associated T cell populations are likely contained in this CM+ cell fraction.

[0158] Antigen-specific FOXP3+ cells are increased with severe milk allergy. To further characterize the CM+ cells, the analysis was focused on clusters 3, 7, and 15 (FIG. 2). FOXP3 expression decisively distinguished cluster 7 (FOXP3+) from clusters 3 and 15 (FOXP3-; FIG. 3A). The FOXP3- clusters had high expression of T helper (Th) activation markers CD40LG, NFKB1, and MIR155HG, it was next determined that these clusters were likely composed of antigen-specific Th cells (FIG. 3A). The FOXP3+ cluster 7 also had high expression of TIGIT and IL2RA, as well as activation markers SI 004, 6,10 and HLA- DRB1 (FIG. 3A). Examining the association of these FOXP3+ and FOXP3- cell clusters with allergic status of the donor, an increase in FOXP3+ (cluster 7) was identified in CMA-S donors compared to non-allergic controls (FIG. 3B). CMA-M individuals showed an intermediary distribution (FIG. 3B). Coincidingly, the ratio of CM+ FOXP3- to FOXP3+ cells was significantly decreased in CMA-S donors compared to controls where CMA-M again showed an intermediary phenotype (FIG. 3C).

[0159] Next, the inventors established an assay that could enumerate CM+ FOXP3+ cells by cell surface marker expression without requiring an intracellular stain as FOXP3 itself does. Such an assay has the potential to serve as a correlate of allergic status, which has potential diagnostic applications. The inventors selected gene markers based on availability of antibodies and a significant difference in expression between the CM+ FOXP3+ and FOXP3- clusters. This identified CCR7, IL7R (CD 127), IL2RA (CD25), HLA- DRB1, and TIGIT (FIG. 3D). These genes were expressed in more than 30% of cells in their respective cluster and the difference in percentage to expression of cells in the opposing cluster was more than 20%. To test this assay, the same gating strategy described above was used, and profiled the indicated surface markers on CM+ cells in an independent cohort of milk allergic donors after ARTE with the Mi l l megapool. Decreased percentages of CCR7+CD 127+ cells were found (proxy for FOXP3-) and coinciding increased CCR7-CD127- populations (proxy for FOXP3+) after Mi l l stimulation in CMA-S donors compared to controls (FIGS. 3E-3G). Further, the percentage of CD25+CD127- cells (proxy for FOXP3+) was increased in CMA-S donors (FIGS. 3H, 31). Next, HLA-DR and TIGIT were also tested as surface markers, and while some donors had similar trends compared to CD25+CD127-, did not as clearly differentiate a distinct population of cells in this assay. These findings demonstrate that the surface marker expression assays in FIG. 3E-I can be used to differentiate patients’ allergic status based on their T cell response phenotype to CM peptides.

[0160] CM+ FOXP3+ cells have a strong interferon-responsive signature with severe milk allergy. To better characterize the transcriptional profile of the CM+ fractions, the inventors first re-clustered the antigen-specific FOXP3+ (C7; FIG. 3 A) cells which resulted in 3 transcriptionally distinct clusters (FIG. 4A). Cluster CO was defined by S100A4 and S100A10, Cl by TNFRSF9 (CD137) and MIR155HG, and C2 by interferon responsive genes IFIT3 and STAT1 (FIG. 4A). It was found that while CO and Cl were fairly evenly distributed between control and CMA cells, a majority of cells in the C2 interferon cluster were from CMA-S donors (FIG. 4B). This trend was also visible on a per donor basis, but did not reach statistical significance. The inventors also performed a differential analysis on total CM+ FOXP3+ cells between CMA-S and control donors. Going with the cluster-based findings, a number of differentially expressed genes (DEG) upregulated with CMA-S that were significantly enriched for genes annotated with GO terms ‘defense response to virus’ and ‘type-I interferon production’ including STAT1, ERAP2, and GBP1 (FIG. 4C) were identified. In addition, allergy-associated FOXP3+ cells (Both CMA-S and CMA-M) were found that had high expression of inflammatory and activation markers TL32 and CD247 compared to controls (FIG. 4D). This was also accompanied by shifts in chemokine receptor expression where allergy-associated FOXP3+ cells had higher expression of CXCR4. but lower expression of CCR6, CXCR6. and CCR8 (FIG. 4D).

[0161] To determine whether these allergy-associated CM+ FOXP3+ signatures were restricted to antigen-specific cells and not all memory CD4 FOXP3+ cells, the three CM+ FOXP3+ clusters were integrated with the CM- FOXP3+ cluster 3 (FIG. 4E). It was found that the CM+ FOXP3+ cells are transcriptionally distinct from CM- FOXP3+ cells, where Cl and C2 are more separated from the CM- cells in UMAP space (FIG. 4E). FOXP3 expression was found to be higher in the CM+ clusters, pointingto an activation-associated role. Importantly, the interferon signature associated with CM+ F0XP3+ cluster C2 and CMA-S donors was not present in the CM- FOXP3+ population (FIG. 4F). This was also true of the cytokine and chemokine receptor expression, which was not apparent in the CM- FOXP3+ cells. These transcriptional differences in allergy-associated CM+ FOXP3+ cells compared to resting CM- FOXP3+ cells support the notion that there are also functional differences in the allergy-associated FOXP3+ cells compared to conventional FOXP3+ Tregs.

[0162] Pathogenic Th2 signature in CM+ FOXP3- cells from CMA donors. As conventionally Th2 cells are associated with allergy, the inventors assessed differences within the CM+ Th cells between CMA and non-allergic donors at higher resolution. Thus, the inventors re-clustered the cells from CM+ FOXP3- clusters 3 and 15 (FIG. 2D and FIG. 5 A), resulting in seven clusters, each with a unique gene expression signature (FIG. 5B). Antigen-specific Thl7 (CCL20. RORC) and Th2 (GATA3, IL17RB) clusters were identified (FIG. 5B). The other clusters had various activation-associated signatures including expression of MIR155HG, NFKB1, TNI-', and CD69 (FIG. 5B). While it was noted that trending decreases in Activated clusters 1 and 4 with CMA-S (back-calculated from total CM+), the inventors did not see major cluster redistribution with allergy in the CM+ FOXP3- cells. Next, the inventors examined more closely the expression of Th2 cytokines (IL4, IL5, IL9, IL13) and pathogenic Th2 markers (IL1RL1, PTGS2, PPARG). It was discovered that while the Th2 cluster had high expression of canonical Th2 markers (GATA3, IL17RB FIG. 5B), there was expression of the more functional and pathogenic Th2 markers at the edge of Activated cluster 3 (FIG. 5C). Further examining these markers, it was found that the expression, while limited to few cells, almost exclusively resided within cells from CMA-S and CMA-M donors (FIG. 5D). More specifically, a small population of CMA-S cells from Activated cluster 3 expressed high levels of II.4. IL5, IL9, IL1RL1, PTGS2, and PPARG (FIG. 5D). Finally, the inventors examined differentially expressed genes in this total CM+ FOXP3- population and found significant upregulation of genes involved in T cell activation, response to IFNy, and migration with CMA-S compared to controls (FIG. 5E). These findings show that there are transcriptional differences between CM+ FOXP3- cells associated with CMA status, reflected in a small subset of Th2 cells that expressed a high level of Th2 cytokine and other pathogenic markers.

[0163] Using the present invention it was possible to identify cow milk (CM) reactive T cells in allergic individuals and to characterize their disease-specific phenotypes. This was done in two distinct steps; first a pool of CM derived peptide epitopes was identified that contained the specific molecular targets of T cell responses against CM in allergic individuals. Second, that pool was used to target CM-specific T cells and characterize disease-associated differences using single cell RNA-Seq and flow cytometry. Together this revealed a set of phenotypic characteristics of the T cell response to CM in allergic individuals that have potential clinical applications as diagnostics, opened hypothesis on the nature of T cells driving disease pathology, and provided a set of reagents that can be used to directly test these hypotheses in future studies.

[0164] The methods used to identify T cell epitopes have been well established8, 18, 19, 28-32. The inventors started from total cow milk extract and identified 27 immune reactive proteins using mass spectrometry. Screening those proteins for peptides with T cell reactivity revealed a tight focus of the response on 9antigens, 7 of which were known IgE reactive, suggesting that there is a strong association of T cell and IgE reactivity in milk allergy. Comparing the sequences of reactive peptides in CM with their human homologs revealed that reactivity had pronounced requirement for dissimilarity to the host. While this is expected based on the deletion of self-reactive T cells during thymic development34, most previous epitope mapping studies found relatively small effect sizes for this non-selection35'37. The difference with the results herein is that CM contains many peptides that are highly identical to human proteins, and none of these were reactive, making the effect of self-tolerance much more apparent. Notably, it was found that the sequence similarity cutoff the inventors previously established for cross-reactive T cell responses in allergy (<3 residue difference in a 15 -mer peptide)38was also predictive for tolerance to human self-peptides.

[0165] Compared to full cow milk extract, the Ml 11 pool of peptides from antigens with demonstrated T cell reactivity is highly enriched for peptides covering the components of cow milk that differ from their human homolog, and that are also predicted to bind multiple HLA class II molecules. This provides a molecularly well-defined stimulus of the allergy relevant T cell response to CM.

[0166] Using the Ml 11 pool to characterize CM specific T cells using single cell transcriptomics, it was found that the CM+ FOXP3+ cells were increased significantly in donors with severe CMA. This was initially counterintuitive as FOXP3+ cells are normally termed Tregs, associated with an anti-inflammatory phenotype, and in the past, their increase has been associated with disease remission39, 40. However, there have been many previous reports of FOXP3+ cells that do not have a suppressive phenotype. While FOXP3 is a hallmark of Tregs in resting cells, it is also an activation marker studies for T effector cells with no regulatory or suppressor function41'43. The functional role of antigen-specific FOXP3+ cells is also up for debate. Studies on aero-antigens (HDM, plant pollen, fungi) and peanut allergy have characterized CD137+CD154- memory CD4 T cells as antigen-reactive Tregs13, 24, 33. However, in the case of food allergens, these cells were rare, more variable between donors, and not shown to increase over the course of oral immunotherapy (OIT) for peanut allergy.24, 33As it was found that the expression of FOXP3 and CD25( / / .2 / ?4) to be higher on the CM+ FOXP3+ cells than the CM- FOXP3+ cells, the increase in this population was associated with CMA could be a differentially activated subset of effector cells.

[0167] To further support this, the inventors looked at the transcriptional profiles of these cells and found that while the CM+ FOXP3+ cells do express some “Treg” markers (FOXP3, IL2RA(CD25), TIGIT) they also express other markers of activation (IL32, HLA-DRB1, S100A6, S100A10, ISG20, TNFRSF1B). Even more interesting, is that there is a viral response / interferon signature associated with CMA in these cells that was not found in CM- FOXP3+ cells. Studies have found interferon signatures in antigen specific cells in the setting of HDM allergy and asthma13, and there have been connections between interferon signaling and T cell function without active viral infection44. The CM+ FOXP3+ cells from CMA donors also had higher expression of inflammatory cytokine IL32 and TCR activation associated ('1)247. and could be differentiated by expression of chemokine receptors (increased CXCR4, decreased CCR6, CXCR6, and CCR8). These findings show that even though the CM+ FOXP3+ population is found in both CMA and control donors, there may still be functional response and migration issues with these cells that could exacerbate CMA disease.

[0168] As CMA and other allergies have been associated with antigen specific Th2 cells1, 2, 7, the inventors looked more closely at the seven clusters associated with the CM+ FOXP3- cells. Recent studies have identified Th2 subtypes associated with allergy (Th2A, peTh2)12, 15, 16. From these data, the inventors were able to identify a Th2 cluster that had high expression of GA / A 3 and IL17RB. However, upon closer examination, there was a small population of cells within a different cluster (Activated 3) that had very high expression of Th2 cytokines IL13, IL5, IL4, and IL9, along with pathogenic Th2 markers IL1RL1, PTGS2, and PPARG. It was found that this very small population of cells were coming from CMA-S donors.

[0169] The differences in T cell responses observed between allergic and non-allergic individuals were most pronounced in the frequency of FOXP3+ CM+ cells, which can be used to directly quantify the T cell response.

[0170] The inventors wanted to avoid intracellular staining that is required to detect F OXP3 , as that makes the assay more complex to perform and complicates downstream use of the identified cell materials for RNA-Seq. Using highly upregulated and downregulated markers from CM+ FOXP3+ compared to the CM+ FOXP3- clusters, it was found that the inventors could differentiate these cell populations by a relatively simple (9 color) flow cytometry panel. And in fact, an increase in CD25+CD127- and a corresponding decrease in CD127+CCR7+ CM+ cells differentiated CMA from non-allergic individuals in an independent cohort. This assay of total PBMC stimulation with the Ml 11 peptide pool for 6 hours followed by flow cytometry could be used to assess the disease status of CMA patients, and can be used to track natural outgrowth of disease and / or tolerance induction through immunotherapy.

[0171] The epitope megapool can be used to characterize and quantify antigen-specific T cells from CMA donors. It was found that specific transcriptional programs associated with CMA that point to an antigenspecific, allergy-associated FOXP3+ T cell population that is distinct from conventional Tregs and has a characteristic interferon-associated signature.

[0172] Embodiments.

[0173] As embodied and broadly described herein, an aspect of the present disclosure relates to a composition comprising: one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more or more peptides comprising, consisting of, or consisting essentially of amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof. In one aspect, one or more peptides or proteins comprises, or wherein the fusion protein comprises two or more or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence,portion, homologue, variant or derivative thereof. In any of the embodiments hereinabove, the amino acid sequence is selected from a cow milk T cell epitope selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In any of the embodiments hereinabove, the composition comprises one or more cow milk peptides amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: I to 1081); or apool of2 or more peptides selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof. In any of the embodiments hereinabove, the peptide or protein comprises a cow milk T cell epitope. In any of the embodiments hereinabove, the one or more peptides or proteins comprises a cow milk CD8+ or CD4+ T cell epitope. In any of the embodiments hereinabove, the one or more peptides or proteins has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids. In any of the embodiments hereinabove, the one or more peptides or proteins elicits, stimulates, induces, promotes, increases, or enhances a regulatory T cell response to a cow milk protein or peptide. In any of the embodiments hereinabove, the one or more peptides or proteins that elicits, stimulates, induces, promotes, increases or enhances the regulatory T cell response to the cow milk protein or peptide, or a variant, homologue, derivative or subsequence thereof. In any of the embodiments hereinabove, the composition further comprises the one or more peptides or proteins formulated into an immunoregulatory formulation with an adjuvant. In any of the embodiments hereinabove, the adjuvant is selected from the group consisting of aluminum hydroxide, calcium phosphate, microcrystalline tyrosine (MCT), and monophosphoryl lipid A (MPL). In any of the embodiments hereinabove, the composition further comprises a downregulator of immune response.

[0174] As embodied and broadly described herein, an aspect of the present disclosure relates to a composition comprising monomers or multimers of: peptides or proteins comprising, consisting of, or consisting essentially of: one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3,concatemers, subsequences, portions, homologues, variants or derivatives thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

[0175] As embodied and broadly described herein, an aspect of the present disclosure relates to a composition comprising one or more peptide-major histocompatibility complex (MHC) monomers or multimers, wherein the peptide-MHC monomer or multimer comprises a peptide comprising, consistingof, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), in a groove of the MHC monomer or multimer.

[0176] As embodied and broadly described herein, an aspect of the present disclosure relates to a composition comprising: one or more peptides or proteins comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); a pool of 2 or more peptides selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof. In one aspect, the one or more peptides or proteins comprises, or wherein the fusion protein comprises, 2 or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof. In any of the embodiments hereinabove, the protein or peptide comprises cow milk T cell epitope. In any of the embodiments hereinabove, the one or more peptides or proteins comprises a cow milk CD8+ or CD4+ T cell epitope. In any of the embodiments hereinabove, the one or more peptides or proteins has a length from about 9-15, 15-20, 20- 25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids. In any of the embodiments hereinabove, the one or more peptides or proteins elicits, stimulates, induces, promotes, increases or enhances a regulatory T cell response to comprises cow milk peptides or proteins. In any of the embodiments hereinabove, the one or more peptides or proteins that elicits, stimulates, induces, promotes, increases or enhances the regulatory T cell response to cow milk protein(s) or peptide(s), or a variant, homologue, derivative or subsequence thereof. In any of the embodiments hereinabove, the composition further comprises formulating the one or more peptides or proteins into an immunogenic formulation with an adjuvant. In any of the embodiments hereinabove, the adjuvant is selected from the group consisting of adjuvant is selected from the group consisting of aluminum hydroxide, calcium phosphate, microcrystalline tyrosine (MCT), and monophosphoryl lipid A (MPL). In any of the embodiments hereinabove, the composition further comprises an immunoregulator of immune response.

[0177] As embodied and broadly described herein, an aspect of the present disclosure relates to a composition comprising monomers or multimers of: one or more peptides or proteins comprising, consisting of, or consisting essentially of: one or more cow milk amino acid sequences selected from any one ofthose sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: I to 1081), concatemers, subsequences, portions, homologues, variants or derivatives thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth inTables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

[0178] As embodied and broadly described herein, an aspect of the present disclosure relates to a composition comprising one or more peptide-major histocompatibility complex (MHC) monomers or multimers, wherein the peptide-MHC monomer or multimer comprises a peptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), in a groove of the (MHC) monomer or multimer.

[0179] As embodied and broadly described herein, an aspect of the present disclosure relates to a method for detecting the presence of one or more cow milk antigens, comprising: providing one or more proteins or peptides for detection of an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells; contacting a biological sample suspected of having cow milk antigen-specific T- cells to one or more proteins or peptides for detection; and detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample, wherein the one or more proteins or peptides for detection comprise one or more amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or comprise a pool of 2 or more or more amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In one aspect, detecting the amount or a relative amount of, and / or activity of antigen-specific T-cells comprises one or more steps of identification or detection of the antigen-specific T-cells and measuring the amount of the antigen-specific T-cells. In any of the embodiments hereinabove, the one or more peptides or proteins comprises 2 or more amino acid sequences selected from those set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In any of the embodiments hereinabove, the detecting the amount or a relative amount of, and / or activity of antigen-specific T-cells comprises indirect detection and / or direct detection. In any of the embodiments hereinabove, the method of detecting an immune response to the one or more cow milk antigens comprises the following steps: providing an MHC monomer or an MHC multimer; contacting a population T-cells to the MHC monomer or MHC multimer; and measuring the number, activity or state of T-cells specific for the MHC monomer or MHC multimer. In any of the embodiments hereinabove, the MHC monomer or MHC multimer comprises a protein or peptide of cow milk. In any of the embodiments hereinabove, the protein or peptide comprises a CD8+ or CD4+ T cell epitope. In any of the embodiments hereinabove, the protein or peptide has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, SO- 75 or 75-100 amino acids. In any of the embodiments hereinabove, the proteins or peptides comprise 2 or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof. In any of the embodiments hereinabove, the method further comprises detecting the presence or amount of the one or more peptides in a biological sample, or a response thereto, which is diagnostic of a cow milk allergy. In any of the embodiments hereinabove, the detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample comprises measuring one or more of a cytokine or lymphokine secretion assay, T cell proliferation, immunoprecipitation, immunoassay, ELISA, radioimmunoassay, immunofluorescence assay, Western Blot, FACS analysis, a competitiveimmunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay, a reporter assay, a luciferase assay, a microarray, a surface plasmon resonance detector, a florescence resonance energy transfer, immunocytochemistry, or a cell mediated assay, or a cytokine proliferation assay. In any of the embodiments hereinabove, the method further comprises administering a treatment comprising the composition described hereinabove to the subject from which the biological sample was drawn that increases the amount or relative amount of, and / or activity of the antigenspecific T-cells.

[0180] As embodied and broadly described herein, an aspect of the present disclosure relates to a method for detecting the presence of an allergy to cow milk antigens, comprising: providing one or more proteins or peptides for detection of an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells; contacting a biological sample suspected of having cow milk-specific T-cells to one or more proteins or peptides for detection; and detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample, wherein the one or more proteins or peptides for detection comprise one or more amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or comprise a pool of 2 or more amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In one aspect, the detecting the amount or a relative amount of, and / or activity of antigen-specific T-cells comprises one or more steps of identification or detection of the antigen-specific T-cells and measuring the amount of the antigen-specific T-cells. In any of the embodiments hereinabove, the one or more peptides or proteins comprises 2 or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In any of the embodiments hereinabove, the detecting the amount or a relative amount of, and / or activity of antigen-specific T-cells comprises indirect detection and / or direct detection. In any of the embodiments hereinabove, the method of detecting an immune response to one or more cow milk antigens comprises the following steps: providing an MHC monomer or an MHC multimer; contacting a population T-cells to the MHC monomer or MHC multimer; and measuring the number, activity or state of T-cells specific for the MHC monomer or MHC multimer. In any of the embodiments hereinabove, the MHC monomer or MHC multimer comprises a protein or peptide of cow milk. In any of the embodiments hereinabove, the protein or peptide comprises a B. pertussis CD8+ or CD4+ T cell epitope. In any of the embodiments hereinabove, the protein or peptide has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids. In any of the embodiments hereinabove, the proteins or peptides comprise 2 or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant, or derivative thereof. In any of the embodiments hereinabove, the method further comprises detecting the presence or amount of the one or more peptides in a biological sample, or a response thereto, which is diagnostic of an allergy to cow milk. In any of the embodiments hereinabove, the detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample comprises measuring one or more of a cytokine or lymphokine secretion assay, T cell proliferation, immunoprecipitation, immunoassay, ELISA, radioimmunoassay,immunofluorescence assay, Western Blot, FACS analysis, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay, a reporter assay, a luciferase assay, a microarray, a surface plasmon resonance detector, a florescence resonance energy transfer, immunocytochemistry, or a cell mediated assay, or a cytokine proliferation assay. In any of the embodiments hereinabove, the method further comprises administering a treatment comprising the composition described hereinabove to the subject from which the biological sample was drawn that increases the amount or relative amount of, and / or activity of the antigen-specific T-cells.

[0181] As embodied and broadly described herein, an aspect of the present disclosure relates to a method detecting an allergy to cow milk antigens in a subject, the method comprising, consisting of, or consisting essentially of: contacting a biological sample from a subject with a composition described hereinabove; and determining if the composition elicits an immune response from the contacted cells, wherein the presence of an immune response indicates that the subject has an allergy to cow milk antigens. In one aspect, the sample comprises T cells. In any of the embodiments hereinabove, the response comprises inducing, increasing, promoting, or stimulating a regulatory activity of T cells. In any of the embodiments hereinabove, the T cells are CD8+ or CD4+ T cells. In any of the embodiments hereinabove, the method comprises determining whether the subject has been exposed to the cow milk antigens more than once by determining if the subject elicits a secondary T cell immune response profile that is different from a primary T cell immune response profile. In any of the embodiments hereinabove, the method further comprises diagnosing an allergy to cow milk in a subject, the method comprising contacting a biological sample from a subject with a composition described hereinabove, and determining if the composition elicits a T or B cell immune response, wherein the T cell immune response identifies that the subject is allergic to cow milk. In any of the embodiments hereinabove, the method is conducted three or more days following the date of exposure to cow milk.

[0182] As embodied and broadly described herein, an aspect of the present disclosure relates to a method detecting a cow milk allergy in a subject, the method comprising, consisting of, or consisting essentially of: contacting a biological sample from a subject with a composition described hereinabove; and determining if the composition elicits an immune response from the contacted cells, wherein the presence of an immune response indicates that the subject has an allergy to cow milk. In one aspect, the sample comprises T cells. In any of the embodiments hereinabove, the response comprises inducing, increasing, promoting or stimulating a regulatory activity of T cells. In any of the embodiments hereinabove, the T cells are CD8+ or CD4+ T cells. In any of the embodiments hereinabove, the method comprises determining whether the subj ect has been exposed to cow milk more than once by determining if the subj ect elicits a secondary T cell immune response profile that is different from a primary T or B cell immune response profile. In any of the embodiments hereinabove, the method further comprises diagnosing an allergy to cow milk in a subject, the method comprising contacting a biological sample from a subject with a composition described hereinabove; and determining if the composition elicits a T or B cell immune response, wherein the T or B cell immune response identifies that the subject has an allergy to cow milk.In any of the embodiments hereinabove, the method is conducted three or more days following the date of suspected exposure to cow milk.

[0183] As embodied and broadly described herein, an aspect of the present disclosure relates to a kit for the detection of an allergy to cow milk in a subject comprising, consisting of or consisting essentially of: one or more T cells that specifically detect the presence of: one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; or a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more or more peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In one aspect, the one or more amino acid sequences are selected from a cow milk T cell epitope set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In any of the embodiments hereinabove, the composition comprises: one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In any of the embodiments hereinabove, the fusion protein has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids. In any of the embodiments hereinabove, the kit includes instruction for a diagnostic method, a process, a composition, a product, a service or component part thereof for the detection of an allergy to cow milk. In any of the embodiments hereinabove, the kit includes reagents for detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample comprises measuring one or more of a cytokine or lymphokine secretion assay, T cell proliferation, immunoprecipitation, immunoassay, ELISA, radioimmunoassay, immunofluorescence assay, Western Blot, FACS analysis, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay, a reporter assay, a luciferase assay, a microarray, a surface plasmon resonance detector, a florescence resonance energy transfer, immunocytochemistry, or a cell mediated assay, or a cytokine proliferation assay. In any of the embodiments hereinabove, the kit includes reagents for determining a Human Leukocyte Antigen (HLA) profile of a subject, and selecting peptides that are presented by the HLA profile of the subject for detecting an immune response to cow milk.

[0184] As embodied and broadly described herein, an aspect of the present disclosure relates to a kit for the detection of an allergy to cow milk in a subject comprising, consisting of or consisting essentially of: one or more T cells that specifically detect the presence of: one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from the amino acid sequences set forth in those sequencesset forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In one aspect, the one or more amino acid sequences is selected from one or more cow milk CD4 T cell epitopes selected from any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or both. In any of the embodiments hereinabove, the amino acid sequence comprises one or more cow milk CD8+ or CD4+ T cell epitopes. In any of the embodiments hereinabove, the fusion protein has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids. In any of the embodiments hereinabove, the kit includes instruction for a diagnostic method, a process, a composition, a product, a service or component part thereof for the detection of an allergy to cow milk. In any of the embodiments hereinabove, the kit includes reagents for detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample comprises measuring one or more of a cytokine or lymphokine secretion assay, T cell proliferation, immunoprecipitation, immunoassay, ELISA, radioimmunoassay, immunofluorescence assay, Western Blot, FACS analysis, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay, a reporter assay, a luciferase assay, a microarray, a surface plasmon resonance detector, a florescence resonance energy transfer, immunocytochemistry, or a cell mediated assay, or a cytokine proliferation assay. In any of the embodiments hereinabove, the kit includes reagents for determining a Human Leukocyte Antigen (HLA) profile of a subject, and selecting peptides that are presented by the HLA profile of the subject for detecting an immune response to cow milk.

[0185] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of stimulating, inducing, promoting, increasing, or enhancing an immunoregulatory response against one or more cow milk antigens in a subject, comprising: administering a composition described hereinabove, in an amount sufficient to stimulate, induce, promote, increase, or enhance an immune response against the cow milk in the subject. In one aspect, the immunoregulatory immune response provides the subject with protection against cow milk allergies, or one or more physiological conditions, disorders, illnesses, diseases or symptoms caused by or associated with cow milk allergies. In any of the embodiments hereinabove, the immune response is specific to: one or more cow milk peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

[0186] A method of stimulating, inducing, promoting, increasing, or enhancing an immunoregulatory response against cow milk in a subject, comprising: administering a composition described hereinabove, in an amount sufficient to stimulate, induce, promote, increase, or enhance an immune response against cow milk in the subject. In one aspect, the immunoregulatory response provides the subject with protection against cow milk, or one or more physiological conditions, disorders, illnesses, diseases or symptoms caused by or associated with cow milk allergies. In any of the embodiments hereinabove, the immunoregulatory response is specific to: one or more cow milk peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

[0187] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of stimulating, inducing, promoting, increasing, or enhancing a regulatory T cell response in a subject with an allergy to one or more cow milk antigens, comprising: administering to a subject an amount of a protein or peptide or a polynucleotide that expresses the protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence of two or more cow milk protein or peptide, or a variant, homologue, derivative or subsequence thereof, wherein the protein or peptide comprises at least two peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081)or a subsequence, portion, homologue, variant or derivative thereof, in an amount sufficient to stimulate, induce, promote, increase, or enhance an immunoregulatory immune response to one or more cow milk antigens in the subject. In one aspect, the immune response provides the subject with protection against an allergy to cow milk, or one or more physiological conditions, disorders, illnesses, diseases or symptoms caused by an allergy to cow milk.

[0188] A method of treating, preventing, or immunizing a subject against cow milk allergies, comprising administering to a subject an amount of a protein, peptide or a polynucleotide that expresses the protein or peptide comprising, consisting of, or consisting essentially of an amino acid sequence of a cow milk protein or peptide, or a variant, homologue, derivative or subsequence thereof, wherein the protein or peptide comprises at least one amino acid sequence selected from any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081)or a subsequence, portion, homologue, variant or derivative thereof, in an amount sufficient to treat, prevent, or immunize the subject to prevent the allergy to cow milk, wherein the protein or peptide comprises or consists of cow milk T cell epitope that elicits, stimulates, induces, promotes, increases, or enhances an anti- cow milk regulatory T cell immune response. In one aspect, the one or more amino acid sequences are selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In any of the embodiments hereinabove, the cow milk T cell response is a CD8+, a CD4+ T cell response, or both. In any of the embodiments hereinabove, the subject is a mammal or a human. In any of the embodiments hereinabove, the method reduces one or more adverse physiological conditions, disorders, illness, diseases, symptoms or complications caused by or associated with cow milk allergies. In any of the embodiments hereinabove, the method improves one or more adverse physiological conditions, disorders, illness, diseases, symptoms or complications caused by or associated with cow milk allergies. In any of the embodiments hereinabove, the symptom is fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea or vomiting, or diarrhea. In any of the embodiments hereinabove, the method reduces or inhibits susceptibility to cow milk allergies. In any of the embodiments hereinabove, the protein or peptide, or a subsequence, portion, homologue, variant or derivative thereof, is administered prior to, substantially contemporaneously with or following exposure to cow milk. In any of the embodiments hereinabove, a plurality of cow milk T cellepitopes are administered prior to, substantially contemporaneously with or following exposure to cow milk. In any of the embodiments hereinabove, the protein or peptide, or a subsequence, portion, homologue, variant or derivative thereof is administered within 2-72 hours, 2-48 hours, 4-24 hours, 4-18 hours, or 6- 12 hours after a symptom of cow milk allergy. In any of the embodiments hereinabove, the protein or peptide, or a subsequence, portion, homologue, variant or derivative thereof is administered prior to exposure to cow milk. In any of the embodiments hereinabove, the method further comprises administering a modulator of immune response prior to, substantially contemporaneously with or following the administration to the subject of an amount of a protein or peptide.

[0189] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of treating, preventing, or immunizing a subject against cow milk allergies, comprising administering to a subject the composition described hereinabove in an amount sufficient to treat or prevent the cow milk allergy. In any of the embodiments hereinabove, the method reduces one or more adverse physiological conditions, disorders, illness, diseases, symptoms or complications caused by cow milk allergy. In any of the embodiments hereinabove, the method improves one or more adverse physiological conditions, disorders, illness, diseases, symptoms or complications caused by or associated with cow milk allergies. In any of the embodiments hereinabove, the method reduces or inhibits susceptibility to cow milk allergies. In any of the embodiments hereinabove, the composition is administered prior to, substantially contemporaneously with or following exposure to cow milk allergens. In any of the embodiments hereinabove, the composition is administered prior to, substantially contemporaneously with or following exposure to cow milk allergens. In any of the embodiments hereinabove, the composition is administered within 2-72 hours, 2-48 hours, 4-24 hours, 4-18 hours, or 6-12 hours after a symptom of cow milk allergies. In any of the embodiments hereinabove, the composition is administered prior to exposure to cow milk.

[0190] As embodied and broadly described herein, an aspect of the present disclosure relates to a peptide or peptides that are immunoprevalent or immunodominant in cow milk obtained by a method comprising, consisting of, or consisting essentially of: obtaining an amino acid sequence of one or more cow milk proteins; determining one or more sets of overlapping peptides spanning one or more cow milk antigen using unbiased selection; synthesizing one or more pools of cow milk peptides comprising the one or more sets of overlapping peptides; combining the one or more pools of cow milk peptides with Class I major histocompatibility proteins (MHC), Class II MHC, or both Class I and Class II MHC to form peptide-MHC complexes; contacting the peptide-MHC complexes with T cells from subjects exposed to the cow milk; determining which pools triggered cytokine release by the T cells; and deconvoluting from the pool of peptides that elicited cytokine release by the T cells, which peptide or peptides are immunoprevalent or immunodominant in the pool. In one aspect, the immunodominant peptides are selected from 1, 2 or more peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In any of the embodiments hereinabove, the immunodominant peptides are selected from 1, 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

[0191] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of selecting an immunoprevalent or immunodominant peptide or protein of cow milk comprising, consisting of, or consisting essentially of: obtaining an amino acid sequence of the cow milk; determining one or more sets of overlapping peptides spanning one or more cow milk antigen using unbiased selection; synthesizing one or more pools of cow milk peptides comprising the one or more sets of overlapping peptides; combining the one or more pools of cow milk peptides with Class I major histocompatibility proteins (MHC), Class II MHC, or both Class I and Class II MHC to form peptide-MHC complexes; contacting the peptide-MHC complexes with T cells from subjects exposed to cow milk; determining which pools triggered cytokine release by the T cells; and deconvoluting from the pool of peptides that elicited cytokine release by the T cells, which peptide or peptides are immunoprevalent or immunodominant in the pool. In one aspect, the immunodominant peptides are selected from 1, 2 or more peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In any of the embodiments hereinabove, the immunodominant peptides are selected from 1, 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: I to 1081).

[0192] As embodied and broadly described herein, an aspect of the present disclosure relates to a polynucleotide that expresses one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more or more peptides comprising, consisting of, or consisting essentially of amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

[0193] A vector that comprises the polynucleotide that expresses one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more or more peptides comprising, consisting of, or consisting essentially of amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In one aspect, the vector is a bacterial vector.

[0194] A host cell that comprises the vector that expresses one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more or more peptides comprising, consisting of, or consisting essentially of amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

[0195] A polynucleotide that expresses: one or more peptides or proteins comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables l, 2, and 3 (SEQ ID NOS: I to 1081); or apool of2 or more peptides selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

[0196] As embodied and broadly described herein, an aspect of the present disclosure relates to a vector that comprises a polynucleotide that expresses: one or more peptides or proteins comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables l, 2, and 3 (SEQ ID NOS: I to 1081); or apool of2 or more peptides selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081). In one aspect, the vector is a bacterial vector.

[0197] As embodied and broadly described herein, an aspect of the present disclosure relates to a host cell that comprises the vector that comprises a polynucleotide that expresses: one or more peptides or proteins comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: I to 1081).

[0198] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0199] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0200] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or the variation that exists among the study subjects.

[0201] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. As used herein, the phrase “consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method / process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method / process steps or limitation(s)) only.

[0202] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0203] As used herein, words of approximation such as, without limitation, “about”, "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0204] Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the invention(s) set out in any claims that may issue from this disclosure. Specifically, and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any invention(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the invention(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from thisdisclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure, but should not be constrained by the headings set forth herein.

[0205] For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.

[0206] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.

[0207] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A composition comprising: one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more or more peptides comprising, consisting of, or consisting essentially of amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

2. The composition of claim 1, wherein the one or more peptides or proteins comprises, or wherein the fusion protein comprises two or more or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

3. The composition of claim 1 or claim 2, wherein the amino acid sequence is selected from a cow milk T cell epitope selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: I to 1081).

4. The composition of claim 1 or claim 2, wherein the composition comprises one or more cow milk peptides amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

5. The composition of any one of claims 1 to 4, wherein the peptide or protein comprises a cow milk T cell epitope.

6. The composition of any one of claims 1 to 5, wherein the one or more peptides or proteins comprises a cow milk CD8+ or CD4+ T cell epitope.

7. The composition of any one of claims 1 to 6, wherein the one or more peptides or proteins has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids.

8. The composition of any one of claims 1 to 7, wherein the one or more peptides or proteins elicits, stimulates, induces, promotes, increases, or enhances a regulatory T cell response to a cow milk protein or peptide.

9. The composition of claim 8, wherein the one or more peptides or proteins that elicits, stimulates, induces, promotes, increases or enhances the regulatory T cell response to the cow milk protein or peptide, or a variant, homologue, derivative or subsequence thereof.

10. The composition of any one of claims 1 to 9, further comprising the one or more peptides or proteins formulated into an immunoregulatory formulation with an adjuvant.

11. The composition of claim 10, wherein the adjuvant is selected from the group consisting of aluminum hydroxide, calcium phosphate, microcrystalline tyrosine (MCT), and monophosphoryl lipid A (MPL).

12. The composition of any one of claims 1 to 11, wherein the composition further comprises a downregulator of immune response.

13. A composition comprising monomers or multimers of: peptides or proteins comprising, consisting of, or consisting essentially of: one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), concatemers, subsequences, portions, homologues, variants or derivatives thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

14. A composition comprising one or more peptide-major histocompatibility complex (MHC) monomers or multimers, wherein the peptide-MHC monomer or multimer comprises a peptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), in a groove of the MHC monomer or multimer.

15. A composition comprising: one or more peptides or proteins comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); a pool of 2 or more peptides selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes one or more peptides or proteins, comprising, consisting of, orconsisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

16. The composition of claim 15, wherein the one or more peptides or proteins comprises, or wherein the fusion protein comprises, 2 or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

17. The composition of claim 15 or claim 16, wherein the protein or peptide comprises cow milk T cell epitope.

18. The composition of any one of claims 15 to 17, wherein the one or more peptides or proteins comprises a cow milk CD8+ or CD4+ T cell epitope.

19. The composition of any one of claims 15 to 18, wherein the one or more peptides or proteins has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids.

20. The composition of any one of claims 15 to 19, wherein the one or more peptides or proteins elicits, stimulates, induces, promotes, increases or enhances a regulatory T cell response to comprises cow milk peptides or proteins.

21. The composition of any one of claims 15 to 20, wherein the one or more peptides or proteins that elicits, stimulates, induces, promotes, increases or enhances the regulatory T cell response to cow milk protein(s) or peptide(s), or a variant, homologue, derivative or subsequence thereof.

22. The composition of any one of claims 15 to 21, further comprising the one or more peptides or proteins formulated into an immunogenic formulation with an adjuvant.

23. The composition of claim 22, wherein the adjuvant is selected from the group consisting of adjuvant is selected from the group consisting of aluminum hydroxide, calcium phosphate, microcrystalline tyrosine (MCT), and monophosphoryl lipid A (MPL).

24. The composition of any one of claims 15 to 23, wherein the composition further comprises an immunoregulator of immune response.

25. A composition comprising monomers or multimers of: one or more peptides or proteins comprising, consisting of, or consisting essentially of: one or more cow milk amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), concatemers, subsequences, portions, homologues, variants or derivatives thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a polynucleotide that encodes one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

26. A composition comprising one or more peptide-major histocompatibility complex (MHC)monomers or multimers, wherein the peptide-MHC monomer or multimer comprises a peptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), in a groove of the (MHC) monomer or multimer.

27. A method for detecting the presence of one or more cow milk antigens, comprising: providing one or more proteins or peptides for detection of an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells; contacting a biological sample suspected of having cow milk antigen-specific T-cells to one or more proteins or peptides for detection; and detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigenspecific T-cells in the biological sample, wherein the one or more proteins or peptides for detection comprise one or more amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or comprise a pool of 2 or more or more amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

28. The method of claim 27, wherein detecting the amount or a relative amount of, and / or activity of antigen-specific T-cells comprises one or more steps of identification or detection of the antigen-specific T-cells and measuring the amount of the antigen-specific T-cells.

29. The method of claim 27 or claim 28, wherein the one or more peptides or proteins comprises 2 or more amino acid sequences selected from those set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: I to 1081).

30. The method of any one of claims 27 to 29, wherein the detecting the amount or a relative amount of, and / or activity of antigen-specific T-cells comprises indirect detection and / or direct detection.

31. The method of any one of claims 27 to 30, wherein the method of detecting an immune response to the one or more cow milk antigens comprises the following steps: providing an MHC monomer or an MHC multimer; contacting a population T-cells to the MHC monomer or MHC multimer; and measuring the number, activity or state of T-cells specific for the MHC monomer or MHC multimer.

32. The method of claim 31, wherein the MHC monomer or MHC multimer comprises a protein or peptide of cow milk.

33. The method of claim 32, wherein the protein or peptide comprises a CD8+ or CD4+ T cell epitope.

34. The method of any one of claims 27 to 33, wherein the protein or peptide has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids.

35. The method of any one of claims 27 to 34, wherein the proteins or peptides comprise 2 or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

36. The method of any one of claims 27 to 35, further comprising detecting the presence or amountof the one or more peptides in a biological sample, or a response thereto, which is diagnostic of a cow milk allergy.

37. The method of any one of claims 27 to 36, wherein detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample comprises measuring one or more of a cytokine or lymphokine secretion assay, T cell proliferation, immunoprecipitation, immunoassay, ELISA, radioimmunoassay, immunofluorescence assay, Western Blot, FACS analysis, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay, a reporter assay, a luciferase assay, a microarray, a surface plasmon resonance detector, a florescence resonance energy transfer, immunocytochemistry, or a cell mediated assay, or a cytokine proliferation assay.

38. The method of any one of claims 27 to 37, further comprising administering a treatment comprising the composition of any one of claims 1-26 to the subject from which the biological sample was drawn that increases the amount or relative amount of, and / or activity of the antigen-specific T-cells.

39. A method for detecting the presence of an allergy to cow milk antigens, comprising: providing one or more proteins or peptides for detection of an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells; contacting a biological sample suspected of having cow milk-specific T-cells to one or more proteins or peptides for detection; and detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigenspecific T-cells in the biological sample, wherein the one or more proteins or peptides for detection comprise one or more amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or comprise a pool of 2 or more amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

40. The method of claim 39, wherein detecting the amount or a relative amount of, and / or activity of antigen-specific T-cells comprises one or more steps of identification or detection of the antigen-specific T-cells and measuring the amount of the antigen-specific T-cells.

41. The method of claim 39 or claim 40, wherein the one or more peptides or proteins comprises 2 or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: I to 1081).

42. The method of any one of claims 39 to 41, wherein the detecting the amount or a relative amount of, and / or activity of antigen-specific T-cells comprises indirect detection and / or direct detection.

43. The method of any one of claims 39 to 42, wherein the method of detecting an immune response to one or more cow milk antigens comprises the following steps: providing an MHC monomer or an MHC multimer; contacting a population T-cells to the MHC monomer or MHC multimer; and measuring the number, activity or state of T-cells specific for the MHC monomer or MHC multimer.

44. The method of claim 43, wherein the MHC monomer or MHC multimer comprises a protein orpeptide of cow milk.

45. The method of claim 44, wherein the protein or peptide comprises a B. pertussis CD8+ or CD4+ T cell epitope.

46. The method of any one of claims 39 to 45, wherein the protein or peptide has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids.

47. The method of any one of claims 39 to 46, wherein the proteins or peptides comprise 2 or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant, or derivative thereof.

48. The method of any one of claims 39 to 47, further comprising detecting the presence or amount of the one or more peptides in a biological sample, or a response thereto, which is diagnostic of an allergy to cow milk.

49. The method of any one of claims 39 to 48, wherein detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample comprises measuring one or more of a cytokine or lymphokine secretion assay, T cell proliferation, immunoprecipitation, immunoassay, ELISA, radioimmunoassay, immunofluorescence assay, Western Blot, FACS analysis, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay, a reporter assay, a luciferase assay, a microarray, a surface plasmon resonance detector, a florescence resonance energy transfer, immunocytochemistry, or a cell mediated assay, or a cytokine proliferation assay.

50. The method of any one of claims 39 to 48, further comprising administering a treatment comprising the composition of any one of claims 1-26 to the subject from which the biological sample was drawn that increases the amount or relative amount of, and / or activity of the antigen-specific T-cells.

51. A method detecting an allergy to cow milk antigens in a subject, the method comprising, consisting of, or consisting essentially of: contacting a biological sample from a subject with a composition of any one of claims 1 to 36; and determining if the composition elicits an immune response from the contacted cells, wherein the presence of an immune response indicates that the subject has an allergy to cow milk antigens.

52. The method of claim 51, wherein the sample comprises T cells.

53. The method of claim 51 or claim 52, wherein the response comprises inducing, increasing, promoting, or stimulating a regulatory activity of T cells.

54. The method of claim 51 or claim 52, wherein the T cells are CD8+ or CD4+ T cells.

55. The method of any one of claims 51 to 54, wherein the method comprises determining whether the subject has been exposed to the cow milk antigens more than once by determining if the subject elicits a secondary T cell immune response profile that is different from a primary T cell immune response profile.

56. The method of any one of claims 51 to 55, further comprising diagnosing an allergy to cow milk in a subject, the method comprising contacting a biological sample from a subject with a composition ofany one of claims 1 to 26, and determining if the composition elicits a T or B cell immune response, wherein the T cell immune response identifies that the subject is allergic to cow milk.

57. The method of any one of claims 51 to 56, wherein the method is conducted three or more days following the date of exposure to cow milk.

58. A method detecting a cow milk allergy in a subject, the method comprising, consisting of, or consisting essentially of: contacting a biological sample from a subject with a composition of any one of claims 1 to 26; and determining if the composition elicits an immune response from the contacted cells, wherein the presence of an immune response indicates that the subject has an allergy to cow milk.

59. The method of claim 58, wherein the sample comprises T cells.

60. The method of claim 58 or claim 59, wherein the response comprises inducing, increasing, promoting or stimulating a regulatory activity of T cells.

61. The method of claim 59 or claim 60, wherein the T cells are CD8+ or CD4+ T cells.

62. The method of any one of claims 58 to 61, wherein the method comprises determining whether the subject has been exposed to cow milk more than once by determining if the subject elicits a secondary T cell immune response profde that is different from a primary T or B cell immune response profde.

63. The method of any one of claims 58 to 62, further comprising diagnosing an allergy to cow milk in a subject, the method comprising contacting a biological sample from a subject with a composition of any one of claims 1 to 26; and determining if the composition elicits a T or B cell immune response, wherein the T or B cell immune response identifies that the subject has an allergy to cow milk.

64. The method of any one of claims 58 to 63, wherein the method is conducted three or more days following the date of suspected exposure to cow milk.

65. A kit for the detection of an allergy to cow milk in a subject comprising, consisting of or consisting essentially of: one or more T cells that specifically detect the presence of: one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; or a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more or more peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

66. The kit of claim 65, wherein the one or more amino acid sequences are selected from a cow milk T cell epitope set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

67. The kit of claim 65 or claim 66, wherein the composition comprises: one or more amino acid sequences selected from any one of those sequences set forth in Tables 1,2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

68. The kit of any one of claims 65 to 67, wherein the fusion protein has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids.

69. The kit of any one of claims 65 to 68, wherein the kit includes instruction for a diagnostic method, a process, a composition, a product, a service or component part thereof for the detection of an allergy to cow milk.

70. The kit of any one of claims 65 to 69, wherein the kit includes reagents for detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample comprises measuring one or more of a cytokine or lymphokine secretion assay, T cell proliferation, immunoprecipitation, immunoassay, ELISA, radioimmunoassay, immunofluorescence assay, Western Blot, FACS analysis, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay, a reporter assay, a luciferase assay, a microarray, a surface plasmon resonance detector, a florescence resonance energy transfer, immunocytochemistry, or a cell mediated assay, or a cytokine proliferation assay.

71. The kit of any one of claims 65 to 70, wherein the kit includes reagents for determining a Human Leukocyte Antigen (HLA) profile of a subject, and selecting peptides that are presented by the HLA profile of the subject for detecting an immune response to cow milk.

72. A kit for the detection of an allergy to cow milk in a subject comprising, consisting of or consisting essentially of: one or more T cells that specifically detect the presence of: one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

73. The kit of claim 72, wherein the one or more amino acid sequences is selected from one or more cow milk CD4 T cell epitopes selected from any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or both.

74. The kit of claims 72 to 73, wherein the amino acid sequence comprises one or more cow milk CD8+ or CD4+ T cell epitopes.

75. The kit of any one of claims 72 to 74, wherein the fusion protein has a length from about 9-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids.

76. The kit of any one of claims 72 to 75, wherein the kit includes instruction for a diagnosticmethod, a process, a composition, a product, a service or component part thereof for the detection of an allergy to cow milk.

77. The kit of any one of claims 72 to 76, wherein the kit includes reagents for detecting an amount or a relative amount of, and / or the activity of, and / or the state of antigen-specific T-cells in the biological sample comprises measuring one or more of a cytokine or lymphokine secretion assay, T cell proliferation, immunoprecipitation, immunoassay, ELISA, radioimmunoassay, immunofluorescence assay, Western Blot, FACS analysis, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay, a reporter assay, a luciferase assay, a microarray, a surface plasmon resonance detector, a florescence resonance energy transfer, immunocytochemistry, or a cell mediated assay, or a cytokine proliferation assay.

78. The kit of any one of claims 72 to 77, wherein the kit includes reagents for determining a Human Leukocyte Antigen (HLA) profile of a subject, and selecting peptides that are presented by the HLA profile of the subject for detecting an immune response to cow milk.

79. A method of stimulating, inducing, promoting, increasing, or enhancing an immunoregulatory response against one or more cow milk antigens in a subject, comprising: administering a composition of claims 1 to 26, in an amount sufficient to stimulate, induce, promote, increase, or enhance an immune response against the cow milk in the subject.

80. The method of claim 79, wherein the immunoregulatory immune response provides the subject with protection against cow milk allergies, or one or more physiological conditions, disorders, illnesses, diseases or symptoms caused by or associated with cow milk allergies.

81. The method of claim 79 or claim 80, wherein the immune response is specific to: one or more cow milk peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

82. A method of stimulating, inducing, promoting, increasing, or enhancing an immunoregulatory response against cow milk in a subject, comprising: administering a composition of claims to 1 to 26, in an amount sufficient to stimulate, induce, promote, increase, or enhance an immune response against cow milk in the subject.

83. The method of claim 82, wherein the immunoregulatory response provides the subject with protection against cow milk, or one or more physiological conditions, disorders, illnesses, diseases or symptoms caused by or associated with cow milk allergies.

84. The method of claim 82 or claim 83, wherein the immunoregulatory response is specific to: one or more cow milk peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof.

85. A method of stimulating, inducing, promoting, increasing, or enhancing a regulatory T cell response in a subject with an allergy to one or more cow milk antigens, comprising: administering to a subject an amount of a protein or peptide or a polynucleotide that expresses the protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence oftwo or more cow milk protein or peptide, or a variant, homologue, derivative or subsequence thereof, wherein the protein or peptide comprises at least two peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081)or a subsequence, portion, homologue, variant or derivative thereof, in an amount sufficient to stimulate, induce, promote, increase, or enhance an immunoregulatory immune response to one or more cow milk antigens in the subject.

86. The method of claim 85, wherein the immune response provides the subject with protection against an allergy to cow milk, or one or more physiological conditions, disorders, illnesses, diseases or symptoms caused by an allergy to cow milk.

87. A method of treating, preventing, or immunizing a subject against cow milk allergies, comprising administering to a subject an amount of a protein, peptide or a polynucleotide that expresses the protein or peptide comprising, consisting of, or consisting essentially of an amino acid sequence of a cow milk protein or peptide, or a variant, homologue, derivative or subsequence thereof, wherein the protein or peptide comprises at least one amino acid sequence selected from any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081)or a subsequence, portion, homologue, variant or derivative thereof, in an amount sufficient to treat, prevent, or immunize the subject to prevent the allergy to cow milk, wherein the protein or peptide comprises or consists of cow milk T cell epitope that elicits, stimulates, induces, promotes, increases, or enhances an anti- cow milk regulatory T cell immune response.

88. The method of claim 87, wherein the one or more amino acid sequences are selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

89. The method of claim 88, wherein the cow milk T cell response is a CD8+, a CD4+ T cell response, or both.

90. The method of any one of claims 87 to 89, wherein the subject is a mammal or a human.

91. The method of any one of claims 87 to 90, wherein the method reduces one or more adverse physiological conditions, disorders, illness, diseases, symptoms or complications caused by or associated with cow milk allergies.

92. The method of any one of claims 87 to 91, wherein the method improves one or more adverse physiological conditions, disorders, illness, diseases, symptoms or complications caused by or associated with cow milk allergies.

93. The method of claim 87 or 92, wherein the symptom is fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea or vomiting, or diarrhea.

94. The method of any one of claims 87 to 93, wherein the method reduces or inhibits susceptibility to cow milk allergies.

95. The method of any one of claims 87 to 94, wherein the protein or peptide, or a subsequence, portion, homologue, variant or derivative thereof, is administered prior to, substantially contemporaneously with or following exposure to cow milk.

96. The method of any one of claims 87 to 95, wherein a plurality of cow milk T cell epitopes are administered prior to, substantially contemporaneously with or following exposure to cow milk.

97. The method of any one of claims 87 to 96, wherein the protein or peptide, or a subsequence, portion, homologue, variant or derivative thereof is administered within 2-72 hours, 2-48 hours, 4-24 hours, 4-18 hours, or 6-12 hours after a symptom of cow milk allergy.

98. The method of any one of claims 87 to 97, wherein the protein or peptide, or a subsequence, portion, homologue, variant or derivative thereof is administered prior to exposure to cow milk.

99. The method of any one of claims 87 to 98, wherein the method further comprises administering a modulator of immune response prior to, substantially contemporaneously with or following the administration to the subject of an amount of a protein or peptide.

100. A method of treating, preventing, or immunizing a subject against cow milk allergies, comprising administering to a subject the composition of any one of claims 1-36 in an amount sufficient to treat or prevent the cow milk allergy.

101. The method of claim 100, wherein the method reduces one or more adverse physiological conditions, disorders, illness, diseases, symptoms or complications caused by cow milk allergy.

102. The method of any claims 100 or 101, wherein the method improves one or more adverse physiological conditions, disorders, illness, diseases, symptoms or complications caused by or associated with cow milk allergies.

103. The method of any one of claims 100 to 102, wherein the method reduces or inhibits susceptibility to cow milk allergies.

104. The method of any one of claims 100 to 103, wherein the composition is administered prior to, substantially contemporaneously with or following exposure to cow milk allergens.

105. The method of any one of claims 100 to 104, wherein the composition is administered prior to, substantially contemporaneously with or following exposure to cow milk allergens.

106. The method of any one of claims 100 to 105, wherein the composition is administered within 2- 72 hours, 2-48 hours, 4-24 hours, 4-18 hours, or 6-12 hours after a symptom of cow milk allergies.

107. The method of any one of claims 100 to 106, wherein the composition is administered prior to exposure to cow milk.

108. A peptide or peptides that are immunoprevalent or immunodominant in cow milk obtained by a method comprising, consisting of, or consisting essentially of: obtaining an amino acid sequence of one or more cow milk proteins; determining one or more sets of overlapping peptides spanning one or more cow milk antigen using unbiased selection; synthesizing one or more pools of cow milk peptides comprising the one or more sets of overlapping peptides;combining the one or more pools of cow milk peptides with Class I major histocompatibility proteins (MHC), Class II MHC, or both Class I and Class II MHC to form peptide-MHC complexes; contacting the peptide-MHC complexes with T cells from subjects exposed to the cow milk; determining which pools triggered cytokine release by the T cells; and deconvoluting from the pool of peptides that elicited cytokine release by the T cells, which peptide or peptides are immunoprevalent or immunodominant in the pool.

109. The peptide or peptides of claim 108, wherein the immunodominant peptides are selected from 1, 2 or more peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: I to 1081).

110. The peptide or peptides of any claims 108 or 109, wherein the immunodominant peptides are selected from 1, 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

111. A method of selecting an immunoprevalent or immunodominant peptide or protein of cow milk comprising, consisting of, or consisting essentially of: obtaining an amino acid sequence of the cow milk; determining one or more sets of overlapping peptides spanning one or more cow milk antigen using unbiased selection; synthesizing one or more pools of cow milk peptides comprising the one or more sets of overlapping peptides; combining the one or more pools of cow milk peptides with Class I major histocompatibility proteins (MHC), Class II MHC, or both Class I and Class II MHC to form peptide-MHC complexes; contacting the peptide-MHC complexes with T cells from subjects exposed to cow milk; determining which pools triggered cytokine release by the T cells; and deconvoluting from the pool of peptides that elicited cytokine release by the T cells, which peptide or peptides are immunoprevalent or immunodominant in the pool.

112. The method of claim 111, wherein the immunodominant peptides are selected from 1, 2 or more peptides selected from the amino acid sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: I to 1081).

113. The method of claims 111 or 112, wherein the immunodominant peptides are selected from 1, 2 or more peptides selected from the amino acid sequences set forth in those sequences set forth in any one of Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

114. A polynucleotide that expresses one or more peptides or proteins, comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more or more peptides comprising, consisting of, or consisting essentially of aminoacid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

115. A vector that comprises the polynucleotide of claim 114.

116. The vector of claim 115, wherein the vector is a bacterial vector.

117. A host cell that comprises the vector of claim 115 or claim 116.

118. A polynucleotide that expresses: one or more peptides or proteins comprising, consisting of, or consisting essentially of an amino acid sequence selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081), or a subsequence, portion, homologue, variant or derivative thereof; a fusion protein comprising one or more amino acid sequences selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081); or a pool of 2 or more peptides selected from any one of those sequences set forth in Tables 1, 2, and 3 (SEQ ID NOS: 1 to 1081).

119. A vector that comprises the polynucleotide of claim 118.

120. The vector of claim 119, wherein the vector is a bacterial vector.

121. A host cell that comprises the vector of claim 119 or claim 120.

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