PEPTIDE ANTIGENS AND USES THEREOF

Abstract

The invention provides a new method for identifying peptide antigens relevant to a non-autoimmune disease involving T cell activation as well as novel peptides identified therefrom. The isolated peptides of the invention are useful in the diagnosis, prevention and/or treatment of a cardiovascular disease, more specifically heart failure (HF). The invention further provides a pharmaceutical composition comprising at least one isolated peptide of the invention and a pharmaceutically acceptable carrier, vehicle, excipient and/or diluent. The pharmaceutical composition of the invention is suitable to be orally administered as a tolerizing vaccine.

Claims

1: An isolated peptide consisting of: (a) an amino acid sequence selected from the group consisting of: SEQ ID NO. 1 derived from a human 14-3-3 protein; SEQ ID NO. 2 derived from a human 14-3-3 protein; SEQ ID NO. 3 derived from a human 14-3-3 protein; SEQ ID NO. 4 derived from a human 14-3-3 protein; SEQ ID NO. 5 derived from a human small nuclear ribonucleoprotein Sm D1 (SNRPD1); SEQ ID NO. 6 derived from a human ATP synthase subunit O, mitochondrial precursor (ATP5O); and SEQ ID NO. 7 derived from a human toll-like receptor 5 precursor (TLR5), and any combination thereof; or (b) an amino acid sequence selected from the group consisting of: SEQ ID NO. 1 derived from a canine 14-3-3 protein; SEQ ID NO. 2 derived from a canine 14-3-3 protein; SEQ ID NO. 3 derived from a canine 14-3-3 protein; SEQ ID NO. 4 derived from a canine 14-3-3 protein; SEQ ID NO. 8 derived from a canine monocarboxylate transporter 3 and 4 proteins (MCT3 and MCT4); SEQ ID NO. 5 derived from a canine SNRPD1; SEQ ID NO. 9 derived from a canine ATP50; SEQ ID NO. 10 derived from a canine dihydropyrimidinase (DHP) protein, and any combination thereof; or (c) an amino acid sequence selected from the group consisting of: SEQ ID NO. 1 derived from a feline 14-3-3 protein; SEQ ID NO. 2 derived from a feline 14-3-3 protein; SEQ ID NO. 3 derived from a feline 14-3-3 protein; SEQ ID NO. 4 derived from a feline 14-3-3 protein; SEQ ID NO. 8 derived from a feline MCT3 and MCT4 proteins; SEQ ID NO. 11 derived from a feline vomeronasal 1 receptor felCatV1R6 protein; SEQ ID NO. 12 derived from a feline transmembrane emp24 domain-containing protein 6; SEQ ID NO. 5 derived from a feline SNRPD1; SEQ ID NO. 13 derived from a feline ATP50; SEQ ID NO. 10 derived from a feline DHP protein, and any combination thereof.

2: An in vitro method for the diagnosis and/or for predicting the risk for developing heart failure (HF) in a human subject, said method comprising the steps of: (i) contacting a biological fluid sample containing IgG immunoglobulins derived from a human subject affected by HF or suspected of being at risk for developing HF, with one or more isolated antigenic peptides from one or more human proteins selected from the group consisting of: 14-3-3 protein epsilon (SEQ ID NO. 18), 14-3-3 protein epsilon isoform X1 (SEQ ID NO. 19), 14-3-3 protein epsilon isoform X2 (SEQ ID NO. 20), 14-3-3 protein beta/alpha (SEQ ID NO. 21), 14-3-3 protein theta (SEQ ID NO. 22), 14-3-3 protein zeta/delta (SEQ ID NO. 23), 14-3-3 protein eta (SEQ ID NO. 24), 14-3-3 protein gamma (SEQ ID NO. 25), 14-3-3 protein sigma (SEQ ID NO. 26), small nuclear ribonucleoprotein Sm D1 isoform 1 (SEQ ID NO. 27), ATP synthase subunit O, mitochondrial precursor (SEQ ID NO. 28), toll-like receptor 5 precursor (SEQ ID NO. 29), and any combination thereof, wherein the one or more isolated antigenic peptides are from between about 6 to 50 amino acids in length; and (ii) detecting the formation of one or more immunocomplexes between the IgG immunoglobulins in the biological fluid sample and the one or more isolated antigenic peptides, the formation of the one or more immunocomplexes being indicative of the human subject being affected by HF or being at risk for developing HF.

3: An in vitro method for the diagnosis and/or for predicting the risk for developing heart failure (HF) in a dog, said method comprising the steps of: (i) contacting a biological fluid sample containing IgG immunoglobulins derived from a dog affected by HF or suspected of being at risk for developing HF with one or more isolated antigenic peptides from one or more canine proteins selected from the group consisting of 14-3-3 protein epsilon isoform X1 (SEQ ID NO. 30), 14-3-3 protein epsilon isoform X2 (SEQ ID NO. 31), 14-3-3 protein epsilon isoform X3 (SEQ ID NO. 32), 14-3-3 protein epsilon isoform X4 (SEQ ID NO. 33), 14-3-3 protein theta-like (SEQ ID NO. 34), 14-3-3 protein theta (SEQ ID NO. 35), 14-3-3 protein zeta/delta (SEQ ID NO. 36), 14-3-3 protein beta/alpha (SEQ ID NO. 37), 14-3-3 protein eta (SEQ ID NO. 38), 14-3-3 protein gamma (SEQ ID NO. 39), 14-3-3 protein sigma (SEQ ID NO. 40), monocarboxylate transporter 3 and 4 proteins (SEQ ID NOs. 41 and 42), small nuclear ribonucleoprotein Sm D1 (SEQ ID NO. 43), ATP synthase subunit O, mitochondrial precursor (SEQ ID NO. 44), dihydropyrimidinase protein (SEQ ID NO. 45), and any combination thereof, wherein the one or more isolated antigenic peptides are from 6 to 50 amino acids in length; and (ii) detecting the formation of one or more immunocomplexes between the IgG immunoglobulins in the biological fluid sample and the one or more isolated antigenic peptides, the formation of the one or more immunocomplexes being indicative of the dog being affected by HF or being at risk for developing HF.

4: An in vitro method for the diagnosis and/or for predicting the risk for developing heart failure (HF) in a cat, said method comprising the steps of: (i) contacting a biological fluid sample containing IgG immunoglobulins derived from a cat affected by HF or suspected of being at risk for developing HF with one or more isolated antigenic peptides from one or more feline proteins selected from the group consisting of 14-3-3 protein epsilon isoform X1 (SEQ ID NO. 46), 14-3-3 protein epsilon isoform X2 (SEQ ID NO. 47), 14-3-3 protein epsilon isoform X3 (SEQ ID NO. 48), 14-3-3 protein theta (SEQ ID NO. 49), 14-3-3 protein zeta/delta (SEQ ID NO. 50), 14-3-3 protein beta/alpha (SEQ ID NO. 51), 14-3-3 protein eta (SEQ ID NO. 52), 14-3-3 protein gamma (SEQ ID NO. 53), 14-3-3 protein sigma (SEQ ID NO. 54), monocarboxylate transporter 3 protein (SEQ ID NO. 55), monocarboxylate transporter 4 proteins isoform X2 (SEQ ID NO. 56) and isoform X21 (SEQ ID NO. 57), vomeronasal 1 receptor felCatV1R6 protein (SEQ ID NO. 58), transmembrane emp24 domain-containing protein 6, isoform X1 (SEQ ID NO. 59), transmembrane emp24 domain-containing protein 6, isoform X2 (SEQ ID NO. 60), small nuclear ribonucleoprotein Sm D1, isoform X1 (SEQ ID NO. 61), small nuclear ribonucleoprotein Sm D1, isoform X2 (SEQ ID NO. 62), ATP synthase subunit O, mitochondrial precursor (SEQ ID NO. 63), dihydropyrimidinase protein, isoform X1 (SEQ ID NO. 64), dihydropyrimidinase protein, isoform X2 (SEQ ID NO. 65) and any combination thereof, wherein the one or more isolated antigenic peptides are from 6 to 50 amino acids in length; and (ii) detecting the formation of one or more immunocomplexes between the IgG immunoglobulins in the biological fluid sample and the one or more isolated antigenic peptides, the formation of the one or more immunocomplexes being indicative of the cat being affected by HF or being at risk for developing HF.

5: The in vitro method according to claim 2, wherein the one or more isolated antigenic peptides consist of an amino acid sequence selected from the group consisting of SEQ ID NOs. 1, 2, 3 and 4 of human 14-3-3 protein; SEQ ID NO. 5 of human small nuclear ribonucleoprotein Sm D1 (SNRPD1); SEQ ID NO. 6 of human ATP synthase subunit O, mitochondrial precursor (ATP5O); SEQ ID NO. 7 of human toll-like receptor 5 precursor (TLR5), and any combination thereof.

6: The in vitro method according to claim 3, wherein the one or more isolated antigenic peptides consist of an amino acid sequence selected from the group consisting of SEQ ID NOs. SEQ ID NOs. 1, 2, 3 and 4 of canine 14-3-3 protein; SEQ ID NO. 8 of canine monocarboxylate transporter 3 and 4 proteins (MCT3 and MCT4); SEQ ID NO. 5 of canine SNRPD1; SEQ ID NO. 9 of canine ATP5O; SEQ ID NO. 10 of canine dihydropyrimidinase (DHP) protein, and any combination thereof.

7: The in vitro method according to claim 4, wherein the one or more isolated antigenic peptides consist of an amino acid sequence selected from the group consisting of SEQ ID NOs. 1, 2, 3 and 4 of feline 14-3-3 protein; SEQ ID NO. 8 of feline MCT3 and MCT4 proteins; SEQ ID NO. 11 of feline vomeronasal 1 receptor felCatV1R6 protein; SEQ ID NO. 12 of feline transmembrane emp24 domain-containing protein 6; SEQ ID NO. 5 of feline SNRPD1; SEQ ID NO. 13 of feline ATP5O; SEQ ID NO. 10 of feline DHP protein, and any combination thereof.

8: The in vitro method according to any of claims 2 to 7, wherein the biological fluid sample is selected from the group consisting of whole blood, plasma, serum, synovial fluid, follicular fluid and intraocular fluid.

9: A kit for the diagnosis and/or for predicting the risk for developing heart failure (HF) in a human subject, said kit comprising: (a) one or more isolated antigenic peptides from one or more human proteins selected from the group consisting of 14-3-3 protein epsilon (SEQ ID NO. 18), 14-3-3 protein epsilon isoform X1 (SEQ ID NO. 19), 14-3-3 protein epsilon isoform X2 (SEQ ID NO. 20), 14-3-3 protein beta/alpha (SEQ ID NO. 21), 14-3-3 protein theta (SEQ ID NO. 22), 14-3-3 protein zeta/delta (SEQ ID NO. 23), 14-3-3 protein eta (SEQ ID NO. 24), 14-3-3 protein gamma (SEQ ID NO. 25), 14-3-3 protein sigma (SEQ ID NO. 26), small nuclear ribonucleoprotein Sm D1 isoform 1 (SEQ ID NO. 27), ATP synthase subunit O, mitochondrial precursor (SEQ ID NO. 28), toll-like receptor 5 precursor (SEQ ID NO. 29), and any combination thereof, wherein the one or more isolated antigenic peptides are from 6 to 50 amino acids in length and are immobilized on a solid support, and (b) means suitable for detecting the formation of one or more immunocomplexes between said one or more isolated antigenic peptides and the IgG immunoglobulins in a biological fluid sample of a human subject affected by HF or suspected of being at risk for developing HF.

10: A kit for the diagnosis and/or for predicting the risk for developing heart failure (HF) in a dog, said kit comprising: (a) one or more isolated antigenic peptides from one or more canine proteins selected from the group consisting of 14-3-3 protein epsilon isoform X1 (SEQ ID NO. 30), 14-3-3 protein epsilon isoform X2 (SEQ ID NO. 31), 14-3-3 protein epsilon isoform X3 (SEQ ID NO. 32), 14-3-3 protein epsilon isoform X4 (SEQ ID NO. 33), 14-3-3 protein theta-like (SEQ ID NO. 34), 14-3-3 protein theta (SEQ ID NO. 35), 14-3-3 protein zeta/delta (SEQ ID NO. 36), 14-3-3 protein beta/alpha (SEQ ID NO. 37), 14-3-3 protein eta (SEQ ID NO. 38), 14-3-3 protein gamma (SEQ ID NO. 39), 14-3-3 protein sigma (SEQ ID NO. 40), monocarboxylate transporter 3 and 4 proteins (SEQ ID NOs. 41 and 42), small nuclear ribonucleoprotein Sm D1 (SEQ ID NO. 43), ATP synthase subunit O, mitochondrial precursor (SEQ ID NO. 44), dihydropyrimidinase protein (SEQ ID NO. 45), and any combination thereof, wherein the one or more isolated antigenic peptides are from 6 to 50 amino acids in length and are immobilized on a solid support, and (b) means suitable for detecting the formation of one or more immunocomplexes between said one or more isolated antigenic peptides and the IgG immunoglobulins in a biological fluid sample of a dog subject affected by HF or suspected of being at risk for developing HF.

11: A kit for the diagnosis and/or for predicting the risk for developing heart failure (HF) in a cat, said kit comprising: (a) one or more isolated antigenic peptides from one or more feline proteins selected from the group consisting of 14-3-3 protein epsilon isoform X1 (SEQ ID NO. 46), 14-3-3 protein epsilon isoform X2 (SEQ ID NO. 47), 14-3-3 protein epsilon isoform X3 (SEQ ID NO. 48), 14-3-3 protein theta (SEQ ID NO. 49), 14-3-3 protein zeta/delta (SEQ ID NO. 50), 14-3-3 protein beta/alpha (SEQ ID NO. 51), 14-3-3 protein eta (SEQ ID NO. 52), 14-3-3 protein gamma (SEQ ID NO. 53), 14-3-3 protein sigma (SEQ ID NO. 54), monocarboxylate transporter 3 protein (SEQ ID NO. 55), monocarboxylate transporter 4 proteins isoform X2 (SEQ ID NO. 56) and isoform X21 (SEQ ID NO. 57), vomeronasal 1 receptor felCatV1R6 protein (SEQ ID NO. 58), transmembrane emp24 domain-containing protein 6, isoform X1 (SEQ ID NO. 59), transmembrane emp24 domain-containing protein 6, isoform X2 (SEQ ID NO. 60), small nuclear ribonucleoprotein Sm D1, isoform X1 (SEQ ID NO. 61), small nuclear ribonucleoprotein Sm D1, isoform X2 (SEQ ID NO. 62), ATP synthase subunit O, mitochondrial precursor (SEQ ID NO. 63), dihydropyrimidinase protein, isoform X1 (SEQ ID NO. 64), dihydropyrimidinase protein, isoform X2 (SEQ ID NO. 65) and any combination thereof, wherein the one or more isolated antigenic peptides are from 6 to 50 amino acids in length and are immobilized on a solid support, and (b) means suitable for detecting the formation of one or more immunocomplexes between said one or more isolated antigenic peptides and the IgG immunoglobulins in a biological fluid sample of a cat affected by HF or suspected of being at risk for developing HF.

12: The kit according to claim 9, wherein the one or more isolated antigenic peptides consist of an amino acid sequence selected from the group consisting of SEQ ID NOs. 1, 2, 3 and 4 of a human 14-3-3 protein; SEQ ID NO. 5 of human small nuclear ribonucleoprotein Sm D1 (SNRPD1); SEQ ID NO. 6 of human ATP synthase subunit O, mitochondrial precursor (ATP5O); SEQ ID NO. 7 of human toll-like receptor 5 precursor (TLR5), and any combination thereof.

13: The kit according to claim 10, wherein the one or more isolated antigenic peptides consist of an amino acid sequence selected from the group consisting of SEQ ID NOs. SEQ ID NOs. 1, 2, 3 and 4 of canine 14-3-3 protein; SEQ ID NO. 8 of canine monocarboxylate transporter 3 and 4 proteins (MCT3 and MCT4); SEQ ID NO. 5 of canine SNRPD1; SEQ ID NO. 9 of canine ATP5O; SEQ ID NO. 10 of canine dihydropyrimidinase (DHP) protein, and any combination thereof.

14: The kit according to claim 11, wherein the one or more isolated antigenic peptides consist of an amino acid sequence selected from the group consisting of SEQ ID NOs. 1, 2, 3 and 4 of feline 14-3-3 protein; SEQ ID NO. 8 of feline MCT3 and MCT4 proteins; SEQ ID NO. 11 of feline vomeronasal 1 receptor felCatV1R6 protein; SEQ ID NO. 12 of feline transmembrane emp24 domain-containing protein 6; SEQ ID NO. 5 of feline SNRPD1; SEQ ID NO. 13 of feline ATP5O; SEQ ID NO. 10 of feline DHP protein, and any combination thereof.

15: A method for treating or preventing heart failure in a subject in need thereof, comprising administering orally a peptide to the subject, wherein the peptide comprises a peptide of claim 1, thereby inducing oral tolerance to said peptide and treating or preventing the heart failure.

16: A pharmaceutical composition comprising at least one isolated peptide according to claim 1 or any combination thereof, and a pharmaceutically acceptable carrier, vehicle, excipient and/or diluent.

17: The pharmaceutical composition according to claim 16, which is a tolerizing vaccine formulated for oral administration.

18: The pharmaceutical composition according to claim 17, wherein the tolerizing vaccine does not comprise any adjuvant.

19: A food, feed or drink product, comprising at least one isolated peptide according to claim 1 or any combination thereof.

20: The method of claim 15, wherein the heart failure is not resulting from autoimmune myocarditis.

Description

EXAMPLE 1: Transfer of T Cells are Sufficient to Transfer Heart Failure (HF) from Cardiopathic to Healthy Mice

[0136] In order to assess whether T cells are not only necessary but also sufficient to induce heart failure (HF), the present inventors followed the experimental approach depicted in FIG. 1A. Such approach involved transferring T cells from mice that had undergone transverse aortic constriction (TAC)-operation to induce HF (or sham-operation controls) into healthy recipient mice, and examining whether this transfer was sufficient to transfer the disease. Briefly, T cells isolated from heart-draining lymph nodes (LN) of C57BL6/J 8 week-old male mice 4 weeks after TAC- or sham-surgery were transferred into healthy mouse recipients. Recipient C57BL6/J 8 week-old male mice received 1*10.sup.6 cells via intravenous injection.

[0137] Cells isolated from heart draining lymph nodes were stained via FACS-analysis in order to examine the population of T cells isolated from heart-draining lymph nodes and injected into healthy recipients. As shown in FIG. 1B, T cells isolated from lymph nodes included cytotoxic CD8+, CD4+ conventional naïve T cells and CD4+ conventional effector T cells, and regulatory T cells

[0138] Cardiac function of the recipient mice was assessed by echocardiography performed at baseline (before receiving T cells), and 9 and 12 weeks after cell transfer. Fractional shortening (% FS), measured via echocardiography, is an indicator of the left ventricle functionality and it represents the percentage of shortening of the left ventricular diameter between end-diastole and end-systole. Mice that received cells from TAC-operated mice showed a significant decrease of heart functionality measured as % FS, compared to mice receiving T cells from control (sham-operated) mice, signifying a reduction in heart functionality (FIG. 1C). Data were plotted as mean±SEM; solid line represents mice that received cells from TAC-operated mice, dotted line represents mice that received cells from sham-operated mice, (n=6-7) Two-way ANOVA, Bonferroni post-test. * p<0.05.

EXAMPLE 2: Antigen Discovery Strategy

[0139] Serum samples were collected from 5 sham-operated mice or 5 TAC-operated mice 4 weeks after sham or TAC surgery. The sera were pooled into two samples, respectively (1 TAC and 1 sham). The pooled serum samples were assayed on a commercial mouse autoantigen and random antigen-discovery array (FIG. 2). The bound antibodies were then detected by anti-IgG reagents. The IgG antibody signal was quantified for each hit and the sequence of each recognized peptide (i.e. the sequence of the random oligopeptide spotted on the array) was reported for further analysis, along with the corresponding signal strength. By detecting only IgG antibodies, the inventors narrowed the analysis to the IgG-switched antibodies, which had received T cell help. Importantly, the mice used in the experiment were all congenic, i.e. genetically identical.

EXAMPLE 3: Pipeline for Antigen Discovery. Bioinformatic Analysis Strategy for the Differential Analysis of the Array Readout

[0140] With the aim of identifying peptide antigens driving T cells involved in HF pathology, the present inventors set up the discovery strategy as illustrated in FIG. 3.

[0141] To perform a differential analysis of the array readout, the inventors ordered each random oligopeptide on the basis of differential ability to produce a hit (TAC IgG binding signal—positive (“up”) signals) when tested with TAC pooled serum and no hit when tested with sham pooled serum (sham IgG binding signal—negative “down” signals).

[0142] In detail, epitopes recognized by antibodies were selected with a user-defined threshold of x>10 (x=the average and corrected fluorescence intensity changes between Sham mouse serum and TAC mouse serum). The identified oligopeptides (i.e. epitopes) were used to predict potential antigenic proteins using the PSI-BLAST (Position-Specific Iterative Basic Local Alignment Search Tool) from the National Center for Biotechnology Information, which is a protein sequence similarity search program, and the RefSeq non-redundant proteins database (organism Mus musculus (taxid: 10090)). PSI-BLAST parameters were set as default. Candidate proteins corresponding to each identified epitope were selected in according to an E-value<0.01.

[0143] In order to guarantee that identified proteins were relevant for human disease, the hits were filtered so as to select only those proteins for high homology to human proteins. The list of proteins or protein domains with “up” and no “down” hits was tested for sequence identity (at protein level) to human. The homology with human was evaluated performing PSI-BLAST against the RefSeq non-redundant protein database (organism Human (taxid: 9606)). Proteins with a percentage of alignment higher than 67% were selected. Then, cardiac enrichment of each protein was evaluated using Human Protein Atlas database (https://www.proteinatlas.org/).

[0144] Peptides identified as end-results of the above-described process are listed in the Table of FIG. 4. The table contains the following information: name of the mouse gene encoding for the whole protein, amino acid FASTA sequence of the entire protein of origin (sequence not fully visible in the graph), start and end amino acid number of the identified peptides within each protein of origin, amino acid sequence of the identified peptide, peptide length, number of source peptides spotted on the array generating each peptide hit, percentage of homology of the peptide with the homologous human protein.

[0145] The three peptides composing Group 3 are derived from mouse beta-1 adrenergic receptor protein (SEQ ID NO. 66), and consist of the amino acid sequences designated as SEQ ID NO. 15 (SAPLSQQWTAGMGLLLALIVLL), SEQ ID NO. 16 (KALKTLGIIMGVFTLCWL), and SEQ ID NO. 17 (HRDLVPDRLFVFFNWL), respectively. The beta-1 adrenergic receptor is known to be an autoantigen driving responses in autoimmune myocarditis (Caforio, A L, et al. (2002), “Circulating cardiac autoantibodies in dilated cardiomyopathy and myocarditis: pathogenetic and clinical significance”, Eur J Heart Fail, 4 411-17; Basavalingappa, R H, et al. (2017), “β1-Adrenergic Receptor Contains Multiple IAk and IEk Binding Epitopes That Induce T Cell Responses with Varying Degrees of Autoimmune Myocarditis in A/J Mice”, Front Immunol, 8 1567). As it is known that peptides from this protein can lead to autoimmune response targeting the heart, group 3 functioned as an internal positive control in the discovery method of the invention, validating the functionality of the process.

[0146] As shown in FIG. 4, Group 4 includes a peptide consisting of amino acid sequence SEQ ID NO. 1, which derives from mouse 14-3-3 protein (SEQ ID NO. 70), a peptide consisting of amino acid sequence SEQ ID NO. 5, which derives from mouse Snrpd1 protein (SEQ ID NO. 79), and a peptide consisting of amino acid sequence SEQ ID NO. 14, which derives from mouse Atp50 protein (SEQ ID NO. 80).

EXAMPLE 4: Immunization Protocol for Assessing the Cardio-Specific Immunogenicity of the Peptides

[0147] In order to validate the functional relevance of identified antigens recognized by T cells driving HF, the inventors conducted the experiments as described below and schematically represented in FIG. 5.

[0148] Briefly, C57BL6/J 8-week-old male mice were screened, via echocardiography, at baseline for heart functionality and randomly divided into three experimental groups. Mice belonging to the negative control group (CTR−, only CFA) were injected subcutaneously (immunized) with 100 μl Complete Freud's Adjuvant (CFA) at day 0 and boosted with 100 μl of Incomplete Freud's Adjuvant (IFA) on day 21, without any antigen. Mice belonging to group 3 (CTR+) were injected subcutaneously (immunized) with 100 μg of each of the three peptides derived from beta-adrenergic receptor (SEQ ID NOs. 15-17) emulsified in Complete Freud's Adjuvant (CFA) at day 0 and boosted with 100 μg of the three peptides emulsified in Incomplete Freud's Adjuvant (IFA) on day 21. Mice belonging to group 4 (Group 4) were injected subcutaneously (immunized) with 100 μg of each of the three peptides of SEQ ID NOs. 1, 5 and 14, derived from murine 14-3-3, Snrpd1 and, Atp5o proteins, emulsified in Complete Freud's Adjuvant (CFA) at day 0 and boosted with 100 μg of each of the three peptides emulsified in Incomplete Freud's Adjuvant (IFA) on day 21. On day 0 and day 2, all the mice were intraperitoneally injected with 200 ng of Pertussis Toxin (PTX), as per standard immunization protocols. Heart functionality of all mice was analyzed via echocardiography at 2, 5 and 9 weeks after the first subcutaneous injection.

[0149] The immunization experiments of healthy mice with peptides derived from beta-adrenergic receptor and from 14-3-3, Snrpd1, and Atp5o lead to cardiac dysfunction. As shown in the bar graphs of FIGS. 6A and 6B, mice immunized with peptides derived from beta-adrenergic receptor (CTR+) and from 14-3-3, Snrpd1, and Atp5o proteins (Group 4) displayed a significant reduction of cardiac functionality, measured via echocardiography analysis at 2, 5 and 9 weeks after the first subcutaneous injection. Left ventricle functionality was measured as % FS (FIG. 6A) and Ejection Fraction (% EF) (FIG. 6B), which measure the volumetric capacity of the left ventricle to pump blood. Data were plotted as mean±SEM, (n=4-6). Two-way ANOVA and Tukey's multiple comparison test. * p<0.05, ** p<0.01.

EXAMPLE 5: Immunization of Healthy Mice with Single Peptides Derived from 14-3-3, Snrpd1, and Atp5o Induces Cardiac Dysfunction

[0150] C57BL6/J 8-week-old male mice were screened at baseline for heart functionality, via echocardiography, and randomly divided into 5 experimental groups. Mice belonging to control group (CTR−, only CFA) were injected subcutaneously (immunized, but without antigen: negative control) with 100 μl Complete Freud's Adjuvant (CFA) at day 0 and boosted with 100 μl of Incomplete Freud's Adjuvant (IFA) on day 21. Mice belonging to group 3 (CTR+) were injected subcutaneously (immunized, known antigen: positive control) with 100 μg of each of the three peptides (SEQ ID NOs. 15-17) derived from beta-adrenergic receptor and listed in the Table of FIG. 4, emulsified in Complete Freud's Adjuvant (CFA) at day 0 and boosted with 100 μg of the three peptides emulsified in Incomplete Freud's Adjuvant (IFA) on day 21. Mice belonging to group 4_A (Peptide 4_A) were injected subcutaneously (immunized) with 100 μg of the peptide of SEQ ID NO. 1, derived from 14-3-3 protein, emulsified in Complete Freud's Adjuvant (CFA) at day 0 and boosted with 100 μg of each of the three peptides emulsified in Incomplete Freud's Adjuvant (IFA) on day 21. Mice belonging to group 4_B (Peptide 4_B) were injected subcutaneously (immunized) with 100 μg of the peptide of SEQ ID NO. 5, derived from Snrpd1 protein, emulsified in Complete Freud's Adjuvant (CFA) at day 0 and boosted with 100 μg of each of the three peptides emulsified in Incomplete Freud's Adjuvant (IFA) on day 21. Mice belonging to group 4_C (Peptide 4_C) were injected subcutaneously (immunized) with 100 μg of the peptide of SEQ ID NO. 14, derived from Atp5o protein, emulsified in Complete Freud's Adjuvant (CFA) at day 0 and boosted with 100 g of each of the three peptides emulsified in Incomplete Freud's Adjuvant (IFA) on day 21. On day 0 and day 2, all the mice were intraperitoneally injected with 200 ng of Pertussis Toxin. Heart functionality of all mice was analyzed via echocardiography at 2, 5 and 9 weeks after the first subcutaneous injection. In FIG. 7, the results of echocardiography analysis of cardiac functionality parameters % FS and % EF of C57BL6/J mice immunized with the peptide of SEQ ID NO. 1 (14-3-3 protein), the peptide of SEQ ID NO. 5 (Snrpd1 protein), or the peptide of SEQ ID NO. 14 (Atp5o protein) are shown. Mice immunized with peptides derived from beta-adrenergic receptor and from 14-3-3, Snrpd1, and Atp5o proteins showed a significantly reduced % FS and % EF. Data are plotted as mean±SEM, (n=4-6). Two-way ANOVA and Tukey's multiple comparison test with matching subjects. * p<0.05, ** p<0.01, ***p<0.001.

EXAMPLE 6: Validation of Functional Relevance of Identified Antigens Recognized by T Cells Driving HF

[0151] The present inventors conducted dedicated experiments in order to assess whether human Heart Failure patients develop IgG responses against the newly-identified antigens, and thus whether the T cell-dependent antibody response to the antigens can be used to differentially identify HF patients.

[0152] ELISA assays were performed using as capture molecules the peptides of SEQ ID NO. 1 (peptide 4_A), SEQ ID NO. 5 (peptide 4_B), and SEQ ID NO. 14 (peptide 4_C), either alone or in combination. A total of 100 ng of either the peptide 4_A, peptide 4_B, or peptide 4_C, or of these peptides in combination was coated on 96-well plates overnight and blocking of non-specific binding was performed. Healthy or heart failure patient human serum samples were then applied and incubated for 1 hour. Anti-human IgG-HIRP was used to detect the bound IgG antibodies present in the serum. Addition of TMB (3,3′,5,5′-tetramethylbenzidine) to the wells created a colorimetric reaction, which is proportional to the presence of bound antibodies. The intensity of the colorimetric reaction was measured and the absorbance of each sample, after blank subtraction, was plotted (FIG. 8). Data were plotted as mean±SEM, (n=2); unpaired t-test. * p<0.05.

[0153] The results of the ELISA assays described above show that the peptides according to the invention, either alone or in combination, enabled the detection of a significant difference between HF patients and healthy controls.

EXAMPLE 7: Application of Identified Antigens Recognized by t Cells Driving HF in Preventive Therapy Experiment

[0154] To assess preventive protection potential of oral tolerance with the newly identified peptides from the development of Heart Failure, the inventors pursued an oral tolerance protocol to induce cardio-specific tolerance for the peptides, followed by active induction of Heart Failure via TAC. A schematic representation of the protocol pursued by the inventors is shown in FIG. 9.

[0155] Briefly, C57BL6/J 8-week-old male mice were screened at baseline via echocardiography. Each mouse was orally fed with 0.8 mg of each peptide from group 4: the peptide of SEQ ID NO. 1 (14-3-3 protein), the peptide of SEQ ID NO. 5 (Snrpd1 protein), and the peptide of SEQ ID NO. 14 (Atp5o protein) (i.e. 0.8 mg per day of each peptide, thus 2.4 mg/day in total), diluted in 600 μl of Phosphate Buffered-Saline (PBS) or plain PBS (as control) via oral feeding (oral gavage) for 4 days in a row. Three days after the end of this procedure, on day 7, the mice underwent TAC surgery, to experimentally induce Heart Failure. Echocardiographies were performed on both groups of mice to monitor their cardiac functionality at 2, 4 and 8 weeks after TAC-surgery. As shown in FIG. 10, oral administration of peptides prevented cardiac dysfunction in TAC-operated mice. Mice that orally received the identified peptides (peptides of SEQ ID NO. 1, 5 and 14) with the tolerization protocol depicted above, showed significantly better cardiac functionality (measured as % FS and % EF, solid line) 8 weeks after surgery compared to mice treated with PBS alone (dotted line). Data were plotted as mean±SEM, (n=8-11). Two-way ANOVA and Tukey's multiple comparison test with matching subjects. ** p<0.01, ***p<0.001.

[0156] Moreover, the present inventors found that oral administration of peptides induces oral tolerance towards the fed peptides via retinoic acid production, known to promote the generation of induced immunosuppressive regulatory T cells. Briefly, eight weeks after oral tolerance induction in TAC-operated mice, mesenteric lymph nodes (MLN) were collected and dendritic cells (DC) were isolated for FACS analysis. Aldehyde dehydrogenase (ALDH) production of retinoic acid from the DC was measured via Aldefluor Fluorescent dye. FACS analysis of DC isolated from MLN of mice fed with the peptides of SEQ ID NO. 1, 5 and 14 showed a significant increase in ALDH activity (FIG. 11), which is a proxy for retinoic acid production. Retinoic acid produced by tolerogenic DC contributes to Regulatory T cell (Treg) generation, essential for tolerance induction. The bar graphs in FIG. 11 illustrate that both percentage of ALDH positive DC and the mean fluorescence intensity (MFI), induced by the presence of the fluorescent dye, in mice orally fed with the peptides compared to mice fed with PBS alone showed a significant increase. Data are plotted as mean±SEM, (n=7-8). Unpaired t-test. * p<0.05.