Human APOB100 epitopes, methods and uses for modulating inflammation and treating adverse cardiovascular events, disease and atherosclerosis

Abstract

Provided herein are epitopes of ApoB100 and composition comprising them as well as sub-sequences, portions and modifications thereof, and uses thereof for treating adverse cardiovascular events, cardiovascular disease, atherosclerosis and certain liver disorders.

Description

Docket No. 051501-0585358HUMAN APOB100 EPITOPES, METHODS AND USES FOR MODULATING INFLAMMATION AND TREATING ADVERSE CARDIOVASCULAR EVENTS, DISEASE AND ATHEROSCLEROSISGOVERNMENT SUPPORT

[0001] This invention was made with government support under grant numbers HL136275 and HL145241, awarded by the National Institutes of Health. The government has certain rights in the invention.RELATED APPLICATION

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 789,764, filed on April 16, 2025, the entire disclosure of which is incorporated herein by reference.FIELD OF THE INVENTION

[0003] The invention relates to novel epitopes of ApoBlOO, or sub-sequences, portions or modifications thereof and methods and compounds thereof for treatment an adverse cardiovascular event or cardiovascular disease, including atherosclerosis.INTRODUCTION

[0004] Atherosclerosis is an inflammatory disease of the arterial wall characterized by monocytes entering the subendothelial space where they differentiate into macrophages and foam cells (Lusis, Nature 407: 233 (2000); Glass & Witztum, Cell 104: 503 (2001); and Galkina & Ley, Annu. Rev. Immunol. 27: 165 (2009); Li & Glass, Nat. Med. 8: 1235 (2002)). Foam cell formation induced by oxidized low density lipoprotein (oxLDL) leads to induction of pro-inflammatory factors that initiate plaque formation and finally plaque rupture with deleterious clinical consequences like myocardial infarction or stroke. oxLDL-induced foam cell formation is promoted by scavenger receptors like CD36 and SR-A, which allow uncontrolled accumulation of modified LDL cholesterol in foam cells (Libby et al., Am. J. Med. 104: 14S (1998); and Kunjathoor et al., J. Biol. Chem. 277: 49982 (2002)).

[0005] ApoB-100 is an apolipoprotein of about 4500 amino acids long and has been shown to be a component involved in the development of atherosclerosis. ApoBlOO has previously been suggested as being an autoantigen relevant to atherosclerosis (1).Docket No. 051501-0585358However, the regions of this apolipoprotein that activate T cells through T cell receptors to induce inflammatory responses in atherosclerosis are hitherto not known.SUMMARY

[0006] The embodiments are based, in part, on the discovery of novel ApoBlOO T cell epitopes and use of such epitopes in atherogenic and vaccination methods. In particular embodiments, the invention provides proteins and peptides comprising an amino acid sequence of ApoBlOO, and sub-sequences, portions or modifications, and methods and compounds comprising such protein and peptides for the treatment of an adverse cardiovascular event or cardiovascular disease, including in certain embodiments atherosclerosis.

[0007] Thus, in one aspect there is provided a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1 or a sub-sequence, portion, homologue, variant or derivative thereof. In some embodiments, a peptide comprises the amino acid sequence of one or more of the peptides P4 (TSLLPQLIEV), P5 (SLLPQLIEV), P22 (ADLTLLDSPIKV), P23 (LTLLDSPIKV) and P24 (TLLDSPIKV) (Fig. 4G).

[0008] In different particular embodiments, a protein or peptide of the present invention modifies an immune or inflammatory response; modifies a T cell response; induces, promotes, increases or enhances an immune response; elicits, stimulates, induces, promotes, increases or enhances a T cell response; decreases, reduces, inhibits, suppresses or disrupts an immune or inflammatory response; or elicits, stimulates, induces, promotes, increases or enhances an anti- immune or anti- inflammatory response.

[0009] In other embodiments, a protein or peptide of the present invention has prophylactic properties against an adverse cardiovascular event or cardiovascular disease, including atherosclerosis, or against liver disease, disorder or damage.

[0010] In another aspect, the protein or peptide has a length from about 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids. In another aspect, the protein or peptide further comprises a heterologous domain.

[0011] In another aspect, the protein or peptide has a function or activity distinct from wild-type full length ApoBlOO. In an additional embodiment, the function or activity comprises protecting against or reducing the risk of atherosclerosis or protecting against liver disease, disorder or damage. In a further embodiment, the function or activity comprises protecting against an adverse cardiovascular event or cardiovascular disease orDocket No. 051501-0585358reducing the risk of a subject to an adverse cardiovascular event or cardiovascular disease. In an additional aspect, the protein or peptide is isolated or purified.

[0012] In another aspect, there is provided a nucleic acid encoding a protein or peptide of the present invention. In yet another aspect, there is provided a cell expressing a protein or peptide of the present invention. In different embodiments, the cell may be a eukaryotic or prokaryotic cell including for example a mammalian, insect, fungal or bacterium cell. In another embodiment, the cell is a dendritic cell.

[0013] In another aspect there is provided, a composition comprising a protein or peptide of the present invention. In particular embodiments, a composition of the present invention may comprise an adjuvant. In additional embodiments, the composition may further comprise a statin. In different embodiments, the composition may be a solid or a liquid. In further embodiments, the composition may be a microparticle or a nanoparticle. In particular embodiments, the composition may be a vaccine. In further embodiments, the composition is sterile. In another embodiment, the composition further comprises a salt, a chelating agent, a buffering agent or an anti-microbial agent.

[0014] In yet another aspect, there is provided, a method of vaccinating a subject against an adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage, the method comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to vaccinate the subject against the adverse cardiovascular event or cardiovascular disease.

[0015] In another embodiment, there is provided a method of providing a subject with protection against an adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage, comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to provide the subject with protection against the adverse cardiovascular event or cardiovascular disease.

[0016] In another embodiment, there is provided a method of reducing the risk of an adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage in a subject, comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue,Docket No. 051501-0585358variant or derivative thereof, sufficient to reduce the risk of the adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage in the subject.

[0017] In certain embodiments, the methods of vaccinating a subject against an adverse cardiovascular event or cardiovascular disease described herein comprise prophylactic vaccination against the adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage. In particular embodiments, the methods of the present invention comprise vaccinating the subject against an adverse symptom of the adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage, including for example atherosclerosis.

[0018] In another aspect, there is provided, a method of treating a subject for an adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage, the method comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of: the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to treat the subject for the adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage.

[0019] In certain embodiments, the method of treating a subject for an adverse cardiovascular event or cardiovascular disease described herein comprises prophylactic treatment against the adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage. In certain embodiments, the method of treating a subject for an adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage described herein comprises treating the subject for an adverse symptom of the adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage, including for example atherosclerosis.

[0020] In another embodiment, there is provided, a method of treating a subject for an adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage, the method comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to treat the subject for the adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage. In further aspects, the adverse cardiovascular event or cardiovascular comprises coronary artery disease, peripheral artery disease, cerebrovascular disease, renal artery disease, stroke, myocardial infarction (heart attack), ischemic heart failure, transient ischemic attack or brain trauma.Docket No. 051501-0585358In additional aspects, the method reduces an adverse symptom of the adverse cardiovascular event, cardiovascular disease or liver disease, disorder or damage, including atherosclerosis. In further aspects, the method comprises plaque regression.

[0021] In some aspects, presented herein is a method of determining whether a subject has, has had, is at risk of having or is need of treatment for an adverse cardiovascular event or cardiovascular disease, the method comprising contacting a biological sample of the subject with an agent that comprises a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof and detecting binding of immune cells in the biological sample to the agent, wherein detection of binding of immune cells in the biological sample to the agent indicates that the subject has, has had, is at risk of having or is need of treatment for an adverse cardiovascular event or cardiovascular disease.

[0022] In certain aspects, presented herein is a method of determining the efficacy of the method of any one of claims 33 to 48, the method comprising: a) contacting a biological sample of the subject prior to performance of the method of any one of claims 33 to 48 with an agent that comprises a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof; b) detecting binding of immune cells in the biological sample to the agent; c) performing the method of any one of claims 33 to 48; d) contacting a biological sample of the subject after performance of the method of any one of claims 33 to 48 with the agent; e) detecting binding of immune cells in the biological sample of the subject to the agent after performance of the method of any one of claims 33 to 48; and f) comparing the amount of binding of immune cells to the agent after performance of the method of any one of claims 33 to 48 to the amount of binding of immune cells in the biological sample to the agent prior to performance of the method of any one of claims 33 to 48; wherein an increase in the binding of immune cells to the agent after performance of the method of any one of claims 33 to 48 when compared to the biological sample of the subject prior to performance of the method of any one of claims 33 to 48 indicates that there is efficacy in the method of any one of claims 33 to 48.

[0023] In some embodiments, the agent is a multimer comprising a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth asDocket No. 051501-0585358any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof and appropriate MHC-II molecule.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] In some embodiments, a multimer is a tetramer. In certain embodiments, an agent comprises a protein or peptide comprising, consisting of, or consisting essentially of the amino acid sequence of one or more of the peptides P4 (TSLLPQLIEV), P5 (SLLPQLIEV), P22 (ADLTLLDSPIKV), P23 (LTLLDSPIKV) and P24 (TLLDSPIKV).

[0028] 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.

[0029] 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.Docket No. 051501-0585358It 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.

[0030] 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).

[0031] 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 Arenavirus infection 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 ofDocket No. 051501-0585358the present invention are suitable for treating or preventing an Arenavirus infection in a subject. The MHC Class I or Class II multimers can be administered in soluble form or loaded on nanoparticles.

[0032] In certain embodiments, the method comprises detecting immune cell activity in the biological sample. In certain embodiments, the method comprises detecting the amount of immune cell activation, immune cell exhaustion, immune cell polarization or apoptosis of immune cells in the biological sample. In certain embodiments, the method comprises detecting the amount of T cell activation, T cell exhaustion, T cell polarization or apoptosis of T cells in the biological sample.

[0033] In certain embodiments, a method described herein comprises administering a polypeptide to a subject, the polypeptide comprising, consisting of, or consisting essentially of the amino acid sequence of one or more of P4 (TSLLPQLIEV), P5 (SLLPQLIEV), P22 (ADLTLLDSPIKV), P23 (LTLLDSPIKV) and P24 (TLLDSPIKV).

[0034] In different embodiments of the presently described methods, the treatment may be administered with a statin. In further embodiments of the presently described methods, the subject may be for example a mouse or a human.DRAWINGS

[0035] Figure 1 shows an example of an Activation-induced Marker (AIM) assay to detect APOB-specific CD8+T cell in human blood. A) Schematic diagram depicting the workflow for expansion and subsequent re-stimulation of human PBMCs with human APOB-derived HLA-A-02: 01 -restricted peptide mesopools (Table 1, A - P1-P16, B -P17-P32, C - P33-P48, D - P49-P64). Antigen-dependent expression of T cell activations markers was examined using flow cytometry (FACS). B-C) Representative FACS plots showing %CD25+4-lBB+ (B) and %CD69+4-lBB+ (C) populations among CD8+T cells in unstimulated and individual APOB mesopool stimulated PBMCs. D) Frequencies (mean ± SEM) of %CD25+4-lBB+ (top) and %CD69+4-lBB+ (bottom) CD8+T cells in unstimulated and individual mesopool stimulated PBMCs. N=7 independent donors. E) %CD25+4-lBB+ (left) and %CD69+4-lBB+ (right) cells (mean ± SEM) among CD8+T (red dots) or CD4+T cells (black dots) in APOB stimulated PBMCs. Background subtracted (responses in stimulated minus unstimulated set) frequencies are shown. Combined data from seven independent donors, with four independent mesopool-Docket No. 051501-0585358stimulated sets per donor. CD8+ or CD4+ cells were gated on singlets, live, dump- CD3+ T cells (FACs plots in Figure SI A). Pairwise (D) and unpaired (E) statistical comparisons between two groups were performed using the Wilcoxon matched-pairs signed rank test and the Mann- Whitney U test, respectively. *p<0.05, ****p<0.0001.

[0036] Figure 2 shows and example of expression of inflammatory and cytotoxic markers in human APOB-specific CD8+T cells. A) Diagrammatic scheme showing the workflow for detection of cytokines and cytotoxic markers using the FACS-based intracellular cytokine staining (ICS) assay. B-D) Representative FACS plots showing %TNF+ IFNy+ (B), %Granzyme+ IFNy+ (C) and %Perforin+ IFNy+ (D) CD8+T cells in unstimulated and individual APOB mesopool stimulated PBMCs. E) Frequencies (mean ± SEM) of Granzyme+ IFNy+ (left) and Perforin+ IFNy+ (right) subsets among CD8+T (red dots) or CD4+T cells (black dots) in APOB stimulated PBMCs. Background subtracted (responses in stimulated minus unstimulated set) frequencies are shown.Combined data from eight independent donors, with four independent mesopool-stimulated sets per donor. Statistical tests (E) were performed using the Mann-Whitney U test. ****p<0.0001.

[0037] Figure 3 shows an example of controls in IFNy-ELISpot assay to examine human CD8+T responses to HLA-I restricted APOB peptides. A) Frequencies (mean ± SEM) of IFNy+CD8+T cells that co-expressed Granzyme (left), TNF (middle) or Perforin (right) in response to stimulation with APOB mesopools A (white), B (black), C (light grey) and D (dark grey). Background subtracted (responses in stimulated minus unstimulated set) frequencies are shown. B) Representative images of ELISpot wells (left) and quantification (right) showing IFNy responses to APOB class-I peptides, either alone or in the presence of pan-HLA-I or pan-HLA-II (DP+DQ+DR) blocking antibodies. Data from three independent donors, with four independent mesopool-stimulated sets per donor. Background subtracted are shown (negative values are collapsed to zero).Statistical tests (B) were done with the Kruskal-Wallis’s test and Dunn’s multiple comparison adjustment. *p<0.05.

[0038] Figure 4 shows an example of the identification of HLA-A-02: 01 -restricted dominant human APOB epitope. A) Schematic workflow for deconvolution of cytokine responses to individual HLA-A-02:01-restricted APOB peptides using an IFNy ELISpot assay. B) Representative IFNy ELISpot data from two donors. Responses to individual APOB mesopools A-D (X-axes) were measured as spot forming cells (SFCs) per millionDocket No. 051501-0585358PBMCs (Y -axes). Background subtracted (responses in stimulated minus unstimulated set) frequencies are shown. Red dotted lines depict a threshold of 200 SFCs / million PBMCs. Response values that were below or above the threshold are shown in white and red, respectively. Black bars depict response to the total mesopool. C) Background subtracted SFCs / million PBMCs (mean ± SEM) for each of the 64 APOB-derived peptides. Six independent donors. D) Scatter plots showing Pearson correlation r between average (X-axis) and median (Y -axis) responses (SFCs / million PBMCs) to the 64 APOB Class I epitopes. Background subtracted values are plotted. Black solid lines and red dotted lines correspond to zero and threshold (200) values, respectively. Red shaded area: peptides with >200 mean and median SFCs / million PBMCs (n=6). E) APOB peptides were ranked between 1-64 (X-axis) based on their percent contribution (Y-axis) to the total APOB-specific response as detected in the ELISpot assay (n=6 donors). The serial numbers (from Table SI) of the 13 peptides that accounted for 80% of the total response (indicated as a red dotted line) are labelled on the plot. F) Responses (mean ± SEM) to individual peptides P4 (grey), 5, 8, 13, 22, 23, 24, 28, 53, 54, 55, 60 (red: significantly higher; white: not significant) and to the remaining peptides (black). G) Sequences, start sites, lengths and IEDB score of the dominant APOB epitopes (red and grey bars in F). Statistical tests (F) were performed using the Kruskal-Wallis’s test with Dunn’s multiple comparison adjustment. *p<0.05, **p<0.01.DETAILED DESCRIPTION

[0039] The present inventors have discovered novel epitopes of ApoBlOO. In different embodiments, the proteins and peptides described herein comprise novel ApoBlOO epitopes which may have atherosclerosis promoting (atherogenic) properties or therapeutic (protective) properties.

[0040] T cells are involved in the development of atherosclerosis and all the subsets of T cells are found in the arterial plaques. In different embodiments, the proteins and peptides of the present invention comprise T cell epitopes and may potentiate or suppress plaque formation. Thus, T cell populations reactive to the T cell epitopes of the present invention may be targets for immunotherapy. In certain embodiments of the present invention, using specific peptides comprising T cell epitopes of the present invention instead of whole ApoB-100 or LDL (low-density lipoprotein) enables modulation of the specifically activated T cells that participate in the disease processes. Previous studiesDocket No. 051501-0585358using whole ApoB-100 or LDL for immunization or vaccination involve a collection of various ApoB-100 peptides that might have opposite effects.

[0041] In another aspect, the present inventors have discovered that vaccination with the proteins or peptides of the embodiments may provide protection against adverse cardiovascular event or cardiovascular disease, including in certain embodiments atherosclerosis.

[0042] Thus there are presently provided proteins and peptides comprising an ApoBlOO amino acid sequence, and sub-sequences, portions or modifications thereof, and methods and compounds for treating an adverse cardiovascular event or cardiovascular disease. In certain embodiments the methods of the present invention comprise protecting or vaccinating against an adverse cardiovascular event or cardiovascular disease, including but not limited to atherosclerosis.

[0043] The embodiments are based at least in part on ApoBlOO peptides, subsequences and portions, and amino acid modifications of ApoBlOO peptides, subsequences and portions. ApoBlOO peptides, sub-sequences, portions, homologues, variants or derivatives thereof, including T cell epitopes that elicit, stimulate, induce, promote, increase or enhance an immune or inflammatory response in vitro or in vivo, are useful in vaccination and immunization (e.g., prophylactic), as well as treatment uses and methods. For example, an invention ApoBlOO peptide, sub-sequence, portion or modification thereof, can be used to immunize or vaccinate a subject, or to treat a subject having or at risk of having an adverse cardiovascular event or cardiovascular disease.

[0044] The proteins and peptides of the embodiments, and sub-sequences, portions, homologues, variants or derivatives thereof, include T cell epitopes. As used herein an “epitope” refers to a peptide or part thereof that elicits an immune response when administered to a subject. In certain embodiments of the present invention, the epitope is a region of an ApoB 100 protein that elicits an immune response when administered to a subject. As will be understood by a person skilled in the art, an immune response may be a cellular or humoral immune response and may comprise an antibody response, a T cell response or both an antibody and T cell response. In particular embodiments of the present invention, a protein or peptide of the present invention comprises an amino acid sequence of ApoBlOO that is a T cell epitope.

[0045] A non-limiting example of an ApoBlOO polypeptide sequence from which peptides, sub-sequences, portions, homologues, variants, derivatives and T cell epitopes may be derived is as follows:Docket No. 051501-0585358MDPPRP ALLALLALP ALLLLLLAGARAEEEMLENVSLVCPKDATRFKHLRKYTY NYEAESSSGVPGTADSRSATRINCKVELEVPQLCSFILKTSQCTLKEVYGFNPEGK ALLKKTKNSEEFAAAMSRYELKLAIPEGKQVFLYPEKDEPTYILNIKRGIISALLVP PETEEAKQVLFLDTVYGNCSTHFTVKTRKGNVATEISTERDLGQCDRFKPIRTGIS PLALIKGMTRPLSTLISSSQSCQYTLDAKRKHVAEAICKEQHLFLPFSYKNKYGM VAQVTQTLKLEDTPKINSRFFGEGTKKMGLAFESTKSTSPPKQAEAVLKTLQELK KLTISEQNIQRANLFNKLVTELRGLSDEAVTSLLPQLIEVSSPITLQALVQCGQPQC STHILQWLKRVHANPLLIDWTYLVALIPEPSAQQLREIFNMARDQRSRATLYAL SHAVNNYHKTNPTGTQELLDIANYLMEQIQDDCTGDEDYTYLILRVIGNMGQTM EQLTPELKSSILKCVQSTKPSLMIQKAAIQALRKMEPKDKDQEVLLQTFLDDASP GDKRLAAYLMLMRSPSQADINKIVQILPWEQNEQVKNFVASHIANILNSEELDIQ DLKKLVKEALKESQLPTVMDFRKFSRNYQLYKSVSLPSLDPASAKIEGNLIFDPN NYLPKESMLKTTLTAFGFASADLIEIGLEGKGFEPTLEALFGKQGFFPDSVNKALY WVNGQVPDGVSKVLVDHFGYTKDDKHEQDMVNGIMLSVEKLIKDLKSKEVPEA RAYLRILGEELGFASLHDLQLLGKLLLMGARTLQGIPQMIGEVIRKGSKNDFFLH YIFMENAFELPTGAGLQLQISSSGVIAPGAKAGVKLEVANMQAELVAKPSVSVEF VTNMGIIIPDFARSGVQMNTNFFHESGLEAHVALKAGKLKFIIPSPKRPVKLLSGG NTLHLVSTTKTEVIPPLIENRQSWSVCKQVFPGLNYCTSGAYSNASSTDSASYYPL TGDTRLELELRPTGEIEQYSVSATYELQREDRALVDTLKFVTQAEGAKQTEATMT FKYNRQSMTLSSEVQIPDFDVDLGTILRVNDESTEGKTSYRLTLDIQNKKITEVAL MGHLSCDTKEERKIKGVISIPRLQAEARSEILAHWSPAKLLLQMDSSATAYGSTV SKRVAWHYDEEKIEFEWNTGTNVDTKKMTSNFPVDLSDYPKSLHMYANRLLDH RVPQTDMTFRHVGSKLIVAMSSWLQKASGSLPYTQTLQDHLNSLKEFNLQNMG LPDFHIPENLFLKSDGRVKYTLNKNSLKIEIPLPFGGKSSRDLKMLETVRTPALHF KSVGFHLPSREFQVPTFTIPKLYQLQVPLLGVLDLSTNVYSNLYNWSASYSGGNT STDHFSLRARYHMKADSVVDLLSYNVQGSGETTYDHKNTFTLSCDGSLRHKFLD SNIKFSHVEKLGNNPVSKGLLIFDASSSWGPQMSASVHLDSKKKQHLFVKEVKID GQFRVSSFYAKGTYGLSCQRDPNTGRLNGESNLRFNSSYLQGTNQITGRYEDGTL SLTSTSDLQSGIIKNTASLKYENYELTLKSDTNGKYKNFATSNKMDMTFSKQNAL LRSEYQADYESLRFFSLLSGSLNSHGLELNADILGTDKINSGAHKATLRIGQDGIS TSATTNLKCSLLVLENELNAELGLSGASMKLTTNGRFREHNAKFSLDGKAALTE LSLGSAYQAMILGVDSKNIFNFKVSQEGLKLSNDMMGSYAEMKFDHTNSLNIAG LSLDFSSKLDNIYSSDKFYKQTVNLQLQPYSLVTTLNSDLKYNALDLTNNGKLRL EPLKLHVAGNLKGAYQNNEIKHIYAISSAALSASYKADTVAKVQGVEFSHRLNTDocket No. 051501-0585358DIAGLASAIDMSTNYNSDSLHFSNVFRSVMAPFTMTIDAHTNGNGKLALWGEHT GQLYSKFLLKAEPLAFTFSHD YKGSTSHHLVSRKSISAALEHKVS ALLTP AEQTGT WKLKTQFNNNEYSQDLDAYNTKDKIGVELTGRTLADLTLLDSPIKVPLLLSEPINI IDALEMRDAVEKPQEFTIVAFVKYDKNQDVHSINLPFFETLQEYFERNRQTIIVVL ENVQRNLKHINIDQFVRKYRAALGKLPQQANDYLNSFNWERQVSHAKEKLTAL TKKYRITENDIQIALDDAKINFNEKLSQLQTYMIQFDQYIKDSYDLHDLKIAIANII DEIIEKLKSLDEHYHIRVNLVKTIHDLHLFIENIDFNKSGSSTASWIQNVDTKYQIRI QIQEKLQQLKRHIQNIDIQHLAGKLKQHIEAIDVRVLLDQLGTTISFERINDVLEHV KHFVINLIGDFEVAEKINAFRAKVHELIERYEVDQQIQVLMDKLVELAHQYKLKE TIQKLSNVLQQVKIKDYFEKLVGFIDDAVKKLNELSFKTFIEDVNKFLDMLIKKLK SFDYHQFVDETNDKIREVTQRLNGEIQALELPQKAEALKLFLEETKATVAVYLES LQDTKITLIINWLQEALSSASLAHMKAKFRETLEDTRDRMYQMDIQQELQRYLSL VGQVYSTLVTYISDWWTLAAKNLTDFAEQYSIQDWAKRMKALVEQGFTVPEIK TILGTMPAFEVSLQALQKATFQTPDFIVPLTDLRIPSVQINFKDLKNIKIPSRFSTPE FTILNTFHIPSFTIDFVEMKVKIIRTIDQMLNSELQWPVPDIYLRDLKVEDIPLARIT LPDFRLPEIAIPEFIIPTLNLNDFQVPDLHIPEFQLPHISHTIEVPTFGKLYSILKIQSPL FTLDANADIGNGTTSANEAGIAASITAKGESKLEVLNFDFQANAQLSNPKINPLAL KESVKFSSKYLRTEHGSEMLFFGNAIEGKSNTVASLHTEKNTLELSNGVIVKINNQ LTLDSNTKYFHKLNIPKLDFSSQADLRNEIKTLLKAGHIAWTSSGKGSWKWACPR FSDEGTHESQISFTIEGPLTSFGLSNKINSKHLRVNQNLVYESGSLNFSKLEIQSQV DSQHVGHSVLTAKGMALFGEGKAEFTGRHDAHLNGKVIGTLKNSLFFSAQPFEIT ASTNNEGNLKVRFPLRLTGKIDFLNNYALFLSPSAQQASWQVSARFNQYKYNQN FSAGNNENIMEAHVGINGEANLDFLNIPLTIPEMRLPYTIITTPPLKDFSLWEKTGL KEFLKTTKQSFDLSVKAQYKKNKHRHSITNPLAVLCEFISQSIKSFDRHFEKNRNN ALDFVTKSYNETKIKFDKYKAEKSHDELPRTFQIPGYTVPVVNVEVSPFTIEMSAF GYVFPKAVSMPSFSILGSDVRVPSYTLILPSLELPVLHVPRNLKLSLPDFKELCTIS HIFIPAMGNITYDFSFKSSVITLNTNAELFNQSDIVAHLLSSSSSVIDALQYKLEGTT RLTRKRGLKLATALSLSNKFVEGSHNSTVSLTTKNMEVSVATTTKAQIPILRMNF KQELNGNTKSKPTVSSSMEFKYDFNSSMLYSTAKGAVDHKLSLESLTSYFSIESST KGDVKGSVLSREYSGTIASEANTYLNSKSTRSSVKLQGTSKIDDIWNLEVKENFA GEATLQRIYSLWEHSTKNHLQLEGLFFTNGEHTSKATLELSPWQMSALVQVHAS QPSSFHDFPDLGQEVALNANTKNQKIRWKNEVRIHSGSFQSQVELSNDQEKAHL DIAGSLEGHLRFLKNIILPVYDKSLWDFLKLDVTTSIGRRQHLRVSTAFVYTKNPN GYSFSIPVKVLADKFIIPGLKLNDLNSVLVMPTFHVPFTDLQVPSCKLDFREIQIYKDocket No. 051501-0585358KLRTSSFALNLPTLPEVKFPEVDVLTKYSQPEDSLIPFFEITVPESQLTVSQFTLPKS VSDGIAALDLNAVANKIADFELPTIIVPEQTIEIPSIKFSVPAGIVIPSFQALTARFEV DSPVYNATWSASLKNKADYVETVLDSTCSSTVQFLEYELNVLGTHKIEDGTLAS KTKGTFAHRDFSAEYEEDGKYEGLQEWEGKAHLNIKSPAFTDLHLRYQKDKKGI STSAASPAVGTVGMDMDEDDDFSKWNFYYSPQSSPDKKLTIFKTELRVRESDEE TQIKVNWEEEAASGLLTSLKDNVPKATGVLYDYVNKYHWEHTGLTLREVSSKL RRNLQNNAEWVYQGAIRQIDDIDVRFQKAASGTTGTYQEWKDKAQNLYQELLT QEGQASFQGLKDNVFDGLVRVTQEFHMKVKHLIDSLIDFLNFPRFQFPGKPGIYT REELCTMFIREVGTVLSQVYSKVHNGSEILFSYFQDLVITLPFELRKHKLIDVISMY RELLKDLSKEAQEVFKAIQSLKTTEVLRNLQDLLQFIFQLIEDNIKQLKEMKFTYLI NYIQDEINTIFSDYIPYVFKLLKENLCLNLHKFNEFIQNELQEASQELQQIHQYIMA LREEYFDPSIVGWTVKYYELEEKIVSLIKNLLVALKDFHSEYIVSASNFTSQLSSQ VEQFLHRNIQEYLSILTDPDGKGKEKIAELSATAQEIIKSQAIATKKIISDYHQQFR YKLQDFSDQLSDYYEKFIAESKRLIDLSIQNYHTFLIYITELLKKLQSTTVMNPYM KLAPGELTIIL

[0046] T cell epitopes typically are short amino acid sequences, e.g. about five to 15 amino acids in length (or 5-10 amino acids in length). A T cell epitope can elicit, stimulate, induce, promote, increase, enhance an immune or inflammatory T cell response in vitro (e.g., in solution, in solid phase, in tissue culture) or in vivo. Such T cell responses can be detected using various assays disclosed herein or known to the skilled artisan.

[0047] A protein or peptide of the present invention includes a protein or peptide comprising, consisting or consisting essentially of an amino acid sequence of ApoBlOO, or a sub-sequence, portion, homologue, variant or derivative thereof.

[0048] In different embodiments of the present invention, the protein or peptide, or sub-sequence, portion, homologue, variant or derivative thereof, comprises, consists, or consists essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof.

[0049] As described herein, proteins and peptides include homologues. A polynucleotide sequence or polypeptide sequence is a “homologue” of, or is “homologous” to, another sequence if the two sequences have substantial identity over a specified region and a functional activity of the sequences is preserved or conserved, at least in part (as used herein, the term 'homologous' does not infer evolutionary relatedness). Two polynucleotide sequences or polypeptide sequences are considered toDocket No. 051501-0585358have substantial identity if, when optimally aligned (with gaps permitted), they share at least about 60% sequence identity or greater (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, etc. identify over a specific region), or if the sequences share defined functional motifs (e.g., epitopes). The percent identity can extend over the entire sequence length or a portion of the sequence. In particular aspects, the length of the sequence sharing the percent identity is 2, 3, 4, 5 or more contiguous amino acids, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. contiguous amino acids.

[0050] An “unrelated” or “non-homologous” sequence shares less than 50% identity.

[0051] The term “identity” and grammatical variations thereof, mean that two or more referenced entities are the same. Thus, where two proteins or peptides, or sub-sequences, portions, homologues, variants or derivatives thereof are identical, they have the same amino acid sequence. The identity can be over a defined area (region or domain) of the sequence. “Areas, regions or domains” of homology or identity mean that a portion of two or more referenced entities share homology or are the same.

[0052] The extent of identity between two sequences can be ascertained using a computer program and mathematical algorithm known in the art. Such algorithms that calculate percent sequence identity (homology) generally account for sequence gaps and mismatches over the comparison region or area. For example, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschul et al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI) has exemplary search parameters as follows: Mismatch -2; gap open 5; gap extension 2. For polypeptide sequence comparisons, a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM 100, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantitate the extent of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85: 2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol. Biol. 147: 195 (1981)). Programs for quantitating protein structural similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

[0053] Thus in certain embodiments, the protein or peptide of the present invention comprises, consists, or consists essentially of homologue of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof.

[0054] Additional proteins or peptides, or sub-sequences, portions, homologues, variants or derivatives thereof can be based upon or derived from an ApoBlOO amino acidDocket No. 051501-0585358sequence. For example, a protein or peptide of the present invention can comprise an amino acid sequence having 60% or more (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity with a region of ApoBlOO. Thus, as disclosed herein, in particular embodiments, the proteins or peptides of the present invention, or sub-sequences, portions, homologues, variants or derivatives thereof include those having at least partial sequence identity to one or more ApoBlOO peptides, subsequences, portions, homologues, variants or derivatives thereof set forth as any one of the peptides set forth in in Table 1. The percent identity of such sequences can be as little as 60%, or can be greater (e.g., 60%, 65%, 70%, 75%, 75% 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, etc.). The percent identity can extend over the entire sequence length or a portion of the sequence. In particular aspects, the length of the sequence sharing the percent identity is 2, 3, 4, 5 or more contiguous amino acids, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. contiguous amino acids.

[0055] Thus, in accordance with the invention, there are also provided proteins or peptides or sub-sequences, portions, homologues, variants or derivatives thereof that exhibit sequence identity to a reference ApoBlOO peptide, sub-sequence or portion, or modification thereof set forth as any one of the peptides set forth in in Table 1. In one embodiment, a protein or peptide of the present invention, or a sub-sequence, portion, homologue, variant or derivative thereof comprises, consists or consists essentially of a sequence at least 60% or more (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to a reference ApoBlOO protein or peptide, or sub-sequence, portion, homologue, variant or derivative thereof as set forth as any one of the peptides set forth in in Table 1.

[0056] In another embodiment, proteins or peptides of the present invention, or subsequences, portions, homologues, variants or derivatives thereof, include or consist of a an ApoBlOO peptide, sub-sequence or portion thereof set forth as any one of the peptides set forth in in Table Iwherein the protein or peptide or sub-sequence, portion, homologue, variant or derivative thereof has one or more modifications, such as an amino acid addition to, deletion of, or substitution of any amino acid residue in any peptide set forth as any one of the peptides set forth in in Table 1. In particular aspects, a modified sequence is at least 80% or more, e.g., 80-85%, 85-90%, 90-95%, 95-100% identical, to a ApoBlOO peptide, or sub-sequence, portion, homologue or derivative thereof set forth as any one of the peptides set forth in in Table 1 or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13,Docket No. 051501-058535814, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50-100, or more, additions to, deletions of, or substitutions.

[0057] Thus, in accordance with the invention, modified and variant forms of ApoBlOO peptides, sub-sequences, portions, homologues or derivatives thereof are provided. Such forms, referred to as “modifications” or “variants” and grammatical variations thereof, mean an ApoBlOO peptide, sub-sequence, portion, homologue or derivative thereof that deviates from a reference sequence. Such modifications may have greater or less activity or function than a reference ApoBlOO peptide, sub-sequence or portion thereof, such as ability to elicit, stimulate, induce, promote, increase or enhance T cell response or immune or inflammatory response. Thus, proteins or peptides of the present invention, sub-sequences, portions, homologues, variants or derivatives thereof, include sequences having substantially the same, greater or less relative activity or function as a T cell epitope than a reference T cell epitope set forth as any one of the peptides set forth in in Table 1, for example, an ability to elicit, stimulate, induce, promote, increase or enhance a T response in vitro or in vivo.

[0058] Non-limiting examples of modifications include one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50-100, or more residues), additions and insertions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50-100, or more residues) and deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50-100) of a reference ApoBlOO peptide, sub-sequence, portion, homologue or derivative thereof. In particular embodiments, a modified or variant sequence retains at least part of a function or an activity of unmodified sequence, which can have less than, approximately the same, or greater, but at least a part of, a function or activity of a reference sequence, for example, the ability to elicit, stimulate, induce, promote, increase or enhance a T cell response in vitro or in vivo. Such T cell responses elicited include, for example, among others, induced, increased, enhanced, stimulated or activated expression or production of a cytokine (e.g., IL-la, IL-2, IL-5, IL-6, IL-10, IL-17a, IFN-y a.k.a. IFNg, TNF, GM-CSF).

[0059] An addition can be the covalent or non-covalent attachment of any type of molecule to the sequence. Specific examples of additions include glycosylation, acetylation, phosphorylation, amidation, formylation, ubiquitination, and derivatization by protecting / blocking groups and any of numerous chemical modifications. Additional specific non-limiting examples of an addition is one or more additional amino acidDocket No. 051501-0585358residues. Accordingly, proteins or peptides of the present invention including T cell epitopes, sub-sequences, portions, homologues or derivatives thereof can be a part of or contained within a larger molecule, such as another peptide sequence, such as a fusion, heterologous domain or chimera with another ApoB 100 sequence, or anon-ApoBlOO peptide sequence. In particular embodiments, an addition is a fusion (chimeric) sequence or heterologous domain, an amino acid sequence having one or more molecules not normally present in a reference native (wild type) sequence covalently attached to the sequence.

[0060] The term “chimeric” and grammatical variations thereof, when used in reference to a sequence, means that the sequence contains one or more portions that are derived from, obtained or isolated from, or based upon other physical or chemical entities. For example, a chimera of two or more different proteins may have one part an ApoB 100 peptide, or sub-sequence, portion, homologue, variant or derivative thereof, and a second part of the chimera may be from a non-ApoBlOO sequence.

[0061] Another particular example of a modified sequence having an amino acid addition is one in which a second heterologous sequence, i.e., heterologous functional domain is attached (covalent or non-covalent binding) that confers a distinct or complementary function. Heterologous functional domains are not restricted to amino acid residues. Thus, a heterologous functional domain can consist of any of a variety of different types of small or large functional moieties. Such moieties include nucleic acid, peptide, carbohydrate, lipid or small organic compounds, such as a drug (e.g., an antiviral), a metal (gold, silver), and radioisotope. For example, a tag such as T7 or polyhistidine can be attached in order to facilitate purification or detection of a T cell epitope. Thus, in other embodiments the invention provides a protein or peptide comprising an ApoB 100 peptide, or sub-sequence, portion, homologue, variant or derivative thereof and a heterologous domain, wherein the heterologous functional domain confers a distinct function, on the ApoB 100 peptide, or sub-sequence, portion, homologue, variant or derivative thereof. For example, a fusion protein or chimera may comprise an immunoglobulin fusion protein or a monoclonal antibody fusion protein. Such constructs containing an ApoB 100 peptide, or sub-sequence, portion, homologue, variant or derivative thereof and a heterologous domain are also referred to as chimeras.

[0062] Linkers, such as amino acid or peptidomimetic sequences may be inserted between the sequence and the addition (e.g., heterologous functional domain) so that the two entities maintain, at least in part, a distinct function or activity. Linkers may haveDocket No. 051501-0585358one or more properties that include a flexible conformation, an inability to form an ordered secondary structure or a hydrophobic or charged character which could promote or interact with either domain. Amino acids typically found in flexible protein regions include Gly, Asn and Ser. Other near neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. The length of the linker sequence may vary without significantly affecting a function or activity of the fusion protein (see, e.g., U.S. Patent No. 6,087,329). Linkers further include chemical moieties and conjugating agents, such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB), disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG) and disuccinimidyl tartrate (DST).

[0063] Further non-limiting examples of additions are detectable labels. Thus, in another embodiment, the invention provides the presently described proteins or peptides, or sub-sequences, portions, homologues, variants or derivatives thereof that are detectably labelled. Specific examples of detectable labels include fluorophores, chromophores, radioactive isotopes (e.g., S35, P32, I125), electron-dense reagents, enzymes, ligands and receptors. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert a substrate such as 3,3-’,5,5-’-tetramethylbenzidine (TMB) to a blue pigment, which can be quantified.

[0064] Another non-limiting example of an addition is an insertion of an amino acid within a ApoBlOO peptide, or sub-sequence, portion, homologue, variant or derivative thereof sequence, sub-sequence, portions or modification thereof, set forth as any one of the peptides set forth in in Table 1. In particular embodiments, an insertion is of one or more amino acid residues inserted into an ApoBlOO peptide, or sub-sequence, portion, homologue, variant or derivative thereof, set forth as any one of the peptides set forth in in Table 1.

[0065] Specific non-limiting examples of substitutions include conservative and nonconservative amino acid substitutions. A “conservative substitution” is the replacement of one amino acid by a biologically, chemically or structurally similar residue.Biologically similar means that the substitution does not destroy a biological activity. Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or a similar size. Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic. Particular examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another,Docket No. 051501-0585358such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, serine for threonine, and the like.

[0066] Modified and variant proteins and peptides of the present invention, or subsequences, portions, homologues, variants or derivatives thereof also include one or more D-amino acids substituted for L-amino acids (and mixtures thereof), structural and functional analogues, for example, peptidomimetics having synthetic or non-natural amino acids or amino acid analogues and derivatized forms. Modifications include cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulphide bond. Proteins and peptides of the present invention, or sub-sequences, portions, homologues, variants or derivatives thereof, may be modified in vitro or in vivo, e.g., post-translationally modified to include, for example, sugar residues, phosphate groups, ubiquitin, fatty acids, lipids, etc.

[0067] Specific non-limiting examples of modifications include at least one amino acid deletion from ApoBlOO peptide sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof. In particular embodiments, a peptide sub-sequence or portion is from about 1 to 14 amino acids in length, provided that said sub-sequence or portion is at least one amino acid less in length than the full-length ApoBlOO peptide sequence set forth as any one of the peptides set forth in in Table 1, or a homologue, variant or derivative thereof. In additional particular embodiments, a peptide sub-sequence or portion is from about 1 to 5, 5 to 10, 10 to 14, amino acids in length, provided that said sub-sequence or portion is at least one amino acid less in length than the full-length ApoBlOO peptide sequence set forth as any one of the peptides set forth in in Table 1, or a homologue, variant or derivative thereof.

[0068] As used herein, a sub-sequence of a protein or peptide includes or consists of one or more amino acids less than the full-length protein or peptide. The term “subsequence” means a fragment or part of the full-length molecule. A sub-sequence of a protein or peptide has one or more amino acids less than the full-length protein or peptide (e.g. one or more internal or terminal amino acid deletions from either amino or carboxytermini). Sub-sequences therefore can be any length up to the full-length native molecule, provided said length is at least one amino acid less than full-length native molecule.

[0069] Sub-sequences can vary in size. For example a sub-sequence of a protein or peptide can be as small as an epitope capable of binding an antibody (i.e. about fiveDocket No. 051501-0585358amino acids) up to a polypeptide that is one amino acid less than the entire length of a reference protein or peptide.

[0070] In various embodiments, a sub-sequence of the protein or peptides of the present invention is characterized as including or consisting of a sub-sequence of an ApoB100 peptide set forth as any one of the peptides set forth in in Table 1, or homologue, variant or derivative thereof.

[0071] As used herein, sub-sequences may also include or consist of one or more amino acid additions or deletions, wherein the sub-sequence does not comprise the full-length ApoB100 peptide set forth as any one of the peptides set forth in in Table 1, or ahomologue, variant or derivative thereof. Accordingly, total sub-sequence lengths can be greater than the length of the full-length ApoB100 peptide set forth as any one of the peptides set forth in in Table 1, a homologue, variant or derivative thereof, for example, where a sub-sequence of anApoB100 peptide set forth as any one of the peptides set forth in in Table 1is fused or forms a chimera with another polypeptide.

[0072] Proteins or peptides of the present invention, or sub-sequences, portions, homologues, derivative thereof including variant or modified forms can be produced by any of a variety of standard protein purification or recombinant expression techniques. For example, a peptide, sub-sequence, portion, homologue, derivative or variant thereof can be produced by standard peptide synthesis techniques, such as solid-phase synthesis. A portion of the protein may contain an amino acid sequence such as a T7 tag or polyhistidine sequence to facilitate purification of expressed or synthesized protein. The protein may be expressed in a cell and purified. The protein may be expressed as a part of a larger protein (e.g., a fusion or chimera) by recombinant methods.

[0073] Proteins or peptides of the present invention, or sub-sequences, portions, homologues, derivative or variants thereof can be made using recombinant DNA technology via cell expression or in vitro translation. Polypeptide sequences including modified forms can also be produced by chemical synthesis using methods known in the art, for example, an automated peptide synthesis apparatus (see, e.g., Applied Biosystems, Foster City, CA).

[0074] The invention provides isolated and / or purified protein or peptides comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof. In particular embodiments, the isolated and / or purified proteins orDocket No. 051501-0585358peptides, or sub-sequence, portion, homologue, variant or derivative thereof includes a T cell epitope.

[0075] The term “isolated,” when used as a modifier of a composition (e.g., ApoB100 peptides, or sub-sequences, portions, homologues, variants or derivatives thereof, nucleic acids encoding same, etc.), means that the compositions are made by the hand of man or are separated, completely or at least in part, from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane. The term “isolated” does not exclude alternative physical forms of the composition, such as fusions / chimeras, multimers / oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host cells produced by the hand of man.

[0076] An “isolated” composition (e.g., ApoB100 peptides, or sub-sequences,portions, homologues, variants or derivatives thereof) can also be “substantially pure” or “purified” when free of most or all of the materials with which it typically associates with in nature. Thus, an isolated ApoB100 peptide, or sub-sequence, portion, homologue, variant or derivative thereof that also is substantially pure or purified does not include polypeptides or polynucleotides present among millions of other sequences, such as peptides of a peptide library or nucleic acids in a genomic or cDNA library, for example.

[0077] A “substantially pure” or “purified” composition can be combined with one or more other molecules. Thus, “substantially pure” or “purified” does not exclude combinations of compositions, such as combinations of ApoB100 peptides, or subsequences, portions, homologues, variants or derivatives thereof (e.g., multiple T cell epitopes), and combination of an ApoB100 peptide, or sub-sequence, portion, homologue, variant or derivative thereof with other antigens, agents, drugs or therapies.

[0078] The invention also provides nucleic acids encoding proteins or peptides comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof. Such nucleic acid sequences encode a peptide sequence at least 75% or more (e.g., 75%, 80%, 85%, 90%, 95%, etc.) identical to an ApoB100 amino acid sequence set forth as any one of the peptides set forth in in Table 1. In an additional embodiment, a nucleic acid encodes a sequence having a modification, such as one or more amino acid additions (insertions), deletions or substitutions of an ApoB100 amino acid sequence set forth as any one of the peptides set forth in in Table 1.Docket No. 051501-0585358

[0079] The terms “nucleic acid,” “polynucleotide” and “polynucleoside” and the like refer to at least two or more ribo- or deoxy-ribonucleic acid base pairs (nucleotides / nucleosides) that are linked through a phosphoester bond or equivalent. Nucleic acids include polynucleotides and polynucleosides. Nucleic acids include single, double or triplex, circular or linear, molecules. Exemplary nucleic acids include but are not limited to: RNA, DNA, cDNA, genomic nucleic acid, naturally occurring and non naturally occurring nucleic acid, e.g., synthetic nucleic acid.

[0080] Nucleic acids can be of various lengths. Nucleic acid lengths typically range from about 20 bases to 20 Kilobases (Kb), or any numerical value or range within or encompassing such lengths, 10 bases to 10Kb, 1 to 5 Kb or less, 1000 to about 500 bases or less in length. Nucleic acids can also be shorter, for example, 100 to about 500 bases, or from about 12 to 25, 25 to 50, 50 to 100, 100 to 250, or about 250 to 500 bases in length, or any numerical value or range or value within or encompassing such lengths. In particular aspects, a nucleic acid sequence has a length from about 10-20, 20-30, 30-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-1000, 1000-2000 bases, or any numerical value or range within or encompassing such lengths. Shorter nucleic acids are commonly referred to as “oligonucleotides” or “probes” of single- or double-stranded DNA. However, there is no upper limit to the length of such oligonucleotides.

[0081] Nucleic acid sequences further include nucleotide and nucleoside substitutions, additions and deletions, as well as derivatized forms and fusion / chimeric sequences (e.g., encoding recombinant polypeptide). For example, due to the degeneracy of the genetic code, nucleic acids include sequences and sub-sequences degenerate with respect to nucleic acids that encode an ApoB100 peptide comprising, consisting or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, as well as variants and modifications thereof (e.g., substitutions, additions, insertions and deletions).

[0082] Nucleic acids can be produced using various standard cloning and chemical synthesis techniques. Techniques include, but are not limited to nucleic acid amplification, e.g., polymerase chain reaction (PCR), with genomic DNA or cDNA targets using primers (e.g., a degenerate primer mixture) capable of annealing to the encoding sequence. Nucleic acids can also be produced by chemical synthesis (e.g., solid phase phosphoramidite synthesis) or transcription from a gene. The sequences produced can then be translated in vitro, or cloned into a plasmid and propagated and thenDocket No. 051501-0585358expressed in a cell (e.g., a host cell such as eukaryote or mammalian cell, yeast or bacteria, in an animal or in a plant).

[0083] Nucleic acid may be inserted into a nucleic acid construct in which expression of the nucleic acid is influenced or regulated by an “expression control element.” An “expression control element” refers to a nucleic acid sequence element that regulates or influences expression of a nucleic acid sequence to which it is operatively linked.Expression control elements include, as appropriate, promoters, enhancers, transcription terminators, gene silencers, a start codon (e.g., ATG) in front of a protein-encoding gene, etc.

[0084] An expression control element operatively linked to a nucleic acid sequence controls transcription and, as appropriate, translation of the nucleic acid sequence.Expression control elements include elements that activate transcription constitutively, that are inducible (i.e., require an external signal for activation), or derepressible (i.e., require a signal to turn transcription off; when the signal is no longer present, transcription is activated or “derepressed”), or specific for cell-types or tissues (i.e., tissue-specific control elements).

[0085] Nucleic acid can also be inserted into a plasmid for propagation into a host cell and for subsequent genetic manipulation. A plasmid is a nucleic acid that can be propagated in a host cell, plasmids may optionally contain expression control elements in order to drive expression of the nucleic acid encoding an ApoB100 peptide comprising, consisting or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or sub-sequence, portion, homologue, variant orderivative thereof, in the host cell. A vector is used herein synonymously with a plasmid and may also include an expression control element for expression in a host cell (e.g., expression vector). Plasmids and vectors generally contain at least an origin of replication for propagation in a cell and a promoter. Plasmids and vectors are therefore useful for genetic manipulation and expression of proteins or peptides of the present invention, or sub-sequences, portion, homologues, variants or derivatives thereof.Accordingly, vectors that include nucleic acids encoding or complementary to an ApoB100 peptide comprising, consisting or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or sub-sequence,portion, homologue, variant or derivative thereof are provided.

[0086] In accordance with the invention, there are provided particles (e.g., viral particles) and transformed host cells that express and / or are transformed with a nucleicDocket No. 051501-0585358acid that encodes and / or express an ApoB100 peptide comprising, consisting orconsisting essentially of an amino acid sequence set forth as any one of the peptides setforth in in Table 1, or sub-sequence, portion, homologue, variant or derivative thereof. Particles and transformed host cells include but are not limited to virions, and prokaryotic and eukaryotic cells such as bacteria, fungi (yeast), plant, insect, and animal (e.g., mammalian, including primate and human, CHO cells and hybridomas) cells. For example, bacteria transformed with recombinant bacteriophage nucleic acid, plasmid nucleic acid or cosmid nucleic acid expression vectors; yeast transformed with recombinant yeast expression vectors; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid); insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); and animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus), or transformed animal cell systems engineered for stable expression. The cells may be a primary cell isolate, cell culture (e.g., passaged, established or immortalized cell line), or part of a plurality of cells, or a tissue or organ ex vivo or in a subject (in vivo). Transfected or transformed cells of the invention may include tolerogenic dendritic cells or other antigen presenting cells that may enhance the disease reducing activity of the ApoB100 protein or peptide of the invention.

[0087] The term “transformed” or “transfected” when used in reference to a cell (e.g., a host cell) or organism, means a genetic change in a cell following incorporation of an exogenous molecule, for example, a protein or nucleic acid (e.g., a transgene) into the cell. Thus, a “transfected” or “transformed” cell is a cell into which, or a progeny thereof in which an exogenous molecule has been introduced by the hand of man, for example, by recombinant DNA techniques.

[0088] The nucleic acid or protein can be stably or transiently transfected or transformed (expressed) in the host cell and progeny thereof. The cell(s) can be propagated and the introduced protein expressed, or nucleic acid transcribed. A progeny of a transfected or transformed cell may not be identical to the parent cell, since there may be mutations that occur during replication.

[0089] Expression of an ApoB100 peptide comprising, consisting or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in inTable 1, or sub-sequence, portion, homologue, variant or derivative thereof and nucleic acid encoding such peptides in particles or introduction into target cells (e.g., host cells)Docket No. 051501-0585358can also be carried out by methods known in the art. Non-limiting examples include osmotic shock (e.g., calcium phosphate), electroporation, microinjection, cell fusion, etc. Introduction of nucleic acid and polypeptide in vitro, ex vivo and in vivo can also be accomplished using other techniques. For example, a polymeric substance, such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide / glycolide copolymers, polylactide / glycolide copolymers, or ethylenevinylacetate copolymers. A nucleic acid can be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatinmicrocapsules, or poly (methylmethacrolate) microcapsules, respectively, or in a colloid system. Colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0090] Liposomes for introducing various compositions into cells are known in the art and include, for example, phosphatidylcholine, phosphatidylserine, lipofectin and DOTAP (e.g., U. S. Patent Nos. 4,844,904, 5,000,959, 4,863,740, and 4,975,282; and GIBCO-BRL, Gaithersburg, MD). Piperazine based amphilic cationic lipids useful for gene therapy also are known (see, e.g., U. S. Patent No. 5,861,397). Cationic lipid systems also are known (see, e.g., U. S. Patent No. 5,459,127). Polymeric substances, microcapsules and colloidal dispersion systems such as liposomes are collectively referred to herein as “vesicles.” Accordingly, viral and non-viral vector means delivery into cells are included.

[0091] As disclosed herein, an ApoB100 peptide comprising, consisting or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or sub-sequence, portion, homologue, variant or derivative thereof may elicit a cellular or humoral immune response. In particular embodiments of the present invention, the proteins or peptides of the present invention may elicit, stimulate, induce, promote, increase or enhance an immune or inflammatory response. In certain embodiments, the proteins or peptides of the present invention may elicit, stimulate induce, promote, increase or enhance a T cell response.

[0092] However, the present inventors have found that an ApoB100 peptide comprising, consisting or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or sub-sequence, portion, homologue, variant or derivative thereof may produce a pro-inflammatory or atherogenic response orDocket No. 051501-0585358alternatively an anti-inflammatory or protective response depending on the manner in which the peptide is administered. For example, changes in the type of adjuvant administered with the peptides, the timing of administration, the amount of peptide administered may all effect whether a peptide produces an atherogenic or protective response.

[0093] As used herein an atherogenic response refers to a inducing, promoting, increasing or enhancing the development, severity, or symptoms of an adverse cardiovascular event or cardiovascular disease, including for example, atherosclerosis. In certain embodiments, an atherogenic response comprises an increase in the occurrence or size of atherosclerosis lesions.

[0094] As used herein, a protective response refers to decreasing, reducing, inhibiting, suppressing or disrupting the development, severity, or symptoms of an adverse cardiovascular event or cardiovascular disease, including for example, atherosclerosis. In certain embodiments, an atherogenic response comprises a decrease in the occurrence or size of atherosclerosis lesions.

[0095] Thus in certain embodiments of the present invention, a protein or peptide comprising, consisting or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or sub-sequence, portion, homologue, variant or derivative thereof may produce an atherogenic response. In other embodiments, of the present invention, a protein or peptide comprising, consisting or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or subsequence, portion, homologue, variant or derivative thereof may produce a protective response. Such responses can provide protection against (e.g., prophylaxis) an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis) or a secondary or subsequent occurrence of an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis). Such responses can also be effective in treatment (e.g., therapeutic) of an initial adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis), or a secondary or subsequent adverse cardiovascular event or cardiovascular disease.

[0096] In particular embodiments, the proteins or peptides of the present invention may decrease, reduce, inhibit, suppress or disrupt an immune or inflammatory response. In still further embodiments, the proteins or peptides of the present invention may elicit, stimulate, induce, promote, increase or enhance an anti- immune or anti- inflammatory response.Docket No. 051501-0585358

[0097] The proteins or peptides of the present invention, or sub-sequences, portions, homologues, variants or derivatives thereof can be employed in various methods, uses and compositions. In different embodiments, the methods, uses and compositions of the present invention comprise a protein or peptide comprising, consisting or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or sub-sequence, portion, homologue, variant or derivative thereof.

[0098] Such methods and uses include, for example, use, contact or administration of one or more proteins or peptides of the present invention, or sub-sequences, portions, homologues, variants or derivatives in vitro and in vivo. Such methods are applicable to providing a subject with protection vaccinate against an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis), and also are applicable to providing treatment to a subject for an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis).

[0099] In certain embodiments of the present invention, proteins or peptides of the present invention, or sub-sequences, portions, homologues, variants or derivatives thereof may be used as vaccine antigens to vaccinate against an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis), or one or more physiological conditions, disorders, illness, diseases or symptoms caused by or associated with an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis).

[0100] Thus, in accordance with one aspect of the present invention, there are provided methods for vaccination and immunization to protect against an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis).

[0101] In one embodiment, there is provided a method of vaccinating a subject against an adverse cardiovascular event or cardiovascular disease, the method comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to vaccinate the subject against the adverse cardiovascular event or cardiovascular disease.

[0102] As used herein, the terms “vaccination”, “vaccinate”, “protection”, “protect” and grammatical variations thereof, when used in reference to an adverse cardiovascular event or cardiovascular disease, means preventing an adverse cardiovascular event or cardiovascular disease, or reducing or decreasing susceptibility to an adverse cardiovascular event or cardiovascular disease, or reducing or decreasing severity of anDocket No. 051501-0585358adverse cardiovascular event or cardiovascular disease, or preventing or reducing one or more symptoms or pathologies caused by or associated with an adverse cardiovascular event or cardiovascular disease. In particular embodiments, the method of vaccinating a subject against an adverse cardiovascular event or cardiovascular disease of the present invention comprises a method of vaccinating a subject against atherosclerosis.

[0103] In another aspect of the present invention there are provided methods for treatment of an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis).

[0104] Thus, in one embodiment of the present invention there is provided a method of treating a subject for an adverse cardiovascular event or cardiovascular disease, the method comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to treat the subject for the adverse cardiovascular event or cardiovascular disease.

[0105] As will be understood by a person skilled in the art, treating a subject for an adverse cardiovascular event or cardiovascular disease may include decreasing, reducing, inhibiting, suppressing, limiting, controlling or eliminating an adverse cardiovascular event or cardiovascular disease. In other embodiments, a method of treating a subject for an adverse cardiovascular event or cardiovascular disease comprises reducing the frequency, severity, progression, or duration of the adverse cardiovascular event or cardiovascular disease in the subject. In yet another embodiment, a method of treating a subject for an adverse cardiovascular event or cardiovascular disease comprises maintaining the severity of an adverse cardiovascular event or cardiovascular disease in a subject by preventing an increase in the occurrence, frequency, severity, progression, or duration of the adverse cardiovascular event or cardiovascular disease in the subject. In still further embodiments, a method of treating a subject for an adverse cardiovascular event or cardiovascular disease comprises eliminating, reducing or maintaining the occurrence, frequency, severity, progression, or duration of physiological conditions, disorders, illnesses, diseases, symptoms or complications caused by or associated with the adverse cardiovascular event or cardiovascular disease.

[0106] In certain embodiments, the subject of the methods provided herein may have been previously had an adverse cardiovascular event or cardiovascular disease. Thus, in certain embodiments, the present methods may be used for treating or protecting a subjectDocket No. 051501-0585358from a secondary or subsequent adverse cardiovascular event or cardiovascular disease. Thus in different embodiments, the presently described methods of vaccination and treatment may be used for prophylactic vaccination against or treatment of an adverse cardiovascular event or cardiovascular disease or can be used to vaccinate against or treatment of a secondary or subsequent occurrence of an adverse cardiovascular event or cardiovascular disease. In particular embodiments, the methods may be used to vaccinate against or treat an adverse symptom of the adverse cardiovascular event or cardiovascular disease.

[0107] In accordance with different embodiments of the present invention, the therapeutic and prophylactic methods of vaccinating against and treating a subject for an adverse cardiovascular event or cardiovascular disease include but are not limited to treatment of a subject having or at risk of having an adverse cardiovascular event or cardiovascular disease, treating a subject with an adverse cardiovascular event or cardiovascular disease, and methods of protecting a subject from an adverse cardiovascular event or cardiovascular disease (e.g., provide the subject with protection against the development or incidence of an adverse cardiovascular event or cardiovascular disease), to decrease or reduce the probability of an adverse cardiovascular event or cardiovascular disease in a subject, to decrease or reduce susceptibility of a subject to an adverse cardiovascular event or cardiovascular disease and to inhibit or prevent an adverse cardiovascular event or cardiovascular disease in a subject. In particular embodiments of the methods described herein, one or more disorders, diseases, physiological conditions, pathologies and symptoms associated with or caused by an adverse cardiovascular event or cardiovascular disease will respond to vaccination or treatment.

[0108] Non-limiting examples of an adverse cardiovascular event or cardiovascular disease are atherosclerosis, coronary artery disease, peripheral artery disease, cerebrovascular disease, renal artery disease, stroke, myocardial infarction (heart attack), ischemic heart failure, transient ischemic attack or brain trauma, artherosclerotic plaque formation, foam cells or foam cell formation, or one or more adverse symptoms, disorders, illnesses, pathologies, diseases, or complications caused by or associated with an adverse cardiovascular event or cardiovascular disease such as atherosclerosis, coronary artery disease, peripheral artery disease, cerebrovascular disease, renal artery disease, stroke, myocardial infarction (heart attack), ischemic heart failure, transientDocket No. 051501-0585358ischemic attack or brain trauma), artherosclerotic plaque formation or foam cells or foam cell formation.

[0109] In certain embodiments of the presently described methods, two or more proteins or peptides comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, may be administered to a subject. As will be understood by a skilled person two or more proteins or peptides comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, may be administered as a combination composition, or administered separately, such as concurrently or in series or sequentially. Different proteins or peptides of the present invention, or a sub-sequence, portion, homologue, variant or derivative thereof, may be administered to a subject in the same amount, volume or concentration or different amounts, volumes or concentrations. Thus in certain embodiments, the subject may be administered the same amount of two or more different proteins or peptides of the present invention, or a sub-sequence, portion, homologue, variant or derivative thereof. In other embodiments, the subject may be administered one a protein or peptide of the present invention, or a sub-sequence, portion, homologue, variant or derivative thereof, in a amount, volume or concentration greater than one or more other protein or peptide of the present invention, or a sub-sequence, portion, homologue, variant or derivative thereof, administered to the subject.

[0110] Methods of the invention include methods of vaccination or treatment that result in any therapeutic or beneficial effect. In various methods embodiments, an adverse cardiovascular event or cardiovascular disease is reduced, decreased, inhibited, limited, delayed or prevented, or a method decreases, reduces, inhibits, suppresses, prevents, controls or limits one or more adverse (e.g., physical) symptoms, disorders, illnesses, diseases or complications caused by or associated with adverse cardiovascular event or cardiovascular disease. In additional various particular embodiments, methods of the present invention include reducing, decreasing, inhibiting, delaying or preventing onset, progression, frequency, duration, severity, probability or susceptibility of one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with an adverse cardiovascular event or cardiovascular disease. In further various particular embodiments, methods of the present invention include improving, accelerating, facilitating, enhancing, augmenting, or hastening recovery of a subject fromDocket No. 051501-0585358an adverse cardiovascular event or cardiovascular disease, or one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with an adverse cardiovascular event or cardiovascular disease. In yet additional various embodiments, methods of treatment include stabilizing an adverse cardiovascular event or cardiovascular disease, or an adverse symptom, disorder, illness, disease or complication caused by or associated with an adverse cardiovascular event or cardiovascular disease.

[0111] A therapeutic or beneficial effect of vaccination or treatment is therefore any objective or subjective measurable or detectable improvement or benefit provided to a particular subject. A therapeutic or beneficial effect can but need not be complete ablation of or protection from all or any particular adverse symptom, disorder, illness, disease or complication caused by or associated with adverse cardiovascular event or cardiovascular disease Thus, a satisfactory clinical endpoint is achieved when there is an incremental improvement or a partial reduction in an adverse symptom, disorder, illness, disease or complication caused by or associated with an adverse cardiovascular event or cardiovascular disease or an inhibition, decrease, reduction, suppression, prevention, limit or control of worsening or progression of one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with an adverse cardiovascular event or cardiovascular disease over a short or long duration (hours, days, weeks, months, etc.).

[0112] A therapeutic or beneficial effect also includes reducing or eliminating the need, dosage frequency or amount of a second active such as another drug or other agent used for treating a subject having or at risk of having an adverse cardiovascular event or cardiovascular disease For example, reducing an amount of an adjunct therapy, for example, a reduction or decrease of a treatment for an adverse cardiovascular event or cardiovascular disease, or a vaccination or immunization protocol for an adverse cardiovascular event or cardiovascular disease is considered a beneficial effect. In addition, reducing or decreasing an amount of protein or peptide used for vaccination or immunization of a subject to provide protection to the subject is considered a beneficial effect.

[0113] In another aspect of the present invention there is provided a composition comprising a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a subsequence, portion, homologue, variant or derivative thereof. In different embodiments, the composition of the present invention may comprise an adjuvant. In certainDocket No. 051501-0585358embodiment, the composition of the present invention may be a solid or a liquid. In particular embodiments, the composition of the present invention is a vaccine.

[0114] Methods and compositions of the invention include administration of a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, to a subject prior to development of an adverse cardiovascular event or cardiovascular disease, administration substantially contemporaneously with development of an adverse cardiovascular event or cardiovascular disease, and administration after development of an adverse cardiovascular event or cardiovascular disease. Methods and compositions of the invention also include administration of a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, to a subject prior to, substantially contemporaneously with or following development of an adverse symptom, disorder, illness or disease caused by or associated with an adverse cardiovascular event or cardiovascular disease. A subject suffering from an adverse cardiovascular event or cardiovascular disease or an adverse symptom, disorder, illness or disease caused by or associated with an adverse cardiovascular event or cardiovascular disease have the adverse cardiovascular event, cardiovascular disease or symptom over a period of 1-5, 5-10, 10-20, 20-30, 30-50, 50-100 hours, days, months, or years.

[0115] Methods and compositions of the invention include administration of a protein or peptide comprising, consisting or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, to a subject alone or in combination with any compound, agent, drug, treatment or other therapeutic regimen or protocol having a desired therapeutic, beneficial, additive, synergistic or complementary activity or effect.

[0116] Exemplary combination compositions and treatments include multiple proteins or peptides comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, such as T cell epitopes as described herein, and second actives, such as compounds, agents, drugs, treatments and therapies for the treatment of an adverse cardiovascular event or cardiovascular disease (e.g. statins, fibrate), as well as agents that assist, promote, stimulate or enhance efficacy. Such compounds, agents, drugs, treatments and therapies can be administered or performedDocket No. 051501-0585358prior to, substantially contemporaneously with or following any composition or method of the invention, for example, a therapeutic use or method of vaccinating or treating a subject for an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis), or a method of prophylactic vaccination or treatment of a subject for an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis).

[0117] Invention compositions comprising a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, and methods described herein can be combined with any compound, agent, drug, treatment or other therapeutic regimen or protocol having a desired therapeutic, beneficial, additive, synergistic or complementary activity or effect. Exemplary combination compositions and treatments include multiple proteins or peptides comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, such as T cell epitopes as described herein, and second actives, such as compounds, agents, drugs, treatments and therapies for the treatment of an adverse cardiovascular event or cardiovascular disease (e.g. statins, fibrate), as well as agents that assist, promote, stimulate or enhance efficacy. Such compounds, agents, drugs, treatments and therapies can be administered or performed prior to, substantially contemporaneously with or following any composition or method of the invention, for example, a therapeutic use or method of vaccinating or treating a subject for an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis), or a method of prophylactic vaccination or treatment of a subject for an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis).

[0118] The invention therefore provides combinations of one or more proteins or peptides comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, in combination with a second active, including but not limited to any compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition, such as a treatment or vaccination protocol set forth herein or known in the art. The compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition can be administered or performed prior to, substantially contemporaneously with or following administration of one or more proteins or peptides comprising, consisting of or consisting essentially of an amino acid sequenceDocket No. 051501-0585358set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, or a nucleic acid encoding all or a portion (e.g., a T cell epitope) of proteins or peptides comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, to a subject. Specific non-limiting examples of combination embodiments therefore include the foregoing or other compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition.

[0119] In invention methods, uses and compositions for which there is a desired outcome, such as a therapeutic or prophylactic method that provides a benefit from treatment, vaccination or immunization, a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, can be administered in a sufficient or effective amount.

[0120] As used herein, a “sufficient amount” or “effective amount” or an “amount sufficient” or an “amount effective” refers to an amount that provides, in single (e.g., primary) or multiple (e.g., booster) doses, alone or in combination with one or more other compounds, treatments, therapeutic regimens or agents (e.g., a drug), a long term or a short term detectable or measurable improvement in a given subject or any objective or subjective benefit to a given subject of any degree or for any time period or duration (e.g., for minutes, hours, days, months, years, or cured).

[0121] An amount sufficient or an amount effective can but need not be provided in a single administration and can but need not be achieved by a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, alone, optionally in a combination composition or method that includes a second active. In addition, an amount sufficient or an amount effective need not be sufficient or effective if given in single or multiple doses without a second or additional administration or dosage, since additional doses, amounts or duration above and beyond such doses, or additional antigens, compounds, drugs, agents, treatment or therapeutic regimens may be included in order to provide a given subject with a detectable or measurable improvement or benefit to the subject. For example, to increase, enhance, improve or optimize immunization and / or vaccination, after an initial or primary administration of one or more proteins or peptides comprising, consisting of or consistingDocket No. 051501-0585358essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, to a subject, the subject can be administered one or more additional “boosters” of one or more proteins or peptides comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof. Such subsequent “booster” administrations can be of the same or a different formulation, dose or concentration, route, etc.

[0122] An amount sufficient or an amount effective need not be therapeutically or prophylactically effective in each and every subject treated, nor a majority of subjects treated in a given group or population. An amount sufficient or an amount effective means sufficiency or effectiveness in a particular subject, not a group of subjects or the general population. As is typical for such methods, different subjects will exhibit varied responses to a method of the invention, such as immunization, vaccination and therapeutic treatments.

[0123] The term “subject” refers includes but is not limited to a subject at risk of an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis), as well as a subject that has already developed an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis). Such subjects, include mammalian animals (mammals), such as a non human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans. Subjects include animal disease models, for example, mouse and other animal models of an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis) known in the art.

[0124] Accordingly, subjects appropriate for vaccination or treatment include those having or at risk of an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis), also referred to as subjects in need of treatment. Subjects in need of vaccination or treatment therefore include subjects that have been previously had an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis) or that have an ongoing adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis) or have developed one or more adverse symptoms caused by or associated with an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis), regardless of the type, timing or degree of onset, progression, severity, frequency, duration of the symptoms.Docket No. 051501-0585358

[0125] Prophylactic uses and methods are therefore included. Target subjects for prophylaxis may be at increased risk (probability or susceptibility) of developing an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis). Such subjects are considered in need of treatment due to being at risk.

[0126] Subjects for prophylaxis need not be at increased risk but may be from the general population in which it is desired to vaccinate or immunize a subject against an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis), for example. Such a subject that is desired to be vaccinated or immunized against an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis) can be administered a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof. In another non-limiting example, a subject that is not specifically at risk for an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis), but nevertheless desires protection against an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis), can be administered a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a subsequence, portion, homologue, variant or derivative thereof. Such subjects are also considered in need of treatment.

[0127] “Prophylaxis” and grammatical variations thereof mean a method in which contact, administration or in vivo delivery to a subject is prior to development of an adverse cardiovascular event or cardiovascular disease. In certain situations it may not be known that a subject has developed an adverse cardiovascular event or cardiovascular disease, but administration or in vivo delivery to a subject can be performed prior to manifestation of disease pathology or an associated adverse symptom, condition, complication, etc. caused by or associated with an adverse cardiovascular event or cardiovascular disease. In such case, a composition or method of the present invention can eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility to an adverse cardiovascular event or cardiovascular, or an adverse symptom, condition or complication associated with or caused by an adverse cardiovascular event or cardiovascular disease.

[0128] “Prophylaxis” can also refer to a method in which contact, administration or in vivo delivery to a subject is prior to a secondary or subsequent exposure or infection. In such a situation, a subject may have had a prior adverse cardiovascular event orDocket No. 051501-0585358cardiovascular disease or prior adverse symptom, condition or complication associated with or caused by an adverse cardiovascular event or cardiovascular disease. Vaccination or treatment by administration or in vivo delivery to such a subject, can be performed prior to a secondary or subsequent adverse cardiovascular event or cardiovascular disease. Such a method can eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility towards a secondary or subsequent adverse cardiovascular event or cardiovascular disease, or an adverse symptom, condition or complication associated with or caused by or associated with a secondary or subsequent adverse cardiovascular event or cardiovascular disease.

[0129] Treatment of an adverse cardiovascular event or cardiovascular disease can be at any time during the adverse cardiovascular event or cardiovascular disease. A protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, can be administered as a combination (e.g., with a second active), or separately concurrently or in sequence (sequentially) in accordance with the methods described herein as a single or multiple dose e.g., one or more times hourly, daily, weekly, monthly or annually or between about 1 to 10 weeks, or for as long as appropriate, for example, to achieve a reduction in the onset, progression, severity, frequency, duration of one or more symptoms or complications associated with or caused by an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis), or an adverse symptom, condition or complication associated with or caused by an adverse cardiovascular event or cardiovascular disease (e.g. atherosclerosis). Thus, a method can be practiced one or more times (e.g., 1-10, 1-5 or 1-3 times) an hour, day, week, month, or year. The skilled artisan will know when it is appropriate to delay or discontinue administration. A non-limiting dosage schedule is 1-7 times per week, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more weeks, and any numerical value or range or value within such ranges.

[0130] Methods of the invention may be practiced by any mode of administration or delivery, or by any route, systemic, regional and local administration or delivery.Exemplary administration and delivery routes include intravenous (i.v.), intraperitoneal (i.p.), intrarterial, intramuscular, parenteral, subcutaneous, intra-pleural, topical, dermal, intradermal, transdermal, transmucosal, intra-cranial, intra-spinal, rectal, oral (alimentary), mucosal, inhalation, respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity,Docket No. 051501-0585358iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, or intralymphatic.

[0131] Doses can be based upon current existing protocols, empirically determined, using animal disease models or optionally in human clinical trials. Initial study doses can be based upon animal studies, e.g. a mouse, and the amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, administered that is determined to be effective. Exemplary non-limiting amounts (doses) are in a range of about 0.1 mg / kg to about 100 mg / kg, and any numerical value or range or value within such ranges. Greater or lesser amounts (doses) can be administered, for example, 0.01-500 mg / kg, and any numerical value or range or value within such ranges. The dose can be adjusted according to the mass of a subject, and will generally be in a range from about 1-10 ug / kg, 10-25 ug / kg, 25-50 ug / kg, 50-100 ug / kg, 100-500 ug / kg, 500-1,000 ug / kg, 1-5 mg / kg, 5-10 mg / kg, 10-20 mg / kg, 20-50 mg / kg, 50-100 mg / kg, 100-250 mg / kg, 250-500 mg / kg, or more, two, three, four, or more times per hour, day, week, month or annually. A typical range will be from about 0.3 mg / kg to about 50 mg / kg, 0-25 mg / kg, or 1.0-10 mg / kg, or any numerical value or range or value within such ranges.

[0132] Doses can vary and depend upon whether the treatment is prophylactic or therapeutic, whether a subject has previously had an adverse cardiovascular event or cardiovascular disease, the onset, progression, severity, frequency, duration probability of or susceptibility of the symptom, condition, pathology or complication, the vaccination protocol and compositions, the clinical endpoint desired, the occurrence of previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.

[0133] The dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by the status of the subject. For example, whether the subject has previously had an adverse cardiovascular event or cardiovascular disease, whether the subject is merely at risk of an adverse cardiovascular event or cardiovascular disease, exposure or infection, whether the subject has been previously vaccinated against an adverse cardiovascular event or cardiovascular disease. The dose amount, number,Docket No. 051501-0585358frequency or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy.

[0134] In the methods of the invention, the route, dose, number and frequency of administrations, treatments, immunizations or vaccinations, and timing / intervals between treatment, immunization and vaccination, and disease development can be modified. In certain embodiments, a desirable vaccine of the present invention will elicit robust, long-lasting immunity against an adverse cardiovascular event or cardiovascular disease. Thus, in certain embodiments, invention methods, uses and compositions provide long-lasting immunity to an adverse cardiovascular event or cardiovascular disease such as atherosclerosis.

[0135] In certain embodiments of the present invention, a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, may be provided as pharmaceutical compositions.

[0136] As used herein the term “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact. Such formulations include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions.

[0137] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes. Exemplary routes of administration for contact or in vivo delivery which a composition can optionally be formulated include inhalation, respiration, intranasal, intubation, intrapulmonary instillation, oral, buccal, intrapulmonary, intradermal, topical, dermal, parenteral, sublingual, subcutaneous, intravascular, intrathecal, intraarticular, intracavity,Docket No. 051501-0585358transdermal, iontophoretic, intraocular, opthalmic, optical, intravenous (i.v.), intramuscular, intraglandular, intraorgan, or intralymphatic.

[0138] Formulations suitable for parenteral administration comprise aqueous and nonaqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient. Nonlimiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.

[0139] To increase an vaccination, a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, can be coupled to another protein such as ovalbumin or keyhole limpet hemocyanin (KLH), thyroglobulin or a toxin such as tetanus or cholera toxin. Invention proteins or peptides, or a sub-sequence, portion, homologue, variant or derivative thereof, can also be mixed with adjuvants. As demonstrated herein, in certain embodiments, the form of adjuvant with which the invention proteins or peptides are mixed may change whether the protein or peptide elicits an atherogenic or protective response in a subject.

[0140] Adjuvants include, for example: Oil (mineral or organic) emulsion adjuvants such as Freund's complete (CFA) and incomplete adjuvant (IFA) (WO 95 / 17210; WO 98 / 56414; WO 99 / 12565; WO 99 / 11241; and U. S. Patent No. 5,422,109); metal and metallic salts, such as aluminum and aluminum salts, such as aluminum phosphate or aluminum hydroxide, alum (hydrated potassium aluminum sulfate); bacterially derived compounds, such as Monophosphoryl lipid A and derivatives thereof (e.g., 3 De-O-acylated monophosphoryl lipid A, aka 3D-MPL or d3-MPL, to indicate that position 3 of the reducing end glucosamine is de-O-acylated, 3D-MPL consisting of the tri and tetra acyl congeners), and enterobacterial lipopolysaccharides (LPS); plant derived saponins and derivatives thereof, for example Quil A (isolated from the Quilaja Saponaria Molina tree, see, e.g., “Saponin adjuvants”, Archiv. fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254; U. S. Patent No. 5,057,540), and fragments of Quil A which retain adjuvant activity without associated toxicity, for example QS7 and QS21 (also known as QA7 and QA21), as described in WO96 / 33739, for example; surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone; oligonucleotides such as CpG (WO 96 / 02555, and WO 98 / 16247), polyriboA and polyriboU; block copolymers; and immunostimulatory cytokines such as GM-CSF and IL-1, and Muramyl tripeptide (MTP). Additional examples of adjuvants areDocket No. 051501-0585358described, for example, in “Vaccine Design— the subunit and adjuvant approach” (Edited by Powell, M. F. and Newman, M. J.; 1995, Pharmaceutical Biotechnology (Plenum Press, New York and London, ISBN 0-306-44867-X) entitled “Compendium of vaccine adjuvants and excipients” by Powell, M. F. and Newman M.

[0141] Salts may be added to a composition of the present invention, the composition comprising a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in Table 1 or a subsequence, portion, homologue, variant or derivative thereof, composition or formulation. Non-limiting examples of salts include acetate, benzoate, besylate, bitartate, bromide, carbonate, chloride, citrate, edetate, edisylate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulphate, mucate, napsylate, nitrate, pamoate (embonate, phosphate, diphosphate, salicylate and disalicylate, stearate, succinate, sulphate, tartrate, tosylate, triethiodide, valerate, aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, megluminie, potassium, procaine, sodium, tromethyamine or zinc.

[0142] Chelating agents may be added to a composition of the present invention, the composition comprising a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in Table 1 or a sub-sequence, portion, homologue, variant or derivative thereof, composition or formulation. Non-limiting examples of chelating agents include ethylenediamine, ethylene glycol tetraacetic acid, l,2-bis(o-aminophenoxy)ethane- N, N, N', N'-tetraacetic acid, Penicillamine, Deferasirox, Deferiprone, Deferoxamine, 2,3-Disulfanylpropan-l-ol, Dexrazoxane, Iron(II, III) hexacyanoferrate(II, III), (R)-5-(l,2-dithiolan-3-yl)pentanoic acid, 2,3 -Dimercapto- 1 -propanesulfonic acid, Dimercaptosuccinic acid, or diethylene triamine pentaacetic acid.

[0143] Buffering agents may be added to a composition of the present invention, the composition comprising a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in Table 1 or a sub-sequence, portion, homologue, variant or derivative thereof, composition or formulation. Non-limiting examples of buffering agents include phosphate, citrate, acetate, borate, TAPS, bicine, tris, tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, SSC, MES or succinic acid.Docket No. 051501-0585358

[0144] Cosolvents may be added to a composition of the present invention, the composition comprising a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, composition or formulation. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.

[0145] Supplementary compounds (e.g., preservatives, antioxidants, antimicrobial agents including biocides and biostats such as antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions. Pharmaceutical compositions may therefore include preservatives, anti-oxidants and antimicrobial agents.

[0146] Preservatives can be used to inhibit microbial growth or increase stability of ingredients thereby prolonging the shelflife of the pharmaceutical formulation. Suitable preservatives are known in the art and include, for example, EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate.Antioxidants include, for example, ascorbic acid, vitamin A, vitamin E, tocopherols, and similar vitamins or provitamins.

[0147] An antimicrobial agent or compound directly or indirectly inhibits, reduces, delays, halts, eliminates, arrests, suppresses or prevents contamination by or growth, infectivity, replication, proliferation, reproduction, of a pathogenic or non- pathogenic microbial organism. Classes of antimicrobials include antibacterial, antiviral, antifungal and antiparasitics. Antimicrobials include agents and compounds that kill or destroy (-cidal) or inhibit (-static) contamination by or growth, infectivity, replication, proliferation, reproduction of the microbial organism.

[0148] Exemplary antibacterials (antibiotics) include penicillins (e.g., penicillin G, ampicillin, methicillin, oxacillin, and amoxicillin), cephalosporins (e.g., cefadroxil, ceforanid, cefotaxime, and ceftriaxone), tetracyclines (e.g., doxycycline, chlortetracycline, minocycline, and tetracycline), aminoglycosides (e.g., amikacin, gentamycin, kanamycin, neomycin, streptomycin, netilmicin, paromomycin andDocket No. 051501-0585358tobramycin), macrolides (e.g., azithromycin, clarithromycin, and erythromycin), fluoroquinolones (e.g., ciprofloxacin, lomefloxacin, and norfloxacin), and other antibiotics including chloramphenicol, clindamycin, cycloserine, isoniazid, rifampin, vancomycin, aztreonam, clavulanic acid, imipenem, polymyxin, bacitracin, amphotericin and nystatin.

[0149] Particular non-limiting classes of anti-virals include reverse transcriptase inhibitors; protease inhibitors; thymidine kinase inhibitors; sugar or glycoprotein synthesis inhibitors; structural protein synthesis inhibitors; nucleoside analogues; and viral maturation inhibitors. Specific non-limiting examples of anti-virals include nevirapine, delavirdine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, zidovudine (AZT), stavudine (d4T), lamivudine (3TC), didanosine (DDI), zalcitabine (ddC), abacavir, acyclovir, penciclovir, ribavirin, valacyclovir, ganciclovir, 1,-D-ribofuranosyl-l,2,4-triazole-3 carboxamide, 9->2 -hydroxy-ethoxy methylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon and adenine arabinoside.

[0150] Pharmaceutical formulations and delivery systems appropriate for the compositions and methods of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20thed., Mack Publishing Co., Easton, PA;Remington’s Pharmaceutical Sciences (1990) 18thed., Mack Publishing Co., Easton, PA;The Merck Index (1996) 12thed., Merck Publishing Group, Whitehouse, NJ;Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel ad Soklosa, Pharmaceutical Calculations (2001) 11thed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N. Y., pp. 253-315).

[0151] A protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a subsequence, portion, homologue, variant or derivative thereof, along with any adjunct agent, compound drug, composition, whether active or inactive, etc., can be packaged in unit dosage form (capsules, tablets, troches, cachets, lozenges) for ease of administration and uniformity of dosage. A “unit dosage form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active ingredient optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g.,Docket No. 051501-0585358prophylactic or therapeutic effect). Unit dosage forms also include, for example, ampules and vials, which may include a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Unit dosage forms additionally include, for example, ampules and vials with liquid compositions disposed therein. Individual unit dosage forms can be included in multidose kits or containers. Pharmaceutical formulations can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.

[0152] In some embodiments, the agent is a multimer comprising a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof and appropriate MHC-II molecule.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] In some embodiments, a multimer is a tetramer. In certain embodiments, an agent comprises a protein or peptide comprising, consisting of, or consisting essentially of the amino acid sequence of one or more of P4 (TSUUPQUIEV), P5 (SUUPQUIEV), P22 (ADLTLLDSPIKV), P23 (LTLLDSPIKV) and P24 (TLLDSPIKV).

[0157] 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)Docket No. 051501-0585358and 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.

[0158] 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.

[0159] 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).

[0160] 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 Arenavirus infection 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 / orDocket No. 051501-0585358expansion 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 an Arenavirus infection in a subject. The MHC Class I or Class II multimers can be administered in soluble form or loaded on nanoparticles.

[0161] In certain embodiments, the method comprises detecting immune cell activity in the biological sample. In certain embodiments, the method comprises detecting the amount of immune cell activation, immune cell exhaustion, immune cell polarization or apoptosis of immune cells in the biological sample. In certain embodiments, the method comprises detecting the amount of T cell activation, T cell exhaustion, T cell polarization or apoptosis of T cells in the biological sample.

[0162] In certain embodiments, a method described herein comprises administering a polypeptide to a subject, the polypeptide comprising, consisting of, or consisting essentially of the amino acid sequence of one or more of P4 (TSLLPQLIEV), P5 (SLLPQLIEV), P22 (ADLTLLDSPIKV), P23 (LTLLDSPIKV) and P24 (TLLDSPIKV).

[0163] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0164] All applications, publications, patents and other references, GenBank citations and ATCC citations cited herein are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control.

[0165] As used herein, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a “protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence,Docket No. 051501-0585358portion, homologue, variant or derivative thereof,” or an “adverse cardiovascular event or cardiovascular disease” includes a plurality of proteins or peptides comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, or a plurality of adverse cardiovascular events or cardiovascular diseases and reference to an “activity or function” can include reference to one or more activities or functions of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a subsequence, portion, homologue, variant or derivative thereof,” including function as a T cell epitopes; eliciting, stimulating, inducing, promoting, increasing or enhancing an immune or inflammatory response; eliciting, stimulating, inducing, promoting, increasing or enhancing a T cell response; decreasing, reducing, inhibiting, suppressing or disrupting an immune or inflammatory response; eliciting, stimulating, inducing, promoting, increasing or enhancing an anti- immune or anti- inflammatory response, and so forth,

[0166] As used herein, numerical values are often presented in a range format throughout this document. The use of a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the use of a range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, to illustrate, reference to a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94%, 91-93%, and so forth. Reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. Reference to a range of 1-5 fold therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth. Further, for example, reference to a series of ranges of 2-72 hours, 2-48 hours, 4-24 hours, 4-18 hours and 6-12 hours, includes ranges of 2-6 hours, 2, 12 hours, 2-18 hours, 2-24 hours, etc., and 4-27 hours, 4-48 hours, 4-6 hours, etc.

[0167] As also used herein a series of range formats are used throughout this document. The use of a series of ranges includes combinations of the upper and lowerDocket No. 051501-0585358ranges to provide a range. Accordingly, a series of ranges include ranges which combine the values of the boundaries of different ranges within the series. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, for example, reference to a series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, and 150-171, includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, 5-171, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, 10-171, and 20-40, 20-50, 20-75, 20-100, 20-150, 20-171, and so forth.

[0168] The invention is generally disclosed herein using affirmative language to describe the numerous embodiments and aspects. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Thus, even though the invention is generally not expressed herein in terms of what is not included, embodiments and aspects that expressly exclude compositions or method steps are nevertheless disclosed and included in the invention.

[0169] A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate but not limit the scope of invention described in the claims.EXAMPLES AIM+ APOB-specific CD8+ T cells are enriched in T cell effector memory markers

[0170] Antigen-specific human CD8+T responses must be studied in the context of specific HLA-I alleles because a) binding of epitopes to HLA-I molecules is a prerequisite for CD8+T cell activation b) HLA-I molecules bind a largely nonoverlapping epitope repertoire due to their closed peptide -binding pockets (1). To gain a deeper insight into the nature of HLA-A*02:01 restricted human APOB peptides, all potential epitopes, spanning the entire length of human APOB (4563 amino acids), were scored for binding to HLA-A*02:01, the most commonly occurring Class I allele in North America. The highest-scoring 64 epitopes were selected, all of which lie within 0.17% of ranked APOB peptides (See Table 1).

[0171] To identify peptides that could potentially be used in human patients, the 64 peptides were distributed in to 4 mesopools, each containing 16 peptides, in order to screen for APOB-specific CD8+T cell responses in human PBMCs. An activation-Docket No. 051501-0585358induced marker (AIM) assay using flow-cytometry based (FACS) was used to examine each mesopool and CD8+T responses after HLA-A*02: 01+ human healthy adult PBMCs were expanded and restimulated (See Figure 1A). Robust APOB-induced CD8+T responses over unstimulated controls were detected using AIM markers CD25 and CD69 in conjunction with mesopool-induced expression of 4-1BB (see Figure IB, 1C, and IE). Both AIM combinations were expressed at significantly higher responses on APOB mesopool stimulated CD8+T cells (Figure ID).Docket No. 051501-0585358 Table 1: HLA-A*0201 binding human APOB peptidesExample 2: Identification of dominant HLA-A*02:01 -restricted APOB epitopesDocket No. 051501-0585358

[0172] Intracellular cytokine staining (ICS) assay was used to detect and characterize the APOB-reactive activated CD8+T cells as T Cell Receptor (TCR)-mediated activation results in the expression of cytotoxic and inflammatory cytokines in CD8+T cells. The ICS assay for IFNy and TNF shows co-expression in most APOB-responsive CD8 T cells (See Figure 3B). All IFNy+ cells also co-expressed granzyme B and perforin (see Figure 3C and 3D). CD4+T responses to HLA Class I APOB peptides were negligible, confirming the specificity of the detected CD8+T responses (See Figure 3E). HLA Class I restricted human APOB-derived peptides trigger robust expression of IFNy in responding CD8+T cells. Co-expression of GZMB and PRFN suggest that APOB-reactive activated CD8+T cells can mediate both inflammation and cytotoxicity.Example 3: Identification of dominant HLA-A*02:01-restricted APOB epitopes

[0173] ICS assay showed all 4 mesopools (A-D) induced IFNy expression over unstimulated controls (Figure 2B-D) however, the average frequency of APOB-reactive IFNy producing CD8+T cells was highest for Pool B (APOB P17-P32), followed by Pool A (APOB P1-P16), Pool D (APOB P49-P64) and Pool C (APOB P33-P48) (see Figure 3A). To examine responses to each individual peptide, PBMCs were expanded from HLA-A*02:01+ donors with APOB mesopools and then restimulated with each peptide in that mesopool using an expansion based The IFNy ELISpot assay (Figure 4A). Spotforming cells (SFCs) were counted and average spots from replicate wells were determined. Response magnitude (SFCs per million PBMCs) was estimated after subtracting background spot counts from control unstimulated wells. Positive (red bars, Figure 4B, C) and negative (white bars, Figure 4B, C) epitopes were scored based on a threshold of 200 peptide-induced SFC / million PBMCs. Total responses to the each mesopool A-D (black bars, Figure 4B) served as positive controls. Higher peptide-induced IFNy secretion was observed specifically against a select set of APOB epitopes (Figure 4B, representative responses in two donors; Figure 4C, mean responses in six donors). 21 positive epitopes that induced an average response >200 SFC / million PBMCs were identified (red bars, Figure 4C). The response strength and breadth (Figure 4C) were highest for peptides in mesopool B (P171340-1348to P322356-2365) and lowest for mesopool C (P332376-2384to P483734-3743).

[0174] Twelve peptides triggered consistently strong responses across all donors, with both mean and median responses >200 SFCs / million PBMCs and accounted for 80% ofDocket No. 051501-0585358the responses, (see Figure 4D-E). Comparison of the mean responses to each of these 12 epitopes versus those elicited by all the other peptide candidates, revealed significantly higher response to five epitopes (P4, P5, P22, P23 and P24) (red bars, Figure 4F) (see Table 1). These 5 peptides (P4364-373, P5365-373, P222015-2026, P232017-2026 and P2420is-2026) mapped to just two specific sites in human APOB (Figure 4G).Specific Embodiments

[0175] 1. A protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof. 2. The protein or peptide of claim 1, wherein the protein or peptide modifies an immune or inflammatory response. 3. The protein or peptide of claim 1, wherein the protein or peptide modifies a T cell response.

[0176] 4. The protein or peptide of claim 1 wherein the protein or peptide induces, promotes, increases or enhances an immune response. 5. The protein or peptide of claim 1 wherein the protein or peptide elicits, stimulates, induces, promotes, increases or enhances a T cell response. 6. The protein or peptide of claim 1 wherein the protein or peptide decreases, reduces, inhibits, suppresses or disrupts an immune or inflammatory response.

[0177] 7. The protein or peptide of claim 1 wherein the protein or peptide elicits, stimulates, induces, promotes, increases or enhances an anti- immune or antiinflammatory response. 8. The protein or peptide of claim 1 wherein the protein or peptide has prophylactic or therapeutic activity or function against an adverse cardiovascular event or cardiovascular disease or against liver disease, disorder or damage. 9. The protein or peptide of claim 1 wherein the protein or peptide has prophylactic or therapeutic activity or function against atherosclerosis.

[0178] 10. The protein or peptide of claim 1 wherein the protein or peptide has a length from about 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids. 11. The protein or peptide of claim 1, wherein the peptide is set forth in Table 1.12. The protein or peptide of claim 1, wherein the peptide comprises one or more of the peptides P4, P5, P22, P23, or P23. 13. The protein or peptide of claim 1, wherein the peptide comprises two or more of the peptides P4, P5, P22, P23, or P23.

[0179] 14. The protein or peptide of claim 1, further comprising a heterologous domain. 15. The protein or peptide of claim 1 to 14, wherein the protein or peptide has a function or activity distinct from wild-type full length apoB 100. 16. TheDocket No. 051501-0585358protein or peptide of claim 15, wherein the function or activity comprises protecting against or reducing the risk of atherosclerosis or protecting against liver disease, disorder or damage. 17. The protein or peptide of claim 15, wherein the function or activity comprises protecting against an adverse cardiovascular event or cardiovascular disease or reducing the risk of a subject to an adverse cardiovascular event or cardiovascular disease.

[0180] 18. The protein or peptide of any one of claims 1 to 17 wherein the protein or peptide is isolated or purified. 19. A nucleic acid encoding the protein or peptide of any one of claims 1 to 18. 20. A cell expressing the protein or peptide of any one of claims 1 to 18. 21. The cell of claim 20, wherein the cell is a eukaryotic or prokaryotic cell. 22. The cell of claim 20, wherein the cell is a mammalian, insect, fungal or bacterium cell.23. The cell of claim 20, wherein the cell is a dendritic cell.

[0181] 24. A composition comprising the protein or peptide of any one of claims 1 to 17. 25. A pharmaceutical composition comprising the protein or peptide of any one of claims 1 to 18 and a pharmaceutically acceptable carrier or excipient. 26. The composition of claim 24 or 25, further comprising an adjuvant. 27. The composition of any of claims 24 to 25, further comprising a statin.

[0182] 28. The composition of any of claims 24 to 27, wherein the composition is a solid or a liquid. 29. The composition of any of claims 24 to 28, wherein the composition is a microparticle or a nanoparticle. 30. The composition of any one of claims 24 to 29, wherein the composition is a vaccine. 31. The composition of any one of claims 24 to 30, wherein the composition is sterile.

[0183] 32. The composition of any one of claims 24 to 31, wherein the composition further comprises a salt, a chelating agent, a buffering agent, or an anti-microbial agent.33. A method of vaccinating a subject against an adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage, the method comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to vaccinate the subject against the adverse cardiovascular event or cardiovascular disease.

[0184] 34. A method of providing a subject with protection against an adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage, comprising administering to the subject an amount of protein or peptide comprising,Docket No. 051501-0585358consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to provide the subject with protection against the adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage.

[0185] 35. A method of reducing the risk of an adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage in a subject, comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to reduce the risk of the adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage in the subject.

[0186] 36. The method of any one of claims 33 to 35, wherein the method comprises prophylactic vaccination, protection or risk reduction against the adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage. 37. The method of any one of claims 33 to 35, wherein the method comprises vaccinating, protecting or reducing risk of the subject against an adverse symptom of the adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage. 38. The method of any one of claims 33 to 35, wherein the method comprises vaccinating, protecting or reducing risk of the subject against atherosclerosis.

[0187] 39. A method of treating a subject for an adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage, the method comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to treat the subject for the adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage.

[0188] 40. The method of claim 39, wherein the adverse cardiovascular event or cardiovascular disease comprises coronary artery disease, peripheral artery disease, cerebrovascular disease, renal artery disease, stroke, myocardial infarction (heart attack), ischemic heart failure, transient ischemic attack or brain trauma. 41. The method of claim 39, wherein the method reduces an adverse symptom of the adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage. 42. The method of any one of claims 39 to 41, wherein the method comprises treating atherosclerosis. 43. TheDocket No. 051501-0585358method of any one of claims 39 to 42, wherein the method comprises inducing plaque regression.

[0189] 44. The method of any one of claims 33 to 43, further comprising administering a statin. 45. The method of any one of claims 33 to 44, wherein the subject is a mammal. 46. The method of any one of claims 33 to 45, wherein the subject is human.

[0190] 47. The method of any one of claims 33 to 46, wherein the peptide comprises one or more of peptides P4 (TSLLPQLIEV), P5 (SLLPQLIEV), P22 (ADLTLLDSPIKV), P23 (LTLLDSPIKV) and P24 (TLLDSPIKV). 48. The method of any one of claims 33 to 46, wherein the peptide comprises two or more of the peptides (TSLLPQLIEV), P5 (SLLPQLIEV), P22 (ADLTLLDSPIKV), P23 (LTLLDSPIKV) and P24 (TLLDSPIKV).

[0191] 49. A method of determining whether a subject has, has had, is at risk of having or is in need of treatment for an adverse cardiovascular event or cardiovascular disease, the method comprising: contacting a biological sample of the subject with an agent that comprises a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, and detecting binding of immune cells in the biological sample to the agent, wherein detection of binding of immune cells in the biological sample to the agent indicates that the subject has, has had, is at risk of having or is need of treatment for an adverse cardiovascular event or cardiovascular disease.

[0192] 50. A method of determining the efficacy of the method of any one of claims 33 to 48, the method comprising: a) contacting a biological sample of the subject prior to performance of the method of any one of claims 33 to 48 with an agent that comprises a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof; b) detecting binding of immune cells in the biological sample to the agent; c) performing the method of any one of claims 33 to 48; d) contacting a biological sample of the subject after performance of the method of any one of claims 33 to 48 with the agent; e) detecting binding of immune cells in the biological sample of the subject to the agent after performance of the method of any one of claims 33 to 48; and f) comparing the amount of binding of immune cells to the agent after performance of the method of any one of claims 33 to 48 to the amount of binding ofDocket No. 051501-0585358immune cells in the biological sample to the agent prior to performance of the method of any one of claims 33 to 48, wherein an increase in the binding of immune cells to the agent after performance of the method of any one of claims 33 to 48 when compared to the biological sample of the subject prior to performance of the method of any one of claims 33 to 48 indicates that there is efficacy in the method of any one of claims 33 to 48.

[0193] 51. The method of claim 49 or 50, wherein the agent is a multimer comprising a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof and appropriate MHC-II molecule. 52. The method of claim 51, wherein the multimer is a tetramer. 53. The method of any one of claims 49 to 52, wherein the method comprises detecting immune cell activity in the biological sample. 54. The method of claim 53, wherein the method comprises detecting the amount of immune cell activation, immune cell exhaustion, immune cell polarization or apoptosis of immune cells in the biological sample. 55. The method of claim 54, wherein the method comprises detecting the amount of T cell activation, T cell exhaustion, T cell polarization or apoptosis of T cells in the biological sample.

[0194] References1. Libby, P. et al. Atherosclerosis. Nat Rev Dis Primers 5, 56 (2019).2. Roy, P., Orec'chioni. M. & Ley, K. How the immune system shapes atherosclerosis: roles of innate and adaptive immunity. Nat Rev Immunol 22, 251-265 (2022).3. Koltsova, E. K. et al. Dynamic T cell-APC interactions sustain chronic inflammation in atherosclerosis. J Clin Invest 122, 3114-26 (2012).4. Khan, A., Roy, P. & Ley, K. Breaking tolerance: the autoimmune aspect of atherosclerosis. Nat Rev Immunol 24, 670-679 (2024).5. Wang, Z. et al. Pairing of single-cell RNA analysis and T cell antigen receptor profiling indicates breakdown of T cell tolerance checkpoints in atherosclerosis. Nat Cardiovasc Res 2, 290-306 (2023).6. Depuydt, M. A. C. et al. Single-cell T cell receptor sequencing of paired human atherosclerotic plaques and blood reveals autoimmune -like features of expanded effector T cells. Nat Cardiovasc Res 2, 112-125 (2023).Docket No. 051501-05853587. Liblau, R. S., Wong, F. S., Mars, L. T. & Santamaria, P. Autoreactive CD8 T cells in organ-specific autoimmunity: emerging targets for therapeutic intervention. Immunity 17, 1-6 (2002).8. Pugliese, A. Autoreactive T cells in type 1 diabetes. J Clin Invest 127, 2881-2891 (2017).9. Gonzalez-Duque, S. et al. Conventional and Neo-antigenic Peptides Presented by beta Cells Are Targeted by Circulating Naive CD8+ T Cells in Type 1 Diabetic and Healthy Donors. Cell Metab 28, 946-960 e6 (2018).10. Schafer, S. & Zemecke, A. CD8(+) T Cells in Atherosclerosis. Cells 10, (2020).11. Saigusa, R., Winkels, H. & Ley, K. T cell subsets and functions in atherosclerosis. Nat Rev Cardiol 17, 387-401 (2020).12. Hwang, Y. et al. Expansion of CD8(+) T cells lacking the IL-6 receptor alpha chain in patients with coronary artery diseases (CAD). Atherosclerosis 249, 44-51 (2016).13. Tae Yu, H. et al. Characterization of CD8(+)CD57(+) T cells in patients with acute myocardial infarction. Cell Mol Immunol 12, 466-73 (2015).14. Bergstrom, I., Backteman, K., Lundberg, A., Emerudh, J. & Jonasson, L.Persistent accumulation of interferon-gamma-producing CD8+CD56+ T cells in blood from patients with coronary artery disease. Atherosclerosis 224, 515-20 (2012).15. Fernandez, D. M. et al. Single-cell immune landscape of human atherosclerotic plaques. Nat Me d 25, 1576-1588 (2019).16. Chowdhury, R. R. et al. Human Coronary Plaque T Cells Are Clonal and Cross-Reactto Virus and Self. Circ Res 130, 1510-1530 (2022).17. Marchini, T., Hansen, S. & Wolf, D. ApoB-Specific CD4(+) T Cells in Mouse and Human Atherosclerosis. Cells 10, (2021).18. Roy, P. et al. Immunodominant MHC-II (Major Histocompatibility Complex II) Restricted Epitopes in Human Apolipoprotein B. Circ Res 131, 258-276 (2022).19. Roy, P., Suthahar, S. S. A., Makings, J. & Ley, K. Identification of apolipoprotein B-reactive CDR3 motifs allows tracking of atherosclerosis-related memory CD4+T cells in multiple donors. Front Immunol 15, 1302031 (2024).20. Rudolph, M. G., Stanfield, R. L. & Wilson, I. A. How TCRs bind MHCs, peptides, and coreceptors. Annu Rev Immunol 24, 419-66 (2006).21. Sidney, J., Peters, B. & Sette, A. Epitope prediction and identification- adaptive T cell responses in humans. Semin Immunol 50, 101418 (2020).Docket No. 051501-058535822. Sidney, J., Peters, B., Frahm, N., Brander, C. & Sette, A. HLA class I supertypes: a revised and updated classification. BMC Immunol 9, 1 (2008).23. Jurtz, V. et al. NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data. J Immunol 199, 3360-3368 (2017).24. Vita, R. et al. The Immune Epitope Database (IEDB): 2018 update. Nucleic Acids Res 47, D339-D343 (2019).25. Peters, B., Nielsen, M. & Sette, A. T Cell Epitope Predictions. Annu Rev Immunol 38, 123-145 (2020).26. Grifoni, A. et al. Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals. Cell 181, 1489-1501 e15 (2020).27. Mateus, J. et al. Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans. Science 370, 89-94 (2020).28. Szabo, P. A. et al. Single-cell transcriptomics of human T cells reveals tissue and activation signatures in health and disease. Nat Commun 10, 4706 (2019).29. Dimayuga, P. C. et al. Identification of apoB-100 Peptide-Specific CD8+ T Cells in Atherosclerosis. J Am Heart Assoc 6, (2017).30. Schaftenaar, F. H. et al. Induction ofHLA-A2 restricted CD8 T cell responses against ApoBlOO peptides does not affect atherosclerosis in a humanized mouse model. Sci Rep 9, 17391 (2019).31. Chyu, K. Y. et al. Immunization using ApoB-100 peptide-linked nanoparticles reduces atherosclerosis. JCI Insight 7, (2022).32. Iqneibi, S. et al. Single cell transcriptomics reveals recent CD8T cell receptor signaling in patients with coronary artery disease. Front Immunol 14, 1239148 (2023). 33. Elhage, R. et al. Deleting TCR alox beta+ or CD4+ T lymphocytes leads to opposite effects on site-specific atherosclerosis in female apolipoprotein E-deficient mice. Am J Pathol 165, 2013-8 (2004).34. Cochain, C. et al. CD8+ T Cells Regulate Monopoiesis and Circulating Ly6C-high Monocyte Levels in Atherosclerosis in Mice. Circ Res 117, 244-53 (2015).35. Kyaw, T. et al. Cytotoxic and proinflammatory CD8+ T lymphocytes promote development of vulnerable atherosclerotic plaques in apoE-deficient mice. Circulation 127, 1028-39 (2013).Docket No. 051501-058535836. de Jong, M. J. M. et al. Virus-Associated CD8+ T-Cells Are Not Activated Through Antigen-Mediated Interaction Inside Atherosclerotic Lesions. Arterioscler Thromb Vase Biol 44, 1302-1314 (2024).37. Saigusa, R. et al. Single cell transcriptomics and TCR reconstruction reveal CD4 T cell response to MHC -II -restricted APOB epitope in human cardiovascular disease. Nat Cardiovasc Res 1, 462-475 (2022).38. Wolf, D. et al. Pathogenic Autoimmunity in Atherosclerosis Evolves From Initially Protective Apolipoprotein BlOO-Reactive CD4(+) T-Regulatory Cells.Circulation 142, 1279-1293 (2020).39. Kimura, T. et al. Regulatory CD4(+) T Cells Recognize MHC -Il-Restricted Peptide Epitopes of Apolipoprotein B. Circulation 138, 1130-1143 (2018).40. Fredrikson, G. N. et al. Identification of immune responses against aldehyde-modified peptide sequences in apoB associated with cardiovascular disease. Arterioscler Thromb Vase Biol 23, 872-8 (2003).41. Nilsson, J., Bjorkbacka, H. & Fredrikson, G. N. Apolipoprotein B100 autoimmunity and atherosclerosis - disease mechanisms and therapeutic potential. Curr Opin Lipidol 23, 422-8 (2012).42. Sage, A. P., Tsiantoulas, D., Binder, C. J. & Mallat, Z. The role of B cells in atherosclerosis. Nat Rev Cardiol 16, 180-196 (2019).

Claims

Docket No. 051501-0585358WHAT IS CLAIMED:

1. A protein or peptide comprising, consisting of or consisting essentially of an aminoacid sequence set forth as any one of the peptides set forth in in Table 1, or a subsequence, portion, homologue, variant or derivative thereof.

2. The protein or peptide of claim 1, wherein the protein or peptide modifies an immuneor inflammatory response.

3. The protein or peptide of claim 1, wherein the protein or peptide modifies a T cell response.

4. The protein or peptide of claim 1 wherein the protein or peptide induces, promotes,increases or enhances an immune response.

5. The protein or peptide of claim 1 wherein the protein or peptide elicits, stimulates, induces, promotes, increases or enhances a T cell response.

6. The protein or peptide of claim 1 wherein the protein or peptide decreases, reduces,inhibits, suppresses or disrupts an immune or inflammatory response.

7. The protein or peptide of claim 1 wherein the protein or peptide elicits, stimulates, induces, promotes, increases or enhances an anti- immune or anti- inflammatory response.

8. The protein or peptide of claim 1 wherein the protein or peptide has prophylactic ortherapeutic activity or function against an adverse cardiovascular event or cardiovascular disease or against liver disease, disorder or damage.

9. The protein or peptide of claim 1 wherein the protein or peptide has prophylacticDocket No. 051501-0585358ortherapeutic activity or function against atherosclerosis.

10. The protein or peptide of claim 1 wherein the protein or peptide has a length from about 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-75 or 75-100 amino acids.

11. The protein or peptide of claim 1, wherein the peptide is set forth in Table 1.

12. The protein or peptide of claim 1, wherein the peptide comprises one or more of the peptides P4, P5, P22, P23, or P23.

13. The protein or peptide of claim 1, wherein the peptide comprises two or more of the peptides P4, P5, P22, P23, or P23.

14. The protein or peptide of claim 1, further comprising a heterologous domain.

15. The protein or peptide of claim 1 to 14, wherein the protein or peptide has a function or activity distinct from wild-type full length apoB100.

16. The protein or peptide of claim 15, wherein the function or activity comprises protecting against or reducing the risk of atherosclerosis or protecting against liver disease, disorder or damage.

17. The protein or peptide of claim 15, wherein the function or activity comprises protecting against an adverse cardiovascular event or cardiovascular disease or reducing the risk of a subject to an adverse cardiovascular event or cardiovascular disease.

18. The protein or peptide of any one of claims 1 to 17 wherein the protein or peptide is isolated or purified.

19. A nucleic acid encoding the protein or peptide of any one of claims 1 to 18.

20. A cell expressing the protein or peptide of any one of claims 1 to 18.Docket No. 051501-058535821. The cell of claim 20, wherein the cell is a eukaryotic or prokaryotic cell.

22. The cell of claim 20, wherein the cell is a mammalian, insect, fungal or bacterium cell.

23. The cell of claim 20, wherein the cell is a dendritic cell.

24. A composition comprising the protein or peptide of any one of claims 1 to 17.

25. A pharmaceutical composition comprising the protein or peptide of any one of claims 1 to 18 and a pharmaceutically acceptable carrier or excipient.

26. The composition of claim 24 or 25, further comprising an adjuvant.

27. The composition of any of claims 24 to 25, further comprising a statin.

28. The composition of any of claims 24 to 27, wherein the composition is a solid or a liquid.

29. The composition of any of claims 24 to 28, wherein the composition is a microparticle or a nanoparticle.

30. The composition of any one of claims 24 to 29, wherein the composition is a vaccine.

31. The composition of any one of claims 24 to 30, wherein the composition is sterile.

32. The composition of any one of claims 24 to 31, wherein the composition further comprises a salt, a chelating agent, a buffering agent, or an anti-microbial agent.

33. A method of vaccinating a subject against an adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage, the method comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides setDocket No. 051501-0585358forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to vaccinate the subject against the adverse cardiovascular event or cardiovascular disease.

34. A method of providing a subject with protection against an adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage, comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to provide the subject with protection against the adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage.

35. A method of reducing the risk of an adverse cardiovascular event or cardiovasculardisease or liver disease, disorder or damage in a subject, comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to reduce the risk of the adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage in the subject.

36. The method of any one of claims 33 to 35, wherein the method comprises prophylactic vaccination, protection or risk reduction against the adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage.

37. The method of any one of claims 33 to 35, wherein the method comprises vaccinating, protecting or reducing risk of the subject against an adverse symptom of the adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage.

38. The method of any one of claims 33 to 35, wherein the method comprises vaccinating, protecting or reducing risk of the subject against atherosclerosis.

39. A method of treating a subject for an adverse cardiovascular event orDocket No. 051501-0585358cardiovasculardisease or liver disease, disorder or damage, the method comprising administering to the subject an amount of protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, sufficient to treat the subject for the adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage.

40. The method of claim 39, wherein the adverse cardiovascular event or cardiovascular disease comprises coronary artery disease, peripheral artery disease, cerebrovascular disease, renal artery disease, stroke, myocardial infarction (heart attack), ischemic heart failure, transient ischemic attack or brain trauma.

41. The method of claim 39, wherein the method reduces an adverse symptom of the adverse cardiovascular event or cardiovascular disease or liver disease, disorder or damage42. The method of any one of claims 39 to 41, wherein the method comprises treating atherosclerosis.

43. The method of any one of claims 39 to 42, wherein the method comprises inducingplaque regression.

44. The method of any one of claims 33 to 43, further comprising administering a statin.

45. The method of any one of claims 33 to 44, wherein the subject is a mammal.

46. The method of any one of claims 33 to 45, wherein the subject is human.

47. The method of any one of claims 33 to 46, wherein the peptide comprises one or more of peptides P4 (TSLLPQLIEV), P5 (SLLPQLIEV), P22 (ADLTLLDSPIKV), P23 (LTLLDSPIKV) and P24 (TLLDSPIKV).Docket No. 051501-058535848. The method of any one of claims 33 to 46, wherein the peptide comprises two or more of the peptides (TSLLPQLIEV), P5 (SLLPQLIEV), P22 (ADLTLLDSPIKV), P23 (LTLLDSPIKV) and P24 (TLLDSPIKV).

49. A method of determining whether a subject has, has had, is at risk of having or is in need of treatment for an adverse cardiovascular event or cardiovascular disease, the method comprising:contacting a biological sample of the subject with an agent that comprises a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof, anddetecting binding of immune cells in the biological sample to the agent, wherein detection of binding of immune cells in the biological sample to the agent indicates that the subject has, has had, is at risk of having or is need of treatment for an adverse cardiovascular event or cardiovascular disease.

50. A method of determining the efficacy of the method of any one of claims 33 to 48, the method comprising:a) contacting a biological sample of the subject prior to performance of the method of any one of claims 33 to 48 with an agent that comprises a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof;b) detecting binding of immune cells in the biological sample to the agent; c) performing the method of any one of claims 33 to 48;d) contacting a biological sample of the subject after performance of the method of any one of claims 33 to 48 with the agent;e) detecting binding of immune cells in the biological sample of the subject to the agent after performance of the method of any one of claims 33 to 48; andf) comparing the amount of binding of immune cells to the agent after performance of the method of any one of claims 33 to 48 to the amount of binding of immune cells in the biological sample to the agent prior to performance of the method of any one of claims 33 to 48,Docket No. 051501-0585358wherein an increase in the binding of immune cells to the agent after performance of the method of any one of claims 33 to 48 when compared to the biological sample of the subject prior to performance of the method of any one of claims 33 to 48 indicates that there is efficacy in the method of any one of claims 33 to 48.

51. The method of claim 49 or 50, wherein the agent is a multimer comprising a protein or peptide comprising, consisting of or consisting essentially of an amino acid sequence set forth as any one of the peptides set forth in in Table 1, or a sub-sequence, portion, homologue, variant or derivative thereof and appropriate MHC-II molecule.

52. The method of claim 51, wherein the multimer is a tetramer.

53. The method of any one of claims 49 to 52, wherein the method comprises detecting immune cell activity in the biological sample.

54. The method of claim 53, wherein the method comprises detecting the amount of immune cell activation, immune cell exhaustion, immune cell polarization or apoptosis of immune cells in the biological sample.

55. The method of claim 54, wherein the method comprises detecting the amount of T cell activation, T cell exhaustion, T cell polarization or apoptosis of T cells in the biological sample.

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