Multi-epitope constructs

Abstract

Recombinant constructs are provided that contain recombinant molecules that display inserted multiple and different epitopes derived from a variety of antigens. [Solution] A recombinant construct comprising (i) a scaffold portion, (ii) a first type of epitope capable of inducing an anti-atherosclerotic vascular disease response via C5aR interaction, and (iii) a second type of epitope capable of inducing an anti-atherosclerotic vascular disease response via a second pathway independent of the C5aR interaction, said second type of epitope being selected from the group consisting of an apolipoprotein epitope, a heat shock protein epitope, and a Chlamydia pneumoniae epitope, wherein the first type of epitope is a C5a receptor protein epitope comprising or consisting of the amino acid sequence MNSFNYTTPDYGHYDDKDTLDLNTPVDKTSN, or a functional fragment thereof having antigenic activity.

Description

[Technical field]

[0001] The present invention provides recombinant vectors that display multiple and different epitopes inserted from various antigens. The present invention relates to multi-epitope constructs, immunogenic and vaccine compositions comprising such molecules. The invention elicits immune responses against antigens and pathogens involved in the development of atherosclerosis. The present invention relates to such compositions, inter alia, for treating said diseases and / or The present invention includes methods for preventing and treating the disease, as well as recombinant protein products. [Background technology]

[0002] Atherosclerosis is characterized by the proliferation of inflammatory cells and activated plaques in the lesions, all of which indicate involvement of the immune system. Complex chronic inflammation of the arterial wall as evidenced by the presence of activated immune cells and cytokines Dendritic cells (DCs) are involved in the induction of innate and adaptive immunity. A key component of the innate response is the induction of monocytes into the early lesion. and subsequent CD11c uptake of monocytes with macrophage- and DC-like properties. + To cells Atherosclerotic plaques are caused by macrophages interacting with T cells to induce inflammation. Macrophages produce a variety of cytokines that can provide both pro- and anti-inflammatory effects. It is known that foam cells derived from the endothelial cell membrane contain molecules that contribute to the development of atherosclerosis. Although the mechanisms are not fully understood, it is believed that the immune system plays a role in the development of atherosclerotic plaques and their complications. As a result, oxidized low-density lipoprotein (LDLP) plays an important role in Proteins (oxLDLs) [9,10], β2-glycoprotein I (β2GP1), and phosphatases These include tidylcholine (PC) [11, 12] and heat shock proteins (HSPs). Related to the pathogenesis of atherosclerosis based on modified self-molecules or peptides derived from Several antigenic stimulations related to autoantigens have been reported. In addition to the above, more research is being conducted on the topic of vaccination against atherosclerosis. Studies have shown that other antigens are highly effective in preventing the formation of arteriosclerotic lesions. One of the challenges in developing an effective vaccination strategy against atherosclerosis The first is the selection of a specific antigen.

[0003] In the discovery and development of potential antigens, apolipoprotein B (ApoB), heat shock Heat shock protein (HSP) 60 and Chlamydia pneumoniae A recombinant construct called "AHHC" containing epitopes derived from the (Cpn) protein B6 by substance; 129S-Ldlr tm1Her Apob tm2Sgy Immunization of / J mice Treatment reduces atherosclerotic lesion formation in mice fed a high-fat diet (HFD) (Lu et al 2012; Atherosclerosis 225:56-68). In addition, the complement component 5a receptor (C Immunization with a peptide derived from the N-terminus of B6;129S-Ldlr tm1Her Apob tm2Sgy Decreased incidence of early atherosclerotic lesions in / J mice It is known from the prior art that 2:2358-2371). In addition, in preclinical studies, Inhibition of 1 showed a strong antithrombotic effect leading to a significant reduction in platelet aggregation, whereas Primary hemostatic function was maintained (Chintala et al 2010 Arterioscler Thromb Vasc Biol 30 :2143-2149).

[0004] The human complement system is a key component of the innate host defense against invading pathogens. C5a is A protein fragment released from complement component C5 that mediates classical, alternative and rectal complement activation. In humans, it is a 74-amino acid that is generated by cleavage of the C5α chain by the C5a convertase in the C5α pathway. C5a is a peptide of the 5'-amino acid sequence. It binds to its G protein-coupled C5a complex on the plasma membrane of target cells. It mediates its effects through the receptor (C5aR / CD88) and induces chemotaxis, respiratory burst and It induces intracellular signaling that leads to the release of proinflammatory mediators from granulocytes. C5a attracts and activates neutrophils, monocytes, and platelets, and produces reactive oxidants, protein Inflammatory mediators, including proteolytic enzymes, chemokines, and cytokines, as well as complement factors Stimulates the release of C3 and properdin. Secretion of C3 and properdin by neutrophils , as well as the presence of apoptotic and necrotic decidual tissue, accelerates activation of the alternative pathway, at sites of leukocyte infiltration, enhancing C3 activation and deposition and generating additional C5a Human C5aR forms a single polypeptide chain. C5a / C5aR is an integral membrane glycoprotein of 350 amino acids that binds to the C5a receptor. Altering the production of IL-12 and thus regulating Th-1 cell responses, as well as IL -6, IL-8 and TNF-alpha have been shown to enhance the production of cytokines. C5aR is abundantly expressed on leukocytes, including neutrophils, monocytes, eosinophils, and lymphocytes. C5aR is also expressed on a wide range of tissues, including glomerular mesangial and proximal tubular epithelial cells. Parenchymal C5aR expression is increased at sites of acute inflammation. It was shown that:

[0005] Atherosclerotic rupture or vulnerable plaque is usually characterized by the presence of a large lipid core, Decreased number of smooth muscle cells, a thin fibrous capsule, and inflammation including macrophages and T cells Macrophages play a key role in plaque destabilization and rupture. Matrix metalloproteinases (MMPs) are thought to play a key role in By producing collagen, proteoglycans, and elastin, these cells which have the ability to degrade components of the extracellular matrix. and in situ zymography, MMP-1, MMP-3 and MMP- 9 was shown to be present in the shoulder region of atherosclerotic plaques and was also found to be present in apoE-deficient mice. It was shown that overexpression of active MMP-9 induces plaque destruction. in monocyte-derived macrophages isolated from mice and in atherosclerotic plaques On the expression of MMP-1 and MMP-9 mRNA in cultured human macrophages The stimulatory effect of C5a has also been reported.

[0006] Clinical and experimental evidence implicating the anaphylatoxin C5a in the pathogenesis of atherosclerosis Cardiovascular disease in patients with advanced atherosclerosis It has recently been reported that there is a predictive value of C5a plasma levels for events. C5a was detected in atherosclerotic lesions. Furthermore, C5a is an adhesion molecule associated with endothelial cells. C5a activates macrophages and induces their expression of inflammatory mediators TNF-alpha, interleukin 1, is released. TNF-alpha, on the other hand, It directly induces the expression of MMPs in these cells.

[0007] In the discovery and development of potential antigens, apolipoprotein B (ApoB), heat shock Heat shock protein (HSP) 60 and Chlamydia pneumoniae A recombinant construct called "AHHC" containing epitopes derived from the (Cpn) protein B6 by substance; 129S-Ldlr tm1Her Apob tm2Sgy Immunization of / J mice Treatment reduces atherosclerotic lesion formation in mice fed a high-fat diet (HFD) (Lu et al 2012; Atherosclerosis 225:56-68). In addition, the complement component 5a receptor (C Immunization with a peptide derived from the N-terminus of B6;129S-Ldlr tm1Her Apob tm2Sgy Decreased incidence of early atherosclerotic lesions in / J mice It is known from the prior art that 2:2358-2371). In addition, in preclinical studies, Inhibition of 1 showed a strong antithrombotic effect leading to a significant reduction in platelet aggregation, whereas Primary hemostatic function was maintained (Chintala et al 2010 Arterioscler Thromb Vasc Biol 30 :2143-2149). Summary of the Invention [Problem to be solved by the invention]

[0008] There is a need for improved treatment of arteriosclerotic vascular disease. [Means for solving the problem]

[0009] Disclosure Summary According to a first aspect of the present invention, i) scaffolding parts and anything attached thereto; ii) a first species capable of eliciting an anti-atherosclerotic vascular disease response via a first pathway; and iii) Induce anti-atherosclerotic vascular disease responses via a second pathway that is independent of the first pathway. A second species of epitope that can cause The present invention provides a recombinant construct comprising:

[0010] References herein to the first and second pathways being "independent" of each other include Or the formation of atherosclerosis via a second pathway may have a different mechanism of action or cause. The purpose is to show that it depends on the route.

[0011] Preferably, the scaffold moiety is a natural dendroaspin, having the structure represented by SEQ ID NO: 1 (FIG. 14A). Contains a selected amino acid sequence.

[0012] Preferably, the first pathway to the formation of atherosclerosis is via C5 interactions, More preferably, it is via the C5a or C5Ar pathway. Thus, the C5 epitope is The C5 epitope is the C5a receptor (C5aR) epitope. It may contain from 8 to 40, for example from 8 to 40 amino acid residues or from 8 to 35 amino acid residues. The C5a epitope is selected from the C5a sequence (SEQ ID NO: 2) (FIG. 14B). consecutive amino acid residues, for example, 8 to 40 amino acid residues, for example, 8 to 35 amino acids In one embodiment, the C5a epitope may comprise 8 to 10 residues selected from the C5a sequence. 20, e.g., 8 to 15 consecutive amino acid residues.

[0013] Preferably, the C5a epitope is EQRAARISLGPR (SEQ ID NO: 3), RAA RISLGPRCIKAFTE (SEQ ID NO: 4) and CVNNDETCEQ (SEQ ID NO: 5) A polypeptide or an antibody comprising or consisting of an amino acid sequence selected from the group consisting of For example, the epitope is selected from the C5a sequence. and may contain a sequence of 5 to 40 consecutive amino acid residues including SEQ ID NO: 2, 3, or 4. .

[0014] Preferably, the C5aR epitope is selected from the C5aR sequence (SEQ ID NO: 6; FIG. 14C). 5 to 50 consecutive amino acid residues, for example, 10 to 40 amino acid residues or In one embodiment, the C5aR epitope comprises 1 to 31 amino acid residues. The amino acid sequence comprises consecutive amino acid residues or a functional fragment thereof.

[0015] Preferably, the C5aR epitope is located at the N-terminus (containing a free amino group) of the C5aR amino acid sequence. or more preferably at the C-terminus (containing a free carboxyl group). The C5aR epitope is MNSFNYTTPDYGHYDDKDTLD (SEQ ID NO: 7 ), TLDLNTPVDKTSN (SEQ ID NO: 8) and MNSFNYTTPDYGHYD An amino acid sequence selected from the group comprising: DKDTLDLNTPVDKTSN (SEQ ID NO: 9) or a functional fragment thereof having antigenic activity.

[0016] Reference herein to a functional fragment refers to a fragment that comprises and functions as an antigenic determinant. In addition, it has antigenic activity and retains sufficient amino acids to function as an epitope. The functional fragment also includes a portion of the epitope amino acid sequence incorporated in the scaffold portion. For example and without limitation, when presented, the area occupied by atherosclerotic lesions The effect of the enzyme that induces an immune response against atherosclerosis can be measured by a decrease in It includes a portion of the pitope amino acid sequence.

[0017] The reference to first and second species epitopes indicates that they are the first and second species that ultimately contribute to atherosclerosis. This means that each is separately and independently involved in different pathways that lead to disease and related disorders. The purpose is to

[0018] Preferably, the recombinant construct comprises a plurality of epitopes of a first species.

[0019] Preferably, the second species epitope is a human epitope. may contain 5 to 40, for example 9 to 40, for example 9 to 20 amino acid residues. Preferably, the second species of epitope associated with an anti-atherosclerotic vascular disease response is an apolipoprotein. apo epitopes, heat shock protein (HSP) epitopes, pneumonia Chlamydia pneumonia epitope, protease-activated receptor 1 epitope The epitopes are selected from the group consisting of or including the PAR-1 and perilipin epitopes.

[0020] More preferably, the second epitope is a heat shock protein (HSP) epitope, The epitopes are Chlamydia pneumoniae epitopes, tissue factor epitopes, or PAR-1 epitopes.

[0021] Preferably, the heat shock protein (HSP) is HSP60 or HSP65. More preferably, the HSP60 is human HSP60 or Mycobacterium hirsutum. Mycobacterium bovis HSP. More preferably, the reaction against HSP60. The epitope capable of inducing a reaction is peptide 1 (AA) 153-160:A ELKKQSK; (SEQ ID NO: 10), peptide 1 (AA) 153-163: AELKKQ SKPVT; (SEQ ID NO: 11), peptide 1 (AA) 303-312: PGFGDNRK NQ (SEQ ID NO: 12), peptide 2: AA277-286 PGFGDNRKNQ (sequence No. 13), peptide (AA) 516-528 KGIIDPTKVVRTA (SEQ ID NO: 14) and Mycobacterium (AA) 253-268: EGEALSTLVVNKI RGT (SEQ ID NO: 15) It is a polypeptide or a functional fragment thereof that has antigenic activity.

[0022] Preferably, the Chlamydia pneumoniae is Cpn1 or Cpn2. The epitope capable of eliciting a response against Cpn is the major outer membrane protein (MOMP) (amino acid sequence (AA) 67-74: GDYVFDRI (SEQ ID NO: 16)) and Cpn putative outer membrane protein (Pomp) 5 (amino acid sequence (AA) 283–29 1: QAVANGGAI (SEQ ID NO: 17) or a polypeptide consisting of, or a functional fragment thereof having antigenic activity.

[0023] Preferably, the tissue factor epitope is (AA56-67) and has the sequence EWEPKPV NQVYT (sequence number 19).

[0024] Preferably, the PAR-1 epitope is (AA42-55) and has the amino acid sequence SF Contains LLRNPNDKYEPF (sequence number 19).

[0025] Preferably, the recombinant construct of the invention comprises multiple second epitopes, e.g., two, may contain 3, 4, 5, 6, 7, 8, 9 or 10 of the same or different second epitopes For example, in a particularly preferred embodiment, the recombinant construct comprises a C5aR epitope, a Cpn The recombinant construct may contain a C epitope and two different HSP epitopes. 5aR epitope, Cpn epitope, HSP epitope and PAR-1 epitope It may include.

[0026] Preferably, the amino acid sequence of the first and / or second epitope is a dendroaspiral epitope. (a) loop I and / or loop II of the scaffold; (b) loop I and / or loop II of the scaffold; (c) loop II and / or loop III; or (d) loop I, loop II, or loop III. Loop I contains amino acid residues 4 to 16. Loop II contains residues 23 to 36, and loop III contains residues 40 to 50. However, the additional amino acids that are incorporated may increase the residues in the non-loop regions or may include additional amino acids. The amino acid sequence or the sequence to be inserted may be replaced by a residue in the outside loop region, i.e. It may extend to or replace residues 1-3, 17-22, and 37-39. The amino acid residue is preferably incorporated into loop I or loop II. Loop III, which contains the The function is maintained. The preferred position for the additional sequence to be inserted is amino acid residues 4 to 1. Sites in the dendroaspin scaffold between 6, 18–21, 23–36, or 52–59 Each inserted further amino acid sequence or part of a further amino acid sequence is preferably or 3 to 40 amino acid residues, more preferably 3 to 16, and even more preferably 3 to 14 The amino acid sequence in the range of amino acid residues in length. The beginning can be any one of amino acid residues 1 to 57 of the dendroaspin scaffold. The additional amino acid sequence inserted ends at amino acid residues 3-5 of the dendroaspin scaffold. It can be any one of 9.

[0027] When two or more additional amino acid sequences are inserted into the dendroaspin scaffold, The linear distance between them is preferably 1 to 35 amino acids, more preferably 1 to 14 amino acids. If two or more additional amino acid sequences are inserted, the preferred Preferably, at least one naturally occurring dendroaspin amine separates each additional amino acid sequence. The RGD-containing loop can be modified by the insertion or deletion of one or more amino acid residues. It can be modified by deletion or substitution, preferably loop III of the dendroaspin. A maximum of eight or a minimum of one amino acid may be modified in Loop I and / or Loop II. or loop II is modified by the insertion, deletion or substitution of one or more amino acid residues. The insertion at one or more sites in the dendroaspin scaffold can be For this purpose, a number of residues ranging from 14 to 36 is preferred, but any suitable number of amino acids may be used in the dendron. They can be inserted into the aspin scaffold to obtain the desired bi- or multifunctional activity.

[0028] Preferably, in some embodiments of the invention, the first or second epitope is a dendritic cell line. Attached to either or both of the C and / or N termini of the doraspin scaffold protein It is possible to do so.

[0029] In a further aspect of the invention, the protein incorporated in the construct of the first aspect of the invention is An expression vector containing the encoding nucleic acid is provided.

[0030] In a further aspect of the invention, a protein according to the first aspect of the invention and antigenic hydrophobicity An antigenic composition comprising the complex is provided.

[0031] In a further aspect of the invention, the immunosuppressant of the invention is formulated as an injectable or oral formulation. The present invention provides a pharmaceutical composition comprising the active ingredient composition. Preferably, the pharmaceutical composition comprises a suitable adjuvant. The composition further comprises an agent, excipient, diluent and / or carrier.

[0032] In a further aspect of the invention, a protein according to the first aspect of the invention, a vector according to the invention or provides a pharmaceutical composition comprising the immunogenic composition of the invention.

[0033] A further aspect of the invention relates to a protein or a compound according to the first aspect of the invention for use as a medicament. A vector of the present invention is provided.

[0034] In a further aspect of the invention, a recombinant protein according to the first aspect of the invention; the immunogenic composition of the present invention; and the pharmaceutical composition of the present invention. The present invention provides a method for generating an anti-atherosclerotic response in a mammal, the method comprising: do.

[0035] In a further aspect of the invention, a recombinant protein according to the first aspect of the invention; the immunogenic composition of the present invention; and the pharmaceutical composition of the present invention. A method for treating, preventing or reducing atherosclerosis comprising administering to The formulations can be administered in a therapeutically effective or therapeutically acceptable amount.

[0036] In a further aspect of the invention, the subject is an individual with early stage atherosclerosis or an individual with atheroma. A method of treating an individual identified as being at risk for developing arteriosclerosis, comprising the steps of: A recombinant protein according to the first aspect; a vector according to the invention; an immunogenic composition according to the invention; and and administering to an individual a formulation selected from the pharmaceutical compositions of the present invention. The formulations can be administered in a therapeutically effective or therapeutically acceptable amount.

[0037] In a further aspect of the invention, a protein according to the first aspect of the invention or a vector according to the invention is The company will provide vaccines, including those containing vaccinia virus type 2 (VAD).

[0038] In a further aspect of the invention, the present invention provides a method for the preparation of at least two unique 1. A method for eliciting an immune response against an epitope associated with an isolated pathway, comprising: (i) at least one first and at least one second epitaxial layer as described hereinabove; constructing and expressing a dendroaspin scaffold protein containing the tope; (ii) incubating eukaryotic cells with the dendroaspin scaffold protein; And, (iii) preparing microsomes using the eukaryotic cells; and (iv) one or more of the microsomes and dendroaspin scaffold proteins. and mixing the compound with a pharma- ceutically acceptable ingredient to produce an oral or injectable formulation. (v) administering said formulation to a mammal or human. The present invention provides a method comprising:

[0039] Data show that immunization with ApoB / PAR-1 / HSP / Cpn enhances the pathogenesis of sclerotic lesions. It was shown that the expression of C5a in the inflammatory bowel syndrome was not affected by the C5 / C5aR. These results suggest that these are independent pathways that play important roles in the prevention and treatment of early atherosclerosis. To provide improved treatment for the treatment and reduction of C5 and ApoB / P Methods for advantageously simultaneously targeting both the AR-1 / HSP / Cpn-related pathways and A formulation is provided.

[0040] Preferred features attributable to the first aspect of the invention are as follows mutatis mutandis. It will be appreciated that the above teachings are applicable to each and every other aspect.

[0041] Embodiments of the invention are further described below with reference to the accompanying drawings. [Brief description of the drawings]

[0042] [Figure 1] (1A) Schematic representation of the backbone of the dendroaspin structure (1B) Schematic representation of the construct and alignment of the dendroaspin scaffold. [Figure 2-1]Figure 2 shows the levels of constructed protein-induced IgG, IgG1 and IgG2c antibodies in serum of B6;129S-Ldlrtm1HerApobtm2Sgy / J mice and in controls (GST-Den (referred to as GST-den) immunized mice) at 2 and 12 weeks after the first immunization, respectively. Figure 2 shows the mean optical densities (ODs) and SEM obtained from plasma samples of AHHC, RHHC and RPHC construct immunized mice on Cpn peptide (C), hHSP60153-163 (H), hHSP60303-312 (H), C5aR-peptide (R), ApoB peptide (A), PAR-1 (P) coated ELISA plates. (2A) IgG at dilution ratio: 1:100. (2B) IgG1 at dilution ratio: 1:6250. (2C) IgG2c at dilution ratio: 1:50. (2D) Cross-reactivity between human ApoB peptide-induced antisera and Cpn peptides. [Figure 2-2] (2E) Cross-reactions between human ApoB peptide and hHSP60303-312 peptide-induced antisera and their antigens. (2F) Cross-reactions between ApoB peptide-induced antisera and either PAR-1 peptide or C5aR peptide antigens. (2G) Cross-reactions between C5aR peptide-induced antisera and either ApoB peptide or PAR-1 peptide antigens. Antisera used for cross-reactions were taken at 8 weeks. [Figure 3-1]Detection and quantification of lesion area in aortas of high fat diet-fed B6;129S-Ldlrtm1HerApobtm2Sgy / J mice after immunization with constructs versus control (GST-den). (3A) Photomicrographs of lesions observed in atherosclerotic aortas analyzed by elastin / van Gieson staining. (3B) Scatter plot showing mean values ​​of lesion area in aortic sinus of mice immunized with constructs compared to control (GST-den). (N=6-9 mice). Error bars=SEM. (3C) Percentage of reduction in lesion size in aortic sinus (reduction in control [GST-den] group was set to 0). (3D) Representative photomicrographs and quantitative analysis of collagen (Sirius Red staining under polarized light) in atherosclerotic aortas in individual mice. (3E) Quantification of collagen content in lesions in the aorta of B6;129S-Ldlrtm1HerApobtm2Sgy / J mice (N=7 mice). NS: not significant. [Figure 3-2] (3F) Representative Oil Red O stained frontal descending aorta of a mouse. (3G) Percentage of lesion area relative to total area of ​​descending aorta in individual mice (N=6 mice) from different experimental groups. Mean lesion size and difference in lesion size between experimental groups are shown. (3H) Percentage of reduction in lesion size in descending aorta (reduction in control [GST-den] group was set to 0). [Figure 4] (4A) Detection and quantification of lesion area in the aorta of high fat diet-fed B6;129S-Ldlrtm1H erApobtm2Sgy / J mice 9 weeks after immunization with constructs versus control (GST-den), GST and alum. Photomicrographs of lesions observed in atherosclerotic aorta analyzed by elastin / van Gieson staining (N=6-8 mice). (4B) Scatter plot showing mean lesion area in the aortic sinus of mice (N=6-8 mice). [Figure 5-1]Figure 5 shows the evaluation of inflammation-related cells in lesions of high-fat-fed B6;129S-Ldlrtm1HerApobtm2Sgy / J mice after immunization with AHHC, RHHC and RPHC constructs. (5A) Photomicrographs showing IHC staining of CD68 (green) and CD11c (red) markers (scale bar: 100 μm and 12.5 μm for magnification). (5B) Scatter plot showing anti-CD68 stained area in lesions relative to total lesion area. Data are shown as averages for 6 mice. (5C) Scatter plot showing anti-CD11c stained area in lesions relative to total lesion area. Data are shown as averages for 6 mice. (5D) Graph showing colocalization of CD68+ and CD11c+ area (obtained from Figures 3B and 3C). (5E) Photomicrograph showing IHC staining of CD4+ T cells (green) and Foxp3+ Treg cells (red) (scale bar: 100 μm and 12.5 μm for magnification). (5F) Scatter plot showing anti-Foxp3 staining area versus anti-CD4+ staining area in the lesion (N=6). [Figure 5-2] (5G) Representative analysis of Foxp3 expression by CD4+ T cells in lymph nodes of construct-immunized mice fed a high fat diet assessed using flow cytometry. (5H) Graph showing percentage of Foxp3+ cells in CD4+ splenocytes analyzed by flow cytometry. Data are presented as mean ± SEM of triplicate analyses. Differences between groups are indicated. [Figure 6-1]6A shows the evaluation of IL-10 producing T cells, TNF-α expression in lesions and cytokine levels in high fat diet fed B6;129S-Ldlrtm1HerApobtm2Sgy / J mice after immunization with AHHC, RHHC and RPHC constructs. (6A) Photomicrograph showing double IHC staining for IL-10 (red) and CD4 (green) (scale bar: 100 μm and 12.5 μm for magnification). (6B) Scatter plot showing mean IL-10 positive area (%) co-localized with CD4+ area (N=6). (6C) Photomicrograph showing IHC staining for TNF-α (green) in lesions (scale bar: 100 μm and 12.5 μm for magnification). (6D) Scatter plot showing mean anti-TNF-α stained area in lesions relative to total lesion area (N=6). [Figure 6-2] (6E-H) Graphs showing cytokine levels measured in plasma. [Figure 6-3] (6I-L) Graph showing cytokine levels measured in the supernatants of splenocytes stimulated with ConA. [Figure 6-4] (6M) Representative analysis of CD4+IL-4+ T cells in splenocytes of construct-immunized mice fed a high fat diet assessed by flow cytometry. (6N) Graph showing percentage of IL-4+ expressing CD4+ splenocytes. Data are presented as mean±SEM of triplicate analyses. Differences between groups are indicated. [Figure 6-5] (6O) Representative analysis of IL-17A expression by CD4+ T cells in splenocytes of construct-immunized mice fed a high fat diet assessed by flow cytometry. (6P) Graph showing levels of IL-17A expression in splenocytes. Data are presented as mean±SEM of triplicate analyses. Differences between groups are indicated. [Figure 6-6] (6Q) Representative analysis of CD4+IL-2+ T cells in splenocytes of construct-immunized mice fed a high fat diet assessed by flow cytometry. (6R) Graph showing levels of IL-2+ expressing CD4+ in splenocytes. Data are presented as mean±SEM of triplicate analyses. Differences between groups are indicated. [Figure 7] Graph showing measurement of cytokines (IFN-gamma and IL-10) from spleens of immunized mice stimulated with antigen. Splenocytes were cultured in RPMI1640 containing 10% fetal bovine serum and induced with 1 μg / ml antigen (GST-den, GST-AHHC, GST-RHHC, GST-RPHC, ApoB100 peptide, C5aR peptide, PAR-1 peptide, respectively) for 48 hours. IL10 and IFN-γ in the supernatants of cultured cells were then measured using DuoSet Mouse IL-10 Kit and Mouse IFN-γ Quantikine Immunoassay Kit (R&Dsystem, Minneapolis) according to the manufacturer's protocol. [Figure 8] Figure 8: Investigation of antigen-specific regulatory function in antigen-immunized mice. Figure 8: Inhibition of CD4+CD25- effector T cell proliferation by CD4+CD25+ regulatory T cells isolated from the spleens of control (GST-den immunized) and construct-immunized mice using AHHC, RHHC and RPHC as antigens. (8A) Quantitative analysis of CD4+CD25- effector T cell proliferation in the presence of Treg cells by flow cytometry. (8B) Proliferation of effector cells alone isolated from immunized mice is shown in the leftmost bar of each group. Treg cells are also added at different ratios to T effector cells. Data are presented as mean ± SEM of triplicate analyses. [Figure 9]Figure 9 shows the evaluation of smooth muscle alpha actin, vascular cell adhesion molecule (VCAM-1) and matrix metalloproteinase 9 (MMP9) expression at the lesion site. (9A) Photomicrographs showing IHC staining of smooth muscle alpha actin (red), vascular cell adhesion molecule VCAM-1 (green) (scale bar: 100 μm and 12.5 μm for magnification). (9B) Scatter plot showing mean area of ​​anti-SMC staining (N=6). (9C) Scatter plot showing mean area of ​​anti-VCAM-1 staining (N=6). (9D) Photomicrographs showing IHC staining of MMP9 (green) (scale bar: 100 μm and 12.5 μm for magnification). (9E) Scatter plot showing mean area of ​​anti-MMP9 staining (N=6). [Figure 10] (10A) Representative photomicrographs showing immunohistochemical staining of the aortic root showing areas of anti-ApoB antibody staining (red) in the lesion. Scale bar: 150 μm (non-magnified) and 25 μm (magnified). (10B) Graph showing quantitative analysis of ApoB expression in the lesion. N=6 mice. NS: not significant. (10C) Representative photomicrographs showing immunohistochemical staining of the aortic root showing areas of anti-HSP antibody staining (red) in the lesion. Scale bar: 150 μm (non-magnified) and 25 μm (magnified). (10D) Graph showing quantitative analysis of HSp60 expression in the lesion. N=6 mice. NS: not significant. [Figure 11-1] Figure 11 shows the evaluation of monocyte differentiation into macrophages. (11A) Differentiation of PBMCs stimulated with recombinant constructs (1 μg / ml) and inhibition of differentiation in the presence of each construct-derived antibody, as assessed by analysis of CD206 expression by flow cytometry. (11B) Graph showing differentiation of PBMCs stimulated with recombinant constructs (1 μg / ml) and inhibition of differentiation in the presence of each construct-derived antibody, as assessed by analysis of CD206 expression by flow cytometry. [Figure 11-2] (11C) A graph showing inhibition of differentiation by each construct and other construct-induced antibodies. (11D) A graph showing inhibition of differentiation by each construct and other construct-induced antibodies. [Figure 11-3](11E) Individual peptides as stimuli (1 μg / ml) of monocyte differentiation analyzed by CD206 expression. [Figure 11-4] (11F) Graph showing individual peptides as stimuli (1 μg / ml) of monocyte differentiation analyzed by CD206 expression. Data are presented as the mean of triplicate analyses. [Figure 12] Figure 12: Evaluation of TLR4 and MyD88 content in lesion sites. (12A) Photomicrograph showing IHC staining for TLR4 (red) (scale bar: 100 μm and 12.5 μm for magnification). (12B) Scatter plot showing mean area of ​​anti-TLR4 staining (N=6). (12C) Photomicrograph showing IHC staining for MyD88 (red) (scale bar: 100 μm and 12.5 μm for magnification). (12D) Scatter plot showing mean area of ​​anti-MyD88 staining (N=6). [Figure 13-1] (13A) Representative photomicrograph showing immunohistochemical staining of the aortic root showing the area of ​​anti-TLR4 antibody staining (red) and overlapping anti-CD11c antibody staining (green) in the lesion (yellow). Scale bars: 141 μm (non-magnified) and 26 μm (magnified). (13B) Graph showing measurement of the combined area of ​​occupied CD11c and TLR4 (%) in the lesion. [Figure 13-2] Table 1: Statistical analysis of the effect of immunization with peptides. [Figure 14] (14A) shows the natural dendroaspin amino acid sequence SEQ ID NO: 1. (14B) shows the amino acid sequence of C5a SEQ ID NO: 2. (14C) shows the amino acid sequence of C5Ar SEQ ID NO: 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Detailed Description Data suggest that the C5a / C5aR pathway in atherosclerosis is mediated by immunization with Apob t m2Sgy LDlr tm1HerBased on studies of / J mice, PAR-1 / HSP / Cp The present invention shows that the dendrolaspin scaffold is independent of the n pathway. The present invention provides formulations and vaccines that target both pathways simultaneously.

[0044] In our previous study, the construct AHHC[ApoB100 688-707 +hHS P60 303-312 +hHSP60 153-163 + Cpn-derived peptide (C)] (Lu et al. Atherosclerosis 2012, 225:56 -68). The present invention relates to a method for determining whether A in mice is “R” (C5aR 1-31 ) replaced by R HHC and further "H" (hHSP60 303-312 ) "P" (protease activity Sexual receptor 1 42-55 ) by sequential epitope replacement called RPHC Modifications and derivatives of this construct are provided. An alternative embodiment is a construct in which the antigenic epitope is A and Call ApoB (AA688-707), H h Human HSP60 (AA303-312) ) (SEQ ID NO: 12), H m Mycobacterium (AA253-268) (SEQ ID NO: 15) and R, derived from the complement component 5a receptor (AA1-31) (SEQ ID NO: 9); Structure AH h H m R. These are the B6;129S-Ldlr tm1Her Apo b tm2Sgy Immunization of / J mice resulted in the production of high levels of antibodies against each epitope. hHSP60 induced low antibody responses 153-163 and P aside Histological analysis demonstrated that mice immunized with RPHC or RHHC had AHH showed a significant reduction in the size of atherosclerotic lesions compared with mice treated with C. showed (69.5 ± 1.1% vs. 55.7 ± 3.4%, P = 0.0006 or 65.6 ±1.3% vs. 55.7 ±3.4%, P = 0.045). The reduction in plaque size correlated with changes in the cellular immune response when compared to controls. The inventors found that these new recombinant constructs were favorable for a significant reduction in the formation of atherosclerotic lesions. We conclude that the present invention may possess novel antigenic and structural features that are It provides a novel strategy for developing anti-atherosclerotic agents.

[0045] Effects of peptides derived from C5aR and PAR-1 on the reduction of atherosclerotic lesions Based on this, the inventors concluded that the effect of the multi-epitope construct on reducing atherosclerotic lesions is , including C5aR and PAR-1 in the vaccination We hypothesized that the effect of GABA on the reduction of phenotype and pathology could be regulated to enhance the reduction of GABA and pathology. In this study, the inventors demonstrated the efficacy of sequential substitution: AHHC → RHHC for the reduction of atherosclerotic lesions. (R denotes an epitope derived from C5aR) and RHHC → RPHC (P denotes PAR- The efficacy of constructs containing C5aR and PAR-1 by The induced immune response was in the order of RPHC ≥ RHHC ≥ AHHC. As a significant reduction in the size of atherosclerotic lesions in both the aortic sinus and descending aorta It is accompanied by a detectable anti-atherosclerotic effect.

[0046] Natural dendroaspin is a 59 amino acid peptide, and the dendroaspin scaffold is Further functional amino acid sequences, e.g., the nucleotide sequence of a factor in the coagulation cascade, may be added. The design is adapted to incorporate active portions or motifs of agonists, antagonists or inhibitors. When modified with androaspin (containing an RGD motif), the resulting molecule acts as an anticoagulant. These anticoagulants are particularly useful as anticoagulants and do not suffer from the drawbacks associated with existing anticoagulants (WO 01 / 57 (See brochure No. 210.) Such hybrid polypeptides contain an RGD motif. a first amino acid sequence that confers dendroaspin activity and a second amino acid sequence that confers dendroaspin activity; The hybrid denaturant may contain additional amino acid sequences that confer activities other than valency. Droaspin-based molecules can be multifunctional.

[0047] C5a is a protein fragment released from complement component C5. The four amino acid peptide binds to the C5α chain in the classical, alternative, and lectin pathways of complement activation. It is produced by cleavage of 5a-convertase.

[0048] The C5a receptor is associated with either the component 5a receptor (C5AR1) or CD88 (cluster of differentiation 88). Also known as C5aR, it is a G protein-coupled receptor for Ca. Human C5aR is a single-stranded polynucleotide that binds to C5aR is an integral membrane glycoprotein consisting of 350 amino acids that form a peptide. It is not released as a soluble receptor and does not circulate. C5aR is expressed by U937 and HL-60 C5aR is expressed on hepatic parenchymal cells, pulmonary vascular smooth muscle, lung and umbilical cord blood vessels, and on differentiated myeloid cells. Vascular endothelial cells, bronchial and alveolar epithelial cells, HepG2 cells, hepatoma cell lines, mesangial on cultured human fetal astrocytes and astrocyte cell lines. Express.

[0049] Throughout the description and claims of this specification, the terms "comprise" and "contain" are used. The words "including, but not limited to" and variations thereof are used to mean "including, but not limited to" and they are intended to exclude other parts, additives, ingredients, integers or steps. Throughout the description and claims of this specification, the singular forms "a," "an," and "the" are used interchangeably. , including plural words unless the context otherwise requires. In all cases, the specification uses the plural as well as the singular, unless the context requires otherwise. It should be understood that this contemplates a state.

[0050] Any features, integers, properties, or components mentioned in connection with any particular aspect, embodiment, or example of the present invention. Any chemical moiety or group may be used in any other aspect, embodiment or mode described herein, unless incompatible therewith. It should be understood that the present invention is applicable to any embodiment or example. All of the features disclosed in the above (and drawings) and / or the method so disclosed. or all of the steps of a process are such features and / or steps. Any combinations except those where at least some of the groups are mutually exclusive The present invention is not limited to the details of the above-described embodiments. The present invention relates to a method for producing a product comprising the steps of: or any novel or inventive aspect of the method or method steps so disclosed. includes novel combinations.

[0051] The reader's attention is drawn to any and all related patents, trademarks, and other materials filed contemporaneously or prior to this application which relate to this application. to all papers and documents available for public inspection in accordance with the and the contents of the documents are incorporated herein by reference.

[0052] Expression and purification of recombinant glutathione S-transferase constructs Glutathione S-transferase-dendroaspin (GST-tagged den, here 1A) and the GST-recombinant construct AHHC[ApoB100 peptide , amino acids (aa) 688 to 707 (numbering including the signal peptide), human heat shock human heat shock protein (hHSP) 60 peptides, aa303-312 and aa15, respectively 3-163; and Chlamydia pneumoniae (Cpn) The next epitope ("C" is aa 66-73 and and aa283-291 from the outer membrane 5 of Cpn)] The constructs RHHC ("R" means C5aR peptide, aa 1-31) and RPHC ("P" stands for protease-activated receptor 1 (PAR-1) peptide, aa42- 55) were prepared. These constructs were constructed using dendroaspin as a scaffold. A schematic diagram is shown in Figure 1B. These recombinant molecules were transformed into Escherichia coli (BL-21 The plasmid was expressed in Escherichia coli (E. coli) and purified by affinity and ion exchange chromatography. and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. All treatments were similar to those previously described for AHHC [Lu et al 2012; Atherosclerosis 225:56-68].

[0053] Animal testing B6;129S-Ldlr tm1Her Apob tm2Sgy / J mice were used, Each group of mice consisted of males aged 5–6 weeks. Dendroaspin was used as a scaffold. Previous data showed that it has a positive effect on reducing lesions when used for subcutaneous immunization in mice. Since no effect of dendroaspin was observed, dendroaspin was used as a control antigen. .

[0054] The immunizing antigens used were the constructs AHHC, RHHC and RPHC, respectively. Repetitive immunization multiple sites strategy (RIM) The mice were sacrificed at the end of the 12th week of the study (high-fat diet at the end of the 2nd week) and were then fed a 10-day diet. The control group was treated with GST-tagged Den and alum. After immunization with (adjuvant) a feeding program was followed.

[0055] Tissue preparation and measurement of antibody responses Twelve weeks after the first immunization, aortic tissues were harvested and subjected to immunohistochemistry (IHC). Optimal cutting temperature compression media was used for cryosectioning and lesion measurement. The images were then subjected to optical coherence tomography (OCT) and mounted in paraffin. Atherosclerotic lesions in the aortic root Olympus UULH optical microscope (Olympus Optical Co.Lt d., Tokyo, Japan) and Image-Pro Plus TM software Software, version 7.0 (Media Cybernetics, Inc. The descending aorta was cut longitudinally and analyzed using a CT scanner (Bethesda, MD, USA). The degree of atherosclerosis was evaluated after Oil Red O (ORO) staining. Peptide-specific antibody levels in plasma samples were measured by ELISA according to the manufacturer's instructions. Embed one third of the spleen in OCT and pass the remaining part through a 70 μm cell strainer. The mixture was homogenized by centrifugation and frozen for further analysis.

[0056] IHC and morphometric analysis, quantitative measurements of atherosclerosis, and CD4 + narrow spleen IHC analysis of forkhead box protein 3 (Foxp3) expression in blebs The OCT-embedded specimens were analyzed for CD68, CD11c, and interleukin (IL )-10 and tumor necrosis factor (INF)-α, Foxp3, vascular cell adhesion molecule (VCAM ) 1, alpha smooth muscle cells (alpha-SMC), matrix metalloproteinase 9 Paraffin-embedded tissue sections were prepared using an Olympus U -Hematoxylin and eosin (HE) for histological examination by ULH light microscopy and stained with elastin / van Gieson (Sigma).

[0057] CD4 in splenocytes + Foxp3, IL-2, IL-4 and IL-1 in T cells Flow cytometric analysis of 7A expression and PBM to macrophages (CD206) Differentiation of C. Spleen cells were incubated with allophycocyanin-anti-mouse Foxp3, IL-2, IL-4 and IL were processed for staining with -17 antibody (BioLegend, Cambridge, UK). For the cell differentiation assay, mouse (C57BL / 6) PBM Cs were stimulated with various antigens or preincubated with antisera to the antigens (antiserum Antigen-induced differentiation of monocytes into macrophages was performed using flow cytometer. The results were measured by cytometry in uninduced or control antigen (GST-Den)-induced cells. were compared with the cellular population.

[0058] Cytokine measurements IL-10 and TNF-α levels in the lesions were quantified by IHC analysis (Rat Anti-mouse TNF-α and IL-10 were purchased from BioLegend, CA, USA. Cytokines, IL-10, transforming growth factor beta (TGF-β ), TNF-α and interferon gamma (IFN-γ) plasma levels were measured according to the manufacturer's (R&D systems, Abingdon) (UK). Concanavalin A (ConA)-induced IL-10, TGF- Levels of IFN-β, TNF-α and IFN-γ were also measured.

[0059] Antigen-specific regulatory T cell functional assay Immunize subcutaneously with each construct to assess antigen-specific regulatory T cell function. B6;129S-Ldlr tm1Her Apob tm2Sgy Spleen CD of / J mice 4 + From T cells, we used Miltenyi Biotec's regulatory T cell isolation kit (Berg sch Gladbach, Germany) to detect CD4 + CD25 + T Reg cells were isolated. CD4 + CD25 -T effector cells were each Splenic CD4 of immunized mice + T cells (CD4 + CD25 + Binding to cells Not bound to beads, CD4 + 99.5% of cells. CD4 + CD25 - cells (2×10 5 ) CD4 + CD25 + cells (2×10 5 ) and 1 μM of the relevant construct or GST- After 2 days of culture, T effector cell proliferation was assessed by the mean fluorescence Changes in the oral intensity of populations of cells by flow cytometry expressed as mean percentile intensity (MFI) and and measured.

[0060] statistical analysis Data are reported as mean ± standard error of the mean (± SEM) unless otherwise indicated. Figures were generated using graph-pad Prism 5.01 and Sigma plot 9. 0. For atherosclerotic lesion size, one-way analysis was used for multiple comparisons. Data were compared and differences between groups were assessed using ANOVA and post-hoc Bonferroni tests. Other data were analyzed using Student's t test (two-tailed analysis). Parametric distributions are supported by the Mann-Whitney U test for pairwise comparisons and multiple proportions The Kruskal-Wallis test was used for comparison. Differences between groups were considered to be greater than 0.05. P values ​​less than were considered significant. EXAMPLES

[0061] Peptide-specific immunoglobulin G in the serum of immunized mice was assessed. Dendroaspin (GST-tagged) or dendroaspin were administered at 2 and 12 weeks, respectively, after the placement. in the serum of mice immunized with constructs (GST-tagged) in the androaspin scaffold. Antibody levels were measured by ELISA. When used as an ELISA antigen, ApoB peptide, hHSP60 303-312 Oh Antibodies specific for the Cpn peptide were also observed in mice immunized with RHHC (Fig. 2A). In mice, the observed hHSP60 303-312 and Cpn peptide-specific antibodies In addition, C5aR peptide-specific antibodies were detected (Figure 2A). High antibody levels against the R peptide, on the other hand, low antibody levels against the PAR-1 peptide hHSP60 levels were detected in mice immunized with RPHC (Figure 2A). 15 3-163 Low antibody levels against the construct were observed in mice immunized with either construct. The overall observed optical density (OD) values ​​at 100-fold dilution were 2.0–3.2 at week 12. decreased compared to week 1 values.

[0062] Interestingly, hHSP60 was significantly higher than GST-Den control. 153-163 oh All peptide antigen epitopes at high dilutions were stained with 100% TNF-α and 100% TNF-α except for the PAR-1 peptide. Peptide-induced specific immunoglobulins in the serum of mice immunized with peptides against the tope ( IgG1 responses were observed (Figure 2B). Furthermore, when compared to the control, IgG2c responses were A similar pattern was also detected for , but at lower serum dilutions (Fig. 2C). However, based on the optical densities measured at different dilutions of the sample (1:50 vs. 1:6250), Furthermore, the levels of IgG2c detected were much lower than those of IgG1. When the antiserum from week 8 was used, the ApoB peptide-induced antiserum and the Cpn peptide ( Fig. 2D), ApoB peptide and hHSP60 303-312 Peptide-induced antisera and Between these antigens (Fig. 2E), the ApoB peptide-induced antiserum and the PAR-1 peptide or Between the C5aR peptide and either antigen (Fig. 2F) and C5aR peptide-induced antiserum The specificity between the serum and either the ApoB peptide or the PAR-1 peptide antigen (Figure 2G) A certain level of cross-reactivity was observed. Pooled sera were tested, so SD values ​​were not calculated. In the absence of Cpn peptide and ApoB peptide antisera (Fig. 2D), cross-reaction was observed except for As such, other cross-reactions showed significant differences compared to the control (Figures E-G; P < 0.05- <0.001). EXAMPLES

[0063] The reduction in atherosclerotic lesion size in the aortic sinus was evaluated. AHHC, RHHC, RP After immunization with HC and after 10 weeks of high-fat feeding, the aortic sinus of the mice was The degree of atherosclerosis in the immunized animals was evaluated. Representative photomicrographs of lesioned sections from the experimental groups are shown in Figure 3A. The plaque area is shown in Figure 3B. The lesion size was 0.01 vs. 0.01 in the control (70,200 ± 5718 μm 2 ) compared to Compared to 31071±998.7μm 2 , 24123±1967μm 2 and 21386± 2482μm 2 Mice immunized with all three constructs showed (P<0.001) The smaller lesion area was observed in mice immunized with AHHC compared to mice immunized with AHHC. Compared with mice immunized with RHHC or RPHC (P = 0.007 The percentage reduction in lesion size is shown in Figure 3C, which was significantly greater than that in control animals. If we assume a zero percent reduction in the change, the results were 55.7±3.4%, 65.6±1.3%, and and 69.5 ± 1.1%. Control mice immunized with GST-Den showed The lesion formation was similar to that of ST-tag or alum (adjuvant) immunized controls (Figure 4A and 4B). Therefore, GST-Den was used as a control throughout the experiments.

[0064] The effect of treatment with these recombinant constructs on collagen content in these lesions Reduction of atherosclerosis in mice treated with these constructs was also investigated. , respectively, compared with control mice and AHHC- or RHHC-immunized mice. The collagen content in the 14.1–18.2 mm Hg collagen-rich 18.6±1.2% and 19.4±1.0% were significantly increased by 1.2-fold. 0.9% vs. control 5.9 ± 0.3%; P < 0.001) (Figures 3D and 3E). Mice immunized with α-aminobutyric acid (AHHC) showed significantly higher cytotoxicity than mice immunized with α-aminobutyric acid (RHHC) compared with mice immunized with AHHC or RHHC. A significant increase in collagen (24.4±0.9%) was observed in both groups (P=0.003 for each group). and P = 0.007).

[0065] The descending aorta was longitudinally incised and stained with Oil Red O (ORO) on the front side. The area of ​​plaque was measured. Representative en face stained descending aortas from the experimental group are shown in Figure 3F. size in AHHC and control (19.5 ± 1.7%, P < 0.001), respectively. 8.4±0.3%, 6.6±0.3% and 6.4±0 for RHHC and RPHC. The R-cell counts were significantly lower in mice immunized with all constructs (Figure 3G). Both HHC and RPHC immunized mice were significantly smaller than AHHC immunized mice The reduction in lesions, expressed as a percentage, was shown in Figure 2H. The results are shown in Fig. 1. The results are 57.3±1.7% and 6% for AHHC, RHHC, and RPHC, respectively. 6.1±1.6% and 67.3±1.9%. The smallest lesion area was in RPHC. More was observed in immunized mice. EXAMPLES

[0066] Local (aortic lesions) and distant organs: Inflammatory cells and and Foxp3 expressing CD4 + The amount of T cells was assessed. Anti-CD68 staining in the lesions The percentage of area was 43.7 ± 3 in control mice immunized with GST-Den. 2% in mice immunized with AHHC, RHHC, and RPHC, respectively, compared with 2% The results were 14.9 ± 1.6%, 13.6 ± 1.3%, and 10.3 ± 0.8% for the 100-fold increase in β-blockers (P <0.001), smaller anti-CD68 staining areas were observed in R compared with AHHC-immunized mice. This was observed in PHC-immunized mice (P=0.034) (Figures 5A and 5B). As measured by anti-CD11c stained lesion area, this was 38.4±1.9% compared with 38.4±1.9% in control mice. 13.3 in mice immunized with AHHC, RHHC and RPHC, respectively. ±2.1%, 10.5±1.5% and 7.9±0.8% (P<0.001). (FIGS. 5A and 5C). The area of ​​anti-CD11c stained lesions in RPHC-immunized mice was It was significantly smaller when compared with that in AHHC-immunized mice (P = 0.03 9) (Figures 5A and 5C). Double immunostaining for CD68 and CD11c revealed the percentage and CD68 expressed as + Area and colocalized CD11c + The area was 68. 6±4.7% for AHHC, RHHC and RPHC, respectively. 3.7%, 55.2±2.6%, and 50.3±3.3%. More than half of the patients were CD68 + CD11c + It was clearly shown that ; P = 0.046-0.011), suggesting that this cell type in the lesions is of bone marrow origin. Foxp3-expressing CD4+ cells analyzed by IHC staining of aortic sections. + Cell division in AH mice compared with 1.2 ± 0.2% (P < 0.001) in control mice. Approximately 6-8 times higher in mice immunized with HC, RHHC, and RPHC ( 8.2±1.4%, P<0.001;9.4±1.1%, P<0.001;9.9±1. 6%, P<0.001) (Figures 5E and 5F). Furthermore, these three constructs CD4 of immunized mice + The expression of Foxp3 in spleen cells was significantly higher than in controls. and AHHC, 4.0±0.5% vs. 4.0±0.5% in controls (P<0.001). 13.3±0.6%, 15.3±1.5% and 18 for RHHC and RPHC. 0±1.1% (Figures 5G and 5H). However, in RHHC-immunized mice, In comparison, RPHC-immunized mice did not show a significant increase in Foxp3 expression. With the exception of β-lactamase, significantly higher levels of F were observed compared to those in AHHC-immunized mice. oxp3 expression was observed (P=0.002). EXAMPLES

[0067] Expression of anti-inflammatory and pro-inflammatory cytokines at the lesion site and Cytokine levels were assessed in plasma and in the supernatants of stimulated splenocytes by IHC analysis. Detected aortic lesions in mice immunized with AHHC, RHHC, and RPHC IL-10 expression in the lesions is shown in Figure 6A. + Thin The percentage of cells was about six-fold higher in mice immunized with these constructs, FIG. and 6B, when compared with that in the control group (0.9 ± 0.2%). The results were 4.8±0.7%, 5.2±0.8% and 5.0±0.9% for AHHC, RHHC and RPHC, respectively. and 5.9 ± 0.7% (P < 0.001). IHC analysis of TNF-α expression , significantly less TNF-α in the lesions of mice immunized with the construct compared to controls. The α-occupancy area was shown to be (25.7 ± 3.1% in AHHC; 15.2 ± 0.9% in RHHC) 15.5 ± 1.5% in RPHC and 41.3 ± 3.1% in controls (Figures 6C and 6D These data are compared to controls (defined as 100% total lesion area and 0% reduction). The percentage reductions are 37.8%, 63.2% and 62.5%, respectively. RHHC or RPHC resulted in improved reduction compared with C (P = 0.00 8~0.014).

[0068] Plasma levels of the atheroprotective cytokine IL-10 were significantly higher in control The results were significantly increased in mice immunized with these three constructs compared to those in mice immunized with 6E and 6F). Immunization with RPHC stimulated the secretion of IL-10 and TGF-β. immunization with the other two constructs had a greater effect on promoting 0.05~<0.001). Atherogenic cytokine TNF-α Plasma levels of were significantly reduced by immunization with these three constructs (Figure 6G). A similar trend was obtained for these constructs with respect to plasma levels of IFN-γ (Figure 2C). 6H). In particular, immunization with RPHC reduced the secretion of TNF-α and IFN-γ ( P ≦ 0.002) and showed a greater effect than AHHC in reducing TNF-α secretion. The AHHC had a greater effect than the RHHC on the overall survival (P = 0.004). Slightly higher blood levels of IFN-γ in the immunized mice compared to the control mice. Plasma levels were significantly higher in the control group (24 vs. 20.8 pg / ml), but this difference did not reach statistical significance. There wasn't.

[0069] The supernatants from splenocytes of mice immunized with these constructs showed 1 IL-10 (Figure 6I) and TGF-β (Figure 6 J) were individually shown to have significantly higher secretions (P<0.05-0.001). Higher levels of IL-10 or TGF-β were expressed in splenocytes from RPHC-immunized than splenocytes from immunized mice. was produced by splenocytes from treated mice (P<0.05-0.01). NF-α (Figure 4K) and IFN-γ levels (Figure 6L) increased with 10 μg / mL ConA. When stimulated more, the splenocytes of mice immunized with these constructs were increased compared to controls. In particular, TNF-α and IFN-γ in RPHC-immunized mice were significantly decreased. The levels of N-γ were significantly higher in the AHHC-immunized mice than in the control mice when stimulated with 10 μg / mL of ConA, respectively. The results were lower than those in the immunized mice (P<0.05-0.01). The supernatants of splenocytes from immunized mice stimulated with peptides or peptide-containing constructs High levels of the protective cytokine IL-10 and low levels of the proinflammatory cytokine IFN -γ, but stimulation with a different protein, KLH, was also shown to be involved. This was not the case for the ApoB and C5aR proteins (Figure 7A-D). and Cpn peptide for IL-10 production in GST-den immunized mice. When used as a stimulant for the induction of PAR-1 peptides in RPHC-immunized mice, The changes in cytokine production in response to various stimuli were significant, except for acetaminophen.

[0070] Mice immunized with these three constructs showed increased expression of IL-4 (Th2-associated), IL-1 7A (Th17-associated) and IL-2 (Th1-associated) expressing CD4 + The proportion of spleen cells is The results were significantly lower (IL-4 + P<0.001 for IL-17A + About P ≤ 0.001), IL-4 + About 2.74±0.11% (AHHC), 2.85±0.12% (RHHC) and 2.87 ± 0.02% (RPHC) (Figures 4M and 4N) compared with 4.1 ± 0.3% (control). In the case of IL-17A + 2.0±0.1% (AHHC), 1.7±0.1% (RH HC) and 0.9 ± 0.1% (RPHC) (Figures 6O and 6P). A small percentage of CD4 compared with values ​​in HHC or RHHC immunized mice + IL -17A + Spleen cells expressing RPHC were found in RPHC-immunized mice (P ≤ 0.006) (Figure 6O-P). Interestingly, the percentage was higher compared to the values ​​in RPHC-immunized mice. CD4 + IL-17A + Expressing spleen cells were observed in RHHC-immunized mice (P=0 021) (Figure 6P). Furthermore, a significantly lower percentage was observed when compared to the values ​​in the controls. CD4 + IL-2 + Expressing spleen cells were observed in mice immunized with the three constructs (P<0.05). 0.001; Figures 6Q and 6R). Furthermore, in AHHC- or RHHC-immunized mice, A small percentage of CD4 + IL-2 + Spleen cells expressing RPHC were This was observed in the 14 patients with glaucoma (P = 0.019-0.007). EXAMPLES

[0071] We investigated antigen-induced specific Treg cell function. Functional Treg cells were generated by immunization. To evaluate whether antigen-specific Treg cells (CD4 + CD25 + T cells) to CD4 + Effector T cells (CD4 + CD25 - T cells) were co-cultured with F of GST-Den-immunized control mice in response to stimulation with 1 μM GST-Den Proliferation of effector T cells was suppressed in the presence of Treg cells from GST-Den-immunized mice (Figures 8A and 8B). In contrast, CD4 + CD25 - effector CD4 T cells were isolated from these mice. + CD25 + When co-cultured with Treg cells , and are immunized by AHHC, RHHC, and RPHC, respectively, in response to stimulation with the relevant antigen. The proliferation of effector T cells in the immunized mice was suppressed (Fig. 8A and When Treg cells were added to effector cells at ratios of 4:1–16:1, T Compared with the absence of reg cells, the difference was significant (P<0.05~<0.00 1). EXAMPLES

[0072] Smooth muscle alpha-actin, VCAM1, MMP9 and specific antigen Apo in lesions The expression of B and HSP60 was evaluated. To assess whether vascular remodeling and SMC content in the lesions influences vascular behavior and The amount of IL-1 and IL-2, as well as the expression of VCAM1 and MMP9 at the lesion site, were analyzed by IHC analysis. The area of ​​anti-SMC staining was significantly higher than that in the control immunized with GST-Den. When the plaques of mice immunized with AHHC and RPHC were significantly smaller, The results were 5.2±0.7% and 4.9±0.8%, respectively, but the immune response was not improved by RHHC. In mice treated with VC, the levels were not significantly decreased (Figures 9A and 9B). The expression of AM1 was significantly higher than that in controls immunized with dendroaspin (18.0±2 .3%), which were significantly down-regulated in AHHC, RHHC and RP, respectively. The results were 4.5 ± 0.9%, 7.8 ± 0.9%, and 4.8 ± 0.9% in the HC group (Fig. 9A and and 9C). The effects were significantly increased in R compared with AHHC or RPHC immunized mice. A similar trend was observed in HHC-immunized mice (Fig. 9A and 9C). The MMP9 expression in mice immunized with all of the above was 7.9±1 0.0%, 10.1±1.0%, and 7.6±1.0% of the stained area in AHHC, R In HHC and RPHC immunized mice, 18.1 ± 2.5% of the mice were immunized with GST-Den. This was shown in control mice treated with the vaccine (Figures 9D and 9E). The significant differences obtained in this respect between C- and RHHC-immunized mice were Except for the absence of IFN-γ (Figures 6D and 6E). Mouse ApoB (Figures 10A and 10B) and mouse HSP60 tag were mutated between the control and control mice. Since almost no difference in protein (Fig. 10C and 10D) antigens was detected in the lesions, Injection of recombinant constructs containing human ApoB and HSP60 peptides increased their counts. It did not affect the expression of turbatins (ApoB and HSP60). EXAMPLES

[0073] Naïve mice on a C57BL / 6 background responding to treatment with recombinant constructs Assessment of monocyte differentiation into macrophages in PBMCs and construct specific for differentiation The effect of immune serum was examined. Upon stimulation with CSF or proatherogenic antigens, monocytes transform into macrophages (or AHHC converted to RHHC by single domain substitution can be differentiated into In response to stimulation of monocytes (from naive mice C57BL / 6 with the same background), To assess whether the same effect of rheumatoid arthritis could be maintained, PBMCs were cultured in either RHHC or AH. After 3 days, the cell surface marker CD206 (mannose receptor, macrophage) was stimulated with HC. The expression of RHHC and A phage markers was evaluated in comparison to unstimulated cells. Both HHCs induced monocyte differentiation into macrophages (based on changes in cell numbers) ( Figure 1 1A and 11B). Furthermore, the differentiation of PBMCs induced by these two constructs preincubated cells with antisera from mice immunized with these two antigens. Interestingly, the inhibition was abolished by incubation with This can be achieved by preincubation with serum (Figures 11A-7D). Observation of differentiation by individual epitopes or domains as catalyzers allows for the determination of different ratios of The results showed that the α-terminal region of the cytoplasmic reticulum (C-ER) was increased (Figures 11E to 11F). EXAMPLES

[0074] Toll-like receptor 4 (TLR4) at the lesion site and associated with atherosclerosis We investigated the content of myeloid differentiation factor 88 (MyD88), which is involved in the TLR4 signaling pathway. The effect of these recombinant constructs on TLR4 and MyD88 content in the lesions was The effects of treatment with these constructs on atherosclerosis in mice were investigated. The reduction in disease was accompanied by a decrease in both TLR4 and MyD88 content. The color area was 8.1±1.1% of that in the control immunized with dendroaspin. In comparison, in the plaques of mice immunized with AHHC, RHHC, and RPHC, were significantly smaller in the 100% and 200% groups, 4.6±1.0%, 3.4±0.5%, and 4.2±0.6%, respectively. % (Figures 12A and 12B). Similarly, the anti-MyD88 stained area was 100% in the control The mice immunized with these three constructs showed a significantly higher immunization rate than the control mice (18.6 ± 2.4%). were significantly smaller in the 10 lesions, 9.7±1.2%, 9.6±1.6%, and 10 0.2±1.9% (Figures 12C and 12D). The overlap of R4 staining area was significantly greater for the three constructs compared to the control (6.6 ± 0.8%). All showed significantly smaller lesions in mice immunized with 3. 4±0.6%, 2.9±0.7%, and 2.7±0.8% (Figures 13A and 13 B). EXAMPLES

[0075] Two HSP60 peptides, one derived from human and one from mycobacterium, were ob tm2Sgy Ldlr tm1Her Immunization of / J mice increased atherosclerotic disease Experiments were conducted to compare their ability to reduce the phenotype of rheumatoid arthritis. mHSP60 253-268 Mycobacterial heat shock proteins (HSPs) 60 (AA253-268) (SEQ ID NO: 15), hHSP60 516-528 People who call Two types of keyhole mussel hemocytes derived from HSP60 (AA516-528) (SEQ ID NO: 14) Mice were immunized with anthraquinone (KLH)-conjugated peptides. The mice were immunized with 100 mg / kg of IgG and 100 mg / kg of IgG, and 2 weeks after the first immunization, they were placed on a high-fat diet. The results showed that the mHSP60 peptide had a stronger binding affinity to IgG and IgG1 than the hHSP60 peptide. Apart from having low titers against IgG1c and almost no titers against IgG2c, These results indicate that the peptides of the present invention have similar functions. Allergic immune response; Reduced lesions; Increased Treg expression; Atheroprotective cytokines: IL Increased concentrations of TGF-10 and TGF-β, as well as increased levels of proinflammatory cytokines such as TNF-α and and INF-γ levels; CD4 by Treg cells + CD25 - Inhibition of T cell proliferation and downregulation of the TLR4 / MyD88 pathway (data not shown). B6;129S-Ldlrtm1HerAp by SP60 and hHSP60 peptide After immunization of obtm2Sgy / J mice, low sequence homology between the two peptides ( 31%) and the lower immune response obtained with the mHSP60 peptide. Both peptides have similar effects on significantly reducing early atherosclerotic lesions. From the data, it is believed that such immunization with the constructs of the present invention may be effective in preventing atherosclerosis. These findings provide attractive opportunities for the design and development of peptide-based vaccines. It is recognized. EXAMPLES

[0076] The transcription factor FOXP3 (forkhead box P3) regulates mouse CD4 + CD25 + It regulates Treg function (Fontenot et al., Nat Immunol. 2003;4:330-336 ; Hori S, Nomura T, Sakaguchi S. Science. 2003;299:1057-1061), native CD4 + CD twenty five +Treg transfer significantly reduced plaque progression in ApoE-KO mouse model (Ait-Oufella et al., Nat Med. 2006;12:178-180; Mor et al Ar terioscler Thromb Vasc Biol. 2007;27:893-900). Based on this, naturally occurring T It is known that reg can affect the size and composition of atherosclerotic lesions, Several reports have demonstrated that antigen-specific responses can be operative in developing atherosclerotic plaques. The inventors have demonstrated that proatherogenic antigen induction We hypothesized that Tregs may have a specific function in reducing pathology. The effects of IL-1 on humoral immune responses, atherosclerotic lesion size, and local and systemic cellular responses were investigated. By testing the answer, B6;129S-Ldlr tm1Her Apob tm2Sg y Antigen-immunized mice blood samples for atherosclerotic lesion formation in / J mice Experiments were performed to evaluate the effect of adoptively transferred Treg cells.

[0077] The first method to examine immune responses and adoptive transfer in KO mice was to use AH h H m and incorporating the R construct into a dendroaspin scaffold to generate a recombinant construct. The antigenic epitope of ApoB (AA688-707) is called A, and the human HSP60 (AA303-312) (SEQ ID NO: 12) h and Mycobacterium (AA25 3 to 268) (SEQ ID NO: 15) m The complement component 5a receptor (AA1-31) Row number 9) was called R. Mice were immunized with the RIMM (repeated multisite immunization strategy) protocol. By AH h H m R. Treg cells were h H m R (Tr eg S) and dendroaspin (Treg C From the blood of mice immunized with Adoptive transfer was achieved via the retro-orbital plexus of mice.

[0078] The second method for evaluating the effect of Treg cells on atherosclerotic lesion formation is Tregs from the blood of AGD-den (control) and AHhHmR-immunized mice, respectively. The recipients were non-immunized naïve mice of the same strain. The mice were fed a high-fat diet (HFD) for 10 weeks and then sacrificed. and immunohistochemical evaluation, analysis of cytokine levels, Treg activity and foam cell morphology. The evaluation of the results was evaluated.

[0079] Data (not shown) show that T cells isolated from the blood of mice immunized with proatherogenic antigens eg S showed that non-proatherogenic antigen immunization increased the expression of IFN-γ-γ-γ in non-immunized mice after adoptive transfer into the vein of non-immunized mice. The results showed that Tregs from the blood of transplanted mice showed less pathology. Natural CD4 + CD25 + Treg transfer in ApoE-KO mouse model In addition to fewer lesions forming, they also significantly reduced plaque progression at the lesion site. Increased collagen content in the lesions, increased Treg expression in the lesions, and increased atherosclerosis in the plasma. Higher concentrations of immune-protective cytokines (IL-10 and TGF-β) and inflammatory They also showed lower concentrations of proinflammatory cytokines (TNF-α and INF-γ) at the lesion site. Downregulation of αSMC and PECAM expression was also observed in the sarcomere.

[0080] These results indicate that the constructs of the present invention may be useful in cell therapy for the treatment of atherosclerosis. This demonstrates that the 2018 Tokyo Olympics offers an attractive opportunity to The present invention also provides the following. [1] (i) a scaffolding portion; (ii) 1, which can induce anti-atherosclerotic vascular disease responses via the 1 pathway and (iii) Inducing anti-atherosclerotic vascular disease through a second pathway independent of the first pathway A second species of epitope that can cause A recombinant construct comprising: [2] A construct according to [1], comprising multiple first and / or second species epitopes. [3] The first pathway involved in the formation of atherosclerosis is C5a or C5aR interaction. The construct according to [1] or [2], [4] The first species epitope is a C5a or C5a receptor (C5aR) protein. A construct described in any one of [1] to [3]. [5] The C5a epitope is an amino acid sequence of 5 to 40 consecutive amino acid residues in SEQ ID NO: 2. The construct according to [4], which is a polypeptide comprising an amino acid sequence. [6] The C5a epitope is EQRAARISLGPR (SEQ ID NO: 3), RAARISL Contains GPRCIKAFTE (SEQ ID NO: 4) and CVNNDETCEQ (SEQ ID NO: 5) or a polypeptide having antigenic activity comprising an amino acid sequence selected from the group consisting of The construct according to [4] or [5], which is a functional fragment thereof having the above structure. [7] The C5a epitope is an amino acid sequence of 5 to 45 consecutive amino acid residues in SEQ ID NO:6. The construct according to [4], which is a polypeptide comprising an amino acid sequence. [8] The C5aR epitope is a C- or N-terminal sequence, and optionally said epitope is SFNYTTPDYGHYDDKDTLD (SEQ ID NO: 7), TLDLNTPVDKTSN (SEQ ID NO: 8) and MNSFNYTTPDYGHYDDKDTLDLNTPVDKTS N (SEQ ID NO: 9) The construct according to [4] or [5], which is a peptide or a functional fragment thereof having antigenic activity. thing. [9] The second species of epitopes associated with an anti-atherosclerotic vascular disease response is apolipoprotein A Apo epitope, heat shock protein (HSP) epitope, Chlamydia pneumoniae (chlamydia pneumonia) epitope, PAR-1 epitope and perilipin epitope The structure according to any one of [1] to [8], wherein the structure is selected from the group consisting of building.

[10] The HSP is HSP60 or HSP65, and optionally, the HSP is HSP6 If the value is 0, the antibody is either human HSP60 or Mycobacterium bovis. s) A construct described in [9], which is an HSP.

[11] The HSP60 epitope is peptide 1 (AA) 153-160: AELKKQSK ; (SEQ ID NO: 10), peptide 1 (AA) 153-163: AELKKQSKPVT; ( SEQ ID NO: 11), peptide 1 (AA) 303-312: PGFGDNRKNQ (SEQ ID NO: 12), Peptide 2: AA277-286 PGFGDNRKNQ (SEQ ID NO: 13), Peptide (AA) 516-528: KGIIDPTKVVRTA (SEQ ID NO: 14) and Icobacterium (AA) 253-268: EGEALSTLVVNKIRGT (Sequence No. No. 15) or having an amino acid sequence or antigenic activity selected from the group consisting of The construct according to

[10] , comprising a functional fragment thereof.

[12] Optionally, the chlamydia pneumonia is Cpn1 or Cpn2; Selectively, the major outer membrane protein (MOMP) (amino acid sequence (AA) 67-74: GDYV FDRI (SEQ ID NO: 16)) and the putative outer membrane protein (Pomp) 5 (amino acid Select from the group including the sequence (AA) 283-291: QAVANGGAI (SEQ ID NO: 17) A polypeptide comprising or consisting of the selected amino acid sequence or a polypeptide having antigenic activity. The construct described in [9], which is a functional fragment of

[13] The PAR-1 epitope is LLRNPNDKYEPF (SEQ ID NO: 19), A structure on the ground.

[14] The scaffold portion may be a dendroaspin scaffold protein as set forth in SEQ ID NO: 1 or a fragment thereof. The construct according to any one of [1] to

[13] , which is a mutant.

[15] The first and / or second species epitopes are located on (a) of the dendroaspin scaffold. (b) Loop I and / or Loop II; (c) Loop I and / or Loop III; ) Loop II and / or Loop III; (d) Loop I, Loop II and Loop I II; (e) incorporated at one or more positions in either the N- or C-terminus,

[14] The construct described in.

[16] (i) a scaffolding portion; (ii) a Cpn epitope; and (iii) one or more additional epitopes from the same or different proteins P and The construct described in [1], comprising:

[17] The additional epitopes are derived from HSP, PAR-1 and C5aR. The construct described.

[18]

[16] Also, the group consisting of AHHC, RHHC, RPHC and AHHR is selected from the group consisting of The construct described in

[17] .

[19] (i) a scaffolding portion; (ii) two HSP epitopes; (iii) one or more additional epitopes from a different protein; The construct described in [1], comprising:

[20] wherein the one or more further epitopes are ApoB and / or Ca5R;

[19] The construct described. [twenty one] The epitope incorporated into the construct according to any one of [1] to

[20] is An expression vector comprising a nucleic acid to be encoded by the gene. [twenty two] An antigenic composition comprising the construct according to any one of [1] to

[20] , Optionally, (i) isolated microsomes, which are optionally inverted microsomes; or (ii) MHC (iii) a hydrophobic complex containing a protein or (iv) a synthetic product. , the composition. [twenty three] The composition according to any one of claims [1] to

[20] , formulated as an injectable or oral formulation. and optionally a suitable adjuvant, excipient, diluent and / or or a carrier. [twenty four] [1] to

[20] , or the vector or or a pharmaceutical composition comprising the immunogenic composition described in

[22] . [twenty five] The construct according to any one of [1] to

[20] or

[21] , which is used as a medicine. The vector described.

[26] A construct according to any one of [1] to

[20] or

[21] for use in the manufacture of a medicine. The vector described in

[27]

[0026] The method according to any one of [1] to

[20] , for use in treating atherosclerosis. or the vector described in

[21] .

[28] [1] to

[20] or the vector according to

[21] Including vaccines.

[29] [1] to

[20] ; the protein according to any one of [1] to

[20] ; the vector according to

[21] ;

[22] The immunogenic composition according to the present invention and the pharmaceutical composition according to the present invention Anti-atherosclerosis in a mammal, comprising administering to an individual a formulation of choice. How to provoke a reaction.

[30] [1] to

[20] ; the protein according to any one of [1] to

[20] ; the vector according to

[21] ;

[22] The immunogenic composition according to the present invention and the pharmaceutical composition according to the present invention A method for treating or preventing atherosclerosis comprising administering to an individual a formulation of the invention. Methods for reducing or eliminating

[31] [1] to

[20] ; the protein according to any one of [1] to

[20] ; the vector according to

[21] ;

[22] The immunogenic composition according to the present invention and the pharmaceutical composition according to the present invention A method for treating an individual with early atherosclerosis comprising administering to the individual a selected formulation. or a method of treating an individual identified as being at risk for developing atherosclerosis.

[32] For epitopes associated with two independent pathways involved in atherosclerosis formation 20. A method for generating an immune response comprising: 1. At least one first and at least one second peptide according to any one of [1] to

[20] A dendroaspin scaffold protein containing both the first and second epitopes was constructed and expressed. And, 2. Incubating eukaryotic cells with the dendroaspin scaffold protein And, 3. Preparing microsomes using the eukaryotic cells; 4. Injecting the microsomes and dendroaspin scaffold protein into one or more drugs and mixing the compound with a physiologically acceptable ingredient to produce an oral or injectable formulation. 5. Administering the formulation to a mammal or human. The method includes:

Claims

1. (i) a scaffold portion; and (ii) an epitope; Including, said epitopes consist of one or more epitopes of a first species and one or more epitopes of a second species; the first type of epitope is a C5a receptor (C5aR) protein epitope capable of inducing an anti-atherosclerotic vascular disease response via C5aR interaction; The second type of epitope is capable of eliciting an anti-atherosclerotic vascular disease response via a second pathway independent of the C5aR interaction and is a heat shock protein (HSP) epitope and / or a chlamydia pneumonia epitope. Recombinant constructs.

2. The construct of claim 1, wherein the C5aR epitope is a polypeptide comprising an amino acid sequence of 5 to 45 consecutive amino acid residues in SEQ ID NO:6, and / or the C5aR epitope is at the C- or N-terminus of the construct.

3. A construct described in claim 1 or 2, wherein the C5aR epitope is at the C- or N-terminus of the construct.

4. The construct described in claim 3, wherein the epitope is a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of MNSFNYTTPDYGHYDDKDTLD (SEQ ID NO: 7) and TLDLNTPVDKTSN (SEQ ID NO: 8), or a functional fragment thereof having antigenic activity.

5. The construct according to any one of claims 1 to 4, wherein the HSP is HSP60 or HSP65.

6. The construct described in claim 5, wherein the HSP is HSP60, and is human HSP60 or Mycobacterium bovis HSP.

7. The construct according to claim 5 or 6, wherein the HSP60 epitope comprises an amino acid sequence selected from the group comprising peptide 1 (AA) 153-160: AELKKQSK; (SEQ ID NO: 10), peptide 1 (AA) 153-163: AELKKQSKPVT; (SEQ ID NO: 11), peptide 1 (AA) 303-312: PGFGDNRKNQ (SEQ ID NO: 12), peptide 2: AA 277-286 PGFGDNRKNQ (SEQ ID NO: 13), peptide (AA) 516-528: KGIIDPTKVVRTA (SEQ ID NO: 14) and mycobacterium (AA) 253-268: EGEALSTLVVNKIRGT (SEQ ID NO: 15), or a functional fragment thereof having antigenic activity.

8. A construct according to any one of claims 1 to 7, wherein the chlamydia pneumonia epitope is Cpn1 or Cpn2.

9. The construct of claim 8, wherein the chlamydia pneumonia epitope is a polypeptide comprising an amino acid sequence selected from the group consisting of major outer membrane protein (MOMP) (amino acid sequence (AA) 67-74: GDYVFDRI (SEQ ID NO: 16)) and putative outer membrane protein (Pomp) 5 of Cpn (amino acid sequence (AA) 283-291: QAVANGGAI (SEQ ID NO: 17)), or a functional fragment thereof having antigenic activity.

10. The construct of any one of claims 1 to 9, wherein the scaffold moiety is a dendroaspin scaffold protein as shown in SEQ ID NO:

1.

11. The construct of claim 10, wherein the first and / or second species epitopes are incorporated into one or more positions of the dendroaspin scaffold: (a) loop I and / or loop II; (b) loop I and / or loop III; (c) loop II and / or loop III; (d) loop I, loop II and loop III; or (e) the N- or C-terminus.

12. 12. An expression vector comprising a nucleic acid encoding a construct according to any one of claims 1 to 11.

13. An antigenic composition comprising a construct according to any one of claims 1 to 11 and a hydrophobic complex.

14. The composition of claim 13, wherein the hydrophobic complex comprises (i) isolated microsomes, (ii) MHC proteins, or (iii) reverse micelles.

15. The composition of claim 14, wherein the isolated microsomes are inverted microsomes.

16. A pharmaceutical composition comprising a construct according to any one of claims 1 to 11, formulated as an injectable or oral preparation, and further comprising suitable adjuvants, excipients, diluents and / or carriers.

17. A pharmaceutical composition comprising a construct according to any one of claims 1 to 11 or a vector according to claim 12 or an antigenic composition according to any one of claims 13 to 15.

18. A construct according to any one of claims 1 to 11 for use as a medicament, for use in the manufacture of a medicament or for use in treating atherosclerosis.

19. 13. The vector of claim 12 for use as a medicament, for use in the manufacture of a medicament, or for use in treating atherosclerosis.

20. A vaccine comprising a construct according to any one of claims 1 to 11 or a vector according to claim 12.

21. A construct according to any one of claims 1 to 11 for generating an anti-atherosclerotic response in a mammal, for treating, preventing or reducing atherosclerosis, or for treating individuals with early stage atherosclerosis or identified as being at risk of developing atherosclerosis.

22. 13. The vector of claim 12 for generating an anti-atherosclerotic response in a mammal, for treating, preventing or reducing atherosclerosis, or for treating an individual with early stage atherosclerosis or identified as being at risk of developing atherosclerosis.

23. 18. The composition of any one of claims 13 to 17 for generating an anti-atherosclerotic response in a mammal, for treating, preventing or reducing atherosclerosis, or for treating an individual with early stage atherosclerosis or identified as being at risk of developing atherosclerosis.

24. 1. A method for producing a formulation for eliciting an immune response against epitopes associated with two independent pathways involved in the formation of atherosclerosis, comprising:

1. Constructing and expressing a dendroaspin scaffold protein comprising at least one first species epitope and at least one second species epitope according to any one of claims 1 to 11; 2. Incubating eukaryotic cells with the dendroaspin scaffold protein; 3. Preparing microsomes using the eukaryotic cells; 4. Mixing the microsomes and dendroaspin scaffold proteins with one or more pharma- ceutically acceptable ingredients to produce an orally or injectably administrable formulation. The method includes:

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