Multi-epitopic construct

Abstract

To provide multi-epitopic constructs, immunogenic and vaccine compositions comprising recombinant molecules presenting inserted multiple and different epitopes from a variety of antigens.SOLUTION: The antigenic determinants are associated with different pathways leading to atherosclerosis. In particular, the present invention relates to such compositions which induce an immune response against antigens and pathogens involved in manifestation of atherosclerosis, and the present invention includes, inter alia, methods for treating and / or preventing the disease as well as recombinant protein products.SELECTED DRAWING: None

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). Several antigenic stimuli associated with the etiology of atherosclerosis based on modified self-molecules or peptides derived from the molecule of have been reported. In addition to using epitopes derived from these autoantigens, more research related to vaccination against atherosclerosis has shown high efficacy against atherosclerotic lesion formation for other antigens. However, one of the difficulties in developing an effective vaccination strategy against atherosclerosis is the selection of specific antigens. In the discovery and development of potential antigens, a recombinant construct called "AHHC" containing epitopes derived from the proteins of apolipoprotein B (ApoB), heat shock protein (HSP) 60, and Chlamydia pneumoniae (Cpn) was used to immunize B6;129S-Ldlr Apob / J mice, and atherosclerotic lesion formation in mice fed a high-fat diet (HFD) was reduced (Lu et al 2012; Atherosclerosis 225:56-68). Furthermore, it has been previously known from the prior art that immunization with a peptide derived from the N-terminus of complement component 5a receptor (C5aR) reduced the expression of early atherosclerotic lesions in B6;129S-Ldlr

[0003] Apob / J mice (Lu et al 2012; Arterioscler Thromb Vasc Biol 3 2:2358-2371). Additionally, in preclinical trials, inhibition of protease-activated receptor (PAR)- 1 showed a strong antithrombotic effect leading to a significant reduction in platelet aggregation, whereas tm1Her Apob tm2Sgy / J mice treatment reduced atherosclerotic lesion formation in mice fed a high-fat diet (HFD) (Lu et al 2012; Atherosclerosis 225:56-68). Furthermore, immunization with a peptide derived from the N-terminus of complement component 5a receptor (C5aR) reduced the expression of early atherosclerotic lesions in B6;129S-Ldlr / J mice has been known from the prior art (Lu et al 2012; Arterioscler Thromb Vasc Biol 3 2:2358-2371). Additionally, in preclinical trials, inhibition of protease-activated receptor (PAR)- tm1Her Apob tm2Sgy / J mice showed a reduction in the expression of early atherosclerotic lesions. 2:2358-2371). Additionally, in preclinical trials, inhibition of protease-activated receptor (PAR)- 1 showed a strong antithrombotic effect leading to a significant reduction in platelet aggregation, whereas The primary hemostatic function was maintained (Chintala et al 2010 Arterioscler Thromb Vasc Biol 30 :2143-2149).

[0004] The human complement system is an important component of innate host defense against invading pathogens. C5a is a protein fragment released from complement component C5 and is generated by cleavage of C5a convertase on the C5α chain in the classical, alternative, and lectin pathways of complement activation, and is a 74-amino acid peptide in humans. C5a mediates its actions through its G protein-coupled C5a receptor (C5aR / CD88) on the plasma membrane of target cells, inducing intracellular signaling that results in chemotaxis, respiratory burst, and the release of pro-inflammatory mediators from granulocytes and triggers the release of pro-inflammatory mediators from granulocytes. C5a attracts, activates, and stimulates the release of reactive oxygen species, proteolytic enzymes, chemokines, cytokines, and complement factors C3 and properdin from neutrophils, monocytes, and platelets. The secretion of C3 and properdin by neutrophils and the presence of apoptotic and necrotic decidual tissue accelerate the activation of the alternative pathway, enhance the activation and deposition of C3, and generate an inflammatory amplification loop at the site of leukocyte infiltration that results in the production of additional C5a. Human C5aR is an integral membrane glycoprotein consisting of 350 amino acids that forms a single-chain polypeptide. The C5a / C5aR interaction has been shown to alter the production of IL-12 and thus regulate Th-1 cell responses, as well as enhance the production of cytokines such as IL-6, IL-8, and TNF-alpha. C5aR is abundantly expressed on leukocytes, including neutrophils, monocytes, eosinophils, and lymphocytes and platelets. ​​​​​It does. C5aR is also expressed by a wide range of parenchymal cells, including glomerular mesangium and proximal tubular epithelial cells. Parenchymal C5aR expression is enhanced at the site of acute inflammation, as has been shown.

[0005] Ruptured or vulnerable plaques in atherosclerosis usually feature the presence of a large lipid core, a decrease in the number of smooth muscle cells, a thin fibrous capsule, and an increase in the number of inflammatory cells such as macrophages and T cells. Macrophages are thought to play a major role in plaque destabilization and rupture. Through the production of matrix metalloproteinases (MMP s), these cells have the ability to degrade components of the extracellular matrix such as collagen, proteoglycan, and elastin. In several studies, immunohistochemistry and in situ zymography have shown that MMP-1, MMP-3, and MMP- 9 are present in the shoulder region of atherosclerotic plaques, and overexpression of active MMP-9 in apoE-deficient mice has been shown to induce plaque disruption. The stimulatory effect of C5a on the expression of MMP-1 and MMP-9 mRNA in monocytes-derived macrophages isolated from different donors and in human macrophages isolated from atherosclerotic plaques has also been reported.

[0006] Clinical and experimental data indicating the involvement of anaphylatoxin C5a in the etiology of atherosclerosis are accumulating. It has recently been reported that there is a predictive value for plasma levels of C5a regarding cardiovascular events in patients with progressive atherosclerosis. The C5a receptor has been detected in atherosclerotic lesions. Furthermore, C5a promotes the expression of adhesion molecules by endothelial cells induces the expression of C5a, which activates macrophages and causes them to release inflammatory mediators such as TNF-alpha and interleukin-1. TNF-alpha, on the other hand, directly induces the expression of MMP in these cells.

[0007] In the discovery and development of potential antigens, a recombinant construct called "AHHC" containing epitopes derived from the proteins of apolipoprotein B (ApoB), heat shock protein (HSP) 60, and Chlamydia pneumoniae (Cpn) was used to immunize B6;129S-Ldlr Apob / J mice, and this treatment reduced atherosclerotic lesion formation in mice fed a high-fat diet (HFD) (Lu et al 2012; Atherosclerosis 225:56-68). Furthermore, it was previously known from the prior art that immunization with a peptide derived from the N-terminus of the complement component 5a receptor (C5aR) reduced the expression of early atherosclerotic lesions in B6;129S-Ldlr tm1Her Apob tm2Sgy / J mice (Lu et al 2012; Arterioscler Thromb Vasc Biol 32:2358-2371). Additionally, in preclinical trials, inhibition of protease-activated receptor (PAR)-1 showed a strong antithrombotic effect leading to a significant reduction in platelet aggregation, while primary hemostatic function was maintained (Chintala et al 2010 Arterioscler Thromb Vasc Biol 30:2143-2149). tm1Her Apob tm2Sgy

Summary of the Invention

Problems to be Solved by the Invention

[0008] There is a need to improve the treatment of atherosclerotic vascular diseases.

Means for Solving the Problems

[0009] Summary of the Disclosure According to a first aspect of the present invention, i) a scaffold portion and what is incorporated therein, ii) a first species of epitope capable of causing an anti-atherosclerotic vascular disease response via a first pathway, iii) a second species of epitope capable of causing an anti-atherosclerotic vascular disease response via a second pathway independent of the first pathway and A recombinant construct comprising is provided.

[0010] The reference in this specification to the first and second pathways being "independent" of each other is intended to indicate that the formation of atherosclerosis via the first or second pathway is due to different mechanisms of action or causes of the pathway.

[0011] Preferably, the scaffold portion is a native dendroaspin and comprises an amino acid sequence selected from SEQ ID NO: 1 (Figure 14A).

[0012] Preferably, the first pathway to the formation of atherosclerosis is via C5 interaction, more preferably via the C5a or C5Ar pathway. Thus, the C5 epitope is a C5 a epitope or a C5a receptor (C5aR) epitope. The C5 epitope may comprise 5 to 40, for example, 8 to 40 amino acid residues or for example, 8 to 35 amino acid residues. The C5a epitope is 5 to 40 selected from the C5a sequence (SEQ ID NO: 2) (Figure 14B). ​​Individual consecutive amino acid residues, for example, 8 to 40 amino acid residues, for example, 8 to 35 amino acids may be included. In one embodiment, the C5a epitope is 8 to selected from the C5a sequence and contains 20, for example, 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 ), and is a polypeptide or its functional fragment having anti genic activity that includes or consists of an amino acid sequence selected from the group. For example, the epitope may include a sequence of 5 to 40 consecutive amino acid residues selected from the C5a sequence and containing SEQ ID NO: 2, 3 or 4 .

[0014] Preferably, the C5aR epitope is selected from the C5aR sequence (SEQ ID NO: 6; FIG. 14C) and contains 5 to 50 consecutive amino acid residues, for example, 10 to 40 amino acid residues or for example 14 to 35 amino acid residues. In one embodiment, the C5aR epitope contains 1 to 31 consecutive amino acid residues or its functional fragment.

[0015] Preferably, the C5aR epitope may be present at the N-terminus (including the free amino group) or the C-terminus (including the free carboxyl group) of the C5aR amino acid sequence. Alternatively, more preferably is that the C5aR epitope is MNSFNYTTPDYGHYDDKDTLD (SEQ ID NO: 7 ), TLDLNTPVDKTSN (SEQ ID NO: 8) and MNSFNYTTPDYGHYD DKDTLDLNTPVDKTSN (SEQ ID NO: 9), and is an amino acid sequence selected from the group ​A polypeptide comprising or consisting of, or a functional fragment thereof having antigenic activity.

[0016] References herein to functional fragments include those having antigenic determinants and performing their function as such, retaining sufficient amino acids to have antigenic activity and thus function as epitopes, and include a part of the epitope amino acid sequence. The functional fragment is also incorporated into a scaffold moiety and, when presented, can elicit an immune response against atherosclerosis, for example and without limitation, as determined by a decrease in the area occupied by atherosclerotic lesions, and includes a part of the epitope amino acid sequence. References herein to functional fragments include those having antigenic determinants and performing their function as such, retaining sufficient amino acids to have antigenic activity and thus function as epitopes, and include a part of the epitope amino acid sequence. The functional fragment is also incorporated into a scaffold moiety and, when presented, can elicit an immune response against atherosclerosis, for example and without limitation, as determined by a decrease in the area occupied by atherosclerotic lesions, and includes a part of the epitope amino acid sequence. References herein to functional fragments include those having antigenic determinants and performing their function as such, retaining sufficient amino acids to have antigenic activity and thus function as epitopes, and include a part of the epitope amino acid sequence. The functional fragment is also incorporated into a scaffold moiety and, when presented, can elicit an immune response against atherosclerosis, for example and without limitation, as determined by a decrease in the area occupied by atherosclerotic lesions, and includes a part of the epitope amino acid sequence. References herein to functional fragments include those having antigenic determinants and performing their function as such, retaining sufficient amino acids to have antigenic activity and thus function as epitopes, and include a part of the epitope amino acid sequence. The functional fragment is also incorporated into a scaffold moiety and, when presented, can elicit an immune response against atherosclerosis, for example and without limitation, as determined by a decrease in the area occupied by atherosclerotic lesions, and includes a part of the epitope amino acid sequence. References herein to functional fragments include those having antigenic determinants and performing their function as such, retaining sufficient amino acids to have antigenic activity and thus function as epitopes, and include a part of the epitope amino acid sequence. The functional fragment is also incorporated into a scaffold moiety and, when presented, can elicit an immune response against atherosclerosis, for example and without limitation, as determined by a decrease in the area occupied by atherosclerotic lesions, and includes a part of the epitope amino acid sequence. References herein to functional fragments include those having antigenic determinants and performing their function as such, retaining sufficient amino acids to have antigenic activity and thus function as epitopes, and include a part of the epitope amino acid sequence. The functional fragment is also incorporated into a scaffold moiety and, when presented, can elicit an immune response against atherosclerosis, for example and without limitation, as determined by a decrease in the area occupied by atherosclerotic lesions, and includes a part of the epitope amino acid sequence.

[0017] References to the first and second types of epitopes are intended to mean that they are separately and independently related to different pathways that ultimately lead to atherosclerosis and related diseases. References to the first and second types of epitopes are intended to mean that they are separately and independently related to different pathways that ultimately lead to atherosclerosis and related diseases. References to the first and second types of epitopes are intended to mean that they are separately and independently related to different pathways that ultimately lead to atherosclerosis and related diseases.

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

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

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

[0021] Preferably, the heat shock protein (HSP) is HSP60 or HSP65. More preferably, the HSP60 is human HSP60 or Mycobacterium bovis HSP. More preferably, the epitope capable of inducing a response against HSP60 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 (SEQ ID NO: 13), Peptide (AA) 516-528 KGIIDPTKVVRTA (SEQ ID NO: 14) and Mycobacterium (AA) 253-268: EGEALSTLVVNKI RGT (SEQ ID NO: 15), and is a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of, or a functional fragment thereof having antigenic activity.

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

[0023] Preferably, the tissue factor epitope is (AA56 - 67), and the sequence EWEPKPV NQVYT (SEQ ID NO: 19).

[0024] Preferably, the PAR-1 epitope is (AA42 - 55), and the amino acid sequence SF LLRNPNDKYEPF (SEQ ID NO: 19).

[0025] Preferably, the recombinant construct of the present invention contains a plurality of second epitopes, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 same or different second epitopes may be included. For example, in a particularly preferred embodiment, the recombinant construct may contain a C5aR epitope, a Cpn epitope and two different HSP epitopes. Or the recombinant construct may contain a C 5aR epitope, a Cpn epitope, an HSP epitope and a PAR-1 epitope.

[0026] Preferably, the amino acid sequences of the first and / or second epitopes are incorporated into (a) loop I and / or loop II; (b) loop I and / or loop III; (c) loop II and / or loop III; or (d) loop I, loop of the dendroaspin scaffold; or III. Loop I contains amino acid residues 4 - 16, loop II contains residues 23 - 36, and loop III contains residues 40 - 50. However, additional amino acids incorporated are in the extra-loop region, i.e., so that the residues in the non-loop region increase or replace the residues of the additional amino acid sequence or the inserted sequence. ​​​​It may extend to or substitute for residues 1-3, 17-22, and 37-39. Further amino acid residues are preferably incorporated into Loop I or Loop II. Thus, the Loop III containing RGD is invariant, and thus the integrin-binding function of dendroaspin is retained. Preferred positions for the additional sequences to be inserted are sites in the dendroaspin scaffold between amino acid residues: 4-1 6, 18-21, 23-36, or 52-59. Each inserted additional amino acid sequence or a part of the additional amino acid sequence is preferably an amino acid sequence in the range of 3-40 amino acid residues, more preferably 3-16, even more preferably 3-14 amino acid residues in length. The start of the additional amino acid

[0027] sequence to be inserted can be any one of amino acid residues 1-57 of the dendroaspin scaffold. The end of the additional amino acid sequence to be inserted can be any one of amino acid residues 3-5 9 of the dendroaspin scaffold. When two or more additional amino acid sequences are inserted into the dendroaspin scaffold, the linear distance between them is preferably in the range of 1-35 amino acids, more preferably 1-14 amino acids. When two or more additional amino acid sequences are is possible. For insertion at one or more sites in the dendroaspin scaffold, a number of residues in the range of 14 - 36 is preferred, but any suitable number of amino acids can be inserted into the dendro aspin scaffold to obtain the desired dual or multifunctional activity. Preferably, in some embodiments of the invention, the first or second epitope can be bound to either or both of the C and / or N termini of the dendro

[0028] aspin scaffold protein. is possible. In a further aspect of the invention, an expression vector is provided that contains a nucleic acid encoding a protein incorporated into the construct of the first aspect of the invention.

[0029] In a further aspect of the invention, an antigenic composition is provided that contains a protein according to the first aspect of the invention and an antigenic hydrophobic complex.

[0030] In a further aspect of the invention, a pharmaceutical composition is provided that contains the immunogenic composition of the invention formulated as an injectable or oral preparation. Preferably, the pharmaceutical composition further contains a suitable adjuvant , excipient, diluent and / or carrier.

[0031] In a further aspect of the invention, a pharmaceutical composition is provided that contains the protein of the first aspect of the invention, the vector of the invention or the immunogenic composition of the invention. In a further aspect of the invention, the protein of the first aspect of the invention or the vector of the invention for use as a medicament is provided.

[0032] In a further aspect of the invention, a recombinant protein according to the first aspect of the invention; the vector of the invention or the immunogenic composition of the invention is provided.

[0033] In a further aspect of the invention, the protein of the first aspect of the invention or the vector of the invention for use as a medicament is provided. is provided.

[0034] In a further aspect of the invention, a recombinant protein according to the first aspect of the invention; the vector of the invention Administering a formulation selected from a chimeric protein; an immunogenic composition of the present invention; and a pharmaceutical composition of the present invention There is provided a method of eliciting an anti-atherosclerotic response in a mammal, comprising doing so.

[0035] In a further aspect of the present invention, a recombinant protein according to the first aspect of the present invention; a vector of the present invention Administering to an individual a formulation selected from a chimeric protein; an immunogenic composition of the present invention; and a pharmaceutical composition of the present invention There is provided a method of treating, preventing or reducing atherosclerosis, comprising The formulation can be administered in a therapeutically effective amount or a therapeutically tolerable amount.

[0036] In a further aspect of the present invention, there is provided a method of treating an individual having early atherosclerosis or an individual identified as being at risk of developing atherosclerosis, comprising administering to the individual a formulation selected from a recombinant protein according to the first aspect of the present invention; a vector of the present invention; an immunogenic composition of the present invention; and a pharmaceutical composition of the present invention. The formulation can be administered in a therapeutically effective amount or a therapeutically tolerable amount. There is provided a method comprising The formulation can be administered in a therapeutically effective amount or a therapeutically tolerable amount.

[0037] In a further aspect of the present invention, there is provided a vaccine comprising a protein according to the first aspect of the present invention or a vector of the present invention. There is provided a vaccine comprising a protein according to the first aspect of the present invention or a vector of the present invention.

[0038] In a further aspect of the present invention, there is provided a method of eliciting an immune response against epitopes associated with at least two independent pathways associated with atherosclerotic formation, comprising (i) constructing and expressing a dendroaspin scaffold protein comprising at least one first and at least one second epitope as described hereinabove; (i) constructing and expressing a dendroaspin scaffold protein comprising at least one first and at least one second epitope as described hereinabove; and (ii) incubating eukaryotic cells with said dendritic spine scaffold protein; and (iii) preparing microsomes using said eukaryotic cells; (iv) mixing said microsomes and dendritic spine scaffold protein with one or more pharmaceutically acceptable components to produce an orally or injectably administrable formulation; (v) administering said formulation to a mammal or a human and providing a method comprising the same.

[0039] Data showed that immunization with ApoB / PAR-1 / HSP / Cpn did not affect C5a expression in atherosclerotic lesions, suggesting that C5 / C5aR is an independent pathway in lesion formation. The present invention provides methods and formulations for advantageously targeting both the independent C5 and ApoB / P AR-1 / HSP / Cpn-related pathways simultaneously, in order to provide improved therapies for the prevention and reduction of early atherosclerosis. It is well understood that the preferred features attributable to the first aspect of the present invention, which may be modified where appropriate, apply to each and all of the other aspects of the present invention. Embodiments of the present invention will be further described below with reference to the accompanying drawings.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS It is fully understood that the preferred features attributable to the first aspect of the present invention, which may be modified where appropriate, apply to each and all of the other aspects of the present invention.

[0041] Embodiments of the present invention will be further described below with reference to the accompanying drawings.

Brief Description of the Drawings

[0042]

Figure 1

Figure 2-1

Figure 2-2

Figure 3-1

Figure 3-2

Figure 4

Figure 5-1

Figure 5-2

Figure 6-1

Figure 6-2

Figure 6-3

Figure 6-4

Figure 6-5

Figure 6-6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11-1

Figure 11-2

Figure 11-3

Figure 11-4

Figure 12

Figure 13-1

Figure 13-2

Figure 14

Mode for Carrying Out the Invention

[0043] Detailed Description From the data, the C5a / C5aR pathway to atherosclerosis is immunized with Apob t m2Sgy LDlr tm1HerBased on the study of / J mice, it is found to be independent of the PAR-1 / HSP / Cp n pathway. The present invention provides a formulation and a vaccine targeting both pathways that simultaneously use composite epitopes within the dendritic spine scaffold.

[0044] In previous studies by the inventors, the construct AHHC [ApoB100 688-707 +hHS P60 303-312 +hHSP60 153-163 +peptide derived from Cpn (C)] significantly reduced atherosclerotic lesions (Lu et al. Atherosclerosis 2012, 225:56 -68). The present invention provides the regulation of this construct and derivatives by sequential epitope substitution in which A in mice is replaced by "R" (C5aR 1-31 ) and further conversion of "H" (hHSP60 303-312 ) to "P" (protease activation receptor 1 42-55 ) to form RPHC. Alternative embodiments are constructs AH 42-55 h )(SEQ ID NO: 12), H m m 15) and the complement component 5a receptor called R (AA1-31) (SEQ ID NO: 9), h h m m tm1Her tm1Her tm2Sgy tm2Sgy tm2Sgy / J mice immunization induced the production of high levels of antibodies against each epitope (except for hHSP60 153-163 153-163 and P which induced a low antibody response). ​). Histological analysis showed a significant reduction in the size of atherosclerotic lesions in mice immunized with RPHC or RHHC compared to mice treated with AHH C (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 in the aortic sinus and descending aorta was correlated with changes in the cellular immune response when compared to the control. The inventors conclude that these new recombinant constructs may have new antigenic and structural features that are beneficial for a significant reduction in the formation of atherosclerotic lesions. The present invention provides a novel strategy for developing anti-atherosclerotic agents. Based on the effects of peptides derived from C5aR and PAR-1 on the reduction of atherosclerotic lesions, the inventors hypothesized that the effects of multiple epitope constructs on the reduction of atherosclerotic lesions could be regulated by including C5aR and PAR-1 in vaccination to aim for a favorable plaque phenotype and an increase in lesion reduction. In this study, the inventors examined the effects of constructs containing C5aR and PAR-1 by sequential substitution: AHHC → RHHC (R represents an epitope derived from C5aR) and RHHC → RPHC (P represents an epitope derived from PAR- 1). The induced immune response was detected as a significant reduction in the size of atherosclerotic lesions in both the aortic sinus and descending aorta, with an anti-atherosclerotic effect in the order of RPHC ≧ RHHC ≧ AHHC.

[0045]

[0046] ​​​​​​​​​​​​Native dendroaspin is a 59 - amino - acid peptide, and the dendroaspin scaffold is suitable for modification. To incorporate additional functional amino - acid sequences, such as the active moiety or motif of a factor in the coagulation cascade, an agonist, antagonist or inhibitor, when modifying dendroaspin (including the RGD motif), the resulting molecule is particularly useful as an anticoagulant and has none of the drawbacks associated with existing anticoagulants (see WO 01 / 57210 pamphlet). Such hybrid polypeptides contain the RGD motif, a first amino - acid sequence conferring dendroaspin activity, and may contain additional amino - acid sequences conferring activities other than dendroaspin activity. Thus, hybrid dendroaspin - based molecules can be multifunctional.

[0047] C5a is a protein fragment released from complement component C5. This 74 - amino - acid peptide in humans is generated by cleavage of C5α - chain on C5a convertase in the classical, alternative and lectin pathways of complement activation.

[0048] The C5a receptor is also known as the complement component 5a receptor (C5AR1) or CD88 (cluster of differentiation 88), and is a G - protein - coupled receptor for Ca. The human C5aR is an integral membrane glycoprotein consisting of 350 amino acids that form a single - chain polypeptide. C5aR is not released as a soluble receptor and does not circulate. C5aR is expressed on differentiated myeloid cells such as U937 and HL - 60. C5aR is expressed on hepatocytes, pulmonary vascular smooth muscle, pulmonary and umbilical vascular endothelial cells, bronchial and alveolar epithelial cells, HepG2 cells, hepatocellular carcinoma cell lines, mesangial ​​​​​​​​​​​​It is expressed on mucosal cells, stellate cells and microglial cells, and on cultured human fetal stellate cells and stellate cell lines. It is expressed.

[0049] Throughout the description and claims of this specification, the terms "comprise" and "contain" and their variants mean "include but are not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the description and claims of this specification, the singular form includes the plural form of the word, unless the context requires otherwise. In particular, when using an indefinite article, this specification should be understood to contemplate both plural and singular states, unless the context requires otherwise. Throughout the description and claims of this specification, the terms "comprise" and "contain" and their variants mean "include but are not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the description and claims of this specification, the singular form includes the plural form of the word, unless the context requires otherwise. In particular, when using an indefinite article, this specification should be understood to contemplate both plural and singular states, unless the context requires otherwise. Throughout the description and claims of this specification, the terms "comprise" and "contain" and their variants mean "include but are not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the description and claims of this specification, the singular form includes the plural form of the word, unless the context requires otherwise. In particular, when using an indefinite article, this specification should be understood to contemplate both plural and singular states, unless the context requires otherwise. Throughout the description and claims of this specification, the terms "comprise" and "contain" and their variants mean "include but are not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the description and claims of this specification, the singular form includes the plural form of the word, unless the context requires otherwise. In particular, when using an indefinite article, this specification should be understood to contemplate both plural and singular states, unless the context requires otherwise. Throughout the description and claims of this specification, the terms "comprise" and "contain" and their variants mean "include but are not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the description and claims of this specification, the singular form includes the plural form of the word, unless the context requires otherwise. In particular, when using an indefinite article, this specification should be understood to contemplate both plural and singular states, unless the context requires otherwise. Throughout the description and claims of this specification, the terms "comprise" and "contain" and their variants mean "include but are not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the description and claims of this specification, the singular form includes the plural form of the word, unless the context requires otherwise. In particular, when using an indefinite article, this specification should be understood to contemplate both plural and singular states, unless the context requires otherwise. Throughout the description and claims of this specification, the terms "comprise" and "contain" and their variants mean "include but are not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the description and claims of this specification, the singular form includes the plural form of the word, unless the context requires otherwise. In particular, when using an indefinite article, this specification should be understood to contemplate both plural and singular states, unless the context requires otherwise.

[0050] The features, integers, characteristics, components, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are applicable to the other aspects, embodiments or examples described herein, unless incompatible therewith. All of the features disclosed in this specification (the appended claims, abstract and drawings), and / or all of the steps of the methods or processes so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the foregoing embodiments. The invention resides in any novel and / or novel combination of the features disclosed in this specification (the appended claims, abstract and drawings), or in any novel and / or novel combination of the methods or steps of the processes so disclosed. The features, integers, characteristics, components, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are applicable to the other aspects, embodiments or examples described herein, unless incompatible therewith. All of the features disclosed in this specification (the appended claims, abstract and drawings), and / or all of the steps of the methods or processes so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the foregoing embodiments. The invention resides in any novel and / or novel combination of the features disclosed in this specification (the appended claims, abstract and drawings), or in any novel and / or novel combination of the methods or steps of the processes so disclosed. The features, integers, characteristics, components, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are applicable to the other aspects, embodiments or examples described herein, unless incompatible therewith. All of the features disclosed in this specification (the appended claims, abstract and drawings), and / or all of the steps of the methods or processes so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the foregoing embodiments. The invention resides in any novel and / or novel combination of the features disclosed in this specification (the appended claims, abstract and drawings), or in any novel and / or novel combination of the methods or steps of the processes so disclosed. The features, integers, characteristics, components, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are applicable to the other aspects, embodiments or examples described herein, unless incompatible therewith. All of the features disclosed in this specification (the appended claims, abstract and drawings), and / or all of the steps of the methods or processes so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the foregoing embodiments. The invention resides in any novel and / or novel combination of the features disclosed in this specification (the appended claims, abstract and drawings), or in any novel and / or novel combination of the methods or steps of the processes so disclosed. The features, integers, characteristics, components, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are applicable to the other aspects, embodiments or examples described herein, unless incompatible therewith. All of the features disclosed in this specification (the appended claims, abstract and drawings), and / or all of the steps of the methods or processes so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the foregoing embodiments. The invention resides in any novel and / or novel combination of the features disclosed in this specification (the appended claims, abstract and drawings), or in any novel and / or novel combination of the methods or steps of the processes so disclosed. The features, integers, characteristics, components, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are applicable to the other aspects, embodiments or examples described herein, unless incompatible therewith. All of the features disclosed in this specification (the appended claims, abstract and drawings), and / or all of the steps of the methods or processes so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the foregoing embodiments. The invention resides in any novel and / or novel combination of the features disclosed in this specification (the appended claims, abstract and drawings), or in any novel and / or novel combination of the methods or steps of the processes so disclosed. The features, integers, characteristics, components, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are applicable to the other aspects, embodiments or examples described herein, unless incompatible therewith. All of the features disclosed in this specification (the appended claims, abstract and drawings), and / or all of the steps of the methods or processes so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the foregoing embodiments. The invention resides in any novel and / or novel combination of the features disclosed in this specification (the appended claims, abstract and drawings), or in any novel and / or novel combination of the methods or steps of the processes so disclosed. The features, integers, characteristics, components, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are applicable to the other aspects, embodiments or examples described herein, unless incompatible therewith. All of the features disclosed in this specification (the appended claims, abstract and drawings), and / or all of the steps of the methods or processes so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the foregoing embodiments. The invention resides in any novel and / or novel combination of the features disclosed in this specification (the appended claims, abstract and drawings), or in any novel and / or novel combination of the methods or steps of the processes so disclosed. The features, integers, characteristics, components, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are applicable to the other aspects, embodiments or examples described herein, unless incompatible therewith. All of the features disclosed in this specification (the appended claims, abstract and drawings), and / or all of the steps of the methods or processes so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the foregoing embodiments. The invention resides in any novel and / or novel combination of the features disclosed in this specification (the appended claims, abstract and drawings), or in any novel and / or novel combination of the methods or steps of the processes so disclosed. The features, integers, characteristics, components, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are applicable to the other aspects, embodiments or examples described herein, unless incompatible therewith. All of the features disclosed in this specification (the appended claims, abstract and drawings), and / or all of the steps of the methods or processes so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of the foregoing embodiments. The invention resides in any novel and / or novel combination of the features disclosed in this specification (the appended claims, abstract and drawings), or in any novel and / or novel combination of the methods or steps of the processes so disclosed.

[0051] Attention is directed to all papers and documents that are filed simultaneously with or before the present specification and that are published in the public docket associated with the present application, and the contents of all such papers and documents are hereby incorporated by reference into this specification.

[0052] Expression and purification of recombinant glutathione S-transferase constructs Glutathione S-transferase-dendroaspin (GST-tagged den, referred to herein as control, Figure 1A), as well as GST-recombinant constructs AHHC [ApoB100 peptide, amino acids (aa) 688-707 (numbering including signal peptide), human heat shock protein (hHSP) 60 peptide, aa 303-312 and aa 15 3-163, respectively; and epitopes derived from Chlamydia pneumoniae (Cpn) (where "C" means a combination of aa 66-73 derived from the major outer membrane protein (MOMP) and aa 283-291 derived from the outer membrane 5 of Cpn)] have been previously described and constructs RHHC (where "R" means the C5aR peptide, aa 1-31) and RPHC (where "P" means the protease-activated receptor 1 (PAR-1) peptide, aa 42- 55) have been prepared. A schematic of these constructs using dendroaspin as a scaffold is shown in Figure 1B. These recombinant molecules were expressed in Escherichia coli (BL-21 strain), purified by affinity and ion exchange chromatography, and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, all of these procedures being the same as those previously described for AHHC [Lu et al 2012; Atherosclerosis 225:56-68]。

[0053] Animal experiments B6;129S-Ldlr tm1Her Apob tm2Sgy / J mice were used, and each group of mice consisted of male mice at 5-6 weeks of age. Dendroaspin was used as a scaffold, and from previous data, since the effect of dendroaspin on lesion reduction when used in subcutaneous immunization in mice was not shown, dendroaspin was used as a control antigen. 。

[0054] The immunizing antigens used were constructs AHHC, RHHC, and RPHC, respectively. Repetitive immunization multiple sites strategy (RIM MS) was adopted [1], and the mice were sacrificed at the end of the 12th week (high-fat diet was started at the end of the 2nd week and continued for 10 weeks). The control group followed the feeding program after immunization with GST-tagged Den and alum (adjuvant).

[0055] Tissue preparation and measurement of antibody response Twelve weeks after the first immunization, aortic tissue was collected and mounted in optimal cutting temperature compound (OCT) and paraffin for frozen section preparation for immunohistochemical (IHC) analysis and lesion measurement. Atherosclerotic lesions at the aortic base were examined with an Olympus UULH optical microscope (Olympus Optical Co., Ltd. d., Tokyo, Japan) and Image-Pro Plus TM software, version 7.0 (Media Cybernetics, Inc. ). 、 Bethesda, MD, USA) and analyzed using. The descending aorta cut longitudinally was evaluated for the degree of atherosclerosis after Oil Red O (ORO) staining. The peptide-specific antibody levels in plasma samples were measured by ELISA according to the manufacturer's instructions . One-third of the spleen was embedded in OCT, and the remaining part was passed through a 70 μm cell strainer and homogenized and frozen for further analysis.

[0056] IHC and morphometric analysis, quantitative measurement, and IHC analysis of Forkhead box protein 3 (Foxp3) expression in CD4 + spleen cells for atherosclerosis. OCT-embedded samples were used for the detection of CD68, CD11c, interleukin (IL )-10 and tumor necrosis factor (INF)-α, Foxp3, vascular cell adhesion molecule (VCAM )1, alpha-smooth muscle cell (alpha-SMC), matrix metalloproteinase 9 (MMP9) by IHC analysis. Sections of paraffin-embedded tissues were stained with hematoxylin and eosin (HE) and elastin / fangesson (Sigma) for histological examination using an Olympus U -ULH optical microscope.

[0057] CD4 in spleen cells + Foxp3, IL-2, IL-4 and IL-1 in T cells 7A expression by flow cytometry analysis and differentiation of PBM into macrophages (CD206) . Spleen cells were stained with allophycocyanin-anti-mouse Foxp3, IL-2, IL-4 and IL -17 antibodies (BioLegend, Cambridge, UK) for staining. treated (30 minutes at 4°C). For cell differentiation assays, mouse (C57BL / 6) PBM Cs were stimulated with various antigens or pre-incubated with antiserum against the antigens (to confirm the antagonistic effect of the antiserum). Antigen-induced differentiation of monocytes into macrophages was measured by flow cytometry and compared with the cell population of non-induced cells or control antigen (GST-Den)-induced cells.

[0058] Measurement of cytokines 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). Plasma levels of cytokines, IL-10, transforming growth factor beta (TGF-β), TNF-α and interferon gamma (IFN-γ) were measured by ELISA according to the manufacturer's instructions (R&D systems, Abingdon, UK). Levels of concanavalin A (ConA)-induced IL-10, TGF-β, TNF-α and IFN-γ in splenocyte cultures were also measured.

[0059] Antigen-specific regulatory T cell function assay To evaluate antigen-specific regulatory T cell function, CD4+CD25+Treg cells were isolated from the spleens of B6;129S-LdlrApob / J mice immunized subcutaneously with the respective constructs using the regulatory T cell isolation kit from Miltenyi Biotec (Bergisch Gladbach, Germany). tm1Her tm2Sgy + + + + - ​​​​​​​​​​​​​​​​​​​T effector cells were each the same construct isolated from the spleens of immunized mice CD4 + T cells (CD4 + CD25 + cells that bind non-binding to beads, CD4 + cells (99.5% of CD4 + CD25 - cells (2 × 10 5 ) were co-cultured with CD4 + CD25 + cells (2 × 10 5 ) and stimulated with 1 μM of the relevant construct or GST- Den control. After 2 days of culture, the proliferation of T effector cells was measured as the change in the oral intensity of the cell population by flow cytometry expressed as mean fluorescence intensity (MFI) and measured as such.

[0060] Statistical analysis Data are reported as mean ± standard error of the mean (±SEM) unless otherwise indicated . Figures were plotted using graph-pad Prism 5.01 and Sigma plot 9. 0. For atherosclerotic lesion size, data were compared using one-way ANOVA for multiple comparisons and post hoc Bonferroni test to analyze the differences between groups and other data were analyzed using Student's t-test (two-sided analysis). Non parametric distributions were analyzed using the Mann-Whitney U test for pairwise comparisons and the Kruskal-Wallis test for multiple comparisons. Differences between groups were considered significant with a P value of less than 0.05 .

Example

[0061] Peptide-specific immunoglobulin G in the sera of immunized mice was evaluated. The first immunization At two and twelve weeks after placement, sera from mice immunized with dendroaspin (GST-tagged) or constructs within the dendroaspin scaffold (GST-tagged) were assayed for antibody levels by ELISA. In AHHC-immunized mice, when the peptide was used as the ELISA antigen, ApoB peptide, hHSP60 and Cpn peptide-specific antibodies were detected (Figure 2A). Similarly, in mice immunized with RHHC, in addition to the hHSP60 and Cpn peptide-specific antibodies detected, C5aR peptide-specific antibodies were detected (Figure 2A). High antibody levels against Cpn peptide and C5a R peptide, while low antibody levels against PAR-1 peptide, were detected in mice immunized with RPHC (Figure 2A). Low antibody levels against hHSP60 303-312 and were detected in mice immunized with any of the constructs (Figure 2A). The overall observed optical density (OD) values at 100-fold dilution decreased at 12 weeks compared to the values at 2 weeks. 303-312 Interestingly, compared to the GST-Den control, peptide-induced specific immunoglobulin ( Ig)G1 responses in sera from peptide-immunized mice were detected against all peptide antigen epitopes at high dilutions, except for those against hHSP60 and PAR-1 peptide (Figure 2B). Furthermore, a similar pattern was detected for IgG2c responses, but at low serum dilutions, when compared to the control (Figure 2C). However, based on the optical densities measured at different dilutions of the samples (1:50 vs 1:6250), 15 3-163 low antibody levels against hHSP60 were observed in mice immunized with any of the constructs (Figure 2A). The overall observed optical density (OD) values at 100-fold dilution decreased at 12 weeks compared to the values at 2 weeks. Interestingly, compared to the GST-Den control, peptide-induced specific immunoglobulin (

[0062] Ig)G1 responses in sera from peptide-immunized mice were detected against all peptide antigen epitopes at high dilutions, except for those against hHSP60 153-163 and PAR-1 peptide (Figure 2B). Furthermore, a similar pattern was detected for IgG2c responses, but at low serum dilutions, when compared to the control (Figure 2C). However, based on the optical densities measured at different dilutions of the samples (1:50 vs 1:6250), Ig)G1 responses in sera from peptide-immunized mice were detected against all peptide antigen epitopes at high dilutions, except for those against hHSP60 and PAR-1 peptide (Figure 2B). Furthermore, a similar pattern was detected for IgG2c responses, but at low serum dilutions, when compared to the control (Figure 2C). However, based on the optical densities measured at different dilutions of the samples (1:50 vs 1:6250), In addition, the level of IgG2c detected was much lower than that of IgG1. Furthermore , when the antiserum at week 8 was used, between the ApoB peptide-induced antiserum and the Cpn peptide ( Figure 2D), between the ApoB peptide and the hHSP60 303-312 peptide-induced antiserum and those antigens ( Figure 2E), between the ApoB peptide-induced antiserum and either the PAR-1 peptide or either antigen of the C5aR peptide (Figure 2F), and between the C5aR peptide-induced antiserum and either antigen of the ApoB peptide or the PAR-1 peptide (Figure 2G), specific levels of cross-reactivity were observed. Since pooled sera were tested, cross-reactivity between the Cpn peptide and the ApoB peptide antiserum when the SD value could not be calculated (Figure 2D) was regarded separately, and the other cross-reactivities showed significant differences compared with the control (Figure E - G; P < 0.05 - <0.001). <0.001).

Example

[0063] The reduction in atherosclerotic lesion size in the aortic sinus was evaluated. After immunization with AHHC, RHHC, RP HC and after 10 weeks of a high-fat diet, the aortic sinuses of mice were evaluated for the degree of atherosclerosis. The plaque size calculated for immunized animals was compared with that of the control. Representative micrographs of sections with lesions in the experimental groups are shown in Figure 3A. The plaque area is shown in Figure 3B. The lesion size was 31071 ± 998.7 μm compared with the control (70200 ± 5718 μm ) for all three constructs that showed (P < 0.001), 24123 ± 1967 μm 2 and 21386 ± 2 2482 μm 2 in immunized mice with all three constructs. 2 (P < 0.001). It was smaller in the mice. A smaller lesion area was observed in the mice immunized with RHHC or RPHC compared to those immunized with AHHC (P = 0.007 ~0.002) (Figure 2B). The percentage reduction in lesion size is shown in Figure 3C. Assuming a zero percent reduction in lesions in the control animals, 55.7 ± 3.4%, 65.6 ± 1.3% and 69.5 ± 1.1% were shown. Control mice immunized with GST-Den showed lesion formation similar to that of the GST tag or alum (adjuvant) immunization controls (Figure 4A and 4B). Therefore, GST-Den was used as a control throughout the experiment. The effects of treatment with these recombinant constructs on the collagen content in these lesions were also examined. The reduction of atherosclerosis in the mice treated with these constructs was accompanied by an increase in collagen content of approximately three-fold in the AHHC-immunized mice or RHHC-immunized mice compared to the control mice respectively (18.6 ± 1.2% or 19.4 ± 0.9% vs. control 5.9 ± 0.3%; P < 0.001) (Figures 3D and 3E). The mice immunized with RPHC showed a significant increase in collagen (24.4 ± 0.9%) compared to the mice immunized with AHHC or RHHC (P = 0.003 and P = 0.007 respectively).

[0064] The descending aorta incised longitudinally was stained with Oil Red O (ORO) from the front, and the area of the positively stained plaque was measured. A representative front-stained descending aorta of the experimental group is shown in Figure 3F. The lesion size was AHHC, respectively, compared to the control (19.5 ± 1.7%, P < 0.001) The reduction of atherosclerosis in the mice treated with these constructs was accompanied by an increase in collagen content of approximately three-fold in the AHHC-immunized mice or RHHC-immunized mice compared to the control mice respectively (18.6 ± 1.2% or 19.4 ± 0.9% vs. control 5.9 ± 0.3%; P < 0.001) (Figures 3D and 3E). The mice immunized with RPHC showed a significant increase in collagen (24.4 ± 0.9%) compared to the mice immunized with AHHC or RHHC (P = 0.003 and P = 0.007 respectively). The mice immunized with RPHC showed a significant increase in collagen (24.4 ± 0.9%) compared to the mice immunized with AHHC or RHHC (P = 0.003 and P = 0.007 respectively). The descending aorta incised longitudinally was stained with Oil Red O (ORO) from the front, and the area of the positively stained plaque was measured. A representative front-stained descending aorta of the experimental group is shown in Figure 3F. The lesion

[0065] size was AHHC, respectively, compared to the control (19.5 ± 1.7%, P < 0.001) The descending aorta incised longitudinally was stained with Oil Red O (ORO) from the front, and the area of the positively stained plaque was measured. A representative front-stained descending aorta of the experimental group is shown in Figure 3F. The lesion size was, compared to the control (19.5 ± 1.7%, P < 0.001), AHHC, For RHHC and RPHC, it was significantly smaller in mice immunized with all constructs that showed 8.4 ± 0.3%, 6.6 ± 0.3%, and 6.4 ± 0 .4% (Figure 3G). Both RHHC- and RPHC-immunized mice had significantly smaller lesions than AHHC-immunized mice (P = 0.011 - 0.008). The reduction in lesions expressed as a percentage is shown in Figure 2H and was 57.3 ± 1.7%, 6 6.1 ± 1.6%, and 67.3 ± 1.9% for AHHC, RHHC, and RPHC, respectively. The smallest lesion area was observed in mice immunized with RPHC .

Example

[0066] Local (aortic lesions) and remote organs: The amount of inflammatory cells and CD4 + T cells expressing Foxp3 were evaluated. The percentage of anti-CD68 staining area in lesions was 43.7 ± 3. 2% in control mice immunized with GST-Den and 14.9 ± 1.6%, 13.6 ± 1.3%, and 10.3 ± 0.8% in mice immunized with AHHC, RHHC, and RPHC, respectively (P < 0.001). A smaller anti-CD68 staining area was observed in RPHC-immunized mice compared to AHHC-immunized mice (P = 0.034) (Figures 5A and 5B). Similarly, in the measurement of anti-CD11c staining lesion area, 13.3 ± 2.1%, 10.5 ± 1.5%, and 7.9 ± 0.8% were shown in mice immunized with AHHC, RHHC, and RPHC, respectively, compared to 38.4 ± 1.9% in control mice (P<0.001) (Figures 5A and 5C). The anti-CD11c staining lesion area in RPHC-immunized mice was Significantly smaller when compared to that in AHHC-immunized mice (P = 0.03 9) (Figs. 5A and 5C). Double immunostaining for CD68 and CD11c showed that the percentage and CD68 expressed as + area co-localized with the CD11c + area was 54.7 ± 3.7%, 55.2 ± 2.6%, and 50.3 ± 3.3% for AHHC, RHHC, and RPHC, respectively, compared with 68. 6 ± 4.7% in control mice, as shown. Thus, more than half of the macrophages were clearly shown to be CD68 + CD11c + (Figs. 5A - 3D ; P = 0.046 - 0.011), indicating that this cell type in the lesions is bone marrow-derived. Analysis of the percentage of CD4 cells expressing Foxp3 by IHC staining of aortic sections showed that the proportion + was approximately 6 - to 8-fold higher in mice immunized with AH HC, RHHC, and RPHC, respectively, compared with 1.2 ± 0.2% in control mice (P < 0.001) ( 8.2 ± 1.4%, P < 0.001; 9.4 ± 1.1%, P < 0.001; 9.9 ± 1. 6%, P < 0.001) (Figs. 5E and 5F). Furthermore, the expression of Foxp3 in CD4 spleen cells of mice immunized with these three constructs was higher than that in the control (P < 0.001), being 13.3 ± 0.6%, 15.3 ± 1.5%, and 18. 0 ± 1.1% for AHHC, RHHC, and RPHC, respectively, compared with 4.0 ± 0.5% in the control (Figs. 5G and 5H). However, no significant increase in Foxp3 expression was shown in RPHC-immunized mice compared with that in RHHC-immunized mice + (P < 0.001), and compared with that in RHHC-immunized mice, RPHC-immunized mice showed no significant increase in Foxp3 expression. Compared with that in RHHC-immunized mice, RPHC-immunized mice showed no significant increase in Foxp3 expression. Compared with that in RHHC-immunized mice, RPHC-immunized mice showed no significant increase in Foxp3 expression. However, compared with that in RHHC-immunized mice, RPHC-immunized mice showed no significant increase in Foxp3 expression. RPHC-immunized mice Except for, significantly higher levels of F were observed in AHHC-immunized mice compared to that oxp3 expression (P = 0.002).

Example

[0067] The expression of anti-inflammatory cytokines and pro-inflammatory cytokines at the lesion site as well as the levels of cytokines in plasma and the supernatant of stimulated splenocytes were evaluated. By IHC analysis The IL-10 expression in the aortic lesions of mice immunized with AHHC, RHHC, and RPHC detected is shown in Fig. 6A. The proportion of CD4 + cells expressing IL-10 in the lesions was approximately 6-fold higher in mice immunized with these constructs and, as shown in Figs. 6A and 6B, when compared to that (0.9 ± 0.2%) in the control group, it was 4.8 ± 0.7%, 5.2 ± 0.8% and 5.9 ± 0.7% (P < 0.001) for AHHC, RHHC, and RPHC, respectively. By IHC analysis of TNF-α expression, significantly smaller TNF- α occupied areas were shown in the lesions of mice immunized with the constructs compared to the control (25.7 ± 3.1% for AHHC; 15.2 ± 0.9% for RHHC; 15.5 ± 1.5% for RPHC and 41.3 ± 3.1% for the control) (Figs. 6C and 6D ). These data showed reduction percentages of 37.8%, 63.2%, and 62.5%, respectively, compared to the control (defining the total lesion area as 100% and the reduction as 0%). Furthermore, an improvement in the reduction was brought about by RHHC or RPHC compared to AHHC (P = 0.00 8 - 0.014). The plasma levels of the atheroprotective cytokine IL-10 were higher in the control compared to that in AHHC (P = 0.00

[0068] 8), and were further increased in mice immunized with RHHC or RPHC compared to AHHC (P = 0.00 Significantly increased in mice immunized with these three constructs compared to (Figure 6E and 6F). Immunization with RPHC had a greater effect on promoting the secretion of IL-10 and TGF-β than immunization with the other two constructs (P < 0.05 to <0.001). The plasma levels of the atherogenic cytokine TNF-α were significantly decreased by immunization with these three constructs (Figure 6G) . A similar trend was obtained for the plasma levels of IFN-γ for these constructs (Figure 6H). In particular, immunization with RPHC had a greater effect on reducing the secretion of TNF-α and IFN-γ ( P ≤ 0.002) than immunization with AHHC, and a greater effect on reducing the secretion of TNF-α (P = 0.004) than immunization with RHHC. Slightly higher plasma levels of IFN-γ were observed in mice immunized with AHHC compared to control mice (24 vs 20.8 pg / ml), but this difference did not show statistical significance.

[0069] The supernatants of splenocytes from mice immunized with these constructs showed significantly higher secretion of IL-10 (Figure 6I) and TGF-β (Figure 6 J) when stimulated with 10 μg / ml of ConA individually compared to the control (P < 0.05 to 0.001). Furthermore, higher levels of IL-10 or TGF-β were produced by splenocytes from RPHC-immunized mice than from AHHC-immunized mice (P < 0.05 to 0.01). In contrast, the levels of TNF-α (Figure 4K) and IFN-γ (Figure 6L) were lower in splenocytes from mice immunized with these constructs compared to the control when stimulated with 10 μg / mL of ConA . It significantly decreased in the supernatant. In particular, the levels of TNF-α and IFN -γ in RPHC-immunized mice were lower than those in AHHC-immunized mice when stimulated with 10 μg / mL of ConA respectively (P<0.05 - 0.01). Interestingly, the supernatant of splenocytes from immunized mice contained high amounts of the protective cytokine IL-10 and low amounts of the pro-inflammatory cytokine IFN -γ when stimulated with peptides or constructs containing peptides, but this was not the case when stimulated with the different protein KLH (Figures 7A - D). In most cases, changes in cytokine production in response to various stimulants were significant when ApoB, C5aR and Cpn peptides were used as stimulants for the production of IL-10 in GST-den-immunized mice, and except for the PAR-1 peptide in RPHC-immunized mice. The percentages of IL-4 (Th2-related), IL-1 7A (Th17-related) and IL-2 (Th1-related) expressing CD4 spleen cells from mice immunized with these three constructs were significantly lower (P<0.001 for IL-4 and P≤0.001 for IL-17A respectively), 2.74 ± 0.11% (AHHC), 2.85 ± 0.12% (RHHC) and 2.87

[0070] ± 0.02% (RPHC) for IL-4 compared with 8.44 ± 0.21% (control) (Figures 4M and 4N), and 2.0 ± 0.1% (AHHC), 1.7 ± 0.1% (RH when compared with 4.1 ± 0.3% (control) for IL-17A + were significantly lower (P<0.001 for IL-4 and P≤0.001 for IL-17A + respectively), 2.74 ± 0.11% (AHHC), 2.85 ± 0.12% (RHHC) and 2.87 + ± 0.02% (RPHC) for IL-4 compared with 8.44 ± 0.21% (control) (Figures 4M and 4N), and 2.0 ± 0.1% (AHHC), 1.7 ± 0.1% (RH C) and 1.8 ± 0.1% (RPHC) (Figure 4O). + for IL-4 compared with 4.1 ± 0.3% (control) for IL-17A ± 0.02% (RPHC) (Figure 4M and 4N), and 2.0 ± 0.1% (AHHC), 1.7 ± 0.1% (RH C) and 1.8 ± 0.1% (RPHC) (Figure 4O). + for IL-17A HC) and 0.9 ± 0.1% (RPHC) (Figs. 6O and 6P) were shown. Furthermore, A A smaller percentage of CD4 + IL -17A + expressing splenocytes were observed in RPHC-immunized mice (P ≤ 0.006) (Figs. 6O - P). Interestingly, a higher percentage of CD4 + IL-17A + expressing splenocytes were observed in RHHC-immunized mice (P = 0 .021) (Fig. 6P). Furthermore, when compared with the values in the control, a significantly lower percentage of CD4 + IL-2 + expressing splenocytes were observed in mice immunized with the three constructs (P < 0 .001; Figs. 6Q and 6R). Furthermore, a smaller percentage of CD4 + IL-2 + expressing splenocytes were observed in RPHC-immunized mice compared to those in AHHC- or RHHC-immunized mice (P = 0.019 - 0.007).

Example

[0071] Antigen-induced specific Treg cell function was examined. To evaluate whether functional Treg cells were induced by immunization, antigen-specific Treg cells (CD4 CD25 + CD25 + T cells) were co-cultured with CD4 + effector T cells (CD4 + CD25 - T cells). The proliferation of effector T cells from GST-Den-immunized control mice, which responded to stimulation with 1 μM GST-Den, was suppressed in the presence of Treg cells from GST-Den-immunized mice Fig. was not shown (Figs. 8A and 8B). In contrast, CD4 + CD25 - effector T cells isolated from these mice were co-cultured with CD4 + CD25 + Treg cells. When stimulated with the respective relevant antigens, the proliferation of effector T cells in the sampled mice immunized with AHHC, RHHC, and RPHC was suppressed (Figs. 8A and 8B). When Treg cells were added to effector cells at a ratio of 4:1 to 16:1, the difference was significant compared to the case without the addition of Treg cells (P < 0.05 to <0.00 1). 1). 1). When Treg cells were added to effector cells at a ratio of 4:1 to 16:1, the difference was significant compared to the case without the addition of Treg cells (P < 0.05 to <0.00 1). 1).

Example

[0072] The expressions of smooth muscle alpha-actin, VCAM1, MMP9, and specific antigens Apo B and HSP60 in the lesions were evaluated. To evaluate whether immunization with the construct of the present invention affects vascular SMC behavior and vascular remodeling, the SMC content of the lesions and the expressions of VCAM1 and MMP9 at the lesion sites were analyzed by IHC analysis. The anti-SMC stained area was significantly smaller in the plaques of the mice immunized with AHHC and RPHC compared to that in the control immunized with GST-Den, showing 5.2 ± 0.7% and 4.9 ± 0.8% respectively, but did not significantly decrease in the mice immunized with RHHC (Figs. 9A and 9B). Furthermore, the expression of VC AM1 was significantly downregulated compared to that in the control immunized with dendroaspin (18.0 ± 2 2.3%), and was significantly downregulated in AHHC, RHHC, and RP HC respectively HC respectively. In the mice immunized with RHHC, it did not significantly decrease (Figs. 9A and 9B). Furthermore, the expression of VC AM1 was significantly downregulated compared to that in the control immunized with dendroaspin (18.0 ± 2 .3%), and was significantly downregulated in AHHC, RHHC, and RP HC showed 4.5 ± 0.9%, 7.8 ± 0.9% and 4.8 ± 0.9% (Figure 9A and 9C). A significantly increased effect was observed in RHHC-immunized mice compared to AHHC or RPHC-immunized mice (Figure 9A and 9C). A similar trend was observed for MMP9 expression in mice immunized with all three constructs, with stained areas of 7.9 ± 1 .0%, 10.1 ± 1.0% and 7.6 ± 1.0% in AHHC-, RHHC- and RPHC-immunized mice, respectively, and 18.1 ± 2.5% in control mice immunized with GST-Den (Figure 9D and 9E). However, no significant difference was obtained at this point between AHHC or RPHC-immunized mice and RHHC-immunized mice (Figure 6D and 6E), except. Interestingly, little difference in mouse ApoB (Figure 10A and 10B) and mouse HSP60 protein (Figure 10C and 10D) antigens was detected at the lesion site between sampled mice and control mice, so injection of recombinant constructs containing human ApoB and HSP60 peptides did not affect the expression of their counterpart

[0073] (ApoB and HSP60). Evaluation of monocyte differentiation into macrophages in PBMC of C57BL / 6 background naive mice in response to treatment with recombinant constructs and the effect of construct-specific immune sera on differentiation were examined. In vitro, monocytes can differentiate into macrophages (or To evaluate whether the same effect on the stimulation of naive cells (from naive mice C57BL / 6 having the same background) can be maintained, PBMCs were stimulated with RHHC or AH HC. Three days later, the expression of the cell surface marker CD206 (mannose receptor, macrophage marker) was evaluated. When compared with unstimulated cells, both RHHC and A HC induced the differentiation of monocytes into macrophages (based on the change in cell number) (Figure 1 1A and 11B). Furthermore, the differentiation of PBMCs induced by these two constructs ceased to occur when the cells were pre-incubated with the antiserum from mice immunized with these two antigens. Interestingly, the inhibition could be achieved by pre-incubating the cells with each other's ant iserum (Figure 11A - 7D). Observation of the differentiation by individual epitopes or domains as stimulants showed different ratios of differentiat ion (Figure 11E - 11F). ion (Figure 11E - 11F). ion (Figure 11E - 11F). ion (Figure 11E - 11F). ion (Figure 11E - 11F).

Example

[0074] The content of toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) involved in the TLR4 signaling pathway related to atherosclerosis at the lesion site was examined . The effect of treatment with these recombinant constructs on the TLR4 and MyD88 content in the lesion was examined. The reduction of atherosclerosis in mice treated with these constructs was accompanied by a decrease in both TLR4 and MyD88 content. The anti-TLR4 staining area was compared with the value (8.1 ± 1.1%) in the control immunized with dendroaspin, and in the plaques of mice immunized with AHHC, RHHC, and RPHC ​​​​​were significantly smaller, being 4.6±1.0%, 3.4±0.5% and 4.2±0.6 %, respectively (Figs. 12A and 12B). Similarly, the anti-MyD88 stained area was significantly smaller in the lesions of mice immunized with these three constructs compared to the value in the control (18.6±2.4%), being 9.7±1.2%, 9.6±1.6% and 10 .2±1.9%, respectively (Figs. 12C and 12D). Furthermore, the overlap of the anti-CD11c and anti-TLR4 stained areas was significantly smaller in the lesions of mice immunized with all three constructs compared to the control (6.6±0.8%), being 3.

Example

[0075] Two kinds of HSP60 peptides derived from human and Mycobacterium, respectively, were used in an experiment to compare their ability to reduce tm2Sgy atherosclerotic lesions by immunization in Ap tm1Her ob Ldlr / J mice. The mice were immunized with two kinds of peptides, 253-268 a Mycobacterium heat shock protein (HSP) called mHSP60 (AA253 - 268) (SEQ ID NO: 15) and a human 516-528 HSP called hHSP60 (AA516 - 528) (SEQ ID NO: 14), respectively, which are keyhole limpet hemocyanin (KLH)-conjugated peptides. The mice were immunized with these two kinds of peptides, and two weeks after the first immunization, the mice were fed a high-fat diet and bred. The results showed that the except for having a low titer and having almost no titer against IgG2c, the two peptides showed similar functions. Similar functions include induced specific immune responses; reduction of lesions; increased expression of Tregs; atheroprotective cytokines: IL -10 and increased concentrations of TGF-β as well as inflammatory cytokines: TNF-α and decreased concentrations of INF-γ; suppression of CD4 + CD25 - T cell proliferation and downregulation of the TLR4 / MyD88 pathway (data not shown). In conclusion, despite the low sequence homology ( 31%) between the two peptides and the lower immune response obtained with the mHSP60 peptide after immunization of B6;129S-Ldlrtm1HerAp obtm2Sgy / J mice with mHSP60 and hHSP60 peptides, both peptides have a similar effect on the significant reduction of early atherosclerotic lesions. From this data, it is confirmed that such immunization with the constructs of the present invention provides an attractive opportunity for the design and development of peptide-based vaccines against atherosclerosis.

Example

[0076] The transcription factor FOXP3 (forkhead box P3) regulates mouse CD4 + CD25 + Treg function (Fontenot et al., Nat Immunol. 2003;4:330-336 ; Hori S, Nomura T, Sakaguchi S. Science. 2003;299:1057-1061), and natural CD4 + CD 25 +Transfer of Tregs significantly reduced plaque progression in the 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, it is known that naturally occurring T regs can affect the size and composition of atherosclerotic lesions, and in several reports, the theory that antigen-specific responses may be operative in atherosclerotic plaques where antigen-specific responses are progressing has been supported. The inventors hypothesized that antigen-induced atherogenic Tregs may have specific functions related to lesion reduction. The inventors tested the humoral immune response, the effect on atherosclerotic lesion size, and local and systemic cellular responses to evaluate the effect of adoptively transferred Treg cells isolated from the blood of antigen-immunized mice on atherosclerotic lesion formation in B6;129S-Ldlr Apob tm1Her Apob tm2Sg y / J mice. An experiment was conducted to evaluate the effect of adoptively transferred Treg cells isolated from the blood of antigen-immunized mice on atherosclerotic lesion formation in B6;129S-Ldlr

[0077] The first method for examining immune responses and adoptive transfer in KO mice involved constructing a recombinant construct by incorporating an AHH h H m R construct into a dendroaspin scaffold. The antigen epitope of ApoB (AA688-707) was designated as A, human HSP60 (AA303-312) (SEQ ID NO: 12) was designated as H , Mycobacterium (AA25 h 3-268) (SEQ ID NO: 15) was designated as H , and the complement component 5a receptor (AA1-31) (arrangement m ID NO: 15) was designated as H Column number 9) was called R. Mice were subjected to the RIMM (Repeated Multiple Site Immunization Strategy) protocol for AH h H m immunized by R. Treg cells were obtained from the blood of h H m R (Tr eg S) and dendrite spin (Treg C )) immunized mice. Adoptive transfer was achieved via the retro-orbital venous plexus of the mice.

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

[0079] From the data (not shown), T eg S isolated from the blood of atherosclerotic-promoting antigen immunized mice showed less lesion formation when compared with Treg from the blood of non-atherosclerotic-promoting antigen immunized mice after adoptive transfer into the veins of non-immunized mice, as shown . Transfer of natural CD4 + CD25 + Treg significantly reduced the progression of plaques in the ApoE-KO mouse model. In addition to less lesion formation, they had an increase in collagen content at the lesion site, an increase in Treg expression at the lesion site, and a decrease in plasma athero Higher concentrations of anti-inflammatory cytokines (IL-10 and TGF-β) and lower concentrations of pro-inflammatory cytokines (TNF-α and INF-γ) were also shown. Downregulation of the expression of α-SMC and PECAM in the lesion site was also observed.

[0080] These results indicate that the constructs of the present invention provide an attractive opportunity in cell therapy for the treatment of atherosclerosis. The present invention also provides the following. [1] (i) a scaffold portion, (ii) a first type of epitope capable of inducing an anti-atherosclerotic vascular disease response via a first pathway, (iii) a second type of epitope capable of inducing anti-atherosclerotic vascular disease via a second pathway independent of the first pathway and a recombinant construct comprising the same. [2] The construct according to [1], comprising a plurality of first and / or second type of epitopes. [3] The construct according to [1] or [2], wherein the first pathway related to the formation of atherosclerosis is via C5a or C5aR interaction. [4] The construct according to any one of [1] to [3], wherein the first type of epitope is a C5a or C5a receptor (C5aR) protein. [5] The construct according to [4], wherein the C5a epitope is a polypeptide comprising an amino acid sequence of 5 to 40 consecutive amino acid residues in SEQ ID NO: 2. [6] The C5a epitope is EQRAARISLGPR (SEQ ID NO: 3), RAARISL ​​​​​​​​Comprising GPRCIKAFTE (SEQ ID NO: 4) and CVNNDETCEQ (SEQ ID NO: 5) Or a polypeptide comprising an amino acid sequence selected from the group consisting of, or an antigenically active Functional fragment thereof, the construct according to [4] or [5]. [7] Wherein the C5a epitope is a polypeptide comprising an amino acid sequence of 5 to 45 consecutive amino acid residues in SEQ ID NO: 6 The construct according to [4]. [8] The C5aR epitope is a C- or N-terminal sequence, and optionally the epitope is MN SFNYTTPDYGHYDDKDTLD (SEQ ID NO: 7), TLDLNTPVDKTSN (SEQ ID NO: 8) and MNSFNYTTPDYGHYDDKDTLDLNTPVDKTS N (SEQ ID NO: 9), or a polypeptide fragment thereof having antigenic activity, the construct according to [4] or [5]. The construct according to [4] or [5]. Substance. [9] The second type of epitope related to the anti-atherosclerotic vascular disease response is an apolipoprotein (Apo) epitope, heat shock protein (HSP) epitope, Chlamydia pneumoniae (chlamydia pneumonia) epitope, PAR-1 epitope and perilipin epitope Selected from the group consisting of or consisting of, the construct according to any one of [1] to [8]. Building.

[10] The HSP is HSP60 or HSP65, and optionally, when the HSP is HSP6 0, it is human HSP60 or Mycobacterium bovis (Mycobacterium bovi s) HSP, the construct according to [9].

[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 mycobacterium (AA) 253-268: EGEALSTLVVNKIRGT (SEQ ID NO: 15), or an amino acid sequence selected from the group consisting of or having antigenic activity a functional fragment thereof, the construct according to

[10] .

[12] The chlamydia pneumonia is Cpn1 or Cpn2, and optionally selected, major outer membrane protein (MOMP) (amino acid sequence (AA) 67-74: GDYV FDRI (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 polypeptide or antigenic activity having an amino acid sequence selected from the group consisting of a functional fragment thereof, the construct according to [9].

[13]

[13] The PAR-1 epitope contains amino acids selected from EWEPKPVNQVYT (SEQ ID NO: 18) and SF LLRNPNDKYEPF (SEQ ID NO: 19), the construct according to [9].

[14]

[14] The scaffold portion is the dendroaspin scaffold protein shown in SEQ ID NO: 1 or a fragment or mutant thereof, the construct according to any one of [1] to

[13] .

[15] wherein the first and / or second type of epitope is incorporated at one or more positions of (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 I II; (e) either one or more positions of the N or C terminus, as described in

[14] the construct described in

[16] (i) a scaffold portion, (ii) a Cpn epitope, (iii) one or more additional epitopes derived from the same or different proteins and the construct described in [1].

[17] wherein the additional epitope is derived from HSP, PAR-1 and C5aR, as described in

[16] the construct described in

[18] selected from the group consisting of AHHC, RHHC, RPHC and AHHR, as described in

[16] or the construct described in

[17] .

[19] (i) a scaffold portion, (ii) two HSP epitopes, (iii) one or more additional epitopes derived from different proteins and

[20] the construct described in [1]. wherein the one or more additional epitopes are ApoB and / or Ca5R,

[21] an expression vector comprising a nucleic acid encoding the epitope incorporated into the construct according to any one of [1] to

[20] .

[22] an antigenic composition comprising the construct according to any one of [1] to

[20] , optionally Optionally (i) an isolated microsome that is an optionally inverted microsome or (ii) an MHC protein or (iii) an inverse micelle or (iv) a hydrophobic complex comprising a synthetic product is the composition as described above..

[23] Formulated as an injectable or oral preparation, described in any one of [1] to

[20] The immunogenic composition as described above, optionally further comprising a suitable adjuvant, excipient, diluent and / or or carrier, is a pharmaceutical composition.

[24] A pharmaceutical composition comprising a construct described in any one of [1] to

[20] or a vector described in

[21] or an immunogenic composition described in

[22] .

[19]

[25] A construct described in any one of [1] to

[20] or a vector described in

[21] , used as a medicament is described above.

[26] A construct described in any one of [1] to

[20] or a vector described in

[21] , used in the manufacture of a medicament is described above.

[27] A construct described in any one of [1] to

[20] or a vector described in

[21] , used for treating atherosclerosis is described above.

[28] A vaccine comprising a construct described in any one of [1] to

[20] or a vector described in

[21] is described above.

[29] A protein described in any one of [1] to

[20] ; a vector described in

[21] ; An immunogenic composition described in

[22] and a pharmaceutical composition described in

[23] or

[24] Administering a preparation selected therefrom to an individual, anti-atherosclerotic in mammals is a method of eliciting a response.

[30] ​A protein according to any one of [1] to

[20] ; a vector according to

[21] ; from the immunogenic composition according to

[22] and the pharmaceutical composition according to

[23] or

[24] A method of treating, preventing or reducing atherosclerosis, comprising administering to an individual a formulation selected therefrom. A method of treating an individual having early atherosclerosis or an individual identified as having a risk of developing atherosclerosis, comprising administering to the individual a formulation selected from the group consisting of:

[31] A protein according to any one of [1] to

[20] ; a vector according to

[21] ; from the immunogenic composition according to

[22] and the pharmaceutical composition according to

[23] or

[24] A method of treating an individual having early atherosclerosis or an individual identified as having a risk of developing atherosclerosis, comprising administering to the individual a formulation selected from the group consisting of: A method of treating an individual having early atherosclerosis or an individual identified as having a risk of developing atherosclerosis, comprising administering to the individual a formulation selected from the group consisting of:

[32] A method of eliciting an immune response against epitopes associated with two independent pathways related to atherosclerotic plaque formation, comprising: 1. Constructing and expressing a dendroaspin scaffold protein comprising at least one first and at least one second epitope according to any one of [1] to

[20] ; 2. Incubating eukaryotic cells with the dendroaspin scaffold protein; 3. Preparing microsomes using the eukaryotic cells; 4. Mixing the microsomes and the dendroaspin scaffold protein with one or more pharmaceutically acceptable ingredients to produce an orally or injectably administrable formulation; 5. Administering the formulation to a mammal or a human. and 3. Preparing microsomes using the eukaryotic cells; 4. Mixing the microsomes and the dendroaspin scaffold protein with one or more pharmaceutically acceptable ingredients to produce an orally or injectably administrable formulation; 5. Administering the formulation to a mammal or a human. 5. Administering the formulation to a mammal or a human. A method comprising the steps of:

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. The construct of claim 1 or 2, wherein the C5aR epitope is at the C- or N-terminus of the construct.

4. The construct of claim 3, wherein the epitope is a polypeptide comprising or consisting of an amino acid sequence selected from the group comprising 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 of claim 5, wherein the HSP is HSP60, which is human HSP60 or Mycobacterium bovis HSP60.

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. 9. The construct of claim 8, wherein the chlamydia pneumonia epitope is a polypeptide comprising an amino acid sequence selected from the group comprising 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, or (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:

Images