MULTI-EPITOPIC CONSTRUCT

INCORPORATION OF SEQUENCE LISTING

A paper copy of the Sequence Listing and a computer readable form of the Sequence Listing containing the file named “P135165USD-1_Sequence_Listing_ST25”, which is 7,729 bytes in size (as measured in MICROSOFT WINDOWS® EXPLORER), are provided herein and are herein incorporated by reference. This Sequence Listing consists of SEQ ID NOs: 1-19.

BACKGROUND

This invention relates to multiple epitope constructs, immunogenic and vaccine compositions comprising recombinant molecules presenting inserted multiple and different epitopes from a variety of antigens. In particular, the invention relates to such compositions for eliciting an immune response against antigens and pathogens involved in the development of atherosclerosis the invention includes inter alia methods of treating and/or preventing the disease and recombinant protein products.

Atherosclerosis is increasingly recognized as a complex chronic inflammatory disease of the arterial walls as evidenced by the presence of inflammatory cells, activated immune cells and cytokines in lesions, all of which indicate involvement of the immune system. Dendritic cells (DCs) are likely to play a crucial role in directing innate and adaptive immunity. A major component of the innate response involves the entry of monocytes into nascent lesions, followed by differentiation of monocytes into macrophages and CD11c⁺ cells with DC-like properties. Atherosclerotic plaques are known to contain macrophage derived foam cells in which macrophages interact with T cells to produce a wide array of cytokines that can exert both pro- and anti-inflammatory effects. Although the molecular mechanism responsible for the development of atherosclerosis is not completely understood, it is clear that the immune system plays a key role in the development of the atherosclerotic plaque and in its complications. Consequently, several antigenic stimuli that are associated with the pathogenesis of atherosclerosis based on modified self-molecules or peptides derived from these molecules such as oxidized low-density lipoproteins (oxLDLs) [9, 10], (32-glycoprotein I (132GP1), phosphatidylcholine (PC) [11, 12], heat shock proteins (HSPs), have been reported. Many more studies related to vaccination against atherosclerosis apart from using epitopes from these self-antigens have also demonstrated high efficacy against atherosclerotic lesion formation for other antigens. However, one of the difficulties in developing effective vaccination strategies against atherosclerosis is the selection of a specific antigen.

In the discovery and development of potential antigens, it has been demonstrated that immunization of B6; 129S-Ldlr^tm1HerApob^tm2Sgy/J mice with the recombinant construct termed “AHHC” containing epitopes derived from apohpoprotein B (ApoB), heat shock protein (HSP) 60 and proteins of Chlamydia pneumoniae (Cpn), reduced atherosclerotic lesion formation in the mice fed with high-fat diet (HFD) (Lu et al 2012; Atherosclerosis 225: 56-68). In addition, it is known from the prior art that immunization with peptides derived from the N-terminal of complement component 5a receptor (C5aR) reduced early atherosclerotic lesion development in B6; 129S-Ldlr^tm1HerApob^tm2Sgy/J mice (Lu et al 2012; Arterioscler Thromb Vase Biol 32: 23S8-2371). Furthermore, in preclinical studies, protease activated receptor (PAR)-1 inhibition showed a strong antithrombotic effect, leading to a significant decrease in platelet aggregation, whereas primary haemostatic function was preserved (Chintala et al 2010 Arterioscler Thromb Vase Biol 30: 2143-2149).

The human complement system is a key component of the innate host defense directed against invading pathogens. C5a is a protein fragment released from complement component C5 and is a 74 amino acid peptide in humans that is generated by the cleavage of C5a convertase on the C5 α-chain during the classical, alternative, and lectin pathways of complement activation. C5a mediates its effects via its G protein-coupled C5a receptor (C5aR/CD88) on the plasma membrane of target cells triggering intracellular signaling, which results in chemotaxis, a respiratory burst and release of pro-inflammatory mediators from granulocytes. C5a attracts and activates neutrophils, monocytes, and platelets and stimulates the release of inflammatory mediators, including reactive oxidants, proteolytic enzymes, chemokines, cytokines, and complement factors C3 and properdin. Secretion of C3 and properdin by neutrophils, as well as the presence of apoptotic and necrotic decidual tissue, and may accelerate alternative pathway activation creating a proinflammatory amplification loop at sites of leukocyte infiltration that enhances C3 activation and deposition and generates additional C5a. Human C5aR is an integral membrane glycoprotein, consisting of 350 amino acids forming a single poly-peptide chain. C5a/C5aR interactions have been shown to modify the production of IL-12, thus regulating Th-1 cell responses, and to potentiate the production of cytokines such as IL-6, IL-8, and TNF-alpha. The C5aR is expressed abundantly on leukocytes, including neutrophils, monocytes, eosinophils, and lymphocytes. C5aR is also expressed by a wide range of parenchymal cells, including glomerular mesangial and proximal tubular epithelial cells. Parenchymal C5aR expression has been shown to be enhanced in areas of acute inflammation.

Ruptured or vulnerable plaques of atherosclerosis are usually characterized by the presence of a large lipid core, a reduced number of smooth muscle cells, a thin fibrous cap, and an increased 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 (MMPs), these cells are capable of degrading components of the extracellular matrix, such as collagen, proteoglycans, and elastin. Several studies have shown by immunohistochemistry and in situ zymography that MMP-1, MMP-3, and MMP-9 are present in the shoulder region of atherosclerotic plaques, and it has been shown that overexpression of active MMP-9 in apoE-deficient mice induces plaque disruption. The stimulatory effect of C5a on the expression of MMP-1 and MMP-9 mRNA in monocyte-derived macrophages isolated from different donors and in human macrophages isolated from atherosclerotic plaques has also been reported.

There is accumulating clinical and experimental data point to an involvement of the anaphylatoxin C5a in the pathogenesis of atherosclerosis. It has recently been reported that there is a predictive value of C5a plasma levels for cardiovascular events in patients with advanced atherosclerosis. Receptors for C5a were detected in atherosclerotic lesions. Furthermore, C5a induces the expression of adhesion molecules by endothelial cells. C5a activates macrophages, causing them to release the inflammatory mediators TNF-alpha, interleukin-1. TNF-alpha, on the other hand, directly induces MMP expression in these cells.

In the discovery and development of potential antigens, it has been demonstrated that immunization of B6; 129S-Ldlr^tm1HerApob^tm2Sgy/J mice with the recombinant construct termed “AHHC” containing epitopes derived from apolipoprotein B (ApoB), heat shock protein (HSP) 60 and proteins of Chlamydia pneumoniae (Cpn), reduced atherosclerotic lesion formation in the mice fed with high-fat diet (HFD) (Lu et al 2012; Atherosclerosis 225: 56-68). In addition, it is known from the prior art that immunization with peptides derived from the N-terminal of complement component 5a receptor (C5aR) reduced early atherosclerotic lesion development in B6; 129S-Ldlr^tm1HerApob^tm2Sgy/J mice (Lu et al 2012; 20 Arterioscler Thromb Vase Biol 32: 2358-2371). Furthermore, in preclinical studies, proteaseactivated receptor (PAR)-1 inhibition showed a strong antithrombotic effect, leading to a significant decrease in platelet aggregation, whereas primary haemostatic function was preserved (Chintala et al 2010 Arterioscler Thromb Vase Biol 30: 2143-2149).

There is a need for improved treatments for arteriosclerotic vascular disease.

BRIEF SUMMARY OF THE DISCLOSURE

According to a first aspect of the invention there is provided a recombinant construct comprising:

i) a scaffold portion and incorporated therein;

ii) a first species of epitope capable of eliciting an anti-arteriosclerotic vascular disease response via a first pathway; and

iii) a second species of epitope capable of eliciting an anti-arteriosclerotic vascular disease response via a second pathway that is independent from said first pathway.

Reference herein to the first and second pathways being “independent” form one another is intended to infer that the formation of atherosclerosis via either the first or second pathway is by different mechanisms of action or causal routes.

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

Preferably, first pathway to atherosclerosis formation is via a C5 interaction and more preferably by a C5a or C5Ar pathway. The C5 epitope is therefore a C5a epitope or a C5a receptor (C5aR) epitope. The C5 epitope may comprise from 5 to 40, e.g. from 8 to 40 amino acid residues or for example from 8 to 35 amino acid residues. The C5a epitope may 10 comprise from 5 to 40 contiguous amino acid residues selected from the C5a sequence (SEQ ID NO:2) (FIG. 14B), e.g. from 8 to 40 amino acid residues, for example from 8 to 35 amino acid residues. In one embodiment, the C5a epitope comprises from 8 to 20, e.g. 8 to 15 contiguous amino acid residues selected from the C5a sequence.

Preferably, the C5a epitope is a polypeptide comprising, or consisting of, the amino acid sequence selected from the group comprising EQRAARISLGPR (SEQ ID NO:3), RAARISLGPRCIKAFTE (SEQ ID NO:4) and CVNNDETCEQ (SEQ ID NO:5) or a functional fragment thereof that has antigenic activity. For example, the epitope may comprise a sequence of from 5 to 40 contiguous amino acid residues selected from the C5a sequence and comprising SEQ ID NOs: 2, 3 or 4.

Preferably, the C5aR epitope comprises from 5 to 50 contiguous amino acid residues selected from the C5aR sequence (SEQ ID NO:6; FIG. 14C), for example from 10 to 40 amino acid residues or for example from 14 to 35 amino acid residues. In one embodiment, the C5aR epitope comprises from 1 to 31 contiguous amino acid residues or a functional fragment thereof.

Preferably, the C5aR epitope may be at the N-terminal end (containing free amino group) or the C-terminal end (containing free carboxyl group) of the C5aR amino acid sequence. Alternatively and more preferably the C5aR epitope is a polypeptide comprising, or consisting of, an amino acid sequence selected from the group comprising MNSFNYTTPDYGHYDDKDTLD (SEQ ID NO:7), TLDLNTPVDKTSN (SEQ ID NO:8) and MNSFNYTTPDYGHYDDKDTLDLNTPVDKTSN (SEQ ID NO:9) or a functional fragment thereof that has antigenic activity.

Reference herein to a functional fragment encompasses portions of the epitope amino acid sequences that retains sufficient amino acids to provide and act as an antigenic determinant and to possess antigenic activity and thus function as an epitope. A functional fragment also encompasses portions of epitope amino acid sequences that when incorporated and presented in the scaffold portion, elicits an immune response against atherosclerosis that can be determined by, for example and without limitation a reduction in the area occupied by an atherosclerotic lesion.

Reference to a first and second species of epitope is intended to convey that they are each separately and independently associated with different pathways that ultimately lead to atherosclerosis and associated diseases.

Preferably, the recombinant construct comprises more than one first species of epitopes.

Preferably, the second species of epitope is a human epitope. The second species of epitope may comprise from 5 to 40, e.g. from 9 to 40 amino acid residues, for example from 9 to 20 amino acid residues. Preferably, the second species of epitope associated with an anti-arteriosclerotic vascular disease response is selected from the group comprising, or consisting of apolipoprotein (Apo) epitopes, heat-shock protein (HSP) epitopes, chlamydia pneumonia epitopes, protease-activated receptor-1 epitopes (PAR-1) and perilipin epitopes.

More preferably, the second epitope is a heat-shock protein (HSP) epitope, chlamydia pneumonia epitope, tissue factor or PAR-1 epitope.

Preferably, said heat-shock protein (HSP) is a HSP 60 or a HSP 65. More preferably said HSP 60 is a human HSP 60 or a Mycobacterium bovis HSP. More preferably said epitope capable of eliciting a response against HSP 60 is a polypeptide comprising, or consisting of the 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) KGIIDPTKWRTA (SEQ ID NO:14), and mycobacterium (AA) 253-268: EGEALSTLVVNKIRGT (SEQ ID NO: 15) or a functional fragment thereof that has antigenic activity.

Preferably, said chlamydia pneumonia is Cpn1 or Cpn2. More preferably, said epitope capable of eliciting a response against Cpn is a polypeptide comprising, or consisting of the amino acid sequence selected from the group comprising the 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 that has antigenic activity.

Preferably, the tissue factor epitope is (AA56-67) and comprises the sequence EWEPKPVNQVYT (SEQ ID NO:19).

Preferably, the PAR-1 epitope is (AA42-55) and comprises the amino acid sequence SFLLRNPNDKYEPF (SEQ ID NO:19).

Preferably, the recombinant construct of the present invention comprises more than one second epitope for example it may comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 identical or different second epitopes. For example in particularly preferred embodiments the recombinant construct may comprise a C5aR epitope, a Cpn epitope and two different HSP epitopes or it may comprise a C5aR epitope, a Cpn epitope, a HSP epitope and a PAR-1 epitope.

Preferably, the amino acid sequences of the first and/or second epitope are incorporated into (a) loop I and/or loop II; (b) loop I and/or loop III; (c) loop II and/or loop II; or (d) loop I, loop II and loop III of the dendroaspin scaffold. Loop I comprises amino acid residues 4-16, loop II residues 23-36 and loop III residues 40-50. However, the further amino acids being incorporated may extend into or substitute regions external to the loops, i.e. residues 1-3, 17-22 and 37-39 such that residues of the non-loop regions are augmented or substituted for those of the further amino acid sequence or sequences being inserted. The further amino acid residues are preferably incorporated into either loop I or loop II. In this way the RGD-containing loop III is unaltered and so the integrin binding function of dendroaspin is retained. A preferred location for the inserted further sequence is at a site in dendroaspin scaffold between amino acid residues: 4-16, 18-21, 23-36, or 52-59. Each inserted further amino acid sequence or portion of a further amino acid sequence is preferably an amino acid sequence in the range 3-40 amino acid residues, more preferably 3-16, even more preferably 3-14 amino acid residues long. The start of the inserted further amino acid sequence may be at any one of amino acid residues 1-57 of the dendroaspin scaffold. The end of the inserted further amino acid sequence may be at any one of amino acid residues 3-59 of the dendroaspin scaffold.

When two or more further amino acid sequences are inserted into the dendroaspin scaffold then the linear distance between these is preferably in the range 1-35 amino acids, more preferably 1-14 amino acids. When more than two or more further amino acid sequences are inserted then there is preferably at least one native dendroaspin amino acid residue separating each further amino acid sequence. The RGD-containing loop may be modified by insertion, deletion or substitution of one of more amino acid residues, preferably a maximum of 8 or a minimum of 1 amino acids can be modified within loop II of dendroaspin. Loop I and/or loop II may be modified by insertion, deletion or substitution of one or more amino acid residues. Any suitable number of amino acids can be inserted into the dendroaspin scaffold to give the desired bi- or multi-functional activity although a number of residues in the range 14 to 36 are preferred for insertion at one or more sites in the dendroaspin scaffold.

Preferably, in some embodiments of the invention the first or second epitopes may be attached at either or both of the C and/or N terminus ends of the dendroaspin scaffold protein.

In a further aspect of the invention there is provided an expression vector comprising the nucleic acids encoding the proteins incorporated into the construct of the first aspect of the invention.

In a yet further aspect of the invention there is provided an antigenic composition comprising the protein according to the first aspect of the invention and an antigenic hydrophobic complex.

In a yet further aspect of the invention there is provided a pharmaceutical composition comprising the immunogenic composition of the present invention, formulated as an injectable or oral product. Preferably, the pharmaceutical composition further includes a suitable adjuvant, excipient, diluent and/or carrier.

In a yet further aspect of the invention there is provided a pharmaceutical composition comprising the protein of the first aspect of the invention, the vector of the invention or the immunogenic composition of the invention.

In a yet further aspect of the invention there is provided the protein of the first aspect of the invention or the vector of the invention for use as a medicament.

In a yet further aspect of the invention there is provided a method of eliciting an anti-atherosclerosis response in a mammal comprising administering a product selected from: the recombinant protein according to the first aspect of the invention; the vector of the invention; the immunogenic composition of the invention; the pharmaceutical composition of the invention; and the pharmaceutical composition of the invention.

In a yet further aspect of the invention there is provided a method of treating, preventing or reducing atherosclerosis comprising administering to an individual a product selected from: the recombinant protein according to the first aspect of the invention; the vector of the invention; the immunogenic composition of the invention; the pharmaceutical composition of the invention; and the pharmaceutical composition of the invention. The product may be administered in a therapeutically effective amount or therapeutically acceptable amount.

In a yet further aspect of the invention there is provided a method of treating an individual with early stage atherosclerosis or an individual identified as at risk of developing atherosclerosis, the method comprising administering to an individual a product selected from: the recombinant protein according to the first aspect of the invention; the vector of the invention; the immunogenic composition of the invention; the pharmaceutical composition of the invention; and the pharmaceutical composition of the invention. The product may be administered in a therapeutically effective amount or therapeutically acceptable amount.

In a yet further aspect of the invention there is provided a vaccine comprising the protein according to the first aspect of the invention or the vector of the invention.

In a yet further aspect of the invention there is provided a method of eliciting an immune response against epitopes associated with at least two independent pathways associated with atherosclerosis formation, the method comprising:

- (i) constructing and expressing a dendroaspin scaffold protein comprising at least one first and at least one second epitope as herein before described;
- (ii) incubating eukaryotic cells with said dendroaspin scaffold protein;
- (iii) using said eukaryotic cells to prepare microsomes;
- (iv) incorporating said microsomes and dendroaspin scaffold protein with one or more pharmaceutically acceptable constituents to produce an orally or injectable administratable preparation; and
- (v) administering said preparation to a mammal or human.

Data has shown that immunization with ApoB/PAR-1/HSP/Cpn did not affect C5a expression in sclerotic lesion, suggesting that C5/C5aR presents an independent pathway in lesion formation. The present invention provides methods and products for advantageously targeting, simultaneously, both the independent C5 and ApoB/PAR-1/HSP/Cpn related pathways in order to provide improved treatments for the prevention and reduction of early stage atherosclerosis.

It will be appreciated that the preferred features ascribed to the first aspect of the invention are applicable mutatis mutandis to each and every other aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

FIGS. 1A & 1B show a schematic representation of the backbone of the dendroaspin structure (FIG. 1A) and a schematic representation of alignment of constructs and dendroaspin scaffold (FIG. 1B).

FIGS. 2A-2G show levels of constructed protein-induced IgG, lgG1, and lgG2c antibodies in the sera of B6; 129S-Ldlr^tm1HerApob^tm2Sgy/J mice at 2 weeks and 12 weeks respectively, after the first immunization and in controls (GST-Den (referred as GST-den)-immunized mice). The mean optical densities (ODs) and SEM obtained from plasma samples of constructs AHHC, RHHC, and RPHC-immunized mice on Cpn peptide (C)-, hHSP60153-163 (H), hHSP60303-312-(H), C5aR-peptide (R)-, ApoB peptide (A)-, PAR-1 (P)-coated ELISA plates are shown. (FIG. 2A) IgG at the dilution ratio: 1:100. (FIG. 2B) lgG1 at the dilution ratio: 1:6250. (FIG. 2C) lgG2c at the dilution ratio: 1:50. (FIG. 2D) Cross reaction between human ApoB peptide-induced antiserum and Cpn peptide. (FIG. 2E) Cross reaction between human ApoB peptide- and hHSP60303″312 peptide-induced antiserum and their antigens. (FIG. 2F) Cross reaction between ApoB peptide-induced antiserum and antigens of either PAR-1 peptide or C5aR peptide. (FIG. 2G) Cross reaction between C5aR peptide-induced antiserum and antigens of either ApoB peptide or PAR-1 peptide. Antiserum used for cross-reaction was taken at week 8.

FIGS. 3A-3H show detection and quantitation of the lesion areas in the aorta of 86; 129S-Ldlr^tm1HerApob^tm2Sgy/J mice fed on a high-fat diet after immunization with constructs versus controls (GST-den).

FIG. 3A: Photomicrograph of lesions observed in atherosclerotic aortas as analyzed with elastin/van Giesen staining.

FIG. 3B: Scatter plot showing mean of lesion area in the aortic sinus of mice immunized with constructs compared with those in controls (GST-Den). (N=6-9 mice). Error bars=SEM

FIG. 3C: Percentage of reduction in lesion size in the aortic sinus (the reduction of control [GSTden] group was set at zero).

FIG. 3D: Representative photomicrographs and quantitative analysis of collagen (Sirius Red coloration under polarized light) in atherosclerotic aortas in individual mice.

FIG. 3E: Quantification of collagen content at lesion area in the aorta of 86; 129S-Ldlr^tm1HerApob^tm2Sgy/J. NS: not significant.

FIG. 3F: Representative Oil Red O-stained en face descending aorta from mice.

FIG. 3G: Percentage of lesion-occupied area versus total area of descending aortas in individual mice (N=6 mice) of the different experimental groups. The mean lesion size and the difference in lesion size between the experimental groups are shown.

FIG. 3H: Percentage of reduction in lesion size in descending aortas (the reduction of control [GSTden] group was set at zero).

FIGS. 4A & 4B show detection and quantitation of the lesion areas in the aorta of B6; Ldlr^tm1HerApob^tm2Sgy/J mice fed on a high-fat diet at week 9 after immunization with constructs versus controls (GST-den), GST and alum. FIG. 4A is a photomicrograph of lesions observed in atherosclerotic aortas as analyzed with elastin/van Giesen staining (N=6-8 mice). FIG. 4B is a scatter plot showing mean of lesion area in the aortic sinus of mice 35 (N=6-8 mice).

FIGS. 5A-5H show the assessment of inflammation-associated cells in the lesions of 86; 129S-Ldlr^tm1HerApob^tm2Sgy/J mice fed a high-fat diet after immunization with constructs AHHC, RHHC, and RPHC.

FIG. 5A: Photomicrographs showing IHC staining of CD68 (green) and CD11c (red) markers (scale 5 bar: 100 μm and 12.5 μm for magnified ones).

FIG. 5B: Scatter plot showing anti-CD68-stained area in lesion versus total lesion area; Data are given as the mean of 6 mice.

FIG. 5C: Scatter plot showing anti-CD11 c-stained area in lesion versus total lesion area; Data are given as the mean of 6 mice.

FIG. 5D: Co-localization of CD68⁺ and CD11c⁺ areas (derived from FIGS. 3B and 3C).

FIG. 5E: Photomicrographs showing IHC staining of CD4⁺ T-cells (green) and Foxp3⁺ Treg cells (red) (scale bar: 100 μm and 12.5 μm for magnified ones).

FIG. 5F: Scatter plot showing anti-Foxp3-stained area versus anti-CD4⁺ stained area in lesion (N=6).

FIG. 5G: Representative analysis of Foxp3 expression by CD4⁺ T cells in lymph nodes from construct-immunized mice fed on a high-fat diet as assessed using flow cytometry.

FIG. 5H: Percentage of Foxp3⁺ cells among CD4⁺ spleen cells as analyzed by flow cytometry. Data are expressed as mean of 3 analyses±SEM. Differences between groups are shown.

FIGS. 6A-6R show the a assessment of IL-10-producing T cells, TNF-α expression in the lesions and cytokine levels in B6; 129S-LDlr^tm1HerApob^tm2Sgy/J mice fed a high-fat diet after immunization with constructs AHHC,RHHC, and RPHC.

FIG. 6A: Photomicrographs showing dual-IHC staining for IL-10 (red) and CD4 (green) (scale bar: 100 μm and 12.5 μm for magnified ones).

FIG. 6B: Scatter plot showing mean of IL-10-positive area co-localized with CD4⁺ area (%) (N=6).

FIG. 6C: Photomicrographs showing IHC staining for TNF-α (green) of lesions (scale bar: 100 μm and 2.5 μm for magnified ones).

FIG. 6D: Scatter plot showing mean of anti-TNF-α stained area in the lesion versus total lesion area (N=6).

FIGS. 6E-6H: Cytokine levels measured in plasma.

FIGS. 6I-6L: Cytokine levels measured in the supernatant of splenocytes stimulated with ConA.

FIG. 6M: Representative analysis of CD4⁺IL-4⁻T-cells in splenocytes from construct-immunized mice fed on a high-fat diet as assessed by flow cytometer.

FIG. 6N: Percentages of IL-4⁺ expressing CD4⁺ spleen cells. Data are expressed as mean of 3 analyses±SEM. Differences between groups are shown.

FIG. 6O: Representative analysis of IL-17A expression by CD4⁺ T-cells in splenocytes from construct-immunized mice fed on a high-fat diet as assessed by flow cytometry.

FIG. 6P: Levels of IL-17A expression in spleen cells. Data are expressed as mean of 3 analyses±SEM. Differences between groups are shown.

FIG. 6Q: Representative analysis of CD4⁺IL-2⁺ T-cells in splenocytes from construct-immunized mice fed on a high-fat diet as assessed by flow cytometer.

FIG. 6R: Levels of IL-2⁺ expressing CD4⁺ in spleen cells. Data are expressed as mean of 3 analyses±SEM. Differences between groups are shown.

FIGS. 7A-7D show the measurement of cytokines (IFN-gamma and IL-10) from the spleen of immunized mice stimulated by antigens. Splenocytes were cultured in RPMI 1640 with 10% fetal calf serum and induced with 1 μg/ml antigen (GST-den, GST-AHHC, GST-10 RHHC, GST-RPHC, ApoB100 peptide, C5aR peptide, PAR-1 peptide, respectively) for 48 hour. Then IL 10 and IFN-γ in supernatant of cultured cell were measured with DuoSet mouse IL-10 kit and mouse IFN-γ Quantikine immunoassay kit (R&D system, Minneapolis) according to manufactory's protocol.

FIGS. 8A & 8B show the investigation of antigen-specific regulatory function in antigen-immunized mice. Inhibition of CD4⁺CD25⁻ effector T cell proliferation by CD4⁺CD25⁺ regulatory T-cells isolated from the spleen of control (GST-den-immunized) and constructimmunized mice when AHHC, RHHC and RPHC were used as antigens.

FIG. 8A: Quantitative analyses of proliferation of CD4⁺CD25⁻ effector T cells in the presence of Treg cell by flow cytometry is shown.

FIG. 8B: Proliferation of effector cells isolated from immunized mice alone is indicated in the leftmost bar of each group. Addition of Treg cells to T effector cells at different ratios was also shown. Data are given as mean of 3 analyses±SEM.

FIGS. 9A-9E show the evaluation of expression of smooth muscle alpha actins, vascular cell adhesion molecule (VCAM)-1 and matrix metalloproteinase 9 (MMP9) in lesion site. FIG. 9A: Photomicrographs showing IHC staining for smooth muscle alpha actin (red), vascular cell adhesion molecule VCAM-1(green) (scale bar: 100 μm and 12.5 μm for magnified ones). FIGS. 9B and 9C: Scatter plot showing means of anti-SMC stained area (FIG. 9B) and anti-VCAM-1 stained area (FIG. 9C) (N=6). FIG. 9D: Photomicrographs showing IHC staining for MMP9 (green) (scale bar: 100 μm and 12.5 μm for magnified ones). FIG. 9E Scatter plot showing means of anti-MMP9 stained area (N=6).

FIG. 10A shows representative photomicrographs showing immunohistochemical staining of the aortic root showed anti-ApoB antibody stained areas (red) in the lesion. (FIG. 10B) Quantitative analysis of ApoB expression in lesions. (FIG. 10C) Representative photomicrographs showing immunohistochemical staining of the aortic root showed anti-HSP60 antibody stained areas (red) in the lesion. (FIG. 10D) Quantitative analysis of HSp60 expression in lesions. Scale bar: 150 μm (unenlarged) and 25 μm (enlarged). N=6 mice. NS: not significant.

FIGS. 11A-11F show the evaluation of monocyte differentiation into macrophages. FIGS. 11A and 11B: PBMCs differentiation stimulated by recombinant constructs (1 μg/ml) as assessed by analyses of CD206 expression by flow cytometry and inhibition of differentiation in the presence of respective construct-induced antibodies. FIGS. 11C and 11D: Inhibition of differentiation by respective and other construct induced antibodies. FIGS. 11E and 11F: Individual peptide as a stimulator (1 μg/ml) of monocyte differentiation as analysed by CD206 expression. Data are expressed as average values of 3 analyses.

FIGS. 12A-12D show the evaluation of the content of TLR4 and MyD88 contents at the lesion sites. FIG. 12A: Photomicrographs showing IHC staining for TLR4 (red) (scale bar: 100 μm and 12.5 μm for magnified ones). FIG. 12B: Scatter plot showing means of anti-TLR4-stained area (N=6). FIG. 12C: Photomicrographs showing IHC staining for MyD88 (red) (scale bar: 100 μm and 12.5 μm for magnified ones). FIG. 12D: Scatter plot showing means of anti-MyD88-stained area (N=6).

FIGS. 13A & 13B show (FIG. 13A) Representative photomicrographs showing immunohisto-chemical staining of the aortic root showed anti-CD11c antibody stained areas (green) overlapped with anti-TLR4 antibody stained area (red) in the lesion (yellow). (FIG. 13B) Measurement of combined area of CD11c and TLR4) occupied in lesion (%). Scale bar: 141 μm (unenlarged) and 26 μm (enlarged).

FIG. 13C shows the statistical analysis of the effect of immunization with the peptides.

FIG. 14A shows the SEQ ID NO: 1 of the native dendroaspin amino acid sequence, FIG. 14B shows the amino acid sequence of C5a SEQ ID NO:2 and FIG. 14C shows the amino acid sequence of C5Ar SEQ ID NO:6.

DETAILED DESCRIPTION

Data indicates that the C5a/C5aR pathway towards atherosclerosis is independent from that of PAR-1/HSP/Cpn pathway based on studies of immunized Apob^tm2sgyldlr^tm1Her/J mice. The present invention provides products and vaccines for targeting both pathways simultaneously using combined epitopes within a dendroaspin scaffold.

Our previous study demonstrated that construct AHHC [ApoB100^688-707+hHSP6^303-312+hHSP60^153-163Cpn derived peptide (C)] significantly reduced atherosclerotic lesion (Lu et al Atherosclerosis 2012, 225: 56-68). The present invention provides modulations and derivatives of this construct with a sequential epitope-substitution named RHHC in which A was replaced by an “R” (C5aR_1-31) and RPHC with a further “H” (hHSP60^303-312) conversion into “P” (protease-activated receptor-1^42-55) in mice. An alternative embodiment is the construct AH^hH^mR wherein the antigenic epitopes are from ApoB (AA688-707), designated as A, human HSP60 (AA^303-312) (SEQ ID NO: 12) designated as H^h, mycobacterium (AA^253-268) (SEQ ID NO:15) designated as H^mand complement component 5a receptor (AA1-31) (SEQ ID NO:9) designated as R. Immunization of B6; 129S-Ldlr^tm1HerApob^tm2Sgy/J mice with these elicited production of high levels of antibodies against each epitope (apart from hHSP60^153-163and P which induced a low antibody response). Histological analyses demonstrated that the mice immunized with either RPHC or RHHC showed significant reductions in the size of atherosclerostic lesions compared to those with AHHC (69.5±1.1% versus 55. 7±3.4%, P=0.006 or 65.6±1.3% versus 55. 7±3.4%, P=0.045). Reduction of plaque size in the aortic sinus and descending aorta correlated with alterations in cellular immune responses when compared with controls. We conclude that these new recombinant constructs may provide new antigenic and structural features which are favourable for significant reduction in atherosclerotic lesion formation. This present invention offers a novel strategy for developing anti-atherosclerotic agents.

Based on the effects of the peptides derived from C5aR and PAR-1 on reducing the atherosclerotic lesion, we hypothesized that the effect of a multi-epitopic construct on reducing atherosclerotic lesion may be modulated towards favorable plaque phenotype and increased lesion reduction with inclusion of C5aR and PAR-1 in vaccination. In the present study we investigated the effect of C5aR and PAR-1 including constructs through a sequential substitution: AHHC→RHHC (R denotes an epitope derived from C5aR) in RHHC→RPHC (P denotes an epitope derived from PAR-1) on reducing atherosclerotic lesion. The induced immune response is associated with an anti-atherogenic effect, detected as a significant reduction in the size of the atheromatous lesion area both in aorta sinus and descending aortas with a rate in the following order: RPHC≥RHHC>AHHC

Native dendroaspin is a 59 amino acid peptide and the dendroaspin scaffold lends itself to modification. When dendroaspin (including the RGD motif) is modified to incorporate further functional amino acid sequences e.g. active portions or motifs of agonists, antagonists or inhibitors of factors in the clotting cascade, the resulting molecules are particularly useful as anticoagulants and do not suffer from the drawbacks associated with existing anticoagulants (see International application WO 01/57210). Such hybrid polypeptides may comprise a first amino acid sequence including the RGD motif and conferring dendroaspin activity and a further amino acid sequence conferring activity other than that of dendroaspin activity. In this way the hybrid dendroaspin-based molecules molecules may be rendered multifunctional.

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

The C5a receptor also known as component 5a receptor (C5AR1) or CD88 (cluster of differentiation 88) is a G protein-coupled receptor for Ca. Human C5aR is an integral membrane glycoprotein consisting of 350 amino acids forming a single poly-peptide chain. The 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 liver parenchymal cells, lung vascular smooth muscle, lung and umbilical vascular endothelial cells, bronchial and alveolar epithelial cells, HepG2 cells, a hepatoma cell line, mesangial cells, as well as astrocytes and microglial cells, on cultured human fetal astrocytes and astrocyte cell line.

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process 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 restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

Expression and Purification of Recombinant Glutathione S-Transferase Constructs

Glutathione S-transferase-dendroaspin (GST-tagged-den here referred to as a control, FIG. 1A) and GST-recombinant construct AHHC [ApoB100 peptide, amino acids (aa) 688-707 (numbered including signal peptide), human heat shock protein (hHSP) 60 peptide, aa 303-312 and aa153-163, respectively; and Chlamydia pneumoniae (Cpn) derived epitope (“C”, denotes a combination of aa 66-73 from major out membrane protein (MOMP) and aa 283-291 from outer membrane 5 of Cpn)] was described previously and construct RHHC (“R” denotes a C5aR peptide, aa 1-31) and RPHC (“P” denotes an protease activated receptor-1 (PAR-1) peptide, aa 42-55) were generated. Schematic presentation of these constructs using dendroaspin as a scaffold is shown in FIG. 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 these procedures were similar to those described for AHHC previously [Lu et at 2012; Atherosclerosis 225: 56-68].

Animal Experiments

86; 129S-Ldlr^tm1HerApob^tm2Sgy/J mice were used, each group of mice consisting of 5-6-week-old males. Dendroaspin as a control antigen, since dendroaspin was used as a scaffold and previous data showed no effect of dendroaspin on lesion reduction when used for subcutaneous immunization in mice.

The immunizing antigens used were constructs AHHC, RHHC and RPHC, respectively. The repetitive immunization multiple sites strategy (RIMMS) was adopted [1] and mice were sacrificed at the end of week 12 (a high-fat diet was started at the end of week 2 and continued for 10 weeks). The control groups followed the diet program after immunization with GST-tagged Den and Alum (adjuvant).

Tissue Preparation and Antibody Response Measurements

Twelve weeks after the first immunization, aorta tissues were harvested and mounted in optimal cutting temperature compound (OCT) and in paraffin, for immunohistochemical (IHC) analyses and lesion measurement, respectively. Atherosclerotic lesions in aortic roots were examined by an Olympus UULH Optical microscope (Olympus Optical Co. Ltd, Tokyo, Japan) and analyzed with Image-Pro Plus TM software, version 7.0 (Media Cybernetics, Inc., Bethesda, Md., USA). Longitudinally opened descending aortas were evaluated for the extent of atherosclerosis after Oil Red 0 (ORO) staining. The peptide-specific antibody levels in the plasma samples were measured by ELISA following the manufacturer's instructions. One third of spleens were embedded in OCT and the remaining part was homogenized by pressing through 70 μm cell strainer and frozen for further analysis.

IHC and Morphometric Analyses. Quantitative Measurements of Atherosclerosis, and IHC Analysis of Forkhead Box Protein 3 (Foxp3) Expression in CD4⁺ Splenocytes.

OCT-embedded samples were used for detection of CD68, CD11c, Interleukin (IL)-10 and tumour necrosis factor (TNF)-α, Foxp3, vascular cell adhesion molecule (VCAM)1, alpha smooth muscle cell (alpha-SMC), matrix metalloproteinase 9 (MMP9) by IHC analyses. Sections of paraffin-embedded tissues were stained with hematoxylin and eosin (HE) and elastin/van Gieson (Sigma) for histological examination by the Olympus U-ULH Optical microscope.

Flow Cytometric Analysis of Foxp3, IL-2, IL-4 and IL-17A Expression in CD4⁺ T-cells in splenocytes and differentiation of PBMC into macrophage (CD206).

Spleen cells were processed for staining (30 min at 4° C.) using allophycocyaninanti-mouse Foxp3, IL-2, IL4 and IL-17 antibodies (Biolegend, Cambridge, UK). For cell differentiation assay, mouse (C57BL/6) PBMCs were stimulated with different antigens or pre-incubated with antiserum of antigen (in order to see any antagonism of antiserum). Antigen-induced differentiation of monocytes into macrophages was measured by flow cytometry which was compared to cell populations from non-induced cells or control antigen (GST-Den)-induced cells.

Measurement of Cytokines

IL-10 and TNF-α levels in the lesions were quantified by IHC analyses (rat anti-mouse TNF-α and IL-10 purchased from Biolegend, CA, USA). Plasma levels of the cytokines, IL-10, transforming growth factor beta (TGF-β), TNF-α and interferon gamma (IFN-γ) were measured by ELISA following 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.

Antigen-Specific Regulatory T Cell Function Assays

To assess antigen-specific regulatory T cell function, CD4⁺CD25⁺ Treg cells were isolated by using regulatory T cell isolation kit of Miltenyi Biotec (Bergisch Gladbach, Germany) from spleen CD4⁺ T cells of B6; 129S-Ldlr^tm1HerApob^tm2Sgy/J mice immunized subcutaneously with constructs, respectively. CD4⁺CD25⁻ T effector cells were isolated from spleen CD4⁺ T cells (unbound to beads binding CD4⁺CD25⁺ cells, 99.5% of CD4⁺ cells) of same construct-immunized mice respectively. CD4⁺CD25⁻ cells (2×10⁵) were co-cultured with CD4⁺CD25⁺ cells (2×10⁵), and stimulated with 1 μM related construct or with GST-Den control. After 2 days of culture, the proliferation of T effector cells was measured for the shift in fluorescence intensity of a population of cells by flow cytometry expressed as mean fluorescence intensity (MFI).

Statistical Analyses

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 and intergroup differences were analyzed using one-way ANOVA for multiple comparisons and post hoc Bonferroni test. Other data were analyzed using Student's t-test (2-tailed analyses). Non-parametric distributions were analyzed using the Mann-Whitney U test for pair wise comparisons and the Kruskal-Wallis test for multiple comparisons. Differences between groups were considered significant at P values below 0.05.

Example 1

Peptide-specific immunoglobulin G in the sera of immunized mice was assessed. Antibody levels were measured by ELISA test in the sera of mice immunized with either dendroaspin (GST-tagged) or constructs within dendroaspin scaffold (GST-tagged) at weeks 2 and week 12 respectively after first immunization. In AHHC-immunized mice, ApoB peptide-, hHSP60^303-312- and Cpn-peptide-specific antibodies were observed when these peptides were used as ELISA antigens (FIG. 2A). Similarly C5aR peptide-specific antibodies were detected in addition to observed hHSP60^303-312- and Cpn peptide-specific antibodies in mice immunized with RHHC (FIG. 2A). High antibody levels against Cpn peptide and C5aR peptide whereas low antibody level against PAR-1 peptide was detected in mice immunized with RPHC (FIG. 2A). Low antibody level against hHSP60^153-163was observed in mice immunized with any construct (FIG. 2A). Overall observed optical density (OD) values at 100 dilutions were reduced at week 12 compared to those at week 2.

Interestingly, a peptide-induced specific immunoglobulin (Ig)G1 response was observed in serum of peptide-immunized mice against all peptide antigen epitopes at high dilutions except for those against hHSP60^153-163and PAR-1 peptides when compared with GST-Den control (FIG. 2B). In addition, a similar pattern for IgG2c response was also 30 detected but with low serum dilutions when compared with that in control (FIG. 2C). However, the levels of IgG2c detected were much lower than those of IgG1 based on the optical densities measured in different dilutions of samples (1:50 versus 1:6250). Furthermore, using antiserum at week 8, certain levels of cross-reactions were observed between ApoB peptide-induced antiserum and Cpn peptide (FIG. 2D), between ApoB peptide- and hHSP60^303-312peptide-induced antiserum and their antigens (FIG. 2E), between ApoB peptide-induced antiserum and antigens of either PAR-1 peptide or C5aR peptide (FIG. 2F) and between C5aR peptide-induced antiserum and antigens of either ApoB peptide or PAR-1 peptide (FIG. 2G). Apart from cross-reaction between Cpn peptide and ApoB peptide antiserum when pooled sera were tested and thus no SD values were calculated (FIG. 2D), the other cross-reactions showed significant difference compared to the controls (FIGS. 2E-G; P<0.05-<0.001).

Example 2

The reduction of atherosclerotic lesion size in the aortic sinus was assessed. Following immunization with AHHC, RHHC, RPHC, and after a 10-week high-fat diet, the aortic sinuses of the mice were evaluated for the extent of atherosclerosis. The calculated 10 plaque sizes from the immunized animals were compared with those of the controls. Representative photomicrograph of sections with lesions from experimental groups are shown in FIG. 3A. The plaque areas are shown in FIG. 3B. Lesion size was smaller in mice immunized with all three constructs showing 31,071±998.7 μm², 24,123±1967 μm²and 21,386±2482 μm²(P<0.001) compared with controls (70200±5718 μm²). The smaller lesion area was observed in mice immunized with either RHHC or RPHC compared with mice immunized with AHHC (P=0.007-0.002) (FIG. 2B). The percentage of reduction in lesion size is shown in FIG. 3C, showing 55.7±3.4%, 65.6±1.3% and 69.5±1.1% when assuming reduction in lesion of control animals as zero percent. The control mice that were immunized with GST-Den showed similar lesion formation in either GST-tag-or Alum 20 (adjuvant)-immunized controls (FIGS. 4A and 4B). Therefore, GST-Den was used as a control throughout the experiment.

The impact of treatment with these recombinant constructs on the collagen content in these lesions was also examined. The reduction of atherosclerosis in mice treated with these constructs was associated with an increase in collagen content: approximately 3-fold for either AHHC-immunized mice or RHHC-immunized mice versus control mice (18.6±1.2% or 19.4±0.9% versus control 5.9±0.3%; P<0.001), respectively (FIGS. 3D and 3E). Mice immunized with in RPHC showed a significant collagen increase (24.4±0.9%) compared with mice immunized with either AHHC or RHHC (P=0.003, and P=0.007, respectively).

Longitudinally opened descending aortas were stained en face with oil red O (ORO) and positively stained plaques areas were measured. Representative en face stained descending aortas from experimental groups are shown in FIG. 3F. Lesion size was significantly smaller in mice immunized with any construct showing 8.4±0.3%, 6.6±0.3% and 6.4±0.4% for AHHC, RHHC, and RPHC, respectively compared with controls (19.5±35 1.7%, P<0.001) (FIG. 3G). Both RHHC- and RPHC-immunized mice showed significantly less lesion than AHHC-immunized mice (P=0.011-0.008). The reduction of lesions expressed as a percentage is shown in FIG. 2H with 57.3±1.7%, 66.1±1.6% and 67.3±1.9% for AHHC, RHHC, RPHC, respectively. The smallest lesion area was observed in mice immunized with RPHC.

Example 3

The amount of inflammatory cells and CD4⁺ T cells expressing Foxp3 in local (lesions of aortas) and remote organs: splenocytes and lymphocytes was assessed. The percentage of anti-CD68-stained area in the lesions showed 14.9±1.6%, 13.6±1.3% and 10.3±0.8% in mice immunized with AHHC, RHHC and RPHC, respectively compared to 43.7±3.2% in control mice immunized with GST-Den (P<0.001). Lesser anti-CD68-stained area was observed in RPHC-immunized mice compared with that in AHHC-immunized mice (P=0.034) (FIGS. 5A and 5B). Similarly, measurement of anti-CD11c-stained lesion area showed 13.3±2.1%, 10.S±1.S % and 7.9±0.8% in mice immunized with AHHC, RHHC and RPHC, respectively compared to 38.4±1.9% in control mice (P<0.001) (FIGS. 5A and 5C). Anti-CD11c-stained lesion area in RPHC-immunized mice was significantly smaller when compared with that in AHHC-immunized mice (P=0.039) (FIGS. 5A and 5C). Double immunostaining for CD68 and CD11c clearly revealed that more than half of macrophages were CD68⁺CD11c⁺, indicating the myeloid origin of this cell type in lesions as CD11c⁺ area co-localized with CD68⁺ area expressed as percentage showing 54.7±3.7%, 55.2±2.6% and 50.3±3.3% for AHHC, RHHC and RPHC, respectively compared to 68.6±4.7% in control mice (FIGS. 5A-5D; P=0.046-0.011). The proportion of CD4⁺ cells expressing Foxp3 analyzed by IHC staining of aorta sections was approximately 6- to 8-fold higher (8.2±1.4%, P<0.001; 9.4±1.1%, P<0.001; 9.9±1.6%, P<0.001) in mice immunized with AHHC RHHC and RPHC, respectively compared to 1.2±0.2% in control mice (P<0.001) (FIGS. 5E and 5F). In addition, expression of Foxp3 in CD4⁺ spleen cells from mice immunized with these three constructs was higher (P<0.001) than that in controls, showing 13.3±0.6%, 15.3±1.5%, and 18.0±1.1%, for AHHC, RHHC and RPHC respectively versus 4.0±0.5% in controls (FIGS. 5G and 5H). With the exception of RPHC-immunized mice, which did not show a significant increase of Foxp3 expression when compared with that in RHHC-immunized mice, however, significantly higher levels of Foxp3 expression were observed in 30 comparison with that in AHHC-immunized mice (P=0.002).

Example 4

The expression of anti-inflammatory cytokines and proinflammatory cytokines in lesion sites and levels of cytokines in plasma and in the supernatants of stimulated splenocytes was assessed. IL-10 expression in the aortic lesions of mice immunized with AHHC, RHHC and RPHC, detected by IHC analyses 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, showing 4.8±0.7%, 5.2±0.8% and 5.9±0.7% (P<0.001) for AHHC, RHHC and RPHC, respectively when compared with that in the control group (0.9±0.2%) as shown in FIGS. 6A and 6B. IHC analyses of TNF-α expression showed significantly smaller TNF-α-occupied areas in lesions of mice immunized with constructs compared with controls (25.7±3.1% for AHHC; 15.2±0.9% for RHHC; 15.5±1.5% for RPHC and 41.3±3.1% for controls) (FIGS. 6C and 6D). These data represent a percent reduction of 37.8%, 63.2%, and 62.5%, respectively, compared with controls (total lesion area defined as 100%, and 0% reduction). Additionally, enhanced reduction was produced by either RHHC or RPHC versus AHHC (P=0.008-0.014).

Plasma levels of atheroprotective cytokines IL-10 were significantly increased in mice immunized with these three constructs compared with controls (FIGS. 6E and 6F). Immunization with RPHC had more effect than with other two constructs on promoting the secretion of IL-10 and TGF-β (P<0.05 to <0.001). Plasma levels of the atherogenic cytokine TNF-α were significantly reduced by immunization with these three constructs (FIG. 6G). A similar trend was obtained for these constructs in respect of plasma levels of IFN-γ (FIG. 6H). Notably, immunization with RPHC had more effect than with AHHC on reducing the secretion of TNF-α and IFN-γ (P<0.002) and more effect than with RHHC on reducing the secretion of TNF-α (P=0.004). Although slightly higher plasma levels of IFN-γ from mice immunized with AHHC was observed compared to that of control mice (24 versus 20.8 pg/ml), this difference did not show statistical significance.

Supernatants of splenocytes from mice immunized with these constructs individually showed significantly increased secretion of IL-10 (FIG. 6I) and TGF-β (FIG. 6J), stimulated with 10 μg/ml of ConA (P<0.05-0.001) when compared to those of controls. In addition, higher levels of either IL-10 or TGF-β were produced by splenocytes of RPHC-immunized mice than those of AHHC-immunized mice (P<0.05-0.01). In contrast, TNF-α (FIG. 4K) and IFN-γ levels (FIG. 6L) were significantly decreased in the supernatants of splenocytes of mice immunized with these constructs compared with the control when stimulated with 10 μg/ml of ConA. Notably, levels of TNF-α and IFN-γ in RPHC-immunized mice were lower than those in AHHC-immunized mice (P<0.05-0.01) when stimulated with 10 μg/ml of ConA, respectively. Interestingly, supernatants of splenocytes from mice immunized were also shown to contain increased amount of protective cytokine IL-10 and decreased amount of proinflammatory cytokine IFN-γ when stimulated with either peptides or construct containing peptides but not in the case when stimulation was done with KLH, a different protein (FIGS. 7A-D). In most cases, the changes in cytokine productions in response to different stimulators were significant except for the cases when ApoB, C5aR and Cpn peptides were used as stimulators for the production of IL-10 in GST-den-immunized mice, and PAR-1 peptide in RPHC-immunized mice.

The proportion of IL-4 (Th2-related), IL-17A (Th17 related) and IL-2 (Th1-related) expressing CD4⁺ spleen cells from mice immunized with these three constructs was significantly lower (P<0.001 for IL-4⁺ and P≤; 0.001 for IL-17A⁺, respectively), showing 2.74±0.11% (AHHC), 2.S5±0.12% (RHHC), and 2.S7±0.02% (RPHC) for IL-4+ when compared to 8.44±0.21% (control) (FIGS. 4M and 4N); 2.0±0.1% (AHHC), 1.7±0.1% (RHHC), and 0.9±0.1% (RPHC) for IL-17A⁺ when compared to 4.1±0.3% (control) (FIGS. 6O and 6P). In addition, smaller percentage of CD4⁺IL-17A⁺ expressing spleen cells was observed in RPHC-immunized mice compared to that in either AHHC- or RHHC-immunized mice (P≤; 0.006) (FIGS. 6O-P). Interestingly, higher percentage of CD4⁺IL-17A⁺ expressing spleen cells was observed in RHHC-immunized mice compared to that in RPHC-immunized mice (P=0.021) (FIG. 6P). Furthermore, significantly lower percentage of CD4⁺IL-2⁺ expressing spleen cells was observed in three construct-immunized mice when compared 15 to that in control (P<0.001; FIGS. 6Q and 6R). Additionally smaller percentage of CD4⁺IL-2⁺ expressing spleen cells was observed in RPHC-immunized mice compared to that in either AHHC- or RHHC-immunized mice (P=0.019-0.007).

Example 5

Antigen-induced specific Treg cell function was investigated. To assess whether 20 functional Treg cells were induced by immunization, antigen-specific Treg cells (CD4⁺CD25⁺ T cells) were co-cultured with CD4⁺ effector T-cells (CD4⁺CD25⁻ T cells). Proliferation of effector T-cells from control mice immunized with GST-Den in response to stimulation with GST-Den at 1 μM did not show suppression in the presence of Treg cells from GST-Den-immunized mice (FIGS. 8A and 8B). In contrast, proliferation of effector T cells from sampling mice immunized with AHHC, RHHC and RPHC in response to stimulation with related antigen respectively was inhibited (FIGS. 8A and 8B), when CD4⁺CD25⁻ effector T cells were co-cultured with CD4⁺CD25⁺ Treg cells isolated from these mice. The differences were significant when Treg cells were added to the effector cells at the ratios between 4:1˜16:1 (P<0.05˜<0.001) compared with that without the addition of Treg cells.

Example 6

An evaluation of expression of smooth muscle alpha actins, VCAM 1, WP9 and specific antigens Apo8 and HSP60 in the lesions was undertaken. To assess whether immunization with the constructs of the present invention influences vascular SMC behavior and vascular remodeling, the SMC content of lesions and expression of VCAM1 and WP9 at lesion sites was analysed by IHC analyses. Anti-SMC stained area was significantly smaller in the plaques of mice immunized with AHHC and RPHC, showing 5.2±0.7% and 4.9±0.8%, respectively, but it was not reduced significantly in mice immunized with RHHC when compared with that in controls immunized with GST-Oen (FIGS. 9A and 98). In addition, expression of VCAM1 was significantly down-regulated, showing 4.5±0.9%, 7.8±5 0.9%, and 4.8±0.9%, for AHHC, RHHC and RPHC, respectively, compared with that in controls immunized with dendroaspin (18.0±2.3%) (FIGS. 9A and 9C). A significantly increased effect was found in RHHC-immunized mice compared to that in AHHC- or RPHCimmunized mice (FIGS. 9A and 9C). A similar trend was observed for MMP9 expression in mice immunized with all three constructs 7.9±1.0%, 10.1±1.0%, and 7.6±1.0% stained 10 areas were shown in RHHC- and RPHC-immunized mice, respectively, and 18.1±2.5% in control mice immunized with GST-Den (FIGS. 9D and 9E), except for that there is no significant difference obtained in this respect between either AHHC- or RPHC-immunized mice and RHHC-immunized mice (FIGS. 6D and 6E). Interestingly, the injection of recombinant construct containing human ApoB and HSP60 peptides did not affect the expression of their counterparts (ApoB and HSP60) as little difference of mouse ApoB (FIGS. 10A and 108) and mouse HSP60 protein (FIGS. 10C and 10D) antigens was detected at the lesion sites between sampling and control mice.

Example 7

Evaluation of monocyte differentiation into macrophages in PBMC from C57BL/6 background naive mice in response to treatment with recombinant constructs and the effect of construct-specific immune sera on the differentiation was investigated. In vitro, monocytes can differentiate into macrophage (or subsets) upon stimulation with macrophage-colony stimulating factor (M-CSF) or atherogenic antigens. To assess whether AHHC converted into RHHC with a single domain substitution could maintain the same effect on stimulation of monocyte (from naive mice C57BL/6 with same background), PBMCs were stimulated with either RHHC or AHHC. After 3 days, the expression of cell surface marker CD206 (mannose receptor, a macrophage marker) was assessed. Both RHHC- and AHHCinduced monocyte differentiation into macrophages (based on the cell number changes) when compared with non-stimulated cells (FIGS. 11A and 11B). In addition, PBMC differentiation induced by these two constructs was abolished by pre-incubation of the cells with antiserum from mice immunized with these two antigens. Interestingly, the inhibition can be achieved by pre-incubation of the cells with antiserum from each other (FIGS. 11A-11D). Observation of differentiations with individual epitope or domain as a stimulator showed different ratio of differentiation (FIGS. 11E-11F).

Example 8

Evaluation of the contents of Toll-like receptor 4(TLR4) and myeloid differentiation factor 88 (MyD88) which are involved in the TLR4 signal pathway related to atherosclerosis at the lesion sites was investigated. The impact of the treatment with these recombinant constructs on TLR4 and MyD88 contents in lesions was examined. The reduction of atherosclerosis in mice treated with these constructs was associated with a decrease in both TLR4 and MyD88 contents. Anti-TLR4 stained area was significantly smaller in the plaques of mice immunized with AHHC, RHHC and RPHC, showing 4.6±1.0%, 3.4±0.5% and 4.2±0.6%, respectively, compared with that in controls immunized with dendroaspin (8.1±1.1%) (FIGS. 12A and 12B). Similarly, anti-MyD88 stained area was significantly smaller 10 in the lesions of mice immunized with these three constructs, showing 9.7±1.2%, 9.6±1.6% and 10.2±1.9%, respectively, compared with that in controls (18.6±2.4%) (FIGS. 12C and 12D). Additionally, overlapping anti-CD11c and anti-TLR4 stained area showed significantly smaller in the lesions of mice immunized with all three constructs, showing 3.4±0.6%, 2.9±0.7% and 2.7±0.8%, respectively, compared with that in controls (6.6±0.8%) (FIGS. 13A and 13B).

Example 9

Experiments were conducted to compare two HSP60 peptides derived from human and mycobacterium, respectively, for their ability to reduce atherosclerotic lesions through immunization in Apob^tm2sgyLdlr^tm1Her/J Mice. Mice were immunized with two Keyhole limpet hemocyanin (KLH)-conjugated peptides derived from mycobacterial heat shock protein (HSP) 60 (AA253-268) (SEQ ID NO:15) designated as mHSP60^253-268, human HSP60 (AA516-528) (SEQ ID NO:14) designated as hHSP60^516-528, respectively. Mice were immunized with these two peptides and two weeks after the first immunization, mice were placed on a high-fat diet. Results indicated that the two peptides showed similar functions apart from that mHSP60 peptide has lower titres for IgG and IgG1 and little for IgG2c, than those of hHSP60 peptide. Similar functions include: induced specific immune responses; lesion reduction; increased Treg expression; increased concentration of atheroprotective cytokines: IL-10 and TGF-β and decreased concentration of pro-inflammatory cytokines: TNF-α and INF-γ; inhibition of CD4⁺CD25⁻ T-cell proliferation by Treg cells and down-regulation of TLR4/MyD88 pathway (data not shown). In conclusion, after immunization of 86; 129S-Ldlr^tm1HerApob^tm2SgyJ mice with mHSP60 and hHSP60 peptides, in spite of a low sequence homology (31%) between two peptides and lower immune responses obtained from mHSP60 peptide, both peptides have similar effects on significantly reduced early atherosclerotic lesions. This data confirms that such immunization with the constructs of the present invention offers attractive opportunities for the design and development of peptide-based vaccines against atherosclerosis.

Example 10

The transcriptional regulator FOXP3 (forkhead box P3) governs 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 it has been shown that transfer of natural CD4⁺CD25⁺ Tregs significantly reduces plaque progression in the ApoE-KO mouse model (Ait-Oufella et al., Nat Med. 2006; 12:178-180; Mor et al Arteriosc/er Thromb Vase Biol. 2007; 27: 893-900). Accordingly it is known that naturally occurring Tregs are capable of influencing the size and composition of atherosclerotic lesions, several reports support the theory that antigen-specific responses may be operable in the evolving atheromatous plaque. We hypothesized that atherogenic antigen-induced Treg may have a specific function on lesion reduction. We conducted experiments to assess the effect of adoptively transferred Treg cells isolated from the blood of antigen-immunized mice on atherosclerotic lesion formation in B6; 129S-Ldlr^tm1HerApob^tm2Sgy/J mice by testing humeral immune response, the effect on atherosclerotic lesion size and local and systemic cellular responses.

The first method, to look at the immune response and adoptive transfer in KO mice, involved incorporating construct AH^hH^mR into a dendroaspin scaffold to generate a recombinant construct. The antigenic epitopes from ApoB (AA688-707) was designated as A, human HSP60 (AA303-312) (SEQ ID NO: 12) designated as H^h, mycobacterium (AA253-268) (SEQ ID NO:15) designated as H^mand complement component 5a receptor (AA1-31) (SEQ ID NO:9) designated as R. Mice were immunized with AH^hH^mR by RIMM (repetitive, multiple site immunization strategy) protocol. Treg cells were purified from the blood of immunized mice with AH^hH^mR (Treg^S) and with dendroaspin (Tree) respectively). Adoptive transfer was achieved though retro-orbital plexus of the mice.

The second method, to evaluate the effect of Treg cells on atherosclerotic lesion formation, involved adoptive transfer of Treg cells from the blood of AGD-den (Control)- and AH^hH^mR-immunized mice, respectively. Recipients were from the same strain nonimmunized naive mice and were fed with a high-fat diet (HFD) for 10 weeks then sacrificed. Histological and immunohistochemical assessment of lesion development, analysis of cytokine level, assessment of Treg activity and foam cell formation were evaluated.

Data (not shown) indicated that Treg^Sisolated from the blood of atherogenic anti-immunized mice, after adoptively being transferred into the vein of a non-immunized mice when compared to Treg from the blood of non-atherogenic antigen-immunized mice showed lesser lesion formation. Transfer of natural CD4⁺CD25⁺ Tregs significantly reduced plaque progression in the ApoE-KO mouse model. In addition to lesser lesion formation they also showed increased collagen content in lesion sites, increased Treg expression in lesion sites, higher concentration of atheroprotective cytokines (IL-10 and TGF-β) and lower concentration of pro-inflammatory cytokines (TNF-α and INF-γ) in plasma. Down-regulation of expression of αSMC and PECAM at the lesion sites was also observed.

These results show that the constructs of the present invention offer attractive s opportunities in the cell-based therapy for the treatment of atherosclerosis.

Number	Date	Country	Kind
1501354.3	Jan 2015	GB	national
1510195.9	Jun 2015	GB	national

	Number	Date	Country
Parent	15546858	Jul 2017	US
Child	17693785		US

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