Long-Term Efficacy and Safety of Immunomodulatory Therapy for Atherosclerosis.

BACKGROUND AND AIMS: The long-term effect of immune tolerance has not been explored so far in atherosclerosis. In the present study, we assessed the effect of mucosal tolerance to a multi antigenic construct expressing three peptides from ApoB, HSP60, and outer membrane protein from Chlamydia pneumonia (AHC) for 30 weeks at every 6-week interval to understand the kinetics of immune modulation in disease progression. The safety profile of the molecule was also evaluated in mice. METHODS: Apobtm2SgyLdlrtm1Her/J mice (5-6 weeks) were orally dosed with multi antigenic construct (AHC) molecule on alternate days, followed by high-fat diet feeding to initiate atherosclerosis. RESULTS: Treated animals showed an efficient reduction in plaque growth and lipid accumulation at 6 weeks (49%, p < 0.01) and 12 weeks (42.3%, p < 0.01) which decreased to 29% (p = 0.0001) at 18 weeks and at later time points. Macrophage accumulation was significantly lower at all time points (53% at 12 weeks to 27% at 30 weeks). Regulatory T cells increased in the spleen following treatment until 12 weeks (week 0 (2.57 ± 0.18 vs. 6.36 ± 0.03, p = 0.02), week 6 (4.52 ± 0.2 vs. 8.87 ± 0.32, p = 0.02), and week 12 (8.74 ± 0.37 vs. 15.4 ± 0.27, p = 0.02)) but showed a decline later. A similar trend was observed with tolerogenic dendritic cells. We observed an increase in antibody levels to low-density lipoprotein and oxidized LDL at later stages. AHC molecule was found to be safe in acute and repeated dose toxicity studies. CONCLUSIONS: Our results suggest that immune tolerance to AHC protein by oral administration is able to provide efficient atheroprotection up to 18 weeks and moderately at later stages. Apart from immune regulatory cells, protective antibodies may also have a role in controlling atherosclerosis.

Identification of apoB-100 Peptide-Specific CD8+ T Cells in Atherosclerosis.

BACKGROUND: T cells are found in atherosclerotic plaques, with evidence supporting a potential role for CD8+ T cells in atherogenesis. Prior studies provide evidence of low-density lipoprotein and apoB-100 reactive T cells, yet specific epitopes relevant to the disease remain to be defined. The current study was undertaken to identify and characterize endogenous, antigen-specific CD8+ T cells in atherosclerosis. METHODS AND RESULTS: A peptide fragment of apoB-100 that tested positive for binding to the mouse MHC-I allele H2Kb was used to generate a fluorescent-labeled H2Kb pentamer and tested in apoE-/- mice. H2Kb pentamer(+)CD8+ T cells were higher in apoE-/- mice fed an atherogenic diet compared with those fed a normal chow. H2Kb pentamer (+)CD8+ T cells in atherogenic diet-fed mice had significantly increased effector memory phenotype with a shift in Vß profile. H2Kb pentamer blocked lytic activity of CD8+ T cells from atherogenic diet-fed mice. Immunization of age-matched apoE-/- mice with the apoB-100 peptide altered the immune-dominant epitope of CD8+ T cells and reduced atherosclerosis. CONCLUSIONS: Our study provides evidence of a self-reactive, antigen-specific CD8+ T-cell population in apoE-/- mice. Immune modulation using the peptide antigen reduced atherosclerosis in apoE-/- mice.

Immune regulation by oral tolerance induces alternate activation of macrophages and reduces markers of plaque destabilization in Apobtm2Sgy/Ldlrtm1Her/J mice.

Atherosclerosis is the leading cause for cardiovascular mortality. We determined the effect of multi-antigenic construct expressing three peptides AHC (ApoB100, HSP60 and outer membrane protein of chlamydia pneumonia) in stabilizing advanced atherosclerosis in Apobtm2Sgy/Ldlrtm1Her/J mice. Atherosclerosis was induced by feeding high fat diet (HFD) to mice for 10 weeks, followed by five oral dosing with purified AHC or ovalbumin on alternate days and continued on HFD for another 10 weeks. Tolerance was associated with significantly higher numbers of regulatory T cells both in aortic sinus and spleen with higher mRNA expression of CTLA4 (3 fold), Foxp3 (1.4 folds) and TGF-ß (1.62) in aorta. Tregs cells were found to induce alternate activation of macrophages to M2 phenotype, with a reduction in plaque inflammation. AHC treatment showed evidence of plaque stabilization as observed by reduction in plaque necrosis in aortic sinus (35.8%) and in brachiocephalic artery (26%), with reduced expression of Tissue factor and MMP9. Macrophage apoptosis was reduced and collagen content was enhanced by treatment. Our results suggest that tolerance to atherogenic peptides increases regulatory T cells which activate M2 macrophages, prevent T cell proliferation and reduce plaque destabilization and inflammatory markers thus providing evidences for plaque stabilization in mice with advanced atherosclerosis.

2015 Russell Ross Memorial Lecture in Vascular Biology: Protective Autoimmunity in Atherosclerosis.

Atherosclerosis is an inflammatory disease of the arterial wall. It is accompanied by an autoimmune response against apolipoprotein B-100, the core protein of low-density lipoprotein, which manifests as CD4 T cell and antibody responses. To assess the role of the autoimmune response in atherosclerosis, the nature of the CD4 T cell response against apolipoprotein B-100 was studied with and without vaccination with major histocompatibility complex-II-restricted apolipoprotein B-100 peptides. The immunologic basis of autoimmunity in atherosclerosis is discussed in the framework of theories of adaptive immunity. Older vaccination approaches are also discussed. Vaccinating Apoe(-/-) mice with major histocompatibility complex-II-restricted apolipoprotein B-100 peptides reduces atheroma burden in the aorta by ≈40%. The protective mechanism likely includes secretion of interleukin-10. Protective autoimmunity limits atherosclerosis in mice and suggests potential for developing preventative and therapeutic vaccines for humans.

Immune responses elicited by apoB-100-derived peptides in mice.

Peptides derived from apolipoprotein B (apoB)-100 have been previously used in vaccine preparations to treat atherosclerosis. Such vaccines have been shown to reduce atherosclerotic plaque development by 50 % in experimental animals, and this effect is associated with induction of T helper (Th)2 immune responses. In this study we immunised apolipoprotein E-deficient (apoE(-/-)) mice with apoB-100-derived peptides P2, P45 and P210. Animals received BSA-conjugated peptides or peptide-loaded bone marrow-derived dendritic cells (DCs). We used enzyme-linked immunosorbent assays to assess the synthesis of anti-peptide-specific IgG1 and IgG2a as well as the levels of interleukin (IL-)10 and interferon gamma (IFN-γ) in plasma of immunised animals. We also measured the effect of immunisation on the number of spleen-derived CD4(+) and CD8(+) regulatory T cells (Tregs) in these animals. Peptide and peptide-loaded DC immunisation significantly increased the levels of peptide-specific immunoglobulins and the number of Tregs in apoE(-/-) mice. This was accompanied by a significant increase in the secretion of IL-10 with no effect on IFN-γ levels. The results also show that the peptides can modulate the homing properties of DCs. Altogether, this study provides novel evidence for the immune mechanisms excerpted by apoB-100-derived peptides and their effect on Tregs and DCs relevant to their use in vaccine preparations.

Regulatory T-cell response to apolipoprotein B100-derived peptides reduces the development and progression of atherosclerosis in mice.

OBJECTIVE: The immunoinflammatory response plays a critical role in the development and progression of atherosclerosis. Recent studies suggested an important role for regulatory T (Treg) cells in the inhibition of disease-related vascular inflammation. We hypothesized that induction of a specific Treg cell response to atherosclerosis-relevant antigens would be an attractive strategy to limit the development and progression of atherosclerosis through the promotion of immune tolerance. METHODS AND RESULTS: Young or old Apoe-/- mice were subcutaneously infused for 2 weeks with either a control ovalbumin (OVA) peptide or with apolipoprotein B100 (ApoB100)-derived peptides without adjuvant. Atherosclerosis development, progression and immunologic status were assessed at 8 weeks after the end of the infusion. Treatment with ApoB100 peptides led to significant reduction of lesion development in young Apoe-/- mice (P=0.001 versus OVA group) and abrogated atherosclerosis progression in old Apoe-/- mice with already established lesions (0% progression in ApoB100 versus 17% in OVA group, P<0.005). Limitation of plaque progression was associated with reduced vascular inflammation and increased collagen content, indicative of plaque stabilization. Infusion of ApoB100 peptides did not alterantibody production but promoted a specific Treg cell response, which was associated with a reduction of both T helper type 1-related and T helper type 2-related cytokines. Interestingly, depletion of CD4+CD25+ Treg cells abrogated ApoB100 peptides-dependent immune modulation and atheroprotection. CONCLUSION: Subcutaneous infusion of adjuvant-free ApoB100-derived peptides to Apoe-/- mice reduces atherosclerosis through the induction of a specific Treg cell response.

Intranasal immunization with an apolipoprotein B-100 fusion protein induces antigen-specific regulatory T cells and reduces atherosclerosis.

OBJECTIVE: Atherosclerosis is an inflammatory disease. Autoimmune responses to low-density lipoproteins (LDL) contribute to its progression, whereas immunization with LDL may induce atheroprotective or proatherogenic responses. The objective of this study was to develop an atheroprotective vaccine by targeting a peptide of the LDL protein constituent apolipoprotein B-100 (apoB-100) to the nasal mucosa to induce a protective mucosal immune response. METHODS AND RESULTS: A peptide comprising amino acids 3136 to 3155 of apoB-100 (p210) was fused to the B subunit of cholera toxin (CTB), which binds to a ganglioside on mucosal epithelia. The effect of nasal administration of the p210-CTB fusion protein on atherogenesis was compared with that of an ovalbumin peptide fused to CTB and with untreated controls. Immunization with p210-CTB for 12 weeks caused a 35% reduction in aortic lesion size in Apoe(-/-) mice. This effect was accompanied by induction of regulatory T cells that markedly suppressed effector T cells rechallenged with apoB-100 and increased numbers of interleukin (IL)-10(+) CD4(+) T cells. Furthermore, a peptide-specific antibody response was observed. Atheroprotection was also documented in apoe(-/-) mice lacking functional transforming growth factor-beta receptors on T cells. CONCLUSION: Nasal administration of an apoB-100 peptide fused to CTB attenuates atherosclerosis and induces regulatory Tr1 cells that inhibit T effector responses to apoB-100.