Advances in immune-modulating therapies to treat atherosclerotic cardiovascular diseases

Abstract

In addition to hypercholesterolemia, innate and adaptive immune mechanisms play a critical role in atherogenesis, thus making immune-modulation therapy a potentially attractive way of managing atherosclerotic cardiovascular disease. These immune-modulation strategies include both active and passive immunization and confer beneficial reduction in atherosclerosis. Preclinical studies have demonstrated promising results and we review current knowledge on the complex role of the immune system and the potential for immunization as an immune-modulation therapy for atherosclerosis.

Keywords

atherosclerosis immune response immunization

Introduction

Atherosclerotic plaques contain features of an innate and adaptive immune system, such as dendritic cells (DCs), B and T lymphocytes, macrophages, Toll-like receptors, immunoglobulins, inflammatory cytokines and complements. Interested readers can refer to the listed references for an in-depth discussion on the complex role of these immune components in atherogenesis [Andersson et al. 2010; Chyu et al. 2011; Hartvigsen et al. 2009; Lundberg and Hansson, 2010; Yan and Hansson, 2007].

Many reports have described the possibility of active or passive immune-modulation strategy to modulate atherosclerosis. In the English literature, based on a PubMed search using ‘immunization’ and ‘atherosclerosis’ as keywords, the first such possibility was raised by Gero and colleagues in 1959 [Gero et al. 1959]. However, only in the past decade have we seen an explosive increase in the amount of literature published on this subject. This change is most likely due to our better understanding of the role of immune system in atherosclerosis and advances in research tools available to scientists. Some parts of this article were already reported in our previous review [Chyu and Shah, 2013] and are represented here for completeness of this article.

Immunization against atherosclerosis

Immunization, in a classical immunological paradigm, can be classified as active or passive. Active immunization relies on the delivery of antigens in a vaccine platform to induce humoral and cellular immunity against the offending agents in the host. This would involve activation of many arms of the immune system, such as B and T cells, to generate effector cells such as antibody-producing B cells or cytotoxic CD8⁺ T cells. Passive immunization consists of administration of a preformed antibody such as various antitoxins or immunoglobulin against infectious agents to achieves its effect against offending agents.

Search for antigens involved in atherosclerosis

No matter which immunization approach is used for disease prevention or treatment, the most important element of the immunization approach is to know which antigens are responsible for the disease. Many antigens have been implicated and reported to be associated with atherogenesis. These include endogenous antigens such as low-density lipoprotein (LDL) or its related protein apolipoprotein B-100 (apoB-100) [Ameli et al. 1996; Fredrikson et al. 2003b, 2005; Freigang et al. 1998; Palinski et al. 1995], oxidized phospholipid and its phosphorylcholine (PC) head group [Binder et al. 2003, 2004; Binder, 2010; Chou et al. 2009], heat shock protein 65 (HSP65) [Kilic and Mandal, 2012; Mandal et al. 2004, 2005; Xu et al. 2012] and β2 glycoprotein I (β2-GPI) [George et al. 2000a, 2000b; Matsuura et al. 2005; Shoenfeld et al. 2001]. Exogenous infectious agents such as Chlamydia pneumoniae, periodontal pathogens or cytomegalovirus were also reported to play some role in atherogenesis [Armitage, 2000; Ausiello et al. 2005; Beck et al. 1999; Betjes et al. 2007; Buhlin et al. 2003; Halme et al. 1997; Mayr et al. 2000; Movahed, 1999; Olofsson et al. 2005; Zelkha et al. 2010]. The establishment of these molecules as potential antigens in atherogenesis is usually based on the presence of humoral or cellular immune responses against such molecules in animal or human studies and their presence in atherosclerotic plaques.

Circulating antibodies against low-density lipoprotein in humans

Circulating antibodies against LDL (or its oxidized forms, oxLDL) exist in humans. In a prospective study of 30 men with accelerated progression of carotid atherosclerosis in 2 years, higher autoantibodies to malondialdehyde-modified (MDA) LDL were found compared with age-matched controls [Salonen et al. 1992]. In a case control study, patients with early onset of atherosclerotic peripheral vascular disease had higher levels of autoantibodies against oxLDL in addition to higher levels of total cholesterol, LDL and triglycerides [Bergmark et al. 1995]. Another study revealed a higher level of autoantibodies against copper oxLDL in patients with established coronary artery disease compared with controls without disease [Lehtimaki et al. 1999].

The clinical utility and significance of these antibodies is not clearly known. In an observational study with asymptomatic patients from the Atherosclerosis Risk in Communities study cohort, there was no correlation between MDA-LDL autoantibody and intima-media thickness (IMT) [Iribarren et al. 1997]. However, Fukumoto and colleagues found a modest inverse relationship between oxLDL antibody titers and carotid IMT in healthy Japanese people [Fukumoto et al. 2000]. In another study examining patients with a diagnosis of myocardial infarction, ischemia or undergoing coronary revascularization procedures, there was no relationship between the antibody titer and risk of death and cardiovascular disease events [Erkkila et al. 2005].

Taking these together, these data suggest the presence of autoantibodies against LDL is likely a marker for the presence of disease, indicating an activation of immune response against oxLDL.

Passive immunization

Passive immunization using immunoglobulin can largely be divided into an antigen specific or nonspecific approach. Intravenous administration of immunoglobulin (IVIG) is an example of an antigen nonspecific approach. IVIG has been used in clinical practice to treat immune-mediated diseases such as autoimmune diseases or transplant rejection. Since atherosclerosis is viewed as an autoimmune disease of arterial wall, proof-of-concept preclinical studies testing the efficacy of using polyclonal immunoglobulin to reduce atherosclerosis have reported promising results [Nicoletti et al. 1998; Yuan et al. 2003]. This atheroprotection has been attributed to the ability of IVIG to modulate T-cell activities or antibody production [Nicoletti et al. 1998] and a complement system is required for its full effect [Persson et al. 2005]. Our laboratory recently reported the use of a polyclonal immunoglobulin M (IgM) in a different model of atherosclerosis. We observed that treatment with polyclonal IgM weekly for 4 weeks reduced existing spontaneous and accelerated atherosclerotic lesions induced by periarterial cuff injury in the carotid artery [Cesena et al. 2012]. This protective effect was associated with downregulation of CD4⁺ T-cell activity and enhancement of oxLDL IgG titers [Cesena et al. 2012]. Taking these together, polyclonal antibodies likely exert their atheroprotective function by modifying the immune system globally, which could also be a potential weakness of this approach.

Passive immunization using monoclonal antibodies against specific antigens has also been reported to confer atheroprotective effects. Monoclonal IgG preparations reactive to cardiolipin or LDL significantly reduced atherosclerotic lesion formation in LDL receptor (–/–) (LDLR(–/–)) mice [Grosso et al. 2008; Nicolo et al. 2003]. Another monoclonal IgG2b antibody against electronegative LDL was also reported to decrease atherosclerosis in LDLR(–/–) mice, possibly by reducing the circulating level of free electronegative LDL and cholesterol oxides in the plasma and liver [Grosso et al. 2008]. Whether these monoclonal antibodies modulate humoral or cellular immune response against atherosclerosis as a potential mechanism of action was not elucidated. Although these antibodies were monoclonal, the exact antigenic epitope in cardiolipin or LDL that these antibodies recognize are not known.

Recently a recombinant human IgG1 antibody against an MDA-modified apoB-100 derived peptide antigen, named p45, was shown to reduced/regress atherosclerosis in animal models [Schiopu et al. 2004, 2007]. This antibody proved to be safe in a phase I human trial and showed anti-inflammatory activity in a primate model [Li et al. 2013]. However, a subsequent phase IIa clinical study, which used an inflammatory index in the arterial wall measured by 18-fluorodeoxyglucose positron emission tomography/computed tomography imaging as an endpoint, failed to achieve the prespecified endpoint (see http://www.news-medical.net/news/20120711/BioInvent-announces-data-from-BI-204-phase-IIa-study-on-stable-atherosclerotic-vascular-disease.aspx). However, there are considerable limitations in the imaging methodology that was used in this study, making it difficult to rule out the possibility that an inappropriate study design may have contributed to the negative results.

IgM antibody preparation was also effective in conferring atheroprotective effects. Natural antibodies, produced by B1 cells, are primarily of the IgM subtype and their production does not require antigen exposure. One such natural IgM antibodies, discovered by Dr Witztum’s group, is EO6, which targets oxidized lipid products, the PC headgroup in phospholipids and apoptotic cells [Binder, 2012]. EO6 is of the T15 idiotype and a monoclonal preparation of IgM against the PC headgroup with T15 idiotype has been shown to reduce accelerated vein graft atherosclerosis [Faria-neto et al. 2006]. Recently another monoclonal IgM antibody with specificity for hypochlorite oxLDL was produced and found to be a natural IgM. Passive immunization with this preparation led to a reduction of pericarotid cuff induced atherosclerotic lesions in LDLR(–/–) mice [van Leeuwen et al. 2013]. The polyclonal IgM preparation also reduced aortic atherosclerotic lesions with concomitant modulation of CD4⁺ T-cell response [Cesena et al. 2012]

Although these studies suggest the feasibility of passive immunization to modulate atherosclerosis, human application still awaits confirmatory clinical studies. Even with proven clinical efficacy in the future, its clinical application could still be limited due to the following factors: passive immunization lacks memory effect; for a long-term effect, it may require frequent administration of antibody which could be costly; cost and safety of producing such antibody; safety of such an antibody due to frequent administration and many immunosuppressive properties associated with polyclonal immunoglobulin; and lack of knowledge of antigenic epitopes in many of these antibody preparations may hinder the improvement of this strategy.

Active immunization with antigens related to low-density lipoprotein

In the mid 1990s, several publications reported that active immunization using native modified LDL as an antigen reduced atherosclerosis in hypercholesterolemic rabbits [Ameli et al. 1996; Nilsson et al. 1997; Palinski et al. 1995] (Table 1). Several other groups of investigators subsequently reported similar atheroprotective effects by active immunization with cholesterol-rich liposomes in cholesterol-fed rabbits [Alving et al. 1996], MDA-modified LDL in apoE(–/–) mice [George et al. 1998b] or with plaque homogenates or MDA-modified LDL in apoE(–/–) mice [Zhou et al. 2001].

Table 1.

A summary of antigens that have been tested in preclinical active immunization studies and their effects on atherosclerosis.

Antigens	Effects on atherosclerosis
LDL and its modified form	Reduction
apoB-100 peptides	Reduction
Phosphorylcholine on oxidized phospholipid	Reduction
TRVB31 peptide	Reduction
Streptococcus pneumonia	Reduction
CD99 or VEGFR2	Reduction
Cholesteryl ester transfer protein	Reduction
β2 glycoprotein I	Promotion
Heat shock protein	Variable

apoB, apolipoprotein B; LDL, low-density lipoprotein.

LDL is not an ideal antigen because of its large size and its heterogeneous molecular contents including apolipoprotein, cholesteryl ester, triglyceride and phospholipids. This complex molecular structure makes it difficult to determine the exact immunogenic epitopes within the LDL molecule. In the early 2000s, our laboratory in collaboration with Dr Jan Nilsson’s laboratory in Sweden launched an extensive effort to identify potential antigenic epitopes within the protein component of LDL, apoB-100, that could be used as antigens to achieve atheroprotective effects via immunization. We first generated a library of 302 peptides (20 amino acids long with a 5-amino-acid overlap with the preceding sequence) using sequences from the human apoB-100 molecule and identified 102 peptides that can be recognized by antibodies in pooled human plasma from healthy control subjects [Fredrikson et al. 2003a]. Active immunization with vaccine formulations using several such peptides, such as p2, p143 and p210, as antigens resulted in a 40–70% decrease in atherosclerosis and reduction in plaque inflammation in hypercholesterolemic mice [Chyu et al. 2005; Fredrikson et al. 2003b].

After successful demonstration of the ability of specific apoB-100 peptides as antigens in vaccine formulation to reduce atherosclerosis, we and other investigators continued to investigate the humoral and cellular mechanisms responsible for the atheroprotective effect of immunization using such peptides. Most of the mechanistic work used p210 as the model antigen in the vaccine formulation.

Humoral immunity was initially speculated to mediate the protective effect of active immunization. However, the atheroprotection of immunization using apoB peptide as antigen occurred without an increase in peptide-specific IgG [Fredrikson et al. 2008]. Klingenberg and colleagues reported that intranasal immunization with p210 elicited IgG titers against p210 but the titers were not correlated with the lesion size [Klingenberg et al. 2010]. We also reported that mice immunized with p210 vaccine developed smaller aortic atherosclerotic lesions with lower titer of circulating p210 IgG compared with mice in control groups. Additionally, adoptively transferring B cells from p210 immunized mice to nonimmunized mice did not convey an atheroprotective effect [Chyu et al. 2012]. Hence the existing evidence does not support humoral immunity as an important mediator for the atheroprotective effect of active immunization with p210.

The focus of mechanistic work then shifted to cellular immunity. Immunizing apoE(–/–) mice with p210 could activate CD8⁺ T cells with concomitant reduction of DCs at the site of immunization and within the plaques, decrease macrophage immunoreactivity in plaques and significantly reduce aortic atherosclerosis compared with controls. The atheroprotective effect of active immunization could be recapitulated by adoptive transfer of CD8⁺ T cells from p210-immunized mice into naïve, nonimmunized mice. CD8⁺ T cells, as effectors, from p210 immunized mice developed a preferentially higher cytolytic response against p210-loaded DCs in vitro. There was no protective CD4⁺ T-cell response as evidenced by lack of activation of CD4⁺ T cells and transfer of CD4⁺CD25⁺ T cells from p210-immunized donors did not provide atheroprotection in the naïve recipient mice [Chyu et al. 2012].

Immunization with p210 peptide also elicits changes in CD4⁺ T cells. Using an immunization schedule and preparation similar to ours, Wigren and colleagues reported that male apoE(–/–) mice immunized with p210 generated a functional CD4⁺CD25⁺ T-cell response and reduced atherosclerosis. Administration of CD25 blocking antibody ablated the increase of CD4⁺CD25⁺ T cells and the associated atheroprotection by p210 immunization [Wigren et al. 2010]. However, the authors acknowledged several limitations in their study, including lack of a control group of mice receiving adjuvant and the possibility of CD25 blocking antibody ablating both CD25⁺ effector T cells and regulatory T cells together [Wigren et al. 2010]. Klingenberg and colleagues used a different approach to deliver p210 for immunization. They generated a recombinant protein by fusing p210 with cholera toxin B (p210-CTB) and delivered it to female apoE(–/–) intranasally twice weekly for 12 weeks. Mice receiving ovalbumin peptide fused with CTB (OVA-CTB) or phosphate-buffered saline served as control groups. They observed a reduction of atherosclerosis in aortic sinuses in mice receiving p210-CTB treatment compared with the other two groups with no difference in FoxP3, interleukin (IL)-10 or transforming growth factor β (TGFβ) mRNA expression in aorta between p210-CTB and OVA-CTB groups. There was no difference in the numbers of FoxP3⁺ cells in aortic lesions or CD4⁺FoxP3⁺ T cells in lung mucosa. However, there was a higher percentage of IL-10⁺ subset of splenic CD4⁺ T cells from p210-CTB immunized mice and they were functional to suppress the effector CD4⁺ T cells. The authors suggested that these CD4⁺IL-10⁺ T cells (called Tr1 type of regulatory T cells) could potentially mediate the beneficial effect of intranasal immunization with p210-CTB [Klingenberg et al. 2010]. Another group of investigators reported that delivering a mixture of apoB-100 peptides (p210, MDA-p210 and p240, 3.3 µg each, total 10 µg) for 2 weeks subcutaneously via an implanted mini-osmotic pump reduced atherosclerosis in aortic sinus compared with control groups of mice [Herbin et al. 2012]. Additional experiments showed that such protection could be achieved by delivering p210 peptide alone, indicating p210 is the major peptide responsible for the atheroprotective effect among the three tested peptides. Such protection was associated with a decreased activation of CD4⁺ T cells and reduced interferon γ, IL-10 and IL-4 cytokine production upon stimulation ex vivo and increased the CD4⁺CD25⁺FoxP3⁺ subset of T cells in lymph nodes. Ablation of CD25⁺ T cells by CD25-depleting antibody also abrogated the atheroprotection by apoB-100 peptide infusion.

Taking the above reports together, it is clear that the beneficial effect of p210 immunization relies on cellular immune responses. It is possible that both atheroprotective and atheropromoting T cells exist within the CD4⁺ and CD8⁺ T cells. Only through a detailed delineation of subtypes of CD4⁺ and CD8⁺ T cells and testing their roles in atherosclerosis by an adoptive transfer strategy can we fully understand and delineate how cellular immune responses after apoB-100 peptide immunization influence atherogenesis. The overall beneficial effect of p210 immunization likely depends on the balance of quantity, strength and timing of appearance of all the elicited immune responses.

There may not be a universal mechanism to explain the effect of immunization using apoB-100 peptides as different apoB-100 peptides could elicit different immune responses. A strong CD4⁺CD25⁺FoxP3⁺ T-cell response can be induced by p2 immunization whereas immunization with p210 induced the least response. Both p2 and p45 elicited a CD8⁺CD25⁺FoxP3⁺ T-cell response compared with the carrier control group but p210 showed the opposite effect [Pierides et al. 2013]. Nevertheless p210 is currently a strong candidate to be developed into future potential human applications given that immunization with p210 consistently reduces atherosclerosis even when it is administered via different routes and in different forms.

Active immunization with antigens unrelated to low-density lipoprotein

A series of publications from Dr Witztum’s group described a natural IgM antibody recognizing the epitopes in oxLDL [Friedman et al. 2002; Horkko et al. 1999] and PC headgroups on the surface of apoptotic cells and inhibits uptake of oxLDL and apoptotic cells in macrophages(Table 1) [Chang et al. 1999; Horkko et al. 1999; Shaw et al. 2000]. Immunization against PC in apoE(–/–) mice elicited antibodies against PC and oxLDL, resulting in reduced atherosclerotic lesions at the aortic root [Caligiuri et al. 2007]. Given that anti-PC antibodies protect against infection from Streptococcus pneumoniae [Briles et al. 1982; Yother et al. 1982], active immunization in LDLR knockout mice with Streptococcus pneumoniae resulted in a modest reduction of atherosclerosis [Binder et al. 2003]. However, human studies failed to show consistent cardiovascular protective effects of pneumococcal vaccine [Hung et al. 2010; Lamontagne et al. 2008; Siriwardena et al. 2010; Tseng et al. 2010].

Dr Göran Hansson’s group described T-cell clones, obtained by immunizing human apoB-100 transgenic mice with human oxLDL, recognizing only native LDL or purified apoB-100. These T-cell clones showed uniform expression of a single T-cell receptor variable β chain, TRVB31. Anti-TRVB31 antibodies generated from immunization with a peptide from TRVB31 blocked T-cell recognition of apoB-100 and reduced atherosclerosis [Hermansson et al. 2010].

Dr Kuiper’s group reported that an orally administered DNA vaccine against CD99 or VEGFR2 generated antigen-specific cytotoxic CD8⁺ T cells with associated reduction in murine atherosclerosis [Hauer et al. 2007; van Wanrooij et al. 2008].

Cholesteryl ester transfer protein (CETP), a key enzyme involved in high-density lipoprotein (HDL) metabolism, has been used as an antigen in active immunization. Immunization with CETP induced neutralizing antibodies and in turn led to a marked elevation of HDL with concomitant reduction in atherosclerosis in rabbits [Gaofu et al. 2005; Mao et al. 2006; Rittershaus et al. 2000] but human studies were disappointing [Davidson et al. 2003].

HSPs are another groups of proteins that have been implicated in atherogenesis [Almanzar et al. 2012; Grundtman et al. 2011; Grundtman and Wick, 2011; Kilic and Mandal, 2012] but the effect of immunization with HSPs on atherosclerosis has been inconsistent. Immunization with HSP65 induces atherosclerotic lesions in several reports [George et al. 1999; Xu et al. 1992; Zhang et al. 2012]; whereas others reported reduced atherosclerotic lesions [Klingenberg et al. 2012; Long et al. 2012]. Tolerance induction by oral HSP60 or nasal immunization of HSP65 reduced atherosclerosis as well [Maron et al. 2002; van Puijvelde et al. 2007]. Immunization with apoB-100 and HSP60 peptides was also reported to confer atheroprotection [Li et al. 2011; Lu et al. 2010].

β2-GPI, a major target for antiphospholipid antibodies, is another vital molecule that has been delineated in atherogenesis. Active immunization using β2-GPI as antigen enhanced lesion formation and inflammation in atherosclerotic plaques [Afek et al. 1999; Dunoyer-Geindre et al. 2007; George et al. 1998a].

Although anti-CD3 monoclonal antibody is not an atherosclerosis-related antigen, oral administration of low-dose anti-CD3 antibody is known to induce tolerance by eliciting regulatory T cells and anti-inflammatory cytokines [da Cunha and Weiner, 2012]. Sasaki and colleagues reported that such a strategy indeed reduced atherosclerosis with induction of CD4⁺CD25⁺FoxP3⁺ regulatory T cells and anti-inflammatory cytokine TGFβ mediated such an effect in apoE (–/–) mice [Sasaki et al. 2009].

Active immunization with dendritic cells primed with atherosclerosis-related antigens

DCs are ‘nature’s adjuvants’ that can process and deliver antigens to hosts to elicit immune responses [Banchereau and Palucka, 2005; Palucka and Banchereau, 2012]. This approach has been widely used in the field of cancer research and is now being adopted in atherosclerosis research. Dr Kuiper and his group immunized LDLR(–/–) mice with DCs matured with lipopolysaccharide and primed with copper oxLDL and demonstrated reduced carotid artery lesions induced by perivascular collar placement compared with controls [Habets et al. 2010]. Additionally this group transfected DCs with Foxp3 encoding mRNA and injected these DCs into LDLR(–/–) mice and demonstrated that this DC-based immunization strategy resulted in a decrease in FoxP3⁺ regulatory T cells and an increase in atherosclerotic lesions in the aortic root [van Es et al. 2009]

Conclusion

From a global healthcare point of view, it is extremely appealing to develop atherosclerosis vaccines to prevent and/or treat atherosclerotic cardiovascular diseases. Many different types of antigens have been tested and shown to have great potential to be incorporated into vaccine formulations to reduce atherosclerosis. We and other investigators in this field have accumulated encouraging preclinical data, especially with LDL-related antigens. However, this is only the beginning of the endeavor to advance such knowledge from the preclinical area to clinical application. While advancing this concept to the human arena, many questions, such as vaccine safety and stability, efficacy endpoints, optimal schedule of vaccination regimens and durability of effects, need to be answered in future clinical trials. To do so this requires extensive collaborations among academic investigators, government agencies and the pharmaceutical industry. This will be a costly and long-term task but the effort is worthwhile if it succeeds.

Footnotes

Funding

This work was supported by a research grant from Cardiovax to Cedars-Sinai Medical Center in support of projects related to vaccine research. Additional support from the Heart Foundation and Speilberg Cardiovascular Research Fund are also acknowledged.

Conflict of interest statement

KYC and PKS are coinventors, along with Dr Jan Nilsson, on patents for immune-modulation therapies for atherosclerosis.

References

Afek

George

Shoenfeld

Gilburd

Levy

Shaish

. (1999) Enhancement of atherosclerosis in beta-2-glycoprotein I-immunized apolipoprotein E-deficient mice. Pathobiology 67: 19–25.

Almanzar

Ollinger

Leuenberger

Onestingel

Rantner

Zehm

. (2012) Autoreactive HSP60 epitope-specific T-cells in early human atherosclerotic lesions. J Autoimmun 39: 441–450.

Alving

Swartz

Jr Wassef

Ribas

Herderick

Virmani

. (1996) Immunization with cholesterol-rich liposomes induces anti-cholesterol antibodies and reduces diet-induced hypercholesterolemia and plaque formation. J Lab Clin Med 127: 40–49.

Ameli

Hultgardh-Nilsson

Regnstrom

Calara

Yano

Cercek

. (1996) Effect of immunization with homologous LDL and oxidized LDL on early atherosclerosis in hypercholesterolemic rabbits. Arterioscler Thromb Vasc Biol 16: 1074–1079.

Andersson

Libby

Hansson

(2010) Adaptive immunity and atherosclerosis. Clin Immunol 134: 33–46.

Armitage

(2000) Periodontal infections and cardiovascular disease – how strong is the association? Oral Dis 6: 335–350.

Ausiello

Palazzo

Spensieri

Fedele

Lande

Ciervo

. (2005) 60-kDa heat shock protein of Chlamydia pneumoniae is a target of T-cell immune response. J Biol Regul Homeost Agents 19: 136–140.

Banchereau

Palucka

(2005) Dendritic cells as therapeutic vaccines against cancer. Nat Rev Immunol 5: 296–306.

Beck

Pankow

Tyroler

Offenbacher

(1999) Dental infections and atherosclerosis. Am Heart J 138: S528-S533.

10.

Bergmark

de Faire

Lefvert

Swedenborg

(1995) Patients with early-onset peripheral vascular disease have increased levels of autoantibodies against oxidized LDL. Arterioscler Thromb Vasc Biol 15: 441–445.

11.

Betjes

Litjens

Zietse

(2007) Seropositivity for cytomegalovirus in patients with end-stage renal disease is strongly associated with atherosclerotic disease. Nephrol Dial Transplant 22: 3298–3303.

12.

Binder

(2010) Natural IgM antibodies against oxidation-specific epitopes. J Clin Immunol 30(Suppl. 1): S56-S60.

13.

Binder

(2012) Naturally occurring IgM antibodies to oxidation-specific epitopes. Adv Exp Med Biol 750: 2–13.

14.

Binder

Hartvigsen

Chang

Miller

Broide

Palinski

. (2004) IL-5 links adaptive and natural immunity specific for epitopes of oxidized LDL and protects from atherosclerosis. J Clin Invest 114: 427–437.

15.

Binder

Horkko

Dewan

Chang

Kieu

Goodyear

. (2003) Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL. Nat Med 9: 736–743.

16.

Briles

Forman

Hudak

Claflin

(1982) Anti-phosphorylcholine antibodies of the T15 idiotype are optimally protective against Streptococcus pneumoniae. J Exp Med 156: 1177–1185.

17.

Buhlin

Gustafsson

Pockley

Frostegard

Klinge

(2003) Risk factors for cardiovascular disease in patients with periodontitis. Eur Heart J 24: 2099–2107.

18.

Caligiuri

Khallou-Laschet

Vandaele

Gaston

Delignat

Mandet

. (2007) Phosphorylcholine-targeting immunization reduces atherosclerosis. J Am Coll Cardiol 50: 540–546.

19.

Cesena

Dimayuga

Yano

Zhao

Kirzner

Zhou

. (2012) Immune-modulation by polyclonal IgM treatment reduces atherosclerosis in hypercholesterolemic apoE–/– mice. Atherosclerosis 220: 59–65.

20.

Chang

Bergmark

Laurila

Horkko

Han

Friedman

. (1999) Monoclonal antibodies against oxidized low-density lipoprotein bind to apoptotic cells and inhibit their phagocytosis by elicited macrophages: evidence that oxidation-specific epitopes mediate macrophage recognition. Proc Natl Acad Sci U S A 96: 6353–6358.

21.

Chou

Fogelstrand

Hartvigsen

Hansen

Woelkers

Shaw

. (2009) Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans. J Clin Invest 119: 1335–1349.

22.

Chyu

Nilsson

Shah

(2011) Immune mechanisms in atherosclerosis and potential for an atherosclerosis vaccine. Discov Med 11: 403–412.

23.

Chyu

Shah

(2013) Can we vaccinate against atherosclerosis? J Cardiovasc Pharmacol Ther 13 September (Epub ahead of print).

24.

Chyu

Zhao

Dimayuga

Zhou

Yano

. (2012) CD8 T cells mediate the athero-protective effect of immunization with an ApoB-100 peptide. PLoS One 7: e30780.

25.

Chyu

Zhao

Reyes

Babbidge

Dimayuga

Yano

. (2005) Immunization using an Apo B-100 related epitope reduces atherosclerosis and plaque inflammation in hypercholesterolemic apo E (–/–) mice. Biochem Biophys Res Commun 338: 1982–1989.

26.

da Cunha

Weiner

(2012) Induction of immunological tolerance by oral anti-CD3. Clin Dev Immunol 2012: 425021.

27.

Davidson

Maki

Umporowicz

Wheeler

Rittershaus

Ryan

(2003) The safety and immunogenicity of a CETP vaccine in healthy adults. Atherosclerosis 169: 113–120.

28.

Dunoyer-Geindre

Kwak

Pelli

Roth

Satta

Fish

. (2007) Immunization of LDL receptor-deficient mice with beta2-glycoprotein 1 or human serum albumin induces a more inflammatory phenotype in atherosclerotic plaques. Thromb Haemost 97: 129–138.

29.

Erkkila

Narvanen

Lehto

Uusitupa

Yla-Herttuala

(2005) Antibodies against oxidized LDL and cardiolipin and mortality in patients with coronary heart disease. Atherosclerosis 183: 157–162.

30.

Faria-neto

Chyu

Dimayuga

Ferreira

Yano

. (2006) Passive immunization with monoclonal IgM antibodies against phosphorylcholine reduces accelerated vein graft atherosclerosis in apolipoprotein E-null mice. Atherosclerosis 189: 83–90.

31.

Fredrikson

Andersson

Soderberg

Dimayuga

Chyu

Shah

. (2005) Atheroprotective immunization with MDA-modified apo B-100 peptide sequences is associated with activation of Th2 specific antibody expression. Autoimmunity 38: 171–179.

32.

Fredrikson

Bjorkbacka

Soderberg

Ljungcrantz

Nilsson

(2008) Treatment with apo B peptide vaccines inhibits atherosclerosis in human apo B-100 transgenic mice without inducing an increase in peptide-specific antibodies. J Intern Med 264: 563–570.

33.

Fredrikson

Hedblad

Berglund

Alm

Ares

Cercek

. (2003a) Identification of immune responses against aldehyde-modified peptide sequences in apoB associated with cardiovascular disease. Arterioscler Thromb Vasc Biol 23: 872–878.

34.

Fredrikson

Soderberg

Lindholm

Dimayuga

Chyu

Shah

. (2003b) Inhibition of atherosclerosis in apoE-null mice by immunization with apoB-100 peptide sequences. Arterioscler Thromb Vasc Biol 23: 879–884.

35.

Freigang

Horkko

Miller

Witztum

Palinski

(1998) Immunization of LDL receptor-deficient mice with homologous malondialdehyde-modified and native LDL reduces progression of atherosclerosis by mechanisms other than induction of high titers of antibodies to oxidative neoepitopes. Arterioscler Thromb Vasc Biol 18: 1972–1982.

36.

Friedman

Horkko

Steinberg

Witztum

Dennis

(2002) Correlation of antiphospholipid antibody recognition with the structure of synthetic oxidized phospholipids. Importance of Schiff base formation and aldol concentration. J Biol Chem 277: 7010–7020.

37.

Fukumoto

Shoji

Emoto

Kawagishi

Okuno

Nishizawa

(2000) Antibodies against oxidized LDL and carotid artery intima-media thickness in a healthy population. Arterioscler Thromb Vasc Biol 20: 703–707.

38.

Gaofu

Jun

Xin

Wentao

Jie

Xiuyun

. (2005) Vaccinating rabbits with a cholesteryl ester transfer protein (CETP) B-Cell epitope carried by heat shock protein-65 (HSP65) for inducing anti-CETP antibodies and reducing aortic lesions in vivo. J Cardiovasc Pharmacol 45: 591–598.

39.

George

Afek

Gilburd

Blank

Levy

Aron-Maor

. (1998a) Induction of early atherosclerosis in LDL-receptor-deficient mice immunized with beta2-glycoprotein I. Circulation 98: 1108–1115.

40.

George

Afek

Gilburd

Levkovitz

Shaish

Goldberg

. (1998b) Hyperimmunization of apo-E-deficient mice with homologous malondialdehyde low-density lipoprotein suppresses early atherogenesis. Atherosclerosis 138: 147–152.

41.

George

Harats

Gilburd

Afek

Shaish

Kopolovic

. (2000a) Adoptive transfer of beta(2)-glycoprotein I-reactive lymphocytes enhances early atherosclerosis in LDL receptor-deficient mice. Circulation 102: 1822–1827.

42.

George

Harats

Shoenfeld

(2000b) Autoimmunity in atherosclerosis. The role of autoantigens. Clin Rev Allergy Immunol 18: 73–86.

43.

George

Shoenfeld

Afek

Gilburd

Keren

Shaish

. (1999) Enhanced fatty streak formation in C57BL/6J mice by immunization with heat shock protein-65. Arterioscler Thromb Vasc Biol 19: 505–510.

44.

Gero

Gergely

Jakab

Szekely

Virag

Farkas

. (1959) Inhibition of cholesterol atherosclerosis by immunisation with beta-lipoprotein. Lancet 2: 6–7.

45.

Grosso

Ferderbar

Wanschel

Krieger

Higushi

Abdalla

(2008) Antibodies against electronegative LDL inhibit atherosclerosis in LDLr–/– mice. Braz J Med Biol Res 41: 1086–1092.

46.

Grundtman

Kreutmayer

Almanzar

Wick

(2011) Heat shock protein 60 and immune inflammatory responses in atherosclerosis. Arterioscler Thromb Vasc Biol 31: 960–968.

47.

Grundtman

Wick

(2011) The autoimmune concept of atherosclerosis. Curr Opin Lipidol 22: 327–334.

48.

Habets

van Puijvelde

van Duivenvoorde

van Wanrooij

Tervaert

. (2010) Vaccination using oxLDL-pulsed dendritic cells reduces atherosclerosis in LDL receptor-deficient mice. Cardiovasc Res 85: 622–630.

49.

Halme

Syrjala

Bloigu

Saikku

Leinonen

Airaksinen

. (1997) Lymphocyte responses to Chlamydia antigens in patients with coronary heart disease. Eur Heart J 18: 1095–1101.

50.

Hartvigsen

Chou

Hansen

Shaw

Tsimikas

Binder

. (2009) The role of innate immunity in atherogenesis. J Lipid Res 50(Suppl.): S388-S393.

51.

Hauer

van Puijvelde

Peterse

de Vos

van Weel

van Wanrooij

. (2007) Vaccination against VEGFR2 attenuates initiation and progression of atherosclerosis. Arterioscler Thromb Vasc Biol 27: 2050–2057.

52.

Herbin

it-Oufella

Fredrikson

Aubier

Perez

. (2012) Regulatory T-cell response to apolipoprotein B100-derived peptides reduces the development and progression of atherosclerosis in mice. Arterioscler Thromb Vasc Biol 32: 605–612.

53.

Hermansson

Ketelhuth

Strodthoff

Wurm

Hansson

Nicoletti

. (2010) Inhibition of T cell response to native low-density lipoprotein reduces atherosclerosis. J Exp Med 207: 1081–1093.

54.

Horkko

Bird

Miller

Itabe

Leitinger

Subbanagounder

. (1999) Monoclonal autoantibodies specific for oxidized phospholipids or oxidized phospholipid-protein adducts inhibit macrophage uptake of oxidized low-density lipoproteins. J Clin Invest 103: 117–128.

55.

Hung

Leung

Chu

Leung

Cheung

Chan

. (2010) Prevention of acute myocardial infarction and stroke among elderly persons by dual pneumococcal and influenza vaccination: a prospective cohort study. Clin Infect Dis 51: 1007–1016.

56.

Iribarren

Folsom

Jacobs

Gross

Belcher

Eckfeldt

(1997) Association of serum vitamin levels, LDL susceptibility to oxidation, and autoantibodies against MDA-LDL with carotid atherosclerosis. A case-control study. The ARIC Study Investigators. Atherosclerosis Risk in Communities. Arterioscler Thromb Vasc Biol 17: 1171–1177.

57.

Kilic

Mandal

(2012) Heat shock proteins: pathogenic role in atherosclerosis and potential therapeutic implications. Autoimmune Dis 2012: 502813.

58.

Klingenberg

Ketelhuth

Strodthoff

Gregori

Hansson

(2012) Subcutaneous immunization with heat shock protein-65 reduces atherosclerosis in apoE(–)/(–) mice. Immunobiology 217: 540–547.

59.

Klingenberg

Lebens

Hermansson

Fredrikson

Strodthoff

Rudling

. (2010) Intranasal immunization with an apolipoprotein B-100 fusion protein induces antigen-specific regulatory T cells and reduces atherosclerosis. Arterioscler Thromb Vasc Biol 30: 946–952.

60.

Lamontagne

Garant

Carvalho

Lanthier

Smieja

Pilon

(2008) Pneumococcal vaccination and risk of myocardial infarction. CMAJ 179: 773–777.

61.

Lehtimaki

Lehtinen

Solakivi

Nikkila

Jaakkola

Jokela

. (1999) Autoantibodies against oxidized low density lipoprotein in patients with angiographically verified coronary artery disease. Arterioscler Thromb Vasc Biol 19: 23–27.

62.

Zhao

Zhang

Wang

(2011) ApoB-100 and HSP60 peptides exert a synergetic role in inhibiting early atherosclerosis in immunized apoE-null mice. Protein Pept Lett 18: 733–740.

63.

Kievit

Robertson

Koluman

von Wachenfeldt

. (2013) Targeting oxidized LDL improves insulin sensitivity and immune cell function in obese Rhesus macaques. Molecular Metabolism 2: 256–269.

64.

Long

Lin

Yang

Yuan

. (2012) Nasal immunization with different forms of heat shock protein-65 reduced high-cholesterol-diet-driven rabbit atherosclerosis. Int Immunopharmacol 13: 82–87.

65.

Chen

Endresz

Xia

Faludi

Burian

. (2010) Immunization with a combination of ApoB and HSP60 epitopes significantly reduces early atherosclerotic lesion in Apobtm2SgyLdlrtm1Her/J mice. Atherosclerosis 212: 472–480.

66.

Lundberg

Hansson

(2010) Innate immune signals in atherosclerosis. Clin Immunol 134: 5–24.

67.

Mandal

Jahangiri

(2004) Autoimmunity to heat shock proteins in atherosclerosis. Autoimmun Rev 3: 31–37.

68.

Mandal

Jahangiri

(2005) Autoimmune mechanisms of atherosclerosis. Handb Exp Pharmacol (170): 723–743.

69.

Mao

Kai

Gaofu

Zheng

Jie

. (2006) Intramuscular immunization with a DNA vaccine encoding a 26-amino acid CETP epitope displayed by HBc protein and containing CpG DNA inhibits atherosclerosis in a rabbit model of atherosclerosis. Vaccine 24: 4942–4950.

70.

Maron

Sukhova

Faria

Hoffmann

Mach

Libby

. (2002) Mucosal administration of heat shock protein-65 decreases atherosclerosis and inflammation in aortic arch of low-density lipoprotein receptor-deficient mice. Circulation 106: 1708–1715.

71.

Matsuura

Kobayashi

Inoue

Lopez

Shoenfeld

(2005) Oxidized LDL/beta2-glycoprotein I complexes: new aspects in atherosclerosis. Lupus 14: 736–741.

72.

Mayr

Kiechl

Willeit

Wick

(2000) Infections, immunity, and atherosclerosis: associations of antibodies to Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus with immune reactions to heat-shock protein 60 and carotid or femoral atherosclerosis. Circulation 102: 833–839.

73.

Movahed

(1999) Infection with Chlamydia pneumoniae and atherosclerosis: a review. J S C Med Assoc 95: 303–308.

74.

Nicoletti

Kaveri

Caligiuri

Bariety

Hansson

(1998) Immunoglobulin treatment reduces atherosclerosis in apo E knockout mice. J Clin Invest 102: 910–918.

75.

Nicolo

Goldman

Monestier

(2003) Reduction of atherosclerosis in low-density lipoprotein receptor-deficient mice by passive administration of antiphospholipid antibody. Arthritis Rheum 48: 2974–2978.

76.

Nilsson

Calara

Regnstrom

Hultgardh-Nilsson

Ameli

Cercek

. (1997) Immunization with homologous oxidized low density lipoprotein reduces neointimal formation after balloon injury in hypercholesterolemic rabbits. J Am Coll Cardiol 30: 1886–1891.

77.

Olofsson

Jatta

Wagsater

Gredmark

Hedin

Paulsson-Berne

. (2005) The antiviral cytomegalovirus inducible gene 5/viperin is expressed in atherosclerosis and regulated by proinflammatory agents. Arterioscler Thromb Vasc Biol 25: e113–e116.

78.

Palinski

Miller

Witztum

(1995) Immunization of low density lipoprotein (LDL) receptor-deficient rabbits with homologous malondialdehyde-modified LDL reduces atherogenesis. Proc Natl Acad Sci U S A 92: 821–825.

79.

Palucka

Banchereau

(2012) Cancer immunotherapy via dendritic cells. Nat Rev Cancer 12: 265–277.

80.

Persson

Boren

Nicoletti

Hansson

Pekna

(2005) Immunoglobulin treatment reduces atherosclerosis in apolipoprotein E low-density lipoprotein receptor mice via the complement system. Clin Exp Immunol 142: 441–445.

81.

Pierides

Bermudez-Fajardo

Fredrikson

Nilsson

Oviedo-Orta

(2013) Immune responses elicited by apoB-100-derived peptides in mice. Immunol Res 56: 96–108.

82.

Rittershaus

Miller

Thomas

Picard

Honan

Emmett

. (2000) Vaccine-induced antibodies inhibit CETP activity in vivo and reduce aortic lesions in a rabbit model of atherosclerosis. Arterioscler Thromb Vasc Biol 20: 2106–2112.

83.

Salonen

Yla-Herttuala

Yamamoto

Butler

Korpela

Salonen

. (1992) Autoantibody against oxidised LDL and progression of carotid atherosclerosis. Lancet 339: 883–887.

84.

Sasaki

Yamashita

Takeda

Shinohara

Nakajima

Tawa

. (2009) Oral anti-CD3 antibody treatment induces regulatory T cells and inhibits the development of atherosclerosis in mice. Circulation 120: 1996–2005.

85.

Schiopu

Bengtsson

Soderberg

Janciauskiene

Lindgren

Ares

. (2004) Recombinant human antibodies against aldehyde-modified apolipoprotein B-100 peptide sequences inhibit atherosclerosis. Circulation 110: 2047–2052.

86.

Schiopu

Frendeus

Jansson

Soderberg

Ljungcrantz

Araya

. (2007) Recombinant antibodies to an oxidized low-density lipoprotein epitope induce rapid regression of atherosclerosis in apobec-1(-/-)/low-density lipoprotein receptor(–/–) mice. J Am Coll Cardiol 50: 2313–2318.

87.

Shaw

Horkko

Chang

Curtiss

Palinski

Silverman

. (2000) Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity. J Clin Invest 105: 1731–1740.

88.

Shoenfeld

Sherer

George

Harats

(2001) beta 2-glycoprotein I in human and murine atherosclerosis. Isr Med Assoc J 3: 85–87.

89.

Siriwardena

Gwini

Coupland

(2010) Influenza vaccination, pneumococcal vaccination and risk of acute myocardial infarction: matched case-control study. CMAJ 182: 1617–1623.

90.

Tseng

Slezak

Quinn

Van den Eeden

Jacobsen

(2010) Pneumococcal vaccination and risk of acute myocardial infarction and stroke in men. JAMA 303: 1699–1706.

91.

van Es

van Puijvelde

Foks

Habets

Bot

Gilboa

. (2009) Vaccination against Foxp3(+) regulatory T cells aggravates atherosclerosis. Atherosclerosis 209: 74–80.

92.

van Puijvelde

van Es

van Wanrooij

Habets

K.L.

de, Vos

van Der

(2007) Induction of oral tolerance to HSP60 or an HSP60-peptide activates T cell regulation and reduces atherosclerosis. Arterioscler Thromb Vasc Biol 27: 2677–2683.

93.

van Wanrooij

de, Vos

Bixel

Vestweber

van Berkel

Kuiper

(2008) Vaccination against CD99 inhibits atherogenesis in low-density lipoprotein receptor-deficient mice. Cardiovasc Res 78: 590–596.

94.

Van Leeuwen

Kemna

de Winther

Boon

Duijvestijn

Henatsch

. (2013) Passive immunization with hypochlorite-oxLDL specific antibodies reduces plaque volume in LDL receptor-deficient mice. PLoS One 8: e68039.

95.

Wigren

Kolbus

Duner

Ljungcrantz

Soderberg

Bjorkbacka

. (2010) Evidence for a role of regulatory T cells in mediating the atheroprotective effect of apolipoprotein B peptide vaccine. J Intern Med 269: 546–556.

96.

Dietrich

Steiner

Gown

Schoel

Mikuz

. (1992) Induction of arteriosclerosis in normocholesterolemic rabbits by immunization with heat shock protein 65. Arterioscler Thromb 12: 789–799.

97.

Metzler

Jahangiri

Mandal

(2012) Molecular chaperones and heat shock proteins in atherosclerosis. Am J Physiol Heart Circ Physiol 302: H506–H514.

98.

Yan

Hansson

(2007) Innate immunity, macrophage activation, and atherosclerosis. Immunol Rev 219: 187–203.

99.

Yother

Forman

Gray

Briles

(1982) Protection of mice from infection with Streptococcus pneumoniae by anti-phosphocholine antibody. Infect Immun 36: 184–188.

100.

Yuan

Kishimoto

Sano

Shioji

Yokode

(2003) Immunoglobulin treatment suppresses atherosclerosis in apolipoprotein E-deficient mice via the Fc portion. Am J Physiol Heart Circ Physiol 285: H899–H906.

101.

Zelkha

Freilich

Amar

(2010) Periodontal innate immune mechanisms relevant to atherosclerosis and obesity. Periodontol 54: 207–221.

102.

Zhang

Xiong

Sun

Tan

Zhang

. (2012) A novel atherogenic epitope from Mycobacterium tuberculosis heat shock protein 65 enhances atherosclerosis in rabbit and LDL receptor-deficient mice. Heart Vessels 27: 411–418.

103.

Zhou

Caligiuri

Hamsten

Lefvert

Hansson

(2001) LDL immunization induces T-cell-dependent antibody formation and protection against atherosclerosis. Arterioscler Thromb Vasc Biol 21: 108–114.