Inflammation and Atherosclerosis || Vaccination Against Atherosclerosis

Vaccination Against Atherosclerosis 27Cecilia Grundtman

27.1 Vaccination Against Atherosclerosis

The development of a vaccine to prevent the build-up of atherosclerotic plaques

would drastically change the life for millions of individuals and hopefully strongly

reduce the number of fatal and nonfatal cardiovascular events. At present, there are

several treatments for the disease (e.g. statins, acetylsalicylic acid, and ADP-

receptor antagonists) and much can be accomplished through lifestyle changes

such as giving up smoking or switching to a low-fat low-cholesterol diet. A large

number of prospective, randomized, controlled clinical trials have demonstrated

both angiographic and clinical benefits of lipid-lowering therapy. Overall, a signifi-

cant and clinically worthwhile relative risk reduction ranging from 20% to 40% in

major cardiovascular events has been achieved with these strategies, without

significant adverse effects or increased noncardiovascular mortality. However,

around 60–70% of adverse cardiovascular events continue to occur despite oxidized

low-density lipoprotein (oxLDL)-lowering therapies, indicating an obvious need

for new therapeutic interventions. It is also important to note that around 60% of

patients with cardiovascular disease (CVD) do not show increased lipid values.

Established therapies almost exclusively aim at risk factor modification by reducing

dyslipidemia, hypertension, and hyperglycemia, without directly targeting the

actual disease process in the artery wall. It seems likely that to achieve further

improvement in the prevention of cardiovascular events, new approaches must be

developed. Optimally this would consist of specific inhibition of atheropromoting

inflammatory pathways contributing to disease development while sparing or

reinforcing those inflammatory pathways that are atheroprotective. In this chapter,

C. Grundtman (*)

Laboratory of Autoimmunity, Division of Experimental Pathophysiology and Immunology,

Biocenter, Innsbruck Medical University, Sch€opfstraße 41, A-6020 Innsbruck, Austria

e-mail: [email protected]

G. Wick and C. Grundtman (eds.), Inflammation and Atherosclerosis,DOI 10.1007/978-3-7091-0338-8_27, # Springer-Verlag/Wien 2012

529

mailto:[email protected]

the focus is on two new “vaccine” interventions, both of which target the actual

disease process in the arterial wall; one is based on antigens derived from LDL and

the other is based on peptides derived from heat shock proteins (HSPs).

27.2 Oxidized Low-Density Lipoprotein (oxLDL)in Atherosclerosis

Cholesterol is one of the main constituents of atherosclerotic plaques, and thus

provides an obvious target for anti-plaque therapy and/or prevention of CVD. Innate

and adaptive immune responses against oxLDL are believed to play an important

role in the inflammatory process of atherosclerosis (Figs. 27.1, 27.2). The oxidation

of aggregated LDL in the extracellular matrix of the artery wall are typical of both

early and advanced atherosclerotic plaques and the oxidation of LDL leads to the

formation of highly reactive lipid peroxides and aldehydes [1, 2]. Oxidation of

the LDL protein results in structural modifications of the apolipoprotein B-100

(ApoB-100). Aldehydes, for instance, can bind to free amino groups on the peptide

fragments. These structural modifications can lead to the formation of many neo-

epitopes, which renders the modified LDL immunogenic and leads both to cellular

and humoral response. This is associated with the activation of an inflammatory

response, including endothelial expression of adhesion molecules and infiltration of

monocytes/macrophages and T cells [3]. As many as 10–20% of the T cells present

in human atherosclerotic plaques are oxLDL-specific and become activated when

exposed to oxLDL/HLA-DR complexes on antigen-presenting cells (APCs) [4]

(Fig. 27.2). Furthermore, oxLDL can also activate peripheral T cells in the develop-

ment of atherosclerosis by several mechanisms [5]; the T-cell receptor repertoire

expressed by T cells in the plaque is restricted and becomes even more restricted

with disease progression [6, 7]. Macrophages express a family of scavenger

receptors (ScRs), which can bind and ingest oxLDL particles [8]. Continuous

activation of such innate immune responses is believed to be a major cause of

atherosclerotic plaque development [9], and it has been suggested that anti-oxLDL

antibodies might possess pathogenic as well as protective effects [10].

Attempts to characterize the functional importance of autoimmune responses

against homologous LDL in animal models [2, 11, 12] unexpectedly showed that

immunization with oxLDL resulted in partial protection against atherosclerosis. For

example, immunization with oxLDL in hypercholesterolemic rabbits was found to

reduce atherosclerosis by 45–75% [11, 12] and similar observations were subse-

quently also made in hypercholesterolemic apolipoprotein E (ApoE�/�) and LDL

receptor (LDLr�/�)-deficient mice [13, 14], as well as in balloon-injured hypercho-

lesterolic rabbits [15]. Additionally, LDL and oxLDL immunizations have been

shown to reduce the number of T cells, class II antigens, and oxLDL in lesions [15]

(Fig. 27.3) despite the fact that these animals had developed significant antibody

titers of oxLDL. The notion that oxidation of LDL makes the lipoprotein a target for

the immune system is also supported by the existence of circulating autoantibodies

530 C. Grundtman

against LDL [16–19] and the fact that these antibodies bind to oxLDL present in the

plaques [20]. Severe hypercholesterolemia is associated with a switch from TH1 to

TH2, which results not only in the formation of IgG1 autoantibodies to oxLDL, but

also in the appearance of TH2-type cytokines in the atherosclerotic lesion [21].

Circulating autoantibodies against oxLDLs are also abundant in humans with some

studies reporting correlation between levels of autoantibodies and severity of

disease in cardiovascular patients [19, 22–24], and others reporting a lack of such

correlation [25, 26]. The observations that immunization with oxLDL could be

atheroprotective were in apparent conflict with the fact that the net effect of

adaptive autoimmunity induced by hypercholesterolemia is proatherogenic.

Plasma cell

B cell

Treg cellTH1 cell

APC

Macrophage

LDL

LDL

LDL

LDL

oxLDL

IFN-γIL-4

Tr1 cellCD4+CD25+

Foxp3+

IL-10TGF-β

oxLDL

oxLDL

oxLDL

oxLDL

Cytokines can induce a macrophage activation

TCR

MHCII

CD28CD40L

CD86/80CD40

TH0 cell

TH2 cell

TH3 cell

ScR

Fig. 27.1 Immune activation against oxLDL in atherosclerosis. LDL and oxLDL particles are

taken up by LDL receptors and, if modified, by ScRs and/or Fc receptors on APCs. After

endosomal degradation, LDL-derived peptides are transferred into the antigen-presenting pathway

where they bind to MHC class II. The peptide-MHC class II complexes can then be transported to

the cell surface where they are recognized by T cells. T cells with a TCR recognizing the antigen

will become activated. Depending on the antigen that is taken up by the APC, the pattern of

costimulatory molecules and cytokines secreted from the APC will determine the further fate of

the naıve T cell. Differentiation into TH1 cells results in an inflammatory, proatherogenic immune

response. Differentiation into TH2 cells leads to activation of B cells. Plasma cells will secrete

antigen-specific IgG and/or IgM autoantibodies that help to clear the antigen (in this case TH2 and

B cells are considered anti-atherogenic because of their ability to remove oxLDL). Antigen

presented on APCs can also induce T cells with a regulatory phenotype. Regulatory T cells are

immune inhibitory cells that dampen immune responses (Partly adapted from Servier Medical Art)

27 Vaccination Against Atherosclerosis 531

However, it soon became clear that atheroprotective immune responses must exist

and it might be possible to specifically activate these by a vaccine approach.

27.3 oxLDL Immunization

The mechanisms through which these atheroprotective immune responses operate

remain to be fully elucidated. One possibility is that antibodies facilitate removal of

oxidatively damaged LDL particles by macrophage ScRs [27, 28]. Macrophage

ScRs only recognize LDL with extensive oxidative damage [1]. Particles with

minimal oxidative damage are not be recognized by the ScRs, which leads to

Tunica intima

Lumen

2. LDL

2. oxLDL

5. oxLDL

Tunica intima

3. Clearance of oxLDL

Monocyte

1. Hyper-cholesterolemia

Endothelial cell

Monocyte/macrophage

Atherosclerosis – LDL Lymph node

Plasma cell

9. Anti - oxLDL autoantibodies

4. Adhesion molecules

Smooth muscle cell

Lamina elastica

Interna tunica media

MΦ

T cell APC MΦ

6. Macrophage infiltration 7. Foam cell

MΦ

8. Cytokine production

Cytokine production

CD4+

Fig. 27.2 A schematic figure of the oxLDL pathway in atherosclerosis. In hypercholesterolemic

settings (1) LDL can be modified and oxidized into (2) oxLDL. (3) Circulating monocytes/

macrophages can also bind oxLDL through ScRs and thereby clear oxLDL from the circulation.

Macrophages, which are later abundant in atheroma, are recruited from blood monocytes that enter

through the endothelial surface. (4) Adhesion molecules and chemokines govern the recruitment

process, which is followed by differentiation of the monocytes into macrophages. During this

process, pattern recognition receptors, such as ScRs, are upregulated on macrophages. (5–7) ScRs

mediate the uptake of oxLDL, and cause the accumulation of LDL-derived cholesterol and foam

cell formation. (8) Other macrophages are primed for activation when stimulated by the T cell

cytokine IFN-g. As a result of activation, the macrophages and T cells can release a range of

proinflammatory mediators both locally and into the circulation. (9) Activated APCs can migrate

to the secondary lymphoid organs and stimulate B cells, to differentiate into plasma cells that can

produce autoantibodies to oxLDL (Partly adapted from Servier Medical Art)

532 C. Grundtman

increased levels oxLDL in the circulation [29, 30]. Binding of antibodies to

circulating oxLDL particles would help to remove them from the circulation before

they can accumulate in the vascular tissue and cause tissue damage [31]. The

finding that the decrease in antibodies against ApoB-100 peptide that occurs with

age in humans is associated with an increase in plasma level of oxLDL supports this

notion [32]. A second and more extensively studied protective mechanism involves

specific immune responses against epitopes present in oxLDLs. Neonatal tolerance

of ApoE�/� mice to oxLDL leads to a decreased immune response to oxLDL and a

reduced susceptibility to atherosclerosis in adult animals. Injection of oxLDL

induced T cell tolerance due to clonal deletion, rather than anergy of the reactive

T cells. Furthermore, the T cell repertoire was altered in these mice during disease

progression, but was normalized by tolerization [33]. The timing of immunization

seems to be important for its efficacy with better atheroprotective properties when

applied in young (6–7-week-old) ApoE�/� mice compared to older (22-week-old)

mice [34]. By using a polypeptide library covering the complete sequence of the

Smooth muscle cell

Monocyte/ macrophage

Tunica intima

Endothelial cell

Lymph node

Plasma cell

Monocyte

4. Macrophage infiltration

T cell

5. Foam cell

oxLDL immunization

Treg�

Treg�

Treg�

T cell MΦ

3. T cell infiltration

Tr1/ Treg

APC MΦ

MΦ

7. TGF-β1�

8. Anti-oxLDL IgG/M (auto)antibodies�

6.Tregs�

2. Normal cholesterol levels

1. oxLDL injection

Spleen 9. Specific oxLDL T cell response�

T cell

T cell


7. TGF-β1�

T cell

Lamina elastica


Fig. 27.3 A schematic figure of oxLDL immunization. (1) oxLDL injections (2) do not change

serum cholesterol levels (3–6) but lower the expression of adhesion molecules, plaque T cells and

macrophages and induce T regulatory cells (Tregs). (7) Increased levels of TGF-b1 are found in

lymph nodes of immunized animals, indicating an induction of TGF-b1-producing regulatory

T cells, which can dampen the disease. (8) Additionally, increased anti-oxLDL IgG and/or IgM

(auto)antibodies are found in the circulation after immunization, which leads to decreased titers of

circulating oxLDL. (9) Moreover, an increased T cell proliferation specific to oxLDL is described

in the secondary lymphoid organs (Partly adapted from Servier Medical Art)


ApoB-100, a large number of native and malondialdehyde (MDA)-modified pep-

tide sequences have been identified. Antibodies in human plasma have also

recognized two of these ApoB-100 peptide sequences. When ApoE�/� mice were

immunized with a mix of these two ApoB-100 peptides (P143 and P210, homology

85% and 90% between mouse and human, respectively), against which high levels

of IgG and IgM antibodies were present in healthy human controls, a 60% reduction

in en face stained fatty lesions in the aorta was seen; however, no reduction was

found in more advanced plaques in the aortic origin, indicating that the protective

effect is primarily on early lesions [13]. Immunizations with these ApoB-100

peptides did not affect macrophage, collagen, or lipid content of plaques in the

aortic arch. No significant difference in plasma high-density lipoprotein (HDL)

cholesterol levels or IgM against native or MDA peptides was found; however,

IgG antibody levels were increased against MDA peptides [13] suggesting a

specific T cell-dependent antibody response. Similarly, ApoE�/� mice immunized

with either homologous plaque homogenates or MDA-LDL showed reduced

lesion development. The plaques of these mice contained immunogen(s) sharing

epitopes on MDA-LDL, MDA-very low-density lipoprotein (VLDL), and oxidized

cardiolipin (CL), indicating a specific T cell-dependent antibody response. A

specific T cell response to MDA-LDL in lymph nodes was also demonstrated

[35]. Furthermore, immunization with MDA ApoB-100 fragments can induce a

shift from TH1 to a TH2-specific oxLDL antibody expression [36]. Interestingly,

oxLDL pulsed mature dendritic cells (mDCs) can induce a lowered TH1 response,

induce oxLDL-specific T cells, and increase the production of oxLDL-specific

antibodies which can lead to a reduction in up to 87% in lesion size in LDLr�/�

mice [37]. Foam cell formation by the addition of oxLDL was significantly lower

when macrophages were incubated with serum from oxLDL-pulsed mDCs com-

pared with controls, suggesting that treatment with oxLDL-pulsed mDCs results in

the formation of oxLDL-specific antibodies, which can reduce foam cell formation

[37]. mDCs pulsed with MDA-LDL has also been studied in ApoE�/� mice;

however, these mice showed significant larger atherosclerotic lesions, with

increased inflammation and antigen-specific immune responses. Administration

of MDA-LDL in complete Freund’s adjuvant leads to an induction of T regulatory

cells whereas MDA-LDL-DCs treatment did not [38]. As already mentioned,

several studies show that atherosclerosis can be attenuated via a TH1 cytokine

inhibition [39–41] and/or with stimulation of TH2 cytokine production [42, 43].

According to some studies, restoration of the imbalance between TH1 and TH2 cells

may be effective in treating atherosclerosis, whereas others dispute this. For

example, interleukin (IL)-4, a TH2 cytokine, has been shown to be proatherogenic

[44] and an imbalance between pathogenic T cells (TH1 and/or TH2) and regulatory

T cells specific for altered self and nonself antigens could play a central role in

counteracting the initiation and progression of the disease [45, 46]. A possible

mechanism to achieve a beneficial shift in the balance between pathogenic T cells

and regulatory T cells might be through the induction of mucosal tolerance [47].

Oral tolerance induction to oxLDL can attenuate both early and advanced stage

atherosclerosis. The mechanisms underlying this effect might be ascribed to the

534 C. Grundtman

induction of CD4+CD25+Foxp3+ regulatory T cells and their increased production

of transforming growth factor-b (TGF-b), which counteract within the plaque the

oxLDL-specific CD4+ T cells [48]. However, oral tolerance to MDA-LDL did not

affect atherosclerosis [48] (Figs. 27.2, 27.3).

To test the clinical relevance of ApoB-100 peptides in human subjects, 302

peptides (20 amino acids long) corresponding to the entire human ApoB-100 amino

acid sequence, were investigated in patients with acute coronary heart events and

healthy controls [32]. Patient plasma IgM antibody titer levels were decreased with

age and were associated with the intima-media thickness of the carotid artery in

subjects younger than 60 years. There was also an inverse association between IgM

levels and oxLDL in plasma. Antibody levels against several MDA-modified

ApoB-100 sites were also associated with cardiovascular disease [32] including

one peptide, which has shown promising protective effects after immunization in

ApoE�/� mice [13]. Patients with coronary events as well as healthy controls were

found to express autoantibodies recognizing amino acid sequences in the LDL

receptor-binding region of ApoB-100. However, these autoantibodies have no or

only very poor affinity for the LDL receptor-binding site as expressed by intact

LDL, but oxidation appears to change the conformation of this peptide such that it

becomes targeted by autoantibodies [49]. One interpretation of these studies is that

antibodies against native ApoB-100 sequences do not seem to bind to native LDL

particles but only to modified peptides, and it is therefore unlikely that they could

influence the LDL metabolism.

27.4 Single oxLDL Peptide Immunization

The efficacy and feasibility of immunization using a single peptide as an antigen in

modulating atherosclerotic lesions has further been investigated by injecting

ApoE�/� mice with two different peptides, peptide-1 and peptide-2 (selected

from the polypeptide library where 302 peptides were spanning the whole human

sequence of ApoB-100 [32]). Peptide-2 immunization reduced aortic atherosclerosis

by 40% and plaque inflammation by 80% both in young (6/7-week-old) and

older (16-week-old) mice [50], suggesting a possibility of treating established

atherosclerosis. Interestingly, even though both peptides could induce an IgG

response, only peptide-2 showed atheroprotective properties. The difference in

the atheroprotective effect could possible lie in the ability of peptide-2 to induce

an IgM response. It has been suggested by several different studies that IgM isotype

antibody might induce atheroprotective properties. For instance, immunization

of LDLr�/�/human ApoB-100 transgenic mice with native human ApoB-100

peptides, p45 and p210 showed an atheroprotective effect. However, their effects

were independent of preexisting ApoB-100 autoantibodies and while they occurred

without activating an increase in peptide-specific IgG, they were associated with an

increase in IgM recognizing native and copper-oxLDL [51]. This expression pattern

is very similar to that observed in humans suggesting similar immune response to

these oxLDL epitopes in mouse and man [52, 53]. Immunization with pneumococcal


extract and Freund’s adjuvant or Freund’s adjuvant alone was able to induce high

IgM titers against oxLDL. Additionally, pneumococcal extract together with

Freund’s adjuvant showed cross-reactivity to phosphorylcholine, which Freund’s

adjuvant alone lacked. However, both immunizations lead to a reduced atheroscle-

rotic plaque size [54]. MDA-LDL immunization can induce not only adaptive

humoral immunity against MDA-LDL but also innate immune response with high

IgM titers against phosphorylcholine epitopes [55], all of which appear to be

important in modulating atherosclerosis. In conclusion, the protective effect of

immunization with ApoB-100 peptides may be partially mediated by IgM-

recognizing epitopes in oxLDL (Fig. 27.3).

The effect of many vaccines is mediated by generation of antigen-specific IgG

antibodies. The effects of passive immunization with different recombinant human

IgG1 antibodies against MDA-modified ApoB-100 peptides have recently been

investigated. In ApoE�/� mice, three injections administered weekly of one of

the IgG1 antibodies specifically recognizing MDA-modified 661–680 amino acid

sequence (p45) of ApoB-100, significantly and dose-dependently reduced the

extent of atherosclerosis, measured as a 50% reduction in total plaque area using

Red O staining, as well as the plaque content of oxLDL epitopes and macrophages.

The same antibody induced an increase in human monocyte binding and uptake of

oxLDL but not with native LDL [56]. In contrast, another study demonstrated that

IgM directed against oxLDL phospholipids, but not against MDA-LDL, inhibits

oxLDL uptake by macrophages [57]. Taken together, these observations suggest

that IgG mediates uptake of oxLDL through binding to Fc receptors, whereas IgM

may lack this effect. A rapid clearance of oxLDL by antibody binding and Fc

receptor uptake may thus serve to limit the damage produced by oxLDL particles in

the arterial wall. Furthermore, three injections with two different recombinant

human IgG1 antibodies against MDA-ApoB-100 p45 sequence (IEI-E3 and

2D03) in LDLr�/� expressing ApoB-100 (Apobec-1�/�/LDLr�/�) mice resulted

in additional regression of atherosclerosis compared to control IgG1-injected mice.

One of the antibodies, 2D03, also reduced macrophage content in plaque, enhanced

plaque expression of the adenosine triphosphate-binding cassette transporter A1,

and inhibited expression of monocyte chemoattractant protein-1 in cultured

monocytes [58], making this specific antibody treatment not only successful in

reducing aortal plaque formation but also stimulating lipid efflux and inhibiting

macrophage recruitment. An even greater reduction of atherosclerosis after four

immunizations with MDA-ApoB-100 p45 IgG1 treatment (2D03) has been reported

in LDLr�/� mice expressing human ApoB-100 [59], indicating that the antibody

reacts with humanized LDL particles providing support for a potential clinical

application. A human antibody (BI-204), which targets the oxLDL, has recently

been tested in a phase I double-blinded, placebo-controlled, dose escalation study

of 80 healthy volunteers with elevated levels of LDL. Preclinical studies showed

that administering BI-204 could substantially reduce the formation of atheroscle-

rotic plaques by over 50% and reduce the size of existing plaques by 50% in 4

weeks. BI-204 is developed by BioInvent, which has entered into collaboration with

Genentech, Inc. BI-204 will soon enter a phase II study.

536 C. Grundtman

27.5 b2-Glycoprotein I (b2-GPI) and PhosphorylcholineImmunizations

oxLDL can interact with b2-GPI forming oxLDL/b2-GPI complexes [60]. The

exact in vivo mechanism(s) of how these oxLDL/b2-GPI complexes are formed

is not fully understood. It is possible that the interaction between b2-GPI and

oxLDL minimizes the inflammatory properties of oxLDL while promoting its

clearance from circulation. In addition, binding of b2-GPI to oxLDL may likely

occur inside the arterial wall as the intima microenvironment is conductive to

further inflammation, oxidation, cell activation, and macrophage uptake of

oxLDL/b2-GPI complexes. The uptake of oxLDL/b2-GPI complexes by macro-

phages is significantly enhanced when b2-GPI-dependent antiphospholipid or

anti-b2-GPI antibodies are co-incubated with oxLDL/b2-GPI complexes.

The increased uptake of oxLDL/b2-GPI/antibody (immune) complexes suggests

the participation of Fcg receptors. Macrophage Fcg receptors bind circulating

immune complexes via the immunoglobulin Fc region, a process that stimulates

phagocytosis (uptake). Autoantibodies against oxLDL/b2-GPI complexes have

been demonstrated in patients with systemic lupus erythematosus (SLE) and

antiphospolipid syndrome and it has been shown to be significantly associated

with arterial thrombosis [61–63]. Moreover, anti-b2-GPI antibodies and b2-GPI-dependent anticardiolipin antibodies (aCL) are important predictors for arterial

thrombosis (myocardial infarction and stroke) in men [64, 65]. Furthermore, the

observation that monoclonal autoantibodies against oxLDL/b2-GPI complexes

significantly increase the oxLDL uptake by macrophages strongly suggests that

such IgG autoantibodies are proatherogenic. Mice that receive syngenetic

lymphocytes from b2-GPI-immunized LDLr�/�mice developed larger fatty streaks

compared to control mice that received lymphocytes from mice immunized with

human serum albumin [66], indicating that b2-GPI-reactive T cells can promote

atherogenesis. Similarly, ApoE�/� mice immunized with human b2-GPI developedincreased atherosclerosis and anti-b2-GPI antibodies compared to controls [67].

Interestingly, attenuation of early atherosclerosis has been achieved with oral

treatment of human and bovine b2-GPI in LDLr�/� mice [68]. Oral feeding with

b2-GPI inhibited lymph node cell reactivity to b2-GPI in mice immunized against

the human protein. Oral tolerance was also capable of reducing reactivity to

oxidized LDL in mice immunized against oxLDL. Furthermore, upon priming

with the respective protein, IL-4 and IL-10 production was upregulated in lymph

node cells of b2-GPI-tolerant mice immunized against b2-GPI [68].Another important class of oxLDL antigens is phosphorylcholine. Phosphor-

ylcholine is the major phospholipid in cell membranes and lipoproteins and is

known to be one of the neo-antigens exposed by LDL oxidations, which can elicit

an immune response. Phosphorylcholine is exposed on the surface of oxLDL and

apoptotic cells and it is targeted by both ScRs and a type of germ-line encoded IgM

antibodies [69]. A monoclonal anti-phosphorylcholine IgM that bears the TEPC-15

isotype has been cloned from a hypercholesterolemic mouse and found to bind to

phosphorylcholine head groups on oxLDL or phospholipids and thereby blocking


oxLDL uptake by macrophages [57, 70]. Active immunization of ApoE�/� mice

with phosphorylcholine-containing Streptococus pneumoniae vaccine is associatedwith an increase in phosphorylcholine-specific IgM antibodies and reduced athero-

sclerosis [54]. This indicates that there is a molecular mimicry between epitopes of

oxLDL and Streptococus pneumoniae, which could have athero-protective effects.

Furthermore, T15, a specific phosphorylcholine antibody, reduced vein graft ath-

erosclerosis in ApoE�/� mice [71]. Inhibition of atherosclerosis together with

increased generation of both phosphorylcholine-specific IgG and IgM antibodies

has also been observed in ApoE�/�mice immunized with phosphorylcholine linked

to a carrier protein [72]. Taken together, these studies indicate an athero-protective

immune response to phosphorylcholine in atherosclerosis settings.

27.6 Heat Shock Proteins (HSPs) in Atherosclerosis

HSPs are normally expressed in prokaryotic and eukaryotic cells under physiologi-

cal conditions as well as in cells exposed to various form of stress. HSPs were first

detected in Drosophila as a response to heat [73]. They are classified into various

families depending on their molecular weight. HSPs have a wide range of physio-

logical functions. They are involved in intracellular protein transport, protein

folding, cellular signaling, protein degradation, and also possess certain chaperone

functions. The members of the HSP60 (the 60 kDa HSP) family (mammalian

HSP60 (hSP60), the Mycobacterium tuberculosis homologue HSP65 (mHSP65),

Chlamydia pneumoniae (cHSP60), and the Escherichia Coli homologue (GroEL)

are highly conserved between all mammalian and bacterial species. Therefore,

extensive immunological cross-reactions between autologous and pathogenic

HSP60 is possible [74]. HSP60 is a mitochondria-bound protein, which can be

translocated to the cytoplasm and cell surface during different stress conditions.

The exact pathway, however, is still not completely understood (Figs. 27.4, 27.5).

All humans develop protective, beneficial adaptive immunity against the

phylogenetically highly conserved microbial HSP60 antigen via infection or

vaccination in addition to the immunity against organism-specific epitopes.

Under physiological conditions, vascular endothelial cells (ECs) do not express

HSP60. However, HSP60 expression can be induced on the EC surface when

stressed by classical atherosclerosis risk factors, such as mechanical stress, temper-

ature, oxygen radicals, infections, toxins, heavy metals, cigarette smoke, and

proinflammatory cytokines [75, 76]. Interestingly, the same stressors can also

simultaneously induce the expression of both HSP60 and adhesion molecules

(ICAM-1, ELAM-1, and VCAM-1) on the EC surface [77], providing prerequisites

for potentially bacterial/human HSP60 cross-reactive antibodies and destruction of

the EC by preexisting cellular and humoral immunity against HSP60, entailing

intimal infiltration by mononuclear cells. Thus, when HSP60 is expressed on

the cell-surface, it can act as a “danger signal” both for cellular and humoral

immune reactions. Hence protective, preexisting anti-HSP60 immunity may have

to be “paid for” by harmful (auto)immune cross-reactive attack on arterial ECs

538 C. Grundtman

maltreated by atherosclerosis risk factors. If atherosclerosis risk factors persist,

these early, still reversible inflammatory stage of atherosclerosis proceeds to plaque

formation with deleterious consequences and at later stages of atherogenesis,

intralesional T cells, macrophages, DCs, and SMC can express HSP60, and the

anti-HSP60 cellular immune reaction could therefore be perpetuated in situ

(Fig. 27.5). These experimentally and clinically proven findings represent the

basis for the “Autoimmune Concept of Atherosclerosis” [78]. Moreover, during

the last two decades HSP60 has been identified as one of the most important

antigens in early stages of atherosclerosis [76, 78, 79]. Proof of concept for the

presence of antigenic mimicry has been thoroughly investigated in different animal

models.

Early atherosclerotic lesions show a strong upregulation of hHSP60 and the

stress-inducible form hHSP70 in ApoE�/� mice [80]. The increased expression can

already be found before plaque formation is visually detected (3-week-old mice) at

lesion-prone sites, followed by a strong and hererogeneous expression in early to

B cell

Treg cell

APC

Macrophage

IFN-γIL-4

Tr1 cellCD4+CD25+

Foxp3+

IL-10TGF-β

Cytokines can induce a macrophage activation

TCR

MHCII

CD28CD40L

CD86/80CD40

HSP60

HSP60

HSP60 HSP60

HSP60

HSP60

TH1 cell

TH0 cell

TH2 cell

TH3 cell

Plasma cell

HSP60

Fig. 27.4 HSP60 immune activation in atherosclerosis. It is not fully understood to what

receptors HSP60 bind and how it is processed. However, the TLR-4/MyD88 pathway seems to

be important. T cells can react to non-self-HSP60 and self-HSP60 and these HSP60 peptides have

access to both MHC class I and class II molecules. When HSP60 peptide-MHC class I/II

complexes is expressed on the cell surface, they can be recognized by T cells. This leads to a

similar pathway as in Fig. 27.1, except that B cells will secrete HSP60 antigen-specific IgG and/or

IgM autoantibodies (Partly adapted from Servier Medical Art)


advanced fibrofatty plaques (8–20-week-old mice) in lesional ECs, macrophages,

smooth muscle cells (SMCs), and CD3+ T cells, with levels correlating to disease

severity. However, in advanced collagenous, acellular, calcified plaques

(40–69-week-old mice) the expression is markedly down-regulated. No expression

could be found in normocholesterolemic ApoE+/+ mice (3–69 weeks old) [80],

indicating that HSPsmight be a goodmarker for progression stages of atherosclerosis.

Tunica intima

Lumen


Lamina elastica

Smooth muscle cell

Monocyte/macrophage

Tunica intima

Endothelial cell


1. Classicalrisk factors

Lymph node

Plasma cell

Monocyte

10. Endothelial cell damage


T cell

9. sHSP60

Atherosclerosis – HSP60

CD4+

T cell MΦ

2. HSP60 surface expression

4. T cell infiltration

T cell APC MΦ

MΦ

7. Cytokine production

Cytokine production

8. Anti-HSP60 autoantibodies

6. Foam cell

T cell

CD4+

CD4+

Fig. 27.5 A schematic figure of the HSP60 pathway in atherosclerosis. All humans develop

protective, beneficial adaptive immunity against the phylogenetically highly conserved microbial

HSP60 antigen via infection or vaccination in addition to the immunity against organism-specific

epitopes. HSP60 is encoded in the nucleus but is expressed in mitochondria, and under physiolog-

ical conditions vascular ECs do not express HSP60 on the surface. (1) However, when stressed by

classical atherosclerosis risk factors, (2) HSP60 is transported into the cytosol and then appears on

the cell surface. (3) The ECs surface expression of HSP60 appears simultaneously with the

expression of adhesion molecules. (4) Activated T cells (mainly CD4+) are the first invaders of

the arterial intima in early atherosclerotic lesions (5, 6) followed only later by macrophages

and SMCs, the latter two are often transformed into foam cells in late, complicated plaques.

(7) Activated macrophages and T cells can also release a range of different proinflammatory

mediators both locally and into the circulation. (8) Also here, activated APCs can migrate to the

secondary lymphoid organs and stimulate B cells, to differentiate into plasma cells that can

produce autoantibodies to HSP60. (9) Increased soluble HSP60 (sHSP60) titers correlate with

the severity of atherosclerosis. (10) Stressed, but not unstressed ECs can be lysed by anti-HSP60

monoclonal or affinity purified polyclonal human anti-HSP60 antibodies in a complement-mediated

fashion or via antibody-dependent cellular cytotoxicity. Moreover, HSP60 may also function as an

inducer of anti-endothelial cell antibodies able to trigger cytotoxic and apoptotic responses when

recognized by the related autoantibodies (Partly adapted from Servier Medical Art)

540 C. Grundtman

Normocholesterolemic rabbits immunized with mHSP65 (in the present context,

mHSP65 is always used as a paradigmatic and potent representative of bacterial

HSP60) develop atherosclerotic plaques irrespective of their cholesterol levels [81]

and T cells isolated from these lesions specifically respond to HSP65 in vitro [82,

83], a finding similar to that in humans [84, 85]. Both C57BL/6 J mice, fed Western

diet, and LDLr�/� mice, fed normal chow diet, revealed enhanced early atheroscle-

rotic lesions after immunization with mHSP65 [86, 87]. hHSP60 immunization

together with Western diet resulted in an enhanced fatty streak formation in

C56BL/6NJcl mice [88]. Rats immunized with mHSP65 caused a brisk and

sustained humoral response together with increased neointimal growth [89]. Early

inflammatory stages of atherosclerotic lesions induced by mHSP65 immunization

can be regressed in the absence of additional risk factors for atherosclerosis and

T cell activation [90, 91]. Enhanced progression of atherosclerosis and an increase

in intralesional CD3+ T cells have been documented in C57BL/6 J, LDLr�/�, andApoE�/� mice after mHSP65 immunization. Transfer of these mHSP65 reactive

lymphocytes to syngenic mice led to an enhancement of fatty streak formation [92],

supporting a selective immunomodulation of the atherosclerotic plaques. Similarly,

high-titer Ig treatment with human anti-HSP60 autoantibodies can accelerate ath-

erosclerosis in ApoE�/� mice [93]. Furthermore, administration of a specific

monoclonal mouse antibody (II-13) that recognizes amino acid residues 288–266

of human HSP60 effectively induced atherosclerosis in ApoE�/� mice due to the

recognition of specific epitopes expressed on arterial ECs. II-13 injection resulted in

EC damage, followed by increased leukocyte attachment and accumulation

of macrophages and SMC in lesions, whereas another monoclonal antibody

(ML-30), which binds to amino acids 315–318 of HSP60, lacked cytotoxic effects

against cells in vitro [93]. Cross-reactive antibodies between bacterial/human

HSP60 can induce cytotoxic damage of stressed ECs [94, 95], indicating that

humoral immune reactions to bacterial HSPs may play an important role in

the process of vascular endothelial injury, which is believed to be a key event

in the pathogenesis of atherosclerosis (Fig. 27.5). Furthermore, it is important to

remember that HSPs are rather large proteins which, when processed, give rise to a

multitude of potential epitopes of which only a few are dominant. Different

epitopes from the same HSP in the same disease may therefore have very different

functional effects on the immune response, some being proinflammatory and others

tolerogenic. It is therefore essential to identify epitopes, rather than proteins, found

in a majority of patients and to characterize the appropriate immune response in

order to identify the most proinflammatory epitope if the induction of tolerance is

the desired goal.

27.7 HSP Tolerization

This new concept of atherogenesis gave rise to the idea that tolerization against

atherogenic HSP60 epitopes may be a plausible approach to preventing or

even treating atherosclerosis. This principle has been successfully applied in


hypercholesterolemic ApoE�/� and LDLr�/�mouse models by treating these either

intranasally or orally with whole mHSP65 preparations. In one study, LDLr�/�

mice were fed mHSP65 in different concentrations every other day for 10 days, and

after the last feeding, were challenged with either an immunization with Mycobac-terium tuberculosis (containing large amounts of bacterial HSP65) or recombinant

mHSP65 or by high-fat diet [96]. Oral tolerance with mHSP65 significantly

attenuated atherogenesis. Additionally, lymphocyte reactivity in mice fed with

mHSP65 and immunized either against mHSP65 or Mycobacterium tuberculosiswas significantly reduced and the specific HSP65 reactivity in the splenocytes was

reduced in these mice as well. Lymph node cells of treated mice produced more

IL-4 (TH2 cytokine) compared with non-tolerized cells. However, no suppressive

effect was seen on TH1 cytokine secretion, as evidenced by the unaltered interferon-

g (IFN-g) production [96]. These results are consistent with previous reports

suggesting that IL-4 plays a protective role in atherosclerosis [97]. Interestingly,

oral feeding with mHSP65 led to a suppression of high-fat diet-induced atheroscle-

rosis where spontaneous reactivity to mHSP65 was not evident compared to the

Mycobacterium tuberculosis and mHSP65-driven fatty-streak model. In another

study, the effect of both nasal and oral administration of mHSP65 was investigated

using LDLr�/� mice maintained on a high-cholesterol diet. The mice were orally

treated five times on consecutive days before a change of diet and once a week

thereafter. Nasally, the mice received three treatments every other day before the

change of diet and once a week thereafter. A significant decrease in the size of

atherosclerotic plaques, a reduction in macrophage-positive area in the aortic arch,

decreased IFN-g expression (TH1), increased IL-10 expression (TH2), and a

reduced number of CD4+ T cells were found in nasally treated mice. A similar

trend was observed in orally treated mice, even though it never reached significant

levels, except in the reduction of plaque size area [98]. Comparable results have

been shown in adjuvant arthritis (AA) after oral administration of mHSP65. Adju-

vant arthritis was suppressed due to reduced HSP65-specific IFN-g production and

increased IL-10 production [99]. Furthermore, decreased levels of anti-HSP

antibodies were detected in the nasally treated group. The antibodies showed a

TH2-phenotype pattern with significantly increased amounts of IgG1 antibodies,

which also is consistent with the cytokine profile found in these mice [98]. The

authors postulate that mucosal treatment with mHSP65 might stimulate the devel-

opment of adaptive immune cells that secrete anti-inflammatory cytokines (IL-10)

and that these cells can then migrate from mucosal inductive sites to the target

organ, the aorta, where they are restimulated by HSP to secrete anti-inflammatory

cytokines. The anti-inflammatory milieu in the vascular wall then leads to a

decrease in inflammatory IFN-g secreting cells which can result in an enhanced

secretion of IL-10 by macrophages and SMCs (Fig. 27.6). Furthermore, a strong

proinflammatory HSP 15-mer epitope (from the Escherichia Coli HSP dnaJ,

danJPI) has been described in rheumatoid arthritis (RA) patients [100]. This peptide

shares sequence homology with the shared epitope, a five amino acid stretch

common among RA-associated HLA alleles [101–103].

542 C. Grundtman

Recently, oral tolerance against mHSP60, HSP60 peptide 253–268, and HSP70

peptide 111–125 (100 mg/dose, respectively) was scrutinized. LDLr�/� mice were

orally treated with the respective protein/peptide four times in 8 days, after treat-

ment the mice were equipped with collars around both carotid arteries and fed

Western diet [104]. An 80% reduction in the plaque size in carotid arteries and a

27% reduction in the aortic root were achieved in mHSP60 and HSP60-peptide

treated mice. The reduction in plaque size correlated with an increase in

CD4+CD25+Foxp3+ regulatory T cells in several organs and an increased mRNA

expression of Foxp3, CD25, and CTLA-4 was found in atherosclerotic lesions of

treated mice. A T cell response, seen with a 13- and 9-fold increased T cell

proliferation, confirmed that mHSP60 but also the HSP60-peptide can induce a


Lamina elastica

Smooth muscle cell

Monocyte/macrophage

Tunica intima

Endothelial cell

3. Adhesion molecules ?

Lymph node

Plasma cell

T cellMonocyte

11. Endothelial cell damage ?


T cell

10. sHSP60

6. Foam cell

HSP60 tolerization

Treg

Treg

Treg

T cell MΦ

2. HSP60 surface expression ?

4. T cell infiltration/proliferation

Tr1/Treg

APC MΦ

MΦ

8. IL-4, IL-10, TGF-β

9. Anti-HSP60 IgG1(auto)antibodies

7. Tregs

1. Mucosal HSP60 treatment

Spleen 12. Specific HSP60 T cell response

T cell

T cell

8. IL-4, IL-10, TGF-β

Fig. 27.6 A schematic figure of HSP60 tolerization. (1) It is not clear if mucosal administration of

HSP60 leads to a (2, 3) reduced surface expression of HSP60 and adhesion molecule. However, a

(4–7) lower number of plaque T cells and macrophages and an increased number of T regulatory

cells (Tregs) have been documented. (8) Reduced levels of TH1 (IFN-g) and an increased level of

TH2 cytokines (IL-4, IL-10, TGF-b1) can be found locally, in secondary lymphoid organs, or in the

circulation. (9) Also, increased anti-HSP60 IgG1 (auto)antibodies are found in the circulation after

HSP60 treatment, (10) which may lead to a lower titer of sHSP60. (11) It is not yet elucidated if

mucosal tolerization against HSP60 leads to reduced EC damage; however, the increased titers of

anti-IgG1 (auto)antibodies may lead to decreased EC damage from sHSP60. (12) Decreased T cell

reactivity in the secondary lymphoid organs against HSP60 antigens indicates an induction of

tolerance to HSP60 (Partly adapted from Servier Medical Art)


specific T cell response; however, after oral treatment, mice showed a significant

reduction in proliferative responses to HSP60. Production of IL-10 and TGF-b by

lymph nodes cells in response to mHSP60 was also observed after tolerance

induction [104] (Fig. 27.6). Supporting data demonstrate that the induced

oxLDL-specific regulatory T cells after oral administration of oxLDL are responsi-

ble for the reduction in atherosclerotic plaque formation [48].

Nasal immunizations with mHSP65 have also show beneficial effects in rabbits.

Ten nasal administrations can effectively attenuate atherosclerosis in cholesterol-

fed wild-type rabbits. There was a 15% reduction in aortal lesion size after nasal

treatment. Suppression of T cell proliferation, increase of IL-10 production, and

absence of related antibodies implied a tolerance to mHSP65. Simultaneously, the

serum lipid levels were down-regulated in this group [105]. Results from another

group of rabbits nasally immunized with HSP65 + CTB-P277, a conjugated pro-

tein, used as a vaccine against autoimmune diabetes [106, 107], showed a lipid

reduction after immunization; however, no tolerance or reduction in lesion size was

found [105]. Thus, a reduction of lipids is not necessarily associated with immune

tolerance to HSP65 and therefore, the lipid reduction found in the HSP65-treated

group is probably a by-product of HSP65 immune interference, but not a conse-

quence or a combined phenomenon of HSP65-specific tolerance.

27.8 HSP Tolerization in Other Autoimmune Diseases

Mucosal treatment with HSP has been shown to induce tolerance not only in

atherosclerotic settings but also in several autoimmune diseases. For instance,

vaccination with HSPs has demonstrated beneficial effects in different arthritis

models [108]. Immunization experiments with Mycobacterium vaccae (a fast

growing mycobacterial strain in cattle expressing large amounts of HSP65 after

heating) revealed that protection or exacerbation of pristane-induced arthritis was

dependent on the dose (given in an oil or aqueous suspension), route, and time of

immunization [109]. Furthermore, T cell response has also been investigated in AA

after DNA vaccination with human HSP70 and HSP90. T cell immunity to HSP70

and to HSP90 induced an HSP60-specific T cell response that modulated the

arthritogenic response in AA. Similarly, DNA vaccination with HSP60 induced

HSP70-specific T cell immunity. Epitope mapping studies revealed that HSP60-

specific T cells induced by human HSP70 vaccination reacted with known regu-

latory HSP60 epitopes. This suggests that the regulatory mechanisms induced by

HSP60, HSP70, and HSP90 are reinforced by an immune network that connects

their reactivities [110]. HSP70 and its mechanisms have also been studied in

proteoglycan (PG)-induced arthritis (PGIA), a chronic and relapsing, T cell-

mediated murine model of arthritis. A single mHSP70 immunization resulted in

suppressed inflammation and tissue damage and resulted in an enhanced regulatory

response in PGIA as shown by the antigen-specific IL-10 production. Moreover,

544 C. Grundtman

HSP70-induced protection is critically IL-10 dependent [111]. Recently, a clinical

pilot phase II trial with the objection to induce immune deviation by mucosal

peptide-specific immunotherapy in active early RA patients was completed [100].

The peptide used, the dnaJP1 described above, was selected based on its ability to

produce MHC class II restricted antigen-specific T cells involved in the pathogene-

sis of RA. Thus, the patients had to show an in vitro responsiveness to danJP1,

defined as T cell proliferation and/or production of proinflammatory cytokines.

dnaJP1 was given orally for 6 months. Fifteen patients met the inclusion criteria and

were divided into three different dose groups (0.25, 2.5, and 25 mg/day) [100].

Immunological analysis at initial, intermediate, and end treatment points showed a

change from proinflammatory to regulatory T cell function. In fact, dnaJP1-induced

T cell production of IL-4 and IL-10 increased significantly when initial and end

treatment points were compared, whereas dnaJP1-induced T cell proliferation and

production of IL-2, IFN-g, and TNF decreased significantly. The total number of

dnaJP1-specific cells did not change over time, whereas expression of Foxp3 by

CD4+CD25bright cells increased, suggesting that the treatment affected regulatory T

cell function [100]. Conclusively, a T cell-dependent, proinflammatory pathway

can be specifically and safely modulated in patients with RA. Epitope-specific

mucosal therapy does not lead to an increased number of epitope-specific T cells,

but rather to a functional readjustment of the responding antigen-specific T cells.

Furthermore, this study and others [100, 112] show that committed TH1 cells can

still undergo phenotypic change, which previously was considered to be

impossible.

The ability of HSPs as immunogenic carrier molecules to regulate anti-

inflammatory immune response has recently been investigated. Preimmunization

with HSP65 could substitute for Bacillus Calmette-Guerin (BCG) in providing

effective priming for the induction of anti-malaria synthetic peptide (anti-NANP)

antibodies. Interestingly, both HSP65 and HSP70 acted as carrier molecules for

the induction of IgG antibodies to meningococcal group C oligosaccharides, in the

absence of adjuvants. These findings strongly suggest that the use of HSPs

as carriers in conjugated constructs for the induction of anti-peptide and anti-

oligosaccharide antibodies could be of value in the design of new vaccines for

possible use in humans [113]. HSP65 has been found to serve as a carrier for the

diabetogenic peptide P277-based vaccine and intranasal administration with P277

carried by HSP65 has been shown to decrease the incidence of diabetes, inhibit

insulitis, reduce IgG2a isotype antibodies to P277, and proinflammatory cytokines.

Intranasal vaccination with P277, in tandem repeat sequences carried by HSP65,

indicates that HSP65 may serve as a particularly advantageous carrier for P277-

based vaccines and mucosal administration may therefore be a useful therapeutic

approach for treatment of type 1 diabetes [114]. Moreover, there are increasing

numbers of reports suggesting that HSPs could also be beneficial in the treatment of

various forms of cancer.


27.9 Conclusion

Numerous studies have identified a role for the innate and adaptive immune

responses, both pro- and anti-atherogenic, in atherosclerosis. Common autoantigens

against which an immune response has been identified in animal and human models

of atherosclerosis include oxLDL, ApoB-100, b2-GPI, and HSP60. Activation of

atheroprotective adaptive immune responses has been demonstrated for all these

antigens. Conversely, atheroprotection has been demonstrated with the induction of

immune tolerance through activation of cellular and humoral immune reactions to

the same antigens. Recent identification of specific immunoreactive antigenic

epitopes of oxLDL, ApoB-100, and HSP60 antigens, and the induction of

atheroprotection by using them in immunization supports the idea that active

vaccination may emerge as a novel immuno-modulating atheroprotective strategy.

However, it is important to emphasize that the atherosclerotic process is a multifac-

torial phenomenon. Blocking one player in the process does not necessarily mean

that the final lesion can be prevented. Furthermore, we still do not fully understand

the intricate regulatory networks governing these tolerizations. A better understand-

ing of these networks would permit the elicitation of sustained protective immune

responses without causing excessive immune activation or inappropriate immune

tolerance. Our current knowledge of the intricacies characterizing atherosclerosis

and its specific antigen tolerization allows us to have just a glimpse of the tip of the

iceberg.

Acknowledgment This work is supported by the Austrian Research Fund (FWF; P19881-B05),

the EU Framework Program 6 (MOLSTROKE, LSHM-CT-2004-005206, EVGN; LSHM-CT-

2003-S03254), and the EU Framework Program 7, Large Scale Integrated Project: Novel

approaches to reconstitute normal immune function at old age (TOLERAGE Health research

grant; HEALTH-F4-2008-202156).

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