Editorial Slides VP Watch, March 26, 2003, Volume 3, Issue 12

Vaccination Against AtherosclerosisA Dream To Come True - Part I

–The central role of immune system in atherosclerosis is no longer a question.

– The question is how to develop new therapies to intervene with immune mechanisms involved in atherosclerosis and vulnerable plaque.

–Vaccination is the most exciting and challenging one.

– It is obvious developing a vaccine first requires discovering the antigen

– Several antigens have been suggested to trigger the immune response in atherosclerosis:

»Oxidized LDL»Heat Shock Protein»Beta II Glycoprotein»Microbial agents (C pneumonia,

CMV, HSV, H Pylori, influenza A virus,…)

–Out of the suspected list of antigens, modified or oxidized LDL has gained much more credence than others.

–The fact that Ox-LDL links the long-standing lipid hypothesis to the new inflammatory hypothesis, makes it even more suspect, however it is not yest accepted as the antigen.

• Goran Hansson 13 has recently reviewed the topic of vaccination against atherosclerosis: “Science or Fiction”? in an editorial accompanying the paper by Maron et al, 14where the authors describe immunization by mucosal administration of heat shock protein-65 in the low-density lipoprotein receptor-deficient mice.

–In apoE-null mice, induction of hypercholesterolemia by a high-fat diet results in a dramatic increase in autoantibodies against oxidized LDL.

–These autoantibodies are very prevalent in individuals with severe atherosclerosis2

Evidence for Immune Response against Oxidized LDL

Evidence for Immune Response against Oxidized LDL

• Antibodies against oxidized LDL in human serum (Palinski et al PNAS 1989)

• Activation of peripheral T cells by oxidized LDL (Frostegård et al ATVB 1992)

• Activation of T cells from human atherosclerotic plaques by oxidized LDL (Stemme et al PNAS 1995)

From Nilsson et al

Why Does Oxidized LDL Become Immunogenic?

• Fragmentation of apo B-100

• Oxidation of phospholipids

• Formation of covalent aldehyde (MDA and 4-HNE) and apo B-100 amino acid (lysine and histidine) adducts

From Nilsson et al

How Does Immunization with Oxidized LDL Affect Atherosclerosis?

• Reduced development of atherosclerosis in rabbits (Palinski et al 1995, Ameli et al 1996)

• Reduced development of atherosclerosis in apo E knockout mice (George et al 1998, Freigang et al 1998, Zhou et al 2001)

• Reduced intimal plaque development after vascular injury in rabbits (Nilsson et al 1997)

From Nilsson et al

Evidence for Antibody /B cell –Mediated Athero-protective Immunity

• IgG treatment inhibits atherosclerosis in apo E null mice (Nicoletti et al JCI, 1998)

• B cell substitution inhibits atherosclerosis in spleen-ectomized apo E null mice (Caliguri et al, JCI 2002)

• B cell substitution inhibits injury-induced plaque formation in lymphocyte deficient (Rag-1) mice (Dimayuga et al, ATVB 2002)

From Nilsson et al

Atherosclerosis in the absence of adaptive immunity?

(G K Hansson & J Nilsson, in: Crawford & DiMarco, Cardiology)

From G.K. Hannson

• A more recent described mechanism in hypercholesterolemic rabbits includes immunization with oxidized LDL, found to reduce atherosclerosis by 40-60%. 2, 3

• The above results have also been reproduced in the apoE-null 4 LDL receptor-null Mice 5.

• These findings raised the possibility of selective activation of atheroprotective immune response against oxidized LDL antigens.

- Studies of a number of laboratories have found that reactive aldehydes, products of LDL oxidation, form covalent adducts with portions of apoB and become targets for the immune system leading to cell damage and inflammation. 6, 7

As featured in VPWatch of this week, Fredrikson et al10 studied whether immune responses against a certain peptide of apoB-100 is atheroprotective. If positive, this will offer an exciting approach for a vaccine or immune therapy against atherosclerosis.

Aims of the study:

• Identify the structures in oxidized LDL recognized immune responses in man

• Develop ELISAs for analyses of antibodies against these structures

• Determine the relation between these immune responses and cardiovascular disease

• Develop an immunization therapy

From Nilsson et al

Development of ELISAs for analyses of antibodies against native and MDA-modified

apo B-100 peptide sequences

• 302 peptides, corresponding to the complete amino acid sequence of Apo B-100

• Each peptide consists of 20 amino acids with 5 amino acids overlap

• Native or MDA-modified peptides used for coating

• Screening of the ELISA library with pooled healthy control plasma

From Nilsson et al

Antibody binding to native and MDA-modified apo B-100 peptide

sequences• Antibody binding to over 100 different

sites in apo B-100 identified• IgM binds to more sites than IgG• No difference in binding to hydrophobic

and hydrophilic sequences• High covariation in binding to native and

MDA sequences

From Nilsson et al

Antibodies against apoB-100 peptides and CHD –clinical charcteristics of the study group

Subjects Cases Controls Number 78 149 Age (years) 61 (49 –67) 61 (49 –67) Male sex (%) 68 69 Current smokers (%) 27 27 Diabetes mellitus (%)

19

11

Hypertension(%) 64 64

Total cholesterol (mmol/L) 6.25 (3.47 – 8.24) 6.00 (4.08 – 9.90) LDL-cholesterol (mmol/L) 4.4 (1.7 – 6.2) 4.0 (1.6 – 7.6) HDL-cholesterol (mmol/L) 1.1 (0.6 – 2.5) 1.2 (0.6 –2.9) Triglycerides (mmol/L) 1.5 (0.5 – 10.0) 1.2 (0.4 – 7.3) Carotid ultrasonography

Common carotid intima-media thickness (mm)

0.81 (0.36 – 1.67) 0.82 (0.47 – 1.58)

Carotid plaques , (%) 64 40 From Nilsson et al

IgM Antibodies Against MDA-modified apoB-100 Peptides Decrease More

Rapidly with Age in CHD Cases

CHD casesCHD cases ControlsControls

Age

70605040Ig

M a

gain

st M

DA

pep

tide

32 (a

bs 4

05 n

m)

2,0

1,5

1,0

,5

0,0 Rsq = 0,0261

Age

70605040

IgM

aga

inst

MD

A p

eptid

e 32

(abs

405

nm

)

2,0

1,5

1,0

,5

0,0 Rsq = 0,3074

From Nilsson et al

Inverse Correlation Between IgM Antibodies against MDA-modified apoB-100 Peptides and

Oxidized LDL Levels in CHD Cases

Age

70605040

IgM

aga

inst

MD

A p

eptid

e 32

(abs

405

nm

)

2,0

1,5

1,0

,5

0,0 Rsq = 0,3074

Age (years)

70605040

Oxi

dize

d L

DL

-cho

lest

erol

(U /

L)

200

180

160

140

120

100

80

60

40

20

0 Rsq = 0,1984

IgM abIgM ab oxLDLoxLDL

From Nilsson et al

Immunization of apo E null mice with apo B-100 peptide sequences

• Immunization with 100 µg peptides at 6 and 9 weeks of age

• High cholesterol diet at 10 weeks of age

• Atherosclerosis assessed by Oil Red O staining of the descending aorta at 25 weeks of age

From Nilsson et al

Lipids, Body Weight and Serum Amyloid A in Immunized Apo E Null Mice

at the Age of 25 Weeks

Peptides Controls(n = 10) (n = 10)

Plasma-Cholesterol, (mg/mL) 4.38±1.58 4.57±1.33

HDL-Cholesterol, (mg/mL) 0.23±0.04 0.18±0.12

Triglycerides, (mg/mL) 0.76±0.13 0.80±0.12

Body weight, (g) 36.2±4.2 38.4±3.0

SAA, (µg/mL) 227±67 252±94

From Nilsson et al

Reduced plaque area in descending aorta of apoE null mice immunzed with Apo B-100 peptide

sequences

0.0

0.1

0.2

0.3

0.4

0.5

Controls Peptides

From Nilsson et al

• Atherosclerosis, as evaluated by Oil Red O staining was reduced by 60% in the descending aorta

• In the aorta between the left ventricle and the left subclavian, no difference was seen regarding Oil Red O staining, macrophage or lipid content.

• Collagen was increased in the subvalvular region compared to control animals.

Increased collagen content in subvalvular plaques of apoE null mice immunized with Apo B-100

peptide sequences

0.0

0.1

0.2

0.3

0.4

0.5

Controls Peptides

From Nilsson et al

• Total cholesterol and HDL cholesterol did not differ between both groups.

• Likewise, serum amyloid A did not differ between both groups.

• In a separate set of experiments using a peptide mixture (mainly nonhomegeneous) the immunization did not inhibit atherosclerosis suggesting that sequence homology is important for the atheroprotective effect of immunization.

• The peptide sequence used in this study is thought to be similar or identical to the portions of oxidized LDL responsible for activation of atheroprotective immune responses.

Discussion:• The authors speculate that antibodies

might facilitate removal of oxidized particles by macrophage Fc receptors in circulation before they accumulate in the vascular tissues.

• The lack of effect on the more advanced proximal regions of the aorta is interesting, probably suggesting that different mechanisms might play role in different stages of the disease.

• Immunization with a newly defined apoB-100 peptides resulted in an increase of specific IgG antibodies probably suggesting a T-Cell-dependent switch to synthesis of IgG antibodies against epitopes of oxidized LDL.

Conclusion:

• The demonstration of atheroprotection in this study with native peptide sequences adds to earlier studies showing the feasibility of athero-protection with immunization against oxidized LDL. 11, 12

Summary:

• There are encouraging models of vaccination against atherosclerosis.

• Vaccination against ApoB-100 is most promising and is well on its way to early stage of human clinical trials.

Summary:• Although clinical experience with other

diseases such as multiple sclerosis, rheumatoid arthritis and diabetes has not been yet very effective, the potential for reducing the incidence of atherosclerosis or more importantly its transition to vulnerable plaque is significant enough to call to launch an international cooperative project to explore this exciting opportunity that can lead to a heart attack free future for mankind.

• 1. http://www.vp.org/AHA2002/presentations/Towards%20Immune%20Modulation%20VP%2016-11%20GKH.ppt

• 2-Autoantibody against oxidised LDL and progression of carotid atherosclerosis.

• 3. Immunization of low density lipoprotein (LDL) receptor-deficient rabbits with homologous malondialdehyde-modified LDL reduces atherogenesis.

• 4. Effect of immunization with homologous LDL and oxidized LDL on early atherosclerosis in hypercholesterolemic rabbits.• 5. Hyperimmunization of apo-E-deficient mice with homologous malondialdehyde low-density lipoprotein suppresses early

atherogenesis.

• 6. 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.

• 7. Immune responses to oxidative neoepitopes on LDL and phospholipids modulate the development of atherosclerosis.• 8. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes.• 9. Protective immunity against atherosclerosis carried by B cells of hypercholesterolemic mice.• 10. Immunoglobulin treatment reduces atherosclerosis in apo E knockout mice.• 11. Inhibition of Atherosclerosis in ApoE-Null Mice by Immunization with ApoB-100 Peptide Sequences• 12. Effect of immunization with homologous LDL and oxidized LDL on early atherosclerosis in hypercholesterolemic rabbits.• 13. 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.• 14. Vaccination against atherosclerosis: science or fiction?• 15. Mucosal administration of heat shock protein-65 decreases atherosclerosis and inflammation in aortic arch of low-density

lipoprotein receptor-deficient mice.• 16. Immunization of low density lipoprotein (LDL) receptor-deficient rabbits with homologous malondialdehyde-modified LDL

reduces atherogenesis.• 17. Induction of arteriosclerosis in normocholesterolemic rabbits by immunization with heat shock protein 65.• 18. Mucosal administration of heat shock protein-65 decreases atherosclerosis and inflammation in aortic arch of low-density

lipoprotein receptor-deficient mice.• 19. Treatment of adjuvant-induced arthritis by oral administration of mycobacterial Hsp65 during disease.