SHAPE Society

1. Editorial Slides
VP Watch, March 26, 2003, Volume 3, Issue 12
Vaccination Against Atherosclerosis
A Dream To Come True - Part I

2. –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.

3. – 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,…)

4. –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.

5. • 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, 14
where the authors describe
immunization by mucosal administration
of heat shock protein-65 in the low-
density lipoprotein receptor-deficient
mice.

6. –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

7. 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

8. 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

9. 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

10. 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

11. Atherosclerosis in the absence of adaptive
immunity?
(G K Hansson & J Nilsson, in: Crawford & DiMarco, Cardiology)
From G.K. Hannson

12. • 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.

13. - 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

14. 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.

15. 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

16. 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

17. 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

18. 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

19. IgM Antibodies Against MDA-modified
apoB-100 Peptides Decrease More
Rapidly with Age in CHD Cases
CHD casesCHD cases ControlsControls
Age
70605040IgMagainstMDApeptide32(abs405nm)
2,0
1,5
1,0
,5
0,0 Rsq= 0,0261
Age
70605040
IgMagainstMDApeptide32(abs405nm)
2,0
1,5
1,0
,5
0,0 Rsq = 0,3074
From Nilsson et al

20. Inverse Correlation Between IgM Antibodies
against MDA-modified apoB-100 Peptides and
Oxidized LDL Levels in CHD Cases
Age
70605040
IgMagainstMDApeptide32(abs405nm)
2,0
1,5
1,0
,5
0,0 Rsq = 0,3074
Age (years)
70605040
OxidizedLDL-cholesterol(U/L)
200
180
160
140
120
100
80
60
40
20
0 Rsq = 0,1984
IgM abIgM ab oxLDLoxLDL
From Nilsson et al

21. 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

22. 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

23. 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

24. • 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.

25. 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

26. • Total cholesterol and HDL
cholesterol did not differ
between both groups.
• Likewise, serum amyloid A did
not differ between both groups.

27. • 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.

28. 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.

29. • 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.

30. 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

31. 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.

32. 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.

33. • 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.