SHAPE Society

1. P.K.Shah, MD
Director, Division of Cardiology and
Atherosclerosis Research Center
Cedars Sinai Medical Center, Los Angeles
““Immunomodulation of Atherosclerosis”Immunomodulation of Atherosclerosis”
AEHA-AHA-Nov 12-2005, Dallas

2. “Vaccines for infectious diseases
are likely to be the most important
medical contribution to public health
during the last 100 years -------------”
Nilsson J , Hansson G K , Shah PK: ATVB 2004; 25: 1-11
Vaccine for Atherosclerosis

3. Yin and the Yang of Immune System in Atherosclerosis
Adaptive ImmunityInnate Immunity
Toll like receptors
(TLR) Scavenger Receptors
(SR-A, CD 36)
T-cells B-cells
Macrophages
Dendritic Cells
CRP
Natural
Antibody

4. Immune Activation in AtherosclerosisImmune Activation in Atherosclerosis
Auto-antigens Consequences of
Immune Response
Hsp-60: Pro-atherogenic
β2GP1 : Pro-atherogenic
ox-LDL: ???
Both innate and adaptive immune responses modulate atherosclerosis

5. Apo B100
Cholesterol
LDL cholesterol
Apo B100
Cholesterol
Oxidized LDL
Immune Recognition
B-cells T-cells
(antibodies) (cytokines)
Plaque
Formation
Immune Response to Oxidized /MDA-LDL
PhospholipidPhospholipid
Macrophage
Phospholipid Apo B 100
Neoantigens Neoantigens

6. Immunized
N=9
Immunization of Cholesterol-fed Rabbits with Homologous LDL SubstantiallyImmunization of Cholesterol-fed Rabbits with Homologous LDL Substantially
Reduces Aortic Atherosclerosis Despite HypercholesterolemiaReduces Aortic Atherosclerosis Despite Hypercholesterolemia
ExtentofPlaque(mm2
)
Ameli, Shah, Nillson et al :ATVB 1996
Nilsson , Ameli, Shah et al: JACC 1997
Cholesterol 1259mg/dl 1181mg/dl
Control
N=7
Apo B100
Cholesterol
LDL Cholesterol
Apo B100
Cholesterol
Oxidized LDL
Phospholipid Phospholipid
-Antigen: 280 mcg LDL
-Adjuvant: 700 mcg AdjuPrime
-Primary SC Vaccination followed by 1 booster
-Animals euthanzied 16 weeks after vaccination

7. CSMC--PKS
Palinski , Witztum et al : PNAS 1995
0
10
20
30
40
50
60
70
Control MDA-LDL
Rabbits Immunized Rabbits
(N=11) (N=14)
Immunization of LDL-Receptor Deficient (Watanabe Rabbits) with
Homologous Malondialdehyde (MDA) Modified LDL Reduces Atherogenesis
% of Aortic Surface with Plaque
P<0.005

8. Apo B100
LDL Cholesterol
Cholesterol
302 Peptides, 20 amino acids long with 5 amino acid
overlap simulating the entire amino acid sequence of
human Apo B 100 were synthesized.
Using an ELISA with peptides sequences as antigens,
antibodies to 101 of these peptide sequences were
identified in pooled human sera
Several peptide sequences were then used to create vaccines
for Immunization in apo E null mice fed a high
cholesterol diet
( Collaborative Research Program between
Cedars Sinai Medical Center (P.K.Shah) and
University of Lund (Jan Nilsson) )
Hypothesis: Specific antigenic epitopes on Apo B 100 component of
LDL provoke athero-protective immune response
Phospholipid

9. ATVB 2003
Peptide 143 + Peptide 210 Mixture

10. Immunization of Apo E Null Mice with Apo B-100 related
Peptide Sequence Reduces Atherosclerosis
EEEMLENVSLVCPKDATRFK
ATRFKHLRKYTYNYQAQSSS
Peptide 1
Peptide 2
Mouse Apo B 100
Homology
75%
85%
Alum used as adjuvant
6-7 wks 8-9 wks 25 wks
Ist vaccination Booster Sacrifice

11. Immunization of Apo E Null Mice with Apo B-100 related Peptide Sequence :
Effect on Cholesterol Levels and Aortic Atherosclerosis
0
200
400
600
800
1000
1200
1400
1600
Alum
(Control)
Peptide 1 Peptide 2
Serum cholesterol mg/dl
Immunization Group
N=9 N=10 N=10
0
0.5
1
1.5
2
2.5
3
3.5
Alum
(Control)
Peptide 1 Peptide 2
% of Aortic Surface Covered by Plaque
Immunization Group
P<0.01
N=9 N=10 N=10

12. Immunization of Apo E Null Mice with Apo B-100 related
Peptide Sequence Reduces Atherosclerosis

13. Immunization of Apo E Null Mice with Apo B-100 related
Peptide Sequence Reduces Plaque Inflammation and Increases Collagen Content
0
2
4
6
8
10
12
14
Alum
(Control)
Peptide 1 Peptide 2
% Macrophage immunoreactivity
Immunization Group
p<0.05
N=9 N=10 N=10 0
5
10
15
20
25
30
35
40
45
Alum
(Control)
Peptide 1 Peptide 2
% Collagen content (Trichrome)
Immunization Group
p<0.05
N=9 N=10 N=10

14. Immunization Group:
Late Immunization of Apo E Null Mice with Apo B-100 related
Peptide Sequence Attenuates Progression of Atherosclerosis
0
2
4
6
8
10
12
14
Alum Ctl Peptide 2
% Aortic Surface with Plaque
16 wk 30 wk 16 wk 30 wk
Cholesterol (mg/dl): 1274 930 1274 989
P<0.05

15. 0
0.5
1
1.5
2
2.5
3
3.5
Mice receiving
Splenocytes
From Alum
Immunized mice
% of Aortic Surface Covered by Plaque
P<0.01
N=9 N=9
Mice receiving
Splenocytes
From Peptide 2
Immunized mice
Adoptive Transfer of Splenocytes from Peptide 2 Immunized Mice Reduces
Atherosclerosis in Recipient Unimmunized Apo E Null Mice
Mice receiving
Splenocytes
From Peptide 1
Immunized mice
N=9

16. Multiple Apo B-100 Related Peptide Antigens Have
Athero-protective Effects in Apo E Null mice
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Control Peptide 45 Peptide 74 Peptide
210
Peptide
240
Peptides
11, 25,74
Peptides
30-34
Peptides
143,210
% Aortic Atherosclerosis Fredrickson, Shah, Nilsson et al : ATVB 2003

17. Conclusions
Acknowledgements
Kuang-Yuh Chyu , MD, PhD (Cedars Sinai)
Xiaoning Li, PhD(Cedars Sinai)
Juliana Yano,BS (Cedars-Sinai)
Gunilla Nordin-Fredrickson, MD, PhD (Sweden)
Jan Nilsson, MD, PhD (Sweden)
-Immune system plays a complex role in atherosclerosis with pro-atherogenic
and athero-protective effects
-Immunization using LDL/ox-LDL and specific Apo B-100 related peptide
sequences reduces atherosclerosis and favorably modifies plaque composition
- Immunotherapy of atherosclerosis warrants further investigation
Michael and Jane Eisner Foundation