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

CSMC--PKS “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

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

Immune Activation in Atherosclerosis Auto-antigensConsequences of Immune Response Hsp-60:Pro-atherogenic  2GP1 :Pro-atherogenic ox-LDL:??? Both innate and adaptive immune responses modulate atherosclerosis

CSMC--PKS 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 Phospholipid Macrophage Phospholipid Apo B 100 Neoantigens

CSMC--PKS Immunized N=9 Immunization of Cholesterol-fed Rabbits with Homologous LDL Substantially Reduces Aortic Atherosclerosis Despite Hypercholesterolemia Extent of Plaque (mm 2 ) Ameli, Shah, Nillson et al :ATVB 1996 Nilsson, Ameli, Shah et al: JACC 1997 Cholesterol1259mg/dl 1181mg/dl Control N=7 Apo B100 Cholesterol LDL Cholesterol Apo B100 Cholesterol Oxidized LDL Phospholipid -Antigen: 280 mcg LDL -Adjuvant: 700 mcg AdjuPrime -Primary SC Vaccination followed by 1 booster -Animals euthanzied 16 weeks after vaccination

CSMC--PKS Palinski, Witztum et al : PNAS 1995 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

CSMC--PKS 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

ATVB 2003 Peptide Peptide 210 Mixture

CSMC--PKS 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 wks8-9 wks25 wks Ist vaccination Booster Sacrifice

CSMC--PKS Immunization of Apo E Null Mice with Apo B-100 related Peptide Sequence : Effect on Cholesterol Levels and Aortic Atherosclerosis Alum (Control) Peptide 1 Peptide 2 Serum cholesterol mg/dl Immunization Group N=9 N=10 Alum (Control) Peptide 1Peptide 2 % of Aortic Surface Covered by Plaque Immunization Group P<0.01 N=9 N=10

CSMC--PKS Immunization of Apo E Null Mice with Apo B-100 related Peptide Sequence Reduces Atherosclerosis

CSMC--PKS Immunization of Apo E Null Mice with Apo B-100 related Peptide Sequence Reduces Plaque Inflammation and Increases Collagen Content Alum (Control) Peptide 1 Peptide 2 % Macrophage immunoreactivity Immunization Group p<0.05 N=9 N=10 Alum (Control) Peptide 1Peptide 2 % Collagen content (Trichrome) Immunization Group p<0.05 N=9 N=10

Immunization Group: Late Immunization of Apo E Null Mice with Apo B-100 related Peptide Sequence Attenuates Progression of Atherosclerosis Alum CtlPeptide 2 % Aortic Surface with Plaque 16 wk 30 wk Cholesterol (mg/dl): P<0.05

CSMC--PKS Mice receiving Splenocytes From Alum Immunized mice % of Aortic Surface Covered by Plaque P<0.01 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

Multiple Apo B-100 Related Peptide Antigens Have Athero-protective Effects in Apo E Null mice % Aortic Atherosclerosis Fredrickson, Shah, Nilsson et al : ATVB 2003

CSMC--PKS 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