This document discusses research into identifying immune responses against oxidized LDL in humans and developing an immunization therapy to inhibit atherosclerosis. The researchers developed ELISAs to analyze antibodies against native and MDA-modified apo B-100 peptides in the LDL protein. They found over 100 sites in apo B-100 that antibodies bound to, and binding correlated with age and oxidized LDL levels in cardiovascular disease cases. Immunizing apo E null mice with specific MDA-modified peptides reduced atherosclerosis development and plaque formation compared to controls. The findings suggest developing an atherosclerosis vaccine based on activating protective immune responses against oxidized LDL.

1. Inhibition of Atherosclerosis in ApoE-Inhibition of Atherosclerosis in ApoE-
Null Mice by Immunization withNull Mice by Immunization with
ApoB-100 Peptide SequencesApoB-100 Peptide Sequences
Gunilla Nordin FredriksonGunilla Nordin Fredrikson
andand
Jan NilssonJan Nilsson

2. The early atherosclerotic plaque - theThe early atherosclerotic plaque - the
inflammatory responseinflammatory response
LDL oxidation and aggregation
LDL
VCAM-1

3. The atherosclerotic plaque – immuneThe atherosclerotic plaque – immune
responses against oxidized LDLresponses against oxidized LDL
LDL
Oxidized LDL
T cell
Macrophage
MHC II
T cell
receptor
Oxidized LDL
peptide

4. Evidence for immune response againstEvidence for immune response against
oxidized LDLoxidized LDL
Antibodies against oxidized LDL in humanAntibodies against oxidized LDL in human
serum (Palinski et al PNAS 1989)serum (Palinski et al PNAS 1989)
Activation of peripheral T cells by oxidizedActivation of peripheral T cells by oxidized
LDL (Frostegård et al ATVB 1992)LDL (Frostegård et al ATVB 1992)
Activation of T cells from humanActivation of T cells from human
atherosclerotic plaques by oxidized LDLatherosclerotic plaques by oxidized LDL
(Stemme et al PNAS 1995)(Stemme et al PNAS 1995)

5. Why does oxidized LDL becomeWhy does oxidized LDL become
immunogenic?immunogenic?
Fragmentation of apo B-100Fragmentation of apo B-100
Oxidation of phospholipidsOxidation of phospholipids
Formation of covalent aldehyde (MDA and 4-Formation of covalent aldehyde (MDA and 4-
HNE) and apo B-100 amino acid (lysine andHNE) and apo B-100 amino acid (lysine and
histidine) adductshistidine) adducts

6. How does immunization with oxidizedHow does immunization with oxidized
LDL affect atherosclerosis?LDL affect atherosclerosis?
Reduced development of atherosclerosis inReduced development of atherosclerosis in
rabbits (Palinski et al 1995, Ameli et al 1996)rabbits (Palinski et al 1995, Ameli et al 1996)
Reduced development of atherosclerosis in apoReduced development of atherosclerosis in apo
E knockout mice (George et al 1998, FreigangE knockout mice (George et al 1998, Freigang
et al 1998, Zhou et al 2001)et al 1998, Zhou et al 2001)
Reduced intimal plaque development afterReduced intimal plaque development after
vascular injury in rabbits (Nilsson et al 1997)vascular injury in rabbits (Nilsson et al 1997)

7. Evidence for antibody /B cell –Evidence for antibody /B cell –
mediated athero-protective immunitymediated athero-protective immunity
IgG treatment inhibits atherosclerosis in apo EIgG treatment inhibits atherosclerosis in apo E
null mice (Nicoletti et al JCI, 1998)null mice (Nicoletti et al JCI, 1998)
B cell substitution inhibits atherosclerosis inB cell substitution inhibits atherosclerosis in
spleen-ectomized apo E null mice (Caliguri etspleen-ectomized apo E null mice (Caliguri et
al, JCI 2002)al, JCI 2002)
B cell substitution inhibits injury-inducedB cell substitution inhibits injury-induced
plaque formation in lymphocyte deficient (Rag-plaque formation in lymphocyte deficient (Rag-
1) mice (Dimayuga et al, ATVB 2002)1) mice (Dimayuga et al, ATVB 2002)

8. Aims of the projectAims of the project
Identify the structures in oxidized LDLIdentify the structures in oxidized LDL
recognized immune responses in manrecognized immune responses in man
Develop ELISAs for analyses of antibodiesDevelop ELISAs for analyses of antibodies
against these structuresagainst these structures
Determine the relation between these immuneDetermine the relation between these immune
responses and cardiovascular diseaseresponses and cardiovascular disease
Develop an immunization therapyDevelop an immunization therapy

9. Development of ELISAs for analyses ofDevelopment of ELISAs for analyses of
antibodies against native and MDA-antibodies against native and MDA-
modified apo B-100 peptide sequencesmodified apo B-100 peptide sequences
302 peptides, corresponding to the complete302 peptides, corresponding to the complete
amino acid sequence of Apo B-100amino acid sequence of Apo B-100
Each peptide consists of 20 amino acids with 5Each peptide consists of 20 amino acids with 5
amino acids overlapamino acids overlap
Native or MDA-modified peptides used forNative or MDA-modified peptides used for
coatingcoating
Screening of the ELISA library with pooledScreening of the ELISA library with pooled
healthy control plasmahealthy control plasma

10. Antibody binding to native and MDA-Antibody binding to native and MDA-
modified apo B-100 peptide sequencesmodified apo B-100 peptide sequences
Antibody binding to over 100 different sites inAntibody binding to over 100 different sites in
apo B-100 identifiedapo B-100 identified
IgM binds to more sites than IgGIgM binds to more sites than IgG
No difference in binding to hydrophobic andNo difference in binding to hydrophobic and
hydrophilic sequenceshydrophilic sequences
High covariation in binding to native and MDAHigh covariation in binding to native and MDA
sequencessequences

11. Antibodies against apoB-100 peptides andAntibodies against apoB-100 peptides and
CHD –clinical charcteristics of the studyCHD –clinical charcteristics of the study
groupgroup
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

12. IgM antibodies against MDA-modifiedIgM antibodies against MDA-modified
apoB-100 peptides decrease more rapidlyapoB-100 peptides decrease more rapidly
with age in CHD caseswith 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

13. Inverse correlation between IgM antibodiesInverse correlation between IgM antibodies
against MDA-modified apoB-100 peptidesagainst MDA-modified apoB-100 peptides
and oxidized LDL levels in CHD casesand 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

14. Immunization of apo E null mice withImmunization of apo E null mice with
apo B-100 peptide sequencesapo B-100 peptide sequences
Immunization with 100 µg peptides at 6Immunization with 100 µg peptides at 6
and 9 weeks of ageand 9 weeks of age
High cholesterol diet at 10 weeks of ageHigh cholesterol diet at 10 weeks of age
Atherosclerosis assessed by Oil Red OAtherosclerosis assessed by Oil Red O
staining of the descending aorta at 25staining of the descending aorta at 25
weeks of ageweeks of age

15. Lipids, body weight and serum amyloid A inLipids, body weight and serum amyloid A in
immunized apo E null miceimmunized apo E null mice
at the age of 25 weeks.at the age of 25 weeks.
PeptidesPeptides ControlsControls
((nn = 10)= 10) ((nn = 10)= 10)
Plasma-Cholesterol,Plasma-Cholesterol, (mg/mL)(mg/mL) 4.38±1.584.38±1.58 4.57±1.334.57±1.33
HDL-Cholesterol,HDL-Cholesterol, (mg/mL)(mg/mL) 0.23±0.040.23±0.04 0.18±0.120.18±0.12
Triglycerides,Triglycerides, (mg/mL)(mg/mL) 0.76±0.130.76±0.13 0.80±0.120.80±0.12
Body weight,Body weight, (g)(g) 36.2±4.236.2±4.2 38.4±3.038.4±3.0
SAA,SAA, (µg/mL)(µg/mL) 227±67227±67 252±94252±94

16. Reduced plaque area in descending aorta of apoEReduced plaque area in descending aorta of apoE
null mice immunzed with Apo B-100 peptidenull mice immunzed with Apo B-100 peptide
sequencessequences
0.0
0.1
0.2
0.3
0.4
0.5
Controls Peptides
***

17. Increased collagen content in subvalvular plaquesIncreased collagen content in subvalvular plaques
of apoE null mice immunized with Apo B-100of apoE null mice immunized with Apo B-100
peptide sequencespeptide sequences
0
10
20
30
40
50
***
Controls Peptides
Trichromestainedareain
subvalvularplaques(%)

18. Activation of athero-protective immuneActivation of athero-protective immune
responses against MDA-apo B-100responses against MDA-apo B-100
fragmentsfragments
Immunization with oxidized LDL has previouslyImmunization with oxidized LDL has previously
been found to reduce atherosclerosis in animalsbeen found to reduce atherosclerosis in animals
Human oxidized LDL antibodies were found toHuman oxidized LDL antibodies were found to
bind to specific MDA-modified peptidebind to specific MDA-modified peptide
sequences in apo B-100sequences in apo B-100
Antibodies to these sequences correlated to ageAntibodies to these sequences correlated to age
and oxidized LDL in plasmaand oxidized LDL in plasma
Immunization with these MDA apo B-100Immunization with these MDA apo B-100
peptide sequences inhibited atherosclerosis inpeptide sequences inhibited atherosclerosis in
apo E null miceapo E null mice
The findings suggest the possibility to develop aThe findings suggest the possibility to develop a
vaccine-based therapy for atherosclerosisvaccine-based therapy for atherosclerosis

19. Department of Medicine, UMAS, Lund University
Gunilla Nordin Fredrikson
Jan Nilsson
Ragnar Alm
Mikko Ares
Göran Berglund
Paul Dimayuga
Bo Hedblad
Isabel Goncalves
Marie Lindholm
Alexandru Schiopu
Ingrid Söderberg
Division of Cardiology, Cedars-Sinai Medical Center, UCLA
Bojan Cercek
Prediman K Shah
Kuang-Yuh Chyu