SHAPE Society

1. Vaccination against interleukin 12
attenuates atherosclerosis in LDL
receptor deficient mice

2.  Daily administration of IL-12 accelerates atherosclerosis in ApoE-/-
mice
Lee T et al, Arterioscler Thromb
Vasc Biol. (1999); 19:734-42
IL-12 and atherosclerosis
 IL-12-/-
/ApoE-/-
mice show less atherosclerosis in the aortic root
than apoE-/-
mice
Davenport P et al. Am J Pathol (2003); 163:1117-25
250
50
200
150
100 (n=10)
(n=10)
(n=12)
(n=12)
Aortic sinus Arch
*
*
Lesionarea,103
µm2
control
IL-12

3. Role of Il-12 in atherosclerosis
Macrophage
IL-12
Th0
Th2
Th1
IFN-γ
Atherosclerosis
Macrophage
activation
(+ IL-18)
Pro-inflammatory cytokines
Chemokines
• EC
• VSMC
Anti-inflammatory cytokines
IL-10
-
+
+
+
-
Dendritic
cell

4. Aim of study
Inhibition of atherosclerosis by blocking IL-12 function
IL-12
Th0
Th2
Th1
IFN-γ
Atherosclerosis
Macrophage
activation
(+IL-18)
Pro-inflammatory cytokines
Chemokines
• EC
• VSMC
Anti-inflammatory cytokines
IL-10
Macrophage
Dendritic
cell

5. Deceptive antigen presentation by B cells
Anti-
self-IL-12
B cell
MHC II
Anti-non-self
T Cell help
IL-12 (self antigen)
‘Self-peptide complex’
PADRE (T cell epitope)
Anti-IL-12 antibodies
Anti-
self-IL-12
B cell

6. 0.0 0.1 0.2 0.3 0.4 0.5 0.6
p40 + p40 comp
p40 + IL-12 comp
p40
IL-12 + p40 comp
IL-12 + IL-12 comp
IL-12
Absorption at 450 nm
AntiIL-12IgGELISA
titer(x103
)
Anti-IL-12 vaccination induces IL-12 antibodies
Specificity of
anti-IL-12 response
Control Vaccinated
Induction of
anti-IL-12 antibodies

7. ..
Anti-IL-12 vaccination blocks IL-12 function
AntiIL-12inhibition
(reciprocaltiter)
Control Vaccinated
Proliferation inhibition of IL-12
responsive cells
Methods:
IL-12 responsive BaF/3 cells
Incubate with IL-12
Add serum from vaccinated or
control mice
Determine proliferation

8. Anti-IL-12 vaccination blocks IL-12 function
IFN-γ post IL-12
IFN-γ(pg/ml)
0
500
1000
1500
2000
2500
ControlVaccinated
*
Method
IL-12 vaccinated or control
vaccinated mice
IL-12 (500 ng) administration
every day during 3 days
After 1 day:
Collect blood
IFN-γ levels determined

9. Carotid artery
0.5 mm
Collar
placement
2.0 mm
-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7
Western type
diet
Anti-IL-12 vaccination and atherogenesis
weeks
Vaccination
against IL-12
Intra muscular
injections with
PADRE IL-12 in
combination with
adjuvant
LDLr-/-
mice
Sacrifice +
lesion analysis

10. 0
2
4
6
8
10
12
ControlVaccinated
SerumIFN-γlevel(pg/ml)
*
Anti-IL-12 vaccination blocks IL-12 function
Serum IFN-γ levels 8 weeks after the
last vaccination against IL-12

11. Anti-IL-12 vaccination attenuates atherosclerosis
Control IL-12 Vaccinated
Control Vaccinated
Lesionarea(x103
)(µm2
)
0
10
20
30
40
50
60
70 P<0.01
*
LESION AREA
Intima/lumen
0.00
0.25
0.50
0.75
1.00
P<0.01
*
VaccinatedControl
I/L RATIO

12. Anti-IL-12 vaccination
leads to a more stable phenotype
collagen
0.0
2.5
5.0
7.5
10.0
Control Vaccinated
α-Actn/intima(x10-2
)
Control Vaccinated
P<0.01
*
0.0
0.1
0.2
0.3
0.4
Collagen/intima
P<0.01
*
α-actin
Control Vaccinated

13. 100 µm 20 µm
IFN-γ positive
lesions
IFN-γ negative
lesions
Control 6 6
Vaccinated 0 10
Anti-IL-12 vaccination decreases the
inflammatory status of the plaque
p<0.05

14. Conclusions
 Vaccination with PADRE IL-12 complex in combination with the
correct adjuvant emulsion MPL/QS21 induces antibodies that block
the function of IL-12
 Vaccination against IL-12 attenuates atherosclerosis in LDLr-/-
mice
 Functional blockade of IL-12 results in lesions with a more stable
phenotype, illustrated by a higher collagen and smooth muscle cells
content
 Blockade of IL-12 results in attenuated inflammatory status of the
atherosclerotic plaque, reflected by reduced IFN-γ staining

15. Future
 Effect of IL-12 vaccination on pre-existing lesions and long-term
effects of vaccination
 Control the degree and length of vaccination
 Side-effects: infections

16. Vaccination against cells overexpressing
VEGFR2 attenuates atherosclerosis

17. DNA vaccination against VEGFR2
CD8+
T-cell
Breaking of tolerance and
induction of VEGFR2
specific cytotoxic T-cells
M-cell
M∅
Phagocytosis by M-cells in GI tract
and transfer to macrophages (M∅)
Attenuated Salmonella typhimurium
transformed with pcDNA3.1-VEGFR2
Bacterial lysis, activation of
M∅, expression of VEGFR2
M∅
MHC-1

18. Control VEGFR2 vaccinated
Neointimaarea(µm2
)
Control VEGFR2
Vacc.
P=0.03
*
Collar induced carotid lesions
63.3% reduction in neointima area
Vaccination against VEGFR2 and
de novo atherogenesis

19. Acknowledgments
The Scripps Research
Institute (La Jolla)
Ralph Reisfeld
Leiden University
Paula de Vos, Arnaud Hauer, Gijs van Puijvelde, Ingrid Michon, Niels Peterse, Eva
van Wanrooij, Miranda Stitzinger, Thomas van Es, Kim Habets, Theo van
Berkel
Ludwig Institute for Cancer
Research (Brussels, Belgium)
Catherine Uyttenhove
Jean-Christophe Renauld
Vincent Stroobant
Jacques van Snick
The Netherlands Heart Foundation
(Grant 2000D040)

20. Acknowlegdments
Division of Biopharmaceutics (Leiden, The Netherlands)
Arnaud D. Hauer
Paula de Vos
Theo J.C. van Berkel
Ludwig Institute for Cancer Research (Brussels, Belgium)
Catherine Uyttenhove
Jean-Christophe Renauld
Vincent Stroobant
Jacques van Snick
The Netherlands Heart Foundation
(Grant 2000D040)

21. Side effects
Recessively inherited IL-12Rß1 mutations
Disseminated non-tuberculous mycobacterial infections, tuberculosis, and
Salmonella infections occur in the setting of IL-12 deficiency or
unresponsiveness
(BCG vaccination protect them against mycobacteria)
Heterozygous carriers: healthy!
IL-12 deficiency
quite variable: from mild to overwhelming infections
Anti-Il-12 treatment:
7 weeks treatment: anti-IL-12 monoclonal antibody may induce clinical
responses in patients with active Crohn's disease. Associated with
decreases in Th1-mediated inflammatory cytokines at the site of disease.
NO ADVERSE EFFECTS