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  1. 1. Vaccination against interleukin 12 attenuates atherosclerosis in LDL receptor deficient mice
  2. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 16. Vaccination against cells overexpressing VEGFR2 attenuates atherosclerosis
  17. 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. 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. 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. 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. 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