A novel atheroprotective role of MF59-like adjuvant when co-administered with CETP vaccine in rabbit model of atherosclerosis

Document Type: Original Article


1 Nanotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

2 Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

3 Department of Anatomy, Faculty of Medicine, Birjand University of Medical Sciences, Birjand, Iran

4 Medical Toxicology Research Centre, Mashhad University of Medical Sciences, Mashhad, Iran

5 Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

6 Research and Education Department, Razavi Hospital, Mashhad, Iran

7 Hefdahe Shahrivar Hospital, Mashhad, Iran


Objective(s): In this study, for the first time, MF59 adjuvant was used to develop a cholesteryl ester transfer protein (CETP) vaccine. The efficacy of the vaccine was compared with the efficacy of CETP vaccine formulated with Alum/CpG, the formulation that its immunogenicity has been already demonstrated in rabbit and mice.
Materials and Methods: Tetanus toxoid- CETP peptide (TT-CETP) was mixed with Alum/CpG or MF59-like and administered subcutaneously for total five times in rabbit model of atherosclerosis. Anti-TT-CETP specific antibody, CETP activity in sera and mRNA level of cytokine IL-4 and IFN-γ in peripheral mononuclear cells were determined. Therapeutic response was also examined by tracking serum lipoprotein levels and pathologic observation of atherosclerotic lesions at aortic site.
Results: More anti-TT-CETP antibody was found in Alum/CpG vaccinated rabbits compared to buffer (P<0.001). Antibody induced by MF59-like formulation was not significantly higher than buffer. CETP activity and lipoprotein levels were not significantly different between vaccinated and control rabbits. The mRNA level of IL-4 was significantly lower than buffer while, IFN-γ gene expression was significantly higher in both vaccinated groups. Atherosclerosis thickness grade of aorta was dramatically lower than buffer (P<0.01) in both vaccinated groups.
Conclusion: It is concluded that MF59-adjuvanted CETP vaccine showed anti-atherosclerosis properties, but the protective effect could not be directly attributed to the immune response induced by anti TT-CETP antibody and CETP inhibition. Further studies are needed to explain the anti-atherosclerosis properties of MF59 in the presence of TT-CETP peptide.


1. Yu B-l, Wang S-h, Peng D-q, Zhao S-p. HDL and immunomodulation: an emerging role of HDL against atherosclerosis. Immunol cell biol 2010; 88:285-290.

2. Inazu A, Brown ML, Hesler CB, Agellon LB, Koizumi J, Takata K, et al. Increased high-density lipoprotein levels caused by a common cholesteryl-ester transfer protein gene mutation.  N Engl J Med 1990; 323:1234-1238.

3. Ryan US, Rittershaus CW. Vaccines for the prevention of cardiovascular disease. Vascul pharmacol 2006; 45:253-257.

4. Hansen MK, McVey MJ, White RF, Legos JJ, Brusq J-M, Grillot DA, et al. Selective CETP inhibition and PPARα agonism increase HDL cholesterol and reduce LDL cholesterol in human ApoB100/Human CETP transgenic mice. J Cardiovasc Pharmacol Ther 2010; 15:196-202.

5. Charles MA, Kane JP. New molecular insights into CETP structure and function: a review. J lipid res 2012; 53:1451-1458.

6. George M, Selvarajan S, Muthukumar R, Elangovan S. Looking into the crystal ball—upcoming drugs for dyslipidemia. J Cardiovasc Pharmacol Ther 2015:20:11-20.

7. Bell TA, Graham MJ, Lee RG, Mullick AE, Fu W, Norris D, et al. Antisense oligonucleotide inhibition of cholesteryl ester transfer protein enhances RCT in hyperlipidemic, CETP transgenic, LDLr−/− mice. J lipid res 2013; 54:2647-2657.

8. Rader DJ, deGoma EM. Future of cholesteryl ester transfer protein inhibitors. Annu Rrev Med 2014; 65:385-403.

9.   Mohammadpour AH, Akhlaghi F. Future of cholesteryl ester transfer protein (CETP) inhibitors: a pharmacological perspective. Clin Pharmacokinetics 2013; 52:615-626.

10. Rittershaus CW, Miller DP, Thomas LJ, Picard MD, Honan CM, Emmett CD, et al. Vaccine-induced antibodies inhibit CETP activity in vivo and reduce aortic lesions in a rabbit model of atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 2000;20:2106-2112.

11. Davidson MH, Maki K, Umporowicz D, Wheeler A, Rittershaus C, Ryan U. The safety and immuno-genicity of a CETP vaccine in healthy adults. Atherosclerosis. 2003; 169:113-120.

12. O'Hagan DT, Ott GS, Nest GV, Rappuoli R, Giudice GD. The history of MF59® adjuvant: a phoenix that arose from the ashes. Expert Rev Vaccines. 2013; 12:13-30

13. Schultze V, D’Agosto V, Wack A, Novicki D, Zorn J, Hennig R. Safety of MF59™ adjuvant. Vaccine 2008; 26:3209-3222.

14. Thomas LJ, Hammond RA, Forsberg EM, Geoghegan-Barek KM, Karalius BH, Marsh Jr HC, et al. Co-administration of a CpG adjuvant (VaxImmuneTM, CPG 7909) with CETP vaccines increased immunogenicity in rabbits and mice. Hum vaccin 2009; 5:79-84.

15. Calabro S, Tortoli M, Baudner BC, Pacitto A, Cortese M, O’Hagan DT, et al. Vaccine adjuvants alum and MF59 induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes. Vaccine 2011; 29:1812-1823.

16. Agnolon V, Bruno C, Leuzzi R, Galletti B, D’Oro U, Pizza M, et al. The potential of adjuvants to improve immune responses against TdaP vaccines: a preclinical evaluation of MF59 and monophosphoryl lipid A. Int J Pharm 2015;492:169-176.

17. Frey A, Di Canzio J, Zurakowski D. A statistically defined endpoint titer determination method for immunoassays. J immunol methods. 1998;221:35-41.

18. Heinicke E, Kumar U, Munoz D. Quantitative dot-blot assay for proteins using enhanced chemiluminescence. J Immunol Methods 1992; 152: 227-236.

19. Espino AM, Rivera F. Quantitation of cytokine mRNA by real-time RT-PCR during a vaccination trial in a rabbit model of fascioliasis. Vet Parasitol 2010;169:82-92.

20. Leary S, Underwood W, Anthony R, Cartner S, Corey D, Temple G, et al. AVMA guidelines for the euthanasia of animals: 1st edition 2013.

21. Chekanov VS. Low frequency electrical impulses reduce atherosclerosis in cholesterol fed rabbits. Med Sci Monit 2003;9:BR302-BR9.

22. Tzimas G, Afshar M, Emadali A, Chevet E, Vali H, Metrakos P, editors. Correlation of cell necrosis and tissue calcification with ischemia/reperfusion injury after liver transplantation. Transplant Proc 2004; 36:1766-1768.

23. Tzimas GN, Afshar M, Chevet E, Emadali A, Vali H, Metrakos PP. Graft calcifications and dysfunction following liver transplantation. BMC surg 2004; 4:4-9.

24. Gaofu Q, Dan M, Jie W, Liao Z, Li Z, Roque RS, et al. Long-lasting specific antibodies against CETP induced by subcutaneous and mucosal administration of a 26-amino acid CETP epitope carried by heat shock protein 65 kDa in the absence of adjuvants. Vaccine 2004; 22:3187-3194.

25. Gaofu Q, Jun L, Xin Y, Wentao L, Jie W, Xiuyun Z,  et al. Vaccinating rabbits with a cholesteryl ester transfer protein (CETP) B-Cell epitope carried by heat shock protein-65 (HSP65) for inducing anti-CETP antibodies and reducing aortic lesions in vivo. J Cardiovasc Pharmacol 2005; 45:591-598.

26. Gaofu Q, Jun L, Xiuyun Z, Wentao L, Jie W, Jingjing L. Antibody against cholesteryl ester transfer protein (CETP) elicited by a recombinant chimeric enzyme vaccine attenuated atherosclerosis in a rabbit model. Life Sci 2005; 77:2690-2702.

27. Gaofu Q, Rongyue C, Dan M, Xiuyun Z, Xuejun W, Jie W, et al. Asparaginase display of human cholesteryl ester transfer protein (CETP) B cell epitopes for inducing high titers of anti-CETP antibodies in vivo. Protein Pept Lett 2006;13:149-154.

28. Liaw Y-W, Lin C-Y, Lai Y-S, Yang T-C, Wang C-J, Whang-Peng J, et al. A vaccine targeted at CETP alleviates high fat and high cholesterol diet-induced atherosclerosis and non-alcoholic steatohepatitis in rabbit. PloS One 2014;9:e111529.

29. Mao D, Kai G, Gaofu Q, Zheng Z, Li Z, Jie W, et al. Intramuscular immunization with a DNA vaccine encoding a 26-amino acid CETP epitope displayed by HBc protein and containing CpG DNA inhibits atherosclerosis in a rabbit model of atherosclerosis. Vaccine. 2006; 24:4942-4950.

30. Yuan X, Yang X, Cai D, Mao D, Wu J, Zong L, et al. Intranasal immunization with chitosan/pCETP nanoparticles inhibits atherosclerosis in a rabbit model of atherosclerosis. Vaccine 2008; 26:3727-3734.

31. Rittershaus CW. Vaccines for cholesterol management. World J Surg. 2007;31:690-694.

32. Tsai TF. Fluad®-MF59®-adjuvanted influenza vaccine in older adults. Infect Chemother. 2013;45:159-174.

33. Hebsur S, Vakil E, Oetgen WJ, Kumar PN, Lazarous DF. Influenza and coronary artery disease: exploring a clinical association with myocardial infarction and analyzing the utility of vaccination in prevention of myocardial infarction.  Rev Cardiovasc Med 2013; 15:168-175.

34. Futado JJ. Influenza vaccines for preventing cardiovascular disease. Sao Paulo Med J 2015;133: 384.

35. Grau AJ, Fischer B, Barth C, Ling P, Lichy C, Buggle F. Influenza vaccination is associated with a reduced risk of stroke. Stroke. 2005;36:1501-1506.

36. Nichol KL, Nordin J, Mullooly J, Lask R, Fillbrandt K, Iwane M. Influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly.  N Engl J Med. 2003;348:1322-1332.

37. Loomba RS, Aggarwal S, Shah PH, Arora RR. Influenza Vaccination and Cardiovascular Morbidity and Mortality Analysis of 292 383 Patients. J cardiovas pharmacol  ther 2012;17:277-283.

38. Puig-Barberà J, Díez-Domingo J, Varea ÁB, Chavarri GS, Rodrigo JAL, Hoyos SP, et al. Effectiveness of MF59™-adjuvanted subunit influenza vaccine in preventing hospitalisations for cardiovascular disease, cerebro-vascular disease and pneumonia in the elderly. Vaccine. 2007; 25:7313-7321.

39. Podda A. The adjuvanted influenza vaccines with novel adjuvants: experience with the MF59-adjuvanted vaccine. Vaccine 2001; 19:2673-2680.

40. Pellegrini M, Nicolay U, Lindert K, Groth N, Della Cioppa G. MF59-adjuvanted versus non-adjuvanted influenza vaccines: integrated analysis from a large
safety database. Vaccine 2009;27:6959-6965.

41. Tuttolomondo A, Di Raimondo D, Pecoraro R, Arnao V, Pinto A, Licata G. Atherosclerosis as an inflammatory disease. Curr pharm des 2012; 18:4266-4288.

42. Keijzer C, Van Der Zee R, Van Eden W, Broere F. Treg inducing adjuvants for therapeutic vaccination against chronic inflammatory diseases. Front immunol 2013; 4:245.

43. Keijzer C, Spiering R, Silva AL, van Eden W, Jiskoot W, Vervelde L, et al. PLGA nanoparticles enhance the expression of retinaldehyde dehydro-genase enzymes in dendritic cells and induce FoxP3+ T-cells in vitro. J  Control Release 2013;168:35-40.

44. Hjorth M, Axelsson S, Rydén A, Faresjö M, Ludvigsson J, Casas R. GAD-alum treatment induces GAD 65-specific CD4+ CD25 high FOXP3+ cells in type 1 diabetic patients. Clin Immunol 2011; 138:117-126.

45. Fousteri G, Dave A, Bot A, Juntti T, Omid S, Von Herrath M. Subcutaneous insulin B: 9-23/IFA immunisation induces Tregs that control late-stage prediabetes in NOD mice through IL-10 and IFNγ. Diabetologia 2010; 53:1958-1970.

46. Khallou-Laschet J, Tupin E, Caligiuri G, Poirier B, Thieblemont N, Gaston A-T, et al. Atheroprotective effect of adjuvants in apolipoprotein E knockout mice. Atherosclerosis 2006;184:330-341.

47. Wigren M, Bengtsson D, Dunér P, Olofsson K, Björkbacka H, Bengtsson E, et al. Atheroprotective effects of Alum are associated with capture of oxidized LDL antigens and activation of regulatory T cells. Circ res 2009;104:e62-e70.

48. Williams GR, Kubajewska I, Glanville NS, Johnston SL, Mclean GR. The potential for a protective vaccine for rhinovirus infections. Expert Rev Vaccines. 2016;15:569-571.