Pharmaceutical Research

, Volume 26, Issue 6, pp 1477–1485 | Cite as

MF59 Emulsion Is an Effective Delivery System for a Synthetic TLR4 Agonist (E6020)

  • Barbara C. Baudner
  • Vanessa Ronconi
  • Daniele Casini
  • Marco Tortoli
  • Jina Kazzaz
  • Manmohan Singh
  • Lynn D. Hawkins
  • Andreas Wack
  • Derek T. O’Hagan
Research Paper



The effectiveness of vaccines depends on the age and immunocompetence of the vaccinee. Conventional non-adjuvanted influenza vaccines are suboptimal in the elderly and vaccines with improved ability to prevent influenza are required. The TLR4 agonist E6020, either given alone or co-delivered with MF59, was evaluated and compared to MF59 and the TLR9 agonist CpG. Its ability to enhance antibody titres and to modulate the quality of the immune response to a subunit influenza vaccine was investigated.


Mice were immunized with either antigens alone, with MF59 or with the TLR agonists alone, or with a combination thereof. Serum samples were assayed for IgG antibody titres and hemagglutination inhibition (HI) titres. Th1/Th2 type responses were determined by titrating IgG subclasses in serum samples and by T-cell cytokine responses in splenocytes.


MF59 was the best single adjuvant inducing HI and T-cell responses in comparison to all alternatives. The co-delivery of E6020 or CpG with MF59 did not further increase antibody titres however shifted towards a more Th1 based immune response.


Combining adjuvants like E6020 and MF59 allowed a finer tuning of the immune response towards a particular Th bias, thus have significant implications for the development of improved influenza vaccines.


adjuvants influenza vaccine delivery MF59 toll like receptor agonists T-cell cytokine response 



We are grateful to Giorgio Corsi for help with the artwork Gillis Otten and Kathyrn Patton for statistical analysis and to Markus Hilleringmann for critical reading of the manuscript.


  1. 1.
    I. Kang, M. S. Hong, H. Nolasco, S. H. Park, J. M. Dan, J. Y. Choi, and J. Craft. Age-associated change in the frequency of memory CD4+ T cells impairs long term CD4+ T cell responses to influenza vaccine. J. Immunol. 173:673–681 (2004).PubMedGoogle Scholar
  2. 2.
    K. Goodwin, C. Viboud, and L. Simonsen. Antibody response to influenza vaccination in the elderly: a quantitative review. Vaccine. 24:1159–1169 (2006).PubMedCrossRefGoogle Scholar
  3. 3.
    K. G. Nicholson, J. M. Wood, and M. Zambon. Influenza. Lancet. 362:1733–1745 (2003).PubMedCrossRefGoogle Scholar
  4. 4.
    A. Banzhoff, P. Nacci, and A. Podda. A new MF59-adjuvanted influenza vaccine enhances the immune response in the elderly with chronic diseases: results from an immunogenicity meta-analysis. Gerontology. 49:177–184 (2003).PubMedCrossRefGoogle Scholar
  5. 5.
    D. T. O’Hagan, and N. M. Valiante. Recent advances in the discovery and delivery of vaccine adjuvants. Nat. Rev. Drug Discov. 2:727–735 (2003).PubMedCrossRefGoogle Scholar
  6. 6.
    B. Guy. The perfect mix: recent progress in adjuvant research. Nat. Rev. Microbiol. 5:505–517 (2007).PubMedCrossRefGoogle Scholar
  7. 7.
    D. T. O’Hagan. MF59 is a safe and potent vaccine adjuvant that enhances protection against influenza virus infection. Expert Rev. Vaccines. 6:699–710 (2007).PubMedCrossRefGoogle Scholar
  8. 8.
    G. Ott, R. Radhakrishnan, J. H. Fang, and M. Hora. The adjuvant MF59: A 10-year perspective. Methods Mol. Med. 42:211–228 (2000).Google Scholar
  9. 9.
    S. De Donato, D. Granoff, M. Minutello, G. Lecchi, M. Faccini, M. Agnello, F. Senatore, P. Verweij, B. Fritzell, and A. Podda. Safety and immunogenicity of MF59-adjuvanted influenza vaccine in the elderly. Vaccine. 17:3094–3101 (1999).PubMedCrossRefGoogle Scholar
  10. 10.
    A. Podda, G. Del Giudice, and D. T. O’Hagan. MF59: a safe and potent adjuvant for human use. In V. Schijns, and D. T. O’Hagan (eds.), Immunopotentiators in Modern Vaccines, Academic, Oxford, 2006, pp. 149–159.CrossRefGoogle Scholar
  11. 11.
    A. Podda, and G. Del Giudice. MF59-adjuvanted vaccines: increased immunogenicity with an optimal safety profile. Expert Rev. Vaccines. 2:197–203 (2003).PubMedCrossRefGoogle Scholar
  12. 12.
    A. Podda, and G. Del Giudice. MF59 adjuvant emulsion. In M. M. Levine, J. B. Kaper, R. Rappuoli, M. A. Liu, and M. F. Good (eds.), New Generation Vaccines, 3Marcel Dekker, New York, 2004, pp. 225–236.Google Scholar
  13. 13.
    D. K. Mitchell, S. J. Holmes, R. L. Burke, A. M. Duliege, and S. P. Adler. Immunogenicity of a recombinant human cytomegalovirus gB vaccine in seronegative toddlers. Pediatr. Infect. Dis. J. 21:133–138 (2002).PubMedCrossRefGoogle Scholar
  14. 14.
    E. J. McFarland, W. Borkowsky, T. Fenton, D. Wara, J. McNamara, P. Samson, M. Kang, L. Mofenson, C. Cunningham, A. M. Duliege, F. Sinangil, S. A. Spector, E. Jimenez, Y. Bryson, S. Burchett, L. M. Frenkel, R. Yogev, F. Gigliotti, K. Luzuriaga, R. A. Livingston, and AIDS Clinical Trials Group 230 Collaborators. Human immunodeficiency virus type 1 (HIV-1) gp120-specific antibodies in neonates receiving an HIV-1 recombinant gp120 vaccine. J. Infect. Dis. 184:1331–1335 (2001).PubMedCrossRefGoogle Scholar
  15. 15.
    A. Iwasaki, and R. Medzhitov. Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5:987–995 (2004).PubMedCrossRefGoogle Scholar
  16. 16.
    T. Kaisho, and S. Akira. Regulation of dendritic cell function through Toll-like receptors. Curr. Mol. Med. 3:373–385 (2003).PubMedCrossRefGoogle Scholar
  17. 17.
    D. M. Underhill. Toll-like receptors: networking for success. Eur. J. Immunol. 33:1767–1775 (2003).PubMedCrossRefGoogle Scholar
  18. 18.
    J. T. Evans, C. W. Cluff, D. A. Johnson, M. J. Lacy, D. H. Persing, and J. R. Baldridge. Enhancement of antigen-specific immunity via the TLR4 ligands MPL adjuvant and Ribi.529. Expert Rev. Vaccines. 2:219–229 (2003).PubMedCrossRefGoogle Scholar
  19. 19.
    CERVARIX® Product Information Human Papillomavirus Vaccine Types 16 and 18 (Recombinant, AS04 adjuvanted) GlaxoSmithKline Australia Pty Ltd. (2007).Google Scholar
  20. 20.
    M. Kundi. New hepatitis B vaccine formulated with an improved adjuvant system. Expert Rev. Vaccines. 6:133–140 (2007).PubMedCrossRefGoogle Scholar
  21. 21.
    N. Garçon, P. Chomez, and M. Van Mechelen. GlaxoSmithKline Adjuvant Systems in vaccines: concepts, achievements and perspectives. Expert Rev. Vaccines. 6:723–739 (2007).PubMedCrossRefGoogle Scholar
  22. 22.
    C. K. Fraser, K. R. Diener, M. P. Brown, and J. D. Hayball. Improving vaccines by incorporating immunological coadjuvants. Expert Rev. Vaccines. 6:559–578 (2007).PubMedCrossRefGoogle Scholar
  23. 23.
    M. Przetak, J. Chow, H. Cheng, J. Rose, L. D. Hawkins, and S. T. Ishizaka. Novel synthetic LPS receptor agonists boost systemic and mucosal antibody responses in mice. Vaccine. 21:961–970 (2003).PubMedCrossRefGoogle Scholar
  24. 24.
    L. D. Hawkins, S. T. Ishizaka, P. McGuinness, H. Zhang, W. Gavin, B. DeCosta, Z. Meng, H. Yang, M. Mullarkey, D. W. Young, H. Yang, D. P. Rossignol, A. Nault, J. Rose, M. Przetak, J. C. Chow, and F. Gusovsky. A novel class of endotoxin receptor agonists with simplified structure, Toll-like receptor 4-dependent immunostimulatory action, and adjuvant activity. J. Pharmacol. Exp. Ther. 300:655–661 (2002).PubMedCrossRefGoogle Scholar
  25. 25.
    S. T. Ishizaka, and L. D. Hawkins. E6020: a synthetic Toll-like receptor 4 agonist as a vaccine adjuvant. Expert Rev. Vaccines. 6:773–784 (2007).PubMedCrossRefGoogle Scholar
  26. 26.
    A. Wack, B. C. Baudner, A. K. Hilbert, I. Manini, S. Nuti, S. Tavarini, H. Scheffczik, M. Ugozzoli, M. Singh, J. Kazzaz, E. Montomoli, G. Del Giudice, R. Rappuoli, and D. T. O’Hagan. Combination adjuvants for the induction of potent, long-lasting antibody and T-cell responses to influenza vaccine in mice. Vaccine. 26:552–561 (2008).PubMedCrossRefGoogle Scholar
  27. 27.
    M. Dupuis, D. M. McDonald, and G. Ott. Distribution of adjuvant MF59 and antigen gD2 after intramuscular injection in mice. Vaccine. 18:434–439 (1999).PubMedCrossRefGoogle Scholar
  28. 28.
    Session 1, R.B. Couch. Correlates of protection against seasonal influenza and Summary of 1. Day. (accessed 12/17/08).
  29. 29.
    R. A. Bright, D. M. Carter, C. J. Crevar, F. R. Toapanta, J. D. Steckbeck, K. S. Cole, N. M. Kumar, P. Pushko, G. Smith, T. M. Tumpey, and T. M. Ross. Cross-clade protective immune responses to influenza viruses with H5N1 HA and NA elicited by an influenza virus-like particle. PLoS ONE. 3(1):e1501 (2008).PubMedCrossRefGoogle Scholar
  30. 30.
    P. G. Thomas, R. Keating, D. J. Hulse-Post, and P. C. Doherty. Cell-mediated protection in influenza infection. Emerg. Infect. Dis. 12:48–54 (2006).PubMedGoogle Scholar
  31. 31.
    P. C. Doherty, S. J. Turner, R. G. Webby, and P. G. Thomas. Influenza and the challenge for immunology. Nat. Immunol. 7:449–455 (2006).PubMedCrossRefGoogle Scholar
  32. 32.
    J. Bennink. Cell mediated immunity to influenza in mice: T-cell specific responses that correlate with protection. (accessed 12/17/08).
  33. 33.
    E. De Gregorio, E. Tritto, and R. Rappuoli. Alum adjuvanticity: Unraveling a century old mystery. Eur. J. Immunol. 38:2068–2071 (2008).PubMedCrossRefGoogle Scholar
  34. 34.
    M. E. Pichichero. Improving vaccine delivery using novel adjuvant systems. Hum. Vaccin. 4:262–70 (2008).PubMedGoogle Scholar
  35. 35.
    N. Thönes, A. Herreiner, L. Schädlich, K. Piuko, and M. Müller. A direct comparison of human papillomavirus type 16 L1 particles reveals a lower immunogenicity of capsomeres than viruslike particles with respect to the induced antibody response. J. Virol. 82:5472–5485 (2008).PubMedCrossRefGoogle Scholar
  36. 36.
    G. Ott, G. L. Barchfeld, D. Chernoff, R. Radhakrishnan, P. van Hoogevest, and G. Van Nest. Design and evaluation of a safe and potent adjuvant for human vaccines. In M. F. Powell, and M. J. Newman (eds.), Vaccine Design: The Subunit and Adjuvant Approach, Plenum, New York, 1995.Google Scholar
  37. 37.
    M. Singh, M. Ugozzoli, J. Kazzaz, J. Chesko, E. Soenawan, D. Mannucci, F. Titta, M. Contorni, G. Volpini, G. Del Guidice, and D. T. O’Hagan. A preliminary evaluation of alternative adjuvants to alum using a range of established and new generation vaccine antigens. Vaccine. 10:1680–1686 (2006).CrossRefGoogle Scholar
  38. 38.
    G. H. Wong, and D. V. Goeddel. Tumour necrosis factors alpha and beta inhibit virus replication and synergize with interferons. Nature. 323:819–822 (1986).PubMedCrossRefGoogle Scholar
  39. 39.
    J. E. McElhaney, D. Xie, W. D. Hager, M. B. Barry, Y. Wang, A. Kleppinger, C. Ewen, K. P. Kane, and R. C. Bleackley. T cell responses are better correlates of vaccine protection in the elderly. J. Immunol. 176:6333–6339 (2006).PubMedGoogle Scholar
  40. 40.
    A. S. Evans. Serologic studies of acute respiratory infections in military personnel. Yale J. Biol. Med. 48:201–209 (1975).PubMedGoogle Scholar
  41. 41.
    T. Sugai, M. Mori, M. Nakazawa, M. Ichino, T. Naruto, N. Kobayashi, Y. Kobayashi, M. Minami, and S. Yokota. A CpG-containing oligodeoxynucleotide as an efficient adjuvant counterbalancing the Th1/Th2 immune response in diphtheria–tetanus–pertussis vaccine. Vaccine. 23:5450–5456 (2005).PubMedCrossRefGoogle Scholar
  42. 42.
    C. Coban, K. J. Ishii, A. W. Stowers, D. B. Keister, D. M. Klinman, and N. Kumar. Effect of CpG oligodeoxynucleotides on the immunogenicity of Pfs25, a Plasmodium falciparum transmission-blocking vaccine antigen. Infect. Immun. 72:584–588 (2004).PubMedCrossRefGoogle Scholar
  43. 43.
    J. Kazzaz, M. Singh, M. Ugozzoli, J. Chesko, E. Soenawan, and D. T. O’Hagan. Encapsulation of the immune potentiators MPL and RC529 in PLG microparticles enhances their potency. J. Control Release. 110:566–573 (2006).PubMedCrossRefGoogle Scholar
  44. 44.
    A. Seubert, E. Monaci, M. Pizza, D. T. O’Hagan, and A. Wack. The adjuvants aluminum hydroxide and MF59 induce monocyte and granulocyte chemoattractants and enhance monocyte differentiation toward dendritic cells. J. Immunol. 180:5402–5412 (2008).PubMedGoogle Scholar
  45. 45.
    F. Mosca, E. Tritto, A. Muzzi, E. Monaci, F. Bagnoli, C. Iavarone, D. O’Hagan, R. Rappuoli, and E. De Gregorio. Molecular and cellular signatures of human vaccine adjuvants. Proc. Natl. Acad. Sci. U. S. A. 105:10501–10506 (2008).PubMedCrossRefGoogle Scholar
  46. 46.
    N. K. Tong, J. Beran, S. A. Kee, J. L. Miguel, C. Sánchez, J. M. Bayas, A. Vilella, J. R. de Juanes, P. Arrazola, F. Calbo-Torrecillas, E. L. de Novales, V. Hamtiaux, M. Lievens, and M. Stoffel. Immunogenicity and safety of an adjuvanted hepatitis B vaccine in pre-hemodialysis and hemodialysis patients. Kidney Int. 68:2298–2303 (2005).PubMedCrossRefGoogle Scholar
  47. 47.
    N. Bourne, G. N. Milligan, L. R. Stanberry, R. Stegall, and R. B. Pyles. Impact of immunization with glycoprotein D2/AS04 on herpes simplex virus type 2 shedding into the genital tract in guinea pigs that become infected. J. Infect. Dis. 192:2117–2123 (2005).PubMedCrossRefGoogle Scholar
  48. 48.
    T. J. Kemp, A. García-Piñeres, R. T. Falk, S. Poncelet, F. Dessy, S. L. Giannini, A. C. Rodriguez, C. Porras, R. Herrero, A. Hildesheim, L. A. Pinto, and Costa Rica Vaccine Trial (CVT) Group. Evaluation of systemic and mucosal anti-HPV16 and anti-HPV18 antibody responses from vaccinated women. Vaccine. 26:3608–3616 (2008).PubMedCrossRefGoogle Scholar
  49. 49.
    S. L. Giannini, E. Hanon, P. Moris, M. Van Mechelen, S. Morel, F. Dessy, M. A. Fourneau, B. Colau, J. Suzich, G. Losonksy, M. T. Martin, G. Dubin, and M. A. Wettendorff. Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only. Vaccine. 24:5937–5949 (2006).PubMedCrossRefGoogle Scholar
  50. 50.
    S. H. Seo, and R. G. Webster. Tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells. J. Virol. 76:1071–1076 (2002).PubMedGoogle Scholar
  51. 51.
    F. G. Hayden, R. Fritz, M. C. Lobo, W. Alvord, W. Strober, and S. E. Straus. Local and systemic cytokine responses during experimental human influenza A virus infection. Relation to symptom formation and host defense. J. Clin. Invest. 101:643–649 (1998).PubMedCrossRefGoogle Scholar
  52. 52.
    M. R. Betts, M. C. Nason, S. M. West, S. C. De Rosa, S. A. Migueles, J. Abraham, M. M. Lederman, J. M. Benito, P. A. Goepfert, M. Connors, M. Roederer, and R. A. Koup. HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood. 107:4781–4789 (2006).PubMedCrossRefGoogle Scholar
  53. 53.
    S. C. De Rosa, F. X. Lu, J. Yu, S. P. Perfetto, J. Falloon, S. Moser, T. G. Evans, R. Koup, C. J. Miller, and M. Roederer. Vaccination in humans generates broad T cell cytokine responses. J. Immunol. 173:5372–5380 (2004).PubMedGoogle Scholar
  54. 54.
    G. Pantaleo, and R. A. Koup. Correlates of immune protection in HIV-1 infection: what we know, what we don’t know, what we should know. Nat. Med. 10:806–810 (2004).PubMedCrossRefGoogle Scholar
  55. 55.
    A. Harari, V. Dutoit, C. Cellerai, P. A. Bart, R. A. Du Pasquier, and G. Pantaleo. Functional signatures of protective antiviral T-cell immunity in human virus infections. Immunol. Rev. 211:236–254 (2006).PubMedCrossRefGoogle Scholar
  56. 56.
    K. G. Nicholson, A. E. Colegate, A. Podda, I. Stephenson, J. Wood, E. Ypma, and M. C. Zambon. Safety and antigenicity of non-adjuvanted and MF59-adjuvanted influenza A/Duck/Singapore/97 (H5N3) vaccine: a randomised trial of two potential vaccines against H5N1 influenza. Lancet. 357:1937–1943 (2001).PubMedCrossRefGoogle Scholar
  57. 57.
    I. Stephenson, K. G. Nicholson, A. Colegate, A. Podda, J. Wood, E. Ypma, and M. Zambon. Boosting immunity to influenza H5N1 with MF59-adjuvanted H5N3 A/Duck/Singapore/97 vaccine in a primed human population. Vaccine. 21:1687–1693 (2003).PubMedCrossRefGoogle Scholar
  58. 58.
    I. Stephenson, K. G. Nicholson, R. Bugarini, A. Podda, J. Wood, M. Zambon, and J. M. Katz. Cross reactivity to highly pathogenic avian influenza H5N1 viruses following vaccination with non-adjuvanted and MF-59-adjuvanted influenza A/Duck/Singapore/97 (H5N3) vaccine: a potential priming strategy. J. Infect. Dis. 191:4962–4970 (2005).CrossRefGoogle Scholar
  59. 59.
    WHO. 2nd WHO meeting on evaluation of pandemic influenza prototype vaccines in clinical trials. WHO, Geneva, 2006.Google Scholar
  60. 60.
    R. L. Atmar, W. A. Keitel, S. M. Patel, J. M. Katz, D. She, H. El Sahly, J. Pompey, T. R. Cate, and R. B. Couch. Safety and immunogenicity of nonadjuvanted and MF59-adjuvanted influenza A/H9N2 vaccine preparations. Clin. Infect. Dis. 43:1135–1142 (2006).PubMedCrossRefGoogle Scholar
  61. 61.
    D. T. O’Hagan, M. Singh, J. Kazzaz, M. Ugozzoli, M. Briones, J. Donnelly, and G. Ott. Synergistic adjuvant activity of immunostimulatory DNA and oil/water emulsions for immunization with HIV p55 gag antigen. Vaccine. 20:3389–3398 (2002).PubMedCrossRefGoogle Scholar
  62. 62.
    T. C. Heineman, M. L. Clements-Mann, G. A. Poland, R. M. Jacobson, A. E. Izu, D. Sakamoto, J. Eiden, G. A. Van Nest, and H. H. Hsu. A randomized, controlled study in adults of the immunogenicity of a novel hepatitis B vaccine containing MF59 adjuvant. Vaccine. 17:2769–2778 (1999).PubMedCrossRefGoogle Scholar
  63. 63.
    A. G. Langenberg, R. L. Burke, S. F. Adair, R. Sekulovich, M. Tigges, C. L. Dekker, and L. Corey. A recombinant glycoprotein vaccine for herpes simplex virus type 2: safety and immunogenicity [corrected]. Ann. Intern. Med. 122:889–898 (1995).PubMedGoogle Scholar
  64. 64.
    L. Corey, A. G. Langenberg, R. Ashley, R. E. Sekulovich, A. E. Izu, J. M. Douglas Jr., H. H. Handsfield, T. Warren, L. Marr, S. Tyring, R. DiCarlo, A. A. Adimora, P. Leone, C. L. Dekker, R. L. Burke, W. P. Leong, and S. E. Straus. Recombinant glycoprotein vaccine for the prevention of genital HSV-2 infection: two randomized controlled trials. Chiron HSV Vaccine Study Group. JAMA. 282:331–340 (1999).PubMedCrossRefGoogle Scholar
  65. 65.
    W. Borkowsky, D. Wara, T. Fenton, J. McNamara, M. Kang, L. Mofenson, E. McFarland, C. Cunningham, A. M. Duliege, D. Francis, Y. Bryson, S. Burchett, S. A. Spector, L. M. Frenkel, S. Starr, R. Van Dyke, and E. Jimenez. Lymphoproliferative responses to recombinant HIV-1 envelope antigens in neonates and infants receiving gp120 vaccines. AIDS Clinical Trial Group 230 Collaborators. J. Infect. Dis. 181:890–896 (2000).PubMedCrossRefGoogle Scholar
  66. 66.
    C. K. Cunningham, D. W. Wara, M. Kang, T. Fenton, E. Hawkins, J. McNamara, L. Mofenson, A. M. Duliege, D. Francis, E. J. McFarland, W. Borkowsky, and Pediatric AIDS Clinical Trials Group 230 Collaborators. Safety of 2 recombinant human immunodeficiency virus type 1 (HIV-1) envelope vaccines in neonates born to HIV-1-infected women. Clin. Infect. Dis. 32:801–807 (2001).PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Barbara C. Baudner
    • 1
  • Vanessa Ronconi
    • 1
  • Daniele Casini
    • 1
  • Marco Tortoli
    • 1
  • Jina Kazzaz
    • 2
  • Manmohan Singh
    • 2
  • Lynn D. Hawkins
    • 3
  • Andreas Wack
    • 1
  • Derek T. O’Hagan
    • 1
  1. 1.Novartis VaccinesSienaItaly
  2. 2.Novartis VaccinesCambridgeUSA
  3. 3.Eisai Research InstituteAndoverUSA

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