AAPS PharmSciTech

, Volume 13, Issue 2, pp 498–506 | Cite as

Immunomodulatory and Physical Effects of Phospholipid Composition in Vaccine Adjuvant Emulsions

  • Christopher B. FoxEmail author
  • Susan L. Baldwin
  • Malcolm S. Duthie
  • Steven G. Reed
  • Thomas S. Vedvick
Research Article


Egg phosphatidylcholine is commonly used as an emulsifier in formulations administered parenterally. However, synthetic phosphatidylcholine (PC) emulsifiers are now widely available and may be desirable substitutes for egg-derived phospholipids due to stability, purity, and material source considerations. In earlier work, we demonstrated that a squalene–1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) emulsion provided equivalent physical stability compared to a squalene–egg PC emulsion. In the present manuscript, we evaluate the physical stability of vaccine adjuvant emulsions containing a range of other synthetic phosphatidylcholine emulsifiers. Besides the POPC emulsion, the 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) emulsion showed good particle size and visual stability compared to emulsions made with other synthetic phospholipids. Moreover, comparable immune responses were elicited by squalene emulsions employing various synthetic PC or egg PC emulsifiers in combination with an inactivated influenza vaccine or a recombinant malaria antigen, and these responses were generally enhanced compared to antigen without adjuvant. Therefore, we show that (1) some synthetic PCs (DMPC, POPC, and to a lesser extent 1,2-dioleoyl-sn-glycero-3-phosphocholine) are effective stabilizers of squalene emulsion over a range of storage temperatures while others are not (1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dilauroyl-sn-glycero-3-phosphocholine) and (2) the immunogenicity of stable squalene emulsions is similar regardless of PC source.

Key words

oil-in-water emulsion phosphatidylcholine squalene vaccine adjuvant 



The authors wish to thank Susan Lin, Sandra Sivananthan, Tim Dutill, Kristen Forseth, Tony Phan, Farah Mompoint, Tara Evers, Alison Bernard, and Marah Hay for skilled technical assistance and Dr. Martin Friede for helpful discussions. This work was supported in part by National Institutes of Health contract HHSN272200800045C and grant 42387 from the Bill and Melinda Gates Foundation. The authors gratefully acknowledge Dr. Evelina Angov for kindly providing the codon-harmonized PbCSP construct developed by Walter Reed Army Institute of Research.

Supplementary material

12249_2012_9771_MOESM1_ESM.doc (384 kb)
ESM 1 DOC 384 kb


  1. 1.
    Fox CB. Squalene emulsions for parenteral vaccine and drug delivery. Molecules. 2009;14:3286–312.PubMedCrossRefGoogle Scholar
  2. 2.
    Reddy LH, Couvreur P. Squalene: a natural triterpene for use in disease management and therapy. Adv Drug Del Rev. 2009;61:1412–26.CrossRefGoogle Scholar
  3. 3.
    Brito LA, Chan M, Baudner B, Gallorini S, Santos G, O’Hagan DT, et al. An alternative renewable source of squalene for use in emulsion adjuvants. Vaccine. 2011;29:6262–8.PubMedCrossRefGoogle Scholar
  4. 4.
    Yang YW, Wu CA, Morrow WJW. Cell death induced by vaccine adjuvants containing surfactants. Vaccine. 2004;22:1524–36.PubMedCrossRefGoogle Scholar
  5. 5.
    Forchielli ML, Bersani G, Tala S, Grossi G, Puggioli C, Masi M. The spectrum of plant and animal sterols in different oil-derived intravenous emulsions. Lipids. 2010;45:63–71.PubMedCrossRefGoogle Scholar
  6. 6.
    Vernooij EAAM, Kettens-van den Bosch JJ, Crommelin DJA. Rapid determination of acyl chain position in egg phosphatidylcholine by high-performance liquid chromatography/electrospray mass spectrometry. Rapid Comm Mass Spec. 1998;12:83–6.CrossRefGoogle Scholar
  7. 7.
    de Man JM. Chemical and physical properties of fatty acids. In: Chow CK, editor. Fatty acids in foods and their health implications. Boca Raton: CRC Press; 2008. p. 17–46.Google Scholar
  8. 8.
    Fox CB, Baldwin SL, Duthie MS, Reed SG, Vedvick TS. Immunomodulatory and physical effects of oil composition in vaccine adjuvant emulsions. Vaccine. 2011;29:9563–72.PubMedCrossRefGoogle Scholar
  9. 9.
    Fox CB, Lin S, Sivananthan SJ, Dutill TS, Forseth KT, Reed SG, et al. Effects of emulsifier concentration, composition, and order of addition in squalene-phosphatidylcholine oil-in-water emulsions. Pharm Dev Technol. 2011;16:511–9.PubMedCrossRefGoogle Scholar
  10. 10.
    Hilleman MR. Personal historical chronicle of six decades of basic and applied research in virology, immunology, and vaccinology. Immunol Rev. 1999;170:7–27.PubMedCrossRefGoogle Scholar
  11. 11.
    Rosenberg SA, Yang JC, Kammula US, Hughes MS, Restifo NP, Schwarz SL, et al. Different adjuvanticity of incomplete Freund’s adjuvant derived from beef or vegetable components in melanoma patients immunized with a peptide vaccine. J Immunother. 2010;33:626–9.PubMedCrossRefGoogle Scholar
  12. 12.
    Bock TK, Muller BW. A novel assay to determine the hemolytic activity of drugs incorporated in colloidal carrier systems. Pharm Res. 1994;11:589–91.PubMedCrossRefGoogle Scholar
  13. 13.
    Baldwin SL, Shaverdian N, Goto Y, Duthie MS, Raman VS, Evers T, et al. Enhanced humoral and Type 1 cellular immune responses with Fluzone adjuvanted with a synthetic TLR4 agonist formulated in an emulsion. Vaccine. 2009;27:5956–63.PubMedCrossRefGoogle Scholar
  14. 14.
    Amselem S, Zawoznik E, Yogev A, Friedman D. Emulsomes, a new type of lipid assembly. In: Barenholz Y, Lasic DD, editors. Handbook of Nonmedical Applications of Liposomes: From design to microreactors. New York: CRC Press. 1995;209–23.Google Scholar
  15. 15.
    Vernooij EAAM, Brouwers JFHM, Kettenes-van den Bosch JJ, Crommelin DJA. RP-HPLC/ESI MS determination of acyl chain positions in phospholipids. J Sep Sci. 2002;25:285–9.CrossRefGoogle Scholar
  16. 16.
    Fox CB, Uibel RH, Harris JM. Detecting phase transitions in phosphatidylcholine vesicles by Raman microscopy and self-modeling curve resolution. J Phys Chem B. 2007;111(39):11428–36.PubMedCrossRefGoogle Scholar
  17. 17.
    Falsey AR. Half-dose influenza vaccine. Arch Intern Med. 2008;168:2402–3.PubMedCrossRefGoogle Scholar
  18. 18.
    Yasuda T, Dancey GF, Kinsky SC. Immunogenicity of liposomal model membranes in mice: dependence on phospholipid composition. Proc Natl Acad Sci. 1977;74:1234–6.PubMedCrossRefGoogle Scholar
  19. 19.
    Baudner BC, Ronconi V, Casini D, Tortoli M, Kazzaz J, Singh M, et al. MF59 emulsion is an effective delivery system for a synthetic TLR4 agonist (E6020). Pharm Res. 2009;26:1477–85.PubMedCrossRefGoogle Scholar
  20. 20.
    Mikrut B. Case study: formulation of an intravenous fat emulsion. In: Burgess DJ, editor. Injectable dispersed systems: formulation, processing, and performance. Boca Raton: Taylor and Francis; 2005. p. 415–25.CrossRefGoogle Scholar
  21. 21.
    Yoon JK, Burgess DJ. Interfacial properties as stability predictors of lecithin-stabilized perfluorocarbon emulsions. Pharm Dev Tech. 1996;1:333–41.CrossRefGoogle Scholar
  22. 22.
    Kabalnov A, Tarara T, Arlauskas R, Weers J. Phospholipids as emulsion stabilizers: phase behavior versus emulsion stability. J Coll Inter Sci. 1996;184:227–35.Google Scholar
  23. 23.
    Mollet H, Grubenmann A. Formulation technology: emulsions, suspensions, solid forms. New York: Wiley; 2001.Google Scholar
  24. 24.
    Nii T, Ishii F. Properties of various phosphatidylcholines as emulsifiers or dispersing agents in microparticle preparations for drug carriers. Colloids Surf B: Biointerfaces. 2004;39(1–2):57–63.CrossRefGoogle Scholar
  25. 25.
    Fox CB, Anderson RC, Dutill TS, Goto Y, Reed SG, Vedvick T. Monitoring the effects of component structure and source and formulation stability and adjuvant activity of oil-in-water emulsions. Colloids Surf B: Biointerfaces. 2008;65:98–105.CrossRefGoogle Scholar
  26. 26.
    Bibette J, Morse DC, Witten TA, Weitz DA. Stability criteria for emulsions. Phys Rev Lett. 1992;69(16):2439.PubMedCrossRefGoogle Scholar
  27. 27.
    Capek I. Degradation of kinetically-stable o/w emulsions. Adv Coll Inter Sci. 2004;107(2–3):125–55.CrossRefGoogle Scholar
  28. 28.
    Dalgleish DG. Adsorption of protein and the stability of emulsions. Trends Food Sci Technol. 1997;8(1):1–6.CrossRefGoogle Scholar
  29. 29.
    McClements DJ. Critical review of techniques and methodologies for characterization of emulsion stability. Crit Rev Food Sci Nutr. 2007;47:611–49.PubMedCrossRefGoogle Scholar
  30. 30.
    Tatulian SA. Binding of alkaline-earth metal cations and some anions to phosphatidylcholine liposomes. Eur J Biochem. 1987;170:413–20.PubMedCrossRefGoogle Scholar
  31. 31.
    Tatulian SA. Effect of lipid phase transition on the binding of anions to dimyristoylphosphatidylcholine liposomes. Biochim Biophys Acta. 1983;736:189–95.PubMedCrossRefGoogle Scholar
  32. 32.
    Tanaka Y, Mashino K, Inoue K, Nojima S. Mechanism of human erythrocyte hemolysis induced by short-chain phosphatidylcholines and lysophosphatidylcholine. J Biochem. 1983;94:833–40.PubMedGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2012

Authors and Affiliations

  • Christopher B. Fox
    • 1
    Email author
  • Susan L. Baldwin
    • 1
  • Malcolm S. Duthie
    • 1
    • 2
  • Steven G. Reed
    • 1
    • 3
  • Thomas S. Vedvick
    • 1
  1. 1.Infectious Disease Research InstituteSeattleUSA
  2. 2.Protein Advances Inc.SeattleUSA
  3. 3.Immune Design Corp.SeattleUSA

Personalised recommendations