Extraction and Characterization of Vaccine Antigens from Water-in-Oil Adjuvant Formulations

  • Aaron P. Miles
  • Allan Saul
Part of the Methods in Molecular Biology™ book series (MIMB, volume 308)


Water-in-oil adjuvants are currently undergoing experimental testing in human vaccine trials (110). Two such adjuvants are the squalene-based Montanide® ISA 720 and the mineral oil-based Montanide ISA 51 (11). Vaccines containing these adjuvants are intended to provide a lasting depot effect, with vaccine persisting at the injection site for many months (12). Because it is often convenient to formulate these vaccines well ahead of use, and because of the extended residence at the injection site, their stability must be established. Therefore, studies should be designed that will address stability at 4°C (intended storage temperature) and 37°C (body temperature) to ensure that the antigens remain intact and the vaccines consequently remain potent. With the addition of benzyl alcohol, the emulsion is broken, and the antigens are released and recoverable in the resulting aqueous layer. Storage at 4°C and 37°C of fresh formulations of ISA 720 and ISA 51 containing either of two recombinant malarial proteins, followed by extraction at various time points and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, and Western blotting will be used to illustrate the methods involved in monitoring the stability of protein antigens in these water-in-oil formulations.


Glacial Acetic Acid Benzyl Alcohol Vaccine Formulation Antigen Extraction Fresh Formulation 
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  1. 1.
    Genton, B., Al-Yaman, F., Anders, R., Saul, A., Brown, G., Pye, D., et al. (2000) Safety and immunogenicity of a three-component blood-stage malaria vaccine in adults living in an endemic area of Papua New Guinea. Vaccine 18, 2504–2511.PubMedCrossRefGoogle Scholar
  2. 2.
    Lawrence, G., Cheng, Q., Reed, C., Taylor, D., Stowers, A., Cloonan, N., et al. (2000) Effect of vaccination with 3 recombinant asexual-stage malaria antigens on initial growth rates of Plasmodium falciparum in non-immune volunteers. Vaccine 18, 1925–1931.PubMedCrossRefGoogle Scholar
  3. 3.
    Lawrence, G., Saul, A., Giddy, A., Kemp, R., and Pye, D. (1997) Phase I trial in humans of an oil-based adjuvant SEPPIC MONTANIDE ISA 720. Vaccine 15, 176–178.PubMedCrossRefGoogle Scholar
  4. 4.
    Saul, A., Lawrence, G., Smillie, A., Rzepczyk, C. M., Reed, C., Taylor, D., et al. (1999) Human phase I vaccine trials of 3 recombinant asexual stage malaria antigens with Montanide ISA720 adjuvant. Vaccine 17, 3145–3159.PubMedCrossRefGoogle Scholar
  5. 5.
    Toledo, H., Baly, A., Castro, O., Resik, S., Laferté, J., Rolo, F., et al. (2001) A Phase I clinical trial of a multi-epitope polypeptide TAB9 combined with Montanide ISA 720 adjuvant in non-HIV-1 infected human volunteers. Vaccine 19, 4328–4336.PubMedCrossRefGoogle Scholar
  6. 6.
    Yamshchikov, G. V., Barnd, D. L., Eastham, S., Galavotti, H., Patterson, J. W., Deacon, D. H., et al. (2001) Evaluation of peptide vaccine immunogenicity in draining lymph nodes and peripheral blood of melanoma patients. Int. J. Cancer 92, 703–711.PubMedCrossRefGoogle Scholar
  7. 7.
    Pinto, L. A., Berzofsky, J. A., Fowke, K. R., Little, R. F., Merced-Galindez, F., Humphrey, R., et al. (1999) HIV-specific immunity following immunization with HIV synthetic envelope peptides in asymptomatic HIV-infected patients. AIDS 13, 2003–2012.PubMedCrossRefGoogle Scholar
  8. 8.
    Carr, A., Rodríguez, E., del Carmen Arango, M., Camacho, R., Osorio, M., Gabri, M., et al. (2003) Immunotherapy of advanced breast cancer with a heterophilic ganglioside (NeuGcGM3) cancer vaccine. J. Clin. Oncol. 21, 1015–1021.PubMedCrossRefGoogle Scholar
  9. 9.
    Slingluff, C. L., Jr., Yamshchikov, G., Neese, P., Galavotti, H., Eastham, S., Engelhard, V. H., et al. (2001) Phase I trial of a melanoma vaccine with gp100280–288 peptide and tetanus helper peptide in adjuvant: immunologic and clinical outcomes. Clin. Cancer Res. 7, 3012–3024.PubMedGoogle Scholar
  10. 10.
    van Driel, W. J., Ressing M. E., Kenter, G. G., Brandt, R. M. P., Krul, E. J. T., van Rossum, A. B., et al. (1999) Vaccination with HPV16 peptides of patients with advanced cervical carcinoma: clinical evaluation of a phase I–II trial. Eur. J. Cancer 35, 946–952.PubMedCrossRefGoogle Scholar
  11. 11.
    Aucouturier, J., Dupuis, L., Deville, S., Ascarateil, S., and Ganne, V. (2002) Montanide ISA 720 and 51: a new generation of water in oil emulsions as adjuvants for human vaccines. Expert Rev. Vaccines 1, 111–118.PubMedCrossRefGoogle Scholar
  12. 12.
    Herbert, W. J. (1968) The mode of action of mineral-oil emulsion adjuvants on antibody production in mice. Immunology 14, 301–318.PubMedGoogle Scholar
  13. 13.
    Zou, L., Miles, A., Wang, J., and Stowers, A. W. (2003) Expression of malaria transmission-blocking vaccine antigen Pfs25 in Pichia pastoris for use in human clinical trials. Vaccine 21, 1650–1657.PubMedCrossRefGoogle Scholar
  14. 14.
    Kennedy, M. C., Wang, J., Zhang, Y., Miles, A. P., Chitsaz, F., Saul, A., et al. (2002) In vitro studies with recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1): production and activity of an AMA1 vaccine and generation of a multiallelic response. Infect. Immunity 70, 6948–6960.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Aaron P. Miles
    • 1
  • Allan Saul
    • 2
  1. 1.Biochemical Assay Development and Quality Control, Malaria Vaccine Development BranchNational Institute of Allergy and Infectious DiseasesRockville
  2. 2.Malaria Vaccine Development BranchNational Institute of Allergy and Infectious DiseasesRockville

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