Advertisement

Monitoring of Human Immunological Responses to Vaccinia Virus

  • Richard Harrop
  • Matthew G. Ryan
  • Hana Golding
  • Irina Redchenko
  • Miles W. Carroll
Part of the Methods in Molecular Biology book series (MIMB, volume 269)

Abstract

For the last 30 yr, interest in vaccinia virus immune monitoring has focused on the use of the vaccinia virus as a recombinant vaccine vector and the potential detrimental effect of antivector immunity on subsequent vaccination with a recombinant vaccinia virus. However, interest in this area has intensified after the publication of reports suggesting that smallpox may be a major pathogen selected for bioterrorist activities. Owing to the unacceptably high incidence of complications induced by previous effective smallpox vaccine strains, alternative safer strains (e.g., modified vaccinia Ankara [MVA]) are being assessed for their antigenicity in clinical trials. The exact immune effector mechanism responsible for vaccine-induced protection to smallpox infection has not been fully elucidated, although it is believed that neutralizing antibody plays a major role. This chapter describes a simple enzyme-linked immunosorbent assay (ELISA) to quantify vaccinia virus antibody titer. Additionally, to define serum-neutralizing activity, both a classical plaque reduction assay and a high-throughput 96-well plate method based on reduction of recombinant vaccinia virus expressed β-galactosidase is described. Furthermore, details are given for a T-cell proliferation assay, primarily for monitoring T-helper CD4 activity and an enzyme-linked immunospot (ELISPOT) assay for CD8 analysis. The use of reliable immunological assays is vital in assessing the potential efficacy of new vaccines to protect against smallpox infection.

Key Words

Human immune assays vaccinia virus MVA ELISA neutralization proliferation ELISPOT 

References

  1. 1.
    Lane, J. M., Ruben, F. L., Neff, J. M., and Millar, J. D. (1969) Complications of smallpox vaccination N. Engl. J. Med. 281, 1201–1208.CrossRefGoogle Scholar
  2. 2.
    Belyakov, I. M., Earl, P., Dzutsev, A., Kuznetsov, V. A., Lemon, M., Wyatt, L. S., et al. (2003) Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses. Proc. Natl. Acad. Sci. USA 100, 9458–9463.PubMedCrossRefGoogle Scholar
  3. 3.
    Manischewitz, J., King, L. R., Bleckwenn, N. A., Shiloach, J., Taffs, R., Merchlinsky, M., et al. (2003) Development of a novel vaccinia-neutralization assay based on reporter-gene expression. J. Infect. Dis. 188, 440–448.PubMedCrossRefGoogle Scholar
  4. 4.
    Earl, P. L., Americo, J. L., and Moss, B. (2003) Vaccinia virus neutralization assay based on flow cytometric detection of green fluorescent protein: Development and use. J. Virol. 77, 10684–10688.PubMedCrossRefGoogle Scholar
  5. 5.
    Earl, P. L., Wyatt, L. S., Moss, B., and Carroll, M. W. (1998) Generation of vaccinia virus recombinant viruses, in Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, (Suppl 43) 16.17.1–16.17.19.Google Scholar
  6. 6.
    Katz, J. B. (1987) The effect of the virus-serum incubation period upon vaccinia virus serum neutralization titers. J. Biol. Standardization 15, 389–392.CrossRefGoogle Scholar
  7. 7.
    Strober, W. (2002) Immunologic studies in humans, in Current Protocols in Immunology, John Wiley & Sons, Inc., New York, 7.0.1–7.0.7.Google Scholar
  8. 8.
    Mulryan, K., Ryan, M. G., Myers, K. A., Shaw, D., Wang, W., et al. (2002) Attenuated vaccinia virus expressing oncofetal antigen (tumour-associated antigen) 5T4 induces active therapy of established tumors. Mol. Cancer Ther. 12, 1129–1137.Google Scholar
  9. 9.
    Carroll, M. W. and Moss, B. (1997) Host range and cytopathogenicity of the highly attenuated MVA strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line. Virology 238, 198–211.PubMedCrossRefGoogle Scholar
  10. 10.
    Boulter, E. A. and Appleyard, G. (1973) Differences between extracellular and intracellular forms of poxvirus and their implications. Prog. Med. Virol. 16, 86–108.PubMedGoogle Scholar
  11. 11.
    Law, M. and Smith, G. L. (2001) Antibody neutralization of the extracellular enveloped form of vaccinia virus. Virol. 280, 132–142.CrossRefGoogle Scholar
  12. 12.
    Chung, C.-S., Hsiao, J.-C., Chang, Y.-S., and Chang, W. (1998) A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate. J. Virol. 72, 1577–1585.PubMedGoogle Scholar
  13. 13.
    Cardigan, R. A., Mackie, I. J., and Machin, S. J. (1996) The effect of heparin and its neutralisation on functional assays for Factor VIIa, Factor VII and TFPI. Thrombosis Res. 84, 237–242.CrossRefGoogle Scholar
  14. 14.
    Mack, T. M., Noble, J., and Thomas, D. B. (1972) A prospective study of serum antibody and protection against smallpox. Am J. Trop. Med. Hyg. 21, 214–218.PubMedGoogle Scholar
  15. 15.
    Paoletti, E. (1996) Applications of pox virus vectors to vaccination: an update. Proc. Nat. Acad. Sci. USA 93, 11349–11353.PubMedCrossRefGoogle Scholar
  16. 16.
    Chakrabarti, S., Sisler, J. R., and Moss, B. (1997) Compact, synthetic, vaccinia virus early/late promoter for protein expression. Biotechniques 23, 1094–1097.PubMedGoogle Scholar
  17. 17.
    Smith, J. G., Liu, X., Kaufhold, R. M., Clair, J., and Caulfield, M. J. (2001) Development and validation of a gamma interferon ELISPOT assay for quantitation of cellular immune responses to varicella-zoster virus. Clin. Diag. Lab. Immunol. 8, 871–879.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Richard Harrop
    • 1
  • Matthew G. Ryan
    • 1
  • Hana Golding
    • 2
  • Irina Redchenko
    • 1
  • Miles W. Carroll
    • 1
  1. 1.Oxford BioMedica (UK) LtdOxfordUK
  2. 2.Division of Viral ProductsCenter for Biologics Evaluation and Research, Food and Drug AdministrationBethesda

Personalised recommendations