Analyzing Cellular Immunity to AAV in a Canine Model Using ELISPOT Assay

  • Zejing Wang
  • Rainer Storb
  • Stephen J. Tapscott
  • Stanley Riddell
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 792)

Abstract

Adeno-associated viral (AAV) vector-mediated gene transfer represents a promising gene replacement strategy for treating various genetic diseases. One obstacle in using viral-derived vectors for in vivo gene delivery is the development of host immune responses to the vector. Recent studies have demonstrated cellular immune responses specific to capsid proteins of various AAV serotypes in animal models and in human trials for different diseases. We developed a canine-specific ELISPOT assay to detect such immunity in dogs received AAV treatment. Here, we describe in detail the use of a constructed panel of overlapping peptides spanning the entire VP1 sequence of AAV capsid protein to detect specific T-cell responses in peripheral blood in dogs following intramuscular injection of AAV. This high-throughput method allows the identification of T-cell epitopes without the need for large cell numbers and the need for major histocompatibility complex molecule-matched cell lines.

Key words

Adeno-associated virus AAV Peptide library Dog ELISPOT assay 

Notes

Acknowledgments

We thank Drs. Jeffery Chamberlain and Dusty Miller for providing vectors used in the protocols and Dr. Kathy High for providing the cFIX plasmid. We thank Drs. Christine Halbert and Carolina Berger for technical advice, E. Zellmer and E. Finn for technical assistance, A. Joslyn, and the canine team, and M. Spector, DVM, and J Duncan, DVM, for their care of the dogs. We further thank S. Carbonneau, H. Crawford, B. Larson, K. Carbonneau, and D. Gayle for administrative assistance and manuscript preparation.

This work was supported by NIH R01 AR056949-01A1, NIH CA15704, and by Career Development Award for Z. Wang from the Muscular Dystrophy Association (MDA 114979).

References

  1. 1.
    Athanasopoulos, T., Fabb, S., and Dickson, G. (2000) Gene therapy vectors based on adeno-associated virus: characteristics and applications to acquired and inherited diseases (review). Int J Mol Med 6, 363–375.PubMedGoogle Scholar
  2. 2.
    Sun, B., Zhang, H., Franco, L. M., Young, S. P., Schneider, A., Bird, A., et al. (2005) Efficacy of an adeno-associated virus 8-pseudotyped vector in glycogen storage disease type II. Molecular Therapy 11, 57–65.PubMedCrossRefGoogle Scholar
  3. 3.
    Warrington, K. H., Jr., and Herzog, R. W. (2006) Treatment of human disease by adeno-associated viral gene transfer. Hum Genet 119, 571–603.PubMedCrossRefGoogle Scholar
  4. 4.
    Bostick, B., Yue, Y., Lai, Y., Long, C., Li, D., and Duan, D. (2008) Adeno-associated virus serotype-9 microdystrophin gene therapy ameliorates electrocardiographic abnormalities in mdx mice. Hum Gene Ther 19, 851–856.PubMedCrossRefGoogle Scholar
  5. 5.
    Yue, Y., Ghosh, A., Long, C., Bostick, B., Smith, B. F., Kornegay, J. N., et al. (2008) A single intravenous injection of adeno-associated virus serotype-9 leads to whole body skeletal muscle transduction in dogs. Molecular Therapy 16, 1944–1952.PubMedCrossRefGoogle Scholar
  6. 6.
    Athanasopoulos, T., Graham, I. R., Foster, H., and Dickson, G. (2004) Recombinant adeno-associated viral (rAAV) vectors as therapeutic tools for Duchenne muscular dystrophy (DMD) (Review). Gene Ther 11 Suppl 1, S109–S121.Google Scholar
  7. 7.
    Sabatino, D. E., Mingozzi, F., Hui, D. J., Chen, H., Colosi, P., Ertl, H. C., et al. (2005) Identification of mouse AAV capsid-specific CD8+ T cell epitopes. Molecular Therapy 12, 1023–1033.PubMedCrossRefGoogle Scholar
  8. 8.
    Gao, G., Lu Y., Calcedo, R., Grant, R. L., Bell, P., Wang, L., et al. (2006) Biology of AAV ­serotype vectors in liver-directed gene transfer to nonhuman primates. Molecular Therapy 13, 77–87.PubMedCrossRefGoogle Scholar
  9. 9.
    Manno C. S., Pierce G. F., Arruda V. R., Glader B., Ragni M., Rasko J. J., et al. (2006) Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response (erratum appears in Nat Med. 2006 May;12(5):592). Nat Med 12, 342–347.Google Scholar
  10. 10.
    Wang, Z., Allen, J. M., Riddell, S. R., Gregorevic, P., Storb, R., Tapscott, S. J., et al. (2007) Immunity to adeno-associated virus-mediated gene transfer in a random-bred canine model of Duchenne muscular dystrophy. Hum Gene Ther 18, 18–26.PubMedCrossRefGoogle Scholar
  11. 11.
    Brantly, M. L., Chulay, J. D., Wang, L., Mueller, C., Humphries, M., Spencer, L. T., et al. (2009) Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapy (Erratum appears in Proc Natl Acad Sci U S A. 2009 Oct 13;106(41):17606). Proc Natl Acad Sci USA 106, 16363–16368.Google Scholar
  12. 12.
    Halbert, C. L., Madtes, D. K., Vaughan, A. E., Wang, Z., Storb, R., Tapscott, S. J., et al. (2010) Expression of human α1-antitrypsin in mice and dogs following AAV6 vector-mediated gene transfer to the lungs. Molecular Therapy 18, 1165–1172.PubMedCrossRefGoogle Scholar
  13. 13.
    Engers, H. D., Thomas, K., Cerottini, J. C., and Brunner, K. T. (1975) Generation of cytotoxic T lymphocytes in vitro. V. Response of normal and immune spleen cells to subcellular alloantigens. J Immunol 115, 356–360.Google Scholar
  14. 14.
    Altman, J. D., Moss, P. A., Goulder, P. J., Barouch, D. H., McHeyzer-Williams, M. G., Bell, J. I., et al. (1996) Phenotypic analysis of antigen-specific T lymphocytes Science 274, 94–96.Google Scholar
  15. 15.
    Appay, V., and Rowland-Jones, S. L. (2002) The assessment of antigen-specific CD8+ T cells through the combination of MHC class I tetramer and intracellular staining (Review). J Immunol Methods 268, 9–19.PubMedCrossRefGoogle Scholar
  16. 16.
    Murali-Krishna, K., Altman, J. D., Suresh, M., Sourdive, D. J., Zajac, A. J., Miller, J. D., et al. (1998) Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8, 177–187.PubMedCrossRefGoogle Scholar
  17. 17.
    Kern F., Faulhaber, N., Frommel, C., Khatamzas, E., Prosch, S., Schonemann, C., et al. (2000) Analysis of CD8 T cell reactivity to cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides. Eur J Immunol 30, 1676–1682.PubMedCrossRefGoogle Scholar
  18. 18.
    Lalvani, A., Brookes, R., Hambleton, S., Britton, W. J., Hill, A. V., and McMichael, A. J. (1997) Rapid effector function in CD8+ memory T cells. J Exp Med 186, 859–865.PubMedCrossRefGoogle Scholar
  19. 19.
    Tobery, T. W., Wang, S., Wang, X. M., Neeper, M. P., Jansen, K. U., McClements, W. L., et al. (2001) A simple and efficient method for the monitoring of antigen-specific T cell responses using peptide pool arrays in a modified ELISpot assay. J Immunol Methods 254, 59–66.PubMedCrossRefGoogle Scholar
  20. 20.
    Schmittel, A., Keilholz, U., and Scheibenbogen, C. (1997) Evaluation of the interferon-gamma ELISPOT-assay for quantification of peptide specific T lymphocytes from peripheral blood. J Immunol Methods 210, 167–174.PubMedCrossRefGoogle Scholar
  21. 21.
    Kumar, A., Weiss, W., Tine, J. A., Hoffman, S. L., and Rogers, W. O. (2001) ELISPOT assay for detection of peptide specific interferon-gamma secreting cells in rhesus macaques. J Immunol Methods 247, 49–60.PubMedCrossRefGoogle Scholar
  22. 22.
    Tobery, T. W., and Caulfield, M. J. (2004) Identification of T-cell epitopes using ELISpot and peptide pool arrays. Methods in Molecular Medicine 94, 121–132.PubMedGoogle Scholar
  23. 23.
    Maecker, H. T., Moon, J., Bhatia, S., Ghanekar, S. A., Maino, V. C., Payne, J. K., et al. (2005) Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT. BMC Immunology 6, 17.PubMedCrossRefGoogle Scholar
  24. 24.
    Smith, J. G., Joseph, H. R., Green, T., Field, J. A., Wooters, M., Kaufhold, R. M., et al. (2007) Establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions. Clinical and Vaccine Immunology 14, 527–537.PubMedCrossRefGoogle Scholar
  25. 25.
    Arlen, P., Tsang, K. Y., Marshall, J. L., Chen, A., Steinberg, S. M., Poole, D., et al. (2000) The use of a rapid ELISPOT assay to analyze peptide-specific immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines. Cancer Immunol Immunother 49, 517–529.Google Scholar
  26. 26.
    Kaufhold, R. M., Field, J. A., Caulfield, M. J., Wang, S., Joseph, H., Wooters M. A., et al. (2005) Memory T-cell response to rotavirus detected with a gamma interferon enzyme-linked immunospot assay. J Virol 79, 5684–5694.PubMedCrossRefGoogle Scholar
  27. 27.
    Larsson, M., Jin, X., Ramratnam, B., Ogg, G. S., Engelmayer, J., Demoitie, M. A., et al. (1999) A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. AIDS 13, 767–777.PubMedCrossRefGoogle Scholar
  28. 28.
    Wang, R., Richie, T. L., Baraceros, M. F., Rahardjo, N., Gay, T., Banania, J. G., et al. (2005) Boosting of DNA vaccine-elicited gamma interferon responses in humans by exposure to malaria parasites. Infection & Immunity 73, 2863–2872.CrossRefGoogle Scholar
  29. 29.
    Firbas, C., Jilma, B., Tauber, E., Buerger, V., Jelovcan, S., Lingnau, K., et al. (2006) Immunogenicity and safety of a novel therapeutic hepatitis C virus (HCV) peptide vaccine: a randomized, placebo controlled trial for dose optimization in 128 healthy subjects. Vaccine 24, 4343–4353.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Zejing Wang
    • 1
  • Rainer Storb
    • 1
  • Stephen J. Tapscott
    • 2
  • Stanley Riddell
    • 3
  1. 1.Program in Transplantation Biology, Division of Clinical ResearchFred Hutchinson Cancer Research CenterSeattleUSA
  2. 2.Division of Human BiologyFred Hutchinson Cancer Research CenterSeattleUSA
  3. 3.Program in Immunology, Division of Clinical ResearchFred Hutchinson Cancer Research CenterSeattleUSA

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