Methods for Constructing and Evaluating Antitumor DNA Vaccines

  • Brian M. Olson
  • Douglas G. McNeelEmail author
Part of the Methods in Molecular Biology™ book series (MIMB, volume 542)


An antitumor DNA vaccine is a bacterial DNA plasmid that encodes the complementary DNA (cDNA) of a tumor antigen. When injected into recipients, antitumor DNA vaccines have been shown to elicit both humoral and cellular immunity against the encoded tumor antigen. These vaccines represent a relatively new immunotherapeutic technique being investigated as a means to deliver a target antigen and elicit or augment antitumor antigen-specific immune responses. One of the primary advantages of DNA vaccines as opposed to some other methods of antigen delivery is that they can be easily constructed, purified, and delivered to recipients. In this review we describe this process, detailing the procedures used to construct, purify, deliver, and evaluate the efficacy of DNA vaccines. We begin by describing the process of molecularly constructing the vaccine, from selecting a bacterial plasmid to form the backbone of the vaccine, cloning the antigen cDNA into this plasmid, and confirming the sequence and orientation of the completed vaccine. This is then followed by a series of experiments that can be used to ensure that the antigen encoded by the vaccine is transcribed and translated after being taken up by eukaryotic cells. We then describe large-scale purification procedures that can be used to obtain sufficient quantities of plasmid DNA to conduct in vivo immunization experiments. Finally, we provide an immunization protocol that can be used to evaluate the immunological efficacy of the constructed DNA vaccine. By following these protocols, it is possible to construct, purify, deliver, and evaluate the efficacy of antitumor DNA vaccines.

Key words

DNA vaccine immunization immunotherapy plasmid purification tumor antigen 



This work is supported by Department of Defense (DOD) grant W81XWH-07–1–0088 to BMO and by National Institutes of Health (NIH) grant K23 RR16489 to DGM.


  1. 1.
    Liu, M. A. (2003) DNA vaccines: a review. J Intern Med 253, 402–10.PubMedCrossRefGoogle Scholar
  2. 2.
    van der Bruggen, P. and Van den Eynde, B. J. (2006) Curr Opin Immunol 18, 98–104.PubMedCrossRefGoogle Scholar
  3. 3.
    Ciernik, I. F., Berzofsky, J. A., and Carbone, D. P. (1996) Processing and presentation of tumor antigens and vaccination strategies. J Immunol 156, 2369–75.PubMedGoogle Scholar
  4. 4.
    Pilon, S. A., Piechocki, M. P., and Wei, W. Z. (2001) Vaccination with cytoplasmic ErbB-2 DNA protects mice from mammary tumor growth without anti-ErbB-2 antibody. J Immunol 167, 3201–6.PubMedGoogle Scholar
  5. 5.
    Roos, A. K., Pavlenko, M., Charo, J., Egevad, L., and Pisa, P. (2005) Induction of PSA-specific CTLs and anti-tumor immunity by a genetic prostate cancer vaccine. Prostate 62, 217–23.PubMedCrossRefGoogle Scholar
  6. 6.
    Cassaday, R. D., Sondel, P. M., King, D. M., Macklin, M. D., Gan, J., Warner, T. F., Zuleger, C. L., Bridges, A. J., Schalch, H. G., Kim, K. M., Hank, J. A., Mahvi, D. M., and Albertini, M. R. (2007) A phase I study of immunization using particle-mediated epidermal delivery of genes for gp100 and GM-CSF into uninvolved skin of melanoma patients. Clin Cancer Res 13, 540–9.PubMedCrossRefGoogle Scholar
  7. 7.
    Liu, M. A. and Ulmer, J. B. (2005) Human clinical trials of plasmid DNA vaccines. Adv Genet 55, 25–40.PubMedCrossRefGoogle Scholar
  8. 8.
    Pavlenko, M., Roos, A. K., Lundqvist, A., Palmborg, A., Miller, A. M., Ozenci, V., Bergman, B., Egevad, L., Hellstrom, M., Kiessling, R., Masucci, G., Wersall, P., Nilsson, S., and Pisa, P. (2004) A phase I trial of DNA vaccination with a plasmid expressing prostate-specific antigen in patients with hormone-refractory prostate cancer. Br J Cancer 91, 688–94.PubMedGoogle Scholar
  9. 9.
    Triozzi, P. L., Aldrich, W., Allen, K. O., Carlisle, R. R., LoBuglio, A. F., and Conry, R. M. (2005) Phase I study of a plasmid DNA vaccine encoding MART-1 in patients with resected melanoma at risk for relapse. J Immunother (1997) 28, 382–8.CrossRefGoogle Scholar
  10. 10.
    Zlotocha, S., Staab, M. J., Horvath, D., Straus, J., Dobratz, J., Oliver, K., Wasielewski, S., Alberti, D., Liu, G., Wilding, G., Eickhoff, J., and McNeel, D. G. (2005) Phase I study of a plasmid DNA vaccine encoding MART-1 in patients with resected melanoma at risk for relapse. Clin Genitourin Cancer 4, 215–8.PubMedCrossRefGoogle Scholar
  11. 11.
    Nichols, W. W., Ledwith, B. J., Manam, S. V., and Troilo, P. J. (1995) Potential DNA vaccine integration into host cell genome. Ann N Y Acad Sci 772, 30–9.PubMedCrossRefGoogle Scholar
  12. 12.
    Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A., and Felgner, P. L. (1990) Direct gene transfer into mouse muscle in vivo. Science 247, 1465–8.PubMedCrossRefGoogle Scholar
  13. 13.
    Disis, M. L., Shiota, F. M., McNeel, D. G., and Knutson, K. L. (2003) Direct gene transfer into mouse muscle in vivo. Immunobiology 207, 179–86.PubMedCrossRefGoogle Scholar
  14. 14.
    McNeel, D. G., Schiffman, K., and Disis, M. L. (1999) Immunization with recombinant human granulocyte-macrophage colony-stimulating factor as a vaccine adjuvant elicits both a cellular and humoral response to recombinant human granulocyte-macrophage colony-stimulating factor. Blood 93, 2653–9.PubMedGoogle Scholar
  15. 15.
    Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., and Akira, S. (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–5.PubMedCrossRefGoogle Scholar
  16. 16.
    Klinman, D. M., Yamshchikov, G., and Ishigatsubo, Y. (1997) Contribution of CpG motifs to the immunogenicity of DNA vaccines. J Immunol 158, 3635–9.PubMedGoogle Scholar
  17. 17.
    Krieg, A. M., Yi, A. K., Schorr, J., and Davis, H. L. (1998) The role of CpG dinucleotides in DNA vaccines. Trends Microbiol 6, 23–7.PubMedCrossRefGoogle Scholar
  18. 18.
    Garmory, H. S., Brown, K. A., and Titball, R. W. (2003) DNA vaccines: improving expression of antigens. Genet Vaccines Ther 1, 2.PubMedCrossRefGoogle Scholar
  19. 19.
    Weeratna, R. D., Wu, T., Efler, S. M., Zhang, L., and Davis, H. L. (2001) Designing gene therapy vectors: avoiding immune responses by using tissue-specific promoters. Gene Ther 8, 1872–8.PubMedCrossRefGoogle Scholar
  20. 20.
    Hornung, V., Rothenfusser, S., Britsch, S., Krug, A., Jahrsdorfer, B., Giese, T., Endres, S., and Hartmann, G. (2002) Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 168, 4531–7.PubMedGoogle Scholar
  21. 21.
    Liu, M. A., Wahren, B., and Karlsson Hedestam, G. B. (2006) DNA vaccines: recent developments and future possibilities. Hum Gene Ther 17, 1051–61.PubMedCrossRefGoogle Scholar
  22. 22.
    Hengge, U. R., Walker, P. S., and Vogel, J. C. (1996) Expression of naked DNA in human, pig, and mouse skin. J Clin Invest 97, 2911–6.PubMedCrossRefGoogle Scholar
  23. 23.
    Raz, E., Carson, D. A., Parker, S. E., Parr, T. B., Abai, A. M., Aichinger, G., Gromkowski, S. H., Singh, M., Lew, D., Yankauckas, M. A., and et al. (1994) Expression of naked DNA in human, pig, and mouse skin. Proc Natl Acad Sci USA 91, 9519–23.PubMedCrossRefGoogle Scholar
  24. 24.
    Fynan, E. F., Webster, R. G., Fuller, D. H., Haynes, J. R., Santoro, J. C., and Robinson, H. L. (1993) DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc Natl Acad Sci USA 90, 11478–82.PubMedCrossRefGoogle Scholar
  25. 25.
    Pertmer, T. M., Eisenbraun, M. D., McCabe, D., Prayaga, S. K., Fuller, D. H., and Haynes, J. R. (1995) Gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantities of DNA. Vaccine 13, 1427–30.PubMedCrossRefGoogle Scholar
  26. 26.
    Lyerly, H. K. (2003) Quantitating cellular immune responses to cancer vaccines. Semin Oncol 30, 9–16.PubMedCrossRefGoogle Scholar
  27. 27.
    Whiteside, T. L. (2004) Methods to monitor immune response and quality control. Dev Biol (Basel) 116, 219–28; discussion 29–36.Google Scholar
  28. 28.
    Mosca, P., Clay, T., Morse, M., and Lyerly, H. K. (2005) Tumor immunology and cancer vaccines: immune monitoring. Cancer Treat Res 123, 369–88.PubMedCrossRefGoogle Scholar
  29. 29.
    Clay, T. M., Hobeika, A. C., Mosca, P. J., Lyerly, H. K., and Morse, M. A. (2001) Assays for monitoring cellular immune responses to active immunotherapy of cancer. Clin Cancer Res 7, 1127–35.PubMedGoogle Scholar
  30. 30.
    Hernandez-Fuentes, M. P., Warrens, A. N., and Lechler, R. I. (2003) Immunologic monitoring. Immunol Rev 196, 247–64.PubMedCrossRefGoogle Scholar
  31. 31.
    Nestle, F. O., Tonel, G., and Farkas, A. (2005) Cancer vaccines: the next generation of tools to monitor the anticancer immune response. PLoS Med 2, e339.PubMedCrossRefGoogle Scholar
  32. 32.
    Manthorpe, M., Cornefert-Jensen, F., Hartikka, J., Felgner, J., Rundell, A., Margalith, M., and Dwarki, V. (1993) Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice. Hum Gene Ther 4, 419–31.PubMedCrossRefGoogle Scholar
  33. 33.
    Wolff, J. A., Williams, P., Acsadi, G., Jiao, S., Jani, A., and Chong, W. (1991) Conditions affecting direct gene transfer into rodent muscle in vivo. Biotechniques 11, 474–85.PubMedGoogle Scholar
  34. 34.
    Chang, D. Z., Lomazow, W., Joy Somberg, C., Stan, R., and Perales, M. A. (2004) Granulocyte-macrophage colony stimulating factor: an adjuvant for cancer vaccines. Hematology 9, 207–15.PubMedCrossRefGoogle Scholar
  35. 35.
    Pan, C. H., Chen, H. W., and Tao, M. H. (1999) Modulation of immune responses to DNA vaccines by codelivery of cytokine genes. J Formos Med Assoc 98, 722–9.PubMedGoogle Scholar
  36. 36.
    Mattner, F., Fleitmann, J. K., Lingnau, K., Schmidt, W., Egyed, A., Fritz, J., Zauner, W., Wittmann, B., Gorny, I., Berger, M., Kirlappos, H., Otava, A., Birnstiel, M. L., and Buschle, M. (2002) Vaccination with poly-L-arginine as immunostimulant for peptide vaccines: induction of potent and long-lasting T-cell responses against cancer antigens. Cancer Res 62, 1477–80.PubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Department of OncologyUniversity of Wisconsin—MadisonMadisonUSA

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