Abstract
DNA vaccination represents a novel and potentially important approach to induce immune responses against protein antigens. In this approach, the vaccine is a plasmid DNA vector that can be taken up by cells to produce a protein, encoded by the vector, to be targeted for the induction of humoral or cellular responses. Although the intracellular production of the antigen may promote responses, the vectors themselves may display adjuvant activity because of their intrinsic immunostimulatory properties. These properties reflect sequence motifs, centering on an unmethylated CpG dinucleotide, which can trigger the TLR9 pattern recognition receptor. As shown by studies in vitro, plasmid DNA can stimulate B cells, macrophages, and dendritic cells, and trigger a broad range of pro-inflammatory responses. Because this stimulation results from common sequence motifs, the activity of a plasmid vector can be assessed by the in vitro assay of a limited number of responses, including proliferation of B cells as well as production of cytokines by macrophages or dendritic cells.
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References
Vogel, F. R. and Sarver, N. (1995) Nucleic acid vaccines. Clin. Microbiol. Rev. 8, 406ā410.
Pardoll, D. M. and Beckerleg, A. M. (1995) Exposing the immunology of naked DNA vaccines. Immunity 3, 165ā169.
Srivastava, I. K. and Liu, M. A. (2003) Gene Vaccines. Ann. Intern. Med. 138, 550ā559.
Corr, M., Lee, D. J., Carson, D. A., and Tighe, H. (1996) Gene vaccination with naked plasmid DNA: mechanism of CTL priming. J. Exp. Med. 184, 1555ā1560.
Haas, J., Park, E. C., and Seed, B. (1996) Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr. Biol. 6, 315ā324.
zur Megede, J., Chen, M. C., Doe, B., et al. (2000) Increased expression and immunogenicity of sequence-modified human immunodeficiency virus type 1 gag gene. J. Virol. 74, 2628ā2635.
Iwasaki, A., Stiernholm, B. J., Chan, A. K., Berinstein, N. L., and Barber, B. H. (1997) Enhanced CTL responses mediated by plasmid DNA immunogens encoding costimulatory molecules and cytokines. J. Immunol. 158, 4591ā4601.
Sasaki, S., Tsuji, T., Asakura, Y., Fukushima, J., and Okuda, K. (1998) The search for a potent DNA vaccine against AIDS: the enhancement of immunogenicity by chemical and genetic adjuvants. Anticancer Res. 18, 3907ā3915.
Sedegah, M., Weiss, W., Sacci, J. B., Jr., et al. (2000) Improving protective immunity induced by DNA-based immunization: priming with antigen and GMCSF-encoding plasmid DNA and boosting with antigen-expressing recombinant poxvirus. J. Immunol. 164, 5905ā5912.
Barouch, D. H., McKay, P. F., Sumida, S. M., et al. (2003) Plasmid chemokines and colony-stimulating factors enhance the immunogenicity of DNA priming-viral vector boosting human immunodeficiency virus type 1 vaccines. J. Virol. 77, 8729ā8735.
Mendoza, R. B., Cantwell, M. J., and Kipps, T. J. (1997) Immunostimulatory effects of a plasmid expressing CD40 ligand (CD154) on gene immunization. J. Immunol. 159, 5777ā5781.
Santra, S., Barouch, D. H., Jackson, S. S., et al. (2000) Functional equivalency of B7-1 and B7-2 for costimulating plasmid DNA vaccine-elicited CTL responses. J. Immunol. 165, 6791ā6795.
Barnett, S. W., Rajasekar, S., Legg, H., et al. (1997) Vaccination with HIV-1 gp120 DNA induces immune responses that are boosted by a recombinant gp120 protein subunit. Vaccine 15, 869ā873.
Pisetsky, D. S. (1996) The immunologic properties of DNA. J. Immunol. 156, 421ā423.
Pisetsky, D. S. (1996) Immune activation by bacterial DNA: a new genetic code. Immunity 5, 303ā310.
Hemmi, H., Takeuchi, O., Kawai, T., et al. (2000) A toll-like receptor recognizes bacterial DNA. Nature 408, 740ā745.
Bauer, S., Kirschning, C. J., Hacker, H., et al. (2001) Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl. Acad. Sci. USA 98, 9237ā9242.
Krug, A., Towarowski, A., Britsch, S., et al. (2001) Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12. Eur. J. Immunol. 31, 3026ā3037.
Yi, A. K., Tuetken, R., Redford, T., Waldschmidt, M., Kirsch, J., and Krieg, A. M. (1998) CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species. J. Immunol. 160, 4755ā4761.
Yi, A. K. and Krieg, A. M. (1998) Rapid induction of mitogen-activated protein kinases by immune stimulatory CpG DNA. J. Immunol. 161, 4493ā4497.
Hacker, H., Vabulas, R. M., Takeuchi, O., Hoshino, K., Akira, S. and Wagner, H. (2000) Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6. J. Exp. Med. 192, 595ā600.
Chuang, T. H., Lee, J., Kline, L., Mathison, J. C., and Ulevitch, R. J. (2002) Tolllike receptor 9 mediates CpG-DNA signaling. J. Leukoc. Biol. 71, 538ā544.
Yamarmoto, S., Kurarnoto, E., Shimada, S., and Tokunaga, T. (1988) In vitro augmentation of natural killer cell activity and production of interferon-Ī±/Ī² and-Ī³ with deoxyribonucleic acid fraction from Mycobacterium bovis BCG. Jpn. J. Cancer Res. 79, 866ā873.
Yarmarmoto, S., Yarnarnoto, T., Shimada, S., et al. (1992) DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth. Microbiol. Immunol. 36, 983ā997.
Messina, J. P., Gilkeson, G. S., and Pisetsky, D. S. (1991) Stimulation of in vitro murine lymphocyte proliferation by bacterial DNA. J. Immunol. 147, 1759ā1764.
Krieg, A. M., Yi, A.-K., Matson, S., et al. (1995) CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374, 546ā549.
Klinman, D. M., Yi, A.-K., Beaucage, S. L., Conover, J., and Krieg, A. M. (1996) CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon. Proc. Natl. Acad. Sci. USA 93, 2879ā2883.
Halpern, M. D., Kurlander, R. J., and Pisetsky, D. S. (1996) Bacterial DNA induces murine interferon-Ī³ production by stimulation of interleukin-12 and tumor necrosis factor-Ī±. Cell. Immunol. 167, 72ā78.
Hartmann, G. and Krieg, A. M. (2000) Mechanism and function of a newly identified CpG DNA motif in human primary B cells. J. Immunol. 164, 944ā953.
Kataoka, T., Yamamoto, S., Yamamoto, T., et al. (1992) Antitumor activity of synthetic oligonucleotides with sequences from cDNA encoding proteins of Mycobacterium bovis BCG. Jpn. J. Cancer Res. 83, 244ā247.
Krieg, A. M. (1995) CpG DNA: a pathogenic factor in systemic lupus erythematosus? J. Clin. Immunol. 15, 284ā292.
Ballas, Z. K., Rasmussen, W. L., and Krieg, A. M. (1996) Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J. Immunol. 157, 1840ā1845.
Boggs, R. T., McGraw, K., Condon, T., et al. (1997) Characterization and modulation of immune stimulation by modified oligonucleotides. Antisense Nucleic Acid Drug Dev. 7, 461ā471.
Vollmer, J., Weeratna, R., Payette, P., et al. (2004) Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur. J. Immunol. 34, 251ā262.
Yu, D., Kandimalla, E. R., Bhagat, L., et al. (2002) āImmunomersā-novel 3ā²-3ā²-linked CpG oligodeoxyribonucleotides as potent immunomodulatory agents. Nucleic Acids Res. 30, 4460ā4469.
Messina, J. P., Gilkeson, G. S., and Pisetsky, D. S. (1993) The influence of DNA structure on the in vitro stimulation of murine lymphocytes by natural and synthetic polynucleotide antigens. Cell. Immunol. 147, 148ā157.
Pisetsky, D. S. and Reich, C. (1993) Stimulation of in vitro proliferation of murine lymphocytes by synthetic oligodeoxynucleotides. Molec. Biol. Rep. 18, 217ā221.
Kimura, Y., Sonehara, K., Kuramoto, E., et al. (1994) Binding of oligoguanylate to scavenger receptors is required for oligonucleotides to augment NK cell activity and induce IFN. J. Biochem. 116, 991ā994.
Wloch, M. K., Pasquini, S., Ertl, H. C., and Pisetsky, D. S. (1998) The influence of DNA sequence on the immunostimulatory properties of plasmid DNA vectors. Hum. Gene Ther. 9, 1439ā1447.
Krieger, M. and Herz, J. (1994) Structures and functions of multiligand lipoprotein receptors: macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu. Rev. Biochem. 63, 604ā637.
Klinman, D. M., Yamshchikov, G. and Ishigatsubo, Y. (1997) Contribution of CpG motifs to the immunogenicity of DNA vaccines. J. Immunol. 158, 3635ā3639.
Cornelie, S., Poulain-Godefroy, O., Lund, C., et al. (2004) Methylated CpG-containing plasmid activates the immune system. Scand. J. Immunol. 59, 143ā151.
Spies, B., Hochrein, H., Vabulas, M., et al. (2003) Vaccination with plasmid DNA activates dendritic cells via Toll-like receptor 9 (TLR9) but functions in TLR9-deficient mice. J. Immunol. 171, 5908ā5912.
Babiuk, S., Mookherjee, N., Pontarollo, R., et al. (2004) TLR9 and TLR9 mice display similar immune responses to a DNA vaccine. Immunology 113, 114ā120.
Ma, X., Forns, X., Gutierrez, R., et al. (2002) DNA-based vaccination against hepatitis C virus (HCV): effect of expressing different forms of HCV E2 protein and use of CpG-optimized vectors in mice. Vaccine 20, 3263ā3271.
Sato, Y., Roman, M., Tighe, H., et al. (1996) Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science 273, 352ā354.
Klinman, D. M., Yamshchikov, G., and Ishigatsubo, Y. (1997) Contribution of CpG motifs to the immunogenicity of DNA vaccines. J. Immunol. 158, 3635ā3639.
Raz, E., Tighe, H., Sato, Y., et al. (1996) Preferential induction of a Thl immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization. Proc. Natl. Acad. Sci. USA 93, 5141ā5145.
Leifer, C. A., Verthelyi, D., and Klinman, D. M. (2003) Heterogeneity in the human response to immunostimulatory CpG oligodeoxynucleotides. J. Immunol. 26, 313ā331.
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Jiang, W., Reich, C.F., Pisetsky, D.S. (2006). In Vitro Assay of Immunostimulatory Activities of Plasmid Vectors. In: Saltzman, W.M., Shen, H., Brandsma, J.L. (eds) DNA Vaccines. Methods in Molecular Medicineā¢, vol 127. Humana Press. https://doi.org/10.1385/1-59745-168-1:55
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DOI: https://doi.org/10.1385/1-59745-168-1:55
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