Nucleic Acid Immunizations

Harriet L. Robinson1, Tamera M. Pertmer1

1 Yerkes Primate Research Center, Emory University, Atlanta, Georgia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 2.14
DOI:  10.1002/0471142735.im0214s27
Online Posting Date:  May, 2001
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Abstract

This unit provides protocols for two broadly used methods of DNA immunization: saline injections and gene gun deliveries of DNA. Saline injections deliver DNA into extracellular spaces; gene gun deliveries bombard DNA directly into cells. Support protocols present methods for preparation of DNA‚Äźcoated gold beads, creation of cartridges containing these beads, and optimization of gene gun parameters. Issues relating to plasmid vectors, DNA preparation and doses, and immunization regimens are also discussed. Expression library immunizations, genetic and conventional adjuvants, alternative boosts, neonatal immunizations, and approaches to mucosal delivery of DNA are covered in the commentary.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Saline Inoculation of DNA in Mice
  • Basic Protocol 2: Gene Gun Inoculation of DNA in Mice
  • Support Protocol 1: Preparation of DNA‐Coated Gold Beads
  • Support Protocol 2: Preparation of Cartridges Containing DNA‐Coated Gold Beads
  • Support Protocol 3: Optimization of Helium Pressure and Bead Size to be Used for Gene Gun Delivery
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Saline Inoculation of DNA in Mice

  Materials
  • Purified expression plasmid DNA (see and unit 10.3)
  • 0.9% NaCl, sterile
  • Mice of appropriate strain
  • 100 mg/ml ketamine⋅HCl
  • 20 mg/ml xylazine⋅HCl
  • 0.5 or 1‐ml disposable syringes with 27‐ or 30‐G needles
  • Steel surgical staples or sutures
  • Additional reagents and equipment for ethanol precipitation of DNA (unit 10.1, protocol 1, steps to ) and handling and restraint (unit 1.3) and injection (unit 1.6) of mice

Basic Protocol 2: Gene Gun Inoculation of DNA in Mice

  Materials
  • Helios gene gun (Bio‐Rad)
  • Compressed helium—purity grade >4.5 (99.995%); maximum pressure, 2600 psi—with high‐pressure helium tank gas regulator
  • Cartridges containing DNA‐coated gold beads (see protocol 3Support Protocols 1 and protocol 42)
  • Electric clippers
  • Hearing protection (e.g., ear plugs)
  • Additional reagents and equipment for handling and restraint (unit 1.3) and anesthetization (unit 1.4; optional) of mice
NOTE: The gene gun and tank regulator are supplied as part of the complete Helios Gene Gun System (Bio‐Rad) or can be purchased separately from Bio‐Rad.

Support Protocol 1: Preparation of DNA‐Coated Gold Beads

  Materials
  • Purified expression plasmid DNA (see and unit 10.3)
  • 1‐µm (or other appropriate size) gold beads (Bio‐Rad; supplied as part of Helios Gene Gun System or can be purchased separately); see protocol 5 for optimization
  • 0.05 M spermidine free base (Sigma; store up to several months at room temperature)
  • 1 M CaCl 2
  • Fresh 100% ethanol, room temperature
  • Sonicating water bath
  • 15‐ml conical polypropylene tubes

Support Protocol 2: Preparation of Cartridges Containing DNA‐Coated Gold Beads

  Materials
  • Compressed nitrogen—purity grade >4.8 (99.998%); maximum pressure, 2600 psi—with single‐stage, low‐pressure nitrogen tank regulator, maximum pressure reading of 30 to 50 psi (Bio‐Rad)
  • DNA/gold/ethanol bead suspension in 15‐ml conical tube (see protocol 3)
  • TE buffer, pH 8.0 ( appendix 2A; optional)
  • Tubing Prep Station (Bio‐Rad)
  • 10‐ml syringes
  • Silicone adapter tubing (Masterflex silicone tubing; Cole‐Parmer)
  • Sonicating water bath
  • Peristaltic pump with 1/8‐in. (0.32‐cm) i.d. tubing (optional)
  • Teflon tubing (Tefzel tubing, 1/8 in. diameter; McMaster‐Carr)
  • Tubing Cutter Unit (Bio‐Rad) or razor blade
  • Glass scintillation vials (or comparable capped tubes)
  • Desiccant packs
  • Parafilm
  • Spectrophotometer (optional)
NOTE: The tubing prep station, silicone and Teflon tubing, tubing cutter unit, glass vials, and desiccant packs are supplied as part of the complete Helios Gene Gun System (Bio‐Rad) or can be purchased separately from Bio‐Rad (or other suppliers as indicated).

Support Protocol 3: Optimization of Helium Pressure and Bead Size to be Used for Gene Gun Delivery

  Materials
  • Mice of appropriate strain
  • Cartridges containing DNA‐coated gold beads (see protocol 3Support Protocols 1 and protocol 42)
  • 4% (w/v) paraformaldehyde in PBS (see appendix 2A for PBS)
  • Electric clippers
  • Skin punch
  • Index cards cut into 1 × 1–in. (2.5 × 2.5–cm) pieces
  • Histology casettes
  • Additional reagents and equipment for anesthetization of mice (see protocol 1), gene gun injections (see protocol 2), paraffin sectioning (unit 21.4), and histological (eosin only) staining (Watkins, )
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Figures

Videos

Literature Cited

Literature Cited
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   Boyle, C.M., Morin, M., Webster, R., and Robinson, H. 1996. Role of different lymphoid tissues in the initiation and maintenance of DNA‐raised antibody responses to the influenza virus H1 glycoprotein. J. Virol. 70:9074‐9078.
   Casares, S., Inaba, K., Brurneanu, T.‐D., Steinman, R., and Bona, C. 1997. Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class II‐restricted viral epitope. J. Exp. Med. 186:1481‐1486.
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   Chapman, B.S., Thayer, R., Vincent, K., and Haigwood, N. 1991. Effect of intron A from human cytomegalovirus (Towne) immediate‐early gene on heterologous expression in mammalian cells. Nuc. Acids Res. 19:3979‐3986.
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  FDA. 1996. Points to consider on plasmid DNA vaccines for preventative infectious disease indications. U.S. Food and Drug Administration, Docket #96‐N‐0400. Available via FTP; see Internet Resources.
   Fynan, E.F., Webster, R., Fuller, D., Haynes, J., Santoro, J., and Robinson, H. 1993. DNA vaccines: Protective immunizations by parenteral, mucosal, and gene gun inoculations. Proc. Natl. Acad. Sci. U.S.A. 90:11478‐11482.
   Ginsberg, H.S., Brown, F., Chanock, R., and Lerner, R. (eds.). 1993. Vaccines 93: Modern approaches to new vaccines including the prevention of AIDS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
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   Gregoriadis, G., Saffie, R., and de Souza, J. 1997. Liposome‐mediated DNA vaccination. FEBS Lett. 402:107‐110.
   Hartikka, J., Sawdey, M., Cornefert‐Jensen, F., Margalith, M., Barnhart, K., Nolasco, M., Vahlsing, H., Meek, J., Marquet, M., Hobart, P., Norman, J., and Manthorpe, M. 1996. An improved plasmid DNA expression vector for direct injection into skeletal muscle. Hum. Gene Ther. 7:1205‐1217.
   Herrmann, J.E. and Robinson, H.L. 1998. DNA vaccines for mucosal immunity. In Handbook for Mucosal Immunity (J. McGhee, ed.). Academic Press, New York in press.
   Huang, L. and Li, S. 1997. Liposomal gene delivery: A complete package. Nat. Biotechnol. 15:620‐621.
  International Meeting on Nucleic Acid Vaccines. 1997. Vaccine 15:8 (special conference issue).
   Iwasaki, A., Stiernholm, B., Chan, A., Berinstein, N., and Barber, B. 1997. Enhanced CTL responses mediated by plasmid DNA immunogens encoding costimulatory molecules and cytokines. J. Immunol. 158:4591‐4601.
   Jones, D.H., Corris, S., McDonald, S., Clegg, J., and Farrar, G. 1997. Poly(DL‐lactide‐co‐glycolide)‐encapsulated plasmid DNA elicits systemic and mucosal antibody responses to encoded protein after oral administration. Vaccine 15:814‐817.
   Liu, M.A., Hillerman, M., and Kurth, R. (eds.). 1995. DNA vaccines: A new era in vaccinology. Ann. N.Y. Acad. Sci. 772:1‐294.
   Livingston, J.B., Lu, S., Robinson, H., and Anderson, D. 1998. Immunization of the female genital tract with a DNA‐based vaccine. Infect. Immun. 66:322‐329.
   Martinez, X., Brandt, C., Saddallah, F., Tougne, C., Barrios, C., Wild, F., Dougan, G., Lambert, P.‐H., and Siegrest, C.‐A. 1997. DNA immunization circumvents deficient induction of T helper type 1 and cytotoxic T lymphocyte responses in neonates and during early life. Proc. Natl.Acad. Sci. U.S.A. 94:8726‐8731.
   Mathiowitz, E., Jacob, J., Jong, Y., Carino, G., Chickering, D., Chaturvedi, P., Santos, C., Vijayaraghaven, K., Montgomery, S., Bassett, M., and Morrell, C. 1997. Biologically erodable microspheres as potential oral drug delivery systems. Nature 386:410‐414.
   Montgomery, D.L., Shiver, J., Leander, K., Perry, H., Friedman, A., Martinez, D., Ulmer, J., Donnelly, J., and Liu, M. 1993. Heterologous and homologous protection against influenza A by DNA vaccination: Optimization of DNA vectors. DNA Cell Biol. 12:777‐783.
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   Richmond, J.F.L., Lu, S., Santoro, J.C., Weng, J., Hu, S.‐L., Montefiori, D.C., and Robinson, H.L. 1998. Studies on neutralizing activity and avidity of anti‐HIV‐1 env antibody elicited by DNA priming and protein boosting. J. Virol. In press.
   Robinson, H.L., Hunt, L., and Webster, R. 1993. Protection against lethal influenza virus challenge by immunization with a haemagglutinin‐expressing plasmid DNA. Vaccine 11:957‐960.
   Robinson, H.L. and Torres, C. 1997. DNA vaccines. Semin. Immunol. 9:271‐283.
   Rodriguez, F., Zhang, J., and Whitton, J. 1997. DNA immunization: Ubiquitination of a viral protein enhances cytolytic T‐lymphocyte induction and antiviral protection but abrogates antibody induction. J.Virol. 71:8497‐8503.
   Sasaki, S., Tsuji, T., Hamajima, K., Fukushima, J., Ishii, N., Kaneko, T., Xin, K.‐Q., Aoki, I., Okubo, T., Nishioka, K., and Okuda, K. 1997. Monophosphoryl lipid A enhances both humoral and cell‐mediated immune responses to DNA vaccination against human immunodeficiency virus type 1. Infect. Immun. 65:3520‐3528.
   Schneider, J., Gilbert, S.C., Blanchard, T.J., Hanke, T., Robson, K.J., Hannan, C.M., Becker, M., Sinden, R., Smith, G.L., and Hill, A.V.S. 1998. Enhanced immunogenicity for CD8+ T cell induction and complete protective efficacy of malaria DNA vaccination boosting with modified virus Anakara. Nat. Med. 4:397‐402.
   Sizemore, D.R., Branstorm, A., and Sadoff, J. 1995. Attenuated Shigella as a DNA delivery device for DNA‐mediated immunization. Science 270:297‐302.
   Tang, T.C., DeVit, M., and Johnston, S. 1992. Genetic immunization is a simple method for eliciting an immune response. Nature 356:152‐154.
   Templeton, N.S., Lasic, D., Frederik, P., Strey, H., Roberts, D., and Pavlakis, G. 1997. Improved DNA: Liposome complexes for increased systemic delivery and gene expression. Nature Biotechnol. 15:647‐652.
   Torres, C.A.T., Iwasaki, A., Barber, B., and Robinson, H. 1997. Differential dependence on target site tissue for gene gun and intramuscular DNA immunizations. J. Immunol. 158:4529‐4532.
   Ulmer, J.B., Donnelly, J., Parker, S., Rhodes, G., Felgner, P., Dwarki, V., Gromkowski, S., Deck, R., DeWitt, C., Friedman, A., Hawe, L., Leander, K., Martinez, D., Perry, H., Shiver, J., Montgomery, D., and Liu, M. 1993. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259:1745‐1749.
   Vahlsing, H.L., Yankauckas, M., Sawdey, M., Gromkowski, S., and Manthorpe, M. 1994. Immunization with plasmid DNA using a pneumatic gun. J. Immunol. Methods. 175:11‐22.
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   Wolff, J.A., Malone, R., Williams, P., Chong, W., Acsadi, G., Jani, A., and Felgner, P. 1990. Direct gene transfer into mouse muscle in vivo. Science 247:1465‐1468.
   Xiang, Z. and Ertl, H. 1995. Manipulation of the immune response to a plasmid‐encoded viral antigen by coinoculation with plasmids expressing cytokines. Immunity 2:129‐135.
Key References
   Donnelly et al., 1997. See above.
  Recent general review of DNA vaccination.
   Fynan et al., 1993. See above.
  The demonstration of protective immunizations by intradermal, intravenous, intramuscular, intranasal, and gene gun delivery.
   Robinson and Torres, 1997. See above.
  Recent general review of DNA vaccines.
   Tang et al., 1992. See above.
  The first demonstration of the raising of an immune response by DNA.
   Ulmer et al., 1993. See above.
  The first demonstration of the use of DNA to raise Tc and the provision of protective immunity by the Tc.
Internet Resources
  http://www.genweb.com/Dnavax/dnavax.html
  The DNA vaccine web page maintained by Robert Whalen.
  ftp://ftp.fda.gov/CBER/ptc/plasmid.txt
  The FDA's “Points to Consider on Plasmid DNA Vaccines for Preventative Infectious Disease Indications.”
  http://www-ngvl.med.umich.edu/
  National Gene Vector Laboratory web site address.
  http://www.bio-rad.com/60287.html
  Bio‐Rad web site address for Helios gene gun information.
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