Plasmid DNA vaccines: assay for integration into host genomic DNA

Dev Biol (Basel). 2000:104:33-43.

Abstract

The primary safety concern for DNA vaccines is their potential to integrate into host cellular DNA. We describe a sensitive and quantitative assay for investigating the tissue distribution and integration of plasmid DNA vaccines. By including gonadal tissues in the analysis, the potential for germline transmission is also assessed. At various time points after injection, total DNA is isolated from a variety of tissues and assayed by PCR for the presence of plasmid. To test for integration, genomic DNA is first purified away from free plasmid using a series of different gel electrophoresis procedures. The gel-purified genomic DNA is then assayed for integrated plasmid using PCR. Stringent methods are used to prevent contamination. The assay, validated using a variety of positive and negative controls, is capable of detecting one copy of plasmid per ug DNA (approximately 150,000 diploid cells). Using this assay, we have carried out intramuscular studies in mice or guinea pigs for four different DNA vaccine plasmids. There was no evidence of integration to a sensitivity of about one copy/microg DNA, which is at least three orders of magnitude below the spontaneous mutation frequency.

MeSH terms

  • Animals
  • DNA / genetics
  • DNA / isolation & purification
  • DNA Restriction Enzymes
  • Electrophoresis, Agar Gel / methods
  • Female
  • Guinea Pigs
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Mutation
  • Plasmids / genetics*
  • Polymerase Chain Reaction
  • Recombination, Genetic
  • Safety
  • Tissue Distribution
  • Vaccines, DNA / genetics*

Substances

  • Vaccines, DNA
  • DNA
  • DNA Restriction Enzymes