The primary safety concern for DNA vaccines is their potential to integrate into the host cell genome. We describe an integration assay based on purification of high-molecular-weight genomic DNA away from free plasmid using gel electrophoresis, such that the genomic DNA can then be assayed for integrated plasmid using a sensitive PCR method. The assay sensitivity was approximately 1 plasmid copy/microg DNA (representing approximately 150,000 diploid cells). Using this assay, we carried out integration studies of three different plasmid DNA vaccines, containing either the influenza hemagglutinin, influenza matrix or HIV gag gene. Six weeks after intramuscular injection, free plasmid was detected in treated muscle at levels ranging from approximately 1,000 to 4,000 copies/microg DNA. At 6 months, the plasmid levels ranged between 200 and 800 copies/microg DNA. Gel purification of genomic DNA revealed that essentially all of the detectable plasmid in treated quadriceps was extrachromosomal. If integration had occurred, the frequency was </= 1-8 integrations per 150,000 diploid cells, which would be at least three orders of magnitude below the spontaneous mutation rate. Our results suggest that the risk of mutation due to integration of plasmid DNA vaccines following intramuscular injection is negligible.
Copyright 2001 S. Karger AG, Basel.