Plasmid DNA vaccines: assay for integration into host genomic DNA.

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.

The role of natural-enemy escape in a gallmaker host-plant shift.

The successful colonization of novel host-plant species by herbivorous insects may be facilitated by a reduction in natural-enemy attack on insect populations associated with the novel (derived) host plant. This is particularly true if natural enemies use host-plant or habitat cues in searching for their herbivore prey. In order to test whether the acquisition of enemy-free space could have influenced the host shift in the goldenrod ball gallmaker, Eurosta solidaginis, we estimated levels of natural-enemy attack in 25 host-race populations associated with Solidago altissima and S. gigantea (Compositae) spanning the zone of sympatry between S. altissima and S. gigantea host races in New England. Mortality due to attack by the parasitoid wasp Eurytoma obtusiventris was significantly higher for the ancestral than for the derived host race (30.5% versus 0.4%) across the transect, which is consistent with the enemy escape hypothesis. Contrary to this hypothesis, mordellid beetles caused significantly higher mortality on the derived than ancestral host race (17.1% versus 2.6%). Mortality by a second parasitoid wasp and birds showed no significant differences between the two host races. Overall, the derived host race had significantly higher survivorship across the transect (36.6% versus 20.8%). An analysis of survivorship and parasitoid mortality levels from sympatric sites in this study and previous studies showed a highly significant correlation between the levels of Eurytoma obtusiventris attack and the survivorship advantage of the derived host race. Observations of this parasitoid's searching behavior confirmed that it preferentially searches the ancestral host for fly larvae. Current patterns of host-race mortality and naturalenemy behavior and abundance are consistent with the facilitation of the host shift by escape from a specialist parasitoid.