title: Construction and evaluation of a eukaryotic expression plasmid for stable delivery using attenuated Salmonella.
abstract: An approach to enhancing the stability of eukaryotic expression plasmids for delivery using attenuated Salmonella has been evaluated. The expression apparatus and beta-galactosidase gene from the expression plasmid, pCMVbeta, was cloned into the low copy number plasmid pLG339. The resulting construct, pLGbetaGAL, was shown to have a lower copy number than pCMVbeta in Salmonella enterica var Typhimurium aroA strain SL7207. Furthermore, beta-galactosidase-specific antibody was induced in mice following intramuscular inoculation with pLGbetaGAL as naked DNA. Following oral administration of mice with SL7207/pCMVbeta, recombinants could not be detected in tissues 3 days after inoculation. In comparison, SL7207/pLGbetaGAL recombinant bacteria could be detected in the Peyer's patches and spleens indicating that the Salmonella strain was stable. However, both SL7207/pCMVbeta and SL7207/pLGbetaGAL failed to induce beta-galactosidase-specific IgG in vivo. The mechanism by which attenuated Salmonella are able to release heterologous DNA for antigen processing and presentation is not yet understood. These results suggest that the mechanism needs to be further elucidated in order to rationally improve the system.
authors.affiliation: Department of Biomedical Sciences, Dstl Chemical and Biological Sciences, Porton Down, SP4 0JQ, Salisbury, UK. hsgarmory@dstl.gov.uk
authors.forename: Helen S
authors.initials: HS
authors.lastname: Garmory
authors.forename: Richard W
authors.initials: RW
authors.lastname: Titball
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Brown
authors.forename: Alice M
authors.initials: AM
authors.lastname: Bennett
mesh_terms: D000818:Animals; D005057:Eukaryotic Cells; D005260:Female; D018628:Gene Dosage; D005822:Genetic Vectors; D006801:Humans; D007114:Immunization; D007074:Immunoglobulin G / Q000097:blood; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D014613:Vaccines, Attenuated; D019444:Vaccines, DNA; D001616:beta-Galactosidase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D007074:Immunoglobulin G; D014613:Vaccines, Attenuated; D019444:Vaccines, DNA; D001616:beta-Galactosidase