title: In vitro and in vivo stability of recombinant plasmids in a vaccine strain of Salmonella enterica var. Typhimurium.
abstract: This study examined the ability of different plasmid vectors encoding H(C) fragment, the non-toxic binding portion of tetanus toxin, to be stably retained by Salmonella enterica var. Typhimurium (Salmonella typhimurium) vaccine strain BRD509 and, upon immunisation, to induce an antibody response against the carried antigen. The H(C) fragment expression cassette containing the transcription/translation signals, H(C) fragment open reading frame and the downstream TrpA terminator, was excised from pTETtac4 and incorporated into the plasmids pIC20H, pBR322, pACYC184 and pRSF1010. The resulting constructs were transferred into attenuated S. typhimurium, BRD509, and the level of H(C) fragment expression was examined by Western blot analysis. The relative stability of each plasmid in S. typhimurium was determined in vitro in the absence of antibiotic selection, and in vivo following immunisation. The ability of each H(C) fragment-expressing strain to induce lipopolysaccharide- and tetanus toxoid-specific antibody responses was assayed by an enzyme-linked immunosorbent assay. These studies showed that all the vaccine vector constructs, except the S. typhimurium carrying the expression vector based on pIC20H, were able to elicit a high titre immune response. The level of tetanus toxoid-specific antibody induced by S. typhimurium directly correlated with the level of in vitro and in vivo stability of the H(C) fragment expression plasmid carried by the bacterium, and not with an increased copy number of the parent plasmid vector.
authors.affiliation: Department of Microbiology and Immunology, The University of Melbourne, Parkville, Vic. 3052, Australia.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Dunstan
authors.forename: Cameron P
authors.initials: CP
authors.lastname: Simmons
authors.forename: Richard A
authors.initials: RA
authors.lastname: Strugnell
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology; D005822:Genetic Vectors; D008070:Lipopolysaccharides / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D022562:Salmonella Vaccines / Q000008:administration & dosage / Q000276:immunology; D012486:Salmonella typhimurium / Q000235:genetics* / Q000276:immunology*; D013744:Tetanus Toxin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D013745:Tetanus Toxoid / Q000276:immunology; D014613:Vaccines, Attenuated / Q000235:genetics / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D008070:Lipopolysaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D022562:Salmonella Vaccines; D013744:Tetanus Toxin; D013745:Tetanus Toxoid; D014613:Vaccines, Attenuated