== We first analyzed the serologic responses following two mucosal priming doses of live vectors expressing ClyA-PA83 from SSB-encoding plasmids (Table4)
== We first analyzed the serologic responses following two mucosal priming doses of live vectors expressing ClyA-PA83 from SSB-encoding plasmids (Table4). reengineeredS. Typhi strains previously tested in clinical trials, i.e., CVD 908-htrAand its less GTS-21 (DMBX-A) attenuated parent CVD 908. Immunogenicity was examined using a mouse model of intranasal immunization with live vector, followed by parenteral improving with purified PA83. PA-specific antibody responses markedly improved as the copy quantity of the SSB-encoding plasmids decreased, and this effect was dramatically enhanced when the foreign antigen was delivered by the less attenuated live vector CVD 908ssb. These results suggest that antibody responses to antigens delivered byS. Typhi live vectors are inversely related to the metabolic burden imposed by expression of the foreign antigen and that these responses can be improved when antigens are expressed from low-copy-number plasmids and exported out of the cytoplasm of less attenuated live vectors. One of the hallmarks of molecular biology has been the engineering of multicopy plasmids for the expression of proteins from both prokaryotic and eukaryotic organisms. When expression plasmids encoding heterologous antigens are launched into attenuated bacterial vaccine strains, the producing multivalent live vectors can be employed to vaccinate against several unrelated human pathogens. Historically, antibiotic resistance genes have been inserted into these expression plasmids for selection purposes after introduction into live vectors. GTS-21 (DMBX-A) One important disadvantage of selection with antibiotics is that the constant selective pressure required for maintaining potentially unstable plasmids within live vectors is usually absent in the host after immunization, increasing the probability of plasmid loss and reduced immune responses against foreign antigens. Perhaps a more pressing concern with expression plasmids encoding resistance determinants relates to the containment of these plasmids within live vectors to prevent possible spread of antimicrobial resistance genes to other species, such as the commensal intestinal microbiota or transient pathogenic bacteria. Until recently, plasmids encoding resistance genes were considered to pose little or no risk for compromising clinical antimicrobial treatments because (i) such plasmids are designed to be inefficiently mobilized from live vector donors to a recipient (27,31), (ii) the live vectors themselves are also genetically attenuated and have a limited ability to persist in a host long enough to allow transfer of genetic information (23,24,26,39), (iii) the genes encoding resistance to target antibiotics are rarely or never used in human medicine, and (iv) with no relevant antibiotic selective pressure, even rare plasmid transfers would not lead to de novo resistances becoming established within a new bacterial populace (27,31). However, accumulating evidence suggests that conditions within the human gastrointestinal tract are in fact conducive to the transfer of both self-transmissible and nonmobilizable plasmids. The human intestine is usually a reservoir of mobile genetic information that can be transferred between resident microbial flora and from commensal organisms to transient bacterial populations (34). Not surprisingly, plasmids play a prominent role in such GTS-21 (DMBX-A) genetic transfer of information and employ an astonishing variety of tools, including insertion sequences, transposons, and integrons, to capture genes and transfer them to other organisms (2,3). Given sufficient populace densities of both donor and recipient strains, transfer of self-transmissible resistance plasmids (encoding their own conjugation functions) has been demonstrated to occur within only a few days in the human gut, both in the presence (21,27) and in the absence (40) of antibiotic selective pressure. Studies with experimental animal models suggest that gastrointestinal transfer frequencies can, in Rabbit Polyclonal to ARC some cases, be higher than observed forin vitroconventional conjugations, particularly when there is antibiotic pressure (6,10). It has also been exhibited that transfer of designed plasmids, in which all transfer and mobilization functions have presumably been removed, can occur in the presence of conjugative plasmids driving the transfer (27). Moreover, such conjugative plasmids do not have to be present in the same bacterium as the designed plasmid for transfer to occur. Experimentsin vitrohave shown that nonmobilizable plasmids can be captured by other bacteria harboring a conjugative plasmid that initiates.