Optimization of plasmid maintenance in the attenuated live vector vaccine strain Salmonella typhi CVD 908-htrA

The broad objective of the research presented here is to develop a noncatal ytic plasmid maintenance system for the stabilization of multicopy expressi on plasmids encoding foreign antigens in a Salmonella typhi live-vector vac cine strain such as CVD 908-htrA. We have enhanced the maintenance of expre ssion plasmids at two independent levels, First, we removed dependence upon balanced-lethal maintenance systems that involve catalytic enzymes express ed from multicopy plasmids we accomplished this through incorporation into expression plasmids of a postsegregational killing system based on the nonc atalytic hok-sok plasmid addiction system from the antibiotic resistance fa ctor pR1, We also included at least one naturally occurring plasmid partiti on function in our expression plasmids, which eliminates random segregation of these plasmids, thereby enhancing their inheritance and stability; to a ccomplish this, we incorporated either the par locus from pSC101, the parA locus from pR1, or both. We monitored the stability of optimized expression plasmids within CVD 908-htrA by quantitating expression of a variant of gr een fluorescent protein (GFPuv) by using flow cytometry. In this report! we demonstrate the utility of this novel plasmid maintenance system in enhanc ing the stability of our expression plasmids and go on to show that as the copy number of stabilized plasmids increases, the toxicity of GFPuv synthes is also increases. The implications of these observations for the rational design of immunogenic and protective bacterial live vector vaccines are dis cussed.