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Infection and Immunity, December 1999, p. 6424-6433, Vol. 67, No. 12
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Optimization of Plasmid Maintenance in the Attenuated Live Vector Vaccine Strain Salmonella typhi CVD 908-htrAdagger

James E. Galen,1,* Jay Nair, Jin Yuang Wang,1,Dagger Steven S. Wasserman,1 Michael K. Tanner,1,§ Marcelo B. Sztein,1 and Myron M. Levine1,2

Center for Vaccine Development, Division of Geographic Medicine, Department of Medicine,1 and Division of Infectious Diseases and Tropical Pediatrics, Department of Pediatrics,2 University of Maryland School of Medicine, Baltimore, Maryland 21201

Received 6 July 1999/Returned for modification 20 August 1999/Accepted 24 September 1999

The broad objective of the research presented here is to develop a noncatalytic plasmid maintenance system for the stabilization of multicopy expression plasmids encoding foreign antigens in a Salmonella typhi live-vector vaccine strain such as CVD 908-htrA. We have enhanced the maintenance of expression plasmids at two independent levels. First, we removed dependence upon balanced-lethal maintenance systems that involve catalytic enzymes expressed from multicopy plasmids; we accomplished this through incorporation into expression plasmids of a postsegregational killing system based on the noncatalytic hok-sok plasmid addiction system from the antibiotic resistance factor pR1. We also included at least one naturally occurring plasmid partition function in our expression plasmids, which eliminates random segregation of these plasmids, thereby enhancing their inheritance and stability; to accomplish 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 green fluorescent protein (GFPuv) by using flow cytometry. In this report, we demonstrate the utility of this novel plasmid maintenance system in enhancing the stability of our expression plasmids and go on to show that as the copy number of stabilized plasmids increases, the toxicity of GFPuv synthesis also increases. The implications of these observations for the rational design of immunogenic and protective bacterial live vector vaccines are discussed.

* Corresponding author. Mailing address: Center for Vaccine Development, University of Maryland School of Medicine, 685 W. Baltimore St., Baltimore, MD 21201. Phone: (410) 706-5328. Fax: (410) 706-6205. E-mail: jgalen{at}umppa1.ab.umd.edu.

dagger This work is dedicated to the memory of James F. Galen, Jr.

Dagger Present address: Pediatric House Staff, Vanderbilt University Hospital, Nashville, TN 37232-7530.

§ Present address: University of Louisville ICT, Louisville, KY 40202.

Infection and Immunity, December 1999, p. 6424-6433, Vol. 67, No. 12
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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Copyright © 1999 by the American Society for Microbiology. All rights reserved.