PURA COMPLEMENTATION FOR PLASMID SELECTION & MAINTENANCE

LIVE ATTENUATED SALMONELLA STRAINS ARE BEING INVESTIGATED ASVECTORS FOR THE DELIVERY OF FOREIGN ANTIGENS TO STIMULATE A PROTECTIVE IMMUNE RESPONSE AGAINST VARIOUS BACTERIAL, VIRAL,AND PROTOZOAN DISEASES. THESE ORAL VACCINES CURRENTLY UNDERDEVELOPMENT RELY ON EXPRESSION OF FOREIGN ANTIGENS FROM PLASMID VECTORS WHICH ALSO ENCODE AMPICILLIN RESISTANCE. ITIS UNLIKELY THAT A VACCINE STRAIN CARRYING A FUNCTIONAL ANTIBIOTIC GENE WILL BE ACCEPTABLE TO THE REGULATORY AGENCIES OR TO THE PUBLIC AT LARGE. THIS PROPOSAL IS TO EXAMINE THE FEASIBILITY OF AN ALTERNATIVE SELECTION SYSTEM WHICH SHOULD SELECTIVELY MAINTAIN THE PLASMID IN THE ABSENCE OF ANY EXOGENOUSLY APPLIED SELECTIVE PRESSURE. COMPLEMENTATION OF A PURA DEFECT IN THE BACTERIAL HOST CHROMOSOME BY A PLASMID EXPRESSING ACTIVE ADENYLSUCCINATE SYNTHETASE (PURA+) SHOULD PROVIDE A POWERFUL SELECTION FOR MAINTENANCE OF THE PLASMID VECTOR. THIS STUDY WILL DETERMINE WHETHER SELECTION OF PURINE INDEPENDENCE COULD SUBSTITUTE FOR STANDARD AMPICILLINSELECTION IN SMALL SCALE LIQUID CULTURE OF S. TYPHIMURIUM AND S. DUBLIN VACCINE STRAINS IN THE LABORATORY, IN 10L SCALE FERMENTATION, AND IN VIVO IN THE MOUSE MODEL SYSTEM. SUCCESSFUL DEMONSTRATION OF SELECTIVE MAINTENANCE OF PURA PLASMID BEARING STRAINS IN CULTURE AND IN VIVO WILL LEAD TO CLINICAL TRIALS OF PURA PLASMIDS IN LIVE ORAL BACTERIAL VACCINES.