MUTANT NONTOXIC FORMS OF PERTUSS TOXIN FOR VACCINES

PERTUSSIS TOXIN (PT) IS AN IMPORTANT IMMUNOGEN FOR DEVELOPMENT OF IMMUNITY TO PERTUSSIS, AND CHEMICALLY INACTIVATED PT IS A MAJOR COMPONENT OF ACELLULAR PERTUSSIS VACCINES BEING TESTED IN THE CLINIC. A PROBLEM OF CHEMICAL INACTIVATION IS PRESERVATION OF THE TOXIN'S IMMUNOGENIC PROPERTIES, WHILE INACTIVATING ITS TOXIC PROPERTIES IN A MANNER THAT PREVENTS REVERSION OF TOXIC ACTIVITIES. THE BIOLOGIC ACTIVITIES OF PT ASSOCIATED WITH TOXICITY REQUIRE AN ENZYMATICALLY ACTIVE S1 SUBUNIT, WHILE THE B-OLIGOMER PORTION IS REQUIRED FOR BINDING PT TO MAMMALIAN CELLS. FORMS OF PT CONTAINING THE B-OLIGOMER ASSOCIATED WITH AN ENZYMATICALLY INACTIVE S1 SUBUNIT, COULD BE NONTOXIC, IMMUNOGENIC FORMS USEFUL FOR PERTUSSIS VACCINES. THE TOXIN GENES HAVE BEEN CLONED AND SEQUENCED AND INDIVIDUAL SUBUNITSEXPRESSED IN E. COLI. PHASE I STUDIES WILL USE SITE-DIRECTED MUTAGENESIS OF THE S1 GENE TO CREATE ENZYMATICALLY INACTIVE ANTIGENIC FORMS OF THE S1 SUBUNIT. THE ALTERED S1 GENE WILL BE EXPRESSED IN E. COLI AND ENZYMATIC ACTIVITY MONITORED BY ADP-RIBOSYLATION OF TRANSDUCIN. ANTIGENICITY WILL BE ASSAYED BY REACTIVITY WITHMONOCLONAL ANTIBODIES TO S1 USING WESTERN BLOTTING. APPROPRIATELY ALTERED S1 GENES WILL BE PLACED IN A PLASMID CLONING VECTOR FOR GENE REPLACEMENT IN B. PERTUSSIS. TO DETERMINE IF THE S1 SUBUNIT ASSOCIATES WITH THE B-OLIGOMER DURING BIOGENESIS, LYSATES OR CULTURE SUPERNATANTS WILL BE APPLIED TO FETUIN-SEPHAROSE WHICH BINDS THE B-OLIGOMER. BOUND MATERIAL WILL BE ASSAYED FOR S1AND THE B-OLIGOMER BY IMMUNOBLOTTING. PHASE II RESEARCH WILL EVALUATE PURIFIED ENZYMATICALLY INACTIVE FORMS OF HOLOTOXIN FOR BIOLOGICAL ACTIVITIES, IMMUNOGENICITY AND FOR PHYSICAL AND IMMUNOGENIC STABILITY.