The goal of this proposal is to construct an effective but nontoxic acellular vaccine against Bordetella pertussis, the causative agent of whooping cough. Infection by B. pertussis involves several virulence determinants and one, pertussis toxin, also appears to be a protective antigen. Toxoids of pertussis toxin will be made by elimination or substitution of active site residues of the toxin. Two complementary approaches will be used to identify active site residues: (1). Pertussis toxin and NAD undergo a novel photochemical reaction in the presence of ultraviolet light, where the nicotinamide portion of NAD is covalently transferred to the protein's NAD binding site. Photochemically labelled toxin will be digested, and photolabelled peptides purified by reverse phase- HPLC. Purified photolabelled peptide(s) will be analyzed to determine the site of nicotinamide attachment. Using oligonucleotide-directed mutagenesis, photolabelled amino acids will be substituted and mutants tested for the loss of enzymatic activity. (2). Active site regions will also be identified by deletion mapping of the S-1 catalytic subunit. Deletion mutants which lack enzymatic activity but retain the ability to reassemble with the B pentamer of pertussis toxin will be isolated. Nonenzymatic mutants of pertussis toxin will be tested for toxicity and protective properties using tissue culture and mouse potency assays. Mutants which are nontoxic but function as protective antigens will be cloned into expression vectors in Escherichia coli to produce toxoids for vaccine development. Future studies will identify the functional domains of the receptor recognition, mitogenic, and hemagglutination activities of pertussis toxin and determine how these activities relate to the toxicity and protective properties of the toxin.
