The primary goal of this proposal is to construct an effective, nontoxic acellular vaccine against Bordetella pertussis. Conditions will be established to produce a toxoid of pertussis toxin that lacks cytotoxicity but retains immunogenicity. Biochemical and biological analyses will define the role that individual amino acids play in the ADP-ribosyltransferase reaction and will identify amino acid substitutions at these residues that reduce catalytic activity with the least perturbation of protein structure. Noncatalytic mutants of the S-1 subunit will be assembled with native B-oligomer to yield mutants of pertussis toxin. These mutants of pertussis toxin will be examined for cytotoxic activity in tissue culture, and those mutants with reduced cytotoxicity will be tested in a mouse model to determine cytotoxicity and protection to challenge by Bordetella pertussis. These studies will also examine additional biochemical and biological properties of the S-1 subunit, in order to (i) map the active site with respect to both adenine and nicotinamide binding, (ii) identify amino acids involved in the binding of the S-1 subunit to target protein, (iii) characterize the assembly of the S-1 subunit with the B-oligomer, (iv) identify the protease sensitive region of the S-1 subunit, and (v) construct hybrid molecules to study the entry of pertussis toxin into sensitive cells. The detailed mapping of functional residues of the S-1 subunit will assist in the development of a genetic approach to the production of a toxoid of pertussis toxin and permit the prediction of active site residues in other bacterial ADP-ribosylating exotoxins.
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