CHARACTERIZATION OF EPITOPES INVOLVED IN THE NEUTRALIZATION OF PASTEURELLA-HAEMOLYTICA SEROTYPE A1 LEUKOTOXIN

Defined segments of the leukotoxin A gene (IktA) from an A1 serotype o f Pasteurella haemolytica were cloned into a plasmid vector and expres sed as LacZ alpha fusion proteins. These fusion proteins were electrop horesed in SDS-PAGE gels and their immunoblotting reactivities with se veral monoclonal antibodies characterized. The epitope recognized by a strongly neutralizing monoclonal antibody was localized to a 32 amino acid region near the C terminus of the leukotoxin A (LktA) molecule. The epitope recognized by a non-neutralizing antibody was localized to a 33 amino acid region immediately adjacent. Smaller recombinant pept ides containing these epitopes were not antigenic, but a polypeptide e ncompassing 229 amino acids at the C terminus evoked neutralizing anti bodies when used to immunize specific-pathogen-free lambs. The distrib utions of linear epitopes recognized by this antiserum and by antisera raised to full-length recombinant LktA and to native LktA produced by P. haemolytica serotype Al were determined by their reactivities with a set of overlapping 10 amino acid synthetic peptides. This revealed a complex distribution of linear epitopes at the C-terminal end of Lkt A. Toxin-neutralizing antibodies in convalescent sheep serum were show n to be directed against conformational epitopes by selective absorpti on of antibodies directed against linear epitopes.