Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities

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Abstract
The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (Mr 38000, 341 amino acids). To identify antigenic determinants on Hib porin that might be exposed at the bacterial ceil surface, seven mouse monoclonal anti-Hib porin antibodies were generated. The monoclonal antibodies were tested for their binding to intact cells by flow cytometry; all but one bound to the cell surface. Digestions of Hib porin with cyanogen bromide, hydroxylamine or trypsin generated fragments, the identities of which were confirmed by microsequencing of the amino termini. Following electrophoresis and immunoblotting of the fragments, the specificities of the monoclonal antibodies for their cognate sequences were determined. The porin gene ompP2 was expressed in the baculovirus expression vector system; the recombinant porin was recognized by all of the monoclonal antibodies. Deletions were created by omega mutagenesis of ompP2, generating proteins truncated after amino acids 139, 174, 182, and 264. These deletion proteins were tested for reactivities with the monoclonal antibodies, thereby establishing the boundaries of three antigenic determinants that were recognized by the monoclonals: domain (i), amino acids 104–139; domain (ii) amino acids 162–174; and domain (iii), amino acids 267–341. The biological activities of monoclonal antibodies that were representative of these three classes were tested for their bactericidal activity in complement-mediated tysis of whole cells. The monoclonal antibodies were also tested for their immunoprotective properties in the infant rat model of bacteraemia. Although the monoclonal antibodies were surface-binding, they were neither bactericidal nor protective.