Identification of surface-exposed B-cell epitopes on high molecular-weight adhesion proteins of nontypeable...

We previously reported that two surface-exposed high-molecular-weight proteins, HMW1 and HMW2, expressed by a prototypic strain of nontypeable Haemophilus influenzae (NTHI) mediate attachment to human epithelial cells. These proteins are members of a family of highly immunogenic proteins common to most nontypeable Haemophilus strains. We also reported that immunization with an HMW1-HMW2 mixture modified the course of disease in an animal model of otitis media, suggesting the potential usefulness of these proteins as NTHI vaccine components. Identification of surface-accessible B-cell epitopes could be important to efforts to develop recombinant or synthetic peptide vaccines based upon these high-molecular-weight proteins. Thus, the purpose of the present study was to identify surface-accessible epitopes on the HMW1 and HMW2 proteins by using monoclonal antibodies (MAbs) and to determine the prevalence of these epitopes among the high-molecular-weight proteins expressed by heterologous nontypeable Haemophilus strains. MAbs were generated by immunizing mice with high-molecular-weight proteins purified from prototype strains and were screened by immunoelectron microscopy (IEM) for the ability to recognize surface epitopes. Two MAbs, designated AD6 and 10C5, that recognized surface epitopes by IEM were recovered. In order to map the epitopes recognized by these two MAbs, we constructed a set of HMW1 and HMW2 recombinant fusion proteins using the pGEMEX vectors and examined the reactivity of the MAbs with these fusion proteins. MAb AD6 recognized an epitope in both HMW1 and HMW2 which mapped to the last 75 amino acids at the carboxy termini of the two proteins. When examined for reactivity with heterologous strains, MAb AD6 recognized high-molecular-weight proteins in 75% of 125 unrelated nontypeable Haemophilus strains and, in addition, reacted with three of three such strains when examined by IEM. MAb 10C5 recognized an epitope that mapped to a 155-amino-acid segment near the carboxy terminus of HMW1. This epitope was adjacent to but distinct from the AD6 epitope and was absent from HMW2. The 10C5 epitope was expressed by 40% of the AD6 reactive strains. Identification of shared surface-exposed epitopes on the high-molecular-weight adhesion proteins suggests the possibility of developing recombinant or synthetic peptide-based vaccines protective against disease caused by the majority of NTHI strains.