A 16,600-D outer membrane protein is present in all strains of Haemophilus influenzae and antibodies to this protein are present in human serum. This study was designed to assess the role of this outer membrane protein (P6) in nontypeable H. influenzae as a target for human serum bactericidal antibody. P6 was isolated and coupled to an affinity column. Depleting normal human serum of antibodies to P6 by affinity chromatography resulted in reduced bactericidal activity of that serum for nontypeable H. influenzae. Immunopurified antibodies to P6 from human serum were bactericidal. Finally, preincubation of bacteria with a monoclonal antibody that recognizes a surface epitope on P6, inhibited human serum bactericidal killing. Taken together, these experiments establish that P6 is a target for human bactericidal antibodies. This observation provides evidence that P6 plays a potentially important role in human immunity to infection by nontypeable H. influenzae.

The purpose of this study was to characterize the degree of antigenic heterogeneity or conservation of a 16,600-dalton outer membrane protein (P6) among strains of nontypable Haemophilus influenzae. Immunization of rabbits with P6 isolated from individual strains resulted in antibody to P6 of all 25 strains tested. The titers of antibody in the sera were similar among the strains. Whole organisms of two strains were used to immunize rabbits, and antibodies were produced to P6 of all strains tested. Monoclonal antibodies developed to P6 from mice immunized with whole cells of three different strains recognized determinants on P6 of all 25 strains tested. Finally, pooled normal human serum contained antibodies to P6 of all 25 strains assayed. These studies indicate that P6 is a highly conserved antigen on the outer membrane of nontypable H. influenzae.

Branhamella catarrhalis colonizes the respiratory tract of humans and commonly causes otitis media in children and respiratory infections in adults with chronic lung disease. In view of the emergence of this organism as an important human pathogen, we used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to examine the outer membrane proteins (OMPs) of 50 strains of B. catarrhalis. OMPs were isolated from broth culture supernatants. Typical of other gram-negative bacteria, eight proteins ranging in molecular weight (MW) from approximately 98,000 to 21,000 daltons were revealed by SDS-PAGE; these proteins were designated OMP A-OMP H. Of the OMPs identified, four were heat modifiable (C, D, E, and H). The 50 strains were obtained from diverse geographic and clinical sources. The OMP patterns were strikingly homogeneous; there was minimal variability in the MW of OMPs between strains. Future studies should establish whether the similarity in MWs of OMPs is paralleled by their antigenic characteristics.

The outer membrane proteins (OMPs) of Branhamella catarrhalis were studied in an effort to identify surface-exposed determinants that are conserved among strains of the bacterium. Aliquots of polyclonal antiserum were absorbed individually by strains of B. catarrhalis. The absorbed antisera were tested in comparison with unabsorbed antiserum in an immunoblot assay against OMPs of the homologous strain. The absence of a band recognized by antibodies in the absorbed antiserum compared with the unabsorbed antiserum indicated that surface-exposed determinants of the absorbing strain cross-reacted with determinants on the homologous strain. Two antisera were absorbed individually by 20 strains of B. catarrhalis, and the absorbed sera were studied in this way in immunoblot assays. OMP E (molecular weight, ca. 56,000) expresses surface-exposed determinants that are shared among 17 of the 20 strains studied. Antibodies to OMP G (molecular weight, 28,000) were absorbed from both antisera by 14 of the 20 strains. These studies demonstrate that OMP E and OMP G express determinants that are exposed on the surface of the intact bacterium. Furthermore, these determinants are antigenically conserved among a majority of strains of B. catarrhalis. On the basis of these observations, OMPs E and G should be considered when bacterial antigens are evaluated as potential vaccine candidates.

The protein P2 comprises a large proportion of the outer membrane of nontypable Haemophilus influenzae and functions as a porin. In view of the importance of the protein as a surface antigen, the present study was designed to purify and analyze P2 with particular emphasis on detection of antigenic determinants expressed on the bacterial surface and identification of bactericidal targets on P2. The P2 protein was purified by using detergent solubility, anion-exchange chromatography, and gel-filtration chromatography sequentially. Two monoclonal antibodies to P2 were developed. One antibody (2E6) recognized a determinant expressed on the bacterial surface, whereas the other antibody (3F3) recognized an internal epitope. The surface-exposed 2E6 determinant was present on 12% of strains from a nationwide collection. P2 is a bactericidal target for antibody 2E6. Cyanogen bromide cleavage of P2 resulted in two fragments, as in type b strains. Both monoclonal antibodies recognized epitopes on the larger fragment. These observations have potentially important implications regarding the development of vaccines to prevent H. influenzae infections and the development of a serotyping system for epidemiologic studies.