A monoclonal antibody (MAb) to Moraxella catarrhalis O35E bound to a surface-exposed epitope of a proteinaceous antigen of this organism. The antigen, designated UspA, was present in every strain of the pathogen tested in a colony blot RIA. UspA had a molecular mass on SDS-PAGE that varied between 300 and 400 kDa, depending on the individual M. catarrhalis strain. Passive immunization of mice with the UspA-reactive Mab enhanced pulmonary clearance of M. catarrhalis. Use of this Mab to screen a M. catarrhalis genomic DNA library permitted identification of a recombinant bacteriophage expressing the M. catarrhalis UspA protein. The recombinant UspA protein was used in Western blot analysis with sera from patients with M. catarrhalis pneumonia. Convalescent-phase sera but not acute-phase sera from these patients contained antibodies to this M. catarrhalis surface protein, indicating that M. catarrhalis strains growing in vivo express this molecule.

A major outer membrane protein (CopB) of Moraxella catarrhalis is a target for antibodies that enhance clearance of this organism from the lungs of mice. A mini-Tn10kan transposon was inserted into the cloned copB gene from M. catarrhalis O35E, and an isogenic mutant unable to express the CopB protein was constructed by transforming this mutated gene into the wild-type strain. The mutant grew at the same rate as the wild-type parent strain in broth. Unlike the serum-resistant parent strain, this mutant was sensitive to killing by normal human serum, and its ability to survive and grow in the lungs of animals was impaired. Genetic restoration of CopB protein expression resulted in the simultaneous acquisition of wild-type levels of serum resistance and the ability to resist pulmonary clearance in vivo. Thus, the CopB protein of M. catarrhalis may be important in the interaction between this organism and the defense mechanisms of the respiratory tract.

A murine immunoglobulin G monoclonal antibody (MAb) raised against outer membrane vesicles of Moraxella catarrhalis 035E was shown to bind to a surface-exposed epitope of a major outer membrane protein of this organism. This outer membrane protein, which had an apparent molecular weight of approximately 80,000 in sodium dodecyl sulfate-polyacrylamide gels, was designated CopB. MAb 10F3, reactive with CopB, bound to a majority (70%) of M. catarrhalis strains tested. More importantly, mice passively immunized with MAb 10F3 exhibited an enhanced ability to clear a bolus challenge of M. catarrhalis from their lungs, a result which suggested that CopB might have potential as a vaccine candidate. The M. catarrhalis gene encoding CopB was cloned in Escherichia coli, and nucleotide sequence analysis of the copB gene indicated that the CopB protein was synthesized with a leader peptide, a finding confirmed by N-terminal amino acid sequence analysis of the mature CopB protein purified from M. catarrhalis 035E. Southern blot analysis showed that chromosomal DNA from seven different M. catarrhalis strains hybridized with a probe comprising the majority of the copB structural gene from strain 035E. Additional data emphasizing the extent of conservation of the CopB protein among M. catarrhalis strains were obtained from Western immunoblot analyses with polyclonal antisera raised against CopB proteins from different M. catarrhalis strains used to probe the recombinant form of the CopB protein from strain 035E. The ability of the CopB protein to function as a target for biologically active antibodies and its apparent conservation among M. catarrhalis strains warrant further investigation of this outer membrane protein as a potential vaccine candidate.

The major outer membrane protein (MOMP) of Haemophilus ducreyi is an OmpA homolog that migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels as three species with apparent molecular weights ranging from 37,000 to 43,000. Monoclonal antibodies directed against this macromolecule were used to identify recombinant clones containing fragments of the gene encoding this protein. Nucleotide sequence analysis of these fragments confirmed that the MOMP encoded by the intact gene (momp) was a member of the OmpA family of outer membrane proteins. Construction of an isogenic H. ducreyi mutant unable to express the MOMP led to the discovery of a second outer membrane protein which migrated at the same rate on SDS-PAGE gels as the MOMP. N-terminal amino acid sequence analysis of this second protein revealed that its N terminus was nearly identical to that of the MOMP and also had homology with members of the OmpA family. Nucleotide sequence analysis of the region downstream from the momp gene revealed the presence of a partial open reading frame encoding a predicted OmpA-like protein. A modification of anchored PCR technology was used to obtain the nucleotide sequence of this downstream gene which was shown to encode a second OmpA homolog (OmpA2). The N-terminal amino acid sequence of OmpA2 was identical to that of the OmpA-like protein detected in the momp mutant. The H. ducreyi MOMP and OmpA2 proteins, which comigrated on SDS-PAGE gels and which were encoded by the tandem arranged momp and ompA2 genes, were 72% identical.

The high-molecular-weight UspA protein of Moraxella catarrhalis has been described as being both present on the surface of all M. catarrhalis disease isolates examined to date and a target for a monoclonal antibody (MAb 17C7) which enhanced pulmonary clearance of this organism in a mouse model system (M. E. Helminen et al., J. Infect. Dis. 170:867-872, 1994). A recombinant bacteriophage that formed plaques which bound MAb 17C7 was shown to contain a M. catarrhalis gene, designated uspA1, that encoded a protein with a calculated molecular weight of 88,271. Characterization of an isogenic uspA1 mutant revealed that elimination of expression of UspA1 did not eliminate the reactivity of M. catarrhalis with MAb 17C7. In addition, N-terminal amino acid analysis of internal peptides derived from native UspA protein and Southern blot analysis of M. catarrhalis chromosomal DNA suggested the existence of a second UspA-like protein. A combination of epitope mapping and ligation-based PCR methods identified a second M. catarrhalis gene, designated uspA2, which also encoded the MAb 17C7-reactive epitope. The UspA2 protein had a calculated molecular weight of 62,483. Both the isogenic uspA1 mutant and an isogenic uspA2 mutant possessed the ability to express a very-high-molecularweight antigen that bound MAb 17C7. Southern blot analysis indicated that disease isolates of M. catarrhalis likely possess both uspA1 and uspA2 genes. Both UspA1 and UspA2 most closely resembled adhesins produced by other bacterial pathogens.

The virulence mechanisms of Moraxella catarrhalis that are involved in producing pulmonary infection are unknown. A well-characterized murine model was used to study the pulmonary clearance of M. catarrhalis and analyze the histopathologic changes and the role of phagocytic cells in the infected lungs. Ten strains of M. catarrhalis from various isolation sites were evaluated for their ability to resist pulmonary clearance. The rates of clearance of these strains, based on the percentage of the original inoculum remaining at 6 h after challenge, varied considerably. Histopathologic examination of lungs infected with 2 strains that exhibited very different clearance rates revealed similar pathologic responses. Analysis of the phagocytic cell response to these 2 strains revealed significant alveolar recruitment of granulocytes at 3, 6, and 24 h after bacterial challenge. However, granulocyte recruitment in response to strain B22, which was cleared readily, was significantly greater than to strain 035E, which resisted pulmonary clearance. This model system should facilitate investigation of the molecular basis of the interaction between M. catarrhalis and the lower respiratory tract.

The amino acid sequence of the cell-surface-exposed, 81-kDa CopB outer membrane protein of Moraxella catarrhalis was found to be similar to those of TonB-dependent outer membrane proteins of other gramnegative bacteria. Expression of CopB was affected by the availability of iron in the growth medium, and the extent of overexpression of CopB in response to iron limitation varied widely among the M. catarrhalis strains tested. Wild-type M. catarrhalis strains were found to be able to utilize ferric citrate, transferrin, lactoferrin, and heme as sources of iron for growth in vitro. However, an isogenic copB mutant was severely impaired in its ability to utilize transferrin and lactoferrin as sole sources of iron for growth, whereas this same mutant grew similarly to the wild-type parent strain when supplied with ferric citrate as the iron source. The copB mutant was not significantly different from its wild-type parent strain in its ability to bind transferrin and lactoferrin. In addition, the wild-type parent strain and the copB mutant exhibited equivalent rates of uptake of 55 Fe from ferric citrate. However, the copB mutant was markedly less able than the wild-type strain to take up 55 Fe from transferrin and lactoferrin. These results indicate that lack of expression of the CopB protein exerts a direct or indirect effect on the ability of M. catarrhalis to utilize iron bound to certain carrier proteins. Moraxella (Branhamella) catarrhalis is a common respiratory tract pathogen in humans. In children, this gram-negative organism is responsible for up to 20% of cases of acute otitis media (3, 14, 18). In fact, in a recent report, M. catarrhalis DNA could be detected by PCR in middle ear effusions from 46% of patients with chronic otitis media with effusion (40). In adults, M. catarrhalis commonly causes lower respiratory tract infections in patients with chronic obstructive lung disease (13, 20, 31). In immunocompromised hosts, and mainly in patients with hematologic malignancies, M. catarrhalis has been identified as an etiologic agent of bacteremia, meningitis, skeletal infections, endocarditis, and other invasive focal infections (13, 15, 20, 29, 31). The recent recognition of M. catarrhalis as an important pathogen in both the upper and lower respiratory tract has resulted in increased interest in its interactions with the human host and, more specifically, in its antigenic composition. Outer membrane proteins constitute major antigenic determinants of this unencapsulated organism (4), and different strains share remarkably similar outer membrane protein profiles (4, 33). CopB (outer membrane protein B2), a major and antigenically well-conserved 81-kDa outer membrane protein of M. catarrhalis 035E, has been characterized in detail (23). Antibody to this surface-exposed protein is bactericidal and has been shown to enhance the rate of clearance of M. catarrhalis from the lungs of mice (23). A copB mutation rendered M. catarrhalis less able to resist pulmonary clearance in this mouse model and also converted the...

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