Eight MAbs were used to study the antigenic structure of OMP CD and the antigenic conservation of the protein among strains of M. catarrhalis. Three observations led to the mapping of antibody 3.9H to a 20-amino-acid region near the amino terminus of the protein. (i) The epitope recognized by antibody 3.9H was present on OMP CD of 49 of 52 strains of M. catarrhalis, indicating that the sequence of OMP CD of the three nonreactive strains differed from the others. (ii) Antibody 3.9H bound to an epitope in the GST construct corresponding to amino acids 1 to 105 (Fig. ). (iii) Previous work involving the determination of the nucleotide sequence of the ompcd gene from eight strains identified minor sequence differences in amino acids 33 to 37. Based on these three observations, a peptide corresponding to amino acids 25 to 44 was expressed as a fusion protein and was tested in immunoblot assay with antibody 3.9H. Antibody 3.9H recognized the peptide (Fig. ). Since this antibody bound whole bacterial cells in flow cytometry, the results indicate that a surface-exposed epitope is located within amino acids 25 to 44 on the OMP CD.
A second surface-exposed epitope is located in the central region of the OMP CD molecule. This conclusion is based on the observation that antibody 1D3 is reactive with whole bacterial cells in flow cytometry and recognizes a peptide composed of amino acids 261 to 331. Three additional MAbs (5E8, 4G10, and 2D8) recognize surface-exposed epitopes on OMP CD but do not bind any of the six peptides which span the sequence of protein. While it is possible that these antibodies recognize linear peptides which are not present in their entirety on the six peptides, the more likely explanation is that these MAbs recognize conformational epitopes on the molecule.
Assays of the antibody response to OMP CD in 20 patients with COPD revealed variability in the antibody response among individuals. Such variability is also observed in the human antibody response to outer membrane proteins of other gram-negative human pathogens (13
). Patients who experienced exacerbations associated with the recovery of M. catarrhalis
from sputum had a higher prevalence and higher levels of antibodies to OMP CD compared to patients with COPD from whom M. catarrhalis
was not recovered (Table ). However, the levels of antibodies prior to exacerbations and after exacerbations were similar, indicating that no new antibodies to OMP CD were made after infection. Therefore, OMP CD does not appear to be an immunodominant antigen with regard to the human antibody response to natural infection. The increased concentration of antibodies to OMP CD in the exacerbation group may represent a marker for repeated infection with M. catarrhalis
is an exclusively human pathogen. Studies with OMPs have revealed that human antibodies can be directed toward different epitopes compared to those recognized by other mammalian species (3
). Therefore, patient samples were used to identify the important portions of the OMP CD molecule recognized by human antibodies. Three human serum samples which contained antibodies to OMP CD were used to begin to elucidate the important parts of the OMP CD molecule with regard to the human immune response. All three sera contained antibodies exclusively to a single region of the OMP CD molecule (amino acids 203 to 260), as detected by immunoblot assay with fusion peptides. Of interest, this is a proline-rich region and likely encompasses the “hinge” region of the OMP CD molecule, which has characteristics of an OMP A-like protein (32
Adsorption of sera with whole bacterial cells was used to determine the proportion of human serum antibodies which were directed at surface-exposed epitopes. Adsorption of human serum samples with whole bacteria resulted in the removal of most of the OMP CD-specific antibody, as measured by ELISA in serum samples 1E19 and 12E16 (Table ). This result indicates that a large proportion of the antibodies in sera 1E19 and 12E16 bound to OMP CD epitopes which are exposed on the bacterial surface, whereas serum 10E12 contains predominantly antibodies to non-surface-exposed epitopes. In spite of the adsorption of antibodies in sera 1E19 and 12E16 by whole bacteria as measured by ELISA, the adsorbed serum samples retained reactivity to the fusion protein encompassing amino acids 203 to 260 in an immunoblot assay. These data indicate that ELISA detected human antibodies which were not detected in immunoblot assays with fusion proteins. Therefore, human antibodies to surface-exposed epitopes on OMP CD may recognize conformational epitopes.
The observation in this study that human antibodies preferentially bind to a single region of OMP CD raises the possibility that OMP CD has an immunodominant region. The major OMP (P2) of nontypeable H. influenzae
contains an immunodominant region which is the major target of the antibody response after the challenge of experimental animals with whole bacterial cells (43
). The observation in this study of human antibodies to a selected region of the OMP CD is somewhat surprising because two lines of evidence indicate that OMP CD does not appear to be an immunodominant antigen in M. catarrhalis
: (i) The absence of the formation of new antibodies after infection (Table ) and (ii) the high degree of sequence conservation of OMP CD among strains and the stability of the sequence in isolates which colonize the human respiratory tract for months (20
). One would expect that an immunodominant antigen would be subject to immune selective pressure and therefore demonstrate more sequence variability among strains, as seen with P2 of nontypeable H. influenzae
OMP CD has several characteristics which indicate that the protein may be an effective vaccine antigen. The observation that new antibodies are not made to OMP CD after natural infection indicates that OMP CD does not appear to be an immunodominant antigen when the human host is presented with the whole bacterium. This result does not predict the outcome of immunization with a purified antigen. Active immunization with purified OMP CD induces protective immune responses in mice (29
). Future studies are needed to characterize the potential protective effect of immune responses generated by active immunization of humans to purified OMP CD. Such studies are needed to rigorously assess the potential of OMP CD as a vaccine antigen to prevent infections caused by M. catarrhalis