Phage Antibodies Obtained by Competitive Selection on Complement-Resistant Moraxella (Branhamella) catarrhalis Recognize the High-Molecular-Weight Outer Membrane Protein

We used competitive panning to select a panel of 10 different human antibodies from a large semisynthetic phage display library that distinguish between serum complement-resistant and complement-sensitive strains of the gram-negative diplococcus Moraxella (Branhamella) catarrhalis. Western blotting analyses and inhibition enzyme-linked immunosorbent assays showed that all phage antibodies were directed against the same or closely spaced epitopes on the target protein, which is the high-molecular-weight outer membrane protein (HMW-OMP) of M. catarrhalis. HMW-OMP was found in multiple isolates of complement-resistant but not complement-sensitive M. catarrhalis strains. Nucleotide sequence analysis demonstrated that the immunoglobulin heavy- and light-chain variable-region genes encoding the 10 phage antibodies were remarkably similar, with a strong preference for basic amino acid residues in the heavy-chain CDR3 regions. This is the first report showing that competitive panning is a successful procedure to obtain phage antibodies against differentially expressed structures on phenotypically dissimilar strains of prokaryotic cells.

The display of single-chain Fv (scFv) or Fab antibody fragments on the surface of filamentous phage particles and the selection of recombinant phages by binding to a target antigen provide a novel means of isolating antibodies with predetermined specificity (for reviews, see references 5 and 39). Libraries may be assembled from the variable (V) regions expressed by B lymphocytes from either an individual with a particular immune response or a nonimmunized individual in an attempt to recruit the diversity generated by the natural immune system. An alternative approach to create diverse libraries exploits the use of large collections of cloned V genes to which randomized CDR3 regions are fused in vitro by PCR. We recently constructed a large semisynthetic phage library of human scFv fragments with partially randomized heavy-chain CDR3 regions, designed to encode a high frequency of functional antibody molecules (8).Selection of phage antibodies (PhAbs) of desired specificities is conventionally performed by panning of libraries on solid-phase-coated antigens and eluting bound phages with high- or low-pH buffers. Alternative strategies have also been successfully used. For example, PhAbs have been obtained by direct selection on structures expressed on the membranes of eukaryotic and bacterial cells expressing the target antigen as a recombinant fusion protein (3, 9, 25, 30). Furthermore, phage libraries may be preabsorbed to remove unwanted specificities, or alternatively, selections may be performed in the presence of a homologous competitor antigen to enrich for phages directed to nonhomologous regions of the target antigen (6, 8, 25). Finally, phages bound to the target structure may be eluted by competition with ligand or a conventional monoclonal antibody (MAb) (5, 26, 39).In this study, we used our library in a competitive panning procedure to demonstrate the feasibility of obtaining antibodies specific for an unknown target structure predicted to be differentially expressed on two strains of the same bacterial species. Selections were performed on Moraxella (Branhamella) catarrhalis, a gram-negative bacterium that may cause upper respiratory tract disease in children and lower respiratory tract disease in elderly people and patients with chronic obstructive pulmonary disease (2, 7, 12, 28). Resistance against complement-mediated lysis is considered an important virulence factor of this bacterium (15, 17). Complement-resistant bacteria were coated onto a solid support, and phage selections were performed in the presence of a complement-sensitive strain as a particulate competitor antigen. After three rounds of selection, a collection of 10 different monoclonal PhAbs (MPhAbs) with binding specificity for complement-resistant but not complement-sensitive M. catarrhalis isolates was obtained. The molecule recognized was identified as the high-molecular-weight outer membrane protein (HMW-OMP) of M. catarrhalis (22).