Mapping of a protective epitope of the CopB outer membrane protein of Moraxella catarrhalis

Abstract

A monoclonal antibody (MAb) (MAb 10F3) directed against the CopB outer membrane protein of Moraxella catarrhalis previously was found to enhance pulmonary clearance of M. catarrhalis in an animal model (M. Helminen, I. Maciver, J. L. Latimer, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 61:2003-2010, 1993). In the present study, this same MAb was shown to exert complement-dependent bactericidal activity against this pathogen in vitro. Nucleotide sequence analysis of the copB gene from two MAb 10F3-reactive and two MAb 10F3-unreactive strains of M. catarrhalis revealed that the deduced amino acid sequences of these four CopB proteins were at least 90% identical. Comparison of the amino acid sequences of these proteins allowed localization of possible MAb 10F3 binding sites to five relatively small regions of the CopB protein from M. catarrhalis O35E. When five synthetic peptides representing these regions were tested for their ability to bind MAb 10F3 in a direct enzyme-linked immunosorbent assay system, an oligopeptide containing 26 amino acids was shown to bind this MAb. The actual binding region for MAb 10F3 was localized further through the use of overlapping decapeptides that spanned this 26-mer. A fusion protein containing the same 26-mer readily bound MAb 10F3 and was used to immunize mice. The resultant antiserum contained antibodies that reacted with the CopB protein of the homologous M. catarrhalis strain in Western blot analysis and bound to the surface of both homologous and heterologous strains of M. catarrhalis.

FIG. 1

Comparison of the deduced amino acid sequences of the CopB proteins from M. catarrhalis O35E, O12E, TTA24, and O46E. Shaded areas indicate positions of amino acid differences. Regions 1 to 5 demarcate the general areas where the amino acid sequences of the CopB proteins from strains O35E and TTA24 differ. The underlined amino acid sequences correspond to the synthetic peptides R1, R2, R3, R4, and R5, respectively.

FIG. 2

Amino acid sequences and reactivities with antibodies of both region 1 from CopB of four different M. catarrhalis strains and synthetic peptides derived from these sequences. The numbers (i.e., 275 to 300) above the region 1 sequences indicate the amino acid positions in the intact CopB protein. The numbers (i.e., 1 to 26) above the R1 synthetic peptide indicate residue positions in the R1 peptide and its derivatives. Shading indicates residues differing from those in O35E. The reactivities of MAb 10F3, GST-26 antiserum, and GST antiserum with the CopB proteins of these strains in Western blot analysis are indicated on the right; these two antisera were diluted 1:100 for use in this assay. The reactivities of the synthetic peptides with these same antibody preparations in the ELISA are indicated on the right opposite these peptides. The asterisk indicates that the ELISA with MAb 10F3 was performed after adsorption of the peptides to the microtiter wells in the presence of glutaraldehyde. The ELISA readings (optical densities at 410 nm) were recorded after 30 min as described in Materials and Methods.

FIG. 3

Fine mapping of the MAb 10F3-reactive epitope by a dot blot assay involving immobilized synthetic decapeptides. Binding of MAb 10F3 to individual peptides was detected by incubating the membrane with radioiodinated goat anti-mouse immunoglobulin. The autoradiograph of the dot blot (peptides 1 to 8) is shown on the left; the corresponding amino acid sequence of each of the overlapping decapeptides with their respective positions in the CopB protein is listed on the right. The numbers written vertically denote the amino acid positions in the intact CopB protein; the R1 region is underlined.

FIG. 4

Hydropathy plot and secondary-structure analysis of region 1 from the CopB protein of M. catarrhalis O35E. The hydropathy plot was generated by the method of Kyte and Doolittle (31), with a window size of seven residues, and secondary-structure predictions were made by the method of Chou and Fasman (9) as found in MacVector sequence analysis software (version 6.0). The numbers on the horizontal scale denote the amino acid positions in the intact CopB protein; the R1 region is underlined.

FIG. 5

Western blot analysis of the reactivities of MAbs and polyclonal serum antibodies with outer membrane proteins of M. catarrhalis strains, the GST-26 fusion protein, and GST alone. Proteins present in outer membrane vesicles from strains O35E (lane 1), O12E (lane 2), TTA24 (lane 3), and O46E (lane 4) and purified GST-26 (lane 5) and GST (lane 6) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with MAb 10F3 (A) or with mouse GST-26 antiserum (dilution 1:1,000) (B). The CopB protein from strain O35E gave rise to a doublet reactive with MAb 10F3 in this experiment. The positions of the molecular mass markers (in kilodaltons) are indicated to the left of each panel.

FIG. 6

Bactericidal activity of MAb 10F3 against M. catarrhalis strains. Suspensions of the MAb 10F3-reactive strains O35E (A) and O12E (B) and the MAb 10F3-unreactive strains TTA24 (C) and O46E (D) were incubated with normal human serum (solid triangles), normal human serum and MAb 10F3 (open squares), heat-inactivated normal human serum and MAb 10F3 (solid squares), normal human serum and GST-26 antiserum (open circles), or normal human serum and GST antiserum (solid circles). Portions of the reaction mixture were removed over time and spread on BHI agar plates to determine the number of viable bacteria. (B) Note that the open circles and open squares are superimposed.

FIG. 7

Detection of M. catarrhalis-reactive antibodies in normal human serum by Western blot analysis. Proteins present in EDTA-extracted outer membrane vesicles of M. catarrhalis O35E (lane 1) and O12E (lane 2) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed in Western blot analysis with 100 μl of the normal human serum used as the source of complement in the bactericidal activity assay. The positions of the molecular mass markers (in kilodaltons) are shown to the left.