Use of an isogenic mutant constructed in Moraxella catarrhalis To identify a protective epitope of outer membrane protein B1 defined by monoclonal antibody 11C6

Abstract

Moraxella catarrhalis-induced otitis media continues to be a significant cause of infection in young children, prompting increased efforts at identifying effective vaccine antigens. We have previously demonstrated that M. catarrhalis expresses specific outer membrane proteins (OMPs) in response to iron limitation and that this organism can utilize transferrin and lactoferrin for in vitro growth. One of these proteins, which binds human transferrin, is OMP B1. As the human host presents a naturally iron-limited environment, proteins, like OMP B1, which are expressed in response to this nutritional stress are potential vaccine antigens. In this study, we have developed monoclonal antibody (MAb) 11C6, which reacts to a surface-exposed epitope of OMP B1 expressed by M. catarrhalis 7169. This antibody was used to clone ompB1, and sequence analysis suggested that OMP B1 is the M. catarrhalis homologue to the transferrin binding protein B described for pathogenic Neisseriaceae, Haemophilus influenzae, Actinobacillus pleuropneumoniae, and M. catarrhalis. Expression of recombinant OMP B1 on the surface of Escherichia coli confers transferrin binding activity, confirming that this protein is likely involved in iron acquisition. In addition, ompB1 was used to construct an isogenic mutant in M. catarrhalis 7169. This mutant, termed 7169b12, was used as the control in bactericidal assays designed to determine if OMP B1 elicits protective antibodies. In the presence of MAb 11C6 and human complement, wild-type 7169 demonstrated a 99% decline in viability, whereas the ompB1 isogenic mutant was resistant to this bactericidal activity. Further analysis with MAb 11C6 revealed the presence of this OMP B1 epitope on 31% of the clinical isolates tested. These data suggest that OMP B1 is a potential vaccine antigen against M. catarrhalis infections.

FIG. 1

An SDS-PAGE gel (7.5% polycrylamide) (panel 1) showing the OMP profile from M. catarrhalis 7169 grown under iron-replete (lane A) and iron-deficient (lane B) conditions. Panel 2 is the corresponding Western blot probed with MAb 11C6, which detects the presence of OMP B1, with an approximate molecular mass of 82 kDa (asterisk), in outer membranes of cells grown only under iron limitation (lane B). Molecular mass standards (left) are expressed in kilodaltons.

FIG. 2

A Western blot, of an SDS-PAGE gel (7.5% polyacrylamide), probed with MAb 11C6. Total membrane proteins from iron-stressed M. catarrhalis 7169 (lane A), from wild-type E. coli BM25.8 (lane B), and from transformed E. coli BM25.8 expressing rOMP B1 (lane C) were analyzed. Lane D contains the binding fraction from a holotransferrin affinity matrix that was incubated with total membranes of BM25.8 expressing rOMP B1, demonstrating that the recombinant protein retains transferrin binding activity. Molecular mass standards (left) are in kilodaltons.

FIG. 3

A composite of colony lift assays of E. coli expressing OMP B1 (A and C) and wild-type E. coli (B and D). The blots shown in panels A and B were probed with MAb 11C6 and demonstrate that rOMP B1 is expressed on the cell surface (A). The blots shown in panels C and D were probed with biotinylated human holotransferrin and demonstrate that rOMP B1 retains transferrin binding activity.

FIG. 4

An SDS–7% PAGE gel (panel 1) showing iron-stressed OMPs of wild-type M. catarrhalis 7169 (lane A) and those isolated from the isogenic mutant 7169b12. Panel 2 shows a Western blot probed with MAb 11C6, corresponding to the gel shown in panel 1 and confirming the loss of OMP B1 expression (asterisk) in the isogenic mutant 7169b12 (lane B). Molecular mass standards (left) are expressed in kilodaltons.

FIG. 5

A bactericidal assay comparing the sensitivities of wild-type M. catarrhalis 7169 and the ompB1 isogenic mutant 7169b12 to MAb 11C6. In the presence of MAb 11C6 and 15% NHS, the wild type (•) exhibited a 99% decline in viability. In contrast, the viability of 7169b12 (■) was unaffected under the same conditions. As controls, the mutant (×) and the wild type (⧫) were incubated with 15% NHS and the mutant (▵) and the wild type (□) were incubated with 45% heated-inactivated NHS plus MAb 11C6.