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Immunochemical characterization of variable epitopes of outer membrane protein P2 of nontypeable Haemophilus
L van Alphen1, P Eijk, L Geelen-van den Broek
1Department of Medical Microbiology, University of Amsterdam, The Netherlands.
Abstract:
Monoclonal antibodies (MAbs) were elicited to the nontypeable Haemophilus influenzae variants d1 to d4, which differ in the outer membrane protein P2 to analyze the immunological properties of the variable parts of this protein. Five MAbs reacted in a whole-cell enzyme-linked immunosorbent assay (ELISA) only with the homologous strain and in some cases with its variants, but not with 69 unrelated nonencapsulated H. influenzae isolates; nine MAbs also reacted with some other H. influenzae isolates, and four MAbs showed broad cross-reactivity. All of the MAbs reacted with purified protein P2 in ELISAs and immunoblotting. The five MAbs which reacted with the homologous strain d3 and not with the variants d1, d2, and d4 promoted complement-dependent bactericidal activity against strain d3. These and four other MAbs reacted with the intact bacteria of strain d3 in immunogold electron microscopy, indicating that they were directed against surface-exposed epitopes of outer membrane protein P2. A mutant of strain d3 was isolated as a survivor from bacterial killing by complement and MAb 30DA5. This mutant had an altered P2 protein on sodium dodecyl sulfate-polyacrylamide gels and had lost its reactivity with all of the five H. influenzae d3-specific MAbs but not with the other MAbs. From these results, we conclude that the variable parts of outer membrane protein P2 of nonencapsulated H. influenzae from the sputum of patients with chronic obstructive pulmonary disease are immunogenic and mostly surface exposed. Only strain-specific MAbs promoted complement-dependent killing of the bacteria, which was abolished in a spontaneous mutant with an altered P2 protein.
Insights
Monoclonal antibodies targeting variable regions of outer membrane protein P2 in nontypeable Haemophilus influenzae are immunogenic and surface-exposed. Strain-specific antibodies promote bacterial killing, a function lost in mutants with altered P2 protein.
Area of Science:
- Immunology
- Microbiology
- Bacterial Pathogenesis
Background:
- Nontypeable Haemophilus influenzae (NTHi) is a significant cause of respiratory infections, particularly in patients with chronic obstructive pulmonary disease (COPD).
- Outer membrane protein P2 (OMP P2) is a key virulence factor and a target for immune responses, but its variable regions' immunogenicity and surface exposure remain incompletely understood.
Purpose of the Study:
- To investigate the immunological properties of the variable regions of NTHi OMP P2.
- To determine if strain-specific antibodies against OMP P2 can mediate complement-dependent bactericidal activity.
Main Methods:
- Generation of monoclonal antibodies (MAbs) against NTHi variants d1-d4 differing in OMP P2.
- Enzyme-linked immunosorbent assays (ELISA) and immunoblotting to characterize MAb reactivity.
- Complement-dependent bactericidal assays.
- Immunogold electron microscopy to assess epitope surface exposure.
- Isolation and characterization of a MAb-resistant mutant.
Main Results:
- Five MAbs reacted specifically with homologous NTHi strains and showed complement-dependent bactericidal activity.
- These strain-specific MAbs targeted surface-exposed epitopes on OMP P2.
- A spontaneous mutant resistant to MAb-mediated killing exhibited an altered OMP P2 and lost reactivity with strain-specific MAbs.
Conclusions:
- Variable regions of NTHi OMP P2 are immunogenic and predominantly surface-exposed.
- Strain-specific MAbs targeting these variable regions are capable of mediating bacterial killing.
- Alterations in OMP P2 can lead to resistance against antibody-mediated immunity.