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Fine mapping of outer membrane protein P2 antigenic sites which vary during persistent infection by Haemophilus
1Department of Medical Microbiology, University of Amsterdam, The Netherlands.
Abstract:
Antigenic drift of the major outer membrane protein (MOMP) P2 of nonencapsulated Haemophilus influenzae as observed during persistent infections in patients with chronic bronchitis was mimicked in a rabbit model in which H. influenzae persisted in subcutaneous cages. The antigenic drift resulted from amino acid substitutions in potentially surface-exposed loops of MOMP P2. Since in a rabbit model the appearance of antigenic variants was associated with the presence of strain-specific bactericidal antibodies (L. Vogel, B. Duim, F. Geluk, P. Eijk, H. Jansen, J. Dankert, and L. van Alphen, Infect. Immun. 64:980-986, 1996), we determined the epitope specificities of these bactericidal antibodies. The eight loops of MOMP P2 of H. influenzae d1 were separately expressed as fusion proteins with glutathione S-transferase. Sera of rabbits persistently infected with H. influenzae reacted with the loop 5 and loop 6 fusion proteins in immunoblotting and enzyme-linked immunosorbent assay. For fine mapping of the epitopes with pepscan analysis, overlapping synthetic peptides consisting of 12 amino acids were made. Rabbit sera contained antibodies reacting with peptides derived from loop 5 and peptides containing amino acids of the side of loop 6. In addition, MOMP P2 variant-specific reactions with the amino acids located at the tip of loop 6 were detected. The rabbit sera showed variant-specific complement-dependent bactericidal activities, which were eliminated by affinity chromatography with fusion proteins of loop 6 but not of loop 5. We conclude that, during persistence of H. influenzae in rabbits, variant-specific bactericidal antibodies are elicited to the variable tip of MOMP P2 loop 6.
Insights
Antigenic drift in Haemophilus influenzae involves changes in the major outer membrane protein (MOMP) P2. This study found that variant-specific bactericidal antibodies target the variable tip of MOMP P2 loop 6 during persistent infections.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- Nonencapsulated Haemophilus influenzae causes persistent infections, such as chronic bronchitis.
- Antigenic drift in the major outer membrane protein (MOMP) P2 contributes to immune evasion during these infections.
- Previous studies showed antigenic variants emerge during persistent infections and are associated with strain-specific bactericidal antibodies.
Purpose of the Study:
- To mimic antigenic drift of Haemophilus influenzae MOMP P2 in a rabbit model.
- To identify the specific epitopes targeted by bactericidal antibodies during persistent infections.
- To understand the role of these antibodies in the emergence of antigenic variants.
Main Methods:
- A rabbit model was used to mimic persistent Haemophilus influenzae infections and observed antigenic drift.
- Expressed eight loops of MOMP P2 as fusion proteins for immunological assays.
- Utilized immunoblotting, enzyme-linked immunosorbent assay, and pepscan analysis to map antibody specificities.
- Performed affinity chromatography to confirm the target of bactericidal antibodies.
Main Results:
- Antigenic drift in MOMP P2 resulted from amino acid substitutions in surface-exposed loops.
- Rabbit sera from infected animals reacted with fusion proteins of loop 5 and loop 6.
- Antibodies were found to react with peptides from loop 5 and loop 6, with variant-specific reactions at the tip of loop 6.
- Bactericidal activity was specifically eliminated by targeting loop 6.
Conclusions:
- Variant-specific bactericidal antibodies are elicited against the variable tip of MOMP P2 loop 6 during persistent Haemophilus influenzae infections in rabbits.
- This immune response is associated with the emergence of antigenic variants.
- The findings contribute to understanding immune evasion strategies of nonencapsulated H. influenzae.