Immunogenicity of overlapping synthetic peptides covering the entire sequence of Haemophilus influenzae type b outer membrane protein P2
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1Connaught Centre for Biotechnology Research, Willowdale, Ontario, Canada.
Insights
Researchers identified key protective regions on Haemophilus influenzae type b outer membrane protein P2. These findings are crucial for developing a universal vaccine against this common childhood bacterial meningitis pathogen.
Area of Science:
- Microbiology
- Immunology
- Vaccine Development
Background:
- Haemophilus influenzae type b (Hib) is a primary cause of bacterial meningitis in young children.
- Antibodies targeting the P2 outer membrane protein demonstrate protective effects against Hib bacteremia in infant rats.
Purpose of the Study:
- To pinpoint conserved, surface-exposed, and protective B-cell epitopes on the P2 protein.
- To lay the groundwork for a universal Haemophilus influenzae vaccine.
Main Methods:
- Synthesis of 17 overlapping peptides spanning the P2 protein sequence.
- Enzyme-linked immunosorbent assays (ELISA) and immunoblotting to test antibody reactivity.
- Utilizing antisera from various animal models and human convalescent samples.
Main Results:
- Three dominant linear B-cell epitopes were mapped to specific residue ranges (53-81, 241-265, 314-341) of mature P2.
- Human convalescent sera showed strong reactivity to these identified epitopes.
- Peptide immunization elicited strong antibody responses recognizing P2 from both Hib and nontypeable strains.
Conclusions:
- The study successfully identified immunodominant and conserved B-cell epitopes on the P2 protein.
- These identified epitopes are critical targets for the future development of a universal Haemophilus influenzae vaccine.
Abstract:
Haemophilus influenzae type b is a major cause of bacterial meningitis in young children. Antibodies against the outer membrane protein P2 are protective in the infant rat model of bacteremia. To identify conserved, surface-exposed, and protective epitopes of P2, 17 overlapping peptides covering the entire sequence of the protein were synthesized. Antisera from mice, guinea pigs, and rabbits raised against chromatographically purified P2 were tested for their reactivities to the peptides by enzyme-linked immunosorbent assays (ELISA). Three major linear immunodominant B-cell epitopes were mapped to residues 53 to 81, 241 to 265, and 314 to 341 of mature P2. Human convalescent-phase antisera also reacted strongly with these three epitopes. Rabbit antisera against all peptide-keyhole limpet hemocyanin conjugates except two peptides containing residues 8 to 19 and 302 to 319 recognized the corresponding peptides in ELISA and reacted with P2 on immunoblots. Immunization with all unconjugated peptides, except the 19 N-terminal residues, induced very strong peptide-specific antibody responses, and these antisera reacted with P2 on immunoblots. Rabbit antisera raised against peptides corresponding to residues 1 to 14, 125 to 150, 193 to 219, and 241 to 319 also recognized P2 purified from H. influenzae nontypeable isolates. Identification of these immunodominant B-cell epitopes and conserved regions is a first step toward the rational design of a universal H. influenzae vaccine.