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Updated: Oct 16, 2026
Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope
Published on: March 24, 2017
Identification of murine B-cell and T-cell epitopes of Escherichia coli outer membrane protein F with synthetic
K M Williams1, E C Bigley, R B Raybourne
1Immunobiology Branch, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Laurel, Maryland 20708, USA. k2w@cfsan.fda.gov
Abstract:
The major pore-forming outer membrane proteins (Omps) of gram-negative bacteria demonstrate numerous immunomodulating properties and are involved in the virulence of pathogenic strains. Because Escherichia coli OmpF is the best-characterized porin in terms of structural and functional characteristics, in vitro B-cell and T-cell responses to this porin in six different strains of mice were analyzed. Mice were immunized with purified OmpF trimers or overlapping synthetic polypeptides (20-mers) spanning the entire 340-amino-acid sequence of the OmpF monomer. T-cell proliferative responses and immunoglobulin G antibody responses to native OmpF and the peptide analogues were determined. For each strain, patterns of T-cell proliferation were similar regardless of whether native OmpF or synthetic peptides were inoculated, although all strains recognized one or more cryptic determinants. Mice exhibited several haplotype-specific responses, but genetically permissive epitopes were also identified. Four peptides (75-94, 265-284, 295-314, and 305-324) elicited strong T-cell proliferative responses from all strains of mice when mice were presensitized with native OmpF or a homologous peptide. In general, 10 or fewer peptides were recognized by sera from mice immunized with native OmpF or synthetic peptides, and most sera from peptide-immunized mice reacted poorly with the native protein. Four peptides spanning amino acids 45 to 64, 95 to 114, 115 to 134, and 275 to 294 were recognized by sera from all strains immunized with native OmpF but not by sera from peptide-immunized mice. Peptides 245-264 and 305-324 were universally recognized by sera from peptide-immunized mice, but these sera reacted weakly or were negative when tested against the native protein. Based on the pattern of cytokine secretion by proliferating T cells, immunization with native OmpF polarizes T helper cells toward development of a TH1 response. T-cell and B-cell responses have been investigated based on the assumption that differences in epitope specificity could influence protective or pathologic host reactions. Because of the high level of structural homology of OmpF to porins isolated from other enteric pathogens, the identification of T- and B-cell-stimulatory determinants of E. coli OmpF may have broader application.
Insights
Outer membrane proteins (Omps) from E. coli trigger immune responses. Researchers identified specific OmpF peptides that stimulate T-cell proliferation and antibody production across different mouse strains, offering insights into bacterial pathogenesis.
Area of Science:
- Immunology
- Microbiology
- Structural Biology
Background:
- Gram-negative bacteria outer membrane proteins (Omps) possess immunomodulating properties and contribute to virulence.
- Escherichia coli OmpF is a well-characterized porin, making it a suitable model for studying immune responses.
Purpose of the Study:
- To analyze in vitro B-cell and T-cell responses to E. coli OmpF in six different mouse strains.
- To identify specific epitopes within OmpF that elicit T-cell proliferation and antibody production.
Main Methods:
- Mice were immunized with purified OmpF trimers or synthetic peptides spanning the OmpF sequence.
- T-cell proliferative responses and immunoglobulin G antibody responses were measured.
- Cytokine secretion patterns were analyzed to determine T helper cell polarization.
Main Results:
- All mouse strains recognized cryptic determinants within OmpF, with some haplotype-specific and genetically permissive responses.
- Four specific peptides consistently elicited strong T-cell proliferation across all strains.
- Distinct sets of peptides were recognized by antibodies from native OmpF-immunized versus peptide-immunized mice, with some peptides recognized by all mice but poorly by antibodies.
Conclusions:
- Immunization with native OmpF induces a T helper 1 (TH1) response.
- The identified T- and B-cell stimulatory determinants of E. coli OmpF have potential applications for understanding and potentially targeting other enteric pathogens due to structural homology.
