Characterization of oligopeptides that cross-react with carbohydrate-specific antibodies by real time kinetics, in-solution competition enzyme-linked immunosorbent assay, and immunological analyses.
Phage displaying random cyclic 7-mer, and linear 7-mer and 12-mer peptides at the N terminus of the coat protein, pIII, were panned with the murine monoclonal antibody, 9-2-L379 specific for meningococcal lipo-oligosaccharide. Five cyclic peptides with two sequence motifs, six linear 7-mers, and five linear 12-mers with different sequence motifs were identified. Only phage displaying cyclic peptides were specifically captured by and were antigenic for 9-2-L379. Monoclonal antibody 9-2-L379 exhibited "apparent" binding affinities to the cyclic peptides between 11 and 184 nm, comparable with lipo-oligosaccharide. All cyclic peptides competed with the binding of 9-2-L379 to lipo-oligosaccharide with EC(50) values in the range 10-105 microm, which correlated with their apparent binding affinities. Structural modifications of the cyclic peptides eliminated their ability to bind and compete with monoclonal antibody 9-2-L379. Mice (C3H/HeN) immunized with the cyclic peptide with optimal apparent binding affinity and EC(50) of competition elicited cross-reactive antibodies to meningococcal lipo-oligosaccharide with end point dilution serum antibody titers of 3200. Cyclic peptides were converted to T-cell-dependent immunogens without disrupting these properties by C-terminal biotinylation and complexing with NeutrAvidin. The data indicate that constrained peptides can cross-react with a carbohydrate-specific antibody with greater specificity than linear peptides, and critical to this specificity is their structural conformation.
Brett
Paul J
PJ
Department of Medical Microbiology, Royal Free and University College Medical School, Royal Free Campus, University College London, Rowland Hill Street, London NW3 2PF.
Tiwana
Harmale
H
Feavers
Ian M
IM
Charalambous
Bambos M
BM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003429:Cross Reactions; D004279:DNA, Viral; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000276:immunology*; D051379:Mice; D008809:Mice, Inbred C3H; D018716:Molecular Mimicry; D009345:Neisseria meningitidis / Q000276:immunology; D009842:Oligopeptides / Q000276:immunology*; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D004279:DNA, Viral; D000939:Epitopes; D008070:Lipopolysaccharides; D009842:Oligopeptides; C023023:lipid-linked oligosaccharides