Mimicking of discontinuous epitopes by phage-displayed peptides, II. Selection of clones recognized by a protective monoclonal antibody against the Bordetella pertussis toxin from phage peptide libraries.
We have screened phage peptide libraries to establish if clones binding to a monoclonal antibody (mAb), specific for a discontinuous epitope, could be isolated and if the selected phage particles would be able to elicit an in vivo immuno-response against the original antigen. Two phage peptide libraries, consisting of 9 random amino acids inserted in the major coat protein (pVIII), were independently screened with a mAb which is capable of neutralizing the Bordetella pertussis toxin (PTX) in in vitro and in vivo assays. The epitope of PTX recognized by this and other protective mAb has been shown to be discontinuous. Six different positive phage clones were selected; their binding to the mAb could be competed for by PTX, showing that these clones bind to the antigen-binding site of the mAb. Three of the clones were used (alone or as a mixture) to immunize BALB/c mice. The sera showed a good immunoresponse both against the phage bearing the epitopes and against synthetic multiple-antigen peptides of the same sequence. The immune sera, however, showed no detectable signal against PTX and no capacity to neutralize the CHO-cell-clustering activity of the toxin. The results show that the selected recombinant phage are capable of mimicking the discontinuous epitope as far as binding to the corresponding mAb, but they are unable to elicit a detectable production of antibodies specific for the original antigen.
Felici
F
F
Istituto di Ricerche di Biologia Molecolare P. Angeletti, Pomezia, Rome, Italy.
Luzzago
A
A
Folgori
A
A
Cortese
R
R
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001886:Bordetella pertussis / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D003090:Coliphages / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin