IDENTIFICATION OF B-CELL EPITOPES ON THE S4-SUBUNIT OF PERTUSSIS TOXIN

The main purpose of the present study was to identify B-cell epitopes on the S4 subunit of pertussis toxin (PT) by the synthetic peptide app roach. Two strategies were followed: (i) screening of two series of ov erlapping peptides (12- and 25-residue peptides) covering the entire S 4 sequence by a panel of murine monoclonal anti-PT antibodies and vari ous polyclonal anti-PT antisera in an enzyme-linked immunosorbent assa y (ELISA), and (ii) analysis of the S4 amino acid sequence by a predic tive algorithm followed by synthesis and immunization of mice with the predicted peptides coupled to diphtheria toxoid. The anti-peptide con jugate antisera were tested in an ELISA for cross-reactivity with nati ve PT, B oligomer, and S4. Screening of the free peptides in an ELISA by the PT antisera indicated the presence of six B-cell epitope-contai ning domains covered by residues 18 to 32, 33 to 46, 39 to 52, 51 to 6 5, 71 to 84, and 91 to 106. None of the peptides, however, were recogn ized by the monoclonal anti-PT antibodies in an ELISA. Immunization wi th six computer-predicted peptides (B1 to B6) and three potential T-ce ll epitopes (T1 to T3) gave rise to very high antibody responses towar ds the homologous conjugates. With the exception of the anti-T1/diphth eria toxoid antisera, all anti-peptide conjugate antisera cross-reacte d with PT in an ELISA at different levels. None of these anti-peptide conjugate antisera, however, showed any PT-neutralizing effect as meas ured by the Chinese hamster ovary cell assay and the leukocytosis-prom oting activity test. The results of the present study suggest that dis continuous epitopes are predominant in the S4 subunit of native PT.