New Epitopes and Comparative Analysis of Children and Murine Immune Responses to Pertussis Toxin Subunits (S1–S5) Induced by Whole-Cell Pertussis Vaccination

Pertussis toxin (Ptx) is a key virulence factor and protective antigen of Bordetella pertussis. Understanding its antigenic landscape is critical for improving vaccine formulations. This study compared the antigenic mapping of continuous epitopes recognized by children and murine antibodies against Ptx subunits S1–S5. Two libraries of nine overlapping 14-mer peptides, each encompassing the full-length Ptx sequence, were synthesized to identify linear antibody-binding regions. Antibodies from children and mice immunized with whole-cell pertussis vaccine recognized several common, though not identical, epitopes. Some regions were species-specific, while shared epitopes exhibited minor positional shifts, indicating subtle differences in recognition. Eighteen major epitopes were recognized by vaccinated mice and twenty-six by the serum of vaccinated children. Eleven present shared sequences [S1 (Ep3-5,7,9,10); S3 (20,21,25,26); S5 (Ep 30)], and eight were recognized uniquely by the serum of children’s [S1 (Ep1,6,8); S3 (Ep22-Ep24) and S4 (Ep27,29-30)]. Four unique and distinct epitopes were identified in the S2 subunit for each species [mice Ep11,14,16,17 and children Ep12,13,15,18, and only one in the S4 subunit [mice Ep 28]. Structural analysis revealed non-uniform antibody recognition of pertussis toxin, with dominant targeting of the S3 subunit and conserved epitope hotspots, alongside a distinct subset of antibodies that engage the catalytic S1 subunit, providing structural evidence for multiple neutralization mechanisms. These findings provide a mechanistic basis for differences in toxin neutralization and highlight limitations in extrapolating murine epitope data to human immunity. The conserved antigenicity of the subdomains provides valuable insights into Ptx immunogenicity and represents promising candidates for inclusion in next-generation synthetic pertussis vaccine design.