Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin

Biochemistry. 2017 Mar 7;56(9):1324-1336. doi: 10.1021/acs.biochem.6b01163. Epub 2017 Feb 23.


Adenylate cyclase toxin (ACT) is an important Bordetella pertussis virulence factor that is not included in current acellular pertussis vaccines. We previously demonstrated that immunization with the repeat-in-toxin (RTX) domain of ACT elicits neutralizing antibodies in mice and discovered the first two antibodies to neutralize ACT activities by occluding the receptor-binding site. Here, we fully characterize these antibodies and their epitopes. Both antibodies bind ACT with low nanomolar affinity and cross-react with ACT homologues produced by B. parapertussis and B. bronchiseptica. Antibody M1H5 binds B. pertussis RTX751 ∼100-fold tighter than RTX751 from the other two species, while antibody M2B10 has similar affinity for all three variants. To initially map the antibody epitopes, we generated a series of ACT chimeras and truncation variants, which implicated the repeat blocks II-III. To identify individual epitope residues, we displayed randomly mutated RTX751 libraries on yeast and isolated clones with decreased antibody binding by flow cytometry. Next-generation sequencing identified candidate epitope residues on the basis of enrichment of clones with mutations at specific positions. These epitopes form two adjacent surface patches on a predicted structural model of the RTX751 domain, one for each antibody. Notably, the cellular receptor also binds within blocks II-III and shares at least one residue with the M1H5 epitope. The RTX751 model supports the notion that the antibody and receptor epitopes overlap. These data provide insight into mechanisms of ACT neutralization and guidance for engineering more stable RTX variants that may be more appropriate vaccine antigens.

MeSH terms

  • Adenylate Cyclase Toxin / chemistry
  • Adenylate Cyclase Toxin / immunology*
  • Amino Acid Sequence
  • Antibodies, Neutralizing / chemistry
  • Antibodies, Neutralizing / immunology*
  • Bordetella pertussis*
  • Conserved Sequence
  • Epitope Mapping*
  • Models, Molecular
  • Protein Domains


  • Adenylate Cyclase Toxin
  • Antibodies, Neutralizing