Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin.
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 RTX751from 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 RTX751libraries 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 RTX751domain, 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 RTX751model 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.
Department of Chemical Engineering, University of Texas at Austin , Austin, Texas 78712, United States.
Xianzhe
X
Wang
Department of Chemical Engineering and Materials Science, Michigan State University , East Lansing, Michigan 48824, United States.
James A
JA
Stapleton
Department of Biochemistry and Molecular Biology, Michigan State University , East Lansing, Michigan 48824, United States.
Justin R
0000-0003-2908-9355
JR
Klesmith
Department of Medicine, University of Virginia , Charlottesville, Virginia 22906, United States.
Erik L
EL
Hewlett
Department of Chemical Engineering and Materials Science, Michigan State University , East Lansing, Michigan 48824, United States.|Department of Biosystems and Agricultural Engineering, Michigan State University , East Lansing, Michigan 48824, United States.
Timothy A
TA
Whitehead
Department of Chemical Engineering, University of Texas at Austin , Austin, Texas 78712, United States.
Jennifer A
0000-0002-0363-8486
JA
Maynard
D037361:Adenylate Cyclase Toxin / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D057134:Antibodies, Neutralizing / Q000737:chemistry / Q000276:immunology*; D001886:Bordetella pertussis; D017124:Conserved Sequence; D018604:Epitope Mapping; D008958:Models, Molecular; D000072417:Protein Domains
D037361:Adenylate Cyclase Toxin; D057134:Antibodies, Neutralizing