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

Xianzhe Wang, James A. Stapleton, Justin R. Klesmith, Erik L. Hewlett, Timothy A. Whitehead, Jennifer A. Maynard

    Research output: Research - peer-reviewArticle

    • 2 Citations

    Abstract

    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.

    LanguageEnglish (US)
    Pages1324-1336
    Number of pages13
    JournalBiochemistry
    Volume56
    Issue number9
    DOIs
    StatePublished - Mar 7 2017

    Profile

    Adenylate Cyclase Toxin
    Epitope Mapping
    Neutralizing Antibodies
    Antibodies
    Epitopes
    Bordetella pertussis
    Clone Cells
    Bordetella Virulence Factors
    Acellular Vaccines
    Pertussis Vaccine
    Structural Models
    Libraries
    Immunization
    Flow Cytometry
    Vaccines
    Yeasts
    Binding Sites
    Antigens
    Mutation
    Flow cytometry

    ASJC Scopus subject areas

    • Biochemistry

    Cite this

    Wang, X., Stapleton, J. A., Klesmith, J. R., Hewlett, E. L., Whitehead, T. A., & Maynard, J. A. (2017). Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin. Biochemistry, 56(9), 1324-1336. DOI: 10.1021/acs.biochem.6b01163

    Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin. / Wang, Xianzhe; Stapleton, James A.; Klesmith, Justin R.; Hewlett, Erik L.; Whitehead, Timothy A.; Maynard, Jennifer A.

    In: Biochemistry, Vol. 56, No. 9, 07.03.2017, p. 1324-1336.

    Research output: Research - peer-reviewArticle

    Wang, X, Stapleton, JA, Klesmith, JR, Hewlett, EL, Whitehead, TA & Maynard, JA 2017, 'Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin' Biochemistry, vol 56, no. 9, pp. 1324-1336. DOI: 10.1021/acs.biochem.6b01163
    Wang X, Stapleton JA, Klesmith JR, Hewlett EL, Whitehead TA, Maynard JA. Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin. Biochemistry. 2017 Mar 7;56(9):1324-1336. Available from, DOI: 10.1021/acs.biochem.6b01163
    Wang, Xianzhe ; Stapleton, James A. ; Klesmith, Justin R. ; Hewlett, Erik L. ; Whitehead, Timothy A. ; Maynard, Jennifer A./ Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin. In: Biochemistry. 2017 ; Vol. 56, No. 9. pp. 1324-1336
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