The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies

Wang, Xianzhe; Maynard, Jennifer A.

doi:10.1074/jbc.m114.585281

Cited by 30 publications

(53 citation statements)

References 79 publications

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“…14, 15, 21, 28 Both antibodies bind unacylated RTX 751 while the M2B10 antibody also binds the truncated, unacylated RTX 985 variant comprising residues 985–1706 with no apparent loss in affinity. 21 …”

Section: Resultsmentioning

confidence: 99%

“…21 To gain a more quantitative measurement of the antibody binding affinities to guide our epitope mapping experiments, we turned to KinExA analysis, a method that measures equilibrium binding affinities of unmodified molecules in the solution phase. Purified RTX 751 was immobilized on polymethylmethacrylate beads, after which the antibodies alone or antibodies incubated with varying concentrations of soluble RTX 751 were flowed over the beads.…”

Section: Resultsmentioning

confidence: 99%

“…20, 21 Notably, intact ACT is poorly soluble and prone to aggregation as well as proteolytic degradation, while the cyclase domain bears homology to human adenylate cyclase. Antibodies induced by active vaccination with the Bordetella enzyme could cross-react with the intracellular mammalian homolog, 22 thereby inducing undesirable auto-reactive responses.…”

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confidence: 99%

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

et al. 2017

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Adenylate cyclase toxin (ACT) is an important B. 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 characterized these antibodies and their epitopes. Both antibodies bind ACT with low nanomolar affinity and cross-react with ACT homologs 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 based on enrichment of clones with mutations at specific positions. These epitopes form two adjacent surface patches on a 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. A predicted structural model of RTX751 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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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“…The resulting designed variable region genes were synthesized with human IgG1/kappa constant domains by DNA 2.0 in pJ602 or pJ607 vectors. For chimeric constructs, murine variable regions were similarly cloned with human constant regions (39). …”

Section: Methodsmentioning

confidence: 99%

A cocktail of humanized anti–pertussis toxin antibodies limits disease in murine and baboon models of whooping cough

et al. 2015

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In spite of wide-spread vaccination, pertussis rates are rising in industrialized countries and remain high world-wide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. Here, we humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human IgG1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the B. pertussis-induced rise in white blood cell count and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated but not control animals experienced a blunted rise in white blood cell count and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care.

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“…Not all anti-ACT monoclonal antibodies block ACT function, as in the case of N042 (Table 2) (36). The positive anti-ACT ELISA result for N042 reflected binding to the RTX component of ACT, the immunodominant component of the toxin in mice (37), which is common to other bacterial toxins. Thus, the finding of neutralization of ACT is more specific than just binding, as is detected by ELISA.…”

Section: Discussionmentioning

confidence: 99%

Use of a Toxin Neutralization Assay To Characterize the Serologic Response to Adenylate Cyclase Toxin after Infection with Bordetella pertussis

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Gray

Warfel

et al. 2017

Clin Vaccine Immunol

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Adenylate cyclase toxin (ACT) is an essential virulence factor of Bordetella pertussis, and antibodies to ACT protect against B. pertussis infection in mice. The toxin is therefore a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG enzyme-linked immunosorbent assay (ELISA) results neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by PCR, anti-ACT IgG ELISA was positive in 72%, and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA, and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination.

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The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies

Cited by 30 publications

References 79 publications

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

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

A cocktail of humanized anti–pertussis toxin antibodies limits disease in murine and baboon models of whooping cough

Use of a Toxin Neutralization Assay To Characterize the Serologic Response to Adenylate Cyclase Toxin after Infection with Bordetella pertussis

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