Single Amino Acid Polymorphisms of Pertussis Toxin Subunit S2 (PtxB) Affect Protein Function

Millen, Scott H.; Watanabe, Mineo; Komatsu, Eiji; Yamaguchi, Fuminori; Nagasawa, Yuki; Suzuki, Eri; Monaco, Haleigh; Weiss, Alison A.

doi:10.1371/journal.pone.0137379

Cited by 5 publications

(3 citation statements)

References 43 publications

(65 reference statements)

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“…An in vivo leukocytosis assay was performed as previously described (30,31), with the following modifications. First, 2 g PTx or 2 g PTx plus 20 g of total antibody was preincubated for 2 h at room temperature to allow for binding equilibration.…”

Section: Methodsmentioning

confidence: 99%

“…Systemic leukocytosis is a primary outcome of B. pertussis infection and is predictive of severe disease in infants (7). As a proof-of-concept study to assess the therapeutic potential of the hu1B7/hu11E6 bispecific antibody, we turned to an in vivo leukocytosis assay which measures the ability of an antibody to neutralize the increase in white blood cell (WBC) count induced by the injection of purified pertussis toxin (30,31). This assay has previously been shown to be more predictive of protection during bacterial infection than the CHO cell assay (30).…”

Section: Figmentioning

confidence: 99%

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Synergistic Neutralization of Pertussis Toxin by a Bispecific Antibody In Vitro and In Vivo

Wagner

Wang

Bui

et al. 2016

Clin Vaccine Immunol

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Bispecific antibodies are a rapidly growing class of therapeutic molecules, originally developed for the treatment of cancer but recently explored for the treatment of autoimmune and infectious diseases. Bordetella pertussis is a reemerging pathogen, and several of the key symptoms of infection are caused by the pertussis toxin (PTx). Two humanized antibodies, hu1B7 and hu11E6, bind distinct epitopes on PTx and, when coadministered, mitigate disease severity in murine and baboon models of infection. Here we describe the generation of a bispecific human IgG1 molecule combining the hu1B7 and hu11E6 binding sites via a knobs-in-holes design. The bispecific antibody showed binding activity equivalent to that of the antibody mixture in a competition enzyme-linked immunosorbent assay (ELISA). A CHO cell neutralization assay provided preliminary evidence for synergy between the two antibodies, while a murine model of PTx-induced leukocytosis definitively showed synergistic neutralization. Notably, the bispecific antibody retained the synergy observed for the antibody mixture, supporting the conclusion that synergy is due to simultaneous blockade of both the catalytic and receptor binding activities of pertussis toxin. These data suggest that a hu1B7/hu11E6 bispecific antibody is a viable alternative to an antibody mixture for pertussis treatment. Despite vaccination, pertussis infection continues to cause ϳ195,000 deaths worldwide, primarily of infants (1). Of the estimated 16 million cases of pertussis each year, ϳ95% occur in the developing world. Even in developed countries, the disease incidence has increased dramatically over the last decade, reaching prevaccination levels in some countries (2, 3). This rise has been attributed to shortcomings of the current acellular vaccine (4) as well as pathogen adaptation (5). In both cases, high levels of circulating disease place young infants at risk, as this population is the most susceptible to severe disease. An antibody therapeutic could be used to treat seriously ill infants in the developing world and to prevent disease in high-risk areas.Pertussis toxin (PTx) is one of several virulence factors secreted by the Gram-negative bacterium Bordetella pertussis. PTx is directly responsible for suppression of the innate immune system (6) and for systemic leukocytosis, which is the key clinical indicator of severe disease and appears to be directly responsible for pulmonary hypertension and organ failure (7). In addition, low titers of PTx-neutralizing antibodies correlate with susceptibility to clinical infection (8). We previously developed a binary mixture of two humanized anti-pertussis toxin antibodies which was able to mitigate whooping cough in mouse and baboon models of infection (9). The antibody hu11E6 blocks binding of the toxin to host cells, while the antibody hu1B7 interferes with the catalytic pathway. At a dose selected to demonstrate efficacy but not synergy, the individual antibodies and the mixture were able to completely suppress leukocytosis in a murine ...

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Section: Methodsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

Synergistic Neutralization of Pertussis Toxin by a Bispecific Antibody In Vitro and In Vivo

Wagner

Wang

Bui

et al. 2016

Clin Vaccine Immunol

View full text Add to dashboard Cite

show abstract

“…Proteins showcasing this type of fold have the ability to establish protein-DNA, -RNA or -protein interactions and so are usually involved in a number of cellular processes such as DNA replication and repair, and activation of the DNA-damage checkpoint pathways [85] , [86] , [87] . DsbA substrates found to have this type of fold include secreted toxins such as CtxB from V. cholerae [87] , [88] , [89] , EltB from E. coli [87] , [90] , and the N-terminal domain of S2 subunit from B. pertussis [91] , [92] .…”

Section: Structural Variability Of Dsba Substratesmentioning

confidence: 99%