Adenylate cyclase toxin (ACT) is a 177 kDa protein produced by Bordetella pertussis, the bacterium causing whooping cough. US CDC estimates 16 million cases and 195,000 deaths worldwide per year, with infants at the greatest risk. The current acellular vaccines consisting of 2~5 protein toxoids are less effective than the previous whole-cell vaccine. This largely contributes to the resurgence of pertussis in the past two decades. Including more protective antigens is one of the strategies to improve the efficacy of acellular vaccines. ACT, as a protective antigen, is not included due to the lack of understanding of its protective epitopes and poor solubility and stability. ACT recognizes αMβ2 integrin present on leukocytes and generates supraphysiological level of cAMP to subvert host immune response. We aimed to identify potent neutralizing antibodies (nAbs) as therapeutic candidates, and use their epitopes to guide the design of vaccine antigen overcoming the solubility and stability issues. We utilized a murine macrophage cell line (J774.A1) as the in vitro neutralization model, and measured the intracellular cAMP level by competition ELISA as the readout. Using phage display, two nAbs, 2B10 and 1H5, were discovered from antibody libraries generated from ACT-immunized mice. The two nAbs bind non-overlapping epitopes in the C-terminal domain of ACT. Their mode-of-actions were both found to be interrupting the interaction between the toxin and αMβ2 integrin. On the other hand, individual domains of ACT were expressed in E.coli and purified. They demonstrated equivalent antigenicity and superior biophysical properties, comparing to full-length ACT. We further showed that the C-terminal domain elicited 2B10 and 1H5-like nAbs in mice. These nAbs, together with those neutralizing other major toxins of B.pertussis, may constitute a potential passive immunization therapy for pertussis infection; and also provide the basis for epitope-based design of vaccine antigens with excellent stability and immunogenicity.

Discovery of novel neutralizing antibodies against Bordetella adenylate cyclase toxin and its implication for vaccine design

249th National Meeting (2015)

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