32] Bordetella pertussis adenylate cyclase toxin: A vehicle to deliver CD8-positive T-cell epitopes into antigen-presenting cells

Abstract

This chapter describes the construction and production of recombinant Bordetella pertussis (B. pertussis) adenylate cyclase (CyaA) toxins that harbor a single CD8+ T-cell epitope, OVA, derived from chicken ovalbumin. This epitope (amino acid sequence, SIINFEKL, corresponding to residues 257 to 264 of ovalbumin) is the main H-2Kb-restricted CD8+ T-cell epitope derived from ovalbumin when this protein is introduced artificially into or expressed by mouse cells. Analysis of the immunological properties of these recombinant toxins is also described in the chapter. Adenylate cyclase is one of the major toxins produced by B. pertussis, the causative agent of whooping cough, and plays an important role in respiratory tract colonization. This toxin is able to enter into eukaryotic target cells where it is activated by endogenous calmodulin (CaM) to produce supraphysiological levels of intracellular cAMP, thus causing the impairment of cellular functions. This 1706 residues long protein, encoded by the cyaA gene, is synthesized as a protoxin that is converted to the active toxin by palmitoylation of Lys 983, a process dependent on the product of an accessory gene, cyaC, adjacent to cyaA. CyaA can invade a large variety of mammalian cell types through a unique mechanism that involves direct delivery of the catalytic domain across the plasma membrane. The translocation of the catalytic domain into the cell cytosol is calcium and temperature dependent.