Bioengineering of Bordetella pertussis Adenylate Cyclase Toxin for Antigen-Delivery and Immunotherapy

Abstract

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells where, upon activation by endogenous calmodulin, it synthesizes massive amounts of cAMP that alters cellular physiology. The CyaA toxin is a 1706 residues-long bifunctional protein: the catalytic domain is located in the 400 amino-proximal residues, whereas the carboxy-terminal 1306 residues are implicated in toxin binding to the cellular receptor, the α_Mβ₂ (CD11b/CD18) integrin, and subsequently in the translocation of the catalytic domain across the cytoplasmic membrane of the target cells. Indeed, this protein is endowed with the unique capability of delivering its N-terminal catalytic domain directly across the plasma membrane of eukaryotic target cells. These properties have been exploited to engineer the CyaA toxin as a potent non-replicating vector able to deliver antigens into antigen presenting cells and elicit specific cell-mediated immune responses. Antigens of interest can be inserted into the CyaA protein to yield recombinant molecules that are targeted in vivo to dendritic cells, where the antigens are processed and presented by the major class I and class II histocompatibility complexes (MHC-I and II). CyaA turned out to be a remarkably effective and versatile vaccine vector capable of inducing all the components of the immune response (T-CD4, T-CD8, and antibody). In this chapter, we summarize the basic knowledge on the adenylate cyclase toxin and then describe the application of CyaA in vaccinology, including some recent results of clinical trials of immunotherapy using a recombinant CyaA vaccine.

Keywords: Bordetella pertussis; CD11b/CD18 integrin; RTX repeats-in-toxin; adenylate cyclase toxin; antigen delivery; cancer immunotherapy; cell-mediated immunity; cyclic AMP; dendritic cells; toxin translocation.

Figure 1

Structural organization of CyaA and schematic model of toxin entry into target cells. (A) The different domains of the protein are indicated. See text for details. (B) Model for CyaA intoxication of target cells: (1) binding of CyaA to CD11b/CD18 receptor at the target cell surface via its RTX domain; (2) insertion of hydrophobic segments of CyaA into the plasma membrane of target cells; (3) destabilization of the membrane by the translocation domain T; (4) translocation of the AC catalytic domain across the plasma membrane of target cells. In the cytosol CyaA interacts with calmodulin (CaM) that stimulates its catalytic activity to produce supraphysiological amounts of cAMP. This intoxication model and its different steps are still largely speculative, although it is reasonable to assume that binding of CyaA to its receptor (step 1) precedes membrane insertion of the hydrophobic domain (step 2). As discussed in the text, interaction of CyaA with CD11b/CD18 may primarily serve to recruit the toxin to the vicinity of the cell surface and thus facilitates the subsequent insertion of the hydrophobic helices into the membrane. In cells lacking the receptor, CyaA will bind and insert into the plasma membrane with a much lower efficiency. Once the hydrophobic domain is imbedded in the membrane, CyaA is irreversibly bound to the cells (irrespectively of the presence or not of CD11b/CD18) [16,24,38]. After insertion of the hydrophobic domain, the translocation domain (step 3) comes in close proximity to the membrane bilayer and triggers a local and transient destabilization of the membrane through its membrane active properties. The transient perturbation of the membrane bilayer integrity may favor the passage of the AC domain across the lipid bilayer (step 4) as well as a calcium influx that is associated with the entry of the AC domain [29]. Insertion of the hydrophobic domain (step 2) could also transiently destabilize the membrane to allow a K⁺ efflux and/or calcium entry [28,29].

Figure 1

Structural organization of CyaA and schematic model of toxin entry into target cells. (A) The different domains of the protein are indicated. See text for details. (B) Model for CyaA intoxication of target cells: (1) binding of CyaA to CD11b/CD18 receptor at the target cell surface via its RTX domain; (2) insertion of hydrophobic segments of CyaA into the plasma membrane of target cells; (3) destabilization of the membrane by the translocation domain T; (4) translocation of the AC catalytic domain across the plasma membrane of target cells. In the cytosol CyaA interacts with calmodulin (CaM) that stimulates its catalytic activity to produce supraphysiological amounts of cAMP. This intoxication model and its different steps are still largely speculative, although it is reasonable to assume that binding of CyaA to its receptor (step 1) precedes membrane insertion of the hydrophobic domain (step 2). As discussed in the text, interaction of CyaA with CD11b/CD18 may primarily serve to recruit the toxin to the vicinity of the cell surface and thus facilitates the subsequent insertion of the hydrophobic helices into the membrane. In cells lacking the receptor, CyaA will bind and insert into the plasma membrane with a much lower efficiency. Once the hydrophobic domain is imbedded in the membrane, CyaA is irreversibly bound to the cells (irrespectively of the presence or not of CD11b/CD18) [16,24,38]. After insertion of the hydrophobic domain, the translocation domain (step 3) comes in close proximity to the membrane bilayer and triggers a local and transient destabilization of the membrane through its membrane active properties. The transient perturbation of the membrane bilayer integrity may favor the passage of the AC domain across the lipid bilayer (step 4) as well as a calcium influx that is associated with the entry of the AC domain [29]. Insertion of the hydrophobic domain (step 2) could also transiently destabilize the membrane to allow a K⁺ efflux and/or calcium entry [28,29].

Figure 2

Recombinant CyaA proteins can deliver antigens into antigen presenting cells. Recombinant CyaA carrying an antigen genetically inserted into the catalytic domain, dCyaA-Ag, binds to dendritic cells (DC) as a result of its selective interaction with the CD11b/CD18 integrin receptor (ϕ). After translocation across the plasma membrane of the DC (left); the antigen is degraded by the proteasome into peptides that can reach the classical cytosolic MHC class I presentation pathway, to elicit specific CD8⁺ CTL responses. Alternatively, the recombinant CyaA can be endocytosed (right). After proteolysis in endosomes/lysosomes, the released peptides, including antigen-derived CD4⁺ epitopes, can enter the MHC class II presentation pathway, to elicit specific Th1 type CD4⁺ helper T-cell responses.