The Adenylate Cyclase (CyaA) vector is a recombinant bacterial protein originating from Bordetella pertussis (the causative agent of whooping cough) into which large antigens can be inserted by recombinant DNA technology. Consequently, the resulting vaccine products are simple chimerical recombinant proteins.

The possibility to use the CyaA protein as an antigen delivery system was discovered by Genticel’s partner, the Institut Pasteur in Paris, France. The Institut Pasteur continues to explore the properties and the mechanisms of action of the adenylate cyclase, as well as its potential for vaccine development, in collaboration with several research teams including the Institute of Microbiology, Academy of Sciences in Prague, Czech Republic.

GENTICEL has conducted 2 formal GLP toxicology studies in large mammals with a CyaA based vaccine candidate, in association with 2 different adjuvants. Both studies revealed that the CyaA-based vaccines are well tolerated and have an excellent non-toxicity profile.

CyaA targets human and animal cells that carry the CD11b/CD18 integrin (Guermonprez et al, 2001). This receptor is expressed only on dendritic cells (DC), neutrophiles/granulocytes, macrophages, Natural Killer cells (NK), and subsets of B and T CD8+ lymphocytes (Guermonprez et al., 2002). In vivo, CyaA targets DC very efficiently. These cells are the only antigen presenting cells (APC) capable of stimulating naive T cells. After binding of the CyaA molecules to DC, two series of events occur:

• Presentation in association with MHC I molecules

Very rapidly after interaction with its CD11b receptor, the CyaA delivers its antigenic cargo into the cytosol of antigen-presenting cells. The inserted protein segment is subsequently processed by the proteasome of the DC. Released peptides bind to Transporter associated with Antigen Processing (TAP) for classic Endoplasmic Reticulum (ER) traffic and presentation in association with neo-synthesized MHC class I complex on the surface of the DC. Epitopes presented by MHC class I molecules are then recognised by CD8+ T lymphocytes through their T Cell Receptor (TCR). This interaction induces the differenciation of CD8+ T lymphocyte into specific Cytotoxic T Lymphocyte (CTL) (Guermonprez et al., 1999).

• Presentation in association with MHC II molecules

Very rapidly after interaction with DC, the CyaA antigenic cargo is also delivered to the endosomal antigen presentation pathway. MHC II-restricted presentation of the inserted antigen requires the involvement of endocytic proteases activated by vesicle acidification and de novo synthesis of MHC II molecules. Antigen delivery by CyaA yields very efficient MHC II-restricted presentation and in vitro or in vivo activation of specific CD4+ T lymphocytes, Th1 polarized, as shown by potent Interferon-gamma (IFN-γ) secretion (Loucka et al., 2002; Schlecht et al., 2004).

• Dual T cell activation: CD4+ and CD8+ lymphocytes

The activation of antigen-specific CD4+ T cells is an important requirement for the induction and survival of antigen-specific CD8+ T cells. Thus, the induction of both CD8+ and CD4+ T cell responses is important to induce a sustained cytotoxic immune response, able to recognise and kill the target cells, i.e., the infected or malignant cells presenting the specific targeted epitopes.
Targeting of CD11b+ dendritic cells by CyaA leads to simultaneous MHC I and MHC II-restricted presentation of the antigenic cargo and to the induction of specific CD4+ and CD8+ T cell responses. Thus, the CyaA vector bears key attributes for use as a potent antigen delivery vehicule in vaccine applications.
The mode of action of the CyaA system is illustrated in the figure below: