Delivery of CD8(+) T-cell epitopes into major histocompatibility complex class I antigen presentation pathway by Bordetella pertussis adenylate cyclase: Delineation of cell invasive structures and permissive insertion sites

Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (ACT-Hly) can p enetrate a variety of eukaryotic cells. Recombinant AC toxoids have therefo re been recently used for delivery of CD8(+) T-cell epitopes into antigen-p resenting cells in vivo and for induction of protective antiviral, as well as therapeutic antitumor cytotoxic T-cell responses. We have explored the c arrier potential of the ACT molecule by insertional mutagenesis scanning fo r new permissive sites, at which integration of two- to nine-residue-long p eptides does not interfere with membrane interaction and translocation of A CT. A model CD8(+) T-cell epitope of ovalbumin was incorporated at 10 of th ese permissive sites along the toxin molecule, and the capacity of ACT cons tructs to penetrate into cell cytosol and deliver the epitope into the majo r histocompatibility complex (MHC) class I antigen processing and presentat ion pathway was examined. While all six constructs bearing the epitope with in the Hly portion of ACT failed to deliver the epitope to the MHC class I molecules, all four toxoids with inserts within different permissive sites in the AC domain efficiently delivered the epitope into this cytosolic path way, giving rise to stimulation of a specific CD8(+) T-cell hybridoma. The results suggest that, in contrast to the AC domain, the hemolysin moiety of ACT does not reach the cytosolic entry of the MHC class I pathway.