Delivery of multiple epitopes by recombinant detoxified adenylate cyclase of Bordetella pertussis induces protective antiviral immunity

CyaA, the adenylate cyclase toxin from Bordetella pertussis, can deliver it s N-terminal catalytic domain into the cytosol of a large number of eukaryo tic cells and particularly into professional antigen-presenting cells. We h ave previously identified within the primary structure of CyaA several perm issive sites at which insertion of peptides does not alter the ability of t he toxin to enter cells. This property has been exploited to design recombi nant CyaA toxoids capable of delivering major histocompatibility complex (M HC) class I-restricted CD8(+) T-cell epitopes into antigen-presenting cells and to induce specific CD8(+) cytotoxic T-lymphocyte (CTL) responses in vi vo. Here we have explored the capacity of the CyaA vector carrying several different CDS' T-cell epitopes to prime multiple CTL responses. The model v accine consisted of a polyepitope made of three CTL epitopes from lymphocyt ic choriomeningitis virus (LCMV) the V3 region of human immunodeficiency vi rus gp120, and chicken ovalbumin, inserted at three different sites of the catalytic domain of genetically detoxified CyaA. Each of these epitopes was processed on delivery by CyaA and presented in vitro to specific T-cell hy bridomas. Immunization of mice by CyaA toxoids carrying the polyepitope lea d to the induction of specific CTL responses for each of the three epitopes , as well as to protection against a lethal viral challenge. Moreover, mice primed against the vector by mock CyaA or a recombinant toxoid were still able to develop strong CTL responses after subsequent immunization with a r ecombinant CyaA carrying a foreign CDS' CTL epitope. These results highligh t the potency of the adenylate cyclase vector for induction of protective C TL responses with multiple specificity and/or broad MHC restriction.