Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells.

The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

Antigen targeting to CD11b+ dendritic cells in association with TLR4/TRIF signaling promotes strong CD8+ T cell responses.

Deciphering the mechanisms that allow the induction of strong immune responses is crucial to developing efficient vaccines against infectious diseases and cancer. Based on the discovery that the adenylate cyclase from Bordetella pertussis binds to the CD11b/CD18 integrin, we developed a highly efficient detoxified adenylate cyclase-based vector (CyaA) capable of delivering a large variety of Ags to the APC. This vector allows the induction of protective and therapeutic immunity against viral and tumoral challenges as well as against transplanted tumors in the absence of any added adjuvant. Two therapeutic vaccine candidates against human papilloma viruses and melanoma have been developed recently, based on the CyaA vector, and are currently in clinical trials. We took advantage of one of these highly purified vaccines, produced under good manufacturing practice-like conditions, to decipher the mechanisms by which CyaA induces immune responses. In this study, we demonstrate that CyaA binds both human and mouse CD11b(+) dendritic cells (DCs) and induces their maturation, as shown by the upregulation of costimulatory and MHC molecules and the production of proinflammatory cytokines. Importantly, we show that DCs sense CyaA through the TLR4/Toll/IL-1R domain-containing adapter-inducing IFN-ß pathway, independent of the presence of LPS. These findings show that CyaA possesses the intrinsic ability to not only target DCs but also to activate them, leading to the induction of strong immune responses. Overall, this study demonstrates that Ag delivery to CD11b(+) DCs in association with TLR4/Toll/IL-1R domain-containing adapter-inducing IFN-ß activation is an efficient strategy to promote strong specific CD8(+) T cell responses.

Induction of neutralizing antibodies and Th1-polarized and CD4-independent CD8+ T-cell responses following delivery of human immunodeficiency virus type 1 Tat protein by recombinant adenylate cyclase of Bordetella pertussis.

HIV-Tat, a conserved protein playing a key role in the early life cycle of the human immunodeficiency virus (HIV) has been proposed as a potential AIDS vaccine. An HIV-Tat-based vaccine should elicit a broad, long-lasting, and neutralizing immune response. We have previously demonstrated that the adenylate cyclase (CyaA) from Bordetella pertussis targets dendritic cells and delivers CD8(+) and CD4(+) T-cell epitopes into the major histocompatibility complex class I and class II presentation pathways. We have also showed that CyaA induced specific and protective cytotoxic T cell responses in vivo. Here, we designed a prototype vaccine based on the HIV type 1 Tat delivered by CyaA (CyaA-E5-Tat) and tested its capacity to induce HIV-Tat-specific cellular as well as antibody responses. We showed that immunization of mice by CyaA-E5-Tat in the absence of adjuvant elicited strong and long-lasting neutralizing anti-Tat antibody responses more efficient than those obtained after immunization with Tat toxoid in aluminum hydroxide adjuvant. Analyses of the anti-Tat immunoglobulin G isotypes and the cytokine pattern showed that CyaA-E5-Tat induced a Th1-polarized immune response in contrast to the Th2-polarized immune responses obtained with the Tat toxoid. In addition, our data demonstrated that HIV-Tat-specific gamma interferon-producing CD8(+) T cells were generated after vaccination with CyaA-E5-Tat in a CD4(+) T-cell-independent manner. Based on these findings, CyaA-E5-Tat represents an attractive vaccine candidate for both preventive and therapeutic vaccination involving CyaA as an efficient nonreplicative vector for protein delivery.