INTRODUCTION: Tuberculosis (TB) remains the first cause of death from infection caused by a bacterial pathogen. Chemotherapy does not eradicate Mycobacterium tuberculosis (Mtb) from human lungs, and the pathogen causes a latent tuberculosis infection that cannot be prevented by the currently available Bacille Calmette Guerin (BCG) vaccine, which is ineffective in the prevention of pulmonary TB in adults. HLA-E-restricted CD8+ T lymphocytes are essential players in protective immune responses against Mtb. Hence, expanding this population in vivo or ex vivo may be crucial for vaccination or immunotherapy against TB. METHODS: The enzymatically inactive Bordetella pertussis adenylate cyclase (CyaA) toxoid is an effective tool for delivering peptide epitopes into the cytosol of antigen-presenting cells (APC) for presentation and stimulation of specific CD8+ T-cell responses. In this study, we have investigated the capacity of the CyaA toxoid to deliver Mtb epitopes known to bind HLA-E for the expansion of human CD8+ T cells in vitro. RESULTS: Our results show that the CyaA-toxoid containing five HLA-E-restricted Mtb epitopes causes significant expansion of HLA-E-restricted antigen-specific CD8+ T cells, which produce IFN-γ and exert significant cytotoxic activity towards peptide-pulsed macrophages. DISCUSSION: HLA-E represents a promising platform for the development of new vaccines; our study indicates that the CyaA construct represents a suitable delivery system of the HLA-E-binding Mtb epitopes for ex vivo and in vitro expansion of HLA-E-restricted CD8+ T cells inducing a predominant Tc1 cytokine profile with a significant increase of IFN-γ production, for prophylactic and immunotherapeutic applications against Mtb.

• Keywords
• Bordetella pertussis adenylate cyclase, HLA-E, Mycobacterium tuberculosis, cytotoxic t lymphocytes, immunotherapy, peptides, vaccine,
• MeSH
• Adenylyl Cyclases MeSH
• HLA-E Antigens MeSH
• Bordetella pertussis MeSH
• CD8-Positive T-Lymphocytes MeSH
• Epitopes MeSH
• Humans MeSH
• Histocompatibility Antigens Class I MeSH
• Mycobacterium tuberculosis * MeSH
• Peptides MeSH
• Toxoids MeSH
• Tuberculosis * prevention & control   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylyl Cyclases MeSH
• HLA-E Antigens MeSH
• Epitopes MeSH
• Histocompatibility Antigens Class I MeSH
• Peptides MeSH
• Toxoids MeSH

Adenylate cyclase toxin (CyaA) is released in the course of B. pertussis infection in the host's respiratory tract in order to suppress its early innate and subsequent adaptive immune defense. CD11b-expressing dendritic cells (DC), macrophages and neutrophils are professional phagocytes and key players of the innate immune system that provide a first line of defense against invading pathogens. Recent findings revealed the capacity of B. pertussis CyaA to intoxicate DC with high concentrations of 3',5'-cyclic adenosine monophosphate (cAMP), which ultimately skews the host immune response towards the expansion of Th17 cells and regulatory T cells. CyaA-induced cAMP signaling swiftly incapacitates opsonophagocytosis, oxidative burst and NO-mediated killing of bacteria by neutrophils and macrophages. The subversion of host immune responses by CyaA after delivery into DC, macrophages and neutrophils is the subject of this review.

• Keywords
• T-helper cells, immune response, intracellular pathways, phagocytosis,
• MeSH
• Adenylate Cyclase Toxin immunology   MeSH
• Cyclic AMP chemistry   MeSH
• Bordetella pertussis MeSH
• Immunity, Cellular MeSH
• Dendritic Cells immunology   MeSH
• Respiratory System immunology   microbiology   MeSH
• Phagocytosis MeSH
• Host-Pathogen Interactions MeSH
• Humans MeSH
• Macrophages immunology   MeSH
• Neutrophils immunology   MeSH
• Whooping Cough immunology   MeSH
• T-Lymphocytes, Regulatory immunology   MeSH
• Signal Transduction MeSH
• Immunity, Mucosal MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Adenylate Cyclase Toxin MeSH
• Cyclic AMP MeSH

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.

• MeSH
• Adenylate Cyclase Toxin genetics   immunology   MeSH
• Adjuvants, Immunologic genetics   MeSH
• Lymphocyte Activation * MeSH
• Cell Differentiation MeSH
• CD4-Positive T-Lymphocytes immunology   MeSH
• CD8-Positive T-Lymphocytes immunology   MeSH
• Cytokines metabolism   MeSH
• Pore Forming Cytotoxic Proteins genetics   immunology   MeSH
• Dendritic Cells immunology   microbiology   MeSH
• Ion Transport MeSH
• Cells, Cultured MeSH
• p38 Mitogen-Activated Protein Kinases metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Cell Membrane Permeability MeSH
• Protein Domains genetics   immunology   MeSH
• Cancer Vaccines immunology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylate Cyclase Toxin MeSH
• Adjuvants, Immunologic MeSH
• Cytokines MeSH
• Pore Forming Cytotoxic Proteins MeSH
• p38 Mitogen-Activated Protein Kinases MeSH
• Cancer Vaccines MeSH

The choice of tools that enable efficient targeting of exogenous antigens (Ag) for processing and presentation by professional Ag-presenting cells (APC) remains limited. This represents, indeed, a bottleneck in development of vaccines inducing specific T-cell responses. Here, we describe a novel strategy of Ag delivery into APCs. The Ag of choice is fused to the N- or C-terminus of streptavidin (SA) and tetrameric Ag-SA or SA-Ag fusion proteins are produced in E. coli and purified by 2-Iminobiotin-Agarose affinity chromatography. Alternatively, Ag-SA proteins are purified from urea extracts of E. coli inclusion bodies and refolded in vitro into functional tetramers. Complexes with biotinylated antibodies targeting cell surface receptors are formed and used to deliver the Ags of choice for processing and presentation by APCs and induction of Ag-specific CD4+ and CD8+ T-cell responses in vitro and in vivo.

• MeSH
• CD11c Antigen immunology   metabolism   MeSH
• Antigens chemistry   genetics   immunology   metabolism   MeSH
• Biotinylation MeSH
• Cell Line MeSH
• Dendritic Cells immunology   metabolism   MeSH
• Chickens MeSH
• Molecular Sequence Data MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Ovalbumin chemistry   genetics   immunology   metabolism   MeSH
• Receptors, Cell Surface immunology   metabolism   MeSH
• Recombinant Fusion Proteins chemistry   genetics   immunology   metabolism   MeSH
• Solubility MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Streptavidin chemistry   genetics   immunology   metabolism   MeSH
• T-Lymphocytes immunology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CD11c Antigen MeSH
• Antigens MeSH
• Ovalbumin MeSH
• Receptors, Cell Surface MeSH
• Recombinant Fusion Proteins MeSH
• Streptavidin MeSH

The Bordetella adenylate cyclase toxin-hemolysin (CyaA; also called ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) that hijacks cellular signaling by conversion of ATP to cyclic AMP (cAMP). Intriguingly, insertion of large passenger peptides removes the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC(-) toxoids, thus enabling their processing and presentation on major histocompatibility complex (MHC) class I molecules to cytotoxic CD8(+) T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and showed that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) with heterologous polypeptides of 40, 146, or 203 residues yielded CyaAΔAC constructs that delivered passenger CTL epitopes into antigen-presenting cells (APCs) and induced strong antigen-specific CD8(+) CTL responses in vivo in mice and ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (repeats in toxin) hemolysin moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membranes. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into the cytosol of CD11b(+) target cells and pave the way for the construction of CyaAΔAC-based polyvalent immunotherapeutic T cell vaccines.

• MeSH
• Adenylate Cyclase Toxin genetics   metabolism   MeSH
• Antigen-Presenting Cells metabolism   MeSH
• Cell Membrane metabolism   MeSH
• CD8-Positive T-Lymphocytes immunology   MeSH
• Dendritic Cells metabolism   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Recombinant Proteins genetics   metabolism   MeSH
• Sequence Deletion MeSH
• Toxoids genetics   metabolism   MeSH
• Protein Transport MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylate Cyclase Toxin MeSH
• Recombinant Proteins MeSH
• Toxoids MeSH

The Bordetella adenylate cyclase toxoid (CyaA) targets cells expressing the alphaMbeta2 integrin receptor CD11b/CD18 (CR3 or Mac-1) and can penetrate into cytosol of professional antigen-presenting cells, such as dendritic cells. This allows us to use CyaA for delivery of passenger antigens into the cytosolic pathway of processing and MHC class I-restricted presentation, which can promote induction of antigen-specific CD8+ cytotoxic T-lymphocyte immune responses. We show here that vaccination with a genetically detoxified CyaA336/E7 protein, carrying the full-length oncoprotein E7 of the human papilloma virus 16 inserted at position 336 of the cell-invasive AC domain of CyaA, induces an E7-specific CD8+ T-cell immune response and confers on mice protective, as well as therapeutic immunity against challenge with TC-1 tumor cells expressing the E7 oncoprotein. The therapeutic efficacy of priming with the CyaA336/E7 vaccine could further be enhanced by a heterologous booster immunization with a highly attenuated modified vaccinia virus Ankara (MVA) expressing the E7 protein fused to the lysosome-associated membrane protein (LAMP1). These results establish the potential of CyaA as a new antigen delivery tool for prime/boost immunotherapy of tumors.

• MeSH
• Adenylate Cyclase Toxin * MeSH
• Antigens, Neoplasm MeSH
• Antigens, Viral MeSH
• Immunity, Cellular MeSH
• CD8-Positive T-Lymphocytes immunology   MeSH
• Dendritic Cells immunology   MeSH
• Immunotherapy methods   MeSH
• Human papillomavirus 16 pathogenicity   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Tumor Cells, Cultured MeSH
• Oncogene Proteins, Viral immunology   MeSH
• Papillomavirus E7 Proteins MeSH
• Cancer Vaccines immunology   MeSH
• Immunization, Secondary MeSH
• Vaccinia virus * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylate Cyclase Toxin * MeSH
• Antigens, Neoplasm MeSH
• Antigens, Viral MeSH
• oncogene protein E7, Human papillomavirus type 16 MeSH Browser
• Oncogene Proteins, Viral MeSH
• Papillomavirus E7 Proteins MeSH
• Cancer Vaccines MeSH