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

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) is a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA targets myeloid phagocytes expressing the complement receptor 3 (CR3, known as αMβ2 integrin CD11b/CD18 or Mac-1) and translocates by a poorly understood mechanism directly across the cytoplasmic membrane into cell cytosol of phagocytes an adenylyl cyclase(AC) enzyme. This binds intracellular calmodulin and catalyzes unregulated conversion of cytosolic ATP into cAMP. Among other effects, this yields activation of the tyrosine phosphatase SHP-1, BimEL accumulation and phagocyte apoptosis induction. In parallel, CyaA acts as a cytolysin that forms cation-selective pores in target membranes. Direct penetration of CyaA into the cytosol of professional antigen-presenting cells allows the use of an enzymatically inactive CyaA toxoid as a tool for delivery of passenger antigens into the cytosolic pathway of processing and MHC class I-restricted presentation, which can be exploited for induction of antigen-specific CD8(+) cytotoxic T-lymphocyte immune responses.

• Keywords
• adenylate cyclase toxin, antigen delivery tool, membrane penetration, pore-formation,
• MeSH
• Adenylate Cyclase Toxin metabolism   toxicity   MeSH
• Apoptosis * MeSH
• Bordetella pertussis metabolism   MeSH
• CD8-Positive T-Lymphocytes immunology   MeSH
• T-Lymphocytes, Cytotoxic immunology   MeSH
• Phagocytes drug effects   physiology   MeSH
• Drug Carriers metabolism   MeSH
• Th1 Cells immunology   MeSH
• Carrier Proteins metabolism   MeSH
• Vaccines immunology   metabolism   MeSH
• Cell Survival MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Adenylate Cyclase Toxin MeSH
• Drug Carriers MeSH
• Carrier Proteins MeSH
• Vaccines MeSH

Viral CC chemokine inhibitor (vCCI) of the clone P13 vaccinia virus (VACV) strain PRAHA lacks eight amino acids in the signal peptide sequence. To study the influence of vCCI on virus biology, a virus with the vCCI gene coding for a prolonged signal sequence was prepared. We found that secreted vCCI attenuated the virus in vivo, and that it correlated with decreased levels of RANTES, eotaxin, TARC, and MDC in the blood in comparison with the parental virus. We determined the influence of vCCI on the CTL response against VACV E3((140-148)) (VGPSNSPTF) and HPV16 E7((49-57)) (RAHYNIVTF) H-2D(b)-restricted epitopes. The examination of the specific CTL response elicited by immunization with the recombinant VACV-expressing tumor-associated HPV16 E7 antigen by IFN-γ ELISPOT showed that the immunogenicity of the recombinant VACV-producing secretory vCCI was similar to that of the parent virus or deletion mutant in the C23L/B29R locus. Immunization with the secretory vCCI-producing recombinant virus has a lower therapeutic anti-tumor effect against TC-1 tumors. Viral CCI downregulated the E7-specific response induced by gene gun immunization with the DNA vaccines pBSC-SigE7 LAMP and pBSC-vCCI. We also observed that the immune response against vCCI elicited by the DNA vaccine did not affect the multiplication of VACV in vivo.

• MeSH
• Cell Line MeSH
• Chemokine CCL17 blood   MeSH
• Chemokine CCL5 blood   MeSH
• Chemokines, CC antagonists & inhibitors   blood   MeSH
• T-Lymphocytes, Cytotoxic immunology   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Tumor Suppressor Proteins blood   MeSH
• Papillomavirus E7 Proteins genetics   immunology   MeSH
• ADAM Proteins blood   MeSH
• Cancer Vaccines genetics   immunology   MeSH
• Sequence Deletion MeSH
• Vaccines, Synthetic genetics   immunology   MeSH
• Vaccination MeSH
• Viral Proteins genetics   metabolism   MeSH
• Viral Vaccines immunology   MeSH
• Vaccinia virus genetics   immunology   pathogenicity   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
• ADAM11 protein, human MeSH Browser
• Chemokine CCL17 MeSH
• Chemokine CCL5 MeSH
• Chemokines, CC MeSH
• Tumor Suppressor Proteins MeSH
• oncogene protein E7, Human papillomavirus type 16 MeSH Browser
• Papillomavirus E7 Proteins MeSH
• ADAM Proteins MeSH
• Cancer Vaccines MeSH
• Vaccines, Synthetic MeSH
• Viral Proteins MeSH
• Viral Vaccines 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

Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca(2+) ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest.

• MeSH
• Amino Acid Motifs MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• Bacterial Toxins chemistry   genetics   metabolism   MeSH
• Gram-Negative Bacteria chemistry   genetics   metabolism   MeSH
• Multigene Family * MeSH
• Gene Expression Regulation, Bacterial MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Bacterial Toxins MeSH