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Svedova M.,Institute of Microbiology of the ASCR
Immunology and Cell Biology | Year: 2015

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.Immunology and Cell Biology advance online publication, 1 December 2015; doi:10.1038/icb.2015.87. © 2015 Australasian Society for Immunology Inc.

Vetrovsky T.,Institute of Microbiology of the ASCR | Baldrian P.,Institute of Microbiology of the ASCR | Gabriel J.,Institute of Microbiology of the ASCR
Applied Biochemistry and Biotechnology | Year: 2013

Production of the lignocellulose-degrading enzymes endo-1,4-β- glucanase, 1,4-β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, 1,4-β-xylosidase, Mn peroxidase, and laccase was characterized in a common wood-rotting fungus Fomes fomentarius, a species able to efficiently decompose dead wood, and compared to the production in eight other fungal species. The main aim of this study was to characterize the 1,4-β-glucosidase produced by F. fomentarius that was produced in high quantities in liquid stationary culture (25.9 U ml-1), at least threefold compared to other saprotrophic basidiomycetes, such as Rhodocollybia butyracea, Hypholoma fasciculare, Irpex lacteus, Fomitopsis pinicola, Pleurotus ostreatus, Piptoporus betulinus, and Gymnopus sp. (between 0.7 and 7.9 U ml-1). The 1,4-β-glucosidase enzyme was purified to electrophoretic homogeneity by both anion-exchange and size-exclusion chromatography. A single 1,4-β-glucosidase was found to have an apparent molecular mass of 58 kDa and a pI of 6.7. The enzyme exhibited high thermotolerance with an optimum temperature of 60 C. Maximal activity was found in the pH range of 4.5-5.0, and K M and V max values were 62 μM and 15.8 μmol min-1 l-1, respectively, when p-nitrophenylglucoside was used as a substrate. The enzyme was competitively inhibited by glucose with a K i of 3.37 mM. The enzyme also acted on p-nitrophenylxyloside, p-nitrophenylcellobioside, p-nitrophenylgalactoside, and p-nitrophenylmannoside with optimal pH values of 6.0, 3.5, 5.0, and 4.0-6.0, respectively. The combination of relatively low molecular mass and low K M value make the 1,4-β-glucosidase a promising enzyme for biotechnological applications. © 2012 Springer Science+Business Media New York.

Institute Of Microbiology Of The Ascr and Institute Pasteur Paris | Date: 2010-12-12

The present invention provides an innovative versatile system, which allows delivery of one or several antigens or biologically active molecules into or onto targeted subset of cells. The invention is in particular directed to a combination of compounds and in particular to a composition, which comprises: (i) a fusion polypeptide comprising a streptavidin (SA) or avidin polypeptide and one or several effector molecule(s), wherein said fusion polypeptide retains the property of SA and avidin polypeptides to bind biotin; (ii) biotinylated targeting molecule(s), which are capable of targeting subset(s) of cells and/or cell surface molecule(s), and in particular dendritic cells (DC), subsets of DC and/or surface molecule(s) (including surface receptor(s)) of DC. The combination of the invention is suitable for use for targeting, in vivo, in vitro or ex vivo, of one or several effector molecule(s) to subset(s) of cells and/or cell surface molecule(s), and in particular for diagnosing or immunomonitoring a disease in a mammal or in prophylactic treatment and especially in vaccination and in therapy including in immunotherapy. The combination of the invention is also intended for use in vivo or ex vivo, for inducing a T cell immune response in bone marrow of naive donors before transplantation, or for activation and/or expansion of a T cell immune response in bone marrow of already immunized donors. The invention also relates to a method for the production of a fusion polypeptide of the invention and to a kit for a diagnostic test of a disease in a mammal, for immunomonitoring a disease in a mammal or for the prevention or treatment of a disease in a mammal.

Institute Pasteur Paris, Institute Of Microbiology Of The Ascr and Institute Of Physiology Of The Ascr | Date: 2015-12-01

The invention relates to mutant CyaA/E570Q+K860 polypeptides suitable for use as proteinaceous vectors for delivering one or more molecules of interest into a cell, in particular into a cell expressing the CD11b receptor. The invention further relates to polypeptide derivatives suitable for eliciting an immune response in a host. The invention is more particularly directed to polypeptides derived from an adenylate cyclase protein (CyaA) either under the form of a toxin or of a toxoid, which are mutant polypeptides. Said mutant polypeptides are capable of retaining the binding activity of native CyaA to a target cell and preferably of also retaining the translocating activity of native CyaA through its N-terminal domain into target cells and furthermore have a pore-forming activity which is reduced or suppressed as compared to that of the native CyaA toxin.

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