Delivery of proteins into eukaryotic cells with recombinant yersinia
AssigneesLudwig Institute for Cancer Research, Université Catholique De Louvain
InventorsPierre B. van der Bruggen, Guy R. Cornelis, Anne M. Boland, Thierry R. Boon-Falleur
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Abstract
The present invention relates to recombinant Yersinia and the use thereof for delivery of proteins into eukaryotic cells, including related compositions and methods of treatment and related assays.
Claims64
An isolated mutant Yersinia strain having the designation of Y. enterocolitica yopEHOMP or Y. pseudotuberculosis yopEHAMJ.
An expression vector for delivering a heterologous protein into a eukaryotic cell, which comprises in the 5' to 3' direction:
- a promoter from a Yersinia virulon gene;
- a first DNA sequence encoding a delivery signal from a Yersinia effector protein, operably linked to said promoter;
- and
- a second DNA sequence coding for said heterologous protein, fused in frame to the 3' end of said first DNA sequence.
An isolated mutant Yersinia for delivery of a heterologous protein into a eukaryotic cell, wherein said Yersinia comprises at least one mutation in at least one Yersinia effector-encoding gene;
- and wherein said Yersinia is deficient in the production of at least one functional effector protein and is transformed with an expression vector which comprises, in the 5' to 3' direction:
- a promoter from a Yersinia virulon gene;
- a first DNA sequence encoding a delivery signal from a Yersinia effector protein, operably linked to said promoter;
- and
- a second DNA sequence coding for said heterologous protein, fused in frame to the 3' end of said first DNA sequence.
A method for delivering a heterologous protein into a eukaryotic cell in vitro, comprising contacting said eukaryotic cell with a Yersinia, wherein said Yersinia comprises at least one mutation in at least one Yersinia effector-encoding gene;
- and wherein said Yersinia is deficient in the production of at least one functional effector protein and is transformed with an expression vector characterized by, in 5' to 3' direction:
- a promoter from a Yersinia virulon gene;
- a first DNA sequence encoding a delivery signal from a Yersinia effector protein, operably linked to said promoter;
- and
- a second DNA sequence coding for said heterologous protein, fused in frame to the 3' end of said first DNA sequence.
The method of claim wherein 30, said Yersinia is Y. enterocolitica yopEHOMP transformed with said expression vector, or Y. pseudotuberculosis yopEHAOJ transformed with said expression vector.
A method for inducing in vitro, a cell-mediated immune response specific for a heterologous protein, comprising the steps of:
- (a) selecting an antigen presenting cell expressing an MHC molecule capable of presenting at least one epitope of said heterologous protein;
- (b) forming a cell mixture by contacting said antigen presenting cell with a Yersinia, wherein said Yersinia comprises at least one mutation in at least one Yersinia effector-encoding gene;
- and wherein said Yersinia is deficient in the production of at least one functional effector protein and is transformed with an expression vector characterized by, in 5' to 3' direction:
- a promoter from a Yersinia virulon gene;
- a first DNA sequence encoding a delivery signal from a Yersinia effector protein, operably linked to said promoter;
- a second DNA sequence coding for said heterologous protein, fused in frame to the 3' end of said first DNA sequence;
- thereby delivering said heterologous protein into said antigen presenting cell;
- and
- (c) contacting a sample containing peripheral blood lymphocytes taken from a subject, with the cell mixture formed in step (b) thereby inducing in vitro, a cell-mediated response specific for said heterologous protein.
A method for monitoring a cellular immune response in a subject before, during and after a vaccination regimen, comprising the steps of:
- (a) obtaining from said subject an antigen presenting cell expressing an MHC molecule;
- (b) forming a cell mixture by contacting said antigen presenting cell with a Yersinia, wherein said Yersinia comprises at least one mutation in at least one Yersinia effector-encoding gene;
- and wherein said Yersinia is deficient in the production of at least one functional effector protein and is transformed with an expression vector characterized by, in 5' to 3' direction:
- a promoter from a Yersinia virulon gene;
- a first DNA sequence encoding a delivery signal from a yersinia effector protein, operably linked to said promoter;
- and
- a second DNA sequence fused in frame to the 3' end of said first DNA sequence, wherein said second DNA sequence codes for at least one epitope of said antigen which is presented by said MHC molecule of said antigen presenting cell;
- thereby delivering said heterologous protein into said antigen presenting cell;
- and (c) contacting a sample containing peripheral blood lymphocytes taken from said subject, with the cell mixture formed in step (b), and assaying for the presence of a cell-mediated immune response specific for said antigen thereby monitoring a cell-mediated immune response in said subject before, during and after a vaccination regimen.
Dependencies are inferred from the claim text.