Production of infectious influenza viruses
AssigneeSanofi Pasteur Limited
InventorsIsabelle Legastelois, Julie Medina, Catherine Moste
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Abstract
The invention relates to a method for producing influenza infectious viruses wherein CHO cells are infected with a seed of infectious influenza virus which has been generated by transfecting cells with an appropriate set of expression vectors. The invention also relates to a recombination cassette, and to a vector comprising said recombination cassette, that may be used in methods for producing infectious viruses, and particularly in the method according to the invention.
Claims25
A method for producing infectious influenza viruses, wherein said method comprises the steps consisting of:
- a) transfecting cells with a set of expression vectors to generate a seed of infectious influenza virus,
- b) infecting CHO cells with said seed of infectious influenza virus.
A CHO cell comprising:
- a) expression vectors allowing the expression of mRNAs encoding at least influenza PB1, PB2, PA and NP proteins, and
- b) expression vectors allowing the expression of at least influenza PB1, PB2, PA, NP, M, NS, HA and NA vRNAs, or the corresponding eRNAs,
- wherein the expression of said set of expression vectors allows (i) the formation of the ribonucleoprotien complex (RNP) containing the influenza vRNA(s), and (ii) the generation of infectious influenza viruses in said transfected cells.
A vector comprising, in the 5′ to 3′ sense:
- a promoter that binds to RNA polymerase I, or to T7 RNA polymerase;
- a recombination cassette comprising, in the 5′ to 3′ sense:
- an inverted complementary recognition sequence for a first restriction enzyme which has its cutting site outside of its recognition sequence and produces sticky ends;
- a restriction site for a second restriction enzyme which has its cutting site inside of its recognition sequence;
- a restriction site for a third restriction enzyme which has its cutting site inside of its recognition sequence;
- and
- a recognition sequence for said first restriction enzyme which has its cutting site outside of its recognition sequence and produces sticky ends;
- wherein said second and third restriction enzymes are different;
- and
- a terminator sequence;
- when the promoter binds to RNA polymerase I, said terminator sequence is hepatitis delta ribozyme sequence,
- when the promoter binds to T7 RNA polymerase, said terminator sequence is T7 polymerase terminator sequence.
Dependencies are inferred from the claim text.