Production of infectious influenza viruses

A method for producing infectious influenza viruses, wherein said method comprises the steps of:

• a) transfecting cells with a set of expression vectors to generate a seed of infectious influenza virus, and
• b) infecting CHO cells with said seed of infectious influenza virus, wherein said set of expression vectors comprises:
• (aa) expression vectors allowing the expression of one or more mRNAs encoding at least influenza PB1, PB2, PA, NP, M, NS, HA, and NA proteins, and
• (bb) expression vectors allowing the expression of one or more influenza PB1, PB2, PA, NP, M, NS, HA and NA vRNAs, or the corresponding cRNAs,
• wherein the expression of said set of expression vectors allows the formation of the ribonucleoprotein complex (RNP) containing the influenza vRNA(s), and the generation of infectious influenza viruses in the transfected cells, and
• (i) said expression vectors allowing the expression of one or more mRNAs encoding influenza PB1, PB2, PA, NP, M, NS, HA, and NA proteins comprise four different uni directional plasmids, each plasmid containing one or more cDNAs complementary to a mRNA encoding one of the four distinct proteins selected from PB1, PB2, PA and NP influenza proteins, wherein the one or more cDNAs are under the control of a promoter that binds to RNA polymerase II, and
• (ii) said expression vectors allowing the expression of influenza PB1, PB2, PA, NP, M, NS, HA and NA vRNAs, or the corresponding cRNAs, comprise eight different uni directional plasmids, each plasmid containing one or more cDNAs complementary to one of the eight distinct vRNAs selected from said PB1, PB2, PA, NP, M, NS, HA and NA influenza vRNAs, or to the corresponding cRNAs, wherein the one or more cDNAs are under the control of a promoter that binds to RNA polymerase I;
• wherein each plasmid in paragraph (ii) has been obtained by cloning said cDNA into a vector comprising, in the 5′ to 3′ sense:
• a) a promoter that binds to RNA polymerase I, or a T7 RNA polymerase;
• b) 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
• c) a terminator sequence;
• wherein:
• when the promoter binds to RNA polymerase I, said terminator sequence is hepatitis delta ribozyme sequence;
• and
• when the promoter binds to T7 RNA polymerase, said terminator sequence is T7 polymerase terminator sequence.

A method of preparing an influenza vaccine composition, which method comprises:

• a) producing influenza viruses by transfecting cells with a set of expression vectors to generate a seed of infectious influenza virus, and infecting CHO cells with said seed of infectious influenza virus;
• b) harvesting the infectious influenza viruses after multiplication in CHO cells,
• c) purifying the harvested infectious influenza virus,
• d) optionally inactivating the purified virus, and
• e) mixing the purified virus with a pharmaceutically acceptable carrier, wherein at least one of the expression vectors comprises, 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;
• wherein:
• 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.