Method for producing temperature sensitive influenza A viruses
Vectors and methods for the production of influenza viruses suitable as recombinant influenza vaccines in cell culture are provided. Bi-directional expression vectors for use in a multi-plasmid influenza virus expression system are provided. Additionally, the invention provides methods of producing influenza viruses with enhanced ability to replicate in embryonated chicken eggs and/or cells (e.g., Vero and/or MDCK) and further provides influenza viruses with enhanced replication characteristics. A method of producing a cold adapted (ca) influenza virus that replicates efficiently at, e.g., 25° C. (and immunogenic compositions comprising the same) is also provided.
1. A method of producing a temperature sensitive (ts) influenza A virus, the method comprising:
(a) introducing mutations into an influenza A virus polynucleotide resulting in amino acid substitutions in PB1, thereby producing a mutated polynucleotide; and
(b) producing influenza A virus comprising the mutated polynucleotide, wherein the substitutions in the PB1 of the virus produced consist of Lysine to Glutamic Acid (K to E) at position PB1391, Glutamic Acid to Glycine (E to G) at position PB1581, and Alanine to Threonine (A to T) at position PB1661.
2. The method of claim 1 , wherein PB2 in the influenza A virus produced in (b) comprises an Asparagine to Serine (N to S) mutation at position PB2265.
3. The method of claim 1 , wherein the influenza A virus comprises a PR8 polynucleotide.
4. The method of claim 2 , wherein the influenza A virus comprises a PR8 polynucleotide.
5. The method of claim 1 , wherein introducing the mutations is by site-directed mutagenesis.
6. The method of claim 2 , wherein introducing the mutation is by site-directed mutagenesis.
7. The method of claim 1 , comprising producing the influenza A virus in hens' eggs.
8. The method of claim 2 , comprising producing the influenza A virus in hens' eggs.
9. The method of claim 1 , comprising producing the influenza A virus in cell culture.
10. The method of claim 9 , wherein the cell culture comprises one or more of Vero cells, MDCK cells, 293T cells and COS cells.
11. The method of claim 2 , comprising producing the influenza A virus in cell culture.
12. The method of claim 11 , wherein the cell culture comprises one or more of Vero cells, MDCK cells, 293T cells and COS cells.