Multi plasmid system for the production of influenza virus

Abstract

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.

1 . (canceled) 
     
     
         2 . (canceled) 
     
     
         3 . An isolated reassortant influenza B virus, comprising:
 a NP polypeptide comprising a threonine at position 55, and: an alanine at position 114, or a histidine at position 410, or an alanine at position 114 and a histidine at position 410, wherein:   the isolated reassortant influenza B virus has a temperature sensitive phenotype, and   the positions in the NP polypeptide correspond to positions in the full-length NP polypeptide of B/Ann Arbor/1/66.   
     
     
         4 . The isolated reassortant influenza B virus of  claim 3 , wherein the NP polypeptide comprises the alanine at position 114 and the histidine at position 410. 
     
     
         5 . The isolated reassortant influenza B virus of  claim 3 , wherein the NP polypeptide comprises a threonine at position 509. 
     
     
         6 . The isolated reassortant influenza B virus of  claim 3 , comprising a PA polypeptide comprising a methionine at position 431, or a histidine at position 497, or a methionine at position 431 and a histidine at position 497, wherein the positions in the PA polypeptide correspond to positions in the full-length PA polypeptide of B/Ann Arbor/1/66. 
     
     
         7 . The isolated reassortant influenza B virus of  claim 3 , which is a 6:2 reassortant influenza B virus. 
     
     
         8 . The isolated reassortant influenza B virus of  claim 6 , which is a 6:2 reassortant influenza B virus. 
     
     
         9 . The isolated reassortant influenza B virus of  claim 3 , wherein the titer of the virus grown in cell culture at 33 degrees Celsius is at least 2 log 10  greater compared to the same virus grown at 37 degrees Celsius. 
     
     
         10 . The isolated reassortant influenza B virus of  claim 6 , wherein the titer of the virus grown in cell culture at 33 degrees Celsius is at least 2 log 10  greater compared to the same virus grown at 37 degrees Celsius. 
     
     
         11 . The isolated reassortant influenza B virus of  claim 3 , wherein the virus is derived from a B/Ann Arbor/1/66 strain. 
     
     
         12 . The isolated reassortant influenza B virus of  claim 6 , wherein the virus is derived from a B/Ann Arbor/1/66 strain. 
     
     
         13 . A composition comprising the isolated reassortant influenza B virus of  claim 3  and a carrier or excipient. 
     
     
         14 . A composition comprising the isolated reassortant influenza B virus of  claim 6  and a carrier or excipient. 
     
     
         15 . A method for stimulating an immune response, which comprises administering the composition of  claim 13 . 
     
     
         16 . A method for stimulating an immune response, which comprises administering the composition of  claim 14 . 
     
     
         17 . A method for making an isolated reassortant influenza B virus, comprising:
 (a) introducing one or more mutations in an influenza B virus genome that result in a NP polypeptide comprising a threonine at position 55, and: an alanine at position 114, or a histidine at position 410, or an alanine at position 114 and a histidine at position 410, wherein the positions in the NP polypeptide correspond to positions in the full-length NP polypeptide of B/Ann Arbor/1/66;   (b) introducing a plurality of vectors into a population of cultured host cells, wherein the plurality of vectors corresponds to the influenza B virus genome and the plurality of vectors comprises the mutations recited in (a);   (c) culturing the population of host cells; and   (d) recovering the isolated reassortant influenza B virus produced by the host cells of (c), wherein the influenza B virus has a temperature sensitive phenotype.   
     
     
         18 . The method of  claim 17 , which comprises introducing one or more mutations in the influenza B virus genome that result in the NP polypeptide comprising the alanine at position 114 and the histidine at position 410. 
     
     
         19 . The method of  claim 17 , which comprises introducing one or more mutations in the influenza B virus genome that result in the NP polypeptide comprising a threonine at position 509. 
     
     
         20 . The method of  claim 17 , which comprises introducing one or more mutations in the influenza B virus genome that result in a PA polypeptide comprising a methionine at position 431, or a histidine at position 497, or a methionine at position 431 and a histidine at position 497, wherein the positions in the PA polypeptide correspond to positions in the full-length PA polypeptide of B/Ann Arbor/1/66. 
     
     
         21 . The method of  claim 17 , wherein the reassortant influenza B virus is a 6:2 reassortant influenza B virus. 
     
     
         22 . The method of  claim 17 , wherein the titer of the virus grown in cell culture at 33 degrees Celsius is at least 2 log 10  greater compared to the same virus grown at 37 degrees Celsius. 
     
     
         23 . The method of  claim 17 , wherein the virus is derived from a B/Ann Arbor/1/66 strain. 
     
     
         24 . The method of  claim 17 , further comprising, after (d), amplifying the recombinant or reassortant influenza B virus by passage in cultured cells or in hens' eggs.