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. 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.

1 . A method of increasing replication of a reassortant influenza A virus by at least 10%, comprising:
 a) introducing an amino acid substitution to an HA amino acid sequence of the reassortant virus, thereby producing an altered reassortant virus, wherein the amino acid substitution is to threonine at position 196; and   b) growing the altered reassortant virus in eggs.   
     
     
         2 . The method of  claim 1 , wherein step a) comprises introducing a second amino acid substitution to the HA amino acid sequence of the reassortant virus, wherein the second amino acid substitution is to valine at position 186. 
     
     
         3 . The method of  claim 1 , wherein step a) comprises introducing a second amino acid substitution to the HA amino acid sequence of the reassortant virus, wherein the second amino acid substitution is to isoleucine at position 226. 
     
     
         4 . The method of  claim 2 , wherein step a) comprises introducing a third amino acid substitution to the HA amino acid sequence of the reassortant virus, wherein the third amino acid substitution is to isoleucine at position 226. 
     
     
         5 . A replication enhanced reassortant influenza virus produced by the method of  claim 1 . 
     
     
         6 . A replication enhanced reassortant influenza virus produced by the method of  claim 2 . 
     
     
         7 . A replication enhanced reassortant influenza virus produced by the method of  claim 3 . 
     
     
         8 . A replication enhanced reassortant influenza virus produced by the method of  claim 4 . 
     
     
         9 . The method of  claim 1 , wherein the reassortant influenza A virus grows to a titer of at least 8.0 log 10  PFU/ml in embryonated eggs. 
     
     
         10 . The method of  claim 2 , wherein the reassortant influenza A virus grows to a titer of at least 8.0 log 10  PFU/ml in embryonated eggs. 
     
     
         11 . The method of  claim 3 , wherein the reassortant influenza A virus grows to a titer of at least 8.0 log 10  PFU/ml in embryonated eggs. 
     
     
         12 . The method of  claim 4 , wherein the reassortant influenza A virus grows to a titer of at least 8.0 log 10  PFU/ml in embryonated eggs. 
     
     
         13 . The reassortant influenza A virus of  claim 5 , wherein the influenza virus is cold adapted, temperature sensitive and attenuated. 
     
     
         14 . The reassortant influenza A virus of  claim 6 , wherein the influenza virus is cold adapted, temperature sensitive and attenuated. 
     
     
         15 . The reassortant influenza A virus of  claim 8 , wherein the influenza virus is cold adapted, temperature sensitive and attenuated. 
     
     
         16 . An immunogenic composition comprising the replication enhanced reassortant influenza virus of  claim 13 . 
     
     
         17 . An immunogenic composition comprising the replication enhanced reassortant influenza virus of  claim 14 . 
     
     
         18 . An immunogenic composition comprising the replication enhanced reassortant influenza virus of  claim 15 . 
     
     
         19 . The reassortant influenza A virus of  claim 5 , wherein the influenza virus comprises the backbone of the MDA-A strain or PR8 strain. 
     
     
         20 . The reassortant influenza A virus of  claim 6 , wherein the influenza virus comprises the backbone of the MDA-A strain or PR8 strain. 
     
     
         21 . A reassortant influenza virus comprising a substitution at one or more of the following positions: 183, 186, 196, or 226, wherein the influenza virus grows to a titer of at least 8.0 log 10  PFU/ml in embryonated eggs.