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. In addition, the present invention includes an improved method of rescue, wherein animal cells (e.g., SF Vero cells) are electroporated with plasmids and vectors of the invention.

1 . A method of rescue of influenza virus, wherein animal cells are electroporated with plasmids that encode an influenza RNA polymerase and nucleoprotein and wherein the electroporated animal cells are co-cultivated with another cell type.  
     
     
         2 . The method of  claim 1 , wherein the animal cells are Vero cells.  
     
     
         3 . The method of  claim 1 , wherein the animal cells are SF Vero cells.  
     
     
         4 . The method of  claim 1 , wherein said another cell type is CEK cells.  
     
     
         5 . The method of  claim 1 , wherein the influenza virus is an influenza A virus.  
     
     
         6 . The method of  claim 1 , wherein the influenza virus is an influenza B virus.  
     
     
         7 . The method of  claim 1 , wherein the influenza virus is a cold adapted virus.  
     
     
         8 . The method of  claim 1 , wherein the influenza virus is an attenuated virus.  
     
     
         9 . The method of  claim 1 , wherein the number of plasmids electroporated is eight.  
     
     
         10 . The method of  claim 1 , wherein the number of plasmids electroporated is twelve.  
     
     
         11 . The method of  claim 1 , wherein the efficiency of said rescue of influenza virus is at least 90%.  
     
     
         12 . An influenza virus produced by the method of  claim 1 .  
     
     
         13 . The influenza virus of  claim 1 , wherein said rescued influenza virus comprises vRNA segments derived from A/PR/8/34.  
     
     
         14 . The influenza virus of  claim 1 , wherein said rescued influenza virus comprises vRNA segments derived from MDV-A.  
     
     
         15 . A vaccine composition comprising the influenza virus of  claim 1 .  
     
     
         16 . A method for producing influenza viruses in cell culture, the method comprising: 
 i) introducing a plurality of vectors comprising an influenza virus genome into a population of Vero cells by electroporation;    ii) co-cultivating the population of Vero cells with another cell type under conditions permissive for viral replication; and,    iii) recovering a plurality of influenza viruses.    
     
     
         17 . A method of rescue of influenza virus, wherein (a) animal cells are electroporated with cell expression vectors which direct the expression in said cells of genomic or antigenomic vRNA segments, and a nucleoprotein, and an RNA-dependent polymerase, such that ribonucleoprotein complexes can be formed and viral particles can be assembled; and (b) culturing said cells wherein viral particles are packaged and rescued.  
     
     
         18 . The method of  claim 17 , wherein assembly does not require a helper virus.  
     
     
         19 - 25 . (canceled)