Rescue of influenza virus

Abstract

The invention relates to the field of influenza vaccine production. Influenza vaccines have been produced in embryonated hens' eggs for over 50 years, but recently there have been considerable efforts to develop cell culture systems for vaccine production. In one embodiment, the invention provides a nucleic acid comprising an influenza gene segment and a bacteriophage polymerase promoter or a complementary strand of said nucleic acid, and a cell comprising such a nucleic acid capable of producing desired influenza virus. In another embodiment, the invention provides a composition comprising a cell or material derived from a cell according to the invention and a virus or material derived from a viral particle according to the invention.

1 - 20 . (canceled) 
     
     
         21 . A method for producing a pharmaceutical composition for generating immunological protection against infection of a subject with an influenza virus comprising preparing a composition comprising:
 (a) transfecting a cell with at least one nucleic acid, wherein said nucleic acid is chosen from:   (1) an influenza gene segment and a bacteriophage polymerase promoter; and   (2) a complement of an influenza gene segment and a bacteriophage polymerase promoter; and   (b) culturing the transfected cell to form virus particles;   (c) combining the virus particles or viral proteins derived from the virus particles with a pharmaceutical carrier to form the pharmaceutical composition.   
     
     
         22 . A method for producing a pharmaceutical composition for generating immunological protection against infection of a subject with an influenza virus comprising preparing a composition comprising:
 (a) transfecting a cell with at least one nucleic acid, wherein the at least one nucleic acid is chosen from:   (1) an influenza gene segment and a bacteriophage polymerase promoter; and   (2) a complement of an influenza gene segment and a bacteriophage polymerase promoter;   wherein at least one nucleic acid comprises a T7 bacteriophage polymerase promoter and at least one additional guanine residue next to the bacteriophage polymerase promoter, and   (b) culturing the transfected cell to form virus particles;   (c) combining the virus particles or viral proteins derived from the virus particles with a pharmaceutical carrier to form the pharmaceutical composition.   
     
     
         23 . A method for generating immunological protection against infection of a subject with an influenza virus comprising administering to a subject in need thereof a pharmaceutical composition comprising virus particles or viral proteins derived from the virus particles prepared by:
 (a) transfecting a cell with at least one nucleic acid, wherein said nucleic acid is chosen from:   (1) an influenza gene segment and a bacteriophage polymerase promoter; and   (2) a complement of an influenza gene segment and a bacteriophage polymerase promoter; and   (b) culturing the transfected cell to form virus particles;   (c) combining the virus particles or viral proteins derived from the virus particles with a pharmaceutical carrier to form the pharmaceutical composition.   
     
     
         24 . A replicative influenza virus particle produced, without the use of helper virus, by the method comprising:
 (a) transfecting a cell with at least one nucleic acid, wherein said nucleic acid is chosen from:   (1) an influenza gene segment and a bacteriophage polymerase promoter; and   (2) a complement of an influenza gene segment and a bacteriophage polymerase promoter; and   (b) culturing the transfected cell.   
     
     
         25 . A cell transfected with at least one nucleic acid, wherein said nucleic acid is chosen from:
 (1) an influenza gene segment and a bacteriophage polymerase promoter; and   (2) a complement of an influenza gene segment and a bacteriophage polymerase promoter; and   (3) an influenza gene segment or its complement, a T7 bacteriophage polymerase promoter, and at least one additional guanine residue next to said promoter   
     
     
         26 . The cell according to  claim 25 , wherein said cell further comprises a T7 bacteriophage polymerase. 
     
     
         27 . The cell according to  claim 26 , wherein said bacteriophage polymerase comprises a nuclear localization signal. 
     
     
         28 . The cell according to  claim 25 , wherein said cell comprises a non-primate cell. 
     
     
         29 . The cell according to  claim 26 , wherein said cell is chosen from a MDCK cell and a CEF cell. 
     
     
         30 . The cell according to  claim 25 , wherein said cell lacks a helper virus. 
     
     
         31 . A composition comprising virus particles or viral protein derived from the virus particles prepared without the use of helper virus, by the method comprising:
 (a) transfecting a cell with at least one nucleic acid, wherein said nucleic acid is chosen from:   (1) an influenza gene segment and a bacteriophage polymerase promoter; and   (2) a complement of an influenza gene segment and a bacteriophage polymerase promoter; and   (b) culturing the transfected cell.   
     
     
         32 . A nucleic acid comprising:
 an influenza gene segment or its complement, a T7 bacteriophage polymerase promoter, and at least one additional guanine residue next to said promoter.   
     
     
         33 . The nucleic acid according to  claim 32 , wherein said nucleic acid comprises at least two additional guanine residues next to said promoter. 
     
     
         34 . The nucleic acid according to  claim 32 , wherein the influenza gene segment is derived from an influenza virus recommended by WHO for vaccine purposes. 
     
     
         35 . The nucleic acid according to  claim 32 , wherein the influenza gene segment comprises an influenza A gene segment. 
     
     
         36 . The method according to  claim 21 , wherein said cell is transfected with at least seven or eight nucleic acids used in said method. 
     
     
         37 . The method according to  claim 21 , wherein said nucleic acids are respectively comprised in bidirectional plasmids. 
     
     
         38 . The method according to  claim 22 , wherein said cell is transfected with at least seven or eight nucleic acids used in said method. 
     
     
         39 . The method according to  claim 22 , wherein said nucleic acids are respectively comprised in bidirectional plasmids. 
     
     
         40 . The method according to  claim 23 , wherein said cell is transfected with at least seven or eight nucleic acids used in said method. 
     
     
         41 . The method according to  claim 23 , wherein said nucleic acids are respectively comprised in bidirectional plasmids. 
     
     
         42 . The replicative influenza virus particle according to  claim 24 , wherein said cell is transfected with at least seven or eight nucleic acids used in said method. 
     
     
         43 . The replicative influenza virus particle according to  claim 24 , wherein said nucleic acids are respectively comprised in bidirectional plasmids. 
     
     
         44 . The cell according to  claim 25 , wherein said cell is transfected with at least seven or eight nucleic acids used in said method. 
     
     
         45 . The cell according to  claim 25 , wherein said nucleic acids are respectively comprised in bidirectional plasmids. 
     
     
         46 . The composition according to  claim 31 , wherein said cell is transfected with at least seven or eight nucleic acids used in said method. 
     
     
         47 . The composition according to  claim 31 , wherein said nucleic acids are respectively comprised in bidirectional plasmids. 
     
     
         48 . The nucleic acid according to  claim 32 , wherein said nucleic acid is comprised in a bidirectional plasmid.