Recombinant influenza viruses for vaccines and gene therapy

Abstract

The invention provides compositions and methods useful to prepare segmented, negative strand RNA viruses, e.g., orthomyxoviruses such as influenza A viruses, entirely from cloned cDNAs and in the absence of helper virus.

1 - 100 . (canceled) 
     
     
         101 . A method for producing influenza virus particles for preparation of a vaccine, comprising:
 growing reassortant influenza virus to produce virus particles and recovering influenza virus particles produced by said growing for preparation of a vaccine, said reassortant influenza virus being a recovered reassortant influenza virus that was produced in cultured cells by introducing into cultured cells expression vectors which direct the expression of the genomic vRNA segments or antigenomic cRNA segments of said genomic vRNA segments, said cells providing a nucleoprotein, and an RNA dependent RNA polymerase so that ribonucleoprotein complexes containing the genomic vRNA segments of said reassortant influenza virus can be formed and wherein said reassortant influenza virus can be assembled within said cells in the absence of helper virus and wherein said cells were cultured so that said reassortant influenza virus was produced.   
     
     
         102 . The method of  claim 101 , wherein the cultured cells are from a cell line which comprises expression vectors which direct expression of said nucleoprotein and the subunits of said RNA-dependent RNA polymerase. 
     
     
         103 . The method of  claim 101 , wherein said virus is an influenza virus of type A, B or C. 
     
     
         104 . The method of  claim 101 , wherein said reassortant influenza virus contains vRNA segments derived from more than one parent virus. 
     
     
         105 . The method of  claim 101 , wherein said expression vectors were capable of directly expressing genomic vRNA segments of said virus. 
     
     
         106 . The method of  claim 101 , wherein said growing of reassortant influenza virus to produce virus particles occurs in an egg. 
     
     
         107 . The method of  claim 101  further comprising attenuating said virus particles. 
     
     
         108 . The method of  claim 101 , wherein all of said expression vectors are introduced by transfection into said cells by use of a liposomal transfection reagent. 
     
     
         109 . The method of  claim 101 , wherein said expression vectors were all plasmids. 
     
     
         110 . The method of  claim 101 , wherein each genomic vRNA segment of said virus or the corresponding antigenomic cRNAs is encoded on a separate expression vector. 
     
     
         111 . The method of  claim 101 , wherein the expression of each genomic vRNA segment or antigenomic cRNA is under the control of a promoter sequence which is a mammalian RNA polymerase I promoter. 
     
     
         112 . The method of  claim 111 , wherein said mammalian RNA polymerase I promoter sequence is a human RNA polymerase I promoter. 
     
     
         113 . The method of  claim 101 , wherein a coding sequence for each genomic vRNA segment or antigenomic cRNA in said expression vectors is upstream of transcription terminator. 
     
     
         114 . The method of  claim 102 . wherein said component was a viral nucleoprotein. 
     
     
         115 . The method of  claim 114 , wherein said cell is a 293 T cell. 
     
     
         116 . A method for producing an influenza virus comprising:
 (a) producing in a cell all of the genomic vRNA of an influenza virus by introducing into the cell DNA encoding at least one of said genomic vRNA or antigenomic cRNA corresponding to said genomic vRNA of the virus,   (b) producing in the cell RNA dependent polymerase and nucleoprotein and assembling in the cell the influenza virus, said cell being free of helper virus.