Helper-free Rescue of Recombinant Negative Strand RNA Virus Systems

Abstract

The present invention relates methods of generating infectious negative-strand virus in host cells by an entirely vector-based system without the aid of a helper virus. In particular, the present invention relates methods of generating infectious recombinant negative-strand RNA viruses intracellularly in the absence of helper virus from expression vectors comprising cDNAs encoding the viral proteins necessary to form ribonucleoprotein complexes (RNPs) and expression vectors comprising cDNA for genomic viral RNA(s) (vRNAs) or the corresponding cRNA(s). The present invention also relates to methods of generating infectious recombinant negative-strand RNA viruses which have mutations in viral genes and/or which express, package and/or present peptides or polypeptides encoded by heterologous nucleic acid sequences. The present invention further relates the use of the recombinant negative-strand RNA viruses or chimeric negative-strand RNA viruses of the invention in vaccine formulations and pharmaceutical compositions.

1 - 30 . (canceled) 
     
     
         31 . A method for producing a population of infectious influenza virus particles for preparation of a vaccine, comprising:
 growing a selected reassortant influenza virus to produce a population of recombinant infectious virus particles and recovering said population of infectious influenza virus particles,   wherein said reassortant influenza virus was produced in cultured cells in the absence of a helper virus by expression vectors that direct the expression of genomic vRNA or antigenomic vRNA (cRNA) segments derived from at least two different strains of influenza virus to produce a virus particle having a selected genome and that is a reassortant of said at least two different strains   wherein substantially all of the infectious influenza virus particles of the resulting population contain said selected genome.   
     
     
         32 . The method of  claim 31  wherein one or more further expression vectors were employed in said cells to express one or more proteins selected from nucleoprotein and the subunits of RNA-dependent RNA polymerase. 
     
     
         33 . The method of  claim 31  wherein said cultured cells are capable of expressing one or more of nucleoprotein and the subunits of RNA-dependent RNA polymerase. 
     
     
         34 . The method of  claim 31  wherein said grown virus is an influenza virus of type A, B or C. 
     
     
         35 . The method of  claim 31  wherein said cultured cells were selected from Vero cells and other cells which are deficient in interferon activity and capable of supporting growth of said virus. 
     
     
         36 . The method of  claim 31  wherein said expression vectors were capable of directly expressing genomic vRNA segments of said virus. 
     
     
         37 . The method of  claim 31  wherein said growing of reassortant influenza virus to produce virus particles occurs in an egg. 
     
     
         38 . The method of  claim 31  further comprising a viral attenuation step. 
     
     
         39 . The method of  claim 31  further comprising a viral killing step. 
     
     
         40 . The method of  claim 31  wherein all the required expression vectors were cotransfected into said cells by use of a liposomal transfection reagent, calcium phosphate precipitation, or electroporation. 
     
     
         41 . The method of  claim 31  wherein said expression vectors were all plasmids. 
     
     
         42 . The method of  claim 31  wherein each vRNA segment of said virus or the corresponding cRNAs was present in a separate expression vector. 
     
     
         43 . The method of  claim 31  wherein the expression of each vRNA segment or cRNA was under the control of a promoter sequence derived from a mammalian Pol I promoter. 
     
     
         44 . The method of  claim 43  wherein said promoter sequence was a truncated human Pol I promoter sequence consisting of nucleotides −250 to −1 of the corresponding native promoter or a functional derivative thereof. 
     
     
         45 . The method of  claim 31  wherein the coding sequence for each vRNA segment or cRNA in said expression vectors was followed by a ribozyme sequence or transcription terminator to ensure a correct 3′ end of each said RNA. 
     
     
         46 . The method of  claim 32  wherein expression of one or more viral proteins from said further expression vectors was under the control of a regulatory sequence selected from the adenovirus 2 major late promoter linked to the spliced tripartite leader sequence of human adenovirus type 2 or the human cytomegalovirus immediate-early promoter, or a functional derivative of said regulatory sequence. 
     
     
         47 . The method of  claim 33  wherein said component was a viral nucleoprotein. 
     
     
         48 . A method for producing an influenza virus comprising:
 (a) producing in a cell all of the genomic RNA of an influenza virus by introducing into the cell DNA encoding at least one of said genomic RNA or cRNA corresponding to said genomic RNA of the virus, and   (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.   
     
     
         49 . A method for producing a population of infectious influenza virus particles for preparation of a vaccine, comprising:
 growing a selected reassortant influenza virus to produce a population of recombinant infectious virus particles comprising the HA-encoding and NA-encoding vRNA segments of a first influenza strain and the non-HA and non-NA encoding vRNA segments of at least one influenza strain different from said first influenza strain and recovering said population of infectious influenza virus particles,   wherein the infectious influenza virus particles of the resulting population contain substantially the same genome genome.   
     
     
         50 . The population of infectious influenza virus particles produced by the method of  claim 49 .