Recombinant influenza viruses for vaccines and gene therapy
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. A method for producing influenza virus particles for preparation of a vaccine, comprising:
(a) introducing expression vectors into cultured cells in the absence of helper virus, wherein said expression vectors comprise influenza genomic vRNA segments or antigenomic cRNAs of said genomic vRNA segments, sufficient to produce in said cells the genomic vRNA segments or antigenomic cRNAs of said genomic vRNA segments;
(b) introducing into said cultured cells protein expression vectors encoding a nucleoprotein and RNA-dependent RNA polymerase subunits, wherein ribonucleoprotein complexes (RNPs) containing the genomic vRNA segments of said influenza viruses are formed; and
(c) recovering said influenza virus particles.
2. The method of claim 1 , wherein said cultured cells are from a cell line containing protein expression vectors which express said nucleoprotein and said RNA-dependent RNA polymerase subunits.
3. The method of claim 1 , wherein said influenza virus is an influenza virus of type A, B or C.
4. The method of claim 1 , wherein said expression vectors comprise genomic vRNA segments.
5. The method of claim 1 , wherein said influenza virus particles are recovered from an egg.
6. The method of claim 1 , wherein the expression vectors are introduced by transfection into said cells in the presence of a liposomal transfection reagent.
7. The method of claim 1 , wherein said expression vectors are plasmids.
8. The method of claim 1 , wherein said genomic vRNA segments or said antigenomic cRNAs of said genomic vRNA segments are encoded on separate expression vectors.
9. The method of claim 1 , wherein said genomic vRNA segments or antigenomic cRNAs of said genomic vRNA segments are expressed under the control of a mammalian RNA polymerase I promoter.
10. The method of claim 1 , wherein said genomic vRNA segments or antigenomic cRNAs of said genomic vRNA segments in said expression vectors are followed by a transcription terminator.
11. The method of claim 1 , wherein said nucleoprotein and RNA-dependent RNA polymerase subunits are expressed under the control of a cytomegalovirus promoter.
12. The method of claim 1 , wherein said influenza viruses are at least 99% free from contaminating viruses.
13. A method for producing influenza virus particles for preparation of a vaccine, the method comprising:
producing genomic RNA of an influenza virus in cells by introducing influenza genomic vRNA segments or antigenomic cRNAs of said genomic vRNA segments into cells in the absence of helper virus, wherein said cells comprises exogenously-introduced nucleic acid encoding RNA dependent RNA polymerase subunits and nucleoprotein capable of expressing these proteins thereby producing RNA dependent RNA polymerase subunits and nucleoprotein in said cells; and assembling influenza virus in said cells to produce influenza viral particles, wherein the cells support viral growth.
14. The method of claim 13 , wherein said influenza virus is an influenza virus of type A, B or C.
15. A method for producing influenza virus particles for preparation of a vaccine, the method comprising:
(a) growing influenza viral particles in cultured cells in the absence of helper virus, wherein said cultured cells comprise:
i) expression vectors comprising influenza genomic vRNA segments or antigenomic cRNAs of said genomic vRNA segments that encode hemagglutinin (HA), neuraminidase (NA), or genomic vRNA segments or antigenomic cRNAs of said genomic vRNA segments that encode a foreign immunogenic peptide or protein;
ii) a protein expression vector encoding a nucleoprotein; and
iii) protein expression vectors encoding RNA-dependent RNA polymerase subunits, wherein ribonucleoprotein complexes (RNPs) containing the genomic vRNA segments of said influenza viruses are formed; and
(b) recovering said influenza virus particles.