Recombinant influenza viruses for vaccines and gene therapy

Abstract

The invention provides a composition useful to prepare influenza A viruses, e.g., in the absence of helper virus.

1 . An expression plasmid comprising an RNA polymerase I (pol I) promoter, a pol I terminator sequence, and a influenza nucleic acid segment which is selected from the group consisting of PA, PB1, PB2, NP, NA, M, M1, M2, and NS2.  
     
     
         2 . A plasmid-based system for the generation of orthomyxoviruses from cloned viral cDNA comprising a plurality of plasmids sufficient to produce said viruses, said plasmids comprising; 
 (a) plasmids comprising cDNA corresponding to viral genomic segments, and    (b) plasmids comprising cDNA encoding viral polypeptides.    
     
     
         3 . The plasmid-based system of  claim 2 , wherein the viral genomic segment is selected from the group consisting of PA, PB1, PB2, NP, HA, NA, M, and NS2.  
     
     
         4 . The plasmid-based system of  claim 2 , wherein the viral polypeptide is selected from the group consisting of PA, PB1, PB2, NP, HA, NA, M, M1, M2, and NS2.  
     
     
         5 . A host cell comprising the plasmid-based system of  claim 2 .  
     
     
         6 . The host cell of  claim 5  comprising a plurality of plasmids having cDNA corresponding to viral genomic segments of an orthomyxovirus, and plasmids having cDNA encoding viral polypeptides of an orthomyxovirus, wherein the host cell is capable of producing an infectious orthomyxovirus in the absence of helper virus.  
     
     
         7 . The host cell of  claim 6  wherein the viral genomic segment is selected from the group consisting of PA, PB1, PB2, NP, HA, NA, M, and NS2.  
     
     
         8 . The host cell of  claim 6  wherein the viral polypeptide is selected from the group consisting of PA, PB1, PB2, NP, HA, NA, M, M1, M2, and NS2.  
     
     
         9 . A method for producing an orthomyxovirus virion comprising culturing the host cell of  claim 5  under conditions which permit the production of viral proteins and vRNA or cDNA.  
     
     
         10 . A method for preparing an orthomyxovirus-specific immunogenic composition comprising purifying a virion from a host cell having a plurality of plasmids comprising cDNA corresponding to viral genomic segments, and plasmids comprising cDNA encoding viral cDNA encoding viral polypeptides, wherein said plasmids are sufficient to produce said virion when introduced into a host cell.  
     
     
         11 . An immunogenic composition comprising an orthomyxovirus virion, wherein viral internal proteins of the virion are from a virus strain well adapted to grow in culture or from an attenuated strain, or both, and viral antigen proteins of the virion are from a pathogenic virus strain.  
     
     
         12 . An immunogenic composition comprising an orthomyxovirus virion produced in the absence of helper virus.  
     
     
         13 . An immunogenic composition comprising an orthomyxovirus virion produced in the absence of helper virus or an in vitro ribonucleoprotein complex.  
     
     
         14 . A method for immunizing a subject against an orthomyxovirus infection comprising administering the immunogenic composition of  claim 11  to the subject.