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 . A host cell, the genome of which is stably augmented with at least one of the following:
 a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second loxP site linked to an influenza virus HA coding region;   a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus NA coding region;   a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus M1 coding region;   a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising atranscription stop sequence linked to a second lox site linked to an influenza virus NS2 coding region;   a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus M2 coding region;   a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus PA coding region;   a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus PB1 coding region;   a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus PB2 coding region; or   a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus NP coding region.   
     
     
         2 . The host cell of  claim 1  which is augmented with a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus HA coding region; a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus NA coding region; a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus M1 coding region; a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus NS2 coding region; and a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus M2 coding region. 
     
     
         3 . The host cell of  claim 1  wherein the cell is augmented with a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus NS2 coding region. 
     
     
         4 . The host cell of  claim 1  wherein the lox sites are loxP sites. 
     
     
         5 . A method to prepare infectious replication defective influenza virus, comprising:
 i) contacting the host cell of  claim 1  with a recombinant influenza virus comprising: vRNA comprising a Cre open reading frame, and vRNA comprising influenza genes not present in the genome of the host cell; and   ii) recovering virus from the host cell.   
     
     
         6 . A method to prepare infectious replication defective influenza virus, comprising:
 i) contacting the host cell of  claim 3  with a recombinant influenza virus comprising: vRNA comprising a Cre open reading frame, vRNA comprising PA, vRNA comprising PB1, vRNA comprising PB2, vRNA comprising NP, vRNA comprising HA, vRNA comprising NA, vRNA comprising M1, and vRNA comprising M2; and   ii) recovering virus from the host cell.   
     
     
         7 . A method to prepare infectious replication defective influenza virus, comprising:
 i) contacting the host cell of  claim 3  with a recombinant influenza virus comprising: vRNA comprising a Cre open reading frame, vRNA comprising PA, vRNA comprising PB1, vRNA comprising PB2, vRNA comprising NP, vRNA comprising HA, vRNA comprising NA, and vRNA comprising M; and   ii) recovering virus from the host cell.   
     
     
         8 . A method to prepare infectious replication defective virus, comprising:
 i) contacting the host cell of  claim 1  with a vector comprising a promoter functional in the host cell operably linked to a DNA segment encoding Cre, and a plurality of vectors each comprising a promoter operably linked to an influenza gene not expressed by the host cell; and   ii) recovering virus from the host cell.   
     
     
         9 . The method of  claim 5  wherein the recombinant virus further comprises vRNA comprising a desired open reading frame. 
     
     
         10 . The method of  claim 8  wherein the plurality of vectors further comprises a vector comprising a promoter linked to the 5′ non-coding region of influenza virus linked to a desired open reading frame linked to the 3′ noncoding region of influenza virus. 
     
     
         11 . The method of  claim 6  wherein the recombinant virus further comprises vRNA comprising a desired open reading frame. 
     
     
         12 . A host cell, the genome of which is augmented with a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to a host cell surface binding protein coding region; a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to a fusion protein coding region; a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus M1 coding region; a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus NS2 coding region; and a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus M2 coding region. 
     
     
         13 . The host cell of  claim 12  wherein the host cell surface binding protein is HA. 
     
     
         14 . The host cell of  claim 12  wherein the fusion protein is NA. 
     
     
         15 . The host cell of  claim 12  wherein the lox sites are loxP sites. 
     
     
         16 . A host cell, the genome of which is augmented with a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to a host cell surface binding and fusion protein coding region; a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second loxP site linked to an influenza virus M1 coding region; a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first lox site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus NS2 coding region; and a recombinant DNA molecule comprising a promoter functional in the host cell linked to a first loxP site linked to a DNA segment comprising a transcription stop sequence linked to a second lox site linked to an influenza virus M2 coding region. 
     
     
         17 . The host cell of  claim 16  wherein the lox sites are loxP sites. 
     
     
         18 . A method to prepare infectious replication defective virus, comprising:
 i) contacting the host cell of  claim 16  with a recombinant influenza virus comprising: vRNA comprising a Cre open reading frame, vRNA comprising PA, vRNA comprising NP, vRNA comprising PB 1, and vRNA comprising PB2; and   ii) recovering virus from the host cell.   
     
     
         19 . The method of  claim 18  wherein the recombinant virus further comprises vRNA comprising a desired open reading frame.