Recombinant negative strand RNA virus expression systems and vaccines

Abstract

Recombinant negative-strand viral RNA templates are described which may be used with purified RNA-directed RNA polymerase complex to express heterologous gene products in appropriate host cells and/or to rescue the heterologous gene in virus particles. The RNA templates are prepared by transcription of appropriate DNA sequences with a DNA-directed RNA polymerase. The resulting RNA templates are of the negative-polarity and contain appropriate terminal sequences which enable the viral RNA-synthesizing apparatus to recognize the template. Bicistronic mRNAs can be constructed to permit internal initiation of translation of viral sequences and allow for the expression of foreign protein coding sequences from the regular terminal initiation site, or vice versa.

1 - 34 . (Cancelled).  
     
     
         35 . A method for rescuing a recombinant negative strand RNA virus comprising: 
 (a) introducing into a 293 cell expression vectors which direct the expression in said cells of genomic or antigenomic vRNA segments, and a nucleoprotein, and an RNA-dependent polymerase, so that ribonucleoprotein complexes can be formed and viral particles can be assembled in the absence of helper virus; and    (b) culturing said cells wherein viral particles are packaged and rescued.    
     
     
         36 . The method of  claim 35  wherein the recombinant negative strand RNA virus is a segmented virus.  
     
     
         37 . The method of  claim 36  wherein the negative strand RNA virus is  influenza.    
     
     
         38 . A method for generating in cultured cells infectious viral particles of a segmented negative-strand RNA virus having greater than 3 genomic vRNA segments, said method comprising: 
 (a) introducing into cultured cells expression vectors which direct the expression of the genomic or antigenomic vRNA segments of said virus, and a nucleoprotein, and an RNA dependent polymerase so that RNP complexes containing the genomic vRNA segments of said virus can be formed and said viral particles, can be assembled within said cells in the absence of helper virus; and    (b) culturing said cells wherein said viral particles are produced.    
     
     
         39 . The method of  claim 38  wherein one or more further expression vectors are employed in said cells to express one or more proteins selected from said nucleoprotein and the subunits of said RNA-dependent RNA polymerase.  
     
     
         40 . The method of  claim 38  wherein a cell line is employed which contains expression vectors which direct expression of one or more of said nucleoprotein and the subunits of said RNA-dependent RNA polymerase.  
     
     
         41 . The method of claims  38 ,  39  or  40  wherein said virus is an  influenza  virus of type A, B or C.  
     
     
         42 . The method of  claim 38  wherein said virus is a reassortant virus having vRNA segments derived from more than one parent virus  
     
     
         43 . The method of  claim 38  wherein said expression vectors direct expression of genomic vRNA segments of said virus.  
     
     
         44 . The method of  claim 38  which further comprises amplifying viral particles produced by said cells by one or more further cellular infection steps employing cells which are the same or different from said first population of cells.  
     
     
         45 . The method of  claim 38  which further comprises isolating infectious viral particles.  
     
     
         46 . The method of  claim 38  which further comprises a viral attenuation or killing step.  
     
     
         47 . The method of  claim 38  wherein said expression vectors are all plasmids.  
     
     
         48 . The method of  claim 38  wherein said expression vectors consist of a separate expression vector for expression of each vRNA segment of said virus or the corresponding cRNAs.  
     
     
         49 . A method for rescuing a chimeric recombinant negative strand RNA virus, wherein said chimeric virus expresses heterologous nucleic acid sequences, comprising: 
 (a) introducing into a 293 cell, expression vectors which direct the expression in said cells of genomic or antigenomic vRNA segments, and a nucleoprotein, and an RNA-dependent RNA polymerase, so that ribonucleoprotein complexes can be formed and viral particles can be assembled in the absence of helper virus; and    (b) culturing said cells wherein viral particles are packaged and rescued.    
     
     
         50 . The method of  claim 49 , wherein said heterologous nucleic acid sequences are inserted into a DNA complement of a negative strand RNA virus gene, such that said heterologous nucleic acid sequences are flanked by the viral polymerase binding site, and a polyadenylation site.  
     
     
         51 . The method of  claim 49 , wherein oligonucleotides encoding the viral polymerase binding site of the negative strand RNA virus are ligated to said heterologous nucleic acid sequences.  
     
     
         52 . The method of  claim 49 , wherein the chimeric recombinant negative strand RNA virus is a segmented virus.  
     
     
         53 . The method of  claim 52 , wherein the segmented RNA virus is  influenza.    
     
     
         54 . The method of  claim 49 , wherein said heterologous nucleic acid sequences are derived from human immunodeficiency virus (HIV).  
     
     
         55 . The method of  claim 49 , wherein said pathogenic antigens are hepatitis B surface antigen, glycoproteins of herpes virus, or VP1 protein of poliovirus.  
     
     
         56 . The method of  claim 49 , wherein said non-viral pathogens are bacteria or parasites.  
     
     
         57 . The method of  claim 49 , wherein said heterologous nucleic acid sequences encode viral genes from different strains of the negative strand RNA virus.  
     
     
         58 . The method of  claim 49 , wherein said heterologous nucleic acid sequences are antisense nucleic acids.  
     
     
         59 . The chimeric recombinant negative strand RNA virus produced by the method of  claim 49 .  
     
     
         60 . A method for generating in cultured cells infectious viral particles of a chimeric negative strand RNA virus, wherein said chimeric virus expresses heterologous nucleic acid sequences and has greater than 3 genomic vRNA segments, comprising: 
 (a) introducing into cultured cells expression vectors which direct the expression of genomic or antigenomic vRNA segments of said chimeric virus, and a nucleoprotein, and an RNA dependent RNA polymerase so that RNP complexes containing the genomic vRNA segments of said chimeric virus can be formed and said viral particles can be assembled within said cells in the absence of helper virus; and    (b) culturing said cells wherein said viral particles are produced.    
     
     
         61 . The method of  claim 60 , wherein one or more further expression vectors are employed in said cells to express one or more proteins selected from said nucleoprotein and the subunits of said RNA-dependent polymerase.  
     
     
         62 . The method of  claim 60 , wherein a cell line is employed which contains expression vectors which direct expression of one or more of said nucleoprotein and the subunits of said RNA-dependent RNA polymerase.  
     
     
         63 . The method of  claim 60 ,  61 , or  62  wherein said virus is an  influenza  virus of type A, B or C.  
     
     
         64 . The method of  claim 60 , wherein said expression vectors direct expression of genomic vRNA segments of said chimeric virus.  
     
     
         65 . The method of  claim 60  which further comprises amplifying viral particles produced by said cells by one or more further cellular infection steps employing cells which are the same or different from said first population of cells.  
     
     
         66 . The method of  claim 60 , which further comprises isolating infectious viral particles.  
     
     
         67 . The method of  claim 60 , which further comprises a viral attenuation or killing step.  
     
     
         68 . The method of  claim 60 , wherein said expression vectors are all plasmids.  
     
     
         69 . The method of  claim 60 , wherein said expression vectors consist of a separate expression vector for expression of each vRNA segment of said chimeric virus or the corresponding cRNAs.  
     
     
         70 . The method of  claim 60 , wherein said heterologous nucleic acid sequences are engineered into the expression vectors directing the expression of the vRNA segments of said chimeric virus.  
     
     
         71 . The method of  claim 53  which further comprises amplifying viral particles produced by said first population of cells by one or more further cellular infection steps employing cells which are the same or different from said first population of cells.  
     
     
         72 . The method of  claim 53  which further comprises isolating infectious viral particles.  
     
     
         73 . The method of  claim 53  which further comprises an attenuation or viral killing step.  
     
     
         74 . The method of  claim 53  which further comprises incorporating attenuated or killed vial particles into a vaccine composition.  
     
     
         75 . A vaccine formulation comprising the chimeric recombinant negative strand RNA virus produced by the method of  claim 49 .  
     
     
         76 . A vaccine formulation comprising the chimeric recombinant negative strand RNA virus produced by the method of  claim 49 , wherein said heterologous nucleic acid sequences are derived from human immunodeficiency virus.  
     
     
         77 . A vaccine formulation comprising the chimeric recombinant negative strand RNA virus produced by the method of  claim 49 , wherein said pathogenic antigen is hepatitis B surface antigen, glycoprotein of herpes virus, or VP1 protein of poliovirus.  
     
     
         78 . The vaccine formulation comprising the chimeric recombinant negative strand RNA virus produced by the method of  claim 49 , wherein said virus is  influenza.    
     
     
         79 . A pharmaceutical composition comprising, the chimeric recombinant negative strand RNA virus produced by the method of  claim 49 , and a pharmaceutically acceptable carrier.