Recombinant newcastle disease virus RNA expression systems and vaccines

Abstract

This invention relates to genetically engineered Newcastle disease viruses and viral vectors which express heterologous genes or mutated Newcastle disease viral genes or a combination of viral genes derived from different strains of Newcastle disease virus. The invention relates to the construction and use of recombinant negative strand NDV viral RNA templates which may be used with viral RNA-directed RNA polymerase to express heterologous gene products in appropriate host cells and/or to rescue the heterologous gene in virus particles. In a specific embodiment of the invention, the heterologous gene product is a peptide or protein derived from the genome of a human immunodeficiency virus. The RNA templates of the present invention may be prepared by transcription of appropriate DNA sequences using any DNA-directed RNA polymerase such as bacteriophage T7, T3, SP6 polymerase, or eukaryotic polymerase I.

1 - 19 . (canceled) 
     
     
         20 . A vaccine formulation comprising a genetically engineered chimeric Newcastle disease virus, the genome of which encodes a heterologous epitope, and a physiologically acceptable excipient. 
     
     
         21 . The vaccine formulation of  claim 20 , wherein said genetically engineered chimeric Newcastle disease virus comprises a recombinant RNA molecule comprising a binding site for an RNA polymerase of a Newcastle disease virus and signals required for NDV mediated replication and transcription, operatively linked to a heterologous RNA sequence. 
     
     
         22 . The vaccine formulation of  claim 21 , wherein said binding site comprising the polymerase binding site and said signals required for NDV mediated replication and transcription are contained in the 3′ and 5′-noncoding flanking region of a Newcastle disease viral RNA genome. 
     
     
         23 . The vaccine formulation of  claim 22 , wherein said 3′ and 5′-noncoding flanking region has a viral sense sequence of: 
       
         
           
                 
                 
                 
               
                     
                   (3′) 
                   5′ ACCAAACAGA GAAUCCGUAA GGUACGUUAA 
                 
                     
                     
                 
                     
                     
                      AAAGCGAAGG AGCAAUUGAA GUCGCACGGG 
                 
                     
                     
                 
                     
                     
                      UAGAAGGUGU GAAUCUCGAG UGCGAGCCCG 
                 
                     
                     
                 
                     
                     
                      AAGCACAAAC UCGAGAAAGC CUUCUACCAA 
                 
                     
                     
                 
                     
                     
                      C 3′ 
                 
                     
                     
                 
                     
                   (5′) 
                   5′ CGACAAUCAC AUAUUAAUAG GCUCCUUUUC 
                 
                     
                     
                 
                     
                     
                      UGGCCAAUUG UAUCCUUGUU GAUUUAAUCA 
                 
                     
                     
                 
                     
                     
                      UACUAUGUUA GAAAAAAGUU GAACUCCGAC 
                 
                     
                     
                 
                     
                     
                      UCCUUAGGAC UCGAACUCGA ACUCAAAUAA 
                 
                     
                     
                 
                     
                     
                      AUGUCUUAGA AAAAGAUUGC GCACAGUUAU 
                 
                     
                     
                 
                     
                     
                      UCUUGAGUGU AGUCUUGUCA UUCACCAAAU 
                 
                     
                     
                 
                     
                     
                      CUUUGUUUGG U 3′ 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         24 . The vaccine formulation of  claim 20 , wherein said heterologous epitope is a viral antigen. 
     
     
         25 . The vaccine formulation of  claim 20 , wherein said heterologous epitope is an immunopotentiating protein. 
     
     
         26 . The vaccine formulation of  claim 20 , wherein said heterologous epitope is a tumor antigen. 
     
     
         27 . A vaccine formulation comprising a genetically engineered Newcastle disease virus containing modifications which result in an attenuated phenotype or enhanced immunogenicity, and a physiologically acceptable excipient. 
     
     
         28 . The vaccine formulation of  claim 27 , wherein said genetically engineered Newcastle disease virus comprises a recombinant RNA molecule comprising a binding site for an RNA polymerase of a Newcastle disease virus and signals required for NDV mediated replication and transcription, operatively linked to a Newcastle disease viral gene. 
     
     
         29 . The vaccine formulation of  claim 28 , wherein said binding site comprising the polymerase binding site and said signals required for NDV mediated replication and transcription are contained in the 3′ and 5′-noncoding flanking region of a Newcastle disease viral RNA genome. 
     
     
         30 . The vaccine formulation of  claim 29 , wherein said 3′ and 5′-noncoding flanking region has a viral sense sequence of: 
       
         
           
                 
                 
                 
               
                     
                   (3′) 
                   5′ ACCAAACAGA GAAUCCGUAA GGUACGUUAA 
                 
                     
                     
                 
                     
                     
                      AAAGCGAAGG AGCAAUUGAA GUCGCACGGG 
                 
                     
                     
                 
                     
                     
                      UAGAAGGUGU GAAUCUCGAG UGCGAGCCCG 
                 
                     
                     
                 
                     
                     
                      AAGCACAAAC UCGAGAAAGC CUUCUACCAA 
                 
                     
                     
                 
                     
                     
                      C 3′ 
                 
                     
                     
                 
                     
                   (5′) 
                   5′ CGACAAUCAC AUAUUAAUAG GCUCCUUUUC 
                 
                     
                     
                 
                     
                     
                      UGGCCAAUUG UAUCCUUGUU GAUUUAAUCA 
                 
                     
                     
                 
                     
                     
                      UACUAUGUUA GAAAAAAGUU GAACUCCGAC 
                 
                     
                     
                 
                     
                     
                      UCCUUAGGAC UCGAACUCGA ACUCAAAUAA 
                 
                     
                     
                 
                     
                     
                      AUGUCUUAGA AAAAGAUUGC GCACAGUUAU 
                 
                     
                     
                 
                     
                     
                      UCUUGAGUGU AGUCUUGUCA UUCACCAAAU 
                 
                     
                     
                 
                     
                     
                      CUUUGUUUGG U 3′ 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         31 . The vaccine formulation of  claim 27 , wherein said modification is derived from a naturally occurring mutant. 
     
     
         32 . An immunogenic composition comprising a genetically engineered chimeric Newcastle disease virus, the genome of which encodes a heterologous epitope. 
     
     
         33 . The immunogenic composition of  claim 32 , wherein said genetically engineered chimeric Newcastle disease virus comprises a recombinant RNA molecule comprising a binding site for an RNA polymerase of a Newcastle disease virus and signals required for NDV mediated replication and transcription, operatively linked to a heterologous RNA sequence. 
     
     
         34 . The immunogenic composition of  claim 33 , wherein said binding site comprising the polymerase binding site and said signals required for NDV mediated replication and transcription are contained in the 3′ and 5′-noncoding flanking region of a Newcastle disease viral RNA genome. 
     
     
         35 . The immunogenic composition of  claim 34 , wherein said 3′ and 5′-noncoding flanking region has a viral sense sequence of: 
       
         
           
                 
                 
                 
               
                     
                   (3′) 
                   5′ ACCAAACAGA GAAUCCGUAA GGUACGUUAA 
                 
                     
                     
                 
                     
                     
                      AAAGCGAAGG AGCAAUUGAA GUCGCACGGG 
                 
                     
                     
                 
                     
                     
                      UAGAAGGUGU GAAUCUCGAG UGCGAGCCCG 
                 
                     
                     
                 
                     
                     
                      AAGCACAAAC UCGAGAAAGC CUUCUACCAA 
                 
                     
                     
                 
                     
                     
                      C 3′ 
                 
                     
                     
                 
                     
                   (5′) 
                   5′ CGACAAUCAC AUAUUAAUAG GCUCCUUUUC 
                 
                     
                     
                 
                     
                     
                      UGGCCAAUUG UAUCCUUGUU GAUUUAAUCA 
                 
                     
                     
                 
                     
                     
                      UACUAUGUUA GAAAAAAGUU GAACUCCGAC 
                 
                     
                     
                 
                     
                     
                      UCCUUAGGAC UCGAACUCGA ACUCAAAUAA 
                 
                     
                     
                 
                     
                     
                      AUGUCUUAGA AAAAGAUUGC GCACAGUUAU 
                 
                     
                     
                 
                     
                     
                      UCUUGAGUGU AGUCUUGUCA UUCACCAAAU 
                 
                     
                     
                 
                     
                     
                      CUUUGUUUGG U 3′ 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         36 . The immunogenic composition of  claim 32 , wherein said heterologous epitope is a viral antigen. 
     
     
         37 . The immunogenic composition of  claim 32 , wherein said heterologous epitope is an immunopotentiating protein. 
     
     
         38 . The immunogenic composition of  claim 32 , wherein said heterologous epitope is a tumor antigen. 
     
     
         39 . An immunogenic composition comprising a genetically engineered Newcastle disease virus containing modifications which result in an attenuated phenotype or enhanced immunogenicity. 
     
     
         40 . The immunogenic composition of  claim 39 , wherein said genetically engineered Newcastle disease virus comprises a recombinant RNA molecule comprising a binding site for an RNA polymerase of a Newcastle disease virus and signals required for NDV mediated replication and transcription, operatively linked to a Newcastle disease viral gene. 
     
     
         41 . The immunogenic composition of  claim 40 , wherein said binding site comprising the polymerase binding site and said signals required for NDV mediated replication and transcription are contained in the 3′ and 5′-noncoding flanking region of a Newcastle disease viral RNA genome. 
     
     
         42 . The immunogenic composition of  claim 41 , wherein said 3′ and 5′-noncoding flanking region has a viral sense sequence of: 
       
         
           
                 
                 
                 
               
                     
                   (3′) 
                   5′ ACCAAACAGA GAAUCCGUAA GGUACGUUAA 
                 
                     
                     
                 
                     
                     
                      AAAGCGAAGG AGCAAUUGAA GUCGCACGGG 
                 
                     
                     
                 
                     
                     
                      UAGAAGGUGU GAAUCUCGAG UGCGAGCCCG 
                 
                     
                     
                 
                     
                     
                      AAGCACAAAC UCGAGAAAGC CUUCUACCAA 
                 
                     
                     
                 
                     
                     
                      C 3′ 
                 
                     
                     
                 
                     
                   (5′) 
                   5′ CGACAAUCAC AUAUUAAUAG GCUCCUUUUC 
                 
                     
                     
                 
                     
                     
                      UGGCCAAUUG UAUCCUUGUU GAUUUAAUCA 
                 
                     
                     
                 
                     
                     
                      UACUAUGUUA GAAAAAAGUU GAACUCCGAC 
                 
                     
                     
                 
                     
                     
                      UCCUUAGGAC UCGAACUCGA ACUCAAAUAA 
                 
                     
                     
                 
                     
                     
                      AUGUCUUAGA AAAAGAUUGC GCACAGUUAU 
                 
                     
                     
                 
                     
                     
                      UCUUGAGUGU AGUCUUGUCA UUCACCAAAU 
                 
                     
                     
                 
                     
                     
                      CUUUGUUUGG U 3′ 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         43 . The immunogenic composition of  claim 39 , wherein said modification is derived from a naturally occurring mutant.