Replication-Deficient RNA Viruses as Vaccines

Abstract

The present invention relates to a genome-replication-deficient and transcription-competent negative-strand RNA virus, which can be used for the expression of transgenes and in particular for the area of vaccine development.

1 . A recombinant negative-strand RNA virus, containing a viral genome with a mutation in at least one of the genes L and P, which leads to loss of capacity for replication without loss of capacity for secondary transcription. 
     
     
         2 . The virus as claimed in  claim 1 , characterized in that it is a paramyxovirus. 
     
     
         3 . The virus as claimed in  claim 1 , characterized in that it is a Sendai virus. 
     
     
         4 . The virus as claimed in  claim 1 , characterized in that it has a mutation in gene P. 
     
     
         5 . The virus as claimed in  claim 4 , characterized in that the mutation relates to the N-terminal partial sequence of the protein encoded by gene P. 
     
     
         6 . The virus as claimed in  claim 5 , characterized in that the mutation comprises: a deletion of
 (a) amino acids 2-77 of the protein encoded by gene P or   (b) a partial sequence of (a) sufficient for loss of the capacity for replication.   
     
     
         7 . The virus as claimed in  claim 1 , characterized in that the viral genome contains at least one sequence coding for a heterologous gene product. 
     
     
         8 . The virus as claimed in  claim 7 , characterized in that the heterologous gene product is a protein, a ribozyme, an antisense molecule or an siRNA molecule. 
     
     
         9 . The virus as claimed in  claim 7 , characterized in that the heterologous gene product is a reporter protein, an antigen or a therapeutic protein. 
     
     
         10 . The virus as claimed in  claim 7 , characterized in that the heterologous gene product is an antigen of a heterologous pathogen, selected from viruses, bacteria and protozoa. 
     
     
         11 . The virus as claimed in  claim 7 , characterized in that the heterologous gene product is a viral antigen. 
     
     
         12 . The virus as claimed in  claim 11 , characterized in that the viral genome codes for several heterologous antigens from the same or different viruses. 
     
     
         13 . The virus as claimed in  claim 7 , characterized in that the sequence coding for at least one heterologous gene product is inserted in the viral genome and/or replaces sequences coding for a homologous gene product. 
     
     
         14 . The virus as claimed in  claim 1 , characterized in that the virus has a capacity for transcription that is reduced by at most a factor of 20 relative to the wild-type virus. 
     
     
         15 . A nucleocapsid of a negative-strand RNA virus as claimed in  claim 1 . 
     
     
         16 . A genome of a negative-strand RNA virus as claimed in  claim 1 . 
     
     
         17 . A DNA molecule that codes for the genome and/or antigenome of a recombinant negative-strand RNA virus as claimed in  claim 1 . 
     
     
         18 . (canceled) 
     
     
         19 . The DNA molecule as claimed in  claim 18 , characterized in that the transcription signal is a bacteriophage promoter, e.g. a T7 or SP6 promoter. 
     
     
         20 . A cell that contains a virus as claimed in  claim 1 , a nucleocapsid as claimed in  claim 15 , a genome as claimed in  claim 16  or a DNA molecule as claimed in  claim 17 . 
     
     
         21 - 22 . (canceled) 
     
     
         23 . The cell as claimed in claim  22 , characterized in that it further contains a DNA molecule coding for a heterologous DNA-dependent RNA polymerase, which effects the transcription of the DNA molecule coding for the recombinant negative-strand RNA virus. 
     
     
         24 . The cell as claimed in  claim 20 , characterized in that it is a virus multiplying cell. 
     
     
         25 . The cell as claimed in  claim 20 , characterized in that it further contains DNA molecules coding for the viral L and/or P protein. 
     
     
         26 . A method of production of a negative-strand RNA virus as claimed in  claim 1 , comprising the steps:
 (a) preparation of a cell that is transfected with a DNA molecule that codes for the genome of a negative-strand RNA virus, containing a mutation in at least one of the genes L and P, which leads to loss of the capacity for viral genome replication without loss of the capacity for secondary transcription, and optionally at least one sequence coding for a heterologous gene product, and   (b) cultivation of the cell under conditions such that a transcription of the DNA according to (a) takes place and the recombinant negative-strand RNA virus is formed.   
     
     
         27 . The method as claimed in  claim 26 , further comprising the obtaining of the nucleocapsid or of the viral genome from the negative-strand RNA virus. 
     
     
         28 . A method of multiplying a negative-strand RNA virus as claimed in  claim 1 , comprising the steps:
 (a) preparation of a cell that is infected with a negative-strand RNA virus, containing a mutation in at least one of the genes L and P, which leads to loss of the capacity for viral genome replication without loss of the capacity for secondary transcription, and optionally at least one sequence coding for a heterologous gene product, and   (b) cultivation of the cell under conditions such that multiplication of the virus takes place.   
     
     
         29 . A pharmaceutical composition, characterized in that it contains a recombinant negative-strand RNA virus as claimed in  claim 1 , a nucleocapsid as claimed in  claim 15  or a viral genome as claimed in  claim 16  as active substance and optionally pharmaceutically acceptable vehicles and/or excipients. 
     
     
         30 - 35 . (canceled) 
     
     
         36 . The use of a cell which stably expresses, constitutively or inducibly, the proteins L and/or P of a negative-strand RNA virus, for the production or multiplication of recombinant negative-strand RNA viruses as claimed in  claim 1 , of nucleocapsids as claimed in  claim 15  or of viral genomes as claimed in  claim 16 . 
     
     
         37 . (canceled) 
     
     
         38 . The use as claimed in  claim 36 , characterized in that the cell is selected from the H29 cell (DSM ACC2702) or a cell derived therefrom.