Bacterial artificial chromosomes

Abstract

The invention relates to the use of a bacterial artificial chromosome (BAC) for the preparation of a vaccine, wherein the BAC comprises an inducible bacterial ori sequence for amplification of the BAC to more than 10 copies per bacterial cell. Plus a viral expression cassette comprising a cDNA of an attenuated RNA virus genome and comprising cis-regulatory elements for transcription of said viral cDNA in mammalian cells and for processing of the transcribed RNA into infectious viral RNA.

1 - 45 . (canceled) 
     
     
         46 . A method of preparing a vaccine against a positive-strand RNA virus comprising the steps of:
 a) providing a bacterial host transformed with a BAC (bacterial artificial chromosome) which comprises:
 an inducible bacterial ori sequence for amplification of said BAC to more than 10 copies per bacterial cell, and 
 a viral expression cassette comprising a cDNA of an attenuated positive-strand RNA virus genome and comprising cis-regulatory elements for transcription of said viral cDNA in mammalian cells and for processing of the transcribed RNA into infectious viral RNA, 
   b) amplifying the BAC by adding a compound which activates said inducible ori,   c) isolating the amplified BAC, and   d) formulating the BAC into a vaccine.   
     
     
         47 . The method according to  claim 46 , wherein said cDNA of the attenuated positive-strand RNA virus genome is a chimeric viral cDNA construct of a positive-strand RNA virus genome, wherein a heterologous DNA sequence has been inserted or wherein a native viral sequence has been deleted, truncated, or mutated. 
     
     
         48 . The method according to  claim 46 , wherein said viral expression cassette comprises: a cDNA of a positive-strand RNA virus genome, a RNA polymerase driven promoter preceding the 5′ end of said cDNA for initiating the transcription of said cDNA, and an element for RNA self-cleaving following the 3′ end of said cDNA for cleaving the RNA transcript of said viral cDNA at a set position. 
     
     
         49 . The method according to  claim 46 , wherein said viral expression cassette comprises a cDNA of a virus of the family Flaviviridae. 
     
     
         50 . The method according to  claim 46 , wherein said viral expression cassette comprises a cDNA of a virus of the genus Pestivirus. 
     
     
         51 . The method according to  claim 50 , wherein said virus of the genus Pestivirus is bovine viral diarrhea virus or classical swine fever virus. 
     
     
         52 . The method according to  claim 46 , wherein said bacterial artificial chromosome further comprises a yeast autonomously replicating sequence for shuttling to and maintaining said bacterial artificial chromosome in yeast. 
     
     
         53 . The method according to  claim 52 , wherein said yeast autonomously replicating sequence is the 2μ plasmid origin or the ARS1 (autonomously replicating sequence 1) or functionally homologous derivatives thereof. 
     
     
         54 . The method according to  claim 48 , wherein said RNA polymerase driven promoter is an RNA polymerase II promoter. 
     
     
         55 . The method according to  claim 54 , wherein said RNA polymerase II promoter is the Cytomegalovirus Immediate Early (CMV-IE) promoter, the Simian virus 40 promoter or functionally homologous derivatives thereof. 
     
     
         56 . The method according to  claim 48 , wherein said RNA polymerase driven promoter is an RNA polymerase I or III promoter. 
     
     
         57 . The method according to  claim 48 , wherein said element for RNA self-cleaving is the cDNA of the genomic ribozyme of hepatitis delta virus or functionally homologous RNA elements. 
     
     
         58 . The method according to  claim 46 , wherein said viral expression cassette comprises a viral cDNA wherein foreign cDNA sequences are inserted to be heterologously expressed. 
     
     
         59 . A vaccine composition comprising a BAC, said BAC comprising:
 an inducible bacterial ori sequence for amplification of said BAC to more than 10 copies per bacterial cell, and   a viral expression cassette comprising a cDNA of an attenuated positive-strand RNA virus genome and comprising cis-regulatory elements for transcription of said viral cDNA in mammalian cells and for processing of the transcribed RNA into infectious viral RNA.   
     
     
         60 . The composition according to  claim 59 , wherein said positive-strand RNA virus is a virus of the family Flaviviridae. 
     
     
         61 . The composition according to  claim 59 , wherein said positive-strand RNA virus is a virus of the genus Pestivirus. 
     
     
         62 . The composition according to  claim 61 , wherein said virus of the genus Pestivirus is bovine viral diarrhea virus or classical swine fever virus. 
     
     
         63 . A method of vaccination against a positive-strand RNA virus infection comprising the step of administering a BAC, said BAC comprising:
 an inducible bacterial ori sequence for amplification of said BAC to more than 10 copies per bacterial cell, and   a viral expression cassette comprising a cDNA of an attenuated positive-strand RNA virus genome, or a viral expression cassette comprising a cDNA of an attenuated positive-strand RNA virus genome and comprising cis-regulatory elements for transcription of said viral cDNA in mammalian cells and for processing of the transcribed RNA into infectious viral RNA.   
     
     
         64 . The method according to  claim 63 , wherein said positive-strand RNA virus is a virus of the family Flaviviridae. 
     
     
         65 . The method according to  claim 63 , wherein said positive-strand RNA virus is a virus of the genus Pestivirus. 
     
     
         66 . The method according to  claim 65 , wherein said virus of the genus Pestivirus is bovine viral diarrhea virus or classical swine fever virus. 
     
     
         67 . A method for the maintenance of cDNA of a native or recombinant positive-strand RNA virus genome or for the propagation of native or recombinant viruses from the cDNA, the method comprising the step of propagating a BAC in a bacterial host, the BAC comprising:
 an inducible bacterial ori sequence for amplification of said BAC to more than 10 copies per bacterial cell, and   a viral expression cassette comprising a cDNA of an attenuated positive-strand RNA virus genome and comprising cis-regulatory elements for transcription of said viral cDNA in mammalian cells and for processing of the transcribed RNA into infectious viral RNA.   
     
     
         68 . The method according to  claim 67 , wherein said positive-strand RNA virus is a virus of the family Flaviviridae. 
     
     
         69 . The method according to  claim 67 , wherein said positive-strand RNA virus is a virus of the genus Pestivirus. 
     
     
         70 . The method according to  claim 69 , wherein said virus of the genus Pestivirus is bovine viral diarrhea virus or classical swine fever virus.