Recombinant vaccinia virus containing a chimeric gene having foreign DNA flanked by vaccinia regulatory DNA
Inventors
Moss, Bernard • Mackett, Michael • Smith, Geoffrey
Assignees
US Department of Health and Human Services
Publication Number
US-7045313-B1
Publication Date
2006-05-16
Expiration Date
2023-05-16
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Abstract
Methods and compositions are provided for the use of vaccinia virus or other poxviruses as vectors for expression of foreign genes. Expression of foreign genes is obtained by combining vaccinia virus transcriptional regulatory sequence with uninterrupted foreign protein coding sequences in vitro to form a chimeric gene. The chimeric gene is flanked by DNA from a non-essential region of the vaccinia virus genome to provide sites for in vivo homologous recombination. These steps are facilitated by the construction of plasmids that contain multiple restriction endonuclease sites, next to the vaccinia transcriptional regulatory sequences, for insertion of any foreign protein coding sequence. Transfection procedures are used to introduce the DNA into cells where homologous recombination results in the insertion of the chimeric gene into a non-essential region of the vaccinia virus genome. Infectious vaccinia virus recombinants are distinguished or selected by expression of the foreign gene, loss of activity of a vaccinia virus gene, or by DNA—DNA hybridization. Expression of the foreign gene is obtained by infecting cells or animals with the recombinant vaccinia virus. Examples are provided to show expression of prokaryotic, RNA virus and other DNA virus genes in vaccinia recombinants.
Core Innovation
This invention provides recombinant vaccinia virus synthetically modified by insertion of a chimeric gene containing vaccinia regulatory sequences or DNA sequences functionally equivalent thereto flanking DNA sequences which in nature are not contiguous with the flanking vaccinia regulatory DNA sequences. It has been found that the recombinant virus containing such a chimeric gene is surprisingly effective in expressing the foreign gene.
Recombinants of the present invention have been useful as vaccines providing antibodies effective against the organisms from which the foreign DNA of the chimeric gene was derived. Expression of foreign genes is obtained by combining vaccinia virus transcriptional regulatory sequence with uninterrupted foreign protein coding sequences in vitro to form a chimeric gene. The chimeric gene is flanked by DNA from a non-essential region of the vaccinia virus genome to provide sites for in vivo homologous recombination.
The problem being solved is that prior virus vectors such as papovaviruses, papilloma viruses, adenoviruses, and retroviruses have limitations including small genome size limiting accommodation of foreign DNA, limited host range, and loss of infectivity upon insertion of foreign DNA. Larger viruses like poxviruses could have advantages of greater capacity and broad host range but genetic engineering of poxviruses is more difficult. This invention addresses those challenges by providing a system that utilizes vaccinia virus regulatory elements combined with foreign genes flanked by vaccinia DNA from non-essential regions to enable stable insertion and expression without loss of infectivity.
Claims Coverage
The patent claims cover five main inventive features related to recombinant vaccinia virus vectors, plasmids containing chimeric genes with vaccinia promoters, and methods for constructing and using these recombinants.
Plasmid containing foreign polypeptide gene adjacent to vaccinia virus promoter flanked by nonessential vaccinia DNA
A plasmid comprising a segment composed of a first DNA sequence encoding a polypeptide foreign to vaccinia virus and an adjacent vaccinia virus promoter controlling its transcription, wherein this segment is flanked by DNA from a nonessential region of the vaccinia genome.
Recombinant vaccinia virus comprising chimeric gene inserted into nonessential genomic region
A recombinant vaccinia virus containing a segment comprised of a foreign DNA sequence encoding a polypeptide and an adjacent vaccinia promoter sequence that exerts transcriptional control, with the segment positioned within a nonessential genomic region of the virus genome.
Method of preparing vector with chimeric gene flanked by vaccinia DNA for homologous recombination
A method involving providing a plasmid and incorporating into it a chimeric gene containing a foreign polypeptide coding sequence adjacent to a vaccinia virus promoter, flanked by DNA from a nonessential region of the vaccinia genome such that the plasmid can undergo homologous recombination with vaccinia virus genome.
Method of obtaining recombinant vaccinia virus by transfection of infected cells
Transfecting vaccinia virus-infected cells with the plasmid containing the chimeric gene flanked by vaccinia virus DNA to generate recombinant vaccinia virus with the chimeric gene inserted into a nonessential genomic region.
Method for producing foreign protein by infecting cells with recombinant vaccinia virus
Infecting host cells with a recombinant vaccinia virus containing the chimeric gene comprised of foreign polypeptide coding sequence under control of a vaccinia promoter positioned in a nonessential genomic region, thereby expressing the polypeptide which is then separated from the host cells.
The claims define plasmids with chimeric genes having vaccinia promoters and flanking nonessential vaccinia DNA, recombinant vaccinia viruses with these chimeric genes inserted into nonessential genomic regions, and related methods for plasmid construction, recombinant virus generation via transfection, and foreign protein expression by infection of host cells.
Stated Advantages
Recombinant virus containing the chimeric gene is surprisingly effective in expressing the foreign gene.
Recombinants have been useful as vaccines providing antibodies effective against the organisms from which the foreign DNA was derived.
The method allows expression of foreign genes in a broad host range in vitro and in vivo with retention of infectivity.
Reduced virulence of recombinant vaccinia virus is observed, making it suitable for vaccine use.
Documented Applications
Use of vaccinia virus recombinants as vaccines to elicit antibodies against pathogens including hepatitis B virus, influenza virus, and others.
Production of foreign proteins by infecting cells or animals with recombinant vaccinia virus for preparation of gene products.
Development of live virus vaccines using recombinant vaccinia viruses expressing foreign antigenic polypeptides.
Expression of prokaryotic, RNA virus and DNA virus genes for immunogenicity and antibody production in animals including rabbits, chimpanzees and hamsters.
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