# US 7,045,313 B1 · Recombinant vaccinia virus containing a chimeric gene having foreign DNA flanked by vaccinia regulatory DNA > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,045,313 B1 - Document ID: US7045313B1 - Status: Granted - Kind: B1 - Application number: 07/987,546 - Filed: 1992-12-07 - Granted: 2006-05-16 - Earliest claimed: 1982-11-30 - Assignees: The United States of America as represented by the Department of Health and Human Services - Inventors: Bernard Moss; Michael Mackett; Geoffrey Smith - Web page: https://patentgrep.com/patent/US7045313B1 ## CPC - C12N 15/86: Viral vectors - A61K 39/12: Viral antigens - A61K 39/275: Poxviridae, e.g. avipoxvirus - A61K 39/285: Vaccinia virus or variola virus - C07K 14/005: from viruses - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - C12N 9/1033: {Chloramphenicol O-acetyltransferase (2.3.1.28)} - A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53) - A61K 2039/5256: {expressing foreign proteins} - C12N 2710/16622: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2710/24122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2710/24134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein - C12N 2710/24143: viral genome or elements thereof as genetic vector - C12N 2730/10122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2760/20234: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein - Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## 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. ## Claims ### Claim 1 A plasmid that comprises (A) a segment comprised of (i) a first DNA sequence encoding a polypeptide that is foreign to vaccinia virus and (ii) a vaccinia virus promoter sequence, wherein said promoter sequence is adjacent to and exerts transcriptional control over said first DNA sequence; and, flanking said segment, (B) DNA from a nonessential region of a vaccinia genome. ### Claim 2 (depends on 1) A plasmid according to claim 1, wherein said plasmid consists essentially of said segment (A) and said DNA (B) and said first DNA sequence comprises a translational initiator codon. ### Claim 3 (depends on 1) A plasmid according to claim 1, wherein said promoter sequence is not from DNA contained in said nonessential region. ### Claim 4 (depends on 1) A plasmid according to claim 1, wherein said promoter sequence is one that in vaccinia virus regulates the thymidine kinase gene or a vaccinia gene encoding a 7.5 K polypeptide. ### Claim 5 (depends on 1) A plasmid according to claim 1, wherein said nonessential region comprises the vaccinia virus thymidine kinase gene. ### Claim 6 (depends on 1) A plasmid according to claim 1, wherein said polypeptide is immunogenic. ### Claim 7 (depends on 6) A plasmid according to claim 6, wherein said immunogenic polypeptide is a hepatitis B virus surface antigen. ### Claim 8 A recombinant vaccinia virus that comprises a segment comprised of (A) a first DNA sequence encoding a polypeptide that is foreign to vaccinia virus and (B) a vaccinia promoter sequence, wherein (i) said promoter sequence is adjacent to and exerts transcriptional control over said first DNA sequence and (ii) said segment is positioned within a nonessential genomic region of said recombinant vaccinia virus. ### Claim 9 (depends on 8) A recombinant vaccinia virus according to claim 8, wherein said segment consists essentially of said first DNA sequence and said promoter sequence, and wherein said first DNA sequence comprises a translation initiator codon. ### Claim 10 (depends on 8) A recombinant vaccinia virus according to claim 8, wherein said promoter sequence is from DNA not contained in said nonessential region. ### Claim 11 (depends on 10) A recombinant vaccinia virus according to claim 10, wherein said promoter sequence is one that in vaccinia virus regulates the thymidine kinase gene or a vaccinia gene encoding a 7.5 K polypeptide. ### Claim 12 (depends on 11) A recombinant vaccinia virus according to claim 11, wherein said nonessential region comprises a thymidine kinase gene. ### Claim 13 (depends on 8) A recombinant vaccinia virus according to claim 8, wherein said polypeptide is immunogenic. ### Claim 14 (depends on 13) A recombinant virus according to claim 13, wherein said immunogenic protein is a hepatitis B virus surface antigen. ### Claim 15 A method for preparing a vector, comprised of the steps of (A) providing a plasmid and (B) incorporating into said plasmid a segment comprising a chimeric gene that contains (i) a first DNA sequence encoding a polypeptide that is foreign to vaccinia virus; (ii) a vaccinia virus promoter sequence, wherein said promoter sequence is adjacent to and exerts transcriptional control over said first DNA sequence; and (iii) DNA from a nonessential region of a vaccinia virus genome, said DNA flanking said chimeric gene such that said plasmid can undergo homologous recombination with a vaccinia virus genome in a vaccinia virus-infected cell. ### Claim 16 (depends on 15) A method according to claim 15, further comprising the step of transfecting a vaccinia virus-infected cell with said plasmid to obtain a recombinant vaccinia virus wherein said chimeric gene is positioned in a nonessential genomic region thereof. ### Claim 17 A method for producing a protein, comprising the steps of (A) providing a recombinant [poxvirus] vaccinia virus that contains a segment comprised of (i) a first DNA sequence encoding a polypeptide that is foreign to vaccinia virus and (ii) a vaccinia virus promoter sequence, wherein (a) said promoter sequence is adjacent to and exerts transcriptional control over said first DNA sequence and (b) said segment is positioned within a nonessential genomic region of said recombinant vaccinia; (B) infecting host cells with said recombinant vaccinia virus, such that said host cells express said polypeptide; and then (C) separating said polypeptide from said host cells.