EP0404835A1 - Novel recombinant vaccinia virus expression vectors and method of selecting same - Google Patents
Novel recombinant vaccinia virus expression vectors and method of selecting sameInfo
- Publication number
- EP0404835A1 EP0404835A1 EP89904461A EP89904461A EP0404835A1 EP 0404835 A1 EP0404835 A1 EP 0404835A1 EP 89904461 A EP89904461 A EP 89904461A EP 89904461 A EP89904461 A EP 89904461A EP 0404835 A1 EP0404835 A1 EP 0404835A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- virus
- gene
- vaccinia
- recombinant
- vaccinia virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24141—Use of virus, viral particle or viral elements as a vector
- C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24151—Methods of production or purification of viral material
Definitions
- the present invention is related generally to construction of recombinant vaccinia virus vectors. More particularly, the present invention is related to the construction of unique vaccinia virus open-reading-frame expression vectors and a method for dominant selection of the same.
- Vaccinia virus is a useful vector for gene expression in mammalian cells. Advantages include the maintenance of infectivity, wide host range, large DNA capacity and correct synthesis, processing and transport of proteins. Because transcription of vaccinia virus genes is carried out by virus encoded enzymes in the cytoplasm and splicing of RNA does not occur, there are requirements for vaccinia promoters ⁇ nd uninterrupted open-reading-frames. In addition, the large size and lack of infectivity of the vaccinia virus genome prohibits the construction of recombinants by standard in vitro cloning techniques. A two-step procedure has been developed to overcome these difficulties.
- a plasmid is constructed that contains foreign gene(s) controlled by vaccinia promoter(s), flanked by sequences derived from a non-essential site on the vaccinia genome.
- the foreign genetic material in the plasmid vector is inserted into the viral genome by homologous recombination in vivo.
- selection or isolation of the recombinant virus thus produced is not easily accomplished by the presently known techniques such as plaque hybridization, thymidine kinase (tk) negative selection, ⁇ -galactosidase expression and the like. Of these, only insertional inactivation of the tk gene is a true selection step.
- Disadvantages of this method include requirements for: inactivation of the viral tk gene which attenuates virus infectivity, use of special tk cell lines, and use of mutagenic selective agents, such as 5-bromodeoxyuridine.
- spontaneous tk mutants arise at a high frequency, necessitating additional steps to distinguish them from the recombinants.
- an object of the present invention to provide a new recombinant vaccinia virus expression vector allowing dominant selection of the recombinant. It is a further object of the present invention to provide a vaccinia virus recombinant expression vector the genome of which includes E. coli gpt gene allowing formation of the recombinant vaccinia plaques on a plurality of cell lines when replicated in a growth medium comprising mycophenolic acid (MPA) and a substrate for purine metabolism. It is another object of the present invention to provide a novel method for selecting recombinant vaccinia virus expression vectors containing a foreign gene which is desired to be expressed by said recombinant vaccinia virus. Other objects and advantages of the present invention will become evident from the following detailed description of the invention.
- Fig. 1 demonstrates plaque formation of vaccinia virus in the presence and absence of MPA.
- Confluent BSCI cells were preincubated overnight (12-16 hours) in selective medium and subsequently infected with 1,000 PFU of vaccinia wild-type virus (A, B) or a recombinant virus that expresses the E. coli gpt gene (C, D). The cells were incubated for two days in the presence (A, C) or in the absence (B, D) of MPA, xanthine, and hypoxanthine and then stained with crystal violet.
- Fig. 1 demonstrates plaque formation of vaccinia virus in the presence and absence of MPA.
- FIG. 2 shows the results of genomic analysis of viruses from six randomly picked gpt plaques.
- Southern blots were prepared from HindIII digested DNA. On the right side, the fragment sizes (in kbp) of a phage lambda Hindlll digest are indicated by arrows.
- A The Southern blot was hybridized with a [ 32 p]dCTP labeled gpt gene specific probe. Lanes 1-6, DNA of BSCI cells infected with virus clones No. 1-6; lane 7, DNA of uninfected BSCI cells; lanes 8 and 9 have 10 and 100 ng of vaccinia wild-type DNA, respectively.
- FIG. 3 shows schematic construction of the vectors. DNA sequences including the vaccinia tk gene, E. coli gpt gene, and plasmid vector are indicated by filled, empty, and cross-hatched regions on the diagrams, respectively. Arrows show the direction of transcription from the 11 K (P11), the 7.5 K (P7.5), and tk gene promoters. The pUC sequences contain the ampicillin resistance gene and the origin of bacterial replication.
- Fig. 4 shows the sequence of the multiple cloning sites downstream of the 11 K gene initiation codon.
- the frameshift mutations were included by inserting additional G residues (arrows) downstream of the ATG codon. Note that only the restriction sites EcoRI, Sall, Hindi, AccI, BamHI, and Hpal are unique. The initiation and the termination codons are boxed.
- Fig. 5 shows the results of ⁇ -galactosidase expression in cells infected with the recombinant viruses.
- Confluent CVI cells (2.5 ⁇ 10 6 ) were infected with 7.5 PFU/cell of the indicated virus; after about 24 hour incubation, cytoplasmic extracts were prepared and the protein content and specific ⁇ -galactosidase activity were determined.
- the virus vF1sB was derived from, the vector pTKgpt-F1s; the virus voF1sB is a derivative of pTKgpt-oF1s; and vtat was derived from pSC11.
- a method for dominant selection including a vaccinia recombinant expression vector comprising in genome thereof an E. coli gpt gene and one or more foreign genes desired to be expressed by the recombinant virus, said recombinant virus forming plaques on a plurality of infectable cell lines when replicated in a growth medium comprising sufficient amount of mycophenolic acid to inhibit purine metabolism in the presence of sufficient amount of an unphosphorylated purine substrate.
- the vectors of the present invention include in the genome a promoter that provides high level of expression and may include translation initiation and termination codons, and multiple restriction sites in three different frames which permit expression of partial or complete foreign genes.
- Enzymes and chemicals Restriction endonucleases and low melting agarose were obtained from Bethesda Research Laboratories. T4 polymerase was from Pharmacia. Enzymes were used according to the instructions of the suppliers.
- MPA was from CalBiochem.
- Xanthine and hypoxanthine were from Sigma Chemicals. MPA and xanthine were dissolved in 0.1 N NaOH, hypoxanthine was dissolved in water and sterile filtered; the solutions were stored frozen as 10 mg/ml stocks.
- Vaccinia virus (strain WR) was originally from the American Type Culture Collection, replicated in Hela cells, and purified by standard techniques (Macket, et al., DNA Cloning: A Practical Approach, "The Construction and Characterization of Vaccinia Virus Recombinants Expressing Foreign Genes", pp. 191-211, IRL Press, Oxford. 1985).
- Human tk 143 cells were grown in Eagle's medium with 10% fetal bovine serum (FBS).
- CVI and BSCI cells were grown in Dulbecco's modified medium (DMEM) containing 10% FBS.
- Recombinant viruses were prepared by standard procedures as described by Macket, et al., supra with the following modifications: 5 X 10 6 CVI cells (confluent monolayers) were infected with 0.2 plaque forming units (PFU) of vaccinia virus per cell. Two hours after infection, 1 ml of a calcium DNA precipitate (consisting of 5 ⁇ g of supercoiled plasmid DNA, 1 ⁇ g of vacinia virus DNA, and 14 ⁇ g of sheared herring sperm DNA) was added to the cells. After 15 minutes of incubation at room temperature, 9 ml of medium (DMEM, 8% FBS with penicillin and streptomycin) were added. The medium was changed after 4 h and the incubation was continued for another 36 to 48 hours. Virus stocks were prepared by resuspending the infected cells in 1 ml of medium, freezing and thawing three times.
- PFU plaque forming units
- gpt virus For the isolation of gpt recombinants, a plaque assay on BSCI cells was done as follows: confluent BSCI cells were preincubated in the gpt selection medium (DMEM, 2.5% FBS, 25 ⁇ g/ml MPA, 250 ⁇ g/ml xanthine, and 15 jxg/ml hypoxanthine) for 14 to 24 hours. The virus stock was digested with an equal volume of trypsin (0.25 mg/ml) for 30 minutes at 37°C and sonicated for 20 seconds on ice.
- DMEM fetal bovine serum
- Dilutions (10 -3 , 10 -4 , and 10 -5 ) of the trypsinized virus stock were used to infect the BSCI cells. After 1.5 hours of incubation at 37°C, the cells were overlaid with the gpt-selective medium containing 1% of low melting agarose. After 2 days of incubation, the cells were stained with neutral red (Gibco). The plaques were readily visible after overnight incubation.
- Recombinant plasmids were constructed and isolated by standard methodologies as described by Maniatis, et al., Molecular Cloning, Cold Spring Harbor Labs, Cold Spring Harbor, New York, 1982.
- genomic analysis of recombinant viruses 2.5 ⁇ 10 6 BSCI cells were infected with the material obtained from a single plaque and grown for 24 hours in selective medium. Total cellular DNA was extracted, digested with restriction endonuclease Hindlll, electrophoresed through a 1% agarose gel, and subjected to Southern blot analysis following standard procedures.
- pTK61-gpt Hindlll linkers were added to the Hindlll-Hpal fragment of E. coli gpt gene obtained from plasmid pSV2-gpt. Subsequently, the fragment was inserted into the unique Hindlll site of pGS61, resulting in the plasmid pTK61-gpt.
- pP11 The Hindlll and SstI site, flanking the 11 K gene promoter in plasmid pSC42, were converted into Xhol sites by T4 polymerase treatment and ligation of Xhol linkers.
- the plasmid pSC42 contains the Clal-EcoRI 11 K gene promoter fragment cloned into the SphI site of pUC19.
- pTKgpt-F1s and pTKgpt-oF1s The construction of these plasmids is outlined in detail in Figure 3.
- pTKgpt-F2s and pTKgpt-F3s The frameshift mutations were done by standard oligonucleotide directed mutagenesis. To obtain pTKgpt-F2s, a 30-mer oligonucleotide (5' -GACCTGCAGGAATTCCATTTATAGCATAGA-3 ' ) , and to obtain pTKgpt-F3s, another 30-mer ( 5 ' -ACCTGCAGGAATTCCCATTTATAGCATAGA-3'), were used. Screening of the mutants was done by standard plasmid sequencing (Hattori, et al., Anal. Biochem.
- pTKgpt-F1sB and pTKgpt-oF1sB The BamHI fragment of pMC1871 containing the E. coli lacZ gene (Shapira, et al. 1983, Gene 25:71-82) was cloned into the BamHI sites of pTKgpt-F1s and pTKgpt-oF1s.
- a deposit of pTKgpt-F1s, pTKgpt-F2s and pTKgpt-F3s has been made at the ATCC on March 18, 1988 under accession numbers 67,655, 67,656 and 67,657, respectively.
- the deposits shall be viably maintained, replacing if it became non-viable, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and made available to the public without restriction in accordance with the provisions of the law.
- the Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.
- pTKgpt-F2sB The Smal-Sall fragment of pMC1871 was inserted into the Hindi site of pTKgpt-F2s.
- pTKgpt-F3sB The Sall fragment of pMC1871 was cloned into the Sail site of pTKgpt-F3s.
- Mycophenolic acid inhibition of the growth of vaccinia virus The mycotoxin MPA inhibits the enzyme inosine monophosphate dehydrogenase and thereby prevents the formation of xanthine monophosphate. This results in the intracellular depletion of purine nucleotides and in an inhibition of cell growth. Treatment of host cells with MPA, therefore, severely inhibits the growth of viruses. This was established by testing the effect of increasing amounts of MPA on the plaque formation of vaccinia virus. It was found that 25 ⁇ g/ml of MPA in the medium results in a nearly complete inhibition of plaque formation in all cell lines tested (BSCI, CVI, and human tk- 143 cells).
- E. coli gpt gene Expression of the E. coli gpt gene and its effect on plaque formation in the presence of MPA.
- the inhibition of the de novo synthesis of purines by MPA can be overcome by a cell that expresses the E. coli gpt gene, which codes for the enzyme xanthine-guanine-phosphoribosyl-transferase (XGPRT), in the presence of a substrate for purine metabolism such as xanthine and hypoxanthine in the growth medium.
- XGPRT xanthine-guanine-phosphoribosyl-transferase
- the gpt gene is controlled by the promoter from the vaccinia virus 7.5 K gene and is flanked by viral tk sequences.
- the 7.5 K gene promoter was chosen because it is active early and late in infection and might provide continuous production of the bacterial purine salvage enzyme.
- the plasmid was transfected into CVI cells that were infected with wild-type virus so that the gpt gene would be recombined into the viral tk locus. Putative recombinants were detected by a plaque assay on BSCI cells in the presence of MPA, xanthine, and hypoxanthine as described herein.
- plaques formed only when the gpt gene was used for transfection, indicating that the desired recombinants behaved in the desired manner.
- One of the recombinants was plaque purified twice under selective conditions and a small virus stock was grown.
- a plaque assay demonstrated that this recombinant, in contrast to the wild type virus, formed plaques on BSCI cells in the presence of selective medium (Fig. 1C).
- Fig. 2 The genomic analysis of virus grown from six randomly picked plaques that formed during the first selection step is shown in Fig. 2.
- the Hindlll fragments of all six genomes contain the anticipated 2.0 kbp fragment that hybridizes with the gpt probe (Fig. 2A).
- Fig. 2B After washing off the labeled DNA, the same Southern filter was hybridized to a vaccinia virus tk gene specific probe (Fig. 2B).
- the tk sequences can be detected as a large fragment (4.7 kbp) and a small one (1.0 kbp), indicating the integration of the gpt gene into the viral tk locus. Since all plaques picked after the first selection step have integrated the selective marker, no other screening procedures are necessary to identify a viral recombinant.
- the present invention provides a single step procedure for selection of dominant recombinants.
- a series of plasmids was constructed that use the gpt gene as a selective marker and that allow the expression of foreign genes controlled by the promoter of the major late 11 K polypeptide (Fig. 3).
- the 5' regulatory region of the 11 K gene lies within a 30 kbp segment located immediately upstream of the ATG initiation codon. Since the initiation codon forms part of a highly conserved TAAATG sequence within which the 5' ends of late mRNAs map, this region was chosen not be altered.
- E. coli lacZ gene fragments that lack their own initiation codons (but still have their own termination codons) were inserted in frame into an appropriate restriction site of each of the four vectors.
- Four plasmids were obtained that were termed pTKgpt-F1sB, pTKgpt-F2sB, pTKgpt-F3sB, and pTKgpt-oF1sB. These plasmids were employed to construct viral recombinants.
- the same assay was done also with a virus (vtat) based on the vector pSC11 that also expressed the lacZ gene driven by the 11 K gene promoter (but the ⁇ -galactosidase of which has a slightly different N-terminus), and with a vaccinia virus-T7 RNA polymerase hybrid system that expresses the lacZ gene behind the phage T7 promoter after co-infection with a T7 polymerase producing virus.
- vtat virus based on the vector pSC11 that also expressed the lacZ gene driven by the 11 K gene promoter (but the ⁇ -galactosidase of which has a slightly different N-terminus)
- a vaccinia virus-T7 RNA polymerase hybrid system that expresses the lacZ gene behind the phage T7 promoter after co-infection with a T7 polymerase producing virus.
- viruses based on the vectors pTKgpt-F1s, pTKgpt-ofls, and pSC11 express similar amounts of ⁇ -galactosidase, indicating that lacZ gene activity is relatively independent of the orientation and of the kind of neighboring sequences in the virus.
- the specific activity of pure ⁇ -galactosidase is 300,000 units/mg. Based on this number, the bacterial enzyme produced by virus-infected cells is more than 3% of the total cellular protein, an amount that is easily detectable in a Coomassie blue stained gel.
- the gpt gene is incorporated into a plasmid vector that has a vaccinia promoter and unique restriction endonuclease sites for insertion of a foreign gene.
- the entire selection-expression cassette is inserted as a unit into the vaccinia virus genome by homologous recombination.
- all of the gpt + recombinants analyzed also contain the foreign gene that has been inserted into the plasmid vector.
- the flanking sequence used in this study were derived from the vaccinia tk gene; however, since tk selection is no longer required in accordance with the method of the present invention, any non-essential site in the vaccinia genome can be employed.
- the promoter chosen for the vectors was derived from the major 11 k structural protein.
- promoters well known to one of ordinary skill in the art could also be used.
- the mechanism of late transcription is still poorly understood and involves the attachment of a unique 5' poly(A) leader, the important sequences are contained within a relatively small region starting about 30 bp upstream of the SNA start site and include the translation initiation codon which is a part of the conserved TAAATG sequence. For this reason, the insertion sites for foreign genes were placed just downstream of the ATG. Since the vectors may prove useful for expressing open-reading-frames, multiple cloning sites were engineered in all three frames as well as termination codons.
- the efficacy of the system was illustrated, by expression of ⁇ -galactosidase; the yield of enzyme was found to be greater than about 3% of the total cell protein. This expression level is higher than that obtained using the more widely used 7.5 K promoter and exceeded even that obtained with the recent bacteriophage T7 vaccinia hybrid system (Fuerst, et al., Mol. Cell. Biol., 7:2538-2544, 1987).
- the vectors described herein could, of course, also be employed for direct cloning and expression of open-reading-frames in mammalian cells as well, in a manner similar to that used routinely with bacteriophage A in E. coli.
- the gpt selection provides a number of important advantages over previous procedures devised to isolate recombinant vaccinia viruses. These include one-step plaque isolation without need for enrichment, application to a variety of cell lines, use of alternative insertion sites in the vaccinia genome, and absence of spontaneous selectable mutants.
- 5-bromodeoxyuridine which is used for tk selection, is highly mutagenic, whereas mycophenolic acid is non-mutagenic in the Ames test and in the related SOS test. Avoidance of mutagens ensures virus stability.
- gpt selection method could also be used with other virus vectors, including other members of the poxvirus family, herpesviruses, adenoviruses, retroviruses, and baculoviruses. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
Abstract
On a obtenu des plasmagènes recombinants et des vecteurs d'expression du virus vaccinia qui permettent une sélection dominante.Recombinant plasmagenses and vaccinia virus expression vectors were obtained which allow dominant selection.
Description
Claims
Applications Claiming Priority (2)
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US16994988A | 1988-03-18 | 1988-03-18 | |
US169949 | 1988-03-18 |
Publications (2)
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EP0404835A1 true EP0404835A1 (en) | 1991-01-02 |
EP0404835A4 EP0404835A4 (en) | 1991-05-08 |
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EP19890904461 Withdrawn EP0404835A4 (en) | 1988-03-18 | 1989-03-08 | Novel recombinant vaccinia virus expression vectors and method of selecting same |
Country Status (5)
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EP (1) | EP0404835A4 (en) |
JP (1) | JPH03504196A (en) |
AU (1) | AU629828B2 (en) |
IL (1) | IL89601A0 (en) |
WO (1) | WO1989008716A1 (en) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
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US7767449B1 (en) | 1981-12-24 | 2010-08-03 | Health Research Incorporated | Methods using modified vaccinia virus |
US5174993A (en) * | 1981-12-24 | 1992-12-29 | Health Research Inc. | Recombinant avipox virus and immunological use thereof |
US5833975A (en) * | 1989-03-08 | 1998-11-10 | Virogenetics Corporation | Canarypox virus expressing cytokine and/or tumor-associated antigen DNA sequence |
NZ227934A (en) * | 1988-02-12 | 1991-06-25 | Commw Scient Ind Res Org | Fowlpox virus vectors |
DE4002521A1 (en) * | 1990-01-29 | 1991-08-01 | Immuno Ag | Recombinant plasmids for pox virus transformation - contg. multifunctional DNA sequence cassette to facilitate cloning of foreign DNA |
EP1180157B1 (en) | 1999-05-28 | 2012-11-28 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES | A combined growth factor-deleted and thymidine kinase-deleted vaccinia virus vector |
ME03528B (en) | 2005-03-23 | 2020-04-20 | Genmab As | Antibodies against cd38 for treatment of multiple myeloma |
Citations (1)
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WO1988002022A1 (en) * | 1986-09-22 | 1988-03-24 | Commonwealth Scientific And Industrial Research Or | Recombinant poxviruses |
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1989
- 1989-03-08 AU AU34108/89A patent/AU629828B2/en not_active Ceased
- 1989-03-08 JP JP1504278A patent/JPH03504196A/en active Pending
- 1989-03-08 EP EP19890904461 patent/EP0404835A4/en not_active Withdrawn
- 1989-03-08 WO PCT/US1989/000931 patent/WO1989008716A1/en not_active Application Discontinuation
- 1989-03-14 IL IL89601A patent/IL89601A0/en unknown
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WO1988002022A1 (en) * | 1986-09-22 | 1988-03-24 | Commonwealth Scientific And Industrial Research Or | Recombinant poxviruses |
Non-Patent Citations (2)
Title |
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J. gen. Virol. vol. 67, no. 10, 1986, & SMITH, G.L.: "Vaccinia virus expression vectors" * |
See also references of WO8908716A1 * |
Also Published As
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AU629828B2 (en) | 1992-10-15 |
IL89601A0 (en) | 1989-09-10 |
JPH03504196A (en) | 1991-09-19 |
WO1989008716A1 (en) | 1989-09-21 |
AU3410889A (en) | 1989-10-05 |
EP0404835A4 (en) | 1991-05-08 |
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