WO2006013815A1 - SOUCHE Dls DE VIRUS DE VACCIN RECOMBINÉ PORTANT UN ADN CODANT POUR UNE PROTÉINE DE VIRUS VHC ET UTILISATION DE CELLE-CI - Google Patents

SOUCHE Dls DE VIRUS DE VACCIN RECOMBINÉ PORTANT UN ADN CODANT POUR UNE PROTÉINE DE VIRUS VHC ET UTILISATION DE CELLE-CI Download PDF

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WO2006013815A1
WO2006013815A1 PCT/JP2005/014027 JP2005014027W WO2006013815A1 WO 2006013815 A1 WO2006013815 A1 WO 2006013815A1 JP 2005014027 W JP2005014027 W JP 2005014027W WO 2006013815 A1 WO2006013815 A1 WO 2006013815A1
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virus
dis
recombinant
strain
region
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PCT/JP2005/014027
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Japanese (ja)
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Koji Ishi
Tetsuro Suzuki
Tatsuo Miyamura
Sanae Machida
Yoshiharu Matsuura
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The Japan Health Sciences Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a recombinant vaccine virus DIs strain that possesses DNA encoding all or part of the HCV virus protein on genomic DNA and can express the protein.
  • Cuter virus is a virus that has been inoculated to many people as a seed pod and has contributed to the eradication of smallpox. It is a large double-stranded DNA virus with a gene encoding more than 200 viral proteins. The genome has a total length of about 200 kBp, and it itself encodes a group of enzymes involved in DNA and mRNA synthesis to replicate in the cytoplasm. Then, there is a characteristic.
  • vaccinia virus has been widely applied to molecular biology and immunology research as a foreign gene transfer and expression vector, such as the target protein with high expression level being correctly post-translationally modified by cultured cells. It is coming.
  • vaccinia virus has been widely applied to molecular biology and immunology research as a foreign gene transfer and expression vector, such as the target protein with high expression level being correctly post-translationally modified by cultured cells. It is coming.
  • it has a history of being used as a seed for many years, research results on safety when administered to humans have been accumulated. For this reason, many laboratories are also working on the use of recombinant vaccinia virus as a live vaccine.
  • the vaccine strain used in Japan has been subcultured over a long period of time using one-day egg passage, and the established attenuated strain is the DIs strain.
  • the 1-day egg technique is a method of inoculating the avian avian fetus directly with the virus 24 hours after its onset, and was developed by Kamesaburo Yoshino of the National Institute of Preventive Health at that time.
  • the mutant strain of the DI strain obtained with this state-of-the-art technology was named DIs strain, showing a smaller box on the chorioallantoic membrane than the parent strain.
  • the DIs strain unlike the parent strain DI, has the unique ability to proliferate in embryonated chicken egg embryo fibroblasts (CEF). Most other mammalian cells are infected but do not grow in the cell. (See Non-Patent Document 2). Therefore, even if it was inoculated to humans, it did not feel good and could not become a practical attenuated strain (see Non-Patent Document 3).
  • HCV Hepatitis C virus
  • non-A non-B hepatitis virus! / A gene that was cloned in 1989 by Chiron in the United States, plus single-stranded RN A virus whose genome is A (see Non-Patent Document 8). Since then, the entire base sequence has been determined by several groups including Chiron, and it has been revealed that there are three types of structural proteins and six types of nonstructural proteins.
  • interferon has been used for the treatment of hepatitis C.
  • the problem is that the therapeutic effect varies depending on the patient and that side effects such as fever are strong.
  • Non-Patent Document 2 Tagaya, I. and 2 other authors, "A new mutant of dermovaccinia virus.”, Nature, 1961, Vol.192, p.381-382
  • Non-Patent Document 3 Takashi Kitamura, Chuko Shinsho, “Natural Spider Disappears”, Chuo Koronsha, 1982
  • Non-Patent Document 4 Mayr, A. and two others MVA. '', Infection ⁇ 1975, Vol. 3, p.6-14.
  • Non-Patent Document 5 Hanke, T. and other 8 authors, “Development of a DNA- MVA / HIVA vaccine for Kenya”, Vaccine V 2002, Vol. 20, p. 1995-1998.
  • Non-Patent Document 6 Tartaglia, J. and 11 other authors, ⁇ NYVAC: a highly attenuated strain of vaccinia virus.J, Virology, 1992, Vol.188, p.217-232.
  • Patent Document 7 Kazanji, M. 7 others, ⁇ Immunogenicity and protective efficacy of reco mbinant human T-cell leukemia / lymphoma virus type 1 NYVAC and naked DNA vac cine candidates in squirrel monkeys (Saimiri sciureus). '' Virol., 2001, Vol.75, p.5939-5948.
  • Non-Patent Document 8 Science, 1989, Vol. 244, p.359-362
  • Non-Patent Document 9 Hepatology, 1993, Vol. 18, p.1039-1044
  • Non-Patent Document 10 J. Immunol, 1992, Vol.149, p.3339-3344
  • the present invention has been made in view of the above situation, and an object of the present invention is to provide an effective vaccine for preventing infection or eliminating viruses against HCV. More specifically, an object is to provide a useful vaccine capable of strongly inducing CTL against HCV. More specifically, DNA encoding all or part of the HCV virus protein is retained on the genomic DNA. Another object of the present invention is to provide a recombinant vaccine virus DIs strain capable of expressing the protein.
  • the present inventors have intensively studied to solve the above problems. First, the present inventors analyzed the gene structure of the DIs strain and found that there was one large defect at the left end of the genomic DNA of the DIs strain. Then, a transfer vector inserted in the center of the region sandwiching the defective site was constructed, and a viral vector was designed in which the target foreign gene and the drug resistance gene XGPRT derived from E. coli were inserted by homologous recombination.
  • HCV applied the strong immunity-inducing ability of ⁇ virus and applied all of the proteins encoded by HCV. It is considered that a strong or CTL response against the protein encoded by HCV can be induced by incorporating a part or part of the protein into DIs and administering these recombinant viruses.
  • the recombinant virus of the present invention may be an effective live vaccine against hepatitis C.
  • the present inventors succeeded in determining the entire nucleotide sequence of DIs excluding a partial sequence at both ends. This made it possible to produce more efficient thread and DIs.
  • the present invention relates to a recombinant vaccine virus DIs strain that possesses DNA encoding all or a part of the HCV virus protein on the genomic DNA and is capable of expressing the protein.
  • Recombinant vaccinia virus DIs strain that has DNA encoding all or part of HCV virus protein on genomic DNA and can express the protein
  • HCV virus protein is non-HCV structure
  • a virus-specific cytotoxic T cell response inducer comprising the recombinant vaccine virus DIs strain according to any one of [1] to [4] as an active ingredient
  • the present invention provides the following [8] to [11].
  • a method for preventing or treating hepatitis C which comprises the step of administering the recombinant vaccine virus DIs strain to an individual.
  • FIG. 1 is a diagram showing genomic analysis of a cuticle virus DIs strain and the identified defective site.
  • FIG. 2 is a diagram showing the structure of a DIs transfer vector pDIsmH5. Insert the desired foreign gene into the multicloning site (MCS) downstream of the mH5 promoter.
  • MCS multicloning site
  • FIG. 3 Schematic diagram of creation of recombinant DIs by homologous recombination: When a transfer vector is transfected into infected cells after infection of DIs with CEF, the DNA polymerase of the virus in the process of virus replication, although with a low probability Replicates the region of DIs integrated on the vector side, so that DIs with the gene of interest are generated. Since the drug resistance gene XGPRT is also incorporated at this time, only target DIs are amplified when plaque assembly is performed in the presence of MPA.
  • FIG. 4 is a graph showing induction of humoral immunity in mice administered with recombinant DIs / J228 expressing the structural region of HCV.
  • the upper row shows the results using HCV core, and the lower row shows HCV E2.
  • the left column shows the result of virus injection into the vein, and the right column shows the result of virus injection into the abdominal cavity.
  • FIG. 5 is a graph showing the results of induction of cellular immunity in mice administered with recombinant DIs expressing HCV structural and nonstructural regions.
  • the vertical axis shows the number of IFN- ⁇ positive T cells by ELISPOT method. The number of cells secreting IFN-y was measured in T cells stimulated with overlapping peptides corresponding to each region, and the structural region was divided into four and the NS5A region was divided into two. In the structural area, 1 corresponds to core, 2 forces to 1, and 3 and 4 to E2.
  • the present invention has a DNA (which may be described as "HCV viral DNA” in this specification) encoding all or a part of the HCV viral protein on genomic DNA, and the protein A recombinant vaccine virus DIs strain that can be expressed is provided.
  • HCV viral DNA a DNA
  • HCV viral protein a DNA encoding all or a part of the HCV viral protein on genomic DNA
  • protein A recombinant vaccine virus DIs strain that can be expressed.
  • Various types (genotypes) of HCV virus hepatitis C virus
  • the HCV virus in the present invention is not limited to viruses belonging to a specific type, but refers to viruses belonging to all these types.
  • the recombinant virus strain of the present invention is characterized in that the virus virus DIs strain has a genomic structure in which DNA encoding the HCV protein is inserted (integrated) onto the genome of the virus virus DIs strain. It is.
  • the HCV protein used in the present invention refers to a protein encoded by a gene existing on the HCV genome (sometimes referred to as "HCV protein" in the present specification), or the protein of the protein. Refers to a partial peptide.
  • HCV protein a protein encoded by a gene existing on the HCV genome
  • the protein of the protein refers to a partial peptide.
  • Examples of the protein of the present invention include structural proteins, regulatory proteins, and accessory proteins.
  • the DNA encoding the HCV protein retained on the DIs strain genomic DNA is preferably a DNA encoding an HCV-derived protein having CTL activity.
  • the DNA include a region that expresses a core protein, a region that expresses the entire structural protein (core protein and two envelope proteins; core, El, E2), a protease protein (non-structural protein (NS2, NS3, NS4A, NS4B, NS5B)) and the interferon sensitivity referred to as NS5A, and DNA contained in the region can be exemplified.
  • a recombinant vaccine DIs strain capable of expressing the protein by retaining the DNA encoding the protein or the DNA fragment on the genomic DNA. It is.
  • the base sequence described in SEQ ID NO: 11 can be preferably shown.
  • the “deficient region” in the genomic DNA of the Copenhagen strain of ⁇ virus is known (GenBank accession number: M35027).
  • the “defect region” is specifically a region corresponding to positions 17145-32562 (SEQ ID NO: 18) counted from the 5 ′ side in the genomic DNA of Copenno, Igen.
  • Those skilled in the art can appropriately know which region on the genomic DNA of the parent strain (Dalian strain) corresponds to the above-mentioned “deleted region” based on the information disclosed in this specification. is there.
  • the HCV virus DNA strength is a recombinant vaccine virus DI s conserved (inserted) in the above-mentioned paired DI strain deletion region or in the vicinity of the region. Offer stocks.
  • the “defect” region in the present invention for example, a region of 15.4 kbp indicated by a black line in FIG. 1 can be mentioned.
  • this “deficient” region there are no genes that are considered essential for virus propagation, and they are called host range genes. Two of them (K1L and C7L) exist. For example, when K1L is deficient, this deficient virus loses its ability to grow in cells such as RK13.
  • the "deletion" region is not strictly limited to the 15.4 kbp region shown in FIG.
  • the “insertion” of DNA into the “defect” region may be a state in which the DNA force corresponding to the “deletion” region is replaced by the HCV virus DNA of the present invention.
  • the HCV viral DNA may be inserted (added) onto the DNA corresponding to the “deficient” region.
  • the "recombinant vaccine virus” in the present invention usually refers to a recombinant attenuated virus.
  • Recombinant attenuated virus refers to a virus that is less toxic than the wild type by, for example, genetic modification (deleting a specific gene) or by long-term passage.
  • a DIs strain obtained by highly attenuating the Kuchi-Kya virus Dalian strain (DI strain) is preferably used.
  • an MVA attenuated strain is known as an attenuated strain (see Non-Patent Document 4 above).
  • the DIs strain is considered to be superior to the MVA attenuated strain in the following points.
  • DNA that codes all or part of the HCV virus protein (sometimes referred to as "HCV virus DNA”) is inserted into the genome of the DIs strain (The site on the genome during integration) is not particularly limited, but is preferably a “non-conserved region” on the genome. That is, in a preferred embodiment of the present invention, there is provided a recombinant cuticular DIs strain in which HCV viral DNA is conserved (inserted) on a non-conserved region of genomic DNA.
  • the "non-conserved region” refers to a region where there is no gene that is considered essential for virus growth.
  • the genome virus has a total genome length of about 200 kbp, and the virus can grow even if a substantial part of the encoded gene is deleted which is not essential for virus replication.
  • the present inventors determined the entire nucleotide sequence of the DIs strain and revealed that there is a large 15.4 kbp deletion at the left end of the DIs strain genome (sometimes simply referred to as “DIs strain deletion region”). did. Note that this 15.4 kbp deletion region is a region that does not exist in the genomic DNA of the DIs strain described in SEQ ID NO: 11, which is a force present in the normal cuticular virus.
  • the "non-conserved region” preferably refers to, for example, the above-mentioned “deletion” region on the DIs strain genome (for example, the above-mentioned 15.4 kbp deletion region or a region near the region). it can.
  • the “vicinity region” usually refers to a region on the chromosome in the vicinity of the region.
  • “near” is not particularly limited, but usually refers to a region within 5 kbp, more preferably within 1 kbp from the end of the region.
  • selection markers include XGPRT gene, LacZ gene and the like. Methods for selecting recombinant virus (DNA) bodies using these marker genes are also known. In addition, selection of homologous recombinant virus can be performed efficiently by suitably using CEF that can only grow a DIs strain.
  • HCV gene HCV virus DNA
  • amino acid sequence of the protein encoded by the gene are already known.
  • a literature database or Public gene (genome) data It can be easily obtained using the base.
  • the genomic DNA of the recombinant vaccine virus DIs strain provided by the present invention or a partial fragment DNA of the genomic DNA is also included in the present invention.
  • the partial fragment DNA is preferably a partial fragment DNA including a DNA region into which HCV virus DNA is inserted.
  • the present invention provides a hepatitis C virus vaccine comprising the recombinant vaccine virus DIs strain of the present invention as an active ingredient.
  • the vaccine in the present invention is usually used for elimination of hepatitis C virus or treatment of hepatitis C.
  • a vaccine can contain an antigen or express an antigen, which can induce an immune response to the antigen.
  • the vaccine of the present invention can be used in a desired form.
  • the vaccine of the present invention may contain, for example, a carrier.
  • the type of carrier can usually be determined according to the mode of administration.
  • the vaccine of the present invention can be formulated by a method suitable for administration, including, for example, topical, oral, intranasal, intravenous, intraperitoneal, subcutaneous or intramuscular administration.
  • the carrier include water, physiological saline, alcohol, fat, wax, buffer and the like.
  • the aforementioned carriers or solid carriers such as mannitol, ratatoose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate can be used as appropriate. .
  • the vaccine of the present invention is used as a recombinant vaccine virus DIs strain of the present invention, and an adjuvant. It is also possible to formulate as a composition containing a bunt.
  • an adjuvant for example,
  • Adjuvant refers to a substance that increases the immunogenicity of an antigen.
  • Administration of the vaccine of the present invention to an individual is not particularly limited, and can be performed, for example, locally, orally, intranasally, intravenously, intraperitoneally, subcutaneously, or intramuscularly.
  • the dose to be administered can be appropriately determined by those skilled in the art (doctors, veterinarians, etc.) in consideration of the age, weight, symptoms, etc. of the individual (patient).
  • Examples of animals that can be inoculated with the vaccine of the present invention include all host organisms that have an immune system and can be infected with HCV, such as humans and some primates. .
  • the recombinant vaccinia virus DIs strain of the present invention has an action of inducing a response of virus-specific cytotoxic T cells (CTL) when introduced into an individual. Accordingly, the present invention provides a virus-specific cytotoxic T cell (CTL) response inducer comprising the recombinant vaccinia virus DIs strain of the present invention as an active ingredient.
  • CTL virus-specific cytotoxic T cell
  • Cytotoxic T cells are also called cytolytic (killer) T cells or cytotoxic T lymphocytes, and have the ability to destroy target cells. It is thought to be helpful in eliminating tumor cells.
  • the CTL response inducer of the present invention can also contain the above-mentioned carrier and the like as appropriate.
  • the present invention also relates to a virus-specific cytotoxic T cell characterized by administering the recombinant vaccine virus DIs strain of the present invention or the agent of the present invention to an individual (for example, a patient).
  • the present invention relates to a method for inducing a response and a method for preventing or treating hepatitis C.
  • the individual in the prevention or treatment method of the present invention is preferably a human, but is not particularly limited, and may be a non-human animal having an immune system.
  • administration to an individual is, for example, intraarterial injection, intravenous injection, subcutaneous injection, etc. This can be done by methods known to those skilled in the art.
  • the dosage varies depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dosage.
  • the present invention relates to the use of the recombinant vaccine virus DIs strain of the present invention in the production of hepatitis C virus vaccine, and the virus-specific cytotoxicity of the recombinant vaccine virus DIs strain of the present invention.
  • the present invention relates to use in the production of a T cell response inducer.
  • Vac H-C [5′-ATAATGTAGCTCCTTCATCAATCATACATT] / SEQ ID NO: 1
  • Vac H-F [5, -AGGAGGTGGTGTAATAGACGAAGATTATAG] / SEQ ID NO: 2 was synthesized. PCR amplification was performed using the genomic DNA of DIs as a template, and the amplified fragment was sequenced by inserting it into the EcoRI site of plasmid pUC19.
  • host range genes Two of the so-called host range genes (K1L and C7L) are present. Existing. Although the function of host range genes is not yet clear, it is clear that they define the range of cultured cells that can be infected and propagated (Pe rkus, ME, uoebel, SJ , Davis, SW, Jonnson, GP, Limbach, K., Norton, E. K., and Paoletti, E. (1990). Vaccinia virus host range genes. Virology 179, 276-286.). For example, when KIL is deficient, this deficient virus loses the ability to grow in RK13 cells.
  • the genome sequence is exchanged between the sequence on the plasmid and the corresponding sequence on the genome at a certain frequency (this phenomenon is homologous recombination). Therefore, by subcloning a part of the DIs genome into a plasmid and inserting the desired foreign gene into this DIs sequence, this plasmid can be used as a transfer vector for inserting the foreign gene into DIs.
  • GCGAGAAATAATCATAAATAZ SEQ ID NO: 3] was synthesized and inserted, and the resulting vector was cleaved with BssHII. After the 240 bp fragment was purified, the end was converted to Hindlll and inserted into the Hindlll site present in the DIs-derived sequence inserted into PUC19. Smal, BamHI, NotI, Sacl, and SacII existing downstream of the vaccine virus promoter mH5 can be used as insertion sites (multicloning sites) for foreign genes.
  • the E. coli-derived enzyme XGPRT hyperxantine-guanine phosphoribosyl transferase residue is added to the vactenif vinores promoter p7.5, and pUC19 It was inserted into the Mlul site present in the DIs fragment inserted in.
  • the inserted sequence is shown in SEQ ID NO: 4.
  • GMP synthesis is inhibited in the presence of MPA (mycophenolic acid), an inhibitor of purine synthesis, and the DNA synthesis of cuticular virus is also inhibited.
  • the target recombinant virus can be obtained by culturing the virus in a medium containing various drugs (Asato Kojima “Vaccinia virus vector” virus experimental protocol (1995) 343-353 Medical View). It is important to use CEF, which can only grow DIs, for virus homologous recombination and selection of virus.
  • the present inventors have inserted the XGPRT gene downstream of the p7.5 promoter of ⁇ virus and further connected a multicloning site downstream of the mH5 promoter so that foreign genes can be easily inserted. Created a unit. By constructing a transfer vector in which the unit is inserted in the center of the region between which the DIs gene is deleted and the region between them is inserted, and homologous recombination is performed, the XGPRT gene is inserted together with the target foreign gene. ( Figure 2).
  • the completed transfer vector was named pDIsgptmH5.
  • Transfer vectors were prepared by inserting various protein regions of HCV into the above-mentioned cloning site of pDIsgptmH5.
  • recombinant DIs expressing HCV lb core, El, and E2 can be prepared by amplifying and inserting a structural protein region of about 2.4 kbp from HCV lb cDNA by PCR.
  • Recombinant DIs expressing HCV lb NS3 and NS5A can be created by PCR amplification of 1.9 kbp and 1.3 kbp fragments corresponding to non-structural regions NS3 and NS5A. .
  • a list of primers used for amplification is shown below.
  • AAAGATCTCATGTCCGGCTCGTGGCTAAGGGATGTT (SEQ ID NO: 9)
  • AAAGATCTTTAGCAGCAGACGACGTCCTCACTAGCC (SEQ ID NO: 10)
  • the cells were cultured for 2 hours under 2 conditions. Remove the medium, wash with PBS (phosphate-buffered serine), and add 0.5 ml of 0.05% trypsin solution to release the cells. Then, centrifuge the cell suspension at 2000 rpm for 3 minutes to collect the cells. , 400 1 in 13 ⁇ 43. 10 g of the transfer vector was dissolved in this cell suspension, and electroporation was performed by applying a voltage once at 250 v, 500 FD in a 0.4 cm cuvette using Gene Pulser II (Bio Rad). The cells were suspended in 2 ml of DME M containing 10% FCS, seeded in a 35 mm petri dish, and cultured for 7 days at 37 ° C and 5% CO.
  • PBS phosphate-buffered serine
  • Vaccinia virus itself has strong cell-mediated immunity-inducing ability and induces cell-mediated immunity against the exogenous gene product of recombinant virus. Therefore, it is highly likely that DIs, which are attenuated strains, can induce immunity against foreign gene products. Attempts are also being made to apply DIs as live recombinant vaccines for refractory infections.
  • Recombinant DIs expressing 10 6 pfo of HCV structural region were administered to 5-week-old Balb / c mice, It was confirmed whether the antibody value was raised. The same amount of virus was administered 3 times in total 2 weeks and 6 weeks after the first administration, and blood was collected at the 7th week to measure the antibody titer in the blood.
  • antigen HCV core and E2 expressed using recombinant baculovirus were used (Aizaki et al., HCV gene expression using baculovirus expression system, Japanese clinical 1995 extra edition, 80- 84).
  • Each solution was diluted 1000 times with 0.05M carbonate buffer (pH 9.6) and immobilized on an ELISA plate (Sumitomo Bakelite). Diluted blood serum was diluted with the primary antibody and anti-mouse antibody bound with horse radish peroxidase.
  • ELISA was performed according to a conventional method! The antibody titer was measured (FIG. 4). As shown in Fig.
  • Recombinant DIs expressing 10 6 pfo HCV structural and nonstructural regions were administered to 5-week-old Balb / c mice to confirm whether they induce cellular immunity.
  • the same amount of virus was administered three times in total, 2 weeks and 6 weeks after the first administration, and the spleen was removed and the spleen cells were isolated at 7 weeks.
  • 3 ml of lysis buffer (potassium bicarbonate 1.0 mM, ammonium chloride 0.15 M, EDTA-2Na 0.1 mM) was added and mixed gently to cause hemolysis.
  • 5X10- 5 washed with RPMI 1640 medium containing 2 mercaptoethanol of M, 5X10- 2-mercaptoethanol 5 M, lng / ml of P MA, 500 ng / ml of Ionomaishin, were suspended in RPMI1640 containing 10% FCS , the concentration was adjusted, the number of living cells in the measurement to 5xl0 4 / ml. After culturing overnight in 96-well piate coated with anti-interferon- ⁇ antibody, the cell solution was discarded and the number of cells secreting interferon- ⁇ was measured using an ELISPOT kit from DIA CLONE (FIG. 5).
  • Vaccinia virus shares the advantages of strong immunity inducing ability with the disadvantage of neuropathogenicity.
  • the virulence of existing strains of DIs is very low compared to the parent strain, the cuticle virus.
  • DIs strains are attenuated because of large genomic deletions of more than 15 kbp. Can't be thought of first.
  • the recombinant vaccine virus DIs strain of the present invention has a strong immunity-inducing ability, can induce strong CTL responses against HCV virus proteins, and is very useful as a recombinant vaccine.

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  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Communicable Diseases (AREA)
  • Gastroenterology & Hepatology (AREA)
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Abstract

On conçoit un vecteur de virus en construisant un vecteur de transfert à insérer dans le centre d'une région portant le site défectif vis-à-vis des souches Dl des souches Dls de façon à ce qu'un gène étranger souhaité et à ce qu'un gène de la tolérance à un médicament XGPRT provenant de Escherichia coli puissent être insérés via une recombinaison homogène. Ce vecteur de virus de souche Dls ne présente pas d'effet cytopathique de virus (CPE), ne se développe pas dans des cellules infectées et présente une faible cytotoxicité, ce qui indique qu'il est extrêmement utile comme vecteur de virus. Lorsqu'on insère dans celui-ci un ADN codant pour la protéine entière du VHC ou pour une partie de celle-ci en tant que gène étranger, on induit des lymphocytes T cytotoxiques (CTL). Ainsi, on s'attend à ce qu'il soit efficace comme vaccin à virus vivant contre l'hépatite C.
PCT/JP2005/014027 2004-08-02 2005-08-01 SOUCHE Dls DE VIRUS DE VACCIN RECOMBINÉ PORTANT UN ADN CODANT POUR UNE PROTÉINE DE VIRUS VHC ET UTILISATION DE CELLE-CI WO2006013815A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009110644A1 (fr) * 2008-03-07 2009-09-11 財団法人東京都医学研究機構 Virus recombinant de la vaccine ayant un gène du virus de l'hépatite c
JP2013048597A (ja) * 2011-08-31 2013-03-14 Tokyo Metropolitan Institute Of Medical Science 新型インフルエンザウイルス由来ヘマグルチニンタンパク質遺伝子を有するＤＩｓ株由来組換えワクシニアウイルス
WO2020184730A1 (fr) * 2019-03-14 2020-09-17 公益財団法人東京都医学総合研究所 Vaccin contre le virus de la dengue

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4344805B2 (ja) * 2000-07-07 2009-10-14 独立行政法人科学技術振興機構 遺伝子組換えワクシニアウイルスワクチン

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ISHII K. ET AL: "Structural Analysis of Vaccinia Virus DIs Strain: Application as a New Replication-Deficient Viral Vector", VIROLOGY, vol. 302, 2002, pages 433 - 444, XP002993347 *
MATSUURA Y. ET AL: "Biotechnology ni yoru Wakuchin no Sosei to Kairyo Gijitsu no Kaihatsu", SOYAKUTO HUMAN SCIENCE KENKYU JUTEN KENKYU HOKOKUSHO, HEISEI 14 NENDO DAI 5 BUNYA KENKO JUMYO ENSHIN, YOBO SHINDAN, CHIRYOHO NO KAIHATSU NI KANSURU KENKYU, 2003, pages 14 - 19, XP002999526 *
MIYAMURA T.: "2. Virus Dai Ni Bu Kenkyu Gyoseki III. Kan'en Virus ni Kansuru Kenkyu 3. C-gata Kan'en Virus (HCV) ni Kansuru Kenkyu", NATIONAL INSTITUTE OF INFECTIOUS DISEASES NENPO, HEISEI 14 NENDO DAI 56 GO (JAPAN SCIENCE AND TECHNOLOGY AGENCY, UKEIREBI), 17 February 2004 (2004-02-17), pages 53 - 57, XP002999527 *
MIYAMURA T.: "2.Virus Dai Ni Bu Kenkyu Gyoseki III. Kan'en Virus ni Kansuru Kenkyu 3. C-gata Kan'en Virus (HCV) ni Kansuru Kenkyu", NATIONAL INSTITUTE OF INFECTIOUS DISEASES NENPO, HEISEI 15 NENDO DAI 57 GO,(JAPAN SCIENCE AND TECHNOLOGY AGENCY UKEIREBI), 28 February 2005 (2005-02-28), pages 59 - 64, XP002999528 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009110644A1 (fr) * 2008-03-07 2009-09-11 財団法人東京都医学研究機構 Virus recombinant de la vaccine ayant un gène du virus de l'hépatite c
JP2009232836A (ja) * 2008-03-07 2009-10-15 Tokyoto Igaku Kenkyu Kiko Ｃ型肝炎ウイルス遺伝子を有する組換えワクシニアウイルス
CN101965396A (zh) * 2008-03-07 2011-02-02 财团法人东京都医学研究机构 具有丙型肝炎病毒基因的重组痘苗病毒
CN101965396B (zh) * 2008-03-07 2013-08-14 公益财团法人东京都医学综合研究所 具有丙型肝炎病毒基因的重组痘苗病毒
US9000136B2 (en) 2008-03-07 2015-04-07 Tokyo Metropolitan Institute Of Medical Science Recombinant vaccinia virus having hepatitis C virus gene
EP2267120B1 (fr) * 2008-03-07 2016-05-18 Tokyo Metropolitan Institute of Medical Science Virus recombinant de la vaccine ayant un gène du virus de l'hépatite c
JP2013048597A (ja) * 2011-08-31 2013-03-14 Tokyo Metropolitan Institute Of Medical Science 新型インフルエンザウイルス由来ヘマグルチニンタンパク質遺伝子を有するＤＩｓ株由来組換えワクシニアウイルス
WO2020184730A1 (fr) * 2019-03-14 2020-09-17 公益財団法人東京都医学総合研究所 Vaccin contre le virus de la dengue
EP3939607A4 (fr) * 2019-03-14 2023-01-25 Tokyo Metropolitan Institute of Medical Science Vaccin contre le virus de la dengue

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