WO2011040527A1 - TRANSGENIC BIRD CAPABLE OF EXPRESSING α-GALACTOSE EPITOPE, VIRUS, AND VACCINE - Google Patents

TRANSGENIC BIRD CAPABLE OF EXPRESSING α-GALACTOSE EPITOPE, VIRUS, AND VACCINE Download PDF

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WO2011040527A1
WO2011040527A1 PCT/JP2010/067083 JP2010067083W WO2011040527A1 WO 2011040527 A1 WO2011040527 A1 WO 2011040527A1 JP 2010067083 W JP2010067083 W JP 2010067083W WO 2011040527 A1 WO2011040527 A1 WO 2011040527A1
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virus
gal
transgenic
gene
expressing
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Japanese (ja)
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晴子 小川
貴寛 田上
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国立大学法人帯広畜産大学
独立行政法人農業・食品産業技術総合研究機構
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Priority to JP2011534306A priority Critical patent/JP5688373B2/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • 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
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61P31/18—Antivirals for RNA viruses for HIV
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/30—Bird
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16051—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16051—Methods of production or purification of viral material
    • C12N2760/16052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • the present invention relates to a transgenic bird capable of expressing an ⁇ -galactose epitope (Gal ⁇ 1-3Gal ⁇ 1-4GlcNAc-R: hereinafter sometimes abbreviated as ⁇ -Gal), a biological sample obtained from the transgenic bird, for example, an egg
  • ⁇ -Gal ⁇ -galactose epitope
  • the present invention relates to a virus expressing ⁇ -Gal obtained by inoculating the virus, and a vaccine containing the virus.
  • Vaccines made from growing chicken eggs are effective for preventing influenza.
  • Current human influenza vaccines do not contain immune enhancing agents that may cause side effects, but if new influenza occurs in the future, it is assumed that the vaccine will be used urgently.
  • the development of a method for enhancing the vaccine effect is an important issue.
  • by providing a highly effective vaccine even in a small amount it is necessary to secure technology that allows more people to be vaccinated.
  • ⁇ -galactose epitope is a saccharide antigen similar in structure to the ABO blood group antigen, and is activated by ⁇ 1,3-galactose transferase (hereinafter abbreviated as ⁇ 1,3-GT) Expressed on the cell surface.
  • ⁇ -Gal is expressed in most mammals, but ⁇ -Gal is not expressed in humans and birds because the ⁇ 1,3-GT gene does not function. Therefore, humans and birds recognize ⁇ -Gal as a foreign antigen and possess a natural antibody (anti-Gal antibody) against ⁇ -Gal (Non-patent Document 1).
  • Non-Patent Document 2 The reaction between ⁇ -Gal and anti- ⁇ -Gal antibody is a cause of hyperacute rejection in organ transplantation between different species such as pigs and humans. Ogawa, one of the inventors of the present invention, worked on the control technology of ⁇ -Gal and anti- ⁇ -Gal antibody in xenotransplantation, and also engaged in research on the antigenicity enhancement of cancer vaccine using this reaction, and confirmed its effectiveness (Non-Patent Document 2).
  • anti- ⁇ -Gal antibody binds to the virus in humans and birds.
  • Galili et al. Have reported that binding to the Fc ⁇ receptor of antigen-presenting cells results in effective antigen presentation and may enhance the immune response to the virus (opsonization) (non-patented). Reference 3). Galili et al. Also reported an experiment on AIDS virus (Non-patent Document 4).
  • Non-Patent Documents 1 to 10 are specifically incorporated herein by reference.
  • Non-patent Documents 3 and 4 In vitro enzyme reactions have been used as a method for expressing ⁇ -Gal in viruses (Non-patent Documents 3 and 4). However, this method requires the enzyme used for the reaction, requires the enzyme reaction time, requires removal of the enzyme reaction solution, etc. It is considered difficult to prepare a Gal-expressing virus.
  • an object of the present invention is to provide a means for expressing ⁇ -Gal in a virus without using an enzyme, to produce a virus expressing ⁇ -Gal using this means, and to obtain a virus obtained To make a vaccine.
  • the present invention is to provide a highly effective (antigenic) vaccine, particularly an influenza virus vaccine, by using a virus having an enhanced immune response to the virus.
  • the inventors of the present invention introduced a bird that does not naturally express ⁇ -Gal in a state where the ⁇ 1,3-galactosyltransferase gene can be expressed, and obtained a biological sample such as an egg from the resulting transgenic bird, By infecting this biological sample with a virus, it is possible to produce a virus that expresses ⁇ -Gal ( ⁇ -galactose epitope), and furthermore, by using a virus that expresses this ⁇ -Gal, conventional ⁇ -Gal is expressed.
  • the present invention has been completed by finding that a vaccine with an enhanced immune response to the virus can be obtained compared to a vaccine using a virus that does not.
  • the present invention is as follows. [1] A transgenic bird capable of expressing an ⁇ -galactose epitope (Gal ⁇ 1-3Gal ⁇ 1-4GlcNAc-R: hereinafter ⁇ -Gal). [2] The transgenic bird according to [1], wherein the bird is a chicken. [3] The transgenic bird according to [1] or [2], which is G0 or a progeny. [Four] An ⁇ 1,3-GT gene introduction vector containing an ⁇ 1,3-galactosyltransferase ( ⁇ 1,3-GT) gene in a state capable of being expressed is introduced into birds, and any one of [1] to [3] A method for producing a transgenic bird to obtain the described transgenic bird.
  • [Five] The method for producing a transgenic bird according to [4], wherein the ⁇ 1,3-GT gene introduction vector is a lentiviral vector.
  • [7] A biological sample obtained from the transgenic bird according to any one of [1] to [3].
  • [8] The biological sample according to [7], wherein the biological sample is an egg laid by a transgenic bird.
  • the biological sample according to [8], wherein the egg laid by the transgenic bird is a fertilized egg or a developed bird egg.
  • [Ten] Including inoculating the biological sample according to any one of [7] to [9] with a virus, growing a biological sample inoculated with the virus, and obtaining a virus expressing ⁇ -Gal from the grown biological sample, -Gal expression virus production method.
  • a method for producing a vaccine wherein a virus expressing ⁇ -Gal is produced by the method according to [10] or [11], and a vaccine is produced from the obtained virus.
  • a bird capable of expressing ⁇ -Gal can be provided.
  • ⁇ 1,3-GT gene transgenic chicken (G1) ID4964 obtained in Example 2 ⁇ 1,3-GT gene transgenic chicken (G2) derived from ID4964 obtained in Example 3 and untreated chicken (control)
  • the analysis result by the flow cytometer of (alpha) -Gal expression in the blood cell of is shown.
  • the numbers in parentheses in the graph indicate the percentage of red blood cells that express ⁇ -Gal
  • the analysis result by the flow cytometer of (alpha) -Gal expression in the blood cell of (alpha) 1,3-GT gene transgenic chicken (G2) sputum derived from ID4964 obtained in Example 3 is shown.
  • the present invention relates to a transgenic bird capable of expressing an ⁇ -galactose epitope (Gal ⁇ 1-3Gal ⁇ 1-4GlcNAc-R: hereinafter ⁇ -Gal).
  • ⁇ -galactose epitope is a sugar chain represented by Gal ⁇ 1-3Gal ⁇ 1-4GlcNAc-R, and is a glycoprotein that expresses N-acetyllactosamine (Gal ⁇ 1-4GlcNAc-R, R represents protein) It is a sugar chain produced by allowing ⁇ 1,3-galactose transferase to act and galactose as a substrate to be ⁇ 1-3 linked to the galactose group of N-acetyllactosamine.
  • the method for introducing an ⁇ 1,3-GT gene into birds used for obtaining transgenic birds is a method in which the transgene is incorporated into cells of the whole body of the individual and the desired ⁇ -Gal protein is expressed. If there is no particular limitation. Specifically, the ⁇ 1,3-GT gene is introduced into birds using the ⁇ 1,3-GT gene transfer vector, which is capable of expressing the ⁇ 1,3-galactosyltransferase ( ⁇ 1,3-GT) gene. By doing so, this transgenic bird can be produced.
  • the ⁇ 1,3-GT gene is known to be derived from mouse, porcine or bovine, etc., and gene sequence information can be obtained from GenBank [Mouse: GenBank accession number M85153; J. Biol. Chem. 267, 5534-5541 1992 (1992) (SEQ ID NO: 1), Pig: GenBank accession number L36535; Xenotransplantation 1, 81-88, 1994 (SEQ ID NO: 2), Cattle: GenBank accession number J04989; J. Biol. Chem. 264, 14290-14297, 1989 (SEQ ID NO: 3)].
  • Birds to which gene transfer is performed using the vectors of the present invention are intended to refer to any species, subspecies or breed of the taxonomic taxon "Aves" organisms (but not limited to, Such as chickens, turkeys, ducks, geese, quail, pheasants, parrots, finch, hawks, crows, ostriches, emu, and cassowary).
  • the terms include Gallus gallus or chicken (e.g.
  • a lentiviral vector encoding the ⁇ 1,3-GT gene can be used as the ⁇ 1,3-GT gene introduction vector.
  • a lentivirus replication-defective human immunodeficiency virus (HIV) can be used from the viewpoint of safety.
  • the lentiviral vector contains an expression regulatory sequence (promoter) necessary for expressing ⁇ 1,3-galactosyltransferase ( ⁇ 1,3-GT), and is not particularly limited. Also preferred are those that express the transgene. Therefore, CAG promoter, PGK promoter, etc. can be used in place of the EF-1 ⁇ promoter.
  • a plasmid in which a porcine ⁇ 1,3-GT gene sequence is incorporated into a SIN vector construct is prepared, and a virus producing cell (human fetus) is prepared together with a packaging construct (encoding HIV-1 gag and pol only) and an envelope & Rev construct.
  • a virus producing cell human fetus
  • a packaging construct encoding HIV-1 gag and pol only
  • an envelope & Rev construct Into the kidney cell line 293T) to produce recombinant virus.
  • the expression regulatory sequence (promoter) necessary for expressing the transgene is not particularly limited, but it is desirable that the transgene is expressed in any cell throughout the body as described above.
  • Non-patent Document 8 Production of transgenic birds using a replication-defective lentiviral vector has been reported by McGrew et al. (Non-patent Document 8) and Chapman et al. (Non-patent Document 9).
  • a method of introducing a lentivirus into the blastoder on the 0th day of incubation is taken, but in the examples described later, a hole with a diameter of 1 to 1.5 cm is formed in the sharp end of the eggshell, Transgenic engineered embryos were made by injecting 1 ⁇ l of recombinant virus particles with titers of 10 8 -10 9 / ml into the dorsal artery of 2.5 day chicken embryos.
  • Manipulated embryos can be hatched by closing the holes with wraps or tape and culturing them in an incubator.
  • day 0 embryo administration method of lentivirus has been reported as a technique for obtaining a transgenic chicken.
  • administration of day 0 embryos would cause the administered virus to spread into the yolk, whereas administration of the 2.5 day embryo into the blood vessels provided the opportunity for the virus to enter the blood circulation and infect cells throughout the embryo. Therefore, the 2.5 day embryo administration method was adopted as described above.
  • the hatched transgenic engineered birds (referred to herein as G0) have heterogeneous transgenes in individual constituent cells. After G0 reaches sexual maturity, in order to obtain a transgenic chicken in which the ⁇ 1,3-GT gene is uniformly integrated in all cells constituting the body, progeny are taken by mating with other birds. Birds used for mating are not limited and may be the same kind of birds or other kinds of birds. Moreover, it is possible to use both transgenic birds and non-transgenic birds.
  • DNA is extracted from some progeny cells and the presence of the ⁇ 1,3-GT gene is confirmed by PCR.
  • a transgenic bird in which the ⁇ 1,3-GT gene is detected is referred to herein as G1.
  • G1 a transgenic bird in which the ⁇ 1,3-GT gene is detected
  • individuals expressing ⁇ -Gal can be selected and used for vaccine production.
  • Detection of ⁇ -Gal in G1 individuals can be performed using a method such as cell staining using an ⁇ -Gal antigen-specific lectin (Griffonia simplicifolia 1 isolectin B4; GS-IB 4 ).
  • transgenic bird in which the ⁇ 1,3-GT gene is incorporated by selecting a desired ⁇ -Gal expression level and a preferred expression form from a plurality of G1 and mating with other birds Can be obtained.
  • This progeny transgenic bird is designated as G2, and its progeny is designated as G3.
  • Transgenic birds of the present invention are transgenic birds (G0) and progeny G1, G2, G3, etc. (if it is a transgenic bird incorporating the ⁇ 1,3-GT gene, the progeny of the progeny is limited. Not).
  • the present invention includes a method for producing a virus that expresses ⁇ -Gal ( ⁇ -galactose epitope) ( ⁇ -Gal expressing virus).
  • Preparation of the ⁇ -Gal expression virus involves inoculating the biological sample derived from the above-described transgenic bird of the present invention with the virus, and obtaining the ⁇ -Gal expression virus from the bird-derived biological sample infected with the inoculated virus.
  • the bird-derived biological sample can be, for example, an egg born by the above-described transgenic bird of the present invention, and the egg born by the bird can be, for example, a fertilized egg or a developed bird egg.
  • the virus used for inoculation is not particularly limited as long as it has a glycoprotein on the surface of the virus.
  • the mechanism will be described later with reference to FIG. 1.
  • ⁇ -Gal expression virus can be propagated.
  • viruses having glycoproteins on the virus surface examples include influenza virus, smallpox virus, measles virus, epidemic parotitis virus, rubella virus, Newcastle disease virus, Marek's disease virus, and the like.
  • AIDS virus retrovirus
  • examples of influenza viruses include human soluenevirus A soviet type (H1N1 subtype) virus, A Hong Kong type (H3N2 subtype) virus, highly pathogenic avian influenza virus H5N1 subtype virus, and low pathogenic avian virus.
  • H9N2 subtype virus of influenza virus examples include human soluenevirus A soviet type (H1N1 subtype) virus, A Hong Kong type (H3N2 subtype) virus, highly pathogenic avian influenza virus H5N1 subtype virus, and low pathogenic avian virus.
  • H9N2 subtype virus of influenza virus examples include human soluenevirus A soviet type (H1N1 subtype) virus, A Hong Kong type (H3N2 subtype) virus, highly pathogenic
  • inoculation of viruses into eggs of developing birds follows a conventional method, for example, inoculation of influenza virus using eggs of developing chickens can be performed according to the method of WHO (see Non-Patent Document 6). Specifically, growing chicken eggs are cultured for 10 to 11 days, inoculated with the original virus solution that does not express ⁇ -Gal into the allantoic cavity, and cultured at 33 to 37 ° C for 3 to 4 days. Virus inoculation can be performed on chicken eggs.
  • the ⁇ -Gal expression virus can be prepared using a bird-derived biological sample infected with the virus of the present invention. More specifically, a virus-infected bird-derived biological sample is grown, and a virus expressing ⁇ -Gal is obtained from the grown bird-derived biological sample. A virus that expresses ⁇ -Gal has ⁇ -Gal on the surface of the virus, and ⁇ -Gal present on the surface of the virus is a foreign antigen in humans and birds that possess natural antibodies against ⁇ -Gal. Be recognized.
  • Preparation of an ⁇ -Gal expression virus using a bird-derived biological sample infected with a virus can be performed, for example, as follows.
  • the virus inoculated in the ⁇ -Gal-expressing bird-derived biological sample is released to the outside as a virus expressing ⁇ -Gal in the process of being replicated in the cells of the bird-derived biological sample. Therefore, ⁇ -Gal expression virus is included in a biological sample derived from birds. If the bird-derived biological sample is a developing bird egg, inoculate the egg with the virus and incubate for 3-4 days, then leave it overnight at 4 ° C to stop the blood flow of the embryo and be rich in ⁇ -Gal-expressing virus Collect chorioallantoic fluid (urine fluid).
  • urine fluid Collect chorioallantoic fluid
  • an ⁇ -Gal-expressing virus having ⁇ -Gal on the surface of the virus can be prepared by infecting cells of a bird-derived biological sample capable of expressing ⁇ -Gal with a virus.
  • a virus particle component protein is synthesized in a cell inoculated with a virus (upper left in the figure). If it is a ⁇ glycoprotein '', it is synthesized by a glycosyltransferase possessed by the cell. "Sugar" is added to the protein. Therefore, ⁇ -Gal can be added to a glycoprotein synthesized in a cell in which ⁇ -galactosyltransferase functions.
  • ⁇ -Gal expressing cells having ⁇ -galactose transferase
  • a virus influenza virus, measles virus, AIDS virus, etc.
  • the ⁇ -Gal-expressing virus obtained by the production method of the present invention has an immune response against the virus in humans and birds that possess natural antibodies against ⁇ -Gal compared to the same type of virus that does not express ⁇ -Gal. It will be enhanced. It shows a so-called opsonization action. This is because, in the case of ⁇ -Gal-expressing virus, anti- ⁇ -Gal antibody binds to the virus in humans and birds, and this binds to the Fc ⁇ receptor of antigen-presenting cells, resulting in effective antigen presentation. It is done.
  • the present invention includes a method for producing a vaccine using the ⁇ -Gal expression virus obtained by the production method of the present invention.
  • ⁇ -Gal expression virus is recovered as chorioallantoic fluid rich in ⁇ -Gal expression virus.
  • This chorioallantoic fluid can be used as it is for preparation of a vaccine, but can also be purified by a conventional method.
  • the purification method include a method performed by ultracentrifugation using a sucrose solution according to the method described in Non-Patent Document 7.
  • the virus obtained by the production method of the present invention can be, for example, an inactivated virus.
  • the virus contained in the vaccine can also be a viral subunit.
  • the virus contained in the vaccine can be an attenuated virus.
  • Virus inactivation can be appropriately performed using a known method as a virus inactivation treatment method.
  • the inactivation treatment include a method of inactivating the purified virus with formalin, ultraviolet light or ⁇ -propiolactone.
  • Virus subunits can be appropriately prepared using known methods. For example, after mixing the virus solution and 1% Tween 20 at a ratio of 9: 1 and leaving it for 30 minutes, add an equal amount of ether, mix vigorously, and centrifuge the resulting aqueous phase fraction as described above. A subunit vaccine can be obtained by inactivation in the same manner.
  • Attenuated virus can be obtained by reducing pathogenicity by a known method such as gene mutation.
  • viruses that are prone to mutation such as influenza virus
  • vaccines using inactivated viruses and subunit vaccines are used instead of attenuated viruses. .
  • the vaccine obtained by the production method of the present invention may contain the above virus alone or may further contain an adjuvant.
  • the adjuvant include vegetable oils such as sesame oil and rapeseed oil, mineral oils such as light liquid paraffin, aluminum hydroxide gel, and aluminum phosphate gel.
  • the administration route of the vaccine obtained by the production method of the present invention includes instillation, nasal drop, intramuscular, or subcutaneous. Moreover, when administering as an inactivated vaccine, intramuscular, intraperitoneal, or subcutaneous administration is preferable.
  • the vaccine obtained by the production method of the present invention is used for treatment for prevention and / or treatment of humans and birds infected with the virus.
  • Test Method The test method used in the following examples is described. (1) Flow cytometry using Griffonia Simplicifolia lectin I-Isolectin B 4 (GS-IB 4 ) ⁇ -Gal expressed on cells using fluorescein isothiocyanate (FITC) labeled GS-IB 4 (Vector Laboratories Inc.) After washing at 4 ° C. for 30 minutes, the cells were washed and analyzed with a FACSCanto flow cytometer (BD Biosciences).
  • FITC fluorescein isothiocyanate
  • Example 1 ⁇ 1,3-galactosyltransferase ( ⁇ 1,3-GT) gene introduction vector preparation method
  • the packaging construct incorporating the gag-pol gene was purchased from RIKEN.
  • a plasmid was constructed in which the porcine ⁇ 1,3-galactosyltransferase ( ⁇ 1,3-GT) gene was linked downstream of the EF-1 ⁇ promoter of the SIN vector construct.
  • VSV-G gene and rev gene inserted construct (10 ⁇ g), the packaging construct (10 ⁇ g) and the constructed SIN vector plasmid (17 ⁇ g) were both pre-treated with Poly-L-Lysine with a diameter of 10 cm using the calcium phosphate method.
  • the cells were incorporated into 293T cells that had been cultured to a subconfluent state on a petri dish, and cultured at 37 ° C. in a 3% CO 2 incubator for 16 hours. After removing the culture supernatant, the medium was replaced with 10 ml of fresh DMEM medium and cultured in a 10% CO 2 incubator at 37 ° C. for 48 hours to produce a lentiviral vector.
  • the culture broth containing the virus was collected, floating cells were removed through a 0.45 ⁇ m filter, and then the lentivirus was concentrated by centrifugation at 50000 g for 2 hours using an ultracentrifuge. After removing the supernatant, a lentiviral vector of ⁇ 1,3-GT gene for gene transfer was prepared by dissolving in 10 ⁇ l of HBSS solution.
  • Example 2 Production Method of Transgenic Chicken ( G1) Expressing ⁇ -Gal
  • the production method of the transgenic chicken was carried out based on the method described in Patent Document 4. Details are as follows.
  • a white leghorn chick fertilized egg was cultured in an incubator for 2.5 days, and the acute end side of the eggshell was opened when the development stage 14 to 16 of the chick embryo by Hamburger and Hamilton (Non-patent Document 10) was reached.
  • 1 ⁇ l of a lentiviral vector in which the ⁇ 1,3-GT gene sequence having a high virus titer (Note 2) was incorporated was injected into the blood vessel (Note 1) of the embryo located on the yolk.
  • the transgenic embryos were hatched by culturing them in an incubator for 18 days after closing the fenestration with a wrap.
  • transgenic transgenic chicken After breeding a transgenic transgenic chicken (G0) and reaching sexual maturity, a mating test was carried out by artificial insemination with a non-transgenic chicken (white Leghorn species) that is opposite to G0. DNA was collected from progeny cells obtained as a result of this mating test, and individuals (G1) into which the ⁇ 1,3-GT gene had been incorporated were selected by PCR. For PCR, TaKaRa ExTaq Hot Start Version (Takara Bio) was used.
  • the primer sequences used for PCR for detecting the 1,3-GT gene are GT-F (caccatgaatgtcaaaggaagagtgg) (SEQ ID NO: 4) and GT-R2 (tcagatgttatttctaaccaaat) (SEQ ID NO: 5).
  • Total amount of template DNA 100ng, 10 ⁇ Ex Taq Buffer 2 ⁇ l, dNTP mixture 1.6 ⁇ l, GT-F primer (10pmol) 0.4 ⁇ l, GT-R2 primer (10pmol) 0.4 ⁇ l, TaKaRa Ex Taq HS 0.1 ⁇ l and ultrapure water 20 ⁇ l was mixed in a 0.2 ml PCR tube. PCR conditions were as follows: preheating at 94 ° C.
  • G1 ⁇ 1,3-GT gene integrated
  • blood cells collected from the individual show hemagglutination by mixing with lectin (Griffonia Simplicifolia lectin I-Isolectin B 4 (GS-IB 4 )).
  • lectin Giriffonia Simplicifolia lectin I-Isolectin B 4 (GS-IB 4 )
  • Individuals whose blood cells were stained by a staining method using ⁇ were selected as ⁇ -Gal-expressing transgenic chicken (G1) (see Fig. 3).
  • ⁇ -Gal is expressed on the surface of blood cells, blood cells are aggregated and fluorescently stained by the action of lectin.By this method, six ⁇ 1,3-GT gene transgenic chickens (ID 293 ( (Male), ID333 (male), ID429 (female), ID466 (male), ID475 (female), ID4964 (female)), 3 (ID293, ID475, ID4964) expressed ⁇ -Gal in blood cells was.
  • Example 3 Production method of transgenic chicken (G2) expressing ⁇ -Gal 1.
  • ID293 male
  • ID466 male
  • ID4964 female
  • a mating test was performed on these three birds with a non-transgenic chicken (white leghorn species) that had not been subjected to gene transfer.
  • DNA was extracted from the collected blood, and a second-generation transgenic chicken (G2) in which the ⁇ 1,3-GT gene was incorporated was searched for by the same PCR method as in Example 2.
  • transgenic chicken (G2) that expresses ⁇ -Gal in blood cells using a hemagglutination reaction using the same lectin as in Example 2. Searched for. As a result, as shown in Table 2, 44 transgenic chickens (G2) incorporating the ⁇ 1,3-GT gene were obtained from ID4964. No fertilized eggs were obtained from ID293 and ID466. As a result of the hemagglutination reaction using lectin, it was determined that 43 out of 44 G2 obtained from ID4964 (97.7%) expressed ⁇ -Gal in blood cells.
  • ID293 and ID466 were mated with ⁇ 1,3-GT gene transgenic engineered female chickens (G0) (ID0006, ID0007 and ID0008).
  • G0 ⁇ 1,3-GT gene transgenic engineered female chickens
  • 21 hens from ID293 and 18 wings from ID466 were obtained as second-generation transgenic chickens (G2) incorporating the ⁇ 1,3-GT gene.
  • G2 second-generation transgenic chickens
  • all 21 G2 birds obtained from ID293 were determined to be transgenic chickens expressing ⁇ -Gal in blood cells.
  • all of the transgenic chickens (G2) incorporating the ⁇ 1,3-GT gene obtained from ID466 did not express ⁇ -Gal in blood cells.
  • ID333 resulted in 32 transgenic chickens (G2) in which the 1,3-GT gene was integrated, both of which had ⁇ -Gal in their blood cells. It was determined that it was not expressed.
  • Example 4 Method for preparing ⁇ -Gal-expressing influenza virus 1.
  • fertilized eggs obtained by mating an ⁇ -Gal-expressing transgenic female chicken (ID 4964) (G1) with a non-transgenic male Five of them were incubated and used for virus infection experiments.
  • Fertilized eggs (G2) started embryo development by artificially cultivating them at 90 ° C every 30 minutes at 37 ° C to 39 ° C and 60% humidity, and used as embryonated chicken eggs.
  • H9N2 virus was inoculated into the chorioallantoic cavity of 4964E1 to E5 growing chicken eggs that reached 10-11 days of age, and chorioallantoic fluid was collected from each egg 3-4 days later.
  • Virus was purified from chorioallantoic fluid collected from G2 eggs using ultracentrifugation. The obtained virus was analyzed by Western blotting. The results are shown in FIG. It was revealed that ⁇ -Gal is expressed in viruses obtained from 4964E-2, E-3, and E-4. Viruses expressing ⁇ -Gal can be donated for vaccine production. Vaccine production can be carried out by conventional methods.
  • ⁇ -Gal expression vaccine prepared from ⁇ -Gal-expressing hen's eggs is expected to be an influenza vaccine with enhanced efficacy compared to conventional vaccines for people who have anti- ⁇ -Gal antibodies and poultry Is done. 3. It is considered that the ⁇ -Gal expression vaccine can be applied not only to influenza but also to other vaccines prepared using chicken embryos.
  • the present invention is useful in the field related to vaccine production.

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Abstract

Disclosed are: a means for expressing α-Gal in a virus without using any enzyme; the production of a virus having α-Gal expressed therein by utilizing the means; the production of a vaccine from the virus; an influenza virus vaccine having a high effect (antigenicity), which is produced using a virus having enhanced immune response to a virus; and others. Specifically disclosed are: a transgenic bird which can express an α-galactose epitope (Galα1-3Galβ1-4GlcNAc-R: referred to as "α-Gal", hereinafter), wherein the bird is a chicken and may be G0 or a progeny thereof; a method for producing a transgenic bird, which comprises introducing a vector that is used for the introduction of an α1,3-galactose transferase (α1,3-GT) gene and contains an α1,3-GT gene in such a manner that the gene can be expressed, into a bird, thereby producing the transgenic bird; a biological sample, such as an egg, obtained from the transgenic bird; and a method for producing an α-Gal-expressing virus, which comprises inoculating a biological sample with a virus, cultivating the biological sample that has been inoculated with the virus, and obtaining a virus capable of expressing α-Gal from the cultivated biological sample.

Description

α-ガラクトースエピトープを発現するトランスジェニック鳥類、ウイルス及びワクチンTransgenic birds, viruses and vaccines expressing α-galactose epitopes 関連出願の相互参照Cross-reference of related applications
　本出願は、２００９年９月３０日出願の日本特願２００９－２２７７５７号の優先権を主張し、その全記載は、ここに特に開示として援用される。 This application claims the priority of Japanese Patent Application No. 2009-227757 filed on Sep. 30, 2009, the entire description of which is specifically incorporated herein by reference.
　本発明は、α-ガラクトースエピトープ（Galα1-3Galβ1-4GlcNAc-R：以下α-Galと略記することがある）を発現し得るトランスジェニック鳥類、このトランスジェニック鳥類から得られる生体試料、例えば、卵にウイルスを接種して得られるα-Galを発現するウイルス、及びこのウイルスを含有するワクチンに関する。 The present invention relates to a transgenic bird capable of expressing an α-galactose epitope (Galα1-3Galβ1-4GlcNAc-R: hereinafter sometimes abbreviated as α-Gal), a biological sample obtained from the transgenic bird, for example, an egg The present invention relates to a virus expressing α-Gal obtained by inoculating the virus, and a vaccine containing the virus.
　高病原性鳥インフルエンザおよび新型インフルエンザの世界的な発生拡大が続く中、今後、家禽のみならず人における流行の危険性も指摘されている。そのため、これまで以上にインフルエンザの予防と制御への取り組みが求められている。インフルエンザ予防には発育鶏卵で作製されるワクチンが有効とされる。現在のヒト用のインフルエンザワクチンには副作用のおそれのある免疫増強剤が含まれていないが、今後、新型インフルエンザが発生した場合は、ワクチンが緊急的に用いられる事が想定されているため、安全にワクチン効果を増強する手法の開発は重要な課題である。さらに、少量でも効果の高いワクチンを提供することで、より多くの人にワクチンを接種できる技術の確保が必要とされる。 As the global outbreak of highly pathogenic avian influenza and new influenza continues, the risk of epidemics not only in poultry but also in humans has been pointed out. Therefore, there is a need for more efforts to prevent and control influenza. Vaccines made from growing chicken eggs are effective for preventing influenza. Current human influenza vaccines do not contain immune enhancing agents that may cause side effects, but if new influenza occurs in the future, it is assumed that the vaccine will be used urgently. In addition, the development of a method for enhancing the vaccine effect is an important issue. Furthermore, by providing a highly effective vaccine even in a small amount, it is necessary to secure technology that allows more people to be vaccinated.
　α-ガラクトースエピトープ(α-Gal)はABO式血液型抗原に構造が類似した糖抗原であり、α1,3-ガラクトース転移酵素(以下α1,3-GTと略記することがある)が作用して細胞表面に発現する。α-Galは大部分の哺乳動物に発現するが、人と鳥類ではα1,3-GT遺伝子が機能していないためにα-Galの発現が無い。そのため、人と鳥類ではα-Galを外来抗原として認識し、α-Galに対する自然抗体(抗Gal抗体)を保有する(非特許文献1)。α-Galと抗α-Gal抗体の反応は、豚から人等の異種間の臓器移植等における超急性拒絶反応の原因である。本発明者のひとりである小川は、異種移植におけるα-Galと抗α-Gal抗体の制御技術に取り組むとともに、この反応を利用したガンワクチンの抗原性増強に関する研究に携わり、その有効性を確認した(非特許文献2)。 α-galactose epitope (α-Gal) is a saccharide antigen similar in structure to the ABO blood group antigen, and is activated by α1,3-galactose transferase (hereinafter abbreviated as α1,3-GT) Expressed on the cell surface. α-Gal is expressed in most mammals, but α-Gal is not expressed in humans and birds because the α1,3-GT gene does not function. Therefore, humans and birds recognize α-Gal as a foreign antigen and possess a natural antibody (anti-Gal antibody) against α-Gal (Non-patent Document 1). The reaction between α-Gal and anti-α-Gal antibody is a cause of hyperacute rejection in organ transplantation between different species such as pigs and humans. Ogawa, one of the inventors of the present invention, worked on the control technology of α-Gal and anti-α-Gal antibody in xenotransplantation, and also engaged in research on the antigenicity enhancement of cancer vaccine using this reaction, and confirmed its effectiveness (Non-Patent Document 2).
　上記α-Galを用いた抗原性増強方法をインフルエンザに応用し、α-Galを発現させたインフルエンザウイルスを作製することが出来れば、人や鳥類ではウイルスに抗α-Gal抗体が結合し、これが抗原提示細胞のFcγ受容体に結合する結果、抗原提示が効果的に行われ、ウイルスに対する免疫応答が増強される(オプソニン作用)可能性がある事は、Galiliらが報告している(非特許文献3)。エイズウイルスについての実験についてもGaliliらは報告している(非特許文献4)。 If the antigenic enhancement method using α-Gal is applied to influenza and an influenza virus expressing α-Gal can be produced, anti-α-Gal antibody binds to the virus in humans and birds. Galili et al. Have reported that binding to the Fcγ receptor of antigen-presenting cells results in effective antigen presentation and may enhance the immune response to the virus (opsonization) (non-patented). Reference 3). Galili et al. Also reported an experiment on AIDS virus (Non-patent Document 4).
特開２００２－１７６８８０号公報JP 2002-176880 A ＷＯ２００４／０６１０８１WO2004 / 061081 特開２００９－８２０３２号公報JP 2009-82032 A 特開２００６－２７１２６６号公報JP 2006-271266 A
非特許文献１～１０の全記載は、ここに特に開示として援用される。 The entire descriptions of Non-Patent Documents 1 to 10 are specifically incorporated herein by reference.
　ウイルスにα-Galを発現させる方法としては、in vitroにおける酵素反応が用いられてきている（非特許文献3、4）。しかし、この方法は、反応に用いる酵素を必要とする事、酵素反応時間を必要とする事、酵素反応液の除去を必要とする事などの点から、同方法によってワクチン用の大量のα-Gal発現ウイルスを準備する事は困難であると考えられる。 In vitro enzyme reactions have been used as a method for expressing α-Gal in viruses (Non-patent Documents 3 and 4). However, this method requires the enzyme used for the reaction, requires the enzyme reaction time, requires removal of the enzyme reaction solution, etc. It is considered difficult to prepare a Gal-expressing virus.
　そこで、本発明の目的は、酵素を用いることなく、ウイルスにおいてα-Galを発現させる手段を提供すること、この手段を用いてα-Galを発現させたウイルスを作製すること、得られたウイルスからワクチンを作製することにある。特に本発明は、ウイルスに対する免疫応答が増強されたウイルスを用いることで、効果(抗原性)の高いワクチン、特にインフルエンザウイルスワクチンを提供することにある。 Therefore, an object of the present invention is to provide a means for expressing α-Gal in a virus without using an enzyme, to produce a virus expressing α-Gal using this means, and to obtain a virus obtained To make a vaccine. In particular, the present invention is to provide a highly effective (antigenic) vaccine, particularly an influenza virus vaccine, by using a virus having an enhanced immune response to the virus.
　本発明者らは、天然にはα-Galを発現しない鳥類にα1，3-ガラクトース転移酵素遺伝子を発現し得る状態で導入し、得られたトランスジェニック鳥類から、卵等の生体試料を得、この生体試料にウイルス感染させることで、α-Gal(α-ガラクトースエピトープ)を発現するウイルスを作製できること、さらには、このα-Galを発現するウイルスを用いることで、従来のα-Galを発現しないウイルスを用いたワクチンに比べて、ウイルスに対する免疫応答が増強されたワクチンが得られることを見出して、本発明を完成させた。 The inventors of the present invention introduced a bird that does not naturally express α-Gal in a state where the α1,3-galactosyltransferase gene can be expressed, and obtained a biological sample such as an egg from the resulting transgenic bird, By infecting this biological sample with a virus, it is possible to produce a virus that expresses α-Gal (α-galactose epitope), and furthermore, by using a virus that expresses this α-Gal, conventional α-Gal is expressed. The present invention has been completed by finding that a vaccine with an enhanced immune response to the virus can be obtained compared to a vaccine using a virus that does not.
　本発明は以下のとおりである。
[1]
α-ガラクトースエピトープ（Galα1-3Galβ1-4GlcNAc-R：以下α-Gal）を発現し得るトランスジェニック鳥類。
[2]
鳥類がニワトリである[1]に記載のトランスジェニック鳥類。
[3]
G0または後代である、[1]または[2]に記載のトランスジェニック鳥類。
[4]
α1，3-ガラクトース転移酵素(α1，3-GT)遺伝子を発現し得る状態で含む、α1，3-GT遺伝子導入用ベクターを鳥類に導入して、[1]～[3]のいずれかに記載のトランスジェニック鳥類を得る、トランスジェニック鳥類の作製方法。
[5]
α1，3-GT遺伝子導入用ベクターがレンチウイルスベクターである[4]に記載のトランスジェニック鳥類の作製方法。
[6]
α1，3-GT遺伝子がマウス、ブタまたはウシ由来である、[4]または[5]に記載のトランスジェニック鳥類の作製方法。
[7]
[1]～[3]のいずれかに記載のトランスジェニック鳥類から得られる生体試料。
[8]
生体試料がトランスジェニック鳥類の産んだ卵である[7]に記載の生体試料。
[9]
トランスジェニック鳥類の産んだ卵が受精卵または発育鳥類卵である[8]に記載の生体試料。
[10]
[7]～[9]のいずれかに記載の生体試料にウイルスを接種し、ウイルスを接種した生体試料を育成し、育成した生体試料からα-Galを発現するウイルスを得ることを含む、α-Gal発現ウイルスの作製方法。
[11]
前記ウイルスがインフルエンザウイルス、天然痘ウイルス、麻疹ウイルス、流行性耳下腺炎ウイルス、風疹ウイルス、エイズウイルス、ニューカッスル病ウイルス、またはマレック病ウイルスである[10]に記載のα-Gal発現ウイルスの
作製方法。
[12]
[10]または[11]に記載の方法で、α-Galを発現するウイルスを作製し、得られたウイルスからワクチンを作製する、ワクチンの作製方法。 The present invention is as follows.
[1]
A transgenic bird capable of expressing an α-galactose epitope (Galα1-3Galβ1-4GlcNAc-R: hereinafter α-Gal).
[2]
The transgenic bird according to [1], wherein the bird is a chicken.
[3]
The transgenic bird according to [1] or [2], which is G0 or a progeny.
[Four]
An α1,3-GT gene introduction vector containing an α1,3-galactosyltransferase (α1,3-GT) gene in a state capable of being expressed is introduced into birds, and any one of [1] to [3] A method for producing a transgenic bird to obtain the described transgenic bird.
[Five]
The method for producing a transgenic bird according to [4], wherein the α1,3-GT gene introduction vector is a lentiviral vector.
[6]
The method for producing a transgenic bird according to [4] or [5], wherein the α1,3-GT gene is derived from mouse, pig or cow.
[7]
A biological sample obtained from the transgenic bird according to any one of [1] to [3].
[8]
The biological sample according to [7], wherein the biological sample is an egg laid by a transgenic bird.
[9]
The biological sample according to [8], wherein the egg laid by the transgenic bird is a fertilized egg or a developed bird egg.
[Ten]
Including inoculating the biological sample according to any one of [7] to [9] with a virus, growing a biological sample inoculated with the virus, and obtaining a virus expressing α-Gal from the grown biological sample, -Gal expression virus production method.
[11]
Production of α-Gal-expressing virus according to [10], wherein the virus is influenza virus, smallpox virus, measles virus, mumps virus, rubella virus, AIDS virus, Newcastle disease virus, or Marek's disease virus Method.
[12]
A method for producing a vaccine, wherein a virus expressing α-Gal is produced by the method according to [10] or [11], and a vaccine is produced from the obtained virus.
　トランスジェニック鳥類については既にいくつかの報告例がある(例えば、特許文献1～3)。しかし、α1，3-ガラクトース転移酵素遺伝子を発現し得る状態で導入したトランスジェニック鳥類は知られていない。さらに、このα1，3-ガラクトース転移酵素遺伝子を発現し得る状態で導入したトランスジェニック鳥類を用いたα-Galを発現するウイルスの作製、およびこのα-Galを発現するウイルスを用いた、ウイルスに対する免疫応答が増強されたワクチンについても、報告はない。 There are already some reports on transgenic birds (for example, Patent Documents 1 to 3). However, there are no known transgenic birds that have been introduced in a state in which the α1,3-galactosyltransferase gene can be expressed. Furthermore, a virus that expresses α-Gal using a transgenic bird introduced in a state capable of expressing the α1,3-galactosyltransferase gene, and a virus that uses the α-Gal-expressing virus There are no reports of vaccines with enhanced immune responses.
　本発明によれば、α-Galを発現し得る鳥類を提供することができる。 According to the present invention, a bird capable of expressing α-Gal can be provided.
　さらに本発明によれば、α-Galに対する自然抗体を保有する人と鳥類においては外来抗原として認識されるα-Galを発現するウイルスを提供することができる。 Furthermore, according to the present invention, it is possible to provide a virus that expresses α-Gal that is recognized as a foreign antigen in humans and birds that possess natural antibodies against α-Gal.
　加えて本発明によれば、α-Galを発現するウイルスを用いることで、従来のα-Galを発現しないウイルスを用いたワクチンに比べて、α-Galに対する自然抗体を保有する人と鳥類においては、ウイルスに対する免疫応答が増強されたワクチンを提供することができる。 In addition, according to the present invention, by using a virus that expresses α-Gal, compared to conventional vaccines using viruses that do not express α-Gal, humans and birds possessing natural antibodies against α-Gal. Can provide a vaccine with an enhanced immune response to the virus.
ウイルス表面に糖タンパクを保有するウイルスを、α-ガラクトース転移酵素を保有し、α-Galを発現させた細胞系に接種すると、細胞内においてインフルエンザウイルスの複製とα-Galの付加が行われ、α-Gal発現ウイルスを増殖させる事ができることの概略説明図である。When inoculating a cell line that possesses a glycoprotein on the surface of a virus into a cell line that possesses α-galactosyltransferase and expresses α-Gal, replication of influenza virus and addition of α-Gal are performed in the cell, It is a schematic explanatory drawing that an α-Gal expression virus can be propagated. PCR法によりα1,3-GT遺伝子検出して、α1,3-GTトランスジェニックニワトリ(G1)を検索した結果を示す。後代ID429にα1,3-GT遺伝子配列が検出された。The results of detecting α1,3-GT gene by PCR and searching for α1,3-GT transgenic chicken (G1) are shown. The α1,3-GT gene sequence was detected in the progeny ID429. α1,3-GT遺伝子トランスジェニックニワトリ(G1)血球のレクチン染色の結果を示す。The results of lectin staining of α1,3-GT gene transgenic chicken (G1) blood cells are shown. α1,3-GT遺伝子トランスジェニックニワトリ(G1)精子のレクチン染色の結果を示す。The results of lectin staining of α1,3-GT gene transgenic chicken (G1) sperm are shown. 実施例2で得られたα1,3-GT遺伝子トランスジェニックニワトリ(G1)ID4964、実施例3で得られたID4964由来のα1,3-GT遺伝子トランスジェニックニワトリ(G2)及び無処置ニワトリ(コントロール)の血球におけるα-Gal発現のフローサイトメーターによる解析結果を示す。(グラフ内の括弧内の数字はα-Galを発現する赤血球の割合を示す)Α1,3-GT gene transgenic chicken (G1) ID4964 obtained in Example 2, α1,3-GT gene transgenic chicken (G2) derived from ID4964 obtained in Example 3 and untreated chicken (control) The analysis result by the flow cytometer of (alpha) -Gal expression in the blood cell of is shown. (The numbers in parentheses in the graph indicate the percentage of red blood cells that express α-Gal) 実施例3で得られたID4964由来のα1,3-GT遺伝子トランスジェニックニワトリ(G2) の血球におけるα-Gal発現のフローサイトメーターによる解析結果を示す。(グラフ内の括弧内の数字はα-Galを発現する赤血球の割合を示す)The analysis result by the flow cytometer of (alpha) -Gal expression in the blood cell of (alpha) 1,3-GT gene transgenic chicken (G2) sputum derived from ID4964 obtained in Example 3 is shown. (The numbers in parentheses in the graph indicate the percentage of red blood cells that express α-Gal) 実施例3で得られたID4964由来のα1,3-GT遺伝子トランスジェニックニワトリ(G2) の血球におけるα-Gal発現のフローサイトメーターによる解析結果を示す。(グラフ内の括弧内の数字はα-Galを発現する赤血球の割合を示す)The analysis result by the flow cytometer of (alpha) -Gal expression in the blood cell of (alpha) 1,3-GT gene transgenic chicken (G2) sputum derived from ID4964 obtained in Example 3 is shown. (The numbers in parentheses in the graph indicate the percentage of red blood cells that express α-Gal) 実施例3で得られたID4964由来のα1,3-GT遺伝子トランスジェニックニワトリ(G2) の血球におけるα-Gal発現のフローサイトメーターによる解析結果を示す。(グラフ内の括弧内の数字はα-Galを発現する赤血球の割合を示す)The analysis result by the flow cytometer of (alpha) -Gal expression in the blood cell of (alpha) 1,3-GT gene transgenic chicken (G2) sputum derived from ID4964 obtained in Example 3 is shown. (The numbers in parentheses in the graph indicate the percentage of red blood cells that express α-Gal) 実施例3で得られたID4964由来のα1,3-GT遺伝子トランスジェニックニワトリ(G2) の血球におけるα-Gal発現のフローサイトメーターによる解析結果を示す。(グラフ内の括弧内の数字はα-Galを発現する赤血球の割合を示す)The analysis result by the flow cytometer of (alpha) -Gal expression in the blood cell of (alpha) 1,3-GT gene transgenic chicken (G2) sputum derived from ID4964 obtained in Example 3 is shown. (The numbers in parentheses in the graph indicate the percentage of red blood cells that express α-Gal) 実施例4で得られた、α1,3-GT遺伝子トランスジェニックニワトリ胚 (G2）で増殖させたH9N2亜型インフルエンザウイルスをウエスタンブロッティング法にてα-Gal発現の確認　(GS-IB4を用いたWestern blotting による解析）した結果を示す。4964E-2, 3, 4から得られたウイルスにα-Gal発現が確認された。Confirmation of α-Gal expression by Western blotting of H9N2 subtype influenza virus grown in α1,3-GT gene transgenic chicken embryo (G2) obtained in Example 4 (using GS-IB 4 ) (Analysis by Western blotting) shows the results. Α-Gal expression was confirmed in the viruses obtained from 4964E-2, 3, and 4.
[トランスジェニック鳥類]
　本発明は、α-ガラクトースエピトープ（Galα1-3Galβ1-4GlcNAc-R：以下α-Gal）を発現し得るトランスジェニック鳥類に関する。 [Transgenic birds]
The present invention relates to a transgenic bird capable of expressing an α-galactose epitope (Galα1-3Galβ1-4GlcNAc-R: hereinafter α-Gal).
　α-ガラクトースエピトープ(α-Gal)は、Galα1-3Galβ1-4GlcNAc-Rで示される糖鎖であり、N-アセチルラクトサミンを発現する糖タンパク(Galβ1-4GlcNAc-R、Rはタンパクを表す)にα1,3-ガラクトース転移酵素を作用させて、基質としてのガラクトースをN-アセチルラクトサミンのガラクトース基にα1-3結合させることで、生成される糖鎖である。 α-galactose epitope (α-Gal) is a sugar chain represented by Galα1-3Galβ1-4GlcNAc-R, and is a glycoprotein that expresses N-acetyllactosamine (Galβ1-4GlcNAc-R, R represents protein) It is a sugar chain produced by allowing α1,3-galactose transferase to act and galactose as a substrate to be α1-3 linked to the galactose group of N-acetyllactosamine.
　本発明において、トランスジェニック鳥類を得るために用いる、鳥類へのα1,3-GT遺伝子の導入方法は、個体全身の細胞に導入遺伝子が組み込まれ、目的とするα-Galタンパクを発現する方法であれば特に限定されない。具体的には、α1，3-ガラクトース転移酵素(α1，3-GT)遺伝子を発現し得る状態で含む、α1，3-GT遺伝子導入用ベクターを用いて鳥類にα1，3-GT遺伝子を導入することで、このトランスジェニック鳥類を作製できる。 In the present invention, the method for introducing an α1,3-GT gene into birds used for obtaining transgenic birds is a method in which the transgene is incorporated into cells of the whole body of the individual and the desired α-Gal protein is expressed. If there is no particular limitation. Specifically, the α1,3-GT gene is introduced into birds using the α1,3-GT gene transfer vector, which is capable of expressing the α1,3-galactosyltransferase (α1,3-GT) gene. By doing so, this transgenic bird can be produced.
　α1，3-GT遺伝子は、マウス由来、ブタ由来及びウシ由来のもの等が知られており、GenBankから遺伝子配列情報を入手することができる[マウス：GenBank accession number M85153; J. Biol. Chem. 267, 5534-5541 (1992)(配列番号1), ブタ：GenBank accession number L36535；Xenotransplantation 1, 81-88, 1994(配列番号2), ウシ：GenBank accession number J04989; J. Biol. Chem. 264, 14290-14297, 1989(配列番号3)]。 The α1,3-GT gene is known to be derived from mouse, porcine or bovine, etc., and gene sequence information can be obtained from GenBank [Mouse: GenBank accession number M85153; J. Biol. Chem. 267, 5534-5541 1992 (1992) (SEQ ID NO: 1), Pig: GenBank accession number L36535; Xenotransplantation 1, 81-88, 1994 (SEQ ID NO: 2), Cattle: GenBank accession number J04989; J. Biol. Chem. 264, 14290-14297, 1989 (SEQ ID NO: 3)].
　本発明のベクターを用いて遺伝子導入をする鳥類とは、分類学的分類 鳥綱「Ａｖｅｓ」の生物のいずれもの種、亜種または品種を指すことを意図する（限定されるものではないが、ニワトリ、シチメンチョウ、アヒル、ガチョウ、ウズラ、キジ、オウム、フィンチ、タカ、カラス、ダチョウ、エミュー、およびヒクイドリのような生物など）。前記用語は、セキショクヤケイ(Gallus gallus)またはニワトリ（例えば、ホワイトレグホン(White Leghorn)、ブラウンレグホン(Brown Leghorn)、バールロック(Barred-Rock)、サセックス(Sussex)、ニューハンプシャー(New Hampshire)、ロードアイランド(Rhode Island)、オーストラロープ(Australorp)、ミノルカ(Minorca)、アムロックス(Amrox)、カリフォルニアグレイ(California Gray)、イタリアンパーティッジカラード(Italian Partidge-colored)）の様々な系統、ならびに一般に繁殖されるシチメンチョウ、キジ、ウズラ、アヒル、ダチョウ、および他の家禽の系統を包含する。なかでもニワトリやウズラは入手が容易であり産卵種としても多産であり、長年の飼育経験により安全性が認められている点で特に好ましい。 Birds to which gene transfer is performed using the vectors of the present invention are intended to refer to any species, subspecies or breed of the taxonomic taxon "Aves" organisms (but not limited to, Such as chickens, turkeys, ducks, geese, quail, pheasants, parrots, finch, hawks, crows, ostriches, emu, and cassowary). The terms include Gallus gallus or chicken (e.g. White Leghorn, Brown ホ ン Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island ( Rhode Island, Australorp, Minorca, Amrox, California Gray, Italian Partidge-colored), and commonly bred turkeys Includes pheasant, quail, duck, ostrich, and other poultry strains. Of these, chickens and quails are particularly preferred because they are readily available and are prolific as egg-laying species, and have been recognized for safety by many years of breeding experience.
　本発明では、α1，3-GT遺伝子導入用ベクターとしては、例えば、α1,3-GT遺伝子をコードするレンチウイルスベクターを用いることができる。レンチウイルスとしては、安全性の観点から複製能欠損型のヒト免疫不全ウイルス（ＨＩＶ）を用いことができる。 In the present invention, as the α1,3-GT gene introduction vector, for example, a lentiviral vector encoding the α1,3-GT gene can be used. As a lentivirus, replication-defective human immunodeficiency virus (HIV) can be used from the viewpoint of safety.
　レンチウイルスベクターは、α1，3-ガラクトース転移酵素(α1,3-GT)を発現させるために必要な発現調節配列(プロモータ)を含むものであり、特に限定はされないが、全身のいずれの細胞においても導入遺伝子が発現するものが望ましい。従って、EF-1αプロモータのかわりに、CAGプロモータ、PGKプロモータ等も利用可能である。 The lentiviral vector contains an expression regulatory sequence (promoter) necessary for expressing α1,3-galactosyltransferase (α1,3-GT), and is not particularly limited. Also preferred are those that express the transgene. Therefore, CAG promoter, PGK promoter, etc. can be used in place of the EF-1α promoter.
　SINベクターコンストラクトに、例えば、ブタのα1,3-GT遺伝子配列を組み込んだプラスミドを作製し、パッケージングコンストラクト(HIV-１のgag, polのみをコード)およびエンベロープ＆Revコンストラクトと共にウイルス産生細胞(ヒト胎児腎細胞株293T)へ導入し、組換えウイルスを産生させる。この場合、導入遺伝子を発現させるために必要な発現調節配列(プロモータ)は、特に限定はされないが、前述のように、全身のいずれの細胞においても導入遺伝子が発現するものが望ましい。 For example, a plasmid in which a porcine α1,3-GT gene sequence is incorporated into a SIN vector construct is prepared, and a virus producing cell (human fetus) is prepared together with a packaging construct (encoding HIV-1 gag and pol only) and an envelope & Rev construct. Into the kidney cell line 293T) to produce recombinant virus. In this case, the expression regulatory sequence (promoter) necessary for expressing the transgene is not particularly limited, but it is desirable that the transgene is expressed in any cell throughout the body as described above.
　複製能欠損型のレンチウイルスベクターを用いるトランスジェニック鳥類の作製は、ＭｃＧｒｅｗら(非特許文献8)やＣｈａｐｍａｎら(非特許文献9)により報告されている。これらの文献では、レンチウイルスを孵卵0日目の胚盤葉へ導入する方法が採られているが、後述の実施例においては、卵殻鋭端部に直径1～1.5cmの穴を開け、ウイルス力価108-109/mlの組換えウイルス粒子の1μlを鶏2.5日胚の後背動脈中へ注入することによりトランスジェニック操作胚を作製した。操作胚は、穴を開けた部分をラップまたはテープで塞ぎ、孵卵器で培養することにより孵化させることができる。尚、レンチウイルスの0日胚投与法は、遺伝子導入ニワトリが得られる技術として報告されている。しかし、0日胚投与では投与ウイルスが卵黄中に拡散してしまうと考えられ、それに対し、2.5日胚の血管中への投与法はウイルスが血液循環に乗り、胚全体の細胞に感染する機会が増えると考えられることから、上記のように2.5日胚への投与法を採用した。 Production of transgenic birds using a replication-defective lentiviral vector has been reported by McGrew et al. (Non-patent Document 8) and Chapman et al. (Non-patent Document 9). In these documents, a method of introducing a lentivirus into the blastoder on the 0th day of incubation is taken, but in the examples described later, a hole with a diameter of 1 to 1.5 cm is formed in the sharp end of the eggshell, Transgenic engineered embryos were made by injecting 1 μl of recombinant virus particles with titers of 10 8 -10 9 / ml into the dorsal artery of 2.5 day chicken embryos. Manipulated embryos can be hatched by closing the holes with wraps or tape and culturing them in an incubator. In addition, the day 0 embryo administration method of lentivirus has been reported as a technique for obtaining a transgenic chicken. However, administration of day 0 embryos would cause the administered virus to spread into the yolk, whereas administration of the 2.5 day embryo into the blood vessels provided the opportunity for the virus to enter the blood circulation and infect cells throughout the embryo. Therefore, the 2.5 day embryo administration method was adopted as described above.
　孵化したトランスジェニック操作鳥類(本明細書ではG0と称する)では、個体構成細胞に不均一に導入遺伝子を有している。G0が性成熟に達した後、体を構成する全ての細胞に均一にα1,3-GT遺伝子が組み込まれているトランスジェニックニワトリを得るため、他鳥類と交配することにより後代をとる。交配に用いる鳥類は限定されることはなく、同種の鳥類でも他種の鳥類でも良い。また、トランスジェニック鳥類でも非トランスジェニック鳥類でも用いることが可能である。 The hatched transgenic engineered birds (referred to herein as G0) have heterogeneous transgenes in individual constituent cells. After G0 reaches sexual maturity, in order to obtain a transgenic chicken in which the α1,3-GT gene is uniformly integrated in all cells constituting the body, progeny are taken by mating with other birds. Birds used for mating are not limited and may be the same kind of birds or other kinds of birds. Moreover, it is possible to use both transgenic birds and non-transgenic birds.
　後代の一部の細胞からDNAを抽出し、α1,3-GT遺伝子の存在をPCR法により確認する。後代のうちα1,3-GT遺伝子が検出されたトランスジェニック鳥類を本明細書ではG1と称する。このようにして作製したG1のうち、α-Galを発現する個体を選抜し、ワクチン生産に供することができる。G1個体におけるα-Galの検出は、α-Gal抗原特異的レクチン（Griffonia simplicifolia 1 isolectin B4; GS-IB4）を用いた細胞染色等の方法を用いることができる。G1が得られれば、複数のG1の中から所望のα-Gal発現量、発現形式の好ましい個体を選抜し、他鳥類と交配させることによりにα1,3-GT遺伝子が組み込まれたトランスジェニック鳥類を得ることが出来るようになる。この後代トランスジェニック鳥類をG2、さらにその後代をG3などとする。本発明のトランスジェニック鳥類は、トランスジェニック操作鳥類(G0)、および後代であるG1、G2、G3など(α1,3-GT遺伝子が組み込まれたトランスジェニック鳥類であれば、後代の代数に限定はない)である。 DNA is extracted from some progeny cells and the presence of the α1,3-GT gene is confirmed by PCR. Among the progeny, a transgenic bird in which the α1,3-GT gene is detected is referred to herein as G1. Among the G1 thus produced, individuals expressing α-Gal can be selected and used for vaccine production. Detection of α-Gal in G1 individuals can be performed using a method such as cell staining using an α-Gal antigen-specific lectin (Griffonia simplicifolia 1 isolectin B4; GS-IB 4 ). Once G1 is obtained, a transgenic bird in which the α1,3-GT gene is incorporated by selecting a desired α-Gal expression level and a preferred expression form from a plurality of G1 and mating with other birds Can be obtained. This progeny transgenic bird is designated as G2, and its progeny is designated as G3. Transgenic birds of the present invention are transgenic birds (G0) and progeny G1, G2, G3, etc. (if it is a transgenic bird incorporating the α1,3-GT gene, the progeny of the progeny is limited. Not).
[α-Gal発現ウイルスの作製方法]
　本発明は、α-Gal(α-ガラクトースエピトープ）を発現するウイルス(α-Gal発現ウイルス)の作製方法を包含する。 [Method for producing α-Gal expressing virus]
The present invention includes a method for producing a virus that expresses α-Gal (α-galactose epitope) (α-Gal expressing virus).
　α-Gal発現ウイルスの作製は、上記本発明のトランスジェニック鳥類に由来する生体試料にウイルスを接種し、接種したウイルスに感染した鳥類由来生体試料からα-Gal発現ウイルスを得る。 Preparation of the α-Gal expression virus involves inoculating the biological sample derived from the above-described transgenic bird of the present invention with the virus, and obtaining the α-Gal expression virus from the bird-derived biological sample infected with the inoculated virus.
　鳥類由来生体試料とは、例えば、上記本発明のトランスジェニック鳥類の産んだ卵であることができ、鳥類の産んだ卵は、例えば、受精卵または発育鳥類卵であることができる。 The bird-derived biological sample can be, for example, an egg born by the above-described transgenic bird of the present invention, and the egg born by the bird can be, for example, a fertilized egg or a developed bird egg.
　接種に用いるウイルスは、ウイルス表面に糖タンパクを保有するウイルスであれば、特に制限はない。機構の説明は図1を用いて後述するが、ウイルス表面に糖タンパクを保有するウイルスを、α1，3-ガラクトース転移酵素を保有する鳥類由来生体試料に接種すると、この生体試料中の細胞においてα-Gal発現ウイルスを増殖させる事ができる。 The virus used for inoculation is not particularly limited as long as it has a glycoprotein on the surface of the virus. The mechanism will be described later with reference to FIG. 1. When a virus having a glycoprotein on the surface of a virus is inoculated into an avian-derived biological sample having α1,3-galactosyltransferase, α -Gal expression virus can be propagated.
　ウイルス表面に糖タンパクを保有するウイルスとしては、例えば、インフルエンザウイルス、天然痘ウイルス、麻疹ウイルス、流行性耳下腺炎ウイルス、風疹ウイルス、ニューカッスル病ウイルス、マレック病ウイルス等であることができる。尚、エイズウイルス（レトロウイルス）については、エンベロープ上に糖タンパクがあり、手法は本発明と異なるが、α-Galを発現する方法をGaliliが報告している(非特許文献4)。さらに、インフルエンザウイルスとしては、例えば、ヒトインルエンザウイルスのAソ連型(H1N1亜型)ウイルスやA香港型(H3N2亜型)ウイルス、高病原性トリインフルエンザウイルスのH5N1亜型ウイルス、低病原性トリインフルエンザウイルスのH9N2亜型ウイルス等を挙げることができる。 Examples of viruses having glycoproteins on the virus surface include influenza virus, smallpox virus, measles virus, epidemic parotitis virus, rubella virus, Newcastle disease virus, Marek's disease virus, and the like. For AIDS virus (retrovirus), there is a glycoprotein on the envelope, and the method is different from that of the present invention, but Galili has reported a method of expressing α-Gal (Non-patent Document 4). Furthermore, examples of influenza viruses include human soluenevirus A soviet type (H1N1 subtype) virus, A Hong Kong type (H3N2 subtype) virus, highly pathogenic avian influenza virus H5N1 subtype virus, and low pathogenic avian virus. And the H9N2 subtype virus of influenza virus.
　発育鳥類卵等へのウイルスの接種は常法に従うが、例えば発育鶏卵を用いたインフルエンザウイルスの接種は、WHOの方法(非特許文献6参照)に準じて行うことができる。具体的には、発育鶏卵を10～11日間培養し、α-Galを発現していない元のウイルス液を尿膜腔へ接種して33～37℃で3～4日間培養することで、発育鶏卵へのウイルス接種を行うことができる。 Although inoculation of viruses into eggs of developing birds follows a conventional method, for example, inoculation of influenza virus using eggs of developing chickens can be performed according to the method of WHO (see Non-Patent Document 6). Specifically, growing chicken eggs are cultured for 10 to 11 days, inoculated with the original virus solution that does not express α-Gal into the allantoic cavity, and cultured at 33 to 37 ° C for 3 to 4 days. Virus inoculation can be performed on chicken eggs.
　α-Gal発現ウイルスは、上記本発明のウイルスに感染した鳥類由来生体試料を用いて作製することができる。より具体的には、ウイルス感染した鳥類由来生体試料を育成して、育成した鳥類由来生体試料からα-Galを発現するウイルスを得る。α-Galを発現するウイルスとは、ウイルスの表面にα-Galを有するものであり、ウイルスの表面に存在するα-Galは、α-Galに対する自然抗体を保有する人や鳥類では外来抗原として認識される。 The α-Gal expression virus can be prepared using a bird-derived biological sample infected with the virus of the present invention. More specifically, a virus-infected bird-derived biological sample is grown, and a virus expressing α-Gal is obtained from the grown bird-derived biological sample. A virus that expresses α-Gal has α-Gal on the surface of the virus, and α-Gal present on the surface of the virus is a foreign antigen in humans and birds that possess natural antibodies against α-Gal. Be recognized.
　ウイルスに感染した鳥類由来生体試料を用いてのα-Gal発現ウイルスの作製は、例えば、以下のように実施することができる。α-Gal発現鳥類由来生体試料に接種したウイルスは、鳥類由来生体試料の細胞内で複製される過程でα-Galを発現するウイルスとなって細胞外へ放出される。そのため、α-Gal発現ウイルスは鳥類由来生体試料中に含まれる。鳥類由来生体試料が発育鳥類卵の場合、卵へウイルスを接種し3～4日間培養した後に、4℃で一晩置き、胚の血流を止めて、α-Gal発現ウイルスが豊富に含まれる漿尿液(尿液)を回収する。 Preparation of an α-Gal expression virus using a bird-derived biological sample infected with a virus can be performed, for example, as follows. The virus inoculated in the α-Gal-expressing bird-derived biological sample is released to the outside as a virus expressing α-Gal in the process of being replicated in the cells of the bird-derived biological sample. Therefore, α-Gal expression virus is included in a biological sample derived from birds. If the bird-derived biological sample is a developing bird egg, inoculate the egg with the virus and incubate for 3-4 days, then leave it overnight at 4 ° C to stop the blood flow of the embryo and be rich in α-Gal-expressing virus Collect chorioallantoic fluid (urine fluid).
　尚、上記α-Galを発現し得る鳥類由来生体試料の細胞にウイルスを感染させることによって、ウイルスの表面にα-Galを有するα-Gal発現ウイルスを作製できる。この点を、図1を用いて具体的に説明する。一般に、ウイルス(図中の左上)が接種された細胞内では、ウイルス粒子の構成タンパクが合成されるが、それが「糖タンパク」である場合には、細胞が保有する糖転移酵素によって合成された「糖」がタンパクに付加される。そのため、α-ガラクトース転移酵素が機能する細胞内で合成された糖タンパクにはα-Galが付加され得る事になる。従って、本発明のように、ウイルス表面に糖タンパクを保有するウイルス（インフルエンザウイルス、麻疹ウイルス、エイズウイルス等）をα-Gal発現細胞（α-ガラクトース転移酵素を保有）に接種すると、α-Gal発現ウイルスを増殖させる事ができる。 In addition, an α-Gal-expressing virus having α-Gal on the surface of the virus can be prepared by infecting cells of a bird-derived biological sample capable of expressing α-Gal with a virus. This point will be specifically described with reference to FIG. In general, a virus particle component protein is synthesized in a cell inoculated with a virus (upper left in the figure). If it is a `` glycoprotein '', it is synthesized by a glycosyltransferase possessed by the cell. "Sugar" is added to the protein. Therefore, α-Gal can be added to a glycoprotein synthesized in a cell in which α-galactosyltransferase functions. Therefore, as in the present invention, when α-Gal expressing cells (having α-galactose transferase) are inoculated with a virus (influenza virus, measles virus, AIDS virus, etc.) having a glycoprotein on the surface of the virus, α-Gal Expression virus can be propagated.
　本発明の作製方法で得たα-Gal発現ウイルスは、α-Galを発現しない同タイプのウイルスと比較して、α-Galに対する自然抗体を保有する人や鳥類においては、ウイルスに対する免疫応答が増強されたものになる。所謂、オプソニン作用を示す。これは、α-Gal発現ウイルスの場合、人や鳥類ではウイルスに抗α-Gal抗体が結合し、これが抗原提示細胞のFcγ受容体に結合する結果、抗原提示が効果的に行われるためと考えられる。 The α-Gal-expressing virus obtained by the production method of the present invention has an immune response against the virus in humans and birds that possess natural antibodies against α-Gal compared to the same type of virus that does not express α-Gal. It will be enhanced. It shows a so-called opsonization action. This is because, in the case of α-Gal-expressing virus, anti-α-Gal antibody binds to the virus in humans and birds, and this binds to the Fcγ receptor of antigen-presenting cells, resulting in effective antigen presentation. It is done.
[ワクチンの作製方法]
　本発明は、上記本発明の作製方法で得たα-Gal発現ウイルスを用いて、ワクチンを作製する方法を包含する。 [Method for preparing vaccine]
The present invention includes a method for producing a vaccine using the α-Gal expression virus obtained by the production method of the present invention.
　上述のように、α-Gal発現ウイルスは、α-Gal発現ウイルスが豊富に含まれる漿尿液として回収される。この漿尿液は、そのままワクチンの作製に用いることもできるが、常法により精製することもできる。精製方法としては、例えば、非特許文献7に記載の方法に従いスクロース液を用いた超遠心により行う方法を挙げることができる。 As described above, α-Gal expression virus is recovered as chorioallantoic fluid rich in α-Gal expression virus. This chorioallantoic fluid can be used as it is for preparation of a vaccine, but can also be purified by a conventional method. Examples of the purification method include a method performed by ultracentrifugation using a sucrose solution according to the method described in Non-Patent Document 7.
　本発明の作製方法で得られるウイルスは、例えば、不活性化されたウイルスであることができる。また、ワクチンに含有されるウイルスは、ウイルスのサブユニットであることもできる。さらに、ワクチンに含有されるウイルスは、弱毒化ウイルスであることもできる。 The virus obtained by the production method of the present invention can be, for example, an inactivated virus. The virus contained in the vaccine can also be a viral subunit. Furthermore, the virus contained in the vaccine can be an attenuated virus.
　ウイルスの不活性化は、ウイルスの不活性化処理方法として公知の方法を用いて適宜行うことができる。不活性化処理としては、例えば、精製ウイルスをホルマリン、紫外線またはβ-プロピオラクトンにより不活化する方法を挙げることができる。 Virus inactivation can be appropriately performed using a known method as a virus inactivation treatment method. Examples of the inactivation treatment include a method of inactivating the purified virus with formalin, ultraviolet light or β-propiolactone.
　ウイルスのサブユニット(成分)は、公知の方法を用いて適宜作製することができる。例えば、ウイルス液と1％ Tween 20を9：1の割合で混和して30分間放置した後、等量のエーテルを加えて激しく混和し、それを遠心して得られた水相画分を上述と同様の方法で不活化することで、サブユニットワクチンを得ることができる。 Virus subunits (components) can be appropriately prepared using known methods. For example, after mixing the virus solution and 1% Tween 20 at a ratio of 9: 1 and leaving it for 30 minutes, add an equal amount of ether, mix vigorously, and centrifuge the resulting aqueous phase fraction as described above. A subunit vaccine can be obtained by inactivation in the same manner.
　弱毒化ウイルスは、遺伝子変異などの公知の方法によって病原性を低減させることで得ることができる。但し、インフルエンザウイルスのような変異を起こしやすいウイルスについては、弱毒化ワクチンの使用は懸念が多いため、一般には、弱毒化ウイルスではなく、不活性化ウイルスを用いたワクチンやサブユニットワクチンが用いられる。 Attenuated virus can be obtained by reducing pathogenicity by a known method such as gene mutation. However, for viruses that are prone to mutation such as influenza virus, there are many concerns about the use of attenuated vaccines. In general, vaccines using inactivated viruses and subunit vaccines are used instead of attenuated viruses. .
　本発明の作製方法で得られるワクチンは、上記ウイルスを単独で含む場合と、アジュバントをさらに含有する場合がある。アジュバントとしては、例えば、ゴマ油、菜種油等の植物油、軽質流動パラフィン等の鉱物オイル、水酸化アルミニウムゲル、リン酸アルミニウムゲル等を挙げることもできる。 The vaccine obtained by the production method of the present invention may contain the above virus alone or may further contain an adjuvant. Examples of the adjuvant include vegetable oils such as sesame oil and rapeseed oil, mineral oils such as light liquid paraffin, aluminum hydroxide gel, and aluminum phosphate gel.
　本発明の作製方法で得られるワクチンの投与経路としては、点眼、点鼻、筋肉内、又は皮下が挙げられる。また、不活化ワクチンとして投与する場合には筋肉内、腹腔内又は皮下への投与が好ましい。 The administration route of the vaccine obtained by the production method of the present invention includes instillation, nasal drop, intramuscular, or subcutaneous. Moreover, when administering as an inactivated vaccine, intramuscular, intraperitoneal, or subcutaneous administration is preferable.
　本発明の作製方法で得られるワクチンは、前記ウイルスに感染するヒトや鳥類の予防および／または治療のための処置に用いられる。 The vaccine obtained by the production method of the present invention is used for treatment for prevention and / or treatment of humans and birds infected with the virus.
　以下、本発明を実施例によりさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to examples.
試験方法
　以下の実施例で用いた試験方法について記載する。
(1) Griffonia Simplicifolia lectin I-Isolectin B4(GS-IB4)を用いたフローサイトメトリー
　細胞上に発現するα-Galをfluorescein isothiocyanate (FITC) 標識GS-IB4(Vector Laboratories Inc.) を用いて４℃で30分間染色した後に洗浄し、FACSCanto flow cytometer (BD Biosciences) にて解析した。 Test Method The test method used in the following examples is described.
(1) Flow cytometry using Griffonia Simplicifolia lectin I-Isolectin B 4 (GS-IB 4 ) α-Gal expressed on cells using fluorescein isothiocyanate (FITC) labeled GS-IB 4 (Vector Laboratories Inc.) After washing at 4 ° C. for 30 minutes, the cells were washed and analyzed with a FACSCanto flow cytometer (BD Biosciences).
(2) Western blotting法
　ウイルスに発現するα-Galを調べるためのWestern blottingは以下のように行った。ウイルスのタンパクを2-mercaptoethanolの存在下でSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) により分離した後、polyvinylidene difluoride (PVDF) membrane (Bio-Rad) に転写し、1％Alkali-soluble Casein (Novagen) を用いてブロッキングを行った。その後、HRP標識GS-IB4 (Sigma-Aldrich)と室温で１時間反応させ、反応後のPVDF membraneをECL Western blotting analysis system (Amersham Biosciences)によって発光させ、LAS-3000 (FujiFilm)を用いて解析した。 (2) Western blotting method Western blotting for examining α-Gal expressed in virus was performed as follows. Viral proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol, then transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad), and 1% Alkali-soluble Casein ( Novagen) was used for blocking. Then, react with HRP-labeled GS-IB 4 (Sigma-Aldrich) at room temperature for 1 hour. After the reaction, the PVDF membrane was lit by ECL Western blotting analysis system (Amersham Biosciences) and analyzed using LAS-3000 (FujiFilm). did.
実施例1
α1，3-ガラクトース転移酵素(α1,3-GT)遺伝子導入用ベクター作製法
　レンチウイルスベクターの作製に必要なSINベクターコンストラクト、ウイルスベクターのエンベロープとなるVSV-G遺伝子とrev遺伝子が挿入されたコンストラクトおよびgag-pol遺伝子を組み込んだパッケージングコンストラクトは、理化学研究所から購入した。SINベクターコンストラクトのEF-1αプロモーターの下流にブタのα1，3-ガラクトース転移酵素(α1,3-GT)遺伝子を連結したプラスミドを構築した。VSV-G遺伝子とrev遺伝子が挿入されたコンストラクト(10μg)、パッケージングコンストラクト(10μg)および構築したSINベクタープラスミド(17μg)を共にリン酸カルシウム法を用いて、あらかじめ直径10cmのPoly-L-Lysine処理したシャーレ上でサブコンフルエント状態まで培養していた293T細胞へ組み込み、37℃、3% CO2インキュベーターで16時間培養した。培養上清を除去した後、新しいDMEM培地10 mlと置換し、37℃、10% CO2インキュベーターで48時間培養することによりレンチウイルスベクターを産生させた。ウイルスを含む培養液を回収し、0.45μmフィルターを通じして浮遊細胞を除去した後、超遠心機を用いて50000g、2時間遠心することによりレンチウイルスを濃縮した。上清を除去した後、10μlのHBSS溶液に溶解することにより遺伝子導入用のα1,3-GT遺伝子のレンチウイルスベクターを作製した。  Example 1
α1,3-galactosyltransferase (α1,3-GT) gene introduction vector preparation method SIN vector construct necessary for the preparation of lentiviral vector, VSV-G gene that becomes the envelope of the viral vector and rev gene inserted The packaging construct incorporating the gag-pol gene was purchased from RIKEN. A plasmid was constructed in which the porcine α1,3-galactosyltransferase (α1,3-GT) gene was linked downstream of the EF-1α promoter of the SIN vector construct. The VSV-G gene and rev gene inserted construct (10 μg), the packaging construct (10 μg) and the constructed SIN vector plasmid (17 μg) were both pre-treated with Poly-L-Lysine with a diameter of 10 cm using the calcium phosphate method. The cells were incorporated into 293T cells that had been cultured to a subconfluent state on a petri dish, and cultured at 37 ° C. in a 3% CO 2 incubator for 16 hours. After removing the culture supernatant, the medium was replaced with 10 ml of fresh DMEM medium and cultured in a 10% CO 2 incubator at 37 ° C. for 48 hours to produce a lentiviral vector. The culture broth containing the virus was collected, floating cells were removed through a 0.45 μm filter, and then the lentivirus was concentrated by centrifugation at 50000 g for 2 hours using an ultracentrifuge. After removing the supernatant, a lentiviral vector of α1,3-GT gene for gene transfer was prepared by dissolving in 10 μl of HBSS solution.
実施例2
α-Galを発現するトランスジェニックニワトリ(G1) 作製法
　トランスジェニックニワトリの作製法は、特許文献4に記載の方法に基づいて実施した。詳細は以下の通りである。 Example 2
Production Method of Transgenic Chicken ( G1) Expressing α-Gal The production method of the transgenic chicken was carried out based on the method described in Patent Document 4. Details are as follows.
　白色レグホン種のニワトリ受精卵を孵卵器において2.5日培養し、Hamburger およびHamilton(非特許文献10)によるニワトリ胚の発生段階14から16に達したところで卵殻の鋭端側を開窓した。卵黄上に位置する胚の血管中(注1)に、高ウイルス力価(注2)のα1,3-GT遺伝子配列が組み込まれたレンチウイルスベクター 1μlを注入した。遺伝子導入操作胚は、開窓部分をラップで閉じた後に、孵卵器中で18日間培養し、孵化させた。孵化したトランスジェニック操作ニワトリ(G0)を飼養して性成熟に達した後、G0に対して異性の非トランスジェニックニワトリ(白色レグホン種)と人工授精による交配試験を行った。この交配試験の結果得られた後代の細胞からDNAを採取し、PCR法を用いてα1,3-GT遺伝子が組み込まれた個体(G1)を選別した。PCRは、TaKaRa ExTaq Hot Start Version (タカラバイオ)を用いた。1,3-GT遺伝子検出のためのPCRに用いるプライマーの配列は、GT-F(caccatgaatgtcaaaggaagagtgg)(配列番号4)およびGT-R2( tcagatgttatttctaaccaaat)(配列番号5)である。テンプレートDNA 100ng、10×Ex Taq Buffer 2μl、dNTP mixture 1.6μl、GT-F primer (10pmol) 0.4μl、GT-R2 primer(10pmol) 0.4μl 、TaKaRa Ex Taq HS 0.1μlおよび超純水を加えた総量20μlを0.2ml PCR用チューブ内で混合した。PCR条件は、94℃ 1分のプレヒートの後、94℃ 30秒、55℃ 30秒および72℃ 20秒を54回サイクル行い、最後に72℃ 5分の伸長反応を行った。α1,3-GT遺伝子検出のためのPCRの結果を図2に示す。表1に示すように、11羽のG0の交配試験の結果、7羽のα1,3-GT遺伝子配列が組み込まれたトランスジェニックニワトリ(G1)が得られた。 A white leghorn chick fertilized egg was cultured in an incubator for 2.5 days, and the acute end side of the eggshell was opened when the development stage 14 to 16 of the chick embryo by Hamburger and Hamilton (Non-patent Document 10) was reached. 1 μl of a lentiviral vector in which the α1,3-GT gene sequence having a high virus titer (Note 2) was incorporated was injected into the blood vessel (Note 1) of the embryo located on the yolk. The transgenic embryos were hatched by culturing them in an incubator for 18 days after closing the fenestration with a wrap. After breeding a transgenic transgenic chicken (G0) and reaching sexual maturity, a mating test was carried out by artificial insemination with a non-transgenic chicken (white Leghorn species) that is opposite to G0. DNA was collected from progeny cells obtained as a result of this mating test, and individuals (G1) into which the α1,3-GT gene had been incorporated were selected by PCR. For PCR, TaKaRa ExTaq Hot Start Version (Takara Bio) was used. The primer sequences used for PCR for detecting the 1,3-GT gene are GT-F (caccatgaatgtcaaaggaagagtgg) (SEQ ID NO: 4) and GT-R2 (tcagatgttatttctaaccaaat) (SEQ ID NO: 5). Total amount of template DNA 100ng, 10 × Ex Taq Buffer 2μl, dNTP mixture 1.6μl, GT-F primer (10pmol) 0.4μl, GT-R2 primer (10pmol) 0.4μl, TaKaRa Ex Taq HS 0.1μl and ultrapure water 20 μl was mixed in a 0.2 ml PCR tube. PCR conditions were as follows: preheating at 94 ° C. for 1 minute, followed by 54 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 20 seconds, and finally an extension reaction at 72 ° C. for 5 minutes. The results of PCR for detecting the α1,3-GT gene are shown in FIG. As shown in Table 1, as a result of the cross test of 11 G0 birds, 7 transgenic chickens (G1) incorporating the α1,3-GT gene sequence were obtained.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　α1,3-GT遺伝子が組み込まれたG1のうち、その個体より採取した血球がレクチン(Griffonia Simplicifolia lectin I-Isolectin B4(GS-IB4)と混合することにより血球凝集反応を示し、同レクチンを利用した染色法により血球細胞が染色された個体をα-Galを発現するトランスジェニックニワトリ(G1)として選別した(図3参照)。図3において、ID 293は、α1,3-GT遺伝子が導入され血球表面にα-Galを発現しているため、レクチンの作用により血球が凝集し、蛍光染色されている。この方法により、6羽のα1,3-GT遺伝子トランスジェニックニワトリ(ID 293(雄), ID333(雄), ID429(雌), ID466(雄),ID475(雌), ID4964(雌))を解析した結果、3羽(ID293, ID475, ID4964)が血球にα-Galを発現していた。
　また、性成熟に達した雄のG1であるID293,ID333およびID466の精子に対して同レクチンを利用した染色法により染色した結果、ID293およびID466の精子はレクチン染色されており、α-Galが発現していることが示された(図4参照)。ID333の精子はレクチン染色されなかった。以上の結果を総合すると、ID293,ID466,ID475,ID4964においてα-Galが発現していることが示された。 Among G1 with α1,3-GT gene integrated, blood cells collected from the individual show hemagglutination by mixing with lectin (Griffonia Simplicifolia lectin I-Isolectin B 4 (GS-IB 4 )). Individuals whose blood cells were stained by a staining method using γ were selected as α-Gal-expressing transgenic chicken (G1) (see Fig. 3). Since α-Gal is expressed on the surface of blood cells, blood cells are aggregated and fluorescently stained by the action of lectin.By this method, six α1,3-GT gene transgenic chickens (ID 293 ( (Male), ID333 (male), ID429 (female), ID466 (male), ID475 (female), ID4964 (female)), 3 (ID293, ID475, ID4964) expressed α-Gal in blood cells Was.
In addition, as a result of staining the sperm of ID293, ID333 and ID466, which are male G1 which has reached sexual maturity, using the same lectin, the sperm of ID293 and ID466 is stained with lectin, and α-Gal It was shown to be expressed (see FIG. 4). ID333 sperm was not lectin stained. When the above results were put together, it was shown that α-Gal is expressed in ID293, ID466, ID475, and ID4964.
　(注1)上記方法では、主として後背動脈へ注入しているが、血管なら何処でも注入可能。
　(注2)ウイルス力価が1.0x109/mlまたは1.7x109/mlのベクターを利用。表１においてID0004, ID0005が、力価1.0 x109/mlのウイルス、その他は力価1.7 x109/mlのウイルスを注入して作製した。 (Note 1) In the above method, injection is mainly made into the posterior artery, but any blood vessel can be injected.
(Note 2) Use a vector with a virus titer of 1.0x10 9 / ml or 1.7x10 9 / ml. In Table 1, ID0004 and ID0005 were prepared by injecting virus having a titer of 1.0 × 10 9 / ml, and others having a titer of 1.7 × 10 9 / ml.
実施例3
α-Galを発現するトランスジェニックニワトリ(G2) 作製法
　１.実施例1で得られた血球または精子にα-Galを発現するトランスジェニックニワトリ(G1)4羽中3羽(ID293(雄), ID466(雄),ID4964(雌))が性成熟に達した。この3羽について遺伝子導入操作を行っていない非トランスジェニックニワトリ(白色レグホン種)との交配試験を行った。孵化した産子については採取した血液よりDNAを抽出し、実施例2と同様のPCR法によりα1,3-GT遺伝子が組込まれている第二世代のトランスジェニックニワトリ(G2)を検索した。α1,3-GT遺伝子が組込まれたことが確認された個体については、実施例2と同様のレクチンを利用した血球凝集反応を利用して血球にα-Galを発現するトランスジェニックニワトリ(G2)を検索した。その結果、表2に示すとおりα1,3-GT遺伝子が組込まれたトランスジェニックニワトリ(G2)は、ID4964から44羽得られた。ID293およびID466からは受精卵が得られなかった。レクチンを用いた血球凝集反応の結果、ID4964から得られた44羽のG2中43羽(97.7%)が血球にα-Galを発現すると判定した。 Example 3
Production method of transgenic chicken (G2) expressing α-Gal 1. Transgenic chicken (G1) expressing α-Gal in blood cells or sperm obtained in Example 1, 3 out of 4 (ID293 (male), ID466 (male), ID4964 (female)) reached sexual maturity. A mating test was performed on these three birds with a non-transgenic chicken (white leghorn species) that had not been subjected to gene transfer. For hatched offspring, DNA was extracted from the collected blood, and a second-generation transgenic chicken (G2) in which the α1,3-GT gene was incorporated was searched for by the same PCR method as in Example 2. For individuals confirmed to have incorporated the α1,3-GT gene, a transgenic chicken (G2) that expresses α-Gal in blood cells using a hemagglutination reaction using the same lectin as in Example 2. Searched for. As a result, as shown in Table 2, 44 transgenic chickens (G2) incorporating the α1,3-GT gene were obtained from ID4964. No fertilized eggs were obtained from ID293 and ID466. As a result of the hemagglutination reaction using lectin, it was determined that 43 out of 44 G2 obtained from ID4964 (97.7%) expressed α-Gal in blood cells.
　ID293およびID466については、α1,3-GT遺伝子トランスジェニック操作雌ニワトリ(G0)( ID0006, ID0007およびID0008)との交配を行った。その結果、表3に示すとおりα1,3-GT遺伝子が組込まれた第二世代のトランスジェニックニワトリ(G2)は、ID293から21羽、ID466から18羽得られた。レクチンによる血球の凝集試験の結果、ID293から得られたG2の21羽は全て血球においてα-Galを発現するトランスジェニックニワトリであると判定された。一方、ID466から得られたα1,3-GT遺伝子が組込まれたトランスジェニックニワトリ(G2)は全て血球におけるα-Galの発現は認められなかった。なお、ID333は非トランスジェニックニワトリとの交配試験を行った結果、32羽の1,3-GT遺伝子が組込まれたトランスジェニックニワトリ(G2)が得られたが、いずれも血球にα-Galが発現していないと判定された。 ID293 and ID466 were mated with α1,3-GT gene transgenic engineered female chickens (G0) (ID0006, ID0007 and ID0008). As a result, as shown in Table 3, 21 hens from ID293 and 18 wings from ID466 were obtained as second-generation transgenic chickens (G2) incorporating the α1,3-GT gene. As a result of the blood cell agglutination test with lectin, all 21 G2 birds obtained from ID293 were determined to be transgenic chickens expressing α-Gal in blood cells. On the other hand, all of the transgenic chickens (G2) incorporating the α1,3-GT gene obtained from ID466 did not express α-Gal in blood cells. As a result of mating tests with non-transgenic chickens, ID333 resulted in 32 transgenic chickens (G2) in which the 1,3-GT gene was integrated, both of which had α-Gal in their blood cells. It was determined that it was not expressed.
α1,3-GT遺伝子トランスジェニックニワトリ(G1)と非トランスジェニックニワトリの交配試験による第2世代(G2)の生産
Figure JPOXMLDOC01-appb-T000002
 Production of second generation (G2) by cross test of α1,3-GT gene transgenic chicken (G1) and non-transgenic chicken
Figure JPOXMLDOC01-appb-T000002
α1,3-GT遺伝子トランスジェニック雄ニワトリ(G1)とα1,3-GT遺伝子トランスジェニック操作雌ニワトリ(G0)の交配試験による第2世代(G2)の生産
Figure JPOXMLDOC01-appb-T000003
 Production of second generation (G2) by cross test of α1,3-GT gene transgenic male chicken (G1) and α1,3-GT gene transgenic engineered chicken chicken (G0)
Figure JPOXMLDOC01-appb-T000003
　２. ID4964から得られた第2世代のα1,3-GT遺伝子トランスジェニックニワトリ(G2)のうち、性成熟に達した28個体の赤血球についてα-Galを発現しているかを、フローサイトメトリーにより解析した。結果を図5に示す。解析した96.6%のα1,3-GT遺伝子トランスジェニックニワトリ(G2)がα-Gal発現を発現していた。導入遺伝子の発現が抑制されるサイレンシングの発生は、本実施例では非常に低かった。 2. By flow cytometry, whether or not α-Gal is expressed in 28 erythrocytes of sexual maturity among the second generation α1,3-GT gene transgenic chicken (G2) obtained from ID4964 Analyzed. The results are shown in FIG. 96.6% of the α1,3-GT gene transgenic chickens (G2) analyzed expressed α-Gal expression. The occurrence of silencing that suppresses the expression of the transgene was very low in this example.
　上記結果から、α-Galを発現するトランスジェニックニワトリ(G1)から第2世代のα-Gal発現ニワトリ(G2)が多数生産出来る可能性があることが明らかとなった。その結果、α-Gal発現ニワトリ由来の受精卵を産業的に利用できる基盤が作られた。 From the above results, it has been clarified that a large number of second-generation α-Gal expressing chickens (G2) can be produced from transgenic chickens (G1) expressing α-Gal. As a result, a platform was established for industrial use of fertilized eggs derived from α-Gal expressing chickens.
実施例4
α-Galを発現するインフルエンザウイルス作製法
　１. 実施例3において、α-Gal発現トランスジェニック雌ニワトリ(ID 4964)(G1)と非遺伝子導入雄との交配で得られた受精卵(G2)のうち５個を孵卵してウイルス感染実験に用いた。受精卵(G2)は、人工的に37℃～39℃、湿度60%で30分毎に90°転卵させる方法により培養することにより胚発生を開始し、発育鶏卵とした。10～11日齢に達した発育鶏卵4964E1～E 5の漿尿膜腔内へH9N2ウイルスを接種し、その３～４日後に各卵から漿尿液を回収した。得られた漿尿液について赤血球凝集(HA)試験を行ったところ、いずれの漿尿液も2048～4096倍のHA力価を示し、同ウイルスを接種した正常卵におけるHA力価と同等であった(表4)。 Example 4
Method for preparing α-Gal-expressing influenza virus 1. In Example 3, fertilized eggs (G2) obtained by mating an α-Gal-expressing transgenic female chicken (ID 4964) (G1) with a non-transgenic male Five of them were incubated and used for virus infection experiments. Fertilized eggs (G2) started embryo development by artificially cultivating them at 90 ° C every 30 minutes at 37 ° C to 39 ° C and 60% humidity, and used as embryonated chicken eggs. H9N2 virus was inoculated into the chorioallantoic cavity of 4964E1 to E5 growing chicken eggs that reached 10-11 days of age, and chorioallantoic fluid was collected from each egg 3-4 days later. When the chorioallantoic fluid was subjected to a hemagglutination (HA) test, all chorioallantoic fluids showed an HA titer of 2048 to 4096 times, which was equivalent to the HA titer in normal eggs inoculated with the virus. (Table 4).
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　２. G2卵から採取した漿尿液より超遠心法を用いてウイルスを精製した。得られたウイルスをウエスタンブロッティング法にて解析した。結果を図6に示す。4964E-2, E-3, E-4から得られたウイルスにはα-Galが発現することが明らかになった。α-Galが発現するウイルスは、ワクチンの生産に供与できる。ワクチンの生産は常法により実施できる。 2. Virus was purified from chorioallantoic fluid collected from G2 eggs using ultracentrifugation. The obtained virus was analyzed by Western blotting. The results are shown in FIG. It was revealed that α-Gal is expressed in viruses obtained from 4964E-2, E-3, and E-4. Viruses expressing α-Gal can be donated for vaccine production. Vaccine production can be carried out by conventional methods.
　上記結果から以下のことが言える。
　１. α-Gal発現の発育鶏卵から産生されるインフルエンザウイルスの増殖性は正常卵のものと同等であるという知見により、α-Gal発現発育鶏卵を用いたα-Gal発現ワクチンの大量生産化が期待出来る。
　２. α-Gal発現発育鶏卵から作製されたα-Gal発現ワクチンは、抗α-Gal抗体を保有する人や家禽等にとって、従来のワクチンよりも効果が増強されたインフルエンザワクチンとなることが期待される。
　３. α-Gal発現ワクチンはインフルエンザのみならず、鶏胚を用いて作製するその他のワクチンにも応用可能であると考えられる。 The following can be said from the above results.
1. Based on the knowledge that the growth of influenza virus produced from α-Gal-expressing chicken eggs is equivalent to that of normal eggs, mass production of α-Gal-expressing vaccines using α-Gal-expressing chicken eggs has become possible. I can expect.
2. α-Gal expression vaccine prepared from α-Gal-expressing hen's eggs is expected to be an influenza vaccine with enhanced efficacy compared to conventional vaccines for people who have anti-α-Gal antibodies and poultry Is done.
3. It is considered that the α-Gal expression vaccine can be applied not only to influenza but also to other vaccines prepared using chicken embryos.
　本発明は、ワクチン製造に関する分野に有用である。 The present invention is useful in the field related to vaccine production.

Claims (12)

  1. α-ガラクトースエピトープ（Galα1-3Galβ1-4GlcNAc-R：以下α-Gal）を発現し得るトランスジェニック鳥類。 A transgenic bird capable of expressing an α-galactose epitope (Galα1-3Galβ1-4GlcNAc-R: hereinafter α-Gal).
  2. 鳥類がニワトリである請求項1に記載のトランスジェニック鳥類。 2. The transgenic bird according to claim 1, wherein the bird is a chicken.
  3. G0または後代である、請求項1または2に記載のトランスジェニック鳥類。 The transgenic bird according to claim 1 or 2, which is G0 or a progeny.
  4. α1，3-ガラクトース転移酵素(α1，3-GT)遺伝子を発現し得る状態で含む、α1，3-GT遺伝子導入用ベクターを鳥類に導入して、請求項1～3のいずれかに記載のトランスジェニック鳥類を得る、トランスジェニック鳥類の作製方法。 The α1,3-GT gene introduction vector containing the α1,3-galactose transferase (α1,3-GT) gene in a state in which the gene can be expressed, is introduced into birds, and according to any one of claims 1 to 3. A method for producing a transgenic bird to obtain a transgenic bird.
  5. α1，3-GT遺伝子導入用ベクターがレンチウイルスベクターである請求項4に記載のトランスジェニック鳥類の作製方法。 5. The method for producing a transgenic bird according to claim 4, wherein the α1,3-GT gene introduction vector is a lentiviral vector.
  6. α1，3-GT遺伝子がマウス、ブタまたはウシ由来である、請求項4または5に記載のトランスジェニック鳥類の作製方法。 The method for producing a transgenic bird according to claim 4 or 5, wherein the α1,3-GT gene is derived from mouse, pig or cow.
  7. 請求項1～3のいずれかに記載のトランスジェニック鳥類から得られる生体試料。 A biological sample obtained from the transgenic bird according to any one of claims 1 to 3.
  8. 生体試料がトランスジェニック鳥類の産んだ卵である請求項7に記載の生体試料。 8. The biological sample according to claim 7, wherein the biological sample is an egg laid by a transgenic bird.
  9. トランスジェニック鳥類の産んだ卵が受精卵または発育鳥類卵である請求項8に記載の生体試料。 9. The biological sample according to claim 8, wherein the egg laid by the transgenic bird is a fertilized egg or a developing bird egg.
  10. 請求項7～9のいずれかに記載の生体試料にウイルスを接種し、ウイルスを接種した生体試料を育成し、育成した生体試料からα-Galを発現するウイルスを得ることを含む、α-Gal発現ウイルスの作製方法。 10. Inoculating the biological sample according to claim 7 with a virus, growing a biological sample inoculated with the virus, and obtaining α-Gal-expressing virus from the grown biological sample. Method for producing expression virus.
  11. 前記ウイルスがインフルエンザウイルス、天然痘ウイルス、麻疹ウイルス、流行性耳下腺炎ウイルス、風疹ウイルス、エイズウイルス、ニューカッスル病ウイルス、またはマレック病ウイルスである請求項10に記載のα-Gal発現ウイルスの作製方法。 11. The production of an α-Gal-expressing virus according to claim 10, wherein the virus is influenza virus, smallpox virus, measles virus, mumps virus, rubella virus, AIDS virus, Newcastle disease virus, or Marek's disease virus. Method.
  12. 請求項10または11に記載の方法で、α-Galを発現するウイルスを作製し、得られたウイルスからワクチンを作製する、ワクチンの作製方法。 A method for producing a vaccine, wherein a virus expressing α-Gal is produced by the method according to claim 10 or 11, and a vaccine is produced from the obtained virus.
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