WO2011040527A1 - OISEAU TRANSGÉNIQUE CAPABLE D'EXPRIMER UN ÉPITOPE DE α-GALACTOSE, VIRUS ET VACCIN - Google Patents

OISEAU TRANSGÉNIQUE CAPABLE D'EXPRIMER UN ÉPITOPE DE α-GALACTOSE, VIRUS ET VACCIN 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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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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    • 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
    • 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
    • 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
    • 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

La présente invention concerne : un moyen pour exprimer α-Gal dans un virus sans utiliser une enzyme quelconque; la production d'un virus ayant α-Gal exprimé dans celui-ci en utilisant le dit moyen; la production d'un vaccin à partir du virus; un vaccin contre l'influenzavirus ayant un effet élevé (antigénicité), qui est produit en utilisant un virus ayant une réponse immunitaire augmentée à un virus; et d'autres. La présente invention concerne spécifiquement : un oiseau transgénique qui peut exprimer un épitope de α-galactose (Galα1-3Galβ1-4GlcNAc-R : appelé ci-après « α-Gal »), où l'oiseau est un poulet et peut être G0 ou une descendance de celui-ci; un procédé pour créer un oiseau transgénique, qui comprend l'introduction d'un vecteur qui est utilisé pour l'introduction d'un gène de α1,3-galactose transférase (α1,3-GT) et contient un gène α1,3-GT de telle manière que le gène puisse être exprimé, dans un oiseau, de manière à créer l'oiseau transgénique; un échantillon biologique, tel qu'un œuf, obtenu à partir de l'oiseau transgénique; et un procédé pour produire un virus exprimant α-Gal, qui comprend l'inoculation d'un virus à un échantillon biologique, la culture de l'échantillon biologique auquel a été inoculé le virus, et l'obtention d'un virus capable d'exprimer α-Gal à partir de l'échantillon biologique cultivé.
PCT/JP2010/067083 2009-09-30 2010-09-30 OISEAU TRANSGÉNIQUE CAPABLE D'EXPRIMER UN ÉPITOPE DE α-GALACTOSE, VIRUS ET VACCIN WO2011040527A1 (fr)

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Cited By (2)

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EP2230900A2 (fr) * 2008-01-07 2010-09-29 Synageva BioPharma Corp. Glycosylation chez l'oiseau
JP2020521494A (ja) * 2017-05-31 2020-07-27 コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガナイゼーション 鳥類における形質選択

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WO2005007827A2 (fr) * 2003-07-14 2005-01-27 University Of Georgia Research Foundation, Inc. Vecteur du virus associe aux adenovirus
EP1939281A1 (fr) * 2003-11-03 2008-07-02 ProBioGen AG Lignées de cellules aviaires immortalisées pour la production de virus
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2230900A2 (fr) * 2008-01-07 2010-09-29 Synageva BioPharma Corp. Glycosylation chez l'oiseau
EP2230900A4 (fr) * 2008-01-07 2013-01-09 Synageva Biopharma Corp Glycosylation chez l'oiseau
JP2020521494A (ja) * 2017-05-31 2020-07-27 コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガナイゼーション 鳥類における形質選択
JP7493940B2 (ja) 2017-05-31 2024-06-03 コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガナイゼーション 鳥類における形質選択

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