WO2022071492A1 - Protéine de fusion pour supprimer le virus de la grippe, et composition pharmaceutique la contenant - Google Patents

Protéine de fusion pour supprimer le virus de la grippe, et composition pharmaceutique la contenant Download PDF

Info

Publication number
WO2022071492A1
WO2022071492A1 PCT/JP2021/036149 JP2021036149W WO2022071492A1 WO 2022071492 A1 WO2022071492 A1 WO 2022071492A1 JP 2021036149 W JP2021036149 W JP 2021036149W WO 2022071492 A1 WO2022071492 A1 WO 2022071492A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
amino acid
protein
seq
deficient
Prior art date
Application number
PCT/JP2021/036149
Other languages
English (en)
Japanese (ja)
Inventor
賢二 関川
文雄 高岩
統 小林
寛 小宮山
一朗 小原
栄虎 山村
Original Assignee
キリンホールディングス株式会社
株式会社グリーンバイオメッド
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by キリンホールディングス株式会社, 株式会社グリーンバイオメッド filed Critical キリンホールディングス株式会社
Publication of WO2022071492A1 publication Critical patent/WO2022071492A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/11—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a fusion protein for suppressing influenza virus and a pharmaceutical composition containing the same.
  • influenza virus the HA antigen present in the outer membrane of influenza virus particles is marketed as a seasonal influenza virus type A HA split vaccine.
  • Influenza virus HA gene undergoes genetic exchange between subtypes A, and mutations in the base sequence cause antigenic variation, but mutations are concentrated in the head of the three-dimensional structure of HA protein. Since the HA head is the major neutralizing antibody epitope, the HA split vaccines and virus inactivated vaccines on the market have little effect on new mutants.
  • the stem region has few mutations but low immunogenicity, so it has not been used as a vaccine antigen for subtype A.
  • Non-Patent Document 1 An anti-stem antibody capable of neutralizing types 1 and 2 was induced in animals immunized with HA in which the head region of the HA gene was deleted. Since then, the development of a universal vaccine using a stem antigen has been promoted in the world (Non-Patent Documents 3 to 6). On the other hand, since the monomeric antigen protein has low immunogenicity, the weak point of the HA split vaccine is that it is ineffective.
  • Non-Patent Documents 7-9 It has been reported that the fusion protein of HA and ferritin enhances the immunogenicity of HA by forming a multimer by the oligomerization activity of ferritin (Non-Patent Documents 10 and 11).
  • the host immune response with the influenza virus HA split vaccine and inactivated vaccine is humoral immunity that neutralizes the virus primarily by inducing antibodies to the HA antigen, while another that destroys infected cells and suppresses the spread of virus infection.
  • the immune response is cell-mediated immunity.
  • the influenza virus antigen (CTL epitope) that binds to the major histocompatibility complex antigen class 1 molecule of infected cells and is recognized by the T cell receptor of cytotoxic T cells (CTL) is the matrix (M1) protein and nucleocapsid (NP). It has been reported to be found in proteins (Non-Patent Documents 12-14).
  • Non-Patent Documents 15 and 16 For the development status of influenza virus type A vaccine, refer to Non-Patent Documents 17 and 18.
  • the present inventors obtained a protein having excellent characteristics by further deleting the amino acid sequence at a specific site in the head-deficient HA-M1 fusion protein designed based on the HA and M1 of influenza virus.
  • the present invention is based on this finding.
  • the present invention provides a head-deficient HA-M1 fusion protein having excellent characteristics, and a pharmaceutical composition containing the same.
  • the present invention includes the following inventions.
  • the HA protein has a protein having an amino acid sequence represented by the 18th to 566 amino acid residues in SEQ ID NO: 1, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence, and has an amino acid sequence.
  • the M1 protein has a protein having an amino acid sequence represented by the 1st to 252nd amino acid residues in SEQ ID NO: 5, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence, and , A protein that functions as an M1 protein derived from influenza virus.
  • At least the amino acid residues in the regions corresponding to the 59th to 335th amino acid residues and the 529th to 554th amino acid residues in SEQ ID NO: 1 are deleted, and A partial defect in which at least the amino acid residues in the regions corresponding to the 18th to 50th amino acid residues, the 340th to 528th amino acid residues in SEQ ID NO: 1 and the 1st to 252nd amino acid residues in SEQ ID NO: 5 are retained.
  • HA-M1 fusion protein At least the amino acid residues in the regions corresponding to the 59th to 335th amino acid residues and the 529th to 554th amino acid residues in SEQ ID NO: 1 are deleted, and A partial defect in which at least the amino acid residues in the regions corresponding to the 18th to 50th amino acid residues, the 340th to 528th amino acid residues in SEQ ID NO: 1 and the 1st to 252nd amino acid residues in SEQ ID NO: 5 are retained.
  • the partially deficient HA-M1 fusion protein according to any one of (1) to (3) above which comprises the HA protein and the M1 protein in this order from the N-terminal to the C-terminal.
  • the linker is a GSG linker, GSGSG linker, GSGSGSGS linker, GSAGSA linker, or GGGGGSGGGGGSGGGS linker.
  • An expression vector comprising the nucleic acid molecule according to (7) above.
  • a transformant comprising the nucleic acid molecule described in (7) above or the expression vector described in (8) above.
  • a method for producing a partially deficient HA-M1 fusion protein which comprises culturing or growing the transformant according to (9) or (10) above.
  • a pharmaceutical comprising the partially defective HA-M1 fusion protein according to any one of (1) to (6), the nucleic acid molecule according to (7), or the expression vector according to (8).
  • Composition (13) The pharmaceutical composition according to (12) above, for preventing or treating an influenza virus infection.
  • the pharmaceutical composition according to (12) above for use as a vaccine against influenza virus.
  • the partially defective HA-M1 fusion protein according to any one of (1) to (6), the nucleic acid molecule according to (7), or the expression vector according to (8) is administered to the subject.
  • the partially defective HA-M1 fusion protein according to any one of (1) to (6), the nucleic acid molecule according to (7), or the expression vector according to (8) is administered to the subject.
  • a method of inducing a protective immune response against an influenza virus in the subject is administered.
  • a head-deficient HA-M1 fusion protein having excellent characteristics is provided.
  • the fusion protein of the present invention can prevent or treat an influenza virus infection.
  • the fusion protein of the present invention is advantageous in that it can be produced in transformed rice and yeast.
  • FIG. 1 is a diagram showing the structure of an expression vector of a head-deficient HA-M1 fusion protein in rice.
  • FIG. 2 is a photograph showing the results of electrophoresis of proteins in inecalus and culture medium expressing the head-deficient HA-M1 fusion protein gene.
  • FIG. 3 is a diagram showing an outline of the improved head-deficient HA-M1 fusion protein.
  • FIG. 4 is a photograph showing the results of western blotting of Inekals expressing the improved head-deficient HA-M1 protein.
  • FIG. 5 is a diagram showing an outline of a re-improved head-deficient HA-M1 fusion protein rice expression vector.
  • FIG. 6 is a photograph showing the expression of the re-improved head-deficient HA-M1 fusion protein in seeds.
  • FIG. 7 is a diagram showing an outline of vector construction for expression of the re-improved head-deficient HA-M1 fusion protein D in rice callus.
  • FIG. 8 is a photograph showing the results of SDS-PAGE and Western blotting of 6 independent lines of transformants by Vector A and 5 independent lines of transformants by Vector B.
  • FIG. 9 is a photograph showing the results of Western blotting of 5 independent lines of the transformant by Vector B, 4 independent lines of the transformant by Vector Hei, and 5 independent lines of the transformant by Vector Ding. be.
  • FIG. 10 is a diagram showing the structures of the plasmids for yeast expression of the head-deficient HA-M1 fusion protein and the improved head-deficient HA-M1 fusion protein B.
  • FIG. 11 is a photograph showing the expression of the head-deficient HA-M1 fusion protein and the improved head-deficient HA-M1 fusion protein B by Saccharomyces cerevisiae.
  • FIG. 12 is a diagram showing an outline of a vector for expressing various head-deficient fusion proteins in yeast.
  • FIG. 13 is a photograph showing the expression of various head-deficient fusion proteins by yeast.
  • FIG. 14 is a diagram showing an outline of a drug efficacy test using influenza virus-infected mice in Example 7.
  • FIG. 15-1 is a diagram showing the results of a drug efficacy test in influenza virus-infected mice using the improved fusion protein.
  • FIG. 15-2 is a diagram showing the results of a drug efficacy test in influenza virus-infected mice using the improved fusion protein.
  • FIG. 16 is a diagram showing an outline of a drug efficacy test using influenza virus-infected mice in Example 8.
  • FIG. 17-1 is a diagram showing the results of a drug efficacy test in influenza virus-infected mice using the improved fusion protein and the re-improved fusion protein.
  • FIG. 17-2 is a diagram showing the results of a drug efficacy test in influenza virus-infected mice using the improved fusion protein and the re-improved fusion protein.
  • the fusion protein of the present invention is a partially deficient HA-M1 fusion protein in which an amino acid residue in a specific region is deleted in a fusion protein of an HA protein derived from influenza virus and an M1 protein.
  • influenza virus examples include type A, type B, type C, and the like, and any of these may be used, but type A influenza virus is preferable.
  • the HA protein is a protein having an amino acid sequence represented by the 18th to 566 amino acid residues in SEQ ID NO: 1, or at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence.
  • Amino acid sequences other than the amino acid sequences represented by the 18th to 566 amino acid residues in SEQ ID NO: 1 replace amino acid residues in the original amino acid sequence with amino acid residues having the same or similar properties (conservative). It can be mutated by substitution).
  • the M1 protein is a protein having an amino acid sequence represented by the 1st to 252nd amino acid residues in SEQ ID NO: 5, or at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence.
  • Amino acid sequences other than the amino acid sequences represented by the 1st to 252nd amino acid residues in SEQ ID NO: 5 replace amino acid residues in the original amino acid sequence with amino acid residues having the same or similar properties (conservative). It can be mutated by substitution).
  • sequence identity between amino acid sequences can be calculated using a known sequence comparison program such as BLAST. In these programs, the parameters can be changed according to the purpose, but the default parameters may be used.
  • amino acid residues in the regions corresponding to the 59th to 335th amino acid residues and the 529 to 554 amino acid residues in SEQ ID NO: 1 are deleted.
  • amino acid residue in the region corresponding to refers not only to the amino acid residue itself in the specified region in a specific amino acid sequence, but also to the amino acid in the region at the same position in another amino acid sequence. It is also used to mean a residue. That is, the HA protein and M1 protein in the present invention are not only proteins having a specific amino acid sequence (18th to 566 amino acid residues in SEQ ID NO: 1 and 1st to 252 amino acid residues in SEQ ID NO: 5, respectively).
  • proteins having an amino acid sequence having 90% or more sequence identity include proteins having an amino acid sequence having 90% or more sequence identity with these. Therefore, when a protein having an amino acid sequence having a specific sequence identity different from the original amino acid sequence is used in the present invention, the amino acid residue in the region at the same position as the region specified in the original amino acid sequence is used. The group will be missing (or retained).
  • the 59th to 335th amino acid residues, the 51st to 335th amino acid residues, the 59th to 339th amino acid residues, or the 51st to 51st amino acid residues in SEQ ID NO: 1 are used.
  • the amino acid residue in the region corresponding to the 339 amino acid residue is missing.
  • the amino acid residue in the region corresponding to the 555 to 566 amino acid residue in SEQ ID NO: 1 is deleted.
  • the amino acid residue in the region corresponding to the 18th to 50th amino acid residues in SEQ ID NO: 1, the 340th to 528 amino acid residues, and the 1st to 252nd amino acid residues in SEQ ID NO: 5 is at least retained.
  • the amino acid residues in the regions corresponding to the 51st to 339th amino acid residues and the 529th to 554th amino acid residues in SEQ ID NO: 1 are deleted. Moreover, other amino acid residues are retained.
  • An example of the amino acid sequence of this fusion protein is shown in SEQ ID NO: 61 (where the GSGSG linker is inserted in place of the missing 51-339 amino acid residue), and the nucleotide sequence encoding this is shown in SEQ ID NO: 12.
  • amino acid residues in the regions corresponding to amino acid residues 51 to 335 and amino acids 529 to 554 in SEQ ID NO: 1 are deleted. Moreover, other amino acid residues are retained.
  • An example of the amino acid sequence of this fusion protein is shown in SEQ ID NO: 62 (where the GSG linker is inserted in place of the missing 51-335 amino acid residues), and the nucleotide sequence encoding this is shown in SEQ ID NO: 14.
  • the amino acid residues in the regions corresponding to the 59th to 339th amino acid residues and the 529 to 554th amino acid residues in SEQ ID NO: 1 are deleted. Moreover, other amino acid residues are retained.
  • An example of the amino acid sequence of this fusion protein is shown in SEQ ID NO: 63 (where the GSGSG linker is inserted in place of the missing 59-339 amino acid residue), and the nucleotide sequence encoding this is shown in SEQ ID NO: 15.
  • amino acid residues in the regions corresponding to amino acid residues 59 to 335 and amino acids 529 to 554 in SEQ ID NO: 1 are deleted. Moreover, other amino acid residues are retained.
  • An example of the amino acid sequence of this fusion protein is shown in SEQ ID NO: 64 (where a GSG linker is inserted in place of the missing 59-335 amino acid residue), and the nucleotide sequence encoding this is shown in SEQ ID NO: 26.
  • An example of the amino acid sequence of this fusion protein is SEQ ID NO: 65 (GSG linker is inserted in place of the missing 51-335 amino acid residue, and GSAGSA linker is inserted in place of the missing 555-566 amino acid residue. ), And the nucleotide sequence encoding this is shown in SEQ ID NO: 25.
  • An example of the amino acid sequence of this fusion protein is SEQ ID NO: 66 (GSG linker is inserted in place of the missing 59-335 amino acid residue, and GSAGSA linker is inserted in place of the missing 555-566 amino acid residue. ), And the nucleotide sequence encoding this is shown in SEQ ID NO: 27.
  • the HA protein and the M1 protein may be contained in this order from the N-terminal to the C-terminal, or may be contained in the reverse order, but preferably in this order. include.
  • a linker may be inserted in a region lacking an amino acid residue. Further, in the fusion protein of the present invention, a linker may be inserted at the fusion site of the partially defective HA protein and the M1 protein.
  • the length of the linker (linker peptide) is not particularly limited as long as the fusion protein of the present invention has an inhibitory effect on influenza virus, but is usually 1 to 100 amino acids, preferably 1 to 50 amino acids, and more preferably 1 to 25 amino acids. , More preferably set to 1 to 15 amino acids.
  • linker peptide examples include a linker peptide in which a flexible peptide having no secondary structure, which is composed of a peptide bond of glycine, serine, etc., is linked in a plurality of tandems, and more specifically. , (GGGGS) n (eg, GGGGGSGGGGSGGGGS linker, etc.), GSG linker, GSGSG linker, GSGSGSGS linker, GSAGSA linker and the like.
  • the fusion protein of the present invention may have an additional sequence at its N-terminal and / or C-terminal.
  • the length of the additional sequence is not particularly limited as long as the fusion protein of the present invention has an inhibitory effect on influenza virus, but is usually 1 to 100 amino acids, preferably 1 to 50 amino acids, more preferably 1 to 25 amino acids, still more preferable. Is 1 to 15 amino acids.
  • the additional sequence can be a tag sequence for facilitating purification or detection of the fusion protein of the invention.
  • the type of tag sequence is not particularly limited, but consists of, for example, FLAG (Hopp, T.P. et al., BioTechnology (1988) 6, 1204-1210) and 6 to 10 His (histidine) residues. 6 ⁇ His-10 ⁇ His, human c-myc fragment, ⁇ -tubulin fragment, B-tag, Protein C fragment, GST (glutathione-S-transferase), ⁇ -galactosidase, MBP (maltose binding protein) S Examples include tags, T7 tags, Strep tags, Nus tags, Trx tags, GFP tags and the like.
  • a signal sequence that can be actuated in the host expressing the fusion protein of the present invention for example, a secretory signal, an endoplasmic reticulum mooring signal (for example, KDEL sequence) and the like can also be preferably used.
  • the fusion protein of the present invention can be produced by using a well-known method for producing a recombinant protein.
  • a nucleic acid molecule preferably DNA
  • an expression vector is prepared by incorporating the nucleic acid molecule (preferably DNA) into an appropriate vector
  • the nucleic acid molecule or the expression vector is incorporated into a host for transformation.
  • the fusion protein of the present invention can be produced by producing a transformant and culturing or growing the transformant.
  • the host used to generate the transformant may be any host available for recombinant production of the protein, eg, bacteria such as Escherichia coli, fungi such as yeast, plant cells or plants, animal cells or Examples include animals.
  • the host is a yeast (eg, Saccharomyces cerevisiae ) or a plant (eg, rice).
  • the transformant may be prepared by introducing an expression vector into a host cell, or by incorporating a nucleic acid molecule into the genome of the host cell by homologous recombination.
  • the expression vector can be selected to function appropriately in that host, depending on the host used.
  • the nucleic acid molecule encoding the fusion protein of the present invention can be configured to contain many codons that are frequently used in the host to be used.
  • expression control sequences such as promoters, enhancers, and terminators that function appropriately in the host may be arranged before and after the coding sequence.
  • the pharmaceutical composition of the present invention comprises the fusion protein of the present invention.
  • a pharmaceutical composition can be obtained by formulating the fusion protein of the present invention according to a conventional method.
  • the pharmaceutical composition of the present invention may contain the nucleic acid molecule of the present invention encoding the fusion protein of the present invention or the expression vector of the present invention, and such a pharmaceutical composition may also be produced according to conventional means. can.
  • the pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, excipients such as sucrose, starch, mannit, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, gelatin, Arabia.
  • Excipients such as rubber, polyethylene glycol, sucrose, starch, disintegrants such as starch, carboxymethyl cellulose, hydroxypropyl starch, sodium-glycol-starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate, magnesium stearate, aerodil, talc , Lubricants such as sodium lauryl sulfate, fragrances such as citric acid, menthol, glycyrrhizin / ammonium salt, glycine, orange powder, preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, propylparaben, citric acid, citrate Stabilizers such as sodium citrate and acetic acid, suspending agents such as methyl cellulose, polyvinylpyrrolidone and aluminum stearate, dispersants such as surfactants, diluents such as water and physiological saline, cocoa butter, polyethylene glycol, white kerosene and the like.
  • the pharmaceutical composition of the present invention may further contain an adjuvant in order to enhance the immune response-inducing effect of the fusion protein of the present invention.
  • an adjuvant include, but are not limited to, aluminum hydroxide, complete Freund's adjuvant, incomplete Freund's adjuvant, Bordetella pertussis adjuvant, poly (I: C), CpG-DNA, 2', 3'-cGAMP and the like.
  • composition of the present invention is provided as a dosage form suitable for oral or parenteral administration (preferably parenteral administration).
  • composition for parenteral administration for example, an injection, a nasal administration (nasal drop, nasal spray, etc.), a suppository, etc. are used, and the injection is an intravenous injection, a subcutaneous injection, a skin. Dosage forms such as internal injection, intramuscular injection, and drip injection may be included.
  • Such injections and nasal administrations can be prepared according to known methods.
  • the fusion protein of the present invention can be prepared by dissolving or suspending it in a sterile aqueous solvent usually used for injections.
  • aqueous solvent for injection for example, distilled water; physiological saline; a phosphate buffer solution, a carbonate buffer solution, a Tris buffer solution, a citrate buffer solution, a buffer solution such as an acetate buffer solution, or the like can be used.
  • the pH of such an aqueous solvent is 5 to 10, preferably 6 to 8.
  • the prepared injection solution or nasal administration solution is a prefilled type that is filled in an appropriate ampoule or vial, or is previously filled in a syringe or a nasal drop drug injector.
  • a powder formulation of the fusion protein of the present invention can be obtained. It can also be prepared.
  • the fusion protein of the present invention can be used by storing it in a powder state and dispersing the powder in an aqueous solvent for injection or nasal administration at the time of use.
  • the content of the fusion protein of the present invention in the pharmaceutical composition is usually about 0.1 to 100% by mass, preferably about 1 to 99% by mass, more preferably about 10 to 90% by mass, based on the whole pharmaceutical composition. ..
  • the fusion protein of the present invention can be used for the prevention or treatment of influenza virus infections. That is, by administering the fusion protein of the present invention or the pharmaceutical composition of the present invention to an animal (including animals in general, but especially mammals such as primates such as humans and mammals such as rodents such as mice), in the animals. , Can prevent or treat influenza virus infections.
  • the fusion protein of the invention comprises an antigen or fragment thereof containing a B cell epitope conserved between subtypes of the virus and an antigen or fragment thereof containing a T cell epitope conserved between the subtypes of the virus.
  • influenza virus infection by inducing a humoral and cellular immunity against influenza virus, i.e., by inducing a protective immune response in the mammal, by administration to a mammal).
  • influenza virus infection by inducing a humoral and cellular immunity against influenza virus, i.e., by inducing a protective immune response in the mammal, by administration to a mammal).
  • the fusion protein of the present invention or the pharmaceutical composition of the present invention may be administered to animals that may be infected with influenza virus (particularly, primates such as humans and mammals such as rodents such as mice).
  • the target influenza virus is influenza A virus.
  • the fusion protein of the invention or the pharmaceutical composition of the invention can be shown to be effective across a wide range of subtype A influenza viruses, including seasonal influenza virus and the expected highly pathogenic pandemic influenza virus.
  • a fusion protein of the invention for use in therapy, for the prevention or treatment of influenza virus infections, or for use as a vaccine against influenza virus.
  • the nucleic acid molecule of the invention or the expression vector of the invention for use in therapy, for the prevention or treatment of influenza virus infections, or for use as a vaccine against influenza virus. Will be done.
  • a method of preventing or treating an influenza virus infection in a subject comprising administering to the subject (including humans) the fusion protein of the invention.
  • a method of inducing a protective immune response against influenza virus in a subject is provided.
  • the prevention or treatment of an influenza virus infection in a subject comprising administering to the subject (including humans) the nucleic acid molecule of the invention or the expression vector of the invention.
  • Methods, or methods of inducing a protective immune response against influenza virus in the subject are also provided.
  • the use of fusion proteins of the invention is provided in the manufacture of agents for the prevention or treatment of influenza virus infections, or in the production of agents for use as vaccines against influenza virus. Will be done.
  • the nucleic acid molecule of the present invention or the expression vector of the present invention in the production of a drug for preventing or treating an infectious disease of influenza virus, or in the production of a drug for use as a vaccine against influenza virus. Use of is also provided.
  • SEQ ID NO: 1 Michigan strain HA amino acid sequence
  • SEQ ID NO: 2 Headless HA
  • SEQ ID NO: 3 with headless HA linker
  • SEQ ID NO: 4 Secretory signal
  • SEQ ID NO: 5 Michigan strain M1
  • SEQ ID NO: 6 HIS tag
  • SEQ ID NO: 7 HA-M1 for rice expression, no linker. ORF from the 10th A to the 1731th A.
  • SEQ ID NO: 8 HA-M1 for rice expression, with linker. ORF from the 10th A to the 1776th A.
  • SEQ ID NO: 9 Rice ⁇ -amylase 3D promoter
  • SEQ ID NO: 10 Rice ⁇ -amylase 3D terminator
  • SEQ ID NO: 11 GSGSGSGS Linker
  • SEQ ID NO: 12 Improved head defect HA-M1 fusion protein A, HA51-339.
  • 10th A ORF to the 1578th A.
  • SEQ ID NO: 13 GSG linker
  • SEQ ID NO: 14 Improved head defect HA-M1 fusion protein B, HA51-335.
  • SEQ ID NO: 15 Improved head defect HA-M1 fusion protein C, HA59-339. ORF to 10th A to 1602nd A.
  • SEQ ID NO: 16 A / SWINE / IL / 00685/2005 (H1N1) M1 SEQ ID NO: 17: With HA-M1 linker for wheat cell-free system. ORF from 1st G to 1,692th A.
  • SEQ ID NO: 18 Improved head defect HA-M1 for wheat cell-free system. ORF in 1st G to 1,515th A.
  • SEQ ID NO: 19 Head defect HA-M1 fusion protein
  • SEQ ID NO: 20 Improved head defect HA-M1 fusion protein
  • SEQ ID NO: 24 GSAGSA linker
  • SEQ ID NO: 25 Re-improved head defect HA-M1 fusion protein B + KDEL. ORF from the 10th A to the 1,581th A.
  • SEQ ID NO: 26 Improved head defect HA-M1 fusion protein D + KDEL. ORF from the 10th A to the 1623th A.
  • SEQ ID NO: 27 Re-improved head defect HA-M1 fusion protein D + KDEL. ORF from 1st A to 1605th A.
  • SEQ ID NO: 28 Glb-1 promoter SEQ ID NO: 29: Glb-1 terminator SEQ ID NO: 30: Rice Ubi1 promoter SEQ ID NO: 31: NOS terminator SEQ ID NO: 32: RAP 3rd intron SEQ ID NO: 33: GluABC SEQ ID NO: 34: Complementary strand of GluABC SEQ ID NO: 35: Pro13K16K SEQ ID NO: 36: Complementary strand of Pro13K16K SEQ ID NO: 37: TKIWVER3_1 SEQ ID NO: 38: TKIWVER3_2 SEQ ID NO: 39: TKIWVER3_3 SEQ ID NO: 40: TKIWVER3_4 SEQ ID NO: 41: Re-improved head defect HA-M1 fusion protein SEQ ID NO: 42: FLAG tag SEQ ID NO: 43: MFA1 secretory signal SEQ ID NO: 44: GSAGSA linker SEQ ID NO: 45: expression gene of pYHAM1-15.
  • SEQ ID NO: 46 Expression gene of pYHAM1-16. ORF from the 10th A to the 1,812th A.
  • SEQ ID NO: 47 Expression gene of pYHAM1-1. ORF to the 10th A to 1,671st A.
  • SEQ ID NO: 48 Expression gene of pYHAM1-2. ORF from the 10th A to the 1,635th A.
  • SEQ ID NO: 49 GGGGS linker
  • SEQ ID NO: 50 Glb-1 secretion signal
  • SEQ ID NO: 53 Re-improved head-deficient HA-M1 fusion protein on the vector instep.
  • SEQ ID NO: 54 Re-improved head-deficient HA-M1 fusion protein loaded on Vector B. ORF from the 10th A to the 1665th A.
  • SEQ ID NO: 55 Code region of HA335-M1 for the re-improved head defect HA-M1 fusion protein. The first A to ORF.
  • SEQ ID NO: 56 Code region of HA335-M1 of the re-improved head defect HA-M1 fusion protein. Codon modified version. The first A to ORF.
  • SEQ ID NO: 57 Re-improved head-deficient HA-M1 fusion protein loaded on Vector Hei.
  • SEQ ID NO: 58 Re-improved head-deficient HA-M1 fusion protein mounted on Vector Ding. ORF from the 10th A to the 1590th A.
  • SEQ ID NO: 59 Head-deficient HA-M1 fusion protein (N-terminal rice ⁇ -amylase 3D secretory signal, C-terminal 6xHis tag removed from the amino acid sequence translated from SEQ ID NO: 7)
  • SEQ ID NO: 60 Head-deficient HA-M1 fusion protein with linker (N-terminal rice ⁇ -amylase 3D secretory signal, C-terminal 6xHis tag removed from the amino acid sequence translated from SEQ ID NO: 8)
  • SEQ ID NO: 61 Improved head-
  • Example 1 Expression of head-deficient HA-M1 fusion protein in rice 1.
  • Construction of plasmid for rice expression of head-deficient HA-M1 fusion protein A) Design of head-deficient HA-M1 fusion protein and head-deficient HA-M1 fusion protein with linker (residues 76 to 308 and 529 to 554 in SEQ ID NO: 1 are missing)
  • SEQ ID NO: 1 From (SEQ ID NO: 1), a sequence consisting of 17 amino acids from the 1st methionine to the 17th alanine corresponding to the secretory signal, and the 529th methionine to the 554th methionine corresponding to the transmembrane region.
  • a head-deficient HA was obtained in which the sequence consisting of 26 amino acids up to the above was deleted, and the sequence consisting of 233 amino acids from the 76th glycine to the 308th proline in the head was deleted (SEQ ID NO: 2).
  • the head-deficient HA with a linker (SEQ ID NO: 3) in which the sequence consisting of 233 amino acids from the deleted 76th glycine to the 308th proline of the head-deficient HA was replaced with the GS linker shown in SEQ ID NO: 49. was designed.
  • a sequence was designed and designated as a head-deficient HA-M1 fusion protein (SEQ ID NO: 59) or a linker-equipped head-deficient HA-M1 fusion protein (SEQ ID NO: 60).
  • This rice ⁇ -amylase 3D secretory signal, head-deficient HA-M1 fusion protein or head-deficient HA-M1 fusion protein with linker, 6 ⁇ His tag (SEQ ID NO: 6) is directed from the N-terminus to the C-terminus.
  • An amino acid sequence arranged in this order was designed and used as a secretory signal / 6 ⁇ His-tagged head-deficient HA-M1 fusion protein or a secretory signal / 6 ⁇ His-tagged head-deficient fusion protein.
  • the DNA base sequence was obtained from the database of The National Center for Biotechnology Information. As shown in FIG. 1, the ⁇ -amylase 3D gene promoter of about 2 kb of rice, the 5'-UTR derived from the ⁇ -amylase 3D gene of rice, the nucleotide encoding the secretory signal of ⁇ -amylase 3D of rice, and the head.
  • the sequence for amplification of each part is 25 mer so that each part of the defective HA-M1 fusion protein gene and the ⁇ -amylase 3D gene terminator of about 0.5 kb of rice can be connected in this order from the 5'side.
  • Primers were designed so that the overlap sequence for ligation was 15 mer, and each part was amplified using KOD FX Neo (Toyo Spinning Co., Ltd.) according to the attached protocol. After purifying all these parts with the illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare), all the parts and pUC18 digested with SmaI (Takara Bio) are mixed so that they have an equimolar ratio. Then, using the In-Fusion (R) HD Cloning Kit (Clontech), they were ligated according to the attached protocol to obtain Escherichia coli transformants.
  • KOD FX Neo Toyo Spinning Co., Ltd.
  • the target head-deficient HA-M1 fusion protein expression cassette (base which is the base sequence of SEQ ID NOs: 7, 9 and 10 or base which is the base sequence of SEQ ID NOs: 8, 9 and 10) was inserted. Clone with plasmid was selected by sequencing.
  • the target head-deficient HA-M1 fusion protein expression cassette was amplified by PCR using Ex-Taq (Takara Bio) and pKS221MCS (Wakasa et al., 2006, Plant Biotechnol. J. 4: 499). -510) was subcloned. Transfer of the head-deficient HA-M1 fusion protein expression cassette to the rice transformation vector p35SHPTAg7-GW (Wakasa et al., 2006, Plant Biotechnol. J. 4: 499-510) was performed using Gateway TM cloning technology. carried out.
  • Gateway (R) LR between the attL1 and attL2 sequences subcloned across the head-deficient HA-M1 fusion protein expression cassette subcloned into pKS221MCS and the attR1 and attR2 sequences present at p35SHPTAg7-GW, respectively. Recombination was performed by the chronaseTM II enzyme mix (Invitrogen) according to the attached protocol, and the head-deficient HA-M1 fusion protein expression cassette was transferred to the rice transformation vector p35SHPTAg7-GW. It was confirmed by sequencing that the prepared plasmid had the desired shape and sequence. In this way, a plasmid for expressing the head-deficient HA-M1 fusion protein in rice was constructed (Fig. 1).
  • FIG. 2 The results of SDS-PAGE of four independent strains of rice transformant callus expressing the head-deficient HA-M1 fusion protein are shown in FIG. In FIG. 2, the contents of each lane are as follows. Lane 1: A culture medium obtained by culturing transformant strain No. 1 expressing the head-deficient HA-M1 fusion protein in a medium containing no sucrose. Lane 2: A culture medium in which transformant lineage No. 2 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing no sucrose. Lane 3: A culture medium in which transformant lineage No.
  • Lane 4 A culture medium in which transformant lineage No. 4 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing no sucrose.
  • Lane 5 A culture medium in which transformant lineage No. 1 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing sucrose.
  • Lane 6 A culture medium in which transformant lineage No. 2 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing sucrose.
  • Lane 7 A culture medium in which transformant lineage No.
  • Lane 8 A culture medium in which transformant lineage No. 4 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing sucrose.
  • Lane 9 Cell mass extract obtained by culturing transformant lineage No. 1 expressing the head-deficient HA-M1 fusion protein in a medium containing no sucrose.
  • Lane 10 Cell mass extract obtained by culturing transformant lineage No. 2 expressing the head-deficient HA-M1 fusion protein in a medium containing no sucrose.
  • Lane 11 Cell mass extract obtained by culturing transformant lineage No.
  • Lane 12 Cell mass extract obtained by culturing transformant lineage No. 4 expressing the head-deficient HA-M1 fusion protein in a medium containing no sucrose.
  • Lane 13 Cell mass extract obtained by culturing transformant lineage No. 1 expressing the head-deficient HA-M1 fusion protein in a medium containing sucrose.
  • Lane 14 Cell mass extract obtained by culturing transformant lineage No. 2 expressing the head-deficient HA-M1 fusion protein in a medium containing sucrose.
  • Lane 15 Cell mass extract obtained by culturing transformant lineage No.
  • Lane 16 Cell mass extract obtained by culturing transformant lineage No. 4 expressing the head-deficient HA-M1 fusion protein in a medium containing sucrose.
  • Example 2 Production of improved head-deficient HA-M1 fusion protein by rice 1.
  • Construction of a plasmid for rice expression of the improved head-deficient HA-M1 fusion protein As described above, the head-deficient HA-M1 fusion protein was not expressed in rice, so improvement was performed. The outline is shown in FIG.
  • A) Design of improved head defect HA-M1 fusion protein (I) Improved head defect HA-M1 fusion protein A (defective residues 51 to 339 and residues 529 to 554 in SEQ ID NO: 1) Based on the head-deficient HA-M1 fusion protein gene shown in SEQ ID NO: 7, glutamic acid encoded by codons from 184th guanine to 186th guanine to 349th cytosine to 351st adenin.
  • the amino acid sequence of the improved head-deficient HA-M1 fusion protein A with a linker from which the secretory signal 6xHis tag has been removed is SEQ ID NO: 61.
  • Improved head defect HA-M1 fusion protein B lacking a sequence consisting of 52 amino acids up to codon-encoded leucine, with a linker encoded by SEQ ID NO: 13 inserted (improved head defect with linker)
  • a nucleotide (SEQ ID NO: 14) encoding HA-M1 fusion protein B) was designed.
  • the amino acid sequence of the improved head-deficient HA-M1 fusion protein B with a linker from which the secretory signal 6xHis tag has been removed is SEQ ID NO: 62.
  • Improved head defect HA-M1 fusion protein C lacking a sequence consisting of 48 amino acids up to codon-encoded proline, with a linker encoded by SEQ ID NO: 67 inserted (improved head defect with linker)
  • a nucleotide encoding HA-M1 fusion protein C (SEQ ID NO: 15) was designed.
  • the amino acid sequence of the improved head-deficient HA-M1 fusion protein C with linker from which the secretory signal 6xHis tag was removed is SEQ ID NO: 63. Become.
  • Improved head defect HA-M1 fusion protein D lacking a sequence consisting of 44 amino acids up to codon-encoded leucine, with a linker encoded by SEQ ID NO: 13 inserted (improved head defect with linker)
  • a nucleotide encoding the HA-M1 fusion protein D) was designed.
  • the amino acid sequence of the improved head-deficient HA-M1 fusion protein D with a linker from which the secretory signal 6xHis tag has been removed is SEQ ID NO: 64.
  • PCR was performed using a plasmid for expressing the head-deficient HA-M1 fusion protein in rice as a template, using KOD-FX Neo (Toyobo), and according to the attached protocol.
  • the PCR product was electrophoresed on a 0.3% agarose gel, the amplified band was stained with ethidium bromide, and the target band was excised under UV irradiation. This was purified with illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare), and then cyclized using In-Fusion (R) HD Cloning Kit (Clontech) according to the attached protocol.
  • the extracted protein was subjected to SDS-PAGE and Western blotting. Detection was performed by anti-His antibody.
  • the results of SDS-PAGE and Western blotting of 10 independent strains of rice transformant callus expressing the improved head-deficient HA-M1 fusion protein A and the improved head-deficient HA-M1 fusion protein B are shown. Shown in 4. In FIG. 4, the contents of each lane are as follows. Lane 1: Cell mass extract of transformant lineage No. 1 expressing the improved head-deficient HA-M1 fusion protein A. Lane 2: Cell mass extract of transformant lineage No. 2 expressing the improved head-deficient HA-M1 fusion protein A. Lane 3: Cell mass extract of transformant lineage No.
  • Lane 4 Cell mass extract of transformant lineage No. 4 expressing the improved head defect HA-M1 fusion protein A.
  • Lane 5 Cell mass extract of transformant lineage No. 5 expressing the improved head-deficient HA-M1 fusion protein A.
  • Lane 6 Cell mass extract of transformant lineage No. 6 expressing the improved head defect HA-M1 fusion protein A.
  • Lane 7 Cell mass extract of transformant lineage number 7 expressing the improved head-deficient HA-M1 fusion protein A.
  • Lane 8 Cell mass extract of transformant lineage No. 8 expressing the improved head defect HA-M1 fusion protein A.
  • Lane 9 Cell mass extract of transformant lineage No.
  • Lane 10 Cell mass extract of transformant lineage No. 10 expressing the improved head defect HA-M1 fusion protein A.
  • Lane 11 Cell mass extract of transformant lineage No. 1 expressing the improved head-deficient HA-M1 fusion protein B.
  • Lane 12 Cell mass extract of transformant lineage No. 2 expressing the improved head-deficient HA-M1 fusion protein B.
  • Lane 13 Cell mass extract of transformant lineage No. 3 expressing improved head defect HA-M1 fusion protein B.
  • Lane 14 Cell mass extract of transformant lineage No. 4 expressing the improved head defect HA-M1 fusion protein B.
  • Lane 15 Cell mass extract of transformant lineage No.
  • Lane 16 Cell mass extract of transformant lineage No. 6 expressing the improved head defect HA-M1 fusion protein B.
  • Lane 17 Cell mass extract of transformant lineage number 7 expressing the improved head defect HA-M1 fusion protein B.
  • Lane 18 Cell mass extract of transformant lineage No. 8 expressing improved head defect HA-M1 fusion protein B.
  • Lane 19 Cell mass extract of transformant lineage number 9 expressing the improved head defect HA-M1 fusion protein B.
  • Lane 20 Cell mass extract of transformant lineage No. 10 expressing improved head defect HA-M1 fusion protein B.
  • Example 3 Production of re-improved head defect HA-M1 fusion protein by rice 1.
  • nucleotide sequence encoding the Glb-1 secretion signal Substituted with the nucleotide sequence encoding the Glb-1 secretion signal, and further linked the nucleotide sequence encoding the KDEL sequence, which is the vesicle mooring signal, downstream of the sequence encoding the 6xHis tag at the C-terminal (SEQ ID NO: 22).
  • the nucleotide sequence encoding the improved head defect fusion protein B + KDEL was designed (SEQ ID NO: 23).
  • For the improved head defect fusion protein B + KDEL 36 from 775th timine to 810th timine of SEQ ID NO: 23, which encodes a cytoplasmic tail consisting of a 12-amino acid sequence present at the C-terminal of HA.
  • a nucleotide sequence encoding the re-improved head-deficient fusion protein B + KDEL was designed by removing the base sequence and inserting a nucleotide sequence (SEQ ID NO: 24) encoding a linker of GSAGSA having a flexible structure therein (SEQ ID NO: 24). SEQ ID NO: 25).
  • SEQ ID NO: 65 The sequence of the re-improved head defect fusion protein B not linked with the secretory signal, 6xHis tag and KDEL was designated as SEQ ID NO: 65.
  • the sequence consisting of 44 amino acids up to Leucine was deleted, the amino acid sequence encoded by the linker encoded by SEQ ID NO: 13 was inserted (improved head-deficient HA-M1 fusion protein D), and the C-terminal 6xHis tag was added.
  • a nucleotide encoding the improved head-deficient HA-M1 fusion protein D + KDEL (SEQ ID NO: 22), in which a nucleotide sequence encoding the KDEL sequence, which is a vesicle mooring signal, is ligated downstream of the sequence encoding 26) was designed.
  • the improved head-deficient fusion protein D the 36-base sequence from 799th timine to 834th timine of SEQ ID NO: 26, which encodes a cytoplasmic tail consisting of a 12-amino acid sequence, located at the C-terminal of HA.
  • FIG. 5 shows an outline of the construction of a vector for expression in rice. From the database of National Center for Biotechnology Information, the nucleotide sequences of the promoter (SEQ ID NO: 28) and terminator (SEQ ID NO: 29) of the Globulin-1 gene (Glb-1) of rice were obtained. Then, a glutelin expression-suppressing cassette and a prolamin expression-suppressing cassette, which are structures for causing two kinds of RNA interference, having the structure shown in FIG. 5 were designed.
  • a promoter (SEQ ID NO: 30) of about 2 kb of the Ubi1 gene of rice and pRI 101-AN (Takara).
  • a part of the glutelin A, B, and C genes (sequence) is sandwiched between the NOS terminator (SEQ ID NO: 31) derived from bio) and the 3rd intron (RAP 3rd intron, SEQ ID NO: 32) of the rice aspartic promoter.
  • SEQ ID NO: 34 a sequence complementary to the sequence was arranged was designed and named as a glutelin expression-suppressing cassette.
  • the promoter of about 2 kb of the Ubi1 gene of rice (SEQ ID NO: 28) and pRI 101-AN (Takarabio)
  • the 3rd intron (RAP 3rd intron, SEQ ID NO: 32) of the rice aspartic protease is sandwiched between the terminators of the NOS from which it is derived (SEQ ID NO: 29), and a partial sequence of the prolamin gene of 13 KD and 16 KD (SEQ ID NO: 35) is sandwiched.
  • a sequence in which a sequence complementary to the sequence (SEQ ID NO: 36) was arranged was designed and named as a promoter expression inhibitory cassette.
  • These two expression-suppressing cassettes were synthesized by PCR using Ex-Taq (Takara Bio), and the entry vectors pKS4-MCS and pKS221MCS (both Wakasa et al., 2006, Plant Biotechnol. J. 4: 499). Subcloned into -510).
  • an expression cassette of the improved or re-improved head defect fusion protein B or D was synthesized by PCR using Ex-Taq (Takara Bio), and the entry vector pK Subcloned into S2-3MCS (detailed in Wakasa et al., 2006, Plant Biotechnol. J. 4: 499-510). Then, the above three types of cassettes were transferred from the obtained three types of entry vectors to the rice transformation vector p35SHPTAg7-43GW (Wakasa et al., 2006, Plant Biotechnol. J. 4: 499-510) using Gateway TM cloning technology. Transferred.
  • the attL4 and attR1 sequences subcloned into pKS4-MCS sandwiching the gluterin expression-suppressing cassette the attL1 and attL2 sequences subcloned into pKS221MCS, and the attL1 and attL2 sequences, pKS2-3MCS.
  • the attR2 and attL3 sequences that are present across the expression cassette of the improved or re-improved head defect fusion protein B or D subcloned into and the attR4 and attR3 sequences that are present in the rice transformation vector p35SHPTAg7-43GW.
  • AttL1 and attR1, attL2 and attR2, attL3 and attR3, attL4 and attR4 sequences are recombined by Gateway (R) LR clonase TM II enzyme mix (invitrogen) according to the attached protocol, and three cassettes are used.
  • Gateway (R) LR clonase TM II enzyme mix invitrogen
  • three cassettes are used.
  • FIG. 5 As a result, as shown in FIG. 5, of Glbpro: HA52-335 (-linker), Glbpro: HA52-335 (+ linker), Glbpro: HA59-335 (-linker), Glbpro: HA59-335 (+ linker), Four types of plasmids were constructed.
  • urea-SDS buffer 50 mM Tris-HCl pH 6.8, 8 M Urea, 4% SDS, 5% 2-mercaptoethanol,
  • 20% Glycerol 20% Glycerol
  • FIG. 6 the contents of each lane are as follows in order from the left.
  • Glbpro HA52-335-M1 (-linker) transformant lineage # 02, # 03, # 06, # 12, # 18 in Fig. 5.
  • Glbpro HA59-335-M1 (-linker) transformant lineage # 02, # 04, # 16, # 21, # 23 in FIG.
  • Glbpro HA52-335-M1 (+ linker) transformant lineage # 03, # 08, # 14, # 19, # 25 in Fig. 5.
  • Glbpro HA59-335-M1 (+ linker) transformant lineage # 01, # 04, # 05, # 18 in Fig. 5.
  • the difference in the head defect region of HA did not bring about a large difference in the expression level of the head defect fusion protein.
  • removal of the cytoplasmic tail consisting of the 12 amino acid sequence present at the C-terminal of HA and / or insertion of a linker of GSAGSA having a flexible structure has been shown to have the effect of increasing the expression level of the head-deficient fusion protein. was done.
  • FIG. 7 shows an outline of the construction of rice callus expression vectors.
  • the Glb-1 promoter is replaced with the Ubi promoter, and the nucleotide sequence encoding the Glb-1 secretion signal is obtained.
  • the nucleotide sequence encoding 3xFLAG of SEQ ID NO: 52 was inserted in frame between the nucleotide sequences encoding the N-terminal of HA, and a vector instep having the gene coding region described in SEQ ID NO: 53 was constructed.
  • Vector B The nucleotide sequence encoding KDEL was removed from Vector A, and Vector B having the gene coding region set forth in SEQ ID NO: 54 was constructed.
  • the collected callus was frozen in liquid nitrogen, pulverized with a multi-bead shocker (registered trademark, Yasui Instrument), and the protein was extracted with a urea-SDS buffer.
  • the extracted protein was subjected to SDS-PAGE and Western blotting. Detection was performed by anti-FLAG antibody.
  • FIG. 8 The results of SDS-PAGE and Western blotting of 6 independent strains of transformants by Vector A and 5 independent strains of transformants by Vector B are shown in FIG. In FIG. 8, the contents of each lane are as follows. Lanes 1-6: Expression of HA-M1 fusion protein of rice callus expressing vector instep (each lane is an independent clone). Lanes 7-11: Expression of HA-M1 fusion protein of rice callus expressing Vector B (each lane is an independent clone).
  • the expression level of the transformant by the vector B expressing the re-improved head defect HA-M1 fusion protein D to which KDEL was not given is the transformant by the vector A to which KDEL was given. It was shown that the addition of KDEL tended to suppress the expression of the head-deficient HA-M1 fusion protein.
  • FIG. 9 shows the results of Western blotting of 5 independent lines of the transformant by Vector B, 4 independent lines of the transformant by Vector Hei, and 5 independent lines of the transformant by Vector Ding.
  • the contents of each lane are as follows. Lanes 1-5: Expression of HA-M1 fusion protein of rice callus expressing Vector B (each lane is an independent clone). Lanes 6-10: Expression of HA-M1 fusion protein of rice callus expressing Vector Hei (each lane is an independent clone). Lanes 11-14: Expression of the HA-M1 fusion protein of rice callus expressing Vector Ding (each lane is an independent clone).
  • the expression level of the transformant by the vector ⁇ expressing the re-improved head defect fusion protein D in which the codon is changed is significantly different from the expression level of the transformant by the vector B. It was shown that the change of the codon of the gene expressing the head-deficient HA-M1 fusion protein without changing the frequency of codon use does not have a great effect on the expression level.
  • the expression level of the transformant by the vector Ding expressing the re-improved head defect fusion protein D which does not have the Glb-1 secretory signal in the head is the trait by the vector ⁇ which has the Glb-1 secretory signal. It tends to be lower than that of the transformant, and it was shown that the addition of the secretory signal of Glb-1 has the effect of promoting the expression of the head-deficient HA-M1 fusion protein.
  • Example 4 Synthesis of antigen protein for animal testing 1 Construction of a plasmid for antigen protein synthesis, which is a head-deficient HA-M1 fusion protein and an improved head-deficient HA-M1 fusion protein.
  • a plasmid for antigen protein synthesis which is a head-deficient HA-M1 fusion protein and an improved head-deficient HA-M1 fusion protein.
  • the amino acid sequence of M1 was changed to the amino acid sequence of the M1 protein of the (A / swine / IL / 00685/2005 (H1N1)) strain (registration number: ACM17279.1, SEQ ID NO: 16).
  • a nucleotide sequence was designed in which the codons were returned to the codons of the original virus for HA and M1, that is, the sequence of registration number MK622940.1 for HA and the codon of registration number FJ638301.1 for M1 (sequence). Number 17).
  • a plasmid containing a nucleotide sequence encoding an improved head-deficient HA-M1 fusion protein In order to synthesize an improved head-deficient HA-M1 fusion protein in a wheat cell-free protein synthesis system, the following is based on pKBac1199. Modified. Regarding the nucleotide sequence set forth in SEQ ID NO: 17, the nucleotide sequence from the 100th guanine to the 300th guanine was deleted, and the sequence set forth in SEQ ID NO: 11 was inserted therein to design the nucleotide sequence of SEQ ID NO: 18.
  • pEU-E01-MCS CellFree Science
  • pKBac1201 a plasmid for synthetic systems
  • the obtained plasmid was cloned into Escherichia coli DH5 ⁇ (Toyobo Co., Ltd.) and prepared in large quantities using the QIAGEN plasmid Maxi Kit (Qiagen Co., Ltd.).
  • WEPRO7240H CellFree Science
  • WEPRO7240 CellFree Science
  • HA-M1 head-deficient HA-M1 protein
  • WEPRO7240 CellFree Science
  • HA-M1 fusion protein Centrifuge the synthesized protein (total fraction) (21,600xG, 4 ° C, 10 minutes), wash the precipitated fraction twice with a translation buffer for wheat cell-free protein synthesis (CellFree Sciences, Inc.), and then wheat. It was suspended in a translation buffer for a cell-free protein synthesis system to a concentration of about 1 mg / mL.
  • the sequence of the head-deficient HA-M1 fusion protein was SEQ ID NO: 19, and the sequence of the improved head-deficient HA-M1 fusion protein was SEQ ID NO: 20.
  • a plasmid for antigen protein synthesis which is a re-improved head-deficient HA-M1 fusion protein
  • a plasmid containing a nucleotide sequence encoding the re-improved head-deficient HA-M1 fusion protein The following vector preparation for synthesizing the re-improved head-deficient HA-M1 fusion protein in a wheat cell-free protein synthesis system It was carried out as follows.
  • PCR is performed using two primers pointing outward from the region encoding the 12 amino acid sequence of the C-terminal of the HA to be deleted. To prepare a fragment of the ring-opened vector lacking this region.
  • this amino acid sequence is encoded, and at both ends, the two ends of the cyclized vector sequence and 15 mer.
  • Two oligonucleotides having a homologous sequence of No. 2 and having a complementary strand relationship with each other were synthesized to prepare an annealed fragment. These two prepared fragments were ligated using an In-Fusion cloning kit to complete a plasmid.
  • TKIWver3_1 SEQ ID NO: 37
  • TKIWver3_2 SEQ ID NO: 38
  • TKIWver3_3 SEQ ID NO: 39
  • TKIWver3_4 SEQ ID NO: 40
  • PCR was performed using the plasmid pKBac1201 as a template, TKIWver3_1 and TKIWver3_2 as primer sets, and KODOne (Toyo Spinning Co., Ltd.) according to the attached protocol. Purified and recovered with andGelBandPurificationKit (GE Healthcare).
  • mix 40 ⁇ L of TKIWver3_3 and TKIWver3_4 (each prepared as an aqueous solution with a concentration of 0.1 nmol / ⁇ L), boil for 1 minute on water in a pot, and then keep the water in the pot at room temperature. I left it until I returned to, and urged annealing.
  • the obtained fragments were concentrated by ethanol precipitation according to a conventional method.
  • the two types of fragments thus obtained were cyclized using an In-Fusion cloning kit and transformed with Escherichia coli DH5 ⁇ (Toyobo). It was confirmed by sequencing that the plasmid thus prepared was constructed as intended, and the name was pKBac1211.
  • the obtained plasmid was cloned into Escherichia coli DH5 ⁇ (Toyobo) and prepared using QIAGEN Plasmamid MaxiKit (Qiagen).
  • Example 5 Expression of head-deficient HA-M1 fusion protein and improved head-deficient HA-M1 fusion protein B in yeast Head-deficient HA-M1 fusion protein and improved head-deficient HA-M1 fusion protein B ( For B) prepared in the above rice example, expression was attempted by the budding yeast Saccharomyces cerevisiae .
  • the skeletal vector, pYES2 (Invitrogen), was digested with SpeI (New England Biolabs) and NotI (New England Biolabs), electrophoresed on a 1% agarose gel, stained with ethidium bromide, and then the band of the vector was formed. It was cut out under UV irradiation.
  • the TDH3 promoter, head-deficient HA-M1 fusion protein or improved head-deficient HA-M1 fusion protein B is encoded between the SpeI and NotI sites of the yeast plasmid pYES2 from the 5'side.
  • the nucleotides to be subjected to were ligated in this order using Ligation High ver.2 (Toyobo). Sequencing confirmed that the plasmid produced by ligation had the desired shape and sequence.
  • plasmids for expressing the head-deficient HA-M1 fusion protein and the improved head-deficient HA-M1 fusion protein B in yeast were constructed and named pKBac1207 and pKBac1208, respectively. These plasmids are multicopy, of the type that autonomously replicates outside the nucleus of yeast, and carry the URA3 gene, which complements the host's uracil requirement as a selectable marker.
  • yeast is collected by centrifugation and in the presence of 0.1 mL extraction buffer (50 mM Tris-HCl pH 6.8, 8 M Urea, 4% SDS, 50 mM DTT, 20% Glycerol). Cells were disrupted by vigorous stirring with glass beads (diameter: 425-600 ⁇ m, sigma). Cell extracts were collected by centrifugation and tested on SDS-PAGE and Western blotting using Atto's system. Detection was performed by anti-His antibody.
  • extraction buffer 50 mM Tris-HCl pH 6.8, 8 M Urea, 4% SDS, 50 mM DTT, 20% Glycerol.
  • the expression level of the improved head-deficient HA-M1 fusion protein B was higher than the expression level of the head-deficient HA-M1 fusion protein. From this, it was confirmed that the expression level of the head-deficient HA-M1 fusion protein is increased by expanding the head-deficient region of HA not only when rice is used as the host but also when yeast is used as the host. did it. It was
  • Saccharomycetale Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, H. Since there was an effect, it was shown that the expansion of the head-deficient region of HA is effective in increasing the expression level of the head-deficient HA-M1 fusion protein, which is common in both the plant and fungal fields.
  • Example 6 Expression of re-improved head-deficient HA-M1 fusion protein by yeast 1.
  • Design of fusion protein In order to investigate the effect of modification of the HA portion of the head-deficient HA-M1 fusion protein on the expression level in yeast, a new series of expression vectors was constructed as outlined in FIG. That is, based on the vector used for the expression of the head-deficient HA-M1 fusion protein, the selectable marker gene in yeast was replaced with a G418 resistance cassette consisting of a G418 resistance gene sandwiched between the PGK1 promoter and PGK1 terminator from URA3. ..
  • HA heads there are two types of HA heads, one in which the same region as the head-deficient HA-M1 fusion protein is deleted and the other in which the same region as the improved head-deficient HA-M1 fusion protein is deleted.
  • a total of 4 types of genes are synthesized by combining two types of terminals, one with the region encoding the 12-amino acid sequence at the C-terminal, which is the same as the re-improved head-deficient HA-M1 fusion protein, and the other without the deletion. rice field.
  • these genes have a nucleotide sequence in which 3 copies of the FLAG tag-encoding nucleotide sequence (SEQ ID NO: 42) are tandemly linked to the 5'side of the nucleotide sequence encoding each fusion protein, and further to the 5'side.
  • the nucleotide sequence encoding the secretory signal of the MFA1 gene (SEQ ID NO: 43) and the Hind III sequence were added to the 5'side thereof.
  • a nucleotide sequence (SEQ ID NO: 44) encoding an amino acid sequence consisting of GSAGSA was inserted between HA and M1. In this way, four types of fusion proteins were designed (SEQ ID NOs: 45-48).
  • URA3 gene which is a selectable marker for pKBac1207
  • the URA3 gene was obtained by PCR using the plasmid pZNEO described in the paper by Yamano et al. (J. Biotechnol., 32: 173-178, 1994) as a template, and an In- Substituting by Fusion cloning gave pKBac1207NEO.
  • the DNA fragment encoding the secretory signal of the MFA1 gene was obtained by PCR using the chromosomal DNA of BY4741 (Funakoshi) as a template.
  • a DNA fragment in which 3 copies of the nucleotide sequence encoding the FLAG tag were tandemly linked was obtained by artificial synthesis.
  • the DNA fragment encoding the fusion protein is amplified by KOD One (Toyobo) in combination with PCR using pKBac1199, pKBac1201 and pKBac1211 as templates, and finally all the fragments are cloned into pKBac1207NEO.
  • KOD One Toyobo
  • pYHAM1-1, pYHAM1-2, pYHAM1-15, and pYHAM1-16 were obtained. Sequencing confirmed that the nucleotide sequence encoding the fusion protein to be expressed was correct.
  • yeast After culturing, yeast is collected by centrifugation, and glass beads (diameter) are present in the presence of 0.1 mL extraction buffer (50 mM Tris-HCl pH 6.8, 8 M Urea, 4% SDS, 50 mM DTT, 20% Glycerol). : 425-600 ⁇ m, Sigma) was vigorously stirred to disrupt the cells. Cell extracts were collected by centrifugation and tested on SDS-PAGE and Western blotting using Atto's system. Detection was performed by anti-His antibody.
  • extraction buffer 50 mM Tris-HCl pH 6.8, 8 M Urea, 4% SDS, 50 mM DTT, 20% Glycerol. : 425-600 ⁇ m, Sigma
  • each lane contains as follows. 1: pYHAM1-15, 2: pYHAM1-16, 3: pYHAM1-1, 4: pYHAM1-2. From the comparison between the strains introduced with pYHAM1-15 and pYHAM1-16 and the strains introduced with pYHAM1-1 and pYHAM1-2, the expression level of the head-deficient HA-M1 fusion protein was expanded by expanding the head-deficient region of HA. Was confirmed to increase.
  • the expression level of the head-deficient HA-M1 fusion protein was further increased by improving the fusion protein.
  • the expression level of the head-deficient HA-M1 fusion protein is deleted in the sprouting yeast of the family Saccharomycetale, Saccharomycetale, and the phylum Saccharomycetale of the family Saccharomycetale.
  • Example 7 Drug efficacy test using influenza-infected mice (improved type. Subcutaneous / nasal) 1. 1. Outline of the test In order to confirm the inhibitory effect of the test substance on influenza virus, an antigen protein (test substance) was administered to mice in advance (subcutaneous administration or nasal administration), and the reaction to influenza virus was examined. The outline is shown in FIG. The test group was 12 groups (Table 1).
  • Test method A Test substance The improved head-deficient HA-M1 fusion protein prepared in Example 6 was used as an antigen protein. Improved version; HA51-335 fusion protein B (deficient in residues 51-335 and 529-554 in SEQ ID NO: 1)
  • mice BALB / c, females, and 4-week-old animals (at the start of the test) (purchased from Charles River Japan and used after acclimatization) were used.
  • the animals were bred in an environment of 5 animals / cage, room temperature 24 ⁇ 3 ° C, humidity 50 ⁇ 20%, ventilation 10-25 times / hour, and lighting 12 hours.
  • the feed was fed by free intake of MF (Oriental Yeast Co., Ltd.). Groups were grouped based on body weight at the end of acclimation (at the start of the test). The number of animals was 10 per group.
  • Influenza virus Influenza virus is H1N1 (strain name: A / PR / 8/34, ATCC. No .: VR-1469, BSL: 2, virus titer: 1.6 ⁇ 10 8 TCID 50 / mL) and H3N2 (strain). Name: A / Port Chalmers / 1/73, ATCC. No .: VR-810, BSL: 2, virus titer: 1.3 ⁇ 10 7 TCID 50 / mL), both viruses have been reported (Nakano).
  • the above-mentioned administration solution was subcutaneously administered at 0.2 mL per mouse, twice at 7-day intervals, for a total of 0.4 mL (25 ⁇ g / head for mice in the high-dose group, for a total of 50 ⁇ g / head).
  • the low-dose group of mice received 2.5 ⁇ g / head of protein at a time, for a total of 5 ⁇ g / head).
  • the above administration solution was nasally administered at 50 ⁇ L per mouse, twice at 7-day intervals, for a total of 100 ⁇ L (12.5 ⁇ g / head for mice in the high-dose group, for a total of 25 ⁇ g / head).
  • Mice in the low-dose group received 1.25 ⁇ g / head of protein at a time, for a total of 2.5 ⁇ g / head). It was
  • mice 14 days (Day-14), 7 days (Day-7), virus inoculation day (Day 0), 3 days (Day 3), 7 days (Day 7) after virus inoculation to grasp the condition of the evaluated mice. ), 10 days later (Day 10), and 14 days later (Day 14).
  • life and death of mice and the general condition were also evaluated during the period from 14 days before the virus inoculation date to 14 days after the virus inoculation date.
  • the measurement data for each test group obtained as test data are described as mean ⁇ standard deviation.
  • the weight loss of the 3rd group peaked (14.5 ⁇ 0.9g), and the body weights of the 1st and 2nd groups at this time were 17.4 ⁇ 2.3g and 20.4 ⁇ 1.2g, respectively. Met.
  • the suppression of weight loss was significantly different in the 1st group on the 3rd, 7th and 14th days after the inoculation, and in the 2nd group on the 3rd to 14th days after the inoculation, compared with the 3rd group (Vehicle group). That is, the suppression of weight loss was observed in the improved administration group as compared with the vehicle group.
  • the weight loss of the 6th group peaked (14.9 ⁇ 1.2g), and at this time, it was 20.4 ⁇ 1.3g and 21.1 ⁇ 1.4g in the 4th and 5th groups, respectively. ..
  • Coarse coat was observed in many individuals in groups 7 and 8, but recovered afterwards except for dead individuals, and recovered from the 11th day after inoculation in the 7th group and from the 9th day in the 8th group. Coarse coat was observed in all cases in group 9, and most of the individuals recovered on the day of the end of observation (14th day of inoculation). That is, it was confirmed that the improved administration group had at least less coarsening of the coat or faster recovery.
  • the weight loss of the 9th group peaked (17.2 ⁇ 1.0g), and at this time, it was 17.9 ⁇ 1.3g and 18.9 ⁇ 1.3g in the 7th and 8th groups, respectively.
  • Coarse coat was observed in about half of the individuals in the 10th and 11th groups, but most of the individuals recovered on the 9th to 11th days except the dead individuals. In the 12th group (vehicle group), coarse stiffness of the coat was observed in all cases, and most of the individuals recovered on the 13th day after inoculation. That is, it was confirmed that the improved administration group had at least less coarsening of the coat or faster recovery.
  • the weight loss of the 12th group peaked (17.0 ⁇ 2.7g), and at this time, it was 19.2 ⁇ 2.4g and 20.6 ⁇ 1.2g in the 10th and 11th groups, respectively. ..
  • the suppression of weight loss was significantly different between the 12 groups (vehicle group) and the 11 groups (improved 1.25 ⁇ g / head administration group) on the 10th and 14th days after inoculation. That is, the improved administration group showed suppression of weight loss as compared with the vehicle group.
  • mice treated with the improved antigen protein have acquired resistance to influenza virus (H1N1 type and H3N2 type) as compared with the vehicle group not treated with the antigen protein. Therefore, it was shown that the improved antigenic protein is useful as a vaccine capable of imparting cross-immunity between influenza A virus subspecies.
  • Example 8 Drug efficacy test using influenza virus-infected mice (improved / re-improved. Nasal) 1. 1. Outline of the test In order to confirm the inhibitory effect of the test substance on influenza virus, an antigen protein (test substance) was administered nasally to mice in advance, and the reaction to influenza virus was examined. The outline is shown in FIG. The test group was 14 groups (Table 2).
  • Test substance The following three head-deficient HA-M1 fusion proteins prepared in Example 4 were used as antigen proteins.
  • Original type Original fusion protein (deficient in residues 76-308 and 529-554 in SEQ ID NO: 1) Improved version; HA51-335 fusion protein B (deficient in residues 51-335 and 529-554 in SEQ ID NO: 1) Re-improved; HA51-335, 555-566 Fusion Protein B (deficient in residues 51-335, 529-554 and 555-566 in SEQ ID NO: 1)
  • mice BALB / c, females, and 6-week-old animals (at the start of the test) (purchased from Charles River Japan and used after acclimatization) were used.
  • the animals were bred in an environment of 5 animals / cage, room temperature 24 ⁇ 3 ° C, humidity 50 ⁇ 20%, ventilation 10-25 times / hour, and lighting 12 hours.
  • the feed was fed by free intake of MF (Oriental Yeast Co., Ltd.). Groups were grouped based on body weight at the end of acclimation (at the start of the test). The number of animals was 10 per group.
  • Influenza virus Influenza virus is H1N1 (strain name: A / PR / 8/34, ATCC. No .: VR-1469, BSL: 2, virus titer: 1.6 ⁇ 10 8 TCID 50 / mL) and H3N2 (strain). Name: A / Port Chalmers / 1/73, ATCC. No .: VR-810, BSL: 2, virus titer: 1.3 ⁇ 10 7 TCID 50 / mL), both viruses have been reported (Nakano).
  • the administration method of antigen protein is as follows. That is, the original suspension, the improved suspension, or the re-improved suspension prepared to 250 ⁇ g / mL with physiological saline was used as a high-dose group administration solution, and further diluted 10-fold with physiological saline. Was used as a low-dose group administration solution. In addition, the physiological saline solution was used as the vehicle administration solution.
  • the above administration solution was nasally administered at 50 ⁇ L per mouse, twice at 7-day intervals, for a total of 100 ⁇ L (12.5 ⁇ g / head for mice in the high-dose group, for a total of 25 ⁇ g / head).
  • Mice in the low-dose group received 1.25 ⁇ g / head of protein at a time, for a total of 2.5 ⁇ g / head).
  • mice 14 days (Day-14), 7 days (Day-7), virus inoculation day (Day 0), 3 days (Day 3), 7 days (Day 7) after virus inoculation to grasp the condition of the evaluated mice. ), 10 days later (Day 10), and 14 days later (Day 14).
  • life and death of mice and the general condition were also evaluated during the period from 14 days before the virus inoculation date to 14 days after the virus inoculation date.
  • the measurement data for each test group obtained as test data are described as mean ⁇ standard deviation.
  • mice nasally administered with the antigenic protein of the present invention acquired resistance to influenza virus (H1N1 type and H3N2 type) as compared with the group not administered with the antigenic protein. Therefore, it has been shown that the antigenic protein of the present invention is useful as a vaccine capable of imparting cross-immunity between influenza A virus subspecies.
  • Example 9 Influenza virus neutralizing antibody test using test substance-administered mice 1. 1. Outline of the test A test was conducted to confirm that an antibody having an activity of neutralizing influenza virus was produced in the blood of mice to which the test substance was administered. Antigen protein (test substance) was administered to mice in advance (nasal administration), and the reaction to influenza virus was examined. The test group was 9 groups (see Table 3 below).
  • Test substance (I) Test substance The following antigen proteins prepared according to Example 4 were used. That is, each protein synthesized using the wheat cell-free protein synthesis system is recovered as a precipitate by centrifugation at 15,000 rpm for 10 minutes at 4 ° C, and then the physiological saline solution (Otsuka Pharmaceutical Factory) is adjusted to 500 ⁇ g / mL. It was suspended in the water and stored in an ultra-low temperature bath at -80 ° C until it was used for the experiment. Improved: improved head-deficient HA-M1 chimeric protein B (SEQ ID NO: 20); and re-improved: re-improved head-deficient HA-M1 chimeric protein (SEQ ID NO: 41).
  • the improved head-deficient HA-M1 chimeric protein B and the re-improved head-deficient HA-M1 chimeric protein are gently inverted and mixed after thawing, and the protein is diffused into the suspension by ultrasonic treatment (protein).
  • Undiluted solution the dose per animal is protein undiluted solution 40 ⁇ L + Adj A 5 ⁇ L + physiological saline solution 5 ⁇ L (+ Adj A group), protein undiluted solution 40 ⁇ L + Adj B 10 ⁇ L (+ Adj B group) , Or protein stock solution 40 ⁇ L + physiological saline solution 10 ⁇ L (Adj-free group) was prepared (Table 3).
  • Adj A (2', 3'-cGAMP (InvivoGen)
  • Adj B Poly (I: C) (InvivoGen)
  • Adj B is prepared by adding physiological saline and gently mixing by pipetting to a concentration of 1 ⁇ g / ⁇ L to promote annealing. After keeping warm in the range of 70 ° C. for 10 minutes, it was allowed to stand at room temperature for 1 hour before use.
  • Adj A and Adj B were treated as described above and subjected to administration together with the protein stock solution as described in (i) above or as a test solution in which only the adjuvant was mixed with a physiological saline solution.
  • a control test group in which only physiological saline was administered was also provided.
  • mice were bred using autoclave-sterilized heat-resistant polysulfon cages (207W x 365D x 140H mm, TECNIPLAST Ltd) with five mice per cage.
  • the animals were bred under the conditions of room temperature 24 ⁇ 3 ° C, humidity 50 ⁇ 20%, ventilation (10 to 25 times / hour), and lighting for 12 hours (8:00 to 20:00).
  • the feed was MF (Oriental Yeast Co., Ltd.) as a free intake.
  • autoclave-sterilized city water was used as free intake.
  • Influenza virus Influenza virus is H1N1 (strain name: A / PR / 8/34, ATCC. No .: VR-1469, BSL: 2, virus titer: 1.6 ⁇ 10 8 TCID50 / mL) and H3N2 (strain name). : A / Port Chalmers / 1/73, ATCC.
  • test substance was administered nasally at 50 ⁇ L / body using a pipette under isoflurane inhalation anesthesia twice at weekly intervals (day 0, 7).
  • the collected serum is allowed to stand at 56 ° C for 30 minutes before use, and then in a 96-well plate, DMEM medium for measurement (BSA 0.2% (w / v), penicillin 100 units / mL, streptomycin 100 ⁇ g / mL, Dilute to 10, 20, 40, 80, 160, 320, 640, 1280, 2560, 5120, 10240, 20480-fold (50 ⁇ L / hole) with fungizone 0.5 ⁇ g / mL, trypsin 1.0 ⁇ g / mL).
  • a virus solution (10 2 TCID 50/50 ⁇ L / hole) prepared in DMEM medium for measurement was added to each diluted solution, and the mixture was allowed to stand at 37 ° C. for 30 minutes (serum / virus mixed solution).
  • DMEM medium for culture containing 10% FBS, penicillin 100 units / mL, streptomycin 100 ⁇ g / mL
  • DMEM medium for culture containing 10% FBS, penicillin 100 units / mL, streptomycin 100 ⁇ g / mL
  • PBS was added one by one and washed, and then 0.1 mL of serum / virus mixture was inoculated on a 24-well plate.
  • 0.1 mL of medium (cell control) and 0.1 mL of virus / medium equivalent mixture (virus control) were inoculated for 6 holes each. After allowing to stand at 34 ° C for 1 hour, 0.5 mL of the medium was added, and the cells were cultured for about 2 to 3 days to determine the presence or absence of viral replication.
  • the reciprocal of the maximum dilution ratio of serum that completely suppresses viral growth was defined as the neutralizing antibody titer.
  • the geometric mean and 95% confidence interval were calculated.
  • the significance test was performed by using JMP (SAS Institute Japan) and calculating the p-value by Dunnett's test method. When the p-value was calculated, if the neutralizing antibody titer was less than 10, it was read as 10 and calculated.
  • Table 4 shows the neutralizing antibody titers of H1N1 against influenza A virus in the serum of mice to which the test substance was administered.
  • the improved head-deficient HA-M1 chimeric protein B (groups 1, 2, and 3) and the re-improved head-deficient HA-M1 chimeric protein (groups 4, 5, and 6) were used as administration substances.
  • the growth of the virus was still suppressed at a dilution ratio of 40 to 160 times with or without an adjuvant.
  • the improved head-deficient HA-M1 chimeric protein B and the re-improved head-deficient HA-M1 chimeric protein were administered to treated mice by producing an antibody that neutralizes the H1N1 subtype virus. , Showed to confer resistance to this virus.
  • Table 5 shows the neutralizing antibody titers of H3N2 against influenza A virus in the serum of mice to which the test substance was administered.
  • the improved head-deficient HA-M1 chimeric protein B (groups 1, 2, and 3) and the re-improved head-deficient HA-M1 chimeric protein (groups 4, 5, and 6) were used as administration substances.
  • the growth of the virus was still suppressed at a dilution ratio of 40 to 160 times with or without an adjuvant.
  • the improved head-deficient HA-M1 chimeric protein B and the re-improved head-deficient HA-M1 chimeric protein were administered to treated mice by producing an antibody that neutralizes the H3N2 subtype virus. , Showed to confer resistance to this virus.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Pulmonology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Communicable Diseases (AREA)
  • Veterinary Medicine (AREA)
  • Environmental Sciences (AREA)
  • Botany (AREA)
  • Cell Biology (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Oncology (AREA)

Abstract

L'invention concerne une protéine de fusion d'une protéine HA dérivée de la grippe et d'une protéine M1, la protéine de fusion étant une protéine de fusion HA-M1 à délétion partielle dans laquelle un résidu d'acide aminé dans une région spécifique a été supprimé. Cette protéine de fusion HA-M1 à délétion partielle présente d'excellentes caractéristiques.
PCT/JP2021/036149 2020-09-30 2021-09-30 Protéine de fusion pour supprimer le virus de la grippe, et composition pharmaceutique la contenant WO2022071492A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2020166188A JP2023166633A (ja) 2020-09-30 2020-09-30 インフルエンザウイルス抑制用の融合タンパク質およびこれを含む医薬組成物
JP2020-166188 2020-09-30

Publications (1)

Publication Number Publication Date
WO2022071492A1 true WO2022071492A1 (fr) 2022-04-07

Family

ID=80950506

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2021/036149 WO2022071492A1 (fr) 2020-09-30 2021-09-30 Protéine de fusion pour supprimer le virus de la grippe, et composition pharmaceutique la contenant

Country Status (2)

Country Link
JP (1) JP2023166633A (fr)
WO (1) WO2022071492A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012521786A (ja) * 2009-03-30 2012-09-20 モウント シナイ スクール オフ メディシネ インフルエンザウイルスワクチン及びその使用
WO2019124557A1 (fr) * 2017-12-21 2019-06-27 株式会社グリーンバイオメッド Vaccin antigénique à immunisation croisée et son procédé de préparation
WO2019145310A1 (fr) * 2018-01-23 2019-08-01 Janssen Vaccines & Prevention B.V. Vaccins contre le virus de la grippe et leurs utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012521786A (ja) * 2009-03-30 2012-09-20 モウント シナイ スクール オフ メディシネ インフルエンザウイルスワクチン及びその使用
WO2019124557A1 (fr) * 2017-12-21 2019-06-27 株式会社グリーンバイオメッド Vaccin antigénique à immunisation croisée et son procédé de préparation
WO2019145310A1 (fr) * 2018-01-23 2019-08-01 Janssen Vaccines & Prevention B.V. Vaccins contre le virus de la grippe et leurs utilisations

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KAROLINA URANOWSKA, TYBOROWSKA JOLANTA, JUREK ANNA, SZEWCZYK BOGUSŁAW, GROMADZKA BEATA: "Hemagglutinin stalk domain from H5N1 strain as a potentially universal antigen", ACTA BIOCHIMICA POLONICA, POLAND, 1 January 2014 (2014-01-01), Poland , pages 541, XP055213732, Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/pubmed/25210720> [retrieved on 20150916] *
YASSINE HADI M., MCTAMNEY PATRICK M., BOYINGTON JEFFERY C., RUCKWARDT TRACY J., CRANK MICHELLE C., SMATTI MARIA K., LEDGERWOOD JUL: "Use of Hemagglutinin Stem Probes Demonstrate Prevalence of Broadly Reactive Group 1 Influenza Antibodies in Human Sera", SCIENTIFIC REPORTS, vol. 8, no. 1, 1 December 2018 (2018-12-01), XP055920135, DOI: 10.1038/s41598-018-26538-7 *

Also Published As

Publication number Publication date
JP2023166633A (ja) 2023-11-22

Similar Documents

Publication Publication Date Title
JP2020014474A (ja) 呼吸器合胞体ウイルス（ｒｓｖ）ワクチン
KR20170102905A (ko) 신규 다가 나노입자 기반 백신
US9732122B2 (en) Attenuated recombinant vesicular stomatitis viruses comprising modified matrix proteins
CN107223130A (zh) 作为疫苗载体的三片段沙粒病毒
US10435712B2 (en) Evolution of high-titer virus-like vesicles for vaccine applications
JP2015502353A (ja) インフルエンザウイルスワクチンおよびその使用
KR101608121B1 (ko) 노다바이러스의 바이러스 유사입자의 생산방법, 이를 발현하는 효모 세포주 및 이를 포함하는 백신 조성물
CN113666990A (zh) 一种诱导广谱抗冠状病毒的t细胞疫苗免疫原及其应用
US9943593B2 (en) Modified matrix proteins of vesicular stomatitis virus
WO2023051701A1 (fr) Arnm, protéine et vaccin contre l&#39;infection par sars-cov-2
US20160000901A1 (en) Compositions and Methods for the Production of Virus-Like Particles
CN114524862A (zh) 禽流感(h5+h7)三价dna疫苗的构建及其应用
WO2022071492A1 (fr) Protéine de fusion pour supprimer le virus de la grippe, et composition pharmaceutique la contenant
JP6877788B2 (ja) ブタのＦｃ断片と融合した抗原およびこれを含むワクチン組成物
EP0176493B1 (fr) Polypeptides vaccinaux
US10266834B2 (en) Recombinant RNA particles and methods of producing proteins
CN101560247A (zh) 大肠杆菌热敏性肠毒素双突变体作为疫苗黏膜免疫佐剂
CN111166881B (zh) 一种重组呼吸道合胞病毒多表位嵌合疫苗及其制备方法和应用
JP2018052953A (ja) インフルエンザウイルスワクチンおよびその使用
MXPA02003789A (es) Particulas virales con epitopes internos exogenos.
CN101611144B (zh) 重组病毒蛋白及颗粒
US20220033764A1 (en) Engineered microorganisms and methods of making and using same
RU2777061C2 (ru) Живая пробиотическая вакцина для профилактики инфекции, вызванной вирусом гриппа
EP4244363A1 (fr) Plate-forme de levures pour la production de vaccins
WO2023207717A1 (fr) Développement et utilisation d&#39;un vaccin à large spectre contre la grippe aviaire h5n8

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21875788

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21875788

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP