WO2021235503A1 - Conjugated protein monomer supporting coronavirus protein, aggregate of said monomers, and component vaccine comprising said aggregate as active component - Google Patents

Conjugated protein monomer supporting coronavirus protein, aggregate of said monomers, and component vaccine comprising said aggregate as active component Download PDF

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WO2021235503A1
WO2021235503A1 PCT/JP2021/019072 JP2021019072W WO2021235503A1 WO 2021235503 A1 WO2021235503 A1 WO 2021235503A1 JP 2021019072 W JP2021019072 W JP 2021019072W WO 2021235503 A1 WO2021235503 A1 WO 2021235503A1
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amino acid
protein
acid sequence
aggregate
complex
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Japanese (ja)
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隆史 上野
和彦 片山
玲子 戸高
成史 澤田
慧 芳賀
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国立大学法人東京工業大学
学校法人北里研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/12—Aerosols; Foams
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
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    • 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
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
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    • C07—ORGANIC CHEMISTRY
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • 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
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    • 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
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    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • 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
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    • 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
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    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention is an invention relating to a functional protein and a vaccine using the same, and more specifically, a coronavirus which is an immunogen, particularly a beta coronavirus including a new type coronavirus (SARS-CoV-2).
  • the present invention relates to a complex protein (monomer) of a structural protein and a molecular needle, an aggregate of the monomer, and a component virus using the aggregate as an active ingredient (infection defense antigen).
  • Patent Document 1 Prior art documents provide a technique (Patent Document 1) for a "molecular needle” invented by focusing on the excellent gene transfer function of bacteriophage into cells.
  • Patent Document 2 a complex protein in which a structural protein of norovirus is carried on this molecular needle is provided as a component vaccine against norovirus
  • the present inventors have studied to solve the above problems by using a component vaccine using a molecular needle. As a result, it was surprisingly found that it is possible to provide a component vaccine that is effective against COVID-19 and does not require the use of adjuvant, and completed the present invention.
  • the vaccine of the present invention is a component vaccine containing a molecular needle carrying one or more structural proteins of coronavirus as an active ingredient.
  • the molecular needle is an aggregate of the complex protein of the present invention (hereinafter referred to as an aggregate) (hereinafter, also referred to as an aggregate of the present invention).
  • Conjugated protein of the present invention is a complex protein having the amino acid sequence of the following formula (1). That is, W-L 1- X n- Y (1) Wherein, W is the amino acid sequence of part or all of the structural proteins of a coronavirus which are immunogenic, L 1 is the number of amino acids shows a first linker sequence 0-100, X is of SEQ ID NO: 1 The amino acid sequence is shown, Y is the amino acid sequence of the cell introduction region, and n, which is the number of repetitions of X, is an integer of 1-3.
  • the amino acid sequence of the cell introduction region Y is the following formula (2): Y 1- L 2- Y 2- Y 3 (2)
  • Y 1 represents any one amino acid sequence selected from the group consisting of SEQ ID NO: 2-5
  • Y 2 represents any one amino acid sequence selected from the group consisting of SEQ ID NO: 6-9.
  • L 2 indicates a second linker sequence having 0-30 amino acids
  • Y 3 indicates an amino acid sequence for modification
  • Y 2 or Y 3 may not be present.
  • beta coronavirus is effective, and it contains a new type of coronavirus (SARS-CoV-2).
  • the present invention also provides a gene expression vector (hereinafter, also referred to as the vector of the present invention) incorporating a nucleic acid encoding the complex protein of the present invention, and further transforms with the nucleic acid encoding the complex protein of the present invention.
  • the transformed transformant hereinafter, also referred to as the transformant of the present invention
  • the transformant of the present invention can be easily obtained.
  • the complex protein of the present invention can be produced.
  • the aggregate of the present invention which is the active ingredient of the vaccine of the present invention, is an aggregate having the complex protein of the present invention as a monomer, and is a trimer or hexamer of the complex protein. It contains a body or a mixture of the trimer and a hexamer.
  • the substance of the content of the aggregate of the present invention may be collectively referred to as “trimer and / or hexamer”.
  • the aggregate of the present invention is an aggregate containing a trimer or a hexamer having the complex protein of the present invention as a monomer.
  • the aggregate of the present invention is also defined as an aggregate formed by associating the complex protein of the present invention in an aqueous liquid.
  • the aggregate of the present invention can exert its own action of penetrating into cells.
  • the trimer is a trimer protein in which the same or different complex protein of the present invention is used as a monomer protein, and the hexamer is a hexamer formed by associating two molecules of the trimer protein. It is a body protein.
  • the aggregate of the present invention can be produced by contacting the complex protein of the present invention in an aqueous liquid.
  • the above-mentioned aggregate may not be formed, or even if the aggregate is formed, solubility in an aqueous liquid may not be obtained.
  • the target complex protein constitutes a trimer or a hexamer, and the active ingredient of the vaccine of the present invention. It is possible to easily grasp whether or not it can be used as a protein.
  • the vaccine of the present invention is a vaccine containing the aggregate of the present invention as an active ingredient (protective antigen for infection), and is administered mucosally, transdermally, subcutaneously, intradermally, or intramuscularly to coronavirus. It is a component vaccine for. Specifically, among the above-mentioned trimers and / or hexamers, one or two or more of the structural proteins of coronavirus having W as W are used as active ingredients, mucosal, transdermal, and subcutaneous. , A component vaccine for intradermal or intramuscular administration.
  • the adjuvant can be contained in the vaccine of the present invention, for example, as a molecular needle carrying the B subunit of choleratoxin as W described above, but rather the adjuvant is excluded. It is preferable that the ability to obtain a subunit-free vaccine is prioritized as an advantage of the vaccine of the present invention.
  • the animals to which the vaccine of the present invention can be applied are not only humans but also all animals that can be infected with the new coronavirus, for example, dogs or cats, etc., but are limited to these. It's not something.
  • the complex protein itself of the present invention can be produced by expressing the nucleic acid encoding the complex protein by a genetic engineering technique or by synthesizing it by a peptide synthesis technique.
  • a trimer and a hexamer of the complex protein are spontaneously constructed, and a mixture containing the trimer and the hexamer is formed. Further, by selectively separating and collecting the trimer or the hexamer, the trimer and the hexamer can be separated and produced.
  • the complex protein of the present invention when the complex protein of the present invention is produced by a genetic engineering method, the complex protein is biologically expressed, for example, by collecting, disrupting or lysing the expressing cells.
  • the aggregate of the present invention spontaneously associates in an aqueous liquid such as water or various buffers used in the process of exposing the complex protein and further separating the complex protein by a known separation method. A mixture containing a trimeric and a hexamer can be obtained.
  • the complex protein of the present invention produced by performing total chemical synthesis or split synthesis for each part and binding by a chemical modification method is suspended in an aqueous liquid such as water or various buffer solutions. By spontaneously associating, a mixture containing a trimer and a hexamer, which are the aggregates of the present invention, can be obtained.
  • the method for separating and collecting a trimer or a hexamer from the above-mentioned mixture containing a trimer and a hexamer is not particularly limited, and a method for separating by a known molecular weight, for example, gel electrophoresis or affinity. Examples thereof include molecular sieves such as chromatography and molecular exclusion chromatography, ion exchange chromatography and the like.
  • a transformant into which a nucleic acid encoding the complex protein of the present invention has been introduced is cultured in a liquid medium to express the complex protein, and spontaneously.
  • the aggregate of the present invention produced in this way can be used as an active ingredient of the vaccine of the present invention.
  • An aggregate of the complex protein that can be used as the active ingredient (infection defense antigen), and (3) a component virus containing the aggregate as an active ingredient are provided.
  • the present invention is a means for providing an effective vaccine against the new coronavirus (SARS-CoV-2).
  • New coronavirus SARS-CoV-2
  • the new coronavirus is a target that is considered to have the highest degree of contribution by applying the present invention.
  • the new coronavirus (SARS-CoV-2) (hereinafter, also referred to as the new coronavirus) is the SARS coronavirus, which is the causative agent of SARS (severe acute respiratory syndrome), and the human coronavirus OC43 strain that causes upper airway inflammation in humans. It belongs to the antigenic group 2 (beta coronavirus) of the genus Coronavirus, which infects mice, cattle, pigs, etc., including the human coronavirus HKU1 that also causes lower airway inflammation.
  • the shape of the new coronavirus is a spherical particle with a diameter of 100 nm, which has a petal-like spike with a thin root and a bulging tip, similar to other coronavirus genera.
  • the structural proteins of the new coronavirus include S (spike) protein, M (membrane) protein, and E (envelope) protein in the envelope.
  • S protein is a glycoprotein that forms a single petal-like spike as a trimer, and has the ability to adsorb host cells to viral receptors (angiotensin converting enzyme II (ACE2)) and the action of serine protease (TMPRSS2).
  • M protein and E protein are also glycoproteins, most of which are located in the lipid bilayer and play an important role in virus particle formation.
  • the N (nucleocapsid) protein is an RNA-binding phosphorylated protein that binds to viral genomic RNA to form nucleocapsid and is involved in RNA replication, transcription, and translation.
  • the genome of the new coronavirus is also a positive-strand single-stranded RNA, which itself functions as mRNA and is also infectious. Also, at least like the SARS coronavirus, the genome has a cap structure at the 5'end and a poly A at the 3'end, and has a leader sequence and untranslated region that regulate gene replication and transcription at the 5'end. Downstream of this, there are non-structural protein genes encoding enzymes (replicases) essential for viral growth such as RNA polymerase and protease, and structural genes encoding the above-mentioned S, E, M, and N.
  • enzymes replicases
  • the above S protein is specifically composed of 1273 amino acids, SS (signal sequence), NTD (N-terminal region: N-terminal domain), RBD (receptor binding region: receptor-binding domain), SD1.
  • the RBD is configured as a trimer composed of two downprotomers and
  • Conjugated protein of the present invention which is an amino acid sequence formula representing the complex protein of the present invention: W-L 1- X n- Y (1)
  • W is the amino acid sequence of part or all of the structural proteins of a coronavirus which are immunogenic
  • L 1 is the number of amino acids shows a first linker sequence 0-100
  • X is of SEQ ID NO: 1
  • the amino acid sequence is shown
  • Y is the amino acid sequence of the cell introduction region
  • n is an integer of 1-3
  • Y is the following formula (2) :.
  • Y 1 represents any one amino acid sequence selected from the group consisting of SEQ ID NO: 2-5
  • Y 2 represents any one amino acid sequence selected from the group consisting of SEQ ID NO: 6-9.
  • L 2 indicates a second linker sequence having 0-30 amino acids
  • Y 3 indicates an amino acid sequence for modification
  • Y 2 or Y 3 may not be present.
  • W which is an immunogen (epitope)
  • W has an amino acid sequence based on the peptide composition of a part or all of the structural protein of coronavirus as described above.
  • the new coronavirus includes all mutant strains.
  • the structural protein of the coronavirus that can be selected as the immunogen W is a structural protein such as S protein, M protein, E protein, and N protein. Further, it may be a peptide sequence containing a recognizable epitope of the antibody.
  • the immunogen W is selected as the core element of the active ingredient of the vaccine of the present invention, and in that case, it is preferable to select the S protein among the above. It is also preferable to select from the S proteins, including all or part of RBD. This can be said for all coronaviruses that are the target viruses of the present invention, and of course, for beta coronaviruses and new coronaviruses.
  • n which is the number of repetitions of the amino acid sequence X in the above Xn, is preferably 1, but may be 2 or 3.
  • the modified amino acid sequence in which one or more amino acids are deleted, substituted or added among the amino acid sequences represented by X n , Y 1 or Y 2 is included in the above formula (1). Is done. “Deletion” means that any amino acid residue in the amino acid sequence of each SEQ ID NO: defined in the above formula (1) is deleted, and the N-terminal side (previous) of the deleted amino acid residue is deleted. ) And the amino acid residue on the C-terminal side (after) are connected by a peptide bond (in the case of deletion of the N-terminal amino acid residue and the C-terminal amino acid residue, the amino acid residue is simply deleted. The number of the deleted residues is counted as "the number of amino acid deletions".
  • substitution means that any amino acid residue in the amino acid sequence of each SEQ ID NO: defined in the above formula (1) is replaced with "another amino acid residue", and the replaced amino acid residue is It is in a state of being connected to each amino acid residue on the N-terminal side (front) and C-terminal side (rear) by a peptide bond (in the case of substitution of the N-terminal amino acid residue, a peptide bond with the amino acid residue on the C-terminal side). In the case of substitution of a C-terminal amino acid residue, only the peptide bond with the amino acid residue on the N-terminal side), the number of the substituted amino acid residues is counted as "the number of amino acid substitutions".
  • “Addition” means that one or more new amino acid residues are inserted at any one or more peptide bond positions in the amino acid sequence of each SEQ ID NO: defined in the above formula (1). It is a state in which a new peptide bond is formed in the state. The content and number of modifications of these amino acid residues can be determined by aligning the amino acid sequence related to the above formula (1) with the amino acid sequence related to the modification on a computer using human power or software capable of analyzing the amino acid sequence. By doing, it can be clarified.
  • linker sequence L 1 or L 2 defined by the above formula (1) or the amino acid sequence Y 3 for modification is an arbitrary sequence as necessary within the range of the number of amino acid residues defined above. Can be selected.
  • the trimer or hexamer (below) of the modified complex protein of the modified amino acid sequence has substantially the same immunostimulatory activity as the trimer or hexamer of the complex protein of the above formula (1). It is preferable to have.
  • “Substantially equivalent” means that when a method established for confirmation of immunostimulatory activity such as "neutralization test" is used, the significant difference in immunostimulatory activity from the unmodified complex protein of amino acid sequence is used. Equivalence to the extent that it is not observed at the significance level within 5%.
  • the number of modifications is 8 n or less, preferably 4 n or less, more preferably 2 n or less;
  • Y 1 is 30 or less, preferably 20 or less, still more preferably 10 or less; and
  • Y 2 is 15 or less. It is preferably within 10 pieces, preferably within 10 pieces, and more preferably within 5 pieces;
  • L 1 showing a first linker sequence of the formula (1) is necessary to suppress the steric hindrance reasonably keeping the distance immunogen W and molecular needle portion Y, the number of amino acid residues As described above, the number of amino acid residues is 0-100, preferably 4-40.
  • X in the above formula (1) is a sequence of n times (integer times) of X in the amino acid sequence Xn, which is composed of the amino acid of SEQ ID NO: 1.
  • the form of the iteration is a series iteration, for example, for X 2 , "XX"("-" is a schematic peptide bond).
  • Xn the above-mentioned modification of the amino acid sequence is permitted.
  • n is an integer of 1-3 as described above, 1 is preferable, but 2 or 3 may be used.
  • the main purpose is to keep the distance of the molecular needle Y stable and appropriate according to the size and characteristics of the immunogen W.
  • the cell introduction region Y corresponds to the basic structure of the molecular needle and is based on the needle portion (intracellular introduction portion) of the tail of the bacteriophage.
  • Y 1 indicates an amino acid sequence selected from the group consisting of SEQ ID NO: 2-5
  • Y 2 indicates an amino acid sequence selected from the group consisting of SEQ ID NO: 6-9
  • L 2 indicates the number of amino acids. shows a second linker sequence 0-30
  • Y 3 represents the amino acid sequence for a given modification, Y 2 or Y 3 is sometimes not present.
  • Y 1 of the formula (2) up to 32 amino acids (32 Leu) on the N-terminal side are the amino acid sequences of the portion of the triple helix ⁇ -sheet structure of Escherichia virus T4. At least the N-terminal amino acid valine (1Val) may be leucine (1Leu).
  • the remaining C-terminal side is the amino acid sequence of the C-terminal portion of the bacteriophage needle protein. Examples of the amino acid sequence that can be used on the C-terminal side of Y 1 include the amino acid sequence of gp5 of Bacterophage T4, the amino acid sequence of gpV of Bacterophage P2, the amino acid sequence of gp45 of Bacterophage Mu, and gp138 of Bacterophage ⁇ 92.
  • Examples include the amino acid sequence of. More specifically, the amino acid sequence of the Y 1 as SEQ ID NO: 2 having the amino acid sequence of gp5 of bacteriophage T4 in the C-terminal side, SEQ number as Y 1 having the amino acid sequence of the gpV of bacteriophage P2 C-terminal 3 amino acid sequence, the amino acid sequence of SEQ ID NO: 4 as Y 1 having the amino acid sequence of gp45 of bacteriophage Mu to C-terminal side, SEQ number as Y 1 having the amino acid sequence of gp138 of bacteriophage ⁇ 92 to C-terminal
  • the amino acid sequence of 5 is mentioned.
  • the nucleic acid sequence encoding the amino acid sequence of Y 1 can be selected according to the known relationship between the amino acid and the nucleobase.
  • Y 2 is the amino acid sequence of the region called folon of bacteriophage T4, or the amino acid sequence of the region called bacteriophage P2 or bacteriophage Mu or bacteriophage ⁇ 92 tip.
  • the earth or tip is a region constituting the tip of a molecular needle structure called a bacteriophage fibritin.
  • Y 2 is present in the formula (2), by having the amino acid sequence of the foldon or tip, it is possible to improve efficiency of incorporation of molecular needle to the cell membrane, the Y 2 It is preferable to accompany it.
  • the amino acid sequence of folon of Escherichia virus T4 is shown in SEQ ID NO: 6.
  • the nucleic acid sequence encoding this amino acid sequence can be selected according to the known relationship between the amino acid and the nucleobase.
  • the amino acid sequence of the tip of bacteriophage P2 is shown in SEQ ID NO: 7.
  • the nucleic acid sequence encoding this amino acid sequence can be selected according to the known relationship between the amino acid and the nucleobase.
  • the amino acid sequence of the bacteriophage Mu tip is shown in SEQ ID NO: 8.
  • the nucleic acid sequence encoding this amino acid sequence can be selected according to the known relationship between the amino acid and the nucleobase.
  • the amino acid sequence of the tip of bacteriophage ⁇ 92 is shown in SEQ ID NO: 9.
  • the nucleic acid sequence encoding this amino acid sequence can be selected according to the known relationship between the amino acid and the nucleobase.
  • L 2 is a second linker interposed between Y 1 and Y 2.
  • Number of amino acids of the linker L 2 is a 0-30 carbon atoms, preferably a 0-5. The number of amino acids of the linker is zero, is an indication that the second linker L 2 is absent.
  • Y 3 is an amino acid sequence for modification, and can be selectively added and used in Y.
  • the amino acid sequence for the modification is added for the purpose of protein purification, protection, etc., and examples thereof include tag proteins such as histidine tag, GST tag, and FLAG tag.
  • a linker sequence can be appropriately added to Y 3 , and such a linker sequence itself can be a component of the amino acid sequence of Y 3.
  • the complex protein of the present invention can be produced by a known method, specifically, a genetic engineering method or a chemical synthesis method. It is also possible to produce all of the complex proteins of the present invention together, and it is also possible to produce each part by ex post-bonding the parts by a chemical modification method. Bonding of polypeptides to each other via a linker (L 1 or L 2, etc.) can be performed by binding lysine residues or cysteine residues in each other's polypeptides with a linker having a succinimide group or a maleimide group. ..
  • a nucleic acid encoding all or part of the complex protein of the present invention to be produced is used as a transformant of a host cell such as Escherichia coli, yeast, insect cell, animal cell, or an Escherichia coli extract.
  • a host cell such as Escherichia coli, yeast, insect cell, animal cell, or an Escherichia coli extract.
  • Rabbit reticulated erythrocyte extract, wheat germ extract and the like can be expressed in a cell-free expression system.
  • an expression vector into which these nucleic acids are incorporated one corresponding to each expression system can be used, for example, pET, pBR322, pBR325, pUC18, pUC119, pTrcHis, pBlueBacHis, etc. for expression in Escherichia coli; in yeast.
  • Examples include pAUR for expression, YEp13, YEp24, YCp50, pYE52; pIEx-1 for expression in insect cells, pBApo-CMV for expression in animal cells, pF3A for expression in wheat germ extract, and the like. It is not limited, and a vector incorporating elements as required can be constructed and used. For example, it is possible to selectively place various promoters in front of the structural gene, and further place a cis element such as an enhancer, a splicing signal, a poly A addition signal, a ribosome binding sequence (SD sequence), a terminator sequence, and the like. It is also possible. It is also possible to incorporate a marker gene. Of course, it is also possible to use various gene expression kits currently on the market.
  • the chemical synthesis method it is possible to use a known chemical synthesis method for peptides. That is, it is possible to produce all or part of the complex protein of the present invention by using the liquid phase peptide synthesis method or the solid phase peptide synthesis method which has been established as a conventional method.
  • the solid-phase peptide synthesis method which is generally recognized as a suitable chemical synthesis method, can also use the Boc solid-phase method or the Fmoc solid-phase method, and as described above, the ligation method is required. It is also possible to use. Further, each amino acid can be produced by a known method, and a commercially available product can also be used.
  • FIG. 1 shows the process of constructing a trimer and a hexamer, which are aggregates of the present invention, based on the complex protein of the present invention.
  • 10 is the complex protein of the present invention as a monomer
  • 30 is the trimer of the present invention
  • 60 is the hexamer of the present invention.
  • the complex protein 10 of the present invention has "Y of formula (1)” in which "basic portion 131 corresponding to X n and Y 1 of formula (2)” and “foldon 132 corresponding to Y 2 of formula (2)” are bound. the corresponding molecular needle region 13 ', and, "wherein the immunogen 11 corresponding to W (1)” is “is configured attached via a linker 12" corresponding to L 1 of formula (1) There is. And Linker than linkers 12, the modified sequence corresponding to Y 3 in formula (2) are not shown.
  • the complex protein 10 of the present invention does not have a function of passing through the cell membrane of the cell of the target tissue.
  • the trimer 30 is a trimer formed by spontaneously associating the above-mentioned complex protein 10 as three monomers.
  • the trimer 30 has a trimeric parallel ⁇ -sheet structure and a spiral structure (triple helix ⁇ -sheet) due to the trimeric parallel ⁇ -sheet structure and the ⁇ -sheet structure itself by the three molecular needle regions 13 described above gathering together and associating with each other at the C-terminals.
  • a needle-like structure called (structure) is formed, and a molecular needle 13 ⁇ 3 is formed.
  • the molecular needle 13 ⁇ 3 is composed of a basic portion 131 ⁇ 3 and a foldon aggregate 132 ⁇ 3.
  • the hexamer 60 is a hexamer composed of two units of the above-mentioned trimer 30 bonded at the N-terminal of the basic parts ((13 ⁇ 3) 1 and (13 ⁇ 3) 2) of each other's molecular needles. It is a body, and the hexamer 60 also has a cell membrane crossing function of cells of a target tissue.
  • Each linker six derived from the trimer (12 1, 12 2, 12 3 and, 12 5, 12 6:12 4 not shown), immunogens are respectively coupled to these linkers 6 pieces (11 1, 11 2, 11 3 and, 11 5, 11 6:11 4 is not shown), two molecules needle (13 ⁇ 3) 1 and (13 ⁇ 3) located outside the 2 doing.
  • trimerization of the complex protein 10 of the present invention into a trimer 30 and the macroscopic dimerization from the trimer 30 to a hexamer 60 proceed spontaneously in an aqueous liquid.
  • the stability of this trimer or hexamer is extremely strong, for example, in an aqueous liquid environment at a temperature of 100 ° C., in an aqueous liquid environment at pH 2-11, and in an aqueous environment containing 50-70% by volume of an organic solvent. It is stable even in a liquid environment and is also excellent in safety. Even when isolated from an aqueous liquid and dried, the trimer or hexamer has excellent stability and cell membrane permeability.
  • the transition from the complex protein of the present invention to the aggregate progresses spontaneously, usually mostly in the final form, hexamerization, but some remain as trimers.
  • Vaccine of the present invention is administered to target tissues and cells by mucosal administration, transdermal administration, and subcutaneous administration due to the excellent cell permeability and immunogenicity of the aggregate of the present invention, which is an active ingredient thereof. It is possible to efficiently transfer all or part of the structural protein of the coronavirus, which is an immunogen, through intradermal administration or intramuscular administration to perform immunization, whereby mucosal administration or transdermal administration is possible. . .
  • Subcutaneous, intradermal, or intramuscular administration can improve the efficacy and safety of viral component vaccines. The manifestation is that it can be used as an adjuvant-free vaccine.
  • mucosal tissue to be administered to the mucosa examples include nasal mucosa, throat mucosa, airway mucosa, bronchial mucosa, sublingual mucosa, anal mucosa, intestinal mucosa, vaginal mucosa and the like. Among these, it is preferable to select nasal mucosa, throat mucosa, airway mucosa, bronchial mucosa, and sublingual mucosa.
  • the vaccine of the present invention is provided as a pharmaceutical composition for subcutaneous administration, intradermal administration, transdermal administration, mucosal administration or intramuscular administration, which contains the above-mentioned aggregate of the present invention as an active ingredient (infection protective antigen).
  • NS a pharmaceutical composition for subcutaneous administration, intradermal administration, transdermal administration, mucosal administration or intramuscular administration, which contains the above-mentioned aggregate of the present invention as an active ingredient (infection protective antigen).
  • NS infections protective antigen
  • mucosal administration, transdermal administration, subcutaneous administration, intradermal administration, or intramuscular administration is performed as a liquid preparation in which the aggregate is suspended and mixed at the time of use in a buffer solution or the like. Therefore, the form of the aggregate itself is also included in the pharmaceutical composition.
  • Mucosal administration can be easily performed with a spray, an aerosol, a capsule, a liquid, or the like, but is not limited to these forms.
  • Nasal administration (nasal in
  • the vaccine of the present invention is prepared in the form of a pharmaceutical composition by blending an aggregate of the present invention, which is an essential active ingredient (infection protective antigen), and, if necessary, adjuvant and a pharmaceutical pharmaceutical carrier.
  • the pharmaceutical carrier can be selected according to the form of use, and excipients or diluents such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants should be used. Can be done.
  • the form of the composition is basically a liquid preparation, but it can also be a desiccant, a powder preparation, a granule preparation or the like for liquid dilution at the time of use.
  • the amount of the aggregate of the present invention in the vaccine of the present invention is appropriately selected and is not constant, but it is usually preferable to use the aggregate of the present invention as a liquid preparation containing 0.1-10% by mass at the time of administration. be.
  • the appropriate dose (inoculation) is about 0.01 ⁇ g-10 mg per adult, and if necessary, the initial inoculation and the booster inoculation are combined as appropriate, and one or more administrations (inoculation) are performed. It is possible.
  • An object of the present embodiment is to show the usefulness as an active ingredient of a component vaccine targeting a new type coronavirus in the assembly of the present invention.
  • the new coronavirus is a pandemic virus that is prevalent worldwide, and serious cases and fatal cases are conspicuous, and it is a great threat to stop each social system.
  • ORF1 encodes a series of non-structural proteins of norovirus, the N-terminal protein, NTPase (p48), p22 (3A-like), Vpg, protease, and RNA-dependent RNA polymerase (RdRp), respectively.
  • the protease cleaves into each non-structural protein and functions as a mature product.
  • VPg has been demonstrated to play an essential role in norovirus genomic replication by translation from genomic RNA and subgenomic RNA and serves as a cap substitute for ribosome recruitment.
  • the amino acid sequence of Vpg of the immunogen LM14-2 strain used in this reference example is as shown in SEQ ID NO: 10 (however, the N-terminal Met is derived from the start codon ATG).
  • the nucleic acid sequence encoding this can be selected according to the known relationship between amino acids and nucleobases.
  • HNV-VPg is the cDNA portion (7639 bases) of the human Nolouis LM14-2 strain incorporated in the plasmid pHuNoV-LM14-2F (12774 bases: SEQ ID NO: 11) provided by Katayama of the Kitasato University Virus Infection Research Institute. : The one contained in SEQ ID NO: 12) was used.
  • VPg is a sequence of 399 bases (SEQ ID NO: 13) corresponding to 2630 to 3028 bases of the cDNA portion (7639 bases) of this LM14-2 strain. The start codon ATG was added to the 5'end of this sequence and used for expression.
  • the UV-vis spectrum was measured with a SHIMADZU UV-2400PC UV-vis spectrometer.
  • the MALDI-TOF mass spectrum was measured by Bruker ultrafleXtreme.
  • MALDI-TOF-MS measurements were made by measuring the sample with an equal volume of 70% (v / v) acetonitrile / containing 0.03% (w / v) sinapic acid and 0.1% (v / v) trifluoroacetic acid. It was mixed with an aqueous solution.
  • Gel permeation chromatography (GPC) was performed using an HPLC system and a column (Asahipack GF-510HQ, Shodex, Tokyo, Japan).
  • PN-Vpg is the above-mentioned formula (1) :. W-L 1- X n- Y (1) And the formula (2) representing the cell introduction region Y of the formula (1): Y 1- L 2- Y 2- Y 3 (2)
  • W is, be a "LM14-2 strain -Vpg" represented by the amino acid sequence of SEQ ID NO: 10; first linker L 1 is SEQ ID NO: 14 (SNSSSVPGG), 15 (GGGGS ), 16 (PAPAP) be amino acid sequences; repeating unit of a repeating sequence X n is an amino acid sequence of SEQ ID NO: 1, the repetition number n is 1; the amino acid sequence of the main body portion Y 1 molecule needle, the amino acid sequence of SEQ ID NO: 2 The second linker L 2 is "SVE"; the amino acid sequence of Foldon Y 2 is the amino acid sequence of
  • the PN-VPg plasmid is constructed using a flexible linker (FL: SNSSSVPGG (SEQ ID NO: 14)) as a template, and based on this, a short flexible linker (sFL: GGGGS (SEQ ID NO: 15)) and a short rigid linker (sRL: PAPAP). (SEQ ID NO: 16)) was constructed with two types of linkers, these were expressed, and the contents of spontaneously generated aggregates were analyzed, and it was confirmed that trimers and hexamers were contained. ..
  • (B) -2 Construction of template plasmid using flexible linker (FL: SNSSSVPGG (SEQ ID NO: 14))
  • Amplification of VPg segment from LM14-2 plasmid is performed by gene amplification primer VPg_F (with NdeI restriction enzyme site: ACGCCATATGGGCAAGAAAGGGAAGAACAAGTCC).
  • VPg_R with EcoRI restriction enzyme site: GCTCGAATTCGACTCAAAGTTGAGTTTCTCATTGTAGTCAACAC (SEQ ID NO: 19)
  • PCR polymerase chain reaction
  • the plasmid pKN1-1 is obtained by first amplifying the gene corresponding to residues 461 to 484 of the wac protein of the T4 phage by PCR from the T4 phage genome and cloning it into pUC18, and then cloning it into pUC18. I got the gene encoding. Subsequently, this plasmid was cleaved with restriction enzymes EcoRI and SalI and inserted into the plasmid pET29b (Novagen) treated with EcoRI and XhoI to obtain plasmid pMTf1-3.
  • the gene corresponding to residues 474 to 575 of gp5 of the T4 phage was amplified by PCR from the T4 phage genome and cloned into pUC18 to obtain a gene encoding gp5. Subsequently, this plasmid was cleaved with restriction enzymes EcoRI and SalI and inserted into the above-mentioned plasmid pMTf1-3 treated with EcoRI and XhoI to obtain plasmid pKA176.
  • the GFP expression vector provided by Takahashi of Gunma University was cleaved with the restriction enzymes NdeI and EcoRI to obtain a gene encoding GFP, and it was prepared by incorporating it into the above-mentioned plasmid pKA176 treated with the restriction enzymes NdeI and EcoRI. ..
  • the cloned gene fragment was introduced into competent cells of Escherichia coli BL21 (DE3), confirmed by DNA sequencing, and mediated by a flexible linker (SNSSSVPGG: SEQ ID NO: 14), a plasmid construct of PN and VPg "PN-FL-". The existence of "VPg” was confirmed.
  • gene amplification primers VPgGS-F (XhoI restriction sites available: a set of JijieijijishijijijijijititishieishitishijiAGGGAAGCAATACAATATTTGTACG (SEQ ID NO: 22) and VPgGS-R (above VPgPA-R (SEQ ID NO: 21)) to , are used separately as primers for gene amplification of inverted PCR using "PN-FL-VPg" as a template, and the plasmid construct "PN-sRL-VPg” (L 1 is a short rigid linker of SEQ ID NO: 16). And “PN-sFL-VPg” (L 1 is a short flexible linker of SEQ ID NO: 15) was constructed.
  • the immunogen carried by the genetic engineering technique is one of the structural proteins of the new corona virus (SARS-CoV-2).
  • the complex protein of the present invention was prepared as an RBD protein (protein in the receptor binding region) constituting a part of the S protein.
  • the RBD protein is encoded as a template sequence of S protein messenger RNA in the new coronavirus genome.
  • the S protein and the RBD protein are as described above.
  • the supported protein may be a partial sequence containing an antibody binding site capable of suppressing the growth of the virus, and can be selected from other than RBD as long as it is a peptide sequence constituting the virus protein.
  • the above RBD protein was selected as W, which is a structural protein.
  • the amino acid sequence of the RBD protein of the new coronavirus (SARS-CoV-2), which is the immunogen actually used, is the spike gene (S-gene) 21563-25384 base of the prototype SARS-CoV-2 Genbank Accession No. MN908947.
  • Example 1 all reagents used in were purchased from a commercial supplier and used without further purification.
  • RBD internal gene fragment ((SEQ ID NO: 26), the amino acid sequence: corresponding to EmuefuarubuikyuPitiiesuaibuiaruefuPienuaitienuerushiPiefujiibuiefuenueitiaruefueiesubuiwaieidaburyuenuarukeiaruaiesuenushibuieidiwaiesubuieruwaienuesueiesuefuesuianaishiwaijibuiesuPitikeieruenudierushiefutienubuiwaieidiesuefubuiaiarujidiibuiarukyuaieiPijikyutijikeiaieidiwaienuwaikeieruPididiefutijishi
  • the ggagatatacatATG sequence (SEQ ID NO: 28) is added to the 5'side primer for RT-PCR, and the ggaggcgggggttca sequence (SEQ ID NO: 29) (corresponding to the GGGGS linker) is added to the 3'side primer. So I added it.
  • this plasmid was introduced into DH5 ⁇ competent cells.
  • the obtained vector was verified by the DNA sequencing method, and then RBDp1-PN was expressed.
  • Escherichia coli BL21 (DE3) carrying this “RBDp1-PN” plasmid was cultured overnight at 37 ° C. in LB medium containing 30 ⁇ g / ml kanamycin. After the OD 600 of the solution incubated at 37 ° C. reached 0.8, 1 mM isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG) and arabinose were added. After 16-17 hours after adding IPTG and arabinose, the bacteria were collected by centrifugation at 8000 rpm for 5 minutes and incubated at 20 ° C. at a rate of 180 rpm.
  • IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • RBDp1-PN was added to a cobalt affinity column (Talon bead column), urea was added to the elution buffer attached to the Talon bead column kit to a weekly concentration of 6 M, and the mixture was purified according to the attached protocol.
  • the RBDp1-PN aggregate was then dialyzed against 4M urea / PBS for 24 hours and then dialyzed against 2M / PBS for 24 hours.
  • the RBDp1-PN aggregate was further dialyzed against PBS for 24 hours.
  • RBDp1-PN is spontaneously an aggregate containing trimers and / or hexamers. This was used in an immunological test as an "aggregate of RBDp1-PN".
  • a recombinant protein obtained by a conventional method according to a series of processes from the expression to recovery of "RBDp1-PN" was prepared based on the base sequence encoding the above RBDp1 protein.
  • the immunological test was performed by nasal inoculation as a solution (purified antigen) of 20 micrograms / mL PBS.
  • the immunofluorescent antibody method was used for new coronavirus infection after wrapping Vero / E6 cells in a 96-well plate with 90% confluent and exchanging the medium with a virus infection medium containing 2% FBS the next day.
  • the plates were washed 3 times in total with PBST (0.1% Tween20 / PBS), 80 ⁇ L / well of PBSB (1% BSA / PBS) was added to each plate, and the mixture was incubated at room temperature for 2 hours for blocking. ..
  • the above guinea pig test serum was then diluted 2000-fold with PBSB, added to the wells and incubated for 1 hour at 37 ° C. for reaction. 200 ⁇ L of PBS was added to each well of the plate and incubated for 10 minutes at room temperature for washing. This operation was repeated 3 times.
  • a 1500-fold diluted solution of 50 ⁇ L / well was added to the plate, incubated at room temperature for 2 hours, washed 5 times with PBS, and then examined under a fluorescence microscope. The presence or absence of the target antibody was confirmed.
  • (D) Virus Neutralizing Test Next, it was confirmed by a virus neutralizing test whether or not the induced antibody had the effect of preventing the infection of SARS-CoV-2 to cells.
  • To the leftmost well of the 96-well plate was added 100 microliters of the above-mentioned guinea pig test serum diluted 4-fold with DMEM. Then, a 2-fold dilution series up to 512-fold dilution was prepared in DMED medium. Approximately 50,000 SARS-CoV-2 infectious particles were added to all wells, stirred, and then incubated at 37 ° C. for 1 hour and then at 4 ° C. overnight.
  • the medium was removed from all wells of the 96-well plate in which Vero / E6 cells were cultured to be 90% confluent, washed 3 times with PBS, and then 50 microliters of fresh 4% FBS-containing medium was added.
  • FIG. 2 the two convalescent sera (serum at discharge) of SARS-CoV-2 infected patients used as positive controls still completely neutralized the virus even at 512-fold dilution. It is shown.
  • the antigen (RBDp1 protein) is diluted with PBS (-) to 2 ⁇ g / mL, 50 ⁇ L / well is added to a 96-well ELISA plate, and the mixture is incubated overnight at 4 ° C., and PBST (0.1% Tween 20) is used. The plates were washed 3 times in total with / PBS), 80 ⁇ L / well of PBSB (1% BSA / PBS) was added to each plate, and the mixture was incubated at room temperature for 2 hours for blocking.
  • PBSB 1% BSA / PBS
  • test serum was then diluted with PBSB to prepare a test sample (IgG detection: 5-step serial dilution up to 4-64 times, IgA detection: 5-step serial dilution up to 4-64 times).
  • the PBSB in the plate was discarded, 50 ⁇ L / well of each test sample was added to the plate, incubated at room temperature for 2 hours, and then the plate was washed 5 times with PBST.
  • the HRP substrate solution was added to the plate at a rate of 50 ⁇ L / Well, and the incubation was carried out at room temperature in the dark until color development was confirmed. 2M sulfuric acid was added to the plate at a rate of 25 ⁇ L / Well, the reaction was stopped, and the absorbance at 490 nm was measured.
  • FIG. 3 IgG
  • FIG. 4 IgA
  • the vertical axis shows the absorbance
  • the horizontal axis shows the dilution ratio.
  • the results for each individual test cotton rat are shown.
  • IgA in blood was lower than that of IgG, it is considered that most of IgA produced by B cells collected on the mucosal surface of the nasal cavity was not in the blood and most of it was secreted on the mucosal surface of the nasal cavity. ..
  • the antibody titer increased remarkably even without the use of adjuvant at all, and it is repeatedly shown that it can be used as an adjuvant-free vaccine. ..
  • SARS-CoV-2 infection occurs by inhaling the virus by droplet infection or airborne infection. That is, since the main infection routes are the oral cavity and the nasal cavity, aggregates are directly introduced into the nasal mucosal cells through the cell membrane by nasal inoculation, and the RBDp1 protein is injected into the nasal mucosal cells to induce humoral immunity. It was induced and defensive immunity against SARS-CoV-2 was evoked. It has been clarified that by nasal inoculation using a molecular needle carrying a structural protein of SARS-CoV-2 as an immunogen, local immunity is induced and an infection protective effect against SARS-CoV-2 can be obtained. rice field.

Abstract

The present invention addresses the problem of establishing a means for providing a component vaccine that can elicit immunity against coronavirus, especially beta coronaviruses including the novel coronavirus (SARS-CoV-2). The inventors discovered that this problem can be solved by providing a component vaccine which comprises, as an active component, an aggregate that comprises a trimer and/or a hexamer of a molecular needle on which a structural protein of the coronavirus is supported.

Description

コロナウイルスの蛋白質が担持された複合蛋白質単量体、当該単量体の会合体、及び当該会合体を有効成分とするコンポーネントワクチンA complex protein monomer carrying a coronavirus protein, an aggregate of the monomers, and a component vaccine containing the aggregate as an active ingredient.
　本発明は、機能性蛋白質とこれを用いるワクチンについての発明であり、さらに具体的には、免疫原であるコロナウイルス、特に、新型コロナウイルス（ＳＡＲＳ－ＣｏＶ－２）をはじめとするベータコロナウイルスの構造蛋白質と分子針との複合蛋白質（単量体）と当該単量体の会合体、及び、当該会合体を有効成分（感染防御抗原）とする、コンポーネントワクチンに関する発明である。 The present invention is an invention relating to a functional protein and a vaccine using the same, and more specifically, a coronavirus which is an immunogen, particularly a beta coronavirus including a new type coronavirus (SARS-CoV-2). The present invention relates to a complex protein (monomer) of a structural protein and a molecular needle, an aggregate of the monomer, and a component virus using the aggregate as an active ingredient (infection defense antigen).
　２０２０年現在、新型コロナウイルス感染症（ＣＯＶＩＤ－１９）は人類に対する差し迫った脅威であり、急速に感染拡大が進む新型コロナウイルス（ＳＡＲＳ－ＣｏＶ－２）によって、重症例と死亡者の数が世界レベルで急激に増加した。 As of 2020, the new coronavirus infection (COVID-19) is an urgent threat to humankind, and the rapidly spreading new coronavirus (SARS-CoV-2) has caused severe cases and deaths worldwide. Increased sharply at the level.
　このような中で、ＣＯＶＩＤ－１９に対する有効性の高いワクチンの開発が急務となっている。 Under these circumstances, there is an urgent need to develop a highly effective vaccine against COVID-19.
　先行技術文献としては、バクテリオファージの、優れた細胞への遺伝子導入機能に着目して発明された「分子針」についての技術（特許文献１）が提供されている。 Prior art documents provide a technique (Patent Document 1) for a "molecular needle" invented by focusing on the excellent gene transfer function of bacteriophage into cells.
　また、この分子針にノロウイルスの構造蛋白質を担持させた複合蛋白質を、ノロウイルスに対するコンポーネントワクチンとして提供する発明が提供されている（特許文献２）。 Further, an invention is provided in which a complex protein in which a structural protein of norovirus is carried on this molecular needle is provided as a component vaccine against norovirus (Patent Document 2).
特開２０１５－１６３０５６号公報Japanese Unexamined Patent Publication No. 2015-163056 ＷＯ２０１８／０７４５５８号国際公開パンフレットWO2018 / 074558 International Pamphlet
　ベータコロナウイルスに起因する重症呼吸器感染症（ＳＡＲＳ）、中東呼吸器症候群（ＭＥＲＳ）は出現以来８－１８年が経過しているが、いまだ有効なワクチンの開発が進んでいない。ＣＯＶＩＤ－１９に対しては迅速なワクチン開発が必須であり、開発に時間がかかり、慎重な安全性管理を要する不活化ワクチンは採用が困難である。コンポーネントワクチンはこれらの問題点を克服しているが、従来のコンポーネントワクチンにおいては、免疫賦活化のためのアジュバントが必要であり、迅速な開発の足枷となりうる。このような状況の中、コロナウイルス、特にベータコロナウイルス、さらにその中でも未知の要素が多いＣＯＶＩＤ－１９に対して有効なワクチンの開発は困難を極めることが予想される。 8-18 years have passed since the appearance of severe respiratory infection (SARS) and Middle East respiratory syndrome (MERS) caused by betacoronavirus, but the development of effective vaccines has not yet progressed. Rapid vaccine development is essential for COVID-19, and it is difficult to adopt an inactivated vaccine that takes time to develop and requires careful safety management. Although component vaccines overcome these problems, conventional component vaccines require an adjuvant for immunostimulation and can be a hindrance to rapid development. Under such circumstances, it is expected that it will be extremely difficult to develop a vaccine effective against coronavirus, especially betacoronavirus, and COVID-19, which has many unknown factors.
　本発明者らは、上記の問題点を、分子針を用いたコンポーネントワクチンを用いて解決すべく検討を行った。その結果、驚くべきことに、ＣＯＶＩＤ－１９に対して有効であり、かつ、アジュバンドを用いる必要がない、コンポーネントワクチンを提供することが可能であることを見出し、本発明を完成した。 The present inventors have studied to solve the above problems by using a component vaccine using a molecular needle. As a result, it was surprisingly found that it is possible to provide a component vaccine that is effective against COVID-19 and does not require the use of adjuvant, and completed the present invention.
　本発明のワクチンは、コロナウイルスの構造蛋白質の一種又は二種以上を担持させた分子針を有効成分とするコンポーネントワクチンである。 The vaccine of the present invention is a component vaccine containing a molecular needle carrying one or more structural proteins of coronavirus as an active ingredient.
　上記分子針は、本発明の複合蛋白質（下記）の会合体（以下、本発明の会合体ともいう）である。 The molecular needle is an aggregate of the complex protein of the present invention (hereinafter referred to as an aggregate) (hereinafter, also referred to as an aggregate of the present invention).
［１］　本発明の複合蛋白質
　本発明の複合蛋白質は、下記式（１）のアミノ酸配列の複合蛋白質である。すなわち、
　Ｗ－Ｌ１－Ｘｎ－Ｙ　　　（１）
　［式中、Ｗは免疫原であるコロナウイルスの構造蛋白質の一部又は全部のアミノ酸配列を示し、Ｌ１はアミノ酸数が０－１００の第１のリンカー配列を示し、Ｘは配列番号１のアミノ酸配列を示し、Ｙは細胞導入領域のアミノ酸配列を示し、Ｘの繰り返し数であるｎは１－３の整数である。］
であって、
　当該細胞導入領域Ｙのアミノ酸配列は、下記式（２）：
　Ｙ１－Ｌ２－Ｙ２－Ｙ３　　　（２）
　［式中、Ｙ１は配列番号２－５からなる群より選択されるいずれか１つのアミノ酸配列を示し、Ｙ２は配列番号６－９からなる群より選択されるいずれか１つのアミノ酸配列を示し、Ｌ２はアミノ酸数が０－３０の第２のリンカー配列を示し、Ｙ３は修飾用のアミノ酸配列を示し、Ｙ２又はＹ３は存在しない場合もある。］
で表される、複合蛋白質；である。 [1] Conjugated protein of the present invention The conjugated protein of the present invention is a complex protein having the amino acid sequence of the following formula (1). That is,
W-L 1- X n- Y (1)
Wherein, W is the amino acid sequence of part or all of the structural proteins of a coronavirus which are immunogenic, L 1 is the number of amino acids shows a first linker sequence 0-100, X is of SEQ ID NO: 1 The amino acid sequence is shown, Y is the amino acid sequence of the cell introduction region, and n, which is the number of repetitions of X, is an integer of 1-3. ]
And
The amino acid sequence of the cell introduction region Y is the following formula (2):
Y 1- L 2- Y 2- Y 3 (2)
[In the formula, Y 1 represents any one amino acid sequence selected from the group consisting of SEQ ID NO: 2-5, and Y 2 represents any one amino acid sequence selected from the group consisting of SEQ ID NO: 6-9. As shown, L 2 indicates a second linker sequence having 0-30 amino acids, Y 3 indicates an amino acid sequence for modification, and Y 2 or Y 3 may not be present. ]
It is a complex protein represented by.
　なお、上記式（１）、（２）における所定のアミノ酸配列同士を結ぶ「－」は、Ｗ、Ｌ１、Ｘｎ、Ｙ等の概念的に纏まったアミノ酸配列同士の区別を明確にするための、単純な分子結合（実質的にはペプチド結合）の表示である。また、本発明における蛋白質は、ペプチドを含む概念である。 The above formula (1), connecting to each other a predetermined amino acid sequences in (2) "-" is, W, L 1, X n, in order to clarify the distinction between the amino acid sequences between which collectively conceptually such as Y Is a simple molecular binding (substantially a peptide bond) indication. Further, the protein in the present invention is a concept including a peptide.
　上記のコロナウイルスの中でも、ベータコロナウイルスが効果的であり、その中には新型コロナウイルス（ＳＡＲＳ－ＣｏＶ－２）が含まれている。 Among the above coronaviruses, beta coronavirus is effective, and it contains a new type of coronavirus (SARS-CoV-2).
　また本発明は、本発明の複合蛋白質をコードする核酸を組み込んでいる遺伝子発現用ベクター（以下、本発明のベクターともいう）を提供し、さらに、本発明の複合蛋白質をコードする核酸で形質転換された形質転換体（以下、本発明の形質転換体ともいう）を提供する。 The present invention also provides a gene expression vector (hereinafter, also referred to as the vector of the present invention) incorporating a nucleic acid encoding the complex protein of the present invention, and further transforms with the nucleic acid encoding the complex protein of the present invention. The transformed transformant (hereinafter, also referred to as the transformant of the present invention) is provided.
　本発明のベクターを用いて宿主を形質転換させることで、容易に本発明の形質転換体を得ることができる。 By transforming the host with the vector of the present invention, the transformant of the present invention can be easily obtained.
　本発明の形質転換体における遺伝子発現を行うことで、本発明の複合蛋白質を製造することができる。 By expressing the gene in the transformant of the present invention, the complex protein of the present invention can be produced.
［２］　本発明の会合体
　本発明のワクチンの有効成分である本発明の会合体は、本発明の複合蛋白質を単量体とする会合体であり、当該複合蛋白質の三量体又は六量体を含有し、あるいは当該三量体と六量体の混合物を含有する。以下、これら本発明の会合体の含有物の実質を、「三量体及び／又は六量体」と総称する場合も有る。言い換えれば、本発明の会合体は、本発明の複合蛋白質を単量体とする三量体又は六量体を含む会合体である。さらに、後述する本発明の複合体の生産過程を鑑みると、本発明の会合体は、本発明の複合蛋白質を水性液体中で会合させてなる会合体とも定義付けられる。本発明の会合体は、それ自身が細胞内に浸透する作用を発揮することができる。 [2] Aggregate of the present invention The aggregate of the present invention, which is the active ingredient of the vaccine of the present invention, is an aggregate having the complex protein of the present invention as a monomer, and is a trimer or hexamer of the complex protein. It contains a body or a mixture of the trimer and a hexamer. Hereinafter, the substance of the content of the aggregate of the present invention may be collectively referred to as “trimer and / or hexamer”. In other words, the aggregate of the present invention is an aggregate containing a trimer or a hexamer having the complex protein of the present invention as a monomer. Further, in view of the production process of the complex of the present invention described later, the aggregate of the present invention is also defined as an aggregate formed by associating the complex protein of the present invention in an aqueous liquid. The aggregate of the present invention can exert its own action of penetrating into cells.
　上記三量体は、同一又は異なる本発明の複合蛋白質を単量体蛋白質としてなる、三量体蛋白質であり、上記六量体は、当該三量体蛋白質２分子が会合してなる、六量体蛋白質である。 The trimer is a trimer protein in which the same or different complex protein of the present invention is used as a monomer protein, and the hexamer is a hexamer formed by associating two molecules of the trimer protein. It is a body protein.
　本発明の会合体は、水性液体中で本発明の複合蛋白質を接触させることにより製造することができる。担持させる蛋白質と分子針の相性によっては、上記会合体が構成されない場合や、会合体が形成されても水性液体に対する可溶性が得られない場合も考えられる。しかしながら、水性液体中で対象の複合蛋白質を接触させ、その結果をＳＤＳ－ＰＡＧＥ等で確認することにより、対象の複合蛋白質が三量体又は六量体を構成し、本発明のワクチンの有効成分として用いることができるか否かを、容易に把握することができる。また、水性液体についての可溶性は、実際に可溶化試験を行うことで確かめることは容易である。実施例においては、鋭意検討の結果、上記会合体の形成と、その可溶性を得ることができた。 The aggregate of the present invention can be produced by contacting the complex protein of the present invention in an aqueous liquid. Depending on the compatibility between the protein to be carried and the molecular needle, it is conceivable that the above-mentioned aggregate may not be formed, or even if the aggregate is formed, solubility in an aqueous liquid may not be obtained. However, by contacting the target complex protein in an aqueous liquid and confirming the result by SDS-PAGE or the like, the target complex protein constitutes a trimer or a hexamer, and the active ingredient of the vaccine of the present invention. It is possible to easily grasp whether or not it can be used as a protein. Moreover, it is easy to confirm the solubility of an aqueous liquid by actually performing a solubilization test. In the examples, as a result of diligent examination, the formation of the above-mentioned aggregate and its solubility could be obtained.
［３］　本発明のワクチン
　本発明のワクチンは、本発明の会合体を有効成分（感染防御抗原）とするワクチンであり、コロナウイルスに対する、粘膜、経皮、皮下、皮内、又は筋肉内投与用コンポーネントワクチンである。具体的には、上記三量体及び／又は六量体のうち、コロナウイルスの構造蛋白質一部若しくは全部をＷとするものの一種又は二種以上、を有効成分とする、粘膜、経皮、皮下、皮内、又は筋肉内投与用のコンポーネントワクチンである。 [3] Vaccine of the present invention The vaccine of the present invention is a vaccine containing the aggregate of the present invention as an active ingredient (protective antigen for infection), and is administered mucosally, transdermally, subcutaneously, intradermally, or intramuscularly to coronavirus. It is a component vaccine for. Specifically, among the above-mentioned trimers and / or hexamers, one or two or more of the structural proteins of coronavirus having W as W are used as active ingredients, mucosal, transdermal, and subcutaneous. , A component vaccine for intradermal or intramuscular administration.
　免疫原性をさらに高めるために、アジュバンドを、例えば、上記ＷとしてコレラトキシンのＢサブユニットを担持した分子針として、本発明のワクチンに含有させることも可能であるが、むしろアジュバンドを除外したアジュバンドフリーのワクチンとすることが可能であることが、本発明のワクチンの長所として優先されることが好ましい。 In order to further enhance immunogenicity, the adjuvant can be contained in the vaccine of the present invention, for example, as a molecular needle carrying the B subunit of choleratoxin as W described above, but rather the adjuvant is excluded. It is preferable that the ability to obtain a subunit-free vaccine is prioritized as an advantage of the vaccine of the present invention.
　本発明のワクチンの適用可能な動物は、ヒトは勿論のこと、新型コロナウイルスが感染し得る全ての動物に適用可能である、例えば、イヌ又はネコ等も適用対象であるが、これらに限定されるものではない。 The animals to which the vaccine of the present invention can be applied are not only humans but also all animals that can be infected with the new coronavirus, for example, dogs or cats, etc., but are limited to these. It's not something.
［４］　本発明の会合体の生産方法
　本発明の会合体は、本発明の複合蛋白質の３分子以上を、水性液体を介して接触させることにより、当該複合蛋白質同士を単量体として会合させて、三量体と六量体の混合物形成を行い、さらに必要に応じて当該三量体又は六量体を選択的に分離・採取することにより生産できる。 [4] Method for producing aggregate of the present invention In the aggregate of the present invention, three or more molecules of the complex protein of the present invention are brought into contact with each other via an aqueous liquid so that the complex proteins are associated with each other as a monomer. It can be produced by forming a mixture of a trimer and a hexamer, and selectively separating and collecting the trimer or hexamer as needed.
　本発明の複合蛋白質自体は、当該複合蛋白質をコードする核酸を、遺伝子工学的な手法により発現させる、又は、ペプチド合成技術により合成する、ことにより生産することができる。当該複合蛋白質同士を、水性液体中で接触させることにより、自発的に複合蛋白質の三量体、及び、六量体が構築され、三量体と六量体を含有する混合物が形成される。そして、さらに当該三量体又は六量体を選択的に分離・採取することにより、三量体と六量体を分離して生産することができる。 The complex protein itself of the present invention can be produced by expressing the nucleic acid encoding the complex protein by a genetic engineering technique or by synthesizing it by a peptide synthesis technique. By contacting the complex proteins with each other in an aqueous liquid, a trimer and a hexamer of the complex protein are spontaneously constructed, and a mixture containing the trimer and the hexamer is formed. Further, by selectively separating and collecting the trimer or the hexamer, the trimer and the hexamer can be separated and produced.
　「水性液体」に関しては、特に、本発明の複合蛋白質を遺伝子工学的な手法により生産する場合は、当該複合蛋白質を生物学的に発現させて、例えば、発現細胞の収集、破砕又は溶解等による当該複合蛋白質の露出、さらに公知の分離方法による当該複合蛋白質の分離の工程を行う過程において用いる水や各種緩衝液等の水性液体中において、自発的に会合が起こり、本発明の会合体である三量体と六量体を含有する混合物を得ることができる。また、例えば、全化学合成や、パーツ毎の分割合成を行って化学修飾法により結合することにより製造した本発明の複合蛋白質を、水や各種緩衝液等の水性液体中に懸濁することで自発的に会合させて、本発明の会合体である三量体と六量体を含有する混合物を得ることができる。 Regarding the "aqueous liquid", in particular, when the complex protein of the present invention is produced by a genetic engineering method, the complex protein is biologically expressed, for example, by collecting, disrupting or lysing the expressing cells. The aggregate of the present invention spontaneously associates in an aqueous liquid such as water or various buffers used in the process of exposing the complex protein and further separating the complex protein by a known separation method. A mixture containing a trimeric and a hexamer can be obtained. Further, for example, the complex protein of the present invention produced by performing total chemical synthesis or split synthesis for each part and binding by a chemical modification method is suspended in an aqueous liquid such as water or various buffer solutions. By spontaneously associating, a mixture containing a trimer and a hexamer, which are the aggregates of the present invention, can be obtained.
　上記の三量体と六量体を含有する混合物から、三量体又は六量体を分離・採取する方法は、特に限定されず、公知の分子量による分別方法、例えば、ゲル電気泳動法、アフィニティークロマトグラフィー、分子排斥クロマトグラフィー等の分子篩、イオン交換クロマトグラフィー等が挙げられる。 The method for separating and collecting a trimer or a hexamer from the above-mentioned mixture containing a trimer and a hexamer is not particularly limited, and a method for separating by a known molecular weight, for example, gel electrophoresis or affinity. Examples thereof include molecular sieves such as chromatography and molecular exclusion chromatography, ion exchange chromatography and the like.
　従って、上記会合体の生産方法の最も好適な態様の一つとして、「本発明の複合蛋白質をコードする核酸を導入した形質転換体を、液体培地で培養して当該複合蛋白質を発現させ、自発的な会合によって産生される、当該複合蛋白質を単量体とする三量体及び六量体を含む混合物を得る生産方法。さらに当該混合物からさらに当該三量体又は六量体を選択的に分離・採取する、複合蛋白質会合体の生産方法。」が挙げられる。 Therefore, as one of the most preferable aspects of the method for producing the aggregate, "a transformant into which a nucleic acid encoding the complex protein of the present invention has been introduced is cultured in a liquid medium to express the complex protein, and spontaneously. A production method for obtaining a mixture containing a trimer and a hexamer having the complex protein as a monomer, which is produced by a specific association. Further, the trimer or hexamer is selectively separated from the mixture. -A method for producing a complex protein aggregate to be collected. "
　このようにして生産される本発明の会合体を、本発明のワクチンの有効成分として用いることができる。 The aggregate of the present invention produced in this way can be used as an active ingredient of the vaccine of the present invention.
　本発明により、（１）粘膜、経皮、皮下、皮内、又は筋肉内投与により標的組織の細胞に、免疫原であるコロナウイルスの構造蛋白質の一種又は二種以上を効率的に導入するコンポーネントワクチンの有効成分（感染防御抗原）の基本単位として用いることができる、分子針に免疫原であるコロナウイルスの構造蛋白質の一種又は二種以上を担持させた複合蛋白質（単量体）、（２）当該有効成分（感染防御抗原）として用いることができる当該複合蛋白質の会合体、（３）当該会合体を有効成分とするコンポーネントワクチンが提供される。本発明は、新型コロナウイルス（ＳＡＲＳ－ＣｏＶ－２）に対しても効果的なワクチンを提供する手段である。 According to the present invention, (1) a component that efficiently introduces one or more structural proteins of coronavirus, which is an immunogen, into cells of a target tissue by intramucosal, transdermal, subcutaneous, intradermal, or intramuscular administration. A complex protein (monomer) in which one or more structural proteins of coronavirus, which is an immunogen, is carried on a molecular needle, which can be used as a basic unit of an active ingredient (infection defense antigen) of a vaccine, (2). ) An aggregate of the complex protein that can be used as the active ingredient (infection defense antigen), and (3) a component virus containing the aggregate as an active ingredient are provided. The present invention is a means for providing an effective vaccine against the new coronavirus (SARS-CoV-2).
本発明の複合蛋白質を基にした、本発明の会合体である三量体と六量体の構築過程を示した図面である。It is a figure which showed the construction process of the trimer and hexamer which is the aggregate of this invention based on the complex protein of this invention. ＲＢＤｐ１蛋白質を担持した分子ニードルによるウイルス中和試験において、血清の希釈倍率毎の、生細胞の存在を示すウェルにおける、メチレンブルーによる染色状態の変化を示した図面である。It is a figure which showed the change of the staining state by methylene blue in the well which shows the existence of a living cell for every dilution ratio of serum in the virus neutralization test by the molecular needle carrying RBDp1 protein. ＲＢＤｐ１蛋白質を担持した分子ニードルで免疫したモルモットの血清に対してＥＬＩＳＡを行い、ＩｇＧ量を検出した結果を示した図面である。It is a figure which showed the result of having performed the ELISA on the serum of the guinea pig immunized with the molecular needle carrying the RBDp1 protein, and detected the amount of IgG. ＲＢＤｐ１蛋白質を担持した分子ニードルで免疫したモルモットの血清に対してＥＬＩＳＡを行い、ＩｇＡ量を検出した結果を示した図面である。It is a figure which showed the result of having performed the ELISA on the serum of the guinea pig immunized with the molecular needle carrying the RBDp1 protein, and detected the amount of IgA.
［１］新型コロナウイルス（ＳＡＲＳ－ＣｏＶ－２）
　新型コロナウイルスは、本発明が適用されることで最も貢献度が高いと考えられる対象である。 [1] New coronavirus (SARS-CoV-2)
The new coronavirus is a target that is considered to have the highest degree of contribution by applying the present invention.
　新型コロナウイルス（ＳＡＲＳ－ＣｏＶ－２）(以下、新型コロナウイルスとも記載する)は、ＳＡＲＳ(severe acute respiratory syndrome)の病原体であるＳＡＲＳコロナウイルス、ヒトに上気道炎を起こすヒトコロナウイルスＯＣ４３株、下気道炎も起こすヒトコロナウイルスＨＫＵ１をはじめ、マウス、ウシ、ブタ等に感染するものと同じ、コロナウイルス属の抗原性グループ２（ベータコロナウイルス）に属している。 The new coronavirus (SARS-CoV-2) (hereinafter, also referred to as the new coronavirus) is the SARS coronavirus, which is the causative agent of SARS (severe acute respiratory syndrome), and the human coronavirus OC43 strain that causes upper airway inflammation in humans. It belongs to the antigenic group 2 (beta coronavirus) of the genus Coronavirus, which infects mice, cattle, pigs, etc., including the human coronavirus HKU1 that also causes lower airway inflammation.
　新型コロナウイルスの形状は、他のコロナウイルス属と同様に、根元が細く、先端が膨らんだ花弁状のスパイクを有する、直径１００ｎｍ台の球状粒子である。新型コロナウイルスの構造蛋白質は、エンベロープにはＳ(spike)蛋白質、Ｍ(membrane)蛋白質、Ｅ(envelope)蛋白質が存在する。Ｓ蛋白質は糖蛋白質であり、三量体として１本の花弁状のスパイクを形成し、宿主細胞のウイルスレセプター（アンジオテンシン変換酵素II(ＡＣＥ２)）への吸着能とセリンプロテアーゼ（ＴＭＰＲＳＳ２）の作用を介した膜融合能を有しており、中和エピトープ、Ｔ細胞エピトープとして、宿主の免疫応答の標的にもなる。Ｍ蛋白質とＥ蛋白質も糖蛋白質であり、大部分が脂質二重層内に位置しており、ウイルス粒子形成に重要な役割を有している。 The shape of the new coronavirus is a spherical particle with a diameter of 100 nm, which has a petal-like spike with a thin root and a bulging tip, similar to other coronavirus genera. The structural proteins of the new coronavirus include S (spike) protein, M (membrane) protein, and E (envelope) protein in the envelope. The S protein is a glycoprotein that forms a single petal-like spike as a trimer, and has the ability to adsorb host cells to viral receptors (angiotensin converting enzyme II (ACE2)) and the action of serine protease (TMPRSS2). It has the ability to fuse membranes through the membrane, and as a neutralizing epitope and T cell epitope, it can also be a target for the immune response of the host. M protein and E protein are also glycoproteins, most of which are located in the lipid bilayer and play an important role in virus particle formation.
　また、Ｎ(nucleocapsid)蛋白質は、ＲＮＡ結合性リン酸化蛋白質であり、ウイルスゲノムＲＮＡと結合し、ヌクレオカプシドを形成すると共に、ＲＮＡの複製、転写、翻訳に関与している。 The N (nucleocapsid) protein is an RNA-binding phosphorylated protein that binds to viral genomic RNA to form nucleocapsid and is involved in RNA replication, transcription, and translation.
　新型コロナウイルスも、ゲノムはプラス鎖の一本鎖ＲＮＡで、それ自体がｍＲＮＡとして機能すると共に感染性も有している。また少なくともＳＡＲＳコロナウイルスと同様に、ゲノム５’末端にはキャップ構造、３’末端にはポリＡを有し、５’末端には遺伝子複製と転写を調節するリーダー配列と非翻訳領域があり、その下流にＲＮＡポリメラーゼ、プロテアーゼ等のウイルス増殖に必須の酵素（レプリカーゼ）をコードする非構造蛋白質遺伝子、さらに、上記のＳ、Ｅ、Ｍ、Ｎをコードする構造遺伝子が存在する。 The genome of the new coronavirus is also a positive-strand single-stranded RNA, which itself functions as mRNA and is also infectious. Also, at least like the SARS coronavirus, the genome has a cap structure at the 5'end and a poly A at the 3'end, and has a leader sequence and untranslated region that regulate gene replication and transcription at the 5'end. Downstream of this, there are non-structural protein genes encoding enzymes (replicases) essential for viral growth such as RNA polymerase and protease, and structural genes encoding the above-mentioned S, E, M, and N.
　上記のＳ蛋白質は、詳細には、１２７３アミノ酸で構成され、ＳＳ（シグナル配列:signal sequence）、ＮＴＤ（Ｎ末端領域:N-terminal domain）、ＲＢＤ（レセプター結合領域:receptor-binding domain）、ＳＤ１（サブドメイン１:subdomain 1）、ＳＤ２（サブドメイン２:subdomain 2）、Ｓ１／Ｓ２（Ｓ１／Ｓ２プロテアーゼ開裂部位:S1/S2 protease cleavage site）、Ｓ２’（Ｓ２’プロテアーゼ開裂部位:S2’protease cleavage site）、ＦＰ（融合ペプチド:fusion peptide）、ＨＲ１（アミノ酸繰り返し構造１:heptad repeat 1）、ＣＨ（中央螺旋:central helix）、ＣＤ（コネクター領域:connector domain）、ＨＲ２（アミノ酸繰り返し構造２: heptad repeat 1）、ＴＭ（トランスメンブレン領域:transmembrane domain）、ＣＴ（細胞質側末端:cytoplasmic tail）を有している。ＲＢＤは、２つのダウンプロトマーと一つのアッププロトマーで構成される三量体として構成されている（非特許文献１、非特許文献２等）。 The above S protein is specifically composed of 1273 amino acids, SS (signal sequence), NTD (N-terminal region: N-terminal domain), RBD (receptor binding region: receptor-binding domain), SD1. (Subdomain 1: subdomain 1), SD2 (subdomain 2: subdomain 2), S1 / S2 (S1 / S2 protease cleavage site: S1 / S2 protease cleavage site), S2'(S2'protease cleavage site: S2'protease) cleavage site), FP (fusion peptide), HR1 (amino acid repeating structure 1: heptad repeat 1), CH (central spiral: central helix), CD (connector region: connector domain), HR2 (amino acid repeating structure 2: amino acid repeating structure 2: It has heptad repeat 1), TM (transmembrane domain), and CT (cytoplasmic tail). The RBD is configured as a trimer composed of two downprotomers and one upprotomer (Non-Patent Document 1, Non-Patent Document 2, etc.).
　新型コロナウイルス感染症は、多くの感染者が軽症・無症状である反面、一定の割合の重症・死亡例が認められることが特徴の一つであり、重症化の一因としてサイトカインストーム（免疫システムの暴走）が挙げられている。 One of the characteristics of the new coronavirus infection is that many infected people are mild and asymptomatic, but a certain percentage of severe and dead cases are observed, and a cytokine storm (immunity) is one of the causes of the aggravation. System runaway) is mentioned.
［２］　本発明の複合蛋白質
　本発明の複合蛋白質を表すアミノ酸配列式である式（１）：
　Ｗ－Ｌ１－Ｘｎ－Ｙ　　　（１）
　［式中、Ｗは免疫原であるコロナウイルスの構造蛋白質の一部又は全部のアミノ酸配列を示し、Ｌ１はアミノ酸数が０－１００の第１のリンカー配列を示し、Ｘは配列番号１のアミノ酸配列を示し、Ｙは細胞導入領域のアミノ酸配列を示し、ｎは１－３の整数であり、Ｙは、下記式（２）：
　Ｙ１－Ｌ２－Ｙ２－Ｙ３　　　（２）
　［式中、Ｙ１は配列番号２－５からなる群より選択されるいずれか１つのアミノ酸配列を示し、Ｙ２は配列番号６－９からなる群より選択されるいずれか１つのアミノ酸配列を示し、Ｌ２はアミノ酸数が０－３０の第２のリンカー配列を示し、Ｙ３は修飾用のアミノ酸配列を示し、Ｙ２又はＹ３は存在しない場合もある。］
で表される。]
　において、免疫原（エピトープ）であるＷは、上記のようにコロナウイルスの構造蛋白質の一部又は全部のペプチド構成に基づくアミノ酸配列が挙げられる。新型コロナウイルスには、全ての変異株が含まれる。 [2] Conjugated protein of the present invention Formula (1) which is an amino acid sequence formula representing the complex protein of the present invention:
W-L 1- X n- Y (1)
Wherein, W is the amino acid sequence of part or all of the structural proteins of a coronavirus which are immunogenic, L 1 is the number of amino acids shows a first linker sequence 0-100, X is of SEQ ID NO: 1 The amino acid sequence is shown, Y is the amino acid sequence of the cell introduction region, n is an integer of 1-3, and Y is the following formula (2) :.
Y 1- L 2- Y 2- Y 3 (2)
[In the formula, Y 1 represents any one amino acid sequence selected from the group consisting of SEQ ID NO: 2-5, and Y 2 represents any one amino acid sequence selected from the group consisting of SEQ ID NO: 6-9. As shown, L 2 indicates a second linker sequence having 0-30 amino acids, Y 3 indicates an amino acid sequence for modification, and Y 2 or Y 3 may not be present. ]
It is represented by. ]
In, W, which is an immunogen (epitope), has an amino acid sequence based on the peptide composition of a part or all of the structural protein of coronavirus as described above. The new coronavirus includes all mutant strains.
　免疫原Ｗとして選択され得るコロナウイルスの構造蛋白質は、上記したように、Ｓ蛋白質、Ｍ蛋白質、Ｅ蛋白質、Ｎ蛋白質等の構造蛋白質である。さらに、抗体の認識可能なエピトープを含むペプチド配列であっても良い。 As described above, the structural protein of the coronavirus that can be selected as the immunogen W is a structural protein such as S protein, M protein, E protein, and N protein. Further, it may be a peptide sequence containing a recognizable epitope of the antibody.
　本発明のワクチンの有効成分の核心的な要素として、免疫原Ｗは選択されるが、その場合、上記のうちでもＳ蛋白質を選択することが好適である。また、Ｓ蛋白質の中から、ＲＢＤの全部又は一部を含んで選択することも好適である。これは、本発明の対象ウイルスであるコロナウイルス全般に言えることであり、当然、ベータコロナウイルス、さらに新型コロナウイルスについて言えることである。 The immunogen W is selected as the core element of the active ingredient of the vaccine of the present invention, and in that case, it is preferable to select the S protein among the above. It is also preferable to select from the S proteins, including all or part of RBD. This can be said for all coronaviruses that are the target viruses of the present invention, and of course, for beta coronaviruses and new coronaviruses.
　上記Ｘｎにおけるアミノ酸配列Ｘの繰り返し数であるｎは、１であることが好適であるが、２又は３であってもよい。 The n, which is the number of repetitions of the amino acid sequence X in the above Xn, is preferably 1, but may be 2 or 3.
　上記式（１）において、Ｘｎ、Ｙ１、又は、Ｙ２で示されるアミノ酸配列のうち、１個以上のアミノ酸が欠失、置換若しくは付加された改変アミノ酸配列が上記式（１）に含まれる。「欠失」とは、上記式（１）において定義されている各配列番号のアミノ酸配列におけるいずれかのアミノ酸残基が欠失しており、当該欠失したアミノ酸残基のＮ末端側（前）とＣ末端側（後）のアミノ酸残基がペプチド結合で結ばれた状態であり（Ｎ末端アミノ酸残基とＣ末端アミノ酸残基の欠失の場合は、当該アミノ酸残基が単に欠失した状態である）、当該欠失残基の個数が「アミノ酸欠失の個数」として数えられる。「置換」とは、上記式（１）において定義されている各配列番号のアミノ酸配列におけるいずれかのアミノ酸残基が「他のアミノ酸残基」に入れ替わっており、当該入れ替わったアミノ酸残基が、Ｎ末端側（前）とＣ末端側（後）の各アミノ酸残基とペプチド結合で結ばれた状態であり（Ｎ末端アミノ酸残基の置換の場合はＣ末端側のアミノ酸残基とのペプチド結合のみであり、Ｃ末端アミノ酸残基の置換の場合はＮ末端側のアミノ酸残基とのペプチド結合のみである）、当該置換残基の個数が「アミノ酸置換の個数」として数えられる。「付加」とは、上記式（１）において定義されている各配列番号のアミノ酸配列における、いずれか１箇所以上のペプチド結合の位置に、各々１個以上の新たなアミノ酸残基が挿入された状態で新たなペプチド結合が形成された状態である。これらのアミノ酸残基の改変の内容と個数は、上記式（１）に係わるアミノ酸配列と、改変に係わるアミノ酸配列のアライメントを、人力又はアミノ酸配列の解析が可能なソフトウエアを用いてコンピュータ上で行うことにより、明らかにすることができる。 In the above formula (1), the modified amino acid sequence in which one or more amino acids are deleted, substituted or added among the amino acid sequences represented by X n , Y 1 or Y 2 is included in the above formula (1). Is done. “Deletion” means that any amino acid residue in the amino acid sequence of each SEQ ID NO: defined in the above formula (1) is deleted, and the N-terminal side (previous) of the deleted amino acid residue is deleted. ) And the amino acid residue on the C-terminal side (after) are connected by a peptide bond (in the case of deletion of the N-terminal amino acid residue and the C-terminal amino acid residue, the amino acid residue is simply deleted. The number of the deleted residues is counted as "the number of amino acid deletions". "Substitution" means that any amino acid residue in the amino acid sequence of each SEQ ID NO: defined in the above formula (1) is replaced with "another amino acid residue", and the replaced amino acid residue is It is in a state of being connected to each amino acid residue on the N-terminal side (front) and C-terminal side (rear) by a peptide bond (in the case of substitution of the N-terminal amino acid residue, a peptide bond with the amino acid residue on the C-terminal side). In the case of substitution of a C-terminal amino acid residue, only the peptide bond with the amino acid residue on the N-terminal side), the number of the substituted amino acid residues is counted as "the number of amino acid substitutions". “Addition” means that one or more new amino acid residues are inserted at any one or more peptide bond positions in the amino acid sequence of each SEQ ID NO: defined in the above formula (1). It is a state in which a new peptide bond is formed in the state. The content and number of modifications of these amino acid residues can be determined by aligning the amino acid sequence related to the above formula (1) with the amino acid sequence related to the modification on a computer using human power or software capable of analyzing the amino acid sequence. By doing, it can be clarified.
　また、上記式（１）で定義されたリンカー配列Ｌ１又はＬ２、あるいは、修飾用のアミノ酸配列Ｙ３は、上記定義されたアミノ酸残基数の範囲内において、必要に応じて任意の配列を選択することができる。 Further, the linker sequence L 1 or L 2 defined by the above formula (1) or the amino acid sequence Y 3 for modification is an arbitrary sequence as necessary within the range of the number of amino acid residues defined above. Can be selected.
　そして、当該改変アミノ酸配列の改変複合蛋白質の三量体又は六量体（下記）が、上記式（１）の複合蛋白質の三量体又は六量体と、実質的に同等の免疫賦活活性を有することが好ましい。「実質的に同等」とは、「中和試験」等の免疫賦活活性の確認について確立している手法を用いた場合に、アミノ酸配列の非改変複合蛋白質との免疫賦活活性の有意差が、５％以内の有意水準において認められない程度の同等性である。 Then, the trimer or hexamer (below) of the modified complex protein of the modified amino acid sequence has substantially the same immunostimulatory activity as the trimer or hexamer of the complex protein of the above formula (1). It is preferable to have. "Substantially equivalent" means that when a method established for confirmation of immunostimulatory activity such as "neutralization test" is used, the significant difference in immunostimulatory activity from the unmodified complex protein of amino acid sequence is used. Equivalence to the extent that it is not observed at the significance level within 5%.
　上記式（１）の、Ｘｎ、Ｙ１、又は、Ｙ２で示されるアミノ酸配列のうち、１個以上のアミノ酸が欠失、置換若しくは付加された改変アミノ酸配列における、各々のアミノ酸配列におけるアミノ酸改変の数は、Ｘｎが８ｎ個以内、好ましくは４ｎ個以内、さらに好ましくは２ｎ個以内；Ｙ１が３０個以内、好ましくは２０個以内、さらに好ましくは１０個以内；及びＹ２が１５個以内、好ましくは１０個以内、さらに好ましくは５個以内；であることが好適である。 Amino acid in each amino acid sequence in the modified amino acid sequence in which one or more amino acids are deleted, substituted or added among the amino acid sequences represented by X n , Y 1 or Y 2 in the above formula (1). The number of modifications is 8 n or less, preferably 4 n or less, more preferably 2 n or less; Y 1 is 30 or less, preferably 20 or less, still more preferably 10 or less; and Y 2 is 15 or less. It is preferably within 10 pieces, preferably within 10 pieces, and more preferably within 5 pieces;
　上記式（１）の第１のリンカー配列を示すＬ１は、免疫原Ｗと分子針部分Ｙの距離を適度に保って立体障害を抑制するために必要であり、このアミノ酸残基の個数は、上記の通りにアミノ酸残基数は０－１００個であり、好適には４－４０個である。具体的な配列の内容は限定されないが、例えば、（ＧＧＧＧＳ）ｍ、（ＰＡＰＡＰ）ｍ［ｍは繰り返し数であり、１－５の整数であることが好ましく、１－３であることが特に好ましい］等が例示される。ただしこれらはあくまでも例示である。ｍ＝１の場合には４－１２個であることが好適である。 L 1 showing a first linker sequence of the formula (1) is necessary to suppress the steric hindrance reasonably keeping the distance immunogen W and molecular needle portion Y, the number of amino acid residues As described above, the number of amino acid residues is 0-100, preferably 4-40. The specific contents of the sequence are not limited, but for example, (GGGGS) m and (PAPAP) m [m are repetition numbers, preferably an integer of 1-5, and particularly preferably 1-3. ] Etc. are exemplified. However, these are just examples. When m = 1, it is preferable that the number is 4-12.
　上記式（１）のＸは、配列番号１のアミノ酸からなる、アミノ酸配列ＸnにおけるＸのｎ回（整数回）の繰り返し単位の配列である。繰り返しの形式は、直列の繰り返しであり、例えば、Ｘ２であれば、「Ｘ－Ｘ」（「－」は模式化したペプチド結合）である。また、繰り返し配列Ｘｎにおいては、上述したアミノ酸配列の改変が許容される。ここでｎは、上述のように１－３の整数であり、１が好適であるが、２又は３であってもよい。繰り返し配列Ｘｎのｎが２又は３である場合は、免疫原Ｗの大きさや特性に応じて、分子針Ｙの距離を安定して適切な距離に保つことが主要な目的となる。 X in the above formula (1) is a sequence of n times (integer times) of X in the amino acid sequence Xn, which is composed of the amino acid of SEQ ID NO: 1. The form of the iteration is a series iteration, for example, for X 2 , "XX"("-" is a schematic peptide bond). Further, in the repeating sequence Xn , the above-mentioned modification of the amino acid sequence is permitted. Here, n is an integer of 1-3 as described above, 1 is preferable, but 2 or 3 may be used. When n of the repeating sequence X n is 2 or 3, the main purpose is to keep the distance of the molecular needle Y stable and appropriate according to the size and characteristics of the immunogen W.
　細胞導入領域Ｙは、分子針の基礎構造に該当し、バクテリオファージの尾（Ｔａｉｌ）の針部分（細胞内導入部）に基づいたものであり、式（２）：
　Ｙ１－Ｌ２－Ｙ２－Ｙ３　　　（２）
　［式中、Ｙ１は配列番号２－５からなる群より選択されるアミノ酸配列を示し、Ｙ２は配列番号６－９からなる群より選択されるアミノ酸配列を示し、Ｌ２はアミノ酸数が０－３０の第２のリンカー配列を示し、Ｙ３は所定の修飾用のアミノ酸配列を示し、Ｙ２又はＹ３は存在しない場合もある。］
で表される蛋白質である。 The cell introduction region Y corresponds to the basic structure of the molecular needle and is based on the needle portion (intracellular introduction portion) of the tail of the bacteriophage.
Y 1- L 2- Y 2- Y 3 (2)
[In the formula, Y 1 indicates an amino acid sequence selected from the group consisting of SEQ ID NO: 2-5, Y 2 indicates an amino acid sequence selected from the group consisting of SEQ ID NO: 6-9, and L 2 indicates the number of amino acids. shows a second linker sequence 0-30, Y 3 represents the amino acid sequence for a given modification, Y 2 or Y 3 is sometimes not present. ]
It is a protein represented by.
　式（２）のＹ１のうち、Ｎ末端側３２アミノ酸（３２Ｌｅｕ）までが、バクテリオファージＴ４の三重らせんβシート構造の部分のアミノ酸配列である。なお、少なくともＮ末端のアミノ酸であるバリン（１Ｖａｌ）は、ロイシン（１Ｌｅｕ）であってもよい。残りのＣ末端側は、バクテリオファージのニードル蛋白質のＣ末端部分のアミノ酸配列である。このＹ１のＣ末端側に使用可能なアミノ酸配列としては、例えば、バクテリオファージＴ４のｇｐ５のアミノ酸配列、バクテリオファージＰ２のｇｐＶのアミノ酸配列、バクテリオファージＭｕのｇｐ４５のアミノ酸配列、バクテリオファージφ９２のｇｐ１３８のアミノ酸配列等が挙げられる。より具体的には、バクテリオファージＴ４のｇｐ５のアミノ酸配列をＣ末端側に有するＹ１として配列番号２のアミノ酸配列が、バクテリオファージＰ２のｇｐＶのアミノ酸配列をＣ末端側に有するＹ１として配列番号３のアミノ酸配列が、バクテリオファージＭｕのｇｐ４５のアミノ酸配列をＣ末端側に有するＹ１として配列番号４のアミノ酸配列が、バクテリオファージφ９２のｇｐ１３８のアミノ酸配列をＣ末端側に有するＹ１として配列番号５のアミノ酸配列が挙げられる。このＹ１のアミノ酸配列をコードする核酸配列は、アミノ酸と核酸塩基の公知の関係に従って選択することができる。 Of Y 1 of the formula (2), up to 32 amino acids (32 Leu) on the N-terminal side are the amino acid sequences of the portion of the triple helix β-sheet structure of Escherichia virus T4. At least the N-terminal amino acid valine (1Val) may be leucine (1Leu). The remaining C-terminal side is the amino acid sequence of the C-terminal portion of the bacteriophage needle protein. Examples of the amino acid sequence that can be used on the C-terminal side of Y 1 include the amino acid sequence of gp5 of Bacterophage T4, the amino acid sequence of gpV of Bacterophage P2, the amino acid sequence of gp45 of Bacterophage Mu, and gp138 of Bacterophage φ92. Examples include the amino acid sequence of. More specifically, the amino acid sequence of the Y 1 as SEQ ID NO: 2 having the amino acid sequence of gp5 of bacteriophage T4 in the C-terminal side, SEQ number as Y 1 having the amino acid sequence of the gpV of bacteriophage P2 C-terminal 3 amino acid sequence, the amino acid sequence of SEQ ID NO: 4 as Y 1 having the amino acid sequence of gp45 of bacteriophage Mu to C-terminal side, SEQ number as Y 1 having the amino acid sequence of gp138 of bacteriophage φ92 to C-terminal The amino acid sequence of 5 is mentioned. The nucleic acid sequence encoding the amino acid sequence of Y 1 can be selected according to the known relationship between the amino acid and the nucleobase.
　式（２）中、Ｙ２はバクテリオファージＴ４のｆｏｌｄｏｎと呼ばれる領域のアミノ酸配列、又は、バクテリオファージＰ２若しくはバクテリオファージＭｕ若しくはバクテリオファージφ９２のｔｉｐと呼ばれる領域のアミノ酸配列である。ｆｏｌｄｏｎ又はｔｉｐは、バクテリオファージのフィブリチンと呼ばれる分子針構造の先端部を構成する領域である。式（２）においてＹ２が存在することは必須とはいえないが、このｆｏｌｄｏｎ又はｔｉｐのアミノ酸配列を有することにより、細胞膜への分子針の取り込み効率を向上させることができるので、Ｙ２を伴っていることが好適である。バクテリオファージＴ４のｆｏｌｄｏｎのアミノ酸配列を配列番号６に示す。このアミノ酸配列をコードする核酸配列は、アミノ酸と核酸塩基の公知の関係に従って選択することができる。 In formula (2), Y 2 is the amino acid sequence of the region called folon of bacteriophage T4, or the amino acid sequence of the region called bacteriophage P2 or bacteriophage Mu or bacteriophage φ92 tip. The earth or tip is a region constituting the tip of a molecular needle structure called a bacteriophage fibritin. Although is not a mandatory that Y 2 is present in the formula (2), by having the amino acid sequence of the foldon or tip, it is possible to improve efficiency of incorporation of molecular needle to the cell membrane, the Y 2 It is preferable to accompany it. The amino acid sequence of folon of Escherichia virus T4 is shown in SEQ ID NO: 6. The nucleic acid sequence encoding this amino acid sequence can be selected according to the known relationship between the amino acid and the nucleobase.
　バクテリオファージＰ２のｔｉｐのアミノ酸配列を配列番号７に示す。このアミノ酸配列をコードする核酸配列は、アミノ酸と核酸塩基の公知の関係に従って選択することができる。バクテリオファージＭｕのｔｉｐのアミノ酸配列を配列番号８に示す。このアミノ酸配列をコードする核酸配列は、アミノ酸と核酸塩基の公知の関係に従って選択することができる。バクテリオファージφ９２のｔｉｐのアミノ酸配列を配列番号９に示す。このアミノ酸配列をコードする核酸配列は、アミノ酸と核酸塩基の公知の関係に従って選択することができる。 The amino acid sequence of the tip of bacteriophage P2 is shown in SEQ ID NO: 7. The nucleic acid sequence encoding this amino acid sequence can be selected according to the known relationship between the amino acid and the nucleobase. The amino acid sequence of the bacteriophage Mu tip is shown in SEQ ID NO: 8. The nucleic acid sequence encoding this amino acid sequence can be selected according to the known relationship between the amino acid and the nucleobase. The amino acid sequence of the tip of bacteriophage φ92 is shown in SEQ ID NO: 9. The nucleic acid sequence encoding this amino acid sequence can be selected according to the known relationship between the amino acid and the nucleobase.
　Ｌ２は、前記Ｙ１とＹ２の間に介在する第２のリンカーである。リンカーＬ２のアミノ酸数は０－３０個であり、好適には０－５個である。リンカーのアミノ酸数が０個とは、第２のリンカーＬ２は存在しないことを示すものである。 L 2 is a second linker interposed between Y 1 and Y 2. Number of amino acids of the linker L 2 is a 0-30 carbon atoms, preferably a 0-5. The number of amino acids of the linker is zero, is an indication that the second linker L 2 is absent.
　Ｙ３は、修飾用のアミノ酸配列であり、Ｙにおいて選択的に付加して用いることができる。当該修飾用のアミノ酸配列は、蛋白質精製や保護の目的等で付加するものであり、ヒスチジンタグ、ＧＳＴタグ、ＦＬＡＧタグ等のタグ蛋白質等が挙げられる。Ｙ３には、適宜リンカー配列を加えることが可能であり、このようなリンカー配列自体もＹ３のアミノ酸配列の構成要素となり得る。 Y 3 is an amino acid sequence for modification, and can be selectively added and used in Y. The amino acid sequence for the modification is added for the purpose of protein purification, protection, etc., and examples thereof include tag proteins such as histidine tag, GST tag, and FLAG tag. A linker sequence can be appropriately added to Y 3 , and such a linker sequence itself can be a component of the amino acid sequence of Y 3.
　本発明の複合蛋白質は、公知の方法、具体的には、遺伝子工学的手法、又は、化学合成法により生産することができる。また、本発明の複合蛋白質全てを一緒に生産することも可能であり、パーツ毎に生産して当該パーツ同士を化学修飾法により事後的に結合させることにより生産することも可能である。リンカー（Ｌ１又はＬ２等）を介したポリペプチド同士の結合は、互いのポリペプチドにおけるリシン残基又はシステイン残基同士を、スクシンイミド基又はマレイミド基を有するリンカーにより結合させる等が可能である。 The complex protein of the present invention can be produced by a known method, specifically, a genetic engineering method or a chemical synthesis method. It is also possible to produce all of the complex proteins of the present invention together, and it is also possible to produce each part by ex post-bonding the parts by a chemical modification method. Bonding of polypeptides to each other via a linker (L 1 or L 2, etc.) can be performed by binding lysine residues or cysteine residues in each other's polypeptides with a linker having a succinimide group or a maleimide group. ..
　遺伝子工学的手法では、生産対象の本発明の複合蛋白質の全部又は一部をコードする核酸を、例えば、大腸菌、酵母、昆虫細胞、動物細胞等の宿主細胞を形質転換体として、あるいは大腸菌抽出液、ウサギ網状赤血球抽出液、小麦胚芽抽出液等の無細胞発現系で発現させることができる。これらの核酸が組み込まれた発現用ベクターとしては、各発現系に応じたものを用いることができ、例えば大腸菌における発現用の、ｐＥＴ、ｐＢＲ３２２、ｐＢＲ３２５、ｐＵＣ１８、ｐＵＣ１１９、ｐＴｒｃＨｉｓ、ｐＢｌｕｅＢａｃＨｉｓ等；酵母における発現用のｐＡＵＲ、ＹＥｐ１３，ＹＥｐ２４，ＹＣｐ５０，ｐＹＥ５２等；昆虫細胞における発現用のｐＩＥｘ－１、動物細胞における発現用のｐＢＡｐｏ－ＣＭＶ、小麦胚芽抽出液における発現用のｐＦ３Ａ等が挙げられるがこれらに限定されるものではなく、必要に応じた要素を組み込んだベクターを構築して用いることができる。例えば、構造遺伝子の前に、様々なプロモーターを選択配置させることも可能であり、さらにエンハンサー等のシスエレメント、スプライシングシグナル、ポリＡ付加シグナル、リボソーム結合配列（ＳＤ配列）、ターミネーター配列等を配置させることも可能である。また、マーカー遺伝子を組み込むことも可能である。勿論、現在市販されている様々な、遺伝子発現キットを用いることも可能である。 In the genetic engineering method, a nucleic acid encoding all or part of the complex protein of the present invention to be produced is used as a transformant of a host cell such as Escherichia coli, yeast, insect cell, animal cell, or an Escherichia coli extract. , Rabbit reticulated erythrocyte extract, wheat germ extract and the like can be expressed in a cell-free expression system. As an expression vector into which these nucleic acids are incorporated, one corresponding to each expression system can be used, for example, pET, pBR322, pBR325, pUC18, pUC119, pTrcHis, pBlueBacHis, etc. for expression in Escherichia coli; in yeast. Examples include pAUR for expression, YEp13, YEp24, YCp50, pYE52; pIEx-1 for expression in insect cells, pBApo-CMV for expression in animal cells, pF3A for expression in wheat germ extract, and the like. It is not limited, and a vector incorporating elements as required can be constructed and used. For example, it is possible to selectively place various promoters in front of the structural gene, and further place a cis element such as an enhancer, a splicing signal, a poly A addition signal, a ribosome binding sequence (SD sequence), a terminator sequence, and the like. It is also possible. It is also possible to incorporate a marker gene. Of course, it is also possible to use various gene expression kits currently on the market.
　化学合成法は、公知のペプチドの化学合成法を用いることが可能である。すなわち、常法として確立している液相ペプチド合成法、又は、固相ペプチド合成法を用いて、本発明の複合蛋白質の全部又は一部を製造することが可能である。そして、一般的に好適な化学合成法として認識されている固相ペプチド合成法も、Ｂｏｃ固相法又はＦｍｏｃ固相法を用いることが可能であり、上述のように、必要に応じてライゲーション法を用いることも可能である。また、個々のアミノ酸は、公知の方法により製造可能であり、市販品を用いることも可能である。 As the chemical synthesis method, it is possible to use a known chemical synthesis method for peptides. That is, it is possible to produce all or part of the complex protein of the present invention by using the liquid phase peptide synthesis method or the solid phase peptide synthesis method which has been established as a conventional method. The solid-phase peptide synthesis method, which is generally recognized as a suitable chemical synthesis method, can also use the Boc solid-phase method or the Fmoc solid-phase method, and as described above, the ligation method is required. It is also possible to use. Further, each amino acid can be produced by a known method, and a commercially available product can also be used.
［３］本発明の会合体
　図１に、本発明の複合蛋白質を基にした、本発明の会合体である三量体と六量体の構築過程を示す。図１において、１０は、単量体としての本発明の複合蛋白質であり、３０は、本発明の三量体であり、６０は、本発明の六量体である。 [3] Aggregate of the present invention FIG. 1 shows the process of constructing a trimer and a hexamer, which are aggregates of the present invention, based on the complex protein of the present invention. In FIG. 1, 10 is the complex protein of the present invention as a monomer, 30 is the trimer of the present invention, and 60 is the hexamer of the present invention.
　本発明の複合蛋白質１０は、「式（２）のＸｎとＹ１に該当する基本部分１３１」と「式（２）のＹ２に該当するｆｏｌｄｏｎ１３２」が結合した「式（１）のＹに該当する分子針領域１３」、及び、「式（１）のＷに該当する免疫原１１」が、「式（１）のＬ１に該当するリンカー１２」を介して結合して構成されている。リンカー１２以外のリンカーと、式（２）のＹ３に相当する修飾配列については、図示を省略した。本発明複合蛋白質１０自体には、標的組織の細胞の細胞膜を通過する機能は実質的に認められない。 The complex protein 10 of the present invention has "Y of formula (1)" in which "basic portion 131 corresponding to X n and Y 1 of formula (2)" and "foldon 132 corresponding to Y 2 of formula (2)" are bound. the corresponding molecular needle region 13 ', and, "wherein the immunogen 11 corresponding to W (1)" is "is configured attached via a linker 12" corresponding to L 1 of formula (1) There is. And Linker than linkers 12, the modified sequence corresponding to Y 3 in formula (2) are not shown. The complex protein 10 of the present invention does not have a function of passing through the cell membrane of the cell of the target tissue.
　三量体３０は、上記の複合蛋白質１０が、３個の単量体として自発的に会合してなる三量体である。三量体３０は、上記の分子針領域１３が３個纏まり互いのＣ末端同士で会合することによって、三量体平行βシート構造、及び、当該βシート構造自身によるらせん構造（三重らせんβシート構造）と呼ばれる針状構造が形成され、分子針１３×３が形成されている。分子針１３×３は、基本部分１３１×３と、ｆｏｌｄｏｎ集合体１３２×３で構成されている。このように三量体化と自己組織化により標的組織の細胞の細胞膜を通過する機能を有する「分子針」が形成され、それぞれの単量体に由来するリンカー３本（１２１、１２２、１２３）と、これらのリンカーにそれぞれ結合している免疫原３個（１１１、１１２、１１３）が、この分子針１３×３の外側に存在している。 The trimer 30 is a trimer formed by spontaneously associating the above-mentioned complex protein 10 as three monomers. The trimer 30 has a trimeric parallel β-sheet structure and a spiral structure (triple helix β-sheet) due to the trimeric parallel β-sheet structure and the β-sheet structure itself by the three molecular needle regions 13 described above gathering together and associating with each other at the C-terminals. A needle-like structure called (structure) is formed, and a molecular needle 13 × 3 is formed. The molecular needle 13 × 3 is composed of a basic portion 131 × 3 and a foldon aggregate 132 × 3. Thus has the function of passing through the cell membrane of the cells of the target tissue "molecular needle" is formed by trimerization and self-organization, the linker three derived from each monomer (12 1, 12 2, 12 3), immunogen three that are respectively coupled to these linkers (11 1, 11 2, 11 3) is present on the outside of the molecule needle 13 × 3.
　六量体６０は、２単位の上記三量体３０が、互いの分子針の基本部分（（１３×３）１と（１３×３）２）のＮ末端において結合して構成される六量体であり、当該六量体６０もまた、標的組織の細胞の細胞膜通過機能を有している。それぞれの三量体に由来するリンカー６本（１２１、１２２、１２３、及び、１２５、１２６：１２４は図示せず）と、これらのリンカーにそれぞれ結合している免疫原６個（１１１、１１２、１１３、及び、１１５、１１６：１１４は図示せず）が、２本の分子針（１３×３）１と（１３×３）２の外側に存在している。 The hexamer 60 is a hexamer composed of two units of the above-mentioned trimer 30 bonded at the N-terminal of the basic parts ((13 × 3) 1 and (13 × 3) 2) of each other's molecular needles. It is a body, and the hexamer 60 also has a cell membrane crossing function of cells of a target tissue. Each linker six derived from the trimer (12 1, 12 2, 12 3 and, 12 5, 12 6:12 4 not shown), immunogens are respectively coupled to these linkers 6 pieces (11 1, 11 2, 11 3 and, 11 5, 11 6:11 4 is not shown), two molecules needle (13 × 3) 1 and (13 × 3) located outside the 2 doing.
　本発明の複合蛋白質１０から、三量体３０への三量体化、及び、当該三量体３０から六量体６０へのマクロ的な２量体化は、水性液体中において自発的に進行し、三量体又は六量体として安定して存在する。この三量体又は六量体の安定性は極めて強いものであり、例えば、温度１００℃の水性液体環境下、さらにｐＨ２－１１の水性液体環境下、さらに有機溶媒を５０－７０容量％含む水性液体環境下であっても安定であり、その上、安全性にも優れている。水性液体から単離して乾燥させた状態でも、当該三量体又は六量体には優れた安定性と細胞膜透過性が認められる。 The trimerization of the complex protein 10 of the present invention into a trimer 30 and the macroscopic dimerization from the trimer 30 to a hexamer 60 proceed spontaneously in an aqueous liquid. However, it exists stably as a trimer or a hexamer. The stability of this trimer or hexamer is extremely strong, for example, in an aqueous liquid environment at a temperature of 100 ° C., in an aqueous liquid environment at pH 2-11, and in an aqueous environment containing 50-70% by volume of an organic solvent. It is stable even in a liquid environment and is also excellent in safety. Even when isolated from an aqueous liquid and dried, the trimer or hexamer has excellent stability and cell membrane permeability.
　上記のように、本発明の複合蛋白質から会合体への移行は、自発的に進行し、通常は大部分が最終形態である六量体化するが、一部は三量体として残る。 As mentioned above, the transition from the complex protein of the present invention to the aggregate progresses spontaneously, usually mostly in the final form, hexamerization, but some remain as trimers.
［４］　本発明のワクチン
　本発明のワクチンは、その有効成分である本発明の会合体の優れた細胞透過性と免疫原性により、標的組織および細胞に、粘膜投与、経皮投与、皮下投与、皮内投与、又は筋肉内投与を介して免疫原であるコロナウイルスの構造蛋白質の全部又は一部を効率よく移行させ、免疫を行うことが可能であり、これにより、粘膜投与、経皮投与、皮下投与、皮内投与、又は筋肉内投与による、ウイルスのコンポーネントワクチンの有効性と安全性を向上させることができる。その表れがアジュバンドフリーのワクチンとして用いることができる点である。粘膜投与する対象粘膜組織としては、鼻粘膜、喉粘膜、気道粘膜、気管支粘膜、舌下粘膜、肛門粘膜、腸粘膜、膣粘膜等が挙げられる。これらの中でも、鼻粘膜、喉粘膜、気道粘膜、気管支粘膜、舌下粘膜を選択することが好適である。 [4] Vaccine of the present invention The vaccine of the present invention is administered to target tissues and cells by mucosal administration, transdermal administration, and subcutaneous administration due to the excellent cell permeability and immunogenicity of the aggregate of the present invention, which is an active ingredient thereof. It is possible to efficiently transfer all or part of the structural protein of the coronavirus, which is an immunogen, through intradermal administration or intramuscular administration to perform immunization, whereby mucosal administration or transdermal administration is possible. , Subcutaneous, intradermal, or intramuscular administration can improve the efficacy and safety of viral component vaccines. The manifestation is that it can be used as an adjuvant-free vaccine. Examples of the mucosal tissue to be administered to the mucosa include nasal mucosa, throat mucosa, airway mucosa, bronchial mucosa, sublingual mucosa, anal mucosa, intestinal mucosa, vaginal mucosa and the like. Among these, it is preferable to select nasal mucosa, throat mucosa, airway mucosa, bronchial mucosa, and sublingual mucosa.
　本発明のワクチンは、上述した本発明の会合体を有効成分（感染防御抗原）として含有する、皮下投与、皮内投与、経皮投与、粘膜投与又は筋肉内投与用の医薬品組成物として提供される。本発明の会合体の直接投与の場合であっても、当該会合体を緩衝液等において用時懸濁混合した液剤として、粘膜投与、経皮投与、皮下投与、皮内投与、又は筋肉内投与がなされるので、この会合体そのものの形態も医薬品組成物に含まれる。粘膜投与は、スプレー剤、エアロゾル剤、カプセル剤、液剤等により容易に行うことができるが、これらの形態に限定されるものではない。粘膜投与形態としては、経鼻投与（経鼻接種）が好適である。 The vaccine of the present invention is provided as a pharmaceutical composition for subcutaneous administration, intradermal administration, transdermal administration, mucosal administration or intramuscular administration, which contains the above-mentioned aggregate of the present invention as an active ingredient (infection protective antigen). NS. Even in the case of direct administration of the aggregate of the present invention, mucosal administration, transdermal administration, subcutaneous administration, intradermal administration, or intramuscular administration is performed as a liquid preparation in which the aggregate is suspended and mixed at the time of use in a buffer solution or the like. Therefore, the form of the aggregate itself is also included in the pharmaceutical composition. Mucosal administration can be easily performed with a spray, an aerosol, a capsule, a liquid, or the like, but is not limited to these forms. Nasal administration (nasal inoculation) is preferable as the mucosal administration form.
　本発明のワクチンは、必須の有効成分（感染防御抗原）である本発明の会合体、及び必要に応じてアジュバンドや、医薬製剤担体を配合して製剤組成物の形態に調製される。当該製剤担体としては、使用形態に応じて選択することが可能であり、充填剤、増量剤、結合剤、付湿剤、崩壊剤、界面活性剤等の賦形剤ないし希釈剤を使用することができる。組成物の形態は、基本的には液剤であるが、用時液体希釈用の乾燥剤、粉末剤、顆粒剤等とすることも可能である。 The vaccine of the present invention is prepared in the form of a pharmaceutical composition by blending an aggregate of the present invention, which is an essential active ingredient (infection protective antigen), and, if necessary, adjuvant and a pharmaceutical pharmaceutical carrier. The pharmaceutical carrier can be selected according to the form of use, and excipients or diluents such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants should be used. Can be done. The form of the composition is basically a liquid preparation, but it can also be a desiccant, a powder preparation, a granule preparation or the like for liquid dilution at the time of use.
　本発明のワクチン中の本発明の会合体の量は、適宜選択され一定ではないが、通常、本発明の会合体を、投与時に０．１－１０質量％含有する液剤として用いるのが好適である。適切な投与（接種）量は、１回成人１人当たり０．０１μｇ－１０ｍｇ程度であり、必要に応じて初回接種と追加接種を適宜組み合わせて、１回又は２回以上の投与（接種）を行うことが可能である。 The amount of the aggregate of the present invention in the vaccine of the present invention is appropriately selected and is not constant, but it is usually preferable to use the aggregate of the present invention as a liquid preparation containing 0.1-10% by mass at the time of administration. be. The appropriate dose (inoculation) is about 0.01 μg-10 mg per adult, and if necessary, the initial inoculation and the booster inoculation are combined as appropriate, and one or more administrations (inoculation) are performed. It is possible.
　以下、本発明の実施例を開示する。 Hereinafter, embodiments of the present invention will be disclosed.
　本実施例の目的は、本発明の会合体における新型コロナウイルスを対象とするコンポーネントワクチンの有効成分としての有用性、を示すことである。 An object of the present embodiment is to show the usefulness as an active ingredient of a component vaccine targeting a new type coronavirus in the assembly of the present invention.
　上記したとおり、新型コロナウイルスは、世界的な流行をしているパンデミックスウイルスであり、重症例、死亡例が目立ち、各社会システムを停止させるほどの非常な脅威となっている。 As mentioned above, the new coronavirus is a pandemic virus that is prevalent worldwide, and serious cases and fatal cases are conspicuous, and it is a great threat to stop each social system.
［参考例１］　ノロウイルス蛋白質を担持した分子針の構築（鋳型用）
（ａ）前提事項
　本実施例では、遺伝子工学的手法を用いて、担持する蛋白質をヒトノロウイルスＧＩＩ．４であるＬＭ１４－２株の非構造蛋白質の一つである「Ｖｐｇ(viral protein genome-linked)」とする分子針（複合蛋白質）を調製した。Ｖｐｇは、ノロウイルスゲノム内のオープンリーディングフレーム１（ＯＲＦ１）に含まれる非構造蛋白質である。ＯＲＦ１は、ノロウイルスの一連の非構造蛋白質をコードしており、Ｎ末端蛋白質、ＮＴＰａｓｅ（ｐ４８）、ｐ２２（３Ａ様）、Ｖｐｇ、プロテアーゼ、及び、ＲＮＡ依存性ＲＮＡポリメラーゼ（ＲｄＲｐ）がそれぞれコードされ、ＯＲＦ１の全体翻訳後、当該プロテアーゼにより、それぞれの非構造蛋白質に切断され、成熟産物として機能する。これらの成熟産物のうち、ＶＰｇは、ゲノムＲＮＡ及びサブゲノムＲＮＡからの翻訳によってノロウイルスゲノム複製に必須の役割を果たすことが実証されており、リボソーム動員の際のキャップ代用品として機能する。本参考例において用いた免疫原であるＬＭ１４－２株のＶｐｇのアミノ酸配列は、配列番号１０（ただし、Ｎ末端のＭｅｔは、スタートコドンＡＴＧに由来するものである）に示した通りである。これをコードする核酸配列は、アミノ酸と核酸塩基の公知の関係に従って選択することができる。 [Reference Example 1] Construction of a molecular needle carrying a norovirus protein (for a template)
(A) Prerequisites In this example, the protein to be carried is determined by human norovirus GII. A molecular needle (complex protein) called "Vpg (viral protein genome-linked)", which is one of the non-structural proteins of the LM14-2 strain of No. 4, was prepared. Vpg is a non-structural protein contained in the open reading frame 1 (ORF1) in the norovirus genome. ORF1 encodes a series of non-structural proteins of norovirus, the N-terminal protein, NTPase (p48), p22 (3A-like), Vpg, protease, and RNA-dependent RNA polymerase (RdRp), respectively. After total translation of ORF1, the protease cleaves into each non-structural protein and functions as a mature product. Of these mature products, VPg has been demonstrated to play an essential role in norovirus genomic replication by translation from genomic RNA and subgenomic RNA and serves as a cap substitute for ribosome recruitment. The amino acid sequence of Vpg of the immunogen LM14-2 strain used in this reference example is as shown in SEQ ID NO: 10 (however, the N-terminal Met is derived from the start codon ATG). The nucleic acid sequence encoding this can be selected according to the known relationship between amino acids and nucleobases.
　この参考例１で使用したすべての試薬は、商業的供給元から購入し、さらに精製することなく使用した。ＨＮＶ－ＶＰｇの遺伝子断片は、北里大学ウイルス感染研究所の片山から提供されたプラスミドｐＨｕＮｏＶ－ＬＭ１４－２Ｆ（１２７７４塩基：配列番号１１）に組み込まれているヒトノロウイスＬＭ１４－２株のｃＤＮＡ部分（７６３９塩基：配列番号１２）に含まれていたものを用いた。ＶＰｇは、このＬＭ１４－２株のｃＤＮＡ部分（７６３９塩基）の２６３０塩基から３０２８塩基に相当する３９９塩基の配列（配列番号１３）である。この配列の５’末端にスタートコドンＡＴＧを付加して、発現に用いた。 All reagents used in Reference Example 1 were purchased from a commercial supplier and used without further purification. The gene fragment of HNV-VPg is the cDNA portion (7639 bases) of the human Nolouis LM14-2 strain incorporated in the plasmid pHuNoV-LM14-2F (12774 bases: SEQ ID NO: 11) provided by Katayama of the Kitasato University Virus Infection Research Institute. : The one contained in SEQ ID NO: 12) was used. VPg is a sequence of 399 bases (SEQ ID NO: 13) corresponding to 2630 to 3028 bases of the cDNA portion (7639 bases) of this LM14-2 strain. The start codon ATG was added to the 5'end of this sequence and used for expression.
　ＵＶ－ｖｉｓスペクトルは、ＳＨＩＭＡＤＺＵ　ＵＶ－２４００ＰＣ　ＵＶ－ｖｉｓ分光計で測定した。ＭＡＬＤＩ－ＴＯＦ質量スペクトルは、Ｂｒｕｋｅｒ　ｕｌｔｒａｆｌｅＸｔｒｍｅで測定した。ＭＡＬＤＩ－ＴＯＦ－ＭＳの測定は、試料を、０．０３％（ｗ／ｖ）シナピン酸及び０．１％（ｖ／ｖ）トリフルオロ酢酸を含む等容量の７０％（ｖ／ｖ）アセトニトリル／水溶液と混合した。ゲル浸透クロマトグラフィー（ＧＰＣ）は、ＨＰＬＣシステム及びカラム（Ａｓａｈｉｐａｃｋ　ＧＦ－５１０ＨＱ、Ｓｈｏｄｅｘ、東京、日本）を用いて行った。 The UV-vis spectrum was measured with a SHIMADZU UV-2400PC UV-vis spectrometer. The MALDI-TOF mass spectrum was measured by Bruker ultrafleXtreme. MALDI-TOF-MS measurements were made by measuring the sample with an equal volume of 70% (v / v) acetonitrile / containing 0.03% (w / v) sinapic acid and 0.1% (v / v) trifluoroacetic acid. It was mixed with an aqueous solution. Gel permeation chromatography (GPC) was performed using an HPLC system and a column (Asahipack GF-510HQ, Shodex, Tokyo, Japan).
（ｂ）「ＰＮ－Ｖｐｇ」発現用プラスミドの作製と発現
　（ｂ）－１：　総論
　ここで「ＰＮ－Ｖｐｇ」とは、上述した式（１）：
　Ｗ－Ｌ１－Ｘｎ－Ｙ　　　（１）
と、式（１）の細胞導入領域Ｙを表す式（２）：
　Ｙ１－Ｌ２－Ｙ２－Ｙ３　　　（２）
において、
　免疫原Ｗが、配列番号１０のアミノ酸配列で表される「ＬＭ１４－２株－Ｖｐｇ」であり；第１のリンカーＬ１が、配列番号１４（ＳＮＳＳＳＶＰＧＧ）、１５（ＧＧＧＧＳ）、１６（ＰＡＰＡＰ）のアミノ酸配列であり；繰り返し配列Ｘｎの繰り返し単位が、配列番号１のアミノ酸配列であり、繰り返し数ｎは１であり；分子針の本体部分Ｙ１のアミノ酸配列が、配列番号２のアミノ酸配列であり；第２のリンカーＬ２が「ＳＶＥ」であり；フォールドンＹ２のアミノ酸配列が、配列番号６のアミノ酸配列であり；修飾配列Ｙ３のアミノ酸配列が、配列番号１７（ＶＥＨＨＨＨＨＨ）である、複合蛋白質である。 (B) Preparation and expression of a plasmid for expressing "PN-Vpg" (b) -1: General remarks Here, "PN-Vpg" is the above-mentioned formula (1) :.
W-L 1- X n- Y (1)
And the formula (2) representing the cell introduction region Y of the formula (1):
Y 1- L 2- Y 2- Y 3 (2)
In
Immunogen W is, be a "LM14-2 strain -Vpg" represented by the amino acid sequence of SEQ ID NO: 10; first linker L 1 is SEQ ID NO: 14 (SNSSSVPGG), 15 (GGGGS ), 16 (PAPAP) be amino acid sequences; repeating unit of a repeating sequence X n is an amino acid sequence of SEQ ID NO: 1, the repetition number n is 1; the amino acid sequence of the main body portion Y 1 molecule needle, the amino acid sequence of SEQ ID NO: 2 The second linker L 2 is "SVE"; the amino acid sequence of Foldon Y 2 is the amino acid sequence of SEQ ID NO: 6; the amino acid sequence of modified sequence Y 3 is SEQ ID NO: 17 (VEHHHHHH). There is a complex protein.
　ＰＮ－ＶＰｇのプラスミドは、フレキシブルリンカー（ＦＬ：ＳＮＳＳＳＶＰＧＧ（配列番号１４））を鋳型として構築し、これを基に短いフレキシブルリンカー（ｓＦＬ：ＧＧＧＧＳ（配列番号１５））、短いリジットリンカー（ｓＲＬ：ＰＡＰＡＰ（配列番号１６））の２種類のリンカーで構築して、これらを発現させ、自発的に生じる会合体の内容の解析を行い、三量体と六量体が含まれていることを確認した。 The PN-VPg plasmid is constructed using a flexible linker (FL: SNSSSVPGG (SEQ ID NO: 14)) as a template, and based on this, a short flexible linker (sFL: GGGGS (SEQ ID NO: 15)) and a short rigid linker (sRL: PAPAP). (SEQ ID NO: 16)) was constructed with two types of linkers, these were expressed, and the contents of spontaneously generated aggregates were analyzed, and it was confirmed that trimers and hexamers were contained. ..
　（ｂ）－２：　フレキシブルリンカー（ＦＬ：ＳＮＳＳＳＶＰＧＧ（配列番号１４））を用いた鋳型プラスミドの構築
　ＬＭ１４－２プラスミドからのＶＰｇセグメントを増幅は、遺伝子増幅用プライマーＶＰｇ＿Ｆ（ＮｄｅＩ制限酵素部位有り：ACGCCATATGGGCAAGAAAGGGAAGAACAAGTCC（配列番号１８））及びＶＰｇ＿Ｒ（ＥｃｏＲＩ制限酵素部位有り：GCTCGAATTCGACTCAAAGTTGAGTTTCTCATTGTAGTCAACAC（配列番号１９））の組を用いて、ポリメラーゼ連鎖反応（ＰＣＲ）を行うことによって行った。その後、当該ＰＣＲ産物を、ＮｄｅＩ－ＥｃｏＲＩで消化したプラスミドｐＫＮ１－１（ＧＦＰ－ｇｐ５ｆ発現用プラスミド）（特許文献２）にクローニングした。 (B) -2: Construction of template plasmid using flexible linker (FL: SNSSSVPGG (SEQ ID NO: 14)) Amplification of VPg segment from LM14-2 plasmid is performed by gene amplification primer VPg_F (with NdeI restriction enzyme site: ACGCCATATGGGCAAGAAAGGGAAGAACAAGTCC). (SEQ ID NO: 18)) and VPg_R (with EcoRI restriction enzyme site: GCTCGAATTCGACTCAAAGTTGAGTTTCTCATTGTAGTCAACAC (SEQ ID NO: 19)) were used to perform a polymerase chain reaction (PCR). Then, the PCR product was cloned into the plasmid pKN1-1 (plasmid for expressing GFP-gp5f) (Patent Document 2) digested with NdeI-EcoRI.
　当該プラスミドｐＫＮ１－１は、特許文献２における開示の通りに、まず、Ｔ４ファージのｗａｃ蛋白質の４６１から４８４残基目に対応する遺伝子をＴ４ファージゲノムよりＰＣＲで増幅してｐＵＣ１８にクローニングし、ｆｏｌｄｏｎをコードする遺伝子を得た。続いて、このプラスミドを制限酵素ＥｃｏＲＩ及びＳａｌＩで切断し、ＥｃｏＲＩとＸｈｏＩで処理したプラスミドｐＥＴ２９ｂ（Ｎｏｖａｇｅｎ）に挿入し、プラスミドｐＭＴｆ１－３を得た。また、Ｔ４ファージのｇｐ５の４７４から５７５残基目に対応する遺伝子をＴ４ファージゲノムよりＰＣＲにより増幅してｐＵＣ１８にクローニングし、ｇｐ５をコードする遺伝子を得た。続いて、このプラスミドを制限酵素ＥｃｏＲＩ及びＳａｌＩで切断し、ＥｃｏＲＩとＸｈｏＩで処理した上述のプラスミドｐＭＴｆ１－３に挿入し、プラスミドｐＫＡ１７６を得た。また、群馬大・高橋より提供されたＧＦＰ発現ベクターを制限酵素ＮｄｅＩ及びＥｃｏＲＩで切断し、ＧＦＰをコードする遺伝子を得、制限酵素ＮｄｅＩ及びＥｃｏＲＩで処理した上述のプラスミドｐＫＡ１７６に組み込むことで作成された。 As disclosed in Patent Document 2, the plasmid pKN1-1 is obtained by first amplifying the gene corresponding to residues 461 to 484 of the wac protein of the T4 phage by PCR from the T4 phage genome and cloning it into pUC18, and then cloning it into pUC18. I got the gene encoding. Subsequently, this plasmid was cleaved with restriction enzymes EcoRI and SalI and inserted into the plasmid pET29b (Novagen) treated with EcoRI and XhoI to obtain plasmid pMTf1-3. In addition, the gene corresponding to residues 474 to 575 of gp5 of the T4 phage was amplified by PCR from the T4 phage genome and cloned into pUC18 to obtain a gene encoding gp5. Subsequently, this plasmid was cleaved with restriction enzymes EcoRI and SalI and inserted into the above-mentioned plasmid pMTf1-3 treated with EcoRI and XhoI to obtain plasmid pKA176. In addition, the GFP expression vector provided by Takahashi of Gunma University was cleaved with the restriction enzymes NdeI and EcoRI to obtain a gene encoding GFP, and it was prepared by incorporating it into the above-mentioned plasmid pKA176 treated with the restriction enzymes NdeI and EcoRI. ..
　クローニングされた遺伝子断片は大腸菌ＢＬ２１（ＤＥ３）のコンピテント細胞に導入し、ＤＮＡ配列決定によって確認され、フレキシブルリンカー（ＳＮＳＳＳＶＰＧＧ：配列番号１４）を介在させたＰＮ及びＶＰｇのプラスミド構築物「ＰＮ－ＦＬ－ＶＰｇ」の存在が確認された。 The cloned gene fragment was introduced into competent cells of Escherichia coli BL21 (DE3), confirmed by DNA sequencing, and mediated by a flexible linker (SNSSSVPGG: SEQ ID NO: 14), a plasmid construct of PN and VPg "PN-FL-". The existence of "VPg" was confirmed.
　（ｂ）－３：　ｓＦＬ／ｓＲＬリンカーを用いた会合体の生産と、その内容の確認
　配列番号１６の「短いリジットリンカー」（ｓＲＬ：ＰＡＰＡＰ）を、リンカーＬ１として介在させるための、遺伝子増幅用プライマーＶＰｇＰＡ－Ｆ（ＸｈｏＩ制限部位有り：CCGGCTCCGGCCCCACTCGAGGGAAGCAATACAATATTTGTACG（配列番号２０））と、ＶＰｇＰＡ－Ｒ（CTCAAAGTTGAGTTTCTCATTGTAGTCAACAC（配列番号２１））の組、並びに、配列番号１５の「短いフレキシブルリンカー」（ｓＦＬ：ＧＧＧＧＳ）を、リンカーＬ１として介在させるための、遺伝子増幅用プライマーＶＰｇＧＳ－Ｆ（ＸｈｏＩ制限部位有り：GGAGGCGGGGGTTCACTCGAGGGAAGCAATACAATATTTGTACG（配列番号２２）とＶＰｇＧＳ－Ｒ（上記のＶＰｇＰＡ－Ｒ（配列番号２１）と同じ）の組、を「ＰＮ－ＦＬ－ＶＰｇ」を鋳型としたインバーテッドＰＣＲの遺伝子増幅用プライマーとして、それぞれ別個に用いて、プラスミド構築物「ＰＮ－ｓＲＬ－ＶＰｇ」（Ｌ１は配列番号１６の短いリジットリンカー）及び「ＰＮ－ｓＦＬ－ＶＰｇ」（Ｌ１は配列番号１５の短いフレキシブルリンカー）を構築した。 (B) -3: sFL / and aggregate of production using sRL linker, "short rigid linker" confirmation SEQ ID NO: 16 of the contents (sRL: PAPAP) a, for interposing a linker L 1, gene amplification Primer VPgPA-F (with XhoI restriction site: CCGGCTCCGGCCCCACTCGAGGGAAGCAATACAATATTTGTACG (SEQ ID NO: 20)) and VPgPA-R (CTCAAAGTTGAGTTTCTCATTGTAGTCAACAC (SEQ ID NO: 21)), as well as the "short flexible linker" (sFL: GG) of SEQ ID NO: 15. and for interposing a linker L 1, gene amplification primers VPgGS-F (XhoI restriction sites available: a set of JijieijijishijijijijijititishieishitishijiAGGGAAGCAATACAATATTTGTACG (SEQ ID NO: 22) and VPgGS-R (above VPgPA-R (SEQ ID NO: 21)) to , Are used separately as primers for gene amplification of inverted PCR using "PN-FL-VPg" as a template, and the plasmid construct "PN-sRL-VPg" (L 1 is a short rigid linker of SEQ ID NO: 16). And "PN-sFL-VPg" (L 1 is a short flexible linker of SEQ ID NO: 15) was constructed.
　次いで、これらの２種のプラスミドをＤＨ５αコンピテント細胞に導入した。得られたベクターをＤＮＡ塩基配列決定法により検証した後、ＰＮ－ｓＲＬ－ＶＰｇ及びＰＮ－ｓＦＬ－ＶＰｇの発現を行った。 Next, these two plasmids were introduced into DH5α competent cells. The obtained vector was verified by the DNA sequencing method, and then PN-sRL-VPg and PN-sFL-VPg were expressed.
［実施例１］　新型コロナウイルスに対するコンポーネントワクチン
（ａ）前提事項
　本実施例では、遺伝子工学的手法を用いて、担持する免疫原を新型コロナウイルス（ＳＡＲＳ－ＣｏＶ－２）の構造蛋白質の一つであるＳ蛋白質の一部を構成するＲＢＤ蛋白質（レセプター結合領域の蛋白質）とする本発明の複合蛋白質を調製した。ＲＢＤ蛋白質は、新型コロナウイルスゲノム内にＳ蛋白質のメッセンジャーＲＮＡの鋳型配列としてコードされている。Ｓ蛋白質とＲＢＤ蛋白質については上記の通りである。なお、担持蛋白質は、ウイルスの増殖抑制が可能な抗体結合部位を含む部分配列でも良く、ウイルス蛋白質を構成するペプチド配列であれば、ＲＢＤ以外からも選択可能である。 [Example 1] Component vaccine against the new corona virus (a) Prerequisites In this example, the immunogen carried by the genetic engineering technique is one of the structural proteins of the new corona virus (SARS-CoV-2). The complex protein of the present invention was prepared as an RBD protein (protein in the receptor binding region) constituting a part of the S protein. The RBD protein is encoded as a template sequence of S protein messenger RNA in the new coronavirus genome. The S protein and the RBD protein are as described above. The supported protein may be a partial sequence containing an antibody binding site capable of suppressing the growth of the virus, and can be selected from other than RBD as long as it is a peptide sequence constituting the virus protein.
　本実施例においては、構造蛋白質であるＷとして、上記のＲＢＤ蛋白質を選択した。実際に用いた免疫原である新型コロナウイルス（ＳＡＲＳ－ＣｏＶ－２）のＲＢＤ蛋白質のアミノ酸配列は、プロトタイプＳＡＲＳ－ＣｏＶ－２　ジェンバンクアクセッション番号　ＭＮ９０８９４７のスパイク遺伝子（Ｓ－ｇｅｎｅ）２１５６３－２５３８４塩基にコードされるＳ蛋白質のアミノ酸配列　配列番号２３「MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT」のうち、下線部分（310aa-540aa）に相当する｛KGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN｝（配列番号２５）であり、これをコードする核酸配列は、アミノ酸と核酸塩基の公知の関係に従って選択することができる。 In this example, the above RBD protein was selected as W, which is a structural protein. The amino acid sequence of the RBD protein of the new coronavirus (SARS-CoV-2), which is the immunogen actually used, is the spike gene (S-gene) 21563-25384 base of the prototype SARS-CoV-2 Genbank Accession No. MN908947. of the amino acid sequence SEQ ID NO: 23 of the S protein encoded "MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVE KGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN" to a corresponding to underline (310aa-540aa) {KGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN} ( SEQ ID NO: 25), nucleic acid sequence encoding it, and amino acids It can be selected according to the known relationship of nucleic acid bases. Wear.
　本実施例においては、ＭＮ９０８９４７のスパイク遺伝子（Ｓ－ｇｅｎｅ）２１５６３－２５３８４塩基の中の２２４９１－２３１８２の配列「aaggaatctatcaaacttctaactttagagtccaaccaacagaatctattgttagatttcctaatattacaaacttgtgcccttttggtgaagtttttaacgccaccagatttgcatctgtttatgcttggaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataattccgcatcattttccacttttaagtgttatggagtgtctcctactaaattaaatgatctctgctttactaatgtctatgcagattcatttgtaattagaggtgatgaagtcagacaaatcgctccagggcaaactggaaagattgctgattataattataaattaccagatgattttacaggctgcgttatagcttggaattctaacaatcttgattctaaggttggtggtaattataattacctgtatagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtagcacaccttgtaatggtgttgaaggttttaattgttactttcctttacaatcatatggtttccaacccactaatggtgttggttaccaaccatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgtggacctaaaaagtctactaatttggttaaaaacaaatgtgtcaat」（配列番号２４）の内部配列を用いた。 In this example, using an internal sequence of the sequence of 22491-23182 in the spike gene (S-gene) 21563-25384 base MN908947 "aaggaatctatcaaacttctaactttagagtccaaccaacagaatctattgttagatttcctaatattacaaacttgtgcccttttggtgaagtttttaacgccaccagatttgcatctgtttatgcttggaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataattccgcatcattttccacttttaagtgttatggagtgtctcctactaaattaaatgatctctgctttactaatgtctatgcagattcatttgtaattagaggtgatgaagtcagacaaatcgctccagggcaaactggaaagattgctgattataattataaattaccagatgattttacaggctgcgttatagcttggaattctaacaatcttgattctaaggttggtggtaattataattacctgtatagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtagcacaccttgtaatggtgttgaaggttttaattgttactttcctttacaatcatatggtttccaacccactaatggtgttggttaccaaccatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgtggacctaaaaagtctactaatttggttaaaaacaaatgtgtcaat" (SEQ ID NO: 24).
　実施例１は、で使用したすべての試薬は、商業的供給元から購入し、さらに精製することなく使用した。ＲＢＤ内部の遺伝子断片（TTTCGTGTTCAGCCGACCGAAAGCATTGTTCGTTTTCCGAATATCACCAATCTGTGTCCGTTTGGCGAAGTTTTTAATGCAACCCGTTTTGCAAGCGTTTATGCCTGGAATCGTAAACGTATTAGCAATTGCGTTGCCGATTATAGCGTTCTGTATAATAGCGCAAGCTTCAGCACCTTTAAATGCTATGGTGTTAGCCCGACCAAACTGAATGATCTGTGTTTTACCAATGTGTATGCCGATAGCTTTGTGATTCGTGGTGATGAAGTTCGTCAGATTGCACCGGGTCAGACCGGTAAAATTGCAGATTATAACTATAAACTGCCGGATGATTTTACGGGTTGTGTTATTGCATGGAATAGCAATAACCTGGATAGCAAAGTTGGTGGCAACTATAACTATCTGTATCGCCTGTTTCGTAAGAGCAATCTGAAACCGTTTGAACGTGATATTAGCACCGAAATTTATCAGGCAGGTAGCACCCCGTGCAATGGTGTTGAAGGTTTTAATTGTTATTTTCCGCTGCAGAGCTATGGTTTTCAGCCTACCAATGGTGTGGGTTATCAGCCGTATCGTGTTGTTGTTCTGTCATTTGAACTGCTGCATGCACCGGCAACCGTT（配列番号２６）、アミノ酸配列：MFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATV（配列番号２７）に相当する）は、北里大学生命医科学研究所ウイルス感染制御学Ｉで感染患者より分離培養されたウイルス株ＫＵＨ００３株より、ＲＴ－ＰＣＲを用いて、Ｓ蛋白質の遺伝子配列から増幅して得た。この断片にはインフュージョンクローニング用に、ＲＴ－ＰＣＲ用５‘側プライマーにggagatatacatATG配列（配列番号２８）を、３’側プライマーにggaggcgggggttca配列（配列番号２９）（ＧＧＧＧＳリンカーに相当する）を付け加えることで、付加した。 In Example 1, all reagents used in were purchased from a commercial supplier and used without further purification. RBD internal gene fragment ((SEQ ID NO: 26), the amino acid sequence: corresponding to EmuefuarubuikyuPitiiesuaibuiaruefuPienuaitienuerushiPiefujiibuiefuenueitiaruefueiesubuiwaieidaburyuenuarukeiaruaiesuenushibuieidiwaiesubuieruwaienuesueiesuefuesutiefukeishiwaijibuiesuPitikeieruenudierushiefutienubuiwaieidiesuefubuiaiarujidiibuiarukyuaieiPijikyutijikeiaieidiwaienuwaikeieruPididiefutijishibuiaieidaburyuenuesuenuenuerudiesukeibuijijienuwaienuwaieruwaiarueruefuarukeiesuenuerukeiPiefuiarudiaiesutiiaiwaikyueijiesutiPishienujibuiijiefuenushiwaiefuPierukyuesuwaijiefukyuPiTNGVGYQPYRVVVLSFELLHAPATV (SEQ ID NO: 27)), the virus strains isolated cultured from infected patients at Kitasato University Biomedical Science infection control Studies I KUH003 It was obtained by amplifying the strain from the gene sequence of S protein using RT-PCR. For infusion cloning, the ggagatatacatATG sequence (SEQ ID NO: 28) is added to the 5'side primer for RT-PCR, and the ggaggcgggggttca sequence (SEQ ID NO: 29) (corresponding to the GGGGS linker) is added to the 3'side primer. So I added it.
（ｂ）「ＲＤＢｐ１－ＰＮ」発現用プラスミドの作製と発現
　本実施例では、「参考例１（ｂ）－２」にて得られた、鋳型プラスミド構築物「ＰＮ－ＦＬ－ＶＰｇ」を鋳型として、新型コロナウイルス（ＳＡＲＳ－ＣｏＶ－２）ＫＵＨ００３株よりＰＣＲで増幅させたＲＢＤ部分配列（ＲＢＤｐ１蛋白質）をコードし、その両端に小文字で示したインフュージョンクローニング用付加配列を持つ核酸配列（ggagatatacatATGTTTCGTGTTCAGCCGACCGAAAGCATTGTTCGTTTTCCGAATATCACCAATCTGTGTCCGTTTGGCGAAGTTTTTAATGCAACCCGTTTTGCAAGCGTTTATGCCTGGAATCGTAAACGTATTAGCAATTGCGTTGCCGATTATAGCGTTCTGTATAATAGCGCAAGCTTCAGCACCTTTAAATGCTATGGTGTTAGCCCGACCAAACTGAATGATCTGTGTTTTACCAATGTGTATGCCGATAGCTTTGTGATTCGTGGTGATGAAGTTCGTCAGATTGCACCGGGTCAGACCGGTAAAATTGCAGATTATAACTATAAACTGCCGGATGATTTTACGGGTTGTGTTATTGCATGGAATAGCAATAACCTGGATAGCAAAGTTGGTGGCAACTATAACTATCTGTATCGCCTGTTTCGTAAGAGCAATCTGAAACCGTTTGAACGTGATATTAGCACCGAAATTTATCAGGCAGGTAGCACCCCGTGCAATGGTGTTGAAGGTTTTAATTGTTATTTTCCGCTGCAGAGCTATGGTTTTCAGCCTACCAATGGTGTGGGTTATCAGCCGTATCGTGTTGTTGTTCTGTCATTTGAACTGCTGCATGCACCGGCAACCGTTggaggcgggggttca）（配列番号３０）を得た。この断片を、ＩｎＦｕｓｉｏｎクローニング法によりＶｐｇと入れ替えて、所望のプラスミド構築物「ＳＡＲＳ－ＣｏＶ－２　ＲＢＤｐ1－ＰＮ（以下、ＲＢＤｐ1－ＰＮともいう）」を構築した。 (B) Preparation and expression of plasmid for expressing "RDPp1-PN" In this example, the template plasmid construct "PN-FL-VPg" obtained in "Reference Example 1 (b) -2" was used as a template. Nucleic acid sequence () (sequence) encoding an RBD partial sequence (RBDp1 protein) amplified by PCR from the new coronavirus (SARS-CoV-2) KUH003 strain, and having an additional sequence for infusion cloning shown in lower letters at both ends thereof. Number 30) was obtained. This fragment was replaced with Vpg by the Infusion cloning method to construct the desired plasmid construct "SARS-CoV-2 RBDp1-PN (hereinafter, also referred to as RBDp1-PN)".
　次いで、このプラスミドをＤＨ５αコンピテント細胞に導入した。得られたベクターをＤＮＡ塩基配列決定法により検証した後、ＲＢＤｐ1－ＰＮの発現を行った。 Next, this plasmid was introduced into DH5α competent cells. The obtained vector was verified by the DNA sequencing method, and then RBDp1-PN was expressed.
　この「ＲＢＤｐ1－ＰＮ」のプラスミドを保有する大腸菌ＢＬ２１（ＤＥ３）を３０μｇ／ｍｌのカナマイシンを含むＬＢ培地で３７℃で一晩培養した。３７℃でインキュベートした溶液のＯＤ600が０．８に達した後、１ｍＭイソプロピルβ－Ｄ－１－チオガラクトピラノシド（ＩＰＴＧ）とアラビノースを添加した。ＩＰＴＧとアラビノースを添加して１６－１７時間後に８０００ｒｐｍで５分間遠心分離して細菌を回収し、１８０ｒｐｍの速度で２０℃でインキュベートした。次いで、細胞ペレットを、１００ｍＭトリス－ＨＣｌ　ｐＨ８．０、５ｍＭイミダゾールを含有する緩衝液中で氷上で１錠のｃＯｍｐｌｅｔｅ，ＥＤＴＡ－ｆｒｅｅと共に懸濁し、超音波処理によって溶解した。細胞破片を遠心分離（１７，５００ｒｐｍで５０分間）により沈降させ、インクルージョンボディに濃縮されたＲＢＤｐ１－ＰＮをえた。沈渣を８Ｍ尿素を含むＰＢＳで懸濁し、３７℃に加温しつつ、振盪して、完全に可溶化させた。可溶化したＲＢＤｐ１－ＰＮをコバルトアフィニティーカラム（タロンビーズカラム）に添加し、タロンビーズカラムキットに付属の溶出バッファーに週濃度６Ｍになるように尿素を加え、付属プロトコールに従って精製した。次いで、このＲＢＤｐ１－ＰＮ会合体を、４Ｍ尿素／ＰＢＳに２４時間透析し、さらに２Ｍ／ＰＢＳで２４時間透析した。このＲＢＤｐ１－ＰＮ会合体を、さらにＰＢＳに２４時間透析した。この過程において、ＲＢＤｐ１－ＰＮは、自発的に三量体及び／又は六量体を含有する会合体となっている。これを「ＲＢＤｐ１－ＰＮの会合体」として免疫試験に用いた。また対照として、上記のＲＢＤｐ１蛋白質をコードする塩基配列を基に、「ＲＢＤｐ１－ＰＮ」の発現から回収の一連の過程に準じた常法により得られた組換え蛋白質を準備した。 Escherichia coli BL21 (DE3) carrying this “RBDp1-PN” plasmid was cultured overnight at 37 ° C. in LB medium containing 30 μg / ml kanamycin. After the OD 600 of the solution incubated at 37 ° C. reached 0.8, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and arabinose were added. After 16-17 hours after adding IPTG and arabinose, the bacteria were collected by centrifugation at 8000 rpm for 5 minutes and incubated at 20 ° C. at a rate of 180 rpm. Cell pellets were then suspended in buffer containing 100 mM Tris-HCl pH 8.0, 5 mM imidazole on ice with 1 tablet of compacte, EDTA-free and lysed by sonication. Cell debris was settled by centrifugation (17,500 rpm for 50 minutes) to give concentrated RBDp1-PN to the inclusion body. The sediment was suspended in PBS containing 8M urea and shaken while warming to 37 ° C. to completely solubilize. The solubilized RBDp1-PN was added to a cobalt affinity column (Talon bead column), urea was added to the elution buffer attached to the Talon bead column kit to a weekly concentration of 6 M, and the mixture was purified according to the attached protocol. The RBDp1-PN aggregate was then dialyzed against 4M urea / PBS for 24 hours and then dialyzed against 2M / PBS for 24 hours. The RBDp1-PN aggregate was further dialyzed against PBS for 24 hours. In this process, RBDp1-PN is spontaneously an aggregate containing trimers and / or hexamers. This was used in an immunological test as an "aggregate of RBDp1-PN". As a control, a recombinant protein obtained by a conventional method according to a series of processes from the expression to recovery of "RBDp1-PN" was prepared based on the base sequence encoding the above RBDp1 protein.
　免疫試験は、２０マイクログラム／ｍＬＰＢＳの溶液（精製抗原）として、経鼻接種にて実施した。 The immunological test was performed by nasal inoculation as a solution (purified antigen) of 20 micrograms / mL PBS.
（ｃ）免疫試験
　上記のＲＢＤｐ１－ＰＮ会合体をモルモットに経鼻接種を行った（ｎ＝３）。経鼻接種は、一匹あたり１ｍＬ（４０μｇ）の精製抗原を、麻酔下にて鼻腔に吸引させることにより行った。接種後、１週間後に初回接種と同量の追加接種、３週後に更に追加接種を行い、その４週間後に初回接種の半分量（５００μＬ）の追加接種を行った。最終接種後１週間目に採血を行い、その血清内に含まれるにより新型コロナウイルス蛋白質に対するＩｇＧ抗体を免疫染色にて測定した。 (C) Immune test The above RBDp1-PN aggregate was nasally inoculated into guinea pigs (n = 3). Nasal inoculation was performed by inhaling 1 mL (40 μg) of purified antigen per animal into the nasal cavity under anesthesia. One week after the inoculation, the same amount of booster inoculation as the first inoculation was given, three weeks later, another booster inoculation was given, and four weeks later, half the dose (500 μL) of the first inoculation was given. Blood was collected 1 week after the final inoculation, and the IgG antibody against the new coronavirus protein contained in the serum was measured by immunostaining.
　免疫蛍光抗体法は、９６ウェルプレートにＶｅｒｏ／Ｅ６細胞を９０％コンフルーエントで捲きこみ、翌日に２％ＦＢＳを含むウイルス感染用培地に培地交換をした後、新型コロナウイルス感染に使用した。新型コロナウイルスＫＵＨ００３株は、ＭＯＩ＝１以上で細胞に感染させ、２４時間感染させた後、ウイルス感染細胞をプレートごと冷メタノールで固定し、以降の免疫蛍光染色に用いた。 The immunofluorescent antibody method was used for new coronavirus infection after wrapping Vero / E6 cells in a 96-well plate with 90% confluent and exchanging the medium with a virus infection medium containing 2% FBS the next day. The new coronavirus KUH003 strain was infected with cells at MOI = 1 or higher, infected for 24 hours, and then the virus-infected cells were fixed with cold methanol together with the plate and used for subsequent immunofluorescent staining.
　ＰＢＳＴ（０．１％Ｔｗｅｅｎ２０／ＰＢＳ）でプレートを計３回洗浄し、各プレートにＰＢＳＢ（１％ＢＳＡ／ＰＢＳ）を８０μＬ／ｗｅｌｌずつ添加し、室温で２時間インキュベートして、ブロッキングを行った。次に上記のモルモットの被験血清をＰＢＳＢで２０００倍に希釈し、ウェルに添加し、１時間、３７℃でインキュベートして、反応させた。プレートの各ウェルにＰＢＳを２００μＬずつ加え、１０分間室温でインキュベートして洗浄した。この操作を３回繰り返した。蛍光体のラベルされた抗モルモットＩｇＧ二次抗体として用い、１５００倍希釈液５０μＬ／ｗｅｌｌずつ、プレートに添加して、室温で２時間インキュベート後、ＰＢＳでプレートを５回洗浄した後、蛍光顕微鏡で標的抗体の有無を確認した。 The plates were washed 3 times in total with PBST (0.1% Tween20 / PBS), 80 μL / well of PBSB (1% BSA / PBS) was added to each plate, and the mixture was incubated at room temperature for 2 hours for blocking. .. The above guinea pig test serum was then diluted 2000-fold with PBSB, added to the wells and incubated for 1 hour at 37 ° C. for reaction. 200 μL of PBS was added to each well of the plate and incubated for 10 minutes at room temperature for washing. This operation was repeated 3 times. Used as a phosphor-labeled anti-guinea pig IgG secondary antibody, a 1500-fold diluted solution of 50 μL / well was added to the plate, incubated at room temperature for 2 hours, washed 5 times with PBS, and then examined under a fluorescence microscope. The presence or absence of the target antibody was confirmed.
　得られた経鼻免疫したモルモットの血清の免疫蛍光染色の結果を示す、新型コロナウイルス感染細胞の蛍光顕微鏡像において、ＳＡＲＳ－ＣｏＶ－２が感染し新型コロナウイルス蛋白質を発現しているＶｅｒｏ／Ｅ６細胞に対し、免疫後血清に含まれるＩｇＧが反応し、明らかな蛍光シグナルが認められた。それに対して、免疫前血清は、全く反応せず、免疫後血清のような蛍光シグナルは認められなかった。つまり、ＲＢＤｐ１－ＰＮ会合体による免疫によって、ＳＡＲＳ－ＣｏＶ－２に対する抗体の誘導が起きたことが証明された。なお、上記の蛍光顕微鏡像は、イメージ化の際の解像度の問題があるので、図面としての開示を省略するが、必要があれば、参考写真として提出することができる。 In the fluorescence microscopic image of the new coronavirus-infected cells showing the results of immunofluorescent staining of the serum of the obtained nasal-immunized guinea pig, SARS-CoV-2 infected and expressing the new coronavirus protein Vero / E6. IgG contained in post-infection serum reacted with the cells, and a clear fluorescent signal was observed. In contrast, pre-immune serum did not react at all, and no fluorescent signal like post-immune serum was observed. That is, it was proved that immunization with the RBDp1-PN aggregate caused the induction of the antibody against SARS-CoV-2. Since the above fluorescence microscope image has a problem of resolution at the time of imaging, disclosure as a drawing is omitted, but if necessary, it can be submitted as a reference photograph.
（ｄ）ウイルス中和試験
　次に、誘導された抗体にＳＡＲＳ－ＣｏＶ－２の細胞への感染を防ぐ効果があるか否かを、ウイルス中和試験で確認した。９６ウェルプレートの左端のウェルにＤＭＥＭで４倍に希釈した上記のモルモットの被検血清１００マイクロリットルを添加した。その後ＤＭＥＤ培地で５１２倍希釈までの２倍希釈系列を作製した。全てのウェルに約５万個のＳＡＲＳ－ＣｏＶ－２感染性粒子を添加し、攪拌後、３７℃で１時間、その後４℃で一晩インキュベートした。翌日、Ｖｅｒｏ／Ｅ６細胞を９０％コンフルーエントになるように培養した９６ウェルプレートの全てのウェルの培地を除き、ＰＢＳで３回洗浄後、新しい４％ＦＢＳ入り培地５０マイクロリットルを添加した。 (D) Virus Neutralizing Test Next, it was confirmed by a virus neutralizing test whether or not the induced antibody had the effect of preventing the infection of SARS-CoV-2 to cells. To the leftmost well of the 96-well plate was added 100 microliters of the above-mentioned guinea pig test serum diluted 4-fold with DMEM. Then, a 2-fold dilution series up to 512-fold dilution was prepared in DMED medium. Approximately 50,000 SARS-CoV-2 infectious particles were added to all wells, stirred, and then incubated at 37 ° C. for 1 hour and then at 4 ° C. overnight. The next day, the medium was removed from all wells of the 96-well plate in which Vero / E6 cells were cultured to be 90% confluent, washed 3 times with PBS, and then 50 microliters of fresh 4% FBS-containing medium was added.
　このプレートに、中和反応の終了したウイルス液を５０マイクロリットルずつ添加し、６日間培養を実施した。添加した新型コロナウイルスが血清中の抗体に中和されると、細胞がウイルスの感染を受けないため、細胞傷害が起きず、プレート底面に生き残った細胞が観察される。しかし、中和が不十分な場合、新型コロナウイルスの感染が起き、その後の連続的なウイルス感染によりウェル内の細胞は死滅する。ウェル内の細胞が生き残っている場合は、メチレンブルーにより染色される。結果を図２に示す。図２において、メチレンブルーによって染色されているウェルは、黒色ベースの色彩になっている。 To this plate, 50 microliters of the virus solution for which the neutralization reaction had been completed was added, and the cells were cultured for 6 days. When the added new coronavirus is neutralized by the antibody in the serum, the cells are not infected with the virus, so that cell damage does not occur and the surviving cells are observed on the bottom surface of the plate. However, if neutralization is inadequate, infection with the new coronavirus occurs, followed by continuous viral infections that kill cells in the wells. If the cells in the well survive, they are stained with methylene blue. The results are shown in FIG. In FIG. 2, the wells stained with methylene blue have a black-based color.
　図２において、ポジティッブコントロールとして用いたＳＡＲＳ－ＣｏＶ－２感染患者の回復期血清（退院時の血清）２例は、いずれも５１２倍希釈でもなおウイルスを完全に中和していたことが示されている。これに対して、ＲＢＤｐ１－ＰＮ免疫モルモット（先の個体と同一個体）血清２例は、いずれも３２倍希釈まで新型コロナウイルスを中和した。そして、６４倍希釈以降はウイルスの完全中和ができず、細胞が死滅して、図中透明ベースのウェルとして示されている。 In FIG. 2, the two convalescent sera (serum at discharge) of SARS-CoV-2 infected patients used as positive controls still completely neutralized the virus even at 512-fold dilution. It is shown. In contrast, two cases of RBDp1-PN immune guinea pig (same individual as the previous individual) serum neutralized the new coronavirus up to a 32-fold dilution. After the 64-fold dilution, the virus could not be completely neutralized, the cells died, and it is shown as a clear-based well in the figure.
　しかしながら、アジュバンドを全く用いていない経鼻接種で、これだけ顕著にＳＡＲＳ－ＣｏＶ－２の感染性粒子に対する中和抗体価が上昇したことは特筆すべきことであり、この知見により、ＲＢＤｐ１－ＰＮを、アジュバンドフリーのワクチンとして用いることが可能であることを示している。 However, it is noteworthy that the neutralizing antibody titer against infectious particles of SARS-CoV-2 increased remarkably by nasal inoculation without using adjuvant at all, and based on this finding, RBDp1-PN Has been shown to be able to be used as an adjuvant-free vaccine.
（ｅ）血中抗体価の測定試験
　上記の試験に用いたモルモットの血清（ＰＮ（＋））（ｎ＝３）と、ＲＢＤｐ１蛋白質そのものを上記の工程で経鼻接種したモルモットの血清（ＰＮ（－））（ｎ＝３）と、ネガティブコントロールとして、生理食塩水を上記の工程で経鼻接種したモルモットの血清（ＮＣ）（ｎ＝１）、に対して、それぞれＥＬＩＳＡ法により、血中抗体価（ＩｇＧ、ＩｇＡ）の測定を行った。 (E) Measurement test of blood antibody titer The serum of the guinea pig (PN (+)) (n = 3) used in the above test and the serum of the guinea pig nasally infused with the RBDp1 protein itself in the above step (PN (+)). -)) (N = 3) and, as a negative control, blood antibodies against serum (NC) (n = 1) of guinea pigs nasally inoculated with physiological saline in the above step by the ELISA method, respectively. The valence (IgG, IgA) was measured.
　ＥＬＩＳＡ法は、抗原（ＲＢＤｐ１蛋白質）をＰＢＳ（－）で２μｇ／ｍＬに希釈し、９６穴ＥＬＩＳＡプレートに５０μＬ／ｗｅｌｌずつ添加し、４℃で一晩インキュベートを行い、ＰＢＳＴ（０．１％Ｔｗｅｅｎ２０／ＰＢＳ）でプレートを計３回洗浄し、各プレートにＰＢＳＢ（１％ＢＳＡ／ＰＢＳ）を８０μＬ／ｗｅｌｌずつ添加し、室温で２時間インキュベートして、ブロッキングを行った。次に被験血清をＰＢＳＢで希釈し、調製して被験サンプルとした（ＩｇＧ検出：４－６４倍まで５段階の系列希釈、ＩｇＡ検出：４－６４倍まで５段階の系列希釈）。プレート内のＰＢＳＢを捨てて、各被験サンプルを５０μＬ／ｗｅｌｌずつ、プレートに添加して、室温で２時間インキュベート後、ＰＢＳＴでプレートを５回洗浄した。ＨＲＰ基質溶液を５０μＬ／Ｗｅｌｌずつプレートに添加し、発色が確認できるまで、室温で遮光してインキュベートを行った。２Ｍ硫酸を２５μＬ／Ｗｅｌｌずつプレートに添加し反応を停止し、４９０ｎｍの吸光度を測定した。 In the ELISA method, the antigen (RBDp1 protein) is diluted with PBS (-) to 2 μg / mL, 50 μL / well is added to a 96-well ELISA plate, and the mixture is incubated overnight at 4 ° C., and PBST (0.1% Tween 20) is used. The plates were washed 3 times in total with / PBS), 80 μL / well of PBSB (1% BSA / PBS) was added to each plate, and the mixture was incubated at room temperature for 2 hours for blocking. The test serum was then diluted with PBSB to prepare a test sample (IgG detection: 5-step serial dilution up to 4-64 times, IgA detection: 5-step serial dilution up to 4-64 times). The PBSB in the plate was discarded, 50 μL / well of each test sample was added to the plate, incubated at room temperature for 2 hours, and then the plate was washed 5 times with PBST. The HRP substrate solution was added to the plate at a rate of 50 μL / Well, and the incubation was carried out at room temperature in the dark until color development was confirmed. 2M sulfuric acid was added to the plate at a rate of 25 μL / Well, the reaction was stopped, and the absorbance at 490 nm was measured.
　結果を、図３（ＩｇＧ）と図４（ＩｇＡ）に示す。両図とも、縦軸は吸光度、横軸は希釈倍率を示している。これらの図においては、被験コットンラットの個体毎の結果を示した。これらの結果は、ＲＢＤｐ１蛋白質を直接免疫（ＰＮ（－））してもＩｇＧ、ＩｇＡ共に抗体価を示す吸光度が上昇しなかったのに対して、ＰＮ（＋）として免疫した場合には、これらの値がそれぞれ明らかに上昇したことを示している。血中ＩｇＡは、ＩｇＧに比べ誘導量は低いが、鼻腔粘膜面に集まったＢ細胞によって産生されたＩｇＡの大部分は、血中ではなく、大部分が鼻腔粘膜面に分泌されたためと考えられる。 The results are shown in FIG. 3 (IgG) and FIG. 4 (IgA). In both figures, the vertical axis shows the absorbance and the horizontal axis shows the dilution ratio. In these figures, the results for each individual test cotton rat are shown. These results show that the absorbance showing antibody titer for both IgG and IgA did not increase even when the RBDp1 protein was directly immunized (PN (-)), whereas when immunized as PN (+), these results were obtained. It shows that the values of are clearly increased. Although the amount of IgA in blood was lower than that of IgG, it is considered that most of IgA produced by B cells collected on the mucosal surface of the nasal cavity was not in the blood and most of it was secreted on the mucosal surface of the nasal cavity. ..
　上述したように、アジュバンドを全く用いていないにもこれだけ顕著に抗体価が上昇したことは特筆すべきことであり、アジュバンドフリーのワクチンとして用いることが可能であることを重ねて示している。 As mentioned above, it is noteworthy that the antibody titer increased remarkably even without the use of adjuvant at all, and it is repeatedly shown that it can be used as an adjuvant-free vaccine. ..
　ＳＡＲＳ－ＣｏＶ－２感染は、飛沫感染、空気感染によりウイルスを吸い込むことにより起こる。すなわち、主な感染経路が口腔、鼻腔であるため、経鼻接種で鼻腔粘膜細胞内に会合体が細胞膜に対して直接貫通導入され、鼻腔粘膜細胞内にＲＢＤｐ１蛋白質が注入され、液性免疫を誘導し、ＳＡＲＳ－ＣｏＶ－２に対する防御免疫が惹起された。このようにＳＡＲＳ－ＣｏＶ－２の構造蛋白を担持した分子ニードルを免疫原として経鼻接種することにより、局所免疫が誘導され、ＳＡＲＳ－ＣｏＶ－２に対する感染防御効果が得られることが明らかになった。 SARS-CoV-2 infection occurs by inhaling the virus by droplet infection or airborne infection. That is, since the main infection routes are the oral cavity and the nasal cavity, aggregates are directly introduced into the nasal mucosal cells through the cell membrane by nasal inoculation, and the RBDp1 protein is injected into the nasal mucosal cells to induce humoral immunity. It was induced and defensive immunity against SARS-CoV-2 was evoked. It has been clarified that by nasal inoculation using a molecular needle carrying a structural protein of SARS-CoV-2 as an immunogen, local immunity is induced and an infection protective effect against SARS-CoV-2 can be obtained. rice field.

Claims (16)

  1. 　下記式（１）のアミノ酸配列：
    　Ｗ－Ｌ１－Ｘｎ－Ｙ　　　（１）
    　［式中、Ｗは免疫原であるコロナウイルスの構造蛋白質の一部又は全部のアミノ酸配列を示し、Ｌ１はアミノ酸数が０－１００の第１のリンカー配列を示し、Ｘは配列番号１のアミノ酸配列を示し、Ｙは細胞導入領域のアミノ酸配列を示し、Ｘの繰り返し数であるｎは１－３の整数である。］
    であって、
    　当該細胞導入領域Ｙのアミノ酸配列は、下記式（２）：
    　Ｙ１－Ｌ２－Ｙ２－Ｙ３　　　（２）
    　［式中、Ｙ１は配列番号２－５からなる群より選択されるいずれか１つのアミノ酸配列を示し、Ｙ２は配列番号６－９からなる群より選択されるいずれか１つのアミノ酸配列を示し、Ｌ２はアミノ酸数が０－３０の第２のリンカー配列を示し、Ｙ３は修飾用のアミノ酸配列を示し、Ｙ２又はＹ３は存在しない場合もある。］
    で表される、複合蛋白質。     Amino acid sequence of the following formula (1):
    W-L 1- X n- Y (1)
    Wherein, W is the amino acid sequence of part or all of the structural proteins of a coronavirus which are immunogenic, L 1 is the number of amino acids shows a first linker sequence 0-100, X is of SEQ ID NO: 1 The amino acid sequence is shown, Y is the amino acid sequence of the cell introduction region, and n, which is the number of repetitions of X, is an integer of 1-3. ]
    And
    The amino acid sequence of the cell introduction region Y is the following formula (2):
    Y 1- L 2- Y 2- Y 3 (2)
    [In the formula, Y 1 represents any one amino acid sequence selected from the group consisting of SEQ ID NO: 2-5, and Y 2 represents any one amino acid sequence selected from the group consisting of SEQ ID NO: 6-9. As shown, L 2 indicates a second linker sequence having 0-30 amino acids, Y 3 indicates an amino acid sequence for modification, and Y 2 or Y 3 may not be present. ]
    A complex protein represented by.
  2. 　前記コロナウイルスは、ベータコロナウイルスである、請求項１に記載の複合蛋白質。 The complex protein according to claim 1, wherein the coronavirus is a betacoronavirus.
  3. 　前記ベータコロナウイルスは、新型コロナコロナウイルス（ＳＡＲＳ－ＣｏＶ－２）である、請求項２に記載の複合蛋白質。 The complex protein according to claim 2, wherein the beta coronavirus is a new type coronavirus (SARS-CoV-2).
  4. 　前記構造蛋白質の一部又は全部は、Ｓ蛋白質の一部又は全部である、請求項１－３のいずれか１項に記載の複合蛋白質。 The complex protein according to any one of claims 1-3, wherein a part or all of the structural protein is a part or all of an S protein.
  5. 　前記Ｓ蛋白質の一部又は全部は、ＲＢＤ蛋白質である、請求項４に記載の複合蛋白質。 The complex protein according to claim 4, wherein a part or all of the S protein is an RBD protein.
  6. 　前記式（１）の、Ｘｎ、Ｙ１、又は、Ｙ２で示されるアミノ酸配列のうち、１個以上のアミノ酸が欠失、置換若しくは付加された改変アミノ酸配列に係わる、請求項１－５のいずれか１項に記載の複合蛋白質。 Claim 1-5 relating to a modified amino acid sequence in which one or more amino acids are deleted, substituted or added among the amino acid sequences represented by X n , Y 1 or Y 2 of the above formula (1). The complex protein according to any one of the above items.
  7. 　前記式（１）の、Ｘｎ、Ｙ１、又は、Ｙ２で示されるアミノ酸配列のうち、１個以上のアミノ酸が欠失、置換若しくは付加された改変アミノ酸配列における、各々のアミノ酸配列におけるアミノ酸改変は、Ｘｎが８ｎ個以内、Ｙ１が３０個以内、及びＹ２が１５個以内である、請求項６に記載の複合蛋白質。 Amino acid in each amino acid sequence in the modified amino acid sequence in which one or more amino acids are deleted, substituted or added among the amino acid sequences represented by X n , Y 1 or Y 2 in the above formula (1). The complex protein according to claim 6, wherein the modification is 8 n or less of X n, 30 or less of Y 1 and 15 or less of Y 2.
  8. 　請求項１－７のいずれか１項に記載の複合蛋白質をコードする核酸を組み込んでいる遺伝子発現用ベクター。 A gene expression vector incorporating a nucleic acid encoding the complex protein according to any one of claims 1-7.
  9. 　請求項１－７のいずれか１項に記載の複合蛋白質をコードする核酸で形質転換された形質転換体。 A transformant transformed with a nucleic acid encoding the complex protein according to any one of claims 1-7.
  10. 　請求項１－７のいずれか１項に記載の１種又は２種以上の複合蛋白質を水性液体中で会合させてなる会合体。 An aggregate obtained by associating one or more of the complex proteins according to any one of claims 1-7 in an aqueous liquid.
  11. 　請求項１－７のいずれか１項に記載の１種又は２種以上の複合蛋白質を単量体蛋白質とする、三量体蛋白質及び／又は六量体蛋白質を含有する会合体。 An aggregate containing a trimer protein and / or a hexamer protein, wherein one or more of the complex proteins according to any one of claims 1-7 is a monomer protein.
  12. 　請求項１－７のいずれか１項に記載の１種又は２種以上の複合蛋白質を水性液体中で会合させてなる会合体を有効成分とする、コンポーネントワクチン。 A component vaccine containing an aggregate obtained by associating one or more of the complex proteins according to any one of claims 1-7 in an aqueous liquid as an active ingredient.
  13. 　請求項１－７のいずれか１項に記載の１種又は２種以上の複合蛋白質を単量体蛋白質とする三量体蛋白質及び／又は六量体蛋白質を含有する会合体を有効成分とする、コンポーネントワクチン。 The active ingredient is a trimer protein and / or an aggregate containing a hexamer protein, which comprises one or more complex proteins according to any one of claims 1-7 as a monomer protein. , Component vaccine.
  14. 　皮下、皮内、経皮、粘膜又は筋肉内投与用のコンポーネントワクチンである、請求項１２又は１３に記載のコンポーネントワクチン。 The component vaccine according to claim 12 or 13, which is a component vaccine for subcutaneous, intradermal, transdermal, mucosal or intramuscular administration.
  15. 　鼻腔粘膜、喉粘膜、気道粘膜、舌下粘膜、又は、気管支粘膜投与用のコンポーネントワクチンである、請求項１３又は１４に記載のコンポーネントワクチン。 The component vaccine according to claim 13 or 14, which is a component vaccine for administration of nasal mucosa, throat mucosa, airway mucosa, sublingual mucosa, or bronchial mucosa.
  16. 　前記コンポーネントワクチンの剤形は、スプレー剤、エアロゾル剤、又は、カプセル剤である、請求項１５に記載のコンポーネントワクチン。 The component vaccine according to claim 15, wherein the dosage form of the component vaccine is a spray agent, an aerosol agent, or a capsule agent.
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