WO2021235503A1 - Monomère protéique conjugué supportant la protéine du coronavirus, agrégat desdits monomères, et composant vaccinal comprenant ledit agrégat comme principe actif - Google Patents

Monomère protéique conjugué supportant la protéine du coronavirus, agrégat desdits monomères, et composant vaccinal comprenant ledit agrégat comme principe actif 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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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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    • 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
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    • A—HUMAN NECESSITIES
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    • A61P37/04—Immunostimulants
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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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
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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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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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.

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Abstract

La présente invention a pour but de fournir un composant vaccinal capable d'induire une immunité contre les coronavirus, en particulier les bêta-coronavirus, y compris le nouveau coronavirus (SARS-CoV-2). Les inventeurs ont découvert que ce problème peut être résolu en fournissant un composant vaccinal comprenant, en tant que principe actif, un agrégat qui comprend un trimère et/ou un hexamère d'une aiguille moléculaire sur laquelle une protéine structurelle du coronavirus est fixée.
PCT/JP2021/019072 2020-05-20 2021-05-19 Monomère protéique conjugué supportant la protéine du coronavirus, agrégat desdits monomères, et composant vaccinal comprenant ledit agrégat comme principe actif WO2021235503A1 (fr)

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WO2024004159A1 (fr) * 2022-06-30 2024-01-04 Ｅｐｓ創薬株式会社 Composition de vaccin pour une administration sublinguale

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