WO2019163781A1 - エンテロウイルスワクチン - Google Patents

エンテロウイルスワクチン Download PDF

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WO2019163781A1
WO2019163781A1 PCT/JP2019/006116 JP2019006116W WO2019163781A1 WO 2019163781 A1 WO2019163781 A1 WO 2019163781A1 JP 2019006116 W JP2019006116 W JP 2019006116W WO 2019163781 A1 WO2019163781 A1 WO 2019163781A1
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Prior art keywords
enterovirus
polypeptide
present
coxsackievirus
protease
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PCT/JP2019/006116
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English (en)
French (fr)
Japanese (ja)
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朋史 中村
落合　晋
智 小池
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一般財団法人阪大微生物病研究会
公益財団法人東京都医学総合研究所
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Priority claimed from JP2018027672A external-priority patent/JP2021070629A/ja
Priority claimed from JP2018060216A external-priority patent/JP2021070634A/ja
Application filed by 一般財団法人阪大微生物病研究会, 公益財団法人東京都医学総合研究所 filed Critical 一般財団法人阪大微生物病研究会
Publication of WO2019163781A1 publication Critical patent/WO2019163781A1/ja

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)

Definitions

  • the present invention relates to a vaccine against enterovirus.
  • Enteroviruses are single-stranded RNA viruses belonging to the Picornaviridae family, and examples of infecting humans include the group of enteroviruses AD.
  • Enteroviruses include poliovirus, coxsackievirus group A, coxsackievirus group B, echoviruses and other enteroviruses.
  • Group A enteroviruses include Coxsackieviruses A2-8, 10, 12, 14, 16 and enterovirus 71 (hereinafter EV71).
  • Enterovirus B group includes Coxsackievirus A9, Coxsackievirus B1-6, Echovirus 1-33, and Enterovirus 69.
  • Enterovirus C group includes poliovirus, Coxsackievirus A1, 11, 13, 15, 17-22 and 24.
  • Enterovirus group D includes enteroviruses 68 and 70. It is well known that enterovirus causes various diseases, and typical examples of such diseases include hand-foot-mouth disease, herpangina, aseptic meningitis and the like. Among enteroviruses, EV71 and Coxsackievirus A group are known as the main causative viruses of hand, foot and mouth disease. Among enteroviruses, Coxsackievirus group A, EV71, Coxsackievirus group B and echovirus are known as the main causative viruses of herpangina. Aseptic meningitis is caused by a wide range of pathogens such as Echovirus, Coxsackievirus Group A, Group B and Enteroviruses such as EV71, as well as parasites.
  • pathogens such as Echovirus, Coxsackievirus Group A, Group B and Enteroviruses such as EV71, as well as parasites.
  • Coxsackievirus group A includes, for example, types A1 to 22 and type 24, which can cause different diseases and symptoms.
  • Coxsackievirus group B includes, for example, types 1-6.
  • Echo viruses include Echo types 1-7, 9, 11-27, and 29-31.
  • Other enteroviruses include enterovirus types 68-71.
  • Each genotype further includes a sub-genotype.
  • EV71 is known to be A type, B1 to B5 type, and C1 to C5 type consisting of only BrCr which is a prototype strain.
  • Hand-foot-mouth disease is generally known as a mild disease in which blistering rash appears on the hands, feet, mouth, etc., and children often develop around summer.
  • the main routes of infection for hand-foot-and-mouth disease are droplet infection and fecal mouth infection.
  • a large-scale epidemic of hand-foot-and-mouth disease has been reported mainly in East Asia. For example, about 2 million people have been reported in China in 2016 and about 500,000 cases in Japan in 2015.
  • Most patients with hand, foot, and mouth disease have a good prognosis, but rarely develop central nervous system complications such as aseptic meningitis, acute encephalitis, or pulmonary edema, and become severe or die. Such complications are often caused by hand-foot-and-mouth disease caused by EV71.
  • hand-foot-and-mouth disease of adults as well as children has been reported, and in adults, it often becomes severe.
  • a treatment method for hand-foot-and-mouth disease has not yet been established, and prevention by vaccine is being investigated or implemented mainly in East Asian countries.
  • Japan the hand-foot-and-mouth disease epidemic is regarded as a public health problem, and a vaccine effective in preventing and preventing its transmission is designated as one of the vaccines with high development priority by the Council of Health and Welfare.
  • Herpangina is generally known as acute viral pharyngitis characterized by fever and a blistering rash appearing on the oral mucosa, and is often prevalent in summer, especially in infants.
  • the main infection routes of herpangina are droplet infection and fecal infection.
  • a treatment method for herpangina has not yet been established, and prevention by a vaccine or the like is expected.
  • Aseptic meningitis has three main signs of fever, headache, and vomiting, and there are signs of meningeal irritation such as posterior stiffness and Kernig's sign. It is a syndrome that is diagnosed by not detecting bacteria by liquid smearing or bacterial culture. Although it is known that aseptic meningitis is caused by various viruses other than enteroviruses, the main causes are enteroviruses, particularly echoviruses and coxsackieviruses. The route of infection and epidemiological properties depend on the pathogen, but in the case of aseptic meningitis caused by enterovirus, as in hand-foot-and-mouth disease, it is caused by droplet infection and faecal infection mainly in children. It is prevalent. An effective treatment method for aseptic meningitis has not yet been established, and prevention by a vaccine or the like is expected.
  • Non-Patent Document 1 and Non-Patent Document 2 report vaccines in which enteroviruses are inactivated with formalin.
  • Patent Document 1 reports a VLP containing enteroviruses VP0 to VP4.
  • Patent Document 2 reports an enterovirus particle and a method for producing the same.
  • Patent Document 3 reports a method for producing VLPs of various viruses including enteroviruses.
  • Non-Patent Document 3 reports the immunogenicity of EV71 F particles.
  • Non-Patent Document 4 reports cross-ability between EV71 genotypes and subgenotypes after EV71 inactivated vaccine administration.
  • Non-Patent Document 5 reports a VLP containing EV71 VP1 to 3 as a vaccine against EV71.
  • Sliveva Inlive is cited as a vaccine against EV71 that has already been put into practical use.
  • EV71 has a structure in which a single-stranded RNA of about 7.4 kb is encapsulated inside a viral capsid having a diameter of 20 to 30 nm, and the capsid has 60 copies of four different structural proteins (VP1 to 4). Consists of. Four structural proteins assemble to form a protomer, five protomers assemble to form a pentamer, and twelve pentamers and the viral genome constitute a mature virion.
  • the subgenotype of EV71 is mainly defined by mutations in VP1 (Non-patent Document 6).
  • the present invention relates to a vaccine against enterovirus.
  • the present invention relates to a polypeptide showing immunogenicity against enterovirus, a method for producing the polypeptide, a vaccine against enterovirus containing the polypeptide, and a method for preventing enterovirus-related diseases using the polypeptide.
  • the polypeptide of the present invention is a polypeptide comprising VP1, VP2, VP3 and VP4 derived from enterovirus.
  • Vaccine effectiveness is generally assessed by immunogenicity.
  • enteroviruses such as hand-foot-and-mouth disease
  • vaccines showing higher immunogenicity against enteroviruses are required.
  • it is more desirable that a high infection prevention effect is shown in animal experiments using a mouse model or the like.
  • the present inventors have manufactured a vaccine having high immunogenicity against enteroviruses. Specifically, focusing on the structure of the enterovirus, particularly that VP0 cleaves into VP2 and VP4, the present inventors have found that the cleaving affects immunogenicity. That is, it was considered that designing a vaccine containing a large amount of VP2 and VP4 as a polypeptide exhibiting immunogenicity leads to the provision of a vaccine exhibiting high immunogenicity. A polypeptide comprising VP1, VP2, VP3 and VP4 derived from enterovirus was produced, and it was confirmed that this polypeptide had a high effect as an immunogen against enterovirus, and the present invention was made.
  • the present invention provides a polypeptide comprising VP1, VP2, VP3 and VP4 derived from enterovirus, wherein the polypeptide comprises an amino acid sequence recognized by a protease at the binding site of VP2 and VP4.
  • a peptide is provided.
  • the enterovirus includes poliovirus, coxsackievirus group A, coxsackievirus group B, echo virus and other enteroviruses.
  • the subject is a group A to group D enterovirus, particularly group A enterovirus.
  • the present invention is particularly directed to viruses that are responsible for enterovirus-related diseases such as hand-foot-and-mouth disease, herpangina, and aseptic meningitis, such as coxsackievirus group A, coxsackievirus group B, echovirus and group A enteroviruses. More specifically, the present invention relates to enteroviruses that cause hand-foot-and-mouth disease, such as group A enteroviruses, in particular EV71, especially Coxsackieviruses A6, 10 and 16.
  • the invention further relates to enteroviruses that cause herpangina, such as Coxsackievirus A group, EV71, Coxsackievirus group B and echoviruses, in particular Coxsackievirus A group, such as Coxsackievirus A2, 3, 4, 5, 6 and 10.
  • enteroviruses that cause herpangina such as Coxsackievirus A group, EV71, Coxsackievirus group B and echoviruses, in particular Coxsackievirus A group, such as Coxsackievirus A2, 3, 4, 5, 6 and 10.
  • the enterovirus genomic RNA has a region encoding a viral structural protein called P1 and a region encoding viral nonstructural proteins called P2 and P3.
  • P1 corresponds to a viral capsid, and this region is cleaved into viral structural proteins of VP0 to VP4 by being processed by a viral protease that is a nonstructural protein.
  • Enterovirus capsids may be formed from structural proteins of VP0, 1 and 3 called procapsids, or VP0 may be cleaved into VP2 and 4 and formed from structural proteins of VP1-4.
  • the former is referred to as an incomplete particle-shaped virus, and the latter is referred to as a complete particle-shaped virus. *
  • enteroviruses VP0, VP1, VP2, VP3 and VP4 and the base sequences encoding them are known, DNA Data Bank of Japan (DDBJ), EMBL-Bank / EBI, GenBank / National Center for Biotechnology (hereinafter referred to as “Biotechnology”). , NCBI) and the like.
  • DDBJ DNA Data Bank of Japan
  • EBI EMBL-Bank / EBI
  • GenBank GenBank / National Center for Biotechnology
  • NCBI NCBI
  • EVCr BrCr sequence information is available from NCBI registration number U22521.
  • the term “derived from” means that the object has immunological homology with the original virus, or has a certain homology with the amino acid sequence of the original virus, It should be understood that it is not tied to.
  • Immunological homology means that an immune response against the original virus is similarly elicited against the object.
  • the method for confirming immunological homology may be any means available to those skilled in the art and is not limited to a specific method. Generally, it can be confirmed by measuring whether the antibody in which the target induces expression can neutralize the original virus, that is, by measuring the neutralizing antibody in which the target induces expression.
  • the description of “derived from” an enterovirus indicates that an immune response against the enterovirus is similarly elicited by an object, or a degree of homology with the amino acid sequence of the enterovirus, for example, microbiologically understood as an enterovirus It means that it has the homology of.
  • a certain degree of homology with the amino acid sequence is typically 80% or more, preferably 90% or more, such as 95% or more, preferably 96% or more, more preferably 97% or more, and even more preferably 98% or more. Means 99% or more homology. Any assay method commonly available to those skilled in the art can be used for homology testing, for example BLAST can be used with default parameters.
  • the amino acid sequence of the polypeptide in question is the amino acid sequence of VP1 of any enterovirus obtained from NCBI and the initial setting parameters.
  • a protein in which one amino acid is replaced by another amino acid having a similar hydrophobicity index and still has a similar biological function eg, a protein that is equivalent in enzyme activity; hereinafter, “biological equivalent”
  • biological equivalent a protein that is equivalent in enzyme activity
  • the hydrophobicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient.
  • Hydrophilic indicators are also considered in the production of biological equivalents. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.
  • the hydrophilicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • the polypeptide may be conservatively substituted.
  • a conservative substitution refers to a substitution in which the hydrophilicity index and / or hydrophobicity index of the original amino acid and the substituted amino acid are similar as described above.
  • Examples of conservative substitutions are well known to those skilled in the art, for example, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; valine, leucine, and isoleucine, etc. However, it is not limited to these. Such conservative substitutions are also included within the scope of the term “derived from” in the present invention.
  • a protease means any enzyme that degrades peptide bonds between amino acids, regardless of whether the target is a protein, a high molecular peptide, or a low molecular peptide.
  • a protease having high substrate specificity such as cysteine protease, 3C protease, Examples include 3CD protease.
  • the protease does not randomly break the peptide bond, but specifically recognizes a specific amino acid sequence and cleaves the peptide bond specifically. Therefore, if one type of protease is selected, the amino acid sequence that can be recognized by the protease can be uniquely determined. What kind of amino acid sequence a certain protease recognizes is described in various documents, for example, instructions of a business operator who sells proteases. In the case of 3C protease or 3CD protease, the recognized amino acid sequence is XX-Gln-Gly-X, and cleavage occurs between Gln and Gly.
  • HRV3C protease it can be understood from the instructions provided by Funakoshi that the recognized amino acid sequence is Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro, and that cleavage occurs between Gln and Gly. It is. X represents any amino acid.
  • the inventors have found that immunogenicity is improved by cleaving VP0 into VP2 and VP4. Furthermore, it has been found that in the production of VLPs using a cell expression system, when the polypeptide of the present invention is produced from a known base sequence, the above cleavage is unlikely to occur.
  • the method for causing cleavage is not particularly limited, but for example, a method using a polypeptide containing an amino acid sequence recognized by a protease is preferable.
  • the amino acid sequence recognized by the protease is determined depending on the protease. Since the genomic RNA of enterovirus contains a base sequence encoding 3C protease or 3CD protease, in the polypeptide of the present invention, glutamine called XX-Gln-Gly-X is used as the amino acid sequence recognized by the protease. And a sequence in which glycine is continuous is preferred. Furthermore, the present inventors have found that the binding site between VP2 and VP4 is preferably an amino acid sequence recognized by the protease.
  • the binding site of VP2 and VP4 is cleaved in a substrate-specific manner with 3C protease or 3CD protease, resulting in glycine at the N-terminus of VP2 and glutamine at the C-terminus of VP4.
  • the amino acid sequence recognized by such protease may have any substitution, insertion, deletion, or addition mutation when compared with the wild-type amino acid sequence.
  • the present invention relates to a polypeptide comprising VP1, VP2, VP3 and VP4 derived from an enterovirus, such as a group AD enterovirus, preferably a group A enterovirus, wherein the protease is present at the binding site of VP2 and VP4.
  • an enterovirus such as a group AD enterovirus, preferably a group A enterovirus, wherein the protease is present at the binding site of VP2 and VP4.
  • a polypeptide comprising the amino acid sequence recognized by is provided.
  • the present invention relates to a polypeptide comprising VP1, VP2, VP3 and VP4 derived from an enterovirus, such as a group AD enterovirus, preferably a group A enterovirus, wherein 3C protease is present at the binding site of VP2 and VP4.
  • an enterovirus such as a group AD enterovirus, preferably a group A enterovirus
  • 3C protease is present at the binding site of VP2 and VP4.
  • a polypeptide comprising an amino acid sequence recognized by 3CD protease is provided.
  • the present invention provides a polypeptide comprising VP1, VP2, VP3 and VP4 derived from an enterovirus, such as a group AD enterovirus, preferably a group A enterovirus, wherein the VP2 and VP4 are glutamine and glycine.
  • an enterovirus such as a group AD enterovirus, preferably a group A enterovirus, wherein the VP2 and VP4 are glutamine and glycine.
  • an enterovirus such as a group AD enterovirus, preferably a group A enterovirus
  • the present invention provides a polypeptide comprising VP1, VP2, VP3 and VP4 from an enterovirus, such as a group AD enterovirus, preferably a group A enterovirus, wherein glutamine is present at the C-terminus of the VP4,
  • an enterovirus such as a group AD enterovirus, preferably a group A enterovirus, wherein glutamine is present at the C-terminus of the VP4
  • glutamine is present at the C-terminus of the VP4
  • the present invention provides a polypeptide comprising VP1, VP2, VP3 and VP4 derived from enterovirus, wherein the polypeptide is substantially free of enterovirus VP0.
  • VP0 is a polypeptide of about 36 kDa and includes about 28 kDa VP2 and about 8 kDa VP4.
  • the term “substantially free” means that the content thereof is 30% or less, 20% or less, 10% or less, preferably 5% or less compared to the main component when measured quantitatively. More preferably, it means 3% or less, most preferably 1% or less.
  • the “main component” here simply means the main component quantitatively and is not intended to be main in nature.
  • the means that can be used for quantitative measurement may be any method that can be normally selected by those skilled in the art, and the content is grasped by using a normal quantitative unit provided by the measurement method.
  • the intensity of the VP0 band detected using the SDS-PAGE method is 30% or less, 20% or less, 10% or less, preferably 5% or less, more preferably 3% compared to the intensity of the VP2 band.
  • VP0 is understood to be “substantially free” when it is less than or equal to%, and most preferably less than or equal to 1%.
  • the SDS-PAGE method is a method in which a protein mixture added to a gel is separated according to molecular weight by electrophoresis, and band intensity is analyzed after staining and decolorization. Those skilled in the art can use gels, reagents, membranes, standards, and apparatuses used. Can be appropriately selected.
  • the present invention is a polypeptide comprising VP1, VP2, VP3 and VP4 from an enterovirus, such as a group AD enterovirus, preferably a group A enterovirus, characterized in that it is substantially free of enterovirus VP0.
  • an enterovirus such as a group AD enterovirus, preferably a group A enterovirus, characterized in that it is substantially free of enterovirus VP0.
  • polypeptide of the present invention can be produced using any of peptide, polypeptide and protein production methods that are usually available to those skilled in the art. Such methods include, but are not limited to, chemical synthesis, virus inactivation and cell expression systems. A specific manufacturing method and reagents, conditions, and apparatuses used therein can be easily selected by those skilled in the art. For example, a commercially available protein expression kit can be used according to the instructions attached to the kit.
  • a preferred method for producing the polypeptide of the present invention is a method using a virus inactivation or cell expression system.
  • examples of cells that can proliferate enterovirus include Vero cells, RD cells (Rhabdomysarcoma cells), RD-A cells, and RD-18S cells.
  • Virus inactivation can be performed by any method such as formalin treatment usually used in vaccines.
  • Group A enteroviruses have problems in proliferation during virus culture using cells, production of VLPs by a cell expression system is more preferable from the viewpoint of quantity and cost.
  • usable expression vectors and cells are not particularly limited. For example, commercially available vectors and cells can be used according to the instructions attached to the product.
  • Such vectors include, but are not limited to, for example, HaloTag vectors, pHEK293 vectors, BacPAK vectors, pRI101 DNA vectors, baculovirus vectors, pcDNA vectors, and the like, and are appropriately selected according to the cells to be used.
  • Available cells include, but are not limited to, CHO cells, HEK293 cells, HeLa cells, Vero cells, MDCK cells, E. coli cells, silkworm cells, tobacco leaf cells, and the like.
  • Examples of the polypeptide produced by the cell expression system include VLP.
  • the polypeptide of the present invention is intended to be administered to humans, it is preferably produced in an expression system using mammalian cells.
  • mammalian cells are preferably used from the viewpoint of post-translational modification.
  • Suitable mammalian cells for producing the polypeptides of the present invention include, for example, CHO cells.
  • the expression system using the cells may be any means available to those skilled in the art, and is not limited to these, but includes, for example, a transient expression system by transfection of a plasmid.
  • a virus inactivation method when using a virus inactivation method to produce a polypeptide of the invention, it can be produced according to the following steps: ⁇ Proliferation of enterovirus in cell culture ⁇ Process of filtering virus solution ⁇ Process of obtaining virus fraction corresponding to complete particle shape by sucrose density gradient centrifugation after virus treatment ⁇ Virus fraction A method comprising the step of obtaining a polypeptide of interest by inactivating is provided.
  • a cell expression system when using a cell expression system to produce a polypeptide of the invention, it can be produced according to the following steps: ⁇ A step of extracting RNA from enterovirus or designing DNA by artificial synthesis ⁇ A step of synthesizing and amplifying a DNA fragment from the extracted RNA by RT-PCR and ligating it into an expression plasmid, or an artificial synthesis in the previous step A step of ligating the obtained DNA fragment directly or after amplification into a plasmid for expression; a step of transfecting the obtained plasmid into cells; a step of growing the transfected cells; a step of recovering the polypeptide of the present invention from the grown cells .
  • any step of the above manufacturing method those skilled in the art can easily select reagents, conditions, devices, and the like.
  • a step for confirming whether the intended result is obtained can be appropriately inserted.
  • the confirmation step for example, the sequence of the extracted RNA is confirmed by an appropriate sequencing method such as a cycle sequencing method or a pyro sequencing method, and the obtained polypeptide is confirmed by Western blotting (WB) or the like. It is included.
  • the means for cleaving VP0 into VP2 and VP4 is not particularly limited, but mutations can be introduced so that the base sequence encodes the amino acid sequence recognized by the protease.
  • the original base sequence may be substituted, inserted, deleted, or added, but is not limited thereto.
  • mutagenesis may be performed at the time of ligation. It may be done before or after.
  • the base sequence encoding the amino acid sequence recognized by the protease to be inserted or substituted can be easily determined using a codon table.
  • mutagenesis may be any means available to those skilled in the art and include, but are not limited to, for example, an inverse PCR method and a complementary primer method.
  • a commercially available mutagenesis kit can be used according to the instructions attached to the kit.
  • the present invention is a method for producing a polypeptide of the present invention using a cell expression system, ⁇ Step of extracting RNA from enterovirus ⁇ Step of synthesizing and amplifying a DNA fragment from the extracted RNA by RT-PCR and ligating it into an expression plasmid ⁇ Amino acid sequence recognized by protease in the relevant portion of the ligated expression plasmid
  • a method comprising a step of mutating a nucleotide sequence coding for a gene, a step of transfecting the obtained plasmid into a cell, a step of growing the transfected cell, and a step of recovering the polypeptide of the present invention from the grown cell.
  • the present invention provides a method for producing the polypeptide of the present invention using a cell expression system, -A step of designing DNA in which a base sequence encoding an amino acid sequence recognized by a protease is introduced by artificial synthesis-A step of ligating an artificially synthesized DNA in the previous step directly or after amplification to an expression plasmid-The resulting plasmid
  • a method comprising the step of transfecting a cell with the cell, the step of growing the transfected cell, and the step of recovering the polypeptide of the present invention from the grown cell are provided.
  • the present invention provides a method for producing a polypeptide of the present invention using a cell expression system, ⁇ Step of extracting RNA from enterovirus ⁇ Step of synthesizing and amplifying a DNA fragment containing a nucleotide sequence encoding VP1 to 4 from RT using the extracted RNA and ligating it to an expression plasmid ⁇ On the ligated expression plasmid A step of mutating a base sequence encoding an amino acid sequence recognized by a protease between a base sequence encoding VP4 and VP2 present in the cell; a step of transfecting the resulting plasmid into a cell; a transfected cell A method comprising the step of proliferating the polypeptide and the step of recovering the polypeptide of the present invention from the expanded expression cells is provided.
  • the present invention provides a method for producing a polypeptide of the present invention using a cell expression system, -Artificial synthesis of DNA containing a base sequence encoding enterovirus-derived VP1 to 4 and containing a base sequence encoding an amino acid sequence recognized by a protease between VP4 and a base sequence encoding VP2.
  • the step of growing the transfected cell. The proliferated expression cell. From which the polypeptide of the present invention is recovered.
  • polypeptide of the present invention thus produced can be modified such as phosphorylation and glycosylation. Such modified polypeptides are still included in the polypeptides of the present invention. Modifications include, for example, post-translational modifications of expressed cells.
  • the polypeptide of the present invention can take any tertiary or quaternary structure by various interactions.
  • the VP1 to 4 subunits are folded by ionic bonds, hydrogen bonds, disulfide bonds, etc. in the polypeptide of the present invention to form tertiary structures, and then the VP1 to 4 subunits are assembled, Quaternary structures can be formed.
  • Such higher-order structures generally exhibit higher immunogenicity as the structure approximates that of natural virus particles. In the present invention, it has been found that the closer the structure is to a perfect particle shape, the higher the immunogenicity.
  • the polypeptide of the invention is a full particle shape-like VLP or a full particle shape inactivated virus.
  • an expression cassette for producing the polypeptide of the present invention.
  • An expression cassette is a combination of a target gene and a promoter operably linked to initiate transcription of the target gene, and is used after being introduced into a vector.
  • an expression cassette comprising an enterovirus, for example, VP1 to 4, derived from group A to D enterovirus, preferably a group A enterovirus, a nucleic acid encoding a protease and an amino acid sequence recognized by the protease, and a promoter that initiates transcription of the nucleic acid. Is also intended.
  • the present inventors have found that the polypeptide of the present invention has high immunogenicity against enteroviruses. Furthermore, the polypeptide of the present invention can also be expected to have cross-reactivity between genotypes and subgenotypes. Accordingly, the present invention provides a vaccine against enteroviruses, such as group AD enteroviruses, preferably group A enteroviruses, more preferably EV71, coxsackievirus A6, coxsackievirus A10 or coxsackievirus A16, more preferably EV71, coxsackievirus A6, coxsackievirus A10 and coxsackievirus A16.
  • a vaccine comprising the polypeptide of the present invention as an antigen is provided.
  • the EV71 genotype may be a B-type vaccine
  • the sub-genotype may be a B5-type vaccine.
  • the term “vaccine” means a pharmaceutical preparation containing an antigen capable of enhancing immunity against a specific pathogen, such as a virus or a bacterium. Whether it is a prophylactic vaccine or a therapeutic vaccine, in the present invention, a prophylactic vaccine is specifically contemplated. Prophylactic vaccines prevent infection and aggravation of a pathogen by administering the vaccine to an unaffected subject and eliciting an immune response against the specific pathogen in the body to increase immunity against the pathogen For pharmaceutical preparations.
  • the route of administration of the vaccine is not limited to these, and examples include subcutaneous administration, intradermal administration, intramuscular administration, nasal administration, transdermal administration, and oral administration.
  • the disease refers to an enterovirus-related disease, such as an enterovirus group A to D-related disease, typically a group A enterovirus-related disease, specifically, for example, hand-foot-and-mouth disease, herpangina and aseptic meningitis, Among them are hand, foot and mouth disease and herpangina.
  • enterovirus-related disease such as an enterovirus group A to D-related disease
  • group A enterovirus-related disease specifically, for example, hand-foot-and-mouth disease, herpangina and aseptic meningitis, Among them are hand, foot and mouth disease and herpangina.
  • the vaccine of the present invention may contain additives commonly used in vaccines in addition to the antigen.
  • additives include carriers, preservatives, adjuvants and the like.
  • the carrier includes water or saline.
  • preservatives include phenol, benzethonium chloride, 2-phenoxyethanol, thimerosal, and the like.
  • adjuvants include aluminum salts, aluminum phosphate, aluminum chloride, aluminum hydroxide, aluminum sulfate, calcium phosphate, Toll-like receptor (TLR) ligand molecules, dsRNA, IL-12, and the like.
  • TLR Toll-like receptor
  • the subject of the vaccine of the present invention includes animals that can be infected with enterovirus, such as mammals such as humans, monkeys, cows, horses, pigs, sheep, and the like.
  • enterovirus such as mammals such as humans, monkeys, cows, horses, pigs, sheep, and the like.
  • a typical subject is a human, particularly but not limited to a human under 5 years who is prone to enterovirus infection.
  • the present invention provides the following aspects: (Aspect 1) A polypeptide comprising VP1, VP2, VP3 and VP4 derived from enterovirus, wherein the VP2 and VP4 are obtained by cleavage of VP0; (Aspect 2) The polypeptide according to Aspect 1, wherein the polypeptide includes VP1, VP2, VP3, and VP4 derived from enterovirus, and the amino acid sequence recognized by a protease at the binding site of VP2 and VP4; (Aspect 3) The polypeptide according to Aspect 2, wherein the protease is 3C or 3CD protease; (Aspect 4) The polypeptide according to Aspect 3, wherein the amino acid sequence recognized by the protease is a sequence in which glutamine and glycine are continuous; (Aspect 5) The polypeptide according to any one of Aspects 1 to 4, wherein the polypeptide includes VP1, VP2, VP3, and VP4 derived from enterovirus, wherein the polypeptide
  • the polypeptide of the present invention not only has high immunogenicity against enteroviruses, but also shows high infection prevention effects in animal tests.
  • the present inventors produced a polypeptide containing VP1 to 4 as an antigen protein, immunized the animal, and measured the neutralizing antibody titer.
  • the polypeptide which contains VP0, 1 and 3 as an antigen protein as a comparison object was manufactured, and the neutralizing antibody titer was measured similarly.
  • a mouse challenge test was performed to confirm the infection prevention effect against enterovirus.
  • the polypeptide of the present invention can also be expected to have cross-reactivity between genotypes and subgenotypes.
  • the polypeptide of the present invention that is, the polypeptide containing VP1 to 4
  • FIG. 6 shows a VLP expression cassette for EV71 comprising EV71 VP1-4, 2A self-cleaving sequence, 3CD protease and CMV promoter. The results of SDS-PAGE and WB of purified VLP are shown.
  • FIG. 6 shows a TEM image of a VLP containing EV71 VP1-4 produced using a CHO cell expression system. The neutralizing antibody titers of complete particle shape-like VLPs containing EV71 VP1-4 and incomplete particle shape-like VLPs containing EV71 VP0, 1 and 3 are shown.
  • Example 1 Production and evaluation of inactivated virus of EV71 Preparation of EV71 Complete Particle Shape and Incomplete Particle Shape Inactivated Virus EV71B5 type (clinical isolate) was cultured using RD-A cells (cell stack, 10 chamber, Corning). Cells in the culture broth were disrupted to obtain a virus solution. The virus solution was centrifuged and filtered (0.45 ⁇ m ⁇ 0.22 ⁇ m) to remove cell debris. The filtrate was ultrafiltered and concentrated using AKTA flux s (molecular weight cut-off value 500 kDa) manufactured by GE Healthcare. Viral particle pellets were prepared using a Hitachi ultracentrifuge CP80wx. The pellet was resuspended overnight in PBS.
  • RD-A cells cell stack, 10 chamber, Corning
  • the suspension was then fractionated using sucrose density gradient centrifugation (10-40%) with an ultracentrifuge CP80wx.
  • the complete particle fraction and the incomplete particle fraction were confirmed by SDS-PAGE and WB. The results are shown in FIG.
  • the complete particle fraction converged to a sucrose density gradient of around 35% (about 1.15 g / mL) and the incomplete particle fraction around 25% (about 1.11 g / mL).
  • the complete particle fraction and the incomplete particle fraction were concentrated using Amicon Ultra from Merck Millipore.
  • Virus was inactivated using formalin, the inactivated virus solution was infected with RD-A cells, and the presence or absence of CPE was measured to confirm the inactivation.
  • Mouse serum was diluted 2-fold to prepare dilution series of 1 / 2-1 / 4096, and after adding EV71 virus solution prepared to 5 ⁇ 10 3 PFU / mL to each dilution series, 37 ° C., 5%
  • the reaction was neutralized with CO 2 for 4 hours. Those without mouse serum were used as negative controls.
  • the neutralization reaction solution was added to the RD-A cells (6-well plate) seeded on the previous day, and the plate was shaken at 37 ° C. for 1 hour every 15 minutes to infect the cells with the virus. After removing the virus solution by suction, methylcellulose was added and overlaid. After culturing at 37 ° C.
  • RNA was extracted from EV71B5 type (clinical isolate) using Roche's High Pure Viral RNA Kit according to the attached instructions.
  • the P1 region and the protease region of the obtained RNA were amplified by RT-PCR according to TaKaRa's PrimeScript II High Fidelity RT-PCR Kit according to the attached instructions.
  • the obtained amplified DNA fragment was ligated to the expression plasmid pcDNA3.4. E. TaKaRa. After the produced plasmid was transformed into E. coli JM109 Competent Cells according to the attached instruction, the cells were seeded in a medium to grow.
  • FIG. 5 shows a VLP expression cassette of EV71 in the obtained plasmid.
  • the VLP expression cassette includes EV71 P1,2A self-cleaving sequence, 3CD protease and CMV promoter.
  • a VLP was produced by transient expression using ThermoCHO's ExpiCHO Expression System. Specifically, ExpiCHO-S cells were cultured with ExpiCHO Expression Medium until a predetermined cell density was reached. Expifectamine CHO Reagent diluted with OptiPRO SFM Complexation Medium and VLP expression plasmid were added to the cultured ExpiCHO-S cells and transfected. The transfected cells were cultured in ExpiCHO Expression Medium, ExpiCHO Feed and Expifectamine CHO Enhancer were added, and the culture was further continued. During the culture period, the cell viability was confirmed, and the culture was stopped and collected 8 days after transfection.
  • the obtained culture broth was centrifuged to separate cells and supernatant, and the supernatant was collected and filtered (0.22 ⁇ m).
  • the obtained supernatant was concentrated by ultrafiltration using GE Healthcare's AKTA fluxs with a molecular weight cut-off value of 500 kDa.
  • VLP pellets were prepared using a Hitachi ultracentrifuge CP80wx.
  • VLP pellet was resuspended in PBS overnight. Further, the suspension was subjected to fractionation by sucrose density gradient centrifugation (10 to 40%) using an ultracentrifuge CP80wx, and each fraction was subjected to SDS-PAGE and WB to confirm the VLP fraction. The results are shown in Lanes 2 and 4 of FIG. From this, it was found that the VLP fraction contains a large amount of VP0 before cleavage.
  • Example 2 Preparation and evaluation of EV71 VLP (with mutation) 1.
  • Preparation and confirmation of VLP of EV71 by cell expression system In the plasmid, a Gln-Gly mutation was inserted between VP4-VP2 in the P1 (virtual capsid) region by an inverse PCR method. Specifically, based on the gene sequence information of EV71, a primer for inserting a mutation at the location was designed. Using TaKaRa BIO's PrimeScript II High Fidelity RT-PCR Kit, reverse transcription and PCR reactions were performed according to the attached instructions. The PCR reaction was repeated 30 cycles of 98 ° C. (10 sec) ⁇ 60 ° C. (15 sec) ⁇ 68 ° C. (7.5 min).
  • a VLP was prepared in the same manner as described above.
  • the prepared VLP fraction was concentrated using Amicon Ultra of Merck Millipore.
  • the particle structure of VLP was confirmed with a transmission electron microscope (FIG. 7).
  • Comparative Example 1 1. Confirmation by SDS-PAGE and WB was performed in the same manner as above. The results are shown in Lanes 1 and 3 in FIG. From FIG. 6, it was found that the cleavage from VP0 to VP2 and VP4 was promoted by inserting the Gln-Gly mutation.

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CN114540315A (zh) * 2021-10-18 2022-05-27 武汉生物制品研究所有限责任公司 一种热稳定的柯萨奇病毒a组2型病毒株
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WO2020067027A1 (ja) * 2018-09-28 2020-04-02 一般財団法人阪大微生物病研究会 Ｖｌｐ発現ｃｈｏ細胞株の構築
JPWO2020067027A1 (ja) * 2018-09-28 2021-08-30 一般財団法人阪大微生物病研究会 Ｖｌｐ発現ｃｈｏ細胞株の構築
CN114540315A (zh) * 2021-10-18 2022-05-27 武汉生物制品研究所有限责任公司 一种热稳定的柯萨奇病毒a组2型病毒株
CN114540315B (zh) * 2021-10-18 2023-05-16 武汉生物制品研究所有限责任公司 一种热稳定的柯萨奇病毒a组2型病毒株
CN117106102A (zh) * 2023-10-23 2023-11-24 中国医学科学院医学生物学研究所 肠道病毒多抗原表位融合蛋白、基因、疫苗及其制备方法
CN117106102B (zh) * 2023-10-23 2024-02-06 中国医学科学院医学生物学研究所 肠道病毒多抗原表位融合蛋白、基因、疫苗及其制备方法

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