WO2023080718A1 - Vaccine composition for prevention or treatment of sars-coronavirus-2 infection - Google Patents
Vaccine composition for prevention or treatment of sars-coronavirus-2 infection Download PDFInfo
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Classifications
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A61P31/12—Antivirals
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- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
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- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
Definitions
- the present invention relates to a vaccine composition used for the purpose of preventing Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection and/or alleviating symptoms expressed during infection.
- SARS-CoV-2 Severe Acute Respiratory Syndrome-Coronavirus-2
- the SARS-CoV-2 virus which has been reported worldwide since December 2019, is a virus belonging to the Coronaviridae family, Betacoronavirus genus Sarbecovirus subgenus, and SARS-CoV-2 virus infection is also called COVID-19 (coronavirus disease 2019).
- the main transmission route is known to be close contact with droplets of an infected person, and direct contact with an infected person or touching a medium such as an item contaminated by droplets of an infected person, then touching the eyes, nose, mouth, etc. without washing hands It is known that viral transmission can be achieved by this.
- the Receptor Binding Domain (RBD) on the surface of the SARS-CoV-2 virus binds to the ACE2 receptor protein on the surface of human cells, causing infection. It is known. Spike glycoprotein monomers are separated into S1 subunits and S2 subunits by host cell proteases.
- SARS-CoV-2 virus causes an increase in deaths and enormous social and economic damage, and as the damage rapidly increases, rapid vaccine development is required.
- Vaccine composition for preventing SARS-CoV-2 infection or alleviating symptoms development is being carried out.
- the present inventors combine the RBD of the SARS-CoV-2 spike glycoprotein with a protein capable of self-assembling to form a nanostructure to prepare RBD nanoparticles that label multivalent RBD antigens,
- an immunostimulant capable of further improving the immunogenicity of the RBD nanoparticles was studied.
- RBD nanoparticles showed excellent immunogenicity at both low and high doses when aluminum hydroxide was used as an immune enhancer.
- Patent Document 1 US Registered Patent Publication US 9630994 B2
- Non-Patent Document 1 Science. 2016 Jul 22;353(6297):389-94.
- a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto is an assembled trimer of three; and an RBD (Receptor Binding Domain) nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer It provides a vaccine composition for preventing or treating SARS-coronavirus-2 infection comprising a.
- RBD Receptor Binding Domain
- Another aspect of the present invention is a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto, a trimer in which three are assembled; And an RBD nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer, SARS - A vaccine composition for preventing or treating coronavirus-2 infection and a kit for preventing or treating SARS-coronavirus-2 infection including instructions for use are provided.
- the vaccine composition according to the present invention has excellent preventive and therapeutic effects against SARS-coronavirus-2 infection because the IgG antibody titer and neutralizing antibody titer increase after vaccine administration and the increased levels are maintained even after the second vaccination.
- the term “about” may be presented before a specific numeric value.
- the term “about” includes not only the exact number that follows the term, but also a range that is or is close to that number. It can be determined whether the number is close to or nearly the specific number mentioned, given the context in which it is presented.
- the term “about” can refer to a range of -10% to +10% of a numerical value.
- the term “about” can refer to a range of -5% to +5% of a given numerical value. However, it is not limited thereto.
- the term “and/or” when used herein is to be understood as specifically disclosing each of the two specified features or elements, either with or without the other.
- the term “and/or” as used herein in phrases such as “A and/or B” includes “A and B”, “A or B”, “A” (alone), and “B” (alone). ) is intended to include.
- the term “and/or” as used in phrases such as "A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
- the term "consisting essentially of” means that a non-specified ingredient may be present if the characteristics of the claimed subject matter are not substantially affected by the presence of the non-specified ingredient.
- the term “consisting of” means that the percentage of the specified component(s) totals 100%. Ingredients or features that come after the term “consisting of” may be essential or mandatory. In some embodiments, in addition to the ingredients or features that come under “consisting of”, any other ingredients or non-essential ingredients may be excluded.
- a polypeptide or protein if it has the same or equivalent activity as the polypeptide consisting of the amino acid sequence of the corresponding sequence number, a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added. It is obvious that can also be used in this application.
- the amino acid sequence has an addition of a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation thereof, or a conservative substitution to the N-terminus and/or C-terminus of the amino acid sequence. It may, but is not limited thereto.
- Vaccine composition for preventing or treating SARS-CoV-2 infection
- a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto is an assembled trimer of three; and an RBD (Receptor Binding Domain) nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer It provides a vaccine composition for preventing or treating SARS-coronavirus-2 infection comprising a.
- RBD Receptor Binding Domain
- the RBD nanoparticle may also be referred to as a "nanostructure", “RBD nanostructure”, or “RBD-NP” as a multimeric protein assembly, and (i) has an amino acid sequence of SEQ ID NO: 1 or at least 75% identity thereto.
- a trimer in which three first polypeptide monomers comprising an amino acid sequence are assembled; and (ii) a second polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto may comprise a five assembled pentamer.
- the first polypeptide comprises a SARS-CoV-2 spike glycoprotein receptor binding domain (receptor binding domain, RBD); A first self-assembling polypeptide serving as a structure of nanoparticles; and a linker connecting the RBD and the first self-assembling polypeptide.
- the first polypeptide monomer may further include an N-terminal phosphatase signal peptide sequence (MGILPSPGMPALLSLVSLLSVLLMGCVA).
- a plurality of isolated first polypeptide monomers may self-assemble through interactions to form a trimer. This assembly can be achieved through non-covalent protein-protein interaction reactions.
- the second polypeptide is a second self-assembling polypeptide monomer serving as a structure of nanoparticles, and a plurality of isolated second polypeptide monomers self-assemble through interactions to form a pentamer. can form This assembly can be achieved through non-covalent protein-protein interaction reactions.
- the first polypeptide may be one of the polypeptides described in International Patent Publication No. 2019-169120.
- the first polypeptide monomer may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto, for example, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 can
- the polypeptide of SEQ ID NO: 1 may be referred to as "RBD-I53-50A" or "RBD-Component A”.
- the first polypeptide may be encoded by a gene consisting of the nucleotide sequence represented by SEQ ID NO: 3.
- the second polypeptide monomer may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, for example, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
- the polypeptide of SEQ ID NO: 2 herein may be referred to as "I53-50B" or "Component B”.
- the second polypeptide monomer may be encoded by a gene consisting of the nucleotide sequence represented by SEQ ID NO: 4.
- the first polypeptide and the second polypeptide may include any modification that does not disrupt assembly into a multimeric protein assembly, for example, may include addition, deletion, insertion, substitution, or modification of amino acids. .
- the first polypeptide and the second polypeptide are International Patent Publication No. 2019-169120, US Patent Publication No. 9630994, International Patent Publication No. 2021-163481, International Patent Publication No. 2021-163438, or International Application No. It may be a protein disclosed in PCT/US2021/037341.
- the first polypeptide is a polypeptide consisting of an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence of SEQ ID NOs: 5 to 9 (SEQ ID NO: 7 of International Patent Publication No. 2019-169120, Corresponding to the amino acid sequence of 29, 30, 31 or 39) may be a polypeptide consisting of.
- the second polypeptide is a polypeptide consisting of an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NOs: 10 to 17 (SEQ ID NOs: 6 and 8 of International Patent Publication No. 2019-169120) , corresponding to the amino acid sequence of 10, 27, 28, 32, 33 or 38).
- the RBD nanoparticles may be formed by self-assembly of a trimer of the first polypeptide and a pentamer of the second polypeptide.
- the RBD nanoparticles of the present invention include (i) 20 trimers of the first polypeptide (Component A) and (ii) 12 pentamers of the second polypeptide (Component B) can have a structure.
- the multimeric protein assembly of the present invention includes 60 copies of the first polypeptide (Component A) and 60 copies of the second polypeptide (Component B).
- the RBD nanoparticles of the present invention may have an icosahedral symmetrical structure.
- the RBD nanoparticles of the present invention have a diameter of about 30 nm to about 70 nm, and in this case, the lumen may be about 15 nm to about 32 nm horizontally.
- the cell line producing the first polypeptide monomer may be a cell line having an expression vector containing a gene encoding a protein represented by the amino acid sequence represented by SEQ ID NO: 1.
- the expression vector may be a plasmid vector, but any vector suitable for expressing the first polypeptide monomer in a cell line may be used without limitation.
- the cell line producing the second polypeptide monomer may be a cell line having an expression vector containing a gene encoding a protein represented by the amino acid sequence represented by SEQ ID NO: 2.
- the expression vector may be a pET-based vector, for example, a pET29b+ vector, but any vector suitable for expressing the second polypeptide monomer in a cell line may be used without limitation.
- an expression vector containing a gene encoding a protein represented by the amino acid sequence represented by SEQ ID NO: 1 in the present invention may have a DNA sequence represented by SEQ ID NO: 18.
- an expression vector containing a gene encoding a protein represented by the amino acid sequence represented by SEQ ID NO: 2 in the present invention may have a DNA sequence represented by SEQ ID NO: 19.
- amino acid sequence or nucleotide sequence used in the present invention includes sequences showing substantial identity to the sequences described in the sequence listing.
- substantially identity is 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, It is a sequence having 98% or 99% or more homology, and amino acid sequences or base sequences within these homology ranges are also construed to be included in the scope of the present invention.
- vector refers to a nucleic acid means comprising a nucleotide sequence that can be introduced into a host cell and recombined and inserted into the genome of the host cell, or can be spontaneously replicated as an episome.
- Suitable expression vectors include expression control elements such as promoters, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. . When a genetic construct encoding a target protein is administered, the initiation codon and stop codon must be functional in the subject and must be in frame with the coding sequence.
- the polynucleotide or vector according to one embodiment of the present invention is an independent molecule outside the genome, specifically a molecule capable of replicating, and may exist in a genetically modified host cell or non-human host organism, or may be present in a host cell or non-human host cell. -Can be stably integrated into the genome of human host organisms.
- the host cell of the cell line producing the first polypeptide monomer according to one embodiment of the present invention is a eukaryotic cell.
- the eukaryotic cells include fungus cells, plant cells or animal cells. Examples of the fungal cell may be yeast, specifically yeast of the genus Saccharomyces (Saccharomyces sp.), more specifically S. cerevisiae.
- animal cells include insect cells or mammalian cells, and specific examples of animal cells include HEK293, 293T, NSO, Chinese hamster ovary cells (CHO), MDCK, U2-OSHela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, C33A, etc.
- suitable cell lines well known in the art can be obtained from cell line depositories such as the American Type Culture Collection (ATCC).
- ATCC American Type Culture Collection
- the host cell of the cell line producing the first polypeptide monomer may be a Chinese Hamster Ovary (CHO) cell, preferably a recombinant Adeno-Associated Virus (rAAV) HD-BIOP3 GS null CHO-K1 cells in which Exon 6 of the Glutamine Synthetase gene was knocked out by the method can be used.
- CHO Chinese Hamster Ovary
- rAAV recombinant Adeno-Associated Virus
- any method for introducing nucleic acid into the cell may be used, and techniques known in the art may be used depending on the host cell.
- methods known in the art may be used depending on the host cell.
- the vaccine composition of the present invention may include a pharmaceutically acceptable carrier together with the RBD nanoparticles.
- pharmaceutically acceptable carrier refers to any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (eg, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof. Except that any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.
- the vaccine composition of the present invention may include a buffer, a stabilizer, an adsorbent (immunity enhancer) and a solvent in addition to RBD nanoparticles as a main component.
- a buffer a stabilizer
- an adsorbent immunophilic adsorbent
- a solvent in addition to RBD nanoparticles as a main component.
- Sodium chloride and tromethamine can be used as a buffer
- L-arginine and sucrose sucrose or sucrose
- aluminum hydroxide can be used as an adsorbent (immune enhancer).
- the "immune enhancer" used in the present invention is a substance that non-specifically promotes an immune response to an antigen during the initial activation of immune cells, and the vaccine composition of the present invention is administered together with RBD nanoparticles to enhance the immune response.
- the immunostimulant may be selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide, and may be aluminum hydroxide (aluminum hydroxide).
- the vaccine composition may further include aluminum hydroxide, and the aluminum hydroxide may be provided in a pre-mixed state in a drug product.
- the adjuvant is aluminum hydroxide
- 750 parts by weight of aluminum hydroxide may be included with respect to 5 parts by weight to 40 parts by weight of the RBD nanoparticles, and 6 to 38 parts by weight and 7 to 36 parts by weight of the RBD nanoparticles. 8 parts by weight to 34 parts by weight, 9 parts by weight to 32 parts by weight, 9 parts by weight to 30 parts by weight, 10 parts by weight to 28 parts by weight, 10 parts by weight to 26 parts by weight, 10 parts by weight to 25 parts by weight
- For the aluminum hydroxide may be included in 750 parts by weight.
- aluminum hydroxide is 1000 ⁇ g to 2000 ⁇ g, 1100 ⁇ g to 1900 ⁇ g, 1150 ⁇ g to 1850 ⁇ g, 1200 ⁇ g to 1800 ⁇ g, 1250 ⁇ g to 1750 ⁇ g, 1300 ⁇ g to 1700 ⁇ g, 1350 ⁇ g to 1650 ⁇ g, 1400 ⁇ g to 1600 ⁇ g, 1450 ⁇ g to 1600 ⁇ g, and 1400 ⁇ g to 16 00 ⁇ g can be included as If the aluminum hydroxide is included in less than 1000 ⁇ g, immune activity may be reduced, and if it is included in excess of 2000 ⁇ g, there is a problem in that the possibility of side effects increases.
- the term "buffer” refers to an agent that serves to maintain the pH of a solution so that the pH does not change rapidly in the solution to which it is added. Buffers applicable to the present invention may include at least one of sodium chloride and tromethamine (Tris). In addition, in addition to sodium chloride and tromethamine, any buffer capable of maintaining pH in a solution can be applied without limitation to the present invention.
- the buffer may be a phosphate buffer such as sodium phosphate or potassium phosphate, HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid), histidine or citrate buffer. can include
- the buffer is sodium chloride
- it may be included in 1 mg to 20 mg, 2 mg to 19 mg, 3 mg for two doses (1 ml) of the RBD nanoparticles, that is, 20 ⁇ g or 50 ⁇ g of the RBD nanoparticles. to 18 mg, 4 mg to 17 mg, 5 mg to 16 mg, 6 mg to 15 mg, 7 mg to 14 mg, 8 mg to 13 mg, 1 mg to 12 mg, 8 mg to 11 mg, 8 mg to 10 mg, 8 mg to 9 mg, and 7 mg to 8.5 mg, for example, 8 mg to 9 mg, 7 mg to 8.5 mg, and 7.5 mg to 8.5 mg.
- sodium chloride is included within the above range, the stability and homogeneity of the antigenic protein are increased, effectively inducing IgG antibodies and neutralizing antibodies, and the resulting SARS-coronavirus-2 infection prevention effect was confirmed (Example 1).
- the buffer when it may be included in 0.1 mg to 10 mg, 0.5 mg to 9.5 mg, for 2 doses (1 ml) of the RBD nanoparticles, that is, 20 ⁇ g or 50 ⁇ g of the RBD nanoparticles, 1 mg to 9 mg, 1.5 mg to 8.5 mg, 2 mg to 8 mg, 2.5 mg to 7.5 mg, 3 mg to 7 mg, 3.5 mg to 6.5 mg, 4 mg to 6 mg, 4.5 mg to 6 mg, 5 mg to It may be included in 6 mg, 5.5 mg to 6 mg, for example, 4 mg to 6 mg, 5 mg to 6 mg.
- stabilizer used in the present invention refers to an agent that allows a poorly soluble immunoactive substance to be easily and reproducibly dispersed in an aqueous solution without a surfactant.
- Buffers applicable to the present invention may include at least one of L-arginine and sucrose.
- any stabilizer capable of dispersing poorly soluble immunoactivating substances in an aqueous solution can be applied without limitation to the present invention.
- the stabilizer may include an amine group in a molecule such as L-lysine or L-histidine and a basic amino acid having alkaline properties, and may include glucose, lactose, maltose, trehalose, sorbitol, mannitol, and combinations thereof. It may contain one or more sugars selected from.
- the stabilizer is L-arginine
- it may be included in 8 mg to 24 mg for two doses (1 ml) of the RBD nanoparticles, that is, 20 ⁇ g or 50 ⁇ g of the RBD nanoparticles, 9 mg to 23 mg, 10 mg to 22 mg, 11 mg to 21 mg, 12 mg to 20 mg, 13 mg to 19 mg, 14 mg to 18 mg, 15 mg to 17 mg, 16 mg to 17 mg, for example, 14 mg to 17 mg, 15 mg to 17 mg.
- L-arginine is included within the above range, the stability and homogeneity of the antigenic protein are increased, effectively inducing IgG antibodies and neutralizing antibodies, and the resulting SARS-coronavirus-2 infection prevention effect was confirmed (Example 1).
- the stabilizer when the stabilizer is sucrose, it may be included in 35 mg to 55 mg, 36 mg to 54 mg, 37 mg for two doses (1 ml) of the RBD nanoparticles, that is, 20 ⁇ g or 50 ⁇ g of the RBD nanoparticles. to 53 mg, 38 mg to 52 mg, 39 mg to 51 mg, 40 mg to 50 mg, 41 mg to 49 mg, 42 mg to 48 mg, 43 mg to 47 mg, 44 mg to 47 mg, For example, 40 mg to 50 mg, 44 mg to 47 mg, and 45 mg to 47 mg may be included.
- sucrose is included within the above range, the stability and homogeneity of the antigenic protein are increased, effectively inducing IgG antibodies and neutralizing antibodies, and the resulting SARS-coronavirus-2 infection prevention effect was confirmed (Example 1).
- the vaccine composition contains 1000 ⁇ g to 2000 ⁇ g of aluminum hydroxide for two doses (1 ml) of the RBD nanoparticles, that is, 20 ⁇ g or 50 ⁇ g of the RBD nanoparticles, and sodium chloride 1 mg to 20 mg of this, 0.1 mg to 10 mg of tromethamine, 8 mg to 24 mg of L-arginine, and 35 mg to 55 mg of sucrose.
- SARS-coronavirus-2 infection includes not only infection with SARS-coronavirus-2 itself, but also various symptoms (eg, respiratory disease, pneumonia, etc.) resulting from infection with the virus.
- prevention used in the present invention refers to any action that suppresses or delays the onset of SARS-coronavirus-2 infection by administration of a vaccine composition.
- the vaccine composition of the present invention contains an immunologically effective amount of RBD nanoparticles.
- Immunologically effective amount means that administration of said amount to a subject as a single dose or as part of a series of doses is effective for prophylaxis. Such amount may depend on the health and physical condition of the individual being treated, the age of the individual being treated, the taxonomic population (eg, non-human primates, primates, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the vaccine formulation, assessment of the medical condition of the treating physician, and other relevant factors. It is expected that these amounts will fall within a relatively wide range and can be determined through routine testing. Dosage treatment can be a single dose schedule or a multiple dose schedule (eg, including booster doses). The composition may be administered with other immunomodulatory agents.
- the vaccine composition contains 5 ⁇ g to 40 ⁇ g, 6 ⁇ g to 38 ⁇ g, 7 ⁇ g to 36 ⁇ g, 8 ⁇ g to 34 ⁇ g, 9 ⁇ g to 32 ⁇ g, 9 ⁇ g to 30 ⁇ g to 30 ⁇ g of the RBD nanoparticles as an antigen per dose.
- the stability and homogeneity of the antigenic protein are increased to effectively induce IgG antibodies and neutralizing antibodies, thereby providing excellent SARS-coronavirus-2 infection prevention effect confirmed (Example 1).
- Excessive immune response may occur at doses higher than the corresponding dose. Less than the corresponding dose may exhibit insignificant immunogenicity.
- RBD nanoparticles were described as the weight of recombinant COVID-19 surface antigen protein (RBD) rather than the total weight of RBD nanoparticles (see [Table 3]).
- the recombinant Corona-19 surface antigen protein (RBD) corresponds to 37% of the RBD nanoparticle weight.
- the RBD weight is 20ug
- the total weight of RBD nanoparticles is 135ug
- the RBD weight is 50ug.
- Those skilled in the art can easily convert the RBD weight from the RBD nanoparticle weight and the RBD nanoparticle weight from the RBD weight.
- a single dose of the RBD nanoparticles can be formulated in 0.5 ml.
- the number of administrations of the vaccine composition is one or more times, preferably twice, and the second inoculation may be performed 4 to 8 weeks after the first inoculation.
- the vaccine composition may be administered as a booster vaccine.
- Booster refers to the second antigen stimulation inoculated after the first antigen stimulation during vaccination with a vaccine, and when antibodies are produced, a faster and higher antibody production rate is achieved by the second antigen stimulation compared to the reaction that appears after the first antigen stimulation.
- the "booster vaccine” is inoculated after the first or second inoculation of the vaccine composition, and may correspond to, for example, the third inoculation, but is not limited thereto, and the immune enhancing effect after the first inoculation It means broadly the inoculation that can be expressed.
- Vaccination schedules including primary inoculations and booster inoculations, may continue over the course of days, weeks, or years, as needed. Due to the vaccination schedule, vaccines are administered at the highest frequency at the beginning of the vaccine regimen, and for booster effects, the administration of booster vaccines can be gradually reduced in frequency.
- the vaccine composition may include one or more doses of the booster vaccine, and may include one, two, three, four or five or more doses.
- the vaccine composition of the present invention can be used to prevent SARS-coronavirus-2 infection in a subject.
- subject refers to an animal.
- An animal may be a mammal.
- Mammal refers to, for example, a primate (eg, human, male or female, infant), cow, sheep, goat, horse, dog, cat, rabbit, rat, mouse, fish, bird, etc. can
- the subject for administration of the booster vaccine may be a subject who has been previously administered and / or administered a vaccine composition for preventing or treating SARS-CoV-2 infection once or twice.
- the vaccine composition may be a vaccine composition according to the present invention, but is not limited thereto.
- the vaccine composition of the present invention can be prepared in a variety of forms.
- the composition may be prepared for injectability as a liquid solution or suspension. Solid forms suitable for solution or suspension in liquid vehicles prior to injection may also be prepared.
- the composition may be prepared for topical administration, for example as an ointment, cream or powder.
- the composition may be prepared for oral administration, for example as a tablet or capsule, or as a syrup (optionally flavored syrup).
- the composition may be prepared for pulmonary administration using a fine powder or spray, eg as an inhaler.
- the composition may be prepared as a suppository or pessary.
- the composition may be prepared for nasal, otic or ocular administration, for example as drops, sprays or powders.
- the composition may be included in a hydration agent.
- the composition may be lyophilized.
- the vaccine composition of the present invention may be administered through intradermal, intramuscular, intraperitoneal, intravenous, intranasal, epidural or other appropriate routes, and may be prepared for intramuscular injection and administered through the intramuscular route.
- composition of the present invention may include one selected from the group consisting of 2-phenoxyethanol, formaldehyde, and mixtures thereof as a preservative, and may include 2-phenoxyethanol.
- Another aspect of the present invention is a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto, a trimer in which three are assembled; and an RBD nanoparticle comprising a pentamer assembled of 5 second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer.
- a vaccine composition for preventing or treating coronavirus-2 infection and a kit for preventing or treating SARS-coronavirus-2 infection including instructions for use are provided.
- the immune enhancer may include aluminum hydroxide.
- the description of the immune enhancer and the content of aluminum hydroxide are described in the description of "1. Vaccine composition for preventing or treating SARS-coronavirus-2 infection", so detailed descriptions are omitted.
- the vaccine composition may further include a buffer and a stabilizer.
- buffers and stabilizers are described in the description of "1. Vaccine composition for preventing or treating SARS-CoV-2 infection", so detailed descriptions are omitted.
- Vaccine booster composition for preventing or treating SARS-CoV-2 infection
- Another aspect of the present invention is a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto, a trimer in which three are assembled; and an RBD (Receptor Binding Domain) nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer It provides a vaccine booster composition for preventing or treating SARS-coronavirus-2 infection comprising a.
- RBD Receptor Binding Domain
- RBD nanoparticles The description and dosage of the RBD nanoparticles are described in the description of "1. Vaccine composition for preventing or treating SARS-coronavirus-2 infection", so detailed descriptions are omitted.
- the immune enhancer may include aluminum hydroxide.
- the description of the immune enhancer and the content of aluminum hydroxide are described in the description of "1. Vaccine composition for preventing or treating SARS-coronavirus-2 infection", so detailed descriptions are omitted.
- the vaccine composition may further include a buffer and a stabilizer.
- buffers and stabilizers are described in the description of "1. Vaccine composition for preventing or treating SARS-CoV-2 infection", so detailed descriptions are omitted.
- vaccine booster composition means a primer vaccine, that is, a composition capable of enhancing, extending or maintaining the therapeutic effect of a previously administered vaccine, and is required at regular intervals after primer vaccination. It is a composition that can be administered according to.
- the booster composition may be administered at least one month after inoculation of a primer vaccine composition for preventing or treating SARS-coronavirus-2 infection, and may be additionally inoculated at least every month.
- boosters may be administered at 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 3 months, 6 months
- Booster doses may be given at 12 months, 9 months, and 12 months.
- the interval between administration of the primer vaccine composition and the booster composition of the present invention may be 1 to 3 weeks, 13 to 18 months, and 2 to 20 years.
- the vaccination schedule may continue over the course of days, weeks, or years, as needed. Due to the vaccination schedule, vaccines are administered at the highest frequency at the beginning of the vaccine regimen, and for booster effects, the administration of booster vaccines can be gradually reduced in frequency.
- the administration may be performed one or more times, and additional administration may be performed several times, if necessary. For example, it may be administered 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times.
- the primer vaccine for preventing or treating SARS-CoV-2 infection may be the "1. Vaccine composition for preventing or treating SARS-CoV-2 infection", and a vaccine for preventing or treating SARS-Coronavirus-2 infection. If it can be applied without limitation to the present invention.
- a subject to be inoculated with the vaccine booster composition, a vaccine booster composition preparation method, an administration route, and a preservative included in the vaccine booster composition are the explanatory part of "1.
- Vaccine composition for preventing or treating SARS-CoV-2 infection It is the same as that described in , so the description is omitted.
- Vaccine booster composition kit for preventing or treating SARS-CoV-2 infection
- vaccine composition for preventing or treating SARS-coronavirus-2 infection
- vaccine booster composition for preventing or treating SARS-coronavirus-2 infection
- SARS -Prov ides a vaccine booster composition kit for preventing or treating coronavirus-2 infection.
- the vaccine composition may include doses required for the first or second inoculation, preferably the first and second inoculations, and the vaccine booster composition may include the doses required for the second or more, preferably the third inoculation, Furthermore, in order to enhance immunogenicity against SARS-coronavirus-2, more additional doses may be optionally included.
- Another aspect of the present invention is a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto, a trimer in which three are assembled; and an RBD (Receptor Binding Domain) nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer
- a method for preventing or treating SARS-coronavirus-2 infection in a subject comprising administering to a subject in need of prevention or treatment of SARS-coronavirus-2 infection.
- the method for preventing or treating SARS-coronavirus-2 infection in the subject may include the step of administering a "vaccine booster composition".
- vaccine composition for preventing or treating SARS-coronavirus-2 infection
- vaccine booster composition for preventing or treating SARS-coronavirus-2 infection
- Another aspect of the present invention is a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto, a trimer in which three are assembled; and an RBD (Receptor Binding Domain) nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer It provides a use for preventing or treating SARS-coronavirus-2 infection of a vaccine composition or vaccine booster composition comprising a.
- RBD Receptor Binding Domain
- vaccine composition for preventing or treating SARS-coronavirus-2 infection
- vaccine booster composition for preventing or treating SARS-coronavirus-2 infection
- the trimer glycoprotein on the surface of the SARS-CoV-2 virus is also called spike glycoprotein, and the receptor binding domain (RBD) at the top of the trimer glycoprotein binds to the ACE2 receptor protein on the surface of human cells. By combining, they cause infection.
- the RBD is combined with nanoparticles to produce RBD nanoparticles.
- the RBD nanoparticles are formed by self-assembly of 20 RBD-Component A and 12 Component B.
- the RBD-Component A is a trimer in which three RBD-I53-50A monomers having the amino acid sequence represented by SEQ ID NO: 1 are assembled, and the RBD-I53-50A monomer is the RBD of SARS-CoV-2 spike glycoprotein, nano It consists of I53-50A, which serves as a particle structure, and a linker (GGSGGSGSGGSGGSGSEKAAKAEEAAR) connecting RBD and I53-50A.
- the component B is a pentamer of I53-50B having an amino acid sequence represented by SEQ ID NO: 2.
- a CHO cell line (Stable cell line) was used for RBD-Component A protein production, and an E. coli cell line was used for Component B protein production.
- RBD-Component A protein production RBD-Component A protein production
- E. coli cell line E. coli cell line
- RBD-Component A used as an antigen for SARS-CoV-2 vaccine
- the HD-BIOP3 cell line distributed from HORIZON Discovery, UK was used as a host cell.
- RBD-Component A cDNA N-SARS-CoV-2RBD-I53-50A-16GS-he
- SEQ ID NO: 18 Information on the location and function of key genes in the recombinant expression vector plasmid (M-2560) is summarized in Table 1.
- the CHO cell line-derived Component A intermediate stock solution was obtained by conventional protein expression and purification methods.
- 100% of the peptide coverage was secured and mutual equivalence to the expected peptide sequence was confirmed, confirming that the intermediate stock solution was the desired Component A protein.
- Component B protein E. coli BL21 strain distributed by Thermofisher was used.
- pET29b+ was used as a vector for expressing Component B protein.
- an I53-50B vector with a total sequence of 5.7 kb (vector: 5.4 kb) is produced.
- the vector is composed of a kanamycin resistance gene, a Lac operon whose transcription is induced by lactose and its derivatives, a T7 promoter region derived from lambda phage, and a multiple cloning site. there is.
- the multicloning site has several restriction enzyme sequences, so it is a site that plays a major role in the introduction of antigenic proteins.
- the I53-50B vector has a DNA sequence represented by SEQ ID NO: 19.
- the main gene information in the I53-50B vector is shown in Table 2 below.
- E. coli-derived Component B intermediate stock solutions were obtained by conventional protein expression and purification methods. It was confirmed that the amino acid sequence of the Component B intermediate stock solution was 100% consistent with the theoretical amino acid sequence.
- a recombinant RBD nanoparticle stock solution (drug substance) was prepared through an assembly process using the prepared Component A intermediate stock solution and Component B intermediate stock solution.
- Component A stock solution and Component B were mixed and assembled so that the reaction ratio was 1:1.1 on a molar basis.
- the reaction conditions were room temperature, 1 hour, reaction at a stirring speed of 80 rpm, and when the reaction was completed, the reaction solution was filtered through a 0.2 ⁇ m filter.
- the filtered assembly reaction solution was concentrated (Ultrafiltration) to 4 kg using a tangential flow filtration system equipped with a 300 kDa MWCO (Molecular weight cut-off) membrane with an area of 0.7 m2, It was recovered by carrying out buffer exchange (Diafiltration) using mM Tris buffer. At this time, the transmembrane pressure (TMP) was maintained at 1 bar or less.
- the ultrafiltration recovered liquid was filtered through a 0.2 ⁇ m filter and stored in a cryogenic freezer below -70°C.
- the recombinant RBD nanoparticle stock solution is a particle-type molecule formed by combining the Component A intermediate stock solution and the Component B intermediate stock solution. Molecules in the form of these particles are computer-designed to form molecules of a certain size when appropriate bonding occurs.
- a commonly used method is a dynamic light scattering method, which measures the molecular size of nanoparticles formed through this method to confirm whether an appropriate structure has been formed.
- a final stock solution was prepared through a formulation process.
- aluminum hydroxide was used as an immune enhancer.
- Aluminum hydroxide was pre-mixed into the final stock solution.
- the final stock solution was named RBD nanoparticle alum formulation.
- Vaccination studies included two intramuscular injections of saline placebo (0.5 mL) or RBD nanoparticle alum formulation (RBD 10 ⁇ g or 25 ⁇ g, total injection volume 0.5 mL). 0.5 mL was withdrawn from a vial containing about 0.65 mL of the RBD nanoparticle alum formulation or a 20 mL ampoule of physiological saline. The remaining volume in the vial or ampoule was discarded. This vaccine study was injected into the deltoid muscle of the upper arm every 28 days on day 0 and day 28.
- Table 5 shows test subjects of alum formulations.
- the assigned group intention-to-treat set, ITT group
- safety group safety set
- all analysis subjects full analysis set, FA group
- protocol-compliant clinical trial subject group per protocol set, PP group
- the analysis population refers to a group of subjects included in statistical analysis among subjects participating in a clinical trial.
- Geometric mean titer is a method of calculating the mean antibody titer for a population of subjects by multiplying all values and taking the nth root of this number, where n is the number of subjects for whom data are available. way.
- Geometric Mean Fold Rise represents the increase in the geometric mean concentration or geometric mean titer after vaccination compared to the baseline before vaccination.
- CI 95% confidence interval
- the GMT of the IgG antibody significantly increased compared to the baseline.
- the GMT increased significantly compared to the baseline 2 weeks after the second inoculation, and the increased GMT was maintained even after 4 weeks after the second inoculation.
- the above level is a significantly higher level of antibody compared to the sera of convalescent patients (WHO Reference Panel, First WHO International Reference Panel for anti-SARS-CoV-2 immunoglubulin, NIBSC code: 20/268) of the National Institute of Biomedicine (NIBSC) in the UK.
- NIBSC National Institute of Biomedicine
- the second administration of the RBD nanoparticle alum formulation increases the GMT of the IgG antibody, so that the RBD nanoparticle alum formulation can be usefully used as an active ingredient in a vaccine composition for preventing or treating SARS-CoV-2 infection.
- Table 7 shows the geometric mean titers and seroconversion rates of neutralizing antibodies for the low-content RBD nanoparticle alum formulation and the high-content RBD nanoparticle alum formulation.
- Neutralizing antibodies are important indicators for confirming immunogenicity because neutralizing antibodies bind to viruses and neutralize them from penetrating into cells.
- RBD nanoparticle alum formulation 10 ⁇ g (N 18)
- RBD nanoparticle alum formulation 25 ⁇ g (N 20)
- Placebo (N 19)
- the GMT of the neutralizing antibody was significantly increased compared to the baseline.
- the vaccine composition of the present invention increased the GMT of neutralizing antibodies to 530.90 ⁇ 2.70 and 708.50 ⁇ 2.83 (IU/ml) at 6 weeks after vaccination when 10 ⁇ g or 25 ⁇ g of RBD nanoparticles were inoculated, compared to the effects of conventional vaccines. .
- the second administration of the RBD nanoparticle alum formulation increases the GMT of neutralizing antibodies, so that the RBD nanoparticle alum formulation can be usefully used as an active ingredient in a vaccine composition for preventing or treating SARS-CoV-2 infection.
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Abstract
The present invention relates to a vaccine composition for prevention or treatment of SARS-Coronavirus-2 infection, comprising: RBD nanoparticles comprising a trimer and a pentamer, the trimer being formed by assembling three first polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto, and the pentamer being formed by assembling five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto; and an adjuvant. The vaccine composition, according to the present invention, exhibits excellent immunogenicity when inoculated, and thus the vaccine composition of the present invention may be usefully employed for the purpose of preventing SARS-Coronavirus-2 infection or alleviating symptoms when infected therewith.
Description
본 발명은 SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus-2) 감염을 예방하고/하거나 감염 시 발현되는 증상을 완화하는 목적으로 사용되는 백신 조성물에 관한 것이다.The present invention relates to a vaccine composition used for the purpose of preventing Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection and/or alleviating symptoms expressed during infection.
2019년 12월 이후 전세계적인 감염이 보고되고 있는 SARS-CoV-2 바이러스는 Coronaviridae family, Betacoronavirus genus Sarbecovirus subgenus에 속하는 바이러스로서, SARS-CoV-2 바이러스 감염은 COVID-19 (coronavirus disease 2019)라고도 불린다. 주된 전파경로는 감염자의 비말과의 밀접접촉인 것으로 알려져 있고, 감염자와 직접 접촉하거나 또는 감염자의 비말 등에 의하여 오염된 물품과 같은 매개체를 만진 후, 손을 씻지 않은 채 눈, 코, 입 등을 만짐으로써 바이러스 전파가 이루어질 수도 있다고 알려져 있다. The SARS-CoV-2 virus, which has been reported worldwide since December 2019, is a virus belonging to the Coronaviridae family, Betacoronavirus genus Sarbecovirus subgenus, and SARS-CoV-2 virus infection is also called COVID-19 (coronavirus disease 2019). The main transmission route is known to be close contact with droplets of an infected person, and direct contact with an infected person or touching a medium such as an item contaminated by droplets of an infected person, then touching the eyes, nose, mouth, etc. without washing hands It is known that viral transmission can be achieved by this.
SARS-CoV-2 바이러스 표면의 삼량체 (trimer) 당단백질 (즉, 스파이크 (Spike) 단백질) 상단의 수용체 결합 도메인 (Receptor Binding Domain, RBD)이 인체 세포 표면의 ACE2 수용체 단백질에 결합함으로써 감염을 일으키는 것으로 알려져 있다. 스파이크 당단백질 단량체 (monomer)는 숙주세포 단백질 분해효소 (protease)에 의하여 S1 서브유닛 및 S2 서브유닛으로 분리된다. The Receptor Binding Domain (RBD) on the surface of the SARS-CoV-2 virus binds to the ACE2 receptor protein on the surface of human cells, causing infection. It is known. Spike glycoprotein monomers are separated into S1 subunits and S2 subunits by host cell proteases.
SARS-CoV-2 바이러스의 범세계적 유행으로 인하여 사망자 증가 및 막대한 사회·경제적 피해가 발생하고, 피해가 급증함에 따라 신속한 백신 개발이 요구되어, SARS-CoV-2 감염 예방 또는 증상 완화를 위한 백신 조성물 개발이 수행되고 있다.The global epidemic of SARS-CoV-2 virus causes an increase in deaths and enormous social and economic damage, and as the damage rapidly increases, rapid vaccine development is required. Vaccine composition for preventing SARS-CoV-2 infection or alleviating symptoms development is being carried out.
본 발명자들은 SARS-CoV-2 스파이크 당단백질의 RBD를, 자가 조립(self-assemble)하여 나노 구조를 형성할 수 있는 단백질과 결합하여 다가 (multivalent)의 RBD 항원을 표지하는 RBD 나노파티클을 제조하고, 상기 RBD 나노파티클의 면역원성을 보다 향상시킬 수 있는 면역증강제에 대해 연구하였다. 그 결과, 면역증강제로 수산화알루미늄을 사용시 RBD 나노파티클 저용량 및 고용량 모두에서 우수한 면역원성을 나타내는 것을 확인하였다. The present inventors combine the RBD of the SARS-CoV-2 spike glycoprotein with a protein capable of self-assembling to form a nanostructure to prepare RBD nanoparticles that label multivalent RBD antigens, In addition, an immunostimulant capable of further improving the immunogenicity of the RBD nanoparticles was studied. As a result, it was confirmed that RBD nanoparticles showed excellent immunogenicity at both low and high doses when aluminum hydroxide was used as an immune enhancer.
[선행기술문헌][Prior art literature]
[특허문헌][Patent Literature]
(특허문헌 1) 미국 등록 특허 공보 US 9630994 B2(Patent Document 1) US Registered Patent Publication US 9630994 B2
[비특허문헌][Non-Patent Literature]
(비특허문헌 1)Science. 2016 Jul 22;353(6297):389-94.(Non-Patent Document 1) Science. 2016 Jul 22;353(6297):389-94.
SARS-CoV-2 바이러스 감염을 예방하고, 감염 시 발현되는 증상을 완화하는 목적으로 사용되는 백신 조성물을 제공하고자 한다.It is intended to provide a vaccine composition used for the purpose of preventing SARS-CoV-2 virus infection and alleviating symptoms expressed during infection.
본 발명의 일 측면은, 서열번호 1의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제1 폴리펩타이드 단량체가 3개 조립된 삼량체(trimer); 및 서열번호 2의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제2 폴리펩타이드 단량체가 5개 조립된 오량체(pentamer)를 포함하는 RBD(Receptor Binding Domain) 나노파티클, 및 면역증강제를 포함하는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물을 제공한다.One aspect of the present invention, a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto is an assembled trimer of three; and an RBD (Receptor Binding Domain) nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer It provides a vaccine composition for preventing or treating SARS-coronavirus-2 infection comprising a.
본 발명의 다른 측면은, 서열번호 1의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제1 폴리펩타이드 단량체가 3개 조립된 삼량체(trimer); 및 서열번호 2의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제2 폴리펩타이드 단량체가 5개 조립된 오량체(pentamer)를 포함하는 RBD 나노파티클, 및 면역증강제를 포함하는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물과, 사용설명서를 포함하는 사스-코로나바이러스-2 감염증 예방 또는 치료용 키트를 제공한다.Another aspect of the present invention is a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto, a trimer in which three are assembled; And an RBD nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer, SARS - A vaccine composition for preventing or treating coronavirus-2 infection and a kit for preventing or treating SARS-coronavirus-2 infection including instructions for use are provided.
본 발명에 따른 백신 조성물은 백신 투여 후 IgG항체가와 중화항체가가 증가하고, 2차 백신 접종 후에도 증가된 정도가 유지되므로 사스-코로나바이러스-2 감염에 대한 예방 및 치료 효과가 우수하다.The vaccine composition according to the present invention has excellent preventive and therapeutic effects against SARS-coronavirus-2 infection because the IgG antibody titer and neutralizing antibody titer increase after vaccine administration and the increased levels are maintained even after the second vaccination.
이하, 본 발명을 보다 구체적으로 설명한다. Hereinafter, the present invention will be described in more detail.
한편, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.
또한, 당해 기술분야의 통상의 지식을 가진 자는 통상의 실험만을 사용하여 본 발명에 기재된 본 발명의 특정 양태에 대한 다수의 등가물을 인지하거나 확인할 수 있다. 또한, 이러한 등가물은 본 발명에 포함되는 것으로 의도된다.In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be included in this invention.
본 명세서에서, 용어 "약(about)"은 특정 숫자 값 앞에 제시될 수 있다. 본 출원에서 사용되는 용어 "약"은 용어 뒤에 기재되는 정확한 숫자뿐만 아니라, 거의 그 숫자이거나 그 숫자에 가까운 범위까지 포함한다. 그 숫자가 제시된 문맥을 고려하여, 언급된 구체적인 숫자와 가깝거나 거의 그 숫자인지 여부를 결정할 수 있다. 일 예로, 용어 "약"은 숫자 값의 -10% 내지 +10% 범위를 지칭할 수 있다. 다른 예로, 용어 "약"은 주어진 숫자 값의 -5% 내지 +5% 범위를 지칭할 수 있다. 그러나 이에 제한되지 않는다.In this specification, the term "about" may be presented before a specific numeric value. As used in this application, the term "about" includes not only the exact number that follows the term, but also a range that is or is close to that number. It can be determined whether the number is close to or nearly the specific number mentioned, given the context in which it is presented. As an example, the term “about” can refer to a range of -10% to +10% of a numerical value. As another example, the term "about" can refer to a range of -5% to +5% of a given numerical value. However, it is not limited thereto.
본 명세서에서, 용어 "및/또는"은 본원에 사용되는 경우에 2가지의 명시된 특색 또는 구성요소 각각을 다른 하나와 함께 또는 다른 하나 없이 구체적으로 개시하는 것으로서 이해되어야 한다. 따라서, 본 명세서에서 "A 및/또는 B"와 같은 어구에 사용된 용어 "및/또는"은 "A 및 B", "A 또는 B", "A" (단독), 및 "B" (단독)를 포함하도록 의도된다. 마찬가지로, "A, B, 및/또는 C"와 같은 어구에 사용된 용어 "및/또는"은 하기 측면 각각을 포괄하도록 의도된다: A, B, 및 C; A, B, 또는 C; A 또는 C; A 또는 B; B 또는 C; A 및 C; A 및 B; B 및 C; A (단독); B (단독); 및 C (단독).In this specification, the term "and/or" when used herein is to be understood as specifically disclosing each of the two specified features or elements, either with or without the other. Thus, the term "and/or" as used herein in phrases such as "A and/or B" includes "A and B", "A or B", "A" (alone), and "B" (alone). ) is intended to include. Likewise, the term "and/or" as used in phrases such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
본 명세서에서, 용어 "본질적으로 이루어지는(consisting essentially of)"은 본 발명에서 청구하는 대상의 특징이 불특정 성분의 존재에 실질적으로 영향을 받지 않는 경우, 상기 불특정 성분이 존재할 수 있음을 의미한다.As used herein, the term "consisting essentially of" means that a non-specified ingredient may be present if the characteristics of the claimed subject matter are not substantially affected by the presence of the non-specified ingredient.
본 명세서에서, 용어 "이루어지는(consisting of)"은 특정 성분(들)의 비율이 총 100%인 것을 의미한다. 용어 "이루어지는" 이하에 오는 성분 또는 특징은 필수적이거나 의무적인 것일 수 있다. 일부 구체예에서, "이루어지는" 이하에 오는 성분 또는 특징 외에, 다른 임의의 성분 또는 필수적이지 않은 성분은 배제될 수 있다.In this specification, the term "consisting of" means that the percentage of the specified component(s) totals 100%. Ingredients or features that come after the term “consisting of” may be essential or mandatory. In some embodiments, in addition to the ingredients or features that come under "consisting of", any other ingredients or non-essential ingredients may be excluded.
본 명세서에서, 용어 "포함하는(comprising)"은 상기 용어 이하에 기재되는 특징, 단계 또는 구성 요소의 존재를 의미하며, 하나 이상의 특징, 단계 또는 구성 요소의 존재 또는 추가를 배제하지 않는다. 본 명세서에서 "포함하는" 이하에 기재되는 성분 또는 특징은 필수적이거나 의무적인 것일 수 있으나, 일부 구체예에서는 다른 임의의 혹은 필수적이지 않은 성분 또는 특징을 더 포함할 수 있다.In this specification, the term "comprising" means the presence of features, steps or components described below the term, and does not exclude the presence or addition of one or more features, steps or components. Components or features described herein under "comprising" may be required or mandatory, but in some embodiments may further include other optional or non-essential components or features.
본 명세서에서, 용어 "포함하는"은, 일부 구체예에서, "본질적으로 이루어지는" 또는 "이루어지는"을 지칭하는 것으로 수정될 수 있다.In this specification, the term “comprising” may be modified to refer to “consisting essentially of” or “consisting of” in some embodiments.
본 명세서에서 아미노산 서열과 관련하여, 특정 서열번호로 기재된 아미노산 서열을 "포함하는" 폴리펩타이드, 특정 서열번호로 기재된 아미노산 서열로 "이루어진" 폴리펩타이드, 또는 특정 서열번호로 기재된 아미노산 서열을 "갖는" 폴리펩타이드 또는 단백질이라고 기재되어 있더라도, 해당 서열번호의 아미노산 서열로 이루어진 폴리펩타이드와 동일 혹은 상응하는 활성을 가지는 경우라면, 일부 서열이 결실, 변형, 치환, 보존적 치환 또는 부가된 아미노산 서열을 갖는 단백질도 본 출원에서 사용될 수 있음은 자명하다. 예를 들어, 상기 아미노산 서열 N-말단 그리고/또는 C-말단에 단백질의 기능을 변경하지 않는 서열 추가, 자연적으로 발생할 수 있는 돌연변이, 이의 잠재성 돌연변이(silent mutation) 또는 보존적 치환을 가지는 경우일 수 있으나, 이에 제한되지는 않는다.With respect to an amino acid sequence herein, a polypeptide "comprising" the amino acid sequence set forth in a particular SEQ ID NO, a polypeptide "consisting of' the amino acid sequence set forth in a particular SEQ ID NO, or a polypeptide "having" an amino acid sequence set forth in a particular SEQ ID NO. Even if it is described as a polypeptide or protein, if it has the same or equivalent activity as the polypeptide consisting of the amino acid sequence of the corresponding sequence number, a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added. It is obvious that can also be used in this application. For example, if the amino acid sequence has an addition of a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation thereof, or a conservative substitution to the N-terminus and/or C-terminus of the amino acid sequence. It may, but is not limited thereto.
1. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물1. Vaccine composition for preventing or treating SARS-CoV-2 infection
본 발명의 일 측면은, 서열번호 1의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제1 폴리펩타이드 단량체가 3개 조립된 삼량체(trimer); 및 서열번호 2의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제2 폴리펩타이드 단량체가 5개 조립된 오량체(pentamer)를 포함하는 RBD(Receptor Binding Domain) 나노파티클, 및 면역증강제를 포함하는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물을 제공한다.One aspect of the present invention, a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto is an assembled trimer of three; and an RBD (Receptor Binding Domain) nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer It provides a vaccine composition for preventing or treating SARS-coronavirus-2 infection comprising a.
상기 RBD 나노파티클은 다량체성 단백질 조립체로서 "나노 구조체", "RBD 나노 구조체", 또는 "RBD-NP"로도 지칭할 수 있으며, (i) 서열번호 1의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제1 폴리펩타이드 단량체가 3개 조립된 삼량체(trimer); 및 (ii) 서열번호 2의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제2 폴리펩타이드 단량체가 5개 조립된 오량체(pentamer)를 포함하는 것일 수 있다.The RBD nanoparticle may also be referred to as a "nanostructure", "RBD nanostructure", or "RBD-NP" as a multimeric protein assembly, and (i) has an amino acid sequence of SEQ ID NO: 1 or at least 75% identity thereto. A trimer in which three first polypeptide monomers comprising an amino acid sequence are assembled; and (ii) a second polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto may comprise a five assembled pentamer.
상기 제1 폴리펩타이드는 SARS-CoV-2 스파이크 당단백질 수용체 결합 도메인(receptor binding domain, RBD); 나노 입자의 구조체 역할을 하는 제1 자가조립 폴리펩타이드; 및 상기 RBD와 상기 제1 자가조립 폴리펩타이드를 연결하는 링커(linker)를 포함하는 것일 수 있다. 상기 제1 폴리펩타이드 단량체는 N-말단 포스파타제 신호 펩타이드 서열 (MGILPSPGMPALLSLVSLLSVLLMGCVA)을 더 포함할 수 있다. 복수개의 단리된 제1 폴리펩타이드 단량체는 상호작용을 통해 자가-조립(self-assembly)되어 삼량체(trimer)를 형성할 수 있다. 이러한 조립은 비공유성 단백질-단백질 상호작용 반응을 통해 이루어질 수 있다.The first polypeptide comprises a SARS-CoV-2 spike glycoprotein receptor binding domain (receptor binding domain, RBD); A first self-assembling polypeptide serving as a structure of nanoparticles; and a linker connecting the RBD and the first self-assembling polypeptide. The first polypeptide monomer may further include an N-terminal phosphatase signal peptide sequence (MGILPSPGMPALLSLVSLLSVLLMGCVA). A plurality of isolated first polypeptide monomers may self-assemble through interactions to form a trimer. This assembly can be achieved through non-covalent protein-protein interaction reactions.
상기 제2 폴리펩타이드는 나노 입자의 구조체 역할을 하는 제2 자가조립 폴리펩타이드 단량체로서, 복수 개의 단리된 제2 폴리펩타이드 단량체는 상호작용을 통해 자가-조립(self-assembly)되어 오량체(pentamer)를 형성할 수 있다. 이러한 조립은 비공유성 단백질-단백질 상호작용 반응을 통해 이루어질 수 있다.The second polypeptide is a second self-assembling polypeptide monomer serving as a structure of nanoparticles, and a plurality of isolated second polypeptide monomers self-assemble through interactions to form a pentamer. can form This assembly can be achieved through non-covalent protein-protein interaction reactions.
본 발명에서 상기 제1 폴리펩타이드는 국제특허공보 제2019-169120호에 기재된 폴리펩타이드 중 하나일 수 있다. 구체적으로, 상기 제1 폴리펩타이드 단량체는 서열번호 1의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열로 이루어진 폴리펩타이드일 수 있고, 예를 들어 서열번호 1로 표시되는 아미노산 서열로 이루어진 폴리펩타이드일 수 있다. 본 명세서에서 서열번호 1의 폴리펩타이드는 "RBD-I53-50A" 또는 "RBD-Component A"로 지칭할 수 있다. 상기 제1 폴리펩타이드는 서열번호 3으로 표시되는 염기서열로 이루어진 유전자에 의해 코딩될 수 있다. In the present invention, the first polypeptide may be one of the polypeptides described in International Patent Publication No. 2019-169120. Specifically, the first polypeptide monomer may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto, for example, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 can In the present specification, the polypeptide of SEQ ID NO: 1 may be referred to as "RBD-I53-50A" or "RBD-Component A". The first polypeptide may be encoded by a gene consisting of the nucleotide sequence represented by SEQ ID NO: 3.
상기 제2 폴리펩타이드 단량체는 서열번호 2의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열로 이루어진 폴리펩타이드일 수 있고, 예를 들어 서열번호 2로 표시되는 아미노산 서열로 이루어진 폴리펩타이드일 수 있다. 본 명세서에서 서열번호 2의 폴리펩타이드는 "I53-50B" 또는 "Component B"로 지칭할 수 있다. 상기 제2 폴리펩타이드 단량체는 서열번호 4로 표시되는 염기서열로 이루어진 유전자에 의해 코딩될 수 있다. The second polypeptide monomer may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, for example, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2. The polypeptide of SEQ ID NO: 2 herein may be referred to as "I53-50B" or "Component B". The second polypeptide monomer may be encoded by a gene consisting of the nucleotide sequence represented by SEQ ID NO: 4.
상기 제1 폴리펩타이드 및 제2 폴리펩타이드는 다량성 단백질 조립체로의 조립을 교란시키지 않는 임의의 변형을 포함할 수 있으며, 예를 들어 아미노산의 추가, 결실, 삽입, 치환 또는 변형을 포함할 수 있다.The first polypeptide and the second polypeptide may include any modification that does not disrupt assembly into a multimeric protein assembly, for example, may include addition, deletion, insertion, substitution, or modification of amino acids. .
상기 제1 폴리펩타이드 및 제2 폴리펩타이드는 국제특허공보 제2019-169120호, 미국 등록 특허 공보 제9630994호, 국제특허공보 제2021-163481호, 국제특허공보 제2021-163438호, 또는 국제출원번호 제PCT/US2021/037341호에 개시된 단백질일 수 있다. 구체적으로, 상기 제1 폴리펩타이드는 서열번호 1의 아미노산 서열과 적어도 85% 동일성을 갖는 아미노산 서열로 이루어진 폴리펩타이드로서 서열번호 5 내지 9의 아미노산 서열(국제 특허 공보 제2019-169120호의 서열번호 7, 29, 30, 31 또는 39의 아미노산 서열에 해당함)로 이루어진 폴리펩타이드일 수 있다. 또한, 상기 제2 폴리펩타이드는 서열번호 2의 아미노산 서열과 적어도 85% 동일성을 갖는 아미노산 서열로 이루어진 폴리펩타이드로서 서열번호 10 내지 17의 아미노산 서열(국제 특허 공보 제2019-169120호의 서열번호 6, 8, 10, 27, 28, 32, 33 또는 38의 아미노산 서열에 해당함)로 이루어진 폴리펩타이드일 수 있다.The first polypeptide and the second polypeptide are International Patent Publication No. 2019-169120, US Patent Publication No. 9630994, International Patent Publication No. 2021-163481, International Patent Publication No. 2021-163438, or International Application No. It may be a protein disclosed in PCT/US2021/037341. Specifically, the first polypeptide is a polypeptide consisting of an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence of SEQ ID NOs: 5 to 9 (SEQ ID NO: 7 of International Patent Publication No. 2019-169120, Corresponding to the amino acid sequence of 29, 30, 31 or 39) may be a polypeptide consisting of. In addition, the second polypeptide is a polypeptide consisting of an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NOs: 10 to 17 (SEQ ID NOs: 6 and 8 of International Patent Publication No. 2019-169120) , corresponding to the amino acid sequence of 10, 27, 28, 32, 33 or 38).
상기 RBD 나노파티클은 제1 폴리펩타이드의 삼량체와 제2 폴리펩타이드의 오량체가 자가조립하여 형성될 수 있다. 구체적으로 본 발명의 RBD 나노파티클은 상기 (i) 제1 폴리펩타이드(Component A)의 삼량체를 20개 포함하고, 상기 (ii) 제2 폴리펩타이드(Component B)의 오량체를 12개 포함하는 구조를 가질 수 있다. 따라서 본 발명의 다량체성 단백질 조립체는 제1 폴리펩타이드(Component A)의 60개 카피 및 제2 폴리펩타이드(Component B)의 60개 카피를 포함한다. The RBD nanoparticles may be formed by self-assembly of a trimer of the first polypeptide and a pentamer of the second polypeptide. Specifically, the RBD nanoparticles of the present invention include (i) 20 trimers of the first polypeptide (Component A) and (ii) 12 pentamers of the second polypeptide (Component B) can have a structure. Accordingly, the multimeric protein assembly of the present invention includes 60 copies of the first polypeptide (Component A) and 60 copies of the second polypeptide (Component B).
본 발명의 RBD 나노파티클은 이십면체의 대칭구조를 가질 수 있다. 본 발명의 RBD 나노파티클은 약 30 nm 내지 약 70 nm의 직경을 가지며, 이때 내강(lumen)은 가로로 약 15 nm 내지 약 32 nm일 수 있다. The RBD nanoparticles of the present invention may have an icosahedral symmetrical structure. The RBD nanoparticles of the present invention have a diameter of about 30 nm to about 70 nm, and in this case, the lumen may be about 15 nm to about 32 nm horizontally.
상기 제1 폴리펩타이드 단량체를 생산하는 세포주는 서열번호 1로 표시되는 아미노산 서열로 표시되는 단백질을 코딩하는 유전자를 포함하는 발현 벡터를 갖는 세포주일 수 있다. 본 발명의 일 구체예에서, 상기 발현 벡터는 플라스미드 벡터일 수 있으나, 세포주에서 제1 폴리펩타이드 단량체를 발현시키기에 적합한 벡터라면 제한없이 사용될 수 있다. The cell line producing the first polypeptide monomer may be a cell line having an expression vector containing a gene encoding a protein represented by the amino acid sequence represented by SEQ ID NO: 1. In one embodiment of the present invention, the expression vector may be a plasmid vector, but any vector suitable for expressing the first polypeptide monomer in a cell line may be used without limitation.
상기 제2 폴리펩타이드 단량체를 생산하는 세포주는 서열번호 2로 표시되는 아미노산 서열로 표시되는 단백질을 코딩하는 유전자를 포함하는 발현 벡터를 갖는 세포주일 수 있다. 본 발명의 일 구체예에서, 상기 발현 벡터는 pET 계열의 벡터일 수 있고, 예를 들어 pET29b+ 벡터일 수 있으나, 세포주에 제2 폴리펩타이드 단량체를 발현시키기에 적합한 벡터라면 제한없이 사용될 수 있다.The cell line producing the second polypeptide monomer may be a cell line having an expression vector containing a gene encoding a protein represented by the amino acid sequence represented by SEQ ID NO: 2. In one embodiment of the present invention, the expression vector may be a pET-based vector, for example, a pET29b+ vector, but any vector suitable for expressing the second polypeptide monomer in a cell line may be used without limitation.
본 발명의 일 구체예에서, 본 발명에서 서열번호 1로 표시되는 아미노산 서열로 표시되는 단백질을 코딩하는 유전자를 포함하는 발현 벡터는 서열번호 18로 표시되는 DNA 서열을 가질 수 있다.In one embodiment of the present invention, an expression vector containing a gene encoding a protein represented by the amino acid sequence represented by SEQ ID NO: 1 in the present invention may have a DNA sequence represented by SEQ ID NO: 18.
본 발명의 일 구체예에서, 본 발명에서 서열번호 2로 표시되는 아미노산 서열로 표시되는 단백질을 코딩하는 유전자를 포함하는 발현 벡터는 서열번호 19로 표시되는 DNA 서열을 가질 수 있다.In one embodiment of the present invention, an expression vector containing a gene encoding a protein represented by the amino acid sequence represented by SEQ ID NO: 2 in the present invention may have a DNA sequence represented by SEQ ID NO: 19.
본 발명에서 사용되는 아미노산 서열 또는 염기 서열은, 서열 목록에 기재된 서열과 실질적인 동일성을 나타내는 서열을 포함하는 것으로 이해된다. 여기서, "실질적인 동일성"은 본 발명의 서열과 임의의 다른 서열로서 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 이상의 상동성을 갖는 서열이며, 이들 상동성 범위에 있는 아미노산 서열 또는 염기서열도 본 발명의 권리범위에 포함되는 것으로 해석된다.It is understood that the amino acid sequence or nucleotide sequence used in the present invention includes sequences showing substantial identity to the sequences described in the sequence listing. Here, "substantial identity" is 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, It is a sequence having 98% or 99% or more homology, and amino acid sequences or base sequences within these homology ranges are also construed to be included in the scope of the present invention.
본 발명에서 사용된 용어, "벡터"는 숙주세포에 도입되어 숙주세포 유전체 내로 재조합 및 삽입될 수 있거나, 또는 에피좀 (episome)으로 자발적으로 복제될 수 있는 뉴클레오티드 서열을 포함하는 핵산 수단을 말한다. 적합한 발현 벡터는 프로모터, 개시코돈, 종결코돈, 폴리아데닐화 신호 및 인핸서 같은 발현 조절 요소 (element) 외에도 막 표적화 또는 분비를 위한 신호서열 또는 리더서열을 포함하며, 목적에 따라 다양하게 제조될 수 있다. 개시코돈 및 종결코돈을 표적 단백질을 암호화하는 유전자 작제물이 투여되었을 때 개체에서 반드시 작용을 나타내야 하며 암호화 서열과 인프레임(in frame)에 있어야 한다.As used herein, the term "vector" refers to a nucleic acid means comprising a nucleotide sequence that can be introduced into a host cell and recombined and inserted into the genome of the host cell, or can be spontaneously replicated as an episome. Suitable expression vectors include expression control elements such as promoters, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. . When a genetic construct encoding a target protein is administered, the initiation codon and stop codon must be functional in the subject and must be in frame with the coding sequence.
본 발명의 일 구체예에 따른 폴리뉴클레오티드 또는 벡터는 게놈 외부의 독립적 분자, 구체적으로는 복제할 수 있는 분자로서 유전적으로 변형된 숙주세포 또는 비-인간 숙주개체 내에 존재할 수 있거나, 또는 숙주세포 또는 비-인간 숙주개체의 게놈으로 안정적으로 삽입될 수 있다.The polynucleotide or vector according to one embodiment of the present invention is an independent molecule outside the genome, specifically a molecule capable of replicating, and may exist in a genetically modified host cell or non-human host organism, or may be present in a host cell or non-human host cell. -Can be stably integrated into the genome of human host organisms.
본 발명의 일 구체예에 따른 상기 제1 폴리펩타이드 단량체를 생산하는 세포주의 숙주세포는 진핵세포이다. 상기 진핵세포는 진균세포 (fungus), 식물세포 또는 동물세포를 포함한다. 진균세포의 예로는 효모, 구체적으로는 사카로마이세스 속(Saccharomyces sp.)의 효모, 더욱 구체적으로는 사카로마이세스 세레비지애 (S. cerevisiae)일 수 있다. 또한, 동물세포의 예로는 곤충세포 또는 포유동물 세포가 있고, 구체적인 동물세포의 예로는 HEK293, 293T, NSO, 중국 햄스터 난소 세포(CHO), MDCK, U2-OSHela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, C33A 등이 있다. 또한, 통상의 기술분야에 잘 알려져 있는 적당한 세포주들은 ATCC (American Type Culture Collection)와 같은 세포주 기탁기관으로부터 수득할 수 있다. The host cell of the cell line producing the first polypeptide monomer according to one embodiment of the present invention is a eukaryotic cell. The eukaryotic cells include fungus cells, plant cells or animal cells. Examples of the fungal cell may be yeast, specifically yeast of the genus Saccharomyces (Saccharomyces sp.), more specifically S. cerevisiae. In addition, examples of animal cells include insect cells or mammalian cells, and specific examples of animal cells include HEK293, 293T, NSO, Chinese hamster ovary cells (CHO), MDCK, U2-OSHela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, C33A, etc. In addition, suitable cell lines well known in the art can be obtained from cell line depositories such as the American Type Culture Collection (ATCC).
본 발명의 일 구체예에 따른 상기 제1 폴리펩타이드 단량체를 생산하는 세포주의 숙주세포는 중국 햄스터 난소 세포 (CHO)일 수 있으며, 바람직하게는 재조합 아데노-관련 바이러스 (recombinant Adeno-Associated Virus, rAAV)에 의하여 Glutamine Synthetase 유전자의 6번 엑손이 넉아웃 (Knock-out)된 HD-BIOP3 GS null CHO-K1 세포를 사용할 수 있다. The host cell of the cell line producing the first polypeptide monomer according to an embodiment of the present invention may be a Chinese Hamster Ovary (CHO) cell, preferably a recombinant Adeno-Associated Virus (rAAV) HD-BIOP3 GS null CHO-K1 cells in which Exon 6 of the Glutamine Synthetase gene was knocked out by the method can be used.
상기 숙주세포에 상기 제1 폴리펩타이드 단량체를 코딩하는 유전 서열을 갖는 발현 벡터를 도입하기 위해서는 핵산을 세포 내로 도입하는 어떤 방법이든 사용할 수 있고, 숙주세포에 따라 통상의 기술분야에 공지된 기술을 사용할 수 있다. 예를 들어, 열충격법 (Heat Shock), 전기천공법 (Electroporation), 인산칼슘 (CaPO4) 침전, 염화칼슘 (CaCl2) 침전, 미세주입법 (microinjection), 폴리에틸렌글리콜 (PEG)법, DEAE-덱스트란법, 양이온 리포좀법, 및 초산 리튬-DMSO법 등이 있으나, 이에 제한되지 않는다. In order to introduce the expression vector having the genetic sequence encoding the first polypeptide monomer into the host cell, any method for introducing nucleic acid into the cell may be used, and techniques known in the art may be used depending on the host cell. can For example, heat shock, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, A cationic liposome method, and a lithium acetate-DMSO method, but are not limited thereto.
본 발명의 백신 조성물은 상기 RBD 나노파티클과 함께 약학적으로 허용되는 담체를 포함할 수 있다. 본 발명에서 사용되는 용어 "약학적으로 허용되는 담체"는 본 기술 분야의 통상의 기술자에게 공지된 바와 같은 임의의 및 모든 용매, 분산 매질, 코팅, 계면활성제, 항산화제, 보존제(예를 들어, 항균제, 항진균제), 등장성 작용제, 흡수 지연제, 염, 보존제, 약물 안정화제, 결합제, 부형제, 붕해제, 윤활제, 감미제, 착향제, 염료 등 및 이들의 조합물을 포함한다. 임의의 통상적인 담체가 활성 성분과 양립되지 않는 것을 제외하고는, 치료 또는 약학적 조성물에서의 이의 용도가 고려된다.The vaccine composition of the present invention may include a pharmaceutically acceptable carrier together with the RBD nanoparticles. As used herein, the term "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (eg, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof. Except that any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.
본 발명의 일 구체예에서, 본 발명의 백신 조성물에는 주성분인 RBD 나노파티클 외에 완충제, 안정제, 흡착제(면역증강제) 및 용제를 포함할 수 있다. 완충제로는 염화나트륨, 트로메타민을 사용할 수 있고, 안정제로는 L-아르기닌, 백당(수크로스 또는 자당)을 사용할 수 있으며, 흡착제(면역증강제)로는 수산화알루미늄을 사용할 수 있다. In one embodiment of the present invention, the vaccine composition of the present invention may include a buffer, a stabilizer, an adsorbent (immunity enhancer) and a solvent in addition to RBD nanoparticles as a main component. Sodium chloride and tromethamine can be used as a buffer, L-arginine and sucrose (sucrose or sucrose) can be used as a stabilizer, and aluminum hydroxide can be used as an adsorbent (immune enhancer).
본 발명에서 사용되는 "면역증강제"는 면역세포의 초기 활성화 과정에서 비특이적으로 항원에 대한 면역 반응을 촉진하는 물질로, 본 발명의 백신 조성물은 RBD 나노파티클과 함께 투여되어 면역 반응을 증강시킬 수 있는 다양한 면역증강제를 포함한다. 본 발명에 있어서, 면역증강제는 알루미늄 포스페이트, 알루미늄 설페이트 및 알루미늄 하이드록사이드로 이루어진 그룹으로부터 선택될 수 있고, 알루미늄 하이드록사이드(수산화알루미늄)일 수 있다. The "immune enhancer" used in the present invention is a substance that non-specifically promotes an immune response to an antigen during the initial activation of immune cells, and the vaccine composition of the present invention is administered together with RBD nanoparticles to enhance the immune response. Contains various immune enhancing agents. In the present invention, the immunostimulant may be selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide, and may be aluminum hydroxide (aluminum hydroxide).
상기 백신 조성물은 수산화알루미늄을 추가로 포함할 수 있고, 상기 수산화알루미늄은 완제의약품에 미리 혼합된 상태로 제공될 수 있다. 상기 면역증강제가 수산화알루미늄인 경우, 상기 RBD 나노파티클 5중량부 내지 40중량부에 대해 수산화알루미늄이 750중량부로 포함될 수 있고, 상기 RBD 나노파티클 6중량부 내지 38중량부, 7중량부 내지 36중량부, 8중량부 내지 34중량부, 9중량부 내지 32중량부, 9중량부 내지 30중량부, 10중량부 내지 28중량부, 10중량부 내지 26중량부, 10중량부 내지 25중량부에 대해 수산화알루미늄은 750중량부로 포함될 수 있다.The vaccine composition may further include aluminum hydroxide, and the aluminum hydroxide may be provided in a pre-mixed state in a drug product. When the adjuvant is aluminum hydroxide, 750 parts by weight of aluminum hydroxide may be included with respect to 5 parts by weight to 40 parts by weight of the RBD nanoparticles, and 6 to 38 parts by weight and 7 to 36 parts by weight of the RBD nanoparticles. 8 parts by weight to 34 parts by weight, 9 parts by weight to 32 parts by weight, 9 parts by weight to 30 parts by weight, 10 parts by weight to 28 parts by weight, 10 parts by weight to 26 parts by weight, 10 parts by weight to 25 parts by weight For the aluminum hydroxide may be included in 750 parts by weight.
상기 면역증강제가 수산화알루미늄인 경우, 상기 RBD 나노파티클의 2회 용량(1ml), 즉 상기 RBD 나노파티클 20 μg 또는 50 μg에 대하여, 수산화알루미늄은 1000 μg 내지 2000 μg, 1100 μg 내지 1900 μg, 1150 μg 내지 1850 μg, 1200 μg 내지 1800 μg, 1250 μg 내지 1750 μg, 1300 μg 내지 1700 μg, 1350 μg 내지 1650 μg, 1400 μg 내지 1600 μg, 1450 μg 내지 1600 μg으로 포함될 수 있고, 1400 μg 내지 1600 μg으로 포함될 수 있다. 만일 상기 수산화알루미늄이 1000 μg 미만으로 포함되면 면역 활성이 감소할 수 있고, 2000 μg을 초과하여 포함되면 부작용 발생 가능성이 증가하는 문제점이 있다.When the immune enhancer is aluminum hydroxide, aluminum hydroxide is 1000 μg to 2000 μg, 1100 μg to 1900 μg, 1150 μg to 1850 μg, 1200 μg to 1800 μg, 1250 μg to 1750 μg, 1300 μg to 1700 μg, 1350 μg to 1650 μg, 1400 μg to 1600 μg, 1450 μg to 1600 μg, and 1400 μg to 16 00 µg can be included as If the aluminum hydroxide is included in less than 1000 μg, immune activity may be reduced, and if it is included in excess of 2000 μg, there is a problem in that the possibility of side effects increases.
흡착제 또는 면역증강제로서 수산화알루미늄을 상기 범위 내로 첨가하면, IgG항체가와 중화항체가가 증가하므로 사스-코로나바이러스-2 감염증 예방 효과가 우수하다(실시예 1).When aluminum hydroxide is added within the above range as an adsorbent or immune enhancer, the IgG antibody titer and the neutralizing antibody titer increase, so the SARS-coronavirus-2 infection prevention effect is excellent (Example 1).
본 발명에서 사용되는 용어 "완충제"는 첨가되는 용액 내에서 pH가 급격히 변화하지 않게 용액의 pH를 유지시키는 역할을 하는 제제를 나타낸다. 본 발명에 적용할 수 있는 완충제는 염화나트륨 및 트로메타민(트리스) 중 적어도 하나를 포함할 수 있다. 또한, 염화나트륨 및 트로메타민 외에도 용액 내에서 pH를 유지시킬 수 있는 완충제라면 본 발명에 제한없이 적용될 수 있다. 예컨대, 상기 완충제는 인산나트륨 또는 인산칼륨과 같은 인산염 완충제, HEPES(2-[4-(2-하이드록시에틸)피페라진-1-일]에탄-1-설폰산), 히스티딘 또는 시트르산염 완충제를 포함할 수 있다.As used herein, the term "buffer" refers to an agent that serves to maintain the pH of a solution so that the pH does not change rapidly in the solution to which it is added. Buffers applicable to the present invention may include at least one of sodium chloride and tromethamine (Tris). In addition, in addition to sodium chloride and tromethamine, any buffer capable of maintaining pH in a solution can be applied without limitation to the present invention. For example, the buffer may be a phosphate buffer such as sodium phosphate or potassium phosphate, HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid), histidine or citrate buffer. can include
상기 완충제가 염화나트륨인 경우, 상기 RBD 나노파티클의 2회 용량(1ml), 즉 상기 RBD 나노파티클 20 μg 또는 50 μg 에 대하여, 1 mg 내지 20 mg로 포함될 수 있고, 2 mg 내지 19 mg, 3 mg 내지 18 mg, 4 mg 내지 17 mg, 5 mg 내지 16 mg, 6 mg 내지 15 mg, 7 mg 내지 14 mg, 8 mg 내지 13 mg, 1 mg 내지 12 mg, 8 mg 내지 11 mg, 8 mg 내지 10 mg, 8 mg 내지 9 mg, 7 mg 내지 8.5 mg로 포함될 수 있으며, 예를 들어 8 mg 내지 9 mg, 7 mg 내지 8.5 mg, 7.5 mg 내지 8.5mg로 포함될 수 있다. 염화나트륨이 상기 범위 내로 포함되면, 항원단백질의 안정성 및 균질성이 증가하여 IgG항체 및 중화항체를 효과적으로 유도하며 그에 따른 우수한 사스-코로나바이러스-2 감염증 예방 효과를 확인하였다(실시예 1).When the buffer is sodium chloride, it may be included in 1 mg to 20 mg, 2 mg to 19 mg, 3 mg for two doses (1 ml) of the RBD nanoparticles, that is, 20 μg or 50 μg of the RBD nanoparticles. to 18 mg, 4 mg to 17 mg, 5 mg to 16 mg, 6 mg to 15 mg, 7 mg to 14 mg, 8 mg to 13 mg, 1 mg to 12 mg, 8 mg to 11 mg, 8 mg to 10 mg, 8 mg to 9 mg, and 7 mg to 8.5 mg, for example, 8 mg to 9 mg, 7 mg to 8.5 mg, and 7.5 mg to 8.5 mg. When sodium chloride is included within the above range, the stability and homogeneity of the antigenic protein are increased, effectively inducing IgG antibodies and neutralizing antibodies, and the resulting SARS-coronavirus-2 infection prevention effect was confirmed (Example 1).
상기 완충제가 트로메타민인 경우, 상기 RBD 나노파티클의 2회 용량(1ml), 즉 상기 RBD 나노파티클 20 μg 또는 50 μg에 대하여, 0.1 mg 내지 10 mg로 포함될 수 있고, 0.5 mg 내지 9.5 mg, 1 mg 내지 9 mg, 1.5 mg 내지 8.5 mg, 2 mg 내지 8 mg, 2.5 mg 내지 7.5 mg, 3 mg 내지 7 mg, 3.5 mg 내지 6.5 mg, 4 mg 내지 6 mg, 4.5 mg 내지 6 mg, 5 mg 내지 6 mg, 5.5 mg 내지 6 mg로 포함될 수 있고, 예를 들어 4 mg 내지 6 mg, 5 mg 내지 6 mg으로 포함될 수 있다. 트로메타민이 상기 범위 내로 포함되면, 항원단백질의 안정성 및 균질성이 증가하여 IgG항체 및 중화항체를 효과적으로 유도하며 그에 따른 우수한 사스-코로나바이러스-2 감염증 예방 효과를 확인하였다(실시예 1).When the buffer is tromethamine, it may be included in 0.1 mg to 10 mg, 0.5 mg to 9.5 mg, for 2 doses (1 ml) of the RBD nanoparticles, that is, 20 μg or 50 μg of the RBD nanoparticles, 1 mg to 9 mg, 1.5 mg to 8.5 mg, 2 mg to 8 mg, 2.5 mg to 7.5 mg, 3 mg to 7 mg, 3.5 mg to 6.5 mg, 4 mg to 6 mg, 4.5 mg to 6 mg, 5 mg to It may be included in 6 mg, 5.5 mg to 6 mg, for example, 4 mg to 6 mg, 5 mg to 6 mg. When tromethamine is included within the above range, the stability and homogeneity of the antigenic protein are increased, effectively inducing IgG antibodies and neutralizing antibodies, and the resulting SARS-coronavirus-2 infection prevention effect was confirmed (Example 1).
본 발명에서 사용되는 용어 "안정제"는 난용성 면역활성화 물질이 계면활성제 없이 수용액에 용이하면서 재현성 있게 분산될 수 있도록 하는 제제를 나타낸다. 본 발명에 적용할 수 있는 완충제는 L-아르기닌 및 백당 중 적어도 하나를 포함할 수 있다. 또한, 염화나트륨 및 트로메타민 외에도 난용성 면역활성화 물질을 수용액에 분산시킬 수 있는 안정제라면 본 발명에 제한없이 적용될 수 있다. 예컨대, 상기 안정제는 L-리신 또는 L-히스티딘과 같은 분자내 아민 그룹 및 알칼리 특성을 갖는 염기성 아미노산을 포함할 수 있고, 글루코스, 락토스, 말토스, 트레할로스, 소르비톨, 만니톨 및 이들의 조합으로 이루어진 군으로부터 선택되는 하나 이상의 당을 포함할 수 있다.The term "stabilizer" used in the present invention refers to an agent that allows a poorly soluble immunoactive substance to be easily and reproducibly dispersed in an aqueous solution without a surfactant. Buffers applicable to the present invention may include at least one of L-arginine and sucrose. In addition, in addition to sodium chloride and tromethamine, any stabilizer capable of dispersing poorly soluble immunoactivating substances in an aqueous solution can be applied without limitation to the present invention. For example, the stabilizer may include an amine group in a molecule such as L-lysine or L-histidine and a basic amino acid having alkaline properties, and may include glucose, lactose, maltose, trehalose, sorbitol, mannitol, and combinations thereof. It may contain one or more sugars selected from.
상기 안정제가 L-아르기닌인 경우, 상기 RBD 나노파티클의 2회 용량(1ml), 즉 상기 RBD 나노파티클 20 μg 또는 50 μg에 대하여, 8 mg 내지 24 mg로 포함될 수 있고, 9 mg 내지 23 mg, 10 mg 내지 22 mg, 11 mg 내지 21 mg, 12 mg 내지 20 mg, 13 mg 내지 19 mg, 14 mg 내지 18 mg, 15 mg 내지 17 mg, 16 mg 내지 17 mg로 포함될 수 있으며, 예를 들어 14 mg 내지 17 mg, 15 mg 내지 17 mg으로 포함될 수 있다. L-아르기닌이 상기 범위 내로 포함되면, 항원단백질의 안정성 및 균질성이 증가하여 IgG항체 및 중화항체를 효과적으로 유도하며 그에 따른 우수한 사스-코로나바이러스-2 감염증 예방 효과를 확인하였다(실시예 1).When the stabilizer is L-arginine, it may be included in 8 mg to 24 mg for two doses (1 ml) of the RBD nanoparticles, that is, 20 μg or 50 μg of the RBD nanoparticles, 9 mg to 23 mg, 10 mg to 22 mg, 11 mg to 21 mg, 12 mg to 20 mg, 13 mg to 19 mg, 14 mg to 18 mg, 15 mg to 17 mg, 16 mg to 17 mg, for example, 14 mg to 17 mg, 15 mg to 17 mg. When L-arginine is included within the above range, the stability and homogeneity of the antigenic protein are increased, effectively inducing IgG antibodies and neutralizing antibodies, and the resulting SARS-coronavirus-2 infection prevention effect was confirmed (Example 1).
상기 안정제가 백당인 경우, 상기 RBD 나노파티클의 2회 용량(1ml), 즉 상기 RBD 나노파티클 20 μg 또는 50 μg에 대하여, 35 mg 내지 55 mg로 포함될 수 있고, 36 mg 내지 54 mg, 37 mg 내지 53 mg, 38 mg 내지 52 mg, 39 mg 내지 51 mg, 40 mg 내지 50 mg, 41 mg 내지 49 mg, 42 mg 내지 48 mg, 43 mg 내지 47 mg, 44 mg 내지 47 mg으로 포함될 수 있으며, 예를 들어 40 mg 내지 50 mg, 44 mg 내지 47 mg, 45 mg 내지 47 mg로 포함될 수 있다. 백당이 상기 범위 내로 포함되면, 항원단백질의 안정성 및 균질성이 증가하여 IgG항체 및 중화항체를 효과적으로 유도하며 그에 따른 우수한 사스-코로나바이러스-2 감염증 예방 효과를 확인하였다(실시예 1).When the stabilizer is sucrose, it may be included in 35 mg to 55 mg, 36 mg to 54 mg, 37 mg for two doses (1 ml) of the RBD nanoparticles, that is, 20 μg or 50 μg of the RBD nanoparticles. to 53 mg, 38 mg to 52 mg, 39 mg to 51 mg, 40 mg to 50 mg, 41 mg to 49 mg, 42 mg to 48 mg, 43 mg to 47 mg, 44 mg to 47 mg, For example, 40 mg to 50 mg, 44 mg to 47 mg, and 45 mg to 47 mg may be included. When sucrose is included within the above range, the stability and homogeneity of the antigenic protein are increased, effectively inducing IgG antibodies and neutralizing antibodies, and the resulting SARS-coronavirus-2 infection prevention effect was confirmed (Example 1).
본 발명의 일 구체예에서, 상기 백신 조성물에 상기 RBD 나노파티클의 2회 용량(1ml), 즉 상기 RBD 나노파티클 20 μg 또는 50 μg에 대하여, 수산화알루미늄이 1000 μg 내지 2000 μg으로 포함되고, 염화나트륨이 1 mg 내지 20 mg으로 포함되며, 트로메타민이 0.1 mg 내지 10 mg으로 포함되고, L-아르기닌이 8 mg 내지 24 mg으로 포함되며, 백당이 35 mg 내지 55 mg으로 포함될 수 있다. 상기 RBD 나노파티클 및 백신 조성물에 사용되는 면역증강제, 완충제 및 안정제를 상기 범위 내로 첨가하면, 항원단백질의 안정성 및 균질성이 증가하여 IgG항체 및 중화항체를 효과적으로 유도하며 그에 따른 우수한 사스-코로나바이러스-2 감염증 예방 효과를 확인하였다(실시예 1).In one embodiment of the present invention, the vaccine composition contains 1000 μg to 2000 μg of aluminum hydroxide for two doses (1 ml) of the RBD nanoparticles, that is, 20 μg or 50 μg of the RBD nanoparticles, and sodium chloride 1 mg to 20 mg of this, 0.1 mg to 10 mg of tromethamine, 8 mg to 24 mg of L-arginine, and 35 mg to 55 mg of sucrose. When the immune enhancers, buffers, and stabilizers used in the RBD nanoparticles and vaccine compositions are added within the above range, the stability and homogeneity of the antigenic protein are increased to effectively induce IgG antibodies and neutralizing antibodies, resulting in an excellent SARS-Coronavirus-2 The infection prevention effect was confirmed (Example 1).
상기 "사스-코로나바이러스-2 감염증"이라 함은 사스-코로나바이러스-2 자체의 감염뿐만 아니라, 상기 바이러스의 감염으로부터 발생되는 여러가지 병증 (예를 들어, 호흡기 질환, 폐렴 등)을 포함한다.The term "SARS-coronavirus-2 infection" includes not only infection with SARS-coronavirus-2 itself, but also various symptoms (eg, respiratory disease, pneumonia, etc.) resulting from infection with the virus.
본 발명서 사용되는 용어 "예방"은 백신 조성물의 투여에 의해 사스-코로나바이러스-2 감염증을 억제시키거나 발병을 지연시키는 모든 행위를 나타낸다.The term "prevention" used in the present invention refers to any action that suppresses or delays the onset of SARS-coronavirus-2 infection by administration of a vaccine composition.
본 발명의 백신 조성물은 면역학적 유효량의 RBD 나노파티클을 포함한다. "면역학적 유효량"은 단일 용량 또는 일련의 용량의 일부로서 개체로의 상기 양의 투여가 예방에 효과적임을 의미한다. 이러한 양은 치료되는 개체의 건강 및 신체 상태, 치료되는 개체의 연령, 분류 집단(예를 들어, 비-인간 영장류, 영장류 등), 항체를 합성하는 개체의 면역계의 능력, 요망되는 보호의 정도, 백신의 제형화, 치료 의사의 의학적 상태의 평가, 및 다른 관련 요인에 따라 변한다. 상기 양은 비교적 광범위한 범위 내에 해당될 것이며, 이는 통상적인 시험을 통해 결정될 수 있음이 예상된다. 투여량 처리는 단일 용량 스케줄 또는 다수 용량 스케줄(예를 들어, 부스터 용량을 포함함)일 수 있다. 조성물은 다른 면역조절 작용제와 함께 투여될 수 있다.The vaccine composition of the present invention contains an immunologically effective amount of RBD nanoparticles. "Immunologically effective amount" means that administration of said amount to a subject as a single dose or as part of a series of doses is effective for prophylaxis. Such amount may depend on the health and physical condition of the individual being treated, the age of the individual being treated, the taxonomic population (eg, non-human primates, primates, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the vaccine formulation, assessment of the medical condition of the treating physician, and other relevant factors. It is expected that these amounts will fall within a relatively wide range and can be determined through routine testing. Dosage treatment can be a single dose schedule or a multiple dose schedule (eg, including booster doses). The composition may be administered with other immunomodulatory agents.
상기 백신 조성물은 항원으로서 상기 RBD 나노파티클을 1회 투여량으로 5㎍ 내지 40㎍, 6㎍ 내지 38㎍, 7㎍ 내지 36㎍, 8㎍ 내지 34㎍, 9㎍ 내지 32㎍, 9㎍ 내지 30㎍, 10㎍ 내지 28㎍, 10㎍ 내지 26㎍, 10㎍ 내지 25㎍으로 포함할 수 있고, 10㎍ 내지 25㎍으로 포함할 수 있다. 상기 RBD 나노파티클을 1회 투여량으로 10㎍ 내지 25㎍으로 포함하면, 항원단백질의 안정성 및 균질성이 증가하여 IgG항체 및 중화항체를 효과적으로 유도하며 그에 따른 우수한 사스-코로나바이러스-2 감염증 예방 효과를 확인하였다(실시예 1). 상기 해당하는 용량보다 높은 용량에서 과도한 면역 반응이 발생할 수 있다. 상기 해당하는 용량 미만에서는 유의하지 않은 면역원성을 나타낼 수 있다.The vaccine composition contains 5 μg to 40 μg, 6 μg to 38 μg, 7 μg to 36 μg, 8 μg to 34 μg, 9 μg to 32 μg, 9 μg to 30 μg to 30 μg of the RBD nanoparticles as an antigen per dose. μg, 10 μg to 28 μg, 10 μg to 26 μg, 10 μg to 25 μg, or 10 μg to 25 μg. When 10 μg to 25 μg of the RBD nanoparticles are included in a single dose, the stability and homogeneity of the antigenic protein are increased to effectively induce IgG antibodies and neutralizing antibodies, thereby providing excellent SARS-coronavirus-2 infection prevention effect confirmed (Example 1). Excessive immune response may occur at doses higher than the corresponding dose. Less than the corresponding dose may exhibit insignificant immunogenicity.
본 명세서 전반에서, RBD 나노파티클의 함량은 RBD 나노파티클 전체 중량이 아닌 재조합 코로나-19 표면항원단백질(RBD)로서의 중량으로 기술하였다 ([표 3] 참조). 본 발명의 RBD 나노파티클에 있어서, 재조합 코로나-19 표면항원단백질(RBD)는 RBD 나노파티클 중량의 37%에 해당한다. [표 3]에 나타낸 바와 같이, 본 발명의 일 구체예에서 RBD 나노파티클 전체 중량이 54ug인 경우 RBD 중량은 20ug이고, RBD 나노파티클 전체 중량이 135ug인 경우 RBD 중량은 50ug임을 기술하고 있으므로, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자는 RBD 나노파티클 중량으로부터 RBD 중량을, RBD 중량으로부터 RBD 나노파티클 중량을 용이하게 환산할 수 있다. Throughout this specification, the content of RBD nanoparticles was described as the weight of recombinant COVID-19 surface antigen protein (RBD) rather than the total weight of RBD nanoparticles (see [Table 3]). In the RBD nanoparticles of the present invention, the recombinant Corona-19 surface antigen protein (RBD) corresponds to 37% of the RBD nanoparticle weight. As shown in [Table 3], in one embodiment of the present invention, when the total weight of RBD nanoparticles is 54ug, the RBD weight is 20ug, and when the total weight of RBD nanoparticles is 135ug, the RBD weight is 50ug. Those skilled in the art can easily convert the RBD weight from the RBD nanoparticle weight and the RBD nanoparticle weight from the RBD weight.
상기 RBD 나노파티클의 1회 용량은 0.5ml 중에 제형화될 수 있다. A single dose of the RBD nanoparticles can be formulated in 0.5 ml.
상기 백신 조성물의 투여 횟수는 1회 이상, 바람직하게는 2회이며, 1차 접종 4주 내지 8주 후에 2차 접종을 수행하는 것일 수 있다.The number of administrations of the vaccine composition is one or more times, preferably twice, and the second inoculation may be performed 4 to 8 weeks after the first inoculation.
상기 백신 조성물은 부스터(booster) 백신으로 투여될 수 있다. "부스터"는 백신에 의한 예방접종시 첫번째 항원 자극 이후에 접종되는 두번째 항원 자극을 나타내며, 항체가 생성될 때, 첫번째 항원 자극 후에 나타나는 반응에 비해서 두번째 항원 자극에 의해 보다 빠르고, 높은 항체 생성율을 달성하게 된다. 본 발명에서 "부스터 백신"은 상기 백신 조성물의 1차 또는 2차 접종 후 접종되며, 예를 들어 3차 접종에 해당할 수 있으나, 이에 제한되는 것은 아니며 1차 접종 이후 회차인, 면역 증강 효과를 나타낼 수 있는 접종을 넓게 의미한다.The vaccine composition may be administered as a booster vaccine. "Booster" refers to the second antigen stimulation inoculated after the first antigen stimulation during vaccination with a vaccine, and when antibodies are produced, a faster and higher antibody production rate is achieved by the second antigen stimulation compared to the reaction that appears after the first antigen stimulation. will do In the present invention, the "booster vaccine" is inoculated after the first or second inoculation of the vaccine composition, and may correspond to, for example, the third inoculation, but is not limited thereto, and the immune enhancing effect after the first inoculation It means broadly the inoculation that can be expressed.
1차 접종 및 부스터 접종을 포함한 백신 접종 일정은, 필요에 따라, 수일, 수주, 수년의 과정에 걸쳐 계속 진행될 수 있다. 백신 접종 일정상 백신은 백신 요법의 시작시에 가장 높은 빈도로 투여되며, 부스터 효과를 위한 부스터 백신의 투여는 점차 빈도를 줄일 수 있다.Vaccination schedules, including primary inoculations and booster inoculations, may continue over the course of days, weeks, or years, as needed. Due to the vaccination schedule, vaccines are administered at the highest frequency at the beginning of the vaccine regimen, and for booster effects, the administration of booster vaccines can be gradually reduced in frequency.
상기 백신 조성물은 상기 부스터 백신 1회 용량 이상을 포함할 수 있고, 1회, 2회, 3회, 4회 또는 5회 용량 이상을 포함할 수 있다. 본 발명의 백신 조성물은 대상체에서 사스-코로나바이러스-2 감염증을 예방하기 위해 사용될 수 있다. 본 발명에서 사용되는 용어 "대상체"는 동물을 나타낸다. 동물은 포유 동물일 수 있다. 포유 동물은, 예를 들어, 영장류(예를 들어, 인간, 남성 또는 여성, 유아), 소, 양, 염소, 말, 개, 고양이, 토끼, 래트, 마우스, 어류, 조류 등을 나타내며, 영장류일 수 있다.The vaccine composition may include one or more doses of the booster vaccine, and may include one, two, three, four or five or more doses. The vaccine composition of the present invention can be used to prevent SARS-coronavirus-2 infection in a subject. As used herein, the term "subject" refers to an animal. An animal may be a mammal. Mammal refers to, for example, a primate (eg, human, male or female, infant), cow, sheep, goat, horse, dog, cat, rabbit, rat, mouse, fish, bird, etc. can
본 발명의 일 구체예에서, 상기 부스터 백신의 투여 대상은 이전에 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물이 1회 또는 2회 투여되고/거나 투여되었던 대상체일 수 있다. 상기 백신 조성물은 본 발명에 따른 백신 조성물일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the subject for administration of the booster vaccine may be a subject who has been previously administered and / or administered a vaccine composition for preventing or treating SARS-CoV-2 infection once or twice. The vaccine composition may be a vaccine composition according to the present invention, but is not limited thereto.
병원체 감염은 신체의 다양한 영역에 영향을 미치고, 따라서 본 발명의 백신 조성물은 다양한 형태로 제조될 수 있다. 예를 들어, 조성물은 액체 용액 또는 현탁액으로 주사 가능하게 제조될 수 있다. 주사 전 액체 비히클 내 용액 또는 현탁액에 적합한 고체 형태가 또한 제조될 수 있다. 조성물은, 예를 들어, 연고, 크림 또는 분말로서 국소 투여를 위해 제조될 수 있다. 조성물은, 예를 들어, 정제 또는 캡슐, 또는 시럽(임의로 착향된 시럽)으로서 경구 투여를 위해 제조될 수 있다. 조성물은 미세 분말 또는 스프레이를 이용하여, 예를 들어, 흡입기로서 폐 투여를 위해 제조될 수 있다. 조성물은 좌약 또는 페서리로서 제조될 수 있다. 조성물은, 예를 들어, 점적, 스프레이 또는 분말로서 코, 귀 또는 눈 투여를 위해 제조될 수 있다. 조성물은 함수제에 포함될 수 있다. 조성물은 동결 건조될 수 있다.Pathogen infection affects various areas of the body, and therefore the vaccine composition of the present invention can be prepared in a variety of forms. For example, the composition may be prepared for injectability as a liquid solution or suspension. Solid forms suitable for solution or suspension in liquid vehicles prior to injection may also be prepared. The composition may be prepared for topical administration, for example as an ointment, cream or powder. The composition may be prepared for oral administration, for example as a tablet or capsule, or as a syrup (optionally flavored syrup). The composition may be prepared for pulmonary administration using a fine powder or spray, eg as an inhaler. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, otic or ocular administration, for example as drops, sprays or powders. The composition may be included in a hydration agent. The composition may be lyophilized.
본 발명의 백신 조성물은 진피내, 근육내, 복막내, 정맥내, 비강, 경막외(eidural) 또는 다른 적절한 경로를 통하여 투여를 수행할 수 있으며, 근육 주사용으로 제조되어 근육내 경로로 투여될 수 있다.The vaccine composition of the present invention may be administered through intradermal, intramuscular, intraperitoneal, intravenous, intranasal, epidural or other appropriate routes, and may be prepared for intramuscular injection and administered through the intramuscular route. can
본 발명의 조성물은 보존제로 2-페녹시에탄올, 포름알데히드, 및 이들의 혼합물로 이루어지는 군으로부터 선택되는 하나를 포함할 수 있고, 2-페녹시에탄올을 포함할 수 있다. The composition of the present invention may include one selected from the group consisting of 2-phenoxyethanol, formaldehyde, and mixtures thereof as a preservative, and may include 2-phenoxyethanol.
2. 사스-코로나바이러스-2 감염증 예방 또는 치료용 키트2. Kits for the prevention or treatment of SARS-CoV-2 infection
본 발명의 다른 측면은, 서열번호 1의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제1 폴리펩타이드 단량체가 3개 조립된 삼량체(trimer); 및 서열번호 2의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제2 폴리펩타이드 단량체가 5개 조립된 오량체(pentamer)를 포함하는 RBD 나노파티클, 및 면역증강제를 포함하는 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물과, 사용설명서를 포함하는 사스-코로나바이러스-2 감염증 예방 또는 치료용 키트를 제공한다.Another aspect of the present invention is a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto, a trimer in which three are assembled; and an RBD nanoparticle comprising a pentamer assembled of 5 second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer. A vaccine composition for preventing or treating coronavirus-2 infection and a kit for preventing or treating SARS-coronavirus-2 infection including instructions for use are provided.
상기 면역증강제는 수산화알루미늄를 포함할 수 있다. 면역증강제에 대한 설명 및 수산화알루미늄의 함량에 대해서는 상기 "1. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물"의 설명 부분에 기재하였으므로 상세한 설명을 생략한다.The immune enhancer may include aluminum hydroxide. The description of the immune enhancer and the content of aluminum hydroxide are described in the description of "1. Vaccine composition for preventing or treating SARS-coronavirus-2 infection", so detailed descriptions are omitted.
상기 백신 조성물은 완충제 및 안정제를 추가로 포함할 수 있다. 완충제 및 안정제의 종류 및 함량에 대해서는 상기 "1. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물"의 설명 부분에 기재하였으므로 상세한 설명을 생략한다.The vaccine composition may further include a buffer and a stabilizer. The types and contents of buffers and stabilizers are described in the description of "1. Vaccine composition for preventing or treating SARS-CoV-2 infection", so detailed descriptions are omitted.
3. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 부스터(booster) 조성물3. Vaccine booster composition for preventing or treating SARS-CoV-2 infection
본 발명의 또 다른 측면은, 서열번호 1의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제1 폴리펩타이드 단량체가 3개 조립된 삼량체(trimer); 및 서열번호 2의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제2 폴리펩타이드 단량체가 5개 조립된 오량체(pentamer)를 포함하는 RBD(Receptor Binding Domain) 나노파티클, 및 면역증강제를 포함하는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 부스터(booster) 조성물을 제공한다.Another aspect of the present invention is a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto, a trimer in which three are assembled; and an RBD (Receptor Binding Domain) nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer It provides a vaccine booster composition for preventing or treating SARS-coronavirus-2 infection comprising a.
상기 RBD 나노파티클에 대한 설명 및 투여량에 대해서는 상기 "1. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물"의 설명 부분에 기재하였으므로 상세한 설명을 생략한다.The description and dosage of the RBD nanoparticles are described in the description of "1. Vaccine composition for preventing or treating SARS-coronavirus-2 infection", so detailed descriptions are omitted.
상기 면역증강제는 수산화알루미늄를 포함할 수 있다. 면역증강제에 대한 설명 및 수산화알루미늄의 함량에 대해서는 상기 "1. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물"의 설명 부분에 기재하였으므로 상세한 설명을 생략한다.The immune enhancer may include aluminum hydroxide. The description of the immune enhancer and the content of aluminum hydroxide are described in the description of "1. Vaccine composition for preventing or treating SARS-coronavirus-2 infection", so detailed descriptions are omitted.
상기 백신 조성물은 완충제 및 안정제를 추가로 포함할 수 있다. 완충제 및 안정제의 종류 및 함량에 대해서는 상기 "1. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물"의 설명 부분에 기재하였으므로 상세한 설명을 생략한다.The vaccine composition may further include a buffer and a stabilizer. The types and contents of buffers and stabilizers are described in the description of "1. Vaccine composition for preventing or treating SARS-CoV-2 infection", so detailed descriptions are omitted.
본 발명에서 사용되는 "백신 부스터 조성물"은 프라이머(primer) 백신, 즉 먼저 투여된 백신의 치료 효과를 증진, 연장 또는 유지시킬 수 있는 조성물을 의미하며, 프라이머 백신 접종을 마친 후, 일정 간격으로 필요에 따라 투여될 수 있는 조성물이다.As used in the present invention, "vaccine booster composition" means a primer vaccine, that is, a composition capable of enhancing, extending or maintaining the therapeutic effect of a previously administered vaccine, and is required at regular intervals after primer vaccination. It is a composition that can be administered according to.
상기 부스터 조성물은 사스-코로나바이러스-2 감염증 예방 또는 치료용 프라이머(primer) 백신 조성물의 접종 후 적어도 1개월 이후에 투여될 수 있고, 적어도 1개월마다 추가 접종될 수 있다. 예를 들면, 1개월, 2개월, 3개월, 4개월, 5개월, 6개월, 7개월, 8개월, 9개월, 10개월, 11개월, 12개월에 추가 접종될 수 있고, 3개월, 6개월, 9개월, 12개월에 추가 접종될 수 있다. 본 발명의 일 구체예에서, 상기 프라이머 백신 조성물 투여와 본 발명의 부스터 조성물 사이의 투여 간격은, 1주 내지 3주, 13개월 내지 18개월, 2년 내지 20년일 수 있다. 이와 같이, 백신 접종 일정은, 필요에 따라, 수일, 수주, 수년의 과정에 걸쳐 계속 진행될 수 있다. 백신 접종 일정상 백신은 백신 요법의 시작시에 가장 높은 빈도로 투여되며, 부스터 효과를 위한 부스터 백신의 투여는 점차 빈도를 줄일 수 있다.The booster composition may be administered at least one month after inoculation of a primer vaccine composition for preventing or treating SARS-coronavirus-2 infection, and may be additionally inoculated at least every month. For example, boosters may be administered at 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 3 months, 6 months Booster doses may be given at 12 months, 9 months, and 12 months. In one embodiment of the present invention, the interval between administration of the primer vaccine composition and the booster composition of the present invention may be 1 to 3 weeks, 13 to 18 months, and 2 to 20 years. As such, the vaccination schedule may continue over the course of days, weeks, or years, as needed. Due to the vaccination schedule, vaccines are administered at the highest frequency at the beginning of the vaccine regimen, and for booster effects, the administration of booster vaccines can be gradually reduced in frequency.
상기 투여는 1회 이상일 수 있고, 필요에 따라, 수회 추가 투여될 수 있다. 예를 들면, 2회, 3회, 4회, 5회, 6회, 7회, 8회, 9회, 10회로 투여될 수 있다.The administration may be performed one or more times, and additional administration may be performed several times, if necessary. For example, it may be administered 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times.
상기 사스-코로나바이러스-2 감염증 예방 또는 치료용 프라이머 백신은 상기 "1. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물"일 수 있고, 사스-코로나바이러스-2 감염증 예방 또는 치료를 위한 백신이라면 본 발명에 제한없이 적용될 수 있다.The primer vaccine for preventing or treating SARS-CoV-2 infection may be the "1. Vaccine composition for preventing or treating SARS-CoV-2 infection", and a vaccine for preventing or treating SARS-Coronavirus-2 infection. If it can be applied without limitation to the present invention.
상기 백신 부스터(booster) 조성물이 접종되는 대상체, 백신 부스터 조성물 제조 방법, 투여 경로, 백신 부스터 조성물에 포함되는 보존제는 상기 "1. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물"의 설명 부분에 기재된 것과 동일하므로 기재를 생략한다.A subject to be inoculated with the vaccine booster composition, a vaccine booster composition preparation method, an administration route, and a preservative included in the vaccine booster composition are the explanatory part of "1. Vaccine composition for preventing or treating SARS-CoV-2 infection" It is the same as that described in , so the description is omitted.
4. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 부스터 조성물 키트4. Vaccine booster composition kit for preventing or treating SARS-CoV-2 infection
본 발명의 또 다른 측면은, 상기 "1. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물" 및 "3. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 부스터 조성물"을 포함하는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 부스터 조성물 키트를 제공한다. 상기 백신 조성물은 1차 이상, 바람직하게는 1차 및 2차 접종에 필요한 용량을 포함할 수 있고, 상기 백신 부스터 조성물은 2차 이상, 바람직하게는 3차 접종에 필요한 용량을 포함할 수 있으며, 나아가 사스-코로나바이러스-2에 대한 면역원성을 증진시키기 위해서, 그 이상의 추가 용량을 선택적으로 포함할 수 있다.Another aspect of the present invention, including the "1. vaccine composition for preventing or treating SARS-coronavirus-2 infection" and "3. vaccine booster composition for preventing or treating SARS-coronavirus-2 infection", SARS -Provides a vaccine booster composition kit for preventing or treating coronavirus-2 infection. The vaccine composition may include doses required for the first or second inoculation, preferably the first and second inoculations, and the vaccine booster composition may include the doses required for the second or more, preferably the third inoculation, Furthermore, in order to enhance immunogenicity against SARS-coronavirus-2, more additional doses may be optionally included.
5. 사스-코로나바이러스-2 감염증을 예방하거나 치료하는 방법5. How to prevent or treat SARS-CoV-2 infection
본 발명의 또 다른 측면은, 서열번호 1의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제1 폴리펩타이드 단량체가 3개 조립된 삼량체(trimer); 및 서열번호 2의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제2 폴리펩타이드 단량체가 5개 조립된 오량체(pentamer)를 포함하는 RBD(Receptor Binding Domain) 나노파티클, 및 면역증강제를 사스-코로나바이러스-2 감염증의 예방 또는 치료가 필요한 대상체에게 투여하는 단계를 포함하는, 대상체에서 사스-코로나바이러스-2 감염증을 예방하거나 치료하는 방법을 제공한다. Another aspect of the present invention is a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto, a trimer in which three are assembled; and an RBD (Receptor Binding Domain) nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer Provides a method for preventing or treating SARS-coronavirus-2 infection in a subject, comprising administering to a subject in need of prevention or treatment of SARS-coronavirus-2 infection.
상기 대상체에서 사스-코로나바이러스-2 감염증을 예방하거나 치료하는 방법은, 상기 "1. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물" 또는 "3. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 부스터 조성물"을 투여하는 단계를 포함할 수 있다. The method for preventing or treating SARS-coronavirus-2 infection in the subject, the above "1. SARS-coronavirus-2 infection prevention or treatment vaccine composition" or "3. SARS-coronavirus-2 infection prevention or treatment It may include the step of administering a "vaccine booster composition".
백신 조성물 및 백신 부스터 조성물에 관한 설명은, 상기 "1. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물" 및 "3. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 부스터 조성물" 항목에 기재된 바와 같다. The description of the vaccine composition and vaccine booster composition is given in the above "1. Vaccine composition for preventing or treating SARS-coronavirus-2 infection" and "3. Vaccine booster composition for preventing or treating SARS-coronavirus-2 infection". As described.
6. 백신 조성물 또는 백신 부스터 조성물의 사스-코로나바이러스-2 감염증 예방 또는 치료 용도6. Use of vaccine composition or vaccine booster composition for prevention or treatment of SARS-CoV-2 infection
본 발명의 또 다른 측면은, 서열번호 1의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제1 폴리펩타이드 단량체가 3개 조립된 삼량체(trimer); 및 서열번호 2의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제2 폴리펩타이드 단량체가 5개 조립된 오량체(pentamer)를 포함하는 RBD(Receptor Binding Domain) 나노파티클, 및 면역증강제를 포함하는 백신 조성물 또는 백신 부스터 조성물의 사스-코로나바이러스-2 감염증을 예방 또는 치료하기 위한 용도를 제공한다. Another aspect of the present invention is a first polypeptide monomer comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto, a trimer in which three are assembled; and an RBD (Receptor Binding Domain) nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer It provides a use for preventing or treating SARS-coronavirus-2 infection of a vaccine composition or vaccine booster composition comprising a.
백신 조성물 및 백신 부스터 조성물에 관한 설명은, 상기 "1. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물" 및 "3. 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 부스터 조성물" 항목에 기재된 바와 같다. The description of the vaccine composition and vaccine booster composition is given in the above "1. Vaccine composition for preventing or treating SARS-coronavirus-2 infection" and "3. Vaccine booster composition for preventing or treating SARS-coronavirus-2 infection". As described.
실시예Example
이하, 본 발명을 하기 실시예에 의하여 더욱 상세하게 설명한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for exemplifying the present invention, and the scope of the present invention is not limited only to these.
제조예 1. RBD 나노파티클의 제조 Preparation Example 1. Preparation of RBD nanoparticles
SARS-CoV-2 바이러스의 표면의 삼량체(trimer) 당단백질은 스파이크 당단백질이라고도 불리며, 삼량체 당단백질의 상단에 있는 수용체 결합도메인(Receptor binding domain, RBD)이 인체 세포 표면의 ACE2 수용체 단백질에 결합함으로써 감염을 일으킨다. 상기 RBD는 나노파티클과 조합되어 RBD 나노파티클로 제조된다. 상기 RBD 나노파티클은 20개의 RBD-Component A와 12개의 Component B의 자가조립(self-assemble)에 의하여 형성된다. 상기 RBD-Component A는 서열번호 1로 표시되는 아미노산 서열을 갖는 RBD-I53-50A 단량체가 3개 조립된 삼량체이며, RBD-I53-50A 단량체는 SARS-CoV-2 스파이크 당단백질의 RBD, 나노입자의 구조체 역할을 하는 I53-50A, RBD와 I53-50A를 연결하는 링커 (GGSGGSGSGGSGGSGSEKAAKAEEAAR)로 구성된다. 또한, 상기 Component B는 서열번호 2로 표시되는 아미노산 서열을 갖는 I53-50B의 오량체(pentamer)이다.The trimer glycoprotein on the surface of the SARS-CoV-2 virus is also called spike glycoprotein, and the receptor binding domain (RBD) at the top of the trimer glycoprotein binds to the ACE2 receptor protein on the surface of human cells. By combining, they cause infection. The RBD is combined with nanoparticles to produce RBD nanoparticles. The RBD nanoparticles are formed by self-assembly of 20 RBD-Component A and 12 Component B. The RBD-Component A is a trimer in which three RBD-I53-50A monomers having the amino acid sequence represented by SEQ ID NO: 1 are assembled, and the RBD-I53-50A monomer is the RBD of SARS-CoV-2 spike glycoprotein, nano It consists of I53-50A, which serves as a particle structure, and a linker (GGSGGSGSGGSGGSGSEKAAKAEEAAR) connecting RBD and I53-50A. In addition, the component B is a pentamer of I53-50B having an amino acid sequence represented by SEQ ID NO: 2.
재조합 나노파티클 코로나바이러스 백신을 제조하기 위해, RBD-Component A 단백질 제조에는 CHO 세포주(Stable cell line)를 사용하고, Component B 단백질 제조를 위해 대장균 세포주를 사용하였으며, 각 성분의 제조에 대해서는 하기 제조예 1-1 및 1-2에 상세하게 설명하였다.To prepare a recombinant nanoparticle coronavirus vaccine, a CHO cell line (Stable cell line) was used for RBD-Component A protein production, and an E. coli cell line was used for Component B protein production. For the production of each component, the following Preparation Examples 1-1 and 1-2 were described in detail.
1-1. RBD-Component A 단백질 제조1-1. Preparation of RBD-Component A protein
SARS-CoV-2 백신의 항원으로 사용되는 RBD-Component A 생산을 위해 영국 HORIZON Discovery 사에서 분양 받은 HD-BIOP3 세포주를 숙주세포로 사용하였다. 또한, Donor plasmid (pJV145)에 SalI/NotI 제한 효소 반응을 이용해 합성한 RBD-Component A cDNA (NT-SARS-CoV-2RBD-I53-50A-16GS-he)를 삽입하여 재조합 발현 벡터 plasmid (M-2560)를 제작하였다. 상기 M-2560 벡터는 서열번호 18로 표시되는 DNA 서열을 갖는다. 재조합 발현 벡터 plasmid (M-2560) 내 주요 유전자 위치 및 기능에 대한 정보는 표 1에 요약되어 있다.For the production of RBD-Component A used as an antigen for SARS-CoV-2 vaccine, the HD-BIOP3 cell line distributed from HORIZON Discovery, UK was used as a host cell. In addition, by inserting RBD-Component A cDNA (NT-SARS-CoV-2RBD-I53-50A-16GS-he) synthesized using SalI/NotI restriction enzyme reaction into Donor plasmid (pJV145), a recombinant expression vector plasmid (M- 2560) was produced. The M-2560 vector has a DNA sequence represented by SEQ ID NO: 18. Information on the location and function of key genes in the recombinant expression vector plasmid (M-2560) is summarized in Table 1.
| 발현 벡터 내 유전자 위치Location of genes in expression vectors | 정보information |
| Nucleotide position 440-593Nucleotide position 440-593 | SV40 early promoterSV40 early promoter |
| Nucleotide position 668-1,789Nucleotide position 668-1,789 | GS geneGS gene |
| Nucleotide position 2,257-2,319Nucleotide position 2,257-2,319 | 3' PiggyBac transposon-specific right ITR (inverted terminal repeat sequence)3' PiggyBac transposon-specific right ITR (inverted terminal repeat sequence) |
| Nucleotide position 2,951-2,985Nucleotide position 2,951-2,985 | 5' PiggyBac transposon-specific left ITR5' PiggyBac transposon-specific left ITR |
| Nucleotide position 3,275-4,477Nucleotide position 3,275-4,477 | cHS4 insulator genecHS4 insulator gene |
| Nucleotide position 4,484-4,955Nucleotide position 4,484-4,955 | mCMV promotermCMV promoter |
| Nucleotide position 6,264-7,667Nucleotide position 6,264-7,667 | NT-SARS-CoV-2RBD-I53-50A-16GS-heNT-SARS-CoV-2RBD-I53-50A-16GS-he |
CHO 세포주 유래 Component A 중간체 원액은 통상의 단백질 발현 및 정제 방법에 의해 얻었다. CHO 세포주 유래 Component A 중간체 원액에 대한 peptide mapping 분석 결과 100 %의 peptide coverage를 확보하였으며 예상되는 펩티드 서열과 상호 동등함을 확인하여, 중간체 원액이 목적하는 Component A 단백질임을 확인하였다.The CHO cell line-derived Component A intermediate stock solution was obtained by conventional protein expression and purification methods. As a result of peptide mapping analysis on the CHO cell line-derived Component A intermediate stock solution, 100% of the peptide coverage was secured and mutual equivalence to the expected peptide sequence was confirmed, confirming that the intermediate stock solution was the desired Component A protein.
1-2. Component B 단백질 제조1-2. Component B protein manufacturing
Component B 단백질 제조를 위해 Thermofisher 사에서 분양받은 대장균 BL21 균주를 사용하였다. Component B 단백질 발현을 위한 벡터로 pET29b+를 사용하였다. pET29b+에 Component B 단백질의 유전자가 삽입되면 총 5.7kb의 서열 (벡터: 5.4kb)의 I53-50B 벡터가 제조된다. 상기 벡터는 카나마이신 저항성 유전자 및 락토즈(Lactose) 및 그 유도체에 의해 전사가 유도되는 Lac 오페론(Operon) 및 람다 파지유래의 T7 프로모터(Promoter) 영역, 다중클로닝부위 (Multiple cloning site) 등으로 구성되어 있다. 다중클로닝 부위에는 여러 제한효소 서열을 가지고 있어서 항원단백질의 도입을 위한 주요한 역할을 하는 부위이다. 상기 I53-50B 벡터는 서열번호 19로 표시되는 DNA 서열을 갖는다. I53-50B 벡터 내 주요 유전자 정보를 하기 표 2에 나타낸다.For the preparation of Component B protein, E. coli BL21 strain distributed by Thermofisher was used. pET29b+ was used as a vector for expressing Component B protein. When the Component B protein gene is inserted into pET29b+, an I53-50B vector with a total sequence of 5.7 kb (vector: 5.4 kb) is produced. The vector is composed of a kanamycin resistance gene, a Lac operon whose transcription is induced by lactose and its derivatives, a T7 promoter region derived from lambda phage, and a multiple cloning site. there is. The multicloning site has several restriction enzyme sequences, so it is a site that plays a major role in the introduction of antigenic proteins. The I53-50B vector has a DNA sequence represented by SEQ ID NO: 19. The main gene information in the I53-50B vector is shown in Table 2 below.
| 발현 벡터 내 유전자 위치Location of genes in expression vectors | 정보information |
| Nucleotide position 12-467Nucleotide position 12-467 | f1 originf1 origin |
| Nucleotide position 560-1375Nucleotide position 560-1375 | KanRKanR |
| Nucleotide position 1471-2144Nucleotide position 1471-2144 | pUCoriginpUCorigin |
| Nucleotide position 4598-4675Nucleotide position 4598-4675 | LacI promoterLacI promoter |
| Nucleotide position 4988-5007Nucleotide position 4988-5007 | T7promotorT7promotor |
| Nucleotide position 5007-5031Nucleotide position 5007-5031 | Lac operatorLac operator |
| Nucleotide position 5076-5549Nucleotide position 5076-5549 | I53-50BI53-50B |
| Nucleotide position 5645-5688Nucleotide position 5645-5688 | T7terminatorT7terminator |
대장균 유래 Component B 중간체 원액은 통상 단백질 발현 및 정제 방법에 의해 얻었다. Component B 중간체 원액의 아미노산 서열은 이론적 아미노산 서열과 100 % 일치함을 확인하였다.E. coli-derived Component B intermediate stock solutions were obtained by conventional protein expression and purification methods. It was confirmed that the amino acid sequence of the Component B intermediate stock solution was 100% consistent with the theoretical amino acid sequence.
1-3. 재조합 RBD 나노파티클 원액 제조1-3. Preparation of recombinant RBD nanoparticle stock solution
제조된 Component A 중간체 원액 및 Component B 중간체 원액을 이용하여 조립 공정을 통해 재조합 RBD 나노파티클 원액(원료의약품)을 제조하였다. A recombinant RBD nanoparticle stock solution (drug substance) was prepared through an assembly process using the prepared Component A intermediate stock solution and Component B intermediate stock solution.
1-3-1. 재조합 RBD 나노파티클 원액 제조1-3-1. Preparation of recombinant RBD nanoparticle stock solution
Component A 원액 및 Component B 의 반응 비율이 몰 농도 기준으로 1 : 1.1 이 되도록 혼합하여 조립하였다. 이때 반응조건은 상온, 1시간, 80 rpm의 교반속도로 반응하며 반응이 완료되면 반응액은 0.2 μm 필터로 여과하였다. 여과된 assembly 반응액을 0.7m2 면적의 300kDa MWCO(Molecular weight cut-off) 멤브레인이 장착된 접선유동여과(Tangential flow filtration)시스템을 이용하여 4 kg으로 농축(Ultrafiltration)하고, 농축액 10배 부피의 50 mM 트리스 완충액을 이용하여 완충액교환(Diafiltration)을 실시하여 회수하였다. 이 때, 막간차압(Transmembrane pressure, TMP)은 1 bar 이하로 유지하였다. 한외여과 회수액을 0.2 μm 필터로 여과하여 -70℃ 이하 초저온 냉동고에서 보관하였다. Component A stock solution and Component B were mixed and assembled so that the reaction ratio was 1:1.1 on a molar basis. At this time, the reaction conditions were room temperature, 1 hour, reaction at a stirring speed of 80 rpm, and when the reaction was completed, the reaction solution was filtered through a 0.2 μm filter. The filtered assembly reaction solution was concentrated (Ultrafiltration) to 4 kg using a tangential flow filtration system equipped with a 300 kDa MWCO (Molecular weight cut-off) membrane with an area of 0.7 m2, It was recovered by carrying out buffer exchange (Diafiltration) using mM Tris buffer. At this time, the transmembrane pressure (TMP) was maintained at 1 bar or less. The ultrafiltration recovered liquid was filtered through a 0.2 μm filter and stored in a cryogenic freezer below -70°C.
1-3-2. 재조합 RBD 나노파티클 원액의 입자 형태 확인1-3-2. Particle morphology confirmation of recombinant RBD nanoparticle stock solution
재조합 RBD 나노파티클 원액은 Component A 중간체 원액 및 Component B 중간체 원액의 결합에 의해 형성되는 입자 형태의 분자이다. 이러한 입자 형태의 분자는 컴퓨터적으로 설계되어 적절한 결합이 발생하였을 경우 일정한 크기의 분자를 형성하게 된다. 단백질의 분자크기를 측정하는 방법 중, 일반적으로 이용되고 있는 방법은 동적광산란(Dynamic Light Scattering) 방법으로 이를 통해 형성된 나노파티클 분자 크기를 측정하여 적절한 구조를 형성하였는지 확인할 수 있다. Component A 중간체 원액 및 Component B 중간체 원액을 이용하여 조립 공정을 통해 제조된 나노파티클 원액의 크기를 동적광산란법에 의해 측정한 결과 이론적 예측 수치인 40 ~ 70 nm의 크기를 갖는 것을 확인하였다.The recombinant RBD nanoparticle stock solution is a particle-type molecule formed by combining the Component A intermediate stock solution and the Component B intermediate stock solution. Molecules in the form of these particles are computer-designed to form molecules of a certain size when appropriate bonding occurs. Among the methods for measuring the molecular size of a protein, a commonly used method is a dynamic light scattering method, which measures the molecular size of nanoparticles formed through this method to confirm whether an appropriate structure has been formed. As a result of measuring the size of the nanoparticle stock solution prepared through the assembly process using the Component A intermediate stock solution and the Component B intermediate stock solution by the dynamic light scattering method, it was confirmed to have a theoretically predicted size of 40 to 70 nm.
1-4. RBD 나노파티클 알럼 제형의 제조1-4. Preparation of RBD nanoparticle alum formulations
제형(formulation) 공정을 통해서 최종 원액을 제조하였다. 상기 제형 공정에서 면역 증강제로 수산화알루미늄(Aluminum hydroxide)을 사용하였다. 수산화알루미늄은 최종 원액에 미리 혼합하였다. 최종 원액은 RBD 나노파티클 알럼 제형으로 명명하였다. A final stock solution was prepared through a formulation process. In the formulation process, aluminum hydroxide was used as an immune enhancer. Aluminum hydroxide was pre-mixed into the final stock solution. The final stock solution was named RBD nanoparticle alum formulation.
실시예 1. RBD 나노파티클 알럼 제형의 면역원성 확인Example 1. Confirmation of immunogenicity of RBD nanoparticle alum formulations
19세 내지 85세의 80명의 성인을 대상으로 제조예 1에 따른 RBD 나노파티클 알럼 제형의 면역원성을 확인하였다. 연구 개입(study intervention)은 본 연구 프로토콜에 따른 연구 참가자들에게 부여된다고 여겨지는 임의의 조사 개입, 제품, 플라시보 또는 의료 장비로 정의된다. 하기 표 3에 1mL당 원료의약품의 분량 및 규격을 나타내고, 표 4에 개입된 원료의약품의 정보를 나타낸다.Immunogenicity of the RBD nanoparticle alum formulation according to Preparation Example 1 was confirmed in 80 adults aged 19 to 85 years. A study intervention is defined as any investigational intervention, product, placebo, or medical device considered to be afforded to study participants pursuant to this study protocol. Table 3 below shows the amount and specifications of drug substances per 1 mL, and Table 4 shows information on drug substances involved.
| 배합목적Blending purpose | 원료명raw material name | 분량amount | 규격standard | |
|
고함량 (25 μg /dose1))high content (25 μg/dose 1) ) |
저함량 (10 μg/dose1))low content (10 μg/dose 1) ) |
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| 주성분chief ingredient | 재조합 코로나-19 표면 항원 단백질 나노파티클(제조예 1의 RBD 나노파티클)Recombinant COVID-19 surface antigen protein nanoparticles (RBD nanoparticles of Preparation Example 1) |
135 μg (50 μg1))135 µg (50 μg 1) ) |
54 μg (20 μg1))54 µg (20 μg 1) ) |
자사company |
| 완충제buffer | 염화나트륨sodium chloride | 8.106 mg8.106mg | 8.106 mg8.106mg | EPEP |
| 완충제buffer | 트로메타민tromethamine | 5.601 mg5.601mg | 5.601 mg5.601mg | EPEP |
| 안정제stabilizator | L-아르기닌L-Arginine | 16.11 mg16.11mg | 16.11 mg16.11mg | EPEP |
| 안정제stabilizator | 백당per hundred | 46.24 mg46.24mg | 46.24 mg46.24mg | EPEP |
| 흡착제(면역증강제)Adsorbent (Immune Booster) | 수산화알루미늄aluminum hydroxide | 1,500 μg1,500 µg | 1,500 μg1,500 µg | EPEP |
| 용제solvent | 주사용수water for injection | 적량Appropriate amount | 적량Appropriate amount | KPKP |
1) 재조합 코로나-19 표면항원단백질(RBD)로서 분량 1) Amount as recombinant COVID-19 surface antigen protein (RBD)
| 개입 명칭name of the intervention | SARS-CoV-2 재조합 단백질 나노파티클 백신(RBD 나노파티클)-알럼(Alum)SARS-CoV-2 Recombinant Protein Nanoparticle Vaccine (RBD Nanoparticle) - Alum |
| 유형category | 백신vaccine |
| 용량(dose) 제형Dose Formulation | SARS-CoV-2의 외부 표면에 드러난 RBD, 즉 자가조립 단백질 나노파티클RBD, self-assembling protein nanoparticles, revealed on the outer surface of SARS-CoV-2 |
| 1회 투여량1 dose | 알럼으로 보조된 RBD 나노파티클(RBD 10 μg 또는 25 μg/용량); 총 주사 부피는 0.5mL임.RBD nanoparticles supplemented with alum (RBD 10 μg or 25 μg/dose); Total injection volume is 0.5 mL. |
| 정량(dosage) 수준Dosage level | SARS-CoV-2 RBD 나노파티클: 10 μg 또는 25 μg수산화알루미늄: 750 μgSARS-CoV-2 RBD nanoparticles: 10 μg or 25 μg Aluminum hydroxide: 750 μg |
| 투여 경로route of administration | 근육내 주사intramuscular injection |
| 보관 조건storage conditions | 2~8℃2~8℃ |
백신 접종 연구는 식염수 플라시보(0.5mL) 또는 RBD 나노파티클 알럼 제형(RBD 10 μg 또는 25 μg, 총 주사 부피는 0.5mL)를 근육내로 2회 주사하는 것을 포함한다. 0.5mL을 RBD 나노파티클 알럼 제형의 약 0.65mL을 함유하는 바이알 또는 생리식염수의 20mL 앰플로부터 빼내었다. 상기 바이알 또는 앰플 내의 잔여 부피는 폐기하였다. 본 백신 연구는 팔 윗쪽의 어깨세모근에 28일 간격으로 0일차, 28일차에 주사하였다.Vaccination studies included two intramuscular injections of saline placebo (0.5 mL) or RBD nanoparticle alum formulation (RBD 10 μg or 25 μg, total injection volume 0.5 mL). 0.5 mL was withdrawn from a vial containing about 0.65 mL of the RBD nanoparticle alum formulation or a 20 mL ampoule of physiological saline. The remaining volume in the vial or ampoule was discarded. This vaccine study was injected into the deltoid muscle of the upper arm every 28 days on day 0 and day 28.
표 5에 알럼 제형의 실험 대상을 나타낸다. 표 5의 배정된 군(intention-to-treat set, ITT군), 안정성군(safety set), 모든 분석 대상자군(full analysis set, FA군), 계획서 순응 임상시험대상자군(per protocol set, PP군)은 의약품 임상시험에서 널리 알려진 분석 모집단이다. 분석 모집단은 임상시험에 참여한 피험자 중에 통계분석에 포함되는 피험자들의 집단을 의미한다.Table 5 shows test subjects of alum formulations. In Table 5, the assigned group (intention-to-treat set, ITT group), safety group (safety set), all analysis subjects (full analysis set, FA group), protocol-compliant clinical trial subject group (per protocol set, PP) group) is a widely known analysis population in drug clinical trials. The analysis population refers to a group of subjects included in statistical analysis among subjects participating in a clinical trial.
|
RBD 나노파티클 알럼 제형 10 μgRBD nanoparticle alum formulation 10 µg |
RBD 나노파티클 알럼 제형 25 μgRBD nanoparticle alum formulation 25 µg |
플라시보placebo | 합계Sum | |
| 무작위배정, nrandomization, n | 2020 | 2020 | 2020 | 6060 |
| ITT군, n (%)ITT group, n (%) | 20 (100.00)20 (100.00) | 20 (100.00)20 (100.00) | 20 (100.00)20 (100.00) | 60 (100.00)60 (100.00) |
| 안정성군, n (%)Stability group, n (%) | 20 (100.00)20 (100.00) | 20 (100.00)20 (100.00) | 20 (100.00)20 (100.00) | 60 (100.00)60 (100.00) |
| FA군, n (%)FA group, n (%) | 20 (100.00)20 (100.00) | 20 (100.00)20 (100.00) | 20 (100.00)20 (100.00) | 60 (100.00)60 (100.00) |
| PP군으로부터 제외된 기준, n (%)Criterion for exclusion from PP group, n (%) | 2 (10.00)2 (10.00) | 0 (0.00)0 (0.00) | 1 (5.00)1 (5.00) | 3 (5.00)3 (5.00) |
| 혈액 샘플 중 어느 하나가 채취되지 못했거나, 7차 방문까지 적절한 시간대에 채취되지 못 함Any of the blood samples were not collected, or were not collected in an appropriate time frame by Visit 7 | 2 (10.00)2 (10.00) | 0 (0.00)0 (0.00) | 1 (5.00)1 (5.00) | 3 (5.00)3 (5.00) |
| PP군, n (%)PP group, n (%) | 18 (90.00)18 (90.00) | 20 (100.00)20 (100.00) | 19 (95.00)19 (95.00) | 57 (95.00)57 (95.00) |
|
ITT군 (%):(ITT군의 수/무작위배정된 참가자의 수)*100 안정성군(%):(안전성군의 수/ITT군의 수)*100 FA군(%):(FA군의 수/안전성군의 수)*100 PP군으로부터 제외된 기준(%):(PP군으로부터 제외된 각 기준의 수/FA군의 수)*100 PP군(%):(PP군의 수/ FA군의 수)*100ITT group (%): (number of ITT group/number of randomized participants)*100 Stability group (%): (number of safety groups/number of ITT groups)*100 FA group (%): (number of FA groups/number of safety groups)*100 Criteria excluded from the PP group (%): (number of criteria excluded from the PP group/number of FA groups)*100 PP group (%): (Number of PP group/ Number of FA group)*100 |
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1-1. IgG 항체의 기하평균역가 및 혈청변환율로 면역원성 확인1-1. Immunogenicity confirmed by geometric mean titer and seroconversion rate of IgG antibody
상기 표 5에 기재된 실험 대상에 RBD 나노파티클 알럼 제형 10μg 및 25 μg를 각각 투여한 후 ELISA로 SARS-CoV-2-RBD에 대한 IgG 항체의 기하평균역가(Geometric Mean titer) 및 혈청변환율(Seroconversion rate)을 확인하였다. 기하평균역가(Geometric mean titer, GMT)는 모든 값을 곱하고 이 수치의 n차 루트 값을 취해(여기서 n은 가용한 자료가 있는 시험대상자의 수), 시험대상자 집단에 대한 평균 항체 역가를 계산하는 방법이다. 기하평균증가비율(Geometric Mean Fold Rise, GMFR)은 백신 접종 전 베이스라인 대비 백신 접종 후 기하평균농도 또는 기하평균역가의 증가비율을 나타낸다. 95% 신뢰구간(confidence interval, CI)이며, 신뢰구간은 모집단 평균과 목표값의 차이가 될 수 있는 값의 범위를 제공한다. 표 6에 저함량 RBD 나노파티클 알럼 제형 및 고함량 RBD 나노파티클 알럼 제형에 대한 IgG 항체의 기하평균역가 및 혈청변환율을 나타내며, 표 6에서 "P-값 vs 플라시보"의 결과의 "t"는 "t-검정(t-test)"결과를 나타낸다. t-검정은 평균을 비교할 수 있는 통계 가설 검정이다.After administering 10 μg and 25 μg of the RBD nanoparticle alum formulation, respectively, to the experimental subjects described in Table 5, the geometric mean titer and seroconversion rate of the IgG antibody against SARS-CoV-2-RBD were determined by ELISA. ) was confirmed. Geometric mean titer (GMT) is a method of calculating the mean antibody titer for a population of subjects by multiplying all values and taking the nth root of this number, where n is the number of subjects for whom data are available. way. Geometric Mean Fold Rise (GMFR) represents the increase in the geometric mean concentration or geometric mean titer after vaccination compared to the baseline before vaccination. It is a 95% confidence interval (CI), and the confidence interval provides a range of values that can be the difference between the population mean and the target value. Table 6 shows the geometric mean titer and seroconversion rate of the IgG antibody for the low-content RBD nanoparticle alum formulation and the high-content RBD nanoparticle alum formulation, and in Table 6, "t" in the result of "P-value vs placebo" is "t" -Test (t-test)" shows the results. A t-test is a statistical hypothesis test that allows means to be compared.
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저함량 RBD 나노파티클 알럼 제형 10 μg (N=18)low content RBD nanoparticle alum formulation 10 μg (N=18) |
고함량 RBD 나노파티클 알럼 제형 25 μg (N=20)high content RBD nanoparticle alum formulation 25 μg (N=20) |
플라시보(N=19)Placebo (N=19) | |
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기하평균역가(GMT) 및 혈청변환율 베이스라인 Geometric mean titer (GMT) and seroconversion rate baseline |
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| nn | 1818 | 2020 | 1919 |
| GMT±표준 편차GMT ± standard deviation | 12.95±1.6812.95±1.68 | 13.43±1.5913.43±1.59 | 12.45±1.6912.45±1.69 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (10.01, 16.75)(10.01, 16.75) | (10.81, 16.69)(10.81, 16.69) | (9.67, 16.03)(9.67, 16.03) |
| P-값 vs 플라시보P-value vs placebo | 0.8195 t0.8195t | 0.6348 t0.6348 t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.8201 t0.8201t | ||
| 4차 방문(1차 백신 접종 4주 후)Visit 4 (4 weeks after vaccination 1) | |||
| nn | 1818 | 2020 | 1919 |
| GMT±표준 편차GMT ± standard deviation | 75.37±2.7175.37±2.71 | 69.91±1.8969.91±1.89 | 11.40±1.7711.40±1.77 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (45.93, 123.68)(45.93, 123.68) | (51.94, 94.11)(51.94, 94.11) | (8.66, 15.02)(8.66, 15.02) |
| P-값 vs 플라시보P-value vs placebo | <0.0001 t<0.0001 t | <0.0001 t<0.0001 t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.7810 t0.7810t | ||
| GMFR±표준 편차GMFR ± standard deviation | 5.82±3.405.82±3.40 | 5.21±2.065.21±2.06 | 0.92±1.400.92±1.40 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (3.17, 10.69)(3.17, 10.69) | (3.71, 7.31)(3.71, 7.31) | (0.78, 1.08)(0.78, 1.08) |
| P-값 vs 플라시보P-value vs placebo | <0.0001 t<0.0001 t | <0.0001 t<0.0001 t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.7385 t0.7385t | ||
| 6차 방문(2차 백신 접종 2주 후)Visit 6 (2 weeks after 2nd vaccination) | |||
| nn | 1818 | 2020 | 1919 |
| GMT±표준 편차GMT ± standard deviation | 718.99±2.43718.99±2.43 | 736.67±1.85736.67±1.85 | 9.11±1.599.11±1.59 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (462.13, 1,118.60)(462.13, 1,118.60) | (552.45, 982.31)(552.45, 982.31) | (7.28, 11.39)(7.28, 11.39) |
| P-값 vs 플라시보P-value vs placebo | <0.0001 t<0.0001 t | <0.0001 t<0.0001 t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.9218 t0.9218t | ||
| GMFR±표준 편차GMFR ± standard deviation | 55.52±3.0255.52±3.02 | 54.85±2.1754.85±2.17 | 0.73±1.570.73±1.57 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (32.05, 96.18)(32.05, 96.18) | (38.16, 78.84)(38.16, 78.84) | (0.59, 0.91)(0.59, 0.91) |
| P-값 vs 플라시보P-value vs placebo | <0.0001 t<0.0001 t | <0.0001 t<0.0001 t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.9686 t0.9686 t | ||
| 7차 방문(2차 백신 접종 4주 후)Visit 7 (4 weeks after 2nd vaccination) | |||
| nn | 1818 | 2020 | 1919 |
| GMT±표준 편차GMT ± standard deviation | 587.33±2.42587.33±2.42 | 576.60±1.86576.60±1.86 | 9.41±1.589.41±1.58 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (378.09, 912.37)(378.09, 912.37) | (431.40, 770.67)(431.40, 770.67) | (7.54, 11.74)(7.54, 11.74) |
| P-값 vs 플라시보P-value vs placebo | <0.0001 t<0.0001 t | <0.0001 t<0.0001 t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.9407 t0.9407 t | ||
| GMFR±표준 편차GMFR ± standard deviation | 45.35±3.0945.35±3.09 | 42.93±2.1442.93±2.14 | 0.76±1.520.76±1.52 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (25.88, 79.48)(25.88, 79.48) | (30.06, 61.32)(30.06, 61.32) | (0.62, 0.93)(0.62, 0.93) |
| P-값 vs 플라시보P-value vs placebo | <0.0001 t<0.0001 t | <0.0001 t<0.0001 t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.8601 t0.8601t | ||
표 6에 기재된 바와 같이, 본 발명의 RBD 나노파티클 알럼 제형을 1차 및 2차 백신 접종한 후 IgG 항체의 GMT가 베이스라인 대비 현저히 증가하였다. RBD 나노파티클 알럼 제형 저함량 및 고함량 모두, GMT가 2차 접종 2주 후 베이스라인 대비 크게 증가하였고, 2차 접종 후 4주가 경과되어도 증가된 GMT가 유지되는 것이 확인되었다. 상기 수치는 영국 국립바이오의약품연구소(NIBSC)의 회복기 환자 혈청 (WHO Reference Panel, First WHO International Reference Panel for anti-SARS-CoV-2 immunoglubulin, NIBSC code: 20/268)과 비교 시 상당히 높은 수준의 항체가로서, 본 발명의 백신 조성물은 사스-코로나바이러스-2를 대상으로 하는 백신으로서의 효과가 현저히 우수한 것을 확인하였다.As shown in Table 6, after the first and second vaccination with the RBD nanoparticle alum formulation of the present invention, the GMT of the IgG antibody significantly increased compared to the baseline. In both low and high RBD nanoparticle alum formulations, it was confirmed that GMT increased significantly compared to the baseline 2 weeks after the second inoculation, and the increased GMT was maintained even after 4 weeks after the second inoculation. The above level is a significantly higher level of antibody compared to the sera of convalescent patients (WHO Reference Panel, First WHO International Reference Panel for anti-SARS-CoV-2 immunoglubulin, NIBSC code: 20/268) of the National Institute of Biomedicine (NIBSC) in the UK. As a, it was confirmed that the vaccine composition of the present invention is remarkably effective as a vaccine against SARS-coronavirus-2.
이를 통해, RBD 나노파티클 알럼 제형의 2차 투여로 인해 IgG 항체의 GMT가 증가되므로 RBD 나노파티클 알럼 제형을 SARS-CoV-2 감염증 예방 또는 치료용 백신 조성물의 유효성분으로 유용하게 사용할 수 있는 것을 알 수 있다.From this, it can be seen that the second administration of the RBD nanoparticle alum formulation increases the GMT of the IgG antibody, so that the RBD nanoparticle alum formulation can be usefully used as an active ingredient in a vaccine composition for preventing or treating SARS-CoV-2 infection. can
1-2. 중화 항체의 기하평균역가 및 혈청변환율로 면역원성 확인1-2. Immunogenicity confirmed by geometric mean titer of neutralizing antibody and seroconversion rate
표 5에 기재된 실험 대상에 RBD 나노파티클 알럼 제형 10μg 및 25 μg를 각각 투여한 후 ELISA로 SARS-CoV-2-RBD에 대한 중화 항체의 기하평균역가(Geometric Mean titer) 및 혈청변환율(Seroconversion rate)을 확인하였다. 표 7에 저함량 RBD 나노파티클 알럼 제형 및 고함량 RBD 나노파티클 알럼 제형에 대한 중화 항체의 기하평균역가 및 혈청변환율을 나타낸다. 중화 항체는 바이러스와 결합하여 세포로 침투하지 못하도록 무력화시키는 항체이므로 중화항체가는 면역원성 확인에 중요한 지표이다.Geometric mean titer and seroconversion rate of neutralizing antibody against SARS-CoV-2-RBD by ELISA after administration of 10 μg and 25 μg of RBD nanoparticle alum formulation, respectively, to the experimental subjects listed in Table 5 confirmed. Table 7 shows the geometric mean titers and seroconversion rates of neutralizing antibodies for the low-content RBD nanoparticle alum formulation and the high-content RBD nanoparticle alum formulation. Neutralizing antibodies are important indicators for confirming immunogenicity because neutralizing antibodies bind to viruses and neutralize them from penetrating into cells.
| RBD 나노파티클 알럼 제형 10 μg (N=18)RBD nanoparticle alum formulation 10 μg (N=18) | RBD 나노파티클 알럼 제형 25 μg(N=20)RBD nanoparticle alum formulation 25 μg (N=20) | 플라시보(N=19)Placebo (N=19) | |
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기하평균역가(GMT) 및 혈청변환율 베이스라인 Geometric mean titer (GMT) and seroconversion rate baseline |
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| nn | 1818 | 2020 | 1919 |
| GMT±표준 편차GMT ± standard deviation | 18.71±1.5018.71±1.50 | 20.01±1.6520.01±1.65 | 20.31±1.7020.31±1.70 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (15.29, 22.89)(15.29, 22.89) | (15.82, 25.32)(15.82, 25.32) | (15.71, 26.25)(15.71, 26.25) |
| P-값 vs 플라시보P-value vs placebo | 0.6024 t0.6024t | 0.9306 t0.9306t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.6532 t0.6532t | ||
| 4차 방문(1차 백신 접종 4주 후)Visit 4 (4 weeks after vaccination 1) | |||
| nn | 1818 | 2020 | 1919 |
| GMT±표준 편차GMT ± standard deviation | 49.08±3.1649.08±3.16 | 40.50±2.5040.50±2.50 | 24.73±2.2024.73±2.20 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (27.69, 87.00)(27.69, 87.00) | (26.35, 62.23)(26.35, 62.23) | (16.93, 36.13)(16.93, 36.13) |
| P-값 vs 플라시보P-value vs placebo | 0.0407 t0.0407 t | 0.0805 t0.0805t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.5708 t0.5708 t | ||
| GMFR±표준 편차GMFR ± standard deviation | 2.62±3.222.62±3.22 | 2.02±2.482.02±2.48 | 1.22±2.781.22±2.78 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (1.47, 4.69)(1.47, 4.69) | (1.32, 3.10)(1.32, 3.10) | (0.74, 1.99)(0.74, 1.99) |
| P-값 vs 플라시보P-value vs placebo | 0.0403 t0.0403 t | 0.1091 t0.1091t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.4468 t0.4468 t | ||
| 6차 방문(2차 백신 접종 2주 후)Visit 6 (2 weeks after 2nd vaccination) | |||
| nn | 1818 | 2020 | 1919 |
| GMT±표준 편차GMT ± standard deviation | 530.90±2.70530.90±2.70 | 708.50±2.83708.50±2.83 | 21.58±1.7921.58±1.79 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (323.85, 870.33)(323.85, 870.33) | (435.50, 1,152.66)(435.50, 1,152.66) | (16.30, 28.57)(16.30, 28.57) |
| P-값 vs 플라시보P-value vs placebo | <0.0001 t<0.0001 t | <0.0001 t<0.0001 t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.3889 t0.3889 t | ||
| GMFR±표준 편차GMFR ± standard deviation | 28.38±2.9228.38±2.92 | 35.40±3.1135.40±3.11 | 1.06±2.321.06±2.32 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (16.64, 48.40)(16.64, 48.40) | (20.83, 60.18)(20.83, 60.18) | (0.71, 1.60)(0.71, 1.60) |
| P-값 vs 플라시보P-value vs placebo | <0.0001 t<0.0001 t | <0.0001 t<0.0001 t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.5423 t0.5423 t | ||
| 7차 방문(2차 백신 접종 4주 후)Visit 7 (4 weeks after 2nd vaccination) | |||
| nn | 1818 | 2020 | 1919 |
| GMT±표준 편차GMT ± standard deviation | 317.72±3.22317.72±3.22 | 402.75±2.52402.75±2.52 | 19.21±1.3419.21±1.34 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (177.75, 567.91)(177.75, 567.91) | (261.39, 620.56)(261.39, 620.56) | (16.69, 22.12)(16.69, 22.12) |
| P-값 vs 플라시보P-value vs placebo | <0.0001 t<0.0001 t | <0.0001 t<0.0001 t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.4898 t0.4898 t | ||
| GMFR±표준 편차GMFR ± standard deviation | 16.99±3.4516.99±3.45 | 20.12±2.8420.12±2.84 | 0.95±1.900.95±1.90 |
| 95% CI (하한, 상한)95% CI (lower limit, upper limit) | (9.18, 31.43)(9.18, 31.43) | (12.34, 32.81)(12.34, 32.81) | (0.69, 1.29)(0.69, 1.29) |
| P-값 vs 플라시보P-value vs placebo | <0.0001 t<0.0001 t | <0.0001 t<0.0001 t | |
| P-값 vs RBD 나노파티클 알럼 제형 25 μgP-Value vs. RBD Nanoparticle Alum Formulation 25 μg | 0.6497 t0.6497 t | ||
표 7에 기재된 바와 같이, 본 발명의 RBD 나노파티클 알럼 제형을 1차 및 2차 백신 접종한 후 중화 항체의 GMT가 베이스라인 대비 현저히 증가하였다. RBD 나노파티클 알럼 제형 저함량 및 고함량 모두, GMT가 2차 접종 2주 후 베이스라인 대비 크게 증가하였고, 2차 접종 후 4주가 경과되어도 증가된 GMT가 유지되는 것이 확인되었다. 구체적으로, 본 발명의 백신 조성물은 기존 백신의 효과 대비, RBD 나노파티클 10 μg 또는 25 μg 접종시 백신 접종 6주차에 중화 항체의 GMT가 530.90±2.70, 708.50±2.83 (IU/ml)까지 증가하였다. 상기 수치는 영국 국립바이오의약품연구소(NIBSC)의 회복기 환자 혈청 (WHO Reference Panel, First WHO International Reference Panel for anti-SARS-CoV-2 immunoglubulin, NIBSC code: 20/268)과 비교 시 상응하거나 보다 높은 수준의 항체가로서, 본 발명의 백신 조성물은 사스-코로나바이러스-2를 대상으로 하는 백신으로서의 효과가 현저히 우수한 것을 확인하였다.As shown in Table 7, after the first and second vaccination with the RBD nanoparticle alum formulation of the present invention, the GMT of the neutralizing antibody was significantly increased compared to the baseline. In both low and high RBD nanoparticle alum formulations, it was confirmed that GMT increased significantly compared to the baseline 2 weeks after the second inoculation, and the increased GMT was maintained even after 4 weeks after the second inoculation. Specifically, the vaccine composition of the present invention increased the GMT of neutralizing antibodies to 530.90±2.70 and 708.50±2.83 (IU/ml) at 6 weeks after vaccination when 10 μg or 25 μg of RBD nanoparticles were inoculated, compared to the effects of conventional vaccines. . These values are equivalent or higher than those of convalescent patient sera (WHO Reference Panel, First WHO International Reference Panel for anti-SARS-CoV-2 immunoglubulin, NIBSC code: 20/268) of the National Institute of Biomedicine (NIBSC) in the UK. As an antibody titer of , it was confirmed that the vaccine composition of the present invention is remarkably effective as a vaccine against SARS-coronavirus-2.
이를 통해, RBD 나노파티클 알럼 제형의 2차 투여로 인해 중화 항체의 GMT가 증가되므로 RBD 나노파티클 알럼 제형을 SARS-CoV-2 감염증 예방 또는 치료용 백신 조성물의 유효성분으로 유용하게 사용할 수 있는 것을 알 수 있다.Through this, it can be seen that the second administration of the RBD nanoparticle alum formulation increases the GMT of neutralizing antibodies, so that the RBD nanoparticle alum formulation can be usefully used as an active ingredient in a vaccine composition for preventing or treating SARS-CoV-2 infection. can
Claims (16)
- 서열번호 1의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제1 폴리펩타이드 단량체가 3개 조립된 삼량체(trimer); 및 서열번호 2의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제2 폴리펩타이드 단량체가 5개 조립된 오량체(pentamer)를 포함하는 RBD(Receptor Binding Domain) 나노파티클, 및 면역증강제를 포함하는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물.A trimer in which three first polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto are assembled; and an RBD (Receptor Binding Domain) nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer A vaccine composition for preventing or treating SARS-coronavirus-2 infection comprising a.
- 제1항에 있어서,According to claim 1,상기 면역증강제가 수산화알루미늄을 포함하는 것인, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물.The vaccine composition for preventing or treating SARS-coronavirus-2 infection, wherein the immune enhancer contains aluminum hydroxide.
- 제2항에 있어서,According to claim 2,상기 면역증강제가 수산화알루미늄인 경우, 상기 RBD 나노파티클 20 μg 또는 50 μg에 대해, 수산화알루미늄이 1000 μg 내지 2000 μg으로 포함되는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물.When the adjuvant is aluminum hydroxide, aluminum hydroxide is contained in an amount of 1000 μg to 2000 μg with respect to 20 μg or 50 μg of the RBD nanoparticles, a vaccine composition for preventing or treating SARS-coronavirus-2 infection.
- 제1항에 있어서,According to claim 1,상기 백신 조성물은 완충제 및 안정제를 추가로 포함하는 것인, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물.The vaccine composition further comprises a buffer and a stabilizer, a vaccine composition for preventing or treating SARS-coronavirus-2 infection.
- 제4항에 있어서,According to claim 4,상기 완충제는 염화나트륨 및 트로메타민 중 적어도 하나를 포함하는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물.Wherein the buffer comprises at least one of sodium chloride and tromethamine, a vaccine composition for preventing or treating SARS-coronavirus-2 infection.
- 제4항에 있어서,According to claim 4,상기 안정제는 L-아르기닌 및 백당 중 적어도 하나를 포함하는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물.The stabilizer comprises at least one of L-arginine and sucrose, a vaccine composition for preventing or treating SARS-coronavirus-2 infection.
- 제1항 내지 제6항 중 어느 한 항에 있어서,According to any one of claims 1 to 6,상기 백신 조성물에, 상기 RBD 나노파티클 20 μg 또는 50 μg에 대하여, 수산화알루미늄이 1000 μg 내지 2000 μg으로 포함되고, 염화나트륨이 1 mg 내지 20 mg으로 포함되며, 트로메타민이 0.1 mg 내지 10 mg으로 포함되고, L-아르기닌이 8 mg 내지 24 mg으로 포함되며, 백당이 35 mg 내지 55 mg으로 포함되는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물.The vaccine composition contains 1000 μg to 2000 μg of aluminum hydroxide, 1 mg to 20 mg of sodium chloride, and 0.1 mg to 10 mg of tromethamine, based on 20 μg or 50 μg of the RBD nanoparticles. A vaccine composition for preventing or treating SARS-coronavirus-2 infection, comprising 8 mg to 24 mg of L-arginine and 35 mg to 55 mg of white sugar.
- 제1항에 있어서,According to claim 1,상기 백신 조성물이 항원으로서 상기 RBD 나노파티클을 1회 투여량으로 5μg 내지 40μg 포함하는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물.A vaccine composition for preventing or treating SARS-coronavirus-2 infection, wherein the vaccine composition comprises 5 μg to 40 μg of the RBD nanoparticles as an antigen in a single dose.
- 제1항에 있어서,According to claim 1,상기 RBD 나노파티클의 1회 용량이 0.5ml 중에 제형화되는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물.A vaccine composition for preventing or treating SARS-coronavirus-2 infection, wherein a single dose of the RBD nanoparticles is formulated in 0.5 ml.
- 제1항에 있어서,According to claim 1,상기 백신 조성물의 투여 횟수는 2회이며, 1차 접종 4주 내지 8주 후에 2차 접종을 수행하는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물.The number of administrations of the vaccine composition is twice, and the second vaccination is performed 4 to 8 weeks after the first vaccination, a vaccine composition for preventing or treating SARS-coronavirus-2 infection.
- 제1항에 있어서,According to claim 1,상기 백신 조성물이 근육내 투여되는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물.A vaccine composition for preventing or treating SARS-coronavirus-2 infection, wherein the vaccine composition is administered intramuscularly.
- 서열번호 1의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제1 폴리펩타이드 단량체가 3개 조립된 삼량체(trimer); 및 서열번호 2의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제2 폴리펩타이드 단량체가 5개 조립된 오량체(pentamer)를 포함하는 RBD 나노파티클, 및 면역증강제를 포함하는, 사스-코로나바이러스-2 감염증 예방 또는 치료용 백신 조성물과, 사용설명서를 포함하는 사스-코로나바이러스-2 감염증 예방 또는 치료용 키트.A trimer in which three first polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto are assembled; And an RBD nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer, SARS - A vaccine composition for preventing or treating coronavirus-2 infection and a kit for preventing or treating SARS-coronavirus-2 infection, including instructions for use.
- 제12항에 있어서,According to claim 12,상기 면역증강제가 수산화알루미늄을 포함하는 것인, 사스-코로나바이러스-2 감염증 예방 또는 치료용 키트.The kit for preventing or treating SARS-coronavirus-2 infection, wherein the immune enhancer contains aluminum hydroxide.
- 제12항에 있어서,According to claim 12,상기 백신 조성물은 완충제 및 안정제를 추가로 포함하는 것인, 사스-코로나바이러스-2 감염증 예방 또는 치료용 키트.The vaccine composition further comprises a buffer and a stabilizer, SARS-coronavirus-2 infection prevention or treatment kit.
- 서열번호 1의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제1 폴리펩타이드 단량체가 3개 조립된 삼량체(trimer); 및 서열번호 2의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제2 폴리펩타이드 단량체가 5개 조립된 오량체(pentamer)를 포함하는 RBD(Receptor Binding Domain) 나노파티클, 및 면역증강제를 사스-코로나바이러스-2 감염증의 예방 또는 치료가 필요한 대상체에게 투여하는 단계를 포함하는, 대상체에서 사스-코로나바이러스-2 감염증을 예방하거나 치료하는 방법. A trimer in which three first polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto are assembled; and an RBD (Receptor Binding Domain) nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer A method for preventing or treating SARS-coronavirus-2 infection in a subject, comprising administering to a subject in need of prevention or treatment of SARS-coronavirus-2 infection.
- 서열번호 1의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제1 폴리펩타이드 단량체가 3개 조립된 삼량체(trimer); 및 서열번호 2의 아미노산 서열 또는 이와 적어도 75% 동일성을 갖는 아미노산 서열을 포함하는 제2 폴리펩타이드 단량체가 5개 조립된 오량체(pentamer)를 포함하는 RBD(Receptor Binding Domain) 나노파티클, 및 면역증강제를 포함하는 백신 조성물 또는 백신 부스터 조성물의 사스-코로나바이러스-2 감염증을 예방 또는 치료하기 위한 용도. A trimer in which three first polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75% identity thereto are assembled; and an RBD (Receptor Binding Domain) nanoparticle comprising a pentamer in which five second polypeptide monomers comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 75% identity thereto, and an immune enhancer Use of a vaccine composition or vaccine booster composition comprising a for preventing or treating SARS-coronavirus-2 infection.
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