WO2022164208A1 - Protéine de fusion du coronavirus-2 du sars et composition immunogène la comprenant - Google Patents

Protéine de fusion du coronavirus-2 du sars et composition immunogène la comprenant Download PDF

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WO2022164208A1
WO2022164208A1 PCT/KR2022/001416 KR2022001416W WO2022164208A1 WO 2022164208 A1 WO2022164208 A1 WO 2022164208A1 KR 2022001416 W KR2022001416 W KR 2022001416W WO 2022164208 A1 WO2022164208 A1 WO 2022164208A1
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fusion protein
protein
immunogenic composition
sars
region
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PCT/KR2022/001416
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Korean (ko)
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류동균
김판겸
나완근
노한미
박근수
임복현
조경민
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(주)셀트리온
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a SARS-coronavirus-2 fusion protein and an immunogenic composition comprising the same, and more particularly, to a SARS-coronavirus-2 (SARS-CoV-2) spike protein (S protein) and It relates to a fusion protein comprising the constant region of an immunoglobulin molecule and an immunogenic composition comprising the same.
  • SARS-CoV-2 SARS-CoV-2
  • S protein spike protein
  • HCoV human coronaviruses
  • HCoV-229E and HCoV-NL63 belong to alphaCoV and cause common respiratory infections.
  • HCoV-OC43, SARS-CoV, and MERS-CoV belong to beta-coronavirus (betaCoV), which also induces upper respiratory infections such as colds and digestive disorders.
  • SARS-CoV and MERS-CoV belonging to betaCoV cause severe respiratory infectious diseases characterized by acute respiratory symptoms.
  • SARS-Coronavirus-2 severe Acute Respiratory Syndrome Coronavirus-2, SARS-CoV-2
  • SARS-CoV-2 severe Acute Respiratory Syndrome Coronavirus-2, SARS-CoV-2
  • betaCoV betaCoV as a result of homology analysis of a virus isolated from atypical pneumonia patients in Wuhan, China. It has been found, and it has high infectivity and transmission ability, and causes acute respiratory symptoms, leading to death. About 90 million people have been infected worldwide and about 1.9 million have died.
  • SARS-coronavirus-2 consists of about 30,000 RNAs, and the SARS-coronavirus-2 spike protein binds to the cell's ACE2 receptor (angiotensin converting enzyme 2), resulting in SARS-coronavirus- The bivalent enters the cell and causes viral replication. Crown-shaped projections (spikes) are characteristically expressed on the surface of the coronavirus.
  • the spike protein consists of about 1,200 amino acids, and consists of a spike 1 (S1) and a spike 2 domain (S2 domain). Functionally, S1 is known to be involved in cell receptor binding and S2 is involved in fusion. It is known that the SARS-coronavirus-2 genome encodes not only structural proteins such as spike proteins, but also proteins involved in viral replication and infection, such as viral polymerases and proteolytic enzymes.
  • Vaccines currently used to prevent COVID-19 are largely mRNA vaccines, and virus vectored vaccines are being used with emergency use approval.
  • the mRNA vaccine is a vaccine in which the mRNA encoding the spike protein is mixed with lipids, etc., and is administered intramuscularly to induce an immune response by expressing the spike protein in the human body.
  • the viral vector vaccine is a vaccine in which the adenovirus surface protrusion is replaced with the SARS-coronavirus-2 spike protein.
  • these two vaccines are novel vaccines and their efficacy and safety have been verified through clinical trials. Meanwhile, the use of vaccines due to virus mutations is also emerging.
  • An object of the present invention is to provide a fusion protein comprising a spike protein (S protein) of SARS-CoV-2 and a constant region of an immunoglobulin molecule.
  • Another problem to be solved by the present invention is to provide a nucleic acid molecule encoding the fusion protein.
  • Another problem to be solved by the present invention is to provide an expression vector comprising the nucleic acid molecule.
  • Another problem to be solved by the present invention is to provide a host cell transformed with the above expression vector.
  • Another problem to be solved by the present invention is to provide a method for preparing the fusion protein.
  • Another problem to be solved by the present invention is to provide an immunogenic composition comprising the fusion protein.
  • Another problem to be solved by the present invention is to provide a kit comprising the above immunogenic composition.
  • Another problem to be solved by the present invention is to provide a method of inducing an immune response by administering the immunogenic composition.
  • Another problem to be solved by the present invention is to provide a method for preventing SARS-coronavirus infection (COVID-19) by administering the immunogenic composition.
  • the present invention provides a fusion protein comprising a spike protein (S protein) of SARS-CoV-2 and a constant region of an immunoglobulin molecule.
  • the spike protein comprises: i) a full length S protein or a fragment thereof; ii) S1 protein or fragment thereof; iii) S2 protein or fragment thereof; Or iv) a receptor binding domain (RBD) region or a fragment thereof, but is not limited thereto.
  • the full-length S protein or S1 protein may include a receptor binding domain (RBD) region.
  • the spike protein comprises: i) a fusion protein of a) a S1 protein or a fragment thereof and b) an S2 protein or a fragment thereof; or ii) a) a fusion protein of a receptor binding domain (RBD) region or a fragment thereof and b) an S2 protein or a fragment thereof, but is not limited thereto.
  • a fusion protein of a) a S1 protein or a fragment thereof and b) an S2 protein or a fragment thereof or ii) a) a fusion protein of a receptor binding domain (RBD) region or a fragment thereof and b) an S2 protein or a fragment thereof, but is not limited thereto.
  • RBD receptor binding domain
  • the spike protein may include any one of SEQ ID NOs: 1 to 4, but is not limited thereto.
  • the constant region (eg, Fc region) of the immunoglobulin molecule may bind to an Fc receptor to enhance immunogenicity.
  • the constant region of the immunoglobulin molecule of the fusion protein according to the present invention may be an immunoglobulin heavy chain constant region, but is not limited thereto.
  • the immunoglobulin may be any one selected from the group consisting of IgG, IgM, IgA, IgD and IgA, but is not limited thereto.
  • the immunoglobulin may be IgG, but is not limited thereto.
  • the IgG may be any one selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, but is not limited thereto.
  • the constant region of the immunoglobulin molecule may be a constant region of an IgG1 heavy chain, but is not limited thereto.
  • the constant portion of the immunoglobulin molecule may include a hinge region, a CH2 domain, and a CH3 domain of IgG1, and in another embodiment, the constant portion of the immunoglobulin molecule includes a CH1 domain of IgG1 may include, but is not limited thereto.
  • the constant region of the immunoglobulin molecule may include an Fc region. In one embodiment, the Fc region can enhance immunogenicity by binding to an Fc receptor.
  • the Fc region may include an Fc variant.
  • the Fc variant may include one or more Fc variants capable of enhancing binding to an Fc receptor.
  • the Fc variant may include one or more Fc variants, which may enhance immunogenicity by enhancing binding to Fc receptors.
  • the Fc variant may include one or more mutations selected from the group consisting of G236A, S239D, A330L, and I332E.
  • the Fc variant may include all of the G236A / S239D / A330L / I332E mutation, but is not limited thereto.
  • the Fc region may include the sequence of SEQ ID NO: 5, but is not limited thereto.
  • the Fc variant may include the sequence of SEQ ID NO: 6, but is not limited thereto.
  • the fusion protein may include any one of SEQ ID NOs: 7 to 18, but is not limited thereto.
  • the spike protein in the fusion protein comprising a spike protein (S protein) of SARS-CoV-2 and a constant region of an immunoglobulin molecule, is a) S1 protein or In the case of a fusion protein of a fragment thereof and b) an S2 protein or a fragment thereof, the sequence of SEQ ID NO: 19 may be included between the S1 protein and the S2 protein, but the sequence is not limited thereto.
  • the present invention also provides a nucleic acid molecule encoding the fusion protein.
  • the present invention provides an expression vector comprising the nucleic acid molecule.
  • the present invention provides a host cell transformed with the expression vector.
  • It provides a method for producing a fusion protein comprising a.
  • the present invention provides an immunogenic composition comprising the fusion protein.
  • the fusion protein may be a monomer, but is not limited thereto.
  • the fusion protein may be a dimer, but is not limited thereto.
  • the dimer may be formed by a disulfide bond in the hinge region of the two fusion proteins, but is not limited thereto.
  • the immunogenic composition according to the present invention may further include an adjuvant, wherein the adjuvant is one selected from the group consisting of a Toll-like receptor 4 (TLR4) agonist, aluminum salt, saponin, and liposome. It may be above, but is not limited thereto.
  • TLR4 Toll-like receptor 4
  • the Toll-like receptor 4 (TLR4) agonist may be at least one selected from the group consisting of Monophosphoryl Lipid A (MPL) and 3D-MPL, but is not limited thereto.
  • the aluminum salt may be at least one selected from the group consisting of aluminum hydroxide, aluminum phosphate, and aluminum sulfate, but is not limited thereto.
  • the saponin may be any one or more selected from the group consisting of QS21, QS17 and QuilA, but is not limited thereto.
  • the present invention provides a kit comprising the immunogenic composition.
  • the present invention provides a method of inducing an immune response by administering the immunogenic composition.
  • the present invention provides a method for preventing SARS-coronavirus infection (COVID-19) by administering the immunogenic composition.
  • a fusion protein comprising a spike protein, S protein of SARS-CoV-2 and a constant region of an immunoglobulin molecule, and an immunogenic composition comprising the same, are SARS-coronavirus It can induce -2 specific neutralizing antibodies as well as significantly increase cell-mediated immune responses. Accordingly, the fusion protein according to the present invention and the immunogenic composition comprising the same can exhibit a long-term sustainable effect as well as a corresponding immune response to the mutant virus, thus preventing SARS-coronavirus infection (COVID-19). It can be useful for prevention.
  • Figure 2 shows the production amount of SARS-coronavirus-2 specific total IgG antibody.
  • the present invention relates to a fusion protein comprising a spike protein (S protein) of SARS-CoV-2 and a constant region of an immunoglobulin molecule.
  • S protein spike protein
  • Fc immunoglobulin heavy chain constant region
  • the spike protein is the spike protein
  • RBD Receptor binding domain
  • the full-length S protein or S1 protein may include a receptor binding domain (RBD) region.
  • the spike protein may include any one of SEQ ID NOs: 1 to 4, but is not limited thereto.
  • the immunoglobulin may be any one selected from the group consisting of IgG, IgM, IgA, IgD and IgA, but is not limited thereto.
  • the immunoglobulin may be IgG, but is not limited thereto.
  • the IgG may be any one selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, but is not limited thereto.
  • the constant region of the immunoglobulin molecule may be a constant region of an IgG1 heavy chain, but is not limited thereto.
  • the constant portion of the immunoglobulin molecule may include a hinge region, a CH2 domain and a CH3 domain of IgG1, the constant portion of the immunoglobulin molecule may further include a CH1 domain of IgG1,
  • the present invention is not limited thereto.
  • the constant region of the immunoglobulin molecule may include an Fc region, but is not limited thereto.
  • the Fc region may enhance immunogenicity by binding to an Fc receptor, but is not limited thereto.
  • the Fc region may include the sequence of SEQ ID NO: 5, but is not limited thereto.
  • the Fc region may include an Fc variant, but is not limited thereto.
  • the Fc variant may include one or more mutations selected from the group consisting of G236A, S239D, A330L, and I332E.
  • the Fc variant may include all G236A/S239D/A330L/I332E mutations, but is not limited thereto.
  • the numbering of the Fc mutation position is according to EU numbering, but is not limited thereto.
  • the Fc variant may include the sequence of SEQ ID NO: 6, but is not limited thereto.
  • the fusion protein may include any one of SEQ ID NOs: 7 to 18, but is not limited thereto.
  • the present invention also provides a nucleic acid molecule encoding the fusion protein.
  • the present invention provides an expression vector comprising the nucleic acid molecule.
  • the present invention provides a host cell transformed with the expression vector.
  • It provides a method for producing a fusion protein comprising a.
  • the present invention provides an immunogenic composition comprising the above fusion protein.
  • the fusion protein may be a monomer, but is not limited thereto.
  • the fusion protein may be a dimer, but is not limited thereto.
  • the dimer may be formed by a disulfide bond in the hinge region of the two fusion proteins, but is not limited thereto.
  • the immunogenic composition according to the present invention may further include an adjuvant, wherein the adjuvant is one selected from the group consisting of a Toll-like receptor 4 (TLR4) agonist, aluminum salt, saponin, and liposome. It may be above, but is not limited thereto.
  • TLR4 Toll-like receptor 4
  • the Toll-like receptor 4 (TLR4) agonist may be at least one selected from the group consisting of Monophosphoryl Lipid A (MPL) and 3D-MPL, but is not limited thereto.
  • the aluminum salt may be at least one selected from the group consisting of aluminum hydroxide, aluminum phosphate, and aluminum sulfate, but is not limited thereto.
  • the saponin is a tripertene glycoside substance widely distributed in plant and marine animal kingdoms, and is an adjuvant mainly accepted in the art.
  • the saponin may be any one or more selected from the group consisting of QS21, QS17 and QuilA, but is not limited thereto.
  • it may be preferably QS21, but is not limited thereto.
  • QS21 uses an extract derived from the bark of Quillaja saponaria by HPLC purification, and is also denoted as acylated 3,28-bisdesmodic triterpene glycosides (1,3).
  • the liposome is a lipid-based oval structure formed of a phospholipid bilayer, and is typically included in a vaccine composition or a pharmaceutical composition to serve as a drug delivery vehicle.
  • the liposome is dimethyldioctadecylammonium bromide (DDA), 1,2-dioleoyl-3-trimethyl ammonium propane (DOTAP), 3 ⁇ -[N-(N′,N) '-Dimethylaminoethane carbamoyl cholesterol (3 ⁇ -[N-(N,N-dimethylaminoethane) carbamoyl cholesterol, DC-Chol), 1,2-dioleoyloxy-3-dimethylammonium propane (DODAP), 1, 2-di-O-octadecenyl-3-triethylammonium propane (1,2-di-O-octadecenyl-3-trimethylammonium propane, DOTMA), 1,2-dimyristoreo
  • DDA dimethyl
  • the liposome is additionally 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (1,2-Dimyristoyl-snglycero-3-phosphorylcholine, DMPC), 1,2-dioleoyl- sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) , 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DS
  • the present invention provides a kit comprising the above immunogenic composition.
  • the present invention provides a method of inducing an immune response by administering the immunogenic composition.
  • the present invention provides a method for preventing SARS-coronavirus infection (COVID-19) by administering the immunogenic composition.
  • S1-Fc, S1-Fc', S2-Fc' fusion proteins were prepared by the following method.
  • the DNA of a fusion protein in which the S protein of SARS-coronavirus-2 and the Fc region of human IgG are bound was synthesized and cloned into an animal cell expression vector, pCT146 vector.
  • the synthesized S1-Fc contains wild-type human IgG Fc, and S1-Fc' and S2-Fc' contain mutant Fc in which amino acids of the human IgG Fc region are mutated (G236A/S239D/A330L/I332E), have.
  • the synthesized S1-Fc, S1-Fc', and S2-Fc' DNA was digested with two restriction enzymes, ie, Nhe I and Pme I, and the pCT146 vector was also digested with the same restriction enzyme.
  • the cleaved S1-Fc, S1-Fc', and S2-Fc' DNA and vector DNA were ligated using T4 ligase.
  • the cloned pCT146 DNA was transformed into E. coli , colony was analyzed, and the S1-Fc, S1-Fc', S2-Fc' genes were inserted into the pCT146 vector bacterial clone (bacteria). clone) was selected.
  • These vectors were named pCT665, pCT666, and pCT667, respectively.
  • the DNA was purified by culturing the selected colonies.
  • Transient transfection was performed using purified high-purity DNA in ExpiCHO-S animal cells, one of the Chinese hamster ovary cell lines (CHO). These ExpiCHO-S cells were fed-batch cultured to produce S1-Fc, S1-Fc', and S2-Fc' fusion proteins. The S1-Fc, S1-Fc', and S2-Fc' proteins released out of the cell were purified using Protein A affinity chromatography.
  • MPL and aluminum hydroxide as adjuvants were prepared in the following manner.
  • Monophosphoryl Lipid A also known as MPL or MPLA
  • MPL Monophosphoryl Lipid A
  • MPLA-SM VacciGrade TM Catalog code: vac-mpla
  • MPL is extracted from lipopolysaccharide (LPS) produced from Re mutant of Salmonella Minnesota R595 strain, lipid A is mainly involved in the endotoxic activity of LPS and MPL is one in which one phosphate group has been removed from lipid A, which refers to a substance that has reduced toxicity and maintained the immune-enhancing effect (Infect Immun. 71 (5): 2498-507, Int Immunol. 14(11): 1325-32, J. Biol. Chem. 257(19): 11808-15, Infect Immun. 79(9): 3576-3587). Like LPS, MPL functions as a Toll-like receptor 4 (TLR4 agonist) and is known to strongly induce a Th1 immune response (Science 316(5831):1628-32, Infect Immun. 75 (12): 5939-46).
  • TLR4 agonist Toll-like receptor 4
  • InvivoGen's alhydrogel adjuvant 2% (Alhydrogel® adjuvant 2%, catlog# vac-alu-250) product was dispensed and used.
  • an aluminum-based adjuvant increases antigen uptake of antigen presenting cells (APCs), induces a Th2 immune response, but does not induce a Th1 immune response (Immunity 33(4): 492-503) .
  • the antigen and fusion protein prepared in Example 1 were mixed with MPL and aluminum hydroxide at a specific concentration using a PBS solution (phosphate-buffered saline) to prepare the immunogenic composition of the present invention.
  • PBS solution phosphate-buffered saline
  • SARS-Coronavirus-2 surface spike (Spike, S) protein domain S1 or S2 domain
  • a fusion protein antigen in which the Fc region of a human IgG1 antibody is bound was immunized by intramuscular injection.
  • orbital blood was collected for serum isolation from mice 2 weeks (14 days) after the second immunization. .
  • S1-Fc in Table 1 is an antigen in which the surface protein S1 domain of SARS-Coronavirus-2 and Fc of a human IgG1 antibody are fused, MPL is monophosphoryl lipid A as an adjuvant (immune adjuvant), alum is aluminum hydroxyl seed (see Examples 1 and 2).
  • S1-Fc' is an antigen in which the surface glycoprotein S1 domain of SARS-coronavirus-2 and Fc of an engineered human IgG1 antibody are fused, MPL is monophosphoryl lipid A as an adjuvant (immune adjuvant) , alum means aluminum hydroxide.
  • S2-Fc' is an antigen in which the surface protein S2 domain of SARS-coronavirus-2 and Fc of an engineered human IgG1 antibody are fused
  • MPL is monophosphoryl lipid A as an adjuvant (immune adjuvant)
  • alum means aluminum hydroxide.
  • the final volume of each composition in groups 1 to 9 was adjusted and administered by 100 ⁇ l.
  • FRNT Fluorescence Reduction Neutralization Test
  • Vero cells (Vero cells) were dispensed in 96-well plates and cell culture was performed for 16-18 hours. Using DMEM medium containing 2% FBS, mouse serum was serially diluted two-fold, and SARS-Coronavirus-2 (3.51 x 10 6 PFU/ml, BetaCoV/Korea/KCDC03/2020, National Resources Bank of Korea) was prepared by diluting to 300 PFU using the same medium. A serum sample prepared by dilution and virus were mixed and reacted for 30 minutes at 37° C. in a CO 2 cell incubator. The serum-virus mixture reacted in this way was added to Vero cells cultured in a 96-well plate, and reacted for 4 hours at 37° C. in a CO 2 cell incubator. After the reaction, PBS was washed and formaldehyde was added to fix the cells at 4oC for 16-18 hours.
  • SARS-Coronavirus-2 3.51 x 10 6 PFU/ml, BetaCoV/Korea/K
  • KPL TrueBlue Peroxidase Substrate (Seracare, Cat:5510-0030) was added to the plate, it was reacted for 30 minutes under dark conditions and room temperature, washed with distilled water, and dried. Foci stained with TrueBlue TS were measured using an immunospot reader (C.T.L.). Percentages were evaluated based on viral infection without serum.
  • the composition containing the S1, S1-Fc, and S1-Fc' antigens and MPL/alum adjuvant showed a significant neutralizing immune response (Table 2, FIG. 1).
  • the composition containing the S1 protein and MPL/alum adjuvant showed the highest neutralizing antibody titer, but it was about 2-3 times different from the compositions containing the S1-Fc or S1-Fc' fusion protein and MPL/alum adjuvant. did not show any significant difference.
  • SARS-CoV-2 S protein (Acro Biosystem, SPN-C52H8) at a concentration of 0.5 ⁇ g/ml and SARS S1 protein (Acro Biosystem, S1N-852H5) at a concentration of 0.5 ⁇ g/ml were mixed with a coating buffer (coating buffer, carbonate/bicarbonate buffer) , Sigma, catalog number C3041-100CAP) was applied to a 96-well plate at 4° C. for 16 to 18 hours.
  • a coating buffer coating buffer, carbonate/bicarbonate buffer
  • Sigma catalog number C3041-100CAP
  • washing solution 0.05 % tween 20 in 1x PBS, PBS-Tween tablet; Calbiochem, catalog# 524653 with 1L PW
  • diluent Dioxide, Teknova, catalog# 2D5120-1L; 1% BSA, 0.05
  • % Tween-20 consisting of 1X PBS
  • detection antibody i) Goat anti-mouse IgG conjugated to HRP (SOUTHERN BIOTECH, Catalog# 1030-05, ii) horseradish peroxidase-conjugated goat anti-mouse IgG1 (BioRad, Catalog# STAR132P) or iii) horseradish Peroxidase-conjugated goat anti-mouse IgG2a (BioRad, Catalog# STAR133P) was added and reacted at room temperature for 1 hour.
  • TMB (3,3', 5,5'-tetramethylbenzidine, Sigma, Catalog# T0440) was put on the plate and reacted for 5 minutes, and sulfuric acid solution (H 2 SO 4 , Merck, Catalog# 109072) was added The reaction was stopped. The absorbance (optical density) of each sample was measured using a plate reader. And the SARS-coronavirus-2 fusion protein, which is the result of the test, the total amount of IgG specific for coronavirus, and the production amount of IgG1 and IgG2a antibodies were measured.
  • the amount of IgG1 produced refers to the product of a humoral immune response
  • the amount of IgG2a produced refers to the product of a cellular immune response.
  • Total IgG refers to all IgG antibodies such as IgG1, IgG2a, etc.
  • the IgG1 antibody was generated in all experimental groups compared to the PBS group (negative control group), and a significant neutralizing antibody was generated in the composition including the S1 domain and MPL/alum adjuvant, especially S1 including the engineered Fc. -Fc' and the MPL/alum mixed composition induced a cellular immune response significantly.
  • Tables 3, 4, and 5 below correspond to FIGS. 2, 3, and 4, respectively.

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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne une protéine de fusion du coronavirus-2 du SARS et une composition immunogène la comprenant et, plus spécifiquement, une protéine de fusion comprenant une protéine de spicule (protéine S) du coronavirus-2 du SARS (SRAS-CoV-2) et une région constante d'une molécule d'immunoglobuline, et une composition immunogène le comprenant. La présente invention peut induire la production d'un anticorps neutralisant spécifique du coronavirus-2 du SRAS et aussi augmenter remarquablement les réponses immunitaires à médiation cellulaire. En conséquence, la présente invention peut non seulement donner lieu à une réponse immunitaire à des virus mutants, mais présente également un effet durable à long terme, et trouve donc des applications avantageuses dans la prévention d'infections par le coronavirus du SRAS (COVID-19).
PCT/KR2022/001416 2021-01-29 2022-01-27 Protéine de fusion du coronavirus-2 du sars et composition immunogène la comprenant WO2022164208A1 (fr)

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KR10-2021-0013681 2021-01-29

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