WO2023080328A1 - Vaccine adjuvant composition comprising agrimonia pilosa extract and galla rhois extract - Google Patents

Vaccine adjuvant composition comprising agrimonia pilosa extract and galla rhois extract Download PDF

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WO2023080328A1
WO2023080328A1 PCT/KR2021/018808 KR2021018808W WO2023080328A1 WO 2023080328 A1 WO2023080328 A1 WO 2023080328A1 KR 2021018808 W KR2021018808 W KR 2021018808W WO 2023080328 A1 WO2023080328 A1 WO 2023080328A1
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vaccine
extract
virus
present
composition
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PCT/KR2021/018808
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French (fr)
Korean (ko)
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강세찬
최순호
김준호
박대원
김경미
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에이피알지 주식회사
경희대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • the present invention relates to a vaccine adjuvant composition
  • a vaccine adjuvant composition comprising a dragon plant extract and a gall nut extract.
  • Influenza is a virus that causes epidemics all over the world, killing thousands to tens of thousands of people every season. In particular, in 2009, the world suffered great social and economic chaos due to the pandemic of the new influenza.
  • Influenza viruses are RNA viruses, and these RNA viruses have characteristics of easily mutating during replication because the RNA polymerase of the virus does not have a proofreading mechanism.
  • COVID-19 which is currently a global problem, is defined as a respiratory syndrome caused by SARS-CoV-2 virus infection, and the disease classification is the first class infectious disease, the new infectious disease syndrome, and the disease code is U071.
  • the pathogen is SARS-CoV-2: an RNA virus belonging to Coronaviridae, and the transmission route is known to be spread through droplets (saliva droplets) and contact. Severe illness or death mainly occurs in elderly patients, patients with reduced immune function, and patients with underlying diseases, but currently there is no suitable treatment other than conservative treatment such as fluid supplementation and antipyretics.
  • the present inventors completed the present invention by conducting research to develop a virus vaccine adjuvant using Yongacho and gall nut.
  • One object of the present invention is to provide a vaccine adjuvant (adjuvant) composition containing the extract of Yongacho and nut gall extract.
  • Another object of the present invention is to provide a vaccine composition comprising the above composition and a viral antigen.
  • Another object of the present invention is to provide a vaccine immunity enhancement method comprising administering the composition to a subject.
  • Another object of the present invention is to provide a virus prevention method comprising administering the composition to a subject.
  • One aspect of the present invention provides a vaccine adjuvant (adjuvant) composition comprising a dragon plant extract and nut gall extract.
  • the weight ratio of the yongacho extract and nut gall extract may be 1:10 to 10:1.
  • the vaccine may be a coronavirus vaccine.
  • the coronavirus may be a SARS-CoV-2 virus.
  • the vaccine may be an influenza virus vaccine.
  • the extraction solvent of the extract may be an aqueous ethanol solution.
  • the volume ratio of ethanol and water in the aqueous ethanol solution may be 5:5.
  • Another aspect of the invention provides a vaccine composition comprising the composition and a viral antigen.
  • the virus may be a coronavirus.
  • the virus may be an influenza virus.
  • Another aspect of the present invention provides a method for enhancing vaccine immunity comprising administering the composition to a subject.
  • Another aspect of the present invention provides a method for preventing a virus comprising administering the composition to a subject.
  • the vaccine adjuvant composition containing the dragon fruit extract and gall nut extract since it can increase the effect of a virus vaccine, it is effective as an adjuvant for increasing the immune response of various vaccines such as SARS-CoV-2 virus and influenza virus vaccine. can be used
  • FIG. 1 is a schematic diagram showing an experimental plan for confirming the effect of APRG64 as a SARS-CoV-2 virus vaccine adjuvant according to the increase in the amount of SARS-CoV-2 RBD protein neutralizing antibody production.
  • Figure 2 is a graph showing the amount of SARS-CoV-2 RBD protein neutralizing antibodies produced 10 days (A) and 15 days (B) after vaccine administration in mice administered with APRG64.
  • Figure 3 is a schematic diagram showing an experimental plan for confirming the effect of SARS-CoV-2 virus vaccine adjuvant of APRG64 according to the increase in the amount of SARS-CoV-2 S1 protein neutralizing antibody production.
  • Figure 4 is a graph showing the amount of SARS-CoV-2 S1 protein neutralizing antibody production in APRG64-administered mice 10 days (A), 15 days (B), and 20 days (C) after vaccine administration.
  • FIG. 5 is a schematic diagram showing an experimental plan for confirming the effect of APRG64 as a SARS-CoV-2 virus vaccine adjuvant on CD8 + T lymphocytes.
  • FIG. 6 is a graph showing the ratio of CD8 + T lymphocytes among CD3 + cells in blood of mice 20 days after vaccine administration in APRG64-administered mice.
  • FIG. 7 is a graph showing T lymphocytes expressing IFN-gamma in blood of mice 20 days after vaccine administration in APRG64-administered mice.
  • FIG. 8 is a schematic diagram showing an experimental plan for confirming the effect of APRG64 as a SARS-CoV-2 virus vaccine adjuvant on blood inflammatory cytokines.
  • Figure 9 shows the blood inflammatory cytokines GM-CSF (A), IL-23 (B), IL-1 ⁇ (C), IFN- ⁇ (D), TNF- ⁇ (E), MCP-1(F), IL-12p70(G), IL-1 ⁇ (H), IL-10(I), IL-6(J), IL-27(K), IL-17A (L) is a graph showing the concentration (pg/ml) of IFN- ⁇ (M).
  • FIG. 10 is a schematic diagram showing an experimental plan for confirming the influenza virus vaccine adjuvant effect of APRG64 according to the increase in influenza-specific antibody production.
  • Figure 11 is a graph showing the amount of influenza-specific antibodies produced in APRG64-administered mice 10 days (A) and 20 days (B) after vaccine administration.
  • One aspect of the present invention provides a vaccine adjuvant (adjuvant) composition comprising a dragon plant extract and nut gall extract.
  • adjuvant refers to a substance or composition that is added to a vaccine or pharmaceutically active ingredients to increase and/or affect an immune response, and is incorporated into an adjuvant or It encompasses a wide range of substances or strategies that can enhance the immunogenicity of antigens administered together.
  • the extract included in the composition according to the present invention can be obtained as follows. After washing Yongacho and nut galls with water to remove foreign substances, dry them in the shade, and add an appropriate amount of solvent to each of Yongacho and nut galls so that they are completely immersed. It is available in powder form.
  • Yongacho and gallbladder can be extracted using a common extraction solvent, respectively, and preferably, (a) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (eg, methanol, ethanol, propanol, butanol, normal- propanol, iso-propanol and normal-butanol, etc.), (b) a mixed solvent of the lower alcohol and water, (c) acetone, (d) ethyl acetate, (e) chloroform, (f) 1,3-butylene It can be extracted using glycol, (g) hexane, (h) diethyl ether, (i) butyl acetate, (j) chloroform-methanol or (k) water, more preferably 50% (v/v) ethanol It can be extracted as an aqueous solution. Extraction can be impregnated at room temperature or warmed.
  • anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms eg, m
  • the extraction solvent of the extract may be an aqueous ethanol solution.
  • the volume ratio of ethanol and water in the aqueous ethanol solution may be 5:5.
  • a vaccine immunity enhancement method comprising administering the composition to a subject.
  • the adjuvant composition according to one embodiment of the present invention when administered in combination with a vaccine, exhibits an excellent effect on enhancing immune activity by a virus vaccine, such as an increase in antibody production and an increase in immune cells.
  • the vaccine adjuvant composition of the present invention may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the composition of the present invention are commonly used in the preparation of drugs, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, silicic acid including, but not limited to, calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; It is not.
  • the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.
  • lubricants wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like.
  • suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: the science and practice of pharmacy 22nd edition (2013).
  • the vaccine adjuvant composition of the present invention may include various bases and/or additives necessary and appropriate for formulating the formulation, and may include nonionic surfactants, silicone polymers, extender pigments, flavorings, Preservatives, bactericides, oxidative stabilizers, organic solvents, ionic or nonionic thickeners, softeners, antioxidants, free radical destroyers, opacifying agents, stabilizers, emollients, silicones, ⁇ -hydroxy acids, antifoaming agents, Known compounds such as moisturizers, vitamins, insect repellents, fragrances, preservatives, surfactants, anti-inflammatory agents, substance P antagonists, fillers, polymers, propellants, basicizing or acidifying agents, or colorants may be further included.
  • nonionic surfactants such as moisturizers, vitamins, insect repellents, fragrances, preservatives, surfactants, anti-inflammatory agents, substance P antagonists, fillers, polymers, propellants, basicizing or acidifying agents, or colorants
  • the appropriate dosage of the vaccine adjuvant composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity. may be prescribed.
  • the dosage of the vaccine adjuvant composition of the present invention may be 0.001 to 1000 mg/kg based on adults.
  • the vaccine adjuvant composition of the present invention may be administered orally or parenterally.
  • the vaccine adjuvant composition of the present invention can be administered in various formulations for oral administration, such as tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, troches, etc. It may further include various excipients, for example, a wetting agent, a sweetening agent, a flavoring agent, a preservative, and the like. Specifically, when the composition of the present invention is formulated into an oral dosage form, suitable carriers, excipients and diluents commonly used in the preparation thereof may be further included.
  • Such carriers, excipients and diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and/or mineral oil may be used, but are not limited thereto.
  • it may be prepared by including diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants commonly used in formulation, and may further include a lubricant such as magnesium stearate or talc in addition to the above excipients. .
  • the vaccine adjuvant composition of the present invention may be administered parenterally, for example, through subcutaneous injection, intravenous injection, or intramuscular injection, but is not limited thereto.
  • Formulation into a formulation for parenteral administration may be, for example, mixing the vaccine adjuvant composition of the present invention in water together with a stabilizer or buffer to prepare a solution or suspension, and prepare it in an ampoule or vial unit dosage form.
  • the composition may be sterilized, and may further include adjuvants such as preservatives, stabilizers, hydration agents or emulsification accelerators, salts and buffers for osmotic pressure control, and other therapeutically useful substances, formulated by conventional methods. It can be.
  • compositions may be presented in unit-dose (single-dose) or multi-dose (several-dose) containers, such as sealed ampoules and vials, and added immediately before use in a sterile liquid carrier, such as water for injection. It can be stored under freeze-drying conditions requiring only the addition of Extemporaneous preparations and suspensions may be prepared from sterile powders, granules and tablets.
  • the weight ratio of the dragon fruit extract and the nut gall extract may be 1:10 to 10:1, and preferably, the weight ratio of the dragon fruit extract and the gall nut extract may be 6:4.
  • the vaccine may be a coronavirus vaccine.
  • the coronavirus may be an RNA virus belonging to the Coronavirinae family, specifically, including HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, MERS-CoV and SARS-CoV-2, etc. , but is not limited thereto.
  • the coronavirus may be the SARS-CoV-2 virus.
  • the weight ratio of the dragon fruit extract and gall nut extract contained in the vaccine adjuvant composition of the present invention is 1:10 to 10:1, preferably 6:4, the immune response of the coronavirus, particularly SARS-CoV-2 virus vaccine can be effectively induced within, for example, 10 to 20 days.
  • the vaccine may be an influenza virus vaccine.
  • the influenza may be a virus included in Orthomyxoviridae, which is an RNA virus, and specifically, may be a virus of the genus influenza A, influenza B and / or influenza C, and on the surface of the virus particle, hemagglutinin (HA) and viruses with a glycoprotein called neuraminidase (NA), such as, but not limited to, H1N1 subtype, H3N2 subtype, H5N1 subtype and H7N7 subtype, etc.
  • HA hemagglutinin
  • NA neuraminidase
  • the immune response of the influenza virus vaccine is, for example, 10 days to 10 days. Effective induction within 20 days.
  • Another aspect of the present invention provides a vaccine composition comprising the composition and a viral antigen.
  • Another aspect of the present invention provides a method for preventing a virus comprising administering the composition to a subject.
  • the vaccine composition according to one embodiment of the present invention includes an adjuvant composition to increase the immune activity of the vaccine, thereby exhibiting excellent effects in preventing viruses such as SARS-CoV-2 virus and influenza virus.
  • the dosage of the vaccine composition according to the present invention can be easily determined by a person skilled in the art in consideration of the purpose of use, target disease (type thereof, etc.), patient age, weight, medical history, etc., and the frequency of administration of the subject (or patient), It can be easily determined by those skilled in the art in consideration of the purpose of use, the target disease (type, severity, etc.), patient's age, weight, medical history, progress, and the like.
  • the frequency of administration of the vaccine composition according to the present invention may be, for example, administered every day to several months or administered once or twice before each epidemic epidemic, but is not limited thereto, and those skilled in the art while examining the progress of maintaining immunogenicity can easily determine an additional immunization interval.
  • the virus may be a coronavirus.
  • the virus may be an influenza virus.
  • the viral antigen of the present invention can be used without limitation as long as it is a substance capable of inducing an immune response to infection with coronavirus and/or influenza virus, and can be easily selected by those skilled in the art.
  • the vaccine composition of the present invention may further comprise one or more pharmaceutically acceptable vaccine adjuvants.
  • vaccine adjuvants for example, aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virosome, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (DL- ⁇ tocopherol, squalene, and a mixture of polysorbate 80 as an emulsifier), CpG, flagellin, Poly I:C, AS01, AS02, ISCOMs, and ISCOMMATRIX, but are not limited thereto.
  • MPL monophosphoryl lipid A
  • the dose of the vaccine composition of the present invention may be 0.001 to 1000 mg/kg for adults, but is not limited thereto, and the dose may be appropriately adjusted by a person skilled in the art according to the age, sex and condition of the patient.
  • Agrimonia pilosa The leaves of Agrimonia pilosa were purchased from BioKorea Co LTd (Seoul), and the voucher specimen (BMRI-RG-1601) was deposited at the Biomedical Research Center of Kyunghee University, Yongin, Korea.
  • a dried sample (20 kg) was extracted with a 50% aqueous ethanol solution at 80 ⁇ 2° C. for 6 hours and then filtered. Then, it was concentrated using a rotary evaporator and lyophilized to finally obtain 1.57 kg of Yongacho extract, which was stored at 4° C. until use.
  • Galla rhois leaves were purchased from BioKorea Co LTd (Seoul), and voucher specimens (BMRI-RG-1602) were deposited at the Biomedical Research Center of Kyunghee University, Yongin, Korea.
  • a dried sample (20 kg) was extracted with a 50% aqueous ethanol solution at 80 ⁇ 2° C. for 6 hours and then filtered. Then, it was concentrated using a rotary evaporator and lyophilized to finally obtain 11.59 kg of nut gall extract, which was stored at 4° C. until use.
  • a 6:4 mixture (APRG64) of Yongacho extract and Nut gall extract was prepared by mixing the Yongacho extract prepared in Preparation Example 1 and the gall nut extract prepared in Preparation Example 2 at a weight ratio of 6:4.
  • Example 1 Confirmation of SARS-CoV-2 virus vaccine adjuvant effect of APRG64 according to increase in SARS-CoV-2 RBD protein neutralizing antibody production
  • APRG64 of Preparation Example 3 was orally administered to Balb/C mice at a dose of 50 mg/kg or 100 mg/kg for 10 days, and on day 6 of oral administration, AstraZeneca (Lot. 210298, AstraZeneca) vaccine was administered to mice 6.02 ⁇ 10 9 virus particles were administered intramuscularly.
  • the 96-well plate was coated with SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Cat# 40592-V08B, Sinobiological) (100 ng/ml), After incubation with mouse serum (1:200-1:500), secondary antibody (1:5000, Goat anti-mouse IgG F(ab')2, polyclonal antibody (HRP conjugate), Cat# ADI-SAB-100 After reacting with -J, Enzo Lifescience), ELISA analysis was performed by measuring color development. Through this, the amount of neutralizing antibodies to the SARS-CoV-2 RBD protein present in the blood of mice was measured (Fig. 1), and compared with the amount of neutralizing antibodies in mice not administered with APRG64, SARS-CoV-2 by APRG64 2 Whether or not the amount of RBD protein neutralizing antibody production increased was analyzed.
  • SARS-CoV-2 RBD protein neutralizing antibodies increased 10 days after vaccine administration (Fig. 2A) and 15 days after vaccine administration (Fig. 2B) in APRG64-administered mice in a concentration-dependent manner of APRG64. In particular, 100 mg It was confirmed that neutralizing antibodies increased statistically significantly in the /kg administration group.
  • APRG64 of Preparation Example 3 was orally administered to Balb/C mice at a dose of 50 mg/kg or 100 mg/kg for 10 days, and on day 6 of oral administration, AstraZeneca (Lot. 210298, AstraZeneca) vaccine was administered to mice 6.02 ⁇ 10 9 virus particles were administered intramuscularly.
  • SARS-CoV-2 2019-nCoV Spike S1-His Recombinant Protein (Cat# 40591-V08B1, Sinobiological) (100 ng/ml)
  • mouse serum 1:200-1:500
  • secondary antibody 1:5000, Goat anti-mouse IgG F(ab')2, polyclonal antibody (HRP conjugate)
  • HRP conjugate Cat# ADI
  • ELISA analysis was performed by measuring color development. Through this, the amount of neutralizing antibodies to the SARS-CoV-2 S1 protein present in the blood of mice was analyzed (FIG. 3), and this was compared with the amount of neutralizing antibodies in mice not administered with APRG64. 2 Whether or not the amount of S1 neutralizing antibody production increased was analyzed.
  • SARS-CoV-2 S1 protein neutralizing antibody increased 10 days (Fig. 4A) and 15 days (Fig. 4B) after vaccine administration in the 100 mg/kg administration group, in particular, 20 days after vaccine administration (Fig. 4B). 4C) A statistically significant increase in neutralizing antibodies was confirmed.
  • APRG64 of Preparation Example 3 was orally administered to Balb/C mice at a dose of 50 mg/kg or 100 mg/kg for 10 days, and on day 6 of oral administration, AstraZeneca (Lot. 210298, AstraZeneca) vaccine was administered to mice 6.02 ⁇ 10 9 virus particles were administered intramuscularly. 20 days after vaccination, blood monocytes were isolated from the blood of mice, reacted with anti-CD3 and anti-CD8 antibodies, and the ratio of CD8 + T lymphocytes to CD3 + cells was analyzed using flow cytometry (Fig. 5), by comparing this with the ratio of CD8 + T lymphocytes in mice not administered with APRG64, the increase in CD8 + T lymphocytes by APRG64 was analyzed.
  • T lymphocytes expressing IFN-gamma were analyzed.
  • APRG64 of Preparation Example 3 was orally administered to Balb/C mice at a dose of 50 mg/kg or 100 mg/kg for 10 days, and on day 6 of oral administration, AstraZeneca vaccine was administered at a dose of 6.02 ⁇ 10 per mouse. 9 virus particles were administered intramuscularly.
  • the number of cells producing IFN-gamma in response to the SARS-CoV-2 S1 peptide was measured by ELISPOT (Mouse IFN-gamma ELISpotBASIC (ALP) kit, cat# 3321-2A, Mabtech)
  • ELISPOT Me IFN-gamma ELISpotBASIC (ALP) kit, cat# 3321-2A, Mabtech
  • the number of T lymphocytes expressing IFN-gamma in the blood of mice was analyzed (Fig. Compared to gamma-expressing T lymphocytes, the increase in IFN-gamma-expressing T lymphocytes by APRG64 was analyzed.
  • concentrations of 12p70, IL-17A, IL-23, IL-27, MCP-1, IFN- ⁇ , IFN- ⁇ , TNF- ⁇ and GM-CSF were analyzed (FIG. 9), which were analyzed without APRG64 administration.
  • the effect of reducing blood inflammatory cytokine concentration by APRG64 was analyzed.
  • GM-CSF (FIG. 9A), IL-23 (FIG. 9B), IL-1 ⁇ (FIG. 9C), IFN- ⁇ (FIG. 9D), TNF- ⁇ (FIG. 9E), MCP-1 (FIG. 9F), IL- 12p70 (Fig. 9G), IL-1 ⁇ (Fig. 9H), IL-10 (Fig. 9I), IL-6 (Fig. 9J), IL-27 (Fig. 9K), IL-17A (Fig. 9L), IFN- ⁇ ( As a result of analyzing the concentration of (Fig. 9A), IL-23 (FIG. 9B), IL-1 ⁇ (FIG. 9C), IFN- ⁇ (FIG. 9D), TNF- ⁇ (FIG. 9E), MCP-1 (FIG. 9F), IL- 12p70 (Fig. 9G), IL-1 ⁇ (Fig. 9H), IL-10 (Fig. 9I), IL-6 (Fig. 9J), IL-27 (Fig. 9K), IL-17
  • Example 5 Confirmation of influenza vaccine adjuvant effect of APRG64 according to the increase in influenza-specific antibody production
  • influenza-specific antibody production was analyzed.
  • APRG64 of Preparation Example 3 was orally administered to Balb/C mice at a dose of 50 mg/kg or 100 mg/kg for 10 days, and on day 6 of oral administration, influenza vaccine per mouse (Lot. Q022024, SKY Cellflu Quadrivalent ) 500 ⁇ l was administered intramuscularly.
  • 96-well plates were coated (300 ng/50 ⁇ l) with inactivated influenza vaccine (IIV) in PBS for 2 hours at room temperature, 1% BSA and PBS- 150 ⁇ l of T was treated and blocked at room temperature for 2 hours, then mouse serum (1:200) was added and incubated overnight at 4°C, secondary antibody (1:5000, Goat anti-mouse IgG (HRP conjugate), BML After reacting with -SA204-0100, Enzo Lifescience), ELISA analysis was performed by measuring color development. Through this, the amount of neutralizing antibodies to the SARS-CoV-2 S1 protein present in the blood of mice was analyzed (FIG. 10), and compared with the amount of influenza-specific antibodies in mice not administered with APRG64, influenza-specific antibodies by APRG64 were analyzed. Whether or not an increase in the amount of enemy antibody production was analyzed.
  • influenza-specific antibodies increased statistically significantly in the 50 mg/kg and 100 mg/kg administration groups 10 days (FIG. 11A) and 20 days (FIG. 11B) after vaccine administration.
  • the 6: 4 mixture (APRG64) of the extract of dragon plant extract and gall nut extract can be used as an effective adjuvant for viral vaccines such as SARS-CoV-2 virus vaccine and influenza virus vaccine.

Abstract

The present invention relates to a vaccine adjuvant composition comprising an Agrimonia pilosa extract and a Galla rhois extract, wherein the vaccine adjuvant composition can increase the effect of virus vaccines and can thus be effectively used as an adjuvant for increasing the immune response of various vaccines, such as SARS-CoV-2 virus and influenza virus vaccines.

Description

용아초 추출물 및 오배자 추출물을 포함하는 백신 아쥬반트 조성물Vaccine adjuvant composition containing extracts of Yongacho and nut gall extracts
본 발명은 용아초 추출물 및 오배자 추출물을 포함하는 백신 아쥬반트 조성물에 관한 것이다.The present invention relates to a vaccine adjuvant composition comprising a dragon plant extract and a gall nut extract.
인플루엔자는 계절마다 수 천에서 수 만명이 사망하는 유행성 독감을 전 세계에 걸쳐 일으키는 바이러스로, 특히, 2009년 전 세계는 신종 인플루엔자의 대유행으로 사회, 경제적으로 큰 혼란을 겪었다. 인플루엔자 바이러스는 RNA 바이러스로, 이러한 RNA 바이러스들은 바이러스의 RNA 중합효소(polymerase)가 교정 기작(proofreading mechanism)을 가지고 있지 않기 때문에 복제과정에서 쉽게 변이가 일어나는 특성을 가지고 있다.Influenza is a virus that causes epidemics all over the world, killing thousands to tens of thousands of people every season. In particular, in 2009, the world suffered great social and economic chaos due to the pandemic of the new influenza. Influenza viruses are RNA viruses, and these RNA viruses have characteristics of easily mutating during replication because the RNA polymerase of the virus does not have a proofreading mechanism.
한편, 현재 전세계적으로 문제되고 있는 COVID-19는 SARS-CoV-2 바이러스 감염에 의한 호흡기 증후군이라 정의되고 있으며, 질병 분류는 법정감염병 제1급감염병 신종감염병증후군 및 질병 코드는 U071이다. 병원체는 SARS-CoV-2: Coronaviridae에 속하는 RNA 바이러스로, 전파 경로는 현재까지 비말(침방울), 접촉을 통한 전파로 알려져 있다. 고령, 면역기능이 저하된 환자, 기저질환을 가진 환자에서 주로 중증 또는 사망이 발생하는데, 현재 수액 보충, 해열제 등 보존적 치료를 하는 것 외에 마땅한 치료제는 없는 상황이다.Meanwhile, COVID-19, which is currently a global problem, is defined as a respiratory syndrome caused by SARS-CoV-2 virus infection, and the disease classification is the first class infectious disease, the new infectious disease syndrome, and the disease code is U071. The pathogen is SARS-CoV-2: an RNA virus belonging to Coronaviridae, and the transmission route is known to be spread through droplets (saliva droplets) and contact. Severe illness or death mainly occurs in elderly patients, patients with reduced immune function, and patients with underlying diseases, but currently there is no suitable treatment other than conservative treatment such as fluid supplementation and antipyretics.
이와 같은 바이러스의 감염을 차단하기 위하여, 백신이 개발되어 투여되고 있으나, 백신 투여 후 시간이 지남에 따라 면역성이 급격하게 낮아져, 계절별로, 또는 일정 주기별로 지속적으로 추가적인 접종(부스터샷)이 필요하다는 문제점이 나타나고 있는 상황이다.In order to block infection with such a virus, vaccines have been developed and administered, but immunity rapidly decreases over time after vaccine administration, and additional vaccinations (booster shots) are needed continuously seasonally or at regular intervals. A problem is appearing.
따라서, 바이러스 백신의 면역 반응을 증가시키기 위한 백신 아쥬반트(adjuvant)를 개발하기 위한 연구가 활발히 수행되고 있으나, 한국, 일본, 중국, 인도 등지에 분포하는 여러해살이풀인 용아초(Agrimonia pilosa), 및 옻나무과(Anacardiaceae)에 속하는 붉나무(Rhus javanica L)의 잎에 오배자진딧물(Aphis chinensis J Bell)이 자상을 주어생긴 벌레집인 오배자(Galla rhois)를 백신 아쥬반트로 개발하기 위한 연구에 대해서는 전혀 보고된 바가 없다.Therefore, research is being actively conducted to develop a vaccine adjuvant for increasing the immune response of a virus vaccine, but Agrimonia pilosa, a perennial plant distributed in Korea, Japan, China, and India, And sumac ( Anacardiaceae ) belonging to the red tree ( Rhus javanica L ) on the leaves of the gall gall aphid ( Aphis chinensis J Bell ), which is a worm nest caused by giving a stab wound ( Galla rhois ) No report on studies to develop vaccine adjuvants. Nothing happened.
이에, 본 발명자들은 용아초 및 오배자를 활용하여 바이러스 백신 아쥬반트를 개발하기 위한 연구를 수행하여 본 발명을 완성하였다.Accordingly, the present inventors completed the present invention by conducting research to develop a virus vaccine adjuvant using Yongacho and gall nut.
본 발명의 하나의 목적은 용아초 추출물 및 오배자 추출물을 포함하는 백신 아쥬반트(adjuvant) 조성물을 제공하는 것이다.One object of the present invention is to provide a vaccine adjuvant (adjuvant) composition containing the extract of Yongacho and nut gall extract.
본 발명의 다른 목적은 상기 조성물 및 바이러스 항원을 포함하는 백신 조성물을 제공하는 것이다.Another object of the present invention is to provide a vaccine composition comprising the above composition and a viral antigen.
본 발명의 또 다른 목적은 상기 조성물을 대상체에 투여하는 단계를 포함하는 백신 면역 증강 방법을 제공하는 것이다.Another object of the present invention is to provide a vaccine immunity enhancement method comprising administering the composition to a subject.
본 발명의 또 다른 목적은 상기 조성물을 대상체에 투여하는 단계를 포함하는 바이러스 예방 방법을 제공하는 것이다.Another object of the present invention is to provide a virus prevention method comprising administering the composition to a subject.
본 발명의 일 양상은 용아초 추출물 및 오배자 추출물을 포함하는 백신 아쥬반트(adjuvant) 조성물을 제공한다.One aspect of the present invention provides a vaccine adjuvant (adjuvant) composition comprising a dragon plant extract and nut gall extract.
본 발명의 일 구체예에 따르면, 상기 용아초 추출물 및 오배자 추출물의 중량비는 1:10 내지 10:1인 것일 수 있다.According to one embodiment of the present invention, the weight ratio of the yongacho extract and nut gall extract may be 1:10 to 10:1.
본 발명의 일 구체예에 따르면, 상기 백신은 코로나바이러스 백신인 것일 수 있다.According to one embodiment of the present invention, the vaccine may be a coronavirus vaccine.
본 발명의 일 구체예에 따르면, 상기 코로나바이러스는 SARS-CoV-2 바이러스인 것일 수 있다.According to one embodiment of the present invention, the coronavirus may be a SARS-CoV-2 virus.
본 발명의 일 구체예에 따르면, 상기 백신은 인플루엔자 바이러스 백신인 것일 수 있다.According to one embodiment of the present invention, the vaccine may be an influenza virus vaccine.
본 발명의 일 구체예에 따르면, 상기 추출물의 추출용매는 에탄올수용액인 것일 수 있다.According to one embodiment of the present invention, the extraction solvent of the extract may be an aqueous ethanol solution.
본 발명의 일 구체예에 따르면, 상기 에탄올수용액의 에탄올 및 물의 부피비는 5:5인 것일 수 있다.According to one embodiment of the present invention, the volume ratio of ethanol and water in the aqueous ethanol solution may be 5:5.
발명의 다른 양상은 상기 조성물 및 바이러스 항원을 포함하는 백신 조성물을 제공한다.Another aspect of the invention provides a vaccine composition comprising the composition and a viral antigen.
본 발명의 일 구체예에 따르면, 상기 바이러스는 코로나바이러스인 것일 수 있다.According to one embodiment of the present invention, the virus may be a coronavirus.
본 발명의 일 구체예에 따르면, 상기 바이러스는 인플루엔자 바이러스인 것일 수 있다.According to one embodiment of the present invention, the virus may be an influenza virus.
본 발명의 또 다른 양상은 상기 조성물을 대상체에 투여하는 단계를 포함하는 백신 면역 증강 방법을 제공한다.Another aspect of the present invention provides a method for enhancing vaccine immunity comprising administering the composition to a subject.
본 발명의 또 다른 양상은 상기 조성물을 대상체에 투여하는 단계를 포함하는 바이러스 예방 방법을 제공한다.Another aspect of the present invention provides a method for preventing a virus comprising administering the composition to a subject.
용아초 추출물 및 오배자 추출물을 포함하는 백신 아쥬반트 조성물에 따르면, 바이러스 백신의 효과를 증대시킬 수 있으므로, SARS-CoV-2 바이러스 및 인플루엔자 바이러스 백신 등 다양한 백신의 면역 반응을 증가시키기 위한 아쥬반트로 효과적으로 사용될 수 있다.According to the vaccine adjuvant composition containing the dragon fruit extract and gall nut extract, since it can increase the effect of a virus vaccine, it is effective as an adjuvant for increasing the immune response of various vaccines such as SARS-CoV-2 virus and influenza virus vaccine. can be used
도 1은 SARS-CoV-2 RBD 단백질 중화 항체 생성량 증가에 따른 APRG64의 SARS-CoV-2 바이러스 백신 아쥬반트 효과를 확인하기 위한 실험계획을 나타낸 모식도이다.1 is a schematic diagram showing an experimental plan for confirming the effect of APRG64 as a SARS-CoV-2 virus vaccine adjuvant according to the increase in the amount of SARS-CoV-2 RBD protein neutralizing antibody production.
도 2는 APRG64를 투여한 마우스에서 백신 투여 10일 후(A) 및 15일 후(B), SARS-CoV-2 RBD 단백질 중화 항체의 생성량을 나타낸 그래프이다.Figure 2 is a graph showing the amount of SARS-CoV-2 RBD protein neutralizing antibodies produced 10 days (A) and 15 days (B) after vaccine administration in mice administered with APRG64.
도 3은 SARS-CoV-2 S1 단백질 중화 항체 생성량 증가에 따른 APRG64의 SARS-CoV-2 바이러스 백신 아쥬반트 효과를 확인하기 위한 실험계획을 나타낸 모식도이다.Figure 3 is a schematic diagram showing an experimental plan for confirming the effect of SARS-CoV-2 virus vaccine adjuvant of APRG64 according to the increase in the amount of SARS-CoV-2 S1 protein neutralizing antibody production.
도 4는 APRG64를 투여한 마우스에서 백신 투여 10일 후(A), 15일 후(B) 및 20일 후(C), SARS-CoV-2 S1 단백질 중화 항체의 생성량을 나타낸 그래프이다.Figure 4 is a graph showing the amount of SARS-CoV-2 S1 protein neutralizing antibody production in APRG64-administered mice 10 days (A), 15 days (B), and 20 days (C) after vaccine administration.
도 5는 CD8+ T 림프구에 대한 APRG64의 SARS-CoV-2 바이러스 백신 아쥬반트 효과를 확인하기 위한 실험계획을 나타낸 모식도이다.5 is a schematic diagram showing an experimental plan for confirming the effect of APRG64 as a SARS-CoV-2 virus vaccine adjuvant on CD8 + T lymphocytes.
도 6은 APRG64를 투여한 마우스에서 백신 투여 20일 후 마우스 혈액 내 CD3+ 세포 중 CD8+ T 림프구의 비율을 나타낸 그래프이다.6 is a graph showing the ratio of CD8 + T lymphocytes among CD3 + cells in blood of mice 20 days after vaccine administration in APRG64-administered mice.
도 7은 APRG64를 투여한 마우스에서 백신 투여 20일 후 마우스 혈액 내 IFN-gamma를 발현하는 T 림프구를 나타낸 그래프이다.7 is a graph showing T lymphocytes expressing IFN-gamma in blood of mice 20 days after vaccine administration in APRG64-administered mice.
도 8은 혈중 염증유발 사이토카인에 대한 APRG64의 SARS-CoV-2 바이러스 백신 아쥬반트 효과를 확인하기 위한 실험계획을 나타낸 모식도이다.8 is a schematic diagram showing an experimental plan for confirming the effect of APRG64 as a SARS-CoV-2 virus vaccine adjuvant on blood inflammatory cytokines.
도 9는 APRG64를 투여한 마우스에서 백신 투여 5일 후 혈중 염증유발 사이토카인인 GM-CSF(A), IL-23(B), IL-1α(C), IFN-γ(D), TNF-α(E), MCP-1(F), IL-12p70(G), IL-1β(H), IL-10(I), IL-6(J), IL-27(K), IL-17A(L), IFN-β(M)의 농도(pg/㎖) 나타낸 그래프이다.Figure 9 shows the blood inflammatory cytokines GM-CSF (A), IL-23 (B), IL-1α (C), IFN-γ (D), TNF- α(E), MCP-1(F), IL-12p70(G), IL-1β(H), IL-10(I), IL-6(J), IL-27(K), IL-17A (L) is a graph showing the concentration (pg/ml) of IFN-β(M).
도 10은 인플루엔자 특이적 항체 생성량 증가에 따른 APRG64의 인플루엔자 바이러스 백신 아쥬반트 효과를 확인하기 위한 실험계획을 나타낸 모식도이다.10 is a schematic diagram showing an experimental plan for confirming the influenza virus vaccine adjuvant effect of APRG64 according to the increase in influenza-specific antibody production.
도 11은 APRG64를 투여한 마우스에서 백신 투여 10일 후(A) 및 20일 후(B), 인플루엔자 특이적 항체의 생성량을 나타낸 그래프이다.Figure 11 is a graph showing the amount of influenza-specific antibodies produced in APRG64-administered mice 10 days (A) and 20 days (B) after vaccine administration.
본 발명의 일 양상은 용아초 추출물 및 오배자 추출물을 포함하는 백신 아쥬반트(adjuvant) 조성물을 제공한다.One aspect of the present invention provides a vaccine adjuvant (adjuvant) composition comprising a dragon plant extract and nut gall extract.
본 발명에서 사용되는 용어, "아쥬반트(adjuvant)"는 백신 또는 약학적으로 활성 있는 성분들에 첨가되어 면역 반응을 증가시키거나 및/또는 영향을 주는 물질 또는 조성물을 말하며, 아쥬반트에 통합되거나 함께 투여된 항원의 면역원성(immunogenicity)을 증진시킬 수 있는 넓은 범위의 물질 또는 책략(stratagem)을 포함한다.As used herein, the term "adjuvant" refers to a substance or composition that is added to a vaccine or pharmaceutically active ingredients to increase and/or affect an immune response, and is incorporated into an adjuvant or It encompasses a wide range of substances or strategies that can enhance the immunogenicity of antigens administered together.
본 발명에 따른 조성물에 포함되는 추출물은 하기와 같이 수득될 수 있다. 용아초 및 오배자를 물로 세척하여 이물질을 제거한 후 그늘에서 건조하고, 용아초 및 오배자에 각각 적당한 양의 용매를 첨가하여 완전히 침지되도록 하며, 이때, 용아초 및 오배자는 건조된 상태 그대로 사용하거나 분쇄하여 분말 형태로 사용할 수 있다. 용아초 및 오배자는 각각 통상의 추출용매를 이용하여 추출할 수 있으며, 바람직하게는, (a) 탄소수 1~4의 무수 또는 함수 저급 알코올(예를 들어, 메탄올, 에탄올, 프로판올, 부탄올, 노말-프로판올, 이소-프로판올 및 노말-부탄올 등), (b) 상기 저급 알코올과 물과의 혼합용매, (c) 아세톤, (d) 에틸 아세테이트, (e) 클로로포름, (f) 1,3-부틸렌글리콜, (g) 헥산, (h) 디에틸에테르, (i) 부틸아세테이트 (j) 클로로포름-메탄올 또는 (k) 물을 이용하여 추출할 수 있고, 더욱 바람직하게는 50%(v/v) 에탄올수용액으로 추출할 수 있다. 추출시 실온에서 함침하거나 가온할 수 있다.The extract included in the composition according to the present invention can be obtained as follows. After washing Yongacho and nut galls with water to remove foreign substances, dry them in the shade, and add an appropriate amount of solvent to each of Yongacho and nut galls so that they are completely immersed. It is available in powder form. Yongacho and gallbladder can be extracted using a common extraction solvent, respectively, and preferably, (a) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (eg, methanol, ethanol, propanol, butanol, normal- propanol, iso-propanol and normal-butanol, etc.), (b) a mixed solvent of the lower alcohol and water, (c) acetone, (d) ethyl acetate, (e) chloroform, (f) 1,3-butylene It can be extracted using glycol, (g) hexane, (h) diethyl ether, (i) butyl acetate, (j) chloroform-methanol or (k) water, more preferably 50% (v/v) ethanol It can be extracted as an aqueous solution. Extraction can be impregnated at room temperature or warmed.
본 발명의 일 구체예에 따르면, 상기 추출물의 추출용매는 에탄올수용액일 수 있다.According to one embodiment of the present invention, the extraction solvent of the extract may be an aqueous ethanol solution.
본 발명의 일 구체예에 따르면, 상기 에탄올수용액의 에탄올 및 물의 부피비는 5:5일 수 있다.According to one embodiment of the present invention, the volume ratio of ethanol and water in the aqueous ethanol solution may be 5:5.
본 발명의 또 다른 양상에 따르면, 상기 조성물을 대상체에 투여하는 단계를 포함하는 백신 면역 증강 방법을 제공한다. According to another aspect of the present invention, there is provided a vaccine immunity enhancement method comprising administering the composition to a subject.
본 발명의 일 구체예에 따른 아쥬반트 조성물은, 백신과 병용 투여 시 항체 생성량 증가, 면역세포 증가 등 바이러스 백신에 의한 면역 활성 증강에 우수한 효과를 나타낸다.The adjuvant composition according to one embodiment of the present invention, when administered in combination with a vaccine, exhibits an excellent effect on enhancing immune activity by a virus vaccine, such as an increase in antibody production and an increase in immune cells.
본 발명의 백신 아쥬반트 조성물은 약학적으로 허용되는 담체를 포함할 수 있다. 본 발명의 조성물에 포함되는 약학적으로 허용되는 담체는 약제의 제조에 통상적으로 이용되는 것으로써, 락토오스, 덱스트로스, 수크로오스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로오스, 폴리비닐피롤리돈, 셀룰로오스, 물, 시럽, 메틸 셀룰로오스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제는 Remington: the science and practice of pharmacy 22nd edition (2013)에 상세히 기재되어 있다.The vaccine adjuvant composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the composition of the present invention are commonly used in the preparation of drugs, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, silicic acid including, but not limited to, calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; It is not. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: the science and practice of pharmacy 22nd edition (2013).
본 발명의 백신 아쥬반트 조성물은 그 제형의 제제화에 필요하고 적절한 각종 기제 및/또는 첨가물을 포함할 수 있으며, 그 효과를 떨어트리지 않는 범위 내에서 비이온 계면활성제, 실리콘 폴리머, 체질안료, 향료, 방부제, 살균제, 산화 안정화제, 유기 용매, 이온성 또는 비이온성 증점제, 유연화제, 산화방지제, 자유 라디칼 파괴제, 불투명화제, 안정화제, 에몰리언트(emollient), 실리콘, α-히드록시산, 소포제, 보습제, 비타민, 곤충 기피제, 향료, 보존제, 계면활성제, 소염제, 물질 P 길항제, 충전제, 중합체, 추진제, 염기성화 또는 산성화제, 또는 착색제 등 공지의 화합물을 더 포함하여 제조될 수 있다.The vaccine adjuvant composition of the present invention may include various bases and/or additives necessary and appropriate for formulating the formulation, and may include nonionic surfactants, silicone polymers, extender pigments, flavorings, Preservatives, bactericides, oxidative stabilizers, organic solvents, ionic or nonionic thickeners, softeners, antioxidants, free radical destroyers, opacifying agents, stabilizers, emollients, silicones, α-hydroxy acids, antifoaming agents, Known compounds such as moisturizers, vitamins, insect repellents, fragrances, preservatives, surfactants, anti-inflammatory agents, substance P antagonists, fillers, polymers, propellants, basicizing or acidifying agents, or colorants may be further included.
본 발명의 백신 아쥬반트 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 본 발명의 백신 아쥬반트 조성물의 투여량은 성인 기준으로 0.001 내지 1000㎎/㎏일 수 있다.The appropriate dosage of the vaccine adjuvant composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity. may be prescribed. The dosage of the vaccine adjuvant composition of the present invention may be 0.001 to 1000 mg/kg based on adults.
본 발명의 백신 아쥬반트 조성물은 경구 또는 비경구 투여될 수 있다.The vaccine adjuvant composition of the present invention may be administered orally or parenterally.
본 발명의 백신 아쥬반트 조성물은 경구 투여시 다양한 제형으로 투여될 수 있는데, 정제, 환제, 경/연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 과립제, 엘릭시르제, 트로키제 등의 형태로 투여될 수 있으며, 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등을 더 포함할 수 있다. 구체적으로, 본 발명의 조성물을 경구투여 제형으로 제형화할 경우, 이의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 예를 들어, 락토오스, 덱스트로스, 수크로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알기네이트, 젤라틴, 인산칼슘, 규산칼슘, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및/또는 광물유가 사용될 수 있으나 이에 한정되지 않는다. 또한, 제제화에 일반적으로 사용되는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 포함하여 조제될 수 있으며, 상기 부형제 이외에 마그네슘 스테아레이트 또는 탈크 같은 윤활제를 더 포함할 수 있다.The vaccine adjuvant composition of the present invention can be administered in various formulations for oral administration, such as tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, troches, etc. It may further include various excipients, for example, a wetting agent, a sweetening agent, a flavoring agent, a preservative, and the like. Specifically, when the composition of the present invention is formulated into an oral dosage form, suitable carriers, excipients and diluents commonly used in the preparation thereof may be further included. Such carriers, excipients and diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and/or mineral oil may be used, but are not limited thereto. In addition, it may be prepared by including diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants commonly used in formulation, and may further include a lubricant such as magnesium stearate or talc in addition to the above excipients. .
본 발명의 백신 아쥬반트 조성물은 비경구 투여될 수 있으며, 예를 들어, 피하주사, 정맥주사 또는 근육 내 주사 등의 방법을 통하여 투여되는 것일 수 있으나, 이에 한정되지 않는다.The vaccine adjuvant composition of the present invention may be administered parenterally, for example, through subcutaneous injection, intravenous injection, or intramuscular injection, but is not limited thereto.
비경구 투여용 제형으로의 제제화는, 예를 들어, 본 발명의 백신 아쥬반트 조성물을 안정제 또는 완충제와 함께 물에 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알 단위 투여형으로 제조하는 것일 수 있다. 또한, 상기 조성물은 멸균되고, 방부제, 안정화제, 수화제 또는 유화 촉진제, 삼투압 조절을 위한 염 및 완충제 등의 보조제, 및 기타 치료적으로 유용한 물질을 추가로 포함할 수 있으며, 통상적인 방법에 의해 제제화될 수 있다.Formulation into a formulation for parenteral administration may be, for example, mixing the vaccine adjuvant composition of the present invention in water together with a stabilizer or buffer to prepare a solution or suspension, and prepare it in an ampoule or vial unit dosage form. there is. In addition, the composition may be sterilized, and may further include adjuvants such as preservatives, stabilizers, hydration agents or emulsification accelerators, salts and buffers for osmotic pressure control, and other therapeutically useful substances, formulated by conventional methods. It can be.
이러한 조성물은 단위-용량(1회분) 또는 다중-용량(수 회분) 용기, 예를 들면, 밀봉된 앰풀 및 바이알에 제시될 수 있고, 사용 직전에 멸균성 액상 담체, 예를 들면, 주사용 수의 부가만을 요구하는 동결-건조 조건하에 저장할 수 있다. 즉석의 사용제 및 현탁제는 멸균성 산제, 과립제 및 정제로부터 제조될 수 있다.Such compositions may be presented in unit-dose (single-dose) or multi-dose (several-dose) containers, such as sealed ampoules and vials, and added immediately before use in a sterile liquid carrier, such as water for injection. It can be stored under freeze-drying conditions requiring only the addition of Extemporaneous preparations and suspensions may be prepared from sterile powders, granules and tablets.
본 발명의 일 구체예에 따르면, 용아초 추출물 및 오배자 추출물의 중량비는 1:10 내지 10:1일 수 있으며, 바람직하게는 용아초 추출물 및 오배자 추출물의 중량비는 6:4일 수 있다.According to one embodiment of the present invention, the weight ratio of the dragon fruit extract and the nut gall extract may be 1:10 to 10:1, and preferably, the weight ratio of the dragon fruit extract and the gall nut extract may be 6:4.
본 발명의 일 구체예에 따르면, 상기 백신은 코로나바이러스 백신일 수 있다.According to one embodiment of the present invention, the vaccine may be a coronavirus vaccine.
상기 코로나바이러스는 코로나바이러스아과(Coronavirinae)에 속하는 RNA 바이러스일 수 있고, 구체적으로, HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, MERS-CoV 및 SARS-CoV-2 등을 포함하나, 이에 한정되는 것은 아니다.The coronavirus may be an RNA virus belonging to the Coronavirinae family, specifically, including HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, MERS-CoV and SARS-CoV-2, etc. , but is not limited thereto.
본 발명의 일 구체예에 따르면, 상기 코로나바이러스는 SARS-CoV-2 바이러스일 수 있다.According to one embodiment of the present invention, the coronavirus may be the SARS-CoV-2 virus.
본 발명의 백신 아쥬반트 조성물에 포함되는 용아초 추출물 및 오배자 추출물의 중량비가 1:10 내지 10:1, 바람직하게는 6:4일 경우, 코로나바이러스, 특히 SARS-CoV-2 바이러스 백신의 면역 반응을 예를 들어, 10일 내지 20일 내에 효과적으로 유도할 수 있다.When the weight ratio of the dragon fruit extract and gall nut extract contained in the vaccine adjuvant composition of the present invention is 1:10 to 10:1, preferably 6:4, the immune response of the coronavirus, particularly SARS-CoV-2 virus vaccine can be effectively induced within, for example, 10 to 20 days.
본 발명의 일 구체예에 따르면, 상기 백신은 인플루엔자 바이러스 백신일 수 있다.According to one embodiment of the present invention, the vaccine may be an influenza virus vaccine.
상기 인플루엔자는 RNA 바이러스인 오르토믹소바이러스과(Orthomyxoviridae)에 포함되는 바이러스일 수 있고, 구체적으로, 인플루엔자 A, 인플루엔자 B 및/또는 인플루엔자 C 속의 바이러스일 수 있으며, 바이러스 입자의 표면에, 헤마글루티닌(HA) 및 뉴라미니다아제(NA)라고 하는 당단백질 갖는 바이러스, 예를 들어, H1N1 아형, H3N2 아형, H5N1 아형 및 H7N7 아형 등을 포함하나, 이에 한정되는 것은 아니다.The influenza may be a virus included in Orthomyxoviridae, which is an RNA virus, and specifically, may be a virus of the genus influenza A, influenza B and / or influenza C, and on the surface of the virus particle, hemagglutinin ( HA) and viruses with a glycoprotein called neuraminidase (NA), such as, but not limited to, H1N1 subtype, H3N2 subtype, H5N1 subtype and H7N7 subtype, etc.
본 발명의 백신 아쥬반트 조성물에 포함되는 용아초 추출물 및 오배자 추출물의 중량비가 1:10 내지 10:1, 바람직하게는 6:4일 경우, 인플루엔자 바이러스 백신의 면역 반응을 예를 들어, 10일 내지 20일 내에 효과적으로 유도할 수 있다.When the weight ratio of the dragon fruit extract and the gall nut extract contained in the vaccine adjuvant composition of the present invention is 1:10 to 10:1, preferably 6:4, the immune response of the influenza virus vaccine is, for example, 10 days to 10 days. Effective induction within 20 days.
본 발명의 다른 양상은 상기 조성물 및 바이러스 항원을 포함하는 백신 조성물을 제공한다.Another aspect of the present invention provides a vaccine composition comprising the composition and a viral antigen.
본 발명의 또 다른 양상은 상기 조성물을 대상체에 투여하는 단계를 포함하는 바이러스 예방 방법을 제공한다.Another aspect of the present invention provides a method for preventing a virus comprising administering the composition to a subject.
본 발명의 일 구체예에 따른 백신 조성물은 아쥬반트 조성물을 포함하여 백신의 면역 활성 효과가 증대되어, SARS-CoV-2 바이러스 및 인플루엔자 바이러스 등 바이러스 예방에 우수한 효과를 나타낸다.The vaccine composition according to one embodiment of the present invention includes an adjuvant composition to increase the immune activity of the vaccine, thereby exhibiting excellent effects in preventing viruses such as SARS-CoV-2 virus and influenza virus.
본 발명에 따른 백신 조성물의 투여량은, 사용 목적, 대상 질병(그 종류 등), 환자 연령, 체중, 병력 등을 고려하여 당업자가 용이하게 결정할 수 있으며, 대상체(또는 환자)의 투여 빈도 또한, 사용 목적, 대상 질병(그 종류, 경중도 등), 환자 연령, 체중, 병력, 경과 등을 고려하여 당업자가 용이하게 결정할 수 있다.The dosage of the vaccine composition according to the present invention can be easily determined by a person skilled in the art in consideration of the purpose of use, target disease (type thereof, etc.), patient age, weight, medical history, etc., and the frequency of administration of the subject (or patient), It can be easily determined by those skilled in the art in consideration of the purpose of use, the target disease (type, severity, etc.), patient's age, weight, medical history, progress, and the like.
본 발명에 따른 백신 조성물의 투여 빈도는 예를 들어, 매일 내지 수개월마다 투여되거나 또는 매 전염병 유행기 전에 한번 내지 두번 투여될 수 있으나, 이에 한정되는 것은 아니며, 면역원성이 유지되는 경과를 살피면서 당업자가 추가 면역 간격을 용이하게 결정할 수 있다.The frequency of administration of the vaccine composition according to the present invention may be, for example, administered every day to several months or administered once or twice before each epidemic epidemic, but is not limited thereto, and those skilled in the art while examining the progress of maintaining immunogenicity can easily determine an additional immunization interval.
본 발명의 일 구체예에 따르면, 상기 바이러스는 코로나바이러스일 수 있다.According to one embodiment of the present invention, the virus may be a coronavirus.
본 발명의 일 구체예에 따르면, 상기 바이러스는 인플루엔자 바이러스일 수 있다.According to one embodiment of the present invention, the virus may be an influenza virus.
본 발명의 바이러스 항원은 코로나바이러스 및/또는 인플루엔자 바이러스 감염에 면역 반응을 유도할 수 있는 물질이라면 제한 없이 사용될 수 있으며, 당업자에 의해 용이하게 선택될 수 있다.The viral antigen of the present invention can be used without limitation as long as it is a substance capable of inducing an immune response to infection with coronavirus and/or influenza virus, and can be easily selected by those skilled in the art.
본 발명의 백신 조성물은 하나 이상의 약학적으로 허용 가능한 백신 아쥬반트를 더 포함할 수 있다. 예를 들어, 알루미늄 하이드록사이드, 알루미늄 포스페이트, 알룸(포타슘 알루미늄 설페이트), MF59, 비로좀(virosome), AS04[알루미늄 하이드록사이드 및 모노포스포릴 리피드 A(MPL)의 혼합물], AS03(DL-αtocopherol, squalene 및 유화제인 polysorbate 80의 혼합물), CpG, 플라겔린(flagellin), Poly I:C, AS01, AS02, ISCOMs 및 ISCOMMATRIX 일 수 있으나, 이에 한정되는 것은 아니다.The vaccine composition of the present invention may further comprise one or more pharmaceutically acceptable vaccine adjuvants. For example, aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virosome, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (DL- αtocopherol, squalene, and a mixture of polysorbate 80 as an emulsifier), CpG, flagellin, Poly I:C, AS01, AS02, ISCOMs, and ISCOMMATRIX, but are not limited thereto.
본 발명의 백신 조성물의 투여량은 성인 기준으로 0.001 내지 1000㎎/㎏일 수 있으나, 이에 한정되는 것은 아니며, 상기 투여량은 환자의 나이, 성별 및 상태에 따라 당업자에 의하여 적절히 조절될 수 있다.The dose of the vaccine composition of the present invention may be 0.001 to 1000 mg/kg for adults, but is not limited thereto, and the dose may be appropriately adjusted by a person skilled in the art according to the age, sex and condition of the patient.
이하 본 발명을 하나 이상의 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through one or more embodiments. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.
제조예 1. 용아초 추출물의 제조Preparation Example 1. Preparation of Yongacho extract
용아초(Agrimonia pilosa)의 잎은 서울의 BioKorea Co LTd(Seoul)에서 구입하고, 바우처 표본(BMRI-RG-1601)은 한국 용인 경희대학교 바이오메디컬연구센터에 기탁하였다. 건조된 샘플(20㎏)을 80±2℃에서 6시간 동안 50% 에탄올수용액으로 추출한 다음 여과하였다. 그 후, 회전 증발기를 사용하여 농축시키고 동결건조하여 최종적으로 용아초 추출물 1.57㎏을 수득하였으며, 사용전까지 4℃에서 보관하였다.The leaves of Agrimonia pilosa were purchased from BioKorea Co LTd (Seoul), and the voucher specimen (BMRI-RG-1601) was deposited at the Biomedical Research Center of Kyunghee University, Yongin, Korea. A dried sample (20 kg) was extracted with a 50% aqueous ethanol solution at 80±2° C. for 6 hours and then filtered. Then, it was concentrated using a rotary evaporator and lyophilized to finally obtain 1.57 kg of Yongacho extract, which was stored at 4° C. until use.
제조예 2. 오배자 추출물의 제조Preparation Example 2. Preparation of nut gall extract
오배자(Galla rhois)의 잎은 서울의 BioKorea Co LTd(Seoul)에서 구입하였고, 바우처 표본(BMRI-RG-1602)은 한국 용인 경희대학교 바이오메디컬연구센터에 기탁하였다. 건조된 샘플(20㎏)을 80±2℃에서 6시간 동안 50% 에탄올수용액으로 추출한 다음 여과하였다. 그 후, 회전 증발기를 사용하여 농축시키고 동결건조하여 최종적으로 오배자 추출물 11.59㎏을 수득하였으며, 사용전까지 4℃에서 보관하였다. Galla rhois leaves were purchased from BioKorea Co LTd (Seoul), and voucher specimens (BMRI-RG-1602) were deposited at the Biomedical Research Center of Kyunghee University, Yongin, Korea. A dried sample (20 kg) was extracted with a 50% aqueous ethanol solution at 80±2° C. for 6 hours and then filtered. Then, it was concentrated using a rotary evaporator and lyophilized to finally obtain 11.59 kg of nut gall extract, which was stored at 4° C. until use.
제조예 3. 용아초 추출물 및 오배자 추출물의 6:4 혼합물(APRG64) 제조Preparation Example 3. Preparation of a 6:4 mixture (APRG64) of Yongacho extract and Nut gall extract
제조예 1에서 제조한 용아초 추출물과 제조예 2에서 제조한 오배자 추출물을 6:4의 중량비로 혼합하여 용아초 추출물 및 오배자 추출물의 6:4 혼합물(APRG64)을 제조하였다.A 6:4 mixture (APRG64) of Yongacho extract and Nut gall extract was prepared by mixing the Yongacho extract prepared in Preparation Example 1 and the gall nut extract prepared in Preparation Example 2 at a weight ratio of 6:4.
실시예 1. SARS-CoV-2 RBD 단백질 중화 항체 생성량 증가에 따른 APRG64의 SARS-CoV-2 바이러스 백신 아쥬반트 효과 확인Example 1. Confirmation of SARS-CoV-2 virus vaccine adjuvant effect of APRG64 according to increase in SARS-CoV-2 RBD protein neutralizing antibody production
SARS-CoV-2 바이러스 감염에 대한 APRG64의 백신 아쥬반트 효과를 확인하기 위하여, SARS-CoV-2 RBD 단백질에 대한 중화 항체 생성량을 분석하였다.In order to confirm the vaccine adjuvant effect of APRG64 against SARS-CoV-2 virus infection, the amount of neutralizing antibody against SARS-CoV-2 RBD protein was analyzed.
구체적으로, Balb/C 마우스에 10일 동안 제조예 3의 APRG64를 50㎎/㎏ 또는 100㎎/㎏의 용량으로 경구 투여하였으며, 경구 투여 6일 째 아스트라제네카(Lot. 210298, AstraZeneca) 백신을 마우스 당 6.02×109 바이러스 입자수로 근육투여하였다. 백신 투여 10일 후 및 15일 후, 96-well plate를 SARS-CoV-2(2019-nCoV) Spike RBD-His Recombinant Protein(Cat# 40592-V08B, Sinobiological)으로 코팅 (100ng/㎖)한 후, mouse serum(1:200-1:500)을 넣어 인큐베이션하고, 2차 항체(1:5000, Goat anti-mouse IgG F(ab')2, polyclonal antibody(HRP conjugate), Cat# ADI-SAB-100-J, Enzo Lifescience)와 반응시킨 후, 발색을 측정하여 ELISA 분석을 수행하였다. 이를 통하여 마우스의 혈액내에 존재하는 SARS-CoV-2 RBD 단백질에 대한 중화 항체량을 측정하였으며(도 1), 이를 APRG64를 투여하지 않은 마우스의 중화 항체량과 비교하여, APRG64에 의한 SARS-CoV-2 RBD 단백질 중화 항체 생성량 증가 여부를 분석하였다.Specifically, APRG64 of Preparation Example 3 was orally administered to Balb/C mice at a dose of 50 mg/kg or 100 mg/kg for 10 days, and on day 6 of oral administration, AstraZeneca (Lot. 210298, AstraZeneca) vaccine was administered to mice 6.02×10 9 virus particles were administered intramuscularly. After 10 and 15 days after vaccine administration, the 96-well plate was coated with SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Cat# 40592-V08B, Sinobiological) (100 ng/ml), After incubation with mouse serum (1:200-1:500), secondary antibody (1:5000, Goat anti-mouse IgG F(ab')2, polyclonal antibody (HRP conjugate), Cat# ADI-SAB-100 After reacting with -J, Enzo Lifescience), ELISA analysis was performed by measuring color development. Through this, the amount of neutralizing antibodies to the SARS-CoV-2 RBD protein present in the blood of mice was measured (Fig. 1), and compared with the amount of neutralizing antibodies in mice not administered with APRG64, SARS-CoV-2 by APRG64 2 Whether or not the amount of RBD protein neutralizing antibody production increased was analyzed.
그 결과, APRG64 농도의존적으로, APRG64를 투여한 마우스에서 백신 투여 10일 후(도 2A) 및 15일 후(도 2B) SARS-CoV-2 RBD 단백질 중화 항체가 증가한 것으로 확인되었으며, 특히, 100㎎/㎏ 투여군에서 통계적으로 유의하게 중화 항체가 증가한 것으로 확인되었다.As a result, it was confirmed that SARS-CoV-2 RBD protein neutralizing antibodies increased 10 days after vaccine administration (Fig. 2A) and 15 days after vaccine administration (Fig. 2B) in APRG64-administered mice in a concentration-dependent manner of APRG64. In particular, 100 mg It was confirmed that neutralizing antibodies increased statistically significantly in the /kg administration group.
실시예 2. SARS-CoV-2 S1 단백질 중화 항체 생성량 증가에 따른 APRG64의 SARS-CoV-2 바이러스 백신 아쥬반트 효과 확인Example 2. Confirmation of SARS-CoV-2 virus vaccine adjuvant effect of APRG64 according to the increase in SARS-CoV-2 S1 protein neutralizing antibody production
SARS-CoV-2 바이러스 감염에 대한 APRG64의 백신 아쥬반트 효과를 확인하기 위하여, SARS-CoV-2 S1 단백질에 대한 중화 항체 생성량을 분석하였다.In order to confirm the vaccine adjuvant effect of APRG64 against SARS-CoV-2 virus infection, the amount of neutralizing antibody against SARS-CoV-2 S1 protein was analyzed.
구체적으로, Balb/C 마우스에 10일 동안 제조예 3의 APRG64를 50㎎/㎏ 또는 100㎎/㎏의 용량으로 경구 투여하였으며, 경구 투여 6일 째 아스트라제네카(Lot. 210298, AstraZeneca) 백신을 마우스 당 6.02×109 바이러스 입자수로 근육투여하였다. 백신 투여 10일 후, 15일 후 및 20일 후, 96-well plate를 SARS-CoV-2(2019-nCoV) Spike S1-His Recombinant Protein(Cat# 40591-V08B1, Sinobiological)으로 코팅(100ng/㎖)한 후, mouse serum(1:200-1:500)을 넣어 인큐베이션하고, 2차 항체(1:5000, Goat anti-mouse IgG F(ab')2, polyclonal antibody(HRP conjugate), Cat# ADI-SAB-100-J, Enzo Lifescience)와 반응시킨 후, 발색을 측정하여 ELISA 분석을 수행하였다. 이를 통하여 마우스의 혈액내에 존재하는 SARS-CoV-2 S1 단백질에 대한 중화 항체량을 분석하였으며(도 3), 이를 APRG64를 투여하지 않은 마우스의 중화 항체량과 비교하여, APRG64에 의한 SARS-CoV-2 S1 단백질 중화 항체 생성량 증가 여부를 분석하였다.Specifically, APRG64 of Preparation Example 3 was orally administered to Balb/C mice at a dose of 50 mg/kg or 100 mg/kg for 10 days, and on day 6 of oral administration, AstraZeneca (Lot. 210298, AstraZeneca) vaccine was administered to mice 6.02×10 9 virus particles were administered intramuscularly. After 10 days, 15 days and 20 days after vaccine administration, a 96-well plate was coated with SARS-CoV-2 (2019-nCoV) Spike S1-His Recombinant Protein (Cat# 40591-V08B1, Sinobiological) (100 ng/ml) ), followed by incubation with mouse serum (1:200-1:500), secondary antibody (1:5000, Goat anti-mouse IgG F(ab')2, polyclonal antibody (HRP conjugate), Cat# ADI After reacting with -SAB-100-J, Enzo Lifescience), ELISA analysis was performed by measuring color development. Through this, the amount of neutralizing antibodies to the SARS-CoV-2 S1 protein present in the blood of mice was analyzed (FIG. 3), and this was compared with the amount of neutralizing antibodies in mice not administered with APRG64. 2 Whether or not the amount of S1 neutralizing antibody production increased was analyzed.
그 결과, 100㎎/㎏ 투여군에서 백신 투여 10일 후(도 4A) 및 15일 후(도 4B) SARS-CoV-2 S1 단백질 중화 항체가 증가한 것으로 확인되었으며, 특히, 백신 투여 20일 후(도 4C) 통계적으로 유의하게 중화 항체가 증가한 것으로 확인되었다.As a result, it was confirmed that the SARS-CoV-2 S1 protein neutralizing antibody increased 10 days (Fig. 4A) and 15 days (Fig. 4B) after vaccine administration in the 100 mg/kg administration group, in particular, 20 days after vaccine administration (Fig. 4B). 4C) A statistically significant increase in neutralizing antibodies was confirmed.
실시예 3. CD8Example 3. CD8 ++ T 림프구에 대한 APRG64의 SARS-CoV-2 바이러스 백신 아쥬반트 효과 확인 Confirmation of SARS-CoV-2 antivirus adjuvant effect of APRG64 on T lymphocytes
3-1. CD83-1. CD8 ++ T 림프구 증가 효과 확인 Confirmation of the effect of increasing T lymphocytes
SARS-CoV-2 바이러스 감염에 대한 APRG64의 백신 아쥬반트 효과를 확인하기 위하여, CD3+ 세포에 대한 CD8+ T 림프구의 비율을 분석하였다.To confirm the vaccine adjuvant effect of APRG64 against SARS-CoV-2 virus infection, the ratio of CD8 + T lymphocytes to CD3 + cells was analyzed.
구체적으로, Balb/C 마우스에 10일 동안 제조예 3의 APRG64를 50㎎/㎏ 또는 100㎎/㎏의 용량으로 경구 투여하였으며, 경구 투여 6일 째 아스트라제네카(Lot. 210298, AstraZeneca) 백신을 마우스 당 6.02×109 바이러스 입자수로 근육투여하였다. 백신 투여 20일 후, 마우스의 혈액에서 혈액단핵구를 분리하고, anti-CD3 및 anti-CD8 항체와 반응시킨 다음, 유세포 분석기를 이용하여 CD3+ 세포에 대한 CD8+ T 림프구의 비율을 분석하였으며(도 5), 이를 APRG64를 투여하지 않은 마우스의 CD8+ T 림프구의 비율과 비교하여, APRG64에 의한 CD8+ T 림프구의 증가 여부를 분석하였다.Specifically, APRG64 of Preparation Example 3 was orally administered to Balb/C mice at a dose of 50 mg/kg or 100 mg/kg for 10 days, and on day 6 of oral administration, AstraZeneca (Lot. 210298, AstraZeneca) vaccine was administered to mice 6.02×10 9 virus particles were administered intramuscularly. 20 days after vaccination, blood monocytes were isolated from the blood of mice, reacted with anti-CD3 and anti-CD8 antibodies, and the ratio of CD8 + T lymphocytes to CD3 + cells was analyzed using flow cytometry (Fig. 5), by comparing this with the ratio of CD8 + T lymphocytes in mice not administered with APRG64, the increase in CD8 + T lymphocytes by APRG64 was analyzed.
그 결과, 50㎎/㎏ 및 100㎎/㎏ 투여군 모두에서 백신 투여 20일 후 마우스 혈액 내 CD8+ T 림프구의 비율이 현저히 증가한 것으로 확인되었다(도 6).As a result, it was confirmed that the ratio of CD8 + T lymphocytes in mouse blood increased significantly 20 days after vaccine administration in both the 50 mg/kg and 100 mg/kg administration groups (FIG. 6).
3-2. IFN-gamma 발현 T 림프구 증가 효과 확인3-2. Confirmation of the effect of increasing IFN-gamma-expressing T lymphocytes
SARS-CoV-2 바이러스 감염에 대한 APRG64의 백신 아쥬반트 효과를 확인하기 위하여, IFN-gamma를 발현하는 T 림프구를 분석하였다.To confirm the vaccine adjuvant effect of APRG64 against SARS-CoV-2 virus infection, T lymphocytes expressing IFN-gamma were analyzed.
구체적으로, Balb/C 마우스에 10일 동안 제조예 3의 APRG64를 50㎎/㎏ 또는 100㎎/㎏의 용량으로 경구 투여하였으며, 경구 투여 6일 째 아스트라제네카(AstraZeneca) 백신을 마우스 당 6.02×109 바이러스 입자수로 근육투여하였다. 백신 투여 20일 후, SARS-CoV-2 S1 peptide(SARS-CoV-2 S1 scanning pool, Cat# 3629-1, Mabtech)에 반응하여 IFN-gamma를 생산하는 세포 수를 ELISPOT(Mouse IFN-gamma ELISpotBASIC (ALP) kit, cat# 3321-2A, Mabtech)을 통해 분석함으로써, 마우스의 혈액에서 IFN-gamma를 발현하는 T 림프구의 수를 분석하였으며(도 7), 이를 APRG64를 투여하지 않은 마우스의 IFN-gamma를 발현하는 T 림프구와 비교하여, APRG64에 의한 IFN-gamma를 발현하는 T 림프구 증가 여부를 분석하였다.Specifically, APRG64 of Preparation Example 3 was orally administered to Balb/C mice at a dose of 50 mg/kg or 100 mg/kg for 10 days, and on day 6 of oral administration, AstraZeneca vaccine was administered at a dose of 6.02 × 10 per mouse. 9 virus particles were administered intramuscularly. 20 days after vaccine administration, the number of cells producing IFN-gamma in response to the SARS-CoV-2 S1 peptide (SARS-CoV-2 S1 scanning pool, Cat# 3629-1, Mabtech) was measured by ELISPOT (Mouse IFN-gamma ELISpotBASIC (ALP) kit, cat# 3321-2A, Mabtech), the number of T lymphocytes expressing IFN-gamma in the blood of mice was analyzed (Fig. Compared to gamma-expressing T lymphocytes, the increase in IFN-gamma-expressing T lymphocytes by APRG64 was analyzed.
그 결과, APRG64 농도의존적으로, 50㎎/㎏ 및 100㎎/㎏ 투여군 모두에서 백신 투여 20일 후 마우스 혈액 내 IFN-gamma를 발현하는 T 림프구의 수가 현저히 증가한 것으로 확인되었다(도 8).As a result, it was confirmed that the number of IFN-gamma-expressing T lymphocytes in mouse blood increased significantly in both the 50 mg/kg and 100 mg/kg administration groups 20 days after vaccine administration in an APRG64 concentration-dependent manner (FIG. 8).
실시예 4. 혈중 염증유발 사이토카인에 대한 APRG64의 SARS-CoV-2 바이러스 백신 아쥬반트 효과 확인Example 4. Confirmation of SARS-CoV-2 virus vaccine adjuvant effect of APRG64 on blood inflammatory cytokines
SARS-CoV-2 바이러스 감염에 대한 APRG64의 백신 아쥬반트 효과를 확인하기 위하여, 혈중 염증유발 사이토카인의 농도를 분석하였다.In order to confirm the vaccine adjuvant effect of APRG64 against SARS-CoV-2 virus infection, the concentration of pro-inflammatory cytokines in blood was analyzed.
구체적으로, Balb/C 마우스에 5일 동안 제조예 3의 APRG64를 경구 투여한 후, 마우스 혈액 내 존재하는 혈중 염증유발 사이토카인인 IL-1α, IL-1β, IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, MCP-1, IFN-β, IFN-γ, TNF-α 및 GM-CSF의 농도를 분석하였으며(도 9), 이를 APRG64를 투여하지 않은 마우스의 혈중 염증유발 사이토카인과 비교하여, APRG64에 의한 혈중 염증유발 사이토카인 농도 감소 효과를 분석하였다.Specifically, after orally administering APRG64 of Preparation Example 3 to Balb/C mice for 5 days, proinflammatory cytokines IL-1α, IL-1β, IL-6, IL-10, and IL present in mouse blood - The concentrations of 12p70, IL-17A, IL-23, IL-27, MCP-1, IFN-β, IFN-γ, TNF-α and GM-CSF were analyzed (FIG. 9), which were analyzed without APRG64 administration. Compared to blood inflammatory cytokines of mice, the effect of reducing blood inflammatory cytokine concentration by APRG64 was analyzed.
GM-CSF(도 9A), IL-23(도 9B), IL-1α(도 9C), IFN-γ(도 9D), TNF-α(도 9E), MCP-1(도 9F), IL-12p70(도 9G), IL-1β(도 9H), IL-10(도 9I), IL-6(도 9J), IL-27(도 9K), IL-17A(도 9L), IFN-β(도 9M)의 농도를 분석한 결과, 혈중 염증유발 사이토카인 중 GM-CSF, MCP-1, IL-1α 및 IL-10에서 APRG64의 투여에 의한 농도 감소 효과가 확인되었으며, 그 외의 혈중 염증유발 사이토카인 수준은 APRG64 무처리군과 유사한 것으로 확인되었다.GM-CSF (FIG. 9A), IL-23 (FIG. 9B), IL-1α (FIG. 9C), IFN-γ (FIG. 9D), TNF-α (FIG. 9E), MCP-1 (FIG. 9F), IL- 12p70 (Fig. 9G), IL-1β (Fig. 9H), IL-10 (Fig. 9I), IL-6 (Fig. 9J), IL-27 (Fig. 9K), IL-17A (Fig. 9L), IFN-β ( As a result of analyzing the concentration of (Fig. 9M), the effect of reducing the concentration of APRG64 was confirmed in GM-CSF, MCP-1, IL-1α and IL-10 among the cytokines inducing inflammation in the blood, and other cytokines inducing inflammation in the blood The caine level was confirmed to be similar to that of the APRG64 untreated group.
실시예 5. 인플루엔자 특이적 항체 생성량 증가에 따른 APRG64의 인플루엔자 백신 아쥬반트 효과 확인Example 5. Confirmation of influenza vaccine adjuvant effect of APRG64 according to the increase in influenza-specific antibody production
인플루엔자 바이러스 감염에 대한 APRG64의 백신 아쥬반트 효과를 확인하기 위하여, 인플루엔자 특이적 항체 생성량을 분석하였다.In order to confirm the vaccine adjuvant effect of APRG64 on influenza virus infection, the amount of influenza-specific antibody production was analyzed.
구체적으로, Balb/C 마우스에 10일 동안 제조예 3의 APRG64를 50㎎/㎏ 또는 100㎎/㎏의 용량으로 경구 투여하였으며, 경구 투여 6일 째 마우스 당 인플루엔자 백신(Lot. Q022024, SKY　Cellflu　Quadrivalent) 500㎕를 근육투여하였다. 백신 투여 10일 후 및 20일 후, 실온에서 2시간 동안 96-well plate를 PBS 중 불활성화된 인플루엔자 백신(inactivated influenza vaccine, IIV)으로 코팅(300ng/50㎕)하고, 1% BSA 및 PBS-T 150㎕를 처리하여 실온에서 2시간 동안 블로킹한 다음, mouse serum(1:200)을 넣어 4℃에서 밤새 인큐베이션하고, 2차 항체(1:5000, Goat anti-mouse IgG(HRP conjugate), BML-SA204-0100, Enzo Lifescience)와 반응시킨 후, 발색을 측정하여 ELISA 분석을 수행하였다. 이를 통하여 마우스의 혈액내에 존재하는 SARS-CoV-2 S1 단백질에 대한 중화 항체량을 분석하였으며(도 10), 이를 APRG64를 투여하지 않은 마우스의 인플루엔자 특이적 항체량과 비교하여, APRG64에 의한 인플루엔자 특이적 항체 생성량 증가 여부를 분석하였다.Specifically, APRG64 of Preparation Example 3 was orally administered to Balb/C mice at a dose of 50 mg/kg or 100 mg/kg for 10 days, and on day 6 of oral administration, influenza vaccine per mouse (Lot. Q022024, SKY Cellflu Quadrivalent ) 500 μl was administered intramuscularly. 10 and 20 days after vaccine administration, 96-well plates were coated (300 ng/50 μl) with inactivated influenza vaccine (IIV) in PBS for 2 hours at room temperature, 1% BSA and PBS- 150 μl of T was treated and blocked at room temperature for 2 hours, then mouse serum (1:200) was added and incubated overnight at 4°C, secondary antibody (1:5000, Goat anti-mouse IgG (HRP conjugate), BML After reacting with -SA204-0100, Enzo Lifescience), ELISA analysis was performed by measuring color development. Through this, the amount of neutralizing antibodies to the SARS-CoV-2 S1 protein present in the blood of mice was analyzed (FIG. 10), and compared with the amount of influenza-specific antibodies in mice not administered with APRG64, influenza-specific antibodies by APRG64 were analyzed. Whether or not an increase in the amount of enemy antibody production was analyzed.
그 결과, 50㎎/㎏ 및 100㎎/㎏ 투여군에서 백신 투여 10일 후(도 11A) 및 20일 후(도 11B) 통계적으로 유의하게 인플루엔자 특이적 항체가 증가한 것으로 확인되었다.As a result, it was confirmed that influenza-specific antibodies increased statistically significantly in the 50 mg/kg and 100 mg/kg administration groups 10 days (FIG. 11A) and 20 days (FIG. 11B) after vaccine administration.
이와 같은 결과를 통하여, 용아초 추출물 및 오배자 추출물의 6:4 혼합물(APRG64)은 SARS-CoV-2 바이러스 백신 및 인플루엔자 바이러스 백신 등 바이러스 백신의 효과적인 아쥬반트로 사용될 수 있음이 확인되었다.Through these results, it was confirmed that the 6: 4 mixture (APRG64) of the extract of dragon plant extract and gall nut extract can be used as an effective adjuvant for viral vaccines such as SARS-CoV-2 virus vaccine and influenza virus vaccine.
이제까지 본 발명에 대하여 그 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been examined focusing on the embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be construed as being included in the present invention.

Claims (12)

  1. 용아초 추출물 및 오배자 추출물을 포함하는 백신 아쥬반트(adjuvant) 조성물.Vaccine adjuvant (adjuvant) composition comprising a dragon plant extract and nut gall extract.
  2. 제 1 항에 있어서, 용아초 추출물 및 오배자 추출물의 중량비는 1:10 내지 10:1인 것인 백신 아쥬반트 조성물.The vaccine adjuvant composition according to claim 1, wherein the weight ratio of the dragon fruit extract and the nut gall extract is 1:10 to 10:1.
  3. 제 1 항에 있어서, 상기 백신은 코로나바이러스 백신인 것인 백신 아쥬반트 조성물.The vaccine adjuvant composition according to claim 1, wherein the vaccine is a coronavirus vaccine.
  4. 제 3 항에 있어서, 상기 코로나바이러스는 SARS-CoV-2 바이러스인 것인 백신 아쥬반트 조성물.4. The vaccine adjuvant composition according to claim 3, wherein the coronavirus is the SARS-CoV-2 virus.
  5. 제 1 항에 있어서, 상기 백신은 인플루엔자 바이러스 백신인 것인 백신 아쥬반트 조성물.The vaccine adjuvant composition according to claim 1, wherein the vaccine is an influenza virus vaccine.
  6. 제 1 항에 있어서, 상기 추출물의 추출용매는 에탄올수용액인 것인 백신 아쥬반트 조성물.The vaccine adjuvant composition according to claim 1, wherein the extraction solvent of the extract is an aqueous ethanol solution.
  7. 제 6 항에 있어서, 상기 에탄올수용액의 에탄올 및 물의 부피비는 5:5인 것인 백신 아쥬반트 조성물.The vaccine adjuvant composition according to claim 6, wherein the volume ratio of ethanol and water in the aqueous ethanol solution is 5:5.
  8. 제 1 항의 조성물 및 바이러스 항원을 포함하는 백신 조성물.A vaccine composition comprising the composition of claim 1 and a viral antigen.
  9. 제 8 항에 있어서, 상기 바이러스는 코로나바이러스인 것인 백신 조성물.9. The vaccine composition according to claim 8, wherein the virus is a coronavirus.
  10. 제 8 항에 있어서, 상기 바이러스는 인플루엔자 바이러스인 것인 백신 조성물.The vaccine composition according to claim 8, wherein the virus is an influenza virus.
  11. 제 1 항의 조성물을 대상체에 투여하는 단계를 포함하는 백신 면역 증강 방법.A vaccine immunity enhancement method comprising administering the composition of claim 1 to a subject.
  12. 제 8 항의 조성물을 대상체에 투여하는 단계를 포함하는 바이러스 예방 방법.A method for preventing a virus comprising administering the composition of claim 8 to a subject.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20090072527A (en) * 2007-12-28 2009-07-02 삼광생명과학 주식회사 A composition comprising the extract of galla rhois or the compounds isolated therefrom showing antibacterial activity
CN111529702A (en) * 2020-05-27 2020-08-14 山东绿都生物科技有限公司 Traditional Chinese medicine composite newcastle disease vaccine and preparation method thereof
KR20200130177A (en) * 2019-05-09 2020-11-18 경희대학교 산학협력단 A composition for the prevention or treatment of hepatitis C virus infection disease comprising a compound isolated from Agrimonia pilosa extract and Galla rhois extract
US20200397711A1 (en) * 2019-06-22 2020-12-24 Gregory Brian LEE Microparticle compositions for treatment of infection or disease, methods of making the same, and methods of treating subjects with microparticle compositions

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180008702A1 (en) 2014-12-05 2018-01-11 Celltrion Inc. Adjuvant composition containing at least one influenza virus neutralizing and binding molecule and vaccine composition containing same

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20090072527A (en) * 2007-12-28 2009-07-02 삼광생명과학 주식회사 A composition comprising the extract of galla rhois or the compounds isolated therefrom showing antibacterial activity
KR20200130177A (en) * 2019-05-09 2020-11-18 경희대학교 산학협력단 A composition for the prevention or treatment of hepatitis C virus infection disease comprising a compound isolated from Agrimonia pilosa extract and Galla rhois extract
US20200397711A1 (en) * 2019-06-22 2020-12-24 Gregory Brian LEE Microparticle compositions for treatment of infection or disease, methods of making the same, and methods of treating subjects with microparticle compositions
CN111529702A (en) * 2020-05-27 2020-08-14 山东绿都生物科技有限公司 Traditional Chinese medicine composite newcastle disease vaccine and preparation method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LEE YEONG-GEUN, KANG KYUNG WON, HONG WOOJAE, KIM YEON HWA, OH JEN TAEK, PARK DAE WON, KO MINSUNG, BAI YUN-FENG, SEO YOUNG-JIN, LEE: "Potent antiviral activity of Agrimonia pilosa, Galla rhois, and their components against SARS-CoV-2", BIOORGANIC & MEDICINAL CHEMISTRY, ELSEVIER, AMSTERDAM, NL, vol. 45, 1 September 2021 (2021-09-01), AMSTERDAM, NL, pages 116329, XP093028750, ISSN: 0968-0896, DOI: 10.1016/j.bmc.2021.116329 *

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