WO2024066288A1 - 一种疫苗佐剂、疫苗组合物及其应用 - Google Patents

一种疫苗佐剂、疫苗组合物及其应用 Download PDF

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WO2024066288A1
WO2024066288A1 PCT/CN2023/087304 CN2023087304W WO2024066288A1 WO 2024066288 A1 WO2024066288 A1 WO 2024066288A1 CN 2023087304 W CN2023087304 W CN 2023087304W WO 2024066288 A1 WO2024066288 A1 WO 2024066288A1
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vaccine
aluminum salt
adjuvant
gaps
hepatitis
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PCT/CN2023/087304
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English (en)
French (fr)
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翟健秀
殷军
廖辉
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沈阳药科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55583—Polysaccharides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to the field of medical technology, and in particular to a ginseng acid polysaccharide (GAPS) vaccine adjuvant, a vaccine composition and applications thereof.
  • GAPS ginseng acid polysaccharide
  • Vaccines can initiate humoral immune responses in organisms to produce antibodies, or activate cytotoxic T cells and other lymphocytes through cellular immune responses to resist invading foreign pathogens and prevent the occurrence of diseases (Cavallo Fet al., Vaccination for treatment and prevention of cancer in animal models. Adv Immunol. 2006.90:175-213. Review). Although vaccines have the effect of activating the immune system, in clinical use, it is often found that they cannot exert their due effectiveness in certain groups with weak immune systems, such as the elderly and children. Therefore, it is necessary to add an appropriate amount of vaccine adjuvant.
  • Vaccine adjuvants refer to substances that can non-specifically change or enhance the body's specific immune response to antigens. They are required to be non-toxic, high-purity, have a certain adsorption capacity and stable properties.
  • the mechanism of action of vaccine adjuvants is mainly to increase the surface area of antigens and improve immunogenicity; to have a sustained release effect on antigens and prolong the retention time of antigens in tissues; to promote inflammatory responses and stimulate active immune responses.
  • adjuvants can usually be divided into two categories. The first is to adsorb antigens to help them be phagocytosed by cells, such as aluminum salts and M59 emulsifiers (O'Hagan DT, Wack A, Podda A. MF59 is a safe and potent vaccine adjuvant for flu vaccines in humans: what did we learn during its development? Clin Pharmacol Ther. 2007Dec; 82(6): 740-4; 4. Clapp T, Siebert P, Chen D, Jones Braun L. Vaccines with aluminum-containing adjuvants: optimizing vaccine efficacy and thermal stability. J Pharm Sci.
  • CFA-mycobacteria mycobacteria
  • Hoft DF Blazevic A, Abate G, Hanekom WA, Kaplan G, Soler JH, Weichold F, Geiter L, Sadoff JC, Horwitz MA.
  • a new recombinant bacille Calmette-Guérin vaccine safely induces significantly enhancedtuberculosis-specific immunity in human volunteers. J Infect Dis. 2008 Nov 15; 198(10): 1491-501).
  • vaccine adjuvants are aluminum salt adjuvants, but aluminum salts can cause inflammation at the injection site and stimulate local erythema, granulomas and subcutaneous nodules.
  • aluminum salt adjuvants may delay the production of neutralizing antibodies in some vaccines and are subject to various restrictions in their application. Therefore, it is urgent to develop novel adjuvants to enhance the antigen specificity or anti-tumor and anti-infection capabilities of vaccines.
  • the present application provides an application of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt as a vaccine adjuvant, which has good activity and can improve the immune effect of the vaccine on the body.
  • GAPS ginseng acid polysaccharide
  • the vaccines involved include but are not limited to: rabies vaccine, influenza vaccine, hepatitis B vaccine, hepatitis A vaccine, hepatitis C vaccine, hand, foot and mouth disease vaccine, HPV vaccine, and new coronavirus vaccine.
  • a vaccine adjuvant comprising a ginseng acid polysaccharide adjuvant and an aluminum salt adjuvant.
  • the mass ratio of the ginseng acid polysaccharide adjuvant to the aluminum salt adjuvant is 0.0001:1 to 1000:1.
  • the vaccine composition also includes physiological saline or water for vaccine injection.
  • the extracted total ginseng polysaccharides were dissolved in deionized water.
  • the eluate was dialyzed against distilled water.
  • the column for column chromatography is a DEAE cellulose column or a macroporous resin column, preferably a DEAE cellulose column, and further preferably, the column chromatography is loaded at 2 to 8 mL/min.
  • the eluent is water and an alkaline solution, a borax solution or a saline solution
  • the saline solution is preferably a NaCl solution
  • the concentration of the eluent is 0.3 to 0.7 mol/L
  • the elution flow rate is 0.5 to 2 mL/min.
  • the step further comprises detecting A 490 of the eluate with a phenol-sulfuric acid method, collecting the absorption peak, and then dialyzing.
  • the dialysis time is 24 to 72 hours.
  • a vaccine composition comprising the vaccine adjuvant described in any one of items 1 to 8 and an antigen or a DNA encoding the antigen.
  • the ratio of the sum of the amounts of GAPS and aluminum salt adjuvant to the vaccine antigen is: ( ⁇ g/IU or ⁇ g: ⁇ g),
  • the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g); preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g); preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g); preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g); preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g); preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g); preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g); preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g); preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g); preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g). preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g).
  • the vaccine composition according to item 10 further comprising a pharmaceutical excipient and a second vaccine adjuvant.
  • a method for preventing and/or treating a disease comprising administering the vaccine composition described in any one of items 9 to 13 to a subject.
  • the disease is selected from rabies, influenza, hepatitis B, hepatitis A, hepatitis C, hand, foot and mouth disease, HPV disease or new coronavirus disease.
  • the active ingredient ginseng acidic polysaccharide GAPS disclosed in the present application is mixed with aluminum salt for use as a vaccine adjuvant, which has a high adjuvant activity.
  • Acidic polysaccharides have a high negative charge density due to their high content of sulfate and carboxylate groups, and therefore may participate in a variety of enzymatic reactions and immune activities in the body (for example, heparin, which is widely present in animals, is a small molecule acidic polysaccharide).
  • Acidic polysaccharides are widely derived from animals, plants, and microorganisms, and their monosaccharide composition, the type and number of acidic groups on the sugar chain vary depending on the source species.
  • acidic polysaccharides from plants are macromolecular structures, and are mostly used as water-soluble dietary fibers, and research on their adjuvant activity is very limited.
  • the present application has undergone a large number of screening tests, and it was found in the activity screening process that ginseng acid polysaccharide has high adjuvant activity.
  • ginseng acid polysaccharide GAPS and aluminum salt adjuvant are mixed and used together as vaccine adjuvants, the immune response activity of various vaccines can be effectively enhanced.
  • Aluminum salt adjuvants are commonly used vaccine adjuvants with mature technology and stable effects, but limited effects.
  • ginseng acid polysaccharide GAPS and aluminum salt are used together as adjuvants in the present application, their adjuvant activity is significantly better than that of aluminum salt vaccine adjuvants, especially ginseng acid polysaccharide comes from natural plants, with a clear source, abundant resources, and the ingredients have good safety and stability.
  • the mixture of GAPS and aluminum salt described in the present application can significantly increase the titer of specific antibodies (or neutralizing antibodies) after antigen immunization, and can effectively enhance the body's immune response level to the vaccine. Its activity is significantly stronger than that of a single aluminum salt adjuvant.
  • the vaccine adjuvant of the present application is a mixture of GAPS and aluminum salt, which has the advantages of good immune effect and easy use, and provides a new adjuvant option for vaccines.
  • Figure 1 is the elution-absorbance curve of ginseng polysaccharide
  • Fig. 2 is a standard curve of absorbance-glucose content
  • Figure 3 is an absorbance-protein content standard curve
  • FIG 4 shows the effects of the mixture of "GAPS + aluminum salt” and “GPS + aluminum salt” on the neutralizing antibody level in mice immunized with influenza vaccine
  • FIG. 5 shows the effects of different doses of aluminum salt on the neutralizing antibody levels in mice immunized with influenza vaccine
  • FIG 6 shows the effect of different total doses of "GAPS + aluminum salt” mixture on neutralizing antibodies in mice immunized with influenza vaccine The impact of level
  • Figure 7 shows the effects of different ratios of "GAPS+aluminum salt” mixture on the neutralizing antibody levels in mice immunized with influenza vaccine
  • Figure 8 shows the effects of the mixture of "GAPS + aluminum salt” and "GPS + aluminum salt” on the IgG antibody level of mice immunized with rabies vaccine
  • Figure 9 shows the effects of the mixture of "GAPS + aluminum salt” and “GPS + aluminum salt” on the neutralizing antibody titer in mice immunized with rabies vaccine
  • Figure 10 shows the effects of the mixture of "GAPS + aluminum salt” and “GPS + aluminum salt” on the IgG antibody titer of mice immunized with hepatitis B antigen;
  • Figure 11 shows the effects of the mixture of "GAPS + aluminum salt” and “GPS + aluminum salt” on the neutralizing antibody level in mice immunized with hepatitis A vaccine
  • Figure 12 shows the effects of different total doses of the mixture of "GAPS + aluminum salt” on the neutralizing antibody levels in mice immunized with hepatitis A vaccine
  • Figure 13 shows the effects of different ratios of "GAPS+aluminum salt” mixture on the neutralizing antibody level in mice immunized with hepatitis A vaccine
  • Figure 14 shows the effects of the mixture of "GAPS + aluminum salt” and “GPS + aluminum salt” on the IgG antibody level of mice immunized with hepatitis C vaccine;
  • Figure 15 shows the effects of the mixture of "GAPS + aluminum salt” and “GPS + aluminum salt” on the neutralizing antibody level in mice immunized with hand, foot and mouth disease vaccine;
  • Figure 16 shows the effects of the mixture of "GAPS + aluminum salt” and “GPS + aluminum salt” on the IgG antibody level of mice immunized with hand, foot and mouth disease vaccine;
  • Figure 17 shows the effects of different total doses of "GAPS + aluminum salt” mixture on the neutralizing antibody level in mice immunized with hand, foot and mouth disease vaccine;
  • Figure 18 shows the effects of different ratios of "GAPS + aluminum salt” mixture on the neutralizing antibody level in mice immunized with hand, foot and mouth disease vaccine
  • Figure 19 shows the effects of the mixture of "GAPS + aluminum salt” and “GPS + aluminum salt” on the IgG antibody titer of HPV vaccine-immunized mice;
  • Figure 20 shows the effects of different total doses of the mixture of "GAPS + aluminum salt” on the neutralizing antibody levels in HPV vaccine-immunized mice;
  • Figure 21 shows the effects of different ratios of "GAPS+aluminum salt” mixture on the neutralizing antibody levels in HPV vaccine-immunized mice;
  • Figure 22 shows the effects of the mixture of "GAPS+aluminum salt” and "GPS+aluminum salt” on the IgG antibody levels of mice immunized with the new coronavirus vaccine.
  • Figure 23 shows the effects of different total doses of a mixture of "GAPS + aluminum salt" on the neutralizing antibody levels of mice immunized with the COVID-19 vaccine;
  • Figure 24 shows the effects of different proportions of "GAPS+aluminum salt” mixture on the neutralizing antibody levels of mice immunized with the new crown vaccine
  • * indicates P ⁇ 0.05 compared with the positive control group; ** indicates P ⁇ 0.01 compared with the positive control group; *** indicates P ⁇ 0.005 compared with the positive control group; # indicates P ⁇ 0.05 compared with the negative control group; ## indicates P ⁇ 0.01 compared with the negative control group; ### indicates P ⁇ 0.005 compared with the negative control group.
  • the present application discloses a vaccine adjuvant, comprising a ginseng acid polysaccharide adjuvant and an aluminum salt adjuvant.
  • vaccine refers to any preparation of antigenic or immunogenic substance suitable for stimulating active immunity in animals or humans.
  • adjuvant refers to any substance or mixture of substances that enhances, increases, upregulates, alters or otherwise promotes an immune response (eg, a humoral or cellular immune response) to an antigen in an animal.
  • the term "antigen" refers to any substance that stimulates a humoral and/or cell-mediated immune response when introduced into an immunocompetent human or animal.
  • the antigen may be a pure substance, a mixture of substances, or particulate matter (including cells, cell fragments, or cell-derived fragments) or a live (usually attenuated) organism or virus.
  • suitable antigens include, but are not limited to, proteins, glycoproteins, lipoproteins, peptides, carbohydrates/polysaccharides, lipopolysaccharides, toxins, viruses, bacteria, fungi, and parasites.
  • suitable antigens include the smallest component of an antigen, such as, but not limited to, an antigenic determinant, epitope, or peptide. Still other suitable antigens include those described in U.S. Pat. No. 5,855,894
  • the antigen may be natural (naturally expressed or produced), synthetic, or derived by recombinant DNA methodology familiar to those skilled in the art.
  • the vaccine adjuvant further comprises physiological saline or water for vaccine injection or pharmaceutical excipients.
  • the vaccine adjuvant consists of a ginseng acid polysaccharide adjuvant, an aluminum salt adjuvant and physiological saline or water for vaccine injection or a pharmaceutical excipient, such as, the vaccine adjuvant consists of a ginseng acid polysaccharide adjuvant, an aluminum salt adjuvant and physiological saline, such as, the vaccine adjuvant consists of a ginseng acid polysaccharide adjuvant, an aluminum salt adjuvant and water for vaccine injection, such as, the ginseng acid polysaccharide adjuvant, an aluminum salt adjuvant and pharmaceutical excipients.
  • the term "pharmaceutical excipient” refers to a general term for all medicinal materials other than the main drug added to the prescription to solve the formability, effectiveness, stability and safety of the preparation when producing medicines and preparing prescriptions. It has been reasonably evaluated in terms of safety and is contained in the drug preparation.
  • pharmaceutical excipients serving as carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, solubilization, and sustained release. They are important ingredients that may affect the quality, safety and effectiveness of drugs.
  • the pharmaceutical excipients described in this application can be appropriate carriers or excipients, emulsifiers, wetting agents, preservatives, stabilizers, antioxidants, adjuvants (such as aluminum hydroxide adjuvants, oil adjuvants, Freund's complete adjuvants and Freund's incomplete adjuvants), etc.
  • adjuvants such as aluminum hydroxide adjuvants, oil adjuvants, Freund's complete adjuvants and Freund's incomplete adjuvants
  • the mass ratio of the ginseng acid polysaccharide adjuvant to the aluminum salt adjuvant is 0.0001:1 to 1000:1, for example, it can be 0.0001:1, 0.0005:1, 0.001:1, 0.005:1, 0.01:1, 0.05:1, 0.1:1, 0.5:1, 1:1, 5:1, 10:1, 50:1, 100:1, 500:1, 1000:1.
  • ginseng acid polysaccharide refers to an acid polysaccharide extracted from the Araliaceae plant ginseng.
  • the extraction from the Araliaceae plant ginseng can be extracted from raw ginseng, or from processed ginseng, such as from secondary development products of ginseng, such as from total ginseng polysaccharides extracted from ginseng, such as from other parts remaining after the total ginseng polysaccharides extracted from ginseng, such as from ginseng extraction waste, such as from ginseng dregs, etc.
  • the ginseng acid polysaccharide is extracted from any ginseng product containing ginseng acid polysaccharide components, it can be used as the vaccine adjuvant of the present application.
  • the ginseng acid polysaccharide is purchased, in a preferred embodiment, the ginseng acid polysaccharide is extracted from ginseng, in a preferred embodiment, the ginseng acid polysaccharide is extracted from ginseng polysaccharide, in a preferred embodiment In the method, the ginseng acid polysaccharide is extracted by the following method:
  • the extracted total ginseng polysaccharides were dissolved in deionized water.
  • the eluate was dialyzed against distilled water.
  • ginseng polysaccharide refers to the total polysaccharide extracted from the Araliaceae plant ginseng. In this application, it has the same meaning as the term “ginseng total polysaccharide” and can be used interchangeably.
  • the method for extracting ginseng total polysaccharide from ginseng can be any method in the art that can extract ginseng total polysaccharide from ginseng, and can also be extracted by the following method:
  • the second extract is extracted with ethanol and water to obtain crude ginseng polysaccharides.
  • the retentate after protein removal is extracted with ethanol to obtain total ginseng polysaccharides.
  • the step of obtaining the first extract comprises:
  • the protease incubation temperature is 30° C. to 40° C.
  • the incubation time is 1 to 3 hours.
  • the step of obtaining the second extract comprises:
  • Neutral protease was added to the first extract, cultured at 40°C for 3h, and then inactivated at 100°C for 30min.
  • the mixed solution was centrifuged at 10000rpm to obtain a supernatant, which was evaporated to dryness to obtain a second extract.
  • the step of obtaining crude ginseng polysaccharide comprises:
  • the molecular weight cutoff during dialysis is 800-2500 kDa, for example, it can be 900 kDa, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa, 2100 kDa, 2200 kDa, 2300 kDa, 2400 kDa, preferably 1000-1500 kDa.
  • Sevag reagent is used to remove proteins from the retentate.
  • the steps of dissolving the crude ginseng polysaccharide and then dialyzing and removing the protein in the retentate include:
  • the step of extracting the retentate after removing the protein with ethanol to obtain ginseng total polysaccharides comprises:
  • the column when eluting with an eluent after column chromatography, can be any column in the art that can achieve column chromatography.
  • the column chromatography column is a DEAE cellulose column or a macroporous resin column, preferably a DEAE cellulose column. It is further preferred that the column chromatography is loaded at 2 to 8 mL/min, for example, it can be 3 mL/min, 4 mL/min, 5 mL/min, 6 mL/min, and 7 mL/min.
  • the eluent can be any one or more solutions in the art that can achieve the purpose of elution.
  • the eluent is water and an alkaline solution, or water and a borax solution, or water and a salt solution.
  • the eluent is water and a salt solution.
  • the eluent is water and a NaCl solution.
  • the concentration of the eluent may be any concentration in the art that can achieve the purpose of elution.
  • the concentration of the eluent is 0.3-0.7 mol/L, for example, 0.4 mol/L, 0.5 mol/L, or 0.6 mol/L.
  • the elution flow rate can be any flow rate in the art that can achieve the elution purpose.
  • the elution flow rate is 0.5 to 2 mL/min. 0.6mL/min, 0.7mL/min, 0.8mL/min, 0.9mL/min, 1.0mL/min, 1.1mL/min, 1.2mL/min, 1.3mL/min, 1.4mL/min, 1.5mL/min, 1.6mL/min, 1.7mL/min, 1.8mL/min, 1.9mL/min.
  • the process before dialyzing the eluate with distilled water, the process further comprises detecting A 490 of the eluate by a phenol-sulfuric acid method, collecting absorption peaks, and then dialyzing.
  • the dialysis time is 24 to 72 hours, for example, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 65 hours, or 70 hours.
  • aluminum salt adjuvant conforms to the general definition in the art and refers to a class of inorganic salt adjuvants containing aluminum ions, mainly aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum and potassium alum, etc.
  • the aluminum salt adjuvant described in this application is not limited and can be any aluminum salt adjuvant used in the art.
  • the present application further provides a vaccine composition, comprising the vaccine adjuvant containing ginseng acid polysaccharide and aluminum salt adjuvant in the present application and an antigen or a DNA encoding the antigen.
  • the amount of the vaccine adjuvant in the vaccine composition is an effective amount for therapeutic effect, and the effective amount is an amount that improves, increases, upwardly regulates, changes or otherwise promotes the immune response to the antigen.
  • the therapeutically effective amount is an amount that induces immunity in animals susceptible to diseases caused by pathogens, cancer cells or allergens.
  • the therapeutically effective amount will vary and be determined on a case-by-case basis.
  • the dosage of the vaccine adjuvant and the vaccine antigen is not particularly limited, and is appropriately selected according to the method of administration, the subject, the age of the subject, the dosage form, the route of administration, etc.
  • the ratio of the sum of the amounts of the ginseng acid polysaccharide and the aluminum salt adjuvant to the amount of the vaccine antigen is: ( ⁇ g/IU or ⁇ g: ⁇ g), for example, the dosage ratio can be 0.005, 0.05, 0.5, 1, 1.25, , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 166.7, 170, 180, 200, 500, 1000, 10000 ( ⁇ g/IU or ⁇ g: ⁇ g), preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g), preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g); preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g); preferably, the dosage ratio is: ( ⁇ g/IU or ⁇ g: ⁇ g); preferably, the dosage ratio
  • the The dosage ratio of the sum of ginseng acid polysaccharide and aluminum salt adjuvant to vaccine antigen is: For example, it can be 17 ( ⁇ g: ⁇ g), 20 ( ⁇ g: ⁇ g), 25 ( ⁇ g: ⁇ g), 30 ( ⁇ g: ⁇ g), 35 ( ⁇ g: ⁇ g), 40 ( ⁇ g: ⁇ g), 45 ( ⁇ g: ⁇ g), 50 ( ⁇ g: ⁇ g), 55 ( ⁇ g: ⁇ g), 60 ( ⁇ g: ⁇ g), 65 ( ⁇ g: ⁇ g), 70 ( ⁇ g: ⁇ g), 75 ( ⁇ g: ⁇ g), 80 ( ⁇ g: ⁇ g), 90 ( ⁇ g: ⁇ g), 100 ( ⁇ g: ⁇ g), 110 ( ⁇ g: ⁇ g), 120 ( ⁇ g: ⁇ g).
  • the dosage may be 6 ( ⁇ g:IU), 7 ( ⁇ g:IU), 8 ( ⁇ g:IU), 9 ( ⁇ g:IU), 10 ( ⁇ g:IU), 15 ( ⁇ g:IU), 20 ( ⁇ g:IU), 25 ( ⁇ g:IU), 30 ( ⁇ g:IU), 35 ( ⁇ g:IU), 40 ( ⁇ g:IU), 45 ( ⁇ g:IU), 49 ( ⁇ g:IU), 50 ( ⁇ g:IU), 55 ( ⁇ g:IU), 60 ( ⁇ g:IU), 65 ( ⁇ g:IU), 70 ( ⁇ g:IU), 75 ( ⁇ g:IU), 80 ( ⁇ g:IU), 85 ( ⁇ g:IU), 90 ( ⁇ g:IU), and 95 ( ⁇ g:IU), which has a better effect.
  • the ratio of the sum of the amount of ginseng acid polysaccharide and the aluminum salt adjuvant to the amount of the vaccine antigen is:
  • the amount of the dosage may be 1.5 ( ⁇ g:IU), 2 ( ⁇ g:IU), 2.5 ( ⁇ g:IU), 3 ( ⁇ g:IU), 4 ( ⁇ g:IU), 5 ( ⁇ g:IU), 6 ( ⁇ g:IU), 7 ( ⁇ g:IU), 8 ( ⁇ g:IU), 9 ( ⁇ g:IU), 10 ( ⁇ g:IU), 15 ( ⁇ g:IU), 16 ( ⁇ g:IU), 17 ( ⁇ g:IU), 18 ( ⁇ g:IU), 19 ( ⁇ g:IU), 20 ( ⁇ g:IU), 21 ( ⁇ g:IU), 22 ( ⁇ g:IU), 23 ( ⁇ g:IU), and 24 ( ⁇ g:IU), which may have a better effect.
  • the ratio of the sum of the amount of ginseng acid polysaccharide and the aluminum salt adjuvant to the amount of the vaccine antigen is: For example, it can be 13 ( ⁇ g: ⁇ g), 14 ( ⁇ g: ⁇ g), 15 ( ⁇ g: ⁇ g), 16 ( ⁇ g: ⁇ g), 17 ( ⁇ g: ⁇ g), 18 ( ⁇ g: ⁇ g), 19 ( ⁇ g: ⁇ g), 20 ( ⁇ g: ⁇ g), 25 ( ⁇ g: ⁇ g), 30 ( ⁇ g: ⁇ g), 35 ( ⁇ g: ⁇ g), 40 ( ⁇ g: ⁇ g), 50 ( ⁇ g: ⁇ g), 60 ( ⁇ g: ⁇ g), 70 ( ⁇ g: ⁇ g), g), 80( ⁇ g: ⁇ g), 90( ⁇ g: ⁇ g), 100( ⁇ g: ⁇ g), 110( ⁇ g: ⁇ g), 120( ⁇ g: ⁇ g), 121( ⁇ g: ⁇ g), 124( ⁇ g: ⁇ g), 125( ⁇ g: ⁇ g), 150(
  • the vaccine composition provided in the present application is used for a novel coronavirus vaccine, and the ratio of the sum of the amounts of ginseng acid polysaccharide and aluminum salt adjuvant to the vaccine antigen is: For example, it may be 11 ( ⁇ g: ⁇ g), 12 ( ⁇ g: ⁇ g), 13 ( ⁇ g: ⁇ g), 14 ( ⁇ g: ⁇ g), 15 ( ⁇ g: ⁇ g), 16 ( ⁇ g: ⁇ g), 17 ( ⁇ g: ⁇ g), 18 ( ⁇ g: ⁇ g), 19 ( ⁇ g: ⁇ g), 20 ( ⁇ g: ⁇ g), 25 ( ⁇ g: ⁇ g), 30 ( ⁇ g: ⁇ g), 35 ( ⁇ g: ⁇ g), 40 ( ⁇ g: ⁇ g), 45 ( ⁇ g: ⁇ g), 46 ( ⁇ g: ⁇ g), ⁇ g), 47( ⁇ g: ⁇ g), 48( ⁇ g: ⁇ g), 49( ⁇ g: ⁇ g), 50( ⁇ g: ⁇ g), 100( ⁇ g: ⁇ g), 150( ⁇ g: ⁇ g), 200( ⁇ g: ⁇ g), 250( ⁇
  • the therapeutic dosage and length of treatment may vary depending on the type, weight and condition of the patient to be treated, their individual response to the vaccine composition and the particular route of administration selected. In some cases, dosage levels below the lower limit of the aforementioned range may be therapeutically effective, while in other cases, still larger doses may be used without causing any harmful side effects, provided that such larger doses are first divided into several smaller doses for administration throughout the day. Whenever secondary stress or exposure is likely to occur, it is considered ideal to have a provocative dose.
  • the vaccine adjuvant and the DNA encoding the antigen in the vaccine combination of the present application can be included in a composition together and can be prepared in a separate composition.
  • the route of administration of the vaccine adjuvant and the antigen or the DNA encoding the antigen can be the same or different.
  • the vaccine adjuvant and the antigen or the DNA encoding the antigen can be given simultaneously or with time difference, that is, the vaccine adjuvant and the antigen or the DNA encoding the antigen can be given simultaneously or separately (for example, the vaccine adjuvant is given before or after the vaccine antigen is given).
  • the vaccine adjuvant and the antigen or the DNA encoding the antigen can be provided as a kit comprising them.
  • the vaccine adjuvant and the antigen or the DNA encoding the antigen are included in a composition so that it can be given simultaneously when administered.
  • the administration mode of the vaccine combination can be any appropriate approach, and the approach, whether it is co-administration or simultaneous administration, all delivers the vaccine combination to the host.
  • the vaccine adjuvant or vaccine composition of the present application further includes a pharmaceutical excipient and a second vaccine adjuvant.
  • the pharmaceutical excipients are as described above.
  • the vaccine adjuvant of the present application can be administered as part of a vaccine formulation, which optionally contains an additional second vaccine adjuvant.
  • the second vaccine adjuvant is another adjuvant different from the ginseng acid polysaccharide adjuvant of the present application, which can be one or more than two. Examples of suitable second vaccine adjuvants include those known in the art,
  • the vaccine adjuvant or vaccine composition of the present application may further comprise one or more antioxidants,
  • the one or more antioxidants are selected from the group consisting of sodium bisulfite, sodium sulfite, sodium metabisulfite, sodium thiosulfate, sodium methyloxysulfate, L-ascorbic acid, isoascorbic acid, acetylcysteine, cysteine, monothioglycerol, mercaptoacetic acid, thiolactic acid, thiothreitol, dithiothreitol, dithioerythrocyanate, glutathione, ascorbyl palmitate, butylated hydroxytoluene, nordihydroguaiaretic acid, propyl gallate, alpha-tocopherol, and mixtures thereof.
  • the vaccine adjuvant or vaccine composition of the present application may further include one or more preservatives, and examples of suitable preservatives include (but are not limited to): benzalkonium chloride, benzyl chloride, benzoic acid, benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, sodium benzoate, phenol, and mixtures thereof.
  • suitable preservatives include (but are not limited to): benzalkonium chloride, benzyl chloride, benzoic acid, benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, sodium benzoate, phenol, and mixtures thereof.
  • suitable preservatives include (but are not limited to): benzalkonium chloride, benzyl chloride
  • the vaccine adjuvant or vaccine composition of the present application can be used to prevent or treat diseases caused by pathogens, cancer cells or allergens in humans or animals by administering a therapeutically effective amount of the adjuvant composition or vaccine to humans or animals susceptible to the disease.
  • the pathogen may be any pathogen, including but not limited to bacteria, protozoa, worms, viruses and fungi.
  • Diseases in animals caused by the pathogen include but are not limited to bovine respiratory disease, porcine respiratory disease, pneumonia, pasteurella disease, coccidiosis, anaplasmosis, and infectious keratitis.
  • the cancer cell may be any type of cancer cell in the art.
  • the allergen may be any allergen known in the art.
  • the vaccine composition of the present application can be a rabies vaccine, influenza vaccine, hepatitis B vaccine, hepatitis A vaccine, hepatitis C vaccine, hand, foot and mouth disease vaccine, HPV vaccine or new coronavirus vaccine, etc.
  • the vaccine type of the vaccine composition of the present application can be an inactivated virus vaccine, an attenuated vaccine, an inactivated vaccine, a protein vaccine, a DNA vaccine or a polypeptide vaccine, etc.
  • the vaccine adjuvant or vaccine composition of the present application can be used to protect or treat humans and non-human animals such as livestock and domestic animals, including (but not limited to) cattle, horses, sheep, pigs, goats, rabbits, cats, dogs and other mammals in need of treatment.
  • the vaccine adjuvant or vaccine composition of the present application is used to protect or treat humans.
  • the vaccine adjuvant or vaccine composition of the present application to be administered can be selected based on the patient to be protected or treated.
  • composition of the present application can be prepared by a general method, wherein one or more pharmaceutically Acceptable diluents or carriers are, for example, in the form of oral drugs, such as tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions, etc., or parenteral drugs, such as external drugs, suppositories, injections, eye drops, intranasal agents, transpulmonary agents, etc.
  • oral drugs such as tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions, etc.
  • parenteral drugs such as external drugs, suppositories, injections, eye drops, intranasal agents, transpulmonary agents, etc.
  • Preferred examples of the preparation include injectable or intranasal solutions, or lyophilized preparations prepared by lyophilizing the solution.
  • injectable solutions include emulsions and liposomes containing aqueous solutions and oily compositions, such as aqueous solution preparations or aqueous suspension preparations in which the vaccine adjuvant and the antigen or the DNA encoding the antigen are dissolved or dispersed in water, or oily solution preparations or oily suspension preparations in which the vaccine adjuvant and the antigen or the DNA encoding the antigen are dissolved or dispersed in oil.
  • aqueous solution preparations or aqueous suspension preparations in which the vaccine adjuvant and the antigen or the DNA encoding the antigen are dissolved or dispersed in water
  • oily solution preparations or oily suspension preparations in which the vaccine adjuvant and the antigen or the DNA encoding the antigen are dissolved or dispersed in oil.
  • aqueous solution, aqueous solution preparation or aqueous suspension preparation examples include an aqueous solution or aqueous suspension containing distilled water for injection and optionally containing a buffer, a pH adjuster, a stabilizer, an isotonic agent and/or an emulsifier, and the like.
  • the vaccine adjuvant or vaccine composition of the present application can be administered orally, intramuscularly, intravenously, subcutaneously, intraocularly, parenterally, topically, intravaginally or rectally.
  • the adjuvant composition or vaccine adjuvant can be administered orally as a feed or as a gavage composition.
  • the vaccine adjuvant or vaccine composition of the present application is injected intramuscularly, intravenously or subcutaneously.
  • the vaccine adjuvant provided by the present application comprises ginseng acid polysaccharide and aluminum salt adjuvant.
  • the combination of the two substances can significantly improve the titer of specific antibodies (or neutralizing antibodies) after antigen immunization, and can be used in rabies vaccine, influenza vaccine, hepatitis B vaccine, hepatitis A vaccine, hepatitis C vaccine, hand, foot and mouth disease vaccine, HPV vaccine, new coronavirus vaccine and other vaccines, and can effectively enhance the immune response activity.
  • the vaccine adjuvant comprising ginseng acid polysaccharide adjuvant and aluminum salt adjuvant of the present application can significantly improve the neutralizing antibody level in the blood of mice vaccinated with rabies vaccine, influenza vaccine, hepatitis B vaccine, hepatitis A vaccine, hepatitis C vaccine, hand, foot and mouth disease vaccine, HPV vaccine, and new coronavirus vaccine (P ⁇ 0.01); for some vaccines, such as hepatitis B vaccine, the antibody level in the experimental mice is more significantly improved (P ⁇ 0.001).
  • the present application explored the effects of different doses of adjuvants on the adjuvant activity of vaccines, and found that different doses have a certain effect on the adjuvant activity, and have different effects on different types of vaccines.
  • the dosage of vaccine adjuvants with better effects, as well as the ratio of ginseng polysaccharide vaccine adjuvant and aluminum salt adjuvant were optimized.
  • the present application further optimized the dosage and ratio of vaccine adjuvants with specific and more excellent effects for different vaccines, such as the present invention.
  • the results of the examples in the application show that for influenza vaccines, the total amount of ginseng acid polysaccharide and aluminum salt adjuvant is used in the ratio of the amount of vaccine antigen: When the mass ratio of GAPS and aluminum salt is between 0.5 and 2, the effect is more excellent; for hepatitis A vaccine, the total amount of ginseng acid polysaccharide and aluminum salt adjuvant and the amount of vaccine antigen are as follows: When the mass ratio of GAPS and aluminum salt is between 0.1 and 2, the effect is better; when used for hand, foot and mouth disease vaccine, the total amount of ginseng acid polysaccharide and aluminum salt adjuvant and the amount of vaccine antigen are in the ratio of: When the mass ratio of GAPS to aluminum salt is between 0.1 and 2, the effect is more excellent; when used for HPV vaccine, the total amount of ginseng acid polysaccharide and aluminum salt adjuvant is in the ratio of the total amount of vaccine antigen: It has a better effect, and when the mass ratio of GAPS and aluminum salt is between 0.
  • Aluminum salt adjuvant produced by Croda
  • Centrifuge for 20 minutes (5000 rpm), collect the precipitate, add 400mL of distilled water to the precipitate, and then add 95% ethanol to the supernatant to a final concentration of 80%, let it stand for 1 hour, centrifuge for 20 minutes (5000 rpm), let it stand overnight at room temperature, and collect the precipitate.
  • Pretreatment Add distilled water to the column tube to prevent bubbles, add DEAE filler to the column tube, and let it stand for 24 hours; rinse with 0.5M hydrochloric acid for 1 column volume; and then balance with ultrapure water for 4 column volumes;
  • ginseng polysaccharide Take the total ginseng polysaccharide, dissolve it in distilled water, load it at a speed of 4mL/min, and elute it with water and 0.3mol/L NaCl solution respectively, with the flow rate set to 0.5mL/min.
  • Detect A 490 of the eluate by phenol-sulfuric acid method collect the absorption peak, dialyze it with distilled water for 72h, and freeze-dry it. Ginseng acidic polysaccharide is obtained, and the elution curve is shown in Figure 1.
  • the phenol-sulfuric acid method was used to determine the content of ginseng acid polysaccharides.
  • a standard curve was drawn with different concentrations of glucose as the horizontal axis and the absorbance value at OD490nm as the vertical axis, as shown in Figure 2.
  • the polysaccharide content was calculated based on the glucose standard curve and the sample absorbance.
  • the content of ginseng acid polysaccharides was 97.3%.
  • the protein content in ginseng acid polysaccharide was determined by Bradford method.
  • the standard curve was drawn with bovine serum albumin as the horizontal axis and OD595nm as the vertical axis, as shown in Figure 3.
  • the protein content was calculated based on the bovine serum albumin standard curve and the sample absorbance.
  • the protein content of ginseng acid polysaccharide was 0.3%.
  • Example 1 Immunological adjuvant effect of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt on influenza vaccine Agent activity
  • Influenza vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • GPS and aluminum salt mixed adjuvant vaccine composition measure appropriate amounts of GPS, aluminum salt and influenza vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GPS, 125 ⁇ g of aluminum salt and 15 ⁇ g of influenza vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • GAPS and aluminum salt mixed adjuvant vaccine composition measure appropriate amounts of GAPS, aluminum salt and influenza vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GAPS, 125 ⁇ g of aluminum salt and 15 ⁇ g of influenza vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Positive control group - aluminum salt adjuvant vaccine composition take appropriate amounts of aluminum salt and influenza vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of aluminum salt and 15 ⁇ g of influenza vaccine, filter with a 0.22 ⁇ m microporous membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant Take an appropriate amount of influenza vaccine, dissolve it with physiological saline so that each milliliter of solution contains 15 ⁇ g of influenza vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 4 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected, and the neutralizing antibody level of the mouse serum influenza vaccine was detected by hemagglutination inhibition test.
  • GAPS and aluminum salt mixed group 1.5 ⁇ g influenza vaccine +
  • Negative control group 1.5 ⁇ g influenza vaccine/animal.
  • the "GPS+aluminum salt” mixture and the “GAPS+aluminum salt” mixture can significantly increase the level of neutralizing antibodies in mice immunized with influenza vaccine (P ⁇ 0.05).
  • the adjuvant activity of GAPS mixed with aluminum salt was significantly higher than that of single aluminum salt adjuvant (P ⁇ 0.01).
  • Example 2 Immunoadjuvant activity of different doses of aluminum salts as vaccine adjuvants on influenza vaccines
  • Influenza vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • Positive control group with different doses - aluminum salt adjuvant vaccine composition take appropriate amounts of aluminum salt and influenza vaccine respectively, dissolve them in physiological saline, so that each milliliter of solution contains:
  • Negative control group - vaccine without adjuvant Take an appropriate amount of influenza vaccine, dissolve it with physiological saline so that each milliliter of solution contains 15 ⁇ g of influenza vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 7 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected, and the neutralizing antibody level of the mouse serum influenza vaccine was detected by a hemagglutination inhibition test.
  • Aluminum salt adjuvant group 1 1.5 ⁇ g influenza vaccine + 5 ⁇ g aluminum salt adjuvant;
  • Aluminum salt adjuvant group 2 1.5 ⁇ g influenza vaccine + 25 ⁇ g aluminum salt adjuvant;
  • Aluminum salt adjuvant group 3 1.5 ⁇ g influenza vaccine + 125 ⁇ g aluminum salt adjuvant;
  • Aluminum salt adjuvant group 4 1.5 ⁇ g influenza vaccine + 250 ⁇ g aluminum salt adjuvant;
  • Aluminum salt adjuvant group 5 1.5 ⁇ g influenza vaccine + 500 ⁇ g aluminum salt adjuvant;
  • Negative control group 1.5 ⁇ g influenza vaccine/animal.
  • Example 3 Immune adjuvant activity of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt on influenza vaccine at different total doses
  • Influenza vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • Vaccine compositions with different total doses of GAPS and aluminum salt adjuvants Take appropriate amounts of GAPS, aluminum salt and influenza vaccine, respectively, and dissolve them in physiological saline to make each milliliter of solution contain:
  • the mixture was filtered through a 0.22 ⁇ m microporous filter membrane and aseptically packaged.
  • the ratio of the total amount of vaccine adjuvant to influenza vaccine in each vaccine composition was 3.33, 16.67, 83.33, 166.67, and 333.33 ( ⁇ g: ⁇ g), respectively.
  • Positive control group - aluminum salt adjuvant vaccine composition take appropriate amounts of aluminum salt and influenza vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of aluminum salt and 15 ⁇ g of influenza vaccine, filter with a 0.22 ⁇ m microporous membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant Take an appropriate amount of influenza vaccine, dissolve it with physiological saline so that each milliliter of solution contains 15 ⁇ g of influenza vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 7 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected, and the neutralizing antibody level of the mouse serum influenza vaccine was detected by a hemagglutination inhibition test.
  • GAPS mixed with aluminum salt group 1 1.5 ⁇ g influenza vaccine +
  • GAPS and aluminum salt mixed group 2 1.5 ⁇ g influenza vaccine +
  • GAPS and aluminum salt mixed group 3 1.5 ⁇ g influenza vaccine +
  • GAPS and aluminum salt mixed group 4 1.5 ⁇ g influenza vaccine +
  • GAPS and aluminum salt mixed group 5 1.5 ⁇ g influenza vaccine +
  • Negative control group 1.5 ⁇ g influenza vaccine/rat.
  • the ratio of the total dose of the vaccine adjuvant to the amount of the vaccine has a more excellent effect when it is between 16.67 and 333.33 ( ⁇ g: ⁇ g).
  • Example 4 Immunological adjuvant activity of mixtures of ginseng acid polysaccharide (GAPS) and aluminum salt in different proportions on influenza vaccine
  • Influenza vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • Vaccine compositions with mixed adjuvants of GAPS and aluminum salt in different ratios According to the optimal total adjuvant dose in Example 3 (250 ⁇ g/animal, 0.1 mL per animal), appropriate amounts of GAPS, aluminum salt and influenza vaccine were measured and dissolved in physiological saline to make the sum of GAPS and aluminum salt doses in each milliliter of solution 2500 ⁇ g:
  • the mixture was filtered through a 0.22 ⁇ m microporous filter membrane and aseptically packaged.
  • the mass ratios of GAPS and aluminum salt in each group of vaccine compositions were 0.1, 0.3, 0.5, 1, and 2, respectively.
  • Positive control group - aluminum salt adjuvant vaccine composition take appropriate amounts of aluminum salt and influenza vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of aluminum salt and 15 ⁇ g of influenza vaccine, filter with a 0.22 ⁇ m microporous membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant Take an appropriate amount of influenza vaccine, dissolve it with physiological saline so that each milliliter of solution contains 15 ⁇ g of influenza vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 7 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected, and the neutralizing antibody level of the mouse serum influenza vaccine was detected by a hemagglutination inhibition test.
  • GAPS mixed with aluminum salt group 1 1.5 ⁇ g influenza vaccine +
  • GAPS and aluminum salt mixed group 2 1.5 ⁇ g influenza vaccine +
  • GAPS and aluminum salt mixed group 3 1.5 ⁇ g influenza vaccine +
  • GAPS and aluminum salt mixed group 4 1.5 ⁇ g influenza vaccine +
  • GAPS and aluminum salt mixed group 5 1.5 ⁇ g influenza vaccine +
  • Negative control group 1.5 ⁇ g influenza vaccine/rat.
  • Example 5 Immune adjuvant activity of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt on rabies vaccine
  • GPS and aluminum salt mixed adjuvant vaccine composition measure appropriate amounts of ginseng total polysaccharide (GPS), aluminum salt and diploid inactivated rabies vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GPS, 125 ⁇ g of aluminum salt and 2.5IU of rabies vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • GPS ginseng total polysaccharide
  • aluminum salt and diploid inactivated rabies vaccine dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GPS, 125 ⁇ g of aluminum salt and 2.5IU of rabies vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • GAPS and aluminum salt mixed adjuvant vaccine composition Take appropriate amounts of ginseng acid polysaccharide (GAPS), aluminum salt and diploid inactivated rabies vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GAPS, 125 ⁇ g of aluminum salt and 2.5IU of rabies vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • GAPS ginseng acid polysaccharide
  • aluminum salt and diploid inactivated rabies vaccine composition Take appropriate amounts of ginseng acid polysaccharide (GAPS), aluminum salt and diploid inactivated rabies vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GAPS, 125 ⁇ g of aluminum salt and 2.5IU of rabies vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Positive control group - aluminum salt adjuvant vaccine composition measure appropriate amounts of GPS and diploid inactivated rabies vaccine respectively, dissolve them with physiological saline to make each milliliter of solution contain 250 ⁇ g of aluminum salt and 2.5 IU of rabies vaccine, filter with a 0.22 ⁇ m microporous membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant an appropriate amount of diploid inactivated rabies vaccine was dissolved in physiological saline to make each milliliter of solution contain 2.5 IU of rabies vaccine, filtered through a 0.22 ⁇ m microporous filter membrane, and packaged aseptically.
  • mice were randomly divided into 4 groups, each with 10 mice. 0.1 ml of the above composition was injected intramuscularly per mouse. One week after the first immunization, the second immunization was performed. The serum of mice in each group was collected 14 days after the second immunization, and the neutralizing antibody and IgG specific antibody titer of each serum were tested by RFFIT and ELISA.
  • GPS and aluminum salt mixed group 0.25IU rabies vaccine + 12.5 ⁇ g aluminum salt + 12.5 ⁇ g GPS/animal;
  • GAPS and aluminum salt mixed group 0.25 IU rabies vaccine + 12.5 ⁇ g aluminum salt + 12.5 ⁇ g GAPS/animal;
  • Positive control group 0.25 IU rabies vaccine + 25 ⁇ g aluminum salt adjuvant/rabbit;
  • Negative control group 0.25 IU rabies vaccine/animal.
  • Example 6 Immune adjuvant activity of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt on hepatitis B vaccine
  • Hepatitis B vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • GPS and aluminum salt mixed adjuvant vaccine composition measure appropriate amounts of GPS, aluminum salt and hepatitis B vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GPS, 125 ⁇ g of aluminum salt and 25 ⁇ g of hepatitis B vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • GAPS and aluminum salt mixed adjuvant-vaccine composition measure appropriate amounts of GAPS, aluminum salt and hepatitis B vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GAPS, 125 g of aluminum salt and 25 ⁇ g of hepatitis B vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Positive control group - aluminum salt adjuvant vaccine composition measure appropriate amounts of aluminum salt and hepatitis B vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of aluminum salt and 25 ⁇ g of hepatitis B vaccine, filter with a 0.22 ⁇ m microporous membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant take an appropriate amount of hepatitis B vaccine, dissolve it with physiological saline so that each milliliter of solution contains 25 ⁇ g of hepatitis B vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 4 groups, 10 in each group. 0.1 ml/mouse was injected intramuscularly. The second immunization was performed 2 weeks after the first immunization. On the 14th day after the second immunization, the blood of the mice was collected and the level of hepatitis B antibody in the serum of the mice was detected by ELISA experiment.
  • GPS and aluminum salt mixed group 2.5 ⁇ g hepatitis B vaccine + 12.5 ⁇ g aluminum salt adjuvant + 12.5 ⁇ g GPS;
  • GAPS and aluminum salt mixed group 2.5 ⁇ g hepatitis B vaccine + 12.5 ⁇ g aluminum salt adjuvant + 12.5 ⁇ g GAPS;
  • Positive control group 2.5 ⁇ g hepatitis B vaccine + 25 ⁇ g aluminum salt adjuvant/rat;
  • Negative control group 2.5 ⁇ g hepatitis B vaccine/animal.
  • Example 7 Adjuvant activity of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt on hepatitis A vaccine
  • Diploid hepatitis A vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • GPS and aluminum salt mixed adjuvant vaccine composition measure appropriate amounts of GPS, aluminum salt and hepatitis A vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GPS, 125 ⁇ g of aluminum salt and 50IU of hepatitis A vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • GAPS and aluminum salt mixed adjuvant vaccine composition measure appropriate amounts of GAPS, aluminum salt and hepatitis A vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GAPS, 125 ⁇ g of aluminum salt and 50 IU of hepatitis A vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Positive control group - aluminum salt adjuvant vaccine composition take appropriate amounts of aluminum salt and hepatitis A vaccine respectively, dissolve them with physiological saline to make each milliliter of solution contain 250 ⁇ g of aluminum salt and 50 IU of hepatitis A vaccine, filter with a 0.22 ⁇ m microporous membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant take an appropriate amount of hepatitis A vaccine, dissolve it with physiological saline so that each milliliter of solution contains 50 IU of hepatitis A vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 4 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected and the level of serum neutralizing antibodies was detected.
  • GPS and aluminum salt mixed group 5IU hepatitis A vaccine + 12.5 ⁇ g aluminum salt + 12.5 ⁇ g GPS/mouse;
  • GAPS and aluminum salt mixed group 5IU hepatitis A vaccine + 12.5 ⁇ g aluminum salt + 12.5 ⁇ g GAPS/mouse;
  • Positive control group 5 IU hepatitis A vaccine + 25 ⁇ g aluminum salt adjuvant/mouse;
  • Negative control group 5 IU hepatitis A vaccine/animal.
  • Example 8 Immune adjuvant activity of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt at different total doses on hepatitis A vaccine
  • Diploid hepatitis A vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • Vaccine compositions with different total doses of GAPS and aluminum salt adjuvants Take appropriate amounts of GAPS, aluminum salt and hepatitis A vaccine, dissolve them in physiological saline, and make each milliliter of solution contain:
  • the mixture was filtered through a 0.22 ⁇ m microporous filter membrane and aseptically packaged. At this time, the ratio of the total amount of vaccine adjuvant to the hepatitis A vaccine in each group of vaccine compositions was 2.5, 5, 25, 50, and 100 ( ⁇ g:IU), respectively.
  • Positive control group - aluminum salt adjuvant vaccine composition take appropriate amounts of aluminum salt and hepatitis A vaccine respectively, dissolve them with physiological saline to make each milliliter of solution contain 250 ⁇ g of aluminum salt and 50 IU of hepatitis A vaccine, filter with a 0.22 ⁇ m microporous membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant take an appropriate amount of hepatitis A vaccine, dissolve it with physiological saline so that each milliliter of solution contains 50 IU of hepatitis A vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 7 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected and the level of serum neutralizing antibodies was detected.
  • GAPS and aluminum salt mixed group 1 5IU hepatitis A vaccine +
  • GAPS and aluminum salt mixed group 2 5IU hepatitis A vaccine +
  • GAPS and aluminum salt mixed group 3 5IU hepatitis A vaccine +
  • GAPS and aluminum salt mixed group 4 5IU hepatitis A vaccine +
  • GAPS and aluminum salt mixed group 5 5IU hepatitis A vaccine +
  • Negative control group 5 IU hepatitis A vaccine/animal.
  • the ratio of the total dose of vaccine adjuvant to the amount of vaccine has a more excellent effect when it is between 5 and 100 ( ⁇ g:IU).
  • Example 9 Immune adjuvant activity of mixtures of ginseng acid polysaccharide (GAPS) and aluminum salt in different proportions on hepatitis A vaccine
  • Diploid hepatitis A vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • Vaccine compositions with mixed adjuvants of GAPS and aluminum salt in different ratios According to the optimal total adjuvant dose in Example 8 (250 ⁇ g/animal, 0.1 mL per animal), appropriate amounts of GAPS, aluminum salt and hepatitis A vaccine were measured and dissolved in physiological saline to make the sum of GAPS and aluminum salt doses in each milliliter of solution 2500 ⁇ g:
  • the mixture was filtered through a 0.22 ⁇ m microporous filter membrane and aseptically packaged.
  • the mass ratios of GAPS and aluminum salt in each group of vaccine compositions were 0.1, 0.3, 0.5, 1, and 2, respectively.
  • Positive control group - aluminum salt adjuvant vaccine composition measure appropriate amounts of aluminum salt and hepatitis A vaccine respectively, dissolve them with physiological saline to make each milliliter of solution contain 250 ⁇ g of aluminum salt and 50 IU of hepatitis A vaccine, filter with a 0.22 ⁇ m microporous membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant take an appropriate amount of hepatitis A vaccine, dissolve it with physiological saline so that each milliliter of solution contains 50 IU of hepatitis A vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 7 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected and the level of serum neutralizing antibodies was detected.
  • GAPS and aluminum salt mixed group 1 5IU hepatitis A vaccine +
  • GAPS and aluminum salt mixed group 2 5IU hepatitis A vaccine +
  • GAPS and aluminum salt mixed group 3 5IU hepatitis A vaccine +
  • GAPS and aluminum salt mixed group 4 5IU hepatitis A vaccine +
  • GAPS and aluminum salt mixed group 5 5IU hepatitis A vaccine +
  • Positive control group 5 IU hepatitis A vaccine + 25 ⁇ g aluminum salt adjuvant/rat;
  • Negative control group 5 IU hepatitis A vaccine/animal.
  • Example 10 Adjuvant activity of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt on hepatitis C vaccine
  • Hepatitis C antigen produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • GPS and aluminum salt mixed adjuvant vaccine composition measure appropriate amounts of GPS, aluminum salt and hepatitis C vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of GPS, 250 ⁇ g of aluminum salt and 50 ⁇ g of hepatitis C vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • GAPS and aluminum salt mixed adjuvant-vaccine composition measure appropriate amounts of GAPS, aluminum salt and hepatitis C vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of GAPS, 250 ⁇ g of aluminum salt and 50 ⁇ g of hepatitis C vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Aluminum salt adjuvant vaccine composition for the positive control group appropriate amounts of aluminum salt and hepatitis C vaccine were measured respectively, dissolved with physiological saline to make each milliliter of solution contain 500 ⁇ g of aluminum salt and 50 ⁇ g of hepatitis C vaccine, filtered with a 0.22 ⁇ m microporous membrane, and packaged aseptically.
  • Negative control group - vaccine without adjuvant measure an appropriate amount of hepatitis C vaccine, dissolve it with physiological saline so that each milliliter of solution contains 50 ⁇ g of hepatitis C vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 4 groups, 10 in each group. 0.1 ml of the above composition was injected intramuscularly. Two weeks after the first immunization, the second immunization was performed. Blood samples were collected from each mouse on the 14th day after the second immunization. Fluid was collected and the level of specific antibody IgG in serum was detected.
  • GPS and aluminum salt mixed group 5 ⁇ g hepatitis C vaccine + 25 ⁇ g aluminum salt + 25 ⁇ g GPS/rat;
  • GAPS and aluminum salt mixed group 5 ⁇ g hepatitis C vaccine + 25 ⁇ g aluminum salt + 25 ⁇ g GAPS/mouse;
  • Positive control group 5 ⁇ g hepatitis C vaccine + 50 ⁇ g aluminum salt adjuvant/rat;
  • Negative control group 5 ⁇ g hepatitis C vaccine/rat.
  • Example 11 Adjuvant activity of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt on hand, foot and mouth disease vaccine
  • Hand, foot and mouth disease vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • GPS and aluminum salt mixed adjuvant vaccine composition respectively measure appropriate amounts of GPS, aluminum salt and hand, foot and mouth disease vaccine, dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GPS, 125 ⁇ g of aluminum salt and 200IU of hand, foot and mouth disease vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • GAPS and aluminum salt mixed adjuvant vaccine composition measure appropriate amounts of GAPS, aluminum salt and hand, foot and mouth disease vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GAPS, 125 ⁇ g of aluminum salt and 200IU of hand, foot and mouth disease vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Positive control group - aluminum salt adjuvant vaccine composition measure appropriate amounts of aluminum salt and hand, foot and mouth disease vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of aluminum salt and 200 IU of hand, foot and mouth disease vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant measure an appropriate amount of hand, foot and mouth disease vaccine, dissolve it with physiological saline so that each milliliter of solution contains 200 IU of hand, foot and mouth disease vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 4 groups, 10 mice in each group. 0.1% of the above composition was injected intramuscularly ml/mouse. The second immunization was performed 2 weeks after the first immunization. The blood of each mouse was collected on the 14th day after the second immunization, and the levels of serum neutralizing antibodies and specific antibody IgG were detected.
  • GPS and aluminum salt mixed group 20IU hand, foot and mouth disease vaccine +
  • GAPS and aluminum salt mixed group 20IU hand, foot and mouth disease vaccine +
  • Positive control group 20 IU hand, foot and mouth disease vaccine + 25 ⁇ g aluminum salt adjuvant/rat;
  • Negative control group 20 IU hand, foot and mouth disease vaccine/animal.
  • aluminum salt, "GPS+aluminum salt” mixture, and “GAPS+aluminum salt” mixture can significantly increase the levels of neutralizing antibodies and specific IgG antibodies in mice immunized with hand, foot and mouth disease vaccine (P ⁇ 0.05).
  • the adjuvant activity of GAPS mixed with aluminum salt is significantly higher than that of a single aluminum salt adjuvant (P ⁇ 0.05).
  • Example 12 Immune adjuvant activity of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt at different total doses on hand, foot and mouth disease vaccine
  • Hand, foot and mouth disease vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • Vaccine compositions with different total doses of GAPS and aluminum salt adjuvants Take appropriate amounts of GAPS, aluminum salt and hand, foot and mouth disease vaccine, respectively, and dissolve them in physiological saline to make each milliliter of solution contain:
  • the vaccine was filtered through a 0.22 ⁇ m microporous filter membrane and aseptically packaged. At this time, the ratio of the total amount of vaccine adjuvant in each group of vaccine composition to the hand, foot and mouth disease vaccine was 0.625, 1.25, 6.25, 12.5, and 25 ( ⁇ g:IU), respectively.
  • Positive control group - aluminum salt adjuvant vaccine composition measure appropriate amounts of aluminum salt and hand, foot and mouth disease vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of aluminum salt and 200 IU of hand, foot and mouth disease vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant measure an appropriate amount of hand, foot and mouth disease vaccine, dissolve it with physiological saline so that each milliliter of solution contains 200 IU of hand, foot and mouth disease vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 7 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected and the level of serum neutralizing antibodies was detected.
  • GAPS and aluminum salt mixed group 1 20IU hand, foot and mouth disease vaccine +
  • GAPS and aluminum salt mixed group 2 20IU hand, foot and mouth disease vaccine +
  • GAPS and aluminum salt mixed group 3 20IU hand, foot and mouth disease vaccine +
  • GAPS and aluminum salt mixed group 4 20IU hand, foot and mouth disease vaccine +
  • GAPS and aluminum salt mixed group 5 20IU hand, foot and mouth disease vaccine +
  • Positive control group 20 IU hand, foot and mouth disease vaccine + 25 ⁇ g aluminum salt adjuvant/rat;
  • Negative control group 20 IU hand, foot and mouth disease vaccine/animal.
  • Example 13 Immune adjuvant activity of mixtures of ginseng acid polysaccharide (GAPS) and aluminum salt in different proportions on hand, foot and mouth disease vaccine
  • Hand, foot and mouth disease vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • Vaccine compositions with mixed adjuvants of GAPS and aluminum salt in different ratios According to the optimal total adjuvant dose in Example 12 (250 ⁇ g/mouse, 0.1 mL per mouse), appropriate amounts of GAPS, aluminum salt and hand, foot and mouth disease vaccine were measured and dissolved in physiological saline to make the sum of GAPS and aluminum salt doses in each milliliter of solution 2500 ⁇ g:
  • the mixture was filtered through a 0.22 ⁇ m microporous filter membrane and aseptically packaged.
  • the mass ratios of GAPS and aluminum salt in each group of vaccine compositions were 0.1, 0.3, 0.5, 1, and 2, respectively.
  • Positive control group - aluminum salt adjuvant vaccine composition measure appropriate amounts of aluminum salt and hand, foot and mouth disease vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of aluminum salt and 200 IU of hand, foot and mouth disease vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant measure an appropriate amount of hand, foot and mouth disease vaccine, dissolve it with physiological saline so that each milliliter of solution contains 200 IU of hand, foot and mouth disease vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 7 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected and the level of serum neutralizing antibodies was detected.
  • GAPS and aluminum salt mixed group 1 20IU hand, foot and mouth disease vaccine +
  • GAPS and aluminum salt mixed group 2 20IU hand, foot and mouth disease vaccine +
  • GAPS and aluminum salt mixed group 3 20IU hand, foot and mouth disease vaccine +
  • GAPS and aluminum salt mixed group 4 20IU hand, foot and mouth disease vaccine +
  • GAPS and aluminum salt mixed group 5 20IU hand, foot and mouth disease vaccine +
  • Positive control group 20 IU hand, foot and mouth disease vaccine + 25 ⁇ g aluminum salt adjuvant/rat;
  • Negative control group 20 IU hand, foot and mouth disease vaccine/animal.
  • Example 14 Adjuvant activity of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt on HPV vaccine
  • HPV vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • GPS and aluminum salt mixed adjuvant vaccine composition measure appropriate amounts of GPS, aluminum salt and HPV vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GPS, 12 ⁇ 5g of aluminum salt and 20 ⁇ g of HPV vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • GAPS and aluminum salt mixed adjuvant vaccine composition measure appropriate amounts of GAPS, aluminum salt and HPV vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 125 ⁇ g of GAPS, 125 ⁇ g of aluminum salt and 20 ⁇ g of HPV vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Positive control group - aluminum salt adjuvant vaccine composition take appropriate amounts of aluminum salt and HPV vaccine, respectively, and dissolve them in physiological saline to make each milliliter of solution contain 250 ⁇ g of aluminum salt and 20 ⁇ g of HPV vaccine. Filter with a 0.22 ⁇ m microporous membrane and package aseptically.
  • Negative control group - vaccine without adjuvant Take an appropriate amount of HPV vaccine, dissolve it with physiological saline so that each milliliter of solution contains 20 ⁇ g of HPV vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 4 groups, each with 10 mice. 0.1 ml of the above composition was injected intramuscularly. Two weeks after the first immunization, the second immunization was performed. On the 14th day after the second immunization, the blood of each mouse was collected and the titer level of specific antibody IgG in the serum was detected.
  • GPS and aluminum salt mixed group 2 ⁇ g HPV vaccine + 12.5 ⁇ g aluminum salt + 12.5 ⁇ g GPS/mouse;
  • GAPS and aluminum salt mixed group 2 ⁇ g HPV vaccine + 12.5 ⁇ g aluminum salt + 12.5 ⁇ g GAPS/mouse;
  • Positive control group 2 ⁇ g HPV vaccine + 25 ⁇ g aluminum salt adjuvant/rat;
  • Negative control group 2 ⁇ g HPV vaccine/animal.
  • Example 15 Immunological adjuvant activity of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt on HPV vaccine at different total doses
  • HPV vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • Vaccine compositions with different total doses of GAPS and aluminum salt adjuvants Take appropriate amounts of GAPS, aluminum salt and HPV vaccine, respectively, and dissolve them in physiological saline, so that each milliliter of the solution contains:
  • the mixture was filtered through a 0.22 ⁇ m microporous filter membrane and aseptically packaged.
  • the ratio of the total amount of vaccine adjuvant to HPV vaccine in each group of vaccine compositions was 6.25, 12.5, 62.5, 125, and 250 ( ⁇ g: ⁇ g), respectively.
  • Positive control group - aluminum salt adjuvant vaccine composition measure appropriate amounts of aluminum salt and HPV vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of aluminum salt and 20 ⁇ g of HPV vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant Take an appropriate amount of hand, foot and mouth disease vaccine, dissolve it with physiological saline so that each milliliter of solution contains 20 ⁇ g of HPV vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 7 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected and the level of serum specific antibodies was detected.
  • GAPS and aluminum salt mixed group 1 2 ⁇ g HPV vaccine +
  • GAPS and aluminum salt mixed group 2 2 ⁇ g HPV vaccine +
  • GAPS and aluminum salt mixed group 3 2 ⁇ g HPV vaccine +
  • GAPS and aluminum salt mixed group 4 2 ⁇ g HPV vaccine +
  • GAPS and aluminum salt mixed group 5 2 ⁇ g HPV vaccine +
  • Positive control group 2 ⁇ g HPV vaccine + 25 ⁇ g aluminum salt adjuvant/rat;
  • Negative control group 2 ⁇ g HPV vaccine/animal.
  • the ratio of the total dose of vaccine adjuvant to the amount of vaccine used was between 12.5 and 250 ( ⁇ g: ⁇ g) and had a more excellent effect.
  • Example 16 Immunological adjuvant activity of mixtures of GAPS and aluminum salts in different proportions on HPV vaccines
  • HPV vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • Vaccine compositions with mixed adjuvants of GAPS and aluminum salt in different ratios According to the optimal total adjuvant dose in Example 12 (25 ⁇ g/mouse, 0.1 mL per mouse), appropriate amounts of GAPS, aluminum salt and hand, foot and mouth disease vaccine were measured and dissolved in physiological saline to make the sum of GAPS and aluminum salt doses in each milliliter of solution 2500 ⁇ g:
  • the mixture was filtered through a 0.22 ⁇ m microporous filter membrane and aseptically packaged.
  • the mass ratios of GAPS and aluminum salt in each group of vaccine compositions were 0.1, 0.3, 0.5, 1, and 2, respectively.
  • Positive control group - aluminum salt adjuvant vaccine composition measure appropriate amounts of aluminum salt and hand, foot and mouth disease vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of aluminum salt and 20 ⁇ g of HPV vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant Take an appropriate amount of hand, foot and mouth disease vaccine, dissolve it with physiological saline so that each milliliter of solution contains 20 ⁇ g of HPV vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 7 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected and the level of serum specific antibodies was detected.
  • GAPS and aluminum salt mixed group 1 2 ⁇ g HPV vaccine +
  • GAPS and aluminum salt mixed group 2 2 ⁇ g HPV vaccine +
  • GAPS and aluminum salt mixed group 3 2 ⁇ g HPV vaccine +
  • GAPS and aluminum salt mixed group 4 2 ⁇ g HPV vaccine +
  • GAPS and aluminum salt mixed group 5 2 ⁇ g HPV vaccine +
  • Positive control group 2 ⁇ g HPV vaccine + 25 ⁇ g aluminum salt adjuvant/rat;
  • Negative control group 2 ⁇ g HPV vaccine/rat.
  • Example 17 Adjuvant activity of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt on the new coronavirus vaccine (COVID-19 vaccine)
  • New coronavirus vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • GPS and aluminum salt mixed adjuvant vaccine composition respectively measure appropriate amounts of GPS, aluminum salt and new coronavirus vaccine, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of GPS, 250 ⁇ g of aluminum salt and 50 ⁇ g of new coronavirus vaccine. Filter with a 0.22 ⁇ m microporous filter membrane and package aseptically.
  • GAPS and aluminum salt mixed adjuvant vaccine composition Take appropriate amounts of GAPS, aluminum salt and new coronavirus vaccine respectively, dissolve them in physiological saline, and make each milliliter of solution contain 250 ⁇ g of GAPS, 250 ⁇ g of aluminum salt and 50 ⁇ g of new coronavirus vaccine are filtered through a 0.22 ⁇ m microporous filter membrane and packaged aseptically.
  • Positive control group - aluminum salt adjuvant vaccine composition measure appropriate amounts of aluminum salt and new coronavirus vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 500 ⁇ g of aluminum salt and 50 ⁇ g of new coronavirus vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant Measure an appropriate amount of the new coronavirus vaccine, dissolve it with physiological saline so that each milliliter of solution contains 50 ⁇ g of the new coronavirus vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 4 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected and the level of specific antibody IgG in the serum was detected.
  • GPS aluminum salt mixed adjuvant group 5 ⁇ g COVID-19 vaccine + 25 ⁇ g GPS + 25 ⁇ g aluminum salt/rat;
  • GAPS aluminum salt mixed adjuvant group 5 ⁇ g COVID-19 vaccine + 25 ⁇ g GAPS + 25 ⁇ g aluminum salt/animal;
  • Aluminum salt positive control group 5 ⁇ g COVID-19 vaccine + 50 ⁇ g aluminum salt adjuvant/rat;
  • Negative control group 5 ⁇ g of new coronavirus vaccine/animal.
  • Example 18 Immune adjuvant activity of a mixture of ginseng acid polysaccharide (GAPS) and aluminum salt at different total doses on the new coronavirus vaccine (COVID-19 vaccine)
  • New coronavirus vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • Vaccine compositions with different total doses of GAPS and aluminum salt adjuvants Take appropriate amounts of GAPS, aluminum salt and novel coronavirus vaccine, respectively, and dissolve them in physiological saline solution so that each milliliter of the solution contains:
  • the ratio of the total amount of vaccine adjuvant in each group of vaccine composition to the new crown vaccine is 4, 10, 20, 100, 200, and 400 ( ⁇ g: ⁇ g), respectively.
  • Positive control group - aluminum salt adjuvant vaccine composition measure appropriate amounts of aluminum salt and new crown vaccine respectively, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of aluminum salt and 50 ⁇ g of new crown vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant Measure an appropriate amount of hand, foot and mouth disease vaccine, dissolve it with physiological saline so that each milliliter of solution contains 50 ⁇ g of new crown vaccine, filter it with a 0.22 ⁇ m microporous filter membrane, and package it aseptically.
  • mice were randomly divided into 8 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected and the level of serum specific antibodies was detected.
  • GAPS and aluminum salt mixed group 1 5 ⁇ g COVID-19 vaccine +
  • GAPS and aluminum salt mixed group 2 5 ⁇ g COVID-19 vaccine +
  • GAPS and aluminum salt mixed group 3 5 ⁇ g COVID-19 vaccine +
  • GAPS and aluminum salt mixed group 4 5 ⁇ g COVID-19 vaccine +
  • GAPS and aluminum salt mixed group 5 5 ⁇ g COVID-19 vaccine +
  • GAPS and aluminum salt mixed group 6 5 ⁇ g COVID-19 vaccine +
  • Positive control group 5 ⁇ g COVID-19 vaccine + 25 ⁇ g aluminum salt adjuvant/rat;
  • Negative control group 5 ⁇ g of new coronavirus vaccine/animal.
  • the ratio of the total dose of vaccine adjuvant to the amount of vaccine used is between 10 and 400 ( ⁇ g: ⁇ g). It has a better effect.
  • Example 19 Immune adjuvant activity of mixtures of ginseng acid polysaccharide (GAPS) and aluminum salt in different proportions on COVID-19 vaccines
  • New coronavirus vaccine produced by Liaoning Chengda Biological Co., Ltd.
  • mice Female C57BL/6 mice, 6-8 weeks old, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • Vaccine compositions with mixed adjuvants of GAPS and aluminum salt in different ratios According to the optimal total adjuvant dose in Example 12 (1000 ⁇ g/animal, 0.1 mL per animal), appropriate amounts of GAPS, aluminum salt and hand, foot and mouth disease vaccine were measured and dissolved in physiological saline to make the sum of GAPS and aluminum salt doses in each milliliter of solution 2500 ⁇ g:
  • the mixture was filtered through a 0.22 ⁇ m microporous filter membrane and aseptically packaged.
  • the mass ratios of GAPS and aluminum salt in each group of vaccine compositions were 0.1, 0.3, 0.5, 1, and 2, respectively.
  • Positive control group - aluminum salt adjuvant vaccine composition separately measure aluminum salt and new crown vaccine, dissolve them with physiological saline so that each milliliter of solution contains 250 ⁇ g of aluminum salt and 50 ⁇ g of new crown vaccine, filter with a 0.22 ⁇ m microporous filter membrane, and package aseptically.
  • Negative control group - vaccine without adjuvant Take an appropriate amount of COVID-19 vaccine and dissolve it in physiological saline. Each milliliter of solution contains 50 ⁇ g of the new coronavirus vaccine, which is filtered through a 0.22 ⁇ m microporous filter membrane and packaged aseptically.
  • mice were randomly divided into 7 groups, each with 10 mice.
  • 0.1 ml of the above composition was injected intramuscularly per mouse.
  • the second immunization was performed.
  • the blood of each mouse was collected and the level of serum specific antibodies was detected.
  • GAPS and aluminum salt mixed group 1 5 ⁇ g COVID-19 vaccine +
  • GAPS and aluminum salt mixed group 2 5 ⁇ g COVID-19 vaccine +
  • GAPS and aluminum salt mixed group 3 5 ⁇ g COVID-19 vaccine +
  • GAPS and aluminum salt mixed group 4 5 ⁇ g COVID-19 vaccine +
  • GAPS and aluminum salt mixed group 5 5 ⁇ g COVID-19 vaccine +
  • Positive control group 5 ⁇ g COVID-19 vaccine + 25 ⁇ g aluminum salt adjuvant/rat;
  • Negative control group 5 ⁇ g of new coronavirus vaccine/animal.

Abstract

一种疫苗佐剂，包括人参酸性多糖佐剂（GAPS）和铝盐佐剂。人参酸性多糖（GAPS）与铝盐的混合物能显著地提高抗原免疫后的特异性抗体（或中和抗体）滴度，可以有效地增强机体对疫苗的免疫应答水平，其活性显著强于单一铝盐佐剂。

Description

一种疫苗佐剂、疫苗组合物及其应用 技术领域
本申请涉及医药技术领域，具体涉及一种人参酸性多糖(GAPS)疫苗佐剂、疫苗组合物及其应用。
背景技术
疫苗可以在生物体中启动体液性免疫反应而产生抗体，或者透过细胞性免疫反应而活化毒杀性T细胞等淋巴细胞，以抵抗入侵的外来病原菌，并预防疾病发生(Cavallo Fet al.,Vaccination for treatment and prevention of cancerin animal models.Adv Immunol.2006.90:175-213.Review)。虽然疫苗具有活化免疫系统的效果，但在临床使用上常发现其对特定自身免疫系统太弱的族群，例如老人与小孩无法发挥其应有的效能，因此适量疫苗佐剂的添加有其必要性。
疫苗佐剂是指能够非特异性改变或增强机体对抗原的特异性免疫应答的物质，要求无毒性、纯度高、有一定的吸附能力性质稳定。疫苗佐剂的作用机理主要是可以增加抗原表面积，提高免疫原性；对抗原起缓释作用，延长抗原在组织中的滞留时间；促进炎症反应，刺激主动免疫应答。
佐剂的功能，通常可区分为两大类。第一种为吸附抗原，协助抗原被细胞吞噬，如铝盐及M59乳化剂等(O'Hagan D T,Wack A,Podda A.MF59is a safeand potent vaccine adjuvant for flu vaccines in humans:what did we learn duringits development？Clin Pharmacol Ther.2007Dec；82(6):740-4；4.Clapp T,Siebert P，Chen D,Jones Braun L.Vaccines with aluminum-containing adjuvants:optimizing vaccine efficacy and thermal stability.J Pharm Sci.2011Feb；100(2):388-401)；另一种则是免疫调节因子，如弗氏佐剂含结合分枝杆菌(CFA-mycobacteria)等(Hoft DF，Blazevic A,Abate G，Hanekom WA,KaplanG，Soler JH,Weichold F,Geiter L,Sadoff JC,Horwitz MA.A new recombinantbacille Calmette-Guérin vaccine safely induces significantly  enhancedtuberculosis-specific immunity in human volunteers.J Infect Dis.2008Nov15；198(10):1491-501)。目前市售疫苗佐剂多为铝盐佐剂，但是铝盐可引起注射部位的炎症并刺激局部红斑、肉芽肿和皮下结节，同时，铝盐佐剂可能会延缓部分疫苗的中和抗体的产生，在应用时受到多种限制，因此亟需开发新颖的佐剂以提升疫苗的抗原专一性或抗肿瘤抗感染的能力。
发明内容
本申请提供了一种人参酸性多糖(GAPS)与铝盐的混合物作为疫苗佐剂的应用，其具有良好的活性，可以提高疫苗对机体的免疫效果，所涉及疫苗包括但不限于：狂犬疫苗、流感疫苗、乙肝疫苗甲肝疫苗、丙肝疫苗、手足口疫苗、HPV疫苗、新型冠状病毒疫苗。
具体的，本申请采用如下技术方案
1、一种疫苗佐剂，包括人参酸性多糖佐剂和铝盐佐剂。
2、根据项1所述的疫苗佐剂，所述的人参酸性多糖佐剂与铝盐佐剂的质量比为0.0001：1～1000：1。
3、根据项1或2所述的疫苗佐剂，所述疫苗组合物还包括生理盐水或疫苗注射用水。
4、根据项1-3中任一项所述的疫苗佐剂，所述人参酸性多糖是由人参提取得到的。
5、根据项4所述的疫苗佐剂，所述提取过程包括，
取人参，提取人参总多糖，
将提取的人参总多糖，溶于去离子水，
进行柱层析后用洗脱液进行洗脱，
将洗脱液用蒸馏水透析，
冷冻干燥，获得到人参酸性多糖。
6、根据项5-7中任一项所述的疫苗佐剂，所述柱层析的柱为DEAE纤维素柱或大孔树脂柱，优选为DEAE纤维素柱，进一步优选所述柱层析以2～8mL/min上样。
7、根据项5-8中任一项所述的疫苗佐剂，所述洗脱液为水和碱溶液、硼砂溶液或盐溶液，所述盐溶液优选为NaCl溶液，进一步优选，所述洗脱液的浓度为0.3～0.7mol/L的，进一步优选所述洗脱流速为0.5～2mL/min。
8、根据项5-9中任一项所述的疫苗佐剂，所述将洗脱液用蒸馏水透析之前还包括将洗脱液以苯酚硫酸法检测A490，收集吸收峰，然后进行透析，优选，所述透析时间为24～72小时。
9、一种疫苗组合物，包括项1-8中任一项所述的疫苗佐剂和抗原或编码所述抗原的DNA。
10、根据项9所述的疫苗组合物，所述的GAPS、铝盐佐剂用量之和与疫苗抗原的用量比为：(μg/IU或μg:μg)，
优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)。
11、根据项10所述的疫苗组合物，所述疫苗组合物还包括药用辅料和第二疫苗佐剂。
12、根据项9-11中任一项所述的疫苗组合物，所述疫苗组合物为狂犬疫苗、流感疫苗、乙肝疫苗、甲肝疫苗、丙肝疫苗、手足口疫苗、HPV疫苗或新型冠状病毒疫苗。
13、根据项9-12中任一项所述的疫苗组合物，所述疫苗类型为灭活病毒疫苗、减毒疫苗、灭活疫苗、蛋白质疫苗、DNA疫苗或多肽疫苗。
14、项1-8中任一项所述的疫苗佐剂或项1-8中任一项涉及的人参酸性多糖(GAPS)和铝盐在制备疫苗制剂、疫苗组合物或抗体中的用途。
15、一种预防和/或治疗疾病的方法，包括向受试者施用项9-13中任一项所述的疫苗组合物。
16、根据项15所述的方法，所述疾病选自狂犬病、流感病、乙肝病、甲肝病、丙肝病、手足口病、HPV病或新型冠状病毒病。
发明效果
(1)本申请公开的活性成分人参酸性多糖GAPS和铝盐混合作为疫苗佐剂使用，其具有很高的佐剂活性。酸性多糖由于其高含量的硫酸根例子及羧酸根例子，具有较高的负电荷密度，因此可能参与体内的多种酶促反应及免疫活动(例如动物体内广泛具有的肝素即为小分子酸性多糖)，酸性多糖广泛来源于动物、植物和微生物，其单糖组成、糖链上酸性基团的种类和多少随来源种属不同而各有差异。但植物来源的酸性多糖为大分子结构，其多作为水溶性膳食纤维使用，佐剂活性方面的研究非常有限。本申请经过大量筛选试验，在活性筛选过程中发现人参酸性多糖具有很高的佐剂活性，当人参酸性多糖GAPS和铝盐佐剂混合后共同作为疫苗佐剂使用时，可以有效地增强多种疫苗的免疫应答活性，铝盐佐剂作为常用的疫苗佐剂技术成熟效果稳定，但是效果有限，通过本申请将人参酸性多糖GAPS和铝盐共同作为佐剂使用后，其佐剂活性要明显优于铝盐疫苗佐剂，尤其是人参酸性多糖来自于天然植物，来源明确，资源丰富，且成分具有较好的安全性和稳定性。
(2)本申请所述的GAPS与铝盐的混合物能显著地提高抗原免疫后的特异性抗体(或中和抗体)滴度，可以有效地增强机体对疫苗的免疫应答水平，其活性显著强于单一铝盐佐剂。
(3)本申请的疫苗佐剂为GAPS与铝盐的混合物，具有免疫效果好、使用方便等优点，为疫苗提供了新的佐剂选择。
附图说明
附图用于更好地理解本申请，不构成对本申请的不当限定。其中：
图1为人参多糖洗脱-吸光度曲线；
图2为吸光度-葡萄糖含量标准曲线；
图3为吸光度-蛋白质含量标准曲线；
图4为“GAPS+铝盐”及“GPS+铝盐”混合物对流感疫苗免疫小鼠中和抗体水平的影响；
图5为不同剂量铝盐对流感疫苗免疫小鼠中和抗体水平的影响；
图6为不同总剂量“GAPS+铝盐”混合物对流感疫苗免疫小鼠中和抗体 水平的影响；
图7为不同比例“GAPS+铝盐”混合物对流感疫苗免疫小鼠中和抗体水平的影响；
图8为“GAPS+铝盐”及“GPS+铝盐”混合物对狂犬疫苗免疫小鼠IgG抗体水平的影响；
图9为“GAPS+铝盐”及“GPS+铝盐”混合物对狂犬疫苗免疫小鼠中和抗体效价的影响；
图10为“GAPS+铝盐”及“GPS+铝盐”混合物对乙肝抗原免疫小鼠IgG抗体效价的影响；
图11为“GAPS+铝盐”及“GPS+铝盐”混合物对甲肝疫苗免疫小鼠中和抗体水平的影响；
图12为不同总剂量“GAPS+铝盐”混合物对甲肝疫苗免疫小鼠中和抗体水平的影响；
图13为不同比例“GAPS+铝盐”混合物对甲肝疫苗免疫小鼠中和抗体水平的影响；
图14为“GAPS+铝盐”及“GPS+铝盐”混合物对丙肝疫苗免疫小鼠IgG抗体水平的影响；
图15为“GAPS+铝盐”及“GPS+铝盐”混合物对手足口疫苗免疫小鼠中和抗体水平的影响；
图16为“GAPS+铝盐”及“GPS+铝盐”混合物对手足口疫苗免疫小鼠IgG抗体水平的影响；
图17为不同总剂量“GAPS+铝盐”混合物对手足口疫苗免疫小鼠中和抗体水平的影响；
图18为不同比例“GAPS+铝盐”混合物对手足口疫苗免疫小鼠中和抗体水平的影响；
图19为“GAPS+铝盐”及“GPS+铝盐”混合物对HPV疫苗免疫小鼠IgG抗体效价的影响；
图20为不同总剂量“GAPS+铝盐”混合物对HPV疫苗免疫小鼠中和抗体水平的影响；
图21为不同比例“GAPS+铝盐”混合物对HPV疫苗免疫小鼠中和抗体水平的影响；
图22为“GAPS+铝盐”及“GPS+铝盐”混合物对新型冠状病毒疫苗免疫小鼠IgG抗体水平的影响。
图23为不同总剂量“GAPS+铝盐”混合物对新冠疫苗免疫小鼠中和抗体水平的影响；
图24为不同比例“GAPS+铝盐”混合物对新冠疫苗免疫小鼠中和抗体水平的影响；
其中，*表示与阳性对照组相比，P<0.05；**表示与阳性对照组相比P<0.01；***表示与阳性对照组相比P<0.005；#表示与阴性对照组相比，P<0.05；##表示与阴性对照组相比P<0.01；###表示与阴性对照组相比P<0.005。
具体实施方式
以下对本申请的示范性实施例做出说明，其中包括本申请实施例的各种细节以助于理解，应当将它们认为仅仅是示范性的。因此，本领域普通技术人员应当认识到，可以对这里描述的实施例做出各种改变和修改，而不会背离本申请的范围和精神。同样，为了清楚和简明，以下的描述中省略了对公知功能和结构的描述。
本申请公开了一种疫苗佐剂，包括人参酸性多糖佐剂和铝盐佐剂。
如本文所用，术语“疫苗“是指任何适于刺激动物或人类中的活性免疫性的抗原或致免疫性物质的制剂。
如本文所用，术语“佐剂＂，是指提高、增加、向上调节、改变或以其它方式促进动物中对抗原的免疫反应(例如，体液或细胞免疫反应)的任何物质或物质的混合物。
如本文所用，术语“抗原”是指当被引入至免疫活性的人或动物中时，刺激体液及/或细胞介导的免疫反应的任何物质。该抗原可为纯物质、物质的混合物或微粒物质(包括细胞、细胞片段或细胞衍生片段)或活的(通常经减毒)的生物或病毒。适当抗原的实例包括但不限于：蛋白质、糖蛋白、脂蛋白、肤、碳水化合物/多糖、脂多糖、毒素、病毒、细菌、真菌及寄生物。其它适当抗原包括抗原的最小组分，例如(但不限于)，抗原决定簇、表位或肤。仍然适当的其它抗原包括那些描述于美国专利笫5,855,894 号中的抗原。抗原可以为天然(自然表达或制得)的、合成的，或由那些本领域的技术人员熟悉的重组DNA方法学所衍生的。
一种优选的实施方式，所述疫苗佐剂还包括生理盐水或疫苗注射用水或药用辅料。
进一步优选，所述疫苗佐剂由人参酸性多糖佐剂、铝盐佐剂和生理盐水或疫苗注射用水或药用辅料组成，如，所述疫苗佐剂由人参酸性多糖佐剂、铝盐佐剂和生理盐水组成，如，所述疫苗佐剂由人参酸性多糖佐剂、铝盐佐剂和疫苗注射用水组成，如，所述人参酸性多糖佐剂、铝盐佐剂和药用辅料组成。
如本文所用，术语“药用辅料”指生产药品和调配处方时，为解决制剂的成型性、有效性、稳定性、安全性加入处方中除主药以外的一切药用物料的统称，在安全性方面已进行了合理的评估，且包含在药物制剂中的物质。药用辅料除了赋形、充当载体、提高稳定性外，还具有增溶、助溶、缓控释等重要功能，是可能会影响到药品的质量、安全性和有效性的重要成分。本申请所述药用辅料可以是适当的载体或赋形剂、乳化剂、润湿剂、防腐剂、稳定剂、抗氧化剂、佐剂(例如氢氧化铝佐剂、油剂佐剂、弗氏完全佐剂和弗氏不完全佐剂)等。
一种优选的实施方式，所述的人参酸性多糖佐剂与铝盐佐剂的质量比为0.0001：1～1000：1，例如可以为0.0001：1、0.0005：1、0.001：1、0.005：1、0.01：1、0.05：1、0.1：1、0.5：1、1：1、5：1、10：1、50：1、100：1、500：1、1000：1。
如本文所用，术语“人参酸性多糖”是指从五加科植物人参中提取的酸性多糖。所述从五加科植物人参中提取可以是从原始人参中提取，也可以从如从经过加工后的人参提取，如从人参的二次开发制品中提取，如从人参中提取的人参总多糖中提取，如从人参中提取的人参总多糖后剩余的其他部分提取，如从人参提取废料中提取，如从含有人参的药渣中提取等等，只要含有人参酸性多糖的成分的人参任何制品中提取的人参酸性多糖都可以作为本申请疫苗佐剂使用。
在一种优选的实施方式中，所述人参酸性多糖是购买所得，在一种优选的实施方式中，所述人参酸性多糖是从人参中提取，在一种优选的实施方式中，所述人参酸性多糖是从人参多糖中提取，在一种优选的实施方式 中，所述人参酸性多糖是通过如下方式进行提取：
取人参，提取人参总多糖，
将提取的人参总多糖，溶于去离子水，
进行柱层析后用洗脱液进行洗脱，
将洗脱液用蒸馏水透析，
冷冻干燥，获得到人参酸性多糖。
如本文所用，术语“人参多糖”是指从五加科植物人参中提取的总多糖。在本申请中，与术语“人参总多糖”的含义相同，可以互换使用，所述从人参中提取人参总多糖的方法可以是本领域内任何能够从人参中提取到人参总多糖的方法，也可以通过如下方法进行提取：
将人参用水提取，得到第一提取物，
向第一提取物中加入蛋白酶培养后提取，得到第二提取物，
将第二提取物通过乙醇和水提取，得到人参粗多糖，
将人参粗多糖溶解后透析，并去除截留液中的蛋白质，
将去除蛋白质后的截留液使用乙醇提取，得到人参总多糖。
在一个具体的实施方式中，得到第一提取物的步骤包括：
干燥的人参根1kg粉碎至10-20目，加入8倍量水(g:mL)，沸水浴煮8h，过滤并收集滤液；向滤渣中继续加入10倍量水(g:mL)，沸水浴加热8h。合并两次滤液，6000rpm离心10min。取上清蒸干，得到第一提取物。
在一个具体的实施方式中，得到第二提取物的步骤中，蛋白酶培养温度为30℃～40℃，培养时间为1～3h。
在一个具体的实施方式中，得到第二提取物的步骤包括：
向第一提取物加入中性蛋白酶，40℃培养3h，后以100℃灭活30min，将混合溶液以10000rpm离心，得到上清液，蒸干，得到第二提取物。
在一个具体的实施方式中，得到人参粗多糖的步骤包括：
将第二提取物使用10倍体积的95％的乙醇低温浸泡2.0h。过滤，取滤渣，加2倍体积的蒸馏水溶解，沸水回流提取3次，每次1.0h，120目尼龙布过滤，合并提取液，浓缩至500mL，离心20分钟(5000rpm)，弃去沉淀。向上清液加入95％的乙醇至乙醇终浓度为90％，静置1h。离心20分钟(5000转/分钟)，收集沉淀，再向沉淀中加入400mL蒸馏水，再向上清液加入95％的乙醇至终浓度为80％，静置1h，离心20分钟(5000转/ 分钟)，室温静置过夜，收集沉淀。次日，将沉淀依次用无水乙醇、95％乙醇洗涤两次。并冷冻干燥得人参粗多糖，
在一个具体的实施方式中，所述透析时的截取分子量为800～2500kDa，例如可以为900kDa、1000kDa、1100kDa、1200kDa、1300kDa、1400kDa、1500kDa、1600kDa、1700kDa、1800kDa、1900kDa、2000kDa、2100kDa、2200kDa、2300kDa、2400kDa，优选为1000～1500kDa。
在一个具体的实施方式中，使用Sevag试剂去除截留液中的蛋白质。
在一个具体的实施方式中，将人参粗多糖溶解后透析，并去除截留液中的蛋白质的步骤包括：
将人参粗多糖配制备成30％的水溶液，加入1000kDa透析袋进行透析，静置过夜。取透析袋内液体，向其中加入1/4体积的Sevag试剂，静置离心后除去凝胶状沉淀。重复上述操作5次。
在一个具体的实施方式中，将去除蛋白质后的截留液使用乙醇提取，得到人参总多糖的步骤包括：
合并去除蛋白质的截留液上清液，减压浓缩除去有机试剂。加入95％的乙醇至终浓度为80％，4℃静置过夜，离心，除上清。沉淀依次用95％乙醇、无水乙醇洗涤两次。冷冻干燥得人参总多糖。
本申请的人参酸性多糖提取过程中，在进行柱层析后用洗脱液进行洗脱时，所述柱可以是本领域内任何能够实现柱层析的柱，一种优选的实施方式，所述柱层析的柱为DEAE纤维素柱或大孔树脂柱，优选为DEAE纤维素柱，进一步优选所述柱层析以2～8mL/min上样，例如可以为3mL/min、4mL/min、5mL/min、6mL/min、7mL/min。
所述洗脱液可以是本领域内任何一种能够达到洗脱目的的一种或两种以上的溶液，一种优选的实施方式，所述洗脱液为水和碱溶液，或者水和硼砂溶液，或者水和盐溶液，进一步优选的实施方法，所述洗脱液为水和盐溶液，进一步优选的实施方法，所述洗脱液为水和NaCl溶液。
所述洗脱液的浓度可以是本领域内任何一种能够达到洗脱目的的浓度，一种优选的实施方式，所述洗脱液的浓度为0.3～0.7mol/L的，例如可以为0.4mol/L、0.5mol/L、0.6mol/L。
所述洗脱流速可以是本领域内任何一种能够达到洗脱目的的流速，一种优选的实施方式，所述洗脱流速为0.5～2mL/min，例如可以为 0.6mL/min、0.7mL/min、0.8mL/min、0.9mL/min、1.0mL/min、1.1mL/min、1.2mL/min、1.3mL/min、1.4mL/min、1.5mL/min、1.6mL/min、1.7mL/min、1.8mL/min、1.9mL/min。
一种优选的实施方式，所述将洗脱液用蒸馏水透析之前还包括将洗脱液以苯酚硫酸法检测A490，收集吸收峰，然后进行透析，优选，所述透析时间为24～72小时，例如可以为25小时、26小时、27小时、28小时、29小时、30小时、35小时、40小时、45小时、50小时、55小时、60小时、65小时、70小时。
如本文所用，术语“铝盐佐剂”符合本领域内的一般定义，是指一类含铝离子的无机盐佐剂，主要有氢氧化铝凝胶，磷酸铝、硫酸铝、铵明矾及钾明矾等，本申请所述铝盐佐剂不受限制，可以是任何本领域内用到的铝盐佐剂。
本申请进一步提供了一种疫苗组合物，包括本申请中含有人参酸性多糖和铝盐佐剂的疫苗佐剂和抗原或编码所述抗原的DNA。
所述疫苗组合物中疫苗佐剂的用量为以起到治疗效果的有效量，所述有效量是提高、增加、向上调节、改变或以其它方式促进对抗原的免疫反应的量。具体地说，治疗有效量是诱导易感于由病原体、癌细胞或过敏原引起的疾病的动物中的免疫性的量。如本领域的技术人员将了解，治疗有效量将有所不同且在个案的基础上进行判定，在本申请中，不特别限制疫苗佐剂和疫苗抗原的剂量，并且根据给药方法、受试者、受试者的年龄、剂型、给药路线等适当地选择。一种优选的实施方式中，所述人参酸性多糖与铝盐佐剂用量之和与疫苗抗原的用量比为：(μg/IU或μg:μg)，例如，所述用量比可以为0.005、0.05、0.5、1、1.25、、2、3、4、5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、105、110、115、120、125、130、135、140、145、150、160、166.7、170、180、200、500、1000、10000(μg/IU或μg:μg)，优选地，所述用量比为：(μg/IU或μg:μg)，优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)； 优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；(μg/IU或μg:μg)；优选地，所述用量比为1.25～125(μg/IU或μg:μg)；优选地，所述用量比为1.25～50(μg/IU或μg:μg)；优选地，所述用量比为1.25～25(μg/IU或μg:μg)；优选地，所述用量比为1.25～12.5(μg/IU或μg:μg)；优选地，所述用量比为1.25～10(μg/IU或μg:μg)；优选地，所述用量比为1.25～5(μg/IU或μg:μg)；优选地，所述用量比为5～166.7(μg/IU或μg:μg)；优选地，所述用量比为5～125(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为：(μg/IU或μg:μg)；优选地，所述用量比为5～50(μg/IU或μg:μg)；优选地，所述用量比为5～25(μg/IU或μg:μg)；优选地，所述用量比为5～12.5(μg/IU或μg:μg)；优选地，所述用量比为5～10(μg/IU或μg:μg)；优选地，所述用量比为10～166.7(μg/IU或μg:μg)；优选地，所述用量比为10～125(μg/IU或μg:μg)；优选地，所述用量比为10～50(μg/IU或μg:μg)；优选地，所述用量比为10～25(μg/IU或μg:μg)；优选地，所述用量比为10～12.5(μg/IU或μg:μg)；优选地，所述用量比为12.5～166.7(μg/IU或μg:μg)；优选地，所述用量比为12.5～125(μg/IU或μg:μg)；优选地，所述用量比为12.5～50(μg/IU或μg:μg)；优选地，所述用量比为12.5～25(μg/IU或μg:μg)；优选地，所述用量比为25～166.7(μg/IU或μg:μg)；优选地，所述用量比为25～125(μg/IU或μg:μg)；优选地，所述用量比为25～50(μg/IU或μg:μg)；优选地，所述用量比为12.5～25(μg/IU或μg:μg)；优选地，所述用量比为50～166.7(μg/IU或μg:μg)；优选地，所述用量比为50～125(μg/IU或μg:μg)；优选地，所述用量比为125～166.7(μg/IU或μg:μg)。
一种优选的实施方式，本申请提供的疫苗组合物用于流感疫苗时，所述 人参酸性多糖与铝盐佐剂用量之和与疫苗抗原的用量比为：例如可以为17(μg:μg)、20(μg:μg)、25(μg:μg)、30(μg:μg)、35(μg:μg)、40(μg:μg)、45(μg:μg)、50(μg:μg)、55(μg:μg)、60(μg:μg)、65(μg:μg)、70(μg:μg)、75(μg:μg)、80(μg:μg)、90(μg:μg)、100(μg:μg)、110(μg:μg)、120(μg:μg)、130(μg:μg)、140(μg:μg)、150(μg:μg)、160(μg:μg)、170(μg:μg)、180(μg:μg)、190(μg:μg)、200(μg:μg)、220(μg:μg)、240(μg:μg)、260(μg:μg)、280(μg:μg)、300(μg:μg)、310(μg:μg)、320(μg:μg)、330(μg:μg)，具有更佳的效果。
一种优选的实施方式，本申请提供的疫苗组合物用于甲肝疫苗时，所述人参酸性多糖与铝盐佐剂用量之和与疫苗抗原的用量比为：例如可以为6(μg:IU)、7(μg:IU)、8(μg:IU)、9(μg:IU)、10(μg:IU)、15(μg:IU)、20(μg:IU)、25(μg:IU)、30(μg:IU)、35(μg:IU)、40(μg:IU)、45(μg:IU)、49(μg:IU)、50(μg:IU)、55(μg:IU)、60(μg:IU)、65(μg:IU)、70(μg:IU)、75(μg:IU)、80(μg:IU)、85(μg:IU)、90(μg:IU)、95(μg:IU)，具有更佳的效果。
一种优选的实施方式，本申请提供的疫苗组合物用于手足口疫苗时，所述人参酸性多糖与铝盐佐剂用量之和与疫苗抗原的用量比为：例如可以为1.5(μg:IU)、2(μg:IU)、2.5(μg:IU)、3(μg:IU)、4(μg:IU)、5(μg:IU)、6(μg:IU)、7(μg:IU)、8(μg:IU)、9(μg:IU)、10(μg:IU)、15(μg:IU)、16(μg:IU)、17(μg:IU)、18(μg:IU)、19(μg:IU)、20(μg:IU)、21(μg:IU)、22(μg:IU)、23(μg:IU)、24(μg:IU)，具有更佳的效果。
一种优选的实施方式，本申请提供的疫苗组合物用于HPV疫苗时，所述人参酸性多糖与铝盐佐剂用量之和与疫苗抗原的用量比为：例如可以为13(μg:μg)、14(μg:μg)、15(μg:μg)、16(μg:μg)、17(μg:μg)、18(μg:μg)、19(μg:μg)、20(μg:μg)、25(μg:μg)、30(μg:μg)、35(μg:μg)、40(μg:μg)、50(μg:μg)、60(μg:μg)、70(μg:μg)、80(μg:μg)、90(μg:μg)、100(μg:μg)、110(μg:μg)、120(μg:μg)、121(μg:μg)、124(μg:μg)、125(μg:μg)、150(μg:μg)、175(μg:μg)、200(μg:μg)、225(μg:μg)、249(μg:μg)，具有更佳的效果。
一种优选的实施方式，本申请提供的疫苗组合物用于新型冠状病毒疫苗，所述人参酸性多糖与铝盐佐剂用量之和与疫苗抗原的用量比为： 例如可以为11(μg:μg)、12(μg:μg)、13(μg:μg)、14(μg:μg)、15(μg:μg)、16(μg:μg)、17(μg:μg)、18(μg:μg)、19(μg:μg)、20(μg:μg)、25(μg:μg)、30(μg:μg)、35(μg:μg)、40(μg:μg)、45(μg:μg)、46(μg:μg)、47(μg:μg)、48(μg:μg)、49(μg:μg)、50(μg:μg)、100(μg:μg)、150(μg:μg)、200(μg:μg)、250(μg:μg)、300(μg:μg)、350(μg:μg)、360(μg:μg)、370(μg:μg)、380(μg:μg)、390(μg:μg)，具有更佳的效果。
本领域的技术人员将易于认识到，治疗剂量及治疗长度可依待治疗患者的种类、重量及病症、其对疫苗组合物的个体反应及所选择的特定给药途径的不同而发生改变。在一些情况下，低于前述范围的下限的剂量水平可以是治疗有效的，而在其它情形下，可采用仍然较大的剂量而不引起任何有害副作用，其条件为，将此等较大剂量首先分为数个小剂量以在一天内给药。无论何时可能发生继发应激或曝露，认为激发剂量是理想的。
本申请的疫苗组合中的疫苗佐剂和抗原或编码所述抗原的DNA可以共同包含在一个组合物中并且可以在单独的组合物中配制，在单独的组合物中配制时，疫苗佐剂和抗原或编码所述抗原的DNA的给药路线可以相同或不同。在本申请中，疫苗佐剂和抗原或编码所述抗原的DNA可以同时给予或有时间差异地给予，即，疫苗佐剂和抗原或编码所述抗原的DNA可以同时或分开给予(例如，疫苗佐剂在给予疫苗抗原之前或之后给予)。疫苗佐剂和抗原或编码所述抗原的DNA可以作为包含它们的试剂盒提供。但是从降低患者负担的角度，优选疫苗佐剂和抗原或编码所述抗原的DNA包含在一个组合物中，使得在给药时可以同时给予。无论是共给药或同时给药，所述疫苗组合物的给药模式均可为任何适当途径，该途径无论是在共给药或同时给药情况下，均将所述疫苗组合物输送至宿主。
一种优选的实施方式中，本申请的疫苗佐剂或者疫苗组合物还包括药用辅料和第二疫苗佐剂。
所述药用辅料如前所述。
本申请的疫苗佐剂可作为疫苗制剂的一部份给药，其任选含有额外的第二疫苗佐剂。所述第二疫苗佐剂是不同于本申请的人参酸性多糖佐剂的其他佐剂，可以是一种或者两种以上，适当的第二疫苗佐剂的实施例包括那些本领域中已知的佐剂，
本申请的疫苗佐剂或者疫苗组合物可进一步包含一种或多种抗氧化剂， 所述一种或多种抗氧化剂选自由以下各物组成的组：亚硫酸氢钠、亚硫酸钠、偏亚硫酸氢钠、硫代硫酸钠、次硫酸甲酪钠、L-抗坏血酸、异抗坏血酸、乙酰半胱氨酸、半胱氨酸、单硫代丙三醇、琉基乙酸、硫代乳酸、硫脉、二硫苏糖醇、二硫赤鲜醇、谷胱甘肤、抗坏血酸棕桐酸酣、丁基化胫基苗香醒、丁基化轻基甲苯、去甲二氢愈创木酸、没食子酸丙酣、a-生育酚，以及其混合物。
本申请的疫苗佐剂或者疫苗组合物可进一步包含一种或多种防腐剂，适当防腐剂的实例包括(但不限于)：苯扎氯绥、爷索氯绥、苯甲酸、爷醇、对轻基苯甲酸甲酣、对轻基苯甲酸乙酣、对轻基苯甲酸丙酣、对轻基苯甲酸丁酣、苯甲酸钠、苯酚，以及其混合物。如本领域的技术人员将了解，防腐剂的存在与否将依抗原而定。举例来说，若抗原是活细菌抗原，则无需添加防腐剂。
本申请的疫苗佐剂或者疫苗组合物可用于预防或治疗人类或动物中由病原体、癌细胞或过敏原引起的疾病，其通过对易感于该疾病的人或动物给予治疗有效量的佐剂组合物或疫苗。
根据本申请，该病原体可为任何病原体，其包括(但不限于)：细茵、原生动物、蠕虫、病毒及真菌。通过所述病原体引起的动物中的疾病包括(但不限于)：牛呼吸道疾病、猪呼吸道疾病、肺炎、巴斯德菌病、球虫病、无形体病、及感染性角膜炎。
根据本申请，该癌细胞可为本领域中任何类型的癌细胞。根据本申请，该过敏原可为本领域中已知的任何过敏原。
例如，本申请的疫苗组合物可以是狂犬疫苗、流感疫苗、乙肝疫苗、甲肝疫苗、丙肝疫苗、手足口疫苗、HPV疫苗或新型冠状病毒疫苗等。
例如，本申请的疫苗组合物的疫苗类型可以为灭活病毒疫苗、减毒疫苗、灭活疫苗、蛋白质疫苗、DNA疫苗或多肽疫苗等。
本申请的疫苗佐剂或者疫苗组合物可用于保护或治疗人类和诸如牲畜及家畜动物的非人类动物，包括(但不限于)牛、马、绵羊、猪、山羊、兔、猫、狗及需要治疗的其它哺乳动物。优选的，本申请的疫苗佐剂或者疫苗组合物用于保护或治疗人类。如本领域的技术人员所了解，可基于待保护或治疗的患者来选择待给药的本申请的疫苗佐剂或者疫苗组合物。
本申请的组合物可以通过通用方法来制备，其中加入一种或多种药学上 可接受的稀释剂或载体，例如，呈口服药物形式，例如片剂、胶囊剂、颗粒剂、散剂、锭剂、糖浆剂、乳剂、混悬剂等，或肠胃外药物，例如外用药物、栓剂、注射剂、滴眼剂、鼻内剂、经肺剂等。制剂的优选的实例包括可注射的或鼻内溶液剂，或通过冻干所述溶液剂而制备的冻干的制剂。
可注射的溶液剂的实例包括包含水性溶液和油质组合物的乳剂和脂质体，例如疫苗佐剂和抗原或编码所述抗原的DNA溶解或分散在水中的水性溶液剂制剂或水性混悬剂制剂，或疫苗佐剂和抗原或编码所述抗原的DNA溶解或分散在油中的油质溶液剂制剂或油质混悬剂制剂。
水性溶液剂、水性溶液剂制剂或水性混悬剂制剂的实例包括包含用于注射的蒸馏水并任选包含缓冲液、pH调节剂、稳定剂、等渗剂和/或乳化剂的水性溶液剂或水性混悬剂等。
本申请的疫苗佐剂或者疫苗组合物可通过经口、肌肉内、静脉内、皮下、眼内、肠胃外、局部、阴道内或直肠途径给予本申请的疫苗佐剂或者疫苗组合物。对于对牛、猪或其它家畜的给药，可以饲料或作为灌服组合物经口给药所述佐剂组合物或疫苗佐剂。一种优选的实施方式中，以经肌肉内、静脉内或皮下注射本申请的疫苗佐剂或者疫苗组合物。
本申请提供的疫苗佐剂，包含人参酸性多糖和铝盐佐剂，通过两种物质配合，能显著地提高抗原免疫后的特异性抗体(或中和抗体)滴度，用于狂犬疫苗、流感疫苗、乙肝疫苗甲肝疫苗、丙肝疫苗、手足口疫苗、HPV疫苗、新型冠状病毒疫苗等疫苗中，可以有效地增强免疫应答活性，如本申请的实验结果显示，相对于现有技术中常用的疫苗佐剂，如铝盐疫苗佐剂，在同等实验条件下，本申请的包含人参酸性多糖佐剂和铝盐佐剂的疫苗佐剂对狂犬疫苗、流感疫苗、乙肝疫苗甲肝疫苗、丙肝疫苗、手足口疫苗、HPV疫苗、新型冠状病毒疫苗均能显著提升接种小鼠血液的中和抗体水平(P＜0.01)；对于有些疫苗，如乙肝疫苗等，实验小鼠体内的抗体水平具有更加显著的提高(P＜0.001)。
同时，本申请经过大量的试验，探索了使用不同剂量的佐剂对疫苗的佐剂活性的影响，发现不同的剂量对于对佐剂活性的有一定影响，对于不同种类的疫苗也具有不一样的效果，经过多次尝试，也优化出了效果更好的疫苗佐剂用量，以及人参多糖疫苗佐剂和铝盐佐剂的配比，以及，本申请对不同的疫苗进一步优化了其特定的更加优异的效果的疫苗佐剂用量和配比，如本 申请中实施例结果显示，对于流感疫苗，所述人参酸性多糖和铝盐佐剂总用量与疫苗抗原的用量比为：时具有更佳的效果，且，当GAPS及铝盐的用量质量比在0.5-2之间具有更加优异的效果；对于甲肝疫苗时，所述人参酸性多糖和铝盐佐剂总用量与疫苗抗原的用量比为：时具有更佳的效果，且，当GAPS及铝盐的用量质量比为0.1-2之间具有更加优异的效果；用于手足口疫苗时，所述人参酸性多糖和铝盐佐剂总用量与疫苗抗原的用量比为：时具有更佳的效果，且，当GAPS及铝盐的用量质量比为0.1-2之间具有更加优异的效果；用于HPV疫苗时，所述人参酸性多糖和铝盐佐剂总用量与疫苗抗原的用量比为：时具有更佳的效果，且，当GAPS及铝盐的用量质量比为0.5-2之间具有更加优异的效果；用于新型冠状病毒疫苗，所述人参酸性多糖和铝盐佐剂总用量与疫苗抗原的用量比为：时具有更佳的效果，且，当GAPS及铝盐的用量质量比为0.1-2之间具有更加优异的效果。
实施例
实验材料
铝盐佐剂：禾大Croda公司生产
人参多糖的制备：
取干燥的人参根1kg粉碎至10-20目，加入8倍量水(g:mL)，沸水浴煮8h，过滤并收集滤液；向滤渣中继续加入10倍量水(g:mL)，沸水浴加热8h。合并两次滤液，6000rpm离心10min。取上清蒸干，向提取物加入中性蛋白酶，40℃培养3h，后以100℃灭活30min，将混合溶液以10000rpm离心，得到上清液，蒸干。
10倍体积的95％的乙醇低温浸泡2.0h。过滤，取滤渣，加2倍体积的蒸馏水溶解，沸水回流提取3次，每次1h，120目尼龙布过滤，合并提取液，浓缩至500mL，离心20分钟(5000rpm)，弃去沉淀。向上清液加入95％的乙醇至乙醇终浓度为90％，静置1h。离心20分钟(5000转/分钟)，收集沉淀，再向沉淀中加入400mL蒸馏水，再向上清液加入95％的乙醇至终浓度为80％，静置1h，离心20分钟(5000转/分钟)，室温静置过夜，收集沉淀。
次日，将沉淀依次用无水乙醇、95％乙醇洗涤两次。并冷冻干燥得人参粗多糖，将其配置成30％的水溶液，加入1000kDa透析袋进行透析，静置过夜。取透析袋内液体，向其中加入1/4体积的Sevag试剂，静置离心后除去凝胶状沉淀。重复上述操作5次，合并上清液，减压浓缩除去有机试剂，加入95％的乙醇至终浓度为80％，4℃静置过夜，离心，除上清。沉淀依次用95％乙醇、无水乙醇洗涤两次。冷冻干燥得人参多糖。
人参酸性多糖的制备：
称取50g上述制备的人参多糖，溶于500mL去离子水进行DEAE纤维素柱层析，具体步骤如下：
预处理：向柱管中加入用于防止气泡的蒸馏水，将DEAE填料加入柱管中，静置24小时；用0.5M的盐酸冲洗1个柱体积；再用超纯水平衡4个柱体积；
取人参总多糖，溶于蒸馏水中，以速度4mL/min上样，分别用水及0.3mol/L的NaCl溶液洗脱，流速设置为0.5mL/min。将洗脱液以苯酚硫酸法检测A490，收集吸收峰，蒸馏水透析72h，冷冻干燥。得到人参酸性多糖，洗脱曲线见图1。
人参酸性多糖的含量测定
应用苯酚-硫酸法测定人参酸性多糖的含量。以不同浓度的葡萄糖为横坐标，以在OD490nm的吸光度值为纵坐标，绘制标准曲线，如图2所示，根据葡萄糖标准曲线和样品吸光度计算多糖含量。人参酸性多糖的含量为97.3％。
人参酸性多糖中蛋白质含量测定
人参酸性多糖中蛋白质含量的测定采用Bradford法。以牛血清白蛋白为横坐标，以在OD595nm为纵坐标，绘制标准曲线，如图3所示，根据牛血清白蛋白标准曲线和样品吸光度计算蛋白质含量。人参酸性多糖的蛋白质含量为0.3％。
实施例1：人参酸性多糖(GAPS)与铝盐的混合物对流感疫苗的免疫佐 剂活性
流感疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
GPS与铝盐混合佐剂疫苗组合物：分别量取GPS、铝盐和流感疫苗适量，以生理盐水溶解，使每毫升溶液中含有125μg的GPS、125μg的铝盐及15μg的流感疫苗，以0.22μm微孔滤膜滤过，无菌分装。
GAPS与铝盐混合佐剂疫苗组合物：分别量取GAPS、铝盐和流感疫苗适量，以生理盐水溶解，使每毫升溶液中含有125μg的GAPS、125μg的铝盐及15μg的流感疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阳性对照组-铝盐佐剂疫苗组合物：分别量取铝盐和流感疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及15μg的流感疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取流感疫苗适量，以生理盐水溶解，使每毫升溶液中含有15μg的流感疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成4组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫1周后，进行第2次免疫。第2次免疫后的第14天，采集每只小鼠的血液，并通过血凝抑制实验检测小鼠血清流感疫苗的中和抗体水平。
实验分组与剂量：
GPS与铝盐混合组：   1.5μg流感疫苗+
                    12.5μg铝盐佐剂+12.5μg GPS；
GAPS与铝盐混合组：  1.5μg流感疫苗+
                    12.5μg铝盐佐剂+12.5μg GAPS；
阳性对照组:         1.5μg流感疫苗+
                    25μg铝盐佐剂/只；
阴性对照组:         1.5μg流感疫苗/只。
3、结果：
如图4所示，与阴性对照组相比，“GPS+铝盐”混合物、“GAPS+铝盐”混合物均能显著提高流感疫苗免疫小鼠体内中和抗体的水平(P<0.05)。 同时，在总剂量相同的情况下，GAPS与铝盐混合，其佐剂活性显著高于单一铝盐佐剂(P<0.01)。
实施例2：不同剂量铝盐作为疫苗佐剂对流感疫苗的免疫佐剂活性
流感疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
不同剂量的阳性对照组-铝盐佐剂疫苗组合物：分别量取铝盐和流感疫苗适量，以生理盐水溶解，使每毫升溶液中含有：
(1)50μg的铝盐及15μg的流感疫苗；
(2)250μg的铝盐及15μg的流感疫苗；
(3)1250μg的铝盐及15μg的流感疫苗；
(4)2500μg的铝盐及15μg的流感疫苗；
(5)5000μg的铝盐及15μg的流感疫苗；
配制后以0.22μm微孔滤膜滤过，分别进行无菌分装。
阴性对照组-不含佐剂的疫苗：量取流感疫苗适量，以生理盐水溶解，使每毫升溶液中含有15μg的流感疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成7组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫1周后，进行第2次免疫。第2次免疫后的第14天，采集每只小鼠的血液，并通过血凝抑制实验检测小鼠血清流感疫苗的中和抗体水平。
实验分组与剂量：
铝盐佐剂组1：1.5μg流感疫苗+5μg铝盐佐剂；
铝盐佐剂组2：1.5μg流感疫苗+25μg铝盐佐剂；
铝盐佐剂组3：1.5μg流感疫苗+125μg铝盐佐剂；
铝盐佐剂组4：1.5μg流感疫苗+250μg铝盐佐剂；
铝盐佐剂组5：1.5μg流感疫苗+500μg铝盐佐剂；
阴性对照组：1.5μg流感疫苗/只。
3、结果：
如图5所述，与阴性对照组相比，几种剂量的铝盐对流感疫苗免疫小鼠体内中和抗体的水平均无显著影响。几个不同剂量下，25μg/只铝盐给药的小鼠抗体水平较高，且个体差异较小；铝盐佐剂组4、5虽剂量较大，但抗体水平并无显著改变。因此，确定25μg/只的剂量是较为适宜的铝盐佐剂剂量。
实施例3：不同总剂量下人参酸性多糖(GAPS)与铝盐的混合物对流感疫苗的免疫佐剂活性
流感疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
不同总剂量的GAPS与铝盐混合佐剂疫苗组合物：分别量取GAPS、铝盐和流感疫苗适量，以生理盐水溶解，使每毫升溶液中分别含有：
(1)25μg的GAPS、25μg的铝盐及15μg的流感疫苗；
(2)125μg的GAPS、125μg的铝盐及15μg的流感疫苗；
(3)625μg的GAPS、625μg的铝盐及15μg的流感疫苗；
(4)1250μg的GAPS、1250μg的铝盐及15μg的流感疫苗；
(5)2500μg的GAPS、2500μg的铝盐及15μg的流感疫苗；
配制后以0.22μm微孔滤膜滤过，分别进行无菌分装，此时，每组疫苗组合物中疫苗佐剂总量与流感疫苗之比分别为3.33、16.67、83.33、166.67、333.33(μg:μg)。
阳性对照组-铝盐佐剂疫苗组合物：分别量取铝盐和流感疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及15μg的流感疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取流感疫苗适量，以生理盐水溶解，使每毫升溶液中含有15μg的流感疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成7组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫1周后，进行第2次免疫。第2次免疫后的第14天，采集每只小鼠的血液，并通过血凝抑制实验检测小鼠血清流感疫苗的中和抗体水平。
实验分组与剂量：
GAPS与铝盐混合组1：1.5μg流感疫苗+
                   2.5μg铝盐佐剂+2.5μg GAPS；
GAPS与铝盐混合组2：1.5μg流感疫苗+
                   12.5μg铝盐佐剂+12.5μg GAPS；
GAPS与铝盐混合组3：1.5μg流感疫苗+
                   62.5μg铝盐佐剂+62.5μg GAPS；
GAPS与铝盐混合组4：1.5μg流感疫苗+
                   125μg铝盐佐剂+125μg GAPS；
GAPS与铝盐混合组5：1.5μg流感疫苗+
                   250μg铝盐佐剂+250μg GAPS；
阳性对照组:1.5μg流感疫苗+
                   25μg铝盐佐剂/只；
阴性对照组:1.5μg流感疫苗/只。
3、结果：
如图6所述，在GAPS和铝盐以相同比例混合，但疫苗佐剂总剂量与疫苗用量的比例不同的情况下作为佐剂给药时，可发现，对于流感疫苗，GAPS与铝盐混合组2、3、4、5小鼠体内抗体水平显著高于阴性对照组及阳性对照组。采用不同剂量的疫苗佐剂对流感疫苗的免疫活性具有不同的影响，在本实施例中，疫苗佐剂总剂量与疫苗用量的比例在16.67～333.33(μg:μg)之间均具有更加优异的效果。
实施例4：不同比例的人参酸性多糖(GAPS)与铝盐的混合物对流感疫苗的免疫佐剂活性
流感疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
不同比例的GAPS与铝盐混合佐剂疫苗组合物：根据实施例3中最佳总佐剂剂量(250μg/只，每只给药0.1mL)分别量取GAPS、铝盐和流感疫苗适量，以生理盐水溶解，使每毫升溶液中GAPS与铝盐剂量之和为2500μg：
(1)227.3μg的GAPS、2272.7μg的铝盐及15μg的流感疫苗；
(2)576.9μg的GAPS、1923.1μg的铝盐及15μg的流感疫苗；
(3)833.3μg的GAPS、1666.7μg的铝盐及15μg的流感疫苗；
(4)1250μg的GAPS、1250μg的铝盐及15μg的流感疫苗；
(5)1666.7μg的GAPS、833.3μg的铝盐及15μg的流感疫苗；
配制后以0.22μm微孔滤膜滤过，分别进行无菌分装，此时，每组疫苗组合物中GAPS和铝盐的用量质量比分别为0.1、0.3、0.5、1、2。
阳性对照组-铝盐佐剂疫苗组合物：分别量取铝盐和流感疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及15μg的流感疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取流感疫苗适量，以生理盐水溶解，使每毫升溶液中含有15μg的流感疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成7组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫1周后，进行第2次免疫。第2次免疫后的第14天，采集每只小鼠的血液，并通过血凝抑制实验检测小鼠血清流感疫苗的中和抗体水平。
实验分组与剂量：
GAPS与铝盐混合组1：1.5μg流感疫苗+
                 227.27μg铝盐佐剂+22.73μg GAPS；
GAPS与铝盐混合组2：1.5μg流感疫苗+
                 192.31μg铝盐佐剂+57.69μg GAPS；
GAPS与铝盐混合组3：1.5μg流感疫苗+
                 166.67μg铝盐佐剂+83.33μg GAPS；
GAPS与铝盐混合组4：1.5μg流感疫苗+
                 125μg铝盐佐剂+125μg GAPS；
GAPS与铝盐混合组5：1.5μg流感疫苗+
                 83.33μg铝盐佐剂+166.67μg GAPS；
阳性对照组:1.5μg流感疫苗+
                 25μg铝盐佐剂/只；
阴性对照组:1.5μg流感疫苗/只。
3、结果：
如图7所述，在总的疫苗佐剂剂量和疫苗用量比例相同的情况下，使用 不同比例的GAPS及铝盐作为佐剂给药，可发现，对于流感疫苗，GAPS与铝盐混合组3、4、5小鼠体内抗体水平显著高于阴性对照组及阳性对照组(即GAPS与铝盐用量之比为0.5～2(μg:μg))，采用不同比例的GAPS及铝盐的疫苗佐剂对流感疫苗的免疫活性具有不同的影响，在本实施例中，GAPS及铝盐的用量质量比在0.5-2之间均具有更加优异的效果。
实施例5：人参酸性多糖(GAPS)与铝盐的混合物对狂犬疫苗的免疫佐剂活性
1、制备疫苗组合物：
GPS与铝盐混合佐剂疫苗组合物：分别量取人参总多糖(GPS)、铝盐和二倍体灭活狂犬疫苗适量，以生理盐水溶解，使每毫升溶液中含有125μg的GPS、125μg的铝盐及2.5IU的狂犬疫苗，以0.22μm微孔滤膜滤过，无菌分装。
GAPS与铝盐混合佐剂疫苗组合物：分别量取人参酸性多糖(GAPS)、铝盐和二倍体灭活狂犬疫苗适量，以生理盐水溶解，使每毫升溶液中含有125μg的GAPS、125μg的铝盐及2.5IU的狂犬疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阳性对照组-铝盐佐剂疫苗组合物：分别量取GPS和二倍体灭活狂犬疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及2.5IU的狂犬疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：二倍体灭活狂犬疫苗适量，以生理盐水溶解，使每毫升溶液中含2.5IU的狂犬疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成4组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫一周后，进行第二次免疫。第二次免疫后14天采集各组小鼠血清，每份血清分别通过RFFIT、ELISA法检测其中的中和抗体及IgG特异性抗体效价。
实验分组及剂量：
GPS与铝盐混合组：0.25IU狂犬疫苗+12.5μg铝盐+12.5μg GPS/只；
GAPS与铝盐混合组：0.25IU狂犬疫苗+12.5μg铝盐+12.5μg GAPS/只；
阳性对照组：0.25IU狂犬疫苗+25μg铝盐佐剂/只；
阴性对照组：0.25IU狂犬疫苗/只。
3、结果：
如图8、图9所示，与阴性对照组相比，铝盐、“GPS+铝盐”混合物、“GAPS+铝盐”混合物均能显著提高狂犬疫苗免疫小鼠体内中和抗体及特异性抗体IgG的水平(P<0.05)。同时，在总剂量相同的情况下，GAPS与铝盐混合，其佐剂活性显著高于单一铝盐佐剂(P<0.001)。
实施例6：人参酸性多糖(GAPS)与铝盐的混合物对乙肝疫苗的免疫佐剂活性
乙肝疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
GPS与铝盐混合佐剂疫苗组合物：分别量取GPS、铝盐和乙肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有125μg的GPS、125μg的铝盐及25μg的乙肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
GAPS与铝盐混合佐剂-疫苗组合物：分别量取GAPS、铝盐和乙肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有125μg的GAPS、125g的铝盐及25μg的乙肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阳性对照组-铝盐佐剂疫苗组合物：分别量取铝盐和乙肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及25μg的乙肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取乙肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有25μg的乙肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成4组，每组10只。肌肉注射0.1ml/只，初次免疫2周后，进行第2次免疫。第2次免疫后第14天，采集小鼠血液，并通过ELISA实验检测小鼠血清乙肝抗体水平。
实验分组及剂量：
GPS与铝盐混合组：2.5μg乙肝疫苗+12.5μg铝盐佐剂+12.5μg GPS；
GAPS与铝盐混合组：2.5μg乙肝疫苗+12.5μg铝盐佐剂+12.5μg GAPS；
阳性对照组：2.5μg乙肝疫苗+25μg铝盐佐剂/只；
阴性对照组：2.5μg乙肝疫苗/只。
3、结果：
如图10所示，与阴性对照组相比，铝盐、“GPS+铝盐”混合物、“GAPS+铝盐”混合物均能显著提高乙肝疫苗免疫小鼠体内中和抗体的水平(P<0.001)。同时，在总剂量相同的情况下，GAPS与铝盐混合，其佐剂活性显著高于单一铝盐佐剂(P<0.05)。
实施例7：人参酸性多糖(GAPS)与铝盐的混合物对甲肝疫苗的佐剂活性
二倍体甲肝疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
GPS与铝盐混合佐剂疫苗组合物：分别量取GPS、铝盐和甲肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有125μg的GPS、125μg的铝盐及50IU的甲肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
GAPS与铝盐混合佐剂疫苗组合物：分别量取GAPS、铝盐和甲肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有125μg的GAPS、125μg的铝盐及50IU的甲肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阳性对照组-铝盐的佐剂疫苗组合物：分别量取铝盐和甲肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及50IU的甲肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取甲肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有50IU的甲肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成4组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫2周后，进行第二次免疫，第二次免疫后的第14天采集各个小鼠的血液，并检测血清中和抗体的水平。
实验分组及剂量：
GPS与铝盐混合组：5IU甲肝疫苗+12.5μg铝盐+12.5μg GPS/只；
GAPS与铝盐混合组：5IU甲肝疫苗+12.5μg铝盐+12.5μg GAPS/只；
阳性对照组：5IU甲肝疫苗+25μg铝盐佐剂/只；
阴性对照组：5IU甲肝疫苗/只。
3、结果：
如图11所示，与阴性对照组相比，铝盐、“GPS+铝盐”混合物、“GAPS+铝盐”混合物均能显著提高甲肝疫苗免疫小鼠体内中和抗体的水平(P<0.05)。同时，在总剂量相同的情况下，GAPS与铝盐混合，其佐剂活性显著高于单一铝盐佐剂(P<0.01)。
实施例8：不同总剂量下人参酸性多糖(GAPS)与铝盐的混合物对甲肝疫苗的免疫佐剂活性
二倍体甲肝疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
不同总剂量的GAPS与铝盐混合佐剂疫苗组合物：分别量取GAPS、铝盐和甲肝疫苗适量，以生理盐水溶解，使每毫升溶液中分别含有：
(1)62.5μg的GAPS、62.5μg的铝盐及50IU的甲肝疫苗；
(2)125μg的GAPS、125μg的铝盐及50IU的甲肝疫苗；
(3)625μg的GAPS、625μg的铝盐及50IU的甲肝疫苗；
(4)1250μg的GAPS、1250μg的铝盐及50IU的甲肝疫苗；
(5)2500μg的GAPS、2500μg的铝盐及50IU的甲肝疫苗；
配制后以0.22μm微孔滤膜滤过，分别进行无菌分装，此时，每组疫苗组合物中疫苗佐剂总量与甲肝疫苗之比分别为2.5、5、25、50、100(μg:IU)。
阳性对照组-铝盐的佐剂疫苗组合物：分别量取铝盐和甲肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及50IU的甲肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取甲肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有50IU的甲肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成7组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫1周后，进行第2次免疫。第二次免疫后的第14天，采集各个小鼠的血液，并检测血清中和抗体的水平。
实验分组与剂量：
GAPS与铝盐混合组1：5IU甲肝疫苗+
                   6.25μg铝盐佐剂+6.25μg GAPS；
GAPS与铝盐混合组2：5IU甲肝疫苗+
                  12.5μg铝盐佐剂+12.5μg GAPS；
GAPS与铝盐混合组3：5IU甲肝疫苗+
                  62.5μg铝盐佐剂+62.5μg GAPS；
GAPS与铝盐混合组4：5IU甲肝疫苗+
                  125μg铝盐佐剂+125μg GAPS；
GAPS与铝盐混合组5：5IU甲肝疫苗+
                  250μg铝盐佐剂+250μg GAPS；
阳性对照组:5IU甲肝疫苗+
                  25μg铝盐佐剂/只；
阴性对照组:5IU甲肝疫苗/只。
3、结果：
如图12所述，在GAPS和铝盐以相同比例混合，但疫苗佐剂总剂量与疫苗用量的比例不同的情况下作为佐剂给药，可发现，对于甲肝疫苗，GAPS与铝盐混合组2、3、4、5小鼠体内抗体水平显著高于阴性对照组及阳性对照组，采用不同剂量的疫苗佐剂对甲肝疫苗的免疫活性具有不同的影响，在本实施例中，疫苗佐剂总剂量与疫苗用量的比例在5～100(μg:IU)之间均具有更加优异的效果。
实施例9：不同比例的人参酸性多糖(GAPS)与铝盐的混合物对甲肝疫苗的免疫佐剂活性
二倍体甲肝疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
不同比例的GAPS与铝盐混合佐剂疫苗组合物：根据实施例8中最佳总佐剂剂量(250μg/只，每只给药0.1mL)分别量取GAPS、铝盐和甲肝疫苗适量，以生理盐水溶解，使每毫升溶液中GAPS与铝盐剂量之和为2500μg：
(1)227.3μg的GAPS、2272.7μg的铝盐及50IU的甲肝疫苗；
(2)576.9μg的GAPS、1923.1μg的铝盐及50IU的甲肝疫苗；
(3)833.3μg的GAPS、1666.7μg的铝盐及50IU的甲肝疫苗；
(4)1250μg的GAPS、1250μg的铝盐及50IU的甲肝疫苗；
(5)1666.7μg的GAPS、833.3μg的铝盐及50IU的甲肝疫苗；
配制后以0.22μm微孔滤膜滤过，分别进行无菌分装，此时，每组疫苗组合物中GAPS和铝盐的用量质量比分别为0.1、0.3、0.5、1、2。
阳性对照组-铝盐佐剂疫苗组合物：分别量取铝盐和甲肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及50IU的甲肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取甲肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有50IU的甲肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成7组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫1周后，进行第2次免疫。第2次免疫后的第14天，采集各个小鼠的血液，并检测血清中和抗体的水平。
实验分组与剂量：
GAPS与铝盐混合组1：5IU甲肝疫苗+
              227.27μg铝盐佐剂+22.73μg GAPS；
GAPS与铝盐混合组2：5IU甲肝疫苗+
              192.31μg铝盐佐剂+57.69μg GAPS；
GAPS与铝盐混合组3：5IU甲肝疫苗+
              166.67μg铝盐佐剂+83.33μg GAPS；
GAPS与铝盐混合组4：5IU甲肝疫苗+
              125μg铝盐佐剂+125μg GAPS；
GAPS与铝盐混合组5：5IU甲肝疫苗+
              83.33μg铝盐佐剂+166.67μg GAPS；
阳性对照组:5IU甲肝疫苗+25μg铝盐佐剂/只；
阴性对照组:5IU甲肝疫苗/只。
3、结果：
如图13所述，在总的疫苗佐剂剂量和疫苗用量比例相同的情况下，使用不同比例的GAPS及铝盐作为佐剂给药，可发现，对于甲肝疫苗，GAPS与铝盐混合组1～5小鼠体内抗体水平显著高于阴性对照组及阳性对照组(即GAPS与铝盐用量之比为0.1～2(μg:μg))，采用不同比例的GAPS及铝盐的疫苗佐剂对甲肝疫苗的免疫活性具有不同的影响，在本实施例中，GAPS及铝盐的用量质量比在0.1-2之间均具有更加优异的效果。
实施例10：人参酸性多糖(GAPS)与铝盐的混合物对丙肝疫苗的佐剂活性
丙肝抗原：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠，6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
GPS与铝盐混合佐剂疫苗组合物：分别量取GPS、铝盐和丙肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的GPS、250μg的铝盐及50μg的丙肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
GAPS与铝盐混合佐剂-疫苗组合物：分别量取GAPS、铝盐和丙肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的GAPS、250μg的铝盐及50μg的丙肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阳性对照组铝盐佐剂疫苗组合物：分别量取铝盐和丙肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有500μg的铝盐及50μg的丙肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取丙肝疫苗适量，以生理盐水溶解，使每毫升溶液中含有50μg的丙肝疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成4组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫2周后，进行第二次免疫，第二次免疫后的第14天采集各个小鼠的血 液，并检测血清中特异性抗体IgG的水平。
实验分组：
GPS与铝盐混合组：5μg丙肝疫苗+25μg铝盐+25μg GPS/只；
GAPS与铝盐混合组：5μg丙肝疫苗+25μg铝盐+25μg GAPS/只；
阳性对照组：5μg丙肝疫苗+50μg铝盐佐剂/只；
阴性对照组：5μg丙肝疫苗/只。
3、结果：
如图14所示，与阴性对照组相比，铝盐、“GPS+铝盐”混合物、“GAPS+铝盐”混合物均能显著提高丙肝疫苗免疫小鼠体内中和抗体的水平(P<0.001)。同时，在总剂量相同的情况下，GAPS与铝盐混合，其佐剂活性显著高于单一铝盐佐剂(P<0.05)。
实施例11：人参酸性多糖(GAPS)与铝盐的混合物对手足口疫苗的佐剂活性
手足口疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
GPS与铝盐混合佐剂疫苗组合物：分别量取GPS、铝盐和手足口疫苗适量，以生理盐水溶解，使每毫升溶液中含有125μg的GPS、125μg的铝盐及200IU的手足口疫苗，以0.22μm微孔滤膜滤过，无菌分装。
GAPS与铝盐混合佐剂疫苗组合物：分别量取GAPS、铝盐和手足口疫苗适量，以生理盐水溶解，使每毫升溶液中含有125μg的GAPS、125μg的铝盐及200IU的手足口疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阳性对照组-铝盐佐剂疫苗组合物：分别量取铝盐和手足口疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及200IU的手足口疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取手足口疫苗适量，以生理盐水溶解，使每毫升溶液中含有200IU的手足口疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：小鼠随机分成4组，每组10只。肌肉注射上述组合物0.1 ml/只，初次免疫2周后，进行第二次免疫，第二次免疫后的第14天采集各个小鼠的血液，并检测血清中和抗体及特异性抗体IgG的水平。
实验分组：
GPS与铝盐混合组：20IU手足口疫苗+
                 12.5μg铝盐+12.5μg GPS/只；
GAPS与铝盐混合组：20IU手足口疫苗+
                 12.5μg铝盐+12.5μg GAPS/只；
阳性对照组：20IU手足口疫苗+25μg铝盐佐剂/只；
阴性对照组：20IU手足口疫苗/只。
3、结果：
如图15、图16所示，与阴性对照组相比，铝盐、“GPS+铝盐”混合物、“GAPS+铝盐”混合物均能显著提高手足口疫苗免疫小鼠体内中和抗体及特异性IgG抗体的水平(P<0.05)。同时，在总剂量相同的情况下，GAPS与铝盐混合，其佐剂活性显著高于单一铝盐佐剂(P<0.05)。
实施例12：不同总剂量下人参酸性多糖(GAPS)与铝盐的混合物对手足口疫苗的免疫佐剂活性
手足口疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
不同总剂量的GAPS与铝盐混合佐剂疫苗组合物：分别量取GAPS、铝盐和手足口疫苗适量，以生理盐水溶解，使每毫升溶液中分别含有：
(1)62.5μg的GAPS、62.5μg的铝盐及200IU的手足口疫苗；
(2)125μg的GAPS、125μg的铝盐及200IU的手足口疫苗；
(3)625μg的GAPS、625μg的铝盐及200IU的手足口疫苗；
(4)1250μg的GAPS、1250μg的铝盐及200IU的手足口疫苗；
(5)2500μg的GAPS、2500μg的铝盐及200IU的手足口疫苗；
配制后以0.22μm微孔滤膜滤过，分别进行无菌分装，此时，每组疫苗组合物中疫苗佐剂总量与手足口疫苗之比分别为0.625、1.25、6.25、12.5、25(μg:IU)。
阳性对照组-铝盐的佐剂疫苗组合物：分别量取铝盐和手足口疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及200IU的手足口疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取手足口疫苗适量，以生理盐水溶解，使每毫升溶液中含有200IU的手足口疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成7组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫1周后，进行第2次免疫。第二次免疫后的第14天，采集各个小鼠的血液，并检测血清中和抗体的水平。
实验分组与剂量：
GAPS与铝盐混合组1：20IU手足口疫苗+
                   6.25μg铝盐佐剂+6.25μg GAPS；
GAPS与铝盐混合组2：20IU手足口疫苗+
                   12.5μg铝盐佐剂+12.5μg GAPS；
GAPS与铝盐混合组3：20IU手足口疫苗+
                   62.5μg铝盐佐剂+62.5μg GAPS；
GAPS与铝盐混合组4：20IU手足口疫苗+
                   125μg铝盐佐剂+125μg GAPS；
GAPS与铝盐混合组5：20IU手足口疫苗+
                   250μg铝盐佐剂+250μg GAPS；
阳性对照组:20IU手足口疫苗+25μg铝盐佐剂/只；
阴性对照组:20IU手足口疫苗/只。
3、结果：
如图17所述，在GAPS和铝盐以相同比例混合，但其总剂量与疫苗用量的比例不同的情况下作为佐剂给药时，可发现，对于手足口疫苗，GAPS与铝盐混合组2～5小鼠体内抗体水平显著高于阴性对照组及阳性对照组，采用不同剂量的疫苗佐剂对手足口疫苗的免疫活性具有不同的影响，在本实施例中，疫苗佐剂总剂量与疫苗用量的比例在1.25～25(μg:IU)之间均具有更加优异的效果。
实施例13：不同比例的人参酸性多糖(GAPS)与铝盐的混合物对手足口疫苗的免疫佐剂活性
手足口疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
不同比例的GAPS与铝盐混合佐剂疫苗组合物：根据实施例12中最佳总佐剂剂量(250μg/只，每只给药0.1mL)分别量取GAPS、铝盐和手足口疫苗适量，以生理盐水溶解，使每毫升溶液中GAPS与铝盐剂量之和为2500μg：
(1)227.3μg的GAPS、2272.7μg的铝盐及200IU的手足口疫苗；
(2)576.9μg的GAPS、1923.1μg的铝盐及200IU的手足口疫苗；
(3)833.3μg的GAPS、1666.7μg的铝盐及200IU的手足口疫苗；
(4)1250μg的GAPS、1250μg的铝盐及200IU的手足口疫苗；
(5)1666.7μg的GAPS、833.3μg的铝盐及200IU的手足口疫苗；
配制后以0.22μm微孔滤膜滤过，分别进行无菌分装，此时，每组疫苗组合物中GAPS和铝盐的用量质量比分别为0.1、0.3、0.5、1、2。
阳性对照组-铝盐佐剂疫苗组合物：分别量取铝盐和手足口疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及200IU的手足口疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取手足口疫苗适量，以生理盐水溶解，使每毫升溶液中含有200IU的手足口疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成7组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫1周后，进行第2次免疫。第2次免疫后的第14天，采集各个小鼠的血液，并检测血清中和抗体的水平。
实验分组与剂量：
GAPS与铝盐混合组1：20IU手足口疫苗+
                   227.27μg铝盐佐剂+22.73μg GAPS；
GAPS与铝盐混合组2：20IU手足口疫苗+
                   192.31μg铝盐佐剂+57.69μg GAPS；
GAPS与铝盐混合组3：20IU手足口疫苗+
                   166.67μg铝盐佐剂+83.33μg GAPS；
GAPS与铝盐混合组4：20IU手足口疫苗+
                   125μg铝盐佐剂+125μg GAPS；
GAPS与铝盐混合组5：20IU手足口疫苗+
                   83.33μg铝盐佐剂+166.67μg GAPS；
阳性对照组:20IU手足口疫苗+25μg铝盐佐剂/只；
阴性对照组:20IU手足口疫苗/只。
3、结果：
如图18所述，在总的疫苗佐剂剂量和疫苗用量比例相同的情况下，使用不同比例的GAPS及铝盐作为佐剂给药，可发现，对于手足口疫苗，GAPS与铝盐混合组2～5小鼠体内抗体水平显著高于阴性对照组及阳性对照组，采用不同比例的GAPS及铝盐的疫苗佐剂对手足口疫苗的免疫活性具有不同的影响，在本实施例中，GAPS及铝盐的用量质量比在0.1-2之间均具有更加优异的效果。
实施例14：人参酸性多糖(GAPS)与铝盐的混合物对HPV疫苗的佐剂活性
HPV疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备佐剂疫苗组合物：
GPS与铝盐混合佐剂疫苗组合物：分别量取GPS、铝盐和HPV疫苗适量，以生理盐水溶解，使每毫升溶液中含有125μg的GPS、12μ5g的铝盐及20μg的HPV疫苗，以0.22μm微孔滤膜滤过，无菌分装。
GAPS与铝盐混合佐剂疫苗组合物：分别量取GAPS、铝盐和HPV疫苗适量，以生理盐水溶解，使每毫升溶液中含有125μg的GAPS、125μg的铝盐及20μg的HPV疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阳性对照组-铝盐佐剂疫苗组合物：分别量取铝盐和HPV疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及20μg的HPV疫苗， 以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取HPV疫苗适量，以生理盐水溶解，使每毫升溶液中含有20μg的HPV疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：小鼠随机分成4组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫2周后，进行第二次免疫，第二次免疫后的第14天采集各个小鼠的血液，并检测血清中特异性抗体IgG的效价水平。
实验分组及剂量：
GPS与铝盐混合组：2μg HPV疫苗+12.5μg铝盐+12.5μg GPS/只；
GAPS与铝盐混合组：2μg HPV疫苗+12.5μg铝盐+12.5μg GAPS/只；
阳性对照组：2μg HPV疫苗+25μg铝盐佐剂/只；
阴性对照组：2μg HPV疫苗/只。
3、结果：
如图19所示，与阴性对照组相比，铝盐、“GPS+铝盐”混合物、“GAPS+铝盐”混合物均能显著提高HPV疫苗免疫小鼠体内中和抗体的水平(P<0.001)。同时，在总剂量相同的情况下，GAPS与铝盐混合，其佐剂活性显著高于单一铝盐佐剂(P<0.01)。
实施例15：不同总剂量下人参酸性多糖(GAPS)与铝盐的混合物对HPV疫苗的免疫佐剂活性
HPV疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
不同总剂量的GAPS与铝盐混合佐剂疫苗组合物：分别量取GAPS、铝盐和HPV疫苗适量，以生理盐水溶解，使每毫升溶液中分别含有：
(1)62.5μg的GAPS、62.5μg的铝盐及20μg的HPV疫苗；
(2)125μg的GAPS、125μg的铝盐及20μg的HPV疫苗；
(3)625μg的GAPS、625μg的铝盐及20μg的HPV疫苗；
(4)1250μg的GAPS、1250μg的铝盐及20μg的HPV疫苗；
(5)2500μg的GAPS、2500μg的铝盐及20μg的HPV疫苗；
配制后以0.22μm微孔滤膜滤过，分别进行无菌分装，此时，每组疫苗组合物中疫苗佐剂总量与HPV疫苗之比分别为6.25、12.5、62.5、125、250(μg:μg)。
阳性对照组-铝盐的佐剂疫苗组合物：分别量取铝盐和HPV疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及20μg的HPV疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取手足口疫苗适量，以生理盐水溶解，使每毫升溶液中含有20μg的HPV疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成7组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫1周后，进行第2次免疫。第二次免疫后的第14天，采集各个小鼠的血液，并检测血清特异性抗体的水平。
实验分组与剂量：
GAPS与铝盐混合组1：2μg HPV疫苗+
                    6.25μg铝盐佐剂+6.25μg GAPS；
GAPS与铝盐混合组2：2μg HPV疫苗+
                    12.5μg铝盐佐剂+12.5μg GAPS；
GAPS与铝盐混合组3：2μg HPV疫苗+
                    62.5μg铝盐佐剂+62.5μg GAPS；
GAPS与铝盐混合组4：2μg HPV疫苗+
                    125μg铝盐佐剂+125μg GAPS；
GAPS与铝盐混合组5：2μg HPV疫苗+
                    250μg铝盐佐剂+250μg GAPS；
阳性对照组:2μg HPV疫苗+25μg铝盐佐剂/只；
阴性对照组:2μg HPV疫苗/只。
3、结果：
如图20所述，在GAPS和铝盐以相同比例混合，但疫苗佐剂总剂量与疫苗用量的比例不同的情况下作为佐剂给药，可发现，对于HPV疫苗，GAPS与铝盐混合组2～5小鼠体内抗体水平显著高于阴性对照组及阳性对照组，采用不同剂量的疫苗佐剂对HPV疫苗的免疫活性具有不同的影响，在本实施 例中，疫苗佐剂总剂量与疫苗用量的比例在12.5～250(μg:μg)之间均具有更加优异的效果。
实施例16：不同比例的参酸性多糖(GAPS)与铝盐的混合物对HPV疫苗的免疫佐剂活性
HPV疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
不同比例的GAPS与铝盐混合佐剂疫苗组合物：根据实施例12中最佳总佐剂剂量(25μg/只，每只给药0.1mL)分别量取GAPS、铝盐和手足口疫苗适量，以生理盐水溶解，使每毫升溶液中GAPS与铝盐剂量之和为2500μg：
(1)22.7μg的GAPS、227.3μg的铝盐及20μg的HPV疫苗；
(2)57.7μg的GAPS、192.3μg的铝盐及20μg的HPV疫苗；
(3)83.3μg的GAPS、166.7μg的铝盐及20μg的HPV疫苗；
(4)125μg的GAPS、125μg的铝盐及20μg的HPV疫苗；
(5)166.7μg的GAPS、83.3μg的铝盐及20μg的HPV疫苗；
配制后以0.22μm微孔滤膜滤过，分别进行无菌分装，此时，每组疫苗组合物中GAPS和铝盐的用量质量比分别为0.1、0.3、0.5、1、2。
阳性对照组-铝盐佐剂疫苗组合物：分别量取铝盐和手足口疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及20μg的HPV疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取手足口疫苗适量，以生理盐水溶解，使每毫升溶液中含有20μg的HPV疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成7组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫1周后，进行第2次免疫。第2次免疫后的第14天，采集各个小鼠的血液，并检测血清特异性抗体的水平。
实验分组与剂量：
GAPS与铝盐混合组1：2μgHPV疫苗+
                   22.73μg铝盐佐剂+2.27μg GAPS；
GAPS与铝盐混合组2：2μgHPV疫苗+
                   19.23μg铝盐佐剂+5.77μg GAPS；
GAPS与铝盐混合组3：2μgHPV疫苗+
                   16.67μg铝盐佐剂+8.33μg GAPS；
GAPS与铝盐混合组4：2μgHPV疫苗+
                   12.5μg铝盐佐剂+12.5μg GAPS；
GAPS与铝盐混合组5：2μgHPV疫苗+
                   8.33μg铝盐佐剂+16.67μg GAPS；
阳性对照组:2μgHPV疫苗+25μg铝盐佐剂/只；
阴性对照组:2μgHPV疫苗/只。
3、结果：
如图21所述，在总的疫苗佐剂剂量和疫苗用量比例相同的情况下，使用不同比例的GAPS及铝盐作为佐剂给药，可发现，对于HPV疫苗，GAPS与铝盐混合组3～5小鼠体内抗体水平显著高于阴性对照组及阳性对照组，采用不同比例的GAPS及铝盐的疫苗佐剂对HPV疫苗的免疫活性具有不同的影响，在本实施例中，GAPS及铝盐的用量质量比在0.5-2之间均具有更加优异的效果。
实施例17：人参酸性多糖(GAPS)与铝盐的混合物对新型冠状病毒疫苗(新冠疫苗)的佐剂活性
新冠疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备佐剂疫苗组合物：
GPS与铝盐混合佐剂疫苗组合物：分别量取GPS、铝盐和新型冠状病毒疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的GPS、250μg的铝盐及50μg的新型冠状病毒疫苗，以0.22μm微孔滤膜滤过，无菌分装。
GAPS与铝盐混合佐剂疫苗组合物：分别量取GAPS、铝盐和新型冠状病毒疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的GAPS、 250μg的铝盐及50μg的新型冠状病毒疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阳性对照组-铝盐佐剂疫苗组合物：分别量取铝盐和新型冠状病毒疫苗适量，以生理盐水溶解，使每毫升溶液中含有500μg的铝盐及50μg的新型冠状病毒疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取新型冠状病毒疫苗适量，以生理盐水溶解，使每毫升溶液中含有50μg的新型冠状病毒疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成4组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫2周后，进行第二次免疫，第二次免疫后的第14天采集各个小鼠的血液，并检测血清中特异性抗体IgG的水平。
实验分组：
GPS铝盐混合佐剂组：5μg新冠疫苗+25μg GPS+25μg铝盐/只；
GAPS铝盐混合佐剂组：5μg新冠疫苗+25μg GAPS+25μg铝盐/只；
铝盐阳性对照组：5μg新冠疫苗+50μg铝盐佐剂/只；
阴性对照组：5μg新冠疫苗/只。
3、结果：
如图22所示，与阴性对照组相比，铝盐、“GPS+铝盐”混合物、“GAPS+铝盐”混合物均能显著提高新冠疫苗免疫小鼠体内中和抗体的水平(P<0.01)。同时，在总剂量相同的情况下，GAPS与铝盐混合，其佐剂活性显著高于单一铝盐佐剂(P<0.01)。
实施例18：不同总剂量下人参酸性多糖(GAPS)与铝盐的混合物对新型冠状病毒疫苗(新冠疫苗)的免疫佐剂活性
新冠疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
不同总剂量的GAPS与铝盐混合佐剂疫苗组合物：分别量取GAPS、铝盐和新型冠状病毒疫苗适量，以生理盐水溶解，使每毫升溶液中分别含有：
(1)100μg的GAPS、100μg的铝盐及50μg的新冠疫苗；
(2)250μg的GAPS、250μg的铝盐及50μg的新冠疫苗；
(3)500μg的GAPS、500μg的铝盐及50μg的新冠疫苗；
(4)2500μg的GAPS、2500μg的铝盐及50μg的新冠疫苗；
(5)5000μg的GAPS、5000μg的铝盐及50μg的新冠疫苗；
(6)10000μg的GAPS、10000μg的铝盐及50μg的新冠疫苗；
配制后以0.22μm微孔滤膜滤过，分别进行无菌分装，此时，每组疫苗组合物中疫苗佐剂总量与新冠疫苗之比分别为4、10、20、100、200、400(μg:μg)。
阳性对照组-铝盐的佐剂疫苗组合物：分别量取铝盐和新冠疫苗适量，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及50μg的新冠疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取手足口疫苗适量，以生理盐水溶解，使每毫升溶液中含有50μg的新冠疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成8组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫1周后，进行第2次免疫。第二次免疫后的第14天，采集各个小鼠的血液，并检测血清特异性抗体的水平。
实验分组与剂量：
GAPS与铝盐混合组1：5μg新冠疫苗+
                   10μg铝盐佐剂+10μg GAPS；
GAPS与铝盐混合组2：5μg新冠疫苗+
                   25μg铝盐佐剂+25μg GAPS；
GAPS与铝盐混合组3：5μg新冠疫苗+
                   50μg铝盐佐剂+50μg GAPS；
GAPS与铝盐混合组4：5μg新冠疫苗+
                   250μg铝盐佐剂+250μg GAPS；
GAPS与铝盐混合组5：5μg新冠疫苗+
                   500μg铝盐佐剂+500μg GAPS；
GAPS与铝盐混合组6：5μg新冠疫苗+
                   1000μg铝盐佐剂+1000μg GAPS；
阳性对照组:5μg新冠疫苗+25μg铝盐佐剂/只；
阴性对照组:5μg新冠疫苗/只。
3、结果：
如图23所述，在GAPS和铝盐以相同比例混合，但疫苗佐剂总剂量与疫苗用量的比例不同的情况下作为佐剂给药时，可发现，对于新冠疫苗，GAPS与铝盐混合组2～6小鼠体内抗体水平显著高于阴性对照组及阳性对照组，采用不同剂量的疫苗佐剂对新冠疫苗的免疫活性具有不同的影响，在本实施例中，疫苗佐剂总剂量与疫苗用量的比例在10～400(μg:μg)之间均具有更加优异的效果。
实施例19：不同比例的人参酸性多糖(GAPS)与铝盐的混合物对新冠疫苗的免疫佐剂活性
新冠疫苗：辽宁成大生物股份有限公司生产。
动物：雌性C57BL/6小鼠,6-8周龄，购自北京华阜康生物科技股份有限公司。
1、制备疫苗组合物：
不同比例的GAPS与铝盐混合佐剂疫苗组合物：根据实施例12中最佳总佐剂剂量(1000μg/只，每只给药0.1mL)分别量取GAPS、铝盐和手足口疫苗适量，以生理盐水溶解，使每毫升溶液中GAPS与铝盐剂量之和为2500μg：
(1)909.1μg的GAPS、9090.9μg的铝盐及50μg的新冠疫苗；
(2)2307.7μg的GAPS、7692.3μg的铝盐及50μg的新冠疫苗；
(3)3333.3μg的GAPS、6666.7μg的铝盐及50μg的新冠疫苗；
(4)5000μg的GAPS、5000μg的铝盐及50μg的新冠疫苗；
(5)6666.7μg的GAPS、3333.3μg的铝盐及50μg的新冠疫苗；
配制后以0.22μm微孔滤膜滤过，分别进行无菌分装，此时，每组疫苗组合物中GAPS和铝盐的用量质量比分别为0.1、0.3、0.5、1、2。
阳性对照组-铝盐佐剂疫苗组合物：分别量取铝盐和新冠疫苗，以生理盐水溶解，使每毫升溶液中含有250μg的铝盐及50μg的新冠疫苗，以0.22μm微孔滤膜滤过，无菌分装。
阴性对照组-不含佐剂的疫苗：量取新冠疫苗适量，以生理盐水溶解，使 每毫升溶液中含有50μg的新冠疫苗，以0.22μm微孔滤膜滤过，无菌分装。
2、免疫方案：
小鼠随机分成7组，每组10只。肌肉注射上述组合物0.1ml/只，初次免疫1周后，进行第2次免疫。第2次免疫后的第14天，采集各个小鼠的血液，并检测血清特异性抗体的水平。
实验分组与剂量：
GAPS与铝盐混合组1：5μg新冠疫苗+
                   909.1μg铝盐佐剂+90.9μg GAPS；
GAPS与铝盐混合组2：5μg新冠疫苗+
                   769.2μg铝盐佐剂+230.8μg GAPS；
GAPS与铝盐混合组3：5μg新冠疫苗+
                   666.7μg铝盐佐剂+333.3μg GAPS；
GAPS与铝盐混合组4：5μg新冠疫苗+
                   500μg铝盐佐剂+500μg GAPS；
GAPS与铝盐混合组5：5μg新冠疫苗+
                   666.7μg铝盐佐剂+333.3μg GAPS；
阳性对照组:5μg新冠疫苗+25μg铝盐佐剂/只；
阴性对照组:5μg新冠疫苗/只。
3、结果：
如图24所述，在总的疫苗佐剂剂量和疫苗用量比例相同的情况下，使用不同比例的GAPS及铝盐作为佐剂给药，可发现，对于新冠疫苗，GAPS与铝盐混合组1～5小鼠体内抗体水平显著高于阴性对照组及阳性对照组，采用不同比例的GAPS及铝盐的疫苗佐剂对新冠疫苗的免疫活性具有不同的影响，在本实施例中，GAPS及铝盐的用量质量比在0.1～2之间均具有更加优异的效果。
尽管以上结合对本申请的实施方案进行了描述，但本申请并不局限于上述的具体实施方案和应用领域，上述的具体实施方案仅仅是示意性的、指导性的，而不是限制性的。本领域的普通技术人员在本说明书的启示下和在不脱离本申请权利要求所保护的范围的情况下，还可以做出很多种的形式，这 些均属于本申请保护之列。

Claims (16)

  1. 一种疫苗佐剂，其特征在于，包括人参酸性多糖佐剂和铝盐佐剂。
  2. 根据权利要求1所述的疫苗佐剂，其特征在于，所述的人参酸性多糖佐剂与铝盐佐剂的质量比为0.0001：1～1000：1。
  3. 根据权利要求1或2所述的疫苗佐剂，其特征在于，所述疫苗组合物还包括生理盐水或疫苗注射用水。
  4. 根据权利要求1-3中任一项所述的疫苗佐剂，其特征在于，所述人参酸性多糖是由人参提取得到的。
  5. 根据权利要求4所述的疫苗佐剂，其特征在于，所述提取过程包括，
    取人参，提取人参总多糖，
    将提取的人参总多糖，溶于去离子水，
    进行柱层析后用洗脱液进行洗脱，
    将洗脱液用蒸馏水透析，
    冷冻干燥，获得到人参酸性多糖。
  6. 根据权利要求5-7中任一项所述的疫苗佐剂，其特征在于，所述柱层析的柱为DEAE纤维素柱或大孔树脂柱，优选为DEAE纤维素柱，进一步优选所述柱层析以2～8mL/min上样。
  7. 根据权利要求5-8中任一项所述的疫苗佐剂，其特征在于，所述洗脱液为水和碱溶液、硼砂溶液或盐溶液，所述盐溶液优选为NaCl溶液，进一步优选，所述洗脱液的浓度为0.3～0.7mol/L的，进一步优选所述洗脱流速为0.5～2mL/min。
  8. 根据权利要求5-9中任一项所述的疫苗佐剂，其特征在于，所述将洗脱液用蒸馏水透析之前还包括将洗脱液以苯酚硫酸法检测A490，收集吸收峰，然后进行透析，优选，所述透析时间为24～72小时。
  9. 一种疫苗组合物，其特征在于，包括权利要求1-8中任一项所述的疫苗佐剂和抗原或编码所述抗原的DNA。
  10. 根据权利要求9所述的疫苗组合物，其特征在于，所述的GAPS、铝盐佐剂用量之和与疫苗抗原的用量比为：
    优选地，所述用量比为：优选地，所述用量比为：优选地，所述用量比为：优选地，所述用量比为： 优选地，所述用量比为：优选地，所述用量比为：优选地，所述用量比为：优选地，所述用量比为：优选地，所述用量比为：
  11. 根据权利要求10所述的疫苗组合物，其特征在于，所述疫苗组合物还包括药用辅料和第二疫苗佐剂。
  12. 根据权利要求9-11中任一项所述的疫苗组合物，其特征在于，所述疫苗组合物为狂犬疫苗、流感疫苗、乙肝疫苗、甲肝疫苗、丙肝疫苗、手足口疫苗、HPV疫苗或新型冠状病毒疫苗。
  13. 根据权利要求9-12中任一项所述的疫苗组合物，其特征在于，所述疫苗类型为灭活病毒疫苗、减毒疫苗、灭活疫苗、蛋白质疫苗、DNA疫苗或多肽疫苗。
  14. 权利要求1-8中任一项所述的疫苗佐剂或权利要求1-8中任一项涉及的人参酸性多糖(GAPS)和铝盐在制备疫苗制剂、疫苗组合物或抗体中的用途。
  15. 一种预防和/或治疗疾病的方法，包括向受试者施用权利要求9-13中任一项所述的疫苗组合物。
  16. 根据权利要求15所述的方法，所述疾病选自狂犬病、流感病、乙肝病、甲肝病、丙肝病、手足口病、HPV病或新型冠状病毒病。
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