CN114010778A - Oil-in-water type vaccine adjuvant - Google Patents

Oil-in-water type vaccine adjuvant Download PDF

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Publication number
CN114010778A
CN114010778A CN202111231493.6A CN202111231493A CN114010778A CN 114010778 A CN114010778 A CN 114010778A CN 202111231493 A CN202111231493 A CN 202111231493A CN 114010778 A CN114010778 A CN 114010778A
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oil
water
emulsion
buffer solution
squalene
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陈先东
许煜华
李捍雄
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Guangzhou Lianrui Pharmaceutical Co ltd
Guangzhou Runlin Pharmaceutical Technology Co ltd
South China Vaccine Co ltd
GUANGZHOU YIPINHONG PHARMACEUTICAL CO Ltd
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Guangzhou Lianrui Pharmaceutical Co ltd
Guangzhou Runlin Pharmaceutical Technology Co ltd
South China Vaccine Co ltd
GUANGZHOU YIPINHONG PHARMACEUTICAL CO Ltd
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Priority to CN202111231493.6A priority Critical patent/CN114010778A/en
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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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • 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
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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

Abstract

The invention provides an oil-in-water emulsion which has excellent immune effect and very good safety, and no side effect is found in experiments such as acute toxicity, anaphylactic reaction, hemolytic experiment, vascular irritation experiment, long-term toxicity experiment and teratogenesis. In addition, the product has the advantages of surprising stability, long storage life of more than 3 years, and convenient use and storage.

Description

Oil-in-water type vaccine adjuvant
Technical Field
The invention relates to the field of medicaments, in particular to an oil-in-water type vaccine adjuvant.
Background
The conventional concept is that when a substance is injected into an animal before or mixed with an antigen, it can nonspecifically change or enhance the specific immune response of the body to the antigen, and thus exert its adjuvant effect, and is called adjuvant. The latest concept holds that all substances that enhance an antigen-specific immune response are called adjuvants. It may or may not have antigenicity. There is no specific concept for the novel immunoadjuvant at present, and the novel immunoadjuvant is generated in response to a novel vaccine, and refers to an adjuvant capable of enhancing the immune response of the novel vaccine in a narrow sense.
The novel vaccine adjuvant comprises a plant-derived adjuvant, an animal-derived adjuvant, a bacterial-derived adjuvant, an artificial synthetic adjuvant and the like. Among them, as for plant-derived adjuvants, it has been found that at least 200 or more plants have an immunoregulatory function, and are classified into low molecular weight and high molecular weight according to molecular weight. Low molecular weight: alkylamine, phenolic components, quinine, saporin, etc. High molecular weight: protein, polypeptide, polysaccharide, glycolipid, phytohemagglutinin, etc. The immune mechanism is as follows: enhancing the phagocytic function of cells; stimulating immune cells to produce various cytokines; stimulating the production of antibodies. For animal-derived adjuvants, there are mainly chitosan: has serial biological effects of activating body system and mediating body system, and can raise the systemic function of phagocyte. Receptors for bacterial polysaccharides exist on the macrophage surface, and chitosan, as an analog of bacterial polysaccharides, stimulates macrophage activation, producing the following response: promoting the phagocytic function of the immune response protein, and enhancing the synergistic effect of the immune response protein in other immune responses, thereby realizing the regulation of the organism on T cells, NK cells and B cells, and mediating the cellular immune response and the humoral immune response of the organism. For adjuvants of bacterial origin, mainly bacterial Lipopolysaccharides (LPS): it is present in the cell wall of G-bacteria and is composed of a unique hydrophobic lipid and a long repetitive multiple chain linkage. Comprising an O-specific polysaccharide domain, a core domain and a lipid A domain. The lipid a domain has the major biological activity of LPS (including adjuvant activity), acting mainly on lymphocytes and macrophages. For artificially synthesized adjuvants, mainly include liposomes, including immunostimulating complex, etc., which are artificially prepared concentric phospholipid bilayer spheres separated by water. The main components are lecithin (phosphatidylcholine), cholesterol, stearamide, phosphatidic acid and the like.
Various polysaccharides such as: astragalus polysaccharides, angelica polysaccharides, lycium polysaccharides, pachyman, chitosan, etc. all have the function of immunological adjuvant.
Because different adjuvants have different mechanisms of action, in order to design a safer and more effective vaccine, the combined application of two or more adjuvants is attempted, and it is expected that a series of immune cells and various immune mechanisms are activated to induce a specific type of immune response or to exert additive/synergistic effects. For example, AS04, developed by Kurarin Stecke, combines an aluminum adjuvant that produces only a Th 2-type immune response with MPL, which promotes the production of interferon-gamma (IFN-gamma) and elicits a Th 1-type immune response. For the elderly with reduced immune system function, infants with underdeveloped immune system, individuals with chronic diseases, and other infectious disease susceptible people, the immune response to the traditional vaccine is reduced, and adjuvant is needed to improve the vaccine efficacy.
An ideal vaccine adjuvant is required to have high safety, good stability, easy storage and transportation, substantially no irritation, and be effective in stimulating immune response in vivo. There is therefore an urgent need to develop a vaccine adjuvant with the above characteristics.
Disclosure of Invention
The invention provides an oil-in-water type vaccine adjuvant which is characterized in that:
the raw materials are as follows according to volume ratio:
4.3% of squalene;
800.5% of polysorbate;
span 850.5%;
the balance of citrate buffer solution;
wherein the squalene is 2, 6, 10, 15, 19, 23-hexamethyl-2, 6, 10, 14, 18, 22-tetracosahexaene, and has molecular formula of C30H50；
The preparation method of the citrate buffer solution comprises the steps of dissolving a proper amount of citric acid and sodium citrate into water for injection, wherein the pH value is 6.5-6.8;
the preparation method comprises dissolving polysorbate 80 in sodium citrate-citric acid buffer solution, dissolving span 85 in squalene, treating coarse emulsion at 10000 rpm in homogenizer, feeding the coarse emulsion into microfluidizer, further treating under 1000-1200bar (14500-17400 psi) to obtain stable submicron emulsion, and passing the emulsion through 0.22 μm polycarbonate membrane under nitrogen;
the average particle size is about 75nm, D90 is less than 121nm, and D100 is less than 178 nm.
The invention also provides an oil-in-water type vaccine adjuvant which is characterized in that:
the raw materials are as follows according to volume ratio:
4.3% of squalene;
800.5% of polysorbate;
span 850.5%;
0.2 to 0.8 percent of angelica polysaccharide solution with the mass concentration of 20 percent;
the balance of citrate buffer solution;
the preparation method of the citrate buffer solution comprises the steps of dissolving a proper amount of citric acid and sodium citrate into water for injection, wherein the pH value is 6.5-6.8;
the preparation method comprises dissolving radix Angelicae sinensis polysaccharide and polysorbate 80 in sodium citrate-citric acid buffer solution, and dissolving span 85 in squalene; the crude emulsion was processed at 10000 rpm in the homogenizer and was sent to a microfluidizer for further processing at 1000-1200bar (14500-17400 psi) to obtain a stable submicron emulsion which was passed through a 0.22 μm polycarbonate membrane under nitrogen.
The invention also provides an oil-in-water type vaccine adjuvant which is characterized in that:
the raw materials are as follows according to volume ratio:
4.3% of squalene;
800.5% of polysorbate;
span 850.5%;
0.2 to 0.8 percent of angelica polysaccharide solution with the mass concentration of 20 percent;
4000.5% -1.0% of polyethylene glycol;
the balance of citrate buffer solution;
the preparation method of the citrate buffer solution comprises the steps of dissolving a proper amount of citric acid and sodium citrate into water for injection, wherein the pH value is 6.5-6.8;
the preparation method comprises dissolving Angelica sinensis polysaccharide, polyethylene glycol 400, and polysorbate 80 in sodium citrate-citric acid buffer solution, and heating to 60 deg.C; dissolving span 85 in squalene, and heating to 60 deg.C; the crude emulsion was treated at 20000 rpm in a homogenizer at 60 ℃ and sent to a microfluidizer at 1000-1200bar (14500-17400 psi) for further processing to obtain a stable submicron emulsion which was passed through a 0.22 μm polycarbonate membrane under nitrogen.
An oil-in-water vaccine adjuvant as described above, characterized in that: the content of 20 wt% of the Angelica polysaccharide solution is 0.2%, 0.5% or 0.8%.
An oil-in-water vaccine adjuvant as described above, characterized in that: the average particle size of the oil-in-water emulsion is 50-100 nm.
An oil-in-water vaccine adjuvant as described above, characterized in that: the average particle size of the oil-in-water emulsion is about 65nm, D90 is less than 99nm, and D100 is less than 134 nm.
The invention also provides the use of an oil-in-water vaccine adjuvant as defined in any of the above in the preparation of a vaccine formulation.
The preparation method of the angelica polysaccharide comprises the following steps: taking 100g of angelica, crushing, sieving by a 40-mesh sieve, adding 300mL of 95% ethanol, refluxing at 70 ℃ for 2h, filtering, and drying in vacuum at 50 ℃ to obtain pretreated angelica powder. Adding distilled water, heating and refluxing at 80 deg.C for 2 hr, vacuum filtering, collecting liquid, concentrating, adding 500mL of anhydrous ethanol, and standing for 24 hr. Filtering, precipitating, sequentially rinsing with anhydrous alcohol, acetone and diethyl ether, and oven drying the solid in a vacuum drying oven to obtain crude polysaccharide. Dissolving the crude polysaccharide in distilled water, fully dissolving, centrifuging to remove impurities, deproteinizing by combining a Savage method with a papain method, decoloring by using a hydrogen peroxide decoloring method, dialyzing by using a pretreated dialysis bag, concentrating liquid in the bag, precipitating by using ethanol, standing overnight, filtering by suction to obtain precipitate, fully rinsing by using absolute ethyl alcohol, acetone and diethyl ether in sequence, and drying in vacuum to obtain the refined angelica polysaccharide.
The smaller the particle size of the oil-in-water emulsion is, the more stable the oil-in-water emulsion prepared in the present invention is, the average particle size of the oil-in-water emulsion is 100nm or less, and the oil-in-water emulsion has excellent stability.
The oil-in-water emulsion of the invention has no side effect in the experiments such as acute toxicity, anaphylaxis, hemolytic experiment, vascular irritation experiment, long-term toxicity experiment and teratogenesis.
After the angelica polysaccharide is added into the oil-in-water emulsion, the immune effect is obviously enhanced, and the oil-in-water emulsion is successfully prepared and is convenient to use.
After the polyethylene glycol 400 is added into the oil-in-water emulsion, the side effect of the adjuvant is obviously reduced, and the particle size and the long-term stability are not influenced.
The invention also finds that the heating operation is carried out during the preparation process while the polyethylene glycol 400 is added, which is beneficial to reducing the generation of side effects.
Therefore, the oil-in-water emulsion prepared by the method has excellent immune effect and very good safety, and no side effect is found through acute toxicity, anaphylactic reaction, hemolytic experiment, vascular irritation experiment, long-term toxicity experiment, teratogenesis experiment and other experiments. In addition, the product has the advantages of surprising stability, long storage life of more than 3 years, and convenient use and storage.
Drawings
FIG. 1: particle size dynamic distribution of oil-in-water vaccine adjuvants:
FIG. 2: a dynamic particle size distribution of an oil-in-water vaccine adjuvant comprising angelicae polysaccharide;
FIG. 3: IgG antibody titer profiles induced by the first and second needle oil-in-water vaccine adjuvants.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: preparation of oil-in-water vaccine adjuvant:
the raw materials are as follows according to volume ratio:
4.3 percent of squalene
Polysorbate 800.5%
Span 850.5%
Citrate buffer balance
Wherein squalene (2, 6, 10, 15, 19, 23-hexamethyl-2, 6, 10, 14, 18, 22-tetracosahexaene, olive oil origin, molecular formula C30H50And the relative molecular mass was 410.7).
The preparation method of the citrate buffer solution comprises the steps of dissolving a proper amount of citric acid and sodium citrate into water for injection, and adjusting the pH value to 6.5-6.8.
The preparation method comprises the following steps: polysorbate 80 was dissolved in sodium citrate-citric acid buffer solution, span 85 was dissolved in squalene, the crude emulsion was treated in a homogenizer (10000 rpm) and sent to a microfluidizer at 1000-1200bar (14500-17400 psi) for further processing to obtain a stable submicron emulsion which was passed through a 0.22 μm polycarbonate membrane under nitrogen.
The number of large particles in the adjuvant emulsion was monitored in addition to the average particle size during each stage of microfluidization to give an average particle size of 75m, D90 < 121nm, and D100 < 178 nm. The particle size dynamic distribution is shown in figure 1
Example 2: preparation of oil-in-water vaccine adjuvant comprising polysaccharide:
astragalus polysaccharide: purchased from hey oriental plant health technologies ltd;
wolfberry polysaccharide: purchased from jiehui biotechnology limited;
pachyman: obtained by referring to the preparation method on page 97 of 387 examples of saccharide products;
and (3) chitosan: purchased from bio-products, inc.
Angelica polysaccharide: the preparation method comprises the following steps: taking 100g of angelica, crushing, sieving by a 40-mesh sieve, adding 300mL of 95% ethanol, refluxing at 70 ℃ for 2h, filtering, and drying in vacuum at 50 ℃ to obtain pretreated angelica powder. Adding distilled water, heating and refluxing at 80 deg.C for 2 hr, vacuum filtering, collecting liquid, concentrating, adding 500mL of anhydrous ethanol, and standing for 24 hr. Filtering, precipitating, sequentially rinsing with anhydrous alcohol, acetone and diethyl ether, and oven drying the solid in a vacuum drying oven to obtain crude polysaccharide. Dissolving the crude polysaccharide in distilled water, fully dissolving, centrifuging to remove impurities, deproteinizing by combining a Savage method with a papain method, decoloring by using a hydrogen peroxide decoloring method, dialyzing by using a pretreated dialysis bag, concentrating liquid in the bag, precipitating by using ethanol, standing overnight, filtering by suction to obtain precipitate, fully rinsing by using absolute ethyl alcohol, acetone and diethyl ether in sequence, and drying in vacuum to obtain the refined angelica polysaccharide.
The raw materials are as follows according to volume ratio:
4.3 percent of squalene
Polysorbate 800.5%
Span 850.5%
0.5 percent of polysaccharide solution with the mass concentration of 20 percent
The balance of citrate buffer.
Polysorbate 80 and polysaccharide solutions were dissolved in sodium citrate-citric acid buffer solution, span 85 was dissolved in squalene, the crude emulsion was treated in a homogenizer (10000 rpm), sent to a microfluidizer at 1000-1200bar (14500-17400 psi) and further treated to obtain a stable submicron emulsion which was passed through a 0.22 μm polycarbonate membrane under nitrogen.
Figure BDA0003315496490000061
The experiments show that the angelica polysaccharide is suitable for preparing oil-in-water type emulsion, and other polysaccharides do not have the condition for preparing the oil-in-water type emulsion.
Example 3: optimization of oil-in-water vaccine adjuvant comprising angelicae sinensis polysaccharide:
the raw materials are as follows according to volume ratio:
4.3 percent of squalene
Polysorbate 800.5%
Span 850.5%
0.2%, 0.5%, 0.8%, 1.0%, 1.5% of Angelica polysaccharide solution with mass concentration of 20%
The balance of citrate buffer.
Polysorbate 80 and polysaccharide solutions were dissolved in sodium citrate-citric acid buffer solution, span 85 was dissolved in squalene, the crude emulsion was treated in a homogenizer (10000 rpm), sent to a microfluidizer at 1000-1200bar (14500-17400 psi) and further treated to obtain a stable submicron emulsion which was passed through a 0.22 μm polycarbonate membrane under nitrogen.
The number of large particles in the adjuvant emulsion was monitored in addition to the average particle size during each stage of microfluidization to obtain the average particle size. And placing the prepared adjuvant at 2-8 deg.C for 1 month, 6 months, 12 months, 18 months, 24 months, 36 months, observing appearance, and determining average particle diameter (nm).
Angelica polysaccharide 1 month 3 months old 6 months old 12 months old 24 months 36 months old
0.2％ 68 68 69 69 69 69
0.5％ 65 67 67 68 68 69
0.8％ 70 76 76 80 82 85
1.0％ 78 136 187 296 With breaking of emulsion Producing stratification
1.5％ 135 203 289 With breaking of emulsion Producing stratification
The smaller the particle size of the oil-in-water emulsion, the greater its stability; the larger the particle diameter, the more likely it is that droplets thereof are polymerized and broken, and the stability is lowered. According to experimental analysis, when the dosage of the 20% angelica polysaccharide solution is less than 0.8%, the influence on the particle size of the oil-in-water emulsion is small, and the stability is good, and when the dosage is more than 1.0%, the particle size of the oil-in-water emulsion is increased, the stability is reduced, and the long-term storage is not facilitated. Therefore, the addition amount of 20% of angelica polysaccharide cannot be higher than 0.8%.
Example 4: side effect testing of oil-in-water vaccine adjuvants comprising angelicae sinensis polysaccharide:
20 white rabbits were used as experimental subjects (divided into 4 groups, each group consisting of 5 rabbits), 0.5mL of adjuvant was intravenously injected through one side of the ear margin, 0.5mL of sodium chloride injection was injected through the other side of the ear margin, 1 time per day, 5 days of continuous administration were carried out, changes in appearance of the administered site were observed every day during the administration, animals were sacrificed after 2 hours of last administration, the injected site and the week were taken as tissues, sectioned, and HE-stained for observation of thrombosis, endothelial injury and other physiological changes.
Figure BDA0003315496490000071
The experiments show that the injection of the adjuvant every day has no obvious abnormality when the dosage of the 20% angelica polysaccharide solution is below 0.8%, only a little inflammatory infiltration is found during dissection, which indicates that the polysaccharide can generate irritation to blood vessels and muscles when the dosage is high.
In addition to this experiment, acute toxicity, allergic reaction, hemolytic experiment, long-term toxicity experiment and teratogenesis experiment were also carried out, and no side effect was observed in the above experiments. The analysis of the irritation of blood vessels may be related to the uneven distribution of polysaccharide in oil-in-water emulsion, so that other components are added to improve the irritation of the oil-in-water type adjuvant.
Example 5: improved testing of oil-in-water vaccine adjuvants comprising angelicae polysaccharide:
the raw materials are as follows according to volume ratio:
4.3% of squalene;
800.5% of polysorbate;
span 850.5%;
0.8 percent of angelica polysaccharide solution with the mass concentration of 20 percent;
0.5 percent of auxiliary solvent
The balance of citrate buffer.
The auxiliary solvents are respectively: propanol, propylene glycol, glycerol, polyethylene glycol 400, DMSO.
Dissolving polysaccharide, auxiliary solvent, and polysorbate 80 in sodium citrate-citric acid buffer solution, and heating to 60 deg.C; dissolving span 85 in squalene, and heating to 60 deg.C; the crude emulsion was treated in a homogenizer at 60 deg.C (10000 rpm) and fed to a microfluidizer at 1000-1200bar (14500-17400 psi) for further processing to obtain a stable submicron emulsion which was passed through a 0.22 μm polycarbonate membrane under nitrogen.
Experiments show that the oil-in-water type vaccine adjuvant prepared by adding 0.5% of polyethylene glycol 400 does not cause irritation to blood vessels and muscles, and does not change the particle size and long-term storage stability of the oil-in-water type emulsion.
Example 6: formulation testing of oil-in-water vaccine adjuvants comprising angelicae polysaccharide:
the raw materials are as follows according to volume ratio:
4.3% of squalene;
800.5% of polysorbate;
span 850.5%;
0.5 percent of angelica polysaccharide solution with the mass concentration of 20 percent;
4000.5 percent of polyethylene glycol
The balance of citrate buffer.
Dissolving polysaccharide, polyethylene glycol 400, and polysorbate 80 in sodium citrate-citric acid buffer solution, and heating to 60 deg.C; dissolving span 85 in squalene, and heating to 60 deg.C; the crude emulsion was treated in a homogenizer at 60 deg.C (10000 rpm) and fed to a microfluidizer at 1000-1200bar (14500-17400 psi) for further processing to obtain a stable submicron emulsion which was passed through a 0.22 μm polycarbonate membrane under nitrogen.
The prepared oil-in-water vaccine adjuvant meets the requirements of particle size, long-term stability test and vascular irritation test. The particle size of the oil-in-water vaccine adjuvant is analyzed in detail in FIG. 2, and the average particle size is 65m, D90 < 99nm, and D100 < 134 nm.
Example 7: immunological activity comprising an oil-in-water vaccine adjuvant:
in the same manner as in example 6, a 20% angelicae sinensis polysaccharide solution 0.2% and 0.8% oil-in-water adjuvant were prepared.
Diluting rabies vaccine for human to 1.751IU/mL by using physiological saline, mixing an adjuvant and a vaccine solution in equal volume to serve as an immunization group, and randomly dividing experimental mice into the following groups:
group 1: blank control group;
group 2: group of example 1;
group 3: 20% of angelica polysaccharide solution 0.2%;
group 4: 20% of angelica polysaccharide solution 0.5%;
group 5: 20% of angelica polysaccharide solution 0.8%;
group 6: Martix-M adjuvant group;
injecting two needles for 2 times respectively on day 0 and day 14, and drawing blood 2 weeks after the first needle for enzyme-linked immunosorbent assay (ELISA); the second 2-week blood draw was performed by enzyme-linked immunosorbent assay (ELISA) for IgG antibody titer test, respectively, and the test results are shown in FIG. 3.
It can be seen that the 20% solution of Angelica polysaccharide can obtain better effect of immunological adjuvant when the dosage is 0.2% -0.8%.
The foregoing description is a general description of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation, as form changes and equivalents may be employed. Various changes or modifications may be effected therein by one skilled in the art and equivalents may be made thereto without departing from the scope of the invention as defined in the claims appended hereto.

Claims (7)

1. An oil-in-water vaccine adjuvant characterized by:
the raw materials are as follows according to volume ratio:
4.3% of squalene;
800.5% of polysorbate;
span 850.5%;
the balance of citrate buffer solution;
wherein the squalene is 2, 6, 10, 15, 19, 23-hexamethyl-2, 6, 10, 14, 18, 22-tetracosahexaene, and has molecular formula of C30H50；
The preparation method of citrate buffer solution comprises dissolving appropriate amount of citric acid and sodium citrate in injectable water, with pH of 6.5-6.8
The preparation method comprises dissolving polysorbate 80 in sodium citrate-citric acid buffer solution, dissolving span 85 in squalene, treating coarse emulsion at 10000 rpm in homogenizer, feeding the coarse emulsion into microfluidizer, further treating under 1000-1200bar (14500-17400 psi) to obtain stable submicron emulsion, and passing the emulsion through 0.22 μm polycarbonate membrane under nitrogen;
the average particle size of the oil-in-water emulsion is about 75nm, D90 is less than 121nm, and D100 is less than 178 nm.
2. An oil-in-water vaccine adjuvant characterized by:
the raw materials are as follows according to volume ratio:
4.3% of squalene;
800.5% of polysorbate;
span 850.5%;
0.2 to 0.8 percent of angelica polysaccharide solution with the mass concentration of 20 percent;
the balance of citrate buffer solution;
the preparation method of the citrate buffer solution comprises the steps of dissolving a proper amount of citric acid and sodium citrate into water for injection, wherein the pH value is 6.5-6.8;
the preparation method comprises dissolving radix Angelicae sinensis polysaccharide and polysorbate 80 in sodium citrate-citric acid buffer solution, and dissolving span 85 in squalene; the crude emulsion was processed at 10000 rpm in the homogenizer and was sent to a microfluidizer for further processing at 1000-1200bar (14500-17400 psi) to obtain a stable submicron emulsion which was passed through a 0.22 μm polycarbonate membrane under nitrogen.
3. An oil-in-water vaccine adjuvant characterized by:
the raw materials are as follows according to volume ratio:
4.3% of squalene;
800.5% of polysorbate;
span 850.5%;
0.2 to 0.8 percent of angelica polysaccharide solution with the mass concentration of 20 percent;
4000.5% -1.0% of polyethylene glycol;
the balance of citrate buffer solution;
the preparation method of the citrate buffer solution comprises the steps of dissolving a proper amount of citric acid and sodium citrate into water for injection, and adjusting the pH value to 6.5-6.8;
the preparation method comprises dissolving Angelica sinensis polysaccharide, polyethylene glycol 400, and polysorbate 80 in sodium citrate-citric acid buffer solution, and heating to 60 deg.C; dissolving span 85 in squalene, and heating to 60 deg.C; the crude emulsion was treated at 20000 rpm in a homogenizer at 60 ℃ and sent to a microfluidizer at 1000-1200bar (14500-17400 psi) for further processing to obtain a stable submicron emulsion which was passed through a 0.22 μm polycarbonate membrane under nitrogen.
4. An oil-in-water vaccine adjuvant according to claim 2 or 3, characterized in that: the content of 20 wt% of the Angelica polysaccharide solution is 0.2%, 0.5% or 0.8%.
5. An oil-in-water vaccine adjuvant according to claim 2 or 3, characterized in that: the average particle size of the oil-in-water emulsion is 50-100 nm.
6. An oil-in-water vaccine adjuvant according to claim 2 or 3, characterized in that: the average particle size of the oil-in-water emulsion is about 65nm, D90 is less than 99nm, and D100 is less than 134 nm.
7. Use of an oil-in-water vaccine adjuvant according to any of claims 1-6 for the preparation of a vaccine formulation.
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