Oil-in-water type vaccine adjuvant, emulsion and preparation method and application of oil-in-water type vaccine adjuvant

Abstract

The invention discloses an oil-in-water vaccine adjuvant, an emulsion as well as a preparation method and application of the oil-in-water vaccine adjuvant, and relates to the technical field of vaccine adjuvant preparation. The adjuvant obtained by the invention is still not layered after being subjected to high-temperature and high-pressure moist heat sterilization. The contamination risk when the adjuvant is uniformly mixed after being subjected to high-temperature and high-pressure damp-heat degerming and layering is reduced, and vaccine manufacturers can conveniently carry out next-step production. The antigen and the adjuvant are mixed, and the prepared vaccine has higher immune efficacy than similar oil-in-water adjuvant vaccines. The oil-in-water adjuvant provided by the invention has good vaccine production and application prospects. When a vaccine composition is prepared on the basis of the oil-in-water vaccine adjuvant, high kinetic energy, high shearing, high-speed stirring emulsification and high-pressure homogeneous emulsification are not needed either, the emulsification process is greatly simplified, emulsification can be successful through low kinetic energy stirring, and vaccine production and use are more convenient and efficient.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine adjuvant preparation, and in particular to an oil-in-water vaccine adjuvant, an emulsion, and a preparation method and application thereof. Background Art

[0002] At present, the use of veterinary vaccines has effectively reduced the occurrence of major animal diseases, which is of great significance to the healthy development of animal husbandry. Adjuvants are substances that are injected into the body before or at the same time as antigens, and can non-specifically enhance or change the body's immune response to antigens. Choosing a suitable adjuvant can improve the immune protection effect of veterinary vaccines with half the effort. At present, in the design of veterinary vaccines, adding adjuvants has become a mainstream trend. Although there are many types of adjuvants for vaccines in various countries around the world, the most widely used adjuvant for animal vaccines is still oil-emulsion adjuvant.

[0003] Commonly used oil-emulsion adjuvant vaccines can be divided into three dosage forms: water-in-oil (W / O), oil-in-water (O / W), and water-in-oil-in-water (W / O / W). Water-in-oil (W / O) vaccines can usually obtain a higher level of immune response and a long immune protection period, but the vaccine has a high viscosity, is not easy to inject, and is not easy to disperse and metabolize in the body. The high viscosity leads to large animal side effects and even ulceration at the injection site; water-in-oil-in-water (W / O / W) vaccines have the advantages of both water-in-oil (W / O) and oil-in-water (O / W) vaccines. This type of vaccine has low viscosity, is easy to inject, has a high immune level, and a long immune protection period, but its preparation technology is difficult and the vaccine stability is poor; water-in-oil (O / W) vaccines usually have a simple preparation process, low viscosity, are easy to disperse after injection into the body, are not easy to remain, and have basically no side effects in animals. It has become the focus of research in the veterinary vaccine manufacturing industry in recent years.

[0004] The most widely used water-in-oil adjuvant is still the oil-emulsion adjuvant designed in the 1980s. This adjuvant not only has large side effects, but also has poor stability, which brings certain difficulties to large-scale promotion and application. First of all, the stability of water-in-oil adjuvants is poor when stored at room temperature. In addition, the current climate warming often causes extreme hot weather of about 40°C or even higher in most parts of the country in summer. Water-in-oil adjuvants with poor stability cannot adapt to such climate changes, and not only will the shelf life of their products be greatly shortened. In order to create a suitable environment for the transportation and storage of water-in-oil adjuvants, the transportation and storage costs of water-in-oil adjuvant products are increased. Secondly, in the high-temperature and wet heat sterilization (121°C, 30 minutes (or even longer)) of water-in-oil adjuvants, most water-in-oil adjuvants on the market will be stratified during high-temperature and wet heat sterilization (121°C, 30 minutes (or even longer)), and then need to be cooled and stirred evenly before continuing to the next step. Stirring again after sterilization not only increases the risk of contamination, but also consumes time and effort, and undoubtedly increases the cost of adjuvant use. Therefore, the market urgently needs a new generation of novel water-in-oil adjuvants developed independently to fill the demand.

[0005] Based on the above background and current situation, it is imperative to develop an oil-in-water emulsion adjuvant with good stability, simple preparation process, few side effects and wide application.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide an oil-in-water vaccine adjuvant, an emulsion, and a preparation method and application thereof to solve the above technical problems.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides an oil-in-water vaccine adjuvant, comprising: a surfactant and an oil phase in a mass ratio of 1:(1.0-6.0), wherein the surfactant mass ratio is a lipophilic surfactant and a hydrophilic surfactant in a mass ratio of 1:(2-20);

[0010] The lipophilic surfactant comprises a first lipophilic surfactant and a second lipophilic surfactant in a mass ratio of 1:(1.5-4.0); the first lipophilic surfactant is selected from any one of polyglycerol ricinoleate, glyceryl monostearate, and lecithin; the second lipophilic surfactant is selected from any one or any two of soybean lecithin, span, and glyceryl monooleate;

[0011] The hydrophilic surfactant comprises a first hydrophilic surfactant and a second hydrophilic surfactant in a mass ratio of 1:(0.6-2.5); the first hydrophilic surfactant is selected from any one of stearic acid polyoxyethylene ester, fatty alcohol polyoxyethylene ether, and fatty amide polyoxyethylene ether, and the second hydrophilic surfactant is selected from any one or any two of oleic acid polyoxyethylene ester, lauryl glucoside, lauryl alcohol polyoxyethylene ether, and Tween;

[0012] The oil phase comprises a mixed oil and a liquid bioprotectant in a mass ratio of (4-8):1, the liquid bioprotectant is selected from at least one of liquid paraffin or glycerol, and the mixed oil is selected from at least one of medicinal mineral oil and squalene.

[0013] In a second aspect, the present invention also provides a method for preparing a vaccine adjuvant, which comprises mixing the above-mentioned surfactant and oil phase in proportion.

[0014] In a third aspect, the present invention further provides an emulsion, which comprises: the above-mentioned vaccine adjuvant and an antigen or a simulated aqueous phase.

[0015] In a fourth aspect, the present invention also provides a method for preparing a vaccine composition, wherein the vaccine adjuvant and the antigen are mixed in aqueous phase and emulsified by low-energy stirring.

[0016] In a fifth aspect, the present invention also provides the use of the vaccine adjuvant or the above-mentioned emulsion in the preparation of a composition for preventing and / or treating animal diseases, wherein the composition is a vaccine composition or a pharmaceutical composition.

[0017] The present invention has the following beneficial effects:

[0018] The present invention provides a vaccine adjuvant that can maintain good stability under high temperature and high pressure conditions. The lipophilic surfactant, the hydrophilic surfactant and the oil phase are compounded and mixed in a specific ratio, and the obtained adjuvant remains unstratified after high temperature and high pressure wet heat sterilization. This not only reduces the risk of contamination when the adjuvant is mixed after being stratified by high temperature and high pressure wet heat sterilization, but also facilitates the vaccine manufacturer to carry out the next step of production.

[0019] In addition, the emulsion (or emulsion) obtained by mixing the adjuvant provided by the present invention with the water phase has a uniform particle size distribution, and the particle size distribution is normally distributed; the emulsion breaks at 37°C for more than 15 days, and remains stable for a long time in a refrigerator at 4-8°C; the emulsion is uniform milky white, has no wall adhesion, and has good fluidity; the emulsion is centrifuged at 3500rpm for 15 minutes, and the precipitated water phase is less than 0.5ml, and the emulsion has good stability.

[0020] The vaccine antigen is mixed with the adjuvant, and the prepared vaccine has a slightly stronger immune efficacy than similar adjuvant vaccines, indicating that the adjuvant provided by the present invention has a good prospect for vaccine production and application.

[0021] When preparing a vaccine composition based on the vaccine adjuvant of the present invention, there is no need for high kinetic energy, high shear, high-speed stirring emulsification, nor is there a need for high-pressure homogenization emulsification, which greatly simplifies the emulsification process. Emulsification can be successfully achieved with low kinetic energy stirring, making vaccine production and use more convenient and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 The changes of the adjuvant prepared in Example 1-6 of the invention before and after sterilization by high temperature, high pressure and wet heat (121°C, 30min);

[0024] Figure 2 This is a particle size distribution diagram of the emulsion after the adjuvant prepared in Example 4 of the present invention is emulsified with a phosphate buffer solution as the aqueous phase;

[0025] Figure 3 This is a particle size distribution diagram of the emulsion after the adjuvant prepared in Example 4 of the present invention is emulsified with an acetate solution as the aqueous phase;

[0026] Figure 4 This is a particle size distribution diagram of the emulsion after the adjuvant prepared in Example 4 of the present invention is emulsified with a citrate solution as the aqueous phase;

[0027] Figure 5 This is a particle size distribution diagram of the emulsion after the adjuvant prepared in Example 4 of the present invention is emulsified with a 0.9% sodium chloride solution as the aqueous phase;

[0028] Figure 6 This is a particle size distribution diagram of the emulsion after the adjuvant prepared in Example 4 of the present invention is stored at -20°C and emulsified with a 0.9% sodium chloride solution as the aqueous phase;

[0029] Figure 7 This is a particle size distribution diagram of the emulsion after the adjuvant prepared in Example 4 of the present invention is stored at room temperature and emulsified with a 0.9% sodium chloride solution as the aqueous phase;

[0030] Figure 8 This is a particle size distribution diagram of the emulsion after the adjuvant prepared in Example 4 of the present invention is stored at 37° C. and emulsified with a 0.9% sodium chloride solution as the aqueous phase;

[0031] Fig. 9 This is a particle size distribution diagram of the mycoplasma vaccine prepared by storing the adjuvant prepared in Example 4 of the present invention at -20°C;

[0032] Fig.10 This is a particle size distribution diagram of the mycoplasma vaccine prepared by storing the adjuvant prepared in Example 4 of the present invention at room temperature;

[0033] Fig.11 This is a particle size distribution diagram of the mycoplasma vaccine prepared by storing the adjuvant prepared in Example 4 of the present invention at 37°C;

[0034] Fig.12 This is the particle size distribution diagram of a mycoplasma vaccine prepared with a similar oil-in-water adjuvant on the market;

[0035] Fig.13 The dynamic changes of ELISA antibodies in the serum of piglets after immunization with Mycoplasma hyopneumoniae vaccine in Experimental Example 5;

[0036] Fig.14 This is a diagram showing the stratification results of the water-in-oil-in-water adjuvant product provided in Comparative Example 1 before and after high-temperature, high-pressure, wet-heat sterilization (121°C, 30 min). DETAILED DESCRIPTION

[0037] In order to more clearly illustrate the purpose, technical solutions and advantages of the present invention, further description will be made below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following drawings and specific embodiments are only used to explain the present invention, and therefore should not be regarded as limiting the scope of the present invention. For those of ordinary skill in the art, other relevant embodiments can also be obtained according to the present invention without paying creativity and innovative work, all of which belong to the scope of protection of the present invention. Those who do not specify specific conditions in the embodiments are carried out according to normal conditions or conditions recommended by the manufacturer. Those who do not specify the manufacturer of reagents or instruments used are all conventional products that can be purchased commercially.

[0038] In a first aspect, the present invention provides a vaccine adjuvant, comprising: a surfactant and an oil phase in a mass ratio of 1:(1.0-6.0), wherein the surfactant mass ratio is a lipophilic surfactant and a hydrophilic surfactant in a mass ratio of 1:(2-20);

[0039] The lipophilic surfactant comprises a first lipophilic surfactant and a second lipophilic surfactant in a mass ratio of 1:(1.5-4.0); the first lipophilic surfactant is selected from any one of polyglycerol ricinoleate, glyceryl monostearate, and lecithin; the second lipophilic surfactant is selected from any one or any two of soybean lecithin, span, and glyceryl monooleate;

[0040] The hydrophilic surfactant comprises a first hydrophilic surfactant and a second hydrophilic surfactant in a mass ratio of 1:(0.6-2.5); the first hydrophilic surfactant is selected from any one of stearic acid polyoxyethylene ester, fatty alcohol polyoxyethylene ether, and fatty amide polyoxyethylene ether, and the second hydrophilic surfactant is selected from any one or any two of oleic acid polyoxyethylene ester, lauryl glucoside, lauryl alcohol polyoxyethylene ether, and Tween;

[0041] The oil phase comprises a mixed oil and a liquid bioprotectant in a mass ratio of (4-8):1, the liquid bioprotectant is selected from at least one of liquid paraffin or glycerol, and the mixed oil is selected from at least one of medicinal mineral oil and squalene.

[0042] Compared with similar oil-in-water adjuvants, the vaccine adjuvant provided by the present invention has better stability, is easy for environmental management during transportation and storage, and remains unstratified after high-temperature, high-pressure, and wet-heat sterilization. It not only reduces the risk of contamination when the adjuvant is mixed after stratification after high-temperature, high-pressure, and wet-heat sterilization, but also facilitates vaccine manufacturers to carry out the next step of production. The vaccine adjuvant provided by the present invention has good stability at both room temperature (about 25°C) and 37°C. Although the adjuvant is solidified in the form of lard at low temperatures (-20°C, or even lower), it can be used normally after being thawed and melted into a liquid. The emulsion after the adjuvant is mixed with the aqueous phase such as the antigen breaks the emulsion at 37°C for more than 15 days, and remains stable for a long time in a refrigerator at 4-8°C, and has good stability.

[0043] In a preferred embodiment of the present invention, the hydrophilic surfactant includes a first hydrophilic surfactant and a second hydrophilic surfactant in a mass ratio of 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.5, 1:2 or 1:2.5.

[0044] In a preferred embodiment of the present invention, the first hydrophilic surfactant is polyoxyethylene stearate, and the second hydrophilic surfactant is polyoxyethylene oleate and Tween.

[0045] In a preferred embodiment of the present invention, the mass ratio of stearic acid polyoxyethylene ester, oleic acid polyoxyethylene ester and Tween in the hydrophilic surfactant is (2-4.5): (2.5-4): 1, preferably 2: 3: 1. For example, the mass ratio of stearic acid polyoxyethylene ester, oleic acid polyoxyethylene ester and Tween is 2: 2.5: 1, 4: 3: 1 or 4.5: 2.5: 1.

[0046] In a preferred embodiment of the present invention, the first hydrophilic surfactant is fatty alcohol polyoxyethylene ether, and the second hydrophilic surfactant is Tween.

[0047] The lipophilic surfactant includes a first lipophilic surfactant and a second lipophilic surfactant in a mass ratio of 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5 or 1:4.0, or any point value selected from the above point value range.

[0048] In a preferred embodiment of the present invention, the first lipophilic surfactant is polyglycerol ricinoleate; the second lipophilic surfactant is Span and glyceryl monooleate.

[0049] In a preferred embodiment of the present invention, the first lipophilic surfactant is polyglycerol ricinoleate, and the second lipophilic surfactant is Span 80. When the lipophilic surfactant is selected from the above two substances, it has the effect of better improving the stability and immune efficacy of the vaccine composition.

[0050] In a preferred embodiment of the present invention, the mass ratio of polyglycerol ricinoleate, monooleyl glycerol and Span in the lipophilic surfactant is (1.7-2):(1.5-5):1; for example, 1.7:1.5:1, 1.8:2:1, 2:2:1, 2:3:1, 2:4:1 or 2:5:1.

[0051] Preferably, the mass ratio of polyglycerol ricinoleate, glyceryl monooleate and Span in the lipophilic surfactant is 1:2:1.

[0052] Under the above mixing ratio, the adjuvant has better stability and has stronger immune efficacy after being added into the aqueous phase.

[0053] In a preferred embodiment of the present invention, Tween is selected from Tween 40, Tween 60, Tween 80, Tween 81 or Tween 85.

[0054] The Span is selected from Span 60, Span 65, Span 80, Span 83 or Span 85.

[0055] In a preferred embodiment of the present invention, the mass ratio of the surfactant to the oil phase is 1:1.5, 1:2-3, 1:3.5 or 1:4.

[0056] In a preferred embodiment of the present invention, the mass ratio of the surfactant to the oil phase is 1:1.5, 1:2, 3:7, 1:2.5, 1:3, 1:3.5 or 1:4.

[0057] In a preferred embodiment of the present invention, the surfactant includes a lipophilic surfactant and a hydrophilic surfactant in a mass ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20, or any point value selected from the above point value range.

[0058] In a preferred embodiment of the present invention, the surfactant includes a lipophilic surfactant and a hydrophilic surfactant in a mass ratio of 1:6, 1:10 or 1:15.

[0059] In a preferred embodiment of the present invention, the mixed oil comprises medicinal mineral oil and squalene in a mass ratio of (80-100):(0-20); for example, the mixed oil comprises a mass ratio of (80-90):(0-20), (80-100):(1-10), (80-100):(5-15) or (80-100):(10-20).

[0060] In an optional embodiment of the present invention, the mixed oil includes medicinal mineral oil and squalene in a mass ratio of 80:20. When squalene is contained, the stability of the vaccine composition of the adjuvant and the aqueous phase is higher.

[0061] In a preferred embodiment of the present invention, the oil phase comprises a mixed oil and a liquid bioprotectant in a mass ratio of 4:1, 5:1, 6:1, 7:1 or 8:1.

[0062] In a preferred embodiment of the present invention, the oil phase comprises a mixed oil and a liquid bioprotectant in a mass ratio of 5-6:1.

[0063] In a preferred embodiment of the present invention, when the mixed oil in the oil phase is entirely medicinal mineral oil (without squalene), the adjuvant has good stability.

[0064] In a second aspect, the present invention further provides a method for preparing a vaccine adjuvant, which comprises mixing the above-mentioned surfactant and oil phase in proportion and stirring the mixture until the mixture is clear and uniform.

[0065] In a preferred embodiment of the present invention, the surfactant and the oil phase are mixed according to any of the following methods:

[0066] (1) firstly mixing a lipophilic surfactant and a hydrophilic surfactant to obtain a surfactant mixture, and then mixing the mixture with an oil phase;

[0067] (2) firstly mixing the lipophilic surfactant with the oil phase, and then mixing the obtained mixture with the hydrophilic surfactant;

[0068] (3) firstly mixing a hydrophilic surfactant with an oil phase, and then mixing the obtained mixture with a lipophilic surfactant;

[0069] (4) Add the lipophilic surfactant, hydrophilic surfactant and oil phase into a container and stir to mix.

[0070] As long as the surfactant and the oil phase are stirred and mixed according to the proportion, no matter which surfactant and oil phase are mixed first, it is within the protection scope of the present invention.

[0071] In a preferred embodiment of the present invention, the surfactant and the oil phase are mixed in proportion to form a clear and uniform liquid, without the need for high-energy excessive stirring and mixing.

[0072] In a preferred embodiment of the present invention, during the stirring and mixing process, the container is sealed, filled with inert gas, and preferably an appropriate negative air pressure is maintained, so that the prepared adjuvant has better stability.

[0073] In an optional embodiment, the vaccine adjuvant prepared by the present invention does not stratify after being treated with high temperature moist heat (121° C., 30 min) or high temperature moist heat (121° C., 90 min).

[0074] In an optional embodiment, the oil-in-water vaccine adjuvant prepared by the present invention can be stored at low temperature (-20°C, or even lower temperature). Although the adjuvant is solidified in the form of lard at low temperature (-20°C, or even lower temperature), it can be used normally after being thawed and melted into liquid at the same temperature.

[0075] In an optional embodiment, the oil-in-water vaccine adjuvant prepared by the present invention can be stored in an environment of 37° C. after being sealed and packaged, and will not delaminate for 24 months, and can still be used in normal emulsification.

[0076] The vaccine adjuvant prepared by the invention has good stability and is easy to manage in the environment during transportation and storage. The vaccine adjuvant can be used in, but not limited to, tropical, subtropical, temperate, subarctic and arctic regions.

[0077] In a third aspect, the present invention further provides an emulsion, which comprises: the above-mentioned vaccine adjuvant and an aqueous phase.

[0078] In a preferred embodiment of the present invention, the aqueous phase is selected from a vaccine antigen or a simulated aqueous phase reagent;

[0079] In a preferred embodiment of the present invention, the simulated aqueous phase reagent is selected from any one of phosphate buffer, 0.9% sodium chloride solution, acetate solution, citrate solution, purified water and water for injection;

[0080] In a preferred embodiment of the present invention, the oil-in-water emulsion may further include at least one of the following substances: an immunopotentiating agent, a solubilizing drug, a thickener, a suspending agent, a pH adjusting agent, a dispersant, a buffer and a preservative.

[0081] In an optional embodiment, the immune enhancing agent includes an immune enhancing fat-soluble drug and an immune enhancing water-soluble drug.

[0082] In an optional embodiment, the solubilizing agent is selected from urea, guanidine hydrochloride, sodium deoxycholate, sodium dodecyl sulfate, glycerol, protein degrading enzymes, albumin, lecithin, 0.1-2000 mg / mL inorganic salts, Triton, Tween, acetic acid, cholesterol, amino acids, glycosides, choline, Brij TM-35, octaethylene glycol monolauryl ether, 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt, digitonin, lauryl dimethylamine oxide, CA-630, dimethyl sulfoxide, acetonitrile, ethanol, methanol, N,N-dimethylformamide, isopropanol, dichloromethane, propanol and ethyl acetate. One or more.

[0083] Suspending agents are also called suspending agents, such as polyvinyl pyrrolidone, aluminum stearate, preservatives such as sodium benzoate, sodium bisulfite, parabens (such as methyl paraben, ethyl paraben, propyl paraben, butyl paraben).

[0084] The viscosity can be adjusted by a pharmaceutically acceptable thickener such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like.

[0085] Buffers include, but are not limited to, phosphate solutions, carbonate solutions, and citrate solutions.

[0086] The pH adjuster is selected from HEPES, citrate, histidine, calcium carbonate, sodium carbonate, potassium carbonate, sodium bicarbonate, calcium bicarbonate, potassium bicarbonate, aluminum hydroxide, and magnesium hydroxide.

[0087] In a preferred embodiment of the present invention, the mass ratio of the vaccine adjuvant to the aqueous phase is 1:(9-13). The emulsified vaccine at this ratio has better stability, suitable viscosity, and good safety. The mass ratio of the vaccine adjuvant to the aqueous phase includes but is not limited to 1:9, 1:10, 1:11, 1:12 or 1:13.

[0088] The active ingredients of the vaccine can come from poultry or pet animals, such as pigs, sheep, cattle, horses, chickens, ducks, geese, cats, dogs, rabbits or monkeys.

[0089] In an optional embodiment, the vaccine active ingredient is selected from any one or any two of porcine diarrhea virus antigen, porcine rotavirus antigen, porcine circovirus antigen, inactivated foot-and-mouth disease virus antigen and Mycoplasma hyopneumoniae antigen.

[0090] The oil-in-water vaccine adjuvant prepared by the present invention is easy to use, can be successfully emulsified with a variety of aqueous phase reagents, and can also be successfully emulsified with vaccine antigens from multiple vaccine manufacturers. It can not only be convenient for vaccine manufacturers to independently add immune-enhancing substances, but also appropriately adjust the emulsification ratio. At the same time, they can also independently choose to produce monovalent vaccines or multivalent combination vaccines.

[0091] In a fourth aspect, the present invention also provides a method for preparing a vaccine composition, wherein the vaccine adjuvant and the antigen are mixed in aqueous phase and emulsified by low-energy stirring.

[0092] The vaccine adjuvant emulsion prepared by the preparation method of the present invention does not require high kinetic energy, high shear, high-speed stirring emulsification, nor does it require high-pressure homogenization emulsification, which greatly simplifies the emulsification process. Emulsification can be successfully achieved with low kinetic energy stirring, making vaccine production and use more convenient and efficient.

[0093] In a preferred embodiment of the present invention, mixing and emulsification are performed under magnetic stirring or mechanical stirring;

[0094] In a preferred embodiment of the present invention, the emulsification speed during magnetic stirring is 500-600 rpm, and the emulsification time is 5-15 min; the emulsification speed during mechanical stirring is 400-500 rpm, and the emulsification time is 5-15 min.

[0095] In a preferred embodiment of the present invention, magnetic stirring is preferably selected during small-scale emulsification, and the adjuvant is added to the vaccine active ingredient or the aqueous phase reagent under stirring conditions;

[0096] In a preferred embodiment of the present invention, when a mechanical stirring paddle is used for stirring in a small-scale emulsification, the vaccine antigen or aqueous phase reagent is added to the adjuvant under stirring conditions;

[0097] In a preferred embodiment of the present invention, a mechanical stirring paddle is preferably used for large-scale emulsification, and the adjuvant is added to the vaccine antigen or aqueous phase reagent under stirring conditions, and it is preferable to avoid adding the adjuvant directly to the vortex, the mechanical stirring shaft and the inner wall of the stirring container.

[0098] The vaccine adjuvant prepared by the preparation method of the present invention does not require high-energy, high-shear, high-speed stirring emulsification, nor does it require high-pressure homogenization emulsification, which greatly simplifies the emulsification process. Emulsification can be successfully achieved with low energy, making vaccine production and use more convenient and efficient.

[0099] In a fifth aspect, the present invention also provides the use of the vaccine adjuvant or the above-mentioned emulsion in the preparation of a composition for preventing and / or treating animal diseases, wherein the composition is a vaccine composition or a pharmaceutical composition.

[0100] The vaccine composition prepared by the vaccine adjuvant provided by the present invention has good stability, low viscosity, easy injection, small side effects and good immune effect.

[0101] Animal diseases include but are not limited to: diseases of livestock, pet animals, or aquatic animals caused by pathogenic microorganisms. Pathogenic microorganisms include but are not limited to: diseases caused by pathogenic microorganisms such as fungi, bacteria, and viruses.

[0102] Pathogenic microorganisms are pathogenic Gram-positive bacteria and Gram-negative bacteria.

[0103] Pathogenic Gram-negative bacteria are Riemerella, Escherichia coli, Pasteurella, Salmonella, Haemophilus, and Brucella. Gram-positive bacteria are Staphylococcus aureus, Streptococcus, Erysipelothrix, Mycobacterium, and Bacillus anthracis.

[0104] In addition, pathogenic microorganisms include but are not limited to diseases caused by Haemophilus parasuis, enterotoxigenic Escherichia coli, pseudorabies virus, porcine circovirus or porcine reproductive and respiratory syndrome virus.

[0105] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0106] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0107] Example 1

[0108] This embodiment provides a novel oil-in-water vaccine adjuvant for veterinary use.

[0109] The composition of the novel veterinary vaccine adjuvant includes an oil phase, a hydrophilic surfactant and a lipophilic surfactant. The mixture of the lipophilic surfactant and the hydrophilic surfactant is defined as a mixture (Smix). The mass ratio of the mixture (Smix) to the oil phase is 2:5.

[0110] The hydrophilic surfactant is a mixture of polyoxyethylene stearate, polyoxyethylene oleate and Tween 80, and the mass ratio of polyoxyethylene stearate, polyoxyethylene oleate and Tween 80 is 2:3:1.

[0111] The lipophilic surfactant is a mixture of polyglycerol ricinoleate, glyceryl monooleate and Span 80, and the mass ratio of polyglycerol ricinoleate, glyceryl monooleate and Span 80 is 1:2:1.

[0112] The oil phase is a mixture of pharmaceutical grade mineral oil and liquid paraffin in a mass ratio of 5:1.

[0113] In this embodiment, the mass ratio of the lipophilic surfactant to the hydrophilic surfactant is 1:7.

[0114] The preparation method is as follows:

[0115] The oil phase, lipophilic surfactant and hydrophilic surfactant in the above proportions are mixed and stirred until a clear and uniform liquid is obtained.

[0116] Example 2

[0117] Compared with Example 1, the only difference is that the mass ratio of the lipophilic surfactant to the hydrophilic surfactant is different, which is 1:9 in this example.

[0118] Example 3

[0119] Compared with Example 1, the only difference is that the mass ratio of the lipophilic surfactant to the hydrophilic surfactant is different, which is 1:11 in this example.

[0120] Example 4

[0121] Compared with Example 1, the only difference is that the mass ratio of the mixture (Smix) to the oil phase is different. In this example, the mass ratio of the mixture (Smix) to the oil phase is 3:7.

[0122] Example 5

[0123] Compared with Example 1, the only difference is that the mass ratio of the mixture (Smix) to the oil phase is different. In this example, the mass ratio of the mixture (Smix) to the oil phase is 4:7.

[0124] Example 6

[0125] Compared with Example 5, the only difference is that the mass ratio of the lipophilic surfactant to the hydrophilic surfactant is different, which is 1:8 in this example.

[0126] Comparative Example 1

[0127] Compared with Example 1, the only difference is that the composition of the hydrophilic surfactant is different. In this comparative example, the hydrophilic surfactant is a mixture of polyglycerol-2-dioleate, decaglycerol monolaurate, and TWeen 85, and the mass ratio of polyglycerol-2-dioleate, decaglycerol monolaurate and TWeen 85 is 2:1:1.

[0128] The adjuvant product type corresponding to patent CN202311370067.X is water-in-oil-in-water (W / O / W), and its adjuvant will be separated after high temperature and high pressure wet heat sterilization (121°C, 30 minutes). And all adjuvant formulas included in the patent will be separated after 121°C, 30 minutes ( Fig.14 , wherein A, B, C, D, E, and F are all water-in-oil-in-water (W / O / W) adjuvants provided by the patent).

[0129] Comparative Example 2

[0130] Compared with Example 1, the only difference is that the composition of the hydrophilic surfactant is different. In this comparative example, the hydrophilic surfactant is a mixture of polyoxyethylene stearate and polyoxyethylene oleate, and the mass ratio of polyoxyethylene stearate to polyoxyethylene oleate is 2:3.

[0131] After deleting Tween 80, the adjuvant stability is better, but the stability of the vaccine composition (including emulsion) will be reduced.

[0132] Experimental Example 1

[0133] The adjuvants prepared in Examples 1-6 were sterilized by high temperature and moist heat (121°C, 30 min). The results showed that none of the adjuvants prepared in Examples 1-6 was stratified, while a similar oil-in-water adjuvant on the market was stratified after the same treatment ( Figure 1 ), indicating that the stability of the oil-in-water adjuvant prepared by the present invention is superior to that of similar oil-in-water adjuvants.

[0134] Figure 1 The numbers 1-6 represent Examples 1-6, respectively, and 15A represents a similar oil-in-water adjuvant on the market.

[0135] Experimental Example 2

[0136] 10 mM phosphate buffer, acetate solution, and citrate solution were prepared respectively, and the adjuvant prepared in Example 4 was emulsified with 10 mM phosphate buffer, acetate solution, citrate solution, and 0.9% sodium chloride solution (Sichuan Kelun Pharmaceutical Co., Ltd.). (The particle size diagram corresponds to Figure 2 , Figure 3 , Figure 4 , Figure 5 shown).

[0137] The emulsification method is as follows: 15 g of the prepared adjuvant and 135 g of the aqueous phase are weighed separately into a 250 ml beaker, and the adjuvant is added to the aqueous phase at a stirring speed of 600 rpm using a magnetic stirrer (completed within 3 seconds, 5-10 L / min), and stirred for 10 min.

[0138] Stop stirring, aliquot the resulting emulsion and perform the following quality controls:

[0139] (1) Appearance: The prepared emulsions are uniformly milky white, with a light blue color on the wall when shaken in a beaker. The emulsions have no wall adhesion and have good fluidity.

[0140] (2) Water drop test: The prepared blank emulsion was dropped drop by drop into cold water placed in a beaker. It can be seen that the emulsion showed uniform solubility and diffusion, indicating that the emulsion dosage form was an oil-in-water (O / W) type.

[0141] (3) Particle size distribution: Take 20 μL of O / W emulsion and dilute it with 1980 μL of PBS buffer and mix it thoroughly. Use a laser particle size analyzer to measure its average particle size and particle size distribution. The test temperature is 25°C. The average particle size is basically around 130 nm, and the particle size distribution is normally distributed ( Figure 2 , Figure 3 , Figure 4 , Figure 5 shown).

[0142] (4) Viscosity measurement: The viscosity of the above emulsions at 25°C was measured using a Brookfield DVNext rheometer and was found to be less than 10 mPa·s.

[0143] (5) Centrifugal stability: 10 mL of the prepared emulsion was added to a centrifuge tube and centrifuged at 3500 rpm for 15 min. The amount of water precipitated at the bottom was less than 0.5 mL, indicating that the emulsion had good stability.

[0144] (6) Stability test at different temperatures: Place the above emulsions in 37°C and 4-8°C environments respectively and observe them regularly. The above emulsions will demulsify in more than 15 days at 37°C and remain stable for a long time in a refrigerator at 4-8°C.

[0145] Experimental Example 3

[0146] The adjuvant prepared in Example 4 was packaged and stored at -20°C, room temperature (about 25°C), and 37°C. After 6 months, it was taken out and warmed up at room temperature as the oil phase, and emulsified with 0.9% sodium chloride solution (Sichuan Kelun Pharmaceutical Co., Ltd.) and Mycoplasma hyopneumoniae antigen as the aqueous phase to prepare a blank O / W emulsion and a vaccine composition. Among them, the emulsification method of the adjuvant and the 0.9% sodium chloride solution is consistent with that of Experimental Example 2. The method for emulsifying the adjuvant and the Mycoplasma hyopneumoniae antigen to prepare the vaccine composition is as follows. At the same time, a similar oil-in-water adjuvant 15A on the market was also emulsified with the Mycoplasma hyopneumoniae antigen according to the following emulsification method:

[0147] i: sterilize the adjuvant with high temperature and wet heat at 121℃ for 30min, shake well after cooling and set aside for use;

[0148] ii: Weigh 15 g of the prepared adjuvant and 135 g of the aqueous phase (mycoplasma antigen) into a sterilized 250 ml beaker;

[0149] iii: Add the adjuvant to the aqueous phase (mycoplasma antigen) at a stirring speed of 600 rpm using a magnetic stirrer (3 seconds, 5-10 L / min), and stir for 10 min;

[0150] iv: Stop stirring and aseptically distribute the obtained vaccine composition.

[0151] The following quality controls were performed on the blank O / W emulsions (emulsions) and vaccine compositions: (1) Appearance: The prepared emulsions and vaccine compositions were uniformly milky white, with a light blue color on the wall when shaken in a beaker. The emulsions had no wall adhesion and had good fluidity.

[0152] (2) Water drop test: The prepared blank emulsion and vaccine composition were dropped drop by drop into cold water placed in a beaker. The emulsion was observed to be evenly soluble and diffused, indicating that the emulsion and vaccine composition were in the oil-in-water (O / W) formulation.

[0153] (3) Particle size distribution: 20 μL of O / W emulsion and vaccine composition were diluted with 1980 μL of PBS buffer and mixed thoroughly. The average particle size and particle size distribution were measured by laser particle size analyzer. The test temperature was 25°C. The average particle size was basically around 130 nm, and the particle size distribution was normal. Figure 6 , Figure 7 , Figure 8 They are the particle size distribution diagrams of the emulsions after the adjuvants are stored at -20°C, room temperature and 37°C and emulsified with a 0.9% sodium chloride solution as the aqueous phase; Fig. 9 , Fig.10 and Fig.11 The particle size distribution diagrams of the emulsions after the adjuvant was stored at -20°C, room temperature and 37°C and emulsified with the mycoplasma antigen are shown in Figure 1. The particle size distribution diagrams of the 15A vaccine composition are shown in Figure 2. Fig.12 The results show that the emulsion prepared by using the adjuvant provided by the present invention has good particle size stability at different temperatures.

[0154] (4) Viscosity measurement: The viscosity of the above emulsions and vaccine compositions at 25°C was measured using a Brookfield DVNext rheometer and was less than 10 mPa·s.

[0155] (5) Centrifugal stability: 10 mL of the prepared emulsion and vaccine composition were added to a centrifuge tube and centrifuged at 3500 rpm for 15 min. The aqueous phase precipitated at the bottom was less than 0.5 mL, indicating that the emulsion and vaccine composition had good stability.

[0156] (6) Stability test at different temperatures: The above-packaged emulsions and vaccine compositions were placed in 37°C and 4-8°C environments, respectively, and observed regularly. The above emulsions and vaccine compositions demulsified in more than 15 days at 37°C, and remained stable for a long time in a refrigerator at 4-8°C.

[0157] Experimental Example 4

[0158] A safety test was conducted on the rewarmed adjuvant and the prepared vaccine composition in Experimental Example 3.

[0159] In Experimental Example 3, the adjuvant prepared in Example 4 was packaged and stored at -20°C, room temperature (about 25°C), and 37°C for 6 months. The adjuvants that were warmed up were labeled as Adjuvant A, Adjuvant B, and Adjuvant C, respectively. The vaccine compositions prepared by emulsifying adjuvant A, adjuvant B, adjuvant C and Mycoplasma hyopneumoniae antigen were labeled as Vaccine A, Vaccine B, and Vaccine C, respectively.

[0160] (1) Forty-five healthy female 2-week-old BALB / c mice weighing 18-22 g were randomly divided into groups 1-9, with 5 mice in each group. Mice in groups 1, 2, 3, and 4 were subcutaneously injected with 1 mL of adjuvant A, adjuvant B, adjuvant C, and 15A adjuvant, respectively; mice in groups 5, 6, 7, and 8 were subcutaneously injected with 1 mL of vaccine A, vaccine B, vaccine C, and 15A mycoplasma vaccine, respectively; mice in group 9 were subcutaneously injected with 1 mL of 0.9% sodium chloride injection (Sichuan Kelun Pharmaceutical Co., Ltd.) as a blank control group. After injection, the mice were observed for 7 consecutive days and their clinical status was recorded. It was found that no death or obvious local adverse reactions or systemic reactions caused by the injection of adjuvants or vaccines occurred in all groups, indicating that the adjuvants and vaccines were safe.

[0161] (2) 45 healthy unweaned piglets aged 2 to 3 weeks with negative antigen and antibody test results for Mycoplasma hyopneumoniae were randomly divided into groups 1 to 9, with 5 piglets in each group. Piglets in groups 1, 2, 3, and 4 were injected with 2 mL of adjuvant A, adjuvant B, adjuvant C, and 15A adjuvant into the neck muscle, respectively; piglets in groups 5, 6, 7, and 8 were injected with 2 mL of vaccine A, vaccine B, vaccine C, and 15A mycoplasma vaccine into the neck muscle, respectively; piglets in group 9 were injected with 2 mL of 0.9% sodium chloride injection (Sichuan Kelun Pharmaceutical Co., Ltd.) into the neck muscle as a blank control group. After injection, the piglets were observed for 14 days and their clinical status was recorded. It was found that no death or obvious local adverse reactions or systemic reactions caused by the injection of vaccines or adjuvants occurred in all groups, and no other stress reactions occurred, indicating that the adjuvants and vaccines were safe.

[0162] Experimental Example 5

[0163] This example conducts an immune efficacy test on pigs using the four groups of mycoplasma vaccine compositions (vaccine A, vaccine B, vaccine C, and 15A mycoplasma vaccine) in Experimental Example 4.

[0164] 25 healthy unweaned piglets aged 2 to 3 weeks with negative antigen and antibody of Mycoplasma hyopneumoniae were randomly divided into 1-5 groups, 5 in each group. The piglets in groups 1, 2, 3, and 4 were injected with 2mL of vaccine A, vaccine B, vaccine C, and 15A Mycoplasma vaccine into the neck muscle, respectively. The piglets in group 5 were injected with 2mL of 0.9% sodium chloride injection (Sichuan Kelun Pharmaceutical Co., Ltd.) into the neck muscle as a blank control group. The rectal temperature of each piglet was measured every day for two days before immunization. The rectal temperature was measured at 24h, 48h, and 72h after immunization, and the clinical status of the piglets was observed and recorded. It was found that no death or obvious local adverse reactions or systemic reactions caused by injection of vaccines or adjuvants occurred in all groups, and there were no other side effects. There was no significant change in body temperature at 24h, 48h, and 72h, indicating that it was safe.

[0165] After immunization, blood was collected from all piglets every two weeks to separate serum. The serum was tested for Mycoplasma hyopneumoniae ELISA antibody using a competitive ELISA antibody detection kit (Biowater (Tianjin) Biotechnology Co., Ltd., catalog number: MHYOPC-5P) to evaluate the immune efficacy of the vaccine ( Fig.13 ).

[0166] The results showed that the Mycoplasma O / W adjuvant vaccine provided by the present invention has a slightly stronger immune efficacy than the Mycoplasma 15A adjuvant vaccine, and after the adjuvant provided by the present invention was stored in a large difference in ambient temperature for half a year, there was no obvious difference in the immune efficacy of the Mycoplasma vaccine formulated with the adjuvant.

[0167] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An oil-in-water vaccine adjuvant, characterized in that It comprises: a surfactant and an oil phase in a mass ratio of 1:(1.0-6.0), wherein the surfactant mass ratio is a lipophilic surfactant and a hydrophilic surfactant in a mass ratio of 1:(2-20); The lipophilic surfactant comprises a first lipophilic surfactant and a second lipophilic surfactant in a mass ratio of 1:(1.5-4.0); the first lipophilic surfactant is selected from any one of polyglycerol ricinoleate, glyceryl monostearate, and lecithin; the second lipophilic surfactant is selected from any one or any two of soybean lecithin, span, and glyceryl monooleate; The hydrophilic surfactant comprises a first hydrophilic surfactant and a second hydrophilic surfactant in a mass ratio of 1:(0.6-2.5); the first hydrophilic surfactant is selected from any one of stearic acid polyoxyethylene ester, fatty alcohol polyoxyethylene ether, and fatty amide polyoxyethylene ether, and the second hydrophilic surfactant is selected from any one or any two of oleic acid polyoxyethylene ester, lauryl glucoside, lauryl alcohol polyoxyethylene ether, and Tween; The oil phase comprises a mixed oil and a liquid bioprotectant in a mass ratio of (4-8):1, wherein the liquid bioprotectant is selected from at least one of liquid paraffin or glycerol, and the mixed oil is selected from at least one of medicinal mineral oil and squalene.

2. The oil-in-water vaccine adjuvant according to claim 1, characterized in that The first hydrophilic surfactant is polyoxyethylene stearate, and the second hydrophilic surfactant is polyoxyethylene oleate and Tween; Preferably, the mass ratio of the polyoxyethylene stearate, polyoxyethylene oleate and Tween in the hydrophilic surfactant is (2-4.5): (2.5-4): 1; Preferably, the mass ratio of the polyoxyethylene stearate, polyoxyethylene oleate and Tween in the hydrophilic surfactant is 2:3:

1.

3. The oil-in-water vaccine adjuvant according to claim 1, characterized in that The first hydrophilic surfactant is fatty alcohol polyoxyethylene ether, and the second hydrophilic surfactant is Tween; Preferably, the first lipophilic surfactant is polyglycerol ricinoleate; the second lipophilic surfactant is span and glyceryl monooleate; Preferably, the mass ratio of polyglycerol ricinoleate, glyceryl monooleate and Span in the lipophilic surfactant is (1.7-2): (1.5-5): 1; Preferably, the mass ratio of polyglycerol ricinoleate, glyceryl monooleate and Span in the lipophilic surfactant is 1:2:

1.

4. The oil-in-water vaccine adjuvant according to claim 1, characterized in that The Tween is selected from Tween 40, Tween 60, Tween 80, Tween 81 or Tween 85; The Span is selected from Span 60, Span 65, Span 80, Span 83 or Span 85.

5. The oil-in-water vaccine adjuvant according to claim 1, characterized in that The mass ratio of the surfactant to the oil phase is 1:1.5, 1:2-3, 1:3.5 or 1:4; Preferably, the mass ratio of the surfactant to the oil phase is 1:1.5, 1:2, 3:7, 1:2.5, 1:3, 1:3.5 or 1:4; Preferably, the surfactant comprises a lipophilic surfactant and a hydrophilic surfactant in a mass ratio of 1:6, 1:10 or 1:

15.

6. The oil-in-water vaccine adjuvant according to claim 1, characterized in that The mixed oil comprises medicinal mineral oil and squalene in a mass ratio of (80-100):(0-20); Preferably, the oil phase comprises a mixed oil and a liquid bioprotectant in a mass ratio of 5-6:1; Preferably, the mixed oil is medicinal mineral oil.

7. A method for preparing an oil-in-water vaccine adjuvant, characterized in that: The method comprises the following steps: mixing the surfactant described in any one of claims 1 to 6 with an oil phase.

8. An emulsion, characterized in that: It comprises: the oil-in-water vaccine adjuvant according to any one of claims 1 to 6 and an aqueous phase; Preferably, the emulsion is an oil-in-water emulsion; Preferably, the aqueous phase is selected from an antigen or a simulated aqueous phase reagent; Preferably, the simulated aqueous phase reagent is selected from any one of phosphate buffer, 0.9% sodium chloride solution, acetate solution, citrate solution, purified water and water for injection; Preferably, the oil-in-water emulsion further contains at least one of the following substances: an immunopotentiating agent, a solubilizing drug, a thickener, a suspending agent, a pH regulator, a dispersant, a buffer and a preservative; Preferably, the mass ratio of the oil-in-water vaccine adjuvant to the aqueous phase is 1:(9-13).

9. The method for preparing an emulsion according to claim 8, characterized in that: The oil-in-water vaccine adjuvant is mixed with water and emulsified by low-energy stirring; Preferably, mixing and emulsification are carried out under conditions of magnetic stirring or mechanical stirring; Preferably, the emulsification speed during the magnetic stirring is 500-600 rpm, and the emulsification time is 5-15 min; the emulsification speed during the mechanical stirring is 400-500 rpm, and the emulsification time is 5-15 min.

10. Use of the oil-in-water vaccine adjuvant according to any one of claims 1 to 6 or the emulsion according to claim 8 in preparing a composition for preventing and / or treating animal diseases, characterized in that: The composition is a vaccine composition or a pharmaceutical composition.

Images