An oil-in-water vaccine adjuvant, emulsion, and methods of making and using the same

Abstract

The application discloses an oil-in-water vaccine adjuvant, an emulsified product and a preparation method and application thereof, and relates to the technical field of vaccine adjuvant preparation.The adjuvant obtained by the application is not delaminated after high-temperature high-pressure moist heat sterilization, thereby reducing the risk of bacterial contamination when the delaminated adjuvant is mixed again after high-temperature high-pressure moist heat sterilization and facilitating the next production of the vaccine manufacturer.The antigen is mixed with the adjuvant, and the prepared vaccine has stronger immunopotency than the same kind of oil-in-water adjuvant vaccine, which indicates that the oil-in-water adjuvant provided by the application has a good application prospect in vaccine production.The vaccine composition prepared based on the oil-in-water vaccine adjuvant does not need high-energy high-shear, high-speed stirring emulsification, and high-pressure homogenization emulsification, thereby greatly simplifying the emulsification process, successfully emulsifying by low-energy stirring, and making the vaccine production more convenient and efficient.

Description

Technical Field

[0001] This invention relates to the field of vaccine adjuvant preparation technology, and more specifically, to an oil-in-water vaccine adjuvant, an emulsion, a preparation method thereof, and its application. Background Technology

[0002] Currently, the use of veterinary vaccines has effectively reduced the incidence of major animal diseases and is of great significance to the healthy development of animal husbandry. Adjuvants are substances that are injected into the body before or simultaneously with the antigen, and can non-specifically enhance or alter the body's immune response to the antigen. Choosing the right adjuvant can significantly improve the immunoprotective effect of veterinary vaccines. Currently, adding adjuvants has become the mainstream trend in veterinary vaccine design. Although there are many types of adjuvants used in vaccines worldwide, oil-emulsion adjuvants remain the most widely used in animal vaccines.

[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 typically elicit a high level of immune response and provide long-lasting protection, but their high viscosity makes them difficult to inject and disperse and metabolize in the body, leading to significant side effects in animals and even injection site ulceration. Water-in-oil-in-water (W / O / W) vaccines combine the advantages of both W / O and O / W vaccines: low viscosity, easy injection, high immune levels, and long-lasting protection. However, their preparation technology is more challenging, resulting in poorer vaccine stability. Oil-in-water (O / W) vaccines are generally simple to prepare, have low viscosity, disperse easily in the body, leave little residue, and cause virtually no side effects in animals, making them a key focus of research in the veterinary vaccine manufacturing industry in recent years.

[0004] Currently, the most widely used oil-in-water adjuvants are still the oil-emulsion adjuvants designed in the 1980s. These adjuvants not only have significant side effects, but also exhibit poor stability, posing challenges to large-scale application. Firstly, oil-in-water adjuvants have poor stability at room temperature. Coupled with global warming and the frequent occurrence of extreme heat (around 40°C or higher) in most parts of the country during summer, the unstable oil-in-water adjuvants cannot adapt to these climate changes, significantly shortening their shelf life. Furthermore, creating suitable environments for the transportation and storage of oil-in-water adjuvants increases transportation and warehousing costs. Secondly, regarding the high-temperature, moist heat sterilization (121°C, 30 minutes (or even longer)) method for oil-in-water adjuvants, most commercially available oil-in-water adjuvants will separate into layers during this process, requiring subsequent cooling and re-stirring before further use. This re-stirring after sterilization not only increases the risk of contamination but is also time-consuming and labor-intensive, undoubtedly increasing the cost of using the adjuvant. Therefore, the market urgently needs to develop a new generation of oil-in-water adjuvants 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 applicability.

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

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

[0008] This invention is implemented as follows:

[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 comprises a lipophilic surfactant and a hydrophilic surfactant in a mass ratio of 1:(2-20);

[0010] The lipophilic surfactant includes 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 includes 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 polyoxyethylene stearate, fatty alcohol polyoxyethylene ether, and fatty amide polyoxyethylene ether; the second hydrophilic surfactant is selected from any one or any two of polyoxyethylene oleate, lauryl glucoside, lauryl alcohol polyoxyethylene ether, and Tween.

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

[0013] Secondly, the present invention also provides a method for preparing a vaccine adjuvant, which includes mixing the above-mentioned surfactant and oil phase in a certain proportion.

[0014] Thirdly, the present invention also provides an emulsion comprising: the above-described vaccine adjuvant and antigen or a simulated aqueous phase.

[0015] Fourthly, the present invention also provides a method for preparing a vaccine composition, wherein the vaccine adjuvant and the antigen are mixed in an aqueous phase and emulsification is completed by low-energy stirring.

[0016] Fifthly, the present invention also provides the use of vaccine adjuvants or the above-described emulsions in the preparation of compositions for the prevention and / or treatment of animal diseases, wherein the compositions are vaccine compositions or pharmaceutical compositions.

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

[0018] This invention provides a vaccine adjuvant that maintains good stability under high temperature and high pressure conditions. A lipophilic surfactant, a hydrophilic surfactant, and an oil phase are compounded and mixed in a specific ratio. The resulting adjuvant does not separate into layers after high-temperature, high-pressure, and moist heat sterilization. This reduces the risk of contamination when the adjuvant separates into layers after high-temperature, high-pressure, and moist heat sterilization and is also convenient for vaccine manufacturers to proceed with subsequent production steps.

[0019] Furthermore, the emulsion (or emulsion) obtained by mixing the adjuvant with the aqueous phase provided by this invention has a uniform particle size distribution, and the particle size distribution is normally distributed. The emulsion breaks down at 37°C for more than 15 days and remains stable in a refrigerator at 4-8°C for a long time. The emulsion is uniformly milky white, does not adhere to the wall, and has good fluidity. After centrifugation at 3500 rpm for 15 minutes, less than 0.5 ml of aqueous phase is separated, and the emulsion has good stability.

[0020] The vaccine prepared by mixing the vaccine antigen with an adjuvant exhibits slightly stronger immunogenicity than similar adjuvanted vaccines. This indicates that the adjuvant provided by this invention has promising prospects for application in vaccine production.

[0021] When preparing vaccine compositions based on the vaccine adjuvant of the present invention, high-energy, high-shear, high-speed stirring emulsification and high-pressure homogenization emulsification are not required, which greatly simplifies the emulsification process. Low-energy stirring is sufficient for successful emulsification, making vaccine production and use more convenient and efficient. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Changes in the adjuvants prepared in Examples 1-6 of the invention before and after sterilization by high temperature, high pressure, and moist heat (121°C, 30 min);

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

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

[0026] Figure 4 This is a particle size distribution diagram of the emulsion prepared by emulsifying the adjuvant with an aqueous citrate solution in Example 4 of the present invention.

[0027] Figure 5 The particle size distribution diagram is shown for the emulsion prepared in Example 4 of this invention after emulsification with a 0.9% sodium chloride solution in aqueous phase.

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

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

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

[0031] Figure 9 The particle size distribution diagram of the mycoplasma vaccine prepared after storing the adjuvant prepared in Example 4 of the present invention at -20°C;

[0032] Figure 10 This is a particle size distribution diagram of a mycoplasma vaccine prepared using the adjuvant prepared in Example 4 of the present invention after storage at room temperature;

[0033] Figure 11 The 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] Figure 12 Particle size distribution of a mycoplasma vaccine prepared using a similar oil-in-water adjuvant on the market;

[0035] Figure 13 The dynamic changes in serum ELISA antibodies in piglets immunized with Mycoplasma pneumoniae vaccine in Experiment Example 5;

[0036] Figure 14 The image shows the stratification results of the water-in-oil-in-water adjuvant product provided in Comparative Example 1 before and after high-temperature, high-pressure, and moist heat sterilization (121°C, 30 min). Detailed Implementation

[0037] To more clearly illustrate the purpose, technical solution, and advantages of the present invention, a further description will be provided below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following drawings and specific embodiments are only for explaining the present invention and should not be considered as limiting the scope of the invention. For those skilled in the art, other related embodiments can be obtained based on the present invention without creative or innovative effort, and all of these fall within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified 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 comprises a lipophilic surfactant and a hydrophilic surfactant in a mass ratio of 1:(2-20);

[0039] The lipophilic surfactant includes 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 includes 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 polyoxyethylene stearate, fatty alcohol polyoxyethylene ether, and fatty amide polyoxyethylene ether; the second hydrophilic surfactant is selected from any one or any two of polyoxyethylene oleate, lauryl glucoside, lauryl alcohol polyoxyethylene ether, and Tween.

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

[0042] Compared with similar oil-in-water adjuvants, the vaccine adjuvant provided by this invention has better stability, is easier to manage during transportation and storage, and does not separate after high-temperature, high-pressure, and moist heat sterilization. This reduces the risk of contamination when the adjuvant separates after high-temperature, high-pressure, and moist heat sterilization and is also convenient for vaccine manufacturers to proceed with the next stage of production. The vaccine adjuvant provided by this invention has good stability at both room temperature (around 25°C) and 37°C. Although the adjuvant is in a lard-like solidified state at low temperatures (-20°C or even lower), it can be used normally after thawing at room temperature. The emulsion formed by mixing the adjuvant with the antigen and other aqueous phases breaks down after more than 15 days at 37°C and remains stable in a refrigerator at 4-8°C for a long time, demonstrating 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 polyoxyethylene stearate, polyoxyethylene oleate, and Tween in the hydrophilic surfactant is (2-4.5):(2.5-4):1, preferably 2:3:1. For example, the mass ratio of polyoxyethylene stearate, polyoxyethylene oleate, 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 a fatty alcohol polyoxyethylene ether, and the second hydrophilic surfactant is Tween.

[0047] The lipophilic surfactants include a first lipophilic surfactant and a second lipophilic surfactant with 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 a better effect on improving the stability and immunogenicity of the vaccine composition.

[0050] In a preferred embodiment of the present invention, the mass ratio of polyglycerol ricinoleate, glyceryl monooleate 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] At the above mixing ratio, the adjuvant exhibits better stability and stronger immunogenicity when added to 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 surfactant to 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 surfactant to 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 lipophilic surfactants and hydrophilic surfactants with 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 selected from any of the above-mentioned point values.

[0058] In a preferred embodiment of the present invention, the surfactant includes lipophilic surfactants and hydrophilic surfactants 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 pharmaceutical mineral oil and squalene in a mass ratio of (80-100):(0-20); for example, the mixed oil comprises (80-90):(0-20), (80-100):(1-10), (80-100):(5-15), or (80-100):(10-20) in a mass ratio.

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

[0061] In a preferred embodiment of the present invention, the oil phase comprises a mixture of 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 mixture of 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 pharmaceutical mineral oil (excluding squalene), the adjuvant exhibits good stability.

[0064] Secondly, the present invention also provides a method for preparing a vaccine adjuvant, which includes mixing the above-mentioned surfactant and oil phase in a certain proportion and stirring until the mixture is clear and homogeneous.

[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) First, mix the lipophilic surfactant and the hydrophilic surfactant to obtain a surfactant mixture, and then mix it with the oil phase;

[0067] (2) First, mix the lipophilic surfactant with the oil phase, and then mix the resulting mixture with the hydrophilic surfactant;

[0068] (3) First, the hydrophilic surfactant is mixed with the oil phase, and then the resulting mixture is mixed with the lipophilic surfactant;

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

[0070] As long as the surfactant and oil phase are mixed in the correct proportions, regardless of which surfactant and oil phase are mixed first, they are all within the scope of protection of this invention.

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

[0072] In a preferred embodiment of the present invention, it is better to seal the container during the stirring and mixing process, fill it with inert gas, and maintain an appropriate negative air pressure, so that the resulting adjuvant has better stability.

[0073] In one alternative embodiment, the vaccine adjuvant prepared by the present invention does not separate into layers after being treated with high temperature and humidity (121°C, 30 min) or high temperature and humidity (121°C, 90 min).

[0074] In one optional embodiment, the water-in-oil vaccine adjuvant prepared by the present invention can be stored at low temperatures (-20°C or even lower). Although the adjuvant is in the form of solidified lard at low temperatures (-20°C or even lower), it can be used normally after thawing and melting back into liquid at warm temperatures.

[0075] In one optional embodiment, the water-in-oil vaccine adjuvant prepared by the present invention can be stored at 37°C for 24 months without separation and can still be used normally after emulsification.

[0076] The vaccine adjuvant prepared by this invention exhibits good stability and is easy to manage environmental conditions during transportation and storage. This vaccine adjuvant can meet the requirements for use in tropical, subtropical, temperate, subarctic, and frigid zones, among others.

[0077] Thirdly, the present invention also provides an emulsion comprising: the above-described vaccine adjuvant and an aqueous phase.

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

[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 also contain at least one of the following substances: immune enhancers, solubilizing agents, thickeners, suspending agents, pH adjusters, dispersants, buffers, and preservatives.

[0081] In one alternative implementation, the immune-enhancing agent includes an immune-enhancing lipid-soluble drug and an immune-enhancing water-soluble drug.

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

[0083] Suspending agents, also known as suspending agents, include polyvinylpyrrolidone and aluminum stearate. Preservatives include sodium benzoate, sodium bisulfite, and parabens (e.g., methylparaben, ethylparaben, propylparaben, butylparaben).

[0084] Thickening agents can be adjusted using pharmaceutically acceptable thickeners such as methylcellulose. Other suitable thickeners include xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc.

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

[0086] pH adjusters are 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 vaccine adjuvant to aqueous phase is 1:(9-13). Vaccines emulsified at this ratio exhibit better stability, suitable viscosity, and good safety. The mass ratio of vaccine adjuvant to aqueous phase includes, but is not limited to, 1:9, 1:10, 1:11, 1:12, or 1:13.

[0088] The active ingredients in vaccines can be derived from livestock or pet animals, such as pigs, sheep, cattle, horses, chickens, ducks, geese, cats, dogs, rabbits, or monkeys.

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

[0090] The water-in-oil vaccine adjuvant prepared by this invention is easy to use, can be successfully emulsified with a variety of aqueous reagents, and can also be successfully emulsified with vaccine antigens from multiple vaccine manufacturers. It allows vaccine manufacturers to easily add immune-enhancing substances, adjust the emulsification ratio appropriately, and choose to produce monovalent or multivalent vaccines.

[0091] Fourthly, the present invention also provides a method for preparing a vaccine composition, wherein the vaccine adjuvant and the antigen are mixed in an aqueous phase and emulsification is completed by low-energy stirring.

[0092] The vaccine adjuvant preparation emulsion prepared by the method of this 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 stirring, making vaccine production and use more convenient and efficient.

[0093] In a preferred embodiment of the present invention, mixing and emulsification are carried out under conditions of 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 preferred for small-scale emulsification, and the adjuvant is added to the vaccine active ingredient or aqueous phase reagent under stirring conditions.

[0096] In a preferred embodiment of the present invention, when using a mechanical agitator for small-scale emulsification, the vaccine antigen or aqueous reagent is added to the adjuvant under stirring conditions.

[0097] In a preferred embodiment of the present invention, it is preferable to use a mechanical stirring paddle for large-scale emulsification. 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 method of the present invention can be used to prepare vaccine compositions or blank O / W type emulsions without the need for high-energy, high-shear, high-speed stirring emulsification or 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] Fifthly, the present invention also provides the use of vaccine adjuvants or the above-described emulsions in the preparation of compositions for the prevention and / or treatment of animal diseases, wherein the compositions are vaccine compositions or pharmaceutical compositions.

[0100] The vaccine compositions prepared with the vaccine adjuvant provided by this invention have good stability, low viscosity, are easy to inject, have few side effects, and have good immunization effects.

[0101] Animal diseases include, but are not limited to, diseases of poultry, livestock, pets, 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 include Riemerella, Escherichia coli, Pasteurella, Salmonella, Haemophilus, and Brucella. Gram-positive bacteria include Staphylococcus aureus, Streptococcus, Erysipelothrix rhusiopathiae, 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] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0106] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0107] Example 1

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

[0109] The novel veterinary vaccine adjuvant consists of an oil phase, a hydrophilic surfactant, and a lipophilic surfactant. A mixture of the lipophilic and hydrophilic surfactants 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, with a mass ratio of polyoxyethylene stearate, polyoxyethylene oleate, and Tween 80 of 2:3:1.

[0111] The lipophilic surfactant is a mixture of polyglycerol ricinoleate, glyceryl monooleate, and Span 80, with a mass ratio of 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] Mix the oil phase, lipophilic surfactant, and hydrophilic surfactant in the above proportions and stir until a clear and homogeneous 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; in this example, it is 1:9.

[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; in this example, it is 1:11.

[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; in this example, it is 1:8.

[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 separate into layers after high-temperature, high-pressure, and moist heat sterilization (121°C, 30 min). Furthermore, all adjuvant formulations included in this patent will separate into layers after 121°C, 30 min. Figure 14 Among them, A, B, C, D, E, and F are all water-in-oil-in-water (W / O / W) adjuvants provided by this 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] Removing Tween 80 improves adjuvant stability, but reduces the stability of the vaccine composition (including emulsions).

[0132] Experimental Example 1

[0133] The adjuvants prepared in Examples 1-6 were subjected to high-temperature moist heat sterilization (121°C, 30 min). The results showed that none of the adjuvants prepared in Examples 1-6 separated into layers, while a similar oil-in-water adjuvant on the market separated into layers after the same treatment. Figure 1 This indicates that the water-in-oil adjuvant prepared by the present invention has certain advantages in stability compared with similar water-in-oil 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] Experiment Example 2

[0136] Prepare 10 mM phosphate buffer, acetate solution, and citrate solution respectively. Emulsify the adjuvant prepared in Example 4 with 10 mM phosphate buffer, acetate solution, citrate solution, and 0.9% sodium chloride solution (Sichuan Kelun Pharmaceutical Co., Ltd.), respectively. (Particle size distribution diagrams correspond to...) Figure 2 , Figure 3 , Figure 4 , Figure 5 (As shown).

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

[0138] Stop stirring, dispense the resulting emulsion, and perform the following quality control:

[0139] (1) Appearance: The prepared emulsions are all uniformly milky white. When shaken in a beaker, the walls are slightly blue. The emulsions do not stick to the walls and have good fluidity.

[0140] (2) Water drop test: The prepared blank emulsion was dropped drop by drop into cold water that was standing in a beaker. The emulsion showed uniform solubility and diffusion, indicating that the emulsion formulation was oil-in-water (O / W).

[0141] (3) Particle size distribution: 20 μL of O / W emulsion was diluted with 1980 μL of PBS buffer and mixed thoroughly. The average particle size and particle size distribution were measured using a laser particle size analyzer at 25℃. The average particle size was approximately 130 nm, and the particle size distribution was normally distributed. Figure 2 , Figure 3 , Figure 4 , Figure 5 (As shown).

[0142] (4) Viscosity measurement: The viscosity of the above emulsions at 25℃ was measured using a Brookfield DVNext rheometer and was 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 aqueous phase precipitated at the bottom was less than 0.5 mL, indicating that the emulsion had good stability.

[0144] (6) Stability test at different temperatures: The above-packaged emulsions were placed in environments of 37℃ and 4-8℃ respectively, and observed at regular intervals. The above emulsions all broke down in more than 15 days at 37℃, and remained stable in a refrigerator at 4-8℃ for a long time.

[0145] Experimental Example 3

[0146] The adjuvant prepared in Example 4 was dispensed and stored at -20°C, room temperature (around 25°C), and 37°C, respectively. After 6 months, it was taken out and warmed to room temperature as the oil phase. This oil phase was then emulsified with 0.9% sodium chloride solution (Sichuan Kelun Pharmaceutical Co., Ltd.) and Mycoplasma hyopneumoniae antigen as the aqueous phase to prepare blank O / W emulsions and vaccine compositions. The emulsification method of the adjuvant with 0.9% sodium chloride solution was the same as in Experiment 2. The method for emulsifying the adjuvant with Mycoplasma hyopneumoniae antigen to prepare the vaccine composition is as follows. Simultaneously, a similar oil-in-water adjuvant 15A on the market was also emulsified with Mycoplasma hyopneumoniae antigen using the following method:

[0147] i: Sterilize the adjuvant at 121℃ for 30 minutes, cool and shake well before use;

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

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

[0150] iv: Stop stirring and aseptically dispense the resulting vaccine composition.

[0151] The following quality control measures were performed on the above blank O / W emulsion (emulsion) and vaccine composition: (1) Appearance: The prepared emulsion and vaccine composition were uniformly milky white, with a light blue stain on the wall when shaken in the beaker, and the emulsion did not stick to the wall and had good fluidity.

[0152] (2) Water drop test: The prepared blank emulsion and vaccine composition were dropped drop by drop into cold water that was standing in a beaker. The emulsion showed uniform solubility and diffusion, indicating that the dosage form of the emulsion and vaccine composition was oil-in-water (O / W).

[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 determined using a laser particle size analyzer at 25℃. The average particle size was approximately 130 nm, and the particle size distribution was normally distributed. Figure 6 , Figure 7 , Figure 8 The particle size distribution diagrams are shown for the emulsions formed after the adjuvants were stored at -20℃, room temperature and 37℃ and then emulsified with a 0.9% sodium chloride solution in aqueous phase. Figure 9 , Figure 10 and Figure 11 The images show the particle size distribution of the emulsions after the adjuvant was stored at -20℃, room temperature, and 37℃ and then emulsified with mycoplasma antigen. The particle size distribution of the 15A vaccine composition is shown in the figures below. Figure 12 As shown in the figure. The results indicate that the emulsions prepared using the adjuvants provided in this invention exhibit good particle size stability at different temperatures.

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

[0155] (5) Centrifugal stability: Take 10 mL of the prepared emulsion and vaccine composition and add them to centrifuge tubes respectively. Centrifuge at 3500 rpm for 15 min. The aqueous phase precipitated at the bottom is less than 0.5 mL, indicating that the emulsion and vaccine composition have good stability.

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

[0157] Experiment Example 4

[0158] Safety tests were conducted on the rewarmed adjuvant and the prepared vaccine composition in Experiment Example 3.

[0159] In Experiment 3, the adjuvants prepared in Example 4 were aliquoted and stored at -20°C, room temperature (around 25°C), and 37°C for 6 months, respectively, and then thawed. The adjuvants were labeled as adjuvant A, adjuvant B, and adjuvant C. The vaccine compositions prepared by emulsifying adjuvant A, adjuvant B, and adjuvant C with porcine mycoplasma pneumoniae antigen were labeled as vaccine A, vaccine B, and vaccine C, respectively.

[0160] (1) Forty-five healthy female BALB / c mice weighing 18-22g at 2 weeks of age 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. The mice were observed for 7 consecutive days after injection 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 any group, indicating that the adjuvants and vaccines were safe.

[0161] (2) Forty-five healthy, unweaned piglets aged 2-3 weeks that were negative for both Mycoplasma pneumoniae antigen and antibody were randomly divided into groups 1-9, with 5 piglets in each group. Piglets in groups 1, 2, 3, and 4 were injected intramuscularly into the neck with 2 mL of adjuvant A, adjuvant B, adjuvant C, and 15A adjuvant, respectively. Piglets in groups 5, 6, 7, and 8 were injected intramuscularly into the neck with 2 mL of vaccine A, vaccine B, vaccine C, and 15A Mycoplasma vaccine, respectively. Piglets in group 9 were injected intramuscularly into the neck with 2 mL of 0.9% sodium chloride injection (Sichuan Kelun Pharmaceutical Co., Ltd.) as a blank control group. The piglets were observed for 14 consecutive days after injection and their clinical status was recorded. It was found that no deaths, obvious local adverse reactions, or systemic reactions caused by the injection of vaccines or adjuvants occurred in any group, and there were no other stress reactions, indicating that the adjuvants and vaccines were safe.

[0162] Experimental Example 5

[0163] This embodiment tests the immunogenicity of the four mycoplasma vaccine compositions (vaccine A, vaccine B, vaccine C, and 15A mycoplasma vaccine) in Experiment 4 on pigs.

[0164] Twenty-five healthy, unweaned piglets aged 2-3 weeks that were negative for both Mycoplasma pneumoniae antigen and antibody were randomly divided into five groups (groups 1-5), with five piglets in each group. Piglets in groups 1, 2, 3, and 4 were injected intramuscularly into the neck with 2 mL of vaccine A, vaccine B, vaccine C, and Mycoplasma 15A vaccine, respectively. Piglets in group 5 were injected intramuscularly into the neck with 2 mL of 0.9% sodium chloride injection (Sichuan Kelun Pharmaceutical Co., Ltd.) as a blank control group. Rectal temperature was measured daily for each piglet two days prior to immunization. Rectal temperature was measured at 24h, 48h, and 72h post-immunization, and the clinical status of the piglets was observed and recorded. No deaths, significant local adverse reactions, or systemic reactions caused by the injected vaccine or adjuvant occurred in any group, and no other side effects were observed. No significant changes in body temperature were observed at 24h, 48h, and 72h, indicating good safety.

[0165] Blood samples were collected from all piglets every two weeks after immunization to separate serum. The serum was then used to detect Mycoplasma hyopneumoniae competitive ELISA antibody detection kits (Biotec (Tianjin) Biotechnology Co., Ltd., catalog number: MHYOPC-5P) to assess the immunogenicity of the vaccine. Figure 13 ).

[0166] The results showed that the mycoplasma O / W adjuvant vaccine provided by the present invention has slightly stronger immunogenicity than the mycoplasma 15A adjuvant vaccine, and after the adjuvant provided by the present invention was stored at significantly different ambient temperatures for six months, the mycoplasma vaccines prepared with the adjuvant did not show significant differences in immunogenicity.

[0167] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An oil-in-water vaccine adjuvant, characterized in that, It includes: a surfactant and an oil phase in a mass ratio of 3:7, wherein the surfactant is a lipophilic surfactant and a hydrophilic surfactant in a mass ratio of 1:7; The lipophilic surfactant is a mixture of polyglycerol ricinoleate, glyceryl monooleate, and Span 80; the mass ratio of polyglycerol ricinoleate, glyceryl monooleate, and Span 80 is 1:2:

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

1. The oil phase comprises pharmaceutical-grade mineral oil and liquid paraffin in a mass ratio of 5:

1.

2. A method for preparing an oil-in-water vaccine adjuvant, characterized in that, It includes the following steps: mixing the surfactant of claim 1 with the oil phase.

3. An emulsion, characterized in that, It includes: The oil-in-water vaccine adjuvant and aqueous phase as described in claim 1.

4. The emulsion according to claim 3, characterized in that, The emulsion is an oil-in-water emulsion.

5. The emulsion according to claim 3, characterized in that, The aqueous phase is selected from antigens or simulated aqueous phase reagents.

6. The emulsion according to claim 5, characterized in that, 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.

7. The emulsion according to claim 4, characterized in that, The oil-in-water emulsion also contains at least one of the following substances: immune enhancers, solubilizing agents, thickeners, suspending agents, buffers, and preservatives.

8. The emulsion according to claim 3, characterized in that, The mass ratio of the adjuvant to the aqueous phase in the oil-in-water vaccine is 1:(9-13).

9. The method for preparing the emulsion according to any one of claims 3-8, characterized in that, The oil-in-water vaccine adjuvant is mixed with the aqueous phase, and emulsification is completed by low-energy stirring.

10. The method for preparing the emulsion according to claim 9, characterized in that, Mixing and emulsification are carried out under magnetic or mechanical stirring conditions.

11. The method for preparing the emulsion according to claim 10, characterized in that, 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.

12. The use of the oil-in-water vaccine adjuvant as described in claim 1 or the emulsion as described in any one of claims 3-8 in the preparation of a vaccine composition for the prevention of Mycoplasma hyopneumoniae.

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