Vaccine adjuvant, vaccine composition, preparation method and application thereof

Abstract

The present invention discloses a vaccine adjuvant, a vaccine composition, and a preparation method and application thereof, relating to the field of veterinary technology. The present invention uses safe, stable, and efficient lipophilic and hydrophilic surfactants, which can reduce the body's side effects and accumulation in the body, and has the technical advantage of high safety. The invention helps to improve the quality of veterinary vaccines and reduce the production costs of veterinary vaccine manufacturers. After the vaccine adjuvant is prepared into a vaccine composition, it can effectively enhance the immunogenicity of the vaccine, improve the vaccine's immune effect, and provide long-term and stable immune protection.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary medicine, and in particular to a vaccine adjuvant, a vaccine composition, and a preparation method and application thereof. Background Art

[0002] my country's animal vaccine market has entered a period of rapid growth, with an increasing number of vaccine types and a constant stream of new vaccines. Adjuvants, as a crucial component in improving vaccine quality, play an irreplaceable role in vaccines. They can significantly enhance the body's immune response and have evolved in tandem with vaccines. Their usage is enormous, and the market is promising. With the exception of a few products, most inactivated vaccines use aluminum adjuvants and oil adjuvants, with oil adjuvants being the most widely used adjuvant in the global animal vaccine industry.

[0003] Currently, there are three formulations of oil-adjuvanted vaccines: single-phase water-in-oil (W / O), oil-in-water (O / W), and biphasic water-in-oil-in-water (W / O / W). Water-in-oil (W / O) vaccines can achieve a high level of immune response and a longer period of immune protection, but their high viscosity makes them difficult to inject and often leads to local granulomas and pain, as well as side effects, including fever and loss of appetite in immunized animals. Unlike W / O vaccines, oil-in-water (O / W) vaccines have a lower viscosity, are easier to inject, are well tolerated, and induce short-term immune responses. However, their adjuvant activity is weak, limiting their ability to enhance vaccine immunity and making it difficult to achieve a satisfactory immune response. Biphasic (W / O / W) adjuvants combine the advantages of both oil-in-water (O / W) and water-in-oil (W / O) adjuvants, capable of inducing both short-term and long-term immune responses, and are a key development direction for adjuvants in the future.

[0004] Although domestic companies and institutions are increasingly researching animal vaccine adjuvants, no single adjuvant has yet been widely used. Currently, the most widely used adjuvant is an oil adjuvant designed in the 1980s. Vaccine manufacturers use white oil and Span-Tween to prepare emulsions, which have poor stability, high viscosity, and strong local reactions. Furthermore, only the oil adjuvant series from the French company SEPPIC has been recognized and used by some companies in China. SEPPIC's biphasic adjuvant product, ISA206, is primarily used in foot-and-mouth disease vaccines. Foot-and-mouth disease vaccines are the leading veterinary vaccines, and the adjuvants used in these vaccines are currently completely monopolized by SEPPIC, which holds a large share of the adjuvant market. Therefore, the domestic market urgently needs a new generation of adjuvants independently developed in my country to fill this demand.

[0005] Based on the above background and current situation, it is imperative to develop a biphasic oil-emulsion adjuvant that can be widely used, has a simple preparation process, low viscosity, good stability, and few side effects.

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

[0007] The present invention aims to provide a vaccine adjuvant, vaccine composition, preparation method, and application thereof, thereby improving the quality of veterinary vaccines and reducing production costs for veterinary vaccine manufacturers. This will enable veterinary vaccine companies to utilize domestically developed, safe, stable, and highly effective animal vaccine adjuvants. This invention will provide a solid foundation for the prevention and control of major animal epidemics and enhance animal and human food safety.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides a vaccine adjuvant comprising: a surfactant and an oil phase in a mass ratio of 9:(1-81);

[0010] Wherein, the surfactant comprises a lipophilic surfactant and a hydrophilic surfactant in a mass ratio of 1:(1-3); the lipophilic surfactant comprises a first lipophilic surfactant and a second lipophilic surfactant in a mass ratio of 1:(2-3), the first lipophilic surfactant is different from the second lipophilic surfactant, and the first lipophilic surfactant or the second lipophilic surfactant is selected from any one of polyglycerol ricinoleate, Span, lecithin, soybean lecithin, glyceryl monostearate and glyceryl monooleate;

[0011] The oil phase comprises a medicinal mineral oil and a liquid bioprotectant in a mass ratio of (2-4):1, and the liquid bioprotectant is selected from liquid paraffin or glycerol.

[0012] After extensive and extensive experimental screening, the inventors discovered that the vaccine adjuvant containing the aforementioned raw material combination exhibits superior stability, safety, and immunogenicity compared to the ISA206 adjuvant produced by SEPPIC. Specifically, the vaccine adjuvant utilizes safe, stable, and highly effective lipophilic and hydrophilic surfactants, which can reduce side effects and accumulation in the body, offering a high safety advantage.

[0013] After the vaccine adjuvant is prepared into a vaccine composition, it can effectively enhance the immunogenicity of the vaccine, improve the immune effect of the vaccine, and provide long-term and stable immune protection.

[0014] The stability of the vaccine composition prepared by using the vaccine adjuvant of the present invention is significantly improved, which is beneficial for the use and storage of vaccine manufacturers and is also beneficial for providing long-term protection to immunized animals.

[0015] The above-mentioned vaccine adjuvant of the present invention is an emulsion, which can form a W / O / W emulsion after adding an aqueous phase (such as an aqueous phase containing a vaccine or an aqueous phase simulating a vaccine). By setting a specific ratio and raw materials, the inventor can directly prepare a W / O / W emulsion by emulsification in one step without the need for a two-step emulsification method. Compared with the two-step emulsification method of the prior art (first preparing a W / O emulsion, and then adding the W / O emulsion to an aqueous phase containing a hydrophilic emulsifier to prepare a W / O / W emulsion), the vaccine adjuvant provided by the present invention is simpler and easier to prepare a vaccine composition. In addition, the vaccine adjuvant provided by the present invention can effectively enhance the immunogenicity of the antigen, improve the immune effect of the vaccine, and can provide long-term and stable immune protection; it does not require high shear and is easy to inject, making vaccine production and use more convenient and efficient; it has strong stability and low viscosity, which is more conducive to the use of vaccine manufacturers and provides long-term protection to immunized animals when emulsified into a vaccine.

[0016] The combination of mineral oil and liquid paraffin or glycerol is beneficial to maintaining the stability of the antigen and preventing it from degrading.

[0017] In an optional embodiment, the mass ratio of surfactant to oil phase is 9:1, or 9:2, or 9:3, or 9:4, or 9:41; or 1:5; or 9:(10-41); or 2:8; or 3:7; or 1:9; or 9:(12-45).

[0018] In an alternative embodiment, the oil phase comprises a pharmaceutical mineral oil and a liquid bioprotectant in a mass ratio of 3: 1. Under the above mass ratio conditions, the adjuvant has a higher stability.

[0019] In an optional embodiment, the surfactant includes a lipophilic surfactant and a hydrophilic surfactant in a mass ratio of 1:1, or 1:2, or 1:3.

[0020] In a preferred embodiment of the present invention, the Span is selected from any one of Span 60, Span 65, Span 80, Span 83 and Span 85.

[0021] In a preferred embodiment of the present invention, the first lipophilic surfactant is polyglycerol ricinoleate, and the second lipophilic surfactant is Span.

[0022] In an optional embodiment, the first lipophilic surfactant is polyglycerol ricinoleate, and the second lipophilic surfactant is Span 85. 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.

[0023] In a preferred embodiment of the present invention, the mass ratio of the surfactant to the oil phase is 9:(30-45). In a preferred embodiment of the present invention, the mass ratio of the surfactant to the oil phase is 9:36.

[0024] In a second aspect, the present invention further provides a method for preparing a vaccine adjuvant, which comprises: mixing a surfactant and an oil phase according to a proportion.

[0025] The vaccine adjuvant of the present invention adopts self-emulsification technology, which simplifies the emulsification process, does not require high shear, and is easy to inject, making vaccine production and use more convenient and efficient.

[0026] In a preferred embodiment of the present invention, the surfactant and the oil phase are mixed according to any of the following methods:

[0027] (1) firstly mixing a lipophilic surfactant and a hydrophilic surfactant to obtain a surfactant mixture, and then mixing it with an oil phase;

[0028] (2) first mixing the lipophilic surfactant with the oil phase, and then mixing the obtained mixture with the hydrophilic surfactant;

[0029] (3) The hydrophilic surfactant is first mixed with the oil phase, and the resulting mixture is then mixed with the lipophilic surfactant.

[0030] As long as the surfactant and the oil phase are mixed in proportion, no matter which surfactant is mixed first, it is within the scope of protection of the present invention.

[0031] In a third aspect, the present invention further provides a vaccine adjuvant or use of the vaccine adjuvant prepared by the preparation method in preparing an animal vaccine composition, a blank W / O / W emulsion or a medicine.

[0032] In an alternative embodiment, a blank W / O / W emulsion comprises a vaccine adjuvant and an aqueous phase; the aqueous phase is a reagent capable of simulating a vaccine. Uses of the blank W / O / W emulsion include, but are not limited to, serving as a control reagent for vaccine compositions, cleaning injection equipment, and screening adjuvant formulations.

[0033] In an optional embodiment, the aqueous phase reagent is at least one of PBS, sodium chloride solution, sodium acetate solution and sodium phosphate solution.

[0034] In an optional embodiment, the animal vaccine composition comprises a vaccine and a vaccine adjuvant; the animal is livestock or a pet animal.

[0035] In an alternative embodiment, the animal is a pig, sheep, cow, chicken, duck, fish, goose, cat, dog, monkey or human.

[0036] In a fourth aspect, the present invention further provides a composition comprising the above-mentioned vaccine adjuvant or the vaccine adjuvant prepared by the above-mentioned preparation method;

[0037] In an optional embodiment, the composition is a vaccine composition, a blank W / O / W emulsion or a drug; the blank W / O / W emulsion comprises a vaccine adjuvant and an aqueous phase; the aqueous phase is an aqueous phase reagent capable of simulating a vaccine;

[0038] In an optional embodiment, the aqueous phase reagent is at least one of PBS, sodium chloride solution, sodium acetate solution and sodium phosphate solution;

[0039] In an optional embodiment, the drug further comprises a pharmaceutically acceptable excipient.

[0040] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipient is selected from at least one of a filler, a disintegrant, a lubricant, a flavoring agent, a binder, a suspending agent and a flavor.

[0041] Pharmaceutically acceptable excipients include, but are not limited to, pharmaceutically acceptable carriers, auxiliary substances or solvents. Pharmaceutically acceptable excipients include various organic or inorganic carriers and / or auxiliary materials as they are commonly used for pharmaceutical purposes, particularly for solid pharmaceutical preparations. Examples include: excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropyl cellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch; disintegrants such as starch, hydrolyzed starch, carboxymethyl cellulose, carboxymethyl cellulose calcium salt, hydroxypropyl starch, ethylene glycol starch sodium, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants such as magnesium stearate, talc, sodium lauryl sulfate; flavors such as citric acid, menthol, glycine , orange powder; preservatives such as sodium benzoate, sodium bisulfite, parabens (such as methyl paraben, ethyl paraben, propyl paraben, butyl paraben); stabilizers such as citric acid, sodium citrate, acetic acid and polycarboxylic acids from the titriplex series, such as diethylenetriaminepentaacetic acid (DTPA); suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents such as water, organic solvents; waxes, fats and oils such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0042] In a preferred embodiment of the present invention, the dosage form of the drug is tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections or sprays.

[0043] In an optional embodiment, the above-mentioned medicine is a liquid pharmaceutical preparation (such as a kind of as injection), and for example solution, suspension and gel usually contain liquid carrier, for example water and / or pharmaceutically acceptable organic solvent.In addition, this type of liquid preparation can also include pH adjusting agent, emulsifying agent or dispersant, buffer, preservative, wetting agent, gelling agent (such as methylcellulose), dye and / or flavoring, for example as defined above. Medicine can be isotonic, that is, they can have the same osmotic pressure as blood. The isotonicity of medicine can be regulated by using sodium chloride and other pharmaceutically acceptable reagents, and these reagents are such as glucose, maltose, boric acid, sodium tartrate, propylene glycol and other inorganic or organic soluble substances. The viscosity of liquid composition can be regulated by pharmaceutically acceptable thickening agent such as methylcellulose. Other suitable thickening agents include such as xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer etc. The preferred concentration of thickening agent depends on selected reagent.

[0044] In a preferred embodiment of the present invention, the drug is formulated for oral administration or injection administration. Injection administration includes but is not limited to subcutaneous injection, intramuscular injection, intravenous injection, and intradermal injection.

[0045] In an optional embodiment, the vaccine composition also includes a vaccine. The vaccine is produced by attenuating, inactivating, or genetically engineering pathogenic microorganisms and / or their metabolites. Genetic engineering methods include, but are not limited to, producing vaccines through genetic modification (such as gene mutation), modifying pure vaccine molecules, introducing vaccine genes into microbial vectors, or modifying DNA.

[0046] In an optional embodiment, the mass ratio of vaccine adjuvant to vaccine in the vaccine composition is 1:(1-1.1). At this ratio, the emulsified vaccine has better stability, suitable viscosity, and less stress.

[0047] In an optional embodiment, the vaccine is prepared by attenuation, inactivation or genetic engineering of pathogenic microorganisms and / or their metabolites;

[0048] The vaccine is selected from attenuated vaccines, inactivated vaccines, subunit vaccines or nucleic acid vaccines;

[0049] In an alternative embodiment, the vaccine is derived from pathogenic microorganisms that infect pigs, sheep, cattle, chickens, ducks, fish, geese, cats, dogs, monkeys or humans;

[0050] In an optional embodiment, the vaccine is selected from at least one of porcine diarrhea virus antigen, porcine transmissible gastroenteritis virus antigen, porcine rotavirus antigen, porcine circovirus antigen, inactivated foot-and-mouth disease virus antigen, and Mycoplasma hyopneumoniae antigen;

[0051] In an optional embodiment, the vaccine composition includes an external aqueous phase, an intermediate oil phase and an internal aqueous phase, wherein the external aqueous phase and the internal aqueous phase are vaccines, and the intermediate oil phase includes an oil phase, a lipophilic surfactant and a hydrophilic surfactant.

[0052] In a fifth aspect, the present invention further provides a method for preparing a vaccine composition or a blank W / O / W emulsion, comprising the following steps:

[0053] The vaccine adjuvant or the vaccine adjuvant prepared by the above-mentioned preparation method is mixed with the vaccine and emulsified to prepare a vaccine composition;

[0054] Alternatively, the above-mentioned vaccine adjuvant or the vaccine adjuvant prepared by the above-mentioned preparation method is mixed with an aqueous phase reagent capable of simulating a vaccine, and emulsified to prepare a blank W / O / W emulsion;

[0055] In an alternative embodiment, the vaccine adjuvant is mixed with the vaccine or an aqueous reagent capable of simulating a vaccine under stirring; the emulsification conditions are: 32°C ± 1°C, emulsification for 1-15 minutes, for example, 1-5 minutes, or 5-10 minutes, and the emulsification speed is 300-400 rpm.

[0056] In an optional embodiment, the aqueous phase reagent is at least one of PBS, sodium chloride solution, sodium acetate solution and sodium phosphate solution.

[0057] In an optional embodiment, the vaccine composition includes an external aqueous phase, an intermediate oil phase and an internal aqueous phase, wherein the external aqueous phase and the internal aqueous phase are vaccines, and the intermediate oil phase includes an oil phase, a lipophilic surfactant and a hydrophilic surfactant.

[0058] In a sixth aspect, the present invention further provides use of the vaccine composition in the preparation of a medicament for preventing and / or treating diseases in livestock or pet animals, including but not limited to pigs, sheep, cattle, chickens, ducks, fish, geese, cats, dogs, monkeys, or humans.

[0059] The present invention has the following beneficial effects:

[0060] (1) The present invention provides a vaccine adjuvant. Compared with the ISA206 adjuvant produced by SEPPIC, the prepared vaccine composition has higher stability, safety and immune efficacy.

[0061] (2) Vaccine adjuvants use safe, stable, and highly effective lipophilic and hydrophilic surfactants, which can reduce the body's side effects and accumulation, and have the technical advantage of high safety.

[0062] (3) After the vaccine adjuvant is prepared into a vaccine composition, it can effectively enhance the immunogenicity of the vaccine, improve the immune effect of the vaccine, and provide long-term and stable immune protection.

[0063] (4) The stability of the vaccine composition prepared using the vaccine adjuvant of the present invention is significantly improved, which is beneficial for the use and storage of vaccine manufacturers and is also beneficial for providing long-term protection to immunized animals.

[0064] (5) The vaccine adjuvant provided by the present invention is an emulsion, which can form a W / O / W emulsion after adding an aqueous phase (such as an aqueous phase containing a vaccine or an aqueous phase simulating a vaccine). By setting a specific ratio and raw materials, the inventor can directly prepare a W / O / W emulsion by emulsification in one step without the need for a two-step emulsification method. Compared with the two-step emulsification method of the prior art (first preparing a W / O emulsion, and then adding the W / O emulsion to an aqueous phase containing a hydrophilic emulsifier to prepare a W / O / W emulsion), the vaccine adjuvant provided by the present invention is simpler and easier to prepare a vaccine composition. It is easy to inject, making vaccine production and use more convenient and efficient.

[0065] (6) The vaccine adjuvant provided by the present invention has strong stability and low viscosity, which is more convenient for vaccine manufacturers to use and can provide long-term protection to immunized animals when emulsified into vaccines.

[0066] (7) The preparation method of the vaccine adjuvant of the present invention adopts self-emulsification technology, which simplifies the emulsification process, does not require high shear, and is easy to inject, making vaccine production and use more convenient and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0068] Figure 1 To optimize the emulsification temperature of blank W / O / W submicroemulsion animal adjuvant;

[0069] Figure 2 To optimize the emulsification time (within 60 min) of blank W / O / W submicroemulsion animal adjuvant;

[0070] Figure 3 To optimize the emulsification time (within 10 minutes) of blank W / O / W submicroemulsion animal adjuvant;

[0071] Figure 4 To optimize the emulsification speed of blank W / O / W submicron emulsion animal adjuvant;

[0072] Figure 5 Particle size distribution of W / O / W submicroemulsion adjuvanted FMD vaccine (a) and ISA206 adjuvanted FMD vaccine (b);

[0073] Figure 6 This is the change of antibody titer in pigs immunized with foot-and-mouth disease W / O / W submicroemulsion adjuvant vaccine and foot-and-mouth disease ISA 206 adjuvant vaccine. DETAILED DESCRIPTION

[0074] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0075] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0076] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0077] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0078] Example 1

[0079] This embodiment provides a vaccine adjuvant.

[0080] Composition of vaccine adjuvants. Vaccine adjuvants include an oil phase, a hydrophilic surfactant, and a lipophilic surfactant.

[0081] The lipophilic surfactant (ie, emulsifier) ​​is a mixture of polyglycerol ricinoleate and Span85, with a mass ratio of polyglycerol ricinoleate to Span85 of 1:2, and is named surfactant 1.

[0082] The hydrophilic surfactant (i.e., co-emulsifier) ​​is a mixture of polyglycerol-2-dioleate, decaglycerol monolaurate, and TWeen 85. The mass ratio of polyglycerol-2-dioleate, decaglycerol monolaurate, and TWeen 85 is 2:1:1, and it is named surfactant 2.

[0083] The oil phase is a mixture of pharmaceutical grade mineral oil and liquid paraffin in a mass ratio of 3:1.

[0084] In this embodiment, the mass ratio of surfactant 1 to surfactant 2 is 1:3. The mixture of surfactant 1 and surfactant 2 is defined as a mixture (Smix).

[0085] The mass ratio of the mixture (Smix) to the oil phase is 2:8.

[0086] The preparation method is as follows:

[0087] The oil phase, hydrophilic surfactant and lipophilic surfactant in the above proportions are mixed.

[0088] Example 2

[0089] Compared with Example 1, the only difference is that the mass ratio of surfactant 1 to surfactant 2 is different, which is 1:2 in this example.

[0090] Example 3

[0091] Compared with Example 1, the only difference is that the mass ratio of surfactant 1 to surfactant 2 is different, which is 1:1 in this example.

[0092] Example 4

[0093] 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 1:9.

[0094] Example 5

[0095] Compared with Example 1, the only difference is that the mass ratio of the mixture (Smix) to the oil phase is different, which is 3:7 in this example.

[0096] Example 6

[0097] Compared with Example 4, the only difference is that the mass ratio of surfactant 1 to surfactant 2 is different, which is 1:1 in this example.

[0098] Example 7

[0099] Compared with Example 4, the only difference is that the mass ratio of surfactant 1 to surfactant 2 is different, which is 1:2 in this example.

[0100] Example 8

[0101] Compared with Example 5, the only difference is that the mass ratio of surfactant 1 to surfactant 2 is different, which is 1:1 in this example.

[0102] Example 9

[0103] Compared with Example 5, the only difference is that the mass ratio of surfactant 1 to surfactant 2 is different, which is 1:2 in this example.

[0104] Example 10

[0105] This example provides a blank W / O / W vaccine composition and a preparation method thereof.

[0106] (1) 0.01 M phosphate buffered saline (PBS) simulated antigen was used as the aqueous phase, a mixture of pharmaceutical grade mineral oil and liquid paraffin in a mass ratio of 3:1 was used as the oil phase, and surfactant 1 and surfactant 2 were mixed according to the adjuvant formula shown in Table 1 to prepare a water-in-oil-in-water (W / O / W) emulsion.

[0107] Specifically, the lipophilic surfactant (ie, emulsifier) ​​is a mixture of polyglycerol ricinoleate and Span85, with a mass ratio of polyglycerol ricinoleate to Span85 being 1:2, and is named surfactant 1.

[0108] The hydrophilic surfactant (i.e., co-emulsifier) ​​is a mixture of polyglycerol-2-dioleate, decaglycerol monolaurate, and TWeen 85. The mass ratio of polyglycerol-2-dioleate, decaglycerol monolaurate, and TWeen 85 is 2:1:1, and it is named surfactant 2.

[0109] The mixture of surfactant 1 and surfactant 2 is defined as mixture (Smix).

[0110] The preparation method is as follows:

[0111] PBS was added dropwise with slow stirring in a 25°C water bath until a uniform W / O / W emulsion was formed. The formulation composition was determined by identifying the W / O / W emulsion formulation, particle size distribution, observing the submicroemulsion microstructure, and examining sample stability. The appropriate amounts of oil phase, surfactant 1, surfactant 2, and aqueous phase were determined.

[0112] Surfactant 1 and surfactant 2 were mixed at different mass ratios of 1:1, 1:2, and 1:3 to form a mixture (Smix). The oil phase was then mixed with Smix at different mass ratios of 9:1, 8:2, and 7:3 to form a W / O / W submicroemulsion animal adjuvant. PBS was added dropwise in a 25°C water bath with slow stirring (emulsification speed of 350 rpm) until a uniform W / O / W emulsion was formed. By combining different mass ratios of surfactant 1 and surfactant 2, and different mass ratios of oil phase and Smix, the W / O / W emulsion formulations were identified, the particle size distribution, the submicroemulsion micromorphology, and the sample stability were observed. This allowed the determination of a suitable adjuvant formulation.

[0113] The results are shown in Table 1. The results show that after mixing different ratios of surfactant 1, surfactant 2, and the oil phase, it was found that the mixed adjuvant had a turbid appearance at some ratios. Therefore, adjuvants with different ratios that were clear and translucent after mixing were selected for subsequent emulsification tests. The dosage form, particle size distribution, and stability of the emulsions after emulsification were subsequently measured, and the optimal W / O / W submicroemulsion animal adjuvant formula was ultimately screened. In the optimal formula, the mass ratio of surfactant 1 to surfactant 2 was 1:3, and the mass ratio of the mixture of surfactant 1 and surfactant 2 to the oil phase was 2:8.

[0114] Table 1 Screening of blank W / O / W submicroemulsion animal adjuvant formula

[0115]

[0116] (2) Optimization of emulsification conditions of blank W / O / W submicroemulsion animal adjuvant.

[0117] A water-in-oil-in-water (W / O / W) emulsion is prepared using the W / O / W submicroemulsion animal adjuvant formulated in step (1) as the oil phase and PBS as the simulated antigen as the aqueous phase. By adjusting emulsification factors (emulsification ratio, sample addition method, sample addition speed, emulsification speed, emulsification temperature, and emulsification time), measuring the particle size distribution of the W / O / W emulsion, observing the microscopic morphology of the submicroemulsion, and examining the sample stability, suitable emulsification conditions are ultimately determined, thereby preparing a water-in-oil-in-water (W / O / W) emulsion with higher stability.

[0118] By measuring the particle size distribution, stability, etc. of W / O / W type emulsions obtained under different emulsification conditions ( Figure 1-Figure 4 ), and finally determined the optimal emulsification conditions of W / O / W submicroemulsion animal adjuvant, Figure 1 As shown, the optimal emulsification temperature is 32℃±1℃. Figure 2 and Figure 3 As shown in the figure, the blank W / O / W submicron emulsion animal adjuvant has better stability and particle size distribution after emulsification for about 5 minutes. It also has better stability and particle size distribution when the emulsification speed is 300-400 rpm.

[0119] Experimental Example 1

[0120] In this experimental example, a foot-and-mouth disease W / O / W submicroemulsion adjuvant vaccine (i.e., vaccine composition) was prepared and its quality was evaluated.

[0121] (1) Preparation of foot-and-mouth disease W / O / W submicroemulsion adjuvant vaccine (i.e., vaccine composition).

[0122] The optimal vaccine adjuvant formula screened in Example 11 was used as the oil phase, and the inactivated foot-and-mouth disease antigen solution was used as the aqueous phase to prepare a foot-and-mouth disease submicroemulsion vaccine. The specific method is as follows:

[0123] The optimal vaccine adjuvant formula includes: a surfactant and an oil phase in a mass ratio of 9:36, the surfactant includes a lipophilic surfactant and a hydrophilic surfactant in a mass ratio of 1:3; the lipophilic surfactant (i.e., emulsifier) ​​is a mixture of polyglycerol ricinoleate and Span85, the mass ratio of polyglycerol ricinoleate to Span85 is 1:2, and it is named surfactant 1.

[0124] The hydrophilic surfactant (i.e., co-emulsifier) ​​is a mixture of polyglycerol-2-dioleate, decaglycerol monolaurate, and TWeen 85. The mass ratio of polyglycerol-2-dioleate, decaglycerol monolaurate, and TWeen 85 is 2:1:1, and it is named surfactant 2.

[0125] The oil phase is a mixture of pharmaceutical grade mineral oil and liquid paraffin in a mass ratio of 3:1.

[0126] The preparation method is as follows:

[0127] (a) First, a lipophilic surfactant and a hydrophilic surfactant are mixed, and then the surfactant mixture is mixed with an oil phase to prepare a vaccine adjuvant. The mixture is then sterilized at 121°C and autoclaved for 30 minutes, followed by cooling.

[0128] (b) Weigh the foot-and-mouth disease virus antigen solution and the W / O / W submicroemulsion animal adjuvant (foot-and-mouth disease virus antigen solution: W / O / W submicroemulsion animal adjuvant = 1:1 mass ratio), place them in a 250 mL glass beaker, and heat to 32°C ± 1°C for use.

[0129] (c) Slowly add the foot-and-mouth disease virus antigen solution to the adjuvant at a stirring speed of 350 rpm / min (3 seconds, 5-10 L / min) and stir for 5 minutes.

[0130] (e) Stop stirring and store the prepared emulsion at low temperature (<15° C.) for 24 h without movement and stirring, and then perform quality control.

[0131] (2) Preparation of foot-and-mouth disease ISA206 vaccine composition

[0132] The preparation of the foot-and-mouth disease ISA206 vaccine composition is consistent with the preparation method of the W / O / W submicroemulsion animal adjuvant vaccine in step (1).

[0133] (a) ISA206 adjuvant was sterilized by autoclaving at 121°C for 30 min and cooled.

[0134] (b) Weigh the FMDV antigen solution and ISA206 adjuvant (FMDV antigen solution:ISA206 adjuvant = 1:1 mass ratio) separately, place them in a 250 mL glass beaker, and heat to 32°C ± 1°C for use.

[0135] (c) Slowly add the foot-and-mouth disease virus antigen solution to the adjuvant at a stirring speed of 300-400 rpm / min (3 seconds, 5-10 L / min) and stir for 5 minutes.

[0136] (d) Stop stirring and store the prepared emulsion at low temperature (<15° C.) for 24 h, avoiding movement and stirring, and then perform quality control.

[0137] (3) Quality evaluation of W / O / W submicroemulsion adjuvanted foot-and-mouth disease vaccine

[0138] (a) Water drop test: When the prepared W / O / W submicroemulsion adjuvanted FMD vaccine is dropped into cold water, the emulsion partially self-dilutes and makes the water appear milky white, indicating that the vaccine is a water-in-oil-in-water (W / O / W) vaccine.

[0139] (b) Appearance: The prepared FMD W / O / W submicroemulsion adjuvanted vaccine and the blank W / O / W submicroemulsion were both milky white and uniform. There was no sticking to the wall when shaking in the beaker, and the fluidity was good.

[0140] (c) Particle size distribution: 100 μL of the W / O / W submicroemulsion adjuvanted FMD vaccine was diluted with distilled water, and the average particle size and intensity particle size distribution were measured using a laser particle size analyzer. The average particle size of the W / O / W submicroemulsion adjuvanted FMD vaccine was 210 nm, the polydispersity index was less than 0.15, and the particle size range was between 200 nm and 250 nm, which was basically normally distributed ( Figure 5 ).

[0141] (d) Viscosity measurement: The viscosity of the prepared foot-and-mouth disease W / O / W submicroemulsion adjuvanted vaccine was measured using a Brookfield DVNext rheometer and was 30 cp at 25°C.

[0142] (e) Centrifugation test: 10 mL of the prepared FMD W / O / W submicroemulsion adjuvanted vaccine was added to a centrifuge tube. After centrifugation at 3000 rpm for 30 min, the tube still maintained a milky white uniform appearance with no stratification and no aqueous phase precipitation at the bottom, indicating that the vaccine had good stability.

[0143] (f) Stability Testing: Three batches of prepared foot-and-mouth disease W / O / W submicroemulsion adjuvanted vaccine were stored at 4°C for one year, 25°C for three weeks, and 37°C for three days. The vaccine appearance was observed daily. The vaccine remained a milky white, homogeneous liquid, without flocculence, stratification, or demulsification. After centrifugation at 3000 rpm for 30 minutes, the vaccine maintained a milky white, homogeneous appearance, without stratification or aqueous precipitation at the bottom, indicating good stability.

[0144] Experimental Example 2

[0145] A safety test was conducted on the foot-and-mouth disease W / O / W submicroemulsion adjuvant vaccine of Experimental Example 1.

[0146] (a) Twenty-five healthy, two-week-old female BALB / c mice weighing 18-22 g were randomly divided into groups 1-5, with five mice in each group. Groups 1 and 2 received subcutaneous injections of 1 mL of a W / O / W submicroemulsion animal adjuvant or ISA206 adjuvant, respectively. Groups 3 and 4 received subcutaneous injections of 1 mL of a W / O / W submicroemulsion foot-and-mouth disease vaccine or ISA 206 adjuvant vaccine, respectively. Group 5 received a subcutaneous injection of 1 mL of PBS, serving as a blank control. The mice were observed for 7 consecutive days after injection, and their clinical status was recorded. No deaths or significant local or systemic adverse reactions due to vaccine or adjuvant injection occurred in any of the groups, demonstrating a favorable safety profile.

[0147] (b) Twenty-five healthy, 50- to 60-day-old weaned piglets that were double negative for FMD antigens and antibodies were randomly divided into groups 1 to 5, with 5 piglets in each group. Groups 1 and 2 received intramuscular injections of 2 mL of a W / O / W submicroemulsion animal adjuvant or ISA206 adjuvant, respectively. Groups 3 and 4 received intramuscular injections of 2 mL of a W / O / W submicroemulsion FMD vaccine or ISA206 adjuvant, respectively. Group 5 received intramuscular injections of 2 mL of PBS, serving as a blank control group. The piglets were observed for 14 days after injection, and their clinical status was recorded. No mortality, significant local adverse reactions, or systemic reactions due to vaccine or adjuvant injection occurred in any of the groups, indicating a good safety profile.

[0148] Experimental Example 3

[0149] This experimental example conducts an immune efficacy test on pigs based on the foot-and-mouth disease W / O / W submicroemulsion adjuvant vaccine of Experimental Example 1.

[0150] Twenty healthy weaned piglets aged 50 to 60 days with double negative foot-and-mouth disease antigen and antibody were randomly divided into 1-4 groups, with 5 pigs in each group. The first group of piglets received an injection of 2 mL of foot-and-mouth disease W / O / W submicron emulsion adjuvanted vaccine into the neck muscle; the second group of piglets received an injection of 2 mL of foot-and-mouth disease ISA206 adjuvanted vaccine into the neck muscle; the third group of piglets received an injection of 2 mL of Tiantiqing (Tiankang foot-and-mouth disease type O and type A bivalent inactivated vaccine) into the neck muscle; the fourth group of piglets received an injection of 2 mL of PBS into the neck muscle, which served as a blank control group. The rectal temperature of each piglet was measured every day for two days before vaccination. The rectal temperature was measured 3, 6, 12, 18, and 24 hours after vaccination, and the clinical status of the piglets was observed and recorded. It was found that no deaths or obvious local adverse reactions or systemic reactions caused by the injection of vaccines or adjuvants occurred in all groups, indicating that it was safe. After vaccination, blood was collected from all piglets every week to separate serum, and the serum was subjected to liquid-phase blocking ELISA test to evaluate the immune efficacy of the vaccine ( Figure 6 ).

[0151] Results showed that the W / O / W submicroemulsion adjuvanted FMD vaccine provided by the present invention had stronger immune efficacy than the ISA206 adjuvanted FMD vaccine, with the effect being particularly pronounced after two weeks. Commercial inactivated FMD vaccines, because they include an immunopotentiator, have a higher immune efficacy than the W / O / W submicroemulsion adjuvanted FMD vaccine provided by the present invention.

[0152] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A vaccine adjuvant, characterized in that It includes: A surfactant and an oil phase in a mass ratio of 9:36, wherein the surfactant comprises a lipophilic surfactant and a hydrophilic surfactant in a mass ratio of 1:3; The lipophilic surfactant is a mixture of polyglycerol ricinoleate and Span 85, and the mass ratio of polyglycerol ricinoleate to Span 85 is 1:

2. The hydrophilic surfactant is a mixture of polyglycerol-2-dioleate, decapolyglycerol monolaurate, and TWeen 85, and the mass ratio of polyglycerol-2-dioleate, decapolyglycerol monolaurate to TWeen 85 is 2:1:

1. The oil phase comprises a medicinal mineral oil and a liquid bioprotectant in a mass ratio of (2-4):1, and the liquid bioprotectant is selected from liquid paraffin or glycerol.

2. The vaccine adjuvant according to claim 1, characterized in that The oil phase includes medicinal mineral oil and a liquid bioprotectant in a mass ratio of 3:

1.

3. The method for preparing the vaccine adjuvant according to any one of claims 1 to 2, wherein: It includes: The surfactant and oil phase are mixed according to the proportion.

4. The method for preparing the vaccine adjuvant according to claim 3, wherein The surfactant and the oil phase are mixed according to any of the following methods: (1) firstly mixing the lipophilic surfactant and the hydrophilic surfactant to obtain a surfactant mixture, and then mixing the mixture with the oil phase; (2) firstly mixing the lipophilic surfactant with the oil phase, and then mixing the obtained mixture with the hydrophilic surfactant; (3) The hydrophilic surfactant is first mixed with the oil phase, and the obtained mixture is then mixed with the lipophilic surfactant.

5. Use of the vaccine adjuvant according to any one of claims 1 to 2 or the vaccine adjuvant prepared by the preparation method according to any one of claims 3 to 4 in preparing an animal vaccine composition or a blank W / O / W emulsion.

6. The use according to claim 5, characterized in that The blank W / O / W emulsion includes the vaccine adjuvant and an aqueous phase; the aqueous phase is an aqueous phase reagent that can simulate vaccine antigens.

7. The use according to claim 6, characterized in that The aqueous phase reagent is at least one of PBS, sodium chloride solution, sodium acetate solution and sodium phosphate solution.

8. The use according to claim 5, characterized in that The animal vaccine composition includes a vaccine antigen and the vaccine adjuvant; The animal is a pig, sheep, cow, chicken, duck, fish, goose, cat, dog, monkey or human.

9. Use of the vaccine adjuvant according to any one of claims 1 to 2 or the vaccine adjuvant prepared by the preparation method according to any one of claims 3 to 4 in the preparation of medicines.

10. A composition, characterized in that The vaccine adjuvant comprises the vaccine adjuvant according to any one of claims 1 to 2 or the vaccine adjuvant prepared by the preparation method according to any one of claims 3 to 4.

11. The composition according to claim 10, characterized in that The composition is a vaccine composition or a blank W / O / W emulsion; the blank W / O / W emulsion comprises the vaccine adjuvant and an aqueous phase; the aqueous phase is an aqueous phase reagent capable of simulating vaccine antigens.

12. The composition according to claim 11, characterized in that The aqueous phase reagent is at least one of PBS, sodium chloride solution, sodium acetate solution and sodium phosphate solution.

13. The composition according to claim 11, characterized in that The vaccine composition also includes a vaccine antigen.

14. The composition according to claim 13, characterized in that The mass ratio of the vaccine adjuvant to the vaccine antigen in the vaccine composition is 1:(1-1.1).

15. The composition according to claim 13, characterized in that The vaccine antigen is prepared from pathogenic microorganisms and / or their metabolites through attenuation, inactivation or genetic engineering methods.

16. The composition according to claim 15, characterized in that The vaccine antigens are derived from pathogenic microorganisms that infect pigs, sheep, cattle, chickens, ducks, fish, geese, cats, dogs, monkeys or humans.

17. The composition according to claim 15, characterized in that The vaccine antigen is selected from at least one of porcine diarrhea virus antigen, porcine transmissible gastroenteritis virus antigen, porcine rotavirus antigen, porcine circovirus antigen, inactivated foot-and-mouth disease virus antigen and Mycoplasma hyopneumoniae antigen.

18. The composition according to claim 11, characterized in that The vaccine composition comprises an external aqueous phase, an intermediate oil phase and an internal aqueous phase, wherein the external aqueous phase and the internal aqueous phase are vaccine antigens, and the intermediate oil phase comprises an oil phase, a lipophilic surfactant and a hydrophilic surfactant.

19. The composition according to claim 10, characterized in that The composition is a medicine.

20. The composition according to claim 19, characterized in that The drug also includes pharmaceutically acceptable excipients.

21. A method for preparing a vaccine composition or a blank W / O / W emulsion, characterized in that: It includes the following steps: The vaccine adjuvant according to any one of claims 1 to 2 or the vaccine adjuvant prepared by the preparation method according to any one of claims 3 to 4 is mixed with the vaccine and emulsified to obtain the vaccine composition; Alternatively, the vaccine adjuvant according to any one of claims 1 to 2 or the vaccine adjuvant prepared by the preparation method according to any one of claims 3 to 4 is mixed with an aqueous phase reagent capable of simulating vaccine antigens, and emulsified to obtain the blank W / O / W emulsion.

22. The method for preparing the vaccine composition or blank W / O / W emulsion according to claim 21, characterized in that: The vaccine adjuvant and the vaccine antigen or an aqueous phase reagent that can simulate the vaccine antigen are mixed under stirring conditions; the emulsification conditions are: emulsification at 32°C±1°C for 5-15 min; and the emulsification speed is 300-400 rpm.

23. The method for preparing the vaccine composition or blank W / O / W emulsion according to claim 22, characterized in that: The aqueous phase reagent is at least one of PBS, sodium chloride solution, sodium acetate solution and sodium phosphate solution.

24. The method for preparing the vaccine composition or blank W / O / W emulsion according to claim 22, characterized in that: The vaccine composition comprises an external aqueous phase, an intermediate oil phase and an internal aqueous phase, wherein the external aqueous phase and the internal aqueous phase are vaccine antigens, and the intermediate oil phase comprises an oil phase, a lipophilic surfactant and a hydrophilic surfactant.

25. Use of the composition according to any one of claims 10 to 20 or the vaccine composition prepared by the preparation method according to any one of claims 21 to 24 in the preparation of a medicament for preventing and / or treating foot-and-mouth disease.

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