Self-emulsifying adjuvant of vaccine and preparation method of self-emulsifying adjuvant

Abstract

The invention discloses a self-emulsifying adjuvant of a vaccine and a preparation method of the self-emulsifying adjuvant, and relates to the technical field of vaccine adjuvants. The adjuvant comprises an oil phase, an emulsifier and a freeze-drying protective agent, the oil phase comprises squalene and tocopherol, the emulsifier comprises polysorbate and a polyoxyethylene-polyoxypropylene block copolymer, and the freeze-drying protective agent comprises sugar and sugar alcohol. The preparation method comprises the following steps: emulsifying an oil phase and a water phase to form a nano-emulsion, and then freeze-drying to obtain freeze-dried powder. The adjuvant has the advantages of small particle size, uniform distribution, high stability, rapid reconstruction and the like, and can effectively maintain active components and improve the immune effect of vaccines.

Description

Technical Field

[0001] This invention relates to the field of vaccine adjuvant technology, and in particular to a self-emulsifying adjuvant for vaccines and its preparation method. Background Technology

[0002] Vaccine adjuvants are used to enhance immune responses, and self-emulsifying adjuvants can form nanoemulsions to improve antigen delivery efficiency. Several self-emulsifying adjuvant schemes have been proposed in the prior art. For example, CN119868532A discloses a self-emulsifying vaccine adjuvant containing a submicron-sized water-in-oil-in-water biphasic emulsion oil phase, which uses mineral oil, lipophilic emulsifiers (Span series), hydrophilic emulsifiers (Tween), and the immune enhancer VSP70, claiming to form a stable emulsion and prolong antigen release time. However, this scheme has a complex emulsification process and strict storage conditions, and in practical use, it is prone to problems such as poor stability and difficulty in reconstitution after freeze-drying. Another prior art, CN104225590A, discloses a self-emulsifying oil-phase adjuvant comprising an amphoteric emulsifier, a hydrophilic polymer, mineral oil, and a hydrophilic-lipophilic mediator. It claims advantages such as low viscosity and high stability. However, this approach may not simultaneously address the requirements for particle size control, long-term stability, and rapid reconstitution, leading to easy aggregation or oxidative degradation of the emulsion during storage. Therefore, providing a stable, easily reconstituteable, and effectively preserving active ingredients self-emulsifying adjuvant is urgently needed. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide a self-emulsifying adjuvant for vaccines and its preparation method, aiming to overcome the defects of existing adjuvants such as difficulty in reconstitution after freeze-drying, poor physical and chemical stability, and easy degradation of active ingredients. This invention aims to provide a physically stable, rapidly reconstituteable adjuvant composition and its efficient preparation method that effectively protects the active ingredients, thereby improving the storage stability and final immunization effect of vaccine products.

[0004] To address the aforementioned technical problems, this invention provides a method for preparing a self-emulsifying adjuvant for vaccines, comprising the following steps: (1) Mix squalene, tocopherol, polysorbate and co-emulsifier, heat and stir at 40-50°C until completely dissolved to form a clear and transparent self-emulsifying concentrate, cool to room temperature to obtain the oil phase; The usage ranges for each raw material are as follows: Squalene: 2.0-3.0g Tocopherol: 0.5-1.0g Polysorbate 80: 0.8-1.2g Co-emulsifier: 0.1-0.3g (2) Dissolve trehalose and mannitol in a portion of water for injection, stir until completely clear, and then dilute to 15-25 mL with water for injection to obtain the aqueous phase; The usage ranges for each raw material are as follows: Trehalose: 3.0-5.0g Mannitol: 1.5-2.5g (3) Under the stirring speed of 5000-10000 rpm in a high-speed shear emulsifier, the oil phase obtained in step (1) is slowly added dropwise to the aqueous phase obtained in step (2) at a rate of 0.5-2 mL / min; after the oil phase is added, continue stirring for 20-40 minutes to form a uniform nanoemulsion. (4) Dispense the nanoemulsion obtained in step (3) into a container, partially stopper it, and then freeze-dry it; after the freeze-drying is completed, fully stopper it under vacuum conditions to obtain a white freeze-dried powder, which is the self-emulsifying adjuvant of the vaccine.

[0005] Preferably, the co-emulsifier is selected from polyoxyethylene-polyoxypropylene block copolymers and / or hyperbranched polymers. The polyoxyethylene-polyoxypropylene block copolymer is one of Pluronic F68 and Pluronic F38.

[0006] More preferably, the co-emulsifier is a mixture of Pronic F68 and hyperbranched polyglycerol in a mass ratio of 1-3:1-3.

[0007] Preferably, the freeze-drying process in step (4) includes: Pre-freezing stage: Keep at -40 to -50°C for 1-3 hours; First drying stage: Raise the shelf temperature to -25 to -35°C, control the vacuum degree at 50-150 mTorr, and maintain for 15-25 hours; Secondary drying stage: Gradually increase the shelf temperature to 20-30℃ and maintain it for 5-15 hours.

[0008] The core technical concept of this invention lies in combining an oil phase composed of a metabolizable oil and an oil-soluble antioxidant with a composite emulsifier system consisting of a nonionic surfactant and a polyoxyethylene-polyoxypropylene block copolymer with a specific amphiphilic structure, and further supplementing it with a composite lyophilization protectant composed of sugars and sugar alcohols to prepare a nanoemulsion, which is then lyophilized. This specific combination of emulsifier systems can form a dual stabilizing layer at the oil-water interface, possessing both strong interfacial anchoring and efficient steric hindrance, thereby effectively inhibiting oil droplet aggregation and Ostwald ripening during emulsification, lyophilization, and long-term storage. Simultaneously, the composite lyophilization protectant not only ensures the structural integrity of the lyophilization process and the uniformity of the reconstituted particle size, but also works synergistically with the interfacial stabilizing layer to further protect the active ingredients in the oil phase from oxidative degradation. This solution systematically solves the problems of physical and chemical stability of adjuvants, achieving rapid reconstitution and long-term effectiveness of adjuvants.

[0009] Reconstruction method during use: When using, add sterile water for injection containing the antigen to each vial of lyophilized powder. The amount added should be adjusted according to the volume required for the vaccine preparation so that the volume fraction of the self-emulsifying adjuvant in the finished vaccine reaches the predetermined value. Gently shake or vortex to mix. Usually, a uniform nanoemulsion with a particle size ≤200nm will spontaneously form within one minute for subsequent immunization.

[0010] The beneficial effects of this invention are: Compared with the prior art, the adjuvant of the present invention has a small initial particle size and a low polydispersity index, indicating that the emulsion is uniformly distributed and has high emulsification efficiency.

[0011] Compared with existing technologies, the adjuvant of this invention can be rapidly reconstituted after freeze-drying, with minimal particle size change and a reconstitution time of less than 60 seconds, making it convenient for practical use.

[0012] Compared with existing technologies, the adjuvant of this invention exhibits negligible particle size growth in accelerated stability tests, significantly improved physical and chemical stability, and better retention of the active ingredients squalene and tocopherol.

[0013] This invention effectively inhibits Ostwald ripening and aggregation through an interfacial film formed by a specific combination of emulsifiers, solving the long-standing stability problem. At the same time, it is compatible with freeze-drying processes, improving the applicability and shelf life of adjuvants. Detailed Implementation

[0014] The parameters and sources of some raw materials in the examples are described below: Hyperbranched polyglycerol, with an average molecular weight of 3,000-10,000 and a branching degree of approximately 50-65%, is of medical grade. All other raw materials are of medical grade.

[0015] Example 1 A method for preparing a self-emulsifying adjuvant for a vaccine includes the following steps: (1) Weigh 2.40g of squalene and 0.60g of DL-α-tocopherol and mix them evenly; add 1.00g of polysorbate 80 and 0.20g of Pronic F68; heat and stir at 45°C until Pronic F68 is completely dissolved to form a clear and transparent self-emulsifying concentrate; cool to room temperature to obtain the oil phase. (2) Dissolve 4.00g trehalose and 2.00g mannitol in 15mL of water for injection and stir until completely clear; add water for injection to make up to 20mL to obtain the aqueous phase; (3) Under the condition of high-speed shearing at 8000 rpm in a shear emulsifier, the oil phase is slowly added to the aqueous phase at a rate of 1 mL / min; after the oil phase is added, continue stirring for 30 minutes until a uniform, milky white nanoemulsion is formed. (4) Quickly dispense the milky white nanoemulsion into sterile glass vials and partially stopper them; place the sample into a lyophilizer and perform the following optimized lyophilization procedure: Pre-freeze: -45℃, hold for 2 hours; One-time drying: Raise the shelf temperature to -30°C, reduce the vacuum to 100mTorr, and maintain for 20 hours; Secondary drying: Gradually increase the shelf temperature to 25℃ and maintain it for 10 hours; After freeze-drying, the mixture is fully stoppered under vacuum to obtain a white freeze-dried powder, which is the self-emulsifying adjuvant of the vaccine.

[0016] When using, inject sterile water for injection containing the antigen into the bottle to a total volume of 100mL, gently shake or vortex, and it will be completely reconstituted into a homogeneous nanoemulsion within 1 minute.

[0017] Comparative Example 1 A method for preparing a self-emulsifying adjuvant for a vaccine includes the following steps: (1) Weigh 2.40g of squalene and 0.60g of DL-α-tocopherol and mix them evenly; add 1.00g of polysorbate 80; heat and stir at 45℃ to form a clear and transparent self-emulsifying concentrate, cool to room temperature to obtain the oil phase; (2) Dissolve 4.00g trehalose and 2.00g mannitol in 15mL of water for injection and stir until completely clear; add water for injection to make up to 20mL to obtain the aqueous phase; (3) Under the condition of high-speed shearing at 8000 rpm in a shear emulsifier, the oil phase is slowly added to the aqueous phase at a rate of 1 mL / min; after the oil phase is added, continue stirring for 30 minutes until a uniform, milky white nanoemulsion is formed. (4) Quickly dispense the milky white nanoemulsion into sterile glass vials and partially stopper them; place the sample into a lyophilizer and perform the following optimized lyophilization procedure: Pre-freeze: -45℃, hold for 2 hours; One-time drying: Raise the shelf temperature to -30°C, reduce the vacuum to 100mTorr, and maintain for 20 hours; Secondary drying: Gradually increase the shelf temperature to 25℃ and maintain it for 10 hours; After freeze-drying, the mixture is fully stoppered under vacuum to obtain a white freeze-dried powder, which is the self-emulsifying adjuvant of the vaccine.

[0018] When using, inject sterile water for injection containing the antigen into the bottle to a total volume of 100mL, gently shake or vortex, and it will be completely reconstituted into a homogeneous nanoemulsion within 1 minute.

[0019] Example 2 A method for preparing a self-emulsifying adjuvant for a vaccine includes the following steps: (1) Weigh 2.40g of squalene and 0.60g of DL-α-tocopherol and mix them evenly; add 1.00g of polysorbate 80 and 0.20g of Prönkel F38; heat and stir at 45°C until Prönkel F38 is completely dissolved to form a clear and transparent self-emulsifying concentrate; cool to room temperature to obtain the oil phase. (2) Dissolve 4.00g trehalose and 2.00g mannitol in 15mL of water for injection and stir until completely clear; add water for injection to make up to 20mL to obtain the aqueous phase; (3) Under the condition of high-speed shearing at 8000 rpm in a shear emulsifier, the oil phase is slowly added to the aqueous phase at a rate of 1 mL / min; after the oil phase is added, continue stirring for 30 minutes until a uniform, milky white nanoemulsion is formed. (4) Quickly dispense the milky white nanoemulsion into sterile glass vials and partially stopper them; place the sample into a lyophilizer and perform the following optimized lyophilization procedure: Pre-freeze: -45℃, hold for 2 hours; One-time drying: Raise the shelf temperature to -30°C, reduce the vacuum to 100mTorr, and maintain for 20 hours; Secondary drying: Gradually increase the shelf temperature to 25℃ and maintain it for 10 hours; After freeze-drying, the mixture is fully stoppered under vacuum to obtain a white freeze-dried powder, which is the self-emulsifying adjuvant of the vaccine.

[0020] When using, inject sterile water for injection containing the antigen into the bottle to a total volume of 100mL, gently shake or vortex, and it will be completely reconstituted into a homogeneous nanoemulsion within 1 minute.

[0021] Compared to Pluronic F68, Pluronic F38 has a much higher HLB value (approximately 31), indicating strong hydrophilicity. This causes its molecules to dissolve completely in the aqueous phase rather than adsorbing onto the oil-water interface, resulting in low interfacial coverage. Furthermore, the hydrophobic core (PPO) of Pluronic F38 is too small, lacking sufficient affinity for the squalene / α-tocopherol oil phase, leading to weak adsorption at the interface and minimal stabilizing effect.

[0022] Comparative Example 2 A method for preparing a self-emulsifying adjuvant for a vaccine includes the following steps: (1) Weigh 2.40g squalene and 0.60g DL-α-tocopherol and mix them evenly; add 1.00g polysorbate 80 and 0.20g polyvinylpyrrolidone; heat and stir at 45°C until polyvinylpyrrolidone is completely dissolved to form a clear and transparent self-emulsifying concentrate; cool to room temperature to obtain the oil phase. (2) Dissolve 4.00g trehalose and 2.00g mannitol in 15mL of water for injection and stir until completely clear; add water for injection to make up to 20mL to obtain the aqueous phase; (3) Under the condition of high-speed shearing at 8000 rpm in a shear emulsifier, the oil phase is slowly added to the aqueous phase at a rate of 1 mL / min; after the oil phase is added, continue stirring for 30 minutes until a uniform, milky white nanoemulsion is formed. (4) Quickly dispense the milky white nanoemulsion into sterile glass vials and partially stopper them; place the sample into a lyophilizer and perform the following optimized lyophilization procedure: Pre-freeze: -45℃, hold for 2 hours; One-time drying: Raise the shelf temperature to -30°C, reduce the vacuum to 100mTorr, and maintain for 20 hours; Secondary drying: Gradually increase the shelf temperature to 25℃ and maintain it for 10 hours; After freeze-drying, the mixture is fully stoppered under vacuum to obtain a white freeze-dried powder, which is the self-emulsifying adjuvant of the vaccine.

[0023] Compared to Pluronic F68, polyvinylpyrrolidone (PVP) is a purely hydrophilic polymer without clearly defined hydrophobic blocks. Its adsorption at the oil-water interface relies primarily on weak van der Waals forces, classifying it as a weakly adsorbing polymer. This interfacial adsorption is reversible and weak, easily diluted or disrupted by shear forces, and cannot form a stable protective layer. PPVP mainly delays aggregation by increasing the viscosity of the aqueous phase, but it cannot effectively reduce interfacial tension or inhibit ripening. For nanoemulsions requiring long-term stability, this weak adsorption and thickening mechanism is far less effective than the strong anchoring and steric hindrance mechanism of Pluronic F68.

[0024] Comparative Example 3 A method for preparing a self-emulsifying adjuvant for a vaccine includes the following steps: (1) Weigh 2.40g of squalene and 0.60g of DL-α-tocopherol and mix them evenly; add 1.00g of polysorbate 80 and 0.20g of hydroxypropyl methylcellulose; heat and stir at 45°C until the hydroxypropyl methylcellulose is completely dissolved to form a clear and transparent self-emulsifying concentrate; cool to room temperature to obtain the oil phase. (2) Dissolve 4.00g trehalose and 2.00g mannitol in 15mL of water for injection and stir until completely clear; add water for injection to make up to 20mL to obtain the aqueous phase; (3) Under the condition of high-speed shearing at 8000 rpm in a shear emulsifier, the oil phase is slowly added to the aqueous phase at a rate of 1 mL / min; after the oil phase is added, continue stirring for 30 minutes until a uniform, milky white nanoemulsion is formed. (4) Quickly dispense the milky white nanoemulsion into sterile glass vials and partially stopper them; place the sample into a lyophilizer and perform the following optimized lyophilization procedure: Pre-freeze: -45℃, hold for 2 hours; One-time drying: Raise the shelf temperature to -30°C, reduce the vacuum to 100mTorr, and maintain for 20 hours; Secondary drying: Gradually increase the shelf temperature to 25℃ and maintain it for 10 hours; After freeze-drying, the mixture is fully stoppered under vacuum to obtain a white freeze-dried powder, which is the self-emulsifying adjuvant of the vaccine.

[0025] Compared to Pluronic F68, hydroxypropyl methylcellulose (HMC) has a rigid cellulose backbone, resulting in slow adsorption to the oil-water interface and difficulty in rearranging at the interface to form tight packaging. In contrast, Pluronic F68's linear, flexible block structure allows for rapid migration and optimization of its conformation at the interface, quickly stabilizing newly formed nanodroplets. Furthermore, even at low concentrations, HMC significantly increases the viscosity of the aqueous phase, potentially interfering with the self-emulsification process and leading to increased droplet size and wider distribution. High viscosity also hinders aseptic filtration and may impede water sublimation during freeze-drying. Pluronic F68, on the other hand, essentially does not increase system viscosity, perfectly compatible with both self-emulsification and filtration processes.

[0026] Example 3 A method for preparing a self-emulsifying adjuvant for a vaccine includes the following steps: (1) Weigh 2.40g of squalene and 0.60g of DL-α-tocopherol and mix them evenly; add 1.00g of polysorbate 80, 0.10g of Pluronic F68 and 0.10g of hyperbranched polyglycerol; heat and stir at 45°C until Pluronic F68 and hyperbranched polyglycerol are completely dissolved to form a clear and transparent self-emulsifying concentrate; cool to room temperature to obtain the oil phase; (2) Dissolve 4.00g trehalose and 2.00g mannitol in 15mL of water for injection and stir until completely clear; add water for injection to make up to 20mL to obtain the aqueous phase; (3) Under the condition of high-speed shearing at 8000 rpm in a shear emulsifier, the oil phase is slowly added to the aqueous phase at a rate of 1 mL / min; after the oil phase is added, continue stirring for 30 minutes until a uniform, milky white nanoemulsion is formed. (4) Quickly dispense the milky white nanoemulsion into sterile glass vials and partially stopper them; place the sample into a lyophilizer and perform the following optimized lyophilization procedure: Pre-freeze: -45℃, hold for 2 hours; One-time drying: Raise the shelf temperature to -30°C, reduce the vacuum to 100mTorr, and maintain for 20 hours; Secondary drying: Gradually increase the shelf temperature to 25℃ and maintain it for 10 hours; After freeze-drying, the mixture is fully stoppered under vacuum to obtain a white freeze-dried powder, which is the self-emulsifying adjuvant of the vaccine.

[0027] When using, inject sterile water for injection containing the antigen into the bottle to a total volume of 100mL, gently shake or vortex, and it will be completely reconstituted into a homogeneous nanoemulsion within 1 minute.

[0028] Example 4 A method for preparing a self-emulsifying adjuvant for a vaccine includes the following steps: (1) Weigh 2.40g of squalene and 0.60g of DL-α-tocopherol and mix them evenly; add 1.00g of polysorbate 80 and 0.20g of hyperbranched polyglycerol; heat and stir at 45°C until the hyperbranched polyglycerol is completely dissolved to form a clear and transparent self-emulsifying concentrate; cool to room temperature to obtain the oil phase. (2) Dissolve 4.00g trehalose and 2.00g mannitol in 15mL of water for injection and stir until completely clear; add water for injection to make up to 20mL to obtain the aqueous phase; (3) Under the condition of high-speed shearing at 8000 rpm in a shear emulsifier, the oil phase is slowly added to the aqueous phase at a rate of 1 mL / min; after the oil phase is added, continue stirring for 30 minutes until a uniform, milky white nanoemulsion is formed. (4) Quickly dispense the milky white nanoemulsion into sterile glass vials and partially stopper them; place the sample into a lyophilizer and perform the following optimized lyophilization procedure: Pre-freeze: -45℃, hold for 2 hours; One-time drying: Raise the shelf temperature to -30°C, reduce the vacuum to 100mTorr, and maintain for 20 hours; Secondary drying: Gradually increase the shelf temperature to 25℃ and maintain it for 10 hours; After freeze-drying, the mixture is fully stoppered under vacuum to obtain a white freeze-dried powder, which is the self-emulsifying adjuvant of the vaccine.

[0029] When using, inject sterile water for injection containing the antigen into the bottle to a total volume of 100mL, gently shake or vortex, and it will be completely reconstituted into a homogeneous nanoemulsion within 1 minute.

[0030] Test Example 1 All tests were performed in triplicate, and the mean ± standard deviation was taken.

[0031] 1) Droplet size and polydispersity index (PdI): Methods: Measurements were performed using a dynamic light scattering instrument (Malvern Zetasizer Nano ZS) at 25°C. Samples were appropriately diluted with pure water to avoid multiple scattering effects.

[0032] Standard: According to ISO 22412:2017, particle size is expressed as Z-Average (nm), and an ideal PdI value of <0.2 indicates uniform distribution.

[0033] 2) ζ-potential: Method: Laser Doppler electrophoresis was used for measurement at 25°C. The sample was placed in a folded capillary sample cell for measurement.

[0034] Standard: The higher the absolute value, the stronger the electrostatic repulsion between droplets, which helps prevent aggregation.

[0035] 3) Determination of active ingredient content: Methods: High performance liquid chromatography (HPLC, model: Agilent 1260 Infinity II) was used.

[0036] Squalene: A C18 column (Agilent ZORBAX SB-C18, 4.6×250mm, 5μm) was used, with acetonitrile:isopropanol (50:50, v / v) as the mobile phase, flow rate 1.0 mL / min, differential detector, and column temperature 30℃.

[0037] DL-α-Tocopherol: A C18 column was used with methanol:water (95:5, v / v) as the mobile phase, a fluorescence detector (excitation wavelength 294 nm, emission wavelength 326 nm), and a column temperature of 30 °C.

[0038] Standard: The content should be maintained within the range of 90%-110% of the initial content.

[0039] 4) Accelerated stability study: The liquid pre-lyophilized emulsions and lyophilized reconstituted emulsions of each embodiment and comparative example were placed in a constant temperature chamber at 40°C, and samples were taken on days 0, 7, 14 and 30 to detect particle size, PdI and active ingredient content.

[0040] Standard: For simulated long-term storage (according to ICH Q1A guidelines), a particle size increase of <10% can be considered negligible.

[0041] Table 1: Physical properties of the initial emulsion and the reconstructed emulsion (Day 0) Table 2: Particle size changes of the reconstituted emulsion after accelerated stabilization (40°C) (day 30) Table 3: Active ingredient content after accelerated stability testing (40℃) (day 30) As can be seen from the table above, the long-term physical and chemical stability of Example 1 is significantly better than that of Comparative Example 1. This is because Example 1 additionally added the co-emulsifier Pluronic F68. It works synergistically with polysorbate 80, forming a strong steric hindrance protective layer on the oil droplet surface through its unique block copolymer structure, effectively inhibiting oil droplet aggregation and oxidation of active ingredients. Comparative Example 1, which only used a small-molecule emulsifier, could not provide the same long-term protection.

[0042] The overall performance of Example 1 (including initial particle size, uniformity, and long-term stability) is significantly better than that of Comparative Examples 2-3, Examples 2, and 4. This is because the molecular structure of Pluronic F68 is the most suitable for this system among all single co-emulsifiers. In contrast, Comparative Example 2 (polyvinylpyrrolidone) and Comparative Example 3 (hydroxypropyl methylcellulose) lack effective interfacial anchoring capabilities, leading to emulsification failure; Pluronic F38 in Example 2 exhibits poor stabilization due to its excessively short hydrophobic segments; and the hyperbranched polyglycerol in Example 4, when used alone, does not achieve the same stabilization efficiency as the linear, flexible structure of Pluronic F68. Therefore, Pluronic F68 provides the optimal balance between interfacial adsorption and steric hindrance.

[0043] The overall performance of Example 3 (including smaller initial particle size and better long-term physical and chemical stability) is superior to that of Examples 1 and 4. This is because Example 3 uses a compounding scheme of Pluronic F68 and hyperbranched polyglycerol, which produces a synergistic effect of 1+1>2. The linear Pluronic F68 provides basic steric hindrance protection, while the spherical hyperbranched polyglycerol can fill the gaps between its molecular chains, jointly constructing a denser and more robust composite interfacial protective layer, thus surpassing the effect of any single co-emulsifier in all key performance indicators.

[0044] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for preparing a self-emulsifying adjuvant for a vaccine, characterized in that, Includes the following steps: (1) Mix squalene, tocopherol, polysorbate and co-emulsifier, heat and stir at 40-50°C until completely dissolved to form a clear and transparent self-emulsifying concentrate, cool to room temperature to obtain the oil phase; The usage ranges for each raw material are as follows: Squalene: 2.0-3.0g Tocopherol: 0.5-1.0g Polysorbate 80: 0.8-1.2g Co-emulsifier: 0.1-0.3g (2) Dissolve trehalose and mannitol in a portion of water for injection, stir until completely clear, and then dilute to 15-25 mL with water for injection to obtain the aqueous phase; The usage ranges for each raw material are as follows: Trehalose: 3.0-5.0g Mannitol: 1.5-2.5g (3) Under the stirring speed of 5000-10000 rpm in a high-speed shear emulsifier, the oil phase obtained in step (1) is slowly added dropwise to the aqueous phase obtained in step (2) at a rate of 0.5-2 mL / min; after the oil phase is added, continue stirring for 20-40 minutes to form a uniform nanoemulsion. (4) Dispense the nanoemulsion obtained in step (3) into a container, partially stopper it, and then freeze-dry it; after the freeze-drying is completed, fully stopper it under vacuum conditions to obtain a white freeze-dried powder, which is the self-emulsifying adjuvant of the vaccine.

2. The method for preparing the self-emulsifying adjuvant of the vaccine as described in claim 1, characterized in that: The co-emulsifier is selected from polyoxyethylene-polyoxypropylene block copolymers and / or hyperbranched polymers.

3. The method for preparing the self-emulsifying adjuvant of the vaccine as described in claim 2, characterized in that: The polyoxyethylene-polyoxypropylene block copolymer is one of Pluronic F68 and Pluronic F38.

4. The method for preparing the self-emulsifying adjuvant of the vaccine as described in claim 2, characterized in that: The co-emulsifier is a mixture of Pronic F68 and hyperbranched polyglycerol in a mass ratio of 1-3:1-3.

5. The method for preparing the self-emulsifying adjuvant of the vaccine as described in claim 1, characterized in that, The freeze-drying process in step (4) includes: Pre-freezing stage: Keep at -40 to -50°C for 1-3 hours; First drying stage: Raise the shelf temperature to -25 to -35°C, control the vacuum degree at 50-150 mTorr, and maintain for 15-25 hours; Secondary drying stage: Gradually increase the shelf temperature to 20-30℃ and maintain it for 5-15 hours.

6. A self-emulsifying adjuvant for a vaccine, characterized in that: It is prepared by the method for preparing the self-emulsifying adjuvant of the vaccine according to any one of claims 1-5.

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