Compound oil adjuvant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of vaccine immunization, in particular to a compound oil adjuvant as well as a preparation method and application thereof. The compound oil adjuvant is prepared from the following components in percentage by mass: 1 to 6 percent of squalene, 0.5 to 3.0 percent of a surfactant, 0.02 to 0.5 percent of a nano slow-release agonist, 0.1 to 1.2 percent of a biodegradable polymer, 3 to 7 percent of an isotonic agent and the balance of a buffer solution, wherein the nano sustained-release agonist jointly encapsulates a Mincle agonist trehalose 6, 6 '-behenate and a TLR4 agonist CRX-675 through a lipid carrier, a fucoidin and sodium hyaluronate stabilizing system is adopted, and the nano sustained-release agonist is integrated into the squalene emulsion after being subjected to surface modification by a biodegradable polymer. Targeted delivery and slow release of double agonists are realized, and an immune signal channel is synergistically activated, so that the immune response of vaccine body fluid and cells is remarkably improved, and excellent physical stability and safety are achieved. The invention further discloses a preparation method of the adjuvant and application of the adjuvant in preparation of inactivated vaccines.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of vaccine immunization technology, and in particular to a complex oil adjuvant, a preparation method and application thereof. BACKGROUND

[0002] The complex oil adjuvant is a modern vaccine preparation, especially a core technical component of inactivated vaccines and subunit vaccines, and its essence is to form a stable system by emulsifying the immune-enhancing components, oil carriers and surfactants, etc. This kind of adjuvant realizes the slow release of antigens by forming an antigen depot effect, and activates the natural immune system by using its own physical properties. Although the oil-in-water emulsion represented by MF59 and AS03 has been successfully applied to human vaccines, the existing technology generally relies on the physical immune enhancement of emulsion, and there are bottlenecks such as limited immune activation intensity and difficulty in effectively inducing Th1-oriented cellular immunity, which limits its application in complex pathogens, especially diseases that require strong T cell immune response.

[0003] At present, the complex oil adjuvant still has great defects in stability and immunization performance. The emulsion is a thermodynamically unstable system in nature, and there is a risk of creaming, demulsification and particle size increase during storage, which directly affects its potency and safety. At the same time, if the release of strong agonists is not properly controlled, it is easy to cause local intense inflammation or systemic reaction. Secondly, the production process and quality control are extremely complex: the emulsification process parameters are sensitive, and it is difficult to ensure batch consistency when scaling up from laboratory to large-scale production; and the synergistic mechanism between the components of the complex system is often unclear, which brings great difficulties to its rational design and universal application. Therefore, to realize the stable, safe and efficient production of complex oil adjuvant, it is still necessary to carry out deep integration and continuous innovation in the fields of pharmaceutical technology and immunology. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application provides a complex oil adjuvant, a preparation method and application thereof.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is:

[0006] A complex oil adjuvant, consisting of the following components by mass percentage: 1-6% of squalene, 0.5-3.0% of surfactant, 0.02-0.5% of nano-sustained agonist, 0.1-1.2% of biodegradable polymer, 3-7% of isotonic agent, and the balance of buffer; wherein the nano-sustained agonist comprises a lipid carrier and Mincle agonist and TLR4 agonist wrapped therein.

[0007] Traditional oil adjuvants generally suffer from limited immune activation intensity, a single response type, and insufficient immune persistence. Therefore, it is necessary to add agonists to compound oil adjuvants. By simultaneously targeting two key innate immune receptors, Mincle and TLR4, multiple immune signaling pathways can be synergistically activated, significantly enhancing the activation and maturation of antigen-presenting cells, thereby more effectively inducing T cell immune responses, especially the Th1 / Th17 response, which is crucial for clearing intracellular pathogens. Furthermore, encapsulating these two agonists in nanoliposomes cleverly achieves slow release, which not only prolongs the duration of immune stimulation to maintain lasting immune memory but also avoids the excessive inflammatory response that might be triggered by instantaneous agonist release, thus improving vaccine efficacy while ensuring safety.

[0008] The Mincle agonist is trehalose 6,6'-dibenzyl ester, and the TLR4 agonist is CRX-675; and the mass ratio of the Mincle agonist to the TLR4 agonist is 1:(2-4).

[0009] Trehalose 6,6'-disorbate activates Mincle receptors, primarily driving a robust Th1 / Th17 cellular immune response. CRX-675, as a highly effective TLR4 agonist, significantly enhances the maturation and activation of antigen-presenting cells, strongly promoting the production of protective antibodies and Th1 cytokines, providing a powerful initiation signal for the immune response. The two work synergistically, inducing a more balanced and robust Th1 and Th17 cellular immune response, thereby more effectively combating intracellular pathogens.

[0010] The surfactant is selected from one or more of sorbitan trioleate, Tween 80, polyoxyethylene sorbitan fatty acid ester, PEG400, and amino acid surfactants; and / or the isotonic agent is selected from at least one of sucrose and trehalose; and / or the biodegradable polymer is polylactic acid-polyethylene glycol.

[0011] In the polylactic acid-polyethylene glycol mixture, the molar ratio of polylactic acid segments to polyethylene glycol segments is 75:25.

[0012] The buffer solution is a 5-20 mM histidine buffer solution with a pH of 6.5-7.0.

[0013] The method for preparing the nano-sustained-release agonist includes the following steps: (a1) preparing an agonist solution and a fucoidan aqueous solution, wherein the agonist comprises Mincle agonist trehalose 6,6'-dibenzyl ester and TLR4 agonist CRX-675 in a mass ratio of 1:(2-4);

[0014] (a2) Dissolve the lipid mixture in anhydrous ethanol to obtain a lipid mixture solution;

[0015] (a3) The agonist solution prepared in (a1), the fucoidan aqueous solution and the lipid mixture are mixed and vortexed, and then sodium hyaluronate aqueous solution is added and vortexed again; the resulting mixture is dialyzed and freeze-dried to obtain the nano-sustained-release agonist.

[0016] Preferably, the preparation method of the nano-sustained-release agonist includes the following steps: (a1) premixing 3-8 mg of the agonist with 8-14 mL of 0.05-0.2 M Na2CO3 / NaHCO3 solution with pH=9-9.7 to obtain an agonist solution of 400-600 μg / mL; dissolving 3-6 mg of fucoidan in water to prepare a fucoidan aqueous solution of 400-480 μg / mL; the agonist includes a mixture of Mincle agonist trehalose 6,6'-dibenzyl behenate and TLR4 agonist CRX-675 in a mass ratio of 1:2-4;

[0017] (a2) Take 8-15 mg of the lipid mixture and dissolve it in 0.5-2 mL of anhydrous ethanol to obtain a lipid mixture solution;

[0018] (a3) Take 2-5 parts by weight of the above agonist solution, 2-5 parts by weight of fucoidan aqueous solution and 1-3 parts by weight of lipid mixture, mix them, and vortex mix at 1000-1400 rpm for 3-8 min at room temperature. Then add 0.2-0.4 parts by weight of sodium hyaluronate aqueous solution with a mass concentration of 12-250 mg / mL, and continue to vortex mix for 5-15 min. Pass the resulting mixture through a dialysis bag with a molecular weight cutoff of 1000 kDa and dialyze it with ultrapure water at 2-8℃ for 18-36 h. Change the dialysate every 6-10 h to remove ethanol, unencapsulated agonist and sodium hyaluronate. Collect the liquid in the dialysis bag, add 3-8% mannitol as a freeze-drying protectant, and freeze-dry to obtain nano-sustained-release agonist.

[0019] The nano-sustained-release agonist prepared by this invention has a good effect on improving the immune response. First, nano-encapsulation enables the slow release of agonists. By encapsulating Mincle and TLR4 agonists in a lipid carrier, it effectively prevents their rapid clearance or degradation in vivo, thereby continuously activating antigen-presenting cells, prolonging the duration of immune stimulation, and contributing to the establishment of durable and high-quality immune memory. Second, nano-encapsulation technology significantly improves the targeted delivery efficiency of drugs. Due to the ease with which nano-encapsulated agonists are phagocytosed by antigen-presenting dendritic cells and macrophages, it can precisely enrich and deliver agonists to key sites of immune action, thereby triggering a more potent immune response with lower doses and reducing the risk of side effects from systemic exposure. Finally, this sustained-release structure also plays a synergistic and stabilizing role. By co-locating the two agonists in the same nanocarrier, it ensures that they can be simultaneously taken up by the same cells, synergistically activating the Mincle and TLR4 signaling pathways within the cell, producing a "1+1>2" immune enhancement effect. At the same time, the lipid carrier also protects the encapsulated material from premature inactivation during formulation storage or in vivo circulation, improving the stability of the entire adjuvant system.

[0020] The lipid mixture is selected from at least one of ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), methoxy polyethylene glycol bistetradecylacetamide, and distearyl phosphatidylethanolamine-polyethylene glycol-azide MW2000; preferably, the lipid mixture is composed of ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) and distearyl phosphatidylethanolamine-polyethylene glycol-azide MW2000 in a mass ratio of 1:1.

[0021] The preparation method of the compound oil adjuvant includes the following steps:

[0022] (1) Dissolve the freeze-dried nano-release agonist in an appropriate amount of ultrapure water to obtain an agonist aqueous dispersion of 1-10 mg / mL; separately, dissolve the biodegradable polymer in an appropriate amount of isopropanol to obtain a polymer alcohol solution of 10-50 mg / mL; add the polymer alcohol solution dropwise to the agonist aqueous dispersion at 300-500 rpm, and stir in a water bath at 40-60℃ for 0.5-2 h to allow the polymer to adsorb onto the surface of the nanoparticles, thereby obtaining a surface-modified nano suspension for later use;

[0023] (2) Aqueous phase preparation: Tween 80, sucrose and the nano suspension prepared in step (1) are dissolved in histidine buffer and stirred at 1200-2000 rpm for 20-50 min to form a homogeneous aqueous phase;

[0024] (3) Primary emulsification: Squalene is slowly added dropwise to the aqueous phase at a homogenization speed of 10,000-15,000 rpm and emulsification is continued for 5-10 min to form a primary emulsion;

[0025] (4) Maturation and stabilization: Transfer the colostrum into a magnetic stirrer and stir continuously at 1000-1500 rpm at room temperature and in the dark for 36-48 hours to mature and stabilize the milk droplets and obtain a uniform and stable mixture.

[0026] (5) Sterilization and finished product: The mixture obtained in step (4) is filtered through a 0.22μm filter for sterilization to obtain the compound oil adjuvant.

[0027] The application of the compound oil adjuvant in the preparation of inactivated vaccines includes mixing the compound oil adjuvant with the vaccine inactivated antigen at a volume ratio of (1-10):(1-10) to obtain an inactivated vaccine.

[0028] The beneficial effects of this invention are:

[0029] 1. This invention provides a composite oil adjuvant, its preparation method, and its application. In preparing a nano-sustained-release agonist, this invention combines the Mincle agonist trehalose 6,6'-dibenzyl behenate and the TLR4 agonist CRX-675 in a specific mass ratio and encapsulates them together in the same nanocarrier. This synergistically activates two key innate immune signaling pathways, significantly stronger than the effect of a single agonist. It not only induces high levels of neutralizing antibodies but also efficiently stimulates Th1 / Th17 cellular immunity, crucial for clearing intracellular pathogens, overcoming the shortcomings of traditional oil adjuvants, such as limited response types and insufficient cellular immune activation.

[0030] 2. In the preparation process of the nano-sustained-release agonist of the present invention, two agonists are first mixed with fucoidan, and then the agonists are encapsulated in a lipid membrane structure spontaneously formed when the lipid material is dispersed in ethanol, forming the prototype of nanoparticles. Finally, sodium hyaluronate is added and dialyzed and lyophilized to stabilize the structure and remove impurities, ultimately obtaining a dry nano-sustained-release agonist powder. The present invention uses a specific lipid mixture combination and combines it with biodegradable polymers to modify the surface of the nanoparticles, forming nanoparticles with uniform particle size and stable structure in an oil emulsion. This composite adjuvant system has good physical stability, is resistant to freeze-thaw cycles, does not easily delaminate or break down during long-term storage, and has low viscosity, is easy to inject, and leaves no visible residue, providing reliable assurance for quality control in industrial production and convenience in clinical use.

[0031] 3. This nano-sustained-release agonist powder achieves targeted delivery and slow release of the agonist. On the one hand, it can continuously activate antigen-presenting cells, prolong the duration of immune stimulation, and help establish a lasting and high-quality immune memory. On the other hand, it effectively avoids the severe inflammatory response caused by the instantaneous release of free agonists, significantly reduces local irritation at the injection site, and has better safety while greatly improving the immunogenicity of the vaccine.

[0032] 4. The preparation method of this invention is simple, and the raw materials are readily available. By combining nano-predispersion technology with conventional emulsification processes, stable integration of functional nanounits and oil adjuvant matrix is ​​achieved. The composite oil adjuvant prepared by this invention is stable and highly efficient, and can significantly improve the immunization effect. Compared with known vaccine adjuvants, it has the characteristics of safety and high efficiency, simple preparation process, convenient use, easy product process quality control, and broad application prospects. Detailed Implementation

[0033] The invention will now be described in further detail with reference to specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments.

[0034] The raw materials described in this application are partially described; all other raw materials not described are commercially available.

[0035] Squalene was purchased from Kunshan Shengan Biotechnology Co., Ltd., model number: D1UL.

[0036] Polylactic acid-polyethylene glycol was purchased from Shanghai Ziyi Reagent Factory, item number: 103741.

[0037] ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), CAS: 2036272-55-4.

[0038] Methoxylated polyethylene glycol bis(tetradecyl)acetamide, CAS: 1849616-42-7.

[0039] Distearate phosphatidylethanolamine-polyethylene glycol-azide MW2000, CAS: 1938081-39-0.

[0040] Trehalose 6,6'-dibenzyl ester was purchased from Chengdu Chaojiuba Biotechnology Co., Ltd., product number: WKQ-0008719.

[0041] Example 1

[0042] A compound oil adjuvant is composed of the following raw materials in weight percentages: 4% squalene, 2% surfactant, 0.4% nano-slow-release agonist, 0.8% biodegradable polymer, 5% isotonic agent, and the balance being buffer solution.

[0043] The isotonic agent is sucrose.

[0044] The surfactant is Tween 80.

[0045] The biodegradable polymer is polylactic acid-polyethylene glycol, and the molar ratio of polylactic acid to polyethylene glycol is 75:25.

[0046] The buffer solution is a histidine buffer solution with a concentration of 10 mM and a pH of 6.7.

[0047] The preparation method of the nano-sustained-release agonist is as follows: (a1) 5 mg of agonist is premixed with 10 mL of 0.1 M Na2CO3 / NaHCO3 solution at pH=9.4 to obtain a 500 μg / mL agonist solution; 5 mg of fucoidan is dissolved in water to prepare a 500 μg / mL fucoidan aqueous solution; the agonist comprises a mixture of Mincle agonist trehalose 6,6'-dibenzyl behenate and TLR4 agonist CRX-675 in a mass ratio of 1:3;

[0048] (a2) Take 12 mg of the lipid mixture, which is composed of ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), distearate phosphatidylethanolamine-polyethylene glycol-MW2000 azide in a mass ratio of 1:1, and dissolve it in 1 mL of anhydrous ethanol to obtain the lipid mixture.

[0049] (a3) Take 3 parts by mass of the above agonist solution, 3 parts by mass of fucoidan aqueous solution and 2 parts by mass of lipid mixture, mix them, and vortex mix at 2000 rpm for 5 min at room temperature. Then add 0.375 parts by mass of sodium hyaluronate aqueous solution with a mass concentration of 20 mg / mL, and continue to vortex mix for 10 min. Pass the resulting mixture through a dialysis bag with a molecular weight cutoff of 1000 kDa and dialyze it with ultrapure water at 4 °C for 24 h. Change the dialysate every 8 h to remove ethanol, unencapsulated agonist and sodium hyaluronate. Collect the liquid in the dialysis bag, add 5% mannitol as a freeze-drying protectant, and freeze-dry to obtain nano-sustained-release agonist.

[0050] The preparation method of the compound oil adjuvant includes the following steps:

[0051] (1) The freeze-dried nano-release agonist was dissolved in an appropriate amount of ultrapure water to obtain a 5 mg / mL agonist aqueous dispersion; the biodegradable polymer was dissolved in an appropriate amount of isopropanol to obtain a 20 mg / mL polymer alcohol solution; the polymer alcohol solution was added dropwise to the agonist aqueous dispersion at 400 rpm, and stirred in a 50°C water bath for 1 h to allow the polymer to be adsorbed onto the surface of the nanoparticles, thus obtaining a surface-modified nano suspension for later use.

[0052] (2) Aqueous phase preparation: Tween 80, sucrose and the nano suspension prepared in step (1) were dissolved in histidine buffer and stirred at 1500 rpm for 30 min to form a homogeneous aqueous phase;

[0053] (3) Primary emulsification: Squalene was slowly added to the aqueous phase at a homogenization speed of 12000 rpm and emulsified for 8 min to form a primary emulsion;

[0054] (4) Maturation and stabilization: The colostrum was transferred to a magnetic stirrer and stirred continuously at 1200 rpm, room temperature and in the dark for 42 hours to mature and stabilize the milk droplets and obtain a uniform and stable mixture.

[0055] (5) Sterilization and finished product: The mixture obtained in step (4) is filtered through a 0.22μm filter for sterilization to obtain the compound oil adjuvant.

[0056] Example 2

[0057] The method is basically the same as in Example 1, except that the agonist in the preparation method of the nano-sustained-release agonist is the Mincle agonist trehalose 6,6'-dibenzyl ester.

[0058] Example 3

[0059] The method is basically the same as in Example 1, except that the agonist used in the preparation method of the nano-sustained-release agonist is the TLR4 agonist CRX-675.

[0060] Comparative Example 1

[0061] The method is basically the same as Example 1, except that: a compound oil adjuvant is composed of the following raw materials in the following mass percentages: 4% squalene, 2% surfactant, 0.4% agonist, 0.8% biodegradable polymer, 5% isotonic agent, and the balance is buffer solution. The agonist is a mixture of free Mincle agonist trehalose 6,6'-dibenzyl ester and TLR4 agonist CRX-675 in a mass ratio of 1:3.

[0062] The preparation method of the compound oil adjuvant is the same as that in Example 1.

[0063] Comparative Example 2

[0064] The method is basically the same as in Example 1, except that the preparation method of the compound oil adjuvant includes the following steps:

[0065] (1) Dissolve the freeze-dried nano-release agonist in an appropriate amount of ultrapure water to obtain an unmodified agonist aqueous dispersion of 5 mg / mL for later use;

[0066] (2) Aqueous phase preparation: The biodegradable polymer was added to the histidine buffer, and Tween 80, sucrose and the unmodified agonist aqueous dispersion prepared in step (1) were added to the histidine buffer under stirring at 1500 rpm. The mixture was stirred at 1500 rpm for 30 min to form a homogeneous aqueous phase.

[0067] (3) Primary emulsification: Squalene was slowly added to the aqueous phase at a homogenization speed of 12000 rpm and emulsified for 8 min to form a primary emulsion;

[0068] (4) Maturation and stabilization: The colostrum was transferred to a magnetic stirrer and stirred continuously at 1200 rpm, room temperature and in the dark for 42 hours to mature and stabilize the milk droplets and obtain a uniform and stable mixture.

[0069] (5) Sterilization and finished product: The mixture obtained in step (4) is filtered through a 0.22μm filter for sterilization to obtain the compound oil adjuvant.

[0070] Comparative Example 3

[0071] The method is basically the same as in Example 1, except that the agonist in the preparation method of the nano-sustained-release agonist is TLR4 agonist lipid monophosphate A.

[0072] Comparative Example 4

[0073] The method is basically the same as Example 1, except that the agonist in the preparation method of the nano-sustained-release agonist is a mixture of Mincle agonist trehalose 6,6'-dibenzyl ester and TLR4 agonist monophospholipid A in a mass ratio of 1:3.

[0074] Comparative Example 5

[0075] It is basically the same as Example 1, except that: a compound oil adjuvant is composed of the following raw materials in mass percentage: 4% squalene, 2% surfactant, 0.8% biodegradable polymer, 5% isotonic agent, and the balance is buffer solution.

[0076] Comparative Example 6

[0077] The method is basically the same as Example 1, except that the lipid mixture in the preparation method of the nano-sustained-release agonist is composed of ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) and methoxy polyethylene glycol bistetradecylacetamide in a mass ratio of 1:1.

[0078] Test Example 1

[0079] Preparation of inactivated vaccine: Take 50 mL of inactivated chicken infectious bursal virus solution, stir evenly at 600 r / min, add 10 mg of the compound oil adjuvant prepared in the above examples and comparative examples to the inactivated virus solution, stir at 600 r / min for 20 min to prepare 50 mL of aqueous phase, aseptically dispense into 2 mL / bottle, label, store in warehouse at 2-8℃.

[0080] Serological test: Several 7-day-old SPF chickens, 10 chickens per group, were inoculated nasally and ocularly with 0.5 ml of the inactivated infectious bursal disease (IBD) vaccine prepared in the examples and comparative examples. A second immunization with the IBD nano-adjuvant inactivated vaccine prepared in the examples and comparative examples was performed 15 days after the second immunization. A third immunization with the same vaccine was performed 15 days after the second immunization. Blood samples were collected from chickens under the same conditions on days 21 and 42 after the three immunizations. Serum was separated, and the titer of neutralizing antibodies against IBD virus was measured according to the 2020 edition of the Chinese Pharmacopoeia. Injection site damage was observed, and the average value of each group of 10 chickens was taken. The results are shown in Table 1.

[0081] Table 1. Results of titer testing

[0082]

[0083] Test Example 2

[0084] Physical stability test: The composite oil adjuvants prepared in the examples and comparative examples were frozen at -20℃ for 12 hours and then thawed at 25℃ for 12 hours, which constituted one freeze-thaw cycle. After repeated freeze-thaw cycles 20 times, the particle size was measured in nm. Five tests were performed for each group, and the average value was taken. The results are shown in Table 2 below.

[0085] Table 2 Stability performance test results

[0086]

[0087] The experimental results above show that the composite oil adjuvant prepared in this invention has good immunomodulatory effects and good stability. Example 1 exhibits superior performance in both immunopotency and physical stability. This is because the TLR4 agonist CRX-675 and the Mincle agonist trehalose disorbate produce a strong synergistic signal at a specific mass ratio of 1:3. Compared with the single agonist groups in Examples 2 and 3, this combination can more comprehensively activate antigen-presenting cells, inducing more balanced and robust Th1 / Th17 cellular immunity and highly efficient humoral immunity, with an antibody titer of 1:64237. Furthermore, the unique nano-sustained-release agonist encapsulates the dual agonists in a specific lipid carrier and is stabilized by fucoidan, sodium hyaluronate, and surface modified with biodegradable polymers, ensuring the uniformity and stability of the nano-sustained-release agonist. The particle size is 121.6 nm, and the PDI is 0.101. This not only achieves targeted delivery and slow release of the agonist, prolonging the duration of immune stimulation, but also significantly avoids the local inflammation caused by the free agonist used in Comparative Example 1, thus improving efficacy while ensuring safety.

[0088] Secondly, in Comparative Examples 3 and 4, the use of the traditional adjuvant MPL instead of CRX-675 resulted in a sharp drop in titer due to the weaker agonist activity. In Comparative Example 2, the use of an unmodified agonist in aqueous dispersion led to decreased stability of the nano-sustained-release agonist, affecting its in vivo delivery efficiency. In Comparative Example 6, only the lipid mixture was modified, resulting in severely compromised physical stability, an increased particle size of 165.7 nm, a PDI of 0.185, and an antibody titer of only 1:12698. This demonstrates that this specific lipid formulation is the cornerstone for maintaining nanostructure stability and ensuring immune activation efficiency. Modifications to it may disrupt the integrity of the nanoparticles, leading to decreased encapsulation efficiency, abnormal release behavior, or even mild immunosuppression, thus completely losing its adjuvant function.

Claims

1. A compound oil adjuvant, characterized in that, It is composed of the following components by mass percentage: 1-6% squalene, 0.5-3.0% surfactant, 0.02-0.5% nano-sustained-release agonist, 0.1-1.2% biodegradable polymer, 3-7% isotonic agent, and the balance being buffer solution; wherein the nano-sustained-release agonist comprises a lipid carrier and a Mincle agonist and a TLR4 agonist encapsulated therein; the Mincle agonist is trehalose 6,6'-dibenzyl ester, and the TLR4 agonist is CRX-675.

2. The compound oil adjuvant as described in claim 1, characterized in that, The mass ratio of the Mincle agonist to the TLR4 agonist is 1:(2-4).

3. The compound oil adjuvant as described in claim 1, characterized in that, The surfactant is selected from one or more of sorbitan trioleate, Tween 80, polyoxyethylene sorbitan fatty acid ester, PEG400, and amino acid surfactants; and / or the isotonic agent is selected from at least one of sucrose and trehalose; and / or the biodegradable polymer is polylactic acid-polyethylene glycol.

4. The compound oil adjuvant as described in claim 3, characterized in that, In the polylactic acid-polyethylene glycol mixture, the molar ratio of polylactic acid segments to polyethylene glycol segments is 75:

25.

5. The compound oil adjuvant as described in claim 1, characterized in that, The buffer solution is a 5-20 mM histidine buffer solution with a pH of 6.5-7.

0.

6. The compound oil adjuvant as described in claim 1, characterized in that, The method for preparing the nano-sustained-release agonist includes the following steps: (a1) preparing an agonist solution and a fucoidan aqueous solution, wherein the agonist comprises Mincle agonist trehalose 6,6'-dibenzyl ester and TLR4 agonist CRX-675 in a mass ratio of 1:(2-4); (a2) Dissolve the lipid mixture in anhydrous ethanol to obtain a lipid mixture solution; (a3) The agonist solution prepared in (a1), the fucoidan aqueous solution and the lipid mixture are mixed and vortexed, and then sodium hyaluronate aqueous solution is added and vortexed again; the resulting mixture is dialyzed and freeze-dried to obtain the nano-sustained-release agonist.

7. The compound oil adjuvant as described in claim 6, characterized in that, The lipid mixture is selected from at least one of ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), methoxy polyethylene glycol bistetradecyl acetamide, distearate phosphatidylethanolamine-polyethylene glycol-azide MW2000.

8. A method for preparing the composite oil adjuvant as described in any one of claims 1-7, characterized in that, Includes the following steps: (1) Aqueous dispersion of nano-slow-release agonist and alcoholic solution of biodegradable polymer were mixed and incubated to obtain surface-modified nano-suspension; (2) The surfactant, isotonic agent and nano suspension obtained in step (1) are mixed with buffer solution to form an aqueous phase; (3) Squalene is primary emulsified with the aqueous phase to form a primary emulsion; (4) The colostrum is matured and shaped to obtain a stable mixture; (5) The mixture is sterilized and filtered to obtain the composite oil adjuvant.

9. The method as described in claim 8, characterized in that, In step (1), the concentration of the aqueous dispersion of the nano-sustained-release agonist is 1-10 mg / mL; the concentration of the alcohol solution of the biodegradable polymer is 5-50 mg / mL, and the alcohol is isopropanol; the incubation conditions are: stirring in a water bath at 40-60℃ for 0.5-2h.

10. The use of the compound oil adjuvant as described in any one of claims 1-7 in the preparation of inactivated vaccines, characterized in that, The application includes mixing the compound oil adjuvant with the vaccine inactivated antigen at a volume ratio of (1-10):(1-10) to obtain an inactivated vaccine.

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