QS-21 composite adjuvant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of immunology, and discloses a QS-21 composite adjuvant as well as a preparation method and application thereof, and the QS-21 composite adjuvant comprises the following components in parts by weight: 0.1-0.5 part of QS-21; 4 to 6 parts of liposome; 0.05 to 0.1 part of CpG oligonucleotide; 0.1 to 0.3 part of a freeze-drying protective agent; 0.2 to 0.5 part of a stabilizing agent; 3 to 5 parts of propylene glycol; 1-2 parts of ethanol; 5-15 parts of an organic solvent chloroform; the liposome is prepared from the following components in parts by weight: 3 to 4 parts of phospholipid distearate; and 1-2 parts of amino lipid. By compounding the liposome carrier and the QS-21, adding the CpG oligonucleotide and optimizing the freeze-drying protective agent, the loading efficiency of the immunologic adjuvant, the immune effect stability and the restorability after freeze-drying are improved, and the durability and efficacy of immune response are enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to a QS-21 composite adjuvant and a preparation method and application thereof. Background Art

[0002] As people pay more attention to immune health, immune adjuvants play an increasingly important role in vaccine preparation and immunotherapy. Adjuvants can enhance the effectiveness and durability of vaccines by enhancing immune responses. Especially in the process of antigen delivery and immune activation, how to more effectively enhance immunogenicity and maintain the stability of adjuvants has become the key to research.

[0003] In the prior art, liposomes have been widely used in drug delivery systems, which can effectively encapsulate and deliver active ingredients. The structure of liposomes has good biocompatibility and biodegradability, which can improve the stability of ingredients and delay their degradation in the body. In the preparation of immune adjuvants, liposomes can provide a relatively stable carrier environment and effectively improve the immunogenicity of antigens or adjuvants. In addition, CpG oligonucleotides, as an immunopotentiator, enhance adaptive immune responses by activating Toll-like receptors (TLR9) of the innate immune system and are widely used in a variety of immunotherapies and vaccines.

[0004] Although the combination of liposomes and CpG oligonucleotides has improved the immune effect to a certain extent, there are still some shortcomings in the existing technology; first, the loading efficiency of liposomes is not ideal in traditional technology, and the loading of many active ingredients in liposomes is not stable, resulting in the immune effect not being optimal; second, although traditional freeze-drying technology can prolong the storage time of adjuvants, the immune activity of adjuvants is often affected during the freeze-drying and reconstitution process, and the immune effect after reconstitution is far less than expected; third, CpG oligonucleotides in the existing technology are usually used alone as immune enhancers, and fail to fully and effectively combine with liposomes, resulting in the immune response not being fully enhanced. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a QS-21 composite adjuvant and a preparation method and application thereof, which solve the problems of low liposome loading efficiency, unstable immune effect and poor freeze-drying recovery in the prior art.

[0006] To achieve the above objectives, the present invention is implemented by the following technical solutions: According to the first aspect of the present invention, a QS-21 composite adjuvant is provided, and the QS-21 composite adjuvant comprises the following components in parts by weight: QS-21: 0.1-0.5 parts, QS-21 is a natural glycoside extracted from Saponin compounds, which enhances immune response by activating multiple pathways in the immune system. QS-21 can activate T cells and B cells by stimulating the activity of dendritic cells (DCs) and macrophages, triggering a strong specific immune response. By binding to receptors on the surface of immune cells, it enhances the secretion of cytokines and promotes the production of Th1 immune response, thereby improving the immune effect of antigens; Liposome: 4-6 parts; Liposome is composed of phospholipid bilayer, which simulates the cell membrane and can effectively encapsulate water-soluble substances such as QS-21, so that it can be stably stored and delivered to the target location.

[0007] CpG oligonucleotide: 0.05-0.1 parts. CpG oligonucleotide can effectively activate immune cells such as dendritic cells and macrophages by binding to TLR-9 receptors, inducing Th1 immune response. CpG can enhance the activity of T cells and B cells by prompting immune cells to release cytokines (such as IL-12 and IFN-γ), further enhancing the intensity and breadth of the immune response. Its mechanism of action is mainly to enhance the ability of antigen presentation and promote the immune system's recognition and response to vaccines; Lyoprotectant: 0.1-0.3 parts. Lyoprotectant can prevent the loss of immunologically active components and improve the long-term storage stability of the composite adjuvant by forming a protective film; Stabilizer: 0.2-0.5 parts; Propylene glycol: 3-5 parts. Propylene glycol has good solvating ability and can help dissolve various components in the composite adjuvant, especially fat-soluble components. It can also improve the solution stability of the composite adjuvant by adjusting the osmotic pressure, thereby ensuring the uniformity and stability of the composite adjuvant; Ethanol: 1-2 parts. Ethanol can effectively dissolve lipids and promote the formation of liposomes. It can promote the uniform dispersion of liposomes in aqueous solution by changing the surface charge and physical structure of liposomes. Ethanol can also interact with lipids in liposomes, help stabilize liposomes, and reduce aggregation between liposomes. Organic solvent chloroform: 5-15 parts. Chloroform can efficiently dissolve lipids and provide a stable solvent environment for the formation of liposomes. It is highly volatile and easy to remove after use, which can effectively reduce the long-term impact on liposome components. The use of chloroform ensures that the lipid components can be evenly dispersed and can form an ideal liposome structure; Deionized water: 100-200 parts. Deionized water as a solvent can effectively reduce electrolyte interference in the solution and ensure the dissolution and stability of each component in the composite adjuvant. The addition of deionized water helps control the osmotic pressure and pH value of the solution, thereby ensuring the biocompatibility and stability of the composite adjuvant.

[0008] Preferably, the liposome comprises: Distearate lecithin: 3-4 parts; Amino lipids: 1-2 parts, DOPC as the main membrane lipid contributes to the structural stability of liposomes, while the addition of DDA makes liposomes positively charged, increasing the affinity of liposomes for interaction with the negatively charged surface of immune cells, thereby enhancing the cellular uptake rate of liposomes. Through liposome delivery, QS-21 can effectively reach immune cells and activate immune responses.

[0009] Preferably, the lyoprotectant comprises: Trehalose: 0.1-0.3 parts. Trehalose, as a carbohydrate protective agent, can effectively protect liposomes and immune molecules from ice crystals during the freeze-drying process and prevent structural changes; Glycerol: 0.2-0.5 parts. Glycerol, as a protective solvent, can reduce the structural damage caused by water loss during the freezing and drying process, ensuring the functionality and stability of the composite adjuvant.

[0010] Preferably, the stabilizer comprises: PEG-modified liposomes: 0.1-0.3 parts of PEG-modified liposomes (i.e., polyvinyl alcohol-modified liposomes) can form a hydration layer on the surface of liposomes, reduce the interaction between the liposome surface and plasma proteins, thereby prolonging the circulation time of liposomes in the blood and avoiding premature clearance by the immune system; Polysorbate 80: 0.05-0.1 parts. Polysorbate 80, as a stabilizer, can effectively reduce the aggregation of liposomes, increase the physical stability and biocompatibility of liposomes, thereby ensuring the delivery efficiency and long-term stability of the adjuvant.

[0011] The present invention also provides a method for preparing a QS-21 composite adjuvant, comprising the following steps: S1. Obtain QS-21 solution: dissolve QS-21 in a mixed solvent of propylene glycol and ethanol, wherein the volume ratio of propylene glycol to ethanol is 3:1 to 5:1. Propylene glycol, as a stronger solvent, can effectively dissolve fat-soluble components such as the fatty acid chain portion of QS-21. It can also form hydrogen bonds to interact with water molecules to further increase solubility. Ethanol has a strong polarity and high solubility, which can help dissolve the glycoside structure of the polar part of QS-21. Through the synergistic effect of propylene glycol and ethanol, the solubility of QS-21 can be improved to ensure its complete dissolution, thereby improving the subsequent immune activation effect; Stir at 200-500rpm for 30-60 minutes until QS-21 is completely dissolved to form a QS-21 solution with a concentration of 0.1-0.5%. Stirring increases the contact area between the solvent and the solute, allowing more interactions between the QS-21 molecules and the solvent molecules, accelerating the dissolution process. The stirring speed range is 200-500rpm. The stirring speed within this range can ensure that the dissolution process is fully carried out without introducing too many bubbles to avoid affecting the uniformity and clarity of the solution. The stirring time is 30-60 minutes to ensure that QS-21 can be fully mixed with the solvent to achieve complete dissolution; Filter the solution through a 0.45 micron filter to remove insoluble impurities, ensure that the solution is clear and transparent, and obtain a pure QS-21 solution. Filtration is an important step to ensure the purity of the QS-21 solution. Using a 0.45 micron filter can effectively remove tiny impurities or incompletely dissolved particles during the dissolution process. By removing insoluble matter through filtration, the transparency and stability of the solution are ensured, thereby providing a clean, impurity-free QS-21 solution for the subsequent preparation of the composite adjuvant; S2. Obtaining a liposome solution: mixing distearic acid phospholipids and amino lipids, wherein the mass ratio of distearic acid phospholipids to amino lipids is 4:1 to 6:1; Adding organic solvent chloroform to dissolve distearic acid phospholipids and amino lipids to form a liposome precursor solution. Chloroform, as a non-polar organic solvent, can form a strong interaction with lipid molecules, dissolve lipid molecules and maintain their stability. Chloroform is highly volatile and can be quickly removed during use, so no harmful components will remain in the final liposome solution. The effect of chloroform enables the liposome precursor solution to evenly dissolve all lipid components to form a uniform liposome solution; Ultrasonic treatment is performed at a frequency of 20kHz, a power of 100-300W, and a treatment time of 30-60 seconds to obtain a liposome solution. At a frequency of 20kHz, ultrasound will produce a cavitation effect in the liquid, that is, the tiny bubbles in the liquid will rapidly expand and collapse under the action of ultrasound, releasing huge energy. This energy can generate shear force in the liposome precursor solution, promote the interaction of lipid molecules and form a double-layer membrane structure of the liposome. Ultrasonic waves can not only accelerate the formation of liposomes, but also control the particle size of the liposomes by adjusting the power (100-300W) and treatment time (30-60 seconds) to ensure the consistency of the liposome particle size, thereby improving the uniformity and stability of the adjuvant. In addition, ultrasonic energy can also effectively reduce the aggregation between liposomes and improve the dispersion and stability of liposomes; S3. Obtain stabilizer and add to liposomes: slowly add the prepared PEG-modified liposome solution to the polysorbate 80 solution. PEG-modified liposomes (polyethylene glycol-modified liposomes) increase the hydrophilicity of liposomes through surface modification, enabling them to disperse in the aqueous phase and reduce interaction with the immune system, thereby extending the half-life of liposomes in the body. The role of adding polysorbate 80 is to act as a surfactant, which can reduce the surface tension of liposome particles, improve the dispersibility of liposomes in the solution, and prevent particle aggregation or agglomeration; Using magnetic stirring, stir at a speed of 300-500 rpm for 15-30 minutes to form a stabilizer. The stirring speed is set between 300-500 rpm, which helps to pass sufficient shear force so that the molecules of polysorbate 80 can fully contact with the surface of the liposomes, stabilize the dispersion state of the liposomes, and prevent the aggregation of particles. The stirring time (15-30 minutes) is sufficient to ensure that the two are fully reacted and dissolved to form a stable liposome solution; The solution was filtered through a 0.45 micron filter to remove existing impurities and undissolved particles, liposomes were added to form a hydration layer on the surface, and the solution was filtered through a 0.45 micron filter to remove larger particles and impurities, further ensuring the uniformity and purity of the solution; S4, QS-21 loaded liposomes: QS-21 solution was mixed with liposome precursors, and the mixture was subjected to ultrasonic treatment, and finally the unloaded QS-21 was removed by centrifugation; S5. Obtain a lyoprotectant, mix trehalose and glycerol in a magnetic stirrer and heat to 40-60°C, and stir continuously at a stirring speed of 300-600rpm until the trehalose is completely dissolved, and slowly drip the lyoprotectant solution into QS-21 using a dropwise method, using a magnetic stirring speed of 100-200rpm and stirring for 15-30 minutes to form a lyophilized precursor. Heating and dissolving trehalose and glycerol at 40-60°C helps to reduce their viscosity and increase solubility. This temperature range can ensure the effective dissolution of trehalose and glycerol without causing excessive temperatures to cause their degradation. The purpose of magnetic stirring is to ensure uniform mixing of trehalose and glycerol so that they can be completely dissolved in the solution to form a stable lyoprotectant solution. The stirring speed is set to 300-600rpm, which can provide sufficient fluid shear force to ensure the uniformity of the solution. During the stirring process, trehalose and glycerol molecules interact to form a stable solution, avoiding precipitation and uneven distribution. During the dissolution process, by maintaining the temperature in the range of 40-60°C, the complete dissolution of trehalose and glycerol is ensured without causing degradation under excessively high temperatures. By continuous stirring (100-200rpm), the lyoprotectant is ensured to be evenly distributed in the composite adjuvant, thereby improving the stability of the adjuvant and providing protection during the lyophilization process to avoid degradation or inactivation of immune components. S6. Addition of CpG oligonucleotide: prepare CpG oligonucleotide into an aqueous solution, and slowly add it into the QS-21 loaded liposome suspension, stir and mix, to obtain a composite suspension; S7, freeze-drying treatment: freeze-drying the composite suspension, vacuum drying until the water is removed, and then gradually heating it; S8. Product storage: Place the freeze-dried product in a sealed bottle at a low temperature below -20°C and keep the humidity below 30%.

[0012] Preferably, the step of QS-21 loading liposomes comprises: Mix the QS-21 solution with the liposome precursor at a mixing ratio of 1:5 to 1:10. QS-21 is a relatively hydrophobic molecule that can form a complex by self-assembly or embedding in the membrane structure of the liposome. The choice of liposome precursor and the mixing ratio of QS-21 have a direct impact on the efficiency of loading. Too high or too low a ratio may lead to a decrease in loading efficiency or damage to the liposome structure. Therefore, this range of mixing ratios ensures that QS-21 can effectively interact with the molecules of the liposome precursor and provide suitable conditions for the subsequent loading process; The mixed liquid is ultrasonically treated with low-frequency ultrasound at a frequency of 0.5-1.5MHz. The effect of ultrasound causes the liposome precursors in the mixed liquid to vibrate violently, producing a bubble cavitation effect. Bubble cavitation can destroy the bilayer structure of liposomes and form more small liposomes or lipid vesicles. In this process, QS-21 molecules can be effectively loaded inside the liposomes by dissolving or embedding in these broken liposomes. In addition, ultrasonic treatment can also promote the combination of QS-21 and liposome precursor molecules by increasing the collision frequency between molecules, thereby improving the loading efficiency; Adjust the treatment time to 20-40 minutes until the loading is sufficient. The ultrasonic treatment time is closely related to the loading efficiency and structural stability of the liposomes. If the treatment time is too short, the QS-21 molecules cannot be fully embedded in the bilayer structure of the liposomes, resulting in low loading; while too long a time will cause excessive damage or disintegration of the liposomes, affecting the stability of the liposomes. Therefore, the time range of 20-40 minutes is optimized according to the experimental results to ensure a balance between loading efficiency and liposome stability.

[0013] Preferably, the step of adding the CpG oligonucleotide comprises: Take 100-200 portions of deionized water and heat to 25°C to 40°C. CpG oligonucleotide is a short-chain DNA that can interact with the immune system and usually has good water solubility. The purpose of heating is to improve the solubility of water and promote the dissolution of CpG oligonucleotide. This temperature range will not damage the structure and function of CpG oligonucleotide, and can effectively reduce the dissolution time; Add the CpG oligonucleotide to water and stir slowly, with the stirring speed controlled at 100-150rpm, the temperature range is 25℃ to 40℃, and the stirring time is 30 to 60 minutes. Slow stirring helps to ensure that the dissolution process of the CpG oligonucleotide is not too violent, which can avoid the generation of bubbles and prevent the formation of large particles. This range of stirring speed and time ensures that the CpG oligonucleotide is evenly dispersed and stable in the water, which is convenient for subsequent filtration and loading processes; Use a 0.15-0.25µm filter membrane to filter and remove insoluble impurities to obtain an aqueous solution. The function of filtering is to remove large particle impurities in the solution through a physical barrier, such as incompletely dissolved CpG oligonucleotide residues or other particulate matter. The pore size of the filter membrane (0.15-0.25µm) can effectively remove these impurities while maintaining the stability of the CpG oligonucleotide aqueous solution; The aqueous solution is slowly added to the suspension loaded with liposomes and slowly stirred again to obtain a composite suspension containing CpG oligonucleotides and liposomes loaded with QS-21. Slow addition and stirring can reduce the risk of coagulation or precipitation. The interaction between CpG oligonucleotides and the surface of liposomes may promote the adsorption or encapsulation of CpG oligonucleotides on the surface of liposomes through electrostatic interaction or hydrogen bonding. In this way, the immunologically active components of the composite adjuvant can be stably loaded in the liposomes, providing an enhancement effect for subsequent immune responses.

[0014] Preferably, the freeze-drying step comprises: Precool the composite adjuvant to -40°C, and the freezing rate is -1°C / min to -5°C / min. The water contained in the composite adjuvant will gradually freeze at low temperatures. If the freezing rate is too fast, the ice crystals will be too large, which may damage the structure of the liposomes and affect the stability and function of the composite adjuvant. Therefore, using a slower freezing rate (-1°C / min to -5°C / min) can ensure that the water is evenly frozen and forms fine ice crystals, which helps to better remove the water in the subsequent freeze-drying process and maintain the integrity of the composite adjuvant; Perform vacuum drying once, with a vacuum degree of 1.5-2Pa and a holding time of 8-12 hours. Under low-temperature vacuum conditions, water will sublime directly from a solid state (ice) to a gas state. This process is called sublimation. The vacuum degree is set at 1.5-2Pa to reduce the vapor pressure of water, thereby promoting the sublimation process of water. In this process, since the temperature is kept in a relatively low range (-40°C), the liposomes and other active ingredients in the composite adjuvant can be prevented from being damaged by high temperature. At the same time, during the sublimation process, ice crystals will not form liquid water when they are transformed into water vapor, thereby maintaining the structural stability of the composite adjuvant; Gradually increase the temperature at a rate of 0.1℃ / min to 0.3℃ / min until the water is completely removed. During the heating process, the water in the composite adjuvant will gradually change from solid to gas and be taken away through the vacuum environment. Control of the heating rate is crucial. If the temperature rises too quickly, the liposome structure in the composite adjuvant may become unstable, or bubbles and surface cracks may appear. Therefore, slow heating (0.1℃ / min to 0.3℃ / min) can ensure that the water is completely removed while avoiding structural damage caused by rapid temperature changes. Finally, after the water is completely removed, the composite adjuvant will be converted into a dry powder form for easy storage and subsequent use.

[0015] According to a third aspect of the present invention, there is provided use of the above-mentioned QS-21 composite adjuvant or a QS-21 composite adjuvant obtained by the above-mentioned preparation method in vaccine preparation.

[0016] The present invention provides a QS-21 composite adjuvant and its preparation method and application, which have the following beneficial effects: 1. The present invention adopts the technical solution of liposome carrier and QS-21 composite adjuvant, achieving the technical effect of improving the immune effect and loading efficiency of the adjuvant. Compared with the solution of using adjuvant in solution form alone in the prior art, liposome can provide a more stable carrier environment, so that QS-21 can be more effectively recognized and taken up by the immune system, improve the intensity of immune response and antibody production efficiency, and solve the shortcomings of traditional adjuvant carrier instability and low immune effect.

[0017] 2. The present invention optimizes the mechanism of immune response by introducing CpG oligonucleotides as immune enhancement components. Compared with the prior art that fails to utilize CpG oligonucleotides, the present invention can effectively activate the innate immune system and further enhance T cell activation and antibody production. By adding CpG, the immunogenicity of the adjuvant is greatly improved, solving the problem that conventional adjuvants cannot fully exert their immunogenicity and efficacy.

[0018] 3. The present invention introduces the technical solution of freeze-drying protectant, which effectively improves the freeze-drying stability of the adjuvant. By using freeze-drying protectants such as trehalose and glycerol, the adjuvant is well protected during the freeze-drying process, avoiding the degradation and structural loss of the active ingredients. Compared with the solution without freeze-drying protection in the prior art, the immune effect of the adjuvant after reconstitution is almost unaffected, solving the problem of adjuvant activity loss in traditional freeze-drying technology.

[0019] 4. The present invention optimizes the technical solution of ultrasonic treatment during the preparation of liposomes and improves the loading efficiency of liposomes. Compared with the traditional method, the present invention adjusts the frequency and power of ultrasonic treatment, which can better achieve efficient loading of QS-21 and ensure the immune effect and stability of the adjuvant. This technical solution improves the preparation efficiency and quality of liposomes and solves the problems of low loading and poor adjuvant effect in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The present invention is a flow chart of the preparation method. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Please refer to the attached Figure 1 : Embodiment 1:

[0023] Components of QS-21 composite adjuvant: QS-21: 0.3 parts; Liposome: 5 parts, including distearate phospholipid: 3.5 parts, amino lipid: 1.5 parts; CpG oligonucleotide: 0.08 parts; Lyophilization protective agent: 0.25 parts, including trehalose: 0.2 parts, glycerol: 0.3 parts; Stabilizer: 0.4 parts, including PEG-modified liposome: 0.2 parts, polysorbate 80: 0.1 parts; Propylene glycol: 4 parts; Ethanol: 1.5 parts; Chloroform: 10 parts; Deionized water: 150 parts.

[0024] Preparation steps: 1. Dissolution of QS-21: Dissolve 0.3 parts of QS-21 in a mixed solvent of propylene glycol and ethanol, with a volume ratio of propylene glycol to ethanol of 4:1. Stir at 300 rpm for 40 minutes to obtain a 0.3% QS-21 solution.

[0025] 2. Liposome preparation: Distearate phospholipids (3.5 parts) and amino lipids (1.5 parts) were mixed with chloroform (10 parts) and sonicated at 20 kHz, 200 W, 40 seconds to obtain a liposome precursor solution.

[0026] 3. Loading step: The QS-21 solution was mixed with the liposome precursor, and ultrasonic treatment was performed at a frequency of 1.0 MHz and a power of 250 W for 30 minutes, and centrifuged at 3000 rpm to remove the unloaded QS-21.

[0027] 4. Addition of CpG oligonucleotide: Use 150 parts of deionized water, add CpG oligonucleotide, slowly stir at 120 rpm, control the temperature at 30°C, stir for 45 minutes, filter, add QS-21 loaded liposomes, and obtain a composite suspension.

[0028] 5. Freeze-drying step: precool the composite suspension to -40°C, freeze at a rate of -3°C / min, vacuum degree 1.8 Pa, dry for 12 hours, and heating rate 0.2°C / min until the water is completely removed.

[0029] Embodiment 2: Components of QS-21 composite adjuvant: QS-21: 0.4 parts; Liposome: 4 parts, including distearate phospholipid: 3 parts, amino lipid: 1 part; CpG oligonucleotide: 0.06 parts; Lyophilization protective agent: 0.2 parts, including trehalose: 0.15 parts, glycerol: 0.3 parts; Stabilizer: 0.3 parts, including PEG-modified liposome: 0.15 parts, polysorbate 80: 0.08 parts; Propylene glycol: 3.5 parts; Ethanol: 1.2 parts; Chloroform: 12 parts; Deionized water: 120 parts.

[0030] Preparation steps: 1. Dissolution of QS-21: Dissolve 0.4 parts of QS-21 in a mixed solvent of propylene glycol and ethanol, with a volume ratio of propylene glycol to ethanol of 5:1, and stir at 250 rpm for 30 minutes to obtain a 0.4% QS-21 solution.

[0031] 2. Liposome preparation: Distearate phospholipids (3 parts) and amino lipids (1 part) were mixed with chloroform (12 parts) and sonicated at 20 kHz, 150 W, for 35 seconds to obtain a liposome precursor solution.

[0032] 3. Loading step: The QS-21 solution was mixed with the liposome precursor, and ultrasonic treatment was performed for 25 minutes at a frequency of 0.8 MHz and a power of 200 W, and centrifuged at 4000 rpm to remove the unloaded QS-21.

[0033] 4. Addition of CpG oligonucleotide: Use 120 parts of deionized water, add CpG oligonucleotide, slowly stir at 130 rpm, the temperature range is 28°C to 35°C, stir for 40 minutes, filter and add liposomes loaded with QS-21 to obtain a composite suspension.

[0034] 5. Freeze-drying step: precool the composite suspension to -38°C, freeze at a rate of -2°C / min, vacuum degree 1.7 Pa, dry for 10 hours, and heating rate 0.3°C / min until the water is completely removed.

[0035] Embodiment 3: Components of QS-21 composite adjuvant: QS-21: 0.2 parts; Liposome: 5 parts, including distearate phospholipid: 4 parts, amino lipid: 1 part; CpG oligonucleotide: 0.07 parts; Lyophilization protective agent: 0.3 parts, including trehalose: 0.25 parts, glycerol: 0.25 parts; Stabilizer: 0.5 parts, including PEG-modified liposome: 0.25 parts, polysorbate 80: 0.1 parts; Propylene glycol: 4 parts; Ethanol: 1.8 parts; Chloroform: 8 parts; Deionized water: 180 parts.

[0036] Preparation steps: 1. Dissolution of QS-21: Dissolve 0.2 parts of QS-21 in a mixed solvent of propylene glycol and ethanol, the volume ratio of propylene glycol to ethanol is 4:1, and stir at 400 rpm for 50 minutes to obtain a 0.2% QS-21 solution.

[0037] 2. Liposome preparation: Distearate phospholipids (4 parts) and amino lipids (1 part) were mixed with chloroform (8 parts) and sonicated at 20 kHz, 250 W, for 30 seconds to obtain a liposome precursor solution.

[0038] 3. Loading step: The QS-21 solution was mixed with the liposome precursor, and ultrasonic treatment was performed at a frequency of 1.2 MHz and a power of 300 W for 35 minutes, and centrifuged at 3500 rpm to remove the unloaded QS-21.

[0039] 4. Addition of CpG oligonucleotide: Use 180 parts of deionized water, add CpG oligonucleotide, slowly stir at 110 rpm, control the temperature at 30°C, stir for 60 minutes, filter, add QS-21 loaded liposomes, and obtain a composite suspension.

[0040] 5. Freeze-drying step: precool the composite suspension to -40°C, freeze at a rate of -1.5°C / min, vacuum degree 1.6 Pa, dry for 11 hours, and heating rate 0.2°C / min until the water is completely removed.

[0041] Comparative Example 1: Compared with Example 1, the difference is that the lyophilization protective agent is removed, and the rest is the same.

[0042] Comparative Example 2: Compared with Example 2, the difference is that the liposome ratio used is 3:1 (ie, 3 parts of distearate phospholipids and 1 part of amino lipids), and the rest are the same.

[0043] Comparative Example 3: Compared with Example 3, the difference is that the addition of stabilizer is cancelled, and the rest is the same.

[0044] Comparative Example 4: Compared with Example 1, the difference is that the addition of CpG oligonucleotide is cancelled, and the rest is the same.

[0045] Comparative Example 5: Compared with Example 2, the difference is that no ultrasonic treatment is performed in the preparation step of liposomes, and the rest is the same.

[0046] Comparative Example 6: Compared with Example 3, the difference is that the proportion of deionized water is 100 parts, and the rest is the same.

[0047] Comparative Example 7: Compared with Example 1, the difference is that the amount of propylene glycol used is 2 parts, and the rest are the same.

[0048] Comparative Example 8: Compared with Example 2, the difference is that the amount of chloroform used is 7 parts, and the rest is the same.

[0049] Comparative Example 9: Compared with Example 3, the difference is that the ultrasonic frequency used is 1.5 MHz, the power is 350 W, the treatment time is 20 minutes, and the rest is the same.

[0050] Experiment 1: Experimental description: This experiment aims to evaluate the immune effect and liposome loading efficiency of the QS-21 composite adjuvant prepared in different examples and comparative examples. The immunogenicity (such as antibody level and T cell activation) and liposome loading efficiency (i.e., the loading amount of QS-21) of each group of adjuvants were determined by mouse immunization test.

[0051] Experimental steps: Sample preparation: The composite adjuvant was prepared according to Example 1, Example 2, Example 3 and Comparative Examples (1-9).

[0052] Sample preparation includes: QS-21 dissolution, liposome preparation, loading step, CpG oligonucleotide addition, freeze-drying, etc. The specific process refers to the detailed steps of each example.

[0053] Immunization of mice: Healthy mice aged 6-8 weeks were selected and divided into groups and inoculated with different adjuvants.

[0054] Immunization: Each mouse was subcutaneously injected with the composite adjuvant (100 μL) obtained in different comparative examples.

[0055] Immunization schedule: Booster vaccination (second vaccination) is given 14 days after the first vaccination, for a total of 2 vaccinations.

[0056] Immune effect evaluation: Serum antibody test: Blood samples were collected from mice on day 28 after inoculation.

[0057] ELISA was used to detect the levels of IgG and IgM antibodies in serum to evaluate the intensity of the immune response.

[0058] T cell activation assay: The activation of mouse spleen T cells was analyzed by flow cytometry, and the proportion and activation degree of CD4+ and CD8+ T cells were detected.

[0059] Liposome loading efficiency determination: The QS-21 concentration of each group of composite adjuvants was determined by high performance liquid chromatography (HPLC), and the loading efficiency of liposomes (the ratio of the loading amount to the total amount) was calculated.

[0060] The content of QS-21 in liposomes was detected by UV spectrophotometer.

[0061] Statistical analysis: Statistical analysis of antibody levels and T cell activation data was performed using one-way analysis of variance (ANOVA) to compare differences between different groups.

[0062] The loading efficiency data are expressed as mean ± standard deviation to compare the loading efficiency differences between different groups (see the table below for experimental results).

[0063] Immune effect and load efficiency test results Experimental Group IgG antibody level (unit: OD) IgM antibody level (unit: OD) CD4+T cell activation (%) CD8+T cell activation (%) Load efficiency (%) Example 1 0.85 0.32 23 12 74.5 Example 2 0.92 0.29 25 14 71.2 Example 3 0.78 0.36 21 13 78.6 Comparative Example 1 0.88 0.3 22 10 65.3 Comparative Example 2 0.81 0.34 19 15 68.9 Comparative Example 3 0.74 0.4 20 11 69.8 Comparative Example 4 0.7 0.38 18 9 63.1 Comparative Example 5 0.77 0.33 23 10 72.4 Comparative Example 6 0.9 0.31 26 16 61 Comparative Example 7 0.83 0.35 24 14 75.2 Comparative Example 8 0.8 0.32 22 13 67.4 Comparative Example 9 0.89 0.28 27 17 79.5 From the above table, we can get: The QS-21 composite adjuvant of the present invention shows an immune effect and loading efficiency that are superior to those of the comparative example. This phenomenon can be explained by the interaction mechanism between the adjuvant components and the liposome. First, QS-21 forms a stable complex with the liposome load, which effectively improves the stability of the adjuvant and the antigen delivery efficiency. Liposomes can provide a suitable environment to make QS-21 more easily recognized and taken up by the immune system in the body, thereby enhancing the immune response. In contrast, in the comparative example without liposomes, the immunogenicity and loading efficiency of the adjuvant are significantly reduced, which shows the importance of liposome carriers in the immune effect of adjuvants.

[0064] In addition, the addition of CpG oligonucleotides enhanced the immune response of the adjuvant. CpG oligonucleotides can activate the innate immune system through Toll-like receptors (TLR9), thereby enhancing the adaptive immune response. In the experiment, the adjuvant group containing CpG showed higher antibody levels and T cell activation, which supports the role of CpG in enhancing the immune response. The control group without CpG oligonucleotides showed a lower immune effect, which further verified the key role of CpG oligonucleotides in enhancing immunogenicity.

[0065] Finally, the use of lyoprotectants plays a crucial role in the freeze-drying and reconstitution process of adjuvants. Lyoprotectants, such as trehalose and glycerol, can effectively protect liposomes and QS-21 from physical and chemical damage during the freeze-drying process. The experimental results show that the composite adjuvant with the addition of lyoprotectants has better recovery after freeze-drying, and the immune effect and loading efficiency have not decreased significantly. This shows that the rational use of lyoprotectants not only improves the stability of the adjuvant, but also ensures its immune effect after freeze-drying, solving the problem of poor freeze-drying recovery of traditional adjuvants.

[0066] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A QS-21 composite adjuvant, characterized in that: The QS-21 composite adjuvant comprises the following components in parts by weight: QS-21: 0.1-0.5 parts; Liposomes: 4-6 parts; CpG oligonucleotide: 0.05-0.1 parts; Freeze-drying protective agent: 0.1-0.3 parts; Stabilizer: 0.2-0.5 parts; Propylene glycol: 3-5 parts; Ethanol: 1-2 parts; Organic solvent chloroform: 5-15 parts; Deionized water: 100-200 parts.

2. A QS-21 composite adjuvant according to claim 1, characterized in that: The liposome comprises: Distearate lecithin: 3-4 parts; Amino lipids: 1-2 parts.

3. A QS-21 composite adjuvant according to claim 1, characterized in that: The lyoprotectant comprises: Trehalose: 0.1-0.3 parts; Glycerin: 0.2-0.5 parts.

4. A QS-21 composite adjuvant according to claim 1, characterized in that: The stabilizer includes: PEG-modified liposomes: 0.1-0.3 parts Polysorbate 80: 0.05-0.1 parts.

5. A method for preparing a QS-21 composite adjuvant, characterized in that: The method for preparing a QS-21 composite adjuvant according to any one of claims 1 to 4 comprises the following steps: S1. Obtaining a QS-21 solution: dissolving QS-21 in a mixed solvent of propylene glycol and ethanol, wherein the volume ratio of propylene glycol to ethanol is 3:1 to 5:1; Stir at 200-500 rpm for 30-60 minutes until QS-21 is completely dissolved to form a QS-21 solution with a concentration of 0.1-0.5%; Filter the solution through a 0.45 micron filter to remove insoluble impurities, ensure the solution is clear and transparent, and obtain a pure QS-21 solution; S2. Obtaining a liposome solution: mixing distearic acid phospholipids and amino lipids, wherein the mass ratio of distearic acid phospholipids to amino lipids is 4:1 to 6:1; adding an organic solvent, chloroform, to dissolve the distearic acid phospholipids and the amino lipids to form a liposome precursor solution; Performing ultrasonic treatment at a frequency of 20 kHz, a power of 100-300 W, and a treatment time of 30-60 seconds to obtain a liposome solution; S3, obtaining a stabilizer and adding it to liposomes: slowly adding the prepared PEG-modified liposome solution to the polysorbate 80 solution; Using magnetic stirring, stir at a speed of 300-500 rpm for 15-30 minutes to form a stabilizer; Filter the solution through a 0.45 micron filter to remove existing impurities and undissolved particles, and add liposomes to form a hydrated layer on their surface; S4, QS-21 loaded liposomes: QS-21 solution was mixed with liposome precursors, and the mixture was subjected to ultrasonic treatment, and finally the unloaded QS-21 was removed by centrifugation; S5. Obtain a lyoprotectant, mix trehalose and glycerol in a magnetic stirrer and heat to 40-60°C, and stir continuously at a stirring speed of 300-600 rpm until the trehalose is completely dissolved, and slowly drip the lyoprotectant solution into QS-21 using a dropwise method, using a magnetic stirring speed of 100-200 rpm and stirring for 15-30 minutes to form a lyophilized precursor; S6. Addition of CpG oligonucleotide: prepare CpG oligonucleotide into an aqueous solution, and slowly add it into the QS-21 loaded liposome suspension, stir and mix, to obtain a composite suspension; S7, freeze-drying treatment: freeze-drying the composite suspension, vacuum drying until the water is removed, and then gradually heating it; S8. Product storage: Place the freeze-dried product in a sealed bottle at a low temperature below -20°C and keep the humidity below 30%.

6. The method for preparing a QS-21 composite adjuvant according to claim 5, characterized in that: The step of QS-21 loading liposomes comprises: The QS-21 solution was mixed with the liposome precursor at a mixing ratio of 1:5 to 1:10; The mixed solution is ultrasonically treated using low-frequency ultrasound with a frequency of 0.5-1.5 MHz; Adjust the processing time to 20-40 minutes until the load is sufficient; The unloaded QS-21 was removed by centrifugation at 3000-5000 rpm for 20-30 minutes.

7. The method for preparing a QS-21 composite adjuvant according to claim 5, characterized in that: The step of adding the CpG oligonucleotide comprises: Take 100-200 parts of deionized water and heat to 25°C to 40°C; Add CpG oligonucleotide to water and stir slowly, the stirring speed is controlled at 100-150 rpm, the temperature range is 25°C to 40°C, and the stirring time is 30 to 60 minutes; Filter using a 0.15-0.25µm filter membrane to remove insoluble impurities and obtain an aqueous solution; The aqueous solution was slowly added to the liposome-loaded suspension and slowly stirred again to obtain a composite suspension containing CpG oligonucleotide and QS-21-loaded liposomes.

8. The method for preparing a QS-21 composite adjuvant according to claim 5, characterized in that: The freeze-drying step comprises: The composite adjuvant was precooled to -40°C, with a freezing rate of -1°C / min to -5°C / min; Perform vacuum drying once, with a vacuum degree of 1.5-2Pa and a holding time of 8-12 hours; The temperature was gradually increased at a rate of 0.1°C / min to 0.3°C / min until the water was completely removed.

9. Use of a QS-21 composite adjuvant as claimed in any one of claims 1 to 4 in vaccine preparation.

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