An MPL composite adjuvant and its preparation method

Abstract

This invention relates to an MPL composite adjuvant and its preparation method, belonging to the field of vaccine adjuvant technology. The composite adjuvant comprises neutral phospholipids, anionic phospholipids, cholesterol, monophospholipid A, QS-21, and surface-modified lipids. The preparation method includes: dissolving the lipid components in an organic solvent to form a mixed solution, evaporating the solvent to form a lipid film, adding buffer solution for hydration to form a liposome suspension, sonicating to obtain small monolayer liposomes, and then mixing with a QS-21 micelle solution. This invention significantly improves the stability of the adjuvant by introducing surface-modified lipids, exhibiting small particle size, uniform distribution, high negative zeta potential, good stability in serum environments, and the ability to induce high-level antibody immune responses, making it suitable for vaccine development.

Description

Technical Field

[0001] This invention relates to the field of vaccine adjuvant technology, and in particular to an MPL composite adjuvant and its preparation method. Background Technology

[0002] Liposome adjuvants, as vaccine carriers, enhance antigen delivery and immunostimulatory effects, and are widely used in vaccine development. In existing technologies, composite liposome adjuvants typically contain phospholipids, cholesterol, and immunostimulants. For example, CN115737800A discloses a composite liposome adjuvant prepared using a combination of DOPC, DOTAP, cholesterol, 3D-MPL, and PolyI:C via microfluidic technology, aiming to improve stability and immunogenicity. However, these adjuvants still suffer from insufficient stability, wide particle size distribution, and limited immune response strength during long-term storage and in vivo application. In particular, the lack of surface modification makes them prone to aggregation and degradation, affecting adjuvant potency. Therefore, there is an urgent need to develop a novel composite adjuvant with high stability, uniform particle size, and strong immunostimulatory capabilities. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide an MPL composite adjuvant and its preparation method, so as to overcome the defects of existing adjuvants such as poor stability and poor immune effect.

[0004] To achieve the above objectives, this invention provides a method for preparing an MPL composite adjuvant, comprising the following steps: dissolving neutral phospholipids, anionic phospholipids, cholesterol, monophospholipid A, and surface-modified lipids in an organic solvent to form a mixed lipid solution; evaporating the organic solvent to form a lipid film; adding a buffer solution to the lipid film for hydration to form a liposome suspension; sonicating the liposome suspension to form small monolayer liposomes; dissolving QS-21 in a buffer solution to form a micelle solution, and mixing the micelle solution with the small monolayer liposome suspension to form a composite adjuvant. This technical solution optimizes the liposome structure and enhances its stability and immunostimulatory properties by introducing surface-modified lipids.

[0005] Preferably, the preparation method of the MPL complex adjuvant includes the following steps, in parts by weight:

[0006] S1. Dissolve 5-7 parts of anionic phospholipids and 20-30 parts of cholesterol in 2000-3500 parts of chloroform to obtain solution A; dissolve 45-55 parts of neutral phospholipids, 15-20 parts of monophospholipid A, and 4-6 parts of surface-modified lipids in 2000-3500 parts of chloroform-methanol solution to obtain solution B; mix solution A and solution B thoroughly.

[0007] S2 evaporates organic solvents to form a lipid film;

[0008] S3 is added to the lipid membrane in 8000-12000 parts of buffer solution, and hydrated to form a liposome suspension;

[0009] S4 is used to sonicate the liposome suspension to form a small monolayer liposome suspension.

[0010] S5 dissolves 6-10 parts of QS-21 in 800-1200 parts of buffer solution to form a micelle solution, and mixes the micelle solution with a small monolayer liposome suspension to form a composite adjuvant.

[0011] QS-21 is a purified saponin compound extracted from the bark of the Chilean soapberry tree (Quillaja saponaria), and has become a highly effective and widely used vaccine adjuvant. Due to its unique immunostimulatory properties, it plays a crucial role in many marketed and developing vaccines; CAS No.: 141256-04-4.

[0012] Preferably, the anionic phospholipid is dimyristoylphosphatidylglycerol.

[0013] Preferably, the neutral phospholipid is dimyristoylphosphatidylcholine.

[0014] Preferably, the surface-modified lipid is a polyethylene glycol-modified lipid or DSPE-PMPC; more preferably, the polyethylene glycol-modified lipid is selected from at least one of DSPE-PEG2000, DSPE-PEG-PSA, DSPE-Hydrazone-PEG, and DSPE-PEG-Mannose.

[0015] DSPE, or 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, is a highly hydrophobic phospholipid with two stearic acid chains.

[0016] Preferably, the volume ratio of chloroform to methanol in the chloroform-methanol solution is 9:1.

[0017] Preferably, the buffer in steps S3 and S5 is 10mM pH7.2 PBS buffer.

[0018] Preferably, the ultrasonic treatment in step S4 is performed in an ice-water bath, using pulse mode with a power of 150-250W, an ultrasonic time of 1-3 seconds, an interval of 3-5 seconds, and a total ultrasonic time of 5-15 minutes. The ultrasonic treatment uses a probe-type ultrasonic instrument, with the probe inserted 1-3 cm below the liquid surface.

[0019] The beneficial effects of this invention are:

[0020] This invention's MPL composite adjuvant, through the combined use of surface-modified lipids, achieves particle size control below 100 nm, a polydispersity index ≤0.15, and uniform distribution, which is beneficial for lymph node targeting. The increased negative zeta potential enhances electrostatic repulsion and improves colloidal stability. Serum stability is significantly improved, with a 48-hour particle size change rate of less than 10%, reducing in vivo aggregation. Immune response is enhanced, with antibody titers more than doubled compared to unmodified adjuvants. This invention's adjuvant outperforms existing technologies in terms of stability, particle size control, and immune activation, solving the problems of instability and insufficient potency of liposomal adjuvants. Detailed Implementation

[0021] The parameters and sources of some raw materials in the examples are as follows:

[0022] Monophospholipid A, brand name: MPLA-SM VacciGrade TM This comes from InvivoGen.

[0023] QS-21, Brand: STIMULON ® , derived from Agenus.

[0024] DSPE-PEG2000, full name: distearylphosphatidylethanolamine-polyethylene glycol 2000, is an amphiphilic material formed by connecting 1,2-distearyl-sn-glycerol-3-phosphoethanolamine (DSPE) and polyethylene glycol (PEG2000) with an average molecular weight of about 2000 Da through amide bonds. DSPE provides the two C18 hydrophobic chains, and PEG2000 is the hydrophilic chain segment.

[0025] DSPE-PEG-PSA, full name: distearate phosphatidylethanolamine-polyethylene glycol-polysialic acid, with a PEG molecular weight of 2000, is sourced from Xi'an Qiyue Biotechnology Co., Ltd.

[0026] DSPE-PMPC, a phospholipid-polymer amphiphilic material formed by using 1,2-distearate-sn-glycerol-3-phosphoethanolamine (DSPE) as a hydrophobic group and poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) as a hydrophilic side chain, and connected by covalent bonds, is derived from Xi'an Qiyue Biotechnology Co., Ltd.

[0027] DSPE-Hydrazone-PEG is a lipid derivative in which hydrazone bonds are introduced between DSPE and PEG chains. The PEG has a molecular weight of 2000 and is derived from Nanosoft Polymers, Inc.

[0028] DSPE-PEG-Mannose, full name: distearate phosphatidylethanolamine-PEG-mannose, with a PEG molecular weight of 2000, is sourced from Xi'an Qiyue Biotechnology Co., Ltd.

[0029] Example 1

[0030] A method for preparing an MPL complex adjuvant includes the following steps:

[0031] S1. Dissolve 68.9 mg of myristoyl phosphatidylglycerol and 295 mg of cholesterol in 20 mL of freshly distilled chloroform to obtain solution A; dissolve 610.1 mg of myristoyl phosphatidylcholine, 195.6 mg of monophosphatidyllipid A, and 56.0 mg of DSPE-PEG2000 in 20 mL of chloroform-methanol solution to obtain solution B; the volume ratio of chloroform to methanol is 9:1; mix solutions A and B thoroughly.

[0032] S2 was rotary evaporated in a 45°C water bath to slowly evaporate the organic solvent until a uniform, translucent lipid film was formed on the bottle wall; the lipid film was then vacuum dried for 4 hours to completely remove any residual organic solvent.

[0033] S3. Add 100 mL of 10 mM pH 7.2 PBS buffer preheated to 40 °C to the dried lipid membrane; rotate to form a multi-layered liposome suspension of varying sizes; at this point, the solution is milky white.

[0034] S4 The liposome suspension was placed in an ice-water bath to prevent local overheating; the probe of the probe-type ultrasonic processor was inserted 2 cm below the surface of the suspension, and ultrasonic treatment was performed in pulse mode with a power of 200W, ultrasonic for 2 seconds, with a 4-second interval, for a total ultrasonic time of 10 minutes; after the treatment, the solution became translucent and formed a small monolayer liposome suspension with a particle size of less than 100 nm.

[0035] S5. Dissolve 88 mg of QS-21 in 10 mL of 10 mM pH 7.2 PBS buffer and gently vortex until completely dissolved to form a QS21 micelle solution. At room temperature, add 10 mL of the QS21 micelle solution dropwise to 100 mL of small monolayer liposome suspension. After mixing, the solution immediately changes from clear to milky white / turbid, which is a direct indicator of liposome fusion and GUVs / TIMs formation. Incubate at room temperature for 30 minutes to allow the fusion process to complete. The resulting MPL complex adjuvant is then stored at 4 °C.

[0036] Compare with Example 1

[0037] A method for preparing an MPL complex adjuvant includes the following steps:

[0038] S1. Dissolve 68.9 mg of myristoyl phosphatidylglycerol and 295 mg of cholesterol in 20 mL of freshly distilled chloroform to obtain solution A; dissolve 610.1 mg of myristoyl phosphatidylcholine and 195.6 mg of monophosphatidyllipide A in 20 mL of chloroform-methanol solution to obtain solution B; the volume ratio of chloroform to methanol is 9:1; mix solutions A and B thoroughly.

[0039] S2 was rotary evaporated in a 45°C water bath to slowly evaporate the organic solvent until a uniform, translucent lipid film was formed on the bottle wall; the lipid film was then vacuum dried for 4 hours to completely remove any residual organic solvent.

[0040] S3. Add 100 mL of 10 mM pH 7.2 PBS buffer preheated to 40 °C to the dried lipid membrane; rotate to form a multi-layered liposome suspension of varying sizes; at this point, the solution is milky white.

[0041] S4 The liposome suspension was placed in an ice-water bath to prevent local overheating; the probe of the probe-type ultrasonic processor was inserted 2 cm below the surface of the suspension, and ultrasonic treatment was performed in pulse mode with a power of 200W, ultrasonic for 2 seconds, with a 4-second interval, for a total ultrasonic time of 10 minutes; after the treatment, the solution became translucent and formed a small monolayer liposome suspension with a particle size of less than 100 nm.

[0042] S5. Dissolve 88 mg of QS-21 in 10 mL of 10 mM pH 7.2 PBS buffer and gently vortex until completely dissolved to form a QS21 micelle solution. At room temperature, add 10 mL of the QS21 micelle solution dropwise to 100 mL of small monolayer liposome suspension. After mixing, the solution immediately changes from clear to milky white / turbid, which is a direct indicator of liposome fusion and GUVs / TIMs formation. Incubate at room temperature for 30 minutes to allow the fusion process to complete. The resulting MPL complex adjuvant is then stored at 4 °C.

[0043] Example 2

[0044] A method for preparing an MPL complex adjuvant includes the following steps:

[0045] S1. Dissolve 68.9 mg of myristoyl phosphatidylglycerol and 295 mg of cholesterol in 20 mL of freshly distilled chloroform to obtain solution A; dissolve 610.1 mg of myristoyl phosphatidylcholine, 195.6 mg of monophosphatidyllipid A, and 56.0 mg of DSPE-PEG-PSA in 20 mL of chloroform-methanol solution to obtain solution B; the volume ratio of chloroform to methanol is 9:1; mix solutions A and B thoroughly.

[0046] S2 was rotary evaporated in a 45°C water bath to slowly evaporate the organic solvent until a uniform, translucent lipid film was formed on the bottle wall; the lipid film was then vacuum dried for 4 hours to completely remove any residual organic solvent.

[0047] S3. Add 100 mL of 10 mM pH 7.2 PBS buffer preheated to 40 °C to the dried lipid membrane; rotate to form a multi-layered liposome suspension of varying sizes; at this point, the solution is milky white.

[0048] S4 The liposome suspension was placed in an ice-water bath to prevent local overheating; the probe of the probe-type ultrasonic processor was inserted 2 cm below the surface of the suspension, and ultrasonic treatment was performed in pulse mode with a power of 200W, ultrasonic for 2 seconds, with a 4-second interval, for a total ultrasonic time of 10 minutes; after the treatment, the solution became translucent and formed a small monolayer liposome suspension with a particle size of less than 100 nm.

[0049] S5. Dissolve 88 mg of QS-21 in 10 mL of 10 mM pH 7.2 PBS buffer and gently vortex until completely dissolved to form a QS21 micelle solution. At room temperature, add 10 mL of the QS21 micelle solution dropwise to 100 mL of small monolayer liposome suspension. After mixing, the solution immediately changes from clear to milky white / turbid, which is a direct indicator of liposome fusion and GUVs / TIMs formation. Incubate at room temperature for 30 minutes to allow the fusion process to complete. The resulting MPL complex adjuvant is then stored at 4 °C.

[0050] Example 3

[0051] A method for preparing an MPL complex adjuvant includes the following steps:

[0052] S1. Dissolve 68.9 mg of myristoyl phosphatidylglycerol and 295 mg of cholesterol in 20 mL of freshly distilled chloroform to obtain solution A; dissolve 610.1 mg of myristoyl phosphatidylcholine, 195.6 mg of monophosphatidyllipid A, and 56.0 mg of DSPE-PMPC in 20 mL of chloroform-methanol solution to obtain solution B; the volume ratio of chloroform to methanol is 9:1; mix solutions A and B thoroughly.

[0053] S2 was rotary evaporated in a 45°C water bath to slowly evaporate the organic solvent until a uniform, translucent lipid film was formed on the bottle wall; the lipid film was then vacuum dried for 4 hours to completely remove any residual organic solvent.

[0054] S3. Add 100 mL of 10 mM pH 7.2 PBS buffer preheated to 40 °C to the dried lipid membrane; rotate to form a multi-layered liposome suspension of varying sizes; at this point, the solution is milky white.

[0055] S4 The liposome suspension was placed in an ice-water bath to prevent local overheating; the probe of the probe-type ultrasonic processor was inserted 2 cm below the surface of the suspension, and ultrasonic treatment was performed in pulse mode with a power of 200W, ultrasonic for 2 seconds, with a 4-second interval, for a total ultrasonic time of 10 minutes; after the treatment, the solution became translucent and formed a small monolayer liposome suspension with a particle size of less than 100 nm.

[0056] S5. Dissolve 88 mg of QS-21 in 10 mL of 10 mM pH 7.2 PBS buffer and gently vortex until completely dissolved to form a QS21 micelle solution. At room temperature, add 10 mL of the QS21 micelle solution dropwise to 100 mL of small monolayer liposome suspension. After mixing, the solution immediately changes from clear to milky white / turbid, which is a direct indicator of liposome fusion and GUVs / TIMs formation. Incubate at room temperature for 30 minutes to allow the fusion process to complete. The resulting MPL complex adjuvant is then stored at 4 °C.

[0057] Example 4

[0058] A method for preparing an MPL complex adjuvant includes the following steps:

[0059] S1. Dissolve 68.9 mg of myristoyl phosphatidylglycerol and 295 mg of cholesterol in 20 mL of freshly distilled chloroform to obtain solution A; dissolve 610.1 mg of myristoyl phosphatidylcholine, 195.6 mg of monophosphatidyllipid A, and 56.0 mg of DSPE-Hydrazone-PEG in 20 mL of chloroform-methanol solution to obtain solution B; the volume ratio of chloroform to methanol is 9:1; mix solutions A and B thoroughly.

[0060] S2 was rotary evaporated in a 45°C water bath to slowly evaporate the organic solvent until a uniform, translucent lipid film was formed on the bottle wall; the lipid film was then vacuum dried for 4 hours to completely remove any residual organic solvent.

[0061] S3. Add 100 mL of 10 mM pH 7.2 PBS buffer preheated to 40 °C to the dried lipid membrane; rotate to form a multi-layered liposome suspension of varying sizes; at this point, the solution is milky white.

[0062] S4 The liposome suspension was placed in an ice-water bath to prevent local overheating; the probe of the probe-type ultrasonic processor was inserted 2 cm below the surface of the suspension, and ultrasonic treatment was performed in pulse mode with a power of 200W, ultrasonic for 2 seconds, with a 4-second interval, for a total ultrasonic time of 10 minutes; after the treatment, the solution became translucent and formed a small monolayer liposome suspension with a particle size of less than 100 nm.

[0063] S5. Dissolve 88 mg of QS-21 in 10 mL of 10 mM pH 7.2 PBS buffer and gently vortex until completely dissolved to form a QS21 micelle solution. At room temperature, add 10 mL of the QS21 micelle solution dropwise to 100 mL of small monolayer liposome suspension. After mixing, the solution immediately changes from clear to milky white / turbid, which is a direct indicator of liposome fusion and GUVs / TIMs formation. Incubate at room temperature for 30 minutes to allow the fusion process to complete. The resulting MPL complex adjuvant is then stored at 4 °C.

[0064] Example 5

[0065] A method for preparing an MPL complex adjuvant includes the following steps:

[0066] S1. Dissolve 68.9 mg of myristoyl phosphatidylglycerol and 295 mg of cholesterol in 20 mL of freshly distilled chloroform to obtain solution A; dissolve 610.1 mg of myristoyl phosphatidylcholine, 195.6 mg of monophosphatidyllipide A, and 56.0 mg of DSPE-PEG-Mannose in 20 mL of chloroform-methanol solution to obtain solution B; the volume ratio of chloroform to methanol is 9:1; mix solutions A and B thoroughly.

[0067] S2 was rotary evaporated in a 45°C water bath to slowly evaporate the organic solvent until a uniform, translucent lipid film was formed on the bottle wall; the lipid film was then vacuum dried for 4 hours to completely remove any residual organic solvent.

[0068] S3. Add 100 mL of 10 mM pH 7.2 PBS buffer preheated to 40 °C to the dried lipid membrane; rotate to form a multi-layered liposome suspension of varying sizes; at this point, the solution is milky white.

[0069] S4 The liposome suspension was placed in an ice-water bath to prevent local overheating; the probe of the probe-type ultrasonic processor was inserted 2 cm below the surface of the suspension, and ultrasonic treatment was performed in pulse mode with a power of 200W, ultrasonic for 2 seconds, with a 4-second interval, for a total ultrasonic time of 10 minutes; after the treatment, the solution became translucent and formed a small monolayer liposome suspension with a particle size of less than 100 nm.

[0070] S5. Dissolve 88 mg of QS-21 in 10 mL of 10 mM pH 7.2 PBS buffer and gently vortex until completely dissolved to form a QS21 micelle solution. At room temperature, add 10 mL of the QS21 micelle solution dropwise to 100 mL of small monolayer liposome suspension. After mixing, the solution immediately changes from clear to milky white / turbid, which is a direct indicator of liposome fusion and GUVs / TIMs formation. Incubate at room temperature for 30 minutes to allow the fusion process to complete. The resulting MPL complex adjuvant is then stored at 4 °C.

[0071] Example 6

[0072] A method for preparing an MPL complex adjuvant includes the following steps:

[0073] S1. Dissolve 68.9 mg of myristoyl phosphatidylglycerol and 295 mg of cholesterol in 20 mL of freshly distilled chloroform to obtain solution A; dissolve 610.1 mg of myristoyl phosphatidylcholine, 195.6 mg of monophosphatidyllipid A, 28.0 mg of DSPE-PEG-Mannose, and 28.0 mg of DSPE-Hydrazone-PEG in 20 mL of chloroform-methanol solution to obtain solution B; the volume ratio of chloroform to methanol is 9:1; mix solutions A and B thoroughly.

[0074] S2 was rotary evaporated in a 45°C water bath to slowly evaporate the organic solvent until a uniform, translucent lipid film was formed on the bottle wall; the lipid film was then vacuum dried for 4 hours to completely remove any residual organic solvent.

[0075] S3. Add 100 mL of 10 mM pH 7.2 PBS buffer preheated to 40 °C to the dried lipid membrane; rotate to form a multi-layered liposome suspension of varying sizes; at this point, the solution is milky white.

[0076] S4 The liposome suspension was placed in an ice-water bath to prevent local overheating; the probe of the probe-type ultrasonic processor was inserted 2 cm below the surface of the suspension, and ultrasonic treatment was performed in pulse mode with a power of 200W, ultrasonic for 2 seconds, with a 4-second interval, for a total ultrasonic time of 10 minutes; after the treatment, the solution became translucent and formed a small monolayer liposome suspension with a particle size of less than 100 nm.

[0077] S5. Dissolve 88 mg of QS-21 in 10 mL of 10 mM pH 7.2 PBS buffer and gently vortex until completely dissolved to form a QS21 micelle solution. At room temperature, add 10 mL of the QS21 micelle solution dropwise to 100 mL of small monolayer liposome suspension. After mixing, the solution immediately changes from clear to milky white / turbid, which is a direct indicator of liposome fusion and GUVs / TIMs formation. Incubate at room temperature for 30 minutes to allow the fusion process to complete. The resulting MPL complex adjuvant is then stored at 4 °C.

[0078] Example 7

[0079] A method for preparing an MPL complex adjuvant includes the following steps:

[0080] S1. Dissolve 68.9 mg of myristoyl phosphatidylglycerol and 295 mg of cholesterol in 20 mL of freshly distilled chloroform to obtain solution A; dissolve 610.1 mg of myristoyl phosphatidylcholine, 195.6 mg of monophosphatidyllipid A, 28.0 mg of DSPE-PMPC, and 28.0 mg of DSPE-PEG2000 in 20 mL of chloroform-methanol solution to obtain solution B; the volume ratio of chloroform to methanol is 9:1; mix solutions A and B thoroughly.

[0081] S2 was rotary evaporated in a 45°C water bath to slowly evaporate the organic solvent until a uniform, translucent lipid film was formed on the bottle wall; the lipid film was then vacuum dried for 4 hours to completely remove any residual organic solvent.

[0082] S3. Add 100 mL of 10 mM pH 7.2 PBS buffer preheated to 40 °C to the dried lipid membrane; rotate to form a multi-layered liposome suspension of varying sizes; at this point, the solution is milky white.

[0083] S4 The liposome suspension was placed in an ice-water bath to prevent local overheating; the probe of the probe-type ultrasonic processor was inserted 2 cm below the surface of the suspension, and ultrasonic treatment was performed in pulse mode with a power of 200W, ultrasonic for 2 seconds, with a 4-second interval, for a total ultrasonic time of 10 minutes; after the treatment, the solution became translucent and formed a small monolayer liposome suspension with a particle size of less than 100 nm.

[0084] S5. Dissolve 88 mg of QS-21 in 10 mL of 10 mM pH 7.2 PBS buffer and gently vortex until completely dissolved to form a QS21 micelle solution. At room temperature, add 10 mL of the QS21 micelle solution dropwise to 100 mL of small monolayer liposome suspension. After mixing, the solution immediately changes from clear to milky white / turbid, which is a direct indicator of liposome fusion and GUVs / TIMs formation. Incubate at room temperature for 30 minutes to allow the fusion process to complete. The resulting MPL complex adjuvant is then stored at 4 °C.

[0085] Test Example 1

[0086] Particle size and zeta potential analysis: Dynamic light scattering (DLS) instrument (Malvern Zetasizer NanoZS) was used according to ISO 22412:2025 Particle size analysis – Dynamic light scattering (DLS) standard. After dilution with PBS, the mean particle size, polydispersity index (PDI), and zeta potential were measured at 25°C.

[0087] In vitro stability test: The sample was incubated with 50% serum (derived from fetal bovine serum, Sigma-Aldrich) at 37°C, and particle size changes were monitored by DLS at 0, 24, and 48 hours. Stability is expressed as the percentage of particle size change (%).

[0088] Verification of Immunization Efficacy: In animal immunization experiments, the adjuvant was mixed with a model antigen (such as OVA) and subcutaneously injected into mice. Serum was collected 14 days later, and IgG antibody titers (expressed as OD450 values) were detected using ELISA. Details are as follows:

[0089] 1) Laboratory animals

[0090] SPF-grade female BALB / c mice, aged 6-8 weeks and weighing 18-22g, were selected from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed under suitable temperature and humidity conditions and a 12-hour light / dark cycle, with free access to food and water. They were acclimatized for 7 days prior to the experiment in the animal facility. All animal experimental procedures complied with the requirements of the institution's animal experiment ethics committee.

[0091] 2) Preparation of immunomodulators

[0092] The MPL complex adjuvant prepared in Examples 1-7 and Control Example 1 was used as the adjuvant, and ovalbumin (OVA, Sigma-Aldrich, purity ≥98%) was used as the model antigen. OVA was prepared into a stock solution of 1 mg / mL using 10 mM PBS buffer at pH 7.2.

[0093] Mix the ovalbumin stock solution with each group's MPL adjuvant at a volume ratio of 1:1, gently vortex to mix, and let stand at room temperature for 30 minutes to obtain the immunomodulator. Each 100 μL of immunomodulator contains 10 μg of ovalbumin. The amounts of MPLA and QS-21 contained correspond to the MPL adjuvant formulation, ensuring that mice in each group receive the same dose of antigen and immunostimulant, with only the adjuvant formulation differing.

[0094] 3) Animal grouping and immunization program

[0095] Mice were randomly divided into 8 groups (n=6 per group), corresponding to the MPL adjuvant in Examples 1-7 and Control Example 1, respectively. Mice in each group were weighed on the day of immunization and randomly assigned according to body weight to avoid bias caused by body weight differences.

[0096] The immunization route was subcutaneous (sc). Each mouse received a subcutaneous injection of 100 μL of the corresponding group's immunizing agent in the nape of the neck. Unless otherwise stated, this test case was a single immunization regimen, i.e., immunization was performed once on day 0. The mice's general condition (activity, mental state, coat color, and appetite) was observed daily after immunization, and local redness and swelling were recorded.

[0097] 4) Serum collection

[0098] Blood samples were collected from mice on day 14 post-immunization (D14). Mice were fasted for 4 hours prior to collection. Approximately 0.8-1.0 mL of blood was collected from the retroorbital venous plexus and placed in an anticoagulant-free centrifuge tube. After clotting at room temperature for 30 minutes, the blood was centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was collected as serum. The serum was aliquoted into 0.5 mL EP tubes and stored at -20°C for later use, avoiding repeated freeze-thaw cycles.

[0099] 5) ELISA antibody detection

[0100] The level of anti-OVA IgG antibodies in mouse serum was detected using an indirect ELISA method. The specific steps are as follows:

[0101] Coating antigen:

[0102] Dissolve OVA in carbonate coating buffer (0.05 mol / L, pH 9.6) to prepare an antigen solution of 2 μg / mL. Add 100 μL to each well of a 96-well high-binding ELISA plate and coat overnight at 4°C.

[0103] Plate washing and sealing:

[0104] Discard the coating solution, add 300 μL of PBST (PBS containing 0.05% Tween-20) to each well for washing, let stand for 3 min, then discard the solution, blot dry, and repeat 3 times. Next, add 200 μL of blocking buffer (5% skim milk powder dissolved in PBST) to each well, incubate at 37°C for 1 h, and wash the plate 3 times with PBST as described above after incubation.

[0105] Serum incubation:

[0106] The mouse serum to be tested was serially diluted with PBST containing 1% BSA, starting at a 1:100 ratio and proceeding in a 2-fold gradient (e.g., 1:100-1:102400). 100 μL of the diluted serum was added to each well, and the plate was incubated at 37°C for 1 h. After incubation, the plate was washed 5 times with PBST.

[0107] Secondary antibody incubation:

[0108] HRP-labeled goat anti-mouse IgG (H+L) (Jackson ImmunoResearch) was diluted to 1:5000 with PBST containing 1% BSA. 100 μL was added to each well and incubated at 37°C for 1 h. After incubation, the plate was washed 5 times with PBST.

[0109] Color development and termination:

[0110] Add 100 μL of TMB chromogenic substrate solution to each well and incubate at room temperature in the dark for 5-10 min. When a moderate blue color appears in the control well, add 50 μL of 2 mol / L H2SO4 to each well to terminate the reaction. The color changes from blue to yellow.

[0111] Readings and results representation:

[0112] The optical density (OD450) of each well was read using a microplate reader at a wavelength of 450 nm. Negative controls were serum from unimmunized mice or PBS.

[0113] Antibody titers can be calculated based on serum dilution curves: a positive criterion is defined as an OD450 value ≥ twice the mean value of blank wells, and the reciprocal of the highest serum dilution factor that meets this criterion is taken as the antibody titer.

[0114] In this embodiment, to facilitate comparison of differences between different groups, the OD value of a fixed dilution was used to evaluate the antibody response intensity. That is, the serum of each group was uniformly diluted at 1:1000 and the OD450 was measured. The "antibody titer (OD450)" listed in Table 1 is the mean under this condition (n=6, the average value of 3 parallel wells for each mouse was taken and then the mean within the group was calculated).

[0115] Table 1

[0116]

[0117] As can be seen from the table above, Examples 1-5 are significantly superior to Control Example 1. The possible reason is that PEGylation and a reasonable lipid ratio result in sub-100 nm, low PDI, and more negative Zeta, which are generally considered to be beneficial for reducing aggregation, improving structural stability in the serum environment and lymph node homing efficiency, thereby obtaining a higher humoral immune response; the control, due to the lack of surface modification, has a large particle size, wide distribution, poor stability, and lower immune activity.

[0118] Of Examples 1-5, Example 4 showed the best results. This is likely because the DSPE-Hydrazone-PEG used in Example 4 has a more compatible hydrophobic chain and head group arrangement with the main phase phospholipids, forming a denser and more uniform PEG layer on the liposome surface. This results in lower bilayer interfacial tension and more stable particles, leading to a smaller PDI and a lower particle size variation rate at the same main phase / cholesterol ratio, thus resulting in a higher antibody response.

[0119] Under the same main phase, MPL, and QS-21 ratio, Example 6, through the combination of DSPE-PEG-Mannose and DSPE-Hydrazone-PE, resulted in particles exhibiting smaller and more uniform particle size, a more negative surface potential, and a lower particle size variation rate in neutral PBS and serum environments. During immunization, DSPE-PEG-Mannose can mediate dendritic cell receptor recognition and uptake, while DSPE-Hydrazone-PE deshields and promotes membrane fusion in weakly acidic endosomes. The synergistic effect of these two mechanisms is presumably beneficial for improving the effective delivery and presentation efficiency of antigens, thereby obtaining higher antibody titers.

[0120] 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 an MPL composite adjuvant, characterized in that, Includes the following steps, in parts by weight: S1. Dissolve 5-7 parts of anionic phospholipids and 20-30 parts of cholesterol in 2000-3500 parts of chloroform to obtain solution A; dissolve 45-55 parts of neutral phospholipids, 15-20 parts of monophospholipid A, and 4-6 parts of surface-modified lipids in 2000-3500 parts of chloroform-methanol solution to obtain solution B; mix solution A and solution B thoroughly. S2 evaporates organic solvents to form a lipid film; S3 is added to the lipid membrane in 8000-12000 parts of buffer solution, and hydrated to form a liposome suspension; S4 is used to sonicate the liposome suspension to form a small monolayer liposome suspension. S5 dissolves 6-10 parts of QS-21 in 800-1200 parts of buffer solution to form a micelle solution, and mixes the micelle solution with a small monolayer liposome suspension to form a composite adjuvant; The anionic phospholipid is dimyristoylphosphatidylglycerol; The neutral phospholipid is dimyristoylphosphatidylcholine; The surface-modified lipid is at least one of DSPE-PMPC, DSPE-PEG2000, DSPE-PEG-PSA, DSPE-Hydrazone-PEG, and DSPE-PEG-Mannose; The ultrasonic treatment in step S4 is carried out in an ice-water bath, using pulse mode, with a power of 150-250W, an ultrasonic time of 1-3 seconds, an interval of 3-5 seconds, and a total ultrasonic time of 5-15 minutes; the ultrasonic treatment uses a probe-type ultrasonic instrument, with the probe inserted 1-3 cm below the liquid surface.

2. The method for preparing the MPL composite adjuvant as described in claim 1, characterized in that: The volume ratio of chloroform to methanol in the chloroform-methanol solution is 9:

1.

3. The method for preparing the MPL composite adjuvant as described in claim 1, characterized in that: The buffer in steps S3 and S5 is 10mM pH7.2 PBS buffer.

4. An MPL composite adjuvant, characterized in that: It is prepared by the method for preparing the MPL complex adjuvant according to any one of claims 1-3.

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