Adjuvant composition as well as preparation method and application thereof

Abstract

The invention provides an adjuvant composition as well as a preparation method and application thereof, the adjuvant composition comprises squalene, Tween 80 and a specific type of poloxamer, and the adjuvant composition is a novel oil-in-water nano-emulsion type adjuvant prepared by taking a biodegradable squalene substance as a matrix and matching with a nano-emulsion processing technology; the adjuvant composition has the characteristics of simplicity in preparation, good stability, quick immune effect, low side reaction and the like, and can be used for preparing an antigen adjuvant with a specific immunological function.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to an adjuvant composition and a preparation method and application thereof. Background Art

[0002] Modern vaccines are increasingly inseparable from the role of adjuvants, especially antigens characterized by genetic engineering products must be supplemented with adjuvants to achieve good immune effects. It can be said that vaccines are a general term for antigens + adjuvants. What is an adjuvant? The earliest English name of adjuvant, Adjuvant, comes from the Latin word "adjuvare", which means "help", so immune adjuvants refer to preparations that are injected simultaneously with antigens or in advance, which can enhance the body's immune response to antigens, prolong the vaccine protection time, reduce the dosage of antigens, or induce specific immune response types. The main functions of adjuvants in vaccine applications are: enhancing the immunogenicity of antigens, turning antigenic substances with no or poor immunogenicity into effective immunogens; enhancing the body's responsiveness to antigen stimulation, increasing the titer of antibodies produced by immune responses, reducing the amount of antigens used, and prolonging the memory of immune responses; changing the type of antibodies, converting the production of IgM to the production of IgG; inducing immune responses of specific immune types, such as aluminum adjuvants can induce Th2 immune responses.

[0003] With the in-depth understanding of vaccines and the human immune system, researchers have fully realized that vaccine development is often inseparable from the study of adjuvants. Not only is adjuvant the most direct and easy way to improve vaccine efficacy and quickly stimulate the host's immune response, but it is also an important strategy to change the type of immune response and avoid pathological immune damage. In the past few decades, adjuvant research and application have been upgraded from the empirical trial and error model to the height of theoretical design. These early studies have provided important scientific data and theoretical basis for future vaccine development, especially for the development of emergency vaccines. The pathways of adjuvant activation of immune response can be generally summarized as intracellular and extracellular receptors such as TLR, CLR, NLR, RLR and signal receptors such as NLRP3, STING, MARVs and RANKs, as well as the downstream signal pathways and amplification loops connected to these substances. Due to the existence of these material bases, the design of modern vaccine adjuvants is inseparable from the use of ligand substances for these receptors and receptors. Despite these directional guidances in modern adjuvant research, the adjuvants and combinations under research often fail to achieve the desired results, and sometimes the side effects of the new adjuvants have the opposite effect on the vaccine effect. For example, the combination of Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA) has been used to produce antiserum in animals for more than half a century and is currently recognized as a relatively effective adjuvant in the laboratory. However, its toxicity and side effects, coupled with the growing concern for the welfare of experimental animals, have put increasing pressure on banning or restricting the use of CFA / IFA, which is also the main reason for the uncertainty in current adjuvant research and application.

[0004] The development of a new adjuvant often requires a very long research and development process, which not only requires an in-depth understanding of the new adjuvant components, but also requires a more in-depth and systematic analysis and research on its physical and chemical properties, stability, immunogenicity, toxic and side effects in combination with antigen molecules. At the same time, it also requires personnel with different professional knowledge backgrounds to participate in the research and development, and provide a more comprehensive and systematic study of the new adjuvant, so that it is possible to develop an innovative adjuvant for clinical application of vaccines. In view of this, the present invention is specially proposed. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide an adjuvant composition with simple preparation, good stability, rapid immune effect, low side effects and wide application range, as well as a preparation method and application thereof.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an adjuvant composition, comprising squalene, Tween 80 and poloxamer; the poloxamer is selected from poloxamer 401, poloxamer 407 or poloxamer 188.

[0008] In an optional embodiment, the squalene accounts for 0.2%-4% of the total volume of the adjuvant composition, Tween 80 accounts for 0.02%-0.4% of the total volume of the adjuvant composition, and poloxamer accounts for 0.1%-2% of the total volume of the adjuvant composition.

[0009] Preferably, the squalene accounts for 0.8%-4% of the total volume of the adjuvant composition, Tween 80 accounts for 0.064%-0.32% of the total volume of the adjuvant composition, and poloxamer accounts for 0.2%-1% of the total volume of the adjuvant composition.

[0010] In an optional embodiment, the adjuvant composition further comprises one or more of monophosphatidyl A, oligonucleotide CpG, R848, PolyI:C, dioctadecyl dimethyl ammonium bromide and 2'3'cGAMP.

[0011] In an optional embodiment, the amount of monophosphatidyl A is 10 μg / mL-100 μg / mL, the amount of oligonucleotide CpG is 200 μg / mL-6000 μg / mL, the amount of R848 is 100 μg / mL-1000 μg / mL, the amount of PolyI:C is 200 μg / mL-2000 μg / mL, the amount of dioctadecyl dimethyl ammonium bromide is 100 μg / mL-1000 μg / mL, and the amount of 2'3'cGAMP is 200 μg / mL-2000 μg / mL;

[0012] The dosage of the adjuvant composition for mice was verified by experiments, and based on the dosage for mice combined with the principle of equivalent body surface area, it was converted into the dosage for future clinical use in humans (conversion according to FDA draft guidelines), that is, the content of oligonucleotide CpG1018 is 200-6000μg / ml (w / v). The body surface area of ​​humans is about 1.67m^2, and that of mice is 0.007m^2; the former is about 230 times the latter.

[0013] Preferably, the dosage of monophosphatidyl A is 10 μg / mL-40 μg / mL, the dosage of oligonucleotide CpG is 400 μg / mL-4000 μg / mL, the dosage of R848 is 100 μg / mL-500 μg / mL, the dosage of PolyI:C is 200 μg / mL-1000 μg / mL, the dosage of dioctadecyl dimethyl ammonium bromide is 100 μg / mL-500 μg / mL, and the dosage of 2'3'cGAMP is 500 μg / mL-1200 μg / mL.

[0014] In a second aspect, the present invention provides a method for preparing the adjuvant composition according to the above technical solution, the preparation method comprising the following steps:

[0015] Among them, for poloxamer, it is necessary to select, according to the characteristics of different models, to mix with squalene to prepare the oil phase in S1 (for example, for poloxamer in the form of a solvent, it is suitable to be added to the prepared oil phase), or to mix with Tween 80 and a buffer to prepare the water phase in S2 (for example, for poloxamer in powder form, it is suitable to be added to the prepared water phase);

[0016] S1: preparing an oil phase, preparing squalene into an oil phase;

[0017] S2: preparing the aqueous phase, mixing Tween 80 with the buffer to prepare the aqueous phase;

[0018] S3: slowly adding the aqueous phase prepared in S2 to the oil phase prepared in S1, and adjusting the volume with a buffer solution, stirring at high speed at room temperature to obtain a colostrum solution;

[0019] S4: the colostrum solution is poured into a microfluidizer to prepare a refined milk solution;

[0020] S5: sterilizing and filtering the refined emulsion solution obtained in S4 to obtain the adjuvant composition;

[0021] The squalene accounts for 0.2%-4% of the total volume of the adjuvant composition, Tween 80 accounts for 0.02%-0.4% of the total volume of the adjuvant composition, and poloxamer accounts for 0.1%-2% of the total volume of the adjuvant composition;

[0022] Preferably, the squalene accounts for 0.8%-4% of the total volume of the adjuvant composition, Tween 80 accounts for 0.064%-0.32% of the total volume of the adjuvant composition, and poloxamer accounts for 0.2%-2% of the total volume of the adjuvant composition.

[0023] Preferably,

[0024] In step S1, the oil phase is prepared by stirring at 18-45° C. for 0-20 minutes;

[0025] Preferably, in step S2, stirring is performed at 18-55° C. for 5-30 minutes to prepare an aqueous phase;

[0026] Preferably, in step S3, a high-speed stirrer is used to stir at a speed of 8000-20000 rpm for 20-40 minutes to prepare a colostrum solution;

[0027] Preferably, in step S4, the colostrum solution prepared in S3 is poured into a microfluidizer, the pressure is adjusted to 15000-20000 psi, the temperature of the refrigerator is adjusted, the outlet temperature of the emulsion is controlled to 20-30° C., and the high-pressure homogenization is cycled 2-10 times to obtain a refined emulsion solution;

[0028] Preferably, in step S5, the refined milk solution obtained in S4 is sterilized and filtered using a 0.22 μm sterilizing filter;

[0029] Preferably, the buffer is selected from but not limited to citrate or phosphate buffer.

[0030] In an optional embodiment, the squalene accounts for 0.2%-4% of the total volume of the adjuvant composition, Tween 80 accounts for 0.02%-0.4% of the total volume of the adjuvant composition, and poloxamer accounts for 0.1%-2% of the total volume of the adjuvant composition.

[0031] Preferably, the squalene accounts for 0.8%-4% of the total volume of the adjuvant composition, Tween 80 accounts for 0.064%-0.32% of the total volume of the adjuvant composition, and poloxamer accounts for 0.2%-1% of the total volume of the adjuvant composition.

[0032] In an optional embodiment, the method for preparing the adjuvant composition further comprises mixing the adjuvant composition prepared in S5 of the above method for preparing the adjuvant composition with one or more of monophosphatidyl A, oligonucleotide CpG, R848, PolyI:C, dioctadecyl dimethyl ammonium bromide and 2'3'cGAMP.

[0033] In an optional embodiment, the amount of monophosphatidyl A is 10 μg / mL-100 μg / mL, the amount of oligonucleotide CpG is 200 μg / mL-3000 μg / mL, the amount of R848 is 100 μg / mL-1000 μg / mL, the amount of PolyI:C is 200 μg / mL-2000 μg / mL, the amount of dioctadecyl dimethyl ammonium bromide is 100 μg / mL-1000 μg / mL, and the amount of 2'3'cGAMP is 200 μg / mL-2000 μg / mL;

[0034] Preferably, the dosage of monophosphatidyl A is 10 μg / mL-40 μg / mL, the dosage of oligonucleotide CpG is 400 μg / mL-2000 μg / mL, the dosage of R848 is 100 μg / mL-500 μg / mL, the dosage of PolyI:C is 200 μg / mL-1000 μg / mL, the dosage of dioctadecyl dimethyl ammonium bromide is 100 μg / mL-500 μg / mL, and the dosage of 2'3'cGAMP is 500 μg / mL-1200 μg / mL.

[0035] Preferably, the particle size of the adjuvant composition described in the first aspect or the adjuvant composition prepared in the second aspect is 100 nm-200 nm, and the pH value is 6-7.5, for example, 6.3-6.7 or 7.0-7.4.

[0036] In a third aspect, the present invention provides an antigen adjuvant composition, which comprises the adjuvant composition described in the first aspect or the adjuvant composition prepared in the second aspect, and an antigen capable of inducing an immune response; the antigen comprises one or more of proteins, polypeptides or nucleic acids.

[0037] Preferably, the antigen includes but is not limited to an antigen selected from varicella-zoster virus (VZV), respiratory syncytial virus (RSV), severe acute respiratory syndrome coronavirus type 1 (SARS-CoV-1), severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), human papillomavirus (HPV), hepatitis B virus (HBV), influenza virus, rotavirus (RV), and pneumococcal polysaccharide vaccine (PCV); further preferably, the varicella-zoster virus (VZV) antigen is gE protein.

[0038] In a fourth aspect, the present invention further provides the use of the adjuvant composition described in the first aspect, the method for preparing the adjuvant composition described in the second aspect, or the antigen adjuvant composition described in the third aspect in preparing a vaccine adjuvant.

[0039] For example, the adjuvant composition of the present invention can also be used as a mother adjuvant by adding components with immunomodulatory functions, agonist molecules such as TLR1, 2, 3, 4, 5, 7, 8, or 9, to expand the function and use of the adjuvant composition, thereby preparing an antigen adjuvant with specific immunological function.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The invention provides an adjuvant composition and a preparation method and application thereof. The adjuvant composition comprises squalene, Tween 80 and poloxamer. The adjuvant composition is a new adjuvant of water-in-oil nanoemulsion type prepared by using a biodegradable squalene substance as a matrix and in cooperation with a nanoemulsification process. The adjuvant composition can be further used together with components having an immunomodulatory function, such as monophosphatidyl A, oligonucleotide CpG, R848, PolyI:C, dioctadecyl dimethyl ammonium bromide or 2'3'cGAMP, so as to prepare various types of antigen adjuvants with specific immunological functions. The adjuvant composition can not only enhance the reactivity of the body to antigen stimulation, improve the titer of antibodies produced by immune response, reduce the amount of antigen used, prolong the memory of immune response, and realize the efficient enhancement of the immune response level of the antigen, but more importantly, it has extremely low toxic and side effects, and has the characteristics of simple preparation, good stability, wide application range, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The antibody titer results against OVA antigen after mice were immunized with the AD20 series adjuvants provided in Example 2;

[0043] Figure 2 The antibody titer results against the new coronavirus S1 antigen after mice were immunized with the AD20G adjuvant provided in Example 3;

[0044] Figure 3 The antibody titer results against the new coronavirus S1 antigen after the AD20G adjuvant immunization experimental rabbits provided in Example 3;

[0045] Figure 4 The effects of the AD20 series adjuvants provided in Example 4 on the antibody titer results of mouse anti-OVA antigen;

[0046] Figure 5 The effects of the AD20 series adjuvants provided in Example 4 on the secretion of anti-OVA antigen-specific IFN-γ in mice;

[0047] Figure 6 The effects of the AD20 series adjuvants provided in Example 4 on the results of anti-OVA antigen-specific CD4 IFN-γT cell subsets in mice;

[0048] Figure 7The antibody titer results against OVA antigen in the serum of immunized mice at different time points provided in Example 5;

[0049] Figure 8 The antibody titer results against OVA antigen in the serum of immunized mice at different time points provided in Example 6;

[0050] Fig. 9 The antibody titer results against influenza antigens in the sera of immunized mice at different time points provided in Example 7;

[0051] Fig.10 The antibody titer results of anti-HBsAg antigen in the serum of immunized mice at different time points provided in Example 7;

[0052] Fig.11 The antibody titer results against RSV Pre F5 antigen in the sera of immunized mice at different time points provided in Example 7. DETAILED DESCRIPTION

[0053] The technical solution of the present invention will be clearly described below in conjunction with the accompanying drawings. Obviously, the described embodiments are not all embodiments of the present invention, and all other embodiments obtained by ordinary technicians in the field without making creative work are within the protection scope of the present invention.

[0054] Example 1. Preparation of adjuvant composition

[0055] The composition of each adjuvant composition is shown in Table 1, and the amount of each component is shown in Table 2.

[0056] Table 1. AD20 series adjuvant ingredients

[0057]

[0058]

[0059] Table 2. Amount of adjuvant components

[0060]

[0061] Note: When adjuvant and antigen are used together, the volume ratio is 1:1, that is, 50 μl of adjuvant is mixed with 50 μl of antigen, and the final volume of 100 μl of adjuvant-antigen mixture is injected into experimental animals.

[0062] Preparation process of each series of adjuvants:

[0063] 1. Preparation of AD20:

[0064] According to the characteristics of different types of poloxamer, it is selected to be mixed with squalene to prepare an oil phase, or mixed with Tween 80 and buffer to prepare an aqueous phase;

[0065] 1.1. Preparation of AD20 adjuvant composition containing poloxamer 401:

[0066] (1) Place all materials in a biosafety cabinet and wait until the temperature of the 1x PBS buffer returns to room temperature before starting the operation;

[0067] (2) Use a pipette to draw 8 mL (optional range: 2 mL-40 mL) of squalene into a 1000 mL beaker;

[0068] (3) Replace the pipette tip and pipette 4 mL (optional range: 1 mL-20 mL) of Poloxamer 401 into the beaker containing squalene in (2); heat and stir with a magnetic stirrer at 40° C. for 15 minutes to prepare an oil phase;

[0069] (4) Measure 500 mL of 1x PBS buffer and add it to another 1000 mL empty beaker. Use a pipette to draw 0.64 mL of Tween 80 (optional range: 0.2 mL-4 mL) and add it to the beaker. Heat at 50° C. with magnetic stirring for 20 minutes to prepare the aqueous phase.

[0070] (5) slowly adding the aqueous phase prepared in (4) to the oil phase prepared in (3), and diluting the volume to 1000 mL with 1x PBS solution, stirring at room temperature with a high-speed stirrer at a speed of 8000-20000 rpm for 30 minutes to prepare a colostrum solution;

[0071] (6) Turn on the microfluidizer. Before formal use, first clean the volumetric cup and pipeline with 800 mL of 20% alcohol, then clean the volumetric cup and pipeline with 1 L of ultrapure water, and finally rinse with 500 mL of sterilized 1xPBS buffer. After cleaning, pour the colostrum solution prepared in (5) into the microfluidizer, adjust the pressure to 15,000 to 20,000 psi, adjust the refrigerator temperature, control the emulsion outlet temperature at 20 to 30° C., and cycle the high-pressure homogenization for 3 times to obtain a refined milk solution.

[0072] (7) sampling and observing the properties of the refined milk solution, which is milky white, and the particle size of the refined milk solution is measured by a Malvern particle size detector to be between 100 nm and 200 nm, and the pH value of the refined milk solution is detected to be between 7.0 and 7.4;

[0073] (8) The concentrated milk solution was sterilized and filtered in a biological safety cabinet using a 0.22 μm sterile filter. The concentrated milk solution (named AD20) was labeled and stored in a refrigerator at 2 to 8°C for future use.

[0074] 1.2. Preparation of AD20 adjuvant composition containing poloxamer 407:

[0075] (1) Place all materials in a biosafety cabinet and wait until the phosphate buffer temperature returns to room temperature before starting the operation;

[0076] (2) using a pipette to draw 8 mL of squalene into a 1000 mL beaker, heating at 40° C. with magnetic stirring to prepare an oil phase;

[0077] (3) Measure 500 mL of phosphate buffer and add it to another 1000 mL empty beaker, weigh 4 g of poloxamer 407 into the beaker, and use a pipette to take 0.64 mL of Tween 80 and add it to the beaker again; heat at 50° C. with magnetic stirring for 20 minutes to prepare an aqueous phase;

[0078] (4) slowly adding the aqueous phase prepared in (3) to the oil phase prepared in (2), and diluting the volume to 1000 mL with a phosphate solution, stirring at room temperature with a high-speed stirrer at a speed of 18000-20000 rpm for 30 min to obtain a colostrum solution;

[0079] (5) Turn on the microfluidizer. Before formal use, first use 800 mL of 20% alcohol to clean the volumetric cup and pipeline, then use 1 L of ultrapure water to clean the volumetric cup and pipeline, and finally rinse with 500 mL of sterilized phosphate buffer. After cleaning, pour the colostrum solution prepared in (4) into the microfluidizer, adjust the pressure to 15,000-20,000 psi, adjust the temperature of the refrigerator, control the outlet temperature of the emulsion at 20-30° C., and cycle the high-pressure homogenization for 3 times to obtain a refined milk solution (named AD20);

[0080] (6) sampling and observing the properties of the refined milk solution, which is milky white, and the particle size of the refined milk solution is measured by a Malvern particle size detector to be between 100 nm and 200 nm, and the pH value of the refined milk solution is detected to be between 7.0 and 7.4;

[0081] (7) Aseptically filter the concentrated milk solution in a biological safety cabinet using a 0.22 μm sterilizing filter, label the concentrated milk solution, and store it in a refrigerator at 2 to 8°C for later use.

[0082] 1.3. Preparation of each group of AD20 adjuvant compositions containing poloxamer 188: AD20 (TB3-1), AD20 (TB3-2), AD20 (TB4-1) and AD20 (TB4-high concentration):

[0083] Among them, for the preparation method of AD20 (TB3-1):

[0084] (1) Place all materials in a biosafety cabinet and wait until the phosphate buffer temperature returns to room temperature before starting the operation;

[0085] (2) using a pipette to draw 8 mL of squalene into a 1000 mL beaker, heating at 40° C. with magnetic stirring to prepare an oil phase;

[0086] (3) Measure 500 mL of phosphate buffer and add it to another 1000 mL empty beaker, weigh 2 g of poloxamer 188 into the beaker, and use a pipette to draw 0.64 mL of Tween 80 into the beaker again; heat at 50° C. with magnetic stirring for 20 minutes to prepare an aqueous phase;

[0087] (4) slowly adding the aqueous phase prepared in (3) to the oil phase prepared in (2), and diluting the volume to 1000 mL with a phosphate solution, stirring at room temperature with a high-speed stirrer at a speed of 18000-20000 rpm for 30 min to obtain a colostrum solution;

[0088] (5) Turn on the microfluidizer. Before formal use, first clean the volumetric cup and pipeline with 800 mL of 20% alcohol, then clean the volumetric cup and pipeline with 1 L of ultrapure water, and finally rinse with 500 mL of sterilized phosphate buffer. After cleaning, pour the colostrum solution prepared in (4) into the microfluidizer, adjust the pressure to 15,000 to 20,000 psi, adjust the temperature of the refrigerator, control the outlet temperature of the emulsion at 20 to 30° C., and cycle the high-pressure homogenization for 3 times to obtain a refined milk solution.

[0089] (6) sampling and observing the properties of the refined milk solution, which is milky white, and the particle size of the refined milk solution is measured by a Malvern particle size detector to be between 100 nm and 200 nm, and the pH value of the refined milk solution is detected to be between 7.0 and 7.4;

[0090] (7) The concentrated milk solution was aseptically filtered in a biological safety cabinet using a 0.22 μm sterilizing filter. The concentrated milk solution (named AD20 (TB3-1), and the same for other groups) was labeled and stored in a refrigerator at 2 to 8°C for future use.

[0091] Similarly, for the AD20 adjuvant composition containing poloxamer 188: AD20 (TB3-2), AD20 (TB4-1) AD20 (TB4-high concentration); the preparation method is similar to that of the AD20 (TB3-1) group, only the amount of each component is different. The amount of each component in each group is shown in Table 3.

[0092] Table 3 Amounts of adjuvant components

[0093]

[0094] Note: When adjuvant and antigen are used together, the volume ratio is 1:1, that is, 50 μl of the prepared adjuvant stock solution is taken and mixed with 50 μl of antigen, and the final volume of 100 μl of adjuvant-antigen mixture is injected into experimental animals.

[0095] 2. Preparation of AD20G: Pipette 20 mg (optional range: 10 mg-100 mg) of monophosphatidyl A (MPL-A) into the above 1000 mL of refined emulsion AD20, vortex mix for at least 30 seconds to prepare AD20G, and store in a refrigerator at 2-8°C for later use.

[0096] 3. Preparation of AD20P: Pipette 1000 mg of CpG (optional range: 200 mg-3000 mg) into the above 1000 mL of the refined emulsion AD20, vortex mix for at least 30 seconds to prepare AD20P, and store in a refrigerator at 2-8°C for later use.

[0097] 4. Preparation of AD20T: Pipette 200 mg (optional range 100 mg-1000 mg) of resiquimod (R848) into the above 1000 mL of the refined emulsion solution AD20, vortex mix for at least 30 seconds to prepare AD20T, and store it in a refrigerator at 2-8°C for later use.

[0098] 5. Preparation of AD20S: Pipette 500 mg (optional range: 200 mg-2000 mg) of PolyI:C (polyinosinic acid-polycytosine nucleotide) into the above 1000 mL of the refined emulsion AD20, vortex mix for at least 30 seconds to prepare AD20S, and store it in a refrigerator at 2-8°C for later use.

[0099] 6. Preparation of AD20B: Pipette 200 mg (optional range: 100 mg-1000 mg) of dimethyl dioctyldecyl ammonium bromide into the above 1000 mL of the refined emulsion AD20, vortex mix for at least 30 seconds to prepare AD20B, and store it in a refrigerator at 2-8°C for later use.

[0100] 7. Preparation of AD20D: Pipette 1000 mg of CpG (optional range 200 mg-3000 mg) and 200 mg of resiquimod (R848) (optional range 100 mg-1000 mg) into the above 1000 mL of the refined emulsion solution AD20, vortex mix for at least 30 seconds to prepare AD20D, and store in a refrigerator at 2-8°C for later use.

[0101] 8. Preparation of AD20C: Pipette 1000 mg (optional range: 200 mg-2000 mg) of 2'3'cGAMP into the above 1000 mL of the refined emulsion AD20, vortex mix for at least 30 seconds to prepare AD20C, and store in a refrigerator at 2-8°C for later use.

[0102] Example 2: Immune effects of AD20 series adjuvants

[0103] In order to verify the effect of each adjuvant prepared in Example 1, ovalbumin (OVA) was used as a model antigen (Sigma, 9006-59-1) in the experiment, and its extremely low dose of 1 μg (1 microgram) / mouse was used. In general laboratories, mice immunized with OVA antigens use a dosage range of 10-100 μg / mouse, but this dose is not obvious for evaluating the effect of adjuvants. Therefore, this experiment uses an extremely low dose of 1 μg of antigen, which can more easily evaluate whether the adjuvant has a great immune enhancement effect on antigens with weak antigenicity. The poloxamer used in the AD20 adjuvant of this embodiment is poloxamer 401.

[0104] Immunization procedure: 6-8 week old female BALB / c mice (10 mice in each group) were treated with 1 μg OVA (very low dose antigen) and various adjuvants to prepare vaccines as required (50 μl adjuvant mixed with 50 μl antigen) and intramuscular immunization was performed. Immunization groups: (1) AD20G (1μg OVA + AD20G), (2) AD20B (1μg OVA + AD20B), (3) AD20P (1μg OVA + AD20P), (4) AD20T (1μg OVA + AD20T), (5) AD20S (1μg OVA + AD20S), (6) AD20D (1μg OVA + AD20D), (7) CFA / IFA (only CFA and 1μg OVA antigen for the first immunization, only IFA and 1μg OVA antigen for the second immunization), (8) Alum (1μg OVA + 100μg Alum), (9) PBS (100μl 1xPBS), (10) AD20 (1μg OVA + AD20); the total immunization volume was 100μl / mouse, each group was immunized twice with an interval of 14 days, and blood was collected before and after each immunization. The titer of OVA-specific IgG antibody was determined by ELISA using a 96-well microplate. CFA (Sigma, F5881) and IFA (Sigma, F5506) were mixed with the antigen in a ratio of 1:1 (50 μl: 50 μl) and then emulsified and injected into mice.

[0105] After immunizing animals with adjuvant combined with OVA antigen, the titer of antigen-specific antibodies in mouse serum was determined by ELISA. The ELISA detection method process: Nunc 96-well ELISA plates were coated with 2μg / mL OVA overnight at 4°C. The plates were washed 3 times and then blocked with PBS containing 5% bovine serum albumin (BSA) (containing 0.05% Tween 20, i.e., PBST buffer) at 37°C for 1 hour. Washed 3 times with 250μL PBST added to each well, and then three times of serial dilution of mouse serum was added to each well at a volume of 100μL per well (the initial dilution ratio of serum was 1:200) and incubated at 37°C for 1 hour. The plates were washed again 6 times, and then goat anti-mouse IgG-HRP with an original concentration of 2mg / mL and a dilution of 1:20000 was added at 37°C for 1 hour, washed 6 times, and then the bound antibodies were detected, the plates were developed using TMB substrate, and the reaction was terminated with 50μl / well 2M H2SO4. The samples were read at 450 nm and 620 nm using a microplate reader, and the data were processed and plotted using GraphPad Prism.

[0106] The experimental results are as follows Figure 1 As shown, the results of ELISA detection of anti-OVA antibody titers in mouse serum are shown, by comparing the antibody results 14, 21 and 28 days after immunization with Freund's adjuvant (CFA / IFA), aluminum adjuvant (Alum) and 7 groups of AD20 series adjuvants (AD20, AD20 G, AD20B, AD20 P, AD20T, AD20S, AD20D). As can be seen from the figure, 14 days after the primary immunization, no anti-OVA antibodies were detected in the CFA / IFA group; 21 and 28 days after immunization, the anti-OVA antibody titer in the Alum group was only about 1:10,000 (21 days after immunization), and it did not rise again on 28 days after immunization. The CFA / IFA immunization group only reached 1:10,000 on 21 days, and dropped to 1:2,400 on 28 days; for the AD20 series adjuvant groups, the AD20 adjuvant group reached about 1:20,000 on 21 days, and the antibody titer could reach about 1:45,000 on 28 days. The antibody titers of the other 6 groups (AD20G, AD20B, AD20 P, AD20T, AD20S, and AD20D) could reach about 1:10,0000 on 21 days, and AD20 G, AD20B, AD20 P, AD20T, AD20S, and AD20D on 28 days P. The antibody titer of the AD20T adjuvant group can reach above 1:20,0000, and the antibody titer of the AD20T adjuvant group can even reach about 1:35,0000. This group has the highest antibody titer and better immune effect.

[0107] The results of this example show that the AD20 series adjuvant group can efficiently enhance the antigen to achieve a high level of immune effect under low-dose immunization, which is significantly better than traditional adjuvants.

[0108] Example 3: Immune response effect of AD20G adjuvant against COVID-19 antigens

[0109] 3.1. Effect of AD20G adjuvant on immune response against COVID-19 antigen in mice

[0110] Immunization procedure: 6-8 week old female BALB / c mice (5 mice per group) were used to prepare vaccines with the new crown S1 antigen and various adjuvants according to their respective requirements, and intramuscular immunization was performed. Immunization grouping: (1) 1μg S1+AD20G, (2) 5μg S1+AD20G, (3) 1μg S1+Alum, (4) 5μg S1+Alum, (5) PBS, (6) 1μg S1+CFA / IFA, (7) 5μg S1+CFA / IFA; the immunization volume was 100μl / mouse, and each group was immunized twice with an interval of 14 days, and blood was collected before and after each immunization. ELISA was used to determine the titer of IgG antibodies specific to the new crown S1 antigen using a 96-well microplate. The ELISA method for determining serum antibodies was consistent with the method in Example 2, in which the coating antigen was the new crown S1 antigen and the coating concentration was 1μg / ml. The poloxamer used in the AD20G adjuvant in this example is poloxamer 401.

[0111] Results: The specific antibody titers against the new coronavirus S1 antigen in the sera of immunized mice are as follows Figure 2 As shown in the figure, it can be seen that for the 1μg S1 antigen immunization group, compared with the 1μg S1+Alum group and the 1μg S1+CFA / IFA group, the 1μg S1+AD20G group can detect specific antibodies to the S1 antigen 7 and 14 days after the primary immunization. At 21 days and 28 days, the antibody titer was significantly higher than that of the other two groups, much higher than the other two groups; for the 5μg S1 antigen immunization group, at 21 days and 28 days after immunization, the antibody level of the 5μg S1+AD20G group increased significantly, and the antibody titer was much higher than that of the 5μg S1+Alum group and the 5μgS1+CFA / IFA group.

[0112] 3.2. Effect of AD20G adjuvant on immune response to COVID-19 antigen in experimental rabbits

[0113] Immunization procedure: New Zealand white rabbits were respectively prepared with the new coronavirus S1 antigen and various adjuvants according to their respective requirements, and intramuscular immunization was performed. Immunization grouping: (1) 1μg S1+AD20G, (2) 1μg S1+CFA / IFA; the immunization volume was 100μl / rabbit, each group was immunized twice with an interval of 14 days, and blood was collected before and after each immunization. ELISA was performed using a 96-well microplate to determine the titer of IgG antibodies specific to the new coronavirus S1 antigen. The experimental process was as described above.

[0114] Results: The specific antibody titers against the new coronavirus S1 antigen in the immunization experiment rabbit serum are as follows Figure 3 As shown, specific antibodies to the S1 antigen could be detected in the AD20G group 7 and 14 days after primary immunization, while no anti-S1 antibodies were detected in the CFA / IFA group; on the 21st day after immunization, the antibody titer in the CFA / IFA group was around 1:50,000, and the antibody level in the AD20G group increased rapidly, reaching about 1:150,000, which was more than 3 times higher than that in the CFA / IFA group.

[0115] The results of this example further illustrate that the AD20G adjuvant can efficiently enhance the immune effect of antigens to reach a high level under low-dose immunization, and can not only enhance the immune effect of the model antigen OVA, but also enhance the immune effect of the new coronavirus antigen.

[0116] The above examples all show that the AD20 adjuvant of the present application can efficiently enhance the immune effect of antigens at extremely low doses, and when used simultaneously with monophosphatidyl A, CpG, R848, PolyI:C or dioctadecyl dimethyl ammonium bromide, the effect is better and the antibody level is higher.

[0117] Example 4: Immune response effect of AD20 adjuvant prepared with poloxamer 407

[0118] The poloxamer used in the AD20 series adjuvants of this example is poloxamer 407. Immunization procedure: 6-8 week old female BALB / c mice (5 mice in each group) were respectively treated with 1 μg OVA (very low dose antigen) and various adjuvants to prepare vaccines (50 μl adjuvant mixed with 50 μl antigen) as required, and then intramuscularly immunized. Immunization groups: (1) PBS, (2) AD20, (3) AD20P (CpG 50 μg / mouse), (4) AD20S (PolyI:C 50 μg / mouse), (5) AD20C (2'3'cGAMP 50 μg / mouse), (6) Alum 100 μg; total immunization volume 100 μl / mouse, each group was immunized twice, with an interval of 14 days, and blood was collected before and after each immunization. ELISA was performed using a 96-well microplate to determine the OVA-specific IgG antibody titer.

[0119] 4.1 Humoral Immune Response

[0120] The ELISA method for measuring serum antibodies was consistent with the method in Example 2, and the results were as follows Figure 4 As shown in the figure, the antibody titers of mice were detected 14 days, 21 days and 28 days after immunization. It can be seen from the results that the AD20 adjuvant configured with poloxamer 407 can still efficiently enhance the immune effect of OVA antigen at low doses. At the same time, the antibody titers of the AD20P adjuvant group, AD20S adjuvant group and AD20C adjuvant group were significantly increased compared with the AD20 adjuvant group, especially the antibody titer of the AD20S adjuvant group at 21 and 28 days after immunization can reach between 1:10,0000 and 1:20,0000.

[0121] 4.2 Cellular Immune Response

[0122] 1) ELISpot detection of INF-γ secretion

[0123] 14 days after the second immunization, the mice were euthanized in a sterile environment and the spleen was removed and ground into a cell suspension. The cells were harvested by centrifugation, resuspended in red blood cell lysis buffer, lysed and filtered to prepare a single cell suspension. According to the instructions of the Mouse IFN-γ Precoated ELISpot Kit (strips), the single cell suspension was diluted to 5×10 5 The cells were inoculated into the ELISpot pre-coated plate wells at a volume of 100 μL / well, and OVA protein with a final concentration of 10 μg / mL was added for stimulation, 100 μL each, and duplicate wells were set. Negative and positive control wells were set at the same time according to the instructions. The positive wells were stimulated with PMA (final concentration 0.1 μg / mL) and Iono (final concentration 1 μg / mL), and the number of cells was 5×10 4 The pre-coated plate with cells was placed in a cell culture incubator at 37°C and 5% CO2 for 18 to 22 hours. The experimental operation was carried out according to the instructions of the kit, and quantitative detection was performed by fluorescent enzyme-linked spot analyzer to obtain the number of cells per 5×10 per mouse. 5 The average number of spots per spleen T lymphocyte was calculated based on the number of spots per 1×10 6 The number of INF-γ spots produced by each spleen T lymphocyte was calculated, and the data were processed and plotted using GraphPadPrism.

[0124] Conclusion: If Figure 5 As shown, AD20P adjuvant group, AD20S adjuvant group and AD20C adjuvant group can effectively induce high levels of antigen-specific IFN-γ response to OVA protein antigen, especially the AD20C adjuvant group secreted the highest level of IFN-γ, which was better than the Alum adjuvant group.

[0125] 2) Flow cytometry to detect CD4 + INF-γ + T cell subsets

[0126] 14 days after the second immunization, the mice were euthanized in a sterile environment and the spleen was removed and ground into a cell suspension. The cells were harvested by centrifugation, resuspended in red blood cell lysis buffer, lysed and filtered to prepare a single cell suspension, and 1×10 ^ 6 cells were plated in a 96-well cell culture plate and stimulated with 10 μg / mL of OVA protein or PMA / Iono at 37°C and 5% CO2, and blocked with 1 μg / ml of Brefeldin A (BD, CA, USA) for 6 hours. Extracellular and intracellular cytokine staining was performed on spleen cells. Stimulated spleen cells were stained with FVD-eFluor780, then washed and stained with anti-mouse CD4 fluorescent antibody for 30 minutes at room temperature in the dark. Cells were permeabilized with fixation / permeabilization buffer and intracellular staining was performed with anti-mouse IFN-γ fluorescent antibody at 4°C for 45 minutes. Cells were washed twice and resuspended with 200 μL PBS, then collected using a flow cytometer (ThermoFisher, MA, USA), and finally analyzed using FlowJo software (BD, CA, USA).

[0127] The results are as follows Figure 6 As shown, for OVA protein antigen, the AD20 adjuvant group, AD20P adjuvant group, AD20S adjuvant group and AD20C adjuvant group were all able to significantly induce the production of antigen-specific CD4 IFNγT cell subsets, and the AD20S adjuvant group had the highest proportion of CD4 IFNγT cell subsets induced and the best effect.

[0128] The results of this example show that the AD20 adjuvant configured with poloxamer 407 can still efficiently enhance the immune effect of OVA antigen at extremely low doses, not only enhance the production of antibodies, but also promote the level of cellular immunity, and when used simultaneously with CpG, PolyI:C or 2'3'cGAMP, the effect is better and can promote immune response at a higher level.

[0129] Example 5: Immune response effect of AD20 adjuvant prepared with poloxamer 188

[0130] The poloxamer used in the AD20 adjuvant of this example is poloxamer 188, and the preparation process is referred to Example 1. Immunization procedure: 6-8 week old female BALB / c mice were respectively treated with 1 μg OVA (extremely low dose antigen) and various adjuvants to prepare vaccines (50 μl adjuvant mixed with 50 μl antigen) as required, and then intramuscularly immunized. Immunization groups: (1) PBS (100 μl 1xPBS), (2) AD20 (TB3-1) adjuvant group, (3) AD20 (TB3-2) adjuvant group, (4) Alum (100 μg Alum), wherein the adjuvant configuration components and dosages of Group (2) and Group (3) are shown in Table 3; the total immunization volume is 100 μl / mouse, each group was immunized twice with an interval of 14 days, and blood was collected before and after each immunization. ELISA was performed using a 96-well microplate to determine the titer of OVA-specific IgG antibodies, and the ELISA method for determining serum antibodies was consistent with the method in Example 2.

[0131] The results are as follows Figure 7 As shown, it is the result of ELISA detection of anti-OVA antibody titer in mouse serum, by detecting the antibody results on days 14, 21, 28, 35 and 42 after immunization of aluminum adjuvant (Alum), AD20 (TB3-1) adjuvant group and AD20 (TB3-2) adjuvant group. As can be seen from the figure, 14 days after the primary immunization, no anti-OVA antibodies were detected in the AD20 (TB3-2) adjuvant group; 21, 28, 35, and 42 days after immunization, the aluminum adjuvant (Alum), AD20 (TB3-1) adjuvant group, and AD20 (TB3-2) adjuvant group were able to induce high levels of antibody titers, and the binding antibody levels produced by the AD20 (TB3-1) adjuvant group on days 28, 35, and 42 after immunization were stable and sustained, and were comparable to the duration and level induced by the aluminum adjuvant group; it can be seen that the formulations of the AD20 (TB3-1) adjuvant group and the AD20 (TB3-2) adjuvant group can efficiently enhance the antigen to achieve a high level of immune effect under low-dose immunization, and the formulation of the AD20 (TB3-1) adjuvant group is better and comparable to the aluminum adjuvant.

[0132] Example 6: Comparison of immune effects of different formulations of AD20 adjuvants

[0133] The poloxamer used in the AD20 adjuvant of this example is poloxamer 188, and the preparation process is referred to Example 1. Immunization procedure: 6-8 week old female BALB / c mice were respectively treated with 1 μg OVA (extremely low dose antigen) and various adjuvants to prepare vaccines (50 μl adjuvant mixed with 50 μl antigen) as required, and then intramuscularly immunized. Immunization groups: (1) PBS (100 μl 1xPBS), (2) AD20 (TB3-1) adjuvant group, (3) AD20 (TB4-1) adjuvant group, (4) AD20 (TB4-high concentration) adjuvant group, (5) Alum (100 μg Alum), wherein the adjuvant configuration components and dosages of groups (2), (3) and (4) are shown in Table 3; the total immunization volume was 100 μl / mouse, each group was immunized twice with an interval of 14 days, and blood was collected before and after each immunization. The OVA-specific IgG antibody titer was determined by ELISA using a 96-well microplate. The serum antibody titer was determined by ELISA according to the method in Example 2.

[0134] The antibody results of mice immunized with different formulations of AD20 adjuvant are as follows Figure 8 As shown in the figure, it can be seen that after the adjuvant formula was changed, specific antibodies to the OVA antigen could be detected in both the AD20 (TB4-1) adjuvant group and the AD20 (TB4-high concentration) adjuvant group 14 days after the primary immunization, and the antibody levels at 21, 28, 35, and 42 days after immunization were significantly increased, which was comparable to that of the AD20 (TB3-1) adjuvant group.

[0135] Example 7: Evaluation of immunogenicity of AD20 adjuvant combined with different antigens

[0136] The mice were immunized with vaccines prepared with influenza antigens (quadrivalent influenza virus split vaccine), HBsAg antigen (blood-borne hepatitis B surface antigen), and RSV Pre F5 protein antigen (respiratory syncytial virus Pre F protein antigen) using AD20 (TB3-1) adjuvant. The immunogenicity enhancement effect of AD20 (TB3-1) adjuvant on different antigens was detected by binding antibodies at different time points. Immunization procedure: 6-8 week old female BALB / c mice were prepared with different antigens and adjuvants as required, and intramuscular immunization was performed. Immunization groups: (1) PBS (100 μl 1xPBS), (2) influenza antigen group, (3) AD20 (TB3-1) + influenza antigen group, (4) Alum + influenza antigen group, (5) HBsAg antigen group, (6) AD20 (TB3-1) + HBsAg antigen group, (7) Alum + HBsAg antigen group, (8) Pre F5 antigen group, (9) AD20 (TB3-1) + Pre F5 antigen group, (10) Alum + Pre F5 antigen group, where the influenza antigen dosage for groups (2)-(4) was 0.3 μg per mouse, the HBsAg antigen dosage for groups (5)-(7) was 1 μg per mouse, the Pre F5 antigen dosage for groups (8)-(10) was 1 μg per mouse, and the dosage for the Alum group was 50 μg per mouse. Alum; total immunization volume 100 μl / mouse, each group was immunized twice with an interval of 14 days, and blood was collected before and after each immunization. ELISA was performed using a 96-well microplate to determine the antigen-specific IgG antibody titer. The ELISA method for determining serum antibodies was the same as the method in Example 2, except that the coated antigens were different, and the coating concentrations of influenza antigens, HBsAg antigens or Pre F5 antigens were all 1 μg / mL.

[0137] For influenza antigens, the results were Fig. 9 As shown in Figure 2, compared with the influenza antigen alone immunization group, the antibody levels of the AD20 (TB3-1) + influenza antigen group with adjuvant were significantly better than those of the influenza antigen alone immunization group at 14, 21, and 28 days after immunization; for HBsAg antigen, the results are as follows: Fig.10 As shown in Figure 2, compared with the group immunized with HBsAg antigen alone, the antibody levels of the group immunized with AD20 (TB3-1) + HBsAg antigen with adjuvant were significantly better than those of the group immunized with HBsAg antigen alone at 14, 21, and 28 days after immunization; for RSV Pre F5 antigen, the results are as follows: Fig.11As shown, compared with the group immunized with Pre F5 antigen alone, the antibody levels of the AD20 (TB3-1) + Pre F5 antigen group with adjuvant added were significantly better than those of the group immunized with Pre F5 antigen alone at 14 days, 21 days, and 28 days after immunization, indicating that the AD20 adjuvant configured in the present invention can be applied to different antigens and has a broad spectrum of improving immune responses to different antigens.

[0138] The above specific implementation methods are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. An adjuvant composition, characterized in that The adjuvant composition comprises squalene, Tween 80 and poloxamer; preferably, the poloxamer is selected from poloxamer 401, poloxamer 407 or poloxamer 188.

2. The adjuvant composition according to claim 1, wherein the squalene accounts for 0.2%-4% of the total volume of the adjuvant composition, Tween 80 accounts for 0.02%-0.4% of the total volume of the adjuvant composition, and poloxamer accounts for 0.1%-2% of the total volume of the adjuvant composition; Preferably, the squalene accounts for 0.8%-4% of the total volume of the adjuvant composition, Tween 80 accounts for 0.064%-0.32% of the total volume of the adjuvant composition, and poloxamer accounts for 0.2%-1% of the total volume of the adjuvant composition.

3. The adjuvant composition according to claim 1 or 2, further comprising one or more of monophosphatidyl A, oligonucleotide CpG, R848, PolyI:C, dioctadecyl dimethyl ammonium bromide and 2'3'cGAMP.

4. The adjuvant composition according to claim 3, wherein the amount of monophosphatidyl A is 10 μg / mL-100 μg / mL, the amount of oligonucleotide CpG is 200 μg / mL-6000 μg / mL, the amount of R848 is 100 μg / mL-1000 μg / mL, the amount of PolyI:C is 200 μg / mL-2000 μg / mL, the amount of dioctadecyl dimethyl ammonium bromide is 100 μg / mL-1000 μg / mL, and the amount of 2'3'cGAMP is 200 μg / mL-2000 μg / mL; Preferably, the dosage of monophosphatidyl A is 10 μg / mL-40 μg / mL, the dosage of oligonucleotide CpG is 400 μg / mL-4000 μg / mL, the dosage of R848 is 100 μg / mL-500 μg / mL, the dosage of PolyI:C is 200 μg / mL-1000 μg / mL, the dosage of dioctadecyl dimethyl ammonium bromide is 100 μg / mL-500 μg / mL, and the dosage of 2'3'cGAMP is 500 μg / mL-1200 μg / mL.

5. The adjuvant composition according to any one of claims 1 to 4, characterized in that The adjuvant composition has a particle size of 100 nm-200 nm and a pH value of 6-7.5, such as 6.3-6.7 or 7.0-7.

4.

6. The method for preparing the adjuvant composition according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: S1: preparing an oil phase, preparing squalene into an oil phase; or adding poloxamer suitable for preparing an oil phase at the same time; S2: preparing an aqueous phase, mixing Tween 80 with a buffer solution to prepare an aqueous phase; or simultaneously adding poloxamer suitable for preparing an aqueous phase; S3: slowly adding the aqueous phase prepared in S2 to the oil phase prepared in S1, and adjusting the volume with a buffer solution, stirring at high speed at room temperature to obtain a colostrum solution; S4: the colostrum solution is poured into a microfluidizer to prepare a refined milk solution; S5: sterilize and filter the refined emulsion solution obtained in S4 to obtain the adjuvant composition.

7. The method for preparing the adjuvant composition according to claim 6, characterized in that: In step S3, a high-speed stirrer is used to stir at a speed of 8000-20000 rpm for 20-40 minutes to obtain a colostrum solution; in step S4, the colostrum solution prepared in S3 is poured into a microfluidizer, the pressure is adjusted to 15000-20000 psi, the temperature of the refrigerator is adjusted, the outlet temperature of the emulsion is controlled at 20-30°C, and the high-pressure homogenization is cycled 2-10 times to obtain a refined milk solution; in step S5, a 0.22 μm sterilizing filter is used to sterilize and filter the refined milk solution obtained in S4; preferably, the buffer solution is selected from but not limited to citrate or phosphate buffer.

8. The method for preparing the adjuvant composition according to claim 6 or 7, characterized in that: The adjuvant composition prepared in step S5 is further mixed with one or more of monophosphatidyl A, oligonucleotide CpG, R848, PolyI:C, dioctadecyl dimethyl ammonium bromide and 2'3'cGAMP to obtain the adjuvant composition.

9. An antigen adjuvant composition, characterized in that: The antigen-adjuvant composition comprises the adjuvant composition according to any one of claims 1 to 5 or the adjuvant composition prepared according to any one of claims 6 to 8, and an antigen capable of inducing an immune response; specifically, the antigen comprises one or more of proteins, polypeptides or nucleic acids; Preferably, the antigen includes but is not limited to an antigen selected from varicella-zoster virus, respiratory syncytial virus, severe acute respiratory syndrome coronavirus type 1, severe acute respiratory syndrome coronavirus type 2, human papillomavirus, hepatitis B virus, influenza virus, rotavirus, and pneumonia polysaccharide vaccine; further preferably, the varicella-zoster virus antigen is gE protein.

10. Use of the adjuvant composition according to any one of claims 1 to 5 or the adjuvant composition prepared according to any one of claims 6 to 8 in preparing a vaccine adjuvant.

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