Oil-in-water vaccine adjuvant composition containing saponin and preparation method of oil-in-water vaccine adjuvant composition

Abstract

The invention belongs to the technical field of vaccine adjuvants, and particularly relates to a saponin-containing oil-in-water vaccine adjuvant combination which comprises an internal oil phase, an external water phase and an immunostimulant, the internal oil phase comprises the following components in percentage by weight: 0.1-2% of a lipophilic surfactant and 0.1-5% of squalene; the external water phase comprises 0.1-5% of a hydrophilic surfactant and a citric acid buffer solution; and the immune stimulant comprises 0 to 100 [mu] g / mL of QS-21 and 0 to 100 [mu] g / mL of 3D-MPL. According to the oil-in-water vaccine adjuvant composition containing the saponin, the cellular immune effect can be remarkably improved, and the influence of heating on QS-21 degradation in the micro-jet process is effectively avoided in the preparation process.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine adjuvants, and in particular to a saponin-containing oil-in-water vaccine adjuvant combination and a preparation method thereof. Background Art

[0002] Adjuvants are immune stimulants. Vaccine prescriptions are usually composed of antigens, immunogenic compounds and vaccine adjuvants. The immunogenic compounds induce protection against the target disease, and the vaccine adjuvants can amplify the immune response of the vaccinated animal to the antigen. The use of adjuvants in vaccine prescriptions can increase the intensity of the humoral or cellular immune response conferred by a dose of vaccine, thereby ensuring a better level of protection; prolong the protection period conferred by a dose of vaccine; obtain the same efficacy as the full dose without adjuvant at a lower antigen dose; and reduce the number of immunizations required to ensure vaccine protection. Among them, oil-water adjuvants include water-in-oil (w / o) type, water-in-oil-in-water (w / o / w) type and water-in-oil (o / w) type.

[0003] Currently available on the market is the MF59 water-in-oil adjuvant, which uses squalene as the main raw material, but its immune stimulation is not strong.

[0004] The Chinese patent CN 116077635 A specification discloses the application of the novel coronavirus fusion protein vaccine. The specific implementation method of the specification records that the vaccine formula contains: citrate buffer, water-in-oil emulsion adjuvant. The emulsion adjuvant includes 3D MPL, QS-21 saponin and tocopherol, as well as Tween 80, squalene and other ingredients. It also discloses that the surfactant is polysorbate-80, and the surfactant concentration is 0.01-2mg / ml, optimized to 0.01-0.3mg / ml, and contains cholesterol and other contents. However, there are still technical problems that vaccine adjuvants generally have poor cellular immunity effects, and are prone to stratification and precipitation during the storage period. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a saponin-containing oil-in-water vaccine adjuvant combination and a preparation method, which can significantly enhance the cellular immunity and humoral immunity effects, while solving the problems of 3D-MPL being difficult to dissolve in the aqueous phase during preparation and the degradation of QS-21 during preparation.

[0006] The present invention solves the above technical problems by providing a saponin-containing oil-in-water vaccine adjuvant combination, the combination comprising an internal oil phase, an external water phase and an immunostimulant; wherein, based on the total mass of the adjuvant, The internal oil phase includes 0.1-2% lipophilic surfactant and 0.1-5% squalene; The external aqueous phase includes 0.1-5% hydrophilic surfactant and citric acid buffer (pH 6.2); The immunostimulants include QS-21 0-100 μg / mL and 3D-MPL (3-O-deacylated monophosphoryl lipid A) 0-100 μg / mL. QS-21 is a saponin extracted from the plant Quillaja saponaria. The addition of component QS-21 plays an important role in enhancing the cellular immune effect and can promote both humoral and cellular immunity. QS-21 can not only stimulate antibody-based humoral immune responses, but also stimulate cellular immune responses. QS-21 can be used alone as an immune adjuvant or in combination with other immune adjuvants to improve the immune effect.

[0007] In the optimized solution, the lipophilic surfactant includes sorbitan trioleate. Sorbitan trioleate is a surfactant (Span 85) to maintain the stability of the oil-in-water dosage form.

[0008] The hydrophilic surfactant includes polysorbate.

[0009] The polysorbate includes polysorbate-80.

[0010] In some embodiments, the pH of the citrate buffer is 6.2.

[0011] The method for preparing a saponin-containing oil-in-water vaccine adjuvant combination of the present invention comprises the following steps: (1) Thoroughly mix the hydrophilic surfactant and citrate buffer to obtain an external aqueous phase; (2) dissolving 3D-MPL in an organic solvent, and then thoroughly mixing the organic solvent containing 3D-MPL, squalene and a lipophilic surfactant to obtain an internal oil phase; (3) Add the inner oil phase to the outer water phase for dispersion to obtain stable colostrum; (4) The colostrum is homogenized in a microfluidizer to obtain a nano-oil-in-water emulsion.

[0012] (5) The citrate buffer solution of QS-21 was added to the nano-oil-in-water emulsion, incubated with stirring in the dark, and then sterilized and filtered to obtain a stable nano-oil-in-water adjuvant containing QS-21 and 3D-MPL.

[0013] In the optimized solution, in step (3), after the internal oil phase is added to the external water phase, the dispersion is performed at 4000-5000 rpm.

[0014] In the further optimized scheme, the dispersion time was 30~50 min.

[0015] The pressure during homogenization in step (4) is 800-1300 bar.

[0016] The particle size of the stable nano-oil-in-water emulsion in step (4) is 100-150 nm, which is the particle size range in which the vaccine adjuvant has a relatively good immune effect.

[0017] The adjuvant of the present invention is added with QS-21 saponin, which can significantly improve the cellular immune effect; the preparation process adopts microfluidization preparation, the texture is uniform, it is not easy to be stratified, it is easy to scale up production, and the process is stable. The preparation process of the present invention effectively avoids the influence of heat generation during the microfluidization process on the degradation of QS-21. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A particle size information diagram of the prescription IV provided in an embodiment of the present invention; Figure 2 A particle size information diagram of a prescription V provided in an embodiment of the present invention; Figure 3 A particle size information diagram of the prescription VI provided in an embodiment of the present invention; Figure 4 Electron microscopic image of the adjuvant provided in the embodiment of the present invention; Figure 5 Different prescription adjuvants provided in the embodiments of the present invention stimulated mice to produce IFN-γ antibody levels at D28; Figure 6 Different prescription adjuvants provided in the embodiments of the present invention stimulated mice to produce IgG antibody levels at D28; Figure 7 It is a comparison chart of QS-21 of two adjuvants in Example 10 of the present invention. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with specific embodiments: Among them, squalene, cholesterol, citric acid, saponin QS-21, immunostimulant 3D-MPL, hydrophilic surfactant polysorbate-80 and lipophilic surfactant sorbitan trioleate were all purchased.

[0020] Example 1 The saponin-containing oil-in-water vaccine adjuvant combination of the present invention is composed of an internal oil phase, an external water phase and an immunostimulant, wherein the internal oil phase is 0.1% sorbitan trioleate and 0.1% squalene, the external water phase is 0.1% polysorbate 80 and a citrate buffer with a pH value of 6.2, and the immunostimulant is 50 μg / mL of QS-21 and 50 μg / mL of 3D-MPL.

[0021] The preparation method is as follows: (1) Thoroughly mix the hydrophilic surfactant and citrate buffer to obtain an external aqueous phase.

[0022] (2) dissolving 3D-MPL in an organic solvent, and then thoroughly mixing the organic solvent containing 3D-MPL, squalene and a lipophilic surfactant to obtain an internal oil phase; (3) Add the internal oil phase to the external water phase and disperse at 4000 rpm to obtain stable colostrum. The dispersion time is 30 to 50 minutes.

[0023] (4) The colostrum was homogenized in a microfluidizer at a pressure of 800 bar to obtain a stable nano-oil-in-water emulsion with a particle size of 100-150 nm.

[0024] (5) The citrate buffer solution of QS-21 was added to the nano-oil-in-water emulsion, incubated with stirring in the dark, and then sterilized and filtered to obtain a stable nano-oil-in-water adjuvant containing QS-21 and 3D-MPL.

[0025] The immunogenicity and safety of the oil-in-water emulsion of the present invention depend on the interaction between the particle size of the oil-in-water adjuvant and the immune system. The particle size affects the distribution, uptake, processing and presentation of antigens by antigen presenting cells (APCs), as well as the activation and polarization of innate and adaptive immune responses. Generally speaking, smaller particles (less than 200nm) have better biodistribution and cellular uptake effects than larger particles (greater than 500nm), and can induce stronger humoral and cellular immune responses. However, particles with smaller particle sizes may also cause more local inflammation and systemic adverse reactions than particles with larger particle sizes.

[0026] Example 2 The saponin-containing oil-in-water vaccine adjuvant combination of the present invention consists of an internal oil phase, an external water phase and an immunostimulant, wherein the internal oil phase is 2% sorbitan trioleate and 5% squalene, the external water phase is 5% polysorbate-80 and a citrate buffer with a pH value of 6.2, and the immunostimulant is 70 μg / mL of QS-21 and 30 μg / mL of 3D-MPL.

[0027] The preparation method is as follows: (1) Thoroughly mix the hydrophilic surfactant and citrate buffer to obtain an external aqueous phase.

[0028] (2) dissolving 3D-MPL in an organic solvent, and then thoroughly mixing the organic solvent containing 3D-MPL, squalene and a lipophilic surfactant to obtain an internal oil phase; (3) Add the inner oil phase into the outer water phase and disperse at 5000 rpm to obtain stable colostrum. The dispersion time is 50 minutes.

[0029] (4) The colostrum is homogenized in a microfluidizer at a pressure of 800-1300 bar to obtain a nano-oil-in-water emulsion with a particle size of 100-150 nm.

[0030] (5) The citrate buffer solution of QS-21 was added to the nano-oil-in-water emulsion, incubated with stirring in the dark, and then sterilized and filtered to obtain a stable nano-oil-in-water adjuvant containing QS-21 and 3D-MPL.

[0031] Example 3 The saponin-containing oil-in-water vaccine adjuvant combination of the present invention consists of an internal oil phase, an external water phase and an immunostimulant, wherein the internal oil phase is 1% sorbitan trioleate and 2% squalene, the external water phase is 2% polysorbate-80 and a citrate buffer with a pH value of 6.2, and the immunostimulant is 50 μg / mL of QS-21 and 50 μg / mL of 3D-MPL.

[0032] The preparation method is as follows: (1) Thoroughly mix the hydrophilic surfactant and citrate buffer to obtain an external aqueous phase.

[0033] (2) dissolving 3D-MPL in an organic solvent, and then thoroughly mixing the organic solvent containing 3D-MPL, squalene and a lipophilic surfactant to obtain an internal oil phase; (3) Add the inner oil phase into the outer water phase and disperse at 4500 rpm to obtain stable colostrum. The dispersion time is 40 minutes.

[0034] (4) The colostrum is homogenized in a microfluidizer at a pressure of 1000 bar to obtain a nano-oil-in-water emulsion with a particle size of 100-150 nm.

[0035] (5) The citrate buffer solution of QS-21 was added to the nano-oil-in-water emulsion, incubated with stirring in the dark, and then sterilized and filtered to obtain a stable nano-oil-in-water adjuvant containing QS-21 and 3D-MPL.

[0036] Example 4 The saponin-containing oil-in-water vaccine adjuvant combination of the present invention consists of an internal oil phase, an external water phase and an immunostimulant, wherein the internal oil phase is 0.5% sorbitan trioleate and 0.6% squalene, the external water phase is 0.6% polysorbate-80 and a citrate buffer with a pH value of 6.2, and the immunostimulant is 100 μg / mL QS-21.

[0037] The preparation method is as follows: (1) Thoroughly mix the hydrophilic surfactant and citrate buffer to obtain an external aqueous phase.

[0038] (2) fully mixing squalene and a lipophilic surfactant to obtain an internal oil phase; (3) Add the internal oil phase to the external water phase and disperse at 4800 rpm to obtain stable colostrum. The dispersion time is 30 to 50 minutes.

[0039] (4) The colostrum is homogenized in a microfluidizer at a pressure of 900 bar to obtain a nano-oil-in-water emulsion with a particle size of 100-150 nm.

[0040] (5) Add the citrate buffer of QS-21 to the nano-oil-in-water emulsion, incubate with stirring in the dark, and then sterilize and filter to obtain a stable nano-oil-in-water adjuvant containing QS-21.

[0041] Example 5 The saponin-containing oil-in-water vaccine adjuvant combination of the present invention is composed of an internal oil phase, an external water phase and an immunostimulant, wherein the internal oil phase is 1.5% sorbitan trioleate and 1.5% squalene, the external water phase is 3% polysorbate 80 and a citrate buffer with a pH value of 6.2, and the immunostimulant is 100 μg / mL 3D-MPL.

[0042] The preparation method is as follows: (1) Thoroughly mix the hydrophilic surfactant and citrate buffer to obtain an external aqueous phase.

[0043] (2) dissolving 3D-MPL in an organic solvent, and then thoroughly mixing the organic solvent containing 3D-MPL, squalene and a lipophilic surfactant to obtain an internal oil phase; (3) Add the inner oil phase into the outer water phase and disperse at 4200 rpm to obtain stable colostrum. The dispersion time is 35 minutes.

[0044] (4) The colostrum was homogenized in a microfluidizer at a pressure of 1200 bar to obtain a stable nano-water-in-oil adjuvant containing 3D-MPL with a particle size of 100-150 nm.

[0045] Example 6 The saponin-containing oil-in-water vaccine adjuvant combination of the present invention is composed of an internal oil phase, an external water phase and an immunostimulant, wherein the internal oil phase is 1.2% sorbitan trioleate and 3% squalene, the external water phase is 4% polysorbate-80 and a citrate buffer with a pH value of 6.2, and the immunostimulant is 40 μg / mL of QS-21 and 40 μg / mL of 3D-MPL.

[0046] The preparation method is as follows: (1) Thoroughly mix the hydrophilic surfactant and citrate buffer to obtain an external aqueous phase.

[0047] (2) dissolving 3D-MPL in an organic solvent, and then thoroughly mixing the organic solvent containing 3D-MPL, squalene and a lipophilic surfactant to obtain an internal oil phase; (3) Add the inner oil phase into the outer water phase and disperse at 4600 rpm to obtain stable colostrum. The dispersion time is 45 minutes.

[0048] (4) The colostrum was homogenized in a microfluidizer at a pressure of 1100 bar to obtain a stable nano-oil-in-water emulsion with a particle size of 100-150 nm.

[0049] (5) The citrate buffer solution of QS-21 was added to the nano-oil-in-water emulsion, incubated with stirring in the dark, and then sterilized and filtered to obtain a stable nano-oil-in-water adjuvant containing QS-21 and 3D-MPL.

[0050] Example 7 The saponin-containing oil-in-water vaccine adjuvant combination of the present invention is composed of an internal oil phase, an external water phase and an immunostimulant, wherein the internal oil phase is 0.8% sorbitan trioleate and 4.3% squalene, the external water phase is 2.5% polysorbate-80 and a citrate buffer with a pH value of 6.2, and the immunostimulant is QS-21 100 μg / mL.

[0051] The preparation method is as follows: (1) Thoroughly mix the hydrophilic surfactant and citrate buffer to obtain an external aqueous phase.

[0052] (2) fully mixing squalene and a lipophilic surfactant to obtain an internal oil phase; (3) Add the inner oil phase into the outer water phase and disperse at 4600 rpm to obtain stable colostrum. The dispersion time is 45 minutes.

[0053] (4) The colostrum was homogenized in a microfluidizer at a pressure of 1100 bar to obtain a stable nano-oil-in-water emulsion with a particle size of 100-150 nm.

[0054] (5) Add the citrate buffer of QS-21 to the nano-oil-in-water emulsion, incubate with stirring in the dark, and then sterilize and filter to obtain a stable nano-oil-in-water adjuvant containing QS-21.

[0055] Example 8 The saponin-containing oil-in-water vaccine adjuvant combination of the present invention consists of an internal oil phase, an external water phase and an immunostimulant, wherein the internal oil phase is 0.8% sorbitan trioleate and 4.3% squalene, the external water phase is 2.5% polysorbate 80 and a citrate buffer with a pH value of 6.2, and the immunostimulant is 100 μg / mL of QS-21 and 100 μg / mL of 3D-MPL.

[0056] The preparation method is as follows: (1) Thoroughly mix the hydrophilic surfactant and citrate buffer to obtain an external aqueous phase.

[0057] (2) dissolving 3D-MPL in an organic solvent, and then thoroughly mixing the organic solvent containing 3D-MPL, squalene and a lipophilic surfactant to obtain an internal oil phase; (3) Add the inner oil phase into the outer water phase and disperse at 4600 rpm to obtain stable colostrum. The dispersion time is 45 minutes.

[0058] (4) The colostrum was homogenized in a microfluidizer at a pressure of 1100 bar to obtain a stable nano-oil-in-water emulsion with a particle size of 100-150 nm.

[0059] (5) The citrate buffer solution of QS-21 was added to the nano-oil-in-water emulsion, incubated with stirring in the dark, and then sterilized and filtered to obtain a stable nano-oil-in-water adjuvant containing QS-21 and 3D-MPL.

[0060] Example 9 The saponin-containing oil-in-water vaccine adjuvant combination of the present invention is composed of an internal oil phase, an external water phase and an immunostimulant, wherein the internal oil phase is 0.8% sorbitan trioleate and 4.3% squalene, the external water phase is 2.5% polysorbate-80 and a citrate buffer solution with a pH value of 6.2, and the immunostimulant is 100 μg / mL 3D-MPL.

[0061] The preparation method is as follows: (1) Thoroughly mix the hydrophilic surfactant and citrate buffer to obtain an external aqueous phase.

[0062] (2) dissolving 3D-MPL in an organic solvent, and then thoroughly mixing the organic solvent containing 3D-MPL, squalene and a lipophilic surfactant to obtain an internal oil phase; (3) Add the inner oil phase into the outer water phase and disperse at 4600 rpm to obtain stable colostrum. The dispersion time is 45 minutes.

[0063] (4) The colostrum was homogenized in a microfluidizer at a pressure of 1100 bar to obtain a stable nano-water-in-oil adjuvant containing 3D-MPL with a particle size of 100-150 nm.

[0064] Test 1 In clinical applications, the adjuvant combination of the present invention is prepared into three different formulations, namely, formulation I, formulation II and formulation III, which are prepared in the same manner as in the above examples. Table 1 shows the material composition of the three formulations (other components and dosages are the same, and the preparation method is the same), see Table 1 and Table 2, and Figure 1-Figure 4 : Table 1 Composition ratio of prescription I, prescription II and prescription III

[0065] Table 2 shows the particle size information of Prescription I, Prescription II and Prescription III, see Table 2: Table 2 Particle size information of Prescription I, Prescription II, and Prescription III

[0066] like Figure 1 , Figure 2 and Figure 3 As shown, the particle size information of prescription IV, prescription II and prescription III are shown respectively. Figure 4 As shown, an electron microscope image of the adjuvant provided in an embodiment of the present invention is shown.

[0067] The particle sizes of the oil-in-water adjuvant particles prepared by Prescription I, Prescription II, and Prescription III were all less than 200 nm, and most of them were kept at around 150 nm; the PDI was less than 0.010, indicating that the particles had good dispersibility and were not easy to aggregate.

[0068] Test 2 The amounts of other raw materials are the same, but the preparation methods are different. Prescription II can be further divided into four dosage forms: ①, ②, ③, and ④, as shown in Table 3 and Table 4. Table 3 shows the composition ratios of the four dosage forms, as shown in Table 3 below: Table 3 Ratio of components ①, ②, ③, and ④ in prescription II

[0069] Table 4 shows the main physicochemical parameters of the four dosage forms, see Table 4: Table 4 Physicochemical parameters of the four adjuvants ①, ②, ③, and ④ in Formulation II

[0070] The only difference among prescriptions ①, ②, ③, and ④ is the preparation method. It can be seen that the particle size results are related to the preparation method. The PDI of the nanoadjuvant prepared by microfluidization is lower than that of the adjuvant prepared by other methods; and its D90 (nm) is smaller than that of the adjuvant prepared by other methods.

[0071] Test 3 In order to verify the efficacy of the immune adjuvant provided by the present invention, animal experiments were conducted on the immune adjuvant provided by the present invention.

[0072] Forty female BALB / c mice that passed the quarantine were randomly divided into 5 groups: Groups 1 to 5 were negative control group, prescription I, prescription II, prescription III, and control group, with 5 animals in each group. Each animal was marked during immunization.

[0073] After grouping, the mice were immunized by intramuscular injection on D0 and D21, with a dosage of 0.1 mL per mouse.

[0074] The first immunization is recorded as D0, and so on for subsequent immunizations.

[0075] The specific grouping information is shown in Table 5: Table 5 Grouping information of pharmacological and pharmaceutical tests

[0076] The control group was a commercially available vaccine (recombinant herpes zoster vaccine (CHO cells)) + adjuvant AS01, which was homemade by Huanuotai.

[0077] Immunoassay sample preparation Before each immunization, take 5 vials of freeze-dried gE protein, reconstitute them with 0.5 mL of sterile water for injection, collect them together and mix them gently to obtain 100 μg / mL of antigen.

[0078] G1: Take 1 ml of sterile PBS before each immunization.

[0079] G2: Prepare 1 mL each time: 0.5 mL antigen + 0.5 mL adjuvant (Formula I), and mix gently.

[0080] G3: Prepare 1 mL each time: 0.5 mL antigen + 0.5 mL adjuvant (Formula II), and mix gently.

[0081] G4: Prepare 1 mL each time: 0.5 mL antigen + 0.5 mL adjuvant (Formula III), and mix gently.

[0082] G5: Prepare 1 mL each time: 0.5 mL antigen + 0.5 mL AS01 adjuvant, and mix gently.

[0083] Figure 5 The levels of IFN-γ produced by mouse splenocytes stimulated by different adjuvant formulations at D28 are shown in Figure 5 .

[0084] At D28, the antibody titer was able to maintain the same level as that of the control group, indicating that the adjuvant of the present invention has a very good effect.

[0085] Test 4: ELISPOT method to detect IFN-γ cytokines One week after the second immunization (D28), spleens of 5 animals in each group were collected, lymphocytes were separated for IFN-γ ELISPOT detection, and the number of IFN-γ spots produced by animals in each group was calculated (the mean number of spots in the antigen peptide stimulation wells - the mean number of spots in the negative wells). This result was used to evaluate the cellular immunity level of the test substance.

[0086] See also Figure 5The number of cells secreting specific IFN-γ in each group of animals was 2±3, 169±132, 337±37, 128±69, 286±76, 415±44, 423±28, and 431±47, respectively. Compared with the negative control group, each group had a significant increase (P<0.05). There were significant differences between prescriptions I to III (P<0.05), and there were also significant differences compared with the control group (P<0.05).

[0087] One week after the second vaccination (D28): Figure 6 The results show that different adjuvant formulations (formulations in Table 3) stimulated the mice to produce IgG antibodies at D28.

[0088] Figure 6 The data in the figure are the results of humoral immunity, indicating that the water-in-oil adjuvant containing QS-21 and 3D-MPL (Formulation III) can significantly enhance the level of humoral immunity in mice (antibody titer (valence) in serum).

[0089] Example 10 The saponin-containing oil-in-water vaccine adjuvant combination of the present invention is composed of an internal oil phase, an external water phase and an immunostimulant, wherein the internal oil phase is 0.8% sorbitan trioleate and 4.3% squalene, the external water phase is 2.5% polysorbate-80 and a citrate buffer with a pH value of 6.2, and the immunostimulant is QS-21 100 μg / mL.

[0090] The preparation method is as follows: Experiment 1: (1) Thoroughly mix the hydrophilic surfactant and citrate buffer to obtain an external aqueous phase.

[0091] (2) fully mixing squalene and a lipophilic surfactant to obtain an internal oil phase; (3) Add the inner oil phase into the outer water phase and disperse at 4600 rpm to obtain stable colostrum. The dispersion time is 45 minutes.

[0092] (4) The colostrum was homogenized in a microfluidizer at a pressure of 1100 bar to obtain a stable nano-oil-in-water emulsion with a particle size of 100-150 nm.

[0093] (5) Add the citrate buffer of QS-21 to the nano-oil-in-water emulsion, incubate with stirring in the dark, and then sterilize and filter to obtain a stable nano-oil-in-water adjuvant containing QS-21.

[0094] Experiment 2: (1) Thoroughly mix the hydrophilic surfactant and the citric acid buffer solution containing QS-21 to obtain an external aqueous phase.

[0095] (2) fully mixing squalene and a lipophilic surfactant to obtain an internal oil phase; (3) Add the inner oil phase into the outer water phase and disperse at 4600 rpm to obtain stable colostrum. The dispersion time is 45 minutes.

[0096] (4) The colostrum was homogenized in a microfluidizer at a pressure of 1100 bar to obtain a stable nano-oil-in-water emulsion with a particle size of 100-150 nm.

[0097] (5) The QS-21 content in the two groups of adjuvants was detected by HPLC, and the two groups were sampled three times in parallel.

[0098] After the experiment, the QS-21 content of the two adjuvants was compared. Figure 7 The figure is a comparison of QS-21 with two adjuvants. The experimental results show that adding QS-21 after microfluidization can effectively reduce the loss of QS-21 during the preparation process due to excessive heat generated during microfluidization.

[0099] The above implementation / test examples are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A saponin-containing oil-in-water vaccine adjuvant combination, characterized in that: The oil-in-water vaccine adjuvant combination comprises an internal oil phase, an external water phase and an immunostimulant; wherein, based on the total mass of the adjuvant, The internal oil phase includes 0.1-2% of lipophilic surfactant and 0.1-5% of squalene; The external aqueous phase includes 0.1-5% of a hydrophilic surfactant and a citrate buffer; Immunostimulants included QS-21 0-100 μg / mL and 3D-MPL 0-100 μg / mL.

2. The oil-in-water vaccine adjuvant combination according to claim 1, characterized in that: Lipophilic surfactants include sorbitan trioleate.

3. The oil-in-water vaccine adjuvant combination according to claim 1, characterized in that: Hydrophilic surfactants include polysorbates.

4. The oil-in-water vaccine adjuvant combination according to claim 3, characterized in that: Polysorbates include polysorbate 80.

5. The oil-in-water vaccine adjuvant combination according to claim 1, characterized in that: The pH of the citrate buffer is 6.

2.

6. A method for preparing a saponin-containing oil-in-water vaccine adjuvant combination according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) Thoroughly mix the hydrophilic surfactant and the citrate buffer to obtain an external aqueous phase; (2) Dissolving 3D-MPL in an organic solvent and then mixing it thoroughly with a lipophilic surfactant and squalene to obtain an internal oil phase; (3) Add the inner oil phase to the outer water phase for dispersion to obtain stable colostrum; (4) homogenizing the colostrum in a microfluidizer to obtain a nano-oil-in-water emulsion; (5) The citrate buffer solution of QS-21 was added to the nano-oil-in-water emulsion, incubated with stirring in the dark, and then sterilized and filtered to obtain a stable nano-oil-in-water adjuvant containing QS-21 and 3D-MPL.

7. The method for preparing a saponin-containing oil-in-water vaccine adjuvant combination according to claim 6, characterized in that In the step (2), 3D-MPL is dissolved in an organic solvent, wherein the organic solvent is selected from dichloromethane, chloroform, glacial acetic acid, and ethyl acetate.

8. The method for preparing a saponin-containing oil-in-water vaccine adjuvant combination according to claim 6, characterized in that :rIn step (3), after the internal oil phase is added to the external water phase, the dispersion is performed at 4000-5000 rpm.

9. The method for preparing a saponin-containing oil-in-water vaccine adjuvant combination according to claim 7, characterized in that :Dispersion time is 30~50 min.

10. The method for preparing a saponin-containing oil-in-water vaccine adjuvant combination according to claim 6, characterized in that: The pressure during homogenization in step (4) is 800-1300 bar.

11. The method for preparing a saponin-containing oil-in-water vaccine adjuvant combination according to claim 6, characterized in that: The incubation time after adding QS-21 in step (5) is 2-8 hours, and the stirring speed is 400-800 rpm / min.

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