Composite adjuvant

Abstract

The invention discloses a composite adjuvant, and belongs to the field of vaccine adjuvants. The main active components of the composite adjuvant are squalene, polysorbate 80 and monophosphoryl ester A, the composite adjuvant is in an oil-in-water emulsion form, nano-scale emulsion droplet particles are uniformly distributed in a water phase, and the system is stable. The invention also discloses a preparation method of the composite adjuvant based on a stirring method. The composite adjuvant comprehensively utilizes the dual characteristics of the oil-in-water emulsion and the immunopotentiator, and can induce a higher level of immunoreaction when being combined with the antigen, so that the composite adjuvant can be used for preparing vaccines and improving the effectiveness of the vaccines.

Description

Technical Field

[0001] The invention belongs to the field of vaccine adjuvants, and particularly relates to the composition, preparation method and application of a composite adjuvant. Background Art

[0002] In the development of recombinant protein vaccines, since the immune stimulation of individual protein components is weak, it is usually necessary to combine with appropriate adjuvant components to enhance the immune response level of the vaccine. Therefore, adjuvants are an indispensable and important component in the development of recombinant protein vaccines. From the discovery of aluminum adjuvants in 1926 to the present, less than 10 adjuvants have been approved for use in marketed human vaccines worldwide, mainly divided into mineral salt adjuvants (such as aluminum hydroxide, aluminum phosphate), water-in-oil adjuvants (such as MF59, AS03), liposome carrier adjuvants (such as AS01), composite adjuvants (such as AS04, aluminum adjuvant adsorbed CpG1018 adjuvant), etc. At present, human vaccines are still dominated by traditional aluminum adjuvants. In order to adapt to the development of the new situation and solve unmet clinical needs, it is urgent to develop new adjuvants.

[0003] Oil-in-water (O / W) adjuvant is a very important adjuvant. Its main active ingredient is squalene. By adding suitable surfactant molecules, nanoparticles of a certain size are formed in the aqueous phase. After the O / W adjuvant is injected into the body by muscle, it can quickly stimulate macrophages, dendritic cells, etc. to secrete a variety of inflammatory cytokines and chemokines (such as CCL2, CCL4, CCL5, CXCL8) at the injection site, and further recruit granulocytes, monocytes, etc. to the injection site, thereby amplifying the immune response at the injection site and activating the adaptive immune response, thereby enhancing the protective effect of the vaccine. At present, in addition to vaccines for emergency use, only three O / W adjuvants for human use have been approved for use: MF59, AS03 and AF03, all of which are used for influenza vaccines, including seasonal influenza vaccines and pandemic A / H1N1 influenza vaccines, and their application range is relatively limited. Among them, (1) MF59 was originally developed by Chiron (WO1990014837A1). In 2006, Novartis acquired Chiron and used MF59 to develop influenza vaccines. In 2014, it was spun off to Seqirus. Its approved quadrivalent influenza vaccine product FLUAD QUADRIVALENT uses MF59 adjuvant. A single dose of 0.5 mL contains 9.75 mg of squalene, 1.175 mg of polysorbate 80, and 1.175 mg of Span 85 (https: / / www.fda.gov / media / 135432 / download). (2) AS03 adjuvant was developed by GSK (WO2008043774A1), mainly containing about 4.5% (v / v) squalene, about 4.85 mg Tween 80 and about 11.86 mg α-tocopherol (Brito LA et al., Semin Immunol. 2013; 25(2): 130-45), which is mixed with antigen in a 1:1 ratio to make the finished vaccine. (3) AF03 adjuvant was developed by Sanofi Pasteur and is used in influenza vaccine Humenza. Its main ingredients are quite different from the first two adjuvants, including squalene, Montane 80, Eumulgin B1-PH and Mannitol.

[0004] Toll-like receptors (TLR) are an important class of protein molecules involved in nonspecific immunity (natural immunity). They are the bridge between nonspecific immunity and specific immunity, and are also the main stimulatory molecules for many adjuvants to stimulate natural immune responses. For example, the CpG1018 adjuvant molecule that has been marketed mainly activates the TLR9 molecular pathway; Monophosphoryl lipid A (MPLA), one of the main components of the AS04 and AS01 adjuvants that have been marketed, mainly activates the TLR4 molecular pathway. Other adjuvants such as PolyI:C, Flagelin, and Imiquimod activate TLR3, TLR5, and TLR7 / 8 molecular pathways, respectively, and are also used in the clinical development stage of various vaccines. After the adjuvant components activate the TLR molecular pathway, they usually further induce a series of downstream cascade reactions, and ultimately induce the secretion of multiple inflammatory-related factors and type I interferons, thereby further enhancing the level of vaccine-induced immune response.

[0005] As can be seen from the above, adjuvant research has a certain foundation. However, it is still difficult to develop a new adjuvant combination (compound adjuvant) that has an immune-boosting effect and does not cause adverse reactions. Even a slight modification on the basis of the existing adjuvant system is not easy:

[0006] (1) First, the disclosed composite adjuvant system is a whole, in which the various components cooperate with each other and are inseparable, so one or more of the components cannot be omitted or replaced at will. For example, the review article published by Novartis emphasized that MF59 is an optimized emulsion formula, which is controlled by multiple factors: including the type and concentration of oil, the selection of surfactants and preparation conditions; among them, the selection of surfactants is a key feature of the development of MF59, which helps to form small and stable emulsion droplets that can be sterile filtered: Experience has shown that the combination of surfactants can achieve the best emulsion stability, which may be due to the formation of a harder film at the interface. Therefore, a surfactant combination of a high HLB value surfactant (such as Tween) and a low HLB value surfactant (such as Span) is usually used to prepare a stable emulsion (Derek TO'Hagan et al., Expert Rev Vaccines. 2013 Jan;12(1):13-30). It can be seen that although MF59 contains two surfactants (Tween 80 and Span 85), their properties are different. The two are used in combination to achieve the effect of stabilizing the emulsion, so one of the surfactants cannot be omitted or replaced at will. Correspondingly, although the main components of GSK's AS03 adjuvant seem to be similar to MF59 (both contain squalene and Tween 80, but the difference is that the former contains Span 85 and the latter contains α-tocopherol), the main substances and mechanisms of action are actually different. This is because: in AS03, in addition to the existing squalene (metabolizable oil), another "degradable oil" is introduced, namely α-tocopherol, and its content is the highest - this is the biggest difference between AS03 adjuvant and other water-in-oil adjuvants. α-Tocopherol is an immunostimulant. Its introduction into AS03 can promote the uptake of antigens by monocytes and promote the production of cytokines (Nathalie Garçon et al., Expert Rev. Vaccines 11(3), 349–366 (2012)). It can be seen that AS03 is not simply based on MF59 by "replacing" Span 85 with α-tocopherol. Therefore, the AS03 adjuvant patent application (WO2008043774A1) filed later than MF59 has been authorized in many countries or regions.

[0007] (2) Secondly, the immune mechanisms of different ingredients in the body may be different, and some may even conflict with each other. In addition, the human immune mechanism is complex. Therefore, it is not possible to simply add a certain adjuvant component (such as an immunostimulant) to the publicly available composite adjuvant formula and assume that the overall effect of the new adjuvant combination is linearly related to the diversity or dosage of the components used. For example, when Chiron was developing the MF59 vaccine in the early stage, it used the immunostimulant MTP-PE (muramyltripeptide) as one of the important ingredients (WO1990014837A1). However, HIV vaccine research found that the addition of MTP-PE to squalene, Tween 80 and Span 85 did not further enhance the immune response (JO Kahn et al., J Infect Dis. 1994 Nov;170(5):1288-91). In fact, the addition of MTP-PE to water-in-oil emulsions would result in an unacceptable degree of reactogenicity. In addition, clinical experience with the addition of TLR4 stimulator E6020 to MF59 has not achieved the expected positive effect. Therefore, the article believes that the clinical experience of using MF59 and adding immunostimulants can be summarized as a very simple equation: 1 plus 1 usually equals 1 (meaning that the combination has no benefit), or even <1 (Derek T O'Hagan et al., Expert Rev Vaccines. 2013 Jan;12(1):13-30). It can be seen that further adding immunostimulants to the composite adjuvant may not further improve the immune effect, but may bring side effects.

[0008] (3) Furthermore, the preparation process of composite adjuvants is somewhat difficult. How to select the appropriate component ratio and obtain a composite adjuvant with stable physical and chemical properties and immune-enhancing effects through a simple and effective process requires repeated exploration in the formulation and preparation process. In addition, new adjuvant ingredients are usually expensive, and the overall economic cost must also be considered when preparing composite adjuvants based on them.

[0009] Therefore, the development of new composite adjuvants is not an easy task. It cannot be easily achieved by simply replacing, adding or removing components based on existing technologies. It is necessary to consider the properties of each component itself and the coordination relationship between them, the mechanism of joint action, safety and effectiveness, etc. Although the development of composite adjuvants is difficult, in the face of emerging infectious disease threats, it is still necessary to develop new adjuvant combinations to lay the foundation for the development of new vaccines. Summary of the invention

[0010] The purpose of the present invention is to provide a new type of highly efficient composite adjuvant with simpler components and a simpler preparation method, which can induce vaccines to produce better immune effects when used in combination with antigens, and a preparation method thereof.

[0011] The present invention provides a composite adjuvant, which comprises squalene, polysorbate 80, and TLR4 agonist monophosphoryl lipid A (MPLA) as an immunopotentiator; the composite adjuvant is in the form of an oil-in-water emulsion, wherein nanoemulsion droplet particles formed by an oil phase are uniformly distributed in an aqueous phase, and the particle size of the emulsion droplet particles is 150nm±30nm, preferably 150nm±20nm, and more preferably 150nm±10nm.

[0012] Preferably, the composite adjuvant does not contain Span 85 or tocopherol; further preferably, the main active ingredients of the composite adjuvant consist of squalene, polysorbate 80 and MPLA.

[0013] Preferably, the oil-in-water emulsion is obtained by mixing an oil phase and an aqueous phase, wherein the oil phase contains MPLA and squalene, and the aqueous phase contains polysorbate 80; that is, MPLA and squalene are located in the oil phase, and polysorbate 80 is located in the aqueous phase.

[0014] Preferably, in the composite adjuvant, the squalene content is 1.0%-10.0% w / v, the polysorbate 80 content is 0.4%-6.4% w / v, and the MPLA content is 0.04-0.4 mg / mL; further preferably, the squalene content is 1.0%-6.0% w / v, the polysorbate 80 content is 0.5%-3.2% w / v, and the MPLA content is 0.1-0.4 mg / mL (for example, the formula of adjuvant four shown in Table 2.5 is used); further preferably, the squalene content is 4%-6% w / v, the polysorbate 80 content is 0.8%-3.2% w / v, and the MPLA content is 0.1-0.4 mg / mL; further preferably, the squalene content is 4%-6% w / v, the polysorbate 80 content is 1%-1.6% w / v, and the MPLA content is 0.1-0.4 mg / mL; further preferably, the squalene content is 4% w / v, the polysorbate 80 content is 1%-1.5% w / v, and the MPLA content is 0.2-0.4 mg / mL; further preferably, the squalene content is 4% w / v, the polysorbate 80 content is 1% w / v or 1.5% w / v, and the MPLA content is 0.2 mg / mL or 0.4 mg / mL (for example, using the formulation of adjuvant one, adjuvant two or adjuvant three shown in Table 2.5).

[0015] Preferably, the MPLA can be selected from chemically synthesized 3D (6-acyl)-PHAD ® PHAD ® 、3D-PHAD ® , and / or PHAD-504 ® ; Further preferably, MPLA selects 3D (6-acyl) -PHAD® .

[0016] Preferably, the aqueous phase is prepared in a citric acid-sodium citrate buffer solution with a molar concentration of 4mM-11mM and a pH of 6.8±1; further preferably, the molar concentration of the citric acid-sodium citrate buffer solution is 5mM-10mM and a pH of 6.8±0.5.

[0017] Preferably, the particle size distribution PDI (Polymer dispersity index) of the emulsion particles is less than 0.3 (preferably less than 0.2), and the Zeta potential is less than 0.

[0018] The present invention also provides the use of the composite adjuvant in preparing an immunogenic composition. Preferably, the composite adjuvant and the antigen solution are mixed at a volume ratio of (1-10):(1-10) to prepare the immunogenic composition; further preferably, the composite adjuvant and the antigen solution are mixed at a volume ratio of 1:1.

[0019] The present invention also provides an immunogenic composition comprising the composite adjuvant, and an antigen and / or an antigen composition.

[0020] Preferably, the type of the antigen and / or antigen composition is selected from one or more of the following: recombinant protein antigen, polypeptide antigen, polysaccharide antigen, polysaccharide-protein conjugate antigen, nucleic acid antigen, inactivated or attenuated pathogen; preferably, the type of the antigen and / or antigen composition is selected from recombinant protein antigen; further preferably, the type of the antigen and / or antigen composition can be selected from recombinant herpes zoster antigen protein (such as gE protein) or respiratory syncytial virus antigen protein (such as RSV F protein).

[0021] Preferably, the immunogenic composition is prepared by mixing the composite adjuvant with a solution containing the antigen and / or antigen composition at a volume ratio of (1-10):(1-10); further preferably, mixing at a volume ratio of 1:1.

[0022] The present invention also provides a method for preparing the composite adjuvant and related vaccine preparations thereof, wherein the method is a stirring method and comprises the following steps:

[0023] (1) Preparation of oil phase: 1) Weigh the required amount of squalene and MPLA respectively and add them to container A; 2) Stir and mix to prepare the oil phase: the stirring parameters are selected as 1500 rpm ± 500 rpm (preferably 1500 rpm) and stirring for 1 h ± 0.5 h (preferably 0.5 h - 1 h);

[0024] (2) Preparation of aqueous phase: 1) Weigh the required amount of polysorbate 80 and add it to container B, and make up the balance with 4 mM-11 mM, pH 6.8±1 (preferably 5 mM-10 mM, pH 6.8±0.5) citric acid-sodium citrate buffer; 2) Stir and mix to prepare the aqueous phase: the stirring parameters are selected as 1500 rpm±500 rpm (preferably 1500 rpm) and stirring for 30 min±10 min (e.g. 30 min);

[0025] (3) Oil phase-water phase mixing: slowly add the oil phase dropwise into the water phase, and then stir continuously: the stirring parameters are selected as 1500 rpm ± 500 rpm (preferably 1500 rpm), not exceeding 40°C (preferably room temperature), and stirring is continued for more than 40 hours (preferably 48 hours ± 8 hours) in the dark to obtain a milky white emulsion;

[0026] (4) Filtration sterilization: The milky white emulsion is filtered and sterilized through a 0.22 μm filter to obtain the composite adjuvant.

[0027] In step 1) of the above step (1), the preferred method is as follows: a) weigh squalene and add it to container A, b) weigh MPLA and add it to container A already filled with squalene. Regarding the weighing of MPLA: MPLA can be weighed and then added directly to container A, or MPLA can be dissolved in an organic reagent before adding it to container A (so as to better perform intermediate control quantification). Therefore, further preferably, the specific method of the above step b) is as follows: dissolve MPLA in an organic solvent (such as chloroform, dimethyl sulfoxide or ethanol) to prepare an MPLA solution of a certain concentration, calculate the volume of the MPLA solution to be added according to the required amount of MPLA in the composite adjuvant, and then add it to container A already filled with squalene, and after stirring and mixing in step 2), add the following operation: remove the organic solvent from the oil phase (for example, using a centrifugal concentrator, a vacuum dryer or a nitrogen blower) until the solution is clear.

[0028] In step 1) of the above step (2), polysorbate 80 may be weighed and then directly added to the container B, or polysorbate 80 may be weighed and first dissolved in a certain volume of citric acid-sodium citrate buffer and then added to the container B.

[0029] In addition, the prepared composite adjuvant is mixed evenly with a solution containing an antigen and / or an antigen composition in a volume ratio of (1-10):(1-10) to prepare an immunogenic composition, or directly used for immunization injection; preferably, the mixture is mixed evenly in a volume ratio of 1:1.

[0030] In the above content, when a numerical range is involved, it means that the end values ​​and the values ​​in between can be taken.

[0031] The beneficial technical effects brought by the present invention include:

[0032] Product aspects:

[0033] (1) The composite adjuvant of the present invention makes comprehensive use of two components: oil-in-water (O / W) emulsion and immunopotentiator MPLA. It can retain the original function of O / W adjuvant to recruit immune cells to the injection site, and stimulate the TLR4 natural immune pathway through MPLA to better stimulate the natural immune response, thereby achieving a better adjuvant effect. The composite adjuvant can induce a higher level of immune response after combining with antigens, and is expected to be applied to the development of more diverse vaccines besides influenza, such as recombinant herpes zoster vaccine, recombinant respiratory syncytial virus vaccine, etc.

[0034] (2) The O / W components used in the composite adjuvant of the present invention are different from those of the existing O / W adjuvants on the market. For example: 1) Compared with AF03, the main components of the emulsion only include squalene and polysorbate-80, plus an immunostimulant MPLA, which is simpler in composition and easier to prepare and control in quality. 2) Compared with MF59, although the three main active ingredients are the same, when the amount of squalene is the same or similar, comparative experiments show that the composite adjuvant provided by the present invention has better immune effect, and the effect is comparable even when the amount of squalene is halved. 3) Compared with AS03 (which uses two oil substances, α-tocopherol and squalene, and the former is used in the largest amount), which brings obvious adverse reactions such as redness, swelling, and pain (Baay M et al., Vaccine. 2018 Jul 5; 36 (29): 4207-4214), the present invention significantly reduces the amount of oil substances, which can reduce the side effects such as pain and redness at the injection site caused by excessive oil substances, and reduce the reactogenicity of the composite adjuvant.

[0035] (3) In the oil-in-water composite adjuvant provided by the present invention, the particle size of the emulsion droplet particles is 150nm±30nm (preferably 150nm±20nm), the particle size distribution is less than 0.3 (preferably less than 0.2), and the Zeta potential is less than 0. It can be seen that it has a nanometer-level, uniformly distributed particle size range and the system is stable.

[0036] Preparation process:

[0037] (1) The present invention provides a method for preparing a small volume (2ml~30ml) of the composite adjuvant by continuous stirring. Although the preparation time of this method is relatively long, it does not require the use of a microfluidizer or a high-pressure homogenizer, which greatly reduces the experimental cost. Compared with the homogenizer, which loses a large amount of sample volume (about 25ml) due to pipeline residues and other reasons, the sample recovery rate during the stirring method experiment is high (more than 95%), and it has outstanding advantages in laboratory-scale small-volume preparation. In addition, if you want to increase the preparation volume, you can slightly modify the parameters in the oil phase-water phase mixing step on the basis of the stirring method (reduce the stirring speed and duration), and add a microfluidizer homogenization treatment after this step and before filtration and sterilization. It can be seen that the two methods have good compatibility.

[0038] (2) Since MPLA has a hydrophilic head (which carries a negative charge due to the phosphate group in a neutral or weakly acidic environment) and multiple long hydrophobic carbon chains in its molecular structure, it mainly exhibits fat-soluble hydrophobic properties (compatible with the oil phase) and is insoluble in water (https: / / avantilipids.com / product / 699855); therefore, the present invention adds MPLA to squalene (metabolizable oil) to prepare the oil phase, which is feasible in terms of process and helps MPLA to be quickly and evenly dispersed in squalene; then the oil phase and the water phase are fully mixed to obtain an oil-in-water emulsion. Based on its aforementioned physical and chemical properties, MPLA is distributed at the interface between the emulsion droplets (oil phase) and the water phase - the long carbon chains are buried inside the oil droplets and the hydrophilic heads are exposed at the oil-water interface (Anderson RC et al., Colloids Surf B Biointerfaces.123–132, 2010), the obtained water-in-oil composite adjuvant has good stability, while improving immunogenicity, promoting Th1 cell immune response, and reducing pyrogenicity (Jean Haensler, International Journal of Pharmaceutics 486 (2015) 99–111). BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Particle size intensity distribution diagrams of the three groups of adjuvant samples prepared in Section 2.4 of Example 2, wherein the upper, middle and lower figures correspond to groups 1, 3 and 5 in Table 2.4, respectively.

[0040] Figure 2 : In Example 3, the composite adjuvant stimulated cells to secrete TNF-α and IL-6 cytokines in vitro.

[0041] Figure 3 : In Section 4.1 of Example 4, whether the composite adjuvant contains the MPLA component affects the vaccine-induced antibody response.

[0042] Figure 4 : In Section 4.2 of Example 4, the effect of the ratio of squalene to MPLA in the composite adjuvant on the vaccine-induced antibody response.

[0043] Figure 5 : In Section 4.3 of Example 4, the vaccine prepared with a composite adjuvant combined with different doses of antigen induced antibody response in mice.

[0044] Figure 6 : In Section 4.4 of Example 4, different formulas of composite adjuvants were used to compare the antibody response induced by the vaccine formulated with homemade MF59 adjuvant combined with gE antigen.

[0045] Figure 7 : In Example 5, the total antibody response level induced by vaccines prepared with different adjuvants combined with F01 or F02 antigen in mice.

[0046] Figure 8 : In Example 5, the antibody typing distribution of mice induced by vaccines prepared with different adjuvants combined with F01 or F02 antigens.

[0047] Fig. 9 : In Example 5, the vaccines prepared with different adjuvants combined with F01 or F02 antigens induced the antibody IgG1 / IgG2a and IgG1 / IgG2b ratios produced in mice. DETAILED DESCRIPTION

[0048] In order to better present the purpose, technical scheme and beneficial effects of the present invention, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. Example 1 studies the condition parameters for preparing the composite adjuvant by the stirring method, Example 2 studies the components and proportions of the composite adjuvant, Example 3 studies the in vitro biological activity of the composite adjuvant, and finally, Examples 4 and 5 respectively study the immune-enhancing effect of the composite adjuvant in combination with varicella-zoster virus gE protein antigen and respiratory syncytial virus F protein antigen. The following is a detailed description.

[0049] Example 1: Exploration of condition parameters for preparing composite adjuvant by stirring method

[0050] The basic process of preparing 2mL~30mL volume composite adjuvant by stirring method is as follows:

[0051] (1) Preparation of oil phase: Dissolve solid powder MPLA (3D (6-acyl) -PHAD, Avanti Polar Lipids, 699855) in anhydrous ethanol solution and detect the MPLA concentration in the MPLA solution; accurately weigh the required amount of squalene (Hubei Gedian Renfu Pharmaceutical Excipients Co., Ltd.) into a 5 mL or 10 mL container; accurately measure the required amount of MPLA solution and add it to the container containing squalene; place a small magnetic stirrer of appropriate size and stir at 1500 rpm for 30 min; place the mixed solution in a centrifugal concentrator and centrifuge at 40 °C for 30 min until the solution is clear, and a uniform oil phase can be obtained.

[0052] (2) Preparation of aqueous phase: Accurately weigh the required amount of polysorbate 80 (Sigma) into a 20 mL or 100 mL container and add citric acid-sodium citrate buffer to make up the remaining volume; stir at 1500 rpm for 30 min to obtain a uniform aqueous phase.

[0053] (3) Oil-water phase mixing: Slowly add the uniformly mixed oil phase to the water phase; the mixed solution is placed in a 20 mL or 100 mL container and stirred continuously at 1500 rpm using a magnetic stirrer in a stoppered container protected from light; during the stirring process, fix the container in the center of the magnetic stirrer to avoid affecting the stirring effect; after the stirring is completed, observe the appearance of the composite adjuvant, which should be a milky white emulsion.

[0054] (4) Filtration and sterilization: The milky white emulsion obtained in the previous step is sterilized by filtration through a 0.22 μm pore size filter to obtain a composite adjuvant sample. The particle size, particle size distribution (PDI) and Zeta potential of the composite adjuvant sample can be measured and analyzed by a Malvern laser particle size analyzer. The samples to be used are stored in a refrigerator at 2-8°C away from light.

[0055] For the preparation of composite adjuvants by the stirring method, in this example, the effects of citric acid-sodium citrate buffer concentration (Section 1.1), stirring time (Section 1.2), and stirring temperature (Section 1.3) on the particle size distribution of the composite adjuvant were explored through gradient experiments.

[0056] Regarding the composite adjuvant sample, the present invention is based on previous theoretical research and determines that squalene, Tween 80 and MPLA are the main components of the composite adjuvant. Regarding the specific formula of the composite adjuvant, this study refers to the relative amounts of squalene and Tween in the MF59 formula of the listed influenza vaccine FLUADQUADRIVALENT. At the same time, considering that MPLA is expensive and the amount of MPLA added in the composite adjuvant is very small, its effect on particle size distribution is not expected to be significant. Therefore, in this embodiment, squalene and polysorbate 80 are used as the main components to configure the composite adjuvant. The following is a detailed description.

[0057] 1.1 Condition Parameter 1: Citric Acid-Sodium Citrate Buffer Concentration

[0058] Study the effect of different buffer concentrations on sample particle size and particle size distribution: According to the basic process of preparing composite adjuvants by the stirring method mentioned above, two batches of adjuvant samples were prepared under different concentrations of citric acid-sodium citrate buffer, and the particle size and particle size distribution were measured respectively. The specific scheme and results are shown in Table 1.1-1 below. Referring to the MF59 formula in the marketed vaccine and the commercially available MF59 formula, the adjuvant formula used in this study is: squalene 4% w / v, polysorbate 80 0.5% w / v, citric acid-sodium citrate buffer 5mM or 10mM.

[0059] Table 1.1-1: Adjuvants prepared with different concentrations of citric acid-sodium citrate buffer

[0060]

[0061] The results showed that the particle size of the adjuvant samples prepared under the conditions of 5mM and 10mM citric acid-sodium citrate buffer was 150nm±20nm, and the particle size distribution was less than 0.2, with no significant difference.

[0062] In order to further compare the stability differences of adjuvant samples prepared under two buffer concentration conditions, the above two batches of water-in-oil adjuvants were placed in a stability incubator at 2~8℃ and 25℃ for one month. Samples were taken on the 0th day, 4th day, 7th day, 14th day and 1st month to monitor the changes in appearance, pH, particle size and particle size distribution of the samples, and the pH and particle size change CV% were calculated. The results are shown in Tables 1.1-2 and 1.1-3 respectively.

[0063] Table 1.1-2: Stability of composite adjuvants prepared in 5 mM citric acid-sodium citrate buffer

[0064]

[0065] The results showed that for samples prepared with a citric acid-sodium citrate buffer concentration of 5 mM, (1) under storage conditions of 2-8°C: there was no significant change in appearance after one month; the CV% values ​​of the five monitoring points of pH and particle size were all less than 5%, with no significant change; and the particle size distribution was less than 0.2. (2) under storage conditions of 25°C: there was no significant change in appearance in the first 14 days, but after one month of storage, the sample showed stratification in appearance; the CV% values ​​of the five monitoring points of pH and particle size were all less than 5%, with no significant change; and the particle size distribution was less than 0.2.

[0066] Table 1.1-3: Stability of composite adjuvants prepared in 10.0 mM citric acid-sodium citrate buffer

[0067]

[0068] The results showed that for samples prepared with a citric acid-sodium citrate buffer concentration of 10 mM, (1) under 2-8°C conditions: there was no significant change in appearance after one month of storage; the CV% values ​​of the five pH monitoring points were less than 5%, and the CV% values ​​of the five particle size monitoring points were slightly greater than 5%, with no significant change; and the particle size distribution was less than 0.3. (2) Under 25°C storage conditions: there was no significant change in appearance in the first 14 days, but after one month of storage, the appearance showed stratification; the CV% values ​​of the five pH and particle size monitoring points were less than 5%, with no significant change; and the particle size distribution was less than 0.3.

[0069] In summary, the adjuvant samples prepared at 5mM or 10mM citric acid-sodium citrate buffer concentrations showed relatively consistent physicochemical properties and stability characteristics, but the CV% of the five monitoring points of particle size at 5mM citric acid-sodium citrate buffer concentration was relatively lower. Therefore, the citric acid-sodium citrate buffer can be selected from 5mM±1mM ~10mM±1mM (4mM-11mM), and the pH is controlled at 6.8±1. Preferably, the concentration of the citric acid-sodium citrate buffer is 5mM~10mM, and the pH is controlled at 6.8±0.5.

[0070] 1.2 Condition parameter 2: stirring time

[0071] Study the effect of different stirring times on sample particle size and particle size distribution [Note: The stirring time here refers to the continuous stirring time after the oil phase-water phase is mixed in step (3)]: According to the basic process of preparing composite adjuvants by the stirring method mentioned above, four batches of adjuvants were prepared based on different stirring times (16~48h), and their particle sizes and particle size distributions were measured respectively. The specific scheme and results are shown in Table 1.2 below. Referring to the MF59 formula in the marketed vaccine, the adjuvant formula used in this study is: squalene 4% w / v, polysorbate 80 0.5% w / v, citric acid buffer 5mM.

[0072] Table 1.2: Adjuvants prepared with different stirring times

[0073]

[0074] The results showed that when the stirring time of the stirring method was ≥40h, the particle size was 150nm±10nm, the particle size distribution was less than 0.2, and the particle uniformity was higher. Therefore, the stirring time was not less than 40h. In order to shorten the preparation cycle and ensure the full reaction of the adjuvant, the stirring time was preferably 48±8h.

[0075] 1.3 Condition parameter three: stirring temperature

[0076] Study the effect of different stirring temperatures on sample particle size and particle size distribution: According to the basic process of preparing composite adjuvants by the stirring method mentioned above, two batches of adjuvants were prepared at different stirring temperatures (room temperature and 40°C), and the particle size and particle size distribution were measured respectively. The specific scheme and results are shown in Table 1.3. The initial exploratory formula used in this study is: squalene 4% w / v, polysorbate 80 0.5% w / v, citric acid-sodium citrate buffer 5mM.

[0077] Table 1.3: Adjuvants prepared at different stirring temperatures

[0078]

[0079] The results showed that the particle size of the adjuvant samples prepared at room temperature or 40°C was 150nm±10nm, the particle size distribution was less than 0.3, and there was no significant difference between the two temperatures. In order to simplify the operation steps, room temperature reaction is preferred.

[0080] Based on the above research results, in the following studies, the stirring method for preparing the composite adjuvant adopted the following conditions and parameters: citric acid buffer concentration: 5mM; stirring time: 48h; stirring temperature: room temperature.

[0081] Example 2: Exploration of the content of key components in preparing composite adjuvants by stirring method

[0082] Based on the basic determination of the condition parameters for preparing the composite adjuvant by the stirring method in Example 1, this example further explored the effects of key components and their contents or ratios on the effects of the composite adjuvant, including squalene content (Section 2.1), polysorbate 80 content (Section 2.2), MPLA content (Section 2.3), and different ratios of squalene and MPLA (Section 2.4). Considering the high price of MPLA and the small amount of MPLA added to the composite adjuvant, it has little effect on the particle size distribution. Therefore, in this example, MPLA is not introduced when studying the contents of squalene and polysorbate 80. In the subsequent study of the content of MPLA and its ratio with squalene, all of the above three key components are introduced to configure the composite adjuvant.

[0083] 2.1 Squalene content

[0084] Study the effect of different squalene contents on the physical parameters of adjuvant samples: According to the basic process of preparing composite adjuvants by the stirring method mentioned above, adjuvant samples with different squalene contents were prepared, and the particle size and particle size distribution were measured respectively. The specific scheme and results are shown in Table 2.1. The adjuvant formula used in this study is as follows: squalene content 1%~10% w / v, polysorbate 80 0.5% w / v, citric acid buffer 5mM.

[0085] Table 2.1: Composite adjuvants with different squalene contents

[0086]

[0087] The results show that when the squalene content is 1%, 2%, 4%, 6%, 8%, and 10%, respectively, the particle size of the prepared adjuvant is 150nm±10nm, and the particle size distribution is less than 0.3. Among them, when the squalene content is 1%, 2%, 4%, and 6%, the particle size is 150nm±10nm, and the particle size distribution is less than 0.25. Further, when the squalene content is 4% or 6%, the particle size is 150nm±10nm, the particle size distribution is less than 0.2, and the particle uniformity is better. Therefore, the squalene content can be 1%~10%, preferably 1%~6%, and further preferably 4%~6%.

[0088] 2.2 Polysorbate 80 (Tween 80) content

[0089] Study the effect of different polysorbate 80 contents on the physical parameters of adjuvant samples: According to the basic process of preparing composite adjuvants by the stirring method mentioned above, samples with different polysorbate 80 contents were prepared, and the particle size and particle size distribution were measured respectively. The specific scheme and results are shown in Table 2.2. The adjuvant formula used in this study is as follows: squalene 4% w / v, polysorbate 80 0.4%~6.4% w / v, citric acid buffer 5mM.

[0090] Table 2.2: Composite adjuvants with different polysorbate 80 contents

[0091]

[0092] The results showed that when the content of polysorbate 80 was 0.4%, 0.5%, 0.8%, 1.0%, 1.2%, 1.6%, 3.2%, and 6.4%, respectively, the particle size of the prepared composite adjuvant was 150nm±30nm, and the particle size distribution was less than 0.3. Among them, when the content of polysorbate 80 was 0.5%, 0.8%, 1.0%, 1.2%, 1.6%, and 3.2%, the particle size was 150nm±20nm, and the particle size distribution was less than 0.25. Further, when the content of polysorbate 80 was 0.8%, 1.0%, 1.2%, 1.6%, and 3.2%, the particle size was 150nm±20nm, the particle size distribution was less than 0.2, and the particle uniformity was better. Further, when the content of polysorbate 80 was 1.0%, 1.2%, and 1.6%, the particle size was 150nm±10nm, the particle size distribution was less than 0.2, and the particle size was closer to 150nm. Therefore, the content of polysorbate 80 may be 0.4% to 6.4%, preferably 0.5% to 3.2%, further preferably 0.8% to 3.2%, and further preferably 1.0% to 1.6%.

[0093] 2.3 MPLA content

[0094] Study the effect of different MPLA contents on the physical parameters of adjuvant samples: According to the basic process of preparing composite adjuvants by the stirring method mentioned above, adjuvant samples with different MPLA contents were prepared, and the particle size, particle size distribution and Zeta potential were measured respectively. The specific scheme and results are shown in Table 2.3. The adjuvant formula used in this study is as follows: squalene 4% w / v, polysorbate 80 1% w / v, MPLA 0.02~0.4 mg / mL, citric acid buffer 5mM.

[0095] Table 2.3: Composite adjuvants with different MPLA contents

[0096]

[0097] The results show that the Zeta potential of the particle surface flips from positive charge to negative charge as the MPLA content increases. In view of the fact that the MPLA molecular structure has 6 long carbon chains and a head with a certain hydrophilicity and negative charge, it is speculated that in the O / W emulsion prepared by the present invention, MPLA basically exists in the dispersed system in the form of inserting into the surface of the oil droplet particles, thereby exposing the negatively charged head, making the Zeta potential negative (Anderson RC et al., Colloids Surf B Biointerfaces. 123–132, 2010). At the same time, after the Zeta potential is negative, as the MPLA content increases, the absolute value of the potential increases, indicating that the higher the concentration of MPLA, the more helpful it is to maintain the stability of the adjuvant O / W nanoparticle system.

[0098] According to the above table, when the MPLA content in the prepared oil-in-water adjuvant is 0.04 mg / mL, 0.1 mg / mL, 0.2 mg / mL, and 0.4 mg / mL, the particle size is 150 nm ± 10 nm, the particle size distribution is less than 0.3, and the potential is less than 0 mV. Further, when the MPLA content is 0.1 mg / mL, 0.2 mg / mL, and 0.4 mg / mL, the particle size is 150 nm ± 10 nm, and the particle size distribution is less than 0.2. Therefore, the MPLA content can be 0.04 mg / mL to 0.4 mg / mL, preferably 0.1 mg / mL to 0.4 mg / mL.

[0099] 2.4 Ratio of Squalene and MPLA

[0100] In the invented composite adjuvant, since the main biologically active ingredients are the two core components of squalene and MPLA, it is necessary to further explore the dosage ratio of the two components. According to the basic process of preparing composite adjuvants by the aforementioned stirring method, composite adjuvants with different ratios of squalene and MPLA were prepared, and their particle size, particle size distribution, Zeta potential and recovery rate (reflecting the content of effective ingredients) were measured respectively, and the effect of the ratio on various quality parameters of the composite adjuvant was evaluated. Combined with the results of the previous formula screening, the adjuvant formula used in this study is as follows: 2%~6% w / v for squalene, 0.1~0.4 mg / mL for MPLA, 1% w / v for polysorbate 80, and 5mM for citric acid-sodium citrate buffer. The specific scheme and results are shown in Table 2.4 below, among which the particle size distribution test results of some representative adjuvant samples are shown in Table 2.4. Figure 1 shown.

[0101] Table 2.4: Composite adjuvants with different ratios of squalene and MPLA

[0102]

[0103] The results showed that except for the highest dose of squalene and MPLA at the same time (i.e., Group 7, 6% and 0.4 mg / mL, respectively), the adjuvant samples prepared with different dose ratios in the remaining groups (Group 1 to Group 6) were in line with expectations, with particle sizes within the range of 150nm±30nm, particle size distribution less than 0.3, and the recovery rate of the active ingredient content in each group met the initial expectations (100%±30%). Further, when the squalene content was not 2% of the lowest dose (Group 2 to Group 6), the sample particle size was 150nm±20nm, and the particle size distribution was less than 0.3. Furthermore, when the squalene content was 4% (Group 2 to Group 4), the sample particle size was 150nm±10nm, the particle size distribution was less than 0.2, and the recovery rate of the active ingredient content of squalene and MPLA was 100%±10%.

[0104] According to the above research results, in the subsequent study of the in vitro and in vivo biological activities of the composite adjuvants, the representative composite adjuvant formula shown in Table 2.5 was used and compared with the commercialized or homemade MF59 adjuvant.

[0105] Table 2.5: Representative formulations of composite adjuvants

[0106]

[0107] Example 3: Study on the biological activity of composite adjuvants in vitro

[0108] In the process of vaccine development, the mechanism of action of adjuvants is complex, and the mechanism of action of different adjuvants is also different. However, in general, in addition to partial adsorption and sustained release effects, more of them stimulate the body's natural immune response, thereby assisting antigens to produce better immunogenicity and enhancing the immune effect of vaccines. The composite adjuvant of the present invention comprehensively utilizes two components: oil-in-water (O / W) emulsion and immunopotentiator MPLA. In theory, it not only retains the recruitment and stimulation of natural immune cells by O / W adjuvants, but also can stimulate the TLR4 natural immune pathway through MPLA, and better stimulate the natural immune response. Specifically, it can be manifested as a significant increase in the expression level of some immune cytokines after stimulation, such as IL-6, TNF-α, etc., thereby achieving a better adjuvant effect.

[0109] Therefore, in order to study the natural immunostimulatory effect of the composite adjuvant, five different groups of samples were prepared as shown in Table 3.1, including the composite adjuvant (code: Adjuvant 1), and the following four samples as controls: single-component control (MPLA only, code: "MPLA"), two-component control (squalene + polysorbate 80, code: squalene control), commercial MF59 adjuvant (Invivogen, Addavax ＠ , code: MF59), blank stimulation negative control (code: blank control), and the in vitro stimulatory activity effect of the adjuvant was studied based on THP-1 cells.

[0110] Table 3.1: Formulations of different natural immune response stimulators

[0111]

[0112] ※ Regarding the commercial MF59 adjuvants listed in the above table (Invivogen, AddaVax TM ) was prepared based on the density of each ingredient in its product information (squalene 5% v / v, Span 85 0.5% w / v, Tween 80 0.5% w / v) and the density of squalene (0.858 g / mL at 25°C): mass-to-volume ratio (w / v) = volume-to-volume ratio (v / v) × density.

[0113] The specific method is as follows: resuspend THP-1 cell line in RPMI 1640 complete medium, after cell counting, plate cells at a certain density in 96-well cell culture plates, and add Phorbol-12-Myristate-13-acetate (PMA) (Sigma, Cat. no. 79346) at a concentration of 100 ng / mL for stimulation and induction for 24 hours. After the induction, wash the plate 3-5 times with RPMI 1640 complete medium to remove PMA stimulants, and then add RPMI 1640 complete medium for overnight culture. After overnight culture, wash the plate 3-5 times with serum-free RPMI 1640 medium. Serum-free RPMI 1640 medium is used as a diluent to dilute MPLA single component and composite adjuvants at a certain MPLA final concentration, and at the same time dilute the commercial MF59 adjuvant and squalene control adjuvant at the same dilution multiple, add equal volumes of each stimulant to each cell culture well, and serum-free RPMI 1640 medium is used as a blank stimulation negative control. After 24 hours of stimulation culture, the culture supernatant was collected and the content of cytokine TNF-α in the supernatant was detected by ELISA; after 48 hours of stimulation culture, the culture supernatant was collected and the content of cytokine IL-6 in the supernatant was detected by ELISA.

[0114] The results are as follows Figure 2 As shown, compared with the single-component control (MPLA only), squalene control (squalene + polysorbate 80), commercial adjuvant MF59 or blank stimulation negative control, the composite adjuvant 1 can stimulate the mononuclear macrophages differentiated from THP-1 cells induced by PMA to secrete and express significantly higher levels of TNF-α and IL-6 cytokines, reflecting better in vitro stimulation activity of natural immunity.

[0115] Example 4: Preparation of recombinant herpes zoster vaccine by mixing composite adjuvant with varicella-zoster virus gE protein antigen

[0116] Preparation of gE antigen: The varicella-zoster virus gE protein antigen stock solution was prepared using the insect cell-baculovirus expression system. The amino acid sequence of the gE antigen (GenBank: AXF42733.1, amino acid sequence: 1-546, N-terminal truncated) was codon-optimized and cloned into the pFastBac vector by gene synthesis, and then integrated into the baculovirus genome by Bac-to-Bac (Invitrogen, Cat. no.A11100). After the baculovirus was amplified in a shake flask and infected with insect Hi5 cells, the expression supernatant was harvested and purified by strong anion column and weak hydrophobic column chromatography to obtain the gE protein antigen stock solution. The results showed that the purity of the prepared gE protein was higher than 90% and was stored in 5mM citric acid buffer.

[0117] Preparation and immunization: In order to study the immunogenicity of the recombinant herpes zoster vaccine prepared by mixing different formulas of composite adjuvants with gE antigen, the homemade gE antigen stock solution was mixed with the prepared composite adjuvants of different formulas in a mixing ratio of 1:1 and then injected intramuscularly into C57 / BL6 mice. The immunization interval was 2 weeks, and the immunization was performed twice in total. The mouse serum was collected 2 weeks after the last immunization, and the gE protein-specific antibody response level was detected by ELISA to evaluate the effect of different formulas of adjuvants on the immunogenicity of the vaccine.

[0118] 4.1 Comparison of compound adjuvants with and without MPLA

[0119] As shown in Table 4.1 below, a composite adjuvant containing 0.2 mg / mL MPLA and a control adjuvant containing no MPLA component were prepared by the stirring method, and the gE antigen was mixed at a volume ratio of 1:1 to immunize mice, and the gE-specific antibody reaction level was detected by ELISA. Figure 3 The results showed that the antibody level induced by the composite adjuvant with added MPLA component combined with antigen was significantly higher than that of the control adjuvant without MPLA component, indicating that MPLA is an important adjuvant active component in the composite adjuvant and can significantly enhance the immune response of the vaccine.

[0120] Table 4.1: Dosage information for adjuvant-gE antigen formulations with and without MPLA components

[0121]

[0122] 4.2 Comparison of different ratios of squalene and MPLA as composite adjuvants

[0123] As shown in Table 4.2 below, composite adjuvants containing different proportions of squalene and MPLA were prepared by stirring method, mixed with gE antigen at a volume ratio of 1:1, and then immunized mice, and the gE-specific antibody reaction level was detected by ELISA. Figure 4 The results showed that the level of antibody response induced by the vaccine increased with the increase of MPLA dose, and higher concentration of squalene dose of composite adjuvant was also conducive to the vaccine inducing higher levels of antibody response, and the overall immunogenicity showed a good dose-dependent effect.

[0124] Table 4.2: Dosage information of adjuvants with different ratios of squalene and MPLA combined with gE antigen

[0125]

[0126] 4.3 Study on the immunogenicity of vaccines prepared with composite adjuvants mixed with different doses of antigens

[0127] As shown in Table 4.3 below, the composite adjuvant prepared by the stirring method was mixed with different doses of gE antigen at a volume ratio of 1:1 and then immunized into mice, and the gE-specific antibody reaction level was detected by ELISA. Figure 5 It showed that the antibody titer induced by immunizing mice with the composite adjuvant combined with different gE antigen doses showed a good dose-dependent effect.

[0128] Table 4.3: Information on the preparation of composite adjuvants combined with different doses of gE antigen

[0129]

[0130] 4.4 Comparison of different compound adjuvants with homemade MF59 adjuvant

[0131] As shown in Table 4.4 below, MF59 adjuvant was prepared according to the commercial MF59 adjuvant formula. At the same time, composite adjuvants with different formulas were prepared by stirring method. The commercial gE protein (Bipsis, catalog number GLE-V52H3) was mixed at a volume ratio of 1:1 and then immunized with mice. There were 8 mice in each group. Blood was collected from mice in each group 14 days after the last immunization, serum was separated, and the gE-specific antibody reaction level was detected by ELISA. The results are shown in Figure 6 The results showed that the antibody response levels induced by different formulas of composite adjuvants combined with antigens were significantly higher than those induced by MF59 adjuvant, and the antibody level gradually increased with the increase of squalene and / or MPLA doses, further illustrating the significant enhancing effect of composite adjuvants on vaccine immunogenicity during vaccine development.

[0132] Table 4.4: Dosage information of different formula composite adjuvants and homemade MF59 adjuvant combined with gE antigen

[0133]

[0134] Example 5: Preparation of recombinant respiratory syncytial virus vaccine by mixing composite adjuvant with respiratory syncytial virus F protein antigen

[0135] F antigen preparation: The respiratory syncytial virus surface fusion protein (Fusion, F) is the most important antigen protein for the development of recombinant respiratory syncytial virus vaccine. The wild-type F protein amino acid sequence (GenBank: AEQ63389.1) was modified to a certain extent (point mutation, truncation of the C-terminal transmembrane region and intracellular region, and addition of the Foldon trimerization motif and histidine purification tag at the C-terminus), so that the F protein can maintain a stable pre-fusion trimer conformation after expression and can be secreted and expressed in the cell culture supernatant, obtaining two antigen designs, F01 and F02. After the antigen amino acid sequence was codon-optimized, it was cloned into the pFastBac vector by gene synthesis and integrated into the baculovirus genome by Bac-to-Bac (Invitrogen, Cat. no. A11100). After the baculovirus was amplified in a shake flask and infected with insect Hi5 cells, the expression supernatant was harvested and then purified by nickel column to obtain the two protein antigen stock solutions of F01 and F02. The results showed that the purity of the prepared proteins was higher than 90% and was stored in phosphate buffer.

[0136] Vaccine preparation and immunization: Adjuvant-compound adjuvant was prepared by stirring method. According to Table 5.1, the compound adjuvant or commercial MF59 adjuvant (Aveto, Cat. no. 172889-84-8, containing squalene 4.3% w / v, Tween 80 0.5% w / v) was mixed with F01 or F02 antigen at a volume ratio of 1:1 to prepare recombinant respiratory syncytial virus vaccine, and a control group without adjuvant vaccine was set up. Balb / c mice were immunized by intramuscular injection twice with an interval of three weeks. The mouse serum was collected 2 weeks after the last immunization, and the serum antigen-specific total antibody response level was detected by ELISA. At the same time, the response level of different subtype antibodies in the serum was detected by a kit (Invitrogen, Cat. no. 88-50630-86).

[0137] Table 5.1: Dosage information for different respiratory syncytial virus vaccines for major immunization groups

[0138]

[0139] The total IgG antibody response level test results are as follows Figure 7 As shown in the figure, the antibody titer of mice induced by RSV-F01 protein combined with adjuvant-compound adjuvant group was higher than that of MF59 group, and both groups were much higher than that of the no adjuvant group. The antibody titer of mice induced by RSV-F02 protein combined with adjuvant-compound adjuvant group was equivalent to that of MF59 group, and both groups were much higher than that of the no adjuvant group.

[0140] Antibody typing test results Figure 8As shown in the figure, RSV-F01 and RSV-F02 antigens showed a relatively consistent trend, that is, overall, the adjuvant group had significantly higher antibody levels of each subtype than the non-adjuvant group. Except for IgG1, where the levels of adjuvant 1 and MF59 were comparable, and IgA levels in all groups were lower, for the remaining antibody subtypes, the antibody levels induced by adjuvant 1 and composite adjuvant were significantly higher than those of commercial MF59.

[0141] In addition, in the mouse model, the ratios of serum antibody IgG1 / IgG2a and IgG1 / IgG2b can indirectly reflect the bias of CD4+ T cell subtypes induced by different vaccines. Specifically, when the ratio is significantly less than 1, it shows a T helper 1 (Th1) bias; when the ratio is approximately equal to 1, it shows a Th1 / Th2 balance; when the ratio is significantly greater than 1, it shows a Th2 bias. Different biased Th responses have different applicability in the development of different vaccines. Therefore, in order to further study the bias of CD4+ T cell subtypes of the vaccine prepared based on the composite adjuvant of the present invention, the ratios of antibody IgG1 / IgG2a and IgG1 / IgG2b induced by each immunization group were calculated, and the results are shown in the following figure. Fig. 9 As shown in the figure, RSV-F01 and RSV-F02 antigens showed relatively consistent trends, that is, in the immunization groups without adjuvant and with commercial MF59 adjuvant, the IgG1 / IgG2a and IgG1 / IgG2b ratios were significantly greater than 1, showing a clear Th2 bias; in the group with adjuvant-compound adjuvant, the IgG1 / IgG2a and IgG1 / IgG2b ratios were close to 1, showing a Th1 / Th2 balanced T cell response, which was also more conducive to inducing a variety of high-titer antibody responses of different subtypes.

[0142] In summary, the recombinant respiratory syncytial virus vaccine prepared by mixing the composite adjuvant with the respiratory syncytial virus F protein antigen can significantly enhance the response levels of total IgG antibodies and antibodies of various subtypes compared with the commercial MF59 adjuvant or no adjuvant, and shows a relatively balanced Th1 / Th2 bias. It can be used as a simple and efficient composite adjuvant to develop vaccine products with stronger and more lasting protection.

[0143] The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art can make conventional modifications or equivalent substitutions based on the technical solutions provided by the present invention, and such conventional modifications or equivalent substitutions should also be deemed to fall within the protection scope of the present invention.

Claims

1. A composite adjuvant comprising squalene, polysorbate 80 and monophosphoryl lipid A (MPLA); the composite adjuvant is in the form of an oil-in-water emulsion, wherein the emulsion droplets are formed by distributing the oil phase in the aqueous phase, and the particle size of the emulsion droplets is 150nm±30nm, preferably 150nm±20nm, and more preferably 150nm±10nm.

2. The composite adjuvant according to claim 1, characterized in that The composite adjuvant does not contain Span 85 or tocopherol.

3. The composite adjuvant according to any one of claims 1 to 2, characterized in that In the oil-in-water emulsion, the oil phase contains squalene and MPLA, and the water phase contains polysorbate 80.

4. The composite adjuvant according to any one of claims 1 to 3, characterized in that In the composite adjuvant, the content of squalene is 1.0%-10.0% w / v, the content of polysorbate 80 is 0.4%-6.4% w / v, and the content of MPLA is 0.04-0.4 mg / mL; Preferably, the squalene content is 1.0%-6.0% w / v, the polysorbate 80 content is 0.5%-3.2% w / v, and the MPLA content is 0.1-0.4 mg / mL; Further preferably, the squalene content is 4%-6% w / v, the polysorbate 80 content is 0.8%-3.2% w / v, and the MPLA content is 0.1-0.4 mg / mL; Further preferably, the squalene content is 4%-6% w / v, the polysorbate 80 content is 1%-1.6% w / v, and the MPLA content is 0.1-0.4 mg / mL; Further preferably, the squalene content is 4% w / v, the polysorbate 80 content is 1%-1.5% w / v, and the MPLA content is 0.2-0.4 mg / mL; Further preferably, the content of squalene is 4% w / v, the content of polysorbate 80 is 1% w / v or 1.5% w / v, and the content of MPLA is 0.2 mg / mL or 0.4 mg / mL.

5. The composite adjuvant according to any one of claims 1 to 4, characterized in that The MPLA may be selected from: 3D (6-acyl) -PHAD, PHAD, 3D-PHAD, and / or PHAD-504; preferably, the MPLA is 3D (6-acyl) -PHAD.

6. The composite adjuvant according to any one of claims 1 to 5, characterized in that The aqueous phase is prepared with citric acid-sodium citrate buffer.

7. The composite adjuvant according to claim 6, characterized in that The molar concentration of the citric acid-sodium citrate buffer is 4mM-11mM, pH 6.8±1; the molar concentration of the citric acid-sodium citrate buffer is preferably 5mM-10mM, pH 6.8±0.

5.

8. The composite adjuvant according to any one of claims 1 to 7, characterized in that The particle size distribution of the emulsion particles is less than 0.3, preferably less than 0.2, and the Zeta potential is less than 0.

9. Use of the composite adjuvant according to any one of claims 1 to 8 in the preparation of an immunogenic composition.

10. The use according to claim 9, characterized in that The composite adjuvant and the antigen solution are mixed in a volume ratio of (1-10):(1-10) to prepare the immunogenic composition.

11. An immunogenic composition, characterized in that The method comprises an antigen and / or an antigen composition, and the composite adjuvant according to any one of claims 1 to 8.

12. The immunogenic composition according to claim 11, characterized in that The type of the antigen and / or antigen composition is selected from one or more of the following: recombinant protein antigen, polypeptide antigen, polysaccharide antigen, polysaccharide-protein conjugate antigen, nucleic acid antigen, inactivated or attenuated pathogen.

13. The immunogenic composition according to claim 12, characterized in that The type of the antigen and / or antigen composition is selected from recombinant protein antigens; preferably, the antigen and / or antigen composition is selected from varicella-zoster virus recombinant protein antigen (such as gE protein), or respiratory syncytial virus recombinant protein antigen (such as F protein antigen).

14. The immunogenic composition according to any one of claims 11 to 13, characterized in that The immunogenic composition is prepared by mixing the composite adjuvant with a solution containing the antigen and / or antigen composition at a volume ratio of (1-10):(1-10).

15. A method for preparing the composite adjuvant according to any one of claims 1 to 8, wherein the method is a stirring method, comprising the following steps: (1) Preparation of oil phase: 1) Weigh the required amount of squalene and MPLA respectively and add them into container A; 2) Stir and mix to prepare the oil phase: the stirring parameters are selected as 1500 rpm ± 500 rpm and stirring for 1 h ± 0.5 h; (2) Preparation of aqueous phase: 1) Weigh the required amount of polysorbate 80 and add it to container B, and make up the balance with 4 mM-11 mM, pH 6.8±1 citric acid-sodium citrate buffer; 2) Stir and mix to prepare the aqueous phase: the stirring parameters are selected as 1500 rpm±500 rpm and stirring for 30 min±10 min; (3) Oil phase-water phase mixing: Slowly add the uniformly mixed oil phase to the water phase, and then stir continuously: the stirring parameters are selected as 1500 rpm ± 500 rpm, not exceeding 40°C (preferably room temperature), and stirring is continued for more than 40 hours in the dark. After the stirring is completed, a milky white emulsion is obtained; (4) Filtration and sterilization: The milky white emulsion obtained in the previous step is filtered and sterilized through a 0.22 μm filter to obtain the composite adjuvant; In the above step (1), preferably, the specific method of step 1) is as follows: a) weighing squalene and adding it to container A, b) weighing MPLA and adding it to container A containing squalene; further preferably, the specific method of step b) is as follows: dissolving MPLA in an organic solvent to prepare an MPLA solution of a certain concentration, calculating the volume of the MPLA solution to be added according to the required amount of MPLA in the composite adjuvant, and then adding it to container A containing squalene, and adding the following operation after stirring and mixing in step 2): removing the organic solvent from the oil phase until the solution is clear.

16. The method for preparing the composite adjuvant according to claim 15, characterized in that: The stirring parameters in step (1) are: 1500 rpm, stirring for 0.5 h to 1 h.

17. The method for preparing the composite adjuvant according to any one of claims 15 to 16, characterized in that: In the step (2), a 5 mM-10 mM citric acid-sodium citrate buffer solution with a pH of 6.8±0.5 is used.

18. The method for preparing the composite adjuvant according to any one of claims 15 to 17, characterized in that: The stirring parameters in step (2) are: 1500 rpm, stirring for 30 min ± 10 min.

19. The method for preparing the composite adjuvant according to any one of claims 15 to 18, characterized in that: The stirring parameters in step (3) are as follows: 1500 rpm, continuous stirring at room temperature in the dark for 48 h±8 h.

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