Safe and efficient nanoemulsion / aluminum gel composite adjuvant system for whole immune response and application thereof

Abstract

The present application relates to the field of vaccine adjuvants, in particular to a safe and efficient comprehensive immune response nanoemulsion / aluminum gel composite adjuvant system and its application, the composite adjuvant system can significantly enhance the humoral immune response and obviously have the release characteristics of aluminum salt gel adjuvant and the rapid enhancement of cellular immune response of nanoemulsion adjuvant. The composite adjuvant system adopts the combination of sustained release and immediate release preparation of aluminum salt and nanoemulsion in the adjuvant, and the synergistic effect of efficient humoral and cellular immune response can promote the delivery of antigen presenting cells such as dendritic cells and macrophages, more efficiently start immune response, form more stable, more durable, more comprehensive immune memory. The composite adjuvant system has simple preparation process, easy to obtain raw materials, and is convenient for large-scale industrial application. After being combined with recombinant subunit vaccine, the effect is better than that of single aluminum salt and nanoemulsion adjuvant widely used in clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a safe and efficient nanoemulsion / aluminum gel composite adjuvant system for comprehensive immune response, and also relates to the application of the composite nano-adjuvant system and recombinant subunit vaccine. BACKGROUND

[0002] Vaccines play an extremely important role in the immunoprophylaxis of major infectious diseases, but the immune adjuvants, which are an important component of vaccines, greatly limit the full play of the immunoprophylaxis of modern new vaccines due to their relatively single composition and immune response effect. Adjuvants play an important role in the composition of vaccines. At present, aluminum salt gel and MF59 as representative emulsion, high molecular polymer microspheres, gel preparation, sustained-release stents and implants have the function of drug reservoir, can maintain the natural structure of antigens, and can slow down the release of antigens, achieve high-efficiency and low-toxicity immune effect, and have certain sustained-release or rapid-release effect. However, the immune response triggered by the above-mentioned single adjuvant system does not last long enough, and the durability and long-term memory of the vaccine cannot be achieved at all, which greatly limits the application of vaccines. The single adjuvant system has low immune response level, biased Th1 or Th2 type reaction, weak presentation and delivery cell ability, toxicity and large side effects, so it limits its application and development as another "immunogen" to assist antigens to achieve double stimulation of antibody production, and the composite adjuvant, especially the composite adjuvant system, has become a new development trend of adjuvant.

[0003] Aluminum salts have been used in clinic for more than 80 years, and it is the first classic adjuvant approved by FDA for human use. Many vaccines contain aluminum salts, such as DPT vaccine and H. influenzae vaccine. According to the preparation process, aluminum salt adjuvanted vaccines can be divided into two types: aluminum adsorbed vaccine and aluminum precipitated vaccine. Aluminum adsorbed vaccine is to add antigen into aluminum hydroxide or aluminum phosphate solution; while aluminum precipitated vaccine is to add aluminum agent suspension into antigen solution. Aluminum hydroxide or aluminum phosphate is the most commonly used aluminum adjuvant. Studies have found that aluminum adjuvant vaccine can reduce the amount of antigen used, and enhance the intensity and duration of the body's immune response. The mechanism of action of aluminum salts is not yet fully understood, and it is generally believed that the antigen adsorbed aluminum salt particles form a gel state, which forms an antigen storage depot after injection into the animal body. These insoluble particles can adsorb antigenic material, increasing the antigenic surface area. In addition, adjuvants can form granulomas rich in macrophages at the injection site, delaying antigen absorption, thereby prolonging the stimulation time of the antigen, leaving the antigen that would normally exist for several days for several weeks, and the ability of the injection site to take up the antigen is enhanced. Studies have shown that aluminum hydroxide as an adjuvant can also activate Th2 cells to secrete IL-4, induce the expression of CD83, CD86 and MHC-II molecules, and thus produce Th2 type humoral immune response. Aluminum salts as vaccine adjuvants have many advantages, but also have disadvantages. Although it can effectively induce humoral immune response, it does not work on cellular immunity and cannot induce cellular immune response.

[0004] Italy was the first to approve the influenza subunit vaccine containing MF59 adjuvant in 1997; the European Union approved the H1N1 vaccine containing AS03 (Montanide) W / O oil-in-water adjuvant with vitamin E in 2009; the human papilloma virus containing AS04 adjuvant (aluminum hydroxide and 3-O-deacylated monophosphoryl lipid A) was approved in 2009. The emulsion with a particle size less than 100 nm is called nanoemulsion, which is composed of oil phase-water phase-surfactant-co-surfactant. The nanoemulsion with continuous phase of water phase and dispersed phase of oil phase is O / W type, and the nanoemulsion with continuous phase of oil phase and dispersed phase of water phase is W / O type. Nanoemulsion has the advantages of small and uniform particle size, easy preparation, good stability and flowability, and easy injection, and can be used as a drug carrier in various drug preparations. The transparent or translucent nanoemulsion is a thermodynamically stable, isotropic, transparent or translucent dispersion system formed spontaneously after mixing oil phase, water phase, surfactant and co-surfactant in a certain proportion. Nanoemulsion can be used as a new type of vaccine carrier to promote the absorption of vaccine protein molecules in vivo, thereby improving the immune effect of the vaccine, and has the advantages of high safety and good stability, and will show unique advantages in enhancing the immune effect of the vaccine.

[0005] The research and development strategy and advantages of the AS series of compound adjuvants lie in their effective combination with immune enhancers or immunostimulatory molecules, including MPL, QS21, and CpG, while focusing on vaccine protein delivery systems. This results in a novel, diverse, and complementary compound immune adjuvant and drug delivery system that not only provides sustained release and antigen delivery at the vaccination site but also fully leverages the immune enhancers to stimulate the body to generate highly efficient protective humoral and cellular immune responses. Numerous human clinical and preclinical studies have confirmed its stable immune response and safe, effective immunoprotective effects. Immunostimulatory molecules, as a crucial component of the AS series adjuvants, primarily function to enhance immune response. Currently, the development of compound adjuvants is relatively limited. Compound traditional Chinese medicine adjuvants, Western medicine adjuvants, compound mucosal immune adjuvants, ISCOM (Immunostimulating complex), aluminum salt adjuvants, and polylactic-co-glycolic acid (PLGA) all have their own limitations. For example, ISCOM can only form complexes with antigens or immunogens containing many hydrophobic groups. In clinical use, certain adjuvants have also presented numerous problems, causing local inflammation, granulomas, or aseptic abscesses in vaccinated animals, as well as systemic reactions such as discomfort, fever, and adjuvant-induced arthritis. Most importantly, while these compound adjuvants can enhance the immune response of vaccines to varying degrees, they fail to address the key issues currently limiting vaccine application—immune persistence and long-lasting immune memory. Therefore, exploring the promoting effects of novel composite nanoemulsion adjuvant systems on release efficacy, immunoprotective effects, and immune memory is crucial for the application of vaccine adjuvants. Globally, there is no combination of emulsions and aluminum salt adjuvants to form compound adjuvants; therefore, this patent combines an aluminum salt adjuvant that significantly enhances humoral immune responses with a nanoemulsion that enhances cellular immune responses. Summary of the Invention

[0006] In view of this, one objective of the present invention is to provide a safe and efficient nanoemulsion / aluminum gel composite adjuvant system for a comprehensive immune response; a second objective of the present invention is to provide the application of the composite adjuvant system as a vaccine adjuvant; and a third objective of the present invention is to provide a vaccine containing the composite adjuvant system.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] 1. A safe and efficient nanoemulsion / aluminum gel composite adjuvant system for a comprehensive immune response, the composite adjuvant system comprising an aluminum salt adjuvant that enhances humoral immune response and a nanoemulsion adjuvant that enhances cellular immune response.

[0009] In this invention, the aluminum salt adjuvant includes at least one of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum sulfate adjuvant, ammonium alum adjuvant, or potassium alum adjuvant.

[0010] In the present application, the nanoemulsion adjuvant composition is composed of a surfactant, a co-surfactant and an oil phase.

[0011] In the present application, the surfactant is any one or a combination of Tween-80, Tween-85, Span 85, Tween 60, polyoxyethylene hydrogenated castor oil and polyoxyethylene castor oil; the co-surfactant is any one or a combination of sorbitan fatty acid ester, C2-C4 monohydric alcohol and C2-C4 polyhydric alcohol; and the oil phase is any one or a combination of paraffin oil, ethyl acetate, myristyl isopropyl ester, squalane, squalene, ethyl oleate and capryl / caprylic triglyceride.

[0012] In the present application, the mass fraction of the aluminum salt adjuvant is 50-80%, and the mass fraction of the nanoemulsion adjuvant is 20-50%.

[0013] In the present application, the composite adjuvant system is composed of an aluminum hydroxide adjuvant and a nanoemulsion adjuvant in a mass ratio of 8:2, and the mass fractions are 80% and 20%, respectively.

[0014] 2. A vaccine containing the composite adjuvant system.

[0015] Preferably, the adjuvant is used in an amount of 50 μg / ml-1.5 mg / ml, more preferably 200 μg / ml-800 μg / ml.

[0016] 3. The vaccine antigen content is 15 μg / ml-1.5 mg / ml, more preferably 60 μg / ml-240 μg / ml.

[0017] 4. The vaccine is a Staphylococcus aureus recombinant subunit vaccine.

[0018] The present application has the advantages that: the present application provides a safe, efficient and comprehensive nanoemulsion / aluminum salt gel composite adjuvant system, which is composed of an aluminum salt adjuvant for enhancing humoral immune response and a nanoemulsion adjuvant for enhancing cellular immune response. The synergistic effect of the aluminum salt adjuvant and the nanoemulsion adjuvant can promote the delivery of antigen-presenting cells such as dendritic cells and macrophages, more efficiently initiate immune response, and form more stable, persistent and comprehensive immune memory. The composite adjuvant system has simple preparation process, readily available raw materials and is easy to industrialize and apply. When used in combination with a recombinant subunit vaccine, the effect is better than that of the single aluminum salt adjuvant widely used in the clinic. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application is described below with reference to the following drawings:

[0020] Figure 1Antibody titers 7 days after immunization with different complex adjuvant systems combined with HI antigen

[0021] Figure 2 Antibody titers 14 days after immunization with different complex adjuvant systems combined with HI antigen

[0022] Figure 3 Antibody titers 21 days after immunization with different complex adjuvant systems combined with HI antigen

[0023] Figure 4 Particle size analysis detection results of different complex adjuvant system prescriptions

[0024] Figure 5 Adsorption of HI antigen results chart for different complex adjuvant system prescriptions (Note: amount of precipitated protein; H1 is the mass ratio of NEA: Alun 0:10; H2 is the mass ratio of NEA: Alun 2:8; H3 is the mass ratio of NEA: Alun 4:6; H4 is the mass ratio of NEA: Alun 5:5; H5 is the mass ratio of NEA: Alun 6:4; H6 is the mass ratio of NEA: Alun 8:2; H7 is the mass ratio of NEA: Alun 10:0, and H8 is HI)

[0025] Figure 6 Unadsorbed HI antigen results chart for different complex adjuvant system prescriptions (Note: amount of precipitated protein; H1 is the mass ratio of NEA: Alun 0:10, H2 is the mass ratio of NEA: Alun 2:8; H3 is the mass ratio of NEA: Alun 4:6, H4 is the mass ratio of NEA: Alun 5:5; H5 is the mass ratio of NEA: Alun 6:4, H6 is the mass ratio of NEA: Alun 8:2; H7 is the mass ratio of NEA: Alun 10:0, and H8 is HI)

[0026] Figure 7 Antibody titers 7 days after immunization with different complex adjuvant systems combined with HI antigen

[0027] Figure 8 Antibody titers 14 days after immunization with different complex adjuvant systems combined with HI antigen

[0028] Figure 9 Antibody titers 21 days after immunization with different complex adjuvant systems combined with HI antigen

[0029] Figure 10 Th1 cytokines after immunization with different complex adjuvant systems combined with HI antigen

[0030] Figure 11 Th2 cytokines after immunization with different complex adjuvant systems combined with HI antigen

[0031] Figure 12 Antibody titers 7 days after immunization with HI antigen combined with the three complex adjuvant systems optimized for formulation

[0032] Figure 13 Antibody titers 14 days after immunization with HI antigen combined with the three complex adjuvant systems optimized for formulation

[0033] Figure 14 Antibody titers 21 days after immunization with HI antigen combined with the three complex adjuvant systems optimized for formulation

[0034] Figure 15 Antibody titers 7 days after immunization with HI antigen combined with the optimal complex adjuvant system

[0035] Figure 16 Antibody titers 14 days after immunization with HI antigen combined with the optimal complex adjuvant system

[0036] Figure 17 Antibody titers 21 days after immunization with HI antigen combined with the optimal complex adjuvant system

[0037] Figure 18 Th1 cytokines after immunization with HI antigen combined with the optimal complex adjuvant system

[0038] Figure 19 Th2 cytokines after immunization with HI antigen combined with the optimal complex adjuvant system. DETAILED DESCRIPTION

[0039] The present application will be further described below in conjunction with the drawings and specific examples so that those skilled in the art can better understand the present application and implement it.

[0040] The nanoemulsion adjuvant used in the present application is prepared from a surfactant, a co-surfactant, an oil phase and water. First, the surfactant and the co-surfactant are mixed uniformly at a mass ratio (Km value) of 2:1, 3:1, 4:1 or 5:1 to form a mixed surfactant. Then, the surfactant and the oil phase are precisely weighed at a mass ratio of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 or 1:9, mixed uniformly in a flat-bottomed glass beaker, stirred at room temperature with a constant-temperature magnetic stirrer, and distilled water is added dropwise while stirring until a clear and transparent nanoemulsion is formed. The prepared nanoemulsion adjuvant has a blue opalescence. After high-speed centrifugation (13 000 r / m, 30 min), the nanoemulsion is stable and does not separate into layers. After parallel light is incident, the nanoemulsion exhibits the Tyndall effect. The particle size of the nanoemulsion is detected to be between 1 and 100 nm by transmission electron microscopy, atomic force microscopy and a laser particle size analyzer.

[0041] The surfactant in the embodiment is any one or a combination of Tween-80, Tween-85, Span 85, Tween 60, polyoxyethylene hydrogenated castor oil and polyoxyethylene castor oil; the co-surfactant is any one or a combination of sorbitan fatty acid ester, C2-C4 monohydric alcohol and C2-C4 polyhydric alcohol; the oil phase is any one or a combination of paraffin oil, ethyl acetate, myristyl isopropyl ester, squalane, squalene, ethyl oleate and caprylic / capric acid triglyceride. The formulations of the nanoemulsion adjuvants disclosed in Chinese Patent Publications CN101961314A and CN105251002A can also be used to achieve the purpose of the application.

[0042] The preparation method of the composite adjuvant is as follows:

[0043] (1) The surfactant, co-surfactant and oil phase are taken according to the prescription, mixed uniformly, and then stirred uniformly at a temperature of 4-37°C and a rotation speed of 50-500 rpm, and water is added dropwise to prepare a clear and transparent nanoemulsion adjuvant by low-energy emulsification or phase inversion method.

[0044] (2) According to the dosage of the composite adjuvant, the aluminum salt gel adjuvant stock solution and the nanoemulsion adjuvant stock solution (NEA) are sucked into a plastic sterile and pyrogen-free centrifuge tube, mixed uniformly by a vortex shaker (4-6 gears, 15s-1min), and sealed.

[0045] (3) The composite adjuvant is prepared by adsorbing at 4-25°C using a shaking bed (100-200 rpm) for 12-14h, and then labeled, divided, and sealed.

[0046] The present application takes Staphylococcus aureus (MRSA) recombinant subunit HI antigen as an example.

[0047] Example 1, Detection of the immune response of different composite adjuvants combined with Staphylococcus aureus (MRSA) recombinant subunit vaccine HI antigen to normal mice

[0048] Take in the SPF environment BALB / c mice 32, average random into 4 groups, each group of 8, each group is numbered 1~4, respectively, muscle injection contains comprehensive stimulation immune response reaction of nano-emulsion / aluminum salt gel complex adjuvant system combined with staphylococcus aureus recombinant(HI antigen) vaccine, the proportion of 4 groups of complex adjuvant is 1:1.Each mouse injection HI antigen is 60μg(antigen final concentration 240μg / ml), the total amount of complex adjuvant is 200μg(adjuvant final concentration 800μg / ml), the injection volume is 250μL, respectively, in 0, 0, 7 days, three times of Blab / c mice are injected into the muscle of both sides each injection immune 125μl.In the first immunization after 7 days, 14 days, 21 days, the serum specific IgG antibody titer of each group is detected, and the specific prescription of each group of complex adjuvant is shown in table 1.

[0049] Table 1, the specific prescription of each group of injection complex adjuvant

[0050]

[0051]

[0052] The average serum specific antibody of each experimental group is shown in Figures 1-3 It can be seen from Figures 1-3 that among the immune response effects of normal mice immunized by the four kinds of complex adjuvant combined with staphylococcus aureus(MRSA) recombinant subunit vaccine HI antigen, NEA / Al(OH)3 can induce higher immune response effect than the other three kinds of complex adjuvant.

[0053] Example 2, particle size analysis of different complex adjuvant system prescriptions

[0054] After preparing the complex adjuvant by different proportions of NEA and AL(OH)3, adsorbing HI antigen to prepare 9 different prescription groups with different antigen amounts, total adjuvant amounts and adjuvant proportions, each group is numbered F1~F9. After diluting 200 times of 9 kinds of prescription stock solution with pure water, the particle size is detected, and the specific prescription of each group of 250μL vaccine complex adjuvant antigen is shown in table 2.

[0055] Table 2, the specific prescription table of each group of particle size detection

[0056]

[0057] The average particle size analysis and detection of each experimental group are shown in Figure 4 It can be seen from Figure 4 that compared with other groups of complex adjuvant prescription, the third group of complex adjuvant prescription has the smallest particle size, and in the subsequent experiment, the complex adjuvant prescription with smaller particle size can better deliver antigen and promote immune response effect.

[0058] Example 3: Adsorption efficiency of different compound adjuvant system formulations for HI antigen

[0059] NEA and Al(OH)3 were prepared into composite adjuvants in different ratios: 0:10, 2:8, 4:6, 5:5, 6:4, 8:2, and 10:0. Each of these seven composite adjuvants adsorbed 120 μg of antigen (final antigen concentration: 600 μg / ml) and a total adjuvant volume of 40 μg (final adjuvant concentration: 200 μg / ml). After forming 200 μl of adjuvant-antigen complex, the supernatant and precipitate were separated by centrifugation at 12000 rpm for 10 min. The supernatant was diluted 40-fold with pure water, and the precipitate was resuspended in 200 μl of water to prepare the sample. 5 μl of 5× denaturing protein loading buffer was added to 20 μl of the sample, and the mixture was boiled for 5 minutes. Take 10 μl of the boiled sample and 5 μl of pre-stained protein marker (10-245 kDa, PM22010, Daco) onto an SDS-PAGE protein gel (protein gel kit, PG12101, Daco). Electrophoresis is performed at a constant voltage of 80 V to the separating gel, and then at a constant voltage of 120 V until the bromophenol blue indicator reaches the bottom of the separating gel. Instant blue staining is performed for 15-30 min, followed by overnight destaining. Images are taken using a protein gel electrophoresis apparatus to detect the HI antigen content in the supernatant and precipitate, evaluating the adsorption efficiency of different ratios of the combined adjuvant system for HI antigen.

[0060] The adsorption efficiencies of different adjuvant formulations for HI antigen in each experimental group are as follows: Figures 5-6 As shown. (Through) Figures 5-6 It can be seen that as the proportion of AL(OH)3 decreases, the adsorption of HI antigen decreases. When the mass ratio of NEA:Al(OH)3 is 2:8, the maximum adsorption efficiency of HI antigen can be achieved.

[0061] Example 4: Detection of the immune response of Staphylococcus aureus (MRSA) recombinant subunit vaccines (HI antigen) with different adjuvant ratios in normal mice.

[0062] Forty-five BALB / c mice in SPF environment were randomly divided into nine groups of five each, numbered F1 to F9. Each group received an intramuscular injection of a nanoemulsion / aluminum salt gel adjuvant system combined with a Staphylococcus aureus recombinant (HI antigen) vaccine that fully stimulates the immune response. The different adjuvant formulations for the nine groups are shown in Table 2, with an NEA:Al(OH)3 adjuvant ratio of 2:8. The injection volume was 250 μL, administered to both sides of the BALB / c mice on days 0, 0, and 7 (125 μl / site). Serum-specific IgG antibody titers were measured on days 7, 14, and 21 post-immunization. The average serum-specific antibody levels in each experimental group are detailed below. Figures 7-9 As shown.

[0063] pass Figures 7-9 It is evident that, among the recombinant subunit vaccines with different antigen-adjuvant ratios, the addition of the composite adjuvant of this invention at an antigen amount of 60 μg (final antigen concentration of 240 μg / ml), an adjuvant amount of 50-200 μg (final adjuvant concentration of 200-800 μg / ml), and a NEA:Al(OH)3 ratio of 2:8 to 5:5 can induce higher antibody titers. Among these conditions, the highest antibody levels were observed at 7, 14, and 21 days when the adjuvant amount was 50 μg, the antigen amount was 60 μg, and the NEA:Al(OH)3 adjuvant formulation (F3) ratio was 2:8, indicating a longer and higher level of immune response.

[0064] Example 5: Detection of cytokine levels in mice immunized with Staphylococcus aureus (MRSA) recombinant subunit vaccine (HI antigen) with different adjuvant ratios.

[0065] Twenty-seven BALB / c mice in SPF environment were randomly divided into nine groups of three (n=1-12). Each group received an intramuscular injection of a nanoemulsion / aluminum salt gel adjuvant system combined with a recombinant Staphylococcus aureus (HI antigen) vaccine that fully stimulates the immune response. The antigen and adjuvant formulations for each group are shown in Table 3. The injection volume was 250 μL, administered to both sides of the BALB / c mice on days 0, 0, and 7 (125 μL / site). Serum cytokine levels were measured 14 days after the initial immunization.

[0066] Table 3. Specific prescriptions for cytokine detection in each group

[0067]

[0068] The specific details of the average serum cytokine levels in each experimental group are as follows: Figures 10-11 As shown. Serum cytokines Figures 10-11 It is evident that the composite adjuvant antigen of the present invention, with an antigen content of 60 μg, an adjuvant dose of 50-200 μg, and a NEA:Al(OH)3 ratio of 2:8 to 5:5, can induce higher Th1 cellular immune and Th2 humoral immune responses.

[0069] Example 6: Verification of the immune response of NEA / Al(OH)3 combined adjuvant in a recombinant subunit vaccine (HI antigen) for Staphylococcus aureus (MRSA).

[0070] Take 15 BALB / c mice in SPF environment, average randomly divided into 3 groups, 5 in each group, each group is numbered F1~F3, respectively, muscle injection containing comprehensive immune response of nano-emulsion / aluminum salt gel complex adjuvant system combined with staphylococcus aureus recombinant (HI antigen) vaccine, each mouse muscle injection 250 μl. Respectively, at 0 days, 0 days, 7 days, three times of Blab / c mice were immunized by bilateral muscle injection (125 μl / site). The serum specific IgG antibody titer was detected at 7 days, 14 days, 21 days after the first immunization. The average serum specific antibody of each group in different proportions of complex double adjuvant is shown in the following table 4; the average serum specific antibody of each experimental group is shown in the following table 4. Figures 12-14

[0071] Table 4, the injection of complex adjuvant prescription table of each group

[0072]

[0073]

[0074] By Figures 12-14 It can be seen that, in the immune response effect of mice combined with HI antigen after different proportions of complex adjuvant system, when the adjuvant amount of the complex adjuvant of the application is 60 μg, the antigen amount is 200 μg, and the mass ratio of NEA: Al(OH)3 adjuvant is 2:8, a higher and more comprehensive immune response effect can be induced, and the immune antibody level is the highest at 7 days, 14 days and 21 days, with a longer and higher immune response level.

[0075] Example 7, different antigen adjuvant staphylococcus aureus (MRSA) recombinant subunit vaccine (HI antigen) verification of NEA / Al(OH)3 complex adjuvant immune response

[0076] Take 20 BALB / c mice in SPF environment, average randomly divided into 4 groups, 5 in each group, each group is HI simple antigen control group, Al(OH)3 adjuvant group, NEA adjuvant group and NEA / Al(OH)3 complex adjuvant group.

[0077] Among them, the HI simple antigen group does not add any adjuvant (antigen 60 μg, antigen final concentration 240 μg / ml)

[0078] The Al(OH)3 adjuvant group is Al(OH)3+HI antigen (adjuvant: 200 μg, adjuvant final concentration 800 μg / ml; antigen: 60 μg, antigen final concentration 240 μg / ml);

[0079] The NEA adjuvant group is NEA+HI antigen (adjuvant: 200 μg, adjuvant final concentration 800 μg / ml; antigen: 60 μg, antigen final concentration 240 μg / ml);​

[0080] The NEA / Al(OH)3 compound adjuvant group consists of NEA / Al(OH)3 compound adjuvant (mass ratio of 2:8) + HI antigen (adjuvant: 200 μg, final adjuvant concentration 800 μg / ml; antigen: 60 μg, final antigen concentration 240 μg / ml);

[0081] In each of the above groups, mice were intramuscularly injected with a recombinant Staphylococcus aureus (HI antigen) vaccine containing a nanoemulsion / aluminum salt gel adjuvant system that fully stimulates the immune response, with 250 μl injected into each mouse. Blab / c mice were immunized bilaterally via intramuscular injection three times on days 0, 0, and 7 (125 μl / site). Serum-specific IgG antibody titers were measured on days 7, 14, and 21 post-initial immunization. The average serum-specific antibody levels in each experimental group are detailed below. Figures 15-17 As shown.

[0082] pass Figures 15-17 It is evident that, among the groups with added antigens, the immune responses of the groups with added adjuvants were stronger than those of the experimental groups without added adjuvants; and among the groups with added adjuvants, the immune response effect of the compound adjuvant of this invention was stronger than that of the single adjuvant experimental group.

[0083] Example 7: Detection of mouse cytokine levels in combination with a compound adjuvant system and Staphylococcus aureus (MRSA) recombinant subunit vaccine (HI antigen).

[0084] Twelve BALB / c mice in SPF environment were randomly divided into four groups of three mice each: HI antigen control group, Al(OH)3 adjuvant group, NEA adjuvant group, and NEA / Al(OH)3 combined adjuvant group.

[0085] The HI simple antigen group did not contain any adjuvants (antigen 30 μg, final antigen concentration 120 μg / ml);

[0086] Al(OH)3 adjuvant group consists of Al(OH)3+HI antigen (adjuvant: 100μg, final adjuvant concentration 400μg / ml; antigen: 30μg, final antigen concentration 120μg / ml);

[0087] The NEA adjuvant group consists of NEA+HI antigen (adjuvant: 100 μg, final adjuvant concentration 400 μg / ml; antigen: 30 μg, final antigen concentration 120 μg / ml);

[0088] The NEA / Al(OH)3 adjuvant group consists of NEA / Al(OH)3 adjuvant (mass ratio of 2:8) + HI antigen.

[0089] In each group, the HI antigen with the composite adjuvant was 50 μg, the total amount of the adjuvant was 100 μg, and each Blab / c mouse was injected with 250 μl of the nanoemulsion / aluminum salt gel composite adjuvant system combined with the Staphylococcus aureus recombinant (HI antigen) vaccine to stimulate the immune response. The Blab / c mice were injected with the vaccine in both sides of the muscle (125 μl / site) at 0 day, 0 day, and 7 days, respectively, and the serum cytokine levels were detected at the 14th day after the first injection. The serum cytokine levels in each experimental group are shown in Table 1. Figures 18-19

[0090] The serum cytokine levels of the mice injected with the composite adjuvant Figures 18-19 It can be seen that the composite adjuvant antigen amount of the present application can induce higher Th1 cell immunity and Th2 humoral immune response.

[0091] The above examples are only the preferred embodiments for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Any equivalent substitution or transformation of the present application based on the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.​

Claims

1. The application of a compound adjuvant system as a subunit vaccine adjuvant, characterized in that: The composite adjuvant system comprises an aluminum salt adjuvant that enhances humoral immune responses and a nanoemulsion adjuvant that enhances cellular immune responses; the aluminum salt adjuvant is aluminum hydroxide adjuvant, and the mass ratio of the aluminum salt adjuvant to the nanoemulsion adjuvant is 2:8; the nanoemulsion adjuvant is composed of a surfactant, a co-surfactant, and an oil phase; the surfactant is any one or a combination of Tween-80, Tween-85, Spand 85, Tween 60, polyoxyethylene hydrogenated castor oil, and polyoxyethylene castor oil; the co-surfactant is any one or a combination of sorbitan fatty acid esters, C2-C4 monohydric alcohols, and C2-C4 polyhydric alcohols; the oil phase is any one or a combination of paraffin oil, ethyl acetate, isopropyl myristate, squalane, squalene, ethyl oleate, and caprylic / capric triglycerides.

2. A recombinant subunit vaccine containing the complex adjuvant system of claim 1.

3. The recombinant subunit vaccine according to claim 2, characterized in that: The dosage of the compound adjuvant is 50 μg / ml to 1.5 mg / ml.

4. The recombinant subunit vaccine according to claim 2, characterized in that: The antigen content of the vaccine is 15 μg / ml to 1.5 mg / ml.

5. The recombinant subunit vaccine according to claim 2, characterized in that: The vaccine in question is a recombinant subunit vaccine against Staphylococcus aureus.

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