Preparation method and application of vaccine based on aluminum adjuvant and phosphate composite adjuvant

Abstract

The invention discloses a preparation method and application of a vaccine based on an aluminum adjuvant and a phosphate composite adjuvant. By changing the adding sequence of the phosphate adjuvant and the antigen in the formula, the composite adjuvant vaccine with different adsorption parameters is finally obtained. The formula for regulating and controlling the adsorption parameters is simple in preparation method and easy to operate, physicochemical characteristics of the aluminum adjuvant and the antigen are not changed, the strength of the aluminum adjuvant for adsorbing the antigen can be controlled within different ranges, and the formula has a good application prospect in vaccine formula optimization.

Description

Technical Field

[0001] The present invention relates to a preparation method and adsorption application of a vaccine based on an aluminum adjuvant and a phosphate composite adjuvant. Specifically, it is a vaccine preparation method that changes the addition sequence of a CpG immunoadjuvant and an antigen, and can be used for the development of vaccines containing composite adjuvants for the prevention and treatment of various infectious diseases. Background Art

[0002] Vaccination is one of the most effective ways to prevent the spread of infectious diseases. New-generation vaccines increasingly use highly purified antigens, which improves vaccine safety and tolerance but usually has low immunogenicity. A method to increase the immunogenicity of antigens in vaccines is to use adjuvants. To date, most licensed vaccines use classical aluminum adjuvants. Aluminum salt adjuvants have been shown to have a bias towards humoral immune responses. This type of immune response is effective against extracellular pathogens but ineffective against intracellular pathogens. The most important limitation of aluminum adjuvants is their inability to support strong cell-mediated immune responses. Therefore, many current studies are dedicated to enabling aluminum adjuvants to achieve both cellular and humoral immune responses. Utilizing the extensive antigen adsorption ability of aluminum adjuvants, aluminum adjuvants are combined with other immune stimulants such as Toll-like receptor agonists to improve their immune bias. However, the introduction of other adjuvants will have a certain impact on antigen adsorption, and the adsorption of antigens by aluminum adjuvants plays a crucial role in their immune effects.

[0003] According to relevant research reports, the interaction between aluminum adjuvants and antigens will affect the immune effect. Therefore, it is necessary to explore the interaction between aluminum adjuvants and antigens. However, current studies have all achieved the regulation of the interaction between the two by changing the properties of the adjuvant and the antigen, which has increased the complexity of the formulation to a certain extent and cannot establish a connection between the immune effect and the interaction. Therefore, starting from the composite adjuvant formulation, without changing the physical and chemical properties of the adjuvant and the antigen, by changing the addition sequence between the adjuvant and the antigen to achieve the regulation of adsorption parameters, this is of great significance for establishing the connection between the interaction and the immune effect and optimizing the vaccine formulation. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method and application of a vaccine based on an aluminum adjuvant and a phosphate composite adjuvant. By changing the addition sequence of components, the adsorption parameters are further regulated to obtain different composite adjuvants. The present invention regulates the adsorption parameters of aluminum adjuvants for adsorbing antigens by changing the order of adding antigens and phosphate adjuvants during the preparation process. The aluminum adjuvants in the prepared composite adjuvant formulation have different adsorption parameters to achieve the optimization of the aluminum adjuvant-containing vaccine formulation and provide a new perspective for the design of aluminum adjuvant composite adjuvant vaccines.

[0005] A method for preparing a vaccine based on an aluminum adjuvant and a phosphate-based composite adjuvant, wherein the composite adjuvant vaccine comprises an aluminum adjuvant, a phosphate-based adjuvant and an antigen. Utilizing the advantage of the strong adsorption ability of the aluminum adjuvant, the phosphate-based adjuvant can be adsorbed on the surface of the aluminum adjuvant, and the antigen will also be adsorbed on the surface of the adjuvant. During the preparation process, the addition sequence of the phosphate-based adjuvant and the antigen is changed to formulate different composite adjuvant vaccines;

[0006] The said addition sequence includes (i) or (ii);

[0007] (i) Mixed adsorption of antigen and phosphate-based adjuvant: According to the chronological order of the addition of antigen and phosphate-based composite adjuvant, it is divided into: sequential mixed adsorption, reverse mixed adsorption, and same-order mixed adsorption;

[0008] The said sequential mixed adsorption is: first mix the antigen and the aluminum adjuvant to prepare a suspension mixture of aluminum pre-adsorbed with the antigen, and then add the unadsorbed phosphate-based adjuvant for mixing to prepare a composite adjuvant vaccine;

[0009] The said reverse mixed adsorption is: first mix the phosphate-based adjuvant and the aluminum adjuvant to prepare a suspension mixture of aluminum pre-adsorbed with the phosphate adjuvant, and then add the unadsorbed antigen for mixing to prepare a composite adjuvant vaccine;

[0010] The said same-order mixed adsorption is: first mix the phosphate-based adjuvant and the antigen, and then add them to the aluminum adjuvant for mixing, so that the aluminum adjuvant adsorbs the antigen and the phosphate-based adjuvant simultaneously to prepare a composite adjuvant vaccine;

[0011] (ii) Pre-adsorption of antigen and phosphate-based adjuvant and then mixed adsorption: Mix the phosphate-based adjuvant and the antigen with the aluminum adjuvant respectively, so that the aluminum adjuvant adsorbs the antigen and the phosphate-based adjuvant respectively, prepare a suspension mixture of aluminum pre-adsorbed with the antigen and a suspension mixture of aluminum pre-adsorbed with the phosphate adjuvant, and then mix the two to prepare a composite adjuvant vaccine.

[0012] Further, the aluminum adjuvant is mainly but not limited to commercially available aluminum hydroxide adjuvant, and can also be various aluminum adjuvants such as aluminum hydroxide and aluminum phosphate. The concentration of the aluminum adjuvant in the composite adjuvant vaccine is 0.05 - 2 mg Al / ml, preferably 0.05 - 0.5 mg Al / ml.

[0013] Further, the phosphate-based adjuvant is mainly but not limited to cytosine-phosphate-guanosine-oligodeoxynucleotide (CpG oligodeoxynucleotides, CpG ODN, hereinafter simply referred to as CpG) immune adjuvant. The concentration of the phosphate-based adjuvant in the composite adjuvant vaccine is 0.03 - 0.25 mg / ml, and the mass ratio of aluminum to CpG is 2.0 - 1.0:1, preferably 2.0 - 1.5:1.

[0014] Further, the antigen is mainly but not limited to ovalbumin (OVA), hepatitis B surface antigen (HBsAg). When using OVA, the concentration of the antigen in the combined adjuvant vaccine is 0.01 - 0.4 mg / ml, preferably 0.05 - 0.4 mg / ml, more preferably 0.05 - 0.19 mg / ml. When using HBsAg, the concentration of the antigen in the combined adjuvant vaccine is 0.01 - 0.1 mg / ml.

[0015] Further, the preparation method of the combined adjuvant vaccine includes the following preparation steps:

[0016] (i) Mixing and adsorbing the antigen and phosphate adjuvant:

[0017] The sequential mixing and adsorption is as follows: Mix the antigen solution and the aluminum adjuvant suspension to prepare an aluminum suspension complex pre-adsorbed with the antigen, and then add the solution of the unadsorbed phosphate adjuvant (such as CpG) for mixing to prepare the combined adjuvant vaccine;

[0018] The reverse mixing and adsorption is as follows: Mix the solution of the phosphate adjuvant (such as CpG) and the aluminum adjuvant suspension to prepare an aluminum suspension complex pre-adsorbed with CpG, and then add the unadsorbed antigen solution for mixing to prepare the combined adjuvant vaccine;

[0019] The same-order mixing and adsorption is as follows: Mix the solution of the phosphate adjuvant (such as CpG) and the antigen solution to prepare a mixed solution of the phosphate adjuvant (such as CpG) and the antigen, and then add it to the aluminum adjuvant suspension for mixing to prepare the combined adjuvant vaccine;

[0020] (ii) Pre-adsorbing the antigen and the phosphate adjuvant and then mixing and adsorbing: Mix the solution of the phosphate adjuvant (such as CpG) and the antigen solution with the aluminum adjuvant suspension respectively to prepare an aluminum suspension complex pre-adsorbed with the antigen and an aluminum suspension complex pre-adsorbed with the phosphate adjuvant (such as CpG), and then mix the two to prepare the combined adjuvant vaccine.

[0021] Further, the mixing is carried out under stirring conditions, and the stirring rate for each mixing is 600 - 1000 rpm, and the stirring time is 30 - 60 min.

[0022] Further, the antigen solution includes an OVA solution or an HBsAg solution; when using OVA, the concentration of the antigen solution is 1 mg / ml; when using HBsAg, the concentration of the antigen solution is 215 μg / ml; the concentration of the aluminum adjuvant suspension is 1 - 10 mg Al / ml; the concentration of the solution of the phosphate adjuvant (such as CpG) is 0.5 - 5 mg / ml.

[0023] Further, the solution is prepared using a buffer solution, which is a MOPS buffer solution, preferably a 10 mM MOPS buffer solution with a pH of 7.3 - 7.5.

[0024] Further, a vaccine based on an aluminum adjuvant and a phosphate composite adjuvant is prepared by the above preparation method.

[0025] Further, the above-mentioned vaccine based on an aluminum adjuvant and a phosphate composite adjuvant can be applied in prophylactic and therapeutic adjuvant vaccines.

[0026] Further, the vaccine includes a composite adjuvant vaccine and a combined vaccine.

[0027] The beneficial effects of the present invention are as follows:

[0028] The present invention provides a preparation method for a vaccine formulation based on an aluminum adjuvant and a phosphate composite adjuvant. Through adsorption experiments, formulations with different adsorption parameters are obtained, realizing the regulation of adsorption parameters. The preparation method of the formulation that can regulate adsorption parameters is simple and easy to operate, and does not change the physical and chemical properties of the aluminum adjuvant and the antigen. It can control the strength of the aluminum adjuvant adsorbing the antigen within different ranges, and is a controllable method for regulating the binding ability and strength between the adjuvant and the antigen, having good application prospects in vaccine formulation optimization. Description of the Drawings

[0029] The attached drawings of the present invention Figure 6 drawings.

[0030] Figure 1 Schematic diagrams of different composite adjuvant vaccine mixing strategies. Among them, (a) sequential mixing adsorption, (b) reverse mixing adsorption, (c) same-order mixing adsorption, and (d) mixing adsorption after pre-adsorption.

[0031] Figure 2 Potential diagrams of different composite adjuvant vaccines containing antigens. Among them, (a) OVA, (b) HBsAg.

[0032] Figure 3 Adsorption isotherm diagrams of aluminum adjuvant adsorbing CpG. Among them, (a) Langmuir adsorption isotherm, (b) linearized Langmuir equation.

[0033] Figure 4 Adsorption isotherm diagrams of aluminum adjuvant adsorbing OVA in composite adjuvant vaccines with different CpG addition amounts. Among them, (a) low CpG addition amount, (b) high CpG addition amount.

[0034] Figure 5 Significance analysis of the adsorption coefficient of aluminum adjuvant adsorbing OVA in composite adjuvant vaccines with different CpG addition amounts. Among them, (a) low CpG addition amount, (b) high CpG addition amount.

[0035] Figure 6 To study the adsorption characteristics of aluminum adjuvant in the composite adjuvant vaccine for adsorbing HBsAg. Among them, (a) Langmuir adsorption isotherm, (b) significance analysis of adsorption coefficient. Specific implementation mode

[0036] The following non-limiting examples can enable those of ordinary skill in the art to more comprehensively understand the present invention, but do not limit the present invention in any way.

[0037] In the following examples, the aluminum adjuvant is a commercial aluminum adjuvant (InvivoGen), which is used as the aluminum adjuvant in the composite adjuvant vaccine and is denoted as Adjuvant.

[0038] Example 1

[0039] A preparation method of an aluminum adjuvant-CpG composite adjuvant OVA vaccine, the method comprising the following specific steps:

[0040] (1) Prepare the required sample stock solution: Prepare 1 L of 10 mM MOPS buffer solution with a pH of 7.4; Weigh a certain mass of OVA, add 10 mM MOPS buffer solution with a pH of 7.4, and prepare 10 ml of 1 mg / ml OVA solution; Weigh a certain mass of CpG, add 10 mM MOPS buffer solution with a pH of 7.4, and prepare 0.1 ml of 5 mg / ml CpG solution.

[0041] (2) Prepare the composite adjuvant vaccine by adding Adjuvant, OVA solution, and CpG solution in different addition sequences. In the final composite adjuvant vaccine, the aluminum concentration is 0.5 mg Al / ml, the OVA antigen concentration is 10 μg / ml, and the CpG concentration is 0.25 mg / ml;

[0042] Among them, the sequential mixing adsorption formula: Add 15 μl Adjuvant (10 mg Al / ml) to a 1.5 ml centrifuge tube, add 105 μl of 10 mM MOPS solution with a pH of 7.4, add 30 μl of OVA solution (1 mg / ml) to prepare 0.15 ml Suspension mixture (1 mg Al / ml), stir at room temperature for 30 min at 1000 rpm to obtain 0.15 ml of aluminum suspension mixture pre-adsorbed with antigen; Add 15 μl of CpG solution (5 mg / ml) to the above mixture, add 135 μl of 10 mM MOPS solution with a pH of 7.4 to make the final volume of the mixture 0.3 ml, and stir at room temperature for 30 min at 1000 rpm to obtain 0.3 ml of sequentially mixed adsorbed composite adjuvant vaccine;

[0043] Reverse sequential adsorption formulation: Add 15 μl adjuvant (10 mg Al / ml) to a 1.5 ml centrifuge tube, add 120 μl of 10 mM MOPS solution with a pH of 7.4, add 15 μl of CpG solution (5 mg / ml) to prepare 0.15 ml suspension mixture (1 mg Al / ml), stir at room temperature at 1000 rpm for 30 min to obtain 0.15 ml of aluminum suspension mixture pre-adsorbed with CpG; add 30 μl of OVA solution (1 mg / ml) to the above mixture, add 120 μl of 10 mM MOPS solution with a pH of 7.4 until the final volume of the mixture is 0.3 ml, stir at room temperature at 1000 rpm for 30 min to obtain 0.3 ml of reverse sequential adsorption combined adjuvant vaccine;

[0044] Sequential adsorption formulation: Add 15 μl adjuvant (10 mg Al / ml) to a 1.5 ml centrifuge tube, add 135 μl of 10 mM MOPS solution with a pH of 7.4 to obtain 0.15 ml suspension (1 mg Al / ml); add 15 μl of CpG solution (5 mg / ml) and 30 μl of OVA solution (1 mg / ml) to a 1.5 ml centrifuge tube, add 105 μl of 10 mM MOPS solution with a pH of 7.4 to prepare 0.15 ml of CpG and OVA mixture, stir at room temperature at 1000 rpm for 30 min to obtain 0.15 ml of mixed solution of OVA and CpG; add it to 0.15 ml of suspension (1 mg Al / ml), stir at room temperature at 1000 rpm for 30 min to obtain 0.3 ml of sequential adsorption combined adjuvant vaccine;

[0045] Pre-adsorption followed by sequential adsorption formulation: Add 15 μl adjuvant (10 mg Al / ml) to a 1.5 ml centrifuge tube, add 105 μl of 10 mM MOPS solution with a pH of 7.4, add 30 μl of OVA solution (1 mg Al / ml) to prepare 0.15 ml suspension mixture (1 mg Al / ml), stir at room temperature at 1000 rpm for 30 min to obtain 0.15 ml of aluminum suspension mixture pre-adsorbed with antigen; add 15 μl adjuvant (10 mg Al / ml) to a 1.5 ml centrifuge tube, add 120 μl of 10 mM MOPS solution with a pH of 7.4, add 15 μl of CpG solution (5 mg / ml) to prepare 0.15 ml Suspension mixture (1 mg Al / ml), stirred at room temperature at 1000 rpm for 30 min to obtain 0.15 ml of aluminum suspension mixture pre-adsorbed with CpG; the above mixtures were mixed in equal volumes and stirred at room temperature at 1000 rpm for 30 min to obtain 0.3 ml of composite adjuvant vaccine with post-pre-adsorption mixed adsorption.

[0046] Example 2

[0047] A preparation method of an aluminum adjuvant-CpG composite adjuvant HBsAg vaccine, replacing the antigen with HbsAg, and preparing by the method described in Example 1. Among them, the HBsAg solution is provided by North China Pharmaceutical Group, with a concentration of 215 μg / ml. Therefore, during the preparation process, the added volume of HBsAg changes correspondingly. In the final composite adjuvant HBsAg vaccine, the aluminum concentration is 0.5 mg Al / ml, the HBsAg concentration is 10 μg / ml, and the CpG concentration is 0.25 mg / ml. After preparation, 0.3 ml of composite adjuvant HBsAg vaccines with sequential mixed adsorption, reverse mixed adsorption, same-order mixed adsorption, and post-pre-adsorption mixed adsorption are all obtained.

[0048] Example 3

[0049] Detection of the physicochemical properties of the composite adjuvant vaccine formulations prepared in Example 1 and Example 2, and comparison with the single adjuvant vaccine formulation containing only , denoted as Among them, The preparation process of the single adjuvant vaccine formulation is as follows: Add 15 μl of adjuvant (10 mg Al / ml) to a 1.5 ml centrifuge tube, add 135 μl of 10 mM MOPS solution with a pH of 7.4 to obtain 0.15 ml of suspension (1 mg Al / ml), add 30 μl of OVA solution (1 mg / ml, prepared using 10 mM MOPS buffer solution with a pH of 7.4), add 120 μl of 10 mM MOPS solution with a pH of 7.4, and stir at room temperature at 1000 rpm for 1 h to prepare single adjuvant complex. The complexes prepared with different addition sequences in Example 1 and Example 2 and the single adjuvant complex were respectively added to 1.2 ml of 10 mM MOPS buffer solution with a pH of 7.4 for dilution, and 1.5 ml of a mixture with an aluminum concentration of 0.1 mg Al / ml was prepared for physicochemical property detection.

[0050] The hydrodynamic size and polymer dispersity index (PDI) are shown in Table 1, and the Zeta potential is shown in Figure 2。With the addition of CpG, the Zeta potential of various formulations containing the complex adjuvant in water changed from positive charge to negative charge, and the hydrated particle size of the formulations containing antigen reached the micron level.

[0051] Table 1 Characterization of the physicochemical properties of the complex adjuvant vaccine.

[0052]

[0053]

[0054] Example 4

[0055] Study on the adsorption characteristics of aluminum adjuvant adsorbing CpG.

[0056] Measure the adsorption of a series of concentration gradients of aluminum adjuvant mixtures containing CpG at 10, 20, 30, 40, 50, and 60 μg / ml. The specific steps are as follows:

[0057] (1) Mix and dilute 5 μl aluminum adjuvant (10 mg Al / ml) with 10 mM MOPS solution at pH 7.4 to prepare a 0.25 ml suspension with an aluminum concentration of 0.1 mg Al / ml. suspension;

[0058] (2) Add different volumes of 0.5 mg / ml CpG solution to the suspension, and add different volumes of 10 mM MOPS buffer at pH 7.4 to prepare a series of concentration gradient complexes containing 10, 20, 30, 40, 50, and 60 μg / ml of CpG in 0.5 ml, and finally the aluminum concentration in the suspension is 0.05 mg Al / ml.

[0059] (3) Continuously oscillate at room temperature for 30 min, centrifuge to retain the supernatant, and use Nanodrop to detect the concentration of unadsorbed CpG in the supernatant. The amount of CpG adsorbed on the aluminum adjuvant is determined by subtracting the amount in the supernatant from the initially added amount.

[0060] The experimental results (as Figure 3 shown) indicate that CpG can bind to the aluminum adjuvant.

[0061] Example 5

[0062] Effect of CpG addition dose on the adsorption characteristics of OVA antigen.

[0063] Determine the adsorption of OVA antigen adsorbed by aluminum adjuvant at two CpG addition doses. Prepare different composite adjuvant vaccine mixtures according to the method described in Example 1, with slightly changed concentrations of the components, and finally formulate composite adjuvant vaccines; among them, in the low-dose CpG composite adjuvant vaccine formulation (the mass ratio of aluminum to CpG is 2:1), the aluminum concentration is 0.1 mg Al / ml, the CpG concentration is 50 μg / ml, and the series concentration gradients of OVA are 150, 200, 250, 300, 350, 370 μg / ml respectively; in the high-dose CpG composite adjuvant vaccine formulation (the mass ratio of aluminum to CpG is 1.7:1), the aluminum concentration is 0.1 mg Al / ml, the CpG concentration is 50 μg / ml, and the series concentration gradients of OVA are 50, 80, 110, 140, 170, 190 μg / ml respectively. And compare with the single adjuvant vaccine formulation containing only the aluminum adjuvant single adjuvant vaccine formulation, denoted as

[0064] Take the single adjuvant vaccine formulation containing only the aluminum adjuvant as an example to introduce, and the specific steps are as follows:

[0065] (a) Comparison of the low-dose CpG composite adjuvant vaccine formulation, the single adjuvant vaccine formulation containing only the aluminum adjuvant:

[0066] (1) Mix and dilute 10 μl aluminum adjuvant (10 mg Al / ml) with 10 mM MOPS solution at pH 7.4 to prepare 0.5 ml of suspension with an aluminum concentration of 0.2 mg / ml ;

[0067] (2) Add a series of different volumes of OVA solution (1 mg / ml) to the suspension, and add different volumes of 10 mM MOPS buffer at pH 7.4 to prepare 1 ml of composite with a series of concentration gradients of OVA (150, 200, 250, 300, 350, 370 μg / ml respectively). In the final formulation, the aluminum concentration is 0.1 mg Al / ml;

[0068] (3) Continuously stir at room temperature for 1 h under the condition of 1000 rpm, and a series of aluminum suspension mixtures adsorbed with OVA can be obtained (that is, the single adjuvant vaccine containing only the aluminum adjuvant) 1 ml. Centrifuge to retain the supernatant, and use the BCA kit to detect the concentration of unadsorbed antigen in the supernatant. The amount of antigen adsorbed on the aluminum adjuvant is determined by subtracting the amount in the supernatant from the initially added amount.

[0069] (4) The adsorption process of OVA antigen by aluminum adjuvant was described by the Langmuir isothermal adsorption equation. The Langmuir linearization equation was used to calculate the correlation coefficient, adsorption coefficient and monolayer adsorption capacity. The correlation coefficient indicated the degree of conformity to the Langmuir adsorption isotherm.

[0070] (b) Comparison of the high-dose CpG combined adjuvant formulations, containing only The single adjuvant vaccine formulation with aluminum adjuvant:

[0071] (1) Mix and dilute 5 μl aluminum adjuvant (10 mg Al / ml) and 10 mM MOPS solution with pH 7.4 to prepare a 0.25 ml suspension with an aluminum concentration of 0.2 mg / ml ;

[0072] (2) Add a series of different volumes of OVA solution (1 mg / ml) to the suspension, and add different volumes of 10 mM MOPS buffer with pH 7.4 to prepare a series of complexes with concentration gradients of OVA (50, 80, 110, 140, 170, 190 μg / ml) of 0.5 ml. Finally, the aluminum concentration in the suspension is 0.05 mg Al / ml;

[0073] (3) Continuously stir at room temperature for 1 h under the condition of 1000 rpm to obtain a series of aluminum suspension mixtures adsorbed with OVA (i.e., obtain the single adjuvant vaccine containing only aluminum adjuvant) of 0.5 ml. Centrifuge and retain the supernatant. The BCA kit was used to detect the concentration of unadsorbed antigen in the supernatant. The amount of antigen adsorbed on the aluminum adjuvant was determined by subtracting the amount in the supernatant from the initially added amount.

[0074] (4) The same as step (4) of the low-dose CpG combined adjuvant vaccine.

[0075] As Figure 4 、 Figure 5 and Table 2 show, the results indicate that compared with the traditional antigen formulation, the adsorption capacity and adsorption coefficient of the combined adjuvant containing CpG adjuvant decreased significantly. There was no obvious difference in the adsorption coefficient of the low-dose CpG for different combined adjuvant formulations. It is worth noting that there were significant differences in the adsorption coefficients of different combined adjuvant formulations with high-dose CpG.

[0076] Table 2 shows the adsorption parameters of aluminum adjuvant for OVA adsorption in the CpG combined adjuvant vaccine with different dosages.

[0077]

[0078] Example 6

[0079] Study on the adsorption characteristics of aluminum adjuvant adsorbed HBsAg antigen.

[0080] The adsorption of aluminum adjuvant adsorbed HBsAg in the CpG-containing combined adjuvant vaccine formulation was determined by the method of Example 4. Different combined adjuvant vaccine mixtures were prepared according to the method described in Example 2. Among them, the aluminum concentration in the combined adjuvant vaccine formulation was 0.05 mg Al / ml, the HBsAg concentrations were 10, 30, 50, 70, 90, 100 μg / ml respectively, and the CpG concentration was 30 μg / ml. In this example, 30 μg / ml of CpG was added to each formulation to achieve the CpG saturated adsorption state, so as to better observe the effect of CpG on antigen adsorption.

[0081] Taking the single adjuvant vaccine formulation containing only aluminum adjuvant as an example, the specific steps are as follows:

[0082] (1) Mix and dilute 5 μl aluminum adjuvant (10 mg Al / ml) with 10 mM MOPS solution at pH 7.4 to prepare a 0.25 ml suspension with an aluminum concentration of 0.2 mg / ml; suspension;

[0083] (2) Add a series of different volumes of HBsAg solution (215 μg / ml) to the suspension, and add different volumes of 10 mM MOPS buffer at pH 7.4 to prepare a series of concentration gradient mixtures containing HBsAg (10, 30, 50, 70, 90, 100 μg / ml respectively) with a volume of 0.5 ml. Finally, the aluminum concentration in the suspension was 0.05 mg Al / ml;

[0084] (3) Continuously stir at room temperature for 1 h at 1000 rpm to obtain a series of aluminum suspension mixtures adsorbed with HBsAg (that is, obtain a single adjuvant vaccine containing only aluminum adjuvant) with a volume of 0.5 ml. Centrifuge and retain the supernatant. The concentration of unadsorbed antigen in the supernatant was detected by BCA kit. The amount of antigen adsorbed on the aluminum adjuvant was determined by subtracting the amount in the supernatant from the initially added amount.

[0085] (4) The same as step (4) of Example 5.

[0086] As Figure 6 shown in Antigen combinations with significantly reduced adsorption coefficients. In sequential mixing adsorption, reverse mixing adsorption, and co-sequential mixing adsorption, the adsorption coefficients are reduced by approximately 5-fold, 192-fold, and 58-fold respectively. In pre-adsorption and then mixing adsorption, the adsorption coefficient is reduced by approximately 6-fold. Notably, the adsorption parameter results of different complex adjuvant HBsAg formulations indicate that the adsorption capacity of sequential mixing adsorption containing HBsAg is not significantly affected by CpG.

[0087] Table 3 Adsorption parameters of aluminum adjuvant adsorbed HBsAg in complex adjuvant vaccines

[0088]

[0089] Example 7

[0090] Using the different vaccine formulations in Example 5 (high-dose CpG) and Example 6 to explore the adsorption rate of aluminum adjuvant adsorbed CpG in different formulations. The determination of CpG adsorption amount is the same as in Example 4.

[0091] As shown in Table 4 and Table 5, in the complex adjuvant formulations containing OVA antigen, most of the CpG is in the adsorbed state, indicating that the presence of OVA has no effect on the adsorption of CpG. While in the complex adjuvant formulations containing hepatitis B surface antigen, CpG is in the free state, indicating that the presence of HBsAg affects the adsorption of CpG. By comparing the antigen adsorption capacities under different formulations, it is found that there are differences in the antigen adsorption capacities under different formulations. In the co-sequential mixing and reverse mixing formulations, the antigen adsorption capacity is greatly affected, while for the sequential mixing and pre-adsorption and then mixing formulations, the degree of influence on the antigen adsorption capacity is smaller. The adsorption of each component is different under different formulations. The binding mechanism of each component to the aluminum adjuvant should be considered to achieve the optimal selection of the formulation. This adsorption situation indicates that in order to ensure that various components can be better adsorbed on the surface of the adjuvant, the formulation should be prepared according to the sequential or pre-adsorption and then mixing formulation scheme.

[0092] Table 4 CpG adsorption rate in complex adjuvant vaccines containing OVA antigen

[0093]

[0094] Table 5 CpG adsorption rate in complex adjuvant vaccines containing HBsAg

[0095]

Claims

1. A preparation method of a vaccine based on an aluminum adjuvant and a phosphate-based composite adjuvant, characterized in that: The composite adjuvant vaccine includes an aluminum adjuvant, a phosphate adjuvant, and an antigen, and the addition order of the antigen and the phosphate adjuvant is changed during the preparation process. The addition order described includes (i) or (ii); (i) Mixing and adsorption of the antigen and the phosphate adjuvant: It also includes the time sequence of adding the antigen and the phosphate composite adjuvant: sequential mixing and adsorption, reverse mixing and adsorption, or same-sequence mixing and adsorption; The sequential mixing and adsorption is: first mix the antigen and the aluminum adjuvant, and then add the phosphate adjuvant for mixing to prepare the composite adjuvant vaccine; The reverse mixing and adsorption is: first mix the phosphate adjuvant and the aluminum adjuvant, and then add the antigen for mixing to prepare the composite adjuvant vaccine; The same-sequence mixing and adsorption is: first mix the phosphate adjuvant and the antigen, and then add them to the aluminum adjuvant for mixing to prepare the composite adjuvant vaccine; (ii) Pre-adsorption and then mixing and adsorption of the antigen and the phosphate adjuvant: Mix the phosphate adjuvant and the antigen with the aluminum adjuvant separately, and then mix the two to prepare the composite adjuvant vaccine.

2. The preparation method of the aluminum adjuvant and phosphate compound adjuvant-based vaccine according to claim 1, wherein: The antigen includes ovalbumin or hepatitis B surface antigen; when ovalbumin is used, the concentration of the antigen in the composite adjuvant vaccine is 0.01 - 0.4 mg / ml; when hepatitis B surface antigen is used, the concentration of the antigen in the composite adjuvant vaccine is 0.01 - 0.1 mg / ml.

3. The preparation method of the aluminum adjuvant and phosphate composite adjuvant-based vaccine according to claim 1, characterized in that: The aluminum adjuvant includes hydroxyaluminum oxide adjuvant, and the concentration of the aluminum adjuvant in the composite adjuvant vaccine is: 0.05 - 2 mg Al / ml; the phosphate adjuvant includes CpG immune adjuvant, and the concentration of the phosphate adjuvant in the composite adjuvant vaccine is 0.03 - 0.25 mg / ml, and the mass ratio of aluminum to the phosphate adjuvant is 2.0 - 1.0:

1.

4. The preparation method of the aluminum adjuvant and phosphate compound adjuvant-based vaccine according to claim 1, wherein: It includes the following preparation steps: (i) Mixing and adsorption of the antigen and the phosphate adjuvant: The sequential mixing and adsorption is: mix the antigen solution and the aluminum adjuvant suspension to prepare a pre-adsorbed antigen aluminum suspension complex, and then add the unadsorbed phosphate adjuvant solution for mixing to prepare the composite adjuvant vaccine; The reverse mixing and adsorption is: mix the phosphate adjuvant solution and the aluminum adjuvant suspension to prepare a pre-adsorbed phosphate adjuvant aluminum suspension complex, and then add the unadsorbed antigen solution for mixing to prepare the composite adjuvant vaccine; The same-sequence mixing and adsorption is: mix the phosphate adjuvant solution and the antigen solution to prepare a mixed solution of the phosphate adjuvant and the antigen, and then add it to the aluminum adjuvant suspension for mixing to prepare the composite adjuvant vaccine; (ii) Pre-adsorption and then mixing and adsorption of the antigen and the phosphate adjuvant: Mix the phosphate adjuvant solution and the antigen solution with the aluminum adjuvant suspension separately to prepare a pre-adsorbed antigen aluminum suspension complex and a pre-adsorbed phosphate adjuvant aluminum suspension complex, and then mix the two to prepare the composite adjuvant vaccine.

5. The preparation method of the aluminum adjuvant and phosphate compound adjuvant-based vaccine according to claim 1 or 4, characterized in that The mixing is carried out under stirring conditions, and the stirring rate for each mixing is 600 - 1000 rpm, and the stirring time is 30 - 60 min.

6. The preparation method of the aluminum adjuvant and phosphate composite adjuvant-based vaccine according to claim 4, wherein The antigen solution includes ovalbumin solution or hepatitis B surface antigen solution; when ovalbumin is used, the concentration of the antigen solution is 1 mg / ml; when hepatitis B surface antigen is used, the concentration of the antigen solution is 215 μg / ml; the concentration of the aluminum adjuvant suspension is 1-10 mg Al / ml; the concentration of the phosphate adjuvant solution is 0.5-5 mg / ml.

7. The preparation method of the aluminum adjuvant and phosphate compound adjuvant-based vaccine according to claim 1 or 4, characterized in that The solution is prepared using a buffer solution, and the buffer solution is MOPS buffer solution.

8. A vaccine based on an aluminum adjuvant and a phosphate composite adjuvant prepared by the preparation method according to any one of claims 1-7.

9. Use of the vaccine based on an aluminum adjuvant and a phosphate composite adjuvant according to claim 8 in prophylactic and therapeutic adjuvant vaccines.

10. The application according to claim 9, characterized in that, The vaccine includes a composite adjuvant vaccine and a combined vaccine.

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