Adjuvant composition for vaccine as well as preparation method and application of adjuvant composition

Abstract

The invention belongs to the technical field of medicines, particularly relates to the field of immunology, and more particularly relates to an adjuvant composition for vaccines as well as a preparation method and application of the adjuvant composition. The adjuvant is prepared from squalene, Tween 80, poloxamer 188 and oligonucleotide CpG1018. Experiments show that the adjuvant disclosed by the invention is obviously superior to an adjuvant in the prior art, so that the adjuvant has an application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, specifically to the field of immunology, and more specifically to an adjuvant composition and a preparation method and application thereof. Background Art

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

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

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

[0005] The purpose of the present invention is to propose an excellent vaccine adjuvant through research.

[0006] Specifically, the present invention provides an adjuvant composition for vaccine, characterized in that it comprises squalene, Tween 80, poloxamer 188 and oligonucleotide CpG1018.

[0007] Preferably, the content of squalene is 0.2-4% (v / v), the content of Tween 80 is 0.02-0.4% (v / v), the content of poloxamer 188 is 0.1-2% (w / v), and the content of oligonucleotide CpG1018 is 200-6000 μg / ml (w / v).

[0008] More preferably, the content of squalene is 0.8-4% (v / v), the content of Tween 80 is 0.064-0.32% (v / v), the content of poloxamer 188 is 0.2-1% (w / v), and the content of oligonucleotide CpG1018 is 300-5000 μg / ml (w / v); preferably, the content of oligonucleotide CpG1018 is 400-4000 μg / ml (w / v).

[0009] The dosage of the adjuvant composition for mice was verified by experiments, and based on the dosage for mice combined with the principle of equivalent body surface area, it was converted into the dosage for future clinical use in humans (conversion according to FDA draft guidelines), that is, the content of oligonucleotide CpG1018 is 200-6000μg / ml (w / v). The body surface area of ​​humans is about 1.67m^2, and that of mice is 0.007m^2; the former is about 230 times the latter.

[0010] In a specific embodiment, poloxamer 188 adopts pharmaceutical grade or injection grade excipients.

[0011] The present invention also provides a method for preparing the adjuvant composition, which comprises the following steps:

[0012] S1: Take squalene to prepare an oil phase; take poloxamer 188 and Tween 80 in a buffer solution and stir to prepare an aqueous phase;

[0013] S2: slowly adding the prepared aqueous phase to the prepared oil phase, and then adjusting the volume with a buffer solution, stirring at high speed at room temperature to obtain a colostrum solution;

[0014] The S3 colostrum solution was poured into a microfluidizer to obtain a refined milk solution;

[0015] S4 draws oligonucleotide CpG 1018 into the above-mentioned refined milk solution and mixes well.

[0016] Preferably, the buffer is selected from but not limited to citrate or phosphate buffer.

[0017] Preferably, in step S1, poloxamer 188 and Tween 80 are added to a buffer solution and stirred at 18-55° C. to prepare an aqueous phase;

[0018] Preferably, in step S2, a high-speed stirrer is used to stir at a speed of 8000-20000 rpm for 15-30 minutes to obtain a colostrum solution; the colostrum solution prepared in step S3 is poured into a microfluidizer, the pressure is adjusted to 15000-20000 psi, the temperature of the refrigerator is adjusted, the outlet temperature of the emulsion is controlled at 20-30°C, and the high-pressure homogenization is cycled 3-10 times to obtain a refined emulsion solution.

[0019] Preferably, in step S3, the particle size of the refined milk solution is measured by a Malvern particle size detector to be between 100nm and 200nm, and the pH value of the refined milk solution is detected to be between 6.3-6.7 or 7.0-7.4; in addition, preferably, sterilizing filtration is also included, specifically, filtering with a 0.22μm sterilizing filter to obtain a clear liquid as the refined milk solution.

[0020] The present invention provides application of the adjuvant composition in preparing an immune system.

[0021] Furthermore, the present invention provides an antigen adjuvant composition, which comprises the adjuvant composition according to any one of claims 1 to 4 and an antigen capable of inducing an immune response; the antigen comprises one or more of proteins, polypeptides or nucleic acids.

[0022] Experiments have shown that the adjuvant of the present invention is significantly superior to the adjuvant of the prior art. Specifically, the adjuvant of the present invention can enhance the body's responsiveness to antigen stimulation, increase the titer of antibodies produced by immune response, reduce the amount of antigen used, prolong the memory of immune response, and achieve efficient enhancement of the immune response level of the antigen. At the same time, the toxic and side effects are extremely low. It has the characteristics of simple preparation, good stability, and a wide range of applications, and therefore has application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of anti-VZV gE antigen-specific antibodies after mouse immunization provided in Example 2. A is the test result 21 days after immunization, and B is the test result 42 days after immunization.

[0024] Figure 2 The results of anti-VZV gE antigen-specific IFN-γ and IL-2 secretion 7 days after mouse immunization provided in Example 2. Among them, A is the detection result of IFN-γ, and B is the detection result of IL-2.

[0025] Figure 3 The results of anti-VZV gE antigen-specific IFN-γ and IL-2 secretion 12 days after mouse immunization provided in Example 2. Among them, A is the detection result of IFN-γ, and B is the detection result of IL-2.

[0026] Figure 4 The results of anti-VZV gE antigen-specific IFN-γ and IL-2 secretion 21 days after mouse immunization provided in Example 2. Among them, A is the detection result of IFN-γ, and B is the detection result of IL-2.

[0027] Figure 5 The results of anti-VZV gE antigen-specific IFN-γ and IL-2 secretion 42 days after mouse immunization provided in Example 2. Among them, A is the detection result of IFN-γ, and B is the detection result of IL-2.

[0028] Figure 6 Anti-VZV gE antigen-specific CD4 7 days after mouse immunization provided in Example 2 + INF-γ + 、CD4 + IL-2 + T cell subset results. A is CD4 + INF-γ + The results of T cell subset detection, B is CD4 + IL-2 + T cell subset detection results.

[0029] Figure 7 Anti-VZV gE antigen-specific CD4 12 days after mouse immunization provided in Example 2 + INF-γ + 、CD4 + IL-2 + T cell subset results. A is CD4 + INF-γ + The results of T cell subset detection, B is CD4 + IL-2 + T cell subset detection results.

[0030] Figure 8 Anti-VZV gE antigen-specific CD4 21 days after mouse immunization provided in Example 2 + INF-γ + 、CD4 + IL-2 + T cell subset results. A is CD4 + INF-γ + The results of T cell subset detection, B is CD4 + IL-2 + T cell subset detection results.

[0031] Fig. 9 Anti-VZV gE antigen-specific CD4 42 days after mouse immunization provided in Example 2 + INF-γ + 、CD4 + IL-2 + T cell subset results. A is CD4 + INF-γ + The results of T cell subset detection, B is CD4 + IL-2 + T cell subset detection results.

[0032] Fig.10 The results of anti-VZV gE antigen-specific antibodies after mouse immunization provided in Example 3. A is the test result 21 days after immunization, and B is the test result 42 days after immunization.

[0033] Fig.11 The effect of anti-VZV gE antigen-specific IFN-γ and IL-2 secretion on mice 12 days after immunization provided in Example 3. Wherein, A is the detection result of IFN-γ, and B is the detection result of IL-2.

[0034] Fig.12 The effect of anti-VZV gE antigen-specific IFN-γ and IL-2 secretion on 42 days after mouse immunization provided in Example 3. Wherein, A is the detection result of IFN-γ, and B is the detection result of IL-2.

[0035] Fig.13 Anti-VZV gE antigen-specific CD4 12 days after mouse immunization provided in Example 3 + INF-γ + 、CD4 + IL-2 + T cell subset results. A is CD4 + INF-γ + The results of T cell subset detection, B is CD4+ IL-2 + T cell subset detection results.

[0036] Fig.14 Anti-VZV gE antigen-specific CD4 42 days after mouse immunization provided in Example 3 + INF-γ + 、CD4 + IL-2 + T cell subset results. A is CD4 + INF-γ + The results of T cell subset detection, B is CD4 + IL-2 + T cell subset detection results. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be clearly described below in conjunction with the accompanying drawings. Obviously, the described embodiments are not all embodiments of the present invention, and all other embodiments obtained by ordinary technicians in the field without making creative work are within the protection scope of the present invention.

[0038] The main materials used in the examples include: Squalene, Tween 80, Poloxamer 188 (injection grade), Poloxamer 188 (pharmaceutical grade) and CpG (oligonucleotide CpG) 1018.

[0039] Example 1. Preparation of adjuvant composition

[0040] For the TB3-C2, TB3-C2-Hi group, TB4-C2, TB4-C2-Hi group, and TB3-C3 group, TB3-C3-Hi group, TB4-C3, TB4-C3-Hi group of AD20P named in the present invention, the dosage of each component is shown in Table 1 and Table 2 respectively.

[0041] (1) Place all materials in a biosafety cabinet and wait until the phosphate buffer temperature returns to room temperature before starting the operation;

[0042] (2) using a pipette to draw a certain amount of squalene into a 1000 mL beaker, heating at 40° C. with magnetic stirring to prepare an oil phase;

[0043] (3) Measure 500 mL of phosphate buffer and add it to another 1000 mL empty beaker, weigh a certain amount of poloxamer 188 and add it to the beaker, and use a pipette to take a certain amount of Tween 80 and add it to the beaker; heat at 50° C. with magnetic stirring for 20 minutes to prepare an aqueous phase;

[0044] (4) slowly adding the aqueous phase prepared in (3) to the oil phase prepared in (2), and diluting the volume to 1000 mL with a phosphate solution, stirring at room temperature with a high-speed stirrer at a speed of 18000-20000 rpm for 30 min to obtain a colostrum solution;

[0045] (5) Turn on the microfluidizer. Before formal use, first clean the volumetric cup and pipeline with 800 mL of 20% alcohol, then clean the volumetric cup and pipeline with 1 L of ultrapure water, and finally rinse with 500 mL of sterilized phosphate buffer. After cleaning, pour the colostrum solution prepared in (4) into the microfluidizer, adjust the pressure to 15,000 to 20,000 psi, adjust the temperature of the refrigerator, control the outlet temperature of the emulsion at 20 to 30° C., and cycle the high-pressure homogenization for 3 times to obtain a refined milk solution.

[0046] (6) sampling and observing the properties of the refined milk solution, which is milky white, and the particle size of the refined milk solution is measured by a Malvern particle size detector to be between 100 nm and 200 nm, and the pH value of the refined milk solution is detected to be between 7.0 and 7.4;

[0047] (7) Aseptically filter the concentrated milk solution in a biological safety cabinet using a 0.22 μm sterilizing filter, label the concentrated milk solution, and store it in a refrigerator at 2 to 8°C for later use.

[0048] (8) Pipette a certain amount of oligonucleotide CpG 1018 into the 1000 mL of the refined milk solution, vortex mix for at least 30 seconds, and store in a refrigerator at 2-8°C for later use.

[0049] For the TB3-C2, TB3-C2-Hi group, TB4-C2, TB4-C2-Hi group, and TB3-C3 group, TB3-C3-Hi group, TB4-C3, TB4-C3-Hi group of AD20P, the dosage of each component is shown in Tables 2 and 3 respectively.

[0050] For the preparation of AD20P TB3-C2 group:

[0051] (1) Place all materials in a biosafety cabinet and wait until the phosphate buffer temperature returns to room temperature before starting the operation;

[0052] (2) Pipette 8 mL of squalene into a 1000 mL beaker, heat at 40° C. with magnetic stirring to prepare an oil phase;

[0053] (3) Measure 500 mL of phosphate buffer and add it to another 1000 mL empty beaker, weigh 2 g of poloxamer 188 into the beaker, and use a pipette to draw 0.64 mL of Tween 80 into the beaker again; heat at 50° C. with magnetic stirring for 20 minutes to prepare an aqueous phase;

[0054] (4) slowly adding the aqueous phase prepared in (3) to the oil phase prepared in (2), and diluting the volume to 1000 mL with a phosphate solution, stirring at room temperature with a high-speed stirrer at a speed of 18000-20000 rpm for 30 min to obtain a colostrum solution;

[0055] (5) Turn on the microfluidizer. Before formal use, first clean the volumetric cup and pipeline with 800 mL of 20% alcohol, then clean the volumetric cup and pipeline with 1 L of ultrapure water, and finally rinse with 500 mL of sterilized phosphate buffer. After cleaning, pour the colostrum solution prepared in (4) into the microfluidizer, adjust the pressure to 15,000 to 20,000 psi, adjust the temperature of the refrigerator, control the outlet temperature of the emulsion at 20 to 30° C., and cycle the high-pressure homogenization for 3 times to obtain a refined milk solution.

[0056] (6) sampling and observing the properties of the refined milk solution, which is milky white, and the particle size of the refined milk solution is measured by a Malvern particle size detector to be between 100 nm and 200 nm, and the pH value of the refined milk solution is detected to be between 7.0 and 7.4;

[0057] (7) Aseptically filter the concentrated milk solution in a biological safety cabinet using a 0.22 μm sterilizing filter, label the concentrated milk solution, and store it in a refrigerator at 2 to 8°C for later use.

[0058] (8) Pipette oligonucleotide CpG 1018 at a concentration of 400 ug / ml into the 1000 mL of the above refined milk solution and vortex mix for at least 30 seconds to obtain (named AD20PTB3-C2 group), which is then stored in a refrigerator at 2-8°C for future use.

[0059] Similarly, for the TB3-C2-Hi group, TB4-C2, TB4-C2-Hi group, and TB3-C3 group, TB3-C3-Hi group, TB4-C3, TB4-C3-Hi group of AD20P; the preparation method is similar to that of the AD20PTB3-C2 group, only the amount of each component is different. The amount of each component in each group is shown in Table 1 and Table 2, respectively.

[0060] Table 1 Amounts of various adjuvant composition ingredients

[0061]

[0062] Table 2 Amount of adjuvant components

[0063]

[0064] Note: When AD20P adjuvant and antigen are used together, the volume ratio is 1:1, that is, 50 μl of the prepared adjuvant is taken and mixed with 50 μl of antigen, and the final volume of 100 μl of the adjuvant-antigen mixture is injected into the experimental animals.

[0065] Example 2: Effects of different formulations of AD20P on immune response to VZV gE antigen

[0066] Immunization procedure: 6-8 week old female C57BL / 6 mice were immunized intramuscularly with 2 μg of varicella-zoster virus (VZV) gE protein (Sino Biological, 40907-V08H) antigen and various adjuvants as required (50 μl adjuvant mixed with 50 μl antigen). Immunization groups: (1) PBS (100 μl 1xPBS), (2) TB3-C2 adjuvant group, (3) TB3-C2-Hi adjuvant group, (4) TB4-C2 adjuvant group, (5) TB4-C2-Hi adjuvant group, (6) AS01 B Adjuvant group: The dosage of adjuvant components in groups (2)-(5) is shown in Table 2. The CpG dose for each mouse immunization is 20 μg. Each group was immunized twice, with an interval of 21 days. Blood was collected before and after each immunization. ELISA was performed using a 96-well microplate to detect the VZV gE-specific IgG antibody titer. AS01 B The adjuvant comes from GSK, product number RE-1125.

[0067] 1. Humoral immune response

[0068] After immunization of animals, antigen-specific antibody titers in mouse serum were determined by ELISA. The ELISA detection method process: Nunc 96-well ELISA plates were coated with 1 μg / mL VZVgE eggs at 4°C overnight. The plates were washed 3 times and then blocked with PBS containing 5% bovine serum albumin (BSA) (containing 0.05% Tween 20, i.e., PBST buffer) at 37°C for 1 hour. Washed 3 times with 250 μL PBST added to each well, and then three times of serial dilution of mouse serum was added to each well at a volume of 100 μL per well (the initial serum dilution ratio was 1:20000) and incubated at 37°C for 1 hour. The plates were washed again 6 times, and then goat anti-mouse IgG-HRP with an original concentration of 2 mg / mL and a dilution of 1:20000 was added for incubation at 37°C for 1 hour, washed 6 times, and then the bound antibodies were detected, developed by using TMB substrate, and the reaction was terminated with 50 μl / well 2M H2SO4. The OD was read at 450 nm and 620 nm using a microplate reader, and the data were processed and plotted using GraphPad Prism.

[0069] The results are as follows Figure 1As shown, the antibody titers against VZVgE antigen were detected 21 days and 42 days after mice were immunized. Figure 1 The results in Figure 2A show that the antibody titers 21 days after immunization showed that the four groups of composite adjuvants TB3-C2 adjuvant group, TB3-C2-Hi adjuvant group, TB4-C2 adjuvant group and TB4-C2-Hi adjuvant group could effectively induce high levels of antigen-specific antibodies, and the antibody levels of the three groups of TB3-C2-Hi adjuvant group, TB4-C2 adjuvant group and TB4-C2-Hi adjuvant group were comparable to those of the existing adjuvant AS01. B Group equivalent; 42 days after immunization, the results are as follows Figure 1 As shown in B, the antibody levels of the TB3-C2 adjuvant group and the TB4-C2 adjuvant group were compared with the existing adjuvant AS01. B The antibody levels of the TB3-C2-Hi adjuvant group and the TB4-C2-Hi adjuvant group were higher than those of the existing adjuvant AS01. B Group.

[0070] 2. Cellular Immune Response

[0071] 1) ELISpot detection of INF-γ and IL-2 secretion

[0072] 7, 12, 21, and 42 days after the first immunization, the mice were euthanized in a sterile environment, the spleens were removed and ground into cell suspensions, the cells were harvested by centrifugation, the cells were resuspended in red blood cell lysis buffer, lysed and filtered to prepare single cell suspensions. According to the instructions of the Mouse IFN-γ Precoated ELISpot Kit and the Mouse IL-2 Precoated ELISpot Kit, the single cell suspensions were concentrated to 5×10 ^ 5 cells / well were added to the ELISpot pre-coated plate wells, and 100 μL of gE protein peptide library with a final concentration of 10 μg / mL was added for stimulation, and duplicate wells were set. Negative and positive control wells were set at the same time according to the instructions. The positive wells were stimulated with PMA (final concentration 0.1 μg / mL) and Iono (final concentration 1 μg / mL), and the number of cells was 5×10 ^ 4 / well. Place the pre-coated plate with cells in a cell culture incubator at 37°C and 5% CO2 for 18 to 22 hours. Perform the experiment according to the instructions of the kit and perform quantitative detection by enzyme-linked spot analyzer to obtain the number of cells per 5×10 ^ The average number of spots of 4 spleen T lymphocytes was used to calculate the number of T lymphocytes per 1×10 ^ The number of INF-γ and IL-2 spots produced by 6 spleen T lymphocytes was processed and plotted using GraphPad Prism.

[0073] The results are as follows Figure 2 As shown in the figure, 7 days after the first immunization, the four groups of composite adjuvants TB3-C2 adjuvant group, TB3-C2-Hi adjuvant group, TB4-C2 adjuvant group and TB4-C2-Hi adjuvant group can effectively induce high levels of antigen-specific IFN-γ and IL-2 response to VZV gE antigen, and the levels of IFN-γ and IL-2 produced are significantly better than those of the existing adjuvant AS01. B , especially the TB4-C2-Hi adjuvant group secreted the highest levels of IFN-γ and IL-2; AS01 B The adjuvant is a vaccine adjuvant developed by GSK for the VZV vaccine that has been on the market. As an adjuvant for the VZV vaccine, it can greatly improve the immune effect of the vaccine. It can be seen that the composite adjuvant configured in the present invention is better than the existing VZV vaccine adjuvant AS01 B . Figure 3 , Figure 4 and Figure 5 The results of IFN-γ and IL-2 detection at 12, 21 and 42 days after immunization are shown respectively, which are basically consistent with the results at 7 days after the first immunization. The levels of IFN-γ and IL-2 secreted by the composite adjuvant group configured by the present invention are significantly better than those of the existing adjuvant AS01. B .

[0074] 2) Flow cytometry to detect CD4 + INF-γ + 、CD4 + IL-2 + T cell subsets

[0075] 7, 12, 21, and 42 days after the first immunization, the mice were euthanized in a sterile environment, the spleens were removed and ground into cell suspensions, the cells were harvested by centrifugation, the cells were resuspended in red blood cell lysis buffer, lysed and filtered, and single cell suspensions were prepared. 1 × 10 ^ 6 cells were plated in a 96-well cell culture plate and stimulated with 10 μg / mL of gE protein peptide library or PMA / Iono for 1 h at 37°C and 5% CO2, and then blocked with 1 μg / ml of Brefeldin A (BD, CA, USA) for 5 h. Extracellular and intracellular cytokine staining of spleen cells was performed. Stimulated spleen cells were stained with FVD-eFluor780, then washed and stained with anti-mouse CD4 fluorescent antibody for 30 min at room temperature in the dark. Cells were permeabilized with fixation / permeabilization buffer and intracellular staining was performed with anti-mouse IFN-γ and IL-2 fluorescent antibodies at 4°C for 45 min. Cells were washed twice and resuspended with 200 μL PBS, then collected using a flow cytometer (ThermoFisher, MA, USA), and finally analyzed using FlowJo software (BD, CA, USA).

[0076] The results are as follows Figure 6 As shown, 7 days after the first immunization, the results were basically consistent with the above ELISpot test results. For VZV gE antigen, the four groups of composite adjuvants TB3-C2 adjuvant group, TB3-C2-Hi adjuvant group, TB4-C2 adjuvant group and TB4-C2-Hi adjuvant group could significantly induce antigen-specific CD4 + INF-γ + 、CD4 + IL-2 + The production of T cell subsets, and the TB3-C2-Hi adjuvant group and the TB4-C2-Hi adjuvant group were significantly better than the existing adjuvant AS01 B , especially the TB4-C2-Hi adjuvant group induced CD4 + INF-γ + 、CD4 + IL-2 + The proportion of T cell subsets is the highest and the effect is the best. Figure 7 , Figure 8 and Fig. 9 The CD4 + INF-γ + 、CD4 + IL-2 + The results of T cell detection were basically consistent with the results described 7 days after the first immunization. The TB3-C2-Hi adjuvant group and the TB4-C2-Hi adjuvant group induced antigen-specific CD4 + INF-γ + 、CD4 + IL-2 + The proportion of T cell subsets is significantly better than that of the existing adjuvant AS01 B .

[0077] Example 3: Effects of different formulations of AD20P on immune response to VZV gE antigen

[0078] Immunization procedure: 6-8 week old female C57BL / 6 mice were immunized intramuscularly with 2 μg of varicella-zoster virus (VZV) gE protein (Sino Biological, 40907-V08H) antigen and various adjuvants as required (50 μl adjuvant mixed with 50 μl antigen). Immunization groups: (1) PBS (100 μl 1xPBS), (2) TB3-C3 adjuvant group, (3) TB3-C3-Hi adjuvant group, (4) TB4-C3 adjuvant group, (5) TB4-C3-Hi adjuvant group, (6) AS01 BAdjuvant group: The dosage of adjuvant components in groups (2) to (5) is shown in Table 3. The CpG dose for each mouse immunization is 50 μg. Each group was immunized twice, with an interval of 21 days. Blood was collected before and after each immunization. ELISA was performed using a 96-well microplate to detect the VZV gE-specific IgG antibody titer.

[0079] 1. Humoral immune response

[0080] ELISA method was used to measure serum antibodies in Example 2. The results are as follows Fig.10 As shown, the antibody titers against VZV gE antigen were detected 21 days and 42 days after the mice were immunized. Fig.10 Middle A and Fig.10 The results in Figure B show the antibody titers 21 days and 42 days after immunization. It can be seen from the figure that the four groups of composite adjuvants TB3-C2 adjuvant group, TB3-C2-Hi adjuvant group, TB4-C2 adjuvant group and TB4-C2-Hi adjuvant group can effectively induce high levels of antigen-specific antibodies after immunization; at 42 days after immunization, the antibody level increased overall, and the antibody levels of the four adjuvant groups were higher than the existing adjuvant AS01 B Group.

[0081] 2. Cellular Immune Response

[0082] 1) ELISpot detection of INF-γ and IL-2 secretion

[0083] The experimental method was similar to that of Example 2, and the secretion of INF-γ and IL-2 in mouse spleen cells was detected 12 and 42 days after immunization.

[0084] The results are as follows Fig.11 As shown in the figure, 12 days after the first immunization, the four groups of composite adjuvants TB3-C3 adjuvant group, TB3-C3-Hi adjuvant group, TB4-C3 adjuvant group and TB4-C3-Hi adjuvant group could effectively induce high levels of antigen-specific IFN-γ and IL-2 response to VZV gE antigen, and the levels of IFN-γ and IL-2 produced were significantly better than those of the existing adjuvant AS01. B , especially the TB3-C3-Hi adjuvant group secreted the highest levels of IFN-γ and IL-2; AS01 B The adjuvant is a vaccine adjuvant developed by GSK for the VZV vaccine that has been on the market. As an adjuvant for the VZV vaccine, it can greatly improve the immune effect of the vaccine. It can be seen that the composite adjuvant configured in the present invention is better than the existing VZV vaccine adjuvant AS01 B . Fig.12 The test results of IFN-γ and IL-2 at 42 days are basically consistent with the results at 12 days after the first immunization. The composite adjuvant configured by the present invention is superior to the existing VZV vaccine adjuvant AS01.B .

[0085] 2) Flow cytometry to detect CD4 + INF-γ + 、CD4 + IL-2 + T cell subsets

[0086] The experimental method refers to Example 2, and the percentages of INF-γ and IL-2 in CD4 T cells of mice were detected 12 and 42 days after immunization.

[0087] The results are as follows Fig.13 As shown, 12 days after the first immunization, the results were basically consistent with the above ELISpot test results. For VZV gE antigen, the four groups of composite adjuvants TB3-C3 adjuvant group, TB3-C3-Hi adjuvant group, TB4-C3 adjuvant group and TB4-C3-Hi adjuvant group could significantly induce antigen-specific CD4 + INF-γ + 、CD4 + IL-2 + The production of T cell subsets, and the TB3-C3-Hi adjuvant group and the TB4-C3-Hi adjuvant group were significantly better than the existing adjuvant AS01 B , the effect is best. Fig.14 CD4 42 days after immunization + INF-γ + 、CD4 + IL-2 + The detection results of T cells are basically consistent with the results 12 days after the first immunization. It can be seen that the composite adjuvant configured by the present invention is superior to the existing VZV vaccine adjuvant AS01. B .

[0088] From the results of all the above embodiments, it can be seen that compared with existing adjuvants, the composite adjuvant configured in the present invention can significantly improve antigen-specific humoral immunity and cellular immune response; especially for cellular immunity, the composite adjuvant configured in the present invention is far superior to existing adjuvants, which shows that the present invention has certain application prospects.

[0089] The above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, a person skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. An adjuvant composition for a vaccine, characterized in that: It includes squalene, Tween 80, poloxamer 188 and the oligonucleotide CpG1018.

2. The adjuvant composition according to claim 1, characterized in that The content of squalene is 0.2-4% (v / v), the content of Tween 80 is 0.02-0.4% (v / v), the content of poloxamer 188 is 0.1-2% (w / v), and the content of oligonucleotide CpG1018 is 200-6000 μg / ml (w / v).

3. The adjuvant composition according to claim 1 or 2, characterized in that The content of squalene is 0.8-4% (v / v), the content of Tween 80 is 0.064-0.32% (v / v), the content of poloxamer 188 is 0.2-1% (w / v), and the content of oligonucleotide CpG1018 is 300-5000 μg / ml (w / v); preferably, the content of oligonucleotide CpG1018 is 400-4000 μg / ml (w / v).

4. The adjuvant composition according to claim 1 or 2, characterized in that Poloxamer 188 is of pharmaceutical grade or injection grade.

5. The method for preparing the adjuvant composition according to any one of claims 1 to 4, characterized in that: The steps include: S1: Take squalene to prepare an oil phase; take poloxamer 188 and Tween 80 in a buffer solution and stir to prepare an aqueous phase; S2 slowly adding the prepared water phase to the prepared oil phase, and after adjusting the volume with a buffer solution, stirring at high speed at room temperature to obtain a colostrum solution; The S3 colostrum solution was poured into a microfluidizer to obtain a refined milk solution; S4 draws oligonucleotide CpG into the above-mentioned refined milk solution and mixes well.

6. The method according to claim 5, characterized in that The buffer is selected from but not limited to phosphate or citrate buffer.

7. The method according to claim 5, characterized in that In step S2, a high-speed stirrer is used to stir at a speed of 8000-20000 rpm for 15-30 minutes to obtain a colostrum solution; the colostrum solution prepared in step S3 is poured into a microfluidizer, the pressure is adjusted to 15000-20000 psi, the temperature of the refrigerator is adjusted, the outlet temperature of the emulsion is controlled at 20-30°C, and the high-pressure homogenization is cycled 3-10 times to obtain a refined milk solution.

8. The method according to claim 5, characterized in that In step S3, the particle size of the refined milk solution is measured by a Malvern particle size detector to be between 100nm and 200nm, and the pH value of the refined milk solution is detected to be between 6.3-6.7 or 7.0-7.4; preferably, sterilization filtration is also included, and specifically, a clear liquid is obtained by filtering with a 0.22μm sterilization filter as the refined milk solution.

9. Use of the adjuvant composition according to any one of claims 1 to 4 in the preparation of an immunization system.

10. An antigen adjuvant composition, characterized in that: The invention comprises the adjuvant composition as claimed in any one of claims 1 to 4 and an antigen capable of inducing an immune response; the antigen comprises one or more of proteins, polypeptides or nucleic acids.

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