A nanoemulsion adjuvant

Abstract

The present invention belongs to the field of immunology and provides a nanoemulsion adjuvant. The nanoemulsion adjuvant contains a bifunctional emulsifier. The bifunctional emulsifier used in the nanoemulsion adjuvant provided by the present invention, which has both emulsifying ability and can enhance the immune response, can completely replace emulsifiers with greater toxic and side effects such as Tween 80 and Span 85, is safer for the human body, and can significantly enhance the immunogenicity of vaccines. The production process of the nanoemulsion adjuvant provided by the present invention is simple, has good stability, is efficient and has good safety.

Description

Technical Field

[0001] The present invention belongs to the field of immunology, and particularly relates to a nanoemulsion adjuvant. Background Art

[0002] An adjuvant, also known as an immunomodulator or immunopotentiator, can enhance the immune response of the body to an antigen or change the type of immune response when it is injected into the body prior to or mixed with the antigen, and it itself has no antigenicity. In recent years, new vaccines with good antigenicity and low toxicity, including subunit vaccines, recombinant protein vaccines, and nucleic acid vaccines, have received extensive attention from researchers. However, these new vaccines usually have low immunogenicity and urgently need to develop highly efficient immune adjuvants for combined use.

[0003] Oil-in-water (O / W) nanoemulsion adjuvants can simultaneously induce Th1 and Th2 responses of the human immune system and have been proposed as adjuvant compositions. WO 95 / 17210 discloses an oil-in-water emulsion containing 2-10% squalene, 2-10% α-tocopherol, and 0.3-3% Tween 80, and its use alone or in combination with QS21 and / or 3D-MPL. WO99 / 12565 discloses an oil-in-water emulsion composition containing a metabolizable oil, saponin, and sterol, and the oil-in-water emulsion further contains 3D-MPL. WO99 / 11241 discloses an oil-in-water emulsion containing a metabolizable oil and saponin, wherein the oil and saponin are present in a ratio between 1:1 and 200:1. The most typical representative is MF59, which is an O / W adjuvant composed of 5% squalene, 0.5% polysorbate 80 (Tween 80), 0.5% sorbitan trioleate (Span 85), and 94% sodium citrate buffer (10 mM). Among them, squalene is a completely metabolizable lipid synthesized by the human body through the cholesterol synthesis pathway. It is emulsified into a milky state in an aqueous buffer by the surfactant Tween 80 and the co-surfactant Span 85, and then processed by high-pressure microfluidics to form a stable emulsion system with a particle size of about 160 nm. MF59 first appeared in a licensed product called Fluad in Europe, which is a trivalent inactivated vaccine against seasonal influenza. MF59 is outstanding in enhancing the immunogenicity of vaccines. The reason is its unique O / W emulsion form, and its individual components cannot cause a considerable adjuvant effect. This is mainly because the emulsifiers Tween 80 and Span 85 selected by MF59 are polyol non-ionic surfactants and cannot effectively stimulate immune cells. In addition, clinical studies have shown that when the Tween 80 in an injection exceeds 0.2%, it is likely to cause adverse reactions such as skin rash, hemolysis, and muscle pain, and the Tween 80 in MF59 is 0.5%, which poses a certain safety risk. Therefore, it is of great significance to develop a new nanoemulsion adjuvant using a bifunctional emulsifier that has both emulsifying ability and can stimulate immune cells. Summary of the Invention

[0004] To solve the above problems existing in the prior art, the present invention provides a nanoemulsion adjuvant.

[0005] The present invention provides a nanoemulsion adjuvant containing a bifunctional emulsifier.

[0006] The nanoemulsion adjuvant provided by the present invention uses a bifunctional emulsifier that not only has emulsifying ability but also can enhance the immune response, can completely replace emulsifiers with relatively large toxic and side effects such as Tween 80 and Span 85, is safer for the human body, and can significantly enhance the immunogenicity of vaccines. At the same time, the production process of this adjuvant is simple, has good stability, high efficiency and good safety. Brief Description of the Drawings

[0007] Figure 1 Shows the appearance of six nanoemulsion adjuvants in Example 1 of the present invention.

[0008] Figure 2 Shows the relationship between the particle size distribution of six nanoemulsion adjuvants in Example 1 of the present invention and the dosage of TPGS.

[0009] Figure 3 Shows the particle size distribution of the nanoemulsion adjuvant with a 2% TPGS dosage in Example 1 of the present invention.

[0010] Figure 4 Shows the change of IgG level against pro-03 over the immunization time in Example 3 of the present invention.

[0011] Figure 5 Shows the change of IFN-γ level in Example 3 of the present invention; in the figure, pool1, pool2, and pool 3 respectively represent three polypeptide segments in pro-03.

[0012] Figure 6 Shows the change of IgG level against pro-03 over the immunization time in Example 4 of the present invention.

[0013] Figure 7 Shows the change of IFN-γ level in Example 4 of the present invention. Detailed Description of the Invention

[0014] To make the technical solutions, objectives and advantages of the present invention clearer, the following further describes the present invention in detail through specific examples. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0015] The present invention provides a novel nanoemulsion adjuvant, and the nanoemulsion adjuvant contains a bifunctional emulsifier (the bifunctional emulsifier has both emulsifying properties and adjuvant properties).

[0016] Furthermore, the nanoemulsion adjuvant provided by the present invention further includes an oil phase and water.

[0017] According to the present invention, the oil phase can be at least one selected from peanut oil, soybean oil, coconut oil, olive oil, jojoba oil, safflower oil, cottonseed oil, sunflower oil, sesame oil, corn oil, cod liver oil, whale oil, tocopherol, squalene, and squalane.

[0018] According to the present invention, the content of the oil phase in the nanoemulsion adjuvant is 1% - 50% (w / v, weight - volume ratio, that is, the ratio of the weight of the oil phase to the volume of the entire nanoemulsion adjuvant. In the present invention, unless otherwise specified, the content of each component is the weight - volume ratio). Preferably, the content of the oil phase in the nanoemulsion adjuvant is 3% - 15%, such as any one or the range between two of 3%, 5%, 7%, 9%, 11%, 13%, 15%, for example, 3% - 7%; 5% - 11%, 7% - 13%, 9% - 15%. Further preferably, the content of the oil phase in the nanoemulsion adjuvant is 5%.

[0019] According to the present invention, the bifunctional emulsifier can be a non - ionic surfactant. The hydrophilic - lipophilic balance value (HLB) of the non - ionic surfactant ≥8, and it also has the function of enhancing the immune response.

[0020] Preferably, the non - ionic surfactant can be at least one selected from PEG (polyethylene glycol) derivatives containing vitamin E succinate (i.e., α - tocopherol ester structure) (abbreviated as TPGS), PEG derivatives containing QS - 21 structure, PEG derivatives containing MPL structure, and other non - ionic surfactants containing immune - stimulating molecular structures. The PEG (polyethylene glycol) can be at least one of PEG450, PEG600, PEG750, PEG800, PEG1000, PEG1200, etc.

[0021] Further preferably, the non - ionic surfactant is TPGS 1000 (vitamin E polyethylene glycol 1000 succinate).

[0022] According to the present invention, the content of the bifunctional emulsifier in the nanoemulsion adjuvant is 0.1%-5% (w / v). Preferably, the content of the bifunctional emulsifier in the nanoemulsion adjuvant is 1%-4%, such as any one or the range between two of 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, such as 1%-2.5%; 1.5%-3.5%, 2%-4%, 1%-3%. Further preferably, the content of the bifunctional emulsifier in the nanoemulsion adjuvant is 2%.

[0023] According to the present invention, the nanoemulsion adjuvant further contains a buffer. The buffer can be any one selected from citric acid buffer, phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer.

[0024] According to the present invention, in a preferred specific embodiment, the oil phase in the nanoemulsion adjuvant is squalene, and the weight ratio of squalene to the bifunctional emulsifier is (1-5):1, such as any one or the range between two of 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, such as (1-3):1, (1.5-3.5):1, (2-4):1, (2.5-4.5):1, (2-5):1. Further preferably, the weight ratio of squalene to the bifunctional emulsifier is 2.5:1, and the content of squalene in the finally formed nanoemulsion adjuvant is 5% (w / v).

[0025] In another preferred specific embodiment, the oil phase in the nanoemulsion adjuvant is squalene, the bifunctional emulsifier is TPGS 1000, and the buffer is citric acid buffer; wherein, the concentration of the citric acid buffer is 10 mM.

[0026] The present invention also provides a preparation method of the above nanoemulsion adjuvant, and the method includes the operation of fully mixing the bifunctional emulsifier, the oil phase and water to form a homogeneous emulsion.

[0027] Specifically, the nanoemulsion adjuvant can be prepared by any one of the following methods:

[0028] Method 1: High-speed stirring method. The high-speed stirring method can include: according to the ratio of the oil phase, water, and bifunctional emulsifier in the nanoemulsion adjuvant provided by the present invention, first dissolve the bifunctional emulsifier in water (if necessary, a buffer can also be added) to obtain an aqueous phase; then, at a stirring speed of 800-2500 rpm (preferably 1500 rpm), add the oil phase to the aqueous phase and stir for 0.5-6 h (preferably 4 h) to make the oil phase uniformly disperse in the aqueous phase.

[0029] Method 2: High-speed shear emulsification method. The high-speed shear emulsification method may include: according to the ratio of the oil phase, water, and bifunctional emulsifier in the nanoemulsion adjuvant provided by the present invention, first dissolve the bifunctional emulsifier in water (a buffer can be added if necessary) to obtain an aqueous phase; then, at a shear emulsification speed of 10,000 - 30,000 rpm (preferably 12,000 rpm), add the oil phase to the aqueous phase and shear for 5 - 30 min (preferably 10 min) to make the oil phase uniformly dispersed in the aqueous phase.

[0030] Method 3: Microfluidic high-pressure homogenization method. The microfluidic high-pressure homogenization method may include: according to the ratio of the oil phase, water, and bifunctional emulsifier in the nanoemulsion adjuvant provided by the present invention, first dissolve the bifunctional emulsifier in water (a buffer can be added if necessary) to obtain an aqueous phase; add the oil phase to the aqueous phase and stir slightly, and then perform homogenization treatment 1 - 10 times (preferably 5 times) at a homogenization pressure of 5,000 - 35,000 PSI (preferably 10,000 PSI) to make the oil phase uniformly dispersed in the aqueous phase.

[0031] After the operation of the above method, in the obtained nanoemulsion adjuvant, the particle size of the oil phase dispersed in the aqueous phase is 50 - 500 nm, preferably 140 - 180 nm. The prepared nanoemulsion adjuvant has no change in particle size after being placed at 2 - 8 °C for 6 months and has excellent stability.

[0032] To make the above objects, features, and advantages of the present invention more clear, the following specific embodiments will be used to describe the specific implementation manners of the present invention in detail.

[0033] For all kinds of reagents, materials, etc. used in the following embodiments, if not otherwise specified, they are all products that can be obtained from commercial channels; for all kinds of testing and detection methods used in the following embodiments, if not otherwise specified, they are all conventional testing and detection methods in the art and can be obtained from textbooks, reference books, or academic journals.

[0034] Example 1

[0035] In the nanoemulsion adjuvant provided in this example, the oil phase is squalene, and the content is fixed at 5% (w / v); the bifunctional emulsifier is TPGS 1000, and the contents are 0.5%, 1%, 2%, 3%, 4%, and 5% respectively (see Table 1).

[0036] The specific operation steps for preparing the nanoemulsion adjuvant (total volume 100 mL) are as follows:

[0037] (1) Weigh accurately 0.5 g, 1.0 g, 2.0 g, 3.0 g, 4.0 g and 5.0 g of TPGS 1000 respectively, place them in six 200 mL beakers, then add the corresponding volume of ultrapure water according to the content requirements in Table 1, stir magnetically at room temperature until completely dissolved, filter through a 0.22 μm filter membrane, and set aside.

[0038] (2) Prepare the oil phase: Weigh accurately six portions of 5.0 g of squalene respectively, filter through a 0.22 μm filter membrane, and set aside.

[0039] (3) High-speed stirring method: At room temperature, use a rotary magnetic stirrer to maintain a rotation speed of 1500 rpm, stir the solution prepared in step (1) until it is transparent and uniform. Then, under the condition of magnetic stirring at 1500 rpm, slowly add the oil phase prepared in step (2) to the solution prepared in step (1). Stir for 4 hours to form a milky white system. Filter through a 0.22 μm PES filter membrane, collect the filtrate in a storage bottle, seal it with sealing gum, and store it at 2 - 8 °C.

[0040] Step (3) can also be carried out in any of the following ways:

[0041] High-speed shear emulsification method: At room temperature, use a high-speed shear emulsifier, stir the solution prepared in step (1) until it is transparent and uniform at a rotation speed of 12000 rpm. Maintain a shear state of 12000 rpm, and slowly add the oil phase prepared in step (2) to the solution prepared in step (1). Shear for 10 min to form a milky white system. Filter through a 0.22 μm PES filter membrane, collect the filtrate in a storage bottle, seal it with sealing gum, and store it at 2 - 8 °C.

[0042] Microfluidic high-pressure homogenization method: At room temperature, add the oil phase prepared in step (2) to the solution prepared in step (1) and stir slightly to make it uniform. Then use a microfluidic high-pressure homogenizer to carry out homogenization treatment 5 times under the condition of a homogenization pressure of 10000 PSI to form a milky white system. Filter through a 0.22 μm PES filter membrane, collect the filtrate in a storage bottle, seal it with sealing gum, and store it at 2 - 8 °C.

[0043] Table 1 Nanoemulsion adjuvant formulation table (w / v)

[0044]

[0045]

[0046] The physicochemical properties of the above-prepared nanoemulsion adjuvant are as follows:

[0047] I. Appearance and pH value

[0048] Such as Figure 1As shown, from left to right are the appearances of the squalene with a content of 5% and the TPGS 1000 nanoemulsion adjuvants with contents of 0.5%, 1%, 2%, 3%, 4%, and 5% prepared above. Among them, the nanoemulsion adjuvants with 1%, 2%, 3%, and 4% TPGS 1000 contents form milky white liquids; among them, the nanoemulsion adjuvant with a 2% TPGS content (the third from the left) is a milky white homogeneous and stable liquid with the best appearance and no stratification; the nanoemulsion adjuvant with a 2% TPGS 1000 content has the best fluidity, and the kinematic viscosity is less than 10 cP (mPa·s); the pH value is in the range of 6.5 - 8.0. It shows that the optimal dosage range of TPGS 1000 in the present invention is between 1% and 4%, and the nanoemulsion adjuvant with a 2% TPGS 1000 content has the best appearance.

[0049] II. Particle Size and Its Distribution

[0050] Take 1 mL of the six nanoemulsion adjuvant samples prepared above respectively, dilute them 100 times with ultrapure water, then take 1.0 mL, and use a Zetasizer nano Pro dynamic laser scattering instrument to measure the particle size (Z - average) and polydispersity index (PDI) of the emulsion.

[0051] Table 2 shows the particle sizes and polydispersity indices of the six nanoemulsion adjuvants above. It can be seen from Table 2 that the particle size distributions of the nanoemulsion adjuvants of squalene with a content of 5% and TPGS 1000 with contents of 0.5%, 1%, 2%, 3%, 4%, and 5% are between 72.92 nm and 467.2 nm, and the PDI distributions are between 0.09 and 0.43.

[0052] Table 2 Particle Sizes and Polydispersity Indices of Nanoemulsion Adjuvants

[0053]

[0054]

[0055] As Figure 2 shown, the particle sizes of the six nanoemulsion adjuvants above have a positive correlation with the dosage of TPGS 1000 (0.5% - 5%), and the particle size increases with the increase of the dosage of TPGS 1000; the correlation between the PDI of the six nanoemulsion adjuvants and the dosage of TPGS 1000 presents a V - shape, and the PDI first decreases and then increases with the increase of the dosage of TPGS 1000. This indicates that when the dosage ratio of squalene to TPGS 1000 is 2.5∶1, the new nanoemulsion adjuvant has a high dispersion uniformity and reaches the best stable dispersion state.

[0056] As Figure 3As shown, the nanoemulsion adjuvant with a 2% dosage of TPGS 1000 has the best particle size distribution, with a particle size of 151.3 nm; the PDI is 0.09 (a PDI of nanoparticles ≤ 0.20 indicates high dispersion uniformity); the number of particles in the range of 140 - 180 nm is greater than 95%, and the number of particles exceeding 200 nm is less than 2%.

[0057] Example 2:

[0058] This example is used to illustrate the stability of the nanoemulsion adjuvant with a 2% concentration of TPGS 1000 prepared in Example 1.

[0059] Centrifugal stability: The nanoemulsion adjuvant with a 2% concentration of TPGS 1000 prepared in Example 1 was centrifuged at 3500 rpm for 30 min, and it still maintained a milky white and uniform appearance, without phenomena such as stratification, flocculation, and precipitation.

[0060] Long-term, accelerated, and high-temperature stability: The nanoemulsion adjuvant with a 2% concentration of TPGS 1000 prepared in Example 1 was sealed and packaged, and placed in a constant temperature and humidity chamber at 4°C (long-term), 25°C (accelerated), and 37°C (high-temperature) for 3 months. The particle size, PDI, and appearance of the nanoemulsion adjuvant were measured and observed at 0 month, 1 month, 2 months, and 3 months according to the method in Example 1. The results are shown in Table 3.

[0061] Table 3 Particle size distribution and appearance changes under different storage conditions

[0062]

[0063]

[0064] As can be seen from the data in Table 3, during the placement at 4°C, the nanoemulsion adjuvant was a milky white and uniform liquid, without demulsification phenomena including stratification, flocculation, and precipitation. The particle size was maintained at about 150 nm, and the PDI was less than 0.2.

[0065] When the nanoemulsion adjuvant was placed at 25°C for 2 - 3 months, it was a milky white and uniform liquid, without demulsification phenomena including stratification, flocculation, and precipitation, but the particle size and PDI both increased slightly.

[0066] When the nanoemulsion adjuvant was placed at 37°C for 3 months, there were no demulsification phenomena including stratification, flocculation, and precipitation, and both the particle size and PDI increased slightly.

[0067] The above results indicate that the centrifugal stability, long-term, accelerated, and high-temperature stability of the nanoemulsion adjuvant provided by the present invention are all very high, especially the most stable under the storage environment of 4°C.

[0068] Using the same method as above, the stability of the nanoemulsion adjuvants with 1% and 3% TPGS 1000 prepared in Example 1 was determined.

[0069] It was found that when the nanoemulsion adjuvants with 1% and 3% TPGS 1000 were centrifuged at 3500 rpm for 30 min, they could also basically maintain a milky white and uniform appearance, and no observable phenomena such as stratification, flocculation, and precipitation occurred.

[0070] Long-term, accelerated, and high-temperature stability: The nanoemulsion adjuvants with 1% and 3% TPGS 1000 could also always remain as milky white and uniform liquids during storage at 4°C, and no observable phenomena such as stratification, flocculation, and precipitation occurred. When stored at 25°C for more than 3 months, a little flocculation and oil layer could be observed, and both the particle size and PDI increased. The above phenomena were more obvious in the nanoemulsion adjuvant with 3% TPGS 1000. When stored at 37°C for 3 months, a little stratification and demulsification occurred, the particle size and PDI increased, and the original stable emulsified dispersion system became unstable.

[0071] Example 3:

[0072] This example is used to illustrate the application of the nanoemulsion adjuvant prepared in Example 1 in enhancing the immunogenicity of recombinant protein vaccines.

[0073] Take 500 μL of the nanoemulsion adjuvant (Nano-Emulsion Adjuvant, NEA) with 2% TPGS 1000 prepared in Example 1 and 500 μL of the respiratory syncytial virus antigen (pro-03Antigen) solution, and place them in separate 1.5 mL sterile centrifuge tubes; take another 250 μL of NEA and 250 μL of the respiratory syncytial virus antigen, place them in a 1.5 mL sterile centrifuge tube and pipette to mix evenly to prepare the NEA-antigen solution (pro-03+NEA); then take 250 μL of SWE TM (a product purchased from SEPPIC, France, with the trade name SEPIVAC SWE, containing Tween 80 and Span 85) and 250 μL of the respiratory syncytial virus antigen, place them in a 1.5 mL sterile centrifuge tube and pipette to mix evenly to prepare the SWE TM -antigen solution (pro-03+SWE TM ).

[0074] Take 20 female BALB / C SPF mice, 5 in each group, and divide them into four groups: A, B, C, and D. Each mouse in group A was inoculated with 100 μL of NEA (corresponding to the negative control in Figure 4 ), and each mouse in group B was inoculated with 100 μL of the respiratory syncytial virus antigen solution (corresponding to Figure 4in pro-03), each mouse in group C was inoculated with 100 μL of the prepared NEA-antigen solution (corresponding to Figure 4 in pro-03 + NEA) solution, and each mouse in group D was inoculated with 100 μL of the SWE TM -antigen solution (corresponding to Figure 4 in pro-03 + SWE TM ). Four weeks after the primary immunization, a secondary immunization was carried out. Blood and spleens were collected at the planned time, and IgG and IFN-γ were detected respectively to evaluate the humoral and cellular immune capabilities.

[0075] It can be seen from Figure 4 that six weeks after the immunization, the immunoglobulin (IgG) level of the mice that used the NEA vaccine (NEA-antigen solution) was 1.2 times that of the mice that used the SWE TM -antigen solution and 1.4 times that of the mice that only used the pro-03 antigen. This indicates that the NEA containing the bifunctional emulsifier provided by the present invention can significantly enhance the humoral immunity.

[0076] It can be seen from Figure 5 that six weeks after the immunization, the IFN-γ level of the mice that used the NEA vaccine (NEA-antigen solution) was 4.7 times that of the mice that used the SWE TM -antigen solution and 80 times that of the mice that only used the pro-03 antigen, which indicates that the NEA containing the bifunctional emulsifier provided by the present invention can significantly enhance the cellular immunity.

[0077] Example 4

[0078] This example is used to illustrate the application of the nanoemulsion adjuvant prepared in Example 1 in enhancing the immunogenicity of the recombinant protein vaccine.

[0079] The nanoemulsion adjuvant with a TPGS 1000 concentration of 2% in Example 3 was replaced with nanoemulsion adjuvants with TPGS 1000 concentrations of 1% and 3% respectively, and the tests were carried out according to the same operation method as above.

[0080] It was found that for the mice that used the NEA vaccine (corresponding to Figure 6 in pro-03 + NEA-1%) formulated with the nanoemulsion adjuvant with a TPGS 1000 concentration of 1%, the IgG level was 1.07 times that of the mice that used the SWE TM -antigen solution and 1.28 times that of the mice that only used the pro-03 antigen; the IFN-γ level was 1.6 times that of the mice that used the SWE TM -antigen solution and 26 times that of the mice that only used the pro-03 antigen ( Figure 7 ).

[0081] For the NEA vaccine formulated with a nanoemulsion adjuvant containing 3% TPGS 1000 (corresponding to pro-03 + NEA-3% in Figure 6 ), the level of mouse IgG was 1.18 times that of the SWE TM -antigen solution and 1.33 times that of the pro-03 antigen alone; the level of IFN-γ was 2.4 times that of the SWE TM -antigen solution and 38 times that of the pro-03 antigen alone.

[0082] Example 5

[0083] Prepare Prescription 7 according to the method of Example 1; it is the same as Example 1 Prescription 3 (TPGS1000 content 2%) except for the following parameters:

[0084] Adjust the content of the oil phase squalene from 5% (w / v) to 2%, so that the weight ratio of squalene to TPGS 1000 is changed from 2.5:1 in Example 1 Prescription 3 to 1:1.

[0085] Prepare Prescription 8 according to the method of Example 1; it is the same as Example 1 Prescription 3 (TPGS1000 content 2%) except for the following parameters:

[0086] Adjust the content of the oil phase squalene from 5% (w / v) to 10%, so that the weight ratio of squalene to TPGS 1000 is changed from 2.5:1 in Example 1 Prescription 3 to 5:1.

[0087] Using the same method as in Example 2 above, determine the stability of the nanoemulsion adjuvants of the above Prescriptions 7 and 8.

[0088] It was found that when the nanoemulsion adjuvants of Prescriptions 7 and 8 were centrifuged at 3500 rpm for 30 min, they both maintained a milky homogeneous appearance and no phenomena such as stratification, flocculation, and precipitation occurred.

[0089] Long-term, accelerated, and high-temperature stability: The nanoemulsion adjuvants of Prescriptions 7 and 8 remained milky homogeneous liquids during storage at 4°C, and no phenomena such as stratification, flocculation, and precipitation occurred.

[0090] When stored at 25°C for more than 3 months, almost no flocculation and oil layer phenomena were observed in the nanoemulsion adjuvants of Prescriptions 7 and 8, and the particle size and PDI increased slightly. When stored at 37°C for 3 months, no demulsification phenomena such as stratification and flocculation were found in the nanoemulsion adjuvants of Prescriptions 7 and 8, and the particle size and PDI both increased slightly, and a stable emulsified dispersion system was basically maintained.

[0091] When testing the application effect of the nanoemulsion adjuvants of Formulation 7 and Formulation 8 in enhancing the immunogenicity of the recombinant protein vaccine according to the operation method of Example 3, it was found that for the mice treated with the nanoemulsion adjuvants of Formulation 7 and Formulation 8, the IgG level and IFN-γ level were basically the same as those of Formulation 3, with no obvious difference.

[0092] In summary, by using the NEA containing the bifunctional emulsifier provided by the present invention, Tween 80 and Span 85 can be reduced or not used, its preparation process is simple and efficient, and at the same time, it can significantly enhance the immunogenicity of the recombinant protein vaccine.

[0093] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A nanoemulsion adjuvant, characterized in that: The nanoemulsion adjuvant is composed of a bifunctional emulsifier, an oil phase and water; The bifunctional emulsifier is TPGS 1000; The oil phase is squalene; The content of the bifunctional emulsifier in the nanoemulsion adjuvant is 0.1% - 5% w / v; The content of the oil phase in the nanoemulsion adjuvant is 1% - 50% w / v; The weight ratio of the oil phase to the bifunctional emulsifier is (1 - 5)∶1.

2. The nanoemulsion adjuvant according to claim 1, characterized in that: The content of the bifunctional emulsifier in the nanoemulsion adjuvant is 1% - 4% w / v.

3. The nanoemulsion adjuvant according to claim 1, characterized in that: The content of TPGS 1000 in the nanoemulsion adjuvant is 2% w / v.

4. The nanoemulsion adjuvant according to claim 1, characterized in that: The content of squalene in the nanoemulsion adjuvant is 5% w / v.

5. The nanoemulsion adjuvant according to claim 1, characterized in that: The weight ratio of squalene to the bifunctional emulsifier is 2.5∶1.

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