Composition, adjuvant, vaccine and use thereof

Abstract

The present invention relates to the field of biological vaccines, and in particular to compositions, adjuvants, vaccines and their applications. The present invention provides a composition, comprising: squalane and squalene, wherein the mass ratio of the squalane to the squalene is: (1-10): (10-1). The novel nanoemulsion adjuvant provided by the present invention has low cost, good stability and safety, and can be used in combination with influenza antigens to effectively enhance the immune response of the vaccine. It can be used as a candidate adjuvant for a variety of vaccines such as influenza vaccines, human papillomavirus vaccines, and rabies vaccines.

Description

Technical Field

[0001] The present invention relates to the field of biological vaccines, and in particular to compositions, adjuvants, vaccines and applications thereof. Background Art

[0002] Vaccines are undoubtedly one of the most remarkable health achievements in human history.

[0003] With the rapid development of modern biotechnology, new vaccine antigens have high purity, small relative molecular weight, and low adverse reactions. However, the immunogenicity of these antigens is generally weak and cannot induce sufficient immune protection. Therefore, they usually need to be used in combination with adjuvants. Adjuvants can effectively enhance the body's specific immune response to antigens, affect the type of immune response, speed up the immune response, reduce the antigen vaccination dose and the number of vaccinations, etc.

[0004] At present, the most widely used adjuvant is still aluminum adjuvant, including aluminum hydroxide, aluminum phosphate, etc. Since Glenny first used aluminum salts to adhere diphtheria toxoid in 1926, aluminum adjuvants have been used as vaccine adjuvants for a variety of vaccines in the United States and Europe, and have accumulated an impressive safety record. Billions of doses have been administered over the decades. It can trigger a strong humoral immune response (i.e., antibody production), but aluminum adjuvants also have many shortcomings, such as little effect on enhancing antibody-dependent cellular cytotoxicity (ADCC) and cytotoxic T lymphocyte (CTL) activity, which are essential for protection from various pathogens and cross-reactions to heterologous influenza viruses; there are large differences between different batches of products, and quality is difficult to control; it can cause local adverse reactions, form granulomas, and even local sterile abscesses.

[0005] At present, in addition to aluminum adjuvants, MF59, AS03 and AF03 are the influenza vaccine adjuvants approved for human use. MF59 was originally developed as an antigen delivery buffer and was first tested in combination with antigens and the immunopotentiator MTP-PE. Surprisingly, MF59 itself has good adjuvant activity. The seasonal influenza vaccine (Fluad) containing MF59 adjuvant was first approved for use in the elderly in Italy in 1997, and was subsequently approved by other countries, the European EMA and the US FDA. In addition to seasonal influenza vaccines, it is also widely used in influenza pandemic vaccines, such as the Audenz vaccine for H5N1 influenza virus approved by the US FDA in 2020. All of its ingredients are biodegradable natural derivatives, safe and well tolerated. In addition, it is usually prepared by microfluidization. The method is to disperse Span85 in the squalene phase, disperse another surfactant Tween80 in the aqueous phase, and then homogenize to form a coarse emulsion. The prepared crude emulsion was repeatedly passed through a high-pressure homogenizer to produce a uniform emulsion with an oil droplet size of about 160 nm, which was sterile filtered for clinical use.

[0006] AS03 is a squalene oil-in-water emulsion adjuvant, which has similar ingredients to MF59, except that α-tocopherol (vitamin E), a cytokine secretion enhancer and innate immune positive regulator, is added. AS03 has been approved for use in pandemic influenza vaccines. Although the molecular mechanism of AS03-induced immunity has not been fully disclosed, it is known to have a dose-saving effect. For influenza vaccines, AS03-adjuvanted vaccines require lower antigen loads than other squalene-based adjuvanted vaccines. However, during the 2009-2010 influenza A (H1N1) epidemic, several European countries reported neurological adverse reactions in recipients of the vaccine Pandemrix, mainly drowsiness and excessive sleep in people aged 4-19 years. It was later withdrawn from the market, but a retrospective study showed that Pandemrix was associated with an increased risk of narcolepsy and multiple sclerosis, which may be unrelated to AS03.

[0007] AF03 adjuvant is an oil-in-water emulsion containing squalene, polyoxyethylene cetearyl ether, mannitol and sorbitan oleate. It is worth noting that it is not prepared by the commonly used microfluidization, but by the phase inversion temperature method. AF03 adjuvant was included in the influenza pandemic vaccine, but the vaccine has never been marketed. Its disadvantage is that the preparation process is complicated and the quality is difficult to control.

[0008] For the MF59 adjuvant, which has been used in marketed vaccines, its oil phase component is squalene, a natural biodegradable oil that is found in plants, animals and humans. In humans, squalene is an intermediate in the biosynthesis of steroid hormones and a precursor for cholesterol synthesis. In addition, it is currently used in many vaccines and drug delivery emulsions due to its enhanced stability and biocompatibility properties. Its hydrogenated form, squalane, is also a natural product that is secreted in small amounts in the sebaceous glands of the human body. Squalane can be prepared by hydrogenation of squalene and has been widely used in cosmetics and as a carrier for lipophilic drugs. Both squalene and squalane can be metabolized and have a good record in toxicological studies, but squalane is a saturated hydrocarbon with more stable chemical properties and is more popular. Summary of the invention

[0009] In view of this, the present invention provides a composition, an adjuvant, a vaccine and uses thereof.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The invention provides a composition, comprising: squalane and squalene, wherein the mass ratio of the squalane to the squalene is: (1-10): (10-1).

[0012] In some embodiments of the present invention, in the above composition, the mass ratio of the squalane to the squalene is: (1-7): (7-1).

[0013] In some embodiments of the present invention, in the above composition, the mass ratio of the squalane to the squalene is: 1:3, 1:1, 3:1, 1:7 and / or 7:1.

[0014] The present invention also provides application of the above composition in preparing adjuvant.

[0015] The present invention also provides an adjuvant, comprising the above composition, a buffer system and a surfactant.

[0016] In some embodiments of the present invention, in the above-mentioned adjuvant, the content of the composition accounts for less than or equal to 10% (m / v) of the total volume of the adjuvant; the content of the emulsifier accounts for 0.3-3% (m / v) of the total volume of the adjuvant.

[0017] In some embodiments of the present invention, in the above-mentioned adjuvant, the content of the composition accounts for 3-5% (m / v) of the total volume of the adjuvant.

[0018] In some embodiments of the present invention, in the above-mentioned adjuvant, the content of the composition accounts for 3.9% (m / v) of the total volume of the adjuvant.

[0019] In some embodiments of the present invention, in the above adjuvant, the content of the emulsifier accounts for 0.47% (m / v) of the total volume of the adjuvant.

[0020] In some embodiments of the present invention, in the above-mentioned adjuvant, the buffer system includes: one or more of phosphate buffer, phosphate buffer, Tris buffer and / or sodium citrate buffer.

[0021] In some embodiments of the present invention, in the above adjuvant, the pH value of the buffer system is 5.0-8.5.

[0022] In some embodiments of the present invention, in the above adjuvant, the pH value of the buffer system is 6.0-8.0.

[0023] In some embodiments of the present invention, in the above adjuvant, the pH value of the buffer system is 6.0-7.5.

[0024] In some embodiments of the present invention, in the above-mentioned adjuvant, the buffer system comprises: sodium citrate buffer.

[0025] In some embodiments of the present invention, the above-mentioned adjuvant, the surfactant includes: one or more of Pluronic L121, mannitol monooleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan laurate, sorbitan trioleate, sorbitan fatty acid ester, triethylene glycol monolauryl ether, polyethylene glycol hexadecyl ether and / or polyethylene glycol octylphenyl ether.

[0026] In some embodiments of the present invention, the above-mentioned adjuvant, the surfactant includes: polyoxyethylene sorbitan monooleate and sorbitan trioleate.

[0027] The present invention also provides a method for preparing the adjuvant, which comprises mixing the composition, the buffer system and the surfactant, homogenizing and emulsifying to obtain the adjuvant.

[0028] In some embodiments of the present invention, the method for preparing the above adjuvant comprises the following steps:

[0029] S1: dispersing the sorbitan trioleate in the squalene and the squalane to obtain an oil phase;

[0030] S2: mixing the polyoxyethylene sorbitan monooleate and the sodium citrate buffer to obtain an aqueous phase;

[0031] S3: The oil phase and the water phase are mixed, homogenized, emulsified and filtered to obtain the adjuvant.

[0032] The present invention also provides the use of the above composition, the above adjuvant and / or the adjuvant obtained by the above preparation method in any of the following items:

[0033] (I), preparing a vaccine; and / or

[0034] (II), preparing a drug delivery vehicle; and / or

[0035] (III) preparing a drug sustained-release carrier.

[0036] In some embodiments of the present invention, in the above-mentioned application, the antigens of the vaccine include: one or more of influenza virus, new coronavirus, human papillomavirus, rabies virus, hepatitis B virus, polio virus and / or human immunodeficiency virus.

[0037] In some embodiments of the present invention, in the above application, the antigen of the vaccine includes: influenza virus.

[0038] In some embodiments of the present invention, in the above application, the antigen content of the vaccine does not exceed 1% (m / v).

[0039] The present invention also provides a vaccine, comprising the above composition, the above adjuvant and / or the adjuvant obtained by the above preparation method.

[0040] In some embodiments of the present invention, the above-mentioned vaccines include: adjuvants of different vaccines such as inactivated vaccines, attenuated vaccines, split vaccines, DNA vaccines, recombinant vaccines, subunit vaccines, polypeptide protein vaccines, including but not limited to one or more of influenza vaccines, human papillomavirus vaccines, hepatitis B vaccines, rabies vaccines and / or polio vaccines.

[0041] In some embodiments of the present invention, the administration of the above-mentioned vaccine includes: one or more of parenteral injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, inhalation through the respiratory tract, rectal administration, vaginal administration, nasal administration, oral administration, ocular administration, topical administration, transdermal and / or intradermal administration.

[0042] The invention provides a composition, comprising: squalane and squalene, wherein the mass ratio of the squalane to the squalene is: (1-10): (10-1).

[0043] The novel nanoemulsion adjuvant provided by the present invention has low cost, good stability and safety, and can be used in combination with influenza antigens to effectively enhance the immune response of vaccines. It can be used as a candidate adjuvant for various vaccines such as influenza vaccine, novel coronavirus vaccine, human papillomavirus vaccine, rabies vaccine, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0045] Figure 1 2 shows the characterization results of Example 2, wherein A and B are the particle size and Zeta potential characterization diagrams of ZSE-12.5, respectively;

[0046] Figure 2 2 shows the characterization results of Example 2, wherein A and B are the particle size and Zeta potential characterization diagrams of ZSE-25, respectively;

[0047] Figure 3 2 shows the characterization results of Example 2, wherein A and B are the particle size and Zeta potential characterization diagrams of ZSE-50, respectively;

[0048] Figure 4 2 shows the characterization results of Example 2, wherein A and B are the particle size and Zeta potential characterization diagrams of ZSE-75, respectively;

[0049] Figure 5 2 shows the characterization results of Example 2, wherein A and B are the particle size and Zeta potential characterization diagrams of ZSE-87.5, respectively;

[0050] Figure 6 2 shows the characterization results of Example 2, wherein A and B are the particle size and Zeta potential characterization diagrams of ZSE-100, respectively;

[0051] Figure 7 The stability results of Example 3 are shown, wherein A, B, and C are the particle size, PDI, and Zeta potential of ZSE-12.5, respectively;

[0052] Figure 8 The stability results of Example 3 are shown, wherein A, B, and C are the particle size, PDI, and Zeta potential of ZSE-25, respectively;

[0053] Fig. 9 The stability results of Example 3 are shown, wherein A, B, and C are the particle size, PDI, and Zeta potential of ZSE-50, respectively;

[0054] Fig.10 The stability results of Example 3 are shown, wherein A, B, and C are the particle size, PDI, and Zeta potential of ZSE-75, respectively;

[0055] Fig.11 The stability results of Example 3 are shown, wherein A, B, and C are the particle size, PDI, and Zeta potential of ZSE-87.5, respectively;

[0056] Fig.12The stability results of Example 3 are shown, wherein A, B, and C are the particle size, PDI, and Zeta potential of ZSE-100, respectively;

[0057] Fig.13 The figure shows the changes in body weight and body temperature after vaccination with the novel nanoemulsion combined with monovalent B / Victoria influenza vaccine in Example 4 of the present invention; wherein: A is the measurement result of body temperature, and B and C are the measurement results of body weight;

[0058] Fig.14 The present invention shows the serum antigen-specific IgG level induced by the novel nanoemulsion combined with the monovalent B / Victoria influenza vaccine in Example 4; wherein: A, B, and C are the results of 1 week after the first immunization, 2 weeks after the second immunization, and 4 weeks after the second immunization, respectively; the IgG titer is displayed as GMT±SD;

[0059] Fig.15 4 shows the IgG typing antibody levels induced by the novel nanoemulsion combined with the monovalent B / Victoria influenza vaccine in Example 4 of the present invention; wherein: A is the result of serum IgG2c 2 weeks after the second immunization, B is the result of serum IgG1 2 weeks after the second immunization, and C is the result of the ratio of serum IgG2c / IgG1 2 weeks after the second immunization; the IgG typing antibody titer is displayed as GMT±SD; One-wayANOVA is used for statistical comparison;

[0060] Fig.16 The HI levels induced by the novel nanoemulsion combined with the monovalent B / Victoria influenza vaccine in Example 4 of the present invention are shown; wherein: A, B, C, and D are the HI levels 2 weeks after the first immunization, 2 weeks after the second immunization, 4 weeks after the second immunization, and 8 weeks after the second immunization, respectively; HI titers are shown as GMT±SD; One-way ANOVA was used for statistical comparison; *, P<0.05; **, P<0.01, ***, P<0.001, ****, P<0.0001. DETAILED DESCRIPTION

[0061] The invention discloses a composition, an adjuvant, a vaccine and applications thereof.

[0062] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0063] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0064] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0065] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0066] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0067] The novel nanoemulsion adjuvant of the present invention is mainly composed of an aqueous phase component, an oil phase component and an emulsifier. All the components are mixed in proportion and emulsified by homogenization and high-pressure homogenization to form an emulsion suspension with uniform and stable particles.

[0068] Preferably, in the nanoemulsion of the present invention, the aqueous phase component is a pH buffer system, which is selected from but not limited to phosphate buffered saline (PBS), phosphoric acid (PB) buffer, Tris buffer, sodium citrate buffer, etc. Particularly preferably, in the nanoemulsion of the present invention, the aqueous phase component is composed of sodium citrate buffer. The pH value of the aqueous phase component is controlled within the range that the human body can tolerate, preferably, the range is pH 5.0 to 8.5, more preferably pH 6.0 to 8.0, and particularly preferably pH 6.0 to 7.5.

[0069] Preferably, the nanoemulsion of the present invention, the emulsifier is a surfactant composition, selected from but not limited to Pluronic L121, mannitol monooleate, polyoxyethylene sorbitan monooleate (Tween 80 / polysorbate 80 / Tween-80), polyoxyethylene sorbitan laurate (Tween 20 / Tween-20), sorbitan trioleate (Span 85 / Span-85), sorbitan fatty acid ester (Span 80 / Span-80), triethylene glycol monolauryl ether (Brij 30 / Brij 30), polyethylene glycol hexadecyl ether (Brij 56 / Brij 56), polyethylene glycol octylphenyl ether (Triton X-100) etc.; more preferably, the emulsifier is a combination of Tween-80 and Span-85, and the content of the emulsifier is 0.01% to 10% (m / v) of the total volume of the emulsion; preferably, the content of the emulsifier is 0.3% to 3% (m / v) of the total volume of the emulsion; particularly preferably, the content of Tween-80 and the content of Span-85 in the emulsifier are both 0.47% (m / v) of the total volume of the emulsion.

[0070] Preferably, the oil phase component of the nanoemulsion of the present invention is a metabolizable oil selected from but not limited to squalene, squalane, etc.; particularly preferably, the oil phase component of the nanoemulsion of the present invention is squalane, squalane and squalene mixed oil in different mass ratios. The oil content does not exceed 10% (m / v) of the total volume of the emulsion, preferably 3% to 5% (m / v) of the total volume of the emulsion, and particularly preferably, its content is 3.9% (m / v).

[0071] Preferably, in the mixed oil of squalane and squalene in the nanoemulsion of the present invention, the mass ratio of squalane to squalene is between 1:7 and 7:1, and particularly preferably, the mass ratio of squalane to squalene is 1:3, 1:1 and 3:1.

[0072] The method for preparing a novel nanoemulsion adjuvant is a microfluidization method well known to those skilled in the art. Specifically, Span85 is dispersed in squalane or squalane / squalene mixed oils that have been mixed in different mass ratios to obtain an oil phase, and another surfactant Tween80 is dispersed in a sodium citrate buffer to obtain an aqueous phase, and then homogenized to form a crude emulsion. The prepared crude emulsion is repeatedly passed through a high-pressure homogenizer until a uniform emulsion containing oil droplets of the desired diameter is obtained, and then filtered through a 0.22 μm filter membrane. In the oil-in-water emulsion described in the present invention, the oil droplets contained have a submicron diameter, and the average diameter is less than 1 μm; preferably, the oil droplet diameter is less than 500 nm; more preferably, it is less than 220 nm; particularly preferably, its diameter ranges from 140 to 180 nm.

[0073] The nanoemulsion adjuvant of the present invention in which the oil phase component is entirely squalane is abbreviated as ZSE-100; the nanoemulsion adjuvant in which the mass ratio of squalane to squalene in the oil phase component is 1:7 is abbreviated as ZSE-12.5; the nanoemulsion adjuvant of 1:3 is abbreviated as ZSE-25; the nanoemulsion adjuvant in which the mass ratio of squalane to squalene in the oil phase component is 1:1 is abbreviated as ZSE-50; the nanoemulsion adjuvant in which the mass ratio of squalane to squalene in the oil phase component is 3:1 is abbreviated as ZSE-75; and the nanoemulsion adjuvant of 7:1 is abbreviated as ZSE-87.5.

[0074] The series of novel nanoemulsion adjuvants described in the present invention can also be combined with antigens to obtain immune compositions, and the amount of antigen used is conventional, preferably not more than 1% (m / v).

[0075] Preferably, the antigen of the immune composition is a viral antigen, which can be selected from but not limited to different forms of antigens of viruses such as influenza virus, human papillomavirus, rabies virus, hepatitis B virus, polio virus, human immunodeficiency virus, etc. Particularly preferably, the antigen of the immune composition of the present invention is an influenza virus antigen.

[0076] Preferably, the immune composition of the present invention can be mixed with the adjuvant and antigen before use or the adjuvant and antigen can be mixed and potted during the production of the antigen for clinical use. Particularly preferably, the nanoemulsion adjuvant and antigen are prepared immediately before use and then stored at 4°C for later use.

[0077] The adjuvant ZSE-25 combined with influenza antigen of the present invention is abbreviated as HA / ZSE-25; the adjuvant ZSE-50 combined with influenza antigen is abbreviated as HA / ZSE-50; the adjuvant ZSE-75 combined with influenza antigen is abbreviated as HA / ZSE-75; and the adjuvant ZSE-100 combined with influenza antigen is abbreviated as HA / ZSE-100.

[0078] The reagents and raw materials used in the invention are all commercially available and have low cost.

[0079] In particular, the series of novel nanoemulsion adjuvants of the present invention all have the appearance of a uniform milky white liquid; the average particle size distribution is between 140 and 170 nm, which is similar to the particle size of MF59 that has been used in marketed vaccines; the Zeta potential is between -20 and -35 mV; the polydispersity index PDI is less than 0.2, indicating that the emulsion has good uniformity; the viscosity is close to that of water, indicating that the emulsion is easy to inject; and the pH is within the range that can be tolerated by the human body.

[0080] The series of novel nanoemulsion adjuvants of the present invention have good stability and can be stored for at least 1 year at 2°C to 8°C.

[0081] The series of novel nanoemulsion adjuvants of the present invention have good safety. After being combined with influenza antigens and injected into mice through intramuscular injection, the body temperature and weight of the mice are not affected; and there is no obvious inflammatory reaction at the injection site of the mice for a long time.

[0082] The series of novel nanoemulsion adjuvants invented in this patent can effectively improve the immunogenicity of the vaccine when combined with monovalent influenza B / Victoria antigens. Among them, the adjuvant effect of ZSE-75 is particularly obvious, followed by ZSE-50. In addition, HA / ZSE-100 and HA / ZSE-50 have Th1 immune bias; HA / ZSE-75, HA / ZMF59, and HA / ZSE-25 are more biased towards humoral immunity. However, compared with ZMF59, the Th1 immunity of HA / ZSE-75 has been improved.

[0083] The present invention provides a variety of application methods, including but not limited to: (1) independent packaging, mixed with vaccine antigen before use; (2) combined packaging, mixed with vaccine antigen, filled, and stored at 2-8°C until clinical use.

[0084] The novel nanoemulsion adjuvant and immune composition of the present invention can be inoculated in a form acceptable to the human body, and the inoculation sites include but are not limited to parenteral injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, inhalation through the respiratory tract, rectal administration, vaginal administration, nasal administration, oral administration, ocular administration, topical administration, transdermal or intradermal administration, etc.

[0085] The novel nanoemulsion adjuvants of the present invention can be used as adjuvants for different vaccines such as inactivated vaccines, attenuated vaccines, split vaccines, DNA vaccines, recombinant vaccines, subunit vaccines, polypeptide protein vaccines, including but not limited to influenza vaccines, human papillomavirus vaccines, hepatitis B vaccines, rabies vaccines, polio vaccines, etc.

[0086] The novel nanoemulsion of the present invention can also be used as a drug delivery or sustained-release carrier.

[0087] In Examples 1 to 4 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0088] The present invention will be further described below in conjunction with embodiments:

[0089] Example 1 Preparation of a novel nanoemulsion adjuvant

[0090] 1. Preparation of ZSE-12.5

[0091] (1) Preparation of oil phase: Use a precision balance to accurately weigh 0.141 g Span 85, 0.1463 g squalane and 1.0237 g squalene to prepare an oil phase mixture.

[0092] (2) Preparation of aqueous phase: Use a precision balance to accurately weigh 0.141 g of Tween 80 and completely dissolve it in 28.356 mL of citric acid buffer.

[0093] (3) Preparation of colostrum: The aqueous phase and the oil phase were mixed, and the mixture was homogenized in a homogenizer for 5 minutes to obtain colostrum.

[0094] (4) Preparation of emulsion: The colostrum is repeatedly passed through a high-pressure homogenizer until a uniform emulsion containing oil droplets of the desired diameter is obtained.

[0095] (5) Sterilization: Filter using a 0.22 μm filter membrane to obtain ZSE-12.5.

[0096] 2. Preparation of ZSE-25

[0097] (1) Preparation of oil phase: Use a precision balance to accurately weigh 0.141 g Span 85, 0.2925 g squalane and 0.8775 g squalene to prepare an oil phase mixture.

[0098] (2) Preparation of aqueous phase: Use a precision balance to accurately weigh 0.141 g of Tween 80 and completely dissolve it in 28.356 mL of citric acid buffer.

[0099] (3) Preparation of colostrum: The aqueous phase and the oil phase were mixed, and the mixture was homogenized in a homogenizer for 5 minutes to obtain colostrum.

[0100] (4) Preparation of emulsion: The colostrum is repeatedly passed through a high-pressure homogenizer until a uniform emulsion containing oil droplets of the desired diameter is obtained.

[0101] (5) Sterilization: Filter using a 0.22 μm filter membrane to obtain ZSE-25.

[0102] 3. Preparation of ZSE-50

[0103] (1) Preparation of oil phase: Use a precision balance to accurately weigh 0.141 g Span85, 0.585 g squalene and 0.585 g squalane to prepare an oil phase mixture.

[0104] Steps (2)(3)(4)(5) are the same as those of ZSE-25.

[0105] 4. Preparation of ZSE-75

[0106] (1) Preparation of oil phase: Use a precision balance to accurately weigh 0.141 g Span85, 0.8775 g squalane and 0.2925 g squalene to prepare an oil phase mixture.

[0107] Steps (2)(3)(4)(5) are the same as those of ZSE-25.

[0108] 5. Preparation of ZSE-87.5

[0109] (1) Preparation of oil phase: Use a precision balance to accurately weigh 0.141 g Span 85, 1.0237 g squalane and 0.1463 g squalene to prepare an oil phase mixture.

[0110] Steps (2)(3)(4)(5) are the same as those of ZSE-25.

[0111] 6. Preparation of ZSE-100

[0112] (1) Preparation of oil phase: Use a precision balance to accurately weigh 0.141 g of Span 85 and 1.17 g of squalane to prepare a squalane / Span 85 mixed solution.

[0113] Steps (2)(3)(4)(5) are the same as those of ZSE-25.

[0114] Example 2 Characterization of novel nanoemulsion adjuvants

[0115] The six new nanoemulsion adjuvants prepared in Example 1 were characterized, including appearance, particle size, Zeta potential, polydispersity index PDI, viscosity and pH. The emulsion was diluted 1:20 with sodium citrate buffer solution, 1 mL of the diluted sample was transferred to a particle size cup, and the particle size and polydispersity index (PDI) were detected using a Malvern particle size analyzer; the new nanoemulsion adjuvant was diluted 1:200 with 1 mM sodium chloride solution, 1 mL of the diluted sample was transferred to a potential cup for Zeta potential detection; a DV3T rheometer was used for viscosity detection; and a pH meter was used to measure the pH of the nanoemulsion. The characterization results are shown in Table 1 below:

[0116] Table 1 Characterization of novel nanoemulsion adjuvants (values ​​are expressed as mean ± standard deviation, n = 3)

[0117]

[0118] Example 3 Investigation of the stability of the novel nanoemulsion adjuvant

[0119] The six novel nanoemulsion adjuvants prepared in Example 1 were stored at 2-8°C and subjected to stability tests, including appearance, particle size, Zeta potential, polydispersity index PDI, viscosity and pH, and the determination method was as in Example 2. The results are shown in Tables 2, 3, 4, 5, 6 and 7. The results show that the series of novel nanoemulsion adjuvants of the present invention were placed at 2-8°C for at least 1 year, and the appearance was uniform milky white liquid without discoloration and stratification; the pH was always within the range of 6-7 that the human body can tolerate; and the viscosity was always close to the viscosity of water. In addition, the particle size of ZSE-12.5 is stable at about 150nm, the PDI is stable at about 0.1, and the Zeta potential is always between -20 and -35mV; the particle size of ZSE-25 is stable at about 150nm, the PDI is stable at about 0.1, and the Zeta potential is always between -20 and -35mV; the particle size of ZSE-50 is stable at about 155nm, the PDI is stable at about 0.1, and the Zeta potential is always between -20 and -35mV The particle size of ZSE-75 is stable at about 160nm, PDI is stable at about 0.1, and Zeta potential is always between -20 and -35mV; the particle size of ZSE-87.5 is stable at about 170nm, PDI is stable at about 0.1, and Zeta potential is always between -20 and -35mV; the particle size of ZSE-100 is stable at about 170nm, PDI is always less than 0.2, and Zeta potential is always between -20 and -35mV.

[0120] Table 2 Characterization of long-term stability of the novel nanoemulsion adjuvant ZSE-12.5 (values ​​are expressed as mean ± standard deviation, n = 3)

[0121] Time(months) Z-average(nm) Pdl Zeta(mV) 0 148.7±2.652 0.098±0.012 -23.1±1.67 1 146.2±3.05 0.125±0.015 -22.7±0.389 4.5 146.9±2.95 0.127±0.026 -26.96±0.63 6.5 149.3±0.765 0.117±0.013 -25.2±0.829 8 148.1±1.04 0.109±0.016 -24.5±0.72 9 148±2.86 0.105±0.012 -25.8±0.12 13 150.1±2.588 0.103±0.008 -30.0±0.818 16 148.93±1.72 0.114±0.016 -26.73±0.472

[0122] Table 3 Characterization of long-term stability of the novel nanoemulsion adjuvant ZSE-25 (values ​​are expressed as mean ± standard deviation, n = 3)

[0123] Time(months) Z-average(nm) Pdl Zeta(mV) 0 151.7±2.702 0.104±0.01 -24.1±1.07 3 149.9±1.56 0.096±0.025 -29.5±0.638 5 149.6±2.43 0.085±0.017 -28.9±2.53 6.5 152.1±5.72 0.109±0.027 -25.8±0.22 8 153.1±2.28 0.106±0.028 -23.4±0.33 11 149.7±1.53 0.101±0.025 -28.3±2.08 14 149.2±0.98 0.0825±0.022 -30.6375±2.14

[0124] Table 4 Characterization of long-term stability of novel nanoemulsion adjuvant ZSE-50 (values ​​are expressed as mean ± standard deviation, n = 3)

[0125] Time(months) Z-average(nm) Pdl Zeta(mV) 0 160.8±0.9074 0.131±0.023 -21.8±0.451 4 154.3±1.14 0.098±0.014 -26.9±1.41 6 155.47125±0.67 0.101±0.020 -27.3±1.10 7 154.2±1.95 0.069±0.021 -29±1.00 8.5 156.2±3.55 0.072±0.017 -27.4±3.27 12 156.1±2.35 0.099±0.035 -28.4±2.40 15 155.7±0.65 0.102±0.018 -26.9±3.56

[0126] Table 5 Characterization of long-term stability of novel nanoemulsion adjuvant ZSE-75 (values ​​are expressed as mean ± standard deviation, n = 3)

[0127] Time(months) Z-average(nm) Pdl Zeta(mV) 0 166.6±2.684 0.144±0.009 -23.0±3.63 3 157±1.96 0.1±0.007 -29±0.86 5 156.9±1.87 0.093±0.017 -28.5±1.20 7 157.6±4.10 0.074±0.014 -29.3±2.43 8 159±1.023 0.088±0.025 -26.6±2.77 12 160.9±3.43 0.117±0.021 -28.3±0.80 15 156.7±1.27 0.074±0.018 -28.6±1.81

[0128] Table 6 Characterization of long-term stability of the novel nanoemulsion adjuvant ZSE-87.5 (values ​​are expressed as mean ± standard deviation, n = 3)

[0129]

[0130]

[0131] Table 7 Characterization of long-term stability of novel nanoemulsion adjuvant ZSE-100 (values ​​are expressed as mean ± standard deviation, n = 3)

[0132] Time(months) Z-average(nm) Pdl Zeta(mV) 0 168.1±2.639 0.123±0.019 -23.4±0.987 1 166.2±3.15 0.115±0.017 -23.7±0.379 4.5 166.9±2.91 0.137±0.026 -26.96±0.52 6.5 166.3±0.755 0.107±0.012 -29.2±0.819 8 181.1±1.04 0.199±0.015 -28.5±1.72 9 168±2.86 0.109±0.014 -27.8±0.1 13 165.1±2.587 0.113±0.004 -30.1±0.808 16 168.93±1.71 0.154±0.017 -25.73±0.462

[0133] Example 4 In vivo evaluation of novel nanoemulsion adjuvant combined with monovalent B / Victoria influenza vaccine

[0134] 1. Animal grouping, immunization and blood collection process

[0135] C57BL / 6 mice (45); SPF grade; 19-21 weeks old; female; weight 22-24g. The experiment was randomly divided into 9 groups, 5 mice in each group. The specific grouping and dosage of the mice in the immunization experiment are shown in Table 8. The vaccine was prepared according to Table 8, in which the antigen HA was a monovalent split B-type Victoria protein. C57BL / 6 mice were immunized by intramuscular injection on days 0 and 14, 50 μL each in each left and right leg, a total of 100 μL. Bleeding was performed on the cheeks of mice 1 week after the first immunization, 2 weeks after the first immunization, 1 week after the second immunization, 2 weeks after the second immunization, 4 weeks after the second immunization, and 8 weeks after the second immunization, and the serum was separated. Note: ZMF59 and monovalent split B-type Victoria protein were both made by Changchun Baike Biotechnology Co., Ltd.

[0136] Table 8 Grouping of mice and vaccine preparation in the immunization experiment of novel nanoemulsion adjuvant combined with monovalent B / Victoria influenza vaccine

[0137]

[0138]

[0139] Mouse husbandry and the above experimental procedures were carried out in accordance with animal ethical review requirements.

[0140] 1. Safety evaluation

[0141] Table 9 shows the changes in the abdominal temperature of mice 1 to 4 hours after vaccination. The results show that the body temperature of mice is within a reasonable fluctuation range, indicating that vaccination with different new nanoemulsion influenza adjuvant vaccines will not affect the body temperature of mice.

[0142] Table 9 Changes in abdominal temperature of mice 1 to 4 hours after vaccination

[0143] As can be seen from Table 10, compared with the control group ZMF59, the body weight of mice in the ZSE-100 group decreased slightly one week after the second immunization, but the body weight recovered three weeks after the second immunization. Compared with the control group HA / ZMF59, the body weight of mice in the HA / ZSE-50 and ZSE-87.5 groups decreased slightly two weeks after the first immunization, but the body weight recovered three weeks after the first immunization. It is worth noting that among all groups, the weight increase and decrease trend of mice in the HA / ZSE-75 group was consistent with that in the PBS group. The above results show that the effect of the new nanoemulsion combined with the monovalent B / Victoria influenza vaccine on the body weight of mice after immunization is transient and will not have a significant effect on the normal growth of mice.

[0144] Table 10 Changes in body weight of mice 1-6 weeks after vaccination

[0145]

[0146]

[0147] 2. Evaluation of antigen-specific antibody IgG

[0148] The antigen-specific IgG titer in mouse serum was detected by indirect ELISA, and the results are shown in Table 11. At all test time points, the six new nanoemulsion adjuvant influenza vaccine groups were higher than the single antigen group, and they could all increase the specific IgG level induced by the vaccine, indicating that they had a good adjuvant effect. One week after the first immunization, the HA / ZSE-75 and HA / ZSE-50 groups were slightly higher than the HA / ZMF59 group, indicating that ZSE-75 and ZSE-50 caused a faster immune response than ZMF59. At all test points, the antibody level of HA / ZSE-75 was always the highest, especially 2 weeks after the second immunization, which was higher than all other groups, and there was an extremely significant difference in statistics. In addition, it is also worth noting that at 2 weeks after the first immunization and 2 weeks after the second immunization, the IgG level of HA / ZSE-50 was equivalent to that of HA / ZMF59, and at 4 weeks after the second immunization, the IgG level of HA / ZSE-50 was slightly higher than that of HA / ZMF59, but there was no statistical difference. It is worth noting that the antibody titer of HA / ZSE-12.5 is close to that of MF59, and the antibody titer of HA / ZSE-87.5 is close to that of ZSE-100, and both are inferior to HA / ZSE-75 and HA / ZSE-50.

[0149] Table 11 Antigen-specific antibody titers produced at different time points after vaccination

[0150]

[0151]

[0152] 3. IgG typing antibody evaluation

[0153] Vaccine adjuvants usually change the body's Th1 / Th2 immune bias, which in turn causes changes in antibody subtypes. In mice, Th2-type reactions are usually associated with IgG1 antibodies, while Th1-type reactions are associated with IgG2a, IgG2b, and IgG2c antibodies. For inbred C57BL / 6 mice, due to the absence of the IgG2a genotype, it is necessary to detect the IgG2c subtype in mouse serum to reflect the Th1-type reaction of mice. This part of the experiment used an indirect ELISA method to detect the titer of typing antibodies in mouse serum 2 weeks after the second vaccine, and the results are shown in Table 12.

[0154] Table 12 Antigen-specific IgG antibody typing titers produced 2 weeks after vaccination

[0155] IgG1 IgG2c IgG2C / IgG1 PBS 0 200±0 ZMF59 0 400±244.949 ZSE-100 0 480±178.885 HA 92340±73705.549 53460±26619.316 0.73±0.365 HA / ZMF59 831060±663349.943 393660±239573.847 0.6±0.365 HA / ZSE-100 831060±663349.944 743580±71872.54 1.4±1.461 HA / ZSE-25 918540±586833.68 481140±239573.8467 0.6±0.365 HA / ZSE-50 568620±195611.227 568620±195611.227 1.27±1.011 HA / ZSE-75 1180980±718721.54 831060±663349.944 0.822±0.398

[0156] Regardless of IgG1 or IgG2c, the antibody level of HA / ZSE-75 was higher than that of other groups. According to the ratio of IgG2c to IgG1, the HA / ZSE-100 and HA / ZSE-50 groups were more inclined to Th1 immunity. HA / ZSE-75, HA / ZMF59, and HA / ZSE-25 were more inclined to Th2 immunity. However, compared with HA / ZMF59, the Th1 immunity of HA / ZSE75 was improved.

[0157] 4. Hemagglutination inhibition titer (HI) evaluation

[0158] At present, HI is internationally recognized as the gold standard for influenza vaccine antibody detection. Therefore, this part of the experiment uses a hemagglutination inhibition test to detect the level of anti-HA antibodies in serum. The results are shown in Table 13. Consistent with the results of antigen-specific IgG, at all test time points, the four new nanoemulsion adjuvant influenza vaccine groups were higher than the single antigen group, which once again shows that they have a good adjuvant effect; one week after the first immunization, the HI titer of HA / ZSE-75 was the highest, followed by ZSE-50, and higher than HA / ZMF59. Although there was no statistical difference, it also once again showed that they had a faster immune response than ZMF59. Two weeks after the second immunization, the HI level of the HA / ZMF59 group was the highest, and was slightly higher than the HA / ZSE-50 and HA / ZSE-75 groups, but there was no statistical difference. It is worth noting that in addition to the extremely significant difference between the HA / ZMF59 group and the single antigen HA group, the HA / ZSE-50 group and the HA group also had extremely significant differences, and HA+ZSE-75 also had extremely significant differences with the HA group. At 4 weeks and 8 weeks after the second vaccination, the HI titer of HA / ZMF59 was still the highest, but there was no statistical difference with other groups, including the single antigen HA group.

[0159] Table 13 HI titers at different time points after vaccination

[0160] time 2 weeks after first dose 2 weeks after second free 4 weeks after second vaccination 8 weeks after second vaccination PBS 0 0 0 0 ZMF59 0 0 0 0 ZSE-100 0 0 0 0 HA 56±21.909 256±87.636 128±43.818 26±31.305 HA / ZMF59 352±175.271 2304±572.433 1024±943.864 132±116.276 HA / ZSE-100 256±87.636 1152±286.217 512±175.271 64±21.909 HA / ZSE-25 448±175.271 1024±858.650 512±175.271 112±117.983 HA / ZSE-50 576±143.108 2048±701.085 832±429.325 104±121.984 HA / ZSE-75 576±417.229 1792±701.085 960±905.097 80±48.990

[0161] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition, characterized in that include: Squalane and squalene, the mass ratio of the squalane to the squalene is: (1-10): (10-1).

2. Use of the composition as claimed in claim 1 in the preparation of an adjuvant.

3. An adjuvant, characterized in that The method comprises the composition as claimed in claim 1, a buffer system and a surfactant.

4. The adjuvant according to claim 3, characterized in that The content of the composition accounts for less than or equal to 10% (m / v) of the total volume of the adjuvant; the content of the emulsifier accounts for 0.3-3% (m / v) of the total volume of the adjuvant.

5. The adjuvant according to claim 3 or 4, characterized in that The buffer system includes: one or more of phosphate buffer, phosphate buffer, Tris buffer and / or sodium citrate buffer.

6. The adjuvant according to any one of claims 3 to 5, characterized in that The surfactant comprises: one or more of Pluronic L121, mannitol monooleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan laurate, sorbitan trioleate, sorbitan fatty acid ester, triethylene glycol monolauryl ether, polyethylene glycol hexadecyl ether and / or polyethylene glycol octylphenyl ether.

7. The method for preparing the adjuvant according to any one of claims 3 to 6, characterized in that: The composition, the buffer system and the surfactant are mixed, homogenized and emulsified to obtain the adjuvant.

8. Use of the composition according to claim 1, the adjuvant according to any one of claims 3 to 6 and / or the adjuvant obtained by the preparation method according to claim 7 in any of the following items; (I), preparing a vaccine; and / or (II), preparing a drug delivery vehicle; and / or (III) preparing a drug sustained-release carrier.

9. The use according to claim 8, characterized in that The antigens of the vaccine include: one or more of influenza virus, new coronavirus, human papillomavirus, rabies virus, hepatitis B virus, polio virus and / or human immunodeficiency virus.

10. A vaccine, characterized in that It comprises the composition according to claim 1, the adjuvant according to any one of claims 3 to 6 and / or the adjuvant obtained by the preparation method according to claim 7.

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