Natural-like lipid droplet nanoemulsion adjuvants and methods of making same

Abstract

The application discloses a kind of lipid nanoemulsion adjuvant, including oil phase part and water phase part, the oil phase part of the lipid nanoemulsion is composed of vitamin E and lecithin and / or nonionic surfactant.The application is based on the purpose of using a safer and more environmentally friendly source of material, and selects vitamin E with good controllability, which can be chemically synthesized or extracted from plants, as the oil phase, while selecting nonionic surfactant and natural zwitterionic surfactant to reduce the oil-water interface surface tension.Using the characteristics of the hydrophilic head of natural lecithin, which has both positive and negative groups, it is easy to use with immunostimulants, and can be better adsorbed through electrostatic interaction.Meanwhile, the lipid nanoemulsion obtained by the application has simple composition, excellent stability, excellent adjuvant effect, is much better than aluminum adjuvant, and can induce humoral immunity and cellular immunity level not inferior to the oil-in-water emulsion MF59 as adjuvant, showing good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a natural-like lipid droplet nanoemulsion adjuvant and a preparation method thereof. BACKGROUND

[0002] Adjuvants are common components in vaccines in addition to antigens, which mainly play an auxiliary role. This auxiliary role can occur at various stages from the vaccine entering the body to taking effect: when using non-invasive inoculation (such as spraying, oral administration), some adjuvants can protect the antigen or mediate the antigen to enter the body; after entering the body, the adjuvant can regulate the release or distribution of the antigen; after being taken up by antigen-presenting cells, the adjuvant can regulate the intracellular behavior of the antigen. This auxiliary role can affect the type, intensity or immune memory of the immune response induced by the antigen. Adjuvants can change or enhance the induction of the antigen to the body's immunity, thereby achieving the effects of reducing the amount of antigen, prolonging the immune memory and expanding the protection spectrum of the vaccine, and have become the key to the development of new vaccines or the upgrading of existing vaccines. Adjuvants can be roughly divided into two categories: immunostimulants and delivery systems. The development trends of adjuvant delivery systems are roughly as follows: modification of marketed adjuvant delivery systems, development of composite adjuvants, and application of bionics to adjuvants. For example, aluminum adsorption or coupling with TLR agonists can compensate for the weak cellular immunity of aluminum adjuvants; nanofication can improve the adsorption capacity of aluminum adjuvants; freeze-durable aluminum adjuvants can compensate for the defects of traditional aluminum adjuvants that cannot be used for freeze-dried antigens; adjuvant bionization can comprehensively simulate natural microorganisms in terms of particle size, morphology, fluidity, surface morphology, charge and surface material type, so as to achieve better immune effect. Traditional aluminum adjuvants have been used in vaccines for more than a hundred years, and their safety has been recognized by everyone, but they can only induce limited humoral immunity and weak cellular immunity, which is not sufficient for the development of some new vaccines.

[0003] Oil-in-water emulsion is another kind of adjuvant widely used in addition to aluminum adjuvant, which often can induce stronger humoral immunity and cellular immunity than aluminum adjuvant when combined with antigens. As a delivery system, the oil-in-water emulsion currently on the market (including MF59, AS03, AF02) or disclosed in patents mostly uses squalene as the oil phase. For example, patent documents WO9014837, EP0399843A2, US6299884B1, US6451325B1, US20090191226A2 disclose the prescription of MF59; the adjuvant contains 4.0% to 5.0% squalene, 0.5% Tween 80, and 0.5% Span 85. Patent documents US2007141078A1, US2010189741A1, US2010183667A1 disclose the prescription of AS03, and a single dose of AS03 contains squalene 10.68 mg, DL-alpha-tocopherol 11.86 mg, and Tween 80 4.85 mg; patent documents US2007014805A1, US2007191314A1, EP2080522A1 disclose the prescription and preparation method of AF03. The adjuvant contains 2.5% squalene, about 0.48% ceteareth-12, and about 0.37% Span 80, and further contains a Toll-like receptor 4 (TLR-4) agonist. Patent document CN108368260A discloses an oil-in-water emulsion using an amphiphilic polyamino acid polymer as an emulsifier and squalene as the oil phase; patent document CN201010247976.0 discloses a submicron emulsion adjuvant containing squalene, polyether, and polyoxyethylene castor oil; and patent document CN103784953A discloses a high-temperature sterilizable emulsion using squalene or squalane as the oil phase, and a complex oil-in-water emulsion.

[0004] Although squalene can be extracted from plants, the content is low, and the low yield results in high price. Therefore, in order to reduce the price and meet the demand, almost all squalene is currently extracted from the livers of marine animals. The animal-derived adjuvant has been questioned for safety due to poor controllability of raw materials, complex production process, easy spoilage, and risk of endogenous and exogenous contaminant residues. Based on the above problems, it is urgent to replace the raw material and find and prepare a new immunological adjuvant which can effectively induce humoral immunity and cellular immunity and has good controllability and can be produced on a large scale. SUMMARY

[0005] An aspect of the present application is to provide a preparation method and application of a natural lipid droplet nanoemulsion adjuvant to solve the problems of poor controllability of raw materials and difficulty in large-scale industrial production of the oil-in-water emulsion adjuvant in the prior art.

[0006] The technical scheme provided by the present application is:

[0007] A lipid nanoemulsion adjuvant, comprising an oil phase portion and an aqueous phase portion, the oil phase portion of the lipid nanoemulsion being vitamin E, the aqueous phase being composed of a buffer, lecithin and / or a non-ionic surfactant

[0008] The lipid nanoemulsion in the present application is based on the purpose of using a safer and more environmentally friendly source of material, and selects to use fat-soluble vitamin E with good controllability, which can be chemically synthesized on a large scale or extracted from plants as the oil phase, and selects non-ionic surfactant (Tween 80) and / or natural zwitterionic surfactant (lecithin) to reduce the oil-water interface surface tension.

[0009] The addition of lecithin allows the emulsion prepared in the present application to be coated with a single layer of phospholipid film on the outside, similar to a natural structure of lipid droplets (Lipid droplet), which may have better biocompatibility as a delivery carrier.

[0010] Lecithin is a natural zwitterionic surfactant, which is a natural component of cell membranes and has better biocompatibility. Its hydrophilic end has both positively charged groups and negatively charged groups, and the presence of both charges allows it to simultaneously adsorb positively charged and negatively charged components through electrostatic interaction, which facilitates better adsorption through electrostatic interaction when the immunostimulant is used later.

[0011] In the present application, any suitable non-ionic surfactant can achieve the purpose of the present application. However, as a preferred, in some embodiments of the present application, the above-mentioned non-ionic surfactant is polyethylene glycol alkyl ether, Tween, polyoxyethylene castor oil or polyoxyethylene hydrogenated castor oil.

[0012] Among them, the alkyl includes lauryl ether alkyl, oleyl ether alkyl, stearyl ether alkyl or cetyl alkyl, and the Tween includes Tween 20, Tween 40, Tween 60 or Tween 80.

[0013] More preferably, in an embodiment of the present application, the above-mentioned non-ionic surfactant is Tween 80.

[0014] As a preferred, in some embodiments of the present application, the mass ratio of the above-mentioned vitamin E, lecithin and non-ionic surfactant is 2:(0-1.35):(0.2-0.75).

[0015] More preferably, in an embodiment of the present application, the above-mentioned mass ratio is 2:(0.1-0.65):(0.2-0.4).

[0016] In the present application, the above-mentioned lipid nanoemulsion can also be a particle-adsorbed lipid nanoemulsion, and the particles used include, for example, aluminum hydroxide, aluminum phosphate, aluminum sulfate, calcium carbonate, calcium phosphate, calcium oxalate, ferroferric oxide, ferrous sulfate, ferric phosphate, or silicon dioxide, etc. As a preferred embodiment, in the present application, the above-mentioned particles are aluminum hydroxide.

[0017] Another aspect of the present application is to provide a method for preparing the above-mentioned lipid nanoemulsion, in which the raw materials of the oil phase and the aqueous phase are mixed in an appropriate amount of buffer, and then stirred, sheared or ultrasonically mixed until all the components are completely dissolved. After constant volume, the mixture is homogenized to obtain the lipid nanoemulsion.

[0018] When the lipid nanoemulsion is a particle-adsorbed lipid nanoemulsion, the particles are added to the mixture after the raw materials are dissolved or to the mixture after homogenization.

[0019] Specifically, the above-mentioned method comprises the following steps:

[0020] Step 1) accurately weigh Tween 80, lecithin, and vitamin E, and add them into the same container;

[0021] Step 2) add an appropriate amount of buffer to the container in Step 1;

[0022] Step 3) stir, shear or ultrasonically mix until all the components are completely dissolved, and then constant volume the buffer to the target volume and mix uniformly;

[0023] Step 4) high-pressure homogenization to obtain the target lipid nanoemulsion;

[0024] Step 5) high-pressure homogenization after adding a certain amount of aluminum adjuvant in Step 3, or adding a certain amount of aluminum adjuvant to the lipid nanoemulsion after homogenization to prepare a particle-adsorbed lipid nanoemulsion;

[0025] Step 6) adding an immunostimulant to the obtained lipid nanoemulsion or particle-adsorbed lipid nanoemulsion and mixing uniformly to obtain a composite adjuvant based on the lipid nanoemulsion.

[0026] Step 7) mixing the lipid nanoemulsion, the particle-adsorbed lipid nanoemulsion or the composite adjuvant based on the lipid nanoemulsion with the corresponding antigen and mixing uniformly to prepare the corresponding vaccine, which can be used for prevention or treatment.

[0027] In the present application, the aqueous phase of the lipid nanoemulsion can be any suitable solution. For example, purified water, water for injection, glycerol aqueous solution, 0.9% physiological saline, phosphate buffer, citric acid buffer or Tris buffer.

[0028] The inventors have found that, after the lipid nanoemulsion is obtained, further high-temperature treatment of the lipid nanoemulsion can unexpectedly produce better effects. Therefore, as a preferred embodiment, the preparation method further comprises high-temperature treatment of the lipid nanoemulsion at 120°C for 30 min after the lipid nanoemulsion is obtained.

[0029] Another aspect of the present application is to provide the use of the lipid nanoemulsion in the preparation of a vaccine.

[0030] As a preferred embodiment, the vaccine is a human vaccine.

[0031] More preferably, in one embodiment of the present application, the human vaccine is a human hepatitis B vaccine, and in another embodiment of the present application, the human vaccine is a herpes zoster vaccine.

[0032] Another aspect of the present application is to provide a prophylactic or therapeutic vaccine comprising the lipid nanoemulsion as an adjuvant.

[0033] As a preferred embodiment, the vaccine further comprises an immunostimulant, such as a TLR agonist.

[0034] The present application has the following advantages:

[0035] The present application is based on the use of vitamin E as an oil phase, which is controllable, can be chemically synthesized in large quantities, or extracted from plants, and the use of non-ionic surfactants (Tween 80) and natural zwitterionic surfactants (lecithin) to reduce the oil-water interfacial surface tension. The natural lecithin has both positive and negative groups on the hydrophilic head, which can be easily adsorbed by electrostatic interaction when used with immunostimulants. The lipid nanoemulsion obtained by the present application has simple components, excellent stability, and excellent adjuvant effect, which is much better than aluminum adjuvant. The induced humoral and cellular immune levels are not inferior to (equivalent or better than) the marketed oil-in-water emulsion MF59 as an adjuvant, which shows good application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The above row is the state diagram without high-temperature treatment, and the lower row is the state diagram after high-temperature treatment. The two rows from left to right are V3, V11, V12, Addavax, and AddaS03, respectively.

[0037] Figure 2The scanning electron microscope analysis result chart and the transmission electron microscope analysis result chart of the emulsion prepared in the embodiment of the present application and the commercial emulsion Addavax, wherein A is the Addavax scanning electron microscope chart, B is the V3 scanning electron microscope chart, C is the V11 scanning electron microscope chart, D is the V12 scanning electron microscope chart, E is the Addavax transmission electron microscope chart, F is the V3 transmission electron microscope chart, G is the V11 transmission electron microscope chart, and H is the V12 transmission electron microscope chart;

[0038] Figure 3 The immunization result chart of the emulsion prepared in the embodiment of the present application and the commercial emulsion Addavax and SERVA in combination with HBsAg, wherein A is the ELISA experiment result chart, and B is the ELISPOT experiment result chart;

[0039] Figure 4 The long-term thermal stability analysis result chart of the emulsion prepared in the embodiment of the present application;

[0040] Figure 5 The immunization result chart of the emulsion prepared in the embodiment of the present application and the commercial emulsion Addavax and Aldrogel Al in combination with HBsAg, wherein A is the ELISA experiment result chart, and B is the ELISPOT experiment result chart;

[0041] Figure 6 The immunization result chart of the emulsion prepared in the embodiment of the present application and the commercial emulsion Addavax and Aldrogel Al in combination with VZVgE, wherein A is the ELISA experiment result chart, and B is the ELISPOT experiment result chart;

[0042] Figure 7 The immunization result chart of the emulsion prepared in the embodiment of the present application and the commercial emulsion Addavax in combination with VZV gE;

[0043] Figure 8 The schematic diagram of the natural lipid droplet-like lipid nanoemulsion structure with a single layer of phospholipid molecules. DETAILED DESCRIPTION

[0044] The present application discloses a natural lipid droplet-like lipid nanoemulsion and a preparation method thereof, and those skilled in the art can refer to the content herein and appropriately improve the process parameters for implementation. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as included in the present application, and the relevant personnel can obviously make changes or appropriate changes and combinations to the content described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0045] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise specified. Unless otherwise explicitly indicated, throughout the specification and claims, the term "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element or group of elements but not the exclusion of any other element or group of elements. The terms "such as", "for example", and the like are intended to mean illustrative embodiments, and are not intended to limit the scope of the disclosure.

[0046] Some terms appearing in the present application are explained as follows.

[0047] The term "adjuvant" refers to a substance added to a vaccine other than an antigen that can modulate the immune response, or a substance that can protect the antigen or deliver the antigen.

[0048] The term "oil-in-water emulsion" refers to a system formed by mixing oil and water, with oil as the dispersed phase and water as the dispersion medium.

[0049] The term "immunostimulant" refers to a substance that can stimulate the immune system, which can enhance the immune response of the body, and can be used for antiviral or antitumor purposes, and is often used as an adjuvant component of a vaccine to enhance the body's response to an antigen.

[0050] The term "lipid droplet" refers to an important subcellular organelle in cells, which is formed by a single layer of phospholipids surrounding lipids, with a particle size ranging from 20 nm to 100 μm. It is generally believed to be related to the metabolism of lipid substances and energy supply in the body. Meanwhile, natural lipid droplets rely on different proteins carried to contact different cells or organelles to play different roles.

[0051] The term "homogenization" refers to a process of breaking up solid particles in a liquid material to achieve ultra-fining of the solid particles and form a uniform suspension emulsion. High-pressure homogenization technology is a process in which the material undergoes a series of physical, chemical, and structural changes under high pressure, ultimately achieving homogenization.

[0052] The term "aluminum adjuvant" is the most widely used immunoadjuvant, mainly including aluminum hydroxide and aluminum phosphate, among which aluminum hydroxide is more widely used. Currently, it is believed that the vaccine adjuvant effect mechanism of aluminum adjuvant mainly includes "depot effect" and "immune stimulation effect". Although conventional aluminum adjuvants can enhance the humoral immune response, they often have difficulty in effectively inducing cellular immune responses in the body. In addition, aluminum adjuvants can also induce Ig E-mediated type I hypersensitivity.

[0053] In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be further described in detail below with reference to specific embodiments.

[0054] Example 1: Preparation of lipid nanoemulsion

[0055] Weigh a certain amount of surfactant (Tween 80 and / or lecithin), add buffer and ultrasonic or stir until fully dissolved, then add a certain amount of vitamin E, and then use ultrasonic, high pressure homogenization or membrane emulsification method to obtain the target emulsion.

[0056] Or

[0057] Weigh a certain amount of surfactant (Tween 80 and / or lecithin), add buffer and ultrasonic or stir until fully dissolved, then add a certain amount of vitamin E, and then use ultrasonic, high pressure homogenization or membrane emulsification method to obtain the target emulsion.

[0058] The specific formulations are as follows:

[0059] 1. Preparation method of oil-in-water emulsion V1: 0.2g Tween 80 is dissolved in 50ml citric acid buffer (10mM, pH 6.4), 2g vitamin E is added, citric acid buffer (10mM, pH 6.4) is added to 100ml, high speed (10000rpm / min) shear for 5min to form a primary emulsion, and then high pressure homogenization.

[0060] Or

[0061] 0.2g Tween 80, 2g vitamin E is added to citric acid buffer 50ml, mixed evenly, then add citric acid buffer (10mM, pH 6.4) to 100ml, high speed (10000rpm / min) shear for 5min to form a primary emulsion, and then high pressure homogenization.

[0062] 2. Preparation method of oil-in-water emulsion V3: 0.2g Tween 80, 0.25g lecithin, is dissolved in 50ml citric acid buffer (10mM, pH 6.4), 2g vitamin E is added, citric acid buffer (10mM, pH 6.4) is added to 100ml, 10000rpm / min, high speed shear for 5min to form a primary emulsion, and then high pressure homogenization to obtain microemulsion.

[0063] Or

[0064] 0.2g Tween 80, 0.25g lecithin, 2g vitamin E is added to citric acid buffer 50ml, mixed evenly, then add citric acid buffer (10mM, pH 6.4) to 100ml, high speed (10000rpm / min) shear for 5min to form a primary emulsion, and then high pressure homogenization.

[0065] 3. Preparation method of oil-in-water emulsion V4: 0.2 g Tween 80, 0.25 g lecithin, dissolved in 50 ml citric acid buffer (10 mM, pH 6.4), 2 g vitamin E, 2 ml aluminum adjuvant with a concentration of 1 mg / ml, add citric acid buffer (10 mM, pH 6.4) to 100 ml, high speed shearing at 10000 rpm / min for 5 min to form the primary emulsion, and then high pressure homogenization to obtain the microemulsion.

[0066] or

[0067] 0.2 g Tween 80, 0.25 g lecithin, 2 g vitamin E, 2 ml aluminum adjuvant with a concentration of 1 mg / ml, add citric acid buffer 50 ml, mix well, then add to 100 ml, high speed shearing (10000 rpm / min) for 5 min to form the primary emulsion, and then high pressure homogenization.

[0068] 4. Preparation method of oil-in-water emulsion V6: 0.4 g Tween 80, 0.35 g lecithin, dissolved in 50 ml citric acid buffer (10 mM, pH 6.4), 2 g vitamin E, add citric acid buffer (10 mM, pH 6.4) to 100 ml, high speed shearing at 10000 rpm / min for 5 min to form the primary emulsion, and then high pressure homogenization to obtain the microemulsion.

[0069] or

[0070] 0.4 g Tween 80, 0.35 g lecithin, 2 g vitamin E, add citric acid buffer 50 ml, mix well, then add to 100 ml, high speed shearing (10000 rpm / min) for 5 min to form the primary emulsion, and then high pressure homogenization.

[0071] 5. Preparation method of oil-in-water emulsion V7: 0.75 g Tween 80, 0.65 g lecithin, dissolved in 50 ml citric acid buffer (10 mM, pH 6.4), 2 g vitamin E, add citric acid buffer (10 mM, pH 6.4) to 100 ml, high speed shearing at 10000 rpm / min for 5 min to form the primary emulsion, and then high pressure homogenization to obtain the microemulsion.

[0072] or

[0073] 0.75 g Tween 80, 0.65 g lecithin, 2 g vitamin E, add citric acid buffer 50 ml, mix well, then add to 100 ml, high speed shearing (10000 rpm / min) for 5 min to form the primary emulsion, and then high pressure homogenization.

[0074] 6. Preparation method of oil-in-water emulsion V8: 0.4 g Tween 80, 0.65 g lecithin, dissolved in 50 ml citric acid buffer (10 mM, pH 6.4), 2 g vitamin E, 0.4 g PEG400, add citric acid buffer (10 mM, pH 6.4) to 100 ml, 10000 rpm / min, high speed shearing for 5 min to form the initial emulsion, and then high pressure homogenization to obtain microemulsion.

[0075] or

[0076] 0.4 g Tween 80, 0.65 g lecithin, 2 g vitamin E added to 50 ml citric acid buffer, mix well, then add to 100 ml, high speed (10000 rpm / min) shearing for 5 min to form the initial emulsion, and then high pressure homogenization.

[0077] 7. Preparation method of oil-in-water emulsion V9: 0.65 g Tween 80, 0.35 g lecithin, dissolved in 50 ml citric acid buffer (10 mM, pH 6.4), 2 g vitamin E, 0.4 g PEG400, add citric acid buffer (10 mM, pH 6.4) to 100 ml, 10000 rpm / min, high speed shearing for 5 min to form the initial emulsion, and then high pressure homogenization to obtain microemulsion.

[0078] or

[0079] 0.65 g Tween 80, 0.35 g lecithin, 2 g vitamin E added to 50 ml citric acid buffer, mix well, then add to 100 ml, high speed (10000 rpm / min) shearing for 5 min to form the initial emulsion, and then high pressure homogenization.

[0080] 8. Preparation method of oil-in-water emulsion V10: 0.4 g Tween 80, 1.35 g lecithin, dissolved in 50 ml citric acid buffer (10 mM, pH 6.4), 2 g vitamin E, add citric acid buffer (10 mM, pH 6.4) to 100 ml, 10000 rpm / min, high speed shearing for 5 min to form the initial emulsion, and then high pressure homogenization to obtain microemulsion.

[0081] or

[0082] 0.4 g Tween 80, 1.35 g lecithin, 2 g vitamin E added to 50 ml citric acid buffer, mix well, then add to 100 ml, high speed (10000 rpm / min) shearing for 5 min to form the initial emulsion, and then high pressure homogenization.

[0083] 9. Preparation method of oil-in-water emulsion V11: 0.2 g Tween 80, 0.65 g lecithin, dissolved in 50 ml citric acid buffer (10 mM, pH 6.4), 2 g vitamin E, add citric acid buffer (10 mM, pH 6.4) to 100 ml, 10000 rpm / min, high speed shearing for 5 min to form the initial emulsion, and then high pressure homogenization to obtain microemulsion.

[0084] or

[0085] 0.2 g Tween 80, 0.65 g lecithin, 2 g vitamin E added to citric acid buffer 50 ml, mix well, then add citric acid buffer (10 mM, pH 6.4) to 100 ml, high speed (10000 rpm / min) shearing for 5 min to form the initial emulsion, and then high pressure homogenization.

[0086] 10. Preparation method of oil-in-water emulsion V12: 0.4 g Tween 80, 0.16 g lecithin, dissolved in 50 ml citric acid buffer (10 mM, pH 6.4), 2 g vitamin E, add citric acid buffer (10 mM, pH 6.4) to 100 ml, 10000 rpm / min, high speed shearing for 5 min to form the initial emulsion, and then high pressure homogenization to obtain microemulsion.

[0087] or

[0088] 0.4 g Tween 80, 0.16 g lecithin, 2 g vitamin E added to citric acid buffer 50 ml, mix well, then add citric acid buffer (10 mM, pH 6.4) to 100 ml, high speed (10000 rpm / min) shearing for 5 min to form the initial emulsion, and then high pressure homogenization.

[0089] 11. Preparation method of oil-in-water emulsion V13: 0.75 g Tween 80, 0.15 g lecithin, dissolved in 50 ml citric acid buffer (10 mM, pH 6.4), 2 g vitamin E, add citric acid buffer (10 mM, pH 6.4) to 100 ml, 10000 rpm / min, high speed shearing for 5 min to form the initial emulsion, and then high pressure homogenization to obtain microemulsion.

[0090] or

[0091] 0.75 g Tween 80, 0.15 g lecithin, 2 g vitamin E added to citric acid buffer 50 ml, mix well, then add citric acid buffer (10 mM, pH 6.4) to 100 ml, high speed (10000 rpm / min) shearing for 5 min to form the initial emulsion, and then high pressure homogenization.

[0092] The schematic diagram of the natural lipid droplet-like lipid nanoemulsion structure with a single layer of phospholipid molecules prepared in the above examples is shown in Figure 8 .

[0093] Experimental Example 1:

[0094] The emulsion obtained in Example 1 was subjected to high temperature treatment (120°C, 30 min), and then the particle size of the emulsion before and after high temperature treatment was detected to investigate the stability. 10 μl of each sample was taken and diluted with 990 μl of ultrapure water, and the particle size was determined using a Malvern Zetasizer Nano ZS90 laser particle size analyzer. The results showed that V3 and V11 had good thermal stability, and the particle size changed little before and after high temperature. Meanwhile, V12 had a small particle size, and although the particle size increased after high temperature treatment, no delamination or emulsion breaking occurred. The experimental results are shown in Table 1 and Figure 1 .

[0095] Table 1 Particle size and phenomenon of different formulations before and after high temperature treatment

[0096]

[0097] 3 ml of each of the prepared V3, V11, V12, and commercial MF59 (Addavax) and AS03 (AddaS03) were taken, sealed with a cap after dispensing, and subjected to high temperature (120°C, 30 min) treatment, and then observed. V3, V11 and V12 were the same as before high temperature treatment, and no obvious delamination or emulsion breaking was observed. However, MF59 and AS03 showed emulsion breaking and delamination. These results prove that the lipid nanoemulsion prepared by the present application has excellent thermal stability.

[0098] Experimental Example 2

[0099] V3, V11 and V12 were observed using an electron microscope, and commercial MF59 (Addavax) was also detected. 50 μl of each sample was taken and then diluted 100 times with water and mixed uniformly. The scanning electron microscope (SEM) detection process: an appropriate amount of liquid was dropped on a silicon wafer, dried, and gold sprayed for detection. The transmission electron microscope (TEM) detection process: an appropriate amount of liquid was dropped on a copper mesh, dried, negatively stained, dried, and then detected on the machine.

[0100] The results are shown in Table 2. Figure 2 The results show that the various emulsions under the electron microscope have a circular overall shape, but there are slight differences. The lipid nanoemulsion prepared by the present application has a radial antenna under the scanning electron microscope, and the surface is rough, while the MF59 is smoother.

[0101] Example 2: Preparation method of particle type emulsion based on V3

[0102] It has been reported that particle type emulsion has a certain rigidity compared with ordinary emulsion, and when used as a vaccine adjuvant, it can induce a stronger immune level.

[0103] Patent document CN 104013955 B reports an oil-in-water emulsion without surfactant. Patent document CN 108324938 B reports a granular emulsion. Both of these patents utilize the property of particles to reduce surface tension, and add particles during emulsion preparation to prepare an oil-in-water emulsion.

[0104] In addition to adding particles during emulsion preparation, we also investigated the preparation of a granular emulsion by adding particles after preparation.

[0105] V4 is a granular emulsion prepared by adding aluminum adjuvant particles during preparation of V3. In addition, granular lipid nanoemulsions can also be prepared by adding aluminum particles after preparation. The specific process is as follows: 100 ml of V3 emulsion is added with 2 ml of aluminum adjuvant at a concentration of 1 mg / ml, and mixed thoroughly to prepare V31.

[0106] Experimental Example 3:

[0107] To test whether the different effects of heating high temperature (high temperature is 120℃, 30min) and the different ways of adding particles (adding during preparation, adding after preparation) of the granular emulsion based on V3 have an impact on the adjuvant effect, mice were used to evaluate the adjuvant effect. The evaluation process with recombinant hepatitis B surface antigen (HBsAg) is as follows:

[0108] Thirty-five 6-8 week old Babl / c mice were randomly divided into 7 groups, as shown in Table 2.

[0109] Table 2 V3, V3Δ with hepatitis B surface antigen (HBsAg) immunization dose

[0110]

[0111]

[0112] Immunization procedure and detection time points:

[0113] Three intramuscular immunizations were performed at 0 weeks, 2 weeks, and 4 weeks. Blood was collected at 5 weeks, and serum was separated for antibody titer measurement using enzyme-linked immunosorbent assay (ELISA); at the same time, mouse spleens were taken, and lymphocytes were separated for antigen-specific cytokine secretion using enzyme-linked immunospot assay (ELISPOT).

[0114] The results are as follows: Figure 3The results show that V3 induces humoral immunity better than its granular emulsion V4, and is comparable to Addavax (MF59). Surprisingly, V3Δ, which is obtained by heating V3 at high temperature, does not decrease the level of induced humoral immunity, but the average is greater than V3, and the cellular immunity induced by V3Δ is stronger than that induced by V3. The addition of particles has only a slight effect on the effect of the adjuvant, and V31 is comparable to V4.

[0115] Experimental Example 4

[0116] In order to compare the immune effects of V4 and V31, and considering the convenience of preparation, the granular emulsions V11 and V12 were prepared by the method of V31 (mixed after preparation), and V111 and V121 were prepared, respectively. The specific process is as follows:

[0117] V111: After the preparation of V11 is completed, 2 ml of aluminum adjuvant with a concentration of 1 mg / ml is added to 100 ml of V11, and mixed thoroughly.

[0118] V121: After the preparation of V12 is completed, 2 ml of aluminum adjuvant with a concentration of 1 mg / ml is added to 100 ml of V12, and mixed thoroughly.

[0119] Then 10 μl of each sample is taken and diluted with 990 μl of ultrapure water, and the particle size is measured using a Malvern Zetasizer Nano ZS90 laser particle size analyzer, and the zeta potential is analyzed. The results are shown in Table 3.

[0120] Table 3 Particle size and zeta potential of granular emulsions V31, V111, and V121

[0121]

[0122] Experimental Example 5: Long-term thermal stability evaluation

[0123] The lipid nanoemulsions prepared in Examples 1 and 2 and the corresponding granular lipid nanoemulsions are divided into 3 ml / bottle, and then placed in 4°C, 25°C, and 37°C environments, respectively. Regular sampling is performed, and the particle size value is detected to evaluate the long-term thermal stability of the lipid nanoemulsion. The results are shown in Table 4. Figure 4 The results show that, except for V31 (slight delamination), the stability of each lipid nanoemulsion is good at different temperatures, and all the lipid nanoemulsions have been stably stored at 37°C for more than 8 weeks.

[0124] Experimental Example 6: Immune effect evaluation

[0125] To investigate the immune enhancement effect of lipid nanoemulsion as vaccine adjuvant, we evaluated the immune effect of emulsion with different antigens. To comprehensively evaluate the effect of lipid nanoemulsion as vaccine adjuvant, we selected two forms of antigens, one of which is recombinant hepatitis B surface antigen (HBsAg), which is a virus-like particle formed by aggregation of antigen monomers. The other is VZV gE, which is a free antigen monomer. These two antigen forms encompass most of the forms of antigens in marketed vaccines, and have a certain representativeness.

[0126] 1. Recombinant hepatitis B surface antigen

[0127] The evaluation process of each adjuvant with recombinant hepatitis B surface antigen (HBsAg) is as follows:

[0128] Forty-five 6-8-week-old Babl / c mice were randomly divided into 9 groups, as shown in Table 4.

[0129] Table 4: Immunization dose of each emulsion mixed with HBsAg

[0130]

[0131] Commercial aluminum adjuvant (Aldrogel Al) and commercial oil-in-water emulsion AddaVax (MF59) were used as positive controls, and antigen alone (S) was used as a negative control.

[0132] Immunization program and detection time points:

[0133] Three intramuscular immunizations were performed at 0, 2, and 4 weeks, respectively. Blood was collected at 5 weeks, and serum was separated for antibody titer determination using enzyme-linked immunosorbent assay (ELISA); at the same time, mouse spleen was taken, and lymphocytes were separated for antigen-specific cytokine secretion using enzyme-linked immunospot assay (ELISPOT).

[0134] The results are shown in Figure 5 ELISA results showed that, with hepatitis B virus surface antigen, each lipid nanoemulsion (V3, V31, V11, V111, V12, V121) could induce a higher level of humoral immunity than aluminum adjuvant, and V3, V11, and V12 could induce a humoral immunity level comparable to MF59. This showed a good humoral immunity enhancement effect.

[0135] ELISPOT results showed that, with hepatitis B virus surface antigen, each lipid nanoemulsion (V3, V31, V11, V111, V12, V121) could induce a higher level of cytokine than aluminum adjuvant, and more surprisingly, V31, V11, and V12 could induce a higher level of cytokine secretion than adjuvant MF59.

[0136] In summary, the lipid nanoemulsion of the present application can induce stronger humoral immunity and cellular immunity than traditional aluminum adjuvant when used in hepatitis B vaccine, and can induce humoral immunity level comparable to MF59 and cellular immunity level higher than MF59.

[0137] 2. VZV gE

[0138] Each adjuvant was combined with VZV gE antigen (gE) for animal immunization evaluation, and the evaluation process was as follows:

[0139] Forty-five 6-8-week-old Babl / c mice were randomly divided into 9 groups, and the grouping was as shown in Table 5.

[0140] Table 5: Immunization dose of each emulsion combined with VZV gE

[0141]

[0142] Commercial aluminum adjuvant (Aldrogel Al) and commercial oil-in-water emulsion AddaVax (MF59) were used as positive controls, and the antigen (VZV gE) alone was used as a negative control.

[0143] Immunization procedure and detection time points:

[0144] After the mice were initially immunized with attenuated herpes zoster vaccine for 1 month, they were twice muscle injected with recombinant herpes zoster antigen combined with each adjuvant for booster immunization.

[0145] Two weeks after completion of the immunization procedure, the serum was separated, and the antibody titer was measured by enzyme-linked immunosorbent assay (ELISA); at the same time, the mouse spleen was separated, and the lymphocytes were separated, and the antigen-specific cytokine secretion was measured by enzyme-linked immunospot assay (ELISPOT).

[0146] The results are shown in Table 6. Figure 6 The ELISA results show that, combined with VZV gE antigen, both MF59 and the lipid nanoemulsion of the present application show significantly higher antibody induction level than aluminum adjuvant. At the same time, different from when combined with hepatitis B surface antigen, when combined with VZV gE, the lipid nanoemulsion of the present application can induce stronger humoral immunity level than MF59.

[0147] The ELISPOT results show that, combined with VZV gE antigen, both MF59 and the lipid nanoemulsion of the present application can induce stronger cytokine level than aluminum adjuvant, and the lipid nanoemulsion V111 can induce stronger cytokine secretion than the adjuvant MF59.

[0148] From the above comparison, we can find that the lipid nanoemulsion of the present application has different degrees of strengthening effect on the induced humoral immunity or cellular immunity when matched with different forms of antigens. Specifically, when used for monomeric antigens (such as VZV gE), it can induce stronger humoral immunity than MF59.

[0149] Experimental Example 7: Preparation method and effect evaluation of the composite adjuvant based on lipid nanoemulsion

[0150] When used as a vaccine adjuvant, nanoemulsion is often used together with an immune stimulator to achieve a synergistic effect.

[0151] To test the effect of the lipid nanoemulsion of the present application matched with an immune stimulator as a vaccine adjuvant, V3 and V31 were matched with poly(I:C) for immune effect evaluation. VZV gE antigen was used, and 35 Babl / c mice aged 6-8 weeks were randomly divided into 7 groups, as shown in Table 6.

[0152] Table 6: Composite adjuvant mixed with VZV gE immune dose table with emulsion added with immune stimulator

[0153]

[0154] Immune procedure and detection time points:

[0155] The mice were initially immunized with attenuated herpes zoster vaccine for 1 month, and then twice muscle injection was performed for booster immunization using recombinant herpes zoster antigen matched with each adjuvant. Two weeks after the completion of the immune procedure, the spleen was taken, lymphocytes were separated, and then enzyme-linked immunospot method (ELISPOT) was used to detect antigen-specific cytokine secretion.

[0156] The results are shown in Table 7. Figure 7 As can be seen from the results, the lipid nanoemulsion is similar to Addavax, and the addition of poly(I:C) can enhance the adjuvant effect of the emulsion alone, but the mixture of Addavax or V3 with poly(I:C) does not have a significant synergistic effect. However, the mixture of the granular lipid nanoemulsion V31 with poly(I:C) can have a significant synergistic effect, which may be related to the adsorption of aluminum particles on the surface of these granular emulsions to enhance the adsorption of poly(I:C).

[0157] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A lipid nanoemulsion adjuvant comprising an oil phase portion and an aqueous phase portion, characterized in that: The lipid nanoemulsion is prepared by the following method: 0.2g Tween 80, 0.25g lecithin, dissolved in 50ml 10mM, pH 6.4 citric acid buffer, 2g vitamin E, 10mM, pH 6.4 citric acid buffer to 100ml, high-speed shearing at 10000rpm / min for 5min to form colostrum, and then high-pressure homogenization to obtain microemulsion; or 0.2g Tween 80, 0.25g lecithin, and 2g vitamin E were added to 50ml of citric acid buffer, mixed thoroughly, and then the volume was adjusted to 100ml. High-speed shearing at 10,000 rpm / min was performed for 5 minutes to form colostrum, and then high-pressure homogenization was performed. The product obtained after the above high-pressure homogenization was subjected to high-temperature treatment at 120°C for 30 minutes.

2. A lipid nanoemulsion adjuvant comprising an oil phase portion and an aqueous phase portion, characterized in that: The lipid nanoemulsion is prepared by the following method: 0.2g Tween 80, 0.65g lecithin, dissolved in 50ml 10mM, pH 6.4 citric acid buffer, 2g vitamin E, 10mM, pH 6.4 citric acid buffer to 100ml, high-speed shearing at 10000rpm / min for 5min to form colostrum, and then high-pressure homogenization to obtain microemulsion; or 0.2g Tween 80, 0.65g lecithin, and 2g vitamin E were added to 50ml of citric acid buffer, mixed thoroughly, and then the volume was adjusted to 100ml. High-speed shearing at 10,000 rpm / min was performed for 5 minutes to form colostrum, and then high-pressure homogenization was performed. The product obtained after the above high-pressure homogenization was subjected to high-temperature treatment at 120°C for 30 minutes.

3. A lipid nanoemulsion adjuvant comprising an oil phase portion and an aqueous phase portion, characterized in that: The lipid nanoemulsion is prepared by the following method: 0.4g Tween 80, 0.16g lecithin, dissolved in 50ml 10mM, pH 6.4 citric acid buffer, 2g vitamin E, 10mM, pH 6.4 citric acid buffer to 100ml, high-speed shearing at 10000rpm / min for 5min to form colostrum, and then high-pressure homogenization to obtain microemulsion; or 0.4g Tween 80, 0.16g lecithin, and 2g vitamin E were added to 50ml of citric acid buffer, mixed thoroughly, and then the volume was adjusted to 100ml. High-speed shearing at 10,000 rpm / min was performed for 5 minutes to form colostrum, and then high-pressure homogenization was performed. The product obtained after the above high-pressure homogenization was subjected to high-temperature treatment at 120°C for 30 minutes.

4. The lipid nanoemulsion according to any one of claims 1 to 3, characterized in that The lipid nanoemulsion is a particle adsorption type lipid nanoemulsion adjuvant.

5. The lipid nanoemulsion according to claim 4, characterized in that The particles are aluminum hydroxide.

6. A preventive or therapeutic vaccine, characterized in that The vaccine comprises the lipid nanoemulsion as claimed in any one of claims 1 to 5 as an adjuvant, and human hepatitis B antigen or human herpes zoster antigen.

7. The preventive or therapeutic vaccine according to claim 6, characterized in that The vaccine also includes an immunostimulant.

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