Emulsion with long-acting immune response as well as preparation method and application thereof

Abstract

The invention relates to an emulsion with long-acting immune response and a preparation method and application thereof, the emulsion comprises a water phase, an oil phase and particles, the water phase comprises an inner water phase and an outer water phase, the inner water phase contains the particles, the oil phase wraps the inner water phase, and the outer water phase wraps the oil phase. According to the emulsion provided by the invention, the particles are introduced into the emulsion, the deformability of the emulsion and the bionic characteristics of the particles are utilized, cellular uptake and efficient lymph node enrichment are remarkably enhanced, rapid and powerful hair growth center reaction is induced, a body can be induced to generate strong cellular and humoral immune response on a mouse model, and a protective antibody is continuously generated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to an emulsion with long-term immune response and a preparation method and application thereof. BACKGROUND

[0002] Vaccines are considered to be the most effective and economical way to prevent infectious diseases, and show an extremely important role in the prevention and control of diseases. However, most vaccines can only induce short-term protection, and multiple vaccinations are needed to produce long-term immune protection. However, it is a challenge to complete such multiple immunizations in developing countries. Due to limited health care channels and lack of funds in developing countries, multiple vaccinations and full immune effects bring great obstacles. In addition, multiple immunizations bring pain problems, side reaction problems and cost problems for injected vaccines, and also bring challenges to the compliance of the vaccinated. Therefore, the development of new safe and effective vaccine adjuvants to stimulate the body to produce efficient natural and acquired immune responses, improve the level of humoral and cellular immune responses, and achieve more long-term immune protection against infectious diseases is an important development direction of future new immune adjuvant research.

[0003] The key to inducing an effective long-term immune response is the strong reaction of the germinal center (GC). In the process of immune response, activated antigen-specific B cells can either differentiate into short-lived plasma cells that produce low-affinity antibodies, or initiate a germinal center reaction to produce high-affinity memory B cells and long-lived plasma cells (LLPCs) that secrete high-affinity antibodies. LLPCs migrate to the bone marrow and obtain a bone marrow niche that carries them, where they can reside for a long time and produce antigen-specific antibodies, maintain the level of antibodies in the serum, induce long-lasting protective humoral immunity, and prevent subsequent infection in the case of new antigen exposure. The formation of follicular helper T (Tfh) cells is a key step to promote the germinal center reaction. Tfh cells mainly locate in lymphoid follicles, can continuously express CXCR5 and transcription factor Bcl-6, produce cytokine IL-21, provide assistance for the differentiation and maturation process of B cells, and are very important for the formation of long-lived plasma cells, the establishment of long-term humoral immunity, and the improvement of function. Therefore, enhancing the uptake of cells to antigens and efficient lymph node enrichment are problems to be solved for inducing strong germinal center reaction and producing long-term immune response.

[0004] At present, although the conventional vaccine formulations such as aluminum salt adjuvants and oil emulsion adjuvants can effectively induce the body to produce a humoral immune response, the long-term immune response effect is poor. The antigens in the two systems are all in the form of soluble free antigens and are taken up by antigen presenting cell (APC) with low efficiency, and the adjuvant does not enter the cell together with the antigen, which cannot realize efficient lymph node enrichment and maintain the differentiation of Tfh cells. Therefore, it is urgent to develop a new type of safe, efficient and long-term protective adjuvant to solve the deficiency of the existing vaccine adjuvant.

[0005] At present, there are few reports on the development of a water-in-oil-in-water emulsion containing particles as a long-acting vaccine adjuvant. Therefore, it is particularly important to develop a water-in-oil-in-water emulsion adjuvant containing particles to induce the body to produce a high, balanced and long-lasting immune response. SUMMARY

[0006] In view of the deficiency of the prior art, the purpose of the present application is to provide an emulsion with long-term immune response and a preparation method and application thereof.

[0007] To achieve the purpose of the present application, the following technical solutions are adopted:

[0008] In a first aspect, the present application provides an emulsion with long-term immune response, which comprises a water phase, an oil phase and particles, the water phase comprises an inner water phase and an outer water phase, the inner water phase contains particles, the oil phase wraps the inner water phase, and the outer water phase wraps the oil phase.

[0009] The particles have biomimetic properties and can mimic the physical properties of natural pathogens. The surface of the particles can adsorb, couple target substances, fluorescent markers, isotope markers, environmental response substances, cytokines, antibodies or immunomodulators and other functional substances.

[0010] We designed a water-in-oil-in-water emulsion containing particles, which introduced particles into the interior of the emulsion. Compared with the traditional formulation, it has the following significant advantages: (1) the particles have biomimetic properties and can mimic the size, charge, shape and other physical properties of natural pathogens, mimic the arrangement mode of pathogen surface characteristic proteins and danger signal molecules, and improve the recognition and uptake efficiency of APCs; (2) the emulsion has deformability, which can exhibit stress deformation when contacting with cells, the contact area expands from a point to a plane, obtaining a larger immune recognition interface and enhancing cell uptake; at the same time, it can deform through the intercellular gap of lymphatic endothelial cells into the lymphatic vessels, realizing efficient lymph node enrichment through the paracellular pathway, and then inducing rapid and potent GC response. The particles have biomimetic properties and can mimic the physical properties of natural pathogens.

[0011] The emulsion of the present application can produce higher levels of antibody titers, and the long-acting refers to maintaining high levels of antibody titers for more than 6 months without significant reduction, achieving more efficient, long-term and safe immune protection for infectious diseases.

[0012] Preferably, the emulsion comprises 0.1-50 parts of the inner water phase, 0.1-90 parts of the outer water phase, 0.1-60 parts of the oil phase and 0.1-45 parts of the particles by mass fraction.

[0013] The mass fraction of the inner water phase can be selected from 0.1 parts, 0.3 parts, 0.5 parts, 0.8 parts, 1 part, 2 parts, 5 parts, 8 parts, 10 parts, 12 parts, 15 parts, 18 parts, 20 parts, 22 parts, 25 parts, 28 parts, 30 parts, 32 parts, 35 parts, 38 parts, 40 parts, 42 parts, 45 parts, 48 parts, 50 parts, etc., the mass fraction of the outer water phase can be selected from 0.1 parts, 0.3 parts, 0.5 parts, 1 parts, 5 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, 35 parts, 40 parts, 45 parts, 50 parts, 55 parts, 60 parts, 65 parts, 70 parts, 75 parts, 80 parts, 85 parts, 90 parts, etc., the mass fraction of the oil phase can be selected from 0.1 parts, 0.3 parts, 0.5 parts, 0.8 parts, 1 parts, 2 parts, 5 parts, 8 parts, 10 parts, 12 parts, 15 parts, 18 parts, 20 parts, 22 parts, 25 parts, 28 parts, 30 parts, 32 parts, 35 parts, 38 parts, 40 parts, 42 parts, 45 parts, 48 parts, 50 parts, 52 parts, 55 parts, 58 parts, 60 parts, etc., and the mass fraction of the particles can be selected from 0.1 parts, 0.3 parts, 0.5 parts, 0.8 parts, 1 parts, 2 parts, 5 parts, 8 parts, 10 parts, 12 parts, 15 parts, 18 parts, 20 parts, 22 parts, 25 parts, 28 parts, 30 parts, 32 parts, 35 parts, 38 parts, 40 parts, 42 parts, 45 parts, etc., and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0014] Preferably, the particles comprise any one or a combination of at least two of aluminum hydroxide, aluminum phosphate, aluminum sulfate, calcium carbonate, calcium phosphate, calcium oxalate, ferroferric oxide, ferrous sulfate, ferric phosphate, silicon dioxide, chitosan, alginate, polylactic acid, poly-lactic-glycolic acid copolymer or polyethylene glycol-lactic acid copolymer.

[0015] Preferably, the shape of the particles comprises spherical, rod-shaped, sheet-shaped, spindle-shaped, disc-shaped, cubic, polyhedral or amorphous.

[0016] Preferably, the average particle size of the particles is 20 nm-10 μm, for example 20 nm, 30 nm, 50 nm, 80 nm, 200 nm, 500 nm, 1 μm, 2 μm, 5 μm, 6 μm, 8 μm or 10 μm, etc., and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0017] Preferably, the particles have a zeta potential of -19 mV to 47 mV, for example -19 mV, -15 mV, -10 mV, -5 mV, 2 mV, 7 mV, 12 mV, 18 mV, 22 mV, 27 mV, 33 mV, 40 mV, 47 mV, and the like, and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0018] Preferably, the particles have a polydispersity coefficient of 0.5 or less, for example 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, and the like, and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0019] Preferably, the chitosan has a molecular weight of 20,000 to 1,000,000, for example 20,000, 50,000, 100,000, 200,000, 400,000, 700,000, 800,000, 1,000,000, and the like, and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0020] Preferably, the oil phase comprises an oily substance.

[0021] Preferably, the oily substance comprises any one or a combination of at least two of squalene, squalane, soybean oil, miglitol, medium-chain oil, fish oil, cod liver oil, vitamin E, vitamin E succinate, vitamin E acetate, safflower oil, corn oil, sea buckthorn oil, linseed oil, peanut oil, tea oil, sunflower seed oil, wheat germ oil, almond oil, coix seed oil, evening primrose oil, sesame oil, cottonseed oil, castor oil, canola oil, ethyl oleate, oleic acid, ethyl linoleate, isopropyl laurate, isopropyl myristate, ethyl butyrate, ethyl lactate, caprylic triglyceride, capric triglyceride, white oil, white vaseline, or light liquid paraffin.

[0022] Preferably, the oily substance is any one or a combination of at least two of squalene, tocopherol, or white oil.

[0023] The squalene according to the present application is a triterpenoid compound, the English name of which is Squalene, the molecular structure of which is isoprene with thirty carbon and fifty hydrogen, the molecular formula of which is 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, the CAS: 111-02-4, and the molecular mass: 410.72. Squalene is a metabolizable oil because it is an intermediate in the biosynthesis of cholesterol (Merck Index, 10th edition, registration number 8619). It is a natural secretion of oil in all higher organisms, including humans (found in sebum). Emulsions containing squalene (with surfactants) have shown excellent immune-enhancing effects in animal experiments and clinical trials.

[0024] The tocopherol is a fat-soluble vitamin, mainly four derivatives, divided into α, β, γ and δ four kinds according to the methyl position, among which α-tocopherol is the most active. As a vaccine component, it can induce the secretion of interleukin 6 (IL-6), interleukin 1β (IL-1β), chemokine 1 (CXCL1), chemokine 11 (CCL11), chemokine 24 (CCL24) and other cytokines, increase the uptake of mononuclear and macrophages to lymph nodes, and has immune enhancement effect.

[0025] The white oil is a refined liquid hydrocarbon mixture from petroleum, also known as paraffin oil, mineral oil, and the main component is C16-C31 normal isomer alkane. Because of its low price and wide source, white oil is widely used in animal vaccine production and is the main component of commercially available oil emulsion vaccines, which can enhance the immunogenicity of antigens and induce the body to produce effective immune response.

[0026] Preferably, the oil phase further comprises an oleophilic emulsifier.

[0027] Preferably, the oleophilic emulsifier comprises any one or a combination of at least two of gum arabic, lecithin, soybean phospholipid, sorbitan sesquioleate, sorbitan monostearate, PO500 emulsifier, sorbitan trioleate, sorbitan tristearate, sorbitan sesquioleate, sorbitan monooleate, glyceryl monostearate, polyethylene glycol dioleate, anionic emulsifying wax, polyoxyethylene stearate, polyoxyethylene fatty alcohol ether, polyoxyethylene (30) dipolyhydroxystearate, cetyl polyethylene glycol / polypropylene glycol-10 / 1 dimethyl silicone, and fatty acid methyl ester.

[0028] Preferably, the mass fraction of the oleophilic emulsifier in the oil phase is 1-20%, for example, 1%, 2%, 4%, 6%, 8%, 10%, 12%, 15%, 18%, 20%, and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0029] Preferably, the water phase comprises any one or a combination of at least two of purified water, water for injection, glycerol aqueous solution, 0.9% physiological saline, phosphate buffer, citric acid buffer, and Tris buffer.

[0030] Preferably, the pH value of the phosphate buffer, citric acid buffer or Tris buffer is independently 5.5-8.5, for example, 5.5, 5.8, 6, 6.2, 6.5, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8.2, 8.5, and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0031] Preferably, the outer aqueous phase comprises any one or a combination of at least two of phosphate buffer, citric acid buffer or Tris buffer.

[0032] Preferably, the inner aqueous phase comprises any one or a combination of at least two of 0.9% normal saline, phosphate buffer or water for injection.

[0033] Preferably, the outer aqueous phase further comprises a hydrophilic surfactant.

[0034] Preferably, the hydrophilic surfactant comprises any one or a combination of at least two of Tween 60, Tween 65, Tween 80, Tween 85, poloxamer, self-polyoxyethylene monolaurate, polyoxyethylene lauryl ether, polyoxyethylene monopalmitate, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ether, polyvinyl alcohol, ethanol, propylene glycol, sodium octadecyl sulfate, caprylic capric acid polyethylene glycol glycerin ester, polyethylene glycol-15 hydroxystearate, polyethylene glycol 300 or polyethylene glycol 400.

[0035] Preferably, the mass fraction of the hydrophilic surfactant in the outer aqueous phase is 3-30%, such as 3%, 5%, 6%, 8%, 10%, 12%, 15%, 20%, 24%, 26%, 28%, 30% and the like, and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0036] Preferably, the average particle size of the emulsion is 80 nm-350 μm, such as 80 nm, 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 20 μm, 50 μm, 70 μm, 90 μm, 100 μm, 120 μm, 150 μm, 200 μm, 300 μm, 350 μm and the like, and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0037] Preferably, the Span value of the emulsion is 1.5 or less, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5 and the like, and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0038] Preferably, the emulsion has an average particle size of 200 nm to 250 μm, such as 200 nm, 500 nm, 800 nm, 1 μm, 10 μm, 20 μm, 50 μm, 80 μm, 100 μm, 120 μm, 150 μm, 180 μm, 200 μm, 205 μm, 210 μm, 215 μm, 220 μm, 225 μm, 230 μm, 235 μm, 240 μm, 245 μm, 250 μm, and the like, with other specific point values within the above numerical range being optional and not listed here.

[0039] Preferably, the emulsion further comprises a water-soluble substance and / or an oil-soluble substance.

[0040] Preferably, the water-soluble substance is loaded in the inner aqueous phase and / or the outer aqueous phase.

[0041] Preferably, the oil-soluble substance is loaded in the oil phase.

[0042] Preferably, the water-soluble substance loaded in the inner aqueous phase has an encapsulation efficiency of 85% to 100%, such as 85%, 86%, 88%, 89%, 90%, 91%, 93%, 95%, 97%, 98%, 99%, 100%, and the like, with other specific point values within the above numerical range being optional and not listed here.

[0043] Preferably, the water-soluble substance comprises any one or a combination of at least two of a biologic macromolecule active substance, a hydrophilic immunostimulant, an immunomodulator, and a cytokine.

[0044] Preferably, the biologic macromolecule active substance comprises any one or a combination of at least two of a protein, a polypeptide, or a nucleic acid.

[0045] Preferably, the protein comprises any one or a combination of at least two of an antigen, an enzyme, a monoclonal antibody, a medicinal protein.

[0046] Preferably, the antigen comprises any one or a combination of at least two of a human antigen, a non-human animal antigen, a plant antigen, a bacterial antigen, a fungal antigen, and a viral antigen.

[0047] Typical but non-limiting combinations include: a combination of a human antigen and a non-human animal antigen; a mixture of a plant antigen and a bacterial antigen; a combination of a fungal antigen, a viral antigen, and a parasitic antigen; a combination of a tumor antigen, a human antigen, a non-human animal antigen, a plant antigen, a bacterial antigen, and a fungal antigen; a combination of a viral antigen, a parasitic antigen, a tumor antigen, a human antigen, a non-human animal antigen, and a plant antigen; a combination of a bacterial antigen, a fungal antigen, a viral antigen, a parasitic antigen, and a tumor antigen.

[0048] The antigen of the present application can be a monovalent antigen or a multivalent antigen; the antigen can be derived from, but not limited to, chicken embryo culture, cell culture, carrier body fluid, organ or tissue purification and isolation, recombinant gene expression or chemical synthesis, preferably the antigen includes, but is not limited to, any one of or a combination of at least two of attenuated vaccine, inactivated vaccine, split vaccine, subunit vaccine, polysaccharide conjugate vaccine, recombinant vaccine or DNA vaccine, for example, a combination of attenuated vaccine and inactivated vaccine, a combination of split vaccine and subunit vaccine, a combination of polysaccharide conjugate vaccine, recombinant vaccine, a combination of DNA vaccine and attenuated vaccine, a combination of inactivated vaccine and split vaccine, a combination of subunit vaccine, polysaccharide conjugate vaccine, recombinant vaccine and DNA vaccine.

[0049] Preferably, the hydrophilic immunostimulant and immunomodulator includes any one of or a combination of at least two of saponin, flagellin, lipopolysaccharide, triacyl lipopeptide, DNA containing CpG motif.

[0050] Preferably, the cytokine includes any one of or a combination of at least two of granulocyte macrophage colony-stimulating factor, interleukin-2, interleukin-4, interleukin-12, interleukin-15, interleukin-18, interleukin-21, interferon, tumor necrosis factor-α.

[0051] The oil-soluble substance includes any one of or a combination of at least two of lipid, hydrophobic immunostimulant or immunomodulator.

[0052] Preferably, the hydrophobic immunostimulant and immunomodulator includes any one of or a combination of at least two of monophosphoryl lipid A, gardiquimod, resiquimod, imiquimod, single-stranded RNA or double-stranded RNA.

[0053] In a second aspect, the present application provides a preparation method of the emulsion with long-lasting immune response according to the first aspect, the preparation method comprising:

[0054] (1) dispersing the particles in the inner water phase and mixing with the oil phase to obtain a water-in-oil emulsion;

[0055] (2) adding the water-in-oil emulsion into the outer water phase and dispersing, and the preparation is completed.

[0056] Preferably, the particle size of the emulsion droplets in the water-in-oil emulsion is 50 nm-20 μm, for example, 50 nm, 100 nm, 200 nm, 500 nm, 1 μm, 3 μm, 5 μm, 10 μm, 12 μm, 15 μm, 20 μm, etc., and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0057] Preferably, the mixing method of step (1) comprises ultrasonic, homogenization, mechanical stirring, magnetic stirring, vortex or oscillation.

[0058] Each mixing method requires setting specific operating parameters, for example, the parameters to be set for ultrasonic include ultrasonic time, ultrasonic power, etc., the parameters to be set for homogenization include homogenization time, homogenization speed, homogenization interval, etc. Different mixing methods and selected operating parameters will affect the particle size, dispersion coefficient and droplet stability of the emulsion.

[0059] Preferably, the dispersion method of step (2) comprises stirring, syringe double-push emulsification, inversion or oscillation.

[0060] The dispersion method is determined according to the batch size, for example, syringe double-push emulsification is often selected for preparing 1-5 mL emulsion system, and stirring method can be selected for preparing 0.5-50 mL emulsion system.

[0061] Compared with the prior art, the present application has the following beneficial effects:

[0062] The emulsion provided by the present application introduces particles into the interior of the emulsion, significantly enhances cell uptake and efficient lymph node enrichment by utilizing the deformability of the emulsion and the biomimetic characteristics of the particles, induces rapid and efficient GC reaction, and can induce strong cellular and humoral immune response of the body and continuously produce protective antibodies in a mouse model. Compared with commercial aluminum adjuvant and MF59 adjuvant, the above-mentioned emulsion of the present application can produce higher levels of antibody titers, and maintain high levels of antibody titers for more than 6 months without significant reduction, achieving more efficient, long-term and safe immune protection against infectious diseases. The present application provides experimental basis and theoretical guidance for the design of new safe and effective vaccine immune adjuvant, and has great development and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 is an electron micrograph of the aluminum hydroxide particles prepared in Preparation Example 1.

[0064] Figure 2 is a particle size distribution graph of the PLGA particles prepared in Preparation Example 5.

[0065] Figure 3 is a particle size distribution graph of the emulsion prepared in Example 2.

[0066] Figure 4 is a laser confocal photograph of the emulsion prepared in Example 2.

[0067] Figure 5 is the test result of antibody titer.

[0068] Figure 6 is the distribution graph of the emulsion prepared in Example 4 in the body after subcutaneous injection. DETAILED DESCRIPTION

[0069] The technical solutions of the present application are further illustrated below by means of specific embodiments. Those skilled in the art should understand that the embodiments are only used for understanding the present application and should not be regarded as specific limitations of the present application.

[0070] Raw materials:

[0071]

[0072]

[0073] Instrument:

[0074] The particle size detection method of the particles or emulsion droplets in the following examples is as follows:

[0075] Name Specification Manufacturer Electronic balance TB-214 Beijing Sartorius Instrument System Co., Ltd. Magnetic stirrer ColorSquid IKA Cold field emission scanning electron microscope JSM-6700F JEOL Laser confocal microscope TCPSP5 Leica Homogenizer T18 Basic IKA Pipette 200 μL, 1 mL, 5 mL, 10 mL Eppendorf Dynamic light scattering particle size instrument Zetasizer Nano ZS Malvern Ultrasonic cell disruptor S-450D Branson Inc. Vortex shaker Vortex-Genie 2 Scientific Industries, Inc. Flow cytometer Arial III BD Microplate reader Infinite M200 Tecan

[0076] The particle size distribution of the nano-sized particles or emulsion droplets is determined by dynamic light scattering particle size analyzer. The specific determination steps are as follows: 1 mg of nano-sized particles is added into 10 mL of deionized water, ultrasonic treatment is performed for 5 min to make it uniformly dispersed, and then 1 mL of the obtained solution is taken and added into a sample cell for detection; the prepared water-in-oil emulsion is dispersed in 10 times the volume of oil phase, mixed uniformly, and then 1 mL of the obtained solution is taken and added into a sample cell for detection; the prepared water-in-oil-in-water emulsion containing particles is dispersed in 10 times the volume of external water phase, mixed uniformly, and then 1 mL of the obtained solution is taken and added into a sample cell for determination. The particle size uniformity is indicated by the polydispersity index (PDI) value. The smaller the PDI value, the more uniform the particle size.

[0077] The particle size of the micron-sized particles and emulsion droplets is detected by a laser particle size analyzer. The specific operation is as follows: 5 mg of particles is dispersed in 5 mL of deionized water, ultrasonic treatment is performed for 5 min to make it uniformly dispersed, and then the obtained solution is mixed and placed into a sample cell for detection; 1 mL of the prepared water-in-oil-in-water emulsion containing particles is added dropwise into a sample cell for detection. The particle size uniformity is indicated by the span value. The smaller the span value, the more uniform the particle size.

[0078] Determination of the embedding rate of antigen or drug:

[0079] Accurately take 1 mL of the emulsion embedding antigen or drug, and place it in a dialysis bag. Accurately take 10 mL of deionized water, and place the dialysis bag in the deionized water. Mix and suspend overnight at 4°C, and then take the solution outside the dialysis bag on the second day. At the same time, a group of emulsions without embedding antigen or drug of the same volume is set as a blank control. The hydrophilic compound is determined by a micro-BCA kit, a Lorry reagent or other suitable detection method. The hydrophobic compound is determined by high performance liquid chromatography (HPLC).

[0080] The antigen or drug embedding rate is calculated according to the following formula:

[0081] Embedding rate = (antigen or drug amount before embedding - antigen or drug amount in supernatant outside the dialysis bag) / antigen or drug amount before embedding x 100%

[0082] Animal experiment determination:

[0083] The BALB / c mice used in the experiment were provided by Vivotecnia. The immunization steps are as follows: first, the mice were randomly divided into groups, and more than 12 mice were used in each group. The mice were grouped and immunized according to the specific instructions in the examples. Before vaccination, 200 μL of blood was taken and immediately centrifuged at 12,000 rpm for 5 min to separate the serum. The IgG antibody level was determined by enzyme-linked immunoassay, and the IgG antibody level at this time was taken as the initial value. Then the mice were immunized. After immunization, blood was taken from the eye or tail tip of the mouse at regular intervals, and the IgG antibody level was determined. Two weeks later, the mice were immunized again. Thirty-five days after immunization, six mice from each group were sacrificed, and the inguinal lymph nodes were taken. The proportion of GC B cells and Tfh cells in lymphocytes, as well as the mRNA levels of chemokines Bcl-6 and IL-21 in B cells and Tfh cells, were analyzed by flow cytometry. Mouse spleen cells were cultured, and the secretion of IFN-γ cytokine in the supernatant of the mouse spleen cell culture was detected by enzyme-linked immunosorbent assay (ELISA). Mouse bone marrow lymphocytes were isolated, and the proportion of B220-CD138+ plasma cells was detected by flow cytometry. The remaining mice continued to take blood at specified times (42, 60, 90, 120, 150, 180 days after immunization) and determine the IgG antibody level.

[0084] Preparation Example 1

[0085] The present preparation example provides an aluminum hydroxide particle prepared by an alkoxide hydrolysis-hydrothermal method.

[0086] Specifically, deionized water was placed in a 100 mL three-necked flask, and the oil bath was heated to 85°C. Then aluminum isopropoxide powder was added. The aluminum isopropoxide and deionized water were mixed in a molar ratio of 1:100, and stirred for 2 h. The reaction solution was then naturally cooled to room temperature. The above reaction solution was placed in a stainless steel high-pressure container with a polytetrafluoroethylene liner and hydrothermally treated at a certain temperature. After the hydrothermal reaction was completed, the material was naturally cooled, and the solid phase product was separated by centrifugation. The product was washed with deionized water three times and vacuum dried to obtain aluminum hydroxide particles. The electron micrograph is shown in Figure 1 The average particle size of the particles was 40 nm, and the PDI value of the particles was 0.128.

[0087] Preparation Example 2

[0088] The present preparation example provides a chitosan particle prepared by a nano-precipitation method.

[0089] Specifically, 0.15 g of chitosan (molecular weight of 50,000, degree of deacetylation of 90%) is dissolved in 2.0 mL of 1 mol / L acetic acid solution, and after being fully dissolved, mixed with 6.0 mL of deionized water. The pH of the chitosan solution is adjusted to 6.5 with 1 mol / L sodium hydroxide solution, centrifuged at 20,000 g for 20 min, and after the supernatant is discarded, the precipitate is freeze-dried to obtain chitosan particles. The average particle size of the particles is 89 nm, and the PDI value of the particles is 0.179.

[0090] Preparation Example 3

[0091] The present preparation example provides a hollow calcium phosphate particle prepared by a template method.

[0092] Specifically, 3.0 g of Tween 80, 0.25 g of PEG 6000, 3.0 mL of 0.5 mol / L Tris-HCl (pH 8.0), and 1.5 mL of deionized water are mixed and stirred uniformly, and then ultrasonicated for 20 min to form non-ionic surfactant vesicles. Then 3.5 mL of CaCl2 (0.18 mol / L) solution is added dropwise, and stirred for 0.5 h. Then 3.5 mL of Na2HPO4 (0.18 mol / L) solution is added to obtain a suspension of sodium phosphate particles. Then 0.8 mL of MgCl2 solution (0.07 mmol / L) is added to stabilize the just-formed calcium phosphate particles. After continuing to stir for 2 h, centrifugal washing and vacuum drying are performed to obtain hollow calcium phosphate particles. The average particle size of the particles is 205 nm, the average thickness of the shell layer is 35 nm, and the PDI value of the particles is 0.319. The prepared particles are hollow spheres.

[0093] Preparation Example 4

[0094] The present preparation example provides a calcium carbonate particle prepared by a liquid-phase direct mixing precipitation method.

[0095] Specifically, calcium acetate and trisodium citrate are weighed and dissolved in 200 mL of distilled water (the mass concentration of calcium acetate and trisodium citrate is 10 wt.% and 30 wt.% respectively). Then 10 wt.% sodium carbonate aqueous solution (50 mL) is added to the solution, and stirred (300 rpm) for 3 h. After filtration, the precipitate is washed with distilled water and anhydrous ethanol for three times respectively, and then dried at 70°C to obtain peanut-shaped calcium carbonate particles. The length of the particles is 1.2 μm, the ratio of long axis to short axis is 2:1, and the morphology of the prepared particles is peanut-shaped.

[0096] Preparation Example 5

[0097] The preparation example provides a PLGA particle prepared by a nano-precipitation method.

[0098] Specifically, 0.30 g of PLGA (LA:GA is 50:50, and the molecular weight is 119 kilodaltons) is weighed and dissolved in 1 mL of acetone, the solution is added to 20 mL of an aqueous solution (containing 1 wt.% of PVA (alcoholysis degree is 99%, and the viscosity is 5.0 mPa·s)) at a speed of 1 drop per second by using a 9-gauge needle, magnetic stirring is performed at a speed of 500 rpm, stirring is performed overnight at 25°C, centrifugation is performed at 20,000 g for 20 min, the supernatant is discarded, 5 mL of deionized water is added to the precipitate, ultrasonic dispersion is performed, centrifugation is performed at 20,000 g for 5 min, the supernatant is discarded, and the precipitate is freeze-dried to obtain PLGA particles, which are stored in a refrigerator at 4°C. The particle size distribution of the prepared PLGA particles is shown in Figure 2 The average particle size is 368 nm, and the PDI is 0.202.

[0099] Example 1

[0100] In this example, a water-in-oil emulsion containing particles is prepared by using the particles prepared in Preparation Examples 1-5.

[0101] 5% of an oily surfactant sorbitan trioleate is added to 5 mL of an oily substance squalene, stirring is performed to uniformly mix the oily phase, and 3 mL of the oily phase is taken for standby. 0.3 g of the chitosan particles prepared in Preparation Example 2 is accurately weighed by using an electronic balance and added to 2 mL of deionized water, the mixture is uniformly dispersed, and then 3 mL of the oily phase is added. Homogenization is performed by using an IKA homogenizer (12000 rpm, 4 min) to obtain a water-in-oil emulsion containing particles. The average particle size of the prepared water-in-oil emulsion containing particles is 720 nm, and the PDI is 0.121.

[0102] The water-in-oil emulsion containing particles can also be prepared by using different processes. The preparation method is as described above, and specific parameters and results are shown in Table 1.

[0103] Table 1

[0104]

[0105] Example 2

[0106] In this example, a water-in-oil-in-water emulsion containing particles is prepared by using the water-in-oil emulsion prepared in Example 1.

[0107] In 2 mL of water for injection, 8% of water-soluble surfactant Tween 80 was added, and stirring was performed to obtain an external water phase. The external water phase was placed on a magnetic stirrer and stirred at a speed of 400 rpm. 1 mL of the water-in-oil emulsion No. 1 in Example 1 was taken with a pipette and slowly added to the external water phase, and stirring was performed for 6 min to prepare a water-in-oil-in-water emulsion containing particles. The emulsion particle size distribution is shown in Figure 3 The laser confocal phase distribution is shown in Figure 4

[0108] When the laser confocal phase distribution was observed, the components of the emulsion needed to be labeled. The labeling steps were as follows:

[0109] Chitosan particle labeling - the weighed chitosan particle powder was dispersed in deionized water, a certain volume of Cy5-SE fluorescent dye (1 mL / mL dissolved in DMSO) was taken with a pipette and added to the chitosan particle suspension, and stirring was performed to obtain uniform mixing. The mixture was shaken overnight at 4°C in the dark on a shaker.

[0110] Composite oil phase labeling - an appropriate amount of Dil dye was dispersed in the oil phase, and shaking was performed overnight at 4°C in the dark.

[0111] The water-in-oil-in-water emulsion containing particles can also be prepared by different processes in this example. The preparation method is referred to the above steps, and the specific parameters and results are shown in Table 2.

[0112] Table 2

[0113]

[0114]

[0115] Example 3

[0116] This example prepared a water-in-oil-in-water emulsion containing particles loaded with a substance

[0117] An electronic balance was used to accurately weigh 0.15 g of the PLGA particles prepared in Preparation Example 5 and disperse them in 2 mL of a phosphate buffer. An electronic balance was used to accurately weigh 10 mg of the hydrophilic substance OVA and dissolve it in 5 mL of a phosphate buffer to obtain a hydrophilic substance OVA dispersion. The PLGA particle suspension and the OVA dispersion were mixed in a volume ratio of 1:1 to obtain an internal water phase loaded with the hydrophilic substance OVA.

[0118] 10% of the oil-soluble surfactant PO500 was added to white oil, and stirring was performed to obtain oil phase I. An electronic balance was used to accurately weigh 1 mg of monophosphoryl lipid A and disperse it in 1 mL of white oil, and ultrasonic dispersion was performed for 30 min at 4°C to obtain oil phase II. Oil phase I and oil phase II were mixed in a volume ratio of 1:1 to obtain an oil phase loaded with the hydrophobic substance monophosphoryl lipid A.​

[0119] Take 0.4 mL of the inner water phase loaded with OVA, and drop it into 0.6 mL of the oil phase loaded with monophosphoryl lipid A under stirring (600 rpm, 10 min) at 4°C to obtain a water-in-oil emulsion containing particles. Add 10% of the water-soluble surfactant Tween 80 in 2 mL of water for injection, and mix uniformly under stirring to obtain an outer water phase. Add the outer water phase to the water-in-oil emulsion, and continue to oscillate (frequency 20 Hz, 15 min) to prepare a water-in-oil-in-water emulsion containing particles. The average particle size of the emulsion is 1463 nm, and the embedding rate of OVA is 92.9%, and the embedding rate of monophosphoryl lipid A is 95.8%. The embedding of other substances is similar. In addition, the loaded substances in the outer water phase of the water-in-oil-in-water emulsion containing particles are similar to the inner water phase, which is a simple mixture, and the embedding rate is not considered here.

[0120] The inner particles containing the loaded substances in the water-in-oil-in-water emulsion can also be prepared by different processes. The preparation method is referred to the above steps, and the specific parameters and results are shown in Table 3.

[0121] Table 3

[0122]

[0123] Example 4

[0124] The present example provides a water-in-oil-in-water emulsion containing fluorescently labeled OVA particles

[0125] The preparation method is as follows: accurately weigh 0.15 g of chitosan particles on an electronic balance, and disperse them in 2 mL of phosphate buffer. Accurately weigh 10 mg of OVA on an electronic balance, and dissolve it in 5 mL of phosphate buffer. Label the OVA with Cy7. Mix the labeled OVA and the chitosan particle suspension uniformly to prepare an inner water phase. Add 5% of the oil-soluble surfactant lecithin to squalene to obtain an oil phase. Take 0.4 mL of the inner water phase with a pipette, and drop it into 0.6 mL of the oil phase under ultrasonication (80 W, 120 s) at 4°C to obtain a water-in-oil emulsion containing particles. Add 5% of the water-soluble surfactant Tween 80 in 2 mL of water for injection, and mix uniformly under stirring to obtain an outer water phase. Add the outer water phase to the water-in-oil emulsion, and continue to oscillate (frequency 30 Hz, 15 min) to prepare a water-in-oil-in-water emulsion containing particles.

[0126] The average particle size of the emulsion is 1091 ± 25 nm, the PDI is 0.287, and the embedding rate of OVA is 95.2%.

[0127] Test Example 1

[0128] Stability investigation

[0129] A water-in-oil-in-water emulsion containing particles was prepared by a similar method as in Example 3. Control A was a commercial ISA206 double emulsion. Stability was investigated at 25°C. As can be seen from Table 4, the emulsion prepared by optimization had good stability.

[0130] Table 4

[0131] 0 months 1 month 2 months 3 months Control A 243 ± 5 nm 355 ± 13 nm 1256 ± 52 nm 2272 ± 85 nm Example 3 No. 2 emulsion 2087 ± 10 nm 2120 ± 19 nm 2092 ± 32 nm 2193 ± 54 nm Example 3 No. 11 emulsion 3101 ± 65 nm 3172 ± 43 nm 3183 ± 50 nm 3188 ± 23 nm Example 3 No. 3 emulsion 5302 ± 12 nm 5398 ± 98 nm 5467 ± 48 nm 5466 ± 102 nm

[0132] Test Example 2

[0133] Safety Evaluation

[0134] Test sample: emulsion obtained in Example 3, No. 1

[0135] Test method:

[0136] BALB / c mice were administered by subcutaneous injection, intramuscular injection and intraperitoneal injection respectively, the administration dose was 100 μL per mouse, and the administration was performed once at day 0 and day 14, and the body weight change of the mice was measured at 0, 14 and 28 days, as shown in Table 5. Blood was taken at 35 days, centrifuged at 10000g for 10 min to separate serum, and the biochemical indexes in the serum were detected, including myocardial index (lactate dehydrogenase LDH), renal function index (urea nitrogen BUN), and liver function index (alanine aminotransferase ALT, aspartate aminotransferase AST, and alkaline phosphatase ALP), as shown in Table 6. Table 5 shows that the water-in-oil-in-water emulsion containing particles does not cause the body weight of the mice to decrease, and Table 6 shows that it has no obvious toxicity to the main organs and has good safety.

[0137] Table 5

[0138] Blank group Subcutaneous injection Intramuscular injection Intraperitoneal injection 0 days 17.1±0.5g 16.6±0.9g 17.2±0.3g 16.9±0.5g 14 days 19.0±0.7g 18.4±0.5g 18.8±0.7g 18.7±0.8g 28 days 20.1±0.3g 19.9±0.6g 20.4±0.5g 20.5±0.7g

[0139] Table 6

[0140] Group ALT ALP LDH BUN AST Blank group 40.4±7.8 232.4±16.2 1141.4±117.2 7.6±2.8 80.2±7.8 Subcutaneous injection 40.9±5.8 222.9±11.3 1056.2±127.5 8.1±1.9 87.5±9.8 Intramuscular injection 41.7±5.0 235.7±16.9 999.3±217.8 8.5±0.6 82.3±12.0 Intraperitoneal injection 42.1±4.7 238.4±20.4 1094.9±117.8 7.8±0.8 84.7±8.7

[0141] Test Example 3

[0142] Evaluation of Immunization Effect

[0143] Test method:

[0144] 0.15 g of chitosan particles were accurately weighed using an electronic balance and dispersed in 2 mL of phosphate buffer. 5 mL of an aqueous solution of antigen at a certain concentration was prepared. The antigen was mixed uniformly with the chitosan particle suspension to prepare an inner water phase containing particles. 0.1 g of sorbitan trioleate was dissolved in 2 mL of squalene to prepare an oil phase. 1 mL of the inner water phase was added to 2 mL of the oil phase to obtain a water-in-oil emulsion containing particles under different preparation conditions. Different doses of hydrophilic surfactant were weighed and dissolved in 2 mL of phosphate buffer to obtain an outer water phase. Under magnetic stirring at 400 rpm, the water-in-oil emulsion containing particles was added to the outer water phase and stirred for 10 min to prepare a water-in-oil-in-water emulsion containing particles. The average particle size of the emulsion was detected to be 1630 nm. BALB / c mice (6-8 weeks old) were selected for the experiment, the gender was female, and the mice were adapted to the environment for 3 days with free feeding and drinking. The first and second immunizations were performed on day 0 and day 14, respectively, with 100 μL of each muscle injection. The mice were sacrificed on day 35, and the spleen, lymph nodes and tibial bone were taken for culture of spleen cells, lymph node cells and bone marrow cells for a period of time. The relevant indicators were detected, and the results are shown in Table 7. At the same time, blood was taken at regular intervals, and the antibody level in the serum was detected by ELISA, and the results are shown in Figure 5

[0145] Table 7

[0146]

[0147]

[0148] As can be seen from Table 7, the water-in-oil-in-water emulsion containing particles can promote the secretion of IL-4 and IFN-γ, promote the cellular and humoral immune response; at the same time, it can significantly induce higher levels of GC B cells and Tfh cells, under the action of transcription factors, the B cells are efficiently converted into plasma blast cells, and then the number of long-lived plasma cells in the bone marrow is increased, and the long-acting antibody persistence is maintained. Figure 5 It is shown that the emulsion group can produce higher levels of antibody titers compared with commercial aluminum adjuvant and MF59, and maintain high levels of antibody titers for more than 6 months without significant reduction. Therefore, the water-in-oil-in-water emulsion containing particles can simultaneously enhance cellular and humoral immunity and induce long-acting immune response.

[0149] Test Example 4

[0150] The enrichment of the water-in-oil-in-water emulsion containing particles in the inguinal lymph nodes was evaluated. Test sample: the emulsion obtained in Example 4

[0151] ​The experiment selects BALB / c mice (6-8 weeks old), the gender is female mouse, each mouse is injected 200ug protein amount of emulsion subcutaneously in the back, 12h after injection, the fluorescence distribution of OVA in vivo is observed by using small animal imaging instrument, as shown in Figure 6 Compared with the blank group, the antigen of the emulsion group can have more aggregation in the lymph node. It is proved that it has great potential to stimulate immune cells in the lymph node and enhance immune activation.

[0152] The applicant declares that the emulsion with long-acting immune response, the preparation method and application of the emulsion are illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned embodiments, that is, it does not mean that the present application must rely on the above-mentioned embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0153] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-mentioned embodiments, within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0154] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined by any suitable method without contradiction, in order to avoid unnecessary repetition, the present application will not further describe various possible combination methods.

Claims

1. An emulsion with a long-lasting immune response, characterized in that, The emulsion comprises an aqueous phase, an oil phase, and particles. The aqueous phase includes an inner aqueous phase and an outer aqueous phase. The inner aqueous phase contains particles. The oil phase encapsulates the inner aqueous phase, and the outer aqueous phase encapsulates the oil phase.

2. The emulsion with a long-lasting immune response according to claim 1, characterized in that, The emulsion comprises, by mass parts, 0.1-50 parts of internal aqueous phase, 0.1-90 parts of external aqueous phase, 0.1-60 parts of oil phase, and 0.1-45 parts of particles.

3. The emulsion with a long-lasting immune response according to claim 1 or 2, characterized in that, The particles include any one or a combination of at least two of the following: aluminum hydroxide, aluminum phosphate, aluminum sulfate, calcium carbonate, calcium phosphate, calcium oxalate, ferric oxide, ferrous sulfate, ferric phosphate, silica, chitosan, alginate, polylactic acid, polylactic acid-glycolic acid copolymer, or polyethylene glycol-lactic acid copolymer. Preferably, the shape of the particles includes spherical, rod-shaped, plate-shaped, spindle-shaped, disc-shaped, cubic, polyhedral, or amorphous. Preferably, the average particle size is 20 nm-10 μm; Preferably, the polydispersity index of the particles is less than 0.

5.

4. The emulsion with a long-lasting immune response according to any one of claims 1-3, characterized in that, The oil phase includes oily substances; Preferably, the oily substance includes any one or a combination of at least two of the following: squalene, squalane, soybean oil, miglitol, medium-chain triglycerides, fish oil, cod liver oil, vitamin E, vitamin E succinate, vitamin E acetate, safflower oil, corn oil, sea buckthorn oil, linseed oil, peanut oil, tea oil, sunflower seed oil, wheat germ oil, almond oil, coix seed oil, evening primrose oil, sesame oil, cottonseed oil, castor oil, low erucic acid rapeseed oil, ethyl oleate, oleic acid, ethyl linoleate, isopropyl laurate, isopropyl myristate, ethyl butyrate, ethyl lactate, caprylic / capric triglyceride, caprylic / capric triglyceride, white oil, white petrolatum, or light liquid paraffin. Preferably, the oily substance includes any one or a combination of at least two of squalene, tocopherol, or white oil; Preferably, the oil phase further includes a lipophilic emulsifier; Preferably, the lipophilic emulsifier includes any one or a combination of at least two of the following: gum arabic, lecithin, soybean lecithin, sorbitan sesquioleate, sorbitan monostearate, PO500 emulsifier, dehydrated sorbitan trioleate, dehydrated sorbitan tristearate, dehydrated sorbitan sesquioleate, dehydrated sorbitan monooleate, glyceryl monostearate, polyethylene glycol dioleate, anionic emulsifying wax, polyoxyethylene stearate, polyoxyethylene fatty alcohol ether, polyoxyethylene (30) dihydroxystearate, cetyl polyethylene glycol / polypropylene glycol-10 / 1 dimethylsiloxane, and fatty acid methyl ester. Preferably, the lipophilic emulsifier has a mass percentage content of 1-20% in the oil phase.

5. The emulsion with a long-lasting immune response according to any one of claims 1-4, characterized in that, The aqueous phase includes any one or a combination of at least two of the following: purified water, water for injection, aqueous glycerol solution, 0.9% physiological saline, phosphate buffer, citrate buffer, or Tris buffer. Preferably, the external aqueous phase includes any one or a combination of at least two of phosphate buffer, citrate buffer, or Tris buffer; Preferably, the external aqueous phase further includes a hydrophilic surfactant; Preferably, the hydrophilic surfactant comprises any one or a combination of at least two of the following: Tween 60, Tween 65, Tween 80, Tween 85, poloxamer, polyoxyethylene monolaurate, polyoxyethylene lauryl ether, polyoxyethylene monopalmitate, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ether, polyvinyl alcohol, ethanol, propylene glycol, sodium octadecanol dodecyl sulfate, polyethylene glycol glycerol caprylate, polyethylene glycol-15 hydroxystearate, polyethylene glycol 300, or polyethylene glycol 400. Preferably, the hydrophilic surfactant has a mass percentage content of 3%-30% in the external aqueous phase; Preferably, the internal aqueous phase includes any one or a combination of at least two of 0.9% physiological saline, phosphate buffer, or water for injection.

6. The emulsion with a long-lasting immune response according to any one of claims 1-5, characterized in that, The average particle size of the emulsion is 80 nm-350 μm; Preferably, the average particle size of the emulsion is 200nm-250μm.

7. The emulsion with a long-lasting immune response according to any one of claims 1-6, characterized in that, The emulsion also includes water-soluble substances and / or oil-soluble substances; Preferably, the water-soluble substance includes any one or a combination of at least two of the following: bioactive macromolecules, hydrophilic immunostimulants and immunomodulators, and cytokines. Preferably, the bioactive macromolecules include any one or a combination of at least two of the following: proteins, polypeptides, or nucleic acids. Preferably, the hydrophilic immunostimulant and immunomodulator include any one or a combination of at least two of the following: saponins, flagellin, lipopolysaccharides, triacyl lipopeptides, and DNA containing CpG motifs. Preferably, the cytokines include any one or a combination of at least two of the following: granulocyte-macrophage colony-stimulating factor, interleukin-2, interleukin-4, interleukin-12, interleukin-15, interleukin-18, interleukin-21, interferon, and tumor necrosis factor-α. Preferably, the oil-soluble substance includes any one or a combination of at least two of lipids, hydrophobic immunostimulants, or immunomodulators; Preferably, the hydrophobic immunostimulant and immunomodulator include any one or a combination of at least two of monophosphatidyl A, gademolide, resimimolide, imiquimolide, single-stranded RNA, or double-stranded RNA.

8. A method for preparing an emulsion with a long-lasting immune response according to any one of claims 1-7, characterized in that, The preparation method includes: (1) Disperse the particles in the aqueous phase and then mix them with the oil phase to obtain a water-in-oil emulsion; (2) Add the water-in-oil emulsion to the external aqueous phase and disperse it to obtain the emulsion.

9. The method for preparing an emulsion with a long-lasting immune response according to claim 8, characterized in that, The droplet size in the water-in-oil emulsion is 50 nm-20 μm.

10. The use of an emulsion having a long-lasting immune response according to any one of claims 1-7 in the preparation of a vaccine for an infectious disease.

Images