Vaccine for inducing immunological tolerance, preparation method therefor, and the use thereof

Abstract

The present invention relates to a vaccine for inducing immunological tolerance, a preparation method therefor, and the use thereof, and belongs to the technical field of biological medicines. The provided vaccine for inducing immunological tolerance comprises a polymer carrier, and an antigen and immunomodulator which are loaded on the polymer carrier. The vaccine for inducing immunological tolerance comprises a nano vaccine and / or a micron vaccine, the particle size of the nano vaccine being 150-1000 nm and the particle size of the micron vaccine being 1μm-5μm. The surface potential of the vaccine for inducing immunological tolerance is -3mV to -150mV. The nano vaccine can more rapidly and efficiently penetrate into lymph nodes by itself and activate the immune system, and the micron vaccine is first phagocytosed by an antigen-presenting cell at an injection site and then enters the lymph nodes by means of homing effects and thus slowly acts, and therefore the long-term effect and the short-term effect cooperate to better exert the functions. The nano vaccine or micron vaccine which is prepared by using PEMA as an emulsifier and carries more negative electric charges can release the antigen rapidly and thus takes effect quickly, and the nano vaccine or micron vaccine which is prepared by using PVA as an emulsifier and carries less negative electric charges releases the antigen slowly, and thus has long-lasting effects, and therefore using the vaccines which act rapidly and act slowly in combination can exert functions in both short term and long term.

Description

A vaccine for inducing immune tolerance and its preparation method and application Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a vaccine for inducing immune tolerance and a preparation method and application thereof. Background Art

[0002] The primary function of the human immune system is to protect the host from microbial and parasitic infections, performing roles in immune surveillance, defense, and regulation. However, sometimes the immune system can become dysfunctional, misidentifying "self" as "non-self" and attacking its own tissues, leading to autoimmune diseases. Autoimmune diseases are chronic conditions related to the immune system that can lead to progressive debilitating or even fatal consequences. The most common of these diseases include multiple sclerosis, type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus.

[0003] Traditional treatments for autoimmune diseases rely primarily on broad-spectrum immunosuppressive drugs, which are often non-disease-specific. Beginning in the 1940s, glucocorticoids and other immunosuppressive drugs have been the mainstay of treatment for various autoimmune diseases, demonstrating initial success in alleviating disease progression and ameliorating exacerbations. While widely used and somewhat effective, glucocorticoids can gradually diminish their therapeutic efficacy over time, often causing significant toxicity to the nerves, bones, blood, kidneys, and gastrointestinal tract. They also work by dampening autoimmune responses at the expense of normal immunity to infection and cancer. Furthermore, current therapies for autoimmune diseases are non-antigen or disease-specific and cannot completely cure the disease. Even the latest monoclonal antibodies cannot distinguish between healthy and autoreactive cells, leading to severe side effects and systemic immunosuppression. Therefore, the development of antigen- or disease-specific therapeutic strategies for the treatment of autoimmune diseases is urgently needed.

[0004] Therefore, recent research has focused on inducing antigen-specific immune tolerance, training the immune system to recognize pathogenic antigens as "self" while avoiding nonspecific immunosuppression. Restoring tolerance to self-antigens allows the immune system to control infection and cancer. This ongoing research has led to the development of numerous antigen-specific therapies.

[0005] Tolerogenic DNA or RNA vaccines have been shown to be well tolerated in multiple autoimmune disease models. Lentivirus-mediated expression of insulin B chain 9-23 (Ins 9-23 ) epitope, inducing the production of antigen-specific FoxP3 +Tregs cells, when combined with anti-CD3 monoclonal antibodies, can prevent the onset of diabetes and promote disease reversal. Pere Santamaria's laboratory delivered nanoparticles loaded with major histocompatibility class I or class II complexes and autoimmune disease-related peptides to induce CD8 + T or CD4 + T cell subsets can slow the progression of type 1 diabetes. Miller discovered that delivering antigens in an apoptotic environment can highly effectively induce antigen-specific immune tolerance. Miller's laboratory used ECDI to conjugate autoantigens to the surface of splenocytes. ECDI promotes splenocyte apoptosis and releases autoantigens. Macrophages then present the apoptotic cell-conjugated autoantigens on MHC class I or MHC class II molecules and upregulate the negative co-stimulatory molecule (PD-L1), promoting tolerance to relapsing-remitting experimental encephalomyelitis (EAE). However, the preparation of antigen-cell conjugates is complex and extremely expensive, making large-scale production and clinical application impossible. Therefore, Miller used 500 nm diameter, highly negatively charged carboxylated polystyrene (PS) nanoparticles instead of apoptotic cells to deliver autoantigens in a tolerogenic manner. Pathogenic antigens conjugated to the surface of PS nanoparticles can prevent the onset of EAE after intravenous injection. Given the safety concerns raised by the in vivo non-degradability of PS nanoparticles, Miller used biodegradable materials such as poly(lactide-co-ethylene glycol) (PLGA) and polylactic acid (PLA). Using poly(ethylene-alt-maleic anhydride) (PEMA) as a surfactant, PLGA nanoparticles with a highly negative surface charge were prepared using an emulsification solvent evaporation method. PLP antigen peptides were conjugated to the carboxyl groups on the surface of the PLGA nanoparticles and were well tolerated by intravenous injection in EAE. Antigen modification of the nanoparticle surface was well tolerated but could cause adverse reactions upon injection.

[0006] Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a vaccine for inducing immune tolerance and a preparation method and application thereof.

[0008] The first object of the present invention is to provide a vaccine for inducing immune tolerance, comprising a polymer carrier, and an antigen and an immunomodulator loaded on the polymer carrier; the vaccine for inducing immune tolerance comprises a nano vaccine and / or a micro vaccine, the particle size of the nano vaccine is 150nm-1000nm; the particle size of the micro vaccine is 1μm-5μm; the surface potential of the vaccine for inducing immune tolerance is -3mV to -150mV.

[0009] Furthermore, the effect is better when the nanovaccine and the microvaccine are administered in combination, and the mass ratio of the nanovaccine to the microvaccine is 1:10-10:1; the preferred mass ratio of the nanovaccine to the microvaccine is 1:5-5:1; more preferably, the mass ratio of the nanovaccine to the microvaccine is 1:2-2:1; most preferably, the mass ratio of the nanovaccine to the microvaccine is 1:1.

[0010] Furthermore, the antigen and immunomodulator are loaded inside the polymer carrier.

[0011] In one embodiment of the present invention, the material of the polymer carrier is selected from one or more of poly(lactic acid) (PLA), poly(lactic-glycolic acid) (PLGA), polyglycolic acid (PGA), protein, cholesterol, lipid, carbohydrate and polypeptide.

[0012] In one embodiment of the present invention, the antigen is an autoantigen associated with an autoimmune disease.

[0013] Furthermore, the antigen is selected from one or more of β-cell-associated antigens, glutamic acid decarboxylase-associated antigens, rheumatoid arthritis-associated nuclear antigens, myelin basic protein, DNA, RNA, phospholipids and polypeptides.

[0014] In one embodiment of the present invention, the immunomodulator is selected from one or more of cyclosporine, rapamycin, tacrolimus, fingolimod, methylprednisolone, tripterygium wilfordii, mycophenolate mofetil, cyclophosphamide, azathioprine, everolimus, sangiomide, cesil, cyclosporin A, cyclosporine, nefotaxime, anti-IL-2 receptor monoclonal antibody, TGFβ, mRNA encoding TGF-β, interleukin, mRNA encoding interleukin, ginseng and astragalus.

[0015] In one embodiment of the present invention, the surface potential of the vaccine for inducing immune tolerance is achieved by an emulsifier.

[0016] In one embodiment of the present invention, the emulsifier is selected from one or more of polyvinyl alcohol (PVA), polyethylene-maleic anhydride (PEMA), phospholipids, glycerides, fatty acid esters, sugar esters, polyoxyethylene ethers, polyoxypropylene ethers, ethylene oxide and propylene oxide block copolymers, dodecyldimethylamine and other amine derivatives, quaternary ammonium salts, fatty acid soaps, alkyl sulfates, and alkylbenzene sulfonates.

[0017] Furthermore, the emulsifier is selected from polyvinyl alcohol (PVA) and / or polyethylene-maleic anhydride (PEMA).

[0018] In one embodiment of the present invention, the mass ratio of the polymer carrier, antigen and immunomodulator is 1-1000:1:1.

[0019] In one embodiment of the present invention, the surface of the polymer carrier is modified with polyethylene glycol (PEG); PEG is modified on its surface to avoid unnecessary immune activation.

[0020] The second object of the present invention is to provide a method for preparing the vaccine for inducing immune tolerance, comprising the following steps:

[0021] S1, ultrasonically treating the antigen solution, the mixed solution of the polymer and the immunomodulator, and the modified polymer solution to obtain a primary emulsion;

[0022] S2, mixing the first emulsifier solution and the primary water-in-oil emulsion, and performing ultrasonic treatment to obtain a secondary emulsion;

[0023] S3. Add the secondary emulsion to the second emulsifier solution and stir evenly. After standing, centrifuge, wash and dry to obtain the vaccine for inducing immune tolerance.

[0024] The third object of the present invention is to provide a use of the vaccine for inducing immune tolerance in the preparation of drugs for treating or preventing autoimmune diseases.

[0025] The technical solution of the present invention has the following advantages over the prior art:

[0026] (1) The vaccine described in the present invention uses a combination of nano-vaccines and micro-vaccines. The nano-vaccine can penetrate into the lymph nodes faster and more by itself to activate the immune system and take effect faster. The micro-vaccine enters the lymph nodes through the homing effect after being phagocytosed by antigen-presenting cells at the injection site and takes effect more slowly. The long-term and short-term effects are coordinated to better exert their efficacy.

[0027] (2) The vaccine described in the present invention uses PEMA as an emulsifier to prepare a nano-vaccine or micro-vaccine with more negative charge, which can release antigens faster and take effect faster; the nano-vaccine or micro-vaccine with less negative charge prepared using PVA as an emulsifier releases antigens slower and has a longer-lasting effect; by combining the fast-acting and slow-acting vaccines, both short-term and long-term effects can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0029] Figure 1 is a characterization of the vaccine in Test Example 1 of the present invention; wherein, a is the particle size distribution of the vaccine measured by DLS method, b is the zeta potential of the vaccine measured by DLS method, and the data are expressed as mean ± SD (n = 3), c is the morphology of the vaccine measured by TEM, d is the encapsulation efficiency and antigen loading of the vaccine, and e is the in vitro release of the loaded antigen;

[0030] FIG2 shows the cellular uptake and lysozyme escape capabilities of the vaccine in Test Example 2 of the present invention;

[0031] FIG3 shows the uptake of the DID-loaded vaccine by antigen-presenting cells in vivo in Test Example 2 of the present invention; data are expressed as mean ± SEM (n = 3), P < 0.05 (*), P < 0.01 (**);

[0032] FIG4 shows the in vitro induction of dendritic cell immune tolerance by the vaccine in Test Example 2 of the present invention; data are expressed as mean ± SD (n = 3), P < 0.05 (*), P < 0.01 (**), P < 0.0001 (***);

[0033] Figure 5 shows the efficacy of the vaccine in Test Example 3 of the present invention in treating the autoimmune disease model EAE; wherein, a is the treatment regimen, bk is the analysis and comparison of different treatment regimens, and the data are expressed as mean ± SEM, P < 0.05 (*), P < 0.01 (**). DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0036] In order to further understand the present invention, the present invention is described in detail below with reference to the embodiments. The meanings of the abbreviations used in the specification are shown in Table 1:

[0037] Table 1

[0038] In the present invention, unless otherwise specified, the mice used in the EAE model were wild-type 10-week-old SPF female C57BL / 6J mice weighing 18 g to 20 g, purchased from Shanghai Jihui Experimental Animal Co., Ltd.

[0039] In the present invention, unless otherwise specified, PLA and PEG-PLA were purchased from Xi'an Ruixi Biotechnology Co., Ltd.; PVA and PEMA were purchased from Sigma.

[0040] Example 1 Preparation of 200 nm PVA Nanoparticles

[0041] The vaccine for inducing immune tolerance and the preparation method thereof of this embodiment specifically comprise the following steps:

[0042] S1. Solution preparation

[0043] Solution 1: Myelin oligodendrocyte peptide MOG 35-55 A 20 mg / mL aqueous solution was prepared by mixing MOG 35-55 Prepared by dissolving in ultrapure water;

[0044] Solution 2: PLA at 20 mg / mL and rapamycin at 2 mg / mL in dichloromethane; this solution was prepared by dissolving PLA and rapamycin in pure dichloromethane;

[0045] Solution 3: PEG-PLA at 20 mg / mL in dichloromethane; this solution was prepared by dissolving PEG-PLA in pure dichloromethane;

[0046] Solution 4: PVA was a 20 mg / mL aqueous solution, which was prepared by dissolving PVA in ultrapure water;

[0047] Solution 5: PVA was a 5 mg / mL aqueous solution prepared by dissolving PVA in ultrapure water.

[0048] S2. Vaccine Preparation

[0049] Preparation of primary emulsion W1 / O1: Solution 1 (0.1 mL), solution 2 (0.75 mL), and solution 3 (0.25 mL) were mixed in a glass bottle and sonicated using ultrasound at 20% power (on 1 s, off 2 s) for 1 min 30 s to prepare primary emulsion W1 / O1;

[0050] Preparation of secondary emulsion W1 / O1 / W2: Solution 4 (3 mL) was mixed with primary emulsion W1 / O1, vortexed for 10 s, and sonicated for 1 min 30 s at 20% power (on 1 s, off 2 s) to prepare secondary emulsion W1 / O1 / W2;

[0051] Preparation of 200 nm PVA nanoparticles: Secondary emulsion W1 / O1 / W2 was added to a 40 mL beaker containing solution 5 and stirred overnight at room temperature using a magnetic stirrer at 750 rpm. The dichloromethane was evaporated to solidify the nanoparticles. The nanoparticle suspension was transferred to a 2 mL centrifuge tube and centrifuged at 15,000 g and 4°C for 30 min. The supernatant was removed, and the pellet was resuspended in phosphate buffered saline and washed twice by centrifugation at 15,000 g and 4°C for 30 min. After the final centrifugation, the pellet was resuspended in 2 mL of 4% trehalose solution. For storage, the nanoparticles were frozen at -20°C overnight, stored at -80°C for at least 8 h, and then freeze-dried in a freeze dryer for 48 h. After drying, the pellet was stored at -80°C until use.

[0052] Example 2 Preparation of 500nm PVA Nanoparticles

[0053] The vaccine for inducing immune tolerance and the preparation method thereof of this embodiment specifically comprise the following steps:

[0054] S1. Solution preparation

[0055] Solution 1: Myelin oligodendrocyte peptide MOG 35-55 A 20 mg / mL aqueous solution was prepared by mixing MOG 35-55 Prepared by dissolving in ultrapure water;

[0056] Solution 2: PLA at 20 mg / mL and rapamycin at 2 mg / mL in dichloromethane; this solution was prepared by dissolving PLA and rapamycin in pure dichloromethane;

[0057] Solution 3: PEG-PLA at 20 mg / mL in dichloromethane; this solution was prepared by dissolving PEG-PLA in pure dichloromethane;

[0058] Solution 4: PVA was a 10 mg / mL aqueous solution, which was prepared by dissolving PVA in ultrapure water;

[0059] Solution 5: PVA was a 5 mg / mL aqueous solution prepared by dissolving PVA in ultrapure water.

[0060] S2. Vaccine Preparation

[0061] Preparation of primary emulsion W1 / O1: Solution 1 (0.1 mL), solution 2 (0.75 mL), and solution 3 (0.25 mL) were mixed in a glass bottle and sonicated using ultrasound at 50% power (on for 1 s, off for 2 s) for 30 s to prepare primary emulsion W1 / O1;

[0062] Preparation of secondary emulsion W1 / O1 / W2: Solution 4 (10 mL) was mixed with primary emulsion W1 / O1, vortexed for 10 s, and homogenized using a homogenizer at setting 8 for 2 min to prepare secondary emulsion W1 / O1 / W2;

[0063] Preparation of 500 nm PVA nanoparticles: Secondary emulsion W1 / O1 / W2 was added to a beaker containing solution 5 (30 mL) and stirred overnight at room temperature using a magnetic stirrer at 750 rpm. The dichloromethane was evaporated to solidify the nanoparticles. The nanoparticle suspension was transferred to a 2 mL centrifuge tube and centrifuged at 11,000 g and 4°C for 20 min. The supernatant was removed, and the pellet was resuspended in phosphate buffered saline and washed twice by centrifugation at 11,000 g and 4°C for 20 min. After the final centrifugation, the pellet was resuspended in 2 mL of 4% trehalose solution. For storage, the nanoparticles were frozen at -20°C overnight, stored at -80°C for at least 8 h, lyophilized in a freeze dryer for 48 h, and then stored at -80°C until use.

[0064] Example 3 Preparation of 1 μm PVA microparticles

[0065] The vaccine for inducing immune tolerance and the preparation method thereof of this embodiment specifically comprise the following steps:

[0066] S1. Solution preparation

[0067] Solution 1: Myelin oligodendrocyte peptide MOG 35-55 A 20 mg / mL aqueous solution was prepared by mixing MOG 35-55 Prepared by dissolving in ultrapure water;

[0068] Solution 2: PLA at 20 mg / mL and rapamycin at 2 mg / mL in dichloromethane; this solution was prepared by dissolving PLA and rapamycin in pure dichloromethane;

[0069] Solution 3: PEG-PLA at 20 mg / mL in dichloromethane; this solution was prepared by dissolving PEG-PLA in pure dichloromethane;

[0070] Solution 4: PVA was a 40 mg / mL aqueous solution, which was prepared by dissolving PVA in ultrapure water;

[0071] Solution 5: PVA was a 1 mg / mL aqueous solution prepared by dissolving PVA in ultrapure water.

[0072] S2. Vaccine Preparation

[0073] Preparation of primary emulsion W1 / O1: Solution 1 (0.1 mL), solution 2 (0.75 mL), and solution 3 (0.25 mL) were mixed in a glass bottle and sonicated using ultrasound at 50% power (on for 1 s, off for 2 s) for 1 min to prepare primary emulsion W1 / O1;

[0074] Preparation of secondary emulsion W1 / O1 / W2: Solution 4 (10 mL) was mixed with primary emulsion W1 / O1, vortexed for 10 s, and homogenized for 2 min using a homogenizer at setting 6 to prepare secondary emulsion W1 / O1 / W2;

[0075] Preparation of 1 μm PVA microparticles: Secondary emulsion W1 / O1 / W2 was added to a 30 mL beaker containing solution 5 and stirred at room temperature overnight with a magnetic stirrer at 750 rpm. The dichloromethane was evaporated to solidify the nanoparticles. The microparticle suspension was transferred to a 2 mL centrifuge tube and centrifuged at 6000 rpm and 4°C for 25 min. The supernatant was removed, and the pellet was resuspended in phosphate buffered saline and washed twice by centrifugation at 6000 rpm and 4°C for 25 min. After the final centrifugation, the pellet was resuspended in 2 mL of 4% trehalose solution. For storage, the microparticles were frozen at -20°C overnight, stored at -80°C for at least 8 h, lyophilized in a freeze dryer for 48 h, and then stored at -80°C until use.

[0076] Example 4 Preparation of 3 μm PVA Microparticles

[0077] The vaccine for inducing immune tolerance and the preparation method thereof of this embodiment specifically comprise the following steps:

[0078] S1. Solution preparation

[0079] Solution 1: Myelin oligodendrocyte peptide MOG 35-55 A 20 mg / mL aqueous solution was prepared by mixing MOG 35-55 Prepared by dissolving in ultrapure water;

[0080] Solution 2: PLA at 20 mg / mL and rapamycin at 2 mg / mL in dichloromethane; this solution was prepared by dissolving PLA and rapamycin in pure dichloromethane;

[0081] Solution 3: PEG-PLA at 20 mg / mL in dichloromethane; this solution was prepared by dissolving PEG-PLA in pure dichloromethane;

[0082] Solution 4: PVA was a 2 mg / mL aqueous solution, which was prepared by dissolving PVA in ultrapure water;

[0083] Solution 5: PVA was a 5 mg / mL aqueous solution prepared by dissolving PVA in ultrapure water.

[0084] S2. Vaccine Preparation

[0085] Preparation of primary emulsion W1 / O1: Solution 1 (0.1 mL), solution 2 (0.75 mL), and solution 3 (0.25 mL) were mixed in a glass bottle and sonicated using ultrasound at 35% power (on for 1 s, off for 2 s) for 30 s to prepare primary emulsion W1 / O1;

[0086] Preparation of secondary emulsion W1 / O1 / W2: Solution 4 (10 mL) was mixed with primary emulsion W1 / O1, vortexed for 10 s, and homogenized for 2 min using a homogenizer at setting 2 to prepare secondary emulsion W1 / O1 / W2;

[0087] Preparation of 3 μm PVA microparticles: Secondary emulsion W1 / O1 / W2 was added to a 30 mL beaker containing solution 5 and stirred overnight at room temperature using a magnetic stirrer at 750 rpm. The dichloromethane was evaporated to solidify the nanoparticles. The microparticle suspension was transferred to a 2 mL centrifuge tube and centrifuged at 4000 rpm and 4°C for 20 min. The supernatant was removed, and the pellet was resuspended in phosphate buffered saline and washed twice by centrifugation at 4000 rpm and 4°C for 20 min. After the final centrifugation, the pellet was resuspended in 2 mL of 4% trehalose solution. For storage, the microparticles were frozen at -20°C overnight, stored at -80°C for at least 8 h, lyophilized in a freeze dryer for 48 h, and then stored at -80°C until use.

[0088] Example 5 Preparation of 5 μm PVA microparticles

[0089] The vaccine for inducing immune tolerance and the preparation method thereof of this embodiment specifically comprise the following steps:

[0090] S1. Solution preparation

[0091] Solution 1: Myelin oligodendrocyte peptide MOG 35-55 A 20 mg / mL aqueous solution was prepared by mixing MOG 35-55 Prepared by dissolving in ultrapure water;

[0092] Solution 2: PLA at 20 mg / mL and rapamycin at 2 mg / mL in dichloromethane; this solution was prepared by dissolving PLA and rapamycin in pure dichloromethane;

[0093] Solution 3: PEG-PLA at 20 mg / mL in dichloromethane; this solution was prepared by dissolving PEG-PLA in pure dichloromethane;

[0094] Solution 4: PVA was a 2 mg / mL aqueous solution, which was prepared by dissolving PVA in ultrapure water;

[0095] Solution 5: PVA was a 5 mg / mL aqueous solution prepared by dissolving PVA in ultrapure water.

[0096] S2. Vaccine Preparation

[0097] Preparation of primary emulsion W1 / O1: Solution 1 (0.1 mL), solution 2 (0.75 mL), and solution 3 (0.25 mL) were mixed in a glass bottle and sonicated using ultrasound at 20% power (on for 1 s, off for 2 s) for 30 s to prepare primary emulsion W1 / O1;

[0098] Preparation of secondary emulsion W1 / O1 / W2: Solution 4 (10 mL) was mixed with primary emulsion W1 / O1, vortexed for 10 s, and homogenized for 1 min using a homogenizer at setting 2 to prepare secondary emulsion W1 / O1 / W2;

[0099] Preparation of 5 μm PVA microparticles: Secondary emulsion W1 / O1 / W2 was added to a 30 mL beaker containing solution 5 and stirred overnight at room temperature using a magnetic stirrer at 750 rpm. The dichloromethane was evaporated to solidify the nanoparticles. The microparticle suspension was transferred to a 2 mL centrifuge tube and centrifuged at 3000 rpm and 4°C for 20 min. The supernatant was removed, and the pellet was resuspended in phosphate buffered saline and washed twice by centrifugation at 3000 rpm and 4°C for 20 min. After the final centrifugation, the pellet was resuspended in 2 mL of 4% trehalose solution. For storage, the microparticles were frozen at -20°C overnight, stored at -80°C for at least 8 h, lyophilized in a freeze dryer for 48 h, and then stored at -80°C until use.

[0100] Example 6 Preparation of 500 nm PEMA Nanoparticles

[0101] The vaccine for inducing immune tolerance and the preparation method thereof of this embodiment specifically comprise the following steps:

[0102] S1. Solution preparation

[0103] Solution 1: Myelin oligodendrocyte peptide MOG 35-55 A 20 mg / mL aqueous solution was prepared by mixing MOG 35-55 Prepared by dissolving in ultrapure water;

[0104] Solution 2: PLA at 20 mg / mL and rapamycin at 2 mg / mL in dichloromethane; this solution was prepared by dissolving PLA and rapamycin in pure dichloromethane;

[0105] Solution 3: PEG-PLA at 20 mg / mL in dichloromethane; this solution was prepared by dissolving PEG-PLA in pure dichloromethane;

[0106] Solution 4: PEMA at 8 mg / mL in water, prepared by dissolving PEMA in ultrapure water;

[0107] Solution 5: PEMA was a 5 mg / mL aqueous solution prepared by dissolving PEMA in ultrapure water.

[0108] S2. Vaccine Preparation

[0109] Preparation of primary emulsion W1 / O1: Solution 1 (0.1 mL), solution 2 (0.75 mL), and solution 3 (0.25 mL) were mixed in a glass bottle and sonicated at 50% power (on for 1 s, off for 2 s) for 2 min to prepare primary emulsion W1 / O1;

[0110] Preparation of secondary emulsion W1 / O1 / W2: Solution 4 (3 mL) was mixed with primary emulsion W1 / O1, vortexed for 10 s, and sonicated for 3 min at 50% power (on for 1 s, off for 2 s) to prepare secondary emulsion W1 / O1 / W2;

[0111] Preparation of 500 nm PEMA nanoparticles: Secondary emulsion W1 / O1 / W2 was added to a beaker containing solution 5 (40 mL) and stirred overnight at room temperature using a magnetic stirrer at 750 rpm. The dichloromethane was evaporated to solidify the nanoparticles. The nanoparticle suspension was transferred to a 2 mL centrifuge tube and centrifuged at 11,000 g and 4°C for 20 min. The supernatant was removed, and the pellet was resuspended in phosphate buffered saline and washed twice by centrifugation at 11,000 g and 4°C for 20 min. After the final centrifugation, the pellet was resuspended in 2 mL of 4% trehalose solution. For storage, the nanoparticles were frozen at -20°C overnight, stored at -80°C for at least 8 h, lyophilized in a freeze dryer for 48 h, and then stored at -80°C until use.

[0112] Example 7 Preparation of 1 μm PEMA Nanoparticles

[0113] The vaccine for inducing immune tolerance and the preparation method thereof of this embodiment specifically comprise the following steps:

[0114] S1. Solution preparation

[0115] Solution 1: Myelin oligodendrocyte peptide MOG 35-55 A 20 mg / mL aqueous solution was prepared by mixing MOG 35-55 Prepared by dissolving in ultrapure water;

[0116] Solution 2: PLA at 20 mg / mL and rapamycin at 2 mg / mL in dichloromethane; this solution was prepared by dissolving PLA and rapamycin in pure dichloromethane;

[0117] Solution 3: PEG-PLA at 20 mg / mL in dichloromethane; this solution was prepared by dissolving PEG-PLA in pure dichloromethane;

[0118] Solution 4: PEMA at 4 mg / mL in water, prepared by dissolving PEMA in ultrapure water;

[0119] Solution 5: PEMA was a 5 mg / mL aqueous solution prepared by dissolving PEMA in ultrapure water.

[0120] S2. Vaccine Preparation

[0121] Preparation of primary emulsion W1 / O1: Solution 1 (0.1 mL), solution 2 (0.75 mL), and solution 3 (0.25 mL) were mixed in a glass bottle and sonicated at 50% power (on for 1 s, off for 2 s) for 2 min to prepare primary emulsion W1 / O1;

[0122] Preparation of secondary emulsion W1 / O1 / W2: Solution 4 (3 mL) was mixed with primary emulsion W1 / O1, vortexed for 10 s, and sonicated for 3 min at 50% power (on for 1 s, off for 2 s) to prepare secondary emulsion W1 / O1 / W2;

[0123] Preparation of 1 μm PEMA nanoparticles: Secondary emulsion W1 / O1 / W2 was added to a beaker containing solution 5 (40 mL) and stirred overnight at room temperature with a magnetic stirrer at 750 rpm to allow the dichloromethane to evaporate and solidify the nanoparticles. The nanoparticle suspension was transferred to a 2 mL centrifuge tube and centrifuged at 11,000 g and 4°C for 20 min. The supernatant was removed, and the pellet was resuspended in phosphate buffered saline and washed twice by centrifugation at 6,000 rpm and 4°C for 20 min. After the final centrifugation, the pellet was resuspended in 2 mL of 4% trehalose solution. For storage, the nanoparticles were frozen at -20°C overnight, stored at -80°C for at least 8 h, lyophilized in a freeze dryer for 48 h, and then stored at -80°C until use.

[0124] Example 8 Preparation of 3 μm PEMA Microparticles

[0125] The vaccine for inducing immune tolerance and the preparation method thereof of this embodiment specifically comprise the following steps:

[0126] S1. Solution preparation

[0127] Solution 1: Myelin oligodendrocyte peptide MOG 35-55 A 20 mg / mL aqueous solution was prepared by mixing MOG 35-55 Prepared by dissolving in ultrapure water;

[0128] Solution 2: PLA at 20 mg / mL and rapamycin at 2 mg / mL in dichloromethane; this solution was prepared by dissolving PLA and rapamycin in pure dichloromethane;

[0129] Solution 3: PEG-PLA at 20 mg / mL in dichloromethane; this solution was prepared by dissolving PEG-PLA in pure dichloromethane;

[0130] Solution 4: PEMA at a 5 mg / mL aqueous solution, which was prepared by dissolving PEMA in ultrapure water;

[0131] Solution 5: PEMA was a 5 mg / mL aqueous solution prepared by dissolving PEMA in ultrapure water.

[0132] S2. Vaccine Preparation

[0133] Preparation of primary emulsion W1 / O1: Solution 1 (0.1 mL), solution 2 (0.75 mL), and solution 3 (0.25 mL) were mixed in a glass bottle and sonicated using ultrasound at 50% power (on for 1 s, off for 2 s) for 30 s to prepare primary emulsion W1 / O1;

[0134] Preparation of secondary emulsion W1 / O1 / W2: Solution 4 (8 mL) was mixed with primary emulsion W1 / O1, vortexed for 10 s, and homogenized using a homogenizer at setting 6 for 3 min to prepare secondary emulsion W1 / O1 / W2;

[0135] Preparation of 3 μm PEMA microparticles: Secondary emulsion W1 / O1 / W2 was added to a 30 mL beaker containing solution 5 and stirred at room temperature overnight with a magnetic stirrer at 750 rpm. The dichloromethane was evaporated to solidify the nanoparticles. The microparticle suspension was transferred to a 2 mL centrifuge tube and centrifuged at 4000 rpm and 4°C for 20 min. The supernatant was removed, and the pellet was resuspended in phosphate buffered saline and washed twice by centrifugation at 4000 rpm and 4°C for 20 min. After the final centrifugation, the pellet was resuspended in 2 mL of 4% trehalose solution. For storage, the microparticles were frozen at -20°C overnight, stored at -80°C for at least 8 h, lyophilized in a freeze dryer for 48 h, and then stored at -80°C until use.

[0136] Example 9 Preparation of 5 μm PEMA Microparticles

[0137] The vaccine for inducing immune tolerance and the preparation method thereof of this embodiment specifically comprise the following steps:

[0138] S1. Solution preparation

[0139] Solution 1: Myelin oligodendrocyte peptide MOG 35-55 A 20 mg / mL aqueous solution was prepared by mixing MOG 35-55 Prepared by dissolving in ultrapure water;

[0140] Solution 2: PLA at 20 mg / mL and rapamycin at 2 mg / mL in dichloromethane; this solution was prepared by dissolving PLA and rapamycin in pure dichloromethane;

[0141] Solution 3: PEG-PLA at 20 mg / mL in dichloromethane; this solution was prepared by dissolving PEG-PLA in pure dichloromethane;

[0142] Solution 4: PEMA at 10 mg / mL in water, which was prepared by dissolving PEMA in ultrapure water;

[0143] Solution 5: PEMA was a 5 mg / mL aqueous solution prepared by dissolving PEMA in ultrapure water.

[0144] S2. Vaccine Preparation

[0145] Preparation of primary emulsion W1 / O1: Solution 1 (0.1 mL), solution 2 (0.75 mL), and solution 3 (0.25 mL) were mixed in a glass bottle and sonicated using ultrasound at 50% power (on for 1 s, off for 2 s) for 30 s to prepare primary emulsion W1 / O1;

[0146] Preparation of secondary emulsion W1 / O1 / W2: Solution 4 (8 mL) was mixed with primary emulsion W1 / O1, vortexed for 10 s, and homogenized using a homogenizer at setting 6 for 3 min to prepare secondary emulsion W1 / O1 / W2;

[0147] Preparation of 5 μm PEMA microparticles: Secondary emulsion W1 / O1 / W2 was added to a 30 mL beaker containing solution 5 and stirred overnight at room temperature using a magnetic stirrer at 750 rpm. The dichloromethane was evaporated to solidify the nanoparticles. The microparticle suspension was transferred to a 2 mL centrifuge tube and centrifuged at 3000 rpm and 4°C for 20 min. The supernatant was removed, and the pellet was resuspended in phosphate buffered saline and washed twice by centrifugation at 3000 rpm and 4°C for 20 min. After the final centrifugation, the pellet was resuspended in 2 mL of 4% trehalose solution. For storage, the microparticles were frozen at -20°C overnight, stored at -80°C for at least 8 h, lyophilized in a freeze dryer for 48 h, and then stored at -80°C until use.

[0148] Test Example 1 Characterization of the physical and chemical properties of the vaccine

[0149] (1) The particle sizes of the vaccines prepared in Examples 1-5 (PVA as an emulsifier) ​​and Examples 4-9 (PEMA as an emulsifier) ​​were measured using a Malvern laser particle size analyzer. The particle sizes of the vaccines in Examples 1-5 were approximately 192±3 nm, 444±17 nm, 1013±57 nm, 2917±93 nm, and 4094±98 nm, respectively. The particle sizes of the vaccines in Examples 4-9 were approximately 504±14 nm, 985±18 nm, 3004±31 nm, and 4277±111 nm, respectively.

[0150] The particle size and surface potential of the vaccines prepared in Examples 1-5 (PVA as an emulsifier) ​​and Examples 4-9 (PEMA as an emulsifier) ​​were measured using a DLS particle size analyzer, and the results are shown in Figures 1a-1b. As can be seen from Figure 1a, the nine vaccines were of suitable size, uniform particle size, and low polydispersity index (PDI) (all <0.3). As can be seen from Figure 1b, the zeta potential of each vaccine varied depending on the choice of emulsifier, with the surface potentials of the vaccines in Examples 1-5 ranging from approximately -15mV to -25mV, respectively; and the surface potentials of the vaccines in Examples 4-9 ranging from approximately -40mV to -50mV, respectively.

[0151] (2) The surface morphology and size of the vaccines were observed using a transmission electron microscope (TEM), and the results are shown in Figure 1c. As can be seen from Figure 1c, all nine vaccines have a complete, regular spherical structure and uniform particle size.

[0152] (3) The antigen content and encapsulation efficiency of the vaccine were determined by the BCA method, and the results are shown in Figure 1d. As can be seen from Figure 1d, the total amount of antigen loaded by the nine vaccines was similar, with an antigen content of approximately 8 μg to 15 μg per milligram of polymer and an encapsulation efficiency of approximately 60% to 90%.

[0153] (4) Determination of in vitro antigen release: First, weigh 15 mg of different vaccines, dissolve them in 1 mL of ultrapure water, wash them, and centrifuge them at 15,000 g at 4°C for 20 min. Wash them three times. Dissolve the vaccine in 750 μL of PBS to a concentration of 20 mg / mL. Place the vaccine in a 37°C incubator and mix it at 100 rpm on a rotary mixer. At the designated time points (2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h, 216 h, 240 h), centrifuge them at 15,000 g for 20 min, collect the supernatant, and place it at -80°C for testing. Resuspend the nanoparticle pellet in 750 μL of fresh PBS. Continue to mix it at 37°C at 100 rpm. Prepare a mixed solution: Add 450 μL of Tween-20 and 50 μL of 10 M NaOH to 9.5 mL of ultrapure water and mix thoroughly. At 240 hours, collect the supernatant by centrifugation, dissolve the vaccine precipitate in the mixed solution, and measure the unreleased peptides. Peptide content in the supernatant was measured using a micro-BCA protein assay kit. The vaccine loading was calculated by adding the peptide content at each time point to the unreleased peptides divided by the vaccine weight. The results are shown in Figure 1e. As shown in Figure 1e, the vaccine prepared with PEMA as an emulsifier (NPs / MPs-PEMA) exhibited a faster in vitro release rate than the vaccine prepared with PVA as an emulsifier (NPs / MPs-PVA). On day 1, NPs / MPs-PEMA released approximately 30% of the antigen, and NPs / MPs-PVA released approximately 20%. Subsequently, the nine NPs / MPs slowly released the antigen over days 2-10, with a total release of approximately 10% over 9 days. The cumulative antigen release rate of NPs / MPs-PEMA on day 10 was approximately 40%, and the cumulative antigen release rate of NPs / MPs-PVA on day 10 was approximately 25%. This indicates that the prepared vaccine can slowly release antigens in vivo, which is conducive to inducing immune tolerance to antigens.

[0154] Test Example 2

[0155] (1) Study the uptake capacity of DC2.4 cells for different vaccines: 24-well plates were incubated with 0.01% poly-L-lysine solution at 37°C for 30 min and then rinsed twice with sterile water. 5×10 DC2.4 cells were placed in the wells of the plate. 4 / well were spread on the plate and allowed to settle and adhere to the wall for 4 hours. 100 μg / mL of the vaccine prepared in Example 1-9 (the vaccine was labeled with coumarin 6 so that the particles could emit green fluorescence for observing the localization of the particles in the cells) was added to the plate and incubated for 6 hours. After removing the culture medium, wash 3 times with PBS. Then take a small amount of Lyso-Tracker Red and add it to the cell culture medium at a ratio of 1:20000 (1 μL Lyso-Tracker Red was added to 20mL RPMI-1640 complete culture medium) and incubate for 30 minutes. After removing the culture medium, wash 3 times with PBS. The cells were then fixed with 4% paraformaldehyde for 20 minutes and washed 3 times with PBS. 200 μL Hoechst was added for staining for 15 minutes, washed 3 times with PBS, and anti-fluorescence quenching sealing liquid was added to seal the slices. The samples were imaged and analyzed on a confocal microscope, and the results are shown in Figure 2. As can be seen from Figure 2, by staining the cell nucleus (DAPI) with Hochest, Lyso-Tracker TM Lysosomes in cells were stained red (Lyso-Trac). After DC2.4 cells were co-incubated with the vaccine, granular fluorescence was observed throughout the cytoplasm, most of which did not overlap with the Lyso-Trac fluorescence. This indicates that after being taken up by antigen-presenting cells, the nine vaccines were partially retained in the lysosomes while others escaped from the lysosomes and were released into the cytoplasm. This demonstrates that the vaccines can effectively deliver antigens to both the lysosomes and the cytoplasm, thereby enabling antigen presentation via both the MHC I and MHC II pathways.

[0156] (2) Study the in vivo uptake of different vaccines: DID dye probes were loaded onto NPs / MPs as a model vaccine to analyze the uptake of NPs / MPs by antigen-presenting cells. 3 mg of DID-loaded vaccine (DID-NPs / MPs, DID was dissolved in dichloromethane during preparation (DID concentration was 100 μg / mL), the vaccine preparation was the same as in the example) was subcutaneously injected into C57BL / 6J mice. Five days later, the mice were killed, and single cell suspensions from the spleen or inguinal lymph nodes were collected. The cells were then stained with live-dead cell dyes, Fc block, CD45 antibodies, etc., and then flow cytometry was performed to analyze the uptake of the vaccine. The results are shown in Figure 3. As can be seen from Figure 3, after 5 days, the DID dye-containing antigen-presenting cells (APCs) in the lymph nodes and spleen were analyzed. This indicates that NPs / MPs can be efficiently phagocytosed by APCs in lymph nodes and spleen. In practical applications, NPs / MPs can effectively deliver loaded adjuvants and antigens to APCs, and then induce antigen-specific immune tolerance through APCs.

[0157] (3) Study on the internalization ability of BMDCs to different vaccines: After inducing BMDCs from bone marrow cells in C57BL / 6J mice, BMDCs were collected on day 8, and 1×10 6 BMDCs were seeded in a 24-well low-attachment plate containing 2 mL of culture medium. After 3–4 hours, different NPs / MPs were added to the wells at a final concentration of 500 μg / mL. BMDCs were incubated with NPs / MPs for 48 hours and then incubated with 1 μg / mL LPS for 24 hours. Cells were harvested by gentle pipetting and centrifuged at 350 g for 5 minutes. The supernatant was removed and the cells were resuspended in 1 mL of PBS. 1 μL of live-dead dye was added, mixed thoroughly, and the cells were incubated on ice in the dark for 30 minutes before centrifugation at 350 g for 5 minutes. The cells were then resuspended in 1 mL of PBS and centrifuged at 350 g for 5 minutes to remove any residual live-dead dye. The cells were resuspended in 100 μL of FACS buffer and incubated with 2 μL of Fc Blocker for 10 minutes to block Fc receptors. Following incubation, 2 μL of antibodies against CD11c, CD80, CD86, and MHC II were added and incubated on ice in the dark for 30 minutes. The cells were then fixed with 500 μL of 4% paraformaldehyde and incubated on ice for 30 minutes in the dark. After centrifugation at 350 g for 5 minutes, the supernatant was discarded and the cells were resuspended in 200 μL of FACS buffer. Cellular uptake of the NPs / MPs was analyzed by flow cytometry, as shown in Figure 4. As shown in Figure 4, compared to the LPS-treated control group, cells treated with all nine NPs / MPs significantly reduced the expression of all evaluated surface molecules, with 3 μm PEMA microparticles showing the greatest effect.

[0158] Test Example 3

[0159] The experimental encephalomyelitis (EAE) model in mice was used as a model of autoimmune disease to study the ability of different vaccines to induce immune tolerance.

[0160] S1. The induction and preparation of the EAE mouse autoimmune disease model and the experimental results are as follows:

[0161] S11. Solution preparation

[0162] MOG 35-55 Antigen solution: Accurately weigh MOG using a precision balance 35-55 40 mg of peptide was dissolved in 10 mL of PBS solution to prepare 4 mg / mL MOG. 35-55 Antigen solution;

[0163] Complete Freund's adjuvant solution: Mix 6.7 mL of 10 mg / mL complete Freund's adjuvant solution and 3.3 mL of 4 mg / mL complete Freund's adjuvant solution to prepare 8 mg / mL complete Freund's adjuvant solution.

[0164] Pertussis toxin solution: Dissolve pertussis toxin in ultrapure water to prepare a 3 μg / mL pertussis toxin solution.

[0165] S12, MOG 35-55 The antigen solution and complete Freund's adjuvant were placed in a screw-capped syringe at a ratio of 1:1, and the three-way valve and syringe were repeatedly pushed and pulled to prepare a water-in-oil emulsion (the first push and pull was from water to oil). The whole process was carried out on ice, and the push and pull were repeated for more than 2 hours until the droplet did not disperse on the water. 35-55 The antigen concentration was 2 mg / mL, and the complete Freund's adjuvant concentration was 4 mg / mL.

[0166] S13. Intraperitoneally inject mice with 4% chloral hydrate (150 μL / mouse). After the tail stimulation shows no response and the mouse is fully anesthetized, wipe the skin around the front and back of the mouse and its limbs with 75% alcohol for disinfection.

[0167] S14. Each mouse was injected with 50 μL of emulsion at four points on the front and back of the skin, for a total of 200 μL. A total of 200 μg of MOG was injected. 35-55 and 400 μg of CFA emulsion.

[0168] S15. Inject 300 ng of pertussis toxin PTX into the abdominal cavity on the other side of the chloral hydrate injection. In order to enhance immunity, inject 300 ng of PTX again after 48 hours.

[0169] S16. After model establishment, the body weight of mice was measured and recorded daily, and behavioral scores were performed daily according to Table 2 for 40 days;

[0170] Table 2

[0171] S17. Experimental results: C57BL / 6J mice were exposed to MOG 35-55 Clinical symptoms appeared on the 10th day after immunization, including dull fur color and tail weakness, and the EAE model in mice could be induced very efficiently.

[0172] S2. The use of different vaccines to treat EAE and the experimental results are as follows:

[0173] S21. As shown in FIG5a , 14 days after the successful induction of the EAE mouse model, the mice in the treatment group were subcutaneously injected with 2 mg of different vaccines (NPs, MPs, NPs+MPs and the mass ratio of the two was 1:1), the control group was subcutaneously injected with PBS, and the blank particle group was subcutaneously injected with blank particles (basically the same as in Example 1, except that the myelin oligodendrocyte peptide MOG was not added to Solution 1). 35-55The mice were injected with 100 μg of MOG35-55 free polypeptide antigen on the other side of the mouse where blank particles were injected. The body weight and behavioral scores of the mice were recorded.

[0174] S22. Experimental results, as shown in Figures 5b-5k, show rapid clinical progression in the PBS and blank particle groups, reaching a peak onset between days 22 and 25, with typical symptoms such as complete hindlimb paralysis and tail weakness. The highest clinical score reached 3.1 ± 0.2. Compared with the PBS and blank particle groups, different NPs / MPs alone or in combination with different NPs and MPs were all effective in treating EAE. Among them, NPs / MPs with a particle size of 200 nm, 500 nm, or 3 μm were most effective when used alone. NPs / MPs prepared using PEMA as an emulsifier had a faster onset and suppressed EAE symptoms early, while NPs / MPs prepared using PVA as an emulsifier had a slower onset and more effective suppression of EAE symptoms later. Nanoparticles or microparticles with higher negative charges induced antigen-specific immune tolerance more quickly, while those with lower negative charges induced antigen-specific immune tolerance more slowly. The duration of antigen-specific immune tolerance induced by these two methods was similar.

[0175] Mixing 500nm PEMA and 3μm PEMA did not significantly improve the efficacy of either alone, nor did mixing 200nm PVA and 3μm PVA. However, mixing 500nm PEMA and 3μm PVA, or mixing 200nm PVA and 3μm PEMA, achieved better results than either nanoparticle alone, suggesting that mixing particles of different sizes prepared with different emulsifiers can achieve better results. This is because particles prepared with PVA and PEMA, respectively, can suppress the symptoms of autoimmune diseases at different speeds.

[0176] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A vaccine for inducing immune tolerance, characterized in that: It includes a polymer carrier, and an antigen and an immunomodulator loaded on the polymer carrier; the vaccine for inducing immune tolerance includes a nano vaccine and / or a micro vaccine, the particle size of the nano vaccine is 150nm-1000nm; the particle size of the micro vaccine is 1μm-5μm; the surface potential of the vaccine for inducing immune tolerance is -3mV to -150mV.

2. The vaccine for inducing immune tolerance according to claim 1, characterized in that The material of the polymer carrier is selected from one or more of poly(lactic acid), poly(lactic-glycolic acid), polyglycolic acid, protein, cholesterol, lipid, sugar and polypeptide.

3. The vaccine for inducing immune tolerance according to claim 1, characterized in that The antigen is an autoantigen associated with an autoimmune disease.

4. The vaccine for inducing immune tolerance according to claim 1, characterized in that The immunomodulator is selected from one or more of cyclosporine, rapamycin, tacrolimus, fingolimod, methylprednisolone, tripterygium wilfordii, mycophenolate mofetil, cyclophosphamide, azathioprine, everolimus, sandylimine, cesilpine, cyclosporin A, cyclosporine, nefotaxime, anti-IL 2 receptor monoclonal antibody, TGFβ, mRNA encoding TGF-β, interleukin, mRNA encoding interleukin, ginseng and astragalus.

5. The vaccine for inducing immune tolerance according to claim 1, characterized in that The surface potential of the vaccine inducing immune tolerance is achieved through an emulsifier.

6. The vaccine for inducing immune tolerance according to claim 5, characterized in that The emulsifier is selected from one or more of vinyl alcohol, polyethylene-maleic anhydride, phospholipids, glycerides, fatty acid esters, sugar esters, polyoxyethylene ethers, polyoxypropylene ethers, ethylene oxide and propylene oxide block copolymers, dodecyldimethylamine and other amine derivatives, quaternary ammonium salts, fatty acid soaps, alkyl sulfates, and alkylbenzene sulfonates.

7. The vaccine for inducing immune tolerance according to claim 1, characterized in that The mass ratio of the polymer carrier, antigen and immunomodulator is 1-1000:1:

1.

8. The vaccine for inducing immune tolerance according to claim 1, characterized in that The surface of the polymer carrier is modified with polyethylene glycol.

9. The method for preparing the vaccine for inducing immune tolerance according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1, ultrasonically treating the antigen solution, the mixed solution of the polymer and the immunomodulator, and the modified polymer solution to obtain a primary emulsion; S2, mixing the first emulsifier solution and the primary water-in-oil emulsion, and performing ultrasonic treatment to obtain a secondary emulsion; S3. Add the secondary emulsion to the second emulsifier solution and stir evenly. After standing, centrifuge, wash and dry to obtain the vaccine for inducing immune tolerance.

10. Use of the immune tolerance-inducing vaccine according to any one of claim 9 in the preparation of a drug for treating or preventing autoimmune diseases.

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