Toleranogen vaccine for treating multiple sclerosis as well as preparation method and application of toleranogen vaccine

Abstract

The invention belongs to the technical field of tolerogen vaccines, and particularly relates to a tolerogen vaccine for treating multiple sclerosis and a preparation method and application of the tolerogen vaccine. The tolerogen vaccine comprises myelin basic protein, protein lipid protein and an antigen peptide fragment composition of myelin oligodendroglia glycoprotein, the preparation method comprises the following steps: S1, preparing a polypeptide mixture solution and an anionic liposome solution; s2, preparing a polypeptide mixture liposome preparation from the polypeptide mixture solution and the anionic liposome solution by adopting an injection method; s3, the polypeptide mixture lipidosome preparation is diluted, filtered and purified, and the lipidosome entrapped tolerogen vaccine is obtained.The multi-antigen tolerogen vaccine can be delivered through lipidosome, the limitation of a single antigen therapy is overcome, the immune tolerance induction effect is enhanced by means of the delivery advantage of lipidosome, and the application prospect is wide. And an efficient and safe novel precise therapy method is provided for MS treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of tolerogen vaccines, and particularly relates to a tolerogen vaccine for the treatment of multiple sclerosis, a preparation method thereof, and an application thereof. Background Art

[0002] Multiple sclerosis (MS) is an autoimmune disease caused by the migration of myelin antigen-specific T cells to the central nervous system (CNS), resulting in CNS demyelination and progressive neuropathy. It is the most common chronic inflammatory demyelinating disease of the central nervous system, with more than 3 million patients globally. The disease is characterized by a high disability rate, a complex relapse-remission pattern, and chronic irreversible progression, which not only severely impairs the motor and cognitive functions of patients, but also brings a huge social and economic burden. At present, the pathogenesis of MS has not been fully elucidated, but studies have shown that antigen-specific CD4+ T cells in the central nervous system migrate to the brain and spinal cord after being activated peripherally, mediate an inflammatory cascade reaction, and release pro-inflammatory cytokines (such as IL-6, IL-1β, IFN-γ), thereby triggering demyelination lesions and neurodegenerative damage.

[0003] Currently, the main treatment methods for MS are immunosuppression and immunomodulation. Immunomodulatory therapies reduce the inflammatory response through systemic immunosuppression. However, such methods have significant limitations, such as severe side effects, an increased risk of infection, and the inevitable "overall immunosuppression" effect, which impairs the normal immune defense function of the body and may lead to adverse effects such as lifelong immunosuppression. Although traditional therapies can temporarily relieve symptoms, they cannot fundamentally block the progression of the disease. Existing treatment methods mainly rely on broad-spectrum immunosuppression. This "overall immunosuppression" strategy not only leads to a high risk of severe infection, but also cannot solve the core problem of disease recurrence, that is, the continuous attack of autoreactive T cells on myelin proteins. In contrast, antigen-specific treatment strategies have high targeting, can precisely induce immune tolerance against specific autoantigens without inhibiting the systemic immune response, and retain the overall immune function while eliminating pathogenic immune responses. It is an ideal immunotherapy strategy for MS and thus has become a more promising treatment direction.

[0004] Currently, the research and development of antigen-specific immunotherapy cover a variety of strategies, including whole antigen / peptide delivery, modified peptides, MHC peptide complexes, cell-based therapies, and DNA vaccines. However, existing clinical trials mostly use single antigens or epitope peptides, and their efficacy is limited by factors such as antigen epitope spreading, individual HLA polymorphism, and local persistent inflammation in the central nervous system, which restricts the clinical translation effect. Summary of the Invention

[0005] To solve the problems in the prior art, the present invention provides a tolerogen vaccine for the treatment of multiple sclerosis, and its preparation method and application, aiming to deliver a multi-antigen tolerogen vaccine through liposomes, which not only overcomes the limitations of single antigen therapy, but also utilizes the delivery advantages of liposomes to enhance the effect of immune tolerance induction, providing an efficient, safe and novel precision therapy for MS treatment.

[0006] The present invention solves its technical problems by adopting the following technical solutions:

[0007] The present invention aims to provide a tolerogen vaccine for the treatment of multiple sclerosis, and the tolerogen vaccine includes an antigen peptide fragment composition of myelin basic protein, proteolipid protein and myelin oligodendrocyte glycoprotein.

[0008] Studies have found that the pathogenic T cells infiltrating in the lesions of MS patients mainly target three myelin proteins: myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG). Based on this pathological feature, the present invention innovatively proposes a multi-antigen tolerogen vaccine, which overcomes the limitations of single antigen therapy by integrating the key T cell epitopes of the above three myelin proteins, thereby more effectively inducing immune tolerance and inhibiting disease progression. This design is expected to provide a novel precision therapy for MS treatment with high efficiency and low toxicity.

[0009] Further, the antigen peptide fragment composition includes any 3 or 4 of SEQ ID NO: 1-4, such as sequence 1, 2, 3; sequence 1, 2, 4; sequence 2, 3, 4; sequence 1, 3, 4; sequence 1, 2, 3, 4.

[0010] Further, each antigen peptide fragment in the antigen peptide fragment composition is composed in an equal proportion.

[0011] Further, the tolerogen vaccine can be encapsulated in liposomes, and the liposomes adopt anionic liposomes, and the liposomes include anionic phospholipids and cholesterol.

[0012] Liposomes, as spherical vesicles based on cholesterol and natural non-toxic phospholipids, can protect antigen peptides from enzymatic degradation, prolong their half-life in vivo, and improve bioavailability due to their biocompatibility, appropriate size, and the presence of hydrophobic and hydrophilic layers capable of loading drugs. They can enhance the delivery efficiency to specific immune cells (such as dendritic cells), promote antigen presentation, and induce immune tolerance. They can mimic the structure of pathogens and are more easily taken up by antigen-presenting cells (APCs), thereby enhancing the immunomodulatory effect of tolerogens. They can also reduce the toxic and side effects of drugs, increase the drug concentration in vivo, and improve the therapeutic effect. By using anionic liposomes to encapsulate tolerogenic polypeptides, efficient loading of polypeptide drugs can be achieved. At the same time, liposome nanoparticles can prevent polypeptide degradation, and the slow erosion / degradation of liposomes and the slow diffusion of drug molecules enable sustained release.

[0013] The object of the present invention also lies in providing a preparation method for a tolerogen vaccine for the treatment of multiple sclerosis, comprising the following steps:

[0014] S1. Prepare a polypeptide mixture solution and an anionic liposome solution;

[0015] S2. Prepare a polypeptide mixture liposome preparation by using the injection method with the polypeptide mixture solution and the anionic liposome solution;

[0016] S3. Dilute, filter, and purify the polypeptide mixture liposome preparation to obtain a tolerogen vaccine encapsulated by liposomes.

[0017] Furthermore, the polypeptide mixture solution comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 with a mass ratio of 1:1:1, or SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 with a mass ratio of 1:1:1:1, and the final concentration of the polypeptide mixture solution is 2 mg / mL.

[0018] Furthermore, the anionic liposome solution comprises cholesterol, DPPG, DSPC, and absolute ethanol, and the final concentration of the anionic liposome solution is 10 mg / mL.

[0019] Furthermore, the method for preparing the polypeptide mixture liposome preparation comprises: while stirring the polypeptide mixture solution at a rotation speed of 700 - 800 rpm / min, adding the anionic liposome solution. After the addition of the anionic liposome solution is completed, continue to stir at a rotation speed of 1200 - 1800 rpm / min, and maintain heating at 35 - 40 °C. After 5 - 7 h, stop heating and stirring, and perform homogenization by ultrasonic treatment for 8 - 12 min.

[0020] Further, in S3, the liposome preparation of the polypeptide mixture is diluted 4 - 6 times with pure water, then filtered through a 0.40 - 0.5 μm PVDF membrane, and finally purified by centrifugation at 10000 - 14000 rpm for 10 - 20 minutes.

[0021] The present invention also aims to provide the use of a tolerogen vaccine for the treatment of multiple sclerosis or a preparation method of a tolerogen vaccine for the treatment of multiple sclerosis in the preparation of a drug for the treatment or prevention of multiple sclerosis.

[0022] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0023] 1. The multiple immunotolerogen vaccine of the present invention is composed of the main T cell epitope functional regions of three proteins: myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). The tolerogen sequence is associated with multiple HLA genotypes, including the main susceptible HLA genotypes of MS patients: HLA - DRB1*15:01, HLA - DRB5*01:01, etc. Compared with the antigenic peptides derived from a single myelin antigen used in previous clinical trials, it has many advantages, including: being able to induce apoptosis of spinal cord - infiltrating autoreactive T cells, up - regulating the frequency of Treg cells, being able to reduce the frequency of spinal cord - infiltrating inflammatory monocytes, significantly reducing the levels of spinal cord inflammatory cytokines, increasing the levels of anti - inflammatory cytokines such as IL - 10, and alleviating CNS inflammation.

[0024] 2. The polypeptide composition in the polypeptide composition solution of the present invention shows a positive charge on the surface. The tolerogen vaccine is encapsulated and delivered using anionic liposomes. Its main raw materials are anionic phospholipids, cholesterol, and other auxiliary phospholipids, all of which are commonly used pharmaceutical excipients registered with CDE. The anionic liposomes of the present invention have a good encapsulation rate, and the encapsulated liposome solution has good stability, and can also play a role in protecting the polypeptide from degradation.

[0025] The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly, it can be implemented according to the content of the specification. And in order to make the above content, objects, features, and advantages of the present invention more obvious and understandable, the following specifically illustrates the specific embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the particle size diagram of the liposome solution obtained by filtration in the present invention.

[0027] Figure 2 It is the particle size diagram of the liposome solution obtained by filtration in the present invention placed in a 4°C refrigerator for 2, 5, and 9 days respectively.

[0028] Figure 3Particle size diagram of the liposome precipitate obtained by centrifugation after filtration in the present invention.

[0029] Figure 4 Particle size diagrams of the liposome precipitate obtained by centrifugation after filtration in the present invention at 4°C for 1, 2, and 9 days respectively.

[0030] Figure 5 Free polypeptide content in the present invention without using Triton for demulsification.

[0031] Figure 6 Polypeptide content after using 2% Triton for demulsification in the present invention.

[0032] Figure 7 Low-voltage transmission electron microscope image of LNP-3P in the present invention.

[0033] Figure 8 Evaluation diagram of the therapeutic effect of LNP-3P in the RR-EAE model in the present invention.

[0034] Figure 9 HE staining diagram of the spinal cord of mice after treatment with LNP-3P in the RR-EAE model in the present invention.

[0035] Figure 10 LFB staining diagram of the spinal cord after treatment with LNP-3P in the RR-EAE model in the present invention.

[0036] Figure 11 Diagram of the apoptosis of infiltrating T cells in the spinal cord after treatment with LNP-3P in the RR-EAE model in the present invention.

[0037] Figure 12 Evaluation diagram of the therapeutic effect of 3P and LNP-3P in the classical EAE model in the present invention.

[0038] Figure 13 Result diagram of the antigen recall experiment after treatment with LNP-3P in the classical EAE model in the present invention.

[0039] Figure 14 Surface zeta potential values of LNP, 3P, and LNP-3P in the present invention.

[0040] Figure 15 Result diagram of the LNP cytotoxicity experiment in the present invention.

[0041] Figure 16 Diagram of DC2.4 cell uptake of LNP-3P in the present invention.

[0042] Figure 17 Evaluation diagram of the therapeutic effect of the tolerogen vaccine in the EAE model of C57BL / 6 mice in the present invention.

[0043] Figure 18 This is the evaluation chart of the treatment effect of the tolerogen vaccine in the RR-EAE model in the present invention.

[0044] Figure 19 This is the chart of the proportions of infiltrated mononuclear cells and microglial cells in the spinal cord after treatment with the tolerogen vaccine in the present invention.

[0045] Figure 20 This is the HE staining chart of the spinal cord after treatment with the tolerogen vaccine in the present invention.

[0046] Figure 21 This is the LFB staining result chart of the spinal cord after treatment with the tolerogen vaccine in the present invention.

[0047] Figure 22 This is the chart of the apoptosis of infiltrated T cells in the spinal cord after treatment with the tolerogen vaccine in the present invention.

[0048] Figure 23 This is the chart of the level of IL10 secreted by splenocytes after restimulation with antigen after treatment with the tolerogen vaccine in the present invention.

[0049] Figure 24 This is the chart showing the upregulation of the proportion of Treg cells induced by restimulation of splenocytes with antigen after treatment with the tolerogen vaccine in the present invention. Detailed implementation manners

[0050] The technical solution of the present invention will be further described in detail below in combination with specific embodiments. It should be understood that the following embodiments are only for illustrative explanation of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0051] In addition, unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or prepared by existing methods.

[0052] Design and synthesis of a tolerogen vaccine for the treatment of multiple sclerosis

[0053] The tolerogen vaccine is a composition of major antigenic peptide fragments of three myelin proteins: myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). The tolerogen vaccine of the present invention is composed of 3 or 4 of the following 4 antigenic peptide fragments in equal proportion: MOG35-55 (Sequence 1), PLP139-154 (Sequence 2), MBP84-103 (Sequence 3), MBP143-163 (Sequence 4). The proportion of each peptide can be adjusted accordingly according to the patient's specific leukocyte-associated antigen (HLA) typing. The 4 antigenic peptide components of the tolerogen vaccine are related to the disease-susceptible alleles HLA genes of MS (HLA-DRB1*15:01, HLA-DRB5*01:01, etc.). The 4 antigenic peptides of the tolerogen vaccine are all synthesized by solid-phase synthesis technology.

[0054] The antigenic peptide sequences used in the present invention are as follows:

[0055] Sequence 1 (SEQ ID NO:1): MEVGWYRPPFSRVVHLYRNGK;

[0056] Sequence 2 (SEQ ID NO:2): HCLGKWLGHPDKFVGI;

[0057] Sequence 3 (SEQ ID NO:3): NPVVHFFKNIVTPRTPPPSQ;

[0058] Sequence 4 (SEQ ID NO:4): GVDAQGTLSKIFKLGGRDSRS.

[0059] Example 1

[0060] A method for preparing a tolerogen vaccine for the treatment of multiple sclerosis, comprising the following steps:

[0061] 1. Weigh and prepare a polypeptide mixture solution (Sequences 1, 2, 3) with a final concentration of 2 mg / mL. Take 2 mg of each polypeptide and dissolve it in 3 mL of physiological saline, and ultrasonicate until completely dissolved.

[0062] 2. Weigh and prepare an anionic liposome solution with a final concentration of 10 mg / mL. Take 10 mg of cholesterol, 8 mg of DPPG (dipalmitoyl phosphatidylglycerol), and 2 mg of DSPC (distearoyl phosphatidylcholine) and dissolve them in 2 mL of absolute ethanol, and ultrasonicate until completely dissolved.

[0063] 3. The liposomal preparation of the polypeptide mixture is prepared by the injection method. While stirring the polypeptide mixture solution at a speed of 750 rpm / min, the liposome solution is added. After the addition of the liposome solution is completed, stirring is continued at a speed of 1500 rpm / min, and heating is maintained at 37°C. After 6 h, heating and stirring are stopped, and it is ultrasonically homogenized for 10 min.

[0064] 4. The prepared liposomal preparation of the polypeptide mixture is diluted several times (5 times) with pure water to reduce the concentration of absolute ethanol in it. Further, a PVDF membrane (0.45 μm) is used to filter out the absolute ethanol, and filtering through this membrane can achieve the purpose of reducing the particle size of LNP-3P, obtaining a polypeptide mixture encapsulated by liposomes.

[0065] 5. Since the polypeptide mixture is a water-soluble drug, it is further purified by high-speed centrifugation: 12000 rpm / min, centrifuged for 15 minutes, and finally a tolerogen vaccine encapsulated by liposomes (LNP-3P) is obtained.

[0066] Example 2

[0067] A method for preparing a tolerogen vaccine for the treatment of multiple sclerosis, comprising the following steps:

[0068] 1. Weigh and prepare a polypeptide mixture solution (sequences 1, 3, 4) with a final concentration of 2 mg / mL. Take 2 mg of each polypeptide and dissolve it in 3 mL of normal saline, and ultrasonically dissolve it until completely dissolved.

[0069] 2. Weigh and prepare an anionic liposome solution with a final concentration of 10 mg / mL. Take 10 mg of cholesterol, 8 mg of DPPG (dipalmitoyl phosphatidylglycerol), and 2 mg of DSPC (distearoyl phosphatidylcholine) and dissolve them in 2 mL of absolute ethanol, and ultrasonically dissolve them until completely dissolved.

[0070] 3. The liposomal preparation of the polypeptide mixture is prepared by the injection method. While stirring the polypeptide mixture solution at a speed of 700 rpm / min, the liposome solution is added. After the addition of the liposome solution is completed, stirring is continued at a speed of 1200 rpm / min, and heating is maintained at 35°C. After 5 h, heating and stirring are stopped, and it is ultrasonically homogenized for 8 min.

[0071] 4. The prepared liposomal preparation of the polypeptide mixture is diluted several times (4 times) with pure water to reduce the concentration of absolute ethanol in it. Further, a PVDF membrane (0.4 μm) is used to filter out the absolute ethanol, and filtering through this membrane can achieve the purpose of reducing the particle size of LNP-3P, obtaining a polypeptide mixture encapsulated by liposomes.

[0072] 5. Since the polypeptide mixture is a water-soluble drug, it is further purified by high-speed centrifugation: 10,000 rpm for 10 minutes, and finally the tolerogen vaccine encapsulated by liposomes (LNP-3P) is obtained.

[0073] Example 3

[0074] A method for preparing a tolerogen vaccine for the treatment of multiple sclerosis, comprising the following steps:

[0075] 1. Weigh and prepare a polypeptide mixture solution (sequences 1, 2, 4) with a final concentration of 2 mg / mL. Take 2 mg of each polypeptide and dissolve it in 3 mL of physiological saline, and ultrasonicate until completely dissolved.

[0076] 2. Weigh and prepare an anionic liposome solution with a final concentration of 10 mg / mL. Take 10 mg of cholesterol, 8 mg of DPPG (dipalmitoyl phosphatidylglycerol), and 2 mg of DSPC (distearoyl phosphatidylcholine) and dissolve them in 2 mL of absolute ethanol, and ultrasonicate until completely dissolved.

[0077] 3. Prepare a polypeptide mixture liposome preparation by the injection method. While stirring the polypeptide mixture solution at a speed of 800 rpm, add the liposome solution. After the liposome solution is added completely, continue to stir at a speed of 1800 rpm and keep heating at 40 °C. After 7 h, stop heating and stirring, and ultrasonicate it for 12 min for homogenization.

[0078] 4. The prepared polypeptide mixture liposome preparation is diluted several times (6 times) with pure water to reduce the concentration of absolute ethanol in it, and further filtered through a PVDF membrane (0.5 μm) to remove absolute ethanol and to reduce the particle size of LNP-3P by filtering through this membrane, and a polypeptide mixture encapsulated by liposomes is obtained.

[0079] 5. Since the polypeptide mixture is a water-soluble drug, it is further purified by high-speed centrifugation: 14,000 rpm for 20 minutes, and finally the tolerogen vaccine encapsulated by liposomes (LNP-3P) is obtained.

[0080] Example 4

[0081] A method for preparing a tolerogen vaccine for the treatment of multiple sclerosis, comprising the following steps:

[0082] 1. Weigh and prepare a polypeptide mixture solution (sequences 1, 2, 3, 4) with a final concentration of 2 mg / mL. Take 2 mg of each polypeptide and dissolve it in 3 mL of physiological saline, and ultrasonicate until completely dissolved.

[0083] 2. Weigh and prepare an anionic liposome solution with a final concentration of 10 mg / mL. Dissolve 10 mg of cholesterol, 8 mg of DPPG (dipalmitoyl phosphatidylglycerol), and 2 mg of DSPC (distearoyl phosphatidylcholine) in 2 mL of absolute ethanol, and ultrasonicate until completely dissolved.

[0084] 3. Prepare a liposomal formulation of the polypeptide mixture by the injection method. While stirring the polypeptide mixture at a speed of 750 rpm / min, add the liposome solution. After the addition of the liposome solution is complete, continue to stir at a speed of 1600 rpm / min and maintain heating at 37 °C. After 6 h, stop heating and stirring, and ultrasonicate it for 11 min for homogenization.

[0085] 4. Dilute the prepared liposomal formulation of the polypeptide mixture several times (5 times) with pure water to reduce the concentration of absolute ethanol. Further filter it using a PVDF membrane (0.45 μm) to remove absolute ethanol and to reduce the particle size of LNP-3P by filtration through this membrane, obtaining a polypeptide mixture encapsulated in liposomes.

[0086] 5. Since the polypeptide mixture is a water-soluble drug, further purify it by high-speed centrifugation: at 13000 rpm / min for 18 minutes, finally obtaining a tolerogen vaccine encapsulated in liposomes (LNP-4P).

[0087] Experimental Example 1

[0088] Conduct the following experimental studies on the tolerogen vaccine prepared in Example 1:

[0089] 1. Study on the particle size and stability of liposomes encapsulating the tolerogen vaccine after filtration and purification

[0090] Place the filtered liposome solution in a 4 °C refrigerator for 2, 5, and 9 days, and detect its particle size. Refer to Figure 1 and Figure 2 , the particle size is between 324.4 nm - 330.7 nm, and the PDI value is less than 0.2. After storage, the particle size of LNP-3P shows no significant change, indicating good stability.

[0091] 2. Study on the size and stability of liposomes encapsulating the tolerogen vaccine after centrifugation purification after filtration

[0092] Redissolve the liposome precipitate obtained after centrifugation after filtration into a solution, place it in a 4 °C refrigerator for 1, 2, and 9 days, and detect its particle size. Refer to Figure 3 and Figure 4 , the particle size is between 237.6 nm - 240.8 nm, and the PDI value is less than 0.2. After storage, the particle size of LNP-3P shows no significant change, indicating good stability.

[0093] 3. Determination of the encapsulation efficiency of the polypeptide composition

[0094] First, a standard curve was drawn using a standard substance, including a standard curve containing 2% Triton (Y = 0.7311X + 0.586, R 2 = 0.9905) and a standard curve without Triton (Y = 0.5846X + 0.7058, R 2 = 0.9901). Refer to Figure 5 and Figure 6 . Subsequently, the content of free polypeptide without using Triton for demulsification after filtration and the content of polypeptide after demulsification with 2% Triton were measured respectively.

[0095] Furthermore, the encapsulation efficiency of LNP-3P after filtration and centrifugation was measured. The encapsulation efficiency of the liposomal preparation encapsulating the tolerogen vaccine after filtration was approximately 87.02%. After further centrifugation, the encapsulation efficiency of this liposome was approximately 89.55%. After centrifugation, there was no significant difference in the encapsulation efficiency of this liposome, as shown in Table 1 and Table 2 below.

[0096] Table 1

[0097] Before demulsification After demulsification 0.762 0.634

[0098] Table 2

[0099] Supernatant Precipitate without demulsification Demulsified precipitate 0.725 0.753 0.681

[0100] 4. Photographing of low-voltage transmission electron micrograph of LNP-3P

[0101] The prepared LNP-3P was dropped on a copper mesh, and a staining agent such as phosphotungstic acid or uranyl acetate was quickly dropped. After drying, it was photographed using a low-voltage transmission electron microscope. Refer to Figure 7 .

[0102] 5. Therapeutic effect of LNP-3P in the RR-EAE model. Refer to Figures 8 - 11 .

[0103] 6. Therapeutic effect of 3P (polypeptide composition not encapsulated by liposome, including sequences 1, 2, and 3) and LNP-3P in the classical EAE model. Refer to Figures 12 - 16 .

[0104] Experimental Example 2

[0105] I. Establishment of the EAE model of mice with multiple sclerosis (EAE) and the relapsing-remitting EAE model and the administration method of the polypeptide drug

[0106] 1. Female C57BL / 6 mice (8 weeks old) were immunized subcutaneously with 200 μl of an emulsion containing complete Freund's adjuvant (CFA, Sigma-Aldrich), MOG 35-55Peptide (300 μg) and Mycobacterium tuberculosis extract H37Ra (Difco) (400 μg). Mice were injected with 300 ng of pertussis toxin (PTX) (List Biological Laboratories) at 4 hours and 24 hours after immunization, respectively.

[0107] 2. Relapsing-remitting model: SJL / C57BL / 6 hybrid F1 mice (8 - 10 weeks old) were subcutaneously immunized with 200 μL of an emulsion containing complete Freund's adjuvant (CFA, Sigma - Aldrich), PLP 139-151 peptide (40 μg), MOG 35-55 peptide (200 μg) and Mycobacterium tuberculosis extract H37Ra (Difco) (400 μg). Mice were injected with 100 ng of pertussis toxin (PTX) (List Biological Laboratories) at 4 hours and 24 hours after immunization, respectively.

[0108] II. Administration methods of polypeptide vaccines

[0109] 1. On the peak day of disease onset after C57BL / 6 mice were modeled, the tolerogen vaccine tripeptide composition (3P) or liposome - encapsulated tripeptide composition (LNP - 3P), both containing 50 μg of each polypeptide of sequences 1, 2, and 3, or the tetrapeptide composition (4P), containing 50 μg of each polypeptide of sequences 1, 2, 3, and 4, were subcutaneously injected. The drug was administered every other day for a total of three times. The evaluation of the treatment effect is shown in Figure 17 .

[0110] 2. On the peak day of disease onset after SJL / C57BL / 6 hybrid F1 mice were modeled, the tolerogen vaccine tripeptide composition (3P) or liposome - encapsulated tripeptide composition (LNP - 3P), both containing 50 μg of each polypeptide of sequences 1, 2, and 3, were subcutaneously injected. The drug was administered every other day for a total of four times. The evaluation of the treatment effect is shown in Figure 18 .

[0111] III. Evaluation of the spinal cord status of mice after treatment

[0112] 1. After treatment, spinal cords of C57BL / 6 mice were used to prepare single - cell suspensions, and the proportions of cells in each infiltrating subgroup in the spinal cord were analyzed by flow cytometry. The results are shown in Figure 19 .

[0113] 2. After treatment, spinal cords of C57BL / 6 mice were fixed with paraformaldehyde, stained with HE and LFB, and the inflammatory infiltration and demyelination in the spinal cord were observed. Immunofluorescence was used to detect CD4 + T cell infiltration. The results are shown in Figure 20 , Figure 21 , Figure 22 .

[0114] III. Antigen recall experiment

[0115] 1. Spleen cells were collected from the F1 mice after treatment. The spleen cells were stimulated with the tolerogen vaccine tripeptide combination in vitro for 3 days. The cell culture supernatants at 48 hours and 72 hours were collected, and the secretion of IL-10 was detected by ELISA. The experimental data are shown in Figure 23 .

[0116] 2. Spleen cells were collected from the C57 mice after treatment. The spleen cells were stimulated with the polypeptide mixture (containing MOG, PLP, MBP) in vitro for 3 days. The proportion of treg was detected by flow cytometry of the spleen cells. The experimental data are shown in Figure 24 .

[0117] The multi-antigen tolerogen vaccine based on the liposome delivery system of the present invention has the following innovative advantages compared with the traditional immunosuppressive therapy: First, we first integrated the major T cell epitopes of three myelin antigens (MBP, PLP, MOG) of MS into a single vaccine system; Second, the liposome delivery technology was innovatively adopted to achieve efficient delivery of antigens and precise immune regulation.

[0118] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages and disadvantages of the embodiments.

[0119] The embodiments of the present invention have been described above with reference to the drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are only illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the present invention and the claims. All of these are within the protection scope of the present invention.

Claims

1. An tolerogen vaccine for the treatment of multiple sclerosis, characterized in that, The tolerogen vaccine comprises an antigenic peptide fragment composition of myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein.

2. The tolerogen vaccine for treating multiple sclerosis according to claim 1, characterized in that: The antigenic peptide fragment composition comprises any 3 or 4 of SEQ ID NO: 1-4.

3. The tolerogen vaccine for treating multiple sclerosis according to claim 2, wherein: Each antigenic peptide fragment in the antigenic peptide fragment composition is composed in equal proportion.

4. The tolerogen vaccine for treating multiple sclerosis according to claim 1, characterized in that: The tolerogen vaccine is encapsulated in liposomes, and the liposomes used are anionic liposomes, which comprise anionic phospholipids and cholesterol.

5. The preparation method of a tolerogen vaccine for the treatment of multiple sclerosis according to any one of claims 1-4, characterized in that: It includes the following steps: S1. Prepare a polypeptide mixture solution and an anionic liposome solution; S2. Use the injection method to prepare a polypeptide mixture liposome preparation from the polypeptide mixture solution and the anionic liposome solution; S3. Dilute, filter, and purify the polypeptide mixture liposome preparation to obtain the tolerogen vaccine encapsulated in liposomes.

6. The preparation method of a tolerogen vaccine for the treatment of multiple sclerosis according to claim 5, characterized in that: The polypeptide mixture solution comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 in a mass ratio of 1:1:1, or SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 in a mass ratio of 1:1:1:1, and the final concentration of the polypeptide mixture solution is 2 mg / mL.

7. The preparation method of a tolerogen vaccine for the treatment of multiple sclerosis according to claim 5, characterized in that: The anionic liposome solution comprises cholesterol, DPPG, DSPC, and absolute ethanol, and the final concentration of the anionic liposome solution is 10 mg / mL.

8. The preparation method of a tolerogen vaccine for the treatment of multiple sclerosis according to claim 5, characterized in that: The method for preparing the polypeptide mixture liposome preparation includes: while stirring the polypeptide mixture solution at a rotation speed of 700-800 rpm / min, adding the anionic liposome solution. After the addition of the anionic liposome solution is completed, continue to stir at a rotation speed of 1200-1800 rpm / min, and maintain heating at 35-40 °C. After 5-7 h, stop heating and stirring, and ultrasonicate it for 8-12 min for homogenization.

9. The preparation method of a tolerogen vaccine for the treatment of multiple sclerosis according to claim 5, characterized in that: In S3, the polypeptide mixture liposome preparation is diluted 4-6 times with pure water, then filtered through a 0.4-0.5 μm PVDF membrane, and finally centrifuged at 10000-14000 rpm / min for 10-20 minutes for purification.

10. Use of the tolerogen vaccine for the treatment of multiple sclerosis according to any one of claims 1-4 or the preparation method of the tolerogen vaccine for the treatment of multiple sclerosis according to any one of claims 5-9 in the preparation of a drug for the treatment or prevention of multiple sclerosis.

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