Tolerant peptide vaccine for treating multiple sclerosis and preparation method thereof

Abstract

The invention belongs to the technical field of peptide vaccines, and discloses a tolerant peptide vaccine for treating multiple sclerosis and a preparation method thereof. The tolerance vaccine is prepared from anionic liposome Lip, myelin sheath oligodendroglia glycoprotein MOG and artesunate ART, and the mass ratio of the Lip to the ART to the MOG is (50 to 150) to (5 to 15) to (1 to 10). The particle size of the tolerance vaccine is 100-200nm, the Zeta potential is-10 to-30mV, the form of the tolerance vaccine is sphere-like, the encapsulation efficiency is 70-90%, and the MOG drug loading capacity is 2-10%. The peptide vaccine constructed by the invention can be administered in a subcutaneous / vein / muscle way, and can target a central nervous system, induce DCs cell tolerance, block a complement signal channel, inhibit the phenotype of microglial cells M1, inhibit the damage of proinflammatory factors to neurons together with MOG, and enhance the treatment effect of multiple sclerosis.

Description

Technical Field

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

[0002] Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) caused by the imbalance of CD4 + T cell subsets and the overactivation of complement, and at least 2.5 million people worldwide are affected by MS. Tolerogenic immunotherapy, as a new immune therapy strategy, brings hope for autoimmune diseases, and can utilize the characteristics and functions of immune cells to down-regulate the autoreactive immune response that damages tissues and organs. Polypeptide vaccines can induce antigen-presenting cells (APCs) to express low levels of co-stimulatory molecules by introducing MS autoantigens or related antigens into the body, and further convert naive T cells into regulatory T cells (Tregs) and anergic T cells, so as to achieve the purpose of alleviating multiple sclerosis. Compared with traditional therapies, polypeptide vaccine therapy has more advantages in terms of effectiveness and safety. It should be noted that the polypeptide vaccine strategy also faces some challenges. Recent studies have found that it is difficult for self-antigen peptides alone to cross the blood-brain barrier and are easily degraded by in vivo biological enzymes. It is necessary to seek a suitable delivery carrier to avoid the above defects and induce stronger immune tolerance. The use of nanocarrier technology can effectively solve the problem of delivering polypeptide vaccines.

[0003] There is a great correlation between the onset of multiple sclerosis and the overactivation of complement. Overactivated complement mediates the phagocytosis of myelin by microglia, damaging neurons. In addition, microglia are overly polarized towards the M1 type, producing pro-inflammatory factors such as IFN-γ and TNF-α, which exacerbate neuronal damage. Therefore, inhibiting the overactivation of complement and effectively inducing the polarization of microglia from the M1 type to the M2 type can be used as a therapeutic target for MS.

[0004] Liposomes have a biomimetic membrane structure and can encapsulate water-soluble and lipophilic drugs. They have high selectivity, no toxicity, no immunogenicity, and are suitable for biodegradation in vivo. The anionic liposome delivery system has the ability to target inflammatory sites. Adding distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) can enhance the ability to cross the blood-brain barrier and prolong the in vivo circulation. Artesunate (ART) can inhibit complement activation, thereby inhibiting the complement-mediated phagocytosis of myelin by microglia; and polarize multiple sclerosis-related microglia from pro-inflammatory M1-type microglia to anti-inflammatory M2-type microglia. At the same time, in combination with the autoantigen peptide MOG, it can regulate the CD4 + T cell subsets and play a better role in treating multiple sclerosis.

[0005] In summary, how to construct a peptide vaccine for treating multiple sclerosis is a technical problem that those skilled in the art urgently need to solve. Summary of the Invention

[0006] In view of this, the present invention provides a combined polypeptide and chemical drug composite liposome tolerance peptide vaccine for treating multiple sclerosis, a preparation method thereof, and an application thereof. The vaccine can achieve the co-loading of MOG and ART, and synergistically play a role in treating multiple sclerosis. Among them, ART can inhibit the over-activation of complement and reprogram microglia to the M2 state; the MOG polypeptide can induce tolerogenic dendritic cells, regulate T cell subsets to promote immune tolerance, and synergistically with ART reduce the pro-inflammatory factors IFN-γ and TGF-β, enhancing the therapeutic effect of multiple sclerosis.

[0007] One of the objectives of the present invention is to provide a tolerance peptide vaccine for treating multiple sclerosis, and the composition of the tolerance peptide vaccine includes: anionic liposome Lip, myelin oligodendrocyte glycoprotein MOG, and artesunate ART;

[0008] Among them, the mass ratio of Lip, ART, and MOG is 50-150:5-15:1-10.

[0009] The beneficial effect of the above technical solution is: the above mass ratio is the raw material ratio during preparation. The weighed materials are dissolved in an organic solvent, and the solution is precisely aspirated during preparation, and the loss of raw materials can be ignored. The ART self-assembles and is encapsulated in the lipid bilayer, and the MOG is adsorbed in the hydrophilic core of the liposome.

[0010] The technical concept of the present invention is: Lip is used as a vaccine carrier, which can achieve the co-loading of the immunomodulator artesunate ART and the multiple sclerosis autoantigen MOG polypeptide, protect the myelin oligodendrocyte protein peptide from protease degradation, and synergistically play a role in treating multiple sclerosis. Among them, MOG can induce tolerogenic dendritic cells to inhibit the immune effect of T cells; ART can induce microglia to polarize into the M2 anti-inflammatory subtype by inhibiting complement activation, synergistically enhance the ability to inhibit demyelination, and enhance the therapeutic effect of multiple sclerosis.

[0011] Furthermore, the mass ratio of Lip, ART, and MOG is 100:10:2.

[0012] Preferably, the encapsulation efficiency of both MOG and ART is 70-90%, the drug loading of MOG is 1-10%, and the drug loading of ART is 5-15%;

[0013] The particle size of the tolerance peptide vaccine is 100-200 nm, and the Zeta potential is -10 MV to -30 MV.

[0014] Preferably, the MOG is an autoantigen of multiple sclerosis, containing antigenic determinants for recognition by T cell antigen receptors in the immune response, with a molecular weight of 2000-3000 g / mol.

[0015] The beneficial effects of the above technical solution are as follows: MOG contains antigenic determinants for recognition by T cell antigen receptors in the immune response. After being presented by tolerogenic dendritic cells lacking the second signal for T cell activation, it can be recognized by specific T cells expressing myelin oligodendrocyte glycoprotein, causing the activated T cells to enter a hyporesponsive or functionally inactivated state and ultimately die, thereby treating multiple sclerosis.

[0016] Preferably, the MOG is selected from 35-55 MOG 89-113 MOG 37-52 and MOGG 30-154 one or more of them.

[0017] Furthermore, the amino acid sequences are respectively:

[0018] The MOG 35-55 : MEVGWYRPPFSRVVHLYRNGK

[0019] MOG 89-113 : RFSDEGGFTCFFRDHSYQEEAAMEL

[0020] MOG 37-52 : EEEEEMAVVPQGLFRG-NH2

[0021] MOG G30-154 :

[0022] GQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVGWYRPPFS

[0023] RVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVR

[0024] FSDEGGFTCFFRDHSYQEEAAMELKVEDPFYWVSPG

[0025] Furthermore, the MOG is preferably the MOG 35-55 fragment, with a molecular weight of 2581.9, highly expressed in multiple sclerosis tissues, and the induced autoimmune experimental autoimmune encephalomyelitis is extremely similar to human multiple sclerosis.

[0026] Preferably, the Lip is selected from one or more of dioleoyl phosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), dioleoyl L-α-phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and cholesterol (CHOL).

[0027] More preferably, the Lip consists of dioleoyl phosphatidylglycerol (DOPG), dioleoyl L-α-phosphatidylethanolamine (DOPE), and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000).

[0028] Preferably, the molar ratio of DOPG, DOPE, and DSPE-PEG2000 is 5 - 15:1 - 10:0.1 - 1.

[0029] More preferably, the molar ratio of DOPG, DOPE, and DSPE-PEG2000 is 12:1:0.2.

[0030] The second object of the present invention is to provide a method for preparing a tolerogenic peptide vaccine for treating multiple sclerosis, which can adopt one or more of reverse phase evaporation method, thin film dispersion method, freeze-thaw method, and ethanol / ether injection method.

[0031] During the preparation process, taking lipidosome characteristics such as particle size, encapsulation efficiency, drug loading, and Zeta potential as the investigation indexes, the main influencing factors, including rotary evaporation temperature, rotation speed, type and pH of the hydration medium, hydration temperature, hydration method, and other conditions, are screened one by one and optimized to obtain the polypeptide vaccine with the best performance.

[0032] Preferably, the thin film dispersion method is adopted, which includes the following steps:

[0033] (1) Weigh ART and Lip precisely, dissolve them in an organic solvent, vortex and mix well, then rotate and evaporate at 25 - 55 °C to form a lipidosome film, and fill with nitrogen to remove the organic solvent for standby.

[0034] The organic solvent is one or more of chloroform, methanol, ethanol, and acetone.

[0035] (2) Weigh MOG precisely, dissolve it in the hydration medium for standby.

[0036] The hydration medium is one or more of phosphate buffer solution, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution, and tris(hydroxymethyl)aminomethane buffer solution.

[0037] (3) Add the hydration medium containing MOG into the round-bottom flask with the formed liposome film, hydrate it in a water bath environment at 25-55 °C for 30-60 min, and extrude it with a liposome extruder to obtain the tolerogenic peptide vaccine co-loaded with MOG and ART.

[0038] Further, in step (1), the organic solvent is a mixture of chloroform and methanol, and the volume ratio thereof is 9:1 to 1:1.

[0039] More preferably, the volume ratio of chloroform to methanol is 1:1, and the film-forming temperature is 40 °C.

[0040] Preferably, the mass of MOG contained in 1 mL of the hydration medium is 1 mg.

[0041] Further, in step (2), the hydration medium is phosphate buffer solution with a pH value of 5-8.

[0042] The third object of the present invention is to provide an application of a tolerogenic peptide vaccine for treating multiple sclerosis. The administration route of the tolerogenic peptide vaccine is any one of subcutaneous injection, intravenous injection, and intramuscular injection, and immunotherapy is performed 5 times, with an interval of 1-3 days between each immunotherapy.

[0043] Preferably, it is intravenous injection.

[0044] The tolerogenic peptide vaccine can effectively induce tolerogenic dendritic cells, inhibit inflammatory infiltration in the central nervous system, and regulate CD4 + T cell subsets, and respond to pro-inflammatory cells as early as possible.

[0045] The tolerogenic peptide vaccine can induce an immune tolerance response. During the treatment of multiple sclerosis, with the synergistic effect of dendritic cells, microglia, and astrocytes, it promotes immune tolerance combined with complement inhibition for the synergistic treatment of multiple sclerosis.

[0046] It can be seen from the above technical solutions that compared with the prior art, the beneficial effects obtained by the present invention are as follows:

[0047] (1) The polypeptide vaccine has low immunogenicity, does not have the risks of gene insertion and anti-rejection reaction like DNA vaccines or cell vaccines, has higher safety, and is more widely used; polypeptide synthesis is rapid, with high purity, simplicity, and low cost. The present invention utilizes the advantages of high biocompatibility, biodegradability, and low toxicity of anionic liposomes, and their good inflammatory targeting and potential to cross the blood-brain barrier in the delivery of drugs / vaccines, constructs an anionic liposome delivery carrier for co-loading MOG and ART to achieve the immunotherapy of multiple sclerosis.

[0048] (2) In the present invention, artesunate ART, an immunomodulator, is inserted into the phospholipid layer of anionic liposomes, and myelin oligodendrocyte glycoprotein MOG, an autoantigen peptide, is inserted into the hydrophilic core of anionic liposomes to prepare a polypeptide vaccine for treating multiple sclerosis (for the structural schematic diagram, see Figure 1 A). By high performance liquid chromatography (HPLC) and BCA protein assay, the encapsulation efficiencies of artesunate ART and the autoantigen MOG polypeptide are detected to be 80.25% and 80.51% respectively, and the drug loadings are 8.51% and 1.61. In addition, transmission electron microscopy and Malvern particle size analyzer are used to detect the morphology, particle size, zeta potential and short-term storage stability of the polypeptide vaccine (see Figure 1 ).

[0049] (3) The results of in vitro dendritic cell stimulation tolerance experiment of the polypeptide vaccine for treating multiple sclerosis prepared in the present invention show that the vaccine can significantly inhibit the expression levels of co-stimulatory molecules CD80, CD86 and MHC-II on the surface of dendritic cells, indicating that it can effectively induce the tolerance of immature dendritic cells in mouse bone marrow (see Figure 2 ); the results of in vitro microglia induction polarization experiment of the polypeptide vaccine show that the vaccine can induce the polarization of M1 microglia into M2 microglia in microglia, endowing an anti-inflammatory effect, and further indicating that the polypeptide vaccine for treating multiple sclerosis in the present invention is a good therapeutic drug for multiple sclerosis (see Figure 3 ).

[0050] The mice with multiple sclerosis are treated by intravenous administration, and the body weight changes and paralysis degree of the mice are monitored within 24 days. It can be significantly found that the polypeptide vaccine has a significant inhibitory effect on mouse paralysis, and through HE staining and LFB staining of spinal cord tissue, it can be known that the polypeptide vaccine can significantly inhibit the inflammatory infiltration of the spinal cord and reduce the demyelination condition (see Figure 4 ). Meanwhile, through flow cytometry analysis, the proportion of CD4 + T subsets in the central nervous system is analyzed. The results show that the addition of the polypeptide vaccine of the present invention significantly increases the proportion of anti-inflammatory Tregs, inhibits the pro-inflammatory Th1 and Th17 subsets, and induces immune tolerance in the body (see Figure 5 ). More importantly, through immunofluorescence experiment, it can be known that the polypeptide vaccine constructed in the present invention can significantly inhibit the activation of microglia and the levels of complement C3 / C3aR, and at the same time inhibit the secretion of inflammatory factors by M1 microglia, regulate T cell subsets together with MOG, assist the immune response, and enhance the therapeutic effect of multiple sclerosis. These effective results may be attributed to the ability of the anionic nanocarrier to target the inflammatory site (see Figure 4 B).

[0051] (4) CD4 +The imbalance of T cell subsets and the overactivation of complement are considered the main pathogenesis of MS. MOG is a myelin oligodendrocyte glycoprotein peptide that contains antigenic determinants recognized by T cell antigen receptors in the immune response. After being presented by dendritic cells with low expression of the second signal for T cell activation, it can be recognized by specific T cells expressing myelin oligodendroprotein, resulting in the activated T cells entering a low-response or inactivated state and eventually dying. ART is an immunomodulator with anti-inflammatory and complement activation inhibitory effects. It can induce the transformation of microglia into the M2 phenotype, especially inhibiting the role of the complement C3 / C3aR pathway in interfering with microglial phagocytosis of myelin. It synergistically inhibits the secretion of inflammatory factors with MOG, reduces the damage of inflammatory factors and microglia to neurons, and jointly treats MS. After the tolerance peptide vaccine prepared in the present invention is administered by intravenous injection, compared with liposomes loaded with MOG or ART alone, the liposomes co-loaded with MOG and ART have stronger targeting ability, can significantly reduce the clinical score of mice with experimental autoimmune encephalomyelitis induced by myelin protein and increase the body weight of mice, and enhance the therapeutic effect of MS. Brief Description of the Drawings

[0052] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0053] Figure 1 It is a schematic structural diagram and related characteristic characterization diagram of a tolerance peptide vaccine for treating multiple sclerosis of the present invention.

[0054] Among them, A is a schematic diagram for preparing a polypeptide vaccine, B is a transmission electron micrograph of the vaccine prepared in Example 3, 1) MOG-Lip is a liposome loaded with MOG, 2) ART-Lip is a liposome loaded with ART, 3) MOG-ART-Lip is a liposome co-loaded with MOG and ART, C is a particle size diagram of the vaccine prepared in Example 2 (Lip is a blank anionic liposome), D is a potential diagram of the vaccine prepared in Example 2, and E is a short-term stability diagram of the vaccine prepared in Example 2.

[0055] Figure 2 It is a flow cytometry measurement result diagram of the tolerance peptide vaccine of the present invention inducing tolerance dendritic cells;

[0056] Figure 3 It is a flow cytometry measurement result diagram of the tolerance peptide vaccine of the present invention inducing the phenotypic transformation of microglia;

[0057] Figure 4This is the evaluation diagram of the therapeutic effect of the tolerogenic peptide vaccine of the present invention on MS in EAE mice. Among them, A is the schematic diagram of modeling and drug administration, B is the in vivo distribution diagram, C is the ex vivo in vivo distribution diagram, D is the morphological difference diagram of mice, E is the body weight change diagram, F is the clinical score diagram, and G is the spinal cord hematoxylin-eosin (HE) staining and luxol fast blue (LFB) staining;

[0058] Figure 5 This is the flow cytometry measurement diagram of CD4 + T cell subsets in the central nervous system of different formulation groups after drug administration to animals of the present invention. Among them, A is the Th1 subset (IFN-γ), Th2 subset (IL-4), and Th17 subset (IL-17A), and B is its semi-quantification diagram. C is the Treg subset (FOXP3), and D is its semi-quantification diagram.

[0059] Figure 6 This is the immunofluorescence measurement diagram of microglial activation and polarization, and the C3 / C3aR pathway in different formulation groups after drug administration to animals of the present invention. Among them, A is the activation of microglia (Iba1), B is the activated numbers of M1 (CD86) and M2 (CD206), and C is the inhibition of the C3 / C3aR pathway.

[0060] Figure 7 This is the ELISA measurement result diagram of the secretion of inflammatory factors in different formulation groups after drug administration to animals of the present invention (where A is IFN-γ and B is TNF-α). Detailed implementation mode

[0061] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0062] Example 1

[0063] Prepare MOG-ART-Lip liposomal tolerogenic peptide vaccine by the thin film hydration method.

[0064] (1) Weigh accurately the prescription amounts of Lip (prepared from DOPG, DOPE, and DSPE-PEG2000) and ART, where the mass ratio of Lip:ART is 100:10;

[0065] The molar ratio of DOPG:DOPE:DSPE-PEG2000 is 12:1:0.2; dissolve in a mixed solution of methanol:chloroform = 1:1, vortex and mix well, and then at 55 °C, rotate and evaporate to form a liposomal film, and fill with nitrogen to remove methanol and chloroform;

[0066] (2) Weigh accurately the prescribed amount of MOG 35-55 (the mass ratio of Lip to MOG is 100:2), dissolve it in phosphate buffer (PH = 7.4), and the concentration of MOG 35-55 is 1 mg / mL;

[0067] (3) Add the phosphate buffer containing MOG 35-55 to the round-bottom flask with the formed liposome film, hydrate it in a water bath at 37 °C for 30 min, and extrude it with a liposome extruder to obtain the MOG-ART-Lip liposome tolerogenic peptide vaccine.

[0068] Example 2

[0069] Preparation of MOG-ART-Lip liposome tolerogenic peptide vaccine by thin film hydration method

[0070] (1) Weigh accurately the prescribed amount of Lip (prepared from DOPG, DOPE, and DSPE-PEG2000) and ART, where the mass ratio of Lip to ART is 100:10;

[0071] The molar ratio of D0PG:DOPE:DSPE-PEG2000 is 12:1:0.2. Dissolve it in an organic solvent of methanol:chloroform = 1:1, vortex and mix well, and then rotate and evaporate at 40 °C to form a liposome film, and fill it with nitrogen to remove the organic solvent;

[0072] (2) Weigh accurately the prescribed amount of MOG 35-55 (the mass ratio of Lip to MOG is 100:2), dissolve it in phosphate buffer (PH = 7.4), and the concentration of MOG 35-55 is 1 mg / mL;

[0073] (3) Add the phosphate buffer containing MOG to the round-bottom flask with the formed liposome film, hydrate it in a water bath at 40 °C for 60 min, and extrude it with a liposome extruder to obtain the MOG-ART-Lip liposome tolerogenic peptide vaccine.

[0074] Example 3

[0075] Preparation of MOG-ART-Lip liposome tolerogenic peptide vaccine by thin film hydration method

[0076] (1) Weigh accurately the prescribed amount of Lip (prepared from DOPG, DOPE, and DEPE-PEG2000) and ART, where the mass ratio of Lip to ART is 100:10;

[0077] The molar ratio of DOPG:DOPE:DEPE-PEG2000 was 12:1:0.2, which was dissolved in a mixed organic solvent of methanol:chloroform = 1:1. After vortex mixing, a liposome film was formed by rotary evaporation at 40 °C, and nitrogen was filled to remove the organic solvent.

[0078] (2) Weigh accurately the prescription amount of MOG 35-55 (the mass ratio of Lip:MOG was 100:2), dissolve it in phosphate buffer (PH = 7.4), and the concentration of MOG 35-55 was 1 mg / mL.

[0079] (3) Add the phosphate buffer containing MOG to the round-bottom flask with the formed liposome film, hydrate it in a water bath environment at 40 °C for 30 min, and extrude it with a liposome extruder, namely the MOG-ART-Lip liposome tolerance peptide vaccine.

[0080] The morphology of the vaccine was observed by transmission electron microscopy, and the particle size, polydispersity index, zeta potential, and short-term stability were measured by a Malvern laser particle size analyzer.

[0081] The preparations of Examples 1-3 were screened, and the results are shown in Table 1, and the transmission electron micrographs are as Figure 1 shown in the accompanying drawings.

[0082] Table 1 Particle size, PDI, zeta potential, EE, and DL of MOG 35-55 -ART-Lip

[0083]

[0084] It can be seen from Table 1 that the preparation of Example 3 has a higher encapsulation efficiency, appropriate particle size and zeta potential, which is helpful for stability, and the comprehensive data is the best. The results screened by this example were used for in vitro and in vivo experiments.

[0085] Experiment 1

[0086] MOG 35-55 -ART-Lip liposome vaccine induces tolerogenic dendritic cells

[0087] To study the induction of tolerogenic dendritic cells by the MOG 35-55 -ART-Lip liposome vaccine, 7 groups of control experiments were set up, and PBS, LPS, Lip+LPS, MOG 35-55 -ART+LPS, MOG 35-55 -Lip+LPS, ART-Lip+LPS, and MOG-ART-Lip+LPS were added to each well of dendritic cells respectively, and incubated in 5% CO 2, After culturing in a 37°C cell incubator for 24 h, the cells were collected, labeled with the flow antibodies CD80, CD86, and MHC-II, and the expression levels of the surface molecules CD80, CD86, and MHC-II were measured using a flow cytometer.

[0088] The results are as Figure 2 shown in the attached figure: The results showed that the MOG-ART-Lip liposome vaccine could significantly downregulate the expression levels of the co-stimulatory molecules CD80, CD86, and MHC-II on the surface of LPS-stimulated dendritic cells, indicating that the MOG-ART-Lip liposome vaccine could induce tolerogenic dendritic cells.

[0089] Experiment 2

[0090] The MOG-ART-Lip liposome vaccine regulates the ability of microglia to polarize into the M2 phenotype.

[0091] To study the regulation of MOG 35-55 -ART-Lip liposome vaccine on the polarization of microglia into the M2 phenotype, BV2 cells with a cell density of over 80% were collected and seeded in 24-well plates (adjusted to a concentration of 1×10 5 cells / mL). After 24 h, different formulation groups were added to each well, namely MOG-ART + LPS, MOG-Lip + LPS, ART-Lip + LPS group, MOG-ART-Lip + LPS, and LPS. They were cultured in a 5% CO 2 2, 37°C cell incubator for 24 h. Then, the cells in each well were collected. After adding the APC-CD11b flow antibody, the flow antibodies 1PE-CD86 and PE-CD206 were added respectively, and the expression of the surface factors CD11c / CD86 and CD11c / 206 double positivity of BV2 was measured using a flow cytometer.

[0092] The results are as Figure 3 shown in the attached figure. The MOG-ART-Lip liposome significantly polarized M1-type BV2 cells into M2-type BV2 cells, which may be due to the role of ART in promoting the polarization of microglia.

[0093] Experiment 3

[0094] Study on the effect of the MOG-ART-Lip liposome vaccine in treating MS

[0095] A multiple sclerosis model induced by polypeptide antigen was established, and the successfully modeled mice were randomly divided into 7 groups: Free-DIR group, Lip-DIR group, PBS group, free MOG-ART group, MOG-Lip group, ART-Lip group, and MOG-ART-Lip group, as well as a blank control group without any treatment, with 6 mice in each group. The Free-DIR group and Lip-DIR group were injected with 100 μL via the tail vein on the 12th day, and small animal in vivo imaging was used to observe the biodistribution. The other groups were injected with 100 μL via the tail vein of each mouse on the 12th, 14th, 16th, 18th, and 20th days of the induction of the MS mouse model (EAE), for a total of 5 injections. During the experiment, the body weight of the mice was measured every other day, and the disease clinical score was performed according to the degree of paralysis of the mice.

[0096] The results are as Figure 4 shown in the attached figure. The results show that the Lip-DIR group can significantly enhance the drug distribution in the brain and spinal cord compared with the Free-DIR group, indicating that Lip may have the ability to target the lesion sites of the central nervous system. The MOG-ART-Lip liposome vaccine can enhance the body weight of EAE mice and reduce the clinical score, indicating that the MOG-ART-Lip liposome vaccine can enhance the therapeutic effect of MS.

[0097] Experiment 4

[0098] The MOG-ART-Lip liposome vaccine regulates CD4 + T cell subsets

[0099] After taking the central nervous system (CNS) tissue of the mice after the treatment, monocytes were isolated from the CNS, and Th1 cells were labeled with the flow antibody CD3CD4IFN-γ, Th17 cells were labeled with CD3CD4IL-17A, and Treg cells were labeled with CD3CD4FOXP3. Then, the proportions of Th1 cells, Th17, and Treg cells were measured by flow cytometry.

[0100] The results are as Figure 5 shown in the attached figure. The MOG-ART-Lip liposome vaccine can reduce the proportions of pro-inflammatory Th1 and Th17 cells and increase the proportion of anti-inflammatory Treg cells, indicating that the MOG-ART-Lip liposome vaccine can treat MS by regulating the proportion of CD4 + T cell subsets.

[0101] Experiment 5

[0102] The MOG-ART-Lip liposome vaccine regulates microglia

[0103] After the treatment, the spinal cord tissues of mice were taken, and monocytes were isolated. Microglia were labeled with the immunofluorescent antibody Iba1, M1 microglia were labeled with Iba1 / CD86, M2 microglia were labeled with Iba1 / CD206, astrocytes were labeled with GFAP, complement C3 protein was labeled with C3, and complement C3 receptor was labeled with C3aR. The activation and polarization of microglia were measured by immunofluorescence, and the inhibition of the inflammatory complement C3 / C3aR pathway was also measured.

[0104] The results are as Figure 6 shown in the attached figures. The MOG-ART-Lip liposome vaccine can significantly inhibit the activation of microglia and has a tendency to increase the polarization of the M2 subtype. In addition, the MOG-ART-Lip liposome vaccine can inhibit abnormally activated microglia through the C3 / C3aR pathway, thereby treating MS.

[0105] Experiment 6

[0106] The MOG-ART-Lip liposome vaccine inhibits the secretion of pro-inflammatory factors

[0107] After the treatment, the peripheral blood of mice was taken and centrifuged. The supernatant was taken to measure the expression levels of IFN-γ and TNF-α according to the steps of the ELISA kit instructions.

[0108] The results are as Figure 7 shown in the attached figures. The results show that the MOG-ART-Lip liposome vaccine can down-regulate the secretion of the pro-inflammatory factors IFN-γ and TNF-α, indicating that the MOG-ART-Lip liposome vaccine can enhance the therapeutic effect of MS by inhibiting the secretion of pro-inflammatory factors.

[0109] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A tolerogenic peptide vaccine for treating multiple sclerosis, characterized in that: The tolerance vaccine comprises: anionic liposome Lip, myelin oligodendrocyte glycoprotein MOG and artesunate ART; The mass ratio of Lip, ART and MOG is 50-150:5-15:1-10.

2. A tolerogenic peptide vaccine for treating multiple sclerosis according to claim 1, characterized in that: The encapsulation rates of MOG and ART are both 70-90%, the drug loading of MOG is 1-10%, and the drug loading of ART is 5-15%; The particle size of the tolerant peptide vaccine is 100-200 nm, and the Zeta potential is -10 MV to -30 MV.

3. A tolerogenic peptide vaccine for treating multiple sclerosis according to claim 1, characterized in that: The MOG contains an antigenic determinant recognized by a T cell antigen receptor in an immune response, and has a molecular weight of 2000 to 3000 g / mol.

4. A tolerogenic peptide vaccine for treating multiple sclerosis according to claim 3, characterized in that: The MOG is selected from MOG 35-55 、MOG 89-113 、MOG 37-52 and MOGG 30-154 One or more of .

5. A tolerogenic peptide vaccine for treating multiple sclerosis according to claim 1, characterized in that: The Lip is selected from one or more of dioleoylphosphatidylglycerol DOPG, 1,2-dioleoyl-sn-glycero-3-phosphocholine DOPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine DLPC, dioleoyl L-α-phosphatidylethanolamine DOPE, distearoylphosphatidylethanolamine-polyethylene glycol 2000 DSPE-PEG2000, and cholesterol CHOL.

6. A tolerogenic peptide vaccine for treating multiple sclerosis according to claim 5, characterized in that: The Lip is composed of dioleoylphosphatidylglycerol DOPG, dioleoyl L-α-phosphatidylethanolamine DOPE, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 DSPE-PEG2000.

7. A tolerogenic peptide vaccine for treating multiple sclerosis according to claim 6, characterized in that: The molar ratio of DOPG, DOPE and DSPE-PEG2000 is 5-15:1-10:0.1-1.

8. A method for preparing a tolerogenic peptide vaccine for treating multiple sclerosis according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) Accurately weigh ART and Lip, dissolve them in an organic solvent, vortex mix them, and then rotary evaporate them at 25-55°C to form a liposome film. Fill them with nitrogen to remove the organic solvent and set aside; (2) Accurately weigh MOG, dissolve it in a hydration medium, and set aside; (3) Adding a hydration medium containing MOG into the liposome film, hydrating it at 25-55° C. for 30-60 min, and extruding it through a liposome extruder to obtain a tolerable peptide vaccine for treating multiple sclerosis.

9. The method for preparing a tolerogenic peptide vaccine for treating multiple sclerosis according to claim 8, characterized in that: In step (1), the organic solvent is one or more of chloroform, methanol, ethanol and acetone.

10. The method for preparing a tolerogenic peptide vaccine for treating multiple sclerosis according to claim 8, characterized in that: In step (2), the hydration medium is one or more of phosphate buffer, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer and tris(hydroxymethyl)aminomethane buffer; The mass of MOG contained in 1 mL of the hydration medium is 1 mg.

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