A manganese-based hydrogel rabies vaccine and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological preparations, and particularly relates to a manganese-based hydrogel rabies vaccine and a preparation method and application thereof. The manganese-based hydrogel rabies vaccine is based on modified chitosan hydrogel, in which inactivated rabies virus and manganese ions are added. The adjuvant and antigen are co-delivered through injection, slowly dissolve with tissue effusion infiltration, gradually release manganese ions to activate cGAS-STING, enhance the activity of immune cells and stimulate the production of germinal centers. At the same time, the released antigens are processed by immune cells or immune organs to produce antibodies to block viral infection. The manganese-based hydrogel rabies vaccine can significantly improve the duration of antibody action, prolong the antibody protection time, enhance the activity of immune cells and stimulate the production of germinal centers, and improve the antibody production level.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological agents, and particularly relates to a manganese-based hydrogel rabies vaccine and a preparation method and application thereof. BACKGROUND

[0002] Rabies is a high mortality rate of zoonosis, and the number of people who die from rabies infection worldwide can reach 60,000 per year, most of whom die in Asia and Africa. After being scratched or bitten by a host carrying the rabies virus, the virus will replicate and spread at the wound site, eventually entering the central nervous system and salivary glands from the peripheral motor neurons against the axon, causing a series of clinical symptoms such as fever, vomiting, neurological dysfunction, respiratory failure, etc. At present, there is no effective treatment for rabies, and only immediate and periodic vaccination can reduce the spread of the virus.

[0003] The multi-dose rabies vaccination program and the prophylactic strategy of immunoglobulin inoculation greatly increase the vaccination cost, thereby limiting the vaccination rate of rabies vaccine in underdeveloped countries. Therefore, researchers are committed to improving the vaccination strategy of rabies vaccine by improving the immune effect of the vaccine and optimizing the vaccination method, so as to reduce the vaccination cost and expand the vaccination coverage.

[0004] The main component of the existing rabies vaccine for human and animals on the market is inactivated rabies virus, which is mostly treated by injection. There is also a kind of oral rabies virus-like particle vaccine and its preparation method provided in the Chinese patent with the publication number CN114272367A, which uses RVLPs self-assembled from the glycoprotein RVGP and matrix protein RVMP of CVS strain rabies virus as antigens, uses L100 and PLGA as main materials to prepare oral acid-resistant slow microspheres, and uses mucosal immunoadjuvant LTB to improve the antigenicity of oral RVLPs and enhance the immune effect. By designing a protein drug carrier suitable for oral delivery to the intestinal tract, it can successfully overcome various physiological obstacles in the gastrointestinal tract and be directly delivered to the intestinal tract, and the antigen is released under the pH condition of the intestinal tract, and the immune effect induced thereby is preliminarily immunologically evaluated. However, whether it is a direct injection type or an oral type, the drug stays in the body for a short time, and the immune effect is limited, and a lasting therapeutic effect cannot be produced. SUMMARY

[0005] In view of the above-mentioned problems existing in the current rabies vaccine, the application provides a manganese-based hydrogel rabies vaccine and a preparation method and application thereof. The manganese-based hydrogel rabies vaccine is based on modified chitosan hydrogel, inactivated rabies virus and manganese ions loaded therein, and realizes the co-delivery of adjuvant and antigen through injection, which can significantly improve the duration of antibody action, enhance the activity of immune cells and stimulate the production of antibody in the germinal center. The specific technical scheme is as follows:

[0006] First, the present invention provides a method for preparing a manganese-based hydrogel rabies vaccine. The method comprises the following steps: firstly, modifying chitosan to prepare a modified chitosan solution; then, adding inactivated rabies virus to the modified chitosan solution, uniformly dispersing the virus, and then adding manganese ions to react to obtain a manganese-based hydrogel rabies vaccine; the chitosan modification step comprises dissolving chitosan acid salt in water, adding a polyphenol compound with activated carboxyl groups thereto, stirring the mixture for reaction at room temperature, and lyophilizing the reaction product after purification by dialysis to obtain the modified chitosan, wherein the polyphenol compound comprises any one of gallic acid, dihydrocaffeic acid, and protocatechuic acid.

[0007] Furthermore, the preparation method comprises the following steps:

[0008] S110 Chitosan modification: Chitosan acid salt is dissolved in water, and a polyphenol compound with activated carboxyl groups is added thereto. The reaction is stirred at room temperature. The reaction product is purified by dialysis and then freeze-dried to obtain modified chitosan;

[0009] S120 Preparation of inactivated rabies virus: Collect the supernatant of BSR cells infected with LBNSE strain, remove cell debris by centrifugation, inactivate the cells with a vaccine inactivator, and collect virus particles by centrifugation after inactivation. The collected virus particles are resuspended after centrifugation to obtain inactivated rabies virus;

[0010] Preparation of S130 manganese-based hydrogel rabies vaccine: Modified chitosan is prepared into a solution, inactivated rabies virus is added thereto, dispersed evenly under ice bath, and then manganese ions are added thereto, stirred evenly and allowed to stand to form a gel.

[0011] Furthermore, in step S110, the ratio of the amount of chitosan salt to the amount of polyphenol compound added is 1-2:1; and the stirring reaction time is 24-36 hours.

[0012] Furthermore, in the step S130, the mass fraction of the modified chitosan in the modified chitosan solution is 2% to 5%; the amount of manganese ions added is 100 μM to 250 μM; and the manganese ions are selected from any one of manganese chloride and manganese sulfate.

[0013] The present invention also provides a manganese-based hydrogel rabies vaccine, which is prepared by the above-mentioned preparation method.

[0014] Furthermore, the manganese-based hydrogel rabies vaccine includes modified chitosan, rabies virus and manganese ions, and the mass ratio of the modified chitosan, inactivated rabies virus and manganese ions is 300:1:32.

[0015] The present invention also provides an application of a manganese-based hydrogel rabies vaccine in the preparation of a rabies treatment drug.

[0016] Furthermore, the dosage form of the manganese-based hydrogel rabies vaccine is an injection.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention uses chitosan as a matrix and modifies it with polyphenolic compounds that activate carboxyl groups. The modified chitosan is enriched with phenolic hydroxyl groups, which can cross-link with manganese ions through coordination bonds to form a phenolic network hydrogel. The resulting hydrogel is then loaded with inactivated rabies virus through physical encapsulation to produce a manganese-based hydrogel rabies vaccine.

[0019] The manganese-based hydrogel rabies vaccine provided by the present invention can achieve co-delivery of manganese ions and inactivated antigens. As the hydrogel dissolves, manganese ions are gradually released. Manganese ions can activate the cGAS pathway and produce a synergistic effect with the inactivated antigen, enhancing the maturation of immune cells, promoting the production of germinal centers and antibody secretion, and sustainably producing high levels of antibodies, thereby improving the level of antibody production.

[0020] At the same time, the manganese-based hydrogel rabies vaccine provided by the present invention gradually dissolves and releases in the body, thereby controlling the release rate of antigens and manganese ions and increasing their action time in the body. Compared with direct injection of pure virus, the manganese-based hydrogel rabies vaccine provided by the present invention can produce high levels of antibodies in the body for at least 10 weeks, significantly prolonging the antibody protection time and improving the immunotherapy effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the preparation process of a manganese-based hydrogel rabies vaccine according to an embodiment of the present invention;

[0022] Figure 2 Fourier transform infrared spectra (a), ultraviolet absorption spectra (b) and fulin phenol color development images (c) of modified chitosan synthesized at different mass ratios in Experimental Example 1 of the present invention;

[0023] Figure 3 Figure 1 shows the injectability characterization of the manganese-based hydrogel rabies vaccine in Experimental Example 1 of the present invention (a) and the gel performance characterization (b).

[0024] Figure 4 This is a SEM morphology image of a manganese-based hydrogel rabies vaccine in an embodiment of the present invention;

[0025] Figure 5 This is a statistical graph of CD80 / CD86 expression levels in the DC2.4 cell activation evaluation experiment in Experimental Example 1 of the present invention;

[0026] Figure 6 This is a statistical graph of MHC-Ⅱ expression in the DC2.4 cell activation evaluation experiment in Experimental Example 1 of the present invention;

[0027] Figure 7 This is a Western Blot image of the cGAS-STING pathway activation experiment in Experimental Example 1 of the present invention;

[0028] Figure 8 This is a fluorescence retention imaging image of a mouse in vivo in the in vivo fluorescence imaging experiment of Experimental Example 2 of the present invention;

[0029] Figure 9 The rabies-specific neutralizing antibody titer (left) and its statistical graph (right) in the antibody detection experiment 1 of Experimental Example 2 of the present invention;

[0030] Figure 10 The titer of rabies-specific neutralizing antibodies in the antibody detection experiment 2 of Experimental Example 2 of the present invention;

[0031] Figure 11 This is a graph showing the survival rate and weight changes of mice in Experimental Example 3 of the present invention;

[0032] Figure 12 This is a scoring diagram of the clinical behavioral changes of mice in Experimental Example 3 of the present invention;

[0033] Figure 13 This is the in vitro release curve of the hydrogel in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments. Example

[0035] like Figure 1 As shown, this embodiment provides a method for preparing a manganese-based hydrogel rabies vaccine, comprising the following steps:

[0036] S110 Chitosan modification: Chitosan salt was dissolved in water, and carboxyl-activated gallic acid was added thereto. The reaction was stirred at room temperature. The reaction product was purified by dialysis and then freeze-dried to obtain modified chitosan.

[0037] Specifically, 1 g of chitosan hydrochloride was dissolved in 100 mL of water. 1 g of gallic acid, which had activated carboxyl groups, was added. The reaction was stirred at room temperature for 24 hours. The reaction product was dialyzed against ultrapure water to remove unreacted monomers and then freeze-dried to obtain modified chitosan (CS-GA).

[0038] S120 Preparation of inactivated rabies virus: Collect the supernatant of BSR cells infected with LBNSE strain, remove cell debris by centrifugation, inactivate the cells with a vaccine inactivator, and collect virus particles by centrifugation after inactivation. The collected virus particles are resuspended after centrifugation to obtain inactivated rabies virus;

[0039] Specifically, supernatants from BSR cells infected with the LBNSE strain were collected and centrifuged at 10,000 rpm to remove cell debris. Cells were then inactivated with β-propiolactone at 4°C for 24 hours and treated at 37°C for 2 hours. The cells were then centrifuged at 30,000 rpm at 4°C for 3 hours and resuspended in PBS. Viral particles were then collected by sucrose density gradient centrifugation, centrifuged at 30,000 rpm for 3 hours, and resuspended in PBS to obtain inactivated rabies virus (iRABV).

[0040] Preparation of S130 manganese-based hydrogel rabies vaccine: Modified chitosan is prepared into a solution, inactivated rabies virus is added thereto, dispersed evenly under ice bath, and then manganese ions are added thereto, stirred evenly and allowed to stand to form a gel.

[0041] Specifically, CS-GA was completely dissolved in ultrapure water to a 3% solution by mass. iRABV was added to 1 mL of the CS-GA solution and stirred on ice to disperse evenly. Then, 200 μL of a 1M manganese chloride solution was added, stirred, and allowed to stand to form the manganese-based hydrogel rabies vaccine (CGMR).

[0042] This embodiment also provides a manganese-based hydrogel rabies vaccine, which includes modified chitosan, rabies virus and manganese ions, wherein the mass ratio of the modified chitosan, inactivated rabies virus and manganese ions is 300:1:32. The characterization diagram of the prepared manganese-based hydrogel rabies vaccine is shown in the attached figure. Figure 4 shown.

[0043] The manganese-based hydrogel rabies vaccine provided in this embodiment can be used in rabies treatment, such as rabies treatment injection.

[0044] Experimental Example 1: In vitro immunization experiment of manganese-based hydrogel rabies vaccine

[0045] Experimental method: Modified chitosan (CG) was synthesized according to different mass ratios of chitosan and gallic acid. The Fourier transform infrared spectroscopy and ultraviolet absorption spectroscopy of the products with different ratios of CG (2:1), CG (1.5:1) and CG (1:1) were characterized as shown in the attached figure. Figure 2 As shown in (a) and (b), the color development of modified chitosan with different gallic acid grafting is shown in the attached figure. Figure 2 As shown in (c), CS represents chitosan without grafted gallic acid, and GA represents gallic acid. The successful grafting of gallic acid was confirmed by the color development experiment of phenol and spectral characterization, among which CG (1:1) was the optimal synthesis ratio. The functionality and micromorphology of the hydrogel prepared according to the above ratio are shown in the attached figure. Figure 3 and 4 As shown, Figure 3 In (a), G' is greater than G", which indicates that the addition of hydrogel transforms the solution into gel form; Figure 3 (b) The hydrogel can be continuously extruded from a 0.45 micron syringe, demonstrating the injectability of the hydrogel; Figure 3 (b) The upper right corner of the figure demonstrates that the addition of manganese ions forms a gel that coagulates at the bottom of the centrifuge tube (right), while the one without manganese ions is in solution form and cannot be retained at the bottom (left).

[0046] The CGMR prepared in the example was taken as the experimental group, PBS was taken as the blank control group, and bacterial lipopolysaccharide (LPS) and iRABV were taken as the control group to carry out the in vitro dendritic cell (DC2.4) activation experiment and the cGAS-STING pathway activation experiment. Among them, the in vitro dendritic cell (DC2.4) activation experiment tests the expression amount of CD80 / CD86 and MHC-II, and the cGAS-STING pathway activation experiment extracts the whole protein of the cells after incubation of the experimental group and the control group, and carries out Western Blot analysis.

[0047] The results of the DC2.4 activation evaluation experiment are shown in Figure 5 and 6 The results of the cGAS-STING pathway activation experiment are shown in Figure 7 .

[0048] Analysis of experimental results: from Figure 5 and 6 It can be seen that compared with the control group, CGMR significantly increased the expression of CD80 / CD86 by about 15%, which indicates that CGMR can effectively stimulate the maturation of DC2.4 and enhance the immunogenicity of the rabies vaccine. In addition, the expression of MHC-II in DC2.4 in the experimental group is about 22% higher than that in the control group, which indicates that CGMR can promote antigen cross-presentation through MHC-II, indicating that CGMR can promote the activation of subsequent immune cells and the generation of germinal centers.

[0049] From Figure 7 It can be seen that compared with the control group, CGMR increases the content of p-STING and p-IRF3 in the cGAS pathway, which can enhance the activation effect of immune cells.

[0050] Experimental Example 2: In vivo immunization experiment of manganese-based hydrogel rabies vaccine

[0051] Experimental method: fluorescence imaging experiment: manganese-based hydrogel (CGM) without iRABV was taken as the experimental group, and uncrosslinked hydrogel monomer (CG) was taken as the control group, wherein the preparation method of CGM refers to the example, and CGM and CG are both loaded with Cy7 for fluorescence imaging. The experimental results are shown in Figure 8 .

[0052] Antibody detection experiment 1: Six-week-old C57BL / 6 mice were randomly divided into three groups and injected intramuscularly with inactivated virus (iRABV) and intramuscularly with aluminum adjuvant + inactivated virus (Al TM R) was used as the control group, and the CGMR prepared in Example 1 was used as the experimental group. Each vaccine group contained the same amount of antigen. After immunization, the serum of the mice was isolated within 6 weeks for antibody analysis. The experimental results are shown in the attached Figure 9 shown.

[0053] Antibody detection experiment 2: Six-week-old C57BL / 6 mice were randomly divided into three groups and injected intramuscularly with inactivated virus (iRABV), intramuscularly with aluminum adjuvant (Al TM ) and manganese-based hydrogel (CGM) without iRABV were used as experimental groups, wherein the CGM preparation method was referred to Example 1. After immunization, serum was isolated from mice within 6 weeks for antibody analysis. The experimental results are shown in the attached Figure 10 shown.

[0054] Analysis of experimental results: Figure 8 It can be seen that the fluorescence signal of CGM loaded with Cy7 (CGM-Cy7) can still be observed in the animal body 14 days after injection, while the fluorescence signal of CG loaded with Cy7 (CG-Cy7) can no longer be observed one day after injection, indicating that the manganese-based hydrogel prepared by the present invention can be slowly released in the animal body, significantly prolonging the duration of vaccine action.

[0055] From the attached Figure 9 It can be seen that the specific neutralizing antibody titer induced in mice injected with CGMR increased by at least 3 times compared with the control group 2 weeks after immunization, and was able to maintain a high level for 6 weeks, indicating that the CGMR prepared by the present invention can effectively induce rabies-specific humoral immune response.

[0056] From the attached Figure 10 It can be seen that aluminum adjuvant Al does not contain inactivated virus (antigen) TM Neither the CGM nor the manganese-based hydrogel CGM could activate the immune system to produce specific neutralizing antibodies in vivo. The antigen-loaded manganese-based hydrogel produced much higher levels of specific antibodies than the control group, indicating that the manganese ions in the CGMR prepared by the present invention can activate the cGAS pathway and synergize with the inactivated antigen, enhancing immune cell maturation, promoting the formation of germinal centers and antibody secretion, and continuously producing high levels of antibodies, thereby improving the therapeutic effect.

[0057] Experimental Example 3: Manganese-based hydrogel rabies vaccine immune protection experiment

[0058] Experimental method: In this experiment, mice were selected for challenge protection experiment after 6 weeks of immunization, blank mice (PBS) were used as negative control, inactivated virus (iRABV) and aluminum adjuvant + inactivated virus (AlTM R) as the control group, 30 μL of 50×LD 50 The mice were challenged intracranially with the CVS-24 strain and their weight changes, survival status, and behavioral changes were observed and recorded over a 3-week period. Figure 11 and 12 shown.

[0059] Analysis of experimental results: Figure 11 It can be seen that all unvaccinated mice died of rabies within 3 weeks, while mice injected with iRABV and Al TM R mice partially survived, while all CGMR-immunized mice survived within 3 weeks after infection, and the mice did not lose significant weight, while the other groups lost significant weight within 6 to 7 days.

[0060] According to the international behavioral evaluation standard for rabies virus infection, the clinical score is 0-5 points, 0 points means no clinical symptoms, 1 point means movement disorder, 2 points means ruffled hair and hunchback, 3 points means trembling, 4 points means complete loss of movement ability, and 5 points means death. Figure 12 It can be seen that except for the CGMR immunization group, whose clinical behavior score was always 0, the mice in the other control groups all showed behavioral manifestations such as movement disorders, epilepsy, and arched back, indicating that CGMR immunization can protect mice from rabies virus infection.

[0061] Experimental Example 4: Manganese-based hydrogel rabies vaccine release time experiment

[0062] Experimental Method: In this experiment, a manganese-based hydrogel (CGM) loaded with a fluorescent protein (Cy7-OVA) was placed in a 12-well plate. 2 ml of PBS buffer was added and the plate was placed in a 37°C incubator. At different time points, 100 μl of supernatant was aspirated and replaced with an equal volume of PBS buffer. Finally, the cumulative release amount and release ratio were calculated by measuring fluorescence intensity.

[0063] Analysis of experimental results: Figure 13 It can be seen that the manganese-based hydrogel rabies vaccine provided in the present invention completed 100% drug release after 216 hours in the simulated drug release, indicating that the manganese-based hydrogel rabies vaccine in the present invention can continuously release immunotherapy drugs in the body after injection, prolong the immunotherapy time, and improve the treatment effect.

[0064] It will be obvious to a person skilled in the art that the application is not limited to the details of the foregoing exemplary embodiments and can be implemented in other concrete forms without departing from the spirit or essential characteristics of the application. The embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, and all changes coming within the meaning and equivalency range of the appended claims are therefore intended to be embraced therein. Furthermore, it is to be understood that the application is defined by the claims and not only by the description of the embodiments contained herein, the disclosure of which is made by way of example only and thus is not limiting upon the scope of the application.

Claims

1. A method for preparing a manganese-based hydrogel rabies vaccine, characterized in that: Chitosan is first modified to prepare a modified chitosan solution, inactivated rabies virus is added to the modified chitosan solution, and after uniform dispersion, manganese ions are added to react to obtain a manganese-based hydrogel rabies vaccine. The preparation steps of the modified chitosan are as follows: dissolving chitosan acid salt in water, adding a polyphenol compound with activated carboxyl groups thereto, stirring and reacting at room temperature, and dialysis-purifying the reaction product and then freeze-drying it to obtain the modified chitosan, wherein the polyphenol compound includes any one of gallic acid, dihydrocaffeic acid, and protocatechuic acid.

2. The method for preparing a manganese-based hydrogel rabies vaccine according to claim 1, characterized in that: The preparation method comprises the following steps: S110 Chitosan modification: Chitosan acid salt is dissolved in water, and a polyphenol compound with activated carboxyl groups is added thereto. The reaction is stirred at room temperature. The reaction product is purified by dialysis and then freeze-dried to obtain modified chitosan; S120 Preparation of inactivated rabies virus: Collect the supernatant of BSR cells infected with LBNSE strain, remove cell debris by centrifugation, inactivate the cells with a vaccine inactivator, and collect virus particles by centrifugation after inactivation. The collected virus particles are resuspended after centrifugation to obtain inactivated rabies virus; Preparation of S130 manganese-based hydrogel rabies vaccine: Modified chitosan is prepared into chitosan solution, inactivated rabies virus is added thereto, dispersed evenly under ice bath, and then manganese ions are added thereto, stirred evenly and allowed to stand to form a gel.

3. The method for preparing a manganese-based hydrogel rabies vaccine according to claim 2, characterized in that: The steps The ratio of the added amount of chitosan salt to the polyphenol compound in S110 is 1 to 2:1; and the stirring reaction time is 24 to 36 hours.

4. The method for preparing a manganese-based hydrogel rabies vaccine according to claim 2, characterized in that: The steps The mass fraction of modified chitosan in the S130 chitosan solution is 2% to 5%.

5. The method for preparing a manganese-based hydrogel rabies vaccine according to claim 4, characterized in that: The amount of manganese ions added in step S130 is 100 μM to 250 μM; the manganese ions are selected from any one of manganese chloride and manganese sulfate.

6. A manganese-based hydrogel rabies vaccine, characterized in that: The vaccine is prepared using the method for preparing a manganese-based hydrogel rabies vaccine according to any one of claims 1 to 5.

7. The manganese-based hydrogel rabies vaccine according to claim 6, characterized in that: The manganese-based hydrogel rabies vaccine comprises modified chitosan, inactivated rabies virus and manganese ions, and the mass ratio of the modified chitosan, inactivated rabies virus and manganese ions is 300:1:

32.

8. Use of the manganese-based hydrogel rabies vaccine prepared by the preparation method according to any one of claims 1 to 5 or the manganese-based hydrogel rabies vaccine according to any one of claims 6 to 7 in the preparation of a rabies therapeutic drug.

9. Use of the manganese-based hydrogel rabies vaccine according to claim 8 in the preparation of a rabies treatment drug, characterized in that: The dosage form of the manganese-based hydrogel rabies vaccine is an injection.

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