Manganese-based hydrogel rabies vaccine as well as preparation method and application thereof

Abstract

The invention 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.The manganese-based hydrogel rabies vaccine is based on modified chitosan hydrogel, inactivated rabies viruses and manganese ions are added into the modified chitosan hydrogel, co-delivery of adjuvants and antigens is achieved through injection, and the rabies vaccine is prepared. Along with infiltration of tissue effusion, the cGAS-STING is slowly dissolved, manganese ions are gradually released to activate cGAS-STING, the activity of immune cells is enhanced, and generation of a hair growth center is stimulated; meanwhile, immune cells or immune organs which release antigens are processed to generate antibodies, so that virus infection is blocked. The manganese-based hydrogel rabies vaccine disclosed by the invention can be used for remarkably prolonging the continuous acting time of an antibody, prolonging the protection time of the antibody, enhancing the activity of immune cells, stimulating the generation of a hair growth center and improving the generation level of the antibody.

Description

Technical Field

[0001] The invention belongs to the technical field of biological preparations, and specifically relates to a manganese-based hydrogel rabies vaccine and a preparation method and application thereof. Background Art

[0002] Rabies is a highly lethal zoonosis, with up to 60,000 deaths worldwide each year, most of which occur in Asia and Africa. After being scratched or bitten by a host carrying the rabies virus, the virus replicates and spreads at the wound, eventually entering the central nervous system and salivary glands via the axons of peripheral motor neurons, causing a series of clinical symptoms such as fever, vomiting, neurological dysfunction, and respiratory failure. Currently, there is no effective treatment for rabies, and the only way to reduce the spread of the virus is through immediate and periodic vaccination.

[0003] The multi-dose rabies vaccination program and immunoglobulin vaccination prevention strategy have greatly increased the cost of vaccination, which in turn limits the vaccination rate of rabies vaccines in underdeveloped countries. Therefore, researchers are committed to improving the immune effect of vaccines, combining with optimizing vaccination methods, continuously improving rabies vaccination strategies, reducing vaccination costs and expanding vaccination coverage.

[0004] The main component of the human and animal rabies vaccines on the market in the prior art is the inactivated rabies virus, which is mostly treated by injection. There is also a Chinese patent with publication number CN1 14272367A that provides an oral rabies virus-like particle vaccine and a preparation method thereof, which uses the glycoprotein RVGP and matrix protein RVMP of the CVS strain rabies virus to self-assemble into RVLPs as antigens, uses L100 and PLGA as the main materials to prepare oral acid-resistant slow microspheres, and uses mucosal immune adjuvant 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 smoothly overcome various physiological obstacles of the gastrointestinal tract and be directly delivered to the intestinal tract, respond to the release of antigens under intestinal pH conditions, and conduct a preliminary immunological evaluation of the immune effect it induces. However, whether it is a direct injection type or an oral type, the drug stays in the body for a short time, the effect of inducing immunity is limited, and it cannot produce a lasting therapeutic effect. Summary of the invention

[0005] In view of the above-mentioned problems existing in the current rabies vaccine, the present invention provides a manganese-based hydrogel rabies vaccine and its preparation method and application. The manganese-based hydrogel rabies vaccine is based on modified chitosan hydrogel and its loaded inactivated rabies virus and manganese ions. The adjuvant and antigen are co-delivered by injection, which can significantly increase the duration of antibody action, enhance the activity of immune cells and stimulate the germinal center to produce antibodies. The specific technical scheme is as follows:

[0006] First, the present invention provides a method for preparing a manganese-based hydrogel rabies vaccine. 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.

[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 group is added thereto, and 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: collecting the supernatant of BSR cells infected with LBNSE strain, centrifuging to remove cell debris, inactivating the cells with a vaccine inactivator, and collecting 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 polyphenol compound includes any one of gallic acid, caffeic acid, and protocatechuic acid, and the ratio of chitosan salt to polyphenol compound addition is 1 to 2:1; and the stirring reaction time is 24 to 36 hours.

[0012] Furthermore, in the step S130, the mass fraction of 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 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 comprises 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 treatment of rabies.

[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] The invention uses chitosan as a matrix, and modifies chitosan by activating a carboxyl polyphenol compound. The modified chitosan is rich in phenolic hydroxyl groups, so that it can be cross-linked with manganese ions in the form of coordination bonds to form a phenolic network hydrogel. The formed hydrogel is loaded with inactivated rabies virus by physical encapsulation to prepare a manganese-based hydrogel rabies vaccine.

[0019] The manganese-based hydrogel rabies vaccine provided by the present invention can achieve the co-delivery of manganese ions and inactivated antigens. As the hydrogel dissolves, the manganese ions are gradually released. The manganese ions can activate the cGAS pathway and produce a synergistic effect with the inactivated antigens, enhance the maturation of immune cells, promote the production of germinal centers and antibody secretion, sustainably produce high levels of antibodies, and improve 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 This is a schematic diagram of a preparation process of a manganese-based hydrogel rabies vaccine in an embodiment of the present invention;

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

[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 figure (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 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] Fig. 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] Fig.10 The rabies-specific neutralizing antibody titer in the antibody detection experiment 2 of Experimental Example 2 of the present invention;

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

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

[0033] Fig.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 solution and advantages of the present invention more clear, the technical solution of the present invention will be clearly and completely described below in conjunction with embodiments.

[0035] Example

[0036] like Figure 1 As shown, this embodiment provides a method for preparing a manganese-based hydrogel rabies vaccine, comprising the following steps:

[0037] S110 Chitosan modification: Chitosan acid 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.

[0038] Specifically, 1 g of chitosan hydrochloride was dissolved in 100 mL of water. 1 g of gallic acid activated by carboxyl group was added thereto. The reaction was stirred at room temperature for 24 h. The reaction product was purified by ultrapure water dialysis to remove unreacted monomers, and then freeze-dried to obtain modified chitosan (CS-GA).

[0039] S120 Preparation of inactivated rabies virus: collecting the supernatant of BSR cells infected with LBNSE strain, centrifuging to remove cell debris, inactivating the cells with a vaccine inactivator, and collecting virus particles by centrifugation after inactivation. The collected virus particles are resuspended after centrifugation to obtain inactivated rabies virus;

[0040] Specifically, the supernatant of BSR cells infected with LBNSE strain was collected and centrifuged at 10,000 rpm to remove cell debris. The cells were inactivated with β-propiolactone at 4°C for 24 hours and treated at 37°C for 2 hours. The cells were centrifuged at 30,000 rpm and 4°C for 3 hours and suspended with PBS. The virus particles were then collected by sucrose density gradient centrifugation, centrifuged at 30,000 rpm for 3 hours, and resuspended with PBS to obtain inactivated rabies virus (iRABV).

[0041] 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.

[0042] Specifically, CS-GA was completely dissolved in ultrapure water to prepare a solution with a mass fraction of 3%, iRABV was added to 1 mL of CS-GA solution, and the solution was stirred and dispersed evenly in an ice bath, and 200 μL of 1 M manganese chloride solution was added, stirred, and then allowed to stand to form a manganese-based hydrogel rabies vaccine (CGMR).

[0043] 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.

[0044] The manganese-based hydrogel rabies vaccine provided in this embodiment can be used in rabies treatment, such as rabies treatment injection, etc.

[0045] Experimental Example 1: In vitro immunization experiment of manganese-based hydrogel rabies vaccine

[0046] Experimental method: Modified chitosan (CG) was synthesized according to different mass ratios of chitosan and gallic acid. The Fourier transform infrared spectra and ultraviolet absorption spectra 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 1 As shown in (a) and (b), the color development of modified chitosan after different gallic acid grafting is shown in the attached figure. Figure 1 As shown in (c), CS represents chitosan grafted with gallic acid, and GA represents gallic acid. The gallic acid grafting was successful through the Folin phenol colorimetric experiment 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 As shown, Figure 3 In (a), G' is greater than G" which indicates that the addition of hydrogel transforms the solution into gel state; Figure 3(b) The hydrogel can be continuously extruded from a 0.45 μm syringe, demonstrating the injectability of the hydrogel. Figure 3 (b) The small picture in the upper right corner of y proves that after adding manganese ions, a gel is formed and solidified at the bottom of the centrifuge tube (right side), while without adding manganese ions, the solution is in the form and cannot be retained at the bottom (left side).

[0047] The CGMR prepared in the example was used as the experimental group, PBS was used as the blank control group, and bacterial lipopolysaccharide (LPS) and iRABV were used as the control groups for in vitro dendritic cell (DC2.4) activation experiments and cGAS-STING pathway activation experiments. The in vitro dendritic cell (DC2.4) activation experiment tested the expression of CD80 / CD86 and MHC-II, and the cGAS-STING pathway activation experiment extracted the whole cell protein after incubation in the experimental group and the control group, and performed Western Blot analysis.

[0048] The results of the DC2.4 activation evaluation experiment are shown in the attached Figure 4 and 5 The results of the cGAS-STING pathway activation experiment are shown in the attached Figure 6 .

[0049] Experimental results analysis: From the attached Figure 4 and 5 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 rabies vaccine. In addition, the expression of MHC-II in DC2.4 in the experimental group was 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 subsequent immune cell activation and germinal center generation.

[0050] From the attached Figure 6 It can be seen that compared with the control group, CGMR increased the content of p-STING and p-IRF3 in the cGAS pathway, and can enhance the activation effect of immune cells.

[0051] Experimental Example 2: In vivo immunization experiment of manganese-based hydrogel rabies vaccine

[0052] Experimental method: Fluorescence imaging experiment: Manganese-based hydrogel (CGM) without iRABV was used as the experimental group, and non-crosslinked hydrogel monomer (CG) was used as the control group. The preparation method of CGM was referenced to the example, and both CGM and CG were loaded with Cy7 for fluorescence imaging. The experimental results are shown in the attached Figure 7 shown.

[0053] 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 an equal amount of antigen. Serum was isolated from mice within 6 weeks after immunization for antibody analysis. The experimental results are shown in the attached Figure 8 shown.

[0054] 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 Fig.11 shown.

[0055] Experimental results analysis: From the attached Figure 7 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 not be observed 1 day after injection, indicating that the manganese-based hydrogel prepared by the present invention can be slowly released in the animal body and significantly prolong the action time of the vaccine.

[0056] From the attached Figure 8 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.

[0057] From the attached Fig.11 It can be seen that the aluminum adjuvant Al without inactivated virus (antigen) TM And manganese-based hydrogel CGM cannot activate the immune system in vivo to produce specific neutralizing antibodies. And from the following Table 1, it can be seen that the level of specific antibodies produced by the antigen-loaded manganese-based hydrogel is much higher than that of the control group, indicating that the manganese ions in the CGMR prepared by the present invention can activate the cGAS pathway and produce synergistic effects with inactivated antigens, enhance immune cell maturation, promote the production of germinal centers and antibody secretion, and can continuously produce high levels of antibodies, thereby improving the therapeutic effect.

[0058] Experimental Example 3: Manganese-based hydrogel rabies vaccine immune protection experiment

[0059] Experimental method: In this experiment, mice 6 weeks after immunization were selected for challenge protection experiment, blank mice (PBS) were used as negative control, inactivated virus (iRABV) and aluminum adjuvant + inactivated virus (Al TM R) as the control group, 30 μL of 50×LD 50 The mice were challenged intracranially with the CVS-24 strain and their weight, survival, and behavioral changes were observed and recorded over a 3-week period. Fig. 9 shown.

[0060] Experimental results analysis: From the attached Fig. 9 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 without significant weight loss, while the other groups had significant weight loss within 6 to 7 days.

[0061] According to the international behavioral evaluation standard for rabies virus infection, the clinical score is 0-5 points, with 0 points indicating no clinical symptoms, 1 point indicating movement disorder, 2 points indicating ruffled hair and hunchback, 3 points indicating trembling, 4 points indicating complete loss of movement ability, and 5 points indicating death. Fig.10 It can be seen that except for the CGMR immunization group mice whose clinical behavior scores were always 0, the mice in the other control groups all showed behavioral manifestations such as movement disorders, epilepsy, and hunched backs, indicating that CGMR immunization can protect mice from rabies virus infection.

[0062] Experimental Example 4: Manganese-based hydrogel rabies vaccine release time experiment Experimental method: In this experimental example, a manganese-based hydrogel (CGM) loaded with fluorescent protein (Cy7-OVA) was placed in a 12-well plate, 2 ml of PBS buffer was added, and the well plate was placed in a 37-degree Celsius incubator. 100 microliters of supernatant was drawn at different time points and an equal volume of PBS buffer was added. Finally, the cumulative release amount and release ratio were calculated by measuring the fluorescence intensity.

[0063] Experimental results analysis: From the attached Fig.12 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 is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-restrictive from any point of view. In addition, it should be understood that although this specification is described in accordance with the embodiments, it does not contain only one technical solution. This narrative of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

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.

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 group is added thereto, and 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: collecting the supernatant of BSR cells infected with LBNSE strain, centrifuging to remove cell debris, inactivating the cells with a vaccine inactivator, and collecting 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 gel.

3. The method for preparing a manganese-based hydrogel rabies vaccine according to claim 2, characterized in that: The steps The polyphenol compound in S110 includes any one of gallic acid, caffeic acid and protocatechuic acid, and the ratio of the added amount of chitosan salt to the polyphenol compound 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 2, characterized in that: In the step S130, the amount of manganese ions added is 100 μM to 250 μM; the manganese ions are from any one of manganese chloride and manganese sulfate.

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

7. A manganese-based hydrogel rabies vaccine according to claim 6, characterized in that: The manganese-based hydrogel rabies vaccine comprises 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.

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 treatment of rabies.

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

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