CN113117060A

CN113117060A - Encephalitis B-A + C group epidemic encephalitis combined vaccine administrated by combining nanocapsule with microneedle

Info

Publication number: CN113117060A
Application number: CN201911400122.9A
Authority: CN
Inventors: 张庶民; 周荔葆; 马凤森; 刘苗苗; 廖辉; 修雪亮; 徐国标; 吴铮; 王一平; 杨文腰
Original assignee: Liaoning Chengda Biological Co ltd
Current assignee: Liaoning Chengda Biological Co ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2021-07-16

Abstract

The invention discloses a encephalitis B-A + C group epidemic encephalitis combined vaccine for administration by combining nanocapsules with microneedles, which comprises a needle body and a back lining, wherein the needle body consists of encephalitis B vaccine liposome, A + C group polysaccharide combined epidemic encephalitis vaccine and a matrix material, and the lipid nanocapsule material for loading the encephalitis B vaccine consists of neutral phospholipid and cholesterol. According to the invention, the Japanese encephalitis vaccine and the A + C group epidemic encephalitis combined vaccine soluble microneedle with proper mechanical properties is prepared by selecting a liposome material (phosphatidylcholine and cholesterol) with specific components to load the Japanese encephalitis vaccine, screening the Japanese encephalitis vaccine with proper drug loading ratio and the A + C group polysaccharide combined epidemic encephalitis vaccine, and controlling the combined vaccine and the matrix material to be in a specific content ratio, so that the immune cycles of the A + C group polysaccharide combined epidemic encephalitis vaccine and the Japanese encephalitis vaccine in the prepared soluble microneedle are balanced, and the safety is good.

Description

Encephalitis B-A + C group epidemic encephalitis combined vaccine administrated by combining nanocapsule with microneedle

Technical Field

The invention relates to the technical field of delivery of a encephalitis B-epidemic encephalitis B combined vaccine, in particular to an encephalitis B-A + C group epidemic encephalitis B combined vaccine administrated by combining nanocapsules with microneedles.

Background

Encephalitis b is a disease of acute viral infection of the central nervous system, occurring mostly in children. At present, no medicine for treating Japanese encephalitis diseases exists, and no effective method for controlling the spreading of the Japanese encephalitis diseases from the aspect of environment exists. The injection for Japanese encephalitis vaccine immunization is the most important means for controlling Japanese encephalitis diseases.

Neisseria meningitidis caused diseases are a significant cause of serious illness and high mortality worldwide. The fatality rate of the disease can be as high as 10% even in developed countries, and the fatality rate of sepsis caused by the disease can exceed 50%. Neisseria meningitidis causes 500,000 cases and 50,000 deaths worldwide each year. The present large increase in resistance of meningococci to antibiotics in many parts of the world, including china, has made the prevention and control of this disease by vaccination an urgent task.

The existing encephalitis B vaccines and A + C group epidemic encephalitis vaccines are mainly injections, and although subcutaneous or intramuscular injection administration has a short treatment period and a faster curative effect, professional medical staff are required to have high operation technical requirements and obvious pain, so that for main administration objects (infants and children) of two vaccines, immunization of the two vaccines needs to be carried out in the same time period, the injection times are increased, and the compliance of patients is poor. Therefore, there is a need to develop a new administration technique to improve patient compliance.

The combined vaccine is always a development target encouraged by the WHO, and has the advantages of reducing the injection times of inoculated objects, reducing the incidence rate of side reactions, improving the vaccination rate and saving social resources. For the epidemic encephalitis B vaccine and the A + C group epidemic encephalitis vaccine, the immune cycles of the vaccine are inconsistent, the immune cycle of the epidemic encephalitis B vaccine is two times of immunization in 0 and 7 days, and then the immunization is strengthened after one year; and the epidemic encephalitis vaccine is immunized twice in 0 and 1 month.

In order to solve the problem, the patent CN200310119414.8 provides a preparation method of a Japanese encephalitis vaccine and A + C group polysaccharide combined epidemic encephalitis vaccine combined vaccine, the scheme prolongs the immune cycle of Japanese encephalitis by improving the antigen content of the Japanese encephalitis vaccine, but the technical defects are as follows: the safety of the combination vaccine was effective only for the first immunization, and it was not known whether the safety could be continued after the second immunization (as can be seen from the 7-day mouse and guinea pig combination vaccine safety evaluation experiment in example 3 of the specification). It is well known that an increase in the content of vaccine antigens increases the incidence of adverse reactions, which increases with the number of immunizations (xiekang. adverse reactions of epidemic encephalitis b vaccine and their prevention [ J ] shanghai preventive medicine, 2006 (3)). That is to say, the technical scheme of patent CN200310119414.8 has uncertainty of safety of secondary immunization and hidden danger of adverse reaction of combined vaccine.

Therefore, how to provide a new technical scheme to balance the immune cycle of the encephalitis B vaccine and the A + C group polysaccharide combined epidemic encephalitis vaccine in the combined vaccine, and the technical problem which is not solved by the technical personnel in the field is that the primary immunity and the secondary immunity are safe and effective.

Lipid nanocapsules consist of natural or synthetic phospholipids, in particular phosphatidylcholine, spherical microcapsules with at least one lipid bilayer. Compared with other materials, the liposome nanocapsule is easier to be absorbed by immune cells, has good biocompatibility and biodegradability, has trapping capacity and drug slow-release capacity on both hydrophilic drugs and lipophilic drugs, and can effectively delay the release of the drugs in vivo. Therefore, the combined vaccine of the Japanese encephalitis vaccine and the A + C group epidemic encephalitis vaccine can be considered to be delivered by combining a nano-capsule with a microneedle administration technology.

At present, the relevant documents at home and abroad are consulted to know that no relevant document report of the combined vaccine soluble microneedle of the Japanese encephalitis vaccine and the A + C group epidemic encephalitis vaccine exists at present.

The technology of combining nanocapsule with microneedle administration only relates to encephalitis B vaccine; there are some documents on liposomes related to Japanese encephalitis Vaccine (Wu S C, Yu C H, Lin C W, et al, the domain III fragment of Japanese encephalitis virus enveloppe protein: mouse immunogenicity and lipid affinity [. J ]. Vaccine,2003,21(19-20):0-2522.) this document mainly relates to cationic liposome-loaded pET32a recombinant protein and adjuvant which are combined with four of PC, PA, SA and cholesterol, and the effect on the delivery and immunogenicity of the Vaccine is examined, and the sustained-release capability is not examined indefinitely, so the sustained-release effect of the liposome still exists. Meanwhile, the encephalitis B vaccine has hybrid protein components besides main active components, the physicochemical properties are more complex, and the uncertainty is still provided whether the encephalitis B vaccine has sustained-release delivery by simply replacing pET32a recombinant protein with the encephalitis B vaccine to load the liposome.

In conclusion, the lipid nanocapsule loaded vaccine is selected to balance the immune cycle of the encephalitis B vaccine and the A + C group polysaccharide combined epidemic encephalitis vaccine in the combined vaccine, so that the safety is met, and the development of the soluble micro-needle of the encephalitis B-A + C group epidemic encephalitis combined vaccine meeting the mechanical performance requirement is a technical problem which needs to be solved in the field urgently.

Disclosure of Invention

The invention aims to solve the technical problem of providing a encephalitis B-A + C group epidemic encephalitis combined vaccine for administration by combining nanocapsules with microneedles, and solving the defects in the prior art by selecting a proper lipid nanocapsule loaded vaccine.

Therefore, the invention adopts the following technical scheme:

a combined vaccine of Japanese encephalitis-A + C group epidemic encephalitis administered by combining nanocapsule with microneedle comprises a needle body and a back lining, wherein the needle body is composed of Japanese encephalitis vaccine liposome, A + C group polysaccharide combined epidemic encephalitis vaccine and a matrix material, and the liposome material for loading the Japanese encephalitis vaccine is composed of neutral phospholipid and cholesterol.

Preferably, in the liposome material loaded with the Japanese encephalitis vaccine, the neutral phospholipid is distearoyl phosphatidylcholine, and the content ratio of the distearoyl phosphatidylcholine to cholesterol is 3: 1.

Preferably, the content ratio of the Japanese encephalitis-A + C group epidemic encephalitis combined vaccine to the matrix material in the needle body is 1: 2.

Preferably, the content ratio of the A + C group epidemic encephalitis vaccine to the Japanese encephalitis vaccine liposome in the needle body is 1: 2.

In summary, compared with the prior art, the invention has the beneficial effects that:

according to the invention, the liposome material (distearoyl phosphatidylcholine and cholesterol) with specific components is selected to load the encephalitis vaccine, the encephalitis vaccine with a proper drug-loading ratio and the A + C group polysaccharide combined epidemic encephalitis vaccine are screened, and the combined vaccine and the matrix material are controlled to have a specific content ratio, so that the soluble microneedle of the encephalitis-A + C group epidemic encephalitis combined vaccine with appropriate mechanical properties is prepared, and the immune cycle of the A + C group polysaccharide combined epidemic encephalitis vaccine and the epidemic encephalitis vaccine in the prepared soluble microneedle is balanced, and the safety is good.

Drawings

Figure 1 is a graph of the cumulative release profile of vaccines under different liposome materials.

Detailed Description

The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.

Example 1 preparation of soluble microneedles for JEOL-A + C group JEOL combination vaccine

The Japanese encephalitis-A + C group epidemic encephalitis combined vaccine soluble microneedle to be prepared by the invention comprises a needle body and a back lining, wherein the needle body consists of Japanese encephalitis vaccine liposome, A + C group polysaccharide combined epidemic encephalitis vaccine and a suitable matrix material, and the liposome material for loading the Japanese encephalitis vaccine consists of neutral phospholipid and cholesterol.

The method for preparing the Japanese encephalitis vaccine liposome comprises the following steps:

controlling the particle size of the lipid nanocapsule by polycarbonate membrane filtration, taking the Japanese encephalitis vaccine solution as a water phase for later use, and dissolving the liposome material in chloroform as an organic phase for later use. 40ml of the organic phase was removed by rotary evaporation of chloroform, which resulted in a thin lipid film in the flask. Hydrating 10ml of vaccine solution for 0.5h, then placing into a high-pressure homogenizer, mixing uniformly for three times under the pressure of 300 +/-100 bar, filtering by a small-sized extruder through a polycarbonate membrane with 100nm membrane pores, and controlling the particle size of the liposome to be uniform, thereby obtaining the Japanese encephalitis vaccine liposome.

The method for preparing the soluble microneedle of the epidemic encephalitis B-A + C group epidemic encephalitis combined vaccine comprises the following steps:

mixing the Japanese encephalitis vaccine liposome suspension, the A + C group epidemic encephalitis vaccine and the micro-needle substrate material according to the formula ratio to form uniform needle fluid. Pouring a proper amount of needle body liquid into a polydimethylsiloxane female die to enable the needle body liquid to completely immerse the holes of the female die, then putting the female die into a high-speed centrifuge, setting the rotating speed to be 4000r/min, and centrifuging for 5min to enable the needle body liquid to fully enter the holes of the female die. Curing for more than 12 hours under normal pressure, demolding, and continuing to dry to obtain the epidemic encephalitis B-A + C group epidemic encephalitis vaccine soluble microneedle.

Example 2 relevant testing of soluble microneedles for JEV-A + C group

1. Method for measuring encapsulation efficiency of Japanese encephalitis vaccine liposome

The evaluation index of the Japanese encephalitis vaccine liposome is the entrapment rate, and the method for measuring the entrapment rate comprises the following steps: the protein content of 10ml of vaccine solution was determined using the BCA assay kit and was designated W1. The prepared encephalitis B vaccine lipid solution is then subjected to ultracentrifugation at 18000rpm for 30 min. The supernatant was collected and assayed for protein content by BCA assay kit, designated W2. Namely, the encapsulation efficiency is EE (W1-W2)/W1.

The Chinese pharmacopoeia (2015 edition) stipulates that the encapsulation efficiency of the liposome is not lower than 80% (general rule 9014).

2. Method for measuring release amount of Japanese encephalitis vaccine

The liposome-soluble microneedle of the encephalitis b vaccine prepared by the method of example 1 was added with 1mL of PBS buffer solution using PBS buffer solution with pH of 7.4 as release medium, and shaken at constant temperature of 37 ℃. At predetermined time intervals (0, 1, 2, 4, 8, 16, 24, 32, 40 days), 2ml of supernatant was withdrawn and supplemented with an equal amount of fresh PBS buffer, protein release was measured using BCA kit, and the mass percent of released protein was calculated to generate cumulative release curves.

3. Method for characterizing mechanical properties of microneedles

Method 1): pressure change performance investigation of microneedle by using physical property analyzer

And (3) placing the soluble microneedle with the surface upward, enabling the direction of the microneedle to be parallel to the axial direction of the probe, performing pressure change measurement, and recording the breaking force of the microneedle.

Method 2): investigation of skin puncture Performance by microneedle

Applying the prepared soluble microneedle on the back of the mouse by using a drug delivery device, uncovering after 1min, removing redundant dye liquor after dyeing with methylene blue, recording the number of holes, and calculating the skin puncture rate, wherein the higher the value is, the better the mechanical property is. The puncture rate is calculated as follows: the puncture rate (number of holes/number of soluble microneedle bodies) was 100%.

When the breaking force of the microneedle is greater than 3N, the puncture rate reaches more than 80%, and effective transdermal delivery can be realized, which is considered to meet the requirements.

4. Method for measuring titer of Japanese encephalitis vaccine

The neutralizing antibody was determined in a plaque reduction neutralization assay using an immunized mouse neutralizing antibody assay. Reference vaccines (RA and RB) and neutralization test positive sera are provided by the national drug certification authority.

The reference vaccines (R) are respectively diluted by 1:32, and 10 mice with the body weight of 12-14 g are respectively immunized in the abdominal cavity. And immunizing 10 mice with the weight of 12-14 g by the combined vaccine soluble microneedle (T) through an administration device, collecting blood after immunization, separating serum, mixing the serum of the same group of mice in equal amount, inactivating the mixture at 56 ℃ for 30 minutes, diluting the positive serum, the T serum and the R serum, and mixing the diluted positive serum, the T serum and the R serum in equal amount with the diluted virus (about 200PFU/0.4 ml). Simultaneously diluting the diluted virus at a ratio of 1:2 to serve as a virus control, placing the diluted virus in a water bath at 37 ℃ for 90 minutes, inoculating 6-pore plate BHK2 cells, culturing the cells at 37 ℃ for 90 minutes at a concentration of 0.4m1 per pore, adding a culture medium cover of methyl cellulose, and placing the cells in CO₂Culturing in an incubator for 5 days, staining, counting plaques, calculating the plaque reduction rate of the T group and the R group to the virus control group, wherein the average number of plaques of the virus control group is 50-150, and evaluating the titer (T value) of the encephalitis B vaccine of the microneedle group.

The Chinese pharmacopoeia stipulates that: when the titer T of the Japanese encephalitis vaccine is more than or equal to (RA + RB)/2-0.33, the Japanese encephalitis vaccine is qualified (RA and RB are the titers of the reference vaccine), and the Japanese encephalitis vaccine is determined to meet the requirement when the titer T value of the Japanese encephalitis vaccine is more than or equal to 1.35 through the experiment.

Method for measuring titer of epidemic encephalitis vaccine in A + C group

Immunizing 12-14 g of NIH (or Balb/C) mice by a combined vaccine soluble microneedle (T) through a dosing device, taking 10 mice in the same batch as a control group, dosing a physiological sodium chloride solution, taking blood after immunization, measuring the IgG antibody titer of the anti-A group polysaccharide and the anti-C group polysaccharide in serum by an ELISA method, and calculating the Cutoff value by the absorbance value of the serum of the mice in the control group of the physiological sodium chloride solution.

The Chinese pharmacopoeia stipulates that: the positive conversion rate of the antibodies in the vaccine group should not be lower than 80% (page 75 "3.5.4 efficacy experiment" in the third section).

Example 3 Effect of the composition and content of Liposome Material on the Loading Effect and sustained Release Effect of Japanese encephalitis vaccine

From example 1, it can be seen that the liposome material loaded with Japanese encephalitis vaccine consists of neutral phospholipids and cholesterol.

The liposome material loaded with the Japanese encephalitis vaccine is set to be a certain value, and only the components of the neutral phospholipid and the content ratio of the neutral phospholipid to the cholesterol are used for researching the influence of the loading effect (the evaluation index is the entrapment rate) and the slow release effect (the evaluation index is the cumulative release curve) of the Japanese encephalitis vaccine.

In this example, neutral phospholipids were screened as follows: distearoyl phosphatidylcholine (DSPC), hydrogenated egg yolk lecithin (HEPC), dimyristoyl phosphatidylcholine (DMPC) and dipalmitoyl phosphatidylcholine (DPPC), the content ratio of neutral phospholipid to cholesterol is (1:1) to (4:1), and the corresponding experimental group number is 1-16.

In this example, the preparation process of the liposome of the Japanese encephalitis vaccine is shown in example 1. The encapsulation efficiency and the release amount of the Japanese encephalitis vaccine liposome are tested in example 2, and the test results are shown in Table 1.

TABLE 1 influence of the composition and content of liposome material on the encapsulation efficiency of Japanese encephalitis vaccine liposomes (mean% + -SD%, n ═ 6)

As can be seen from Table 1:

1) the different content ratio of the same neutral phospholipid and cholesterol also affects the encapsulation efficiency of the Japanese encephalitis vaccine liposome, and the two conditions of meeting the test requirement and not meeting the test requirement exist.

2) The same content ratio of neutral phospholipid to cholesterol and different components of neutral phospholipid can also influence the encapsulation efficiency of the encephalitis B vaccine liposome, and the conditions of meeting the test requirements and not meeting the test requirements exist.

3) When only the experiment group 2(DSPC: cholesterol: 2:1), the experiment group 3(DSPC: cholesterol: 3:1) and the experiment group 6(HEPC: cholesterol: 2:1) are used, the encapsulation rate of the liposome of the Japanese encephalitis vaccine is more than 80 percent, and the test requirement is met.

Furthermore, the release amount of the Japanese encephalitis vaccine liposome in experimental groups 2, 3 and 6 is tested to screen out the optimal liposome material composition. The test results are shown in FIG. 1.

As can be seen from fig. 1 in conjunction with table 1:

1) when the content ratio of the DSPC to the cholesterol is 3:1 (experimental group 3), the sustained-release time of the Japanese encephalitis vaccine is 32 days, and the immune cycle of the Japanese encephalitis vaccine and the A + C group epidemic encephalitis vaccine tend to be balanced and meet the requirements.

2) The slow release time of the encephalitis B vaccines of the experimental group 2 and the experimental group 6 is too short, and the encephalitis B vaccines cannot reach the balance with the immune cycle of the epidemic encephalitis A and C vaccine groups, and do not meet the requirements.

The loading effect and the sustained release effect of the Japanese encephalitis vaccine are comprehensively considered, the mixture of DSPC and cholesterol is selected as a liposome material, and the content ratio of the DSPC to the cholesterol is 3: 1.

Example 4 influence of content ratio of Japanese encephalitis-group A + group C epidemic encephalitis combined vaccine and matrix material on mechanical property of microneedle

On the basis of example 3, the content ratio of the liposome material loaded with the Japanese encephalitis vaccine is determined, and the influence of different content ratios on the mechanical properties of the microneedles is researched by only changing the content ratio of the Japanese encephalitis-A + C group epidemic encephalitis combined vaccine to the matrix material.

In this example, the preparation process of the soluble microneedle of the epidemic encephalitis B-A + C group epidemic encephalitis vaccine is the same as that of example 1. The mechanical performance of the Japanese encephalitis-A + C group epidemic encephalitis combined vaccine soluble microneedle is tested, see example 2, and the test results are shown in Table 2.

TABLE 2 Effect of the content ratio of combination vaccine and matrix material on the mechanical Properties of microneedles (mean% + -SD%, n ═ 6)

NO.	Content ratio of combined vaccine and matrix material	Breaking force (N)	Skin puncture Rate (%)
				1	2:1	2.58±0.16	64.2±1.4
2	1:1	2.64±0.15	73.4±1.6
				3	1:2	4.84±0.36	98.7±1.2
4	1:3	3.26±0.12	76.8±2.1

As can be seen from Table 2: when the content ratio of the epidemic encephalitis B-A + C group epidemic encephalitis combined vaccine to the matrix material is 1:2, the breaking force and the skin puncture rate both meet the test requirements.

Example 5 Effect of the content ratio of group A + C epidemic encephalitis vaccine and group B encephalitis vaccine liposomes on the immune Effect of the vaccine

On the basis of example 4, the content ratio of the epidemic encephalitis B-A + C group epidemic encephalitis combined vaccine to the matrix material is set to be a certain value (1:2), and the content ratio of the epidemic encephalitis B vaccine A + C group epidemic encephalitis B vaccine to the epidemic encephalitis B vaccine liposome is only changed to explore the influence of different content ratios on the immune effect of the epidemic encephalitis B vaccine or the epidemic encephalitis B vaccine.

In this example, the preparation process of the soluble microneedle of the epidemic encephalitis B-A + C group epidemic encephalitis vaccine is the same as that of example 1. The titer of the Japanese encephalitis vaccine and the A + C group epidemic encephalitis vaccine in the Japanese encephalitis-A + C group epidemic encephalitis combined vaccine soluble microneedle is tested, see example 2, and the test results are shown in Table 3.

TABLE 3 potency test results for encephalitis B vaccine and group A + C epidemic encephalitis vaccine

NO.	Content ratio of epidemic encephalitis A + C and epidemic encephalitis B	Japanese encephalitis vaccine titer T value	Group A positive conversion rate	Positive conversion rate of group C
					1	2:1	1.24	100％	100％
2	1:1	1.31	96％	95％
					3	1:2	1.89	93％	94％
4	1:3	1.92	73％	70％

From table 3, it can be seen that:

1) from the titer (T value) of the Japanese encephalitis vaccine, the T values of the experimental groups 3 and 4 are 1.89 and 1.92 respectively, and the test requirements are met.

2) From the titer of the epidemic encephalitis vaccine of the A + C group, the positive conversion rates of the A group and the C group of the experimental groups 2 and 3 are both more than 80%, and the test requirements are met.

3) The immune effects of the encephalitis B vaccine and the epidemic encephalitis vaccine are comprehensively considered, and only the experimental group 3 meets the requirements, namely the content ratio of the epidemic encephalitis A vaccine of the A + C group to the encephalitis B vaccine liposome is 1: 2.

In summary, in the needle body of the soluble microneedle of the epidemic encephalitis B-A + C group epidemic encephalitis B combined vaccine, the optimal formula of the components and the mass ratio thereof is as follows: the ratio of the encephalitis B vaccine liposome is 11.1 percent, the ratio of the A + C group epidemic encephalitis vaccine is 22.2 percent, and the ratio of the matrix material is 66.7 percent. Wherein, in the liposome material loaded with the Japanese encephalitis vaccine, the content ratio of distearoyl phosphatidylcholine to cholesterol is 3: 1.

Example 6 evaluation of safety of soluble microneedles of epidemic encephalitis B-A + C group epidemic encephalitis vaccine

Blank microneedles (without the addition of Japanese encephalitis-A + C group epidemic encephalitis combined vaccine) and combined vaccine soluble microneedles (the optimal formula) were prepared according to the preparation process of example 1, and safety evaluation was performed on the microneedles. The evaluation method is an active systemic anaphylaxis test, the test steps are as follows, and the test results are shown in table 5.

A method for determining the safety of the soluble microneedle of the epidemic encephalitis B-A + C group epidemic encephalitis B combined vaccine (active systemic anaphylaxis test): guinea pigs with the weight of 300-400 g are selected and randomly divided into 3 groups of 6 animals each, and the animals are numbered. Setting a blank microneedle control group (a negative control group) (No. 1-6), a positive control group (a commercial product, a Japanese encephalitis vaccine and an A + C group epidemic encephalitis vaccine are jointly administered by adopting an injection mode) (No. 7-12), and a combined vaccine soluble microneedle group (a test group) (No. 13-18). After immunization, challenge assays were performed.

The observation method comprises the following steps: during the excitation period: immediately to 30 minutes after intravenous injection, each animal was observed for response, appearance and disappearance of symptoms, which were observed as: 0. normal, 1. restlessness, 2. piloerection, 3. shiver, 4. nasal pruritus, 5. sneezing, 6. cough, 7. shortness of breath, 8. urination, 9. defecation, 10. lacrimation, 11. dyspnea, 12. wheezing, 13. purpura, 14. gait instability, 15. jumping, 16. wheezing, 17. spasm, 18. twirling, 19. tidal breathing, 20. death. Judging the occurrence degree of the anaphylactic reaction according to the table 1, calculating the occurrence rate of the anaphylactic reaction, carrying out comprehensive judgment according to the occurrence rate and the occurrence degree of the anaphylactic reaction, and the evaluation indexes are shown in the table 4.

TABLE 4 evaluation criteria for systemic sensitization

The symptoms appear	Extent of reaction	Result judgment
			0	－	Negative allergic reaction
1～4	+	Weak positive of anaphylaxis
			5～10	++	Positive allergic reaction
11～19	+++	Strong positive of allergic reaction
			20	++++	Very strong positive of allergic reaction

TABLE 5 results of allergic reactions in various groups of animals after challenge

From table 5, it can be seen that:

1) after the excitation, 6 guinea pigs in the negative control group have no allergic symptoms, and the allergic reactions are negative; in the positive control group, 6 guinea pigs have allergic symptoms of different degrees, such as restlessness, hair erection, nasal pruritus, cough, tachypnea, wheeze, gait instability, spasm, death and the like, and the allergic reaction is positive by 2, strong positive by 2 and extremely strong positive by 2; in the test sample group, 6 guinea pigs showed allergic symptoms such as pilosis, nasal pruritus, cough, and unstable gait, and the allergic reaction was weak and positive for 2 guinea pigs, and positive for 4 guinea pigs.

2) Compared with the positive control group, the allergic symptoms of the test sample group have no extremely positive allergic symptoms, and are obviously weaker than those of the positive control group. Therefore, compared with the products sold in the market, the soluble microneedle of the epidemic encephalitis B-A + C group epidemic encephalitis vaccine provided by the invention has higher safety.

3) Through analysis, the existence of protein substances and the amount of antigen in the vaccine are main reasons for influencing whether guinea pigs can generate anaphylactic reaction. Normally vaccinated vaccines are administered at a safe dose, and therefore the vaccine has a low probability of producing an allergic reaction when vaccinated.

Claims

1. A combined Japanese encephalitis-A + C group epidemic encephalitis vaccine administered by combining nanocapsules with microneedles comprises a needle body and a back lining, and is characterized in that the needle body consists of Japanese encephalitis vaccine lipid nanocapsules, A + C group polysaccharide combined epidemic encephalitis vaccine and a matrix material, wherein the liposome material for loading the Japanese encephalitis vaccine consists of neutral phospholipid and cholesterol.

2. The encephalitis-A + C group epidemic encephalitis combined vaccine delivered by combining nanocapsules and microneedles in claim 1, wherein the neutral phospholipid in the liposome material loaded with the encephalitis vaccine is distearoyl phosphatidylcholine, and the content ratio of distearoyl phosphatidylcholine to cholesterol is 3: 1.

3. The nanocapsule-microneedle array encephalitis conjugate vaccine as claimed in claim 1, wherein the content ratio of the group encephalitis-a + C vaccine to the matrix material is 1: 2.

4. The nanocapsule-microneedle-coupled encephalitis B-A + C group vaccine combination according to claim 1, wherein the content ratio of the group A + C vaccine to the liposome of the encephalitis B vaccine in the needle body is 1: 2.