An intradermal immune enhancer for veterinary vaccines and its application

Abstract

The present invention provides an intradermal immunopotentiator for veterinary vaccines and its application, and relates to the field of biopharmaceuticals. The intradermal immunopotentiator for veterinary vaccines is an aqueous solution containing chondroitin sulfate, chitosan oligosaccharides, all-trans retinoic acid liposomes and cyclic diguanylic acid. The preparation method of the intradermal immunopotentiator is as follows: chondroitin sulfate, chitosan oligosaccharides, all-trans retinoic acid liposomes and cyclic diguanylic acid are dissolved in a buffer solution with a pH of 7-8 to obtain the intradermal immunopotentiator. The preparation method of the immunopotentiator of the present invention is simple, convenient and low in cost. The porcine epidemic diarrhea inactivated vaccine prepared by the intradermal immunopotentiator of the present invention is used to intradermally immunize pregnant sows twice, and there is no adverse reaction at the skin injection site, the safety is high, and the level of anti-PEDV IgA antibodies in sow milk is significantly increased, which can better protect piglets.

Description

Technical Field

[0001] The invention relates to the field of biopharmaceuticals, and in particular to an intradermal immunopotentiator for veterinary vaccines and application thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious enteric disease caused by porcine epidemic diarrhea virus (PEDV). Clinically, it manifests as vomiting, diarrhea, dehydration and other symptoms in piglets, with a high mortality rate. Currently, the main means of preventing and controlling PED is vaccination.

[0003] The current method of preventing PED in piglets in breeding production is to vaccinate pregnant sows with epidemic diarrhea vaccine to increase the level of anti-PEDVIgA antibodies in the sow's milk after delivery to passively protect piglets. The inactivated porcine epidemic diarrhea vaccine refers to the preparation of inactivated porcine epidemic diarrhea virus after emulsification with mineral oil or multiple emulsion adjuvant. In clinical use, the level of IgA antibodies in the milk of sows after immunization is low.

[0004] Existing public literature reports that intradermal immunization with inactivated antigens can induce the animal body to produce mucosal IgA antibodies. However, due to the limitations of the animal's anatomical structure, the immunization dose is too small, and multiple immunizations or the use of immunopotentiators are required. Clinical trials have found that re-immunization with the same antigen or the use of immunopotentiators can lead to varying degrees of side effects (redness, nodules, skin ulcers, etc.) at the skin injection site, and secondary skin infections in sows cause huge economic losses. Summary of the invention

[0005] The purpose of the present invention is to provide an intradermal immune enhancer for veterinary vaccines, which can be used for repeated intradermal immunization, has no side effects at the skin injection site, is highly safe, and can significantly improve the level of mucosal antibodies.

[0006] Another object of the present invention is to provide a method for preparing the intradermal immunopotentiator of the veterinary vaccine, which method is simple, convenient and low in cost.

[0007] Another object of the present invention is to provide the use of the intradermal immunopotentiator in the preparation of veterinary vaccines. When the veterinary vaccine is an inactivated vaccine for porcine epidemic diarrhea, pregnant sows are immunized intradermally twice, and there is no adverse reaction at the skin injection site, which is highly safe, and the level of anti-PEDVIgA antibodies in sow milk is significantly increased, which can better protect piglets.

[0008] The purpose of the present invention is achieved by the following technical solutions:

[0009] The invention discloses an intradermal immune enhancer for veterinary vaccines, which is an aqueous solution containing chondroitin sulfate, chitosan oligosaccharide, all-trans retinoic acid liposome and cyclic diguanylic acid.

[0010] In the present invention, the concentration of chondroitin sulfate is 5-25 mg / mL, the concentration of chitosan oligosaccharide is 5-40 mg / mL, the concentration of all-trans retinoic acid liposome is 0.5-4 ug / mL, and the concentration of cyclic diguanylate is 0.05-1 ug / mL.

[0011] In the present invention, the pH of the intradermal immunopotentiator is 7-8.

[0012] The present invention also provides a method for preparing the intradermal immunopotentiator, which comprises dissolving chondroitin sulfate, chitosan oligosaccharide, all-trans retinoic acid liposome and cyclic diguanylic acid in a buffer solution with a pH of 7-8 to obtain the intradermal immunopotentiator.

[0013] In the present invention, the buffer is a PBS buffer with a pH of 7-7.5.

[0014] The present invention also provides application of the intradermal immunopotentiator in preparing veterinary vaccines.

[0015] In the present invention, the vaccine is an inactivated porcine epidemic diarrhea vaccine.

[0016] In the present invention, the inactivated porcine epidemic diarrhea virus liquid and the intradermal immunopotentiator are uniformly mixed in a volume ratio of 1:0.5-1.5 to obtain a veterinary vaccine.

[0017] The immunopotentiator of the present invention has no side effects at the skin injection site during intradermal immunization, is highly safe, and significantly improves the level of mucosal antibodies. The preparation method of the intradermal immunopotentiator of the present invention is simple, convenient, and low-cost. The porcine epidemic diarrhea inactivated vaccine prepared by the intradermal immunopotentiator of the present invention is used to intradermally immunize pregnant sows twice, has no side effects at the skin injection site, is highly safe, significantly improves the level of anti-PEDVIgA antibodies in the sow's milk, and can better protect piglets. The final users of the inactivated vaccine containing the immunopotentiator of the present invention include, but are not limited to, sows, and the immunization procedure is intradermal immunization, including, but not limited to, intradermal immunization methods such as microneedles and needle-free injections. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The anti-PEDV IgA antibody levels in the milk of sows in Example 1 are shown.

[0019] Figure 2 The anti-PEDV IgA antibody levels in the milk of sows in Example 2 are shown. DETAILED DESCRIPTION

[0020] All reagents in the present invention are endotoxin-free.

[0021] The PBS with pH 7.2 in the present invention has a concentration of 0.01 M and is purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number: P2272.

[0022] Chondroitin sulfate: purchased from Wuhan Rongcan Biotechnology Co., Ltd., CAS No. 9007-28-7 (porcine source).

[0023] Chitosan oligosaccharide (molecular weight ≤ 1000Da): purchased from Qingdao Hailongyuan Biotechnology Co., Ltd.

[0024] c-di-GMP (cyclic diguanylate): purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., CAS No. 61093-23-0.

[0025] c-di-AMP (cyclic di-AMP): purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., CAS No. 54447-84-6.

[0026] All-trans retinoic acid liposomes: purchased from Huzhou Purui Biopharmaceutical Technology Co., Ltd.

[0027] Porcine epidemic diarrhea virus PEDV-NJ strain, disclosed in CN201611248419.4, with the deposit number: CGMCCNO.13283.

[0028] Example 1 Preparation and Immunization Experiment of Porcine Epidemic Diarrhea Inactivated Vaccine

[0029] 1. Preparation of immune enhancers

[0030] Preparation of immunopotentiator 1: Chondroitin sulfate was dissolved in PBS at pH 7.2 to a concentration of 20 mg / mL to obtain immunopotentiator 1.

[0031] Preparation of immunopotentiator 2: Dissolve chitosan oligosaccharide in PBS at pH 7.2 to make the concentration of chitosan oligosaccharide 20 mg / mL to obtain immunopotentiator 2.

[0032] Preparation of immunopotentiator 3: All-trans retinoic acid liposomes were dissolved and dispersed in PBS at pH 7.2 to make the concentration of all-trans retinoic acid liposomes 2 ug / mL to obtain immunopotentiator 3.

[0033] Preparation of immunopotentiator 4: c-di-GMP was dissolved in PBS at pH 7.2 to a concentration of 0.1 ug / mL to obtain immunopotentiator 4.

[0034] Preparation of immunopotentiator 5: c-di-AMP was dissolved in PBS at pH 7.2 to a concentration of 0.1 ug / mL to obtain immunopotentiator 5.

[0035] Preparation of immunopotentiator 6: Chondroitin sulfate and chitosan oligosaccharide were dissolved in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL and the concentration of chitosan oligosaccharide 20 mg / mL, to obtain immunopotentiator 6.

[0036] Preparation of immunopotentiator 7: Chondroitin sulfate and all-trans retinoic acid liposomes were dissolved in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL and the concentration of all-trans retinoic acid liposomes 2 ug / mL, to obtain immunopotentiator 7.

[0037] Preparation of immunopotentiator 8: Chondroitin sulfate and c-di-GMP were dissolved in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL and the concentration of c-di-GMP 0.1 ug / mL, to obtain immunopotentiator 8.

[0038] Preparation of immunopotentiator 9: Chondroitin sulfate and c-di-AMP were dissolved in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL and the concentration of c-di-AMP 0.1 ug / mL, to obtain immunopotentiator 9.

[0039] Preparation of immunopotentiator 10: Dissolve chitosan oligosaccharide and all-trans retinoic acid liposome in PBS at pH 7.2 to make the concentration of chitosan oligosaccharide 20 mg / mL and the concentration of all-trans retinoic acid liposome 2 ug / mL, to obtain immunopotentiator 10.

[0040] Preparation of immunopotentiator 11: Dissolve chitosan oligosaccharide and c-di-GMP in PBS at pH 7.2 to make the concentration of chitosan oligosaccharide 20 mg / mL and the concentration of c-di-GMP 0.1 ug / mL to obtain immunopotentiator 11.

[0041] Preparation of immunopotentiator 12: Dissolve chitosan oligosaccharide and c-di-AMP in PBS at pH 7.2 to make the concentration of chitosan oligosaccharide 20 mg / mL and the concentration of c-di-AMP 0.1 ug / mL to obtain immunopotentiator 12.

[0042] Preparation of immunopotentiator 13: Dissolve all-trans retinoic acid liposomes and c-di-GMP in PBS at pH 7.2 to make the concentration of all-trans retinoic acid liposomes 2 ug / mL and the concentration of c-di-GMP 0.1 ug / mL to obtain immunopotentiator 13.

[0043] Preparation of immunopotentiator 14: Dissolve all-trans retinoic acid liposomes and c-di-AMP in PBS at pH 7.2 to make the concentration of all-trans retinoic acid liposomes 2 ug / mL and the concentration of c-di-AMP 0.1 ug / mL to obtain immunopotentiator 14.

[0044] Preparation of immunopotentiator 15: c-di-GMP and c-di-AMP were dissolved in PBS at pH 7.2 to make the concentration of c-di-GMP 0.1 ug / mL and the concentration of c-di-AMP 0.1 ug / mL to obtain immunopotentiator 15.

[0045] Preparation of immunopotentiator 16: dissolve chondroitin sulfate, chitosan oligosaccharide and all-trans retinoic acid liposomes in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL, the concentration of chitosan oligosaccharide 20 mg / mL and the concentration of all-trans retinoic acid liposomes 2 ug / mL to obtain immunopotentiator 16.

[0046] Preparation of immunopotentiator 17: dissolve chondroitin sulfate, chitosan oligosaccharide and c-di-GMP in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL, the concentration of chitosan oligosaccharide 20 mg / mL and the concentration of c-di-GMP 0.1 ug / mL to obtain immunopotentiator 17.

[0047] Preparation of immunopotentiator 18: dissolve chondroitin sulfate, chitosan oligosaccharide and c-di-AMP in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL, the concentration of chitosan oligosaccharide 20 mg / mL and the concentration of c-di-AMP 0.1 ug / mL to obtain immunopotentiator 18.

[0048] Preparation of immunopotentiator 19: dissolve chondroitin sulfate, all-trans retinoic acid liposomes and c-di-GMP in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL, the concentration of all-trans retinoic acid liposomes 2 ug / mL, and the concentration of c-di-GMP 0.1 ug / mL to obtain immunopotentiator 19.

[0049] Preparation of immunopotentiator 20: dissolve chondroitin sulfate, all-trans retinoic acid liposomes, and c-di-AMP in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL, the concentration of all-trans retinoic acid liposomes 2 ug / mL, and the concentration of c-di-AMP 0.1 ug / mL to obtain immunopotentiator 20.

[0050] Preparation of immunopotentiator 21: Dissolve chitosan oligosaccharide, all-trans retinoic acid liposomes, and c-di-GMP in PBS at pH 7.2 to make the concentration of chitosan oligosaccharide 20 mg / mL, the concentration of all-trans retinoic acid liposomes 2 ug / mL, and the concentration of c-di-GMP 0.1 ug / mL to obtain immunopotentiator 21.

[0051] Preparation of immunopotentiator 22: Dissolve chitosan oligosaccharide, all-trans retinoic acid liposomes, and c-di-AMP in PBS at pH 7.2 to make the concentration of chitosan oligosaccharide 20 mg / mL, the concentration of all-trans retinoic acid liposomes 2 ug / mL, and the concentration of c-di-AMP 0.1 ug / mL to obtain immunopotentiator 22.

[0052] Preparation of immunopotentiator 23: Dissolve all-trans retinoic acid liposomes, c-di-GMP, and c-di-AMP in PBS at pH 7.2 to make the concentration of all-trans retinoic acid liposomes 2 ug / mL, the concentration of c-di-GMP 0.1 ug / mL, and the concentration of c-di-AMP 0.1 ug / mL to obtain immunopotentiator 23.

[0053] Preparation of immunopotentiator 24: Dissolve chitosan oligosaccharide, c-di-GMP, and c-di-AMP in PBS at pH 7.2 to make the concentration of chitosan oligosaccharide 20 mg / mL, the concentration of c-di-GMP 0.1 ug / mL, and the concentration of c-di-AMP 0.1 ug / mL to obtain immunopotentiator 24.

[0054] Preparation of immunopotentiator 25: dissolve chondroitin sulfate, c-di-GMP, and c-di-AMP in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL, the concentration of c-di-GMP 0.1 ug / mL, and the concentration of c-di-AMP 0.1 ug / mL to obtain immunopotentiator 25.

[0055] Preparation of immunopotentiator 26: dissolve chondroitin sulfate, chitosan oligosaccharide, all-trans retinoic acid liposomes and c-di-GMP in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL, the concentration of chitosan oligosaccharide 20 mg / mL, the concentration of all-trans retinoic acid liposomes 2 ug / mL, and the concentration of c-di-GMP 0.1 ug / mL to obtain immunopotentiator 26.

[0056] Preparation of immunopotentiator 27: dissolve chondroitin sulfate, chitosan oligosaccharide, all-trans retinoic acid liposomes and c-di-AMP in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL, the concentration of chitosan oligosaccharide 20 mg / mL, the concentration of all-trans retinoic acid liposomes 2 ug / mL, and the concentration of c-di-AMP 0.1 ug / mL to obtain immunopotentiator 27.

[0057] Preparation of immunopotentiator 28: dissolve chondroitin sulfate, chitosan oligosaccharide, c-di-GMP and c-di-AMP in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL, the concentration of chitosan oligosaccharide 20 mg / mL, the concentration of c-di-GMP 0.1 ug / mL, and the concentration of c-di-AMP 0.1 ug / mL to obtain immunopotentiator 28.

[0058] Preparation of immunopotentiator 29: dissolve chondroitin sulfate, all-trans retinoic acid liposomes, c-di-GMP and c-di-AMP in PBS at pH 7.2 to make the concentration of chondroitin sulfate 20 mg / mL, the concentration of all-trans retinoic acid liposomes 2 ug / mL, the concentration of c-di-GMP 0.1 ug / mL, and the concentration of c-di-AMP 0.1 ug / mL to obtain immunopotentiator 29.

[0059] Preparation of immunopotentiator 30: Dissolve chitosan oligosaccharide, all-trans retinoic acid liposomes, c-di-GMP and c-di-AMP in PBS at pH 7.2 to make the concentration of chitosan oligosaccharide 20 mg / mL, the concentration of all-trans retinoic acid liposomes 2 ug / mL, the concentration of c-di-GMP 0.1 ug / mL, and the concentration of c-di-AMP 0.1 ug / mL to obtain immunopotentiator 30.

[0060] 2. Preparation of inactivated porcine epidemic diarrhea vaccine

[0061] Porcine epidemic diarrhea inactivated vaccine 1 (referred to as vaccine 1): inactivated porcine epidemic diarrhea virus liquid (the strain is PEDV-NJ strain, the virus titer before inactivation is 10 8.0 TCID 50 / mL) and immunopotentiator 1 were mixed at a volume ratio of 1:1 to obtain vaccine 1. According to the preparation method of vaccine 1, immunopotentiator 2-30 and inactivated porcine epidemic diarrhea virus liquid (the strain is PEDV-NJ strain, the virus titer before inactivation is 10 8.0 TCID 50 / mL) were mixed at a volume ratio of 1:1 to prepare vaccines 2-30. The number of the vaccine corresponds to the number of the immunopotentiator used.

[0062] Porcine epidemic diarrhea inactivated vaccine without enhancer (referred to as vaccine 31): inactivated porcine epidemic diarrhea virus liquid (strain is PEDV-NJ strain, virus titer before inactivation is 10 8.0 TCID 50 / mL) and PBS with pH 7.2 were mixed at a volume ratio of 1:1 to obtain vaccine 31.

[0063] 3. Animal grouping, immune protection experiment and results

[0064] (1) Animal grouping and immune protection experiment

[0065] 160 pregnant sows (PEDV antigen and antibody negative) were randomly divided into 32 groups, numbered as Groups 1-32, with 5 sows in each group. Immunization procedure: Sows were first immunized two months before farrowing and again one month before farrowing. For sows in Groups 1-31, each sow was immunized with the vaccine corresponding to the group number (see Table 1). At each immunization, 100uL of the corresponding vaccine was injected into the inner thigh of each sow's hind leg, and the immunization route was intradermal injection. Group 32 was the PBS control group. At each immunization, each sow was intradermally injected with 100uL of PBS, pH 7.2.

[0066] After each immunization, observe the injection site and record the adverse reactions after injection. No adverse reaction: no changes in the skin at the injection site (same as normal skin); Mild adverse reaction: redness, swelling and scab at the injection site; Moderate adverse reaction: nodules or mild ulceration at the injection site; Severe adverse reaction: swelling or severe ulceration at the injection site.

[0067] Sampling: Collect milk from sows during farrowing.

[0068] Sample testing: The IgA antibody level in the milk of the experimental sows at farrowing was tested using the Korean Anigen Porcine Epidemic Diarrhea IgA Detection Kit. The test results were expressed as OD values, and the average IgA level in the milk of each group of sows was calculated.

[0069] (2) Results of immune protection experiments

[0070] The results of the adverse reactions at the injection site of each group of sows are shown in Table 1. After the first immunization, there were no adverse reactions at the injection site of all the test sows immunized with vaccine 1, vaccine 6, vaccine 8, vaccine 9, vaccine 19, vaccine 20, vaccine 26, vaccine 27, and vaccine 31 without immunopotentiator, while sows immunized with other vaccines had adverse reactions to varying degrees. After the second immunization, there were no adverse reactions in all the sows immunized with vaccine 26 alone, while sows in other experimental groups had adverse reactions to varying degrees. The results of the injection site adverse reaction test showed that vaccine 26 showed good safety in both the first immunization and the second immunization, especially the safety of the immunopotentiator in the second immunization, which provides a good application prospect for it in animal vaccine booster immunization.

[0071] The results of ELISA test on IgA antibody in milk of experimental sows at farrowing are shown in Figure 1. Compared with the antibody levels after immunization with vaccine 1-5 and vaccine 31, the antibody level after immunization with vaccine 1 was close to that of vaccine 31, indicating that the immunopotentiator contained in vaccine 1 had no immunopotentiating effect. The antibody levels after immunization with vaccines 2 and 3 were slightly increased, but the effect was not obvious. The antibody levels after immunization with vaccines 4 and 5 increased by about 1 times. The immunopotentiators used in vaccines 6-15 contain two components. Only the antibody level after immunization with vaccine 10 was slightly higher than that of vaccines 1-5, and the improvement effect was not obvious. In addition, after immunization with vaccines 11 and 12, the antibody level decreased, indicating that the two components in the immunopotentiator had an antagonistic effect. Immunpotentiators containing three components were used in vaccines 16-25. Among them, after immunization with vaccine 16, due to the obvious antagonism between the three components in the immunopotentiator, its immune effect was lower than that of vaccine 31 without an enhancer. The components contained in the immunopotentiators in vaccines 17, 18, and 24 also had an antagonistic effect. The immunopotentiators used in vaccines 26-30 contain four components. Compared with vaccine 31 without the immunopotentiator, the antibody level after immunization with vaccine 29 increased by about 1 times; the antibody levels after immunization with vaccines 26 and 27 increased by about 8-10 times compared with vaccine 31 without the immunopotentiator, and increased by about 3-4 times compared with the antibody levels after immunization with vaccines 28 and 30.

[0072] Based on the above experimental results, the immunopotentiator used in vaccine 26 contains four components, which can be repeatedly used in intradermal immunization without adverse reactions at the injection site; and can significantly increase the level of anti-PEDVIgA antibodies in sow milk. Therefore, immunopotentiator 26 is an excellent intradermal immunopotentiator.

[0073] Table 1. Statistics of sow groups and injection site adverse reactions after immunization (number of sows)

[0074]

[0075]

[0076] Example 2 Intradermal Immunopotentiator 26 High, Medium and Low Doses Immunization Experiment

[0077] 1. Preparation of intradermal immune enhancer 26 high, medium and low (H, M, L) dose formula

[0078] Preparation of intradermal immune enhancer 26-H: dissolve chondroitin sulfate, chitosan oligosaccharide, all-trans retinoic acid liposomes and c-di-GMP in PBS at pH 7.2 to make the concentration of chondroitin sulfate 25 mg / mL, chitosan oligosaccharide 40 mg / mL, all-trans retinoic acid liposomes 4 ug / mL, and c-di-GMP 1 ug / mL to obtain intradermal immune enhancer 26-H.

[0079] Preparation of intradermal immune enhancer 26-M: dissolve chondroitin sulfate, chitosan oligosaccharide, all-trans retinoic acid liposomes and c-di-GMP in PBS at pH 7.2 to make the concentration of chondroitin sulfate 15 mg / mL, the concentration of chitosan oligosaccharide 15 mg / mL, the concentration of all-trans retinoic acid liposomes 1.5 ug / mL, and the concentration of c-di-GMP 0.1 ug / mL to obtain intradermal immune enhancer 26-M.

[0080] Preparation of intradermal immunopotentiator 26-L: dissolve chondroitin sulfate, chitosan oligosaccharide, all-trans retinoic acid liposomes and c-di-GMP in PBS at pH 7.2 to make the concentration of chondroitin sulfate 5 mg / mL, the concentration of chitosan oligosaccharide 5 mg / mL, the concentration of all-trans retinoic acid liposomes 0.5 ug / mL, and the concentration of c-di-GMP 0.05 ug / mL to obtain intradermal immunopotentiator 26-L.

[0081] 2. Preparation of inactivated porcine epidemic diarrhea vaccine

[0082] The inactivated porcine epidemic diarrhea virus liquid (the strain is PEDV-NJ strain, the virus titer before inactivation is 10 8.0 TCID 50 / mL) and the intradermal immune enhancer 26-H were mixed at a volume ratio of 1:1 to obtain vaccine 26-H.

[0083] The inactivated porcine epidemic diarrhea virus liquid (the strain is PEDV-NJ strain, the virus titer before inactivation is 10 8.0 TCID 50 / mL) and the intradermal immune enhancer 26-M were mixed at a volume ratio of 1:1 to obtain vaccine 26-M.

[0084] The inactivated porcine epidemic diarrhea virus liquid (the strain is PEDV-NJ strain, the virus titer before inactivation is 10 8.0 TCID 50 / mL) and intradermal immune enhancer 26-L were mixed at a volume ratio of 1:1 to obtain vaccine 26-L.

[0085] 3. Animal grouping, immune protection experiment and results

[0086] (1) Animal grouping and immune protection experiment

[0087] 25 pregnant sows (PEDV antigen antibody negative) were randomly divided into 5 groups, 5 in each group. Immunization procedure: Sows were first immunized two months before delivery and again one month before delivery. Among them, 4 groups of sows were immunized with vaccine 26-H, vaccine 26-M, vaccine 26-L, and vaccine 31 (preparation method see Example 1), and the specific grouping is shown in Table 2. At each immunization, 100uL of the corresponding vaccine was injected into the inner thigh of the hind limb of each sow, and the immunization route was intradermal injection. The last group was the PBS control group. At each immunization, each sow was intradermally injected with 100uL of PBS with pH 7.2.

[0088] After each immunization, observe the injection site and record the adverse reactions after injection. No adverse reaction: no changes in the skin at the injection site (same as normal skin); Mild adverse reaction: redness, swelling and scab at the injection site; Moderate adverse reaction: nodules or mild ulceration at the injection site; Severe adverse reaction: swelling or severe ulceration at the injection site.

[0089] Sampling: Collect milk from sows during farrowing.

[0090] Sample testing: The IgA antibody level in the milk of the experimental sows at farrowing was tested using the Korean Anigen Porcine Epidemic Diarrhea IgA Detection Kit. The test results were expressed as OD values, and the average IgA level in the milk of each group of sows was calculated.

[0091] (2) Results of immune protection experiments

[0092] The adverse reactions at the injection site of each group of sows are shown in Table 2. Vaccine 26-H, Vaccine 26-M, and Vaccine 26-L containing intradermal immune enhancers at high, medium, and low doses were used to immunize pregnant sows. There were no adverse reactions at the injection site after the first intradermal immunization and re-immunization, and the vaccine had good safety.

[0093] The results of ELISA test on IgA antibody in milk of experimental sows at farrowing are shown in Figure 2 Compared with vaccine 31, vaccine 26-H, vaccine 26-M, and vaccine 26-L containing intradermal immune enhancers with high, medium, and low doses significantly increased the level of IgA antibodies in sow milk, and there was no significant difference among the vaccine 26-H, vaccine 26-M, and vaccine 26-L groups.

[0094] Table 2. Statistics of sow groups and injection site adverse reactions after immunization (number of sows)

[0095]

Claims

1. An intradermal immunopotentiator for veterinary vaccines, characterized in that An aqueous solution containing chondroitin sulfate, chitosan oligosaccharide, all-trans retinoic acid liposomes and cyclic diguanylic acid; the veterinary vaccine is an inactivated vaccine for porcine epidemic diarrhea; the concentration of chondroitin sulfate is 5-25 mg / mL, the concentration of chitosan oligosaccharide is 5-40 mg / mL, the concentration of all-trans retinoic acid liposomes is 0.5-4 ug / mL, and the concentration of cyclic diguanylic acid is 0.05-1 ug / mL.

2. The intradermal immunopotentiator according to claim 1, characterized in that The pH of the intradermal immunopotentiator is 7-8.

3. The method for preparing the intradermal immunopotentiator according to claim 1, characterized in that Chondroitin sulfate, chitosan oligosaccharide, all-trans retinoic acid liposome and cyclic diguanylic acid are dissolved in a buffer solution with a pH of 7-8 to obtain the intradermal immune enhancer.

4. The method for preparing the intradermal immunopotentiator according to claim 3, characterized in that The buffer is PBS buffer with a pH of 7-7.

5.

5. Use of the intradermal immunopotentiator according to claim 1 in the preparation of veterinary vaccines, wherein the vaccine is an inactivated porcine epidemic diarrhea vaccine.

6. The use according to claim 5, characterized in that The inactivated porcine epidemic diarrhea virus liquid and the intradermal immunopotentiator are evenly mixed in a volume ratio of 1:0.5-1.5 to obtain a veterinary vaccine.

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