Adjuvant suitable for intradermal immunization and application thereof

Abstract

The invention provides an adjuvant suitable for intradermal immunization and application thereof, and belongs to the field of pharmaceutical preparations. The adjuvant is prepared from the following components in parts by mass: 10 to 25 parts of squalene, 10 to 25 parts of sea buckthron oil, 25 to 35 parts of caprylic / capric polyethylene glycol glyceride and 25 to 45 parts of Tween 80. The adjuvant suitable for intradermal immunization is low in viscosity, easy to inject and absorb and capable of remarkably improving the immune efficacy of vaccines.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and in particular relates to an adjuvant suitable for intradermal immunization and application thereof. Background Art

[0002] Intradermal immunization is a new approach to animal vaccination. The skin is the most superficial organ of the body, with a large area, and is easy to immunize and observe. At the same time, because it is relatively far away from the important organs of the body, it has a high safety. The skin and mucous membranes are easily exposed to various foreign substances and have a strong ability to recognize and process antigens, so they are ideal vaccination sites. In addition, the skin contains abundant immune cells that can produce an effective immune response to the vaccine. The use of intradermal immunization can significantly (10 times) reduce the amount of vaccine (antigen). Intradermal immunization is not only an ideal choice in theory, but also has been verified in practice. For example, the BCG vaccine used to prevent tuberculosis is administered by intradermal immunization, which has been adopted in many countries around the world. In addition, intradermal immunization has also been used for rabies vaccination, further proving its effectiveness and safety in vaccination. In summary, intradermal immunization is considered to be an ideal vaccine immunization route due to its many advantages.

[0003] However, in the prior art, in the practice of intradermal immunization of veterinary vaccines, since commonly used adjuvants such as oil adjuvants have high viscosity, it will increase the difficulty of injection and absorption of vaccine components in the animal body, thereby affecting the immune effect of the vaccine and the convenience of the vaccination process. Summary of the invention

[0004] The purpose of the present invention is to provide an adjuvant suitable for intradermal immunization, which has low viscosity, is easy to inject and absorb, and can significantly improve the immune efficacy of the vaccine.

[0005] Another object of the present invention is to provide a use of an adjuvant.

[0006] The purpose of the present invention is achieved by the following technical solutions:

[0007] An adjuvant suitable for intradermal immunization, comprising the following components in parts by weight:

[0008] Squalene 10-25 parts,

[0009] Sea buckthorn oil 10-25 parts,

[0010] 25-35 parts of caprylic and capric macrogol glycerides,

[0011] Tween 80 25-45 parts.

[0012] In a preferred technical solution, the adjuvant is composed of the following components in parts by mass:

[0013] Squalene 15-20 parts,

[0014] 15-20 parts of sea buckthorn oil,

[0015] 28-32 parts of caprylic acid and capric acid macrogol glycerides,

[0016] Tween 80 30-40 parts.

[0017] The present invention also provides application of the adjuvant in preparing an inactivated vaccine of porcine pseudorabies virus.

[0018] In the present invention, the inactivated porcine pseudorabies virus vaccine is an intradermal immunization vaccine.

[0019] In the present invention, the adjuvant and the antigen are mixed at a volume ratio of 1:8-10 and emulsified to obtain the vaccine.

[0020] Compared with the existing adjuvants, the present invention has the following beneficial effects: the present invention prepares the adjuvant by mixing the immune enhancing substance (sea buckthorn oil, squalene) and the surfactant (caprylic acid capric acid macrogol glyceride, Tween 80). The adjuvant and the inactivated antigen can be made into a stable O / W type vaccine by hand shaking, magnetic stirring and machine stirring. The preparation process does not require precision instruments, is easy to operate, and has low preparation cost.

[0021] In addition, the raw materials used for adjuvants are all safe or low-toxic substances and have no toxic side effects on animals.

[0022] The vaccine prepared by using the adjuvant suitable for intradermal immunization of the present invention has low viscosity, can be used for intradermal immunization through equipment, is convenient to inject, is easy to absorb and has good immunogenicity, and can significantly improve the immune efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Results of the pseudorabies blocking ELISA antibody test in pigs 28 days after immunization, where GEL01 refers to control vaccine 1, ns indicates no significant difference, **** indicates extremely significant difference, p<0.001. Note: The blocking ELISA antibody OD value is inversely proportional to the titer. DETAILED DESCRIPTION

[0024] Caprylic / capric macrogol glycerides were purchased from Guangzhou Tianrun Pharmaceutical Co., Ltd., Labrasol, CAS No: 85536-07-8.

[0025] Propylene glycol picaprate, purchased from Guangzhou Tianrun Pharmaceutical Co., Ltd., Propylene glycol picaprate, CAS No: 56519-72-3.

[0026] Tween 80 was purchased from Croda Chemicals (Shanghai) Co., Ltd. TM 80-LQ-(RB), CAS No:9005-65-6.

[0027] Sea buckthorn oil was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; cinnamon essential oil was purchased from Taizhou Chundao Spice Co., Ltd.

[0028] Squalene was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0029] Example 1 Preparation of O / W adjuvant

[0030] Adjuvant 1 formulation:

[0031] Sea buckthorn oil 40g,

[0032] Caprylic / capric macrogol glycerides 30g, Tween 80 30g.

[0033] Adjuvant 2 formulation:

[0034] Squalene 40g,

[0035] Caprylic / capric macrogol glycerides 30g, Tween 80 30g.

[0036] Adjuvant 3 formulation:

[0037] Sea buckthorn oil 40g,

[0038] Squalene 10g,

[0039] Caprylic / capric macrogol glycerides 25g, Tween 80 25g.

[0040] Adjuvant 4 formulation:

[0041] Squalene 30g,

[0042] Sea buckthorn oil 20g,

[0043] Caprylic / capric macrogol glycerides 25g, Tween 80 25g.

[0044] Adjuvant 5 formulation:

[0045] Squalene 20g,

[0046] Sea buckthorn oil 20g,

[0047] Caprylic / capric macrogol glycerides 30g, Tween 80 30g.

[0048] Adjuvant 6 formulation:

[0049] Squalene 15g,

[0050] Sea buckthorn oil 15g,

[0051] Caprylic / capric macrogol glycerides 30g, Tween 80 40g.

[0052] Adjuvant 7 formulation:

[0053] 20g cinnamon essential oil,

[0054] Vitamin E 10g,

[0055] Squalene 10g,

[0056] Caprylic / capric acid macrogol glyceride 30g,

[0057] Propylene glycol dicaprate 30g.

[0058] Adjuvant 8 formulation:

[0059] 40g cinnamon essential oil,

[0060] Caprylic / capric macrogol glycerides 30g,

[0061] Propylene glycol dicaprate 30g.

[0062] Adjuvant 9 formulation:

[0063] 20g cinnamon essential oil,

[0064] Squalene 10g,

[0065] Sea buckthorn oil 10g,

[0066] Caprylic / capric macrogol glycerides 30g,

[0067] Propylene glycol dicaprate 30g.

[0068] Adjuvant 10 Formulation:

[0069] Squalene 20g,

[0070] Sea buckthorn oil 20g,

[0071] Caprylic / capric macrogol glycerides 30g,

[0072] Glycerin formal 30g.

[0073] Weigh each substance according to the formula of adjuvants 1 to 10, stir or shake at low shear force for 5 to 10 minutes to form a uniform system, and sterilize at 115° C. for 20 minutes to obtain adjuvants 1 to 10, all of which are O / W adjuvants and are stored at room temperature away from light.

[0074] Example 2 Inactivated Pseudorabies Virus Vaccine and Its Viscosity and Stability

[0075] 1. Inactivated pseudorabies virus vaccine

[0076] The inactivated pseudorabies virus vaccine was prepared using the adjuvants in Example 1. The antigen in the vaccine was the pseudorabies virus PRV-NJ strain (disclosed in Chinese invention patent ZL201710943404.8).

[0077] Preparation of inactivated pseudorabies virus vaccine 1: Inactivated pseudorabies virus PRV-NJ strain virus solution (the virus content before inactivation is 10 8.0 TCID 50 / mL) was mixed with adjuvant 1 at a volume ratio of 9:1 and stirred evenly to obtain vaccine 1.

[0078] According to the preparation method of vaccine 1, adjuvants 2 to 10 were added to the inactivated porcine pseudorabies virus PRV-NJ strain virus solution (the virus content before inactivation was 10 8.0 TCID 50 / mL) were mixed at a volume ratio of 1:9 and stirred evenly to obtain vaccines 2 to 10. The vaccine numbers correspond to the adjuvant numbers.

[0079] 2. Vaccine viscosity

[0080] During the vaccine production process, it is crucial to ensure that the viscosity of the vaccine is controlled within an appropriate range. Viscosity is not only a key indicator of vaccine quality standards, but also directly related to the safety and effectiveness of vaccine injection. Excessive viscosity may cause serious problems such as necrosis of the skin at the injection site, affecting the immune effect of the vaccine and the health of the vaccine recipient. Vaccine viscosity testing is usually carried out at a standard temperature of 25°C.

[0081] Viscosity measurement: The viscosity of each vaccine at 25°C was measured using a Brookfield DV2T cone-plate viscometer from the United States. The test results are shown in Table 1. The viscosities of vaccines 1, 2, 5, and 6 were all lower than 2 mPa·s; the viscosities of vaccines 3 and 4 were 8.5 mPa·s and 4.4 mPa·s; the viscosities of vaccines 7, 8, 9, and 10 were 10.5 mPa·s, 11.2 mPa·s, 10.2 mPa·s, and 10.6 mPa·s, respectively, which were significantly higher than those of other vaccines.

[0082] Table 1 Vaccine viscosity test results

[0083]

[0084] 3. Vaccine stability

[0085] Each vaccine was subjected to centrifugation test and 37°C accelerated aging stability test.

[0086] Centrifugation test: When evaluating the stability of animal vaccines, observing the stratification of O / W vaccines under the action of centrifugal force is a simple and quick method. The longer the stratification time, the better the stability of the oil emulsion; on the contrary, if the stratification is rapid and the amount of stratification is large, it means that the stability of the oil emulsion is poor. The specific method of vaccine centrifugation test is as follows: take 10mL of vaccine and centrifuge at 3000r / min for 15 minutes. The water precipitated at the bottom of the tube should not exceed 0.5mL.

[0087] 37℃ accelerated aging stability test: Place vaccines 1 to 10 in a 37℃ incubator and observe regularly every day. Demulsification is judged as the oil phase and the water phase separating.

[0088] The results are shown in Table 2. Vaccines 1 to 10 are all O / W vaccines. After centrifugation, no water phase was precipitated. Therefore, the centrifugation test of vaccines 1 to 10 was qualified. During the storage of vaccines 1 to 10 at 37°C for 30 days, vaccines 3 and 4 had water and oil phase separation and demulsification on the fifth day; vaccines 1, 2, 5, 6, 7, 8, 9, and 10 groups had no water and oil phase separation after 30 days of storage, and there was no obvious change in appearance. The heat stability was qualified.

[0089] Table 2 Vaccine stability test results

[0090]

[0091]

[0092] Therefore, the vaccine groups 1, 2, 5, 6, 7, 8, 9, and 10 all passed the tests, the vaccine groups 3 and 4 failed the 37°C accelerated aging test in the stability test, and passed the viscosity and centrifugation tests.

[0093] Example 3 Vaccine Immunological Safety Test

[0094] The safety of intradermal immunization of the inactivated pseudorabies virus vaccines 1, 2, 5, 6, 7, 8, 9, and 10 prepared in Example 2 was tested.

[0095] Piglet safety test: 45 PRV-negative piglets of about 30 days old were randomly divided into 9 groups. Each group was intradermally injected with inactivated pseudorabies virus vaccine 1, 2, 5, 6, 7, 8, 9, 10, 0.5 mL / head in the neck; another control group was intradermally injected with the same volume of PBS buffer in the neck.

[0096] After intradermal immunization of each vaccine in the neck, observe the skin at the injection site and record the adverse reactions after injection. The criteria for judging adverse reactions are: ① No adverse reaction: No changes in the skin at the injection site (same as normal skin); ② Mild adverse reaction: Redness, swelling and scab at the injection site; ③ Moderate adverse reaction: Nodules appear on the skin at the injection site; ④ Severe adverse reaction: Ulceration or above of the skin at the injection site.

[0097] 1 to 3 days after vaccination, the absorption of the injection site was observed and recorded, and the results are shown in Table 3. It can be seen from Table 3 that vaccines 1, 2, 5, 6, 7, 8, 9, and 10 were injected intradermally into the neck of PRV-negative piglets, and skin papules (a sign of intradermal injection) appeared after injection. Vaccines 1, 2, 5, and 6 were absorbed by the body within 1 day, and the skin papules disappeared. Only piglet No. 2, which was vaccinated with vaccine 1, absorbed it in 2 days, with a mild side effect of slight redness and swelling. Among the piglets vaccinated with vaccine 7, 2 / 5 piglets had redness and swelling at the injection site, which disappeared and was completely absorbed in 2-3 days; among the piglets vaccinated with vaccine 8, 3 / 5 piglets had redness and swelling, which disappeared and was completely absorbed in 2-3 days, and 1 / 5 piglets had persistent hard nodules; among the piglets vaccinated with vaccine 9, 2 / 5 piglets had redness and swelling, which disappeared and was completely absorbed in 2 days; among the piglets vaccinated with vaccine 10, 2 / 5 piglets had redness and swelling, which disappeared and was completely absorbed in 2 days. The control group vaccinated with PBS buffer was absorbed within 2 hours after intradermal injection of the neck, without side effects.

[0098] Table 3 Results of piglet intradermal immunization vaccine safety test

[0099]

[0100]

[0101]

[0102] Note: “+” in the table indicates yes, “-” indicates no.

[0103] In addition, all the experimental pigs were observed for 14 days after intradermal vaccination, and their feeding and drinking, body weight loss, local reactions, allergic reactions and body temperature were observed. The results are shown in Table 4. Table 4 shows that all the experimental pigs had normal feeding and drinking, and no weight loss; the piglets vaccinated with vaccines 2, 7, 8, 9 and 10 had redness, swelling or hard nodules at the vaccination site, and no abnormal reactions were found in other groups; the average body temperature was tested 7 days after vaccination, and the temperature difference did not exceed 0.5℃.

[0104] Table 4 Results of the piglet overdose vaccination safety test

[0105]

[0106]

[0107] Note: The temperature (℃) in the table is expressed as mean ± standard deviation.

[0108] The inactivated pseudorabies virus vaccines 1, 2, 5, 6, 7, 8, 9, and 10 prepared in Example 2 were subjected to the above-mentioned safety test on pigs, respectively. Vaccines 1, 2, 5, and 6 met the requirements of the current "Chinese Veterinary Pharmacopoeia" for the safety of veterinary vaccines, proving that intradermal immunization with adjuvants 1, 2, 5, and 6 is safe for pigs.

[0109] Example 4 Intradermal Immunization Efficacy Test of Inactivated Pseudorabies Virus Vaccine

[0110] Preparation method of control vaccine 1: inactivated pseudorabies virus PRV-NJ strain virus liquid (the virus content before inactivation is 10 8.0 TCID 50 / mL) and GEL01 adjuvant (purchased from France Cepico) were mixed at a volume ratio of 9:1 and stirred evenly to obtain control vaccine 1. After testing, the viscosity of control vaccine 1 was 4-5 mPa·s, which was significantly higher than vaccines 5 and 6 in Example 2.

[0111] The pig immune efficacy test was conducted by intradermal immunization with the inactivated pseudorabies virus vaccines 5 and 6 prepared in Example 2 and the control vaccine 1. The specific method is as follows: 15 healthy, 30-day-old PRV-negative pigs were randomly divided into 3 groups, with 5 pigs in each group. Pigs in groups 1 and 2 were injected intradermally in the neck with vaccines 5 and 6, respectively, and the vaccination dose was 0.5 mL / head; the other group was injected intradermally in the neck with the control vaccine 1, and the vaccination dose was 0.5 mL / head. After intradermal immunization of the neck with each vaccine, the skin at the injection site was observed and the adverse reactions after injection were recorded. Judgment criteria for adverse reactions: ① No adverse reactions: no changes in the skin at the injection site (the same as normal skin); ② Mild adverse reactions: redness, swelling and scab at the injection site; ③ Moderate adverse reactions: nodules are produced on the skin at the injection site; ④ Severe adverse reactions: ulceration and above of the skin at the injection site. After 28 days of immunization, blood was collected to separate serum, and the PRV antibody level was detected using a pseudorabies ELISA antibody detection kit (purchased from Wuhan Keqian Biological Co., Ltd.).

[0112] Vaccines 5 and 6 were absorbed by the body within 1 day, the papules disappeared, and there were no adverse reactions on the skin at the injection site. After immunization with control vaccine 1, 2 / 5 of the 5 pigs developed hard nodules at the injection site, indicating moderate adverse reactions.

[0113] The results of the pig immune efficacy test showed that the antibody titers of pigs vaccinated with vaccines 5 and 6 were significantly higher than those of control vaccine 1 (a vaccine prepared with imported adjuvant GEL01), among which vaccine 5 had the highest antibody titer. The main immune components of vaccines 5 and 6 are sea buckthorn oil and squalene. After immunization, the titers of vaccines 5 and 6 were better than those of control vaccine 1, proving that immunization with sea buckthorn oil and squalene together has an immune-boosting effect.

Claims

1. An adjuvant suitable for intradermal immunization, comprising the following components in parts by weight: Squalene 10-25 parts, Sea buckthorn oil 10-25 parts, 25-35 parts of caprylic and capric macrogol glycerides, Tween 80 25-45 parts.

2. The adjuvant according to claim 1, characterized in that The components are as follows according to mass: Squalene 15-20 parts, 15-20 parts of sea buckthorn oil, 28-32 parts of caprylic acid and capric acid macrogol glycerides, Tween 80 30-40 parts.

3. Use of the adjuvant according to claim 1 or 2 in the preparation of an inactivated vaccine of pseudorabies virus.

4. The use according to claim 3, characterized in that The porcine pseudorabies virus inactivated vaccine is an intradermal immunization vaccine.

5. The use according to claim 4, characterized in that The adjuvant according to claim 1 or 2 and the antigen are mixed in a volume ratio of 1:8-10, and emulsified to obtain the vaccine.

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