Adjuvant suitable for intradermal immunization and application thereof

Abstract

Provided are an adjuvant suitable for intradermal immunization and an application thereof, relating to the field of pharmaceutical formulations. The adjuvant consists of the following components in parts by mass: 10-25 parts of squalene, 10-25 parts of sea buckthorn oil, 25-35 parts of polyethylene glycol caprylic / capric glycerides, and 25-45 parts of Tween 80. The adjuvant suitable for intradermal immunization has a low viscosity, is easy to inject and absorb, and can also significantly improve the immune efficacy of vaccines.

Description

An adjuvant for intradermal immunization and its use TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical preparations, and particularly relates to an adjuvant for intradermal immunization and its use. BACKGROUND

[0002] Intradermal immunization is a new route for animal vaccine immunization. Skin is the most superficial organ of the body, and has a large area, is easy to be immunized and observed. Meanwhile, it is relatively far from important organs of the body, and thus has high safety. Skin and mucosa are easy to contact various foreign substances, and have strong ability to identify and process antigens, and thus they are ideal sites for vaccination. In addition, skin contains rich immune cells, and can produce effective immune response to the inoculated vaccine. The use of intradermal immunization route can greatly (10 times) reduce the amount of vaccine (antigen). Intradermal immunization is not only a theoretically ideal choice, but also has been verified in practice. For example, the Bacillus Calmette-Guerin vaccine for preventing tuberculosis is inoculated by intradermal immunization, and this method is adopted in many countries in the world. In addition, intradermal immunization is also used for the prophylactic inoculation of rabies, further proving its effectiveness and safety in vaccination. In summary, intradermal immunization is considered as an ideal route for vaccine immunization due to its various advantages.

[0003] However, in the practice of intradermal immunization of veterinary vaccines, the commonly used adjuvants such as oil adjuvants have high viscosity, which increases the difficulty of injection and the absorption of vaccine components in the animal body, thereby affecting the immunization effect of the vaccine and the convenience of the inoculation process.

[0004] SUMMARY

[0005] The purpose of the present application is to provide an adjuvant for intradermal immunization, which has low viscosity, is easy to inject and absorb, and can significantly improve the immunization efficacy of the vaccine.

[0006] Another purpose of the present application is to provide an application of the adjuvant.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] An adjuvant for intradermal immunization, which is composed of the following components by mass fraction:

[0009] Squalene 10-25 parts,

[0010] Sea buckthorn oil 10-25 parts,

[0011] Caprylocaproyl macrogolglycerides 25-35 parts,

[0012] Tween 80 25-45 parts.

[0013] In a preferred technical solution, the adjuvant consists of the following components by mass fraction:

[0014] Squalene 15-20 parts,

[0015] Sea buckthorn oil 15-20 parts,

[0016] Caprylocaproyl macrogolglycerides 28-32 parts,

[0017] Tween 80 30-40 parts.

[0018] The application also provides use of the adjuvant in preparation of a porcine pseudorabies virus inactivated vaccine.

[0019] In the application, the porcine pseudorabies virus inactivated vaccine is an intradermal immunization vaccine.

[0020] In the application, the adjuvant and the antigen are mixed in a volume ratio of 1:8-10, emulsified, to obtain the vaccine.

[0021] Compared with the prior art, the application has the following beneficial effects: the adjuvant is prepared by mixing immune enhancer substances (sea buckthorn oil, squalene) and surfactants (caprylocaproyl macrogolglycerides, Tween 80). The adjuvant and the inactivated antigen can be mixed by hand shaking, magnetic stirring and machine stirring to prepare a stable O / W type vaccine. The preparation process does not require precision instruments, is easy to operate, and has low preparation cost.

[0022] In addition, the raw materials of the adjuvant are all safe or low-toxicity substances, and have no toxic side effects on animals.

[0023] The vaccine prepared by the adjuvant suitable for intradermal immunization has low viscosity, can be used for intradermal immunization by equipment, is convenient to inject, easy to absorb and has good immunogenicity, and can significantly improve the immunization efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a result of porcine pseudorabies blocking ELISA antibody detection 28 days after immunization, wherein GEL01 refers to a control vaccine 1, ns indicates that the difference is not significant, **** indicates that the difference is extremely significant, and p<0.001. Note: the blocking ELISA antibody OD value is inversely proportional to the titer. DETAILED DESCRIPTION

[0025] Caprylocaproyl macrogolglycerides, purchased from Guangzhou Tianlun Pharmaceutical Co., Ltd., Labrasol, CAS No: 85536-07-8.

[0026] Propylene glycol picaprate, purchased from Guangzhou Tianlun Pharmaceutical Co., Ltd., Propylene glycol picaprate, CAS No: 56519-72-3.

[0027] Tween 80, purchased from Hualda Chemicals (Shanghai) Co., Ltd., TWEEN TM 80-LQ-(RB), CAS No: 9005-65-6.

[0028] Sea buckthorn oil, purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.; Cinnamon essential oil, purchased from Taizhou Chun Dao Spice Co., Ltd.

[0029] Squalene, purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.

[0030] Preparation of O / W type adjuvant in Example 1

[0031] Adjuvant 1 formula:

[0032] Sea buckthorn oil 40g,

[0033] Caprylic capric acid polyethylene glycol glyceride 30g, Tween 80 30g.

[0034] Adjuvant 2 formula:

[0035] Squalene 40g,

[0036] Caprylic capric acid polyethylene glycol glyceride 30g, Tween 80 30g.

[0037] Adjuvant 3 formula:

[0038] Sea buckthorn oil 40g,

[0039] Squalene 10g,

[0040] Caprylic capric acid polyethylene glycol glyceride 25g, Tween 80 25g.

[0041] Adjuvant 4 formula:

[0042] Squalene 30g,

[0043] Sea buckthorn oil 20g,

[0044] Caprylic capric acid polyethylene glycol glyceride 25g, Tween 80 25g.

[0045] Adjuvant 5 formula:

[0046] Squalene 20g,

[0047] Sea buckthorn oil 20g,

[0048] Caprylic capric acid polyethylene glycol glyceride 30g, Tween 80 30g.

[0049] Adjuvant 6 formula:

[0050] Squalene 15g,

[0051] Sea buckthorn oil 15g,

[0052] Caprylocaproyl macrogolglycerides 30g, Tween 80 40g.

[0053] Adjuvant 7 formula:

[0054] Cinnamon essential oil 20g,

[0055] Vitamin E 10g,

[0056] Squalene 10g,

[0057] Caprylocaproyl macrogolglycerides 30g,

[0058] Propylene glycol dicaprylate 30g.

[0059] Adjuvant 8 formula:

[0060] Cinnamon essential oil 40g,

[0061] Caprylocaproyl macrogolglycerides 30g,

[0062] Propylene glycol dicaprylate 30g.

[0063] Adjuvant 9 formula:

[0064] Cinnamon essential oil 20g,

[0065] Squalene 10g,

[0066] Sea buckthorn oil 10g,

[0067] Caprylocaproyl macrogolglycerides 30g,

[0068] Propylene glycol dicaprylate 30g.

[0069] Adjuvant 10 formula:

[0070] Squalene 20g,

[0071] Sea buckthorn oil 20g,

[0072] Caprylocaproyl macrogolglycerides 30g,

[0073] Glycerol formal 30g.

[0074] Each substance is weighed according to the formula of adjuvant 1-10, stirred or shaken for 5-10 minutes under low shear force to form a uniform system, sterilized at 115℃ for 20 minutes under high pressure, and then adjuvant 1-10 is obtained, which is O / W type adjuvant, and is stored at room temperature in the dark.

[0075] Example 2 Inactivated porcine pseudorabies virus vaccine and its viscosity and stability

[0076] 1. Inactivated vaccine of porcine pseudorabies virus

[0077] An inactivated vaccine of porcine pseudorabies virus was prepared using each adjuvant in Example 1. The antigen in the vaccine was porcine pseudorabies virus PRV-NJ strain (disclosed in Chinese invention patent ZL201710943404.8).

[0078] Preparation of inactivated vaccine of porcine pseudorabies virus 1: the inactivated porcine pseudorabies virus PRV-NJ strain virus liquid (virus content before inactivation was 10 8.0 TCID 50 / mL) was mixed with adjuvant 1 at a volume ratio of 9:1, and stirred uniformly to obtain vaccine 1.

[0079] According to the preparation method of vaccine 1, adjuvants 2-10 were mixed with the inactivated porcine pseudorabies virus PRV-NJ strain virus liquid (virus content before inactivation was 10 8.0 TCID 50 / mL) at a volume ratio of 1:9, and stirred uniformly to obtain vaccines 2-10, the number of which corresponded to the number of adjuvants.

[0080] 2. Viscosity of vaccine

[0081] During the production of vaccines, it is crucial to ensure that the viscosity of the vaccine is controlled within an appropriate range. Viscosity not only serves as a key indicator of vaccine quality standards, but also directly relates to the safety and effectiveness of vaccine injection. Excessive viscosity can lead to necrosis of the skin at the injection site and other serious problems, affecting the immune effect of the vaccine and the health of the inoculator. The viscosity of the vaccine is usually detected at a standard temperature of 25℃.

[0082] Viscosity determination: The viscosity of each vaccine at 25℃ was measured using a Brookfield DV2T cone and plate viscometer from the United States. The detection 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, respectively; 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 the other vaccines.

[0083] Table 1: Viscosity detection results of vaccines

[0084] 3. Stability of vaccine

[0085] Each vaccine was subjected to centrifugal test and 37℃ accelerated aging stability test.

[0086] Centrifugal test: when evaluating the stability of animal vaccines, observing the delamination of O / W type vaccines under centrifugal force is a simple and rapid method. The longer the time required for delamination, the better the stability of the oil emulsion; conversely, if the delamination is rapid and the amount of delamination is large, the stability of the oil emulsion is poor. The specific method of the vaccine centrifugal test is as follows: take 10 mL of the vaccine and centrifuge at 3000 r / min for 15 minutes, and the water phase separated at the bottom should not exceed 0.5 mL.

[0087] 37℃ accelerated aging stability test: place the vaccines 1-10 in a 37℃ incubator, observe at regular intervals every day, and judge as emulsion breaking if the oil phase and water phase separate.

[0088] The results are shown in Table 2. Vaccines 1-10 are all O / W type vaccines, and after centrifugation, no water phase is separated, so the centrifugal test of vaccines 1-10 is qualified. During the 30-day placement of vaccines 1-10 at 37℃, vaccines 3 and 4 have water phase and oil phase separation on the fifth day of placement, and emulsion breaking; vaccines 1, 2, 5, 6, 7, 8, 9, and 10 have no water phase and oil phase separation after 30 days of placement, and no obvious changes in appearance, and the heat stability is qualified.

[0089] Table 2: Results of vaccine stability test

[0090] Therefore, the tests of vaccines 1, 2, 5, 6, 7, 8, 9, and 10 are all qualified, and the stability tests of vaccines 3 and 4 are not qualified in the 37℃ accelerated aging test, but are qualified in the viscosity and centrifugal tests.

[0091] Example 3: Immune safety test of the vaccine

[0092] Test the safety of intradermal immunization of pig pseudorabies virus inactivated vaccines 1, 2, 5, 6, 7, 8, 9, and 10 prepared in Example 2.

[0093] Piglet safety test: 45 PRV-negative piglets of about 30 days old were randomly divided into 9 groups. Each group was injected with pig pseudorabies virus inactivated vaccines 1, 2, 5, 6, 7, 8, 9, and 10, 0.5 mL per head, intradermally in the neck; another group was set as a control group, and the same volume of PBS buffer was injected intradermally in the neck.

[0094] After intradermal immunization in the neck of each vaccine, the injection site skin was observed, and the post-injection reactions were recorded. The judgment criteria for side reactions are as follows: ① no side reaction: no change in the skin of the injection site (same as normal skin); ② mild side reaction: redness and scabbing at the injection site; ③ moderate side reaction: nodules on the skin of the injection site; ④ severe side reaction: skin ulceration at the injection site and above.

[0095] The absorption of injection site was observed and recorded 1-3 days after inoculation, and the results are shown in Table 3. As shown in Table 3, the piglets inoculated with vaccines 1, 2, 5, 6, 7, 8, 9 and 10 respectively showed skin bumps (a sign of intradermal injection) after injection, and the skin bumps of the piglets inoculated with vaccines 1, 2, 5 and 6 disappeared within 1 day, and only the piglet No. 2 inoculated with vaccine 1 showed slight redness and swelling after 2 days. Among the piglets inoculated with vaccine 7, 2 / 5 of the piglets showed redness and swelling of the injection site, which disappeared completely after 2-3 days; among the piglets inoculated with vaccine 8, 3 / 5 of the piglets showed redness and swelling, which disappeared completely after 2-3 days, and 1 / 5 of the piglets had hard nodules that persisted; among the piglets inoculated with vaccine 9, 2 / 5 of the piglets showed redness and swelling, which disappeared completely after 2 days; among the piglets inoculated with vaccine 10, 2 / 5 of the piglets showed redness and swelling, which disappeared completely after 2 days. The control group inoculated with PBS buffer was absorbed within 2 hours after intradermal injection of the neck, and showed no side effects.

[0096] Table 3 Results of safety test of intradermal immunization of piglets with vaccines

[0097] Note: "+" in the table means "yes", and "-" means "no".

[0098] In addition, all test pigs were observed for 14 days after intradermal inoculation, and the feeding and drinking, body weight loss, local reaction, allergic reaction and body temperature of all test pigs were observed, and the results are shown in Table 4. As shown in Table 4, all test pigs had normal feeding and drinking, and no weight loss; the piglets inoculated with vaccines 2, 7, 8, 9 and 10 showed redness and swelling or hard nodules at the inoculation site, and no abnormal reactions were observed in the other groups; the average body temperature was detected 7 days after inoculation, and the temperature difference was not more than 0.5°C.

[0099] Table 4 Results of safety test of piglets inoculated with vaccines at an overdose

[0100] Note: The representation method of body temperature (°C) in the table is: mean ± standard deviation.

[0101] The pig pseudorabies virus inactivated vaccines 1, 2, 5, 6, 7, 8, 9 and 10 prepared in Example 2 were subjected to the above safety test on pigs, and vaccines 1, 2, 5 and 6 met the current safety requirements of "Chinese Veterinary Pharmacopoeia" for veterinary vaccines, which proved that the adjuvants 1, 2, 5 and 6 were safe for intradermal immunization of pigs.

[0102] Example 4 Intradermal immunization efficacy test of pig pseudorabies virus inactivated vaccine

[0103] Control vaccine 1 was prepared as follows: 10 mL of inactivated pig pseudorabies virus PRV-NJ strain virus solution (virus content before inactivation was 10 8.0 TCID50 / mL) and GEL01 adjuvant (purchased from France Sipco Company) were mixed at a volume ratio of 9:1, stirred uniformly to obtain control vaccine 1. The viscosity of control vaccine 1 was 4-5 mPa·s, which was significantly higher than that of vaccines 5 and 6 in Example 2.

[0104] The pig immunization efficacy test was carried out by intradermal immunization of the inactivated porcine pseudorabies virus vaccines 5 and 6 and the control vaccine 1 prepared in Example 2. The specific method was as follows: 15 healthy, 30-day-old PRV-negative pigs were randomly divided into 3 groups, 5 pigs in each group. The pigs in groups 1 and 2 were injected intradermally in the neck with vaccines 5 and 6, respectively, at a dosage of 0.5 mL per pig; the pigs in the other group were injected intradermally in the neck with the control vaccine 1 at a dosage of 0.5 mL per pig. After intradermal immunization of the vaccines in the neck, the injection sites were observed, and the post-injection reactions were recorded. The judgment criteria for the reactions were as follows: ① no reaction: no change in the skin at the injection site (the same as normal skin); ② mild reaction: redness and scabbing at the injection site; ③ moderate reaction: nodules on the skin at the injection site; and ④ severe reaction: ulceration of the skin at the injection site and above. After 28 days of immunization, blood was collected to separate serum, and the PRV antibody level was detected using a porcine pseudorabies ELISA antibody detection kit (purchased from Wuhan Keqian Biological Co., Ltd.).

[0105] The vaccines 5 and 6 were absorbed by the body within 1 day, the skin bumps disappeared, and there was no reaction at the injection site. After immunization with the control vaccine 1, 2 / 5 of the pigs had hard nodules at the injection site, i.e., moderate reactions.

[0106] The results of the pig immunization efficacy test showed that the antibody titers of the pigs inoculated with the vaccines 5 and 6 were significantly higher than those of the pigs inoculated with the control vaccine 1 (the vaccine prepared using the imported adjuvant GEL01), and the antibody titer of the vaccine 5 was the highest. The main immunizing components of the vaccines 5 and 6 were seabuckthorn oil and squalene. The titers of the vaccines 5 and 6 after immunization were better than those of the control vaccine 1, which proved that seabuckthorn oil and squalene together had an immune-promoting effect.

Claims

1. An adjuvant suitable for intradermal immunization, consisting of the following components by mass parts: Squalene 10-25 parts, Sea buckthorn oil 10-25 parts, Caprylic capric acid polyethylene glycol glyceride 25-35 parts, Tween 80 25-45 parts.

2. The adjuvant of claim 1, wherein Squalene 15-20 parts, Sea buckthorn oil 15-20 parts, Caprylic capric acid polyethylene glycol glyceride 28-32 parts, Tween 80 30-40 parts.

3. Use of the adjuvant of claim 1 or 2 in the preparation of an inactivated vaccine of porcine pseudorabies virus. The inactivated vaccine of porcine pseudorabies virus is an intradermal immunization vaccine.

4. Use according to claim 3, characterized in that The adjuvant of claim 1 or 2 and the antigen are mixed at a volume ratio of 1:8-10, emulsified, to obtain the vaccine.

5. Use according to claim 4, characterized in that ​

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