A self-emulsifying adjuvant for veterinary vaccines, preparation method and application thereof

Abstract

The present invention provides a kind of self-emulsifying adjuvant, preparation method and application of veterinary vaccine, veterinary vaccine adjuvant field.The self-emulsifying adjuvant of the veterinary vaccine is composed of the following materials according to mass parts: 5-25 parts of mineral oil, 3-10 parts of squalene, 2-10 parts of medium-chain triglycerides, 10-25 parts of lecithin, 2-20 parts of Tween80, 10-30 parts of polyethylene glycol monooleate, 5-15 parts of polyoxyethylene pyrrolidone, and 15-35 parts of diethylene glycol monoethyl ether.The self-emulsifying adjuvant of the veterinary vaccine of the present invention, when mixed with aqueous antigen, is gently stirred with a glass rod or shaken by hand, and O / W type nanoemulsion can be spontaneously formed, and the vaccine has low viscosity, good stability, is easy to inject, has small side effects, and has good immune effect.

Description

Technical Field

[0001] The present invention relates to the field of veterinary vaccine adjuvants, and in particular to a self-emulsifying adjuvant for veterinary vaccines, a preparation method and application thereof. Background Art

[0002] Vaccine adjuvants can non-specifically increase the immune response type or immune response intensity of vaccine antigens, prolong the immune protection period, and improve the cross-protection ability of vaccines against heterologous strains. Oil-containing adjuvants are the most commonly used in animal vaccine products at home and abroad. Among them, the amount of oil and surfactant used in oil-in-water (O / W) vaccines is relatively small, the viscosity is extremely low, and it is easy to inject. However, in the prior art, the preparation of O / W vaccines requires emulsification equipment that can provide high shear force or high mechanical energy, which has high technical barriers, complex operation, long time consumption, and high cost. For example, O / W emulsions MF59 and AS03 developed by Novartis, GlaxoSmithKline and other companies prepare squalene, Tween 80, Span 85, vitamin E, etc. in appropriate proportions, and are homogenized by high-pressure microfluidization to form a uniform emulsion. Invention patent 201310021011.3 provides a ready-to-use oil-in-water emulsion prepared by colloid mill extrusion, microfluidization or high-pressure homogenization, which can significantly improve the safety, stability and effectiveness of vaccines. However, the above-mentioned process of preparing oil-in-water emulsion still requires the investment of high-value equipment, and the preparation process still requires high mechanical power. For animal vaccines, the production cost is still high. Summary of the invention

[0003] The purpose of the present invention is to provide a self-emulsifying adjuvant for veterinary vaccines. When mixed with an aqueous antigen, an O / W type nanoemulsion can be spontaneously formed by gently stirring with a glass rod or shaking by hand. The vaccine has low viscosity, good stability, easy injection, small side effects and good immune effect.

[0004] Another object of the present invention is to provide a method for preparing a self-emulsifying adjuvant for veterinary vaccines, which can spontaneously form an O / W type nanoemulsion by gently stirring with a glass rod or shaking by hand, without the need for emulsification equipment, simple operation, short time consumption and low cost.

[0005] Another object of the present invention is to provide the use of the self-emulsifying adjuvant in the preparation of veterinary vaccines, which have low viscosity, good stability, easy injection, small side effects and good immune effect.

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

[0007] A self-emulsifying adjuvant for veterinary vaccines, comprising the following substances in parts by weight:

[0008] 5-25 parts of mineral oil,

[0009] 3-10 parts of squalene,

[0010] Medium chain triglycerides 2-10 parts,

[0011] 10-25 parts of lecithin,

[0012] Tween80 2-20 servings,

[0013] 10-30 parts of polyethylene glycol monooleate,

[0014] 5-15 parts of polyoxyethylene pyrrolidone,

[0015] 15-35 parts of diethylene glycol monoethyl ether.

[0016] The present invention also provides a method for preparing the self-emulsifying adjuvant of the veterinary vaccine, comprising the following steps: mixing the components to obtain the self-emulsifying adjuvant of the veterinary vaccine.

[0017] The present invention also provides an oil-in-water vaccine containing the self-emulsifying adjuvant.

[0018] In the present invention, the oil-in-water vaccine comprises a self-emulsifying adjuvant and an inactivated antigen aqueous solution.

[0019] In the present invention, the volume ratio of the self-emulsifying adjuvant to the inactivated antigen aqueous solution is 10-25:75-90.

[0020] In the present invention, the inactivated antigen aqueous solution is a swine foot-and-mouth disease inactivated virus solution.

[0021] Beneficial effects: When the self-emulsifying adjuvant of the present invention is mixed with an aqueous antigen, it can spontaneously form an O / W type nanoemulsion by gently stirring with a glass rod or shaking it by hand, and the vaccine has low viscosity, good stability, easy injection, small side effects, and good immune effect. The preparation method of the self-emulsifying adjuvant of the present invention is simple. It can spontaneously form an O / W type nanoemulsion by gently stirring with a glass rod or shaking it by hand. It does not require high-value emulsification equipment, is simple to operate, takes a short time, and has low production cost. When the self-emulsifying adjuvant of the present invention is mixed with an aqueous antigen, the volume ratio of the adjuvant to the antigen is 10~25:75~90, which increases the proportion of the antigen in the vaccine (more than 75%), provides a larger antigen space for the preparation of multivalent multi-unit vaccines, and can reduce production costs by reducing the concentration multiple of the antigen. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the scope of protection of the present invention.

[0023] Medium chain triglycerides (MCT), product number S25953, were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0024] Lecithin (for injection) was purchased from Nanjing Well Pharmaceutical Group Co., Ltd.

[0025] Polyethylene glycol monooleate, CAS No. 9004-96-0, was purchased from Nanjing Well Pharmaceutical Group Co., Ltd.

[0026] Polyvinylpyrrolidone K-30, CAS No. 9003-39-8, purchased from Sigma-Aldrich, Product No. 81420.

[0027] Diethylene glycol monoethyl ether, product number D806762, was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Example 1

[0028] (1) Self-emulsifying adjuvant 1

[0029] The self-emulsifying adjuvant 1 is composed of the following components in parts by weight:

[0030] 9 parts of mineral oil,

[0031] 3 parts of squalene,

[0032] 3 parts of medium chain triglycerides,

[0033] 18 parts of lecithin,

[0034] Tween80 15 servings,

[0035] 17 parts of polyethylene glycol monooleate,

[0036] 5 parts of polyoxyethylene pyrrolidone,

[0037] 30 parts of diethylene glycol monoethyl ether.

[0038] (2) Self-emulsifying adjuvant 2

[0039] The self-emulsifying adjuvant 2 is composed of the following components in parts by weight:

[0040] 15 parts of mineral oil,

[0041] 5 parts of squalene,

[0042] Medium chain triglycerides 5 parts,

[0043] 20 parts of lecithin,

[0044] Tween80 3 parts,

[0045] 22 parts of polyethylene glycol monooleate,

[0046] 10 parts of polyoxyethylene pyrrolidone,

[0047] 20 parts of diethylene glycol monoethyl ether.

[0048] (3) Self-emulsifying adjuvant 3

[0049] The self-emulsifying adjuvant 3 is composed of the following components in parts by weight:

[0050] 24 parts of mineral oil,

[0051] 4 parts of squalene,

[0052] 3 parts of medium chain triglycerides,

[0053] 12 parts of lecithin,

[0054] Tween80 16 servings,

[0055] 10 parts of polyethylene glycol monooleate,

[0056] 6 parts of polyoxyethylene pyrrolidone,

[0057] 25 parts of diethylene glycol monoethyl ether.

[0058] The preparation method of each emulsified adjuvant is as follows: weigh the above ingredients according to the formula amount, gently stir with a glass rod or shake by hand, sterilize with high pressure, and store at room temperature for use.

[0059] Example 2 Preparation, property evaluation and application of self-emulsifying inactivated vaccine for swine foot-and-mouth disease

[0060] (1) Swine foot-and-mouth disease inactivated virus solution

[0061] Swine foot-and-mouth disease inactivated virus solution (containing inactivated foot-and-mouth disease O type O / MYA98 / BY / 2010 strain and A type Re-A / WH / 09 strain virus) was provided by Inner Mongolia Jinyu Baoling Biological Pharmaceutical Co., Ltd. Before inactivation, the virus content of O type and A type in each 0.2 ml of virus solution was not less than 10 7.5 TCID 50. The inactivated virus liquid meets the relevant requirements of the "Compilation of Quality Standards for Veterinary Biological Products" (2018).

[0062] (2) Preparation of self-emulsifying inactivated swine foot-and-mouth disease vaccine

[0063] Under sterile conditions, 25 mL of the self-emulsifying adjuvant 1 prepared in Example 1 was mixed with 50 mL of inactivated swine foot-and-mouth disease virus solution and 25 mL of PBS buffer (concentration of 0.01 M, pH 7.2), and shaken by hand to obtain a uniform system, which was named self-emulsifying swine foot-and-mouth disease inactivated vaccine 1.

[0064] Under sterile conditions, 20 mL of the self-emulsifying adjuvant 2 prepared in Example 1 was mixed with 50 mL of inactivated swine foot-and-mouth disease virus solution and 30 mL of PBS buffer (concentration of 0.01 M, pH 7.2), and shaken by hand to obtain a uniform system, which was named self-emulsifying swine foot-and-mouth disease inactivated vaccine 2.

[0065] Under sterile conditions, 10 mL of the self-emulsifying adjuvant 3 prepared in Example 1 was mixed with 50 mL of inactivated swine foot-and-mouth disease virus solution and 40 mL of PBS buffer (concentration of 0.01 M, pH 7.2), and shaken by hand to obtain a uniform system, which was named self-emulsifying swine foot-and-mouth disease inactivated vaccine 3.

[0066] Preparation of control vaccine A: Under sterile conditions, first mix 50 mL of inactivated swine foot-and-mouth disease virus solution with 25 mL of PBS buffer (concentration of 0.01 M, pH 7.2) (antigen phase), add the mixed antigen phase to 25 mL of ISA 25 adjuvant under stirring, and stir continuously for 10 minutes with a magnetic stirrer at a stirring speed of 2500 rpm to obtain control vaccine A.

[0067] Preparation of control vaccine B: According to the patent number 201310021011.3, the formula 4 in the invention patent entitled "A ready-to-use adjuvant for veterinary vaccine, preparation and application thereof": squalane 15g, white oil 15g, Pluronic 121 0.2g, Tween 808.8g, Speran 80 0.2g, Speran 85 8.8g, hydroxymethyl cellulose 2.0g, carbomer 971P 3.0g, water 47.0g. Weigh each component according to the formula amount, mix evenly after ice water bath overnight, emulsify by colloid mill extrusion method, filter and sterilize with 0.22μm filter membrane to obtain a ready-to-use water-in-oil adjuvant, recorded as adjuvant EAO. The control vaccine B was prepared by replacing the self-emulsifying adjuvant of the present invention with adjuvant EAO, wherein the mass ratio of EAO adjuvant to the inactivated virus liquid of swine foot-and-mouth disease was 1:1.

[0068] (3) Testing of the physical properties of vaccines

[0069] The physical properties of the self-emulsifying swine foot-and-mouth disease inactivated vaccines 1-3, control vaccine A and control vaccine B were tested by taking 1 mL of the vaccine and dropping it drop by drop onto the surface of clean cold water in a beaker to observe whether it can be quickly dispersed in the water.

[0070] Self-emulsifying swine foot-and-mouth disease inactivated vaccines 1-3, control vaccine A and control vaccine B were stored at 4°C and 37°C, respectively, and their appearance changes were observed to compare the stability of each vaccine.

[0071] The viscosity was measured using the rotational viscometer method specified in Appendix 3102 of the current Veterinary Pharmacopoeia of the People’s Republic of China.

[0072] The specific results are shown in Table 1.

[0073] Table 1 Comparison of physical properties of self-emulsifying swine foot-and-mouth disease inactivated vaccines 1-3, control vaccine A, and control vaccine B

[0074]

[0075] It can be seen from Table 1 that the vaccine prepared using the self-emulsifying adjuvant of the present invention has a stability of more than 19 months at 4°C, which is more stable than the control vaccine A and the control vaccine B.

[0076] According to the requirements of the Chinese Veterinary Pharmacopoeia, the viscosity of all vaccines for injection should not exceed 200 cP. The vaccines prepared using the self-emulsifying adjuvant of the present invention all meet the requirements, and it can be seen from Table 1 that the viscosity of the vaccine prepared using the self-emulsifying adjuvant of the present invention is significantly lower than that of the control vaccine A and the control vaccine B, which is convenient for clinical injection.

[0077] The particle size distribution of the above-mentioned inactivated vaccines was detected using a Malvern Zetasizer Nano ZS particle size analyzer, and the results are shown in Table 2. The vaccines prepared using the self-emulsifying adjuvants 1-3 of the present invention have uniform particle size distribution, and the particle size is significantly lower than that of the control vaccine A and the control vaccine B, which is beneficial to reduce the viscosity of the vaccine, improve the stability, and facilitate clinical injection.

[0078] Table 2 Comparison of particle size, Zeta potential and dispersion coefficient of each vaccine

[0079]

[0080] Example 2 Evaluation of the immune efficacy and safety of self-emulsifying inactivated swine foot-and-mouth disease vaccine in piglets

[0081] 60 piglets weighing 15-20 kg with negative antigen and antibody of swine foot-and-mouth disease virus were selected and randomly divided into 6 groups, 10 piglets in each group. One group of piglets was immunized with each emulsified swine foot-and-mouth disease inactivated vaccine, control vaccine A, and control vaccine B, with an immunization dose of 2.0 ml / head, and a blank control group was set up without immunization. Blood was collected 4 weeks after immunization, serum was separated, and the titers of swine foot-and-mouth disease O type antibody and foot-and-mouth disease A type antibody liquid phase blocking ELISA detection kit (provided by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were respectively detected. The positive rates of antibodies of different dilutions 4 weeks after immunization were statistically analyzed. The results are shown in Tables 3 and 4.

[0082] Table 3 Comparison of the immune efficacy of each vaccine (FMD O type antibody)

[0083]

[0084] Table 4 Comparison of the immune efficacy of each vaccine (FMD A antibody)

[0085]

[0086] According to the requirements of the "Compilation of Quality Standards for Veterinary Biological Products (2010)", the antibody positive rate ≥ 80% is qualified. Antibody titer ≥ 1:64 can provide more than 99% immune protection to pigs. It can be seen from Tables 3 and 4 that four weeks after immunization of piglets with vaccines prepared with self-emulsifying adjuvants 1-3, the proportion of antibody titers ≥ 1:64 is above 80%, which is significantly better than vaccines prepared with ISA25 adjuvant and EAO adjuvant. Vaccines prepared with self-emulsifying adjuvants 1-3 can significantly increase the titers of FMD O and A antibodies, thereby providing effective immune protection for animals.

[0087] (5) Safety evaluation of self-emulsifying inactivated swine foot-and-mouth disease vaccine for piglets

[0088] The above-mentioned immunized piglets were observed for their growth, mental state, changes in the appearance of the injection site, and abnormal behavior every day. Except for the lumps at the injection site of 2 pigs in the control vaccine A immunization group, which disappeared after 1-2 days, no side effects were observed in the other immunization groups. The body temperature of the immunized piglets in this embodiment was tested for 7 consecutive days, and the specific results are shown in Table 4. As can be seen from Table 5, the body temperature of the pigs after immunization with each emulsified swine foot-and-mouth disease inactivated vaccine was within the normal range of 39.0-40.0°C, without fever symptoms, while the body temperature of the pigs in the control vaccine A was greater than 40.0 degrees on the 2nd-3rd day after immunization, and then returned to normal. At the same time, the injection site of the pigs was observed with the naked eye, and no abnormal phenomena such as injury and ulcers at the injection site were found in the pigs injected with each emulsified swine foot-and-mouth disease inactivated vaccine; and 2 pigs immunized with the control vaccine A had lumps at the injection site, which disappeared in about 1-2 days. The above results indicate that the vaccine prepared with self-emulsifying adjuvants 1-3 has minimal side effects after immunizing piglets and is safe for piglets.

[0089] Table 5 Average body temperature changes of piglets in each group 7 days after immunization (℃)

[0090]

Claims

1. A self-emulsifying adjuvant for veterinary vaccines, comprising the following components in parts by weight: 5-25 parts of mineral oil, 3-10 parts of squalene, Medium chain triglycerides 2-10 parts, 10-25 parts of lecithin, Tween80 2-20 parts, 10-30 parts of polyethylene glycol monooleate, 5-15 parts of polyoxyethylene pyrrolidone, 15-35 parts of diethylene glycol monoethyl ether; The components are mixed evenly to obtain the self-emulsifying adjuvant of the veterinary vaccine; the veterinary vaccine is an inactivated vaccine for swine foot-and-mouth disease.

2. An oil-in-water vaccine containing the self-emulsifying adjuvant according to claim 1, characterized in that: The oil-in-water vaccine is composed of a self-emulsifying adjuvant and an inactivated antigen aqueous solution in a volume ratio of 10-25:75-90, and the oil-in-water vaccine is an inactivated vaccine for swine foot-and-mouth disease.

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