阅读说明：本技术 双相油佐剂口蹄疫疫苗的稳定剂及其应用 (Stabilizer of foot-and-mouth disease vaccine with biphasic oil adjuvant and application thereof ) 是由 潘京学 严石 黄书林 潘春刚 于 2021-09-30 设计创作，主要内容包括：本发明公开了双相油佐剂口蹄疫疫苗的稳定剂及其应用。所述双相油佐剂口蹄疫疫苗的稳定剂,由酸、醇、胺、聚合物、糖、电荷调节剂和水组成,所述酸的用量为0.1～1％,所述醇的用量为0.1～2％,所述胺的用量为0.1～2％,所述电荷调节剂的用量为0.1～10％,所述聚合物用量为0.1～15％,所述糖用量为0.1～20％,余量为水。本发明的稳定剂提高了双相油佐剂口蹄疫疫苗的保存效果、免疫效力和安全水平。(The invention discloses a stabilizer of a foot-and-mouth disease vaccine with a dual-phase oil adjuvant and application thereof. The stabilizer of the foot-and-mouth disease vaccine with the two-phase oil adjuvant comprises 0.1-1% of acid, 0.1-2% of alcohol, 0.1-2% of amine, 0.1-10% of charge regulator, 0.1-15% of polymer, 0.1-20% of sugar and the balance of water. The stabilizer of the invention improves the preservation effect, the immune efficacy and the safety level of the foot-and-mouth disease vaccine with the dual-phase oil adjuvant.)

1. A stabilizer of a foot-and-mouth disease vaccine with a two-phase oil adjuvant comprises 0.1-1% of acid, 0.1-2% of alcohol, 0.1-2% of amine, 0.1-10% of charge regulator, 0.1-15% of polymer, 0.1-20% of sugar and the balance of water.

2. The stabilizer according to claim 1, wherein the acid is selected from one or more of tartaric acid, citric acid, lactic acid, palmitic acid, myristic acid, malic acid, sorbic acid, maleic acid, succinic acid, lauric acid, linoleic acid, oleic acid, ricinoleic acid, citric acid and stearic acid in any combination.

3. The stabilizer of claim 1, wherein the alcohol is selected from octanol, nonanol, decanol, undecanol, dodecanol, tetradecanol, hexadecanol, octadecanol, eicosanol, docosanol, or any combination of two or more thereof.

4. The stabilizer according to claim 1, wherein the amine is one or more selected from glutamine, asparagine and nicotinamide in any combination.

5. The stabilizer according to claim 1, wherein the polymer is selected from one or more of cellulose, acrylate and copolymers thereof, povidone, polyvinyl alcohol, soluble starch, sodium hydroxymethyl starch, sodium polyacrylate salt, chitosan, polyethylene glycol (40) hydrogenated castor oil, natural gum and modified product thereof, hyaluronic acid and sodium salt thereof, guar gum, acacia, pectin and carbomer;

the cellulose is preferably selected from one or more of ethyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose and sodium carboxymethyl cellulose in any combination; the natural gum and the modified product thereof are preferably alginic acid or salt thereof;

the sugar is selected from one or more of sucrose, trehalose, heparin, arabinose, stachyose, verbascose, lactose, glucose, sorbitol, dextran, maltose, mannose, cellobiose, inositol, raffinose, inulin, xylitol, fructose, dextrin, cyclodextrin and derivatives thereof;

the cyclodextrin derivative is preferably sulfobutyl ether-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, glucosyl-beta-cyclodextrin, or dimethyl-beta-cyclodextrin.

6. The stabilizer according to claim 1, wherein the charge regulator is selected from one or more of sodium chloride, potassium chloride, ammonium chloride, monoethanolamine chloride, diethanolamine chloride, triethanolamine, sodium sulfate, sodium phosphate, disodium phosphate, pentasodium triphosphate, sodium caprylate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium citrate, sodium acetate, sodium stearate, zinc gluconate, and magnesium stearate.

7. The stabilizer according to any one of claims 1 to 6, wherein the stabilizer consists of the following components in percentage by mass: 0.1 to 0.6% of the acid, 0.2 to 0.5% of the alcohol, 0.2 to 1.2% of the amine, 0.3 to 1.95% of the charge control agent, 0.2 to 3.5% of the polymer, 2 to 12% of the sugar, and the balance of water.

8. A biphasic oil-adjuvanted foot-and-mouth disease vaccine comprising a stabiliser according to any one of claims 1 to 8, a biphasic oil adjuvant and a foot-and-mouth disease antigen.

9. The biphasic oil-adjuvanted foot-and-mouth disease vaccine of claim 8, wherein said foot-and-mouth disease antigen is a foot-and-mouth disease type a, O, C, SAT1, SAT2, SAT3 or Asia1 inactivated virus antigen;

the two-phase oil adjuvant is an oil adjuvant capable of forming a water-in-oil-in-water dosage form, and is preferably one or any combination of more than two of a mineral oil component adjuvant, a vegetable oil component adjuvant and an animal oil component adjuvant.

10. Use of the stabilizer of any one of claims 1-7 in the preparation of a two-phase oil-adjuvanted foot-and-mouth disease vaccine.

Technical Field

The invention belongs to the technical field of animal biological products, and particularly relates to a stabilizer of a foot-and-mouth disease vaccine with a dual-phase oil adjuvant and application thereof.

Background

Foot-and-Mouth Disease is an acute, hot and highly contact infectious Disease of artiodactyls such as cattle, pigs and sheep caused by Foot and Mouth Disease Virus (FMDV), can reduce the production performance of animals and cause huge direct economic loss, and is listed as the first of 16A-type animal diseases by OIE. Foot-and-mouth disease virus belongs to the family picornaviridae, the genus foot-and-mouth disease virus, which has seven serotypes: type A, type O, type C, type SAT1, type SAT2, type SAT3 and type Asia1, and the foot-and-mouth disease virus antigen has weak immunogenicity and no cross protection reaction among the types of the antigen. The prevention and control measures of the foot-and-mouth disease mainly aim at immune prevention, and safe and effective vaccines are key conditions for successfully preventing, controlling and finally eliminating the foot-and-mouth disease. The vaccine widely applied to the foot-and-mouth disease prevention and immunization at present is an inactivated virus vaccine, and is prepared by emulsifying an inactivated foot-and-mouth disease complete virus antigen and a dual-phase oil adjuvant. An adjuvant is a substance that is applied simultaneously or in advance with an antigen, and that enhances the body's ability to respond to the antigen or alters the type of immune response. Adjuvants play a crucial role in vaccines, acting both on antigens and on the body, and can be used to modulate and even alter the type of humoral and/or cellular immune response of the body's immune system that generates antigen specificity. The adjuvant mainly comprises mineral adjuvant, oil adjuvant, microbial adjuvant and the like, wherein the oil adjuvant is a common adjuvant in veterinary biological products, and can improve the immune efficacy and prolong the immune period of the vaccine through the following action mechanisms: the oil emulsion contains oil and emulsifier, and the antigen is coated in the microstructure formed by the oil phase to form a reservoir for slow release and stimulate body cells to generate antibodies; the oil emulsion stimulates local production of granuloma or inflammatory reaction, attracting aggregation of macrophages and the like, these cells producing large quantities of active mediators which in turn enhance humoral and cellular immunity. The types of oil adjuvants currently available on the market can be classified into water-in-oil (W/O), oil-in-water (O/W), water-in-oil-in-water (W/O/W), etc. according to the form of the emulsion forming the vaccine, and the type of vaccine used in each form is different. The adjuvant widely applied to the foot-and-mouth disease inactivated virus vaccine at present is a water-in-oil-in-water (W/O/W) biphasic oil adjuvant.

The complete foot-and-mouth disease virus particles (146S) in the foot-and-mouth disease inactivated antigen are immune antigens determining the immune effectiveness, the quantity and the stability of the complete foot-and-mouth disease virus particles determine the effect of the vaccine, the complete foot-and-mouth disease virus particles (146S) have obvious instability, and can be decomposed into pentamers under the conditions of isoelectric points, pH value change or temperature rise, and the immune effect of the complete foot-and-mouth disease virus particles can be greatly reduced. Meanwhile, the safety and the efficacy of the vaccine are high and low, and the safety and the efficacy are also related to the composition of an adjuvant and the stability of a vaccine emulsion. The stability of the vaccine emulsion prepared by emulsifying the antigen and the adjuvant is very important, the protective power of the emulsified vaccine and the slow release capability and safety of the vaccine injected into an organism are reflected, and the good stable form of the emulsion is an important mark for achieving immune expectation. However, in the production and application processes of the current foot-and-mouth disease vaccine, the problems of unstable immune effect, no antibody detection or low antibody generation level after immunization, short immune period and the like exist, and one of the important reasons of the problems is that the oil-water emulsion of the current dual-phase oil adjuvant foot-and-mouth disease vaccine is a thermodynamically unstable system, the vaccine is very easy to be layered or demulsified after the emulsification of the commercial oil adjuvant, and the integrity and the immunogenicity of the effective antigen cannot be fully protected. Meanwhile, when the two-phase emulsion oil adjuvant vaccine is injected into an animal body, if the emulsion is decomposed and destroyed by body fluid quickly and the effective antigen of the foot-and-mouth disease is not protected by the emulsion, the original antigen wrapped in the emulsion can enter the body fluid to be decomposed quickly, the time of stimulating the organism by the antigen is shortened, the effective antigen cannot be presented fully by cells, the immune effect is greatly reduced, the immune failure is caused, the immune effect of the vaccine cannot be fully embodied, and the current situations have adverse effects on the development of the foot-and-mouth disease vaccine industry. Meanwhile, in the existing foot-and-mouth disease vaccine composition with the two-phase oil adjuvant, the oil adjuvant component occupies a considerable proportion, and the occurrence of animal side reactions is inevitable. Therefore, how to maintain the stability of the emulsion and perform effective antigen protection, and further provide a safe and effective dual-phase oil adjuvant foot-and-mouth disease vaccine is a very key technical demand in the field of animal epidemic prevention.

Disclosure of Invention

The invention aims to provide a stabilizer applied to a dual-phase oil adjuvant foot-and-mouth disease vaccine, which can enhance the emulsion stability of the finished dual-phase oil adjuvant foot-and-mouth disease vaccine, and the obtained stable water-in-oil-in-water emulsion can ensure that the dual-phase oil adjuvant foot-and-mouth disease vaccine is in a uniform physical form when being stored.

Based on the above purposes, the stabilizer applied to the dual-phase oil adjuvant foot-and-mouth disease vaccine provided by the invention comprises acid, alcohol, amine, polymer, sugar, charge regulator and water.

The amount of the acid is 0.1-1%, the amount of the alcohol is 0.1-2%, the amount of the amine is 0.1-2%, the amount of the charge regulator is 0.1-10%, the amount of the polymer is 0.1-15%, the amount of the sugar is 0.1-20%, and the balance is water.

Wherein the acid is selected from one or more of tartaric acid, citric acid, lactic acid, palmitic acid, myristic acid, malic acid, sorbic acid, maleic acid, succinic acid, lauric acid, linoleic acid, oleic acid, ricinoleic acid, citric acid and stearic acid.

The alcohol is one or more of octanol, nonanol, decanol, undecanol, dodecanol (lauryl alcohol), tetradecanol (myristyl alcohol), hexadecanol (palmityl alcohol), octadecanol (stearyl alcohol), eicosanol (arachidyl alcohol) and docosanol (behenyl alcohol) in any combination.

The amine is one or any combination of more than two of glutamine, asparagine and nicotinamide.

The polymer is selected from one or more of cellulose, acrylate and copolymers thereof, polyvidone, polyvinyl alcohol, soluble starch, polyacrylic acid sodium salt, chitosan, polyethylene glycol (40) hydrogenated castor oil, natural gum and modified substances thereof such as alginic acid and (ammonium, calcium and potassium) salts thereof, hyaluronic acid and sodium salts thereof, guar gum, acacia, pectin and carbomer.

The sugar is selected from one or more of sucrose, trehalose, heparin, arabinose, stachyose, verbascose, lactose, glucose, maltose, mannose, cellobiose, inositol, raffinose, inulin, sorbitol, dextran, xylitol, fructose, dextrin, cyclodextrin and derivatives thereof. The cyclodextrin derivative is preferably sulfobutyl ether-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, glucosyl-beta-cyclodextrin, or dimethyl-beta-cyclodextrin.

The charge regulator is selected from one or more of sodium chloride, potassium chloride, ammonium chloride, monoethanolamine chloride, diethanolamine chloride, triethanolamine, sodium sulfate, sodium phosphate, disodium phosphate, pentasodium triphosphate, sodium caprylate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium citrate, sodium acetate, sodium stearate, zinc gluconate and magnesium stearate.

As a preferred embodiment of the present invention, the stabilizer is composed of the following components by mass: 0.1 to 0.6% of the acid, 0.2 to 0.5% of the alcohol, 0.2 to 1.2% of the amine, 0.3 to 1.95% of the charge control agent, 0.2 to 3.5% of the polymer, 2 to 12% of the sugar, and the balance of water.

In one embodiment of the present invention, the stabilizer consists of the following components in percentage by mass: 0.2 to 0.6% of the acid, 0.2 to 0.5% of the alcohol, 0.2 to 1.2% of the amine, 0.3 to 1.95% of the charge control agent, 0.2 to 3.2% of the polymer, 2 to 12% of the sugar, and the balance of water.

The second purpose of the invention is to provide a foot-and-mouth disease vaccine with a biphasic oil adjuvant.

The dual-phase oil adjuvant foot-and-mouth disease vaccine provided by the invention comprises the stabilizer and the dual-phase oil adjuvant foot-and-mouth disease vaccine.

Wherein, the foot-and-mouth disease antigen of the two-phase oil adjuvant foot-and-mouth disease vaccine is an inactivated virus antigen of foot-and-mouth disease A type, O type, C type, SAT1 type, SAT2 type, SAT3 type or Asia1 type.

In the conventional invention, protection against the inactivated foot-and-mouth disease antigen is simply provided, and both the emulsion form and the antigen stability are not considered. In the technology adopted by the invention, the foot-and-mouth disease antigen can be protected under the condition of maintaining the stability of the two-phase emulsion, the integrity of the foot-and-mouth disease effective antigen can be protected, the degradation of the effective components 146S of the foot-and-mouth disease antigen can be inhibited, the immunogenicity of the foot-and-mouth disease antigen can be ensured, and the foot-and-mouth disease vaccine can be suitable for long-term storage and complex field conditions of the foot-and-mouth disease vaccine.

The stabilizer of the invention improves the preservation effect, the immune efficacy and the safety level of the foot-and-mouth disease vaccine with the dual-phase oil adjuvant.

Detailed Description

The methods in the following examples are conventional methods unless otherwise specified.

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.

As a first aspect of the present invention, there is provided a stabilizer for a foot-and-mouth disease vaccine for use in a biphasic oil adjuvant, said stabilizer comprising (or consisting of): acid, alcohol, amine, polymer, sugar, charge regulator and balance water.

Wherein, acid, alcohol, amine, etc. are used to enhance the material composition of oil-water (or water-oil) interface film, to improve oil-water phase separation, and to prevent the formation of particle clusters. The acid includes, but is not limited to, one or any combination of more than two of tartaric acid, citric acid, lactic acid, palmitic acid, myristic acid, malic acid, sorbic acid, maleic acid, succinic acid, lauric acid, linoleic acid, oleic acid, ricinoleic acid, citric acid and stearic acid. . The alcohol includes, but is not limited to, one or any combination of two or more of octanol, nonanol, decanol, undecanol, dodecanol (lauryl alcohol), tetradecanol (myristyl alcohol), hexadecanol (palmityl alcohol), octadecanol (stearyl alcohol), eicosanol (arachidyl alcohol), and docosanol (behenyl alcohol). Such amines include, but are not limited to, glutamine, asparagine, nicotinamide. The sugar comprises one or more of sucrose, trehalose, heparin, arabinose, stachyose, verbose, lactose, glucose, maltose, mannose, cellobiose, inositol, raffinose, inulin, sorbitol, xylitol, fructose, dextrin, cyclodextrin and derivatives thereof in any combination; wherein the cyclodextrin derivative is preferably sulfobutyl ether-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, glucosyl-beta-cyclodextrin, or dimethyl-beta-cyclodextrin. The charge regulator includes, but is not limited to, sodium chloride, potassium chloride, ammonium chloride, monoethanolamine chloride, diethanolamine chloride, triethanolamine, sodium sulfate, sodium phosphate, disodium phosphate, pentasodium triphosphate, sodium caprylate, dipotassium phosphate, potassium dihydrogen phosphate, sodium citrate, sodium acetate, sodium stearate, zinc gluconate, magnesium stearate. Such polymers include, but are not limited to, hydroxyethyl cellulose, polyethylene glycol (40) hydrogenated castor oil, dextran, acrylates and copolymers thereof, povidone, polyvinyl alcohol; one or more of polyacrylic acid sodium salt, natural gum and its modifier such as alginic acid and its (ammonium, calcium, potassium) salt, hyaluronic acid and its sodium salt, guar gum, acacia, pectin, and carbomer; preferably, the cellulose is selected from one or more of ethyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose and sodium carboxymethyl cellulose in any combination; the natural gum and its modified product are preferably alginic acid or its salt. The polymer is used for adjusting the viscosity of the emulsion and enhancing the elasticity of an oil-water (or water-oil) interface film of the emulsion. The saccharide in the stabilizer and the polymer and the amine, alcohol, acid and other matters act synergistically to raise the stability of the effective antigen in the foot-and-mouth disease vaccine and prolong the storage time of the effective antigen in the vaccine for production and use.

In order to reduce the instability, the existence of polar substances such as acid, alcohol, amine and the like in the stabilizer greatly enhances the strength of an oil-water interfacial film and enhances the stability of the emulsion. Generally, most stable emulsion droplets are charged, and emulsion droplets in a system having the same sign of charge repel each other when they are in close proximity to each other, thereby preventing coalescence and also improving emulsion stability, so that charge control agents may be added by controlling the interfacial charge. In the emulsion system, the larger the viscosity of the dispersion medium, the slower the moving speed of the dispersed phase liquid beads, which is more favorable for the stability of the emulsion, and a high molecular polymer can be added for adjusting the viscosity of the emulsion. The presence of saccharides and polymers, and amines, alcohols, acids, etc. in the stabilizer may improve the stability of the effective antigen in the foot and mouth disease vaccine. The synergistic effect of the above factors can prolong the storage time of the vaccine and keep the product quality and the stability of the immune effect in the production and use processes.

As a preferred embodiment of the stabilizer of the present invention, the acid includes tartaric acid, citric acid, lactic acid, malic acid, maleic acid, sorbic acid, succinic acid, ricinoleic acid, citric acid; the alcohol comprises decanol, undecanol, dodecanol (lauryl alcohol), tetradecanol (myristyl alcohol), hexadecanol (palmityl alcohol), octadecanol (stearyl alcohol), eicosanol (arachidyl alcohol), docosanol (behenyl alcohol); amines such as glutamine, asparagine, nicotinamide; the charge regulator comprises sodium chloride, potassium chloride, ammonium chloride, monoethanolamine chloride, diethanolamine chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, pentasodium triphosphate, sodium stearate, magnesium stearate, gluconate, acetate and citrate; the polymer comprises hydroxyethyl cellulose, polyethylene glycol (PEG), dextran, acrylate and its copolymer, polyvidones, polyvinyl alcohol, polyacrylic acid and its sodium salt, chitosan, natural gum and its modifier such as alginic acid and its (ammonium, calcium, potassium) salt, and sodium hyaluronate; the sugar includes sucrose, trehalose, mannose, lactose, glucose, sorbitol, maltose, cellobiose, raffinose, dextrin, cyclodextrin and derivatives thereof.

As a more preferred embodiment of the stabilizer of the present invention, the acid includes, but is not limited to, tartaric acid, lactic acid, sorbic acid, succinic acid, linoleic acid, stearic acid, maleic acid; the alcohol includes decanol, undecanol, dodecanol (lauryl alcohol), tetradecanol (myristyl alcohol), hexadecanol (palmityl alcohol), and behenyl alcohol. The amine comprises glutamine, asparagine, nicotinamide; the charge regulator comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, potassium chloride, sodium caprylate, sodium stearate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, gluconate, acetate and citrate; the polymer comprises hydroxyethyl cellulose, polyethylene glycol (PEG), dextran, acrylate and its copolymer, polyvidone, polyvinyl alcohol, polyacrylic acid sodium salt, natural gum and its modifier such as alginic acid and its (ammonium, calcium, potassium) salt, and sodium hyaluronate; the sugar includes sucrose, trehalose, lactose, glucose, maltose, mannose, sorbitol, dextrin, cyclodextrin and its derivatives, etc.

As an embodiment of the stabilizer of the present invention, the stabilizer is composed of the following components by mass: 0.1-1% of the acid, 0.1-2% of the alcohol, 0.1-2% of the amine, 0.1-10% of the charge regulator, 0.1-15% of the polymer, 0.1-20% of the sugar, and the balance of water.

The stabilizer preferably comprises the following components in percentage by mass: 0.2 to 0.6% of the acid, 0.2 to 0.5% of the alcohol, 0.2 to 1.2% of the amine, 0.3 to 1.95% of the charge control agent, 0.2 to 3.2% of the polymer, 2 to 12% of the sugar, and the balance of water.

The second aspect of the invention provides a dual-phase oil adjuvant foot-and-mouth disease vaccine containing a stabilizer, wherein the foot-and-mouth disease vaccine comprises a foot-and-mouth disease antigen, a dual-phase oil adjuvant and the stabilizer provided by the invention. The foot-and-mouth disease antigen of the invention can be foot-and-mouth disease A type, O type, C type, SAT1 type, SAT2 type, SAT3 type, Asia1 type foot-and-mouth disease inactivated virus antigen or a multi-type combined antigen. The two-phase oil adjuvant is a commercially available oil adjuvant capable of forming a water-in-oil-in-water dosage form, and can be a mineral oil component adjuvant, a vegetable oil component adjuvant, an animal oil component adjuvant or a mixture of the above components.

In the conventional invention, protection against the inactivated foot-and-mouth disease antigen is simply provided, and both the emulsion form and the antigen stability are not considered. In the technology adopted by the invention, the foot-and-mouth disease antigen can be protected under the condition of maintaining the stability of the two-phase emulsion, the integrity of the foot-and-mouth disease effective antigen can be protected, the degradation of the effective components 146S of the foot-and-mouth disease antigen can be inhibited, the immunogenicity of the foot-and-mouth disease antigen can be ensured, and the foot-and-mouth disease vaccine can be suitable for long-term storage and complex field conditions of the foot-and-mouth disease vaccine.

The stabilizer improves the safety of the existing dual-phase oil adjuvant foot-and-mouth disease vaccine, the biocompatibility of the components of the stabilizer is good, more importantly, the stabilizer can replace a part of the dual-phase oil adjuvant to a certain extent, so that the dosage of the oil adjuvant is reduced while the stability of the vaccine emulsion is improved, the stress reaction of animals can be effectively reduced, the occurrence of side reactions is reduced, and the safety is higher.

The experimental reagents and consumables used in the following examples and experimental examples are all conventional reagent consumables and are commercially available, unless otherwise specified, and the experimental methods are all conventional methods.

Example 1 stabilizer for foot-and-mouth disease vaccine applied to biphasic oil adjuvant

The stabilizer applied to the foot-and-mouth disease vaccine of the two-phase oil adjuvant comprises the following substances in percentage by mass:

acid: tartaric acid 0.2%, sorbic acid 0.1%,

alcohol: decanol 0.3%, dodecanol (lauryl alcohol) 0.2%,

amine: 0.3 percent of glutamine,

polymer (b): sodium carboxymethylcellulose 1%, potassium alginate 0.5%,

charge regulators: potassium chloride 0.75%, sodium caprylate 0.5%,

sugar: 2 percent of lactose, 2 percent of mannose, 5 percent of dimethyl-beta-cyclodextrin,

water for injection: 87.15 percent.

Example 2 stabilizers for foot and mouth disease vaccine applied to biphasic oil adjuvant

The stabilizer applied to the foot-and-mouth disease vaccine of the two-phase oil adjuvant comprises the following substances in percentage by mass:

acid: linoleic acid 0.2%, succinic acid 0.1%,

alcohol: stearyl alcohol (stearyl alcohol) 0.2%,

amine: nicotinamide 0.3%, glutamine 0.1%,

polymer (b): 1 percent of povidone (K30), 1.5 percent of polyethylene glycol (40) hydrogenated castor oil,

charge regulators: dipotassium phosphate 0.5%, potassium dihydrogen phosphate 0.04%, potassium chloride 0.1%, zinc gluconate 0.5%,

sugar: maltose 2%, cellobiose 2%, hydroxypropyl-beta-cyclodextrin 3%,

water for injection: 88.46 percent of the total weight of the steel,

example 3 stabilizer for foot and mouth disease vaccine applied to two-phase oil adjuvant

The stabilizer applied to the foot-and-mouth disease vaccine of the two-phase oil adjuvant comprises the following substances in percentage by mass:

acid: 0.2 percent of lauric acid, 0.1 percent of stearic acid, 0.1 percent of tartaric acid,

alcohol: behenyl alcohol (behenyl alcohol) 0.2%, cetyl alcohol (palmityl alcohol) 0.1%,

amine: 0.3 percent of nicotinamide, 0.1 percent of asparagine,

polymer (b): 0.2 percent of hydroxymethyl starch sodium, 3 percent of polyethylene glycol (8000),

charge regulators: 0.75 percent of potassium chloride, 1 percent of sodium tartrate, 0.2 percent of sodium caprylate,

sugar: 5 percent of maltose, 5 percent of sulfobutyl ether-beta-cyclodextrin, 2 percent of cane sugar,

water for injection: 81.75 percent.

Example 4 stabilizers for foot and mouth disease vaccines applied to two-phase oil adjuvants

The stabilizer applied to the foot-and-mouth disease vaccine of the two-phase oil adjuvant comprises the following substances in percentage by mass:

acid: oleic acid 0.1%, ricinoleic acid 0.1%,

alcohol: 0.2 percent of dodecanol (lauryl alcohol), 0.1 percent of eicosanol (arachidyl alcohol),

amine: 0.3 percent of nicotinamide,

polymer (b): 1 percent of polyethylene glycol (6000), 1 percent of sodium hyaluronate,

charge regulators: 0.5 percent of disodium hydrogen phosphate, 0.05 percent of monopotassium phosphate, 0.1 percent of sodium caprylate,

sugar: 1 percent of sorbitol, 3 percent of maltosyl-beta-cyclodextrin,

water for injection: 92.55 percent.

Example 5 stabilizers for foot and mouth disease vaccine applied in biphasic oil adjuvant

The stabilizer applied to the foot-and-mouth disease vaccine of the two-phase oil adjuvant comprises the following substances in percentage by mass:

acid: palmitic acid 0.2%, myristic acid 0.4%,

alcohol: stearyl alcohol (stearyl alcohol) 0.1%, behenyl alcohol (behenyl alcohol) 0.4%,

amine: 0.3 percent of nicotinamide,

polymer (b): carbomer 0.1%, sodium polyacrylate 0.2%,

charge regulators: 0.1 percent of triethanolamine, 0.2 percent of sodium acetate,

sugar: mannose, glucosyl-beta-cyclodextrin 3%,

water for injection: 94 percent.

Example 6 stabilizer for foot and mouth disease vaccine applied to biphasic oil adjuvant

The stabilizer applied to the foot-and-mouth disease vaccine of the two-phase oil adjuvant comprises the following substances in percentage by mass:

acid: 0.2 percent of lauric acid, 0.1 percent of linoleic acid,

alcohol: cetyl alcohol (palmityl alcohol) 0.1%, arachidyl alcohol (arachidyl alcohol) 0.4%,

amine: 0.2 percent of glutamine,

polymer (b): 0.1 percent of hydroxyethyl cellulose, 0.1 percent of sodium hyaluronate,

charge regulators: dipotassium phosphate 0.4%, sodium dihydrogen phosphate 0.04%, sodium caprylate 0.1%,

sugar: 1 percent of sucrose, 1 percent of dimethyl-beta-cyclodextrin,

water for injection: 96.26 percent.

Example 7 stabilizers for foot and mouth disease vaccine applied in biphasic oil adjuvant

The stabilizer applied to the foot-and-mouth disease vaccine of the two-phase oil adjuvant comprises the following substances in percentage by mass:

acid: 0.1 percent of ricinoleic acid, 0.2 percent of citric acid,

alcohol: docosanol (behenyl alcohol) 0.4%,

amine: 0.3 percent of asparagine,

polymer (b): 0.2 percent of polyethylene glycol PEG (8000), 0.1 percent of sodium alginate,

charge regulators: 0.1 percent of sodium chloride, 0.2 percent of sodium citrate,

sugar: 1 percent of lactose, 2 percent of trehalose,

water for injection: 95.4 percent.

Example 8 stabilizer for foot and mouth disease vaccine applied to two-phase oil adjuvant

The stabilizer applied to the foot-and-mouth disease vaccine of the two-phase oil adjuvant comprises the following substances in percentage by mass:

acid: maleic acid 0.1%, succinic acid 0.2%,

alcohol: decanol 0.1%, docosanol (behenyl alcohol) 0.3%,

amine: asparagine 1%, nicotinamide 0.1%, glutamine 0.1%,

polymer (b): 0.2% of povidone (k90), 0.2% of hyaluronic acid, 0.2% of guar gum and 2% of ethyl cellulose,

charge regulators: 0.1 percent of ammonium chloride, 0.1 percent of sodium acetate, 0.2 percent of magnesium stearate,

sugar: 1 percent of cellobiose, 0.2 percent of inositol, 2 percent of sulfobutyl ether-beta-cyclodextrin,

water for injection: 91.9 percent.

The following examples of the invention are the stabilizers of examples 1 to 8, and the foot-and-mouth disease vaccine without the stabilizer is the control, and the adjuvants and antigens used in the experimental examples and the control are the same.

Preparation of the stabilizers

The invention provides a preparation method of a foot-and-mouth disease vaccine stabilizer, which comprises the steps of adding the charge regulator in the embodiments 1-8 into water for injection, mixing uniformly, then adding acid, alcohol, amine, polysaccharide and polymer components in sequence under the condition of uniform stirring, stirring and dispersing uniformly, and sterilizing by adopting a 0.22 micron microporous filtering membrane to obtain the stabilizer. Wherein, the components of the electrolyte regulator, the salt, the polysaccharide and the polymer are added into the water for injection while stirring, and the stirring speed is controlled within 2000 rpm. Heating is carried out in the mixing process, and the temperature is controlled to be 40-60 ℃.

The preparation method is simple and controllable, and is beneficial to large-scale industrial production and product process quality control.

Physical examination index of stabilizer

The stabilizers were tested according to the test standards to obtain physical indexes as shown in table 1:

TABLE 1 physical test indices for stabilizers

Preparation of foot-and-mouth disease vaccine with biphasic oil adjuvant

The preparation method of the animal vaccine applying the stabilizer comprises the following steps:

(1) the 206VG biphasic oil adjuvant (batch No. 190424010722) sold in Seppic company on the market is sterilized and then added into a vaccine preparation tank, the mixture is stirred and mixed evenly, the stirring speed is controlled to 2000 r/m,

(2) with stirring, a certain amount of the stabilizer prepared in examples 1, 2, 3, 4, 5, 6, 7, 8 was added.

(3) Slowly adding inactivated foot-and-mouth disease O-type MYA98/BY/2010 strain virus antigen meeting the production regulation requirements into the stabilizer-containing biphasic oil adjuvant prepared in the step (2), controlling the amount of the foot-and-mouth disease antigen to be the sum of the weights of the biphasic oil adjuvant and the stabilizer, uniformly shearing and stirring, and then carrying out labeling and subpackaging to obtain the foot-and-mouth disease vaccine respectively containing the stabilizer of the embodiment 1-the embodiment 8, wherein the foot-and-mouth disease vaccine is used as the invention embodiment 1-the invention embodiment 8.

Meanwhile, according to the production procedure of the foot-and-mouth disease vaccine, a conventional foot-and-mouth disease vaccine without a stabilizer is prepared as a comparison example without adding a stabilizer.

Foot and mouth disease vaccine quality control test

1. Dosage form detection

The foot-and-mouth disease vaccine prepared by adding the stabilizer of the invention is dispersed in a cloud form after being dropped into cold water, and is a water-in-oil-in-water dosage form.

2. Centrifugal assay

By adopting a table centrifuge, the vaccine sample is centrifuged for 15 minutes at 3500 rpm, and the phenomena of oil production, water discharge, layering, emulsion breaking and the like are avoided.

3. Particle size determination

The vaccine is measured by a nanometer particle size analyzer, the size distribution of emulsion particles is uniform, and the characteristic parameter D60 of particle size is below 500 nanometers.

4. Viscosity detection

The viscosity was measured by using a1 ml pipette (1.2 mm inner diameter at the outlet of the pipette), sucking 1 ml of a vaccine sample at 25 ℃, setting the pipette upright, allowing the vaccine sample to flow out naturally, recording the time with a stopwatch when 0.4 ml of the vaccine sample flows out, continuously measuring the time for three times, and averaging the values. The numerical value meets the standard requirements of veterinary drug code.

5. Sterility testing

10g of casein peptone, 1000ml of meat extract broth culture medium, 5g of sodium chloride and 15-20 g of agar, adding the casein peptone and the sodium chloride into the meat extract broth culture medium, dissolving at a low temperature, adjusting the pH value to be alkalescent, boiling, filtering, adjusting the pH value to be 7.2 +/-0.2 after sterilization, subpackaging and sterilizing. After the vaccine and the culture medium are inoculated, the culture is carried out for 48 hours at the temperature of 30-35 ℃, and the observation result is aseptic.

Foot and mouth disease vaccine stability test

Stability test at 1.4 deg.C

The prepared vaccine is stable and effective after being placed for 12 months at 4 ℃; the vaccine has stable form and does not have the phenomenon of demulsification and water yielding. The effective antigen retention of the aftosa vaccines containing the stabilizers of examples 1-8 was between 94.6% and 92.6%, much higher than 73.3% of the control, as shown in table 2 below:

TABLE 2 effective antigen (146S) retention after 12 months at 4 ℃

Group of	146S preservation Rate in 12 months
		Stabilizer-free foot and mouth disease vaccine	73.3％
Foot and mouth disease vaccine containing the stabilizer of example 1	94.6％
		Foot and mouth disease vaccine containing the stabilizer of example 2	93.7％
Foot and mouth disease vaccine containing the stabilizer of example 3	94.1％
		Foot and mouth disease vaccine containing the stabilizer of example 4	92.6％
Foot and mouth disease vaccine containing the stabilizer of example 5	93.2％
		Foot and mouth disease vaccine containing the stabilizer of example 6	92.8％
Foot and mouth disease vaccine containing the stabilizer of example 7	94.2％
		Foot and mouth disease vaccine containing the stabilizer of example 8	93.5％

2.37 ℃ vaccine accelerated aging test

TABLE 3 vaccine morphology after incubation at 37 deg.C

Group of	9 days	Day 12	15 days	18 days	Day 21
						Stabilizer-free foot and mouth disease vaccine	Demulsification	Demulsification	Demulsification	Demulsification	Demulsification
Foot and mouth disease vaccine containing stabilizer (examples 1-8)	Is normal	Is normal	Is normal	Is normal	Is normal

Table 3 shows the forms of the invention example and the comparison example after accelerated aging at 37 ℃, and it can be observed from experiments that the foot-and-mouth disease vaccine of the comparison example breaks the water within 2 days, while the invention example still maintains the stability of the emulsion within 21 days.

Table 4 shows the residual amounts of effective antigens of the aftosa vaccines measured by HPLC at 37 ℃ for 9 days, 12 days, 14 days, 18 days and 21 days, and the residual amounts of effective antigens of the aftosa vaccines containing the stabilizers of examples 1 to 8 were significantly higher than those of the control examples at each of the 9 days, 12 days, 15 days, 18 days and 21 days.

TABLE 4 determination of the content of effective antigens of vaccines after leaving at 37 deg.C

Group of	9 days	Day 12	15 days	18 days	Day 21
						Control two-phase oil-adjuvanted vaccine	71％	54％	27％	12％	0
Foot and mouth disease vaccine containing the stabilizer of example 1	85％	78％	64％	55％	46％
						Foot and mouth disease vaccine containing the stabilizer of example 2	81％	77％	66％	51％	43％
Foot and mouth disease vaccine containing the stabilizer of example 3	82％	72％	63％	50％	41％
						Foot and mouth disease vaccine containing the stabilizer of example 4	84％	76％	65％	53％	44％
Foot and mouth disease vaccine containing the stabilizer of example 5	86％	74％	62％	51％	42％
						Foot and mouth disease vaccine containing the stabilizer of example 6	80％	71％	63％	52％	40％
Foot and mouth disease vaccine containing the stabilizer of example 7	81％	72％	63％	51％	42％
						Foot and mouth disease vaccine containing the stabilizer of example 8	83％	75％	63％	51％	43％

Safety testing of foot and mouth disease vaccines

2 guinea pigs weighing 350-450 g were injected into each abdominal cavity with 2ml of each of the vaccines in the invention and control groups: each of 5 mice weighing 18-22 g was injected subcutaneously with 0.5ml of each of the vaccines of the invention and the control. After continuously observing for 7 days, no abnormal clinical reaction appears in all the observations.

2 healthy susceptible piglets (without foot-and-mouth disease neutralizing antibody measured by a suckling mouse neutralization test) with the age of 30-40 days are injected with 2 vaccine parts of each group of the invention example and the control example through points after the ear roots on two sides, and the continuous observation is carried out for 14 days one by one. No foot-and-mouth disease symptoms or obvious abnormal toxic reaction caused by vaccine injection appear.

1. Cell safety assay

Cell Counting Kit-8(CCK-8) Kit is adopted for Cell safety evaluation and determination. Using PK15 adherent cells, the number of the cells is 100000 per milliliter, inoculating the cells into a 96-well plate, DMEM culture medium is added to the outermost circle of the pore plate as a blank pore, 100 mu L of cell sap is added to each of the experimental pore and the blank control pore, 10 mu L of the inventive vaccine and the comparative vaccine are respectively added to each of the experimental pore and the blank control pore, 10 mu L of culture medium is added to each of the blank control pore, each group adopts 5 times of repetition, after 48 hours of incubation at 37 ℃ in a carbon dioxide incubator, the cell solution in the 96-well plate was aspirated, washed three times with 300. mu.L/well of PBS, tapped dry, 100. mu.L of medium and 10. mu.L of CCK-8 were added to each well, and after further incubation at 37 ℃ in a carbon dioxide incubator for 4 hours, absorbance at a wavelength of 450nm was read in each well using a microplate reader, and the data of each set was calculated according to the following formula for cell viability [ (%) [ A (load) -A (blank) ]/[ A (load) -A (blank) as shown in Table 5 below:

TABLE 5 cell viability of different vaccines

Grouping	Cell viability
		Inventive example 1	75.5％
Inventive example 2	73.9％
		Inventive example 3	71.2％
Inventive example 4	72.4％
		Inventive example 5	73.1％
Inventive example 6	72.8％
		Inventive example 7	74.8％
Inventive example 8	75.1％
		Comparative example	54.3％

The experimental result shows that the cell viability of each group in the invention example is obviously higher than that of the comparative example, and the stabilizer vaccine provided by the invention has good cell safety.

Efficacy testing of foot and mouth disease vaccines

Preferably, the formula of the vaccine of the invention example 1, the vaccine of the invention example group prepared by the preparation method, the vaccine of the control example group and the blank group are selected, the skeleton pig with the weight of about 40kg is selected, the foot-and-mouth disease-free neutralizing antibody is determined by a suckling mouse neutralization test, three groups of 13 heads are used, 2 ml/head of each of the invention example group and the control example group, 5 heads of each of the invention example group and the control example group and 10 heads of each of the invention example group and the control example group are respectively injected, and 3 heads of the blank group are arranged. After inoculation, blood is collected every seven days, serum is separated, and the antibody level is determined by adopting a foot-and-mouth disease antibody liquid blocking ELISA detection kit. The immunized mount pigs were tested for antibodies and the data are shown in table 6 below.

TABLE 6 foot and mouth disease antibody level assay

As can be seen from the test table 6 of the foot-and-mouth disease antibody level, the antibody level of the invention group is higher, the high antibody maintenance time is longer, and long-term protection can be obtained; the control group had low antibody levels and short high antibody retention times, and could not achieve long-term protection.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.