Composite dual-phase adjuvant for swine foot-and-mouth disease animal vaccine as well as preparation method and application of composite dual-phase adjuvant

Abstract

The invention relates to the technical field of animal vaccine adjuvants, and discloses a composite biphasic adjuvant for a swine foot-and-mouth disease animal vaccine as well as a preparation method and application of the composite biphasic adjuvant, and the composite biphasic adjuvant comprises the following components in parts by weight: 50-65 parts of squalene; 0.1 to 0.5 part of phytosterol; 0.05 to 0.2 part of an antioxidant; 1 to 3 parts of chitosan oligosaccharide; 0.5 to 1 part of trehalose; 3-7 parts of an emulsifier; the deionized water is supplemented to 100 parts, and the antioxidant comprises 2-4 parts of a segmented copolymer PluronicF68; 0.5 to 1.5 parts of Tween 80; the antioxidant comprises the following components in parts by weight: 0.03 to 0.1 part of vitamin E (alpha-tocopherol); and 0.02 to 0.1 part of ascorbyl palmitate. By optimizing the squalene oil phase, the dual-antioxidant system and the synergistic emulsifier formula, the technical effects of high emulsion stability, high antigen delivery efficiency and remarkably enhanced immune response are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of animal vaccine adjuvants, in particular to a composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine and a preparation method and application thereof. Background Art

[0002] Foot-and-mouth disease is an acute, highly contagious disease caused by the foot-and-mouth disease virus, which has caused serious economic losses to the animal husbandry industry. Pig vaccination is one of the animals susceptible to foot-and-mouth disease and is currently an important means of preventing and controlling the epidemic. The immune effect of the vaccine is closely related to the performance of its adjuvant. Composite biphasic adjuvants have gradually become a research hotspot in vaccine development because of their ability to enhance the immune response of the vaccine.

[0003] In the prior art, the common composite biphasic adjuvant for swine foot-and-mouth disease animal vaccines uses white oil as the main oil phase, combined with a certain proportion of emulsifiers (such as Tween 80 and Span 80) and a single antioxidant (such as vitamin E), and forms an emulsion structure through an emulsification process. This adjuvant can improve the delivery efficiency of antigens to a certain extent, and at the same time has a good enhancing effect on antibody titer. In addition, the existing composite biphasic adjuvant has a certain storage stability and can be stored at room temperature for several months without stratification. After being applied to animal vaccines, it can significantly reduce the incidence of epidemics.

[0004] Although the existing technology has achieved certain results, it still has many shortcomings, which are mainly reflected in the following aspects: First, the existing adjuvant uses white oil as the main oil phase. Although it has a certain chemical stability, its biocompatibility is poor and may cause redness, swelling or allergic reactions at the injection site; second, the antioxidant system is single and cannot comprehensively inhibit the oxidative degradation of the oil phase components, resulting in the emulsion being easy to stratify and demulsify during storage, affecting the effectiveness of the vaccine; third, the design of the emulsifier is mostly a simple combination, which makes it difficult to take into account the uniformity of particle size and the stability of the emulsion structure; in addition, the immune enhancing components lack targeting and delivery efficiency, the antibody titer is low and the protection time is limited. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine and a preparation method and application thereof, which solves the problem in the prior art of reduced safety due to high toxicity and side effects of the white oil phase and poor biocompatibility.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine, the composite biphasic adjuvant comprising the following components in parts by weight: Squalene: 50-65 parts; Phytosterols: 0.1-0.5 parts; Antioxidant: 0.05-0.2 parts; Chitosan oligosaccharide: 1-3 parts; Trehalose: 0.5-1 part; Emulsifier: 3-7 parts; Deionized water is added to make up to 100 parts.

[0007] Preferably, the antioxidant comprises: Block copolymer Pluronic F68: 2 to 4 parts; Tween 80: 0.5-1.5 parts; Span 80: 0.5~1.5 servings.

[0008] Preferably, the antioxidant comprises: Vitamin E (α-tocopherol): 0.03-0.1 parts; Ascorbyl palmitate: 0.02-0.1 parts.

[0009] The present invention also provides a method for preparing a composite biphasic adjuvant for a swine foot-and-mouth disease animal vaccine, comprising the following steps: S1. preparing an oil phase, wherein the oil phase comprises squalene, phytosterols and antioxidants; S2, preparing an aqueous phase, wherein the aqueous phase comprises chitosan oligosaccharides, trehalose and an emulsifier; S3, mixing the oil phase with the water phase and emulsifying the mixture to form an emulsion; S4, homogenizing the emulsion so that the particle size of the emulsion reaches 120-150 nm; S5. Sterilize the homogenized emulsion and store it in subpackages.

[0010] Preferably, in the step of preparing the oil phase, squalene, phytosterols and antioxidants are mixed at a temperature of 45 to 55° C. and stirred until uniform.

[0011] Preferably, in the step of preparing the aqueous phase, chitosan oligosaccharides and trehalose are dissolved in deionized water, and the pH is adjusted to 6.8-7.2.

[0012] Preferably, the emulsification treatment includes high-speed shear emulsification, the shear speed is 4000-6000 rpm, and the emulsification time is 8-12 minutes.

[0013] Preferably, the homogenization treatment includes ultra-high pressure homogenization, the homogenization pressure is 100-150 MPa, the number of cycles is 2-3 times, the homogenization time is 5-10 minutes, and the homogenization temperature is 20-30°C.

[0014] Preferably, the sterilization treatment is a filtration sterilization process through a 0.22 μm filter membrane, and after filtration, the mixture is packaged into sterile glass bottles and stored at 4°C.

[0015] The present invention also provides an application method of the composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine, characterized in that it comprises the following steps: The vaccine emulsion was injected at a dose of 1.5 to 2.5 mL per pig; Vaccination is given once every 14 days, for a total of two vaccinations; The level of type O antibodies was detected 21 days and 28 days after immunization using a liquid-phase blocking ELISA kit.

[0016] The present invention provides a composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine and its preparation method and application. It has the following beneficial effects: 1. The present invention adopts an optimized composite formula design, with squalene as the main oil phase, supplemented with phytosterols and emulsifiers, and introduces a dual antioxidant system of vitamin E and ascorbyl palmitate, achieving the technical effects of improving the stability of the emulsion, extending the storage period, and enhancing biocompatibility. Compared with the technical solution of using white oil and a single antioxidant in the prior art, the shortcomings of high toxicity and side effects of white oil, easy oxidation of the emulsion, and short storage time are solved, and the stability and safety of the composite adjuvant are improved.

[0017] 2. The present invention adopts an innovative formula of chitosan oligosaccharides and trehalose as immune enhancing components, combined with optimized emulsification and ultra-high pressure homogenization processes, to prepare an emulsion with uniform particle size distribution, thereby achieving the technical effects of enhancing antigen delivery efficiency and improving immune response effects. Compared with the problems of poor emulsion stability and uneven immune effects caused by the single emulsifier design and traditional processes in the prior art, the present invention solves the problems of insufficient immune enhancing effect and short maintenance time of antibody levels, and provides technical support for the high efficiency of vaccines.

[0018] 3. The present invention adopts optimized vaccination doses and immunization procedures, combined with efficient sterilization and aseptic packaging technology, to achieve the technical effects of reducing toxic side effects, improving immune safety and extending the storage period of vaccines. Compared with the prior art that has unsatisfactory immune effects and safety issues caused by improper dosage design or insufficient sterilization process, it solves the technical bottlenecks of frequent allergic reactions after vaccination and excessive endotoxins, and provides a guarantee for the safety and long-term application of vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a flow chart of the preparation method of the present invention. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Please see attached Figure 1 The embodiment of the present invention provides a composite biphasic adjuvant for a swine foot-and-mouth disease animal vaccine, wherein the composite biphasic adjuvant comprises the following components in parts by weight: Squalene: 50-65 parts. Squalene is the main component of the oil phase, which is used to enhance the antigen delivery ability and provide long-term immune protection. The amount of squalene is between 50 and 65 parts, which ensures that the oil phase ratio of the emulsion is sufficient to maintain the stability of the emulsion, while avoiding problems such as increased emulsion viscosity and difficulty in emulsification due to excessive use. In general, the purity of squalene is required to reach more than 95% to avoid impurities affecting the stability and safety of the adjuvant. As an option, squalene can be purified by distillation or selected as a pharmaceutical grade finished product; Phytosterols: 0.1-0.5 parts. Phytosterols are used as the immune-enhancing component of the adjuvant. Their function is to enhance the biocompatibility of the adjuvant by synergizing with squalene, and to provide support for the targeting during antigen delivery. The dosage of phytosterols is 0.1-0.5 parts, which is lower than that of squalene, but it is effective in synergistically enhancing immunogenicity. Specifically, β-sitosterol or similar plant-derived sterols can be selected for phytosterols, and the purity is required to reach more than 90% to ensure its immune-adjuvant effect; Antioxidant: 0.05-0.2 parts. The dosage of antioxidant is 0.05-0.2 parts, which is composed of vitamin E (α-tocopherol) and ascorbyl palmitate. This dual antioxidant system can effectively inhibit the oxidative degradation of squalene and phytosterols during storage and ensure the long-term stability of the emulsion. In a possible implementation method, the dosage of vitamin E is 0.03-0.1 parts, and the dosage of ascorbyl palmitate is 0.02-0.1 parts. The two are used together to form a synergistic antioxidant effect. In general, the purity of vitamin E is required to be above 95%, and the purity of ascorbyl palmitate should reach 99% to reduce the impact of by-products on the stability of the emulsion; Chitosan oligosaccharide: 1-3 parts. Chitosan oligosaccharide is an important component of the aqueous phase and is used in an amount of 1-3 parts. Chitosan oligosaccharide can not only improve the biocompatibility of the emulsion, but also serve as an immunopotentiator to stimulate the efficiency of antigen delivery. Specifically, the molecular weight of chitosan oligosaccharide ranges from 1 to 10 kDa to ensure its solubility and activity. In a possible implementation method, chitosan oligosaccharide can be obtained by enzymatic hydrolysis of deacetylated chitosan raw materials, and high molecular weight impurities are removed by gradient filtration technology; Trehalose: 0.5-1 part. The dosage of trehalose is 0.5-1 part. Its main function is to stabilize the emulsion structure and improve the stability of the adjuvant during the freeze-thaw process. In general, trehalose should be selected from pharmaceutical grade raw materials with a purity requirement of more than 99%. As an option, trehalose can be obtained by enzymatic method or purification method and finally used in the aqueous phase system of the emulsion; Emulsifier: 3-7 parts, the emulsifier includes block copolymer Pluronic F68, Tween 80 and Span 80, wherein Pluronic F68 is the main emulsifier, the dosage is 2-4 parts, and it is used to form a stable emulsion interface, Tween 80 and Span 80 are auxiliary emulsifiers, the dosage is 0.5-1.5 parts respectively, specifically, the molecular weight range of Pluronic F68 is 8400-13000, which can effectively control the emulsion particle size and form a stable interface structure, Tween 80 and Span 80 can work synergistically to improve the dispersion performance of the emulsion and enhance the emulsification efficiency; Deionized water is added to 100 parts. Deionized water is used as the solvent of the aqueous phase to dissolve chitosan oligosaccharides, trehalose and emulsifiers, and is added to 100 parts of the total weight. Generally, the conductivity of deionized water should be less than 10 μS / cm to avoid impurity ions affecting the stability of the emulsion; Through the configuration and optimization of the above components, the composite biphasic adjuvant can form a stable water-in-oil-in-water (WOW) emulsion structure with a particle size distribution range of 120 to 150 nm and a polydispersity index (PDI) of less than 0.2. It has good immune enhancement effect and storage stability. In some embodiments, the adjuvant can adjust the emulsifier ratio according to actual needs to meet the needs of different vaccine preparations, thereby achieving a wider range of application scenarios.

[0022] Please see attached Figure 1 In a preferred embodiment of the present invention, the antioxidant comprises: Block copolymer Pluronic F68: 2 to 4 parts. In this embodiment, the block copolymer Pluronic F68 is used as the core component of the antioxidant system, and its usage is 2 to 4 parts. Generally, Pluronic F68 is a polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO) triblock copolymer with a molecular weight range of 8400 to 13000, which can form a stable interfacial film in the emulsion system. As an option, after Pluronic F68 is dissolved in the water phase, it will be arranged on the oil-water interface to form a double-layer membrane structure, which can not only effectively reduce the interfacial tension, but also protect the oil phase components from oxidation through its oxidation stability; In a possible implementation, the molecular weight of Pluronic F68 can be adjusted according to the control requirements of the emulsion particle size. For example, selecting a high molecular weight Pluronic F68 close to 13,000 can show a better effect on the uniformity of the emulsion particle size distribution; Tween 80: 0.5-1.5 parts. Tween 80 is a polyoxyethylene sorbitan monooleate, which contains a large number of hydrophilic groups in its molecular structure and can effectively stabilize the water-in-oil structure of the emulsion. Generally, the purity of Tween 80 should be above 99% to reduce the influence of impurities on the antioxidant performance. In some embodiments, Tween 80 can enhance the dispersibility of the emulsion and synergize with Pluronic F68 to enable the oil phase and the water phase to be more fully mixed during the emulsification process. In a possible implementation, the step of adding Tween 80 is combined with Pluronic F68, and the water phase is added after premixing, which helps to form a more stable emulsion. Span 80: 0.5-1.5 parts. The dosage of Span 80 is 0.5-1.5 parts. It acts as a lipophilic emulsifier to further enhance the stability of the emulsion and improve the synergistic effect of the antioxidant system. Generally, Span 80 is sorbitan monooleate. Its structural characteristics determine its excellent solubility in the oil phase, enabling it to form a protective film on the surface of the oil phase components (such as squalene and phytosterols) to avoid oxidative degradation. As an option, Span 80 can be combined with other components by pre-dissolving in the oil phase. In a possible implementation method, the addition ratio of Span 80 can be fine-tuned according to the mixing ratio of the oil phase and the water phase, thereby further optimizing the stability of the emulsion; As a possible optimization, Pluronic F68 in the antioxidant system can also form a temperature-sensitive reversible micelle structure in the emulsion. This structure is a stable emulsion at room temperature, and can further promote the efficiency of antigen delivery through the release mechanism under body temperature conditions after immunization. In addition, by adjusting the ratio of Tween 80 to Span 80, the viscosity of the emulsion can be effectively regulated to meet the process requirements of different vaccine preparations. Through the above-mentioned technical design and optimization, the antioxidant system of the present invention can ensure the stability of the emulsion while improving the storage period and application range of the adjuvant.

[0023] Please see attached Figure 1 In a preferred embodiment of the present invention, the antioxidant comprises: Vitamin E (α-tocopherol): 0.03-0.1 parts. In this embodiment, vitamin E (α-tocopherol) is a commonly used fat-soluble antioxidant, and its main function is to inhibit the propagation of free radical chain reactions, thereby protecting the stability of unsaturated lipid components such as squalene. Generally, the dosage of vitamin E is 0.03-0.1 parts, and its specific selection can be determined according to the oxidation potential of the oil phase components. As an option, vitamin E can use high-purity α-tocopherol, and the purity requirement is not less than 95% to avoid secondary oxidation reactions caused by impurities. Specifically, the molecular structure of vitamin E has a highly reactive hydroxyl group, which can react quickly with free radicals to generate relatively stable intermediates, thereby interrupting the oxidation chain reaction; In some embodiments, in order to enhance the solubility and uniformity of distribution of vitamin E, vitamin E can be added first in the mixing step of squalene and phytosterols, and stirred at 45-55°C. In this way, vitamin E can be fully dissolved in the oil phase and form a stable interface protection layer with the aqueous phase component in the subsequent emulsification process. In a possible implementation, the amount of vitamin E can be maintained in a ratio of 1:500 to 1:200 to squalene, thereby providing sufficient antioxidant protection without adversely affecting the emulsion structure; Ascorbyl palmitate is a water-soluble and fat-soluble antioxidant that can work synergistically with vitamin E to further improve the overall efficiency of the composite antioxidant. Generally, the dosage of ascorbyl palmitate is 0.02 to 0.1 parts. The specific dosage is optimized based on the oxidation characteristics of the oil phase and storage conditions. As an option, the purity of ascorbyl palmitate is required to be no less than 99% to ensure its stability and antioxidant activity. Specifically, the chemical structure of ascorbyl palmitate contains ascorbic acid groups and palmitate groups, in which the ascorbic acid group can react with lipid peroxides to generate inactive intermediates, while the palmitate group makes it highly lipophilic and can be embedded in the oil-water interface; In some embodiments, ascorbyl palmitate is added before emulsification, that is, it is first dissolved in the oil phase before the oil phase and the water phase are mixed, and then mixed with other components for emulsification. In a possible implementation, in order to further improve the antioxidant efficiency, it can be used in combination with vitamin E in a weight ratio of 1:1 to 1:3, thereby providing both short-term and long-term antioxidant protection in the emulsion; Ascorbyl palmitate: 0.02-0.1 parts. The antioxidant system of the present invention can effectively delay the oxidative degradation of the oil phase component in the composite biphasic adjuvant through the synergistic effect of vitamin E and ascorbyl palmitate, and improve the stability of the emulsion. In some experiments, when the total amount of the antioxidant is 0.15 parts, wherein the ratio of vitamin E to ascorbyl palmitate is 2:1, after the adjuvant is stored at 4°C for 6 months, the oxidative degradation rate of squalene is less than 2%, the emulsion particle size distribution is stable, and the polydispersity index (PDI) is less than 0.2.

[0024] Please see attached Figure 1 The present invention also provides a method for preparing a composite biphasic adjuvant for a swine foot-and-mouth disease animal vaccine, comprising the following steps: S1. preparing an oil phase, wherein the oil phase comprises squalene, phytosterols and antioxidants; S2, preparing an aqueous phase, wherein the aqueous phase comprises chitosan oligosaccharides, trehalose and an emulsifier; S3, mixing the oil phase with the water phase and emulsifying the mixture to form an emulsion; S4, homogenizing the emulsion so that the particle size of the emulsion reaches 120-150 nm; S5, sterilizing the homogenized emulsion and storing it in subpackages; In the embodiment, an oil phase is first prepared, and the oil phase includes squalene, phytosterols and antioxidants (vitamin E and ascorbyl palmitate). Generally, the oil phase components are weighed according to the proportion, placed in a constant temperature water bath at 45 to 55°C, and continuously stirred until completely dissolved to form a uniform oil phase mixture. As an option, vitamin E and ascorbyl palmitate can be dissolved in squalene first, and then phytosterols are added, which can more effectively avoid local precipitation of phytosterols. In one possible implementation, the oil phase mixing time is controlled at 10 to 15 minutes to ensure uniform distribution of the antioxidant; The preparation steps of the aqueous phase include the dissolution of chitosan oligosaccharides, trehalose and emulsifiers. Generally, chitosan oligosaccharides and trehalose are dissolved in deionized water respectively, stirred evenly and then combined, and the pH is adjusted to 6.8-7.2. As an option, dilute hydrochloric acid or dilute sodium hydroxide solution can be used to adjust the pH to avoid strong acid or strong base causing the degradation of chitosan oligosaccharides. Subsequently, the emulsifiers (Pluronic F68, Tween 80 and Span 80) are gradually added to the aqueous phase solution, stirred at room temperature until completely dissolved, filtered and set aside. In one possible implementation, the aqueous phase solution is filtered using a 0.45μm filter membrane to remove insoluble impurities and improve the emulsification efficiency; Mixing the oil phase with the water phase is one of the key steps in preparing the emulsion. In this embodiment, the oil phase and the water phase are mixed in a ratio of 4:6 or 3:7, and a high-speed shear emulsifier is used for emulsification. Specifically, the rotation speed of the high-speed shear emulsification is 4000-6000rpm, and the emulsification time is 8-12 minutes. This treatment process can make the oil phase evenly dispersed in the water phase to form colostrum. Generally, in order to avoid the influence of the temperature increase on the stability of the emulsion during the emulsification process, a cooling water bath can be used to maintain the emulsification temperature between 20 and 25°C. As an option, the uniformity of the emulsion can be observed under a microscope after emulsification to ensure that there are no obvious oil phase particles remaining; Homogenization is a core step to further optimize the particle size distribution of the emulsion. In this embodiment, the colostrum is homogenized by an ultra-high pressure homogenizer. Generally, the homogenization pressure is 100-150 MPa, the number of homogenizations is 2-3 times, and the homogenization time is controlled at 5-8 minutes each time. In a possible implementation, the pressure should be gradually increased during the homogenization process to avoid instantaneous destruction of the emulsion interface. Specifically, the homogenization process can reduce the particle size of the emulsion and reduce the particle size distribution range, so that the polydispersity index (PDI) of the emulsion is maintained below 0.2. In addition, the particle size distribution of the homogenized emulsion can be tested again after cooling to room temperature to confirm the stability of the emulsion. In this embodiment, the emulsion is filtered and sterilized through a 0.22 μm filter membrane and then dispensed into sterile glass bottles for storage. Generally, in order to prevent the emulsified structure of the emulsion from being damaged during the filtration process, it is recommended to use a low-pressure filtration process, and the filtration pressure is controlled within the range of 0.1 to 0.2 MPa. In one possible implementation, the dispensed emulsion should be stored at 4°C away from light to extend the shelf life of the product; In some embodiments, the performance of the emulsion can be further optimized by adjusting the type and proportion of the emulsifier. For example, if the freeze-thaw resistance of the emulsion needs to be improved, the proportion of Pluronic F68 can be appropriately increased, and if the viscosity of the emulsion needs to be reduced, the amount of Tween 80 can be increased. In another possible implementation, the particle size distribution of the emulsion can be adjusted to meet the requirements of different vaccine preparations by optimizing the homogenization pressure and emulsification time.

[0025] Please see attached Figure 1 The present invention also provides an application method of a composite biphasic adjuvant for a swine foot-and-mouth disease animal vaccine, comprising the following steps: The vaccine emulsion was injected at a dose of 1.5 to 2.5 mL per pig; Vaccination is given once every 14 days, for a total of two vaccinations; O-type antibody levels were measured 21 and 28 days after immunization using a liquid-phase blocking ELISA kit; Specifically: In this embodiment, firstly, an inactivated swine foot-and-mouth disease vaccine emulsion is prepared. Generally, an inactivated swine foot-and-mouth disease virus antigen solution is mixed with a composite biphasic adjuvant at a volume ratio of 1:1. The final concentration of the obtained emulsion antigen is 5-10 μg / mL. Specifically, the preparation of the antigen solution should meet the sterilization conditions. The inactivation method can be chemical inactivation (such as β-propiolactone treatment) or physical inactivation (such as high temperature inactivation). In a possible implementation, in order to ensure the uniformity of the emulsion, 0.5-1.5 μg / mL of an immunopotentiator, such as CpG oligonucleotide, can be added during the mixing process and stirred until uniform; In some embodiments, the preparation of the emulsion can be completed by a low-speed stirring process to avoid the destruction of the emulsion structure by high-speed stirring. Generally, the stirring speed is controlled at 300-500 rpm, and the stirring time is 10-15 minutes. As an option, in order to improve the stability of the emulsion, the temperature can be appropriately adjusted during the stirring process, for example, the temperature is maintained between 20 and 25°C to avoid the influence of low or high temperature on the viscosity of the emulsion and the stability of the antigen; In this embodiment, the prepared vaccine emulsion needs to be packaged into sterile glass bottles and stored under refrigeration at 4°C. Specifically, aseptic operation should be used during the packaging process to avoid secondary contamination of the vaccine. As an option, in order to further extend the shelf life of the vaccine, a protective agent, such as a stabilizer trehalose, can be added to the emulsion to improve the anti-freeze and thaw ability of the emulsion; During the vaccination process, generally, the vaccine emulsion is injected into the pig at a dose of 1.5-2.5 mL / head. Specifically, the injection site can be selected as the subcutaneous or muscular site of the neck behind the ear to ensure rapid absorption of the vaccine. In some embodiments, in order to improve the immune effect, a two-inoculation procedure can be adopted, that is, the same dose of vaccine emulsion is injected again 14 days after the initial vaccination. In a possible implementation method, the vaccination interval can be adjusted according to the immune status of the animal, for example, shortened to 10 days to cope with the epidemic in high-risk areas; In this embodiment, the antibody level of the animal needs to be tested after immunization. Generally, the antibody level test should use a liquid-phase blocking ELISA kit with high specificity, and the critical value of the O-type antibody detected is 1.050. As an option, blood samples can be collected for testing on the 21st and 28th days after immunization, and the results show that the OD450nm value is less than 1.050, which is considered positive. Specifically, animal groups with a positive rate of more than 90% can be considered to have effective immune protection; In some embodiments, in order to evaluate the safety and effectiveness of the vaccine emulsion, the health status of the immunized animals can be observed under experimental conditions, for example, whether redness, swelling or allergic reaction occurs at the injection site is recorded daily, and the observation is continued until the 28th day. In general, the composite biphasic adjuvant of the present invention exhibits low toxicity and side effects in practical applications, and no obvious adverse reactions occur; Through the above application method, the composite biphasic adjuvant of the present invention is successfully applied to the preparation and vaccination of swine foot-and-mouth disease inactivated vaccine, which improves the immune effect of the vaccine and meets the needs of the animal vaccine industry for efficient, safe and low-cost adjuvants.

[0026] In order to better understand the present invention, the above contents are described in detail below in conjunction with specific embodiments.

[0027] Example 1 (Optimized formula and preparation process of the adjuvant of the present invention) formula: Squalene: 60 parts Phytosterols: 0.3 parts Antioxidants (Vitamin E: 0.05 parts, Ascorbyl Palmitate: 0.03 parts) Chitosan oligosaccharide: 2 parts Trehalose: 0.8 parts Emulsifier (Pluronic F68: 3 parts, Tween 80: 1 part, Span 80: 1 part) Deionized water to make up to 100 parts Preparation process: Preparation of oil phase: Squalene, phytosterols and antioxidants were mixed at 50°C and stirred until uniform.

[0028] Preparation of aqueous phase: chitosan oligosaccharides and trehalose were dissolved in deionized water, pH was adjusted to 7.0, and emulsifier was gradually added; Emulsification treatment: the oil phase and the water phase were mixed in a ratio of 4:6, and high-speed shear emulsification was performed (5000 rpm, 10 minutes); Homogenization: ultra-high pressure homogenization (120MPa, 3 cycles, 25°C); Sterilization and packaging: Sterilize by filtering with a 0.22μm filter membrane, and store in sterile glass bottles.

[0029] Test results: Particle size distribution: particle size is 125nm, PDI is 0.18; Storage stability: No stratification or demulsification after storage at 4°C for 6 months; Immune effect: The positive rate of type O antibody exceeded 95% 21 days and 28 days after immunization; Safety: No obvious redness, swelling or allergic reaction at the injection site.

[0030] Example 2 (Control group without adding phytosterols and ascorbyl palmitate) formula: Squalene: 60 parts Antioxidant (Vitamin E: 0.05 parts) Chitosan oligosaccharide: 2 parts Trehalose: 0.8 parts Emulsifier (Pluronic F68: 3 parts, Tween 80: 1 part, Span 80: 1 part) Deionized water to make up to 100 parts Preparation process: Same as Example 1.

[0031] Test results: Particle size distribution: particle size is 135nm, PDI is 0.25; Storage stability: Slight stratification occurred after 3 months of storage at 4°C, and the emulsion broke after 5 months; Immune effect: The positive rate of type O antibody was 75% 21 days after immunization and 68% 28 days after immunization; Safety: Slight redness and swelling occurred at the injection site, and some animals showed allergic reactions.

[0032] Example 3 (control group without adding chitosan oligosaccharide) formula: Squalene: 60 parts Phytosterols: 0.3 parts Antioxidants (Vitamin E: 0.05 parts, Ascorbyl Palmitate: 0.03 parts) Trehalose: 0.8 parts Emulsifier (Pluronic F68: 3 parts, Tween 80: 1 part, Span 80: 1 part) Deionized water to make up to 100 parts Preparation process: Same as Example 1.

[0033] Test results: Particle size distribution: particle size is 128nm, PDI is 0.21; Storage stability: No stratification after 5 months at 4°C, but a small amount of stratification occurs after 6 months; Immune effect: The positive rate of type O antibody was 82% 21 days after immunization and 77% 28 days after immunization. Safety: No obvious toxic side effects, slight redness and swelling may occasionally occur at the injection site.

[0034] Example 4 (control group without homogenization) Formula: Same as Example 1.

[0035] Preparation process: After emulsification, the product was sterilized and packaged directly without homogenization.

[0036] Test results: Particle size distribution: particle size is 180nm, PDI is 0.35; Storage stability: After storage at 4°C for 3 months, stratification and oil droplet precipitation occurred.

[0037] Immune effect: The positive rate of type O antibody was 70% 21 days after immunization and 62% 28 days after immunization; Safety: Obvious redness and swelling occurred at the injection site, and some animals developed nodules.

[0038] Comparative experiment 1: Particle size distribution and polydispersity index test Experimental purpose: To verify the particle size distribution uniformity and polydispersity index (PDI) control ability of the adjuvant of the present invention.

[0039] Experimental setup Group design: Experimental group (the group of the present invention): prepared according to the optimized formula of the present invention, including a complete formula of squalene, phytosterols, vitamin E, ascorbyl palmitate, chitosan oligosaccharides, trehalose and emulsifier.

[0040] Control group 1 (without phytosterols and ascorbyl palmitate): phytosterols and ascorbyl palmitate were removed, and the remaining components and processes were the same.

[0041] Control group 2 (no chitosan oligosaccharide added): chitosan oligosaccharide was removed, and the remaining components and processes were the same.

[0042] Control group 3 (no homogenization): The formula was the same as that of the experimental group, but no homogenization was performed, only high-speed shear emulsification was performed.

[0043] Experimental procedures Recipe preparation: According to the formula designed by each group, the components were weighed to prepare the oil phase and the water phase; The oil phase and the water phase were mixed in proportion and emulsified using a high-speed shear emulsifier (5000 rpm, 10 min); Control group 3 was not homogenized, and the remaining groups were homogenized with ultrahigh pressure (120 MPa, 3 cycles).

[0044] Particle size measurement: The prepared emulsions were diluted to 1:100, and the particle size distribution and PDI of each group were measured using a dynamic light scattering (DLS); The test was repeated 3 times for each group and the average value was recorded.

[0045] The comparative experimental data are shown in Table 1: Table 1 Particle size distribution and PDI test data From the data in Table 1, we can get: Experimental group (present invention): The present invention group showed that the particle size distribution was concentrated in the range of 118-135 nm, the average particle size was 125 nm, and the polydispersity index (PDI) was 0.19, showing excellent particle size uniformity and stability. This was due to the synergistic effect of phytosterols and chitosan oligosaccharides, which enhanced the interface stability, and the optimization of the homogenization process, which further reduced the distribution range of the particle size; Control group 1 (without phytosterols and ascorbyl palmitate): the particle size distribution range expanded to 130-160 nm, and the PDI increased to 0.27. Due to the lack of the interfacial enhancement effect of phytosterols and the antioxidant function of ascorbyl palmitate, the interfacial stability of the emulsion decreased, the particle size distribution became wider, and the uniformity was poor; Control group 2 (no chitosan oligosaccharide added): The particle size distribution further expanded to 125-170 nm, the average particle size reached 145 nm, and the PDI was 0.24. The lack of chitosan oligosaccharide resulted in insufficient protection of the emulsion interface and decreased antigen delivery stability; Control group 3 (no homogenization): The particle size range is the widest, ranging from 140 to 200 nm, with an average particle size of 160 nm and a PDI of 0.38. The particle distribution of the emulsion without homogenization is extremely uneven, indicating the key role of the homogenization process in controlling the particle size distribution.

[0046] Comparative experiment 2: Antioxidant performance test Experimental purpose: To verify the effect of the present invention on improving the antioxidant performance of the emulsion by optimizing the antioxidant system (vitamin E and ascorbyl palmitate).

[0047] Experimental setup Group design: Experimental group (the present invention): The formula includes squalene, phytosterols, vitamin E (0.05 parts), ascorbyl palmitate (0.03 parts), chitosan oligosaccharides, trehalose and emulsifiers.

[0048] The preparation process included emulsification treatment (5000 rpm, 10 min) and ultrahigh pressure homogenization (120 MPa, 3 cycles).

[0049] Control group 1 (no ascorbyl palmitate added): Ascorbyl palmitate was removed from the formula, and the remaining components and processes were the same as those of the experimental group.

[0050] Control group 2 (no vitamin E added): Vitamin E was removed from the formula, and the remaining components and processes were the same as those of the experimental group.

[0051] Control group 3 (no antioxidant added): Vitamin E and ascorbyl palmitate were removed from the formula, and the remaining components and processes were the same as those of the experimental group.

[0052] Experimental procedures Emulsion preparation: Each group prepared the emulsion according to the formula, strictly controlling the emulsification and homogenization process parameters to ensure that the initial state of the emulsion was consistent.

[0053] Antioxidant test conditions: Each group of emulsions was placed in a 50°C environment for accelerated oxidation. The test period was 30 days, with sampling every 7 days. After each sampling, the remaining amount of squalene was determined using high performance liquid chromatography (HPLC) to evaluate the antioxidant properties of the emulsion; At the same time, observe whether the emulsion undergoes stratification or demulsification.

[0054] The comparative experimental data are shown in Table 2: Table 2 Antioxidant performance test data From the data in Table 2, we can get: Experimental group (the present invention): The experimental group showed excellent antioxidant performance, and the residual rate of squalene on the 28th day was still as high as 92.8%, and the emulsion did not stratify or break during the entire test period. This is due to the synergistic effect of vitamin E and ascorbyl palmitate, which significantly improved the antioxidant stability of the emulsion by scavenging free radicals and inhibiting peroxidation reactions; Control group 1 (no ascorbyl palmitate added): The residual rate of squalene dropped to 78.4% on the 28th day, and the emulsion began to show slight stratification on the 21st day, and the antioxidant performance was significantly lower than that of the experimental group. This indicates that the lack of ascorbyl palmitate reduces the ability to clear intermediate oxidation products, resulting in an accelerated oxidation process; Control group 2 (no vitamin E added): The residual rate of squalene was 72.5% on the 28th day, and the emulsion showed obvious stratification on the 21st day. Since vitamin E could not interrupt the free radical chain reaction, the oxidation rate was accelerated and the antioxidant performance of the emulsion decreased; Control group 3 (no antioxidant added): The residual rate of squalene was only 45.2% on the 28th day, the emulsion was severely stratified after the 14th day, and completely demulsified on the 28th day. The emulsion without antioxidant added quickly oxidized and failed in a high temperature environment, and had the worst storage stability.

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine, characterized in that: The composite biphasic adjuvant comprises the following components in parts by weight: Squalene: 50-65 parts; Phytosterols: 0.1-0.5 parts; Antioxidant: 0.05-0.2 parts; Chitosan oligosaccharide: 1-3 parts; Trehalose: 0.5-1 part; Emulsifier: 3-7 parts; Deionized water is added to make up to 100 parts.

2. The composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine according to claim 1, characterized in that: The antioxidants include: Block copolymer Pluronic F68: 2 to 4 parts; Tween 80: 0.5-1.5 parts; Span 80: 0.5~1.5 servings.

3. The composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine according to claim 1, characterized in that: The antioxidants include: Vitamin E (α-tocopherol): 0.03-0.1 parts; Ascorbyl palmitate: 0.02-0.1 parts.

4. A method for preparing a composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine, characterized in that: Using the composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine according to any one of claims 1 to 3 comprises the following steps: preparing an oil phase, wherein the oil phase comprises squalene, phytosterols and an antioxidant; preparing an aqueous phase, wherein the aqueous phase comprises chitosan oligosaccharide, trehalose and an emulsifier; The oil phase is mixed with the water phase and emulsified to form an emulsion; The emulsion is homogenized so that the particle size of the emulsion reaches 120 to 150 nm; The homogenized emulsion is sterilized and stored in aliquots.

5. The method for preparing a composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine according to claim 4, characterized in that: In the step of preparing the oil phase, squalene, phytosterols and antioxidants are mixed at a temperature of 45 to 55° C. and stirred until uniform.

6. The method for preparing a composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine according to claim 4, characterized in that: In the step of preparing the aqueous phase, chitosan oligosaccharides and trehalose are dissolved in deionized water, and the pH is adjusted to 6.8-7.

2.

7. The method for preparing a composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine according to claim 1, characterized in that: The emulsification treatment includes high-speed shear emulsification, the shear speed is 4000-6000 rpm, and the emulsification time is 8-12 minutes.

8. The method for preparing a composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine according to claim 1, characterized in that: The homogenization treatment includes ultra-high pressure homogenization, the homogenization pressure is 100-150 MPa, the number of cycles is 2-3 times, the homogenization time is 5-10 minutes, and the homogenization temperature is 20-30°C.

9. The method for preparing a composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine according to claim 1, characterized in that: The sterilization treatment is a filtration sterilization process using a 0.22 μm filter membrane. After filtration, the solution is dispensed into sterile glass bottles and stored at 4° C.

10. A method for applying a composite biphasic adjuvant for a swine foot-and-mouth disease animal vaccine, characterized in that: Using the composite biphasic adjuvant for swine foot-and-mouth disease animal vaccine according to any one of claims 1 to 3 comprises the following steps: The vaccine emulsion was injected at a dose of 1.5 to 2.5 mL per pig; Vaccination is given once every 14 days, for a total of two vaccinations; The level of type O antibodies was detected 21 days and 28 days after immunization using a liquid-phase blocking ELISA kit.

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