A water-in-oil-in-water adjuvant for animal vaccines and methods of making and using the same

Abstract

The application provides a water-in-oil-in-water adjuvant for livestock vaccines and a preparation method and application thereof, and belongs to the technical field of animal vaccines of biological products. The raw materials of the water-in-oil-in-water adjuvant include, in percentage by mass, 75wt%-85wt% of injection oil, 1wt%-5wt% of refined Span-80 and 10wt%-24wt% of refined Tween-80, and can further include 1wt%-3wt% of an immunostimulating complex. The vaccine prepared by using the water-in-oil-in-water adjuvant provided by the application is stable in quality, high in safety, and can induce the body to produce an immune response with a longer duration and higher efficiency, and therefore can be used as a safe and effective adjuvant for livestock vaccines and the like.

Description

Technical Field

[0001] This invention belongs to the field of veterinary vaccine technology, specifically relating to a water-in-oil-in-water adjuvant for livestock vaccines, its preparation method, and its application. Background Technology

[0002] Immunological adjuvants are components that, when injected into an animal before or simultaneously with the antigen, can non-specifically alter or enhance the body's specific immune response to that antigen, thus playing a supportive role. Currently, the immunological adjuvants widely used and researched in veterinary vaccines mainly include inorganic adjuvants, such as aluminum hydroxide and alum; organic adjuvants, microorganisms and their products, such as mycobacteria, lipopolysaccharides, cell wall phthalopeptides, cytokines, lipid-soluble waxes, short corynebacteria, Bordetella pertussis, endotoxins, etc.; synthetic adjuvants, such as levamisole, levamisole, and liposomes; and oil adjuvants, such as Freund's adjuvant, mineral oil, peanut oil emulsion, and animal oil adjuvants. Among the many immunological adjuvants, oil adjuvants (such as mineral oil) are the most widely used adjuvants in veterinary vaccines, especially livestock and poultry vaccines. In the preparation of vaccines using oil adjuvants as immunizing adjuvants, the antigens are encapsulated in oil droplets through an emulsification process. The oil droplets can act as an antigen reservoir, continuously releasing antigens at the injection site and preventing rapid clearance of antigens from the injection site. Therefore, oil adjuvants can induce a sustained level of immune response.

[0003] Oil adjuvants commonly used in animal vaccine preparation can be classified into three dosage forms: water-in-oil (W / O), oil-in-water (O / W), and water-in-oil-in-water (W / O / W). Among them, water-in-oil-in-water (W / O / W) adjuvants are more widely used due to their low viscosity, ease of emulsification, and safety and efficacy.

[0004] Water-in-oil-in-water (W / O / W) adjuvants are an important component of inactivated vaccines for livestock. Each dose of finished vaccine contains approximately 1 ml of adjuvant, accounting for about 50% of the total vaccine volume, and plays a crucial role in the sustained immune response. However, existing inactivated vaccines prepared with W / O / W adjuvants still have shortcomings in various aspects, mainly manifested in poor quality stability and safety, short duration of immunity, and insufficient immunogenicity. In complex breeding environments, illness or death often occurs due to individual differences. Summary of the Invention

[0005] To address one or more problems existing in the prior art, one aspect of the present invention provides a water-in-oil-in-water adjuvant for livestock vaccines, which is prepared from raw materials, wherein the raw materials, by weight percentage, comprise: 75wt%-85wt% of injectable oil, 1wt%-5wt% of refined Span-80, and 10wt%-24wt% of refined Tween-80; wherein:

[0006] The refined Span-80 is a product obtained by processing Span-80 in the following manner:

[0007] 1) Treating Span-80 with H2O2 causes the color of Span-80 to change from dark to light. When the color of Span-80 no longer changes, the first treated product is obtained.

[0008] 2) Remove unreacted H2O2 from the first treated product to obtain the second treated product;

[0009] 3) Filter the second processed product to obtain the purified Span-80;

[0010] The refined Tween-80 is a product obtained by treating Tween-80 in the following manner:

[0011] (1) Tween-80 is treated with H2O2 to change the color of Tween-80 from dark to light. When the color of Tween-80 no longer changes, the third treatment product is obtained.

[0012] (2) Remove unreacted H2O2 from the third processed product to obtain the fourth processed product;

[0013] (3) The fourth processed product is filtered to obtain the refined Tween-80.

[0014] In some embodiments, the raw materials comprise, by weight percentage: 80wt%-85wt% of injectable oil, 2wt%-3wt% of refined Span-80, and 12wt%-18wt% of refined Tween-80.

[0015] In some embodiments, the filtration described in steps 3) and (3) is to first perform coarse filtration using a filter device with a pore size of 0.40μm-0.65μm, and then perform fine filtration using a filter device with a pore size of 0.10μm-0.22μm.

[0016] In some embodiments, the injectable oil includes injectable mineral oil, injectable vegetable oil, or a combination thereof;

[0017] Optionally, the mineral oil for injection includes white oil;

[0018] Further optionally, the white oil includes Marcol-52 white oil, Primol 352 white oil, Total130# white oil, Total150# white oil, Total170# white oil, Drakeol-5 white oil, Drakeol-7 white oil, Sonneborn 4# white oil, Sonneborn 10# white oil, Paracos KF40, Paracos KF50, squalene, and squalane.

[0019] In some embodiments, the raw materials further include 1 wt% to 3 wt% of an immunostimulatory complex, the immunostimulatory complex comprising phospholipids, cholesterol, tocopherols and saponins in a mass ratio of (0.5-2):(0.5-2):(0.5-2):(0.5-3).

[0020] In another aspect, the present invention provides a method for preparing a water-in-oil-in-water adjuvant for livestock vaccines, comprising the following steps:

[0021] S1: Heat 75wt%-85wt% of the injection oil to 30℃-40℃ by weight percentage;

[0022] S2: 1wt%-5wt% of refined Span-80 and 10wt%-24wt% of refined Tween-80 (by weight percentage) are added to the heated injection oil from step S1, mixed thoroughly, and then filtered to obtain the water-in-oil-in-water adjuvant for the animal vaccine; wherein:

[0023] The refined Sben-80 is obtained by processing Sben-80 in the following way:

[0024] 1) Treating Span-80 with H2O2 causes the color of Span-80 to change from dark to light. When the color of Span-80 no longer changes, the first treated product is obtained.

[0025] 2) Remove unreacted H2O2 from the first processed product (e.g., by vacuum dehydration) to obtain the second processed product;

[0026] 3) Filter the second processed product to obtain the purified Span-80;

[0027] The refined Tween-80 is obtained by processing Tween-80 in the following way:

[0028] (1) Tween-80 is treated with H2O2 to change the color of Tween-80 from dark to light. When the color of Tween-80 no longer changes, the third treatment product is obtained.

[0029] (2) Remove unreacted H2O2 from the third processed product (e.g., by vacuum dehydration) to obtain the fourth processed product;

[0030] (3) The fourth processed product is filtered to obtain the refined Tween-80.

[0031] In some embodiments, the filtration described in steps 3) and (3) is to first perform coarse filtration using a filter device with a pore size of 0.40μm-0.65μm, and then perform fine filtration using a filter device with a pore size of 0.10μm-0.22μm.

[0032] In some embodiments, in step S2, an immunostimulatory complex of 1 wt% to 3 wt% by weight is added to the heated injection oil. The immunostimulatory complex comprises phospholipids, cholesterol, tocopherols, and saponins in a mass ratio of (0.5-2):(0.5-2):(0.5-2):(0.5-3).

[0033] In some embodiments, the preparation of the immunostimulatory complex includes the following steps:

[0034] T1: Dissolve a mixture of phospholipids, cholesterol, tocopherols and saponins in a mass percentage of (0.5-2):(0.5-2):(0.5-2):(0.5-3) in a chloroform / methanol solvent to obtain a lipid-like organic solution; optionally, the chloroform / methanol solvent is composed of chloroform, methanol and ultrapure water in a volume ratio of 20:60:20.

[0035] T2: Remove the organic solvent from the organic solution of the lipids obtained in step T1 to obtain solids;

[0036] T3: Dissolve the solid obtained in step T2 with PBS solution to obtain the first mixed solution;

[0037] T4: The first mixed solution obtained in step T3 is subjected to ultrasonic treatment to obtain the second mixed solution;

[0038] T5: Filter the second mixed solution obtained in step T4 to obtain the immunostimulatory complex.

[0039] In some embodiments, the conditions for ultrasonic treatment in step T4 include: a temperature of 20-30°C, a power of 50-70W, a total ultrasonic treatment time of 5-15 minutes, and an ultrasonic mode of 10 seconds of ultrasonic treatment followed by a 10-second pause.

[0040] In another aspect, the present invention provides a livestock vaccine comprising the above-mentioned livestock vaccine water-in-oil-in-water adjuvant.

[0041] The water-in-oil-in-water adjuvant for livestock vaccines provided by the above technical solutions uses purified Sben-80 and purified Tween-80 as raw materials, and can further utilize immunostimulatory complexes. This results in vaccine products prepared using the water-in-oil-in-water adjuvant provided by the present invention exhibiting stable quality and high safety. Surprisingly, it can also induce a longer-lasting (e.g., the immune protection period for piglets using the foot-and-mouth disease type O inactivated vaccine prepared with it can be up to 150 days) and a more efficient (e.g., the antibody titer after immunizing piglets with the foot-and-mouth disease type O inactivated vaccine prepared with it can reach up to 1:2880) specific immune response. Therefore, the water-in-oil-in-water adjuvant provided by the present invention can help develop livestock vaccine products with better stability and safety, longer duration of immunity, and better immunization efficacy. Attached Figure Description

[0042] Figure 1 This is a particle size distribution diagram of the foot-and-mouth disease type O inactivated vaccine prepared in Example 1. Detailed Implementation

[0043] To address the shortcomings of existing water-in-oil-in-water adjuvants in vaccine preparation, such as poor stability and safety, short duration of immunity, and insufficient immunogenicity, this invention aims to provide a water-in-oil-in-water adjuvant for livestock vaccines. When used in vaccine preparation, this adjuvant effectively improves vaccine quality stability and safety, extends the duration of immunity, and enhances the body's specific immune response to antigens in the vaccine. This invention also provides a method for preparing this water-in-oil-in-water adjuvant.

[0044] The present invention will be described in detail below through specific embodiments. The terms "first," "second," "third," and "fourth," etc., used in this document are for distinguishing similar objects and are not intended to limit a specific order or sequence, nor are they intended to limit the number of objects.

[0045] In a first aspect of the present invention, a water-in-oil-in-water adjuvant for livestock vaccines is provided, wherein the raw materials, by weight percentage, may include: 75wt%-85wt% of injectable oil, 1wt%-5wt% of refined Span-80, and 10wt%-24wt% of refined Tween-80; wherein:

[0046] The refined Span-80 may be a product obtained by processing Span-80 in the following ways, wherein Span-80 may be commercially available or obtained according to conventional Span-80 preparation methods:

[0047] 1) Treating Span-80 with H2O2 causes the color of Span-80 to change from dark to light. When the color of Span-80 no longer changes, the first treated product is obtained. This step may specifically include the following operations: Weighing Span-80 into a stainless steel reactor, starting the stirrer until uniform stirring, then introducing nitrogen gas to reduce the pressure and raising the temperature to 80±5℃ / 700mmHg, with an internal pressure of 0.15-0.25MPa. Slowly adding H2O2, to control the amount of H2O2 added, measuring a small amount of H2O2 and connecting it to a vacuum pump for slow dripping, observing the color change of Span-80 in the reactor. When the color changes from dark to light, stop adding H2O2 and maintain the reaction time for 10-15 minutes.

[0048] 2) Remove unreacted H2O2 from the first processed product (e.g., by vacuum dehydration) to obtain the second processed product. This step may specifically include the following operations: when the color of Span-80 in the reactor no longer changes and it is slightly yellow, clear, and transparent, heat the reactor to 110±5℃, and vacuum dehydrate to remove excess unreacted H2O2 from the reactor. The vacuum dehydration time is 3-5 minutes.

[0049] 3) Filter the second processed product to obtain the purified Sben-80. This step may specifically include the following operations: stop the vacuuming in step 2), continue to introduce nitrogen to maintain pressure and cool down, and discharge the second processed product from the reactor; connect the discharge to a 0.40μm-0.65μm filter device to coarsely filter the liquid to remove unreacted substances, and then transfer the filtrate to a sterile environment and filter it again with a 0.10μm-0.22μm filter membrane to obtain purified Sben-80.

[0050] The refined Tween-80 can be a product obtained by treating Tween-80 in the following ways, wherein Tween-80 can be commercially available or obtained according to conventional Tween-80 preparation methods:

[0051] (1) Tween-80 is treated with H2O2 to change its color from dark to light. When the color of Tween-80 no longer changes, the third treated product is obtained. This step may include the following operations: weigh Tween-80 and put it into a stainless steel reactor. Start stirring until the mixture is uniformly stirred. Then, introduce nitrogen gas to reduce the pressure and raise the temperature to 80±5℃ / 700mmHg. The pressure inside the reactor is 0.15-0.25MPa. Slowly add H2O2. To control the amount of H2O2 added, measure a small amount of H2O2 and slowly drip it into the reactor using a vacuum pump. Observe the color change of Tween-80 in the reactor. When the color changes from dark to light, stop adding H2O2 and maintain the reaction time for 10-15 minutes.

[0052] (2) Remove the unreacted H2O2 from the third processed product (e.g., by vacuum dehydration) to obtain the fourth processed product. This step may specifically include the following operations: when the color of Tween-80 in the reactor no longer changes and is slightly yellow, clear and transparent, heat the reactor to 110±5℃, and vacuum dehydrate to remove the excess unreacted H2O2 from the reactor. The vacuum dehydration time is 3-5 minutes.

[0053] (3) The fourth processed product is filtered to obtain the refined Tween-80. This step may specifically include the following operations: stop the vacuuming in step (2), continue to introduce nitrogen to maintain pressure and cool down, and discharge the fourth processed product from the reactor; connect the discharge to a 0.40μm-0.65μm filter device to coarsely filter the liquid to remove unreacted substances, and then transfer the filtrate to a sterile environment and filter it again with a 0.10μm-0.22μm filter membrane to obtain refined Tween-80.

[0054] In this invention, compared to vaccine adjuvants formulated using commercially available untreated Span-80 and / or Tween-80, as well as ISA206 adjuvant, vaccine adjuvants formulated using purified Span-80 and purified Tween-80 can effectively improve the quality stability and safety of the prepared vaccine, and can further enhance the specific immune response level of animals to antigens in the vaccine. This is likely because it removes excess byproducts and impurities introduced from the raw materials, thereby improving the stability and safety of the vaccine, and simultaneously enhancing its immunogenicity.

[0055] In some embodiments, the injectable oil may include injectable mineral oil, injectable vegetable oil, or a combination thereof.

[0056] In some embodiments, the injectable mineral oil may include white oil.

[0057] In some embodiments, the white oil includes, but is not limited to, Marcol-52 white oil, Primol 352 white oil, Total130# white oil, Total150# white oil, Total170# white oil, Drakeol-5 white oil, Drakeol-7 white oil, and Sonneborn 4# white oil, Sonneborn 10# white oil, Paracos KF40, Paracos KF50, squalene, and squalane, all of which are available from any commercial source.

[0058] In a preferred embodiment, the raw materials for the vaccine adjuvant may include, by weight percentage: 80wt%-85wt% of injectable oil, 2wt%-3wt% of refined Span-80, and 12wt%-18wt% of refined Tween-80; the water-in-oil-in-water vaccine adjuvant obtained under these conditions may further enhance the level of specific immune response of the animal to the antigens in the vaccine.

[0059] The animal vaccine product prepared using the water-in-oil-in-water adjuvant provided by this invention is a water-in-oil-in-water particle. This particle has a three-layered structure: the outermost layer is the aqueous phase (i.e., antigen), the middle layer is the oil phase (i.e., the adjuvant of this invention), and the innermost layer is the aqueous phase (i.e., antigen). When the vaccine is injected into the body, the particle ruptures due to the animal's body temperature, releasing the antigenic substances. The antigen is then presented to antigen-presenting cells, which process it before presenting it to T cells, inducing a specific immune response in the body.

[0060] In some embodiments, the vaccine adjuvant may further include 1 wt% to 3 wt% of an immunostimulatory complex by weight percentage, said immunostimulatory complex comprising phospholipids, cholesterol, tocopherols, and saponins in a mass ratio of (0.5-2):(0.5-2):(0.5-2):(0.5-3). The further inclusion of 1 wt% to 3 wt% of the immunostimulatory complex in the vaccine adjuvant can further enhance the body's specific immune response to antigens in the vaccine containing it.

[0061] When a vaccine is prepared using a vaccine adjuvant containing an immunostimulatory complex, the finished vaccine is a water-in-oil-in-water particle containing the immunostimulatory complex. This particle has a three-layered structure: the outermost layer is an aqueous phase (i.e., a combination of antigen and immunostimulatory complex), the middle layer is an oil phase (i.e., the adjuvant of this invention), and the innermost layer is an aqueous phase (i.e., a combination of antigen and immunostimulatory complex). When the vaccine is injected into the body, the particles rupture due to the animal's body temperature, releasing the antigen bound to the immunostimulatory complex. The immunostimulatory complex is a highly efficient immune presentation system that activates antigen-presenting cells. Through the water-in-oil-in-water adjuvant, various inflammatory cells are generated at the antigen injection site, further increasing the uptake of antigenic substances.

[0062] In a second aspect of the invention, a method for preparing a vaccine adjuvant is provided, which may include the following steps:

[0063] S1: Heat 75wt%-85wt% of the injection oil to 30℃-40℃ by weight percentage;

[0064] S2: 1wt%-5wt% of refined Span-80 and 10wt%-24wt% of refined Tween-80 (by weight percentage) are added to the heated injection oil from step S1, mixed thoroughly, and then filtered to obtain the vaccine adjuvant; wherein:

[0065] Both the refined Sben-80 and the refined Tween-80 can be obtained by the processing method described in the first aspect of the invention.

[0066] In some embodiments, in step S2, an immunostimulatory complex of 1 wt% to 3 wt% by weight may be added to the heated injection oil. The immunostimulatory complex comprises phospholipids, cholesterol, tocopherols and saponins in a mass ratio of (0.5-2):(0.5-2):(0.5-2):(0.5-3).

[0067] In some embodiments, the preparation of the immunostimulatory complex includes the following steps:

[0068] T1: Dissolve a mixture of phospholipids, cholesterol, tocopherols and saponins in a mass percentage of (0.5-2):(0.5-2):(0.5-2):(0.5-3) in a chloroform / methanol solvent to obtain a lipid-like organic solution; optionally, the chloroform / methanol solvent is composed of chloroform, methanol and ultrapure water in a volume ratio of 20:60:20.

[0069] T2: Remove the organic solvent from the organic solution of the lipids obtained in step T1 to obtain solids;

[0070] T3: Dissolve the solid obtained in step T2 with PBS solution to obtain the first mixed solution;

[0071] T4: The first mixed solution obtained in step T3 is subjected to ultrasonic treatment to obtain the second mixed solution;

[0072] T5: Filter the second mixed solution obtained in step T4 to obtain the immunostimulatory complex.

[0073] In a preferred embodiment, the conditions for ultrasonic treatment in step T4 include: a temperature of 20-30°C, a power of 50-70W, a total ultrasonic treatment time of 5-15 minutes, and an ultrasonic mode of 10 seconds of ultrasonic treatment followed by a 10-second pause.

[0074] Example

[0075] Unless otherwise specified, the methods used in the following embodiments are conventional methods. The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials for this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0076] The embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. The embodiments will help to understand the present invention, but should not be regarded as limiting the content of the present invention.

[0077] Example 1:

[0078] In this embodiment, a water-in-oil-in-water adjuvant is prepared as a vaccine adjuvant, and the vaccine adjuvant and the foot-and-mouth disease type O whole virus inactivated antigen MYA98 strain (provided by Jinyu Baoling Biopharmaceutical Co., Ltd.) are formulated into a foot-and-mouth disease type O inactivated vaccine at a mass ratio of 1:1.

[0079] 1.1 The preparation method of vaccine adjuvant includes the following steps:

[0080] S1: Heat 80 wt% Marcol-52 white oil to 35°C;

[0081] S2: Add 3 wt% refined Span-80 and 17 wt% refined Tween-80 (by weight percentage) to the heated injection oil from step S1, mix well, and then filter (e.g., using a 0.22 μm filter membrane) to obtain the vaccine adjuvant. Wherein:

[0082] The refined Span-80 mentioned in step S2 is obtained by the following method:

[0083] 1) Weigh out Span-80 (purchased from Guangzhou Qiaoling'er Biotechnology Co., Ltd.) and put it into a stainless steel reactor. Start stirring until the mixture is uniformly stirred. Then, introduce nitrogen gas to reduce the pressure and raise the temperature to 80±5℃ / 700mmHg. The pressure inside the reactor is 0.2MPa. Slowly add H2O2. To control the amount of H2O2 added, measure a small amount of H2O2, connect it to a vacuum pump and slowly drip it in. Observe the color change of Span-80 in the reactor. When the color changes from dark to light, stop adding H2O2 and maintain the reaction time for 10-15 minutes.

[0084] 2) When the color of Span-80 in the reactor no longer changes and becomes slightly yellow, clear and transparent (the first processed product is obtained), the reactor is heated to 110°C, and the unreacted excess H2O2 is extracted from the reactor under vacuum for 3 minutes to obtain the second processed product.

[0085] 3) Stop the vacuuming in step 2), continue to purge with nitrogen to maintain pressure and cool down, and discharge the second processed product from the reactor. Connect the discharge to a 0.45μm filter to coarsely filter the liquid to remove unreacted substances, and then transfer the filtrate to a sterile environment and filter it again with a 0.22μm filter membrane to obtain purified Span-80.

[0086] The refined Tween-80 mentioned in step S2 is obtained by the following method:

[0087] (1) Weigh out Tween-80 (purchased from Guangzhou Qiaolinger Biotechnology Co., Ltd.), put it into a stainless steel reactor, turn on the stirrer until it is uniformly stirred, then introduce nitrogen gas to reduce the pressure and raise the temperature to 80±5℃ / 700mmHg. The pressure inside the reactor is 0.2MPa. Slowly add H2O2. In order to control the amount of H2O2 added, measure a small amount of H2O2, connect the vacuum pump and slowly drip it in. Observe the color change of Tween-80 in the reactor. When the color changes from dark to light, stop adding H2O2 and maintain the reaction time for 10-15min.

[0088] (2) When the color of Tween-80 in the reactor no longer changes and becomes slightly yellow, clear and transparent (the third processed product is obtained), the reactor is heated to 110°C, and the unreacted excess H2O2 is extracted from the reactor under vacuum for 3 minutes (the fourth processed product is obtained).

[0089] (3) Stop the vacuuming in step (2), continue to purge with nitrogen to maintain pressure and cool down, and discharge the fourth processing product from the reactor. Connect the discharge to a 0.45μm filter device to coarsely filter the liquid to remove unreacted substances, and then transfer the filtrate to a sterile environment and filter it again with a 0.22μm filter membrane to obtain purified Tween-80.

[0090] 1.2 The preparation method of foot-and-mouth disease type O inactivated vaccine includes the following steps:

[0091] The vaccine adjuvant was mixed with the foot-and-mouth disease type O inactivated vaccine at a 1:1 mass ratio and emulsified at 30-32℃ and 600 rpm / min for 15 minutes to prepare the foot-and-mouth disease type O inactivated vaccine. Figure 1 As shown, the particle size distribution of the foot-and-mouth disease type O inactivated vaccine obtained in Example 1 is illustrated, showing that its particle size is approximately 0.100-0.500 μm.

[0092] Example 2-3

[0093] Examples 2-3 follow the same procedure as Example 1 to prepare vaccine adjuvants and foot-and-mouth disease type O inactivated vaccines, differing only in the content of each component in the raw materials used to prepare the vaccine adjuvants. Specifically:

[0094] The raw materials for preparing the vaccine adjuvant in Example 2 were: 75 wt% Marcol-52 white oil, 1 wt% purified Span-80 and 24 wt% purified Tween-80.

[0095] The raw materials for preparing the vaccine adjuvant in Example 3 were: 85 wt% Marcol-52 white oil, 5 wt% purified Span-80 and 10 wt% purified Tween-80.

[0096] Example 4

[0097] Example 4: Following the steps of Example 1, a vaccine adjuvant and a foot-and-mouth disease type O inactivated vaccine were prepared. The difference was that in step S2, 2 wt% of an immunostimulatory complex was added to the heated injection oil. The raw materials for preparing the vaccine adjuvant were: 80 wt% Marcol-52 white oil, 3 wt% purified Span-80, 15 wt% purified Tween-80, and 2 wt% immunostimulatory complex. The immunostimulatory complex comprised phospholipids, cholesterol, tocopherol, and saponins in a mass ratio of 1:0.5:0.5:1. The preparation method of this immunostimulatory complex included the following steps:

[0098] T1. Weigh soybean lecithin HSPC, cholesterol CHOL, α-tocopherol, and saponins, and prepare mixture A in a mass fraction ratio of 1:0.5:0.5:1. Dissolve the obtained mixture A in a chloroform / methanol solvent in a 1:2 ratio to obtain a lipid-like organic solution for later use, thus preparing mixture B. The chloroform / methanol solvent is composed of chloroform, methanol, and ultrapure water in a volume ratio of 20:60:20.

[0099] T2. Transfer the mixture B from step T1 to a 250ml round-bottom beaker, place the round-bottom flask on a rotary evaporator, and remove the organic solvent by vacuum rotary evaporation under constant temperature water bath conditions. Solid C is formed on the inner wall of the rotary evaporator flask. The rotary evaporation temperature is 30-40℃, the rotation speed is 80-150r / min, and the rotary evaporation time is 60-90min.

[0100] T3. After the organic solvent in step T2 has been completely evaporated, remove the round-bottom flask and dissolve solid C in 0.01 mol / L PBS solution to obtain mixed solution D.

[0101] T4. The mixed solution D obtained in step T3 is ultrasonically treated at a temperature of 20-30℃ and a power of 60W to obtain mixed solution E. The ultrasonic treatment conditions are: ultrasonic for 10 minutes, ultrasonic for 10 seconds, and pause for 10 seconds.

[0102] T5. The resulting mixed solution E was sterilized by filtration through a 0.22 μm filter membrane and stored in a sealed container at -70 °C to obtain the immunostimulatory complex.

[0103] Comparative Examples 1-3

[0104] Comparative Examples 1-3 prepared vaccine adjuvants and foot-and-mouth disease type O inactivated vaccines according to the operating steps of Example 1, the difference being that the raw materials used to prepare the vaccine adjuvants were different, specifically:

[0105] The raw materials for preparing the vaccine adjuvant in Comparative Example 1 were: 80 wt% Marcol-52 white oil, 3 wt% Span-80 and 17 wt% Tween-80.

[0106] The raw materials for preparing the vaccine adjuvant in Comparative Example 2 were: 80 wt% Marcol-52 white oil, 3 wt% purified Span-80 (obtained in Example 1) and 17 wt% Tween-80.

[0107] The raw materials for preparing the vaccine adjuvant in Comparative Example 3 were: 80 wt% Marcol-52 white oil, 3 wt% Span-80 and 17 wt% purified Tween-80 (obtained in Example 1).

[0108] Comparative Example 4

[0109] Comparative Example 4 prepared a foot-and-mouth disease type O inactivated vaccine by mixing SEPPIC ISA206 vaccine adjuvant with foot-and-mouth disease type O inactivated vaccine at a mass ratio of 1:1 and emulsifying at 30-32°C and 600 rpm / min for 15 minutes.

[0110] Forty 28-day-old antibody-negative piglets were selected and divided into 8 groups of 5 piglets each as the experimental group. Each group of piglets was injected with the foot-and-mouth disease type O inactivated vaccine prepared in Examples 1-4 and Comparative Examples 1-4 at a dose of 5 μg / piglet of antigen (single-point intramuscular injection in the buttock). Five 28-day-old antibody-negative piglets were selected as the blank control group. On days 28, 60, 90, 120, 150, and 180 post-injection, antibody titers in each experimental group were measured using a liquid-phase blocking ELISA kit (Lanzhou Veterinary Research Institute; antibody titer greater than 1:180 was considered positive). Body temperature was monitored in the morning and afternoon of each experimental group and the blank control group three days before injection, and on days 1, 2, 3, and 14 post-injection. Feeding activity and any swelling, nodules, or ulceration at the injection site were also observed and recorded. The antibody titer results of piglets in each experimental group are shown in Table 1 below, and the body temperature monitoring results of piglets in each group are shown in Table 2 below.

[0111] Table 1: Results of antibody titer determination in piglets of each group

[0112]

[0113]

[0114] Table 2: Body temperature monitoring results of piglets in each group (based on the average body temperature of piglets in each group, in °C)

[0115]

[0116] As shown in Table 1 above, when the antigen immunization content is 5 μg / dose, the inactivated foot-and-mouth disease (FMD) type O vaccines of Examples 1-4 and Comparative Examples 1-4 can induce a specific immune response to the antigen in the vaccine in piglets 28 days after immunization, resulting in an antibody titer greater than 1:180. However, the FMD type O inactivated vaccine of Comparative Examples 1-3 showed a generally low antibody titer level after immunization, with a maximum of only 1:720, and a significant decrease in antibody titer level appeared as early as 90 days after immunization, indicating a short duration of action and poor uniformity. Comparative Example 4, prepared using ISA206 adjuvant, showed some improvement in immunoprotective effect compared to the FMD type O inactivated vaccines of Comparative Examples 1-3, with a maximum antibody titer of 1:1024, but antibodies did not appear until 120 days after immunization. While the antibody titer level decreased significantly, the immunoprotective effect remained insufficient, and the uniformity was also poor. Compared to the foot-and-mouth disease type O inactivated vaccine in Comparative Examples 1-4, the immunoprotective effect of the foot-and-mouth disease type O inactivated vaccine in Examples 1-4 was significantly improved. Its antibody titer after immunization reached a maximum of 1:2880, and it maintained a high antibody titer level of over 1:360 even 150 days after immunization, demonstrating a long duration of immunity and good uniformity (indicating stable vaccine quality). The results show that, compared to the vaccines prepared using adjuvants containing Span-80 and / or Tween-80 in Comparative Examples 1-3, and the vaccine prepared using ISA206 adjuvant in Comparative Example 4, the vaccine products prepared using adjuvants containing purified Span-80 and purified Tween-80 in Examples 1-4 of this invention better enhance the specific immune response level of piglets to the antigens in the vaccine, and have a longer duration of immunity and better uniformity.

[0117] In addition, compared with Examples 1-3, the vaccine product prepared using a vaccine adjuvant containing an immunostimulatory complex in Example 4 can further enhance the specific immune response level of piglets to the antigens in the vaccine. Its antibody titer after immunization can reach up to 1:2880, and it remains at a high level of more than 1:360 180 days after immunization.

[0118] As shown in Table 2 above, piglets immunized with the inactivated foot-and-mouth disease (FMD) type O vaccine in Comparative Examples 1-3 exhibited significant and prolonged fever 1-4 days after immunization, along with slight swelling at the injection site and decreased feed intake and activity. Piglets immunized with the inactivated FMD type O vaccine in Comparative Example 4 showed a brief, slight fever on the first day after immunization, which returned to normal on the second day without other adverse reactions. Piglets immunized with the inactivated FMD type O vaccine in Examples 1-4 generally maintained normal body temperature after immunization, without significant fever, and no local or systemic adverse reactions were observed. The injection sites remained normal. Therefore, compared to the inactivated FMD type O vaccine in Comparative Examples 1-4, the inactivated FMD type O vaccine in Examples 1-4 demonstrated higher safety.

[0119] Examples 5-6

[0120] Examples 5-6 follow the procedures outlined in Example 1 to prepare vaccine adjuvants and foot-and-mouth disease type O inactivated vaccines, differing only in the content of each component in the raw materials used to prepare the vaccine adjuvants. Specifically:

[0121] The raw materials for preparing the vaccine adjuvant in Example 5 were: 80 wt% Marcol-52 white oil, 2 wt% purified Span-80 and 18 wt% purified Tween-80.

[0122] The raw materials for preparing the vaccine adjuvant in Example 6 were: 85 wt% Marcol-52 white oil, 3 wt% purified Span-80 and 12 wt% purified Tween-80.

[0123] Examples 7-8

[0124] Examples 7-8 describe the preparation of vaccine adjuvants and foot-and-mouth disease type O inactivated vaccines following the procedures outlined in Example 4, with the only difference being the content of each component in the raw materials used to prepare the vaccine adjuvants. Specifically:

[0125] Example 7 The raw materials for preparing the vaccine adjuvant were: 80 wt% Marcol-52 white oil, 4 wt% purified Span-80, 15 wt% purified Tween-80 and 1 wt% immunostimulatory complex.

[0126] Example 8 The raw materials for preparing the vaccine adjuvant were: 80 wt% Marcol-52 white oil, 3 wt% purified Span-80, 14 wt% purified Tween-80 and 3 wt% immunostimulatory complex.

[0127] Twelve 28-day-old antibody-negative piglets were selected and divided into four groups of three piglets each as the experimental group. Each group was injected with the foot-and-mouth disease type O inactivated vaccine prepared in Examples 5-8 above (single-point intramuscular injection of 5 μg / piglet of antigen). Antibody titers in each experimental group were measured using a liquid-phase blocking ELISA kit (Lanzhou Veterinary Research Institute; antibody titer greater than 1:180 was considered positive) on days 28, 60, 90, 120, 150, and 180 post-injection. The antibody titer results for each experimental group are shown in Table 3 below.

[0128] Table 3: Results of antibody titer determination in piglets of each group

[0129]

[0130] As shown in Table 3 above, when the antigen immunization content is 5 μg / dose, the foot-and-mouth disease type O inactivated vaccines of Examples 5-8 were able to induce a significant specific immune response in piglets to the antigens in the vaccine 28 days after immunization. The antibody titer reached a maximum of 1:2880 after immunization, and maintained a high antibody titer level of over 1:360 even 150 days after immunization, demonstrating a long duration of immunity and good uniformity. In addition, compared with Examples 5-6, the vaccine products prepared using vaccine adjuvants containing further immunostimulatory complexes in Examples 7-8 can further enhance the specific immune response level of piglets to the antigens in the vaccine. The antibody titer reached a maximum of 1:2880 after immunization, and remained at a relatively high level even 180 days after immunization.

[0131] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A water-in-oil-in-water adjuvant for livestock vaccines, prepared from raw materials, wherein the raw materials comprise, by weight percentage: 75wt%-85wt% of injectable oil, 1wt%-5wt% of refined Span-80 and 10wt%-24wt% of refined Tween-80; wherein: The refined Span-80 is a product obtained by processing Span-80 in the following manner: 1) Treating Span-80 with H2O2 causes the color of Span-80 to change from dark to light. When the color of Span-80 no longer changes, the first treated product is obtained. 2) Remove unreacted H2O2 from the first treated product to obtain the second treated product; 3) Filter the second processed product to obtain the purified Span-80; The refined Tween-80 is a product obtained by treating Tween-80 in the following manner: (1) Tween-80 is treated with H2O2 to change the color of Tween-80 from dark to light. When the color of Tween-80 no longer changes, the third treatment product is obtained. (2) Remove unreacted H2O2 from the third processed product to obtain the fourth processed product; (3) The fourth processed product is filtered to obtain the refined Tween-80.

2. The water-in-oil-in-water adjuvant for livestock vaccines according to claim 1, wherein the raw materials comprise, by weight percentage: 80wt%-85wt% of injectable oil, 2wt%-3wt% of refined Span-80, and 12wt%-18wt% of refined Tween-80.

3. The water-in-oil-in-water adjuvant for animal vaccines according to claim 1, wherein the oil for injection includes mineral oil for injection, vegetable oil for injection, or a combination thereof.

4. The water-in-oil-in-water adjuvant for animal vaccines according to claim 3, wherein the mineral oil for injection includes white oil.

5. The water-in-oil-in-water adjuvant for animal vaccines according to claim 4, wherein the white oil comprises Marcol-52 white oil, Primol 352 white oil, Total130# white oil, Total150# white oil, Total170# white oil, Drakeol-5 white oil, Drakeol-7 white oil, Sonneborn 4# white oil, Sonneborn 10# white oil, Paracos KF40, Paracos KF50, squalene, and squalane.

6. The water-in-oil-in-water adjuvant for animal vaccines according to any one of claims 1-5, wherein the raw material further comprises 1wt%-3wt% of an immunostimulatory complex by weight percentage, wherein the immunostimulatory complex comprises phospholipids, cholesterol, tocopherols and saponins in a mass ratio of (0.5-2):(0.5-2):(0.5-2):(0.5-3).

7. A method for preparing a water-in-oil-in-water adjuvant for livestock vaccines, comprising the following steps: S1: Heat 75wt%-85wt% of the injection oil to 30℃-40℃ by weight percentage; S2: 1wt%-5wt% of refined Span-80 and 10wt%-24wt% of refined Tween-80 (by weight percentage) are added to the heated injection oil from step S1, mixed thoroughly, and then filtered to obtain the water-in-oil-in-water adjuvant for the animal vaccine; wherein: The refined Sben-80 is obtained by processing Sben-80 in the following way: 1) Treating Span-80 with H2O2 causes the color of Span-80 to change from dark to light. When the color of Span-80 no longer changes, the first treated product is obtained. 2) Remove unreacted H2O2 from the first treated product to obtain the second treated product; 3) Filter the second processed product to obtain the purified Span-80; The refined Tween-80 is obtained by processing Tween-80 in the following way: (1) Tween-80 is treated with H2O2 to change the color of Tween-80 from dark to light. When the color of Tween-80 no longer changes, the third treatment product is obtained. (2) Remove unreacted H2O2 from the third processed product to obtain the fourth processed product; (3) The fourth processed product is filtered to obtain the refined Tween-80.

8. The preparation method according to claim 7, wherein the filtration in steps 3) and (3) is first coarse filtration using a filter device with a pore size of 0.40μm-0.65μm, and the filtrate is then fine filtration using a filter device with a pore size of 0.10μm-0.22μm.

9. The preparation method according to claim 7 or 8, wherein in step S2, an immunostimulatory complex of 1 wt% to 3 wt% by weight is added to the heated injection oil, the immunostimulatory complex comprising phospholipids, cholesterol, tocopherols and saponins in a mass ratio of (0.5-2):(0.5-2):(0.5-2):(0.5-3).

10. The preparation method according to claim 9, wherein the preparation of the immunostimulatory complex comprises the following steps: T1: Dissolve a mixture of phospholipids, cholesterol, tocopherols and saponins in chloroform / methanol solvent in a mass percentage of (0.5-2):(0.5-2):(0.5-2):(0.5-3) to obtain an organic solution of lipids; T2: Remove the organic solvent from the organic solution of the lipids obtained in step T1 to obtain solids; T3: Dissolve the solid obtained in step T2 with PBS solution to obtain the first mixed solution; T4: The first mixed solution obtained in step T3 is subjected to ultrasonic treatment to obtain the second mixed solution; T5: Filter the second mixed solution obtained in step T4 to obtain the immunostimulatory complex.

11. The preparation method according to claim 10, wherein the chloroform / methanol solvent is composed of chloroform, methanol and ultrapure water in a volume ratio of 20:60:

20.

12. The preparation method according to claim 10, wherein the conditions for the ultrasonic treatment in step T4 include: The temperature is 20-30℃, the power is 50-70W, the total ultrasonic treatment time is 5-15min, and the ultrasonic mode is: 10s of ultrasonic treatment followed by 10s of pause.

13. A livestock vaccine comprising the water-in-oil-in-water adjuvant of any one of claims 1-6.

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