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

Abstract

The application provides a water-in-oil adjuvant for poultry animal vaccines and a preparation method and application thereof, and belongs to the technical field of biological product animal vaccines. The raw materials of the water-in-oil adjuvant provided by the application include, in percentage by mass, 85wt%-90wt% of injection oil, 5wt%-10wt% of refined Span-80 and 1wt%-5wt% of refined Tween-80, and can further include 0.1wt%-1wt% of an immune stimulating complex. The vaccine prepared by using the water-in-oil 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 poultry animal vaccines and the like.

Description

Technical Field

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

[0002] Poultry vaccines mainly fall into three categories: live attenuated vaccines, genetically engineered vaccines, and inactivated vaccines, with inactivated vaccines being the most numerous. Among inactivated vaccines for poultry, there are two main types: oil-emulsion inactivated vaccines and aluminum hydroxide inactivated vaccines. Aluminum hydroxide inactivated vaccines have not been widely adopted due to their high cost of antigen adsorption and short duration of immunity. Oil-emulsion inactivated vaccines, on the other hand, induce highly effective and long-lasting immunity and are less affected by maternal antibodies, making them the most widely used.

[0003] Currently, the oil-emulsion inactivated vaccines used in poultry are mainly water-in-oil vaccines, with water-in-oil adjuvants accounting for 2 / 3-3 / 4 of the vaccine by weight, playing a particularly important role. However, due to the drawbacks of traditional white oil adjuvants or commercially available water-in-oil adjuvants, such as high viscosity, slow absorption, poor safety, and slow antibody production, some vaccines prepared from them often result in decreased feed intake and activity, occasional outbreaks of infectious diseases (such as atypical Newcastle disease and avian influenza) or other illnesses, and short periods of immune protection after immunization of poultry (e.g., chickens and ducks). Therefore, in order to achieve effective control of infectious diseases in poultry, it is necessary to provide adjuvants that can make poultry vaccines safer and provide better immune protection. Summary of the Invention

[0004] To address one or more problems existing in the prior art, one aspect of the present invention provides an oil-in-water adjuvant for avian animal vaccines, which is prepared from raw materials, wherein the raw materials comprise, by weight percentage: 85wt%-90wt% of injectable oil, 5wt%-10wt% of refined Span-80, and 1wt%-5wt% of refined Tween-80; wherein:

[0005] The refined Span-80 is a product obtained by processing Span-80 in the following manner:

[0006] 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.

[0007] 2) Remove unreacted H2O2 from the first treated product to obtain the second treated product;

[0008] 3) Filter the second processed product to obtain the purified Span-80;

[0009] The refined Tween-80 is a product obtained by treating Tween-80 in the following manner:

[0010] (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.

[0011] (2) Remove unreacted H2O2 from the third processed product to obtain the fourth processed product;

[0012] (3) The fourth processed product is filtered to obtain the refined Tween-80.

[0013] 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.

[0014] In some embodiments, the injectable oil includes injectable mineral oil, injectable vegetable oil, or a combination thereof.

[0015] In some embodiments, the injectable mineral oil includes white oil.

[0016] In some embodiments, 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.

[0017] In some embodiments, the raw material further comprises 0.5 wt% to 1 wt% of an immunostimulatory complex, 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).

[0018] Another aspect of the present invention provides a method for preparing an oil-in-water adjuvant for avian animal vaccines, comprising the following steps:

[0019] S1: Heat 85wt%-90wt% of the injection oil to 30℃-40℃ by weight percentage;

[0020] S2: 5wt%-10wt% of refined Span-80 and 1wt%-5wt% of refined Tween-80 are added to the heated injection oil from step S1, mixed thoroughly, and then filtered to obtain the water-in-oil adjuvant for poultry vaccines; wherein:

[0021] The refined Sben-80 is obtained by processing Sben-80 in the following way:

[0022] 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.

[0023] 2) Remove unreacted H2O2 from the first treated product to obtain the second treated product;

[0024] 3) Filter the second processed product to obtain the purified Span-80;

[0025] The refined Tween-80 is obtained by processing Tween-80 in the following way:

[0026] (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.

[0027] (2) Remove unreacted H2O2 from the third processed product to obtain the fourth processed product;

[0028] (3) The fourth processed product is filtered to obtain the refined Tween-80.

[0029] 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.

[0030] In some embodiments, in step S2, 0.1 wt% to 1 wt% of an immunostimulatory complex 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).

[0031] In some embodiments, the preparation of the immunostimulatory complex includes the following steps:

[0032] 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.

[0033] T2: Remove the organic solvent from the organic solution of the lipids obtained in step T1 to obtain solids;

[0034] T3: Dissolve the solid obtained in step T2 with PBS solution to obtain the first mixed solution;

[0035] T4: The first mixed solution obtained in step T3 is subjected to ultrasonic treatment to obtain the second mixed solution;

[0036] T5: Filter the second mixed solution obtained in step T4 to obtain the immunostimulatory complex.

[0037] 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.

[0038] In another aspect, the present invention provides a poultry vaccine comprising the above-mentioned water-in-oil adjuvant for poultry vaccines.

[0039] The oil-in-water adjuvant for poultry 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 oil-emulsion inactivated vaccine products prepared using the oil-in-water adjuvant provided by the present invention exhibiting high safety and, surprisingly, the ability to induce a longer-lasting (e.g., the duration of immunity to the bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) prepared using this adjuvant can be up to 3 months) and more effective (e.g., the bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) prepared using this adjuvant can still provide more than 90% protection 90 days after immunization). Therefore, the oil-in-water adjuvant provided by the present invention can help develop poultry vaccine products with better safety, longer duration of immunity, and better immunization efficacy. Attached Figure Description

[0040] Figure 1 The particle size distribution diagram is shown for the bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) prepared in Example 1. Detailed Implementation

[0041] To address the shortcomings of existing water-in-oil adjuvants in vaccine preparation, such as poor safety, short duration of immunity, and insufficient immunogenicity, this invention aims to provide a water-in-oil adjuvant for avian vaccines. When used in vaccine preparation, this adjuvant effectively improves vaccine 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 adjuvant.

[0042] 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.

[0043] In a first aspect of the invention, an oil-in-water adjuvant for avian animal vaccines is provided, wherein the raw materials, by weight percentage, may include: 85wt%-90wt% of injectable oil, 5wt%-10wt% of refined Span-80, and 1wt%-5wt% of refined Tween-80; wherein:

[0044] 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:

[0045] 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.

[0046] 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.

[0047] 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 purge 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 for further filtration with a 0.10μm-0.22μm filter membrane to obtain purified Sben-80.

[0048] 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:

[0049] (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.

[0050] (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.

[0051] (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.

[0052] In this invention, compared to vaccine adjuvants formulated using commercially available untreated Span-80 and / or Tween-80, vaccine adjuvants formulated using purified Span-80 and purified Tween-80 can effectively improve the safety of the prepared vaccine and further enhance the specific immune response of animals to the antigens in the vaccine. This is likely because it removes excess byproducts and impurities introduced from the raw materials, thereby improving vaccine safety and simultaneously enhancing its immunogenicity.

[0053] In some embodiments, the injectable oil may include injectable mineral oil, injectable vegetable oil, or a combination thereof.

[0054] In some embodiments, the injectable mineral oil may include white oil.

[0055] 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.

[0056] In a preferred embodiment, the raw materials may include, by weight percentage: 85wt%-90wt% of injectable oil, 6wt%-10wt% of refined Span-80 and 1wt%-5wt% of refined Tween-80; the water-in-oil vaccine adjuvant obtained under these conditions may further enhance the level of specific immune response of the animal to the antigens in the vaccine.

[0057] The animal vaccine product prepared using the water-in-oil adjuvant provided by this invention is a water-in-oil particle. This particle has a bilayer structure: the outermost layer is the oil phase (i.e., the adjuvant of this invention), and the inner layer is the aqueous phase (i.e., the antigen). When the vaccine is injected into the body, the particles rupture with changes in body temperature, releasing the antigenic substances within. 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.

[0058] In some embodiments, the vaccine adjuvant may further include 0.1 wt% to 1 wt% of an immunostimulatory complex by weight, 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 0.1 wt% to 1 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.

[0059] When a vaccine is prepared using a vaccine adjuvant containing an immunostimulatory complex, the finished vaccine is a water-in-oil particle containing the immunostimulatory complex. This particle has a water-in-oil bilayer structure: the outermost layer is the oil phase (i.e., the adjuvant of this invention), and the inner layer is the aqueous phase (i.e., the combination of antigen and the immunostimulatory complex). When the vaccine is injected into the body, the particles rupture with changes in 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 adjuvant, various inflammatory cells are generated at the antigen injection site, enhancing the phagocytic activity of macrophages and thereby increasing the uptake of antigenic substances.

[0060] In a second aspect of the present invention, a method for preparing a vaccine adjuvant is provided, which may include the following steps:

[0061] S1: Heat 85wt%-90wt% of the injection oil to 30℃-40℃ by weight percentage;

[0062] S2: Add 5wt%-10wt% refined Span-80 and 1wt%-5wt% refined Tween-80 (by weight percentage) to the heated injection oil from step S1, mix well, and then filter to obtain the vaccine adjuvant; wherein:

[0063] 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.

[0064] In some embodiments, in step S2, 0.1 wt% to 1 wt% of an immunostimulatory complex may be 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).

[0065] In some embodiments, the preparation of the immunostimulatory complex includes the following steps:

[0066] 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.

[0067] T2: Remove the organic solvent from the organic solution of the lipids obtained in step T1 to obtain solids;

[0068] T3: Dissolve the solid obtained in step T2 with PBS solution to obtain the first mixed solution;

[0069] T4: The first mixed solution obtained in step T3 is subjected to ultrasonic treatment to obtain the second mixed solution;

[0070] T5: Filter the second mixed solution obtained in step T4 to obtain the immunostimulatory complex.

[0071] 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.

[0072] In a third aspect of the invention, a poultry vaccine comprising the water-in-oil adjuvant of the first aspect of the invention is also provided, such as a vaccine for poultry such as chickens and ducks.

[0073] Example

[0074] 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.

[0075] 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.

[0076] Example 1:

[0077] This embodiment prepares a water-in-oil adjuvant as a vaccine adjuvant, and uses this vaccine adjuvant and duck infectious serositis bivalent inactivated antigen (type 1 SG4 strain + type 2 ZZY7 strain) to prepare the vaccine with a bacterial suspension concentration of 1×10⁻⁶. 10 CFU / ml (provided by Liaoning Yikang Biological Pharmaceutical Co., Ltd., which is no less than 2.5 × 10⁻⁶ CFU / ml, according to the Ministry of Agriculture's Announcement No. 1893 regarding the manufacturing and testing procedures for duck infectious serositis bivalent inactivated vaccine (type 1 SG4 + type 2 ZZY7 strain)). 11 The CFU / ml was 20 times lower, and the adjuvant and antigen were formulated into a bivalent inactivated vaccine for duck infectious serositis (type 1 SG4 strain + type 2 ZZY7 strain) at a mass ratio of 2:1.

[0078] 1.1 The preparation method of vaccine adjuvant includes the following steps:

[0079] S1: Heat 90 wt% Marcol-52 white oil to 35°C;

[0080] S2: 6 wt% refined Span-80 and 4 wt% refined Tween-80 are added to the heated injection oil from step S1, mixed thoroughly, and then filtered (e.g., using a 0.22 μm filter membrane) to obtain the vaccine adjuvant. Wherein:

[0081] The refined Span-80 mentioned in step S2 is obtained by the following method:

[0082] 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.

[0083] 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.

[0084] 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.

[0085] The refined Tween-80 mentioned in step S2 is obtained by the following method:

[0086] (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 heat 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.

[0087] (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).

[0088] (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.

[0089] 1.2 The preparation method of the bivalent inactivated vaccine for duck infectious serositis (type 1 SG4 strain + type 2 ZZY7 strain) includes the following steps:

[0090] The vaccine adjuvant and duck infectious serositis bivalent inactivated antigen (type 1 SG4 strain + type 2 ZZY7 strain) were prepared at a mass ratio of 2:1. At 30-32℃, the vaccine adjuvant was first placed in an emulsification tank and stirred at 2000 rpm / min. The aqueous antigen was then slowly added, followed by high-speed stirring at 8000 rpm / min for 30 minutes. This yielded the duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain + type 2 ZZY7 strain). Figure 1 As shown, the particle size distribution of the bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) obtained in Example 1 is shown, and its volume average particle size is approximately 0.630 μm.

[0091] Example 2-3

[0092] Examples 2-3 describe the preparation of vaccine adjuvants and bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) following the procedures outlined in Example 1. The only difference lies in the content of each component in the raw materials used to prepare the vaccine adjuvants. Specifically:

[0093] The raw materials for preparing the vaccine adjuvant in Example 2 were: 90 wt% Marcol-52 white oil, 5 wt% purified Span-80 and 5 wt% purified Tween-80.

[0094] The raw materials for preparing the vaccine adjuvant in Example 3 were: 85 wt% Marcol-52 white oil, 10 wt% purified Span-80 and 5 wt% purified Tween-80.

[0095] Example 4

[0096] Example 4: Following the procedures of Example 1, a vaccine adjuvant and bivalent inactivated duck infectious serositis antigen (type 1 SG4 strain + type 2 ZZY7 strain) were prepared. The difference was that in step S2, 0.2 wt% of an immunostimulatory complex was added to the heated injection oil. The raw materials for preparing the vaccine adjuvant were: 90 wt% Marcol-52 white oil, 6 wt% purified Span-80, 3.8 wt% purified Tween-80, and 0.2 wt% of the 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:

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] Comparative Example 1

[0103] The method for preparing a bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) in Comparative Example 1 includes: taking 90 parts by weight of Marcol-52 white oil and 6 parts by weight of Span-80, mixing and stirring evenly, and sterilizing at 115°C for 40 minutes to prepare an oil phase; taking qualified inactivated duck infectious serositis bivalent inactivated antigen (type 1 SG4 strain + type 2 ZZY7 strain), mixing equal volumes to obtain 100 parts by weight of a mixture, adding 8 parts by weight of Tween-80 to it, and mixing thoroughly to prepare an aqueous phase; taking the oil phase and aqueous phase at a mass ratio of 2:1, at 30-32°C, first placing the oil phase into an emulsification tank, stirring at 2000 rpm / min, slowly adding the aqueous phase, and then stirring at 8000 rpm / min for 30 minutes after the addition is complete, thus preparing the duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain + type 2 ZZY7 strain).

[0104] Compare 2-4

[0105] Comparative Examples 2-4 prepared vaccine adjuvants and duck infectious serositis bivalent inactivated vaccines (type 1 SG4 strain + type 2 ZZY7 strain) according to the operating steps of Example 1, the difference being that the raw materials used to prepare the vaccine adjuvants were different, specifically:

[0106] The raw materials for preparing the vaccine adjuvant in Comparative Example 2 were: 90 wt% Marcol-52 white oil, 6 wt% purified Span-80 (obtained in Example 1) and 4 wt% Tween-80.

[0107] The raw materials for preparing the vaccine adjuvant in Comparative Example 3 were: 90 wt% Marcol-52 white oil, 6 wt% Span-80 and 4 wt% purified Tween-80 (obtained in Example 1).

[0108] The raw materials for preparing the vaccine adjuvant in Comparative Example 4 were: 90 wt% Marcol-52 white oil, 6 wt% Span-80 and 4 wt% Tween-80.

[0109] The following immunization challenge test and vaccine safety test were conducted using the bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) prepared in Examples 1-4 and Comparative Examples 1-4.

[0110] (1.1) Immunochaete test

[0111] Five hundred and forty healthy, susceptible ducks aged 5-10 days (negative for duck plague Riegeria type 1 and type 2 antibodies) were selected and divided into nine groups of 60 ducks each. Eight groups (480 ducks in total) served as the experimental group, receiving a subcutaneous injection in the neck of 0.25 ml of the bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) prepared in Examples 1-4 and Comparative Examples 1-4. The remaining group of 60 ducks served as the control group, without vaccination. At 21, 60, and 90 days post-vaccination, ten immunized ducks and ten control ducks from each group were intramuscularly injected with 0.4 ml of Riegeria duck plague Riegeria SG4 strain bacterial suspension (containing approximately 1.0 × 10⁻⁶ live bacteria). 10 CFU), then take 10 immunized ducks and 10 control ducks, and inject each duck intramuscularly with 0.4 ml of Rhizobium anguillarum strain ZZY7 (containing approximately 1.0 × 10⁻⁶ live bacteria), one dose of which is sufficient to cause disease. 5 CFU was administered, and the patients were observed for 7 days. At least 9 mice in the control group should develop the disease, and at least 9 mice in the immunized group should remain healthy. The results of post-immunization challenge protection in each experimental and control group are shown in Table 1 below.

[0112] Table 1: Post-immunization challenge protection results of ducks in each group

[0113]

[0114] As shown in Table 1 above, the blank control group showed that inoculation with Rhizobium anatipestifer SG4 and ZZY7 strains at different time points resulted in a morbidity rate of over 9 / 10, confirming the validity of the experiment. The bivalent inactivated vaccine in Comparative Example 1 was prepared using the traditional bivalent inactivated vaccine preparation method for duck infectious serositis (i.e., first preparing an oil phase by mixing injection oil with Span-80 and an aqueous phase by mixing antigen with Tween-80, then emulsifying and mixing the aqueous and oil phases). Inoculation with Rhizobium anatipestifer SG4 and ZZY7 strains 21 days post-immunization only achieved a protection rate of over 7 / 10; challenge at 60 days post-immunization only achieved a maximum protection rate of 5 / 10; and challenge at 90 days post-immunization only achieved a maximum protection rate of 3 / 10. This vaccine showed poor protective efficacy and a short duration of immunity. Comparative Examples 2-4 involved first preparing vaccine adjuvants using Span-80 and / or Tween-80, and then using these adjuvants to formulate vaccines with antigens. The vaccine in Comparative Example 4 failed to provide effective immune protection to the ducks 21 days post-immunization, achieving only a maximum protection rate of 8 / 10. Comparative Examples 2-3 used either purified Span-80 or purified Tween-80 in the preparation of the adjuvant, showing a slight improvement in immune protection compared to Comparative Example 4, but only achieved a maximum protection rate of 6 / 10 upon challenge at 60 days post-immunization, also demonstrating a short duration of efficacy. Compared to Comparative Examples 1-4, Examples 1-4 used both purified Span-80 and purified Tween-80 in the preparation of the adjuvant. The vaccine prepared with this adjuvant achieved a protection rate of over 9 / 10 upon inoculation with *Riebelella anatipestifer* strains SG4 and ZZY7 at 21, 60, and 90 days post-immunization, meaning the vaccine's efficacy could last for more than 3 months. The above results demonstrate that, compared to the vaccines prepared using adjuvants containing Span-80 and / or Tween-80 in Comparative Examples 1-4, the vaccine products prepared using adjuvants containing purified Span-80 and purified Tween-80 in Examples 1-4 of the present invention significantly enhance the specific immune response level of ducks to the antigens in the vaccine, and the duration of immunity lasts for more than 3 months, with good uniformity (indicating stable vaccine product quality).

[0115] In addition, the bacterial concentration used in Examples 1-4 for preparing the bivalent inactivated vaccine for duck infectious serositis (type 1 SG4 strain + type 2 ZZY7 strain) was 1×10⁻⁶. 10 The CFU / ml concentration is no less than 2.5 × 10⁻⁶, as specified in the Ministry of Agriculture's Announcement No. 1893 regarding the manufacturing and testing procedures for the bivalent inactivated duck infectious serositis vaccine (type 1 SG4 + type 2 ZZY7 strain). 11Although the CFU / ml was 20 times lower, according to the results shown in Table 1 above, the bivalent inactivated vaccine for duck infectious serositis (type 1 SG4 strain + type 2 ZZY7 strain) obtained in Examples 1-4 still had good immunoprotective effect, with an immunity duration of more than 3 months and good uniformity.

[0116] (1.2) Vaccine safety testing

[0117] Ninety healthy, susceptible ducks aged 5-10 days (negative for duck plague type 1 and type 2 antibodies) were selected and divided into 9 groups of 10 each. Eight groups (80 ducks in total) served as the experimental group, receiving a subcutaneous injection of 1 ml of the bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) prepared in Examples 1-4 and Comparative Examples 1-4. The remaining group (10 ducks) served as the control group, without vaccination. All ducks were observed for 7 consecutive days, and all were expected to remain healthy without any local or systemic adverse reactions caused by the vaccine. The results are shown in Table 2 below.

[0118] Table 2: Safety results of ducks after immunization in each group

[0119]

[0120]

[0121] As shown in Table 2 above, ducks immunized with the bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) of Comparative Examples 1-4 experienced anorexia, decreased feed intake, and reduced activity within one day, with some ducks exhibiting disheveled feathers. These adverse reactions were prolonged, but recovered to normal within 48 hours post-immunization, with slight swelling at the injection site in a few cases. In contrast, ducks immunized with the bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) of Examples 1-4 generally returned to normal, with no significant adverse reactions or local or systemic adverse reactions caused by the vaccine, and all injection sites remained normal. These results indicate that the bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) of Examples 1-4 has a higher safety profile compared to the bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) of Comparative Examples 1-4.

[0122] Examples 5-6

[0123] Examples 5-6 follow the procedures outlined in Example 1 to prepare vaccine adjuvants and a bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain). The only difference lies in the content of each component in the raw materials used to prepare the vaccine adjuvants. Specifically:

[0124] The raw materials for preparing the vaccine adjuvant in Example 5 were: 89 wt% Marcol-52 white oil, 10 wt% purified Span-80 and 1 wt% purified Tween-80.

[0125] The raw materials for preparing the vaccine adjuvant in Example 6 were: 85 wt% Marcol-52 white oil, 10 wt% purified Span-80 and 5 wt% purified Tween-80.

[0126] Examples 7-8

[0127] Examples 7-8 describe the preparation of vaccine adjuvants and bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) following the procedures outlined in Example 4. The only difference lies in the content of each component in the raw materials used to prepare the vaccine adjuvants. Specifically:

[0128] Example 7 The raw materials for preparing the vaccine adjuvant were: 86.9 wt% Marcol-52 white oil, 9 wt% purified Span-80, 4 wt% purified Tween-80 and 0.1 wt% immunostimulatory complex.

[0129] Example 8 The raw materials for preparing the vaccine adjuvant were: 87 wt% Marcol-52 white oil, 8 wt% purified Span-80, 4 wt% purified Tween-80 and 1 wt% immunostimulatory complex.

[0130] Three hundred healthy, susceptible ducks aged 5-10 days (negative for duck plague Riegeria type 1 and type 2 antibodies) were selected and divided into five groups of 60 ducks each. Four groups (240 ducks in total) served as the experimental group, receiving a subcutaneous injection of 0.25 ml of the bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) described in Examples 5-8 above in their necks. The remaining group (60 ducks) served as the control group and was not vaccinated. At 21, 60, and 90 days post-immunization, 10 immunized ducks and 10 control ducks from each group were intramuscularly injected with 0.4 ml of Riegeria duck plague Riegeria SG4 strain bacterial suspension (containing approximately 1.0 × 10⁻⁶ live bacteria). 10 CFU), and then 10 immunized ducks and 10 control ducks were each intramuscularly injected with 0.4 ml of Rhizobium anguillicarbosus strain ZZY7 (containing approximately 1.0 × 10⁻⁶ live bacteria), one dose of the disease-causing agent. 5 CFU was administered, and the patients were observed for 7 days. At least 9 mice in the control group should develop the disease, and at least 9 mice in the immunized group should remain healthy. The results of post-immunization challenge protection in each experimental and control group are shown in Table 3 below.

[0131] Table 3: Post-immunization challenge protection results of ducks in each group

[0132]

[0133] As shown in Table 3 above, the blank control group showed that inoculation with Rhizobium anatipestifer SG4 and ZZY7 strains at different time points resulted in a morbidity rate of over 9 / 10, confirming the validity of the experiment. The bivalent inactivated duck infectious serositis vaccine (type 1 SG4 strain + type 2 ZZY7 strain) in Examples 5-8 achieved a protection rate of over 9 / 10 when inoculated with Rhizobium anatipestifer SG4 and ZZY7 strains from 21 to 90 days post-immunization, and maintained a protective level for at least 3 months, demonstrating good immunization effect and long duration of immunity. Furthermore, the vaccine exhibited excellent uniformity, indicating high product quality stability.

[0134] 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. Use of a water-in-oil adjuvant in the manufacture of a vaccine for avian animals, wherein the water-in-oil adjuvant is prepared from a raw material comprising, by weight percentage: 85 wt% to 90 wt% of an injection oil, 5 wt% to 10 wt% of refined Solutol-80, and 1 wt% to 5 wt% of refined Tween-80; wherein: the refined Solutol-80 is a product obtained by treating Solutol-80 by: 1) treating Solutol-80 with H2O2 to change the color of the Solutol-80 from dark to light, and obtaining a first treatment product when the color of the Solutol-80 no longer changes; 2) removing unreacted H2O2 in the first treatment product to obtain a second treatment product; 3) filtering the second treatment product to obtain the refined Solutol-80; the refined Tween-80 is a product obtained by treating Tween-80 by: (1) treating Tween-80 with H2O2 to change the color of the Tween-80 from dark to light, and obtaining a third treatment product when the color of the Tween-80 no longer changes; (2) removing unreacted H2O2 in the third treatment product to obtain a fourth treatment product; (3) filtering the fourth treatment product to obtain the refined Tween-80; wherein the antigen in the avian animal vaccine is a bivalent inactivated antigen of duck infectious serositis of type 1 SG4 strain and type 2 ZZY7 strain.

2. The use according to claim 1, wherein the filtering in step 3) and step (3) is first coarsely filtering using a filtering device with a pore size of 0.40 μm to 0.65 μm, and then finely filtering the filtrate using a filtering device with a pore size of 0.10 μm to 0.22 μm.

3. The use according to claim 1 or 2, wherein the injection oil comprises an injection mineral oil, an injection vegetable oil, or a combination thereof.

4. The use according to claim 3, wherein the injection mineral oil comprises white oil.

5. The use according to claim 4, wherein the white oil comprises Marcol-52 white oil, Primol 352 white oil, Total 130# white oil, Total 150# white oil, Total 170# white oil, Drakeol-5 white oil, Drakeol-7 white oil, and Sonneborn 4# white oil, Sonneborn 10# white oil PARACOS KF40, PARACOS KF50, squalene, squalane.

6. The use according to claim 1 or 2, wherein the raw material further comprises 0.5 wt% to 1 wt% of an immune stimulatory complex, the immune stimulatory complex comprising phospholipid, cholesterol, tocopherol, and saponin in a mass ratio of 0.5-2:0.5-2:0.5-2:0.5-3.

7. An avian animal vaccine comprising the water-in-oil adjuvant and the antigen mentioned in any one of claims 1-6, wherein the antigen is a bivalent inactivated antigen of duck infectious serositis of type 1 SG4 strain and type 2 ZZY7 strain.

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