WO2023231333A1 - Adjuvant eau dans huile pour vaccin destiné à une volaille, son procédé de préparation et son utilisation - Google Patents

Adjuvant eau dans huile pour vaccin destiné à une volaille, son procédé de préparation et son utilisation Download PDF

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WO2023231333A1
WO2023231333A1 PCT/CN2022/134909 CN2022134909W WO2023231333A1 WO 2023231333 A1 WO2023231333 A1 WO 2023231333A1 CN 2022134909 W CN2022134909 W CN 2022134909W WO 2023231333 A1 WO2023231333 A1 WO 2023231333A1
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oil
tween
siben
refined
vaccine
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PCT/CN2022/134909
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English (en)
Chinese (zh)
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李晓艳
刘超
刘国英
舒秀伟
马振宇
陈坚
宋庆庆
赵丽霞
关平原
李超
齐志涛
杨青春
贺瑶
宋志刚
王�华
张建宏
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金宇保灵生物药品有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine

Definitions

  • the invention belongs to the technical field of veterinary vaccines, and specifically relates to a water-in-oil adjuvant for poultry animal vaccines and its preparation method and application.
  • Poultry vaccines mainly include attenuated vaccines, genetically engineered vaccines, and inactivated vaccines, among which inactivated vaccines have the largest number of strains.
  • Inactivated vaccines for poultry animals mainly include oil emulsion inactivated vaccines and aluminum hydroxide inactivated vaccines. Among them, due to the high cost of adsorbing antigens by aluminum hydroxide and the short duration of immunity, they have not been widely used, while oil emulsions Inactivated vaccines can induce efficient and long-lasting immunity and are less affected by maternal antibodies, so they are the most widely used.
  • the oil emulsion inactivated vaccines used in poultry vaccines are mainly water-in-oil vaccines.
  • the water-in-oil adjuvant accounts for 2/3-3/4 of the vaccine in terms of weight percentage, and its role is particularly important.
  • some vaccines prepared from them can immunize poultry animals (such as chickens, ducks, etc.)
  • the occasional occurrence of infectious diseases such as atypical Newcastle disease, avian influenza, etc.
  • infectious diseases such as atypical Newcastle disease, avian influenza, etc.
  • one aspect of the present invention provides a water-in-oil adjuvant for poultry animal vaccines, which is prepared from raw materials, and the raw materials include in terms of weight percentage: 85wt%-90wt % injection oil, 5wt%-10wt% refined Siben-80 and 1wt%-5wt% refined Tween-80; among which:
  • the refined Siben-80 is a product obtained by processing Siben-80 in the following manner:
  • the refined Tween-80 is a product obtained by processing Tween-80 in the following manner:
  • the filtration described in step 3) and step (3) is to first use a filtration device with a filtration pore size of 0.40 ⁇ m-0.65 ⁇ m for rough filtration, and then use a filtration device with a filtration pore size of 0.10 ⁇ m-0.22 ⁇ m for the filtrate. Perform fine filtration.
  • the injectable oil includes injectable mineral oil, injectable vegetable oil, or a combination thereof.
  • the injectable mineral oil includes white oil.
  • 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 and Sonneborn 4# white oil, Sonneborn 10# white oil PARACOS KF40, PARACOS KF50, squalene, squalane.
  • the raw material also includes 0.5wt%-1wt% of an immune-stimulating complex by weight, and the immune-stimulating complex includes a mass ratio of (0.5-2):(0.5-2):( 0.5-2): (0.5-3) of phospholipids, cholesterol, tocopherols and saponins.
  • Another aspect of the present invention provides a method for preparing a water-in-oil adjuvant for poultry vaccines, which includes the following steps:
  • step S2 Add 5wt%-10wt% of refined Siben-80 and 1wt%-5wt% of refined Tween-80 to the heated injection oil in step S1, mix and filter to obtain The water-in-oil adjuvant for poultry vaccine; wherein:
  • the refined Siben-80 is obtained by processing Siben-80 in the following ways:
  • the refined Tween-80 is obtained by processing Tween-80 in the following manner:
  • the filtration described in step 3) and step (3) is to first use a filtration device with a filtration pore size of 0.40 ⁇ m-0.65 ⁇ m for rough filtration, and then use a filtration device with a filtration pore size of 0.10 ⁇ m-0.22 ⁇ m for the filtrate. Perform fine filtration.
  • an immunostimulatory complex of 0.1wt%-1wt% is also added to the heated injection oil in terms of weight percentage, and the immunostimulatory complex includes a mass ratio of ( 0.5-2):(0.5-2):(0.5-2):(0.5-3) of phospholipids, cholesterol, tocopherols and saponins.
  • the preparation of the immunostimulatory complex includes the following steps:
  • T1 Dissolve a mixture of phospholipids, cholesterol, tocopherol and saponin with a mass percentage of (0.5-2):(0.5-2):(0.5-2):(0.5-3) in a chloroform/methanol solvent to obtain a similar Organic solution of lipid;
  • the chloroform/methanol solvent is composed of chloroform, methanol and ultrapure water with a volume ratio of 20:60:20;
  • T2 Remove the organic solvent from the lipid organic solution obtained in step T1 to obtain a solid;
  • T3 Dissolve the solid obtained in step T2 with PBS solution to obtain the first mixed solution
  • T4 Perform ultrasonic treatment on the first mixed solution obtained in step T3 to obtain a second mixed solution
  • T5 Filter the second mixed solution obtained in step T4 to obtain the immunostimulatory complex.
  • the conditions for ultrasonic treatment in step T4 include: temperature is 20-30°C, power is 50-70W, total ultrasonic treatment time is 5-15 minutes, and ultrasonic mode is: ultrasonic for 10 seconds, pause for 10 seconds.
  • the present invention also provides a poultry vaccine, which contains the above-mentioned water-in-oil adjuvant for poultry vaccines.
  • the raw materials of the water-in-oil adjuvant for poultry animal vaccines provided based on the above technical solution use refined Siben-80 and refined Tween-80, and immune stimulating complexes can also be further used to utilize the water-in-oil adjuvant provided by the present invention.
  • the oil emulsion inactivated vaccine product prepared with the adjuvant has high safety, and surprisingly, it can also induce the body to produce a longer-lasting vaccine (for example, the duck infectious serositis bivalent inactivated vaccine prepared using the same (1 type SG4 strain + type 2 ZZY7 strain) the duration of immune efficacy can be as long as more than 3 months) and more efficient (for example, the duck infectious serositis bivalent inactivated vaccine prepared by using it (type 1 SG4 strain + 2 Type ZZY7 strain) can still provide a specific immune response of more than 90% protection rate 90 days after vaccination). Therefore, the water-in-oil adjuvant provided by the present invention can contribute to the development of poultry animal vaccine products with better safety, longer duration of immunity, and better immune effect.
  • a longer-lasting vaccine for example, the duck infectious serositis bivalent inactivated vaccine prepared using the same (1 type SG4 strain + type 2 ZZY7 strain) the duration of immune efficacy can be as long as more than 3 months
  • Figure 1 is a particle size distribution diagram of the duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain + type 2 ZZY7 strain) prepared in Example 1.
  • the present invention aims to provide a water-in-oil adjuvant for poultry animal vaccines. , when used to prepare vaccines, it can effectively improve the safety of the vaccine, effectively improve the immunity duration of the vaccine, and enhance the body's specific immune response level to the antigens in the vaccine.
  • the invention also provides a preparation method of the water-in-oil adjuvant.
  • a water-in-oil adjuvant for poultry animal vaccines the raw materials of which may include, in terms of weight percentage: 85wt%-90wt% injection oil, 5wt%-10wt% refined Siben- 80 and 1wt%-5wt% refined Tween-80; where:
  • the refined Siben-80 can be a product obtained by processing Siben-80 in the following manner, wherein Siben-80 can be commercially available, or can be obtained according to a conventional preparation method of Siben-80:
  • H 2 O 2 Use H 2 O 2 to treat Siben-80 so that the color of Siben-80 changes from dark to light.
  • This step may specifically include the following operations: Weigh Siben-80 and put it into a stainless steel reaction kettle, turn on the stirring until evenly stirred, then add nitrogen to reduce the pressure and raise the temperature to 80 ⁇ 5°C/700mmHg, the pressure in the kettle is 0.15-0.25MPa, slowly Add H 2 O 2.
  • H 2 O 2 measure a small amount of H 2 O 2 , connect the vacuum pump and slowly drip it in. Observe the color change of Siben-80 in the reaction kettle. Stop when the color changes from deep to shallow. Add H 2 O 2 and keep the reaction time for 10-15 minutes;
  • This step may specifically include the following operations: When the color of Siban-80 in the reaction kettle no longer changes and becomes slightly yellow and clear and transparent, heat the reaction kettle to 110 ⁇ 5°C, vacuum and dehydrate the unreacted excess H 2 O2 is taken out of the reaction kettle, and the vacuum dehydration time is 3-5 minutes;
  • This step may specifically include the following operations: stop vacuuming in step 2), continue to pass in nitrogen to maintain pressure and cool down, and discharge the second treatment product from the reaction kettle; connect the discharge to a 0.40 ⁇ m-0.65 ⁇ m filter device, and filter the feed liquid Carry out coarse filtration to remove unreacted substances, then transfer the filtrate to a sterile environment, and filter and sterilize it again with a 0.10 ⁇ m-0.22 ⁇ m filter membrane to obtain refined Siben-80.
  • the refined Tween-80 can be a product obtained by processing Tween-80 in the following manner, wherein Tween-80 can be commercially available, or can be obtained according to a conventional Tween-80 preparation method:
  • This step may specifically include the following operations: weigh Tween-80 and put it into a stainless steel reaction kettle, turn on the stirring until evenly stirred, then add nitrogen to reduce the pressure and raise the temperature to 80 ⁇ 5°C/700mmHg, the pressure in the kettle is 0.15-0.25MPa, slowly Add H 2 O 2.
  • H 2 O 2 In order to control the amount of H 2 O 2 added, measure a small amount of H 2 O 2 , connect the vacuum pump and slowly drip it in. Observe the color change of Tween-80 in the reaction kettle. Stop when the color changes from deep to shallow. Add H 2 O 2 and keep the reaction time for 10-15 minutes;
  • This step may specifically include the following operations: when the color of the Tween-80 in the reaction kettle no longer changes and becomes slightly yellow and clear and transparent, heat the reaction kettle to 110 ⁇ 5°C, evacuate and dehydrate the unreacted excess H 2 O2 is taken out of the reaction kettle, and the vacuum dehydration time is 3-5 minutes;
  • This step may specifically include the following operations: stop vacuuming in step (2), continue to pass in nitrogen to maintain pressure and cool down, and discharge the fourth treatment product from the reaction kettle; connect the discharge to a 0.40 ⁇ m-0.65 ⁇ m filter device, and The liquid is coarsely filtered to remove unreacted matter, and then the filtrate is transferred to a sterile environment, and filtered and sterilized again with a 0.10 ⁇ m-0.22 ⁇ m filter membrane to obtain refined Tween-80.
  • the vaccine adjuvant formulated with purified Siben-80 and purified Tween-80 can It effectively improves the safety of the prepared vaccine and can further enhance the specific immune response level of the animal body to the antigens in the vaccine.
  • the possible reason is that excess by-products and impurities introduced from raw materials are removed, thereby improving the safety of the vaccine and simultaneously improving the immune efficacy of the vaccine.
  • the injectable oil may include injectable mineral oil, injectable vegetable oil, or a combination thereof.
  • the injectable mineral oil may include white oil.
  • 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 Oil and Sonneborn 4# white oil, Sonneborn 10# white oil PARACOS KF40, PARACOS KF50, squalene, squalane, these components can be obtained from any commercial source.
  • the raw materials may include, in terms of weight percentage: 85wt%-90wt% injection oil, 6wt%-10wt% refined Tween-80 and 1wt%-5wt% refined Tween-80 ;
  • the water-in-oil vaccine adjuvant obtained in this case can further enhance the specific immune response level of the animal body to the antigens in the vaccine.
  • the animal vaccine product prepared from the water-in-oil adjuvant provided by the present invention is a water-in-oil particle.
  • the particle has a double-layer structure of water-in-oil: the outermost layer is an oil phase (i.e., the adjuvant of the present invention), and the inner layer is a water phase. (i.e. antigen).
  • the particles When the vaccine is injected into the body, the particles will rupture as the body temperature changes, releasing the antigenic substances in them, and then presenting the antigen to antigen-presenting cells. After being processed by the antigen-presenting cells, they will be presented to T cells, inducing the body to produce Specific immune response.
  • the raw materials of the vaccine adjuvant may also include 0.1wt%-1wt% of an immune-stimulating complex by weight, and the immune-stimulating complex includes a mass ratio of (0.5-2):(0.5-2 ):(0.5-2):(0.5-3) of phospholipids, cholesterol, tocopherols and saponins.
  • the vaccine adjuvant further includes 0.1wt%-1wt% immune stimulating complex, which can further enhance the body's specific immune response level to the antigen in the vaccine containing it.
  • the finished vaccine is a water-in-oil particle containing an immune stimulating complex.
  • the particle has a double-layer structure of water-in-oil: the outermost layer is an oil phase (i.e., the present invention adjuvant), and the inner layer is an aqueous phase (i.e., a combination of antigen and immunostimulatory complex).
  • the vaccine is injected into the body, the particles will rupture as body temperature changes, releasing antigenic substances bound by immune-stimulating complexes.
  • the immune-stimulating complex is an efficient immune presentation system that activates antigen-presenting cells. Water-in-oil adjuvants generate various types of inflammatory cells at the antigen injection site, enhance the phagocytosis of macrophages, and thereby increase the intake of antigen substances.
  • a method for preparing a vaccine adjuvant which may include the following steps:
  • step S2 Add 5wt%-10wt% of refined Siben-80 and 1wt%-5wt% of refined Tween-80 to the heated injection oil in step S1, mix and filter to obtain Vaccine adjuvants; including:
  • Both the refined Siben-80 and the refined Tween-80 can be obtained according to the processing method described in the first aspect of the present invention.
  • an immunostimulatory complex of 0.1wt%-1wt% in weight percentage can also be added to the heated injection oil, and the immunostimulatory complex includes a mass ratio of (0.5-2):(0.5-2):(0.5-2):(0.5-3) of phospholipids, cholesterol, tocopherols and saponins.
  • the preparation of the immunostimulatory complex includes the following steps:
  • T1 Dissolve a mixture of phospholipids, cholesterol, tocopherol and saponin with a mass percentage of (0.5-2):(0.5-2):(0.5-2):(0.5-3) in a chloroform/methanol solvent to obtain a similar Organic solution of lipid;
  • the chloroform/methanol solvent is composed of chloroform, methanol and ultrapure water with a volume ratio of 20:60:20;
  • T2 Remove the organic solvent from the lipid organic solution obtained in step T1 to obtain a solid;
  • T3 Dissolve the solid obtained in step T2 with PBS solution to obtain the first mixed solution
  • T4 Perform ultrasonic treatment on the first mixed solution obtained in step T3 to obtain a second mixed solution
  • T5 Filter the second mixed solution obtained in step T4 to obtain the immunostimulatory complex.
  • the conditions for ultrasonic treatment in step T4 include: temperature is 20-30°C, power is 50-70W, total ultrasonic treatment time is 5-15 minutes, ultrasonic mode is: ultrasonic for 10 seconds, pause for 10 seconds .
  • a poultry animal vaccine comprising the water-in-oil immune adjuvant of the first aspect of the present invention is also provided, such as a vaccine for poultry animals such as chickens and ducks.
  • This example prepares a water-in-oil adjuvant as a vaccine adjuvant, and uses the vaccine adjuvant and duck infectious serositis bivalent inactivated antigen (type 1 SG4 strain + type 2 ZZY7 strain) to prepare vaccines.
  • the concentration of bacterial liquid was 1 ⁇ 10 10 CFU/ml (provided by Liaoning Yikang Biopharmaceutical Co., Ltd., compared with the duck infectious serositis bivalent inactivated vaccine (type 1 SG4 + type 2 ZZY7 strain) announced by the Ministry of Agriculture in 1893 )
  • the concentration of bacterial solution for vaccine preparation specified in the manufacturing and inspection regulations is not less than 2.5 ⁇ 10 11 CFU/ml (20 times lower), and the bivalent vaccine for duck infectious serositis is prepared according to the mass ratio of adjuvant and antigen of 2:1.
  • Live vaccine type 1 SG4 strain + type 2 ZZY7 strain).
  • the preparation method of vaccine adjuvant includes the following steps:
  • step S2 Add 6wt% of refined Siben-80 and 4wt% of refined Tween-80 to the heated injection oil in step S1, mix well and then filter (for example, use a 0.22 ⁇ m filter membrane) Filtration) to obtain the vaccine adjuvant.
  • filter for example, use a 0.22 ⁇ m filter membrane
  • step S2 The refined Siben-80 described in step S2 is obtained in the following way:
  • step 2) Stop the vacuuming in step 2), continue to pass in nitrogen to maintain pressure and cool down, and discharge the second treatment product from the reaction kettle.
  • the discharge is connected to a 0.45 ⁇ m filtration device to coarsely filter the feed liquid to remove unreacted matter, then transfer the filtrate to a sterile environment, and filter and sterilize it again with a 0.22 ⁇ m filter membrane to obtain refined Siben-80.
  • the refined Tween-80 described in step S2 is obtained by:
  • Tween-80 purchased from Guangzhou Qiao Linger Biotechnology Co., Ltd.
  • Tween-80 put it into a stainless steel reaction kettle, start stirring until evenly stirred, then add nitrogen to reduce the pressure and raise the temperature to 80 ⁇ 5°C/700mmHg, and the kettle
  • the internal pressure is 0.2MPa, and H 2 O 2 is slowly added.
  • H 2 O 2 is slowly added.
  • a small amount of H 2 O 2 is measured, connected to the vacuum pump and slowly dripped in. Observe the color change of Tween-80 in the reaction kettle. The color is from When the depth changes, stop adding H 2 O 2 and keep the reaction time for 10-15 minutes.
  • step (3) Stop the vacuuming in step (2), continue to pass in nitrogen to maintain pressure and cool down, and discharge the fourth treatment product from the reaction kettle.
  • the discharge is connected to a 0.45 ⁇ m filtration device to coarsely filter the feed liquid to remove unreacted matter, then transfer the filtrate to a sterile environment, and filter and sterilize it again with a 0.22 ⁇ m filter membrane to obtain refined Tween-80.
  • the preparation method of duck infectious serositis bivalent inactivated vaccine includes the following steps:
  • the vaccine adjuvant and duck infectious serositis bivalent inactivated antigen are prepared according to a mass ratio of 2:1. At 30-32°C, first put the vaccine adjuvant into the emulsified In the tank, stir at 2000rpm/min, slowly add the aqueous antigen, and then stir at 8000rpm/min for 30 minutes to prepare a bivalent inactivated vaccine for duck infectious serositis (type 1 SG4 strain + type 2 ZZY7 strain). As shown in Figure 1, the particle size distribution diagram of the duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain + type 2 ZZY7 strain) obtained in Example 1 is shown. It can be seen that the volume average particle size is about is 0.630 ⁇ m.
  • Example 2-3 The vaccine adjuvant and duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain + type 2 ZZY7 strain) were prepared according to the operating steps of Example 1. The only difference lies in the preparation of the vaccine adjuvant. The content of each ingredient in the raw materials is different, specifically:
  • Example 2 The raw materials for preparing vaccine adjuvant are: 90wt% Marcol-52 white oil, 5wt% refined Siban-80 and 5wt% refined Tween-80.
  • the raw materials for preparing vaccine adjuvant in Example 3 are: 85 wt% Marcol-52 white oil, 10 wt% refined Siben-80 and 5 wt% refined Tween-80.
  • Example 4 Prepare vaccine adjuvant and duck infectious serositis bivalent inactivated antigen (type 1 SG4 strain + type 2 ZZY7 strain) according to the operating steps of Example 1, except that in step S2, the heated Add 0.2wt% immune stimulating complex to the oil for injection, and the raw materials for preparing the vaccine adjuvant are: 90wt% Marcol-52 white oil, 6wt% refined Shiben-80, 3.8wt% Refined Tween-80 and 0.2wt% immune-stimulating complex, wherein the immune-stimulating complex includes phospholipids, cholesterol, tocopherols and saponins in a mass ratio of 1:0.5:0.5:1, and the preparation method of the immune-stimulating complex includes Following steps:
  • mixture A weigh soybean phospholipid HSPC, cholesterol CHOL, ⁇ -tocopherol, and saponin, and prepare mixture A according to the mass fraction of 1:0.5:0.5:1. Dissolve the obtained mixture A with the chloroform/methanol solvent in a ratio of 1:2 to obtain an organic solution of lipids for later use, to prepare mixture B.
  • the chloroform-methanol solvent is composed of chloroform, methanol and ultrapure liquid with a volume ratio of 20:60:20. Composed of water.
  • step T2 Transfer the mixture B in step T1 to a 250ml round-bottomed beaker, place the round-bottomed flask on a rotary evaporator, and perform vacuum rotary evaporation to remove the organic solvent under the conditions of a constant temperature water bath.
  • a solid will form on the inner wall of the rotary evaporator flask.
  • C where the rotary evaporation temperature is 30-40°C, the rotation speed is 80-150r/min, and the rotary evaporation time is 60-90min.
  • step T3 After the organic solvent is completely rotary evaporated in step T2, remove the round-bottomed flask and dissolve solid C with 0.01 mol/L PBS solution to obtain mixed solution D.
  • step T4 Ultrasonicate the mixed solution D obtained in step T3 at a temperature of 20-30°C and a power of 60W to obtain mixed solution E.
  • the ultrasonic treatment conditions are: ultrasonic for 10 minutes, ultrasonic for 10s, and pause for 10s.
  • T5. Filter and sterilize the obtained mixed solution E with a 0.22 ⁇ m filter membrane, and seal and store it at -70°C to obtain an immune stimulating complex.
  • the method for preparing a bivalent inactivated duck infectious serositis vaccine includes: taking 90 parts by weight of Marcol-52 white oil and 6 parts by weight of Shiben-80, Mix and stir evenly, sterilize at 115°C for 40 minutes to make an oil phase; take the bivalent inactivated antigen of duck infectious serositis (type 1 SG4 strain + type 2 ZZY7 strain) that has passed the inactivation test, and mix it in equal volumes to obtain 100 parts by weight of the mixed solution, add 8 parts by weight of Tween-80, mix thoroughly to form a water phase; take the oil phase and water phase at a mass ratio of 2:1, at 30-32°C, first mix the oil and Put the phase into the emulsification tank, stir at 2000rpm/min, slowly add the water phase, and then stir at 8000rpm/min for 30 minutes to prepare the duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain+ Type 2
  • Comparative Examples 2-4 prepared vaccine adjuvant and duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain + type 2 ZZY7 strain) according to the operating procedures of Example 1. The difference lies in the preparation of vaccine adjuvant.
  • the raw materials are different, specifically:
  • the raw materials for preparing the vaccine adjuvant in Comparative Example 2 are: 90wt% Marcol-52 white oil, 6wt% refined Siban-80 (prepared in Example 1) and 4wt% Tween-80.
  • the raw materials for preparing the vaccine adjuvant in Comparative Example 3 are: 90wt% Marcol-52 white oil, 6wt% Spon-80 and 4wt% refined Tween-80 (prepared in Example 1).
  • the raw materials for preparing the vaccine adjuvant in Comparative Example 4 are: 90wt% Marcol-52 white oil, 6wt% Siben-80 and 4wt% Tween-80.
  • a total of 540 healthy susceptible ducks aged 5 to 10 days were selected and divided into 9 groups, with 60 ducks in each group. Among them, 8 groups with a total of 480 animals were used as test groups.
  • the duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain + type 2 ZZY7) prepared in the above-mentioned Examples 1-4 and Comparative Examples 1-4 was subcutaneously injected into the neck respectively. strain)0.25ml, and another group of 60 animals without vaccination served as the control group.
  • the bivalent inactivated vaccine in Comparative Example 1 adopts the traditional preparation method of the bivalent inactivated vaccine for duck infectious serositis (i.e., first prepare the oil for injection with Siben-80 into an oil phase and mix the antigen with Tween-80). 80 was prepared into an aqueous phase, and then the aqueous phase and the oil phase were emulsified and mixed), and the protection rate of R.
  • Comparative Example 2-4 is to first use Siben-80 and/or Tween-80 to prepare a vaccine adjuvant, and then use the vaccine adjuvant and antigen to formulate a vaccine.
  • the vaccine in Comparative Example 4 cannot respond to the vaccine on the 21st day after vaccination. This group of ducks provides effective immune protection and can only achieve the highest protection rate of 8/10.
  • Comparative Examples 2-3 one of refined Siben-80 and refined Tween-80 was used to prepare the immune adjuvant.
  • the immune protective effect of Comparative Example 4 was slightly improved, but the highest protection rate of 6/10 could only be achieved by challenge 60 days after vaccination, which also showed the shortcoming of the short duration of immune efficacy.
  • Comparative Examples 1-4 used both purified Siben-80 and purified Tween-80 in the preparation of immune adjuvants.
  • the vaccines prepared from the immune adjuvants were effective on days 21 and 60 after immunization. Inoculation of R.
  • anatipestifer SG4 strain and ZZY7 strain on the 90th and 90th day can achieve a protection rate of more than 9/10, that is, the duration of the vaccine's effectiveness can be as long as more than 3 months.
  • the above results prove that compared to the vaccines prepared using vaccine adjuvants containing Siben-80 and/or Tween-80 in Comparative Examples 1-4, the vaccines containing refined Siben-80 and/or Tween-80 were used in Examples 1-4 of the present invention.
  • the vaccine product prepared with the refined Tween-80 vaccine adjuvant can significantly enhance the specific immune response level of ducks to the antigens in the vaccine, and the immune duration is as long as more than 3 months, and the uniformity is good (showing the quality of the vaccine product Stablize).
  • the bacterial liquid concentration used in Examples 1-4 when preparing the duck infectious serositis bivalent inactivated vaccine was 1 ⁇ 10 10 CFU/ml, and the ratio was according to the Ministry of Agriculture
  • the manufacturing and inspection regulations for the bivalent inactivated duck infectious serositis vaccine (Type 1 SG4 + Type 2 ZZY7 strain) announced in 1893 stipulates that the concentration of the bacterial solution used for vaccine preparation is not less than 2.5 ⁇ 10 11 CFU/ml, which is 20 times lower.
  • the duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain + type 2 ZZY7 strain) obtained in Examples 1-4 still has a good immune protection effect and sustained immunity.
  • the period is more than 3 months, and the uniformity is good.
  • the duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain + type 2 ZZY7) prepared in the above embodiments 1-4 and comparative examples 1-4 was subcutaneously injected into the neck respectively. strain) 1ml, and another group of 10 animals without vaccination as the control group. Observe continuously for 7 days. All animals should be healthy and alive, and there should be no local or systemic adverse reactions caused by the vaccine. The observation results are shown in Table 2 below.
  • Examples 5-6 Prepare vaccine adjuvant and duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain + type 2 ZZY7 strain) according to the operating steps of Example 1, the only difference lies in the preparation of vaccine adjuvant The content of each ingredient in the raw materials is different, specifically:
  • the raw materials for preparing vaccine adjuvant in Example 5 are: 89wt% Marcol-52 white oil, 10wt% refined Siban-80 and 1wt% refined Tween-80.
  • the raw materials for preparing vaccine adjuvant in Example 6 are: 85wt% Marcol-52 white oil, 10wt% refined Siban-80 and 5wt% refined Tween-80.
  • Examples 7-8 The vaccine adjuvant and duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain + type 2 ZZY7 strain) were prepared according to the operating steps of Example 4. The only difference lies in the preparation of the vaccine adjuvant. The content of each ingredient in the raw materials is different, specifically:
  • the raw materials for preparing vaccine adjuvant in Example 7 are: 86.9wt% Marcol-52 white oil, 9wt% refined Spon-80, 4wt% refined Tween-80 and 0.1wt% immune stimulating complex.
  • Example 8 The raw materials for preparing the vaccine adjuvant are: 87wt% Marcol-52 white oil, 8wt% refined Spon-80, 4wt% refined Tween-80 and 1wt% immune stimulating complex.
  • 300 healthy susceptible ducks aged 5 to 10 days were selected and divided into 5 groups, with 60 ducks in each group.
  • 4 groups of 240 animals were used as test groups, and 0.25 ml of the duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain + type 2 ZZY7 strain) of the above embodiments 5-8 was injected subcutaneously into the neck respectively.
  • a total of 60 animals were not vaccinated as a control group.

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Abstract

L'invention concerne un adjuvant eau dans l'huile pour un vaccin destiné à une volaille, son procédé de préparation et son utilisation, qui appartiennent au domaine technique des vaccins animaux dans la catégorie des produits biologiques. Les matières premières de l'adjuvant eau dans l'huile selon l'invention comprennent, en pourcentage en poids : 85 % en poids à 90 % en poids d'huile pour injection, 5 % en poids à 10 % en poids de Span raffiné 80 et 1 % en poids à 5 % en poids de Tween 80 raffiné, et peut en outre comprendre 0,1 % en poids à 1 % en poids d'un complexe immunostimulateur. Un vaccin préparé à l'aide de l'adjuvant eau dans l'huile présentement décrit est stable en termes de qualité et présente une sécurité élevée, et peut induire un corps pour générer une immunoréaction plus longue et plus efficace, de telle sorte que l'adjuvant eau dans l'huile peut être utilisé en tant qu'adjuvant sûr et efficace pour un vaccin destiné à une volaille et similaire.
PCT/CN2022/134909 2022-06-02 2022-11-29 Adjuvant eau dans huile pour vaccin destiné à une volaille, son procédé de préparation et son utilisation WO2023231333A1 (fr)

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