CN117618553A - Oil-in-water adjuvant and preparation method and application thereof - Google Patents
Oil-in-water adjuvant and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an oil-in-water adjuvant and a preparation method and application thereof, belonging to the technical field of biological product animal vaccines. The provided oil-in-water adjuvant comprises the following raw materials in percentage by mass: 75-83 wt% of oil for injection, 11-15 wt% of refined span-80, 6-9 wt% of refined tween-80 and 0.1-1 wt% of immunopotentiator. The vaccine prepared by the oil-in-water adjuvant provided by the invention has low viscosity, stable quality and high safety, can induce organisms to generate long-duration and efficient immune response, and can be used as a safe and effective adjuvant for animal vaccines and the like.
Description
Technical Field
The invention belongs to the technical field of veterinary vaccines, and particularly relates to an oil-in-water adjuvant and a preparation method and application thereof.
Background
The immunoadjuvant refers to a component which is injected into an animal body before or simultaneously with an antigen, can nonspecifically change or enhance specific immune response of the body to the antigen, and plays an auxiliary role. The animal vaccine mainly uses oil adjuvant, the oil adjuvant vaccine is a vaccine using mineral oil and surfactant as adjuvants, the oil adjuvant can enable antigens to be released continuously at an injection site of an organism, a carrier is provided for transferring the antigens in a lymphatic system, the formation and aggregation of mononuclear cells are increased, and the immune efficacy is improved. Oil adjuvants can be divided into three dosage forms: water-in-oil (W/O) type, water-in-oil-in-water (W/O/W) type, and oil-in-water (O/W) type.
At present, domestic animal inactivated vaccines mainly comprise two dosage forms of water-in-oil and water-in-oil, and the finished vaccine has higher adjuvant content (50-75%), and has the following defects: the viscosity of the finished vaccine is high, the side effect of individual oil adjuvant is serious, and the cost is continuously increased along with the continuous increase of the price of petroleum. The research and application of new formulation are widely paid attention to, and the vaccine new formulation, in particular various antigen controlled release systems, can obviously improve the immunity efficacy of the vaccine, and is safer and more convenient to use. In particular, oil-in-water dosage forms, biphasic microemulsion dosage forms and water adjuvants greatly reduce the consumption of mineral oil, and the vaccine has small viscosity and light immune side effect and becomes the main stream of high-quality vaccines.
The mass ratio of the oil-in-water (O/W) adjuvant in the vaccine is 10% -25%, so that the use of the oil adjuvant can be greatly reduced, the veterinary vaccine is changed from the original W/O agent (high toxicity) vaccine to O/W type (low toxicity or non-toxicity), the viscosity of the finished vaccine is greatly reduced, and the safety of the finished vaccine is improved. However, oil-in-water (O/W) adjuvants have not been widely used due to their short immunization duration.
Disclosure of Invention
In view of one or more problems of the prior art, one aspect of the present invention provides an oil-in-water adjuvant prepared from raw materials comprising, in weight percent: 75-83 wt% of oil for injection, 11-15 wt% of refined span-80, 6-9 wt% of refined tween-80 and 0.1-1 wt% of immunopotentiator, wherein the immunopotentiator comprises phospholipid, pollen Pini, tocopherol and saponin with the mass ratio of (0.5-2): 2-4): 0.5-2): 0.5-3, wherein:
the refined span-80 is a product obtained by processing span-80 in the following way:
1) Using H 2 O 2 To the departmentThe method comprises the steps of processing the span-80 to enable the color of the span-80 to change from dark to light, and obtaining a first processing product when the color of the span-80 is not changed any more;
2) Removing unreacted H from the first treated product 2 O 2 Obtaining a second treatment product;
3) Filtering the second treated product to obtain the refined span-80;
the purified tween-80 is a product obtained by treating tween-80 in the following way:
(1) Using H 2 O 2 Treating the Tween-80 to change the color of the Tween-80 from dark to light, and obtaining a third treated product when the color of the Tween-80 is not changed any more;
(2) Removing unreacted H in the third treated product 2 O 2 Obtaining a fourth treatment product;
(3) And filtering the fourth processed product to obtain the refined Tween-80.
In some embodiments, the filtration in step 3) and step (3) is performed by first performing a rough filtration using a filtration device having a filtration pore size of 0.40 μm to 0.65 μm, and then performing a fine filtration of the filtrate using a filtration device having a filtration pore size of 0.10 μm to 0.22 μm.
In some embodiments, the injection oil comprises injection mineral oil, injection vegetable oil, or a combination thereof; optionally, the mineral oil for injection comprises white oil.
In some embodiments, 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, and Sonneborn 4# white oil, sonneborn10# white oil PARACOS KF40, PARACOS KF50, squalene, squalane.
In another aspect, the present invention provides a method for preparing an oil-in-water adjuvant comprising the steps of:
s1: heating 75-83 wt% of injection oil to 30-40 ℃;
s2: adding 11-15 wt% of refined span-80, 6-9 wt% of refined tween-80 and 0.1-1 wt% of immunopotentiator into the oil for injection heated in the step S1, uniformly mixing, and filtering to obtain the oil-in-water adjuvant; wherein:
the refined span-80 is obtained by processing span-80 in the following way:
1) Using H 2 O 2 Processing the span-80 to change the color of the span-80 from dark to light, and obtaining a first processing product when the color of the span-80 is not changed any more;
2) Removing unreacted H from the first treated product 2 O 2 Obtaining a second treatment product;
3) Filtering the second treated product to obtain the refined span-80;
the refined tween-80 is obtained by treating tween-80 in the following way:
(1) Using H 2 O 2 Treating the Tween-80 to change the color of the Tween-80 from dark to light, and obtaining a third treated product when the color of the Tween-80 is not changed any more;
(2) Removing unreacted H in the third treated product 2 O 2 Obtaining a fourth treatment product;
(3) And filtering the fourth processed product to obtain the refined Tween-80.
In some embodiments, the filtration in step 3) and step (3) is performed by first performing a rough filtration using a filtration device having a filtration pore size of 0.40 μm to 0.65 μm, and then performing a fine filtration of the filtrate using a filtration device having a filtration pore size of 0.10 μm to 0.22 μm.
In some embodiments, the immunopotentiator comprises phospholipids, pollen Pini, tocopherol, and saponins in a mass ratio of (0.5-2): (2-4): (0.5-2): (0.5-3).
In some embodiments, the immunopotentiator is prepared by the steps of:
t1: dissolving a mixture of phospholipid, pollen pini, tocopherol and saponin with the mass percentage of (0.5-2), (2-4), and (0.5-2) in an ethanol solution to obtain a lipoid organic solution; optionally, the ethanol solution is prepared from the following components in a volume ratio of 80:20, ethanol and ultrapure water;
t2: shaking and uniformly mixing the lipoid organic solution obtained in the step T1, standing and filtering, and collecting filtrate and precipitate; adding the precipitate into the ethanol solution, shaking, mixing, standing, filtering, collecting filtrate, mixing the two filtrates, centrifuging, and collecting supernatant;
t3: removing the organic solvent in the supernatant obtained in the step T2 to obtain a solid;
t4: dissolving the solid obtained in the step T3 by using PBS solution to obtain a first mixed solution;
t5: performing ultrasonic treatment on the first mixed solution obtained in the step T4 to obtain a second mixed solution;
t6: and (3) filtering the second mixed solution obtained in the step (T5) to obtain the immunopotentiator.
In some embodiments, the conditions of the sonication in step T5 include: the temperature is 20-30 ℃, the power is 50-70W, the total ultrasonic treatment time is 5-15min, and the ultrasonic mode is as follows: ultrasound for 10s and pause for 10s.
In yet another aspect, the invention provides an animal vaccine comprising the above oil-in-water adjuvant.
According to the technical scheme, the refined span-80 and the refined tween-80 are used in the raw materials of the oil-in-water adjuvant, and the immunopotentiator is further used, so that the vaccine product prepared by the oil-in-water adjuvant provided by the invention has low viscosity, stable quality and high safety, and surprisingly, can induce organisms to generate specific immune response with longer duration (for example, the immune protection period of the bovine epidemic heat inactivated vaccine (ZJ strain) prepared by the aid of the vaccine for calves can be longer than 150 days) and higher efficiency (for example, the antibody titer of the bovine epidemic heat inactivated vaccine (ZJ strain) prepared by the aid of the vaccine for calves after immunization of the calves can be up to more than 1:8192). Therefore, the oil-in-water adjuvant provided by the invention can be helpful for developing animal vaccine products with better stability and safety, longer immunization duration and better immunization effect.
Drawings
FIG. 1 is a graph showing the particle size distribution of a bovine circulating heat-inactivated vaccine (ZJ strain) obtained in example 1.
FIG. 2 is a graph showing the time course of the level of neutralizing antibodies induced after immunization for the vaccines of examples 1-3 and comparative examples 1-3.
Detailed Description
The present invention will be described in detail with reference to the following specific examples. The terms "first," "second," "third," and "fourth," and the like, herein are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order, or for limiting the number of objects.
In a first aspect of the present invention, there is provided an oil-in-water adjuvant comprising the following raw materials in weight percent: 75-83 wt% of oil for injection, 11-15 wt% of refined span-80, 6-9 wt% of refined tween-80 and 0.1-1 wt% of immunopotentiator, wherein the immunopotentiator comprises phospholipid, pollen Pini, tocopherol and saponin with the mass ratio of (0.5-2): 2-4): 0.5-2): 0.5-3; wherein:
the refined span-80 can be a product obtained by processing span-80 in the following way, wherein the span-80 can be obtained commercially or according to a conventional span-80 preparation method:
1) Using H 2 O 2 Processing the span-80 to change the color of the span-80 from dark to light, and obtaining a first processing product when the color of the span-80 is not changed any more. This step may specifically comprise the following operations: weighing span-80, putting into a stainless steel reaction kettle, stirring until stirring uniformly, introducing nitrogen, heating to 80+ -5deg.C/700 mmHg under reduced pressure, and slowly adding H under pressure of 0.15-0.25MPa 2 O 2 To control H 2 O 2 The amount of H added is small 2 O 2 Slowly dripping the mixture by connecting a vacuum pump, observing the color change of the span-80 in the reaction kettle, and stopping adding H when the color changes from deep to shallow 2 O 2 The reaction time is kept for 10-15min;
2) Removing unreacted H from the first treated product 2 O 2 (e.g., by vacuum dehydration) to yield a second treated product. This step may specifically comprise the following operations: when the color of the span-80 in the reaction kettle is unchanged and is slightly yellow, clear and transparent, the reaction kettle is heated to 110+/-5 ℃, and the unreacted excessive H is dehydrated by vacuumizing 2 O 2 Pumping out the reaction kettle, and vacuumizing and dehydrating for 3-5min;
3) And filtering the second treated product to obtain the refined span-80. This step may specifically comprise the following operations: stopping vacuumizing in the step 2, continuously introducing nitrogen, maintaining pressure and cooling, and discharging the second treatment product from the reaction kettle; the discharged material is connected with a 0.40-0.65 mu m filtering device to carry out rough filtration on the feed liquid to remove unreacted substances, then the filtrate is transferred into a sterile environment, and the filter membrane with the thickness of 0.10-0.22 mu m is used for filtering and sterilizing to obtain the refined span-80.
The purified tween-80 can be obtained by treating tween-80 in the following way, wherein tween-80 can be obtained commercially or according to the conventional tween-80 preparation method:
(1) Using H 2 O 2 And (3) treating the Tween-80 to change the color of the Tween-80 from dark to light, and obtaining a third treated product when the color of the Tween-80 is not changed any more. This step may specifically comprise the following operations: weighing Tween-80, putting into a stainless steel reaction kettle, stirring, introducing nitrogen, heating to 80+ -5deg.C/700 mmHg under reduced pressure, adding 0.15-0.25MPa, and slowly adding H 2 O 2 To control H 2 O 2 The amount of H added is small 2 O 2 Slowly dripping with vacuum pump, observing the change of Tween-80 color in the reaction kettle, and stopping adding H when the color changes from deep to light 2 O 2 The reaction time is kept for 10-15min;
(2) Removing unreacted H from the third treated product 2 O 2 (e.g., by vacuum dehydration) to yield the fourth treated product. This step may specifically comprise the following operations: heating the reaction kettle when the color of Tween-80 in the reaction kettle is not changed and is slightly yellow, clear and transparentVacuum dehydration to 110+ -5deg.C 2 O 2 Pumping out the reaction kettle, and vacuumizing and dehydrating for 3-5min;
(3) And filtering the fourth processed product to obtain the refined Tween-80. This step may specifically comprise the following operations: stopping vacuumizing in the step (2), continuously introducing nitrogen, maintaining pressure and cooling, and discharging the fourth processed material from the reaction kettle; the discharged material is connected with a 0.40-0.65 mu m filtering device to carry out rough filtration on the feed liquid to remove unreacted substances, then the filtrate is transferred into a sterile environment, and the filtered and sterilized solution is filtered and sterilized by a 0.10-0.22 mu m filter membrane to obtain refined Tween-80.
In the invention, compared with SEPPIC ISA25VG vaccine adjuvant or SEPPIC ISA AVG vaccine adjuvant, the vaccine adjuvant prepared by using refined span-80, refined tween-80 and immunopotentiator can effectively improve the quality stability and safety of the prepared vaccine, and can further enhance the specific immune response level of an animal organism to antigens in the vaccine. The possible reason is that the extra by-products and impurities introduced from the raw materials are removed, thereby improving the stability and safety of the vaccine and simultaneously improving the immune efficacy of the vaccine by adding the immune enhancer.
In some embodiments, the injectable oil may include injectable mineral oil, injectable vegetable oil, or a combination thereof.
In some embodiments, the mineral oil for injection may include white oil.
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, sonneborn10# white oil PARACOS KF40, PARACOS KF50, squalene, squalane, which may be obtained from any commercial source.
The animal vaccine product prepared by the oil-in-water adjuvant provided by the invention is an oil-in-water particle which has an oil-in-water two-layer structure: the outermost layer is the aqueous phase (i.e., the combination of antigen and immunopotentiator) and the inner layer is the oil phase (i.e., the adjuvant of the present invention). When the vaccine is injected into a body, particles are broken along with the body temperature change of the body, antigen substances combined by an immunopotentiator are released, the immunopotentiator is a high-efficiency immune presentation system, antigen presentation cells are activated, various inflammatory cells are generated at an antigen injection position through an oil-in-water adjuvant, and the intake of the antigen substances can be further improved.
In a second aspect of the invention, there is provided a method of preparing a vaccine adjuvant, which may comprise the steps of:
s1: heating 75-83 wt% of injection oil to 30-40 ℃;
s2: adding 11-15 wt% of refined span-80, 6-9 wt% of refined tween-80 and 0.1-1 wt% of immunopotentiator into the oil for injection heated in the step S1, uniformly mixing, and filtering to obtain a vaccine adjuvant; wherein:
both refined span-80 and refined tween-80 are obtainable according to the treatment method described in the first aspect of the invention.
In some embodiments, the immunopotentiator comprises phospholipids, pollen Pini, tocopherol, and saponins in a mass ratio of (0.5-2): (2-4): (0.5-2): (0.5-3).
In some embodiments, the immunopotentiator is prepared by the steps of:
t1: dissolving a mixture of phospholipid, pollen pini, tocopherol and saponin with the mass percentage of (0.5-2), (2-4), and (0.5-2) in an ethanol solution to obtain a lipoid organic solution; optionally, the ethanol solution is prepared from the following components in a volume ratio of 80:20, ethanol and ultrapure water;
t2: shaking and uniformly mixing the lipoid organic solution obtained in the step T1, standing and filtering, and collecting filtrate and precipitate; adding the precipitate into the ethanol solution, shaking, mixing, standing, filtering, collecting filtrate, mixing the filtrates, centrifuging (e.g. 6000-8000r/min for 10-20 min), and collecting supernatant;
t3: removing the organic solvent in the supernatant collected in the step T2 to obtain a solid;
t4: dissolving the solid obtained in the step T3 by using PBS solution to obtain a first mixed solution;
t5: performing ultrasonic treatment on the first mixed solution obtained in the step T4 to obtain a second mixed solution;
t6: and (3) filtering the second mixed solution obtained in the step (T5) to obtain the immunopotentiator.
In a preferred embodiment, the conditions of the ultrasonic treatment in step T5 include: the temperature is 20-30 ℃, the power is 50-70W, the total ultrasonic treatment time is 5-15min, and the ultrasonic mode is as follows: ultrasound for 10s and pause for 10s.
Examples
The methods used in the examples described below are conventional methods unless otherwise specified. The various biomaterials described in the examples were obtained by merely providing an experimental route for achieving the objectives of the specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples.
Examples detailed embodiments and specific operation procedures are given on the premise of the technical scheme of the present invention, and examples are helpful for understanding the present invention, but should not be construed as limiting the present invention.
Example 1:
the embodiment prepares an oil-in-water adjuvant as a vaccine adjuvant, prepares the bovine epidemic heat inactivated vaccine by using the vaccine adjuvant and bovine epidemic heat (ZJ strain) whole virus inactivated antigen according to the mass ratio of 1:3, and the titer of the vaccine preparation virus liquid is 10 7.5 TCID 50/ ml, prepared and stored by Jin Yubao Ling Bio-medicine Co.
1.1, the preparation method of the vaccine adjuvant comprises the following steps:
s1: heating 81.5wt% of Marcol-52 white oil to 35 ℃;
s2: adding 12wt% of refined span-80, 6wt% of refined tween-80 and 0.5wt% of immunopotentiator (comprising phospholipid, pollen Pini, tocopherol and saponin in a mass ratio of 1:3:1:1) into the oil for injection heated in the step S1, mixing uniformly, and filtering (for example, filtering with a 0.22 μm filter membrane) to obtain the vaccine adjuvant. Wherein:
the refined span-80 in step S2 is obtained by:
1) Weighing span-80 (purchased from Guangzhou Qiaoling Biotechnology Co., ltd.), putting into a stainless steel reaction kettle, stirring until uniform stirring, introducing nitrogen, heating under reduced pressure to 80+ -5deg.C/700 mmHg, and slowly adding H under pressure of 0.2MPa 2 O 2 To control H 2 O 2 The amount of H added is small 2 O 2 Slowly dripping the mixture by connecting a vacuum pump, observing the color change of the span-80 in the reaction kettle, and stopping adding H when the color changes from deep to shallow 2 O 2 The reaction time is kept for 10-15min.
2) When the color of the span-80 in the reaction kettle is not changed any more and is slightly yellow, clear and transparent (the first treatment product is obtained), the reaction kettle is heated to 110 ℃, and the unreacted excess H is dehydrated by vacuumizing 2 O 2 And (5) extracting the reaction kettle, and vacuumizing and dehydrating for 3min to obtain a second treatment product.
3) Stopping vacuumizing in the step 2), continuously introducing nitrogen, maintaining pressure and cooling, and discharging the second treatment product from the reaction kettle. The discharged material is connected with a 0.45 mu m filter device to carry out rough filtration on the feed liquid to remove unreacted substances, then the filtrate is transferred into a sterile environment, and the filter membrane with the thickness of 0.22 mu m is used for filtration and sterilization again to obtain the refined span-80.
The refined tween-80 in step S2 is obtained by:
(1) Weighing Tween-80 (purchased from Qiaoling Biotechnology Co., ltd., guangzhou), putting into a stainless steel reaction kettle, stirring until uniform stirring, introducing nitrogen, heating under reduced pressure to 80+ -5deg.C/700 mmHg, pressure in the kettle being 0.2MPa, and slowly adding H 2 O 2 To control H 2 O 2 The amount of H added is small 2 O 2 Slowly dripping with vacuum pump, observing the change of Tween-80 color in the reaction kettle, and stopping adding H when the color changes from deep to light 2 O 2 Maintaining the reactionThe time is 10-15min.
(2) When the color of Tween-80 in the reaction kettle is not changed and is slightly yellow, clear and transparent (the third treatment product is obtained), heating the reaction kettle to 110 ℃, vacuumizing and dehydrating to remove unreacted excessive H 2 O 2 And (3) pumping out the reaction kettle, and vacuumizing and dehydrating for 3min (obtaining a fourth treatment product).
(3) Stopping vacuumizing in the step (2), continuously introducing nitrogen, maintaining pressure and cooling, and discharging the fourth treated product from the reaction kettle. The discharged material is connected with a 0.45 mu m filter device to carry out rough filtration on the feed liquid to remove unreacted substances, then the filtrate is transferred into a sterile environment, and the purified Tween-80 is obtained by filtration sterilization with a 0.22 mu m filter membrane.
The preparation method of the immunopotentiator in the step S2 comprises the following steps:
t1: dissolving a mixture of phospholipid, pollen pini, tocopherol and saponin with the mass percentage of 1:3:1:1 in ethanol solution to obtain a lipoid organic solution; optionally, the ethanol is prepared from the following components in volume ratio of 80:20, ethanol and ultrapure water;
t2: shaking and uniformly mixing the lipoid organic solution obtained in the step T1, standing for 6 hours, filtering, and collecting filtrate and precipitate; adding ethanol solution into the precipitate, shaking, mixing, standing for 6 hr, filtering, collecting filtrate, mixing the two filtrates, centrifuging at 8000r/min for 10min, and collecting supernatant;
t3: removing the organic solvent in the supernatant obtained in the step T2 to obtain a solid;
t4: dissolving the solid obtained in the step T3 by using PBS solution to obtain a first mixed solution;
t5: performing ultrasonic treatment on the first mixed solution obtained in the step T4 to obtain a second mixed solution;
t6: and (3) filtering the second mixed solution obtained in the step (T5) to obtain the immunopotentiator.
1.2, the preparation method of the bovine ephemeral heat inactivated vaccine (ZJ strain) comprises the following steps:
mixing the vaccine adjuvant with bovine epidemic heat-inactivated antigen (ZJ strain) according to a mass ratio of 1:3, and emulsifying at 25-30 ℃ and 500rpm/min for 15 minutes to prepare the bovine epidemic heat-inactivated vaccine. As shown in FIG. 1, the particle size distribution diagram of the bovine ephemeral heat-inactivated vaccine obtained in example 1 shows that the particle size is about 0.080 to 0.600. Mu.m, and the average particle size is 0.168. Mu.m.
Examples 2 to 3
Examples 2-3 following the procedure of example 1, a vaccine adjuvant was prepared using bovine ephemeral heat-inactivated antigen of the same viral content as the adjuvant prepared in examples 2-3, except that the content of each component in the raw materials used to prepare the vaccine adjuvant was different, specifically:
example 2 the vaccine adjuvant was prepared from the following raw materials: 82.9wt% of Marcol-52 white oil, 11wt% of refined span-80, 6wt% of refined tween-80 and 0.1wt% of immunopotentiator (comprising phospholipid, pollen Pini, tocopherol and saponin in a mass ratio of 1:3:1:1);
example 3 the vaccine adjuvant was prepared from the following raw materials: 75wt% of Marcol-52 white oil, 15wt% of refined span-80, 9wt% of refined tween-80 and 1wt% of immunopotentiator (comprising phospholipid, pollen Pini, tocopherol and saponin in a mass ratio of 1:3:1:1).
Comparative example 1
Comparative example 1 a vaccine adjuvant and a bovine ephemeral heat inactivated vaccine were prepared according to the procedure of example 1, except that the vaccine adjuvant was prepared using different raw materials, specifically: comparative example 1 the vaccine adjuvant was prepared from the following raw materials: 81.5wt% of Marcol-52 white oil, 12wt% of refined span-80, 6wt% of refined tween-80 and 0.5wt% of immunostimulating complex (comprising phospholipids, cholesterol, tocopherols and saponins in a mass ratio of 1:3:1:1). Wherein the preparation method of the immunostimulating complex comprises the following steps:
t1 weighing soybean phospholipid HSPC, cholesterol CHOL, alpha-tocopherol and saponin, and preparing a mixture A according to the mass fraction of 1:3:1:1. The obtained mixture A and chloroform/methanol solvent are dissolved in a ratio of 1:2 to obtain lipoid organic solution for standby, and a mixture B is prepared, wherein the chloroform/methanol solvent consists of chloroform, methanol and ultrapure water in a volume ratio of 20:60:20.
And T2, transferring the mixture B in the step T1 into a 250ml round bottom beaker, placing the round bottom flask on a rotary evaporator, and performing vacuum rotary evaporation under the condition of constant-temperature water bath to remove the organic solvent, wherein the inner wall of the rotary evaporator flask forms a solid C, the rotary evaporation temperature is 30-40 ℃, the rotating speed is 80-150r/min, and the rotary evaporation time is 60-90min.
And T3, taking down the round bottom flask after the organic solvent is completely distilled in the step T2, and dissolving the solid C by using 0.01mol/L PBS (phosphate buffered saline) to obtain a mixed solution D.
And T4, carrying out ultrasonic treatment on the mixed solution D obtained in the step T3 at the temperature of 20-30 ℃ and the power of 60W to obtain a mixed solution E, wherein the ultrasonic treatment conditions are as follows: ultrasound for 10 minutes, ultrasound for 10s, pause for 10s.
T5 filtering the obtained mixed solution E with 0.22 μm filter membrane, sterilizing, and sealing at-70deg.C to obtain immunostimulating complex.
Comparative examples 2 to 3
Comparative examples 2-3 bovine circulating heat-inactivated vaccines were prepared using bovine circulating heat-inactivated antigens with the same virus content as the vaccine adjuvants according to the procedure of example 1, except that the vaccine adjuvants and vaccine preparation parameters used were different, in particular:
comparative example 2 a bovine ephemeral heat-inactivated vaccine was prepared by mixing SEPPIC ISA25VG vaccine adjuvant with bovine ephemeral heat-inactivated antigen in a mass ratio of 1:3, emulsifying at room temperature at 600rpm/min for 15 minutes;
comparative example 3 a bovine ephemeral heat-inactivated vaccine was prepared by mixing SEPPIC ISA AVG vaccine adjuvant with bovine ephemeral heat-inactivated antigen in a mass ratio of 1:3, emulsifying at room temperature at 600rpm/min for 15 minutes.
(1) Physical and chemical property test of vaccine of examples 1-3 and comparative examples 1-3
The physicochemical properties of the vaccines of examples 1 to 3 and comparative examples 1 to 3 prepared above were examined, including observing the appearance of the vaccine, the formulation and detecting the pH of the vaccine, and examining the viscosity, stability and particle size distribution of the vaccine by:
and (3) viscosity test: 1mL of each of the bovine popular heat inactivated vaccines (ZJ strain) prepared in examples 1-3 and comparative examples 1-3 was taken up, and the samples were slowly added into a sample cup, and the viscosity of the vaccine was measured by a viscometer under conditions of a torque of 40% -60% and a rotor rotation speed of 6-16 r/min;
particle size distribution inspection: 1mL of each of the bovine circulating heat inactivated vaccines (ZJ strain) prepared in examples 1 to 3 and comparative examples 1 to 3 was taken, and the intensity of scattered light was detected by a Beckmann particle sizer detector in the United states, and the particle size distribution was calculated;
stability test: bovine circulating heat inactivated vaccine (ZJ strain) prepared in examples 1-3 and comparative examples 1-3 was treated with centrifugation at 8000rpm for 15min, respectively.
The physical and chemical properties of the vaccines are shown in the following Table 1, and the vaccines of examples 1 to 3 and comparative examples 1 to 3 are each in the form of milky slightly viscous emulsion, O/W type in formulation, and pH value in the range of 7 to 8. Compared with the vaccine of comparative examples 1-3, the bovine ephemeral heat inactivated vaccine (ZJ strain) prepared in examples 1-3 has lower viscosity, which is beneficial to the injection of the vaccine and reduces stress reaction; the vaccines of examples 1-3 are all free of water phase after centrifugal treatment, have the advantage of high stability, are favorable for long-time storage of the vaccine, and can provide convenience for needleless injection of the oil adjuvant vaccine.
Table 1: results of physical and chemical property test of vaccine
(2) Immunopotentiation test of the vaccines of examples 1-3 and comparative examples 1-3
5-6 month old BEFV antigen and 5-head groups of healthy susceptible cattle with negative antibodies (serum neutralization antibody titer is not higher than 1:2) were selected, each group was intramuscular injected with 2.0ml of the bovine epidemic heat (ZJ strain) inactivated vaccine prepared in the above examples 1-3 and comparative examples 1-3, and 5-6 month old susceptible cattle with negative antibodies were used as blank control groups. Boosting once on 21 days after immunization, taking blood from the vein of the cattle on 21 days after one-day immunity, 28 days after two-day immunity, 90 days, 150 days and 180 days, separating serum, and detecting the serum neutralizing antibody by using a cell neutralization experimental method. The inoculated 5 cattle should have at least 4 cattle neutralizing antibodies not less than 1:64 (not less than 2) 6 ). And split intoThe body temperature of each test group and the blank control group test cattle are monitored in the morning and afternoon from the day of injection to the day of 14 after injection, and feeding activity of each group test cattle and the conditions of swelling, nodules, fester and the like of the post-injection parts are observed and recorded. The results of the antibody titer measurements for each group of test cattle are shown in table 2 below, and the average body temperature monitoring results for each group of test cattle at noon and afternoon are shown in table 3 below.
Table 2: results of measurement of neutralizing antibody titers in each group of test cattle (2 n )
Table 3: body temperature monitoring results (unit degree C) of each group of test cattle
From the results shown in Table 2, it is found that the antigen content was reduced at the same level (10 7.5 TCID 50/ ml), the vaccine antibody titers of immunization examples 1-3 rise rapidly, reaching the level of potency requirements (1) on 21 days of immunization, relative to the vaccine of comparative examples 1-3: 256 The highest antibody titer can reach 1:8192 after 90 days of secondary immunization, and still maintain a higher antibody titer level at 150 days after secondary immunization, which is more than 1:1024, and the antibody level is still more than or equal to 1 after 180 days of secondary immunization: 256, shows the characteristic of long immune duration, and has the advantage of good uniformity (showing stable vaccine quality); and the antibody levels detected at the same time points were significantly higher than those induced by the vaccines of comparative examples 1 to 3Body level (all raised by more than 1 titer). In contrast, the antibody titer rises more slowly after immunization with the vaccine of comparative example 1, and the induced antibody level is lower, and the efficacy level after 21 days of one immunization is at most 1:256, and the antibody level of the partially immunized cattle after 180 days of secondary immunization can not reach the efficacy requirement (more than or equal to 1:64), the immunization duration is shorter; comparative examples 2-3 are vaccines prepared using ISA25VG and 15AVG immunoadjuvants, which reach a potency level of only up to 1 after 21 days of one immunization: 128, 20% -40% of individuals were unable to reach the level of efficacy requirements 180 days after the second immunization, antibody values below 1:64, the immune duration is short, the results are shown in figure 2. The results demonstrate that the vaccine products prepared in examples 1-3 of the present invention using the vaccine adjuvants comprising purified span-80, purified tween-80 and immunopotentiator more enhance the specific immune response level of the antigen in the vaccine, and have a long immune duration and good uniformity, compared to the vaccines prepared in comparative examples 1-3.
As is clear from the results shown in Table 3, the calves immunized with the heat inactivated vaccine of the bovine popular type of examples 1-3 and comparative examples 1-3 were almost normal in body temperature after immunization, and had no significant increase in body temperature, no local or systemic adverse reaction caused by the vaccine, and normal injection sites. Therefore, the bovine ephemeral heat-inactivated vaccine provided by the invention has good safety.
Examples 4 to 5
Examples 4-5 vaccine adjuvants and bovine popular heat inactivated vaccines (ZJ strain) were prepared following the procedure of example 1, except that the content of each component in the raw material for preparing the vaccine adjuvants was different, specifically:
example 4 the vaccine adjuvant was prepared from the following raw materials: 81.5wt% of Marcol-52 white oil, 12wt% of refined span-80, 6wt% of refined tween-80 and 0.5wt% of immunopotentiator (comprising phospholipid, pollen Pini, tocopherol and saponin in a mass ratio of 0.5:2:0.5:0.5);
example 5 the vaccine adjuvant was prepared from the following raw materials: 81.5wt% of Marcol-52 white oil, 12wt% of refined span-80, 6wt% of refined tween-80 and 0.5wt% of immunopotentiator (comprising phospholipid, pollen Pini, tocopherol and saponin in a mass ratio of 2:4:2:3).
3 healthy susceptible cattle with BEFV antigen and antibody negative (serum neutralization antibody titer is not higher than 1:2) of 5-6 months old are selected, each cattle is intramuscular injected with 2.0ml of the bovine epidemic heat (ZJ strain) inactivated vaccine prepared in the embodiment 4-5, and 3 susceptible cattle with antibody negative of 5-6 months old are selected as blank control groups. Boosting once at 21 days after immunization, and taking blood from the vein of the cattle on 21 days after one-day, 28 days after two-day, 90 days, 150 days and 180 days, separating serum, and detecting serum neutralizing antibodies by using a cell neutralization experimental method, and the results are shown in the following table 4.
Table 4: results of the neutralizing antibody titer assay for each group of immunized cattle (2 n )
From the results shown in Table 4, it is found that the antigen content was reduced at the same level (10 7.5 TCID 50/ ml), the bovine circulating heat-inactivated vaccines of examples 4-5 all produced higher specific neutralizing antibodies 21 days after the first immunization, and the average antibody titer could reach about 2 after 28 days after the second immunization 11 The average titer of the 90 balance after the second time can reach about 2 12 The weight of the product can be up to 2 after 180 days 8 The advantages of good immune effect, long immune duration and good uniformity are presented.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or that equivalents may be substituted for part of the technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An oil-in-water adjuvant, which is prepared from the following raw materials in percentage by weight: 75-83 wt% of oil for injection, 11-15 wt% of refined span-80, 6-9 wt% of refined tween-80 and 0.1-1 wt% of immunopotentiator, wherein the immunopotentiator comprises phospholipid, pollen Pini, tocopherol and saponin with the mass ratio of (0.5-2): 2-4): 0.5-2): 0.5-3, wherein:
the refined span-80 is a product obtained by processing span-80 in the following way:
1) Using H 2 O 2 Processing the span-80 to change the color of the span-80 from dark to light, and obtaining a first processing product when the color of the span-80 is not changed any more;
2) Removing unreacted H from the first treated product 2 O 2 Obtaining a second treatment product;
3) Filtering the second treated product to obtain the refined span-80;
the purified tween-80 is a product obtained by treating tween-80 in the following way:
(1) Using H 2 O 2 Treating the Tween-80 to change the color of the Tween-80 from dark to light, and obtaining a third treated product when the color of the Tween-80 is not changed any more;
(2) Removing unreacted H in the third treated product 2 O 2 Obtaining a fourth treatment product;
(3) And filtering the fourth processed product to obtain the refined Tween-80.
2. The oil-in-water adjuvant according to claim 1, wherein the filtration in step 3) and step (3) is performed by first performing rough filtration using a filtration device having a filtration pore size of 0.40 μm to 0.65 μm, and then performing fine filtration of the filtrate using a filtration device having a filtration pore size of 0.10 μm to 0.22 μm.
3. The oil-in-water adjuvant of claim 1 or 2, wherein the oil for injection is mineral oil for injection, vegetable oil for injection, or a combination thereof; optionally, the mineral oil for injection comprises white oil.
4. An oil-in-water adjuvant according to claim 3, 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 and sonne born 4# white oil, sonne born10# white oil PARACOS KF40, PARACOS KF50, squalene, squalane.
5. A method of preparing an oil-in-water adjuvant comprising the steps of:
s1: heating 75-83 wt% of injection oil to 30-40 ℃;
s2: adding 11-15 wt% of refined span-80, 6-9 wt% of refined tween-80 and 0.1-1 wt% of immunopotentiator into the oil for injection heated in the step S1, uniformly mixing, and filtering to obtain the oil-in-water adjuvant; wherein:
the refined span-80 is obtained by processing span-80 in the following way:
1) Using H 2 O 2 Processing the span-80 to change the color of the span-80 from dark to light, and obtaining a first processing product when the color of the span-80 is not changed any more;
2) Removing unreacted H from the first treated product 2 O 2 Obtaining a second treatment product;
3) Filtering the second treated product to obtain the refined span-80;
the refined tween-80 is obtained by treating tween-80 in the following way:
(1) Using H 2 O 2 Treating the Tween-80 to change the color of the Tween-80 from dark to light, and obtaining a third treated product when the color of the Tween-80 is not changed any more;
(2) Removing unreacted H in the third treated product 2 O 2 Obtaining a fourth treatment product;
(3) And filtering the fourth processed product to obtain the refined Tween-80.
6. The production method according to claim 5, wherein the filtration in step 3) and step (3) is performed by first performing rough filtration using a filtration device having a filtration pore size of 0.40 μm to 0.65 μm, and then performing fine filtration of the filtrate using a filtration device having a filtration pore size of 0.10 μm to 0.22 μm.
7. The preparation method according to claim 5 or 6, wherein the immunopotentiator comprises phospholipids, pollen Pini, tocopherol and saponin in a mass ratio of (0.5-2): (2-4): (0.5-2): (0.5-3).
8. The method of claim 7, wherein the immunopotentiator is prepared by the steps of:
t1: dissolving a mixture of phospholipid, pollen pini, tocopherol and saponin with the mass percentage of (0.5-2), (2-4), and (0.5-2) in an ethanol solution to obtain a lipoid organic solution; optionally, the ethanol solution is prepared from the following components in a volume ratio of 80:20, ethanol and ultrapure water;
t2: shaking and uniformly mixing the lipoid organic solution obtained in the step T1, standing and filtering, and collecting filtrate and precipitate; adding the precipitate into the ethanol solution, shaking, mixing, standing, filtering, collecting filtrate, mixing the two filtrates, centrifuging, and collecting supernatant;
t3: removing the organic solvent in the supernatant obtained in the step T2 to obtain a solid;
t4: dissolving the solid obtained in the step T3 by using PBS solution to obtain a first mixed solution;
t5: performing ultrasonic treatment on the first mixed solution obtained in the step T4 to obtain a second mixed solution;
t6: and (3) filtering the second mixed solution obtained in the step (T5) to obtain the immunopotentiator.
9. The production method according to claim 8, wherein the conditions of the ultrasonic treatment in step T5 include: the temperature is 20-30 ℃, the power is 50-70W, the total ultrasonic treatment time is 5-15min, and the ultrasonic mode is as follows: ultrasound for 10s and pause for 10s.
10. An animal vaccine comprising the oil-in-water adjuvant of any one of claims 1-4.
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