CN114209822A - Adjuvant for pig Japanese encephalitis inactivated vaccine composition, composition and application thereof - Google Patents
Adjuvant for pig Japanese encephalitis inactivated vaccine composition, composition and application thereof Download PDFInfo
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- CN114209822A CN114209822A CN202210075918.7A CN202210075918A CN114209822A CN 114209822 A CN114209822 A CN 114209822A CN 202210075918 A CN202210075918 A CN 202210075918A CN 114209822 A CN114209822 A CN 114209822A
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- japanese encephalitis
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- A61K2039/55511—Organic adjuvants
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Abstract
The invention discloses an adjuvant for a swine Japanese encephalitis inactivated vaccine composition, and the composition and application thereof. The adjuvant comprises 77-90% of mineral oil MARCOL52 for injection; 1-4% of xylitol monolaurate; 8-15% of triglycerol trioleate; 0.5-2% of alkyl glycoside (APG 0816); 0.5-2% of n-decanol. The adjuvant can be used for the inactivated swine Japanese encephalitis vaccine composition, so that the inactivated swine Japanese encephalitis vaccine composition has good immunogenicity and safety.
Description
Technical Field
The invention relates to the field of biological products for livestock, in particular to an adjuvant for a swine Japanese encephalitis inactivated vaccine composition, and the composition and application thereof.
Background
Japanese Encephalitis (JE), also known as Japanese encephalitis, is called Japanese encephalitis for short, and is a zoonosis caused by Japanese Encephalitis Virus (JEV), and China classifies pig Japanese encephalitis as a second type animal epidemic disease and pig Japanese encephalitis Virus as a third type animal pathogenic microorganism. The disease is transmitted through mosquito vector, has strict seasonality, is popular for 6-10 months, and is concentrated for 7, 8 and 9 months. Meanwhile, mosquitoes are also the long-term storage hosts of the virus, pigs are the most main proliferation hosts and infection sources, and the incidence rate of infection of children in people is high. The symptoms of animal infection are acute onset, high temperature and high fever, abortion, dead fetus and higher disease death rate. During the epidemic period, the infection rate of the pigs to the Japanese encephalitis virus can almost reach 100 percent, but the pigs are in a hidden infection state in many times and become a main source of the Japanese encephalitis infection of human beings.
Currently, there are 4 different types of JEV vaccines: inactivated mouse brain vaccine, inactivated cell culture vaccine, live attenuated cell culture vaccine and live attenuated genetic engineering vaccine. The encephalitis B vaccine for animals mainly comprises a mouse brain inactivated vaccine and a hamster kidney cell encephalitis B live vaccine. The existing pig Japanese encephalitis vaccines have certain problems, such as the disadvantages of high cost, much foreign protein, easy anaphylactic reaction and the like of vaccines prepared from rat brain, and the disadvantages of high cost, complex operation, batch difference and the like of hamster kidney cell live vaccines.
The Vero cell is derived from the kidney passage cell of the African green monkey, has the advantages of easy large-scale culture, stable cytozoology, no exogenous factor pollution and no tumorigenicity, is a human vaccine production matrix recommended by WHO, and is suitable for a cell source for large-scale vaccine production. In addition, the domestic commercial swine Japanese encephalitis vaccine adopts a traditional rotary bottle culture mode, and has the defects of easy cell pollution, high labor intensity and the like. Compared with the rotary bottle culture process, the reactor adopts serum-free culture for suspension culture, the obtained cell density, virus titer and product quality are obviously improved, the pollution of the exogenous virus without serum is avoided, and the production energy consumption is greatly reduced.
Disclosure of Invention
The invention aims to provide an adjuvant for a swine Japanese encephalitis inactivated vaccine composition, which can enhance the immunogenicity of the vaccine.
The adjuvant for the inactivated Japanese encephalitis vaccine composition comprises the following components in percentage by weight:
the adjuvant provided by the invention can be used for preparing a pig Japanese encephalitis inactivated vaccine composition.
The invention also aims to provide a swine Japanese encephalitis inactivated vaccine composition, which comprises the adjuvant and a swine Japanese encephalitis virus inactivated antigen.
Wherein the mass ratio of the adjuvant to the inactivated antigen of the porcine Japanese encephalitis virus is (0.5-1.5): (0.5 to 1.5).
Wherein the mass ratio of the adjuvant to the inactivated antigen of the porcine Japanese encephalitis virus is 1: 1.
wherein the inactivated antigen of the porcine Japanese encephalitis virus is prepared by the following steps: mixing beta-propiolactone and the porcine Japanese encephalitis virus antigen, stirring and inactivating at 4 ℃, and hydrolyzing in water bath at 37 ℃.
The beta-propiolactone and the porcine encephalitis B antigen are mixed at a ratio of 1: (3800-4200), stirring and inactivating at 4 ℃ for 22-26 hours, and hydrolyzing in water bath at 37 ℃ for 1.5-2.5 hours.
Wherein the Japanese encephalitis virus antigen is from a culture of Japanese encephalitis virus SA 14-14-2.
The culture of the pig encephalitis B virus SA14-14-2 is obtained by culturing the pig encephalitis B virus SA14-14-2 on a full-suspension cell line Vero-zm of Vero kidney cells, wherein the Vero-zm is preserved in the China general microbiological culture Collection center (address: Beijing city rising area, North Chen West Lu No. 1 Hospital No. 3) in 26 months at 2021, and the preservation number is CGMCC No. 23098.
Wherein the porcine Japanese encephalitis virus antigen is obtained by concentrating and purifying the culture of the porcine Japanese encephalitis virus SA 14-14-2.
Wherein the Vero-zm is cultured on a V-SFM medium.
The adjuvant can be used as an immunologic adjuvant in a swine Japanese encephalitis inactivated vaccine composition, can enhance the immunogenicity of the vaccine, and enables the swine Japanese encephalitis inactivated vaccine composition to have good immunogenicity and safety.
The invention utilizes the concentrated and purified Japanese encephalitis virus SA14-14-2 antigen to produce the live Japanese encephalitis vaccine composition, so that the immune side reaction is obviously reduced, and the quality of the vaccine product is obviously improved.
Detailed Description
In the following description, for purposes of explanation, specific details are set forth in order to provide an understanding of the present disclosure.
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Unless otherwise specified, the percentages referred to herein are mass percentages.
Example 1 preparation of Vero Total suspension cell line
Adherent culture Vero cells adapted to the encephalitis B virus SA14-14-2 strain are used as candidate suspension domesticated cells. The adherent Vero cells are prepared into a full-suspension adaptive cell strain through two stages of domestication by a special step-by-step suspension culture medium and serum-reducing adaptation.
1. Stepwise suspension acclimation process
Digesting the adherent culture Vero cells with trypsin, and then using the cells to culture the Vero cells in a 1-2 multiplied by 10 mode6Cell/ml density, adding newborn bovine serum according to 8% -10% concentration to inoculate suspension cell bottle, placing in constant temperature oscillator, culturing at 37 deg.C, 120rpm/min, 5% carbon dioxide concentration. Respectively taking the proportion of an original culture medium (DMEM) to a suspension special culture medium as 75%: 25% and 50%: 50% and 25%: 75% and 0: domesticating the four culture mediums with a proportion of 100 percent, carrying out the proportion of each culture medium for more than 3 generations of subculture cells to reach a normal suspension adaptation state (the cells reach a normal multiplication rate, the survival rate reaches more than 90 percent), and carrying out the next proportion of cell subculture until the cells are completely adapted to the special suspension culture medium for culture and can be stably subcultured.
2. Low serum suspension acclimatization process
Domesticating the suspension cells cultured in the suspension special culture medium of the newborn calf serum with the concentration of 8% -10% in a manner of gradually reducing the serum. Gradually reducing (respectively 8%, 6%, 4% and 2%) bovine serum dosage of suspension passage cells, stably passing each serum concentration cell for more than 3 generations to reach a normal suspension adaptation state of the cell (the cell reaches a normal multiplication rate, and the survival rate reaches more than 90%), carrying out next serum concentration cell passage until the cell is completely adapted to low serum suspension culture, and stably passing. The Vero whole suspension cell strain adapting to low serum is named as Vero-zm.
Vero-zm has been preserved in China general microbiological culture Collection center (address: Xilu No. 1 Hospital No. 3, North Cheng, south China, Beijing city) at 26.10.2021 with the preservation number of CGMCC No. 23098.
Adaptability of Vero-zm to different media
Four kinds of commercial Vero suspension culture special culture medium (A, B, C, D) are selected from the market for the adaptability comparison test of Vero full suspension cell strain.
Vero suspension culture special culture medium A: vero602 (Zhejiang Yishengke Biotechnology Co., Ltd., product code: Y1131);
vero suspension culture special culture medium B: V-SFM medium (Volme Biotechnology Co., Ltd., product No. M21216101-2);
special culture medium C for Vero suspension culture: CD medium (Gibco, product number: A16277-03);
special culture medium D for Vero suspension culture: CDPK15 (Jianshun organism, trade name: 10701).
Cell recovery and cell passage are carried out according to the following method, after the cells are passaged in a corresponding culture medium for 3 generations, the porcine encephalitis B SA14-14-2 virus is inoculated under the same condition, after the virus inoculation, cell staining counting (0.4 percent trypan blue staining) is carried out, after the cell survival rate is lower than 20 percent, virus liquid is harvested and sampled, and the virus content of the vaccine is determined by adopting a virus plaque test.
1. Cell resuscitation
The frozen Vero-zm was taken out from the liquid nitrogen and placed in a 37 ℃ water bath to be rapidly thawed. The thawed cells are transferred to a centrifuge tube, a fresh Vero suspension culture special culture medium (A, B, C or D) is added, the mixture is stood for 5min, then the mixture is centrifuged for 5min at 800rpm, the supernatant is discarded, and the mixture is resuspended by the fresh Vero suspension culture special culture medium. At an initial density of 0.8X 106Each cell/ml is inoculated into 50ml of suspension culture special medium which is added with 4 percent newborn bovine serum and has the total volume of 50ml in a 250ml cell shake flask, and the suspension culture special medium is placed in a constant temperature oscillator to be cultured under the conditions of 37 ℃ and 120rpm/min and the carbon dioxide concentration of 5 percent. Cells were sampled and counted daily and analyzed for cell viability and cell doubling rate. The cells are normally cultured for 48 hours to reach the normal multiplication rate of 3-6 multiplied by 106The cell viability rate of each cell/ml is more than 95 percent.
2. Cell passage
Sampling and counting after 48 hours of cell culture until the cell density reaches 3-6 multiplied by 106The cell viability rate of each cell/ml is more than 95 percent. Diluting the cells with Vero suspension culture special culture medium, and then setting the final concentration of the cells to be 0.8 multiplied by 106Each cell/ml is inoculated into 50ml of Vero suspension culture special culture medium which is added with 4 percent newborn bovine serum and has the total volume of 50ml in a 250ml cell shake flask, and the Vero suspension culture special culture medium is placed in a constant temperature oscillator to be cultured under the conditions of 37 ℃, 120rpm/min and 5 percent of carbon dioxide concentration. Cells were sampled and counted daily and analyzed for cell viability and cell doubling rate.
3. Incubation for virus inoculation
The virus inoculation amount is 0.5M.O.I, after virus inoculation, the virus adsorption process is carried out in a bioreactor under the conditions of low stirring speed of 60rpm/min, the temperature of 37 ℃, the pH value of 7.2, 50 percent of Dissolved Oxygen (DO) and the ventilation amount of 0.1-0.5 SPLM, and the adsorption time is 60 min.
4. Determination of viral content
Serial 10-fold dilution of the virus was performed to obtain 10-2、10-3、10-43 dilutions were seeded in 12-well cell plates grown with monolayers of BHK-21 cells, each dilution being provided with 3 replicate wells, 0.1 mL/well. After virus inoculation, the cells were adsorbed at 37 ℃ for 90min, covered with a cover (DMEM containing 1.5% methylcellulose and 2% calf serum), stained with crystal violet 5 days later, fixed for 30min, washed with running water, dried, and the number of plaques in each well was counted to determine the virus content (Log PFU/mL). And respectively recording parameters such as cell growth morphology, cell density and the like after 3 generations of cell culture. Specific data for the four media are detailed in table 1 below:
TABLE 1 results of four suspension media in terms of culture characteristics and toxigenic effects of Vero-zm cell lines
Remarks are as follows: cell viability was determined by counting with 0.4% trypan blue cell stain.
As can be seen from the above test results, for the four commercial culture media, the culture of Vero-zm cells in Vero suspension culture special medium B is superior to that in other 3 culture media in terms of cell density, morphology and virus production of porcine Japanese encephalitis virus SA 14-14-2. Therefore, Vero suspension culture special medium B (V-SFM medium (Volme Biotechnology Co., Ltd., product No. M21216101-2)) is preferable as a candidate medium for preparing the swine Japanese encephalitis virus antigen.
Example 2 inactivated Japanese encephalitis vaccine composition
Revivification and subculture of Vero-zm cell strain
1. Suspension seed cell resuscitation
The frozen Vero-zm cell line was taken out from the liquid nitrogen and placed in a water bath at 37 ℃ to be rapidly thawed. And transferring the thawed cells into a centrifuge tube, adding a fresh special suspension cell culture medium B, standing for 5min, centrifuging at 800rpm for 5min, discarding the supernatant, and resuspending with the fresh special suspension cell culture medium. At an initial density of 0.8X 106Each cell/ml is inoculated into 50ml of suspension culture special medium which is added with 4 percent newborn bovine serum and has the total volume of 50ml in a 250ml cell shake flask, and the suspension culture special medium is placed in a constant temperature oscillator to be cultured under the conditions of 37 ℃ and 120rpm/min and the carbon dioxide concentration of 5 percent. Cells were sampled and counted daily and analyzed for cell viability and cell doubling rate. The cells are normally cultured for 48 hours to reach the normal multiplication rate of 3-6 multiplied by 106The cell viability rate of each cell/ml is more than 95 percent.
2. Cell passage
Sampling and counting after 48 hours of cell culture until the cell density reaches 3-6 multiplied by 106The cell viability rate of each cell/ml is more than 95 percent. Diluting the cells with a special culture medium for suspension culture, and then setting the final concentration of the cells to be 0.8 multiplied by 106Each cell/ml is inoculated into 50ml of suspension culture special medium which is added with 4 percent newborn bovine serum and has the total volume of 50ml in a 250ml cell shake flask, and the suspension culture special medium is placed in a constant temperature oscillator to be cultured under the conditions of 37 ℃ and 120rpm/min and 5 percent carbon dioxide concentration. Cells were sampled and counted daily and analyzed for cell viability and cell doubling rate.
Step-by-step amplification culture of Vero-zm suspension cells in bioreactor
Suspension culture of Vero-zm suspension cells in 10L bioreactor
After the cell strain is subjected to suspension culture in a shake flask for 1-3 generations, the cell strain is transferred to a 10L bioreactor, and a suspension culture medium is supplemented to ensure that the cell concentration is 0.6 multiplied by 106Adding 4% newborn calf serum into each cell/ml, stirring at 37 deg.C, pH7.2, Dissolved Oxygen (DO) 50%, stirring at 100rpm, and introducingCulturing for 48-72 h under the gas volume of 0.1-0.3, sampling and counting cells every day, and analyzing the cell viability and cell multiplication rate. After culturing for 48-72 h, the cells reach the normal multiplication rate of 3-6 multiplied by 106cells/ml, cell viability up to more than 95%, can further expand the culture.
Continuous amplification culture of Vero-zm suspension cells in 10L-40L-100L bioreactor
Transferring Vero-zm suspension cells cultured in a 10L bioreactor to a 40L bioreactor through connection, and adding a special culture medium for suspension culture to adjust the cell density to 0.6 multiplied by 106Adding 4% of newborn bovine serum into each cell/ml, culturing at 37 ℃, pH value of 7.2, dissolved oxygen DO value of 50%, stirring speed of 100rpm and ventilation volume of 0.1-0.3 for 48-72 h, sampling and counting cells every day, and analyzing cell viability and cell multiplication rate. After culturing for 48-72 h, the cells reach the normal multiplication rate of 3-6 multiplied by 106The cell viability reaches more than 95 percent per ml, and the cells can be further expanded and cultured. With this culture process, cells were scaled up to a 100L bioreactor for culture.
Production of 100L-grade reactor of porcine Japanese encephalitis virus SA14-14-2 strain
The 100L suspension culture cell prepared by the method is subjected to cell sedimentation and liquid exchange stage, virus inoculation and incubation stage, and virus culture to harvest stage to complete the generation of the porcine Japanese encephalitis virus SA14-14-2 strain:
1. cell sedimentation liquid changing stage
And (3) standing and settling the prepared 100L of suspension culture cells in a low-temperature cold water bath at 10-20 ℃ for 4-8 h, settling most of the cells, and discharging a cell growth solution with half of the total volume through a reactor cleaning device.
2. Incubation phase for virus inoculation
After the cells are settled and changed, the porcine encephalitis B SA14-14-2 virus is inoculated, the virus inoculation amount is 0.5M.O.I, and after the virus is inoculated, the virus adsorption process is carried out in a bioreactor under the conditions of low stirring speed of 60rpm/min, the temperature of 37 ℃, the pH value of 7.2, Dissolved Oxygen (DO)50 percent and the ventilation volume of 0.1-0.5 SPLM, and the adsorption time is 60 min.
3. Culturing virus to harvest stage
After the inoculation and incubation of the virus, a suspension cell special culture medium B (pH7.8) containing 4% newborn calf serum is added to the original culture volume, and the culture is continued in a reactor at the temperature of 35 ℃, the Dissolved Oxygen (DO) of 50%, the stirring speed of 100rpm and the ventilation amount of 0.1-0.5 SPLM. Observing and counting cells every day, monitoring the growth state of the cells, and adjusting the pH value by using 7.5-10% (w/v) of sodium bicarbonate according to the change of the pH value; according to the consumption of glucose, 20-40% (w/v) of the glucose concentrated solution is used for timely supplementing the glucose in the culture solution. Culturing until the cell viability is lower than 20%, harvesting virus liquid, freezing and thawing once, sampling to detect virus titer, wherein the virus content per mL is 107.5PFU。
Concentration and purification method and process of porcine Japanese encephalitis virus SA14-14-2
1. And (3) treating a virus feed liquid sample:
collecting porcine encephalitis B SA14-14-2 virus liquid, adding protamine sulfate with final concentration of 1mg/ml, shaking up, and standing overnight at 4 ℃.
2. Centrifuging a virus feed liquid sample:
centrifuging the porcine Japanese encephalitis virus at 10000rpm for 30min by adopting a high-speed continuous centrifugation method, discarding the precipitate, collecting the virus supernatant, and storing at 4 ℃ for later use.
3. Virus feed liquid sample ultrafiltration:
3.1 system pretreatment: a300 KD hollow fiber column (Shibi pure company)/membrane-packed Rambo company) is installed in a hollow fiber/membrane-packed control equipment system (comprising a pipeline, a pump and a fiber/membrane packed), and after the assembly is finished, the whole system is flushed for 20min by sterile water for injection, and the flow rate is controlled to be 90L/min.
3.2 System integrity and Water flux detection: and (5) washing the system by sterile water for injection, and detecting the water flux of the hollow fiber column/membrane package and whether the system leaks or not at room temperature.
And (3) online sterilization and cleaning of the system: and (3) carrying out circulating sterilization treatment on the system for 30min by using a sterile 0.2mol/L NaOH solution, then cleaning the ultrafiltration system by using sterile water for injection, and washing away the residual NaOH solution until the pH value reaches 7.0.
3.3 System Balancing: the mixture was equilibrated with sterile 0.01mol/L PBS (pH7.2) for 10min at a flow rate of 60L/min. And (3) allowing the centrifuged feed liquid to pass through a 300KD hollow fiber column/membrane package system, controlling the pump speed of a liquid inlet end at 60L/min, maintaining the transmembrane pressure at a permeation end at 1.5psi, collecting liquid at a backflow end, supplementing 0.01mol/L PBS (phosphate buffer solution) solution to the original volume when 1/20 of the original volume of the feed liquid at the liquid inlet end is remained, continuing ultrafiltration, repeating the ultrafiltration for three times until the original volume reaches 1/20, and finishing the ultrafiltration process.
4. And (3) purifying by molecular sieve chromatography gel:
4.1 filling and pretreatment of chromatographic packing: and (3) installing a 4FF molecular sieve (GE company) prepacked column in place, and detecting the column effect.
4.2 treating the molecular sieve gel chromatographic column with 5-10 column volumes of sterile 0.5mol/L NaOH, then cleaning with sterile injection water to pH7.0, then balancing the chromatographic column with 5-10 column volumes of 0.01 mol/L7.2 PBS solution, wherein the conductivity is 15.45ms/cm, the pH value is stabilized at 7.2, and the ultraviolet UV280 baseline is stable.
4.5, chromatographic purification: and (3) loading the virus sample subjected to ultrafiltration concentration, setting the linear flow rate of loading to be 10ml/min, and controlling the pressure to be less than 2.0 bar. The sample loading amount is 10% of the column volume, after the sample loading is finished, 0.01mol/L PBS buffer solution is switched, the light absorption value is detected by UV280, when the light absorption value curve is in an ascending trend, the first peak appearing as the target sample is collected, the collection is stopped when the light absorption value of the light absorption value curve is reduced to the lowest position, the collected peak sample is subjected to Protein removal rate determination by using BCA Protein Assay Kit (Thermo company), and the Protein removal rate is 98.7%. And (3) continuously treating the molecular sieve gel chromatography column with 5-10 column volumes of sterile 0.5mol/L NaOH, and then washing with sterile injection water to pH7.0 to obtain the concentrated and purified porcine Japanese encephalitis virus SA14-14-2 antigen.
Preparation of pig Japanese encephalitis inactivated vaccine composition
1. Preparation of pig Japanese encephalitis inactivated antigen
Mixing beta-propiolactone and porcine Japanese encephalitis virus SA14-14-2 antigen in a weight ratio of 1: stirring and inactivating at 4000 ℃ for 24 hours, and hydrolyzing in water bath at 37 ℃ for 2 hours to obtain the inactivated antigen of the porcine Japanese encephalitis virus.
2. Preparation of animal vaccine oil adjuvant
Weighing a certain amount of emulsifier and auxiliary agent according to the following weight percentage, adding the emulsifier and auxiliary agent into weighed mineral oil for injection produced by Exxon Mobil oil company, controlling the stirring speed, carrying out physical mixing, adopting a heating and dissolving-assisting mode in the mixing process, controlling the temperature below 80 ℃, uniformly mixing to form liquid, sterilizing at 121 ℃ for 30 minutes, and cooling to the emulsification temperature to obtain the animal vaccine oil adjuvant 1-3.
The oil adjuvant 1 comprises the following components in percentage by weight:
the oil adjuvant 2 comprises the following components in percentage by weight:
the oil adjuvant 3 comprises the following components in percentage by weight:
3. preparation of pig Japanese encephalitis inactivated vaccine composition
Respectively adding the sterilized and cooled animal vaccine oil adjuvants 1-3 into an emulsification tank, stirring and mixing uniformly, controlling the stirring speed to be 1000 revolutions per minute and the emulsification temperature to be 30 ℃; slowly adding the inactivated antigen of the swine Japanese encephalitis virus with the same quantity as the animal vaccine oil adjuvant into the prepared oil adjuvant; after the addition is finished, performing pre-shearing emulsification, controlling the shearing speed to be below 3000 r/min, and controlling the process of pre-shearing emulsification to be 10 minutes; after the pre-shearing is finished, controlling the rotating speed to be 8000 rpm, and carrying out shearing emulsification, wherein the process is 15 minutes; and after emulsification is finished, labeling and subpackaging to obtain the inactivated Japanese encephalitis vaccine composition 1-3.
Safety test of pig Japanese encephalitis inactivated vaccine composition on mice
Safety experiments were performed with 12-14g ICR mice. The experiment was divided into 4 groups (group 1 of inactivated Japanese encephalitis vaccine composition, group 2 of inactivated Japanese encephalitis vaccine composition, group 3 of inactivated Japanese encephalitis vaccine composition and control group), each group consisting of 10 mice.
In the group 1 of the Japanese encephalitis inactivated vaccine composition, the group 2 of the Japanese encephalitis inactivated vaccine composition and the group 3 of the Japanese encephalitis inactivated vaccine composition, 0.03ml of the pig Japanese encephalitis inactivated vaccine composition is injected intracerebrally into each mouse, the death number of the mice within 3 days does not exceed 2, and the rest mice are continuously observed for 14 days to observe the spirit, appetite and injection parts of the mice.
In the control group, each mouse was injected intracerebrally with 0.03ml of the culture medium, and the number of deaths within 3 days was not more than 2, and the remaining mice were continuously observed for 14 days, and the spirit, appetite, and injection site of the mice were observed.
After the inactivated vaccine composition for Japanese encephalitis is injected into a mouse, the mouse is normal in clinical performance, good in mental state and normal in appetite, has no local and systemic adverse reactions, and is completely healthy and alive. The results are shown in Table 1.
TABLE 1 safety test results of inactivated vaccine composition for Japanese encephalitis of pig in ICR mouse
Immune challenge test of pig encephalitis B inactivated vaccine composition on mice
Mice were used for a mouse challenge immunization test with 12-14g ICR mice. The experiment is divided into 5 groups (pig encephalitis B inactivated vaccine composition 1 group, pig encephalitis B inactivated vaccine composition 2 group, pig encephalitis B inactivated vaccine composition 3 group, conventional oil adjuvant pig encephalitis B inactivated vaccine control group, challenge control group), and each group comprises 10 mice.
The conventional oil adjuvant porcine Japanese encephalitis inactivated vaccine control group uses a conventional oil adjuvant (alumina gel adjuvant) porcine Japanese encephalitis inactivated vaccine and is prepared according to the following method: adding the sterilized and cooled conventional oil adjuvant into an emulsification tank, uniformly stirring and mixing, controlling the stirring speed to be 1000 revolutions per minute and the emulsification temperature to be 30 ℃; slowly adding the prepared porcine Japanese encephalitis virus inactivated antigen with the same amount as that of the conventional oil adjuvant into the conventional oil adjuvant; after the addition is finished, performing pre-shearing emulsification, controlling the shearing speed to be below 3000 r/min, and controlling the process of pre-shearing emulsification to be 10 minutes; after the pre-shearing is finished, controlling the rotating speed to be 8000 rpm, and carrying out shearing emulsification, wherein the process is 15 minutes; and after emulsification is finished, labeling and subpackaging to obtain the conventional oil adjuvant porcine Japanese encephalitis inactivated vaccine.
The control group was not immunized and was used 10 days after the 2 nd vaccination of mice5LD50The Japanese encephalitis P3 is strongly toxic and acts as an abdominal cavity.
The porcine encephalitis B inactivated vaccine composition and the conventional oil adjuvant porcine encephalitis B inactivated vaccine are respectively diluted by PBS with pH 7.5 in three dilutions of 50 times, 500 times and 5000 times, 10 mice are respectively inoculated with 10-12g of each dilution through the abdominal cavity, secondary inoculation is carried out at intervals of 7 days, 0.5 ml/mouse is used for each time, and 10 mice are taken as challenge control at the same time. All mice were challenged intraperitoneally 5 days after the 2 nd inoculation 105LD50Encephalitis B P3 was virulent, 0.3ml each, and 0.04ml of virus dilution was injected intracerebrally into each mouse before abdominal challenge to disrupt the blood-brain barrier. After the virus attack, the mice in the virus attack control group are observed for 14 days (death within 3 days), no less than 80 percent of the death should occur, and half protection amount (PD) is calculated after 14 days50)。
And (3) performing immunogenicity evaluation on the inactivated swine Japanese encephalitis vaccine composition through an immune challenge test of a mouse. The result shows that the conventional oil adjuvant pig Japanese encephalitis inactivated vaccine control group PD50Is 10-2 . 030.3ml of pig Japanese encephalitis inactivated vaccine composition 1 group PD50Is 10-2.700.3ml of porcine type B brainInactivated vaccine composition 2 group PD50Is 10-2.600.3ml of pig Japanese encephalitis inactivated vaccine composition 3 groups PD50Is 10-2.37/0.3ml。
Therefore, the inactivated swine Japanese encephalitis vaccine composition 1 has the best immunogenicity, and can still protect 50% of mice after being diluted by 501 times. The inactivated swine Japanese encephalitis vaccine composition 2 can still protect 50% of mice after being diluted 398 times. After the inactivated Japanese encephalitis vaccine composition 3 is diluted 234 times, 50% of mice can still be protected. The group of the inactivated Japanese encephalitis vaccine with the conventional oil adjuvant has poor immune effect, and can protect 50% of mice after being diluted by 107 times, as shown in Table 2.
TABLE 2 porcine Japanese encephalitis inactivated vaccine composition half Protection Dose (PD)50) Measurement of (2)
Example 3 preparation of live porcine encephalitis B vaccine composition
1. Preparation of pig Japanese encephalitis live vaccine
Adding a freeze-drying protective agent (the heat-resistant freeze-drying protective agent for the live swine Japanese encephalitis vaccine described in CN 103656661B, specifically, the heat-resistant freeze-drying protective agent for the live swine Japanese encephalitis vaccine of example 1) into the antigen SA14-14-2 of the swine Japanese encephalitis virus, and performing freeze-vacuum drying to prepare the live swine Japanese encephalitis vaccine.
2. Preparation of pig Japanese encephalitis live vaccine water adjuvant
Weighing the following auxiliary agents and polymer combinations according to a proportion, sequentially adding the auxiliary agents and the polymer combinations into the weighed water solution for injection, controlling the stirring speed, mixing, uniformly mixing into liquid by adopting a heating and dissolving assisting mode in the mixing process, controlling the temperature to be below 60 ℃, sterilizing at 121 ℃ for 30 minutes, and cooling to room temperature to obtain the swine Japanese encephalitis live vaccine aqueous adjuvant 1-3.
The swine Japanese encephalitis live vaccine water-based adjuvant 1 comprises the following components in percentage by weight:
88% of an aqueous solution for injection;
polymer combination:
auxiliary agent:
oleic acid 3%
The swine Japanese encephalitis live vaccine aqueous adjuvant 2 consists of the following components in percentage by weight:
96% of water solution for injection;
polymer combination:
auxiliary agent:
oleic acid 1%
The swine Japanese encephalitis live vaccine aqueous adjuvant 3 consists of the following components in percentage by weight:
80% of water solution for injection;
polymer combination:
auxiliary agent:
oleic acid 4%
3. Preparation of pig encephalitis B live vaccine composition
Diluting pig Japanese encephalitis live vaccine with diluent (1/15mol/L phosphate buffered saline, pH7.4) to 1 part (virus content 10)6.0PFU/ml) is added into the swine B type B provided by the invention with the volume ratio of 20 percent1-3 parts of encephalitis live vaccine aqueous adjuvant, and shaking up to prepare the pig encephalitis B live vaccine composition 1-3.
4. Preparation of Japanese encephalitis live vaccine composition with conventional alumina gel adjuvant
Diluting pig Japanese encephalitis live vaccine with diluent (1/15mol/L phosphate buffered saline, pH7.4) to 1 part (virus content 10)6.0PFU/ml), adding 20% by volume of a conventional commercially available alumina gel adjuvant (purchased from Invivogen), shaking up to prepare a live encephalitis b vaccine composition.
Safety test of pig Japanese encephalitis live vaccine composition on mice
Safety experiments were performed with 12-14g ICR mice. The experiment was divided into 4 groups (pig Japanese encephalitis live vaccine composition 1 group, pig Japanese encephalitis live vaccine composition 2 group, pig Japanese encephalitis live vaccine composition 3 group and control group), each group consisting of 10 mice.
In the group 1 of the pig Japanese encephalitis live vaccine composition, the group 2 of the pig Japanese encephalitis live vaccine composition and the group 3 of the pig Japanese encephalitis live vaccine composition, 0.03ml of the pig Japanese encephalitis live vaccine composition is injected intracerebrally into each mouse, the death number within 3 days does not exceed 2, and the rest mice are continuously observed for 14 days to observe the spirit, the appetite and the injection part of the mice.
In the control group, each mouse was injected intracerebrally with 0.03ml of the culture medium, and the number of deaths within 3 days was not more than 2, and the remaining mice were continuously observed for 14 days, and the spirit, appetite, and injection site of the mice were observed.
After the pig Japanese encephalitis live vaccine composition 1-3 is injected, mice are normal in clinical performance, good in mental state and normal in appetite, local and systemic adverse reactions are not generated, and the mice are all healthy and alive. The results are shown in Table 2.
TABLE 2 ICR mice for Japanese encephalitis vaccine composition safety test results
Immunization challenge test of pig Japanese encephalitis live vaccine composition on mice
Mice were used for a mouse challenge immunization test with 12-14g ICR mice. The experiment was divided into 6 groups (pig encephalitis B live vaccine composition 1 group, pig encephalitis B live vaccine composition 2 group, pig encephalitis B live vaccine composition 3 group, pig encephalitis B live vaccine group, conventional alumina gel adjuvant encephalitis B live vaccine composition group, challenge control group), each group consisting of 10 mice.
DMEM is used for diluting the pig Japanese encephalitis live vaccine composition 1-3 and the pig Japanese encephalitis live vaccine in 10-fold serial dilution. Get 10-2、10-3、10-43 dilutions, respectively subcutaneously inoculating 10-12g ICR mice each with 0.1ml, 14 days after immunization, and injecting 0.3ml of Japanese encephalitis virus virulent P3 strain (containing 500 LD) into each abdominal cavity together with 10 mice of control group with the same conditions50) The observation was continued for 14 days.
And (3) performing immunogenicity evaluation on the pig Japanese encephalitis live vaccine compositions 1-3 through an immune challenge test of a mouse. The result shows that the pig Japanese encephalitis live vaccine group PD50Is 10-3.00.3 ml; japanese encephalitis B live vaccine composition group PD with conventional alumina gel adjuvant50Is 10-3.100.3 ml; pig encephalitis B live vaccine composition 1 group PD50Is 10-3.500.3 ml; pig encephalitis B live vaccine composition 2 group ED50Is 10-3 . 430.3 ml; pig Japanese encephalitis live vaccine composition 3 groups PD50Is 10-3.29/0.3ml。
Therefore, the pig Japanese encephalitis live vaccine composition 1 has the best immunogenicity, and can still protect 50% of mice after being diluted by 3162 times; the swine Japanese encephalitis live vaccine group had the least excellent immune effect, and was diluted 1000 times, and 50% of mice were protected, as shown in Table 4.
TABLE 4 porcine Japanese encephalitis live vaccine composition half Protection Dose (PD)50) Measurement of (2)
Claims (10)
1. An adjuvant for a swine Japanese encephalitis inactivated vaccine composition, wherein the adjuvant comprises:
2. the adjuvant of claim 1 for preparing inactivated Japanese encephalitis vaccine composition of pig.
3. An inactivated swine Japanese encephalitis vaccine composition, comprising the adjuvant of claim 1 and inactivated swine Japanese encephalitis virus antigen.
4. The composition according to claim 3, wherein the mass ratio of the adjuvant to the inactivated antigen of Japanese encephalitis virus of swine is (0.5-1.5): (0.5 to 1.5).
5. The composition according to claim 3, wherein the mass ratio of the adjuvant to the inactivated antigen of Japanese encephalitis virus of pig is 1: 1.
6. the composition according to claim 3, wherein the inactivated antigen of porcine Japanese encephalitis virus is prepared by the following steps: mixing beta-propiolactone and the porcine Japanese encephalitis virus antigen, stirring and inactivating at 4 ℃, and hydrolyzing in water bath at 37 ℃.
7. The composition according to claim 6, wherein the antigen of porcine Japanese encephalitis virus is from a culture of porcine Japanese encephalitis virus SA 14-14-2.
8. The composition according to claim 7, wherein the culture of the porcine encephalitis b virus SA14-14-2 is obtained by culturing the porcine encephalitis b virus SA14-14-2 on Vero-zm, a whole suspension cell line of Vero-cells, which has been deposited at 26 months at 2021 with the China general microbiological culture Collection center (CGMCC) with the accession number of CGMCC No. 23098.
9. The composition according to claim 7, characterized in that the pig Japanese encephalitis virus antigen is obtained by concentration and purification of the culture of the pig Japanese encephalitis virus SA 14-14-2.
10. The composition of claim 8, wherein the Vero-zm is cultured on V-SFM medium.
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