Adjuvant for porcine Japanese encephalitis inactivated vaccine composition and its composition and application

Abstract

The invention discloses an adjuvant for a porcine Japanese encephalitis inactivated vaccine composition, and a 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 (APG0816); and 0.5-2% of n-decanol. The adjuvant of the invention can be used for a porcine Japanese encephalitis inactivated vaccine composition, so that the porcine Japanese encephalitis inactivated vaccine composition has good immunogenicity and safety.

Description

Technical Field

[0001] The present invention relates to the field of veterinary biological products, and in particular to an adjuvant for a porcine Japanese encephalitis inactivated vaccine composition, and a composition and application thereof. Background Art

[0002] Japanese encephalitis, also known as Japanese encephalitis (JE), is a zoonotic infectious disease caused by Japanese encephalitis virus (JEV). my country classifies swine Japanese encephalitis as a Class II animal epidemic disease, and swine Japanese encephalitis virus as a Class III animal pathogenic microorganism. The disease is transmitted by mosquito vectors and has strict seasonality. It is prevalent from June to October, concentrated in July, August, and September. At the same time, mosquitoes are also long-term storage hosts of the virus. Pigs are the most important proliferation hosts and sources of infection. Among the population, the incidence of infection in children is higher. The symptoms of animal infection are acute onset, high temperature, high fever, miscarriage, stillbirth, and high mortality rate. During the epidemic period, the infection rate of pigs to Japanese encephalitis virus can reach almost 100%, but they are often in an invisible infection state, becoming the main source of human Japanese encephalitis infection.

[0003] At present, there are 4 different types of JEV vaccines: mouse brain inactivated vaccine, cell culture inactivated vaccine, cell culture attenuated live vaccine and genetic engineering attenuated live vaccine. The veterinary Japanese encephalitis vaccines are mainly mouse brain inactivated vaccine and hamster kidney cell Japanese encephalitis live vaccine. The existing porcine Japanese encephalitis vaccines all have certain problems. For example, the vaccine prepared from mouse brain has the disadvantages of high cost, high impurity protein and easy to cause allergic reactions, and the hamster kidney cell live vaccine has the disadvantages of high cost, complex operation and batch differences.

[0004] Vero cells are derived from African green monkey kidney passage cells. They are easy to culture on a large scale, have stable nuclei, are free of exogenous factor contamination, and are non-tumorigenic. They are the human vaccine production matrix recommended by WHO and are suitable as a cell source for large-scale vaccine production. In addition, domestic commercialized porcine Japanese encephalitis vaccines all use traditional rotary bottle culture, which has the disadvantages of easy cell contamination and high labor intensity. Compared with the rotary bottle culture process, the reactor suspension culture uses serum-free culture, and the obtained cell density, virus titer, and product quality are significantly improved, there is no serum exogenous virus contamination, and the production energy consumption is greatly reduced. Summary of the invention

[0005] The object of the present invention is to provide an adjuvant for a porcine Japanese encephalitis inactivated vaccine composition which can enhance the immunogenicity of the vaccine.

[0006] The adjuvant for the inactivated porcine Japanese encephalitis vaccine composition provided by the present invention comprises the following components in percentage by weight:

[0007]

[0008] The adjuvant provided by the invention can be used to prepare an inactivated vaccine composition of porcine Japanese encephalitis.

[0009] Another object of the present invention is to provide an inactivated porcine Japanese encephalitis vaccine composition, comprising the adjuvant and inactivated porcine Japanese encephalitis virus antigen.

[0010] Wherein, the mass ratio of the adjuvant to the inactivated porcine Japanese encephalitis virus antigen is (0.5-1.5): (0.5-1.5).

[0011] Wherein, the mass ratio of the adjuvant to the inactivated porcine Japanese encephalitis virus antigen is 1:1.

[0012] The inactivated porcine Japanese encephalitis virus antigen is prepared by the following steps: beta-propiolactone is mixed with porcine Japanese encephalitis virus antigen, then stirred and inactivated at 4°C and then hydrolyzed at 37°C in a water bath.

[0013] The beta-propiolactone and the porcine Japanese encephalitis antigen are mixed at a weight ratio of 1:(3800-4200), then stirred and inactivated at 4°C for 22-26 hours, and then hydrolyzed at 37°C in a water bath for 1.5-2.5 hours.

[0014] Wherein, the porcine Japanese encephalitis virus antigen comes from the culture of porcine Japanese encephalitis virus SA14-14-2.

[0015] Among them, the culture of porcine Japanese encephalitis virus SA14-14-2 is obtained by culturing porcine Japanese encephalitis virus SA14-14-2 on the full suspension cell line Vero-zm of African green monkey kidney cells, wherein the Vero-zm was deposited in the General Microbiology Center of China Culture Collection Administration (address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on ​​October 26, 2021, with the deposit number CGMCC No. 23098.

[0016] Wherein, the porcine Japanese encephalitis virus antigen is obtained by concentrating and purifying the culture of the porcine Japanese encephalitis virus SA14-14-2.

[0017] Wherein, the Vero-zm is cultured in V-SFM culture medium.

[0018] The adjuvant of the invention can be used as an immune adjuvant in a porcine Japanese encephalitis inactivated vaccine composition, and can enhance the immunogenicity of the vaccine, so that the porcine Japanese encephalitis inactivated vaccine composition has good immunogenicity and safety.

[0019] The Japanese encephalitis live vaccine composition produced by the present invention using concentrated and purified Japanese encephalitis virus SA14-14-2 antigen has significantly reduced immune side effects and significantly improved vaccine product quality. DETAILED DESCRIPTION

[0020] In the following description, for purposes of explanation, specific details are set forth in order to provide an understanding of the present disclosure.

[0021] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0022] Unless otherwise specified, the percentages mentioned in this article are by mass.

[0023] Example 1. Preparation of Vero full suspension cell line

[0024] Adherent cultured Vero cells adapted to the Japanese encephalitis virus SA14-14-2 strain were used as candidate suspension-adapted cells. The adherent Vero cells were prepared into fully suspension-adapted cell lines through two stages of gradual suspension-specific medium adaptation and serum reduction adaptation.

[0025] 1. Step-by-step suspension taming process

[0026] Adherent cultured Vero cells were digested with trypsin and the cells were 1-2×10 6 Cells / ml density, add newborn calf serum at a concentration of 8% to 10% to inoculate the suspension cell bottle, place in a constant temperature oscillator, 37 ° C, 120rpm / min, 5% carbon dioxide concentration culture. Four culture medium ratios of 75%:25%, 50%:50%, 25%:75% and 0:100% of the original culture medium (DMEM) and the suspension-specific culture medium were used for acclimatization. Each culture medium ratio was used to pass the cells for more than 3 generations, and the cells reached the normal suspension adaptation state (the cells reached the normal doubling rate and the viability reached more than 90%), and the next ratio of cells was passed until the cells were fully adapted to the suspension-specific culture medium and could be stably passed.

[0027] 2. Low serum suspension acclimation process

[0028] The suspension cells cultured in the suspension-specific culture medium with 8% to 10% newborn calf serum were acclimated by gradually reducing the serum level. The amount of bovine serum used for suspension-passaged cells was gradually reduced (to 8%, 6%, 4%, and 2%, respectively). The cells were stably passaged for more than 3 generations at each serum concentration until the cells reached a normal suspension adaptation state (the cells reached a normal doubling rate and the viability reached more than 90%). The cells were passaged at the next serum concentration until the cells were fully adapted to low-serum suspension culture and could be stably passaged. The Vero full suspension cell strain adapted to low serum was named Vero-zm.

[0029] Vero-zm was deposited at the General Microbiology Center of China Microorganism Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​October 26, 2021, with the deposit number CGMCC No.23098.

[0030] Adaptability of Vero-zm to different culture media

[0031] Four domestically produced commercial Vero suspension culture medium (A, B, C, and D) were selected from the market to conduct a comparative test on the adaptability of Vero full suspension cell lines.

[0032] Vero suspension culture medium A: Vero602 (Zhejiang Yishengke Biotechnology Co., Ltd., product code: Y1131);

[0033] Vero suspension culture special medium B: V-SFM medium (Womei Biotechnology Co., Ltd., product number M21216101-2);

[0034] Vero suspension culture special medium C: CD medium (gibco, product number: A16277-03);

[0035] Vero suspension culture special medium D: CDPK15 (Jian Shun Biological, product number: 10701).

[0036] Cell recovery and cell passaging were performed according to the following method. After the cells were passaged for 3 generations in the corresponding culture medium, they were inoculated with porcine Japanese encephalitis SA14-14-2 virus under the same conditions. After inoculation, the cells were counted by cell staining (0.4% trypan blue staining). When the cell viability was lower than 20%, the virus liquid was harvested and samples were taken to determine the vaccine virus content using the virus plaque test.

[0037] 1. Cell Recovery

[0038] Take out the frozen Vero-zm from liquid nitrogen and thaw it quickly in a 37°C water bath. Transfer the thawed cells to a centrifuge tube, add fresh Vero suspension culture medium (A, B, C or D), let it stand for 5 minutes, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend it in fresh Vero suspension culture medium. 6Cells / ml were inoculated in a 250ml cell shake flask in a total volume of 50ml of a suspension culture medium supplemented with 4% newborn calf serum, and placed in a constant temperature oscillator at 37°C, 120rpm / min, and 5% carbon dioxide concentration. Cells were sampled and counted daily to analyze cell viability and cell doubling rate. The cells were cultured normally for 48h and reached a normal doubling rate of 3 to 6×10 6 cells / ml, and the cell viability is over 95%.

[0039] 2. Cell Passaging

[0040] After 48 hours of cell culture, samples were taken and counted, and the cell density reached 3-6×10 6 cells / ml, and the cell viability was above 95%. The cells were diluted with Vero suspension culture medium, and then the final cell concentration was 0.8×10 6 Cells / ml were inoculated into a 250ml cell shake flask in a total volume of 50ml of Vero suspension culture medium supplemented with 4% newborn calf serum, and cultured in a constant temperature oscillator at 37°C, 120rpm / min, and 5% carbon dioxide concentration. Cells were sampled and counted daily to analyze cell viability and cell doubling rate.

[0041] 3. Virus Inoculation and Incubation

[0042] The virus inoculation amount was 0.5 MOI. After the virus was inoculated, the virus adsorption process was carried out in a bioreactor at a low stirring speed of 60 rpm / min, a temperature of 37° C., a pH value of 7.2, a dissolved oxygen (DO) of 50%, and an aeration volume of 0.1 to 0.5 SPLM. The adsorption time was 60 minutes.

[0043] 4. Determination of Virus Content

[0044] The virus was serially diluted 10-fold and 10 -2 , 10 -3 , 10 -4 3 dilutions were inoculated in 12-well cell plates growing BHK-21 cell monolayers, with 3 replicate wells for each dilution, 0.1 mL / well. After virus inoculation, the virus was adsorbed at 37°C for 90 minutes, and a covering (DMEM containing 1.5% methylcellulose and 2% calf serum) was added. After 5 days, it was stained with crystal violet, fixed for 30 minutes, rinsed with running water, and the number of plaques in each well was counted after drying to determine the virus content (Log PFU / mL). The cell growth morphology, density and other parameters after 3 generations of cell culture were recorded respectively. The specific data of the four culture media are shown in Table 1 below:

[0045] Table 1. Results of four suspension culture media on the culture characteristics and toxin production of Vero-zm cell lines

[0046]

[0047]

[0048] * Note: Cell viability was determined by counting cells with 0.4% trypan blue staining.

[0049] It can be seen from the above test results that, for the four commercial culture media, in terms of cell density, morphology and toxin production of porcine Japanese encephalitis virus SA14-14-2, Vero-zm cells cultured in Vero suspension culture medium B are superior to those cultured in the other three culture media. Therefore, Vero suspension culture medium B (V-SFM medium (Womei Biotechnology Co., Ltd., product number M21216101-2)) is preferred as a candidate culture medium for preparing porcine Japanese encephalitis virus antigens.

[0050] Example 2: Inactivated porcine Japanese encephalitis vaccine composition

[0051] Vero-zm cell line recovery and subculture

[0052] 1. Suspension Cell Recovery

[0053] Take out the frozen Vero-zm cell line from liquid nitrogen and thaw it quickly in a 37°C water bath. Transfer the thawed cells to a centrifuge tube, add fresh suspension cell culture medium B, let it stand for 5 minutes, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend it in fresh suspension culture medium. 6 Cells / ml were inoculated in a 250ml cell shake flask in a total volume of 50ml of a suspension culture medium supplemented with 4% newborn calf serum, and placed in a constant temperature oscillator at 37°C, 120rpm / min, and 5% carbon dioxide concentration. Cells were sampled and counted daily to analyze cell viability and cell doubling rate. The cells were cultured normally for 48h and reached a normal doubling rate of 3 to 6×10 6 cells / ml, and the cell viability is over 95%.

[0054] 2. Cell Passaging

[0055] After 48 hours of cell culture, samples were taken and counted, and the cell density reached 3-6×10 6 cells / ml, and the cell viability was above 95%. The cells were diluted with a suspension culture medium and then the final cell concentration was 0.8×10 6Cells / ml were inoculated into a 250ml cell shake flask in a total volume of 50ml of a suspension culture medium supplemented with 4% newborn calf serum, and cultured in a constant temperature oscillator at 37°C, 120rpm / min, and 5% carbon dioxide concentration. Cells were sampled and counted daily to analyze cell viability and cell doubling rate.

[0056] Vero-zm suspension cells cultured in bioreactors in stages

[0057] 1. Vero-zm suspension cell 10L bioreactor suspension culture

[0058] After 1 to 3 generations of suspension culture in a shake flask, the cell line was transferred to a 10 L bioreactor and supplemented with suspension culture medium to a cell concentration of 0.6 × 10 6 Cells / ml were added with 4% newborn calf serum and cultured at 37°C, pH 7.2, dissolved oxygen (DO) 50%, stirring speed 100rpm, ventilation volume 0.1-0.3, cultured for 48h-72h, cells were sampled and counted daily, and cell viability and cell doubling rate were analyzed. After 48-72h of culture, the cells reached a normal doubling rate of 3-6×10 6 cells / ml, the cell viability is over 95%, and the culture can be further expanded.

[0059] 2. Continuous scale-up of Vero-zm suspension cells in 10L-40L-100L bioreactors

[0060] The Vero-zm suspension cells cultured in the 10L bioreactor were transferred to the 40L bioreactor through a connection, and the suspension culture medium was added to adjust the cell density to 0.6×10 6 cells / ml, add 4% newborn calf serum, culture at 37°C, pH 7.2, dissolved oxygen DO value 50%, stirring speed 100rpm, ventilation volume 0.1-0.3, culture for 48h-72h, sample and count cells every day, analyze cell viability and cell doubling rate. After 48-72h of culture, the cells reach a normal doubling rate of 3-6×10 6 The cells were cultured in a 100L bioreactor using this culture process.

[0061] Production of 100L reactor for porcine Japanese encephalitis virus SA14-14-2 strain

[0062] The 100L suspension culture cells prepared above were used to complete the generation of porcine Japanese encephalitis virus SA14-14-2 strain through the cell sedimentation and liquid replacement stage, virus inoculation and incubation stage, and virus culture to harvest stage:

[0063] 1. Cell sedimentation and medium replacement stage

[0064] The 100 L suspension culture cells prepared above were statically settled in a 10-20° C. low-temperature cold water bath for 4-8 hours, and most of the cells were settled. Half of the total volume of the cell growth liquid was discharged through the reactor clarification device.

[0065] 2. Virus inoculation and incubation stage

[0066] After the above cells were settled and the fluid was changed, they were inoculated with porcine Japanese encephalitis SA14-14-2 virus at a virus inoculation amount of 0.5 MOI. After the virus inoculation, the virus adsorption process was carried out in a bioreactor at a low stirring speed of 60 rpm / min, a temperature of 37°C, a pH value of 7.2, a dissolved oxygen (DO) of 50%, and a ventilation volume of 0.1-0.5 SPLM. The adsorption time was 60 minutes.

[0067] 3. Virus culture to harvest stage

[0068] After the above-mentioned virus inoculation and incubation, add suspension cell-specific culture medium B (pH 7.8) containing 4% newborn calf serum to the original culture volume, and continue to culture in the reactor at a temperature of 35°C, dissolved oxygen (DO) of 50%, stirring speed of 100rpm, and ventilation volume of 0.1-0.5SPLM. Observe and count the cells daily to monitor the cell growth status. At the same time, adjust the pH value according to the change in pH value using 7.5-10% (w / v) sodium bicarbonate; according to the consumption of glucose, use 20-40% (w / v) of glucose concentrate to replenish the glucose in the culture medium in time. Cultivate until the cell viability is less than 20%, harvest the virus solution, freeze and thaw once, take samples to detect the virus titer, and the virus content per mL is 10 7.5 PFU.

[0069] Concentration and purification method and process of porcine Japanese encephalitis virus SA14-14-2

[0070] 1. Treatment of virus liquid samples:

[0071] Collect the Japanese encephalitis SA14-14-2 virus solution, add protamine sulfate with a final concentration of 1 mg / ml, shake well, and incubate at 4°C overnight.

[0072] 2. Centrifugation of virus liquid sample:

[0073] Using high-speed continuous centrifugation, porcine Japanese encephalitis virus was centrifuged at 10,000 rpm for 30 minutes, the precipitate was discarded, the virus supernatant was collected, and stored at 4°C for later use.

[0074] 3. Ultrafiltration of virus liquid sample:

[0075] 3.1 System pretreatment: Install the 300KD hollow fiber column (Spectrum Pure Technology) / membrane package (Millipore) into the hollow fiber / membrane package control equipment system (including pipelines, pumps and fiber / membrane packages). After assembly, flush the entire system with sterile water for injection for 20 minutes, and control the flow rate at 90L / min.

[0076] 3.2 System integrity and water flux testing: Flush the system with sterile water for injection, and test the water flux of the hollow fiber column / membrane package and whether the system is leaking at room temperature.

[0077] Online sterilization and cleaning of the system: Use sterile 0.2 mol / L NaOH solution to circulate and sterilize the system for 30 minutes, then use sterile water for injection to clean the ultrafiltration system to wash away the residual NaOH solution until the pH reaches 7.0.

[0078] 3.3 System balance: Use sterile 0.01mol / L pH7.2 PBS solution to balance for 10 minutes, flow rate 60L / min. After centrifugation, the feed liquid passes through the 300KD hollow fiber column / membrane package system, the pump speed at the inlet end is controlled at 60L / min, the transmembrane pressure at the permeate end is maintained at 1.5psi, the liquid at the reflux end is collected, and when the feed liquid at the inlet end remains 1 / 20 of the original volume, 0.01mol / L PBS solution is added to the original volume, and ultrafiltration is continued. After three times, it reaches 1 / 20 of the original volume, and the ultrafiltration process is completed.

[0079] 4. Molecular sieve chromatography gel purification:

[0080] 4.1 Chromatographic filler filling and pretreatment: Install the 4FF molecular sieve (GE Company) pre-packed column in place and test the column efficiency.

[0081] 4.2 Treat the molecular sieve gel chromatography column with 5 to 10 column volumes of sterile 0.5 mol / L NaOH, then wash with sterile injection water to pH 7.0, and then balance the column with 5 to 10 column volumes of 0.01 mol / L 7.2 PBS solution. The conductivity is 15.45 ms / cm, the pH is stable at 7.2, and the UV280 baseline is stable.

[0082] 4.5 Chromatographic purification: Load the ultrafiltration concentrated virus sample, set the loading linear flow rate to 10ml / min, and control the pressure to less than 2.0bar. The loading volume is 10% column volume. After loading, switch to 0.01mol / L PBS buffer, UV280 ultraviolet detection absorbance, collect the first peak as the target sample when the absorbance curve shows an upward trend, stop collecting when the absorbance curve drops to the lowest point, and use BCA Protein AssayKit (Thermo Company) to measure the protein removal rate of the collected peak sample, and the protein removal rate is 98.7%. Continue to treat the molecular sieve gel chromatography column with 5 to 10 column volumes of sterile 0.5mol / L NaOH, and then wash it with sterile injection water to pH7.0 to obtain the concentrated and purified porcine Japanese encephalitis virus SA14-14-2 antigen.

[0083] Preparation of inactivated vaccine composition for porcine Japanese encephalitis

[0084] 1. Preparation of inactivated porcine Japanese encephalitis antigen

[0085] The inactivated porcine Japanese encephalitis virus antigen was prepared by mixing β-propiolactone and porcine Japanese encephalitis virus SA14-14-2 antigen at a weight ratio of 1:4000 at 4°C for 24 hours with stirring, and then hydrolyzed at 37°C for 2 hours in a water bath.

[0086] 2. Preparation of animal vaccine oil adjuvant

[0087] Weigh a certain amount of emulsifier and adjuvant in the following proportion by weight, add them to the weighed mineral oil for injection produced by ExxonMobil Petroleum Company, control the stirring speed, and perform physical mixing. During the mixing process, heat is used to assist dissolution, and the temperature is controlled below 80°C. After mixing evenly into a liquid, sterilize at 121°C for 30 minutes, and cool to the emulsification temperature to obtain the animal vaccine oil adjuvant 1-3 used.

[0088] Oil adjuvant 1 is composed of the following components in percentage by weight:

[0089]

[0090] Oil adjuvant 2 is composed of the following components in percentage by weight:

[0091]

[0092] Oil adjuvant 3 is composed of the following components in percentage by weight:

[0093]

[0094] 3. Preparation of inactivated porcine Japanese encephalitis vaccine composition

[0095] The sterilized and cooled animal vaccine oil adjuvants 1-3 are added to an emulsification tank respectively, stirred and mixed evenly, and the stirring speed is controlled at 1000 rpm and the emulsification temperature is 30°C; an inactivated porcine Japanese encephalitis virus antigen equal to the animal vaccine oil adjuvant is slowly added to the prepared oil adjuvant; after the addition is completed, pre-shearing emulsification is performed, and the shear speed is controlled below 3000 rpm, and the pre-shearing emulsification process is 10 minutes; after the pre-shearing is completed, the rotation speed is controlled to 8000 rpm, and shearing emulsification is performed, and this process is 15 minutes; after the emulsification is completed, the porcine Japanese encephalitis inactivated vaccine composition 1-3 is obtained.

[0096] Safety test of inactivated porcine Japanese encephalitis vaccine composition on mice

[0097] The safety experiment was conducted using 12-14g ICR mice. The experiment was divided into 4 groups (Japanese encephalitis inactivated vaccine composition group 1, Japanese encephalitis inactivated vaccine composition group 2, Japanese encephalitis inactivated vaccine composition group 3 and control group), with 10 mice in each group.

[0098] In the Japanese encephalitis inactivated vaccine composition group 1, Japanese encephalitis inactivated vaccine composition group 2, and Japanese encephalitis inactivated vaccine composition group 3, 0.03 ml of the inactivated porcine Japanese encephalitis vaccine composition was injected intracerebrally into each mouse. The number of deaths within 3 days should not exceed 2. The remaining mice were observed for 14 consecutive days to observe the mice's spirit, appetite and injection site.

[0099] In the control group, each mouse was injected with 0.03 ml of culture medium into the brain. No more than 2 mice died within 3 days, and the remaining mice were observed for 14 consecutive days to observe their spirits, appetite and injection sites.

[0100] After injection of the inactivated porcine Japanese encephalitis vaccine composition, the mice showed normal clinical manifestations, good mental state, normal appetite, no local and systemic adverse reactions, and all mice were alive and well. The results are shown in Table 1.

[0101] Table 1. Safety test results of inactivated porcine Japanese encephalitis vaccine composition using ICR mice

[0102]

[0103]

[0104] Immunization test of inactivated porcine Japanese encephalitis vaccine composition on mice

[0105] The mouse immune challenge test was conducted using 12-14g ICR mice. The experiment was divided into 5 groups (1 group of porcine Japanese encephalitis inactivated vaccine composition, 2 group of porcine Japanese encephalitis inactivated vaccine composition, 3 group of porcine Japanese encephalitis inactivated vaccine composition, conventional oil adjuvant porcine Japanese encephalitis inactivated vaccine control group, and challenge control group), with 10 mice in each group.

[0106] The conventional oil adjuvant Japanese encephalitis inactivated vaccine control group uses a conventional oil adjuvant (aluminum gel adjuvant) Japanese encephalitis inactivated vaccine and is prepared according to the following method: adding the sterilized and cooled conventional oil adjuvant into an emulsification tank, stirring and mixing evenly, controlling the stirring speed to 1000 rpm, and the emulsification temperature to 30°C; slowly adding the above-prepared Japanese encephalitis virus inactivated antigen equal to the conventional oil adjuvant to the conventional oil adjuvant; after the addition is completed, pre-shearing emulsification is performed, the shearing speed is controlled to be below 3000 rpm, and the process of pre-shearing emulsification is 10 minutes; after the pre-shearing is completed, the rotation speed is controlled to 8000 rpm, and shearing emulsification is performed, and this process is 15 minutes; after the emulsification is completed, marking and packaging are performed to obtain the conventional oil adjuvant Japanese encephalitis inactivated vaccine.

[0107] The challenge control group was not immunized, and mice were vaccinated with 10 5 LD 50 The strong Japanese encephalitis P3 virus was used for intraperitoneal infection.

[0108] The inactivated porcine encephalitis vaccine composition and the conventional oil-adjuvanted porcine encephalitis inactivated vaccine were diluted with PBS at pH 7.5 at three dilutions of 50, 500 and 5000, and 10 mice of 10-12 g were inoculated intraperitoneally at each dilution. A second inoculation was performed 7 days apart, with 0.5 ml / mouse each time. At the same time, 10 mice were taken as challenge control. All mice were challenged intraperitoneally with 10 mice 5 days after the second inoculation. 5 LD 50 Japanese encephalitis P3 strong virus, 0.3 ml per mouse, and 0.04 ml of virus dilution was injected into the brain of each mouse before intraperitoneal challenge to destroy the blood-brain barrier. Observe for 14 days after challenge (death within 3 days is not counted), and the mice in the challenge control group should have no less than 80% mortality. After 14 days, calculate the half protection dose (PD 50 ).

[0109] The immunogenicity of the inactivated porcine encephalitis vaccine composition was evaluated by immune challenge test in mice. The results showed that the control group of the conventional oil adjuvant porcine encephalitis inactivated vaccine PD 50 For 10 -2 . 03 / 0.3ml, porcine Japanese encephalitis inactivated vaccine composition 1 group PD 50 For 10 -2.70 / 0.3ml, porcine Japanese encephalitis inactivated vaccine composition 2 groups PD 50 For 10 -2.60 / 0.3ml, porcine Japanese encephalitis inactivated vaccine composition 3 groups PD 50 For 10 -2.37 / 0.3ml.

[0110] It can be seen that the Japanese encephalitis inactivated vaccine composition 1 has the best immunogenicity. After being diluted 501 times, 50% of mice can still be protected. After being diluted 398 times, the Japanese encephalitis inactivated vaccine composition 2 can still protect 50% of mice. After being diluted 234 times, the Japanese encephalitis inactivated vaccine composition 3 can still protect 50% of mice. The Japanese encephalitis inactivated vaccine group using conventional oil adjuvant has poor immune effect. After being diluted 107 times, it can protect 50% of mice, as shown in Table 2.

[0111] Table 2. Half protective dose (PD) of inactivated porcine Japanese encephalitis vaccine composition 50 )

[0112]

[0113] Example 3: Preparation of live porcine Japanese encephalitis vaccine composition

[0114] 1. Preparation of live porcine Japanese encephalitis vaccine

[0115] The porcine Japanese encephalitis virus SA14-14-2 antigen was added with a lyophilization protectant (the heat-resistant lyophilization protectant for the live porcine Japanese encephalitis vaccine described in CN 103656661B, specifically, the heat-resistant lyophilization protectant for the live porcine Japanese encephalitis vaccine of Example 1) and subjected to freeze vacuum drying to prepare a live porcine Japanese encephalitis vaccine.

[0116] 2. Preparation of aqueous adjuvant for live Japanese encephalitis vaccine

[0117] Weigh the following amounts of adjuvants and polymer combinations in proportion, add them to the weighed aqueous solution for injection in sequence, control the stirring speed and mix. During the mixing process, heating can be used to assist dissolution. The temperature is controlled below 60°C. After mixing evenly into a liquid, sterilize at 121°C for 30 minutes and cool to room temperature to obtain aqueous adjuvant 1-3 for porcine Japanese encephalitis live vaccine.

[0118] The aqueous adjuvant 1 of the live porcine Japanese encephalitis vaccine is composed of the following components in percentage by weight:

[0119] Aqueous solution for injection 88%;

[0120] Polymer Combination:

[0121]

[0122] Additives:

[0123] Oleic acid 3%

[0124] The porcine Japanese encephalitis live vaccine aqueous adjuvant 2 is composed of the following components in percentage by weight:

[0125] Aqueous solution for injection 96%;

[0126] Polymer Combination:

[0127]

[0128] Additives:

[0129] Oleic acid 1%

[0130] The aqueous adjuvant 3 of the live porcine Japanese encephalitis vaccine is composed of the following components in percentage by weight:

[0131] Aqueous solution for injection 80%;

[0132] Polymer Combination:

[0133]

[0134]

[0135] Additives:

[0136] Oleic acid 4%

[0137] 3. Preparation of live porcine Japanese encephalitis vaccine composition

[0138] The porcine Japanese encephalitis live vaccine was diluted with diluent (1 / 15 mol / L phosphate buffered saline, pH 7.4) to 1 aliquot (virus content 10 6.0 PFU / ml), add 20% by volume of the porcine Japanese encephalitis live vaccine aqueous adjuvant 1-3 provided by the present invention, and shake well to prepare the porcine Japanese encephalitis live vaccine composition 1-3.

[0139] 4. Preparation of conventional aluminum gel adjuvant Japanese encephalitis live vaccine composition

[0140] The porcine Japanese encephalitis live vaccine was diluted with diluent (1 / 15 mol / L phosphate buffered saline, pH 7.4) to 1 aliquot (virus content 10 6.0 PFU / ml), added 20% by volume of conventional commercially available aluminum gel adjuvant (purchased from Invivogen), and shook well to prepare a Japanese encephalitis live vaccine composition.

[0141] Safety test of live vaccine composition of porcine Japanese encephalitis in mice

[0142] The safety experiment was conducted using 12-14g ICR mice. The experiment was divided into 4 groups (1 group of live Japanese encephalitis vaccine composition, 2 group of live Japanese encephalitis vaccine composition, 3 group of live Japanese encephalitis vaccine composition and a control group), with 10 mice in each group.

[0143] In the Japanese encephalitis live vaccine composition group 1, the Japanese encephalitis live vaccine composition group 2, and the Japanese encephalitis live vaccine composition group 3, each mouse was injected intracerebrally with 0.03 ml of the Japanese encephalitis live vaccine composition. The number of deaths within 3 days should not exceed 2. The remaining mice were observed for 14 consecutive days to observe the mice's spirit, appetite and injection site.

[0144] In the control group, each mouse was injected with 0.03 ml of culture medium into the brain. No more than 2 mice died within 3 days, and the remaining mice were observed for 14 consecutive days to observe their spirits, appetite and injection sites.

[0145] After injection of live porcine Japanese encephalitis vaccine composition 1-3, the mice showed normal clinical manifestations, good mental state, normal appetite, no local and systemic adverse reactions, and all mice were alive and well. The results are shown in Table 2.

[0146] Table 2 Results of safety test of Japanese encephalitis live vaccine composition using ICR mice

[0147]

[0148]

[0149] Immunization test of porcine Japanese encephalitis live vaccine composition on mice

[0150] The mouse immune challenge test was conducted using 12-14g ICR mice. The experiment was divided into 6 groups (1 group of live Japanese encephalitis vaccine composition, 2 group of live Japanese encephalitis vaccine composition, 3 group of live Japanese encephalitis vaccine composition, live Japanese encephalitis vaccine group, conventional aluminum gel adjuvant Japanese encephalitis live vaccine composition group, and challenge control group), with 10 mice in each group.

[0151] Use DMEM to dilute the live porcine Japanese encephalitis vaccine composition 1-3 and the live porcine Japanese encephalitis vaccine in a 10-fold serial dilution. -2 , 10 -3 , 10 -4 Ten ICR mice (10-12 g) were subcutaneously inoculated with 0.1 ml of the 3 dilutions. Fourteen days after immunization, 10 mice in the control group with the same conditions were intraperitoneally injected with 0.3 ml of the virulent P3 strain of Japanese encephalitis virus (containing 500 LD 50 ) and observed for 14 consecutive days.

[0152] The immunogenicity of porcine Japanese encephalitis live vaccine compositions 1-3 was evaluated by immune challenge test in mice. 50 For 10 -3.0 / 0.3ml; conventional aluminum gel adjuvant Japanese encephalitis live vaccine composition group PD 50 For 10 -3.10 / 0.3ml; Swine Japanese encephalitis live vaccine composition 1 group PD 50 For 10 -3.50 / 0.3ml; Swine Japanese encephalitis live vaccine composition 2 groups ED 50 For 10 -3 . 43 / 0.3ml; Swine Japanese encephalitis live vaccine composition 3 groups PD 50 For 10 -3.29 / 0.3ml.

[0153] It can be seen from this that the Japanese encephalitis live vaccine composition 1 has the best immunogenicity, and even after a 3162-fold dilution, it can still protect 50% of the mice; the Japanese encephalitis live vaccine group has the worst immune effect, and even after a 1000-fold dilution, it can still protect 50% of the mice, as shown in Table 4.

[0154] Table 4 Porcine Japanese encephalitis live vaccine composition half protective dose (PD 50 )

[0155]

[0156]

Claims

1. A porcine Japanese encephalitis inactivated vaccine composition, characterized in that: The composition consists of an adjuvant and an inactivated porcine Japanese encephalitis virus antigen; The adjuvants include: Mineral oil for injection MARCOL 52 85% Xylitol laurate ester 2.5% Triglycerol trioleate 10% Alkyl glycoside APG0816 1.25 % n-Decanol 1.25%; The mass ratio of the adjuvant to the inactivated porcine Japanese encephalitis virus antigen is (0.5-1.5): (0.5-1.5); The inactivated porcine Japanese encephalitis virus antigen is prepared by the following steps: β-propiolactone is mixed with the porcine Japanese encephalitis virus antigen, then stirred and inactivated at 4°C and then hydrolyzed at 37°C in a water bath; The porcine Japanese encephalitis virus antigen comes from the culture of porcine Japanese encephalitis virus SA14-14-2; the culture of porcine Japanese encephalitis virus SA14-14-2 is obtained by culturing porcine Japanese encephalitis virus SA14-14-2 on the full suspension cell line Vero-zm of African green monkey kidney cells, wherein the Vero-zm has been deposited in the General Microbiology Center of China National Culture Collection Administration on October 26, 2021, with the deposit number CGMCC No.23098.

2. The composition according to claim 1, characterized in that The mass ratio of the adjuvant to the inactivated porcine Japanese encephalitis virus antigen is 1:

1.

3. The composition according to claim 2, characterized in that The porcine Japanese encephalitis virus antigen is obtained by concentrating and purifying the culture of the porcine Japanese encephalitis virus SA14-14-2.

4. The composition according to claim 3, characterized in that The Vero-zm was cultured in V-SFM medium.

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