An inactivated vaccine for infectious bovine rhinotracheitis and a method for preparing the same

Abstract

The application optimizes the antigen component of the bovine contagious nasal tracheitis inactivated vaccine, and prepares a new bovine contagious nasal tracheitis inactivated vaccine, which can produce high level of neutralizing antibody after immunizing the bovine; the safety of the vaccine is improved through the suspension culture process promotion and the inactivation agent, adjuvant screening and purification process promotion. The safety and efficacy test results show that: after the bovine is immunized by using the inactivated vaccine, no adverse reaction is produced, and high level of neutralizing antibody is produced. The results show that the immunization effect of the bovine contagious nasal tracheitis inactivated vaccine can be improved by using the application.

Description

Technical Field

[0001] This invention relates to an inactivated vaccine for bovine infectious rhinotracheitis and its preparation method, belonging to the field of veterinary biological products technology. Background Technology

[0002] Infectious bovine rhinotracheitis (IBR) is caused by the infectious bovine rhinotracheitis virus (IBRV). Clinical symptoms mainly include difficulty breathing, nasal discharge, and inflammation of the upper respiratory tract and tracheal mucosa; it can also cause reproductive system symptoms. Besides these clinical symptoms, IBR can lead to decreased milk production in dairy cows, increased abortion rates, and slowed weight gain in beef cattle, causing significant economic losses to farms. This disease is immunosuppressive; while its harm is relatively low when contracted alone, the severity increases dramatically when mixed with other viral or bacterial diseases. The most effective method for controlling this disease is vaccination. Currently, the IBR vaccines used on farms are mainly inactivated vaccines. Monitoring of immunization efficacy shows that the neutralizing antibody titer in cattle after immunization with inactivated vaccines is low, and some allergic reactions occur. Therefore, it is necessary to provide a safe and effective IBR vaccine for the prevention of bovine rhinotracheitis. Summary of the Invention

[0003] The purpose of this invention is to mix bovine infectious rhinotracheitis virus (BRV) J1 strain antigen obtained from MDBK cell suspension culture with bovine BRV J1 strain gB protein obtained from CHO cell culture in a certain proportion after inactivation and purification, and then prepare an inactivated vaccine with appropriate adjuvants. This improves the level of neutralizing antibodies and vaccine safety after bovine immunization, thereby increasing the success rate of preventing bovine infectious rhinotracheitis and ensuring immunization efficacy.

[0004] Technical solution of the present invention

[0005] 1. An inactivated bovine infectious rhinotracheitis vaccine, characterized in that the active ingredient of the vaccine comprises inactivated and purified J1 strain bovine infectious rhinotracheitis virus and purified J1 strain bovine infectious rhinotracheitis virus gB protein.

[0006] 2. The inactivated bovine infectious rhinotracheitis vaccine of the present invention is characterized in that the vaccine preparation steps include:

[0007] (1) A eukaryotic expression vector pcDNA3.1-gB expressing the gB protein of bovine infectious rhinotracheitis virus J1 strain was constructed using the pcDNA3.1(-) vector;

[0008] (2) Semi-finished product preparation:

[0009] 1) J1 strain of bovine infectious rhinotracheitis virus was cultured in MDBK suspension cells, inactivated by BEI, and purified using a membrane package;

[0010] 2) The gB protein of bovine infectious rhinotracheitis virus strain J1 was purified by nickel column affinity chromatography after transfection of CHO cells with the eukaryotic expression vector pcDNA3.1-gB.

[0011] (3) Vaccine preparation: The inactivated and purified J1 strain bovine infectious rhinotracheitis virus liquid and the purified bovine infectious rhinotracheitis virus J1 strain gB protein are mixed in a certain proportion and emulsified with biphasic adjuvant.

[0012] The bioreactor used in this invention is an APPLIKON 30L-2000L cell bioreactor.

[0013] Beneficial effects of the invention

[0014] This invention optimizes the antigenic components of an inactivated bovine infectious rhinotracheitis (BNR) vaccine to prepare a novel BNR vaccine that induces high levels of neutralizing antibodies in immunized cattle. Improvements in suspension culture and the screening and purification processes for inactivating agents and adjuvants enhance the vaccine's safety. Safety and efficacy tests show that no adverse reactions occurred after immunization of cattle with the inactivated vaccine of this invention, and all cattle produced high levels of neutralizing antibodies. These results demonstrate that this invention can improve the immunizing effect of an inactivated bovine BNR vaccine.

[0015] This invention relates to biomaterial resource information.

[0016] The viral strain of this invention, Bovine Infectious Rhinotracheitis Virus J1, was isolated and obtained by the inventors in my country. This strain was deposited on March 15, 2017, at the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 13786.

[0017] Detailed embodiments of the present invention

[0018] 1. Acclimatization of MDBK cell lines in full suspension culture

[0019] (1) Acclimatization of MDBK cells during adherent culture (reducing serum levels)

[0020] MDBK adherent cells were seeded from a liquid nitrogen tank and revived in a T75 culture flask with cell culture medium containing 10% newborn calf serum. The cells were then cultured in an adherent culture medium with progressively decreasing serum concentrations for acclimatization. Serum concentrations were reduced from 10%, 8%, 5%, 3%, 2%, and 1% to 0.5%.

[0021] (2) Suspension acclimatization of MDBK cells (low serum medium)

[0022] MDBK adherent cells, already adapted to low serum conditions, were cultured in Erlenmeyer flasks. When the MDBK cells exhibited consistent cell morphology and proliferation rate for five consecutive passages, suspension acclimatization was completed. Successfully acclimatized MDBK cells (F40) were harvested and cryopreserved. At this point, the cell proliferation efficiency had stabilized at 1.7 × 10⁻⁶. 6 With approximately 100 cells / mL and cell viability exceeding 96%, the MDBK suspension cells have been successfully domesticated.

[0023] Further optimization of the bioreactor full suspension culture process was carried out, and the culture parameters were determined as follows: dissolved oxygen 40%, pH 7.0±0.1, and rotation speed 110-120 r / min, realizing the large-scale culture of MDBK cells from Erlenmeyer flasks to bioreactors.

[0024] 2. Preparation of MDBK whole suspension cells

[0025] (1) Cell shake-flask culture: After cell resuscitation, the cells were cultured at 8.0 × 10⁻⁶ cm⁻¹. 5 Cells were inoculated at a density of [number] cells / mL into 125mL Erlenmeyer flasks and cultured at 120 rpm in a 37°C incubator containing 5% CO2. The cells were cultured until they reached a growth rate of 4.0 × 10⁶ cells / mL. 6 Passage the cells at a density of 8.0 × 10⁶ cells / mL, and replenish with fresh culture medium as needed to achieve an initial cell density of 8.0 × 10⁶ cells / mL. 5 Cells / mL.

[0026] (2) Cell seeding and scale-up culture: Clean the cell culture jar thoroughly and sterilize it at 121℃ for 30 min. After sterilization, perform 100% point calibration of the dissolved oxygen electrode in the empty jar. Select vigorously growing MDBK cells and seed them into the cell culture jar at a seeding density of 8.0 × 10⁶ cells / mL. 5 ~1.3×10 6 Cells / mL, cultured at 37°C until cell density exceeds 4.0 × 10⁻⁶. 6 When the cell density reaches 100 cells / mL, add culture medium and continue culturing until the cell density reaches 4.0 × 10⁶ cells / mL. 6 When the cell density reaches 8.0 × 10⁶ cells / mL, transfer the cell suspension from the cell culture jar to a new cell culture jar for further culture, with a seeding density of 8.0 × 10⁶ cells / mL. 5 ~1.3×10 6 Cells / mL.

[0027] 3. Semi-finished product preparation

[0028] (1) Preparation of bovine infectious rhinotracheitis virus strain J1 viral fluid

[0029] 1) Inoculation: After MDBK is cultured in full suspension to a density of approximately 4.0 × 10⁶ cells / year 6 When the cell density reaches 100 cells / mL, add cell culture medium containing 0.5%–1% newborn calf serum (approximately 1 / 3 of the culture medium volume) to dilute the cell density to 2.0 × 10⁶ cells / mL. 6 Cells / mL ~ 3.0 × 10⁻⁶ 6 1 cell / mL, inoculated with bovine infectious rhinotracheitis virus strain J1 at an MOI of 0.01–0.5.

[0030] 2) Harvesting the virus solution: Continue to culture the suspended cells in the bioreactor. When the cell viability drops below 50%, harvest the cell culture virus solution as a semi-finished product.

[0031] 3) Scale-up culture: Referring to the above parameters, the semi-finished product of bovine infectious rhinotracheitis virus J1 strain was scaled up in tanks from 30L to 2000L using a cell bioreactor from APPLIKON.

[0032] 4) Virus inactivation: The prepared bovine infectious rhinotracheitis virus J1 strain semi-finished product was tested for purity according to the appendix of the "Veterinary Pharmacopoeia of the People's Republic of China". It should be free of bacteria, fungi, and mycoplasma, and the virus content of the semi-finished product should be ≥10. 8.5 TCID 50 / mL.

[0033] The qualified semi-finished products were placed in different containers, and BEI with a working concentration of 0.1-0.5% was added. The mixture was inactivated at 30℃-37℃ for 24-48 hours. After inactivation, sodium thiosulfate solution with a final concentration of 0.2% was added to the virus solution, and the inactivation was stopped by stirring for 1 hour. The inactivated samples were taken and inoculated into MDBK cells at a 10% inoculation rate. The cells were then blindly passaged three times for inactivation testing.

[0034] 5) Virus purification: After centrifuging and filtering the harvested bovine infectious rhinotracheitis virus strain J1, the purified virus solution was obtained by using a membrane with a molecular cutoff value of 750KD.

[0035] (2) Preparation of gB protein of bovine infectious rhinotracheitis virus J1 strain

[0036] 1) Design primers to amplify the gB gene of bovine infectious rhinotracheitis virus strain J1, and add Xho I and Hind III restriction enzyme sites and protective bases to the 5' ends of the upstream and downstream primers. Also, add a His tag to the primers. The primer sequences are as follows:

[0037] gB-F 5'- ccg ctcgaga atgtggccgc tcgcggcggt-3'30(Sequence 1)

[0038] gB-R 5'– ccc aagcttc ta gtgatggt gatggtgatg tcatgccccc ccgacgt-3'47(Sequence 2)

[0039] 2) The gB gene of bovine infectious rhinotracheitis virus J1 strain obtained by PCR amplification was recovered by gel extraction and combined with... -T1 cloning vectors were ligated according to the following system (Table 1).

[0040] Table 1 Cloning Ligation System

[0041]

[0042] 3) The correctly sequenced recombinant cloning vector pEASY-gB and the vector pcDNA3.1(-) were double-digested with enzymes. The enzyme digestion reaction system is shown in Table 2 below.

[0043] Table 2 Enzyme digestion reaction system

[0044]

[0045] After ligation, the eukaryotic expression vector pcDNA3.1-gB was constructed.

[0046] 4) The constructed eukaryotic expression vector pcDNA3.1-gB was transfected into CHO cells via liposomes.

[0047] 5) Culture parameters of CHO cells

[0048] ① Cell culture in shake flasks: After cell resuscitation, cells are cultured at 8.0 × 10⁻⁶ cm⁻¹. 5 Cells were inoculated at a density of [number] cells / mL into 125mL Erlenmeyer flasks and cultured at 120 rpm in a 37°C incubator containing 5% CO2. The cells were cultured until they reached a growth rate of 5.0 × 10⁶ cells / mL. 6 Passage the cells at a density of 8.0 × 10⁶ cells / mL, and replenish with fresh culture medium as needed to achieve an initial cell density of 8.0 × 10⁶ cells / mL. 5 Cells / mL. ② Cell seeding and scale-up culture: Clean the cell culture vessel thoroughly and sterilize at 121℃ for 30 min. After sterilization, perform 100% point calibration of the dissolved oxygen electrode in the empty vessel. Select vigorously growing CHO cells and seed them into the cell culture vessel at a seeding density of 8.0 × 10⁶ cells / mL. 5 ~1.0×10 6 Cells / mL, cultured at 37℃. Starting from day 4, 5% fed-batch medium was added to the culture vessel every day, and glucose content was monitored daily, with glucose solution added as needed to ensure the sugar content of the culture system was not lower than 0.6 g / L. On day 5, the culture temperature was lowered to 33℃ for cryopreservation, and the culture medium was harvested after 15 days.

[0049] 6) Purification of gB protein

[0050] The harvested bovine infectious rhinotracheitis virus J1 strain gB protein was purified using nickel column affinity chromatography.

[0051] 4. Emulsified seedling preparation

[0052] Bovine infectious rhinotracheitis virus strain J1 virus fluid and bovine infectious rhinotracheitis virus strain J1 gB protein were mixed in a 1:1 ratio, and then emulsified with domestic biphasic adjuvant at a mass ratio of 1:1 under isothermal conditions at 35℃. Example

[0053] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0054] The original strain, bovine infectious rhinotracheitis virus J1, was isolated and obtained by the inventors in my country, with accession number CGMCC No. 13786. MDBK cells, MDBK suspension cells, CHO cells, and the vector pcDNA3.1(-) were all preserved in the inventors' laboratory.

[0055] Enzymes and other biochemical reagents: Universal column-based genomic DNA extraction kit, 2×Taq Plus MasterMix (Dye), and gel extraction kit were all purchased from Kangwei Century Biotechnology Co., Ltd.; recombinant cloning vectors -T1 is a product of Beijing TransGen Biotech Co., Ltd.; the fast digestion enzymes Xho I and Hind III are products of TaKaRa.

[0056] The culture medium used for MDBK cell suspension culture was a product of Hyclone; the CHO cell culture medium and fed-batch culture medium were both products of Suzhou Womei Co., Ltd.

[0057] Example 1

[0058] Preparation of semi-finished product of bovine infectious rhinotracheitis virus strain J1

[0059] 1. Cell culture and inoculation

[0060] (1) Shake flask culture

[0061] 1) Effect of different infection multiples on viral load: initial cell density was 2.5 × 10⁻⁶. 6MDBK cells per mL were inoculated with bovine infectious rhinotracheitis virus strain J1 at MOIs of 0.01, 0.05, 0.1, and 0.5, respectively. Samples were taken at 24, 36, and 48 hours after inoculation to determine the viral load.

[0062] 2) Effect of cell density on viral load at inoculation: MDBK cells were inoculated into shake flasks, and the cell density was 2.0 × 10⁻⁶. 6 cells / mL, 2.5 × 10 6 3.0 × 10⁻⁶ cells / mL 6 3.5 × 10⁻⁶ cells / mL 6 4.0 × 10⁻⁶ cells / mL 6 When the viral load was 100 cells / mL, bovine infectious rhinotracheitis virus strain J1 was inoculated at an MOI of 0.05. Samples were taken at 24, 36 and 48 hours after inoculation to determine the viral load.

[0063] 3) Effect of different harvest times on virus content: MDBK cells were inoculated into shake flasks, and when the cell density was 2.0 × 10⁻⁶, the virus content was... 6 3.0×10 6 When the viral load was 100 cells / mL, bovine infectious rhinotracheitis virus strain J1 was inoculated at an MOI of 0.05. Samples were taken at 8, 16, 24, 32, 40 and 48 hours after inoculation to determine the viral load.

[0064] Through experimentation with the shake-flask process, the optimal cell density was determined to be 2.0 × 10⁻⁶. 6 ~3.0×10 6 When the viral load is 1 cell / mL, the virus is inoculated at a multiplicity of infection (MOI) of 0.05, and the cell culture harvested after 48 hours of culture has the highest viral content.

[0065] (2) Cell container inoculation

[0066] Healthy MDBK cells cultured in shake flasks were seeded into 30L cell culture jars, and then scaled up to 2000L cell culture jars. MDBK cells were cultured in 2000L cell culture jars until a density of 2.5 × 10⁶ cells / mL was reached. 6 At a cell / mL concentration, bovine infectious rhinotracheitis virus strain J1 was inoculated at a multiplicity of infection (MOI) of 0.05 and cultured further. Viral content was measured at 72, 96, and 120 hours post-inoculation. The results are shown in Table 3.

[0067] Table 3. Results of virus content determination in the inoculation test of 2000L cell culture tanks

[0068]

[0069] J1 strain of bovine infectious rhinotracheitis virus was propagated using cell culture tanks. Virus production was stable 96–120 hours after inoculation, with a viral load consistently not lower than 10. 8.5 TCID 50 / mL indicates that it is feasible to use cell culture tanks for large-scale culture of J1 strain bovine infectious rhinotracheitis virus.

[0070] 2. Observations and Gains

[0071] After inoculation, sample cells every 24 hours to observe their condition. When cell viability drops below 50%, harvest the viral fluid and store it below -15°C. Perform sterility testing according to the appendix of the *Veterinary Pharmacopoeia of the People's Republic of China*, and sample one portion to determine the viral load. The viral load should not be less than 10... 8.5 TCID 50 / mL.

[0072] 3. Inactivation of semi-finished products

[0073] Bovine infectious rhinotracheitis virus strain J1, prepared in a cell culture vessel, was dispensed into 12 vials, each containing 100 mL. These were then divided into three groups of four samples each. In group 1, BEI was added to each vial to a final concentration of 0.1%; in group 2, the final concentration was 0.2%; and in group 3, the final concentration was 0.5%. The samples were then inactivated in a 37°C shaker. At 24, 32, 40, and 48 hours after inactivation, one vial from each group was taken, and a 2% sodium thiosulfate solution was added to terminate the inactivation process. Samples were then taken for inactivation testing. This experiment was repeated once.

[0074] Take the inactivated virus solution and inoculate it into MDBK cells that have grown into a monolayer at a ratio of 10%. After adsorption for 1 hour, replace it with cell maintenance medium containing 2% newborn calf serum and incubate at 37°C in a 5% CO2 incubator for 5 days. Harvest the cell culture by freeze-thaw and then blindly passage it twice in MDBK cells.

[0075] By comparing the inactivation effects of three concentrations (0.1%, 0.2%, and 0.5%), it was found that 0.1% was slightly less effective and could not inactivate the virus within 24 hours; while 0.2% and 0.5% concentrations could completely inactivate the virus within 24 hours.

[0076] Example 2

[0077] Preparation of gB protein of J1 strain bovine infectious rhinotracheitis virus

[0078] Healthy CHO cells were seeded into 30L cell culture jars from Applikon (Netherlands), and then scaled up to 2000L cell culture jars. CHO cells were cultured in the 2000L jars until a density of 8.0 × 10⁶ cells / year was reached. 6The culture temperature was lowered to 33℃ to express gB protein at a concentration of [number] cells / mL, with 5% fed-batch medium and glucose solution added during the process. After 15 days of culture, the cells were harvested, and the expression level of gB protein of bovine infectious rhinotracheitis virus strain J1 was determined to be 80–120 mg / L. This indicates that it is feasible to prepare gB protein of bovine infectious rhinotracheitis virus strain J1 using cell culture tanks from Applikon, Netherlands.

[0079] Example 3

[0080] Vaccine safety trials

[0081] 1. Safety trials on calves

[0082] Thirteen healthy susceptible cattle aged 1–3 months were randomly divided into three groups: Group 1 (5 cattle) underwent a single-dose repeated safety test (intramuscular injection, 2 mL / cattle, two vaccinations); Group 2 (5 cattle) underwent an overdose safety test (intramuscular injection, 4 mL / cattle); and Group 3 (3 cattle) served as a blank control without immunization.

[0083] Two days prior to immunization, rectal body temperature was measured daily at fixed locations, once a day. Including the temperature measured on the day of immunization (before immunization), a total of three temperature readings were taken. The basal body temperature for each cow was calculated as (sum of the three temperatures / 3). From day 1 to day 14 post-immunization, rectal body temperature was measured daily at fixed locations, and the cows' mental state, appetite, and feces were observed. The results are shown in Table 4.

[0084] Table 4. Results of vaccine safety trials in calves

[0085]

[0086] 2. Safety trials in lactating cows

[0087] Thirty lactating cows were selected and randomly divided into two groups of 15 each. Group 1 received an intramuscular injection of vaccine (2 mL / cow), while Group 2 served as a control. From 1 to 14 days post-immunization, the cows' mental state, appetite, and feces were observed daily, and milk production data were recorded. Combined with pre-immunization milk production data, the decrease in milk production was statistically analyzed. The results are shown in Table 5.

[0088] Table 5. Results of vaccine safety trials in lactating cows

[0089]

[0090] Example 4

[0091] Vaccine efficacy trials

[0092] Thirteen healthy, susceptible cattle aged 1–3 months were randomly divided into three groups: Group 1 (cattle 5) was immunized with the vaccine of this invention; Group 2 (cattle 5) was immunized with a commercially available inactivated bovine infectious rhinotracheitis vaccine; and Group 3 (cattle 3) served as a blank control without immunization. Immunization was administered intramuscularly, 2 mL / cattle. A second immunization was administered 21 days after the first immunization, following the same procedure. Fourteen days after the second immunization, blood samples were collected, serum was separated, and the neutralizing antibody titer for bovine infectious rhinotracheitis was determined. The results are shown in Table 6.

[0093] Table 6. Results of the efficacy test on cattle.

[0094]

Claims

1. An inactivated vaccine against infectious bovine rhinotracheitis, characterised in that, The effective components of the vaccine include inactivated and purified J1 strain of bovine infectious rhinotracheitis virus and purified J1 strain of bovine infectious rhinotracheitis virus gB protein; the J1 strain of bovine infectious rhinotracheitis virus is preserved in the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, with a preservation number of CGMCC No. 13786; the amplification primers of the J1 strain of bovine infectious rhinotracheitis virus gB protein are shown in SEQ ID No. 1 and SEQ ID No. 2; The vaccine preparation steps include: (1) constructing a eukaryotic expression vector pcDNA3.1-gB expressing bovine infectious rhinotracheitis virus J1 strain gB protein by using a pcDNA3.1(-) vector; (2) semi-finished product preparation: 1) J1 strain of bovine infectious rhinotracheitis virus liquid, inactivated by BEI after MDBK suspension cell culture, and purified by membrane bag; 2) J1 strain of bovine infectious rhinotracheitis virus gB protein, purified by nickel column affinity chromatography after transfection of CHO cells by using the eukaryotic expression vector pcDNA3.1-gB; (3) vaccine preparation: mixing the inactivated and purified J1 strain of bovine infectious rhinotracheitis virus liquid and the purified bovine infectious rhinotracheitis virus J1 strain gB protein, and adding biphasic adjuvant emulsion vaccine preparation.

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