Preparation method of akabane virus inactivated vaccine

Abstract

The invention discloses an akabane virus inactivated vaccine preparation method, which comprises: carrying out Vero cell passage in a cell bottle, taking well growing cells until the cells grow to 80-90%, adding into a DMEM culture medium containing 10% of fetal calf serum, taking out the cells growing to 80-90% and having a good state, adding akabane virus CH-JL-01-2022 according to a volume ratio of 0.1%, and carrying out culture at a temperature of 80-90 DEG C so as to obtain the akabane virus inactivated vaccine. Culturing for 2-3 days in an incubator with the temperature of 37 DEG C and 5% CO2; the method comprises the following steps: preparing 0.25 M BEI, 0.1 M BPL and a 40% formaldehyde solution as inactivators, setting five concentration gradients according to the volume ratios of 0.01%, 0.05%, 0.1% and 0.5%, and collecting after 6 hours, 12 hours, 24 hours, 36 hours and 48 hours after adding; the screening of the akabane virus inactivation conditions according to the types, concentrations and inactivation time of inactivators finds that 0.5% formaldehyde shows a complete inactivation effect after inactivation for 36 hours. The inactivated virus liquid is mixed with MONTANIDETM ISA 201, an ImjectI aluminum adjuvant and a manganese adjuvant according to the volume ratio of 1: 1, and then immunization is carried out, and the protection effect of the ImjectI aluminum adjuvant is found to be optimal.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms and biological products, and in particular to a method for preparing an inactivated Akabane virus vaccine. Background Art

[0002] Akabane virus is a single-stranded, negative-sense RNA virus of the Bunyaviridae family, genus Orthobunyavirus, serogroup Simbu. It is primarily transmitted by arthropods, and infection of ruminants can cause miscarriage, stillbirth, and congenital malformations in newborns. Antibodies to Akabane virus have also been detected in wild animals such as buffalo, camels, and wild donkeys. AKAV was first discovered in Japan in 1959 and subsequently in South Korea, Iraq, Iran, China, and other Asian countries. Outside of Asia, it has also been reported in Sudan, Nigeria, Kenya, Egypt, and Turkey, severely impacting the livestock industry. In my country, it has been detected in Yunnan, Guangdong, Inner Mongolia, Shandong, and Jilin, and an Akabane virus epidemic is suspected to have occurred in Northeast my country from 2021 to 2022. Inactivated vaccines are widely used to prevent other diseases such as influenza and smallpox. Despite continuous technological improvements, inactivated vaccines still account for a large proportion of current global vaccine candidates.

[0003] Currently, there are no effective medications or vaccines available for Akabane virus. Foreign vaccines primarily utilize attenuated vaccines generated using HmLu-1 cells, as well as inactivated vaccines developed using Vero and BHK cells. In China, the main inactivated vaccines developed by the Institute of Specialty Products of the Chinese Academy of Agricultural Sciences and the Yunnan Academy of Animal Husbandry and Medical Sciences utilize different inactivators and adjuvants. Therefore, screening of inactivators and adjuvants is necessary to develop a safe and effective vaccine against the predominant strains currently circulating in my country. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing an inactivated Akabane virus vaccine. The inactivation conditions of the Akabane virus are screened by the type of inactivator, the concentration of the inactivator and the inactivation time. It is found that 0.5% formaldehyde shows a complete inactivation effect after 36 hours of inactivation, with high biosafety and no risk of infection and leakage.

[0005] Implementation of the technical solution of the present invention:

[0006] A method for preparing an inactivated Akabane virus vaccine comprises the following steps:

[0007] (1) Vero cells were passaged in a cell flask. Well-grown cells were taken until the cells grew to 80-90%, and DMEM medium containing 10% fetal bovine serum was added. Cells that grew to 80-90% of their normal state were taken out, and Akabane virus CH-JL-01-2022 was added at a volume ratio of 0.1%. The cells were cultured in a 37°C, 5% CO2 incubator for 2-3 days, and the cells were frozen and thawed three times. The cells were centrifuged at 8000 rpm for 20 min, and the supernatant was aspirated and stored at -80°C for later use.

[0008] (2) 0.25 M BEI, 0.1 M BPL, and 40% formaldehyde solution were prepared as inactivators, and five concentration gradients were set according to the volume ratio of 0.01%, 0.05%, 0.1%, and 0.5%, and the samples were collected at 6 h, 12 h, 24 h, 36 h, and 48 h after addition;

[0009] (3) The inactivated virus solution was mixed with Montanide ISA 201, The aluminum adjuvant and the manganese adjuvant are mixed in a volume ratio of 1:1, packaged in a quantitative manner, and sealed to obtain the product.

[0010] Preferably, in step (2), 0.5% formaldehyde can be completely inactivated after 36 hours of inactivation.

[0011] Preferably, Montanide ISA 201 and Aluminum adjuvants need to be emulsified to fully mix the inactivated virus solution with the adjuvant. Complete emulsification should be confirmed by microscopic examination and stored at 4°C for future use.

[0012] The present invention has the following beneficial effects: by screening the inactivation conditions for Akabane virus by inactivating agent type, inactivator concentration, and inactivation time, it was found that 0.5% formaldehyde exhibited a complete inactivation effect after 36 hours of inactivation, with high biosafety and no risk of infection or leakage. Adjuvants were screened under optimal inactivation conditions, and high levels of neutralizing antibodies and low levels of viremia were detected in surrogate animals. Furthermore, pathological changes after challenge with the surrogate animals were alleviated, demonstrating that the vaccine of the present invention is safe and reliable, can prevent Akabane virus infection, and alleviate clinical symptoms following infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0014] Figure 1 for Figure 1This is a cytopathic effect image after infection with the Akabane virus strain (CH-JL-01-2022);

[0015] Figure 2 This is the growth curve of Akabane virus strain (CH-JL-01-2022);

[0016] Figure 3 This is a graph showing the neutralizing antibody titer after immunization. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0018] Example 1

[0019] Screening of inactivation conditions and preparation of inactivated vaccines

[0020] (1) Amplification of Akabane virus

[0021] Cell recovery: Take out the Vero cells frozen in liquid nitrogen, heat at 37°C, stir continuously until thawed, transfer to a T75 cell culture flask, add DMEM medium containing 10% fetal bovine serum, and culture at 37°C, 5% CO2.

[0022] Cell passaging: Remove cells that have grown to 95-100%, discard the culture medium, add 0.25% trypsin solution to digest for 1 min, discard the trypsin, re-add normal culture medium at a ratio of 1:3, discard excess cells, add DMEM culture medium containing 10% fetal bovine serum to the remaining cells, and continue to culture at 37°C, 5% CO2.

[0023] Inoculation and harvesting: Take out cells that have grown to 80-90% of the cells in good condition, add Akabane virus at 0.1% (V:V), and continue culturing at 37°C and 5% CO2 for 2-3 days.

[0024] The cells were transferred to a -80°C refrigerator, frozen and thawed three times, transferred to a 50 mL centrifuge tube, centrifuged at 8000 rpm for 20 min, and the supernatant was collected and stored in a -80°C refrigerator for later use.

[0025] (2) Screening of inactivation conditions

[0026] Preparation of Inactivating Agent: BEI: Add an appropriate amount of BEA to 0.2N sodium hydroxide in a 37°C waterbath, mixing every 15 minutes to form BEI. Prepare the solution immediately. Add sterile sodium thiosulfate to terminate the inactivation. BPL: Add an appropriate amount of BPL to sterile PBS to make 0.1M BPL. Prepare the solution immediately. 40% Formaldehyde: Dilute an appropriate volume of formaldehyde to 40% formaldehyde by volume with sterile water. Prepare the solution immediately.

[0027] Screening of inactivation conditions: Five concentration gradients were set up at volume ratios of 0.01%, 0.05%, 0.1%, and 0.5%, and samples were collected at 6, 12, 24, 36, and 48 hours after addition. Fluorescence quantitative analysis of viral particles revealed that 0.5% formaldehyde completely inactivated viral particles after 36 hours, with no detectable viral particles.

[0028] (3) Preparation of inactivated vaccines

[0029] Montanide ISA 201 is a mineral oil-based adjuvant in the form of water-in-oil-in-water (W / O / W) emulsion that can induce short-term and long-term immunity. Compared with traditional double emulsions, MONTANIDE TM ISA 201VG emulsion is stable, has low viscosity and is easily injectable. Aluminum adjuvants form an antigen reservoir at the injection site, slowly releasing antigens and continuously stimulating the immune system. Manganese adjuvants are novel adjuvants that can activate innate immune pathways to promote antigen uptake, presentation, and germinal center formation, thereby enhancing the immune response.

[0030] According to the above method, a large amount of inactivated antigen was prepared and mixed with adjuvant in a volume ratio of 1:1, wherein Montanide ISA 201 and Aluminum adjuvants must be emulsified to ensure thorough mixing of the antigen and adjuvant. Complete emulsification should be confirmed by microscopic examination. Store at 4°C until ready for use.

[0031] Example 2

[0032] Evaluation of the effectiveness of inactivated vaccines

[0033] (1) Vaccine immunity and safety evaluation

[0034] The above-described vaccine and a single antigen were used as immunogens in five groups: PBS (control), AKAV (no adjuvant), AKAV-AL (AL adjuvant), AKAV-201 (201 adjuvant), and AKAV-Mn (Mn) adjuvant. Nine SPF-grade, 6-week-old Balb / c mice were immunized intramuscularly with 0.2 mL of the vaccine. A booster immunization was performed 21 days later. Post-immunization, the mice were observed for swelling at the injection site and for symptoms such as abnormal feeding, lethargy, matted fur, and weight loss to evaluate vaccine safety.

[0035] (2) Vaccine effectiveness evaluation

[0036] Serum was collected at 0, 7, 14, 21, 28, and 35 days after immunization for neutralizing antibody assay. Neutralizing antibodies were first detected 7 days after immunization, and the antibody titer continued to increase. In the first 21 days, all adjuvant-added immunization groups showed higher antibody levels than the non-adjuvanted groups. Mn adjuvant did not show a significant enhancement effect in the AKAV inactivated vaccine. The antibody level in the late immunization period only reached 1:32-1:64. On the 35th day, there was no significant difference between the AKAV-201 group and the AKAV-AL group, and the antibody titer was about 1:128. Further testing of the lymphocyte level after immunization found that the CD4 + T cells and CD8 + T were higher than those in other immune groups.

[0037] Immunized mice were challenged with the virus by intramuscular injection in the legs. Whole blood was collected 7 and 14 days after the challenge for viremia detection. The main organs were autopsied and collected for tissue viral load detection on the 14th day. The main organs such as the uterus and brain were collected for pathological change detection. Viremia decreased in all immunized groups, and a significant decrease in viral load was observed in the heart, liver, spleen, kidney and brain of the AKAV-AL immunized group. The brain tissue was evaluated by four indicators: neuronal degeneration, tissue edema, necrosis and hemorrhagic brain tissue. The AKAV-AL group had a lower score, showing a good protective effect. Compared with the PBS group, all immunized groups had a protective effect on the uterus, and no uterine epithelial cell shedding occurred.

[0038] The above results all prove that the immune effect of AKAV inactivated vaccine is better after mixing with AL adjuvant, and it has good application and promotion value.

[0039] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing an inactivated Akabane virus vaccine, characterized in that: The following steps are involved: (1) Vero cells were passaged in a cell flask. Well-grown cells were taken until the cells grew to 80-90%, and DMEM medium containing 10% fetal bovine serum was added. Cells that grew to 80-90% of their normal state were taken out, and Akabane virus CH-JL-01-2022 was added at a volume ratio of 0.1%. The cells were cultured in a 37°C, 5% CO2 incubator for 2-3 days, and the cells were frozen and thawed three times. The cells were centrifuged at 8000 rpm for 20 min, and the supernatant was aspirated and stored at -80°C for later use. (2) 0.25 M BEI, 0.1 M BPL, and 40% formaldehyde solution were prepared as inactivators, and five concentration gradients were set according to the volume ratio of 0.01%, 0.05%, 0.1%, and 0.5%, and the samples were collected at 6 h, 12 h, 24 h, 36 h, and 48 h after addition; (3) The inactivated virus solution is mixed with Montanide ISA 201, Imject I aluminum adjuvant, and manganese adjuvant in a volume ratio of 1:1, and the mixture is quantitatively divided and sealed.

2. The method for preparing an inactivated Akabane virus vaccine according to claim 1, wherein: In step (2), 0.5% formaldehyde showed complete inactivation after 36 hours of inactivation.

3. The method for preparing an inactivated Akabane virus vaccine according to claim 2, wherein: In step (3), Montanide ISA 201 and Imject I aluminum adjuvant need to be emulsified to fully mix the inactivated virus solution and the adjuvant. Complete emulsification is confirmed by microscopic examination and the virus is stored at 4°C for later use.

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