WO2024032360A1 - Lumpy skin disease virus strain, inactivated vaccine prepared from same, and method for preparing vaccine - Google Patents

Abstract

The present invention relates to the technical field of veterinary biological products, and specifically, to a lumpy skin disease virus strain, an inactivated vaccine prepared from the strain, and a method for preparing the vaccine. The lumpy skin disease virus strain LSDV/CH/JY/2021 features high virulence, good immunogenicity, high homology with existing strains, and suitability for producing broad-spectrum inactivated vaccines. The inactivated LSDV vaccine features high safety, high immune potency, and suitability for industrial massive production, and is favorable for the prevention and control of lumpy skin disease.

Description

A bovine nodular skin disease virus strain, an inactivated vaccine prepared therefrom and a vaccine preparation method

Cross-references to related applications

This application claims priority and rights to Chinese Provisional Patent Application No. 202210967365.6 submitted on August 12, 2022, the entire content of which is incorporated herein by reference, and claims priority thereto.

Technical field

The present invention relates to the technical field of veterinary biological products, and specifically relates to a bovine nodular skin disease virus strain, an inactivated vaccine prepared from the strain and a preparation method of the vaccine.

Background technique

Lumpy skin disease (LSD) is a viral infectious disease caused by lumpy skin disease virus (LSDV), also known as lumpy skin disease and lumpy skin disease. dermatitis and bovine nodular rash. Generally speaking, LSDV has a high degree of host specificity and mainly infects cattle and buffaloes. It is often more serious in lactating cows and calves, and will seriously affect the production performance, milk production and physical condition of cattle (damage to skin, cause Abortion and infertility, etc.), causing huge losses to the cattle industry.

Safe and effective LSDV vaccine is an important means to prevent and control bovine nodular skin disease. However, there is currently no commercialized LSDV inactivated vaccine for this disease. Only the Moroccan researcher Hamdi et al. used the Neethling strain to inactivate the vaccine with oily adjuvant. Laboratory development of live vaccines (Jihane Hamdi et al., Development and Evaluation of an Inactivated Lumpy Skin Disease Vaccine for Cattle, Veterinary Microbiology 245(2020)108689), immunized animals began to develop specificity on the 7th day after inoculation of the inactivated vaccine Antibody; The results of the virus challenge protection test showed that the 7 cattle vaccinated with the inactivated vaccine were well protected. However, the use of primary testicular cells to propagate the virus in the production of this inactivated vaccine has shortcomings such as difficulty in amplification, unstable sources of raw materials, large differences between batches, and susceptibility to infection with exogenous factors. The risk of serious side effects is high; and Neethling The strain has been isolated for many years, and using this strain to prepare an inactivated vaccine may not be suitable for the current epidemic prevention situation of bovine nodular skin disease at home and abroad.

Contents of the invention

In response to one or more problems existing in the prior art, one aspect of the present invention provides a bovine nodular skin virus virus strain named LSDV/CH/JY/2021 strain, the LSDV/CH/JY The full genome sequence of the /2021 strain is 150606bp, and its difference from the full genome sequence of the LSDV China/GD01/2020 strain represented by the GenBank accession number MW355944.1 is only at positions 671, 149471, 149478 and 150037bp The bases are W(A/T), G, G and T.

In some embodiments, the bovine nodular skin disease virus strain LSDV/CH/JY/2021 was deposited in the General Microbiology Center of the China Microbial Culture Collection Committee on June 7, 2023, and the deposit number is CGMCC No. 25781.

The application of the above-mentioned bovine nodular skin disease virus strain LSDV/CH/JY/2021 in the preparation of bovine nodular skin disease virus inactivated vaccine also belongs to the content of the present invention.

Another aspect of the present invention provides an inactivated bovine nodular skin disease virus vaccine, wherein the antigen of the inactivated vaccine is from bovine nodular skin disease virus strain LSDV/CH/JY/2021.

On the other hand, the present invention also provides a method for preparing an inactivated vaccine for bovine nodular skin disease virus, which includes: preparing a vaccine containing an inactivated antigen from bovine nodular skin disease virus strain LSDV/CH/JY/2021. The aqueous phase is mixed and emulsified with the vaccine adjuvant.

In some embodiments, the vaccine adjuvant includes 206 adjuvant.

In some embodiments, the mass ratio of the aqueous phase to the vaccine adjuvant is 1:1.

In some embodiments, the mixing and emulsifying operation includes: mixing the aqueous phase and the vaccine adjuvant, and then emulsifying at 30-33°C for 30 minutes.

In some embodiments, the inactivated antigen is obtained by:

S1: Cultivate the bovine nodular skin virus virus strain LSDV/CH/JY/2021 to obtain LSDV virus liquid;

S2: After clarifying the LSDV virus liquid obtained in step S1, it is concentrated using a 500KD-750KD hollow fiber column to obtain a concentrated LSDV antigen solution;

S3: After purifying the LSDV antigen concentrated solution obtained in step S2 with PEG, obtain the LSDV purified antigen solution;

S4: Inactivate the LSDV purified antigen solution obtained in step S3 to obtain the inactivated antigen.

In some embodiments, step S1 uses suspended MDBK cells to suspend and culture the bovine nodular skin virus virus strain LSDV/CH/JY/2021, and culture until the cell viability reaches 40%-60% or 5-6 days, and then harvest the culture The substance is used as LSDV virus liquid.

In some embodiments, when inoculating the bovine nodular skin disease virus strain LSDV/CH/JY/2021, the cell density of the suspended MDBK cells is 0.8-1.5×10 ⁶ cells/ml.

In some embodiments, the bovine nodular skin disease virus strain LSDV/CH/JY/2021 is administered at a concentration of 2%-10% (v/v), preferably 2%-5% (v/v). The toxic ratio was inoculated into the suspended MDBK cells.

In some embodiments, during the cultivation of bovine nodular skin disease virus strain LSDV/CH/JY/2021, the rotation speed of the culture container is controlled at 100rpm-120rpm.

In some embodiments, the temperature controlled for culturing bovine nodular skin disease virus strain LSDV/CH/JY/2021 is 36°C-37°C.

In some embodiments, during the cultivation of bovine nodular skin disease virus strain LSDV/CH/JY/2021, the dissolved oxygen content is 30%-60%, preferably 40%-60%.

In some embodiments, the pH of the cultured bovine nodular skin disease virus strain LSDV/CH/JY/2021 is controlled to be 7.2-7.4.

In some embodiments, in step S2, continuous flow centrifugation or positive pressure filtration with a 0.45 μm filter element is used to clarify the LSDV virus liquid obtained in step S1.

In some embodiments, the concentration ratio in step S2 is 5-10 times.

In some embodiments, the PEG in step S3 is selected from PEG6000 or PEG8000.

In some embodiments, the purification operation described in step S3 includes: mixing the LSDV antigen concentrate obtained in step S2 with PEG at 2-8°C, leaving it overnight, centrifuging at 6000-10000 rpm for more than 15 minutes, and discarding Clear, collect the precipitate, and resuspend it in PBS to obtain the LSDV purified antigen solution.

In some embodiments, the antigen content of the LSDV purified antigen solution is 10 ^6.00 TCID ₅₀ /0.1ml or more, optionally 10 ^6.75 TCID ₅₀ /0.1ml or more, and further optionally 10 ^7.00 TCID ₅₀ /0.1ml or more.

In some embodiments, the inactivation operation in step S4 includes: adding 40% formaldehyde to the LSDV purified antigen solution at a ratio of 0.15% (v/v), and performing inactivation at 33-37°C. 44-50h.

The bovine nodular dermatosis virus LSDV/CH/JY/2021 provided based on the above technical solution is a domestic popular strain. This strain has strong virulence and good immunogenicity. After comparison of the whole genome sequence, this strain is similar to The homology of foreign popular strains is higher than 97%, and the homology with domestic popular strains is about 100%. Therefore, this strain is suitable for the production of inactivated vaccines with wide protection range (broad spectrum) and good immune effect. . The preparation method of the LSDV inactivated vaccine provided by the invention utilizes a bioreactor large-scale suspension culture process to cultivate bovine nodular skin disease virus LSDV/CH/JY/2021, by controlling the parameters of the culture (including cell density during inoculation, inoculation Toxin ratio, culture speed, culture temperature, dissolved oxygen content, pH, etc.), higher titers of bovine nodular skin disease virus antigens can be obtained. After concentration and PEG purification through hollow fiber columns, most impurities can be removed. Protein can be obtained to obtain a purer antigen solution for vaccine preparation, thereby improving the safety of the prepared inactivated vaccine. Moreover, this preparation method has a high degree of automation, which can minimize the impact of human factors, has small batch-to-batch differences, is easy to amplify, and facilitates industrial production. It can also reduce the risk of mixing in exogenous factors, and is more conducive to matching existing epidemic prevention policies. The safety test and efficacy test of the finished vaccine show that the LSDV inactivated vaccine prepared by the present invention will not cause obvious local reactions or systemic adverse reactions due to injection, and can achieve 100% protection in the challenge test. Therefore, the LSDV The inactivated vaccine also has the characteristics of high safety and high immune efficacy, and can be used to prevent and control bovine nodular skin disease.

Description of drawings

Figure 1 shows the homology comparison results between the LSDV/CH/JY/2021 strain and other popular LSDV strains at home and abroad.

Figure 2 shows the phylogenetic tree of the LSDV/CH/JY/2021 strain and other popular LSDV strains at home and abroad.

Figure 3 is a photo of LSDV lesions in suspension culture.

Figure 4 is the relationship curve between TCID ₅₀ and culture time under different cell densities when exposed to the virus.

Figure 5 shows the relationship curve between TCID ₅₀ and culture time under different inoculation amounts and different harvesting times.

Figure 6 shows the relationship between TCID ₅₀ and culture time at different rotational speeds.

Figure 7 shows the relationship between TCID ₅₀ and culture time at different culture temperatures.

Figure 8 shows the relationship between TCID ₅₀ and culture time under different dissolved oxygen concentrations.

Figure 9 shows the relationship between TCID ₅₀ and culture time at different pH.

Detailed ways

The present invention will be further described below with reference to specific examples. It should be understood that the specific embodiments are only used to further illustrate the present invention, but are not used to limit the content of the present invention.

The methods used in the examples are all conventional methods unless otherwise specified. For specific steps, please refer to: "Molecular Cloning: A Laboratory Manual" Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

The ways to obtain various biological materials described in the examples are only to provide a way to obtain experimental materials to achieve the specific purpose of disclosure, and should not be used to limit the sources of biological materials of the present invention. In fact, the sources of biological materials used are wide, and any biological materials that can be obtained without violating laws and ethics can be replaced and used according to the tips in the embodiments.

The examples are implemented on the premise of the technical solution of the present invention, and provide detailed implementation modes and specific operating processes. The examples will help understand the present invention, but should not be used to limit the content of the present invention.

Example 1: Isolation and identification of LSDV virus strains

1.1. Isolation of LSDV virus strains

In this example, an LSDV virus strain (named Lumpy Skin Disease Virus LSDV/CH/JY/2021, hereinafter referred to as LSDV/CH/JY/) was isolated from diseased cattle material obtained from a cattle farm in Guangdong Province, China 2021), the specific separation method includes the following operations:

(1) Isolation of LSDV virus strains: The epidermal tissue of cattle affected by bovine nodular dermatosis was taken, and chicken embryos were used to separate and propagate it to obtain virus fluid containing bovine nodular dermatosis virus.

(2) Purification of virus liquid: This step uses plaque cloning to purify the LSDV virus strain, which specifically includes the following operations:

(2.1) Select a monolayer of MDBK adherent cells, discard the original culture medium, add cell maintenance medium containing 10% of the bovine nodular skin disease virus virus liquid obtained in step (1), and place at 37°C , 5% CO ₂ culture, when the cytopathic effect reaches more than 80%, freeze and thaw twice, harvest the virus liquid, which is the F1 generation virus, pass the F1 generation virus on MDBK adherent cells to F3, and perform domestication culture;

(2.2) Dilute the virus liquid containing the F3 generation bovine nodular skin disease virus 10 times with DMEM to 10 ^-1 to 10 ^-5 , then inoculate it into MDBK cells that have grown into a dense monolayer. After adsorption for 1 hour, discard liquid, add a first layer of nutrient agar about 3 mm thick. The first layer of nutrient agar contains a final concentration of 1% low melting point agarose and DMEM containing 2% fetal calf serum. Place it in a CO ₂ incubator at 37°C. After 7 days of inverted culture under 5% CO ₂ conditions, when the cells appear pathological changes, a second layer of nutrient agar is added. The second layer of nutrient agar contains a final concentration of 1% low melting point agarose and 1% fetal bovine serum. ×DMEM, culture at 37°C, 5% CO2, protected from light and inverted;

(2.3) After the plaque is formed, suck it out with a 10μl pipette tip and put 1.0ml of serum-free DMEM together with the agarose. , repeatedly freeze and thaw three times to fully release the virus, inoculate new blank MDBK cells, and propagate the virus. Repeat cloning three times. Randomly select five plaques of the fourth-generation clone, that is, the F9-generation virus, for culture, and measure the virus. content, select the strain with the highest virus content, and continue passage through MDBK cells to obtain the purified bovine nodular skin disease virus, which is named LSDV/CH/JY/2021 strain. The LSDV/CH/JY/2021 strain is classified as bovine nodular dermatosis virus and has been deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 7, 2023. The address is: Beichen, Chaoyang District, Beijing. No. 3, Yard No. 1, West Road, the preservation number is CGMCC No. 25781.

1.2. Identification of LSDV virus strains

Perform whole-genome sequencing on the LSDV/CH/JY/2021 strain isolated in step 1.1, and obtain its full-genome sequence (composed of 150,606 nucleotides) that is consistent with domestic and foreign LSDV epidemic strains (for example: LSDV China/GD01/ 2020, accession number MW355944.1 (150606bp); LSDV/KM/Taiwan/2020, accession number OL752713.2 (150822bp); LSDV/HongKong/2020, accession number MW732649.1 (150513bp); LSDV20L81_Bang-Thanh/VNM/20 , accession number MZ577076.1 (150644bp); LSDV 20L70_Dinh-To/VNM/20, accession number MZ577075.1 (150600bp); LSDV 20L43_Ly-Quoc/VNM/20, accession number MZ577074.1 (150599bp); Neethling-LSD vaccine -OBP, accession number KX764645.1 (150508bp); Neethling-Herbivac vaccine, accession number KX764644.1 (150529bp)) performed homology comparison and constructed a phylogenetic tree. The results are shown in Figures 1 and 2, where Figure 1 represents the homology comparison result between the LSDV/CH/JY/2021 strain and other popular LSDV strains. Figure 2 represents the phylogenetic tree constructed between the LSDV/CH/JY/2021 strain and other popular LSDV strains. It can be seen that The homology of the LSDV/CH/JY/2021 strain with the popular foreign LSDV strains is higher than 97%, and the homology with the domestic LSDV popular strains is about 100% (of which the LSDV/CH/JY/2021 strain Although the full genome length of the LSDV China/GD01/2020 strain is the same as 150606bp, there are multiple mutation sites in the two. For example, at positions 671, 149471, 149478 and 150037bp, the LSDV/CH/JY/2021 strain has The bases are W(A/T)G, G and T respectively, while the bases of the China/GD01/2020 strain are Y(C/T), B(G/T/C) and K(G/ T), proving that this example has indeed isolated an LSDV strain, and it is different from the whole genome sequence of the current domestic and foreign LSDV circulating strains.

Analysis of the full genome sequence of the LSDV/CH/JY/2021 strain shows that the full genome sequence of LSDV/CH/JY/2021 consists of 150,606 nucleotides, from 336bp to 2527bp (2192bp) at the 5' end and from 5' The 148415bp-150606bp (2192bp) at the 'end are palindromic inverted repeats of each other; starting from the 9bp-371bp at the 5' end is the first coding (CDS) sequence, and the 150120bp-150599bp from the 5' end is the 157th coding (CDS) sequence. CDS) sequence, that is, the nucleotide (DNA) sequence of the coding (CDS) sequence of LSDV/CH/JY/2021 predicted based on the whole genome sequence is shown from 9bp to 150599bp at the 5' end, and is 150591bp long.

Example 2: Preparation of LSDV inactivated vaccine

This example uses the LSDV/CH/JY/2021 strain isolated in the above Example 1 to prepare an inactivated vaccine for bovine nodular dermatosis, which specifically includes the following processes:

2.1. Cell culture

2.1.1) Preparation of MDBK suspension cells: Take 1-2 cells of MDBK working seed cells (provided by Jinyu Baoling Biopharmaceutical Co., Ltd.), revive them, and expand the culture step by step (the culture medium is MDBK Medium A, purchased from Jianshun Biotechnology Co., Ltd. Company), sample and count the cells after 36-72 hours of suspension culture. When the density reaches more than 4×10 ⁶ cells/ml and the viability reaches 90%, transfer to a 5L quadruple parallel reactor.

2.1.2) Culture of MDBK suspension cells in 5L reactor: Transfer the MDBK suspension cells that meet the requirements cultured in step 2.1.1 (density reaches more than 4×10 ⁶ cells/ml) into a sterile transfer bottle, and transfer them to In the reactor, add the same cell culture medium at the same time to the specified volume of the reactor. The cell density is: 0.2-0.5×10 ⁶ cells/ml, the temperature is 35-37°C, the pH value is 7.0-7.2, and the dissolved oxygen value is 30%- 60%, the rotation speed is 80-120rpm, and the cell growth is detected daily, as shown in Table 1 below. It can be expanded to a 50L reactor for culture or used to culture bovine nodular skin disease virus LSDV/CH/JY/2021 according to the actual situation. .

Table 1: Growth of MDBK suspension cells cultured in 5L reactor

2.1.3) Culture of MDBK suspension cells in 50L reactor: Transfer the MDBK suspension cells that meet the requirements cultured in step 2.1.2 (density reaches more than 4×10 ⁶ cells/ml) to the 50L reactor, and add the same cell culture at the same time The liquid reaches the specified volume of the reactor, the cell density is: 0.2-0.5×10 ⁶ cells/ml, the temperature is 35-37°C, the pH value is 7.0-7.2, the dissolved oxygen value is 30%-60%, the rotation speed is 30-60rpm, daily Detect the cell growth, as shown in Table 2 below, and expand to a 500L reactor for culture or use to culture bovine nodular skin disease virus LSDV/CH/JY/2021 according to actual conditions.

Table 2: Growth of MDBK suspension cells cultured in 50L reactor

2.1.4) Culture of MDBK suspension cells in 500L reactor: Transfer the MDBK suspension cells that meet the requirements cultured in step 2.1.3 (density reaches 4×10 ⁶ cells/ml or above) to the 500L reactor, and add the same cell culture at the same time The liquid reaches the specified volume of the reactor, the cell density is: 0.2-0.5×10 ⁶ cells/ml, the temperature is 35-37°C, the pH value is 7.0-7.2, the dissolved oxygen value is 30%-60%, the rotation speed is 30-60rpm, daily Detect the cell growth, as shown in Table 3 below, and expand the culture according to the actual situation or use it to culture bovine nodular skin disease virus LSDV/CH/JY/2021.

Table 3: Growth of MDBK suspension cells cultured in 500L reactor

2.2. Culture of bovine nodular dermatosis virus LSDV/CH/JY/2021

2.2.1) Preparation of LSDV/CH/JY/2021 basic seed virus: Take the MDBK adherent cells that have grown to a good monolayer, discard the original culture medium, and add an appropriate amount of maintenance liquid. The maintenance liquid formula is: DMEM medium addition 0.5%-2% (v/v) newborn calf serum, then add LSDV/CH/JY/2021 virus liquid at a ratio of 2%-5% (v/v), and culture at 37°C. When the cells When the lesions are ≥80%, the virus liquid is harvested and frozen and thawed three times, which is the basic seed virus.

2.2.2) Preparation of suspended seed virus for production: Take the MDBK suspension cells prepared in step 2.1 that meet the requirements, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add virus maintenance solution (purchased from Jianshun Biotechnology Co., Ltd.), and mix Dilute the cells to 0.8-1.5×10 ⁶ cells/ml, then add the basic seed virus prepared in step 2.2.1 according to the toxin ratio of 2%-5% (v/v), and incubate at 37°C and 90-110rpm. Culture, when the cell viability reaches 40%-60%, harvest the virus liquid and freeze and thaw 3 times, take samples to detect TCID ₅₀ , the virus content per 0.1ml should be ≥10 ^6.0 TCID ₅₀ . It is a suspended seed virus for production and is stored at minus 20°C or below. According to the different amounts of venom required for production, reactors with different specifications and volumes are used to cultivate LSDV/CH/JY/2021 suspended seed poison. Figure 3 shows photos of lesions of LSDV/CH/JY/2021 cultured in suspension.

2.2.3) Preparation of antigen: Take the MDBK suspension cells prepared in step 2.1 that meet the requirements, discard the original culture medium, add virus maintenance medium (purchased from Jianshun Biotechnology Co., Ltd.), and dilute the cells to 0.8-1.5× 10 ⁶ cells/ml, and then add the suspended seed virus for production prepared in step 2.2.2 at a poisoning ratio of 2%-5% (v/v). Adjust the parameters: temperature 37°C, rotation speed 80-100rpm, pH 7.4 , DO value 30%-60%, when the cell viability reaches 40%-60%, harvest the virus liquid and freeze and thaw 3 times. The same method is used for the 3rd generation. Samples are taken to detect TCID _50. The virus content per 0.1ml should be ≥10 ^6.0 TCID ₅₀ . It is the antigen solution for vaccine preparation and is stored at minus 20°C or below.

In this step, the effects of cell density, virus exposure ratio, rotation speed, culture temperature, dissolved oxygen and pH on virus proliferation were also optimized to determine the optimal cell density, virus exposure ratio, Rotation speed, culture temperature, dissolved oxygen and pH.

(1) Effect of cell density on virus proliferation during exposure to virus

Prepare the virus maintenance solution according to the cell density so that the cell densities transferred to the virus culture reactor are 0.5×10 ⁶ cells/ml, 0.8×10 ⁶ cells/ml, 1.0× ^{10 6} cells/ml and 1.5×10 ⁶ cells. /ml. Inoculate the production suspension seed virus prepared in step 2.2.2 at an inoculation ratio of 2% (v/v). Set the reactor virus culture parameters as follows: temperature 37°C, pH value 7.2, dissolved oxygen value 45%, and rotation speed 100 rpm. Cultivate for 5-6 days, harvest the virus liquid, and measure TCID _50. The results are shown in Table 4 and Figure 4 below. It can be seen that the optimal cell density for inoculation is 0.8-1.5×10 ⁶ cells/ml. After more than 120 hours of inoculation, the obtained The titer of virus fluid can be It reaches more than 10 ^6.00 TCID ₅₀ /0.1ml, and even reaches 10 ^7.00 TCID ₅₀ /0.1ml.

Table 4: Determination results of virus content at different cell densities when exposed to virus

(2) Effects of different virus inoculation ratios on virus proliferation: After diluting the cells according to the optimal cell density for inoculating the virus, stir and raise the temperature to 37°C. The production suspension seed virus prepared in step 2.2.2 is inoculated with the virus proportion. Set the reactor virus culture parameters as follows: temperature 37°C, pH value 7.2, dissolved oxygen value 45%, and rotation speed 100 rpm. After culturing for 5-6 days, harvest the virus liquid and measure TCID _50. The results are shown in Table 5 and Figure 5 below. It can be seen that the suitable harvest time for LSDV is 5-6 days after culturing (120-144h), and the virus content can reach 10 ^6.00 TCID ₅₀ /0.1ml and above, the cell viability rate is 40-60%; the dose of 2%-10% can reach 10 ^6.00 TCID ₅₀ /0.1ml or above. In order to minimize the amount of poison exposure, it is recommended that 2%-5% is the recommended dose range.

Table 5: Determination results of virus content at different virus exposure ratios and different harvest times

(3) Optimization of rotation speed: After diluting the cells according to the optimal cell density for inoculation, inoculate the suspended seed virus for production prepared in step 2.2.2 according to the recommended inoculation ratio (5%). The virus culture parameters of the reactor were set as follows: temperature 37°C, pH value 7.2, dissolved oxygen value 45%, and the rotation speeds were set to 60rpm, 80rpm, 100rpm, and 120rpm respectively. Cultivate for 5-6 days respectively, harvest the virus liquid, and measure TCID _50. The results are shown in Table 6 and Figure 6 below. It can be seen that the culture effect is best when the rotation speed is 100-120 rpm. After 120 hours of culture, the virus content can reach 10 ^6.00 TCID ₅₀ / 0.1ml and above.

Table 6: Virus content test results at different speeds

(4) Optimization of temperature: After diluting the cells according to the optimal cell density for inoculation, inoculate the suspended seed virus for production prepared in step 2.2.2 according to the recommended inoculation ratio (5%). The virus culture parameters of the reactor were set as follows: the temperature was set to 35°C, 36°C, 37°C, and 38°C respectively, the pH value was 7.2, the dissolved oxygen value was 45%, and the rotation speed was 100 rpm. Cultivate for 5-6 days respectively, harvest the virus liquid, and measure TCID _50. The results are shown in Table 7 and Figure 7 below. It can be seen that the virus content is relatively low at 35°C and has a downward trend at 38°C. Under the conditions of 36°C-37°C The virus content is relatively high, and the virus titer can reach 10 ^6.00 TCID ₅₀ /0.1ml and above after 120 hours of culture.

Table 7: Test results of virus content at different culture temperatures

(5) Optimization of dissolved oxygen: After diluting the cells according to the optimal cell density for inoculation, inoculate the suspended seed virus for production prepared in step 2.2.2 according to the recommended inoculation ratio (5%). The virus culture parameters of the reactor were set as follows: temperature 37°C, pH value 7.2, dissolved oxygen content set to 30%, 40%, 50%, and 60% respectively, and rotation speed 100 rpm. Cultivate for 5-6 days respectively, harvest the virus liquid, and measure TCID _50. The results are shown in Table 8 and Figure 8 below. It can be seen that under the conditions of 30%-60% dissolved oxygen, the virus titer can reach 10 ^6.00 TCID after 120 hours of culture. ₅₀ /0.1ml and above, 40%-60% dissolved oxygen is more effective.

Table 8: Test results of virus content at different dissolved oxygen concentrations

(6) Optimization of pH: After diluting the cells according to the optimal cell density for inoculation, inoculate the suspended seed virus for production prepared in step 2.2.2 according to the recommended inoculation ratio (5%). Set the reactor virus culture parameters as follows: temperature 37°C, pH 7.0, 7.2, 7.4, and 7.6, dissolved oxygen value 45%, and rotation speed 100 rpm. Cultivate for 5-6 days respectively, harvest the virus liquid, and measure TCID _50. The results are shown in Table 9 and Figure 9 below. It can be seen that the virus content is relatively low under pH conditions of 7.0 and 7.6. Therefore, it is recommended to control the pH at 7.2-7.4 and culture for 120 hours. The virus titer can reach 10 ^6.00 TCID ₅₀ /0.1ml and above.

Table 9: Virus content test results at different pH

2.3. Antigen concentration and purification

2.3.1) Antigen concentration: Take the antigen solution for vaccine preparation prepared in step 2.2. After thawing, centrifuge in a continuous flow centrifuge to obtain a clear supernatant. Use a 500KD or 750KD hollow fiber column to concentrate the antigen solution for 5- 10 times (during the concentration process, PBS needs to be used for diafiltration to replace the virus maintenance solution) to obtain the antigen concentrate. Table 10 below shows the concentration of the antigen solution for vaccine preparation through 500KD and 750KD hollow fiber columns for three times (NS001-NS003). It can be seen that after the antigen solution for vaccine preparation is concentrated by 500KD or 750KD hollow fiber columns, the virus antigen is basically no longer present. Loss, especially the concentration effect of 500KD hollow fiber column is better.

Table 10: Concentration of antigen solution for vaccine preparation by 500KD and 750KD hollow fiber columns

2.3.2) Antigen purification: Take the antigen concentrate prepared in step 2.3.1, add appropriate concentration of PEG6000, mix at 2-8°C (not less than 2h), let stand overnight, and centrifuge at 6000-10000rpm. After 15 minutes or more, discard the supernatant, collect the precipitate, and resuspend in PBS (pH 7.4, concentration 0.06mol/L) to obtain the purified antigen solution for vaccine preparation. The antigen loss and impurity protein removal after PEG purification of the antigen concentrate is shown in Table 11 below. It can be seen that after the antigen concentrate is purified by PEG, the impurity protein removal rate can reach more than 85%, which can effectively remove impurity proteins in the antigen concentrate. Conducive to the safety of vaccine use.

Table 11: Antigen loss and impurity protein removal after PEG purification of antigen concentrate

2.4. Antigen inactivation

2.4.1) Antigen inactivation: Take the purified vaccine antigen solution prepared in step 2.3.2 and perform the inactivation process. Add 40% formaldehyde to the purified antigen solution for vaccine preparation at a ratio of 0.15% (v/v), and inactivate it at 33-37°C for 44-50 hours (stir evenly every 120 minutes) to obtain the inactivated antigen solution .

2.4.2) Inactivation test: Take the inactivated antigen solution in 2.4.1 and inoculate 3 MDBK good monolayer cells at a ratio of 25% (v/v) (add 2.5ml to each T25 cell bottle), and adsorb at 37°C for 1 hour , then discard the inactivated antigen solution, add 10ml/bottle of cell maintenance solution, and set two controls in the same way, one of which is the normal cell group, and the other bottle is the maintenance solution containing formaldehyde (formaldehyde content and inactivated antigen liquid test sample), place it in a 37°C, 5% CO ₂ incubator and culture it for 2-6 days. Observe daily whether CPE occurs and record the observation results. Harvest the culture after 2-6 days of culture and freeze and thaw the harvested culture. Afterwards, MDBK cells were inoculated at 25% (v/v) and passed blindly for two generations. The occurrence of CPE was observed daily and the observation results were recorded.

2.5. Vaccine preparation

2.5.1) Water phase preparation: Take the qualified inactivated antigen solution prepared in step 2.4.1, centrifuge at 6000rpm-8000rpm for more than 15 minutes, and take the supernatant as the water phase.

2.5.2) Oil phase preparation: Take the qualified 206 adjuvant, sterilize it by high pressure, and then cool it to 30°C to serve as the oil phase.

2.5.3) Emulsification: After the water phase and oil phase are mixed according to the mass ratio of 1:1 (add the water phase to the oil phase), emulsify at 30-33°C for 30 minutes. Pack the emulsified vaccine according to the specified amount and label it. After labeling, store at 2-8°C for later use.

2.6. Inspection of finished seedlings

2.6.1)Characteristics

(1)Appearance: Milky white or light pink viscous emulsion.

(2)Dosage form: water-in-oil-in-water (W/O/W). Take a clean straw, drop a small amount of vaccine on the surface of clean cold water, and spread it like a cloud.

(3) Stability: Add 10.0ml of vaccine into a centrifuge tube, and centrifuge at 3000r/min for 15 minutes. The water precipitated at the bottom of the tube should not exceed 0.5ml.

(4) Viscosity: tested according to the appendix of the current "Chinese Veterinary Pharmacopoeia", it should not exceed 200cP.

2.6.2) Loading quantity inspection: Inspection shall be carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia" and shall comply with regulations.

2.6.3) Sterility test: Test according to the appendix of the current "Chinese Veterinary Pharmacopoeia" and it should grow sterilely.

2.6.4) Endotoxin content detection: Take 12ml of vaccine and put it into a 15ml centrifuge tube, place it in a water bath at 50±5°C for 90 minutes, then centrifuge at 15000g for 10 minutes at 4°C, take 5.0ml of the aqueous phase, and follow the current "China An appendix to the Veterinary Pharmacopoeia Carry out inspection. The endotoxin content of each vaccine dose should not exceed 20EU.

2.6.5) Formaldehyde residue detection: According to the appendix of the current "Chinese Veterinary Pharmacopoeia", the amount should not exceed 0.2% formaldehyde solution (40% formaldehyde). The properties inspection results of the three batches of finished vaccines (ND001-ND003) are shown in Table 12 below. It can be seen that the properties of the vaccines all meet the requirements.

Table 12: Characteristics inspection of finished vaccines

2.6.6) Safety inspection

(1) Test with small animals: Use 5 guinea pigs weighing 350-400g, and subcutaneously inject 0.5ml of vaccine each; use 10 mice weighing 18-22g, and inject 0.2ml subcutaneously each vaccine. After 7 days of daily observation, there should be no death or obvious local reaction or systemic adverse reaction caused by the injection of the vaccine.

(2) This animal test: Use 5 healthy susceptible cattle between 2 and 6 months old. Inject 4.0ml (2 doses) of vaccine into each head and neck muscle. Observe daily for 14 days. There should be no LSD symptoms or symptoms due to injection. Significant local reactions or systemic adverse reactions caused by vaccines.

The safety test results of the finished vaccine are shown in Table 13 below. It can be seen that no matter whether it is tested with small animals or with this animal, there is no death or obvious local reaction or systemic adverse reaction caused by the injected vaccine, indicating that the LSDV prepared by the present invention Inactivated vaccines are safe.

Table 13: Safety inspection status of finished vaccines

2.6.7) Effectiveness test

Cattle immune challenge method: 10 healthy susceptible cattle aged 2-6 months were randomly divided into 2 groups, including 5 in the immune group and 5 in the challenge control group. Cattle in the immune group were injected with 2.0ml of vaccine intramuscularly into each head and neck (1 portion). On the 21st day after vaccination, they were boosted with the same method once. On the 21st day after the second vaccination, LSDV strain (virus content was 10 ^3.5 TCID ₅₀ /ml) was administered. To challenge the virus, inoculate 2.0ml into each head and inject 0.5ml into 4 points intradermally on the neck. After continuous observation for 21 days after challenge, at least 4 cows in the control group should become sick, and at least 4 cows in the immune group should be protected. The efficacy test results are shown in Table 14 below. It can be seen that the LSDV prepared by the present invention is inactivated. Vaccines provide 100% protection.

Table 14: Efficacy testing of finished vaccines

The embodiments described here are for illustration only (as examples), and various modifications or changes made by skilled persons should also be included within the essential scope of the patent application.

Industrial applicability

The invention provides a bovine nodular skin virus virus strain, an inactivated vaccine prepared from the strain, and a preparation method of the vaccine. The inactivated vaccine provided has high safety, strong immune efficacy, and is suitable for industrial application.

Claims (14)

1. A bovine nodular skin disease virus strain is named LSDV/CH/JY/2021 strain. The full genome sequence of the LSDV/CH/JY/2021 strain is 150606bp, and its GenBank accession number is The only difference in the full genome sequence of the LSDV China/GD01/2020 strain represented by MW355944.1 is that the bases at positions 671, 149471, 149478 and 150037bp are W(A/T), G, G and T respectively.
2. The bovine nodular skin disease virus strain according to claim 1 was deposited in the General Microbiology Center of the China Microbial Culture Collection Committee on June 7, 2023, and the preservation number is CGMCC No. 25781.
3. Application of the bovine nodular skin disease virus strain LSDV/CH/JY/2021 described in claim 1 or 2 in the preparation of bovine nodular skin disease virus inactivated vaccine.
4. An inactivated bovine nodular skin disease virus vaccine, wherein the antigen of the inactivated vaccine comes from the bovine nodular skin disease virus strain LSDV/CH/JY/2021 described in claim 1 or 2.
5. A method for preparing an inactivated bovine nodular skin disease virus vaccine, which includes: containing an inactivated antigen from the bovine nodular skin disease virus strain LSDV/CH/JY/2021 described in claim 1 or 2 The aqueous phase is mixed and emulsified with the vaccine adjuvant.
6. The preparation method according to claim 5, wherein the vaccine adjuvant includes 206 adjuvant; and/or

   The mass ratio of the aqueous phase to the vaccine adjuvant is 1:1; and/or

   The mixing and emulsifying operation includes: mixing the aqueous phase and the vaccine adjuvant, and then emulsifying at 30-33°C for 30 minutes.
7. The preparation method according to claim 5 or 6, wherein the inactivated antigen is obtained by:

   S1: Cultivate the bovine nodular skin virus virus strain LSDV/CH/JY/2021 to obtain LSDV virus liquid;

   S2: After clarifying the LSDV virus liquid obtained in step S1, it is concentrated using a 500KD-750KD hollow fiber column to obtain a concentrated LSDV antigen solution;

   S3: After purifying the LSDV antigen concentrated solution obtained in step S2 with PEG, obtain the LSDV purified antigen solution;

   S4: Inactivate the LSDV purified antigen solution obtained in step S3 to obtain the inactivated antigen.
8. According to the preparation method of claim 7, step S1 adopts suspended MDBK cells to suspend and culture bovine nodular skin disease virus strain LSDV/CH/JY/2021, and culture until the cell viability reaches 40%-60% or 5-6 day, harvest the culture as LSDV virus liquid.
9. According to the preparation method of claim 8, when inoculating the bovine nodular skin disease virus strain LSDV/CH/JY/2021, the cell density of the suspended MDBK cells is 0.8-1.5×10 ⁶ cells/ml ;and / or

   The bovine nodular skin disease virus strain LSDV/CH/JY/2021 is inoculated into the described bovine nodular skin disease virus strain LSDV/CH/JY/2021 according to a virus exposure ratio of 2%-10% (v/v), preferably 2%-5% (v/v). suspended in MDBK cells; and/or

   During the cultivation of bovine nodular skin disease virus strain LSDV/CH/JY/2021, the rotation speed of the culture container is controlled at 100rpm-120rpm; and/or

   The temperature control for culturing bovine nodular skin disease virus strain LSDV/CH/JY/2021 is 36℃-37℃; and/or

   During the cultivation of bovine nodular skin disease virus strain LSDV/CH/JY/2021, the dissolved oxygen content is 30%-60%, preferably 40% to 60%; and/or

   The pH control for culturing bovine nodular skin disease virus strain LSDV/CH/JY/2021 is 7.2-7.4.
10. According to the preparation method according to any one of claims 7 to 9, in step S2, continuous flow centrifugation or positive pressure filtration with a 0.45 μm filter element is used to clarify the LSDV virus liquid obtained in step S1.
11. According to the preparation method according to any one of claims 7-10, the concentration ratio in step S2 is 5-10 times.
12. According to the preparation method according to any one of claims 7-11, the antigen content of the LSDV purified antigen liquid in step S3 is 10 ^6.00 TCID ₅₀ /0.1ml or more, optionally 10 ^6.75 TCID ₅₀ /0.1ml or more, Further options are 10 ^7.00 TCID ₅₀ /0.1ml and above.
13. According to the preparation method according to any one of claims 7-12, the PEG in step S3 is selected from PEG6000 or PEG8000; and/or

    The purification operation described in step S3 includes: mixing the LSDV antigen concentrate obtained in step S2 with PEG at 2-8°C, leaving it overnight, centrifuging at 6000-10000 rpm for more than 15 minutes, discarding the supernatant, collecting the precipitate, and Resuspend in PBS to obtain the LSDV purified antigen solution.
14. According to the preparation method according to any one of claims 7-13, the inactivation operation in step S4 includes: adding 40% formaldehyde to the LSDV purified antigen solution at a ratio of 0.15% (v/v) , inactivate at 33-37℃ for 44-50h.