CN113249339B - H3 subtype canine influenza virus strain, inactivated vaccine and preparation method thereof - Google Patents

Abstract

The invention discloses an H3 subtype canine influenza virus strain, an inactivated vaccine and a preparation method thereof. The vaccine is prepared by inactivating the H3 subtype canine influenza virus strain with the deposit number CGMCC NO.20649 as an antigen. The present invention uses SPF chicken embryos or MDCK cells as a carrier for virus propagation, collects virus liquid, inactivates, purifies, and prepares a vaccine. The H3 subtype canine influenza virus inactivated vaccine prepared by the invention has high virus content, good stability and high safety, is an ideal H3 subtype canine influenza virus inactivated vaccine, and has important public health significance.

Description

A kind of H3 subtype canine influenza virus strain, inactivated vaccine and preparation method thereof

technical field

The invention belongs to the field of biotechnology, and relates to a virus strain, a vaccine product thereof and a preparation method thereof, and more particularly to an H3 subtype canine influenza virus strain, an inactivated vaccine thereof and a preparation method thereof.

Background technique

Canine influenza (CI) is a contagious respiratory infection of canine animals caused by Canine influenza virus (CIV) belonging to the genus Influenza A of the family Orthomyxoviridae. CIV is mainly divided into H3N8 and H3N2 subtypes. The H3N8 subtype is a cross-species transmission of equine influenza virus in racing dogs, and the clinical symptoms of infected pet dogs are mild or no obvious clinical symptoms are seen. After H3N2 subtype CIV infection in dogs, the dogs showed obvious clinical symptoms such as fever, depression, cough and runny nose, and some dogs died if they were not treated in time, or were prone to respiratory diseases after being cured.

The H3N2 subtype CIV is mainly transmitted by aerosols and is the main prevalent subtype in my country.

In 2004, the first outbreak of CI in racing dogs in Florida, USA, was found to be H3N8 subtype and originated from Equine influenza virus (EIV). In 2007, an outbreak of canine influenza in South Korea led to the isolation of H3N2 subtype CIV. This subtype was first transferred from poultry to Chinese dogs around 2005, and was easily transmitted between dogs. From 2009 to 2010, avian-derived H3N2 subtype CIV was also isolated in Zhejiang, Jiangsu, Beijing, Liaoning and other places, and it was highly homologous to H3N2 subtype CIV in South China and South Korea. As of 2015, the H3N2 subtype is widely prevalent in Asian regions such as China and South Korea. Epidemiological trends indicate that canine influenza H3N2 subtype has become an endemic zoonotic disease in Asia and the United States including China. In addition, H3N2 subtype CIV has only appeared for more than ten years, and there have been many reassortment events with human influenza virus. In 2012 and 2014, South Korea reported that novel CIV strains isolated from dogs were reassorted with H3N2 subtype CIV and H1N1 subtype human influenza virus.

The research group of Li Shoujun from South China Agricultural University is the most in-depth research on this disease in China. They isolated the H3N2 subtype canine influenza virus in 2010, and then sequenced the whole genome, prokaryotic expression of NS1 gene, diagnosis (HA1 protein indirect ELISA, HI- HA, RT-LAMP), inactivated vaccines, genetically engineered vaccines, pathogenicity (pathogenicity of PB2 gene locus mutation in mice), etc. have been studied in depth. At present, the most effective strategy for preventing CIV infection is vaccination. There are vaccines against this disease on the market in countries with high incidence of canine influenza such as the United States and South Korea. In view of this, the development of a new high-efficiency canine influenza vaccine has important public health significance.

Because the vaccine evaluation model has not yet been established, it restricts the development of the current vaccine. Therefore, the development of a safe and effective canine influenza vaccine to control the prevalence of canine influenza is of great significance for ensuring public health security.

SUMMARY OF THE INVENTION

One of the objects of the present invention is to provide a kind of H3 subtype canine influenza virus inactivated vaccine;

The second purpose of the present invention is to provide a preparation method of the H3 subtype canine influenza virus inactivated vaccine.

The virus strain of the present invention is named H3 subtype canine influenza virus HB strain, and is preserved in the General Microbiology Center of the China Microorganism Culture Collection and Management Committee with the preservation number of CGMCC.NO.20649; the virus belongs to the Orthomyxoviridae family (Orthomyxoviridea). ) Influenza A virus (Influenza A virus) is a single-stranded negative-stranded RNA virus with an envelope; the resistance and thermal stability of the virus to the environment are relatively poor, and in the environment of 56 ° C for 30 minutes, 65 It can be inactivated in a few minutes at ℃～75℃, can only be stored for a short time at 4℃, and can be kept active for several months in a freezer at -20℃, but the ability of the virus to agglutinate red blood cells may disappear; the virus Usually, it can be inoculated into the allantoic cavity of 9-11-day-old chick embryos and harvested after 48 hours of culture; or cultured in MDCK cells.

An H3 subtype canine influenza virus inactivated vaccine, the vaccine is inoculated with SPF chicken embryos or MDCK cells by the H3 subtype canine influenza virus, the chicken embryo allantoic fluid or cell culture supernatant is harvested, inactivated, and an adjuvant is added And get.

The preparation method of the vaccine comprises the following steps:

(1) Inoculation: H3 subtype canine influenza virus was inoculated into 10-11 day-old SPF chicken embryos via the allantoic route at a dose of 2×10 ⁴ EID ₅₀ /piece, incubated at 35°C; The virus content of the harvested chick embryo allantoic fluid is high and stable, and the inoculation dose is one of the important conditions of the present invention.

(2) Incubation and observation: within 48 hours after inoculation, the eggs were irradiated once a day, and the dead embryos within 48 hours were discarded. The selection of the inoculation time of 48h is also one of the important conditions determined by experiments in the present invention, and the virus content of the chicken embryo allantoic fluid harvested at this time is high and stable.

(3) Harvest: take out the cooled chicken embryos, sterilize the eggshells in the air chamber, aseptically strip off the eggshells in the air chamber, absorb the allantoic fluid, centrifuge to get the supernatant and store it at 2-8°C to be the virus stock solution; The virus content was determined according to the half-infectious dose determination method specified in Appendix 7 of the 2015 edition of the Veterinary Pharmacopoeia of the People's Republic of China, and the proliferating virus titer was ≥10 ^7.5 EID ₅₀ /0.1mL.

(4) Concentrating and washing the virus stock solution: the virus stock solution harvested in step (3) is concentrated to 1/5 of the original volume with an ultrafiltration concentration device (100KD～300KD ultrafiltration membrane package) (this concentration level ensures enough virus content to reach the hemagglutination titer, that is, the standard of HA 1:512); then wash with an equal volume of PBS solution with a pH value of 7.0 to 7.2 (to wash away impurities and improve antigen purity), and then concentrate (with ultrafiltration). The equipment is concentrated by 1 times), and this is repeated 1 to 3 times to obtain a concentrated virus solution with a protein content not higher than 3 mg/mL; the concentration of the antigen is increased by concentration and washing, and the antigen is purified.

(5) Virus inactivation: To the concentrated virus solution obtained in step (4), add the inactivator formaldehyde containing 0.1% of the total amount of concentrated virus solution (V/V), and shake and inactivate at 4° C. for 7 days.

(6) Column purification: the inactivated concentrated virus solution obtained in step (5) is purified by Capto Core 700 column (flow rate 1 mL/min) to obtain a purified antigen whose hemagglutination titer is not less than 1:512 , the final protein content is not higher than 400μg/mL (this parameter is a key parameter), freeze-dried storage; column purification can remove impurities and reduce formaldehyde, reducing side reactions to dogs.

(7) Vaccine preparation: before use, the lyophilized antigen obtained in step (6) is diluted with physiological saline, mixed with Montanide ^TM GEL 02 adjuvant in an appropriate proportion (the amount of GEL02 added is 8%), stirred and mixed to obtain a vaccine .

The evaluation method of the vaccine includes the following steps:

(1) Immunization: 5 beagle dogs aged 4-6 months were taken, and 0.5 mL of the vaccine was subcutaneously injected into the neck of each dog. After 28 days of immunization, the same dose was used for secondary immunization, and 5 control groups were set at the same time.

(2) Challenge: 14 to 28 days after the secondary immunization, blood was collected, serum was separated, and hemagglutination inhibitory (HI) antibodies were determined. All dogs were inoculated with H3 subtype canine influenza virus (about 1×10 ^7.0 EID) by intranasal spray. ₅₀ ).

(3) Virus isolation: 5 days after challenge, the nasal swabs of all dogs were collected, and the stock solution, 10- and 100-fold diluted virus solutions were grouped into the allantoic cavity of chicken embryos, and 5 9-11-day-old chickens were inoculated in each group. Embryos were inoculated with 0.2 mL per embryo and cultured at 35°C for 96 hours to determine the HA titer; the immunization group should be significantly lower than the control group.

(4) Necropsy: 7 to 14 days later, the lung consolidation was observed by autopsy. 80% of the non-immune controls had more than 10% consolidation in the necropsy, which was considered effective for the challenge, and 80% of the immunized groups had no obvious consolidation in the necropsy, and they were considered vaccines. Protect.

Canine influenza H3 subtype HI antibody detection method is as follows:

(1) The canine influenza H3 subtype HI antigen was serially diluted with PBS, and 0.5% chicken erythrocyte suspension was added to observe the agglutination reaction.

(2) According to the agglutination test results, 4 to 8 working units of antigen are prepared with PBS.

(3) After the serum to be tested is treated with 10% kaolin and chicken red blood cell suspension, it is regarded as the serum with a dilution of 1:10.

(4) After serially doubling dilution with the serum treated in (3), add 4 to 8 working units of HI antigen for 30 minutes at room temperature, then add 0.5% chicken erythrocyte suspension, mix gently and observe Agglutination inhibition reaction, the test set negative and positive serum controls.

Beneficial effects of the present invention:

1) The vaccine of the present invention has a good protective effect on the lungs of immunized dogs, and the data on lung consolidation shows that the lung morphology of immunized dogs can be protected after injection of the vaccine.

2) The immunization of the vaccine of the present invention is rapid, and the immunized dog can produce an immune response within 2 weeks, and a high level of HI antibody against canine influenza virus H3 subtype can be produced in one month.

3) The vaccine of the present invention is prepared by using a novel polymer adjuvant as a water adjuvant, which is easy to disperse and has high stability, and is an ideal canine influenza virus H3 subtype inactivated vaccine.

4) The vaccine of the present invention has good safety. The dogs were immunized with twice the dose and observed for 2 weeks. No abnormality was found in the mental state, feed intake and the like.

Description of drawings

In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

Figure 1 shows the results of HA gene PCR identification.

1 is the HA PCR product, 2 is the negative control, and M is the DL2000 Marker.

Figure 2 shows the results of NA gene PCR identification.

1 is the negative control, 2 is the NA PCR product, and M is the DL2000 Marker.

Figure 3 shows the changes in protein content during vaccine preparation.

Harvest represents the protein content of the harvest process, concentrated wash represents the protein content of the concentration and 3 washing processes, and purified represents the protein content after column purification.

Detailed ways

The present invention will be further illustrated by the description of specific embodiments below, but it is not intended to limit the present invention.

Example 1

Virus isolation and identification:

(1) Virus isolation: In 2019, nasopharyngeal swabs were collected from dogs with respiratory symptoms in a pet hospital in Yanqing, Beijing. After aseptic processing, the collected nasopharyngeal swabs were inoculated into the allantoic cavity of 10-day-old SPF chicken embryos. 0.2mL, incubate at 37°C, discard dead embryos within 24 hours, collect allantoic fluid of chicken embryos after 72 hours, measure the hemagglutination value, and store at -70°C for future use.

(2) Virus typing: Extract viral RNA from the isolated canine influenza virus HB strain, amplify HA and NA fragments by RT-PCR, and perform sequence analysis. The results showed that the isolated strain was canine influenza virus H3N2 subtype strain, which was named A/Canine/Huabei/2019 (H3N2). The results are shown in Figures 1 and 2. The virus strain was deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee on April 23, 2021, and the deposit number is CGMCC.NO.20649.

(3) Virus specificity: The chicken embryo allantoic fluid with hemagglutination activity was compared with canine influenza virus H3 subtype, swine influenza virus H1 subtype, H3 subtype, avian influenza virus H5 subtype, H7 subtype, H9 subtype positive serum was tested for HI. The results are shown in Table 1. The results show that the isolated strains are all negative for swine influenza virus H1 subtype, H3 subtype, avian influenza virus H5 subtype, H7 subtype, H9 subtype positive serum HI test (HI antibody titer is less than 1:10), the positive serum HI test for H3 subtype canine influenza virus was positive (HI antibody titer was 1:640).

Table 1 Virus specificity test results

Example 2

Culture conditions and inactivation conditions of virus seeds

(1) Selection of the optimum inoculation dose, temperature and time: 10-day-old chicken embryos were randomly divided into 15 pieces/group, and the virus contents were 10 ^3.0 EID ₅₀ /0.1mL and 10 ^4.0 EID ₅₀ /0.1 respectively through the allantoic cavity. mL, 10 ^5.0 EID ₅₀ /0.1mL production toxicity, 0.2mL/embryo, cultured at 37°C and 35°C, harvested at 48 hours, 64 hours, and 72 hours, respectively, and determined the urine of different groups of chicken embryos The HA titer, harvest amount and virus content of the cyst fluid were determined as the inoculation dose of 10 ⁴ EID ₅₀ /0.1mL, the optimum culture temperature of 35°C, and the optimum harvest time of 48 hours.

(2) Optimal inactivation time: add formaldehyde solution at a final concentration of 0.1%, mix well and inactivate at a constant temperature of 4 °C, and inoculate samples on 10-day-old SPF chicken embryos 1 to 7 days after inactivation. The HA titer of chick embryo allantoic fluid was measured embryo by embryo after incubation at 37°C for 120 hours. Blind passage was required for one generation, and if it was still negative, the inactivation was complete, and the optimal inactivation time was determined to be 7 sky.

Example 3

Preparation of Inactivated Vaccine of Canine Influenza Virus H3 Subtype

(1) Inoculation: H3 subtype canine influenza virus was inoculated into 10-day-old SPF chicken embryos via the allantoic route at a dose of 2×10 ⁴ EID ₅₀ /piece, and incubated at 35°C;

(2) Incubation and observation: within 48 hours after inoculation, the eggs were irradiated once a day, the dead embryos within 48 hours were discarded, and the live embryos 48 hours after inoculation were taken out and cooled at 2-8°C overnight for 6-18 hours;

(3) Harvest: take out the cooled chicken embryos, sterilize the eggshells in the air chamber, aseptically strip off the eggshells in the air chamber, absorb the allantoic fluid, centrifuge to get the supernatant and store it at 2-8°C to be the virus stock solution; The virus content was determined in accordance with the half-infectious dose determination method specified in Appendix 7 of the 2015 edition of the "Veterinary Pharmacopoeia of the People's Republic of China"; (Figure 3 harvest)

(4) Concentrating and washing the virus stock solution: the virus stock solution harvested in step (3) is double filtered with a suitable pore size filter membrane, concentrated to 1/5 of the original volume with an ultrafiltration concentration device, and then used an equivalent pH value of 7.0～ The PBS solution of 7.2 was washed, and then concentrated, repeated 3 times to obtain a concentrated virus solution; (Figure 3 concentrated and washed)

(5) Inactivation of virus: to the concentrated virus solution obtained in step (4), add inactivator formaldehyde containing 0.1% of the total amount of concentrated virus solution (V/V), and shake and inactivate at 4° C. for 7 days; after the inactivation is completed , take samples for inactivation test, and store the inactivated virus liquid at 2-8°C for no more than 12 days;

(6) Column purification: filter the inactivated concentrated virus solution obtained in step (5) through a 0.45 μm filter membrane; load the CaptoCore 700 into the AKTA instrument, equilibrate the Core 700 with 5 column volumes of PBS, and the virus solution at 1 mL/min Load the sample, collect the flow-through; combine the samples with high flow-through titer (1:512 and 1:1024), filter and sterilize through a 0.22 μm filter; measure the protein content by BCA method. (Figure 3 after column purification)

(7) Vaccine preparation: First, stir the antigen obtained in step (6), mix with Montanide GEL02 adjuvant in an appropriate proportion (GEL02 addition amount is 8%), and continue stirring at 200 r/min to fully mix to obtain a vaccine.

(8) Sterility test: The test shall be carried out according to the sterility test method stipulated in the appendix of the 2015 edition of the Veterinary Pharmacopoeia of the People's Republic of China, and there should be no bacteria and mold growth.

Example 4

Immune efficacy of canine influenza virus H3 subtype inactivated vaccine

(1) Prepare canine influenza virus H3 subtype inactivated vaccine according to the method of Example 3. The inactivation test and sterility test of the inactivated vaccine were all qualified. The virus content, HA titer and total protein content of the inactivated vaccine are shown in Table 2 below, which meet the qualified standard. Among them, the determination of total protein content is shown in Figure 3. The results show that the concentration washing and inactivating washing process greatly reduce the content of impurity proteins and further improve the purity of the antigen.

Table 2 Virus content, HA titer and total protein content of semi-finished products (not yet emulsified with vaccine)

(2) Safety inspection: 2 Beagle dogs aged 4 to 6 months were immunized with twice the dose, and were observed continuously for 14 days, and the food intake, drinking water and clinical conditions of the experimental dogs were recorded. The results showed that the prepared vaccine had no local and systemic adverse reactions.

(3) Efficacy test: Take 5 beagle dogs aged 4 to 6 months, inject 0.5 mL of the vaccine subcutaneously into the neck, 28 days after immunization with the same dose for secondary immunization, and set up 5 controls at the same time; 14 days after the second immunization , blood was collected to separate serum, and HI antibody detection was carried out. The results are shown in Table 3 below. The results show that the antibodies in the control dogs were all negative, and the antibodies in the immunization group were all positive. At the same time, all dogs were intranasally inoculated with H3 subtype canine influenza virus (about 1×10 ^7.0 EID ₅₀ ), and 11 days later, the lung lesions were observed by autopsy, and the IL-1β levels were measured by ELISA method. Results The 5 control dogs all had clinical symptoms such as runny nose and cough, and all had severe lung lesions. The clinical symptoms of the experimental dogs in the immunization group were normal, and none of the lungs had changes. The IL-1β in the lungs of the immunized group was significantly lower than that of the control group, suggesting that IL-1β could reflect the onset and protection of canine influenza.

Table 3 HB strain challenge protection results

Note: The calculation method of percent consolidation is the ratio of the mass of consolidation to the mass of the whole lung.

Claims (2)

1. a H3 subtype canine influenza virus strain, is characterized in that: be preserved in the General Microorganism Center of China Microorganism Culture Collection Management Committee, and its deposit number is CGMCC NO.20649. 2. an H3 subtype canine influenza virus inactivated vaccine, characterized in that: the vaccine is inoculated with SPF chicken embryos by the H3 subtype canine influenza virus described in claim 1, and the chicken embryo allantoic fluid is harvested, inactivated, and added with adjuvant. obtained from the drug; It includes the following steps: (1) Inoculation: Inoculate H3 subtype canine influenza virus via the allantoic route at a dose of 2×10 ⁴ EID ₅₀ /piece in SPF chicken embryos aged 10-11 days, and incubate at 35°C; (2) Incubation and observation: within 48 hours after inoculation, the eggs were irradiated once a day, and the dead embryos within 48 hours were discarded. (3) Harvest: Take out the cooled chicken embryos, sterilize the eggshells in the air chamber, aseptically peel off the eggshells in the air chamber, absorb the allantoic fluid, centrifuge to get the supernatant and store it at 2~8℃ to be the virus stock solution; (4) Concentrating and washing the virus stock solution: Concentrate the virus stock solution harvested in step (3) to 1/5 of the original volume with an ultrafiltration concentration device, and the ultrafiltration concentration device is a 100KD~300KD ultrafiltration membrane bag; then add Washing with an equal volume of PBS solution with a pH value of 7.0~7.2, and then concentrating, repeating this for 1~3 times to obtain a concentrated virus solution; (5) Virus inactivation: To the concentrated virus solution obtained in step (4), add formaldehyde with 0.1% of the total volume of the concentrated virus solution and inactivate it by shaking at 4°C for 7 days; (6) Column purification: Purify the inactivated concentrated virus solution obtained in step (5) by Capto Core 700 column at a flow rate of 1 mL/min to obtain a purified antigen with a hemagglutination titer of not less than 1:512. The final protein content is not higher than 400μg/mL, freeze-dried storage; (7) Vaccine preparation: before use, the freeze-dried antigen obtained in step (6) is diluted with physiological saline, mixed with Montanide ^TM GEL 02 adjuvant, and stirred to obtain a vaccine.

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