A kind of avian influenza chicken Newcastle disease two-unit inactivated vaccine

Abstract

A bivalent inactivated vaccine for avian influenza and Newcastle disease. The invention discloses an inactivated vaccine using the avian influenza virus strain with a microbiological collection number of CGMCC No.46189 as an immunogen and a bivalent inactivated vaccine composition using the avian influenza virus strain with a microbiological collection number of CGMCC No.46189 and the La Sota strain of Newcastle disease as immunogens. The two vaccine compositions have good specificity, are safe and effective, and have application prospects.

Description

Technical Field

[0001] The invention belongs to the field of preventive veterinary medicine and relates to a bivalent inactivated vaccine for avian influenza and chicken Newcastle disease. Background Art

[0002] Newcastle disease (ND) is highly contagious, spreads rapidly, and has a high mortality rate. It has been proven to infect more than 250 species of birds, including poultry, waterfowl, and wild birds, among which chickens and turkeys are most susceptible. In the past 100 years, the host infection spectrum of ND has continued to expand, and in recent years, outbreaks have frequently occurred in waterfowl, breaking the previous perception that ND is non-pathogenic to waterfowl.

[0003] Influenza A virus is a member of the Orthomyxoviridae family and the influenza virus genus. According to reports, influenza A virus has a wide host spectrum and can infect animals such as horses, pigs, marine mammals, mustelids and birds, and can also infect humans. With the continuous mutation of the epidemic avian influenza strain, a pathogenic pathogen of an infectious disease characterized by eyelid swelling and bleeding, respiratory symptoms, and an acute decrease in egg production in laying hens has occurred in chickens. The morbidity rate of chickens is high, and they are prone to secondary bacterial infection in the later stage, with a mortality rate of 1% to 10%. It was initially diagnosed as H3N8 subtype avian influenza. The new three-source reassortment H3N8 avian influenza virus is highly adapted to chickens and has a strong pathogenicity to the respiratory system. Systematic epidemiological monitoring should be carried out as soon as possible, and the source and transmission route of the virus should be prevented and controlled as early as possible, so as to strive to eliminate this new virus in the early stage of the epidemic. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the first aspect of the present invention provides an avian influenza virus strain, wherein the avian influenza virus strain is selected from S1 and S2;

[0005] S1: avian influenza virus strain with microbiological collection number CGMCC No.46189;

[0006] S2: A virus strain whose clinical pathogenicity and immunogenicity remain unchanged compared to the avian influenza virus strain with the microbiological collection number of CGMCC No.46189.

[0007] The second aspect of the present invention provides a vaccine composition, wherein the vaccine composition uses G1 or G2 as an immunogen;

[0008] G1: the avian influenza virus strain described in the first aspect of the present invention;

[0009] G2: The avian influenza virus strain and the Newcastle disease La Sota strain described in the first aspect of the present invention.

[0010] In some embodiments, the vaccine composition is an inactivated vaccine composition; and / or

[0011] The raw materials of the vaccine composition include the immunogen and adjuvants.

[0012] In some embodiments, in the vaccine composition using G1 as the immunogen, the avian influenza virus strain: lipid adjuvant: first emulsifier: second emulsifier = 94×10 8.0-11.0 EID 50 : 100-300g: 5-15g: 5-8g;

[0013] In the vaccine composition with G2 as the immunogen, the avian influenza virus strain: the La Sota strain of Newcastle disease: the lipid adjuvant: the first emulsifier: the second emulsifier = 96×10 8.0-11.0 EID 50 :96×10 8.0-11.0 EID 50 : 100-300g: 5-15g: 3-5g;

[0014] The avian influenza virus strain is an inactivated avian influenza virus strain; the chicken Newcastle disease La Sota strain is an inactivated chicken Newcastle disease La Sota strain;

[0015] EID 50 It is calculated based on the amount of virus used before inactivation.

[0016] In some embodiments, any one or a combination thereof is selected from the following:

[0017] The oil adjuvant is selected from white oil;

[0018] The first emulsifier is selected from Siben-80;

[0019] The second emulsifier is selected from Tween-80;

[0020] In the vaccine composition with G1 as the immunogen, the avian influenza virus strain: lipid adjuvant: first emulsifier: second emulsifier = 94×10 9.5 EID 50 : 190g: 10g: 6g;

[0021] In the vaccine composition with G2 as the immunogen, the avian influenza virus strain: the La Sota strain of Newcastle disease: the lipid adjuvant: the first emulsifier: the second emulsifier = 96×10 9.5 EID 50 :96×10 9.0 EID 50 : 190g: 10g: 4g;

[0022] In the vaccine composition with G1 as the immunogen, the content of the avian influenza virus strain is 107.0- 10.0 EID 50 / 0.1ml;

[0023] In the vaccine composition with G2 as the immunogen, the content of the avian influenza virus strain is 10 7.0- 10.0 EID 50 / 0.1ml; the content of the chicken Newcastle disease La Sota strain is 10 8.0-10.0 EID 50 / 0.1ml;

[0024] The preparation method of the inactivated avian influenza virus strain is as follows: adding a formaldehyde solution with a final concentration of 0.1-0.2% to the avian influenza virus strain, inactivating the virus at 35-38° C. for 18-24 hours;

[0025] The preparation method of the inactivated La Sota strain of Newcastle disease of chickens is as follows: adding a formaldehyde solution with a final concentration of 0.1-0.2% to the La Sota strain of Newcastle disease of chickens, inactivating it at 35-38° C. for 18-24 hours;

[0026] The EID 50 It is calculated based on chicken embryo culture according to the Reed-Muench method.

[0027] The third aspect of the present invention provides a method for preparing the vaccine composition of the second aspect of the present invention, wherein in the vaccine composition using G1 as an immunogen, the preparation method comprises: using the avian influenza virus strain described in the first aspect of the present invention as an immunogen to prepare the vaccine composition;

[0028] In the vaccine composition using G2 as the immunogen, the preparation method is: using the avian influenza virus strain and the La Sota strain of Newcastle disease described in the first aspect of the present invention as immunogens to prepare the vaccine composition.

[0029] In some embodiments, the preparation method is: mixing the immunogen with an adjuvant to prepare the vaccine composition.

[0030] In some embodiments, the preparation method comprises the following steps:

[0031] (a) Preparation of oil phase adjuvant:

[0032] Mixing the oil adjuvant with the first emulsifier to obtain an oil phase adjuvant;

[0033] (b) Preparation of aqueous phase:

[0034] mixing the immunogen with a second emulsifier to obtain an aqueous phase;

[0035] (c) Mixing:

[0036] The oil phase adjuvant is mixed with the water phase to obtain the vaccine composition.

[0037] The fourth aspect of the present invention provides the use of the avian influenza virus strain described in the first aspect of the present invention or the vaccine composition with G1 as the immunogen described in the second aspect of the present invention in the preparation of a preparation for use alone, in combination with other immune preparations and / or drugs, or as a component of a compound preparation composed of other immune preparations and / or drugs to prevent, mitigate and / or control avian influenza.

[0038] The fifth aspect of the present invention provides the use of the vaccine composition with G2 as the immunogen described in the second aspect of the present invention in the preparation of a preparation for use alone, in combination with other immune preparations and / or drugs, or as a component of a compound preparation composed of other immune preparations and / or drugs to prevent, mitigate and / or control avian influenza and Newcastle disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the evolutionary tree of the HA gene of avian influenza H3 subtype strains.

[0040] Figure 2 This is the result of comparative analysis of the homology of HA gene of avian influenza H3 subtype strains. DETAILED DESCRIPTION

[0041] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0042] For the virus strain claimed for protection in the present invention (the avian influenza virus strain with the microbial collection number of CGMCC NO.46189, AIV-H3-YK strain), the passaged virus strains of the AIV-H3-YK strain with the same genome without mutation should be regarded as the AIV-H3-YK strain preserved by microorganisms. The passaged virus strains with no change in clinical pathogenicity and immunogenicity mainly refer to the virus strains based on the passage of the AIV-H3-YK strain and the accumulation of minor synonymous mutations during the passage.

[0043] The AIV-H3-YK strain inevitably introduces minor mutations through passage and application, and the passaged virus strains with no change in clinical pathogenicity and immunogenicity should fall within the scope of contribution of the present invention. No change in clinical pathogenicity and immunogenicity means no substantial change, for example, within the limitations of detection technology such as detection sensitivity and detection limit and the range of acceptable or unavoidable errors, the clinical pathogenicity and immunogenicity are considered to be the same.

[0044] It is often necessary to use animals to measure the clinical pathogenicity and immunogenicity of the offspring of the AIV-H3-YK strain. Due to differences in animal breed, age, gender, health status, and predictable or unavoidable systematic errors, there is no substantial change in the clinical pathogenicity and immunogenicity of the progeny of the virus strain.

[0045] After multiple generations of passaging, the AIV-H3-YK strain inevitably introduces minor mutations. When mutations occur in non-coding sequence regions or synonymous mutations in coding regions, it can be reasonably expected that when these minor changes do not significantly affect the clinical pathogenicity and immunogenicity of the offspring strains, they are still within the scope of the substantial technical contribution of the present invention. These minor mutations are still non-substantial mutations and should be regarded as mutant virus strains with no changes in clinical pathogenicity and immunogenicity.

[0046] Example 1. Acquisition, isolation and identification of avian influenza H3-YK strain

[0047] 1. Virus isolation and species identification

[0048] (1) Cases

[0049] In 2023, diseased laying hens were collected from Jiangsu Province. The clinical manifestations of the diseased chickens included swollen and bleeding eyelids, respiratory symptoms, and an acute decrease in egg production in laying hens. Preliminary autopsy revealed bleeding spots in the larynx and trachea, swollen spleen and kidneys, and bleeding in the cecum and tonsils. It was preliminarily determined to be the H3 subtype of avian influenza virus.

[0050] (2) Pathogenic material treatment and pathogen isolation

[0051] The oral and cloacal swabs collected from sick chickens were mixed and fully twisted and squeezed in pH 7.2 PBS buffer (containing double antibodies), and the liquid part was retained. After centrifugation at 8000r / min for 10 minutes, the supernatant was collected and inoculated into 5 10-day-old SPF chicken embryos at a dose of 0.2ml / piece. The allantoic fluid (culture a) of the surviving chicken embryos for 24h to 72h was collected to determine the hemagglutination titer HA and the chicken embryo half infectious dose EID 50 , the hemagglutination titer HA ≥ 1:256, the chicken embryo infection dose EID 50 ≥10 8.0 / 0.1ml of allantoic fluid was stored at -70℃ for future use.

[0052] According to the conserved sequences of the genomes of avian influenza virus H9 subtype, avian influenza virus H5 subtype, avian influenza virus H7 subtype, Newcastle disease virus, and infectious bronchitis virus, primer pairs were designed and identified. The nucleic acid in culture a was amplified by conventional RT-PCR. The amplification results were observed by gel electrophoresis. As a result, only the primers for type A avian influenza virus H3 subtype amplified positively. It can be seen that the avian influenza virus H3 subtype exists in culture a. The case of this embodiment suffers from the A avian influenza virus H3 subtype, and the corresponding strain is referred to as the A avian influenza virus H3 subtype YK strain (AIV-H3-YK strain).

[0053] The primer pair for avian influenza A virus H3 subtype is R1 and R2 as described below.

[0054] F1 (SEQ ID NO.1): 5′-TATTCGTCTCAGGGAGCAAAAGCAGGGG-3′

[0055] R1 (SEQ ID NO.2): 5′-ATATCGTCTCGTATTAGTAGAAACAAGGGT GTTTT-3′

[0056] The product of RT-PCR amplification of the nucleic acid in culture a using primers R1 and R2 corresponding to the partial sequence of the HA gene of the H3 subtype of avian influenza virus was sequenced by the Sanger method to obtain a 1700 bp specific nucleic acid fragment. The fragment was compared with the reference avian influenza strains included in GenBank for genetic phylogenetic tree analysis. The results are shown in Figure 1 , statistics of HA gene sequence homology comparison among strains can be found in Figure 2 , Figure 1-2 In the results, the name of the AIV-H3-YK strain was boxed. The results showed that the highest homology between the H3 subtype of avian influenza A virus and the published H3N3 subtype of avian influenza was 99.6%. Therefore, it can be determined that the newly isolated strain is the H3 subtype of avian influenza A virus.

[0057] (II) Virulence test

[0058] (1) Toxicity test on chicken embryos

[0059] Take the virus solution of culture a and use sterile saline to make 10 -5 Dilution, each virus liquid was inoculated into the allantoic cavity of 10 10-11 day old SPF embryos, 0.2 ml / egg, incubated at 35℃, and the embryos were illuminated twice a day. The number of dead embryos within 24-72 hours was recorded, and the dead chicken embryos were dissected to observe whether there were systemic hemorrhage, obvious edema, liver necrosis and other lesions. The results showed that there was no death in 10 chicken embryos, and the HA was measured in the allantoic fluid of each chicken embryo, and the results of 8 / 10 embryos were greater than 1:256.

[0060] (2) Toxicity test on chickens

[0061] Take the virus liquid of culture a, take 10 28-day-old SPF chickens, divide them into the virus attack group, 10 chickens / group, dilute the virus liquid with sterile saline at a volume ratio of 1:10, inoculate through the wing vein, 0.2ml per chicken, and observe daily for 14 days; take another 5 as the control, each chicken is inoculated with 0.2ml sterile saline; 5 days after the virus attack, collect the throat swab and cloaca swab of each chicken respectively, mix them and isolate the virus, each swab is inoculated with 5 10-day-old chicken embryos, 0.2ml / piece, incubate at 35℃, and illuminate the embryos twice a day. After 3 days of culture observation, measure the HA of each chicken embryo allantoic fluid, and the HA result of 1 / 5 pieces is greater than 1:16, indicating that the isolated virus is positive. For samples with negative virus infection, blind transmission should be performed once, and the HA titer should be measured again. The result should be 8 / 10 chickens infected and expelled, and 0 / 5 chickens in the control group should expel toxins. The results show that the positive rate of expelling toxins in chickens is 100%.

[0062] (III) Virus immunogenicity assay

[0063] Take the qualified culture a, add formaldehyde solution with a final concentration of 0.1%, stir and inactivate at 36℃ for 24 hours, and dilute to 10 according to the antigen virus content before inactivation. 8.5 EID 50 / 0.1ml, 10 8.0 EID 50 / 0.1ml, 10 7.5 EID 50 / 0.1ml as antigen, prepare the vaccine according to the following procedure, take 95 parts of injection white oil by volume, heat it, add 5 parts of Siben-80, mix well, and sterilize by high pressure to obtain the oil phase; add 6 parts of sterilized Tween-80 to 94 parts of antigen (diluted inactivated virus solution) by volume, shake well to dissolve Tween-80 completely, and obtain the water phase; pour 2 parts of oil phase into the emulsification tank and stir, then slowly add 1 part of water phase by volume, stir well and emulsify. The vaccines with different antigen contents were subcutaneously inoculated into 10 SPF chickens aged 21 days through the neck, 0.3ml each, and 5 chickens were used as controls, each with 0.3ml of sterilized saline subcutaneously in the neck. 21 days after immunization, blood was collected from each chicken, serum was separated, and the HI antibody titer was determined using the hemagglutination inhibition test antigen of H3 subtype avian influenza virus YK strain. 21 days after immunization, 0.2 ml (containing 100 ID) of AIV-H3-YK strain virus diluted 1:10 was injected intravenously into each chicken. 50). On the 5th day after the virus attack, the throat swab and cloaca swab of each chicken were collected and mixed for virus isolation. Five 10-day-old SPF chicken embryos were inoculated into the allantoic cavity of each chicken sample. After incubation and observation for 3 days, the hemagglutination titer of the chicken embryo fluid was measured for both dead and live embryos. If at least one of the five chicken embryos had a chicken embryo fluid HA titer of no less than 1:16, the virus isolation was positive. For samples that were negative for virus infection, they should be blindly transmitted once and the HA titer should be measured again. Results 10 8.0 EID 50 After immunization with the vaccine prepared with 0.1 ml pathogen, the average antibody HI value of chickens was >1:128, the protection rate against virus attack was 90%, and the immunogenicity was good.

[0064] (IV) Microbial Preservation

[0065] The present invention submits the isolated A-type avian influenza virus H3 subtype strain (AIV-H3-YK strain) to a patent procedure approved depository for deposit; the depository is: General Microbiology Center of China Microbiological Culture Collection Administration; address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its microbiological deposit number is CGMCC No. 46189; the classification name in Chinese is: Avian Influenza Virus H3 subtype; the species in Latin is: Avian InfluenzaVirus; the name of the biological material is: AIV-H3-YK; the deposit time is: September 20, 2024; the proven survival time is: September 20, 2024.

[0066] Example 2: Preparation of avian influenza virus AIV-H3-YK strain vaccine

[0067] (1) Antigen virus preparation: Take the original virus solution and use sterile saline for 10 -5 Dilute, inoculate 10-day-old SPF embryos into the allantoic cavity, 0.2 ml / egg, incubate at 35°C, and illuminate the embryos twice a day. Discard dead embryos within 24 hours, incubate for 72 hours, mix and collect the allantoic fluid of the chicken embryos to obtain the virus strain E4, and determine HA and EID 50 Results A total of 400 ml of E4 virus fluid was harvested and passed the sterility and virus content tests.

[0068] (2) Virus content determination: Avian influenza virus AIV-H3-YK strain E4 seed virus was serially diluted 10 times with sterile saline. -6 , 10 -7 , 10 -8 , 10 -9Four dilutions were inoculated through the allantoic cavity of 10-day-old SPF chicken embryos. Five embryos were inoculated at each dilution, and each embryo was inoculated with 0.1 ml. The embryos were placed at 35°C for further incubation. The chicken embryos that died before 24 hours were discarded, and the remaining chicken embryos were incubated for 72 hours, and all live embryos were removed. The red blood cell agglutination titer was determined for each embryo. Those with HA agglutination titer of not less than 1:16 were considered infected, and the EID was calculated according to the Reed-Muench method. 50 The results showed that the virus HA = 1:256, and the virus content per 0.1 ml was 10 8.63 EID 50 .

[0069] (3) Inactivation and vaccine preparation: Add a 0.1% formaldehyde solution to the virus solution that has passed the test and inactivate it at 36°C for 24 hours. The inactivated virus solution is stored at 2-8°C. After the inactivated antigen has passed the test (sterile, completely inactivated), dilute it to 10% with sterile saline according to the antigen virus content before inactivation. 8.5 EID 50 / 0.1ml, 10 8.0 EID 50 / 0.1ml, 10 7.5 EID 50 / 0.1ml as antigen and prepare the vaccine according to the following procedure.

[0070] Preparation of oil phase: Take 95 parts of injection white oil by volume, heat it, then add 5 parts of Siben-80, mix well, and sterilize it by high pressure for later use.

[0071] Preparation of aqueous phase: Add 94 parts of antigen to 6 parts of sterile Tween-80 by volume and shake thoroughly to dissolve the Tween-80 completely.

[0072] Emulsification: Pour 2 parts of the oil phase into the emulsification tank by volume and stir, then slowly add 1 part of the water phase and stir thoroughly to emulsify.

[0073] The three vaccines with different antigen contents are respectively called A1, A2 and A3.

[0074] Example 3: Performance Characterization of Avian Influenza Virus AIV-H3-YK Strain Vaccine

[0075] (I) HI titer determination

[0076] The vaccines A1, A2, and A3 with different antigen contents were subcutaneously inoculated into 10 21-day-old SPF chickens through the neck, 0.3 ml each, and 5 controls were inoculated with 0.3 ml sterile saline each. 21 days after immunization, blood was collected from each chicken, serum was separated, and the HI antibody titer was determined using the hemagglutination inhibition test antigen of the AIV-H3-YK strain.

[0077] Virus content 108.5 EID 50 / 0.1ml A1 vaccine group HI titer geometric mean is 1:206; virus content 10 8.0 EID 50 The geometric mean of HI titer of A2 vaccine group was 1∶108; the virus content was 10 7.5 EID 50 The geometric mean of HI titer in the A3 vaccine group with 0.1 ml / ml was 1:54, lower than 1:64; the geometric mean of HI titer in the blank control group was less than 1:2. The results are shown in Table 1.

[0078] Table 1. Serum HI antibody titer determination results

[0079]

[0080] (II) Immune attack method

[0081] The vaccines A1, A2, and A3 with different antigen contents were injected subcutaneously into 10 21-day-old SPF chickens, and 0.3 ml of the vaccine was injected subcutaneously into each chicken. The other 10 chickens were used as controls, and each chicken was inoculated with 0.3 ml of sterile saline. 21 days after immunization, AIV-H3-YK virus seed diluted 1:10 in sterile saline was injected intravenously, 0.2 ml (containing 100 ID 50 ). On the 5th day after the virus attack, the throat swab and cloaca swab of each chicken were collected and mixed for virus isolation. Five 10-day-old SPF chicken embryos were inoculated into the allantoic cavity of each chicken sample. After incubation and observation for 3 days, the hemagglutination titer of the chicken embryo fluid should be measured regardless of dead or alive embryos. If the hemagglutination titer of the chicken embryo fluid of at least one of the five chicken embryos is not less than 1:16, it is judged as positive for virus isolation. For samples that are negative for virus infection, blind transmission is performed once, and the HA titer is measured again.

[0082] The virus isolation of group A1 was 0 / 10 positive, that of group A2 was 1 / 10 positive, that of group A3 was 3 / 10 positive, and that of control group was 10 / 10 positive. The results are shown in Table 2. 8.0 EID 50 / 0.1ml to prepare the vaccine, and the immune protection rate was 90%.

[0083] Table 2. Results of immune challenge test

[0084]

[0085] (III) Comparison of HI antibody titer and immune challenge results

[0086] When the geometric mean of HI titer is higher than 1:64, the positive rate of virus isolation after challenge is no higher than 1 / 10; when the geometric mean of HI titer is lower than 1:64, the positive rate of virus isolation after challenge is 3 / 10, and the protection of challenge is lower than 90%; determine the virus content of the seedling 10 8.0 EID 50 The vaccine was prepared with 0.1 ml of HI titer geometric mean higher than 1:64, and the protection rate against virus was 90%, which was the minimum immunization dose. The results are shown in Table 3.

[0087] Table 3. Comparison of HI antibody titer and immune challenge test results

[0088]

[0089] From the above results, it can be seen that when the vaccine is prepared with different antigen content vaccines to immunize 21-day-old SPF chickens, the virus content is 10 8.0 EID 50 / 0.1ml, the HI titer was higher than 1:64, and the positive rate of immune challenge virus isolation was 1 / 10, so the minimum immune dose of the virus for vaccine preparation was determined to be 10 8.0 EID 50 / 0.1ml.

[0090] Example 4: Preparation of a dual inactivated vaccine

[0091] (I) Preparation of virus solution

[0092] (1) Preparation of virus solution for avian influenza AIV-H3-YK vaccine

[0093] Take the AIV-H3-YK virus strain (E5) for production and use sterile saline for 10 -5 Dilution, inoculate 10-day-old healthy susceptible chicken embryos, inoculate 0.2 ml per embryo through the allantoic cavity, seal the pinhole, and continue incubation at 35°C without turning the eggs. Within 72 hours after inoculation, illuminate the eggs once a day, and discard the chicken embryos that died within 72 hours. Collect 72-hour live embryos and cool them at 2-8°C for 4-24 hours. Take out the cooled chicken embryos and aseptically harvest the chicken embryo fluid. Mix several chicken embryos into a group, harvest the allantoic fluid and put it in the same sterilized container, and take samples for inspection.

[0094] According to the current "Chinese Veterinary Pharmacopoeia" (2020 edition, the same below), each group of samples was sampled for 1% hemagglutination titer, and those with a hemagglutination titer lower than 1:256 were discarded. The qualified virus solution was mixed and then subjected to other tests.

[0095] Sterility tests were carried out according to the appendices of Volume 3 of the current "Chinese Pharmacopoeia of Veterinary Medicine", and no sterile growth was found.

[0096] The content determination method of Example 2 was used for determination, and the virus content per 0.1 ml was 108.71 EID 50 .

[0097] (2) Preparation of virus solution for Newcastle disease La Sota strain vaccine

[0098] The Newcastle disease virus used to make the vaccine is the La sota strain (CVCC AV1615) and is kept and supplied by the China Veterinary Drug Administration.

[0099] Take the La Sota strain of Newcastle disease in chickens and use sterile saline for 10 -4 Dilution, inoculate 10-11 day old healthy susceptible chicken embryos, inoculate 0.1 ml into the allantoic cavity of each embryo, seal the pinhole, and continue incubation at 36-37℃ without turning the eggs.

[0100] Within 72 hours after inoculation, the eggs are illuminated once a day, and the dead chicken embryos within 72 hours are discarded. Thereafter, the embryos are illuminated once every 4 to 6 hours until 120 hours, and the dead embryos can be removed at any time. After 120 hours, all dead and alive chicken embryos are removed, the air chambers are upright, and they are cooled at 2 to 8 degrees Celsius for 4 to 24 hours.

[0101] Take out the cooled chicken embryos, separate the live embryos from the dead embryos, and aseptically harvest the chicken embryo fluid. Mix several chicken embryos into a group, harvest the allantoic fluid and put it in the same sterilized container, and take samples for inspection.

[0102] According to the current "Chinese Veterinary Pharmacopoeia" three appendices, each group of samples were tested for 1% hemagglutination titer, and those with hemagglutination titer lower than 1:512 were discarded. The qualified virus solution was mixed and then subjected to other tests.

[0103] After mixing, the samples were tested for sterility according to the appendix to Volume III of the current Chinese Pharmacopoeia of Veterinary Medicine, and all showed no bacterial growth.

[0104] The mixed samples were tested according to the method of Example 2, except that different viruses were used. The results showed that the virus content per 0.1 ml was 10 8.63 EID 50 .

[0105] (II) Concentration and clarification of virus solution

[0106] (1) Concentration of Newcastle disease La Sota strain virus solution

[0107] The qualified Newcastle disease La Sota strain virus liquid is filtered to remove large particle impurities in the virus liquid, making it a transparent or translucent solution. The virus liquid is concentrated 3 to 5 times using an ultrafiltration concentration system and samples are taken for inspection.

[0108] (2) Concentration of avian influenza AIV-H3-YK virus fluid

[0109] The virus liquid of avian influenza AIV-H3-YK strain that has passed the inspection is filtered to remove large particle impurities in the virus liquid, making it a transparent or translucent solution. The virus liquid is concentrated 3 to 5 times using an ultrafiltration concentration system and samples are taken for inspection.

[0110] (III) Virus liquid inactivation

[0111] (1) Inactivation of avian influenza AIV-H3-YK strain virus liquid

[0112] The concentrated avian influenza AIV-H3-YK virus liquid is introduced into the inactivation tank, and a final concentration of 0.1% formaldehyde solution is slowly added to the inactivation tank, stirring while adding to fully mix, and then heated to 36°C for inactivation for 20 to 24 hours (starting from the time when the antigen temperature in the tank reaches 36°C), stirring continuously during the period. After the inactivation is completed, samples are taken for inspection, and the inactivated antigen is stored at 2 to 8°C for no more than 6 months.

[0113] (2) Inactivation of La Sota strain of Newcastle disease virus

[0114] The concentrated La Sota strain of Newcastle disease virus liquid is introduced into the inactivation tank, and a final concentration of 0.1% formaldehyde solution is slowly added to the inactivation tank, stirring while adding to fully mix, and then heated to 36°C for inactivation for 20 to 24 hours (starting from the time when the antigen temperature in the tank reaches 36°C), stirring continuously during the period. After the inactivation is completed, samples are taken for inspection, and the inactivated antigen is stored at 2 to 8°C for no more than 6 months.

[0115] (IV) Inactivation test

[0116] (1) Test for inactivation of concentrated antigen of La Sota strain of Newcastle disease in chickens

[0117] Take the inactivated Newcastle disease La Sota strain antigen liquid and inoculate 10 10-day-old SPF chicken embryos into the allantoic cavity, 0.2 ml per embryo, incubate at 37°C for 120 hours, discard the dead chicken embryos within 24 hours, and there are no non-specific dead chicken embryos. The hemagglutination value is measured for each embryo, and no hemagglutination occurs. Mix the chicken embryo liquid in equal amounts and blindly propagate for 1 generation according to the above method, and measure the hemagglutination value. No hemagglutination occurs, which is judged to be completely inactivated.

[0118] (2) Test for inactivation of concentrated antigen of avian influenza AIV-H3-YK strain

[0119] Take the inactivated avian influenza AIV-H3-YK strain antigen and inoculate 10 10-day-old SPF chicken embryos into the allantoic cavity, 0.2 ml per embryo, incubate at 37°C, discard the dead chicken embryos within 24 hours, observe for 120 hours, there is no non-specific dead chicken embryos, and measure the hemagglutination value of each embryo, and no hemagglutination occurs. Mix the chicken embryo fluid in equal amounts and blindly propagate for 1 generation according to the above method, measure the hemagglutination value, and all hemagglutination occurs, which is judged to be completely inactivated.

[0120] 5. Vaccine preparation

[0121] (1) Oil phase preparation

[0122] Heat 95 parts by volume of injection white oil, add 5 parts by volume of Siben-80, mix thoroughly, and sterilize by high pressure for later use.

[0123] (2) Preparation of aqueous phase

[0124] The inactivated and qualified La Sota strain of Newcastle disease (calculated according to the virus content before inactivation) was diluted to 2×10 8.5 EID 50 / 0.1ml), avian influenza AIV-H3-YK strain (calculated according to the virus content before inactivation and diluted with sterile saline to 2×10 8.0 EID 50 / 0.1ml) antigen solution in a volume ratio of 1:1, take 96 volumes of the mixed antigen solution, add 4 volumes of sterilized Tween-80, and stir thoroughly while adding until Tween-80 is completely dissolved to form an aqueous phase.

[0125] (3) Emulsification

[0126] 2 parts by volume of the oil phase are placed in an emulsification tank, and then 1 part by volume of the water phase is slowly added and stirred thoroughly for emulsification.

[0127] (4) Packaging

[0128] Package in quantitative quantities, seal with a cover, attach a label, and store at 2-8℃.

[0129] Example 5: Experiment on the immune efficacy of three batches of vaccines on chickens of different ages through each vaccination route The following experiment was conducted using the three batches of vaccines (codenamed B1, B2, and B3) prepared in Example 3.

[0130] (I) Grouping

[0131] Each batch of vaccine in B1, B2, and B3 was administered subcutaneously and intramuscularly in the neck, and each method was used to inoculate 7-day-old, 28-day-old, and 120-day-old SPF chickens, for a total of 6 groups. A total of 6 control groups with the same conditions were set up for the three batches of vaccine.

[0132] (II) Immunization

[0133] Each batch of vaccine selected 60 SPF chickens at 7 days, 28 days and 120 days of age, and divided into 2 groups at each age. One group of 7-day-old SPF chickens were subcutaneously vaccinated with 0.3 ml / chicken, and 28-day-old and 120-day-old SPF chickens were subcutaneously vaccinated with 0.5 ml / chicken. The other group of 7-day-old SPF chickens were intramuscularly vaccinated with 0.3 ml / chicken, and 28-day-old and 120-day-old SPF chickens were intramuscularly vaccinated with 0.5 ml / chicken. SPF chickens at 7 days, 28 days and 120 days of age were set up under the same conditions, with 60 chickens at each age, and physiological saline was inoculated according to the above grouping method. The immunization group and the control group were raised under the same conditions.

[0134] (III) Antibody titer determination

[0135] (1) Determination of HI antibody titer of avian influenza AIV-H3-YK

[0136] Blood was collected from the immunization group and the control group 21 days after the first immunization, and the serum was separated for the determination of HI antibody titer, and the geometric mean was calculated.

[0137] The results are as follows: Three batches of vaccines were used to immunize SPF chickens of different ages by subcutaneous injection in the neck and intramuscular injection. Blood was collected 21 days after the first immunization and serum was separated to determine the HI antibody titer. The subcutaneous injection route was used, and the geometric mean values ​​of avian influenza HI antibodies in SPF chickens of 7 days old from three batches of inactivated vaccines were between 1:158 and 1:181; the geometric mean values ​​of avian influenza HI antibodies in SPF chickens of 28 days old were between 1:294 and 1:315; and the geometric mean values ​​of avian influenza HI antibodies in SPF chickens of 120 days old were between 1:274 and 1:315. The intramuscular injection route was used, and the geometric mean values ​​of avian influenza HI antibodies in SPF chickens of 7 days old from three batches of inactivated vaccines were all 1:169; the geometric mean values ​​of avian influenza HI antibodies in SPF chickens of 28 days old were between 1:223 and 1:274; and the geometric mean values ​​of avian influenza HI antibodies in SPF chickens of 120 days old were between 1:239 and 1:294. There was no significant difference in the titer levels of HI antibodies in SPF chickens of different ages due to the vaccination route.

[0138] (2) Determination of HI antibody titers of some Newcastle disease La Sota strains

[0139] Blood was collected from the immunization group and the control group 21 days after the first immunization, and the serum was separated for the determination of HI antibody titer, and the geometric mean was calculated.

[0140] The results are as follows: Three batches of products were used to immunize different age groups by subcutaneous injection in the neck and intramuscular injection. Blood was collected 21 days after the first immunization and serum was separated to determine the HI antibody titer. Subcutaneous injection was used, and the geometric mean values ​​of Newcastle disease HI antibodies in SPF chickens of 7 days old and 3 batches of inactivated vaccines were between 1:24 and 1:26; the geometric mean values ​​of Newcastle disease HI antibodies in SPF chickens of 28 days old and 1:37; the geometric mean values ​​of Newcastle disease HI antibodies in SPF chickens of 120 days old were between 1:34 and 1:42. Intramuscular injection was used, and the geometric mean values ​​of Newcastle disease HI antibodies in SPF chickens of 7 days old and 3 batches of inactivated vaccines were between 1:21 and 1:24; the geometric mean values ​​of Newcastle disease HI antibodies in SPF chickens of 28 days old and 1:37 and 1:42; the geometric mean values ​​of Newcastle disease HI antibodies in SPF chickens of 120 days old were between 1:32 and 1:39. There was no significant difference in the titer levels of HI antibodies in SPF chickens of different ages due to the vaccination route.

[0141] (IV) Attack

[0142] The immunized group and the control group SPF chickens were intravenously injected with 1:10 diluted avian influenza virus AIV-H3-YK strain virus, 0.2 ml per chicken. On the 5th day after the virus attack, the throat swab and cloaca swab of each chicken were collected and mixed for virus isolation. Five 10-day-old SPF chicken embryos were inoculated into the allantoic cavity of each chicken sample. After incubation and observation for 3 days, the hemagglutination titer of the chicken embryo fluid should be measured regardless of dead or alive embryos. The virus isolation was positive if at least one chicken embryo in the 5 chicken embryos had a hemagglutination titer of not less than 1:16. For samples that were negative for virus infection, blind transmission should be performed once and the HA titer should be measured again. The results are shown in Table 4.

[0143] Table 4. Results of challenge with avian influenza AIV-H3-YK strain

[0144]

[0145]

[0146] It can be seen that 9-10 birds died in each control group, and the protection rate of each immunization group was 90-100%, which shows that immunization with the dual vaccine of the present invention can produce good protection against avian influenza virus attack.

[0147] Example 6: Experiment on the duration of immunity of three batches of vaccines

[0148] 80 14-day-old SPF chickens were randomly divided into 3 groups, 20 in each group, and 3 batches of Newcastle disease and avian influenza bivalent inactivated vaccines (B1, B2, B3) were used. The vaccine was injected subcutaneously at 0.3 ml / bird. Another group was used as a blank control and was raised under the same conditions.

[0149] Newcastle disease part: blood was collected from the immunized group and the control group 14, 21, 60, 90, 120, and 150 days after vaccination, and the serum was separated for the HI antibody titer determination, and the geometric mean was calculated.

[0150] Avian influenza part: blood was collected from the immunized group and the control group 14, 21, 60, 90, 120, and 150 days after vaccination, and the serum was separated for the HI antibody titer determination, and the geometric mean was calculated.

[0151] Chickens in the 150-day immunization group and the control group were intravenously injected with a 1:10 dilution of the AIV-H3-YK strain virus, 0.2 ml per chicken. On the 5th day after the virus attack, the throat swab and cloaca swab of each chicken were collected and mixed for virus isolation. Five 10-day-old SPF chicken embryos were inoculated into the allantoic cavity with samples from each chicken. After incubation and observation for 3 days, the hemagglutination titer of the chicken embryo fluid should be measured regardless of dead or alive embryos. If the hemagglutination titer of the chicken embryo fluid of at least one of the five chicken embryos is not less than 1:16, it is judged as positive for virus isolation. For samples that are negative for virus infection, they should be blindly transmitted once and the HA titer should be measured again.

[0152] The results of the antibody titer determination are as follows.

[0153] 14 days after the first vaccination of the three batches of inactivated vaccines, the geometric mean of Newcastle disease HI antibody titer reached above 1:30, while the geometric mean of the control group was <1:4; the geometric mean of avian influenza HI antibody titer reached above 1:158, while the geometric mean of the control group was <1:4.

[0154] 21 days after the first vaccination of the three batches of inactivated vaccines, the geometric mean of Newcastle disease HI antibody titer reached above 1:39, while the geometric mean of the control group was <1:4; the geometric mean of avian influenza HI antibody titer reached above 1:181, while the geometric mean of the control group was <1:4.

[0155] 60 days after the first vaccination with the three batches of inactivated vaccines, the geometric mean of Newcastle disease HI antibody titer reached above 1:34, while the geometric mean of the control group was <1:4; the geometric mean of avian influenza HI antibody titer reached above 1:194, while the geometric mean of the control group was <1:4.

[0156] 90 days after the first vaccination with the three batches of inactivated vaccines, the geometric mean of Newcastle disease HI antibody titer reached above 1:42, while the geometric mean of the control group was <1:4; the geometric mean of avian influenza HI antibody titer reached above 1:223, while the geometric mean of the control group was <1:4.

[0157] 120 days after the first vaccination of the three batches of inactivated vaccines, the geometric mean of Newcastle disease HI antibody titer reached above 1:39, while the geometric mean of the control group was <1:4; the geometric mean of avian influenza HI antibody titer reached above 1:169, while the geometric mean of the control group was <1:4.

[0158] 150 days after the first vaccination with the three batches of inactivated vaccines, the geometric mean of Newcastle disease HI antibody titer reached above 1:28, while the geometric mean of the control group was <1:4; the geometric mean of avian influenza HI antibody titer reached above 1:158, while the geometric mean of the control group was <1:4.

[0159] The three batches of inactivated vaccines were challenged with avian influenza virus 150 days after the first immunization. The protection rate of the immunized group was over 90%, and the virus separation rate was over 90% negative; the virus separation rate of the control group was 100% positive.

[0160] Example 7: Minimum immunization dose of 3 batches of vaccines for SPF chickens of different ages

[0161] (I) 7-day-old SPF chicken

[0162] 120 7-day-old SPF chickens were randomly divided into 9 groups of 10 chickens in each batch of vaccine. Three batches of two-inactivated vaccines (B1, B2, B3) were used. The vaccine was injected subcutaneously or intramuscularly, and immunized with 0.1 ml / chicken, 0.3 ml / chicken, and 0.5 ml / chicken, respectively. Another 30 chickens were taken as blank controls and raised under the same conditions. 21 days after immunization, the Newcastle disease part and the avian influenza part were tested for HI antibody titer, and the avian influenza part was challenged with virus after immunization, and the challenge strain was AIV-H3-YK (the same in this embodiment), and the challenge dose was 100 ID 50 .

[0163] The results are as follows: For avian influenza, three batches of inactivated vaccines were used to immunize 7-day-old SPF chickens with different doses. At 0.1 ml / hen, the geometric mean values ​​of HI antibody titers were 1:97, 1:119, and 1:128, respectively, and the protection rates against virus infection were between 60% and 80%; at 0.3 ml / hen, the geometric mean values ​​of HI antibody titers were 1:169, 1:147, and 1:158, respectively, and the protection rates against virus infection were between 90% and 100%; at 0.5 ml / hen, the geometric mean values ​​of HI antibody titers were 1:294, 1:223, and 1:208, respectively, and the protection rates against virus infection were 100%.

[0164] For Newcastle disease, 7-day-old SPF chickens were immunized with different doses of three batches of inactivated vaccines. The geometric mean HI antibody titers of 0.1 ml / hen were 1:11, 1:11, and 1:11, respectively; the geometric mean HI antibody titers of 0.3 ml / hen were 1:32, 1:30, and 1:30, respectively; the geometric mean HI antibody titers of 0.5 ml / hen were 1:45, 1:39, and 1:37, respectively.

[0165] (ii) 21-day-old SPF chicken

[0166] 120 21-day-old SPF chickens were randomly divided into 9 groups of 10 chickens each, and 3 batches of two-inactivated vaccines (B1, B2, B3) were used for subcutaneous or intramuscular injection, respectively, 0.3ml / chicken, 0.5ml / chicken, 1.0ml / chicken, and another 30 chickens were taken as blank controls and raised under the same conditions. 21 days after immunization, the HI antibody titer of the Newcastle disease part and the avian influenza part was determined, and the avian influenza part was challenged after immunization.

[0167] For avian influenza, three batches of inactivated vaccines were used to immunize 21-day-old SPF chickens with different doses. At 0.3 ml / hen, the geometric mean HI antibody titers were 1:137, 1:147, and 1:137, respectively, and the protection rates against virus were between 90% and 100%; at 0.5 ml / hen, the geometric mean HI antibody titers were 1:169, 1:158, and 1:169, respectively, and the protection rates against virus were between 90% and 100%; at 1.0 ml / hen, the geometric mean HI antibody titers were 1:294, 1:239, and 1:256, respectively, and the protection rates against virus were 100%.

[0168] For Newcastle disease, 21-day-old SPF chickens were immunized with different doses of three batches of inactivated vaccines. The geometric mean HI antibody titers of 0.3 ml / hen were 1:30, 1:32, and 1:30, respectively; the geometric mean HI antibody titers of 0.5 ml / hen were 1:39, 1:34, and 1:37, respectively; the geometric mean HI antibody titers of 1.0 ml / hen were 1:56, 1:52, and 1:60, respectively.

[0169] (III) 120-day-old SPF chicken

[0170] 120 SPF chickens aged 120 days were randomly divided into 9 groups, 10 in each group, and 3 batches of two-inactivated vaccines (B1, B2, B3) were used for subcutaneous or intramuscular injection, respectively, 0.3ml / chicken, 0.5ml / chicken, 1.0ml / chicken, and another 30 chickens were taken as blank controls and raised under the same conditions. 21 days after immunization, the HI antibody titer of the Newcastle disease part and the avian influenza part was determined, and the avian influenza part was challenged after immunization.

[0171] For avian influenza, three batches of inactivated vaccines were used to immunize 120-day-old SPF chickens at different doses. At 0.3 ml per chicken, the geometric mean HI antibody titers were 1:128, 1:158, and 1:137, respectively, and the protection rates against virus infection were between 80% and 100%; at 0.5 ml per chicken, the geometric mean HI antibody titers were 1:158, 1:169, and 1:181, respectively, and the protection rates against virus infection were between 90% and 100%; at 1.0 ml per chicken, the geometric mean HI antibody titers were 1:294, 1:223, and 1:239, respectively, and the protection rates against virus infection were between 90% and 100%.

[0172] For Newcastle disease, 120-day-old SPF chickens were immunized with different doses of three batches of inactivated vaccines. The geometric mean HI antibody titers of 0.3 ml / hen were 1:32, 1:34, and 1:30, respectively; the geometric mean HI antibody titers of 0.5 ml / hen were 1:37, 1:34, and 1:37, respectively; the geometric mean HI antibody titers of 1.0 ml / hen were 1:45, 1:48, and 1:52, respectively.

[0173] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. An avian influenza virus strain, wherein the avian influenza virus strain is selected from S1 and S2; S1: avian influenza virus strain with microbiological collection number CGMCC No.46189; S2: A virus strain whose clinical pathogenicity and immunogenicity remain unchanged compared to the avian influenza virus strain with the microbiological collection number of CGMCC No.46189.

2. A vaccine composition, wherein the vaccine composition uses G1 or G2 as an immunogen; G1: the avian influenza virus strain according to claim 1; G2: The avian influenza virus strain and the Newcastle disease La Sota strain described in claim 1.

3. The vaccine composition according to claim 2, characterized in that The vaccine composition is an inactivated vaccine composition; and / or The raw materials of the vaccine composition include the immunogen and adjuvants.

4. The vaccine composition according to claim 2, characterized in that In the vaccine composition with G1 as the immunogen, the avian influenza virus strain: lipid adjuvant: first emulsifier: second emulsifier = 94×10 8.0-11.0 EID 50 : 100-300g: 5-15g: 5-8g; In the vaccine composition with G2 as the immunogen, the avian influenza virus strain: the La Sota strain of Newcastle disease: the lipid adjuvant: the first emulsifier: the second emulsifier = 96×10 8.0-11.0 EID 50 :96×10 8.0-11.0 EID 50 : 100-300g: 5-15g: 3-5g; The avian influenza virus strain is an inactivated avian influenza virus strain; the chicken Newcastle disease La Sota strain is an inactivated chicken Newcastle disease La Sota strain; EID 50 It is calculated based on the amount of virus used before inactivation.

5. The vaccine composition according to claim 3 or 4, characterized in that Selected from any one or combination of the following: The oil adjuvant is selected from white oil; The first emulsifier is selected from Siben-80; The second emulsifier is selected from Tween-80; In the vaccine composition with G1 as the immunogen, the avian influenza virus strain: lipid adjuvant: first emulsifier: second emulsifier = 94×10 9.5 EID 50 : 190g: 10g: 6g; In the vaccine composition with G2 as the immunogen, the avian influenza virus strain: the La Sota strain of Newcastle disease: the lipid adjuvant: the first emulsifier: the second emulsifier = 96×10 9.5 EID 50 :96×10 9.0 EID 50 : 190g: 10g: 4g; In the vaccine composition with G1 as the immunogen, the content of the avian influenza virus strain is 10 7.0-10.0 EID 50 / 0.1ml; In the vaccine composition with G2 as the immunogen, the content of the avian influenza virus strain is 10 7.0-10.0 EID 50 / 0.1ml; the content of the chicken Newcastle disease La Sota strain is 10 8.0-10.0 EID 50 / 0.1ml; The preparation method of the inactivated avian influenza virus strain is as follows: adding a formaldehyde solution with a final concentration of 0.1-0.2% to the avian influenza virus strain, inactivating the virus at 35-38° C. for 18-24 hours; The preparation method of the inactivated La Sota strain of Newcastle disease of chickens is as follows: adding a formaldehyde solution with a final concentration of 0.1-0.2% to the La Sota strain of Newcastle disease of chickens, inactivating it at 35-38° C. for 18-24 hours; The EID 50 It is calculated based on chicken embryo culture according to the Reed-Muench method.

6. A method for preparing the vaccine composition according to any one of claims 2 to 5, wherein in the vaccine composition using G1 as an immunogen, the preparation method comprises: using the avian influenza virus strain according to claim 1 as an immunogen to prepare the vaccine composition; In the vaccine composition using G2 as an immunogen, the preparation method is: using the avian influenza virus strain described in claim 1 and the La Sota strain of Newcastle disease as immunogens to prepare the vaccine composition.

7. The preparation method according to claim 6, characterized in that: The preparation method comprises: mixing the immunogen with auxiliary materials to prepare the vaccine composition.

8. The preparation method according to claim 6, characterized in that: The preparation method comprises the following steps: (a) Preparation of oil phase adjuvant: Mixing the oil adjuvant with the first emulsifier to obtain an oil phase adjuvant; (b) Preparation of aqueous phase: mixing the immunogen with a second emulsifier to obtain an aqueous phase; (c) Mixing: The oil phase adjuvant is mixed with the water phase to obtain the vaccine composition.

9. Use of the avian influenza virus strain according to claim 1 or the vaccine composition with G1 as the immunogen according to any one of claims 2 to 5 in the preparation of a preparation for use alone, in combination with other immune preparations and / or drugs, or as a component of a compound preparation composed of other immune preparations and / or drugs to prevent, mitigate and / or control avian influenza.

10. Use of the vaccine composition with G2 as the immunogen according to any one of claims 2 to 5 in the preparation of a preparation for use alone, in combination with other immune preparations and / or drugs, or as a component of a compound preparation composed of other immune preparations and / or drugs to prevent, mitigate and / or control avian influenza and Newcastle disease.

Images