Poultry six-joint inactivated vaccine as well as preparation method and application thereof

Abstract

The invention provides a six-joint inactivated vaccine for poultry as well as a preparation method and application of the six-joint inactivated vaccine, and belongs to the technical field of veterinary vaccines. The six-combined inactivated vaccine for poultry is prepared from an inactivated newcastle disease virus antigen concentrated solution, an inactivated infectious bronchitis virus antigen concentrated solution, an inactivated H3 subtype avian influenza virus antigen concentrated solution, an inactivated H9 subtype avian influenza virus antigen concentrated solution, an inactivated I-group 4-type avian adenovirus solution, an inactivated H3 subtype avian influenza virus antigen concentrated solution, an inactivated H3 subtype avian influenza virus antigen concentrated solution, an inactivated H3 subtype avian influenza virus antigen concentrated solution, an inactivated H3 subtype avian influenza virus antigen concentrated solution and an inactivated H3 subtype avian influenza virus antigen concentrated solution. The inactivated avian infectious laryngotracheitis virus gD protein liquid is pretreated, then mixed according to a step-by-step mixing method and emulsified, and the chicken infectious laryngotracheitis virus gD protein liquid is prepared. According to the vaccine, through the innovative preparation process and technical means, the optimization of the antigen proportion and the mixing sequence and the adjustment of reaction environment parameters, the structural change or antigenicity reduction caused by the interaction between antigens is effectively avoided, the stable and effective structure of each antigen in the vaccine can be ensured, and finally the immune effect of the vaccine is not influenced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of veterinary vaccines, and particularly relates to a poultry hexavalent inactivated vaccine and a preparation method and application thereof. BACKGROUND

[0002] There are various infectious diseases of poultry. With the expansion of the breeding scale and the increase of the breeding density of breeding enterprises, the prevention and control pressure of poultry diseases is increasing. In order to cope with the increasing epidemic prevention pressure, the prevention and control procedures formulated by breeding enterprises are becoming more and more complex, and the chicken flock produces a serious immune burden. Taking a layer chicken farm in Hunan as an example, the layer chickens have to undergo 35 times of immunization from birth to 62 weeks of age in the immunization program formulated by the farm. For the five diseases of Newcastle disease, infectious bronchitis, avian influenza, avian adenovirus disease and infectious laryngotracheitis, a total of 10 times of inactivated vaccine immunization and 14 times of live vaccine immunization are required, which is heavy immunization burden and high cost.

[0003] Newcastle disease (ND) is an acute and highly contagious avian infectious disease caused by a virulent strain of Newcastle disease virus, which is a Class II animal disease regulated by the Ministry of Agriculture and Rural Affairs of China. The attenuated strain of genotype II (La Sota strain) can be used as a vaccine for the prevention and control of ND, but the main genotype currently prevalent in China is genotype VII Newcastle disease, which is also the most serious genotype. However, SPF chickens immunized with La Sota strain cannot provide complete protection against the genotype VII virulent strain isolated in China.

[0004] Chicken infectious bronchitis (IB) is a common acute and highly contagious respiratory viral disease of chickens caused by infectious bronchitis virus. Currently, vaccines are still the main measure to control the prevalence of IB. IBV M41 strain has good immunogenicity and stable biological characteristics, and is the main vaccine strain for the production of inactivated vaccines containing IBV antigen components in China. However, during the production of IBV antigen, the problem of low titer often occurs, which leads to low test results of IB vaccine efficacy and seriously affects the production and application of IB vaccine.

[0005] Avian influenza (AI) is a kind of infection or disease syndrome caused by avian influenza virus, which is an important zoonosis and poses a serious threat to poultry industry and human health. H3 subtype avian influenza virus and H9 subtype avian influenza virus are widely prevalent in poultry in China. The antigens of H3 subtype avian influenza virus have irregularity, mainly in the following aspects:

[0006] (1) High variability: The antigens of H3 subtype avian influenza virus (especially the hemagglutinin HA protein) have high variability, which constantly evolves through antigenic drift and antigenic shift, enabling it to evade the recognition of the host immune system.

[0007] (2)Cross-species transmission: H3 subtype avian influenza viruses not only infect birds, but also infect mammals (such as pigs, horses, humans, etc.), and this cross-species transmission ability makes its antigenic characteristics diverse in different hosts.

[0008] (3)Emergence of new strains: Since December 2022, a new H3N8 subtype avian influenza virus has emerged in chicken flocks, and it has been prevalent in chicken flocks in many provinces in China, posing an increasing threat to the poultry industry. There is an urgent need for a vaccine that can prevent new H3 subtype avian influenza.

[0009] The antigen of H3 subtype avian influenza virus has high variability, cross-species transmission ability, and immune escape characteristics, making it difficult to develop a vaccine. In particular, the emergence of new H3N8 subtype avian influenza viruses in recent years has further increased the complexity of vaccine development. Currently, vaccine development against H3 subtype avian influenza virus is still in the exploratory stage, and no mature antigen component has been included in the combined vaccine.

[0010] H9 subtype avian influenza virus evolves into a more pathogenic virus through recombination with other influenza viruses by providing internal genes. Controlling the prevalence of H9 subtype avian influenza virus in poultry is an important measure to prevent the emergence of new avian influenza viruses.

[0011] Fowl adenovirus disease (FAD) is a disease caused by group I fowl adenovirus, among which serotype 4 (FAdV-4) has the characteristics of fast transmission speed and wide transmission range in poultry, causing serious economic losses to the poultry industry. It mainly causes a pericardial effusion-hepatitis syndrome in poultry, characterized by pericardial effusion and hepatitis, leading to high mortality.

[0012] Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease caused by the chicken infectious laryngotracheitis virus, but there is no effective treatment method at present, and it can only be controlled by good biosecurity management and vaccination. Currently, the ILT live attenuated vaccine has been used for decades, but individual chickens may experience adverse reactions such as ophthalmia, cough, and egg production decline after vaccination, posing certain safety risks.

[0013] The currently marketed joint vaccine mainly contains a series of inactivated vaccines of chicken Newcastle disease virus La Sota strain. The antigen matching between the Lasota strain vaccine and the domestic prevalent Newcastle disease virus gene type VII may be relatively low, which may lead to incomplete immune protection efficacy and cannot fully meet the current new demand for vaccines in Newcastle disease prevention and control (such as reducing the excretion of immune chickens). Although the Lasota strain vaccine has been widely used in history, with the variation of Newcastle disease virus and the development of new vaccine technology, it may have some limitations in antigen matching, immune protection efficacy, disease monitoring and prevention and control demand. The currently marketed inactivated vaccine containing Newcastle disease virus gene type VII is mainly triple vaccine and pentavalent vaccine, and there is no marketed hexavalent inactivated vaccine.

[0014] Moreover, the chicken infectious laryngotracheitis virus currently mainly relies on attenuated live vaccines for prevention and control, but such vaccines have certain safety risks, such as the possibility of causing adverse reactions such as ophthalmia, cough and egg production decline after vaccination. The development of joint vaccine needs to ensure the compatibility between each antigen component and avoid immune interference. The antigenic characteristics of H3 subtype avian influenza virus and infectious laryngotracheitis virus are complex, and the synergistic effect with other common avian disease antigens (such as Newcastle disease, H9 subtype avian influenza, avian adenovirus disease and chicken infectious bronchitis) is not fully clear, which increases the technical difficulty of joint vaccine development. Therefore, how to simultaneously contain H3 subtype avian influenza virus and infectious laryngotracheitis virus antigen components in the joint vaccine is imminent. SUMMARY

[0015] The present application provides a kind of poultry hexavalent inactivated vaccine and its preparation method and application. The vaccine is prepared by innovative preparation process and technical means, optimizes antigen proportion and mixing order, adjusts reaction environment parameters, effectively avoids the structural change or antigenicity reduction caused by the interaction between antigens, ensures that each antigen in the vaccine can maintain stable and effective structure, and finally ensures that the immune effect of the vaccine is not affected.

[0016] In order to achieve the above purpose, the present application provides a kind of poultry hexavalent inactivated vaccine, by inactivated chicken Newcastle disease virus antigen concentrate, inactivated chicken infectious bronchitis virus antigen concentrate, inactivated H3 subtype avian influenza virus antigen concentrate, inactivated H9 subtype avian influenza virus antigen concentrate, inactivated group I 4 type avian adenovirus liquid, inactivated chicken infectious laryngotracheitis virus gD protein liquid are pretreated, then mixed by step-by-step mixing method after emulsification, it can be prepared.

[0017] As preferred, the volume ratio of the added inactivated chicken Newcastle disease virus antigen concentrate, inactivated chicken infectious bronchitis virus antigen concentrate, inactivated H3 subtype avian influenza virus antigen concentrate, inactivated H9 subtype avian influenza virus antigen concentrate, inactivated group I 4 type avian adenovirus liquid, and inactivated chicken infectious laryngotracheitis virus gD protein liquid is 3:3:5:3:2:4.

[0018] As preferred, the pretreatment specifically is:

[0019] 0.1% trehalose is added to the H3 subtype avian influenza virus antigen concentrate and the H9 subtype avian influenza virus antigen concentrate respectively, and is pre-equilibrated at 4°C for 1 hour to maintain the stability of the HA protein conformation;

[0020] 5mM EDTA chelated metal ions are added to the chicken infectious laryngotracheitis virus gD protein antigen concentrate to avoid gD protein oxidation dimerization.

[0021] As preferred, the virus used in the inactivated chicken Newcastle disease virus antigen concentrate is the attenuated strain NDV-Ⅶb strain of genotype Ⅶ Newcastle disease virus Avulavirus Newcastle disease virus , which has been preserved in the China Center for Type Culture Collection in Wuhan on October 15, 2019, with the preservation number CCTCC NO: V201968.

[0022] The virus used in the inactivated chicken infectious bronchitis virus antigen concentrate is the infectious bronchitis M41 strain; The virus used in the inactivated H3 subtype avian influenza virus antigen concentrate is the H3 subtype avian influenza virus YB-GD strain Influenza A virus Avian influenza virus , which has been preserved in the China General Microbiological Culture Collection Center in Beijing on March 11, 2025, with the preservation number CGMCC NO: 46406; The virus used in the inactivated H9 subtype avian influenza virus antigen concentrate is the H9 subtype avian influenza virus YBF18 strain Influenza A virus Avian influenza virus , which has been preserved in the China General Microbiological Culture Collection Center in Beijing on March 11, 2025, with the preservation number CGMCC NO: 46405; The virus used in the inactivated group I 4 type avian adenovirus liquid is the avian adenovirus YBAV-4 strain of group I 4 type avian adenovirus Aviadenovirus Fowl adenovirus , which has been preserved in the China Center for Type Culture Collection in Wuhan on October 15, 2015, with the preservation number CCTCC NO: V201541.

[0026] The inactivated chicken infectious laryngotracheitis virus gD protein liquid is obtained by centrifuging the gD protein liquid of the engineering cell strain CHO-gD at 4℃ and 8000-8500 r / min for 10-15 min when the cell viability is reduced to 70-80%, and taking the supernatant.

[0027] The application further provides a preparation method of the avian hexavalent inactivated vaccine according to any one of the above technical solutions, comprising the following steps:

[0028] The chicken Newcastle disease virus antigen, the chicken infectious bronchitis virus antigen, the H3 subtype avian influenza virus antigen, the H9 subtype avian influenza virus antigen, the group I 4-type avian adenovirus antigen and the chicken infectious laryngotracheitis virus gD protein antigen are preheated to 35-36℃ respectively;

[0029] The H3 subtype avian influenza virus antigen and the H9 subtype avian influenza virus antigen are mixed at a volume ratio of 5:3 to obtain a mixed liquid 1;

[0030] The chicken Newcastle disease virus antigen, the chicken infectious bronchitis virus antigen and the group I 4-type avian adenovirus antigen are mixed at a volume ratio of 3:3:2 to obtain a mixed liquid 2, and then the mixed liquid 1 and the mixed liquid 2 are mixed at a volume ratio of 1:1 to obtain a mixed liquid 3;

[0031] The mixed liquid 3 and the chicken infectious laryngotracheitis virus gD protein antigen are mixed at a volume ratio of 4:1 to make the content of the chicken infectious laryngotracheitis virus gD protein in the mixed liquid be 120 μg / ml, and a mixed liquid 4 is obtained;

[0032] 96 parts of white oil and 1 part of aluminum stearate are uniformly mixed in an oil phase preparation tank and heated to 80℃, then 4 parts of Span-80 is added, and when the temperature reaches 115℃, it is maintained for 40 min, and then the temperature is reduced to 25℃ to be used as the oil phase;

[0033] The mixed liquid 4 and Tween-80 are mixed at a volume ratio of 95:5, and when the system temperature is reduced to 25℃, the water phase temperature is controlled at 20-25℃, 3 parts of the oil phase is introduced into an emulsifying tank and stirred, 2 parts of the water phase is added, and low-speed premixing and high-speed premixing are carried out in sequence, then the sample is centrifuged, and after the centrifuged sample is free of water phase, an oil emulsion is obtained, which is sheared to obtain the corresponding vaccine.

[0034] As preferred, the mixing is carried out at a rotation speed of 200-220 r / min and a temperature of 35-36℃, and the mixing and stirring time is 10-15 min.

[0035] As preferred, the low-speed premixing is carried out at 15-25 Hz for 10-15 min, and the high-speed premixing is carried out at 40-45 Hz for 20-25 min.

[0036] As preferred, before obtaining the mixed solution 2, 0.1% hyaluronic acid is added into the mixed solution of the chicken newcastle disease virus antigen, the chicken infectious bronchitis virus antigen and the group I 4 type avian adenovirus antigen as a steric hindrance agent to reduce the collision between virus particles.

[0037] The application also provides application of the poultry hexa-inactivated vaccine according to any one of the above technical solutions in simultaneously preventing and controlling egg chicken newcastle disease, H3N8 subtype avian influenza, H9 subtype avian influenza, avian adenovirus disease, chicken infectious bronchitis, infectious laryngotracheitis.

[0038] As preferred, the vaccine is inoculated through subcutaneous injection or intramuscular injection of the neck, and the inoculation dose is 0.5 ml per chicken. 21 days after inoculation, the geometric mean of the serum HI antibody titer of the partially immunized chickens of the chicken newcastle disease is 8.6 log2; the geometric mean of the serum HI antibody titer of the partially immunized chickens of the chicken infectious bronchitis is 7.7 log2; the geometric mean of the serum HI antibody titer of the partially immunized chickens of the H3 subtype avian influenza is 9.5 log2, and 10 / 10 virus isolation of the immunized chickens is negative 5 days after challenge; the geometric mean of the serum HI antibody titer of the partially immunized chickens of the H9 subtype avian influenza is as high as 9.2 log2, and 10 / 10 virus isolation of the immunized chickens is negative 3 days after challenge; 10 / 10 of the partially immunized chickens of the group I 4 type avian adenovirus disease are protected after challenge; and 10 / 10 of the partially immunized chickens of the chicken infectious laryngotracheitis are protected after challenge.

[0039] Compared with the prior art, the application has the following advantages and positive effects:

[0040] 1. Reducing the immune burden and cost: The current vaccination program of breeding enterprises is complex, and the immune burden and cost are high. The application aims to expand the coverage of the combined vaccine to prevent multiple important poultry diseases at the same time, reduce the number of immunizations in the breeding farm, and reduce the immune burden and cost, thereby providing a more efficient and more economical solution for the prevention and control of poultry diseases.

[0041] 2. Improving the antigen matching and immune protection efficacy of the newcastle disease vaccine: Since the gene II type weak strain (La Sota strain) cannot provide complete protection against the gene VII type newcastle disease strong strain prevalent in China, the antigen matching of the series of inactivated vaccines containing the La Sota strain on the market may be low. The vaccine to be developed in the application needs to improve the prevention and control ability of the gene VII type newcastle disease, meet the new needs of reducing the virus shedding of the immunized chicken population, and enhance the immune protection efficacy.

[0042] 3. Solving the problem of low toxicity of the infectious bronchitis virus antigen: In view of the problem that the toxicity of the IBV antigen is often not high during production, resulting in low test results of the IB vaccine efficacy and seriously affecting the production and application of the vaccine, the application is dedicated to improving the toxicity of the infectious bronchitis virus antigen in the vaccine to ensure the efficacy of the vaccine.

[0043] 4. Fill in the blank of H3 subtype avian influenza virus antigen: In view of the high variability of H3 subtype avian influenza virus antigen, the ability of cross-species transmission and the immune escape characteristics, the present application develops an antigen component with broad-spectrum protective efficacy by screening H3 subtype avian influenza virus antigen, fills in the blank of the lack of H3 subtype avian influenza virus antigen in the existing combined vaccine, and effectively responds to the epidemic threat of new H3N8 subtype avian influenza virus.

[0044] 5. Prevention and control of H9 subtype avian influenza virus: Considering that H9 subtype avian influenza virus can evolve into more pathogenic viruses by providing internal genes and recombination with other influenza viruses, the present application controls the prevalence of H9 subtype avian influenza virus in poultry by including it in the hexavalent vaccine, and prevents and controls the generation of new avian influenza viruses.

[0045] 6. Reduce the loss of avian adenovirus disease: In view of the problem that serotype 4 (FAdV-4) in group I avian adenovirus spreads rapidly and widely in poultry, causes pericardial effusion-hepatitis syndrome, leads to high mortality, and causes serious economic losses to poultry farming, the vaccine of the present application can effectively prevent avian adenovirus disease and reduce economic losses.

[0046] 7. Solve the safety risk of infectious laryngotracheitis vaccine: In view of the fact that individual chickens may develop adverse reactions such as ophthalmia, cough and egg production decline after vaccination with the current live attenuated vaccine for infectious laryngotracheitis, there is a safety risk, the hexavalent inactivated vaccine of the present application does not contain such risks, and there is no infectious laryngotracheitis virus antigen component in the currently marketed combined vaccine, the present application fills in this blank and provides a new choice for the prevention and control of infectious laryngotracheitis.

[0047] 8. Overcome the antigen compatibility problem: The hexavalent inactivated vaccine contains multiple antigens, and there may be interactions between different antigens. It is necessary to determine the optimal content of each antigen so that it can produce sufficient immune response and will not cause excessive immune response or adverse reactions, affecting the immune effect of the vaccine. The present application is dedicated to solving the problem of mutual interference of different pathogen antigens when combined. The vaccine of the present application optimizes the antigen ratio and mixing order, adjusts the reaction environment parameters through innovative preparation process and technical means, effectively avoids the structural change or antigenicity reduction caused by the interaction between antigens, ensures that each antigen in the vaccine can maintain stable and effective structure, and finally ensures that the immune effect of the vaccine is not affected.

[0048] 9. Simplify and optimize the production process: In order to solve the problem of complex production process of hexavalent inactivated vaccine, the present application is dedicated to developing an efficient and integrated production process. The optimal culture conditions, inactivation methods and subsequent treatment requirements of each pathogen are systematically studied, and through technical innovation and equipment upgrading, the organic integration of antigen culture, inactivation, concentration, purification and other links of different pathogens is realized. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 SDS-PAGE results of protein centrifugal purification (left) and chromatography purification (right) provided by the embodiments of the present application;

[0050] Figure 2 The schematic diagram of the production process of the hexavalent inactivated vaccine provided by the embodiments of the present application. DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0052] Example 1: Study on the propagation process of chicken infectious bronchitis virus M41 strain

[0053] 1. Virus and chicken embryo: chicken infectious bronchitis virus M41 strain (CVCC AV1511) was from China Institute for Control of Veterinary Drug; susceptible chicken embryos were provided by Qingdao Chengyang Kangxin Poultry Breeding Farm.

[0054] 1.1 Determination of inoculation day of chicken embryo and dilution multiple of virus: the virus was diluted by 50, 100 and 500 times, respectively, and 100 susceptible chicken embryos of 9, 10 and 11 days old were inoculated in the allantoic cavity, 0.1 ml per embryo, and then incubated at 36-37℃. The chicken embryos were observed once a day for 24 hours, and the dead chicken embryos were removed. Thereafter, the chicken embryos were observed once every 12 hours, and the dead chicken embryos were removed in time. The chicken embryos were incubated until 48 hours, and then all the chicken embryos were removed. The average harvest amount of chicken embryo liquid was calculated, and the EID 50 of the chicken embryo liquid was determined.

[0055] Table 1: Test results of inoculation day of chicken embryo and dilution multiple

[0056]

[0057] From the data in Table 1, it can be seen that there is no obvious difference in virus harvest amount and EID 50 when the chicken infectious bronchitis virus M41 strain is propagated by using 10 and 11 day old chicken embryos, and the virus harvest amount is higher than that when 9 day old chicken embryos are used (10.7-11.0 ml). Therefore, 10 and 11 day old chicken embryos are selected to propagate the chicken infectious bronchitis virus M41 strain.

[0058] 1.2 Determination of the optimal harvest time:

[0059] After diluting the virus according to the dilution factor selected in 1.1, 0.1 ml of the diluted virus was inoculated into the allantoic cavity of 400 susceptible chicken embryos of the selected age in 1.1. 100 embryos were harvested at 36, 48, 50, and 72 hours, and the EID of the embryonic fluid was measured. 50 .

[0060] Table 2 Results of the Optimal Harvest Time Experiment

[0061]

[0062]

[0063] As shown in Table 2, the optimal harvest time for the M41 strain of infectious bronchitis virus in chickens is 48–50 hours.

[0064] 1.3 Determination of Optimal Incubation Humidity: After diluting the virus according to the dilution ratio selected in 1.1, 500 susceptible chicken embryos of the selected age in 1.1 were inoculated into the allantoic cavity at a rate of 0.1 ml / embryo. The embryos were incubated at 36–37°C with incubation humidity conditions of 58%, 60%, 62%, 65%, and 70%, with 100 embryos placed under each humidity condition. Candling was performed once every 24 hours, and dead embryos were discarded. Candling was then performed every 12 hours thereafter until the optimal harvest time determined based on the experimental results in 1.2. All embryos were then removed, and the embryonic fluid was harvested. The state of the embryonic fluid was observed, the average yield of the embryonic fluid was calculated, and the EID of the embryonic fluid was measured. 50 .

[0065] Table 3 Determination of Optimal Incubation Humidity

[0066]

[0067] As shown in Table 3, after inoculating susceptible chicken embryos with the virus strain and incubating them under humidity conditions of 58%–62%, the harvested chicken embryo liquid was clearer and the yield was greater. The EID of the chicken embryo liquid was lower. 50 10 -6.3 ~10 -6.5 / 0.1ml, based on this, the optimal post-incubation humidity after inoculation of chicken embryos with the M41 strain of infectious bronchitis virus was determined to be 60% ± 2%.

[0068] 1.4 Determination of the optimal inoculation dose: The virus was diluted according to the dilution ratio selected in 1.1, and 100 susceptible chicken embryos of the selected age in 1.1 were inoculated intraallantoically at 0.2 ml / embryo and 0.1 ml / embryo, respectively. The embryos were incubated at 36–37°C and the optimal post-incubation humidity selected in 1.3. The embryonic fluid was harvested at the optimal harvest time determined based on the experimental results in 1.2, and the EID of the embryonic fluid was measured. 50 .

[0069] Table 4 Determination of Optimal Vaccine Dosage

[0070]

[0071] From the data of Table 4, it can be seen that the EID50 of the harvested chick embryo fluid of the chicken infectious bronchitis virus M41 strain was 10 50 The difference was not great, and the virus proliferation effect was equivalent, so in large-scale virus culture, it is recommended to inoculate 0.1 ml / embryo.

[0072] Example 2: Study on the expression and purification process of the gD protein of the chicken infectious laryngotracheitis virus:

[0073] 2.1 Determination of the initial cell density for flow culture in the reactor:

[0074] The engineering cell strain CHO-gD was subcultured into the reactor at an initial cell density of 2.0 x 10 6 cells / ml, 2.5 x 10 6 cells / ml, and 3.0 x 10 6 cells / ml, pH 7.0-7.2, temperature 37°C, dissolved oxygen 35%-45%, clean air was supplied to the surface layer to maintain positive pressure, and the rotation speed was adjusted to a suitable value according to the performance requirements of the 5L bioreactor. Cell counting was performed daily, and cell viability and glucose concentration were detected. The glucose concentration was controlled at 4-6 g / L, and if it was lower than this range, flow addition of the medium was performed according to the glucose concentration. When the cell density reached 1.0 x 10 7 -1.2 x 10 7 cells / ml, the temperature was lowered to 32°C for continued culture. Cell counting was performed daily, and cell viability and glucose concentration were detected. Flow addition of the medium was performed daily according to the glucose concentration. If the glucose concentration was lower than 2 g / L, the flow rate of the flow culture medium was adjusted according to the volume ratio to control the glucose concentration at 4-6 g / L. When the cell viability decreased to 70%-80%, part of the culture solution was aseptically harvested. The supernatant was harvested by centrifugation at 8000-8500 r / min at 4°C for 10-15 minutes, and the gD protein solution was obtained. The protein content and purity were detected by the Bradford method and SDS-PAGE method.

[0075] Table 5: Content and purity of gD protein expressed at different initial cell densities for flow culture

[0076]

[0077] As shown in Table 5, the gD protein expressed at an initial cell density of 2.0 x 10 6 cells / ml and 2.5 x 10 6The gD protein content expressed by 2.0 x 10 6 The gD protein content expressed by 3.0 x 10 6 The gD protein content expressed by 2.0 x 10 6 The gD protein content expressed by 2.0 x 10

[0078] 2.2 Determination of the purification method of the gD protein of the chicken infectious laryngotracheitis virus: according to the determined fed-batch culture process, the fed-batch culture of the engineering cell strain CHO-gD was carried out, when the cell viability decreased to the specified range, the rotation speed of the bioreactor was decreased to 0, the temperature was decreased to 4°C, and after the cells were settled, the cell culture supernatant was directly harvested as the unpurified gD protein solution.

[0079] 2.2.1 Centrifugal purification: 300 ml of the unpurified gD protein solution was centrifuged at 8000-8500 r / min for 10-15 minutes at 4°C, and the supernatant was taken as the centrifugally purified gD protein solution.

[0080] 2.2.2 Chromatographic purification: 100 ml of the centrifugally purified gD protein solution in 2.2.1 was subjected to affinity chromatography using a Ni-NTA superflow column at 4°C.

[0081] 2.2.3 Protein content and purity determination: the centrifugally purified gD protein solution in 2.2.1, the chromatographically purified gD protein solution in 2.2.2 and the unpurified gD protein solution were subjected to total protein content and purity determination using the Bradford method and the SDS-PAGE method, respectively.

[0082] Table 6 gD protein content and purity determination results

[0083]

[0084] As shown in Table 6, Figure 1 The data show that the protein purity of the centrifugally purified protein, the chromatographically purified protein and the unpurified protein is 47.8%, 88.2% and 31.5%, respectively, the gD protein content produced is 629.1 μg / ml, 248.4 μg / ml and 651.1 μg / ml, respectively, compared with the unpurified protein, the gD protein purity after centrifugal purification is increased by 16.3%, and the gD protein content is only decreased by 22.0 μg / ml; while compared with the centrifugal purification, the protein purity after chromatographic purification is increased by 40.4%, but the protein content is decreased by 380.7 μg / ml.

[0085] 2.2.4 Preparation of vaccines: The centrifugally purified gD protein solution in 2.1, the chromatographically purified gD protein solution in 2.2 and the unpurified gD protein solution were diluted with physiological saline to a gD protein content of 120 μg / ml, respectively, inactivated, and then emulsified with mineral oil as an adjuvant to prepare vaccines at a water phase:oil phase ratio of 1:2.

[0086] 2.2.5 Safety test: 40 SPF chickens at the age of 21 days were divided into 4 groups, 10 chickens in each group, 5 chickens in each group were injected subcutaneously at the neck and 5 chickens were injected intramuscularly with 1.0 ml of each of the three vaccines prepared in 2.4, respectively, and 10 chickens were not inoculated as a control. The chickens were observed for 14 days, and the general conditions and the injection sites after inoculation were recorded. After 14 days, the chickens were dissected to observe the absorption of the vaccines. The results showed that after the SPF chickens were immunized with the three vaccines, the chickens were normal in terms of food intake, water drinking and spirit, and no adverse reactions were observed at the injection sites, and the injection sites were well absorbed.

[0087] 2.2.6 Immune efficacy test: 40 SPF chickens at the age of 21 days were divided into 4 groups, 10 chickens in each group, 5 chickens in each group were injected subcutaneously at the neck and 5 chickens were injected intramuscularly with 0.25 ml of each of the three vaccines prepared in 2.4, respectively, and the remaining 10 chickens were not immunized as a control. After 21 days, each chicken was injected intratracheally with ILTV Wanggang strain, 0.2 ml per chicken (containing 10 4.0 EID 50 ), and the chickens were continuously observed for 14 days after the challenge, and the disease conditions of the test chickens were recorded.

[0088] The results showed that the 21-day-old SPF chickens were immunized with the three vaccines prepared from chromatographically purified, centrifugally purified and unpurified proteins, respectively, at a dose of 0.25 ml per chicken, and the chickens were challenged after 21 days. The results showed that 8 / 10 or more of the chickens were protected after immunization with the three vaccines for 21 days, and 8 / 10 of the chickens in the control group were sick. The results indicated that there was little difference in the immune efficacy of the vaccines prepared from gD proteins purified by different methods.

[0089] To meet the needs of actual production, the cost, time, equipment and whether it is suitable for large-scale production were comprehensively considered, and finally it was determined that the vaccine protein was purified by centrifugation, i.e. the engineering cell strain CHO-gD was flow-cultured until the cell viability decreased to 70% to 80%, then centrifuged at 4°C and at 8000 to 8500 r / min for 10 to 15 minutes, and the supernatant was taken as the centrifugally purified gD protein solution. As shown in Figure 1 .

[0090] Preparation of hexavalent inactivated vaccine

[0091] 3.1 Preparation method of six antigens in hexavalent inactivated vaccine

[0092] Preparation method of chicken Newcastle disease virus antigen

[0093] Take the gene type VII newcastle disease virus weak strain, use sterile PBS to make 10 -4 Dilution, inoculate 10-11 day-old susceptible chicken embryos, 0.1 ml per embryo, and incubate at 37℃. After inoculation, the embryo was irradiated every day, and the dead embryo was discarded. After that, the dead embryo was taken out at any time, and all the embryos were incubated at 2-8℃ for 16 hours. The cooled embryos were taken out, and the embryo fluid was harvested. Open the concentrator, flush before concentration, and detect the pH value. The pH value of 6.4-7.0 is qualified. Concentrate to 1 / 4 of the original volume, sample the concentrated virus solution, and ensure that the HA titer after concentration should not be less than 1:2048. The concentrated virus solution was introduced into the inactivation tank, 10% formaldehyde solution was added, the final concentration of formaldehyde solution was 0.1%, and stirring for 5 minutes. Inactivate at 35℃-36℃, the virus solution starts to warm up. After inactivation, the intermittent stirring is automatically switched to intermittent stirring with a power of 17 Hz. After inactivation for 14-15 hours, it was taken out and stored at 2-8℃.

[0094] Among them, the gene type VII newcastle disease virus weak strain NDV-Ⅶb strain has been preserved in China Typical Culture Collection Center located in Wuhan on October 15, 2019, and the preservation number is CCTCC NO: V201968.

[0095] Preparation method of chicken infectious bronchitis virus antigen:

[0096] Take chicken infectious bronchitis virus (M41 strain (CVCC AV1511)), use sterile PBS to make 10 -2 Dilution, inoculate 10-11 day-old susceptible chicken embryos, 0.1 ml per embryo, and incubate at 37℃. After inoculation, the embryo was irradiated every day, and the dead embryo was discarded. After that, the dead embryo was taken out at any time, and all the embryos were incubated at 2-8℃ for 16 hours. The cooled embryos were taken out, and the embryo fluid was harvested. Open the concentrator, flush before concentration, and detect the pH value. The pH value of 6.4-7.0 is qualified. Concentrate to 1 / 4 of the original volume, sample the concentrated virus solution, and ensure that the HA titer after concentration should not be less than 1:2048. The concentrated virus solution was introduced into the inactivation tank, 10% formaldehyde solution was added, the final concentration of formaldehyde solution was 0.1%, and stirring for 5 minutes. Inactivate at 35℃-36℃, the virus solution starts to warm up. After inactivation, the intermittent stirring is automatically switched to intermittent stirring with a power of 17 Hz. After inactivation for 14-15 hours, it was taken out and stored at 2-8℃.

[0097] Preparation method of H3 subtype avian influenza virus antigen:

[0098] Take H3 subtype avian influenza virus, use sterile PBS to make 10 -4Dilution, inoculation of 10-11 day old susceptible chicken embryos, each embryo allantoic cavity inoculation of 0.1 ml, placed at 37℃ continue to incubate. After inoculation every day embryo, incubation to 72 hours, discard the dead chicken embryos, live embryos placed at 2-8℃ cooling 16 hours. The cooled chicken embryos were taken out, harvest the chicken embryo fluid, concentrated to the original volume of 1 / 4, sampling of concentrated virus liquid, ensure that the HA titer after concentration should not be less than 1:1024. The concentrated virus liquid was introduced into the inactivation tank, 10% formaldehyde solution was added, the final concentration of formaldehyde solution was 0.1%, and the inactivation was carried out at 35-36℃ for 14-15 hours. Then, the inactivated virus was taken out and stored at 2-8℃.

[0099] The H3 subtype avian influenza virus YB-GD strain is a representative epidemic strain isolated from a chicken flock in Guangdong Province by Qingdao Yibang Biological Engineering Co., Ltd. through molecular epidemiological investigation of H3 subtype avian influenza virus in China. It was deposited at the China General Microbiological Culture Collection Center in Beijing on March 11, 2025, and the deposit number is CGMCC NO: 46406.

[0100] Preparation method of H9 subtype avian influenza virus antigen:

[0101] Take H9 subtype avian influenza virus, use sterile PBS to make 10 -4 Dilution, inoculation of 10-11 day old susceptible chicken embryos, each embryo allantoic cavity inoculation of 0.1 ml, placed at 37℃ continue to incubate. After inoculation every day embryo, incubation to 72 hours, discard the dead chicken embryos, live embryos placed at 2-8℃ cooling 16 hours. The cooled chicken embryos were taken out, harvest the chicken embryo fluid, concentrated to the original volume of 1 / 4, sampling of concentrated virus liquid, ensure that the HA titer after concentration should not be less than 1:2048. The concentrated virus liquid was introduced into the inactivation tank, 10% formaldehyde solution was added, the final concentration of formaldehyde solution was 0.1%, and the inactivation was carried out at 35-36℃ for 14-15 hours. Then, the inactivated virus was taken out and stored at 2-8℃.

[0102] The H9 subtype avian influenza virus YBF18 strain is a representative epidemic strain isolated from a chicken flock in Jiangsu Province by Qingdao Yibang Biological Engineering Co., Ltd. through molecular epidemiological investigation of H9 subtype avian influenza virus in China. It was deposited at the China General Microbiological Culture Collection Center in Beijing on March 11, 2025, and the deposit number is CGMCC NO: 46405.

[0103] Preparation method of group I 4 type avian adenovirus antigen:

[0104] The cell density of the suspended LMH-C cells was adjusted to 2.5x10 6.0The I group 4 type fowl adenovirus is taken at 4.0×10

[0105] The I group 4 type fowl adenovirus YBAV-4 strain is preserved in the China Center for Type Culture Collection in Wuhan on October 15, 2015, and the preservation number is CCTCC NO: V201541.

[0106] The preparation method of the chicken infectious laryngotracheitis virus gD protein antigen is as follows:

[0107] The expression chicken infectious laryngotracheitis virus gD protein engineering cell strain is diluted to 3.0×10 6 ~3.5×10 6 cells / ml and added into the reactor for culture, the pH value is 7.0-7.2, the temperature is 37℃, the dissolved oxygen is 35%-45%, and clean air is supplied to maintain the positive pressure on the surface. The cell counting and glucose concentration detection are performed daily, and the flow rate of the added culture medium is adjusted according to the glucose concentration to control the glucose concentration at 4-6g / L. When the cell density reaches 1.0×10 7 ~1.2×10 7 cells / ml, the temperature is reduced to 32℃ for continuous culture. The cell counting, cell viability and glucose concentration detection are performed daily, and the flow rate of the added culture medium is adjusted according to the glucose concentration, for example, if the glucose concentration is lower than 2g / L, the flow rate of the added culture medium is adjusted according to the volume ratio to control the glucose concentration at 4-6g / L. When the cell viability is reduced to 70%-80%, the harvest is performed. The supernatant is harvested by centrifugation at 8000-8500r / min and 4℃ for 10-15 minutes, 10% formaldehyde solution is added into the harvested gD protein liquid, and the formaldehyde solution is mixed thoroughly, the final concentration of the formaldehyde solution is 0.1%, and the inactivation is performed at 2-8℃ for 22-24 hours, then the inactivated product is taken out and stored at 2-8℃.

[0108] The chicken infectious laryngotracheitis virus gD protein is encoded by the optimized gD gene, the T cell epitope peptide Q-Y-I-K-A-N-S-K-F-I-G-I-T-E of tetanus toxin is specifically introduced into the amino acid sequence, and the 205th amino acid is mutated from N to A.

[0109] 3.2 Pretreatment of six antigens in hexavalent inactivated vaccine

[0110] H3 subtype avian influenza virus antigen and H9 subtype avian influenza virus antigen were added with 0.1% trehalose respectively, and pre-equilibrated at 4°C for 1 hour to maintain the stability of HA protein conformation. Chicken infectious laryngotracheitis virus gD protein antigen was added with 5mM EDTA to chelate metal ions to avoid gD protein oxidation dimerization.

[0111] 3.3 Preparation method of hexavalent inactivated vaccine

[0112] Pre-warming: turn on the constant temperature water circulation and stirring (speed 70r / min) of the liquid preparation tank, and pre-warm the six antigens to 35°C-36°C respectively.

[0113] First mixing (avian influenza virus antigen mixture): mix H3 subtype avian influenza virus antigen and H9 subtype avian influenza virus antigen at a volume ratio of 5:3, stir at a speed of 200-220r / min at 35°C-36°C for 10-15 minutes.

[0114] Second mixing: mix chicken Newcastle disease virus antigen, chicken infectious bronchitis virus antigen, and group I 4 type avian adenovirus antigen at a volume ratio of 3:3:2, add 0.1% hyaluronic acid as a steric hindrance agent to reduce the collision between virus particles. Mix the above mixture with the avian influenza virus antigen mixture at a volume ratio of 1:1, stir at a speed of 200-220r / min at 35°C-36°C for 10-15 minutes.

[0115] Third mixing: mix the above mixture with chicken infectious laryngotracheitis virus gD protein antigen at a volume ratio of 4:1 to make the content of chicken infectious laryngotracheitis virus gD protein in the mixture 120μg / ml, stir at a speed of 200-220r / min at 35°C-36°C for 10-15 minutes.

[0116] Water phase preparation: mix the above mixture with Tween-80 at a volume ratio of 95:5, stir at 35°C-36°C for 15 minutes, turn off the constant temperature water circulation and turn on the refrigerated water circulation, when the temperature of the liquid preparation tank drops to 25°C, turn off the refrigerated water circulation and stirring, and control the temperature of the water phase at 20-25°C.

[0117] The emulsification of the hexavalent inactivated vaccine includes: taking 96 parts of white oil and 1 part of aluminum stearate, mixing them uniformly in an oil phase preparation tank, heating to 80°C, then adding Span-80 4 parts, maintaining the temperature at 115°C for 40 minutes when the temperature reaches 115°C, and cooling to 25°C to be used as the oil phase. Take 3 parts of the oil phase and introduce it into the emulsification tank for stirring. The oil phase is added to the emulsification tank through 3 layers of 240 mesh filter screens. Start the premixer and observe the oil phase through the sight glass to ensure that it is in a gentle rolling state. After 1 minute, start adding the water phase. The water phase is filtered and added to the emulsification tank through the flow guide tube to ensure that the water phase falls at the center of the oil phase vortex. The addition speed is controlled at 100 kg / 2-3 minutes. After the addition is complete, mix at low speed for 10-15 minutes at 15-25 Hz. Mix at high speed for 20-25 minutes at 40-45 Hz. After high-speed mixing is completed, take 10 ml of sample from the sampling port of the emulsification tank and centrifuge it (at 3000 r / min) for 15 minutes. The centrifuged sample should have no water phase separation. After the centrifuged sample is qualified, start the pipeline shear machine at a speed of 45-50 Hz and shear the oil emulsion in the emulsification tank into the finished product tank. Start the finished product tank stirring (at a speed of 70 r / min) and stir for 30 minutes. The temperature is controlled at 20-25°C.

[0118] The hexavalent inactivated vaccine is divided into portions, filtered through 3 layers of 80 mesh filter screens into a filling barrel, and sealed after being quantitatively divided.

[0119] The hexavalent inactivated vaccine is stored at low temperature: the vaccine is placed in a 4°C gradient cooling box, and the temperature is reduced from 25°C to 4°C at a rate of 0.5°C / min, maintained for 24 hours, and the thermodynamic equilibrium of each phase is reached to avoid stratification during storage. If Figure 2

[0120] Example 4 Comparative test research on the sequence of the preparation steps of the hexavalent inactivated vaccine

[0121] 4.1 Vaccine preparation: 3 vaccines with different production processes are prepared

[0122] 4.1.1 Experimental group 1: directly mix the inactivated chicken Newcastle disease virus antigen concentrate, inactivated chicken infectious bronchitis virus antigen concentrate, inactivated H3 subtype avian influenza virus antigen concentrate, inactivated H9 subtype avian influenza virus antigen concentrate, inactivated group 4 avian adenovirus liquid, and inactivated chicken infectious laryngotracheitis virus gD protein liquid in a ratio of 3:3:5:3:2:4, and then perform subsequent emulsification and other steps to prepare the vaccine.

[0123] ​4.1.2 Experimental group 2: The inactivated chicken Newcastle disease virus antigen concentrate, the inactivated chicken infectious bronchitis virus antigen concentrate, the inactivated H3 subtype avian influenza virus antigen concentrate, the inactivated H9 subtype avian influenza virus antigen concentrate, the inactivated group I 4 type avian adenovirus liquid, and the inactivated chicken infectious laryngotracheitis virus gD protein liquid are not pretreated, mixed by the step-by-step mixing method, and then subjected to subsequent emulsification and other steps to prepare the vaccine.

[0124] 4.1.3 Experimental group 3: The inactivated chicken Newcastle disease virus antigen concentrate, the inactivated chicken infectious bronchitis virus antigen concentrate, the inactivated H3 subtype avian influenza virus antigen concentrate, the inactivated H9 subtype avian influenza virus antigen concentrate, the inactivated group I 4 type avian adenovirus liquid, and the inactivated chicken infectious laryngotracheitis virus gD protein liquid are pretreated, mixed by the step-by-step mixing method, and then subjected to subsequent emulsification and other steps to prepare the vaccine.

[0125] 4.2 Vaccine testing

[0126] 4.2.1 Properties

[0127] 4.2.1.1 Appearance: Carefully observe the color, state, and uniformity of the vaccine.

[0128] 4.2.1.2 Dosage form: Take a clean pipette, draw a small amount of vaccine, and drop it on the surface of cold water to observe the diffusion.

[0129] 4.2.1.3 Stability: Take 10 ml of vaccine and add it to a centrifuge tube, centrifuge at 3000 r / min for 15 minutes, and observe the water phase separated at the bottom of the tube.

[0130] 4.2.1.4 Viscosity: Test according to the 2020 edition of Chinese Veterinary Pharmacopoeia, Appendix III.

[0131] In terms of property testing, the three vaccines performed consistently in appearance, dosage form, stability, and viscosity, all showing a milky white uniform emulsion, which was water-in-oil type, no water phase was separated after centrifugation, and the viscosity was 35 cP. This indicates that in these basic physical properties, different preparation processes do not produce significant differences, and changes in production processes do not negatively affect the appearance and physical stability of the vaccine.

[0132] 4.2.2 Safety Testing: Forty 21-day-old SPF chickens were divided into four groups of 10 each. Five chickens in each group received a subcutaneous injection in the neck, and five chickens received an intramuscular injection of one of the three vaccines prepared according to steps 1.1, 1.2, and 1.3, at a dose of 1.0 ml per chicken. Ten chickens served as a control group and were not vaccinated. The chickens were observed for 14 days, and the overall condition and the condition at the injection site were recorded. After 14 days, the chickens were dissected to observe vaccine absorption. The results showed that after immunization with the three vaccines, the chickens' feed intake, water consumption, and overall health were normal. No adverse reactions were observed at the injection sites, and the absorption at the injection sites was good. This indicates that the vaccines prepared using the three methods are all highly safe and will not have adverse effects on the chickens.

[0133] 4.2.3 Immunopotency Test:

[0134] 2.3.1 Newcastle Disease in Chickens: Thirty-five 21-day-old SPF chickens were subcutaneously injected in the neck with 20 μl of each of the three vaccines prepared in sections 1.1, 1.2, and 1.3, with 10 chickens per group. Five chickens were not immunized and served as a control group. Twenty-one days post-vaccination, blood was collected from each chicken, serum was separated, and HI antibody titers were determined. Results showed that the geometric mean HI antibody titer in the serum of immunized chickens in experimental group 3 was 7.0 log2, higher than that of experimental group 1 (6.1 log2) and experimental group 2 (6.8 log2). The HI antibody titers in the control group did not exceed 1:4. This indicates that experimental group 3, which used a stepwise mixing method and pretreated each antigen, more effectively stimulated the chickens to produce an immune response against Newcastle disease virus, resulting in higher levels of antibodies. (See Table 7.)

[0135] 4.2.3.2 Infectious Bronchitis in Chickens: Thirty-five 14-day-old SPF chickens were used. Thirty-ten chickens were each nasally inoculated with one dose (0.05 ml) of live infectious bronchitis vaccine (H120 strain). The remaining five chickens served as a control group. Twenty-one days after inoculation, blood was collected from each chicken, and serum was separated. Simultaneously, the immunized chickens were subcutaneously injected in the neck with 0.3 ml of the three vaccines prepared in sections 1.1, 1.2, and 1.3, respectively, at a rate of 10 chickens per group. Twenty-one days after the second immunization, blood was collected again from both the immunized and control chickens, and serum was separated. The HI antibody titer was measured in both serum samples. The results showed that the geometric mean of the HI antibody titer in the serum of the immunized chickens in experimental group 3 was 7.6 log2, which was higher than the 6.5 log2 in experimental group 1 and the 7.5 log2 in experimental group 2. The HI antibody titer in the serum of the control chickens did not exceed 1:4. This further demonstrates that the preparation process in experimental group 3 is more effective in inducing an immune response in chickens against infectious bronchitis virus, thus helping chickens better resist viral infection. See Table 7.

[0136] 4.2.3.3 H3 subtype avian influenza: Forty 21-day-old SPF chickens were subcutaneously injected in the neck with 0.3 ml of one of the three vaccines prepared in sections 1.1, 1.2, and 1.3, respectively, with 10 chickens per group. Another 10 chickens were not immunized and served as a control group. Twenty-one days after vaccination, blood was collected from 10 chickens in each group, serum was separated, and HI antibody titers were measured. Simultaneously, 10 chickens in each group were randomly selected and intravenously injected with 0.2 ml of H3 subtype avian influenza virus solution (containing 10... 6.0 EID 50 Five days after challenge, throat and cloacal swabs were collected, processed, and used for virus isolation. Results showed that the geometric mean titer of HI antibodies in the serum of immunized chickens in experimental group 3 was 7.6 log2, and all 10 immunized chickens tested negative for virus isolation five days after challenge. This was superior to experimental group 1 (antibody titer 6.6 log2, 8 chickens tested negative for virus isolation), experimental group 2 (antibody titer 7.5 log2, 9 chickens tested negative for virus isolation), and the control group (antibody titers not exceeding 1:4, 10 chickens tested positive for virus isolation). This clearly demonstrates that the vaccine prepared in experimental group 3 is highly effective in preventing H3 subtype avian influenza virus infection, not only inducing high levels of antibodies but also effectively reducing the viral infection rate after challenge.

[0137] 4.2.3.4 H9 subtype avian influenza: Forty 21-day-old SPF chickens were subcutaneously injected in the neck with 0.2 ml of each of the three vaccines prepared in sections 1.1, 1.2, and 1.3, with 10 chickens per group. Ten chickens served as a control group. Twenty-one days post-vaccination, blood was collected from ten chickens in each group, serum was separated, and HI antibody titers were measured. Simultaneously, ten chickens in each group were randomly selected and intravenously injected into each wing with 0.2 ml of H9 subtype avian influenza virus solution (containing 2×10⁻⁶ mmol / L). 7.0 EID 50 Three days after challenge, throat and cloacal swabs were collected, processed, and used for virus isolation. Results showed that experimental group 3 exhibited the best performance against the H9 subtype avian influenza virus, with a geometric mean HI antibody titer in the serum of immunized chickens reaching 8.4 log2, significantly higher than the 6.2 log2 in experimental group 1 and 7.0 log2 in experimental group 2. Three days after challenge, all 10 immunized chickens tested negative for virus isolation, compared to only 5 in experimental group 1 and 8 in experimental group 2. Meanwhile, the HI antibody titers in the control chicken serum did not exceed 1:4, and all 10 chickens tested positive for virus isolation after challenge. This indicates that the vaccine in experimental group 3 had the best immunizing effect against the H9 subtype avian influenza virus, significantly enhancing the chickens' resistance to the virus. (See Table 7.)

[0138] 4.2.3.5 Group I Avian Adenovirus Type 4 Disease: Forty 21-day-old SPF chickens were subcutaneously injected in the neck with 0.2 ml of each of the three vaccines prepared in sections 1.1, 1.2, and 1.3, with 10 chickens per group. Ten chickens served as a control group and were not immunized. Twenty-one days after vaccination, each chicken was intramuscularly injected with 0.2 ml of Group I Avian Adenovirus Type 4 YBAV-4 strain virus solution (containing 2×10⁻⁶ spores). 5.5 The vaccine (TCID50) was administered, and the chickens were observed for 7 days after challenge. Results showed that 10 chickens in experimental group 3 were protected after challenge, achieving a 100% protection rate, while only 6 chickens in experimental group 1 and 8 chickens in experimental group 2 were protected. In the control group, 10 chickens became ill and 8 died. This clearly demonstrates that the vaccine in experimental group 3 was significantly more effective in preventing avian adenovirus type 4 (AAV) infection than the other two groups, providing more effective immune protection for the chickens. (See Table 7.)

[0139] 4.2.3.6 Infectious laryngotracheitis in chickens: Forty 21-day-old SPF chickens were subcutaneously injected in the neck with 0.2 ml of each of the three vaccines prepared in sections 1.1, 1.2, and 1.3, with 10 chickens per group. Ten chickens served as a control group, while the remaining 10 chickens were not immunized. Twenty-one days after vaccination, each chicken was injected intratracheally with 0.2 ml of the Wanggang strain of infectious laryngotracheitis virus (containing 10... 4.0 EID 50 After challenge with the virus, the chickens were observed for 14 consecutive days. The results showed that 10 chickens in experimental group 3 were protected (100% protection rate), 5 chickens in experimental group 1 were protected, 9 chickens in experimental group 2 were protected, and 10 chickens in the control group became ill and 7 died. This further demonstrates that the vaccine in experimental group 3 has a significant advantage in preventing infectious laryngotracheitis in chickens and can effectively reduce the risk of morbidity and mortality after infection. See Table 7.

[0140] Table 7. Comparison of the order of preparation steps for the hexavalent inactivated vaccine - Results of the immunogenicity test.

[0141]

[0142]

[0143] Note: For Group I avian adenovirus type 4 disease: the criteria for determining protection against challenge are the absence of abnormal clinical manifestations (mental state, feed intake, water intake, and behavior), and the absence of pathological changes such as pericardial effusion, hepatomegaly, or renal enlargement upon necropsy. For infectious laryngotracheitis in chickens: the criteria for determining protection against challenge are the absence of abnormal clinical manifestations (mental state, feed intake, water intake, and behavior), and the absence of severe conjunctivitis, cough, dyspnea, hemoptysis, and death.

[0144] As can be seen from the above, experimental group 3 showed significant advantages compared to the other groups. Regarding immunogenicity, experimental group 3 induced higher geometric mean titers of HI antibodies in the serum of immunized chickens against Newcastle disease, infectious bronchitis, H3 subtype avian influenza, H9 subtype avian influenza, group I 4 avian adenovirus disease, and infectious laryngotracheitis. The protective effect of immunized chickens after challenge was better, with a higher rate of virus isolation negativity and fewer morbidities and deaths. This indicates that the vaccine in experimental group 3 can more effectively stimulate the chicken's immune response, enhance the chicken's resistance to multiple viruses, provide more reliable immune protection for the flock, and demonstrate stronger capabilities in controlling poultry diseases, showing significant superiority over the other two groups. In conclusion, the hexavalent inactivated vaccine prepared in experimental group 3 was selected for subsequent trials.

[0145] Example 5: Safety Comparison Trial of Different Immunization Procedures

[0146] Thirty 7-day-old SPF chickens were divided into 3 groups of 10 each. The immunization schedules for breeding and laying hens were designed, and the grouping and immunization programs for the experimental chickens are shown in Table 8. The chickens' mental state, feed intake, water consumption, and feces were observed. The age at first laying of each chicken and the egg production of laying hens from 118 to 201 days of age were recorded. Soft-shelled eggs, sandy-shelled eggs, deformed eggs, or abnormalities at the eggshell tip were observed.

[0147] Table 8. Grouping and Immunization Schedule of Experimental Chickens (Safety Comparison Trial)

[0148]

[0149]

[0150] The results showed that Group A had a relatively later age at first egg production, while Groups B and C had relatively earlier ages. The egg production rate was 53.5% in Group A, 54.4% in Group B, and 54.8% in Group C. The egg production rate in Group C (non-immunized control) was slightly higher than that in the immunized groups A and B, indicating that different immunization programs affect the age at first egg production and egg production rate. Between 118 and 201 days of age, no soft-shelled, sandy-shelled, deformed, or abnormal eggshell tips appeared in any of the three groups during the observation period. This indicates that eggshell quality remained normal regardless of the immunization program or lack thereof in either the immunized or non-immunized control groups. The safety comparison test results are shown in Table 9.

[0151] Table 9. Results of safety comparison trials of different immunization procedures.

[0152]

[0153] As can be seen from the above, Group A's immunization program is more complex, involving the administration of multiple vaccines, which may increase operational complexity and stress on the chickens. Group B's immunization program is relatively simpler, reducing operational complexity and stress on the chickens, and its earlier onset of egg production minimizes the impact on the chickens' egg production performance. From an economic perspective, Group B's immunization program is more advantageous, as it simplifies the process, reduces stress on the chickens, and maintains good egg production performance. Group A may require more resources and time to manage its complex immunization program, which could increase costs. In large commercial laying hen flocks, considering ease of operation and cost-effectiveness, Group B's immunization program is the better choice.

[0154] Example 6: Comparative Test of Immunoefficiency of Different Immunization Procedures

[0155] One hundred and twenty 7-day-old SPF chickens were divided into three groups of 40 each. Following the immunization schedules for breeder and laying hens, the experimental chicken groups and immunization schedules were designed (see Table 10). The efficacy test was conducted 21 days after the last immunization.

[0156] Table 10. Grouping and Immunization Schedule of Experimental Chickens (Comparative Immunization Efficacy Test)

[0157]

[0158] (1) Partial serological efficacy test for Newcastle disease in chickens: Blood was collected from 10 chickens in each group, serum was separated, and HI antibody titer was determined. The results showed that the geometric mean of HI antibody titer in the serum of immunized chickens in group A was 8.6 log2, the geometric mean of HI antibody titer in the serum of immunized chickens in group B was 8.5 log2, and the HI antibody titer in the serum of control chickens in group C did not exceed 1:4. The experimental results are shown in Table 11.

[0159] (2) Partial serological efficacy test for infectious bronchitis in chickens: Blood was collected from 10 chickens in each group, serum was separated, and HI antibody titers were determined. The results showed that the geometric mean of HI antibody titers in the serum of immunized chickens in group A was 7.5 log2, the geometric mean of HI antibody titers in the serum of immunized chickens in group B was 7.7 log2, and the HI antibody titers in the serum of control chickens in group C did not exceed 1:4. The experimental results are shown in Table 11.

[0160] (3) Partial potency test of H3 subtype avian influenza: Blood was collected from 10 chickens in each group, serum was separated, and HI antibody titers were determined. At the same time, 0.2 ml of H3 subtype avian influenza virus solution (containing 106.0 EID) was injected intravenously into each wing of 10 chickens in each group. 50Five days after challenge, throat swabs and cloacal swabs were collected, processed, and used for virus isolation. The results showed that the geometric mean titer of HI antibodies in the serum of immunized chickens in group A was 9.4 log2, the geometric mean titer in the serum of immunized chickens in group B was 9.5 log2, and the titer in the serum of control chickens did not exceed 1:4. Five days after challenge, 10 chickens from each of groups A and B tested negative for virus isolation, while 10 chickens from the control group C tested positive for virus isolation. The experimental results are shown in Table 11.

[0161] (4) Partial potency test of H9 subtype avian influenza: Blood was collected from 10 chickens in each group, serum was separated, and HI antibody titers were determined. At the same time, 0.2 ml of H9 subtype avian influenza virus solution (containing 2×10⁻⁶) was injected intravenously into each wing of 10 chickens in each group. 7.0 EID 50 Three days after challenge, throat swabs and cloacal swabs were collected, processed, and used for virus isolation. The results showed that the geometric mean titer of HI antibodies in the serum of immunized chickens in group A was 9.3 log2, the geometric mean titer in the serum of immunized chickens in group B was 9.2 log2, and the titer in the serum of control chickens did not exceed 1:4. Three days after challenge, 10 chickens from each of groups A and B tested negative for virus isolation, while 10 chickens from the control group C tested positive. The experimental results are shown in Table 11.

[0162] (5) Partial efficacy test for Group I type 4 avian adenovirus infection: Ten chickens were randomly selected from each group and each chicken was injected intramuscularly with 0.2 ml of Group I type 4 avian adenovirus solution (containing 2×10⁻⁶ spores). 5.5 (TCID50). Chickens were observed for 7 days after challenge. Results showed that 10 chickens in both Group A and Group B were protected, while 9 chickens in Group C (control group) became ill and 7 died. The experimental results are shown in Table 11.

[0163] (6) Partial efficacy test of infectious laryngotracheitis virus in chickens: Ten chickens were randomly selected from each group, and 0.2 ml of Wanggang strain of infectious laryngotracheitis virus (containing 10...) was injected into the larynx and trachea of ​​each chicken. 4.0 EID 50 After the virus challenge, the individual was observed for 14 consecutive days.

[0164] Table 11 Results of comparative immunization efficacy tests of different immunization programs

[0165]

[0166] Note: For Group I avian adenovirus type 4 disease: the criteria for determining protection against challenge are the absence of abnormal clinical manifestations (mental state, feed intake, water intake, and behavior), and the absence of pathological changes such as pericardial effusion, hepatomegaly, or renal enlargement upon necropsy. For infectious laryngotracheitis in chickens: the criteria for determining protection against challenge are the absence of abnormal clinical manifestations (mental state, feed intake, water intake, and behavior), and the absence of severe conjunctivitis, cough, dyspnea, hemoptysis, and death.

[0167] The experimental results are shown in Table 11. In both Group A and Group B, 10 chickens were protected against infection, while in Group C (the control group), 10 chickens became ill and 6 died. Both Groups A and B exhibited high antibody titers and similar protective effects against viral challenge, reducing the risk of infection with the corresponding viruses and decreasing the incidence and spread of disease. Compared with existing immunization programs, this hexavalent inactivated vaccine has similar immunogenicity and protective effects against viral challenge, reducing the incidence and mortality of diseases caused by multiple viral infections in chickens, lowering breeding costs, simplifying the immunization process, reducing stress on chickens, and maintaining good egg production performance, leading to higher economic benefits. It also provides a new and effective means of controlling poultry diseases, promoting the stable development of the poultry farming industry.

[0168] Example 7: Study on the cross-protective effect of the hexavalent inactivated vaccine against circulating strains of H3 subtype avian influenza.

[0169] Sixty 21-day-old SPF chickens were used. Thirty chickens were subcutaneously injected with 0.3 ml of a hexavalent inactivated vaccine per chicken via the neck, while the remaining 30 chickens served as a control. Twenty-one days post-immunization, the chickens were challenged with three circulating H3 subtype avian influenza strains isolated in 2021-2022. Each chicken was intravenously inoculated with 0.2 ml of a 10-fold diluted virus solution. Each challenge strain was used to challenge 10 immunized chickens and 10 non-immunized controls. Five days post-challenge, throat swabs and cloacal swabs were collected, processed, and used for virus isolation.

[0170] Table 12 Results of Cross-Protection Test

[0171]

[0172] The results are shown in Table 12. After challenging the experimental chickens with the three prevalent strains, all 10 immunized chickens tested negative for the virus, while all 10 control chickens tested positive. This data clearly demonstrates the strong cross-protective capability of the hexavalent inactivated vaccine against prevalent H3 subtype avian influenza strains, with the H3 subtype avian influenza virus antigen playing a crucial role. Compared to the unimmunized control group, the immune systems of the vaccinated experimental chickens were effectively activated, generating specific immune responses against these prevalent strains, enabling them to recognize and eliminate invading viruses, thereby preventing infection. This advantage is crucial for the poultry industry, significantly reducing the risk of H3 subtype avian influenza infection in flocks, minimizing mortality and performance decline due to infection, and ensuring poultry health and economic benefits. Simultaneously, it provides strong technical support and practical evidence for poultry disease prevention and control, proving the effectiveness and reliability of the hexavalent inactivated vaccine in preventing infection by prevalent H3 subtype avian influenza strains.

Claims

1. A hexavalent inactivated vaccine for poultry, characterized in that, The inactivated Newcastle disease virus antigen concentrate, inactivated infectious bronchitis virus antigen concentrate, inactivated H3 subtype avian influenza virus antigen concentrate, inactivated H9 subtype avian influenza virus antigen concentrate, inactivated group I type 4 avian adenovirus solution, and inactivated infectious laryngotracheitis virus gD protein solution are pretreated, then mixed using a stepwise mixing method and emulsified to obtain the final product.

2. The poultry hexavalent inactivated vaccine according to claim 1, characterized in that, The volume ratio of the added inactivated Newcastle disease virus antigen concentrate, inactivated infectious bronchitis virus antigen concentrate, inactivated H3 subtype avian influenza virus antigen concentrate, inactivated H9 subtype avian influenza virus antigen concentrate, inactivated group I type 4 avian adenovirus solution, and inactivated infectious laryngotracheitis virus gD protein solution was 3:3:5:3:2:

4.

3. The poultry hexavalent inactivated vaccine according to claim 1 or 2, characterized in that, The preprocessing is as follows: Add 0.1% trehalose to the H3 subtype avian influenza virus antigen concentrate and the H9 subtype avian influenza virus antigen concentrate, respectively, and pre-equilibrate at 4°C for 1 hour to maintain the conformational stability of HA protein. Add 5 mM EDTA to the concentrated gD protein antigen solution of chicken infectious laryngotracheitis virus to chelate metal ions to prevent gD protein oxidative dimerization.

4. The poultry hexavalent inactivated vaccine according to claim 3, characterized in that, The virus used in the inactivated Newcastle disease virus antigen concentrate was the attenuated strain NDV-VIIb of Newcastle disease virus type VII, which was deposited on October 15, 2019, at the China Center for Type Culture Collection in Wuhan, with accession number CCTCC NO: V201968. The virus used in the inactivated chicken infectious bronchitis virus antigen concentrate was the infectious bronchitis M41 strain. The virus used in the inactivated H3 subtype avian influenza virus antigen concentrate is the H3 subtype avian influenza virus YB-GD strain, which was deposited on March 11, 2025, at the China General Microbiological Culture Collection Center in Beijing, with the accession number CGMCCNO: 46406. The virus used in the inactivated H9 subtype avian influenza virus antigen concentrate is the H9 subtype avian influenza virus YBF18 strain, which was deposited on March 11, 2025 at the China General Microbiological Culture Collection Center in Beijing, with accession number CGMCCNO: 46405. The virus used in the inactivated Group I type 4 avian adenovirus solution was Group I type 4 avian adenovirus YBAV-4 strain, which was deposited on October 15, 2015 at the China Center for Type Culture Collection in Wuhan, with accession number CCTCC NO: V201541. The inactivated chicken infectious laryngotracheitis virus gD protein solution was obtained by centrifuging the engineered cell line CHO-gD until the cell viability dropped to 70%-80%, centrifuging at 4℃, 8000-8500 r / min for 10-15 minutes, and collecting the supernatant.

5. The method for preparing the poultry hexavalent inactivated vaccine according to any one of claims 1-4, characterized in that, Includes the following steps: First, preheat the Newcastle disease virus antigen, infectious bronchitis virus antigen, H3 subtype avian influenza virus antigen, H9 subtype avian influenza virus antigen, group I type 4 avian adenovirus antigen, and infectious laryngotracheitis virus gD protein antigen to 35℃~36℃ respectively. H3 subtype avian influenza virus antigen and H9 subtype avian influenza virus antigen were mixed in a primary mixture at a volume ratio of 5:3 to obtain mixture 1. Chicken Newcastle disease virus antigen, chicken infectious bronchitis virus antigen and group I type 4 avian adenovirus antigen were thoroughly mixed at a volume ratio of 3:3:2 to obtain mixture 2. Then, mixture 1 and mixture 2 were mixed in a secondary mixture at a volume ratio of 1:1 to obtain mixture 3. Mixture 3 was mixed with chicken infectious laryngotracheitis virus gD protein antigen at a volume ratio of 4:1 in three stages to make the chicken infectious laryngotracheitis virus gD protein content in the mixture 120 μg / ml, thus obtaining mixture 4. Take 96 parts of white oil and 1 part of aluminum stearate, mix them evenly in an oil phase preparation tank and heat to 80°C. Then add 4 parts of Span-80 and maintain the temperature at 115°C for 40 minutes. Cool down to 25°C and use it as the oil phase for later use. Mix 4 with Tween-80 at a volume ratio of 95:

5. When the system temperature drops to 25°C, control the temperature of the aqueous phase at 20-25°C. Take 3 parts of the oil phase and put them into an emulsification tank and stir. Add 2 parts of the aqueous phase and perform low-speed premixing and high-speed premixing in sequence. After the premixing is completed, take a sample and centrifuge it. After no aqueous phase is separated from the centrifuged sample, the oil emulsion is obtained. Cut it to obtain the corresponding vaccine.

6. The preparation method according to claim 5, characterized in that, The above mixing was carried out at a speed of 200-220 r / min and a temperature of 35-36℃, with a mixing time of 10-15 minutes.

7. The preparation method according to claim 5, characterized in that, For low-speed premixing, mix at 15-25Hz for 10-15 minutes; for high-speed premixing, mix at 40-45Hz for 20-25 minutes.

8. The preparation method according to claim 5, characterized in that, Before obtaining Mixture 2, 0.1% hyaluronic acid was added to the mixture of Newcastle disease virus antigen, infectious bronchitis virus antigen and group I type 4 avian adenovirus antigen as a steric hindrance to reduce collisions between virus particles.

9. The application of the poultry hexavalent inactivated vaccine according to any one of claims 1-4 in the simultaneous prevention and control of Newcastle disease in laying hens, H3N8 subtype avian influenza, H9 subtype avian influenza, avian adenovirus disease, infectious bronchitis in chickens, and infectious laryngotracheitis.

10. The application according to claim 9, characterized in that, The inoculation was administered subcutaneously or intramuscularly in the neck, with a dosage of 0.5 ml per bird. 21 days post-inoculation, the geometric mean titer of HI antibodies in serum from partially immunized chickens against Newcastle disease was 8.6 log2; the geometric mean titer of HI antibodies in serum from partially immunized chickens against infectious bronchitis was 7.7 log2; the geometric mean titer of HI antibodies in serum from partially immunized chickens against H3 subtype avian influenza was 9.5 log2, and 10 out of 10 immunized chickens tested negative for virus isolation 5 days after challenge; the geometric mean titer of HI antibodies in serum from partially immunized chickens against H9 subtype avian influenza was as high as 9.2 log2, and 10 out of 10 immunized chickens tested negative for virus isolation 3 days after challenge; 10 out of 10 chickens were protected against group I type 4 avian adenovirus disease after challenge; and 10 out of 10 chickens were protected against infectious laryngotracheitis after challenge.

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