Consensus ha protein of h9 subtype aiv, genes and uses thereof

Abstract

This invention belongs to the field of biology and discloses a consensus HA protein for H9 subtype AIV, the amino acid sequence of which is shown in SEQ ID NO.1. This consensus protein, located at the center of the phylogenetic tree, covers common mutations in H9 subtype AIV and is genetically close to currently circulating strains. Furthermore, this invention also discloses the gene encoding this consensus HA protein and its applications; a recombinant virus developed based on this gene serves as a vaccine candidate strain carrying the common mutant antigen, exhibiting better protective efficacy.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to the consensus HA protein, gene and application of H9 subtype AIV. Background Technology

[0002] Avian influenza belongs to type A influenza. Although the promotion and use of vaccines in recent years have effectively controlled other subtypes of avian influenza, the H9 subtype AIV is the most damaging low-pathogenic avian influenza pathogen to the poultry industry and is gradually becoming the mainstream, causing problems such as decreased production performance, immunosuppression, and secondary infections. AIV encodes eight structural proteins: the surface protein hemagglutinin (HA), neuraminidase (NA), nucleoprotein, matrix proteins M1 and M2, and polymerase proteins (PB1, PB2, and PA). Based on the differences in HA and NA, influenza can be divided into 18 HA subtypes and 11 NA subtypes.

[0003] Currently, there are many types of influenza vaccines available. Inactivated vaccines are the most widely used, and their effectiveness is undeniable. However, many H9 subtype AIV inactivated vaccines are currently ineffective in preventing reinfection of immunized chickens with H9 subtype AIV and in stopping viral shedding. Newcastle disease vectors have been used in the development of H9 subtype AIV vaccines.

[0004] One of the challenges in developing vector vaccines is finding a consensus protein. Due to the rapid rate of antigenic drift and mutation, available vaccines are only effective against specific strains, and antigenic mismatch between vaccine strains and circulating strains makes it easier for pathogens to evade the immune system. Faced with highly variable viruses, new challenges arise regarding the broad-spectrum and long-term effectiveness of vaccines, leading to the concept of a more robust "broad-spectrum vaccine." Currently used vaccines are mostly developed directly from wild-type virus sequences, requiring redesigned antigens. Obtaining the antigen necessitates years of monitoring and analysis of dominant strain changes. Furthermore, vaccine development is lengthy, and this method may result in a development speed that cannot keep pace with the rate of viral mutation. In this context, vaccines with consensus antigens may be the best option.

[0005] However, the prediction and development of effective, broad-spectrum consensus proteins is a challenging task. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a consensus HA protein that is located at the center of the phylogenetic tree, covers common mutations of H9 sub-AIV, and is genetically close to the currently prevalent strain, thus solving the problem that the current vaccine strains cannot cope with the mutations of the prevalent strains, resulting in poor immunization effects.

[0007] In addition, this invention also discloses the gene encoding the consensus HA protein and its applications.

[0008] To achieve this objective, the present invention employs the following technical solution:

[0009] A consensus HA protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] In addition, the present invention also discloses a gene encoding a consensus HA protein, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0011] Furthermore, this invention also discloses the application of the consensus HA protein or the gene described above in the preparation of vaccines.

[0012] In the above applications, the vaccine is an avian influenza vaccine, or a bivalent vaccine; the bivalent vaccine is used for the prevention and treatment of avian influenza.

[0013] In the above applications, the bivalent vaccine is also used to prevent and control Newcastle disease and H9 subtype avian influenza.

[0014] Finally, the present invention also discloses a recombinant virus comprising a vector and the gene as described above inserted into the vector virus and expressing the consensus HA protein as described above.

[0015] In the aforementioned recombinant virus, the vector virus is Newcastle disease virus.

[0016] In the aforementioned recombinant virus, the vector virus of the recombinant virus is Newcastle disease virus; the gene insertion site is between the PM uncoding regions of Newcastle disease virus.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The consensus antigen consensus-HA developed in this invention covers the common HA mutations in H9 subtype AIV; it has broad spectrum in H9 subtype AIV.

[0019] 2. This invention constructs a recombinant Newcastle disease virus carrying consensus-HA, which is a weak virus;

[0020] 3. The vaccine constructed in this invention has a better effect on inhibiting viral shedding compared with the commercially available NDV-AIV bivalent vaccine;

[0021] 4. The vaccine constructed in this invention, wherein QS201 is more advantageous than traditional white oil adjuvant.

[0022] While commercial vaccines can increase antibody titers more quickly, we believe that the antibody-stimulating effect of the vaccine of this invention is significantly superior to that of commercial vaccines in terms of shedding effect, cross-reactivity, and other aspects. Attached Figure Description

[0023] Figure 1 The image shows the results of homology analysis of the HA gene sequence.

[0024] Figure 2 A phylogenetic tree diagram for screening the neoantigen consensus-HA;

[0025] Figure 3 Figure showing the genetic evolution analysis results of the neoantigen consensus-HA;

[0026] Figure 4 A schematic diagram illustrating the method for constructing recombinant viruses;

[0027] Figure 5 Photographs showing the hemagglutination properties of the virus for virus rescue;

[0028] Figure 6A A specific fluorescence image of NP protein in BHK-21 cells 24 h after mGM-conHA infection;

[0029] Figure 6B A specific fluorescence image of HA protein 24 h after mGM-conHA infection of BHK cells;

[0030] Figure 7 A bar chart showing the HI antibody detection results of the mGM-conHA vaccine, using the GM strain as the detection antigen for NDV.

[0031] Figure 8 The bar chart shows the HI antibody detection results of the mGM-conHA vaccine, with H9-1 as the detection antigen for H9 subtype AIV. Detailed Implementation

[0032] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0033] Part 1: Prediction of the Consensus Sequence

[0034] Sequences were downloaded from the National Center for Biotechnology Information (NCBI) and the Global Initiative of Sharing All Influenza Data (GISAID).

[0035] To improve comparability and limit the bias of identical sequences, phylosuite software was used to remove repetitive sequences, and sequences whose length deviated from the median length value were manually removed. Sequences originating from the same epidemic, location, or with the same name and date were also manually removed. A phylogenetic tree was then constructed. To obtain the latest specific grouping of the epidemic type, HA sequence information of H9 subtype AIV isolated from various parts of my country from 2020 to 2022 (up to mid-2022) was downloaded from the NCBI and Gisaid databases. After processing, 71 sequences were finally obtained. Homology analysis was performed with representative H9 subtype AIV HA gene sequences published and uploaded domestically and internationally. The results showed that the current epidemic branch of H9 subtype AIV in my country is h9.4.2.5c (…). Figure 1 ).

[0036] After statistical analysis using PhyloSuite to remove repetitive sequences, and manual comparison to remove sequences whose length deviated from the median length, the final dataset contained 1,387 sequences. Through Clustal Omega and Muscle computational analysis, a novel antigen, consensus-HA, was designed, located precisely at the center of the phylogenetic tree and capable of covering common mutations. Figure 2 ).

[0037] To further analyze whether the consensus sequence can represent the popular strain, the obtained consensus-HA was subjected to genetic evolution analysis based on the standard strain. Figure 3 Consensus-HA is located in the h9.4.2.5c branch, consistent with the current popular version.

[0038] Part II. Construction and Biological Characteristics of NDV Recombinant Viruses Expressing H9 Subtype AIV Consensus Sequence

[0039] To obtain NDV H9-HA vector vaccines, based on existing research, the Newcastle disease vector rGM-VIIm (genotype VII, which has been mutated and attenuated and has good immunogenicity) was selected as the vector, abbreviated as mGM. Previous studies introduced exogenous genes between NDV PMs. This invention inserts conHA between PMs and removes the non-coding region after GS to directly express the consensus antigen. Figure 4 ), in order to obtain the NDV-H9 subtype AIV recombinant virus.

[0040] The Newcastle disease attenuated strain mGM (genotype VII), full-length transcript mGM, helper plasmids pCI-NP, pCI-P, and pCI-L were preserved in our laboratory. BSR-T7 / 5 cells and MVA / T7 virus were kindly donated by Researcher Qiu Xusheng of Shanghai Veterinary Research Institute; 9-11 day old SPF chicken embryos were provided by Guangdong Wenshi Dahua Agricultural Biotechnology Co., Ltd.

[0041] References: [1]SUN M, XIANG B, LI Y, et al. Generation and evaluation of a genetically attenuated Newcastle disease virus rGM-VIIm as a genotype-matched vaccine[J]. Virus Genes, 2017,53(1): 35-43.

[0042] The consensus antigen consensus-HA (hereinafter referred to as conHA, nucleotide sequence as shown in SEQ ID NO.2, amino acid sequence as shown in SEQ ID NO.1) was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The gene fragment was then ligated to the mGM vector using primers with homologous arms to obtain the full-length transcript mGM-conHA. The primers involved in this invention are:

[0043] conHA-F: cgggtagaagtaccgccaccatgGAGACAGTATCACTAATAAC (SEQ ID NO.3)

[0044] conHA-R: gtggatttgcgagggggcctcTTATATACAAATGTTGCATCTGC (SEQ ID NO. 4);

[0045] The lowercase letters represent homologous arms. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0046] Virus rescue

[0047] BSR-T7 / 5 cells were mixed and prepared into a suspension, seeded into 6-well plates, and cultured overnight at 37°C with 5% CO2. When the cells reached 60%–80% confluency, the full-length plasmid mGM-conHA and three helper plasmids pCI-NP, pCI-P, and pCI-L were transfected into the cells at ratios of 0.5, 1.0, 1.0, and 0.5 μg, respectively. 72 h later, the supernatant was collected and inoculated into 9-day-old SPF chicken embryos at a rate of 0.2 mL per embryo, discarding any embryos that died within 24 h. Allantoic fluid was harvested 72 h after inoculation for an HA assay. Figure 5 The allantoic fluid that tested positive for HA was subjected to RT-PCR sequencing, and the resulting recombinant virus strain was named mGM-conHA.

[0048] Identification of biological characteristics of recombinant viruses

[0049] Sterile PBS was used for 10 consecutive times (10... -3 10 -4 ~10 -7 Allantoic fluid with hemagglutination properties was serially diluted and inoculated into 10-day-old SPF chicken embryos at a rate of 0.2 mL per embryo. Hemagglutination-prone embryos were harvested 72 h post-inoculation for RT-PCR sequencing.

[0050] Indirect immunofluorescence identification of recombinant viral strains

[0051] BHK-21 cells were cultured and seeded into 12-well plates. After 24 hours, the cell supernatant was discarded, and the allantoic fluid of the recombinant virus mGM-conHA with hemagglutination properties was aseptically collected at 0.1 MOI to infect the cells. Simultaneously, the cells were seeded with mGM(VII-NDV) and H9-1 (H9 subtype AIV) as positive controls, and the wells with culture medium added only to the cells were used as negative controls. 24 hours after viral infection, the cell supernatant was discarded, and the cells were washed three times with sterile PBS. The cells were then fixed with 4% paraformaldehyde at room temperature for 20-30 minutes, and the paraformaldehyde was discarded. The cells were then washed three times with PBST. Next, PBS containing 0.2% Triton X-100 was added to permeate the cells for 15 minutes. The cells were washed three times with PBST, and then blocked for 1 hour at room temperature with 5% BSA. The blocking solution was removed, and the cells were washed three times with PBST. Rabbit anti-NDV-NP polyclonal antibody was added as the primary antibody to test for NP protein. Chicken anti-H9 subtype AIV fully immunized serum was added as the primary antibody to test for HA protein. After incubation overnight at 4°C, the cells were washed three times with PBST, and the corresponding fluorescent secondary antibodies (488-labeled goat anti-rabbit IgG and 594-labeled goat anti-chicken IgG) were added. The cells were incubated at 37°C in the dark for 1 hour, washed three times with PBST, and the results were observed under a fluorescence microscope.

[0052] The results are as follows Figure 6A and Figure 6B As shown, after 24 hours of mGM-conHA infection of BHK-21 cells, obvious specific green fluorescence (NP protein) was observed in all cells; after 24 hours of mGM-conHA infection of BHK cells, obvious specific red fluorescence (HA protein) was observed in all cells, while no corresponding fluorescence was observed in the blank control group, indicating that the virus rescue was successful and the inserted foreign protein could be expressed normally.

[0053] Biological characteristics determination of recombinant virus mGM and mGM-conHA strains

[0054] Based on WOAH standards, compare the MDT, ICPI, and EID of the recombinant virus mGM-conHA. 50 and TCID 50 .

[0055] According to OIE testing standards, in terms of pathogenicity index, mGM-conHA has an MDT greater than 120 h and an ICPI index of 0, thus meeting the criteria for determining a standard attenuated strain. EID 50 / 0.1mL is 10 7.429 The HA value reaches over 9log2, indicating good proliferation capacity.

[0056] The above results indicate that the obtained recombinant virus mGM-conHA has good replication ability and exhibits the biological characteristics of a weak strain, providing a theoretical basis for the subsequent development of a highly safe NDV-H9 subtype AIV bivalent vaccine.

[0057] Part Three: Evaluation of the Immunopotency of the Gen VII ND-H9 Subtype AI Recombinant Bivalent Vaccine

[0058] 1. Preparation of inactivated vaccines

[0059] Since adjuvants are an important factor affecting the cross-protective efficacy of vaccines, this invention prepares two adjuvants to formulate vaccines (Marcol-52 and QS201 - a novel composite immune adjuvant), and the specific steps are as follows:

[0060] (1) Recombinant viruses mGM-conHA, rGM, and H9-1 were inoculated into the allantoic cavity of chicken embryos. The allantoic fluid was collected and inactivated with 0.1% formaldehyde solution. The incubator was sealed with sealing film and kept at 37°C for 24 hours to ensure complete inactivation. The inactivated allantoic fluid was then inoculated into three SPF chicken embryos, and the embryos were passaged for three consecutive generations to confirm that the virus had completely lost its infectivity. The prepared vaccine antigen (aqueous phase) content was 10. 6 EID 50 / 0.2mL.

[0061] (2) Marcol-52 adjuvant: First, add 2.5% Tween to Marcol-52 white oil and stir. Then, slowly add the aqueous phase and continue stirring to ensure that the oil phase and aqueous phase are fully mixed. Then, emulsify online using a shear machine (22,000 rpm, 3 min). After emulsification, take a drop on the water surface. It should clump together and not spread outward within 1 min, indicating that a qualified oil emulsion vaccine has been successfully prepared. Take 5 ml of vaccine and add it to a centrifuge tube. Centrifuge at 3000 r / min for 15 min. No stratification should occur.

[0062] (3) QS201-novel compound immune adjuvant: Add compound adjuvant QS201 to the aqueous phase, mix and emulsify to make the final concentration 0.4%~0.8%, and the shearing emulsification operation is the same as above.

[0063] 2. Preparation of two adjuvants and grouping of animals immunized with different doses of inactivated vaccine and immunization challenge regimens

[0064] This part of the experiment involved screening two adjuvants and exploring different dosages simultaneously. Immunization and challenge were conducted in two separate isolation chambers. One-day-old chicks were immunized with the corresponding vaccines and three dosages for up to three weeks. Blood samples were collected weekly to measure HI antibodies. After three weeks, animals in the two isolation chambers were challenged with GM strain (genotype VII NDV) and H9-1 (H9 subtype AIV), respectively. Pharyngeal swabs and cloacal swabs were collected on days 3, 5, 7, and 9 to measure viral shedding. Animal mortality was observed during the challenge period.

[0065] The following are the grouping and experimental methods, where Marcol-52 adjuvant is abbreviated as M in the vaccine, and QS201-novel combined immune adjuvant is abbreviated as Q in the vaccine:

[0066] One-day-old chicks were randomly divided into 14 groups. Three doses of two adjuvant inactivated vaccines (12 groups) were administered, named as follows: Experimental group: M-mGM-conHA-1, 70 μL / chick; M-mGM-conHA-2, 140 μL / chick; M-mGM-conHA, 200 μL / chick; Experimental group: Q-mGM-conHA-1, 70 μL / chick; Q-mGM-conHA-2, 140 μL / chick; Q-mGM-conHA, 200 μL / chick; A commercial vaccine group (a mixed vaccine of genotype VII NDV and H9 subtype AIV) and a PBS group were also established, with 12 chicks in each group. All chicks were immunized subcutaneously in the neck. HI antibody titers were measured every 7 days post-immunization. After 21 days, 10... 6 EID 50 Chickens were challenged with the NDV GM strain via nasal and ocular drops. Oropharyngeal and cloacal swabs were collected at 3, 5, and 7 days post-challenge for virus shedding. Three chickens from each group were dissected to examine organ lesions and observed for up to 7 days. Clinical symptoms and mortality were recorded during the challenge period.

[0067] One-day-old chicks were randomly divided into 14 groups. Three doses of two adjuvanted inactivated vaccines (12 groups) were administered, named as follows: Experimental group: M-mGM-conHA-1, 70 μL / chick; M-mGM-conHA-2, 140 μL / chick; M-mGM-conHA, 200 μL / chick; Experimental group: Q-mGM-conHA-1, 70 μL / chick; Q-mGM-conHA-2, 140 μL / chick; Q-mGM-conHA, 200 μL / chick. A commercial vaccine group and a PBS group were also established. Each group consisted of 15 one-day-old SPF chicks. Immunization was performed via subcutaneous injection in the neck. HI antibody titers were measured every 7 days post-immunization. The vaccine was administered 10 days after immunization. 6 EID 50Chickens were tested for viral shedding by nasal and ocular administration of strain H9-1. Oropharyngeal and cloacal swabs were collected at 3, 5, 7, and 9 days post-infection for viral shedding detection. Three chickens from each group were dissected to examine organ lesions and observed until day 9. Clinical symptoms and mortality were recorded during the infection period.

[0068] Blood was collected from each group of experimental chickens in the third week after immunization, and serum was separated and then serum antibodies were detected.

[0069] In the chart, “M”: Marcol-52 adjuvant; “Q”: QS201 adjuvant; “-1”: dose 70 μL; “-2”: dose 140 μL; the dose without a number suffix is ​​200 μL.

[0070] Using the GM strain as the detection antigen for NDV, the HI antibody detection results for the mGM-conHA vaccine are shown below. Figure 7 Tables 1 and 2.

[0071] The results showed that the experimental groups M-mGM-conHA-1, M-mGM-conHA-2, M-mGM-conHA, Q-mGM-conHA-1, Q-mGM-conHA-2, and Q-mGM-conHA had NDV antibody levels approximately 1.5 log2 and 2 log2 lower than the commercial vaccine in weeks 1 and 2, respectively. Then, in week 3, the antibody levels in the experimental and commercial vaccine groups at doses of 140 μL and 200 μL were comparable, and the antibody levels induced by the same dose of vaccine with different adjuvants were not significantly different.

[0072] Table 1. NDV antibody level detection after Marcol-52 adjuvant vaccine immunization (± is standard deviation)

[0073] Immunization Group 1 week (log2) 2 weeks (log2) 3 weeks (log2) M-mGM-conHA-1 3.25±0.38 7.08±0.84 9.33±0.94 M-mGM-conHA-2 3.75±0.63 7.25±0.38 10.33±0.47 M-mGM-conHA 3.83±0.69 7.5±0.5 11±0.82 PBS 0 0 0 Commercial vaccine 5±0.76 9.08±0.93 10.75±0.83

[0074] Table 2. Detection of NDV antibody levels after immunization with QS201 adjuvant vaccine.

[0075] Immunization Group 1 week (log2) 2 weeks (log2) 3 weeks (log2) Q-mGM-conHA-1 3.75±0.63 6.83±0.37 9.17±0.90 Q-mGM-conHA-2 3.92±0.61 7.33±0.47 10±1.29 Q-mGM-conHA 4.01±0.73 7.5±0.5 11±0.58

[0076] Using H9-1 as the detection antigen for H9 subtype AIV, the HI antibody detection results for the mGM-conHA vaccine are shown below. Figure 8 Tables 3 and 4.

[0077] The results showed that in week 1, antibody levels in the experimental groups were lower than those in the vaccine groups, with only the 140 μL and 200 μL doses of the improved adjuvant vaccine mGM-conHA showing higher levels than the other groups. In week 2, the HI titer in all groups rose above 5.0 log2, at which point the antibody level in the commercial vaccine group reached 8.67 ± 0.94 log2. In week 3, the antibody levels induced by the different doses of the adjuvant QS201 group of mGM-conHA vaccine were higher than those induced by the adjuvant Marcol-52 group.

[0078] Table 3. Detection of H9 subtype AIV antibody levels after Marcol-52 adjuvant vaccine immunization.

[0079] Immunization Group 1 week (log2) 2 weeks (log2) 3 weeks (log2) M-mGM-conHA-1 3±0.58 5.67±0.47 6.5±0.5 M-mGM-conHA-2 4.5±0.5 6.5±0.5 7.33±0.75 M-mGM-conHA 4.67±0.47 6.33±0.50.47 8.17±0.90 PBS 0 0 0 Commercial vaccine 6.33±0.47 8.67±0.94 10.92±0.61

[0080] Table 4. Detection of H9 subtype AIV antibody levels after immunization with QS201 adjuvant vaccine.

[0081] Immunization Group 1 week (log2) 2 weeks (log2) 3 weeks (log2) Q-mGM-conHA-1 3.83±0.69 5.67±0.47 7.5±0.76 Q-mGM-conHA-2 4±0.58 5.83±0.69 7.83±1.07 Q-mGM-conHA 4.67±0.47 6.17±0.69 8.33±0.94

[0082] 3. Detoxification testing

[0083] Pharyngeal and cloacal swabs were collected from experimental chickens on days 3, 5, and 7 post-challenge for virus shedding detection. The collected pharyngeal and cloacal swabs were added to EP tubes containing 1 mL of double-antibody PBS solution; the tubes were thoroughly vortexed and centrifuged at 8000×g for 3 min at 4°C. 0.1 mL of the supernatant was collected to extract RNA, which was then reverse transcribed and amplified using identification primers. A positive control well was also included.

[0084] Twenty-one days after immunization, chickens in each group in isolation room 1 were inoculated with the NDV GM strain. On day 3 post-challenge, animals in the PBS-immunized group began to show symptoms of lethargy, ruffled feathers, and open-mouth breathing. By day 4, one chicken had died, and the remaining animals had all died by day 5. Animals in the other immunized groups showed no symptoms or deaths. The survival rate was 100% in the experimental groups with different adjuvants and dosages. Twenty-one days after immunization, chickens in each group in isolation room 2 were inoculated with the H9-1 strain. During the challenge period, the immunized groups showed no symptoms or deaths. The survival rate after challenge was 100%.

[0085] During the observation period after challenge with NDV GM strain, pharyngeal swabs and cloacal swabs were collected from each group on days 3, 5, and 7 for in vitro virus shedding detection. The results are shown in Table 5 below. After immunizing animals with vaccines prepared with different adjuvants and three different doses, NDV was not detected in either pharyngeal swabs or cloaca. In the PBS immunization group, the virus shedding rate was 83.3% in pharyngeal swabs and 66.7% in cloaca on day 3.

[0086] Table 5. NDV shedding after challenge with inactivated vaccine

[0087]

[0088] " / " represents death.

[0089] During the observation period after H9-1 challenge, pharyngeal swabs and cloacal swabs were collected from each group on days 3, 5 and 7 for in vitro virus shedding detection. The results are shown in Tables 6 and 7 (the control groups were the PBS group and the commercial vaccine group in the same group).

[0090] When Marcol-52 was used as an adjuvant for immunization (Table 6), viral shedding was detected in pharyngeal swabs from all groups 3 days after challenge, with a low detection rate in the cloaca. On day 5, viral shedding was low in both oropharyngeal and cloacal swabs from the M-mGM-conHA groups; by day 7, oropharyngeal viral shedding further decreased in the M-mGM-conHA groups. By day 9, no viral shedding occurred in the oropharynx or cloaca of the M-mGM-conHA groups, but it was still detectable in the commercial vaccine group.

[0091] With increasing immunization dose, all three M-mGM-conHA groups showed a decrease in toxin excretion.

[0092] Table 6. H9-1 shedding after challenge with Marcol-52 adjuvanted inactivated vaccine.

[0093]

[0094] Following immunization with the QS201 compound adjuvant vaccine (Table 7), viral shedding was detected in pharyngeal swabs from all groups except for the high-dose Q-mGM-conHA (200 μL) group 3 days post-challenge. At this time, the cloacal detection rate was lower than that of pharyngeal swabs. On day 5, a significant decrease in pharyngeal swab viral shedding was detected in the three Q-mGM-conHA groups, while the cloacal viral shedding was higher in the commercial vaccine group. On day 7, viral shedding was no longer detectable in the oropharynx and cloaca of the three Q-mGM-conHA groups, while the commercial vaccine group continued to shed viral. On day 9, viral shedding was undetectable in the three Q-mGM-conHA groups, while the commercial vaccine group continued to shed viral.

[0095] When comparing the effects of different doses on immunity, all three groups of Q-mGM-conHA showed a decrease in detoxification as the immune dose increased.

[0096] Table 7 H9-1 shedding after QS201 adjuvant-inactivated vaccine immunization challenge

[0097]

[0098] Comparing the viral shedding results from the two tests, on days 3 and 5, the Q-mGM-conHA groups showed less viral shedding in the oropharynx and cloaca, and from day 7 onwards, viral shedding was completely suppressed in all three groups. Regarding viral shedding results, when using Marcol-52 adjuvant to prepare the vaccine, immunization with 140 μL of mGM-conHA vaccine completely suppressed viral shedding within 9 days; when using QS201 compound adjuvant to prepare the vaccine, immunization with 140 μL of mGM-conHA vaccine completely suppressed the virus within 7 days. Overall, the QS201 compound adjuvant is more advantageous than the Marcol-52 adjuvant and superior to commercial vaccines.

[0099] 4. Cross-reactivity of antibodies after immunization

[0100] To compare the cross-reactivity of the generated immune serum against H9.4.2.5c AIV, serum was collected from immunized chickens 21 days later, and HI tests were performed using circulating strains (29, 144, 131, and H9-1) of the h9.4.2.5c branch as detection antigens. The results are shown in Table 8. When H9-1 was used as the detection antigen, the antibody level produced by the mGM-conHA vaccine was 7.4 log2, while that in the commercial vaccine group was 11.4 log2; when 144 was used as the detection antigen, the antibody level produced by the mGM-conHA vaccine was 6.4 log2, while that in the commercial vaccine group was 9.6 log2; when 133 was used as the detection antigen, the antibody level produced by the mGM-conHA vaccine was 11.4 log2, while that in the commercial vaccine group was 7.6 log2; and when 29 was used as the detection antigen, the antibody level produced by the mGM-conHA vaccine was 5 log2, while that in the commercial vaccine group was 6 log2.

[0101] Combined with the viral shedding data of animals after immunization and challenge mentioned above, the fact that mGM-conHA can produce high levels of antibodies and effectively inhibit the virus indicates that the antibodies induced by mGM-conHA can neutralize different H9 subtypes of AIV. High levels of antibodies were detected in the commercial vaccine group against different detection antigens, suggesting that, based on the HI test, the antibodies induced by the commercial vaccine are not entirely effective.

[0102] Table 8. Results of HI tests using different antigens after immunization with different vaccines.

[0103]

[0104] In summary, inactivated vaccines prepared from recombinant virus mGM-conHA using two different adjuvants, Marcol-52 and QS201, were demonstrated to provide 100% protection against virulent NDV in chickens after immunogenicity evaluation. Simultaneously, they effectively induced H9 subtype AIV antibody production. However, from a shedding perspective, the QS201 adjuvant vaccine was more effective than the Marcol-52 vaccine, requiring only a 140 μL immunization dose to completely stop shedding by day 7. Furthermore, since the inserted antigen is a broad-spectrum antigen designed through analysis, HI validation and comprehensive analysis of animal shedding results revealed that the antibodies induced by the mGM-conHA vaccine can neutralize different H9 subtype AIVs, demonstrating its broad-spectrum vaccine significance.

[0105] Summarize:

[0106] 1. The consensus antigen developed in this invention, consensus-HA, has broad-spectrum activity in the H9 subtype of AIV.

[0107] 2. This invention constructs a recombinant Newcastle disease virus carrying consensus-HA, which is a weak virus;

[0108] 3. The bivalent vaccine constructed in this invention has a better effect on inhibiting viral shedding compared to commercial AIV vaccines and commercial NDV vaccines;

[0109] Although commercial vaccines can increase antibody titers more quickly, we believe that the bivalent vaccine of this invention is significantly superior to commercial vaccines in terms of antibody effectiveness in terms of shedding effect and cross-reactivity.

[0110] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, nor does it mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A consensus HA protein of H9 subtype AIV, characterized in that, The amino acid sequence of the consensus HA protein is shown in SEQ ID NO.

1.

2. A gene encoding a consensus HA protein, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. The application of the consensus HA protein as described in claim 1 or the gene as described in claim 2 in the preparation of vaccines.

4. The application according to claim 3, characterized in that, The vaccine is an avian influenza vaccine, or a bivalent vaccine; the bivalent vaccine is used for the prevention and treatment of avian influenza.

5. The application according to claim 3, characterized in that, The bivalent vaccine is also used to prevent Newcastle disease and H9 avian influenza.

6. A recombinant virus, characterized in that, It includes a vector and the gene as described in claim 2 inserted into the vector virus and expresses the consensus HA protein as described in claim 1.

7. The recombinant virus according to claim 6, characterized in that, The vector virus is Newcastle disease.

8. The recombinant virus according to claim 6, characterized in that, The vector virus for the recombinant virus is Newcastle disease virus; the gene insertion site is between the PM uncoding regions of Newcastle disease virus.

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