Construction and application of avian influenza bivalent DNA vaccine

Abstract

The present application relates to construction and application of a bivalent DNA vaccine for avian influenza. The present application belongs to the field of molecular biology, and aims to provide a DNA vaccine for providing efficient immunoprotection against H5 subtype avian influenza virus. Specifically, the present application discloses an avian influenza virus hemagglutinin protein, which has an amino acid sequence as shown in SEQ ID NO: 5 and / or SEQ ID NO: 6. The present application also discloses a polynucleotide encoding the protein, which has a sequence as shown in SEQ ID NO: 1 and / or SEQ ID NO: 2. The polynucleotide can be used to prepare a vaccine against the currently prevalent H5 subtype avian influenza virus, and the vaccine can provide complete immunoprotection against lethal challenge of the currently prevalent H5 subtype avian influenza virus.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology. Specifically, this invention provides a recombinant expression vector for avian influenza hemagglutinin (HA) protein of H5 subtype avian influenza 2.3.4.4h branch virus and H5 subtype avian influenza 2.3.4.4b branch virus, and a DNA vaccine containing an effective amount of the above recombinant expression vector, wherein the vaccine can effectively prevent and / or treat infection with H5 subtype 2.3.4.4h branch and 2.3.4.4b branch avian influenza virus. Background Technology

[0002] The spread and mutation of the H5 subtype of highly pathogenic avian influenza virus continues to threaten the health of poultry, mammals, and humans, posing a significant threat to global livestock farming and public health security. Since 2020, multiple outbreaks of highly pathogenic avian influenza have occurred in poultry and wild birds, causing the deaths of large numbers of poultry and wild birds, and even infecting humans. Among these outbreaks, strains belonging to the 2.3.4.4b branch of the H5 subtype have been isolated from both wild birds and poultry, suggesting that this branch is currently the dominant circulating branch.

[0003] The first case of H5N1 influenza virus infection in dairy cows was reported in Texas, USA, on March 25, 2024. Research indicated that this H5N1 strain belonged to clade 2.3.4.4b of the current major circulating clade. Subsequently, the dairy cow influenza outbreak continued to spread, reaching over 1,000 dairy farms in 17 US states as of June 7, 2025. The main symptoms in infected cows included high fever, high incidence of mastitis, and reduced milk production; high titers of the H5N1 virus were also detected in the milk. Furthermore, in 2024, the US reported 70 cases of human infection with the H5 subtype of influenza virus, 41 of which were related to the dairy processing industry, raising global concerns about public health security.

[0004] The nationwide avian influenza epidemiological survey results from the National Avian Influenza Reference Laboratory show that the H5 subtype avian influenza viruses isolated in 2023-2024 belong to three different evolutionary branches: the 2.3.4.4b branch, the 2.3.4.4h branch, and the low pathogenic H5N3 virus branch from wild birds (see...). Figure 8Systematic analysis shows that the 2.3.4.4b branch virus is widely distributed in 22 provinces across China. Most of the monitored 2.3.4.4b branch viruses are antigenically similar to the currently used H5-Re14 vaccine strain. However, in provinces such as Hunan, Jiangxi, and Guangxi, the antigenic matching of some viruses with the H5-Re14 vaccine strain has decreased. The antiserum of the representative strain from Hunan shows a more balanced reactivity with representative strains from different time periods. The 2.3.4.4h branch virus is sporadically distributed in 11 provinces across China. The monitored 2.3.4.4h branch virus shows significant antigenic differences (8-32 times) compared to the currently used H5-Re13 vaccine strain. To effectively prevent the antigenic variants of the 2.3.4.4h and 2.3.4.4b branch H5 subtypes currently monitored in my country, it is necessary to construct and update vaccine strains with better antigenic matching.

[0005] DNA vaccines have significant advantages over traditional vaccines. They can simultaneously induce cellular and humoral immunity; they are not affected by maternal antibodies and do not interfere with immune detection; they are simple to prepare, and for pathogens with frequent mutations, antigen-specific DNA vaccines can be rapidly constructed. They can also be arbitrarily combined into combination vaccines and multivalent vaccines. Furthermore, they are environmentally friendly and have reliable biosafety, making them one of the important technological development directions for future human and animal preventative and therapeutic vaccines. Therefore, DNA vaccines are considered one of the ideal vaccines for influenza control. The H5 subtype avian influenza trivalent DNA vaccine (pH5-FJ(H5-Re13) strain + pH5-SX(H5-Re14) strain + pH7-YN(H7-Re4) strain) received a new product announcement from the Ministry of Agriculture and Rural Affairs in March 2024. In December 2024, Huapai Biotechnology (Group) Co., Ltd.'s "Nuclear Sensitive" obtained a veterinary drug product approval number (Veterinary Drug Approval Number 221012395). The H5 subtype avian influenza (H5+H7) trivalent DNA vaccine began market application in March 2025.

[0006] Due to the frequent mutations and numerous antigenic subgroups of the H5 subtype avian influenza virus, only vaccines with good antigenic matching can provide robust and effective prevention and control. Currently, there is no effective DNA vaccine targeting the 2.3.4.4h and 2.3.4.4b branch H5 subtype antigenic variants circulating in my country. Therefore, developing a DNA vaccine with better antigenic matching to these variants is an urgent problem to be solved. Summary of the Invention

[0007] To effectively prevent antigenic variants of the 2.3.4.4h and 2.3.4.4b branches of H5 subtype currently monitored in my country, this invention targets the currently prevalent H5 subtype avian influenza virus. Through systematic antigenic analysis, DK / HeN / SD40-1 / 24 (H5N6) and GS / HuN / S10466 / 24 (H5N6) viruses were screened as representative strains. H5 subtype H5-Re15 and H5-Re16 vaccine strains with better antigen matching were constructed respectively, and the immunogenicity of the H5 subtype avian influenza bivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain) was evaluated. The results showed that the two monovalent DNA vaccine plasmids pH5-Re-15 and pH5-Re-16, as well as the bivalent DNA vaccine for H5 subtype avian influenza (pH5-Re15 strain + pH5-Re16 strain), exhibited good immunogenicity and provided complete immune protection against lethal challenges from the currently circulating H5 subtype avian influenza virus. The bivalent DNA vaccine for H5 subtype avian influenza (pH5-Re15 strain + pH5-Re16 strain) will provide necessary technical reserves for the prevention and control of H5 subtype avian influenza in my country.

[0008] Specifically, the present invention relates to the following technical solutions:

[0009] In one aspect, the present invention provides an avian influenza virus hemagglutinin protein having an amino acid sequence as shown in SEQ ID NO:5 and / or SEQ ID NO:6.

[0010] In a second aspect of the invention, an isolated polynucleotide is also provided that encodes the avian influenza virus hemagglutinin protein.

[0011] In a specific embodiment of the present invention, the polynucleotide has the sequences shown as SEQ ID NO:1 and / SEQ ID NO:2.

[0012] In a third aspect of the invention, a recombinant expression plasmid comprising the aforementioned polynucleotide is also provided.

[0013] In a fourth aspect of the invention, a DNA or mRNA vaccine is also provided, wherein the vaccine contains mRNA encoding the avian influenza virus hemagglutinin protein and a DNA expression vector.

[0014] In a specific embodiment of the present invention, when the vaccine is a DNA vaccine, it contains the polynucleotide or the recombinant expression plasmid.

[0015] In a specific embodiment of the present invention, when the vaccine is a DNA vaccine, the DNA expression vector for expressing the avian influenza virus hemagglutinin protein includes the aforementioned polynucleotide, or the DNA expression vector for expressing the avian influenza virus hemagglutinin protein is the aforementioned recombinant expression plasmid.

[0016] In a specific embodiment of the present invention, the DNA or mRNA vaccine further includes an immunologically acceptable carrier or excipient.

[0017] In a specific embodiment of the present invention, the DNA or mRNA vaccine is in a form suitable for immunization by injection (including injection with a needle-free injector), mucosal delivery, or gene gun delivery.

[0018] In a specific embodiment of the present invention, the DNA or mRNA vaccine is in a form suitable for immunization by means of intravenous injection, arterial injection, intramuscular injection, subcutaneous injection, organ injection, intrapleural injection, and intraperitoneal injection.

[0019] In a fifth aspect of the invention, a vaccine composition is also provided, comprising the avian influenza virus hemagglutinin protein, the polynucleotide, or the recombinant expression plasmid, and an immunologically acceptable vector or excipient.

[0020] In a sixth aspect of the invention, the use of the polynucleotide or the recombinant expression plasmid is also provided in the preparation of a DNA or mRNA vaccine for the prevention of avian influenza virus; preferably, the avian influenza virus is an H5 subtype avian influenza virus.

[0021] In a seventh aspect of the invention, the use of the avian influenza virus hemagglutinin protein, the polynucleotide, the recombinant expression plasmid, the DNA or mRNA vaccine, or the vaccine composition thereof is also provided, characterized in that (a) it is used to prepare an antibody against avian influenza virus hemagglutinin; and / or (b) it is used to prepare a medicament for the prevention and / or treatment of avian influenza virus infection, said avian influenza virus including H5 subtype avian influenza virus. Attached Figure Description

[0022] Figure 1 The PCR identification of plasmid pH5-Re15 is shown.

[0023] Figure 2 Enzyme digestion analysis of plasmid pH5-Re15 is shown.

[0024] Figure 3 The PCR identification of plasmid pH5-Re16 is shown.

[0025] Figure 4 Enzyme digestion analysis of plasmid pH5-Re16 is shown.

[0026] Figure 5 The results of indirect immunofluorescence assays of the plasmids are shown; A shows the assay results of 293T cells transfected with plasmid pH5-Re15 for 48 h, B shows the assay results of 293T cells not transfected with the plasmid, C shows the assay results of 293T cells transfected with plasmid pH5-Re16 for 48 h, and D shows the assay results of 293T cells transfected with vector pCAGGs.

[0027] Figure 6 Western blot analysis of the protein expression products of plasmid pH5-Re15 in 293T cells is shown.

[0028] Figure 7 Western blot analysis of the protein expression products of plasmid pH5-Re16 in 293T cells is shown.

[0029] Figure 8 The evolutionary analysis of the HA gene in H5 subtype avian influenza virus isolates from 2023 to 2024 is shown.

[0030] Figure 9 The map of the pCAGGs plasmid is shown. Detailed Implementation

[0031] Materials and Methods

[0032] Strains, cells and plasmids

[0033] E. coli DH5α was purchased from Invitrogen, USA; 293T cells (human embryonic kidney cells (CRL-11268), from ATCC) and plasmid pH5-FJ (H5-Re13) are the active ingredients of avian influenza DNA vaccine (H5 subtype, pH5-FJ (H5-Re13)) (the preparation process can be found in Chinese patent application number 202111547076.2), all of which were preserved by the National Avian Influenza Reference Laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0034] Enzymes and main reagents

[0035] Q5 high-fidelity enzyme, T4 DNA ligase and endonucleases EcoRI, XhoI, BglII and BsaI were purchased from New England Bliolabs; DL10000, DL8000, DL5000 and DL2000 DNA markers were purchased from Dalian Takara Bio Inc.; Trans8K DNA Marker was purchased from Beijing TransGen Biotech Co., Ltd.; Gel DNA recovery kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; BigDye Terminator 3.1 DNA sequencing kit was purchased from Applied Biosystems; Qiagen® Plasmid Mini Kit (100) and Endofree® Plasmid Maxi plasmid extraction kit were purchased. Kit (10) was purchased from Qiagen, Lipofectamine™ 2000 transfection reagent was purchased from Invitrogen, fetal bovine serum, DMEM cell culture medium and serum-free Opti-MEM® I cell culture medium were all purchased from Gibco, and cell lysis buffer, 6× protein loading buffer and SDS-PAGE preparation kit were all purchased from Beijing Beyotime Institute.

[0036] Antibody

[0037] Rabbit anti-chicken IgG-FITC fluorescent secondary antibody was purchased from Sigma-Aldrich; rabbit anti-GAPDH polyclonal antibody was purchased from Santa Cruz Biotechnology; goat anti-rabbit IRDye® 800CW secondary antibody and donkey anti-chicken IRDye® 800CW secondary antibody were purchased from LI-COR; chicken anti-H5 subtype AIV DK / HeN / SD40-1 / 24 (H5N6) and GS / HuN / S10466 / 24 (H5N6) polyclonal antibodies were purchased from Harbin Weike Biotechnology Co., Ltd.

[0038] Virus, antigen

[0039] The H5 subtype HPAIV strains A / duck / Henan / SD40-1 / 2024 (abbreviated as DK / HeN / SD40-1 / 24) (H5N6) and A / goose / Hunan / S10466 / 2024 (abbreviated as GS / HuN / S10466 / 24) (H5N6) used for challenge were obtained from the National Avian Influenza Reference Laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The corresponding standard antigens were purchased from Harbin Weike Biotechnology Co., Ltd.

[0040] Two highly virulent representative strains of H5 avian influenza from the 2.3.4.4h branch were identified: strain A / quail / Anhui / SD46 / 2024 (H5N6) (abbreviated as QA / AH / SD46 / 24) and strain A / duck / Jiangxi / S12031 / 2024 (H5N1) (abbreviated as DK / JX / S12031 / 24). (EID) 50 10 respectively 8.50 / 0.1mL and 10 8.68 / 0.1mL. Two representative strains of H5 subtype 2.3.4.4b avian influenza virus, A / duck / Jiangxi / S10208 / 2024 (abbreviated as DK / JX / S10208 / 24) (H5N6) strain and A / chicken / Shandong / S1275 / 2024 (H5N1) strain (abbreviated as CK / SD / S1275 / 24), with EID50 values ​​of 10. 8.83 / 0.1mL and 10 8.50 / 0.1mL. All the above strains were obtained from the National Avian Influenza Reference Laboratory of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0041] SPF chicken embryos and SPF chicken

[0042] SPF chicken embryos and SPF chickens were obtained from the National Poultry Laboratory Animal Resource Bank of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The 3-week-old SPF chickens were raised in a negative pressure isolator in the poultry disease infection laboratory during the experiment.

[0043] Main instruments

[0044] The small centrifuge and gradient PCR instrument were products of Eppendorf GmbH, Germany; the gel electrophoresis imaging system and inverted fluorescence microscope were products of Zeiss GmbH, Germany; the rest of the instruments were domestically produced.

[0045] Example 1: Codon optimization and synthesis of the HA gene of avian influenza virus

[0046] Using DNAStar software, all codons in the HA gene (SEQ ID NO: 3 or 4) of influenza viruses DK / HeN / SD40-1 / 24 (H5N6) and GS / HuN / S10466 / 24 (H5N6) were replaced with codons most preferred by chickens, thus writing the sequences of the codon-optimized genes (opti-HeNHA5 and opti-HuNHA5, whose nucleotide sequences are shown in SEQ ID NO: 1 or 2). The Kozak sequence gccgccacc was added before the start codon ATG. EcoRI and XhoI restriction enzyme sites were added to both ends of the optimized genes, and then the genes were sent to Jilin Kumei Biotechnology Co., Ltd. for synthesis.

[0047] Example 2: Construction and identification of recombinant plasmids for optimizing HA gene expression

[0048] The pH5-FJ (H5-Re13) plasmid vector and the optimized gene synthesized in the pMD18T vector in Example 1 were treated with restriction endonucleases EcoRI and XhoI, respectively, and ligated at 16°C to obtain recombinant plasmids pH5-Re15 (i.e., the optimized gene containing the HA gene of strain DK / HeN / SD40-1 / 24) and pH5-Re16 (i.e., the optimized gene containing the HA gene of strain GS / HuN / S10466 / 24). The ligation products were transformed into E. coli DH5α competent cells. The recombinant plasmids were identified by restriction endonuclease digestion with EcoRI, Bgl II, and BsaI. Simultaneously, PCR identification of the inserted fragment was performed using upstream primers from the vector and downstream primers from the optimized gene. The primers used are as follows:

[0049] pH5-Re15 strain:

[0050] Upstream primer 5' ~ TTC GGC TTC TGG CGT GTG AC ~ 3' (SEQ ID NO:7)

[0051] Downstream primer 5'~ACG CGC TCC ACG ATG TAG CTC~3' (SEQ ID NO:8)

[0052] pH5-Re16 strain:

[0053] Upstream primer 5'~TTC GGC TTC TGG CGT GTG AC~3' (SEQ ID NO:9)

[0054] Downstream primer 5' ~ TCG TCG GGC TTC AGG ATG GTC ~ 3' (SEQ ID NO:10)

[0055] The results of PCR and restriction enzyme digestion identification of plasmids pH5-Re-15 and pH5-Re-6 are as follows:

[0056] When the plasmid vector pH5-Re15 was identified by PCR using specific upstream and downstream primers, a band of approximately 430 bp was obtained, consistent with the expected result. Figure 1 The plasmid pH5-Re15 was digested with EcoRI to obtain a fragment of approximately 6.5 kb; digestion with BglII yielded fragments of approximately 4.8 kb and 1.7 kb; digestion with BsaI yielded a fragment of approximately 6.5 kb. All results were consistent with the expected size. Figure 2 ).

[0057] When the plasmid vector pH5-Re16 was identified by PCR, a band of approximately 890 bp was obtained, consistent with the expected result. Figure 3 The plasmid pH5-Re16 was digested with EcoRI to obtain a fragment of approximately 6.5 kb; digested with BglII to obtain fragments of approximately 4.8 kb and 1.7 kb; and digested with BsaI to obtain fragments of approximately 3.5 kb and 3 kb. All results were consistent with the expected sizes. Figure 4 ).

[0058] Example 3: Expression of recombinant plasmids pH5-Re15 and pH5-Re16 in 293T cells

[0059] The transfection plasmid was extracted according to the Qiagen® Plasmid Mini Kit (100). Healthy 293T cells were collected, the culture medium was discarded, and the cells were washed twice with serum-free MEM. An appropriate amount of MEM was added for later use. Transfection was performed according to the Lipofectamine™ 2000 transfection kit instructions.

[0060] 1. Indirect immunofluorescence detection

[0061] Forty-eight hours after plasmid transfection into 293T cells, indirect immunofluorescence assay was performed, with a negative control of untransfected 293T cells. The culture medium was discarded, and the cells were washed three times with PBST (0.01 mol / L, pH 7.2) (containing 0.05% Tween 20). After fixation with 3% paraformaldehyde at room temperature for 20 minutes, the cells were washed three times with PBST. Positive serum from H5 subtype Re-15 or Re-16 avian influenza virus was diluted to the working concentration with PBS (0.01 mol / L, pH 7.2) (containing 0.1% fetal bovine serum albumin) and added to the selected central area of ​​a 6-well plate, incubated at room temperature for 30 minutes. After washing 5 times with PBST, the FITC-labeled rabbit anti-chicken IgG secondary antibody was diluted to the working concentration with PBS (0.01 mol / L, pH 7.2) (containing 0.1% fetal bovine serum albumin), and dropped into the selected area in the center of a 6-well plate. After incubation at room temperature for 30 minutes, the plate was washed 5 times with PBST and then observed under a fluorescence microscope (LeicaDMIRE2).

[0062] The results of indirect immunofluorescence (IFA) detection are as follows: After transfection of 293T cells, the specific protein products expressed by the plasmids were mainly distributed on the cell membrane and exhibited specific fluorescence under a fluorescence microscope. At 48 hours post-transfection, both plasmids pH5-Re15 and pH5-Re16 were efficiently expressed in 293T cells. Figure 5 ).

[0063] 2. Western blot detection

[0064] Following standard methods, the pH5-Re15 or pH5-Re16 plasmid and the empty vector pCAGGs (a gift from Professor Yoshihiro Kawaoka of the University of Wisconsin, whose image can be found here) were prepared. Figure 9 293T cells were transfected. 48 hours post-transfection, the supernatant from the six-well plates was removed, and the cells were lysed to extract proteins. The collected proteins were subjected to SDS-PAGE electrophoresis. Filter paper was cut to size according to the gel size. The cut NC membrane, filter paper, and gel were immersed in prepared transfer buffer for 5 minutes, and wet transfer was performed under constant current of 300 mA for 90 minutes. The membrane was washed three times with PBS, then blocked with freshly prepared 5% skim milk at room temperature for 1 hour. H5 subtype polyclonal antibody (1:300 dilution) and rabbit anti-GAPDH internal control antibody (1:5000 dilution) were used as primary antibodies. The membrane was incubated on a shaker at room temperature for 1 hour, followed by washing three times with TBST for approximately 3-5 minutes each time. Then, donkey anti-chicken IRDye® 800CW secondary antibody and goat anti-rabbit IRDye® 800CW secondary antibody were diluted 1:5000 and incubated on a shaker at room temperature for 1 hour. The membrane was then washed 5 times with TBST for 5 minutes each time, and the expression of the target HA protein was observed using an Odyssey laser scanner.

[0065] Western blot results are as follows: 293T cells transfected with plasmids for 48 hours were collected. Cells transfected with pH5-Re-15 and pH5-Re-16 were analyzed by Western blot using their respective H5 subtype-specific antiserums. The results showed that pH5-Re-15 could detect HA protein with a molecular weight of approximately 72 kDa, HA1 protein with a molecular weight of 46 kDa, and HA2 protein with a molecular weight of 26 kDa. Figure 6 pH5-Re-16 can detect HA protein with a molecular weight of approximately 74 kDa, HA1 protein with a molecular weight of 50 kDa, and HA2 protein with a molecular weight of 24 kDa. Figure 7 No target protein was expressed in pCAGGs vector transfection or in 293T cell control wells, but GAPDH protein expression was detected in different 293T cell wells.

[0066] Example 4: Preparation and formulation of DNA plasmids

[0067] Plasmids pH5-Re-15 and pH5-Re-16 were transformed into E. coli DH5α competent cells and evenly spread on solid LB agar plates containing ampicillin resistance. The cells were incubated overnight at 37°C. Single positive colonies were inoculated into 5 ml of liquid LB medium containing ampicillin and cultured at 37°C with shaking for 12 h. Then, they were inoculated into 500 ml of liquid LB medium containing ampicillin and cultured at 37°C with shaking for 16 h. Plasmids were extracted in large quantities according to the instructions of the QIAGEN Maxi Endofree plasmid extraction kit.

[0068] Before immunization, the two DNA plasmids were diluted with sterile PBS, with a single plasmid immunization dose of 30 μg. The two plasmids were then mixed in equal concentrations and volumes to prepare a bivalent avian influenza DNA vaccine (pH5-Re15 strain + pH5-Re16 strain), with an immunization dose of 30 μg for each plasmid and a total immunization dose of 60 μg.

[0069] Example 5: Experimental Grouping

[0070] Homologous and heterologous challenge protection experiments were conducted on the avian influenza DNA vaccine (pH5-Re15 strain), avian influenza DNA vaccine (pH5-Re16 strain), and H5 subtype avian influenza bivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain) according to the table below (see Table 1) to evaluate the protective effect of the vaccine against representative strains of different branches of the currently circulating H5 subtype avian influenza.

[0071] Table 1 Experimental Grouping

[0072]

[0073] Example 6: SPF Chicken Immunization Method and Procedure

[0074] 150 SPF chickens were divided into 18 groups: 12 immunization groups (10 chickens per group) and 6 control groups (5 chickens per group). The vaccine was administered via intramuscular injection at two points, with 0.1 ml of vaccine injected at each site, for a total immunization volume of 0.2 ml. The immunization doses of avian influenza DNA vaccine (pH5-Re15 strain) and avian influenza DNA vaccine (pH5-Re16 strain) were 30 μg each, and the total immunization dose of the H5 subtype avian influenza bivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain) was 60 μg (as shown in Table 1).

[0075] Immunization schedule: All SPF chickens in the experimental groups were first immunized at 3 weeks of age, and a booster immunization was given 3 weeks later at the same dose.

[0076] Example 7: Determination of HI Antibody

[0077] Serum was collected weekly starting from the first immunization; 25 μl of serum was added to a 96-well V-type hemagglutination plate and serially diluted 2-fold with sterile PBS to a final concentration of 2 μl. -12 Positive serum was added to the positive control wells and PBS was added to the negative control wells. Then, 25 μl of 4 units of antigen (each serum group was tested using the antigen corresponding to the challenge strain) was added and incubated at room temperature for 20 min. 25 μl of 1% red blood cell suspension was added and incubated at room temperature for 20 min. The final results were observed and recorded.

[0078] The changes in HI antibodies after immunization and challenge are as follows:

[0079] Seven days after booster immunization, blood was collected from each chicken to separate serum. The titer of HI antibodies in groups V1–V6 was determined using the hemagglutination inhibition test antigen of avian influenza H5 subtype Re-15 strain (purchased from Harbin Weike Biotechnology Co., Ltd.), and the titer in groups V7–V12 was determined using the hemagglutination inhibition test antigen of avian influenza H5 subtype Re-16 strain (purchased from Harbin Weike Biotechnology Co., Ltd.). The results showed that the average titer of HI antibodies in groups V1–V6 was 4 log2 or higher, and the average titer in groups V7–V12 was also 4 log2 or higher. The HI antibody titer in the control group was <1. Detailed results are shown in Table 2.

[0080] Table 2. HI antibody titers induced by monovalent and bivalent DNA vaccines against different antigens.

[0081]

[0082] Example 8 Virus Challenge Protection Experiment

[0083] Both experimental and control chickens were challenged with the virus one week after immunization. The virus strains used for challenge are shown in Table 1. The observation period after challenge was 14 days. During the observation period, the morbidity and mortality of chickens in the immunized and control groups were observed and recorded daily.

[0084] Example 9 Virus isolation and titration

[0085] Laryngeal and cloacal swabs were collected on days 3, 5, and 7 post-challenge. Each swab was serially diluted 10-fold (two dilutions) with sterile PBS containing penicillin (6000 IU / mL) and streptomycin (6000 μg / mL). Each dilution was inoculated into three 9-11 day old SPF chicken embryos and incubated at 37°C for 48 hours. Afterward, 50 μL of allantoic fluid was collected from each embryo, and an equal volume of 1% red blood cells was added for hemagglutination testing. The titer of the isolated virus was calculated using the Reed-Meunch method.

[0086] The results are as follows: During the 14-day observation period after challenge with six H5 subtype highly pathogenic avian influenza viruses, all six control chickens developed the disease and died within 5 days after challenge; all immunized chickens remained healthy, and none of the immunized chickens developed the disease, shed the virus, or died. See Table 3 for details.

[0087] Table 3. Protective efficacy against current circulating strains after immunization with different vaccines

[0088]

[0089] Note: * Swabs were also collected from chickens that died within 3 days on the 3rd day; first-generation positive cases do not require blind transmission; / Chicken deaths.

[0090] discuss

[0091] Avian influenza viruses mutate frequently, with new strains and branches constantly emerging and changing. Real-time monitoring and analysis are essential for the timely development and updating of vaccines targeting circulating strains and antigen-matched strains, ensuring effective and scientific control of avian influenza. DNA vaccines are simple and rapid to prepare, production does not rely on large-scale chicken embryo supplies, and they offer broader cross-protection. Furthermore, DNA vaccine immunization does not interfere with epidemiological surveillance, making DNA vaccine development a crucial area of ​​research in avian influenza vaccine development. Because DNA vaccines are clear, aqueous solutions dissolved in PBS, and DNA plasmids are easily soluble and can be stably stored at high concentrations, allowing for the mixing of any number of DNA plasmids without affecting their properties, DNA vaccines are more easily formulated into multivalent vaccines.

[0092] The nationwide epidemiological survey of avian influenza revealed that the H5 subtype avian influenza viruses isolated in 2023-2024 belonged to three different evolutionary branches: the 2.3.4.4b branch, the 2.3.4.4h branch, and the low pathogenic H5N3 virus branch from wild birds. Systematic analysis found that the 2.3.4.4b branch virus was widely distributed in 22 provinces across China, while the 2.3.4.4h branch virus was sporadically distributed in 11 provinces. The H5 subtype 2.3.4.4h and 2.3.4.4b branches are currently the main circulating strains threatening my country's poultry industry.

[0093] This study is based on the currently prevalent 2.3.4.4h H5 and 2.3.4.4b H5 branches in my country. The strains A / duck / Henan / SD40-1 / 2024 (DK / HeN / SD40-1 / 24) (H5N6) and A / goose / Hunan / S10466 / 2024 (GS / HuN / S10466 / 24) (H5N6) were selected as representative strains of the 2.3.4.4h H5 and 2.3.4.4b H5 branches, respectively. The codons of their hemagglutinin gene (HA) were optimized and rewritten to codons preferred by chickens. DNA vaccines pH5-Re15 and pH5-Re16 were constructed and transfected into 293T cells for in vitro expression verification. Indirect immunofluorescence and Western blotting results showed that the plasmids could specifically react with their corresponding positive sera, and different HA proteins could be efficiently expressed in 293T cells.

[0094] The results of the immunogenicity test showed that the avian influenza DNA vaccine (pH5-Re15 strain), the avian influenza DNA vaccine (pH5-Re16 strain), and the H5 subtype avian influenza bivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain) could induce high levels of HI antibodies after immunization. Moreover, the immunized chickens were able to provide 100% immune protection against challenges from representative isolates from different branches. All immunized chickens did not develop the disease, did not shed the virus, and did not die.

[0095] Following immunization with the avian influenza DNA vaccine (pH5-Re15 strain), challenge against representative isolates of the recently prevalent H5 subtype 2.3.4.4h branch virus was provided with good immune protection. Following immunization with the avian influenza DNA vaccine (pH5-Re16 strain), challenge against representative isolates of the recently prevalent H5 subtype 2.3.4.4b branch virus was provided with good immune protection. Following immunization with the H5 subtype avian influenza bivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain), challenge against representative isolates of both the recently prevalent H5 subtype 2.3.4.4h and 2.3.4.4b branch viruses was provided with good immune protection. The avian influenza DNA vaccine (pH5-Re15 strain), the avian influenza DNA vaccine (pH5-Re16 strain), and the H5 subtype avian influenza bivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain) can be applied to current avian influenza prevention and control practices.

[0096] sequence list

[0097] SEQ ID NO: 1, gene opti-HeNHA5, total length 1701 bp, encoding 566 amino acids:

[0098]

[0099] SEQ ID NO: 2, gene opti-HuNHA5, total length 1704 bp, encoding 567 amino acids:

[0100]

[0101] SEQ ID NO: 3, wild-type HA gene of strain DK / HeN / SD40-1 / 24 (H5N6)

[0102]

[0103] SEQ ID NO: 4, wild-type HA gene of strain GS / HuN / S10466 / 24 (H5N6)

[0104]

[0105] SEQ ID NO: 5, Amino acid sequence of the HA protein of the DK / HeN / SD40-1 / 24 (H5N6) strain

[0106] MEKIVLLLSVVNLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCRLNGVKPLILKDCSVAGWLLGNPMCDEFISVPEWSYIVERVNPANDLCYPGNLNDYEELKHLLSRINHFEKTRIIPKNSWSNHTSSGVSAACPYQGNASFFRNVVWLTKKNDAYPTIKMSYNNTNKEDLLILWGIHHSNSAEEQTKLYKNLITYVSVGTSTLNQRLVPKIATRSQVNGQSGRMDFFWTMLKPNDAIHFESNGNFIAPEYAYKIIKTGDSTIMKSEIEYGHCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPLRERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADRESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLVLAIIVAGLSLWMCSNGSLQCRICI.

[0107] SEQ ID NO: 6, Amino acid sequence of the HA protein of the GS / HuN / S10466 / 24 (H5N6) strain

[0108] MENIVLLLATVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAGWLLGNPMCDEFIRVPEWSYIVERDNPANDLCYPGSLNDYEELKHLLSRINHFEKILIIPKSSWPNHETSLGVSAACPYRGAPSFFRNVVWLIKKDDAYPTIKISYNNTNREDLLILWGIHHSNNAEEQTKLYKNPTTYISVGTSTLNQRLVPKIATRSQVNGQRGRMDFFWTILKPDDAIHFESNGNFIAPEYAYKIVKKGDSTIMKSGVEYGHCNTKCQTPVGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPLREKRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYYYPQYSEEARLKREEISGVKLESIGTYQILSIYSTAASSLALAIMMAGLSLWMCSNGSLQCRICI.

Claims

1. A hemagglutinin protein of avian influenza virus, the amino acid sequence of which is shown in SEQ ID NO:5 or SEQ ID NO:

6.

2. A mixture of avian influenza virus hemagglutinin proteins, comprising avian influenza virus hemagglutinin proteins as shown in SEQ ID NO:5 and avian influenza virus hemagglutinin proteins as shown in SEQ ID NO:

6.

3. An isolated polynucleotide encoding the avian influenza virus hemagglutinin protein of claim 1 or a mixture of avian influenza virus hemagglutinin proteins of claim 2.

4. The polynucleotide according to claim 3, having a sequence as shown in SEQ ID NO:1 and / or SEQ ID NO:

2.

5. A recombinant expression plasmid comprising the polynucleotides described in claim 3 or 4.

6. A DNA or mRNA vaccine, characterized in that, The vaccine contains an mRNA or DNA expression vector encoding the avian influenza virus hemagglutinin protein of claim 1 or the mixture of avian influenza virus hemagglutinin proteins of claim 2.

7. The DNA or mRNA vaccine according to claim 6, further comprising an immunologically acceptable carrier or excipient.

8. The DNA or mRNA vaccine according to claim 6 or 7, wherein it is in a form suitable for immunization by injection, mucosal delivery, or gene gun delivery.

9. A vaccine composition comprising the avian influenza virus hemagglutinin protein of claim 1, the avian influenza virus hemagglutinin protein mixture of claim 2, the polynucleotide of claim 3 or 4, or the recombinant expression plasmid of claim 5, and an immunologically acceptable vector or excipient.

10. Use of the polynucleotide of claim 3 or 4 or the recombinant expression plasmid of claim 5 in the preparation of a DNA or mRNA vaccine for the prevention of H5 subtype avian influenza virus.

11. The use of the avian influenza virus hemagglutinin protein of claim 1, or the avian influenza virus hemagglutinin protein mixture of claim 2, or the polynucleotide of claim 3 or 4, or the recombinant expression plasmid of claim 5, or the DNA or mRNA vaccine of any one of claims 6-8, or the vaccine composition of claim 9, characterized in that, (a) for the preparation of antibodies against hemagglutinin of H5 subtype avian influenza virus; and / or (b) for the preparation of drugs for the prevention of H5 subtype avian influenza virus infection.

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