Construction and application of H7 subtype and H5 and H7 tetravalent avian influenza DNA vaccine

Abstract

The present application relates to construction and application of H7 subtype and H5 and H7 tetravalent avian influenza DNA vaccine. The present application belongs to the field of molecular biology, and aims to provide a DNA vaccine for providing efficient immunoprotection against H7 subtype avian influenza virus. Specifically, the present application discloses a hemagglutinin protein of H7 subtype avian influenza virus, 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 for preparing a vaccine against currently prevalent H7 subtype avian influenza virus. The present application also combines the vaccine with a vaccine against H5 subtype to obtain an H5+H7 tetravalent DNA vaccine, which can be used for prevention and control of current H5 subtype and H7 subtype avian influenza.

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 expressed by H7 subtype avian influenza A branch virus and H7 subtype avian influenza B branch virus, and a DNA vaccine strains pH7-Re5 and pH7-Re6 containing effective amounts of the above recombinant expression vector. The vaccine can effectively prevent and / or treat infection with H7 subtype A and B branch avian influenza viruses.

[0002] The above two H7 subtype avian influenza DNA vaccines, strains pH7-Re5 and pH7-Re6, and avian influenza DNA vaccines expressing avian influenza hemagglutinin protein (HA) from H5 subtype avian influenza 2.3.4.4h and H5 subtype avian influenza 2.3.4.4b branches, were mixed in equal amounts to construct a quadrivalent avian influenza (H5+H7) DNA vaccine (pH5-Re15 + pH5-Re16 + pH7-Re5 + pH7-Re6). This vaccine can effectively prevent and / or treat infections of the currently prevalent H5 subtype 2.3.4.4h and 2.3.4.4b branches of avian influenza virus, as well as H7 subtype A and B branches of avian influenza virus. Background Technology

[0003] Avian influenza is a major and highly contagious infectious disease that affects both humans and zoonots, seriously endangering the poultry industry and public health. Some strains of highly pathogenic avian influenza viruses, specifically the H5 and H7 subtypes, can cause 100% mortality in poultry. Vaccination is a crucial strategy for controlling avian influenza, especially highly pathogenic avian influenza.

[0004] 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.

[0005] my country has a huge poultry population and a wide variety of poultry species. In some areas, there are gaps in or inadequate immunization practices, resulting in the H7N9 virus not being completely eradicated from poultry. From January 2023 to February 2025, the National Avian Influenza Reference Laboratory collected 90,135 poultry swab samples and 756 environmental samples from poultry in 520 markets, 1,275 poultry farms, and 55 poultry slaughterhouses across 27 provinces in China. It also collected 13,992 wild bird droppings from wild bird habitats and hundreds of on-site poultry pathogen samples. Virus isolation results showed that 40 highly pathogenic H7N9 virus strains were isolated from the collected farm chicken samples. Gene sequence analysis showed that the HA gene nucleotide homology between the virus isolates and the currently used H7-Re4 vaccine strain was 95.9%–97.9%, and the viral HA gene gradually evolved into branches A and B (see...). Figure 1The pathogenicity and infection tests of the virus in chickens showed that SPF chickens infected with the representative H7N9 strains experienced partial or complete morbidity and death, and all infected chickens shed the virus through the larynx and cloaca, indicating that all four representative virus strains were highly lethal to chickens. The pathogenicity and infection tests of the virus in ducks showed that the representative strains with highly pathogenic viral molecular characteristics exhibited limited replication at a low level in ducks. Antigenic analysis revealed that the antigenic differences between the virus isolates and the currently used H7-Re4 vaccine strains gradually increased. The vaccine strains showed 2-16 times antigenic differences with the A-branch virus and 2-16 times antigenic differences with the B-branch virus. In summary, the H7N9 subtype avian influenza virus mutates frequently and has many antigenic subgroups; only vaccines with good antigenic matching can provide solid and effective prevention and control. Currently, there are no effective vaccines against the A-branch and B-branch variants currently circulating in my country, and the threat of H7N9 virus continues. Therefore, it is necessary to develop vaccines with antigenic matching to the current H7N9 virus to achieve better immunization and control effects. Summary of the Invention

[0006] To effectively prevent the H7 subtype A and B branch antigen variants currently monitored in my country and ensure long-term effective H7N9 control, this invention screened the two branches of the virus and obtained two representative strains: A / chicken / Hebei / SD016 / 2024 (CK / HeB / SD016 / 2024) (H7N9) (B branch) and A / chicken / Henan / SD018 / 2024 (CK / HeN / SD018 / 2024) (H7N9) (A branch). H7-Re5 and H7-Re6 vaccine strains with better antigen matching were constructed for these strains, and their immunogenicity was evaluated. The results showed that the two monovalent DNA vaccine plasmids, pH7-Re5 and pH7-Re6, have good immunogenicity and can provide complete immune protection against lethal challenges from the currently prevalent H7 subtype avian influenza virus.

[0007] Furthermore, 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. The 2.3.4.4b branch of the H5 subtype avian influenza virus, which emerged in 2020, not only infects poultry and wild birds but has also crossed species boundaries to infect various terrestrial and marine mammals. Since March 2024, the 2.3.4.4b branch of the highly pathogenic H5N1 avian influenza virus has been continuously spreading in cattle herds and causing human infections in the United States. 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. The 2.3.4.4b branch virus is widely distributed in 22 provinces across China, while the 2.3.4.4h branch virus is sporadically distributed in 11 provinces, and both exhibit varying degrees of antigenic variation. To effectively prevent the antigenic variants of the 2.3.4.4h and 2.3.4.4b branches of the H5 subtype currently monitored in my country, this invention also constructs vaccine strains H5-Re15 and H5-Re16, which have better antigenic matching against the H5 subtype.

[0008] To comprehensively control and effectively prevent the H5 subtype antigen variants of the 2.3.4.4h and 2.3.4.4b branches, as well as the H7 subtype antigen variants of the A and B branches currently monitored in my country, and based on the above research on H7 and H5 subtype avian influenza DNA vaccines, we have developed a quadrivalent avian influenza (H5+H7) DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) and evaluated its immunogenicity. The results show that the quadrivalent avian influenza (H5+H7) DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) has good immunogenicity and can provide complete immune protection against lethal challenges from the currently circulating H5 subtype avian influenza virus and H7 subtype avian influenza virus. It can be used in my country's avian influenza prevention and control practices and provide strong technical support for the healthy development of my country's poultry industry.

[0009] Specifically, the present invention provides the following technical solutions:

[0010] In a first aspect of the invention, an H7 subtype avian influenza virus hemagglutinin protein is provided, having an amino acid sequence as shown in SEQ ID NO:5 and / or SEQ ID NO:6.

[0011] In a second aspect of the invention, isolated polynucleotides encoding the H7 subtype avian influenza virus hemagglutinin protein are also provided.

[0012] In a specific embodiment of the present invention, the polynucleotide has the sequences shown as SEQ ID NO:1 and / SEQ ID NO:2.

[0013] In a third aspect of the invention, a recombinant expression plasmid comprising the isolated polynucleotide is also provided.

[0014] In a fourth aspect of the invention, a DNA or mRNA vaccine is also provided, the vaccine containing an mRNA or DNA expression vector encoding the hemagglutinin protein of the H7 subtype avian influenza virus.

[0015] In a specific embodiment of the present invention, when the vaccine is a DNA vaccine, it contains the polynucleotide or the recombinant expression plasmid.

[0016] In a specific embodiment of the present invention, when the vaccine is a DNA vaccine, the DNA expression vector for expressing the H7 subtype avian influenza virus hemagglutinin protein includes the above-mentioned polynucleotide, or the DNA expression vector for expressing the avian influenza virus hemagglutinin protein is the above-mentioned recombinant expression plasmid.

[0017] In a specific embodiment of the present invention, the DNA or mRNA vaccine further includes an mRNA or DNA expression vector for expressing the hemagglutinin protein of H5 subtype avian influenza virus.

[0018] In a specific embodiment of the present invention, the H5 subtype avian influenza virus hemagglutinin protein has an amino acid sequence as shown in SEQ ID NO: 9 and / or 10.

[0019] In a specific embodiment of the present invention, the DNA or mRNA vaccine further includes an immunologically acceptable carrier or excipient.

[0020] 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.

[0021] 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.

[0022] In a fifth aspect of the invention, a vaccine composition is also provided, comprising the H7 subtype avian influenza virus hemagglutinin protein, the polynucleotide, or the recombinant expression plasmid, and an immunologically acceptable vector or excipient.

[0023] In a specific embodiment of the present invention, the vaccine composition further includes H5 subtype avian influenza virus hemagglutinin protein, a polynucleotide encoding the H5 subtype avian influenza virus hemagglutinin protein, or a recombinant expression plasmid for expressing the H5 subtype avian influenza virus hemagglutinin protein.

[0024] The H5 subtype avian influenza virus hemagglutinin protein has an amino acid sequence as shown in SEQ ID NO: 9 and / or 10.

[0025] The polynucleotide encoding the H5 subtype avian influenza virus hemagglutinin protein has a nucleotide sequence as shown in SEQ ID NO: 7 and / or 8.

[0026] 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 and / or an H7 subtype avian influenza virus.

[0027] In a seventh aspect of the invention, the use of the H7 subtype avian influenza virus hemagglutinin protein, the polynucleotide, the recombinant expression plasmid, the DNA or mRNA vaccine, or the vaccine composition of claim 8 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 and / or H7 subtype avian influenza virus. Attached Figure Description

[0028] Figure 1 The evolutionary analysis of the HA gene in H7N9 virus isolates from January 2023 to February 2025 is shown.

[0029] Figure 2 The PCR identification results of plasmid pH7-Re5 are shown. Band 1 shows the results from the DL8000 DNA Marker; band 2 shows the results from the recombinant plasmid pH7-Re5; band 3 shows the results from the ddH2O control; and band 4 shows the results from the pH7-Re5 / PCR product.

[0030] Figure 3The results of enzyme digestion identification of plasmid pH7-Re5 are shown. Band 1 shows the results of DL8000 DNA Marker; Band 2 shows the results of EcoRI digestion of recombinant plasmid pH7-Re5; Band 3 shows the results of BsaI digestion of recombinant plasmid pH7-Re5; Band 4 shows the results of StyI digestion of recombinant plasmid pH7-Re5; Band 5 shows the results of recombinant plasmid pH7-Re5; and Band 6 shows the results of ddH2O control.

[0031] Figure 4 The PCR identification results of plasmid pH7-Re6 are shown. Band 1 shows the results of the DL8000 DNA Marker; band 2 shows the results of the recombinant plasmid pH7-Re6; band 3 shows the results of the ddH2O control; and band 4 shows the results of the pH7-Re6 / PCR product.

[0032] Figure 5 The results of enzyme digestion identification of plasmid pH7-Re6 are shown. Band 1 shows the results of DL8000 DNA Marker; Band 2 shows the results of recombinant plasmid pH7-Re6; Band 3 shows the results of EcoRI digestion of recombinant plasmid pH7-Re6; Band 4 shows the results of BsaI digestion of recombinant plasmid pH7-Re6; Band 5 shows the results of StyI digestion of recombinant plasmid pH7-Re6; Band 6 shows the results of ddH2O control; and Band 7 shows the results of DL5000 DNA Marker.

[0033] Figure 6 The results of indirect immunofluorescence assay for plasmid pH7-Re5 are shown; A shows the assay results of 293T cells transfected with the plasmid for 48 hours, and B shows the assay results of 293T cells not transfected with the plasmid.

[0034] Figure 7 The results of indirect immunofluorescence assay for plasmid pH7-Re6 are shown; A shows the assay results of 293T cells transfected with the plasmid for 48 hours, and B shows the assay results of 293T cells not transfected with the plasmid.

[0035] Figure 8 The map of the pCAGGs plasmid is shown.

[0036] Figure 9 The Western blot analysis results of the protein expression products of plasmid pH7-Re5 in 293T cells are shown. Band 1 shows the results for recombinant plasmid pH7-Re5; band 2 shows the results for plasmid pCAGGs; and band 3 shows the control results.

[0037] Figure 10 The Western blot analysis results of the protein expression products of plasmid pH7-Re6 in 293T cells are shown. Band 1 shows the results for recombinant plasmid pH7-Re6; band 2 shows the results for plasmid pCAGGs; and band 3 shows the control results. Detailed Implementation

[0038] Materials and Methods

[0039] Strains, cells and plasmids

[0040] 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.

[0041] Enzymes and main reagents

[0042] 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.

[0043] Antibody

[0044] 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-H7 subtype AIV CK / HeN / SD018 / 24 (H7N9) and CK / HeB / SD016 / 24 (H7N9) polyclonal antibodies were purchased from Harbin Weike Biotechnology Co., Ltd.

[0045] Virus, antigen

[0046] The H7 subtype highly pathogenic avian influenza virus strains A / chicken / Henan / SD018 / 2024 (abbreviated as CK / HeN / SD018 / 24) (H7N9) and A / chicken / Hebei / SD016 / 2024 (abbreviated as CK / HeB / SD016 / 24) (H7N9) 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.

[0047] 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) (abbreviated as CK / SD / S1275 / 24) strain, EID 50 10 respectively 8.83 / 0.1mL and 10 8.50 / 0.1mL. Two representative isolates of the H7 subtype from 2024, namely, strain A / chicken / Henan / SD021 / 2021 (abbreviated as CK / HeN / SD021 / 2024) (H7N9) (branch A) and strain A / chicken / Hebei / SD014 / 2024 (abbreviated as CK / HeB / SD014 / 2024) (H7N9) (branch B), serve as representative epidemic strains of the H7 subtype, with their EID... 50 10 respectively 8.50 / 0.1ml and 10 8.38 / 0.1ml. The above strains are the challenge strains for the quadrivalent DNA vaccine against avian influenza (H5+H7). Additionally, the H7 subtype A branch avian influenza virus A / chicken / Henan / SD001 / 2024 (abbreviated as CK / HeN / SD001 / 24) strain has an EID of... 50 10 8.38 / 0.1ml, is one of the attack viruses in the H7 subtype avian influenza DNA vaccine (H7-Re5 strain), H7 subtype B branch avian influenza virus A / chicken / Shandong / SD013 / 2024 (abbreviated as CK / SD / SD013 / 24), its EID 50 10 8.63 / 0.1ml, is one of the challenge viruses in the H7 subtype avian influenza DNA vaccine (H7-Re6 strain). All of the above strains were obtained from the National Avian Influenza Reference Laboratory of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0048] SPF chicken embryos and SPF chicken

[0049] 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.

[0050] Main instruments

[0051] 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.

[0052] Example 1: Codon optimization and synthesis of the HA gene of avian influenza virus

[0053] Using DNAStar software, all codons in the HA gene (SEQ ID NO: 3 or 4) of influenza viruses CK / HeN / SD018 / 24 (H7N9) and CK / HeB / SD016 / 24 (H7N9) were replaced with codons most favored by chickens, thus writing the sequences of the codon-optimized genes (opti-HeNHA7 and opti-HeBHA7, whose nucleotide sequences are shown in SEQ ID NO: 1 or 2, and whose encoded HA protein amino acid sequences are shown in SEQ ID NO: 5 or 6, respectively). The Kozak sequence gccaccacc was added before the start codon ATG, and EcoRI and XhoI restriction enzyme sites were added to both ends of the optimized gene, respectively, before being sent to Jilin Kumei Biotechnology Co., Ltd. for synthesis.

[0054] SEQ ID NO: 1, gene opti-HeNHA7, total length 1695 bp, encoding 564 amino acids:

[0055]

[0056] SEQ ID NO: 2, gene opti-HeBHA7, total length 1695 bp, encoding 564 amino acids:

[0057]

[0058] SEQ ID NO:5, Amino acid sequence of HA protein of CK / HeN / SD018 / 24 (H7N9) strain

[0059] MNTQILIFALITIIPTNADKICLGHHSVSNGTKVNTLTEKGVEVVNATETVERTNTPRICSKGKRTVDLGQCGLLGTITGPPQCDRFLTFSADLIVERREGSDVCYPGKFVNEEALRQILRESGGIDKKSMGFNYNGIRTNGTTSACMRSGSSFYAEMKWLLSNKDNATFPQMTKSYKNTRGSPAIVVWGIHHSVSNAEQTKLYGSGNKLVTVESSNYQQSFVPSPGARPQINGQSGRIDFHWLILNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSGVQVDANCEGDCYHSGGTIISNLPFQNIDSRAVGKCPRYVRQRSLLLATGMKNVPEVPKRKRTARGLFGAIAGFIENGWEGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIAKTNQQFELIDNEFNEIEKQIGNVINWTRDSITEVWSYNAEFLVAMENQHTIDLADSEMNKLYERVKKQLRENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHRKYREEAMQNRIQIDPIKLSSGYKDVILWFSFGASCFMLLAIVMGLFFICVKNGNMRCTICI.

[0060] SEQ ID NO: 6, Amino acid sequence of HA protein of CK / HeB / SD016 / 24 (H7N9) strain

[0061] .

[0062] Example 2: Construction and identification of recombinant plasmids for optimizing HA gene expression

[0063] 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 pH7-Re5 (i.e., the recombinant plasmid obtained by ligating the optimized gene of the HA gene of the CK / HeN / SD018 / 24 strain to the pCAGGoptiHA5 vector backbone) and pH7-Re6 (i.e., the recombinant plasmid obtained by ligating the optimized gene of the HA gene of the CK / HeB / SD016 / 24 strain to the pCAGGoptiHA5 vector backbone). The recombinant plasmids were transformed into E. coli DH5α competent cells, and the recombinant plasmids were identified by restriction endonucleases EcoRI, BsaI, and StyI. Simultaneously, PCR identification of the inserted fragment was performed using upstream primers on the vector and downstream primers on the optimized gene. The primers used are as follows:

[0064] pH7-Re5 strain:

[0065] Upstream primer, 5'~TCC TGG GCA CGT GCT GGT ATT GTG~3' (SEQ ID NO: 11)

[0066] Downstream primer, 5'~CTT GCG GTG GTC GTA GGT GT~3' (SEQ ID NO: 12)

[0067] pH7-Re6 strain:

[0068] Upstream primer, 5'~TTC GGC TTC TGG CGT GTG AC~3' (SEQ ID NO: 13)

[0069] Downstream primer, 5'~GAT GGC GCT CTG GGT GCT CT~3' (SEQ ID NO: 14).

[0070] The PCR and restriction enzyme digestion identification results of plasmids pH7-Re5 and pH7-Re6 are as follows:

[0071] When the plasmid vector pH7-Re5 was identified by PCR using specific upstream and downstream primers, a band of approximately 1.6 kb in length was obtained, consistent with the expected result. Figure 2 The plasmid pH7-Re5 was digested with EcoRI to obtain a fragment of approximately 6.5 kb; digested with BsaI to obtain fragments of approximately 3.7 kb and 2.7 kb; and digested with StyI to obtain fragments of approximately 2.5 kb, 1.4 kb, 1.2 kb, 1.1 kb, and 100 bp. All results were consistent with the expected sizes. Figure 3 ).

[0072] When the plasmid vector pH7-Re6 was identified by PCR, a band of approximately 1.3 kb in length was obtained, consistent with the expected result. Figure 4 The plasmid pH7-Re6 was digested with EcoRI to obtain a fragment of approximately 6.5 kb; digestion with BsaI yielded fragments of approximately 3.7 kb and 2.7 kb; digestion with StyI yielded fragments of approximately 2.5 kb, 1.4 kb, 1.1 kb, 650 bp, 300 bp, 250 bp, and 100 bp. All results were consistent with the expected sizes. Figure 5 ).

[0073] Example 3: Expression of recombinant plasmids pH7-Re5 and pH7-Re6 in 293T cells

[0074] 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.

[0075] 1. Indirect immunofluorescence detection

[0076] Forty-eight hours after plasmid transfection into 293T cells, indirect immunofluorescence assay was performed. A negative control of untransfected 293T cells was also included. 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 H7 subtype Re5 or Re6 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 area in the center of a 6-well plate. The plate was incubated at room temperature for 30 minutes. After washing five 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 five times with PBST and then observed under a fluorescence microscope (Leica DMIRE2).

[0077] 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 pH7-Re5 and pH7-Re6 were efficiently expressed in 293T cells. Figure 6 , Figure 7 ).

[0078] 2. Western blot detection

[0079] Following standard methods, the pH7-Re5 or pH7-Re6 plasmid and the empty vector pCAGGs (obtained from Professor Yoshihiro Kawaoka of the University of Wisconsin, whose diagram can be found here) were processed. Figure 8293T cells were transfected. 48 hours post-transfection, the supernatant from the six-well plates was removed, and the cells were collected and 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. H7 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.

[0080] Western blot results are as follows: 293T cells transfected with plasmids for 48 hours were collected. Cells transfected with pH7-Re5 and pH7-Re6 were analyzed by Western blot using their respective H7 subtype-specific antiserums. The results of pH7-Re5 showed that 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 could be detected. Figure 9 pH7-Re6 can 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 10 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.

[0081] Example 4: Preparation and formulation of DNA plasmids

[0082] Plasmids pH7-Re5 and pH7-Re6 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.

[0083] Before immunization, the two DNA plasmids were diluted with sterile PBS, and the immunization dose of a single plasmid was 30 μg.

[0084] In addition, this invention also prepared an H5+H7 quadrivalent DNA vaccine, the specific operations of which are as follows:

[0085] Preparation of pH5-Re15 strain plasmid: The recombinant plasmid of pH5-Re15 strain was constructed according to the methods in Examples 1 and 2. The difference was that the HA gene of influenza virus DK / HeN / SD40-1 / 24 (H5N6) was used and the codons were optimized to obtain the optimized HA gene sequence SEQ ID NO: 7, which was then ligated to the pCAGGoptiHA5 vector backbone.

[0086] Preparation of pH5-Re16 strain plasmid: The recombinant plasmid of pH5-Re16 strain was constructed according to the methods in Examples 1 and 2. The difference was that the HA gene of influenza virus GS / HuN / S10466 / 24 (H5N6) was used and the codons were optimized to obtain the optimized HA gene sequence SEQ ID NO: 8, which was then ligated to the pCAGGoptiHA5 vector backbone.

[0087] Then, the pH5-Re15 strain, pH5-Re16 strain, pH7-Re5 strain, and pH7-Re6 strain were mixed in equal concentrations and volumes to prepare a quadrivalent DNA vaccine for avian influenza (H5+H7) (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain), with an immunization dose of 30 μg for each plasmid and a total immunization dose of 120 μg.

[0088] Example 5: Experimental Grouping

[0089] Homologous and heterologous challenge protection experiments were conducted on avian influenza DNA vaccines (pH7-Re5 strain) and avian influenza DNA vaccines (pH7-Re6 strain) according to Table 1 to evaluate the protective efficacy of the two H7 subtype monovalent DNA vaccines against representative strains of different branches of currently circulating H7 subtype avian influenza.

[0090] Table 1. Experimental grouping of H7 subtype DNA vaccine immunogenicity

[0091]

[0092] According to Table 2, homologous and heterologous challenge protection experiments of the avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) were conducted to evaluate the protective effect of the avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) against representative strains of different branches of currently circulating H5 and H7 subtype avian influenza.

[0093] Table 2. Grouping of the experimental results for the immunogenicity of the quadrivalent DNA vaccine for avian influenza (H5+H7).

[0094]

[0095] Example 6: SPF Chicken Immunization Method and Procedure

[0096] Ninety SPF chickens were divided into 12 groups: 6 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 for the avian influenza DNA vaccine (pH7-Re5 strain) and the avian influenza DNA vaccine (pH7-Re6 strain) were 30 μg each.

[0097] 150 SPF chickens were divided into 20 groups: 10 immunization groups (10 chickens per group) and 10 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 total immunization dose of the avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) was 120 μg.

[0098] 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.

[0099] Example 7: Determination of HI Antibody

[0100] 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.

[0101] Example 8 Virus Challenge Protection Experiment

[0102] 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.

[0103] Example 9 Virus isolation and titration

[0104] 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.

[0105] Example 10: Evaluation of the immunoprotective efficacy of H7 subtype avian influenza DNA vaccine (pH7-Re5 strain) and avian influenza DNA vaccine (pH7-Re6 strain)

[0106] 1. Changes in HI antibodies after immunization and challenge

[0107] Seven days after booster immunization, blood was collected from each chicken to separate serum. The titer of HI antibodies in groups V1-V3 was determined using the hemagglutination inhibition assay antigen of avian influenza H7 subtype Re5 strain (purchased from Harbin Weike Biotechnology Co., Ltd.), and the titer in groups V4-V6 was determined using the hemagglutination inhibition assay antigen of avian influenza H7 subtype Re6 strain (purchased from Harbin Weike Biotechnology Co., Ltd.). The results showed that the average titer of HI antibodies in groups V1-V3 was 4log2 or higher, and the average titer in groups V4-V6 was also 4log2 or higher. The HI antibody titer in the control group was <1. See Table 3 for detailed results.

[0108] Table 3. HI antibody titers against different antigens induced after immunization with H7 subtype monovalent DNA vaccines.

[0109]

[0110] 2. Virus isolation and swab titration

[0111] During the 14-day observation period following challenge with six strains of highly pathogenic avian influenza H7 subtype, all six control chickens developed symptoms and died within 7 days of challenge; all immunized chickens remained healthy, and none developed symptoms, shed the virus, or died. See Table 4 for details.

[0112] Table 4. Protective efficacy against current circulating strains after immunization with different vaccines

[0113]

[0114] 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.

[0115] Example 11 Evaluation of the immunoprotective efficacy of a quadrivalent DNA vaccine against avian influenza (H5+H7) (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) against different HPAIV strains

[0116] 1. Changes in HI antibodies after immunization and challenge

[0117] 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 Re15 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 Re16 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. See Table 5 for detailed results.

[0118] Table 5. HI antibody titers induced by quadrivalent DNA vaccine against different antigens in avian influenza (H5+H7)

[0119]

[0120] 2. Virus isolation and swab titration

[0121] During the 14-day observation period following challenge with 6 strains of H5 subtype highly pathogenic avian influenza virus and 4 strains of H7 subtype highly pathogenic avian influenza virus, all 10 control groups of chickens developed symptoms and died within 5 days of challenge; all immunized groups of chickens remained healthy, and none of the immunized chickens developed symptoms, shed the virus, or died. See Table 6 for details.

[0122] Table 6. Protective efficacy of avian influenza (H5+H7) quadrivalent DNA vaccine against current circulating strains after immunization.

[0123]

[0124] 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.

[0125] discuss

[0126] 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.

[0127] my country has a huge poultry farming population and a wide variety of poultry species. In some areas, there are gaps in or inadequate immunization of poultry flocks, resulting in the H7N9 virus not being completely eradicated from poultry. From January 2023 to February 2025, the National Avian Influenza Reference Laboratory collected 90,135 poultry swab samples and 756 environmental samples from poultry in 520 markets, 1,275 poultry farms, and 55 poultry slaughterhouses across 27 provinces nationwide. It also collected 13,992 wild bird droppings samples from wild bird habitats and hundreds of on-site poultry pathogen samples. Virus isolation results showed that 40 highly pathogenic H7N9 virus strains were isolated from the collected farm chicken samples. Genetic evolution analysis revealed that the isolated H7 subtype avian influenza virus gradually evolved into different A and B branches.

[0128] This study selected the representative strains of the H7 subtype A branch, namely A / chicken / Henan / SD018 / 2024 (hereinafter referred to as CK / HeN / SD018 / 2024 (H7N9)) and the representative strain of the H7 subtype B branch, namely A / chicken / Hebei / SD016 / 2024 (hereinafter referred to as CK / HeB / SD016 / 2024 (H7N9)). From these, CK / HeN / SD018 / 24 and CK / HeB / SD016 / 24 were selected as representative strains of the A and B branches, respectively. The codons of their hemagglutinin gene (HA) were optimized and rewritten to codons preferred by chickens, and DNA vaccines pH7-Re5 and pH7-Re6 were constructed. These vaccines were transfected into 293T cells for in vitro expression verification. Indirect immunofluorescence experiments 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. Immunopotency tests showed that avian influenza DNA vaccines (pH7-Re5 strain) and (pH7-Re6 strain) induced high levels of HI antibodies in immunized chickens, providing 100% protection against challenges from representative isolates of different branches. All immunized chickens did not develop the disease, shed the virus, or die. The avian influenza DNA vaccine (pH7-Re5 strain) provided good protection against challenges from representative isolates of the recently prevalent H7 subtype A branch virus, and the avian influenza DNA vaccine (pH7-Re6 strain) provided good protection against challenges from representative isolates of the recently prevalent H7 subtype B branch virus. Both avian influenza DNA vaccines (pH7-Re5 strain and pH7-Re6 strain) can be applied to current avian influenza prevention and control practices.

[0129] Currently, H5 and H7 subtypes of avian influenza remain important pathogens threatening my country's poultry industry. National epidemiological surveys and systematic analyses have revealed that H5 subtype 2.3.4.4b branch virus is widely distributed in 22 provinces across China, while H5 subtype 2.3.4.4h branch virus is sporadically distributed in 11 provinces. Both have undergone varying degrees of antigenic variation. H5 subtype 2.3.4.4h and 2.3.4.4b branches are currently the main pathogens threatening my country's poultry industry. To comprehensively control and effectively prevent the H5 subtype antigenic variants of the 2.3.4.4h and 2.3.4.4b branches, as well as the H7 subtype antigenic variants of the A and B branches currently monitored in my country, based on the above research on H7 subtype avian influenza DNA vaccines and previous research on H5 subtype avian influenza DNA vaccines, we further constructed a quadrivalent avian influenza (H5+H7) DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain). Immunochaet challenges were conducted using 3 strains from the H5 subtype 2.3.4.4h branch, 3 strains from the H5 subtype 2.3.4.4b branch, 2 strains from the H7 subtype A branch, and 2 strains from the H7 subtype B branch. The results showed that the quadrivalent avian influenza (H5+H7) DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re6 strain) was effective. The pH7-Re5 + pH7-Re6 strain provides complete immune protection against lethal challenges from various currently circulating H5 and H7 subtype avian influenza viruses, exhibiting good immunogenicity. The quadrivalent DNA vaccine for avian influenza (H5+H7) (pH5-Re15 + pH5-Re16 + pH7-Re5 + pH7-Re6 strains) can be used in avian influenza prevention and control practices in my country.

Claims

1. A hemagglutinin protein of H7 subtype avian influenza virus, characterized in that, The amino acid sequence of the H7 subtype avian influenza virus hemagglutinin protein is shown in SEQ ID NO:5 or SEQ ID NO:

6.

2. A mixture of H7 subtype avian influenza virus hemagglutinin proteins, comprising the H7 subtype avian influenza virus hemagglutinin protein as shown in SEQ ID NO: 5 and the H7 subtype avian influenza virus hemagglutinin protein as shown in SEQ ID NO:

6.

3. An isolated polynucleotide, characterized in that, The polynucleotide encodes the H7 subtype avian influenza virus hemagglutinin protein of claim 1 or the H7 subtype avian influenza virus hemagglutinin protein mixture 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, characterized in that, The recombinant expression plasmid comprises the polynucleotide as described in claim 3 or 4.

6. A DNA vaccine, characterized in that, The vaccine contains a DNA expression vector for expressing the hemagglutinin protein of the H7 subtype avian influenza virus as described in claim 1 or a mixture of the hemagglutinin proteins of the H7 subtype avian influenza virus as described in claim 2.

7. The DNA vaccine according to claim 5, further comprising a DNA expression vector for expressing H5 subtype avian influenza virus hemagglutinin protein or a mixture thereof.

8. A vaccine composition comprising the H7 subtype avian influenza virus hemagglutinin protein of claim 1, the H7 subtype 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.

9. The vaccine composition according to claim 8, further comprising H5 subtype avian influenza virus hemagglutinin protein or a mixture thereof, a polynucleotide encoding the H5 subtype avian influenza virus hemagglutinin protein or a mixture thereof, or a recombinant expression plasmid for expressing the H5 subtype avian influenza virus hemagglutinin protein or a mixture thereof. in, The amino acid sequence of the H5 subtype avian influenza virus hemagglutinin protein is shown in SEQ ID NO: 9 or 10; the H5 subtype avian influenza virus hemagglutinin protein mixture is composed of the H5 subtype avian influenza virus hemagglutinin protein shown in SEQ ID NO: 9 and the H5 subtype avian influenza virus hemagglutinin protein shown in SEQ ID NO:

10.

10. Use of the polynucleotide of claim 3 or 4 or the recombinant expression plasmid of claim 5 in the preparation of a DNA vaccine for the prevention of H7 subtype avian influenza virus.

11. The use of the avian influenza virus hemagglutinin protein of claim 1, or the H7 subtype 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 vaccine of claim 6, or the vaccine composition of claim 8, characterized in that, (a) for the preparation of antibodies against hemagglutinin of H7 subtype avian influenza virus; and / or (b) for the preparation of drugs for the prevention of H7 subtype avian influenza virus infection.

12. The use of the DNA vaccine of claim 7 or the vaccine composition of claim 9, characterized in that, (a) for the preparation of antibodies against hemagglutinin of H5 and H7 subtype avian influenza viruses; and / or (b) for the preparation of drugs for the prevention of infection with H5 and H7 subtype avian influenza viruses.

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