Virus-like particles of h5n6, h5n8 and h7n9 and uses, vaccines

Abstract

The application belongs to the technical field of biology, and discloses virus-like particles of H5N6, H5N8 and H7N9 and purposes and vaccines thereof, wherein the virus-like particles of H5N6, H5N8 and H7N9 are obtained by jointly connecting HA, NA and M1 genes of H5N6, H5N8 and H7N9 to plasmids and then being transferred into competent cells to obtain recombinant baculovirus plasmids, and then the recombinant baculovirus plasmids are transfected and expressed to obtain corresponding virus-like particles, and in addition, the trivalent vaccine prepared by using the virus-like particles does not produce antagonism between different antigens.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to virus-like particles of H5N6, H5N8 and H7N9 and their uses, and vaccines. Background Technology

[0002] Inactivated vaccines are a primary strategy for the prevention and control of avian influenza. However, these vaccines have several limitations, including long production cycles, insufficient immunogenicity, suboptimal induction of cellular immune responses, and limited broad-spectrum protection against rapidly evolving viral strains. Furthermore, the production of inactivated vaccines relies on egg-based culture systems, which not only increases production costs but also introduces the risk of antigenic variation due to viral adaptive mutations. Therefore, there is an urgent need to develop novel vaccine platforms that provide enhanced immunogenicity, broader cross-protection, scalable production capacity, and improved safety.

[0003] Virus-like particles (VLPs) have become a cutting-edge vaccine platform due to their unique ability to mimic viral structure and elicit strong immune responses. VLPs are self-assembled from viral structural proteins such as hemagglutinin (HA), neuraminidase (NA), and matrix protein (M1), forming particles that are highly similar in morphology and structure to natural viruses. This high degree of similarity allows VLPs to effectively mimic the viral infection process, thereby inducing strong humoral and cellular immune responses, while the lack of viral genetic material in VLPs further enhances their safety. Furthermore, VLPs can be engineered to develop multivalent vaccines, providing broader cross-protection. HA is a major target for neutralizing antibodies and a key antigen in the design of avian influenza vaccines. NA is crucial for viral release and transmission, exhibits low variability, and contains conserved epitopes, contributing to broader protection. The highly conserved M1 is essential for viral particle assembly and morphology, further enhancing the breadth and durability of the immune response. Furthermore, M1 can act as an immune adjuvant during viral infection, thereby making vaccine design more effective. Due to their superior immunogenicity, safety, and engineering flexibility, VLPs have become ideal candidates for developing multivalent vaccines against various pandemic avian influenza subtypes. The baculovirus expression vector system (BEVS) serves as a highly efficient platform for VLP production, enabling the rapid generation of high-purity and high-yield VLPs in a short time. This capability has significantly facilitated the rapid development and large-scale production of avian influenza vaccines.

[0004] In their paper, "Trivalent Human Papillomavirus (HPV) VLP vaccine covering HPV type 58 can elicit high level of humoral immunity but also induce immune interference among component types," Ting Zhang, Yufei Xu, Liang Qiao, Youchun Wang, Xueling Wu, Dongsheng Fan, Qinglin Peng, Xuemei Xu, and others pointed out that monovalent vaccines typically cannot provide sufficient cross-protection against the antigenically diverse H5 and H7 avian influenza viruses. Therefore, developing multivalent vaccines targeting these key subtypes is crucial for enhancing overall protective efficacy and improving pandemic preparedness. However, immune interference between different antigens in multivalent vaccines is a significant issue. For example, while trivalent vaccines against human papillomavirus (HPV) (containing HPV types 16, 18, and 58) induce high levels of humoral immunity, immune interference between different antigens was also observed, and this interference became more pronounced with an increase in the number of antigens.

[0005] Therefore, interference between different antigens is a crucial issue that requires careful attention during the preparation of multivalent vaccines. Studying immune interference between different antigens is of great significance because it directly affects the overall effectiveness and protective spectrum of the vaccine. Understanding and overcoming this interference can help optimize multivalent vaccine design by adjusting antigen dosage and adding appropriate adjuvants to enhance the vaccine's immune response. This will not only help improve the efficacy of existing vaccines but also promote the development of novel broad-spectrum vaccines, thereby more effectively addressing the threat of multiple pathogens and strengthening public health protection capabilities.

[0006] The technical problem of this solution is: how to develop a virus-like particle for a trivalent avian influenza (H5+H7) vaccine with broad-spectrum protection. Summary of the Invention

[0007] The purpose of this application is to provide a broad-spectrum avian influenza vaccine virus-like particle. Through experiments, it was found that when the trivalent vaccine prepared by using H5N6, H5N8 and H7N9 virus-like particles together showed no significant difference in the immune effect against the parent strains compared with the monovalent vaccine of the three virus-like particles. It can be seen that there is no immune interference between the antigens of the three virus-like particles in the vaccine.

[0008] To achieve the above objectives, this application discloses virus-like particles of H5N6, H5N8 and H7N9, wherein the virus-like particles of H5N6 are obtained by linking the HA, NA and M1 genes of H5N6 virus together to a plasmid and transfecting it into competent cells to obtain a recombinant baculovirus plasmid.

[0009] The recombinant baculovirus plasmid was then transfected into insect cells and expressed to obtain virus-like particles of H5N6.

[0010] The H5N8 virus-like particles are obtained by linking the HA, NA and M1 genes of the H5N8 virus together to a plasmid and then transferring it into competent cells to obtain a recombinant baculovirus plasmid.

[0011] The recombinant baculovirus plasmid was then transfected into insect cells and expressed to obtain H5N8 virus-like particles;

[0012] The H7N9 virus-like particles are obtained by linking the HA, NA and M1 genes of the H7N9 virus together to a plasmid and then transferring it into competent cells to obtain a recombinant baculovirus plasmid.

[0013] The recombinant baculovirus plasmid was then transfected into insect cells and expressed to obtain virus-like particles of H7N9.

[0014] The amino acid sequence of the H5N6 virus HA protein is shown in SEQ ID NO: 1;

[0015] The amino acid sequence of the H5N6 virus NA protein is shown in SEQ ID NO: 2;

[0016] The amino acid sequence of the H5N6 virus M1 protein is shown in SEQ ID NO: 3;

[0017] The amino acid sequence of the H5N8 virus HA protein is shown in SEQ ID NO: 4;

[0018] The amino acid sequence of the H5N8 virus NA protein is shown in SEQ ID NO: 5;

[0019] The amino acid sequence of the H5N8 virus M1 protein is shown in SEQ ID NO: 6;

[0020] The amino acid sequence of the H7N9 virus HA protein is shown in SEQ ID NO: 7;

[0021] The amino acid sequence of the H7N9 virus NA protein is shown in SEQ ID NO: 8;

[0022] The amino acid sequence of the H7N9 virus M1 protein is shown in SEQ ID NO: 9.

[0023] In addition, this application also discloses the use of virus-like particles of H5N6, H5N8 and H7N9 as described above to prepare vaccines.

[0024] In addition, this application also discloses an H5+H7 trivalent vaccine, which is prepared using H5N6 virus-like particles, H5N8 virus-like particles, and H7N9 virus-like particles as antigens.

[0025] Preferably, the H5N6 virus-like particles are obtained by linking the HA, NA and M1 genes of the H5N6 virus together to a plasmid and then transferring it into competent cells to obtain a recombinant baculovirus plasmid.

[0026] The recombinant baculovirus plasmid was then transfected into insect cells and expressed to obtain virus-like particles of H5N6.

[0027] The H5N8 virus-like particles are obtained by linking the HA, NA and M1 genes of the H5N8 virus together to a plasmid and then transferring it into competent cells to obtain a recombinant baculovirus plasmid.

[0028] The recombinant baculovirus plasmid was then transfected into insect cells and expressed to obtain H5N8 virus-like particles;

[0029] The H7N9 virus-like particles are obtained by linking the HA, NA and M1 genes of the H7N9 virus together to a plasmid and then transferring it into competent cells to obtain a recombinant baculovirus plasmid.

[0030] The recombinant baculovirus plasmid was then transfected into insect cells and expressed, yielding virus-like particles of H7N9.

[0031] Preferably, the nucleotide sequence encoding the H5N6 virus HA protein is shown in SEQ ID NO: 10;

[0032] The nucleotide sequence encoding the H5N6 viral NA protein is shown in SEQ ID NO: 11;

[0033] The nucleotide sequence encoding the H5N6 virus M1 protein is shown in SEQ ID NO: 12;

[0034] The nucleotide sequence encoding the H5N8 virus HA protein is shown in SEQ ID NO: 13;

[0035] The nucleotide sequence encoding the H5N8 viral NA protein is shown in SEQ ID NO: 14;

[0036] The nucleotide sequence encoding the H5N8 virus M1 protein is shown in SEQ ID NO: 15;

[0037] The nucleotide sequence encoding the H7N9 virus HA protein is shown in SEQ ID NO: 16;

[0038] The nucleotide sequence encoding the H7N9 virus NA protein is shown in SEQ ID NO: 17;

[0039] The nucleotide sequence encoding the H7N9 virus M1 protein is shown in SEQ ID NO: 18.

[0040] The beneficial effects of this application are: the broad-spectrum avian influenza vaccine virus-like particles provided in this application, through experiments, have shown that when the trivalent vaccine prepared by using H5N6, H5N8 and H7N9 virus-like particles together, there is no significant difference in the immune effect against the parent strains compared to the monovalent vaccines of the three virus-like particles. It can be seen that there is no immune interference between the antigens of the three virus-like particles in the vaccine.

[0041] Furthermore, the trivalent vaccine made from the three types of virus-like particles mentioned above produces antibody titers even higher than those of commercial vaccines in a short period of time, demonstrating that the trivalent vaccine has the advantage of rapidly inducing an immune response. Attached Figure Description

[0042] Figure 1 Schematic diagram of the design of the rH5N6-pFastBac1-NA-HA-M1 plasmid;

[0043] Figure 2 Schematic diagram of the design of the rH5N8-pFastBac1-NA-HA-M1 plasmid;

[0044] Figure 3 Schematic diagram of the design of the rH7N9-pFastBac1-NA-HA-M1 plasmid;

[0045] Figure 4 Fluorescence contrast between Sf9 control cell wells and Sf9 cell wells infected with recombinant baculovirus rBV-H5N6-NA-HA-M172h;

[0046] Figure 5 Fluorescence contrast between Sf9 control cell wells and Sf9 cell wells infected with recombinant baculovirus rBV-H5N8-NA-HA-M172h;

[0047] Figure 6 Fluorescence contrast between Sf9 control cell wells and Sf9 cell wells infected with recombinant baculovirus rBV-H7N9-NA-HA-M172h;

[0048] Figure 7 This is a Western blotting image of H5N6-VLP. Figure 7 In the image, 'a' represents the expression of H5N6-VLP identified by PAGE. Figure 7b is a Western blot image of H5N6-VLP incubated with a His-tagged monoclonal antibody as the primary antibody. Figure 7 In the middle (c), there is a Western blot diagram of H5N6-VLP identified by incubation with H5 subtype HA monoclonal antibody as the primary antibody.

[0049] Figure 8 PAGE was used to identify the expression patterns of H5N8-VLP and H7N9-VLP. Figure 8 In the image, 'a' represents the expression of H5N8-VLP identified by PAGE. Figure 8 In the image, b represents the expression of H7N9-VLP identified by PAGE; lane 1 is the target sample; and lane 2 is a negative control of High Five cell samples.

[0050] Figure 9 To identify the expression patterns of H5N8-VLP and H7N9-VLP using Western blot, Figure 9 In the middle section, 'a' represents the primary antibody, which is a His-tagged monoclonal antibody, used for incubation to identify H5N8-VLP. Figure 9 In the middle section, b is an H5 subtype HA monoclonal antibody used for primary antibody incubation to identify H5N8-VLP; Figure 9 In the middle lane, the primary antibody c is a His-tagged monoclonal antibody used for incubation to identify H7N9-VLP; lane 1 is the target sample; lane 2 is a HighFive cell sample negative control.

[0051] Figure 10 Purification and identification of H5N6-VLP Figure 10 In the diagram, 'a' represents a schematic diagram of sucrose purification. Figure 10 In the middle b, PAGE (20%–30% of the sucrose layer is H5N6-VLP); Figure 10 c represents the incubated His monoclonal antibody; Figure 10 In the middle, d represents the incubated H5 subtype avian influenza HA monoclonal antibody;

[0052] Figure 11 Purification and identification of H5N8-VLP and H7N9-VLP Figure 11 In diagram 'a', we see the purification process of sucrose using H5N8-VLP. Figure 11 In the middle b, PAGE (20%–30% of the sucrose layer is H5N8-VLP); Figure 11 c represents the H5N8-VLP identified by the incubated His monoclonal antibody; Figure 11 In the middle section, d represents the identification of H5N8-VLP using incubated H5 subtype avian influenza HA monoclonal antibodies; Figure 11 The diagram in 'e' represents the purification process of sucrose using H5N8-VLP. Figure 11 f in the middle represents PAGE (20%–30% of the sucrose layer is H7N9-VLP); Figure 11In lane g, the His monoclonal antibody was incubated to identify H7N9-VLP, and lane 1 was the target sample; lane 2 was a High Five cell sample as a negative control.

[0053] Figure 12 Electron micrographs of the morphological characteristics of H5N6-VLP;

[0054] Figure 13 Electron micrographs of the morphological characteristics of H5N8-VLP;

[0055] Figure 14 Electron micrographs of the morphological characteristics of H7N9-VLP;

[0056] Figure 15 The graph shows the HI antibody titer against the H5N6-GD154 strain in SPF chickens immunized with H5N6-VLP vaccine.

[0057] Figure 16 The graph shows the MN antibody titer against the H5N6-GD154 strain in SPF chickens immunized with the H5N6-VLP vaccine.

[0058] Figure 17 A graph showing the survival rate of chickens challenged with the H5N6-GD154 strain after immunization with the H5N6-VLP vaccine at SPF.

[0059] Figure 18 This is a comparison chart of HI antibody titers against parental strains in SPF chickens immunized with H5N6-H5N8-H7N9-VLP, H5N6-VLP, and H7N9-VLP. Figure 18 In the figure, 'a' is a comparison diagram of H5N6-H5N8-H7N9-VLP and H5N6-VLP. Figure 18 Figure b is a comparison diagram of H5N6-H5N8-H7N9-VLP and H5N8-VLP. Figure 18 In the middle, c is a comparison diagram of H5N6-H5N8-H7N9-VLP and H7N9-VLP;

[0060] Figure 19 This image shows the HI antibody titer against circulating H5 subtype strains in SPF chickens immunized with the H5N6-H5N8-H7N9-VLP vaccine. Figure 19 Figure 'a' shows the antibody titer detection graph for the H5N2-GD285 challenge strain. Figure 19 Figure b shows the antibody titer detection graph for H5N1-GD105;

[0061] Figure 20 This is a graph showing the HI antibody titer against circulating H7N9 subtype strains in SPF chickens immunized with the H5N6-H5N8-H7N9-VLP vaccine. Figure 20Figure 'a' shows the antibody titer detection result for the H7N9-C5F-145 challenge strain. Figure 20 Figure b shows the antibody titer detection graph for the H7N9-GD014 challenge strain;

[0062] Figure 21 A comparison of survival rates of chickens challenged with parental strains after immunization with the H5N6-H5N8-H7N9-VLP vaccine at SPF levels. Figure 21 In Figure 'a', the survival rate comparison chart of H5N6-H5N8-H7N9-VLP and H5N6-VLP is shown. Figure 21 Figure b shows a comparison of the survival rates of H5N6-H5N8-H7N9-VLP and H5N8-VLP. Figure 21 In the middle, c is a comparison of the survival rates of H5N6-H5N8-H7N9-VLP and H7N9-VLP. Detailed Implementation

[0063] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0064] Example 1 Construction of recombinant baculovirus plasmid

[0065] 1.1 Design of Recombinant Transfer Plasmids

[0066] BGI Genomics Co., Ltd. codon-optimized and synthesized the HA, NA, and M1 genes from A / Chicken / Guangdong / 154 / 2022 (H5N6-GD154), A / Chicken / Guangdong / 110 / 2022 (H5N8-GD110), and A / Chicken / Guangdong / 144 / 2022 (H7N9-C5E-144), respectively, and ligated them into the baculovirus transfer plasmid pFastBac1 to obtain recombinant transfer plasmids rH5N6-pFastBac1-NA-HA-M1, rH5N8-pFastBac1-NA-HA-M1, and rH7N9-pFastBac1-NA-HA-M1. The plasmid construction diagram is shown below. Figure 1-3 As shown, where, Figure 1 Schematic diagram of the design of the rH5N6-pFastBac1-NA-HA-M1 plasmid;

[0067] Figure 2 Schematic diagram of the design of the rH5N8-pFastBac1-NA-HA-M1 plasmid;

[0068] Figure 3 Schematic diagram of the design of the rH7N9-pFastBac1-NA-HA-M1 plasmid;

[0069] The nucleotide sequences of the HA, NA and M1 genes after codon optimization for the H5N6 subtype are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and their amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0070] The nucleotide sequences of the HA, NA and M1 genes after codon optimization for the H5N8 subtype are shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, and their amino acid sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0071] The nucleotide sequences of the HA, NA and M1 genes after codon optimization for the H7N9 subtype are shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and their amino acid sequences are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[0072] 1.2 Construction of recombinant baculovirus plasmid

[0073] The recombinant transfer plasmids rH5N6-pFastBac1-NA-HA-M1, rH5N8-pFastBac1-NA-HA-M1, and rH7N9-pFastBac1-NA-HA-M1 were transformed into DH10bac competent cells, respectively. The specific steps are as follows: 50 ng of the recombinant transfer plasmid was added to 50 µL of DH10Bac competent cells, gently mixed, and incubated on ice for 30 min. The cells were then heat-shocked at 42°C for 45 s, followed by an ice incubation for 5 min. 900 µL of antibiotic-free LB liquid medium was added to EP tubes, and the cells were incubated at 37°C with shaking at 220 r / min for 4 h. The bacterial suspensions were then diluted 10-fold using antibiotic-free LB liquid medium to obtain bacterial suspensions with dilutions of 10⁻¹, 10⁻², and 10⁻³. 400 µL of bacterial suspension at each dilution was evenly spread onto a blue-white screening LB plate. After incubation at 37 °C for 48 h, white single colonies were picked for expansion culture. After PCR identification, plasmids were extracted to obtain recombinant baculovirus plasmids, which were named Bacmid-H5N6-NA-HA-M1, Bacmid-H5N8-NA-HA-M1, and Bacmid-H7N9-NA-HA-M1, respectively.

[0074] Example 2: Obtaining Recombinant Baculovirus

[0075] 2.1 The recombinant baculovirus plasmid prepared in Example 1 was transfected into sf9 insect cells (fall armyworm cells) (Invitrogen) using liposome-mediated transfection. The specific transfection steps are as follows:

[0076] (1) Sf9 cells in the logarithmic growth phase (1.6~1.8×10⁻⁶) 6 (cells / mL) were seeded in a six-well plate and incubated at 27°C for 1 h. After the cells adhered, they were transfected.

[0077] (2) Take 6-8 µL of Cellfectin® II and dilute it in 100 µL of Grace medium and vortex to mix. Take 1 µL of baculovirus DNA and dilute it in 100 µL of Grace medium and mix gently. Mix the diluted DNA with the diluted Cellfectin® II and incubate at room temperature for 15-30 min to obtain a DNA-liposome mixture.

[0078] (3) Add 800 µL of Grace medium to the liposome mixture to make up to 1 mL. Add the DNA-liposome mixture to the six-well plate and incubate the cells at 27°C for 3–5 h;

[0079] (4) Remove the transfection mixture, add 2 mL of HF-SFM, and incubate at 27°C for 72 h. Observe the cytopathic effect during the period. After 72 h of incubation, collect the cell culture supernatant to obtain the first generation of recombinant baculovirus, named: rBV-H5N6-NA-HA-M1.

[0080] The preparation methods for recombinant baculoviruses of H5N8 and H7N9 are the same as above.

[0081] 2.2 Detection of target gene expression using indirect immunofluorescence assay (IFA)

[0082] sf9 cells were infected with P3 generation recombinant baculoviruses (rBV-H5N6-NA-HA-M1, rBV-H5N8-NA-HA-M1, and rBV-H7N9-NA-HA-M1) with an MOI of 0.1, respectively. Indirect immunofluorescence assays were performed 48 hours after infection. The simplified steps are as follows:

[0083] (1) Discard the liquid in the six-well plate, add 1 mL of cold methanol solution to each well, and fix in a refrigerator at 4°C for 15 min;

[0084] (2) Discard the fixative, add PBS solution, and wash 3 times.

[0085] (3) Add mouse-derived anti-His tag monoclonal antibody diluted 1:200 and incubate overnight at 4°C;

[0086] (4) Recover the primary antibody, add PBST buffer for washing, 5 min each time, repeat 3 times;

[0087] (5) Add diluted FITC-conjugated goat anti-mouse IgG antibody and incubate at 37°C in the dark for 1 h;

[0088] (6) Discard the secondary antibody, add PBST buffer for washing, 5 min each time, repeat 3 times;

[0089] (7) Observation using a fluorescence microscope, with sf9 cells as a control, showed that specific fluorescence could be detected in all wells infected with the recombinant baculovirus, while no specific fluorescence was produced in the wells of the sf9 control cells. Specifically, as follows: Figure 4-6 As shown, where, Figure 4 Fluorescence contrast between Sf9 control cell wells and Sf9 cell wells infected with recombinant baculovirus rBV-H5N6-NA-HA-M172h;

[0090] Figure 5 Fluorescence contrast between Sf9 control cell wells and Sf9 cell wells infected with recombinant baculovirus rBV-H5N8-NA-HA-M172h;

[0091] Figure 6 Fluorescence contrast image of Sf9 control cell wells and Sf9 cell wells infected with recombinant baculovirus rBV-H7N9-NA-HA-M172h.

[0092] Example 3 Assembly and purification of H5N6-VLP, H5N8-VLP and H7N9-VLP

[0093] 3.1 Assembly and harvesting of virus-like particles

[0094] rBV-H5N6-NA-HA-M1 viral suspensions with different MOIs were inoculated onto High Five culture medium in good growth condition. ++ In cells, suspension culture was performed at 27℃ and 115 r / min for 5 days. After centrifugation at 2000 g for 20 min, the supernatant was collected, followed by centrifugation at 12000 g for 60 min. H5N6-VLP was then collected with PBS for later use. Results showed that at an MOI of 0.1, the highest hemagglutination titers were observed in both the extracellular culture supernatant and the intracellular fragmented supernatant, indicating that an MOI of 0.1 was the optimal infection condition for producing virus-like particles (Table 1). Similarly, rBV-H5N8-NA-HA-M1 and rBV-H7N9-NA-HA-M1 with an MOI of 0.1 were inoculated into High Five culture medium. ++Insect cells were used to prepare H5N8-VLP and H7N9-VLP.

[0095] Table 1: Screening MOIs

[0096] MOI High Five Cell Density Culture volume VLP blood clotting titer (log2) 0.05 <![CDATA[2.5×10 6 ]]> 50mL 12 0.1 <![CDATA[2.5×10 6 ]]> 50mL 14 0.5 <![CDATA[2.5×10 6 ]]> 50mL 13 1 <![CDATA[2.5×10 6 ]]> 50mL 13

[0097] 3.2 Identification of virus-like particles

[0098] The virus-like particles harvested in step 1 were analyzed by SDS-PAGE and Western blot. In the Western blot analysis, the primary antibody for identifying the HA protein of H5N6-VLP and H5N8-VLP was an H5 subtype influenza hemagglutinin HA monoclonal antibody (Surpass Biotechnology, Beijing), and the primary antibody for identifying HA, NA, and M1 proteins was His-tag (4C2) mouse monoclonal antibody (Bioword). Normal High Five insect cell supernatant was used as a negative control. Results are as follows: Figure 7-9 As shown, distinct bands are observed at approximately 72 kDa, 60 kDa, and 28.5 kDa in H5N6-VLP, corresponding to HA, NA, and M1 proteins, respectively. Distinct bands are also observed at approximately 72 kDa, 55 kDa, and 28.5 kDa in H5N8-VLP and H7N9-VLP, corresponding to HA, NA, and M1, respectively.

[0099] It should be noted that, Figure 7 The image shows a Western blotting diagram for H5N6-VLP, where a: PAGE; b: H5N6-VLP was identified by incubation with a His-tagged monoclonal antibody as the primary antibody; c: H5N6-VLP was identified by incubation with an H5 subtype HA monoclonal antibody as the primary antibody.

[0100] Figure 8 PAGE was used to identify the expression patterns of H5N8-VLP and H7N9-VLP. Figure 8 In the image, 'a' represents the expression of H5N8-VLP identified by PAGE. Figure 8 In the image, b represents the expression of H7N9-VLP identified by PAGE; lane 1 is the target sample; and lane 2 is a negative control of High Five cell samples.

[0101] Figure 9 To identify the expression patterns of H5N8-VLP and H7N9-VLP using Western blot, Figure 9 In the middle section, 'a' represents the primary antibody, which is a His-tagged monoclonal antibody, used for incubation to identify H5N8-VLP. Figure 9 In the middle section, b is an H5 subtype HA monoclonal antibody used for primary antibody incubation to identify H5N8-VLP; Figure 9In the middle lane, the primary antibody c is a His-tagged monoclonal antibody used for incubation to identify H7N9-VLP; lane 1 is the target sample; lane 2 is a HighFive cell sample negative control.

[0102] 3.3 Purification of virus-like particles

[0103] Preparation of sucrose solutions of different concentrations: 20%, 30%, 45%, and 60% (w / v) sucrose solutions were prepared and filtered through a 0.22 μm filter. The 20%, 30%, 45%, and 60% sucrose solutions were added to centrifuge tubes from top to bottom, with the virus-like particle sample added on top. The tubes were centrifuged at 100,000 × g for 1 h at 4 °C. After centrifugation, the white transparent band between the sucrose layers was collected. The sucrose was removed by centrifugation at 100,000 × g at 4 °C for 1 h. The virus-like particle sample was resuspended in PBS buffer and identified by PAGE and Western blotting. The results are shown below. Figure 10-11 As shown, HA, NA and M1 proteins were confirmed to be expressed in purified H5N6-VLP, H5N8-VLP and H7N9-VLP, and the HA titer of H5N6-VLP, H5N8-VLP and H7N9-VLP could reach 13log2.

[0104] It should be noted that, Figure 10 Purification and identification of H5N6-VLP Figure 10 In the diagram, 'a' represents a schematic diagram of sucrose purification. Figure 10 In the middle b, PAGE (20%–30% of the sucrose layer is H5N6-VLP); Figure 10 c represents the incubated His monoclonal antibody; Figure 10 In the middle, d represents the incubated H5 subtype avian influenza HA monoclonal antibody;

[0105] Figure 11 Purification and identification of H5N8-VLP and H7N9-VLP Figure 11 In diagram 'a', we see the purification process of sucrose using H5N8-VLP. Figure 11 In the middle b, PAGE (20%–30% of the sucrose layer is H5N8-VLP); Figure 11 c represents the H5N8-VLP identified by the incubated His monoclonal antibody; Figure 11 In the middle section, d represents the identification of H5N8-VLP using incubated H5 subtype avian influenza HA monoclonal antibodies; Figure 11 The diagram in 'e' represents the purification process of sucrose using H5N8-VLP. Figure 11 f in the middle represents PAGE (20%–30% of the sucrose layer is H7N9-VLP); Figure 11 In lane g, the His monoclonal antibody was incubated to identify H7N9-VLP, and lane 1 was the target sample; lane 2 was a High Five cell sample as a negative control.

[0106] 3.4 Observation of the morphology and structure of virus-like particles using transmission electron microscopy

[0107] The virus-like particle sample harvested in step 3 was dropped onto a carbon-coated copper grid for adsorption and incubated at room temperature for 2 minutes. Excess liquid on the copper grid was gently blotted away with absorbent paper. After drying, 3% uranium acetate was dropped onto the copper grid, and after 3 minutes, excess staining liquid was blotted away with a small piece of filter paper. The grid was then allowed to air dry at room temperature. The results were observed using a transmission electron microscope as follows: Figure 12-14 As shown ( Figure 12 Electron micrographs of the morphological characteristics of H5N6-VLP. Figure 13 Electron micrographs of the morphological characteristics of H5N8-VLP. Figure 14 (Electron micrograph of the morphological characteristics of H7N9-VLP): Particle size is about 100-120 nm, and fibrous structures are visible on the surface.

[0108] Example 4: Evaluation of the Homologous Protective Efficacy of H5N6-VLP Vaccine

[0109] 4.1 Vaccine preparation

[0110] H5N6-VLP and inactivated vaccines with different gradient hemagglutination titers (7log2, 9log2, 11log2) were prepared in a 1:1 ratio with ISA 78 adjuvant.

[0111] 4.2 Immunization schedule for vaccines

[0112] Forty-five 21-day-old SPF chickens were randomly divided into four groups of ten each, with five chickens receiving PBS buffer. All groups were immunized via subcutaneous injection in the neck. Group 1 received 0.3 mL of H5N6-VLP vaccine with a hemagglutination titer of 7 log2 per chicken. Group 2 received 0.3 mL of H5N6-VLP vaccine with a hemagglutination titer of 9 log2 per chicken. Group 3 received 0.3 mL of H5N6-VLP vaccine with a hemagglutination titer of 11 log2 per chicken. Group 4 received 0.3 mL of H5N6 inactivated vaccine with a hemagglutination titer of 9 log2 per chicken. Group 5 served as a blank control with PBS buffer. The animal groupings and immunization protocols are shown in Table 2.

[0113] Table 2: Animal grouping and immunization regimen for evaluating the homologous protective efficacy of H5N6-VLP vaccine

[0114]

[0115] 4.3 Serum antibody test results

[0116] Blood was collected from all experimental chickens at weeks 2 and 3 post-immunization to separate serum. Antibody detection was performed using a routine serum inhibition (HI) and microneutralization (MN) antibody detection assay. Homologous tetra-unit antigen was prepared from the H5N6-GD154 strain (Tetra-unit antigen preparation: The hemagglutination titer of the avian influenza virus antigen was determined, with the highest dilution factor for complete hemagglutination as the endpoint. The endpoint dilution factor divided by 4 gave the 4 HAU dilution factor. The antigen was diluted to 4 HAU using PBS buffer. 4 HAU verification: A 96-well V-plate was prepared, with 25 µL of PBS buffer added to each well; 25 µL of 4 HAU was added to the first well, and serially diluted 2-fold to the third well; 25 µL of 1% chicken red blood cell suspension was added, and the plate was incubated at room temperature for 30 min. The results were then observed). HI antibody detection results are shown below. Figure 15 ( Figure 15 The graph shows the HI antibody titer against the H5N6-GD154 strain in SPF chickens immunized with the H5N6-VLP vaccine. Results showed that HI antibody titers were detectable in all vaccine groups 2 weeks post-immunization. The average HI titers at 2 weeks post-immunization were 4.3 log2, 5.7 log2, 6.3 log2, and 3.8 log2 in the 7 log2, 9 log2, 11 log2, and inactivated vaccine groups, respectively. The average HI titers at 3 weeks post-immunization were 6.4 log2, 7 log2, 8 log2, and 7.2 log2, respectively. MN antibody detection results are shown below. Figure 16 ( Figure 16 (Graph showing MN antibody titer against H5N6-GD154 strain in SPF chickens immunized with H5N6-VLP vaccine). Results showed that the average MN antibody titer in all groups of experimental chickens was higher at 3 weeks post-immunization than at 2 weeks post-immunization. At 3 weeks post-immunization, the MN antibody titer in the 11log2 group was 1:1920, while the MN antibody titer in the 9log2 group (1:1120) showed no significant difference from that in the inactivated vaccine group (1:960).

[0117] 4.4 Results of Homologous Challenge Protection Experiment

[0118] Three weeks post-immunization, avian influenza virus strain H5N6-GD154 was administered via intranasal challenge at a dose of 0.2 mL / bird (10 avians). 6.0 EID 50 Following viral challenge, the incidence and mortality of experimental chickens were observed and recorded daily for 14 consecutive days. Laryngeal and cloacal swabs were collected on days 3, 5, and 7 post-challenge. Results showed that all chickens in the PBS control group died within two days, exhibiting typical clinical symptoms including lethargy, bradykinesia, and neurological abnormalities. (Reference) Figure 17 ( Figure 17The survival rate of chickens challenged with the H5N6-GD154 strain after immunization with the H5N6-VLP vaccine in SPF chickens was 100%. All chickens in the immunized group survived 14 days after challenge with the lethal dose of H5N6-GD154 AIV, achieving a 100% survival rate. Virus isolation results (Table 3) showed that 2 chickens in the 7log2 vaccine group shed the virus on day 3 post-challenge, and 1 chicken shed the virus on day 5 post-challenge. No virus shedding was detected in the other immunized groups on days 3, 5, and 7 post-challenge.

[0119] The above results indicate that immunization with the 9log2 H5N6-VLP vaccine can effectively protect against lethal doses of H5N6-GD154 virus in SPF chickens.

[0120] Table 3: Virus isolation results in the H5N6-GD154 strain challenge experiment after SPF chickens immunized with H5N6-VLP vaccine.

[0121]

[0122] Note: NA: No samples were collected from dead experimental chickens; dpc: Number of days after infection.

[0123] Example 5: Evaluation of the immunogenicity of a trivalent avian influenza (H5+H7) virus-like particle vaccine

[0124] 5.1 Vaccine Preparation

[0125] The high-speed homogenizer was cleaned with sterile PBS. 150 µL each of the pre-prepared H5N6-VLP, H5N8-VLP, and H7N9-VLP antigens were mixed thoroughly. This step ensured that the amount of each antigen was identical, guaranteeing the homogeneity of the final mixture. The mixed antigens were then mixed with 450 µL of ISA 78 adjuvant at a volume ratio of 1:1. After emulsification in the homogenizer, 10 µL of the vaccine was aspirated for testing. A second drop was added after the first drop formed an oil film; if the film did not disperse within 30 seconds, the vaccine preparation was successful. The resulting vaccine was called the H5N6-H5N8-H7N9-VLP vaccine, and the final hemagglutination titer was 9 log2.

[0126] 5.2 Immunization schedule for vaccines

[0127] Two hundred and five 21-day-old SPF chickens were randomly divided into six groups, as detailed in Table 4. All groups were immunized via subcutaneous injection in the neck. The H5N6-VLP, H5N8-VLP, H7N9-VLP, and PBS groups received 0.3 mL per chicken; the commercially available inactivated vaccine group received 0.3 mL per chicken according to the instructions; and the H5N6-H5N8-H7N9-VLP vaccine group received 0.9 mL per chicken.

[0128] Table 4: Animal grouping and immunization regimen for evaluating the immunogenicity of H5N6-H5N8-H7N9-VLP vaccine

[0129] Group vaccine adjuvant volume ratio Quantity (pieces) 1 H5N6 VLP ISA 78 1:1 10 2 H5N8 VLP ISA 78 1:1 10 3 H7N9 VLP ISA 78 1:1 10 4 H5N6-H5N8-H7N9-VLP ISA 78 1:1 70 5 Commercial inactivated vaccines - - 70 6 PBS - - 35

[0130] 5.3 Serum antibody test results

[0131] 5.3.1 Antibody detection of H5N6-H5N8-H7N9-VLP vaccine against parental strains

[0132] To evaluate the differences in antibody induction between the H5N6-H5N8-H7N9-VLP trivalent vaccine and its corresponding monovalent VLP vaccine and commercially available inactivated vaccine, immunization was conducted in H5N6-VLP, H5N8-VLP, H7N9-VLP, H5N6-H5N8-H7N9-VLP trivalent vaccine, and commercially available inactivated vaccine groups. Serum samples were collected at 2 and 3 weeks post-immunization for HI antibody detection against the parental virus strain. HI antibody test results are shown below. Figure 18 The results showed that, against the H5N6-GD154 strain, the mean HI antibody titers at 2 weeks post-immunization were 5.5 log2, 5.8 log2, and 4.1 log2 in the H5N6-VLP group, H5N6-H5N8-H7N9-VLP group, and the commercially available inactivated vaccine group, respectively; and at 3 weeks post-immunization, the mean HI antibody titers were 7.5 log2, 7.6 log2, and 8 log2, respectively. Figure 18 (a) Against the H5N8-GD110 strain, the mean HI antibody titers at 2 weeks post-immunization were 6.5 log2, 6.7 log2, and 7.1 log2 in the H5N8-VLP group, H5N6-H5N8-H7N9-VLP group, and the commercially available inactivated vaccine group; the mean HI antibody titers at 3 weeks post-immunization were 8.2 log2, 8.5 log2, and 8.6 log2. Figure 18 (b) Regarding the H7N9-C5e-144 strain, the mean HI antibody titers at 2 weeks post-immunization were 5.6 log2, 5.8 log2, and 4.6 log2 in the H7N9-VLP group, H5N6-H5N8-H7N9-VLP group, and the commercially available inactivated vaccine group, respectively; and at 3 weeks post-immunization, the mean HI antibody titers were 7.5 log2, 7.5 log2, and 8 log2, respectively. Figure 18 (c)

[0133] It should be noted that, Figure 18 This is a comparison chart of HI antibody titers against parental strains in SPF chickens immunized with H5N6-H5N8-H7N9-VLP, H5N6-VLP, and H7N9-VLP. Figure 18In the figure, 'a' is a comparison diagram of H5N6-H5N8-H7N9-VLP and H5N6-VLP. Figure 18 Figure b is a comparison diagram of H5N6-H5N8-H7N9-VLP and H5N8-VLP. Figure 18 In the middle, c is a comparison diagram of H5N6-H5N8-H7N9-VLP and H7N9-VLP.

[0134] The above results indicate that all vaccine groups produced high levels of antibodies by week 3 post-immunization, and there was no significant difference in HI antibody titers between the monovalent VLP vaccine and the H5N6-H5N8-H7N9-VLP trivalent vaccine at weeks 2 and 3 post-immunization. At week 3 post-immunization, the HI antibody titer of the H5N6-H5N8-H7N9-VLP vaccine was not significantly different from that of the commercially available inactivated vaccine. This suggests that the H5N6-H5N8-H7N9-VLP vaccine has a good immunogenicity, comparable to that of the corresponding monovalent VLP vaccine and the commercially available inactivated vaccine, and that there is no immune interference between the multiple antigens.

[0135] 5.3.2 Antibody detection of H5N6-H5N8-H7N9-VLP vaccine against circulating strains

[0136] The cross-protection of the H5N6-H5N8-H7N9-VLP vaccine against two predominantly circulating H5 and H7N9 subtype AIV strains in 2024 was assessed using the HI antibody assay. Results are as follows: Figure 19 ( Figure 19 This image shows the HI antibody titer against circulating H5 subtype strains in SPF chickens immunized with the H5N6-H5N8-H7N9-VLP vaccine. Figure 19 Figure 'a' shows the antibody titer detection graph for the H5N2-GD285 challenge strain. Figure 19(Figure b shows the antibody titer detection for H5N1-GD105). Against the H5N2-GD285 strain, at week 2 post-immunization, the mean HI antibody titers for the H5N6-H5N8-H7N9-VLP group and the commercially available inactivated vaccine group were 3.4 log2 and 3.0 log2, respectively; at week 3 post-immunization, the mean HI antibody titers for the two groups were 5.2 log2 and 5.7 log2, respectively. Against the H5N1-GD105 strain, at week 2 post-immunization, the mean HI antibody titers for the H5N6-H5N8-H7N9-VLP group and the commercially available inactivated vaccine group were 2.9 log2 and 2.3 log2, respectively; at week 3 post-immunization, the mean HI antibody titers for the two groups were 4.4 log2 and 5.2 log2, respectively. Regarding the immune response to the prevalent H5 subtype AIV strains, all immunization groups showed a 100% HI antibody seroconversion rate at week 3 post-immunization. In terms of immunization efficacy, the antibody levels in the H5N6-H5N8-H7N9-VLP vaccine group were higher than those in the commercially available inactivated vaccine group at week 2 post-immunization. However, at week 3 post-immunization, the antibody titer in the commercially available inactivated vaccine group was significantly higher than that in the H5N6-H5N8-H7N9-VLP vaccine group. This indicates that the H5N6-H5N8-H7N9-VLP vaccine can induce an immune response relatively quickly and possesses good cross-protection.

[0137] Further evaluation of the cross-protective efficacy of the H5N6-H5N8-H7N9-VLP vaccine against circulating H7N9 subtype AIV strains using HI antibody detection technology yielded the following results: Figure 20 ( Figure 20 The titer of HI antibodies against circulating H7N9 subtype strains was measured in SPF chickens immunized with the H5N6-H5N8-H7N9-VLP vaccine. Figure 20 Figure 'a' shows the antibody titer detection result for the H7N9-C5F-145 challenge strain. Figure 20 Figure b shows the antibody titer detection for the challenge strain H7N9-GD014. For the H7N9-C5F-145 strain, at week 2 post-immunization, the mean HI antibody titers for the H5N6-H5N8-H7N9-VLP group and the commercially available inactivated vaccine group were 4.6 log2 and 5.8 log2, respectively; at week 3 post-immunization, the mean HI antibody titers for the two groups were 5.3 log2 and 6.4 log2, respectively. For the H7N9-GD014 strain, at week 2 post-immunization, the mean HI antibody titers for the H5N6-H5N8-H7N9-VLP group and the commercially available inactivated vaccine group were 3.2 log2 and 4.8 log2, respectively; at week 3 post-immunization, the mean HI antibody titers for the two groups were 4.8 log2 and 5.5 log2, respectively. For different H7N9 subtype strains, all immunization groups achieved a 100% antibody seroconversion rate at week 3 post-immunization.

[0138] In summary, the H5N6-H5N8-H7N9-VLP vaccine can rapidly induce an immune response in the early stages and exhibits good cross-protection. However, in the later stages of immunization (3 weeks), the antibody response against the heterologous strain is weaker compared to commercially available vaccines.

[0139] 5.3.3 Results of the challenge protection experiment of H5N6-H5N8-H7N9-VLP vaccine

[0140] (1) Results of challenge protection against parental strains with H5N6-H5N8-H7N9-VLP vaccine

[0141] The protective efficacy of the H5N6-H5N8-H7N9-VLP trivalent vaccine, its corresponding monovalent vaccine, and commercially available inactivated vaccines against the parent strain was evaluated through a challenge protection test. Chickens were challenged 3 weeks post-vaccination, and mortality was recorded daily for 14 consecutive days. Results are shown below. Figure 21 ( Figure 21 Survival curves of chickens challenged with parental strains after immunization with H5N6-H5N8-H7N9-VLP vaccine at SPF. Figure 21 In the figure, a represents the survival curves of chickens with H5N6-H5N8-H7N9-VLP and H5N6-VLP. Figure 21 In Figure b, the survival curves of chickens in the H5N6-H5N8-H7N9-VLP and H5N8-VLP series are shown. Figure 21 Table 5-7 shows the survival curves of chickens in the H5N6-H5N8-H7N9-VLP and H7N9-VLP groups (c represents the survival curves of chickens in the H5N6-H5N8-H7N9-VLP group). Laryngeal and cloacal swabs were collected from the experimental chickens 5 and 7 days after challenge for virus shedding detection. The results are shown in Table 5-7. The results showed that all chickens in the H5 subtype challenge PBS group died within 2 days after challenge, and all chickens in the H7N9 subtype challenge PBS group died within 3 days after challenge. All chickens exhibited symptoms such as respiratory distress, diarrhea, unsteady gait, lethargy, ruffled feathers, and general malaise. All vaccine groups survived without showing obvious symptoms throughout the observation period, and no virus shedding was detected, resulting in a 100% protection rate against the parental virus strain. This indicates that the H5N6-H5N8-H7N9-VLP trivalent vaccine, along with commercially available inactivated vaccines and corresponding monovalent VLP vaccines, provides 100% complete protection against challenge with the parental virus strain.

[0142] Table 5: Virus isolation results in the challenge experiment of H5N6-GD154 strain in SPF chickens immunized with H5N6-H5N8-H7N9-VLP vaccine.

[0143]

[0144] Note: NA: No samples were collected from dead experimental chickens; dpc: After virus challenge.

[0145] Table 6: Virus isolation results in the challenge experiment of H5N8-GD110 strain in SPF chickens immunized with H5N6-H5N8-H7N9-VLP vaccine.

[0146]

[0147] Note: NA: No samples were collected from dead experimental chickens; dpc: After virus challenge.

[0148] Table 7: Virus isolation results in the H7N9-C5e-114 strain challenge experiment after SPF chickens immunized with H5N6-H5N8-H7N9-VLP vaccine

[0149]

[0150] Note: NA: No samples were collected from dead experimental chickens; dpc: After virus challenge.

[0151] (2) Results of challenge protection against circulating strains with H5N6-H5N8-H7N9-VLP vaccine

[0152] The protective efficacy of the H5N6-H5N8-H7N9-VLP trivalent vaccine and the commercially available inactivated vaccine against the currently prevalent H5 subtypes (H5N2-GD285 and H5N1-GD105) and H7 subtypes (H7N9-C5F-145 and H7N9-GD014) of highly pathogenic avian influenza was evaluated through challenge protection experiments. Throat swabs and cloacal swabs were collected from experimental chickens 5 and 7 days after challenge for virus shedding detection. The results are shown in Tables 8-9: In the PBS group, all chickens died within 3 days after challenge, exhibiting symptoms such as respiratory distress, diarrhea, unsteady gait, lethargy, ruffled feathers, and general malaise. Against the H5 and H7 subtype HPAIV prevalent strains, both the H5N6-H5N8-H7N9-VLP trivalent vaccine and the commercially available inactivated vaccine showed no obvious symptoms throughout the observation period; all chickens survived and no virus shedding was detected. This indicates that the H5N6-H5N8-H7N9-VLP vaccine and the commercially available inactivated vaccine provide broad protection against two circulating H5 subtype and two H7N9 subtype HPAIV strains.

[0153] Table 8: Results of virus isolation after challenge chickens with H5 subtype HPAIV epidemic strains

[0154]

[0155] Note: NA: No samples were collected from dead experimental chickens; dpc: After virus challenge.

[0156] Table 9: Virus isolation results of H7N9 subtype HPAIV epidemic strains 5 and 7 days after challenge to chickens.

[0157]

[0158] Note: NA: No samples were collected from dead experimental chickens; dpc: After virus challenge.

[0159] All the above-mentioned animal test results indicate that the present invention has developed an avian influenza virus-like particle vaccine (H5N6-H5N8-H7N9-VLP) covering three subtypes: H5N6, H5N8, and H7N9. This trivalent vaccine exhibits good immunogenicity, with protective efficacy comparable to monovalent VLP vaccines and commercially available inactivated vaccines, and no immune interference was observed between different antigens. This vaccine provides good cross-protection against the parent strain and the major circulating strains in 2024. The avian influenza (H5+H7) trivalent virus-like particle vaccine prepared by the present invention provides a new vaccine option for the prevention and control of avian influenza.

[0160] Comparative Example 1

[0161] Reference CN113862284B, the subject of which is a gene encoding a recombinant avian influenza virus HA protein, virus-like particles, a vaccine, and its preparation and application. This vaccine provides complete clinical protection and significantly inhibits viral shedding against lethal challenges to homologous and wild-type H7N9 subtype highly pathogenic avian influenza viruses.

[0162] Comparative Example 1 is the applicant's earlier application. A comparison with Comparative Example 1 shows that the virus-like particle vaccine prepared in Comparative Example 1 uses a co-infection expression method, requiring co-infection with three insect baculoviruses during production; the vaccine in Comparative Example 1 is a monovalent virus-like particle vaccine of the H7N9 subtype with limited protective efficacy. In contrast, the virus-like particle vaccine prepared in this application uses a co-expression method, requiring only one insect baculovirus to achieve high-level protein expression during production. Furthermore, this application is a trivalent virus-like particle vaccine for avian influenza (H5+H7), covering three subtypes of avian influenza: H5N6, H5N8, and H7N9. Regarding cross-protection, the challenge strains selected in this application, in addition to the parent strain, also include circulating strains from 2024, better demonstrating the broad-spectrum protective efficacy of this trivalent virus-like particle vaccine.

Claims

1. An H5+H7 trivalent vaccine, characterized in that, It was prepared using virus-like particles of H5N6, H5N8, and H7N9 as antigens. The H5N6 virus-like particles are obtained by linking the HA, NA and M1 genes of the H5N6 virus together to a plasmid and then transferring it into competent cells to obtain a recombinant baculovirus plasmid. The recombinant baculovirus plasmid was then transfected and expressed to obtain virus-like particles of H5N6. The H5N8 virus-like particles are obtained by linking the HA, NA and M1 genes of the H5N8 virus together to a plasmid and then transferring it into competent cells to obtain a recombinant baculovirus plasmid. The recombinant baculovirus plasmid was then transfected and expressed to obtain virus-like particles of H5N8. The H7N9 virus-like particles are obtained by linking the HA, NA and M1 genes of the H7N9 virus together to a plasmid and then transferring it into competent cells to obtain a recombinant baculovirus plasmid. The recombinant baculovirus plasmid was then transfected and expressed to obtain virus-like particles of H7N9. The amino acid sequence of the H5N6 virus HA protein is shown in SEQ ID NO: 1; The amino acid sequence of the H5N6 virus NA protein is shown in SEQ ID NO: 2; The amino acid sequence of the H5N6 virus M1 protein is shown in SEQ ID NO: 3; The amino acid sequence of the H5N8 virus HA protein is shown in SEQ ID NO: 4; The amino acid sequence of the H5N8 virus NA protein is shown in SEQ ID NO: 5; The amino acid sequence of the H5N8 virus M1 protein is shown in SEQ ID NO: 6; The amino acid sequence of the H7N9 virus HA protein is shown in SEQ ID NO: 7; The amino acid sequence of the H7N9 virus NA protein is shown in SEQ ID NO: 8; The amino acid sequence of the H7N9 virus M1 protein is shown in SEQ ID NO:

9.

2. The vaccine according to claim 1, characterized in that, The H5N6 virus-like particles are obtained by linking the HA, NA and M1 genes of the H5N6 virus together to a plasmid and then transferring it into competent cells to obtain a recombinant baculovirus plasmid. The recombinant baculovirus plasmid was then transfected into insect cells and expressed to obtain virus-like particles of H5N6. The H5N8 virus-like particles are obtained by linking the HA, NA and M1 genes of the H5N8 virus together to a plasmid and then transferring it into competent cells to obtain a recombinant baculovirus plasmid. The recombinant baculovirus plasmid was then transfected into insect cells and expressed to obtain H5N8 virus-like particles; The H7N9 virus-like particles are obtained by linking the HA, NA and M1 genes of the H7N9 virus together to a plasmid and then transferring it into competent cells to obtain a recombinant baculovirus plasmid. The recombinant baculovirus plasmid was then transfected into insect cells and expressed, yielding virus-like particles of H7N9.

3. The H5+H7 trivalent vaccine according to claim 2, characterized in that, The nucleotide sequence encoding the H5N6 virus HA protein is shown in SEQ ID NO: 10; The nucleotide sequence encoding the H5N6 viral NA protein is shown in SEQ ID NO: 11; The nucleotide sequence encoding the H5N6 virus M1 protein is shown in SEQ ID NO: 12; The nucleotide sequence encoding the H5N8 virus HA protein is shown in SEQ ID NO: 13; The nucleotide sequence encoding the H5N8 viral NA protein is shown in SEQ ID NO: 14; The nucleotide sequence encoding the H5N8 virus M1 protein is shown in SEQ ID NO: 15; The nucleotide sequence encoding the H7N9 virus HA protein is shown in SEQ ID NO: 16; The nucleotide sequence encoding the H7N9 virus NA protein is shown in SEQ ID NO: 17; The nucleotide sequence encoding the H7N9 virus M1 protein is shown in SEQ ID NO: 18.

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