Broad spectrum anti-influenza vaccine immunogens and uses thereof

Abstract

To provide a novel influenza immunogen with a broad-spectrum anti-influenza virus activity, and to provide an immunization method using the influenza immunogen.SOLUTION: The present invention provides a novel anti-influenza immunogen comprising an amino acid sequence whose sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2, or an immunogenic fragment thereof, or a combination thereof. Furthermore, the present invention also provides use of a recombinant vector vaccine using the immunogen in preparing an anti-influenza vaccine, and an immunization method of a recombinant vector vaccine using the immunogen. By sequentially applying multiple vector vaccines expressing this novel influenza immunogen and using a combination of systemic and local immunization, a high-level T cell immune response can be induced locally in the respiratory tract and produce broad-spectrum protection against multiple influenza virus infections.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Technical Field The present invention relates to the research, design and production of engineered vaccines, in particular broad-spectrum anti-influenza virus vaccine immunogens and their uses, including novel immunogens, recombinant vector vaccines and immunization methods therefor. [Background technology]

[0002] Background technology Influenza is a highly contagious, rapid-transmitting acute respiratory tract infection caused by influenza virus infection. Influenza viruses belong to the Orthomyxoviridae family and are antisense single-stranded RNA viruses. Seasonal influenza and frequent, unpredictable influenza pandemics caused by influenza viruses pose a serious risk to human health and public health. According to statistics reported by the World Health Organization (WHO), influenza A virus infects 3 to 5 million people worldwide each year, resulting in approximately 250,000 to 500,000 deaths. Due to the frequent outbreaks of highly pathogenic influenza strains such as H5N1, H1N1, H3N2, and H7N9 in recent years, research into a universal influenza vaccine with cross-protective effects against different influenza virus subtypes is crucial.

[0003] Vaccination is the most effective and cost-effective means of preventing influenza. Currently, all influenza vaccines approved by the World Health Organization are seasonal influenza vaccines, and many influenza vaccine research hotspots worldwide focus on inducing antibody responses against the influenza virus envelope hemagglutinin (HA) protein to achieve protective effects. The HA head region has a significant immunodominant role in inducing neutralizing antibody production. However, this region is prone to antigen drift. Neutralizing antibodies against the HA head region have strong strain specificity, and viruses may selectively mutate in a way that escapes neutralizing antibodies, making cross-protection difficult. In recent years, several broad-spectrum neutralizing antibodies targeting the HA stem region have been discovered, but their successful application is hindered by poor cross-protection across influenza virus groups. The HA stem region is a less immunogenic region in natural infections, resulting in weak virus neutralization. Currently, influenza-specific CD8 + Studies have confirmed that T cells have broad-spectrum anti-influenza activity and can kill cells infected by different influenza subtypes. Furthermore, after influenza infection, influenza virus antigen-specific CD8 + Memory T cells can persist in the airways for up to one year, and this group has specific CD8 + The number of cells is related to the host's cross-protective ability against influenza infection, providing a theoretical basis for designing a highly efficient and broad-spectrum antiviral influenza vaccine.

[0004] Currently, all influenza vaccines approved by the WHO are seasonal, the most widely used of which is a trivalent inactivated vaccine containing two influenza A viruses (H1N1 and H3N2) and one influenza B virus. Subcutaneously administered vaccines and live-attenuated vaccines administered via nasal spray are also approved. However, all of these vaccines share the common challenge that their protective effect depends on the match between the current circulating influenza virus strain and the vaccine's virulent strain. Influenza viruses continue to mutate, making WHO monitoring and prediction time-consuming, labor-intensive, and inaccurate. Ensuring a supply of vaccines for this season requires production at least seven or eight months in advance, significantly increasing the uncertainty of vaccine predictions and rendering them virtually ineffective against potential pandemic influenza. Current vaccine production still relies primarily on chicken embryos, resulting in a long production cycle, complex processes, time-consuming and labor-intensive processes, and relatively high costs. Currently, there are many strategies for developing influenza vaccines, among which the commonly used inactivated vaccines and live attenuated vaccines lack efficacy and require complex production processes and long production times.

[0005] Currently, DNA vaccines and viral vector vaccines are widely used, and DNA vaccines have proven to be the most effective priming vaccine form. Combined immunization strategies, priming with DNA vaccines and boosting with protein or viral vector vaccines, are also a hot topic of research in vaccine immunization strategies. Currently, the most commonly used adenovirus vaccine vector is human type 5 adenovirus, which has a relatively good ability to express foreign genes. However, because it is a human-derived adenovirus vector, it is easily neutralized by pre-existing adenovirus antibodies in most humans, rendering the vaccine ineffective and thereby limiting the application of this vaccine vector. In recent years, the gorilla-derived type 68 adenovirus vaccine vector has been discovered, successfully overcoming the problem of the human body's lack of antibodies against this adenovirus. Furthermore, gorilla-derived type 68 adenovirus can infect both dividing and non-dividing cells and can transduce lung cells, liver cells, bone cells, blood vessels, muscles, brain, and central nervous system cells. Its excellent gene stability and exogenous gene expression capability allow it to be mass-produced in HEK293 cells and it has been widely used in vaccine research for AIDS, Ebola, influenza, malaria, hepatitis C, and other diseases. The poxvirus Tiantan strain vaccine vector has a wide host range, high replication titer, induced a very long-lasting immune response, and a very large capacity for foreign gene insertion (theoretically reaching 25-50 kb). The poxvirus Tiantan strain can effectively stimulate antibody and T cell immune responses. It has been proven to be very safe and can be used in immunocompromised individuals.

[0006] Therefore, the current broad-spectrum influenza vaccines face a challenging challenge: how to effectively utilize the conserved proteins within the influenza virus, design immunogens against internal CD8 T cell epitopes of the influenza virus, and stimulate the immune system more comprehensively, efficiently, and sustainably through a variety of different vaccine vectors and their immunological combination strategies, thereby providing broader and more effective protective effects. Summary of the Invention

[0007] Summary of the Invention In one aspect of the present invention, there is provided an influenza vaccine immunogen comprising an amino acid sequence selected from the group consisting of: the amino acid sequence set forth in SEQ ID No. 1 and SEQ ID No. 2, or an immunogenic fragment thereof, or a combination of these sequences.

[0008] In certain embodiments of the invention, the immunogen comprises an influenza virus internal conserved protein, or an immunogenic fragment of a conserved protein.

[0009] In another specific embodiment of the invention, the influenza virus internal conserved proteins include influenza virus matrix proteins (M1, M2), nucleoprotein (NP), alkaline polymerase (PB1, PB2) and acid polymerase (PA).

[0010] In yet another specific embodiment of the present invention, the immunogen is derived from recombinant proteins of all influenza virus subtypes, including influenza A virus subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, influenza B virus, or recombinant proteins of shared sequences thereof, or combinations thereof.

[0011] In another aspect of the present invention, there is provided an anti-influenza virus recombinant vector vaccine, which is a recombinant vector vaccine constructed by expressing the above-mentioned anti-influenza vaccine immunogens, or immunogenic fragments thereof, or a combination thereof, in multiple different vectors.

[0012] In a specific embodiment of the present invention, the recombinant vector vaccine comprises a recombinant protein vaccine, a recombinant DNA vaccine, a recombinant viral vector vaccine, a recombinant bacterial vector vaccine, a recombinant yeast vector vaccine, or a recombinant virus-like particle vaccine, and the viral vector comprises an adenovirus vector, a poxvirus vector, an adeno-associated virus vector, a herpes simplex virus vector, a cytomegalovirus vector, or the like.

[0013] In another aspect of the present invention, there is provided an immunization method for immunization using a recombinant influenza vaccine constructed using the anti-influenza vaccine immunogen described above, or an immunogenic fragment thereof, or a combination thereof, the method comprising a step of sequentially administering different recombinant vector vaccines described above for each immunization, wherein each recombinant vector vaccine is administered at least once, and the administration procedure includes at least one intrarespiratory immunization and one systemic immunization.

[0014] In a specific embodiment of the present invention, during the above vaccination process, each needle was immunized with a recombinant vaccine derived from a different vector.

[0015] In a specific embodiment of the present invention, during the above vaccination process, the vaccines are administered using a "prime-boost-re-boost" immunization strategy, each recombinant vaccine is administered at least once, and the vaccination procedure includes at least one intratracheal vaccination and one systemic immunization.

[0016] In a specific embodiment of the present invention, in the above-mentioned vaccination process, the systemic administration of the vaccine includes intramuscular injection, subcutaneous administration, and intradermal administration, and the respiratory administration of the vaccine includes nebulization and nasal drops.

[0017] In a specific embodiment of the present invention, during the above-mentioned vaccination process, the vaccination procedure is to carry out vaccine construction and immunization as a strategy of priming with intramuscular injection of a recombinant DNA vaccine, boosting respiratory tract immunity with a recombinant adenovirus vector vaccine, and reboosting with intramuscular injection of a recombinant poxvirus vaccine.

[0018] In a particular embodiment of the present invention, in the above vaccination process, the vaccination procedure is such that the recombinant poxvirus vaccine is administered as the final vaccination.

[0019] In a particular embodiment of the present invention, the interval between every two inoculations in the above vaccination course is at least one week, preferably two weeks or more.

[0020] The above vaccines and immunization techniques of the present invention can be used to inoculate avian animals to prevent the transmission of avian influenza to humans, to inoculate humans to reduce the pathogenicity of avian influenza to infect humans, to inoculate humans to reduce the pathogenicity of human influenza to infect humans, and to inoculate humans to prevent the transmission of influenza to other humans after the infection.

[0021] In yet another aspect of the present invention, there is provided a therapeutic method comprising treating tumors including lung cancer, liver cancer, kidney cancer, pancreatic cancer, gastric cancer, breast cancer, esophageal cancer, bladder cancer, and osteosarcoma by using the anti-influenza vaccine as described above for intratumoral inoculation.

[0022] In yet another aspect of the present invention, there is provided an immunization method comprising using the above-described anti-influenza vaccine as an adjuvant for other vaccines, including anti-viral vaccines and anti-tumor vaccines, to enhance immune responses to other immunogens. These other vaccines include anti-ZIKV, anti-hepatitis B, anti-hepatitis C, anti-tuberculosis, anti-HIV, anti-malaria, and anti-dengue vaccines. These other vaccines also include vaccines against lung cancer, liver cancer, kidney cancer, pancreatic cancer, gastric cancer, breast cancer, esophageal cancer, bladder cancer, and osteosarcoma.

[0023] In yet another aspect of the present invention, there is provided the use of an anti-influenza vaccine immunogen or immunogenic fragment or a combination thereof according to the present invention in the preparation of an anti-influenza virus vaccine.

[0024] In yet another aspect of the invention, there is provided the use of an anti-influenza vaccine immunogen or immunogenic fragment, or a combination thereof, as an adjuvant for other vaccines.

[0025] The present invention provides broad-spectrum immunogens or immunogenic fragments thereof, or combinations thereof and immunization methods against influenza virus, wherein the immunogenic sequences comprise the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, or immunogenic fragments thereof, wherein the two sequences can be used individually or simultaneously, and either the entire sequence is used or the immunogenic fragments thereof are truncated, and the immunogenic fragments have the same biological activity as the sequences of the present invention, and the immunogenic sequences of the present invention are capable of immunizing influenza virus-specific CD8 antigens that bind with high affinity to human MHC class I molecules. +The recombinant vaccine is characterized by containing a T cell epitope. A recombinant vaccine is constructed using multiple different vaccine vectors, and different recombinant vector vaccines are administered sequentially for each immunization, with each recombinant vaccine administered at least once, and the vaccination procedure includes at least one intrarespiratory vaccination and one systemic vaccination. The combination of the recombinant vector vaccines used and the mode of administration can generate high levels of T cell immune responses in the respiratory tract and the systemic system, thereby enabling the vaccinee to obtain immunity against different influenza subtypes.

[0026] The immunogen of the present invention is a recombinant protein containing influenza virus internal conserved matrix proteins (M1, M2), nucleoprotein (NP), alkaline polymerase (PB1, PB2), and acid polymerase (PA) or immunogenic fragments thereof. The immunogen sequences of the present invention are two sequences designated as SEQ ID NO:1 and SEQ ID NO:2.

[0027] The above immunogens of the present invention can be used to construct recombinant vector vaccines with different vaccine vectors, including but not limited to recombinant protein vaccines, recombinant DNA vaccines, recombinant viral vector vaccines, recombinant bacterial vector vaccines, recombinant yeast vector vaccines, or recombinant virus-like particle vaccines.

[0028] The immunization method using the recombinant vector vaccine of the present invention utilizes a "prime-boost-booster" approach that combines systemic and local respiratory tract immunity, sequentially administering different vector vaccines for each immunization. Based on the characteristics of the different vector vaccines, the present invention preferably employs a strategy of priming with intramuscular injection of a recombinant DNA vaccine to establish a systemic immune response, followed by intramuscular injection of a type 68 adenovirus vaccine to enhance respiratory tract immunity, and finally intramuscular injection of a recombinant poxvirus vaccine to establish and boost the systemic immune response. The present invention preferably employs a third injection of a recombinant poxvirus vector for boosting, which effectively establishes a broad-spectrum influenza-specific immune response in both the local respiratory tract and the systemic system, thereby enhancing the broad-spectrum nature of the vaccine. The interval between two vaccinations is at least one week, but may be two weeks or longer.

[0029] The systemic immunization modes of the present invention include, but are not limited to, intramuscular, subcutaneous, and intradermal injections, and the local airway immunization modes include, but are not limited to, nebulization and nasal drops.

[0030] The immunogens of the present invention can be used in the research, design, and production of vaccines and drugs for preventing or treating influenza virus infection in birds and mammals. Furthermore, the immunization method of the present invention is characterized by its ability to induce high levels of antigen-specific CD8+ T cell responses locally in the respiratory tract, and is therefore expected to be useful for preventing pathogen infection in the respiratory tract, reducing the virulence of pathogens in the respiratory tract, and preventing and treating respiratory tract tumors.

[0031] The advantages of the present invention are that the immunogen contains a highly conserved influenza CD8 T cell epitope that binds with high affinity to human MHC class I molecules, can induce a broad-spectrum, high-level influenza-specific T cell immune response, can effectively counter the influenza virus's attempts to escape the host's existing immune response through antigen drift and antigen transformation, and has a certain cross-protective effect against different influenza virus subtypes.

[0032] An advantage of the present invention is that the immunization method efficiently activates a broad-spectrum T cell immune response in the local airway and systemic systems using different modes of administration by sequentially inoculating with various different vectors to enhance the protective effect of the vaccine against different influenza virus subtypes.

[0033] The advantages of the present invention are that the immunogens and immunization methods can be used for immunization with any respiratory pathogen vaccine, and the immunogens can be prepared into recombinant vaccines with viral vectors and used for intratumoral inoculation to treat tumors, including but not limited to lung cancer, liver cancer, kidney cancer, pancreatic cancer, gastric cancer, breast cancer, esophageal cancer, bladder cancer, osteosarcoma, etc. Further aspects of the present invention are described below: [Section 1] An anti-influenza vaccine immunogen, characterized in that the immunogen comprises the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, or immunogenic fragments thereof, or a combination thereof. [Section 2] Item 1. The anti-influenza vaccine immunogen according to Item 1, wherein the immunogen comprises an influenza virus internal conserved protein or an immunogenic fragment of the conserved protein. [Section 3] Item 2. The anti-influenza vaccine immunogen according to Item 1, wherein the influenza virus internal conserved proteins include influenza virus matrix proteins (M1, M2), nucleoprotein (NP), alkaline polymerase (PB1, PB2), and acid polymerase (PA). [Section 4] Item 4. The anti-influenza vaccine immunogen according to any one of Items 1 to 3, wherein the immunogen is derived from recombinant proteins of all influenza virus subtypes, including influenza A virus subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18, and influenza B virus, or recombinant proteins of shared sequences thereof, or a combination thereof. [Section 5] An anti-influenza vaccine that is a recombinant vector vaccine constructed by using the anti-influenza vaccine immunogen according to any one of Items 1 to 4 and expressing it in a plurality of different vectors. [Section 6] Item 6. The anti-influenza vaccine of Item 5, wherein the recombinant vector vaccine comprises a recombinant protein vaccine, a recombinant DNA vaccine, a recombinant virus vector vaccine, a recombinant bacterial vector vaccine, a recombinant yeast vector vaccine, or a recombinant virus-like particle vaccine. [Section 7] Item 6. The anti-influenza vaccine according to Item 5, wherein the viral vector comprises an adenovirus vector, a poxvirus vector, an adeno-associated virus vector, a herpes simplex virus vector, or a cytomegalovirus vector. [Section 8] Use of the anti-influenza vaccine immunogen according to any one of paragraphs 1 to 4 in the preparation of an anti-influenza virus vaccine. [Section 9] An immunization method for immunization using a recombinant influenza vaccine constructed using the anti-influenza vaccine immunogen described in any one of Items 1 to 4, the immunization method comprising a step of sequentially inoculating a different recombinant vector vaccine described in Item 5 for each immunization, wherein each recombinant vector vaccine is administered at least once, and the inoculation procedure includes at least one intrarespiratory immunization and one systemic immunization. [Section 10] Item 10. The method according to Item 9, wherein during the vaccination process, each needle is immunized with a recombinant vaccine derived from a different vector. [Section 11] Item 10. The method according to item 9, wherein the vaccines are administered using a prime-boost-booster immunization strategy, each recombinant vaccine is administered at least once, and the administration procedure includes at least one intratracheal vaccination and one systemic immunization. [Section 12] Item 10. The method according to Item 9, wherein the mode of systemic administration of the vaccine includes intramuscular injection, subcutaneous administration, and intradermal administration. [Section 13] Item 10. The method according to Item 9, wherein the mode of administration of the vaccine to the respiratory tract includes nebulization and nasal administration. [Section 14] The method described in item 9, wherein the vaccination procedure involves vaccine construction and immunization as a strategy of priming with intramuscular injection of a recombinant DNA vaccine, strengthening airway immunity with a recombinant adenovirus vector vaccine, and re-strengthening with intramuscular injection of a recombinant poxvirus vaccine. [Section 15] Item 10. The method according to Item 9, wherein the vaccination procedure is such that the recombinant poxvirus vaccine is administered as the final vaccination. [Section 16] Item 14. The method according to any one of items 9 to 13, wherein the interval between two inoculations is at least one week, preferably two weeks or more. [Section 17] Use of the anti-influenza vaccine according to paragraph 5 in the preparation of a recombinant vaccine for treating tumors. [Section 18] Item 18. The method of any one of items 9 to 17, wherein the vaccine and immunization techniques can be used to inoculate avian animals to prevent the transmission of avian influenza to humans. [Section 19] Item 18. The method according to any one of items 9 to 17, wherein the vaccine and immunization techniques can be used to inoculate humans to reduce the pathogenicity of avian influenza infection in humans. [Section 20] Item 18. The method of any one of items 9 to 17, wherein the vaccine and immunization techniques can be used to inoculate humans to reduce the pathogenicity of human influenza infections in humans. [Section 21] 18. The method of any one of clauses 9 to 17, wherein the vaccine and immunization techniques can be used to inoculate humans with influenza to prevent transmission to other humans after the human has been infected. [Section 22] Item 6. The anti-influenza vaccine according to Item 5, which is used for intratumoral inoculation to treat a tumor. [Section 23] Item 23. The anti-influenza vaccine according to Item 22, wherein the tumor includes lung cancer, liver cancer, kidney cancer, pancreatic cancer, stomach cancer, breast cancer, esophageal cancer, bladder cancer, and osteosarcoma. [Section 24] 5. The anti-influenza vaccine immunogen of any one of paragraphs 1 to 4, wherein the immunogenic sequence is used as an adjuvant in other vaccines to enhance the immune response to other immunogens. [Section 25] Item 25. The anti-influenza vaccine immunogen according to Item 24, wherein the other vaccines include anti-viral vaccines and anti-tumor vaccines. [Section 26] 26. The anti-influenza vaccine immunogen of paragraph 24 or 25, wherein the other vaccines include anti-ZIKV, anti-hepatitis B, anti-hepatitis C, anti-tuberculosis, anti-HIV, anti-malaria, and anti-dengue vaccines. [Section 27] 26. The anti-influenza vaccine immunogen of paragraph 24 or 25, wherein the other vaccines include anti-lung cancer, liver cancer, kidney cancer, pancreatic cancer, stomach cancer, breast cancer, esophageal cancer, bladder cancer, and osteosarcoma vaccines. [Brief explanation of the drawings]

[0034] [Figure 1]Figure 1 shows the detection of immunogen expression by Western blot experiment. (A) Western blot experiment confirms that the DNA vector pSV1.0, the adenovirus vector AdC68, and the poxvirus vector TTV all effectively express the immunogen SEQ ID NO:1 of the present invention. After incubation with influenza matrix protein 1 antibody, the empty vectors pSV1.0, AdC68, and TTV, which do not contain the immunogen SEQ ID NO:1 of the present invention, all showed no specific bands. However, the vectors pSV1.0-SEQ ID NO:1, AdC68-SEQ ID NO:1, and TTV-SEQ ID NO:1 / 2, which contained the immunogen SEQ ID NO:1 of the present invention, showed significant bands with a protein size of approximately 130 kD, demonstrating that the DNA vector pSV1.0, the adenovirus vector AdC68, and the poxvirus vector TTV all effectively express the immunogen SEQ ID NO:1 of the present invention. Furthermore, after incubation with β-actin antibody, a significant band with a protein size of approximately 42 kD was observed, further demonstrating that each step of the experiment was accurate and the experimental results reliable. (B) Western blot experiments confirm that the DNA vector pSV1.0, the adenovirus vector AdC68, and the poxvirus vector TTV all effectively express the immunogen SEQ ID NO:2 of the present invention. After incubation with influenza matrix protein 2 antibody, the empty vectors pSV1.0, AdC68, and TTV, which do not contain the immunogen SEQ ID NO:2 of the present invention, did not show any specific bands. However, the vectors pSV1.0-SEQ ID NO:2, AdC68-SEQ ID NO:2, and TTV-SEQ ID NO:1 / 2, which contained the immunogen SEQ ID NO:2 of the present invention, showed significant bands with a protein size of approximately 130 kD, demonstrating that the DNA vector pSV1.0, the adenovirus vector AdC68, and the poxvirus vector TTV all effectively express the immunogen SEQ ID NO:2 of the present invention. Furthermore, after incubation with β-actin antibody, a significant band with a protein size of approximately 42 kD was observed, further demonstrating that each step of the experiment was accurate and the experimental results reliable. [Figure 2]Figure 2 shows the detection of influenza-specific T cell immune responses based on immunogens. (A) shows the detection of influenza-specific T cell immune response levels in mouse spleen cells using an enzyme-linked immunospot experiment. The results showed that mice in the control group did not exhibit spot-forming cells and did not demonstrate influenza-specific T cell immune responses. Mice in the adenovirus group had more spot-forming cells against two epitopes, NP-2 and PB2-1, indicating a high T cell immune response. Mice in the poxvirus group had spot-forming cells against epitopes such as NP-2, NP-3, PB1-1, PB1-3, and PA-3, indicating a high T cell immune response. (B) shows the detection of influenza-specific immune response levels in mouse spleen cells using staining for intracellular factors interferon-γ and tumor necrosis factor-α. The results showed that there were no T cells expressing interferon-γ or tumor necrosis factor-α in the control group, but that these T cells were present in both the adenovirus and poxvirus groups, indicating the presence of influenza-specific T cell immune responses. (C) shows the level of influenza-specific immune responses in mouse spleen cells detected by staining for the intracellular factor CD107a. The results showed that there were no T cells expressing CD107a in the control group, but that these T cells were present in the adenovirus group, indicating the presence of influenza-specific T cell immune responses. [Figure 3]Figure 3 shows the evaluation of the protective effect against H1N1 and H7N9 influenza virus challenge based on immunogen. (A) and (B) show the mouse weight curves. After challenge with H1N1 and H7N9 influenza viruses, the weight of the control group mice continued to decrease, while the weight of the adenovirus and poxvirus groups mice initially decreased and then increased. (C) and (D) show the mouse survival curves. After challenge with H1N1 influenza virus, all mice in the control group died, while all mice in the adenovirus and poxvirus groups survived until day 14. (E) and (F) show the detection of viral load in the lungs of mice 5 days after challenge. After challenge with H1N1 and H7N9 influenza viruses, the viral load in the lungs of mice in the adenovirus and poxvirus groups was lower than that of the control group. [Figure 4]Figure 4 shows the detection of influenza-specific T cell immune responses induced by different immunization methods. (A) shows the detection of influenza-specific T cell immune response levels in mouse spleen cells by enzyme-linked immunospot assay. The results showed that mice in control group 1 showed no spot-forming cells, indicating no influenza-specific T cell immune response. Control groups 2 and 3 and experimental groups 1 and 2 all showed spot-forming cells for each monopeptide, indicating high levels of T cell immune responses. (B) shows the detection of influenza-specific immune response levels in mouse lung lavage fluid by enzyme-linked immunospot assay. Under stimulation with the two peptides, NP-2 and PB2-1, no spot-forming cells were observed in control groups 1, 2, and 3, indicating failure to establish influenza-specific immune responses in the lungs. Experimental groups 1 and 2 showed many spot-forming cells, indicating high levels of influenza-specific T cell immune responses. (C) shows the detection of influenza-specific immune response levels in mouse spleen cells by staining for intracellular factors interferon-γ and tumor necrosis factor-α. The results showed that control group 1 did not have T cells expressing interferon-γ or tumor necrosis factor-α, while control groups 2 and 3 and experimental groups 1 and 2 all had T cells expressing interferon-γ and tumor necrosis factor-α, indicating the presence of an influenza-specific T cell immune response. (D) shows the level of influenza-specific immune response in mouse spleen cells detected by staining for the intracellular factor CD107a. The results showed that T cells expressing CD107a were found in control group 3 and experimental group 2, indicating the presence of an influenza-specific T cell immune response. [Figure 5]Figure 5 shows the protective effects of mice against H1N1 and H7N9 influenza viruses after immunization with different methods. (A) and (B) show the weight curves of mice. After H1N1 and H7N9 influenza virus challenge, the weights of mice in experimental groups 1 and 2 initially decreased and then increased, demonstrating superiority over control groups 1, 2, and 3. (C) and (D) show the survival curves of mice. After H1N1 and H7N9 influenza virus challenge, all mice in experimental groups 1 and 2 survived until day 14, while all mice in control groups 1, 2, and 3 died by day 14. (E) and (F) show the viral load in the lungs of mice 5 days after challenge. After H1N1 and H7N9 influenza challenge, the viral loads of mice in experimental groups 1 and 2 were slightly lower than those of control groups 1, 2, and 3. [Figure 6] Figure 6 shows the protective effect of mice in the nasal vaccination-based immune boosting experimental group after influenza virus challenge. (A) and (B) show the weight curves of mice. After challenge with H1N1 and H7N9 influenza viruses, the weight of mice in experimental group 1 + FTY720 and experimental group 2 + FTY720 first decreased and then increased, demonstrating superiority over the control group + FTY720 group. (C) and (D) show the survival curves of mice. After challenge with H1N1 and H7N9 influenza viruses, some mice in experimental group 1 + FTY720 and experimental group 2 + FTY720 survived up to 14 days, demonstrating superiority over the control group + FTY720 group. (E) and (F) show the viral load in the lungs of mice 5 days after challenge. After challenge with H1N1 and H7N9 influenza viruses, the viral load of mice in experimental group 1 + FTY720 and experimental group 2 + FTY720 was lower than that of mice in the control group + FTY720 group. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention will now be described in more detail with reference to the following examples.

[0036] MODE FOR CARRYING OUT THE INVENTION Other aspects of the invention are described in detail below. These and other features and advantages of the invention will become apparent from a review of the following detailed description of the disclosed embodiments and the appended claims.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. [Example]

[0038] Example 1: Design and preparation of anti-influenza vaccine immunogens The GenBank database is a gene sequence database established by the National Center for Biotechnology Information (NCBI) in the United States, and by searching this database, the gene sequences of approximately 40,000 strains of influenza viruses were obtained.

[0039] The amino acid sequences of the M1, M2, NP, PB1, PB2, and PA proteins contained within the approximately 40,000 strains of influenza viruses mentioned above were calculated and analyzed. The most frequently occurring amino acid at each site in the amino acid sequence was determined to be the shared amino acid at that site, and the shared amino acid sequence of this protein was composed of the shared amino acids at each site, thereby obtaining the shared amino acid sequences of the M1, M2, NP, PB1, PB2, and PA proteins.

[0040] The amino acid sequences of PB1, PB2, and PA obtained above were analyzed using online CD8 T cell epitope prediction software (http: / / tools.immuneepitope.org / main / tcell / and http: / / www.syfpeithi.de / ). The common CD8 T cell epitopes predicted by the two software programs were retained and combined to form the amino acid epitope sequences of PB1, PB2, and PA.

[0041] A vaccine sequence was designed based on the amino acid sequence obtained above. The PA and PB1 amino acid epitope sequence obtained by combining the epitopes was combined with the shared amino acid sequence of the M1 protein to obtain a vaccine amino acid sequence designated SEQ ID NO: 1. The PB2 amino acid epitope sequence obtained by combining the epitopes was combined with the shared amino acid sequence of the NP and M2 proteins to obtain another vaccine amino acid sequence designated SEQ ID NO: 2.

[0042] For the above SEQ ID NO: 1 and SEQ ID NO: 2, the amino acid sequences were translated into nucleic acid sequences and the nucleic acid sequences were subjected to eukaryotic codon optimization using the online software http: / / www.jcat.de / to obtain the nucleic acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, which were synthesized by Suzhou GENEWIZ Biotechnology Co., Ltd. The synthesized sequences were sequenced by Suzhou GENEWIZ Biotechnology Co., Ltd. and confirmed to be SEQ ID NO: 1 and SEQ ID NO: 2 described herein.

[0043] Example 2: Construction of a vaccine based on an anti-influenza vaccine immunogen The immunogens of the present invention are used to construct recombinant DNA vector vaccines, recombinant adenovirus vector vaccines, and recombinant poxvirus vector vaccines. The immunogens SEQ ID NO:1 and SEQ ID NO:2 of the present invention were inserted into the pSV1.0 vector (preserved by Shanghai Public Health Clinical Center) to construct recombinant DNA vector vaccines named pSV1.0-SEQ ID NO:1 and pSV1.0-SEQ ID NO:2, respectively.

[0044] The immunogen was inserted into the AdC68 adenovirus vector (purchased from the Shanghai Pasteur Institute, Chinese Academy of Sciences) and transfected into 293a cells (purchased from the Cell Resources Center, Shanghai Institutes for Life Sciences, Chinese Academy of Sciences) to construct recombinant adenovirus vector vaccines designated AdC68-sequence number 1 and AdC68-sequence number 2, respectively.

[0045] The cleaved peptide p2a was used to combine immunogens SEQ ID NO:1 and SEQ ID NO:2, which were then inserted into pSC65 vector (preserved by the Shanghai Public Health Clinical Center) and transfected into TK143 cells (purchased from the Cell Resources Center of the Shanghai Institutes for Life Sciences, Chinese Academy of Sciences) to construct a recombinant poxvirus vector vaccine designated TTV-SEQ ID NO:1 / 2.

[0046] The specific steps for detecting the expression of anti-influenza vaccine immunogens by Western blot experiments are as follows. (1) Preparation of experimental samples pSV1.0-SEQ ID NO:1 and pSV1.0-SEQ ID NO:2 were transfected into 293T cells (purchased from the Cell Resources Center of the Shanghai Institutes of Life Sciences, Chinese Academy of Sciences), respectively. After 48 hours, the 293T cells were harvested and resuspended in 75 μL of cell lysis solution. 25 μL of protein loading buffer was added, and the cells were incubated in a water bath at 100°C for 10 minutes to prepare the samples. 293A cells were infected with AdC68-sequence number 1 and AdC-sequence number 2, respectively, and 24 hours later, the 293A cells were harvested, resuspended in 75 μL of cell lysis solution, 25 μL of protein loading buffer was added, and the cells were incubated in a water bath at 100°C for 10 minutes to prepare samples.

[0047] TK143 cells were infected with TTV-sequence number 1 / 2, and 48 hours later, the TK143 cells were harvested. The cells were resuspended in 75 μL of cell lysis solution, 25 μL of protein loading buffer was added, and the mixture was placed in a water bath at 100°C for 10 minutes to prepare a sample.

[0048] (2) For Western blot experiments, an 8% polyacrylamide separating gel was prepared and allowed to stand at room temperature for 30 minutes. After this, a 10% polyacrylamide concentrating gel was added, and the gel was immediately gently inserted with a comb. After allowing the gel to stand for 30 minutes, the gel was placed in an electrophoresis chamber, electrophoresis buffer was poured in, the comb was slowly removed, and the samples prepared above were added sequentially. The electrophoresis apparatus was connected and electrophoresis was performed at 70 volts for 0.5 hours. The voltage was then adjusted to 90 volts and electrophoresis continued for 1.5 hours before termination. The polyvinylidene fluoride membrane was activated in methanol for 30 seconds. The sponge, filter paper, and polyvinylidene fluoride membrane were then immersed in membrane transfer solution and attached in order. The apparatus and ice bag were then placed in the membrane transfer chamber, filled with pre-cooled membrane transfer buffer, and membrane transfer was performed at a constant current of 200 mA for 2.5 hours. After membrane transfer, the polyvinylidene fluoride membrane was removed and blocked with 5% nonfat dry milk for 1 hour. Influenza matrix protein 1 antibody (purchased from Abcam Shanghai Trading Co., Ltd.) was added at a dilution of 1:1000, and influenza matrix protein 2 antibody (purchased from Santa Cruz Biotechnology Shanghai Co., Ltd.) was added at a dilution of 1:250. The mixture was incubated on a shaker at room temperature for 2 hours, followed by three 5-minute washes with phosphate buffer containing Tween-20. Horseradish peroxidase-conjugated goat anti-mouse IgG antibody was added at a dilution of 1:5000. The mixture was incubated on a shaker at room temperature for 1 hour, followed by five 5-minute washes with phosphate buffer containing Tween-20. A color-developing solution was prepared and applied to a polyvinylidene fluoride membrane for luminescence detection.

[0049] The results of immunogen expression detection by Western blot experiments are shown in Figure 1. The DNA vector pSV1.0, adenovirus vector AdC68, and poxvirus vector TTV all effectively expressed the immunogen SEQ ID NO:1 of the present invention. After incubation with influenza matrix protein 1 antibody, the empty vectors pSV1.0, AdC68, and TTV, which do not contain the immunogen SEQ ID NO:1 of the present invention, did not show any specific bands. However, the vectors pSV1.0-SEQ ID NO:1, AdC68-SEQ ID NO:1, and TTV-SEQ ID NO:1 / 2, which contained the immunogen SEQ ID NO:1 of the present invention, showed significant bands corresponding to a protein size of approximately 130 kD. This demonstrated that the DNA vector pSV1.0, adenovirus vector AdC68, and poxvirus vector TTV all effectively expressed the immunogen SEQ ID NO:1 of the present invention. After incubation with influenza matrix protein 2 antibody, the empty vectors pSV1.0, AdC68, and TTV, which do not contain the immunogen SEQ ID NO:2 of the present invention, showed no specific bands, while the vectors pSV1.0-SEQ ID NO:2, AdC68-SEQ ID NO:2, and TTV-SEQ ID NO:1 / 2, which contained the immunogen SEQ ID NO:2 of the present invention, showed significant bands with a protein size of approximately 130 kD, demonstrating that the DNA vector pSV1.0, the adenovirus vector AdC68, and the poxvirus vector TTV all effectively expressed the immunogen SEQ ID NO:2 of the present invention. Furthermore, after incubation with β-actin antibody, a significant band with a protein size of approximately 42 kD was observed, further demonstrating that each step of the experiment was accurate and the experimental results were reliable.

[0050] Example 3: Detection of vaccine immunogenicity based on anti-influenza vaccine immunogens The immunogens of the present invention were used to construct DNA vaccines, adenovirus vector vaccines, and poxvirus vector vaccines, as described in Example 2. The recombinant influenza vaccines were used to immunize mice, and the immunogenicity of the recombinant influenza vaccines was evaluated 4 weeks after the end of immunization.

[0051] Six-week-old C57BL / 6 mice were randomly divided into three groups: control group, adenovirus group, and poxvirus group. The specific immunization procedure is shown in Table 1. The immunization method was intramuscular injection, the inoculation dose of pSV1.0 was 100 μg, the inoculation dose of AdC68 was 10 viral particles, the inoculation doses of pSV1.0-SEQ ID NO:1 and pSV1.0-SEQ ID NO:2 were 50 μg, and the inoculation doses of AdC68-SEQ ID NO:1 and AdC68-SEQ ID NO:2 were 5×10 10 The inoculation dose of TTV-SEQ ID NO: 1 / 2 was 10 7 plaque-forming units, and the vaccine was administered every two weeks.

[0052] [Table 1]

[0053] The immunogenicity of the recombinant influenza vaccine was assayed using enzyme-linked immunospot assay (ELISpot) and intracellular staining of cytokines (ICS) in mouse spleen cells.

[0054] Based on the epitope predictions for SEQ ID NO:1 and SEQ ID NO:2, as well as reported commonly used influenza T cell epitopes, 16 epitope monopeptides were selected to stimulate mouse T cell immune responses and named M1-1, M1-2, M1-3, M2, NP-1, NP-2, NP-3, PB1-1, PB1-2, PB1-3, PB2-1, PB2-2, PB2-3, PA-1, PA-2, and PA-3, respectively.

[0055] (1) The experimental steps of the enzyme-linked immunospot assay are as follows: One day before the assay, mouse interferon-γ was diluted to a final concentration of 5 μg / mL, and 100 μL was added per well of the assay plate. The plate was coated overnight at 4°C. The next day, the coating solution was discarded, and each well was washed once with 200 μL of complete medium. Then, 200 μL of complete medium was added and blocked for 2 hours at room temperature. After blocking, the concentration of mouse spleen cells was adjusted to 4 x 106 cells per mL, and 50 μL of spleen cells was added to each well, followed by 50 μL of 10 μg / mL monopeptide. The wells were then cultured for approximately 20 hours in an incubator. After incubation, each well was rinsed twice with 200 μL of distilled water, then washed three times with 200 μL of Tween-20 phosphate buffer. Anti-mouse interferon-γ biotin was diluted to a final concentration of 2 μg / μL, and 100 μL was added to each well. The plate was then incubated for 2 hours at room temperature. After incubation, 200 μL of Tween-20-containing phosphate buffer solution was added to each well and washed three times. A 1:100 diluted horseradish peroxidase fluorescent substrate was added to each well (100 μL per well) and incubated at room temperature for 1 hour. After incubation, 200 μL of Tween-20-containing phosphate buffer solution was added to each well and washed four times. 200 μL of phosphate buffer solution was added and washed twice. A color-developing solution was prepared and added to each well (100 μL per well). The plate was incubated in the dark at room temperature for approximately 15 minutes. When a distinct red spot appeared, the plate was gently rinsed with tap water for 5 minutes to terminate the color development reaction. After drying at room temperature, the plate was read in an enzyme-linked immunospot plate reader and the number of positive spots was counted.

[0056] (2) The experimental steps for intracellular factor staining are as follows. 2 x 10 mouse spleen cells per mL 7After dilution, 150 μL of cells and 150 μL of peptide library were added to each well, followed by 1 μL of CD107a antibody. After 1 hour of incubation, 0.3 μL of protein transport inhibitor was added to each well and the wells were placed in an incubator for 6 hours. After incubation, cells were collected in flow tubes and centrifuged at 800 rpm for 3 minutes. 800 μL of staining buffer was added to each tube for washing, followed by centrifugation at 800 rpm for 3 minutes and discarding the supernatant. CD3, CD8, and cell activity / cytotoxicity staining antibody mixtures were prepared, and 40 μL of the antibody mixture was added to each tube. The mixture was stained for 20 minutes at room temperature in the dark. After staining, 800 μL of staining buffer was added to each tube for two washes, followed by centrifugation at 800 rpm for 3 minutes, discarding the wash solution, and 150 μL of fixative solution was added to each tube for 20 minutes at room temperature in the dark. Each tube was washed with 800 μL of staining buffer, centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded. Interferon-γ and tumor necrosis factor-α staining antibody mixture was prepared, and 40 μL of the antibody mixture was added to each tube. Staining was performed for 20 minutes at room temperature in the dark. Each tube was washed with 800 μL of staining buffer, centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded. The cells were then resuspended in 250 μL of staining buffer and detected using a flow cytometer. The results were analyzed statistically.

[0057] The results of detecting vaccine immunogenicity are shown in Figure 2. Enzyme-linked immunospot experiments showed that mice in the control group had no spot-forming cells and no influenza-specific T cell immune responses. Mice in the adenovirus group had more spot-forming cells and higher levels of T cell immune responses to both NP-2 and PB2-1 epitopes. Mice in the poxvirus group had more spot-forming cells and higher levels of T cell immune responses to epitopes such as NP-2, NP-3, PB1-1, PB1-3, and PA-3.

[0058] The level of influenza-specific immune responses in mouse spleen cells was detected by staining for the intracellular factors interferon-γ, tumor necrosis factor-α, and CD107a. The results showed that T cells expressing interferon-γ, tumor necrosis factor-α, and CD107a were not observed in the control group, but were present in both the adenovirus and poxvirus groups, indicating influenza-specific T cell immune responses.

[0059] This experiment demonstrated that the anti-influenza vaccine immunogens SEQ ID NO: 1 and SEQ ID NO: 2, when expressed in different vaccine vectors, can induce significant T cell immune responses.

[0060] Example 4: Evaluation of exposure protection effects based on anti-influenza immunogens The immunogens of the present invention were used to construct DNA vaccines, adenovirus vector vaccines, and poxvirus vector vaccines, as described in Example 2. As described in Example 3, mice were immunized with the recombinant influenza vaccines, and the protective effect of the recombinant influenza vaccines against challenge was evaluated 4 weeks after the end of immunization.

[0061] The protective effects of immunogens were evaluated using H1N1 and H7N9 influenza challenge models. H1N1 challenge experiments were performed in a biosafety level 2 laboratory, and H7N9 challenge experiments were performed in a biosafety level 3 laboratory.

[0062] Each mouse was anesthetized by intraperitoneal injection of 50 μL of 10% chloral hydrate, and 50 μL of influenza virus was instilled into the nose of each mouse for exposure. The exposure dose of H1N1 influenza virus was 500 TCID of virus per mouse. 50 The H7N9 influenza virus challenge dose was 100 viral median tissue infectious doses per mouse. Five days after challenge, five mice from each group were sacrificed, and lungs were harvested to measure viral load.

[0063] The results of the exposure protection experiment are shown in Figure 3.

[0064] Mice in the control group continued to lose weight after exposure to a lethal dose of H1N1 influenza virus, and all died by day 12. Mice in the adenovirus group began to regain weight by day 9, and all survived to 14 days. Mice in the poxvirus group lost weight significantly more slowly, began to regain weight by day 9, and all survived to 14 days.

[0065] After exposure to a non-lethal dose of H7N9 influenza virus, mice in the control group lost approximately 20% of their body weight, which regained on day 9. Mice in the adenovirus and poxvirus groups lost less than 10% of their body weight, which regained rapidly on day 7.

[0066] This experiment demonstrated that the expression of anti-influenza vaccine immunogens SEQ ID NO: 1 and SEQ ID NO: 2 in different vaccine vectors resulted in cross-protection against both H1N1 and H7N9 influenza viruses, i.e., the immunogens of the present invention have broad-spectrum protective effects against different subtypes of influenza viruses.

[0067] Example 5: Detection of influenza vaccine immunogenicity based on different immunization methods The immunogens of the present invention were used to construct DNA vaccines, adenovirus vector vaccines, and poxvirus vector vaccines, as described in Example 2. Mice were immunized using the immunization method of the present invention, and immunogenicity was detected according to the method described in Example 3 four weeks after the end of immunization.

[0068] Six-week-old C57BL / 6 mice were randomly divided into five groups, named control group 1, control group 2, control group 3, experimental group 1, and experimental group 2, respectively. Experimental group 1 and experimental group 2 were immunized with the immunization method of the present invention. The specific immunization procedure is shown in Table 2. The pSV1.0 dose was 100 μg, the AdC68 dose was 10 viral particles, the pSV1.0-SEQ ID NO:1 and pSV1.0-SEQ ID NO:2 doses were 50 μg, the AdC68-SEQ ID NO:1 and AdC68-SEQ ID NO:2 doses were 5×10 viral particles, and the TTV and TTV-SEQ ID NO:1 / 2 doses were 10 plaque-forming units. The vaccines were administered every two weeks.

[0069] [Table 2]

[0070] The results of detecting vaccine immunogenicity are shown in Figure 4. Enzyme-linked immunospot experiments showed that no spot-forming cells were observed in the spleen cells of mice in control group 1, indicating that no influenza-specific T cell immune response was observed. Spot-forming cells against each monopeptide were observed in control groups 2 and 3 and experimental groups 1 and 2, indicating a high level of T cell immune response. In the lung lavage fluid of mice, no spot-forming cells were observed in control groups 1, 2, and 3, indicating that an influenza-specific immune response could not be established in the lungs, while many spot-forming cells were observed in experimental groups 1 and 2. That is, experimental groups 1 and 2, which used the vaccination method of the present invention, showed a very high level of influenza-specific T cell immune response.

[0071] The level of influenza-specific immune response in mouse spleen cells was detected by staining for the intracellular factors interferon-γ, tumor necrosis factor-α, and CD107a. The results showed that no T cells expressing interferon-γ or tumor necrosis factor-α were observed in control group 1, while T cells expressing interferon-γ and tumor necrosis factor-α were observed in both control groups 2 and 3 and experimental groups 1 and 2, indicating the presence of an influenza-specific T cell immune response.

[0072] This experiment demonstrated that sequential immunization with different recombinant vector vaccines combined respiratory tract immunity with systemic immunity, and experimental groups 1 and 2 using the vaccine immunization method of the present invention were able to effectively establish high levels of influenza-specific immune responses both systemically and locally in the lungs, which was superior to the control group.

[0073] Example 6: Evaluation of the protective effect of influenza vaccines based on different immunization methods According to the method described in Example 5, mice were immunized using the immunization method of the present invention, and the protective effects of the immunogens were evaluated using H1N1 and H7N9 influenza challenge models 4 weeks after the mice were immunized with the final vaccine. The H1N1 influenza challenge experiment was conducted in a biosafety level 2 laboratory, and the H7N9 influenza challenge experiment was conducted in a biosafety level 3 laboratory.

[0074] Mice were anesthetized with 50 μL of 10% chloral hydrate intraperitoneally and challenged with 50 μL of influenza virus via nasal instillation. The challenge dose for H1N1 influenza virus was 500 MDIDs per mouse, and the challenge dose for H7N9 influenza virus was 500 MDIDs per mouse. Five days after challenge, five mice from each group were sacrificed, and lungs were harvested to measure viral load.

[0075] The results of the exposure protection experiment are shown in Figure 5.

[0076] After exposure to H1N1 influenza virus, all mice in control group 1 died on day 13, while control groups 2 and 3 showed partial protection, with 80% and 60%, respectively, surviving until day 14. Mice in experimental groups 1 and 2 exposed to the vaccine immunization method of the present invention regained weight on day 10 and all survived until day 14, with the viral load in experimental group 2 decreasing significantly, demonstrating excellent protection.

[0077] After exposure to H7N9 influenza virus, mice in experimental groups 1 and 2 treated with the vaccine immunization method of the present invention rapidly regained weight on day 10, and all mice survived until day 14, demonstrating excellent protective effects. Mice in other groups did not show significant protective effects.

[0078] This experiment demonstrated that sequential immunization with different recombinant vector vaccines combined respiratory tract immunity with systemic immunity, and experimental groups 1 and 2, which used the vaccine immunization method of the present invention, had excellent cross-protective effects against H1N1 and H7N9 influenza viruses, and their protective effects were superior to those of control groups 2 and 3, which were administered via intramuscular injection only. It also demonstrated that the protective effect of the vaccine was best when the recombinant poxvirus vector vaccine was administered as the final injection vaccine.

[0079] Example 7: Evaluation of the protective effect of nasal immunization in experimental mice after exposure to influenza virus According to the method described in Example 5, mice were immunized using the immunization method of the present invention, and four weeks after the mice were immunized with the final vaccine, the protective effects of the immunogen were evaluated using H1N1 and H7N9 influenza challenge models, with the specific influenza challenge method being as described in Example 6. Throughout the entire virus challenge process, mice were continuously given drinking water containing 2 μg / mL of FTY720, an immunosuppressant that effectively reduces the number of peripheral circulating lymphocytes and preserves in situ memory T cells colonizing the lungs established by nasal inoculation. This water was continuously used during the entire challenge with lethal doses of H1N1 and H7N9 influenza viruses to evaluate whether the nasal inoculation method had an enhancing effect.

[0080] The experimental results are shown in Figure 6.

[0081] After challenge with H1N1 and H7N9 influenza viruses, both experimental group 1 + FTY720 and experimental group 2 + FTY720 showed partial protection, with mice regaining weight on day 11, surviving until day 14, and down-regulating viral load, demonstrating superior protective effects compared with control group 1 + FTY720.

[0082] This experiment demonstrated that the respiratory administration mode of the vaccine can effectively enhance the protective effect of vaccination against H1N1 and H7N9 influenza. Those skilled in the art will appreciate that the present invention is not limited to the above-described embodiments, and that various modifications, additions, and substitutions are possible without departing from the scope and spirit of the present invention as disclosed in the appended claims.

Claims

1. An anti-influenza vaccine immunogen comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. An anti-influenza vaccine which is a recombinant vector vaccine comprising a plurality of recombinant vectors each expressing the anti-influenza vaccine immunogen according to claim 1.

3. 3. The anti-influenza vaccine of claim 2, wherein the recombinant vector vaccine comprises a recombinant protein vaccine, a recombinant DNA vaccine, a recombinant virus vector vaccine, a recombinant bacterial vector vaccine, a recombinant yeast vector vaccine, or a recombinant virus-like particle vaccine.

4. The anti-influenza vaccine described in claim 3, wherein the viral vector includes an adenovirus vector, a poxvirus vector, an adeno-associated virus vector, a herpes simplex virus vector, or a cytomegalovirus vector.

5. 2. Use of the anti-influenza vaccine immunogen according to claim 1 in the preparation of an anti-influenza virus vaccine.

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