Universal influenza vaccine construction method based on influenza a virus na protein and m2e epitope chimeric protein and application

Abstract

The application discloses a universal influenza vaccine construction method based on an A-type influenza virus NA protein and M2e epitope chimeric protein and application, and relates to the field of gene recombination technology. The chimeric protein is composed of an amino acid sequence of an A-type influenza virus NA protein and an amino acid sequence of M2e, wherein the amino acid sequence of M2e is inserted into the amino acid sequence corresponding to the stem domain of the NA protein. The chimeric protein can fully display the M2e epitope while keeping the natural conformation and function of the NA protein. The application also provides a nucleic acid molecule encoding the chimeric protein, a recombinant influenza virus carrying the nucleic acid molecule, and a vaccine composition based on the above-mentioned substances. Experiments prove that the chimeric protein and the recombinant virus can be stably expressed in various NA subtypes, the prepared vaccine can simultaneously stimulate specific immune responses to homologous HA and broad-spectrum cross protection to heterologous A-type influenza virus subtypes, and a new technical approach is provided for the development of a universal influenza vaccine.

Description

Technical Field

[0001] This invention relates to the field of gene recombination technology, and in particular to a method for constructing and applying a universal influenza vaccine based on a chimeric protein of influenza A virus NA protein and M2e epitope. Background Technology

[0002] Influenza (flu) is an acute respiratory infectious disease caused by the influenza virus, posing a persistent threat to global public health. Influenza vaccination is the most cost-effective measure to prevent influenza and its serious complications, reducing the risk of contracting influenza by approximately 40% to 60%, and is a core strategy for managing seasonal influenza epidemics.

[0003] Influenza viruses belong to the Orthomyxoviridae family and are single-stranded, negative-sense, segmented RNA viruses. Based on the antigenicity of their nucleoprotein (NP) and matrix protein (M1), they can be classified into four types: A (influenza A), B (influenza B), C (influenza C), and D (influenza D). Type A and B influenza viruses are the primary causes of most symptomatic seasonal epidemics. Type A influenza viruses possess two main glycoproteins on their surface: hemagglutinin (HA) and neuraminidase (NA). The NA protein has neuraminidase activity and plays a crucial role in viral release and transmission. Simultaneously, the NA protein itself has good immunogenicity; its active site region is relatively conserved, enabling it to induce antibodies against NA, synergistically inhibiting viral release. It is an important antigenic component in the development of universal influenza vaccines.

[0004] However, influenza viruses mutate rapidly, undergoing antigenic drift almost every year. This results in limited efficacy of traditional vaccines designed to target the circulating strains of the year, and the antibodies they induce offer weak cross-protection against different strains. Therefore, vaccine strains need to be updated annually based on epidemic forecasts. If the forecasts do not match the circulating strains, the vaccine's protective efficacy drops significantly. Recent studies indicate that the protective efficacy of licensed seasonal influenza vaccines varies from approximately 6% to 40% across different seasons. This situation underscores the urgent importance of developing a universal influenza vaccine that can induce broad cross-protection and is unaffected by annual strain variations.

[0005] Among numerous universal vaccine candidate targets, the extracellular domain (M2e) of influenza A virus matrix protein 2 has attracted significant attention. The M2e sequence is highly conserved across all influenza A viruses, and antibodies against M2e can provide cross-protection through mechanisms such as antibody-dependent phagocytosis (ADCP), antibody-dependent cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). However, the standalone M2e peptide has a small molecular weight and weak immunogenicity, typically requiring combination with potent adjuvants, virus-like particles (VLPs), or vectors to enhance the immune response. To improve the immunogenicity of M2e, tandemly combining multiple M2e copies (e.g., 4M2e, i.e., four M2e repeat sequences tandemly) is a commonly used and effective strategy in this field, significantly enhancing its ability to induce specific antibody production.

[0006] In existing technologies, attempts have been made to fuse M2e multicopy tandem variants with NA proteins at their ends, with the aim of simultaneously presenting two protective antigens. However, this linear fusion method has significant drawbacks: the native conformation of the NA protein in the fusion protein may be disrupted by the addition of exogenous sequences at the ends, leading to a decrease or even loss of its neuraminidase activity, thereby weakening the NA-specific protective immune response; at the same time, the M2e tandem variants located at the ends often fail to induce sufficiently high titers of anti-M2e antibodies due to steric hindrance or differences in immunodominance. Summary of the Invention

[0007] The technical problem to be solved by the present invention is how to fuse the M2e multicopy tandem variant with the NA protein to induce a long-lasting and strong anti-M2e immune response while ensuring the immunogenicity of NA, thus successfully constructing a broad-spectrum influenza vaccine.

[0008] To address the above problems, the present invention proposes the following technical solution: In a first aspect, the present invention provides a chimeric protein whose amino acid sequence comprises the amino acid sequence of influenza A virus NA protein and the amino acid sequence of influenza A virus M2e, wherein the amino acid sequence of M2e is inserted inside the amino acid sequence corresponding to the stem domain of the extracellular region of the NA protein.

[0009] Furthermore, the M2e comprises 2 to 10 (e.g., 4) tandem M2e units, each M2e unit being linked by a linker peptide, and the tandem M2e units being linked to the amino acid sequence of the NA protein also by a linker peptide.

[0010] Furthermore, the linker peptide comprises at least one GGGGS unit, more preferably comprising 2 to 10 directly tandem GGGGS units.

[0011] Furthermore, the NA protein is the NA protein of the N1 influenza virus, and its amino acid sequence is shown in SEQ ID NO: 8. The M2e is inserted at any position within the amino acid sequence interval from the 36th to the 75th position.

[0012] Furthermore, the M2e is inserted at one of the following positions in the amino acid sequence of the N1 influenza virus NA protein: between glycine at position 41 and serine at position 42, between serine at position 42 and glutamine at position 43, between asparagine at position 44 and histidine at position 45, or between serine at position 35 and histidine at position 36.

[0013] Furthermore, the NA protein is the NA protein of the N6 influenza virus, and its amino acid sequence is shown in SEQ ID NO: 10. The M2e is inserted at any position within the amino acid sequence interval from the 34th to the 79th position.

[0014] Furthermore, the M2e is inserted at one of the following positions in the amino acid sequence of the N6 influenza virus NA protein: between glutamic acid at position 49 and threonine at position 50, between asparagine at position 59 and isoleucine at position 60, or between glutamic acid at position 69 and glycine at position 70.

[0015] Furthermore, the NA protein is the NA protein of the N2 influenza virus, and its amino acid sequence is shown in SEQ ID NO: 12. The M2e is inserted at any position within the amino acid sequence interval from the 36th to the 88th position.

[0016] Furthermore, the M2e is inserted at one of the following positions in the amino acid sequence of the N2 influenza virus NA protein: between glutamine at position 49 and alanine at position 50, between glutamic acid at position 59 and arginine at position 60, or between asparagine at position 69 and serine at position 70.

[0017] Furthermore, the NA protein is the NA protein of the N8 influenza virus, and its amino acid sequence is shown in SEQ ID NO: 20. The M2e is inserted at any position within the amino acid sequence interval from the 32nd to the 86th position.

[0018] Furthermore, the M2e is inserted at one of the following positions in the amino acid sequence of the N8 influenza virus NA protein: between valine at position 70 and valine at position 71.

[0019] Secondly, the present invention provides an isolated nucleic acid molecule encoding the chimeric protein. This nucleic acid molecule may be in the form of DNA, RNA, or mRNA.

[0020] Furthermore, the nucleic acid molecule comprises, for example, the nucleotide sequence shown in SEQ ID NO: 4-7.

[0021] Thirdly, the present invention provides a method for constructing a universal influenza vaccine, comprising the step of preparing a vaccine formulation from the chimeric protein, or the nucleic acid molecule, or a recombinant expression system containing the nucleic acid molecule.

[0022] Fourthly, the present invention provides a recombinant influenza virus whose genome contains the aforementioned nucleic acid molecules.

[0023] Furthermore, its genome also contains internal gene segments of the influenza virus and the HA gene.

[0024] Furthermore, the internal gene fragments include NS, NP, PA, PB1, PB2 gene fragments of influenza A virus and M gene fragment, wherein the M gene fragment is an unmodified M gene or a modified M gene, and the HA gene is derived from influenza A virus.

[0025] Furthermore, the internal gene fragments of the influenza virus and the HA gene may originate from the same strain (e.g., both from A / PR / 8 / 34 or both from wild-type H9N2), or they may originate from different strains.

[0026] Fifthly, the present invention provides a method for preparing the recombinant influenza virus, comprising the following steps: (1) Provide a plasmid expressing an internal gene fragment of influenza virus, a plasmid expressing the HA gene of influenza virus, and a plasmid expressing the nucleic acid molecule; (2) Co-transfect the plasmid from step (1) into eukaryotic host cells; (3) Culture the transfected cells to rescue and harvest recombinant influenza virus.

[0027] This invention successfully rescues a recombinant virus that can stably replicate and highly express the chimeric protein by placing the gene encoding the chimeric protein together with the influenza A virus HA gene in the influenza A virus replication backbone and using reverse genetics technology.

[0028] In a sixth aspect, the present invention provides an attenuated live vaccine composition comprising an attenuated recombinant influenza virus and a pharmaceutically acceptable vector.

[0029] In a seventh aspect, the present invention provides an inactivated vaccine composition comprising an inactivated recombinant influenza virus and a pharmaceutically acceptable vector.

[0030] Eighthly, the present invention provides a protein vaccine composition comprising the chimeric protein and a pharmaceutically acceptable carrier.

[0031] The present invention also provides the use of the chimeric protein, or the nucleic acid molecule, or the recombinant influenza virus, or the recombinant viral vector containing the nucleic acid molecule in the preparation of drugs for the prevention or treatment of influenza virus infection.

[0032] Compared with the prior art, the technical effects achieved by the present invention include: The chimeric protein provided by this invention inserts the M2e amino acid sequence of influenza A virus into the stem domain of the extracellular region of the NA protein of different subtypes of influenza A virus, thereby displaying the M2e epitope on the surface of the NA protein. This allows a single chimeric protein to simultaneously display key antigens of two different influenza viruses: its main body can be recognized by the immune system as a target for the corresponding influenza A virus, while the inserted module (influenza A M2e) provides a conserved target for other influenza A subtypes. The chimeric protein of this invention maintains the native conformation and neuraminidase activity of the NA protein while simultaneously inducing specific immune responses against both NA and M2e. This chimeric protein does not affect the neuraminidase activity and viral release function of NA, and can stimulate cross-protective immune responses against multiple influenza A virus subtypes (such as H3N2 and H9N2) through the displayed M2e epitope, exhibiting broad-spectrum protective potential.

[0033] By utilizing the isolated nucleic acid molecule encoding the aforementioned chimeric protein, the chimeric protein can be efficiently expressed in host cells through a recombinant expression system, providing a stable antigen source for the preparation of various forms of vaccines. The universal influenza vaccine construction method provided by this invention prepares vaccine formulations based on the chimeric protein, the aforementioned nucleic acid molecule, or a recombinant expression system containing the nucleic acid molecule. The process steps are mature, the operation is simple, and it possesses universality and industrial applicability.

[0034] The recombinant influenza virus constructed based on the aforementioned nucleic acid molecules contains a genome that encodes a chimeric protein, along with internal gene fragments of the influenza virus and its HA gene. This recombinant virus, through its self-expressed HA protein, can effectively stimulate the body to produce hemagglutination-inhibiting antibodies against its own HA subtype of influenza virus. Simultaneously, through the M2e epitope displayed by the chimeric protein, it can elicit a cross-protective immune response against multiple influenza A virus subtypes (such as H3N2 and H9N2). This recombinant virus can replicate efficiently in MDCK cells or chicken embryos, and its yield characteristics meet the requirements for industrial-scale vaccine production.

[0035] The protein vaccine composition provided by the present invention, with the chimeric protein as the active ingredient, can be directly presented to the immune system as an antigen, inducing a cross-protective immune response against M2e, and has high safety.

[0036] The live attenuated vaccine composition and inactivated vaccine composition provided by this invention use attenuated or inactivated recombinant influenza virus as active ingredients, respectively. The live attenuated vaccine composition can mimic the natural infection route and induce a more comprehensive and durable immune response; the inactivated vaccine composition maintains immunogenicity while having higher safety and is easier to store and transport. Both vaccine compositions can effectively stimulate cross-protective immunity against multiple subtypes of influenza virus.

[0037] The nucleic acid vaccine composition provided by the present invention uses the nucleic acid molecule as the active ingredient. By encapsulating the nucleic acid molecule encoding the chimeric protein in a delivery medium such as liposomes, polymer nanoparticles, exosomes or virus-like particles, or inserting it into a recombinant viral vector, the chimeric protein can be expressed in situ in the host cell in vivo, continuously stimulating the immune system, and providing a new technical approach for the development of influenza vaccines.

[0038] The aforementioned chimeric proteins, nucleic acid molecules, recombinant influenza viruses, and various vaccine compositions can all effectively induce cross-protective immune responses against multiple influenza A virus subtypes through the presentation of the M2e epitope. In the form of recombinant influenza viruses employing internal gene fragments of influenza A virus and the HA gene backbone of influenza A virus, hemagglutination inhibition antibodies against the corresponding HA subtype can also be simultaneously stimulated. Furthermore, the recombinant virus possesses the mass production capability required for industrial-scale production, and has broad application prospects in the preparation of drugs for the prevention or treatment of influenza virus infections. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the chimeric NA-4M2e protein structure provided in an embodiment of the present invention; L represents the linker peptide; Figure 2 A schematic diagram of a chimeric virus genome provided in an embodiment of the present invention; Figure 3 The change in body weight of mice immunized with N1-4M2e recombinant virus after challenge with H3N2 strain; Figure 4 The survival rate of mice immunized with N1-4M2e recombinant virus after challenge with H3N2 strain; Figure 5 The change in body weight of mice immunized with N1-4M2e recombinant virus after challenge with H9N2 strain; Figure 6 The survival rate of mice immunized with N1-4M2e recombinant virus after challenge with H9N2 strain; Figure 7 The change in body weight of mice immunized with N6-4M2e recombinant virus after challenge with H3N2 strain; Figure 8 The results show the survival rate of mice immunized with N6-4M2e recombinant virus after challenge with H3N2 strain. Figure 9 The change in body weight of mice immunized with N6-4M2e recombinant virus after challenge with H9N2 strain; Figure 10 The survival rate of mice immunized with N6-4M2e recombinant virus after challenge with H9N2 strain; Figure 11 The change in body weight of mice immunized with N2-4M2e recombinant virus after challenge with H1N1 strain; Figure 12 The results show the survival rate of mice immunized with N2-4M2e recombinant virus after challenge with H1N1 strain. Figure 13 The change in body weight of mice immunized with N2-4M2e recombinant virus after challenge with H3N2 strain; Figure 14 The results show the survival rate of mice immunized with the N2-4M2e recombinant virus after challenge with the H3N2 strain. Detailed Implementation

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Similar component reference numerals in the drawings represent similar components. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0041] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0042] It should also be understood that the terminology used in the specification of embodiments of the present invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the present invention. As used in the specification of embodiments of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" are intended to include the plural forms.

[0043] like Figure 2As shown, the recombinant viruses constructed in the embodiments of the present invention are all rescued using influenza virus reverse genetics. The internal gene fragments include the NS, NP, PA, PB1, and PB2 gene fragments of influenza A virus, as well as the M gene fragment. The M gene fragment is either unmodified or modified, and the HA gene originates from influenza A virus. In specific implementations, the internal gene fragments of the influenza virus and the HA gene can originate from the same strain (e.g., both from A / PR / 8 / 34 or both from wild-type H9N2), or they can originate from different strains.

[0044] It should be noted that the A / PR / 8 / 34 M gene attenuated virus and wild-type H9N2 virus used in the following examples are only one of the viral backbones selected for the examples. Those skilled in the art will understand that the core of this invention lies in the chimeric protein design strategy of inserting the M2e epitope of influenza A virus into the stem domain of the extracellular region of the NA protein of influenza A virus (various subtypes). Therefore, without departing from the concept of this invention, other influenza virus backbones, such as other attenuated strains of influenza A virus, cold-adapted strains, or other genetically engineered influenza virus vectors, can also be used as expression and delivery vectors for the aforementioned chimeric NA protein.

[0045] Example 1: Construction of chimeric protein-coding gene and rescue of N1-4M2e recombinant virus This embodiment aims to construct and rescue a recombinant influenza virus. The virus uses an attenuated M gene virus of A / PR / 8 / 34 (H1N1) as its genetic backbone (its construction method and attenuation characteristics have been disclosed in patent CN114381439A). An epitope sequence of the extracellular domain (4M2e) of influenza A virus matrix protein 2 was inserted into the stem domain of its neuraminidase (NA) protein extracellular region to screen for viral strains capable of stable replication and expression of the chimeric protein. Figure 1 ).

[0046] 1. Construction of chimeric genes First, a sequence encoding four tandem M2e epitopes (4M2e) was designed and synthesized. Each M2e unit and the N-terminus and C-terminus of the entire tandem sequence are linked by flexible linker peptides (GGGGS). The amino acid sequence (including the flexible linker peptides connecting each M2e at both ends and in the middle) is shown in SEQ ID NO: 1, and the corresponding nucleotide sequence is shown in SEQ ID NO: 2.

[0047] To screen for the optimal insertion site that does not affect the correct folding of the NA protein, enzyme activity, and viral assembly, a systematic construction was performed in this embodiment. Specifically, the extracellular domain of the N1 type NA protein coding region (from amino acid 34 in the stem to amino acid 454 in the head) was selected as the target region. Within this region, tandem sequences encoding four M2e epitopes (4M2e) were inserted at intervals of 10-20 amino acids, resulting in a total of 23 different insertion sites. The amino acid sequence of the coding region of the N1 type (A / PR / 8 / 34 strain) NA protein gene used in this embodiment is SEQ ID NO: 8, and the corresponding nucleotide sequence is SEQ ID NO: 3.

[0048] The 4M2e sequence was synthesized (by Qingke Biotechnology). Specific primers were designed for each pre-defined insertion site, and PCR amplification was performed using the synthesized 4M2e as a template. After separation by agarose gel electrophoresis, correctly sized bands were excised and purified. The purified 4M2e fragments were inserted into the corresponding positions in the cloning vector pUC containing the N1 type NA gene using homologous recombination cloning technology (using the homologous recombination kit from Beijing Jinsha Biotechnology Co., Ltd., catalog number 22SC61234, reaction conditions: 50℃, 15 minutes), thereby constructing a series of recombinant plasmids. All recombinant plasmids were transformed into competent *E. coli* cells. After colony PCR screening and sequencing verification, high-purity expression plasmids were prepared using an endotoxin-free plasmid miniprep kit (Beijing Adley Biotechnology Co., Ltd., catalog number PL10).

[0049] 2. Rescue and Screening of Recombinant Viruses HEK293T cells were evenly seeded into 6-well cells and cultured in a cell culture incubator at 37°C with 5% CO2. Transfection was performed when the cell density reached 70%-80%. Plasmids expressing six internal gene fragments of the A / PR / 8 / 34 strain (NS, NP, PA, PB1, PB2, and the genetically modified M gene), a plasmid expressing A / PR / 8 / 34 HA, and a plasmid expressing N1 NA chimeric with 4M2e were mixed in a 1:1:1:1:1:1:1:1 ratio and transfected using the Lipofectamine™ LTX transfection kit. The culture medium was replaced with fresh medium 6-8 hours after transfection. Forty-eight hours after transfection, the cell culture plates were freeze-thawed three times, and the supernatant was collected by centrifugation. The harvested virus was used to infect MDCK cells, and cytopathic effects (CPE) were observed after 48-72 hours of culture. The viral supernatant showing CPE was then collected and seeded into MDCK cells for amplification. The virus was harvested after 72 hours. The harvested virus was serially diluted 10-fold, resulting in nine dilutions. Each dilution was inoculated into MDCK cells in triplicate. Cytopathic effects were observed after 72 hours, and viral titers were calculated using the Reed-Muench method. RNA was extracted from the amplified virus and subjected to RT-PCR. After amplification with appropriate PCR primers, the amplified virus was subjected to agarose gel electrophoresis and then sequenced for identification. Viruses with correctly identified sequences were considered successfully rescued.

[0050] After attempting to rescue chimeric viruses at all 23 different insertion sites, two live viruses with stable replication capabilities were successfully obtained. Sequencing analysis revealed that the 4M2e sequence insertion sites of both viruses were located within the stem domain (amino acids 36-75) of the extracellular region of the NA protein, specifically between asparagine at position 44 and histidine at position 45 (the complete nucleotide sequence of the chimeric NA protein is SEQ ID NO: 4) and between serine at position 35 and histidine at position 36 (the complete nucleotide sequence of the chimeric NA protein is SEQ ID NO: 7).

[0051] To verify that inserting 4M2e into the stem domain of the NA protein could successfully rescue the virus, this invention further randomly selected two positions within the stem domain for verification: the 4M2e epitope between glycine at position 41 and serine at position 42 (the specific nucleotide sequence of the entire chimeric protein is SEQ ID NO:5) and between serine at position 42 and glutamic acid at position 43 (the specific nucleotide sequence of the entire chimeric NA protein is SEQ ID NO:6). Molecular construction and virus rescue were performed using the same method described above. Sequencing confirmed that both sites successfully rescued live viruses with stable replication capabilities.

[0052] Titer determination showed that the rescued virus, after amplification on MDCK cells, achieved a titer of up to 10. 7.5 TCID 50 / mL, which has the mass production capability to meet the needs of industrial vaccine production.

[0053] Example 2: Construction of chimeric protein-coding gene and rescue of N6-4M2e recombinant virus This embodiment, like Embodiment 1, uses an A / PR / 8 / 34 (H1N1) M gene attenuated virus as the gene backbone, inserts the 4M2e epitope sequence into the stem domain of the extracellular region of the N6 type NA protein, and constructs and rescues a recombinant influenza virus.

[0054] 1. Construction of chimeric genes Following the construction strategy of Example 1, the 4M2e tandem sequence (SEQ ID NO: 2) was inserted into the extracellular domain of the N6 type NA protein. Specifically, 44 different insertion sites were designed, spaced approximately 10 amino acids apart, within the extracellular domain of the N6 type NA protein coding region (from amino acid 35 in the stem to amino acid 459 in the head). The amino acid and nucleotide sequences of the N6 type NA protein used in this example are shown in SEQ ID NO: 10 and SEQ ID NO: 9, respectively.

[0055] Specific primers were synthesized for each pre-defined insertion site. PCR amplification was performed using 4M2e as a template, and the amplified fragment was inserted into the corresponding position of the pUC vector containing the N6 type NA gene using homologous recombination to construct a recombinant plasmid. After transformation, screening, and sequencing verification, high-purity expression plasmids were extracted.

[0056] 2. Rescue and Screening of Recombinant Viruses During transfection, plasmids expressing six internal gene fragments of the A / PR / 8 / 34 strain (PB2, PB1, PA, NP, M (modified), NS), PR8 HA, and N6 NA (chimeric 4M2e) were co-transfected into HEK293T cells at a ratio of 1:1:1:1:1:1:1:1:1. The supernatant was harvested 48 hours post-transfection and used to infect MDCK cells for virus rescue and amplification. Viral titers were calculated using the Reed-Muench method to determine TCID. 50 .

[0057] After attempting to rescue chimeric viruses at all 44 different insertion sites, three live viruses with stable replication capabilities were successfully obtained. Sequencing results showed that the 4M2e insertion sites were all located within the stem domain (amino acids 34-79) of the N6 type NA protein, specifically between glutamic acid at position 49 and threonine at position 50 (the complete nucleotide sequence of the chimeric NA protein is SEQ ID NO:13), between asparagine at position 59 and isoleucine at position 60 (the complete nucleotide sequence of the chimeric NA protein is SEQ ID NO:14), and between glutamic acid at position 69 and glycine at position 70 (the complete nucleotide sequence of the chimeric NA protein is SEQ ID NO:15). The obtained recombinant virus titers reached 10. 7.75 TCID 50 It has a capacity of / mL or higher and is suitable for mass production.

[0058] Example 3: Construction of chimeric protein-coding gene and rescue of N2-4M2e recombinant virus In this embodiment, a 4M2e epitope sequence was inserted into the stem domain of the extracellular region of the N2 type NA protein using wild-type H9N2 influenza virus as the gene backbone, and a recombinant influenza virus was constructed and rescued.

[0059] 1. Construction of chimeric genes In the extracellular domain of the coding region of the N2 type NA protein (from amino acid 35 in the stem to amino acid 469 in the head), 44 different 4M2e insertion sites were designed with intervals of approximately 10 amino acids. The amino acid sequence of the N2 type NA protein is shown in SEQ ID NO: 12, and the nucleotide sequence is shown in SEQ ID NO: 11. The construction of the 4M2e tandem sequence and the preparation of the recombinant plasmid were carried out in the same manner as in Example 1.

[0060] 2. Rescue and Screening of Recombinant Viruses Plasmids expressing six internal gene fragments of wild-type H9N2 influenza virus (PB2, PB1, PA, NP, M, NS), plasmids expressing H9N2 influenza virus HA, and chimeric NA expressing chimeric 4M2e were co-transfected into HEK293T cells at a ratio of 1:1:1:1:1:1:1:1:1. The culture medium was replaced with fresh medium 6 to 8 hours after transfection. Forty-eight hours after transfection, the cell plate was repeatedly frozen and thawed, and the supernatant was collected by centrifugation. The harvested virus solution was used to infect MDCK cells, and CPE was observed after 48 to 72 hours of culture. Virus solutions with obvious pathological changes were collected and inoculated into SPF chicken embryos for amplification. Chicken embryo allantoic fluid was harvested after 72 hours. The amplified virus solution was serially diluted 10-fold in nine gradients, with each gradient inoculated into three SPF chicken embryos. The chicken embryo allantoic fluid virus was harvested after 72 hours, and the virus titer was detected using chicken erythrocytes and calculated using the Reed-Muench method.

[0061] After attempting to rescue all 44 chimeric viruses, three stably replicating live viruses were successfully obtained. Sequencing confirmed that their 4M2e insertion sites were all located within the stem domain (amino acids 36-88) of the N2-type NA protein, specifically between glutamine at position 49 and alanine at position 50 (the complete nucleotide sequence of the chimeric NA protein is SEQ ID NO:16), between glutamic acid at position 59 and arginine at position 60 (the complete nucleotide sequence of the chimeric NA protein is SEQ ID NO:17), and between asparagine at position 69 and serine at position 70 (the complete nucleotide sequence of the chimeric NA protein is SEQ ID NO:18). The viral titer reached 10. 8.25 EID 50 / mL, meeting the requirements for vaccine production.

[0062] Example 4: Construction of chimeric protein-coding gene and rescue of N8-4M2e recombinant virus Examples 1-3 above have demonstrated that inserting the 4M2e epitope tandem sequence into the stem domain of N1, N6, and N2 NA proteins can successfully rescue recombinant viruses with stable replication capabilities. To further verify the universality of this design strategy, this example selects the N8 NA protein, which is more distantly related to the aforementioned three NA subtypes, and performs insertion verification within its stem domain. The amino acid sequence of the N8 NA protein is shown in SEQ ID NO: 20, and the nucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO: 19.

[0063] Based on the stem domain region (amino acids 32 to 86) of the N8 type NA protein, this invention did not perform a systematic full-region screening, but instead randomly selected a site within this region—between valine at position 70 and position 71—for 4M2e insertion, and operated according to the same molecular construction and virus rescue method as in Example 1.

[0064] The results showed that a recombinant virus with stable replication ability was successfully rescued from this site, and the sequence was confirmed to be correct by sequencing, with the viral titer meeting the requirements for vaccine production. The insertion site is located inside the stem domain of the N8 type NA protein.

[0065] Thus far, this invention has successfully achieved the insertion and recombinant virus rescue of 4M2e in the stem domains of four different NA subtypes: N1, N6, N2, and N8. These results fully demonstrate that the chimeric protein design strategy of this invention, which inserts the M2e epitope tandem sequence of influenza A virus into the stem domain of NA proteins, has cross-subtype universality and is applicable to various influenza A virus NA proteins.

[0066] Table 1. Amino acid positions corresponding to the stem domains of different types of NA proteins Example 5: Preliminary evaluation of the immunogenicity of chimeric recombinant virus The immunogenicity of the N1-4M2e, N6-4M2e and N2-4M2e chimeric recombinant viruses obtained in Examples 1-3 in mouse models was initially evaluated, particularly their ability to induce specific antibodies against their respective homologous HA.

[0067] Each chimeric virus vaccine strain rescued in Examples 1-3 was used to administer intranasal immunization to 6-week-old female BALB / c mice. For each chimeric virus vaccine strain, the experiment included a chimeric virus vaccine immunization group, a non-chimeric control group, and a PBS group, with 10 mice in each group.

[0068] The non-chimeric control groups of N1-4M2e and N6-4M2e recombinant viruses were recombinant viruses with an attenuated M gene of A / PR / 8 / 34 (H1N1) as the backbone, expressing PR8 HA and the corresponding NA protein (N1-NA or N6-NA), but without chimeric 4M2e epitope tandem sequences in the NA protein. The non-chimeric control group of N2-4M2e recombinant virus was a recombinant virus with a wild-type H9N2 backbone, expressing H9N2 HA and N2-NA proteins, but without chimeric 4M2e in the NA protein. The construction methods of each control virus were basically the same as those described in the corresponding examples, except that the NA expression plasmid used during transfection was a corresponding NA protein expression plasmid without 4M2e insertion. Each control virus had the same viral backbone and surface antigen as the vaccine strain of this invention, the only difference being that its NA protein did not contain the influenza A virus M2e epitope. Each control virus was also successfully rescued using reverse genetics technology and amplified in MDCK cells / chicken embryos.

[0069] The mice were immunized using a "0+7" two-immunization schedule, meaning they were immunized once on day 0 and once on day 7, with a dose of 10 mg / mL per mouse each time. 6 EID 50 (In 50 μL PBS). The immunization route was intranasal administration. Mouse body weight was monitored daily until day 14 post-immunization. No significant weight loss or abnormal clinical symptoms were observed in any group of mice, indicating that the vaccine strain and control virus of this invention have good safety in mice at this dose.

[0070] Mice serum was collected on day 14 post-immunization to detect the titer of hemagglutination inhibition (HI) antibodies against their respective HA subtypes. The specific detection steps are as follows: 1. Based on the hemagglutination titer of the antigen, prepare four units of antigen and verify them; 2. Serum serial dilution: Inactivate the serum to be tested at 56℃ for 30 min in advance, add PBS to the 96-well hemagglutination plate, and then dilute the serum to be tested, standard negative serum and positive serum according to the 2-fold serial dilution method; 3. Add antigen: Add an equal volume of the validated four-unit antigen solution to the diluted serum, and allow the antibodies in the serum to react with the antigen at room temperature for 30 minutes; 4. Add red blood cells: Add an equal volume of 1% chicken red blood cells to the reaction well, let stand at room temperature for 30 min, and observe the binding of red blood cells with unbound antigens.

[0071] 5. Result Interpretation: Tilt the blood coagulation plate at 45° and read the value on the back. The well from which red blood cells can flow completely is taken as the HI titer of the serum. The prerequisite for result interpretation is that negative and positive sera must be valid, i.e., complete agglutination must be observed in the negative serum well, and the positive serum titer must differ from the labeled titer by no more than one titer.

[0072] On day 14 post-immunization, the specific results of hemagglutination inhibition antibody levels in mice immunized with N1-4M2e, N6-4M2e, and N2-4M2e chimeric recombinant viruses are shown in Table 2-4.

[0073] Table 2. Serum HI antibody levels in mice 14 days after immunization with the N1-4M2e recombinant virus vaccine strain. In Table 2, no hemagglutination inhibition antibodies were detected in the PBS group. The non-chimeric control group (N1-NA) and the chimeric virus vaccine strain induced higher levels of hemagglutination inhibition antibodies against PR8 HA (HI hemagglutination titer > 2) in mice immunized with these antibodies. 4 This result indicates that inserting the 4M2e tandem sequence into the N1 type NA protein does not affect the specific humoral immune response induced by the recombinant virus against PR8 HA. The chimeric viral vaccine strain obtained in this invention can stimulate the body to produce high levels of hemagglutination inhibition (HI) antibodies against the vaccine strain homologous PR8 HA, thus inducing immunogenicity against the PR8 strain.

[0074] Table 3. Serum HI antibody levels on day 14 after immunization of mice with the N6-4M2e recombinant virus vaccine strain. In Table 3, no hemagglutination inhibition antibodies were detected in the PBS group. The non-chimeric control group (N6-NA) and the chimeric virus vaccine strain induced higher levels of hemagglutination inhibition antibodies against PR8 HA (HI hemagglutination titer > 2) in mice immunized with these vaccines. 4This result indicates that inserting the 4M2e tandem sequence into the N6 type NA protein does not affect the specific humoral immune response induced by the recombinant virus against PR8 HA. The chimeric viral vaccine strain obtained in this invention can stimulate the body to produce high levels of hemagglutination inhibition (HI) antibodies against the vaccine strain homologous PR8 HA, thus inducing immunogenicity against the PR8 strain.

[0075] Table 4. Serum HI antibody levels in mice 14 days after immunization with the N2-4M2e recombinant virus vaccine strain. In Table 4, no hemagglutination inhibition antibodies were detected in the PBS group. The non-chimeric control group (N2-NA) and the chimeric virus vaccine strain induced higher levels of hemagglutination inhibition antibodies against H9N2 HA (HI hemagglutination titer > 2) in mice immunized with these vaccines. 4 This result indicates that inserting the 4M2e tandem sequence into the N2 type NA protein does not affect the specific humoral immune response induced by the recombinant virus against H9N2 HA. The chimeric viral vaccine strain obtained in this invention can stimulate the body to produce high levels of hemagglutination inhibition (HI) antibodies against the vaccine strain's homologous H9N2 HA, thus inducing immunogenicity against the H9N2 strain.

[0076] The above results indicate that inserting the 4M2e tandem sequence into type A NA proteins such as N1, N6, and N2 does not affect the specific humoral immune response induced by the recombinant virus against their respective homologous HA. All chimeric viral vaccine strains obtained in this invention can stimulate the body to produce high levels of hemagglutination inhibition (HI) antibodies against the vaccine strain's homologous HA.

[0077] Example 6. Protection against heterologous virus challenge in mice after immunization with chimeric virus vaccine strain 1. N1-4M2e recombinant virus challenge experiment Serum was collected on day 14 after the initial immunization (day 7 after the booster immunization), and mice in each immunization group were challenged with intranasal administration of viral strains. The challenge strains were the H3N2 subtype (A / Aichi / 2 / 1968 (H3N2) mouse-adapted strain) and the H9N2 subtype strain. The PBS group, the non-chimeric control group (N1-NA), and the N1-4M2e chimeric virus vaccine immunization group were all challenged with one of the two strains, with 5 mice challenged per strain-experimental group combination. The challenge dose of H3N2 per mouse was 10... 6 EID 50 (In a 50 μL volume), the challenge dose of H9N2 is 10 6.5 EID 50 (In 50 μL volume). Mouse body weight was monitored daily for 14 consecutive days after challenge, and survival status was recorded. Mice whose body weight dropped below 75% of their initial body weight were considered dead, and the survival rate was calculated accordingly.

[0078] Results of the challenge experiment ( Figures 3-6 The results showed that both the PBS control group and the non-chimeric control group experienced severe weight loss and mortality after being challenged with either the H3N2 or H9N2 strains. Specifically, mice in the PBS group challenged with H3N2 died on day 6, and all died by day 8; mice in the non-chimeric control group challenged with H3N2 died on day 8, with a final survival rate of 60%. In stark contrast, mice in the vaccine-immunized group did not experience significant weight loss after being challenged with the H3N2 strain, and there were no deaths. Mice in the PBS group challenged with H9N2 died on day 4, and all died by day 6; mice in the non-chimeric control group challenged with H9N2 died on day 5, with a final survival rate of 20%. Mice in the vaccine-immunized group did not experience significant weight loss on average after being challenged with the H9N2 strain, and the survival rate of all immunized groups was 80% or higher.

[0079] 2. N6-4M2e recombinant virus challenge experiment The challenge protocol, virus strain, and dosage were the same as in the N1-4M2e challenge experiment. The PBS group, the non-chimeric control group (N6-NA), and the N6-4M2e chimeric virus vaccine immunization group were challenged separately.

[0080] Results of the challenge experiment ( Figures 7-10 The results showed that both the PBS control group and the non-chimeric control group experienced severe weight loss and mortality after being challenged with either the H3N2 or H9N2 strains. Mice in the PBS group challenged with H3N2 died on day 6, and all died by day 8; mice in the non-chimeric control group challenged with H3N2 died on day 8, and all died by day 10. Mice in the vaccine-immunized groups did not experience significant weight loss after being challenged with the H3N2 strain, and there were no deaths. Mice in the PBS group challenged with H9N2 died on day 4, and all died by day 5; mice in the non-chimeric control group challenged with H9N2 died on day 7, and all died by day 8. Mice in the vaccine-immunized groups did not experience significant weight loss on average after being challenged with the H9N2 strain, and there were no deaths.

[0081] 3. N2-4M2e recombinant virus challenge experiment The challenge strains were H1N1 subtype (A / PR / 8 / 34) and H3N2 subtype (A / Aichi / 2 / 1968 (H3N2) mouse-adapted strain). Mice were challenged with the PBS group, the non-chimeric control group (N2-NA), and the N2-4M2e chimeric virus vaccine immunization group, with each mouse receiving a challenge dose of 10 H1N1. 5.0 EID 50 (In a 50 μL volume), the challenge dose of H3N2 is 10 6.0 EID50 (In 50 μL volume). Mouse body weight was monitored daily for 14 consecutive days after challenge, and survival status was recorded. Mice whose body weight dropped below 75% of their initial body weight were considered dead, and the survival rate was calculated accordingly.

[0082] Results of the challenge experiment ( Figures 11-14 The results showed that both the PBS control group and the non-chimeric control group experienced severe weight loss and mortality after being challenged with either the H1N1 or H3N2 strains. Mice in the PBS group challenged with H1N1 died on day 4, and all died on day 5. Mice in the non-chimeric control group challenged with H1N1 died on day 7, and all died on day 8. Mice in the vaccine-immunized groups did not experience significant weight loss after being challenged with the H1N1 strain, and there were no deaths. Mice in the PBS group challenged with H3N2 and the non-chimeric control group died on day 4 after challenge, and all died on day 5. Mice in the vaccine-immunized groups did not experience significant weight loss after being challenged with the H3N2 strain, and the survival rate of all immunized groups was 100%.

[0083] The results of the above-mentioned challenge experiments show that the N1-4M2e, N6-4M2e, and N2-4M2e chimeric virus vaccine strains constructed in this invention can effectively provide cross-protection against heterologous influenza A virus subtypes (including H3N2, H9N2, and H1N1). Compared with the non-chimeric control group, the survival rate of mice in the vaccine-immunized group after challenge with heterologous strains was significantly improved, proving that this cross-protective effect is mediated by the 4M2e epitope chimeric in the NA protein.

[0084] Example 7. Protective effect of inactivated N1-4M2e virus vaccine strain against heterologous strains This embodiment uses the N1-4M2e chimeric recombinant virus obtained in Example 1 as an example to evaluate whether the chimeric recombinant virus still has a protective effect against heterologous influenza A virus subtypes after inactivation treatment.

[0085] The four chimeric virus strains rescued in Example 1 were inactivated. The amplified viral stock solution was collected, and the viral titer was measured on MDCK cells. The viral titer should be greater than 10. 6.5 TCID 50 / mL. The original virus solution was transferred to an inactivation vessel, and β-propiolactone was added to a final concentration of 0.05%. After thorough mixing, the solution was inactivated at 2–8°C and 100 rpm / min for 24 hours. After inactivation, the virus solution was hydrolyzed at 37°C for 2 hours, and then stored at 2–8°C for no more than 2 months. The inactivated virus solution was passaged three times in MDCK cells, and no cytopathic effect was observed, confirming complete inactivation.

[0086] Three-week-old SPF chickens were selected and immunized subcutaneously, with each chicken receiving 300 μL of the vaccine. The experiment included a PBS group and an inactivated virus immunization group, with five chickens in each group. Serum was collected on day 21 post-immunization to detect hemagglutination inhibition (HI) antibodies against PR8 HA, using the detection method described in Example 5.

[0087] Table 5 shows the HI antibody results for each group on day 21 post-immunization. No HI antibodies were detected in the PBS group, while all four inactivated vaccine immunization groups produced high levels of HI antibodies against PR8 HA, with HI titers as high as 2. 10 This result indicates that the chimeric virus retained good immunogenicity after inactivation treatment and was able to induce a high level of specific antibody response.

[0088] On day 21 post-immunization, all chickens were challenged with the H9N2 strain. The challenge was administered intravenously, with each chicken receiving a dose of 2 × 10⁻⁶. 7.5 EID 50 The volume was 200 μL. Pharyngeal swab samples were collected from chickens on days 3 and 5 post-challenge to assess viral shedding. Samples showing hemagglutination activity were considered positive (+), otherwise negative (-). The results of the viral shedding tests are shown in Table 6.

[0089] Table 5. HI antibody levels in SPF chickens immunized with inactivated vaccine Table 6. Virus shedding from throat swabs on days 3 and 5 after immunization with inactivated vaccine in SPF chickens. Table 6 shows that the PBS group had positive results for viral shedding in pharyngeal swabs on days 3 and 5 after challenge (10 / 10). In contrast, all four inactivated vaccine immunization groups of this invention had negative results for viral shedding in pharyngeal swabs on days 3 and 5 after challenge (0 / 10). These results indicate that the N1-4M2e inactivated chimeric virus vaccine can effectively inhibit the replication and shedding of the H9N2 strain in chickens, thus blocking virus transmission.

[0090] The results of this embodiment demonstrate that the immunoprotective effect of chimeric viruses, represented by N1-4M2e, after inactivation treatment does not depend on viral replication activity. Even after viral inactivation, the particle structure formed by this chimeric protein can still effectively stimulate the body to produce cross-protective immunity against heterologous influenza A virus subtypes. It is reasonable to expect that chimeric proteins such as N6-4M2e, N2-4M2e, and N8-4M2e, constructed based on the same design strategy of this invention, can also provide similar cross-protective effects after inactivation treatment. This result further confirms that the chimeric protein obtained by the design strategy of inserting the M2e epitope into the stem domain of the NA protein is itself a potent immunogen, providing a solid experimental basis for developing safer inactivated vaccines and other forms of subunit vaccines or virus-like particle vaccines based on this chimeric protein.

[0091] In summary, this invention provides a chimeric protein design strategy that inserts the tandem sequence of the M2e epitope of influenza A virus into the stem domain of the NA protein. This strategy, by inserting M2e into the amino acid sequence corresponding to the stem domain of the NA protein, achieves the effective integration and synergistic display of two key antigens—NA and M2e—within the same protein molecule. Experiments have confirmed that the above design strategy can be successfully implemented for various NA subtypes of influenza, including N1, N6, N2, and N8. The resulting recombinant viruses, while maintaining the native conformation and function of the NA protein, can effectively display the M2e epitope and can stably replicate in different viral backbones (such as the A / PR / 8 / 34 attenuated backbone and the wild-type H9N2 backbone), demonstrating good universality. Immunogenicity and challenge protection assays further confirm that the chimeric recombinant virus constructed based on this strategy can simultaneously elicit a specific immune response against homologous HA and broad-spectrum cross-protection against heterologous influenza A virus subtypes mediated by M2e, and this cross-protection effect is independent of a specific viral backbone or NA subtype. This invention provides a new technical solution for the development of universal influenza vaccines that is highly versatile and effective.

[0092] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0093] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A chimeric protein, characterized in that, Its amino acid sequence includes the amino acid sequence of the influenza A virus NA protein and the amino acid sequence of the influenza A virus M2e, wherein the amino acid sequence of the M2e is inserted inside the amino acid sequence corresponding to the stem domain of the extracellular region of the NA protein; the M2e comprises 2 to 10 tandem M2e units, each of the M2e units is connected by a linker peptide, and the tandem M2e units are also connected to the amino acid sequence of the NA protein by a linker peptide.

2. The chimeric protein according to claim 1, characterized in that, The linker peptide contains at least one GGGGS unit.

3. The chimeric protein according to claim 1, characterized in that, The NA protein is selected from the NA protein of influenza viruses of type N1, N2, N6 or N8.

4. The chimeric protein according to claim 3, characterized in that, The NA protein is the NA protein of the N1 influenza virus, and the M2e is inserted at any position within the amino acid sequence range from the 36th to the 75th amino acid position. Alternatively, the NA protein is the NA protein of the N6 influenza virus, and the M2e is inserted at any position within the amino acid sequence range from the 34th to the 79th amino acid position. Alternatively, the NA protein is the NA protein of the N2 influenza virus, and the M2e is inserted at any position within the amino acid sequence range from the 36th to the 88th amino acid position corresponding to its amino acid sequence. Alternatively, the NA protein is the NA protein of the N8 influenza virus, and the M2e is inserted at any position within the amino acid sequence range from the 32nd to the 86th amino acid position.

5. The chimeric protein according to claim 4, characterized in that, The M2e is inserted at one of the following positions in the amino acid sequence of the N1 influenza virus NA protein: between glycine at position 41 and serine at position 42, between serine at position 42 and glutamine at position 43, between asparagine at position 44 and histidine at position 45, or between serine at position 35 and histidine at position 36.

6. The chimeric protein according to claim 4, characterized in that, The M2e is inserted at one of the following positions in the amino acid sequence of the N6 influenza virus NA protein: between glutamic acid at position 49 and threonine at position 50, between asparagine at position 59 and isoleucine at position 60, or between glutamic acid at position 69 and glycine at position 70.

7. The chimeric protein according to claim 4, characterized in that, The M2e is inserted at one of the following positions in the amino acid sequence of the N2 influenza virus NA protein: between glutamine at position 49 and alanine at position 50, between glutamic acid at position 59 and arginine at position 60, or between asparagine at position 69 and serine at position 70.

8. The chimeric protein according to claim 4, characterized in that, The M2e is inserted between valine at position 70 and position 71 of the amino acid sequence of the N8 influenza virus NA protein.

9. An isolated nucleic acid molecule, characterized in that, Encoding the chimeric protein as described in any one of claims 1-8.

10. A method for constructing a universal influenza vaccine, characterized in that, The step includes preparing a vaccine formulation from the chimeric protein of any one of claims 1 to 8 or the nucleic acid molecule of claim 9.

11. A recombinant influenza virus, characterized in that, Its genome contains the nucleic acid molecule described in claim 9.

12. The recombinant influenza virus according to claim 11, characterized in that, Its genome also contains internal gene segments of the influenza virus and the HA gene.

13. The recombinant influenza virus according to claim 12, characterized in that, The internal gene fragments include NS, NP, PA, PB1, PB2 gene fragments of influenza A virus and M gene fragment, wherein the M gene fragment is an unmodified M gene or a modified M gene, and the HA gene is derived from influenza A virus.

14. A method for preparing the recombinant influenza virus according to any one of claims 11-13, characterized in that, Includes the following steps: (1) Provides a plasmid expressing an internal gene fragment of influenza virus, a plasmid expressing the HA gene of influenza virus, and a plasmid expressing the nucleic acid molecule of claim 9; (2) Co-transfect the plasmid from step (1) into eukaryotic host cells; (3) Culture the transfected cells to rescue and harvest recombinant influenza virus.

15. A live attenuated vaccine composition, characterized in that, The recombinant influenza virus as described in any one of claims 11-13, which is attenuated, and a pharmaceutically acceptable vector.

16. An inactivated vaccine composition, characterized in that, It comprises the recombinant influenza virus as described in any one of claims 11-13 that has been inactivated, and a pharmaceutically acceptable vector.

17. A protein vaccine composition, characterized in that, It comprises the chimeric protein as described in any one of claims 1-8 and a pharmaceutically acceptable carrier.

18. The use of the chimeric protein of any one of claims 1-8, or the nucleic acid molecule of claim 9, or the recombinant influenza virus of any one of claims 11-13, in the preparation of a medicament for the prevention or treatment of influenza virus infection.

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