Influenza A virus H5 protein mutant and application thereof

Abstract

The invention relates to the technical field of biology, and discloses an influenza A virus H5 protein mutant and application thereof. In order to solve the problems of weak cross immune response to epidemic strains, insufficient stability of wild type HA protein, low immunogenicity and the like of the existing H5N1 vaccine, a stable soluble H5 protein mutant is constructed through a sequence modification strategy. Specifically, the 332th to 380th amino acids in the HA protein of a wild type influenza A virus H5N1 are replaced by SPGCAT, and point mutations T386C, L418P and H456P are introduced to obtain the mutant. The mutant can significantly improve the structural stability of trimer protein, improve the expression level of recombinant protein, broaden the cross protection spectrum, enhance the level of immunogenic hemagglutination inhibition antibodies and improve immune response balance.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to mutant influenza A virus H5 protein and its applications. Background Technology

[0002] The highly pathogenic avian influenza virus H5N1 poses a persistent and serious threat to global public health. In recent years, 14 human H5N1 vaccines have been approved for use or included in stockpiling programs in various countries and regions, including whole-virus inactivated vaccines, split vaccines, subunit vaccines, and live attenuated vaccines. However, existing vaccines exhibit weak cross-immune responses against currently circulating 2.3.4.4b branch viruses, highlighting the urgent need to develop specific vaccines against the novel H5N1 virus. In addition to traditional vaccine platforms, mRNA vaccine technology demonstrates significant advantages due to its rapid development, efficient production, and ability to induce a comprehensive immune response.

[0003] Hemagglutinin (HA) is a key protein mediating viral invasion of host cells and a major target of neutralizing antibodies against influenza virus. HA comprises two subunits, HA1 and HA2, distributed on the viral envelope surface as a trimer spike. Structurally, the HA trimer can be divided into a head and a stem. The HA head contains its receptor binding site, while the HA stem contains a hydrophobic fusion peptide and a transmembrane region formed by the C-terminus of HA2. Upon viral entry into the host cell, the conformation of HA undergoes a series of changes as the pH decreases, ultimately transitioning from a pre-fusion state to a post-fusion state and mediating the fusion of the viral membrane with the host cell membrane. Before fusion, the HA head domain contains multiple immunodominant sites (such as SA, SB, CA1, CA2, and CB), which are the main binding targets of neutralizing antibodies. After HA fusion, its structure undergoes significant changes; some antigenic epitopes may be hidden or exposed, leading to altered immunogenicity. The stem region of the fused HA protein may expose new antigenic epitopes, but these epitopes have relatively weak immunogenicity and induce less neutralizing antibody activity than antibodies targeting the head domain of the unfused HA protein. This limits the immunogenicity of the fused HA protein in vaccine design. Therefore, stabilizing the trimer conformation of the HA protein before fusion is crucial for improving its immunogenicity and vaccine efficacy.

[0004] The H5N1 subtype of influenza A virus (a highly pathogenic avian influenza virus) has the ability to spread across species, causing severe infection and death in humans, posing a serious threat to public health. Current H5N1 vaccines mainly face the following technical bottlenecks: conformational instability: the pre-fusion conformation of wild-type HA protein easily changes to the post-fusion conformation (mediated by membrane fusion-related polybasic amino acid sequences), leading to reduced immunogenicity and insufficient induced neutralizing antibody titers; weak cross-protection: existing vaccines mostly target single circulating strains, with limited cross-neutralizing activity against variant strains (such as the 2.3.4.4b branch); low expression level and solubility: wild-type HA protein easily forms inclusion bodies during recombinant expression, resulting in poor solubility and difficulty meeting the needs of large-scale production. Therefore, developing a stable pre-fusion conformation, high immunogenicity, and broad-spectrum cross-protection H5 trimer protein is crucial to overcoming the technical bottlenecks of existing vaccines. Summary of the Invention

[0005] The purpose of this invention is to provide a mutant of the H5 protein of influenza A virus and its applications.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides a mutant of influenza A virus H5 protein, wherein the mutant is obtained by replacing amino acid residues 332-380 in the hemagglutinin protein (HA protein) of wild-type influenza A virus H5N1 with SPGCAT, and introducing point mutations T386C, L418P and H456P.

[0007] Preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO:4 (i.e. mutant HA-mod0).

[0008] Furthermore, the mutant also contains mutations selected from at least one of the following groups: (1) S219C and S233C; (2) N222C and K228C; (3) R224C and P227C; (4) T231C and H256C.

[0009] More preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO:5, 6, 7 or 8, which corresponds to mutants HA-mod1, HA-mod2, HA-mod3 and HA-mod4, respectively.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the mutant or biological material containing the nucleic acid molecule.

[0011] The biological materials include, but are not limited to, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0012] Thirdly, the present invention provides any of the following applications of the mutant, the nucleic acid molecule, or the biological material: (1) To prepare drugs for the prevention and / or treatment of influenza A virus H5N1 infection; (2) Preparation of antibodies against influenza A virus H5N1; (3) Prepare diagnostic reagents for influenza A virus H5N1.

[0013] Fourthly, the present invention provides a pharmaceutical composition comprising the mutant or the nucleic acid molecule or biological material of claim 5.

[0014] Furthermore, the pharmaceutical composition is a vaccine.

[0015] The vaccines include recombinant protein vaccines, nucleic acid vaccines, vector vaccines, or virus-like particle vaccines.

[0016] For example, the nucleic acid vaccine is an mRNA-LNP vaccine, wherein the mRNA encodes the mutant.

[0017] Preferably, the LNP contains cationic lipids, DSPC, cholesterol and DMG-PEG2000 in a molar ratio of 50:(8-12):(35-40):(2-3), and more preferably a molar ratio of 50:10:38.5:1.5.

[0018] Fifthly, the present invention provides a method for preparing the mutant, comprising the following steps: (1) The nucleic acid molecule encoding the mutant is cloned into an expression vector to construct a recombinant vector; (2) The recombinant vector was transfected into eukaryotic cells at a cell density of 3 × 10⁻⁶ cells / year. 6 / mL, culture temperature 36-38℃, CO2 concentration 5-8%, rotation speed 115-125 rpm; (3) 16 hours after transfection, add 5% (v / v) 293F Hi-exp feed and continue culturing for 48 hours; (4) Collect cells, lyse them using Pierce IP lysis buffer, centrifuge at 4°C and 13000×g for 10 minutes, and take the supernatant to purify the target protein.

[0019] Preferably, the eukaryotic cells are 293F cells, the transfection dose is 4 μg, and the cell viability is ≥95%.

[0020] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) Significantly improves the structural stability of trimeric proteins: Non-reducing Western Blot showed that the mutants formed stable trimeric bands under non-denaturing conditions, while wild-type HA was prone to forming monomers or dimers; proline mutations (L418P, H456P) enhanced the rigidity of the α-helix, disulfide bonds (such as T386C) reduced conformational fluctuations, and segment substitution (332G-380A→SPGCAT) deleted the easily cleaved fusion peptide sequence (SEQ ID NO:2), further reducing structural instability.

[0021] (ii) Improved expression level of recombinant protein: Flow cytometry showed that at a transfection dose of 4 μg, the expression levels of HA-mod1, mod2, and mod4 were significantly higher than those of wild type (WT-HA was hardly expressed); after transfection with 6 μg WT-HA, only weak positive expression was observed; the mutant of the present invention can achieve high-efficiency expression at low doses (2 μg), reducing production costs and making it suitable for industrial production.

[0022] (III) Broadening the cross-protection spectrum: ELISA showed that the mutant produced high-titer antibodies against both homologous H5N1 strains (such as A / chicken / VietNam / NCVD-016 / 2008, A / barnswallow / Hong Kong / D10-1161 / 2010) and heterologous subtypes (H1N1PR8, B / Austria / 1359417 / 2021 (Victoria), B / PHUKET / 3073 / 2013 (Yamagata)): HA-mod1 / mod2 / mod3: broad-spectrum cross-recognition of H5 / H1 subtypes; HA-mod4: recognition of H1 subtype and influenza B strains.

[0023] (iv) Enhanced immunogenic hemagglutination inhibition antibody levels: Antibody binding affinity increased: At a dose of 10 μg / animal, the titers of HA-mod1 and mod2 were 7.5 times higher than those of wild type; Hemagglutination inhibition activity was enhanced: Against strain A / Jiangsu / NJ210 / 2023, the titers of hemagglutination inhibition antibodies of HA-mod1 and mod2 were both >40, which were significantly higher than those of wild type.

[0024] (v) Improved immune response balance: Antibody subtype analysis showed that the level of IgG2a (Th1 type) induced by HA-mod2 was significantly higher than that of wild type, and the level of IgG2b of HA-mod1 / mod2 increased, indicating that the Th1 / Th2 immune balance was tilted towards a protective response. Attached Figure Description

[0025] Figure 1In a preferred embodiment of the present invention, non-reducing Western blotting was used to analyze the binding of wild-type HA (WT-HA) and the mutants of the present invention (HA-mod1 to mod4) to the broad-spectrum neutralizing antibody 86001-RM01 at the protein level and their trimer stability. The results showed that all mutants exhibited clear trimer bands under non-denaturing conditions, while the WT-HA band was weak, indicating that the conformational stability of the mutants of the present invention was significantly improved.

[0026] Figure 2 In a preferred embodiment of the present invention, the representative flow cytometry profiles of HA protein expression levels on the cell surface of 293F cells transfected with 4 μg of mRNA-LNP 24 hours later are presented. AE represent the detection results for WT-HA, HA-mod1, HA-mod2, HA-mod3, and HA-mod4, respectively. WT-HA (A) showed almost no expression, while all mutants (B~E) exhibited high expression, with HA-mod1, HA-mod2, and HA-mod4 (B, C, E) showing the highest expression levels.

[0027] Figure 3 In a preferred embodiment of the present invention, flow cytometry was used to analyze the expression dynamics of HA protein at different transfection time points (24 h, 48 h, and 72 h). A shows the change curve of the average expression level (%) of each sample over time at a transfection dose of 2 μg; B shows the expression dynamics at a transfection dose of 4 μg. The results showed that each mutant maintained high expression at multiple time points, significantly better than WT-HA.

[0028] Figure 4 In a preferred embodiment of the present invention, flow cytometry was used to analyze the effect of different mRNA-LNP transfection doses (2 μg and 4 μg) on ​​HA protein expression levels. The expression levels of each mutant increased with increasing dose, further verifying its highly efficient and soluble expression characteristics.

[0029] Figure 5 In a preferred embodiment of the present invention, flow cytometry was used to detect the expression of WT-HA mRNA-LNP transfected into 293F cells at a dose of 6 μg. After permeabilization treatment with Triton X-100, the total protein of WT-HA (intracellular + cell surface) showed weak positive expression (approximately 48%), indicating that its expression was limited to the intracellular space and at a low level.

[0030] Figure 6In a preferred embodiment of the present invention, the titers of specific IgG antibodies against the HA protein of the homologous H5N1 virus (A / Texas / 37 / 2024) were detected by ELISA in the serum of mice immunized with a 10 μg dose of mRNA-LNP on days 7, 21, and 35 after the second immunization. The antibody titers of the mutant groups (especially HA-mod1 and HA-mod2) were significantly higher than those of the WT-HA group.

[0031] Figure 7 In a preferred embodiment of the present invention, ELISA was used to detect the cross-reactive antibody titers of serum samples taken on day 35 after secondary immunization against the HA protein of various homologous and heterologous influenza virus strains, including the H5N1 homologous strain (A / barnswallow / Hong Kong / D10-1161 / 2010, A / chicken / VietNam / NCVD-016 / 2008), the heterologous H1N1 strain (A / Puerto Rico / 8 / 1934), and influenza B strains (B / Austria / 1359417 / 2021, B / PHUKET / 3073 / 2013). The results showed that the mutants of the present invention have broad-spectrum cross-reactivity.

[0032] Figure 8 In a preferred embodiment of the present invention, the hemagglutination inhibition (HI) assay was used to detect the inhibitory antibody titers against the epidemic strain A / Jiangsu / NJ210 / 2023 (H5N1) in the serum of mice immunized with a 10 μg dose of mRNA-LNP on days 7, 21, and 35 after the second immunization. HA-mod2 induces a high functional antibody response in an early stage (D7).

[0033] Figure 9 In a preferred embodiment of the present invention, the relative levels of key antibody subtypes in serum on day 35 of the second immunization were detected by ELISA. A represents a comparison of IgG2a (Th1 type) levels; B represents a comparison of IgG3 (non-protective antibody) levels. The results show that, compared with WT-HA, HA-mod2 significantly increased IgG2a levels, while all mutants significantly decreased IgG3 levels, indicating that the present invention can guide the immune response towards the more protective Th1 type. Detailed Implementation

[0034] This invention addresses the problems of weak cross-immune response to circulating strains, insufficient stability of wild-type HA protein, and low immunogenicity of existing H5N1 vaccines by constructing a stable soluble H5 protein mutant through sequence modification strategy.

[0035] This invention constructs H5 protein mutants through a multi-dimensional modification strategy involving segment substitution, key site mutation, and trimerization. The specific technical solution is as follows: This invention provides a mutant of influenza A virus H5 protein, the mutant being modified based on wild-type H5N1 hemagglutinin protein (WT-HA, SEQ ID NO:1), comprising: (1) Replace the 332G-380A region (SEQ ID NO:2) in the amino acid sequence with 332S-337T (SPGCAT, SEQ ID NO:3); (2) Introduce point mutations T386C, L418P and H456P.

[0036] Preferably, the amino acid sequence of the influenza A virus H5 protein mutant is shown in SEQ ID NO:4.

[0037] In some embodiments, the mutant further comprises mutations selected from at least one of the following groups: (1) S219C and S233C; (2) N222C and K228C; (3) R224C and P227C; (4) T231C and H256C.

[0038] In some embodiments, the amino acid sequence of the mutant is shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0039] The present invention also provides a nucleic acid molecule encoding the H5 protein mutant, the nucleic acid molecule comprising: Polynucleotides encoding the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8; or Polynucleotides that are complementary to the above polynucleotides and can hybridize under strict conditions.

[0040] The present invention also provides an expression vector comprising the above-mentioned nucleic acid molecule, wherein the vector is a pFastBac1 plasmid or an mRNA expression vector.

[0041] This invention also provides a method for preparing an H5 protein mutant of influenza A virus, comprising the following steps: (1) The above-mentioned nucleic acid molecules were cloned into an expression vector to construct a recombinant vector; (2) The recombinant vector was transfected into eukaryotic cells at a cell density of 3 × 10⁻⁶ cells / year. 6 / mL, incubation temperature 36-38℃, CO2 concentration 5-8%, rotation speed 115-125 rpm; (3) 16 hours after transfection, add 5% (v / v) 293F Hi-exp feed and continue culturing for 48 hours; (4) Collect cells, lyse them using Pierce IP lysis buffer, centrifuge at 4°C and 13000×g for 10 minutes, and take the supernatant to purify the target protein.

[0042] Preferably, the eukaryotic cells are 293F cells, the transfection dose is 4 μg, and the cell viability is ≥95%.

[0043] The present invention also provides a vaccine composition comprising the H5 protein mutant, or the H5 nucleic acid molecule, or the expression vector, adjuvant, or pharmaceutically acceptable vector.

[0044] Preferably, the nucleic acid molecule is mRNA, and an mRNA-LNP complex is formed by encapsulation with lipid nanoparticles (LNPs). The LNPs contain cationic lipids, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 50:10:38.5:1.5.

[0045] The present invention also provides the use of the H5 protein mutant, the nucleic acid molecule, the expression vector, or the vaccine composition in the preparation of a drug for preventing infection with influenza A virus H5N1.

[0046] Furthermore, the drug can induce neutralizing antibodies against H5N1 virus 2.3.4.4b branch and has cross-immune activity against heterologous subtype virus strains (including A / Puerto Rico / 8 / 1934 and A / Darwin / 6 / 2021).

[0047] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0048] The 293F cells used in the following examples were purchased from ATCC; the broad-spectrum monoclonal antibody 86001-RM01 was purchased from Sino Biological; the HA antigens of influenza A virus strains A / barnswallow / Hong Kong / D10-1161 / 2010, A / chicken / VietNam / NCVD-016 / 2008, A / Wiscoonsin / 67 / 2022, A / Puerto Rico / 8 / 1934, A / Darwin / 6 / 2021 and influenza B virus strains B / Austria / 1359417 / 2021 and B / PHUKET / 3073 / 2013 were all purchased from Sino Biological; the experimental animals were 6-8 week old SPF-grade female BALB / c mice.

[0049] Example 1: Design of H5 protein mutants This embodiment provides a structurally stable H5 influenza A virus protein mutant. Using wild-type H5N1 hemagglutinin WT-HA (A / Texas / 37 / 2024(H5N1) strain, GenBank: PP577943.1, its amino acid sequence is shown in SEQ ID NO:1) as a template, a protein mutant stable in the prefusion conformation was constructed through a reasonable antigen design strategy.

[0050] The design strategies mainly include: introducing intramolecular disulfide bonds into the HA protein, substituting proline to enhance helical rigidity, and replacing or truncating specific functional segments, thereby locking the protein conformation in the pre-fusion state with optimal immunogenicity.

[0051] Based on the above strategy, through systematic screening and structural simulation evaluation of key sites, a preferred H5 protein design template was finally obtained, named HA-mod0. This template is based on the sequence shown in SEQ ID NO:1 and contains the following modifications: (1) Replace the segment from glycine at position 332 to alanine at position 380 (SEQ ID NO:2) with the sequence SPGCAT (SEQ ID NO:3); (2) Introduce point mutations T386C, L418P and H456P.

[0052] The complete amino acid sequence of the HA-mod0 template is shown in SEQ ID NO:4.

[0053] To further enhance structural stability and explore its structure-activity relationship, four derived mutants were constructed based on the aforementioned HA-mod0 template (SEQ ID NO:4), each introducing different disulfide bond pairs: HA-mod1 (SEQ ID NO:5): Contains S219C and S233C mutations; HA-mod2 (SEQ ID NO:6): Contains N222C and K228C mutations; HA-mod3 (SEQ ID NO:7): Contains R224C and P227C mutations; HA-mod4 (SEQ ID NO:8): Contains T231C and H256C mutations.

[0054] This invention is based on structural stability design as the initial goal, using the wild-type influenza virus H5N1 hemagglutinin WT-HA (A / Texas / 37 / 2024(H5N1) strain; GenBank: PP577943.1; SEQ ID NO:1) as a template, and applying artificial intelligence (AI)-assisted tools to accelerate antigen design. The main design strategies include, but are not limited to: introducing disulfide bonds into the key H5N1 influenza virus antigen HA; proline substitution mutations; and sequence truncation, attempting to lock it into the pre-fusion conformation with the strongest immunogenicity. By scanning all possible single-point mutations, mutations that can generally improve protein thermostability without affecting its key antigenic epitopes are identified. Then, Alphafold is used to predict and score all designed antigens, ultimately selecting one optimal H5 design template.

[0055] Using WT-HA (SEQ ID NO:1) as a reference sequence, the H5 design template includes: (1) Replace the 332G-380A segment (SEQ ID NO:2) with 332S-337T (SPGCAT, SEQ ID NO:3). (2) Introduce point mutations T386C, L418P and H456P.

[0056] The amino acid sequence of the H5 design template is SEQ ID NO:4.

[0057] To verify the reliability of the template experimentally, different additional mutations were introduced based on the H5 design template (SEQ ID NO:4). These mutants included: HA-mod1 (SEQ ID NO:5): S219C, S233C; HA-mod2 (SEQ ID NO:6): N222C, K228C; HA-mod3 (SEQ ID NO:7): R224C, P227C; HA-mod4 (SEQ ID NO:8): T231C, H256C, Based on the above strategy, this invention constructed a series of mutants, the sequence information of which is shown in Table 1: Table 1 H5 protein mutant sequence

[0058] Example 2: Preparation and in vitro expression verification of mRNA-LNP This embodiment provides a method for preparing mRNA-LNP encoding the H5 trimer protein mutant of the present invention, and verifies its protein expression ability and trimer structure stability through in vitro experiments.

[0059] 2.1 Preparation of mRNA-LNP Following the method described by Andrew J Bett et al., the mRNA-LNPs of each mutant (HA-mod0 to mod4) and wild-type control (WT-HA) described in Example 1 were prepared using the following steps: Plasmid linearization and mRNA transcription: Plasmids containing the coding sequences of each target protein (corresponding to SEQ ID NO: 4-8 and SEQ ID NO: 1) were linearized using the BspQI restriction endonuclease. The linearized plasmids were transcribed using the T7 in vitro transcription kit to obtain 5' capped mRNA transcripts.

[0060] mRNA purification: After transcription, the DNA template was digested with DNase I, and the mRNA was purified by lithium chloride precipitation. The purified mRNA was dissolved in acidic sodium citrate buffer, and its concentration and purity were determined.

[0061] LNP encapsulation: Cationic lipids, DSPC, cholesterol, and DMG-PEG2000 were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to form a lipid solution. The aqueous mRNA solution was then mixed with the lipid ethanol solution using a microfluidic device at a flow rate ratio of 3:1 to achieve mRNA encapsulation.

[0062] Dialysis and finished product: The encapsulated crude product was dialyzed in PBS buffer (pH 7.4) to remove residual ethanol and replace the buffer system, finally obtaining the desired mRNA-LNP, which was stored at 2-8℃ for later use.

[0063] 2.2 Cell transfection and protein expression analysis To assess the function of mRNA-LNP, it was transfected into human embryonic kidney 293F suspension cells for analysis.

[0064] Cell transfection and culture: The five mutants and wild-type H5 mRNA-LNP were transfected into 293F cells. Transfection conditions were as follows: transfection dose 4 μg, cell density 3 × 10⁶ cells / year. 6 / mL, cell viability ≥95%, culture temperature 36-38℃, humidity 80%, CO2 concentration 5-8%, rotation speed 115-125 rpm (amplitude 50 mm). 16 hours after transfection, add 5% (v / v) 293F Hi-exp feed to the shake flask and continue culturing for 48 hours to collect cells.

[0065] Western blot verification of trimer stability: The cell suspension was centrifuged at 1000×g for 5 minutes, the supernatant was discarded, and the cells were washed once with pre-chilled PBS. After recentrifugation, 500 μL of ice-cold Pierce IP lysis buffer (10:1 v / w) was added to every 50 mg of wet cell particles, and the cells were incubated on ice for 5 minutes. Subsequently, the cells were centrifuged at 13000×g for 10 minutes at 4°C, and the supernatant was used for Western blot analysis. H5 wild-type and mutant proteins were subjected to 10% polyacrylamide SDS-PAGE electrophoresis under non-reducing conditions: Samples were mixed with non-denaturing loading buffer and loaded. The initial electrophoresis voltage was 100 V (15 minutes), adjusted to 200 V (30 minutes) after entering the separating gel, and stopped when bromophenol blue migrated to the bottom of the gel. After electrophoresis, the gel was rinsed with pure water and transferred to a PVDF membrane (using an eBlot L1 rapid wet transfer apparatus, 7 minutes). After transfer, the membrane was rinsed in TBS-T buffer for 8 minutes, repeated 3 times; then blocked overnight at 4°C with blocking buffer; after rinsing again, it was incubated with primary antibody (86001-RM01, purchased from SinoBiologico (Beijing, China)) at room temperature for 1 hour, rinsed, and then incubated with the corresponding secondary antibody (Goat Anti-Rabbit IgG Secondary Antibody (HRP) Catalog Number: SSA004, purchased from SinoBiologico (Beijing, China)) at room temperature for 1 hour; finally, chemiluminescence imaging was performed using freshly prepared West Femto ECL ultrasensitive chemiluminescent solution (equal volumes of solution A and solution B), and the signal was acquired using a gel imaging system. Results are as follows: Figure 1 As shown, all mutants of this invention (HA-mod1 to mod4) exhibit clear and strong trimer bands at the expected molecular weight positions, indicating that they can successfully form and stably exist in the prefused trimer conformation. In contrast, the trimer band of wild-type WT-HA is weak, forming monomers or dimers. This result demonstrates that the mutant design of this invention effectively solves the technical problem of conformational instability of wild-type HA protein.

[0066] Flow cytometry Quantitative Analysis of Expression Levels: To quantitatively assess protein expression levels, flow cytometry was used to detect transfected cells. 293F cells were transfected with 2 μg and 4 μg of H5 wild-type and mutant mRNA-LNP, respectively. Cells were collected for analysis at 24 h, 48 h, and 72 h post-transfection. The specific steps are as follows: 1 mL of cell suspension (density 1×10⁻⁶) was taken... 6 Cells were collected by centrifugation at 350×g for 5 minutes at 4°C. The cells were then resuspended and washed with 200 μL of pre-chilled 2% BSA / PBS. The cells were then resuspended in blocking buffer (2% BSA / PBS) and blocked at 4°C for 20 minutes. Primary antibody working solution (diluted with 2% BSA / PBS) was added and incubated at 4°C in the dark for 30 minutes. After washing once with washing buffer, secondary antibody working solution was added and incubated in the dark for 30 minutes. The cells were washed twice, resuspended in washing buffer, and immediately analyzed.

[0067] The results are as follows Figures 2 to 5 As shown: at a transfection dose of 4 μg, all mutants (HA-mod1 to mod4) exhibited high levels of protein expression, while wild-type WT-HA showed extremely low expression levels under the same conditions, almost undetectable. Figure 2 Expression level comparisons showed that the expression levels of HA-mod1, HA-mod2, and HA-mod4 were higher than that of HA-mod3, but all mutants were significantly superior to the wild type. Figure 3 Dose-response analysis showed that the expression levels of each mutant increased with increasing transfection dose (2 μg to 4 μg). Figure 4 To further investigate the expression characteristics of WT-HA, a high-dose transfection of 6 μg followed by Triton X-100 permeabilization was performed. Flow cytometry analysis showed that WT-HA exhibited only weak positive expression in approximately 48% of samples. Figure 5 This indicates that it is expressed in small amounts intracellularly only under high pressure conditions. In summary, the mutant constructed in this invention significantly improves the soluble expression efficiency of H5 protein in eukaryotic cells.

[0068] Example 3: Evaluation of immunogenicity and cross-protection of mutants This embodiment systematically evaluated the strength, breadth, and functionality of the immune response induced by the mRNA-LNP of this invention through mouse immunization experiments, demonstrating its significant advantages over wild-type antigens.

[0069] 3.1 Experimental Design Experimental animals and grouping: 6-8 week old SPF-grade female BALB / c mice were randomly divided into 6 groups, with 6 mice in each group. The groups were as follows: PBS negative control group, wild-type mRNA-LNP group (WT-HA), and the four mutant mRNA-LNP groups of this invention (HA-mod1 to mod4).

[0070] Immunization program: Mice were immunized twice, on day 0 and day 21, via intramuscular injection in the hind limbs. The immunization dose of all mRNA-LNPs was 10 μg / mouse.

[0071] Sample collection: Blood was collected on day 7 (D7), day 21 (D21), and day 35 (D35) after the second immunization. Serum was separated and stored at -20℃ for subsequent testing.

[0072] 3.2 Specific humoral immune response assessment (ELISA) The titer of specific IgG antibodies in mouse serum was detected using an enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows: Antigen coating: Dilute the HA antigen to 2 μg / mL with coating buffer, add 100 μL / well to a 96-well microplate, seal with sealing film, and incubate overnight at 4°C.

[0073] Washing and blocking: Discard the coating solution and pat the plate surface dry on absorbent paper; add 200 μL of washing solution to each well, gently shake for 30 seconds and discard, repeat washing 3 times. Then add 200 μL of blocking solution to each well and incubate at 37°C for 1 hour.

[0074] Serum incubation: The serum to be tested was serially diluted according to the predetermined ratio. The negative control serum was diluted 1:100, and an equal volume of sample diluent was added to the blank control wells. 100 μL of the diluted sample was added to each well, and the sample was incubated at 37°C for 1 hour.

[0075] Secondary antibody incubation: After incubation, discard the liquid in the wells and wash 5 times as described above. Dilute Goat-anti-mouse IgG-HRP at a ratio of 1:4000, add 100 μL to each well, and incubate at 37°C in the dark for 1 hour.

[0076] Color development and detection: Discard the liquid in the wells, wash 5 times, and pat the plate dry on absorbent paper. Add 100 μL of chromogenic substrate solution to each well and incubate at room temperature for 10 minutes in the dark. Then add 50 μL of stop solution to each well to terminate the reaction. Mix well on a micro-shaker and immediately measure the absorbance at 450 nm using a microplate reader. The reference wavelength is set to 630 nm.

[0077] The results are as follows Figure 6As shown, throughout the experimental period, the specific IgG antibody titers induced by the mutant groups of this invention (especially HA-mod1 and HA-mod2) were significantly higher than those of the WT-HA group. At days 7 and 21 post-secondary immunization, the average antibody titers of the HA-mod1 and HA-mod2 groups were 7.5 times higher than those of the WT-HA group. Even at day 35 post-secondary immunization, although antibody levels decreased in all groups, the antibody titer of the HA-mod1 group remained significantly higher than that of the WT-HA group. This indicates that the mutants of this invention can induce a stronger and more durable specific humoral immune response.

[0078] 3.3 Assessment of the breadth of cross-immunity When assessing the breadth of vaccine-induced cross-immunity, ELISA testing should be performed according to the following detailed method: Recombinant HA proteins from the following sources were used as coating antigens: homologous H5N1 strains (A / barnswallow / Hong Kong / D10-1161 / 2010, A / chicken / VietNam / NCVD-016 / 2008), heterologous H1N1 strains (A / Puerto Rico / 8 / 1934, PR8), and influenza B strains (B / Austria / 1359417 / 2021, BV; B / PHUKET / 3073 / 2013, BY). All antigens were uniformly diluted to a working concentration of 2 μg / mL with coating buffer, and 100 μL / well was added to a 96-well ELISA plate and incubated overnight at 4°C. Washing, blocking, serum sample incubation, secondary antibody incubation, color development, and detection procedures were strictly followed.

[0079] The results are as follows Figure 7 As shown in Table 2, the different mutants of this invention exhibit unique and excellent cross-reactivity profiles. HA-mod1 and HA-mod2: produced high-titer cross-antibodies against all tested H5N1 homologous strains and heterologous H1N1 (PR8) strains, demonstrating broad-spectrum cross-protective potential against influenza A. HA-mod4: responded well to homologous H5N1 strains and uniquely induced high-titer cross-antibodies against influenza B virus, showing cross-protective potential across influenza types, a characteristic not possessed by wild-type and other mutants.

[0080] Table 2. Cross-antibody reaction profiles induced by each mutant (secondary immunization D35)

[0081] 3.4 Assessment of functional antibody response (hemagglutination inhibition assay) To assess the functionality of the induced antibodies, the hemagglutination inhibition (HI) assay was used to detect their ability to block viral invasion.

[0082] The hemagglutination inhibition assay was performed according to standard operating procedures. This assay was used to assess the level of functional antibodies against the prevalent strain A / Jiangsu / NJ210 / 2023 (H5N1) in immune serum. First, serum pretreatment was performed: 100 μL of the serum sample to be tested was added to 400 μL of receptor-degrading enzyme solution, thoroughly mixed, and incubated at 37°C for 12–24 hours; an international standard serum was also used as a positive control. The treated serum was cooled to room temperature and incubated in a 56°C water bath for 50 minutes to inactivate residual enzyme activity. After cooling again, 50 μL of packed erythrocytes was added, and the mixture was incubated at 2–8°C for 2 hours. The supernatant was then collected by centrifugation.

[0083] For the preparation of the antigen working solution, based on the hemagglutination titer test results, the antigen was diluted to the working concentration (hemagglutination titer of 1:4) with 0.9% sodium chloride injection. A U-shaped hemagglutination plate was used for testing. Except for the first well, 25 μL of physiological saline was added to each of the remaining wells. 25 μL of the antigen working solution was added to the first and second wells, and serially diluted from the second well to the fourth well. The fifth well was used as a negative control. 25 μL of 1% chicken erythrocyte suspension was added to each well, and the mixture was allowed to stand at room temperature for 30 minutes before observing the agglutination results.

[0084] For antibody testing, take a clean U-shaped blood coagulation plate and add 25 μL of physiological saline to each well except the first well. Add 25 μL of treated serum supernatant to the first, second, and last wells respectively, and serially dilute from the second well to the second to last well. Add 25 μL of antigen working solution to each well except the serum control well. Mix well with a vortex mixer and let stand at room temperature for 45 minutes. Add 25 μL of 1% chicken red blood cell suspension to each well, mix well, and let stand at room temperature for 30 minutes to observe the results.

[0085] Results were determined based on the serum control wells. A complete "teardrop" flow pattern was considered negative (-), indicating complete inhibition; complete agglutination of red blood cells without a teardrop pattern was considered positive (+); and levels in between indicated partial inhibition (±). The HI titer was calculated using the highest dilution of serum showing complete or partial inhibition. Remaining antigen was back-titrated to verify the accuracy of the working concentration.

[0086] The hemagglutination inhibition (HI) test is a key indicator for assessing the functionality of vaccine-induced antibodies; its titer directly reflects the antibody's ability to block viral invasion of host cells. Figure 8As shown, on day 7 after the second immunization, the geometric mean titer (GMT) of HI antibodies in the HA-mod2 group was significantly higher than that in the WT-HA group. Although the HI titers in all groups decreased at subsequent time points, the mutants of this invention (represented by HA-mod2) demonstrated a clear advantage in inducing early and efficient functional antibody responses. According to the influenza vaccine evaluation criteria, an HI titer ≥40 is considered to be associated with protective efficacy (Control & Prevention, 2024), and the mutants of this invention reached this standard early on. The above results confirm that the H5 trimer protein mutants obtained through the structural modification strategy of this invention are significantly superior to wild-type antigens in both the strength and functionality of induced antibodies.

[0087] 3.5 Antibody Subtype Analysis To assess the types of immune responses induced by the vaccine, enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of various IgG subtypes and IgM antibodies in mouse serum on day 35 after secondary immunization.

[0088] The method is as follows: Anti-mouse IgG1, IgG2a, IgG2b, IgG3, and IgM specific antibodies were diluted 1:200 with PBS and coated with 100 μL / well of a 96-well plate, incubated overnight at 4°C. After incubation, the coating solution was discarded, and each well was washed with 300 μL of wash buffer, repeated three times. After washing, 300 μL of blocking buffer was added to each well, and the plate was blocked at room temperature for 1 hour. After washing again, 100 μL of the test sample (including appropriately diluted serum sample, positive control, and negative control) was added to each well, and the plate was incubated at room temperature for 1 hour. After washing, 100 μL / well of HRP-labeled rabbit anti-mouse IgG secondary antibody diluted 1:5000 was added, and the plate was incubated at room temperature in the dark for 1 hour. After washing, 100 μL of substrate solution was added to each well, and the plate was developed at room temperature in the dark for 10 minutes. Finally, 50 μL of stop solution was added to terminate the reaction, and the absorbance was immediately measured at 450 nm using a microplate reader, with a reference wavelength of 630 nm.

[0089] The results are as follows Figure 9 As shown: Compared with wild-type WT-HA, HA-mod2 induced a significant increase in IgG2a levels; HA-mod1 and HA-mod2 induced significant increases in IgG2b levels; and the level of non-protective antibody IgG3 in the WT-HA group was significantly higher than that in each mutant group. There were no significant differences in IgG1 and IgM levels among the groups. These results indicate that the H5 trimer protein mutants constructed in this invention, especially HA-mod1 and HA-mod2, can guide the immune response towards Th1-type immunity represented by IgG2a and IgG2b, demonstrating that this invention has a significant effect on improving antigen-specific Th1 / Th2 immune balance.

[0090] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0091] References: 1. Control, CC f. D., & Prevention. (2024). Technical Guidelines for Influenza Vaccination in China (2023-2024). Chinese Journal of Virology , 14 (01), 1-19. https: / / doi.org / 10.16505 / j.2095-0136.2024.1001.

Claims

1. A mutant of the H5 protein of influenza A virus, characterized in that, The mutant was obtained by replacing amino acid residues 332-380 in the hemagglutinin protein of wild-type influenza A virus H5N1 with SPGCAT and introducing point mutations T386C, L418P and H456P.

2. The mutant according to claim 1, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO:

4.

3. The mutant according to claim 1 or 2, characterized in that, The mutant also includes mutations selected from at least one of the following groups: (1) S219C and S233C; (2) N222C and K228C; (3) R224C and P227C; (4) T231C and H256C.

4. The mutant according to claim 3, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO:5, 6, 7 or 8.

5. A nucleic acid molecule encoding the mutant of any one of claims 1-4 or biological material containing said nucleic acid molecule; The biological materials are expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

6. Any of the following applications of the mutant according to any one of claims 1-4 or the nucleic acid molecule or biological material according to claim 5: (1) To prepare drugs for the prevention and / or treatment of influenza A virus H5N1 infection; (2) Preparation of antibodies against influenza A virus H5N1; (3) Prepare diagnostic reagents for influenza A virus H5N1.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the mutant of any one of claims 1-4 or the nucleic acid molecule or biological material of claim 5.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is a vaccine; The vaccines include recombinant protein vaccines, nucleic acid vaccines, vector vaccines, or virus-like particle vaccines.

9. The pharmaceutical composition according to claim 8, characterized in that, The nucleic acid vaccine is an mRNA-LNP vaccine, wherein the mRNA encodes the mutant described in any one of claims 1-4; and / or, LNP contains cationic lipids, DSPC, cholesterol, and DMG-PEG2000.

10. The pharmaceutical composition according to claim 9, characterized in that, The molar ratio of cationic lipids, DSPC, cholesterol and DMG-PEG2000 in the mRNA-LNP vaccine is 50:(8-12):(35-40):(2-3).

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