Influenza virus neuraminidase mutant, nucleic acid molecule encoding influenza virus neuraminidase mutant, vaccine composition comprising influenza virus neuraminidase mutant, and use of influenza virus neuraminidase mutant for preparing influenza virus vaccine composition

Abstract

This invention provides an influenza virus neuraminidase mutant, a nucleic acid molecule encoding the influenza virus neuraminidase mutant, a vaccine composition containing the influenza virus neuraminidase mutant, and the use of the influenza virus neuraminidase mutant in the preparation of an influenza virus vaccine composition. The vaccine composition of this invention achieves the effect of preventing influenza virus infection through various efficacy experiments.

Description

Influenza virus neuraminidase mutant, nucleic acid molecule encoding influenza virus neuraminidase mutant, vaccine composition containing influenza virus neuraminidase mutant, and use of influenza virus neuraminidase mutant in preparing influenza virus vaccine composition The present invention relates to an influenza virus neuraminidase mutant, a nucleic acid molecule encoding the influenza virus neuraminidase mutant, a vaccine composition comprising the influenza virus neuraminidase mutant, and the use of the influenza virus neuraminidase mutant in preparing an influenza virus vaccine composition. Influenza pandemics cause significant morbidity and mortality, especially among children and the elderly. Influenza viruses are prone to antigenic variation and can cross species, with influenza A being the most important. Due to the rapid mutation of viral strains, once a new influenza virus subtype emerges, the general population lacks immunity to it, making it prone to pandemics. Currently, the only viable preventive measure is vaccination. Marketed vaccines primarily consist of inactivated influenza viruses, attenuated influenza viruses, virus-like particles (VLPs), and recombinant subunit proteins. These vaccines primarily target hemagglutinin (HA) and neuraminidase (NA) to produce specific antibodies. However, there are numerous influenza virus serotypes, and existing vaccines offer poor cross-protection across serotypes, resulting in a lack of cross-protection against novel influenza viruses. Therefore, developing a broadly effective influenza vaccine is a key area of ​​focus. In order to solve the above problems, those skilled in the art are in urgent need of developing novel influenza virus neuraminidase mutants and vaccine compositions comprising influenza virus neuraminidase mutants to benefit the vast population in need. In view of this, the object of the present invention is to provide an influenza virus neuraminidase mutant having an N-glycosylation masked for influenza virus neuraminidase. In one embodiment of the present invention, the influenza virus neuraminidase mutant has a mutation at an amino acid residue position N1 of human influenza virus neuraminidase, wherein the amino acid residue position is selected from the group consisting of amino acid residue position 221, amino acid residue position 223, amino acid residue position 270, amino acid residue position 272, amino acid residue position 273, amino acid residue position 275, amino acid residue position 329, amino acid residue position 331, amino acid residue position 332, amino acid residue position 335, amino acid residue position 337, amino acid residue position 341, and amino acid residue position 343. In one embodiment of the present invention, the influenza virus neuraminidase mutant has a mutation at an amino acid residue at N2 of human influenza virus neuraminidase, wherein the amino acid residue is selected from the group consisting of amino acid residue 93, amino acid residue 245, amino acid residue 247, amino acid residue 267, amino acid residue 269, amino acid residue 331, amino acid residue 336, amino acid residue 338, amino acid residue 348, amino acid residue 368, amino acid residue 370, amino acid residue 401, amino acid residue 403, amino acid residue 463, and amino acid residue 465. In one embodiment of the present invention, the mutation is to substitute an amino acid residue with asparagine (N) or threonine (T). In one embodiment of the present invention, the 221st and 223rd amino acid residues of N1 of the human influenza virus neuraminidase are substituted with asparagine and monothreonine, respectively; the 270th and 272nd amino acid residues are substituted with asparagine and monothreonine, respectively; the 273rd and 275th amino acid residues are substituted with asparagine and monothreonine, respectively. 329 and 331, respectively, with asparagine and monothreonine substitutions; 332, with asparagine substitutions; 335, with asparagine and monothreonine substitutions; or 341, with asparagine and monothreonine substitutions. In one embodiment of the present invention, the 93rd amino acid residue of N2 of the human influenza virus neuraminidase has an asparagine substitution, the 245th amino acid residue and the 247th amino acid residue have an asparagine and a threonine substitution, respectively, the 267th amino acid residue and the 269th amino acid residue have an asparagine and a threonine substitution, respectively, the 331st amino acid residue has a threonine substitution, the 336th amino acid residue and the 338th amino acid residue have a threonine substitution, respectively. The amino acid residue positions 348 and 370 each have an asparagine and a monothreonine substitution, the amino acid residue position 368 and 370 each have an asparagine and a monothreonine substitution, the amino acid residue positions 401 and 403 each have an asparagine and a monothreonine substitution, or the amino acid residue positions 463 and 465 each have an asparagine and a monothreonine substitution. Another object of the present invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding the influenza virus neuraminidase mutant as described above. Another object of the present invention is to provide a use of the aforementioned vector for improving gene delivery efficiency. Another object of the present invention is to provide a vaccine composition comprising the influenza virus neuraminidase mutant as described above. In one embodiment of the present invention, the influenza virus neuraminidase mutant is expressed in a recombinant virus. In one embodiment of the present invention, the recombinant virus comprises the nucleic acid molecule described above. In one embodiment of the present invention, the recombinant virus is a recombinant adenovirus. Another object of the present invention is to provide a use of the influenza virus neuraminidase mutant as described above for preparing an influenza virus vaccine composition. In one embodiment of the present invention, the influenza virus vaccine composition elicits an immune response against multiple influenza virus variants in a subject. In one embodiment of the present invention, the influenza virus vaccine composition elicits high titers of antigen-specific antibodies and / or neuraminidase inhibitory IC 50 potency. In summary, the efficacy of the influenza virus vaccine composition of the present invention lies in its use of a hyperglycosylated influenza virus neuraminidase mutant, which masks non-essential antigenic determinants with sugars, thereby refocusing the antibody response of individual B cells to influenza virus neuraminidase without affecting the overall protein folding structure. The influenza virus neuraminidase mutant of the present invention can effectively induce an inhibitory effect against influenza virus H1N1, H5N1, H3N2, and H7N9 neuraminidase, thereby effectively enhancing an individual's ability to resist infection by different influenza virus variants. The following will further illustrate the embodiments of the present invention. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone skilled in the art may make slight changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the appended patent applications. definition The values ​​used in this article are approximate, and all experimental data are expressed in 20%, preferably within the range 10% range, the best is within the range of 5%. Unless otherwise specified herein, the terms "a", "an", "the" and similar terms used in this specification (especially in the scope of the following patent applications) should be understood to include both singular and plural forms. In this paper, when analyzing the data of anti-influenza virus neuraminidase IgG antibody titers detected by ELISA, statistical tests for multiple comparisons were performed on all groups (except the control group using PBS solution). Experimental data were analyzed with GraphPad Prism v6.01 using the nonparametric Kruskal-Wallis test and corrected with Dunn's multiple comparisons test. Statistical significance is indicated as follows: *p < 0.05; **p < 0.01; and ***p < 0.001. When analyzing the experimental data of neuraminidase antibody titers, a curve of the percentage of inhibition of viral infection was fitted based on the equation of nonlinear regression log(inhibitor) and the slope of the standardized response-variable, and the IC was obtained from the fitted curve. 50 All experiments were performed at least three times and the data are presented as average values. Standard deviation is expressed. As used herein, the term "N-glycosylation" refers to a sugar chain covalently linked to asparagine in a protein via an N-glycosidic bond, comprising at least ten different monosaccharide units. More specifically, the sugar chain is linked to asparagine (N) in an amino acid residue, such as asparagine (N)-any amino acid (X)-threonine (T), represented by NXT. N-glycosylation has varying molecular weights and structures depending on the monosaccharide composition. Herein, unless otherwise specified, the term "hyperglycated" means that in addition to the "natural sugar-masked" amino acid residues on the wild-type protein, there are additional "mutant sugar-masked" amino acid residues. Herein, unless otherwise specified, the term "mutant" is equivalent to the term "variant". Herein, N221 / I223T, N270 / P272T, N273 / H275T, N329 / K331T, T332N, C335N / P337T, and N341 / A343T represent specific amino acid residues of the neuraminidase of the H1N1 influenza virus substituted with asparagine and / or threonine to represent the influenza virus spike protein mutants of the present invention. K93N, S245N / S247T, P267N / S269T, N329 / R331T, Y336N / R338T, N346 / G348T, E368N / S370T, D401N / R403, and D463N / N465T represent substitutions of specific amino acid residues of the neuraminidase of the H3N2 influenza virus with asparagine and / or threonine to represent the influenza virus spike protein mutants of the present invention. In this article, Ad-N1-WT, Ad-N1-N221 / I223T, Ad-N1-N270 / P272T, Ad-N1-N273 / H275T, Ad-N1-N329 / K331T, Ad-N1-T332N, Ad-N1-C335N / P337T or Ad-N1-N341 / A343T; Ad-N2-K93N, Ad-N2-S245N / S247T, Ad-N2-P267N / S269T Ad-N2-N329 / R331T, Ad-N2-Y336N / R338T, Ad-N2-N346 / G348T, Ad-N2-E368N / S370T, Ad-N2-D401N / R403T, and Ad-N2-D463N / N465T represent recombinant adenoviruses expressing different mutants of the influenza virus neuraminidase protein of the present invention. According to the present invention, the operating procedures and parameter conditions for gene cloning are within the professional knowledge and routine skills of those skilled in the art. According to the present invention, the operating procedures and parameter conditions for site-directed mutagenesis are within the professional competence and routine skills of those skilled in the art. According to the present invention, the operating procedures and parameter conditions for adding N-linked glycosylation to amino acid residues of proteins are within the professional knowledge and routine technical scope of those skilled in the art. According to the present invention, the operating procedures and parameter conditions for expressing antigens using adenovirus are within the professional knowledge and routine technical scope of those skilled in the art, and "adenovirus vector" herein refers to recombinant adenoviruses expressing different influenza virus spike protein mutants of the present invention. The present invention is further illustrated by the following examples. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. The scope of the present invention is as set forth in the appended claims. Materials and methods Experimental cells and culture methods In the examples of the present invention, human embryonic kidney cell line 293A (HEK293A) and human embryonic kidney cell line 293T (HEK293T) were used for cell experiments. HEK293A cells and HEK293T cells were obtained from the Bioresource Collection and Research Center (BCRC) and cultured in Dulbecco's modified Eagle medium (DMEM) (Thermo Scientific) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 units / mL penicillin / streptomycin (P / S). o C. Contains 5% CO 2 incubator for cultivation. Preparation of adenoviral vectors In the examples of the present invention, adenovirus expressing influenza virus neuraminidase or a neuraminidase mutant with a sugar masking mutation was used as a vector for immunizing experimental animals. The genes encoding the influenza virus neuraminidase or neuraminidase mutant were first cloned into the pENTR1A vector (Invitrogen). These genes were then cloned into the adenoviral plasmid pAd / CMV / V5-DEST (Invitrogen) using LR Clonase™ II Enzyme Mix (Invitrogen) to produce adenoviral plasmids encoding the influenza virus neuraminidase or neuraminidase mutant. To obtain adenoviral vectors expressing influenza virus neuraminidase or neuraminidase mutants, the adenoviral plasmids were cut with Pac I restriction enzyme to expose the inverted terminal repeats (ITRs). The two adenoviral plasmids were then transfected into 293A cells using TurboFect transfection reagent (Fermentas). 10 to 15 days after transfection, when cytopathic effect (CPE) appeared, the transfected cells and their culture medium were collected. The cells were disrupted by three freeze-thaw cycles to release intracellular viral particles, and the cells were cultured for 4 hr. oThe supernatant of the cell lysate was collected by centrifugation at 3,000 rpm for 15 minutes at 40°C to obtain the adenoviral vector expressing influenza virus neuraminidase or neuraminidase mutant. In addition, in order to prepare adenoviral vectors with higher titers, a 30-kDa Amicon Ultra-15 centrifugal filter (Millipore) was used for concentration. The adenoviral vector stock solution can be stored at -80°C. o C. To further determine the titer of adenoviral vectors, HEK293A cells were cultured at 10 6 Cells were seeded into 6-well culture plates at a density of 10 cells / well and incubated at 37 o After incubation at 37 o Ten-fold serial dilutions of adenoviral vector stock were added to each well at 4°C for 24 hours. The culture medium containing the diluted adenoviral vector was then removed, and 3 mL / well of DMEM supplemented with 0.4% agar and 100 U / mL penicillin / streptomycin was added to the six culture plates to infect the cells. Seven to ten days after infection of HEK293A cells with adenoviral vectors, plaques were visually quantified and counted as plaque-forming units (PFU). Sodium dodecyl sulfate polyacrylamide colloid electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) Operation The SDS-PAGE procedure is briefly described as follows. First, the protein sample was mixed with reducing sample buffer (containing 50 mM Tris-HCl, pH 6.8; 100 mM dithiothreitol (DTT); 2% SDS; 0.1% bromophenol blue; and 10% glycerol) at a ratio of 3:1 and then incubated at 95 o C for 5 minutes. Meanwhile, an electrophoresis colloid was prepared containing a separating colloid (for example, a 12% separating colloid: 2.5 mL of 1 M Tris, pH 8.8; 3.3 mL of deionized water; 4 mL of a 30% acrylamide mix; 0.1 mL of 10% SDS; 0.1 mL of 10% ammonium persulfate (APS); and 0.01 mL of tetramethylethylenediamine (TEMED)) and a focusing colloid (for example, a 5% focusing colloid: 0.63 mL of 1 M Tris, pH 6.8; 3.4 mL of deionized water; 0.83 mL of a 30% acrylamide mix; 0.05 mL of 10% SDS; 0.05 mL of 10% APS; and 0.005 mL of TEMED). Protein electrophoresis was performed at 80 V for focus concentration and 140 V for separation. The duration of electrophoresis depended on the molecular weight of the protein being analyzed. The colloid was then stained with a Coomassie Brilliant Blue solution (containing 0.1% Coomassie R250, 10% acetic acid, and 50% methanol) for 1 hour and then destained with a destaining solution (containing 10% acetic acid and 50% methanol). Western blotting procedure The Western blot procedure is briefly described below. In a transfer tank, a protein sample separated by SDS-PAGE was transferred to a nitrocellulose membrane (NC membrane) at 135 V. The membrane containing the transferred proteins was then immersed in 20 mL of blocking solution and shaken for at least 1 hour to block nonspecific binding. The blocking solution consisted of 5% skim milk in tris-buffered saline with Tween-20 (TBST solution, containing 50 mM Tris, 150 mM NaCl, and 0.05% Tween-20). The NC membrane was then washed three times with TBST solution, and the primary antibody diluted at a specific multiple with TBST solution was added. oThe membrane was shaken at room temperature for approximately 16 hours. The next day, the membrane was washed three times with TBST. The membrane was then treated with a secondary antibody conjugated to horseradish peroxidase (HRP) diluted at a specific dilution in TBST at room temperature for 1 hour. The membrane was then washed three times with TBST. HRP-catalyzed enhanced chemiluminescence (Millipore) was added to the membrane for 1 minute to generate a luminescent signal, which was then developed onto X-ray film, such as medical blue-sensitive X-ray film (Fujifilm). Immunization methods for experimental mice In one embodiment of the present invention, 6-8 week old BALB / c female mice were used for vaccination experiments, wherein the BALB / c female mice were obtained from the National Laboratory Animal Center, National Research Institute of Japan. In the first set of immunization experiments, each dose contained 1×10 8 pfu of Ad-N1, Ad-N1-N221 / I223T, Ad-N1-N270 / P272T, Ad-N1-N273 / H275T, Ad-N1-N329 / K331T, Ad-N1-T332N, Ad-N1-C335N / P337T, or Ad-N1-N341 / A343T vector in PBS (Phosphate buffered saline) solution (pH 7.4) were injected intramuscularly; in the second group of immunization experiments, each dose contained 1×10 8 Mice were immunized intranasally with pfu of Ad-N2, Ad-N2-K93N, Ad-N2-S245N / S247T, Ad-N2- P267N / S269T, Ad-N2-N329 / R331T, Ad-N2-Y336N / R338T, Ad-N2- N346 / G348T, Ad-N2-E368N / S370T, Ad-N2-D401N / R403T, or Ad-N2- D463N / N465T vector in PBS. Each mouse was immunized at week 0 and week 3, and serum was collected 2 weeks after the second immunization. Collection of serum samples from experimental mice Mice were immunized with intranasal spray using the aforementioned method, and serum samples were collected from each mouse 2 weeks after the second intranasal spray. Before sampling, the mice were heated for 10 minutes using a super red light and a heating blanket. After disinfection with 70% ethanol, the lateral tail vein of the mice was cut with a scalpel, and approximately 500 μL of serum was collected. Next, the whole blood was allowed to stand at room temperature for 2 hours to allow the blood to clot, and then centrifuged twice at 800 g for 15 minutes to remove the blood clots. The serum was immediately transferred to a new centrifuge tube and centrifuged at 56 o C for 30 minutes to inactivate complement, and after cooling to room temperature, the serum was distributed and stored at -20 o C. Example 1. Preparation of influenza virus neuraminidase mutants of the present invention In one embodiment of the present invention, based on the three-dimensional structure of influenza virus neuraminidase, target sites suitable for the addition of sugar shielding are selected to mask unimportant antigenic determinants (epitopes), thereby refocusing the B cell antibody response to neuraminidase without affecting the overall folding structure of neuraminidase. Adenovirus vectors are then used to express the neuraminidase antigen with a sugar shielding mutation at the target site, which serves as the main component of the vaccine composition of the present invention. In the present invention, PyMol (The PyMol Molecular Graphics System, version 4.0; Schrödinger, LLC) was used to identify exposed loops or protrusions within the three-dimensional structure of neuraminidase (PDB ID: 4B7R, 3TIA) as target sites for the addition of sugar shields. Sites located less than 5 Å from the native sugar shield and NA were excluded. Ultimately, 16 amino acid residues were screened for the addition of additional sugar shields to generate 16 influenza virus neuraminidase mutants of the present invention. Their N-glycosylation positions are shown in Figures 1A and 3A. Compared to the amino acid sequence of influenza virus neuraminidase N1 (SEQ ID NO: 1) or N2 (SEQ ID NO: 2), the 16 neuraminidase mutants have one or two amino acid substitutions to achieve N-glycosylation (see Table 1). Specifically, the N1 neuraminidase protein has an asparagine substitution at amino acid residue 221 and a threonine substitution at amino acid residue 223, an asparagine substitution at amino acid residue 270 and a threonine substitution at amino acid residue 272, and an asparagine substitution at amino acid residue 273. and a threonine substitution at the 275th amino acid residue position, an asparagine substitution at the 329th amino acid residue position and a threonine substitution at the 331st amino acid residue position, an asparagine substitution at the 332nd amino acid residue position, an asparagine substitution at the 335th amino acid residue position and a threonine substitution at the 337th amino acid residue position, and an asparagine substitution at the 341st amino acid residue position and a threonine substitution at the 343rd amino acid residue position. Specifically, the N2 neuraminidase protein has an asparagine substitution at amino acid residue 93, an asparagine substitution at amino acid residue 245 and an asparagine substitution at amino acid residue 247, an asparagine substitution at amino acid residue 267 and an asparagine substitution at amino acid residue 269, an asparagine substitution at amino acid residue 331, an threonine substitution at amino acid residue 336 and an threonine substitution at amino acid residue 338. The amino acid residues at position 348 have an asparagine and monothreonine substitution, the amino acid residues at position 368 and position 370 have an asparagine and monothreonine substitution, the amino acid residues at position 401 and position 403 have an asparagine and monothreonine substitution, and the amino acid residues at position 463 and position 465 have an asparagine and monothreonine substitution. Table 1 To construct adenoviral expression vectors containing these neuraminidase mutant genes, neuraminidase genes from influenza viruses obtained from GenScript (H1N1 and H3N2 isolates, both codon-optimized) (N1 nucleotide sequence is SEQ ID NO: 3; N2 nucleotide sequence is SEQ ID NO: 4) were subjected to site-directed mutagenesis by polymerase chain reaction (PCR) using the primers shown in Table 2 (SEQ ID NO: 5 to SEQ ID NO: 38). Mutagenesis) was performed to obtain DNA fragments containing 9 neuraminidase mutant genes, and adenoviral vectors expressing these neuraminidase mutants were prepared using the above-mentioned adenoviral vector preparation method, which were respectively labeled as Ad-N1-N221 / I223T, Ad-N1-N270 / P272T, Ad-N1-N273 / H275T, Ad-N1-N329 / K331T, Ad-N1-T332N, Ad-N1-C335N / P337T or Ad-N1-N341 / A343T; Ad-N2-K93N, Ad-N2-S245N / S247T, Ad-N2-P267N / S269T Ad-N2-N329 / R331T, Ad-N2-Y336N / R338T, Ad-N2-N346 / G348T, Ad-N2-E368N / S370T, Ad-N2-D401N / R403T, Ad-N2-D463N / N465T, and adenovirus vectors expressing wild-type influenza virus neuraminidase were prepared as a comparison group. Table 2 To confirm the expression of neuraminidase in the adenoviral vector, the presence of neuraminidase in cell lysates infected with the adenoviral vector was analyzed by SDS-PAGE and Western blotting. First, the virus infection dose (multiplicity of infection, MOI) of Ad-N1 (adenovirus vector expressing wild-type neuraminidase), Ad-N1-N221 / I223T, Ad-N1-N270 / P272T, Ad-N1-N273 / H275T, Ad-N1-N329 / K331T, Ad-N1-T332N, Ad-N1-C335N / P337T or Ad-N1-N341 / A343T; Ad-N2 (adenovirus vector expressing wild-type neuraminidase), Ad-N2-K93N, Ad-N2-S245N / S247T, Ad-N2-P267N / S269T HEK293A cells were infected with Ad-N2-N329 / R331T, Ad-N2-Y336N / R338T, Ad-N2-N346 / G348T, Ad-N2-E368N / S370T, Ad-N2-D401N / R403T, and Ad-N2-D463N / N465T for 48 h. HEK293A cells were then lysed with Glo Lysis buffer (Promega) and cultured at 4 °C. o The cell lysate was centrifuged at 12,000 x g for 5 minutes at 95°C to remove cell debris. o C for 5 minutes and can be heated at 37 o The samples were treated with PNGase F (BioLabs) for 2 hours at 4°C (or without PNGase F treatment). Proteins in the samples were then separated by SDS-PAGE using either a 7% or 8% separating gel. The SDS-PAGE gel was transferred to a NC membrane (Millipore) and incubated with blocking solution for 1 hour at room temperature, followed by three washes with TBST. Primary antibodies: anti-influenza virus neuraminidase (H1N1 and H3N2) antibodies (anti-N1 antibody, ab21304, Abcam; anti-N2 antibody, 40017-T62, SINO Biology) were added and reacted overnight. Secondary antibodies: HRP-conjugated goat anti-rabbit IgG (KPL) were added and incubated for 1 hour at room temperature. Antibody signals were detected using a chemical luminescence reagent and developed on X-ray film. The results are shown in Figures 1B and 3B. As can be seen from Figure 1B, the influenza virus N1 neuraminidase protein is indeed present in cells infected with Ad-N1, Ad-N1-N221 / I223T (#1), Ad-N1-N270 / P272T (#2), Ad-N1-N273 / H275T (#3), Ad-N1-N329 / K331T (#4), Ad-N1-T332N (#5), Ad-N1-C335N / P337T (#6), or Ad-N1-N341 / A343T (#7) adenovirus vectors. As can be seen from Figure 3B, the influenza virus N2 neuraminidase protein is indeed present in cells infected with Ad-N2, Ad-N2-K93N (#1), Ad-N2-S245N / S247T (#2), Ad-N2-P267N / S269T (#3), Ad-N2-N329 / R331T (#4), Ad-N2-Y336N / R338T (#5), Ad-N2-N346 / G348T (#6), Ad-N2-E368N / S370T (#7), Ad-N2-D401N / R403T (#8), and Ad-N2-D463N / N465T (#9) vectors. Example 2. The influenza virus neuraminidase mutant of the present invention increases the titer of antibodies against other influenza virus strains In one embodiment of the present invention, to demonstrate that the influenza virus neuraminidase mutant of the present invention can effectively induce an anti-influenza virus antibody response in mammals, an adenoviral vector expressing the sugar-masked neuraminidase of the present invention was used to prepare a vaccine composition. The vaccine composition was then injected intramuscularly or nasally into experimental mice. An adenoviral vector expressing the native neuraminidase was used as a comparison group. After a period of time, the serum of the mice was collected to analyze the anti-influenza virus antibody titer. First, the adenovirus vector expressing wild-type neuraminidase or neuraminidase mutant was diluted with PBS solution to prepare 50 L vaccine composition, and BALB / c mice (n = 5) were divided into the following groups for immunization experiments: (1) control group (PBS): mice were only immunized with PBS solution by nasal spray or intramuscular injection; (2) comparison group (Ad-N1): mice were intramuscularly injected with 1 10 8 pfu vaccine composition of adenovirus vector expressing wild-type neuraminidase; (3) Experimental group (Ad-N1-N221 / I223T): mice were immunized intramuscularly with 1 10 8 pfu vaccine composition of adenoviral vector expressing Ad-N1-N221 / I223T sugar-masked neuraminidase; (4) Experimental group (Ad-N1-N270 / P272T): mice were immunized intramuscularly with 1 10 8 pfu vaccine composition of adenoviral vector expressing Ad-N1-N270 / P272T sugar-masked neuraminidase; (5) Experimental group (Ad-N1-N273 / H275T): mice were immunized intramuscularly with 1 10 8 pfu vaccine composition of adenoviral vector expressing Ad-N1-N273 / H275T sugar-masked neuraminidase; (6) Experimental group (Ad-N1-N329 / K331T): mice were immunized intramuscularly with 1 10 8 pfu vaccine composition of adenoviral vector expressing Ad-N1-N329 / K331T sugar-masked neuraminidase; (7) Experimental group (Ad-N1-T332N): mice were immunized intramuscularly with 1 10 8 pfu vaccine composition of adenoviral vector expressing Ad-N1-T332N sugar-masked neuraminidase; (8) Experimental group (Ad-N1-C335N / P337T): mice were immunized intramuscularly with 1 10 8 pfu vaccine composition of adenoviral vector expressing Ad-N1-C335N / P337T sugar-masked neuraminidase; (9) Experimental group (Ad-N1-N341 / A343T): mice were immunized intramuscularly with 1 10 8 pfu vaccine composition of adenoviral vector expressing Ad-N1-N341 / A343T sugar-masked neuraminidase. (10) Comparative group (Ad-N2): mice immunized with nasal spray containing 1 10 8 pfu vaccine composition of adenovirus vector expressing wild-type neuraminidase; (11) Experimental group (Ad-N2-K93N): mice immunized with nasal spray containing 1 10 8pfu vaccine composition of adenoviral vector expressing Ad-N2-K93N sugar-masked neuraminidase; (12) Experimental group (Ad-N2-S245N / S247T): mice immunized with nasal spray containing 1 10 8 Vaccine composition of adenovirus vector expressing 245N / S247T sugar-masked neuraminidase; (13) Experimental group (Ad-N2-P267N / S269T): mice immunized with nasal spray containing 1 10 8 pfu vaccine composition of adenovirus vector expressing P267N / S269T sugar-masked neuraminidase; (14) Experimental group (Ad-N2-N329 / R331T): mice immunized with nasal spray containing 1 10 8 pfu vaccine composition of adenovirus vector expressing N329 / R331T sugar-masked neuraminidase; (15) Experimental group (Ad-N2-Y336N / R338T): mice immunized with nasal spray containing 1 10 8 pfu vaccine composition of adenovirus vector expressing Y336N / R338T sugar-masked neuraminidase; (16) Experimental group (Ad-N2-N346 / G348T): mice immunized with nasal spray containing 1 10 8 pfu vaccine composition of adenovirus vector expressing N346 / G348T sugar-masked neuraminidase; (17) Experimental group (Ad-N2-E368N / S370T): mice immunized with nasal spray containing 1 10 8 pfu vaccine composition of adenovirus vector expressing E368N / S370T sugar-masked neuraminidase; (18) Experimental group (Ad-N2-D401N / R403T): mice immunized with nasal spray containing 1 10 8 pfu vaccine composition of adenovirus vector expressing D401N / R403T sugar-masked neuraminidase; (19) Experimental group (Ad-N2-D463N / N465T): mice immunized with nasal spray containing 1 10 8 Each group of mice was immunized with two doses of nasal spray, with an interval of 3 weeks between each dose. Two weeks after the second intramuscular injection or intranasal spray, sera from each group of mice were collected and analyzed for levels of anti-neuraminidase IgG antibodies and neuraminidase inhibitory antibodies against influenza viruses (H1N1 (A / Texas / 05 / 2009); H3N2 (A / Udorn / 307 / 1972)). Enzyme-linked immunosorbent assay (ELISA) was used to detect the anti-neuraminidase IgG antibody titer in serum samples. The detailed method is as follows. First, recombinant neuraminidase of influenza virus H1N1 (A / Texas / 05 / 2009) or H3N2 (A / Udorn / 307 / 1972) was added to each well at 2 μg / mL. The concentration of g / ml was 10 μg / ml in coating buffer solution. L), respectively in 4 o C overnight and fixed in two 96-well culture plates. Aspirate the coating buffer solution in the culture plate and L of PBS solution containing 0.05% Tween-20 (hereinafter referred to as PBST solution) was washed three times to remove excess recombinant protein. 200 μL of PBS solution containing 0.05% Tween-20 was added to each well. L of blocking buffer (PBS solution containing 1% fetal bovine serum albumin (BSA)) was added and blocked at room temperature for 2 hours to avoid non-specific binding. Wash three times with PBST solution. Pre-dilution of each heat-inactivated serum sample was performed at 1:1000, followed by 2-fold serial dilution with dilution buffer (PBST solution containing 1% BSA and 0.05% Tween 20). The serially diluted serum samples were added to 96-well culture plates and incubated at room temperature for 1 hour to allow the antibodies in the serum to bind to the neuraminidase or RBD immobilized on the 96-well culture plates. Wash three times with PBST solution. Add 100 μL of PBST solution to the 96-well culture plate. L HRP conjugated anti-mouse IgG antibody (HRP conjugated anti-mouse IgG antibody, diluted in dilution buffer at a ratio of 1:30000) was incubated at room temperature in the dark for 1 hour. Wash three times with PBST solution. L was added to a 96-well culture plate and the color reaction was carried out in the dark for 15 minutes. L is 2 N sulfuric acid (H 2SO 4) Terminate the reaction. Measure the optical density of each well at 450 nm using a TECAN spectrophotometer. Calculate the endpoint titration value based on the final serial dilution that achieves an optical density greater than 0.2. The neuraminidase inhibition assay (NA inhibition assay) was used to test the titer of influenza virus neuraminidase inhibitory antibodies in serum samples. The detailed method is as follows: A layer of fetuin was attached to the bottom of a 96-well plate. After 16 hours, it was washed three times with PBST solution. Then, a blocking agent was added and shaken for at least 2 hours to block non-specific binding. After washing three times with PBST solution, a mixture of diluted mouse serum and a specific dilution of virus was added at 100 μg / mL. After incubation at 37°C for 1 hour, wash three times with PBST solution. l lectin was cultured at room temperature for 1 hour. After washing three times with PBST solution, 100 l TMB was reacted for 15 minutes, and sulfuric acid was added to terminate the reaction. Neutralization curves and IC were analyzed using GraphPad Prism v6.01 software. 50 Numeric value. The titers of anti-neuraminidase IgG antibodies in mouse sera after intramuscular injection of vaccine compositions containing Ad-N1, Ad-N1-N221 / I223T, Ad-N1-N270 / P272T, Ad-N1-N273 / H275T, Ad-N1-N329 / K331T, Ad-N1-T332N, Ad-N1-C335N / P337T, or Ad-N1-N341 / A343T are shown in FIG2A , where * indicates p < 0.05 and ND indicates not detected. The titers of anti-neuraminidase antibodies against H1N1 (A / California / 07 / 2009) in mouse sera are shown in FIG2B , where ND indicates not detected. The titers of anti-neuraminidase antibodies against H5N1 in mouse sera were shown in FIG2B , where ND indicates not detected. The titers of neuraminidase inhibitory antibodies against H3N2 (A / Vietnam / 1203 / 2004) are shown in Figure 2C , expressed as percentage of inhibition of viral infection. The titers of neuraminidase inhibitory antibodies against H3N2 (A / Udorn / 307 / 1972) in mouse serum are shown in Figure 2D , and the titers of neuraminidase inhibitory antibodies against H7N9 (A / Shanghai / 02 / 2013) in mouse serum are shown in Figure 2E . The values ​​of the experimental groups compared to the control groups are expressed on a linear scale. ND indicates not detected. As shown in Figure 2A , the titer of IgG antibodies against the H1N1 influenza virus (A / Texas / 05 / 2009) neuraminidase protein elicited by mice injected intramuscularly with Ad-N1-N273 / H275T was significantly lower than that of mice injected intramuscularly with Ad-N1, Ad-N1-N221 / I223T, or Ad-N1-N329 / K331T. As shown in Figure 2B, the neuraminidase antibodies induced by intramuscular injection of Ad-N1-N273 / H275T or Ad-N1-C335N / P337T in mice contained relatively low levels of inhibitory antibodies against H1N1 (A / California / 07 / 2009) neuraminidase. The neuraminidase inhibitory antibody titers induced by the other groups were similar to those of the wild-type. As shown in Figure 2C, most of the immunization groups induced inhibition of H5N1 (A / Vietnam / 1203 / 2004) neuraminidase activity at levels close to that of the wild type, but the group immunized with Ad-N1-N329 / K331T elicited neuraminidase-inhibitory antibodies against H5N1 (A / Vietnam / 1203 / 2004) that were 3.74 times higher than those of the wild type. As shown in Figure 2D , sera from mice immunized with Ad-N1-N221 / I223T, Ad-N1-N270 / P272T, Ad-N1-N273 / H275T, and Ad-N1-N329 / K331T showed similar inhibitory activity against influenza virus H3N2 (A / Udorn / 307 / 1972) neuraminidase as wild-type sera. In contrast, sera from mice immunized with Ad-N1-T332N, Ad-N1-C335N / P337T, and Ad-N1-N341 / A343T exhibited stronger inhibitory activity against H3N2 (A / Udorn / 307 / 1972) neuraminidase than wild-type sera. As shown in Figure 2E , the inhibitory activity of mouse sera immunized with Ad-N1-N221 / I223T, Ad-N1-N270 / P272T, Ad-N1-N273 / H275T, and Ad-N1-N329 / K331T against influenza virus H7N9 (A / Shanghai / 02 / 2013) neuraminidase was similar to that of the wild-type virus. However, sera from mice immunized with Ad-N1-T332N, Ad-N1-C335N / P37T, and Ad-N1-N341 / A343T exhibited superior inhibitory activity against H7N9 (A / Shanghai / 02 / 2013). The titers of anti-neuraminidase IgG antibodies in the serum of mice after immunization with the vaccine composition containing Ad-N2, Ad-N2-K93N, Ad-N2-S245N / S247T, Ad-N2-P267N / S269T, Ad-N2-N329 / R331T, Ad-N2-Y336N / R338T, Ad-N2-N346 / G348T, Ad-N2-E368N / S370T, Ad-N2-D401N / R403T, and Ad-N2-D463N / N465T are shown in FIG4A , where * indicates p < 0.05 and ND indicates not detected. ... The titers of neuraminidase inhibitory antibodies against H7N9 (A / Shanghai / 02 / 2013) in mouse serum are shown in Figure 4B , where ND indicates not detected. The titers of neuraminidase inhibitory antibodies against H7N9 (A / Shanghai / 02 / 2013) in mouse serum are shown in Figure 4C , expressed as percentage of inhibition of viral infection. The titers of neuraminidase inhibitory antibodies against H1N1 (A / California / 07 / 2009) in mouse serum are shown in Figure 4D , and the titers of neuraminidase inhibitory antibodies against H5N1 (A / Vietnam / 1203 / 2004) in mouse serum are shown in Figure 4E , with the numerical values ​​of the experimental groups compared to the control groups expressed on a linear scale. ND indicates not detected. As shown in Figure 4A, mice immunized with intranasal sprays of Ad-N2-P267N / S269T, Ad-N2-N346 / G348T, Ad-N2-E368N / S370T, and Ad-N2-D463N / N465T also elicited lower (but not statistically significant) anti-neuraminidase IgG antibody titers against influenza virus H3N2 (A / Udorn / 307 / 1972). Mice immunized with intranasal sprays of Ad-N2, Ad-N2-K93N, Ad-N2-N329 / R331T, and Ad-N2-D401N / R403T elicited anti-neuraminidase IgG antibody titers similar to those elicited by intranasal immunization with Ad-N2, while mice immunized with intranasal sprays of PBS alone did not exhibit this effect. After mice were immunized with nasal spray of Ad-N2-S245N / S247T and Ad-N2-Y336N / R338T, the anti-neuraminidase IgG antibody titer in their serum was higher than that of Ad-N2 (but not statistically significant). As shown in Figure 4B, the neuraminidase inhibitory IC against influenza virus H3N2 (A / Udorn / 307 / 1972) was significantly increased in mice immunized with Ad-N2-S245N / S247T, Ad-N2-E368N / S370T, Ad-N2-N346 / G348T, and Ad-N2-D463N / N465T. 50 The titer is also relatively low. After nasal immunization with Ad-N2, Ad-N2-K93N, Ad-N2-P267N / S269T, Ad-N2-N329 / R331T, Ad-N2-D401N / R403T, and Ad-N2-Y336N / R338T, the IC inhibitory effect against influenza virus H3N2 (A / Udorn / 307 / 1972) was observed. 50 The titer was similar to that of mice immunized with nasal spray of Ad-N2, but this phenomenon did not occur in the control group mice that only received nasal spray of PBS solution. As shown in Figure 4C, the neuraminidase inhibition IC against influenza virus H7N9 (A / Shanghai / 02 / 2013) induced by nasal immunization with Ad-N2, Ad-N2-K93N, Ad-N2-P267N / S269T, Ad-N2-N329 / R331T, Ad-N2-Y336N / R338T, Ad-N2-E368N / S370T, Ad-N2-D401N / R403T, and Ad-N2-D463N / N465T was significantly higher than that of Ad-N2-K93N, Ad-N2-P267N / S269T, Ad-N2-N329 / R331T, Ad-N2-Y336N / R338T, Ad-N2-E368N / S370T, Ad-N2-D401N / R403T, and Ad-N2-D463N / N465T. 50The titer was similar to that of mice immunized with intranasal Ad-N2, while this phenomenon was not seen in the groups of mice immunized with intranasal PBS solution and intranasal Ad-N2-N346 / G348T. 50 The potency is higher than Ad-N2. Neuraminidase inhibition IC 50 The titer is 1.51 times As shown in Figure 4D, after nasal immunization with Ad-N2-D463N / N465T, the neuraminidase inhibitory IC 50 The titer was lower than that of Ad-N2 immunization nasal spray, while the IC inhibitory effect of neuraminidase against influenza virus H1N1 (A / California / 07 / 2009) induced by Ad-N2, Ad-N2-K93N, Ad-N2-N329 / R331T, and Ad-N2-N346 / G348T nasal spray was 50 The titer was similar to that of mice immunized with nasal spray of Ad-N2, but this phenomenon was not seen in the groups of mice immunized with nasal spray of PBS solution and nasal spray of Ad-N2-E368N / S370T and Ad-N2-D401N / R403T. 50 The potency is higher than Ad-N2. Neuraminidase inhibition IC 50 The titers were 7.85-fold and 7.49-fold, respectively. As shown in Figure 4E, the neuraminidase inhibitory IC against influenza virus H5N1 (A / Vietnam / 1203 / 2004) induced by nasal immunization with Ad-N2-P267N / S269T and Ad-N2-E368N / S370T was significantly inhibited. 50 The titer was lower than that of Ad-N2 after nasal immunization. The IC inhibitory effect of neuraminidase against influenza virus H5N1 (A / Vietnam / 1203 / 2004) induced by nasal immunization of Ad-N2, Ad-N2-K93N, Ad-N2-N329 / R331T, Ad-N2-N346 / G348T, Ad-N2-D401N / R403T and Ad-N2-D463N / N465T was 50 The titer was similar to that of mice immunized with Ad-N2 by nasal spray, but this phenomenon was not seen in the group of mice immunized with PBS solution. 50 The potency is higher than Ad-N2. Neuraminidase inhibition IC 50 The titers were 2.21-fold and 2.74-fold, respectively. In summary, the influenza virus vaccine composition of the present invention utilizes a hyperglycosylated influenza virus neuraminidase mutant to mask non-essential antigenic determinants with sugars, thereby refocusing the antibody response of individual B cells to influenza virus neuraminidase without affecting the overall protein folding structure. The influenza virus neuraminidase mutant of the present invention can effectively induce an inhibitory effect against influenza virus H1N1, H5N1, H3N2, and H7N9 neuraminidase, thereby effectively enhancing an individual's ability to resist infection by different influenza virus variants. The above description is for illustrative purposes only and is not intended to be limiting. Any equivalent modifications or variations that do not depart from the spirit and scope of the present invention should be included in the scope of the appended patent applications. none Figure 1A shows a schematic diagram of the complete tetrameric structure of the influenza virus neuraminidase protein N1. Wherein, #1 represents residues N221 / I223T, #2 represents residues N270 / P272T, #3 represents residues N273 / H275T, #4 represents residues N329 / K331T, #5 represents residues T332N, #6 represents residues C335N / P337T, and #7 represents residues N341 / A343T, which have additional glycan-masking sites. Figure 1B shows Western blotting results of the H1N1 influenza virus (A / Texas / 5 / 2009) neuraminidase protein expressed in adenovirus-infected host cells. NA represents the neuraminidase protein. Figure 2A shows the titer of anti-neuraminidase IgG antibodies against influenza virus H1N1 in the serum of mice immunized with the vaccine composition of the present invention (anti-N1 IgG titer). In Figures 2A to 2E, mice were intramuscularly injected with the vaccine composition of the present invention containing Ad-N1-WT, Ad-N1-N221 / I223T, Ad-N1-N270 / P272T, Ad-N1-N273 / H275T, Ad-N1-N329 / K331T, Ad-N1-T332N, Ad-N1-C335N / P337T, or Ad-N1-N341 / A343T. Figure 2B shows the IC of neuraminidase inhibition against influenza virus H1N1 (A / Texas / 5 / 2009) in the serum of mice immunized with the vaccine composition of the present invention. 50 Potency (NA-inhibition IC 50 Figure 2C shows the neuraminidase inhibition IC against H5N1 influenza virus (A / Vietnam / 1203 / 2004) in the serum of mice injected intramuscularly with the H1N1 influenza virus (A / Texas / 5 / 2009) vaccine composition. 50 Potency (NA-inhibition IC 50 Figure 2D shows the neuraminidase inhibition IC against H3N2 influenza virus (A / Udorn / 307 / 1972) in the serum of mice injected intramuscularly with the H1N1 influenza virus (A / Texas / 5 / 2009) vaccine composition. 50 Potency (NA-inhibition IC 50Figure 2E shows the neuraminidase inhibition IC against H7N9 influenza virus (A / Shanghai / 02 / 2013) in the serum of mice injected intramuscularly with the H1N1 influenza virus (A / Texas / 5 / 2009) vaccine composition. 50 Potency (NA-inhibition IC 50Figure 3A shows a schematic diagram of the complete tetrameric structure of influenza virus neuraminidase protein N2. Among them, #1 represents residue K93N, #2 represents residue S245N / S247T, #3 represents residue P267N / S269T, #4 represents residue N329 / R331T, #5 represents residue Y336N / R338T, #6 represents residue N346 / G348T, #7 represents residue E368N / S370T, #8 represents residue D401N / R403T, and #9 represents residue D463N / N465T, which have additional glycan-masking sites. Figure 3B shows the results of Western blotting of H3N2 influenza virus (A / Udorn / 307 / 1972) neuraminidase protein expressed in adenovirus-infected host cells. NA represents the neuraminidase protein, with #1 representing K93N residues, #2 representing S245N / S247T residues, #3 representing P267N / S269T residues, #4 representing N329 / R331T residues, #5 representing Y336N / R338T residues, #6 representing N346 / G348T residues, #7 representing E368N / S370T residues, #8 representing D401N / R403T residues, and #9 representing D463N / N465T residues, having additional glycan-masking sites. Figure 4A shows the titer of IgG antibodies against H3N2 influenza virus neuraminidase (anti-N2 IgG titer) in the serum of mice immunized with the intranasal H3N2 influenza virus (A / Udorn / 307 / 1972) vaccine composition. In Figures 4B to 4E , mice were immunized with intranasal sprays of the vaccine compositions of the present invention containing Ad-N2-K93N, Ad-N2-S245N / S247T, Ad-N2-P267N / S269T, Ad-N2-N329 / R331T, Ad-N2-Y336N / R338T, Ad-N2-N346 / G348T, Ad-N2-E368N / S370T, Ad-N2-D401N / R403T, and Ad-N2-D463N / N465T, respectively. Figure 4B shows the neuraminidase inhibition IC against H3N2 influenza virus (A / Udorn / 307 / 1972) in the serum of mice immunized with intranasal sprays of the vaccine compositions. 50 Potency (NA-inhibition IC 50Figure 4C shows the neuraminidase inhibition IC against H7N9 influenza virus (A / Shanghai / 02 / 2013) in the serum of mice immunized with the intranasal H3N2 influenza virus (A / Udorn / 307 / 1972) vaccine composition. 50 Potency (NA-inhibition IC 50 Figure 4D shows the neuraminidase inhibition IC against H1N1 influenza virus (A / California / 07 / 2009) in the serum of mice immunized with the intranasal H3N2 influenza virus (A / Udorn / 307 / 1972) vaccine composition. 50 Potency (NA-inhibition IC 50 Figure 4E shows the neuraminidase inhibition IC against H5N1 influenza virus (A / Vietnam / 1203 / 2004) in the serum of mice immunized with the intranasal H3N2 influenza virus (A / Udorn / 307 / 1972) vaccine composition. 50 Potency (NA-inhibition IC 50 titer). TW202415769A_112139270_SEQL.xml

Claims

1. An influenza virus neuraminidase mutant having an N-glycosylation of the influenza virus neuraminidase, wherein the influenza virus neuraminidase mutant has a mutation at an amino acid residue position N1 of the human influenza virus neuraminidase, wherein the amino acid residue position is selected from the group consisting of: amino acid residue position 272, amino acid residue position 275, amino acid residue position 331, amino acid residue position 332, amino acid residue position 335, amino acid residue position 337, and amino acid residue position 343; or the influenza virus neuraminidase mutant has a mutation at an amino acid residue position N1 of the human influenza virus neuraminidase. The N2 amino acid residue of the enzyme has a mutation, wherein the amino acid residue is selected from the group consisting of: amino acid residue 267, amino acid residue 269, amino acid residue 331, amino acid residue 336, amino acid residue 338, amino acid residue 348, amino acid residue 368, amino acid residue 370, amino acid residue 401, amino acid residue 403, amino acid residue 463, and amino acid residue 465; the mutation is to replace the amino acid residue with asparagine (N) or threonine (T).

2. The influenza virus neuraminidase mutant as claimed in claim 1, wherein the 272nd amino acid residue of the N1 of the human influenza virus neuraminidase has a threonine substitution, the 275th amino acid residue has a threonine substitution, the 331st amino acid residue has a threonine substitution, the 332nd amino acid residue has an asparagine substitution, the 335th amino acid residue is asparagine, the 337th amino acid residue has a threonine substitution, or the 343rd amino acid residue has a threonine substitution.

3. The influenza virus neuraminidase mutant as claimed in claim 1, wherein the 267th and 269th amino acid residues of the N2 amino acid of the human influenza virus neuraminidase are respectively substituted with asparagine and threonine, the 331st amino acid residue is substituted with threonine, the 336th and 338th amino acid residues are respectively substituted with asparagine and threonine, the 348th amino acid residue is substituted with threonine, the 368th and 370th amino acid residues are respectively substituted with asparagine and threonine, the 401st and 403rd amino acid residues are respectively substituted with asparagine and threonine, or the 463rd and 465th amino acid residues are respectively substituted with asparagine and threonine.

4. A nucleic acid molecule comprising a nucleotide sequence encoding an influenza virus neuraminidase mutant as described in claim 2 or 3.

5. A vaccine composition comprising an influenza virus neuraminidase mutant as claimed in any one of claims 1 to 3.

6. The vaccine composition as claimed in claim 5, wherein the influenza virus neuraminidase mutant is expressed on a recombinant virus.

7. The vaccine composition as claimed in claim 6, wherein the recombinant virus comprises the nucleic acid molecule as claimed in claim 4.

8. The vaccine composition as claimed in claim 6, wherein the recombinant virus is a recombinant adenovirus.

9. Use of an influenza virus neuraminidase mutant as claimed in any one of claims 1 to 3 for the preparation of an influenza virus vaccine composition.

10. The use as claimed in claim 9, wherein the influenza virus vaccine composition induces an immune response against multiple influenza virus variants in an individual.

11. The use as claimed in claim 9, wherein the influenza virus vaccine composition induces a high titer of antigen-specific antibodies and / or neuraminidase inhibitor IC50 titer.

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