WO2024078631A1 - 流感病毒神经氨酸酶突变体、编码流感病毒神经氨酸酶突变体的核酸分子、包含流感病毒神经氨酸酶突变体的疫苗组合物及流感病毒神经氨酸酶突变体用于制备流感病毒疫苗组合物的用途 - Google Patents

流感病毒神经氨酸酶突变体、编码流感病毒神经氨酸酶突变体的核酸分子、包含流感病毒神经氨酸酶突变体的疫苗组合物及流感病毒神经氨酸酶突变体用于制备流感病毒疫苗组合物的用途 Download PDF

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WO2024078631A1
WO2024078631A1 PCT/CN2023/124604 CN2023124604W WO2024078631A1 WO 2024078631 A1 WO2024078631 A1 WO 2024078631A1 CN 2023124604 W CN2023124604 W CN 2023124604W WO 2024078631 A1 WO2024078631 A1 WO 2024078631A1
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amino acid
acid residue
influenza virus
residue position
neuraminidase
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PCT/CN2023/124604
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English (en)
French (fr)
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吴夙钦
张家豪
洪浩展
林伟硕
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吴夙钦
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • 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 a use of the influenza virus neuraminidase mutant for preparing an influenza virus vaccine composition.
  • Pandemics caused by influenza have high morbidity and mortality, especially for children and the elderly.
  • the antigenicity of influenza viruses is prone to mutation and can spread across species, among which influenza A virus is the most important. Due to the rapid mutation of virus strains, once a new influenza virus subtype appears, the general population lacks immunity to it, which can easily cause a pandemic.
  • the vaccines used in the market are mainly inactivated influenza virus, attenuated influenza virus, virus-like particles (VLPs) and recombinant subunit proteins.
  • the above vaccines mainly target hemagglutinin.
  • the influenza virus is a kind of virus that has a lot of serotypes.
  • the existing vaccines have poor cross-protection ability between different serotypes, which makes the influenza vaccine lack cross-protection against new influenza viruses. Therefore, the development of a broad-spectrum influenza vaccine is a part of the current efforts.
  • an object of the present disclosure is to provide an influenza virus neuraminidase mutant having an N-glycosylation masked for the influenza virus neuraminidase.
  • the influenza virus neuraminidase mutant has a mutation at an amino acid residue position of 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.
  • the influenza virus neuraminidase mutant has a mutation at an amino acid residue position of N2 of human influenza virus neuraminidase.
  • the amino acid residue position is selected from the group consisting of: the 93rd amino acid residue position, the 245th amino acid residue position, the 247th amino acid residue position, the 267th amino acid residue position, the 269th amino acid residue position, the 331st amino acid residue position, the 336th amino acid residue position, the 338th amino acid residue position, the 348th amino acid residue position, the 368th amino acid residue position, the 370th amino acid residue position, the 401st amino acid residue position, the 403rd amino acid residue position, the 463rd amino acid residue position, and the 465th amino acid residue position.
  • the mutation is to replace the amino acid residue with asparagine (N) or threonine (T).
  • the 221st and 223rd amino acid residues of N1 of the human influenza virus neuraminidase have an asparagine and a threonine substitution, respectively
  • the 270th and 272nd amino acid residues have an asparagine and a threonine substitution, respectively
  • the 273rd and 275th amino acid residues have an asparagine and a threonine substitution, respectively
  • the 329th and 331st amino acid residues have an asparagine and a threonine substitution, respectively
  • the 332nd amino acid residue has an asparagine substitution
  • the 335th and 337th amino acid residues have an asparagine and a threonine substitution, respectively
  • the 341st and 347th amino acid residues have an asparagine and a threonine substitution, respectively.
  • 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 substitution, respectively.
  • the 267th amino acid residue position and the 269th amino acid residue position have an asparagine and a threonine substitution
  • the 331st amino acid residue position has a threonine substitution
  • the 336th amino acid residue position and the 338th amino acid residue position have an asparagine and a threonine substitution
  • the 348th amino acid residue position has a threonine substitution
  • the 368th amino acid residue position and the 370th amino acid residue position have an asparagine and a threonine substitution
  • the 401st amino acid residue position and the 403rd amino acid residue position have an asparagine and a threonine substitution
  • the 463rd amino acid residue position and the 465th amino acid residue position have an asparagine and a threonine substitution, respectively.
  • Another object of the present disclosure 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 disclosure is to provide a use of the aforementioned vector for improving gene delivery efficiency.
  • Another object of the present disclosure is to provide a vaccine composition comprising the influenza virus neuraminidase mutant as described above.
  • influenza virus neuraminidase mutant is expressed in a recombinant virus.
  • the recombinant virus comprises the nucleic acid molecule as described above.
  • the recombinant virus is a recombinant adenovirus.
  • Another object of the present disclosure is to provide a use of the influenza virus neuraminidase mutant as described above for preparing an influenza virus vaccine composition.
  • influenza virus vaccine composition elicits an immune response against multiple influenza virus variants in a subject.
  • influenza virus vaccine composition elicits high titers of antigen-specific antibodies and/or neuraminidase inhibition IC 50 titers.
  • the efficacy of the influenza virus vaccine composition disclosed in the present invention is that: by using hyperglycosylated influenza virus neuraminidase mutants, sugars are used to shield unimportant antigenic determinants, so that the antibody response of individual B cells to influenza virus neuraminidase can be refocused without affecting the overall folding structure of the protein.
  • the influenza virus neuraminidase mutant disclosed in the present invention can effectively induce an individual's inhibition of H1N1, H5N1, H3N2, and H7N9 neuraminidase against influenza viruses, thereby effectively enhancing the individual's ability to resist infection by different variants of influenza viruses.
  • Figure 1A shows a schematic diagram of the complete tetrameric structure of the influenza virus neuraminidase protein N1, wherein #1 represents the N221/I223T residue position, #2 represents the N270/P272T residue position, #3 represents the N273/H275T residue position, #4 represents the N329/K331T residue position, #5 represents the T332N residue position, #6 represents the C335N/P337T residue position, and #7 represents the N341/A343T residue position with additional added glycan-masking sites.
  • FIG1B shows the results of Western blot detection of neuraminidase protein expressed in adenovirus vector, wherein NA represents neuraminidase protein.
  • FIG. 2A shows the titer of anti-neuraminidase protein IgG antibodies against influenza virus H1N1 in the serum of mice immunized with the vaccine composition of the present invention (anti-N1 IgG titer).
  • 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.
  • FIG. 2B shows the neuraminidase inhibition IC 50 titer (NA-inhibition IC 50 titer) against H1N1 influenza virus (A/Texas/5/2009) in the sera of mice injected intramuscularly with the H1N1 influenza virus (A/Texas/ 5 /2009) vaccine composition.
  • FIG. 2C shows the neuraminidase inhibition IC 50 titer (NA-inhibition IC 50 titer) against H5N1 influenza virus (A/Vietnam/1203/2004) in the sera of mice injected intramuscularly with the H1N1 influenza virus (A/Texas/ 5 /2009) vaccine composition.
  • FIG. 2D shows the neuraminidase inhibition IC 50 titer (NA-inhibition IC 50 titer) against H3N2 influenza virus (A/Udorn/307/1972) in the sera of mice injected intramuscularly with the H1N1 influenza virus (A/Texas/ 5 /2009) vaccine composition.
  • FIG2E shows the expression of H7N9 influenza virus (A/Shanghai) in the serum of mice injected intramuscularly with the vaccine composition of H1N1 influenza virus (A/Texas/5/2009). (Shanghai)/02/2013) neuraminidase inhibition IC 50 titer (NA-inhibition IC 50 titer).
  • Figure 3A shows a schematic diagram of the complete tetrameric structure of influenza virus neuraminidase protein N2, wherein #1 represents K93N residue position, #2 represents S245N/S247T residue position, #3 represents P267N/S269T residue position, #4 represents N329/R331T residue position, #5 represents Y336N/R338T residue position, #6 represents N346/G348T residue position, #7 represents E368N/S370T residue position, #8 represents D401N/R403T residue position, and #9 represents D463N/N465T residue position with additional added glycan-masking sites.
  • FIG3B shows the results of Western blot detection of neuraminidase of H3N2 (A/Udorn/307/1972) influenza virus expressed in adenovirus vector.
  • NA represents neuraminidase protein, wherein #1 represents K93N residue position, #2 represents S245N/S247T residue position, #3 represents P267N/S269T residue position, #4 represents N329/R331T residue position, #5 represents Y336N/R338T residue position, #6 represents N346/G348T residue position, #7 represents E368N/S370T residue position, #8 represents D401N/R403T residue position, and #9 represents D463N/N465T residue position with additional added glycan-masking sites.
  • FIG4A shows the titer of anti-N2 IgG antibody against H3N2 influenza virus neuraminidase in the serum of mice immunized with nasal spray of H3N2 influenza virus (A/Udorn/307/1972) vaccine composition.
  • mice were immunized with nasal spray 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,
  • the vaccine composition of the present disclosure is Ad-N2-D401N/R403T and Ad-N2-D463N/N465T.
  • FIG. 4B shows the neuraminidase inhibition IC 50 titer (NA-inhibition IC 50 titer) against H3N2 influenza virus (A/Udorn/307/1972) in the sera of mice immunized with the nasal spray of the H3N2 influenza virus (A/Udorn/307/ 1972 ) vaccine composition.
  • FIG. 4C shows the neuraminidase inhibition IC 50 titer (NA-inhibition IC 50 titer) against H7N9 influenza virus (A/Shanghai/02/2013) in the sera of mice immunized with the nasal spray of the H3N2 influenza virus (A/Udorn/307/ 1972 ) vaccine composition.
  • FIG. 4D shows the neuraminidase inhibition IC 50 titer (NA-inhibition IC 50 titer) against H1N1 influenza virus (A/California/07/2009) in the sera of mice immunized with the nasal spray of the H3N2 influenza virus (A/Udorn/ 307 /1972) vaccine composition.
  • FIG. 4E shows the neuraminidase inhibition IC 50 titer (NA-inhibition IC 50 titer) against H5N1 influenza virus (A/Vietnam/1203/2004) in the sera of mice immunized with the nasal spray of the H3N2 influenza virus (A/Udorn/307/ 1972 ) vaccine composition.
  • the numerical values used in this article are approximate values, and all experimental data are expressed in the range of ⁇ 20%, preferably in the range of ⁇ 10%, and most preferably in the range of ⁇ 5%.
  • N-glycosylation refers to a carbohydrate chain covalently linked to asparagine of a protein by an N-glycosidic bond, comprising at least about ten different types of monosaccharide units. More specifically, the carbohydrate chain is linked to asparagine (N) in an amino acid residue, which is asparagine (N)-any amino acid (X)-threonine (T), represented by N-X-T. N-glycosylation has different molecular weights and structures depending on the monosaccharide composition.
  • hypoglycosylation means having additional "mutant carbohydrate-masking" amino acid residues in addition to the "natural carbohydrate-masking" amino acid residues on the wild-type protein.
  • N221/I223T, N270/P272T, N273/H275T, N329/K331T, T332N, C335N/P337T, N341/A343T represent specific amino acid residue positions of neuraminidase of H1N1 influenza virus substituted with asparagine and/or threonine to show influenza virus spike protein mutants disclosed herein.
  • K93N, S245N/S247T, P267N/S269T, N329/R331T, Y336N/R338T, N346/G348T, E368N/S370T, D401N/R403, D463N/N465T represent specific amino acid residue positions of neuraminidase of H3N2 influenza virus substituted with asparagine and/or threonine to show influenza virus spike protein mutants disclosed herein.
  • Ad-N2-K93N, Ad-N2-S245N/S24 7T, Ad-N2-P267N/S269TAd-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 disclosed herein.
  • the operating procedures and parameter conditions for site-directed mutagenesis are within the professional qualities and routine technical scope of persons familiar with this technology.
  • the operating procedures and parameter conditions for adding N-linked glycosylation to the amino acid residues of proteins are within the professional qualities and routine technical scope of people familiar with this technology.
  • adenovirus vector is used herein to refer to recombinant adenoviruses that express different influenza virus spike protein mutants of the present disclosure.
  • HEK293A human embryonic kidney cell line 293A
  • HEK293T human embryonic kidney cell line 293T
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin
  • adenovirus expressing neuraminidase of influenza virus or neuraminidase mutant of influenza virus with sugar masking mutation is used as a vector for immunizing experimental animals.
  • the genes encoding neuraminidase of influenza virus or neuraminidase mutant are first cloned into pENTR1A vector (Invitrogen), and then the two genes are cloned into adenovirus plasmid pAd/CMV/V5-DEST (Invitrogen) using LR ClonaseTM II Enzyme Mix (Invitrogen) to produce adenovirus plasmid (adenoviral plasmid) of neuraminidase of influenza virus or neuraminidase mutant.
  • the adenoviral plasmid was cut with Pac I restriction enzyme to expose the inverted terminal repeats (ITR), and then the two adenoviral plasmids were transfected into 293A cells using TurboFect transfection reagent (Fermentas). After 10 to 15 days of transfection, when cytopathic effect (CPE) appeared, the transfected cells and their culture medium were collected.
  • ITR inverted terminal repeats
  • CPE cytopathic effect
  • the cells were disrupted by three freeze-thaw cycles to release intracellular viral particles, and the supernatant of the cell lysate was collected by centrifugation at 3,000 rpm for 15 minutes at 4°C to obtain an adenoviral vector expressing neuraminidase or a neuraminidase mutant of influenza virus.
  • a 30-kDa Amicon Ultra-15 centrifugal filter (Millipore) can be used for concentration.
  • the adenoviral vector stock solution can be stored at -80°C.
  • HEK293A cells were seeded into 6-well culture plates at a density of 106 cells/well and cultured overnight at 37°C. Then, 10-fold serial dilutions of the adenoviral vector stock solution were added to each well at 37°C for 24 hours. The culture medium containing the diluted adenoviral vector was then removed, and 3 mL/well DMEM containing 0.4% agar and 100 U/mL penicillin/streptomycin was added to the 6-well culture plate to infect the cells. Seven to ten days after HEK293A cells were infected with adenoviral vectors, plaques were visually quantified and the counts of plaque-forming units (PFU) were recorded.
  • PFU plaque-forming units
  • SDS-PAGE The operation of SDS-PAGE is briefly described as follows. First, the protein sample is 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) in a ratio of 3:1 and heated at 95°C for 5 minutes.
  • reducing sample buffer containing 50 mM Tris-HCl, pH 6.8; 100 mM dithiothreitol (DTT); 2% SDS; 0.1% bromophenol blue; and 10% glycerol
  • a separation colloid taking 12% separation colloid as an example: comprising 2.5 mL of 1 M Tris, pH 8.8; 3.3 mL of deionized water; 4 mL of 30% acrylamide premix; 0.1 mL of 10% SDS; 0.1 ml of 10% ammonium persulfate (APS); and 0.01 mL of tetramethylethylenediamine (TEMED)) and a coking colloid (taking 5% coking colloid as an example: comprising 0.63 mL of 1 M Tris, pH 6.8; 3.4 mL of deionized water; 0.83 mL of 30% acrylamide premix; 0.05 mL 0.05 mL of 10% SDS; 0.05 mL of 10% APS; and 0.005 mL of TEMED) for electrophoresis colloid.
  • Protein electrophoresis was focused at 80 V and separated at 140 V, where the electrophoresis time depended on the molecular weight of the protein to be tested. Afterwards, the colloid was stained with a Coomassie brilliant blue dye solution (containing 0.1% coomassie R250; 10% acetic acid; and 50% methanol) for 1 hour, and then decolorized with a decolorization solution (containing 10% acetic acid; and 50% methanol).
  • a Coomassie brilliant blue dye solution containing 0.1% coomassie R250; 10% acetic acid; and 50% methanol
  • the operation of Western blot is briefly described as follows.
  • the colloid of the protein sample separated by SDS-PAGE is transferred to a nitrocellulose membrane (hereinafter referred to as NC membrane) at a voltage of 135V, and then the NC membrane containing the transferred protein is immersed in 20mL of blocking solution and shaken for at least 1 hour to block non-specific binding; wherein, the blocking solution is tris-hydroxymethylamine buffered saline containing Tween-20 (hereinafter referred to as TBST solution, containing 50mM Tris; 150mM sodium chloride; and 0.05% Tween-20) added with 5% skim milk.
  • TBST solution tris-hydroxymethylamine buffered saline containing Tween-20
  • the NC membrane was then washed three times with TBST solution, and then the primary antibody diluted with TBST solution was added and shaken at 4°C for about 16 hours. The next day, the membrane was washed three times with TBST solution, and then the secondary antibody linked to horseradish peroxidase (HRP) was diluted with TBST solution for 1 hour at room temperature. The membrane was then washed three times with TBST solution. HRP-catalyzed enhanced chemiluminescence (Millipore) was added to the membrane for 1 minute to generate Cold light signals are detected and developed onto X-ray film, such as medical blue-sensitive X-ray film (Fujifilm).
  • HRP horseradish peroxidase
  • mice were immunized with nasal sprays using the above method, and serum samples were collected from each mouse 2 weeks after the second nasal spray. Before sampling, the mice were heated for 10 minutes using ultra-red light and a heat blanket, and disinfected with 70% ethanol. The lateral tail vein of the mice was cut with a scalpel, and about 500 ⁇ L of blood was collected. Then, the whole blood was left to stand at room temperature for 2 After the blood was allowed to clot, it was centrifuged at 800 g for 15 minutes twice to remove the blood clots, and the serum was immediately transferred to a new centrifuge tube and heated at 56°C for 30 minutes to inactivate complement. After cooling to room temperature, the serum was distributed and stored at -20°C.
  • a target site suitable for the additional addition of a carbohydrate shield is selected to shield unimportant antigenic determinants (epitopes), so that the antibody response of B cells to neuraminidase can be refocused without affecting the overall folding structure of neuraminidase.
  • an adenovirus vector is used to express a neuraminidase antigen with a carbohydrate shielding mutation at the target site as the main component of the vaccine composition of the present disclosure.
  • PyMol The PyMol Molecular Graphics System, version 4.0; LLC identified exposed loops or protruding sites in the three-dimensional structure of neuraminidase (PDB ID: 4B7R, 3TIA) as target sites for the addition of sugar shields, excluding sites with natural sugar shields and NA distances less than Finally, 9 groups of amino acid residues were screened for adding additional sugar masking modifications to prepare 16 influenza virus neuraminidase mutants disclosed herein, and their N-glycosylation positions are shown in FIG. 1A and FIG. 3A .
  • the 16 neuraminidase mutants 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 one or two amino acid substitutions at the 221st amino acid residue.
  • the N2 neuraminidase protein has an asparagine substitution at the 93rd amino acid residue position, an asparagine substitution at the 245th amino acid residue position and a threonine substitution at the 247th amino acid residue position, an asparagine substitution at the 267th amino acid residue position and a threonine substitution at the 269th amino acid residue position, a threonine substitution at the 331st amino acid residue position, an asparagine substitution at the 336th amino acid residue position and a threonine substitution at the 338th amino acid residue position, a threonine substitution at the 348th amino acid residue position, an asparagine substitution at the 368th amino acid residue position and a threonine substitution at the 370th amino acid residue position, an asparagine substitution at the 401st amino acid residue position and a threonine substitution at the 403rd amino acid residue position, and an asparagine substitution at the 463rd amino acid residue
  • the neuraminidase genes H1N1 and H3N2 isolates, both of which were codon-optimized
  • influenza virus obtained from GenScript
  • the nucleotide sequence of N1 is SEQ ID NO: 3
  • the nucleotide sequence of N2 is SEQ ID NO: 4
  • PCR polymerase chain reaction
  • adenovirus expression vectors expressing the neuraminidase mutants were prepared using the above-mentioned adenovirus vector preparation method.
  • Viral vectors 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 vector of wild-type influenza virus neuraminidase were prepared as a comparison group.
  • the virus infection dose (multiplicity of infection, MOI) was 1 with Ad-N1 (adenoviral 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 (adenoviral vector expressing wild-type neuraminidase), Ad-N2-K93N, Ad-N2-S245N/S247T, Ad-N2-P267N/S269T HE
  • HEK293A cells were then lysed with Glo Lysis buffer (Promega) and centrifuged at 12,000 x g for 5 minutes at 4°C to remove cell debris.
  • the cell lysate was mixed with reducing sample buffer and heated at 95°C for 5 minutes, and the mixture was allowed to stand for 37°C for 1 h.
  • PNGase F BioLabs
  • PNGase F treatment was not performed.
  • 7% or 8% separation gel was used to separate the proteins in the sample by SDS-PAGE. After the SDS-PAGE gel was transferred to NC membrane (Millipore), it was treated with blocking solution for 1 hour at room temperature and then washed 3 times with TBST solution.
  • the primary antibody anti-influenza virus neuraminidase (H1N1 and H3N2) antibody (anti-N1antibody, ab21304, Abcam; anti-N2antibody, 40017-V07H, SINO biology) was added to react overnight, and the secondary antibody: HRP-conjugated goat anti-rabbit IgG antibody (HRP-conjugated goat anti-rabbit IgG, KPL) was added to react at room temperature for 1 hour. The antibody signal was detected using a chemical cold light reagent and developed to X-ray film. The results are shown in Figures 1B and 3B.
  • the N1 neuraminidase protein of influenza virus 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.
  • the N2 neuraminidase protein of influenza virus 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 disclosed herein improves the titer of antibodies against other influenza virus strains
  • a vaccine composition was prepared using an adenovirus vector expressing the carbohydrate-shielded neuraminidase of the present disclosure, and injected intramuscularly or nasally into experimental mice.
  • An adenovirus vector expressing 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 therein.
  • mice were immunized with PBS solution only by intranasal spray or intramuscular injection
  • comparison group mice were intramuscularly injected with a vaccine composition containing 1 ⁇ 10 8 pfu of adenovirus vector expressing wild-type neuraminidase
  • experimental group mice were immunized with a vaccine composition containing 1 ⁇ 10 8 pfu of adenovirus vector expressing Ad-N1-N221/I223T sugar-masked neuraminidase by intranasal spray
  • experimental group (Ad-N1-N270/P272T):
  • mice were immunized with a vaccine composition containing 1 ⁇ 10 8 pfu of adenovirus vector expressing wild-type neuraminidase by nasal spray;
  • Experimental group (Ad-N2-K93N) mice were immunized with a vaccine composition containing 1 ⁇ 10 8 pfu of adenovirus vector expressing Ad-N2-K93N sugar-shielded neuraminidase by nasal spray;
  • Experimental group (Ad-N2-S245N/S247T) mice were immunized with a vaccine composition containing 1 ⁇ 10 8 pfu of adenovirus vector expressing 245N/S247T sugar-shielded neuraminidase by nasal spray;
  • Experimental group (Ad-N2-P267N/S269T) mice were immunized with a vaccine composition containing 1 ⁇ 10 8 pfu of adenovirus vector expressing 245N
  • mice in each group were collected and analyzed for the levels of anti-neuraminidase IgG antibodies and neuraminidase inhibitory antibodies against influenza viruses (H1N1 (A/Texas/20172009); H3N2 (A/Udorn/307/1972)).
  • Enzyme-linked immunosorbent assay was used to detect the titer of anti-neuraminidase IgG antibodies in serum samples.
  • the detailed method is as follows. First, recombinant neuraminidase of influenza virus H1N1 (A/Texas/20172009) or H3N2 (A/Udorn/307/1972) was fixed in two 96-well culture plates at a concentration of 2 ⁇ g/ml per well in coating buffer (10 ⁇ L per well) at 4°C overnight. The coating buffer in the culture plate was aspirated and washed three times with 300 ⁇ L of PBS solution containing 0.05% Tween-20 (hereinafter referred to as PBST solution) to remove excess recombinant protein.
  • PBST solution PBS solution containing 0.05% Tween-20
  • PBST solution 200 ⁇ L of blocking buffer (PBS solution containing 1% fetal bovine serum albumin (BSA)) was added to each well and blocked for 2 hours at room temperature to avoid non-specific binding. Washed three times with 300 ⁇ L of PBST solution.
  • the heat-inactivated serum samples of each group were pre-diluted at 1:1000 and then diluted twice with dilution buffer (PBST solution containing 1% BSA and 0.05% Tween 20). Serial dilution.
  • the serially diluted serum samples were added to a 96-well plate and allowed to react at room temperature for 1 hour to allow the antibodies therein to bind to the neuraminidase or RBD fixed on the 96-well plate. Washed three times with 300 ⁇ L of P PBST solution.
  • NA inhibition assay The neuraminidase inhibition assay (NA inhibition assay) was used to detect 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, and 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, 100 ⁇ l of a mixture containing diluted mouse serum and a virus at a specific dilution ratio was added. After incubation at 37 degrees for 1 hour, it was washed three times with PBST solution.
  • the titers of mouse serum H5N1 (A/Vietnam/1203/2004) neuraminidase inhibitory antibodies are shown in Figure 2C, expressed as the percentage of inhibition of viral infection;
  • the titers of mouse serum H3N2 (A/Udorn/307/1972) neuraminidase inhibitory antibodies are shown in Figure 2D
  • the titers of mouse serum H7N9 (A/Shanghai/02/2013) neuraminidase inhibitory antibodies are shown in Figure 2E, and the numerical multiples of the experimental group compared with the comparison group are expressed on a linear scale, and N.D. means not detected.
  • the neuraminidase antibodies induced by intramuscular injection of Ad-N1-N273/H275T or Ad-N1-C335N/P337T in mice contained less inhibitory antibodies against H1N1 (A/Texas/20172009) neuraminidase, and the neuraminidase inhibitory antibody titers induced by the other groups were similar to those of the wild type.
  • the titers of anti-neuraminidase IgG antibodies in mouse serum are shown in FIG4A , where * indicates p ⁇ 0.05 and ND indicates not detected; the titers of H3N2 (A/Udorn/307/1972) neuraminidase inhibitory antibodies in mouse serum are shown in FIG4B , where ND indicates not detected; the titers of H7N9 in mouse serum are shown in FIG4A , where * indicates p ⁇
  • the titer of neuraminidase inhibitory antibodies against (A/Shanghai/02/2013) is shown in FIG4C , expressed as a percentage of inhibition of viral infection; the titer of neuraminidase inhibitory antibodies against mouse serum H1N1 (A/California/07/2009) is shown in FIG4D , and the titer of neuraminidase inhibitory antibodies against (A/Vietnam/1203/2004) in mouse serum is shown in FIG4E , and is expressed on a linear scale as the multiple of the experimental group compared to the comparison group, and ND means not detected.
  • the titer of anti-neuraminidase IgG antibodies against influenza virus H3N2 (A/Udorn/307/1972) induced by nasal spray of Ad-N2, Ad-N2-K93N, Ad-N2-N329/R331T, and Ad-N2-D401N/R403T was similar to that of mice immunized with nasal spray of Ad-N2, while the control group mice nasally sprayed with only PBS solution did not have this phenomenon.
  • 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 without statistical significance).
  • the IC 50 titer of neuraminidase inhibition against influenza virus H3N2 (A/Udorn/307/1972) induced by mice immunized with Ad-N2-S245N/S247T, Ad-N2-E368N/S370T, Ad-N2-N346/G348T, and Ad-N2-D463N/N465T was also relatively low.
  • mice were immunized with Ad-N2-S245N/S247T nasally, the IC 50 titer of neuraminidase inhibition against influenza virus H7N9 (A/Shanghai/02/2013) elicited by their serum was higher than that of Ad-N2, with the IC 50 titer of neuraminidase inhibition being 1.51 times higher.
  • the IC 50 titer of neuraminidase inhibition in serum of mice immunized with Ad-N2-S245N/S247T and Ad-N2-Y336N/R338T was higher than that of Ad-N2-Y336N/R338T.
  • Ad-N2 was high.
  • the neuraminidase inhibition IC50 titers were 7.85-fold and 7.49-fold, respectively.
  • the IC 50 titer of neuraminidase inhibition against influenza virus H5N1 (A/Vietnam/1203/2004) induced by nasal immunization with Ad-N2-P267N/S269T and Ad-N2-E368N/S370T was lower than that induced by nasal immunization with Ad-N2, while the IC 50 titer of neuraminidase inhibition against influenza virus H5N1 (A/Vietnam/1203/2004) induced by nasal immunization with Ad-N2, Ad-N2-K93N, Ad-N2-N329/R331T, Ad-N2-N346/G348T, Ad-N2-D401N/R403T and Ad-N2- D463N /N465T was similar to that induced by nasal immunization with Ad-N2, while this phenomenon was not seen in the group of mice that were only nasally sprayed with PBS solution.
  • mice were immunized with Ad-N2-S245N/S247T and Ad-N2-Y336N/R338T by nasal spray, the neuraminidase inhibition IC 50 titers of their serum were higher than that of Ad-N2, with the neuraminidase inhibition IC 50 titers being 2.21 times and 2.74 times, respectively.
  • influenza virus vaccine composition disclosed herein uses a hyperglycosylated influenza virus neuraminidase mutant to shield unimportant antigenic determinants with sugar, so that the antibody response of individual B cells to influenza virus neuraminidase can be refocused without affecting the overall folding structure of the protein.
  • the influenza virus neuraminidase mutant disclosed herein can effectively induce an individual's inhibitory effect on influenza virus H1N1, H5N1, H3N2, and H7N9 neuraminidase, thereby effectively enhancing the individual's ability to resist infection by different variants of influenza virus.

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Abstract

提供一种流感病毒神经氨酸酶突变体、编码流感病毒神经氨酸酶突变体的核酸分子、包含流感病毒神经氨酸酶突变体的疫苗组合物及流感病毒神经氨酸酶突变体用于制备流感病毒疫苗组合物的用途。疫苗组合物藉由多种功效实验，达到预防流感病毒感染的效果。

Description

流感病毒神经氨酸酶突变体、编码流感病毒神经氨酸酶突变体的核酸分子、包含流感病毒神经氨酸酶突变体的疫苗组合物及流感病毒神经氨酸酶突变体用于制备流感病毒疫苗组合物的用途
相关申请的交叉引用
本公开主张在2022年10月14日在美国提交的专利申请63/416,002的优先权，其全部内容通过引用包含于此。
技术领域
本公开是有关于一种流感病毒神经氨酸酶突变体、编码流感病毒神经氨酸酶突变体的核酸分子、包含流感病毒神经氨酸酶突变体的疫苗组合物及流感病毒神经氨酸酶突变体用于制备流感病毒疫苗组合物的用途。
背景技术
流感引起的大流行发病率和死亡率很高，尤其是对儿童和老年人。流感病毒的抗原性易发生变异并且可跨种传播，其中以A型流感病毒最为重要。由于病毒株快速变异，一旦有新的流感病毒亚型出现，人群普遍对其缺乏免疫力，容易引起大流行。
目前唯一可行的预防措施是疫苗接种，市场上应用的疫苗主要是去活性流感病毒、减毒流感病毒、类病毒颗粒(VLPs)及重组亚单位蛋白，上述疫苗主要针对血球凝集素 (hemagglutinin，HA)和神经氨酸酶(neuraminidase，NA)产生特异性抗体。但是流感病毒血清型众多，现有疫苗在各个血清型之间的交叉保护能力较差，致使流感疫苗缺乏对新型流感病毒的交叉保护功能，因此发展广效性流感疫苗是目前所需努力的部分。
为了解决上述问题，本领域的技术人员亟需研发出新颖的流感病毒神经氨酸酶突变体及包含流感病毒神经氨酸酶突变体的疫苗组合物以造福有此需求的广大族群。
发明内容
有鉴于此，本公开之目的为提供一种流感病毒神经氨酸酶突变体，具有遮蔽流感病毒的神经氨酸酶的一N-醣基化。
在本公开的一实施例中，该流感病毒神经氨酸酶突变体是在人类流感病毒神经氨酸酶的N1的一氨基酸残基位具有一突变，其中该氨基酸残基位是选自于下列所组成的组：第221个氨基酸残基位、第223个氨基酸残基位、第270个氨基酸残基位、第272个氨基酸残基位、第273个氨基酸残基位、第275个氨基酸残基位、第329个氨基酸残基位、第331个氨基酸残基位、第332个氨基酸残基位、第335个氨基酸残基位、第337个氨基酸残基位、第341个氨基酸残基位，及第343个氨基酸残基位。
在本公开的一实施例中，该流感病毒神经氨酸酶突变体是在人类流感病毒神经氨酸酶的N2的一氨基酸残基位具有一突 变，其中该氨基酸残基位是选自于下列所组成的组：第93个氨基酸残基位、第245个氨基酸残基位、第247个氨基酸残基位、第267个氨基酸残基位、第269个氨基酸残基位、第331个氨基酸残基位、第336个氨基酸残基位、第338个氨基酸残基位、第348个氨基酸残基位、第368个氨基酸残基位、第370个氨基酸残基位、第401个氨基酸残基位、第403个氨基酸残基位、第463个氨基酸残基位，及第465个氨基酸残基位。
在本公开的一实施例中，该突变是将氨基酸残基位取代为一天冬酰胺(asparagine，N)或一苏氨酸(threonine，T)。
在本公开的一实施例中，在该人类流感病毒神经氨酸酶的N1的第221个氨基酸残基位与第223个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第270个氨基酸残基位与第272个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第273个氨基酸残基位与第275个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第329个氨基酸残基位与第331个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第332个氨基酸残基位具有一天冬酰胺取代、第335个氨基酸残基位与第337个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代，或第341个氨基酸残基位与第347个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代。
在本公开的一实施例中，在该人类流感病毒神经氨酸酶的N2的第93个氨基酸残基位具有一天冬酰胺取代、第245个氨基酸残基位与第247个氨基酸残基位分别具有一天冬酰胺与一 苏氨酸取代、第267个氨基酸残基位与第269个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第331个氨基酸残基位具有一苏氨酸取代、第336个氨基酸残基位与第338个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第348个氨基酸残基位具有一苏氨酸取代、第368个氨基酸残基位与第370个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第401个氨基酸残基位与第403个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代，或第463个氨基酸残基位与第465个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代。
本公开之另一目的为提供一种核酸分子，包含编码如前所述的流感病毒神经氨酸酶突变体的一核苷酸序列。
本公开之另一目的为提供一种如前所述的载体用于提高基因递送效率的用途。
本公开之另一目的为提供一种疫苗组合物，包含如前所述的流感病毒神经氨酸酶突变体。
在本公开的一实施例中，该流感病毒神经氨酸酶突变体是表达在一重组病毒上。
在本公开的一实施例中，该重组病毒包含如前所述的核酸分子。
在本公开的一实施例中，该重组病毒是一重组腺病毒。
本公开之另一目的为提供一种如前所述的流感病毒神经氨酸酶突变体用于制备流感病毒疫苗组合物的用途。
在本公开的一实施例中，该流感病毒疫苗组合物在一个体中引发抗多个流感病毒变体的免疫反应。
在本公开的一实施例中，该流感病毒疫苗组合物引发高效价的抗原专一性抗体及/或神经氨酸酶抑制IC50效价。
综上所述，本公开流感病毒疫苗组合物的功效在于：使用过度醣化的流感病毒神经氨酸酶突变体，以醣遮蔽不重要的抗原决定位，使个体B细胞对流感病毒神经氨酸酶的抗体反应得以重新聚焦，且不会影响蛋白的整体折叠结构。本公开之流感病毒神经氨酸酶突变体可以有效引发个体针对流感病毒之H1N1、H5N1、H3N2、H7N9神经氨酸酶抑制作用，以有效提升个体抵抗流感病毒之不同变异株感染的能力。
以下将进一步说明本公开的实施方式，下述所列举的实施例系用以阐明本公开，并非用以限定本公开之范围，任何熟习此技艺者，在不脱离本公开之精神和范围内，当可做些许更动与润饰，因此本公开之保护范围当视权利要求书所界定者为准。
附图说明
图1A显示流感病毒之神经氨酸酶蛋白N1的完整四聚体结构的示意图。其中#1表示N221/I223T残基位、#2表示N270/P272T残基位、#3表示N273/H275T残基位、#4表示N329/K331T残基位、#5表示T332N残基位、#6表示C335N/P337T残基位、#7表示N341/A343T残基位具有额外添加的醣遮蔽(glycan-masking sites)。
图1B显示以西方墨点法侦测表达在腺病毒载体中之神经氨酸酶蛋白的结果。其中，NA表示神经氨酸酶蛋白。
图2A显示经免疫注射本公开之疫苗组合物的小鼠血清中针对流感病毒H1N1之抗神经氨酸酶蛋白IgG抗体的效价(anti-N1 IgG titer)。在图2A至图2E中，小鼠经肌肉注射含有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或Ad-N1-N341/A343T的本公开之疫苗组合物。
图2B显示经肌肉注射H1N1流感病毒(A/得克萨斯州(Texas)/5/2009)疫苗组合物的小鼠血清中针对H1N1流感病毒(A/Texas/5/2009)的神经氨酸酶抑制IC50效价(NA-inhibition IC50titer)。
图2C显示经肌肉注射H1N1流感病毒(A/Texas/5/2009)疫苗组合物的小鼠血清中针对H5N1流感病毒(A/越南(Vietnam)/1203/2004)的神经氨酸酶抑制IC50效价(NA-inhibition IC50titer)。
图2D显示经肌肉注射H1N1流感病毒(A/Texas/5/2009)疫苗组合物的小鼠血清中针对H3N2流感病毒(A/乌隆(Udorn)/307/1972)的神经氨酸酶抑制IC50效价(NA-inhibition IC50titer)。
图2E显示经肌肉注射H1N1流感病毒(A/Texas/5/2009)疫苗组合物的小鼠血清中针对H7N9流感病毒(A/上海 (Shanghai)/02/2013)的神经氨酸酶抑制IC50效价(NA-inhibition IC50titer)。
图3A显示流感病毒神经氨酸酶蛋白N2的完整四聚体结构的示意图。其中#1表示K93N残基位、#2表示S245N/S247T残基位、#3表示P267N/S269T残基位、#4表示N329/R331T残基位、#5表示Y336N/R338T残基位、#6表示N346/G348T残基位、#7表示E368N/S370T残基位、#8表示D401N/R403T残基位、#9表示D463N/N465T残基位具有额外添加的醣遮蔽(glycan-masking sites)。
图3B显示以西方墨点法侦测表达在腺病毒载体中之H3N2(A/Udorn/307/1972)流感病毒之神经氨酸酶的结果。其中，
NA表示神经氨酸酶蛋白，其中#1表示K93N残基位、#2表示S245N/S247T残基位、#3表示P267N/S269T残基位、#4表示N329/R331T残基位、#5表示Y336N/R338T残基位、#6表示N346/G348T残基位、#7表示E368N/S370T残基位、#8表示D401N/R403T残基位、#9表示D463N/N465T残基位具有额外添加的醣遮蔽(glycan-masking sites)。
图4A显示经免疫鼻喷H3N2流感病毒(A/Udorn/307/1972)疫苗组合物的小鼠血清中针对H3N2流感病毒神经氨酸酶IgG抗体的效价(anti-N2IgG titer)。在图4B至4E中，小鼠经分别免疫鼻喷含有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的本公开之疫苗组合物。
图4B显示经免疫鼻喷H3N2流感病毒(A/Udorn/307/1972)疫苗组合物的小鼠血清中针对H3N2流感病毒(A/Udorn/307/1972)的神经氨酸酶抑制IC50效价(NA-inhibition IC50titer)。
图4C显示经免疫鼻喷H3N2流感病毒(A/Udorn/307/1972)疫苗组合物的小鼠血清中针对H7N9流感病毒(A/Shanghai/02/2013)的神经氨酸酶抑制IC50效价(NA-inhibition IC50titer)。
图4D显示经免疫鼻喷H3N2流感病毒(A/Udorn/307/1972)疫苗组合物的小鼠血清中针对H1N1流感病毒(A/加利福尼亚(California)/07/2009)的神经氨酸酶抑制IC50效价(NA-inhibition IC50titer)。
图4E显示经免疫鼻喷H3N2流感病毒(A/Udorn/307/1972)疫苗组合物的小鼠血清中针对H5N1流感病毒(A/Vietnam/1203/2004)的神经氨酸酶抑制IC50效价(NA-inhibition IC50titer)。
具体实施方式
定义
本文中所使用数值为近似值，所有实验数据皆表示在±20％的范围内，较佳为在±10％的范围内，最佳为在±5％的范围内。
除非文中有另外说明，于本说明书中(尤其是在权利要求书中)所使用之「一」、「该」及类似用语应理解为包含单数及复数形式。
本文中在分析使用ELISA侦测抗流感病毒神经氨酸酶IgG抗体之效价的数据时，对所有的组别(除了使用PBS溶液的控制组)进行多重比较的统计测试。实验数据以GraphPad Prism v6.01进行分析，使用非参数克-瓦检测(Kruskal-Wallis test)，并以邓恩多重比较检测(Dunn’s multiple comparisons test)进行校正。统计学的显著性表示如下：*p<0.05；**p<0.01；以及***p<0.001。而在分析抑制神经氨酸酶抗体效价的实验数据时，则是根据非线性回归对数(抑制剂)与标准化反应-变量斜率的方程，来拟合抑制病毒感染百分比的曲线，并从拟合曲线中获得IC50的数值。所有的实验皆至少进行三次，数据以平均值±标准偏差表示。
在本文中，用语“N-醣基化”是指以一N-醣苷键(N-glycosidic bond)共价连接一蛋白质的天冬酰胺的醣链，包含约至少十个不同种类的单醣单元。更具体地，该醣链是连接至一氨基酸残基中的天冬酰胺(N)，该氨基酸残基为天冬酰胺(N)-任意氨基酸(X)-苏氨酸(T)，以N-X-T表示。N-醣基化依单醣组成具有不同的分子量与结构。
在本文中，除非特别指明，用语“过度醣化”意指在野生型蛋白质上的“天然醣遮蔽”氨基酸残基外，具有额外的“突变醣遮蔽”氨基酸残基。
在本文中，除非特别指明，用语“突变株”等同于用语“变体”。
在本文中，N221/I223T、N270/P272T、N273/H275T、N329/K331T、T332N、C335N/P337T、N341/A343T表示以天冬酰胺及/或苏氨酸取代H1N1流感病毒之神经氨酸酶的特定氨基酸残基位，以显示本公开流感病毒棘蛋白突变体。K93N、S245N/S247T、P267N/S269T、N329/R331T、Y336N/R338T、N346/G348T、E368N/S370T、D401N/R403、D463N/N465T表示以天冬酰胺及/或苏氨酸取代H3N2流感病毒之神经氨酸酶的特定氨基酸残基位，以显示本公开流感病毒棘蛋白突变体。
在本文中，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或Ad-N1-N341/A343T；Ad-N2-K93N、Ad-N2-S245N/S247T、Ad-N2-P267N/S269TAd-N2-N329/R331T、Ad-N2-Y336N/R338T、Ad-N2-N346/G348T、Ad-N2-E368N/S370T、Ad-N2-D401N/R403T、Ad-N2-D463N/N465T表示表达不同之本公开流感病毒神经氨酸酶蛋白突变体的重组腺病毒。
依据本公开，有关基因选殖(gene cloning)的操作程序与参数条件等是落在熟习此项技术之人士的专业素养与例行技术范畴内。
依据本公开，有关定点突变(site-directed mutagenesis)的操作程序与参数条件等是落在熟习此项技术之人士的专业素养与例行技术范畴内。
依据本公开，有关于蛋白质的氨基酸残基添加N-醣基化(N-linked glycosylation)的操作程序与参数条件等是落在熟习此项技术之人士的专业素养与例行技术范畴内。
依据本公开，有关于以腺病毒表达抗原的操作程序与参数条件等是落在熟习此项技术之人士的专业素养与例行技术范畴内，且在本文中以“腺病毒载体”表示表达不同之本公开流感病毒棘蛋白突变体的重组腺病毒。
兹以下列实施例进一步例示说明本公开。其中该等实施例仅提供作为说明，而非用以限制本公开之保护范围。本公开保护范围系如权利要求书所示。
材料及方法
实验细胞及培养方法
在本公开之实施例中，使用人类胚胎肾细胞株293A(HEK293A)与人类胚胎肾细胞株293T(HEK293T)进行细胞实验。HEK293A细胞与HEK293T细胞皆取自生物资源保存及研究中心(Bioresource Collection and Research Center，BCRC)，并使用添加有10％之胎牛血清(fetal bovine sera，FBS)(Gibco)及100units/mL之青霉素/链霉素(penicillin/streptomycin，P/S)的良伊格尔氏培养基(Dulbecco’s modified Eagle medium，DMEM)(Thermo  Scienific)作为细胞培养液，于37℃、含有5％之CO2的培养箱进行培养。
腺病毒载体的制备
在本公开之实施例中，使用表达流感病毒之神经氨酸酶或具醣遮蔽突变之流感病毒之神经氨酸酶突变体的腺病毒作为免疫实验动物的载体。先将编码流感病毒之神经氨酸酶或神经氨酸酶突变体的基因分别选殖至pENTR1A载体(Invitrogen)中，再使用LR ClonaseTM II Enzyme Mix(Invitrogen)将该二基因分别选殖至腺病毒质体pAd/CMV/V5-DEST(Invitrogen)中，以产生流感病毒之神经氨酸酶或神经氨酸酶突变体的腺病毒质体(adenoviral plasmid)。
为了得到表达流感病毒之神经氨酸酶或神经氨酸酶突变体的腺病毒载体，使用Pac I限制酶切割该腺病毒质体，以使倒转终端重复(inverted terminal repeats，ITR)暴露出来，接着使用TurboFect转染试剂(Fermentas)将该二腺病毒质体分别转染至293A细胞中。于转染10至15天后，待细胞病变效应(cytopathic effect，CPE)出现，便收集经转染的细胞与其培养基。以三次冷冻解冻循环破坏细胞以释放细胞内的病毒颗粒，并在4℃下使用3,000rpm离心15分钟，以收集细胞裂解物的上清液而获取表达流感病毒之神经氨酸酶或神经氨酸酶突变体的腺病毒载体。此外，为了制备具有更高滴度(titer)的腺病毒载体，可以使用30-kDa Amicon Ultra-15离心过滤器(Millipore)进行浓缩。腺病毒载体原液可以储存于-80℃。
为了进一步确定腺病毒载体的滴度，将HEK293A细胞以106个细胞/孔的密度接种至6孔培养盘中，并在37℃下培养过夜后，在37℃下将10倍序列稀释的腺病毒载体原液添加至每孔中作用24小时。接着移除含有稀释腺病毒载体的培养基，并将含有0.4％洋菜胶及100U/mL青霉素/链霉素的3mL/孔DMEM添加至该6培养盘中感染细胞。在以腺病毒载体感染HEK293A细胞7至10天后，对噬菌斑进行可见定量，并记录噬菌斑形成单位(plaque-forming unit，PFU)的计数。
十二烷基硫酸钠聚丙烯酰胺胶体电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE)的操作
SDS-PAGE的操作简述如下。首先，依3:1的比例将蛋白质样品与还原样品缓冲溶液(reducing sample buffer)(包含有50mM的三羟甲基氨基甲烷-氯化氢(Tris-HCl)，pH 6.8；100mM的二硫苏糖醇(dithiothreitol，DTT)；2％的SDS；0.1％的溴酚蓝(bromophenol blue)；以及10％的甘油)混合后于95℃加热5分钟。
与此同时，制备包含分离胶体(以12％的分离胶体为例：包含有2.5mL之1M的Tris，pH 8.8；3.3mL的去离子水；4mL之30％的丙烯酰胺预混液(acrylamide mix)；0.1mL之10％的SDS；0.1ml之10％的过硫酸铵(ammonium persulfate，APS)；以及0.01mL的四甲基乙二胺(TEMED))，及焦集胶体(以5％的焦集胶体为例：包含有0.63mL之1M的Tris，pH 6.8；3.4mL的去离子水；0.83mL之30％的丙烯酰胺预混液；0.05mL 之10％的SDS；0.05mL之10％的APS；以及0.005mL的TEMED)的电泳胶体。
蛋白质电泳在电压80V下进行焦集，并且在140V下进行分离，其中电泳的时间依待测蛋白质的分子量而定。其后，将胶体以考马斯亮蓝染剂溶液(包含有0.1％的coomassie R250；10％的醋酸；以及50％的甲醇)染色1小时，再以脱色溶液(包含有10％的醋酸；以及50％的甲醇)进行脱色。
西方墨点法的操作
西方墨点法(Western blot)的操作简述如下。在转渍槽中，将经SDS-PAGE分离之蛋白质样品的胶体，以电压135V转印至硝化纤维膜(nitrocellulose membrane，以下简称NC膜)，再将该含有转印蛋白质的NC膜浸泡于20mL的阻断溶液中，并震荡至少1小时以阻断非专一性结合；其中，该阻断溶液为添加有5％之脱脂奶的含Tween-20之三羟甲基氨基缓冲食盐水(以下简称TBST溶液，包含有50mM的Tris；150mM的氯化钠；以及0.05％的Tween-20)。
接着使用TBST溶液清洗该NC膜3次后，加入经TBST溶液进行特定倍数稀释的一级抗体，于4℃震荡处理约16小时，隔天再使用TBST溶液清洗3次后，以连接山葵过氧化酶(horseradish peroxidase，HRP)之经TBST溶液进行特定倍数稀释的二级抗体，于室温下震荡处理1小时，而后使用TBST溶液清洗3次。将增强的化学冷光试剂(HRP-catalyzed enhanced chemiluminescence，Millipore)添加至该膜作用1分钟以产生 冷光信号，并显影至X光胶片上，例如医用感蓝X射线胶片(Medical X-ray Film，Fujifilm)。
实验小鼠的免疫方法
在本公开之一实施例中，使用6至8周龄的BALB/c雌性小鼠进行疫苗接种的实验，其中BALB/c雌性小鼠是取自财团法人国家实验研究院国家实验动物中心(National Laboratory Animal Center)。在第一组免疫实验中，使用每剂含有1×108pfu的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、或Ad-N1-N341/A343T载体的PBS(Phosphate buffered saline)溶液(pH 7.4)进行肌肉注射；而在第二组免疫实验中，则使用每剂含有1×108pfu的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或Ad-N2-D463N/N465T载体的PBS溶液进行喷鼻免疫。每只小鼠皆分别于第0周与第3周予以免疫注射，并于第二次免疫注射的2周后收集血清。
实验小鼠血清样品的采集
以前述方法对小鼠进行免疫鼻喷，并于第二次免疫鼻喷的2周后收集各小鼠的血清样品。在取样前透过超红灯及热毯加热小鼠10分钟，并使用70％乙醇消毒后，用手术刀切开小鼠侧尾静脉，采集约500μL的血液。接着，将全血在室温下静置2 小时，使血液凝结后，以800g转速离心15分钟共两次以去除血液凝块，并立即将血清转移到新的离心管中，并在56℃加热处理30分钟以减活补体，而待冷却至室温后，再将血清分配并储存在-20℃。
实施例1.本公开之流感病毒神经氨酸酶突变体的制备
在本公开之一实施例中，基于流感病毒神经氨酸酶的三维结构，挑选适合额外添加醣遮蔽的目标位点，以遮蔽不重要的抗原决定位(epitope)，使B细胞对神经氨酸酶的抗体反应得以重新聚焦，且不会影响神经氨酸酶的整体折叠结构，接着使用腺病毒载体表达在目标位点具有醣遮蔽突变的神经氨酸酶抗原，以作为本公开之疫苗组合物中的主要成分。
在本公开之实施例中，使用PyMol(The PyMol Molecular Graphics System，version 4.0；LLC)确认在神经氨酸酶的三维结构(PDB ID:4B7R，3TIA)中暴露环(loop)或暴露环中突出的位点，以作为醣遮蔽的添加目标位点，其中排除与天然醣遮蔽与NA距离小于的位点。最终筛选出9组氨基酸残基位添加额外的醣遮蔽修饰，以制备16种本公开之流感病毒神经氨酸酶突变体，其N-醣基化位置如图1A与图3A。
相较于流感病毒神经氨酸酶的N1(序列识别号：1)或N2(序列识别号：2)的氨基酸序列，该16种神经氨酸酶突变体具有一或二个氨基酸取代以实现N-醣基化(参见表1)，具体来说，N1神经氨酸酶蛋白的第221个氨基酸残基位具有一天冬 酰胺与第223个氨基酸残基位具有一苏氨酸取代、第270个氨基酸残基位具有一天冬酰胺与第272个氨基酸残基位具有一苏氨酸取代、第273个氨基酸残基位具有一天冬酰胺与第275个氨基酸残基位具有一苏氨酸取代、第329个氨基酸残基位具有一天冬酰胺与第331个氨基酸残基位具有一苏氨酸取代、第332个氨基酸残基位具有一天冬酰胺取代、第335个氨基酸残基位具有一天冬酰胺取代与第337个氨基酸残基位具有一苏氨酸取代、第341个氨基酸残基位具有一天冬酰胺与第343个氨基酸残基位具有一苏氨酸取代。
具体来说，N2神经氨酸酶蛋白的第93个氨基酸残基位具有一天冬酰胺取代、第245个氨基酸残基位与第247个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第267个氨基酸残基位与第269个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第331个氨基酸残基位具有一苏氨酸取代、第336个氨基酸残基位与第338个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第348个氨基酸残基位具有一苏氨酸取代、第368个氨基酸残基位与第370个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第401个氨基酸残基位与第403个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第463个氨基酸残基位与第465个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代。
表1

为建构包含该等神经氨酸酶突变体基因的腺病毒表现载体，将取自GenScript公司流感病毒的神经氨酸酶基因(H1N1及H3N2分离株，皆经人类密码子优化(codon-optimized)(N1的核苷酸序列为序列识别号：3；N2的核苷酸序列为序列识别号：4)，再利用如下表2所示的引子(序列识别号：5至序列识别号：38)进行基于聚合酶链锁反应(polymerase chain reaction，PCR)的定点突变(site-directed mutagenesis)，以获得包含9种神经氨酸酶突变体基因的DNA片段，并以前述腺病毒载体的制备方法，制备表达该等神经氨酸酶突变体的腺 病毒载体，分别标示为Ad-N1-N221/I223T、Ad-N1-N270/P272T、Ad-N1-N273/H275T、Ad-N1-N329/K331T、Ad-N1-T332N、Ad-N1-C335N/P337T或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，同时制备野生型流感病毒神经氨酸酶的腺病毒载体作为比较组。
表2

为了确认表达在腺病毒载体中的神经氨酸酶，使用SDS-PAGE与西方墨点法分析经腺病毒载体感染的细胞裂解物中，是否具有神经氨酸酶。首先，将病毒感染剂量(multiplicity of infection，MOI)为1的Ad-N1(表达野生型神经氨酸酶的腺病毒载体)、Ad-N1-N221/I223T、Ad-N1-N270/P272T、Ad-N1-N273/H275T、Ad-N1-N329/K331T、Ad-N1-T332N、Ad-N1-C335N/P337T或Ad-N1-N341/A343T；Ad-N2(表达野生型神经氨酸酶的腺病毒载体)、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感染HEK293A细胞48小时。接着以Glo Lysis缓冲溶液(Promega)裂解HEK293A细胞，并在4℃下以12,000xg离心5分钟以去除细胞碎片。将细胞裂解物与还原样品缓冲溶液混合后在95℃加热5分钟，且可以在37℃下经 PNGase F(BioLabs)处理2小时，也可以不经PNGase F处理，接着使用7％或8％的分离凝胶，以SDS-PAGE分离样本中的蛋白质。将SDS-PAGE的胶体转印至NC膜(Millipore)后，在室温下以阻断溶液作用1小时，再使用TBST溶液清洗3次。加入一级抗体：抗流感病毒神经氨酸酶(H1N1及H3N2)抗体(anti-N1antibody，ab21304，Abcam；anti-N2antibody，40017-V07H，SINO biology)反应过夜，并加入二级抗体：连接HRP的山羊抗兔IgG抗体(HRP-conjugated goat anti-rabbit IgG，KPL)在室温下作用1小时。使用化学冷光试剂侦测抗体的信号，并显影至X光胶片，结果如图1B及3B所示。
由图1B可以看出，在感染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)或Ad-N1-N341/A343T(#7)腺病毒载体的细胞中，确实存在流感病毒的N1神经氨酸酶蛋白。
图3B可以看出，在感染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)、Ad-N2-D463N/N465T(#9)载体的细胞中，确实存在流感病毒的N2神经氨酸酶蛋白。
实施例2.本公开之流感病毒神经氨酸酶突变体提高针对其他流感病毒株之抗体的效价
在本公开之一实施例中，为证实本公开之流感病毒神经氨酸酶突变体确实能有效地诱发哺乳动物产生对抗流感病毒的抗体反应，以表达本公开之醣遮蔽神经氨酸酶的腺病毒载体制备疫苗组合物，并肌肉注射或鼻喷至实验小鼠体内，且以表达原生型神经氨酸酶的腺病毒载体作为比较组，在经过一段时间后收取小鼠的血清，以分析其中抗流感病毒之抗体效价。
首先，使用PBS溶液将表达野生型神经氨酸酶或神经氨酸酶突变体的腺病毒载体稀释，以分别配制成50μL的疫苗组合物，并将BALB/c小鼠(n＝5)分成以下组别进行免疫实验：(1)控制组(PBS)：小鼠仅鼻喷或肌肉注射免疫PBS溶液；(2)比较组(Ad-N1)：小鼠肌肉注射含有1×108pfu表达野生型神经氨酸酶之腺病毒载体的疫苗组合物；(3)实验组(Ad-N1-N221/I223T)：小鼠鼻喷免疫含有1×108pfu表达Ad-N1-N221/I223T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物；(4)实验组(Ad-N1-N270/P272T)：小鼠鼻喷免疫含有1×108pfu表达Ad-N1-N270/P272T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物；(5)实验组(Ad-N1-N273/H275T)：小鼠鼻喷免疫含有1×108pfu表达Ad-N1-N273/H275T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物；(6)实验组(Ad-N1-N329/K331T)：小鼠鼻喷免疫含有1×108pfu表达Ad-N1-N329/K331T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物；(7)实验组(Ad-N1-T332N)：小鼠鼻喷免疫含有1×108pfu表达Ad-N1-T332N醣遮蔽神经氨酸酶之腺病毒载体 的疫苗组合物；(8)实验组(Ad-N1-C335N/P337T)：小鼠鼻喷免疫含有1×108pfu表达Ad-N1-C335N/P337T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物；(9)实验组(Ad-N1-N341/A343T)：小鼠鼻喷免疫含有1×108pfu表达Ad-N1-N341/A343T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物。(11)比较组(Ad-N2)：小鼠鼻喷免疫含有1×108pfu表达野生型神经氨酸酶之腺病毒载体的疫苗组合物；(12)实验组(Ad-N2-K93N)：小鼠鼻喷免疫含有1×108pfu表达Ad-N2-K93N醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物；(13)实验组(Ad-N2-S245N/S247T)：小鼠鼻喷免疫含有1×108表达245N/S247T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物；(14)实验组(Ad-N2-P267N/S269T)：小鼠鼻喷免疫含有1×108pfu表达P267N/S269T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物；(15)实验组(Ad-N2-N329/R331T)：小鼠鼻喷免疫含有1×108pfu表达N329/R331T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物；(16)实验组(Ad-N2-Y336N/R338T)：小鼠鼻喷免疫含有1×108pfu表达Y336N/R338T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物；(17)实验组(Ad-N2-N346/G348T)：小鼠鼻喷免疫含有1×108pfu表达N346/G348T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物；(18)实验组(Ad-N2-E368N/S370T)：小鼠鼻喷免疫含有1×108pfu表达E368N/S370T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物；(19)实验组(Ad-N2-D401N/R403T)： 小鼠鼻喷免疫含有1×108pfu表达D401N/R403T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物；(20)实验组(Ad-N2-D463N/N465T)：小鼠鼻喷免疫含有1×108pfu表达D463N/N465T醣遮蔽神经氨酸酶之腺病毒载体的疫苗组合物。以上各组小鼠皆免疫鼻喷共二剂且每剂间隔3周。
在肌肉注射或鼻喷第二剂的2周后，采集并收集各组小鼠的血清，用以分析其中针对流感病毒(H1N1(A/Texas/05/2009)；H3N2(A/Udorn/307/1972))抗神经氨酸酶IgG抗体、及神经氨酸酶抑制抗体的含量。
使用酵素结合免疫吸附分析(Enzyme-linked immunosorbent assay，ELISA)侦测血清样本中抗神经氨酸酶IgG抗体效价，详细方法如下。首先，将流感病毒H1N1(A/Texas/05/2009)或H3N2(A/Udorn/307/1972)之重组神经氨酸酶以每孔2μg/ml的浓度，在涂层缓冲溶液(coating buffer，每孔10μL)中，分别在4℃下隔夜固定于二个96孔培养盘。吸出培养盘中的涂层缓冲溶液，并以300μL含有0.05％Tween-20的PBS溶液(以下简称PBST溶液)清洗三次，以移除多余的重组蛋白。在各孔中加入200μL的阻断缓冲溶液(blocking buffer，含1％胎牛血清蛋白(bovine serum albumin，BSA)的PBS溶液)，在室温下阻断2小时，以避免非专一性结合。以300μL的PBST溶液清洗三次。将经热减活的各组血清样本以1:1000进行预稀释，接着以稀释缓冲溶液(dilution buffer，含1％BSA、0.05％Tween 20的PBST溶液)进行2倍 连续稀释。将连续稀释的血清样本分别加入96孔培养盘中，于室温作用1小时，使其中的抗体与固定在96孔培养盘的神经氨酸酶或RBD结合。以300μL的P PBST溶液清洗三次。在96孔培养盘中加入100μL连接HRP的抗小鼠IgG抗体(HRP conjugated anti-mouse IgG antibody，使用稀释缓冲溶液以1:30000比例稀释)，于室温下避光作用1小时。以300μL的P PBST溶液清洗三次。将HRP的受质3,3'，5,5'-四甲基联苯胺(3,3’，5,5’-Tetramethylbenzidine，TMB，BioLegend)以100μL加入96孔培养盘中，并于暗处进行呈色反应15分钟。加入100μL为2N的硫酸(H2SO4)以终止反应。使用TECAN分光光度计测定各孔在450nm的光密度。根据高于0.2之光密度值的最终连续稀释程度计算终点滴定值。
使用神经氨酸酶抑制试验(NA inhibition assay)检测血清样本中，针对流感病毒神经氨酸酶抑制抗体效价，详细方法如下。在96孔盘底部贴附一层胎儿蛋白(Fetuin)，16小时后使用PBST溶液清洗三次，接着加入阻断剂震荡至少2小时以阻断非专一性结合。PBST溶液清洗三次后，加入含有已稀释的小鼠血清与特定稀释倍率的病毒混合物100μl。37度培养1小时后，再以PBST溶液清洗三次。加入100μl lectin室温震荡培养1小时。PBST溶液清洗三次后每孔加入100μl TMB反应15分钟，最终加入硫酸终止反应。使用GraphPad Prism v6.01软件分析中和曲线与IC50数值。
在经肌肉注射含有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、或Ad-N1-N341/A343T之疫苗组合物后，小鼠血清中抗神经氨酸酶IgG抗体的效价如图2A所示，*表示p<0.05、N.D.表示未检出；小鼠血清中H1N1(A/Texas/5/2009)神经氨酸酶抑制抗体的效价如图2B所示，N.D.表示未检出；小鼠血清H5N1(A/Vietnam/1203/2004)神经氨酸酶抑制抗体的效价如图2C所示，以抑制病毒感染的百分比表示；小鼠血清H3N2(A/Udorn/307/1972)神经氨酸酶抑制抗体的效价如图2D所示，小鼠血清H7N9(A/Shanghai/02/2013)神经氨酸酶抑制抗体的效价如图2E所示并以线性标度表示实验组相较于比较组的数值倍数，N.D.表示未检出。
由图2A可以看出，小鼠经肌肉注射Ad-N1-N273/H275T后，所引发针对H1N1流感病毒(A/Texas/05/2009)之神经氨酸酶蛋白IgG抗体效价，显著低于经肌肉注射Ad-N1、Ad-N1-N221/I223T或Ad-N1-N329/K331T的小鼠。
由图2B可以看出，小鼠肌肉注射Ad-N1-N273/H275T或Ad-N1-C335N/P337T所诱发的神经氨酸酶抗体中，含有较少抑制H1N1(A/Texas/05/2009)神经氨酸酶的抑制抗体，其余组别诱发的神经氨酸酶抑制抗体效价与野生型相近。
由图2C可以看出，针对抑制H5N1(A/Vietnam/1203/2004)神经氨酸酶活性中，大部分的免疫组别诱发的抑制能力与野生 型接近，但其中免疫Ad-N1-N329/K331T的组别所引发针对H5N1(A/Vietnam/1203/2004)的神经氨酸酶抑制抗体高于野生型3.74倍。
由图2D可以看出，免疫Ad-N1-N221/I223T、Ad-N1-N270/P272T、Ad-N1-N273/H275T及Ad-N1-N329/K331T后，针对流感病毒H3N2(A/Udorn/307/1972)神经氨酸酶的抑制能力与野生型相近。而与野生型相比，免疫Ad-N1-T332N、Ad-N1-C335N/P337T及Ad-N1-N341/A343T的小鼠血清产生较强的H3N2(A/Udorn/307/1972)神经氨酸酶抑制能力。
由图2E可以看出，免疫Ad-N1-N221/I223T、Ad-N1-N270/P272T、Ad-N1-N273/H275T及Ad-N1-N329/K331T后，针对流感病毒H7N9(A/Shanghai/02/2013)神经氨酸酶的抑制能力与野生型相近。而Ad-N1-T332N、Ad-N1-C335N/P37T及Ad-N1-N341/A343T的小鼠血清针对H7N9(A/Shanghai/02/2013)有较佳的抑制能力。
在经免疫鼻喷含有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、Ad-N2-D463N/N465T之疫苗组合物后，小鼠血清中抗神经氨酸酶IgG抗体的效价如图4A所示，*表示p<0.05、N.D.表示未检出；小鼠血清中H3N2(A/Udorn/307/1972)神经氨酸酶抑制抗体的效价如图4B所示，N.D.表示未检出；小鼠血清H7N9 (A/Shanghai/02/2013)神经氨酸酶抑制抗体的效价如图4C所示，以抑制病毒感染的百分比表示；小鼠血清H1N1(A/California/07/2009)神经氨酸酶抑制抗体的效价如图4D所示，小鼠血清H5N1(A/Vietnam/1203/2004)神经氨酸酶抑制抗体的效价如图4E所示并以线性标度表示实验组相较于比较组的数值倍数，N.D.表示未检出。
由图4A可以看出，小鼠经免疫鼻喷Ad-N2-P267N/S269T、Ad-N2-N346/G348T、Ad-N2-E368N/S370T、Ad-N2-D463N/N465T后，所引发针对流感病毒H3N2(A/Udorn/307/1972)抗神经氨酸酶IgG抗体效价亦较为低(但无统计学上的意义)。免疫鼻喷Ad-N2、Ad-N2-K93N、Ad-N2-N329/R331T、Ad-N2-D401N/R403T后，所引发针对流感病毒H3N2(A/Udorn/307/1972)抗神经氨酸酶IgG抗体效价与经免疫鼻喷Ad-N2相近，而仅鼻喷PBS溶液的控制组小鼠则无此现象。小鼠经免疫鼻喷Ad-N2-S245N/S247T、Ad-N2-Y336N/R338T后，其血清的抗神经氨酸酶IgG抗体效价较Ad-N2为高(但无统计学上的意义)。
由图4B可以看出，小鼠经免疫Ad-N2-S245N/S247T、Ad-N2-E368N/S370T、Ad-N2-N346/G348T、Ad-N2-D463N/N465T后，所引发针对流感病毒H3N2(A/Udorn/307/1972)神经氨酸酶抑制IC50效价亦较为低。免疫鼻喷Ad-N2、Ad-N2-K93N、Ad-N2-P267N/S269T、Ad-N2-N329/R331T、Ad-N2-D401N/R403T、 Ad-N2-Y336N/R338T后，所引发针对流感病毒H3N2(A/Udorn/307/1972)神经氨酸酶抑制IC50效价与经免疫鼻喷Ad-N2相近，而仅鼻喷PBS溶液的控制组小鼠则无此现象。
由图4C可以看出，免疫鼻喷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、Ad-N2-D463N/N465T后，所引发针对流感病毒H7N9(A/Shanghai/02/2013)神经氨酸酶抑制IC50效价与经免疫鼻喷Ad-N2相近，而仅鼻喷PBS溶液以及鼻喷Ad-N2-N346/G348T的组别小鼠则无此现象。小鼠经免疫鼻喷Ad-N2-S245N/S247T后，其血清的所引发针对流感病毒H7N9(A/Shanghai/02/2013)神经氨酸酶抑制IC50效价较Ad-N2为高。神经氨酸酶抑制IC50效价为1.51倍。
由图4D可以看出，免疫鼻喷Ad-N2-D463N/N465T后，所引发针对流感病毒H1N1(A/California/07/2009)神经氨酸酶抑制IC50效价较经免疫鼻喷Ad-N2低，而免疫鼻喷Ad-N2、Ad-N2-K93N、Ad-N2-N329/R331T、Ad-N2-N346/G348T后，所引发针对流感病毒H1N1(A/California/07/2009)神经氨酸酶抑制IC50效价与经免疫鼻喷Ad-N2相近，而仅鼻喷PBS溶液以及鼻喷Ad-N2-E368N/S370T、Ad-N2-D401N/R403T的组别小鼠则无此现象。小鼠经免疫鼻喷Ad-N2-S245N/S247T以及Ad-N2-Y336N/R338T后，其血清的神经氨酸酶抑制IC50效价较 Ad-N2为高。神经氨酸酶抑制IC50效价分别为7.85倍和7.49倍。
由图4E可以看出，免疫鼻喷Ad-N2-P267N/S269T、Ad-N2-E368N/S370T后，所引发针对流感病毒H5N1(A/Vietnam/1203/2004)神经氨酸酶抑制IC50效价较经免疫鼻喷Ad-N2低，而免疫鼻喷Ad-N2、Ad-N2-K93N、Ad-N2-N329/R331T、Ad-N2-N346/G348T、Ad-N2-D401N/R403T后、Ad-N2-D463N/N465T，所引发针对流感病毒H5N1(A/Vietnam/1203/2004)神经氨酸酶抑制IC50效价与经免疫鼻喷Ad-N2相近，而仅鼻喷PBS溶液的组别小鼠则无此现象。小鼠经免疫鼻喷Ad-N2-S245N/S247T以及Ad-N2-Y336N/R338T后，其血清的神经氨酸酶抑制IC50效价较Ad-N2为高。神经氨酸酶抑制IC50效价分别为2.21倍和2.74倍。
综上所述，本公开流感病毒疫苗组合物使用过度醣化的流感病毒神经氨酸酶突变体，以醣遮蔽不重要的抗原决定位，使个体B细胞对流感病毒神经氨酸酶的抗体反应得以重新聚焦，且不会影响蛋白的整体折叠结构。本公开之流感病毒神经氨酸酶突变体可以有效引发个体针对流感病毒之H1N1、H5N1、H3N2、H7N9神经氨酸酶抑制作用，以有效提升个体抵抗流感病毒之不同变异株感染的能力。
以上所述仅为举例性，而非为限制性者。任何未脱离本公开之精神与范畴，而对其进行之等效修改或变更，均应包含于权利要求书之范围中。

Claims (14)

  1. 一种流感病毒神经氨酸酶突变体，具有遮蔽流感病毒的神经氨酸酶的一N-醣基化。
  2. 如权利要求1所述的流感病毒神经氨酸酶突变体，其中该流感病毒神经氨酸酶突变体是在人类流感病毒神经氨酸酶的N1的一氨基酸残基位具有一突变，其中该氨基酸残基位是选自于下列所组成的组：第221个氨基酸残基位、第223个氨基酸残基位、第270个氨基酸残基位、第272个氨基酸残基位、第273个氨基酸残基位、第275个氨基酸残基位、第329个氨基酸残基位、第331个氨基酸残基位、第332个氨基酸残基位、第335个氨基酸残基位、第337个氨基酸残基位、第341个氨基酸残基位，及第343个氨基酸残基位。
  3. 如权利要求1所述的流感病毒神经氨酸酶突变体，其中该流感病毒神经氨酸酶突变体是在人类流感病毒神经氨酸酶的N2的一氨基酸残基位具有一突变，其中该氨基酸残基位是选自于下列所组成的组：第93个氨基酸残基位、第245个氨基酸残基位、第247个氨基酸残基位、第267个氨基酸残基位、第269个氨基酸残基位、第331个氨基酸残基位、第336个氨基酸残基位、第338个氨基酸残基位、第348个氨基酸残基位、第368个氨基酸残基位、第370个氨基酸残基位、第401个氨基酸残基位、第403个氨基酸残基位、第463个氨基酸残基位，及第465个氨基酸残基位。
  4. 如权利要求2或3所述的流感病毒神经氨酸酶突变体， 其中该突变是将氨基酸残基位取代为一天冬酰胺(asparagine，N)或一苏氨酸(threonine，T)。
  5. 如权利要求4所述的流感病毒神经氨酸酶突变体，其中在该人类流感病毒神经氨酸酶的N1的第221个氨基酸残基位与第223个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第270个氨基酸残基位与第272个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第273个氨基酸残基位与第275个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第329个氨基酸残基位与第331个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第332个氨基酸残基位具有一天冬酰胺取代、第335个氨基酸残基位与第337个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代，或第341个氨基酸残基位与第347个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代。
  6. 如权利要求4所述的流感病毒神经氨酸酶突变体，其中在该人类流感病毒神经氨酸酶的N2的第93个氨基酸残基位具有一天冬酰胺取代、第245个氨基酸残基位与第247个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第267个氨基酸残基位与第269个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第331个氨基酸残基位具有一苏氨酸取代、第336个氨基酸残基位与第338个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第348个氨基酸残基位具有一苏氨酸取代、第368个氨基酸残基位与第370个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代、第401个氨基酸残基位与第403个氨基 酸残基位分别具有一天冬酰胺与一苏氨酸取代，或第463个氨基酸残基位与第465个氨基酸残基位分别具有一天冬酰胺与一苏氨酸取代。
  7. 一种核酸分子，包含编码如权利要求5或6所述的流感病毒神经氨酸酶突变体的一核苷酸序列。
  8. 一种疫苗组合物，包含如权利要求1至6中任一项所述的流感病毒神经氨酸酶突变体。
  9. 如权利要求8所述的疫苗组合物，其中该流感病毒神经氨酸酶突变体是表达在一重组病毒上。
  10. 如权利要求9所述的疫苗组合物，其中该重组病毒包含如权利要求7所述的核酸分子。
  11. 如权利要求9所述的疫苗组合物，其中该重组病毒是一重组腺病毒。
  12. 一种如权利要求1至6中任一项所述的流感病毒神经氨酸酶突变体用于制备流感病毒疫苗组合物的用途。
  13. 如权利要求12所述的用途，其中该流感病毒疫苗组合物在一个体中引发抗多个流感病毒变体的免疫反应。
  14. 如权利要求12所述的用途，其中该流感病毒疫苗组合物引发高效价的抗原专一性抗体及/或神经氨酸酶抑制IC50效价。
PCT/CN2023/124604 2022-10-14 2023-10-13 流感病毒神经氨酸酶突变体、编码流感病毒神经氨酸酶突变体的核酸分子、包含流感病毒神经氨酸酶突变体的疫苗组合物及流感病毒神经氨酸酶突变体用于制备流感病毒疫苗组合物的用途 WO2024078631A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5962298A (en) * 1994-01-11 1999-10-05 Vlaams Interuniversitair Instituut Voor Biotechnologie Influenza vaccine
CN101983069A (zh) * 2006-08-09 2011-03-02 米迪缪尼有限公司 流感血凝素和神经氨酸酶变体
CN105400753A (zh) * 2014-09-04 2016-03-16 吴夙钦 重组神经氨酸酶蛋白及其应用
US20160287692A1 (en) * 2012-11-16 2016-10-06 Medimmune, Llc Swine influenza hemagglutinin and neuraminidase variants

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5962298A (en) * 1994-01-11 1999-10-05 Vlaams Interuniversitair Instituut Voor Biotechnologie Influenza vaccine
CN101983069A (zh) * 2006-08-09 2011-03-02 米迪缪尼有限公司 流感血凝素和神经氨酸酶变体
US20160287692A1 (en) * 2012-11-16 2016-10-06 Medimmune, Llc Swine influenza hemagglutinin and neuraminidase variants
CN105400753A (zh) * 2014-09-04 2016-03-16 吴夙钦 重组神经氨酸酶蛋白及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHU, FULU ET AL.: "Role of N-linked glycosylation of the human parainfluenza virus type 3 hemagglutinin-neuraminidase protein", VIRUS RESEARCH, 3 April 2013 (2013-04-03), pages 137 - 147, XP028533805, DOI: 10.1016/j.virusres.2013.03.012 *
ZHENG QIN-MEI: "Progress on Neuraminidase Protein Function of Influenza A Virus ", PROGRESS IN VETERINARY MEDICINE, vol. 42, no. 10, 20 October 2021 (2021-10-20), pages 66 - 72, XP093159344, DOI: 10.16437/j.cnki.1007-5038.2021.10.012 *

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