WO2019076218A1 - H3n2亚型流感病毒血凝素蛋白的突变体及其应用 - Google Patents

H3n2亚型流感病毒血凝素蛋白的突变体及其应用 Download PDF

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WO2019076218A1
WO2019076218A1 PCT/CN2018/109589 CN2018109589W WO2019076218A1 WO 2019076218 A1 WO2019076218 A1 WO 2019076218A1 CN 2018109589 W CN2018109589 W CN 2018109589W WO 2019076218 A1 WO2019076218 A1 WO 2019076218A1
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mutant
protein
amino acid
residue
influenza virus
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PCT/CN2018/109589
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French (fr)
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陈毅歆
沈晨光
陈俊煜
张梦娅
张丽敏
夏宁邵
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厦门大学
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Priority to EP18869363.4A priority Critical patent/EP3699186A4/en
Priority to US16/756,973 priority patent/US11426459B2/en
Priority to JP2020521995A priority patent/JP7009625B2/ja
Priority to AU2018351209A priority patent/AU2018351209B2/en
Priority to CA3079486A priority patent/CA3079486A1/en
Publication of WO2019076218A1 publication Critical patent/WO2019076218A1/zh

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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16171—Demonstrated in vivo effect

Definitions

  • This application relates to the field of virology and immunology.
  • the present application relates to mutants of the H3N2 subtype influenza virus hemagglutinin protein and uses thereof.
  • the present application relates to a pharmaceutical composition (for example, a vaccine) comprising the mutant, a method of preparing the mutant, and the use of the mutant to prevent and/or treat an infection of an influenza virus and/or A method of treating a disease caused by an infection, such as influenza.
  • Influenza viruses are a major threat to human health, and their continued rapid antigenic drift has allowed seasonal influenza to spread widely among people.
  • Common human seasonal influenza viruses include seasonal H1N1, seasonal H3N2, and influenza B viruses.
  • seasonal flu causes at least 250,000-500,000 deaths per year (Peter D.C. et al., J Clin Invest. 2008, 118:3273-3275).
  • the outbreak of influenza is still a major threat to civilization. Since the discovery of the flu virus, there have been five world-wide influenza pandemics in human history, resulting in tens of millions of deaths, including a 1918 Spanish flu outbreak that killed approximately 20-50 million people worldwide. .
  • influenza virus belongs to the Orthomyxoviridae family, an influenza virus genus, and is an enveloped single-stranded negative sense RNA virus.
  • the genome of the influenza virus encodes more than ten viral proteins.
  • influenza viruses are classified into three types: A (A), B (B), and C (C) (Horimoto T. et al. , Nat Rev Microbiol, 2005, 3(8): 591-600).
  • A A
  • B B
  • C C
  • Influenza A Virus has rapid mutation and strong pathogenicity, which can cause pandemics worldwide.
  • Influenza B virus (Flu B) has a slower variation and can only cause a small epidemic in a local area.
  • Influenza C virus has the slowest variation and is weak in pathogenicity. It usually only infects pregnant women and children with low resistance. In nature, Flu A has a wide range of hosts, and in addition to its natural host waterfowl, it can cause infections in a variety of animals such as humans, horses, and pigs. Flu A has many subtypes and large variations, and has become the focus of influenza prevention and control research.
  • Flu A virus can be divided into multiple subtypes depending on the surface antigen hemagglutinin protein (HA) and neuraminidase (NA) antigenicity and gene characteristics. Eighteen HA subtypes (H1-H18) and eleven NA subtypes (N1-N11) have been discovered (Tong S. et al., PLoS Pathog. 2013; 9(10): e1003657).
  • the popular Flu A in the population mainly involves two HA subtypes (H1, H3) and two NA subtypes (N1, N2), while the highly pathogenic avian influenza viruses H5N1 and H7N9 also have occasional infections, and It has received wide attention due to its higher mortality rate.
  • the flu vaccine is the most effective means of fighting the flu virus.
  • the main target of current influenza vaccine-induced antiviral antibodies is the hemagglutinin (HA) protein located on the surface of the virus.
  • the HA protein has a trimer structure on the surface of the virus, wherein each HA monomer consists of two domains, HA1 and HA2.
  • HA1 is located at the head of the trimer and constitutes a globular structure containing a receptor binding site, which is a key region for viral infection of host cells.
  • HA1 as it contains important antigenic sites, induces the production of protective neutralizing antibodies, and has become a key target for vaccine design (Wang T.T. et al., Nat Struct Mol Biol. 2009, 16:233-234).
  • HA2 is located at the base of the trimer and has a stalk-like structure containing a fusion peptide that mediates fusion of the viral envelope with the host cell membrane. It has been reported that some monoclonal antibodies against HA2 are capable of neutralizing the virus by inhibiting viral membrane fusion (Wang T. T. et al., Nat Struct Mol Biol. 2009, 16: 233-234).
  • Influenza viruses are highly variable, with HA mutations being the most rapid.
  • the current traditional vaccine is mainly directed to HA protein. Due to the high variability of the HA gene, the vaccine is ineffective due to antigenic drift.
  • WHO annually selects or establishes new vaccine strains as vaccine candidates for the next season's epidemic season based on the monitoring of the mutations of the epidemic strains in the previous year. New vaccines are vaccinated each year to ensure effective protection against existing strains of the epidemic.
  • current influenza vaccines require adjustment of vaccine candidate strains based on the antigenic variation of the virus strains prevailing in the previous year, which is time consuming and labor intensive. Therefore, the development of "broad-spectrum vaccines" that are not affected by viral mutations has gradually become a hot spot in new vaccine research.
  • unmodified natural HA protein can only induce narrow-band immunoprotection as a vaccine, it has been proposed to modify the natural HA protein to obtain a vaccine that can induce a broad-spectrum immune response, avoiding rapid mutation due to HA. Causes the vaccine to fail quickly.
  • influenza virus HA protein subtypes are numerous and have complex post-translational glycosyl modifications, so no significant progress has been made in this area of research. There is still a need in the art to develop HA mutants that are capable of inducing a broad spectrum of anti-influenza virus protective antibodies in vivo and capable of providing a broad spectrum of anti-influenza virus protection in vivo.
  • HA is a glycoprotein whose both HA1 and HA2 domains contain a glycosylation site carrying an N-linked glycosyl chain (Keil W et al. (1985) EMBO J 4:2711-2720).
  • the resulting HA protein carries an N-linked glycosyl chain in both the HA1 and HA2 domains;
  • the resulting HA trimer will carry an N-linked glycosyl chain in its head and stem regions (Fig. 2A).
  • the inventors of the present application have found through extensive research that the H3N2 subtype influenza virus HA protein is completely modified to completely remove the N-linked glycosyl chain carried thereby, thereby enhancing the HA protein-induced broad-spectrum protective antibody.
  • the ability to induce protective antibodies recognizes more subtypes of influenza viruses with a broader spectrum of protection.
  • the inventors of the present application have developed a mutant of the H3N2 subtype influenza virus hemagglutinin protein which does not contain an N-linked glycosylation site (eg, does not contain the signature sequence NX-(S or T) It can induce a broad spectrum of anti-influenza virus protective antibodies in vivo, and can provide a broad spectrum of anti-influenza virus protection in vivo.
  • mutants disclosed herein are capable of eliciting protective antibodies against different subtypes of influenza virus, achieving protection against influenza viruses of different subtypes, and thus can be used to be resistant to multiple subtypes (eg, at least 2 a broad-spectrum vaccine of influenza virus of at least 3 or more subtypes for the prevention and/or treatment of influenza viruses of various subtypes (eg, at least 2, at least 3 or more subtypes) Infections and diseases associated with the infection (eg, influenza).
  • multiple subtypes eg, at least 2 a broad-spectrum vaccine of influenza virus of at least 3 or more subtypes for the prevention and/or treatment of influenza viruses of various subtypes (eg, at least 2, at least 3 or more subtypes)
  • influenza viruses of various subtypes eg, at least 2, at least 3 or more subtypes
  • the mutants derived from the H3N2 subtype influenza virus HA protein disclosed in the present application are capable of inducing not only protective antibodies against a plurality of H3N2 subtype influenza viruses, particularly a plurality of H3N2 subtype influenza viruses prevalent in different ages. It can protect against multiple H3N2 subtype influenza viruses, and can induce protective antibodies against H7N9 and/or H1N1 subtype influenza viruses, and achieve protection against H7N9 and/or H1N1 subtype influenza viruses.
  • mutants derived from the H3N2 subtype influenza virus HA protein are particularly suitable for use as broad-spectrum vaccines for the prevention and/or treatment of H3N2, H7N9 and/or H1N1 subtype influenza virus infections and diseases associated therewith. .
  • the present application relates to a mutant of the H3N2 subtype influenza virus hemagglutinin protein that does not contain an N-linked glycosylation site. Such mutants do not contain an N-linked glycosyl chain due to the absence of an N-linked glycosylation site.
  • the present application provides a mutant of a H3N2 subtype influenza virus hemagglutinin protein, wherein the wild type hemagglutinin protein of the H3N2 subtype influenza virus is compared to the wild type hemagglutinin protein of the H3N2 subtype influenza virus
  • the mutant does not contain an N-linked glycosylation site, and, optionally, the mutant does not comprise an N-terminal signal peptide and/or a transmembrane region of the wild-type hemagglutinin protein.
  • N-linked glycosylation is a post-translational modification of a polypeptide, which means that the glycosyl chain is linked to a free -NH 2 group on a particular asparagine residue in the polypeptide chain.
  • the N-linked glycosylation process is usually carried out in the endoplasmic reticulum (ER) and the Golgi apparatus (GA).
  • the mutant differs from the wild-type hemagglutinin protein of the H3N2 subtype influenza virus by at least the N-linked glycosylation sites of the wild-type hemagglutinin protein.
  • the asparagine residues at the points are each independently deleted or replaced with one or more additional amino acid residues (eg, a non-N amino acid residue).
  • the N-linked glycosylation site in the influenza virus HA protein can be determined by various known methods (see, Tate MD. et al., Viruses. 6(3): 1294-316). For example, the prediction and determination of N-linked glycosylation sites can be performed using a computer program or software (eg, protein sequence analysis software package Antheprot 5.0).
  • the N-linked glycosylated amino acid is usually the asparagine (N) in the characteristic sequence NX-(S or T), wherein N represents asparagine and X represents deuterium. Any amino acid other than amino acid, S represents serine and T represents threonine.
  • the mutant differs from the wild type hemagglutinin protein of the H3N2 subtype influenza virus by at least that the mutant does not comprise the signature sequence NX-(S or T) Wherein N represents asparagine, X represents any amino acid other than proline, S represents serine, and T represents threonine.
  • the mutant differs from the wild type hemagglutinin protein of the H3N2 subtype influenza virus at least in each of the wild type hemagglutinin proteins NX-(S or T) each independently has one or more mutations selected from the group consisting of:
  • N residue is deleted or substituted with one or more other amino acid residues (eg, a non-N amino acid residue);
  • the (S or T) residue is deleted or substituted with one or more other amino acid residues (eg, a non-S and non-T amino acid residue);
  • N asparagine
  • X represents any amino acid other than proline
  • S represents serine
  • T represents threonine
  • the mutant does not contain any characteristic sequence N-X-(S or T).
  • Each of the wild type hemagglutinin proteins NX-(S or T) can be individually modified in various known ways such that the resulting mutant does not contain any characteristic sequence NX-(S or T).
  • the signature sequence NX-(S or T in wild-type hemagglutinin protein can be engineered by deleting the N residue or replacing the N residue with one or more additional amino acid residues. ) to remove the N-glycosylation site.
  • the N-glycosylation site can be removed by engineering the signature sequence N-X-(S or T) in the wild-type hemagglutinin protein by deleting the N residue.
  • the N-glycosyl group can be engineered by replacing the N-residue with a non-N amino acid residue to modulate the characteristic sequence NX-(S or T) in the wild-type hemagglutinin protein. Chemical site.
  • the signature sequence in the wild-type hemagglutinin protein can be engineered by replacing the N residue with at least two or more (eg, two, three or four) amino acid residues. NX-(S or T), thereby removing the N-glycosylation site, provided that the last amino acid residue of the at least two or more amino acid residues is a non-N amino acid residue.
  • the wild-type hemagglutinin protein can be engineered by deleting the (S or T) residue or replacing the (S or T) residue with one or more additional amino acid residues.
  • the characteristic sequence NX-(S or T) is removed to remove the N-glycosylation site.
  • the N-glycosylation site can be removed by engineering the signature sequence N-X-(S or T) in the wild-type hemagglutinin protein by deleting the (S or T) residue.
  • the signature sequence NX-(S or T) in wild-type hemagglutinin protein can be engineered by replacing the (S or T) residue with a non-S and non-T amino acid residue.
  • the wild-type hemagglutinin protein can be engineered by replacing the (S or T) residue with at least two or more (eg, two, three or four) amino acid residues.
  • a characteristic sequence NX-(S or T) to remove the N-glycosylation site provided that the first amino acid residue of the at least two or more amino acid residues is non-S and non-T Amino acid residues.
  • the signature sequence NX-(S or T) in the wild-type hemagglutinin protein can be engineered by deleting the X residue or replacing the X residue with a proline residue.
  • N-glycosylation site In certain preferred embodiments, the N-glycosylation site can be removed by engineering the signature sequence N-X-(S or T) in the wild-type hemagglutinin protein by deleting the X residue. In certain preferred embodiments, the N-glycosylation site can be removed by engineering the characteristic sequence NX-(S or T) in the wild-type hemagglutinin protein by replacing the X residue with a proline residue. point.
  • the signature sequence NX-(S or T) in the wild-type hemagglutinin protein can be engineered by adding one or more amino acid residues between the N residue and the X residue, thereby The N-glycosylation site is removed.
  • the signature sequence NX-(S or T) in the wild-type hemagglutinin protein can be engineered by adding a non-N amino acid residue between the N residue and the X residue, thereby The N-glycosylation site is removed.
  • wild-type hemagglutinin can be engineered by adding at least two or more (eg, two, three, or four) amino acid residues between the N residue and the X residue. a characteristic sequence NX-(S or T) in the protein, thereby removing the N-glycosylation site, provided that the last amino acid residue of the at least two or more amino acid residues is a non-N amino acid residue .
  • the signature sequence NX-(S or in the wild-type hemagglutinin protein can be engineered by adding one or more amino acid residues between the X residue and the (S or T) residue. T) to remove the N-glycosylation site.
  • the signature sequence NX- in the wild-type hemagglutinin protein can be engineered by adding a non-S and non-T amino acid residue between the X residue and the (S or T) residue. (S or T) to remove the N-glycosylation site.
  • wild-type hemagglutinin can be engineered by adding at least two or more (eg, two, three, or four) amino acid residues between the N residue and the X residue. a characteristic sequence NX-(S or T) in the protein, thereby removing the N-glycosylation site, provided that the first amino acid residue of the at least two or more amino acid residues is non-S and non- Amino acid residue of T.
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus at least in each of the characteristic sequences NX-(S or T) in the wild-type hemagglutinin protein.
  • N residues and/or (S or T) residues are each independently deleted or substituted with one or more additional amino acid residues (eg, another amino acid residue); wherein N represents asparagine and X represents Any amino acid other than proline, S represents serine and T represents threonine, whereby the mutant does not contain any characteristic sequence NX-(S or T).
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus at least in each of the characteristic sequences NX-(S or T) in the wild-type hemagglutinin protein.
  • the N residues are each independently deleted or replaced with one or more additional amino acid residues (eg, a non-N amino acid residue).
  • wild type hemagglutination can be engineered by deleting asparagine residues at each N-linked glycosylation site, particularly in the signature sequence NX-(S or T).
  • the protein is thus produced; thus, the resulting mutant no longer contains any N-linked glycosylation sites and no longer carries any N-linked glycosyl chains.
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus at least in that each of the characteristic sequences NX-(S or T) in the wild-type hemagglutinin protein The asparagine residues in the group were deleted.
  • each of the asparagine residues at each of the N-linked glycosylation sites can be independently replaced by one or Multiple other amino acid residues (eg, a non-N amino acid residue) to engineer the wild-type hemagglutinin protein; thus, the resulting mutant no longer contains any N-linked glycosylation sites and is no longer carried Any N-linked glycosyl chain.
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus at least in that each of the characteristic sequences NX-(S or T) in the wild-type hemagglutinin protein
  • the asparagine residues in each are each independently replaced with one or more additional amino acid residues (eg, a non-N amino acid residue).
  • the asparagine residues at some of the N-linked glycosylation sites may be deleted and the remaining N-
  • the asparagine residues at the linked glycosylation site are each independently replaced with one or more other amino acid residues (eg, a non-N amino acid residue)
  • the resulting mutant no longer contains any N-linked glycosylation sites and no longer carries any N-linked glycosyl chains.
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus by at least some of the characteristic sequences NX-(S or T) in the wild-type hemagglutinin protein.
  • the asparagine residue in the residue is deleted, and the asparagine residues in the remaining characteristic sequences NX-(S or T) are each independently substituted with one or more other amino acid residues (eg, a non-N amino acid) Residues).
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus at least in each of the characteristic sequences NX-(S or T) in the wild-type hemagglutinin protein.
  • the (S or T) residues are each independently deleted or replaced with one or more additional amino acid residues (eg, a non-S and non-T amino acid residue).
  • the wild-type hemagglutinin protein can be engineered by deleting the (S or T) residue in each of the characteristic sequences NX-(S or T); thus, the resulting mutant is not It also contains any N-linked glycosylation sites that no longer carry any N-linked glycosyl chains.
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus at least in that each of the characteristic sequences NX-(S or T) in the wild-type hemagglutinin protein The (S or T) residues in the are deleted.
  • the (S or T) residues in each of the characteristic sequences NX-(S or T) can each be independently replaced with one or more other amino acid residues (eg, one non-S and Non-T amino acid residues) to engineer wild-type hemagglutinin proteins; thus, the resulting mutants no longer contain any N-linked glycosylation sites and no longer carry any N-linked glycosyl chains.
  • one or more other amino acid residues eg, one non-S and Non-T amino acid residues
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus at least in that each of the characteristic sequences NX-(S or T) in the wild-type hemagglutinin protein
  • the (S or T) residues in each are each independently replaced with one or more additional amino acid residues (eg, a non-S and non-T amino acid residue).
  • the (S or T) residues in some of the signature sequences NX-(S or T) may be deleted and the remaining characteristic sequences NX-(S or T) (S or The T) residues are each independently replaced with one or more other amino acid residues (eg, a non-S and non-T amino acid residue) to engineer the wild-type hemagglutinin protein; thus, the resulting mutant is no longer included Any N-linked glycosylation site no longer carries any N-linked glycosyl chains.
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus by at least some of the characteristic sequences NX-(S or T) in the wild-type hemagglutinin protein.
  • (S or T) residues are deleted, and the (S or T) residues in the remaining characteristic sequences NX-(S or T) are each independently replaced with one or more other amino acid residues (eg, one Non-S and non-T amino acid residues).
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus by at least some of the characteristic sequences in the wild-type hemagglutinin protein NX-(S or T)
  • the N residues are each independently deleted or substituted with one or more other amino acid residues (eg, a non-N amino acid residue); and, in the remaining characteristic sequence NX-(S or T) (S or T)
  • the residues are each independently deleted or replaced with one or more other amino acid residues (eg, a non-S and non-T amino acid residue).
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus at least in that each of the characteristic sequences NX-(S or T) in the wild-type hemagglutinin protein is Independently having a mutation selected from the group consisting of:
  • N residue is deleted or replaced with an additional amino acid residue (eg, a non-N amino acid residue);
  • any amino acid residue in a polypeptide chain can be engineered (eg, deleted or replaced) by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • the amino acid residue of the asparagine residue used to replace the N-linked glycosylation site may be selected from One or more of the following amino acid residues: alanine, glycine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, serine, tyrosine, cysteine Amino acid, methionine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.
  • the amino acid residue of the asparagine residue used to replace the N-linked glycosylation site may be selected from One of the following amino acid residues: alanine, glycine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, serine, tyrosine, cysteine, Methionine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.
  • the amino acid residue of the asparagine residue used to replace the N-linked glycosylation site may be alanine Acid residue.
  • the asparagine residue at each N-linked glycosylation site is replaced with alanine.
  • the amino acid residue used to replace the asparagine residue at the N-linked glycosylation site may be glutamine Amide residue.
  • the asparagine residue at each N-linked glycosylation site is replaced with glutamine.
  • the amino acid residue used for the (S or T) residue in the signature sequence NX-(S or T) may be one or more amino acid residues selected from the group consisting of: alanine , glycine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, tyrosine, cysteine, methionine, glutamine, aspartate Acid, glutamic acid, lysine, arginine and histidine.
  • the amino acid residue used to replace the (S or T) residue in the signature sequence NX-(S or T) may be one amino acid residue selected from the group consisting of alanine, glycine , valine, leucine, isoleucine, phenylalanine, valine, tryptophan, tyrosine, cysteine, methionine, glutamine, aspartic acid, Glutamate, lysine, arginine and histidine.
  • HA proteins In addition, with the development of protein crystallization and structural analysis techniques, the research and understanding of the function and properties of HA proteins is also deepening. Thus, by means of a computer program or software (eg PyMol), asparagine residues and (S or T) Position and conformation of residues in the HA trimer. Based on this, in combination with the physicochemical properties of amino acid residues (eg size, shape, charge, ability to form covalent bonds or hydrogen bonds, etc.), suitable amino acid residues can be selected for replacement of asparagine residues and (S or T) residue. For example, it is known in the art that conservative substitutions can be made to a protein or polypeptide without significantly affecting or altering the function and properties of the protein or polypeptide.
  • a computer program or software eg PyMol
  • amino acid residues eg size, shape, charge, ability to form covalent bonds or hydrogen bonds, etc.
  • suitable amino acid residues can be selected for replacement of asparagine residues and
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus at least in that each N-linked glycosylation site in the wild-type hemagglutinin protein
  • the asparagine residues on the upper are each independently conservatively substituted.
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus at least in each of the N-linked glycosylation sites in the wild-type hemagglutinin protein (
  • the asparagine residues in the signature sequence NX-(S or T) are each independently conservatively substituted with amino acid residues selected from the group consisting of alanine, glycine, glutamine, serine, threonine, Tyrosine, cysteine, tryptophan.
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus at least in each of the characteristic sequences NX-(S or T) in the wild-type hemagglutinin protein.
  • the (S or T) residues are each independently conservatively replaced by non-S and non-T amino acid residues.
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus at least in each of the characteristic sequences NX-(S or T) in the wild-type hemagglutinin protein.
  • the (S or T) residues are each independently conservatively substituted with an amino acid residue selected from the group consisting of alanine, glycine, asparagine, glutamine, tyrosine, cysteine, tryptophan.
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus at least in that each of the characteristic sequences NX-(S or T) in the wild-type hemagglutinin protein is Independently having a mutation selected from the group consisting of:
  • N residues are deleted or conservatively replaced
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus by at least some of the characteristic sequences in the wild-type hemagglutinin protein NX-(S or T)
  • the N residues are each independently conservatively substituted; and, the (S or T) residues in the remaining characteristic sequences NX-(S or T) are each independently conservatively replaced by non-S and non-T amino acid residues.
  • the mutant differs from the wild-type hemagglutinin protein of the influenza virus by at least some of the characteristic sequences in the wild-type hemagglutinin protein NX-(S or T)
  • the N residues are each independently conservatively substituted with an amino acid residue selected from the group consisting of alanine, glycine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan;
  • the (S or T) residues in the characteristic sequence NX-(S or T) are each independently conservatively substituted with amino acid residues selected from the group consisting of alanine, glycine, asparagine, glutamine, tyramine Acid, cysteine, tryptophan.
  • the signal peptide of the protein (usually located at the N-terminus of the protein) is capable of directing/promoting protein secretion, and that the signal peptide can be cleaved during secretion or after secretion without affecting the function of the protein.
  • the mutant does not contain an N-linked glycosylation site and does not comprise the wild type hemagglutinin protein of the H3N2 subtype influenza virus.
  • a signal peptide of a wild-type hemagglutinin protein for example, an N-terminal signal peptide).
  • the transmembrane region of a protein typically directs/promotes protein anchoring to the membrane (eg, cell membrane or viral envelope).
  • deletion of the transmembrane region of a protein does not adversely affect the biological activity of the protein (eg, immunogenicity and immunoprotection).
  • the mutant does not contain an N-linked glycosylation site and does not comprise the wild type hemagglutinin protein of the H3N2 subtype influenza virus. Transmembrane region of wild-type hemagglutinin protein.
  • the position and sequence of the signal peptide in the influenza virus HA protein as well as the position and sequence of the transmembrane region can be determined by various known methods (see, for example, TMTumpey et al., Proc. Natl. Acad. Sci. USA 99, 13849 ( 2002)). In addition, signal peptides and transmembrane regions of various HA proteins have also been reported (see, for example, James Stevens et al. Science 312, 404 (2006)). Therefore, the position and sequence of the signal peptide and transmembrane region of various HA proteins can be conveniently determined and modified (for example, deleted).
  • the mutant does not contain an N-linked glycosylation site and does not comprise the wild type compared to the wild type hemagglutinin protein of the H3N2 subtype influenza virus.
  • a signal peptide of a hemagglutinin protein eg, an N-terminal signal peptide
  • a transmembrane region e.g., an N-terminal signal peptide
  • the wild-type hemagglutinin protein is derived from an influenza A virus H3N2 subtype, such as an H3N2 subtype influenza virus that is prevalent after 2005, such as A/WISCONSIN/67/2005 (H3N2) ) and A/HONG_KONG/4801/2014 (H3N2).
  • the wild-type hemagglutinin protein has a sequence selected from the group consisting of SEQ ID NOs: 1 and 6.
  • the amino acid sequence of the wild-type hemagglutinin protein is set forth in SEQ ID NO: 1; and wherein the mutant differs from SEQ ID NO: 1 at least in that the mutant
  • the characteristic sequence NX-(S or T) is not included; wherein N represents asparagine, X represents any amino acid other than proline, S represents serine, and T represents threonine.
  • the amino acid sequence of the wild-type hemagglutinin protein is set forth in SEQ ID NO: 1; and wherein the mutant differs from SEQ ID NO: 1 at least in that SEQ ID NO:
  • Each of the characteristic sequences NX-(S or T) of 1 independently has a mutation selected from the group consisting of: (1) the N residue is deleted or replaced with one or more other amino acid residues (eg, a non-N (2) (S or T) residues are deleted or substituted with one or more other amino acid residues (eg, a non-S and non-T amino acid residue); (3) X residues are Delete or replace with a proline residue; (4) add a non-N amino acid residue between the N residue and the X residue; (5) add between the X residue and the (S or T) residue a non-S and non-T amino acid residue; and, (6) any combination of (1) to (5).
  • the mutant differs from SEQ ID NO: 1 further in that the mutant does not comprise a signal peptide (e.g., amino acids 1-10 of SEQ ID NO: 1). In certain preferred embodiments, the mutant differs from SEQ ID NO: 1 further in that the mutant does not comprise a transmembrane region (e.g., amino acids 504-550 of SEQ ID NO: 1). In certain preferred embodiments, the mutant differs from SEQ ID NO: 1 further in that the mutant does not comprise a signal peptide (eg, amino acids 1-10 of SEQ ID NO: 1) and a transmembrane Region (eg, amino acids 504-550 of SEQ ID NO: 1).
  • the amino acid sequence of the wild-type hemagglutinin protein is set forth in SEQ ID NO: 1; and wherein the mutant differs from SEQ ID NO: 1 at least in that SEQ ID NO: Amino acids 1-10 and 504-550 of 1 are deleted, and asparagine at positions 22, 38, 63, 126, 133, 144, 165, 246, 285 and 483 of SEQ ID NO: 1.
  • the residues are each independently deleted or substituted with one or more additional amino acid residues (eg, a non-N amino acid residue, such as an alanine residue or a glutamine residue).
  • the amino acid sequence of the wild-type hemagglutinin protein is set forth in SEQ ID NO: 6; and wherein the mutant differs from SEQ ID NO: 6 at least in that the mutant
  • the characteristic sequence NX-(S or T) is not included; wherein N represents asparagine, X represents any amino acid other than proline, S represents serine, and T represents threonine.
  • the amino acid sequence of the wild-type hemagglutinin protein is set forth in SEQ ID NO: 6; and wherein the mutant differs from SEQ ID NO: 6 at least in that SEQ ID NO:
  • Each of the characteristic sequences NX-(S or T) of 6 independently has a mutation selected from the group consisting of: (1) the N residue is deleted or replaced with one or more other amino acid residues (eg, a non-N (2) (S or T) residues are deleted or substituted with one or more other amino acid residues (eg, a non-S and non-T amino acid residue); (3) X residues are Delete or replace with a proline residue; (4) add a non-N amino acid residue between the N residue and the X residue; (5) add between the X residue and the (S or T) residue a non-S and non-T amino acid residue; and, (6) any combination of (1) to (5).
  • the mutant differs from SEQ ID NO: 6 in that the mutant does not comprise a signal peptide (e.g., amino acids 1-25 of SEQ ID NO: 6). In certain preferred embodiments, the mutant differs from SEQ ID NO: 6 in that the mutant does not comprise a transmembrane region (e.g., amino acids 518-565 of SEQ ID NO: 6). In certain preferred embodiments, the mutant differs from SEQ ID NO: 1 further in that the mutant does not comprise a signal peptide (eg, amino acids 1-25 of SEQ ID NO: 6) and a transmembrane Region (eg, amino acids 518-565 of SEQ ID NO: 6).
  • a signal peptide e.g., amino acids 1-25 of SEQ ID NO: 6
  • transmembrane Region e.g, amino acids 518-565 of SEQ ID NO: 6
  • the amino acid sequence of the wild-type hemagglutinin protein is set forth in SEQ ID NO: 6; and wherein the mutant differs from SEQ ID NO: 6 at least in that SEQ ID NO:
  • the amino acids at positions 1-25 and 518-565 of 6 are deleted, and the days at positions 37, 53, 60, 78, 137, 141, 148, 180, 261, 300 and 498 of SEQ ID NO: 6.
  • the asparagine residues are each independently deleted or substituted with one or more additional amino acid residues (eg, a non-N amino acid residue, such as an alanine residue or a glutamine residue).
  • the mutant has an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-13.
  • amino acid sequence of a protein or polypeptide can be suitably engineered (e.g., addition, deletion, and/or substitution of amino acid residues) without significantly affecting the function and properties of the protein or polypeptide.
  • additional mutants can be obtained which retain the ability to elicit protective antibodies against different subtypes of influenza virus, with different subtypes The protective effect of the flu virus.
  • the mutant of the invention has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95 with an amino acid sequence selected from the group consisting of %, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity: SEQ ID NO: 12-13; with the proviso that the mutant does not comprise any N-linked glycosylation sites Point (for example, does not contain any feature sequence NX-(S or T)).
  • the mutant of the invention has the addition, deletion or substitution of one or more amino acid residues compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-13; The mutant does not comprise any N-linked glycosylation sites (eg, does not contain any of the signature sequences NX-(S or T)).
  • the mutant of the invention has one or several amino acid residues (eg, 1, 2, 3, 4, 5, compared to an amino acid sequence selected from the group consisting of Addition, deletion or substitution of 6, 7 , 8 , 9 ): SEQ ID NO: 12-13; provided that the mutant does not comprise any N-linked glycosylation sites (eg not included) Any feature sequence NX-(S or T)).
  • the mutant of the invention has one or several amino acid residues (eg, 1, 2, 3, 4, 5, compared to an amino acid sequence selected from the group consisting of Six, seven, eight, nine) substitutions (particularly conservative substitutions): SEQ ID NOs: 12-13; with the proviso that the mutant does not comprise any N-linked glycosylation sites (eg Does not contain any feature sequence NX-(S or T)).
  • amino acid residues eg, 1, 2, 3, 4, 5
  • substitutions particularly conservative substitutions: SEQ ID NOs: 12-13; with the proviso that the mutant does not comprise any N-linked glycosylation sites (eg Does not contain any feature sequence NX-(S or T)).
  • the mutant derived from the H3N2 subtype influenza virus HA protein disclosed in the present application does not contain a glycosylation site (for example, does not contain the signature sequence NX-(S or T)), and is capable of inducing a broad spectrum of anti-influenza virus protection in vivo.
  • the mutants disclosed herein are capable of eliciting protective antibodies against influenza viruses of different subtypes (eg, H3N2, H7N9 and/or H1N1 subtypes), achieving resistance to different subtypes (eg, H3N2, H7N9, and/or H1N1 sub- (type) influenza virus protection, and thus can be used as an influenza virus capable of resisting multiple subtypes (eg, at least 2, at least 3 or more subtypes; eg, H3N2, H7N9, and/or H1N1 subtypes) Broad-spectrum vaccine for the prevention and/or treatment of influenza infections of various subtypes (eg, at least 2, at least 3 or more subtypes; eg, H3N2, H7N9, and/or H1N1 subtypes) and The infection-related disease (eg, influenza). Therefore, the mutants disclosed herein are particularly advantageous.
  • the mutants disclosed herein are particularly advantageous.
  • the present application relates to a recombinant protein comprising a mutant of the H3N2 subtype influenza virus hemagglutinin protein according to the present invention, and an additional peptide segment to which the additional peptide is linked.
  • the additional peptide can be linked to the mutant by various means.
  • the additional peptide is linked directly to the mutant.
  • the additional peptide is directly linked to the mutant by a peptide bond.
  • the additional peptide is linked to the mutant by a linker.
  • Suitable prior art linkers can be composed of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • linkers can be used, such as Alfthan et al. (1995), Protein Eng. 8: 725-731; Choi et al. (2001), Eur. J. Immunol. 31: 94-106; Hu et al. (1996). , Cancer Res. 56: 3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the additional peptide can be ligated to either end of the mutant.
  • the additional peptide is linked to the N-terminus of the mutant.
  • the additional peptide is linked to the C-terminus of the mutant.
  • the recombinant protein according to the invention may comprise one or more additional peptides.
  • a recombinant protein according to the invention may comprise at least 1, at least 2, at least 3, at least 5 or more additional peptides. It will be readily understood that these peptides can each independently be attached to either end of the mutant (N-terminus or C-terminus) in a variety of ways.
  • the recombinant protein of the invention may comprise two additional peptides, wherein one additional peptide is linked to the N-terminus of the mutant via a linker or not via a linker, and Another additional peptide is attached to the C-terminus of the mutant via a linker or not via a linker.
  • the recombinant protein of the invention may comprise two or more additional peptides, wherein the two or more additional peptides each independently pass through a linker or not through a linker Linked to the N-terminus or C-terminus of the mutant.
  • the two or more additional peptides when two or more additional peptides are attached to the N-terminus of the mutant, the two or more additional peptides can be placed in series in any order and then passed through the linker Alternatively, it is not linked to the N-terminus of the mutant by a linker. Similarly, in certain preferred embodiments, when two or more additional peptides are linked to the C-terminus of the mutant, the two or more additional peptides can be joined in series in any order. It is then ligated to the C-terminus of the mutant by a linker or not via a linker.
  • the additional peptide can be a signal peptide (eg, a signal peptide as set forth in SEQ ID NO: 9).
  • a signal peptide eg, a signal peptide as set forth in SEQ ID NO: 9
  • a signal peptide can be ligated to the N-terminus of the mutant.
  • the signal peptide can be cleaved to produce the desired mutant or recombinant protein.
  • the additional peptide can be a tag peptide, for example, a 6*His tag as set forth in SEQ ID NO:11.
  • a tag peptide facilitates the detection, recovery, and purification of recombinant proteins.
  • nickel ions can be used to purify a protein carrying a 6*His tag.
  • the additional peptide segment can be a folding motif that promotes the formation of a trimer of the mutant. Such folding motifs include, but are not limited to, a folding motif as set forth in SEQ ID NO: 10.
  • the additional peptide can be a detectable label, such as a fluorescent protein.
  • the additional peptide is selected from the group consisting of a signal peptide, a tag peptide, a folding motif, a detectable label, and any combination thereof.
  • the signal peptide has the amino acid sequence set forth in SEQ ID NO:9.
  • the signal peptide is linked to the N-terminus of the mutant.
  • the folding motif has the amino acid sequence set forth in SEQ ID NO: 10.
  • the folding motif is linked to the C-terminus of the mutant.
  • the tag peptide has the amino acid sequence set forth in SEQ ID NO:11.
  • the tag peptide is linked to the N-terminus or C-terminus of the mutant.
  • the recombinant protein can have an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 8.
  • amino acid sequence of a protein or polypeptide can be suitably engineered (e.g., addition, deletion, and/or substitution of amino acid residues) without significantly affecting the function and properties of the protein or polypeptide. Therefore, in some cases, by further engineering the amino acid sequence of the above recombinant protein, an additional recombinant protein can be obtained which retains the ability to induce protective antibodies against different subtypes of influenza virus, and has different subtypes. The protective effect of the flu virus.
  • the recombinant protein of the invention has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95 with an amino acid sequence selected from the group consisting of %, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity: SEQ ID NOS: 3 and 8; with the proviso that the recombinant protein or mutant does not comprise any N-linked sugar The basement site (eg, does not contain any feature sequences NX-(S or T)).
  • the recombinant protein of the invention has the addition, deletion or substitution of one or more amino acid residues compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 8;
  • the recombinant protein or the mutant does not comprise any N-linked glycosylation sites (eg, does not comprise any of the signature sequences NX-(S or T)).
  • the recombinant protein of the invention has one or several amino acid residues (eg, 1, 2, 3, 4, 5, compared to an amino acid sequence selected from the group consisting of Addition, deletion or substitution of 6, 7, 8, 9): SEQ ID NOS: 3 and 8; provided that the recombinant protein or the mutant does not comprise any N-linked glycosylation sites Point (for example, does not contain any feature sequence NX-(S or T)).
  • amino acid residues eg, 1, 2, 3, 4, 5, compared to an amino acid sequence selected from the group consisting of Addition, deletion or substitution of 6, 7, 8, 9
  • the recombinant protein of the invention has one or several amino acid residues (eg, 1, 2, 3, 4, 5, compared to an amino acid sequence selected from the group consisting of 6, (6, 8, 9) substitutions (particularly conservative substitutions): SEQ ID NOS: 3 and 8; provided that the recombinant protein or the mutant does not comprise any N-linked glycosylation
  • the site for example, does not contain any feature sequence NX-(S or T)).
  • nucleic acid molecules About nucleic acid molecules, vectors, viruses, and host cells
  • the present application relates to a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding a mutant of the invention or a recombinant protein of the invention.
  • the nucleic acid molecules of the invention are isolated or purified.
  • the present application is directed to a vector comprising a nucleic acid molecule as described above.
  • the vector of the present invention may be a cloning vector, a transfer vector, or an expression vector.
  • the vector of the invention is, for example, a plasmid, a cosmid, a bacteriophage or the like.
  • the vector is capable of expressing a mutant of the invention or a recombinant protein of the invention in a eukaryotic cell, such as an insect cell.
  • the vector is a baculovirus transfer vector that can be used with baculovirus genomic DNA to effect expression of a mutant of the invention or a recombinant protein of the invention in insect cells.
  • the baculovirus is a Spodoptera litura nuclear polyhedrosis virus (AcMNPV).
  • the invention also relates to a host cell comprising a nucleic acid molecule or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, such as mouse cells, human cells, etc.).
  • the host cell of the invention may also be a cell line, such as a 293T cell.
  • the host cell of the invention is a eukaryotic cell, such as an insect cell.
  • the host cell of the invention is an insect cell comprising a baculovirus transfer vector comprising the nucleic acid molecule described above, and a baculovirus genomic DNA.
  • the baculovirus is a Spodoptera litura nuclear polyhedrosis virus (AcMNPV).
  • the invention also relates to a virus (eg, a baculovirus) comprising a nucleic acid molecule or vector as described above.
  • a virus eg, a baculovirus
  • the virus is a baculovirus, such as the Spodoptera litura nuclear polyhedrosis virus (AcMNPV).
  • the present application relates to a multimer comprising a plurality of mutants of the invention or a plurality of recombinant proteins of the invention, or consisting of a plurality of mutants of the invention or a plurality of recombinant proteins of the invention.
  • the multimer is a trimer.
  • the multimer comprises or consists of three mutants or recombinant proteins of the invention.
  • the trimer has the same or similar conformation as the trimer formed from the native HA protein.
  • the present application is also directed to a composition
  • a composition comprising the above mutant, or the above recombinant protein, or the above nucleic acid molecule, or the above vector, or the above host cell, or the above virus, or the above multimer.
  • the composition comprises a mutant or recombinant protein of the invention.
  • the composition comprises a multimer of the invention.
  • the invention also relates to a pharmaceutical composition (eg, a vaccine) comprising a mutant or recombinant protein or multimer of the invention, optionally further comprising a pharmaceutically acceptable carrier and/or excipient .
  • a pharmaceutical composition of the present invention (for example, a vaccine) can be used for preventing or treating an influenza virus infection or a disease caused by an influenza virus infection such as influenza or the like.
  • the mutant or recombinant protein or multimer of the invention is present in an amount effective to prevent or treat an influenza virus infection or a disease caused by an influenza virus infection.
  • the pharmaceutical compositions (e.g., vaccines) of the invention further comprise additional active ingredients.
  • the additional active ingredient is capable of preventing or treating an influenza virus infection or a disease caused by an influenza virus infection.
  • the pharmaceutical compositions (e.g., vaccines) of the invention further comprise an adjuvant, such as an aluminum adjuvant.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, stabilizer or capable of providing advantageous properties for administration (e.g., administration to a human subject) of the pharmaceutical composition.
  • Suitable pharmaceutical carriers include, for example, sterile water, saline, dextrose, a condensation product of castor oil and ethylene oxide, a liquid acid, a lower alcohol (e.g., a C 1-4 alcohol), an oil (e.g., corn oil, peanut oil, sesame oil; It optionally further comprises an emulsifier such as a mono- or di-glyceride of a fatty acid or a phospholipid such as lecithin, ethylene glycol, polyalkylene glycol, sodium alginate, poly(vinylpyrrolidone) and the like.
  • the carrier optionally may further comprise an adjuvant, a preservative, a stabilizer, a wetting agent, an emulsifier, a penetration enhancer, and the like.
  • the pharmaceutical composition is sterile.
  • the viscosity of the pharmaceutical combination can be controlled and maintained by the selection of a suitable solvent or excipient.
  • the pharmaceutical composition is formulated to have a pH of 4.5-9.0, 5.0-8.0, 6.5-7.5, or 6.5-7.0.
  • compositions of the invention can be administered by methods well known in the art, such as, but not limited to, by oral or injection.
  • the pharmaceutical compositions (e.g., vaccines) of the invention are administered in unit dosage form.
  • the amount of the pharmaceutical composition of the invention (e.g., vaccine) required to prevent or treat a particular condition will depend on the route of administration, the severity of the condition being treated, the sex, age, weight and general health of the patient, and the like, which may be According to the actual situation, it is reasonable to determine.
  • the pharmaceutical composition of the invention (eg, a vaccine) comprises a mutant derived from an H3N2 subtype influenza virus HA protein or a recombinant protein or multimer comprising the mutant, which is capable of eliciting Protective antibodies against H3N2, H7N9 and/or H1N1 subtype influenza viruses, which protect against influenza viruses of the H3N2, H7N9 and/or H1N1 subtypes and, therefore, can be used for the prevention and/or treatment of H3N2, H7N9 and/or H1N1 Infection with influenza viruses and diseases associated with them (such as influenza).
  • a vaccine comprises a mutant derived from an H3N2 subtype influenza virus HA protein or a recombinant protein or multimer comprising the mutant, which is capable of eliciting Protective antibodies against H3N2, H7N9 and/or H1N1 subtype influenza viruses, which protect against influenza viruses of the H3N2, H7N9 and/or H1N1 subtypes and, therefore,
  • the invention in another aspect, relates to a method of preventing or treating an influenza virus infection or a disease caused by an influenza virus infection in a subject, comprising administering a prophylactically or therapeutically effective amount of a mutant or recombinant according to the invention
  • a protein or multimer or a pharmaceutical composition of the invention is administered to the subject.
  • the disease caused by infection with an influenza virus is influenza.
  • the subject is a mammal, such as a mouse and a human.
  • the methods of the invention are useful for preventing and/or treating infections of H3N2, H7N9 and/or H1N1 subtype influenza viruses and diseases associated therewith (eg, influenza).
  • H3N2, H7N9 and/or H1N1 subtype influenza viruses and diseases associated therewith eg, influenza.
  • the invention also relates to the use of a mutant or recombinant protein or multimer of the invention in the preparation of a pharmaceutical composition, such as a vaccine, for use in a subject Prevention or treatment of influenza virus infection or diseases caused by influenza virus infection.
  • a pharmaceutical composition such as a vaccine
  • Prevention or treatment of influenza virus infection or diseases caused by influenza virus infection is influenza.
  • the disease caused by infection with an influenza virus is influenza.
  • the subject is a mammal, such as a mouse and a human.
  • the pharmaceutical composition (eg, a vaccine) comprises a mutant derived from an H3N2 subtype influenza virus HA protein or a recombinant protein or multimer comprising the mutant for use in prevention and/or Or treatment of infection with H3N2, H7N9 and/or H1N1 subtype influenza viruses and diseases associated therewith (eg influenza).
  • the present invention also relates to the above mutant or recombinant protein or multimer for use in preventing or treating an influenza virus infection or a disease caused by an influenza virus infection in a subject.
  • the disease caused by infection with an influenza virus is influenza.
  • the subject is a mammal, such as a mouse and a human.
  • the mutant or recombinant protein or multimer is used to prevent and/or treat an infection of a H3N2, H7N9 and/or H1N1 subtype influenza virus and a disease associated therewith (eg, influenza) .
  • the present invention relates to a method of producing the above mutant or recombinant protein, which comprises culturing a host cell or virus of the present invention under conditions permitting expression of the mutant or recombinant protein; and, recycling Expressed mutant or recombinant protein.
  • the method comprises: introducing a vector of the invention (eg, an expression vector) into a host cell (eg, a eukaryotic cell), thereby expressing the mutant or recombinant protein in a host cell; The expressed mutant or recombinant protein is recovered.
  • the method comprises: introducing a baculovirus transfer vector comprising the nucleic acid molecule described above and a baculovirus genomic DNA into an insect cell to thereby express the mutant or recombinant protein in the insect cell; Recover the expressed mutant or recombinant protein.
  • the baculovirus is a Spodoptera litura nuclear polyhedrosis virus (AcMNPV).
  • the invention also relates to a method of preparing a vaccine comprising mixing a mutant or recombinant protein or multimer of the invention with a pharmaceutically acceptable carrier and/or excipient, optionally also Adjuvants such as aluminum adjuvants, and/or additional active ingredients, for example, additional active ingredients that are capable of preventing or treating influenza virus infection or diseases caused by influenza virus infection.
  • the method of preparing a vaccine comprises the step of mixing a mutant or recombinant protein or multimer of the invention with an adjuvant, such as an aluminum adjuvant.
  • the vaccine obtained can be used to prevent or treat influenza virus infection or diseases caused by influenza virus infection such as influenza.
  • identity is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the biological activity of a protein/polypeptide comprising an amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains in place of amino acid residues, for example, physically or functionally similar to corresponding amino acid residues (eg, having similar size, shape, charge, chemical properties, including Substitution of residues by formation of a covalent bond or a hydrogen bond, etc.).
  • a family of amino acid residues having similar side chains has been defined in the art.
  • These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoluminescence) Acid, valine, phenylalanine, methionine), beta branch side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, Amino acids of phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, and histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
  • Phage such as lambda phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus.
  • a prokaryotic cell such as Escherichia coli or Bacillus subtilis
  • a fungal cell such as a yeast cell or an Aspergillus.
  • S2 Drosophila cells or insect cells such as Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • corresponding sequence fragment or “corresponding fragment” means that when the sequences are optimally aligned, ie when the sequences are aligned to obtain the highest percentage identity, the sequences are compared. A fragment that is in the same position.
  • corresponding amino acid position means that when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percentage identity, the amino acid sites at the equivalent positions in the compared sequences are/ Residues.
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope” is also referred to in the art as an "antigenic determinant.”
  • An epitope or antigenic determinant typically consists of a chemically active surface group of a molecule, such as an amino acid or a carbohydrate or sugar side chain, and typically has specific three dimensional structural characteristics as well as specific charge characteristics.
  • an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear" "or” conformational.
  • the term "multimer” refers to a polymer composed of a polypeptide molecule (eg, a mutant or recombinant protein of the invention) as a monomer, which may typically comprise at least two (eg, 3) , 4, 5 or more) polypeptide monomers (eg, mutants or recombinant proteins of the invention).
  • monomer molecules polymerize to form multimers by intermolecular interactions (eg, hydrogen bonding, van der Waals forces, hydrophobic interactions).
  • the multimer is a trimer comprising 3 monomers.
  • the terms "isolated” or “isolated” refer to artificially obtained from a natural state. If a “separated” substance or ingredient appears in nature, it may be that the natural environment in which it is located has changed, or that it has been isolated from the natural environment, or both. For example, a certain living animal has a naturally isolated polynucleotide or polypeptide that is not isolated, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called separation. of.
  • the term “isolated” or “isolated” does not exclude the inclusion of artificial or synthetic materials, nor does it exclude the presence of other impure substances that do not affect the activity of the material.
  • characteristic sequence NX-(S or T) refers to a characteristic motif capable of N-linked glycosylation, wherein N represents asparagine and X represents proline-free Any amino acid other than that, S represents serine and T represents threonine.
  • protective antibody refers to an antibody that has a protective effect against viruses.
  • Protective antibodies include, but are not limited to, antibodies capable of neutralizing viral virulence, antibodies capable of inhibiting viral recognition and binding to host cells, and antibodies capable of inhibiting fusion of the virus with host cells.
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength Enhancer.
  • pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80; adjuvants include, but are not limited to, aluminum adjuvants (eg, hydroxides) Aluminum), Freund's adjuvant (eg complete Freund's adjuvant); ionic strength enhancers include, but are not limited to, sodium chloride.
  • surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80
  • adjuvants include, but are not limited to, aluminum adjuvants (eg, hydroxides) Aluminum), Freund's adjuvant (eg complete Freund's adjuvant); ionic strength enhancers include, but are not limited to, sodium chloride.
  • the term "adjuvant” refers to a non-specific immunopotentiator that, when brought together with an antigen or pre-delivered into the body, enhances the body's immune response to the antigen or alters the type of immune response.
  • adjuvants including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. .
  • Freund's adjuvant is the most commonly used adjuvant in animal testing.
  • Aluminum hydroxide adjuvant is used more in clinical trials. In the present invention, it is particularly preferred that the adjuvant is an aluminum adjuvant.
  • an effective amount refers to an amount that is effective to achieve the intended purpose.
  • an effective amount for preventing or treating a disease means that it is effective to prevent, prevent or delay the occurrence of a disease (for example, an influenza virus infection), or alleviate, alleviate or treat an existing disease (for example, infection by an influenza virus).
  • the amount of severity of the disease caused is well within the abilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.
  • the term "immunogenicity” refers to the ability to stimulate the body to form specific antibodies or sensitize lymphocytes. It means that the antigen can stimulate specific immune cells, activate, proliferate and differentiate immune cells, and finally produce the characteristics of immune effector substances such as antibodies and sensitized lymphocytes. It also means that after the antigen stimulates the body, the body's immune system can form antibodies or A specific immune response to sensitized T lymphocytes. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host, and the mode of immunization.
  • polypeptide and “protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by single letter and three letter abbreviations as are known in the art.
  • alanine can be represented by A or Ala.
  • subject refers to an animal, such as a vertebrate.
  • the subject is a mammal, such as a human, bovine, equine, feline, canine, rodent or primate.
  • the subject is a human.
  • the term can be used interchangeably with "patient.”
  • the present application provides a mutant of the H3N2 subtype influenza virus hemagglutinin protein, which is capable of inducing protective antibodies against influenza viruses of different subtypes (eg, H3N2, H7N9 and/or H1N1 subtypes), achieving different resistance
  • the protective effect of influenza viruses of the type and thus can be used as a broad-spectrum vaccine against influenza viruses capable of resisting multiple subtypes (eg, at least 2, at least 3 or more subtypes) for prevention and/or treatment Infection of influenza viruses of various subtypes (eg, at least 2, at least 3 or more subtypes) and diseases associated with the infection (eg, influenza).
  • the mutants derived from the H3N2 subtype influenza virus HA protein disclosed in the present application are capable of inducing not only protective antibodies against a plurality of H3N2 subtype influenza viruses, particularly a plurality of H3N2 subtype influenza viruses prevalent in different ages. It can protect against multiple H3N2 subtype influenza viruses, and can induce protective antibodies against H7N9 and/or H1N1 subtype influenza viruses, and achieve protection against H7N9 and/or H1N1 subtype influenza viruses.
  • the present application provides a broad-spectrum influenza vaccine capable of providing cross-protection against influenza viruses against various subtypes (eg, H3N2, H7N9, and/or H1N1 subtypes), and having an immune effect that is not readily flu
  • the virus mutates quickly and fails, thereby overcoming the shortcomings of the current influenza vaccine due to frequent mutation of the influenza virus, the loss of immune efficacy, and the unsatisfactory immune effect.
  • the broad-spectrum influenza vaccine of the present application solves the drawbacks of the current influenza vaccine that requires annual changeovers and annual injections.
  • the broad-spectrum influenza vaccine of the present application can effectively suppress the spread of various subtypes of influenza viruses, and reduce economic losses and social panic caused by influenza viruses. Therefore, the broad-spectrum influenza vaccine of the present application has a particularly significant advantage over existing influenza vaccines.
  • Fig. 1 schematically illustrates sequence mutations and N-linked glycosylation of the native HA protein (WI2005-WT-HA), HA-mut1 protein, HA-mut2 protein and HA-mut3 protein used in Example 1.
  • Figure 2 schematically illustrates the formation of the native HA protein (Figure 2A), HA-mut1 protein ( Figure 2B), HA-mut2 protein (Figure 2C) and HA-mut3 protein ( Figure 2D), respectively, used in Example 1.
  • Schematic diagram of the trimer wherein, Figure 2A shows that the trimer formed by the native HA protein contains an N-linked glycosyl chain in both the head and stem regions; Figure 2B shows the three formed by the HA-mut1 protein.
  • the polymer does not contain an N-linked glycosyl chain in both the head and stem regions;
  • Figure 2C shows that the trimer formed by the HA-mut2 protein does not contain an N-linked glycosyl chain in the head region, but in the stem The region still contains an N-linked glycosyl chain;
  • Figure 2D shows that the trimer formed by the HA-mut3 protein does not contain an N-linked glycosyl chain in the stem region, but still contains an N-linked region in the head region. Glycosyl chain.
  • Figure 3 shows the results of SDS-PAGE analysis of the six proteins prepared in Example 1; wherein, Figure 3A shows the results of SDS-PAGE analysis of native HA protein, HA-mut3, HA-mut2 and HA-mut1 proteins; Figure 3B The results of SDS-PAGE analysis of natural HA protein, HAmg protein and HAug protein are shown.
  • Figure 4 shows mouse sera obtained by immunizing mice with native HA protein, HA-mut1, HA-mut2, HA-mut3 and PBS (used as a negative control) as immunogens against influenza A/Wisconsin/67/ 2005 (H3N2 subtype) (Fig. 4A), A/Victoria/361/2011 (H3N2 subtype) (Fig. 4B), A/Beijing/32/1992 (H3N2 subtype) (Fig. 4C), A/Aichi/2 Neutralizing activity of /1968 (H3N2 subtype) (Fig. 4D), A/Shanghai/02/2013 (H7N9 subtype) (Fig. 4E) and A/California/04/2009 (H1N1 subtype) (Fig. 4F).
  • Figure 5 shows mouse sera obtained by immunizing mice with natural HA protein, HA-mut1, HAmg, HAug and PBS (used as a negative control) as immunogens against influenza A/Wisconsin/67/2005 (H3N2 sub- Type) (Fig. 5A), A/Victoria/361/2011 (H3N2 subtype) (Fig. 5B), A/Beijing/32/1992 (H3N2 subtype) (Fig. 5C), A/Aichi/2/1968 (H3N2) Neutralization activity of subtype) (Fig. 5D), A/Shanghai/02/2013 (H7N9 subtype) (Fig. 5E) and A/California/04/2009 (H1N1 subtype) (Fig. 5F).
  • Figure 6 shows that mice immunized with native HA protein, HA-mut1 protein, HA-mut2 protein, HA-mut3 protein or PBS (negative control) were infected with H3N2 subtype influenza A/Beijing/ earlier in the year of infection. Changes in body weight and survival after 32/1992 (H3N2) (Fig. 6A-6B) and A/Aichi/2/1968 (H3N2) (Fig. 6C-6D), wherein Fig. 6A and Fig. 6C show small experiments Changes in body weight of the mice, Figures 6B and 6D show the survival rates of the experimental mice.
  • Figure 7 shows that mice immunized with native HA protein, HA-mut1 protein, HAmg protein, HAug protein or PBS (negative control) were infected with the H3N2 subtype influenza A/Beijing/32/1992 earlier in the year of infection ( Changes in body weight and survival after H3N2) (Figs. 7A-7B) and A/Aichi/2/1968 (H3N2) (Fig. 7C-7D), wherein Fig. 7A and Fig. 7C show changes in body weight of each experimental mouse Figures 7B and 7D show the survival rates of each experimental mouse.
  • Figure 8 shows that mice immunized with native HA protein, HA-mut1 protein, HA-mut2 protein, HA-mut3 protein or PBS (negative control) were infected with non-H3N2 subtype influenza A/Shanghai/02/2013 ( Body weight changes and survival after H7N9) (Figs. 8A-8B) and A/California/04/2009 (H1N1) (Fig. 8C-8D), wherein Fig. 8A and Fig. 8C show changes in body weight of each experimental mouse Figures 8B and 8D show the survival rates of each experimental mouse.
  • Figure 9 shows that mice immunized with native HA protein, HA-mut1 protein, HAmg protein, HAug protein or PBS (negative control) were infected with non-H3N2 subtype influenza A/Shanghai/02/2013 (H7N9) (Fig. Body weight changes and survival after 9A-9B) and A/California/04/2009 (H1N1) (Fig. 9C-9D), wherein Fig. 9A and Fig. 9C show changes in body weight of each experimental mouse, Fig. 9B and Figure 9D shows the survival rate of each experimental mouse.
  • Figure 10 shows the results of SDS-PAGE analysis (left panel) and Western blot analysis (right panel) of HK2014-WT-HA protein; lane M: molecular weight marker; lane 1: non-Ni-NTA nickel ion chromatography Column purified sample; Lane 2: fraction flowing through a Ni-NTA nickel ion chromatography column; Lane 3: fraction eluted with 50 mM imidazole; Lane 4: fraction eluted with 50 mM imidazole; Lane 5: used Fractions eluted with 250 mM imidazole; arrows indicate the location of the protein of interest HK2014-WT-HA.
  • Figure 11 shows the results of SDS-PAGE analysis (left panel) and Western blot analysis (right panel) of HK2014-DG-HA protein; lane M: molecular weight marker; lane 1: non-Ni-NTA nickel ion chromatography Column purified sample; Lane 2: fraction flowing through a Ni-NTA nickel ion chromatography column; Lane 3: fraction eluted with 50 mM imidazole; Lane 4: fraction eluted with 250 mM imidazole; arrow indicating the protein of interest The location of HK2014-DG-HA.
  • Figure 12 shows the results of SDS-PAGE analysis of native HA protein HK2014-WT-HA and deglycosylated protein HK2014-HAug; lanes M: molecular weight marker; lane 1: purified HK2014-WT-HA; lane 2 : HK2014-HAug (obtained by digesting HK2014-WT-HA with endoglycosidase F for 3 hours).
  • Figure 13 shows mouse sera obtained by immunizing mice with HK2014-WT-HA, HK2014-DG-HA and PBS (used as a negative control) as immunogens against influenza A/Wisconsin/67/2005 (H3N2) Results of ELISA analysis of binding activities of A/Xiamen/N794/2013 (H3N2) and A/Shanghai/02/2013 (H7N9).
  • Figure 14 shows mouse sera obtained by immunizing mice with HK2014-WT-HA, HK2014-HAug and PBS (used as a negative control) as immunogens against influenza A/Wisconsin/67/2005 (H3N2), A ELISA analysis results of binding activity of /Xiamen/N794/2013 (H3N2) and A/Shanghai/02/2013 (H7N9).
  • Figure 15 shows that mice (3/group) immunized with HK2014-WT-HA, HK2014-DG-HA or PBS (used as a negative control) after infection with A/Aichi/2/1968 (H3N2) The change in weight (left) and survival (right).
  • Figure 16 shows that mice (3/group) immunized with HK2014-WT-HA, HK2014-DG-HA or PBS (used as a negative control) after infection with A/Shanghai/059/2013 (H7N9) The change in weight (left) and survival (right).
  • Figure 17 shows the body weight of each group of mice (4/group) immunized with HK2014-WT-HA, HK2014-HAug or PBS (used as a negative control) after infection with A/Shanghai/059/2013 (H7N9). Changes.
  • the molecular biology experimental methods and immunoassays used in the present application are basically referred to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Methods in Editing Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995.
  • the restriction enzymes are used in accordance with the conditions recommended by the product manufacturer. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer.
  • the reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.
  • the invention is described by way of example, and is not intended to limit the scope of the invention.
  • the N-linked glycosylated amino acid is usually the asparagine (N) in the characteristic sequence NX-(S or T), wherein N represents asparagine and X represents deuterium ammonia. Any amino acid other than acid, S represents serine and T represents threonine.
  • N-linked glycosylation of HA protein is removed by mutating asparagine (N) in the characteristic sequence NX-(S or T) in the native HA protein to alanine (A). Site.
  • the native HA protein (WI2005-WT-HA) used in this example was derived from the HA protein of the H3N2 subtype influenza virus strain A/Wisconsin/67/2005.
  • the HA protein of the strain comprises the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acids 1-10 of SEQ ID NO: 1 are signal peptides, and the amino acids 504-550 are transmembrane regions, and There are 10 potential N-linked glycosylation sites, ie located at the 22nd, 38th, 63rd, 126th, 133th, 144th, 165th, 246th, Asparagine (N) at position 285 and position 483.
  • the asparagine at position 483 which is located in the HA2 subunit of the HA protein
  • the asparagine at the other sites is located in the HA1 subunit of the HA protein.
  • the 22nd, 38th, 285th, and 483th asparagine are located in the stem region of the HA protein trimer; and the 63rd, 126th, and 133th positions
  • the asparagine at positions 144, 165 and 246 is located in the head region of the HA protein trimer.
  • a native HA protein (WI2005-WT-HA) comprising the amino acid sequence shown as SEQ ID NO: 2, and which differs from SEQ ID NO: 1 in that the 1-10 of SEQ ID NO: And the amino acids 504-550 are deleted, and a peptide comprising a thrombin cleavage site, a folding motif and a 6*His tag (which comprises SEQ ID NO: 10 and Sequence of 11 for promoting protein purification and trimer formation).
  • the trimer formed from the native HA protein contained N-linked glycosyl chains in both the head and stem regions (Fig. 2A).
  • HA-mut1 comprising the amino acid sequence shown as SEQ ID NO: 3, and which differs from the native HA protein (WI2005-WT-HA; SEQ ID NO: 2) in all 10 as described above
  • the asparagine on the N-linked glycosylation sites was mutated to alanine. Accordingly, the trimer formed by HA-mut1 does not contain an N-linked glycosyl chain in both the head and stem regions (Fig. 2B).
  • HA-mut2 which comprises the amino acid sequence shown as SEQ ID NO: 4, and which differs from the native HA protein (WI2005-WT-HA; SEQ ID NO: 2) in that it is located in the head region (ie Asparagine at positions 63, 126, 133, 144, 165 and 246 of SEQ ID NO: 1 was mutated to alanine. Accordingly, the trimer formed by HA-mut2 does not contain an N-linked glycosyl chain in the head region, but still contains an N-linked glycosyl chain in the stem region (Fig. 2C).
  • HA-mut3 which comprises the amino acid sequence shown as SEQ ID NO: 5, and which differs from the native HA protein (WI2005-WT-HA; SEQ ID NO: 2) in that it is located in the stem region (ie Asparagine at positions 22, 38, 285 and 483 of SEQ ID NO: 1 was mutated to alanine. Accordingly, the trimer formed by HA-mut2 does not contain an N-linked glycosyl chain in the stem region, but still contains an N-linked glycosyl chain in the head region (Fig. 2D).
  • a nucleus encoding a signal peptide (SEQ ID NO: 9) was introduced at the 5' end of the nucleotide sequence encoding the native HA protein, HA-mut1 protein, HA-mut2 protein and HA-mut3 protein. Glycosidic acid sequence.
  • the expressed signal peptide will be cleaved during protein secretion. Therefore, the finally obtained natural HA protein, HA-mut1 protein, HA-mut2 protein, and HA-mut3 protein do not contain a signal peptide, and their amino acid sequences are shown in SEQ ID NOS: 2-5.
  • Figure 1 is a schematic illustration of the sequence mutation and N-linked glycosylation of the native HA protein, HA-mut1 protein, HA-mut2 protein and HA-mut3 protein used in Example 1 (Note: the signal peptide will be in the protein Excised during the secretion process).
  • the native HA protein has asparagine at positions corresponding to positions 22, 38, 63, 126, 133, 144, 165, 246, 285, and 483 of SEQ ID NO: 1, and thus The site carries an N-linked glycosyl chain.
  • the HA-mut1 protein has an alanine at positions corresponding to positions 22, 38, 63, 126, 133, 144, 165, 246, 285 and 483 of SEQ ID NO: 1, and thus no longer carries any N- Linked glycosyl chains.
  • the HA-mut2 protein has an asparagine at a position corresponding to positions 22, 38, 285 and 483 of SEQ ID NO: 1, and thus can carry an N-linked glycosyl chain at these positions; Alanine is present at positions 63, 126, 133, 144, 165 and 246 of SEQ ID NO: 1, and thus no N-linked glycosyl chains are carried at these positions.
  • the HA-mut3 protein has an asparagine at a position corresponding to positions 63, 126, 133, 144, 165 and 246 of SEQ ID NO: 1, and thus can carry an N-linked glycosyl chain at these positions.
  • a signal peptide was introduced at the N-terminus of the native HA protein, HA-mut1 protein, HA-mut2 protein and HA-mut3 protein (the amino acid sequence thereof is SEQ ID NO :9, and will be excised during protein secretion), introducing a peptide fragment containing a thrombin cleavage site, a folding motif and a 6*His tag at the C-terminus (which includes SEQ ID NOS: 10 and 11 Amino acid sequence).
  • Figure 2 schematically illustrates the formation of the native HA protein (Figure 2A), HA-mut1 protein ( Figure 2B), HA-mut2 protein (Figure 2C) and HA-mut3 protein ( Figure 2D), respectively, used in Example 1.
  • Schematic diagram of the trimer wherein, Figure 2A shows that the trimer formed by the native HA protein contains an N-linked glycosyl chain in both the head and stem regions; Figure 2B shows the three formed by the HA-mut1 protein.
  • the polymer does not contain an N-linked glycosyl chain in both the head and stem regions;
  • Figure 2C shows that the trimer formed by the HA-mut2 protein does not contain an N-linked glycosyl chain in the head region, but in the stem The region still contains an N-linked glycosyl chain;
  • Figure 2D shows that the trimer formed by the HA-mut3 protein does not contain an N-linked glycosyl chain in the stem region, but still contains an N-linked region in the head region. Glycosyl chain.
  • the natural HA protein, HA-mut1 protein, HA-mut2 protein and HA-mut3 protein were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. (they each introduced a signal peptide (SEQ ID NO: 9) at the N-terminus, A DNA sequence comprising a thrombin cleavage site, a folding motif and a 6*His tag peptide (SEQ ID NOS: 10 and 11) was introduced at the C-terminus, and these DNA sequences were separately cloned into a baculovirus transfer vector.
  • pAcGP67-B (BD, Catalog Number: 554757). Subsequently, the transfer vector carrying the DNA sequence of interest was separately transformed into E.
  • the transfer plasmid containing the DNA sequence of interest was extracted from the transformed E. coli using a plasmid miniprep kit (TIANprep Mini Plasmid Kit; TianGen, Catalog Number: DP103-03), and used.
  • the transfection mixture in each well was removed, and 2 ml of medium containing CCM3 was added to each well to continue culturing the cells.
  • the transfer plasmid carrying the DNA sequence of interest and the linear DNA of baculovirus are transfected into insect cells to produce a recombinant baculovirus.
  • the obtained recombinant baculovirus was passaged to obtain a second generation recombinant baculovirus.
  • 15 ml of the second generation recombinant baculovirus was added to 1200 ml of Sf9 insect cells and cultured at 27 ° C for 48 hours.
  • the cells and the culture supernatant were collected and centrifuged at 11500 rpm for 30 minutes. After centrifugation, the supernatant was collected, which contained the recombinantly produced protein of interest.
  • the supernatant containing the protein of interest was concentrated to 35 ml with an ultrafiltration concentrated centrifuge tube of Millipore, adjusted to pH 7.4, and then centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected, and the target protein in the supernatant was enriched and purified using a Ni-NTA nickel ion chromatography column (NI-sepharose 6 fast flow, GE, Catalog Number: 17-5318-04). The solution was depleted into PBS containing 250 mM imidazole. The eluate containing the desired protein was concentrated to 1 ml, dialyzed into PBS buffer, and stored at 4 ° C until use.
  • purified natural HA protein, HA-mut1 protein, HA-mut2 protein and HA-mut3 protein (the N-terminal signal peptide is cleaved during secretion, and thus the obtained protein retains the folding motif and 6*His tag, but does not contain an N-terminal signal peptide).
  • HA protein (WI2005-WT-HA) was subjected to enzymatic treatment to prepare a HA protein carrying a single glycosyl group (hereinafter abbreviated as HAmg) at the N-linked glycosylation site and an N-linked glycosylation site.
  • HAmg a single glycosyl group
  • a HA protein (hereinafter abbreviated as HAug) which does not substantially carry a glycosyl group.
  • the six proteins (natural HA protein, HA-mut1, HA-mut2, HA-mut3, HAmg, and HAug) prepared in Example 1 were analyzed using polyacrylamide gel electrophoresis (SDS-PAGE).
  • the top coat used was a 5% concentrated gel (prepared as follows: 830 ⁇ l of 30% acrylamide, 630 ⁇ l of 1 M Tris (pH 6.8), 50 ⁇ l of 10% SDS, 50 ⁇ l of 10% ammonium persulfate and 3.4 ml of water were added. 5 ⁇ l TEMED).
  • the lower gum used was a 12% separation gel (prepared as follows: 4 ml of 30% acrylamide, 2.5 ml of 1 M Tris (pH 8.8), 100 ⁇ l of 10% SDS, 100 ⁇ l of 10% ammonium persulfate and 10 ⁇ l were added to 3.3 ml of water. TEMED).
  • the electrophoresis conditions used were electrophoresis at 150 V for 2 hours. After electrophoresis, the polyacrylamide gel was stained with Coomassie Brilliant Blue (Sigma). The experimental results are shown in Figure 3.
  • Figure 3 shows the results of SDS-PAGE analysis of the six proteins prepared in Example 1; wherein, Figure 3A shows the results of SDS-PAGE analysis of native HA protein, HA-mut3, HA-mut2 and HA-mut1 proteins; Figure 3B The results of SDS-PAGE analysis of natural HA protein, HAmg protein and HAug protein are shown.
  • the results in Figure 3 show that the molecular weight of the native HA protein is above 70kD, while the molecular weights of HA-mut3, HA-mut2, HA-mut1, HAmg and HAug proteins are significantly reduced, all below 70kD, and HA-mut1 protein The molecular weight is the smallest.
  • mice 6-week-old, SPF-grade, female Balb/C mice were provided by the Experimental Animal Center of Xiamen University and weighed approximately 20 g.
  • the six proteins (natural HA protein, HA-mut1, HA-mut2, HA-mut3, HAmg, and HAug) prepared in Example 1 and PBS (used as a negative control) were respectively 1:1 volume with aluminum adjuvant. More than mixed, used to immunize mice.
  • the immunization protocol was as follows: 6 mice in each group were immunized by intramuscular injection, the immunization dose was 5 ⁇ g protein/mouse, the injection volume was 100 ⁇ l/mouse, the immunization was performed twice, and the interval between the two immunizations was 14 days. . After 14 days of the second immunization, mouse serum was collected. The collected serum samples were inactivated at 56 ° C for 30 minutes and then stored at -20 ° C until use.
  • Neutralization titer is an important indicator for evaluating whether serum samples have the potential to prevent and treat disease.
  • the influenza virus used is a representative strain of influenza virus isolated from different time, different regions, representing different subtypes (H3N2, H7N9 and H1N1), and the specific virus strain is as follows: A/Wisconsin/67/2005 (H3N2 subtype), A /Victoria/361/2011 (H3N2 subtype), A/Beijing/32/1992 (H3N2 subtype), A/Aichi/2/1968 (H3N2 subtype), A/Shanghai/02/2013 (H7N9 subtype) And A/California/04/2009 (H1N1 subtype).
  • MDCK cells 6 x 10 5 MDCK cells were seeded in a six-well cell culture plate.
  • the influenza virus used was diluted to 50 PFU/50 ⁇ l with MEM medium containing 0.5 ⁇ g/ml TPCK trypsin. Then, serially diluted serum samples were mixed with influenza virus and incubated at 37 ° C for 1 hour, then added to a six-well cell culture plate inoculated with MDCK cells, and incubation was continued at 37 ° C for 1 hour. After the incubation, the cell culture was aspirated and the cells were washed twice with PBS.
  • the cell surface was covered with MEM medium containing 0.5% agarose, and the cells were cultured in a constant temperature incubator at 5% CO 2 at 37 ° C for two days. Thereafter, the cells were stained with 2% crystal violet, and the titer of the influenza virus was determined by counting the number of plaques, thereby calculating the neutralizing activity of each serum sample. The result is shown in Figure 4-5.
  • Figure 4 shows mouse sera obtained by immunizing mice with native HA protein, HA-mut1, HA-mut2, HA-mut3 and PBS (used as a negative control) as immunogens against influenza A/Wisconsin/67/ 2005 (H3N2 subtype) (Fig. 4A), A/Victoria/361/2011 (H3N2 subtype) (Fig. 4B), A/Beijing/32/1992 (H3N2 subtype) (Fig. 4C), A/Aichi/2 Neutralizing activity of /1968 (H3N2 subtype) (Fig. 4D), A/Shanghai/02/2013 (H7N9 subtype) (Fig. 4E) and A/California/04/2009 (H1N1 subtype) (Fig. 4F).
  • mice were immunized with native HA protein, HA-mut1, HA-mut2 or HA-mut3 for the influenza virus strain A/Wisconsin/67/2005 from which the HA protein used in the experiment was derived.
  • the obtained mouse serum had strong neutralizing activity, wherein the serum obtained by immunizing the mouse with the native HA protein and HA-mut1 had the highest neutralizing titer, and the serum obtained by immunizing the mouse with HA-mut3 was in the middle. And the lowest titer.
  • the virus strains A/Shanghai/02/2013 (H7N9 subtype) and A/California/04/2009 (H1N1 subtype) belonging to different subtypes of the HA protein used in this experiment are classified.
  • the serum obtained by immunizing mice with only HA-mut1 had neutralizing activity, while the serum obtained by immunizing mice with other proteins had substantially no neutralizing activity (no significant difference from the negative control).
  • HA-mut1 is particularly suitable for use as a broad-spectrum vaccine for inducing protective antibodies with broad-spectrum neutralizing activity in vivo.
  • Figure 5 shows mouse sera obtained by immunizing mice with natural HA protein, HA-mut1, HAmg, HAug and PBS (used as a negative control) as immunogens against influenza A/Wisconsin/67/2005 (H3N2 sub- Type) (Fig. 5A), A/Victoria/361/2011 (H3N2 subtype) (Fig. 5B), A/Beijing/32/1992 (H3N2 subtype) (Fig. 5C), A/Aichi/2/1968 (H3N2) Neutralization activity of subtype) (Fig. 5D), A/Shanghai/02/2013 (H7N9 subtype) (Fig. 5E) and A/California/04/2009 (H1N1 subtype) (Fig. 5F).
  • the mouse obtained by immunizing the mouse with the native HA protein, HA-mut1, HAmg or HAug was small.
  • the murine serum has a potent neutral neutralizing activity.
  • the serum obtained by immunizing the mouse with HA-mut1 was used.
  • the neutralizing titer was the highest, and the neutralizing titer of the serum obtained by immunizing mice with HAmg or HAug was second (both comparable), while the serum obtained with the natural HA protein mouse had substantially no neutralizing activity ( There was no significant difference from the negative control).
  • the results in Figure 5 indicate that the serum obtained by immunizing mice with native HA protein is only neutralizing the influenza virus of the H3N2 subtype; the serum obtained by immunizing mice with HAmg and HAug can not only neutralize the H3N2 subtype of influenza.
  • Virus and showed a weaker neutralizing activity across the HA subtype (a virus strain capable of neutralizing the H7N9 subtype but not the H1N1 subtype); serum obtained by immunizing mice with HA-mut1 It has the broadest spectrum of neutralizing activity and the highest neutralizing potency. It not only effectively neutralizes multiple strains in the H3N2 subtype (regardless of the evolutionary relationship), but also has a strong neutralization across the HA subtype.
  • HA-mut1 is particularly suitable for use as a broad-spectrum vaccine for inducing protective antibodies with broad-spectrum neutralizing activity in vivo.
  • Example 3 It has been confirmed by PRNT experiment in Example 3 that the antisera induced by the six proteins prepared in Example 1 have different neutralizing titers to the strains of H3N2 subtype, H7N9 subtype and H1N1 subtype, among them, HA
  • the anti-sera induced by -mut1 has the broadest spectrum of neutralizing activity.
  • the inventors based on A/Beijing/32/1992 (H3N2 subtype) and A/Aichi/02/1968 (H3N2 subtype).
  • mice Balb/C mice, SPF grade, 6-8 weeks old, female, weighing approximately 20 g.
  • Vaccine native HA protein, HA-mut1 protein, HA-mut2 protein, HA-mut3 protein, HAmg protein, HAug protein and PBS (used as a negative control).
  • Immune protocol natural HA protein, HA-mut1 protein, HA-mut2 protein, HA-mut3 protein, HAmg protein, HAug protein and PBS negative control were mixed with aluminum adjuvant in a volume ratio of 1:1, respectively.
  • mouse Six mice were used in each group, and the immunization method was intramuscular injection, and the immunization dose was 5 tg protein/mouse, and the injection volume was 100 ⁇ l/mouse. Immunization was performed twice, with an interval of 14 days between the two immunizations. After 14 days of the second immunization, the mice were challenged.
  • the influenza strains used are as follows:
  • mice were sent to the biosafety laboratory one day in advance, grouped in 6 cages, and the body weight of each mouse was recorded.
  • Viral infection The challenge dose of each virus was 25 times the lethal dose (LD 50 ), and the virus inoculation volume was 50 ⁇ l/mouse. Prior to inoculation, the mice were anesthetized with isoflurane and then the mice were inoculated with virus via the nasal cavity.
  • LD 50 lethal dose
  • Figure 6 shows that mice immunized with native HA protein, HA-mut1 protein, HA-mut2 protein, HA-mut3 protein or PBS (negative control) were infected with H3N2 subtype influenza A/Beijing/ earlier in the year of infection. Changes in body weight and survival after 32/1992 (H3N2) (Fig. 6A-6B) and A/Aichi/2/1968 (H3N2) (Fig. 6C-6D), wherein Fig. 6A and Fig. 6C show small experiments Changes in body weight of the mice, Figures 6B and 6D show the survival rates of the experimental mice.
  • mice immunized with HA-mut1 or HA-mut3 began to recover after the lethal dose of virus A/Beijing/32/1992 and returned to the end of the experiment.
  • the mouse survival rate was 100%; however, mice immunized with native HA protein, HA-mut2 or PBS continued to lose weight and all died before the end of the experiment. This result indicates that HA-mut1 and HA-mut3 have complete protection and can be used as a vaccine against A/Beijing/32/1992.
  • the results of Figures 6C-6D show that mice immunized with HA-mut1 began to recover after the lethal dose of virus A/Aichi/2/1968, and the survival rate of the mice at the end of the experiment.
  • HA-mut3 had partial protection against mice infected with lethal dose of virus A/Aichi/2/1968, and the survival rate of the mice at the end of the experiment was 33.3%; however, with natural HA protein, HA- Mice immunized with mut2 or PBS continued to lose weight and all died before the end of the experiment. This result indicates that HA-mut1 has complete protection and can be used as a vaccine against A/Aichi/2/1968.
  • Figure 7 shows that mice immunized with native HA protein, HA-mut1 protein, HAmg protein, HAug protein or PBS (negative control) were infected with the H3N2 subtype influenza A/Beijing/32/1992 earlier in the year of infection ( Changes in body weight and survival after H3N2) (Figs. 7A-7B) and A/Aichi/2/1968 (H3N2) (Fig. 7C-7D), wherein Fig. 7A and Fig. 7C show changes in body weight of each experimental mouse Figures 7B and 7D show the survival rates of each experimental mouse.
  • mice immunized with HA-mut1 protein, HAmg protein or HAug protein began to recover after the seventh day after infection with a lethal dose of virus A/Beijing/32/1992 (with HA).
  • -mut1 immunized mice had the best weight recovery effect, and the mouse survival rate was 100% at the end of the experiment; however, mice immunized with native HA protein or PBS continued to lose weight and ended at the end of the experiment. All died before.
  • This result indicates that HA-mut1 protein, HAmg protein and HAug protein have complete protection and can be used as a vaccine against A/Beijing/32/1992.
  • Figure 8 shows that mice immunized with native HA protein, HA-mut1 protein, HA-mut2 protein, HA-mut3 protein or PBS (negative control) were infected with non-H3N2 subtype influenza A/Shanghai/02/2013 ( Body weight changes and survival after H7N9) (Figs. 8A-8B) and A/California/04/2009 (H1N1) (Fig. 8C-8D), wherein Fig. 8A and Fig. 8C show changes in body weight of each experimental mouse Figures 8B and 8D show the survival rates of each experimental mouse.
  • mice immunized with HA-mut1 began to recover after the lethal dose of virus A/Shanghai/02/2013 (H7N9) after day 6 and were small at the end of the experiment.
  • the mouse survival rate was 100%; however, mice immunized with native HA protein, HA-mut2, HA-mut3 or PBS continued to lose weight and all died before the end of the experiment. This result indicates that HA-mut1 has complete protection and can be used as a vaccine against A/Shanghai/02/2013.
  • Figure 9 shows that mice immunized with native HA protein, HA-mut1 protein, HAmg protein, HAug protein or PBS (negative control) were infected with non-H3N2 subtype influenza A/Shanghai/02/2013 (H7N9) (Fig. Body weight changes and survival after 9A-9B) and A/California/04/2009 (H1N1) (Fig. 9C-9D), wherein Fig. 9A and Fig. 9C show changes in body weight of each experimental mouse, Fig. 9B and Figure 9D shows the survival rate of each experimental mouse.
  • FIGS 9A-9B show that mice immunized with HA-mut1 protein or HAug protein began to recover after 6 or 7 days after infection with a lethal dose of virus A/Shanghai/02/2013 (H7N9).
  • HA-mut1 immunized mice had the best weight recovery effect, and the mouse survival rate was 100% at the end of the experiment; however, mice immunized with native HA protein, HAmg protein or PBS continued to lose weight. And all died before the end of the experiment.
  • This result indicates that the HA-mut1 protein and the HAug protein have complete protection and can be used as a vaccine against A/Shanghai/02/2013 (H7N9).
  • HA-mut1 protein can be used as a vaccine to effectively prevent infection of H3N2 subtype (regardless of evolutionary relationship), H7N9 subtype and H1N1 subtype of influenza virus and diseases caused thereby, and thus can be used as an vaccine.
  • An effective broad-spectrum vaccine that is resistant to multiple subtypes of influenza virus.
  • N-linked glycosylation of HA protein is removed by mutating asparagine (N) in the characteristic sequence NX-(S or T) in the native HA protein to glutamine (Q). Site.
  • the natural HA protein (HK2014-WT-HA) used in the present example was derived from the HA protein of the H3N2 subtype influenza virus strain A/HONG_KONG/4801/2014 (H3N2).
  • the HA protein of the strain comprises the amino acid sequence shown in SEQ ID NO: 6, wherein amino acids 1 to 25 of SEQ ID NO: 6 are signal peptides, and amino acids 518 to 565 are transmembrane regions, and Eleven potential N-linked glycosylation sites, namely asparagine (N) at positions 37, 53, 60, 78, 137, 141, 148, 180, 261, 300 and 498.
  • the native HA protein HK2014-WT-HA and its mutant HK2014-DG-HA were designed in this example:
  • a native HA protein comprising the amino acid sequence shown as SEQ ID NO: 7, and which differs from SEQ ID NO: 6 in that it is 1-25 of SEQ ID NO: 6. And the amino acids 518-565 are deleted, and a peptide comprising a thrombin cleavage site, a folding motif and a 6*His tag (which comprises SEQ ID NO: 10 and Sequence of 11 for promoting protein purification and trimer formation). Accordingly, the trimer formed from the native HA protein (HK2014-WT-HA) contains N-linked glycosyl chains in both the head and stem regions.
  • mutant HK2014-DG-HA which comprises the amino acid sequence shown as SEQ ID NO: 8, and which differs from the native HA protein (HK2014-WT-HA; SEQ ID NO: 7) in the above
  • the asparagine (N) on all 11 N-linked glycosylation sites described was mutated to glutamine (Q). Accordingly, the trimer formed by the mutant HK2014-DG-HA does not contain an N-linked sugar-based chain in both the head and stem regions.
  • a nucleoside encoding a signal peptide (SEQ ID NO: 9) was introduced at the 5' end of the nucleotide sequence encoding the native HA protein HK2014-WT-HA and the mutant protein HK2014-DG-HA. Acid sequence.
  • the expressed signal peptide will be cleaved during protein secretion. Therefore, the finally obtained natural HA protein HK2014-WT-HA and its mutant HK2014-DG-HA did not contain signal peptides, and their amino acid sequences are shown in SEQ ID NOS: 7-8.
  • the native protein HK2014-WT-HA and the mutant protein HK2014-DG-HA are introduced at the C-terminus.
  • the DNA sequences of the sequence and 6*His-tagged peptides (SEQ ID NOS: 10 and 11) were cloned into the baculovirus transfer vector pAcGP67-B (BD Company, Catalog Number: 554757), respectively.
  • the transfer vector carrying the DNA sequence of interest was separately transformed into E. coli competent cell DHSa for amplification.
  • the transfer plasmid containing the DNA sequence of interest was extracted from the transformed E. coli using a plasmid miniprep kit (TIANprep Mini Plasmid Kit; TianGen, Catalog Number: DP103-03), and used.
  • a recombinant baculovirus containing the DNA sequence of interest was constructed using the transfer plasmid prepared above as described in Example 1, and cultured in Sf9 insect cells. After the incubation, the cells and the culture supernatant were collected and centrifuged at 11,500 rpm for 30 minutes. After centrifugation, the supernatant was collected, which contained the recombinantly produced protein of interest.
  • HK2014-WT-HA deglycosylated HA protein
  • Figure 10 shows the results of SDS-PAGE analysis (left panel) and Western blot analysis (right panel) of HK2014-WT-HA protein; lane M: molecular weight marker; lane 1: non-Ni-NTA nickel ion chromatography Column purified sample; Lane 2: fraction flowing through a Ni-NTA nickel ion chromatography column; Lane 3: fraction eluted with 50 mM imidazole; Lane 4: fraction eluted with 50 mM imidazole; Lane 5: used Fractions eluted with 250 mM imidazole; arrows indicate the location of the protein of interest HK2014-WT-HA.
  • Figure 11 shows the results of SDS-PAGE analysis (left panel) and Western blot analysis (right panel) of HK2014-DG-HA protein; lane M: molecular weight marker; lane 1: non-Ni-NTA nickel ion chromatography Column purified sample; Lane 2: fraction flowing through a Ni-NTA nickel ion chromatography column; Lane 3: fraction eluted with 50 mM imidazole; Lane 4: fraction eluted with 250 mM imidazole; arrow indicating the protein of interest The location of HK2014-DG-HA.
  • Figure 12 shows the results of SDS-PAGE analysis of native HA protein HK2014-WT-HA and deglycosylated protein HK2014-HAug; lanes M: molecular weight marker; lane 1: purified HK2014-WT-HA; lane 2 : HK2014-HAug (obtained by digesting HK2014-WT-HA with endoglycosidase F for 3 hours).
  • Fig. 12 show that the molecular weight of HK2014-WT-HA is above 70 KD, and the molecular weight of HK2014-HAug is lowered. These results indicate that the glycosylation modification in HK2014-HAug is effectively removed.
  • the proteins HK2014-WT-HA, HK2014-DG-HA and HK2014-HAug prepared in Example 5 were mixed with Freund's adjuvant to prepare an immunogen, and then used to immunize 6-8 week old Balb/C female mice. (body weight is about 20g).
  • the immunization procedure was as follows: subcutaneous immunization 3 times, each immunization interval of 14 days. After 14 days of the third immunization, mouse sera were collected and the collected serum samples were inactivated at 56 ° C for 30 minutes and then stored at -20 ° C until use.
  • the ELISA assay was used to evaluate whether the mouse serum samples collected as above were for three influenza viruses A/Wisconsin/67/2005 (H3N2), A/Xiamen/N794/2013 (H3N2), and A/Shanghai/02/2013 (H7N9). Has specific binding activity. Briefly, Elisa plates were coated with 100 ul of different types of influenza virus (128 HA), and then gradient diluted mouse serum was added to the virus-coated plates and incubated for 1 h at 37 °C. Subsequently, a 1:5000 dilution of GAM-HRP (provided by the National Engineering Center of Xiamen University) was added and incubated at 37 ° C for 30 min.
  • GAM-HRP provided by the National Engineering Center of Xiamen University
  • Figure 13 shows mouse sera obtained by immunizing mice with HK2014-WT-HA, HK2014-DG-HA and PBS (used as a negative control) as immunogens against influenza A/Wisconsin/67/2005 (H3N2) Results of ELISA analysis of binding activities of A/Xiamen/N794/2013 (H3N2) and A/Shanghai/02/2013 (H7N9).
  • Figure 14 shows mouse sera obtained by immunizing mice with HK2014-WT-HA, HK2014-HAug and PBS (used as a negative control) as immunogens against influenza A/Wisconsin/67/2005 (H3N2), A ELISA analysis results of binding activity of /Xiamen/N794/2013 (H3N2) and A/Shanghai/02/2013 (H7N9).
  • Example 5 To further verify that the protein prepared in Example 5 induced an immunoprotective effect against influenza virus in animals, the following experiment was conducted.
  • the proteins HK2014-WT-HA, HK2014-DG-HA and HK2014-HAug prepared in Example 5 were mixed with Freund's adjuvant to prepare an immunogen, and then used to immunize 6-8 week old Balb/C female mice. (body weight is about 20g).
  • the immunization procedure was as follows: subcutaneous immunization 3 times, each immunization interval of 14 days. After 14 days of the third immunization, each group of mice was subjected to an influenza virus challenge experiment using the influenza virus strain: H3N2 strain A/Aichi/2/1968 (H3N2) which is far from the prevalence year of the immunogen.
  • H7N9 strain A/Shanghai/059/2013 which is popular in recent years, both are lethal strains.
  • the body weight and survival rate of each group of mice were observed and recorded, and the prepared protein was evaluated to protect mice against lethal virus infection. The experimental results are shown in Figure 15-17.
  • Figure 15 shows that mice (3/group) immunized with HK2014-WT-HA, HK2014-DG-HA or PBS (used as a negative control) after infection with A/Aichi/2/1968 (H3N2) The change in weight (left) and survival (right).
  • the experimental results in Figure 15 show that mice immunized with HK2014-WT-HA died after infection with a lethal dose of virus A/Aichi/2/1968 (H3N2), and the remaining mice were in the first day.
  • mice Body weight recovery began at 6 days, and the survival rate of mice at the end of the experiment was 66%; mice immunized with HK2014-DG-HA after infection with a lethal dose of virus A/Aichi/2/1968 (H3N2), all mice Body weight recovery was started on day 5, and the mouse survival rate was 100% at the end of the experiment; the negative control mice all died on the 8th day after infection.
  • H3N2 lethal dose of virus A/Aichi/2/1968
  • Figure 16 shows that mice (3/group) immunized with HK2014-WT-HA, HK2014-DG-HA or PBS (used as a negative control) after infection with A/Shanghai/059/2013 (H7N9) The change in weight (left) and survival (right).
  • the experimental results in Figure 16 show that mice immunized with HK2014-WT-HA lost weight in all mice after infection with a lethal dose of virus A/Shanghai/059/2013 (H7N9), and on day 9 after challenge.
  • mice immunized with HK2014-DG-HA were infected with lethal dose of virus A/Shanghai/059/2013 (H7N9), and one mouse began to recover weight on the 8th day, and The survival rate of the mice at the end of the experiment was 33%.
  • HK2014-WT-HA does not have the protective effect against influenza A/Shanghai/059/2013 (H7N9); in contrast, HK2014-DG-HA is against virus A/Shanghai/059/2013 ( H7N9) exhibits a certain protective effect (a broad-spectrum protection across the type).
  • Figure 17 shows the body weight of each group of mice (4/group) immunized with HK2014-WT-HA, HK2014-HAug or PBS (used as a negative control) after infection with A/Shanghai/059/2013 (H7N9). Changes.
  • the experimental results in Figure 17 show that mice immunized with HK2014-WT-HA, HK2014-HAug or PBS, after infection with a lethal dose of virus A/Shanghai/059/2013 (H7N9), all mice continued to lose weight and The survival rate of mice on the 9th day after challenge was 0%. This result indicates that neither HK2014-WT-HA nor HK2014-HAug have the protective effect of antiviral A/Shanghai/059/2013 (H7N9).
  • HK2014-DG-HA is more suitable for influenza vaccine than HK2014-WT-HA and HK2014-HAug, which can resist the infection of influenza virus of H3N2 subtype (regardless of evolutionary relationship) and H7N9 subtype. It exhibits broad-spectrum protection across regions and better protection.

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Abstract

提供一种H3N2亚型流感病毒血凝素蛋白的突变体及其制备方法，所述突变体用来预防和/或治疗流感病毒的感染和/或由所述感染引起的疾病(例如流感)的方法。还提供了包含所述突变体的药物组合物，如疫苗。

Description

H3N2亚型流感病毒血凝素蛋白的突变体及其应用 技术领域
本申请涉及病毒学和免疫学领域。特别地，本申请涉及H3N2亚型流感病毒血凝素蛋白的突变体及其应用。此外，本申请还涉及，包含所述突变体的药物组合物(例如疫苗)，制备所述突变体的方法，以及使用所述突变体来预防和/或治疗流感病毒的感染和/或由所述感染引起的疾病(例如流感)的方法。
背景技术
流感病毒是人类健康的一大威胁，并且其持续快速的抗原性漂移使得季节性流感在人群中广泛传播。常见的人类季节性流感病毒包括季节性H1N1、季节性H3N2和乙型流感病毒。据WHO统计，季节性流感每年造成至少250,000-500,000人死亡(Peter D.C.等人，J Clin Invest.2008，118：3273-3275)。此外，流感大暴发仍是人类面临的一个重大威胁。自从流感病毒被发现以来，人类历史上共出现了五次世界范围内的流感大流行，共计造成了数千万人的死亡，其中1918年的西班牙流感大暴发造成全球约2000-5000万人死亡。20世纪暴发的其它流感大暴发还包括，1957年的亚洲流感(H2N2)大暴发及1968年的香港流感(H3N2)大暴发，二者均造成了严重的公共卫生威胁及人类社会大恐慌(Xu R.等人，Science.2010，328：357-360)。进入21世纪，流感大暴发仍没有停下脚步。2009年在墨西哥暴发并迅速蔓延全球的新型甲型流感病毒(H1N1)大流行又一次给人类社会敲响警钟。据WHO统计，截至2010年8月6日，全球200多个国家和地区报导的确诊死亡病例共计18449例(WHO Pandemic(h1n1)2009-update 112.6Aug，2010)。当流感病毒大流行结束后，流感病毒往往会演变成季节性流感继续流行，并且在流行过程中通过抗原性漂移，持续危害人类健康。此外，人类还面临着高致病性禽流感的威胁。2003年至今，全球共报告600例人感染H5N1禽流感病毒的病例，其中死亡353例，死亡率接近60％(WHO：http：//www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/index.html)；2013年至今，全球共报告1554例人感染H5N1禽流感病毒的病例，死亡率超过25％(WHO：http：//www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archi ves/en/index.html)。这不禁使人们担心一旦流感病毒在人群中传播，将给人类社会带在致命打击。总之，由流感病毒引起的流感是人类面临的一个重大传染病。
流感病毒属于正粘病毒科(Orthomyxoviridae)，流感病毒属，为有包膜的单链负义RNA病毒。流感病毒的基因组编码十种以上的病毒蛋白。根据病毒核蛋白(NP)及基质蛋白(M)的抗原性及其基因特性的差异，将流感病毒分为甲(A)、乙(B)、丙(C)三型(Horimoto T.等人，Nat Rev Microbiol，2005，3(8)：591-600)。其中甲型流感病毒(Influenza A Virus，简称Flu A)变异快，致病力强，能引起世界范围内的大流行。乙型流感病毒(Influenza B Virus，简称Flu B)变异较慢，只能引起局部范围内的小流行。丙型流感病毒(Influenza C Virus，简称Flu C)变异最慢，致病力弱，通常只能感染抵抗力较低的孕妇和小孩。在自然界中Flu A的宿主范围广，除了其天 然宿主水禽以外，还能够引起人、马、猪等多种动物的感染。Flu A亚型多，变异大，已成为流感防控及疫苗研究的重点。
Flu A病毒根据表面抗原血凝素蛋白(HA)及神经氨酸酶(NA)抗原性及基因特性的不同又可以分多种亚型。目前已发现十八种HA亚型(H1-H18)和十一种NA亚型(N1-N11)(Tong S.等人，PLoS Pathog.2013；9(10)：e1003657)。人群中流行的Flu A主要涉及两种HA亚型(H1、H3)和两种NA亚型(N1、N2)，同时高致病性禽流感病毒H5N1和H7N9也有偶发的感染人疫情发生，并且因其导致的较高致死率而广受关注。
流感疫苗是对抗流感病毒最有效的手段。当今流感疫苗诱导的抗病毒抗体的主要靶点是位于病毒表面的血凝素(HA)蛋白。HA蛋白在病毒表面呈三聚体结构，其中，每个HA单体由HA1和HA2两个结构域组成。HA1位于三聚体的头部，构成球状结构，含有受体结合位点，是病毒感染宿主细胞的关键区域。目前，HA1因含有重要的抗原性位点，可诱导机体产生保护性中和抗体，而成为疫苗设计的关键靶点(Wang T.T.等人，Nat Struct Mol Biol.2009，16：233-234)。HA2位于三聚体的基部，呈柄状结构，含有融合肽，可介导病毒包膜与宿主胞膜的融合。已报道，一些针对HA2的单抗能够通过抑制病毒膜融合从而起到中和病毒的效果(Wang T.T.等人，Nat Struct Mol Biol.2009，16：233-234)。
流感病毒具有高度变异性，其中尤其以HA变异最迅速。目前的传统疫苗主要针对HA蛋白。由于HA基因的高变异性，容易因抗原性漂移而导致疫苗无效。为克服流感疫苗的抗原变异现象，WHO每年均要根据上一年对流行病毒株的变异的监测情况，选择沿用或是确立新的疫苗株用作下一年度流行季节的疫苗候选株，并通过每年接种新疫苗来确保对现有的流行病毒株的有效保护性。换言之，目前的流感疫苗需要每年根据上一年流行的病毒株的抗原性变异情况进行疫苗候选株的调整，这费时费力。因此，研制不受病毒变异影响的“广谱疫苗”逐渐成为新型疫苗研究的热点。
由于未经改造的天然HA蛋白作为疫苗只能诱导窄谱的免疫保护作用，因此，已提出，对天然HA蛋白进行改造，以获得能诱导广谱免疫反应的疫苗，避免因HA的快速变异而导致疫苗迅速失效。然而，流感病毒HA蛋白亚型众多，并且具有复杂的翻译后糖基修饰，因此，此方面的研究尚未取得显著进展。本领域仍需开发能够在体内诱导广谱性抗流感病毒保护性抗体、能够在体内提供广谱的抗流感病毒保护作用的HA突变体。
发明内容
HA是一种糖蛋白，其HA1和HA2结构域均含有糖基化位点，携带有N-连接的糖基链(Keil W et al.(1985)EMBO J 4：2711-2720)。在真核表达(例如使用昆虫-杆状病毒表达系统进行真核表达)的情况下，所产生的HA蛋白在HA1和HA2结构域中均携带有N-连接的糖基链；相应地，由此所形成的HA三聚体在其头部区和茎部区将携带有N-连接的糖基链(图2A)。本申请的发明人经过大量的研究后发现，通过对H3N2亚型流感病毒HA蛋白进行改造来完全去除其所携带的N-连接的糖基链，能够增强HA蛋白诱发广谱的保护性抗体的能力，所诱发的保护性抗体能够识别更多亚 型的流感病毒，具有更加广谱的保护作用。基于此，本申请的发明人开发了一种H3N2亚型流感病毒血凝素蛋白的突变体，其不含有N-连接的糖基化位点(例如不包含特征序列N-X-(S或T))，能够在体内诱导广谱的抗流感病毒保护性抗体，能够在体内提供广谱的抗流感病毒保护作用。特别地，本申请公开的突变体能够诱发抗不同亚型的流感病毒的保护性抗体，实现抗不同亚型的流感病毒的保护作用，并因此可用作能够抗多种亚型(例如至少2种、至少3种或更多种亚型)的流感病毒的广谱疫苗，用于预防和/或治疗多种亚型(例如至少2种、至少3种或更多种亚型)的流感病毒的感染以及与所述感染相关的疾病(例如流感)。
特别地，本申请公开的衍生自H3N2亚型流感病毒HA蛋白的突变体不仅能够诱发针对多株H3N2亚型流感病毒(特别是流行于不同年代的多株H3N2亚型流感病毒)的保护性抗体，实现抗多株H3N2亚型流感病毒的保护作用，而且能够诱发针对H7N9和/或H1N1亚型流感病毒的保护性抗体，实现抗H7N9和/或H1N1亚型流感病毒的保护作用。因此，此类衍生自H3N2亚型流感病毒HA蛋白的突变体特别适合用作广谱疫苗，用于预防和/或治疗H3N2、H7N9和/或H1N1亚型流感病毒的感染以及与之相关的疾病。
关于突变体
因此，在一个方面，本申请涉及一种H3N2亚型流感病毒血凝素蛋白的突变体，其不含有N-连接的糖基化位点。由于不存在N-连接的糖基化位点，此类突变体不含有N-连接的糖基链。在某些优选的实施方案中，本申请提供了一种H3N2亚型流感病毒血凝素蛋白的突变体，其中，与所述H3N2亚型流感病毒的野生型血凝素蛋白相比，所述突变体不含有N-连接的糖基化位点，并且，任选地，所述突变体不包含所述野生型血凝素蛋白的N端信号肽和/或跨膜区。
N-连接的糖基化(N-linked glycosylation)是一种多肽的翻译后修饰，其是指，糖基链与多肽链中的特定天冬酰胺残基上的自由-NH 2基相连接。N-连接的糖基化过程通常在内质网(Endoplasmic reticulum，ER)和高尔基体(Golgi apparatus，GA)中进行。因此，在某些优选的实施方案中，所述突变体与所述H3N2亚型流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白各个N-连接的糖基化位点上的天冬酰胺残基各自独立地被删除或者置换为一个或多个其他的氨基酸残基(例如一个非N的氨基酸残基)。
可通过各种已知的方法来确定流感病毒HA蛋白中的N-连接的糖基化位点(参见，Tate MD.等人，Viruses.6(3)：1294-316)。例如，可利用计算机程序或软件(例如蛋白序列分析软件包Antheprot 5.0)来进行N-连接的糖基化位点的预测和确定。在流感病毒的天然HA蛋白中，发生N-连接的糖基化的氨基酸通常为特征序列N-X-(S或T)中的天冬酰胺(N)，其中N代表天冬酰胺，X代表除脯氨酸以外的任何一种氨基酸，S代表丝氨酸，T代表苏氨酸。因此，在某些优选的实施方案中，所述突变体与所述H3N2亚型流感病毒的野生型血凝素蛋白的区别至少在于，所述突变体不包含特征序列N-X-(S或T)；其中，N代表天冬酰胺，X代表除脯氨酸以外的任何一种氨基酸，S代表丝氨酸，T代表苏氨酸。在某些优选的实施方案中，所述突变体与所述H3N2亚型流 感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的每一个特征序列N-X-(S或T)各自独立地具有选自下列的一项或多项突变：
(1)N残基被删除或者置换为一个或多个其他的氨基酸残基(例如一个非N的氨基酸残基)；
(2)(S或T)残基被删除或者置换为一个或多个其他的氨基酸残基(例如一个非S且非T的氨基酸残基)；
(3)X残基被删除或者置换为脯氨酸残基；
(4)在N残基与X残基之间添加一个或多个氨基酸残基(例如一个非N的氨基酸残基)；和
(5)在X残基与(S或T)残基之间添加一个或多个氨基酸残基(例如一个非S且非T的氨基酸残基)；
其中，N代表天冬酰胺，X代表除脯氨酸以外的任何一种氨基酸，S代表丝氨酸，T代表苏氨酸，
由此，所述突变体不包含任何特征序列N-X-(S或T)。
可通过各种已知的方式来对野生型血凝素蛋白中的每一个特征序列N-X-(S或T)分别进行改造，以使得所产生的突变体不包含任何特征序列N-X-(S或T)。
在某些优选的实施方案中，可通过将N残基删除或者将N残基置换为一个或多个其他的氨基酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点。在某些优选的实施方案中，可通过将N残基删除来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点。在某些优选的实施方案中，可通过将N残基置换为一个非N的氨基酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点。在某些优选的实施方案中，可通过将N残基置换为至少两个或更多个(例如2个，3个或4个)氨基酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点，前提是，所述至少两个或更多个氨基酸残基的最后一个氨基酸残基是非N的氨基酸残基。
在某些优选的实施方案中，可通过将(S或T)残基删除或者将(S或T)残基置换为一个或多个其他的氨基酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点。在某些优选的实施方案中，可通过将(S或T)残基删除来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点。在某些优选的实施方案中，可通过将(S或T)残基置换为一个非S且非T的氨基酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点。在某些优选的实施方案中，可通过将(S或T)残基置换为至少两个或更多个(例如2个，3个或4个)氨基酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点，前提是，所述至少两个或更多个氨基酸残基的第一个氨基酸残基为非S且非T的氨基酸残基。
在某些优选的实施方案中，可通过将X残基删除或者将X残基置换为脯氨酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点。在某些优选的实施方案中，可通过将X残基删除来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点。在某些优选的实施方案中，可通过将X残基置换为脯氨酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基 化位点。
在某些优选的实施方案中，可通过在N残基与X残基之间添加一个或多个氨基酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点。在某些优选的实施方案中，可通过在N残基与X残基之间添加一个非N的氨基酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点。在某些优选的实施方案中，可通过在N残基与X残基之间添加至少两个或更多个(例如2个，3个或4个)氨基酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点，前提是，所述至少两个或更多个氨基酸残基的最后一个氨基酸残基是非N的氨基酸残基。
在某些优选的实施方案中，可通过在X残基与(S或T)残基之间添加一个或多个氨基酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点。在某些优选的实施方案中，可通过在X残基与(S或T)残基之间添加一个非S且非T的氨基酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点。在某些优选的实施方案中，可通过在N残基与X残基之间添加至少两个或更多个(例如2个，3个或4个)氨基酸残基来改造野生型血凝素蛋白中的特征序列N-X-(S或T)，从而去除N-糖基化位点，前提是，所述至少两个或更多个氨基酸残基的第一个氨基酸残基为非S且非T的氨基酸残基。
在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的各个特征序列N-X-(S或T)中的N残基和/或(S或T)残基各自独立地被删除或者置换为一个或多个其他的氨基酸残基(例如另一个氨基酸残基)；其中，N代表天冬酰胺，X代表除脯氨酸以外的任何一种氨基酸，S代表丝氨酸，T代表苏氨酸，由此所述突变体不包含任何特征序列N-X-(S或T)。
在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的各个特征序列N-X-(S或T)中的N残基各自独立地被删除或者置换为一个或多个其他的氨基酸残基(例如一个非N的氨基酸残基)。
在某些优选的实施方案中，可通过将各个N-连接的糖基化位点上(特别是特征序列N-X-(S或T)中)的天冬酰胺残基删除来改造野生型血凝素蛋白；由此，所产生的突变体不再包含任何N-连接的糖基化位点，不再携带任何N-连接的糖基链。因此，在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的各个特征序列N-X-(S或T)中的天冬酰胺残基均被删除。
在某些优选的实施方案中，可以通过将各个N-连接的糖基化位点上(特别是特征序列N-X-(S或T)中)的天冬酰胺残基各自独立地置换为一个或多个其他氨基酸残基(例如一个非N的氨基酸残基)来改造野生型血凝素蛋白；由此，所产生的突变体不再包含任何N-连接的糖基化位点，不再携带任何N-连接的糖基链。因此，在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的各个特征序列N-X-(S或T)中的天冬酰胺残基各自独立地被置换为一个或多个其他的氨基酸残基(例如一个非N的氨基酸残基)。
在某些优选的实施方案中，可以将一些N-连接的糖基化位点上(特别是特征序列N-X-(S或T)中)的天冬酰胺残基删除，并且将剩余的N-连接的糖基化位点上(特别 是特征序列N-X-(S或T)中)的天冬酰胺残基各自独立地置换为一个或多个其他氨基酸残基(例如一个非N的氨基酸残基)来改造野生型血凝素蛋白；由此，所产生的突变体不再包含任何N-连接的糖基化位点，不再携带任何N-连接的糖基链。因此，在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的一些特征序列N-X-(S或T)中的天冬酰胺残基被删除，并且其余特征序列N-X-(S或T)中的天冬酰胺残基各自独立地被置换为一个或多个其他的氨基酸残基(例如一个非N的氨基酸残基)。
在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的各个特征序列N-X-(S或T)中的(S或T)残基各自独立地被删除或者置换为一个或多个其他的氨基酸残基(例如一个非S且非T的氨基酸残基)。
在某些优选的实施方案中，可通过将各个特征序列N-X-(S或T)中的(S或T)残基删除来改造野生型血凝素蛋白；由此，所产生的突变体不再包含任何N-连接的糖基化位点，不再携带任何N-连接的糖基链。因此，在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的各个特征序列N-X-(S或T)中的(S或T)残基均被删除。
在某些优选的实施方案中，可以通过将各个特征序列N-X-(S或T)中的(S或T)残基各自独立地置换为一个或多个其他氨基酸残基(例如一个非S且非T的氨基酸残基)来改造野生型血凝素蛋白；由此，所产生的突变体不再包含任何N-连接的糖基化位点，不再携带任何N-连接的糖基链。因此，在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的各个特征序列N-X-(S或T)中的(S或T)残基各自独立地被置换为一个或多个其他的氨基酸残基(例如一个非S且非T的氨基酸残基)。
在某些优选的实施方案中，可以将一些特征序列N-X-(S或T)中的(S或T)残基删除，并且将剩余的特征序列N-X-(S或T)中的(S或T)残基各自独立地置换为一个或多个其他氨基酸残基(例如一个非S且非T的氨基酸残基)来改造野生型血凝素蛋白；由此，所产生的突变体不再包含任何N-连接的糖基化位点，不再携带任何N-连接的糖基链。因此，在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的一些特征序列N-X-(S或T)中的(S或T)残基被删除，并且其余特征序列N-X-(S或T)中的(S或T)残基各自独立地被置换为一个或多个其他的氨基酸残基(例如一个非S且非T的氨基酸残基)。
在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的一些特征序列N-X-(S或T)中的N残基各自独立地被删除或者置换为一个或多个其他的氨基酸残基(例如一个非N的氨基酸残基)；并且，剩余特征序列N-X-(S或T)中的(S或T)残基各自独立地被删除或者置换为一个或多个其他的氨基酸残基(例如一个非S且非T的氨基酸残基)。
在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的每一个特征序列N-X-(S或T)各自独立地具有选自下列的突变：
(1)N残基被删除或者置换为一个其他的氨基酸残基(例如一个非N的氨基酸残基)；
(2)(S或T)残基被删除或者置换为一个其他的氨基酸残基(例如一个非S且非T的氨基酸残基)；
(3)X残基被删除或者置换为脯氨酸残基；
(4)在N残基与X残基之间添加一个或多个氨基酸残基(例如一个非N的氨基酸残基)；和
(5)在X残基与(S或T)残基之间添加一个或多个氨基酸残基(例如一个非S且非T的氨基酸残基)；和
(6)(1)至(5)的任何组合。
将多肽链中的某一氨基酸残基删除或置换为其他氨基酸残基的方法是本领域技术人员熟知的。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变来对多肽链中的任意氨基酸残基进行改造(例如，删除或置换)。
在某些优选的实施方案中，用于置换N-连接的糖基化位点上(特别是特征序列N-X-(S或T)中)的天冬酰胺残基的氨基酸残基可以为选自下列的一个或多个氨基酸残基：丙氨酸，甘氨酸，缬氨酸，亮氨酸，异亮氨酸，苯丙氨酸，脯氨酸，色氨酸，丝氨酸，酪氨酸，半胱氨酸，甲硫氨酸，谷氨酰胺，苏氨酸，天冬氨酸，谷氨酸，赖氨酸，精氨酸和组氨酸。在某些优选的实施方案中，用于置换N-连接的糖基化位点上(特别是特征序列N-X-(S或T)中)的天冬酰胺残基的氨基酸残基可以为选自下列的一个氨基酸残基：丙氨酸，甘氨酸，缬氨酸，亮氨酸，异亮氨酸，苯丙氨酸，脯氨酸，色氨酸，丝氨酸，酪氨酸，半胱氨酸，甲硫氨酸，谷氨酰胺，苏氨酸，天冬氨酸，谷氨酸，赖氨酸，精氨酸和组氨酸。在某些优选的实施方案中，用于置换N-连接的糖基化位点上(特别是特征序列N-X-(S或T)中)的天冬酰胺残基的氨基酸残基可以为丙氨酸残基。在某些优选的实施方案中，各个N-连接的糖基化位点上(特别是特征序列N-X-(S或T)中)的天冬酰胺残基均被置换为丙氨酸。在某些优选的实施方案中，用于置换N-连接的糖基化位点上(特别是特征序列N-X-(S或T)中)的天冬酰胺残基的氨基酸残基可以为谷氨酰胺残基。在某些优选的实施方案中，各个N-连接的糖基化位点上(特别是特征序列N-X-(S或T)中)的天冬酰胺残基均被置换为谷氨酰胺。
在某些优选的实施方案中，用于特征序列N-X-(S或T)中的(S或T)残基的氨基酸残基可以为选自下列的一个或多个氨基酸残基：丙氨酸，甘氨酸，缬氨酸，亮氨酸，异亮氨酸，苯丙氨酸，脯氨酸，色氨酸，酪氨酸，半胱氨酸，甲硫氨酸，谷氨酰胺，天冬氨酸，谷氨酸，赖氨酸，精氨酸和组氨酸。在某些优选的实施方案中，用于置换特征序列N-X-(S或T)中的(S或T)残基的氨基酸残基可以为选自下列的一个氨基酸残基：丙氨酸，甘氨酸，缬氨酸，亮氨酸，异亮氨酸，苯丙氨酸，脯氨酸，色氨酸，酪氨酸，半胱氨酸，甲硫氨酸，谷氨酰胺，天冬氨酸，谷氨酸，赖氨酸，精氨酸和组氨酸。
此外，随着蛋白质结晶和结构解析技术的发展，对HA蛋白的功能和性质的研究和理解也日益深入。因此，借助于计算机程序或软件(例如PyMol)，可以确定各个N-连接的糖基化位点上(特别是特征序列N-X-(S或T)中)的天冬酰胺残基和(S或T) 残基在HA三聚体中的位置和构象。基于此，结合氨基酸残基的物理化学性质(例如大小、形状、电荷、形成共价键或氢键的能力等)，可选择合适的氨基酸残基用于置换天冬酰胺残基和(S或T)残基。例如，本领域已知，可对蛋白质或多肽进行保守置换，而不显著影响或改变所述蛋白质或多肽的功能和性质。
因此，在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的各个N-连接的糖基化位点上(特别是特征序列N-X-(S或T)中)的天冬酰胺残基各自独立地被保守置换。在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的各个N-连接的糖基化位点上(特别是特征序列N-X-(S或T)中)的天冬酰胺残基各自独立地被保守置换为选自下列的氨基酸残基：丙氨酸、甘氨酸、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸。
在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的各个特征序列N-X-(S或T)中的(S或T)残基各自独立地被非S且非T的氨基酸残基保守置换。在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的各个特征序列N-X-(S或T)中的(S或T)残基各自独立地被保守置换为选自下列的氨基酸残基：丙氨酸、甘氨酸、天冬酰胺、谷氨酰胺、酪氨酸、半胱氨酸、色氨酸。
在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的每一个特征序列N-X-(S或T)各自独立地具有选自下列的突变：
(1)N残基被删除或者被保守置换；
(2)(S或T)残基被删除或者被保守置换；
(3)X残基被删除或者置换为脯氨酸残基；
(4)在N残基与X残基之间添加一个非N的氨基酸残基；
(5)在X残基与(S或T)残基之间添加一个非S且非T的氨基酸残基；和
(6)(1)至(5)的任何组合。
在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的一些特征序列N-X-(S或T)中的N残基各自独立地被保守置换；并且，剩余的特征序列N-X-(S或T)中的(S或T)残基各自独立地被非S且非T的氨基酸残基保守置换。在某些优选的实施方案中，所述突变体与所述流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的一些特征序列N-X-(S或T)中的N残基各自独立地被保守置换为选自下列的氨基酸残基：丙氨酸、甘氨酸、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸；并且，剩余的特征序列N-X-(S或T)中的(S或T)残基各自独立地被保守置换为选自下列的氨基酸残基：丙氨酸、甘氨酸、天冬酰胺、谷氨酰胺、酪氨酸、半胱氨酸、色氨酸。
易于理解，蛋白的信号肽(通常位于蛋白的N端)能够指导/促进蛋白的分泌，并且，在分泌过程中或者分泌之后，信号肽可被切除，而不影响蛋白质的功能。因此，在某些优选的实施方案中，与所述H3N2亚型流感病毒的野生型血凝素蛋白相比，所述突变体不含有N-连接的糖基化位点，且不包含所述野生型血凝素蛋白的信号肽(例 如N端信号肽)。
还易于理解，蛋白的跨膜区通常指导/促进蛋白锚定至膜(例如，细胞膜或病毒包膜)。在某些情况下，将蛋白的跨膜区删除，不会不利地影响蛋白的生物学活性(例如免疫原性和免疫保护性)。因此，在某些优选的实施方案中，与所述H3N2亚型流感病毒的野生型血凝素蛋白相比，所述突变体不含有N-连接的糖基化位点，且不包含所述野生型血凝素蛋白的跨膜区。
可通过各种已知的方法来确定流感病毒HA蛋白中信号肽的位置和序列以及跨膜区位置和序列(参见例如T.M.Tumpey et al.，Proc.Natl.Acad.Sci.U.S.A.99，13849(2002))。此外，还已报道了多种HA蛋白的信号肽和跨膜区(参见例如，James Stevens et al.Science 312，404(2006))。因此，可方便地确定各种HA蛋白的信号肽和跨膜区的位置和序列，并对其进行改造(例如删除)。
在某些优选的实施方案中，与所述H3N2亚型流感病毒的野生型血凝素蛋白相比，所述突变体不含有N-连接的糖基化位点，且不包含所述野生型血凝素蛋白的信号肽(例如N端信号肽)和跨膜区。
在某些优选的实施方案中，所述野生型血凝素蛋白来源于甲型流感病毒H3N2亚型，例如在2005年后流行的H3N2亚型流感病毒，例如A/WISCONSIN/67/2005(H3N2)和A/HONG_KONG/4801/2014(H3N2)。在某些优选的实施方案中，所述野生型血凝素蛋白具有选自下列的序列：SEQ ID NO：1和6。
在某些优选的实施方案中，所述野生型血凝素蛋白的氨基酸序列如SEQ ID NO：1所示；并且，所述突变体与SEQ ID NO：1的区别至少在于，所述突变体不包含特征序列N-X-(S或T)；其中，N代表天冬酰胺，X代表除脯氨酸以外的任何一种氨基酸，S代表丝氨酸，T代表苏氨酸。在某些优选的实施方案中，所述野生型血凝素蛋白的氨基酸序列如SEQ ID NO：1所示；并且，所述突变体与SEQ ID NO：1的区别至少在于，SEQ ID NO：1中的每一个特征序列N-X-(S或T)各自独立地具有选自下列的突变：(1)N残基被删除或者置换为一个或多个其他的氨基酸残基(例如一个非N的氨基酸残基)；(2)(S或T)残基被删除或者置换为一个或多个其他的氨基酸残基(例如一个非S且非T的氨基酸残基)；(3)X残基被删除或者置换为脯氨酸残基；(4)在N残基与X残基之间添加一个非N的氨基酸残基；(5)在X残基与(S或T)残基之间添加一个非S且非T的氨基酸残基；和，(6)(1)至(5)的任何组合。在某些优选的实施方案中，所述突变体与SEQ ID NO：1的区别还在于，所述突变体不包含信号肽(例如SEQ ID NO：1的第1-10位氨基酸)。在某些优选的实施方案中，所述突变体与SEQ ID NO：1的区别还在于，所述突变体不包含跨膜区(例如SEQ ID NO：1的第504-550位氨基酸)。在某些优选的实施方案中，所述突变体与SEQ ID NO：1的区别还在于，所述突变体不包含信号肽(例如SEQ ID NO：1的第1-10位氨基酸)和跨膜区(例如SEQ ID NO：1的第504-550位氨基酸)。
在某些优选的实施方案中，所述野生型血凝素蛋白的氨基酸序列如SEQ ID NO：1所示；并且，所述突变体与SEQ ID NO：1的区别至少在于，SEQ ID NO：1的第1-10位和第504-550位氨基酸被缺失，并且SEQ ID NO：1的第22，38，63，126，133，144，165，246，285和483位上的天冬酰胺残基各自独立地被删除或者置换为一个或 多个其他的氨基酸残基(例如一个非N的氨基酸残基，例如丙氨酸残基或谷氨酰胺残基)。
在某些优选的实施方案中，所述野生型血凝素蛋白的氨基酸序列如SEQ ID NO：6所示；并且，所述突变体与SEQ ID NO：6的区别至少在于，所述突变体不包含特征序列N-X-(S或T)；其中，N代表天冬酰胺，X代表除脯氨酸以外的任何一种氨基酸，S代表丝氨酸，T代表苏氨酸。在某些优选的实施方案中，所述野生型血凝素蛋白的氨基酸序列如SEQ ID NO：6所示；并且，所述突变体与SEQ ID NO：6的区别至少在于，SEQ ID NO：6中的每一个特征序列N-X-(S或T)各自独立地具有选自下列的突变：(1)N残基被删除或者置换为一个或多个其他的氨基酸残基(例如一个非N的氨基酸残基)；(2)(S或T)残基被删除或者置换为一个或多个其他的氨基酸残基(例如一个非S且非T的氨基酸残基)；(3)X残基被删除或者置换为脯氨酸残基；(4)在N残基与X残基之间添加一个非N的氨基酸残基；(5)在X残基与(S或T)残基之间添加一个非S且非T的氨基酸残基；和，(6)(1)至(5)的任何组合。在某些优选的实施方案中，所述突变体与SEQ ID NO：6的区别还在于，所述突变体不包含信号肽(例如SEQ ID NO：6的第1-25位氨基酸)。在某些优选的实施方案中，所述突变体与SEQ ID NO：6的区别还在于，所述突变体不包含跨膜区(例如SEQ ID NO：6的第518-565位氨基酸)。在某些优选的实施方案中，所述突变体与SEQ ID NO：1的区别还在于，所述突变体不包含信号肽(例如SEQ ID NO：6的第1-25位氨基酸)和跨膜区(例如SEQ ID NO：6的第518-565位氨基酸)。
在某些优选的实施方案中，所述野生型血凝素蛋白的氨基酸序列如SEQ ID NO：6所示；并且，所述突变体与SEQ ID NO：6的区别至少在于，SEQ ID NO：6的第1-25位和第518-565位氨基酸被缺失，并且SEQ ID NO：6的第37，53，60，78，137，141，148，180，261，300和498位上的天冬酰胺残基各自独立地被删除或者置换为一个或多个其他的氨基酸残基(例如一个非N的氨基酸残基，例如丙氨酸残基或谷氨酰胺残基)。
在某些优选的实施方案中，所述突变体具有选自下列的氨基酸序列：SEQ ID NO：12-13。
本领域技术人员知晓，可对蛋白质或多肽的氨基酸序列进行适当的改造(例如，氨基酸残基的添加，删除和/或置换)，而不对所述蛋白质或多肽的功能和性质造成显著影响。因此，在某些情况下，通过对上述突变体的氨基酸序列进行进一步的改造，可获得额外的突变体，其保持诱发抗不同亚型的流感病毒的保护性抗体的能力，具有抗不同亚型的流感病毒的保护作用。
因此，在某些优选的实施方案中，本发明的突变体与选自下列的氨基酸序列具有至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％或100％的同一性：SEQ ID NO：12-13；前提条件是所述突变体不包含任何N-连接的糖基化位点(例如不包含任何特征序列N-X-(S或T))。
在某些优选的实施方案中，本发明的突变体与选自下列的氨基酸序列相比，具有一个或多个氨基酸残基的添加、删除或置换：SEQ ID NO：12-13；前提条件是所述突 变体不包含任何N-连接的糖基化位点(例如不包含任何特征序列N-X-(S或T))。在某些优选的实施方案中，本发明的突变体与选自下列的氨基酸序列相比，具有一个或几个氨基酸残基(例如，1个，2个，3个，4个，5个，6个，7个，8个，9个)的添加、删除或置换：SEQ ID NO：12-13；前提条件是所述突变体不包含任何N-连接的糖基化位点(例如不包含任何特征序列N-X-(S或T))。在某些优选的实施方案中，本发明的突变体与选自下列的氨基酸序列相比，具有一个或几个氨基酸残基(例如，1个，2个，3个，4个，5个，6个，7个，8个，9个)的置换(特别是保守置换)：SEQ ID NO：12-13；前提条件是所述突变体不包含任何N-连接的糖基化位点(例如不包含任何特征序列N-X-(S或T))。
本申请公开的衍生自H3N2亚型流感病毒HA蛋白的突变体不包含糖基化位点(例如不包含特征序列N-X-(S或T))，能够在体内诱导广谱的抗流感病毒保护性抗体，能够在体内提供广谱的抗流感病毒保护作用。特别地，本申请公开的突变体能够诱发抗不同亚型(例如H3N2、H7N9和/或H1N1亚型)的流感病毒的保护性抗体，实现抗不同亚型(例如H3N2、H7N9和/或H1N1亚型)的流感病毒的保护作用，并因此可用作能够抗多种亚型(例如至少2种、至少3种或更多种亚型；例如H3N2、H7N9和/或H1N1亚型)的流感病毒的广谱疫苗，用于预防和/或治疗多种亚型(例如至少2种、至少3种或更多种亚型；例如H3N2、H7N9和/或H1N1亚型)的流感病毒的感染以及与所述感染相关的疾病(例如流感)。因此，本申请公开的突变体是特别有利的。
关于重组蛋白
在一个方面，本申请涉及一种重组蛋白，其包含根据本发明的H3N2亚型流感病毒血凝素蛋白的突变体，以及额外的肽段，所述额外的肽段连接至所述突变体。
在本申请的重组蛋白中，所述额外的肽段可通过各种方式与所述突变体相连接。例如，在某些优选的实施方案中，所述额外的肽段直接与所述突变体连接。换言之，所述额外的肽段通过肽键直接与所述突变体连接。在某些优选的实施方案中，所述额外的肽段通过接头连接至所述突变体。合适的现有技术接头可以由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS) 4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90：6444-6448)。此外，还可使用其他接头，例如Alfthan等人(1995)，Protein Eng.8：725-731；Choi等人(2001)，Eur.J.Immunol.31：94-106；Hu等人(1996)，Cancer Res.56：3055-3061；Kipriyanov等人(1999)，J.Mol.Biol.293：41-56和Roovers等人(2001)，Cancer Immunol.中描述的接头。
在本申请的重组蛋白中，所述额外的肽段可连接至所述突变体的任一末端。例如，在某些优选的实施方案中，所述额外的肽段连接至所述突变体的N端。在某些优选的实施方案中，所述额外的肽段连接至所述突变体的C端。
根据本发明的重组蛋白可以包含一个或多个的额外肽段。例如，在某些优选的实施方案中，根据本发明的重组蛋白可以包含至少1个，至少2个，至少3个，至少5个或更多个额外的肽段。易于理解的是，这些肽段各自独立地可以以各种方式连接至 所述突变体的任一末端(N端或C端)。例如，在某些优选的实施方案中，本发明的重组蛋白可以包含两个额外的肽段，其中，一个额外的肽段通过接头或者不通过接头连接至所述突变体的N端，并且，另一个额外的肽段通过接头或者不通过接头连接至所述突变体的C端。在某些优选的实施方案中，本发明的重组蛋白可以包含两个或者更多个额外的肽段，其中，所述两个或者更多个额外的肽段各自独立地通过接头或者不通过接头连接至所述突变体的N端或C端。在某些优选的实施方案中，当两个或者更多个额外的肽段连接至突变体的N端时，所述两个或者更多个额外的肽段可以以任何顺序串联，然后通过接头或者不通过接头连接至所述突变体的N端。类似地，在某些优选的实施方案中，当两个或者更多个额外的肽段连接至突变体的C端时，所述两个或者更多个额外的肽段可以以任何顺序串联，然后通过接头或者不通过接头连接至所述突变体的C端。
可根据实际需要来选择合适的额外的肽段。例如，在某些优选的实施方案中，额外的肽段可以为信号肽(例如，如SEQ ID NO：9所示的信号肽)。不受任何理论约束，通常认为，信号肽的使用可以促进重组蛋白的分泌，从而便于重组蛋白的回收。通常，此类信号肽可连接至突变体的N端。此外，在分泌过程中或者分泌之后，信号肽可被切除，产生期望的突变体或重组蛋白。在某些优选的实施方案中，额外的肽段可以为标签肽，例如，如SEQ ID NO：11所示的6＊His标签。不受任何理论约束，通常认为，标签肽的使用可以便于重组蛋白的检测、回收和纯化。例如，可使用镍离子来纯化携带6＊His标签的蛋白。在某些优选的实施方案中，额外的肽段可以为促进突变体形成三聚体的折叠基序。此类折叠基序包括但不限于，如SEQ ID NO：10所示的折叠基序。在某些优选的实施方案中，额外的肽段可以为可检测标记，例如荧光蛋白。
因此，在某些优选的实施方案中，所述额外的肽段选自信号肽，标签肽，折叠基序，可检测标记，以及其任何组合。在某些优选的实施方案中，所述信号肽具有如SEQ ID NO：9所示的氨基酸序列。在某些优选的实施方案中，所述信号肽连接至所述突变体的N端。在某些优选的实施方案中，所述折叠基序具有如SEQ ID NO：10所示的氨基酸序列。在某些优选的实施方案中，所述折叠基序连接至所述突变体的C端。在某些优选的实施方案中，所述标签肽具有如SEQ ID NO：11所示的氨基酸序列。在某些优选的实施方案中，所述标签肽连接至所述突变体的N端或C端。
在某些优选的实施方案中，所述重组蛋白可具有选自下列的氨基酸序列：SEQ ID NO：3和8。
本领域技术人员知晓，可对蛋白质或多肽的氨基酸序列进行适当的改造(例如，氨基酸残基的添加，删除和/或置换)，而不对所述蛋白质或多肽的功能和性质造成显著影响。因此，在某些情况下，通过对上述重组蛋白的氨基酸序列进行进一步的改造，可获得额外的重组蛋白，其保持诱发抗不同亚型的流感病毒的保护性抗体的能力，具有抗不同亚型的流感病毒的保护作用。
因此，在某些优选的实施方案中，本发明的重组蛋白与选自下列的氨基酸序列具有至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％或100％的同一性：SEQ ID NO：3和8；前提条件是所述重组蛋白或突变体不包含任何N-连接的糖基化位点(例如不包含任何 特征序列N-X-(S或T))。
在某些优选的实施方案中，本发明的重组蛋白与选自下列的氨基酸序列相比，具有一个或多个氨基酸残基的添加、删除或置换：SEQ ID NO：3和8；前提条件是所述重组蛋白或所述突变体不包含任何N-连接的糖基化位点(例如不包含任何特征序列N-X-(S或T))。在某些优选的实施方案中，本发明的重组蛋白与选自下列的氨基酸序列相比，具有一个或几个氨基酸残基(例如，1个，2个，3个，4个，5个，6个，7个，8个，9个)的添加、删除或置换：SEQ ID NO：3和8；前提条件是所述重组蛋白或所述突变体不包含任何N-连接的糖基化位点(例如不包含任何特征序列N-X-(S或T))。在某些优选的实施方案中，本发明的重组蛋白与选自下列的氨基酸序列相比，具有一个或几个氨基酸残基(例如，1个，2个，3个，4个，5个，6个，7个，8个，9个)的置换(特别是保守置换)：SEQ ID NO：3和8；前提条件是所述重组蛋白或所述突变体不包含任何N-连接的糖基化位点(例如不包含任何特征序列N-X-(S或T))。
关于核酸分子、载体、病毒和宿主细胞
在另一个方面，本申请涉及一种核酸分子，其包含或者由编码本发明的突变体或本发明的重组蛋白的核苷酸序列组成。在某些优选的实施方案中，本发明的核酸分子是分离的或经纯化的。
在另一个方面，本申请涉及一种载体，其包含如上所述的核酸分子。本发明的载体可以是克隆载体，转移载体，或表达载体。在一个优选实施方案中，本发明的载体是例如质粒，粘粒，噬菌体等等。在一个优选实施方案中，所述载体能够在真核细胞(例如昆虫细胞)中表达本发明的突变体或本发明的重组蛋白。在一个优选实施方案中，所述载体为杆状病毒的转移载体，其能够与杆状病毒基因组DNA一起使用，实现本发明的突变体或本发明的重组蛋白在昆虫细胞中的表达。在一个优选实施方案中，所述杆状病毒为苜蓿丫纹夜蛾核型多角体病毒(AcMNPV)。
在另一个方面，本发明还涉及包含如上所述的核酸分子或载体的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的宿主细胞还可以是细胞系，例如293T细胞。不受任何理论约束，通常认为，真核细胞的使用有助于维持蛋白的正确构象，促进蛋白的折叠。因此，在某些优选的实施方案中，本发明的宿主细胞是真核细胞例如昆虫细胞。在某些优选的实施方案中，本发明的宿主细胞是昆虫细胞，其包含含有上述核酸分子的杆状病毒转移载体，以及杆状病毒基因组DNA。在一个优选实施方案中，所述杆状病毒为苜蓿丫纹夜蛾核型多角体病毒(AcMNPV)。
在另一个方面，本发明还涉及包含如上所述的核酸分子或载体的病毒(例如杆状病毒)。在某些优选的实施方案中，所述病毒为杆状病毒，例如苜蓿丫纹夜蛾核型多角体病毒(AcMNPV)。
关于多聚体
在一个方面，本申请涉及一种多聚体，其包含多个本发明的突变体或者多个本发 明的重组蛋白，或者由多个本发明的突变体或者多个本发明的重组蛋白组成。在某些优选的实施方案中，所述多聚体为三聚体。换言之，所述多聚体包含或者由三个本发明的突变体或者重组蛋白组成。在某些优选的实施方案中，所述三聚体具有与由天然HA蛋白形成的三聚体相同或类似的构象。
关于组合物
在另一个方面，本申请还涉及包含上述突变体，或上述重组蛋白，或上述核酸分子，或上述载体，或上述宿主细胞，或上述病毒，或上述多聚体的组合物。在某些优选的实施方案中，所述组合物包含本发明的突变体或重组蛋白。在某些优选的实施方案中，所述组合物包含本发明的多聚体。
关于药物组合物、治疗方法和用途
在另一个方面，本发明还涉及一种药物组合物(例如疫苗)，其包含本发明的突变体或重组蛋白或者多聚体，任选地还包含药学可接受的载体和/或赋形剂。本发明的药物组合物(例如疫苗)可以用于预防或治疗流感病毒感染或由流感病毒感染所导致的疾病例如流感等。
在某些优选的实施方案中，本发明的突变体或重组蛋白或者多聚体以预防或治疗流感病毒感染或由流感病毒感染所导致的疾病的有效量存在。在某些优选的实施方案中，本发明的药物组合物(例如疫苗)还包含另外的活性成分。优选地，所述另外的活性成分能够预防或治疗流感病毒感染或由流感病毒感染所导致的疾病。在某些优选的实施方案中，本发明的药物组合物(例如疫苗)还包含佐剂，例如铝佐剂。
在某些优选的实施方案中，所述药物组合物还包含药学可接受的载体，赋形剂，稳定剂或能够为所述药物组合物的施用(例如施用给人受试者)提供有利性质的其他试剂。合适的药物载体包括例如，无菌水，盐水，葡萄糖，蓖麻油和环氧乙烷的缩合产物，液体酸，低级醇(例如C 1-4醇)，油(例如玉米油，花生油，芝麻油；其任选还包含乳化剂例如脂肪酸的单-或二-甘油酯或磷脂例如卵磷脂)，乙二醇，聚亚烷基二醇，藻酸钠，聚(乙烯基吡咯烷酮)等等。所述载体任选地还可包含佐剂，防腐剂，稳定剂，润湿剂，乳化剂，渗透增强剂等。在某些优选的实施方案中，所述药物组合物是无菌的。此外，所述药物组合的粘度可通过选择合适的溶剂或赋形剂来控制和维持。在某些优选的实施方案中，所述药物组合物经配制具有4.5-9.0，5.0-8.0，6.5-7.5，或6.5-7.0的pH。
本发明的药物组合物(例如疫苗)可通过本领域公知的方法进行施用，例如但不限于通过口服或者注射进行施用。在某些优选的实施方案中，本发明的药物组合物(例如疫苗)以单位剂量形式进行施用。
预防或治疗特定病况所需的本发明药物组合物(例如疫苗)的量将取决于施用途径、待治疗的病况的严重程度、患者的性别、年龄、体重和总体健康情况等等，其可由医生根据实际情况合理确定。
在某些优选的实施方案中，本发明的药物组合物(例如疫苗)包含衍生自H3N2亚型流感病毒HA蛋白的突变体或包含所述突变体的重组蛋白或多聚体，其能够诱发 针对H3N2、H7N9和/或H1N1亚型流感病毒的保护性抗体，实现抗H3N2、H7N9和/或H1N1亚型流感病毒的保护作用，因此，可用于预防和/或治疗H3N2、H7N9和/或H1N1亚型流感病毒的感染以及与之相关的疾病(例如流感)。
在另一个方面，本发明涉及一种在受试者中预防或治疗流感病毒感染或由流感病毒感染所导致的疾病的方法，其包括将预防或治疗有效量的根据本发明的突变体或重组蛋白或者多聚体或者本发明的药物组合物施用给所述受试者。在某些优选的实施方案中，所述由流感病毒感染所导致的疾病为流感。在某些优选的实施方案中，所述受试者是哺乳动物，例如小鼠和人。
在某些优选的实施方案中，本发明的方法可用于预防和/或治疗H3N2、H7N9和/或H1N1亚型流感病毒的感染以及与之相关的疾病(例如流感)。
在另一个方面，本发明还涉及本发明的突变体或重组蛋白或者多聚体在制备药物组合物(例如疫苗)中的用途，所述药物组合物(例如疫苗)用于在受试者中预防或治疗流感病毒感染或由流感病毒感染所导致的疾病。在某些优选的实施方案中，所述由流感病毒感染所导致的疾病为流感。在某些优选的实施方案中，所述受试者是哺乳动物，例如小鼠和人。
在某些优选的实施方案中，所述药物组合物(例如疫苗)包含衍生自H3N2亚型流感病毒HA蛋白的突变体或包含所述突变体的重组蛋白或多聚体，用于预防和/或治疗H3N2、H7N9和/或H1N1亚型流感病毒的感染以及与之相关的疾病(例如流感)。
在另一个方面，本发明还涉及上述的突变体或重组蛋白或者多聚体，其用于在受试者中预防或治疗流感病毒感染或由流感病毒感染所导致的疾病。在某些优选的实施方案中，所述由流感病毒感染所导致的疾病为流感。在某些优选的实施方案中，所述受试者是哺乳动物，例如小鼠和人。在某些优选的实施方案中，所述突变体或重组蛋白或多聚体用于预防和/或治疗H3N2、H7N9和/或H1N1亚型流感病毒的感染以及与之相关的疾病(例如流感)。
关于制备方法
在另一个方面，本发明涉及一种制备上述突变体或重组蛋白的方法，其包括，在允许所述突变体或重组蛋白表达的条件下，培养本发明的宿主细胞或病毒；和，回收所表达的突变体或重组蛋白。
在某些优选的实施方案中，所述方法包括：将本发明的载体(例如表达载体)导入宿主细胞(例如真核细胞)，从而在宿主细胞中表达所述突变体或重组蛋白；以及，回收所表达的突变体或重组蛋白。在某些优选的实施方案中，所述方法包括：将含有上述核酸分子的杆状病毒转移载体以及杆状病毒基因组DNA导入昆虫细胞，从而在昆虫细胞中表达所述突变体或重组蛋白；以及，回收所表达的突变体或重组蛋白。在一个优选实施方案中，所述杆状病毒为首蓿丫纹夜蛾核型多角体病毒(AcMNPV)。
在另一个方面，本发明还涉及一种制备疫苗的方法，其包括将本发明的突变体或重组蛋白或者多聚体与药学可接受的载体和/或赋形剂混合，任选地还混合佐剂例如铝佐剂，和/或另外的活性成分，例如能够预防或治疗流感病毒感染或由流感病毒感染所 导致的疾病的另外的活性成分。在某些优选的实施方案中，所述制备疫苗的方法包括下述步骤：将本发明的突变体或重组蛋白或者多聚体与佐剂(例如铝佐剂)混合。
如上所论述的，所获得的疫苗可以用于预防或治疗流感病毒感染或由流感病毒感染所导致的疾病例如流感。
本申请中相关术语的说明及解释
在本申请中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar，Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4：11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48：444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的生物学活性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基 酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32：1180-1187(1993)；Kobayashi等人Protein Eng.12(10)：879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94：412-417(1997)，其通过引用并入本文)。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，表述“相应序列片段”或“相应片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的片段。根据本发明，表述“相应氨基酸位置”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的氨基酸位点/残基。
如本文中所使用的，术语“表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
如本文中所使用的，术语“多聚体”是指，以多肽分子(例如，本发明的突变体或重组蛋白)为单体构成的聚合体，其通常可包含至少2个(例如，3个，4个，5个或更多个)多肽单体(例如，本发明的突变体或重组蛋白)。在此类多聚体中，单体分子通过分子间相互作用(例如氢键、范德华力、疏水相互作用)而聚合形成多聚体。在本发明的某些实施方案中，多聚体是包含3个单体的三聚体。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然 环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，“特征序列N-X-(S或T)”是指，能够发生N-连接的糖基化的特征性基序，其中，N代表天冬酰胺，X代表除脯氨酸以外的任何一种氨基酸，S代表丝氨酸，T代表苏氨酸。
如本文中所使用的，术语“保护性抗体”是指，具有抵抗病毒的保护性作用的抗体。保护性抗体包括但不限于，能够中和病毒毒力的抗体，能够抑制病毒识别并结合宿主细胞的抗体，以及能够抑制病毒与宿主细胞融合的抗体。
如本文中所使用的，术语“药学可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington′s Pharmaceutical Sciences.Edited by Gennaro AR，19th ed.Pennsylvania：Mack Publishing Company，1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；佐剂包括但不限于铝佐剂(例如氢氧化铝)，弗氏佐剂(例如完全弗氏佐剂)；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“佐剂”是指非特异性免疫增强剂，当其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂有很多种，包括但不限于铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。弗氏佐剂是目前动物试验中最常用的佐剂。氢氧化铝佐剂则在临床实验中使用较多。在本发明中，特别优选地，佐剂为铝佐剂。
如本文中所使用的，术语“有效量”是指能够有效实现预期目的的量。例如，预防或治疗疾病(例如流感病毒感染)有效量是指，能够有效预防、阻止或延迟疾病(例如流感病毒感染)的发生、或缓解、减轻或治疗已有的疾病(例如由流感病毒感染所导致的疾病)的严重程度的量。测定这样的有效量在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中使用的，术语“免疫原性(immunogenicity)”是指，能够刺激机体形成特异抗体或致敏淋巴细胞的能力。其既指，抗原能刺激特定的免疫细胞，使免疫细胞活化、增殖、分化，最终产生免疫效应物质如抗体和致敏淋巴细胞的特性，也指抗原刺激机体后，机体免疫系统能形成抗体或致敏T淋巴细胞的特异性免疫应答。免疫原性是抗原最重要的性质，一种抗原能否成功地诱导宿主产生免疫应答取决于三方面的因素：抗原的性质、宿主的反应性和免疫方式。
如本文中所使用的，术语“多肽”和“蛋白质”具有相同的含义，可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸 可用A或Ala表示。
如本文中所使用的，“受试者”是指动物，例如脊椎动物。优选地，受试者为哺乳动物，例如人，牛科动物，马科动物，猫科动物，犬科动物，啮齿类动物或灵长类动物。特别优选地，受试者为人。在本文中，该术语可以与“患者”互换使用。
发明的有益效果
本申请提供了一种H3N2亚型流感病毒血凝素蛋白的突变体，其能够诱发抗不同亚型(例如H3N2、H7N9和/或H1N1亚型)的流感病毒的保护性抗体，实现抗不同亚型的流感病毒的保护作用，并因此可用作能够抗多种亚型(例如至少2种、至少3种或更多种亚型)的流感病毒的广谱疫苗，用于预防和/或治疗多种亚型(例如至少2种、至少3种或更多种亚型)的流感病毒的感染以及与所述感染相关的疾病(例如流感)。
特别地，本申请公开的衍生自H3N2亚型流感病毒HA蛋白的突变体不仅能够诱发针对多株H3N2亚型流感病毒(特别是流行于不同年代的多株H3N2亚型流感病毒)的保护性抗体，实现抗多株H3N2亚型流感病毒的保护作用，而且能够诱发针对H7N9和/或H1N1亚型流感病毒的保护性抗体，实现抗H7N9和/或H1N1亚型流感病毒的保护作用。
由此，本申请提供了一种广谱流感疫苗，其能够提供针对多种亚型(例如H3N2、H7N9和/或H1N1亚型)的流感病毒的交叉保护作用，并且免疫效果理想，不易由于流感病毒变异而快速失效，从而克服了现有流感疫苗因流感病毒频繁变异而导致的免疫效力丧失、免疫效果不理想等缺点。特别地，本申请的广谱流感疫苗解决了现有流感疫苗需要每年换型，每年注射的弊端。此外，本申请的广谱流感疫苗可以有效地扼制多种亚型的流感病毒的蔓延，减少流感病毒造成的经济损失和社会恐慌。因此，本申请的广谱流感疫苗与现有的流感疫苗相比，具有特别显著的优势。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1示意性地说明实施例1中所使用的天然HA蛋白(WI2005-WT-HA)、HA-mut1蛋白、HA-mut2蛋白和HA-mut3蛋白的序列突变和N-连接的糖基化。
图2示意性地说明分别由实施例1中所使用的天然HA蛋白(图2A)、HA-mut1蛋白(图2B)、HA-mut2蛋白(图2C)和HA-mut3蛋白(图2D)形成的三聚体的结构示意图；其中，图2A显示由天然HA蛋白形成的三聚体在头部和茎部区域均含有N-连接的糖基链；图2B显示由HA-mut1蛋白形成的三聚体在头部和茎部区域均不含有N-连接的糖基链；图2C显示由HA-mut2蛋白形成的三聚体在头部区域不含有N-连接的糖基链，而在茎部区域仍然包含N-连接的糖基链；图2D显示由HA-mut3蛋白形成的三聚 体在茎部区域不含有N-连接的糖基链，而在头部区域仍然包含N-连接的糖基链。
图3显示了实施例1制备的六种蛋白的SDS-PAGE分析结果；其中，图3A显示了天然HA蛋白、HA-mut3、HA-mut2和HA-mut1蛋白的SDS-PAGE分析结果；图3B显示了天然HA蛋白，HAmg蛋白和HAug蛋白的SDS-PAGE分析结果。
图4显示了用天然HA蛋白、HA-mut1、HA-mut2、HA-mut3和PBS(用作阴性对照)作为免疫原分别免疫小鼠所获得的小鼠血清对流感病毒A/Wisconsin/67/2005(H3N2亚型)(图4A)、A/Victoria/361/2011(H3N2亚型)(图4B)、A/Beijing/32/1992(H3N2亚型)(图4C)、A/Aichi/2/1968(H3N2亚型)(图4D)、A/Shanghai/02/2013(H7N9亚型)(图4E)和A/California/04/2009(H1N1亚型)(图4F)的中和活性。
图5显示了用天然HA蛋白、HA-mut1、HAmg、HAug和PBS(用作阴性对照)作为免疫原分别免疫小鼠所获得的小鼠血清对流感病毒A/Wisconsin/67/2005(H3N2亚型)(图5A)、A/Victoria/361/2011(H3N2亚型)(图5B)、A/Beijing/32/1992(H3N2亚型)(图5C)、A/Aichi/2/1968(H3N2亚型)(图5D)、A/Shanghai/02/2013(H7N9亚型)(图5E)和A/California/04/2009(H1N1亚型)(图5F)的中和活性。
图6显示了，用天然HA蛋白、HA-mut1蛋白、HA-mut2蛋白、HA-mut3蛋白或PBS(阴性对照)免疫的小鼠在感染流行年份较早的H3N2亚型流感病毒A/Beijing/32/1992(H3N2)(图6A-6B)和A/Aichi/2/1968(H3N2)(图6C-6D)后的体重变化情况和存活情况，其中，图6A和图6C显示了各实验小鼠的体重变化，图6B和图6D显示了各实验小鼠的生存率。
图7显示了，用天然HA蛋白、HA-mut1蛋白、HAmg蛋白、HAug蛋白或PBS(阴性对照)免疫的小鼠在感染流行年份较早的H3N2亚型流感病毒A/Beijing/32/1992(H3N2)(图7A-7B)和A/Aichi/2/1968(H3N2)(图7C-7D)后的体重变化情况和存活情况，其中，图7A和图7C显示了各实验小鼠的体重变化，图7B和图7D显示了各实验小鼠的生存率。
图8显示了，用天然HA蛋白、HA-mut1蛋白、HA-mut2蛋白、HA-mut3蛋白或PBS(阴性对照)免疫的小鼠在感染非H3N2亚型流感病毒A/Shanghai/02/2013(H7N9)(图8A-8B)和A/California/04/2009(H1N1)(图8C-8D)后的体重变化情况和存活情况，其中，图8A和图8C显示了各实验小鼠的体重变化，图8B和图8D显示了各实验小鼠的生存率。
图9显示了，用天然HA蛋白、HA-mut1蛋白、HAmg蛋白、HAug蛋白或PBS(阴性对照)免疫的小鼠在感染非H3N2亚型流感病毒A/Shanghai/02/2013(H7N9)(图9A-9B)和A/California/04/2009(H1N1)(图9C-9D)后的体重变化情况和存活情况，其中，图9A和图9C显示了各实验小鼠的体重变化，图9B和图9D显示了各实验小鼠的生存率。
图10显示了HK2014-WT-HA蛋白的SDS-PAGE分析(左图)和Western blot分析(右图)的结果；其中，泳道M：分子量标记；泳道1：未经Ni-NTA镍离子层析柱纯化的样品；泳道2：流经Ni-NTA镍离子层析柱的级分；泳道3：用50mM咪唑洗脱的级分；泳道4：用50mM咪唑洗脱的级分；泳道5：用250mM咪唑洗脱的级分；箭头指示目的蛋白HK2014-WT-HA的位置。
图11显示了HK2014-DG-HA蛋白的SDS-PAGE分析(左图)和Western blot分析(右图)的结果；其中，泳道M：分子量标记；泳道1：未经Ni-NTA镍离子层析柱纯化的样品；泳道2：流经Ni-NTA镍离子层析柱的级分；泳道3：用50mM咪唑洗脱的级分；泳道4：用250mM咪唑洗脱的级分；箭头指示目的蛋白HK2014-DG-HA的位置。
图12显示了天然HA蛋白HK2014-WT-HA和去糖基化蛋白HK2014-HAug的SDS-PAGE分析结果；其中，泳道M：分子量标记；泳道1：经纯化的HK2014-WT-HA；泳道2：HK2014-HAug(用内切糖苷酶F消化HK2014-WT-HA3小时而获得)。
图13显示了用HK2014-WT-HA、HK2014-DG-HA和PBS(用作阴性对照)作为免疫原分别免疫小鼠所获得的小鼠血清对流感病毒A/Wisconsin/67/2005(H3N2)、A/Xiamen/N794/2013(H3N2)和A/Shanghai/02/2013(H7N9)的结合活性的ELISA分析结果。
图14显示了用HK2014-WT-HA、HK2014-HAug和PBS(用作阴性对照)作为免疫原分别免疫小鼠所获得的小鼠血清对流感病毒A/Wisconsin/67/2005(H3N2)、A/Xiamen/N794/2013(H3N2)和A/Shanghai/02/2013(H7N9)的结合活性的ELISA分析结果。
图15显示了，用HK2014-WT-HA、HK2014-DG-HA或PBS(用作阴性对照)免疫的各组小鼠(3只/组)在感染A/Aichi/2/1968(H3N2)后的体重变化情况(左图)和存活情况(右图)。
图16显示了，用HK2014-WT-HA、HK2014-DG-HA或PBS(用作阴性对照)免疫的各组小鼠(3只/组)在感染A/Shanghai/059/2013(H7N9)后的体重变化情况(左图)和存活情况(右图)。
图17显示了，用HK2014-WT-HA、HK2014-HAug或PBS(用作阴性对照)免疫的各组小鼠(4只/组)在感染A/Shanghai/059/2013(H7N9)后的体重变化情况。
序列信息
本发明涉及的序列的信息提供于下面的表1中。
表1：序列信息
Figure PCTCN2018109589-appb-000001
Figure PCTCN2018109589-appb-000002
Figure PCTCN2018109589-appb-000003
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本申请中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons，Inc.，1995中所述的方法进行。限制性内切酶的使用依照产品制造商推荐的条件。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：H3N2流感病毒HA蛋白及其突变体的制备
(a)HA蛋白突变体的设计和结构
流感病毒的天然HA蛋白中，发生N-连接的糖基化的氨基酸通常为特征序列N-X-(S或T)中的天冬酰胺(N)，其中N代表天冬酰胺，X代表除脯氨酸以外的任何一种氨基 酸，S代表丝氨酸，T代表苏氨酸。在本实施例中，通过将天然HA蛋白中的特征序列N-X-(S或T)中的天冬酰胺(N)突变为丙氨酸(A)来去除HA蛋白的N-连接的糖基化位点。
本实施例中使用的天然HA蛋白(WI2005-WT-HA)来源于H3N2亚型流感病毒株A/Wisconsin/67/2005的HA蛋白。该毒株的HA蛋白包含如SEQ ID NO：1所示的氨基酸序列，其中，SEQ ID NO：1的第1-10位氨基酸为信号肽，第504-550位氨基酸为跨膜区，并且，具有10个潜在的N-连接的糖基化位点，即位于第22位、第38位、第63位、第126位、第133位、第144位、第165位、第246位、第285位和第483位的天冬酰胺(N)。在这些N-连接的糖基化位点中，除了第483位的天冬酰胺位于HA蛋白的HA2亚基之外，其他位点的天冬酰胺均位于HA蛋白的HA1亚基。另外，从空间结构来说，第22位、第38位、第285位和第483位的天冬酰胺位于HA蛋白三聚体的茎部区域；而第63位、第126位、第133位、第144位、第165位和第246位的天冬酰胺位于HA蛋白三聚体的头部区域。
基于上述结构信息，本实施例中设计了以下天然HA蛋白和三个HA蛋白突变体(图1)：
(1)天然HA蛋白(WI2005-WT-HA)，其包含如SEQ ID NO：2所示的氨基酸序列，并且其与SEQ ID NO：1的差异在于，SEQ ID NO：1的第1-10位和第504-550位氨基酸被缺失，并且，在SEQ ID NO：1的C端引入包含凝血酶裂解位点、折叠基序和6＊His标签的肽段(其包含SEQ ID NO：10和11的序列，用于促进蛋白的纯化和三聚体形成)。相应地，由天然HA蛋白(WI2005-WT-HA)形成的三聚体在头部和茎部区域均含有N-连接的糖基链(图2A)。
(2)HA-mut1，其包含如SEQ ID NO：3所示的氨基酸序列，并且其与天然HA蛋白(WI2005-WT-HA；SEQ ID NO：2)的差异在于，如上所述的所有10个N-连接的糖基化位点上的天冬酰胺均被突变为丙氨酸。相应地，由HA-mut1形成的三聚体在头部和茎部区域均不含有N-连接的糖基链(图2B)。
(3)HA-mut2，其包含如SEQ ID NO：4所示的氨基酸序列，并且其与天然HA蛋白(WI2005-WT-HA；SEQ ID NO：2)的差异在于，位于头部区域(即，SEQ ID NO：1的第63位、第126位、第133位、第144位、第165位和第246位)的天冬酰胺被突变为丙氨酸。相应地，由HA-mut2形成的三聚体在头部区域不含有N-连接的糖基链，而在茎部区域仍然包含N-连接的糖基链(图2C)。
(4)HA-mut3，其包含如SEQ ID NO：5所示的氨基酸序列，并且其与天然HA蛋白(WI2005-WT-HA；SEQ ID NO：2)的差异在于，位于茎部区域(即，SEQ ID NO：1的第22位、第38位、第285位和第483位)的天冬酰胺被突变为丙氨酸。相应地，由HA-mut2形成的三聚体在茎部区域不含有N-连接的糖基链，而在头部区域仍然包含N-连接的糖基链(图2D)。
此外，为了便于蛋白的分泌，在编码天然HA蛋白、HA-mut1蛋白、HA-mut2蛋白和HA-mut3蛋白的核苷酸序列的5’端引入编码信号肽(SEQ ID NO：9)的核苷酸序列。所表达的信号肽将在蛋白分泌过程中被切除。因此，最终获得的天然HA蛋白、HA-mut1蛋白、HA-mut2蛋白和HA-mut3蛋白均不包含信号肽，它们的氨基酸序列如SEQ ID NO： 2-5所示。
图1示意性地说明实施例1中所使用的天然HA蛋白、HA-mut1蛋白、HA-mut2蛋白和HA-mut3蛋白的序列突变和N-连接的糖基化(注：信号肽将在蛋白分泌过程中被切除)。具体而言，天然HA蛋白在对应于SEQ ID NO：1的第22、38、63、126、133、144、165、246、285和483位的位置上具有天冬酰胺，并因此可在这些位点上携带N-连接的糖基链。HA-mut1蛋白在对应于SEQ ID NO：1的第22、38、63、126、133、144、165、246、285和483位的位置上具有丙氨酸，并因此不再携带任何N-连接的糖基链。HA-mut2蛋白在对应于SEQ ID NO：1的第22、38、285和483位的位置上具有天冬酰胺，并因此可在这些位点上携带N-连接的糖基链；但在对应于SEQ ID NO：1的第63、126、133、144、165和246位的位置上具有丙氨酸，并因此在这些位点上不再携带任何N-连接的糖基链。HA-mut3蛋白在第对应于SEQ ID NO：1的63、126、133、144、165和246位的位置上具有天冬酰胺，并因此可在这些位点上携带N-连接的糖基链；但在对应于SEQ ID NO：1的第22、38、285和483位的位置上具有丙氨酸，并因此在这些位点上不再携带任何N-连接的糖基链。另外，为了便于蛋白的分泌、纯化和三聚体形成，在天然HA蛋白、HA-mut1蛋白、HA-mut2蛋白和HA-mut3蛋白的N端分别引入了信号肽(其氨基酸序列如SEQ ID NO：9所示，并且将在蛋白分泌过程中被切除)，在C端分别引入了包含凝血酶裂解位点、折叠基序和6＊His标签的肽段(其包含SEQ ID NO：10和11的氨基酸序列)。
图2示意性地说明分别由实施例1中所使用的天然HA蛋白(图2A)、HA-mut1蛋白(图2B)、HA-mut2蛋白(图2C)和HA-mut3蛋白(图2D)形成的三聚体的结构示意图；其中，图2A显示由天然HA蛋白形成的三聚体在头部和茎部区域均含有N-连接的糖基链；图2B显示由HA-mut1蛋白形成的三聚体在头部和茎部区域均不含有N-连接的糖基链；图2C显示由HA-mut2蛋白形成的三聚体在头部区域不含有N-连接的糖基链，而在茎部区域仍然包含N-连接的糖基链；图2D显示由HA-mut3蛋白形成的三聚体在茎部区域不含有N-连接的糖基链，而在头部区域仍然包含N-连接的糖基链。
(b)转移质粒的制备
由上海生工生物工程技术服务有限公司合成分别编码天然HA蛋白、HA-mut1蛋白、HA-mut2蛋白和HA-mut3蛋白(其各自在N端引入了信号肽(SEQ ID NO：9)，在C端引入了包含凝血酶裂解位点、折叠基序和6＊His标签的肽段(SEQ ID NO：10和11))的DNA序列，并将这些DNA序列分别克隆入杆状病毒的转移载体pAcGP67-B(BD公司，Catalog Number：554757)中。随后，将携带有目的DNA序列的转移载体分别转化至大肠杆菌感受态细胞DH5a中，进行扩增。用质粒小量制备试剂盒(TIANprep Mini Plasmid Kit；TianGen公司，Catalog Number：DP103-03)，从经转化的大肠杆菌中提取包含目的DNA序列的转移质粒，备用。
(c)共转染
转染前1小时，把1×10 6个昆虫细胞(Sf9细胞，Invitrogen)铺于6孔培养板上，并用添加了血清的培养基进行培养。把1μg步骤(b)制备的转移质粒、0.1μg杆状病毒线性DNA(BD公司)、1μl脂质体(Sigma公司)和100μl无血清细胞培养基混合，于室温下静置30分钟，从而获得转染混合物。去除各个孔中的含血清培养基，并添加转染 混合物。27℃孵育6小时后，移去各个孔中的转染混合物，并向每个孔中加入2ml含有CCM3的培养基，继续培养细胞。由此，携带有目的DNA序列的转移质粒和杆状病毒线性DNA被转染入昆虫细胞中，产生重组杆状病毒。
(d)目的蛋白的产生和纯化
将获得的重组杆状病毒进行传代，获得第二代的重组杆状病毒。把15ml第二代的重组杆状病毒加入至1200ml的Sf9昆虫细胞中，并在27℃培养48小时。收集细胞和培养上清，并以11500rpm离心30分钟。离心后，收集上清，其包含重组产生的目的蛋白。
将包含目的蛋白的上清用Millipore公司的超滤浓缩离心管浓缩至35ml，调节其pH至7.4，然后以10000rpm离心10分钟。收集上清，并利用Ni-NTA镍离子层析柱(NI-sepharose 6 fast flow，GE公司，Catalog Number：17-5318-04)来富集和纯化上清中的目的蛋白，所使用的洗脱液为包含250mM咪唑的PBS。将包含目的蛋白洗脱液浓缩至1ml，并透析至PBS缓冲液中，于4℃保存备用。由此，获得了经纯化的天然HA蛋白、HA-mut1蛋白、HA-mut2蛋白和HA-mut3蛋白(N端信号肽在分泌过程中被切除，因此，所获得的蛋白保留有折叠基序和6＊His标签，但不包含N端信号肽)。
(e)HAmg和HAug蛋白的制备
另外，还参照Juine-Ruey Chen等人(Proc Natl Acad Sci，USA.2014Feb 18；111(7)：2476-81)中描述的方法，通过用内切糖苷酶H和内切糖苷酶F对天然HA蛋白(WI2005-WT-HA)进行酶处理，制备了在N-连接的糖基化位点上携带单个糖基的HA蛋白(下文简称为HAmg)和在N-连接的糖基化位点上基本上不携带糖基的HA蛋白(下文简称为HAug)。
应当注意的是，由于酶促作用的限制以及部分糖基化位点的不可接近性，HAug不可避免地在N-连接的糖基化位点上仍然携带少量的糖基，这一点也可从Juine-Ruey Chen等人(同上)的Table S1中提供的数据得到确认。相比之下，由于所有N-连接的糖基化位点上的天冬酰胺均已被置换为丙氨酸，HA-mut1蛋白不再携带任何N-连接的糖基。
实施例2：聚丙烯酰胺凝胶电泳(SDS-PAGE)分析
使用聚丙烯酰胺凝胶电泳法(SDS-PAGE)来分析实施例1中制备的六种蛋白(天然HA蛋白、HA-mut1、HA-mut2、HA-mut3、HAmg和HAug)。所使用的上层胶为5％的浓缩胶(如下配制：向3.4ml水中加入830μl 30％的丙烯酰胺，630μl 1M Tris(pH6.8)，50μl 10％的SDS，50μl 10％的过硫酸铵和5μl TEMED)。所使用的下层胶为12％的分离胶(如下配制：向3.3ml水中加入4ml 30％丙烯酰胺，2.5ml 1M Tris(pH8.8)，100μl 10％SDS，100μl 10％的过硫酸铵和10μl TEMED)。所使用的电泳条件为，在150V电压下进行电泳2小时。电泳后，对聚丙烯酰胺凝胶进行考马斯亮蓝(Sigma)染色。实验结果如图3所示。
图3显示了实施例1制备的六种蛋白的SDS-PAGE分析结果；其中，图3A显示了天然HA蛋白、HA-mut3、HA-mut2和HA-mut1蛋白的SDS-PAGE分析结果；图3B显示了天然HA蛋白，HAmg蛋白和HAug蛋白的SDS-PAGE分析结果。图3的结果显示，天然HA蛋白的分子量在70kD以上，而HA-mut3、HA-mut2、HA-mut1、 HAmg和HAug蛋白的分子量均有明显降低，都在70kD以下，并且HA-mut1蛋白的分子量最小。
实施例3：抗血清的中和活性的评估
(a)免疫实验
6周龄、SPF级、雌性Balb/C小鼠由厦门大学实验动物中心提供，体重约为20g。将实施例1中制备的六种蛋白(天然HA蛋白、HA-mut1、HA-mut2、HA-mut3、HAmg和HAug)和PBS(用作阴性对照)分别与铝佐剂按1：1的体积比混合，用于免疫小鼠。免疫方案如下：每组使用6只小鼠，免疫方式为肌肉注射，免疫剂量为5μg蛋白/只小鼠，注射体积为100μl/只小鼠，免疫进行两次，两次免疫之间间隔14天。在第二次免疫14天后，采集小鼠血清。将采集的血清样品在56℃下灭活30分钟，然后放置于-20℃保存备用。
(b)血清样品的中和效价的评估
中和滴度(Neutralization titer)是评价血清样品是否具有预防和治疗疾病潜能的重要指标。在本实验中，利用空斑减少中和实验(Plagues reduction neutralization test，PRNT)来分析采集的血清样品的中和抗体滴度。所使用的流感病毒为分离自不同时间、不同地区、代表不同亚型(H3N2、H7N9和H1N1)的流感病毒代表株，具体病毒株如下：A/Wisconsin/67/2005(H3N2亚型)、A/Victoria/361/2011(H3N2亚型)、A/Beijing/32/1992(H3N2亚型)、A/Aichi/2/1968(H3N2亚型)、A/Shanghai/02/2013(H7N9亚型)和A/California/04/2009(H1N1亚型)。
将6×10 5个MDCK细胞接种于六孔细胞培养板中。将所使用的流感病毒用含有0.5μg/ml TPCK胰酶的MEM培养基稀释至50PFU/50μl。然后，将连续梯度稀释的血清样品与流感病毒混合并在37℃下孵育1小时，随后加入至接种有MDCK细胞的六孔细胞培养板中，并在37℃下继续孵育1小时。孵育后，吸去细胞培养液，并且将细胞用PBS洗涤两遍。然后，用含有0.5％琼脂糖的MEM培养基覆盖细胞表面，并将细胞置于恒温培养箱中在5％CO 2，37℃下培养两天。之后，用2％结晶紫对细胞进行染色，并通过计数空斑数量来确定流感病毒的滴度，进而计算各个血清样品的中和活性。结果如图4-5所示。
图4显示了用天然HA蛋白、HA-mut1、HA-mut2、HA-mut3和PBS(用作阴性对照)作为免疫原分别免疫小鼠所获得的小鼠血清对流感病毒A/Wisconsin/67/2005(H3N2亚型)(图4A)、A/Victoria/361/2011(H3N2亚型)(图4B)、A/Beijing/32/1992(H3N2亚型)(图4C)、A/Aichi/2/1968(H3N2亚型)(图4D)、A/Shanghai/02/2013(H7N9亚型)(图4E)和A/California/04/2009(H1N1亚型)(图4F)的中和活性。
如图4A所示，对于本实验所使用的HA蛋白所源自的流感病毒株A/Wisconsin/67/2005而言，用天然HA蛋白、HA-mut1、HA-mut2或HA-mut3免疫小鼠所获得的小鼠血清均具有强中和活性，其中，用天然HA蛋白和HA-mut1免疫小鼠所获得的血清的中和滴度最高，用HA-mut3免疫小鼠所获得的血清的中和滴度最低。
如图4B所示，对于与本实验所使用的HA蛋白进化关系较近的H3N2亚型病毒株A/Victoria/361/2011而言，用HA-mut1免疫小鼠所获得的血清的中和滴度最高(甚 至高于用天然HA蛋白免疫小鼠所获得的血清)，用HA-mut3免疫小鼠所获得的血清的中和滴度最低。
如图4C所示，对于与本实验所使用的HA蛋白进化关系较远的H3N2亚型病毒株A/Beijing/32/1992而言，用HA-mut3免疫小鼠所获得的血清的中和滴度最高，用HA-mut1免疫小鼠所获得的血清的中和滴度次之(两者均高于用天然HA蛋白免疫小鼠所获得的血清)，用HA-mut2免疫小鼠所获得的血清的中和滴度最低。
如图4D所示，对于与本实验所使用的HA蛋白进化关系最远的H3N2亚型病毒株A/Aichi/2/1968而言，用HA-mut1免疫小鼠所获得的血清的中和滴度最高，用HA-mut3免疫小鼠所获得的血清的中和滴度次之，而用天然HA蛋白或HA-mut2免疫小鼠所获得的血清则基本上不具有中和活性(与阴性对照无显著区别)。
如图4E和4F所示，对于与本实验所使用的HA蛋白分属不同亚型的病毒株A/Shanghai/02/2013(H7N9亚型)和A/California/04/2009(H1N1亚型)而言，仅用HA-mut1免疫小鼠所获得的血清具有中和活性，而用其他蛋白免疫小鼠所获得的血清则基本上不具有中和活性(与阴性对照无显著区别)。
图4的结果表明，用HA-mut1免疫小鼠所获得的血清具有最广谱的中和活性，其不仅能有效中和H3N2亚型中的多个病毒株(无论进化关系远近)，而且还能中和跨亚型的病毒株(例如H7N9和H1N1亚型的病毒株)。相比之下，用天然HA蛋白、HA-mut2和HA-mut3免疫小鼠所获得的血清只对H3N2亚型的部分病毒株具有中和活性，而对跨亚型的病毒株不具有中和活性。由此可见，HA-mut1特别适合用作广谱疫苗，用于在体内诱发具有广谱中和活性的保护性抗体。
图5显示了用天然HA蛋白、HA-mut1、HAmg、HAug和PBS(用作阴性对照)作为免疫原分别免疫小鼠所获得的小鼠血清对流感病毒A/Wisconsin/67/2005(H3N2亚型)(图5A)、A/Victoria/361/2011(H3N2亚型)(图5B)、A/Beijing/32/1992(H3N2亚型)(图5C)、A/Aichi/2/1968(H3N2亚型)(图5D)、A/Shanghai/02/2013(H7N9亚型)(图5E)和A/California/04/2009(H1N1亚型)(图5F)的中和活性。
如图5A所示，对于本实验所使用的HA蛋白所源自的流感病毒株A/Wisconsin/67/2005而言，用天然HA蛋白、HA-mut1、HAmg或HAug免疫小鼠所获得的小鼠血清具有效价相当的强中和活性。
如图5B所示，对于与本实验所使用的HA蛋白进化关系较近的H3N2亚型病毒株A/Victoria/361/2011而言，用HA-mut1免疫小鼠所获得的血清的中和滴度最高，用天然HA蛋白免疫小鼠所获得的血清的中和滴度最低。
如图5C所示，对于与本实验所使用的HA蛋白进化关系较远的H3N2亚型病毒株A/Beijing/32/1992而言，用HA-mut1免疫小鼠所获得的血清的中和滴度最高，用其他蛋白免疫小鼠所获得的血清的中和滴度较低且彼此相当。
如图5D所示，对于与本实验所使用的HA蛋白进化关系最远的H3N2亚型病毒株A/Aichi/2/1968而言，用HA-mut1免疫小鼠所获得的血清的中和滴度最高，用HAmg或HAug免疫小鼠所获得的血清的中和滴度次之(两者相当)，而用天然HA蛋白小鼠所获得的血清则基本上不具有中和活性(与阴性对照无显著区别)。
如图5E所示，对于与本实验所使用的HA蛋白分属不同亚型的病毒株 A/Shanghai/02/2013(H7N9亚型)而言，用HA-mut1免疫小鼠所获得的血清的中和滴度最高，用HAmg或HAug免疫小鼠所获得的血清的中和滴度次之(两者相当)，而用天然HA蛋白小鼠所获得的血清则基本上不具有中和活性(与阴性对照无显著区别)。
如图5F所示，对于与本实验所使用的HA蛋白分属不同亚型的病毒株A/California/04/2009(H1N1亚型)而言，仅用HA-mut1免疫小鼠所获得的血清具有中和活性，而用其他蛋白免疫小鼠所获得的血清则基本上不具有中和活性(与阴性对照无显著区别)。
图5的结果表明，用天然HA蛋白免疫小鼠所获得的血清只对H3N2亚型的流感病毒有中和活性；用HAmg和HAug免疫小鼠所获得的血清不仅能中和H3N2亚型的流感病毒，而且显示出较弱的跨HA亚型的中和活性(能够中和H7N9亚型的病毒株，但不能中和H1N1亚型的病毒株)；用HA-mut1免疫小鼠所获得的血清具有最广谱的中和活性且中和效价最高，其不仅能有效中和H3N2亚型中的多个病毒株(无论进化关系远近)，而且还具有较强的跨HA亚型的中和活性(例如能够中和H7N9和H1N1亚型的病毒株)。由此可见，HA-mut1特别适合用作广谱疫苗，用于在体内诱发具有广谱中和活性的保护性抗体。
实施例4：体内保护活性的评估
实施例3中已通过PRNT实验证实：实施例1中制备的六种蛋白所诱发的抗血清对H3N2亚型、H7N9亚型和H1N1亚型的病毒株的中和滴度存在差异，其中，HA-mut1所诱发的抗血清具有最广谱的中和活性。为进一步验证这六种蛋白在动物体内诱导抗流感病毒的免疫保护的效果，本发明人基于用A/Beijing/32/1992(H3N2亚型)和A/Aichi/02/1968(H3N2亚型)、A/Shanghai/02/2013(H7N9亚型)和A/California/04/2009(H1N1亚型)流感病毒感染的小鼠动物模型，在生物安全实验室内对这六种蛋白的体内抗病毒效果进行了评估。具体方案如下。
材料与方法
动物：Balb/C小鼠，SPF级，6-8周龄，雌性，体重约20g。
疫苗：天然HA蛋白、HA-mut1蛋白、HA-mut2蛋白、HA-mut3蛋白、HAmg蛋白、HAug蛋白和PBS(用作阴性对照)。
免疫方案：将天然HA蛋白、HA-mut1蛋白、HA-mut2蛋白、HA-mut3蛋白、HAmg蛋白、HAug蛋白和PBS阴性对照分别与铝佐剂按1：1的体积比混合，用于免疫小鼠。每组使用6只小鼠，免疫方式为肌肉注射，免疫剂量为5tg蛋白/只小鼠，注射体积为100μl/只小鼠。免疫进行两次，两次免疫之间间隔14天。在第二次免疫14天后，对小鼠进行攻毒。所使用的流感病毒株如下：
H3N2亚型流感病毒的小鼠适应株：A/Beijing/32/1992；
H3N2亚型流感病毒的小鼠适应株：A/Aichi/02/1968；
H7N9亚型流感病毒的小鼠适应株：A/Shanghai/02/2013；
H1N1亚型流感病毒的小鼠适应株：A/California/04/2009。
麻醉剂：Isoflorane(异弗烷)。
动物分组：提前一天将小鼠送入生物安全实验室，按6只一笼分组，并记录下每只小鼠的体重。
病毒感染：各病毒的攻毒剂量均为25倍半数致死剂量(LD 50)，病毒接种体积为50μl/只小鼠。接种前，先用异弗烷麻醉小鼠，然后经鼻腔给小鼠接种病毒。
观察记录：在病毒感染后1-14天，每天记录小鼠的体重变化情况和存活情况。实验结果如图6-9所示。
图6显示了，用天然HA蛋白、HA-mut1蛋白、HA-mut2蛋白、HA-mut3蛋白或PBS(阴性对照)免疫的小鼠在感染流行年份较早的H3N2亚型流感病毒A/Beijing/32/1992(H3N2)(图6A-6B)和A/Aichi/2/1968(H3N2)(图6C-6D)后的体重变化情况和存活情况，其中，图6A和图6C显示了各实验小鼠的体重变化，图6B和图6D显示了各实验小鼠的生存率。图6A-6B的结果显示，用HA-mut1或HA-mut3免疫的小鼠在感染致死剂量的病毒A/Beijing/32/1992后，其体重在第七天后开始恢复，并且在实验结束之时小鼠存活率为100％；然而，用天然HA蛋白、HA-mut2或PBS免疫的小鼠的体重均不断下降，并在实验结束之前全部死亡。这一结果表明，HA-mut1和HA-mut3具有完全保护作用，可用作抗A/Beijing/32/1992的疫苗。图6C-6D的结果显示，用HA-mut1免疫的小鼠在感染致死剂量的病毒A/Aichi/2/1968后，其体重在第四天后开始恢复，并且在实验结束之时小鼠存活率为100％；HA-mut3对感染致死剂量的病毒A/Aichi/2/1968的小鼠具有部分保护作用，在实验结束之时小鼠存活率为33.3％；然而，用天然HA蛋白、HA-mut2或PBS免疫的小鼠的体重均不断下降，并在实验结束之前全部死亡。这一结果表明，HA-mut1具有完全保护作用，可用作抗A/Aichi/2/1968的疫苗。
图7显示了，用天然HA蛋白、HA-mut1蛋白、HAmg蛋白、HAug蛋白或PBS(阴性对照)免疫的小鼠在感染流行年份较早的H3N2亚型流感病毒A/Beijing/32/1992(H3N2)(图7A-7B)和A/Aichi/2/1968(H3N2)(图7C-7D)后的体重变化情况和存活情况，其中，图7A和图7C显示了各实验小鼠的体重变化，图7B和图7D显示了各实验小鼠的生存率。图7A-7B的结果显示，用HA-mut1蛋白、HAmg蛋白或HAug蛋白免疫的小鼠在感染致死剂量的病毒A/Beijing/32/1992后，其体重在第七天后开始恢复(用HA-mut1免疫的小鼠的体重恢复效果最佳)，并且在实验结束之时小鼠存活率为100％；然而，用天然HA蛋白或PBS免疫的小鼠的体重均不断下降，并在实验结束之前全部死亡。这一结果表明，HA-mut1蛋白、HAmg蛋白和HAug蛋白具有完全保护作用，可用作抗A/Beijing/32/1992的疫苗。图7C-7D的结果显示，用HA-mut1、HAmg或HAug免疫的小鼠在感染致死剂量的病毒A/Aichi/2/1968后，其体重在第四或五天后开始恢复(用HA-mut1免疫的小鼠的体重恢复效果最佳)，并且在实验结束之时小鼠存活率为100％；然而，用天然HA蛋白或PBS免疫的小鼠的体重均不断下降，并在实验结束之前全部死亡。这一结果表明，HA-mut1蛋白、HAmg蛋白和HAug蛋白具有完全保护作用，可用作抗A/Aichi/2/1968的疫苗。
图8显示了，用天然HA蛋白、HA-mut1蛋白、HA-mut2蛋白、HA-mut3蛋白或PBS(阴性对照)免疫的小鼠在感染非H3N2亚型流感病毒A/Shanghai/02/2013(H7N9)(图8A-8B)和A/California/04/2009(H1N1)(图8C-8D)后的体重变化情况和存活 情况，其中，图8A和图8C显示了各实验小鼠的体重变化，图8B和图8D显示了各实验小鼠的生存率。图8A-8B的结果显示，用HA-mut1免疫的小鼠在感染致死剂量的病毒A/Shanghai/02/2013(H7N9)后，其体重在第6天后开始恢复，并且在实验结束之时小鼠存活率为100％；然而，用天然HA蛋白、HA-mut2、HA-mut3或PBS免疫的小鼠的体重均不断下降，并在实验结束之前全部死亡。这一结果表明，HA-mut1具有完全保护作用，可用作抗A/Shanghai/02/2013的疫苗。图8C-8D的结果显示，用HA-mut1免疫的小鼠在感染致死剂量的病毒A/California/04/2009(H1N1)后，其体重在第八天后维持稳定，不再下降，并且在实验结束之时小鼠存活率为66.7％；然而，用天然HA蛋白、HA-mut2、HA-mut3或PBS免疫的小鼠的体重均不断下降，并在实验结束之前全部死亡。这一结果表明，HA-mut1针对流感病毒A/California/04/2009(H1N1)具有较强的体内保护效果。
图9显示了，用天然HA蛋白、HA-mut1蛋白、HAmg蛋白、HAug蛋白或PBS(阴性对照)免疫的小鼠在感染非H3N2亚型流感病毒A/Shanghai/02/2013(H7N9)(图9A-9B)和A/California/04/2009(H1N1)(图9C-9D)后的体重变化情况和存活情况，其中，图9A和图9C显示了各实验小鼠的体重变化，图9B和图9D显示了各实验小鼠的生存率。图9A-9B的结果显示，用HA-mut1蛋白或HAug蛋白免疫的小鼠在感染致死剂量的病毒A/Shanghai/02/2013(H7N9)后，其体重在第6或7天后开始恢复(用HA-mut1免疫的小鼠的体重恢复效果最佳)，并且在实验结束之时小鼠存活率为100％；然而，用天然HA蛋白、HAmg蛋白或PBS免疫的小鼠的体重均不断下降，并在实验结束之前全部死亡。这一结果表明，HA-mut1蛋白和HAug蛋白具有完全保护作用，可用作抗A/Shanghai/02/2013(H7N9)的疫苗。图9C-9D的结果显示，用HA-mut1免疫的小鼠在感染致死剂量的病毒A/California/04/2009(H1N1)后，其体重在第八天后维持稳定，不再下降，并且在实验结束之时小鼠存活率为66.7％；然而，用天然HA蛋白、HAmg、HAug或PBS免疫的小鼠的体重均不断下降，并在实验结束之前全部死亡。这一结果表明，HA-mut1针对流感病毒A/California/04/2009(H1N1)具有较强的体内保护效果。
上述实验结果表明，HA-mut1蛋白作为疫苗能够在动物体内有效预防H3N2亚型(无论进化关系远近)、H7N9亚型和H1N1亚型的流感病毒的感染以及由其导致的疾病，从而可用作有效的、能够抵抗多种亚型的流感病毒的广谱疫苗。
实施例5：H3N2流感病毒HA蛋白及其突变体的制备和分析
在本实施例中，通过将天然HA蛋白中的特征序列N-X-(S或T)中的天冬酰胺(N)突变为谷氨酰胺(Q)来去除HA蛋白的N-连接的糖基化位点。
本实施例中所使用的天然HA蛋白(HK2014-WT-HA)来源于H3N2亚型流感病毒株A/HONG_KONG/4801/2014(H3N2)的HA蛋白。该毒株的HA蛋白包含如SEQ ID NO：6所示的氨基酸序列，其中，SEQ ID NO：6的第1-25位氨基酸为信号肽，第518-565位氨基酸为跨膜区，并且具有11个潜在的N-连接的糖基化位点，即位于第37，53，60，78，137，141，148，180，261，300和498位的天冬酰胺(N)。
基于上述结构信息，本实施例中设计了天然HA蛋白HK2014-WT-HA及其突变体 HK2014-DG-HA：
(1)天然HA蛋白(HK2014-WT-HA)，其包含如SEQ ID NO：7所示的氨基酸序列，并且其与SEQ ID NO：6的差异在于，SEQ ID NO：6的第1-25位和第518-565位氨基酸被缺失，并且，在SEQ ID NO：6的C端引入包含凝血酶裂解位点、折叠基序和6＊His标签的肽段(其包含SEQ ID NO：10和11的序列，用于促进蛋白的纯化和三聚体形成)。相应地，由天然HA蛋白(HK2014-WT-HA)形成的三聚体在头部和茎部区域均含有N-连接的糖基链。
(2)突变体HK2014-DG-HA，其包含如SEQ ID NO：8所示的氨基酸序列，并且其与天然HA蛋白(HK2014-WT-HA；SEQ ID NO：7)的差异在于，如上所述的所有11个N-连接的糖基化位点上的天冬酰胺(N)均被突变为谷氨酰胺(Q)。相应地，由突变体HK2014-DG-HA形成的三聚体在头部和茎部区域均不含有N-连接的糖基链。
此外，为了便于蛋白的分泌，在编码天然HA蛋白HK2014-WT-HA和突变体蛋白HK2014-DG-HA的核苷酸序列的5’端引入编码信号肽(SEQ ID NO：9)的核苷酸序列。所表达的信号肽将在蛋白分泌过程中被切除。因此，最终获得的天然HA蛋白HK2014-WT-HA及其突变体HK2014-DG-HA均不包含信号肽，它们的氨基酸序列如SEQ ID NO：7-8所示。
将编码天然蛋白HK2014-WT-HA和突变体蛋白HK2014-DG-HA(其各自在N端引入了信号肽(SEQ ID NO：9)，在C端引入了包含凝血酶裂解位点、折叠基序和6＊His标签的肽段(SEQ ID NO：10和11))的DNA序列分别克隆入杆状病毒的转移载体pAcGP67-B(BD公司，Catalog Number：554757)中。随后，将携带有目的DNA序列的转移载体分别转化至大肠杆菌感受态细胞DHSa中，进行扩增。用质粒小量制备试剂盒(TIANprep Mini Plasmid Kit；TianGen公司，Catalog Number：DP103-03)，从经转化的大肠杆菌中提取包含目的DNA序列的转移质粒，备用。
随后，如实施例1所述，利用如上制备的转移质粒构建包含目的DNA序列的重组杆状病毒，并将其在Sf9昆虫细胞中进行培养。培养后，收集细胞和培养上清，并以11500rpm离心30分钟。离心后，收集上清，其包含重组产生的目的蛋白。然后，如实施例1所述，使用包含咪唑(50mM或250mM)的PBS作为洗脱液，利用Ni-NTA镍离子层析柱(NI-sepharose 6 fast flow，GE公司，Catalog Number：17-5318-04)来富集和纯化上清中的目的蛋白，即，HK2014-WT-HA和HK2014-DG-HA(N端信号肽在分泌过程中被切除，因此，所获得的蛋白保留有折叠基序和6＊His标签，但不包含N端信号肽)。
另外，还参照Juine-Ruey Chen等人(Proc Natl Acad Sci，USA.2014Feb 18；111(7)：2476-81)中描述的方法，通过用内切糖苷酶F对如上获得的天然HA蛋白(HK2014-WT-HA)进行酶处理，制备获得在所有N-连接的糖基化位点上基本上不携带糖基的去糖基化HA蛋白(下文简称为HK2014-HAug)。
使用SDS聚丙烯酰胺凝胶电泳法(SDS-PAGE)和Western blot(所使用的抗体为：HRP-conjugated 6＊His，His-Tag Antibody，Proteintech，Catalog Number：HRP-66005)来分析如上制备的3种蛋白(HK2014-WT-HA，HK2014-DG-HA和HK2014-HAug)。实验结果如图10-12所示。
图10显示了HK2014-WT-HA蛋白的SDS-PAGE分析(左图)和Western blot分析(右图)的结果；其中，泳道M：分子量标记；泳道1：未经Ni-NTA镍离子层析柱纯化的样品；泳道2：流经Ni-NTA镍离子层析柱的级分；泳道3：用50mM咪唑洗脱的级分；泳道4：用50mM咪唑洗脱的级分；泳道5：用250mM咪唑洗脱的级分；箭头指示目的蛋白HK2014-WT-HA的位置。
图11显示了HK2014-DG-HA蛋白的SDS-PAGE分析(左图)和Western blot分析(右图)的结果；其中，泳道M：分子量标记；泳道1：未经Ni-NTA镍离子层析柱纯化的样品；泳道2：流经Ni-NTA镍离子层析柱的级分；泳道3：用50mM咪唑洗脱的级分；泳道4：用250mM咪唑洗脱的级分；箭头指示目的蛋白HK2014-DG-HA的位置。
图10-11的结果显示，蛋白HK2014-WT-HA和HK2014-DG-HA主要包含在用250mM咪唑洗脱的级分中；并且，HK2014-WT-HA的分子量在70KD以上，HK2014-DG-HA的分子量有所降低。这些结果说明，HK2014-DG-HA中的糖基化修饰被有效去除。
图12显示了天然HA蛋白HK2014-WT-HA和去糖基化蛋白HK2014-HAug的SDS-PAGE分析结果；其中，泳道M：分子量标记；泳道1：经纯化的HK2014-WT-HA；泳道2：HK2014-HAug(用内切糖苷酶F消化HK2014-WT-HA 3小时而获得)。
图12的结果显示，HK2014-WT-HA的分子量在70KD以上，HK2014-HAug的分子量有所降低。这些结果说明，HK2014-HAug中的糖基化修饰被有效去除。
实施例6：H3N2流感病毒HA蛋白及其突变体的免疫原性评价
将实施例5制备的蛋白HK2014-WT-HA、HK2014-DG-HA和HK2014-HAug分别与弗氏佐剂混合制备成免疫原，然后用于免疫6-8周龄的Balb/C雌性小鼠(体重约为20g)。免疫程序如下：皮下免疫3次，每次免疫间隔14天。在第三次免疫14天后，采集小鼠血清，并将采集的血清样品在56℃下灭活30分钟，然后于-20℃保存备用。
使用ELISA测定来评价如上采集的小鼠血清样品是否对三种流感病毒A/Wisconsin/67/2005(H3N2)、A/Xiamen/N794/2013(H3N2)和A/Shanghai/02/2013(H7N9)具有特异性结合活性。简言之，用100ul的不同型别的流感病毒(128HA)包被Elisa板，然后将梯度稀释的小鼠血清加入到经病毒包被的板中，并在37℃孵育1h。随后，加入1∶5000稀释的GAM-HRP(由厦门大学国家工程中心提供)，并在37℃孵育30min。孵育后，清洗板，加入显色液A&B(由北京万泰公司提供)显色15min，然后用终止液终止显色反应。最后，使用酶标仪读取各个孔的吸光值，计算小鼠血清对病毒的特异结合活性。ELISA测定结果如图13-14所示。
图13显示了用HK2014-WT-HA、HK2014-DG-HA和PBS(用作阴性对照)作为免疫原分别免疫小鼠所获得的小鼠血清对流感病毒A/Wisconsin/67/2005(H3N2)、A/Xiamen/N794/2013(H3N2)和A/Shanghai/02/2013(H7N9)的结合活性的ELISA分析结果。
图13的结果显示，用HK2014-WT-HA和HK2014-DG-HA免疫小鼠所获得的小 鼠血清对三种流感病毒(A/Wisconsin/67/2005(H3N2)、A/Xiamen/N794/2013(H3N2)、A/Shanghai/02/2013(H7N9))均表现出水平相当的反应滴度。结果表明，HK2014-WT-HA和HK2014-DG-HA均具有良好的免疫原性，能够在小鼠体内触发正常的免疫应答，诱导机体产生特异性抗体，并且这些特异性抗体能够识别并结合多种流感病毒。
图14显示了用HK2014-WT-HA、HK2014-HAug和PBS(用作阴性对照)作为免疫原分别免疫小鼠所获得的小鼠血清对流感病毒A/Wisconsin/67/2005(H3N2)、A/Xiamen/N794/2013(H3N2)和A/Shanghai/02/2013(H7N9)的结合活性的ELISA分析结果。
图14的结果显示，用HK2014-WT-HA和HK2014-HAug免疫小鼠所获得的小鼠血清对三种流感病毒(A/Wisconsin/67/2005(H3N2)、A/Xiamen/N794/2013(H3N2)、A/Shanghai/02/2013(H7N9))均表现出水平相当的反应滴度。结果表明，HK2014-WT-HA和HK2014-HAug均具有良好的免疫原性，能够在小鼠体内触发正常的免疫应答，诱导机体产生特异性抗体，并且这些特异性抗体能够识别并结合多种流感病毒。
实施例7：H3N2流感病毒HA蛋白及其突变体的免疫保护性评价
为进一步验证实施例5制备的蛋白在动物体内诱导抗流感病毒的免疫保护效果，进行如下实验。
将实施例5制备的蛋白HK2014-WT-HA、HK2014-DG-HA和HK2014-HAug分别与弗氏佐剂混合制备成免疫原，然后用于免疫6-8周龄的Balb/C雌性小鼠(体重约为20g)。免疫程序如下：皮下免疫3次，每次免疫间隔14天。在第三次免疫14天后，对各组小鼠进行流感病毒攻毒实验，所使用的流感病毒毒株为：与免疫原流行年份相隔久远的H3N2病毒株A/Aichi/2/1968(H3N2)和近年流行的H7N9病毒株A/Shanghai/059/2013(H7N9)，二者均为致死性毒株。攻毒后，观察和记录各组小鼠的体重和存活率，评估所制备的蛋白保护小鼠抵御致死性病毒感染的功效。实验结果如图15-17显示。
图15显示了，用HK2014-WT-HA、HK2014-DG-HA或PBS(用作阴性对照)免疫的各组小鼠(3只/组)在感染A/Aichi/2/1968(H3N2)后的体重变化情况(左图)和存活情况(右图)。图15的实验结果显示，用HK2014-WT-HA免疫的小鼠在感染致死剂量的病毒A/Aichi/2/1968(H3N2)后，一只小鼠在第五天死亡，剩余小鼠在第6天开始恢复体重，且在实验结束时小鼠存活率为66％；用HK2014-DG-HA免疫的小鼠在感染致死剂量的病毒A/Aichi/2/1968(H3N2)后，所有小鼠在第5天开始恢复体重，且在实验结束时小鼠存活率为100％；阴性对照组小鼠在感染病毒后第8天全部死亡。这一结果表明，与HK2014-WT-HA相比，HK2014-DG-HA对病毒A/Aichi/2/1968(H3N2)具有更好的保护效果。
图16显示了，用HK2014-WT-HA、HK2014-DG-HA或PBS(用作阴性对照)免疫的各组小鼠(3只/组)在感染A/Shanghai/059/2013(H7N9)后的体重变化情况(左图)和存活情况(右图)。图16的实验结果显示，用HK2014-WT-HA免疫的小鼠在 感染致死剂量的病毒A/Shanghai/059/2013(H7N9)后，所有小鼠体重不断下降，且在攻毒后第9天小鼠存活率为0％；用HK2014-DG-HA免疫的小鼠在感染致死剂量的病毒A/Shanghai/059/2013(H7N9)后，一只小鼠在第8天开始恢复体重，且在实验结束时小鼠存活率为33％。这一结果表明，HK2014-WT-HA不具有抗流感病毒A/Shanghai/059/2013(H7N9)的保护效果；相比之下，HK2014-DG-HA则对病毒A/Shanghai/059/2013(H7N9)展现出一定的保护效果(跨型别的广谱保护作用)。
图17显示了，用HK2014-WT-HA、HK2014-HAug或PBS(用作阴性对照)免疫的各组小鼠(4只/组)在感染A/Shanghai/059/2013(H7N9)后的体重变化情况。图17的实验结果显示，用HK2014-WT-HA、HK2014-HAug或PBS免疫的小鼠在感染致死剂量的病毒A/Shanghai/059/2013(H7N9)后，所有小鼠体重不断下降，且在攻毒后第9天小鼠存活率全部为0％。这一结果表明，HK2014-WT-HA和HK2014-HAug均不具有抗病毒A/Shanghai/059/2013(H7N9)的保护效果。
从以上结果可以看出，HK2014-DG-HA比HK2014-WT-HA和HK2014-HAug更适合用作流感疫苗，其能够抵抗H3N2亚型(无论进化关系远近)和H7N9亚型的流感病毒的感染，展现出跨型别的广谱保护作用以及更佳的保护效果。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (12)

  1. 一种H3N2亚型流感病毒血凝素蛋白的突变体，其不含有N-连接的糖基化位点；
    优选地，所述突变体与所述H3N2亚型流感病毒的野生型血凝素蛋白的区别至少在于，所述突变体不包含特征序列N-X-(S或T)；其中，N代表天冬酰胺，X代表除脯氨酸以外的任何一种氨基酸，S代表丝氨酸，T代表苏氨酸；并且，任选地，所述突变体不包含所述野生型血凝素蛋白的N端信号肽和/或跨膜区；
    优选地，所述突变体与所述H3N2亚型流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的每一个特征序列N-X-(S或T)各自独立地具有选自下列的一项或多项突变：
    (1)N残基被删除或者置换为一个或多个其他的氨基酸残基(例如一个非N的氨基酸残基)；
    (2)(S或T)残基被删除或者置换为一个或多个其他的氨基酸残基(例如一个非S且非T的氨基酸残基)；
    (3)X残基被删除或者置换为脯氨酸残基；
    (4)在N残基与X残基之间添加一个或多个氨基酸残基(例如一个非N的氨基酸残基)；和
    (5)在X残基与(S或T)残基之间添加一个或多个氨基酸残基(例如一个非S且非T的氨基酸残基)；
    由此，所述突变体不包含任何特征序列N-X-(S或T)，
    其中，N代表天冬酰胺，X代表除脯氨酸以外的任何一种氨基酸，S代表丝氨酸，T代表苏氨酸，由此所述突变体不包含任何特征序列N-X-(S或T)；
    优选地，所述突变体与所述H3N2亚型流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的每一个特征序列N-X-(S或T)各自独立地具有选自下列的突变：
    (1)N残基被删除或者置换为一个其他的氨基酸残基(例如一个非N的氨基酸残基)；
    (2)(S或T)残基被删除或者置换为一个其他的氨基酸残基(例如一个非S且非T的氨基酸残基)；
    (3)X残基被删除或者置换为脯氨酸残基；
    (4)在N残基与X残基之间添加一个或多个氨基酸残基(例如一个非N的氨基酸残基)；和
    (5)在X残基与(S或T)残基之间添加一个或多个氨基酸残基(例如一个非S且非T的氨基酸残基)；和
    (6)(1)至(5)的任何组合；
    优选地，所述突变体与所述H3N2亚型流感病毒的野生型血凝素蛋白的区别至少在于，野生型血凝素蛋白中的每一个特征序列N-X-(S或T)各自独立地具有选自下列的突变：
    (1)N残基被删除或者被保守置换；
    (2)(S或T)残基被删除或者被保守置换；
    (3)X残基被删除或者置换为脯氨酸残基；
    (4)在N残基与X残基之间添加一个非N的氨基酸残基；
    (5)在X残基与(S或T)残基之间添加一个非S且非T的氨基酸残基；和
    (6)(1)至(5)的任何组合；
    优选地，所述野生型血凝素蛋白来源于H3N2亚型流感病毒，例如A/WISCONSIN/67/2005(H3N2)和A/HONG_KONG/4801/2014(H3N2)；
    优选地，所述野生型血凝素蛋白具有选自下列的序列：SEQ ID NO：1和6；
    优选地，所述野生型血凝素蛋白的氨基酸序列如SEQ ID NO：1所示；并且，所述突变体与SEQ ID NO：1的区别至少在于，所述突变体不包含特征序列N-X-(S或T)；其中，N代表天冬酰胺，X代表除脯氨酸以外的任何一种氨基酸，S代表丝氨酸，T代表苏氨酸；并且，任选地，所述突变体不包含信号肽(例如SEQ ID NO：1的第1-10位氨基酸)和/或跨膜区(例如SEQ ID NO：1的第504-550位氨基酸)；
    优选地，所述野生型血凝素蛋白的氨基酸序列如SEQ ID NO：6所示；并且，所述突变体与SEQ ID NO：6的区别至少在于，所述突变体不包含特征序列N-X-(S或T)；其中，N代表天冬酰胺，X代表除脯氨酸以外的任何一种氨基酸，S代表丝氨酸，T代表苏氨酸；并且，任选地，所述突变体不包含信号肽(例如SEQ ID NO：6的第1-25位氨基酸)和/或跨膜区(例如SEQ ID NO：6的第518-565位氨基酸)；
    优选地，所述突变体具有选自下列的氨基酸序列：SEQ ID NO：12-13；或者，所述突变体与选自下列的氨基酸序列具有至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％或100％的同一性：SEQ ID NO：12-13；前提条件是所述突变体不包含任何N-连接的糖基化位点(例如不包含任何特征序列N-X-(S或T))；或者，所述突变体与选自下列的氨基酸序列相比，具有一个或多个氨基酸残基的添加、删除或置换：SEQ ID NO：12-13；前提条件是所述突变体不包含任何N-连接的糖基化位点(例如不包含任何特征序列N-X-(S或T))。
  2. 一种重组蛋白，其包含权利要求1的流感病毒血凝素蛋白的突变体，以及额外的肽段，所述额外的肽段连接至所述突变体；
    优选地，所述额外的肽段直接与所述突变体连接，或者通过接头连接至所述突变体；
    优选地，所述额外的肽段连接至所述突变体的N端或C端；
    优选地，所述重组蛋白包含至少1个，至少2个，至少3个，至少5个或更多个额外的肽段；
    优选地，所述额外的肽段选自信号肽，标签肽，折叠基序，可检测标记，以及其任何组合；
    优选地，所述信号肽连接至所述突变体的N端；进一步优选地，所述信号肽具有如SEQ ID NO：9所示的氨基酸序列；
    优选地，所述折叠基序连接至所述突变体的C端；进一步优选地，所述折叠基序具有如SEQ ID NO：10所示的氨基酸序列。
  3. 一种核酸分子，其包含或者由编码权利要求1的突变体或权利要求2的重组蛋白的核苷酸序列组成。
  4. 一种载体，其包含权利要求3的核酸分子。
  5. 一种宿主细胞或病毒(例如杆状病毒)，其包含权利要求3的核酸分子或权利要求4的载体。
  6. 一种多聚体，其包含多个权利要求1的突变体或权利要求2的重组蛋白，或者由多个权利要求1的突变体或权利要求2的重组蛋白组成；优选地，所述多聚体为三聚体。
  7. 一种组合物，其包含权利要求1的突变体，或权利要求2的重组蛋白，或权利要求3的核酸分子，或权利要求4的载体，或权利要求5的宿主细胞或病毒，或权利要求6的多聚体。
  8. 一种药物组合物(例如疫苗)，其包含权利要求1的突变体或权利要求2的重组蛋白或权利要求6的多聚体，任选地还包含药学可接受的载体和/或赋形剂。
  9. 一种在受试者中预防或治疗流感病毒感染或由流感病毒感染所导致的疾病的方法，其包括将预防或治疗有效量的权利要求1的突变体或权利要求2的重组蛋白或权利要求6的多聚体或者权利要求8的药物组合物施用给所述受试者；
    优选地，所述流感病毒选自H3N2、H7N9和H1N1亚型流感病毒；
    优选地，所述由流感病毒感染所导致的疾病为流感；
    优选地，所述受试者是哺乳动物，例如小鼠和人。
  10. 权利要求1的突变体或权利要求2的重组蛋白或权利要求6的多聚体在制备药物组合物(例如疫苗)中的用途，所述药物组合物(例如疫苗)用于在受试者中预防或治疗流感病毒感染或由流感病毒感染所导致的疾病；
    优选地，所述流感病毒选自H3N2、H7N9和H1N1亚型流感病毒；
    优选地，所述由流感病毒感染所导致的疾病为流感；
    优选地，所述受试者是哺乳动物，例如小鼠和人。
  11. 一种制备权利要求1的突变体或权利要求2的重组蛋白的方法，其包括，在允许所述突变体或重组蛋白表达的条件下，培养权利要求5的宿主细胞或病毒；和，回收所表达的突变体或重组蛋白。
  12. 一种制备疫苗的方法，其包括将权利要求1的突变体或权利要求2的重组蛋白 或权利要求6的多聚体与药学可接受的载体和/或赋形剂混合；任选地，所述方法还包括，混合佐剂例如铝佐剂，和/或另外的活性成分，例如能够预防或治疗流感病毒感染或由流感病毒感染所导致的疾病的另外的活性成分。
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TATE, M.D. ET AL.: "Glycosylation of the Hemagglutinin Modulates the Sensitivity of H3N2 Influenza Viruses to Innate Proteins in Airway Secretions and Virulence in Mice", VIROLOGY, vol. 413, no. 1, 25 April 2011 (2011-04-25), XP028159150, ISSN: 0042-6822, DOI: doi:10.1016/j.virol.2011.01.036 *
TM TUMPEY ET AL., PROC. NATL. ACAD. SCI. USA, vol. 99, 2002, pages 13849
TONG S. ET AL., PLOS PATHOG., vol. 9, no. 10, 2013, pages e1003657
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WHO PANDEMIC (HLNL) 2009-UPDATE 112, 6 August 2010 (2010-08-06)
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US11426459B2 (en) 2022-08-30
AU2018351209B2 (en) 2022-11-24
CN109678937A (zh) 2019-04-26
EP3699186A4 (en) 2021-12-15
US20210023198A1 (en) 2021-01-28
JP2021509254A (ja) 2021-03-25
CA3079486A1 (en) 2019-04-25
CN109678937B (zh) 2022-06-07
EP3699186A1 (en) 2020-08-26
JP7009625B2 (ja) 2022-01-25

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