WO2022242649A1 - 一种新型冠状病毒SARS-CoV-2突变体疫苗与应用 - Google Patents

一种新型冠状病毒SARS-CoV-2突变体疫苗与应用 Download PDF

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WO2022242649A1
WO2022242649A1 PCT/CN2022/093354 CN2022093354W WO2022242649A1 WO 2022242649 A1 WO2022242649 A1 WO 2022242649A1 CN 2022093354 W CN2022093354 W CN 2022093354W WO 2022242649 A1 WO2022242649 A1 WO 2022242649A1
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ifn
amino acid
vaccine
acid sequence
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林晶晶
陈曦
胡振湘
杨嘉明
唐阳刚
朱保国
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珠海市丽珠单抗生物技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the disclosure belongs to the field of biotechnology, and in particular relates to a novel coronavirus SARS-CoV-2 mutant vaccine and its application.
  • the new coronavirus (2019-nCoV, SARS-CoV-2) is a coronavirus of the genus Beta, which was first discovered in 2019 and is the seventh known coronavirus that can infect humans. It leads to symptoms such as fever, dry cough, and fatigue in patients; some patients will develop severe pneumonia, and then develop into acute respiratory distress syndrome, septic shock, coagulation dysfunction, multiple organ failure, etc., and even die.
  • the new coronavirus is composed of four structural proteins (spike protein, envelope protein, membrane protein and nucleocapsid protein) and RNA nucleic acid chains.
  • Spike Glycoprotein S protein
  • S protein is a glycoprotein located on the surface of the new coronavirus membrane, which mainly plays a role in cell adhesion and cell membrane fusion.
  • the S protein is composed of two subunits, S1 and S2, in which the S1 subunit contains the receptor binding domain (Receptor Binding Domain, RBD), which is responsible for recognizing the receptor ACE2 of the host cell, which is the interaction between the virus and the receptor and the virus A key factor in invading cells and a key target for vaccine design.
  • RBD receptor binding domain
  • the S2 subunit contains the basic elements required for the membrane fusion process, which can promote the fusion of the virus and the host cell membrane.
  • the new coronavirus is constantly mutating, and many popular SARS-CoV-2 mutants are associated with a rapid increase in the number of cases, such as the British mutant B.1.1.7/501Y.V1, the South African mutant B.1.351/501Y .V2, and the Brazilian mutant P.1/501Y.V3.
  • the above-mentioned 2019-nCoV mutants and possibly other 2019-nCoV variants with K417N/T, E484K and N501Y mutations can reduce the neutralizing ability of vaccine-induced plasma neutralizing antibodies.
  • the present disclosure provides a fusion protein vaccine comprising interferon, COVID-19 antigen and immunoglobulin Fc region, which can enhance the immunogenicity and immunity of mutant COVID-19 antigen through fusion expressed IFN.
  • the titer of neutralizing antibodies ensures efficient production of neutralizing antibodies, which can significantly improve the defense against mutant strains.
  • the present disclosure provides a fusion protein comprising:
  • the present disclosure provides a nucleic acid encoding the aforementioned fusion protein.
  • the present disclosure provides a vector comprising the aforementioned nucleic acid.
  • the present disclosure provides a host cell expressing the aforementioned fusion protein, comprising the aforementioned nucleic acid, and/or comprising the aforementioned vector.
  • the present disclosure provides a vaccine for treating and/or preventing novel coronavirus SARS-CoV-2 infection or novel coronavirus disease COVID-19, comprising the aforementioned fusion protein, nucleic acid, vector and/or host cell, And optionally, pharmaceutically acceptable carriers and/or excipients.
  • the present disclosure provides a kind of aforementioned fusion protein, nucleic acid, vector, host cell and/or vaccine in the preparation prevention and/or treatment new coronavirus SARS-CoV-2 infection and/or new coronavirus disease COVID-19 application in drugs or products.
  • the present disclosure provides a preparation method of a vaccine for preventing and/or treating novel coronavirus SARS-CoV-2 infection or coronavirus disease COVID-19, the method comprising expressing the aforementioned fusion protein.
  • the present disclosure provides a method for preventing and/or treating novel coronavirus SARS-CoV-2 infection and/or novel coronavirus disease COVID-19, the method comprising administering to a subject an effective amount of the aforementioned Fusion proteins, nucleic acids, vectors, host cells and/or vaccines.
  • the present disclosure provides a method for inducing a neutralizing antigen-specific immune response in an individual, the method comprising administering to the subject an effective amount of the aforementioned fusion protein, nucleic acid, vector, host cell and/or vaccine.
  • the antigen used in the mutant vaccine of the present disclosure is directed against the antigenic part (especially the RBD part) of the mutant new coronavirus strain, and is combined with IFN that enhances immune activation and the Fc domain to form a fusion protein.
  • the disclosure adopts the method of fusion with the Fc fragment to increase the half-life of the mutant vaccine; through the IFN domain, the immunogenicity of the new crown mutant antigen and the titer of neutralizing antibodies are improved to ensure the efficient production of neutralizing antibodies; through The mutated RBD increases the defense ability against mutant strains; the IgG1 type Fc is used, and the downstream purification method is relatively simple, which is conducive to industrial production.
  • This mutant vaccine can induce a stronger immune response in animals and humans. Therefore, the fusion protein vaccine of the crown mutant strain of the present disclosure has a longer half-life, higher immunogenicity, and stronger defense ability than the traditional single-antigen new crown vaccine.
  • the disclosure also develops a CHO protein expression and preparation method suitable for the transient expression of larger protein macromolecules, which reduces the difficulty of early preparation and can ensure that the product quality meets the scale-up production standards.
  • the mutant vaccine of the present disclosure has a long-lasting effect, is conducive to industrial production, has activity comparable to that of ordinary IFN, higher immunogenicity and neutralizing antibody titer, and can increase the defense ability against mutant strains , can be used as a new generation of mutant strain vaccine drugs to resist the spread of the new crown epidemic.
  • Figure 1 is a schematic diagram of the molecular structure of the parental strain vaccine (V-01) and the mutant strain vaccine (UK strain, South African strain).
  • Figure 2 is the expression plasmid map of parental strain vaccine (V-01) and mutant strain vaccine (UK strain, South African strain).
  • Figure 2a is the map of the parental strain vaccine (V-01);
  • Figure 2b is the map of the British strain vaccine;
  • Figure 2c is the map of the South African strain vaccine.
  • Fig. 3 shows the identification results of expression plasmid enzymes of parental strain vaccine (V-01) and mutant strain vaccine (UK strain, South African strain).
  • M1 DL15000 nucleic acid molecular marker; 1. Parent strain vaccine (V-01) HindIII single enzyme digestion; 2. Parent strain vaccine (V-01) HindIII/PacI double enzyme digestion; 3. British strain vaccine HindIII single enzyme digestion ; 4. British strain vaccine HindIII/PacI double enzyme digestion; 5. South African strain vaccine HindIII single enzyme digestion; 6. South African strain vaccine HindIII/PacI double enzyme digestion; M2: DL15000 nucleic acid molecular marker.
  • Figure 4 is a comparison of the results of the instant method used in the present disclosure and the commercially available method. Among them, Fig. 4a is the detection result of viable cell density and cell viability; Fig. 4b is the result of expression level.
  • Figure 5 is a graph showing the expression levels (Figure 5a) of the parental strain vaccine (V-01) and the mutant strain vaccine (UK strain, South African strain) expressed in Example 1 of the present disclosure ( Figure 5a) and the electrophoresis detection results after purification ( Figure 5b). Among them, M1: 180kDa protein marker.
  • Fig. 6 is an in vitro affinity diagram of parental strain vaccine (V-01), mutant strain vaccine (UK strain, South African strain) and IFNR/ACE2 determined based on SPR method.
  • Fig. 6a is the fitting curve of parental strain (V-01) and ACE2 affinity
  • Fig. 6b is the fitting curve of British strain vaccine and ACE2 affinity
  • Fig. 6c is the fitting curve of South African strain vaccine and ACE2 affinity
  • Fig. 6e is a fitting curve of the affinity between the parent strain (V-01) and IFNAR2
  • Fig. 6e is a fitting curve of the affinity between the British strain vaccine and IFNAR2
  • Fig. 6f is a fitting curve of the affinity between the South African strain vaccine and IFNAR2.
  • Fig. 7 is a graph showing the biological activity of parental strain vaccine (V-01), mutant strain vaccine (UK strain, South African strain) and IFNR determined based on the indicator cell method.
  • Figure 8 is the in vivo efficacy evaluation of parental strain vaccine (V-01) and mutant strain vaccine (British strain, South African strain) in mice.
  • Figure 9 is the evaluation of the neutralizing potency of the parental strain vaccine (V-01) and the mutant strain vaccine (UK strain, South African strain) for three pseudoviruses.
  • Fig. 9a is the neutralizing titer of parental strain vaccine (V-01), mutant strain vaccine (UK strain, South African strain) for three kinds of pseudoviruses
  • Fig. 9 b is parental strain vaccine (V-01), mutant strain vaccine (British strain, South African strain) for the neutralization titer comparison of pseudoviruses.
  • the term “about” or “approximately” means within plus or minus 10% of a given value or range. Where integers are required, the term means rounding up or down to the nearest whole number within plus or minus 10% of a given value or range.
  • fusion protein refers to a natural or synthetic molecule consisting of one or more molecules in which two or more peptide- or protein-based (including glycoprotein)-based molecules with different specificities are optionally synthesized by chemical or Amino acid-based linker molecules are fused together.
  • the linkage can be achieved by C-N fusion or N-C fusion (in 5' ⁇ 3' direction), preferably C-N fusion.
  • Interferon refers to the body is infected by a virus or under the action of other interferon-inducing agents, which is produced under the control of the cell genome and has various biological activities such as anti-virus, anti-tumor and immunomodulatory activities.
  • a class of cytokines. Interferons can be divided into three main classes according to their biological and physical properties: Type I, Type II and Type III interferons.
  • Type I interferons constitute a structurally related family (IFN- ⁇ ( ⁇ ), IFN- ⁇ ( ⁇ ), IFN- ⁇ ( ⁇ ), IFN- ⁇ ( ⁇ ), IFN- ⁇ ( ⁇ ), IFN- ⁇ ( ⁇ ) and IFN- ⁇ ( ⁇ )), of which IFN- ⁇ and IFN- ⁇ do not occur in humans.
  • Human type I interferon (IFN) genes are clustered on human chromosome 9p21, while mouse genes are located in a conserved syntenic region on mouse chromosome 4. To date, 14 IFN- ⁇ genes and 3 pseudogenes have been identified in mice.
  • IFN- ⁇ (or IFNA) genes IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21
  • IFN- ⁇ genes Two of the human IFN- ⁇ genes (IFNA1/IFN- ⁇ 1 and IFNA13/IFN- ⁇ 13) encode the same protein. All human type I interferons bind to a cell surface receptor (IFN ⁇ receptor, IFNAR) composed of two transmembrane proteins (IFNAR-1 and IFNAR-2), which cause JAK-STAT activation, ISGF3 formation and subsequent initiation of gene expression.
  • IFN ⁇ receptor IFNAR
  • Interferon gamma is the only known type II interferon, which is mainly involved in the induction of antibacterial and antitumor mechanisms through macrophage stimulation.
  • the IFN- ⁇ receptor (IFNGR) is a heterodimeric receptor consisting of two ligand-binding IFNGR1 chains associated with two signaling IFNGR2 chains.
  • Type III interferons consist of three subtypes and are also known as IFN ⁇ (IFN ⁇ 1 or IL-29, IFN ⁇ 2 or IL-28A and IFN ⁇ 3 or IL-28B) and possess antiviral, antitumor and immunomodulatory activities.
  • the IFN- ⁇ receptor is also a heterogeneous binary composed of a unique ligand-binding chain, IFN- ⁇ R1 (also designated IL-28R ⁇ ), and a secondary chain, IL-10R2, shared with the receptor for IL-10-related cytokines. polymer complex.
  • coronavirus belongs to the family Coronaviridae, the genus Coronaviridae, can infect mammals and birds, and cause various diseases of the respiratory system, digestion and central nervous system. According to genomic and serological differences, coronaviruses can be divided into four different genera: ⁇ , ⁇ , ⁇ , and ⁇ . Currently, only ⁇ and ⁇ coronaviruses infect humans. Up to now, six human coronaviruses (HCoV) from two genera ( ⁇ and ⁇ ) have been identified. SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Novel Coronavirus (SARS-CoV-2).
  • antibody or “immunoglobulin” has the broadest meaning and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg bispecific antibodies) composed of at least 2 intact antibodies, and antibody fragments , so long as it is shown to have the desired biological activity.
  • the term generally includes hybrid antibodies that are composed of two or more antibodies or antibody fragments with different binding specificities linked together.
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226, or from Pro230, to the carboxy-terminus of the heavy chain.
  • the C-terminal lysine of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody.
  • compositions of intact antibodies may comprise antibody populations from which all K447 residues have been removed, antibody populations from which no K447 residues have been removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • sequence identity has an art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide, or along a region of the molecule. While there are many methods of measuring identity between two polynucleotides or polypeptides, the term “identity” is well known to those of skill (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)) .
  • Th cell helper epitope refers to all epitopes that activate helper T cells, including PADRE.
  • PADRE is a short peptide sequence of 13 amino acids, which can bind to different DR molecules in humans and various animals and present them on the cell surface, thereby activating CD4+ T helper cells and exerting immune regulation.
  • the ability of PADRE to induce T cell response is more than 1000 times that of the natural epitope, so PADRE has some characteristics as an immune adjuvant (PMID: 7895164).
  • PADRE peptides can immunologically activate helper T cells (Th1) in vivo to assist in the activation of CTL, and can activate helper T cells (Th2) to assist B cells to secrete specific antibodies, thereby further enhancing the antigenicity caused by recombinant proteins immune response.
  • treatment refers to any indication of success in treating or ameliorating an injury, disease, lesion or condition, including any objective or subjective parameter such as alleviation; remission; attenuation of symptoms or making the injury, lesion or condition more tolerable to the patient ; slowing the rate of degeneration or decline; making the degeneration endpoint less debilitating; improving the physical or mental health status of the patient.
  • Treatment or amelioration of symptoms may be based on objective or subjective parameters; including results of physical examination, neuropsychiatric testing and/or psychiatric evaluation.
  • an effective amount is an amount sufficient to achieve a stated purpose (e.g., to achieve the effect for which it is administered, to treat a disease, to decrease enzyme activity, to increase enzyme activity, to decrease protein function, to alleviate one or more symptoms of a disease or condition ).
  • An example of an "effective amount” is an amount sufficient to facilitate the treatment, prevention or alleviation of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount”.
  • the present disclosure provides a fusion protein comprising:
  • the fusion protein comprises from N-terminus to C-terminus: interferon or its functional fragment, SARS-CoV-2 or its functional fragment and immunoglobulin Fc region.
  • the fusion protein comprises from N-terminus to C-terminus: SARS-CoV-2 or a functional fragment thereof, interferon or a functional fragment thereof, and an immunoglobulin Fc region.
  • the interferon is selected from Type I interferon, Type II interferon and/or Type III interferon.
  • the interferon may be of human or murine origin.
  • the type I interferon is selected from the group consisting of IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ .
  • the type II interferon is interferon gamma.
  • the type III interferon is selected from IFN- ⁇ 1 (IL-29), IFN- ⁇ 2 (IL-28a), and IFN- ⁇ (IL-28b).
  • the interferon is selected from human IFN- ⁇ 1, IFN- ⁇ 2, IFN- ⁇ 4, IFN- ⁇ 5, IFN- ⁇ 6, IFN- ⁇ 7, IFN- ⁇ 8, IFN- ⁇ 10, IFN- ⁇ 13, IFN - ⁇ 14, IFN- ⁇ 16, IFN- ⁇ 17 and IFN- ⁇ 21.
  • the interferon is IFN- ⁇ 2a; preferably, the amino acid sequence of the IFN- ⁇ 2a comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:1, preferably An amino acid sequence having an identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99%, more preferably an amino acid sequence having an identity of 98% or more; more preferably, the IFN
  • the amino acid sequence of - ⁇ 2a is shown in SEQ ID NO:1.
  • the IFN active domain is preferably connected to the N-terminus behind the linker sequence of the mutant new crown RBD antigen (Gly4Ser) 3.
  • a mutated IFN is used. Compared with the wild-type IFN, the two have only one amino acid sequence difference.
  • GenBank sequence number: AAP20099. 1
  • the position in the complete interferon ⁇ -2b the mutation is named recombinant interferon ⁇ -2b (Q124R).
  • the purpose of this mutation is to realize the partial combination of human IFN ⁇ and heterologous mouse IFN receptor, so as to detect the activity and function of human IFN ⁇ by using the mouse model.
  • this mutation does not affect the affinity of interferon ⁇ -2b to human interferon receptors, and aims to improve the binding activity to mouse interferon receptors, which is conducive to in vivo drug efficacy evaluation in mice.
  • the human IFN- ⁇ 2a extracellular secretion sequence was fused to the C-terminal of the Fc fragment of the targeted IFN bifunctional molecule, and the amino acid sequence was not changed.
  • the structures of the parental strain vaccine (V-01) and the mutant strain vaccine (UK strain, South African strain) made from the fusion protein of IFN ⁇ New Crown RBD domain and Fc are shown in Figure 1. After expression in mammalian cells, the fusion protein self-assembles into a dimer form.
  • the SARS-CoV-2 is a mutant SARS-CoV-2.
  • the mutant SARS-CoV-2 is the British mutant strain B.1.1.7/501Y.V1 or the South African mutant strain 501Y.V2 (South African strain B.1.351).
  • the RBD mutation of the South African strain compared with the wild-type new coronavirus strain, it contains 3 point mutations, namely K417N/T, E484K, and N501Y.
  • the above three point mutations not only changed the conformation of RBD, but also changed the antigenicity of RBD.
  • E484K has been confirmed to increase the immune escape of mutant strains
  • N501Y can significantly enhance the binding force with ACE2
  • K417N affects the generation of salt bridge and Enhanced escape mechanism of E484K.
  • using the RBD domain of the mutant strain as the delivery antigen can make up for the deficiency of the wild-type new crown vaccine.
  • the functional fragment of SARS-CoV-2 is its receptor binding domain RBD.
  • the RBD comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2, preferably having 85%, 90%, 95%, 96%, 97%, 98% %, more than 99% identity amino acid sequence, more preferably 98% or more than 99% identity amino acid sequence; more preferably, the amino acid sequence of the RBD is shown in SEQ ID NO:2.
  • the RBD comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO:3, preferably having 85%, 90%, 95%, 96%, 97%, 98% %, more than 99% identity amino acid sequence, more preferably 98% or more than 99% identity amino acid sequence; more preferably, the amino acid sequence of the RBD is shown in SEQ ID NO:3.
  • the immunoglobulin Fc region is selected from the constant region amino acid sequences of IgG1, IgG2, IgG3 and/or IgG4.
  • the immunoglobulin Fc region is an IgG1 Fc region; preferably, the IgG1 Fc region comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 4, preferably having Amino acid sequences with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more 99% identity; more preferably, the IgG1 Fc region The amino acid sequence is shown in SEQ ID NO:4.
  • IgG1 Fc is selected, wherein IgG Fc has a T250Q/M428L mutation (amino acids are numbered according to EU nomenclature), which increases the affinity of human FcRn and can prolong the half-life in vivo.
  • the fusion protein further comprises one or more Th cell helper epitopes and/or linking fragments.
  • the Th cell auxiliary epitope is PADRE or its derivatives; the amino acid sequence of the PADRE or its derivatives is selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
  • the connecting fragment is a soft polypeptide sequence; preferably, the amino acid sequence of the flexible peptide is selected from SEQ ID NO:11 and SEQ ID NO:12.
  • the linker connection sequence can be 2-8 repeats of (Gly4Ser)x or (GlySer)x linker, and the specific length depends on the size of the actual fusion protein molecule, and the difference between its steric hindrances needs to be considered.
  • the linker connection sequence used in the present disclosure is (Gly4Ser)3 linker, and the sequence information is as follows:
  • the fusion protein comprises an amino acid sequence having 80% or more identity, preferably 85%, with the amino acid sequence selected from SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15. , 90%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; preferably, the amino acid sequence of the fusion protein Selected from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15; more preferably, the amino acid sequence of the fusion protein is SEQ ID NO:14 or SEQ ID NO:15; more preferably Preferably, the amino acid sequence of the fusion protein is SEQ ID NO: 15.
  • the present disclosure provides a nucleic acid encoding the aforementioned fusion protein.
  • the nucleic acid comprises a nucleotide sequence selected from a nucleotide sequence having 80% or more identity with the nucleotide sequences shown in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, preferably A sequence of nucleotides having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity; preferably, Described nucleic acid is selected from the nucleic acid shown in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18; More preferably, described nucleic acid is the nucleic acid shown in SEQ ID NO:17 or SEQ ID NO:18 More preferably, described nucleic acid is the nucleic acid shown in SEQ ID NO:18.
  • the present disclosure provides a vector of the aforementioned nucleic acid.
  • the present disclosure provides a host cell expressing the aforementioned fusion protein, comprising the aforementioned nucleic acid, and/or comprising the aforementioned vector.
  • the host cell is a prokaryotic or eukaryotic cell.
  • the prokaryotic cells are bacterial cells. In some specific embodiments, the prokaryotic cells are E. coli cells.
  • the eukaryotic cells are selected from yeast cells, insect cells, and mammalian cells.
  • the mammalian cell is selected from CHO, HEK293, SP2/0, BHK, C127, and the like.
  • the eukaryotic cells are CHO cells.
  • the present disclosure provides a vaccine for treating and/or preventing novel coronavirus SARS-CoV-2 infection or novel coronavirus disease COVID-19, comprising the aforementioned fusion protein, nucleic acid, vector and/or host cell, And optionally, pharmaceutically acceptable carriers and/or excipients.
  • the pharmaceutically acceptable carrier may contain liquids such as water, saline, glycerol and sorbitol.
  • liquids such as water, saline, glycerol and sorbitol.
  • auxiliary substances in these carriers such as lubricants, glidants, wetting agents or emulsifiers, pH buffer substances and stabilizers, such as albumin.
  • the vaccine is in the form of a recombinant protein subunit vaccine, a recombinant protein mRNA vaccine or a recombinant protein adenovirus vector vaccine.
  • the vaccine can be made into various dosage forms suitable for administration to mammals, and the dosage forms include but are not limited to: injections, capsules, tablets, emulsions, suppositories, lyophilized powders; preferably It is an injection.
  • the present disclosure provides a preparation method of a vaccine for preventing and/or treating novel coronavirus SARS-CoV-2 infection or coronavirus disease COVID-19, the method comprising expressing the aforementioned fusion protein.
  • the present disclosure provides a kind of aforementioned fusion protein, nucleic acid, vector, host cell and/or vaccine in the preparation prevention and/or treatment new coronavirus SARS-CoV-2 infection and/or new coronavirus disease COVID-19 use in medicines or products.
  • the present disclosure provides a method for preventing and/or treating novel coronavirus SARS-CoV-2 infection and/or novel coronavirus disease COVID-19, the method comprising administering to a subject an effective amount of the aforementioned Fusion proteins, nucleic acids, vectors, host cells and/or vaccines.
  • the present disclosure provides a method for inducing a neutralizing antigen-specific immune response in an individual, the method comprising administering to the subject an effective amount of the aforementioned fusion protein, nucleic acid, vector, host cell and/or vaccine.
  • the vaccine is administered intradermally, subcutaneously, intramuscularly or intravenously.
  • the vaccine is administered by injection.
  • Embodiment 1 the preparation of fusion protein
  • the mutant vaccine is a fusion protein of IFN ⁇ New Crown RBD domain and Fc. It consists of genetically fusing the C-terminus of IFN to RBD-Fc via a flexible (Gly4Ser)3 linker ( Figure 1).
  • the mutant vaccine South African strain, British strain
  • the parental strain vaccine V-01
  • All the genes involved in the present disclosure are synthesized through the whole gene, and then linked to the mammalian cell expression vector pCGS3 (purchased from: Sigma-Aldrich) by double restriction enzymes for expression.
  • FIG. 2 shows the diagrams of parental strain vaccine (V-01) and mutant strain vaccine (UK strain, South African strain).
  • Figure 3 shows the results of the electropherogram of the enzyme digestion identification, and the enzyme digestion identification is correct.
  • the plasmid was extracted with Omega’s detoxified plasmid large-scale extraction kit (product number: D6926-03B; purchased from Omega Bio-tek), and stored at -80°C.
  • the amino acid sequence of the parent strain vaccine (V-01) is shown in SEQ ID NO: 13, and its coding nucleic acid sequence is shown in SEQ ID NO: 16; the amino acid sequence of the British strain vaccine is shown in SEQ ID NO: 14, Its coding nucleic acid sequence is shown in SEQ ID NO:17; the amino acid sequence of the South African strain vaccine is shown in SEQ ID NO:15, and its coding nucleic acid sequence is shown in SEQ ID NO:18.
  • the disclosure uses the ExpiCHO expression system to express the protein, which uses a bioreactor to amplify the expression scale (15L), that is, a large The volume transient transfection protocol was used for expression, which improved the commercially available ExpiCHO expression system.
  • the viable cell density should reach 6 ⁇ 10 6 -10 ⁇ 10 6 cells/mL, and the cell viability should not be lower than 95%.
  • the plasmid diluent needs to be sterilized by filtration, then slowly add the diluted transfection reagent to the plasmid, mix well, and incubate at room temperature for 5 minutes. This mixture was then added uniformly to the cell culture.
  • the commercially available method is manual pouring and adding, according to the operating instructions of the commercially available ExpiCHO expression system (purchased from: Life technologies, product number: A29133), and the reaction scale is 1L shake flask production.
  • the disclosed transfection mixture is prepared by using the same ratio as the commercially available instructions for configuration.
  • the present disclosure uses a bioreactor liquid replenishment device to add it at a constant speed.
  • the bioreactor is controlled at 37° C., 5% CO 2 , pH 7.0, dissolved oxygen 40%, rotation speed 150 rpm, and deep air constant flow 20 mL/min.
  • the cell viability rate is lower than 80%, or on the 6th day, the supernatant is collected for protein quantification and further purification.
  • the parental strain vaccine (V-01) expression vector was used to prepare the transfection mixture, and then transient transfection was carried out by using commercially available manual direct addition and the disclosed reactor rehydration device.
  • the change trend of cell viability and living cell density was similar, and the cell viability and cell density could always be kept high (Fig. 4a).
  • the expression level of the parent strain vaccine (V-01) is significantly increased, and the production can be increased by nearly 2 times compared with the traditional commercial mode (Fig. 4b).
  • the transient expression in the bioreactor provided by the present disclosure the production cells can continuously maintain a high activity rate and expression yield, and compared with the traditional commercially available transfection process, the transient expression can be significantly improved.
  • the transient expression system adopted in the present disclosure is suitable for the transient expression production of larger molecules, which reduces the difficulty of early sample preparation.
  • the reaction conditions are precisely controlled by the bioreactor, which is basically consistent with the later commercial scale production control, making the quality of the transient product more representative.
  • the parental strain vaccine (V-01) and the mutant strain vaccine (UK strain, South Africa strain) were transiently expressed through the large-volume transient expression system provided by the present disclosure. Under the conditions of the same expression process, the same signal peptide and expression vector, the expression level of the parental strain vaccine was the highest, followed by the British strain vaccine and the South African strain vaccine ( Figure 5a). The difference in expression level may be caused by different point mutations, especially the South African strain vaccine The vaccine increased the K417T and E484K mutations, and the expression level decreased significantly.
  • the clarified sample of the cell culture fluid is directly captured on a Protein A affinity chromatography column to obtain a purified fusion protein.
  • the purity of the parent strain vaccine (V-01) and the mutant strain vaccine (UK strain, South Africa strain) were both greater than 95% by reducing SDS-PAGE analysis.
  • the affinity between the parental strain vaccine (V-01) and the mutant strain vaccine (UK strain, South African strain) was detected using a molecular interaction instrument (SPR method), including the affinity between the RBD domain and the ACE2 receptor, and the IFN ⁇ domain Affinity for the IFNAR2 receptor. details as follows:
  • HBS-EP+ Product No. BR100826, purchased from GE Healthcare
  • each cycle included capturing different vaccines, injecting and regenerating ACE2 proteins at different concentrations.
  • HBS-EP+ was used as the experimental buffer, and each cycle included capture vaccine, injection of different concentrations of IFNAR2 protein, and regeneration.
  • Embodiment 3 the biological activity of parental strain vaccine (V-01) and mutant strain vaccine (British strain, South African strain)
  • IFN ⁇ -2b in the structure of the parental strain vaccine (V-01) and the mutant strain vaccine (UK strain, South African strain) binds to the endogenous receptors IFNAR2 and IFNAR1 on the cell membrane, it can activate the interferon-stimulated response element through signal transduction, Start the expression of luciferase, and the expression amount is positively correlated with the biological activity of interferon.
  • HEK-Lucia TM Null recombinant cells (product number: hkl-null, purchased from: InvivoGen) were collected, the cell density was adjusted to 8 ⁇ 10 5 cells/mL with assay culture medium, and 50 ⁇ L/well was added to a 96-well whole white blood cell plate. That is, 4 ⁇ 10 4 cells/well were added, and then 50 ⁇ L/well of samples of each dilution gradient were added, and 3 replicate wells were set for each. Set the negative control (NC) without drug addition and the blank control (Blank) of the culture medium. Cultivate in 37°C, 5% CO 2 incubator for 18h-24h.
  • NC negative control
  • Blank blank control
  • the results are shown in Table 3 and Figure 7.
  • the IFN ⁇ domains of the parental strain vaccine (V-01) and the mutant strain vaccine (UK strain, South African strain) can both activate the IFN ⁇ signaling pathway and have the expected cell biological activity.
  • the EC 50 of the two were 15.1ng/mL, 12.4ng/mL and 12.8ng/mL, respectively.
  • the in vivo efficacy evaluation of vaccines is very important, which can directly reflect the immunogenicity of vaccines and is directly related to the protection they produce. Therefore, the in vivo efficacy of the parental strain vaccine (V-01) and the mutant strain vaccine (UK strain, South African strain) was evaluated using C57BL/6 mice.
  • the parental strain vaccine (V-01) and mutant strain vaccine (UK strain, South African strain) prepared in Example 1 were used to immunize 6-8 week-old C57BL/6 mice at a vaccine concentration of 10 ⁇ g/mL.
  • Each mouse was injected with 0.1 mL of thigh intramuscularly, 10 mice/group, and each mouse was immunized twice (one on day 0 and one on day 14).
  • blood was collected from the orbit to collect serum.
  • the blood was allowed to stand at room temperature for coagulation, centrifuged at 4000rpm, 2-8°C for 10min, and the supernatant was taken.
  • GMT geometric mean titer
  • Embodiment 5 Pseudovirus neutralization titer-protection evaluation
  • Serum samples were inactivated in a 56°C water bath for 30 minutes in advance.
  • the anti-RBD mouse neutralizing antibody was firstly diluted to 25 ⁇ g/mL with detection medium (10% FBS DMEM) as a positive quality control (PC).
  • detection medium (10% FBS DMEM) as a positive quality control (PC).
  • PC positive quality control
  • serum sample to be tested and dilute it in a 96-well whole white blood cell plate to a concentration of 5% in the first hole (i.e. 1:20), and then carry out a doubling dilution with the positive quality control product according to 1:3, a total of 8 dilution gradients, Serum samples for each dilution gradient were 100 ⁇ L/well.
  • HEK293T-ACE2 cells Collect HEK293T-ACE2 cells, resuspend them with detection medium, and adjust the cell concentration to 2.5 ⁇ 10 5 cells/mL.
  • the abscissa is the logarithmic value of the multiple dilution factor (Log Titer), and the ordinate is the percentage of inhibition rate (%Inhibition), perform four-parameter fitting analysis, the software automatically calculates the pNT 50 value, rounded That is, the pseudovirus neutralization titer.
  • the serum after immunization with the British strain vaccine can also neutralize the wild-type pseudovirus, the British mutant pseudovirus and the South African mutant pseudovirus, and its pNT 50 is 846, 1535, and 217, respectively. The effect is better than the South African mutant pseudovirus.
  • the design of the British strain vaccine can increase its protection against the British mutation by about 1.8 times, and there is a significant difference, but the protection against the South African mutation is slightly worse.
  • the serum after immunization with the South African strain vaccine can neutralize the wild-type pseudovirus, the British mutant pseudovirus and the South African mutant pseudovirus, and its pNT 50 is 279, 496, and 1136, respectively.
  • Vaccine (V-01) increased by about 4.1 times, and the difference was extremely significant. Therefore, the design of the South African strain vaccine has greatly improved the protection against South African mutations.

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Abstract

本公开涉及一种新型冠状病毒SARS-CoV-2突变体疫苗与应用，所述疫苗包含一种融合蛋白，所示融合蛋白包含：(1)干扰素或其功能片段；(2)新型冠状病毒SARS-CoV-2或其功能片段；(3)免疫球蛋白Fc区。本公开的突变体疫苗具有长效、有利于工业化生产、活性与普通IFN相当、更高的免疫原性和中和抗体滴度、可增加对突变株的防御能力，可作为新一代突变株疫苗药物，用于抵御新冠疫情蔓延。

Description

一种新型冠状病毒SARS-CoV-2突变体疫苗与应用 技术领域
本公开属于生物技术领域，具体涉及一种新型冠状病毒SARS-CoV-2突变体疫苗与应用。
背景技术
新型冠状病毒(2019-nCoV，SARS-CoV-2)是一种β属的冠状病毒，于2019年首次被发现，是目前已知的第七种能感染人的冠状病毒，感染该病毒后可导致患者出现发热、干咳、乏力等症状；部分患者会产生严重的肺炎，进而发展为急性呼吸窘迫综合征、脓毒症休克、出凝血功能障碍及多器官功能衰竭等，甚至死亡。
新型冠状病毒由四种结构蛋白(棘突蛋白、包膜蛋白、膜蛋白和核衣壳蛋白)以及RNA核酸链组成。其中棘突蛋白(Spike Glycoprotein，S蛋白)是一种糖蛋白，位于新冠病毒膜表面，主要作用于细胞粘附和细胞膜融合。S蛋白由S1和S2两个亚基组成，其中S1亚基中包含受体结合结构域(Receptor Binding Domain,RBD)，其负责识别宿主细胞的受体ACE2，是病毒和受体相互作用以及病毒入侵细胞的关键因素，也是疫苗设计的关键靶点。S2亚基含有膜融合过程所需的基本元件，能够促进病毒与宿主细胞膜的融合。
新型冠状病毒在不停地突变之中，许多流行的SARS-CoV-2突变体与病例数迅速增加有关，例如英国突变株B.1.1.7/501Y.V1、南非突变株B.1.351/501Y.V2，以及巴西突变株P.1/501Y.V3。上述新冠病毒突变体以及可能带有K417N/T、E484K和N501Y突变的其他新冠病毒变体，可以降低疫苗诱导的血浆中和抗体的中和能力。
目前市售/在研新冠疫苗，大多采用野生型S蛋白或RBD设计，对野生型或早期新冠突变株病御能力较好，但对目前流行的变异株(主要为B.1.351/501Y.V2南非突变株)已出现不同程度的保护力下降现象。因此，亟需开发新的针对新型冠状病毒，尤其是突变型新型冠状病毒的疫苗。
发明内容
为了避免现有疫苗的局限，本公开提供了一种包含干扰素、新冠抗原和免疫球蛋白Fc区的融合蛋白疫苗，所述疫苗能够通过融合表达的IFN提升突变型新冠抗原的免疫原性和中和抗体的滴度，保证高效产生中和抗体，可显著提升对突变株的防御能力。
在一方面，本公开提供了一种融合蛋白，其包含：
(1)干扰素或其功能片段；
(2)新型冠状病毒SARS-CoV-2或其功能片段；和
(3)免疫球蛋白Fc区。
在一方面，本公开提供了一种编码前述融合蛋白的核酸。
在一方面，本公开提供了一种包含前述核酸的载体。
在一方面，本公开提供了一种表达前述融合蛋白、包含前述核酸和/或包含前述载体的宿主细胞。
在一方面，本公开提供了一种治疗和/或预防新型冠状病毒SARS-CoV-2感染或新型冠状病毒疾病COVID-19的疫苗，其包含前述融合蛋白、核酸、载体和/或宿主细胞，以及任选地，药学上可接受的载体和/或赋形剂。
在一方面，本公开提供了一种前述融合蛋白、核酸、载体、宿主细胞和/或疫苗在制备预防和/或治疗新型冠状病毒SARS-CoV-2感染和/或新型冠状病毒疾病COVID-19的药物或产品中的应用。
在一方面，本公开提供了一种预防和/或治疗新型冠状病毒SARS-CoV-2感染或冠状病毒疾病COVID-19的疫苗的制备方法，所述方法包括表达前述融合蛋白。
在一方面，本公开提供了一种预防和/或治疗新型冠状病毒SARS-CoV-2感染和/或新型冠状病毒疾病COVID-19的方法，所述方法包括向受试者施用有效量的前述融合蛋白、核酸、载体、宿主细胞和/或疫苗。
在一方面，本公开提供了一种诱导个体中和抗原特异性免疫应答的方法，所述方法包括向受试者施用有效量的前述融合蛋白、核酸、载体、宿主细胞和/或疫苗。
与已知的灭活、腺病毒以及mRNA新冠病毒疫苗都不同，本公开的突变株疫苗使用的抗原是针对突变新冠毒株的抗原部分(特别是RBD部分)，并且与增强免疫激活能力的IFN以及Fc结构域形成融合蛋白。本公开为了延长半衰期，采用与Fc片段融合的方式，提升了突变株疫苗的半衰期；通过IFN结构域提升新冠突变抗原的免疫原性和中和抗体的滴度，保证高效产生中和抗体；通过变异型RBD增加对突变株的防御能力；采用IgG1型Fc，下游纯化方法相对简单，利于产业化生产。这种突变株疫苗可在动物体内、人体内诱导更强的免疫应答。所以，本公开的冠突变株融合蛋白疫苗，对比传统单一抗原新冠疫苗，具有更长的半衰期、更高的免疫原性、更强的防御能力。本公开同时开发了适合较蛋白大分子瞬时表达的CHO蛋白表达与制备方法，降低了早期制备的难度，同时可保证产品质量符合放大生产标准。综合以上所有创新设计和优化，本公开的突变株疫苗具有长效、有利于工业化生产、活性与普通IFN相当、更高的免疫原性和中和抗体滴度、可增加对突变株的防御能力，可作为新一代突变株疫苗药物，用于抵御新冠疫情蔓延。
附图说明
图1为亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)的分子结构示意图。
图2为亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)表达质粒图谱。其中，图 2a为亲本株疫苗(V-01)图谱；图2b为英国株疫苗图谱；图2c为南非株疫苗图谱。
图3为亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)表达质粒酶鉴定结果。其中，M1：DL15000核酸分子标记；1.亲本株疫苗(V-01)HindIII单酶切；2.亲本株疫苗(V-01)HindIII/PacI双酶切；3.英国株疫苗HindIII单酶切；4.英国株疫苗HindIII/PacI双酶切；5.南非株疫苗HindIII单酶切；6.南非株疫苗HindIII/PacI双酶切；M2：DL15000核酸分子标记。
图4为本公开采用瞬转方法与市售方法结果对比。其中，图4a为活细胞密度与细胞活率检测结果；图4b为表达量结果。
图5为本公开实施例1所表达亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)表达水平(图5a)与纯化后的电泳检测结果图(图5b)。其中，M1：180kDa蛋白标记。
图6为基于SPR法测定的亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)与IFNR/ACE2体外亲和力图。其中，图6a为亲本株(V-01)与ACE2亲和力拟合曲线图；图6b为英国株疫苗与ACE2亲和力拟合曲线图；图6c为南非株疫苗与ACE2亲和力拟合曲线图；图6d为亲本株(V-01)与IFNAR2亲和力拟合曲线图；图6e为英国株疫苗与IFNAR2亲和力拟合曲线图；图6f为南非株疫苗与IFNAR2亲和力拟合曲线图。
图7为基于指示细胞法测定的亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)与IFNR生物学活性图。
图8为亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)小鼠体内效力评估。
图9为亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)对于三种假病毒的中和效价评估。其中，图9a为亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)对于三种假病毒的中和效价；图9b为亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)对假病毒的中和效价比较。
具体实施方式
I.定义
在本公开中，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，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
如本文使用的和除非另作说明，术语“约”或“大约”是指在给定值或范围的加或减10％之内。在需要整数的情况下，该术语是指在给定值或范围的加或减10％之内、向上或向下舍入到最接近的整数。
如本文使用的和除非另作说明，术语“包含”，“包括”，“具有”，“含有”，包括其语法上的等同形式，通常应当理解为开放式且非限制性的，例如，不排除其他未列举的要素或步骤。
术语“融合蛋白”是指由一种或多种分子组成的天然或合成分子，其中具有不同特异性 的两种或多种基于肽或蛋白质(包括糖蛋白)的分子任选的通过化学的或基于氨基酸的接头分子融合在一起。该连接可通过C-N融合或N-C融合(以5′→3′方向)，优选C-N融合而实现。
术语“干扰素”(Interferon，IFN)指机体受到病毒感染或在其他干扰素诱生剂作用下，由细胞基因组控制产生的具有抗病毒、抗肿瘤和和免疫调节活性等多种生物学活性的一类细胞因子。干扰素可以根据它们的生物和物理性质分成三大类：I型、II型和III型干扰素。
I型干扰素构建了在结构上相关的家族(IFN-α(α)、IFN-β(β)、IFN-κ(κ)、IFN-δ(δ)、IFN-ε(ε)、IFN-τ(τ)、IFN-ω(ω)和IFN-ζ(ζ))，其中IFN-δ和IFN-τ不会在人类中出现。人I型干扰素(IFN)基因簇集在人染色体9p21上，而小鼠基因位于小鼠4号染色体上的保守的共线性区中。迄今为止，已在小鼠中鉴定出14种IFN-α基因和3种伪基因。在人类中，已经鉴定出13种IFN-α(或IFNA)基因(IFNA1、IFNA2、IFNA4、IFNA5、IFNA6、IFNA7、IFNA8、IFNA10、IFNA13、IFNA14、IFNA16、IFNA17和IFNA21)以及1个伪基因，其中两种人IFN-α基因(IFNA1/IFN-α1和IFNA13/IFN-α13)针对相同的蛋白编码。所有的人I型干扰素结合至由两种跨膜蛋白(IFNAR-1和IFNAR-2)组成的细胞表面受体(IFNα受体，IFNAR)，该细胞表面受体引起JAK-STAT活化、ISGF3的形成和随后开始的基因表达。干扰素γ(IFN-γ)是唯一已知II型干扰素，其主要涉及通过巨噬细胞刺激诱导抗菌和抗肿瘤机制。IFN-γ受体(IFNGR)是由与两种信号转导IFNGR2链相关联的两种配体结合IFNGR1链组成的异质二聚体受体。III型干扰素由三种亚型组成，并且还被称为IFNλ(IFNλ1或IL-29、IFNλ2或IL-28A和IFNλ3或IL-28B)，且具有抗病毒、抗肿瘤和免疫调节活性。IFN-λ受体也是由唯一的配体结合链IFN-λR1(也被指定为IL-28Rα)以及与用于IL-10相关细胞因子的受体共享的副链IL-10R2组成的异质二聚体复合物。
术语“冠状病毒(Coronavirus)”属于冠状病毒科，冠状病毒属，可以感染哺乳动物和禽类，引起呼吸系统、消化和中枢神经的各种疾病。根据基因组和血清学差异可以将冠状病毒分成四个不同的属：α、β、γ和δ，目前只有α和β属冠状病毒感染人类。截至目前已鉴定出来自两个属(α和β)的6种人冠状病毒(HCoV)，α属冠状病毒包括NL63和229E，β属冠状病毒包括OC43、HKU1、急性呼吸系统综合征冠状病毒(SARS-CoV)、中东呼吸综合征冠状病毒(MERS-CoV)和新型冠状肺炎病毒(SARS-CoV-2)。
术语“抗体”或“免疫球蛋白”有最广义的含义，特别包括完整的单克隆抗体、多克隆抗体、由至少2个完整抗体构成的多特异性抗体(例如双特异性抗体)以及抗体片段，只要其显示出具有所需的生物学活性即可。此术语一般包括由2个或多个具有不同结合特异性的抗体或抗体片段连接在一起构成的杂合抗体。
术语“Fc区”在本文中用于定义免疫球蛋白重链的C端区，包括天然序列Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以变化，但人IgG重链Fc区通常定义为自位置Cys226，或自Pro230处的氨基酸残基延伸至重链的羧基端。可以除去Fc区的C端赖氨酸(依 照EU编号系统的残基447)，例如在抗体的产生或纯化期间，或通过重组工程化改造编码抗体重链的核酸。因此，完整抗体的组合物可以包含已除去所有K447残基的抗体群体，未除去任何K447残基的抗体群体，和具有有和无K447残基的抗体混合物的抗体群体。
序列“相同性”或“同一性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo,H.&Lipman,D.,SIAM J Applied Math48:1073(1988))。
术语“Th细胞辅助表位”是指使辅助T细胞活化的所有表位，包括PADRE。
PADRE为13个氨基酸的短肽序列，能人和多种动物不同DR分子结合，提呈于细胞表面，进而激活CD4+T辅助细胞，发挥免疫调节作用。PADRE诱导T细胞应答的能力是天然表位的1000倍以上，因此PADRE具备作为免疫佐剂的一些特征(PMID：7895164)。PADRE肽段在体内可以免疫性激活辅助型T细胞(Th1)以协助CTL的激活，并且可以激活辅助型T细胞(Th2)以协助B细胞分泌特异性抗体，进而进一步增强重组蛋白所引起的抗原免疫反应。
术语“治疗”是指在治疗或改善损伤、疾病、病变或病状方面的任何成功迹象，包括任何客观或主观参数，诸如减轻；缓解；减弱症状或使得损伤、病变或病状更可为患者耐受；减缓变性或衰退速率；使得变性终点的致虚弱性较小；改进患者的身体或精神健康状态。症状的治疗或改善可基于客观或主观参数；包括身体检查、神经精神病学测验和/或精神病学评估的结果。
术语“有效量”是足以实现所陈述目的的量(例如实现它被施用来达成的作用，治疗疾病，降低酶活性，增加酶活性，降低蛋白质功能，减轻疾病或病状的一种或多种症状)。“有效量”的一实例是足以促进对疾病的一种或多种症状的治疗、预防或减轻的量，其也可被称为“治疗有效量”。
II.融合蛋白及疫苗
在一方面，本公开提供了一种融合蛋白，其包含：
(1)干扰素或其功能片段；
(2)新型冠状病毒SARS-CoV-2或其功能片段；和
(3)免疫球蛋白Fc区。
在一些实施方案中，所述融合蛋白从N-末端至C-末端包含：干扰素或其功能片段、SARS-CoV-2或其功能片段和免疫球蛋白Fc区。
在一些实施方案中，所述融合蛋白从N-末端至C-末端包含：SARS-CoV-2或其功能片段、干扰素或其功能片段和免疫球蛋白Fc区。
在一些实施方案中，所述干扰素选自I型干扰素、II型干扰素和/或III型干扰素。
在一些实施方案中，所述干扰素可来自人源或鼠源。
在一些实施方案中，所述I型干扰素选自IFN-α、IFN-β、IFN-κ、IFN-δ、IFN-ε、IFN-τ、IFN-ω和IFN-ζ。
在一些实施方案中，所述II型干扰素为干扰素γ。
在一些实施方案中，所述III型干扰素选自IFN-λ1(IL-29)、IFN-λ2(IL-28a)和IFN-λ(IL-28b)。
在一些实施方案中，所述干扰素选自人IFN-α1、IFN-α2、IFN-α4、IFN-α5、IFN-α6、IFN-α7、IFN-α8、IFN-α10、IFN-α13、IFN-α14、IFN-α16、IFN-α17和IFN-α21。
更在一些实施方案中，所述干扰素为IFN-α2a；优选地，所述IFN-α2a的氨基酸序列包含与SEQ ID NO:1所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；更优选地，所述IFN-α2a的氨基酸序列如SEQ ID NO:1所示。
在一个实施方案中，IFN活性域优选地与突变型新冠RBD抗原(Gly4Ser)3接头序列后的N-端相连接。在本公开中的一个优选方案中，使用了突变的IFN，其与野生型IFN相比，二者仅有一个氨基酸序列有差异，根据该突变IFN的突变位点在GenBank(序列号：AAP20099.1)完整干扰素α-2b中的位置，将该突变命名为重组干扰素α-2b(Q124R)。该突变的目的是实现人IFNα与异源的小鼠IFN受体的部分结合，从而利用小鼠模型，检测人IFNα的活性和功能。该突变已有文献证实不影响干扰素α-2b与人干扰素受体的亲和力，旨在提升与鼠源干扰素受体的结合活性，有利于小鼠体内进行体内药效评估。再将人源IFN-α2a胞外分泌序列融合于靶向性IFN双功能分子Fc片段C-端，且未对其氨基酸序列进行更改。由IFN\新冠RBD结构域与Fc的融合蛋白制成的亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)的结构见图1。通过哺乳动物细胞表达后，融合蛋白会自行组装成二聚体形式。
在一些实施方案中，所述SARS-CoV-2为突变型SARS-CoV-2。
在一些实施方案中，所述突变型SARS-CoV-2为英国突变株B.1.1.7/501Y.V1或南非突变株501Y.V2(南非株B.1.351)。尤其针对南非株RBD突变，与野生型新冠毒株相比，含有3个点突变，分别为K417N/T、E484K、N501Y。上述3个点突变，不仅改变了RBD的构象，并且改变了RBD的抗原性，其中E484K已证实可增加突变株的免疫逃逸，N501Y可显著增强与ACE2的结合力，K417N影响盐桥的产生并增强E484K的逃逸机制。基于以上影响，采用突变株RBD结构域作为递程抗原，可弥补野生型新冠疫苗的不足。
在一些实施方案中，所述SARS-CoV-2的功能片段为其受体结合结构域RBD。
在一些实施方案中，所述RBD包含与SEQ ID NO:2所示的氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；更优选地，所述RBD的氨基酸序列如SEQ ID NO:2所示。
在一些实施方案中，所述RBD包含与SEQ ID NO:3所示的氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；更优选地，所述RBD的氨基酸序列如SEQ ID NO:3所示。
在一些实施方案中，所述免疫球蛋白Fc区选自IgG1、IgG2、IgG3和/或IgG4的恒定区氨基酸序列。
在一些实施方案中，所述免疫球蛋白Fc区为IgG1的Fc区；优选地，所述IgG1Fc区包含与SEQ ID NO:4所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；更优选地，所述IgG1Fc区的氨基酸序列如SEQ ID NO:4所示。
在本公开的一种实施方案中，选用IgG1Fc，其中，IgG Fc具有T250Q/M428L突变(氨基酸按照EU命名法编号)，该突变增加了人FcRn亲和力，可延长体内半衰期。
在一些实施方案中，所述融合蛋白还包含一个或多个Th细胞辅助表位和/或连接片段。
在一些实施方案中，所述Th细胞辅助表位为PADRE或其衍生物；所述PADRE或其衍生物的氨基酸序列选自SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10。
在一些实施方案中，所述连接片段为柔多肽序列；优选地，所述柔性肽的氨基酸序列选自SEQ ID NO:11和SEQ ID NO:12。
接头连接序列可以是(Gly4Ser)x或(GlySer)x接头的2-8个重复序列，具体长度依据实际融合蛋白分子大小而定，并且需考虑其空间位阻之间的差异而定。本公开所采用的接头连接序列为(Gly4Ser)3接头，序列信息如下：
GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer(SEQ ID NO:11)
GlySer GlySer GlySer(SEQ ID NO:12)
在一些实施方案中，所述融合蛋白包含与选自SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；优选地，所述融合蛋白的氨基酸序列选自SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的氨基酸序列；更优选地，所述融合蛋白的氨基酸序列为SEQ ID NO:14或SEQ ID NO:15；更优选地，所述融合蛋白的氨基酸序列为SEQ ID NO:15。
在一方面，本公开提供了一种编码前述融合蛋白的核酸。
在一些实施方案中，所述核酸包含选自与SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示核苷酸序列具有80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；优选地，所述核酸选自SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的核酸；更优 选地，所述核酸为SEQ ID NO:17或SEQ ID NO:18所示的核酸；更优选地，所述核酸为SEQ ID NO:18所示的核酸。
在一方面，本公开提供了一种前述核酸的载体。
在一方面，本公开提供了一种表达前述融合蛋白、包含前述核酸和/或包含前述载体的宿主细胞。
在一些实施方案中，所述宿主细胞是原核细胞或真核细胞。
在一些实施方案中，所述原核细胞是细菌细胞。在一些具体实施方案中，所述原核细胞是大肠杆菌细胞。
在一些实施方案中，所述真核细胞选自酵母细胞、昆虫细胞和哺乳动物细胞。在一些实施方案中，所述哺乳动物细胞选自CHO、HEK293、SP2/0、BHK、C127等。在一些具体实施方案中，所述真核细胞为CHO细胞。
在一方面，本公开提供了一种治疗和/或预防新型冠状病毒SARS-CoV-2感染或新型冠状病毒疾病COVID-19的疫苗，其包含前述融合蛋白、核酸、载体和/或宿主细胞，以及任选地，药学上可接受的载体和/或赋形剂。
所述药学上可接受的载体可含有液体，如水、盐水、甘油和山梨醇。另外，这些载体中还可能存在辅助性的物质，如润滑剂、助流剂、润湿剂或乳化剂、pH缓冲物质和稳定剂，如白蛋白等。
在一些实施方案中，所述的疫苗形式为重组蛋白亚单位疫苗、重组蛋白mRNA疫苗或重组蛋白腺病毒载体疫苗。
在一些实施方案中，可以将所述的疫苗制成各种适合于哺乳动物给药的剂型，所述剂型包括但不限于：注射剂、胶囊剂、片剂、乳剂、栓剂、冻干粉剂；优选地为注射剂。
在一方面，本公开提供了一种预防和/或治疗新型冠状病毒SARS-CoV-2感染或冠状病毒疾病COVID-19的疫苗的制备方法，所述方法包括表达前述融合蛋白。
III.治疗方法
在一方面，本公开提供了一种前述融合蛋白、核酸、载体、宿主细胞和/或疫苗在制备预防和/或治疗新型冠状病毒SARS-CoV-2感染和/或新型冠状病毒疾病COVID-19的药物或产品中的用途。
在一方面，本公开提供了一种预防和/或治疗新型冠状病毒SARS-CoV-2感染和/或新型冠状病毒疾病COVID-19的方法，所述方法包括向受试者施用有效量的前述融合蛋白、核酸、载体、宿主细胞和/或疫苗。
在一方面，本公开提供了一种诱导个体中和抗原特异性免疫应答的方法，所述方法包括向受试者施用有效量的前述融合蛋白、核酸、载体、宿主细胞和/或疫苗。
在一些实施方案中，所述疫苗通过皮内注射、皮下注射、肌肉注射或静脉注射的方式进 行接种。
在一些实施方案中，所述疫苗通过注射进行施用。
为了达到清楚和简洁描述的目的，本文中作为相同的或分开的一些实施方案的一部分来描述特征，然而，将要理解的是，本公开的范围可包括具有所描述的所有或一些特征的组合的一些实施方案。
实施例
实施例1：融合蛋白的制备
1.1表达质粒构建
突变株疫苗是IFN\新冠RBD结构域与Fc的融合蛋白。其包含通过遗传学方式，将IFN的C-端经柔性(Gly4Ser)3接头与RBD-Fc融合(图1)。为了对比活性功能，同时构建突变株疫苗(南非株、英国株)与亲本株疫苗(V-01)，区别在于RBD抗原不同的点突变。本公开涉及的所有基因都是通过全基因合成，然后通过双酶切连接到哺乳细胞表达载体pCGS3(购自：Sigma-Aldrich)进行表达。图2示出了亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)图谱。图3示出了酶切鉴定电泳图结果，酶切鉴定正确。用Omega的去内毒质粒大提试剂盒(货号：D6926-03B；购自Omega Bio-tek)提取质粒，-80℃保存。
其中，亲本株疫苗(V-01)的氨基酸序列如SEQ ID NO:13所示，其编码核酸序列如SEQ ID NO:16所示；英国株疫苗的氨基酸序列如SEQ ID NO:14所示，其编码核酸序列如SEQ ID NO:17所示；南非株疫苗的氨基酸序列如SEQ ID NO:15所示，其编码核酸序列如SEQ ID NO:18所示。
1.2蛋白质表达
对于亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)的表达，本公开采用ExpiCHO表达系统对蛋白进行表达，其用生物反应器放大了表达规模(15L)，即采用大体积瞬时转染的方案进行表达，从而对市售的ExpiCHO表达系统进行了改进。
(1)在转染之前24小时，调整Expi-CHO细胞的密度至1.5×10 6-3×10 6个细胞/mL，打入15L规模生物反应器并培养过夜。控制参数设定与后期200L或以上规模的反应器一致，可使表达产物的质量与商业化规模尽可能一致。
(2)转染前1小时，测定活细胞密度和活率，活细胞密度应该达到6×10 6-10×10 6个细胞/mL，细胞活率应为不低于95％。
(3)先用Opti-MEM分别稀释表达质粒和转染试剂，质粒稀释液需过滤除菌，再将稀释后的转染试剂慢慢滴加至质粒中，混和均匀，室温孵育5分钟。然后将该混合物匀速加入至细胞培养物中。市售方法为手动倒入添加，按照市售ExpiCHO表达系统(购自：Life technologies，货号：A29133)操作说明书进行，反应规模为1L摇瓶生产。本公开转染混合物制备，采用市售说明书相同比例进行配置。混合物制备完成后，本公开采用生物反应器补 液装置匀速添加。生物反应器控制在37℃，5％CO 2，pH7.0，溶氧40％，转速150rpm，空气深层恒通20mL/min的培养参数条件下进行培养。
(4)转染24小时后，降温至31℃，并添加适量的丁酸钠；转染后第1、3、5天，分别添加5％的补料(Feed)，糖浓度控制在3-6g/L范围。
(5)转染后细胞活率低于80％，或第6天，收集上清液进行蛋白定量与下一步纯化。
按照上述所示步骤，使用亲本株疫苗(V-01)表达载体进行了转染混合物的制备后，采用市售手动直接添加与本公开反应器补液装置添加的方式，分别进行了瞬时转染。表达培养过程中细胞活率与活细胞密度的变化趋势相当，始终能保持较高的细胞活率与细胞密度(图4a)。本公开采用的生物反应器添加模式下，亲本株疫苗(V-01)表达量显著提升，对比传统市售模式可提升产量近2倍以上(图4b)。本公开提供的生物反应器瞬时表达，生产细胞能持续保持较高活率与表达产率，相比传统市售转染工艺，可显著提升瞬转表达量。本公开采用的瞬时表达系统适合于较大分子的瞬时表达生产，减少了早期样品制备的难度。通过生物反应器精密控制反应条件，基本与后期商业化规模生产控制一致，使瞬转产物质量更具代表性。
通过本公开提供的大体积瞬时表达系统，对亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)进行了瞬转表达。采用相同表达工艺、相同信号肽与表达载体条件下，亲本株疫苗表达量最高，随后依次为英国株疫苗与南非株疫苗(图5a)，表达量差异可能为不同点突变所致，尤其南非株疫苗增加了K417T与E484K突变，表达水平下降明显。
1.3蛋白纯化
亲和层析捕获
细胞培养液经澄清后的样品直接上Protein A亲和层析柱进行捕获，获得纯化的融合蛋白。经还原SDS-PAGE检定分析，结果如图5b所示，亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)纯度均大于95％。
实施例2：亲和力测定
使用分子相互作用仪(SPR法)对亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)的亲和力进行检测，包括RBD结构域与ACE2受体的亲和力、以及IFNα结构域对IFNAR2受体的亲和力。具体如下：
2.1与ACE2亲和力测定
研究方法：使用HBS-EP+(货号BR100826，购自GE Healthcare)作为实验缓冲液，每个循环包括捕获不同疫苗、不同浓度ACE2蛋白的进样及再生。将实施例1制备的突变株(南非、英国)、亲本株(V-01)疫苗分别稀释至1μg/mL后，以10μL/min的流速注入2通道40s，使其通过Protein A捕获在此通道上，1通道作为空白参考通道。使用High Performance模型，将ACE2(200、100、50、20、12.5、0nM)按浓度梯度依次以30μL/min的流速注入芯片1、2通道， 结合时间150s，解离时间300s。将10mM甘氨酸(pH 1.5)以10μL/min的流速分别注入30s，对芯片进行再生。仪器设定温度为25℃。使用Biacore T200分析软件(Version:1.0,General Electric Company)对数据进行分析，1通道作为空白参考通道，扣除背景信号后分析各样品的结合，分析所用的模型为1:1binding。
结果与结论：结果见表1和图6a-6c，亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)均可与ACE2结合，亲和力分别为1.05E-08M、1.65E-08M和7.41E-09M，与ACE2的亲和力基本一致。
表1亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)与ACE2亲和力结果
批号 ka(1/Ms) kd(1/s) KD(M)
亲本株疫苗(V-01) 3.03E+04 3.17E-04 1.05E-08
英国株疫苗 2.31E+04 3.81E-04 1.65E-08
南非株疫苗 4.59E+04 3.40E-04 7.41E-09
2.2与IFNAR2亲和力测定
研究方法：使用HBS-EP+作为实验缓冲液，每个循环包括捕获疫苗、不同浓度IFNAR2蛋白的进样及再生。将实施例1制备的突变株(南非、英国)、亲本株(V-01)疫苗分别稀释至4μg/mL后，以10μL/min的流速注入4通道40s，使其通过Protein A捕获在此通道上，3通道作为空白参考通道。使用High Performance模型，将IFNAR2(100、50、20、12.5、6.25、3.125、0nM)按浓度梯度依次以30μL/min的流速注入芯片3、4通道，结合时间100s，解离时间150s。将10mM甘氨酸(pH1.5)以30μL/min的流速分别注入30s，对芯片进行再生。仪器设定温度为25℃。使用Biacore T200分析软件(Version:1.0,General Electric Company)对数据进行分析，3通道作为空白参考通道，扣除背景信号后分析各样品的结合，分析所用的模型为1:1binding。
结果与结论：结果见表2和图6d-6f，亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)均可与IFNAR2蛋白结合，亲和力分别为1.03E-07M、8.64E-08M和2.95E-07M，与IFNAR2的亲和力基本一致。
表2亲本株(V-01)疫苗、突变株疫苗(英国株、南非株)与IFNAR2亲和力结果
样品 ka(1/Ms) kd(1/s) KD(M)
亲本株疫苗(V-01) 1.61E+05 1.65E-02 1.03E-07
英国株疫苗 2.26E+05 1.96E-02 8.64E-08
南非株疫苗 6.14E+04 1.81E-02 2.95E-07
实施例3：亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)的生物学活性
亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)结构中的IFNα-2b与细胞膜上内源受体IFNAR2和IFNAR1结合后，可以通过信号转导激活干扰素刺激反应元件，启动萤光素酶 的表达，表达量与干扰素的生物学活性成正相关，加入细胞裂解液和萤光素酶底物后，测定其发光强度，以此测定其生物学活性。因此，使用干扰素重组细胞(报告基因法)对实施例1制备的突变株(南非、英国)疫苗与亲本株疫苗(V-01)结构中IFNα结构域的细胞活性进行检测。具体如下：
研究方法：用测定培养液(含1％GlutaMax，10％FBS的DMEM)将亲本株疫苗(V-01)与突变株疫苗(英国株、南非株)稀释至12μg/mL(2×，终浓度为6μg/mL)，加至稀释板第2列作为起始浓度，按照第3-7列约3.5倍、8-12列约6倍梯度稀释至第11个浓度梯度。浓度依次为12、3.43、0.98、0.28、0.080、0.023、0.0065、0.0011、0.00018、0.000030、0.0000050μg/mL。收集HEK-Lucia TM Null重组细胞(货号：hkl-null，购自：InvivoGen)，用测定培养液调整细胞密度至8×10 5个/mL，按50μL/孔加入到96孔全白细胞板中，即细胞4×10 4个/孔，然后以50μL/孔分别加入各稀释梯度的样品，各设3复孔。设置不加药阴性对照(NC)和测定培养液空白对照(Blank)。于37℃、5％CO 2培养箱培养18h～24h。反应结束后取出培养板平衡至室温，以100μL/孔加入Bio-Glo萤光素酶试剂，200～500rpm室温震荡避光反应10min～30min。酶标仪检测化学发光单位RLU值，利用Softmax软件进行四参数拟合分析。并计算EC 50。
结果与结论：结果见表3和图7，亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)的IFNα结构域均能够激活IFNα信号通路，具备预期细胞生物学活性，三者的EC 50分别为15.1ng/mL、12.4ng/mL和12.8ng/mL。
表3亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)IFNα结构域的生物学活性
样品 EC 50(ng/mL)
亲本株疫苗(V-01) 15.1
英国株疫苗 12.4
南非株疫苗 12.8
实施例4：小鼠体内效力评估-滴度
疫苗的动物体内效力评估至关重要，可以直接体现疫苗产生的免疫原性，与其产生的保护力直接相关。因此，采用C57BL/6小鼠对亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)的体内效力进行评估。
研究方法：将实施例1制备的亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)疫苗免疫6～8周龄C57BL/6小鼠，疫苗浓度10μg/mL，每只小鼠每次大腿肌肉注射0.1mL，10只/组，每只小鼠共免疫2次(第0天与第14天各一次)，初免28天(即2免14天)后眼眶采血取血清，血液室温静置待凝固后，4000rpm，2～8℃，离心10min，取上清。
采用酶联免疫吸附法测定抗RBD抗体几何平均滴度(GMT)：使用PBS将各疫苗对应的RBD蛋白(包括野生型RBD、南非突变RBD、英国突变RBD)稀释至1μg/mL，100μL/孔，过 夜包被，PBST洗涤2次后，再使用1％脱脂奶粉或BSA封闭。使用PBST将血清稀释1000倍，再使用96孔板进行2倍梯度稀释，共12个梯度。将稀释好的血清样品加入提前完成RBD蛋白包被和封闭的酶标板中，100μL/孔，200rpm震荡孵育2h。PBST洗涤4次后，加入约1:20000的HRP标记的羊抗鼠IgG二抗，200rpm震荡孵育1h。PBST洗涤4次后，加入100μL/孔的TMB显色液显色10min。使用0.2M H 2SO 4终止后，读取450nm和620nm处吸光值。免疫血清样本的滴度为信号值大于Cut off值的最大稀释倍数，并计算结合效价的几何平均滴度。
结果与结论：结果见表4和图8，亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)均能引起较强的免疫原性，且亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)的平均滴度相当。
表4亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)小鼠体内效力
样品 野生型RBD 英国突变RBD 南非突变RBD
亲本株疫苗(V-01) 406375 322540 237024
英国株疫苗 406375 512000 203187
南非株疫苗 376252 298631 348362
实施例5：假病毒中和效价-保护力评估
疫苗免疫动物后产生特异性的抗体，但该抗体是否能够保护机体不被病毒攻击，还需要进行免疫后血清对于病毒攻击的保护力进行评估。由于新冠病毒的危险性，用真病毒进行攻毒评估非常困难。因此，采用重组的新冠病毒S蛋白、以VSV G为骨架构建包装、并且携带萤光素酶报告基因的假病毒对实施例1制备的亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)的假病毒保护力进行评估。
研究方法：血清样本提前采用56℃水浴灭活30min。取抗RBD小鼠中和抗体，先用检测培养液(10％FBS DMEM)稀释至25μg/mL，作为阳性质控品(PC)。取待测血清样本，在96孔全白细胞板中稀释至首孔浓度为5％(即1:20)，然后与阳性质控品一起按照1:3进行倍比稀释，共8个稀释梯度，各稀释梯度的血清样本为100μL/孔。提前在4℃融化假病毒，用检测培养液将各疫苗对应的假病毒(包括野生型假病毒、南非突变假病毒、英国突变假病毒)稀释至20000TCID 50/mL，在血清稀释板中加入50μL/孔假病毒稀释液，假病毒量即为1000TCID 50/孔，血清初始稀释度即为1:30。同时设置假病毒对照(VC，不含血清)以及细胞对照(CC，不含血清和假病毒)，然后将全白细胞板置于37℃、5％CO 2培养箱孵育1～2h。收集HEK293T-ACE2细胞，用检测培养液重悬计数，并调整细胞浓度至2.5×10 5个/mL，按照100μL/孔加入细胞，即每孔细胞为2.5×10 4个。将全白细胞板置于37℃，5％CO 2培养箱培养20～28h。检测前先吸弃150μL/孔上清，然后按100μL/孔加入萤光素酶检测试剂，并用多道移液器将反应孔中的液体反复吹吸6～8次，室温避光反应5min。酶标仪读取化学发光单位RLU值，按照如下公式进行抑制率计算：抑制率(％)＝[1－(样品组RLU值－细胞对照CC均值)/(假病 毒对照VC均值－细胞对照CC均值)]×100％。
将抑制率数据结果导入软件，横坐标为倍比稀释倍数的对数值(Log Titer)，纵坐标为抑制率百分比(％Inhibition)，进行四参数拟合分析，软件自动计算pNT 50值，取整即为假病毒中和效价。
结果与结论：结果见表5和图9，亲本株疫苗(V-01)免疫后血清能够中和野生型假病毒、英国突变假病毒和南非突变假病毒，其pNT 50分别为1977、1131和418，相比较野生型假病毒来说，其对英国突变假病毒和南非突变假病毒的中和效果分别下降1.7倍和4.7倍，对南非突变假病毒的中和效果有大幅降低，差异显著。
英国株疫苗免疫后血清亦能够中和野生型假病毒、英国突变假病毒和南非突变假病毒，其pNT 50分别为846、1535、217，其对野生型假病毒和英国突变假病毒的中和效果好于南非突变假病毒。相比亲本株疫苗(V-01)，英国株疫苗的设计，可以提高其对于英国突变的保护力约1.8倍，且有显著差异，但对南非突变的保护力略差。
南非株疫苗免疫后血清能够中和野生型假病毒、英国突变假病毒和南非突变假病毒，其pNT 50分别为279、496、1136，其对南非突变假病毒的中和效果明显高于亲本株疫苗(V-01)，提高约4.1倍，且差异极显著。因此南非株疫苗的设计，大幅提高了对于南非突变的保护力。
表5亲本株疫苗(V-01)、突变株疫苗(英国株、南非株)假病毒中和效价
样品 野生型假病毒 英国突变假病毒 南非突变假病毒
亲本株疫苗(V-01) 1977 1131 418
英国株疫苗 846 1535 217
南非株疫苗 279 496 1136

Claims (15)

  1. 一种融合蛋白，其包含：
    (1)干扰素或其功能片段；
    (2)新型冠状病毒SARS-CoV-2或其功能片段；和
    (3)免疫球蛋白Fc区；
    优选地，所述融合蛋白从N-末端至C-末端包含：干扰素或其功能片段、SARS-CoV-2或其功能片段和免疫球蛋白Fc区；
    优选地，所述融合蛋白从N-末端至C-末端包含：SARS-CoV-2或其功能片段、干扰素或其功能片段和免疫球蛋白Fc区。
  2. 权利要求1所述的融合蛋白，其中，所述干扰素选自I型干扰素、II型干扰素和/或III型干扰素；
    优选地，所述干扰素可来自人源或鼠源；
    优选地，所述I型干扰素选自IFN-α、IFN-β、IFN-κ、IFN-δ、IFN-ε、IFN-τ、IFN-ω和IFN-ζ；
    优选地，所述II型干扰素为干扰素γ；
    优选地，所述III型干扰素选自IFN-λ1(IL-29)、IFN-λ2(IL-28a)和IFN-λ(IL-28b)；
    优选地，所述干扰素选自人IFN-α1、IFN-α2、IFN-α4、IFN-α5、IFN-α6、IFN-α7、IFN-α8、IFN-α10、IFN-α13、IFN-α14、IFN-α16、IFN-α17和IFN-α21；
    更优选地，所述干扰素为IFN-α2a；优选地，所述IFN-α2a的氨基酸序列包含与SEQ ID NO:1所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；更优选地，所述IFN-α2a的氨基酸序列如SEQ ID NO:1所示。
  3. 根据权利要求1或2所述融合蛋白，其中，所述SARS-CoV-2为突变型SARS-CoV-2；
    优选地，所述突变型SARS-CoV-2为英国突变株B.1.1.7/501Y.V1或南非突变株501Y.V2；
    优选地，所述SARS-CoV-2的功能片段为其受体结合结构域RBD；
    优选地，所述RBD包含与SEQ ID NO:2所示的氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；更优选地，所述RBD的氨基酸序列如SEQ ID NO:2所示；
    优选地，所述RBD包含与SEQ ID NO:3所示的氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；更优选地，所述RBD的氨基酸序列如SEQ ID NO:3所示。
  4. 根据权利要求1-3任一项所述融合蛋白，其中，所述免疫球蛋白Fc区选自IgG1、IgG2、 IgG3和/或IgG4的恒定区氨基酸序列；
    优选地，所述免疫球蛋白Fc区为IgG1的Fc区；优选地，所述IgG1 Fc区包含与SEQ ID NO:4所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；更优选地，所述IgG1 Fc区的氨基酸序列如SEQ ID NO:4所示。
  5. 根据权利要求1-4任一项所述融合蛋白，其中，所述融合蛋白还包含一个或多个Th细胞辅助表位和/或连接片段；
    优选地，所述Th细胞辅助表位为PADRE或其衍生物；所述PADRE或其衍生物的氨基酸序列选自SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10；
    优选地，所述连接片段为柔多肽序列；优选地，所述柔性肽的氨基酸序列选自SEQ ID NO:11和SEQ ID NO:12。
  6. 根据权利要求1-5任一项所述融合蛋白，其中，所述融合蛋白包含与选自SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；优选地，所述融合蛋白的氨基酸序列选自SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的氨基酸序列；更优选地，所述融合蛋白的氨基酸序列为SEQ ID NO:14或SEQ ID NO:15；更优选地，所述融合蛋白的氨基酸序列为SEQ ID NO:15。
  7. 编码权利要求1-6任一项所述的融合蛋白的核酸；优选地，所述核酸包含选自与SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示核苷酸序列具有80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；优选地，所述核酸选自SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的核酸；更优选地，所述核酸为SEQ ID NO:17或SEQ ID NO:18所示的核酸。
  8. 一种包含权利要求7所述的核酸的载体。
  9. 一种表达权利要求1-6任一项所述的融合蛋白、包含权利要求7所述的核酸和/或包含权利要求8所述的载体的宿主细胞；
    优选地，所述宿主细胞是原核细胞或真核细胞；
    优选地，所述原核细胞是细菌细胞；优选地，所述原核细胞是大肠杆菌细胞；
    优选地，所述真核细胞选自酵母细胞、昆虫细胞和哺乳动物细胞；优选地，所述哺乳动物细胞选自CHO、HEK293、SP2/0、BHK、C127等；更优选地，所述真核细胞为CHO细胞。
  10. 一种治疗和/或预防新型冠状病毒SARS-CoV-2感染或新型冠状病毒疾病COVID-19的疫苗，其包含权利要求1-6任一项所述的融合蛋白、权利要求7所述的核酸、权利要求8 所述的载体和/或权利要求9所述的宿主细胞，以及任选地，药学上可接受的载体和/或赋形剂。
  11. 根据权利要求10所述的疫苗，所述的疫苗形式为重组蛋白亚单位疫苗、重组蛋白mRNA疫苗或重组蛋白腺病毒载体疫苗。
  12. 一种预防和/或治疗新型冠状病毒SARS-CoV-2感染或冠状病毒疾病COVID-19的疫苗的制备方法，所述方法包括表达如权利要求1-6任一项所述的融合蛋白。
  13. 一种权利要求1-6任一项所述的融合蛋白、权利要求7所述的核酸、权利要求8所述的载体、权利要求9所述的宿主细胞和/或权利要求10或11所述的疫苗在制备预防和/或治疗新型冠状病毒SARS-CoV-2感染和/或新型冠状病毒疾病COVID-19的药物或产品中的用途。
  14. 一种预防和/或治疗新型冠状病毒SARS-CoV-2感染和/或新型冠状病毒疾病COVID-19的方法，所述方法包括向受试者施用有效量的权利要求1-6任一项所述的融合蛋白、权利要求7所述的核酸、权利要求8所述的载体、权利要求9所述的宿主细胞和/或权利要求10或11所述的疫苗。
  15. 一种诱导个体中和抗原特异性免疫应答的方法，所述方法包括向受试者施用权利要求1-6任一项所述的融合蛋白、权利要求7所述的核酸、权利要求8所述的载体、权利要求9所述的宿主细胞和/或权利要求10或11所述的疫苗。
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