WO2023036191A1 - 新型冠状病毒SARS-CoV-2的Delta变异株疫苗与应用 - Google Patents

新型冠状病毒SARS-CoV-2的Delta变异株疫苗与应用 Download PDF

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WO2023036191A1
WO2023036191A1 PCT/CN2022/117601 CN2022117601W WO2023036191A1 WO 2023036191 A1 WO2023036191 A1 WO 2023036191A1 CN 2022117601 W CN2022117601 W CN 2022117601W WO 2023036191 A1 WO2023036191 A1 WO 2023036191A1
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vaccine
cov
sars
mutant
fusion protein
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French (fr)
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林晶晶
陈曦
孙艺飞
胡振湘
杨嘉明
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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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • 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
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the disclosure belongs to the field of biotechnology, and in particular relates to a Delta variant vaccine of the novel coronavirus SARS-CoV-2 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 there have been four rounds of large-scale outbreaks around the world.
  • the prevalence of SARS-CoV-2 mutants is related to the rapid increase in the number of cases.
  • the above four rounds of large-scale outbreaks were the first round of outbreaks caused by the wild-type strain, the second round of outbreaks caused by the D614G mutant strain, the third round of outbreaks caused by the Alpha mutant strain (B.1.1.7), and the third round of outbreaks caused by the The fourth round of the outbreak caused by the Delta variant (B.1.617.2).
  • the R0 value of the basic infection number of the Delta mutant strain reached 8.0, far exceeding the 2.5 people of the wild-type strain and other mutant strains, and the viral load of the confirmed patient was 1260 times that of the wild-type strain, which is more likely to cause large-scale transmission and severe illness.
  • the RBD domain of the Delta mutant strain contains the L452R/T478K mutation point, which can significantly enhance the binding ability to the host receptor ACE2 and the immune escape ability.
  • the neutralizing antibodies produced after immunization with commercially available/under-developed COVID-19 vaccines are mainly directed against RBD to block the interaction between RBD and ACE2.
  • Most SARS-CoV-2 mutants acquire mutations in the neutralizing antibody epitope of RBD, which can escape neutralizing antibodies and reduce vaccine efficacy.
  • the latest research has found that the NTD antigen of the S protein is more likely to produce enhanced NTD antibodies, which increases the infectivity of the Delta variant strain, making it easy to infect the Delta variant strain after vaccination with wild-type vaccines.
  • the booster immunization strategy while the total antibody titer increases, the enhanced NTD antibody will also increase. In this regard, only the antigen design of RBD will not induce such enhanced NTD antibodies, which may be a better strategy for vaccine design.
  • COVID-19 vaccines are designed with wild-type full-length S protein antigens, which have better anti-epidemic ability against wild-type or early COVID-19 variants, but have varying degrees of protection against the current mainstream Alpha and Delta variants drop phenomenon.
  • the Delta mutant strain has the strongest immune escape ability and the highest viral load. Even after the wild-type vaccine is injected, reinfection still occurs repeatedly.
  • the present disclosure provides a fusion protein vaccine comprising interferon, Delta variant antigen and immunoglobulin Fc region, which can enhance immunity to variant new crown antigens through fusion expressed IFN
  • the titer of original and 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 a mutant SARS-CoV-2 polypeptide comprising:
  • the disclosure provides a vaccine combination for the treatment or prevention of a disease or condition associated with SARS-CoV-2, the vaccine combination comprising:
  • parent strain vaccine comprises the fusion protein of wild-type SARS-CoV-2 polypeptide, and described wild-type SARS-CoV-2 fusion protein comprises: (1) interferon or its functional fragment; (2) ) RBD of the S protein of wild-type SARS-CoV-2 or a functional fragment thereof; and (3) immunoglobulin Fc region; and
  • mutant vaccine comprising the fusion protein comprising a mutant SARS-CoV-2 polypeptide.
  • the present disclosure provides a use of a parental strain vaccine and a mutant strain vaccine in the preparation of a vaccine combination product for treating or preventing a disease or condition related to SARS-CoV-2, the vaccine combination product comprising:
  • parent strain vaccine comprises the fusion protein of wild-type SARS-CoV-2, and described wild-type SARS-CoV-2 fusion protein comprises: (1) interferon or its functional fragment; (2) RBD of the S protein of wild-type SARS-CoV-2 or a functional fragment thereof; and (3) an immunoglobulin Fc region; and
  • mutant vaccine comprising the fusion protein comprising a mutant SARS-CoV-2 polypeptide.
  • the present disclosure provides a method of inducing an immune response against SARS-CoV-2 in a subject in need thereof, the method comprising:
  • the parental strain vaccine comprising a fusion protein of wild-type SARS-CoV-2, the wild-type SARS-CoV-2 fusion protein comprising: (1) interferon or its function Fragments; (2) RBD of the S protein of wild-type SARS-CoV-2 or functional fragments thereof; and (3) immunoglobulin Fc region; and
  • one of the parent strain vaccine or the mutant strain vaccine is administered as a priming vaccine, and one of the parent strain vaccine or the mutant strain vaccine is administered as a booster vaccine, and the parent strain vaccine and the mutant strain vaccine are administered as a booster vaccine. Both mutant vaccines were administered.
  • the present disclosure provides a nucleic acid encoding the fusion protein or the vaccine combination.
  • the present disclosure provides an expression vector comprising the nucleic acid.
  • the present disclosure provides a host cell expressing the fusion protein, expressing the vaccine combination product, comprising the nucleic acid and/or comprising the expression vector.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein, the vaccine combination product, the nucleic acid, the expression vector and/or the host cell, and one or more A pharmaceutically acceptable carrier, diluent or excipient.
  • the present disclosure provides a vaccine for treating and/or preventing a disease or condition associated with SARS-CoV-2, comprising said fusion protein, said vaccine combination product, said nucleic acid, said expression carrier, the host cell and/or the pharmaceutical composition, and optionally, a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.
  • the present disclosure provides a preparation method of a vaccine for preventing and/or treating a disease or condition related to SARS-CoV-2, said method comprising expressing said fusion protein or said vaccine combination product.
  • the present disclosure provides the fusion protein, the vaccine combination product, the nucleic acid, the expression vector, the host cell, the pharmaceutical composition, the vaccine in the preparation of prevention and/or Or use in a medicament or product for the treatment of a disease or condition associated with SARS-CoV-2.
  • the present disclosure provides a kit comprising the fusion protein, the vaccine combination product, the nucleic acid, the expression vector, the host cell, the pharmaceutical composition and/or the vaccines.
  • kits of parts comprising the following components:
  • parental strain vaccine in combination with a pharmaceutically acceptable adjuvant, diluent or carrier;
  • a mutant vaccine in combination with a pharmaceutically acceptable adjuvant, diluent or carrier;
  • the present disclosure provides a kit or the kit in preparation of a pharmaceutical composition for inducing an immune response against the novel coronavirus SARS-CoV-2 in a subject in need thereof. use.
  • the present disclosure provides a method of preventing and/or treating a disease or condition associated with SARS-CoV-2, the method comprising administering to a subject an effective amount of the fusion protein, the vaccine Combination product, said nucleic acid, said expression vector, said host cell, said pharmaceutical composition, said vaccine, said kit and/or said kit of parts.
  • the present disclosure provides a method of inducing a neutralizing antigen-specific immune response in an individual, the method comprising administering to the subject an effective amount of the fusion protein, the vaccine combination product, the nucleic acid, The expression vector, the host cell, the pharmaceutical composition, the vaccine, the kit and/or the kit.
  • Fig. 1 is a schematic diagram of the molecular structure of the parental strain vaccine (V-01) and the Delta strain vaccine.
  • Fig. 2 is the expression plasmid map of parental strain vaccine (V-01) and Delta strain vaccine.
  • Fig. 2a is the map of the parent strain vaccine (V-01);
  • Fig. 2b is the map of the Delta strain vaccine.
  • Fig. 3 is the identification result of expression plasmid enzymes of parental strain vaccine (V-01) and Delta strain vaccine.
  • M1 DL15000 nucleic acid molecular marker; 1. Parental strain vaccine (V-01) HindIII single enzyme digestion; 2. Parental strain vaccine (V-01) HindIII/PacI double enzyme digestion; 3. Delta strain vaccine HindIII single enzyme 4.Delta strain vaccine HindIII/PacI double enzyme digestion; M2.DL10000 nucleic acid molecule marker.
  • Fig. 4 is the transient expression growth trend of parental strain vaccine (V-01) and Delta strain vaccine. Among them, the left ordinate represents the living cell density; the right ordinate represents the cell viability.
  • Fig. 5 is a graph showing the expression levels of the parent strain vaccine (V-01) and the Delta strain vaccine expressed in Example 1 of the present disclosure (Fig. 5a) and the electrophoresis detection results after purification (Fig. 5b). Among them, M1: 180kDa protein marker.
  • Fig. 6 is an in vitro affinity diagram of parental strain vaccine (V-01), Delta strain vaccine 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 Delta strain vaccine and ACE2 affinity
  • Fig. 6c is the fitting curve of parental strain (V-01) and IFNAR2 affinity Graph
  • Figure 6d is a fitting curve of the affinity between the Delta strain vaccine and IFNAR2.
  • Fig. 7 is a graph showing the biological activity of parental strain vaccine (V-01), Delta strain vaccine and IFNR determined based on the indicator cell method.
  • Figure 8 is the in vivo efficacy evaluation of parental strain vaccine (V-01) and Delta strain vaccine in mice.
  • Fig. 9 is the evaluation of the neutralizing titer of the parental strain vaccine (V-01) and the Delta strain vaccine for the wild-type pseudovirus and the Delta mutant pseudovirus.
  • Figure 10 is the evaluation of the neutralizing titer of the wild-type pseudovirus, Delta mutant pseudovirus, Beta mutant pseudovirus, and Alpha mutant pseudovirus after the second immunization of the parental strain vaccine (V-01) by adding the Delta strain vaccine.
  • 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.
  • Coronaviridae the genus Coronavirus
  • coronaviruses 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).
  • COVID-19 is a viral illness typically characterized by high fever, cough, difficulty breathing, chills, persistent tremors, muscle pain, headache, sore throat, new loss of taste and/or smell, and Other symptoms of viral pneumonia are characteristic.
  • coagulopathy-related symptoms e.g., blood clotting, thrombosis, acute respiratory distress syndrome, seizures, heart attack, stroke, multiple cerebral infarctions, renal failure, diabetes insipidus and/or disseminated intravascular coagulation.
  • rare inflammatory syndromes are sometimes associated with COVID-19 (eg, atypical Kawasaki syndrome, toxic shock syndrome, pediatric multisystem inflammatory disease, and cytokine storm syndrome).
  • the coronavirus SARS-CoV-2 of the genus ⁇ is a causative agent.
  • fusion protein refers to a natural or synthetic molecule consisting of one or more molecules, wherein two or more peptide or protein (including glycoprotein) based molecules with different specificities are either Selected molecules are fused together via chemical or amino acid-based linker molecules.
  • the linkage can be achieved by C-N fusion or N-C fusion (in 5' ⁇ 3' direction), preferably C-N fusion.
  • Interferon refers to a virus-infected or under the action of other interferon-inducing agents, which are produced under the control of the cell genome and have anti-viral, anti-tumor, and immunomodulatory activities.
  • a class of cytokines with biological activity. 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.
  • antibody or “immunoglobulin” has the broadest meaning and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies composed of at least 2 intact antibodies (e.g. bispecific Antibodies) and antibody fragments, as long as they are 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 (residue 447 according to the EU numbering system) may be removed, eg, 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” or “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 of various animals and present on the cell surface, thereby activating CD4+ T helper cells and exerting immune regulation.
  • the ability of PADRE to induce T cell responses is more than 1000 times that of natural epitopes, so PADRE has some characteristics as an immune adjuvant.
  • 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.
  • disease or “condition” refers to the living or healthy state of a patient or individual that can be treated with the fusion proteins, pharmaceutical compositions or methods provided herein.
  • the term "vaccine” is a purified antigen vaccine or immunogenic composition, a subunit vaccine or immunogenic composition, an inactivated whole virus vaccine or immunogenic composition, or an attenuated virus vaccine or immunogenic composition .
  • the vaccine or immunogenic composition is a purified fusion protein.
  • treating or treatment refers to the successful treatment or improvement of any indicator of an injury, disease, pathology or condition, including any objective or subjective parameter, such as, elimination; remission; alleviation of symptoms Or to make the injury, pathology or condition more tolerable to the patient; to slow the rate of degeneration or decline; or to make the end point of degeneration less decaying; to improve the physical or mental health of the patient.
  • Treatment or amelioration of symptoms may be based on objective or subjective parameters; including results of physical examination, neuropsychiatric examination and/or psychiatric evaluation.
  • the term “treating” and its conjugates may encompass preventing injury, pathology, condition or disease.
  • treatment is prophylaxis. In embodiments, treatment does not include prophylaxis.
  • treating also broadly encompasses the process of obtaining a beneficial or desired outcome (including a clinical outcome) in a subject's condition. any method.
  • Beneficial or desired clinical outcomes may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, lessening of disease extent, stabilization (i.e., not worsening) of disease state, prevention of disease transmission or spread, delay or slowing of disease progression , improvement or remission of disease state, reduction of disease recurrence, and remission (whether partial or total, and whether detectable or undetectable).
  • treatment encompasses any cure, amelioration or prevention of disease. Treatment can prevent a disease from occurring; stop the spread of a disease; relieve the symptoms of a disease, completely or partially remove the underlying cause of the disease, shorten the duration of the disease, or a combination of these things.
  • Treating and treatment includes prophylactic treatment.
  • the methods of treatment comprise administering to a subject a therapeutically effective amount of an active agent.
  • the administering step may consist of a single application, or may comprise a series of applications.
  • the length of the treatment period depends on various factors, such as the severity of the condition, the age of the patient, the concentration of the active agent, the activity of the composition used in the treatment, or a combination thereof.
  • effective dosages of agents used for treatment or prophylaxis may be increased or decreased over the course of a particular treatment or prophylaxis regimen. Variations in dosage may occur and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required.
  • the composition is administered to a subject in an amount sufficient to treat the patient, and for a sufficient duration.
  • prevention refers to reducing the occurrence of disease symptoms in a patient.
  • prophylaxis can be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would otherwise occur in the absence of treatment.
  • patient or “subject in need” refers to a living organism suffering from or susceptible to a disease or condition that can be treated by administering a pharmaceutical composition as provided herein.
  • Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals.
  • the patient is a human.
  • combined administration refers to the “combined administration” of the fusion protein or vaccine of the present disclosure with known drugs (or other compounds, or other vaccines), so that both have therapeutic or diagnostic effects.
  • Such concomitant administration may include concurrent (ie simultaneous), prior or sequential administration of the drug (or other compound, or other vaccine) relative to administration of the fusion protein or vaccine of the present disclosure.
  • One of ordinary skill in the art will readily be able to determine the appropriate timing, sequence and dosage of administration for a particular drug (or other compound, or other vaccine) and combinations of the present disclosure.
  • the term "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 a disease or condition one or more symptoms).
  • An example of an “effective amount” is an amount sufficient to effect treatment, prevention or reduction of one or more symptoms of a disease, which amount may also be referred to as a "therapeutically effective amount”.
  • "Reduction" of one or more symptoms means reducing the severity or frequency of one or more symptoms, or eliminating one or more symptoms.
  • a prophylactically effective amount of a drug is that amount of the drug which, when administered to a subject, will have the desired prophylactic effect, such as preventing or delaying the onset (or recurrence) of an injury, disease, pathology or condition or reducing the severity of an injury, disease, pathology or condition.
  • a complete preventive effect does not necessarily occur by administering a single dose, and may occur after administering only a series of doses.
  • a prophylactically effective amount may be administered in one or more administrations.
  • a therapeutically effective amount refers to an amount of a therapeutic agent as described above sufficient to ameliorate a condition.
  • a therapeutically effective amount will exhibit an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.
  • Treatment efficacy can also be expressed as a "fold" increase or decrease.
  • a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold or more effect relative to a control.
  • Dosage can vary according to the needs of the patient and the fusion protein or vaccine employed.
  • the dosage administered to a patient should be sufficient to produce a beneficial therapeutic response in the patient over time.
  • the size of the dose will also be determined by the presence, nature and extent of any adverse side effects. It is within the skill of the practitioner to determine the appropriate dosage for a particular situation.
  • treatment is initiated with smaller doses that are less than optimal for the fusion protein or vaccine. Thereafter, the dosage is increased in small increments until the optimum effect under the circumstances is reached.
  • the amount and interval of administration can be adjusted individually to provide a level of the fusion protein or vaccine administered that is effective for the particular clinical indication being treated. This will provide treatment options commensurate with the severity of the individual disease state.
  • administration means oral administration, administration in suppository form, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration to a subject, Or implant a slow-release device (eg, mini-osmotic pump). Administration is by any route, including parenteral and transmucosal (eg, buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. In embodiments, administering does not comprise administering any active agent other than those recited.
  • the present disclosure provides a fusion protein comprising:
  • the fusion protein comprises from N-terminus to C-terminus: interferon or its functional fragment, Delta mutant S protein or its functional fragment and immunoglobulin Fc region.
  • the fusion protein comprises from N-terminus to C-terminus: Delta strain S protein or functional fragment thereof, interferon or functional fragment thereof and immunoglobulin Fc region.
  • the disclosure provides a vaccine combination for the treatment or prevention of a disease or condition associated with SARS-CoV-2, the vaccine combination comprising:
  • parent strain vaccine comprises the fusion protein of wild-type SARS-CoV-2 polypeptide, and described wild-type SARS-CoV-2 fusion protein comprises: (1) interferon or its functional fragment; (2) ) RBD of the S protein of wild-type SARS-CoV-2 or a functional fragment thereof; and (3) immunoglobulin Fc region; and
  • mutant vaccine comprising said fusion protein comprising a mutant SARS-CoV-2 polypeptide
  • one of the parent strain vaccine or the mutant strain vaccine is administered as a priming vaccine, and one of the parent strain vaccine or the mutant strain vaccine is administered as a booster vaccine, and the parent strain vaccine and the mutant strain vaccine are administered as a booster vaccine. Both mutant vaccines were administered.
  • the present disclosure provides the use of the parental strain vaccine and the mutant strain vaccine in the preparation of a vaccine combination product for the treatment or prevention of a disease or condition associated with SARS-CoV-2, the vaccine combination product comprising:
  • parent strain vaccine comprises the fusion protein of wild-type SARS-CoV-2, and described wild-type SARS-CoV-2 fusion protein comprises: (1) interferon or its functional fragment; (2) RBD of the S protein of wild-type SARS-CoV-2 or a functional fragment thereof; and (3) an immunoglobulin Fc region; and
  • mutant vaccine comprising said fusion protein comprising a mutant SARS-CoV-2 polypeptide
  • one of the parent strain vaccine or the mutant strain vaccine is administered as a priming vaccine
  • one of the parent strain vaccine or the mutant strain vaccine is administered as a booster vaccine
  • the parent strain vaccine and the Both mutant vaccines were administered.
  • the present disclosure provides a method of inducing an immune response against SARS-CoV-2 in a subject in need thereof, the method comprising:
  • the parental strain vaccine comprising a fusion protein of wild-type SARS-CoV-2, the wild-type SARS-CoV-2 fusion protein comprising: (1) interferon or its function Fragments; (2) RBD of the S protein of wild-type SARS-CoV-2 or functional fragments thereof; and (3) immunoglobulin Fc region; and
  • mutant vaccine comprising the fusion protein comprising the mutant SARS-CoV-2 polypeptide
  • one of the parent strain vaccine or the mutant strain vaccine is administered as a priming vaccine
  • one of the parent strain vaccine or the mutant strain vaccine is administered as a booster vaccine
  • the parent strain vaccine and the Both mutant vaccines were administered.
  • the subject is a mammal or an avian.
  • the subject is a human, bovine, canine, feline, goat, sheep, pig, horse, turkey, duck, or chicken.
  • 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 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 IFN- The amino acid sequence of ⁇ -2a is shown in SEQ ID NO:1.
  • the functional fragment of the S protein of the Delta mutant is its receptor binding domain RBD.
  • the RBD of the S protein of the wild-type SARS-CoV-2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2, preferably having 85%, 90% , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; more preferably, the wild-type SARS-CoV-2
  • the amino acid sequence of the RBD of the S protein is shown in SEQ ID NO:2.
  • the RBD of the Delta mutant S protein 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%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; 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 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 IgG1
  • the amino acid sequence of the Fc region is shown in SEQ ID NO:4.
  • 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 fusion protein comprising a mutant SARS-CoV-2 polypeptide comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 14, preferably having 85%, 90%, Amino acid sequences with 95%, 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more 99% identity; preferably, the polypeptide comprising mutant SARS-CoV-2
  • the fusion protein of is SEQ ID NO:14.
  • the fusion protein comprising wild-type SARS-CoV-2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 13, preferably having 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; preferably, the wild-type SARS-CoV-2 polypeptide comprising The fusion protein is SEQ ID NO: 13.
  • the present disclosure provides a nucleic acid encoding the fusion protein or the vaccine combination product.
  • the nucleic acid is mRNA.
  • the nucleic acid encoding the fusion protein comprising the wild-type SARS-CoV-2 polypeptide comprises a nucleotide sequence having 80% or more identity with the nucleotide sequence shown in SEQ ID NO: 15, preferably A sequence of nucleotides having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or more identity of 99% or more; the wild The coding nucleic acid of the fusion protein of type SARS-CoV-2 is the nucleic acid shown in SEQ ID NO:15.
  • the nucleic acid encoding the fusion protein comprising a mutant SARS-CoV-2 polypeptide comprises a nucleotide sequence with 80% or more identity to the nucleotide sequence shown in SEQ ID NO: 16, 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 the nucleic acid shown in SEQ ID NO:16.
  • the present disclosure provides an expression vector comprising the nucleic acid.
  • the expression vector is selected from a plasmid, cosmid, viral vector, RNA vector, or linear or circular DNA or RNA molecule.
  • the plasmid is selected from pCI, puc57, pcDNA3, pSG5, pJ603, and pCMV.
  • the viral vector is selected from the group consisting of adenoviruses, retroviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (eg, rabies and vesicular stomatitis viruses), paramyxoviruses (eg, Machi and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses.
  • orthomyxoviruses e.g., influenza viruses
  • rhabdoviruses eg, rabies and vesicular stomatitis viruses
  • paramyxoviruses eg, Machi and Sendai
  • positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses.
  • the adenovirus vector is selected from human, chimpanzee or rhesus monkey adenovirus; preferably, the adenovirus is selected from serotypes Ad2, Ad4, Ad5, Ad6, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 and Pan9 adenoviruses.
  • Nucleic acid molecules can also be inserted into expression vectors, such as adenoviral vectors, and incorporated into compositions of the present disclosure.
  • adenovirus vector and “adenoviral vector” and “adenoviral particle” are used interchangeably and refer to a genetically engineered adenovirus designed to incorporate a polynucleotide of interest (for example, polynucleotides encoding ZIKVM and Env antigens) are inserted into eukaryotic cells such that the polynucleotides are subsequently expressed.
  • adenoviruses useful as viral vectors of the present disclosure include those having or derived from serotypes Ad2, Ad4, Ad5, Ad6, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (e.g., RhAd52) and Pan9 (also known as AdC68); these vectors can be derived, for example, from humans, chimpanzee (e.g., ChAd1, ChAd3, ChAd7, ChAd8, ChAd21, ChAd22, ChAd23, ChAd24, ChAd25, ChAd26, ChAd27.1, ChAd28.1, ChAd29, ChAd30, ChAd31.1, ChAd32, ChAd33, ChAd34, ChAd35.1, ChAd36, ChAd37.2, ChAd39, ChAd40.1, ChAd41.1, ChAd42.1, ChAd43, ChAd44
  • the retrovirus is selected from the group consisting of avian leukogenic-sarcoma, mammalian C-type, B-type virus, D-type virus, HTLV-BLV pool, lentivirus, and foamy virus.
  • the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, and ovine demyelinating leukoencephalitis lentivirus.
  • the double-stranded DNA viruses include adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus) and poxviruses (e.g., vaccinia virus, chicken poxvirus and canarypox virus), Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, baculovirus, and hepatitis virus.
  • herpesviruses e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus
  • poxviruses e.g., vaccinia virus, chicken poxvirus and canarypox virus
  • Norwalk virus togavirus
  • flavivirus flavivirus
  • reovirus reovirus
  • papovavirus papovavirus
  • hepadnavirus hepadnavirus
  • baculovirus
  • the present disclosure provides a host cell expressing the fusion protein, expressing the vaccine combination product, comprising the nucleic acid and/or comprising the expression vector.
  • the host cell is a prokaryotic or eukaryotic cell.
  • the prokaryotic cells are bacterial cells; preferably, the prokaryotic cells are E. coli cells.
  • the eukaryotic cells are mutant yeast cells, insect cells and mammalian cells; preferably, the mammalian cells are selected from CHO, HEK293, SP2/0, BHK, C127, etc.; more preferably, the The eukaryotic cells described above are CHO cells.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein, the vaccine combination product, the nucleic acid, the expression vector and/or the host cell, and Optionally one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the present disclosure provides a vaccine for treating and/or preventing diseases or conditions related to SARS-CoV-2, which comprises the fusion protein, the vaccine combination product, the nucleic acid, The expression vector, the host cell and/or the pharmaceutical composition, and optionally a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.
  • the parental strain vaccine and/or the mutant strain vaccine independently further comprise one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants.
  • the adjuvant is selected from aluminum hydroxide, aluminum phosphate, saponins such as Quil A, QS-21, GPI-0100, water-in-oil emulsions, oil-in-water emulsions, oil-in-water Aqueous emulsion.
  • the parental strain vaccine and/or the mutant strain vaccine is in the form of a liquid, emulsion, solid, aerosol, mist or gas.
  • the mutant strain vaccine is a priming vaccine and the parental strain vaccine is a boosting vaccine.
  • the parental strain vaccine is a prime vaccine and the mutant strain vaccine is a booster vaccine.
  • 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 present disclosure provides a method for preparing a vaccine for preventing and/or treating a disease or condition related to SARS-CoV-2, said method comprising expressing said fusion protein or said vaccine combination product .
  • the disease or condition associated with SARS-CoV-2 is SARS-CoV-2 infection or COVID-19.
  • the parental strain vaccine and the mutant strain vaccine may be presented in combination kits.
  • the term "combination kit” or “kit of parts” as used herein refers to one or more pharmaceutical compositions for administering the parental strain vaccine and the mutant strain vaccine according to the present disclosure.
  • the combination kit comprises the parental strain vaccine and the mutant strain vaccine in separate pharmaceutical compositions, wherein the parental strain vaccine and the mutant strain vaccine are in separate packaged individual pharmaceutical compositions.
  • the kit includes the following components:
  • parental strain vaccine in combination with a pharmaceutically acceptable adjuvant, diluent or carrier;
  • a mutant vaccine in combination with a pharmaceutically acceptable adjuvant, diluent or carrier;
  • the kit of parts includes:
  • the kit or combination kit can also be in accordance with instructions, such as dosage and administration instructions.
  • dosage and administration instructions may be of the type given to physicians, such as on drug labels, or may be of the type provided by physicians, such as instructions to patients.
  • compositions or vaccines may be presented in separate pharmaceutical combinations or co-formulated in one pharmaceutical composition. Accordingly, the present disclosure also provides a combination of a plurality of pharmaceutical compositions or vaccines, one of which comprises a parental strain vaccine and one or more pharmaceutically acceptable carriers, diluents or excipients. And a pharmaceutical composition comprising the mutant vaccine and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • compositions or vaccines may be presented in unit dosage form containing a predetermined amount of active ingredient per unit dosage.
  • the amount of active ingredient per dose depends on the condition being treated, the route of administration and the age, weight and condition of the patient, as is known to those skilled in the art.
  • Preferred unit dosage compositions contain a daily dose or sub-dose, or an appropriate fraction thereof, of an active ingredient.
  • such pharmaceutical compositions may be prepared by any methods well known in the art of pharmacy.
  • a pharmaceutical composition or vaccine may be administered by any suitable route.
  • Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural). It is understood that each agent administered may be administered by the same or different routes and that the parental strain vaccine and the mutant strain vaccine are in separate pharmaceutical compositions.
  • parenteral administration of a pharmaceutical composition or vaccine includes any route of administration characterized by a physical wound in a subject's tissue and administration of a pharmaceutical composition or vaccine through a wound in a tissue, resulting generally in direct administration into the muscles, or into the internal organs.
  • Parenteral administration thus includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, application of the composition through a surgical incision, application of the composition by tissue penetration into a non-surgical wound, and the like.
  • parenteral administration contemplated includes, but is not limited to, subcutaneous, intramuscular injection or infusion.
  • Formulations of pharmaceutical compositions or vaccines suitable for parenteral administration generally comprise the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be formulated, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations can be formulated, packaged, or sold in unit dosage form, eg, in ampoules or in multi-dose containers, with a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, pastes, and the like in oily or aqueous vehicles. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing or dispersing agents.
  • the active ingredient is presented in dry (i.e. powder or granules) form for reconstitution with a suitable vehicle (e.g. sterile sterile pyrogen water) refactoring.
  • a suitable vehicle e.g. sterile sterile pyrogen water
  • Parenteral formulations also include aqueous solutions, which may contain excipients such as salts, carbohydrates and buffers (preferably to a pH of 3-9), although for some applications they may be more suitable to be formulated as sterile non-aqueous or dry form for use in combination with a suitable vehicle such as sterile, pyrogen-free water.
  • exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, such as propylene glycol or dextrose in water.
  • parenterally administrable formulations which are useful include those containing the active ingredient in microcrystalline form or in liposomal formulations.
  • Formulations for parenteral administration may be formulated to be immediate and/or sustained release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release.
  • sterile injectable solutions can be prepared by incorporating the pharmaceutical composition or vaccine in the required amount in an appropriate solvent with one or a combination of ingredients listed above, if desired, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active pharmaceutical composition or vaccine into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Proper fluidity of the solution can be maintained, for example, by the use of coatings such as lecithin, in the case of dispersions by maintaining the required particle size and by the use of surfactants.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent delaying absorption, for example monostearate and gelatin.
  • the vaccines of the present disclosure may also be administered intranasally or by inhalation, typically as a dry powder (alone, as a mixture, or as granules of mixed components, e.g. mixed with a suitable pharmaceutically acceptable excipient) from a dry powder inhaler , as an aerosol spray from a pressurized container, pump, injector, nebulizer (preferably using an electrohydrodynamic nebulizer to produce a fine mist) or atomizer, with or without a suitable propellant, or as drops nasal agent.
  • a dry powder alone, as a mixture, or as granules of mixed components, e.g. mixed with a suitable pharmaceutically acceptable excipient
  • nebulizer preferably using an electrohydrodynamic nebulizer to produce a fine mist
  • atomizer preferably using an electrohydrodynamic nebulizer to produce a fine mist
  • Pressurized containers, pumps, injectors, nebulizers or atomizers generally contain solutions or suspensions of the vaccines of the present disclosure containing, for example, suitable agents for dispersing, dissolving or prolonging the release of the active, one or more propellants as solvent.
  • Drug products are generally micronized to a size suitable for delivery by inhalation (typically less than 5 microns) prior to use as a dry powder or suspension formulation. This can be achieved by any suitable comminution method such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.
  • Capsules, blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the disclosed pharmaceutical compositions or vaccines, a suitable powder base and performance modifiers.
  • Suitable solution formulations for use in nebulizers that use electrohydrodynamics to generate a fine mist may contain suitable doses of the disclosed vaccine/prime, and the prime volume may vary, for example, from 1 ⁇ L to 100 ⁇ L.
  • Suitable flavoring agents such as peppermint and levomenthol or sweetening agents such as saccharin or sodium saccharin may be added to the formulations of the present disclosure intended for inhaled/intranasal administration.
  • Formulations for inhaled/intranasal administration may be formulated to be immediate and/or sustained release.
  • Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release.
  • the dosage unit is determined by means of a valve delivering a measured amount.
  • a unit according to the present disclosure is generally arranged to administer a measured amount or "puff" of a vaccine of the present disclosure.
  • the total daily dosage will generally be administered in a single dose or, more usually, as divided doses throughout the day.
  • compositions or vaccines of the present disclosure may also be formulated for oral route administration.
  • Pharmaceutical compositions or vaccines suitable for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or creams; or packets of water. Oil liquid emulsions or water-in-oil liquid emulsions are presented.
  • the active drug ingredient can be combined with an oral, nontoxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • an oral, nontoxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • Powders are prepared by comminuting a pharmaceutical composition or vaccine to a suitable fine particle size and mixing with a similarly comminuted pharmaceutical carrier such as an edible carbohydrate (eg starch or mannitol). Flavoring, preservative, dispersing and coloring agents can also be present.
  • Capsules are prepared by preparing the powder mixture as described above and filling the formed capsule sheaths.
  • Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycols can be added to the powder mixture prior to the filling operation.
  • a disintegrating or solubilizing agent such as agar-agar, calcium carbonate or sodium carbonate may also be added to improve the availability of the drug when the capsule is swallowed.
  • suitable binders, lubricants, disintegrants and colorants can also be granulated, if desired or necessary, and the powder mixture can be passed through a tableting machine, with the result that incompletely formed small pieces are broken into granules.
  • the particles can be incorporated into the mixture with lubrication.
  • Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, wax etc.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or impacting, adding lubricants and disintegrants and compressing into tablets.
  • Powder mixtures are prepared by mixing a suitably comminuted pharmaceutical composition or vaccine with a diluent or base as described above, optionally together with a binder such as carboxymethylcellulose, alginate, gelatin or polyvinylpyrrolidone, a solution barrier Retention agents such as paraffin, resorption enhancers such as quaternary salts and/or absorbents such as bentonite, kaolin or dibasic calcium phosphate.
  • the powder mixture can be granulated by moistening with a binder such as syrup, starch paste, acadia mucilage, or a solution of cellulosic or polymeric material, and forcing through a screen.
  • compositions or vaccines of the present disclosure can also be combined with free-flowing inert carriers and compressed directly into tablets without going through granulation or impact steps.
  • a clear or opaque protective coating consisting of a seal coat of shellac, a coating of sugar or polymeric material and a polishing coat of wax may be provided. Dyestuffs can be added to these coatings to distinguish different unit doses.
  • Oral fluids such as solution, syrups and elixirs can be prepared in unit dosage form so that a given quantity contains a predetermined amount of the vaccine.
  • Syrups are prepared by dissolving the vaccine in a suitably flavored aqueous solution
  • elixirs are prepared using a non-toxic alcoholic vehicle.
  • Suspensions can be formulated by dispersing the vaccine in a nontoxic vehicle.
  • Solubilizers and emulsifiers such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ether, preservatives, flavoring additives such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners can also be added.
  • compositions for oral administration can, if appropriate, be microencapsulated.
  • the compositions can also be prepared for prolonged or sustained release, for example by coating or embedding the particulate material in polymers, waxes and the like.
  • Agents for use in accordance with the present disclosure can also be administered in the form of liposomal delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
  • compositions suitable for transdermal administration may be presented as a self-contained patch intended to maintain prolonged, intimate contact with the epidermis of the recipient.
  • the active ingredient can be delivered from the patch by iontophoresis.
  • compositions adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.
  • compositions suitable for topical administration in the oral cavity include lozenges, lozenges and mouthwashes.
  • compositions adapted for rectal administration may be presented as suppositories or enemas.
  • the present disclosure provides a method of preventing and/or treating a disease or condition associated with SARS-CoV-2, the method comprising administering to a subject an effective amount of the fusion protein, the vaccine Combination product, said nucleic acid, said expression vector, said host cell, said pharmaceutical composition, said vaccine, said kit and/or said kit of parts.
  • the disease or condition associated with SARS-CoV-2 is SARS-CoV-2 infection or COVID-19.
  • the present disclosure provides a method of inducing a neutralizing antigen-specific immune response in an individual, the method comprising administering to the subject the fusion protein, the vaccine combination, the nucleic acid, the expressed The carrier, the host cell, the pharmaceutical composition, the vaccine, the kit and/or the kit.
  • the present disclosure provides a use of a parental strain vaccine and a mutant strain vaccine in the preparation of a vaccine combination product for treating or preventing a disease or condition related to the novel coronavirus SARS-CoV-2.
  • the present disclosure provides a method of inducing an immune response against the novel coronavirus SARS-CoV-2 in a subject in need thereof, the method comprising:
  • the parental strain vaccine and the mutant strain vaccine may be administered separately in a sequential fashion.
  • the mutant strain vaccine is a priming vaccine and the parental strain vaccine is a boosting vaccine.
  • the parental strain vaccine is a prime vaccine and the mutant strain vaccine is a booster vaccine.
  • the booster vaccine is administered at intervals after the initial administration of the priming vaccine for immunization.
  • the booster vaccine is administered for the first time at an interval after the initial administration of the priming vaccine for the first booster immunization, and the booster vaccine is administered again at another interval after the initial administration of the priming vaccine for Carry out a second booster immunization;
  • the parental strain vaccine is a priming vaccine
  • the mutant strain vaccine is a booster vaccine
  • the mutant strain vaccine is given for the first time at an interval after the initial administration of the parental strain vaccine, and , the mutant strain vaccine is re-administered at another interval after the initial administration of the parental strain vaccine.
  • the priming vaccine is administered again at an interval after the initial administration of the priming vaccine for the first booster immunization, and the booster vaccine is first administered at another interval after the initial administration of the priming vaccine for the first time.
  • Carry out a second booster immunization preferably, the parental strain vaccine is a priming vaccine, and the mutant strain vaccine is a booster vaccine, and the parental strain vaccine is given again at intervals after the initial administration of the parental strain vaccine, and , the mutant strain vaccine is administered for the first time at another period of time after the initial administration of the parental strain vaccine.
  • the booster vaccine is administered about 1-10 weeks after the initial administration of the priming vaccine, preferably 2-8, 3-8, 1-3, 2-5, 2-3, 3-5, 5-8 weeks of administration, more preferably 2-8, 2-5, 3-5 weeks of administration.
  • the booster vaccine is first administered about 1-10 weeks after the initial administration of the priming vaccine, preferably 2-8, 3-8, 1-3, 2-5, 2-3, 3-5 , 5-8 weeks for the first time, more preferably 2-8, 2-5, 3-5 weeks for the first time; and about 4-72 weeks after the initial administration of the priming vaccine, give again, preferably 6-52, 6-45 , 6-30, 6-25, 8-52, 8-30, 8-25, 4-12 weeks, more preferably 4-12, 6-25, 8-25 weeks to give again.
  • the priming vaccine is administered again about 1-10 weeks after the initial administration of the priming vaccine, preferably 2-8, 3-8, 1-3, 2-5, 2-3, 3-5 , 5-8 weeks re-administration, more preferably 2-8, 2-5, 3-5 weeks re-administration, the booster vaccine is given for the first time about 4-72 weeks after the initial administration of the priming vaccine, preferably 6-52, 6-45, 6-30, 6-25, 8-52, 8-30, 8-25, 4-12 weeks, more preferably 4-12, 6-25, 8-25 weeks for the first administration.
  • the booster vaccine is first administered about 2-5 weeks after the initial administration of the priming vaccine, and is re-administered about 6-25 weeks after the initial administration of the priming vaccine.
  • the priming vaccine is administered again about 2-5 weeks after the initial administration of the priming vaccine, and the booster vaccine is first administered about 6-25 weeks after the initial administration of the priming vaccine.
  • Dosage forms for administration include, for example, oral preparations (e.g., tablets, capsules, granules, powders, oral solutions, syrups, oral jellies, etc.), oromucosal preparations (e.g., tablets for oral mucosal application) , sprays for oral mucosal application, semi-solid preparations for oral mucosal application, mouthwash, etc.), preparations for injection (such as injections, etc.), nasal sprays or nasal drops, preparations for inhalation (such as: nebulized inhalation, etc.), preparations for rectal application (e.g.
  • oral preparations e.g., tablets, capsules, granules, powders, oral solutions, syrups, oral jellies, etc.
  • oromucosal preparations e.g., tablets for oral mucosal application
  • sprays for oral mucosal application e.g., sprays for oral mucosal application, semi-solid preparations for oral mu
  • suppositories preparations for rectal application, enemas for rectal application, etc.
  • preparations for skin application e.g.: solid preparations, liquids and solutions for skin application, sprays, ointments, creams, gels, patches, etc.
  • skin application e.g.: solid preparations, liquids and solutions for skin application, sprays, ointments, creams, gels, patches, etc.
  • the vaccine is vaccinated by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, nasal drop or aerosol inhalation.
  • the vaccine is administered by injection.
  • the experiment of the present disclosure proves that the vaccine can increase the immunogenicity and neutralizing antibody titer of the mutant new crown antigen through the fusion expressed IFN, ensure the efficient production of neutralizing antibodies, and can significantly improve the defense ability against mutant strains.
  • Embodiment 1 the preparation of fusion protein
  • the mutant vaccine is a fusion protein of IFN ⁇ new coronavirus S protein RBD domain and Fc. It involves genetically fusing the C-terminus of IFN to RBD-Fc via a flexible linker (Gly4Ser)3 (SEQ ID NO: 11)-PADRE-flexible linker GSGSGS (SEQ ID NO: 12) ( Figure 1).
  • Gly4Ser flexible linker
  • GSGSGS SEQ ID NO: 12
  • Figure 1 the parental strain vaccine
  • V-01 the Delta strain vaccine were constructed. The difference between the two lies in the different point mutations of the RBD antigen. All the genes involved in this example were synthesized through whole genes, and then linked to the mammalian cell expression vector pCGS3 (purchased from: Sigma-Aldrich) by double restriction enzymes for expression.
  • Figure 2 shows the parental strain vaccine (V-01) and Delta strain vaccine maps.
  • 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.
  • amino acid sequence of the parental strain vaccine (V-01) is shown in SEQ ID NO: 13, and its coding nucleic acid sequence is shown in SEQ ID NO: 15; the amino acid sequence of the Delta strain vaccine is shown in SEQ ID NO: 14 , its coding nucleic acid sequence is shown in SEQ ID NO:16.
  • this embodiment adopts a large-volume transient transfection scheme for expression.
  • the viable cell density should reach 6 ⁇ 10 6 -8 ⁇ 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 transfection mixture was prepared by using the instruction manual of the ExpiCHO expression system (purchased from: Life technologies, product number: A29133), and configured according to the same ratio. After the transfection mixture is prepared, it is added at a constant speed using a bioreactor liquid replenishment device. The bioreactor is controlled at 37° C., 5% CO 2 , pH 7.0, dissolved oxygen 40%, rotating speed 100 rpm, ventilation volume 0.5 mm pore size, and culture parameters.
  • the cell viability rate is lower than 80%, or on the 6th day, the supernatant is collected for protein quantification and further purification.
  • Transient transfection was carried out by using the expression vectors of the parent strain vaccine (V-01) and the Delta strain vaccine according to the steps shown above.
  • V-01 parent strain vaccine
  • Delta strain vaccine the Delta strain vaccine according to the steps shown above.
  • the transient expression system used in this example 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 Delta strain vaccine were transiently expressed through the large-volume transient expression system shown in this example. Under the conditions of the same expression process, the same signal peptide and expression vector, the protein expression levels of the parental strain vaccine (V-01) and the Delta strain vaccine were comparable ( Figure 5a), and there was no difference in expression level caused by different RBD point mutations.
  • 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 Delta strain vaccine were both greater than 95% by reducing SDS-PAGE analysis.
  • the affinity between the parental strain vaccine (V-01) and the Delta strain vaccine was tested using a molecular interaction instrument (SPR method), including the affinity between the RBD domain of the S protein of the new coronavirus and the ACE2 receptor, and the IFN ⁇ domain’s affinity for the IFNAR2 receptor. body affinity. details as follows:
  • HBS-EP+ product number BR100826, purchased from GE Healthcare
  • each cycle included capture vaccine, injection of different concentrations of ACE2 protein, and regeneration.
  • the Delta strain vaccine prepared in Example 1 and the parental strain (V-01) vaccine were diluted to 1 ⁇ g/mL respectively, they were injected into channel 2 for 40 s at a flow rate of 10 ⁇ L/min, so that they were captured on this channel by Protein A, 1 channel as a blank reference channel.
  • ACE2 200, 100, 50, 20, 12.5, 0 nM
  • the binding time was 150 s
  • the dissociation time was 300 s.
  • 10mM glycine (pH1.5) was injected at a flow rate of 10 ⁇ L/min for 30s to regenerate the chip.
  • the set temperature of the instrument is 25°C.
  • Biacore T200 analysis software (Version: 1.0, General Electric Company) was used to analyze the data, channel 1 was used as a blank reference channel, and the binding of each sample was analyzed after subtracting the background signal.
  • the model used for the analysis was 1:1 binding.
  • HBS-EP+ was used as the experimental buffer, and each cycle included capture vaccine, injection of different concentrations of IFNAR2 protein, and regeneration.
  • the Delta strain vaccine prepared in Example 1 and the parental strain (V-01) vaccine were diluted to 4 ⁇ g/mL respectively, they were injected into channel 4 for 40 s at a flow rate of 10 ⁇ L/min, so that they were captured on this channel by Protein A, 3 channel as a blank reference channel.
  • IFNAR2 100, 50, 20, 12.5, 6.25, 3.125, 0 nM
  • Embodiment 3 the biological activity of parental strain vaccine (V-01) and Delta strain vaccine
  • the IFN ⁇ -2b in the parental strain vaccine (V-01) and the Delta strain vaccine structure binds to the endogenous receptors IFNAR2 and IFNAR1 on the cell membrane, it can activate the interferon-stimulated response element through signal transduction and start the production of luciferase.
  • the expression amount is positively correlated with the biological activity of interferon, after adding cell lysate and luciferase substrate, measure its luminescence intensity, so as to measure its biological activity. Therefore, the cell activity of the IFN ⁇ domain in the structures of the Delta strain vaccine prepared in Example 1 and the parent strain vaccine (V-01) was detected using interferon recombinant cells (reporter gene method). details as follows:
  • Collect HEK-Lucia TM Null recombinant cells (product number: hkl-null, purchased from: InvivoGen), adjust the cell density to 8 ⁇ 10 5 cells/mL with assay culture medium, add 50 ⁇ L/well to a 96-well whole white blood cell plate, That is, 4 ⁇ 10 4 cells/well were added to each dilution gradient sample at 50 ⁇ L/well, 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 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), Delta strain vaccine was evaluated using C57BL/6 mice.
  • the parental strain vaccine (V-01) and the Delta strain vaccine prepared in Example 1 were immunized to 6-8 week-old C57BL/6 mice, the vaccine concentration was 20 ⁇ g/mL, and each mouse was injected with 0.1 mL, 10 rats/group, 14 days later, blood was collected from the orbit to get serum, the blood was allowed to stand at room temperature until coagulated, centrifuged at 4000rpm, 2-8°C for 10min, and the supernatant was taken.
  • GMT geometric mean titer
  • Embodiment 5 Pseudovirus neutralization titer-protection evaluation
  • the parental strain vaccine (V-01) and the Delta strain vaccine prepared in Example 1 were immunized to 6-8 week-old C57BL/6 mice, the vaccine concentration was 20 ⁇ g/mL, and each mouse was injected with 0.1 mL, 10 mice/group, each mouse was immunized twice (one time on the 0th day and one time on the 14th day), the orbital blood was collected 14 days after the second immunization, and the serum was collected. °C, centrifuge for 10 min, and take the supernatant. Serum samples to be tested were inactivated in a 56°C water bath for 30 minutes in advance.
  • a pseudovirus control (VC, without serum) and a cell control (CC, without serum and pseudovirus) were set, and then the whole white blood cell plate was placed in a 37° C., 5% CO 2 incubator for 1-2 hours.
  • Collect HEK293T-ACE2 cells (purchased from Jimon Biotechnology (Shanghai) Co., Ltd., Cat. No. GM-C09233), resuspend and count in the detection medium, and adjust the cell concentration to 2.5 ⁇ 10 5 cells/mL, add 100 ⁇ L/well Cells, that is, 2.5 ⁇ 10 4 cells per well. Place the whole white blood cell plate in a 37°C, 5% CO 2 incubator for 20-28 hours.
  • Inhibition rate (%) [1-(RLU value of the sample group-the mean value of the cell control CC)/(the mean value of the VC of the pseudovirus control-the cell control CC Mean value)] ⁇ 100%.
  • 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 neutralization titer of the pseudovirus.
  • the results are shown in Table 5 and Figure 9.
  • the serum of the parental strain vaccine (V-01) after secondary immunization can effectively neutralize the wild-type pseudovirus, and its neutralization titer is 1394, but for the neutralization titer of the Delta mutant pseudovirus degree decreased significantly.
  • the serum after the second immunization of the Delta strain vaccine can effectively neutralize the Delta mutant pseudovirus, with a neutralization titer of 958, compared with a slightly lower neutralization titer for the wild-type pseudovirus.
  • Embodiment 6 Pseudovirus neutralization titer evaluation after adding immunization Delta strain vaccine
  • the parent strain vaccine (V-01) prepared in Example 1 was used to immunize 6-8 week-old C57BL/6 mice, and the immunization methods of the first and second immunizations were the same as in Example 5.
  • the third immunization was carried out with the Delta strain vaccine.
  • blood was collected from the orbit to obtain serum. 14 days after the second immunization (V01+V01) and 14 days after the third immunization (V01+V01+Delta) were detected for the neutralizing titers of the wild-type pseudovirus, the Delta mutant pseudovirus, the Beta mutant pseudovirus, and the Alpha mutant pseudovirus. Spend.
  • the detection method is the same as in Example 5.
  • Example 7 Two doses of marketed wild-type vaccine plus immunization of Delta strain vaccine plan
  • the protective power of the major vaccines decreased more significantly for the mainstream VOC variants (Alpha, Delta, Beta, Gamma) and the VOI variants that may develop into the main epidemic strains.
  • VOC variants Alpha, Delta, Beta, Gamma
  • VOI variants that may develop into the main epidemic strains.
  • the first group of people who have completed full immunization through emergency use has been more than 12 months old, and a considerable number of people have been immunized for more than 6 months.
  • Booster or sequential immunization is imperative.
  • the Delta strain vaccine is planned to be strengthened on the basis of wild-type vaccines. Immunization research predicts that it can increase the neutralizing antibody titer against most VOC/VOI mutants, and can significantly increase the protection and durability against the new coronavirus in the body.

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Abstract

一种新型冠状病毒SARS-CoV-2的Delta变异株疫苗与应用，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 Delta毒株S蛋白或其功能片段；(3)免疫球蛋白Fc区。所述疫苗能够通过融合表达的IFN提升对变异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。

Description

新型冠状病毒SARS-CoV-2的Delta变异株疫苗与应用 技术领域
本公开属于生物技术领域，具体涉及新型冠状病毒SARS-CoV-2的Delta变异株疫苗与应用。
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，全球范围内已经相继出现了4轮大范围疫情，其流行的SARS-CoV-2突变体与病例数迅速增加有关。上述4轮大范围疫情，分别为野生型毒株引发的第一轮疫情，由D614G变异株引发第二轮疫情，由Alpha变异株(B.1.1.7)引发的第三轮疫情、以及由Delta变异株(B.1.617.2)所引发的第四轮疫情。其中Delta变异株的基本传染数R0值达到8.0人，远超野生型毒株的2.5人及其他变异株，且确诊患者的病毒载量为野生型毒株的1260倍，更易引发大范围的传播及重症。另外，Delta变异株RBD结构域含有L452R/T478K突变点，可显著增强与宿主受体ACE2的结合力和免疫逃逸能力。
市售/在研的新冠疫苗免疫后产生的中和抗体主要针对RBD，以阻断RBD与ACE2之间的相互作用。大多数SARS-CoV-2突变体在RBD的中和抗体表位中获得了突变，可逃逸中和抗体，降低疫苗效力。最新研究发现S蛋白的NTD抗原更易产生增强型NTD抗体，增加了Delta变异株的感染力，导致接种野生型疫苗后依旧容易感染Delta变异株。随着加强针免疫策略的实施，总抗体滴度增加的同时，增强型NTD抗体也会随之增加。对此，仅采用RBD的抗原设计，不会诱导此类增强型NTD抗体，可能是疫苗设计的更优策略。
目前市售/在研新冠疫苗，大多采用野生型全长S蛋白抗原设计，对野生型或早期新冠变异株的防疫能力较好，但对目前主流Alpha及Delta变异株已出现不同程度的保护力下降现象。其中Delta变异株的免疫逃逸能力最强且病毒载量最高，即便是注射了野生型疫苗，依然反复发生重复感染。
因此，急需开发新的针对新型冠状病毒，尤其是变异型新型冠状病毒的疫苗，应对变异株的感染。
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、Delta变异株抗原和免疫球蛋白Fc区的融合蛋白疫苗，所述疫苗能够通过融合表达的IFN提升对变异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，本公开提供了一种包含突变型SARS-CoV-2多肽的融合蛋白，其包含：
(1)干扰素或其功能片段；
(2)SARS-CoV-2 Delta突变株S蛋白或其功能片段；和
(3)免疫球蛋白Fc区。
在另一方面，本公开提供了一种治疗或预防与SARS-CoV-2相关的疾病或病状的疫苗组合产品，所述疫苗组合产品包含：
(i)亲本株疫苗，所述亲本株疫苗包含野生型SARS-CoV-2多肽的融合蛋白，所述野生型SARS-CoV-2融合蛋白包含：(1)干扰素或其功能片段；(2)野生型SARS-CoV-2的S蛋白的RBD或其功能片段；和(3)免疫球蛋白Fc区；和
(ii)突变株疫苗，所述突变株疫苗包含所述包含突变型SARS-CoV-2多肽的融合蛋白。
在另一方面，本公开提供了一种亲本株疫苗和突变株疫苗在制备治疗或预防与SARS-CoV-2相关的疾病或病状的疫苗组合产品中的用途，所述疫苗组合产品包含：
(i)亲本株疫苗，所述亲本株疫苗包含野生型SARS-CoV-2的融合蛋白，所述野生型SARS-CoV-2融合蛋白包含：(1)干扰素或其功能片段；(2)野生型SARS-CoV-2的S蛋白的RBD或其功能片段；和(3)免疫球蛋白Fc区；和
(ii)突变株疫苗，所述突变株疫苗包含所述包含突变型SARS-CoV-2多肽的融合蛋白。
在另一方面，本公开提供了一种在有需要的受试者中诱导针对SARS-CoV-2的免疫应答的方法，所述方法包括：
(i)向受试者施用亲本株疫苗，所述亲本株疫苗包含野生型SARS-CoV-2的融合蛋白，所述野生型SARS-CoV-2融合蛋白包含：(1)干扰素或其功能片段；(2)野生型SARS-CoV-2的S蛋白的RBD或其功能片段；和(3)免疫球蛋白Fc区；和
(ii)向受试者施用突变株疫苗，所述突变株疫苗包含所述包含突变型SARS-CoV-2多肽的融合蛋白。
其中，所述亲本株疫苗或所述突变株疫苗中的一种作为引发疫苗施用，所述亲本株疫苗或所述突变株疫苗中的一种作为加强疫苗施用，并且所述亲本株疫苗和所述突变株疫苗两者均被施用。
在另一方面，本公开提供了一种编码所述融合蛋白或所述疫苗组合产品的核酸。
在另一方面，本公开提供了一种包含所述核酸的表达载体。
在另一方面，本公开提供了一种表达所述融合蛋白、表达所述疫苗组合产品、包含所述核酸和/或包含所述表达载体的宿主细胞。
在另一方面，本公开提供了一种药物组合物，其包含所述融合蛋白、所述疫苗组合产品、所述核酸、所述表达载体和/或所述宿主细胞，以及一种或多种药学上可接受载体、稀释剂或赋形剂。
在另一方面，本公开提供了一种治疗和/或预防与SARS-CoV-2相关的疾病或病状的疫苗，其包 含所述融合蛋白、所述疫苗组合产品、所述核酸、所述表达载体、所述宿主细胞和/或所述药物组合物，以及任选地，药学上可接受的载体、赋形剂、稀释剂或佐剂。
在另一方面，本公开提供了一种预防和/或治疗与SARS-CoV-2相关的疾病或病状的疫苗的制备方法，所述方法包括表达如所述融合蛋白或所述疫苗组合产品。
在另一方面，本公开提供了一种所述融合蛋白、所述疫苗组合产品、所述核酸、所述表达载体、所述宿主细胞、所述药物组合物、所述疫苗在制备预防和/或治疗与SARS-CoV-2相关的疾病或病状的药物或产品中的用途。
在另一方面，本公开提供了一种试剂盒，其包含所述融合蛋白、所述疫苗组合产品、所述核酸、所述表达载体、所述宿主细胞、所述药物组合物和/或所述疫苗。
在另一方面，本公开提供了一种成套药盒，其包括以下组分：
亲本株疫苗，与药学上可接受佐剂、稀释剂或载体联合；
突变株疫苗，与药学上可接受佐剂、稀释剂或载体联合；
其中所述组分以分开施用的形式提供。
在另一方面，本公开提供了一种试剂盒或所述成套药盒在制备用于在有需要的受试者中诱导针对新型冠状病毒SARS-CoV-2的免疫应答的药物组合物中的用途。
在另一方面，本公开提供了一种预防和/或治疗与SARS-CoV-2相关的疾病或病状的方法，所述方法包括向受试者施用有效量的所述融合蛋白、所述疫苗组合产品、所述核酸、所述表达载体、所述宿主细胞、所述药物组合物、所述疫苗、所述剂盒和/或所述成套药盒。
在另一方面，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所述融合蛋白、所述疫苗组合产品、所述核酸、所述表达载体、所述宿主细胞、所述药物组合物、所述疫苗、所述剂盒和/或所述成套药盒。
附图说明
图1为亲本株疫苗(V-01)与Delta毒株疫苗的分子结构示意图。
图2为亲本株疫苗(V-01)与Delta毒株疫苗表达质粒图谱。其中，图2a为亲本株疫苗(V-01)图谱；图2b为Delta毒株疫苗图谱。
图3为亲本株疫苗(V-01)与Delta毒株疫苗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.Delta毒株疫苗HindIII单酶切；4.Delta毒株疫苗HindIII/PacI双酶切；M2.DL10000核酸分子标记。
图4为亲本株疫苗(V-01)与Delta毒株疫苗瞬转表达生长趋势。其中，左纵坐标表示活细胞密度；右纵坐标表示细胞活率。
图5为本公开实施例1所表达亲本株疫苗(V-01)与Delta毒株疫苗表达水平(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)、Delta毒株疫苗与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Delta毒株疫苗与ACE2亲和力拟合曲线图；图6c为亲本株(V-01)与IFNAR2亲和力拟合曲线图；图6d为Delta毒株疫苗与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)、Delta毒株疫苗与IFNR生物学活性图。
图8为亲本株疫苗(V-01)与Delta毒株疫苗小鼠体内效力评估。
图9为亲本株疫苗(V-01)与Delta毒株疫苗对于野生型假病毒、Delta突变假病毒的中和效价评估。
图10为亲本株疫苗(V-01)二免后加免Delta毒株疫苗，对于野生型假病毒、Delta突变假病毒、Beta突变假病毒、Alpha突变假病毒的中和效价评估。
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。
如本文所使用的，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)。
如本文所使用的，术语“COVID-19”是一种病毒性疾病，通常以高烧、咳嗽、呼吸困难、寒意、持续震颤、肌肉疼痛、头痛、咽喉痛、新的味觉和/或嗅觉丧失和病毒性肺炎的其他症状为特征。在严重的情况下，会出现大量与COVID-19严重程度相关的凝血病相关症状(例如，血液凝固、血栓形成、急性呼吸窘迫综合征、癫痫发作、心脏病发作、中风、多发性脑梗塞、肾衰竭尿崩症和/或弥散性血管内凝血)。在年轻患者中，罕见的炎症综合征有时与COVID-19相关(例如，非典型川崎综合征、中毒性休克综合征、小儿多系统炎性疾病、和细胞因子风暴综合征)。β属的冠状病毒SARS-CoV-2是致病因子(causative agent)。
如本文所使用的，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。
如本文所使用的，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。
如本文所使用的，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。
如本文所使用的，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。
如本文所使用的，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 Math 48:1073(1988))。
如本文所使用的，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。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。
如本文所使用的，术语“疾病”或“病状”是指能够用本文提供的融合蛋白、药物组合物或方法治疗的患者或个体的生存状态或健康状态。
术语“疫苗”是纯化的抗原疫苗或免疫原性组合物，亚基疫苗或免疫原性组合物，灭活的整体病毒疫苗或免疫原性组合物，或减毒病毒疫苗或免疫原性组合物。在一些实施方案中，疫苗或免疫原性组合物是纯化的融合蛋白。
如本文所使用的，术语“治疗(treating或treatment)”是指成功治疗或改善损伤、疾病、病理或病状(condition)的任何指标，包含任何客观或主观参数，如，消除；缓解；减轻症状或使得损伤、病理或病状对患者而言更易忍受；减缓退化或衰退的速度；或使退化的最终点较少衰退；改善患者的身体或精神健康。症状的治疗或改善可以基于客观或主观参数；包含身体检查、神经精神病学检查和/或精神病学评估的结果。术语“治疗”及其缀合可以包含预防损伤、病理、病状或疾病。在实施例中，治疗是预防。在实施例中，治疗不包含预防。
如本文所使用的(并且在本领域中被充分理解的)，“治疗(treating或treatment)”还广泛地包含用于在受试者的病状中获得有益的或期望的结果(包含临床结果)的任何方法。有益的或期望的临床结果可以包含但不限于：减轻或改善一种或多种症状或病状、减轻疾病程度、稳定(即，不恶化)疾病状态、预防疾病传播或扩散、延迟或减缓疾病进展、改善或缓解疾病状态、减少疾病复发以及缓解(无论是部分的还是全部的，以及无论是可检测的还是不可检测的)。换句话说，如本文所使用的，“治疗”包含对疾病的任何治愈、改善或预防。治疗可以预防疾病发生；抑制疾病扩散；缓解疾病的症状、完全或部分去除疾病的根本原因、缩短疾病的持续时间或这些事物的组合。
如本文所使用的，“治疗(Treating和treatment)”包含预防性治疗。治疗方法包含向受试者施用治疗有效量的活性剂。施用步骤可以由单次施用组成，或者可以包含一系列施用。治疗期的长度取决于多种因素，如病状的严重程度、患者的年龄、活性剂的浓度、在治疗中所使用的组合物的活性或其组合。还应当理解，用于治疗或预防的药剂的有效剂量可以在特定治疗或预防方案的过程中增加或减少。通过本领域中已知的标准诊断测定，剂量的变化可以产生并且变得显而易见。在一些情况下，可能需要慢性施用。例如，以足以治疗患者的量向受试者施用组合物，且持续足够的持续时间。
如本文所使用的，术语“预防”是指减少患者的疾病症状的发生。如上所述，预防可以是完全的(没有可检测的症状)或部分的，使得观察到比不存在治疗时可能发生的症状更少的症状。
如本文所使用的，“患者”或“有需要的受试者”是指遭受或易于遭受可以通过施用如本文所提供的药物组合物进行治疗的疾病或病状的活生物体。非限制性实例包含人、其它哺乳动物、牛科动物、大鼠、小鼠、狗、猴、山羊、绵羊、牛、鹿和其它非哺乳动物。在一些实施例中，患者是人。
术语“联合给药”是指将本公开的融合蛋白或疫苗与已知药物(或其它化合物，或其它疫苗)进行“联合用药”，从而二者都具有治疗或诊断效果。这种联合用药可以包括相对于施用本公开的融合蛋白或疫苗而言对该药物(或其它化合物，或其它疫苗)进行并行(即同时)、在前或相继给药。本领域一般技术人员将能够很容易判断特定药物(或其它化合物，或其它疫苗)以及本公开的联合物的合适的给药时间、顺序和剂量。
如本文所使用的，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”的实例是足以促成治疗、预防或减少疾病的一种或多种症状的量，所述量也可以被称为“治疗有效量”。一种或多种症状的“减少”意指降低一种或多种症状的严重程度或频率，或消除一种或多种症状。 药物的“预防有效量”是当施用于受试者时，将具有预期的预防效果的药物的量，例如预防或延迟损伤、疾病、病理或病状的发作(或复发)或降低损伤、疾病、病理或病状或其症状发作(或复发)的可能性。完全预防效果不一定通过施用一次剂量发生，并且可以在仅施用一系列剂量之后发生。因此，预防有效量可以以一次或多次施用的形式施用。
如本文所使用的，术语“治疗有效量”是指如上文所描述的足以改善病症的治疗剂的量。例如，对于给定参数，治疗有效量将显示增加或降低至少5％、10％、15％、20％、25％、40％、50％、60％、75％、80％、90％或至少100％。治疗功效也可以表示为“倍数”增加或减少。例如，治疗有效量可以相对于对照具有至少1.2倍、1.5倍、2倍、5倍或更多的效果。
剂量可以根据患者的需要和所采用的融合蛋白或疫苗而变化。在本公开的上下文中，向患者施用的剂量应足以随时间推移而在患者体内产生有益治疗反应。剂量的大小也将通过任何不良副作用的存在、性质和程度确定。针对特定情况来确定适当的剂量是在执业者的技能之内的。通常，治疗开始于比融合蛋白或疫苗的最优剂量小的较小剂量。其后，剂量以小增量增加直到达到在这些情况下的最优效果。可以单独调整给药的量和间隔，以提供所施用融合蛋白或疫苗的对所治疗的特定临床适应症有效的水平。这将提供与个体疾病状态的严重程度相称的治疗方案。
如本文所使用的，术语“施用”意指向受试者口服施用、以栓剂形式施用、局部接触、静脉内、肠胃外、腹膜内、肌肉内、病灶内、鞘内、鼻内或皮下施用，或植入缓慢释放装置(例如，微型渗透泵)。通过任何途径进行施用，包含肠胃外和经粘膜(例如，颊、舌下、腭、牙龈、鼻、阴道、直肠或经皮)。肠胃外施用包含例如静脉内、肌肉内、动脉内、皮内、皮下、腹膜内、心室内和颅内施用。其它递送模式包含但不限于使用脂质体调配物、静脉内输注、经皮贴剂等。在实施例中，施用不包含施用除了所叙述的活性剂之外的任何活性剂。
II.融合蛋白、药物组合物、疫苗及制剂
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)SARS-CoV-2 Delta突变株S蛋白或其功能片段；和
(3)免疫球蛋白Fc区；
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、Delta突变株S蛋白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：Delta毒株S蛋白或其功能片段、干扰素或其功能片段和免疫球蛋白Fc区。
在另一方面，本公开提供了一种治疗或预防与SARS-CoV-2相关的疾病或病状的疫苗组合产品，所述疫苗组合产品包含：
(i)亲本株疫苗，所述亲本株疫苗包含野生型SARS-CoV-2多肽的融合蛋白，所述野生型SARS-CoV-2融合蛋白包含：(1)干扰素或其功能片段；(2)野生型SARS-CoV-2的S蛋白的RBD或其功能片段；和(3)免疫球蛋白Fc区；和
(ii)突变株疫苗，所述突变株疫苗包含所述的包含突变型SARS-CoV-2多肽的融合蛋白；
其中，所述亲本株疫苗或所述突变株疫苗中的一种作为引发疫苗施用，所述亲本株疫苗或所述突 变株疫苗中的一种作为加强疫苗施用，并且所述亲本株疫苗和所述突变株疫苗两者均被施用。
在另一方面，本公开提供了亲本株疫苗和突变株疫苗在制备治疗或预防与SARS-CoV-2相关的疾病或病状的疫苗组合产品中的用途，所述疫苗组合产品包含：
(i)亲本株疫苗，所述亲本株疫苗包含野生型SARS-CoV-2的融合蛋白，所述野生型SARS-CoV-2融合蛋白包含：(1)干扰素或其功能片段；(2)野生型SARS-CoV-2的S蛋白的RBD或其功能片段；和(3)免疫球蛋白Fc区；和
(ii)突变株疫苗，所述突变株疫苗包含所述的包含突变型SARS-CoV-2多肽的融合蛋白；
其中所述亲本株疫苗或所述突变株疫苗中的一种作为引发疫苗施用，所述亲本株疫苗或所述突变株疫苗中的一种作为加强疫苗施用，并且所述亲本株疫苗和所述突变株疫苗两者均被施用。
在另一方面，本公开提供了一种在有需要的受试者中诱导针对SARS-CoV-2的免疫应答的方法，所述方法包括：
(i)向受试者施用亲本株疫苗，所述亲本株疫苗包含野生型SARS-CoV-2的融合蛋白，所述野生型SARS-CoV-2融合蛋白包含：(1)干扰素或其功能片段；(2)野生型SARS-CoV-2的S蛋白的RBD或其功能片段；和(3)免疫球蛋白Fc区；和
(ii)向受试者施用突变株疫苗，所述突变株疫苗包含所述的包含突变型SARS-CoV-2多肽的融合蛋白；
其中所述亲本株疫苗或所述突变株疫苗中的一种作为引发疫苗施用，所述亲本株疫苗或所述突变株疫苗中的一种作为加强疫苗施用，并且所述亲本株疫苗和所述突变株疫苗两者均被施用。
在一些实施方案中，所述受试者是哺乳动物或鸟类。
在一些实施方案中，所述受试者是人类、牛、犬、猫、山羊、绵羊、猪、马、火鸡、鸭或鸡。
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。
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-α-2a的氨基酸序列如SEQ ID NO:1所示。
在一些实施方案中，所述Delta突变株S蛋白的功能片段为其受体结合结构域RBD。
在一些实施方案中，所述野生型SARS-CoV-2的S蛋白的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，所述野生 型SARS-CoV-2的S蛋白的RBD的氨基酸序列如SEQ ID NO:2所示。
在一些实施方案中，所述Delta突变株S蛋白的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。
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。
在一些实施方案中，所述包含突变型SARS-CoV-2多肽的融合蛋白包含与SEQ ID NO:14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，所述包含突变型SARS-CoV-2多肽的融合蛋白为SEQ ID NO:14。
在一些实施方案中，所述包含野生型SARS-CoV-2的融合蛋白包含与SEQ ID NO:13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，所述包含野生型SARS-CoV-2多肽的融合蛋白为SEQ ID NO:13。
在另一方面，本公开提供了一种编码所述融合蛋白或所述的疫苗组合产品的核酸。
在一些实施方案中，所述核酸是mRNA。
在一些实施方案中，所述包含野生型SARS-CoV-2多肽的融合蛋白的编码核酸包含与SEQ ID NO:15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；所述野生型SARS-CoV-2的融合蛋白的编码核酸为SEQ ID NO:15所示的核酸。
在一些实施方案中，所述包含突变型SARS-CoV-2多肽的融合蛋白的编码核酸包含与SEQ ID NO:16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，所述核酸为SEQ ID NO:16所示的核酸。
在另一方面，本公开提供了一种包含所述核酸的表达载体。
在一些实施方案中，所述表达载体选自质粒、粘粒、病毒载体、RNA载体或线性或圆形DNA或RNA分子。
在一些实施方案中，所述质粒选自pCI、puc57、pcDNA3、pSG5、pJ603和pCMV。
在一些实施方案中，所述病毒载体选自腺病毒、逆转录病毒、细小病毒(例如，腺伴随病毒)、冠状病毒、负链RNA病毒诸如正粘病毒(例如，流感病毒)、弹状病毒(例如，狂犬病和水疱性口炎病毒)、副粘病毒(例如，麻疼和仙台)、正链RNA病毒诸如小RNA病毒和甲病毒，和双链DNA病毒。
在一些实施方案中，所述腺病毒载体选自人、黑猩猩或恒河猴腺病毒；优选地，所述腺病毒选自血清型Ad2、Ad4、Ad5、Ad6、Ad11、Ad12、Ad24、Ad26、Ad34、Ad35、Ad40、Ad48、Ad49、Ad50、Ad52和Pan9腺病毒。
核酸分子也可以插入表达载体，诸如腺病毒载体中，并掺入本公开的组合物中。术语“腺病毒载体(adenovirus vector)”和“腺病毒载体(adenoviral vector)”和“腺病毒颗粒”可互换使用，是指经基因工程改造的腺病毒，其设计为将目标多核苷酸(例如，编码ZIKVM和Env抗原的多核苷酸)插入真核细胞中，使得该多核苷酸随后表达。可用作本公开的病毒载体的腺病毒的实例包括具有或衍生自血清型Ad2、Ad 4、Ad5、Ad6、Ad11、Ad12、Ad24、Ad26、Ad34、Ad35、Ad40、Ad48、Ad49、Ad50、Ad52(例如，RhAd52)和Pan9(也称为AdC68)的那些腺病毒；这些载体可以源自例如人、黑猩猩(例如，ChAd1、ChAd3、ChAd7、ChAd8、ChAd21、ChAd22、ChAd23、ChAd24、ChAd25、ChAd26、ChAd27.1、ChAd28.1、ChAd29、ChAd30、ChAd31.1、ChAd32、ChAd33、ChAd34、ChAd35.1、ChAd36、ChAd37.2、ChAd39、ChAd40.1、ChAd41.1、ChAd42.1、ChAd43、ChAd44、ChAd45、ChAd46、ChAd48、ChAd49、ChAd49、ChAd50、ChAd67或SA7P)或恒河猴腺病毒(例如rhAd51、rhAd52或rhAd53)。
在一些实施方案中，所述逆转录病毒选自禽造白细胞组织增生-肉瘤、哺乳动物C-型、B-型病毒、D-型病毒、HTLV-BLV集合、慢病毒和泡沫病毒。
在一些实施方案中，所述慢病毒载体选自HIV-1、HIV-2、SIV、FIV、BIV、EIAV、CAEV和绵羊脱髓鞘性脑白质炎慢病毒。
在一些实施方案中，所述双链DNA病毒包括腺病毒、疱疹病毒(例如，单纯疱疹病毒1和2型、愛泼斯坦-巴尔病毒、巨细胞病毒)和痘病毒(例如，牛痘病毒、鸡痘病毒和金丝雀痘病毒)、诺沃克病毒、披膜病毒、黄病毒、呼肠孤病毒、乳多泡病毒、嗜肝DNA病毒、杆状病毒和肝炎病毒。
在另一方面，本公开提供了一种表达所述融合蛋白、表达所述疫苗组合产品、包含所述核酸和/或包含所述表达载体的宿主细胞。
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；优选地，所述原核细胞是大肠杆菌细胞。
在一些实施方案中，所述真核细胞突变酵母细胞、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。
在另一方面，本公开提供了一种药物组合物，其包含所述的融合蛋白、所述的疫苗组合产品、所述的核酸、所述的表达载体和/或所述的宿主细胞，以及任选地一种或多种药学上可接受载体、稀释剂或赋形剂。
在另一方面，本公开提供了一种治疗和/或预防与SARS-CoV-2相关的疾病或病状的疫苗，其包 含所述的融合蛋白、所述的疫苗组合产品、所述的核酸、所述的表达载体、所述的宿主细胞和/或所述的药物组合物，以及任选地药学上可接受的载体、赋形剂、稀释剂或佐剂。
在一些实施方案中，所述亲本株疫苗和/或所述突变株疫苗独立地还包含一种或更多种药学上可接受的载体、赋形剂、稀释剂或佐剂。
在一些实施方案中，所述佐剂选自氢氧化铝、磷酸铝、皂苷例如Quil A、QS-21、GPI-0100、油包水型乳状液、水包油型乳状液、水包油包水型乳状液。
在一些实施方案中，所述亲本株疫苗和/或所述突变株疫苗呈液体、乳液、固体、气雾剂、薄雾或气体的形式。
在一些实施方案中，所述突变株疫苗是引发疫苗，并且所述亲本株疫苗是加强疫苗。
在一些实施方案中，所述亲本株疫苗是引发疫苗，并且所述突变株疫苗是加强疫苗。
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。
在另一方面，本公开提供了一种预防和/或治疗与SARS-CoV-2相关的疾病或病状的疫苗的制备方法，所述方法包括表达如所述融合蛋白或所述的疫苗组合产品。
在一些实施方案中，所述与SARS-CoV-2相关的疾病或病状为SARS-CoV-2感染或COVID-19。
亲本株疫苗和突变株疫苗可以组合药盒呈现。本文所用的术语“组合药盒”或“成套药盒”指一种或多种药物组合物根据本公开用于施用亲本株疫苗和突变株疫苗。所述组合药盒包含分开的药物组合物中的亲本株疫苗和突变株疫苗，其中亲本株疫苗和突变株疫苗在在分开包装的单独药物组合物中。
在本公开的一个实施方式中，所述成套药盒包括以下组分：
亲本株疫苗，与药学上可接受佐剂、稀释剂或载体联合；
突变株疫苗，与药学上可接受佐剂、稀释剂或载体联合；
其中所述组分以适合顺序、分开施用的形式提供。
在一个实施方式中，所述成套药盒包括：
含亲本株疫苗与药学上可接受佐剂、稀释剂或载体的第一容器；和含突变株疫苗与药学上可接受佐剂、稀释剂或载体的第二容器。
所述试剂盒或组合药盒还能根据有说明，如剂量和施用说明。这种剂量和施用说明可以是提供给医生的类型例如药品标签，或可以是由医生提供的类型，如给患者的说明。
所用药物组合物或疫苗的这些要素可以单独药物组合呈现或以共同配制于一种药物组合物中。因此，本公开还提供多个药物组合物或疫苗的组合，药物组合物或疫苗其中之一含有亲本株疫苗和一种或多种药学上可接受载体、稀释剂或赋形剂的药物组合物以及一种药物组合物含有突变株疫苗和一种或多种药学上可接受载体、稀释剂或赋形剂的药物组合物。
药物组合物或疫苗可以每单位剂量含预定量活性成分的单位剂型呈现。如本领域技术人员已知，每剂量的活性成分的量取决于所治疗病症、施用途径以及患者的年龄、体重和状况。优选的单位剂量组合物含有活性成分的日剂量或亚剂量或其合适部分。此外，这种药物组合物可通过制药领域熟知的任何方法制备。
药物组合物或疫苗可通过任何合适途径施用。合适的途径包括口服、直肠、鼻、局部(包括颊和 舌下)、阴道和胃肠外(包括皮下、肌肉、静脉、皮内、鞘内和硬脑膜外)。应理解施用的各试剂可经相同或不同途径施用且亲本株疫苗和突变株疫苗处于分开的药物组合物中。
本公开的药物组合物或疫苗一般适合于肠胃外施用。如本文所使用的，药物组合物或疫苗的“肠胃外施用”包括特征在于受试者组织的物理伤口的任何施用途径和药物组合物或疫苗通过组织中的伤口的施用，从而一般导致直接施用到肌肉内、或到内脏器官内。肠胃外施用因此包括但不限于，通过注射组合物施用药物组合物，通过外科切口应用组合物，通过组织穿透非外科创伤应用组合物等。特别地，肠胃外施用考虑包括但不限于，皮下、肌内注射或输注。
适合于肠胃外施用的药物组合物或疫苗的制剂一般包含与药学上可接受的载体组合的活性成分，所述载体例如无菌水或无菌等渗盐水。此种制剂可以以适合于推注施用或连续施用形式进行配制、包装或销售。可注射制剂可以以单位剂型，例如在包含防腐剂的安瓿或多剂量容器中进行配制、包装或销售。用于肠胃外施用的制剂包括但不限于，在油或水媒介物中的悬浮液、溶液、乳状液、糊剂等。此种制剂可以进一步包含一种或多种另外的成分，包括但不限于悬浮、稳定或分散剂。在用于肠胃外施用的制剂的一个实施方案中，活性成分以干燥(即粉剂或粒剂)形式提供，用于在重构组合物的肠胃外施用前用合适的媒介物(例如灭菌无热原水)重构。肠胃外制剂还包括水溶液，其可以包含赋形剂例如盐、碳水化合物和缓冲剂(优选至3-9的pH)，但对于某些应用，它们可以更适合于配制为无菌非水溶液或干燥形式，以与合适的媒介物例如灭菌、无热原水结合使用。示例性肠胃外施用形式包括在无菌水溶液中的溶液或悬浮液，例如丙二醇或右旋糖水溶液。需要时，此种剂型可以进行适当缓冲。有用的其他肠胃外可施用的制剂包括包含以微晶形式或在脂质体制剂中的活性成分的那些。用于肠胃外施用的制剂可以配制为立即和/或缓和释放的。缓和释放制剂包括延迟、持续、脉冲、控制、靶向和按程序释放。
例如，在一个方面，无菌可注射溶液可以通过将所需量的药物组合物或疫苗与上文列出的成分之一或组合掺入合适溶剂中进行制备，需要时，随后为过滤灭菌。一般地，分散系通过将活性药物组合物或疫苗掺入无菌媒介物中进行制备，所述无菌媒介物包含基本分散介质和来自上文列出那些的所需其他成分。在用于制备无菌可注射溶液的无菌粉剂的情况下，优选制备方法是真空干燥和冷冻干燥，这产生来自其先前无菌过滤溶液的活性成分加任何另外所需成分的粉末。溶液的合适流动性可以这样得到维持：例如通过使用包衣例如卵磷脂、在分散系的情况下通过维持所需粒子大小和通过使用表面活性剂。可注射组合物的延长吸收可以通过在组合物中包括延迟吸收的试剂例如单硬脂酸盐和明胶来达到。
本公开的疫苗还可以鼻内或通过吸入进行施用，一般以来自干粉吸入器的干粉(单独，作为混合物，或作为混合组分颗粒，例如与合适的药学上可接受的赋形剂混合)形式，作为来自增压容器、泵、喷射器、喷雾器(优选使用电流体动力学的喷雾器以产生精细薄雾)或雾化器的气溶胶喷雾，连同使用或不使用合适的推进剂，或作为滴鼻剂。
增压容器、泵、喷射器、喷雾器或雾化器一般包含本公开疫苗的溶液或悬浮液，其包含例如合适的试剂用于分散、溶解或延长活性的释放，一种或多种推进剂作为溶剂。
在以干粉或悬浮制剂使用前，药物产品一般微粒化至适合于通过吸入递送的大小(一般小于5微米)。这可以通过任何合适的粉碎方法来达到，所述方法例如螺旋气流磨、流体床气流磨、超临界流 体加工以形成纳米颗粒、高压均质化、或喷雾干燥。
可以配制用于在吸入器或吹入器中使用的胶囊、水泡和药筒，以包含本公开药物组合物或疫苗、合适的粉基和性能改良剂的粉末混合物。用于在使用电流体动力学以产生精细薄雾的喷雾器中使用的合适的溶液制剂可以包含合适剂量的本公开疫苗/启动，并且启动体积可以例如从1μL到100μL不等。
合适的调味剂例如薄荷和左薄荷脑或甜味剂例如糖精或糖精钠，可以加入预期用于吸入/鼻内施用的本公开的这些制剂中。
用于吸入/鼻内施用的制剂可以配制为立即和/或缓和释放的。缓和释放制剂包括延迟、持续、脉冲、控制、靶向和按程序释放。
在干粉吸入器和气溶胶的情况下，剂量单位借助于递送测定量的阀进行确定。依照本公开的单位一般安排为施用本公开疫苗的测定量或“团(puff)”。总日剂量一般将以单次剂量或更通常地作为全天的分份剂量进行施用。
本公开的药物组合物或疫苗也可以配制用于口部途径施用。适合口服施用的药物组合物或疫苗可作为独立单元，如胶囊剂或片剂；粉末剂或颗粒剂；水性或非水性液体中的溶液剂或混悬剂；可食用泡沫或奶油；或水包油液体乳剂或油包水液体乳剂呈现。
例如，对于片剂或胶囊剂形式的口服施用，活性药物组分能与口服、无毒的药学上可接受惰性载体如乙醇、甘油、水等组合。粉末如下制备：粉碎药物组合物或疫苗至合适细粒度，并与类似粉碎的药物载体如可食用碳水化合物(例如淀粉或甘露醇)混合。增味剂、防腐剂、分散剂和着色剂也能存在。
胶囊如下制备：如上所述制备粉末混合物，填充形成的胶囊套。助流剂和润滑剂如硅胶、滑石、硬脂酸镁、硬脂酸钙或固体聚乙二醇能在填充操作前加入粉末混合物。还可加入崩解剂或增溶剂如琼脂-琼脂、碳酸钙或碳酸钠以改善吞咽胶囊时的药物利用度。
此外，需要或必要时，合适的粘合剂、润滑剂、崩解剂和着色剂也能造粒，粉末混合物可经过压片机，结果是未完全形成的小块会碎成颗粒。颗粒能经润滑纳入混合物。合适的粘合剂包括淀粉、明胶、天然糖如葡萄糖或β-乳糖、玉米甜味剂、天然和合成树胶如阿拉伯胶、黄芪胶或藻酸钠、羧甲基纤维素、聚乙二醇、蜡等。用于这些剂型的润滑剂包括油酸钠、硬脂酸钠、硬脂酸镁、苯甲酸钠、乙酸钠、氯化纳等。崩解剂包括但不限于淀粉、甲基纤维素、琼脂、皂土、黄原胶等。配制片剂，例如通过制备粉末混合物，造粒或冲击，加入润滑剂和崩解剂并压成片剂。粉末混合物如下制备：如上所述将适当粉碎的药物组合物或疫苗与稀释剂或基质混合，可选联同粘合剂如羧甲基纤维素、藻酸盐、明胶或聚乙烯吡咯烷酮，溶液阻滞剂如石蜡，再吸收促进剂如季盐和/或吸收剂如皂土、高岭土或磷酸氢钙。粉末混合物能如下造粒：用粘合剂如糖浆、淀粉糊、阿拉伯胶粘液(acadiamucilage)或者纤维素或聚合材料溶液湿润，并迫使过筛。防止粘住压片模具的替代方式是加入硬脂酸、硬脂酸盐、滑石或矿物油。然后，润滑的混合物压成片剂。本公开药物组合物或疫苗还能与自由流动的惰性载体组合并直接压成片剂，而不需经历造粒或冲击步骤。可提供澄清或不透明的保护包衣，所述包衣由虫胶密封涂层、糖或聚合材料的涂层和蜡的抛光涂层组成。能向这些包衣加入染料以区分不同单位剂量。
口服液体如溶液、糖浆和酏剂能以单位剂型制备，从而给定量包含预定量的疫苗。通过将疫苗溶于适当调味的水溶液可制备糖浆，使用无毒醇载剂制备酏剂。通过将疫苗分散于无毒载剂可配制混悬剂。还能加入增溶剂和乳化剂如乙氧基化异硬脂醇及聚氧乙烯山梨醇醚、防腐剂、香味添加剂如薄荷 油或天然甜味剂或糖精或其它人造甜味剂等。
适当时，口服施用的组合物能装入微胶囊。所述组合物还能制备成延长或维持释放，例如通过将微粒材料包被或包埋于聚合物、蜡等。
根据本公开使用的试剂还可以脂质体递送系统形式施用，如小单层囊泡、大单层囊泡和多层囊泡。脂质体能形成自多种磷脂，如胆固醇、硬脂胺或磷脂酰胆碱。
适合经皮施用的药物组合物可作为独立贴片呈现，贴片旨在维持与受体表皮的长时间密切接触。例如，活性成分可通过离子导入法从贴片递送。
适合局部施用的药物组合物可配制成软膏剂、乳膏剂、混悬剂、洗剂、粉末剂、溶液剂、糊剂、凝胶剂、喷雾剂、气雾剂或油剂。
适合口腔局部施用的药物组合物包括锭剂、糖果锭剂和漱口剂。
适合直肠施用的药物组合物可以为栓剂或灌肠剂。
III.治疗方法
在另一方面，本公开提供了一种预防和/或治疗与SARS-CoV-2相关的疾病或病状的方法，所述方法包括向受试者施用有效量的所述融合蛋白、所述疫苗组合产品、所述核酸、所述表达载体、所述宿主细胞、所述药物组合物、所述疫苗、所述试剂盒和/或所述成套药盒。
在一些实施方案中，所述与SARS-CoV-2相关的疾病或病状为SARS-CoV-2感染或COVID-19。
在另一方面，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所述融合蛋白、所述疫苗组合产品、所述核酸、所述表达载体、所述宿主细胞、所述药物组合物、所述疫苗、所述试剂盒和/或所述成套药盒。
在另一方面，本公开提供了一种亲本株疫苗和突变株疫苗在制备治疗或预防与新型冠状病毒SARS-CoV-2相关的疾病或病状的疫苗组合产品中的用途。
在另一方面，本公开提供了本公开提供了一种在有需要的受试者中诱导针对新型冠状病毒SARS-CoV-2的免疫应答的方法，所述方法包括：
(i)向受试者施用亲本株疫苗；和
(ii)向受试者施用突变株疫苗。
依照本公开，亲本株疫苗和突变株疫苗可以顺序形式分开施用。
在一些实施方案中，所述突变株疫苗是引发疫苗，并且所述亲本株疫苗是加强疫苗。
在一些实施方案中，所述亲本株疫苗是引发疫苗，并且所述突变株疫苗是加强疫苗。
在一些实施方案中，所述加强疫苗在所述引发疫苗初始给予后间隔一段时间给予以进行免疫。
在一些实施方案中，所述加强疫苗在所述引发疫苗初始给予后间隔一段时间首次给予以进行第一次加强免疫，并且，所述加强疫苗在引发疫苗初始给予后间隔另一段时间再次给予以进行第二次加强免疫；优选地，所述亲本株疫苗是引发疫苗，并且所述突变株疫苗是加强疫苗，所述突变株疫苗在所述亲本株疫苗初始给予后间隔一段时间首次给予，并且，所述突变株疫苗在亲本株疫苗初始给予后间隔另一段时间再次给予。
在一些实施方案中，所述引发疫苗在所述引发疫苗初始给予后间隔一段时间再次给予以进行第一次加强免疫，并且，所述加强疫苗在引发疫苗初始给予后间隔另一段时间首次给予以进行第二次加强 免疫；优选地，所述亲本株疫苗是引发疫苗，并且所述突变株疫苗是加强疫苗，所述亲本株疫苗在所述亲本株疫苗初始给予后间隔一段时间再次给予，并且，所述突变株疫苗在亲本株疫苗初始给予后间隔另一段时间首次给予。
在一些实施方案中，所述加强疫苗在所述引发疫苗初始给予后大约1-10周给予，优选2-8、3-8、1-3、2-5、2-3、3-5、5-8周给予，更优选2-8、2-5，3-5周给予。
在一些实施方案中，所述加强疫苗在所述引发疫苗初始给予后大约1-10周首次给予，优选2-8、3-8、1-3、2-5、2-3、3-5、5-8周首次给予，更优选2-8、2-5，3-5周首次给予；并在所述引发疫苗初始给予后大约4-72周再次给予，优选6-52、6-45、6-30、6-25、8-52、8-30、8-25、4-12周，更优选4-12、6-25、8-25周再次给予。
在一些实施方案中，所述引发疫苗在所述引发疫苗初始给予后大约1-10周再次给予，优选2-8、3-8、1-3、2-5、2-3、3-5、5-8周再次给予，更优选2-8、2-5，3-5周再次给予，所述加强疫苗在所述引发疫苗初始给予后大约4-72周首次给予，优选6-52、6-45、6-30、6-25、8-52、8-30、8-25、4-12周，更优选4-12、6-25、8-25周首次给予。
在一些实施方案中，所述加强疫苗在所述引发疫苗初始给予后大约2-5周首次给予，并在所述引发疫苗初始给予后大约6-25周再次给予。
在一些实施方案中，所述引发疫苗在所述引发疫苗初始给予后大约2-5周再次给予，所述加强疫苗在所述引发疫苗初始给予后大约6-25周首次给予。
用于给药的剂型包括，例如，口服制剂(例如：片剂、胶囊、颗粒剂、粉剂、口服溶液、糖浆剂、口服胶冻剂等)、口腔黏膜制剂(例如：口腔黏膜应用的片剂、口腔黏膜应用的喷雾剂、口腔黏膜应用的半固体制剂、漱口剂等)、用于注射的制剂(例如：注射剂等)、鼻喷雾剂或滴鼻剂、用于吸入的制剂(例如：雾化吸入剂等)、用于直肠的制剂(例如：栓剂、用于直肠应用的半固体制剂、用于直肠应用的灌肠剂等)、用于皮肤应用的制剂(例如：用于皮肤应用的固体制剂、用于皮肤应用的液体和溶液、喷雾剂、软膏剂、霜剂、凝胶剂、贴剂等)及类似形式。
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、静脉注射、滴鼻或雾化吸入的方式进行接种。
在一些实施方案中，所述疫苗通过注射进行施用。
本公开实验证明，所述疫苗能够通过融合表达的IFN提升对变异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。
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\新冠病毒S蛋白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-端经柔性接头(Gly4Ser)3(SEQ ID NO:11)-PADRE-柔性接头GSGSGS(SEQ ID NO:12)与RBD-Fc 融合(图1)。为了对比活性功能，构建亲本株疫苗(V-01)与Delta毒株疫苗，两者区别在于RBD抗原不同的点突变。本实施例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Delta毒株疫苗图谱。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:15所示；Delta毒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:16所示。
1.2蛋白质表达
对于亲本株疫苗(V-01)与Delta毒株疫苗的表达，本实施例采用大体积瞬时转染的方案进行表达。
(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 6-8×10 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。本实施例转染混合物制备，采用ExpiCHO表达系统(购自：Life technologies，货号：A29133)操作说明书，按照相同比例进行配置。转染混合物制备完成后，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100rpm，通气量0.5mm孔径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Enhancer；转染后第一、三、五天，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)与Delta毒株疫苗表达载体，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密度与细胞活率(图4)。本实施例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。
通过本实施例中所示的大体积瞬时表达系统，对亲本株疫苗(V-01)与Delta毒株疫苗进行了瞬转表达。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(V-01)与Delta毒株疫苗蛋白表达水平相当(图5a)，未见RBD点突变不同导致的表达水平差异。
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Delta毒株疫苗纯度均大于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Delta毒株疫苗的亲和力进行检测，包括新冠病毒S蛋白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，每个循环包括捕获疫苗、不同浓度ACE2蛋白的进样及再生。将实施例1制备的Delta毒株疫苗、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(pH1.5)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-6b，亲本株疫苗(V-01)、Delta毒株疫苗均可与ACE2结合，亲和力分别为5.41E-09M和4.91E-09M，与ACE2的亲和力基本一致。
表1亲本株(V-01)、Delta毒株疫苗与ACE2亲和力结果
批号 ka(1/Ms) kd(1/s) KD(M)
亲本株(V-01) 6.68E+04 3.61E-04 5.41E-09
Delta毒株疫苗 5.34E+04 2.62E-04 4.91E-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Delta毒株疫苗、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1:1 binding。
结果与结论：结果见表2和图6c-6d，亲本株疫苗(V-01)、Delta毒株疫苗均可与IFNAR2蛋白结合，亲和力分别为1.18E-07M和1.35E-07M，与IFNAR2的亲和力基本一致。
表2亲本株(V-01)、Delta毒株疫苗与IFNAR2亲和力结果
样品 ka(1/Ms) kd(1/s) KD(M)
亲本株(V-01) 1.73E+05 2.04E-02 1.18E-07
Delta毒株疫苗 1.35E+05 1.82E-02 1.35E-07
实施例3：亲本株疫苗(V-01)与Delta毒株疫苗的生物学活性
亲本株疫苗(V-01)与Delta毒株疫苗结构中的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Delta毒株疫苗与亲本株疫苗(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Delta毒株疫苗稀释至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)、Delta毒株疫苗的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，EC 50分别为3.51ng/mL和3.3ng/mL。
表3亲本株疫苗(V-01)、Delta毒株疫苗IFNα结构域的生物学活性
样品 EC 50(ng/mL)
亲本株疫苗(V-01) 3.51
Delta毒株疫苗 3.3
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)、Delta毒株疫苗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)、Delta毒株疫苗免疫6～8周龄C57BL/6小鼠，疫苗浓度20μg/mL，每只小鼠每次大腿肌肉注射0.1mL，10只/组，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新冠病毒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、Delta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8000倍，再使用96孔板进行2倍梯度稀释，共8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TBM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。样品免疫血清样本的滴度为信号值大于Cutoff值的最大稀释倍数，并计算几何平均滴度。
结果与结论：结果见表4和图8，亲本株疫苗(V-01)、Delta毒株疫苗均能引起较强的免疫原性，但亲本株疫苗(V-01)免疫后血清对Delta突变株S蛋白的RBD结合滴度较差，而Delta毒株疫苗免疫后血清对新冠病毒野生型S蛋白的RBD、Delta突变株S蛋白的RBD结合滴度相当，且与亲本株疫苗(V-01)对野生型RBD结合滴度相当。
表4亲本株疫苗(V-01)、Delta毒株疫苗小鼠体内效力
样品 野生型RBD Delta突变RBD
亲本株疫苗(V-01) 55715 32000
Delta毒株疫苗 55715 55715
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/HIV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)、Delta毒株疫苗的假病毒保护力进行评估。
研究方法：将实施例1制备的亲本株疫苗(V-01)、Delta毒株疫苗免疫6～8周龄C57BL/6小鼠，疫苗浓度20μg/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、第14天各一次)，二免后14天眼眶采血取血清，血液室温静置待凝固后，4000rpm，2～8℃，离心10min，取上清。待测血清样本提前采用56℃水浴灭活30min。取抗新冠病毒S蛋白RBD小鼠中和抗体(购自novoprotein，货号DA035)，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孔全白细胞板中用检测培养液稀释至首孔浓度为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，用检测培养液将野生型假病毒(购自北京天坛生物制品股份有限公司，货号80033)、Delta突变假病毒(购自北京天坛生物制品股份有限公司，货号80048)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(购自吉满生物科技(上海)有限公司，货号GM-C09233)，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滴度。
结果与结论：结果见表5和图9，亲本株疫苗(V-01)二免后血清能够有效中和野生型假病毒，其中和滴度为1394，但对于Delta突变假病毒的中和滴度有明显下降。而Delta毒株疫苗二免后血清能够有效中和Delta突变假病毒，其中和滴度为958，相比之下对于野生型假病毒的中和滴度略低。
表5亲本株疫苗(V-01)、Delta毒株疫苗假病毒中和效价
样品 野生型假病毒 Delta突变假病毒
亲本株疫苗(V-01) 1394 98
Delta毒株疫苗 442 958
实施例6：加免Delta毒株疫苗后假病毒中和效价评估
研究方法：将实施例1制备的亲本株疫苗(V-01)免疫6～8周龄C57BL/6小鼠，第一、二免的免疫方式同实施例5。二免后14天(即第28天)用Delta毒株疫苗进行第三免。三免后14天眼眶采血取血清。检测其二免后14天(V01+V01)、三免后14天(V01+V01+Delta)血清对于野生型假病毒、Delta突变假病毒、Beta突变假病毒、Alpha突变假病毒的中和滴度。检测方法同实施例5。
结果与结论：结果见表6和图10。亲本株疫苗(V-01)二免后血清对于各种突变假病毒的中和滴度相比野生型假毒有明显下降。再此基础上用Delta毒株疫苗进行三免，其对于Delta突变假病毒(购自北京天坛生物制品股份有限公司，货号80048)、Beta突变假病毒(购自北京天坛生物制品股份有限公司，货号80044)、Alpha突变假病毒(购自北京天坛生物制品股份有限公司，货号80043)的中和滴度分别提升9.3、9.1和3.2倍，同时对野生型假病毒中和滴度亦有所提高。表明Delta毒株疫苗对设计，大幅提高了对Delta变异株的保护力，且对多种毒株具有交叉中和活性。
表6亲本株疫苗(V-01)、Delta毒株疫苗假病毒中和效价
样品 野生型假病毒 Delta突变假病毒 Beta突变假病毒 Alpha突变假病毒
V01+V01 2630 148 70 739
V01+V01+Delta 4154 1379 634 2401
实施例7：已上市野生型疫苗两针加免Delta毒株疫苗计划
随着全球范围内疫苗接种的普及，已接种疫苗人群正在持续增加，包括我国在内的一些国家已达到群体免疫的水平。国内以野生型灭活疫苗为主，国外以野生型mRNA疫苗或腺病毒疫苗为主。但随着目前各大疫苗已公开的保护力分析数据，已接种人群体内的中和抗体滴度在全程免疫(即完成2针接种)后2个月开始下降，4个月后低于中位标准，6个月后普遍下降近10倍以上。其中，各大疫苗的保护力对于主流的VOC变异株(Alpha、Delta、Beta、Gamma)，以及可能发展为主要流行株的VOI变异株下降更加明显。目前，最先通过紧急使用完成全程免疫的人群已经超过12个月，也有相当数量的人群超过了6个月，加强或序贯免疫势在必行。另外，鉴于新冠病毒不断变异，VOC/VOI种类也再不断增多，为了提高现有疫苗针对突变株的保护效力以及增强免疫持久性，Delta毒株疫苗计划在已接种野生型疫苗人群基础上进行加强免疫研究，预测可提高对大多数VOC/VOI突变株的中和抗体滴度，并且可显著增加体内对抗新冠病毒的保护力及持久性。
研究方法：对已完成全程免疫野生型疫苗(灭活、mRNA、腺病毒等)的人群，进行1针Delta毒 株疫苗加免。加免前后采血，对比加免前后的受试者血清对于野生型假病毒、Delta突变假病毒、Beta突变假病毒、Alpha突变假病毒及其他主流毒株假病毒的中和滴度。

Claims (23)

  1. 一种包含突变型SARS-CoV-2多肽的融合蛋白，其包含：
    (1)干扰素或其功能片段；
    (2)SARS-CoV-2 Delta突变株S蛋白或其功能片段；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、Delta突变株S蛋白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：Delta毒株S蛋白或其功能片段、干扰素或其功能片段和免疫球蛋白Fc区。
  2. 一种治疗或预防与SARS-CoV-2相关的疾病或病状的疫苗组合产品，所述疫苗组合产品包含：
    (i)亲本株疫苗，所述亲本株疫苗包含野生型SARS-CoV-2多肽的融合蛋白，所述野生型SARS-CoV-2融合蛋白包含：(1)干扰素或其功能片段；(2)野生型SARS-CoV-2的S蛋白的RBD或其功能片段；和(3)免疫球蛋白Fc区；和
    (ii)突变株疫苗，所述突变株疫苗包含权利要求1所述的包含突变型SARS-CoV-2多肽的融合蛋白；
    其中，所述亲本株疫苗或所述突变株疫苗中的一种作为引发疫苗施用，所述亲本株疫苗或所述突变株疫苗中的一种作为加强疫苗施用，并且所述亲本株疫苗和所述突变株疫苗两者均被施用。
  3. 亲本株疫苗和突变株疫苗在制备治疗或预防与SARS-CoV-2相关的疾病或病状的疫苗组合产品中的用途，所述疫苗组合产品包含：
    (i)亲本株疫苗，所述亲本株疫苗包含野生型SARS-CoV-2的融合蛋白，所述野生型SARS-CoV-2融合蛋白包含：(1)干扰素或其功能片段；(2)野生型SARS-CoV-2的S蛋白的RBD或其功能片段；和(3)免疫球蛋白Fc区；和
    (ii)突变株疫苗，所述突变株疫苗包含权利要求1所述的包含突变型SARS-CoV-2多肽的融合蛋白；
    其中所述亲本株疫苗或所述突变株疫苗中的一种作为引发疫苗施用，所述亲本株疫苗或所述突变株疫苗中的一种作为加强疫苗施用，并且所述亲本株疫苗和所述突变株疫苗两者均被施用。
  4. 一种在有需要的受试者中诱导针对SARS-CoV-2的免疫应答的方法，所述方法包括：
    (i)向受试者施用亲本株疫苗，所述亲本株疫苗包含野生型SARS-CoV-2的融合蛋白，所述野生型SARS-CoV-2融合蛋白包含：(1)干扰素或其功能片段；(2)野生型SARS-CoV-2的S蛋白的RBD或其功能片段；和(3)免疫球蛋白Fc区；和
    (ii)向受试者施用突变株疫苗，所述突变株疫苗包含权利要求1所述的包含突变型SARS-CoV-2多肽的融合蛋白；
    其中所述亲本株疫苗或所述突变株疫苗中的一种作为引发疫苗施用，所述亲本株疫苗或所述突变株疫苗中的一种作为加强疫苗施用，并且所述亲本株疫苗和所述突变株疫苗两者均被施用；
    优选地，所述受试者是哺乳动物或鸟类；
    优选地，所述受试者是人类、牛、犬、猫、山羊、绵羊、猪、马、火鸡、鸭或鸡。
  5. 权利要求1所述的融合蛋白、权利要求2所述的疫苗组合产品、权利要求3所述的用途或权利要求4所述的方法，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-α-2a的氨基酸序列如SEQ ID NO:1所示。
  6. 根据权利要求1或5所述融合蛋白、权利要求2或5所述的疫苗组合产品、权利要求3或5所述的用途或权利要求4或5所述的方法，其中，所述Delta突变株S蛋白的功能片段为其受体结合结构域RBD；
    优选地，所述野生型SARS-CoV-2的S蛋白的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，所述野生型SARS-CoV-2的S蛋白的RBD的氨基酸序列如SEQ ID NO:2所示；
    优选地，所述Delta突变株S蛋白的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。
  7. 根据权利要求1、2、5或6任一项所述的融合蛋白、权利要求2、5或6任一项所述的疫苗组合产品、权利要求3、5或6任一项所述的用途或权利要求4-6任一项所述的方法，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。
  8. 根据权利要求1、2、5-7任一项所述融合蛋白、权利要求2、5-7任一项所述的疫苗组合产品、权利要求3、5-7任一项所述的用途或权利要求4-7任一项所述的方法，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。
  9. 根据权利要求1、2、5-8任一项所述融合蛋白、权利要求2、5-8任一项所述的疫苗组合产品、权利要求3、5-8任一项所述的用途或权利要求4-8任一项所述的方法，其中，所述包含突变型SARS-CoV-2多肽的融合蛋白包含与SEQ ID NO:14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，所述包含突变型SARS-CoV-2多肽的融合蛋白为SEQ ID NO:14；
    优选地，所述包含野生型SARS-CoV-2的融合蛋白包含与SEQ ID NO:13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，所述包含野生型SARS-CoV-2多肽的融合蛋白为SEQ ID NO:13。
  10. 编码权利要求1、2、5-9任一项所述融合蛋白或权利要求2、5-9任一项所述的疫苗组合产品的核酸；
    优选地，所述包含野生型SARS-CoV-2多肽的融合蛋白的编码核酸包含与SEQ ID NO:15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；所述野生型SARS-CoV-2的融合蛋白的编码核酸为SEQ ID NO:15所示的核酸；
    优选地，所述包含突变型SARS-CoV-2多肽的融合蛋白的编码核酸包含与SEQ ID NO:16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，所述核酸为SEQ ID NO:16所示的核酸。
  11. 一种包含权利要求10所述的核酸的表达载体；
    优选地，所述表达载体选自质粒、粘粒、病毒载体、RNA载体或线性或圆形DNA或RNA分子；
    优选地，所述质粒选自pCI、puc57、pcDNA3、pSG5、pJ603和pCMV；
    优选地，所述病毒载体选自腺病毒、逆转录病毒、细小病毒(例如，腺伴随病毒)、冠状病毒、负链RNA病毒诸如正粘病毒(例如，流感病毒)、弹状病毒(例如，狂犬病和水疱性口炎病毒)、副粘病毒(例如，麻疼和仙台)、正链RNA病毒诸如小RNA病毒和甲病毒，和双链DNA病毒；
    优选地，所述腺病毒载体选自人、黑猩猩或恒河猴腺病毒；优选地，所述腺病毒选自血清型Ad2、Ad4、Ad5、Ad6、Ad11、Ad12、Ad24、Ad26、Ad34、Ad35、Ad40、Ad48、Ad49、Ad50、Ad52和Pan9腺病毒；
    优选地，所述逆转录病毒选自禽造白细胞组织增生-肉瘤、哺乳动物C-型、B-型病毒、D-型病毒、HTLV-BLV集合、慢病毒和泡沫病毒；
    优选地，所述慢病毒载体选自HIV-1、HIV-2、SIV、FIV、BIV、EIAV、CAEV和绵羊脱髓鞘性脑白质炎慢病毒；
    优选地，所述双链DNA病毒包括腺病毒、疱疹病毒(例如，单纯疱疹病毒1和2型、愛泼斯坦 -巴尔病毒、巨细胞病毒)和痘病毒(例如，牛痘病毒、鸡痘病毒和金丝雀痘病毒)、诺沃克病毒、披膜病毒、黄病毒、呼肠孤病毒、乳多泡病毒、嗜肝DNA病毒、杆状病毒和肝炎病毒。
  12. 一种表达权利要求1、2、5-9任一项所述的融合蛋白、表达权利要求2、5-9任一项所述疫苗组合产品、包含权利要求10所述核酸和/或包含权利要求11所述表达载体的宿主细胞；
    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，所述真核细胞突变酵母细胞、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。
  13. 一种药物组合物，其包含权利要求1、2、5-9任一项所述的融合蛋白、权利要求2、5-9任一项所述的疫苗组合产品、权利要求10所述的核酸、权利要求11所述的表达载体和/或权利要求12所述的宿主细胞，以及任选地一种或多种药学上可接受载体、稀释剂或赋形剂。
  14. 一种治疗和/或预防与SARS-CoV-2相关的疾病或病状的疫苗，其包含权利要求1、2、5-9任一项所述的融合蛋白、权利要求2、5-9任一项所述的疫苗组合产品、权利要求10所述的核酸、权利要求11所述的表达载体、权利要求12所述的宿主细胞和/或权利要求13所述的药物组合物，以及任选地，药学上可接受的载体、赋形剂、稀释剂或佐剂；
    优选地，所述亲本株疫苗和/或所述突变株疫苗独立地还包含一种或更多种药学上可接受的载体、赋形剂、稀释剂或佐剂；
    优选地，所述佐剂选自氢氧化铝、磷酸铝、皂苷例如Quil A、QS-21、GPI-0100、油包水型乳状液、水包油型乳状液、水包油包水型乳状液；
    优选地，其中所述亲本株疫苗和/或所述突变株疫苗呈液体、乳液、固体、气雾剂、薄雾或气体的形式；
    优选地，所述突变株疫苗是引发疫苗，并且所述亲本株疫苗是加强疫苗；
    优选地，所述亲本株疫苗是引发疫苗，并且所述突变株疫苗是加强疫苗；
    优选地，所述加强疫苗在所述引发疫苗初始给予后间隔一段时间给予以进行免疫；
    优选地，所述加强疫苗在所述引发疫苗初始给予后间隔一段时间首次给予以进行第一次加强免疫，并且，所述加强疫苗在引发疫苗初始给予后间隔另一段时间再次给予以进行第二次加强免疫；优选地，所述亲本株疫苗是引发疫苗，并且所述突变株疫苗是加强疫苗，所述突变株疫苗在所述亲本株疫苗初始给予后间隔一段时间首次给予，并且，所述突变株疫苗在亲本株疫苗初始给予后间隔另一段时间再次给予；
    优选地，所述引发疫苗在所述引发疫苗初始给予后间隔一段时间再次给予以进行第一次加强免疫，并且，所述加强疫苗在引发疫苗初始给予后间隔另一段时间首次给予以进行第二次加强免疫；优选地，所述亲本株疫苗是引发疫苗，并且所述突变株疫苗是加强疫苗，所述亲本株疫苗在所述亲本株疫苗初始给予后间隔一段时间再次给予，并且，所述突变株疫苗在亲本株疫苗初始给予后间隔另一段时间首次给予；
    优选地，所述加强疫苗在所述引发疫苗初始给予后大约1-10周给予，优选2-8、3-8、1-3、2-5、2-3、3-5、5-8周给予，更优选2-8、2-5，3-5周给予；
    更优选地，所述加强疫苗在所述引发疫苗初始给予后大约1-10周首次给予，优选2-8、3-8、1-3、2-5、2-3、3-5、5-8周首次给予，更优选2-8、2-5，3-5周首次给予；所述加强疫苗在所述引发疫苗初始给予后大约4-72周再次给予，优选6-52、6-45、6-30、6-25、8-52、8-30、8-25、4-12周，更优选4-12、6-25、8-25周再次给予；
    更优选地，所述引发疫苗在所述引发疫苗初始给予后大约1-10周再次给予，优选2-8、3-8、1-3、2-5、2-3、3-5、5-8周再次给予，更优选2-8、2-5，3-5周再次给予；所述加强疫苗在所述引发疫苗初始给予后大约4-72周首次给予，优选6-52、6-45、6-30、6-25、8-52、8-30、8-25、4-12周，更优选4-12、6-25、8-25周首次给予；
    更优选地，所述加强疫苗在所述引发疫苗初始给予后大约2-5周首次给予，并在所述引发疫苗初始给予后大约6-25周再次给予；
    更优选地，所述引发疫苗在所述引发疫苗初始给予后大约2-5周再次给予，所述加强疫苗在所述引发疫苗初始给予后大约6-25周首次给予。
  15. 根据权利要求14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。
  16. 一种预防和/或治疗与SARS-CoV-2相关的疾病或病状的疫苗的制备方法，所述方法包括表达如权利要求1、2、5-9任一项所述融合蛋白或权利要求2、5-9任一项所述的疫苗组合产品；
    优选地，所述与SARS-CoV-2相关的疾病或病状为SARS-CoV-2感染或COVID-19。
  17. 一种权利要求1、2、5-9任一项所述的融合蛋白、权利要求2、5-9任一项所述的疫苗组合产品、权利要求10所述的核酸、权利要求11所述的表达载体、权利要求12所述的宿主细胞、权利要求13所述的药物组合物、权利要求14或15所述的疫苗在制备预防和/或治疗与SARS-CoV-2相关的疾病或病状的药物或产品中的用途；
    优选地，所述与SARS-CoV-2相关的疾病或病状为SARS-CoV-2感染或COVID-19。
  18. 一种试剂盒，其包含权利要求1、2、5-9任一项所述的融合蛋白、权利要求2、5-9任一项所述的疫苗组合产品、权利要求10所述的核酸、权利要求11所述的表达载体、权利要求12所述的宿主细胞、权利要求13所述的药物组合物和/或权利要求14或15所述的疫苗。
  19. 成套药盒，其包括以下组分：
    亲本株疫苗，与药学上可接受佐剂、稀释剂或载体联合；
    突变株疫苗，与药学上可接受佐剂、稀释剂或载体联合；
    其中所述组分以分开施用的形式提供。
  20. 权利要求19所述成套药盒包括：
    含亲本株疫苗与药学上可接受佐剂、稀释剂或载体的第一容器；和含突变株疫苗与药学上可接受佐剂、稀释剂或载体的第二容器。
  21. 权利要求18所述的试剂盒或权利要求19或20所述的成套药盒在制备用于在有需要的受试者中诱导针对新型冠状病毒SARS-CoV-2的免疫应答的药物组合物中的用途；
    优选地，所述与SARS-CoV-2相关的疾病或病状为SARS-CoV-2感染或COVID-19。
  22. 一种预防和/或治疗与SARS-CoV-2相关的疾病或病状的方法，所述方法包括向受试者施用有 效量的权利要求1、2、5-9任一项所述的融合蛋白、权利要求2、5-9任一项所述的疫苗组合产品、权利要求10所述的核酸、权利要求11所述的表达载体、权利要求12所述的宿主细胞、权利要求13所述的药物组合物、权利要求14或15所述的疫苗、权利要求18所述的试剂盒和/或权利要求19或20所述的成套药盒；
    优选地，所述与SARS-CoV-2相关的疾病或病状为SARS-CoV-2感染或COVID-19。
  23. 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、2、5-9任一项所述的融合蛋白、权利要求2、5-9任一项所述的疫苗组合产品、权利要求10所述的核酸、权利要求11所述的表达载体、权利要求12所述的宿主细胞、权利要求13所述的药物组合物、权利要求14或15所述的疫苗、权利要求18所述的试剂盒和/或权利要求19或20所述的成套药盒。
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Families Citing this family (2)

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Publication number Priority date Publication date Assignee Title
CN114717205A (zh) * 2022-03-29 2022-07-08 中国人民解放军军事科学院军事医学研究院 一种冠状病毒RBDdm变异体及其应用
CN117511968B (zh) * 2023-11-06 2024-07-05 军事科学院军事医学研究院军事兽医研究所 表达SARS-CoV-2的S、E和M蛋白的重组金丝雀痘病毒及其构建方法和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004094454A2 (en) * 2003-04-18 2004-11-04 Idm Pharma, Inc. Hla-a2 tumor associated antigen peptides and compositions
WO2021160346A1 (en) * 2020-02-13 2021-08-19 Institut Pasteur Nucleic acid vaccine against the sars-cov-2 coronavirus

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004094454A2 (en) * 2003-04-18 2004-11-04 Idm Pharma, Inc. Hla-a2 tumor associated antigen peptides and compositions
WO2021160346A1 (en) * 2020-02-13 2021-08-19 Institut Pasteur Nucleic acid vaccine against the sars-cov-2 coronavirus

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BARROS-MARTINS, J. ET AL.: "Immune responses against SARS-CoV-2 variants after heterologous and homologous ChAdOx1nCoV-19/BNT162b2 vaccination", NATURE MEDICINE, vol. 27, 14 July 2021 (2021-07-14), XP037562946, DOI: 10.1038/s41591-021-01449-9 *
DATABASE PROTEIN ANONYMOUS : "Surface glycoprotein, partial [Severe acute respiratory syndrome coronavirus 2]", XP093046916, retrieved from NCBI *
LIU ZEZHONG, ET AL: "RBD-Fc-based COVID-19 vaccine candidate induces highly potent SARS-CoV-2 neutralizing antibody response", SIGNAL TRANSDUCTION AND TARGETED THERAPY, vol. 5, no. 1, 1 December 2020 (2020-12-01), pages 1 - 10, XP055950023, DOI: 10.1038/s41392-020-00402-5 *
LIU, XINXUE ET AL.: "Safety and immunogenicity of heterologous versus homologous prime-boost schedules with an adenoviral vectored and mRNA COVID-19 vaccine (Com-COV): a single-blind, randomised, non-inferiority trial", LANCET, vol. 398, 6 August 2021 (2021-08-06), XP086763578, DOI: 10.1016/S0140-6736(21)01694-9 *
MLCOCHOVA, P. ET AL.: "SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion", NATURE, vol. 599, 6 September 2021 (2021-09-06), XP037607898, DOI: 10.1038/s41586-021-03944-y *
SHI QINGFENG, GAO XIAO-DONG; HU BI-JIE: "Research progress on characteristics, epidemiology and control measure of SARS-CoV-2 Delta VOC", ZHONG HUA YI YUAN GAN RAN XUE ZA ZHI= CHINESE JOURNAL OF NOSOCOMICOLOGY, XX, CN, vol. 31, no. 24, 8 July 2021 (2021-07-08), CN , pages 3703 - 3707, XP093046918, ISSN: 1005-4529, DOI: 10.11816/cn.ni.2021-211664 *
SHU YA-JUN, HE JIAN-FENG, PEI RONG-JUAN, HE PENG, HUANG ZHU-HANG, CHEN SHAO-MIN, OU ZHI-QIANG, DENG JING-LONG, ZENG PEI-YU, ZHOU J: "Immunogenicity and safety of a recombinant fusion protein vaccine (V-01) against coronavirus disease 2019 in healthy adults: a randomized, double-blind, placebo-controlled, phase II trial", CHINESE MEDICAL JOURNAL / ZHONGHUA YIXUE ZAZHI YINGWEN BAN., CHINESE MEDICAL ASSOCIATION, BEIJING., CN, vol. 134, no. 16, 1 January 2021 (2021-01-01), CN , pages 1967 - 1976, XP093046922, ISSN: 0366-6999, DOI: 10.1097/CM9.0000000000001702 *
SONG SHUO, ZHOU BING, CHENG LIN, LIU WEILONG, FAN QING, GE XIANGYANG, PENG HUA, FU YANG-XIN, JU BIN, ZHANG ZHENG: "Sequential immunization with SARS-CoV-2 RBD vaccine induces potent and broad neutralization against variants in mice", VIROLOGY JOURNAL, vol. 19, no. 1, 1 December 2022 (2022-12-01), XP093046924, DOI: 10.1186/s12985-021-01737-3 *
SUN SHIYU, CHEN XI, LIN JINGJING, AI JUNWEN, YANG JIAMING, HU ZHENXIANG, FU YANG-XIN, PENG HUA: "Broad neutralization against SARS-CoV-2 variants induced by a next-generation protein vaccine V-01", CELL DISCOVERY, vol. 7, no. 1, XP093046925, DOI: 10.1038/s41421-021-00350-6 *
SUN, SHIYU ET AL.: "Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2", CELL RESEARCH, vol. 31, 15 July 2021 (2021-07-15), XP037566854, DOI: 10.1038/s41422-021-00531-8 *
ZHANG ZHIREN, HE QIAREN, ZHAO WEI, LI YONG, YANG JIAMING, HU ZHENXIANG, CHEN XI, PENG HUA, FU YANG-XIN, CHEN LONG, LU LIGONG: "A Heterologous V-01 or Variant-Matched Bivalent V-01D-351 Booster following Primary Series of Inactivated Vaccine Enhances the Neutralizing Capacity against SARS-CoV-2 Delta and Omicron Strains", JOURNAL OF CLINICAL MEDICINE, vol. 11, no. 14, 1 January 2022 (2022-01-01), pages 4164, XP093046923, DOI: 10.3390/jcm11144164 *

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