WO2021228167A1 - 通过形成Fc片段融合蛋白糖缀合物增强抗原免疫原性的方法 - Google Patents

通过形成Fc片段融合蛋白糖缀合物增强抗原免疫原性的方法 Download PDF

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WO2021228167A1
WO2021228167A1 PCT/CN2021/093470 CN2021093470W WO2021228167A1 WO 2021228167 A1 WO2021228167 A1 WO 2021228167A1 CN 2021093470 W CN2021093470 W CN 2021093470W WO 2021228167 A1 WO2021228167 A1 WO 2021228167A1
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protein
antigen
cancer
peptide antigen
antibody
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PCT/CN2021/093470
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French (fr)
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谢良志
张延静
孙春昀
罗春霞
张建东
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神州细胞工程有限公司
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Priority to CN202180031113.6A priority Critical patent/CN115551896A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • C12N15/63—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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention belongs to the field of immunology. Specifically, it relates to a method for enhancing the immunogenicity of a protein/peptide antigen, wherein the protein/peptide antigen and an Fc fragment, preferably receptor binding/complement binding enhances the Fc fragment to form a fusion protein, and is further combined with a sugar Conjugation to form a fusion protein glycoconjugate.
  • the Fc fragment in the preferred embodiment has an improved binding ability to Fc receptors and or complement protein C1q compared to its natural form due to changes in its amino acid sequence and/or glycosylation form. .
  • SARS-CoV-2RBD region as the antigen of the immune composition, and form a fusion protein with the amino acid sequence and fucose altered form of the Fc region, and further form a fusion protein glycoconjugate with pneumonia polysaccharide , Used to prevent SARS-CoV-2 infection related diseases.
  • Non-live vaccines that is, vaccines that use subunits of infectious microorganisms, toxins, viruses, and tumor cells as antigens, are widely adopted because of their safety.
  • the immunogenicity of subunit antigens is poor, and its ability to induce specific immune responses is weak.
  • the traditional method of enhancing immunogenicity is to add immune adjuvants.
  • New methods of enhancing the immune response are still under constant research and exploration.
  • Fc fusion protein is another new approach, that is, a new type of recombinant protein produced by fusing a functional protein with biological activity with an Fc fragment. It not only retains the biological activity of the functional protein molecule, but also has some antibody properties. Like the binding of FcRs and related biological functions mediated. These two methods will be detailed below.
  • Conventional conjugate vaccines include encephalitis vaccine, haemophilus influenza bacteria b vaccine and pneumonia vaccine. Its purified capsular polysaccharide is combined with carrier protein to produce more effective immunogenic compositions.
  • carrier proteins such as tetanus Toxoid, tetanus toxoid fragment C, tetanus toxin non-toxic mutant, diphtheria toxoid, CRM197, other non-toxic mutants of diphtheria toxin such as CRM176, CRM197, CRM228, CRM45 (Uchida et al. J. Biol. Chem. 218; 3838-3844, 1973); CRM9, CRM45, CRM02, CRM103 and CRM107 and other mutants.
  • the above-mentioned conjugate vaccine uses polysaccharides as antigenic substances.
  • Such polysaccharide antigens are non-thymocyte-dependent antigens and cannot produce cellular immune responses and cannot form immune memory.
  • Protective antibodies cannot be formed in children or people with low immunity.
  • the polysaccharide antigen is conjugated to a protein carrier with T cell epitopes, the antigen-presenting cell or B cell endocytosed and processed the conjugate of sugar and protein, and then the polypeptide fragment of the carrier protein was displayed on the cell surface to activate helper T cells , Causing a series of immune responses to generate protective antibodies and immune memory.
  • US5192540A discloses a vaccine containing an immunogenic conjugate of Haemophilus influenzae type B 38,000 Dalton or 40,000 Dalton outer membrane protein and Haemophilus influenzae type B oxidized polyribose-ribitol-phosphopolysaccharide fragment, which It can be used to immunize diseases caused by Haemophilus influenzae type B.
  • the conjugate vaccine of the present invention has high immunogenicity in animal models. Their antibody response to PRP is significantly higher than the previously reported antibody response.
  • the conjugate vaccine also induces the major protein of Haemophilus influenzae type B (38K or 40k protein) Antibody.”
  • US9296795B discloses the use of an immunogenic polysaccharide-protein conjugate having a polysaccharide antigen (or an oligosaccharide fragment thereof, representing one or more epitopes) derived from a hospital pathogen in an immunogenic composition, the polysaccharide- The protein conjugate is conjugated to the staphylococcal surface adhesion carrier protein to elicit an antibody response to the polysaccharide antigen and the staphylococcal surface adhesion carrier protein.
  • the patent discloses "The conjugate of the present invention has unique advantages: it can induce the production of antibodies against polysaccharide antigens and surface adhesin carrier proteins (both are virulence factors), and confer on diseases caused by hospital pathogens. Immunity.
  • the surface adhesin protein itself can also confer immunity to the body, rather than just acting as a protein carrier for polysaccharide antigens.”
  • “Surface adhesin protein specificity induced by conjugated surface adhesin protein The titer of the antibody is similar to that of the unconjugated surface adhesin protein ( Figure 17-20). This confirms that the epitope is not changed by the binding of the surface adhesin protein and CP.”
  • the name of the inventor's invention is "A method for enhancing the immunogenicity of protein-peptide antigens", the application number is PCT/CN2021/090809 (priority application number CN202010369100.7), and the application date is April 2021
  • the invention patent application on the 29th reported the applicant's pioneering invention: by conjugating protein/peptide antigens with sugars to form sugar-protein/peptide antigen conjugates, the immunogenicity of protein/peptide antigens was improved.
  • the inventor’s invention is titled "Method for enhancing protein/peptide antigen immunogenicity by forming a fusion protein with an altered Fc fragment", and the application number is PCT/CN2021/092013 (priority application number CN202010394463.6), Another pioneering invention of the applicant is reported in the invention patent application filed on May 7, 2021: by combining protein/peptide antigens with Fc fragments, preferably receptor binding/complement binding to enhance Fc fragments to form fusion proteins, Improve the immunogenicity of protein/peptide antigens.
  • B cell-mediated humoral immunity is one of the body's protective mechanisms mediated by vaccines.
  • FcRs Fc receptors
  • CR complement receptors
  • FcRs Fc receptors
  • CR complement receptors
  • FcRs Fc receptors
  • CR complement receptors
  • FcRs Fc receptors
  • CR complement receptors
  • FcRs Fc receptors
  • fDC follicular dendritic cells
  • This part of the antigen is essential for maintaining the long-term existence of the antigen and maintaining the survival of antigen-specific B cells. Very important [ 7 ].
  • cytotoxic T lymphocytes play an important role in resisting virus infection and removing virus-infected cells.
  • CTL cytotoxic T lymphocytes
  • the antigen cross-presentation mechanism allows exogenous antigens to enter the cell’s endogenous processing and presentation mechanism, so that the exogenous antigen peptides are displayed on MHC class I molecules to be recognized by T cells and initiate CTL cell responses.
  • the cross-presentation of foreign antigens is of great significance for effectively activating CTLs and triggering antiviral immune responses. Therefore, enhancing the cross-presentation of subunit vaccines is one of the effective strategies to improve vaccine immunity.
  • Dendritic cells are currently known as the most powerful professional antigen-presenting cells, and they are also the cells that mainly perform cross-presentation.
  • phagocytosis endocytosis
  • pinocytosis receptor-mediated endocytosis.
  • endocytic receptors related to the cross-presentation of foreign antigens, including C-type lectin receptors (CLR), Fc receptors that recognize immune complex IgG (FcRs), and scavengers that recognize apoptotic cells Receptors, chemokine receptors, etc., these receptors mediate the endocytosis of antigens and enter specific endosomes before they can bind to MHC class I molecules, thereby activating CD8 + T cells.
  • CLR C-type lectin receptors
  • FcRs Fc receptors that recognize immune complex IgG
  • scavengers that recognize apoptotic cells Receptors, chemokine receptors, etc.
  • Antigen-antibody complexes can be recognized by the FcRs of DC cells, and the resulting cross-linking can internalize the antigen, and cross-process and present the antigen to specifically activate the CTL response [ 8-10 ]. This cross-presentation of antigens mediated by FcRs has been shown to induce strong CTL responses [ 11 ].
  • the Fc receptors (FcR ⁇ ) that bind to IgG in the human body mainly include Fc ⁇ RI (CD64), Fc ⁇ RIIA (CD32a), Fc ⁇ RIIB (CD32B), Fc ⁇ RIII (CD16), etc. [ 12 ].
  • Fc ⁇ RIIB is an inhibitory receptor, which is mainly expressed on B cells, macrophages and mast cells [ 13 ]. It can be divided into Fc ⁇ RIIB-1 and Fc ⁇ RIIB-2. Fc ⁇ RIIB-1 is only expressed on B cells and controls the excessive activation of B cells and the recognition of self-antigens during the development of B cells.
  • Fc ⁇ RIIB-2 is expressed on immune cells other than NK and T cells, and the cross-linking of receptors can effectively induce the phagocytosis of antigen-antibody complexes [ 14 , 15 ].
  • the other Fc receptors are all activating receptors.
  • CD64 is mainly expressed on monocytes, macrophages, DCs and other cells
  • CD32a is mainly expressed on neutrophils
  • CD16a is mainly It is expressed in NK, monocytes, and macrophages [ 15 ].
  • ITAM intracellular receptor tyrosine activation motif
  • Fc fusion protein refers to a new type of recombinant protein produced by fusing a certain biologically active functional protein with an Fc fragment using genetic engineering and other technologies. It not only retains the biological activity of the functional protein molecule, but also has some antibody properties, such as FcRs binding and related biological functions mediated.
  • Antigen-Fc fusion protein can be used as an antigen delivery vehicle, with the help of Fc fragments to target and bind antigen-presenting cells, shorten the free time of antigen in plasma, increase the half-life of antigen, and strengthen the reaction of antigen presentation and antigen cross-presentation.
  • the number of Fc fusion protein vaccines in clinical trials is relatively small.
  • patients with chronic type B cHBV infection were given a YIC immunogenic complex (the hepatitis B surface antigen HBsAg expressed by yeast and the human serum neutralizing antibody HBIG immunized with HBsAg at a specific ratio of 60 ⁇ g/4 weeks) Mixed incubation and aluminum adjuvant) treatment, compared with the control group given only aluminum adjuvant, the negative rate of serum hepatitis B virus E antigen (HBeAg) was significantly increased (21.8% vs 9%), and the virus titer was significant Decrease, and accompanied by the production of anti-E antigen antibodies [ 22 ].
  • HBeAg serum hepatitis B virus E antigen
  • YIC can increase the maturation (high CD83 expression), antigen recognition and presentation (expression of HLA-II, CD86, CD80, CD40 markers) of DC cells in patients infected with cHBV, and secrete more inflammation Sex factor (IL-12).
  • the patient’s DC-PBMC mixed cells produced more T lymphocyte cytokines (Th1 cells: IL-2, IFN ⁇ ) under YIC stimulation than HBsAg antigen alone (Th2 cells: IL-5, IL-10) [ 23 ] .
  • Th1 cells IL-2, IFN ⁇
  • Th2 cells IL-5, IL-10)
  • the excessive stimulation of YIC may cause the body's immune fatigue, thereby reducing the cellular immune response [ 24 ]. Therefore, in order to achieve a better immune effect, a suitable immunization program is also particularly important.
  • virus antigen-Fc fusion protein vaccines RSV, HBV, DENV, TB
  • RSV virus antigen-Fc fusion protein vaccines
  • AE Phase IIb clinical side effects
  • the Fc fragment is modified to enhance the binding of FcRs, and Fe4-Fc modified molecules can be obtained, which can increase the binding of Fc to complement protein C1q and Fc receptors CD16, CD32, CD64, etc. [ 17-21 ], and it is possible to further enhance Fc Its receptor-mediated antigen capture and presentation, enhance the maturation of B cells and the production of high-affinity antibodies, maintain long-term humoral immunity, and enhance the CTL immune response mediated by antigen cross-presentation.
  • the inventor’s invention is titled "A method for enhancing the immunogenicity of protein/peptide antigens by forming a fusion protein with an altered Fc fragment", the application number is CN202010394463.6, and the application date is May 11, 2020.
  • the applicant’s technological invention is reported in the paper: by forming a fusion protein between a protein/peptide antigen and a modified antibody Fc fragment, the immunogenicity of the protein/peptide antigen is improved, wherein the Fc fragment is due to its amino acid sequence and/or The altered glycosylation form has an improved binding capacity to Fc receptor and/or complement protein C1q compared to its natural form.
  • the present invention provides a method for enhancing the immunogenicity of a protein/peptide antigen, the method comprising
  • the fusion protein is conjugated with a sugar to form a fusion protein glycoconjugate, preferably
  • Fc fragments are receptor binding/complement binding enhancing Fc fragments.
  • the protein/peptide antigen in the method is a pathogen-related protein/peptide antigen or a tumor-related protein/peptide antigen.
  • the pathogen in the method is selected from:
  • Coronavirus human immunodeficiency virus HIV-1, human herpes simplex virus, cytomegalovirus, rotavirus, Epstein-Barr virus, varicella-zoster virus, hepatitis virus, respiratory syncytial virus, parainfluenza virus, measles virus, epidemic Mumps virus, human papilloma virus, flavivirus or influenza virus, Neisseria, Moraxella, Bordetella, Mycobacterium, including Mycobacterium tuberculosis; Escherichia , Including enterotoxin Escherichia coli; Salmonella, Listeria, Helicobacter, Staphylococcus, including Staphylococcus aureus, Staphylococcus epidermidis; Borrelia, Chlamydia, including Chlamydia trachomatis, Chlamydia pneumoniae; Plasmodium , Including Plasmodium falciparum; Toxoplasma gondii, Candida;
  • the tumor is selected from:
  • the protein/peptide protein antigen in the method is selected from a secreted protein or a full-length membrane protein, or a functional domain, a mutein, a truncated protein, or one or more antigen polypeptides.
  • the protein/peptide antigen in the method is selected from
  • coronavirus spike protein Preferably coronavirus spike protein
  • coronavirus spike protein fragment More preferably, the coronavirus spike protein fragment
  • ACE2 receptor binding domain (RBD) of the coronavirus spike protein
  • the S1 subunit of the coronavirus spike protein More preferably, the S1 subunit of the coronavirus spike protein
  • the aforementioned coronavirus is selected from SARS-CoV-2.
  • the Fc fragment in the method is derived from the heavy chain constant region of a human antibody, murine antibody, rabbit antibody or other animal antibody.
  • the Fc fragment in the method is derived from an IgG, IgM or IgA subtype antibody of a human antibody,
  • antibodies of IgG1, IgG2, IgG3 or IgG4 subtype are preferred from antibodies of IgG1, IgG2, IgG3 or IgG4 subtype;
  • it is an IgG1 modified Fc fragment that undergoes amino acid sequence mutations and/or changes in glycosylation for the purpose of improving the Fc receptor/C1q complement binding function.
  • the Fc receptor of the method is selected from CD16, CD32a, CD32b, or CD64.
  • the modified antibody Fc fragments are Fc receptors CD32a, CD32b and CD64 binding enhancing fragments/complement C1q binding enhancing fragments;
  • the modified antibody Fc fragments are Fc receptors CD16a, CD32a, CD32b and CD64 binding enhancing fragments/complement C1q binding enhancing fragments;
  • amino acid sequence is shown in SEQ ID NO: 4, and it is produced by fucose knockout mammalian cells.
  • the mammalian cell is fut8 knockout HEK-293 cell.
  • the antigen in the method is preferably conjugated to other macromolecules through a linker.
  • the other macromolecules are polysaccharides, peptides/proteins.
  • the other macromolecules in the method are selected from polysaccharides, oligosaccharides or monosaccharides;
  • Neisserial encephalitis capsular polysaccharide Haemophilus influenzae b capsular polysaccharide, Streptococcus pneumoniae capsular polysaccharide, group B Staphylococcus aureus capsular polysaccharide, dextran, mannan, starch, inulin , Pectin, carboxymethyl starch, chitosan and its derivatives;
  • the capsular polysaccharide of Streptococcus pneumoniae serotype 14 the capsular polysaccharide of Streptococcus pneumoniae serotype 6B, and the capsular polysaccharide of Streptococcus pneumoniae serotype 7F;
  • capsular polysaccharide of Streptococcus pneumoniae serotype 14 is most preferred.
  • the molecular weight of the conjugate in the method is 800-6000 KDa.
  • the protein/peptide antigen in the form of a fusion protein glycoconjugate and an immune adjuvant are combined in the method.
  • the adjuvant in the method is selected from aluminum adjuvant, MF59 or a mixture thereof.
  • the present invention provides a protein/peptide antigen with enhanced immunogenicity, which comprises
  • Fusion protein/peptide antigen and Fc fragment to form a fusion protein
  • Protein/peptide-Fc fragment fusion protein glycoconjugate formed by conjugation with sugar preferably
  • Fc fragments are receptor binding/complement binding enhancing Fc fragments.
  • the protein/peptide antigen wherein the protein/peptide antigen is a pathogen-related protein/peptide antigen or a tumor-related protein/peptide antigen.
  • the protein/peptide antigen in one embodiment, the protein/peptide antigen, and
  • the pathogen is selected from:
  • Coronavirus human immunodeficiency virus HIV-1, human herpes simplex virus, cytomegalovirus, rotavirus, Epstein-Barr virus, varicella-zoster virus, hepatitis virus, respiratory syncytial virus, parainfluenza virus, measles virus, epidemic Mumps virus, human papilloma virus, flavivirus or influenza virus, Neisseria, Moraxella, Bordetella, Mycobacterium, including Mycobacterium tuberculosis; Escherichia , Including enterotoxin Escherichia coli; Salmonella, Listeria, Helicobacter, Staphylococcus, including Staphylococcus aureus, Staphylococcus epidermidis; Borrelia, Chlamydia, including Chlamydia trachomatis, Chlamydia pneumoniae; Plasmodium , Including Plasmodium falciparum; Toxoplasma gondii, Candida;
  • the tumor is selected from:
  • the protein/peptide antigen wherein the protein/peptide protein antigen is selected from a secreted protein or a full-length membrane protein, or a functional domain, a mutein, a truncated protein, or one or more of them A modified protein composed of a splicing of antigen polypeptide epitopes.
  • the protein/peptide antigen wherein the protein/peptide antigen is selected from
  • coronavirus spike protein Preferably coronavirus spike protein
  • coronavirus spike protein fragment More preferably, the coronavirus spike protein fragment
  • ACE2 receptor binding domain (RBD) of the coronavirus spike protein
  • the S1 subunit of the coronavirus spike protein More preferably, the S1 subunit of the coronavirus spike protein
  • the aforementioned coronavirus is selected from SARS-CoV-2.
  • the Fc fragment in the protein/peptide antigen is derived from the heavy chain constant region of a human antibody, murine antibody, rabbit antibody or other animal antibody.
  • the Fc fragment in the protein/peptide antigen is derived from an IgG, IgM or IgA subtype antibody of a human antibody,
  • antibodies of IgG1, IgG2, IgG3 or IgG4 subtype are preferred from antibodies of IgG1, IgG2, IgG3 or IgG4 subtype;
  • it is an IgG1 modified Fc fragment with amino acid sequence mutations and/or glycosylation changes for the purpose of improving the function of binding to Fc receptors and C1q complement.
  • the protein/peptide antigen, Fc receptor is selected from CD16, CD32a, CD32b or CD64.
  • the protein/peptide antigen in one embodiment, the protein/peptide antigen
  • the modified antibody Fc fragments are Fc receptors CD32a, CD32b and CD64 binding enhancing fragments/complement C1q binding enhancing fragments;
  • the protein/peptide antigen in one embodiment, the protein/peptide antigen
  • the modified antibody Fc fragments are Fc receptors CD16a, CD32a, CD32b and CD64 binding enhancing fragments/complement C1q binding enhancing fragments;
  • amino acid sequence is shown in SEQ ID NO: 4, and it is produced by fucose knockout mammalian cells.
  • the mammalian cell is fut8 knockout HEK-293 cell.
  • the antigen in the protein/peptide antigen is preferably conjugated to other macromolecules through a linker.
  • the other macromolecules are polysaccharides, peptides/proteins.
  • the other macromolecules in the protein/peptide antigen are selected from polysaccharides, oligosaccharides or monosaccharides;
  • Neisserial encephalitis capsular polysaccharide Haemophilus influenzae b capsular polysaccharide, Streptococcus pneumoniae capsular polysaccharide, group B Staphylococcus aureus capsular polysaccharide, dextran, mannan, starch, inulin , Pectin, carboxymethyl starch, chitosan and its derivatives;
  • the capsular polysaccharide of Streptococcus pneumoniae serotype 14 the capsular polysaccharide of Streptococcus pneumoniae serotype 6B, and the capsular polysaccharide of Streptococcus pneumoniae serotype 7F;
  • capsular polysaccharide of Streptococcus pneumoniae serotype 14 is most preferred.
  • the molecular weight of the conjugate in the protein/peptide antigen is 800-6000 KDa.
  • the adjuvant in the protein/peptide antigen is selected from aluminum adjuvant, MF59 or a mixture thereof.
  • the present invention provides an immune composition comprising
  • the protein/peptide antigen of the present invention is the protein/peptide antigen of the present invention.
  • a pharmaceutically acceptable carrier, excipient or stabilizer preferably
  • a pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized formulation or an aqueous solution is provided.
  • the adjuvant in the immune composition may be at least one selected from aluminum adjuvant and MF59.
  • the present invention provides the protein/peptide antigen of the present invention, or the immune composition, which is used to prevent pathogens, preferably coronaviruses, more preferably SARS-CoV-2 caused diseases/tumors. .
  • the present invention provides the protein/peptide antigen of the present invention or the immune composition for preparing a vaccine for preventing pathogens, preferably coronaviruses, more preferably SARS-CoV-2 diseases/tumors Applications.
  • the present invention provides an immune combination comprising
  • the protein/peptide antigen of the present invention or the immune composition of the present invention.
  • One or more additional immunogenic agents are provided.
  • the present invention provides a kit comprising
  • the protein/peptide antigen of the present invention or the immune composition of the present invention.
  • the present invention provides a method for preventing a disease/tumor caused by a pathogen, preferably a coronavirus, more preferably SARS-CoV-2, which comprises administering to a subject the protein/peptide antigen of the present invention, the present invention
  • a pathogen preferably a coronavirus, more preferably SARS-CoV-2
  • the immune composition according to the invention, the immune combination according to the invention or the kit according to the invention comprises administering to a subject the protein/peptide antigen of the present invention, the present invention
  • the immune composition according to the invention, the immune combination according to the invention or the kit according to the invention preferably a coronavirus, more preferably SARS-CoV-2
  • the present invention provides a method of immunizing an animal, which comprises administering to the animal the protein/peptide antigen of the present invention, the immune composition of the present invention, the immune combination of the present invention or the present invention Kit to produce neutralizing antibodies.
  • Figure 1 shows the ratio of IgG2a/IgG1 in mice immunized with RBD-Fc-Fe4 and RBD-Fc-Fe4-PS14 as antigens; aluminum adjuvant, MF59 adjuvant, and MF59 plus aluminum adjuvant mixed adjuvant. Three immunization doses of 1 ⁇ g, 3 ⁇ g, and 10 ⁇ g were set for each adjuvant. After the second immunization, the serum was taken to determine the ratio of IgG2a/IgG1.
  • Figure 2 shows the comparison of the serum IgG2a/IgG1 ratio of RBD-PS14, RBD-Fc-PS14, RBD-Fc-Ce3-PS14 and RBD-Fc-Fe4-PS14.
  • the inventor invented a method to further improve the immunogenicity of protein/peptide antigens: fusion of protein/peptide antigens with Fc fragments, preferably receptor binding/complement binding enhancing Fc fragments, A fusion protein is formed, and then the fusion protein is conjugated with a sugar to form a fusion protein glycoconjugate.
  • the immunogenicity of the protein/peptide antigen modified in this way is enhanced, and the above-mentioned Fc fragment has improved binding ability with Fc receptor and/or complement protein C1q due to changes in its amino acid sequence and/or glycosylation form.
  • the inventor used this invention to prepare a new coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) vaccine.
  • SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, angiotensin converting enzyme 2 (ACE2) [29].
  • ACE2 angiotensin converting enzyme 2
  • the trimeric S protein of the virus binds to the ACE2 receptor and is cleaved by the host protease into the S1 polypeptide containing the receptor binding domain (RBD) and the S2 polypeptide responsible for mediating the fusion of the virus with the cell membrane [30].
  • RBD receptor binding domain
  • the specific interaction between S1 and ACE2 triggers the conformational change of the S2 subunit, which leads to the fusion of the viral envelope and cell membrane or lysosome membrane and release of viral nucleic acid into the cytoplasm [31].
  • RBD protein is an effective target for SARS-CoV-2 vaccine.
  • antigen refers to a foreign substance recognized (specifically bound) by an antibody or T cell receptor, but it cannot definitively induce an immune response. Exogenous substances that induce specific immunity are called “immune antigens” or “immunogens.”
  • a "hapten” refers to an antigen that cannot elicit an immune response by itself (although a combination of several haptens or a combination of a hapten and a macromolecular carrier can elicit an immune response).
  • RBD receptor binding domain specifically refers to the "ACE2 receptor binding domain of coronavirus spike protein (SARS-CoV-2RBD)" in this specification and the appended claims, The above terms are used interchangeably.
  • SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, angiotensin converting enzyme 2 (ACE2).
  • ACE2 angiotensin converting enzyme 2
  • the trimeric S protein of the virus binds to the ACE2 receptor and is cleaved by the host protease into the S1 polypeptide containing the receptor binding domain (RBD) and the S2 polypeptide responsible for mediating the fusion of the virus with the cell membrane.
  • Human immune response is an antibody-mediated immune response and involves the introduction and production of antibodies that recognize and bind to the antigen in the immunogenic composition of the present invention with a certain affinity.
  • the "cell-mediated immune response” is composed of T cells and / Or other leukocyte-mediated immune responses.
  • Cell-mediated immune response is induced by providing epitopes associated with major histocompatibility complex (MHC) class I or class II molecules, CD1 or other atypical MHC-like molecules.
  • MHC major histocompatibility complex
  • polypeptide encompass chains of amino acids of any length, wherein relatively short (eg, shorter than 100 amino acids) amino acid chains are commonly referred to as peptides.
  • the chain may be straight or branched, it may contain modified amino acids, and/or non-amino acids may be intervened.
  • antibody means an immunoglobulin molecule, and refers to any form of antibody that exhibits the desired biological activity. Including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (such as bispecific antibodies), and even antibody fragments.
  • the full-length antibody structure preferably comprises 4 polypeptide chains, usually 2 heavy (H) chains and 2 light (L) chains connected to each other by disulfide bonds. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. Each light chain contains a light chain variable region and a light chain constant region. In addition to this typical full-length antibody structure, its structure also includes other derivative forms.
  • complete antibodies can be classified into five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, among which IgG and IgA can be further divided into subclasses (isotypes), such as IgG1, IgG2 , IgG3, IgG4, IgA1 and IgA2.
  • the heavy chains of the five types of antibodies are classified into ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ chains, respectively.
  • the amino acid sequence of the constant region of its light chain the light chain of an antibody can be classified into ⁇ and ⁇ . .
  • variable region refers to the domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen.
  • constant region refers to such amino acid sequences on the light chain and heavy chain of an antibody, which do not directly participate in the binding of the antibody to the antigen, but exhibit a variety of effector functions, such as antibody-dependent cytotoxicity.
  • Fc region is used to define the C-terminal region of an immunoglobulin heavy chain.
  • the "Fc region” can be a native sequence Fc region or a variant Fc region.
  • the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position 226 of Cys or from Pro230 to its carboxy terminus.
  • the numbering of residues in the Fc region is like the EU index in Kabat. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • the Fc region of IgG usually has two constant regions, CH 2 and CH 3 .
  • Fc receptor refers to a receptor that binds to the Fc region of an antibody.
  • Human FcR of natural sequence is preferred, and receptors ( ⁇ receptors) that bind to IgG antibodies are preferred, which include Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII subtypes, and variants of these receptors.
  • Other FcRs are included in the term “FcR”.
  • the term also includes the neonatal receptor (FcRn), which is responsible for the transport of maternal IgG to the fetus (Guyer et al., Journal of Immunology 117:587 (1976) and Kim et al., Journal of Immunology 24:249 (1994)).
  • FcRn neonatal Fc receptor
  • the neonatal Fc receptor (FcRn) plays an important role in the metabolic fate of IgG antibodies in the body. FcRn functions to rescue IgG from the lysosomal degradation pathway, thereby reducing its clearance in serum and increasing its half-life. Therefore, the in vitro FcRn binding properties/characteristics of IgG indicate its in vivo pharmacokinetic properties in the blood circulation.
  • Fc fusion protein refers to a new type of recombinant protein produced by fusing a certain biologically active functional protein with an Fc fragment using genetic engineering and other technologies. It not only retains the biological activity of the functional protein molecule, but also has some antibodies. Properties, such as the binding of FcRs and related biological functions mediated.
  • engineered Fc polypeptide engineered Fc region
  • engineered Fc engineered Fc
  • effector function refers to those biological activities attributable to the Fc region of an antibody, which differ by antibody isotype.
  • antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor (such as CD16, CD32, CD64) binding, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP) , Cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (such as B cell receptors) and B cell activation.
  • sugar can be used to refer to polysaccharides, oligosaccharides or monosaccharides.
  • Polysaccharides can be separated from organisms, such as bacteria, and can be natural polysaccharides.
  • the size of the polysaccharide can be adjusted to a certain extent using a microfluidization method. Adjusting the size of the polysaccharide can reduce the viscosity of the polysaccharide sample and/or improve the filterability of the conjugated product.
  • Oligosaccharides are hydrolyzed polysaccharides with a small number of repeating units (typically, 5-30 repeating units). Polysaccharides can also be chemically synthesized.
  • conjugate refers to a protein/peptide covalently conjugated to a sugar.
  • the fusion protein glycoconjugate of the present invention and the immunogenic composition containing the same may contain a certain amount of free sugar, protein/peptide.
  • conjugation refers to a process whereby sugars such as bacterial capsular polysaccharides are covalently linked to proteins/peptides.
  • immunogenic composition refers to any pharmaceutical composition containing an antigen, which composition can be used to induce an immune response in an individual.
  • Immunogenicity means an antigen (or epitope of an antigen) such as the Coronavirus Spike Protein Receptor Binding Domain (SARS-CoV-2RBD) or a fusion protein glycoconjugate containing the antigen or immune The ability of the original composition to induce a humoral or cell-mediated immune response or both in a host (e.g., a mammal).
  • SARS-CoV-2RBD Coronavirus Spike Protein Receptor Binding Domain
  • fusion protein glycoconjugate containing the antigen or immune The ability of the original composition to induce a humoral or cell-mediated immune response or both in a host (e.g., a mammal).
  • a “protective” immune response refers to the ability of an immunogenic composition to induce a humoral or cell-mediated immune response, or both, used to protect an individual from infection.
  • the protection provided does not have to be absolute, that is, it does not have to completely prevent or eradicate the infection, as long as there is a statistically significant improvement relative to a control population of individuals (for example, infected animals not administered a vaccine or immunogenic composition) . Protection may be limited to alleviating the severity of infection symptoms or rapid onset.
  • Immunogenic amount and “immunologically effective amount” are used interchangeably herein and refer to an antigen or immunogenic composition sufficient to elicit an immune response (cell (T cell) or humoral (B cell or antibody) response or two Or, as measured by standard determinations known to those skilled in the art).
  • the effectiveness of an antigen as an immunogen can be measured by a proliferation assay, by a cytolysis assay, or by measuring the level of B cell activity.
  • the present invention is based on two pioneering inventions of the inventor.
  • the protein/peptide antigen is fused with the Fc receptor binding/complement binding enhancing fragment to form a fusion protein, and then the fusion protein is conjugated with a sugar to form a fusion protein glycoconjugate.
  • fusion protein glycoconjugates as antigens of the immune composition to immunize animals can maintain long-term humoral and cellular immune responses, have higher cellular immune responses, and produce higher titers of neutralization Antibody.
  • Fusion protein glycoconjugate as protein/peptide antigen of the present invention
  • Coronaviruses mainly mediate virus invasion through the binding of spike protein (S protein) to host cell receptors, and determine the tissue or host tropism of the virus.
  • the host cell receptor protein of SARS-CoV-2 is angiotensin converting enzyme 2 (ACE2).
  • ACE2 angiotensin converting enzyme 2
  • the trimeric spike protein (S protein) of the virus binds to the ACE2 receptor and is cleaved by the host protease into the S1 polypeptide containing the receptor binding domain (SARS-COV-2 RBD) and is responsible for mediating the virus with the cell membrane
  • SARS-COV-2 RBD receptor binding domain
  • SARS-COV-2 RBD selects SARS-COV-2 RBD as the protein/peptide antigen.
  • Antigens can be obtained by extracting natural pathogens or by genetic recombination.
  • the binding ability of SARS-COV-2 RBD and Fc receptor and/or complement protein C1q improves the Fc fragment to form a fusion protein through genetic engineering.
  • the Fc fragment is the Fc receptor CD32a, CD32b, and CD64 binding enhancement fragment/complement C1q binding enhancement fragment; its amino acid sequence is shown in SEQ ID NO: 3.
  • the modified antibody Fc fragments are Fc receptors CD16a, CD32a, CD32b, and CD64 binding enhancing fragments/complement C1q binding enhancing fragments; its amino acid sequence is shown in SEQ ID NO: 4, and is Produced by CHO cells knocked out of alginose. Then, the fusion protein is conjugated with sugars, which can be polysaccharides, oligosaccharides and monosaccharides.
  • the polysaccharide may be a bacterial polysaccharide, such as common Neisserial encephalitis capsular polysaccharide, Haemophilus influenzae b capsular polysaccharide, Streptococcus pneumoniae capsular polysaccharide, group B Staphylococcus aureus capsular polysaccharide, and dextran , Mannan, etc.
  • the polysaccharide can also be a plant-derived polysaccharide, such as starch, inulin, pectin, etc., or a derivative of a chemically modified polysaccharide, such as carboxymethyl starch.
  • the polysaccharide may also be a polysaccharide of animal origin, such as chitosan and its derivatives.
  • Polysaccharides and proteins are conjugated through a chemical reaction.
  • the polysaccharide is activated first, even if the polysaccharide has reactive groups.
  • the active group and the amino group, carboxyl group, sulfhydryl group, imidazole ring on histidine, indole ring of tryptophan, benzene ring on tyrosine, phenyl ring of phenylalanine, and serine on the protein molecule Hydroxyl and glutamine, asparagine and other chemically reactive groups react to form covalent bonds.
  • One method of conjugating polysaccharides and protein molecules is to oxidize polysaccharides with sodium periodate to produce aldehyde groups on polysaccharides.
  • the aldehyde groups react with amino groups on protein molecules to form Schiff bases.
  • it is hoped that The base is reduced to a stable single bond.
  • the polysaccharide and the protein molecule form a covalent connection.
  • the immunogenic composition of the present invention further comprises at least one of adjuvants, buffers, cryoprotectants, salts, divalent cations, non-ionic detergents, free radical oxidation inhibitors, diluents or carriers A sort of.
  • An adjuvant is a substance that enhances the immune response when administered with an immunogen or antigen.
  • the immunogenic composition of the present invention may or may not contain a vaccine adjuvant.
  • Adjuvants that can be used in the composition of the present invention include, but are not limited to: at least one of MF59, QS-21 or MPL.
  • the adjuvant in the immunogenic composition of the present invention is an aluminum-based adjuvant.
  • the adjuvant used will depend on the individual to whom the immunogenic composition is administered, the prescribed route of injection, and the number of injections.
  • the immunogenic composition may optionally include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes the carriers used in the pharmacopoeias of various countries for animals (including humans and non-human mammals).
  • the term carrier can be used to refer to a diluent, adjuvant, excipient or vehicle with which the pharmaceutical composition is administered. Water, saline solutions, and aqueous dextrose and glycerol solutions can be used as liquid carriers especially for injection solutions.
  • the immunogenic composition of the invention may also contain one or more additional immunogenic agents.
  • the immunogenic composition of the present invention for treatment or prophylactic treatment can be administered to the oral cavity/esophagus, respiratory tract, urogenital tract by intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection; or via mucosal administration. Intranasal administration of vaccines is preferred for the treatment of certain diseases, such as pneumonia or otitis media.
  • the vaccine of the present invention can be administered in a single dose, its components can also be co-administered at the same time or in time sharing. In addition to a single route of administration, two different routes of administration can be used.
  • the optimal amount of the components for a particular immunogenic composition can be determined by standard studies involving observation of the appropriate immune response in the individual. After the initial vaccination, the individual can receive one or several well-spaced booster immunizations.
  • the protein/peptide antigen and immune complex of the present invention can prevent or treat diseases caused by pathogens, especially coronaviruses, and more especially diseases caused by SARS-CoV-2 virus.
  • the protein/peptide antigen and immune complex of the present invention can prevent or treat tumor diseases. It can also be used to immunize animals to produce neutralizing antibodies.
  • SARS-CoV-2 RBD SARS-CoV-2 coronavirus spike protein receptor binding domain
  • RBD-Fc A fusion protein between the SARS-CoV-2 coronavirus spike protein receptor binding domain and the Fc fragment of human IgG
  • RBD-Fc-Ce3 A fusion protein between the SARS-CoV-2 coronavirus spike protein receptor binding region and the Fc fragment of human IgG, where the Fc sequence has been modified, and its sequence is shown in SEQ ID NO: 3
  • RBD-Fc-Fe4 The fusion protein of the SARS-CoV-2 coronavirus spike protein receptor binding region and the Fc fragment of human IgG, in which the Fc sequence has been modified, and its sequence is shown in the sequence SEQ ID NO: 4, and is Animal cells are expressed in HEK-293 cells with fut8 gene knockout
  • PS14 Streptococcus pneumoniae serotype 14 capsular polysaccharide
  • RBD-PS14 RBD and PS14 conjugate
  • RBD-Fc-PS14 RBD-Fc and PS14 conjugate
  • RBD-Fc-Fe4-PS14 RBD-Fc-Fe4 and PS14 conjugate
  • RBD-Fc-Ce3-PS14 RBD-Fc-Ce3 and PS14 conjugate
  • Alum aluminum adjuvant, this article is aluminum phosphate adjuvant
  • Example 1 SARS-CoV-2 RBD, RBD-Fc, RBD-Fc-Ce3 and RBD-Fc-Fe4 fusion protein expression vector construction and protein production
  • the SARS-CoV-2-Spike-RBD sequence (SEQ ID NO: 7) was obtained by PCR amplification (the PCR amplification template was from Beijing Yiqiao Shenzhou Technology Co., Ltd., the same below), including the signal peptide sequence (SEQ ID NO: 6) and SARS-CoV-2-Spike-RBD sequence (SEQ ID NO: 5), inserted into Hind III + Xba I (source: Fermentas, the same below) digested pSE vector (source: Shenzhou) by in-fusion method
  • the pSE-CoV-2-RBD expression vector (SEQ ID NO: 7) was obtained from Cell Engineering Co., Ltd., the same below).
  • RBD-2 TATAGAATAGGGCCCTCTAGATTTAGAAGTTCACACACTTGTTCTTCACC (SEQ ID NO: 17)
  • the pSE-CoV-2-RBD plasmid was extracted, transfected into HEK-293 cells (source: Invitrogen, the same below), cultured and expressed for 7 days, and purified to obtain high-purity SARS-CoV-2 RBD protein.
  • the SARS-CoV-2-Spike-RBD sequence was amplified by PCR, and inserted into Afe I digested FastAP dephosphorized by in-fusion method, including signal peptide (SEQ ID NO: 6), linker (SEQ ID NO: 8)
  • the pSE-mFc expression vector (SEQ ID NO: 14) was obtained from the pSE-mFc vector (source: Shenzhou Cell Engineering Co., Ltd.) of the mouse IgG1 constant region sequence (SEQ ID NO: 13).
  • RBD-22GTCATCGTCATCAGCGAAGTTCACACACTGGTTCTTAA (SEQ ID NO: 19) extracts pSE-CoV-2-RBD-mFc plasmid, transfects HEK-293 cells for culture and expression for 7 days, and uses protein A purification column to obtain high-purity RBD-mFc protein.
  • the SARS-CoV-2-Spike-RBD sequence was amplified by PCR, and inserted into Afe I digested FastAP dephosphorized by in-fusion method, including signal peptide (SEQ ID NO: 6), linker (SEQ ID NO: 8)
  • the pSE-CoV-2-RBD-Fc expression vector (SEQ ID NO: 10) was obtained from the pSTEP2-Fc vector (source: Shenzhou Cell Engineering Co., Ltd.) of the human IgG1 constant region sequence (SEQ ID NO: 9).
  • RBD-22GTCATCGTCATCAGCGAAGTTCACACACTGGTTCTTAA (SEQ ID NO: 21) extracted pSE-CoV-2-RBD-Fc plasmid, transfected HEK-293 cells for culture and expression for 7 days, purified by protein A purification column to obtain high purity RBD-Fc protein.
  • nucleotide mutations in the constant region of the IgG1 subtype were carried out with reference to the literature [36, 37] to obtain the genetically engineered heavy chain IgG1 constant region nucleotide sequence (Fc-Ce3, SEQ ID NO: 11).
  • the SARS-CoV-2-Spike-RBD-Ce3-Fc sequence (SEQ ID NO: 12) was amplified by PCR, and inserted into Hind III+Xba I digested pSE vector by in-fusion method to obtain pSE-nCoV-2 -RBD-Fc-Ce3 expression vector (SEQ ID NO: 12).
  • Amplification primers :
  • the pSE-nCoV-2-RBD-Fc-Ce3 plasmid was extracted, transfected into HEK-293 cells, cultured and expressed for 7 days, and purified by a protein A purification column to obtain high-purity RBD-Fc-Ce3.
  • the pSE-nCoV-2-RBD-Fc-Ce3 plasmid was extracted and transfected into HEK-293 cells with knockout fut8 gene (source: Shenzhou Cell Engineering Co., Ltd.) for culture and expression 7
  • knockout fut8 gene source: Shenzhou Cell Engineering Co., Ltd.
  • a protein A purification column was used to obtain high-purity defucosylated RBD-Fc-Fe4.
  • Serotype 14 Streptococcus pneumoniae seeds are ATCC 6314.
  • Centrifuge at 14000g for 30 minutes take the supernatant, and concentrate by 100kDa ultrafiltration to one-tenth of the original volume, about 400ml.
  • the concentrated solution was gradually added with 36% acetic acid to adjust the pH to 3.5. Let stand for 2 hours, centrifuge at 14000g for 30 minutes, take 390ml of supernatant and add 130ml of absolute ethanol to mix, and let stand overnight. Centrifuge at 14000g for 30 minutes the next day, take the supernatant, add 780ml of absolute ethanol to mix, and let it stand overnight. Centrifuge at 14000g for 30 minutes on the next day and discard the supernatant.
  • SARS-CoV-2 RBD, RBD, RBD-Fc, RBD-Fc-Fe4, RBD-Fc-Ce3 or their fusion proteins are selected as protein/peptide antigens, and they are conjugated with S. pneumoniae serotype 14 capsular polysaccharide respectively Together, the steps are as follows:
  • SARS-CoV-2 RBD or its fusion protein Conjugation of SARS-CoV-2 RBD or its fusion protein to polysaccharide: Take 3mg of SARS-CoV-2 RBD or its fusion protein, add 0.5mg of activated Streptococcus pneumoniae capsular polysaccharide, and add 50mM, pH is 7.0 Na 2 HPO 4 buffer solution, to a total final volume of 0.6 ml, then add 3.6 ⁇ l of 5M sodium cyanoborohydride solution, and rotate and mix for 1 h at room temperature and protected from light. Then, 0.15 ml of 10 mg/mL sodium borohydride solution was added to the reaction solution and reacted at room temperature for 2 hours.
  • conjugate sample after ultrafiltration was aseptically filtered on a 0.22um filter and stored at 4°C.
  • Example 5 Preparation of immune composition of SARS-CoV-2 RBD/its fusion protein glycoconjugate and determination of its immunogenicity
  • RBD, RBD-Fc, RBD-Fc-Fe4, RBD-Fc-Ce3 and their corresponding polysaccharide conjugates RBD-PS14, RBD-FC-PS14, RBD-Fe4-FC-Ce3-PS14, RBD-Fc, respectively -Fe4-PS14 is used as an antigen to prepare an immune composition and determine its immunogenicity
  • an immune composition was prepared.
  • Antigen was diluted with PBS to 0.02mg/ml, 0.06mg/ml or 0.2mg/ml (calculated as peptide/protein, the same below), aluminum adjuvant (Beijing Nuoning Biotechnology Co., Ltd.) was diluted with PBS to 1mg/ml .
  • the diluted antigen and aluminum adjuvant are mixed in equal volumes.
  • the protein concentration of the antigen in the immune composition is 0.01 mg/ml, 0.03 mg/ml or 0.1 mg/ml, respectively.
  • the antigen was diluted with PBS to 0.02mg/ml, 0.06mg/ml or 0.2mg/ml, and the diluted antigen was mixed with an equal volume of MF59 adjuvant.
  • the protein concentration of the antigen in the immune composition was 0.01mg/ml, 0.03mg/ml or 0.1mg/ml.
  • the protein concentration of antigen in the immune composition is 0.01mg/ml respectively , 0.03mg/ml or 0.1mg/ml.
  • mice 5.2 Immunization of mice:
  • mice for 4-6 weeks and inject 0.1ml intraperitoneally with the antigen concentration of 0.01mg/ml or 0.03mg/ml or 0.1mg/ml immune composition as described in Example 5.1, and boost the immunization on the 14th day ( Same dose as the first immunization).
  • blood was taken from the orbit to determine the serum titer, neutralization titer and the ratio of IgG2a/IgG1.
  • OD 450 Dilute the serum to be tested (prepared in Example 5.2) to 1:8000 with PBS containing 0.1% bovine serum albumin (BSA), and add the serum to be tested and goat anti-mouse IgG F(ab ) 2 /HRP (Beijing Yiqiao Shenzhou Technology Co., Ltd.) detection secondary antibody, each 100 ⁇ l/well, incubate together for 2h, wash the plate 5 times, add substrate color solution for color development, after termination, read the OD 450 by the microplate reader .
  • the OD 450 at a certain dilution factor represents the antibody titer.
  • the measurement uses RBD, RBD-Fc, RBD-Fc-Ce3, RBD-PS14, RBD-Fc-PS14, RBD-Fc-Ce3-PS14 as antigens and MF59+Alum mixed adjuvant; the immunization doses are 1, 3, and The titer of serum (1:8000 dilution) on the 21st day of mice immunized with 10 ⁇ g of the immune composition is shown in Table 2.
  • Antigen/immunization dose/serum titer of immune composition 1 ⁇ g/OD 450 3 ⁇ g/OD 450 10 ⁇ g/OD 450 RBD 0.988 0.997 1.006
  • RBD-Fc 1.103 1.235 1.298
  • RBD-Fc-Ce3 1.023 1.275 1.906
  • RBD-PS14 1.061 1.272 1.257
  • RBD-Fc-PS14 0.427 0.648 0.827
  • RBD-Fc-Ce3-PS14 0.450 0.469 0.882
  • the measurement uses RBD-Fc-Fe4 and RBD-Fc-Fe4-PS14 as antigens; Alum, MF59, MF59+Alum as adjuvants; immunization doses of 1, 3, and 10 ⁇ g of the immune composition on the 21st day of serum ( 1:500 dilution) of the pseudovirus neutralization titer, the results are shown in Table 3.
  • the measurement uses RBD, RBD-Fc, RBD-Fc-Ce3, RBD-PS14, RBD-Fc-PS14, RBD-Fc-Ce3-PS14 as antigens and MF59+Alum mixed adjuvant; the immunization doses are 1, 3, and The pseudovirus neutralization titer of the serum (diluted 1:500) on the 21st day of immunized mice at 10 ⁇ g is shown in Table 4.
  • Antigen/immunization dose of immune composition 1 ⁇ g inhibition rate% 3 ⁇ g inhibition rate% 10 ⁇ g inhibition rate% RBD 6.2 9.8 14.0 RBD-Fc 26.4 24.0 25.4 RBD-Fc-Ce3 29.6 53.6 71.0 RBD-PS14 44.2 93.2 81.2 RBD-Fc-PS14 36.0 42.8 75.4 RBD-Fc-Ce3-PS14 26.4 44.8 78.8
  • Enzyme-linked immunoassay was used to detect IgG1 antibody in mouse serum. After diluting the coating protein RBD protein to a concentration of 2 ⁇ g/mL, take 100 ⁇ l of the coated ELISA plate and place it at 4°C overnight. Wash the plate, use 100 ⁇ l of TBST buffer containing 2% BSA (bovine serum albumin) to block the ELISA plate for 2 hours at room temperature, and then wash the plate.
  • the mouse serum sample of Example 5.2 was diluted to 1:500,000 using TBST diluent containing 0.1% BSA. Add 100 ⁇ l of the diluted mouse serum sample to the blocked enzyme-labeled plate, and react for 1 hour at room temperature.
  • Enzyme-linked immunoassay was used to detect the content of IgG2a antibody in mouse serum. After diluting the coating protein RBD protein to a concentration of 2 ⁇ g/mL, take 100 ⁇ l of the coated ELISA plate and place it at 4°C overnight. Wash the plate, use 100 ⁇ l TBST buffer containing 2% BSA to block the microtiter plate for 2 hours at room temperature, and then wash the plate. Dilute the mouse serum sample to 1:5000 using TBST diluent containing 0.1% BSA. Add 100 ⁇ l of the diluted mouse serum sample to the blocked enzyme-labeled plate, let it act at room temperature for 1 hour, and then wash the plate.
  • IgG2a/IgG1 (1:5000 serum) OD 450 value/(1:500000 serum) OD 450 value.
  • the measurement results are shown in Table 5-6 and Figure 1-2.
  • the measurement uses RBD, RBD-Fc, RBD-Fc-Ce3, RBD-PS14, RBD-Fc-PS14, RBD-Fc-Ce3-PS14 as the antigen, and the adjuvant is MF59+Alum mixed adjuvant; the immunization dose is 1, respectively
  • the ratio of IgG2a/IgG1 in serum of immunized mice at 3 and 10 ⁇ g on day 21 diluted with 1:5000 serum to measure IgG2a; measured with diluted serum 1:500000 for IgG1). The results are shown in Table 6.
  • Figure 1 shows the results of immunizing mice with two antigens, RBD-Fc-Fe4 and RBD-Fc-Fe4-PS14, respectively using aluminum adjuvant, MF59 adjuvant, and MF59 plus aluminum adjuvant mixed adjuvant.
  • Three immunization doses of 1 ⁇ g, 3 ⁇ g, and 10 ⁇ g were set for each adjuvant.
  • the serum was taken to determine the ratio of IgG2a/IgG1. It can be seen from the figure that when the MF59 adjuvant is used, the ratio of IgG2a/IgG1 in the immune serum of the antigen conjugated with the polysaccharide is greatly increased.
  • a high IgG2a/IgG1 ratio indicates a higher cellular immune response, which also indicates that the vaccine has a better protective effect.
  • Figure 2 shows the comparison of the serum IgG2a/IgG1 ratios of RBD-PS14, RBD-Fc-PS14, RBD-Fc-Ce3-PS14 and RBD-Fc-Fe4-PS14 antigens.
  • the adjuvant is MF59+Alum mixed adjuvant. It can be seen from the figure that RBD-Fc-Fe4-PS14 has a higher IgG2a/IgG1 ratio than other proteoglycan conjugates.
  • the titer of pure protein immune serum is improved compared with polysaccharide conjugate.
  • the antibody titer and pseudovirus neutralization titer of RBD-Fc-Fe4 and its corresponding polysaccharide conjugate immune serum are not much different.
  • the IgG2a/IgG1 ratio of RBD-Fc-Fe4 and polysaccharide conjugate immune serum was significantly increased.
  • the IgG2a/IgG1 ratio reflects the tendency of cellular immunity.
  • a high IgG2a/IgG1 ratio indicates a higher cellular immune response, which also indicates that the vaccine has a better protective effect. Comparing the IgG2a/IgG1 ratios of several antigen proteins and their corresponding polysaccharide conjugates, RBD-Fc-Fe4 polysaccharide conjugate is the highest, so RBD-Fc-Fe4 polysaccharide conjugate is the best vaccine candidate molecule.

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Abstract

提供增强蛋白/肽抗原免疫原性的方法，其中该蛋白/肽抗原与Fc片段，优选受体结合/补体结合增强Fc片段形成融合蛋白，且进一步与糖缀合形成融合蛋白糖缀合物，优选方案中的Fc片段因其氨基酸序列和/或糖基化形式改变与其天然形式相比，具有提高的与Fc受体和或补体蛋白C1q的结合能力。该等疫苗，可维持长时程的体液免疫和细胞免疫反应，且具有更高的细胞免疫反应、免疫动物可产生较高滴度的中和抗体。以SARS-CoV-2 RBD区为免疫组合物的抗原，将其与氨基酸序列及岩藻糖改变的形式的Fc区形成融合蛋白，进一步与肺炎多糖形成融合蛋白糖缀合物，用于预防SARS-CoV-2感染相关疾病。

Description

通过形成Fc片段融合蛋白糖缀合物增强抗原免疫原性的方法
相关申请的交叉引用
本申请要求2020年05月15日提交的中国专利申请202010415139.8的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明属于免疫学领域，具体地，涉及增强蛋白/肽抗原免疫原性的方法，其中该蛋白/肽抗原与Fc片段，优选受体结合/补体结合增强Fc片段形成融合蛋白，且进一步与糖缀合形成融合蛋白糖缀合物，优选方案中的Fc片段因其氨基酸序列和/或糖基化形式改变，与其天然形式相比，具有提高的与Fc受体和或补体蛋白C1q的结合能力。该等疫苗，可维持长时程的体液免疫和细胞免疫反应，且具有更高的细胞免疫反应、免疫动物可产生较高滴度的中和抗体。本发明的一个实例是以SARS-CoV-2RBD区为免疫组合物的抗原，将其与氨基酸序列及岩藻糖改变的形式的Fc区形成融合蛋白，进一步与肺炎多糖形成融合蛋白糖缀合物，用于预防SARS-CoV-2感染相关疾病。
背景技术
安全性及免疫原性是疫苗研发过程中关注的核心问题。非活体疫苗，即以感染性微生物、毒素、病毒及肿瘤细胞的亚单位为抗原的疫苗，因其安全性被广泛地采纳。但是亚单位抗原的免疫原性差，其诱导特异性免疫应答的能力较弱。
传统的增强免疫原性的手段是添加免疫佐剂。新的增强免疫反应的手段仍在不断的研究探索中。
一个重要的手段是将免疫原性差的抗原与用作载体的外源大分子缀合，这一手段已成功应用了数十年。Fc融合蛋白是另一新进的手段，即将某种具有生物活性的功能蛋白与Fc片段融合而产生的新型重组蛋白，其不仅保留了功能蛋白分子的生物学活性，还具有一些抗体的性质，如同FcRs的结合及介导的相关生物学功能等。这两种手段将在下文中详述。
1.缀合物疫苗
习见的缀合物疫苗有脑炎疫苗、嗜血流感细菌b疫苗和肺炎疫苗，以其纯化的荚膜多糖与载体蛋白结合而产生更有效的免疫原性组合物常用的载体蛋白如破伤风类毒素、破伤风类毒素片段C、破伤风毒素非毒性突变体、白喉类毒素、CRM197、白喉毒素的其它非毒性突变体例如CRM176、CRM197、CRM228、CRM45(Uchida等人J.Biol.Chem.218；3838-3844，1973)；CRM9，CRM45，CRM02，CRM103和CRM107以及其它突变体。
值得注意的是，上述缀合物疫苗以多糖为抗原物质。该类多糖抗原是非胸腺细胞依赖性抗原，不能产生细胞免疫反应，不能形成免疫记忆。在儿童或免疫低下人群不能形成保护性抗体。将多糖抗原与具有T细胞表位的蛋白类载体缀合，抗原提呈细胞或B细胞内吞并加工糖与蛋白的缀合物，随后将载体蛋白的多肽片段展示在细胞表面，激活辅助T细胞，引起系列免疫反应生成保护性抗体，和免疫记忆。
但是未见蛋白/肽抗原缀合糖后提高免疫原性的报道。US5192540A公开了含有B型流感嗜血杆菌38,000道尔顿或40,000道尔顿外膜蛋白和B型流感嗜血杆菌氧化聚核糖-核糖醇-磷酸多糖片段的免疫原性缀合物的疫苗，其可用于免疫由B型流感嗜血杆菌引起的疾病。但是“本发明的结合疫苗在动物模型中具有高免疫原性。它们对PRP的抗体反应明显高于之前报道的抗体反应。结合疫苗还诱导B型流感嗜血杆菌主要蛋白质(38K或40k蛋白)抗体。”
US9296795B公开了在免疫原性组合物中使用具有源自医院病原体的多糖抗原(或其寡糖片段，代表一个或多个抗原表位)的免疫原性多糖-蛋白质缀合物，所述多糖-蛋白质缀合物缀合到葡萄球菌表面粘附素载体蛋白，以引起对多糖抗原和葡萄球菌表面粘附载体蛋白的抗体应答。该专利披露“本发明所述的缀合物具有独特的优点：能够诱导产生抗多糖抗原和表面粘附素载体蛋白(二者均为毒力因子)的抗体，并对医院病原体引起的疾病赋予免疫力。也就是说，表面粘附素蛋白本身也能够赋予机体免疫力，而不只是充当多糖抗原的蛋白载体。”“缀合的表面粘附素蛋白诱导产生的表面粘附素蛋白特异性抗体的滴度与没有缀合的表面粘附素蛋白是类似的(图17-20)。这证实了抗原表位没有因表面粘附素蛋白和CP的结合而改变。”
就发明人所知，蛋白/肽多糖缀合物用作抗原时，蛋白部分不是作为载体蛋白以增强多糖的免疫原性，而是作为免疫原性物质存在的报道仅有以上两项研究工作。但是这两项工作止步于蛋白/肽部分亦可以引起抗体产生，但是没有报道其免疫原性增强。发明人的发明名称为《一种增强蛋白-肽抗原免疫原性的方法》、申请号为PCT/CN2021/090809(在先国家申请优先权申请号CN202010369100.7)、申请日为2021年4月29日的发明专利申请中报道了申请人的开创性的发明：通过将蛋白/肽抗原与糖缀合，形成糖-蛋白/肽抗原缀合物，提高了蛋白/肽抗原的免疫原性。
发明人的发明名称为《通过与改变的Fc片段形成融合蛋白增强蛋白/肽抗原免疫原性的方法》、申请号为PCT/CN2021/092013(在先国家申请优先权申请号CN202010394463.6)、申请日为2021年5月7日的发明专利申请中报道了申请人的另一 开创性的发明：通过将蛋白/肽抗原与Fc片段，优选受体结合/补体结合增强Fc片段形成融合蛋白，提高了蛋白/肽抗原的免疫原性。
2.Fc受体融合蛋白疫苗
B细胞介导的体液免疫是疫苗介导的机体保护机制之一。已有研究表明，Fc与Fc受体(FcRs)及补体受体(CR)相互作用可介导更优的抗原捕获及呈递、促进B细胞亲和力成熟及高亲和力抗体的产生[ 1-6]。此外，Fc与滤泡树突细胞(fDC)上的受体结合(通过FcR或CR介导)后，展示于fDC表面，这部分抗原对于维持抗原的长期存在、维持抗原特异性B细胞的存活极为重要[ 7]。
此外，细胞毒性T淋巴细胞(CTL)对抵抗病毒感染，清除病毒感染细胞具有重要的作用。抗原交叉递呈机制可使外源性抗原进入细胞内源性的加工和递呈机制，从而使外源抗原肽展示在MHC I类分子上被T细胞识别，启动CTL细胞应答。外源抗原的交叉递呈对有效激活CTL从而引发抗病毒免疫反应有着重要意义，因此增强亚单位疫苗的交叉递呈是提升疫苗免疫效果的有效策略之一。树突状细胞(DC)是目前已知功能最强的专职抗原递呈细胞，也是主要进行交叉递呈的细胞。外源抗原进入DC的方式主要有三种:吞噬作用(胞吞)、胞饮和受体介导的内吞作用。目前已知的与外源抗原交叉递呈有关的内吞受体，包括C-型凝集素受体(CLR)、识别免疫复合物IgG的Fc受体(FcRs)、识别凋亡细胞的清道夫受体、趋化因子受体等，这些受体介导抗原内吞后进入特定的内涵体才能与MHCⅠ类分子结合，从而激活CD8 +T细胞。抗原抗体复合物(免疫复合物)可被DC细胞的FcRs识别，由此引发的交联可将抗原内化，并将抗原进行交叉加工递呈，特异性激活CTL反应[ 8-10]。这种由FcRs介导的抗原交叉递呈已证实可诱导强CTL反应[ 11]。
人体中与IgG结合的Fc受体(FcRγ)主要包含FcγRI(CD64)、FcγRIIA(CD32a)、FcγRIIB(CD32B)、FcγRIII(CD16)等[ 12]。其中FcγRIIB为抑制性受体，主要表达在B细胞、巨噬及肥大细胞上[ 13]。其又可分为FcγRIIB-1、FcγRIIB-2。FcγRIIB-1仅表达在B细胞上，在B细胞发育过程中控制着B细胞的过度激活以及对自身抗原的识别，其通过胞内的受体酪氨酸抑制基序(ITIM)传递B细胞凋亡信号实现B细胞的阴性选择过程，调控B细胞的发育过程。FcγRIIB-2表达在除NK及T细胞的其它免疫细胞上，通过受体的交联可以有效的诱导抗原抗体复合物的吞噬作用[ 14, 15]。其余几种Fc受体均为激活性受体，其中CD64主要表达在单核、巨噬、DC等细胞上，CD32a主要表达在中性粒、单核、巨噬、DC等细胞上，CD16a主要表达在NK、单核、巨噬细胞[ 15]。激活型受体在 识别抗原抗体复合物后，胞内受体酪氨酸激活基序(ITAM)决定着抗原提呈细胞APC启动抗原的摄取以及摄取后经MHC类分子行使的抗原提呈功能[ 16]。
Fc融合蛋白是指利用基因工程等技术将某种具有生物活性的功能蛋白与Fc片段融合而产生的新型重组蛋白，其不仅保留了功能蛋白分子的生物学活性，还具有一些抗体的性质，如同FcRs的结合及介导的相关生物学功能等。抗原-Fc融合蛋白可作为抗原运载工具，借助Fc片段靶向结合抗原递呈细胞，缩短抗原在血浆中的游离时间，提高抗原半衰期，从而加强抗原递呈和抗原交叉递呈反应。
目前已开展临床实验的Fc融合蛋白类疫苗数量较少。一项IIb期临床中，慢性乙型cHBV感染者，按照60μg/4周给予一种YIC免疫原性复合物(由酵母表达的乙肝表面抗原HBsAg与HBsAg免疫的人血清中和抗体HBIG以特定比例混合孵育，并加入铝佐剂)治疗，与仅给予铝佐剂的对照组相比，其血清乙肝病毒E抗原(HBeAg)转阴率有明显提升(21.8％vs 9％)，病毒滴度显著下降，并且伴随抗E抗原抗体的产生[ 22]。于此同时体外条件下，YIC可增加cHBV感染病人DC细胞的成熟(CD83高表达)、抗原识别与提呈(HLA-II、CD86、CD80、CD40的标志物的表达)，并分泌更多炎性因子(IL-12)。病人的DC-PBMC混合细胞在YIC刺激下比单独HBsAg抗原刺激产生更多的T淋巴细胞细胞因子(Th1细胞：IL-2，IFNγ)(Th2细胞:IL-5,IL-10)[ 23]。然而，YIC的过度刺激可能会引发机体的免疫疲劳，从而降低细胞免疫反应[ 24]。因此，为了实现更好的免疫效果，合适的免疫方案也尤为重要。
其它一些病毒抗原-Fc融合蛋白的疫苗(RSV，HBV，DENV，TB)也在动物模型中展开了尝试，实现了有效的免疫系统激活，同时产生的抗体可降低血清中病毒抗原水平[ 25-28]。
Fc融合蛋白类疫苗对免疫系统的激活所引发的安全性风险也是值得考虑的议题。上文中提到的慢性乙肝cHBV感染者，给予YIC治疗后一段时间内，少量病人出现丙氨酸转移酶(ALT)的瞬时上升。与仅给予铝佐剂的对照组相比，发生ALT上升病人的比例相近。转氨酶水平的上升在一定程度上反映着肝损伤，但随后的观察显示，ALT的可恢复到正常水平[ 24]。其IIa期临床也观察到HBeAg抗原血清转阴的病人中部分观察到ALT升高的现象[ 23]。从YIC的IIb期临床副反应(AE)数据可见，YIC 30μg剂量组，YIC 60μg剂量组，铝佐剂对照组发生严重AE的比例相近(3.6％vs 5％vs 5.1％)，YIC组最常见的AE为注射点相关反应，包括皮疹、肿胀、瘙痒等与炎性相关反应，其他系统性的AE如发烧、头疼、恶心等与对照组均无明显差异[ 22]，因此包含Fc的免疫复合物在临床中 的安全性相对较好，无因过度激活免疫系统产生的相关严重副反应发生。
对Fc片段进行增强FcRs结合的改造，可以获得Fe4-Fc改造分子，实现增加Fc与补体蛋白C1q及Fc受体CD16、CD32、CD64等结合的作用[ 17-21]，则有可能进一步提升Fc及其受体介导的抗原捕获及呈递、提升B细胞成熟及高亲和力抗体的产生、维持长时程的体液免疫、提升抗原交叉递呈介导的CTL免疫反应。
发明人的发明名称为《一种通过与改变的Fc片段形成融合蛋白增强蛋白/肽抗原免疫原性的方法》、申请号为CN202010394463.6、申请日为2020年5月11日的发明专利申请中报道了申请人的开创性的发明：通过将蛋白/肽抗原与经过改造的抗体Fc片段形成融合蛋白，提高了蛋白/肽抗原的免疫原性，其中该Fc片段因其氨基酸序列和/或糖基化形式改变，与其天然形式相比，具有提高的与Fc受体和/或补体蛋白C1q的结合能力。
发明内容
在一个方面，本发明提供一种增强蛋白/肽抗原的免疫原性的方法，该方法包含
将蛋白/肽抗原与Fc片段融合，形成融合蛋白，然后
将该融合蛋白与糖缀合，形成融合蛋白糖缀合物，优选地
Fc片段为受体结合/补体结合增强Fc片段。
在一个实施方式中，所述方法中所述的蛋白/肽抗原为病原体相关的蛋白/肽抗原或肿瘤相关蛋白/肽抗原。
在一个实施方式中，所述方法中所述的病原体选自：
冠状病毒，人免疫缺陷性病毒HIV-1，人单纯疱疹病毒，巨细胞病毒，轮状病毒，EB病毒，水痘带状疱疹病毒，肝炎病毒，呼吸道合胞体病毒，副流感病毒，麻疹病毒，流行性腮腺炎病毒，人乳头瘤病毒，黄病毒或流感病毒，奈瑟氏菌属，莫拉氏菌属，博代氏杆菌属，分枝杆菌属，包括结核分枝杆菌；埃希氏菌属，包括肠毒素大肠埃希菌；沙门氏菌属，李斯特氏菌属，螺杆菌属，葡萄球菌属，包括金黄葡萄球菌，表皮葡萄球菌；疏螺旋体属，衣原体，包括砂眼衣原体，肺炎衣原体；疟原虫，包括恶性疟原虫；弓形虫，念珠菌；
其中的肿瘤选自：
弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、其他淋巴瘤、白血病、多发性骨髓瘤、间皮瘤、胃癌、恶性横纹肌瘤、肝细胞癌、前列腺癌，乳腺癌、胆管癌和胆囊癌、膀胱癌、脑肿瘤包括神经母细胞瘤、神经鞘瘤、胶质瘤、胶质母细胞瘤和星形细胞瘤、宫颈癌、结肠癌、黑色素瘤、子宫内膜癌、食管癌、头颈癌、肺癌、鼻咽癌、卵巢癌、胰腺癌、 肾细胞癌、直肠癌、甲状腺癌、甲状旁腺肿瘤、子宫肿瘤和软组织肉瘤。
在一个实施方式中，所述方法中的蛋白/肽蛋白抗原选自分泌蛋白或全长膜蛋白，或其功能结构域、突变蛋白、截短蛋白，或由其1个或多个抗原多肽表位拼接组成的改造蛋白。
在一个实施方式中，所述方法中的蛋白/肽抗原选自
冠状病毒抗原；
优选冠状病毒棘突蛋白；
更优选冠状病毒棘突蛋白片段；
更优选冠状病毒棘突蛋白的ACE2受体结合结构域(RBD)；
更优选冠状病毒棘突蛋白S1亚单位；
优选地，上述冠状病毒选自SARS-CoV-2。
在一个实施方式中，所述方法中的Fc片段来自人抗体、鼠源抗体、兔源抗体或其他动物抗体的重链恒定区。
在一个实施方式中，所述方法中的Fc片段来自人抗体的IgG、IgM或IgA亚型抗体，
优选地，来自IgG1、IgG2、IgG3或IgG4亚型抗体；
更优选地，是为提高与Fc受体/C1q补体结合功能之目的，而进行氨基酸序列突变和/或糖基化形式改变的IgG1改造Fc片段。
在一个实施方式中，所述方法的Fc受体选自CD16、CD32a、CD32b或CD64。
在一个实施方式中，所述方法中
经过改造的抗体Fc片段为Fc受体CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
其氨基酸序列如SEQ ID NO：3所示。
在一个实施方式中，所述方法中
经过改造的抗体Fc片段为Fc受体CD16a、CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
其氨基酸序列如SEQ ID NO：4所示，且用岩藻糖敲除的哺乳细胞生产，优选地，哺乳动物细胞为fut8基因敲除的HEK-293细胞。
在一个实施方式中，所述方法中所述抗原优选地通过接头和其他大分子缀合，优选地，其他大分子为多糖、肽/蛋白。
在一个实施方式中，所述方法中其他大分子选自多糖、寡糖或单糖；
优选为奈瑟氏脑炎球菌荚膜多糖、嗜血性流感杆菌b荚膜多糖、肺炎链球菌荚膜多糖、B群金黄色葡萄球菌荚膜多糖、葡聚糖、甘露聚糖、淀粉、菊糖、果胶、羧甲基淀粉、壳聚糖及其衍生物；
更优选为肺炎链球菌荚膜多糖；
更优选为肺炎链球菌血清型14荚膜多糖、肺炎链球菌血清型6B荚膜多糖和肺炎链球菌血清型7F荚膜多糖；
最优选为肺炎链球菌血清型14荚膜多糖。
在一个实施方式中，所述方法中缀合物的分子量为800-6000KDa。
在一个实施方式中，所述方法中融合蛋白糖缀合物形式的蛋白/肽抗原和免疫佐剂组合。
在一个实施方式中，所述方法中佐剂选自铝佐剂、MF59或其混合物。
在另一个方面，本发明提供一种免疫原性增强的蛋白/肽抗原，其包含
将蛋白/肽抗原与Fc片段融合形成融合蛋白，然后
与糖缀合形成的蛋白/肽-Fc片段融合蛋白糖缀合物；优选地
Fc片段为受体结合/补体结合增强Fc片段。
在一个实施方式中，所述的蛋白/肽抗原，其中所述的蛋白/肽抗原为病原体相关的蛋白/肽抗原或肿瘤相关蛋白/肽抗原。
在一个实施方式中，所述的蛋白/肽抗原，
其中所述的病原体选自：
冠状病毒，人免疫缺陷性病毒HIV-1，人单纯疱疹病毒，巨细胞病毒，轮状病毒，EB病毒，水痘带状疱疹病毒，肝炎病毒，呼吸道合胞体病毒，副流感病毒，麻疹病毒，流行性腮腺炎病毒，人乳头瘤病毒，黄病毒或流感病毒，奈瑟氏菌属，莫拉氏菌属，博代氏杆菌属，分枝杆菌属，包括结核分枝杆菌；埃希氏菌属，包括肠毒素大肠埃希菌；沙门氏菌属，李斯特氏菌属，螺杆菌属，葡萄球菌属，包括金黄葡萄球菌，表皮葡萄球菌；疏螺旋体属，衣原体，包括砂眼衣原体，肺炎衣原体；疟原虫，包括恶性疟原虫；弓形虫，念珠菌；
其中的肿瘤选自：
弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、其他淋巴瘤、白血病、多发性骨髓瘤、间皮瘤、胃癌、恶性横纹肌瘤、肝细胞癌、前列腺癌，乳腺癌、胆管癌和胆囊癌、膀胱癌、脑肿瘤包括神经母细胞瘤、神经鞘瘤、胶质瘤、胶质母细胞瘤和星形细胞瘤、宫颈癌、 结肠癌、黑色素瘤、子宫内膜癌、食管癌、头颈癌、肺癌、鼻咽癌、卵巢癌、胰腺癌、肾细胞癌、直肠癌、甲状腺癌、甲状旁腺肿瘤、子宫肿瘤和软组织肉瘤。
在一个实施方式中，所述的蛋白/肽抗原，其中蛋白/肽蛋白抗原选自分泌蛋白或全长膜蛋白，或其功能结构域、突变蛋白、截短蛋白，或由其1个或多个抗原多肽表位拼接组成的改造蛋白。
在一个实施方式中，所述的蛋白/肽抗原，其中蛋白/肽抗原选自
冠状病毒抗原；
优选冠状病毒棘突蛋白；
更优选冠状病毒棘突蛋白片段；
更优选冠状病毒棘突蛋白的ACE2受体结合结构域(RBD)；
更优选冠状病毒棘突蛋白S1亚单位；
优选地，上述冠状病毒选自SARS-CoV-2。
在一个实施方式中，所述的蛋白/肽抗原中Fc片段来自人抗体、鼠源抗体、兔源抗体或其他动物抗体的重链恒定区。
在一个实施方式中，所述的蛋白/肽抗原中Fc片段来自人抗体的IgG、IgM或IgA亚型抗体，
优选地，来自IgG1、IgG2、IgG3或IgG4亚型抗体；
更优选地，是为提高与Fc受体、C1q补体结合功能之目的，而进行氨基酸序列突变和/或糖基化形式改变的IgG1改造Fc片段。
在一个实施方式中，所述的蛋白/肽抗原，Fc受体选自CD16、CD32a、CD32b或CD64。
在一个实施方式中，所述的蛋白/肽抗原中
经过改造的抗体Fc片段为Fc受体CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
其氨基酸序列如SEQ ID NO：3所示。
在一个实施方式中，所述的蛋白/肽抗原中
经过改造的抗体Fc片段为Fc受体CD16a、CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
其氨基酸序列如SEQ ID NO：4所示，且用岩藻糖敲除的哺乳细胞生产，优选地，哺乳动物细胞为fut8基因敲除的HEK-293细胞。
在一个实施方式中，所述的蛋白/肽抗原中所述抗原优选地通过接头和其他大分子缀合，优选地，其他大分子为多糖、肽/蛋白。
在一个实施方式中，所述的蛋白/肽抗原中其他大分子选自多糖、寡糖或单糖；
优选为奈瑟氏脑炎球菌荚膜多糖、嗜血性流感杆菌b荚膜多糖、肺炎链球菌荚膜多糖、B群金黄色葡萄球菌荚膜多糖、葡聚糖、甘露聚糖、淀粉、菊糖、果胶、羧甲基淀粉、壳聚糖及其衍生物；
更优选为肺炎链球菌荚膜多糖；
更优选为肺炎链球菌血清型14荚膜多糖、肺炎链球菌血清型6B荚膜多糖和肺炎链球菌血清型7F荚膜多糖；
最优选为肺炎链球菌血清型14荚膜多糖。
在一个实施方式中，所述的蛋白/肽抗原中缀合物的分子量为800-6000KDa。
在一个实施方式中，所述的蛋白/肽抗原中佐剂选自铝佐剂、MF59或其混合物。
在又一个方面，本发明提供一种免疫组合物，其包含
本发明所述的蛋白/肽抗原；
佐剂；和
药学上可接受的载体、赋形剂或稳定剂，优选为
冻干制剂或水溶液形式的药学上可接受的载体、赋形剂或稳定剂。
在一个实施方式中，所述的免疫组合物中佐剂可选自铝佐剂、MF59的至少一种。
在又一个方面，本发明提供本发明所述的蛋白/肽抗原、或所述的免疫组合物，其用于预防病原体，优选冠状病毒、更优选SARS-CoV-2引起的疾病/肿瘤的应用。
在又一个方面，本发明提供本发明所述的蛋白/肽抗原、或所述的免疫组合物用于制备预防病原体，优选冠状病毒、更优选SARS-CoV-2引起的疾病/肿瘤的疫苗中的应用。
在又一个方面，本发明提供一种免疫组合，其包含
本发明所述的蛋白/肽抗原、或本发明所述的免疫组合物；以及
一种或多种另外的免疫原性剂。
在又一个方面，本发明提供一种试剂盒，其包含
本发明所述的蛋白/肽抗原、或本发明所述的免疫组合物；
优选地，
还进一步包含给予免疫组合物的装置。
在又一个方面，本发明提供一种预防病原体，优选冠状病毒、更优选SARS-CoV-2 引起的疾病/预防肿瘤的方法，其包含给予受治疗者本发明所述的蛋白/肽抗原、本发明所述的免疫组合物、本发明所述的免疫组合或本发明所述的试剂盒。
在又一个方面，本发明提供一种免疫动物的方法，其包含给予动物本发明所述的蛋白/肽抗原、本发明所述的免疫组合物、本发明所述的免疫组合或本发明所述的试剂盒，以产生中和抗体。
附图说明
图1显示了采用RBD-Fc-Fe4和RBD-Fc-Fe4-PS14作为抗原；铝佐剂、MF59佐剂、MF59加铝佐剂混合佐剂免疫小鼠IgG2a/IgG1比值。每种佐剂又分别设置1μg,3μg,10μg三个免疫剂量，二免后取血清测定IgG2a/IgG1比值。
图2显示了RBD-PS14、RBD-Fc-PS14、RBD-Fc-Ce3-PS14及RBD-Fc-Fe4-PS14抗原血清IgG2a/IgG1比值的比较。
发明详述
发明人在上述的两项开创性发明的基础上，发明了进一步提高蛋白/肽抗原的免疫原性的方法：将蛋白/肽抗原与Fc片段，优选受体结合/补体结合增强Fc片段融合，形成融合蛋白，然后将该融合蛋白与糖缀合，形成融合蛋白糖缀合物。如此改造过的蛋白/肽抗原的免疫原性得以增强，上述Fc片段由于其氨基酸序列和/或糖基化形式改变具有提高的与Fc受体和/或补体蛋白C1q的结合能力。
发明人将此发明用于制备新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2，SARS-CoV-2)疫苗。
SARS-CoV-2和SARS-CoV具有共同的宿主细胞受体蛋白，即血管紧张素转化酶2(ACE2)[29]。病毒的三聚体S蛋白同ACE2受体结合后被宿主蛋白酶切割为包含受体结合域(Receptor binding domain,RBD)的S1多肽和负责介导病毒同细胞膜融合的S2多肽[30]。S1和ACE2之间的特异性相互作用会触发S2亚基的构象变化，从而导致病毒包膜和细胞膜或溶酶体膜融合并释放病毒核酸进入细胞质[31]。数据表明COVID-19患者，尤其是在重症患者中，伴随肺炎症状，淋巴细胞显著降低，血浆促炎因子显著增加，提示了免疫系统在疾病进程中发挥着重要的作用[32-34]。对23例COVID-19患者出现症状后血清抗体的分析表明，大多数患者在出现症状10天后发生针对RBD蛋白的抗体反应[35]。发病早期血清中RBD蛋白抗体阳性患者比例高于N蛋白抗体阳性患者比例，说明机体可能先产生具有中和作用的抗体，以抑制病毒通过RBD侵入细胞。对细胞免疫的分析表明，刚出院患者针对不同抗原的特异性T细胞与未感染者的T细胞有显著差异，其中RBD特异性T细胞分布最广。康复两周后的随访患者细胞免疫水平则明显降低。RBD不仅可引起体液免疫，产生中和抗体，而且还可诱导T细胞免疫应答，因此RBD蛋白是 SARS-CoV-2疫苗的有效靶标。
定义
除非另有说明，本文使用的所有技术和科学术语具有本发明所属的技术领域的普通技术人员通常理解的含义。为了本发明的目的，进一步定义以下术语。
当用于本文和所附权利要求书中时，单数形式“一”、“一种”、“另一”和“所述”包括复数指代对象，除非上下文明确地另有指示。
术语“包括”、“包含”是指包括具体成分而不排除任何其他的成分。诸如“基本上由……组成”允许包括不损害本发明的新颖或基本特征的其他成分或步骤，即，它们排除损害本发明的新颖或基本的特征的其他未列举的成分或步骤。术语“由……组成”是指包括具体成分或成分组并且排除所有其他成分。
术语“/”包含和、或两种情形。
术语“抗原”是指一种由抗体或T细胞受体所识别(特异性结合)的外源物质，但是其不能确定性地诱导免疫应答。诱导特异性免疫的外源性物质称为“免疫性抗原”或“免疫原”。“半抗原”是指一种本身不能引发免疫应答(尽管几个分子半抗原的结合物，或半抗原与大分子载体的结合物可引发免疫应答)的抗原。
术语“RBD受体结合域(Receptor binding domain,RBD)”在本说明书和所附的权利要求书中特指“冠状病毒棘突蛋白的ACE2受体结合结构域(SARS-CoV-2RBD)”，以上术语互换使用。SARS-CoV-2和SARS-CoV具有共同的宿主细胞受体蛋白，即血管紧张素转化酶2(ACE2)。病毒的三聚体S蛋白同ACE2受体结合后被宿主蛋白酶切割为包含受体结合域(Receptor binding domain,RBD)的S1多肽和负责介导病毒同细胞膜融合的S2多肽。
“体液免疫应答”是抗体介导的免疫应答并且涉及引入和生成以一定亲和力识别和结合本发明的免疫原性组合物中的抗原的抗体，“细胞介导的免疫应答”是由T细胞和/或其他白细胞介导的免疫应答。“细胞介导的免疫应答”是通过提供与主要组织相容性复合物(MHC)的I类或II类分子、CD1或其他非典型MHC样分子相关的抗原表位而诱发的。
术语“多肽”、“寡肽”、“肽”和“蛋白质”，涵盖任何长度的氨基酸的链，其中相对短(例如，短于100个氨基酸)氨基酸链通常称为肽。该链可以是直链或支链的，其可包含修饰氨基酸，和/或可间插非氨基酸。
术语“抗体”意指免疫球蛋白分子，是指表现所需生物学活性的抗体的任何形式。包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体和多特异性抗体(例如双特异性抗体),甚至包括抗体片段。典型地，全长抗体结构优选包含4条多肽链，通常通过二硫键相互连接的2条重(H)链和2条轻(L)链。每条重链包含重链可变区和重链恒定区。每条轻链包含轻链可变区和轻链恒定区。在此典型全长抗体结构外,其结构还包括其他衍生形式。
根据其重链恒定区的氨基酸序列，完整的抗体可归属于IgA、IgD、IgE、IgG和IgM 五类抗体，其中IgG和IgA还可进一步分为亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。相应地，五类抗体的重链分别归入α、δ、ε、γ和μ链。根据其轻链恒定区的氨基酸序列，抗体的轻链可归入κ和λ。。
术语“可变区”指抗体重链或轻链中涉及抗体结合抗原的域。
术语“恒定区”是指抗体的轻链和重链上的这样一些氨基酸序列，不直接参与抗体与抗原的结合，但展现出多种效应子功能，例如抗体依赖性细胞毒性。
术语“Fc区”用于定义免疫球蛋白重链的C-末端区。“Fc区”可以是天然序列Fc区或变体Fc区。尽管免疫球蛋白重链的Fc区的边界可变化，但通常将人IgG重链Fc区定义为从Cys226位处的氨基酸残基或从Pro230延伸至其羧基末端。Fc区中残基的编号如Kabat中的EU索引。(Kabat等人,SequencesofProteinsofImmunologicalInterest,5thEd.PublicHealthService,NationalInstitutesofHealth,Bethesda,Md.,1991)。IgG的Fc区通常具有两个恒定区，CH 2和CH 3。
术语“Fc受体”或“FcR”指与抗体Fc区结合的受体。优选天然序列的人FcR，且优选与IgG抗体结合的受体(γ受体)，其包括FcγRI，FcγRII和FcγRIII亚型，以及这些受体的变体。其它FcR均被包含在术语“FcR”中。该术语也包括新生儿受体(FcRn)，其负责将母体的IgG转运至胎儿(Guyer等，免疫学杂志117：587(1976)和Kim等，免疫学杂志24：249(1994))。
术语“新生儿Fc受体”、简称“FcRn”，其结合IgG抗体Fc区。新生儿Fc受体(FcRn)在体内IgG类抗体的代谢命运中起重要作用。FcRn行使功能以从溶酶体降解途径营救IgG，从而降低其在血清中的清除率并加长半衰期。因此，IgG体外FcRn结合性质/特征指示它在血液循环中的体内药代动力学性质。
术语“Fc融合蛋白”是指利用基因工程等技术将某种具有生物活性的功能蛋白与Fc片段融合而产生的新型重组蛋白，其不仅保留了功能蛋白分子的生物学活性，还具有一些抗体的性质，如同FcRs的结合及介导的相关生物学功能等。
术语“改造的Fc多肽”、“改造的Fc区”和“改造的Fc”在本文中可互换使用，意指包含至少一个氨基酸改变的、或其糖基化修饰改变的Fc多肽或其部分。
术语“效应子功能”指可归因于抗体的Fc区的那些生物学活性，其随抗体同种型而不同。抗体效应子功能的实例包括：C1q结合和依赖补体的细胞毒性(CDC)、Fc受体(如CD16、CD32、CD64)结合、依赖抗体的细胞毒性(ADCC)、依赖抗体的吞噬作用(ADCP)、细胞因子分泌、免疫复合物介导的抗原呈递细胞对抗原的摄取、细胞表面受体(例如B细胞受体)的下调和B细胞激活。
术语“糖”可以用于指多糖、寡糖或单糖。多糖可以自生物体，如细菌分离，可以是天然的多糖，任选地，用微流化方法，将其大小调整至一定程度。将多糖进行大小调整，可降低多糖样品的粘度并且/或者提高缀合的产品的过滤性。寡糖是具有少量重复单元的水解多糖(典型地，5-30个重复单元)。多糖亦可以是化学合成的。
术语“缀合物”是指与糖共价缀合的蛋白/肽。本发明的融合蛋白糖缀合物和包含其的 免疫原性组合物可以包含一定量的游离糖、蛋白/肽。
本文所使用的“缀合”是指借以使例如细菌荚膜多糖的糖与蛋白/肽共价连接的过程。
术语“免疫原性组合物”是指含有抗原的任何药物组合物，该组合物可用于在个体中诱发免疫应答。
如本文所使用的“免疫原性”意指抗原(或抗原的表位)例如冠状病毒棘突蛋白受体结合区(SARS-CoV-2RBD)或包含该抗原的融合蛋白糖缀合物或免疫原性组合物在宿主(例如哺乳动物)中诱发体液或细胞介导的免疫应答或二者的能力。
“保护性”免疫应答是指免疫原性组合物诱发用于保护个体免于感染的体液或细胞介导的免疫应答或两者的能力。所提供的保护不必是绝对的，即，不必完全阻止或根除感染，只要相对于对照个体群体(例如未给药疫苗或免疫原性组合物的受感染动物)存在统计学上显著的改进即可。保护可限于缓和感染症状的严重性或发作快速性。
“免疫原性量”和“免疫有效量”二者在本文可交换使用，是指抗原或免疫原性组合物足以引发免疫应答(细胞(T细胞)或体液(B细胞或抗体)应答或二者，如通过本领域技术人员已知的标准测定所测量的)的量。
抗原作为免疫原的有效性可通过增殖测定、通过细胞溶解测定、或通过测量B细胞活性水平来测量。
本发明的提高蛋白/肽抗原免疫原性的方法
本发明基于发明人的两项开创性的发明做出。将蛋白/肽抗原与Fc受体结合/补体结合增强片段融合，形成融合蛋白，然后将该融合蛋白与糖缀合，形成融合蛋白糖缀合物。以该等融合蛋白糖缀合物做为免疫组合物的抗原免疫动物，可维持长时程的体液免疫和细胞免疫反应，且具有更高的细胞免疫反应、可产生较高滴度的中和抗体。
本发明的作为蛋白/肽抗原的融合蛋白糖缀合物
冠状病毒主要通过棘突蛋白(S蛋白)与宿主细胞受体结合来介导病毒的入侵，并决定病毒的组织或宿主嗜性。SARS-CoV-2的宿主细胞受体蛋白为血管紧张素转化酶2(ACE2)。病毒的三聚体棘突蛋白(S蛋白)同ACE2受体结合后被宿主蛋白酶切割为包含受体结合域(Receptor binding domain,SARS-COV-2 RBD)的S1多肽和负责介导病毒同细胞膜融合的S2多肽，进而侵入体内。
本发明的一个方案选用SARS-COV-2 RBD作为蛋白/肽抗原。抗原可以通过提取天然病原获得，也可以通过基因重组获得。SARS-COV-2 RBD与Fc受体和/或补体蛋白C1q结合能力提高Fc片段通过基因工程手段形成融合蛋白。在一个特别优选的方案中，Fc片段是Fc受体CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；其氨基酸序列SEQ ID NO：3所示。在一个最优选的方案中，经过改造的抗体Fc片段为Fc受体CD16a、CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；其氨基酸序列如SEQ ID NO：4所示，且用岩藻糖敲除的CHO细胞生产。然后，该融合蛋白与糖缀合， 糖可以是多糖、寡糖和单糖。
所述多糖可以是细菌多糖，如常见的奈瑟氏脑炎球菌荚膜多糖、嗜血性流感杆菌b荚膜多糖、肺炎链球菌荚膜多糖、B群金黄色葡萄球菌荚膜多糖以及葡聚糖、甘露聚糖等。多糖也可以是植物来源多糖，如淀粉、菊糖、果胶等，也可以是经化学改性的多糖的衍生物，如羧甲基淀粉。所述多糖也可以是动物来源多糖，如壳聚糖及其衍生物。
多糖与蛋白通过化学反应缀合。首先活化多糖，即使多糖带有反应活性的基团。活性基团再与蛋白质分子上的氨基、羧基、巯基、组氨酸上的咪唑环、色氨酸的吲哚环、酪氨酸上的苯环、苯丙氨酸的苯环、丝氨酸上的羟基以及谷氨酰胺、天冬酰胺等可发生化学反应的基团进行反应而形成共价键。
多糖与蛋白质分子缀合的一种方法是用高碘酸钠间多糖氧化，在多糖上生产醛基，醛基与蛋白质分子上的氨基反应，形成希夫碱，在还原剂的存在下，希夫碱被还原成稳定的单键。从而多糖与蛋白质分子形成共价连接。
本发明的免疫组合物
在一个实施方案中，本发明的免疫原性组合物还包含佐剂、缓冲剂、冷冻保护剂、盐、二价阳离子、非离子清洁剂、自由基氧化抑制剂、稀释剂或载体中的至少一种。
佐剂是当与免疫原或抗原一起给药时增强免疫应答的物质。本发明的免疫原性组合物可含有或不含有疫苗佐剂。可用于本发明组合物的佐剂包括但不限于：MF59、QS-21或MPL的至少一种。
在一个实施方案中，本发明的免疫原性组合物中的佐剂是铝系佐剂。所用的佐剂会取决于被给药所述免疫原性组合物的个体、规定的注射途径及注射次数。
所述免疫原性组合物可任选地包含药学上可接受的载体。所述药学上可接受的载体包括各国药典用于动物(包括人类以及非人类哺乳动物)的载体。术语载体可用于指与药物组合物一起给药的稀释剂、佐剂、赋形剂或媒介物。可采用水、盐水溶液以及含水的右旋糖和甘油溶液作为尤其用于注射溶液剂的液体载体。
本发明的免疫原性组合物还可包含一种或多种额外的免疫原性剂。
本发明的免疫原性组合物的给药形式
用于治疗或预防性治疗的本发明免疫原性组合物可以通过肌内注射、腹膜内注射、皮内注射或皮下注射；或者经由粘膜给药至口腔/食道、呼吸道、泌尿生殖道。鼻内给予疫苗对于治疗某些疾病，例如肺炎或中耳炎是优选的。虽然本发明的疫苗可单剂量给予，但是其组分也可同时或分时共同给予。除了单一给药途径以外，可以使用两种不同的给药途径。
用于特定免疫原性组合物的组分的最佳量可通过涉及在个体中观察适当免疫应答的标准研究来确定。在进行初始疫苗接种后，个体可接受一次或若干次充分间隔的加强免疫。
本发明的免疫原性组合物的用途
本发明的蛋白/肽抗原及免疫复合物可以预防或治疗病原体引起的疾病，尤其是冠状病毒，更尤其是SARS-CoV-2病毒引起的的疾病。本发明的蛋白/肽抗原及免疫复合物可以预防或治疗肿瘤疾病。亦可以用于免疫动物，以产生中和抗体。
本发明的术语简写
RBD或SARS-CoV-2 RBD：SARS-CoV-2冠状病毒棘突蛋白受体结合区
RBD-Fc：SARS-CoV-2冠状病毒棘突蛋白受体结合区与人IgG的Fc片段融合蛋白
RBD-Fc-Ce3：SARS-CoV-2冠状病毒棘突蛋白受体结合区与人IgG的Fc片段融合蛋白，其中Fc序列经过改造，其序列如SEQ ID NO:3所示
RBD-Fc-Fe4：SARS-CoV-2冠状病毒棘突蛋白受体结合区与人IgG的Fc片段融合蛋白，其中Fc序列经过改造，其序列如序列SEQ ID NO:4所示，且由哺乳动物细胞为fut8基因敲除的HEK-293细胞表达
PS14：肺炎链球菌血清型14荚膜多糖
RBD-PS14：RBD与PS14缀合物
RBD-Fc-PS14：RBD-Fc与PS14缀合物
RBD-Fc-Fe4-PS14：RBD-Fc-Fe4与PS14缀合物
RBD-Fc-Ce3-PS14：RBD-Fc-Ce3与PS14缀合物
Alum：铝佐剂，本文为磷酸铝佐剂
MF59：水包油型微乳佐剂，详见实施例
具体实施方式
实施例
实施例1：SARS-CoV-2 RBD、RBD-Fc、RBD-Fc-Ce3及RBD-Fc-Fe4融合蛋白表达载体的构建及蛋白生产
1.1 SARS-CoV-2 RBD表达载体的构建及蛋白生产
通过PCR扩增获得SARS-CoV-2-Spike-RBD序列(SEQ ID NO:7)(PCR扩增模板来源于北京义翘神州科技有限公司，下文同)，包含信号肽序列(SEQ ID NO:6)和SARS-CoV-2-Spike-RBD序列(SEQ ID NO:5)，通过in-fusion方法插入到Hind III+Xba I(来源：Fermentas，下文同)酶切的pSE载体(来源：神州细胞工程有限公司，下文同)中获得pSE-CoV-2-RBD表达载体(SEQ ID NO:7)。
扩增引物：
RBD-1 GTCACCGTCCTGACACGAAGCTT GGTACC(SEQ ID NO:16)
RBD-2 TATAGAATAGGGCCCTCTAGATTTAGAAGTTCACACACTTGTTCTTCACC(SEQ ID NO:17)
提取pSE-CoV-2-RBD质粒，转染HEK-293细胞(来源：Invitrogen，下文同)进行培养表达7天，纯化获得高纯度SARS-CoV-2 RBD蛋白。
1.2 SARS-CoV-2 RBD-mFc表达载体的构建及蛋白生产
通过PCR扩增SARS-CoV-2-Spike-RBD序列，通过in-fusion方法插入到Afe I酶切FastAP去磷的包含信号肽(SEQ ID NO:6)、linker(SEQ ID NO:8)和鼠IgG1恒定区序列(SEQ ID NO:13)的pSE-mFc载体(来源：神州细胞工程有限公司)中获得pSE-CoV-2-RBD-mFc表达载体(SEQ ID NO:14)。
扩增引物：
RBD-21TTTGTCTCTCCCAGCAGGGTGGTGCCATCTGGAGATGT(SEQ ID NO:18)
RBD-22GTCATCGTCATCAGCGAAGTTCACACACTGGTTCTTAA(SEQ ID NO:19)提取pSE-CoV-2-RBD-mFc质粒，转染HEK-293细胞进行培养表达7天，采用蛋白A纯化柱纯化获得高纯度RBD-mFc蛋白。
1.3 SARS-CoV-2 RBD-Fc表达载体的构建及蛋白生产
通过PCR扩增SARS-CoV-2-Spike-RBD序列，通过in-fusion方法插入到Afe I酶切FastAP去磷的包含信号肽(SEQ ID NO:6)、linker(SEQ ID NO:8)和人IgG1恒定区序列(SEQ ID NO:9)的pSTEP2-Fc载体(来源：神州细胞工程有限公司)中获得pSE-CoV-2-RBD-Fc表达载体(SEQ ID NO:10)。
扩增引物：
RBD-21TTTGTCTCTCCCAGCAGGGTGGTGCCATCTGGAGATGT(SEQ ID NO:20)
RBD-22GTCATCGTCATCAGCGAAGTTCACACACTGGTTCTTAA(SEQ ID NO:21)提取pSE-CoV-2-RBD-Fc质粒，转染HEK-293细胞进行培养表达7天，采用蛋白A纯化柱纯化获得高纯度RBD-Fc蛋白。
1.4 SARS-CoV-2 RBD-Fc-Ce3表达载体的构建及蛋白生产
为增强抗体Fc片段介导的CDC功能，参照文献对IgG1亚型的恒定区进行核苷酸突变[36,37]，得到基因工程改造的重链IgG1恒定区核苷酸序列(Fc-Ce3，SEQ ID NO:11)。通过PCR扩增SARS-CoV-2-Spike-RBD-Ce3-Fc序列(SEQ ID NO:12)，通过in-fusion方法插入到Hind III+Xba I酶切的pSE载体中获得pSE-nCoV-2-RBD-Fc-Ce3表达载体(SEQ ID NO:12)。
扩增引物:
RBD-23GTCACCGTCCTGACACGAAGCTTGGTACC(SEQ ID NO:22)
RBD-24
Figure PCTCN2021093470-appb-000001
RBD-25
Figure PCTCN2021093470-appb-000002
提取pSE-nCoV-2-RBD-Fc-Ce3质粒，转染HEK-293细胞进行培养表达7天，采用蛋白A纯化柱纯化获得高纯度RBD-Fc-Ce3。
1.5 SARS-CoV-2 RBD-Fc-Fe4表达载体的构建及蛋白生产
为进一步增强抗体Fc片段介导的免疫功能，提取pSE-nCoV-2-RBD-Fc-Ce3质粒，转染fut8基因敲除的HEK-293细胞(来源：神州细胞工程有限公司)进行培养表达7天，采用蛋白A纯化柱纯化获得去岩藻糖基化的高纯度RBD-Fc-Fe4。
实施例2：肺炎链球菌血清型14荚膜多糖(PS14)的制备
血清型14肺炎链球菌种子为ATCC 6314。
取0.5ml甘油保存的肺炎链球菌种子接入500ml Hoeprich's培养基(V.M.Goncalves, Optimization of medium and cultivation conditions for capsular polysaccharide productionby Streptococcus pneumoniae serotype 23F,AllpMicrobiolBiotechnol(2002)59:713-717)中，37℃摇床培养，转速150rpm，培养10-16小时，待OD 600大于1.0时停止培养。加入0.6g脱氧胆酸钠，混匀，静置2小时以上，使细菌完全裂解。转速14000g条件下离心30分钟，取上清，用100kDa超滤浓缩至原十分之一体积,约400ml。浓缩液逐渐加入36％乙酸，调至pH 3.5。静置2小时，转速14000g离心30分钟，取上清390ml加入130ml无水乙醇混匀，静置过夜。次日在14000g转速条件下离心30分钟后取上清，再加入780ml无水乙醇混匀，静置过夜。次日转速14000g离心30分钟后弃上清，在沉淀中加入300ml75％乙醇溶液，悬浮沉淀，之后再次14000g离心30分钟。弃上清，沉淀用10ml水溶解，控制溶液中多糖浓度大于10mg/ml。所得溶液即为肺炎链球菌荚膜多糖溶液。
实施例3：肺炎链球菌血清型14荚膜多糖的活化
取10mL浓度10mg/ml实施例2制备的多糖溶液，加入100mg高碘酸钠，混匀，避光静置反应1h。取装有5ml Sephadex G 25填料的离心层析柱，加入10ml 50mM，PH＝7.0的Na 2HPO 4缓冲液，缓冲液在重力作用下流过层析柱。然后将层析柱放入离心机，转速1000g条件下离心2min。之后更换新的收集管，取1ml经高碘酸钠氧化的多糖溶液放入离心层析柱，再次1000g离心2min。收集的离心柱流出液，即为活化的多糖溶液。
实施例4:蛋白/肽抗原与多糖缀合
选用RBD、RBD-Fc、RBD-Fc-Fe4、RBD-Fc-Ce3四种SARS-CoV-2 RBD或其融合蛋白为蛋白/肽抗原，将其分别与肺炎链球菌血清型14荚膜多糖缀合，步骤如下：
1.SARS-CoV-2 RBD或其融合蛋白换液：取SARS-CoV-2 RBD或其融合蛋白5mg，用30,000MW超滤管换液至50mM，PH为7.0的Na 2HPO 4缓冲液中，最终浓缩至体积小于0.5mL，既蛋白终浓度≥10mg/mL。
2.SARS-CoV-2 RBD或其融合蛋白与多糖缀合：取3mg SARS-CoV-2 RBD或其融合蛋白，加入0.5mg活化后肺炎链球菌荚膜多糖，并补加50mM,PH为7.0的Na 2HPO 4缓冲液，至总终体积为0.6ml，然后加入5M氰基硼氢化钠溶液3.6μl，在室温避光条件下旋转混合反应1h。然后向反应液中加入10mg/mL的硼氢化钠溶液0.15ml，室温反应2小时。然后将缀合物用100,000MW超滤管，PBS缓冲液换液10倍，最终超滤后体积小于2ml。0.22um滤器无菌过滤超滤后的缀合物样品后于4℃保存。
3.使用HPLC-MALLS测定缀合物的分子量。
缀合物 缀合物分子量kDa
RBD-PS14 6011/1237 *
RBD-FC-PS14 5944/1242/881.9 **
RBD-Fe4-FC-Ce3-PS14 5948/1441/1337 **
RBD-Fc-Fe4-PS14 7850/1886 *
*分子量分布两个峰
**分子量分布三个峰
实施例5.SARS-CoV-2 RBD/其融合蛋白糖缀合物的免疫组合物的制备及其免疫原性的测定
分别使用RBD、RBD-Fc、RBD-Fc-Fe4、RBD-Fc-Ce3和其对应的多糖缀合物RBD-PS14、RBD-FC-PS14、RBD-Fe4-FC-Ce3-PS14、RBD-Fc-Fe4-PS14作为抗原，制备免疫组合物并测定其免疫原性
5.1免疫组合物的制备
采用SARS-CoV-2 RBD蛋白/其融合蛋白或实施例4制备的缀合物作为抗原，制备免疫组合物。
5.1.1 MF59佐剂制备
配制200ml10mM柠檬酸钠溶液(HCl调节PH6.5)，加入1ml Tween 80(南京威尔药业股份有限公司)混匀充分溶解。另取10ml角鲨烯(Merck)并加入1ml Span 85(肇庆市超能实业有限公司)混匀充分溶解。将前面两种溶液混合，用高压匀质机(AH-PILOTATS)设置800bar均质3次，得到均匀的乳液即为MF59佐剂。
5.1.2铝佐剂免疫组合物的制备
抗原分别用PBS稀释成0.02mg/ml、0.06mg/ml或0.2mg/ml(以肽/蛋白计，下同)，铝佐剂(北京诺宁生物科技有限公司)用PBS稀释成1mg/ml。稀释后的抗原和铝佐剂等体积混合。该免疫组合物中抗原的蛋白浓度分别为0.01mg/ml、0.03mg/ml或0.1mg/ml。
5.1.3 MF59佐剂免疫组合物的制备
抗原分别用PBS稀释成0.02mg/ml、0.06mg/ml或0.2mg/ml，稀释后的抗原与等体积的MF59佐剂混合，该免疫组合物中抗原的蛋白浓度分别为0.01mg/ml、0.03mg/ml或0.1mg/ml。
5.1.4 MF59和铝佐剂混合佐剂免疫组合物的制备
取3份1.5ml铝佐剂各加1.5ml MF59佐剂混合后分别加入0.03ml、0.09ml、0.3ml浓度为1mg/ml的抗原，该免疫组合物中抗原的蛋白浓度分别为0.01mg/ml、0.03mg/ml或0.1mg/ml。
5.2免疫小鼠：
选用4-6周Balb/c小鼠，腹腔注射0.1ml如实施例5.1所述的抗原浓度为0.01mg/ml或0.03mg/ml或0.1mg/ml免疫组合物，于第14天加强免疫(与首免相同剂量)。第7天，第21天眼眶取血，测定血清效价、中和滴度以及IgG2a/IgG1的比例。
5.3血清效价的测定
5.3.1抗原为SARS-COV-2 RBD或其融合蛋白时血清效价的测定
将浓度为5μg/mL的SARS-COV-2 RBD-mFc蛋白(神州细胞工程有限公司，全文同)包被于96孔板，100μl/孔，室温放置2小时，洗板后加入2％BSA室温封闭1小时，以CD155(D1)-mFc(神州细胞工程有限公司，全文同)作为相同标签的无关对照。用含0.1％牛血清白蛋白(BSA)的PBS将待检测血清(实施例5.2制备的)稀释到1:8000，向包被好的96孔板加入待测血清和山羊抗鼠IgG F(ab) 2/HRP(北京义翘神州科技有限公司)检测二抗，各100μl/孔，共同孵育2h后洗板5遍，加入底物显色液进行显色，终止后酶标仪读取OD 450。在一定稀释倍数下的OD 450表示抗体滴度。
测量以RBD-Fc-Fe4和RBD-Fc-Fe4-PS14为抗原；Alum、MF59、MF59+Alum 为佐剂；免疫剂量分别为1、3和10μg的免疫组合物免疫小鼠的第21天血清(1:8000稀释)的效价，结果见表1。
表1.以RBD-Fc-Fe4和RBD-Fc-Fe4-PS14为抗原的免疫组合物免疫小鼠血清抗体效价
Figure PCTCN2021093470-appb-000003
测量以RBD、RBD-Fc、RBD-Fc-Ce3、RBD-PS14、RBD-Fc-PS14、RBD-Fc-Ce3-PS14为抗原、以MF59+Alum混合佐剂；免疫剂量分别为1、3和10μg的免疫组合物免疫小鼠的第21天血清(1:8000稀释)的效价，结果见表2。
表2.以RBD-Fc-Ce3和RBD-Fc-Ce3-PS14为抗原的免疫组合物免疫小鼠血清抗体效价
免疫组合物的抗原/免疫剂量/血清效价 1μg/OD 450 3μg/OD 450 10μg/OD 450
RBD 0.988 0.997 1.006
RBD-Fc 1.103 1.235 1.298
RBD-Fc-Ce3 1.023 1.275 1.906
RBD-PS14 1.061 1.272 1.257
RBD-Fc-PS14 0.427 0.648 0.827
RBD-Fc-Ce3-PS14 0.450 0.469 0.882
5.4中和效价的测定
将实施例5.2中获得的小鼠血清样品稀释500倍后与800TCID50/ml假病毒2019-nCoV PSV(中国食品药品检定研究院)等体积混合，37℃孵育1小时后，同步侵染3×10 4/孔Vero E6或293FT/ACE2细胞(神州细胞工程有限公司)。侵染后细胞在37℃，5％CO 2条件下培养20-28小时左右，在微孔板式发光检测仪上检测RLU值。以加假病毒、不加血清样品作为阳性对照，不加假病毒和血清样品作为阴性对照。按照中和抑制率％＝(lg(阳性RLU)-lg(样品RLU))/(lg(阳性RLU)-lg(阴性RLU))x100％,计算中和抑制率。
测量以RBD-Fc-Fe4和RBD-Fc-Fe4-PS14为抗原；Alum、MF59、MF59+Alum为佐剂；免疫剂量为1、3和10μg的免疫组合物免疫小鼠的第21天血清(1:500稀释)的假病毒中和效价，结果见表3。
表3.以RBD-Fc-Fe4和RBD-Fc-Fe4-PS14为抗原的免疫组合物免疫小鼠血清假病毒中和效价
Figure PCTCN2021093470-appb-000004
测量以RBD、RBD-Fc、RBD-Fc-Ce3、RBD-PS14、RBD-Fc-PS14、RBD-Fc-Ce3-PS14为抗原、以MF59+Alum混合佐剂；免疫剂量分别为1、3和10μg时免疫小鼠的第21天血清(1:500稀释)的假病毒中和效价，结果见表4。
表4.以RBD-Fc-Ce3和RBD-Fc-Ce3-PS14为抗原的免疫组合物免疫小鼠血清假病毒中和效价
免疫组合物的抗原/免疫剂量 1μg抑制率％ 3μg抑制率％ 10μg抑制率％
RBD 6.2 9.8 14.0
RBD-Fc 26.4 24.0 25.4
RBD-Fc-Ce3 29.6 53.6 71.0
RBD-PS14 44.2 93.2 81.2
RBD-Fc-PS14 36.0 42.8 75.4
RBD-Fc-Ce3-PS14 26.4 44.8 78.8
5.5测定并计算IgG2a/IgG1
5.5.1小鼠血清中IgG1抗体的测定
采用酶联免疫法(ELISA)检测小鼠血清中IgG1抗体。将包被蛋白RBD蛋白稀释至浓度为2μg/mL后，取100μl包被酶标板，放置于4℃条件下过夜。洗板，在室温下使用100μl含2％BSA(牛血清白蛋白)的TBST缓冲液封闭酶标板2小时，洗板。使用含0.1％BSA的TBST稀释液稀释实施例5.2的小鼠血清样品至1:500000。将100μl稀释好的小鼠血清样品加入到封闭过的酶标板中，室温作用1小时。洗板，将100μl浓度为1μg/mL的标记HRP抗IgG1抗体R-mIgG1-R020-H(来源：北京义翘神州科技有限公司)加入到酶标板中室温作用1小时。加入显色液显色，终止反应后使用酶标仪在450nm下进行读数。以1:500000稀释度血清的OD 450值表示IgG1的量。
5.5.2小鼠血清中IgG2a抗体的定量测定
采用酶联免疫法(ELISA)检测小鼠血清中IgG2a抗体的含量。将包被蛋白RBD蛋白稀释至浓度为2μg/mL后，取100μl包被酶标板，放置于4℃条件下过夜。洗板，在室温下使用100μl含2％BSA的TBST缓冲液封闭酶标板2小时，洗板。使用含0.1％BSA的TBST稀释液稀释小鼠血清样品至1:5000。将100μl稀释好的小鼠血清样品加入到封闭过的酶标板中，室温作用1小时，洗板。将100μl浓度为1μg/mL的标记HRP抗IgG2a抗体R-mIgG2a-R005-H(来源：北京义翘神州科技有限公司)加入到酶标板中室温作用 1小时。加入显色液显色，终止反应后使用酶标仪在450nm下进行读数。以1:5000稀释度血清的OD 450值表示小鼠血清中IgG2a抗体的量。
IgG2a/IgG1＝(1:5000血清)OD 450值/(1:500000血清)OD 450值，测定结果如表5-6和图1-2所示。
测量以RBD-Fc-Fe4和RBD-Fc-Fe4-PS14为抗原；Alum、MF59、MF59+Alum为佐剂；免疫剂量分别为1、3和10μg的免疫组合物免疫小鼠的第21天血清(以1:5000稀释血清测IgG2a；以1:500000稀释血清测IgG1)的IgG2a/IgG1比值，结果见表5。
表5.以RBD-Fc-Fe4和RBD-Fc-Fe4-PS14为抗原的免疫组合物免疫小鼠血清IgG2a/IgG1比值
Figure PCTCN2021093470-appb-000005
测量以RBD、RBD-Fc、RBD-Fc-Ce3、RBD-PS14、RBD-Fc-PS14、RBD-Fc-Ce3-PS14为抗原、佐剂为MF59+Alum混合佐剂；免疫剂量分别为1、3和10μg时免疫小鼠的第21天血清(以1:5000稀释血清测IgG2a；以1:500000稀释血清测IgG1)的IgG2a/IgG1比值，结果见表6。
表6.三种蛋白及其对应的缀合物为抗原的免疫组合物免疫小鼠血清IgG2a/IgG1比值
Figure PCTCN2021093470-appb-000006
Figure PCTCN2021093470-appb-000007
图1显示了RBD-Fc-Fe4和RBD-Fc-Fe4-PS14两种抗原分别使用铝佐剂、MF59佐剂、MF59加铝佐剂混合佐剂免疫小鼠的结果。每种佐剂又分别设置1μg,3μg,10μg三个免疫剂量，二免后取血清测定IgG2a/IgG1比值。由图可以看出，在使用MF59佐剂时，与多糖缀合的抗原免疫血清IgG2a/IgG1比值大幅度提高。高的IgG2a/IgG1比值预示着更高的细胞免疫反应，也就预示着疫苗有更好的保护效果。
图2显示了RBD-PS14、RBD-Fc-PS14、RBD-Fc-Ce3-PS14及RBD-Fc-Fe4-PS14抗原血清IgG2a/IgG1比值的比较，佐剂为MF59+Alum混合佐剂。由图可见RBD-Fc-Fe4-PS14相比其他蛋白多糖缀合物具有更高的IgG2a/IgG1比值。
根据上述数据，对于RBD-Fc-Fe4，在与铝佐剂同时使用时，与多糖的缀合物相比单纯的蛋白免疫血清效价有所提高。但在使用MF59佐剂时，RBD-Fc-Fe4与其对应的多糖缀合物免疫血清的抗体效价和假病毒中和滴度相差不大。但RBD-Fc-Fe4与多糖缀合物免疫血清的IgG2a/IgG1比值明显提高。IgG2a/IgG1比值反映了细胞免疫倾向，高的IgG2a/IgG1比值预示着更高的细胞免疫反应，也就预示着疫苗有更好的保护效果。对比几种抗原蛋白与其对应多糖缀合物的IgG2a/IgG1比值，RBD-Fc-Fe4多糖缀合物最高，所以RBD-Fc-Fe4多糖缀合物是最优的疫苗候选分子。
Figure PCTCN2021093470-appb-000008
Figure PCTCN2021093470-appb-000009
Figure PCTCN2021093470-appb-000010
Figure PCTCN2021093470-appb-000011
Figure PCTCN2021093470-appb-000012
Figure PCTCN2021093470-appb-000013
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Claims (36)

  1. 一种增强蛋白/肽抗原的免疫原性的方法，该方法包含
    将蛋白/肽抗原与Fc片段融合，形成融合蛋白，然后
    将该融合蛋白与糖缀合，形成融合蛋白糖缀合物，优选地
    Fc片段为受体结合/补体结合增强Fc片段。
  2. 如权利要求1所述的方法，其中所述的蛋白/肽抗原为病原体相关的蛋白/肽抗原或肿瘤相关蛋白/肽抗原。
  3. 如权利要求2所述的方法，
    其中所述的病原体选自：
    冠状病毒，人免疫缺陷性病毒HIV-1，人单纯疱疹病毒，巨细胞病毒，轮状病毒，EB病毒，水痘带状疱疹病毒，肝炎病毒，呼吸道合胞体病毒，副流感病毒，麻疹病毒，流行性腮腺炎病毒，人乳头瘤病毒，黄病毒或流感病毒，奈瑟氏菌属，莫拉氏菌属，博代氏杆菌属，分枝杆菌属，包括结核分枝杆菌；埃希氏菌属，包括肠毒素大肠埃希菌；沙门氏菌属，李斯特氏菌属，螺杆菌属，葡萄球菌属，包括金黄葡萄球菌，表皮葡萄球菌；疏螺旋体属，衣原体，包括砂眼衣原体，肺炎衣原体；疟原虫，包括恶性疟原虫；弓形虫，念珠菌；
    其中的肿瘤选自：
    弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、其他淋巴瘤、白血病、多发性骨髓瘤、间皮瘤、胃癌、恶性横纹肌瘤、肝细胞癌、前列腺癌，乳腺癌、胆管癌和胆囊癌、膀胱癌、脑肿瘤包括神经母细胞瘤、神经鞘瘤、胶质瘤、胶质母细胞瘤和星形细胞瘤、宫颈癌、结肠癌、黑色素瘤、子宫内膜癌、食管癌、头颈癌、肺癌、鼻咽癌、卵巢癌、胰腺癌、肾细胞癌、直肠癌、甲状腺癌、甲状旁腺肿瘤、子宫肿瘤和软组织肉瘤。
  4. 如权利要求1-3所述的方法，其中蛋白/肽蛋白抗原选自分泌蛋白或全长膜蛋白，或其功能结构域、突变蛋白、截短蛋白，或由其一个或多个抗原多肽表位拼接组成的改造蛋白。
  5. 如权利要求1-4之任一所述的方法，其中蛋白/肽抗原选自
    冠状病毒抗原；
    优选冠状病毒棘突蛋白；
    更优选冠状病毒棘突蛋白片段；
    更优选冠状病毒棘突蛋白的ACE2受体结合结构域(RBD)；
    更优选冠状病毒棘突蛋白S1亚单位；
    优选地，上述冠状病毒选自SARS-CoV-2。
  6. 如权利要求1-5之任一所述的方法，其中Fc片段来自人抗体、鼠源抗体、兔源抗体或其他动物抗体的重链恒定区。
  7. 如权利要求6所述的方法，其中Fc片段来自人抗体的IgG、IgM或IgA亚型抗体，优选地，来自IgG1、IgG2、IgG3或IgG4亚型抗体；
    更优选地，是为提高与Fc受体/C1q补体结合功能之目的，而进行氨基酸序列突变和/或糖基化形式改变的IgG1改造Fc片段。
  8. 如权利要求1-7之任一所述的方法，Fc受体选自CD16、CD32a、CD32b或CD64。
  9. 如权利要求8所述的方法，其中经过改造的抗体Fc片段为Fc受体CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
    其氨基酸序列如SEQ ID NO：3所示。
  10. 如权利要求8所述的方法，其中经过改造的抗体Fc片段为Fc受体CD16a、CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
    其氨基酸序列如SEQ ID NO：4所示，且用岩藻糖敲除的哺乳细胞生产，优选地，哺乳动物细胞为fut8基因敲除的HEK-293细胞。
  11. 如权利要求1-10所述的方法，其中所述抗原优选地通过接头和其他大分子缀合，优选地，其他大分子为多糖、肽/蛋白。
  12. 如权利要求1-11所述的方法，其中其他大分子选自多糖、寡糖或单糖；
    优选为奈瑟氏脑炎球菌荚膜多糖、嗜血性流感杆菌b荚膜多糖、肺炎链球菌荚膜多糖、B群金黄色葡萄球菌荚膜多糖、葡聚糖、甘露聚糖、淀粉、菊糖、果胶、羧甲基淀粉、壳聚糖及其衍生物；
    更优选为肺炎链球菌荚膜多糖；
    更优选为肺炎链球菌血清型14荚膜多糖、肺炎链球菌血清型6B荚膜多糖和肺炎链球菌血清型7F荚膜多糖；
    最优选为肺炎链球菌血清型14荚膜多糖。
  13. 如权利要求1-11之任一的所述的方法，其中缀合物的分子量为800-6000KDa。
  14. 如权利要求1-12之任一的所述的方法，其中融合蛋白糖缀合物形式的蛋白/肽抗原和免疫佐剂组合。
  15. 如权利要求14所述的方法，其中佐剂选自铝佐剂、MF59或其混合物。
  16. 一种免疫原性增强的蛋白/肽抗原，其包含
    将蛋白/肽抗原与Fc片段融合形成融合蛋白，然后
    与糖缀合形成的蛋白/肽-Fc片段融合蛋白糖缀合物；优选地
    Fc片段为受体结合/补体结合增强Fc片段。
  17. 如权利要求16所述的蛋白/肽抗原，其中所述的蛋白/肽抗原为病原体相关的蛋白/肽抗原或肿瘤相关蛋白/肽抗原。
  18. 如权利要求16或17所述的蛋白/肽抗原，
    其中所述的病原体选自：
    冠状病毒，人免疫缺陷性病毒HIV-1，人单纯疱疹病毒，巨细胞病毒，轮状病毒，EB病毒，水痘带状疱疹病毒，肝炎病毒，呼吸道合胞体病毒，副流感病毒，麻疹病毒，流行性腮腺炎病毒，人乳头瘤病毒，黄病毒或流感病毒，奈瑟氏菌属，莫拉氏菌属，博代氏杆菌属，分枝杆菌属，包括结核分枝杆菌；埃希氏菌属，包括肠毒素大肠埃希菌；沙门 氏菌属，李斯特氏菌属，螺杆菌属，葡萄球菌属，包括金黄葡萄球菌，表皮葡萄球菌；疏螺旋体属，衣原体，包括砂眼衣原体，肺炎衣原体；疟原虫，包括恶性疟原虫；弓形虫，念珠菌；
    其中的肿瘤选自：
    弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、其他淋巴瘤、白血病、多发性骨髓瘤、间皮瘤、胃癌、恶性横纹肌瘤、肝细胞癌、前列腺癌，乳腺癌、胆管癌和胆囊癌、膀胱癌、脑肿瘤包括神经母细胞瘤、神经鞘瘤、胶质瘤、胶质母细胞瘤和星形细胞瘤、宫颈癌、结肠癌、黑色素瘤、子宫内膜癌、食管癌、头颈癌、肺癌、鼻咽癌、卵巢癌、胰腺癌、肾细胞癌、直肠癌、甲状腺癌、甲状旁腺肿瘤、子宫肿瘤和软组织肉瘤。
  19. 如权利要求16-18所述的蛋白/肽抗原，其中蛋白/肽蛋白抗原选自分泌蛋白或全长膜蛋白，或其功能结构域、突变蛋白、截短蛋白，或由其1个或多个抗原多肽表位拼接组成的改造蛋白。
  20. 如权利要求16-19之任一所述的蛋白/肽抗原，其中蛋白/肽抗原选自
    冠状病毒抗原；
    优选冠状病毒棘突蛋白；
    更优选冠状病毒棘突蛋白片段；
    更优选冠状病毒棘突蛋白的ACE2受体结合结构域(RBD)；
    更优选冠状病毒棘突蛋白S1亚单位；
    优选地，上述冠状病毒选自SARS-CoV-2。
  21. 如权利要求16-20之任一所述的蛋白/肽抗原，其中Fc片段来自人抗体、鼠源抗体、兔源抗体或其他动物抗体的重链恒定区。
  22. 如权利要求21所述的蛋白/肽抗原，其中Fc片段来自人抗体的IgG、IgM或IgA亚型抗体，
    优选地，来自IgG1、IgG2、IgG3或IgG4亚型抗体；
    更优选地，是为提高与Fc受体、C1q补体结合功能之目的，而进行氨基酸序列突变和/或糖基化形式改变的IgG1改造Fc片段。
  23. 如权利要求16-22之任一所述的蛋白/肽抗原，Fc受体选自CD16、CD32a、CD32b或CD64。
  24. 如权利要求23所述的蛋白/肽抗原，其中经过改造的抗体Fc片段为Fc受体CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
    其氨基酸序列如SEQ ID NO：3所示。
  25. 如权利要求23所述的蛋白/肽抗原，其中经过改造的抗体Fc片段为Fc受体CD16a、CD32a、CD32b和CD64结合增强片段/补体C1q结合增强片段；
    其氨基酸序列如SEQ ID NO：4所示，且用岩藻糖敲除的哺乳细胞生产，优选地，哺乳动物细胞为fut8基因敲除的HEK-293细胞。
  26. 如权利要求16-25所述的蛋白/肽抗原，其中所述抗原优选地通过接头和其他大分子 缀合，优选地，其他大分子为多糖、肽/蛋白。
  27. 如权利要求26所述的蛋白/肽抗原，其中其他大分子选自多糖、寡糖或单糖；
    优选为奈瑟氏脑炎球菌荚膜多糖、嗜血性流感杆菌b荚膜多糖、肺炎链球菌荚膜多糖、B群金黄色葡萄球菌荚膜多糖、葡聚糖、甘露聚糖、淀粉、菊糖、果胶、羧甲基淀粉、壳聚糖及其衍生物；
    更优选为肺炎链球菌荚膜多糖；
    更优选为肺炎链球菌血清型14荚膜多糖、肺炎链球菌血清型6B荚膜多糖和肺炎链球菌血清型7F荚膜多糖；
    最优选为肺炎链球菌血清型14荚膜多糖。
  28. 如权利要求27所述的蛋白/肽抗原，其中缀合物的分子量为800-6000KDa。
  29. 一种免疫组合物，其包含
    a)权利要求16-28之任一所述的蛋白/肽抗原；
    b)佐剂；和
    c)药学上可接受的载体、赋形剂或稳定剂，优选为
    冻干制剂或水溶液形式的药学上可接受的载体、赋形剂或稳定剂。
  30. 权利要求29所述的免疫组合物，其中佐剂可选自铝佐剂、MF59的至少一种。
  31. 权利要求16-28之任一所述的蛋白/肽抗原、或权利要求29或30所述的免疫组合物，其用于预防病原体，优选冠状病毒、更优选SARS-CoV-2引起的疾病/肿瘤的应用。
  32. 权利要求16-28之任一所述的蛋白/肽抗原、或权利要求29或30所述的免疫组合物用于制备预防病原体，优选冠状病毒、更优选SARS-CoV-2引起的疾病/肿瘤的疫苗中的应用。
  33. 一种免疫组合，其包含
    权利要求16-28之任一所述的蛋白/肽抗原、或权利要求29或30所述的免疫组合物；以及
    一种或多种另外的免疫原性剂。
  34. 一种试剂盒，其包含
    权利要求16-28之任一所述的蛋白/肽抗原、或权利要求29或30所述的免疫组合物；
    优选地，还进一步包含给予免疫组合物的装置。
  35. 一种预防病原体，优选冠状病毒、更优选SARS-CoV-2引起的疾病/预防肿瘤的方法，其包含给予受治疗者权利要求16-28之任一所述的蛋白/肽抗原、权利要求29或30所述的免疫组合物、权利要求33的免疫组合或权利要求34的试剂盒。
  36. 一种免疫动物的方法，其包含给予动物权利要求16-28之任一所述的蛋白/肽抗原、权利要求29或30所述的免疫组合物、权利要求33的免疫组合或权利要求34的试剂盒，以产生中和抗体。
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