WO2021219047A1 - 一种增强蛋白/肽抗原免疫原性的方法 - Google Patents

一种增强蛋白/肽抗原免疫原性的方法 Download PDF

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WO2021219047A1
WO2021219047A1 PCT/CN2021/090809 CN2021090809W WO2021219047A1 WO 2021219047 A1 WO2021219047 A1 WO 2021219047A1 CN 2021090809 W CN2021090809 W CN 2021090809W WO 2021219047 A1 WO2021219047 A1 WO 2021219047A1
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protein
virus
peptide
antigen
seq
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PCT/CN2021/090809
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French (fr)
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谢良志
张延静
张建东
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神州细胞工程有限公司
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Priority to KR1020227041681A priority Critical patent/KR20230005313A/ko
Priority to MX2022013456A priority patent/MX2022013456A/es
Priority to CA3176745A priority patent/CA3176745A1/en
Priority to CN202180030064.4A priority patent/CN115484977A/zh
Priority to BR112022022003A priority patent/BR112022022003A2/pt
Priority to AU2021262999A priority patent/AU2021262999A1/en
Priority to JP2022566202A priority patent/JP2023526780A/ja
Priority to US17/922,335 priority patent/US20230330220A1/en
Priority to EP21797065.6A priority patent/EP4144365A4/en
Publication of WO2021219047A1 publication Critical patent/WO2021219047A1/zh

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Definitions

  • the present invention relates to the field of immunogenic compositions, in particular to a method for enhancing the immunogenicity of protein/peptide antigens by conjugating protein/peptide antigens with sugars, forming sugar-protein/peptide antigen conjugates, Compared with unconjugated protein/peptide antigens, its immunogenicity is improved. More specifically, it involves conjugating pathogens, such as viral surface protein antigens or fragments thereof, with polysaccharides, especially Streptococcus pneumoniae capsular polysaccharides.
  • the immunogenicity-enhanced conjugate can be used to prevent or treat diseases caused by pathogens, especially diseases caused by coronaviruses.
  • the above-mentioned antigen component is a foreign substance that induces or stimulates vertebrates
  • the individual's memory immune response to the foreign molecule so that when the vertebrate is exposed to the foreign molecule again, the immune response is again, and the individual is protected from harm.
  • an "antigen” is a foreign substance recognized (specifically bound) by antibodies or T cell receptors, but not necessarily capable of inducing an immune response.
  • the exogenous substances that can be recognized by antibodies or T cell receptors (specifically bind) and induce specific immunity are called “immune antigens" or “immunogens.”
  • Vaccines that use subunits of infectious microorganisms, toxins, and viruses, that is, a part of the cell structure (bacteria or fungi) or viruses, are non-live vaccines, and are widely adopted due to their safety.
  • the ability of subunits to induce specific immune responses is weak, that is, the antigen has poor immunogenicity.
  • 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. An important method is to conjugate poorly immunogenic antigens with exogenous macromolecules used as carriers to improve the immunogenicity of these molecules. This method has been successfully applied for decades, such as conventional encephalitis vaccines and addictive drugs. Blood influenza bacteria b vaccine and pneumonia vaccine, with its purified capsular polymer combined with carrier protein to produce a more effective immunogenic composition (Schneerson et al. (1984) Infect. Immun.
  • Commonly used carrier proteins such as tetanus toxoid, tetanus toxoid fragment C, non-toxic mutant of tetanus toxin, diphtheria toxoid, CRM197, other non-toxic mutants of diphtheria toxin (e.g. CRM176, CRM197, CRM228, CRM45( Uchida et al. J. Biol. Chem. 218; 3838-3844, 1973); CRM9, CRM45, CRM02, CRM103 and CRM107 and other mutants.
  • Such polysaccharide antigens are non-thymocyte-dependent antigens and cannot produce cellular immune responses and cannot form immune memory.
  • the polysaccharide antigen is conjugated to a protein carrier with T cell epitopes, the antigen-presenting cell or B cell endocytic sugar and the protein conjugate are processed, and the polypeptide fragment of the carrier protein is displayed on the cell surface to activate the helper T Cells cause 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 derived from a hospital pathogen (or its oligosaccharide fragment, which represents one or more epitopes) in an immunogenic composition, the polysaccharide conjugate It is combined with the staphylococcal surface adhesion carrier protein to elicit an antibody response to the polysaccharide antigen and the staphylococcal surface adhesion carrier protein.
  • 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 immunity to diseases caused by hospital pathogens In other words, the surface adhesin protein itself can also confer immunity to the body, rather than just acting as a protein carrier for polysaccharide antigens.
  • the drop of surface adhesin protein-specific antibodies induced by the conjugated surface adhesin protein The degree is similar to that of unconjugated surface adhesin protein ( Figure 17-20). This confirmed that the epitope was not changed by the binding of surface adhesin protein and CP.
  • the inventor's pioneering discovery includes the improvement of the immunogenicity of the protein/peptide antigen by conjugating the protein/peptide antigen with the sugar to form a sugar-protein/peptide antigen conjugate.
  • most of the pattern recognition receptors on the surface of antigen-presenting cells of the animal immune system are related to sugars, and the sugars produced by bacteria are important signals to stimulate the immune system.
  • the present invention is not bound by this theory.
  • One aspect of the present invention relates to a method for improving the immunogenicity of a protein/peptide antigen, the method comprising forming a sugar-protein/peptide antigen conjugate by conjugating the protein/peptide antigen to a sugar.
  • the sugar is 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;
  • capsular polysaccharide of Streptococcus pneumoniae serotype 14 the capsular polysaccharide of Streptococcus pneumoniae serotype 6B, and the capsular polysaccharide of Streptococcus pneumoniae serotype 7F,
  • the protein/peptide antigen is selected from pathogen-related protein/peptide antigen or tumor-related protein/peptide antigen,
  • 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;
  • proteins/peptides related to pathogens invading the host Preferably proteins/peptides related to pathogens invading the host;
  • the above-mentioned pathogen is a virus
  • the above-mentioned virus is selected from the virus of the coronavirus family, paramyxoviridae, orthomyxoviridae, filoviridae or flaviviridae, and
  • the tumor is selected from:
  • the protein/peptide antigen is a virus antigen of the coronavirus family
  • coronavirus spike protein Preferably coronavirus spike protein
  • the S1 subunit of the coronavirus spike protein More preferably, the S1 subunit of the coronavirus spike protein
  • RBD the receptor binding region of coronavirus spike protein
  • the coronavirus is SARS-CoV-2 or Middle East respiratory syndrome coronavirus.
  • the protein/peptide antigen is a fusion protein of the aforementioned antigen and other proteins or peptides,
  • the fusion protein is selected from SARS-CoV-2 RBD-mFc; or
  • Fc is preferably an IgG Fc fragment, more preferably a human or murine IgG Fc fragment.
  • the protein/peptide antigen comprises the sequence described in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.
  • the molecular weight of the sugar-protein/peptide antigen conjugate is 400-14000 KDa.
  • the protein/peptide antigen comprises a Paramyxoviridae virus antigen
  • the paramyxovirus glycoprotein receptor binding region Preferably, the paramyxovirus glycoprotein receptor binding region
  • Paramyxovirus glycoprotein F and glycoprotein G Preferably, Paramyxovirus glycoprotein F and glycoprotein G;
  • the Paramyxoviridae virus is human respiratory syncytial virus.
  • the protein/peptide antigen is a fusion protein of the aforementioned antigen and other proteins or peptides; preferably, the fusion protein is RSV-gpG-his.
  • protein/peptide antigen comprises the sequence set forth in SEQ ID NO: 4 and/or SEQ ID NO: 12.
  • the protein/peptide antigen comprises an Orthomyxoviridae virus antigen
  • Preferred orthomyxovirus glycoprotein receptor binding region Preferred orthomyxovirus glycoprotein receptor binding region
  • HA hemagglutinin
  • NA neuraminidase
  • the orthomyxovirus is influenza B virus and/or influenza A H5N1 virus.
  • the protein/peptide antigen is a fusion protein of the aforementioned antigen and other proteins or peptides,
  • the fusion protein is Flu-B-HA1-his or H5N1-HA-his.
  • the protein/peptide antigen comprises the sequence described in any one of SEQ ID NO: 7 and/or SEQ ID NO: 15, SEQ ID NO: 8 and/or SEQ ID NO: 16 .
  • the protein/peptide antigen is a virus antigen of the family Filoviridae
  • the filovirus envelope glycoprotein receptor binding region Preferably, the filovirus envelope glycoprotein receptor binding region
  • the envelope glycoprotein of filovirus GP1 and/or GP2 Preferably, the envelope glycoprotein of filovirus GP1 and/or GP2;
  • envelope glycoprotein of filovirus GP1 More preferably, the envelope glycoprotein of filovirus GP1;
  • the filovirus is Ebola virus.
  • the protein/peptide antigen is a fusion protein of the aforementioned antigen and other proteins or peptides,
  • the fusion protein is Ebola-GP-Fc or Ebola-GP1-his;
  • Fc is preferably an IgG Fc fragment, more preferably a human or murine IgG Fc fragment.
  • the protein/peptide antigen comprises the sequence described in any one of SEQ ID NO: 9 and/or SEQ ID NO: 17, SEQ ID NO: 10 and/or SEQ ID NO: 18.
  • the protein/antigen comprises an antigen of a Flaviviridae virus
  • Flavivirus or Hepatitis virus antigen Preferably Flavivirus or Hepatitis virus antigen
  • the receptor binding region of the envelope protein of the Flavivirus genus virus Preferably, the receptor binding region of the envelope protein of the Flavivirus genus virus;
  • At least one of the EDI, EDII and EDIII domains of the envelope protein of the Flavivirus virus are preferably at least one of the EDI, EDII and EDIII domains of the envelope protein of the Flavivirus virus;
  • the EDIII domain of the envelope protein of Flavivirus More preferably, the EDIII domain of the envelope protein of Flavivirus.
  • the envelope glycoprotein receptor binding region of the hepatitis virus genus virus Preferably, the envelope glycoprotein receptor binding region of the hepatitis virus genus virus;
  • hepatitis virus envelope glycoprotein E1 and/or E2 Preferably hepatitis virus envelope glycoprotein E1 and/or E2;
  • the Flavivirus genus virus is preferably Zika virus; the Hepatitis virus genus virus is preferably hepatitis C virus.
  • the protein/peptide antigen is a fusion protein of the aforementioned antigen and other proteins or peptides,
  • the fusion protein is ZIKV-E-Fc; wherein
  • Fc is preferably an IgG Fc fragment, more preferably a human or murine IgG Fc fragment; or
  • the fusion protein is HCV-E2-his and/or HCV-E1-his.
  • the protein/peptide antigen comprises SEQ ID NO: 11 and/or SEQ ID NO: 19, SEQ ID NO: 6 and/or SEQ ID NO: 14, SEQ ID NO: 5 and/ Or the sequence described in any one of SEQ ID NO: 13.
  • the sugar-protein/peptide antigen is further conjugated with a protein carrier.
  • the protein carrier is tetanus toxoid, tetanus toxoid fragment C, non-toxic mutant of tetanus toxin, diphtheria toxoid, CRM197, other non-toxic mutants of diphtheria toxin, preferably CRM197 .
  • the second aspect of the present invention relates to a sugar-protein/peptide antigen conjugate, which has improved immunogenicity compared with unconjugated protein/peptide antigen.
  • the sugar is 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; wherein the protein/peptide antigen is selected from pathogen-related protein/peptide antigen or tumor-related Protein/peptide antigen,
  • 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;
  • proteins/peptides related to pathogens invading the host Preferably proteins/peptides related to pathogens invading the host;
  • the above-mentioned pathogen is a virus
  • the above-mentioned virus is selected from the virus of the coronavirus family, paramyxoviridae, orthomyxoviridae, filoviridae or flaviviridae, and
  • the tumor is selected from:
  • the protein/peptide antigen is a virus antigen of the coronavirus family
  • coronavirus spike protein Preferably coronavirus spike protein
  • the S1 subunit of the coronavirus spike protein More preferably, the S1 subunit of the coronavirus spike protein
  • RBD the receptor binding region of coronavirus spike protein
  • the coronavirus is SARS-CoV-2 or Middle East respiratory syndrome coronavirus.
  • the protein/peptide antigen is a fusion protein of the aforementioned antigen and other proteins or peptides,
  • the fusion protein is selected from SARS-CoV-2 RBD-mFc; or
  • Fc is preferably an IgG Fc fragment, more preferably a human or murine IgG Fc fragment.
  • the protein/peptide antigen comprises the sequence described in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.
  • the protein/peptide antigen comprises
  • the paramyxovirus glycoprotein receptor binding region Preferably, the paramyxovirus glycoprotein receptor binding region
  • Paramyxovirus glycoprotein F and glycoprotein G Preferably, Paramyxovirus glycoprotein F and glycoprotein G;
  • the paramyxovirus is human respiratory syncytial virus.
  • the protein/peptide antigen is a fusion protein of the aforementioned antigen and other proteins or peptides,
  • the fusion protein is RSV-gpG-his.
  • the protein/peptide antigen comprises the sequence set forth in SEQ ID NO: 4 and/or SEQ ID NO: 12.
  • the protein/peptide antigen comprises
  • Preferred orthomyxovirus glycoprotein receptor binding region Preferred orthomyxovirus glycoprotein receptor binding region
  • HA hemagglutinin
  • NA neuraminidase
  • the orthomyxovirus is influenza B virus and/or influenza A H5N1 virus.
  • the protein/peptide antigen is a fusion protein of the aforementioned antigen and other proteins or peptides,
  • the fusion protein is Flu-B-HA1-his or H5N1-HA-his.
  • the protein/peptide antigen comprises the sequence described in any one of SEQ ID NO: 7 and/or SEQ ID NO: 15, SEQ ID NO: 8 and/or SEQ ID NO: 16.
  • the protein/peptide antigen comprises
  • the filovirus envelope glycoprotein receptor binding region Preferably, the filovirus envelope glycoprotein receptor binding region
  • the envelope glycoprotein of filovirus GP1 and/or GP2 Preferably, the envelope glycoprotein of filovirus GP1 and/or GP2;
  • envelope glycoprotein of filovirus GP1 More preferably, the envelope glycoprotein of filovirus GP1;
  • the filovirus is Ebola virus.
  • the protein/peptide antigen is a fusion protein of the aforementioned antigen and other proteins or peptides,
  • the fusion protein is Ebola-GP-Fc or Ebola-GP1-his;
  • Fc is preferably an IgG Fc fragment, more preferably a human or murine IgG Fc fragment.
  • the protein/peptide antigen comprises the sequence described in any one of SEQ ID NO: 9 and/or SEQ ID NO: 17, SEQ ID NO: 10 and/or SEQ ID NO: 18.
  • the protein/peptide antigen comprises
  • Flavivirus or Hepatitis virus antigen Preferably Flavivirus or Hepatitis virus antigen
  • the receptor binding region of the envelope protein of the Flavivirus genus virus Preferably, the receptor binding region of the envelope protein of the Flavivirus genus virus;
  • At least one of the EDI, EDII and EDIII domains of the envelope protein of the Flavivirus virus are preferably at least one of the EDI, EDII and EDIII domains of the envelope protein of the Flavivirus virus;
  • the EDIII domain of the envelope protein of Flavivirus More preferably, the EDIII domain of the envelope protein of Flavivirus.
  • the envelope glycoprotein receptor binding region of the hepatitis virus genus virus Preferably, the envelope glycoprotein receptor binding region of the hepatitis virus genus virus;
  • hepatitis virus envelope glycoprotein E1 and/or E2 Preferably hepatitis virus envelope glycoprotein E1 and/or E2;
  • the Flavivirus virus is preferably Zika virus; or
  • the hepatitis virus genus virus is preferably hepatitis C virus.
  • the protein/peptide antigen is a fusion protein of the aforementioned antigen and other proteins or peptides,
  • the fusion protein is ZIKV-E-Fc; wherein
  • Fc is preferably an IgG Fc fragment, more preferably a human or murine IgG Fc fragment; or
  • the fusion protein is HCV-E2-his and/or HCV-E1-his.
  • the protein/peptide antigen comprises SEQ ID NO: 11 and/or SEQ ID NO: 19, SEQ ID NO: 6 and/or SEQ ID NO: 14, SEQ ID NO: 5 and/ Or the sequence described in any one of SEQ ID NO: 13.
  • the molecular weight of the sugar-protein/peptide antigen conjugate is 400-14000 KDa.
  • the sugar-protein/peptide antigen is further conjugated with a protein carrier.
  • the protein carrier is tetanus toxoid, tetanus toxoid fragment C, non-toxic mutant of tetanus toxin, diphtheria toxoid, CRM197, other non-toxic mutants of diphtheria toxin, preferably CRM197 .
  • the third aspect of the present invention relates to an immune complex comprising the aforementioned sugar-protein/peptide antigen conjugate, an immune adjuvant and an excipient.
  • the adjuvant is selected from aluminum adjuvant, oil-in-water emulsion adjuvant, MF59, QS-21 and lipid A monophosphate.
  • the fourth aspect of the present invention relates to the use of glyco-protein/peptide antigen conjugates or immune complexes to prevent or treat diseases caused by pathogen-related protein/peptide antigens or tumor-related proteins/peptides as defined above.
  • the preferred pathogen is
  • Coronavirus more preferably SARS-CoV-2 and/or MERS-CoV;
  • Paramyxovirus more preferably human respiratory syncytial virus
  • Orthomyxovirus more preferably influenza B virus and/or influenza A H5N1 virus;
  • Filovirus more preferably Ebola virus
  • Flavivirus preferably Zika virus; or hepatitis C virus.
  • the fifth aspect of the present invention relates to the use of glyco-protein/peptide antigen conjugates or immune complexes in the preparation of vaccines or drugs for the prevention and treatment of diseases caused by pathogen-related protein/peptide antigens or tumor-related proteins/peptides as defined above ,
  • the preferred pathogen is
  • Coronavirus more preferably SARS-CoV-2 and/or MERS-CoV;
  • Paramyxovirus more preferably human respiratory syncytial virus
  • Orthomyxovirus more preferably influenza B virus and/or influenza A H5N1 virus;
  • Filovirus more preferably Ebola virus
  • Flavivirus preferably Zika virus; or hepatitis C virus.
  • Figure 1 depicts the anti-SARS-COV-2 RBD antibody titers of mice immunized with the immune composition with SARS-COV-2 RBD-mFc as the antigen.
  • the value is the absorbance detected when the serum is diluted 8000 times.
  • Figure 2 depicts the comparison of different adjuvants.
  • the antigen is SARS-COV-2 RBD-his-PS14 conjugate
  • the serum dilution factor is 32000 times
  • the immunization dose is 3 ⁇ g/mouse.
  • Figure 3 depicts the comparison of the serum neutralization activity of SARS-COV-2 RBD protein-PS14 polysaccharide conjugate and SARS-COV-2 RBD and CRM197 co-conjugated PS14 conjugate as antigen.
  • the serum dilution factor is 1500 times, and the immunization dose is 3 ⁇ g/mouse.
  • Figure 4 depicts the immunization results of the conjugates of PS7F, PS14 and dextran as conjugating agents.
  • antigen is a foreign substance recognized (specifically bound) by antibodies or T cell receptors, but 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 molecules of hapten, or a combination of a hapten and a macromolecular carrier can elicit an immune response).
  • 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.
  • Cell-mediated immune response is composed of T cells and /Or other leukocyte-mediated immune responses, which are induced by providing epitopes related to major histocompatibility complex (MHC) class I or class II molecules, CD1 or other atypical MHC-like molecules.
  • MHC major histocompatibility complex
  • 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 in this specification and the appended claims refers to a protein/peptide covalently conjugated to a sugar.
  • the sugar-protein/peptide conjugate 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 such as a microorganism or its components, which composition can be used to induce an immune response in an individual.
  • 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.
  • Immunogenicity means an antigen (or epitope of an antigen) such as the coronavirus spike protein receptor binding region or a glycoconjugate or immunogenic composition containing the antigen in a host (e.g., lactating The ability of animals) to induce humoral or cell-mediated immune responses or both.
  • 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 can be limited to alleviating the severity of infection symptoms or rapid onset.
  • Immunogenic amount and “immune 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, for example, by a proliferation assay, by a cytolysis assay, or by measuring the level of B cell activity.
  • the present invention is a technological invention.
  • the inventors discovered that the immunogenicity of the protein/peptide antigen is improved by conjugating the protein/peptide antigen with the sugar to form a sugar-protein/peptide antigen conjugate.
  • Carbohydrate-conjugated protein/peptide antigen of the present invention is Carbohydrate-conjugated protein/peptide antigen of the present invention
  • the coronavirus family includes Orthocoronavirinae and Letovirinae.
  • the Betacoronaviru of the orthocoronavirus subfamily includes the well-known severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (Middle East respiratory syndrome-related coronavirus).
  • SARS-CoV Severe Acute Respiratory Syndrome Coronavirus 2
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
  • S protein spike protein
  • ACE2 angiotensin converting enzyme 2
  • the spike protein (S protein) is the same as the ACE2 receptor After binding, it is cleaved by the host protease into the S1 polypeptide containing the receptor binding domain (SARS-COV-2 RBD) and the S2 polypeptide responsible for mediating the fusion of the virus with the cell membrane, and then invades the body.
  • S protein S1 polypeptide containing the receptor binding domain (SARS-COV-2 RBD) and the S2 polypeptide responsible for mediating the fusion of the virus with the cell membrane, and then invades the body.
  • a scheme of the present invention selects the coronavirus SARS-CoV-2 spike protein (SARS-CoV-2S protein), its extracellular domain, S1 subunit or receptor binding domain as the antigen.
  • SARS-CoV-2S protein coronavirus SARS-CoV-2 spike protein
  • S1 subunit or receptor binding domain as the antigen.
  • One solution of the present invention selects Middle East Respiratory Syndrome Coronavirus as the antigen. Such as its extracellular domain, S1 subunit or receptor binding domain as an antigen.
  • the Paramyxoviridae family includes two subfamilies, Paramyxivirinae and Pneumovirinae.
  • Human Respiratory Syncytial Virus (RSV) is one of the Respiroviruses of the Paramyxovirus subfamily Respirovirus.
  • RSV encodes two major transmembrane surface glycoproteins, glycoprotein G (adsorption protein) and glycoprotein F (fusion protein) .
  • Glycoprotein G mediates the binding of virus to cell receptors, while glycoprotein F promotes the fusion of virus and cell membrane, allowing viral ribonucleoprotein to invade the cytoplasm (Lopez et al. (1998) J. Virology 72:6922-6928).
  • RSV envelope proteoglycan as the antigen.
  • the human RSV envelope protein sugar can be selected, such as RSV glycoprotein F or glycoprotein G.
  • Orthomyxoviridae includes Influenzavirus A, Influenzavirus B, Influenzavirus C and other genera.
  • Influenza virus A, B and C infection mainly rely on two envelope protein sugars: hemagglutinin (HA) and neuraminidase (NA), which are responsible for virus attachment and virus particle invasion into cells. Influenza virus infection is triggered by the attachment of the virion surface hemagglutinin (HA) protein to the sialic acid-containing cell receptors (glycoprotein and glycolipid).
  • HA hemagglutinin
  • NA neuraminidase
  • the type A H5N1 influenza virus hemagglutinin (HA) protein is used as the antigen, and the type B influenza virus hemagglutinin protein (HA1 subunit) can also be used as the antigen.
  • Representative species of the filovirus family are Ebolaviruses of Ebolavirus and marburgviruses of Marburgvirus.
  • GP1 glycoprotein
  • GP2 glycoprotein
  • disulfide bonds Volchkova, VA ef a/., (1998), Virology 250:408- 414; Falzarano, D. et al., (2006), Chembiochem 7:1605-161 1). It is known that GP1 mediates virus attachment to host cells, and GP2 participates in membrane fusion (Sanchez, A. et al., (1996), Proc Natl Acad Sci USA 93: 3602-3607; Alazard-Dany, N. et al. 2006), J.Gen.Virol.87:1247-1257).
  • One aspect of the present invention selects Ebola virus glycoprotein (GP) as the antigen.
  • GP Ebola virus glycoprotein
  • Flaviviridae viruses mainly include Flavivirus, Pestivirus, Pegivirus
  • the Flavivirus includes Zika virus (ZIKV), dengue fever (DV), West Nile virus, Japanese encephalitis virus and yellow fever virus.
  • the hepatitis virus genus includes hepatitis C virus (HCV).
  • the flavivirus envelope protein plays an important role in the host cell virus infection, mediating the virus into the host cell. It consists of three independent structural envelope domains I, II and III (EDI, EDII and EDIII).
  • EDI is a structural central domain of the envelope protein, which stabilizes the overall direction of the protein.
  • the glycosylation site in EDI is related to virus production, pH sensitivity and neural invasion. Due to the immunological advantages of fusion ring epitopes and envelope dimer epitopes, EDII plays an important role in membrane fusion.
  • EDIII is the main target of neutralizing antibodies (Xingcui Zhang.et al., (2017) Viruses. 2017 Nov; 9(11): 338. Structures and Functions of the Envelope Glycoprotein in Flavivirus Infections).
  • E domain I is the central domain that organizes the entire E protein structure.
  • E domain II is formed by two extended loops protruding from E-DI and is located in the pocket at E-DI and E domain III (E-DIII).
  • E-DIII is an immunoglobulin-like domain that forms small protrusions on the surface of otherwise smooth spherical mature virus particles and is thought to interact with cell receptors on target cells (CN109996560A).
  • the HCV RNA genome encodes a single polyprotein, which is cleaved into three structural proteins (core, glycoprotein E1 and E2) and seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and E2) during or after translation. NS5B).
  • Envelope protein sugars E1 and E2 form a heterodimer to form a viral envelope protein, which plays an important role when the virus enters the host cell, mediating virus entry and morphogenesis.
  • the envelope protein sugar of hepatitis C virus binds to a specific protein on the surface of the host's liver cells to initiate the entry process. This process involves a large number of host receptors/co-receptors.
  • E2 is the main HCV envelope proteoglycan, which directly interacts with the receptor/co-receptor.
  • E1 does not directly interact with the host receptor during this process. It maintains the functional E2 conformation required for receptor binding and co-induced membrane fusion with E2 (Yimin, Tong. Et al., ( 2018) Front Immunol. 2018; 9: 1411. Role of Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly).
  • a scheme of the present invention selects the Zika virus envelope protein E-DIII as the antigen.
  • hepatitis C virus envelope protein sugars E1 and/or E2 are used as antigens.
  • the viral antigens mentioned in this chapter can be obtained by extracting natural pathogens or by genetic recombination. All can be further modified, for example, their immunogenic fragments or variants, as well as proteins with purification tags or fusion proteins with antibody Fc fragments, can all be used in the present invention.
  • the polysaccharide is 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 and so on.
  • 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.
  • the conjugation process of polysaccharide and protein is as follows: the polysaccharide has reactive groups through chemical reaction.
  • the active groups are combined with the amino, carboxyl, sulfhydryl, imidazole ring on histidine, indole ring of tryptophan, benzene ring on tyrosine, phenyl ring of phenylalanine and serine on the protein molecule.
  • the hydroxyl group, glutamine, asparagine, and other chemically reactive groups react to form a covalent bond.
  • One method of conjugating polysaccharides and protein molecules is to oxidize the polysaccharides with sodium periodate to produce aldehyde groups on the polysaccharides.
  • the aldehyde groups react with amino groups on the protein molecules to form a Schiff base.
  • a reducing agent such as sodium cyanoborohydride can be added to the reaction system.
  • Another method for conjugating polysaccharides and protein molecules is to react polysaccharides with cyanogen bromide or 1-cyano-4-dimethylaminopyridine tetrafluoroborate to produce reactive cyanate esters.
  • the cyanate ester group reacts with the amino group on the protein surface to form a covalent.
  • the activated polysaccharide can also react with hexamethylene diamine, adipic hydrazide and other linking arms first, and then the product reacts with the protein in the presence of a shrinking agent to form a covalent connection.
  • Polysaccharides can also be activated with other chemical reagents, and then react with proteins to form conjugations.
  • Other chemical reagents such as epichlorohydrin, triazine, diazine, divinyl sulfone and other reagents well known in the art.
  • a protein carrier may be further added to form a sugar-protein/peptide antigen-protein carrier conjugate during the conjugation reaction of the sugar with the protein/peptide antigen.
  • the protein carrier can be tetanus toxoid, tetanus toxoid fragment C, non-toxic mutant of tetanus toxin, diphtheria toxoid, CRM197, other non-toxic mutants of diphtheria toxin commonly used in the vaccine industry, preferably CRM197.
  • 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.
  • the adjuvant in the immunogenic composition of the present invention is an aluminum-based adjuvant.
  • the adjuvant is an aluminum-based adjuvant selected from aluminum phosphate, aluminum sulfate, and aluminum hydroxide.
  • the adjuvant is aluminum phosphate.
  • An adjuvant is a substance that enhances the immune response when administered with an immunogen or antigen.
  • the composition used in 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:
  • Oil emulsion composition includes squalene-water emulsion, such as MF59; complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); saponin preparation; combined use of saponin and cholesterol to form an immunostimulatory complex (ISCOM) unique particles; virosomes and virus-like particles; the adjuvant used will depend on the individual to whom the immunogenic composition is administered, the prescribed injection route and the number of injections.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • saponin preparation combined use of saponin and cholesterol to form an immunostimulatory complex (ISCOM) unique particles
  • virosomes and virus-like particles the adjuvant used will depend on the individual to whom the immunogenic composition is administered, the prescribed injection route and the number of injections.
  • the immunogenic composition may optionally include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes a carrier for animals (including humans and non-human mammals) described in or to be described in the pharmacopoeias of various countries.
  • 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 present invention may also contain one or more additional immunomodulators, which are substances that disrupt or alter the immune system so that up- or down-regulation of humoral and/or cell-mediated immunity is observed.
  • additional immunomodulators are substances that disrupt or alter the immune system so that up- or down-regulation of humoral and/or cell-mediated immunity is observed.
  • an up-regulation of the humoral and/or cell-mediated arms of the immune system is provided. This includes, for example, adjuvants or cytokines.
  • the immunogenic composition of the present invention used 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 conjugate and immune complex of the present invention can prevent or treat diseases caused by pathogens, such as coronavirus, paramyxovirus, orthomyxovirus, filovirus and flavivirus, and more particularly SARS-CoV-2 And/or diseases caused by MERS-CoV virus, human respiratory syncytial virus, influenza B virus, influenza A H5N1 virus, Ebola virus, Zika virus and/or hepatitis C virus.
  • pathogens such as coronavirus, paramyxovirus, orthomyxovirus, filovirus and flavivirus
  • SARS-CoV-2 And/or diseases caused by MERS-CoV virus human respiratory syncytial virus
  • influenza B virus influenza A H5N1 virus
  • Ebola virus Zika virus
  • Zika virus Zika virus
  • SARS-COV-2 RBD-mFc SARS-CoV-2 coronavirus spike protein receptor binding domain mouse Fc fusion protein
  • SARS-COV-2 RBD-his SARS-CoV-2 coronavirus spike protein receptor binding region and 6-histidine tag fusion protein
  • MERS-COV RBD-his A fusion protein of the receptor binding domain of the spike protein receptor of the Middle East respiratory syndrome coronavirus and the 6-histidine tag;
  • RSV-gpG Human respiratory syncytial virus glycoprotein G/Human respiratory syncytial virus glycoprotein G;
  • HCV-E1 Hepatitis C virus Envelope Glycoprotein E1/Hepatitis C virus envelope glycoprotein E1;
  • HCV-E2 Hepatitis C virus Envelope E2 Protein/hepatitis C virus envelope glycoprotein E2;
  • flu-B-HA1 Influenza B Hemagglutinin Protein (HA1 Subunit)/Type B influenza virus hemagglutinin (HA) protein (HA1 subunit);
  • H5N1-HA Influenza A H5N1 Hemagglutinin/A H5N1 influenza virus hemagglutinin (HA) protein;
  • Ebola-GP Ebola virus Glycoprotein (Receptor Binding Domain,) Ebola virus glycoprotein (receptor binding domain);
  • Ebola-GP1 Ebola virus Glycoprotein GP1/Ebola virus glycoprotein GP1;
  • ZIKV-E Zika virus Envelope protein (Domain III)/Zika virus envelope protein (III domain);
  • PS14 Streptococcus pneumoniae serotype 14 capsular polysaccharide
  • PS7F Streptococcus pneumoniae serotype 7F capsular polysaccharide
  • Alum aluminum adjuvant, this article is aluminum phosphate adjuvant
  • Example 1 Preparation of capsular polysaccharides of Streptococcus pneumoniae serotype 14 (PS14) and 7F (PS7F)
  • the seeds of serotype 14 Streptococcus pneumoniae are ATCC 6314, and the seeds of serotype 7F Streptococcus pneumoniae are ATCC 10351.
  • 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 it stand for 2 hours, centrifuge at 14000g for 30 minutes, take 390ml of supernatant and add 130ml of absolute ethanol to mix, and let it 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.
  • the polysaccharide is the capsular polysaccharide of Streptococcus pneumoniae serotype 14 (PS14) and 7F (PS7F) prepared in Example 1 or dextran (dextran, Sigma, the same below 00894).
  • the molecular weight of PS14 capsular polysaccharide is about 500KDa, and it is about 300KDa after hydrolysis.
  • PS7F capsular polysaccharide has a molecular weight of about 700KDa and has not been hydrolyzed.
  • Glucan is not hydrolyzed.
  • the protein/peptide antigen used is the receptor binding region of the coronavirus spike protein, and the polysaccharide is the capsular polysaccharide of Streptococcus pneumoniae or dextran.
  • the specific components, amounts and volumes are shown in Table 1 respectively.
  • Coronavirus spike receptor protein exchange Take 5 mg of the coronavirus spike receptor protein, exchange the solution with a 30,000MW ultrafiltration tube to 50mM Na 2 HPO 4 buffer, pH 7.0, and exchange the protein at the end The concentration requirement is ⁇ 10mg/mL.
  • Coronavirus spike receptor protein and polysaccharide conjugation Take 3 mg of coronavirus spike receptor protein, add activated Streptococcus pneumoniae capsular polysaccharide or dextran according to Table 1, and add 50mM, pH 7.0 Na 2 HPO 4 buffer solution, the final volume is shown in Table 1, and then 5M sodium cyanoborohydride solution is added according to the volume in Table 1, and the reaction is rotated and mixed for 1 h at room temperature and protected from light. Then, 10 mg/mL sodium borohydride solution was added to the reaction solution (0.15 ml was added to the 0.6 ml reaction system, and 0.375 ml was added to the 1.5 ml reaction system), and the reaction was carried out at room temperature for 2 hours.
  • conjugate sample after ultrafiltration was aseptically filtered on a 0.22um filter and stored at 4°C.
  • MERS-COV RBD-his source: Beijing Yiqiao Shenzhou Technology Co., Ltd., 40071-V08B1
  • other proteins were prepared by the inventor himself.
  • the protein/peptide antigen used is the SARS-COV-2 RBD of the coronavirus spike protein receptor binding region
  • the protein carrier is CRM197
  • the polysaccharide is the capsular polysaccharide of Streptococcus pneumoniae.
  • the specific components, amounts and volumes are shown in Table 2 respectively.
  • CRM197 is a variant of diphtheria toxin (Geert J. Schenk, Efficient CRM197-mediated dr ⁇ g targeting to monocytes, Journal of Controlled Release 158 (2012) 139-147). Coronavirus spike protein receptor binding domain and CRM197 are jointly conjugated to the capsular polysaccharide of Streptococcus pneumoniae serotype 14. The activation process of polysaccharide is the same as in Example 2.2. Take 1.5 mg of polysaccharide, 2.7 mg of coronavirus spike protein receptor binding region protein, add 0.3 mg of CRM197, and conjugate according to the same process as in Example 3. The reaction conditions of the conjugate and the molecular weight of the product are shown in Table 2.
  • coronavirus spike protein receptor binding domain-polysaccharide conjugates are shown in Table 3-6.
  • an immune composition was prepared.
  • MPL MPL
  • sodium citrate buffer 10 mM, pH 6.5.
  • the antigen was diluted with PBS to 0.02mg/ml or 0.06mg/ml (calculated as peptide/protein, the same below), and 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 or 0.03 mg/ml, respectively.
  • the antigen was diluted with PBS to 0.02 mg/ml or 0.06 mg/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 0.01 mg/ml or 0.03 mg/ml, respectively.
  • the antigen was diluted with PBS to 0.02mg/ml or 0.06mg/ml, and the diluted antigen was mixed with an equal volume of MPL-containing MF59 adjuvant.
  • the protein concentration of the antigen in the immune composition is 0.01 mg/ml or 0.03 mg/ml, respectively.
  • the protein concentration of the antigen in the immune composition is 0.03mg/ml.
  • mice 5.2 Immunization of mice:
  • mice were selected from Balb/c mice for 4-6 weeks, and 0.1ml of the immune composition as described in Example 5.1 was injected intraperitoneally at a concentration of 0.01mg/ml or 0.03mg/ml, and the immunization was boosted on the 14th day and the 28th day, respectively .
  • Blood was taken from the orbit on the 7th day, the 21st day, and the 35th day, and the serum antibody titers and neutralization titers were determined.
  • MERS-COV RBD-his When detecting MERS-COV RBD-his immune serum, use MERS-COV RBD-his (Beijing Yiqiao Shenzhou Technology Co., Ltd., 40071-V08B1) coating. There is no positive control and negative control. The operation steps are exactly the same as 5.3.1.
  • Table 3-Table 5 and Figures 1-4 all show the results of serum titer determination.
  • Table 3 shows the titer results of serum dilution 8000 times after immunization of mice with aluminum adjuvant immunization composition 35 days.
  • SARS-COV-2 RBD-his-PS14 aluminum/MF59/MF59-aluminum/MF59-MPL adjuvant immunization composition 35 days immunized serum diluted 32000 times the titer results are shown in Table 4.
  • Example 5.2 Dilute the mouse serum sample obtained in Example 5.2 to a certain multiple (for example, 500-fold dilution) based on experience, and mix it with the pseudovirus 2019-nCoV PSV (from China Institute for Food and Drug Control) in equal volume, without adding the serum sample as positive As a control, no false virus was added as a negative control.
  • Vero E6 or 293FT/ACE2 cells were simultaneously infected. After the infection, the cells were cultured at 37°C and 5% CO 2 for about 20-28 hours, and the RLU value was measured on a microplate luminescence detector. according to
  • Neutralization inhibition rate % (lg(positive RLU)-lg(sample RLU))/(lg(positive RLU)-lg(negative RLU)) ⁇ 100%, calculate the neutralization inhibition rate.
  • Serotype 14 (PS14) of Streptococcus pneumoniae was prepared according to Example 1, and activated according to the steps of Example 2.2.
  • the amount of sodium periodate added was adjusted according to Table 8.
  • PS14 was conjugated with the carrier protein CRM197.
  • the ratio of protein to polysaccharide during conjugation is shown in Table 8.
  • an aluminum-containing adjuvant immune composition was prepared. Mice were immunized according to Example 5.2, and the immunization dose was 3 ⁇ g antigen per mouse.
  • Table 8 shows the immune serum titers of various antigen conjugates. It can be seen that the immunogenicity of most antigens and polysaccharides is significantly improved after conjugation.
  • the antigens used in Table 8 are all described in Table 7, but His-tag is not shown.
  • the immunogenicity of the protein antigen and the capsular polysaccharide of Streptococcus pneumoniae is significantly improved after conjugation.
  • the antibody titer of the conjugate immune serum can reach 2.3 times the original.
  • the neutralizing activity of the conjugate is also greatly improved compared to the neutralizing activity of the corresponding protein.
  • MF59 adjuvant, MF59 adjuvant mixed with aluminum adjuvant, MF59 adjuvant plus MPL adjuvant can further improve the immune effect of the conjugate.

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Abstract

一种提高蛋白/肽抗原免疫原性的方法，该方法包括通过将蛋白/肽抗原与糖缀合，形成糖-蛋白/肽抗原缀合物，与未经缀合的蛋白/肽抗原相比，其免疫原性提高。具体涉及将病原体，例如病毒表面蛋白抗原或其片段与多糖，尤其是肺炎链球菌荚膜多糖缀合。该免疫原性增强的缀合物可以用于预防或治疗病原体引起的疾病，尤其是冠状病毒引起的疾病。

Description

一种增强蛋白/肽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01日提交的中国专利申请202010369100.7的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明涉及免疫原性组合物领域，具体地，涉及一种通将蛋白/肽抗原与糖缀合而增强蛋白/肽抗原免疫原性的方法，形成的糖-蛋白/肽抗原缀合物，与未经缀合的蛋白/肽抗原相比，其免疫原性提高。更具体地，涉及将病原体，例如病毒表面蛋白抗原或其片段与多糖，尤其是肺炎链球菌荚膜多糖缀合。该免疫原性增强的缀合物可以用于预防或治疗病原体引起的疾病，尤其是冠状病毒引起的疾病。
背景技术
将感染性微生物、毒素、病毒或其亚单位作为疫苗的抗原组分，将脊椎动物个体接种疫苗，上述的抗原组分相对于个体而言，是一种外源物质，其诱导或刺激脊椎动物个体针对外源分子的记忆免疫应答，从而在脊椎动物再次暴露于外源分子时再次免疫应答，保护个体免受侵害。
“抗原”是由抗体或T细胞受体所识别(特异性结合)的外源物质，但不一定能诱导免疫应答。而能够由抗体或T细胞受体所识别(特异性结合)、且诱导特异性免疫的外源性物质称为“免疫性抗原”或“免疫原”。
采用感染性微生物、毒素、病毒的亚单位，即细胞结构(细菌或真菌)或病毒的一部分为抗原的疫苗，为非活体疫苗，因其安全性被广泛地采纳。但是亚单位诱导特异性免疫应答的能力较弱，即抗原的免疫原性差。
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从而提高这些分子的免疫原性，这一方法已成功应用了数十年，例如习见的脑炎疫苗、嗜血流感细菌b疫苗和肺炎疫苗，以其纯化的荚膜多糖(capsularpolymer)与载体蛋白结合而产生更有效的免疫原性组合物(Schneerson等.(1984)Infect.Immun.45：582-591)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。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细胞内吞糖与蛋白的缀合物进行处理，并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。
但是很少报道细菌多糖对蛋白/肽抗原的免疫原性的影响。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。虽然“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。”
以上两项研究工作中，仅报道与多糖缀合的蛋白/肽抗原亦可以引起抗体产生，但是没有报道其免疫原性增强。
发明人开创性地发现包括通过将蛋白/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。
发明人推测其原理是，蛋白聚集体比蛋白单体更容易刺激机体产生免疫反应，产生抗体。此外，动物免疫系统的抗原提呈细胞表面的模式识别受体多数与糖有关，细菌产生的糖类是刺激免疫系统的重要信号。但是，本发明不受此理论的束缚。
发明内容
本发明的一个方面涉及一种提高蛋白/肽抗原免疫原性的方法，该方法包括通过将蛋白/肽抗原与糖缀合，形成糖-蛋白/肽抗原缀合物。
在本发明的一个具体实施方案中，糖选自多糖、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，
最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，
其中蛋白/肽抗原选自病原体相关的蛋白/肽抗原或肿瘤相关蛋白/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；
优选与病原体侵入宿主相关的蛋白/肽；
更优选上述病原体为病毒；
更优选上述病毒选自冠状病毒科、副黏液病毒科、正黏液病毒科、丝状病毒科或黄病毒科病毒，以及
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。
在本发明的一个具体实施方案中，蛋白/肽抗原是包含冠状病毒科病毒抗原；
优选冠状病毒棘突蛋白；
更优选冠状病毒棘突蛋白S1亚单位；
更优选冠状病毒棘突蛋白受体结合区RBD；
或具有免疫原性的上述所有蛋白/肽抗原的片段或变体的蛋白/肽。
在本发明的一个具体实施方案中，冠状病毒为SARS-CoV-2或中东呼吸综合征冠状病毒。
在本发明的一个具体实施方案中，蛋白/肽抗原为前述定义的抗原与其他蛋白或肽的融合蛋白，
优选地，融合蛋白选自SARS-CoV-2 RBD-mFc；或
SARS-CoV-2 RBD-his；或
MERS-COV RBD-his；以及
Fc优选为IgG Fc片段、更优选人或鼠IgG Fc片段。
在本发明的一个具体实施方案中，蛋白/肽抗原包含SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3之任一所述的序列。
在本发明的一个具体实施方案中，糖-蛋白/肽抗原缀合物的分子量为400-14000KDa。
在本发明的一个具体实施方案中，蛋白/肽抗原是包含副黏液病毒科病毒抗原；
优选副黏液病毒糖蛋白受体结合区；
优选副黏液病毒糖蛋白F、糖蛋白G；
或具有免疫原性的上述所有蛋白/肽抗原的片段或变体的蛋白/肽。
在本发明的一个具体实施方案中，副黏液病毒科病毒为人呼吸道合胞病毒。
在本发明的一个具体实施方案中，蛋白/肽抗原为前述定义的抗原与其他蛋白或肽的融合蛋白；优选融合蛋白为RSV-gpG-his。
在本发明的一个具体实施方案中，其中蛋白/肽抗原包含SEQ ID NO:4和/或SEQ ID NO:12所述的序列。
在本发明的一个具体实施方案中，蛋白/肽抗原是包含正黏液病毒科病毒抗原；
优选正黏液病毒糖蛋白受体结合区；
优选是血凝集素(HA)蛋白和/或神经氨酸酶(NA)蛋白；
或具有免疫原性的上述所有蛋白/肽抗原的片段或变体的蛋白/肽。
在本发明的一个具体实施方案中，正黏液病毒为B型流感病毒和/或A型H5N1流感病毒。
在本发明的一个具体实施方案中，蛋白/肽抗原为前述定义的抗原与其他蛋白或肽的融合蛋白，
优选融合蛋白为Flu-B-HA1-his或H5N1-HA-his。
在本发明的一个具体实施方案中，其中蛋白/肽抗原包含SEQ ID NO:7和/或SEQ ID NO:15、SEQ ID NO:8和/或SEQ ID NO:16之任一所述的序列。
在本发明的一个具体实施方案中，蛋白/肽抗原是包含丝状病毒科病毒抗原；
优选丝状病毒包膜糖蛋白受体结合区；
优选丝状病毒包膜糖蛋白GP1和/或GP2；
更优选丝状病毒包膜糖蛋白GP1；
或具有免疫原性的上述所有蛋白/肽抗原的片段或变体的蛋白/肽。
在本发明的一个具体实施方案中，丝状病毒为埃博拉病毒。
在本发明的一个具体实施方案中，蛋白/肽抗原为前述定义的抗原与其他蛋白或肽的融合蛋白，
优选融合蛋白为Ebola-GP-Fc或Ebola-GP1-his；
其中Fc优选IgG Fc片段、更优选人或鼠IgG Fc片段。
在本发明的一个具体实施方案中，蛋白/肽抗原包含SEQ ID NO:9和/或SEQ ID NO:17、SEQ ID NO:10和/或SEQ ID NO:18之任一所述的序列。
在本发明的一个具体实施方案中，蛋白/抗原是包含黄病毒科病毒抗原；
优选黄病毒属病毒或肝炎病毒属病毒抗原；
优选黄病毒属病毒被膜蛋白受体结合区；
优选黄病毒属病毒被膜蛋白EDI、EDII和EDIII结构域之至少之一；
更优选黄病毒属病毒被膜蛋白EDIII结构域；或
优选肝炎病毒属病毒被膜糖蛋白受体结合区；
优选肝炎病毒属病毒被膜糖蛋白E1和/或E2；
或具有免疫原性的上述所有蛋白/肽抗原的片段或变体的蛋白/肽。
在本发明的一个具体实施方案中，黄病毒属病毒优选寨卡病毒；肝炎病毒属病毒优选丙型肝炎病毒。
在本发明的一个具体实施方案中，蛋白/肽抗原为前述定义的抗原与其他蛋白或肽的融合蛋白，
优选融合蛋白为ZIKV-E-Fc；其中
Fc优选IgG Fc片段、更优选人或鼠IgG Fc片段；或
优选融合蛋白为HCV-E2-his和/或HCV-E1-his。
在本发明的一个具体实施方案中，蛋白/肽抗原包含SEQ ID NO:11和/或SEQ ID NO:19、SEQ ID NO:6和/或SEQ ID NO:14、SEQ ID NO:5和/或SEQ ID NO:13之任一所述的序列。
在本发明的一个具体实施方案中，糖-蛋白/肽抗原进一步缀合蛋白载体。
在本发明的一个具体实施方案中，蛋白载体是破伤风类毒素、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，优选CRM197。
本发明的第二个方面涉及一种糖-蛋白/肽抗原缀合物，与未经缀合的蛋白/肽抗原相比，其免疫原性提高。
在本发明的一个具体实施方案中，糖选自多糖、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，
最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；其中蛋白/肽抗原选自病原体相关的蛋白/肽抗原或肿瘤相关蛋白/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；
优选与病原体侵入宿主相关的蛋白/肽；
更优选上述病原体为病毒；
更优选上述病毒选自冠状病毒科、副黏液病毒科、正黏液病毒科、丝状病毒科或黄病毒科病毒，以及
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。
在本发明的一个具体实施方案中，蛋白/肽抗原是包含冠状病毒科病毒抗原；
优选冠状病毒棘突蛋白；
更优选冠状病毒棘突蛋白S1亚单位；
更优选冠状病毒棘突蛋白受体结合区RBD；
或具有免疫原性的上述所有蛋白/肽抗原的片段或变体的蛋白/肽。
在本发明的一个具体实施方案中，冠状病毒为SARS-CoV-2或中东呼吸综合征冠状病毒。
在本发明的一个具体实施方案中，蛋白/肽抗原为前述定义的抗原与其他蛋白或肽的融合蛋白，
优选地，融合蛋白选自SARS-CoV-2 RBD-mFc；或
SARS-CoV-2 RBD-his；或
MERS-COV RBD-his；以及
Fc优选为IgG Fc片段、更优选人或鼠IgG Fc片段。
在本发明的一个具体实施方案中，蛋白/肽抗原包含SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3之任一所述的序列。
在本发明的一个具体实施方案中，蛋白/肽抗原是包含
副黏液病毒科病毒抗原；
优选副黏液病毒糖蛋白受体结合区；
优选副黏液病毒糖蛋白F、糖蛋白G；
或具有免疫原性的上述所有蛋白/肽抗原的片段或变体的蛋白/肽。
在本发明的一个具体实施方案中，副黏液病毒为人呼吸道合胞病毒。
在本发明的一个具体实施方案中，蛋白/肽抗原为前述定义的抗原与其他蛋白或肽的融合蛋白，
优选融合蛋白为RSV-gpG-his。
在本发明的一个具体实施方案中，蛋白/肽抗原包含SEQ ID NO:4和/或SEQ ID NO:12所述的序列。
在本发明的一个具体实施方案中，蛋白/肽抗原是包含
正黏液病毒科病毒抗原；
优选正黏液病毒糖蛋白受体结合区；
优选是血凝集素(HA)蛋白和/或神经氨酸酶(NA)蛋白；
或具有免疫原性的上述所有蛋白/肽抗原的片段或变体的蛋白/肽。
在本发明的一个具体实施方案中，正黏液病毒为B型流感病毒和/或A型H5N1流感病毒。
在本发明的一个具体实施方案中，蛋白/肽抗原为前述定义的抗原与其他蛋白或肽的融合蛋白，
优选融合蛋白为Flu-B-HA1-his或H5N1-HA-his。
在本发明的一个具体实施方案中，蛋白/肽抗原包含SEQ ID NO:7和/或SEQ ID NO:15、SEQ ID NO:8和/或SEQ ID NO:16之任一所述的序列。
在本发明的一个具体实施方案中，蛋白/肽抗原是包含
丝状病毒科病毒抗原；
优选丝状病毒包膜糖蛋白受体结合区；
优选丝状病毒包膜糖蛋白GP1和/或GP2；
更优选丝状病毒包膜糖蛋白GP1；
或具有免疫原性的上述所有蛋白/肽抗原的片段或变体的蛋白/肽。
在本发明的一个具体实施方案中，丝状病毒为埃博拉病毒。
在本发明的一个具体实施方案中，蛋白/肽抗原为前述定义的抗原与其他蛋白或肽的融合蛋白，
优选融合蛋白为Ebola-GP-Fc或Ebola-GP1-his；
其中Fc优选IgG Fc片段、更优选人或鼠IgG Fc片段。
在本发明的一个具体实施方案中，蛋白/肽抗原包含SEQ ID NO:9和/或SEQ ID NO:17、SEQ ID NO:10和/或SEQ ID NO:18之任一所述的序列。
在本发明的一个具体实施方案中，蛋白/肽抗原是包含
黄病毒科病毒抗原；
优选黄病毒属病毒或肝炎病毒属病毒抗原；
优选黄病毒属病毒被膜蛋白受体结合区；
优选黄病毒属病毒被膜蛋白EDI、EDII和EDIII结构域之至少之一；
更优选黄病毒属病毒被膜蛋白EDIII结构域；或
优选肝炎病毒属病毒被膜糖蛋白受体结合区；
优选肝炎病毒属病毒被膜糖蛋白E1和/或E2；
或具有免疫原性的上述所有蛋白/肽抗原的片段或变体的蛋白/肽。
在本发明的一个具体实施方案中，黄病毒属病毒优选寨卡病毒；或
肝炎病毒属病毒优选丙型肝炎病毒。
在本发明的一个具体实施方案中，蛋白/肽抗原为前述定义的抗原与其他蛋白或肽的融合蛋白，
优选融合蛋白为ZIKV-E-Fc；其中
Fc优选IgG Fc片段、更优选人或鼠IgG Fc片段；或
优选融合蛋白为HCV-E2-his和/或HCV-E1-his。
在本发明的一个具体实施方案中，蛋白/肽抗原包含SEQ ID NO:11和/或SEQ ID NO:19、SEQ ID NO:6和/或SEQ ID NO:14、SEQ ID NO:5和/或SEQ ID NO:13之任一所述的序列。
在本发明的一个具体实施方案中，糖-蛋白/肽抗原缀合物的分子量为400-14000KDa。
在本发明的一个具体实施方案中，糖-蛋白/肽抗原进一步缀合蛋白载体。
在本发明的一个具体实施方案中，蛋白载体是破伤风类毒素、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，优选CRM197。
本发明的第三方面涉及一种免疫复合物，包含前述糖-蛋白/肽抗原缀合物、免疫佐剂和赋形剂。
在本发明的一个具体实施方案中，佐剂选自铝佐剂、水包油乳液佐剂、MF59、QS-21和单磷酸脂质A。
本发明的第四方面涉及糖-蛋白/肽抗原缀合物或免疫复合物预防或治疗前述定义的病原体相关的蛋白/肽抗原或肿瘤相关蛋白/肽引起的疾病的用途，优选的病原体为
冠状病毒，更优选为SARS-CoV-2和/或MERS-CoV；
副黏液病毒，更优选为人呼吸道合胞病毒；
正黏液病毒，更优选为B型流感病毒和/或A型H5N1流感病毒；
丝状病毒，更优选为埃博拉病毒；
黄病毒，优选寨卡病毒；或丙型肝炎病毒。
本发明的第五方面涉及糖-蛋白/肽抗原缀合物或免疫复合物在制备预防治疗前述定义的病原体相关的蛋白/肽抗原或肿瘤相关蛋白/肽引起的疾病的疫苗或药物中的用途，优选的病原体为
冠状病毒，更优选为SARS-CoV-2和/或MERS-CoV；
副黏液病毒，更优选为人呼吸道合胞病毒；
正黏液病毒，更优选为B型流感病毒和/或A型H5N1流感病毒；
丝状病毒，更优选为埃博拉病毒；
黄病毒，优选寨卡病毒；或丙型肝炎病毒。
附图说明
图1描述了以SARS-COV-2 RBD-mFc为抗原的免疫组合物免疫小鼠血清的抗SARS-COV-2 RBD抗体滴度。数值为血清稀释8000倍时检测到的吸光度。
图2描述了不同佐剂的比较，抗原为SARS-COV-2 RBD-his-PS14缀合物，血清稀释倍数为32000倍，免疫剂量为3μg/鼠。
图3描述了SARS-COV-2 RBD蛋白-PS14多糖缀合物，以及SARS-COV-2 RBD与CRM197共同缀合PS14的缀合物为抗原的免疫组合物免疫小鼠血清中和活性的比较，血清稀释倍数是1500倍，免疫剂量是3μg/鼠。
图4描述了PS7F、PS14和葡聚糖作为缀合剂的缀合物的免疫结果。
具体实施方式
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。在本说明书及所附的权利要求书中，“其中的蛋白/肽抗原是包含X的蛋白/肽”是指蛋白/肽抗原的氨基酸序列包含X的蛋白/肽序列。
术语“抗原”是一种由抗体或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。
“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，其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“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”在本说明书及所附的权利要求书中是指与糖共价缀合的蛋白/肽。本发明的糖-蛋白/肽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”是指含有抗原如微生物或其组分的任何药物组合物，该组合物可用于在个体中诱发免疫应答。
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。
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或包含该抗原的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例如增殖测定、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
本发明是一个开创性的发明，发明人发现包括通过将蛋白/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。
在此发明之前，没有研究报道糖-蛋白/肽抗原缀合物中蛋白/肽抗原的免疫原性的提高。与此相反，正如背景技术中所提及的，之前的研究对于缀合物中蛋白/肽抗原的免疫原性的描述是，保留了免疫原性(US5192540A/US9296795B)或蛋白/肽抗原表位没有因缀合而改变(US9296795B)。这些教导与本发明的宗旨相背离。
本发明的糖缀合蛋白/肽抗原
1.冠状病毒科(Coronaviridae)病毒作为抗原
冠状病毒科包括正冠状病毒亚科(Orthocoronavirinae)及勒托病毒亚科(Letovirinae)。
正冠状病毒亚科的乙型冠状病毒属(Betacoronaviru)包含为人熟知的严重急性呼吸道综合征冠状病毒(respiratory syndrome coronavirus(SARS-CoV))，中东呼吸综合症冠状病毒(Middle East respiratory syndrome-related coronavirus(MERS-CoV)，严重急性呼吸系统综合征冠状病毒2(Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2))。这三种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)。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冠状病毒SARS-CoV-2棘突蛋白(SARS-CoV-2S蛋白)、其胞外区、S1亚单位或受体结合区作为抗原。
本发明的一个方案选用中东呼吸综合症冠状病毒作为抗原。如其胞外区、S1亚单位或受体结合区作为抗原。
2.副黏液病毒科(Paramyxoviridae)病毒作为抗原
副黏液病毒科包括二个亚科，副黏液病毒亚科(Paramyxivirinae)及肺炎病毒亚科(Pneumovirinae)。人呼吸道合胞病毒(RSV)是副黏液病毒亚科呼吸道病毒属(Respirovirus)病毒之一，RSV编码两种主要跨膜表面糖蛋白，糖蛋白G(吸附蛋白)和糖蛋白F(融合蛋 白)。糖蛋白G介导病毒结合至细胞受体，而糖蛋白F促进病毒与细胞膜的融合，允许病毒核糖核蛋白侵入细胞质(Lopez et al.(1998)J.Virology72:6922-6928)。
本发明的一个方案选用RSV被膜蛋白糖作为抗原。可以选用人RSV被膜蛋白糖，如RSV糖蛋白F或糖蛋白G。
3.正黏液病毒科(Orthomyxoviridae)病毒作为抗原
正黏液病毒科包括甲型流行性感冒病毒属(Influenzavirus A)、乙型流行性感冒病毒属(Influenzavirus B)、丙型流行性感冒病毒属(Influenzavirus C)及其他属。
流感病毒A、B和C感染主要依赖两种被膜蛋白糖：血凝素(HA)和神经氨酸酶(NA),负责病毒附着和病毒颗粒侵入细胞。通过病毒体表面血凝素(HA)蛋白附着于含唾液酸细胞受体(糖蛋白和糖脂)而引发流感病毒感染。神经氨酸酶(NA)蛋白介导唾液酸受体的加工，且病毒对细胞的侵入依赖于HA依赖性受体介导的胞吞作用(CN103865892B)。
本发明的一个方案选用A型H5N1流感病毒血凝集素(HA)蛋白作为抗原，也可以选用B型流感病毒血凝集素蛋白(HA1亚单位)作为抗原。
4.丝状病毒科(Filoviridae)病毒作为抗原
丝状病毒科代表性的种属有埃博拉病毒属(Ebolavirus)的埃博拉病毒(ebolaviruses)和马尔堡病毒属(Marburgvirus)的马尔堡病毒(marburgviruses)。
存在于埃博拉病毒表面的唯一蛋白质是糖蛋白(GP)。GP1,2的三聚体形成病毒的表面刺突，GP1,2由两个亚单位GP1和GP2通过二硫键连接而成(Volchkova,VA ef a/.,(1998),Virology 250:408-414；Falzarano,D.et al.,(2006),Chembiochem 7:1605-161 1)。已知GP1介导病毒附着在宿主细胞上，GP2参与膜融合(Sanchez,A.et al.,(1996),Proc Natl Acad Sci USA 93:3602-3607；Alazard-Dany,N.et al.(2006),J.Gen.Virol.87:1247-1257).
本发明的一个方案选用埃博拉病毒糖蛋白(GP)作为抗原。例如GP胞外结构域、亚基GP蛋白(GP1和/或GP2)。
5.黄病毒科(Flaviviridae)病毒作为抗原
黄病毒科病毒主要包括黄病毒属(Flavivirus)、瘟疫病毒属(Pestivirus)、Pegivirus
和肝炎病毒属(Hepacivirus)，其中黄病毒属包括寨卡病毒(ZIKV)、登革热(DV)、西尼罗河病毒、日本脑炎病毒和黄热病病毒等。肝炎病毒属包括丙型肝炎病毒(HCV)。
黄病毒被膜蛋白在宿主细胞病毒感染中起着重要作用，介导病毒进入宿主细胞。它由三个独立的结构包膜域I、II和III(EDI、EDII和EDIII)组成。EDI是被膜蛋白的一个结构中心域，它稳定了蛋白的整体方向，EDI中的糖基化位点与病毒的产生、pH敏感性和神经侵入性有关。由于融合环表位和包膜二聚体表位的免疫优势，EDII在膜融合中发挥重要作用。此外，EDIII是中和抗体的主要目标(Xingcui Zhang.et al.,(2017)Viruses.2017Nov；9(11):338.Structures and Functions of the Envelope Glycoprotein in Flavivirus Infections)。
寨卡病毒被膜蛋白("E"或"EP")由三个不同的结构域组成。E结构域I(E-DI)是组织整个E蛋白结构的中心结构域。E结构域II(E-DII)由从E-DI突出的两个延伸环形成并且位于E-DI和E结构域III(E-DIII)处的口袋中。E-DIII是免疫球蛋白样结构域，其在另外平滑的球形成熟病毒颗粒的表面上形成小突起，并且被认为与靶细胞上的细胞受体相互作用(CN109996560A)。
HCV RNA基因组编码一个单一的多聚蛋白，翻译时或翻译后裂解为三个结构蛋白(核 心、糖蛋白E1和E2)和七个非结构蛋白(p7、NS2、NS3、NS4A、NS4B、NS5A和NS5B)。被膜蛋白糖E1和E2形成异源二聚体，构成病毒被膜蛋白，在病毒进入宿主细胞时时发挥重要作用，介导病毒进入和形态发生。丙型肝炎病毒的被膜蛋白糖与宿主肝细胞表面的特定蛋白结合，启动进入过程。这个过程涉及大量的宿主受体/共受体。其中，E2是主要的HCV被膜蛋白糖，直接与受体/共受体相互作用。长期以来，人们认为在这一过程中E1不直接与宿主受体相互作用，其通过维持受体结合所需的功能性E2构象，与E2共同诱导膜融合(Yimin,Tong.Et al.,(2018)Front Immunol.2018；9:1411.Role of Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly)。
本发明的一个方案选用寨卡病毒被膜蛋白E-DIII作为抗原。
本发明的一个方案选用丙型肝炎病毒被膜蛋白糖E1和/或E2作为抗原。
本章上述的病毒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。均可进一步修饰，如它们具有免疫原性的片段或变体以及如带纯化标签或其与抗体Fc片段的融合蛋白，均可用于本发明。
所述多糖是细菌多糖，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。
多糖与蛋白缀合过程如下：通过化学反应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将多糖氧化，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。反应体系中可以加入还原剂如氰基硼氢化钠。
多糖与蛋白质分子缀合的另一种方法是多糖与溴化氰或1-氰基-4-二甲氨基吡啶四氟硼酸盐反应，生产具有反应活性的氰酸酯。氰酸酯基团与蛋白质表面的氨基反应形成共价。活化的多糖也可以先与己二胺，己二酰肼等连接臂反应，其产物再与蛋白在缩水剂存在下反应形成共价连接。
多糖也可以用其它化学试剂活化，再与蛋白质反应形成缀合。如环氧氯丙烷、三嗪、二嗪、二乙烯基砜等本领域公知的试剂。
为提高本发明的糖-蛋白/肽抗原缀合物的免疫原性，可以在糖与蛋白/肽抗原缀合反应时，进一步加入蛋白载体形成糖-蛋白/肽抗原-蛋白载体缀合物。蛋白载体可采用疫苗工业中常用的破伤风类毒素、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，优选CRM197。
本发明的免疫原性组合物
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。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。在一个实施方案中，所述佐剂是选自磷酸铝、硫酸铝和氢氧化铝的铝系佐剂。在一实施方案中，所述佐剂为磷酸铝。
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。本发明所用的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：
油乳剂组合物包括角鲨烯-水乳剂、例如MF59；完全弗氏佐剂(CFA)和不完全弗氏佐剂(IFA)；皂苷制剂；联用皂苷和胆固醇来形成称为免疫刺激复合物(ISCOM)的独特颗粒；病毒体和病毒样颗粒；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记载的或将记载的动物(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调节剂，其是扰乱或改变免疫系统从而观察到体液和/或细胞介导的免疫的上调或下调的物质。在一个实施方案中，提供了免疫系统的体液和/或细胞介导的能力(arms)的上调。包括例如佐剂或细胞因子。
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
本发明蛋白/肽抗原缀合物及免疫复合物可以预防或治疗病原体引起的疾病，例如是冠状病毒、副黏液病毒、正黏液病毒、丝状病毒和黄病毒，更尤其是SARS-CoV-2和/或MERS-CoV病毒、人呼吸道合胞病毒、B型流感病毒、A型H5N1流感病毒、埃博拉病毒、寨卡病毒和/或丙型肝炎病毒引起的疾病。
本发明的术语简写
SARS-COV-2 RBD-mFc：SARS-CoV-2冠状病毒棘突蛋白受体结合区小鼠Fc融合蛋白；
SARS-COV-2 RBD-his：SARS-CoV-2冠状病毒棘突蛋白受体结合区与6组氨酸标签融合蛋白；
MERS-COV RBD-his：中东呼吸综合征冠状病毒棘突蛋白受体结合区与6组氨酸标签融合蛋白；
RSV-gpG：Human respiratory syncytial virus glycoprotein G/人呼吸道合胞病毒糖蛋白G；
HCV-E1：Hepatitis C virus Envelope Glycoprotein E1/丙型肝炎病毒被膜糖蛋白E1；
HCV-E2：Hepatitis C virus Envelope E2 Protein/丙型肝炎病毒被膜糖蛋白E2；
flu-B-HA1：Influenza B Hemagglutinin Protein(HA1 Subunit)/B型流感病毒血凝集素(HA)蛋白蛋白(HA1亚单位)；
H5N1-HA：Influenza A H5N1 Hemagglutinin/A型H5N1流感病毒血凝集素(HA)蛋白；
Ebola-GP：Ebola virus Glycoprotein(Receptor Binding Domain,)埃博拉病毒糖蛋白 (受体结合区)；
Ebola-GP1：Ebola virus Glycoprotein GP1/埃博拉病毒糖蛋白GP1；
ZIKV-E：Zika virus Envelope protein(Domain III)/寨卡病毒被膜蛋白(III结构域)；
PS14：肺炎链球菌血清型14荚膜多糖
PS7F：肺炎链球菌血清型7F荚膜多糖
Alum：铝佐剂，本文为磷酸铝佐剂
实施例1：肺炎链球菌血清型14(PS14)和7F(PS7F)荚膜多糖的制备
血清型14肺炎链球菌种子为ATCC 6314、血清型7F肺炎链球菌种子为ATCC 10351。
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pH3.5。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300ml 75％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2：多糖的水解、高活化度活化和低活化度活化
多糖为实施例1中制备的肺炎链球菌血清型14(PS14)和7F(PS7F)荚膜多糖或葡聚糖(dextran,Sigma,00894下同)。
2.1多糖水解：
取10mL 10mg/ml纯化后荚膜多糖,加入36％乙酸0.86ml,溶液中乙酸终浓度为500mM，90℃条件水浴2h后，加入1M NaOH中和至pH6-7，得到水解多糖样品。
经HPLC-MALS测定，PS14荚膜多糖分子量约为500KDa，水解后约为300KDa。
PS7F荚膜多糖分子量约为700KDa，未经水解。
葡聚糖未经水解。
2.2多糖的高活化度活化：
向10mL10mg/ml多糖溶液中加入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，缓冲液在其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，即为高活化度活化的多糖溶液。
2.3多糖的低活化度活化：
向10mL 10mg/ml多糖溶液中加入30mg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10ml 50mM，pH＝7.0的Na 2HPO 4缓冲液，缓冲 液在其重力作用下流过层析柱。然后将层析柱放入离心机，1000g转速条件下离心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，即为低活化度活化的多糖溶液。
实施例3:蛋白/肽抗原与多糖缀合
所用蛋白/肽抗原为冠状病毒棘突蛋白受体结合区、多糖为肺炎链球菌荚膜多糖或葡聚糖。其具体成分、量及体积分别如表1所示。
1.冠状病毒棘突受体蛋白换液：取冠状病毒棘突受体蛋白5mg，用30,000MW超滤管换液至50mM，pH为7.0的Na 2HPO 4缓冲液中，且换液蛋白终浓度要求≥10mg/mL。
2.冠状病毒棘突受体蛋白与多糖缀合：取3mg冠状病毒棘突受体蛋白，按照表1加入活化后肺炎链球菌荚膜多糖或葡聚糖，并补加50mM,pH为7.0的Na 2HPO 4缓冲液，终体积见表1，然后按表1的体积加入5M氰基硼氢化钠溶液，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(0.6ml反应体系中加入0.15ml，1.5ml反应体系加入0.375ml)，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。结果见表1。
表1.几种缀合物反应条件及其产物的分子量
Figure PCTCN2021090809-appb-000001
Figure PCTCN2021090809-appb-000002
*表示HPLC-MALLS测定缀合物三个峰值的分子量
MERS-COV RBD-his(来源：北京义翘神州科技有限公司，40071-V08B1)，其它蛋白由发明人自己制备。
实施例4.蛋白/肽抗原及蛋白载体与多糖缀合
所用蛋白/肽抗原为冠状病毒棘突蛋白受体结合区SARS-COV-2 RBD，蛋白载体为CRM197、多糖为肺炎链球菌荚膜多糖。其具体成分、量及体积分别如表2所示。
CRM197为一种白喉毒素的变体(Geert J.Schenk，Efficient CRM197-mediated drμg targeting to monocytes，Journal of Controlled Release 158(2012)139–147)。冠状病毒棘突蛋白受体结合区及CRM197共同与肺炎链球菌血清型14荚膜多糖缀合。多糖的活化过程与实施例2.2相同。取1.5mg多糖，2.7mg冠状病毒棘突蛋白受体结合区蛋白，加0.3mg CRM197，按实施例3相同过程缀合。缀合物反应条件及其产物的分子量如表2所示。
表2 SARS-COV-2 RBD与CRM197共同缀合物反应条件及其产物的分子量
Figure PCTCN2021090809-appb-000003
实施例5.冠状病毒棘突蛋白受体结合区-多糖缀合物的免疫原性
所用的冠状病毒棘突蛋白受体结合区-多糖缀合物见表3-6。
5.1免疫组合物的制备
采用冠状病毒棘突蛋白受体结合区蛋白或实施例3或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200ml 10mM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含单磷酰脂质A(MPL)的MF59佐剂的制备
将10mg MPL(MERCK L6895)分散于10ml柠檬酸钠缓冲液(10mM,pH 6.5)。另取4ml MF59佐剂，加入1ml MPL分散液，混匀得到含MPL的MF59佐剂。
5.1.3铝佐剂免疫组合物的制备
抗原分别用PBS稀释成0.02mg/ml或0.06mg/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或0.03mg/ml。
5.1.4 MF59佐剂免疫组合物的制备
抗原分别用PBS稀释成0.02mg/ml或0.06mg/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或0.03mg/ml。
5.1.5含有MPL的MF59佐剂免疫组合物的制备
抗原分别用PBS稀释成0.02mg/ml或0.06mg/ml，稀释后的抗原与等体积的含MPL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或0.03mg/ml。
5.1.6 MF59和铝佐剂混合佐剂免疫组合物的制备
取1.5ml铝佐剂和1.5ml MF59佐剂混合后再加入0.18ml浓度为1mg/ml的抗原，该免疫组合物中抗原的蛋白浓度为0.03mg/ml。
5.2免疫小鼠：
小鼠选用4-6周Balb/c小鼠，腹腔注射0.1ml如实施例5.1所述的浓度为0.01mg/ml或0.03mg/ml免疫组合物，分别于第14天和第28天加强免疫。第7天，第21天，第35天眼眶取血，测定血清抗体滴度和中和滴度。
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。使用含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)稀释到不同稀释度(具体稀释倍数根据免疫采血时间设定有不同，如1000x、8000x、16000x、32000x稀释)，实验设定SARS-COV-2 RBD-mFc免疫的小鼠血清为阳性对照，无关免疫靶点的小鼠血清(抗CD70血清，北京义翘科技有限公司)为阴性对照，同时加入不同稀释度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。
5.3.2抗原为MERS-COV RBD或其融合蛋白时血清效价的测定
检测MERS-COV RBD-his免疫血清时使用MERS-COV RBD-his(北京义翘神州科技有限公司，40071-V08B1)包被，不设阳性对照和阴性对照，操作步骤与5.3.1完全相同。
5.3.3血清效价测定结果
表3-表5及附图1-4均展示了血清效价的测定结果。
铝佐剂免疫组合物免疫小鼠35天后血清稀释8000倍测定的效价结果如表3所示。
表3铝佐剂免疫组合物免疫小鼠35天血清效价(免疫35天结果)
抗原 免疫剂量(μg) 血清稀释8000倍(OD 450)
SARS-COV-2 RBD-mFc 1 0.319
SARS-COV-2 RBD-mFc 3 0.591
SARS-COV-2 RBD-mFc-PS14Conj-1 3 0.609
SARS-COV-2 RBD-mFc-PS14Conj-2 1 1.232
SARS-COV-2 RBD-mFc-PS14Conj-2 3 0.844
SARS-COV-2 RBD-mFc-PS14Conj-3 3 1.348
SARS-COV-2 RBD-mFc-PS14Conj-4 1 1.384
SARS-COV-2 RBD-mFc-PS14Conj-4 3 0.950
SARS-COV-2 RBD-his 3 0.171
SARS-COV-2 RBD-his-PS14 3 1.694
SARS-COV-2 RBD-his-CRM197-PS14 3 1.403
SARS-COV-2 RBD-mFc-PS14 3 1.224
SARS-COV-2 RBD-mFc-CRM197-PS14 3 0.839
SARS-COV-2 RBD-his-PS14铝/MF59/MF59-铝/MF59-MPL佐剂免疫组合物免疫35天血清稀释32000倍测定的效价结果如表4所示。
表4使用不同佐剂组合时缀合物SARS-COV-2 RBD-his-PS14血清效价(免疫35天结果,免疫剂量3μg)
佐剂 血清稀释32000倍(OD 450)
铝佐剂 0.423
MF59 1.052
MF59-铝 1.114
MF59-MPL 1.111
MF59佐剂免疫组合物免疫小鼠21天后血清稀释8000倍测定的血清效价结果如表5所示。
表5 MF59佐剂免疫组合物血清效价(免疫21天结果,免疫剂量3μg)
抗原 血清稀释8000倍(OD 450)
SARS-COV-2 RBD-his-PS7F 1.382
SARS-COV-2 RBD-his-葡聚糖 1.040
MERS-COV RBD-his-PS14 1.325
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根据经验稀释一定倍数(例如，稀释500倍)后与假病毒2019-nCoV PSV(来自中国食品药品检定研究院)等体积混合，不加血清样品作为阳性对照，不加假病毒作为阴性对照，37℃孵育1小时后，同步侵染Vero E6或293FT/ACE2细胞(来自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。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。
不同免疫组合物免疫小鼠后血清的中和效价结果见表6和图3。
表6免疫组合物中和效价(第35天结果,免疫剂量3μg)
Figure PCTCN2021090809-appb-000004
实施例6.多糖增强几种病毒抗原的免疫原性
选取几种病毒抗原，如表7所示(均来自北京义翘神州科技有限公司)，与PS14多糖缀合，步骤如下：
按照实施例1制备肺炎链球菌血清型14(PS14)，按照实施例2.2步骤进行活化，其中加入高碘酸钠的量按照表8进行调整。按照实施例3步骤将PS14与载体蛋白CRM197缀合，缀合时蛋白和多糖的比例如表8所示。按照实施例5.1.3制备含铝佐剂免疫组合物。按照实施例5.2免疫小鼠，免疫剂量为每只小鼠3μg抗原。
检测免疫血清效价，使用对应的抗原包被，不设阳性对照和阴性对照，操作步骤与5.3.1完全相同。
各种抗原缀合物的免疫血清效价如表8所示。可见对于大多数抗原与多糖缀合后免疫原性都有明显提高。
表7几种病毒抗原名称货号和对应简写
Figure PCTCN2021090809-appb-000005
Figure PCTCN2021090809-appb-000006
表8几种病毒抗原与多糖缀合条件和缀合物免疫血清效价
Figure PCTCN2021090809-appb-000007
表8中所用的抗原均记载于表7，其中，His-tag未示出。
根据上述数据，蛋白抗原与肺炎链球菌荚膜多糖缀合后，其免疫原性明显提高。在铝佐剂条件下，缀合物免疫血清的抗体效价最高可达到原来的2.3倍。缀合物的中和活性比对应的蛋白的中和活性也大幅度提高。和铝佐剂相比，MF59佐剂，MF59与铝佐剂混合佐剂，MF59佐剂加MPL佐剂都能进一步提高缀合物的免疫效果。使用肺炎链球菌血清型14荚膜多糖、肺炎链球菌血清型7F荚膜多糖以及葡聚糖为缀合剂时免疫效果相近。
Figure PCTCN2021090809-appb-000008
Figure PCTCN2021090809-appb-000009
Figure PCTCN2021090809-appb-000010
Figure PCTCN2021090809-appb-000011

Claims (53)

  1. 一种提高蛋白/肽抗原免疫原性的方法，该方法包括通过将蛋白/肽抗原与糖缀合，形成糖-蛋白/肽抗原缀合物。
  2. 如权利要求1所述的方法，其中糖选自多糖、寡糖或单糖；
    优选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；
    更优选肺炎链球菌荚膜多糖；
    最优选肺炎链球菌血清型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,
    其中蛋白/肽抗原选自病原体相关的蛋白/肽抗原或肿瘤相关蛋白/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；
    优选与病原体侵入宿主相关的蛋白/肽；
    更优选上述病原体为病毒；
    更优选上述病毒选自冠状病毒科、副黏液病毒科、正黏液病毒科、丝状病毒科或黄病毒科病毒，以及
    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。
  3. 如权利要求1或2所述的方法，其中的蛋白/肽抗原是包含
    冠状病毒科病毒抗原；
    优选冠状病毒棘突蛋白；
    更优选冠状病毒棘突蛋白S1亚单位；
    更优选冠状病毒棘突蛋白受体结合区RBD；
    或具有免疫原性的上述所有蛋白/肽抗原的片段或变体
    的蛋白/肽。
  4. 如权利要求3所述的方法，其中冠状病毒为SARS-CoV-2或中东呼吸综合征冠状病 毒。
  5. 如权利要求3或4所述的方法，其中蛋白/肽抗原为权利要求3或4中定义的抗原与其他蛋白或肽的融合蛋白，
    优选融合蛋白选自SARS-CoV-2 RBD-mFc；SARS-CoV-2 RBD-his；或MERS-COV RBD-his；以及
    Fc片段优选为IgG Fc片段、更优选为人或鼠IgG Fc片段。
  6. 如权利要求4或5所述的方法，其中蛋白/肽抗原
    包含SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3之任一所述的序列。
  7. 如权利要求1-6之任一的所述的方法，其中缀合物的分子量为400-14000KDa。
  8. 如权利要求1或2所述的方法，其中的蛋白/肽抗原是包含
    副黏液病毒科病毒抗原；
    优选副黏液病毒糖蛋白受体结合区；
    优选副黏液病毒糖蛋白F、糖蛋白G；
    或具有免疫原性的上述所有蛋白/肽抗原的片段或变体
    的蛋白/肽。
  9. 如权利要求8所述的方法，其中的副黏液病毒为人呼吸道合胞病毒。
  10. 如权利要求8或9所述的方法，其中蛋白/肽抗原为权利要求8或9中定义的抗原与其他蛋白或肽的融合蛋白，
    优选融合蛋白为RSV-gpG-his。
  11. 如权利要求9或10所述的方法，其中蛋白/肽抗原包含SEQ ID NO:4和/或SEQ ID NO:12所述的序列。
  12. 如权利要求1或2所述的方法，其中的蛋白/肽抗原是包含
    正黏液病毒科病毒抗原；
    优选正黏液病毒糖蛋白受体结合区；
    优选是血凝集素(HA)蛋白和/或神经氨酸酶(NA)蛋白；
    或具有免疫原性的上述所有蛋白/肽抗原的片段或变体
    的蛋白/肽。
  13. 如权利要求12所述的方法，其中的正黏液病毒为B型流感病毒和/或A型H5N1流感病毒。
  14. 如权利要求12或13所述的方法，其中蛋白/肽抗原为权利要求12或13中定义的抗原与其他蛋白或肽的融合蛋白，
    优选融合蛋白为Flu-B-HA1-his或H5N1-HA-his。
  15. 如权利要求13或14所述的方法，其中的蛋白/肽抗原包含SEQ ID NO:7和/或SEQ ID NO:15、SEQ ID NO:8和/或SEQ ID NO:16之任一所述的序列。
  16. 如权利要求1或2所述的方法，其中蛋白/肽抗原是包含
    丝状病毒科病毒抗原；
    优选丝状病毒包膜糖蛋白受体结合区；
    优选丝状病毒包膜糖蛋白GP1和/或GP2；
    更优选丝状病毒包膜糖蛋白GP1；
    或具有免疫原性的上述所有蛋白/肽抗原的片段或变体
    的蛋白/肽。
  17. 如权利要求16所述的方法，其中的丝状病毒为埃博拉病毒。
  18. 如权利要求16或17所述的方法，其中蛋白/肽抗原为权利要求16或17中定义的抗原与其他蛋白或肽的融合蛋白，
    优选融合蛋白为Ebola-GP-Fc或Ebola-GP1-his；
    其中Fc优选IgG Fc片段、更优选人或鼠IgG Fc片段。
  19. 如权利要求17或18所述的方法，其中蛋白/肽抗原包含SEQ ID NO:9和/或SEQ ID NO:17、SEQ ID NO:10和/或SEQ ID NO:18之任一所述的序列。
  20. 如权利要求1或2所述的方法，其中蛋白/肽抗原是包含
    黄病毒科病毒抗原；
    优选黄病毒属病毒或肝炎病毒属病毒抗原；
    优选黄病毒属病毒被膜蛋白受体结合区；
    优选黄病毒属病毒被膜蛋白EDI、EDII和EDIII结构域之至少之一；
    更优选黄病毒属病毒被膜蛋白EDIII结构域；或
    优选肝炎病毒属病毒被膜糖蛋白受体结合区；
    优选肝炎病毒属病毒被膜糖蛋白E1和/或E2；
    或具有免疫原性的上述所有蛋白/肽抗原的片段或变体
    的蛋白/肽。
  21. 如权利要求20所述的方法，其中的黄病毒属病毒优选寨卡病毒；
    其中肝炎病毒属病毒优选丙型肝炎病毒。
  22. 如权利要求20或21所述的方法，其中蛋白/肽抗原为权利要求20或21中定义的抗原与其他蛋白或肽的融合蛋白，
    优选融合蛋白为ZIKV-E-Fc；其中
    Fc优选IgG Fc片段、更优选人或鼠IgG Fc片段；或
    优选融合蛋白为HCV-E2-his和/或HCV-E1-his。
  23. 如权利要求21或22所述的方法，其中蛋白/肽抗原包含SEQ ID NO:11和/或SEQ ID NO:19、SEQ ID NO:6和/或SEQ ID NO:14、SEQ ID NO:5和/或SEQ ID NO:13之任一所述的序列。
  24. 如权利要求1-23之任一所述的方法，其中的糖-蛋白/肽抗原进一步缀合蛋白载体。
  25. 如权利要求24述的方法，其中的蛋白载体是破伤风类毒素、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，优选CRM197。
  26. 一种糖-蛋白/肽抗原缀合物，与未经缀合的蛋白/肽抗原相比，其免疫原性提高。
  27. 如权利要求26所述的缀合物，其中糖选自多糖、寡糖或单糖；
    优选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；
    更优选肺炎链球菌荚膜多糖；
    最优选肺炎链球菌血清型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,
    其中蛋白/肽抗原选自病原体相关的蛋白/肽抗原或肿瘤相关蛋白/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；
    优选与病原体侵入宿主相关的蛋白/肽；
    更优选上述病原体为病毒；
    更优选上述病毒选自冠状病毒科、副黏液病毒科、正黏液病毒科、丝状病毒科或黄病毒科病毒，以及
    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。
  28. 如权利要求26或27所述的缀合物，其中的蛋白/肽抗原是包含
    冠状病毒科病毒抗原；
    优选冠状病毒棘突蛋白；
    更优选冠状病毒棘突蛋白S1亚单位；
    更优选冠状病毒棘突蛋白受体结合区RBD；
    或具有免疫原性的上述所有蛋白/肽抗原的片段或变体
    的蛋白/肽。
  29. 如权利要求28所述的缀合物，其中冠状病毒为SARS-CoV-2或中东呼吸综合征冠状病毒。
  30. 如权利要求28或29所述的缀合物，其中蛋白/肽抗原为权利要求29或30中定义的抗原与其他蛋白或肽的融合蛋白，
    优选融合蛋白选自SARS-CoV-2 RBD-mFc；SARS-CoV-2 RBD-his；或MERS-COV RBD-his；以及
    Fc片段优选为IgG Fc片段、更优选为人或鼠IgG Fc片段。
  31. 如权利要求29或30所述的缀合物，其中蛋白/肽抗原包含SEQ ID NO:1、SEQ ID  NO:2、SEQ ID NO:3之任一所述的序列。
  32. 如权利要求26或27所述的缀合物，其中的蛋白/肽抗原是包含
    副黏液病毒科病毒抗原；
    优选副黏液病毒糖蛋白受体结合区；
    优选副黏液病毒糖蛋白F、糖蛋白G；
    或具有免疫原性的上述所有蛋白/肽抗原的片段或变体
    的蛋白/肽。
  33. 如权利要求32所述的缀合物，其中的副黏液病毒为人呼吸道合胞病毒。
  34. 如权利要求32或33所述的缀合物，其中蛋白/肽抗原为权利要求32或33中定义的抗原与其他蛋白或肽的融合蛋白，
    优选融合蛋白为RSV-gpG-his。
  35. 如权利要求33或34所述的缀合物，其中蛋白/肽抗原包含SEQ ID NO:4和/或SEQ ID NO:12所述的序列。
  36. 如权利要求26或27所述的缀合物，其中的蛋白/肽抗原是包含
    正黏液病毒科病毒抗原；
    优选正黏液病毒糖蛋白受体结合区；
    优选是血凝集素(HA)蛋白和/或神经氨酸酶(NA)蛋白；
    或具有免疫原性的上述所有蛋白/肽抗原的片段或变体
    的蛋白/肽。
  37. 如权利要求36所述的缀合物，其中的正黏液病毒为B型流感病毒和/或A型H5N1流感病毒。
  38. 如权利要求36或37所述的缀合物，其中蛋白/肽抗原为权利要求37或38中定义的抗原与其他蛋白或肽的融合蛋白，
    优选融合蛋白为Flu-B-HA1-his或H5N1-HA-his。
  39. 如权利要求37或38所述的缀合物，其中的蛋白/肽抗原包含SEQ ID NO:7和/或SEQ ID NO:15、SEQ ID NO:8和/或SEQ ID NO:16之任一所述的序列。
  40. 如权利要求26或27所述的缀合物，其中蛋白/肽抗原是包含
    丝状病毒科病毒抗原；
    优选丝状病毒包膜糖蛋白受体结合区；
    优选丝状病毒包膜糖蛋白GP1和/或GP2；
    更优选丝状病毒包膜糖蛋白GP1；
    或具有免疫原性的上述所有蛋白/肽抗原的片段或变体
    的蛋白/肽。
  41. 如权利要求40所述的缀合物，其中的丝状病毒为埃博拉病毒。
  42. 如权利要求40或41所述的缀合物，其中蛋白/肽抗原为权利要求40或41中定义的抗原与其他蛋白或肽的融合蛋白，
    优选融合蛋白为Ebola-GP-Fc或Ebola-GP1-his；
    其中Fc优选IgG Fc片段、更优选人或鼠IgG Fc片段。
  43. 如权利要求41或42所述的缀合物，其中蛋白/肽抗原包含SEQ ID NO:9和/或SEQ ID NO:17、SEQ ID NO:10和/或SEQ ID NO:18之任一所述的序列。
  44. 如权利要求26或27所述的缀合物，其中蛋白/肽抗原是包含
    黄病毒科病毒抗原；
    优选黄病毒属病毒或肝炎病毒属病毒抗原；
    优选黄病毒属病毒被膜蛋白受体结合区；
    优选黄病毒属病毒被膜蛋白EDI、EDII和EDIII结构域之至少之一；
    更优选黄病毒属病毒被膜蛋白EDIII结构域；或
    优选肝炎病毒属病毒被膜糖蛋白受体结合区；
    优选肝炎病毒属病毒被膜糖蛋白E1和/或E2；
    或具有免疫原性的上述所有蛋白/肽抗原的片段或变体
    的蛋白/肽。
  45. 如权利要求44所述的缀合物，其中的黄病毒属病毒优选寨卡病毒；或
    其中肝炎病毒属病毒优选丙型肝炎病毒。
  46. 如权利要求44或45所述的缀合物，其中蛋白/肽抗原为权利要求45或46中定义的抗原与其他蛋白或肽的融合蛋白，
    优选融合蛋白为ZIKV-E-Fc；其中
    Fc优选IgG Fc片段、更优选人或鼠IgG Fc片段；或
    优选融合蛋白为HCV-E2-his和/或HCV-E1-his。
  47. 如权利要求45或46所述的缀合物，其中蛋白/肽抗原包含SEQ ID NO:11和/或SEQ ID NO:19、SEQ ID NO:6和/或SEQ ID NO:14、SEQ ID NO:5和/或SEQ ID NO:13之任一所述的序列。
  48. 如权利要求26-47之任一所述的糖-蛋白/肽抗原缀合物，其中的糖-蛋白/肽抗原进一步缀合蛋白载体。
  49. 如权利要求48所述的糖-蛋白/肽抗原缀合物，其中的蛋白载体是破伤风类毒素、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，优选CRM197。
  50. 一种免疫复合物，包含权利要求26-49之任一的糖-蛋白/肽抗原缀合物、免疫佐剂及赋形剂。
  51. 如权利要求50所述免疫复合物，佐剂选自铝佐剂、水包油乳液佐剂、MF59、QS-21和单磷酸脂质A。
  52. 权利要求26-49之任一所述的糖-蛋白/肽抗原缀合物或权利要求50或51所述的免疫复合物预防或治疗由权利要求27中定义的病原体相关的蛋白/肽抗原或肿瘤相关蛋白/肽抗原引起的疾病的用途，优选的病原体为
    冠状病毒，更优选SARS-CoV-2和/或MERS-CoV；
    副黏液病毒，更优选为人呼吸道合胞病毒；
    正黏液病毒，更优选为B型流感病毒和/或A型H5N1流感病毒；
    丝状病毒，更优选为埃博拉病毒；
    黄病毒，优选寨卡病毒；或丙型肝炎病毒。
  53. 权利要求26-49之任一所述的糖-蛋白/肽抗原缀合物或权利要求50或51所述的免疫复合物在制备预防治疗由权利要求27中定义的病原体相关的蛋白/肽抗原或肿瘤相关蛋白/肽抗原引起的疾病的疫苗或药物中的用途，优选的病原体为
    冠状病毒，更优选SARS-CoV-2和/或MERS-CoV；
    副黏液病毒，更优选为人呼吸道合胞病毒；
    正黏液病毒，更优选为B型流感病毒和/或A型H5N1流感病毒；
    丝状病毒，更优选为埃博拉病毒；
    黄病毒，优选寨卡病毒；或丙型肝炎病毒。
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WO2023236726A1 (zh) * 2022-06-07 2023-12-14 中国人民解放军军事科学院军事医学研究院 基于金属硫蛋白家族的免疫佐剂及其应用

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