A method for enhancing the immunogenicity of protein / peptide antigens

Abstract

A method for enhancing the immunogenicity of protein / peptide antigens, which comprises conjugating the protein / peptide antigen with a sugar to form a glyco-protein / peptide antigen conjugate, whose immunogenicity is enhanced compared to the unconjugated protein / peptide antigen. Specifically, it involves conjugating a pathogen, such as a viral surface protein antigen or a fragment thereof, with a polysaccharide, especially the capsular polysaccharide of Streptococcus pneumoniae. The conjugate with enhanced immunogenicity can be used for preventing or treating diseases caused by pathogens, especially diseases caused by coronaviruses.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202010369100.7, filed on May 1, 2020, the content of which is incorporated herein by reference. Technical field

[0003] The present invention relates to the field of immunogenic compositions. Specifically, it relates to a method for enhancing the immunogenicity of a protein / peptide antigen by conjugating the protein / peptide antigen with a sugar, forming a sugar - protein / peptide antigen conjugate, whose immunogenicity is improved compared with the unconjugated protein / peptide antigen. More specifically, it relates to conjugating a pathogen, such as a viral surface protein antigen or a fragment thereof, with a polysaccharide, especially a Streptococcus pneumoniae capsular polysaccharide. The immunogenicity - enhanced conjugate can be used for preventing or treating diseases caused by pathogens, especially diseases caused by coronaviruses. Background art

[0004] Using an infectious microorganism, toxin, virus or its subunit as the antigen component of a vaccine to vaccinate a vertebrate individual, the above - mentioned antigen component is an exogenous substance relative to the individual, which induces or stimulates the vertebrate individual to produce a memory immune response against the exogenous molecule, so that when the vertebrate is exposed to the exogenous molecule again, a secondary immune response occurs to protect the individual from being invaded.

[0005] An "antigen" is an exogenous substance recognized (specifically bound) by an antibody or a T - cell receptor, but it does not necessarily induce an immune response. An exogenous substance that can be recognized (specifically bound) by an antibody or a T - cell receptor and induces a specific immune response is called an "immunogenic antigen" or an "immunogen".

[0006] Vaccines using subunits of infectious microorganisms, toxins, viruses, that is, parts of cell structures (bacteria or fungi) or viruses as antigens are non - live vaccines and are widely adopted because of their safety. However, the ability of subunits to induce specific immune responses is weak, that is, the immunogenicity of the antigen is poor.

[0007] Traditional means of enhancing immunogenicity involve adding immunoadjuvants. New means of enhancing the immune response are still being continuously researched and explored. An important means 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. For example, common encephalitis vaccines, Haemophilus influenzae type b vaccines, and pneumococcal vaccines use their purified capsular polymers conjugated with carrier proteins to produce more effective immunogenic compositions (Schneerson et al. (1984) Infect. Immun. 45: 582-591). Commonly used carrier proteins such as tetanus toxoid, fragment C of tetanus toxoid, non-toxic mutants of tetanus toxin, diphtheria toxoid, CRM197, and 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]. Such polysaccharide antigens are thymus-independent antigens, unable to generate a cellular immune response or form immune memory. Protective antibodies cannot be formed in children or immunocompromised individuals. By conjugating the polysaccharide antigen with a proteinaceous carrier having T cell epitopes, antigen-presenting cells or B cells endocytose the conjugate of the sugar and the protein for processing, and display the polypeptide fragments of the carrier protein on the cell surface, activating helper T cells and triggering a series of immune responses to generate protective antibodies and immune memory.

[0008] However, there are few reports on the effect of bacterial polysaccharides on the immunogenicity of protein / peptide antigens. US5192540A discloses a vaccine containing an immunogenic conjugate of the 38,000 Dalton or 40,000 Dalton outer membrane protein of Haemophilus influenzae type b and a fragment of oxidized polyribosyl-ribitol-phosphate polysaccharide of Haemophilus influenzae type b, which can be used to immunize against diseases caused by Haemophilus influenzae type b. However, "the conjugate vaccines of the present invention have high immunogenicity in animal models. Their antibody responses to PRP are significantly higher than the previously reported antibody responses. The conjugate vaccines also induce antibodies against the major proteins (38K or 40k proteins) of Haemophilus influenzae type b."

[0009] US9296795B discloses the use of an immunogenic polysaccharide - protein conjugate having a polysaccharide antigen (or its oligosaccharide fragment representing one or more antigenic epitopes) derived from a nosocomial pathogen in an immunogenic composition, wherein the polysaccharide is conjugated to a staphylococcal surface adhesin carrier protein to elicit an antibody response against the polysaccharide antigen and the staphylococcal surface adhesin carrier protein. Although "the conjugate described in the present invention has unique advantages: it can induce the production of antibodies against both the polysaccharide antigen and the surface adhesin carrier protein (both are virulence factors) and confer immunity against diseases caused by nosocomial pathogens. That is to say, the surface adhesin protein itself can also confer immunity on the body, not just acting as a protein carrier for the polysaccharide antigen." The titer of the surface adhesin protein - specific antibodies induced by the conjugated surface adhesin protein is similar to that of the non - conjugated surface adhesin protein (Figures 17 - 20). This confirms that the antigenic epitopes have not been altered due to the binding of the surface adhesin protein and CP.

[0010] In the above two research works, only the report shows that the protein / peptide antigen conjugated with polysaccharide can also induce antibody production, but there is no report on the enhanced immunogenicity.

[0011] The inventors have pioneered the discovery that by conjugating a protein / peptide antigen with a sugar to form a sugar - protein / peptide antigen conjugate, the immunogenicity of the protein / peptide antigen is enhanced.

[0012] The inventors speculate that the principle is that protein aggregates are more likely to stimulate the body to produce an immune response and generate antibodies than protein monomers. In addition, most of the pattern recognition receptors on the surface of antigen - presenting cells in the animal immune system are related to sugars, and the sugars produced by bacteria are important signals for stimulating the immune system. However, the present invention is not bound by this theory. Summary of the Invention

[0013] One aspect of the present invention relates to a method for enhancing the immunogenicity of a protein / peptide antigen, which method comprises conjugating the protein / peptide antigen with a sugar to form a sugar - protein / peptide antigen conjugate.

[0014] In a specific embodiment of the present invention, the sugar is selected from polysaccharides, oligosaccharides or monosaccharides;

[0015] Preferably, it is Neisseria meningitidis capsular polysaccharide, Haemophilus influenzae type b capsular polysaccharide, Streptococcus pneumoniae capsular polysaccharide, Staphylococcus aureus group B capsular polysaccharide, dextran, mannan, starch, inulin, pectin, carboxymethyl starch, chitosan and its derivatives;

[0016] More preferably, it is Streptococcus pneumoniae capsular polysaccharide,

[0017] Most preferably, it is Streptococcus pneumoniae serotype 14 capsular polysaccharide, Streptococcus pneumoniae serotype 6B capsular polysaccharide and Streptococcus pneumoniae serotype 7F capsular polysaccharide,

[0018] Wherein the protein / peptide antigen is selected from pathogen-related protein / peptide antigens or tumor-related protein / peptide antigens,

[0019] Wherein the pathogens are selected from:

[0020] 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, mumps virus, human papillomavirus, flavivirus or influenza virus, Neisseria, Moraxella, Bordetella, Mycobacterium, including Mycobacterium tuberculosis; Escherichia, including enterotoxigenic 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;

[0021] Preferably a protein / peptide related to pathogen invasion of the host;

[0022] More preferably, the above pathogens are viruses;

[0023] More preferably, the above viruses are selected from Coronaviridae, Paramyxoviridae, Orthomyxoviridae, Filoviridae or Flaviviridae viruses, and

[0024] Wherein the tumors are selected from:

[0025] Diffuse large B-cell lymphoma, follicular lymphoma, other lymphomas, leukemia, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdomyoma, hepatocellular carcinoma, prostate cancer, breast cancer, cholangiocarcinoma and gallbladder cancer, bladder cancer, brain tumors including neuroblastoma, schwannoma, glioma, glioblastoma and astrocytoma, cervical cancer, colon cancer, melanoma, endometrial cancer, esophageal cancer, head and neck cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, renal cell carcinoma, rectal cancer, thyroid cancer, parathyroid tumor, uterine tumor and soft tissue sarcoma.

[0026] In a specific embodiment of the present invention, the protein / peptide antigen is a protein containing a Coronaviridae virus antigen;

[0027] Preferably a coronavirus spike protein;

[0028] More preferably the S1 subunit of the coronavirus spike protein;

[0029] More preferably the receptor-binding domain RBD of the coronavirus spike protein;

[0030] Or a protein / peptide that is a fragment or variant of the above all protein / peptide antigens with immunogenicity.

[0031] In a specific embodiment of the present invention, the coronavirus is SARS-CoV-2 or Middle East Respiratory Syndrome Coronavirus.

[0032] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of the antigen defined above and other proteins or peptides.

[0033] Preferably, the fusion protein is selected from SARS-CoV-2 RBD-mFc; or

[0034] SARS-CoV-2 RBD-his; or

[0035] MERS-COV RBD-his; and

[0036] Fc is preferably an IgG Fc fragment, more preferably a human or murine IgG Fc fragment.

[0037] In a specific embodiment of the present invention, the protein / peptide antigen comprises any one of the sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0038] In a specific embodiment of the present invention, the molecular weight of the sugar-protein / peptide antigen conjugate is 400-14000 KDa.

[0039] In a specific embodiment of the present invention, the protein / peptide antigen comprises a Paramyxoviridae virus antigen;

[0040] Preferably the Paramyxovirus glycoprotein receptor-binding region;

[0041] Preferably Paramyxovirus glycoprotein F, glycoprotein G;

[0042] Or a protein / peptide which is a fragment or variant of any of the above protein / peptide antigens having immunogenicity.

[0043] In a specific embodiment of the present invention, the Paramyxoviridae virus is human respiratory syncytial virus.

[0044] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of the antigen defined above and other proteins or peptides; preferably the fusion protein is RSV-gpG-his.

[0045] In a specific embodiment of the present invention, wherein the protein / peptide antigen comprises the sequence of SEQ ID NO:4 and / or SEQ ID NO:12.

[0046] In a specific embodiment of the present invention, the protein / peptide antigen comprises an Orthomyxoviridae virus antigen;

[0047] Preferably, it is the receptor-binding region of the orthomyxovirus glycoprotein;

[0048] Preferably, it is the hemagglutinin (HA) protein and / or the neuraminidase (NA) protein;

[0049] Or a protein / peptide which is a fragment or variant of all the above-mentioned protein / peptide antigens with immunogenicity.

[0050] In a specific embodiment of the present invention, the orthomyxovirus is influenza B virus and / or influenza A H5N1 virus.

[0051] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of the antigen defined above and other proteins or peptides,

[0052] Preferably, the fusion protein is Flu-B-HA1-his or H5N1-HA-his.

[0053] In a specific embodiment of the present invention, the protein / peptide antigen contains any one of the sequences of SEQ ID NO:7 and / or SEQ ID NO:15, SEQ ID NO:8 and / or SEQ ID NO:16.

[0054] In a specific embodiment of the present invention, the protein / peptide antigen contains a filovirus antigen;

[0055] Preferably, it is the receptor-binding region of the filovirus envelope glycoprotein;

[0056] Preferably, it is the filovirus envelope glycoprotein GP1 and / or GP2;

[0057] More preferably, it is the filovirus envelope glycoprotein GP1;

[0058] Or a protein / peptide which is a fragment or variant of all the above-mentioned protein / peptide antigens with immunogenicity.

[0059] In a specific embodiment of the present invention, the filovirus is Ebola virus.

[0060] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of the antigen defined above and other proteins or peptides,

[0061] Preferably, the fusion protein is Ebola-GP-Fc or Ebola-GP1-his;

[0062] Wherein Fc is preferably an IgG Fc fragment, more preferably a human or murine IgG Fc fragment.

[0063] In a specific embodiment of the present invention, the protein / peptide antigen comprises the sequence of any one of SEQ ID NO:9 and / or SEQ ID NO:17, SEQ ID NO:10 and / or SEQ ID NO:18.

[0064] In a specific embodiment of the present invention, the protein / antigen comprises a Flaviviridae virus antigen;

[0065] Preferably, it is a Flavivirus or Hepacivirus antigen;

[0066] Preferably, it is the receptor-binding region of the envelope protein of a Flavivirus;

[0067] Preferably, it is at least one of the EDI, EDII, and EDIII domains of the envelope protein of a Flavivirus;

[0068] More preferably, it is the EDIII domain of the envelope protein of a Flavivirus; or

[0069] Preferably, it is the receptor-binding region of the envelope glycoprotein of a Hepacivirus;

[0070] Preferably, it is the envelope glycoprotein E1 and / or E2 of a Hepacivirus;

[0071] Or a protein / peptide that is a fragment or variant of any of the above protein / peptide antigens with immunogenicity.

[0072] In a specific embodiment of the present invention, the Flavivirus is preferably Zika virus; the Hepacivirus is preferably hepatitis C virus.

[0073] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of the antigen defined above and other proteins or peptides,

[0074] Preferably, the fusion protein is ZIKV-E-Fc; wherein

[0075] Fc is preferably an IgG Fc fragment, more preferably a human or murine IgG Fc fragment; or

[0076] Preferably, the fusion protein is HCV-E2-his and / or HCV-E1-his.

[0077] In a specific embodiment of the present invention, the protein / peptide antigen comprises the sequence of any one of 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 SEQ ID NO:13.

[0078] In a specific embodiment of the present invention, the glyco-protein / peptide antigen is further conjugated to a protein carrier.

[0079] In a specific embodiment of the present invention, the protein carrier is tetanus toxoid, fragment C of tetanus toxoid, non-toxic mutant of tetanus toxin, diphtheria toxoid, CRM197, other non-toxic mutants of diphtheria toxin, preferably CRM197.

[0080] A second aspect of the present invention relates to a sugar-protein / peptide antigen conjugate, which has enhanced immunogenicity compared to the unconjugated protein / peptide antigen.

[0081] In a specific embodiment of the present invention, the sugar is selected from polysaccharides, oligosaccharides or monosaccharides;

[0082] Preferably it is Neisseria meningitidis capsular polysaccharide, Haemophilus influenzae type b capsular polysaccharide, Streptococcus pneumoniae capsular polysaccharide, Staphylococcus aureus group B capsular polysaccharide, dextran, mannan, starch, inulin, pectin, carboxymethyl starch, chitosan and its derivatives;

[0083] More preferably it is Streptococcus pneumoniae capsular polysaccharide,

[0084] Most preferably it is Streptococcus pneumoniae serotype 14 capsular polysaccharide, Streptococcus pneumoniae serotype 6B capsular polysaccharide and Streptococcus pneumoniae serotype 7F capsular polysaccharide; wherein the protein / peptide antigen is selected from pathogen-related protein / peptide antigens or tumor-related protein / peptide antigens,

[0085] wherein the pathogens are selected from:

[0086] 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, mumps virus, human papillomavirus, flavivirus or influenza virus, Neisseria, Moraxella, Bordetella, Mycobacterium, including Mycobacterium tuberculosis; Escherichia, including enterotoxigenic Escherichia coli; Salmonella, Listeria, Helicobacter, Staphylococcus, including Staphylococcus aureus, Staphylococcus epidermidis; Borrelia, Chlamydia, including Chlamydia trachomatis, Chlamydia pneumoniae; Plasmodium, including Plasmodium falciparum; Toxoplasma, Candida;

[0087] Preferably proteins / peptides related to pathogen invasion of the host;

[0088] More preferably the above pathogens are viruses;

[0089] More preferably the above viruses are selected from Coronaviridae, Paramyxoviridae, Orthomyxoviridae, Filoviridae or Flaviviridae viruses, and

[0090] wherein the tumors are selected from:

[0091] Diffuse large B-cell lymphoma, follicular lymphoma, other lymphomas, leukemia, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdomyoma, hepatocellular carcinoma, prostate cancer, breast cancer, cholangiocarcinoma and gallbladder cancer, bladder cancer, brain tumors including neuroblastoma, schwannoma, glioma, glioblastoma and astrocytoma, cervical cancer, colon cancer, melanoma, endometrial cancer, esophageal cancer, head and neck cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, renal cell carcinoma, rectal cancer, thyroid cancer, parathyroid tumor, uterine tumor and soft tissue sarcoma.

[0092] In a specific embodiment of the present invention, the protein / peptide antigen comprises a Coronaviridae virus antigen;

[0093] Preferably a coronavirus spike protein;

[0094] More preferably the S1 subunit of the coronavirus spike protein;

[0095] Even more preferably the receptor-binding domain RBD of the coronavirus spike protein;

[0096] Or a protein / peptide that is a fragment or variant of any of the above protein / peptide antigens having immunogenicity.

[0097] In a specific embodiment of the present invention, the coronavirus is SARS-CoV-2 or Middle East respiratory syndrome coronavirus.

[0098] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of the antigen defined above with other proteins or peptides,

[0099] Preferably, the fusion protein is selected from SARS-CoV-2 RBD-mFc; or

[0100] SARS-CoV-2 RBD-his; or

[0101] MERS-COV RBD-his; and

[0102] Fc is preferably an IgG Fc fragment, more preferably a human or murine IgG Fc fragment.

[0103] In a specific embodiment of the present invention, the protein / peptide antigen comprises any one of the sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3.

[0104] In a specific embodiment of the present invention, the protein / peptide antigen comprises

[0105] Paramyxoviridae virus antigen;

[0106] Preferably the receptor-binding domain of the paramyxovirus glycoprotein;

[0107] Preferably, the paramyxovirus glycoprotein F and glycoprotein G;

[0108] Or a protein / peptide which is a fragment or variant of any of the above protein / peptide antigens having immunogenicity.

[0109] In a specific embodiment of the present invention, the paramyxovirus is human respiratory syncytial virus.

[0110] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of the antigen defined above and other proteins or peptides.

[0111] Preferably, the fusion protein is RSV-gpG-his.

[0112] In a specific embodiment of the present invention, the protein / peptide antigen comprises the sequence described in SEQ ID NO:4 and / or SEQ ID NO:12.

[0113] In a specific embodiment of the present invention, the protein / peptide antigen is one comprising

[0114] Orthomyxovirus antigen;

[0115] Preferably, the orthomyxovirus glycoprotein receptor-binding region;

[0116] Preferably, it is hemagglutinin (HA) protein and / or neuraminidase (NA) protein;

[0117] Or a protein / peptide which is a fragment or variant of any of the above protein / peptide antigens having immunogenicity.

[0118] In a specific embodiment of the present invention, the orthomyxovirus is influenza B virus and / or influenza A H5N1 virus.

[0119] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of the antigen defined above and other proteins or peptides.

[0120] Preferably, the fusion protein is Flu-B-HA1-his or H5N1-HA-his.

[0121] In a specific embodiment of the present invention, 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.

[0122] In a specific embodiment of the present invention, the protein / peptide antigen is one comprising

[0123] Filovirus antigen;

[0124] Preferred filovirus envelope glycoprotein receptor-binding region;

[0125] Preferred filovirus envelope glycoproteins GP1 and / or GP2;

[0126] More preferably, filovirus envelope glycoprotein GP1;

[0127] Or a protein / peptide that is a fragment or variant of any of the above protein / peptide antigens having immunogenicity.

[0128] In a specific embodiment of the present invention, the filovirus is Ebola virus.

[0129] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of the antigen defined above and other proteins or peptides,

[0130] Preferably, the fusion protein is Ebola-GP-Fc or Ebola-GP1-his;

[0131] Wherein Fc is preferably an IgG Fc fragment, more preferably a human or murine IgG Fc fragment.

[0132] In a specific embodiment of the present invention, the protein / peptide antigen comprises any one of the sequences of SEQ ID NO:9 and / or SEQ ID NO:17, SEQ ID NO:10 and / or SEQ ID NO:18.

[0133] In a specific embodiment of the present invention, the protein / peptide antigen is a protein / peptide antigen comprising

[0134] Flaviviridae virus antigen;

[0135] Preferably, flavivirus or hepacivirus antigen;

[0136] Preferably, the flavivirus envelope protein receptor-binding region;

[0137] Preferably, at least one of the EDI, EDII, and EDIII domains of the flavivirus envelope protein;

[0138] More preferably, the EDIII domain of the flavivirus envelope protein; or

[0139] Preferably, the hepacivirus envelope glycoprotein receptor-binding region;

[0140] Preferably, the hepacivirus envelope glycoproteins E1 and / or E2;

[0141] Or a protein / peptide that is a fragment or variant of any of the above protein / peptide antigens having immunogenicity.

[0142] In a specific embodiment of the present invention, the flavivirus is preferably Zika virus; or

[0143] The virus of the genus Hepacivirus is preferably hepatitis C virus.

[0144] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of the antigen defined above and other proteins or peptides.

[0145] Preferably, the fusion protein is ZIKV-E-Fc; wherein

[0146] Fc is preferably an IgG Fc fragment, more preferably a human or murine IgG Fc fragment; or

[0147] Preferably, the fusion protein is HCV-E2-his and / or HCV-E1-his.

[0148] In a specific embodiment of the present invention, the protein / peptide antigen comprises any one of the sequences of 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 SEQ ID NO:13.

[0149] In a specific embodiment of the present invention, the molecular weight of the sugar-protein / peptide antigen conjugate is 400 - 14000 KDa.

[0150] In a specific embodiment of the present invention, the sugar-protein / peptide antigen is further conjugated to a protein carrier.

[0151] In a specific embodiment of the present invention, the protein carrier is tetanus toxoid, tetanus toxoid fragment C, a non-toxic mutant of tetanus toxin, diphtheria toxoid, CRM197, other non-toxic mutants of diphtheria toxin, preferably CRM197.

[0152] 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.

[0153] In a specific embodiment of the present invention, the adjuvant is selected from aluminum adjuvants, oil-in-water emulsion adjuvants, MF59, QS-21 and monophosphoryl lipid A.

[0154] The fourth aspect of the present invention relates to the use of the sugar-protein / peptide antigen conjugate or the immune complex for preventing or treating diseases caused by the protein / peptide antigen related to the pathogen or the tumor-related protein / peptide defined above. The preferred pathogens are

[0155] coronaviruses, more preferably SARS-CoV-2 and / or MERS-CoV;

[0156] paramyxoviruses, more preferably human respiratory syncytial virus;

[0157] Orthomyxovirus, more preferably influenza B virus and / or influenza A H5N1 virus;

[0158] Filovirus, more preferably Ebola virus;

[0159] Flavivirus, preferably Zika virus; or hepatitis C virus.

[0160] The fifth aspect of the present invention relates to the use of a sugar-protein / peptide antigen conjugate or immune complex in the preparation of a vaccine or drug for preventing and treating diseases caused by the protein / peptide antigen related to the pathogen defined above or the tumor-related protein / peptide. The preferred pathogens are

[0161] Coronavirus, more preferably SARS-CoV-2 and / or MERS-CoV;

[0162] Paramyxovirus, more preferably human respiratory syncytial virus;

[0163] Orthomyxovirus, more preferably influenza B virus and / or influenza A H5N1 virus;

[0164] Filovirus, more preferably Ebola virus;

[0165] Flavivirus, preferably Zika virus; or hepatitis C virus. Description of the Drawings

[0166] Figure 1 Describes the anti-SARS-COV-2 RBD antibody titer of the serum of mice immunized with an immunocomposition with SARS-COV-2 RBD-mFc as the antigen. The value is the absorbance detected when the serum is diluted 8000-fold.

[0167] Figure 2 Describes the comparison of different adjuvants, with the antigen being the SARS-COV-2 RBD-his-PS14 conjugate, the serum dilution factor being 32000-fold, and the immunization dose being 3 μg / mouse.

[0168] Figure 3 Describes the comparison of the neutralizing activity of the serum of mice immunized with an immunocomposition with the SARS-COV-2 RBD protein-PS14 polysaccharide conjugate and the conjugate of SARS-COV-2 RBD and CRM197 conjugated to PS14 together as the antigen. The serum dilution factor is 1500-fold, and the immunization dose is 3 μg / mouse.

[0169] Figure 4 Describes the immunization results of conjugates with PS7F, PS14, and dextran as conjugating agents. Detailed Description of the Invention

[0170] Definitions

[0171] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. For the purposes of this invention, the following terms are further defined.

[0172] As used herein and in the appended claims, the singular forms "a", "an", "another", and "the" include plural referents unless the context clearly dictates otherwise.

[0173] The terms "comprising" and "including" mean including the specific components without excluding any other components. Terms such as "consisting essentially of" allow the inclusion of other components or steps that do not impair the novel or essential features of the invention, i.e., they exclude other unrecited components or steps that would impair the novel or essential features of the invention. The term "consisting of" means including the specific components or group of components and excluding all other components. In this specification and the appended claims, "the protein / peptide antigen therein is a protein / peptide comprising X" means that the amino acid sequence of the protein / peptide antigen comprises the protein / peptide sequence of X.

[0174] The term "antigen" is an exogenous substance that is recognized (specifically bound) by an antibody or a T cell receptor, but does not necessarily induce an immune response. An exogenous substance that induces a specific immune response is called an "immunogenic antigen" or "immunogen". A "hapten" is an antigen that by itself cannot elicit an immune response (although a conjugate of several hapten molecules, or a conjugate of a hapten with a macromolecular carrier can elicit an immune response).

[0175] A "humoral immune response" is an antibody-mediated immune response and involves the introduction and generation of antibodies that recognize and bind to the antigen in the immunogenic composition of the present invention with a certain affinity. A "cell-mediated immune response" is an immune response mediated by T cells and / or other white blood cells, which is induced by presenting antigenic epitopes associated with class I or class II molecules of the major histocompatibility complex (MHC), CD1, or other non-classical MHC-like molecules.

[0176] The term "sugar" can be used to refer to polysaccharides, oligosaccharides, or monosaccharides. Polysaccharides can be isolated from organisms such as bacteria and can be natural polysaccharides. Optionally, the size of the polysaccharides can be adjusted to a certain extent using a microfluidization method. Adjusting the size of the polysaccharides 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.

[0177] The term "conjugate" as used in this specification and the appended claims refers to a protein / peptide covalently conjugated to a sugar. The sugar-protein / peptide conjugates of the present invention and the immunogenic compositions containing the same may contain a certain amount of free sugar, protein / peptide.

[0178] As used herein, "conjugation" refers to the process by which a sugar, such as a bacterial capsular polysaccharide, is covalently linked to a protein / peptide.

[0179] The term "immunogenic composition" refers to any pharmaceutical composition containing an antigen, such as a microorganism or a component thereof, which can be used to induce an immune response in an individual.

[0180] The term "carrier" can be used to refer to a diluent, adjuvant, excipient, or vehicle administered together with a pharmaceutical composition. Water, saline solutions, and aqueous dextrose and glycerol solutions can be used as liquid carriers, especially for injectable solutions.

[0181] As used herein, "immunogenicity" means the ability of an antigen (or an epitope of an antigen), such as the receptor-binding domain of a coronavirus spike protein or a sugar conjugate or an immunogenic composition containing the antigen, to induce a humoral or cell-mediated immune response or both in a host (such as a mammal).

[0182] A "protective" immune response refers to the ability of an immunogenic composition to induce a humoral or cell-mediated immune response or both that protects an individual from infection. The protection provided need not be absolute, i.e., it need not completely prevent or eradicate the infection, as long as there is a statistically significant improvement relative to a control population of individuals (such as infected animals that have not been administered a vaccine or immunogenic composition). The protection can be limited to moderating the severity or rapidity of onset of the symptoms of the infection.

[0183] Both "immunogenic amount" and "immunologically effective amount" are used interchangeably herein and refer to the amount of an antigen or immunogenic composition sufficient to elicit an immune response (a cellular (T cell) or humoral (B cell or antibody) response or both, as measured by standard assays known to those skilled in the art).

[0184] 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.

[0185] Method for enhancing the immunogenicity of protein / peptide antigens of the present invention

[0186] The present invention is a pioneering invention. The inventors have found that by conjugating a protein / peptide antigen with a sugar to form a sugar-protein / peptide antigen conjugate, the immunogenicity of the protein / peptide antigen is enhanced.

[0187] Prior to this invention, there were no research reports on the improvement of the immunogenicity of protein / peptide antigens in sugar-protein / peptide antigen conjugates. On the contrary, as mentioned in the background art, previous studies described the immunogenicity of protein / peptide antigens in conjugates as retaining immunogenicity (US5192540A / US9296795B) or the protein / peptide antigen epitopes not being altered by conjugation (US9296795B). These teachings are contrary to the purpose of the present invention.

[0188] The glycoconjugated protein / peptide antigen of the present invention

[0189] 1. Coronaviridae virus as an antigen

[0190] The Coronaviridae family includes the subfamily Orthocoronavirinae and the subfamily Letovirinae.

[0191] The genus Betacoronavirus of the subfamily Orthocoronavirinae contains the well-known Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome-related Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). These three viruses mainly mediate virus invasion by binding the spike protein (S protein) to the host cell receptor 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). After the spike protein (S protein) binds to the ACE2 receptor, it is cleaved by host proteases into an S1 polypeptide containing the receptor binding domain (Receptor binding domain, SARS-COV-2 RBD) and an S2 polypeptide responsible for mediating virus-cell membrane fusion, and then invades the body.

[0192] One embodiment of the present invention selects the spike protein of SARS-CoV-2 (SARS-CoV-2 S protein), its extracellular region, S1 subunit, or receptor binding region as an antigen.

[0193] One embodiment of the present invention selects the Middle East Respiratory Syndrome Coronavirus as an antigen, such as its extracellular region, S1 subunit, or receptor binding region as an antigen.

[0194] 2. Paramyxoviridae virus as an antigen

[0195] The Paramyxoviridae family includes two subfamilies, Paramyxovirinae and Pneumovirinae. Human respiratory syncytial virus (RSV) is one of the viruses in the Respirovirus genus of the Paramyxovirinae subfamily. RSV encodes two major transmembrane surface glycoproteins, glycoprotein G (attachment protein) and glycoprotein F (fusion protein). Glycoprotein G mediates virus binding to cell receptors, while glycoprotein F promotes virus fusion with the cell membrane, allowing the viral ribonucleoprotein to invade the cytoplasm (Lopez et al. (1998) J. Virology 72:6922 - 6928).

[0196] One embodiment of the present invention selects RSV envelope protein glycans as antigens. Human RSV envelope protein glycans can be selected, such as RSV glycoprotein F or glycoprotein G.

[0197] 3. Orthomyxoviridae virus as an antigen

[0198] The Orthomyxoviridae family includes the genera Influenzavirus A, Influenzavirus B, Influenzavirus C, and other genera.

[0199] Infection with influenza viruses A, B, and C mainly depends on two envelope protein glycans: hemagglutinin (HA) and neuraminidase (NA), which are responsible for virus attachment and virus particle invasion of cells. Influenza virus infection is initiated by the attachment of the hemagglutinin (HA) protein on the virion surface to sialic acid-containing cell receptors (glycoproteins and glycolipids). The neuraminidase (NA) protein mediates the processing of sialic acid receptors, and virus entry into cells depends on HA-dependent receptor-mediated endocytosis (CN103865892B).

[0200] One embodiment of the present invention selects the hemagglutinin (HA) protein of influenza A virus H5N1 as an antigen, or the hemagglutinin protein (HA1 subunit) of influenza B virus can also be selected as an antigen.

[0201] 4. Filoviridae virus as an antigen

[0202] The representative genera of the Filoviridae family include Ebola virus (ebolaviruses) of the genus Ebolavirus and Marburg virus (marburgviruses) of the genus Marburgvirus.

[0203] The only protein present on the surface of the Ebola virus is the glycoprotein (GP). Trimers of GP1,2 form the surface spikes of the virus, and GP1,2 is composed of two subunits, GP1 and GP2, linked by disulfide bonds (Volchkova, V. A. et al., (1998), Virology 250:408 - 414; Falzarano, D. et al., (2006), Chembiochem 7:1605 - 1611). It is known that GP1 mediates the attachment of the virus to host cells, and GP2 is involved 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).

[0204] One embodiment of the present invention selects Ebola virus glycoprotein (GP) as an antigen. For example, the extracellular domain of GP, subunit GP proteins (GP1 and / or GP2).

[0205] 5. Viruses of the Flaviviridae family as antigens

[0206] Viruses of the Flaviviridae family mainly include the genus Flavivirus, the genus Pestivirus, Pegivirus

[0207] and the genus Hepacivirus. Among them, the genus Flavivirus includes Zika virus (ZIKV), dengue fever (DV), West Nile virus, Japanese encephalitis virus, yellow fever virus, etc. The genus Hepacivirus includes hepatitis C virus (HCV).

[0208] The flavivirus envelope protein plays an important role in virus infection of host cells and mediates virus entry into host cells. 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 orientation of the protein. The glycosylation sites in EDI are related to virus production, pH sensitivity, and neuroinvasiveness. Due to the immunodominance of the fusion loop epitope and the envelope dimer epitope, EDII plays an important role in membrane fusion. In addition, EDIII is the main target of neutralizing antibodies (Xingcui Zhang.et al.,(2017)Viruses.2017Nov;9(11):338.Structures and Functions of the Envelope Glycoprotein in FlavivirusInfections).

[0209] The Zika virus envelope protein ("E" or "EP") consists of three distinct domains. The E domain I (E-DI) is the central domain that organizes the entire E protein structure. The E domain II (E-DII) is formed by two extended loops protruding from E-DI and is located in a pocket at E-DI and the E domain III (E-DIII). E-DIII is an immunoglobulin-like domain that forms small protrusions on the surface of the otherwise smooth spherical mature virus particles and is thought to interact with cellular receptors on target cells (CN109996560A).

[0210] The HCV RNA genome encodes a single polyprotein that is cleaved into three structural proteins (core, glycoprotein E1, and E2) and seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) during or after translation. The envelope glycoproteins E1 and E2 form a heterodimer that constitutes the virus envelope protein and plays an important role in virus entry into host cells, mediating virus entry and morphogenesis. The envelope glycoprotein of hepatitis C virus binds to specific proteins on the surface of host hepatocytes to initiate the entry process. This process involves a large number of host receptors / coreceptors. Among them, E2 is the main HCV envelope glycoprotein that directly interacts with the receptor / coreceptor. For a long time, it has been thought that E1 does not directly interact with host receptors during this process and that it co-induces membrane fusion with E2 by maintaining the functional E2 conformation required for receptor binding (Yimin,Tong.Etal.,(2018)Front Immunol.2018;9:1411.Role of Hepatitis C Virus EnvelopeGlycoprotein E1 in Virus Entry and Assembly).

[0211] One embodiment of the present invention selects the Zika virus envelope protein E-DIII as an antigen.

[0212] One embodiment of the present invention selects the hepatitis C virus envelope glycoproteins E1 and / or E2 as antigens.

[0213] The viral antigens described above in this chapter can be obtained by extracting natural pathogens or by gene recombination. They can all be further modified, such as fragments or variants with immunogenicity, as well as fusion proteins with purification tags or their Fc fragments of antibodies, and all can be used in the present invention.

[0214] The polysaccharides are bacterial polysaccharides, such as common Neisseria meningitidis capsular polysaccharides, Haemophilus influenzae type b capsular polysaccharides, Streptococcus pneumoniae capsular polysaccharides, group B Staphylococcus aureus capsular polysaccharides, as well as dextran, mannan, etc. The polysaccharides can also be plant-derived polysaccharides, such as starch, inulin, pectin, etc., or derivatives of polysaccharides modified chemically, such as carboxymethyl starch. The polysaccharides can also be animal-derived polysaccharides, such as chitosan and its derivatives.

[0215] The process of conjugating polysaccharides with proteins is as follows: making the polysaccharides carry reactive groups through chemical reactions. The reactive groups then react with the amino groups, carboxyl groups, sulfhydryl groups, imidazole rings of histidine, indole rings of tryptophan, benzene rings of tyrosine, benzene rings of phenylalanine, hydroxyl groups of serine, and reactive groups such as glutamine and asparagine on the protein molecule to form covalent bonds.

[0216] One method of conjugating polysaccharides with protein molecules is to oxidize the polysaccharides with sodium periodate to produce aldehyde groups on the polysaccharides. The aldehyde groups react with the amino groups on the protein molecules to form Schiff bases, which are reduced to stable single bonds in the presence of reducing agents. Thus, covalent connections are formed between the polysaccharides and the protein molecules. Reducing agents such as sodium cyanoborohydride can be added to the reaction system.

[0217] Another method of conjugating polysaccharides with protein molecules is to react the polysaccharides with cyanogen bromide or 1-cyano-4-dimethylaminopyridine tetrafluoroborate to produce reactive cyanate esters. The cyanate ester groups react with the amino groups on the protein surface to form covalent bonds. The activated polysaccharides can also first react with linkers such as hexamethylenediamine and adipic dihydrazide, and the products then react with the protein in the presence of condensing agents to form covalent connections.

[0218] The polysaccharides can also be activated with other chemical reagents and then react with proteins to form conjugates. Such as reagents well-known in the art, such as epichlorohydrin, triazine, diazine, divinyl sulfone, etc.

[0219] To improve the immunogenicity of the sugar-protein / peptide antigen conjugates of the present invention, a protein carrier can be further added during the conjugation reaction of the sugar with the protein / peptide antigen to form a sugar-protein / peptide antigen-protein carrier conjugate. The protein carrier can be tetanus toxoid, fragment C of tetanus toxoid, non-toxic mutants of tetanus toxin, diphtheria toxoid, CRM197, other non-toxic mutants of diphtheria toxin commonly used in the vaccine industry, preferably CRM197.

[0220] The immunogenic composition of the present invention

[0221] In one embodiment, the immunogenic composition of the present invention further comprises at least one of an adjuvant, a buffer, a cryoprotectant, a salt, a divalent cation, a non-ionic detergent, a free radical oxidation inhibitor, a diluent or a carrier. In one embodiment, the adjuvant in the immunogenic composition of the present invention is an aluminum-based adjuvant. In one embodiment, the adjuvant is an aluminum-based adjuvant selected from aluminum phosphate, aluminum sulfate and aluminum hydroxide. In one embodiment, the adjuvant is aluminum phosphate.

[0222] An adjuvant is a substance that enhances the immune response when administered together with an immunogen or an antigen. The compositions used in the present invention may or may not contain a vaccine adjuvant. Adjuvants that can be used in the compositions of the present invention include, but are not limited to:

[0223] Oil emulsion compositions include squalene-water emulsions, such as MF59; complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); saponin preparations; using saponin and cholesterol together to form unique particles called immunostimulating complexes (ISCOMs); virosomes and virus-like particles; 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.

[0224] The immunogenic composition may optionally comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes carriers of animals (including humans and non-human mammals) recorded or to be recorded in various pharmacopoeias. The term "carrier" can be used to refer to a diluent, an adjuvant, an excipient or a vehicle administered together with a pharmaceutical composition. Water, saline solutions and aqueous dextrose and glycerol solutions can be used as liquid carriers especially for injectable solutions.

[0225] The immunogenic composition of the present invention may further comprise one or more additional immunomodulators, which are substances that disrupt or alter the immune system so as to observe an up-regulation or down-regulation of the humoral and / or cell-mediated immunity. In one embodiment, an up-regulation of the humoral and / or cell-mediated capabilities (arms) of the immune system is provided. This includes, for example, adjuvants or cytokines.

[0226] The dosage form of the immunogenic composition of the present invention

[0227] The immunogenic compositions of the present invention for therapeutic or prophylactic treatment can be administered by intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection; or via mucosal administration to the oral / esophagus, respiratory tract, urogenital tract. Intranasal administration of the vaccine is preferred for the treatment of certain diseases, such as pneumonia or otitis media. Although the vaccines of the present invention can be administered as a single dose, their components can also be co-administered simultaneously or at different times. In addition to a single administration route, two different administration routes can be used.

[0228] The optimal amounts of the components for a particular immunogenic composition can be determined by standard studies involving the observation of an appropriate immune response in an individual. After the initial vaccination, the individual can receive one or several booster immunizations at appropriate intervals.

[0229] Use of the immunogenic compositions of the present invention

[0230] The protein / peptide antigen conjugates and immunocomplexes of the present invention can prevent or treat diseases caused by pathogens, such as coronaviruses, paramyxoviruses, orthomyxoviruses, filoviruses and flaviviruses, more particularly diseases caused by SARS-CoV-2 and / or MERS-CoV viruses, human respiratory syncytial virus, influenza B virus, influenza A H5N1 virus, Ebola virus, Zika virus and / or hepatitis C virus.

[0231] Abbreviations of the terms of the present invention

[0232] SARS-COV-2 RBD-mFc: Mouse Fc fusion protein of the receptor-binding domain of the spike protein of SARS-CoV-2 coronavirus;

[0233] SARS-COV-2 RBD-his: Fusion protein of the receptor-binding domain of the spike protein of SARS-CoV-2 coronavirus and a 6-histidine tag;

[0234] MERS-COV RBD-his: Fusion protein of the receptor-binding domain of the spike protein of Middle East respiratory syndrome coronavirus and a 6-histidine tag;

[0235] RSV-gpG: Human respiratory syncytial virus glycoprotein G;

[0236] HCV-E1: Hepatitis C virus Envelope Glycoprotein E1;

[0237] HCV-E2: Hepatitis C virus Envelope E2 Protein / Hepatitis C virus envelope glycoprotein E2;

[0238] flu-B-HA1: Influenza B Hemagglutinin Protein (HA1 Subunit) / Influenza B virus hemagglutinin (HA) protein (HA1 subunit);

[0239] H5N1-HA: Influenza A H5N1 Hemagglutinin / Influenza A H5N1 virus hemagglutinin (HA) protein;

[0240] Ebola-GP: Ebola virus Glycoprotein (Receptor Binding Domain,) / Ebola virus glycoprotein (receptor binding domain);

[0241] Ebola-GP1: Ebola virus Glycoprotein GP1 / Ebola virus glycoprotein GP1;

[0242] ZIKV-E: Zika virus Envelope protein (Domain III) / Zika virus envelope protein (domain III);

[0243] PS14: Streptococcus pneumoniae serotype 14 capsular polysaccharide

[0244] PS7F: Streptococcus pneumoniae serotype 7F capsular polysaccharide

[0245] Alum: Aluminum adjuvant, aluminum phosphate adjuvant in this article

[0246] Example 1: Preparation of Streptococcus pneumoniae serotype 14 (PS14) and 7F (PS7F) capsular polysaccharides

[0247] The seed of Streptococcus pneumoniae serotype 14 is ATCC 6314, and the seed of Streptococcus pneumoniae serotype 7F is ATCC 10351.

[0248] Inoculate 0.5 mL of pneumococcus seeds preserved with glycerol into 500 mL of Hoeprich's medium (V.M. Goncalves, Optimization of medium and cultivation conditions for capsular polysaccharide production by Streptococcus pneumoniae serotype 23F, Allp Microbiol Biotechnol (2002) 59:713 - 717), culture in a shaker at 37 °C with a rotation speed of 150 rpm for 10 - 16 hours. Stop culturing when OD 600 is greater than 1.0. Add 0.6 g of sodium deoxycholate, mix well, and let stand for more than 2 hours to completely lyse the bacteria. Centrifuge at 14000g for 30 minutes, take the supernatant, and concentrate it to one - tenth of the original volume (about 400 mL) using a 100 kDa ultrafiltration. Gradually add 36% acetic acid to the concentrated solution to adjust the pH to 3.5. Let stand for 2 hours, centrifuge at 14000g for 30 minutes, take 390 mL of the supernatant, add 130 mL of absolute ethanol, mix well, and let stand overnight. The next day, after centrifuging at 14000g for 30 minutes, take the supernatant, add another 780 mL of absolute ethanol, mix well, and let stand overnight. The next day, after centrifuging at 14000g for 30 minutes, discard the supernatant, add 300 mL of 75% ethanol solution to the precipitate to suspend the precipitate, and then centrifuge at 14000g for 30 minutes again. Discard the supernatant, dissolve the precipitate in 10 mL of water, and control the polysaccharide concentration in the solution to be greater than 10 mg / mL. The resulting solution is the pneumococcus capsular polysaccharide solution.

[0249] Example 2: Hydrolysis, high - activation - degree activation, and low - activation - degree activation of polysaccharides

[0250] The polysaccharides are the capsular polysaccharides of Streptococcus pneumoniae serotype 14 (PS14) and 7F (PS7F) prepared in Example 1 or dextran (Sigma, 00894, the same below).

[0251] 2.1 Polysaccharide hydrolysis:

[0252] Take 10 mL of 10 mg / mL purified capsular polysaccharide, add 0.86 mL of 36% acetic acid, so that the final concentration of acetic acid in the solution is 500 mM. After water - bathing at 90 °C for 2 h, add 1M NaOH to neutralize to pH 6 - 7 to obtain a hydrolyzed polysaccharide sample.

[0253] Determined by HPLC - MALS, the molecular weight of the PS14 capsular polysaccharide is about 500 KDa, and after hydrolysis, it is about 300 KDa.

[0254] The molecular weight of the PS7F capsular polysaccharide is about 700 KDa and is not hydrolyzed.

[0255] The dextran is not hydrolyzed.

[0256] 2.2 High-activation activation of polysaccharides:

[0257] Add 100 mg of sodium periodate to 10 mL of a 10 mg / mL polysaccharide solution, mix well, and let it react in the dark for 1 h. Take a centrifugal chromatography column filled with 5 mL of Sephadex G 25 packing material, add 10 mL of 50 mM Na2HPO4 buffer solution with pH = 7.0. The buffer solution flows through the chromatography column under its own gravity. Then place the chromatography column in a centrifuge and centrifuge at 1000 g for 2 min. After that, replace the collection tube with a new one, take 1 mL of the polysaccharide solution oxidized by sodium periodate and put it into the centrifugal chromatography column, and centrifuge again at 1000 g for 2 min. The eluate collected from the centrifugal column is the polysaccharide solution with high-activation activation.

[0258] 2.3 Low-activation activation of polysaccharides:

[0259] Add 30 mg of sodium periodate to 10 mL of a 10 mg / mL polysaccharide solution, mix well. Let it react in the dark for 1 h. Take a centrifugal chromatography column filled with 5 mL of Sephadex G 25 packing material, add 10 mL of 50 mM Na2HPO4 buffer solution with pH = 7.0. The buffer solution flows through the chromatography column under its own gravity. Then place the chromatography column in a centrifuge and centrifuge at 1000 g for 2 min. After that, replace the collection tube with a new one, take 1 mL of the polysaccharide solution oxidized by sodium periodate and put it into the centrifugal chromatography column, and centrifuge again at 1000 g for 2 min. The eluate collected from the centrifugal column is the polysaccharide solution with low-activation activation.

[0260] Example 3: Conjugation of protein / peptide antigen and polysaccharide

[0261] The protein / peptide antigen used is the receptor-binding domain of the coronavirus spike protein, and the polysaccharide is the polysaccharide of Streptococcus pneumoniae capsule or dextran. The specific components, amounts and volumes are shown in Table 1 respectively.

[0262] 1. Buffer exchange of coronavirus spike receptor protein: Take 5 mg of the coronavirus spike receptor protein and exchange the buffer to 50 mM Na2HPO4 buffer solution with pH = 7.0 using a 30,000 MW ultrafiltration tube, and the final concentration of the exchanged protein is required to be ≥10 mg / mL.

[0263] 2. Conjugation of Coronavirus Spike Receptor Protein with Polysaccharide: Take 3 mg of coronavirus spike receptor protein, add the activated Streptococcus pneumoniae capsular polysaccharide or dextran according to Table 1, and supplement with 50 mM Na2HPO4 buffer at pH 7.0. The final volume is shown in Table 1. Then add 5 M sodium cyanoborohydride solution according to the volume in Table 1, and rotate and mix the reaction in the dark at room temperature for 1 h. Then add 10 mg / mL sodium borohydride solution to the reaction solution (add 0.15 ml to a 0.6 ml reaction system and 0.375 ml to a 1.5 ml reaction system), and react at room temperature for 2 hours. Then exchange the buffer of the conjugate 10 times with a 100,000 MW ultrafiltration tube and PBS buffer, and the final volume after ultrafiltration is less than 2 ml. Sterile filter the ultrafiltered conjugate sample with a 0.22 μm filter and store it at 4°C.

[0264] 3. Determine the molecular weight of the conjugate using HPLC-MALLS. The results are shown in Table 1.

[0265] Table 1. Reaction conditions of several conjugates and the molecular weights of their products

[0266]

[0267]

[0268] * indicates the molecular weights of the three peaks of the conjugate determined by HPLC-MALLS

[0269] MERS-COV RBD-his (Source: Beijing Protein Innovation Co., Ltd., 40071-V08B1), and other proteins were prepared by the inventors themselves.

[0270] Example 4. Conjugation of Protein / Peptide Antigen and Protein Carrier with Polysaccharide

[0271] The protein / peptide antigen used was the receptor-binding domain of the coronavirus spike protein SARS-COV-2 RBD, the protein carrier was CRM197, and the polysaccharide was Streptococcus pneumoniae capsular polysaccharide. Their specific components, amounts, and volumes are shown in Table 2 respectively.

[0272] CRM197 is a variant of diphtheria toxin (Geert J. Schenk, Efficient CRM197-mediated drug targeting to monocytes, Journal of Controlled Release 158 (2012) 139–147). The receptor-binding domain of the coronavirus spike protein and CRM197 were conjugated to Streptococcus pneumoniae serotype 14 capsular polysaccharide together. The activation process of the polysaccharide was the same as that in Example 2.2. Take 1.5 mg of polysaccharide, 2.7 mg of the coronavirus spike protein receptor-binding domain protein, and 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 its product are shown in Table 2.

[0273] Table 2 Reaction conditions of the conjugate of SARS-COV-2 RBD and CRM197 and the molecular weight of its product

[0274]

[0275] Example 5. Immunogenicity of the conjugate of the coronavirus spike protein receptor-binding domain and polysaccharide

[0276] The conjugates of the coronavirus spike protein receptor-binding domain and polysaccharide used are shown in Tables 3-6.

[0277] 5.1 Preparation of the immunocomposition

[0278] The conjugate prepared using the coronavirus spike protein receptor-binding domain protein or in Example 3 or 4 was used as the antigen to prepare the immunocomposition.

[0279] 5.1.1 Preparation of MF59 adjuvant

[0280] Prepare 200 ml of 10 mM sodium citrate solution (pH 6.5 adjusted with HCl), add 1 ml of Tween 80 (Nanjing Weier Pharmaceutical Co., Ltd.) and mix well to dissolve completely. Take another 10 ml of squalene (Merck) and add 1 ml of Span 85 (Zhaoqing Chaoneng Industry Co., Ltd.) and mix well to dissolve completely. Mix the above two solutions and homogenize 3 times with a high-pressure homogenizer (AH-PILOT ATS) at 800 bar to obtain a uniform emulsion, which is the MF59 adjuvant.

[0281] 5.1.2 Preparation of MF59 adjuvant containing monophosphoryl lipid A (MPL)

[0282] Disperse 10 mg of MPL (MERCK L6895) in 10 ml of sodium citrate buffer (10 mM, pH 6.5). Take another 4 ml of MF59 adjuvant, add 1 ml of the MPL dispersion, and mix well to obtain the MF59 adjuvant containing MPL.

[0283] 5.1.3 Preparation of Aluminum Adjuvant Immunocomposition

[0284] The antigens were respectively diluted with PBS to 0.02 mg / ml or 0.06 mg / ml (calculated as peptide / protein, the same below), and the aluminum adjuvant (Beijing Nuoning Biotechnology Co., Ltd.) was diluted with PBS to 1 mg / ml. The diluted antigens and aluminum adjuvant were mixed in equal volume. The protein concentrations of the antigens in the immunocomposition were 0.01 mg / ml or 0.03 mg / ml respectively.

[0285] 5.1.4 Preparation of MF59 Adjuvant Immunocomposition

[0286] The antigens were respectively diluted with PBS to 0.02 mg / ml or 0.06 mg / ml, and the diluted antigens were mixed with an equal volume of MF59 adjuvant. The protein concentrations of the antigens in the immunocomposition were 0.01 mg / ml or 0.03 mg / ml respectively.

[0287] 5.1.5 Preparation of MF59 Adjuvant Immunocomposition Containing MPL

[0288] The antigens were respectively diluted with PBS to 0.02 mg / ml or 0.06 mg / ml, and the diluted antigens were mixed with an equal volume of MF59 adjuvant containing MPL. The protein concentrations of the antigens in the immunocomposition were 0.01 mg / ml or 0.03 mg / ml respectively.

[0289] 5.1.6 Preparation of Immunocomposition with MF59 and Aluminum Adjuvant Mixture

[0290] Take 1.5 ml of aluminum adjuvant and 1.5 ml of MF59 adjuvant, mix them, and then add 0.18 ml of antigen with a concentration of 1 mg / ml. The protein concentration of the antigen in the immunocomposition is 0.03 mg / ml.

[0291] 5.2 Immunization of Mice:

[0292] 4 - 6-week-old Balb / c mice were selected and intraperitoneally injected with 0.1 ml of the immunocomposition with a concentration of 0.01 mg / ml or 0.03 mg / ml as described in Example 5.1, and boosted on the 14th day and the 28th day respectively. Blood was collected from the orbital cavity on the 7th day, the 21st day, and the 35th day to measure the serum antibody titer and neutralization titer.

[0293] 5.3 Determination of Serum Titer

[0294] 5.3.1 Determination of Serum Titer When the Antigen is SARS-COV-2 RBD or Its Fusion Protein

[0295] Coat a 96-well plate with 5 μg / mL of SARS-COV-2 RBD-mFc protein (the same throughout, Shenzhou Cell Engineering Co., Ltd.), 100 μl / well, and place it at room temperature for 2 hours. After washing the plate, add 2% BSA and block at room temperature for 1 hour. Use CD155(D1)-mFc (the same throughout, Shenzhou Cell Engineering Co., Ltd.) as an irrelevant control with the same label. Dilute the serum to be tested (prepared in Example 5.2) to different dilutions (specific dilution multiples vary according to the immunization blood collection time, such as 1000x, 8000x, 16000x, 32000x dilution) using PBS containing 0.1% bovine serum albumin (BSA). Set the serum of mice immunized with SARS-COV-2 RBD-mFc as the positive control, and the serum of mice with an irrelevant immune target (anti-CD70 serum, Beijing Sino Biological Inc.) as the negative control. At the same time, add the serum to be tested at different dilutions and goat anti-mouse IgG F(ab)2 / HRP (Beijing Sino Biological Inc.) as the detection secondary antibody, 100 μl / well each, incubate together for 2 h, then wash the plate 5 times, add the substrate chromogenic solution for color development, and after termination, read the OD using an enzyme-linked immunosorbent assay (ELISA) reader. 450 . The OD at a certain dilution factor 450 represents the antibody titer.

[0296] 5.3.2 Determination of serum titer when the antigen is MERS-COV RBD or its fusion protein

[0297] When detecting the immune serum of MERS-COV RBD-his, use MERS-COV RBD-his (Beijing Sino Biological Inc., 40071-V08B1) for coating, without setting positive and negative controls, and the operation steps are exactly the same as in 5.3.1.

[0298] 5.3.3 Results of serum titer determination

[0299] Tables 3 - 5 and the appendix Figures 1-4 all show the results of serum titer determination.

[0300] The titer results of the serum diluted 8000-fold after immunizing mice with the aluminum adjuvant immunocomposition for 35 days are shown in Table 3.

[0301] Table 3 Serum titer of mice immunized with the aluminum adjuvant immunocomposition for 35 days (results of immunization for 35 days)

[0302] Antigen Immunization dose (μg) <![CDATA[Serum diluted 8000-fold (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

[0303] The titer results of the serum diluted 32000-fold after immunizing with the SARS-COV-2 RBD-his-PS14 aluminum / MF59 / MF59-aluminum / MF59-MPL adjuvant immunocomposition for 35 days are shown in Table 4.

[0304] Table 4 Serum titers of conjugate SARS-COV-2 RBD-his-PS14 when using different adjuvant combinations (results on day 35 of immunization, immunization dose 3 μg)

[0305] Adjuvant <![CDATA[Serum diluted 32,000 times (OD 450 )]]> Aluminum adjuvant 0.423 MF59 1.052 MF59-Aluminum 1.114 MF59-MPL 1.111

[0306] The serum titer results measured after diluting the serum of mice immunized with the MF59 adjuvant immunocomposition 8000-fold on day 21 are shown in Table 5.

[0307] Table 5 Serum titers of the MF59 adjuvant immunocomposition (results on day 21 of immunization, immunization dose 3 μg)

[0308] Antigen <![CDATA[Serum diluted 8000 times (OD 450 )]]> SARS-COV-2 RBD-his-PS7F 1.382 SARS-COV-2 RBD-his-Dextran 1.040 MERS-COV RBD-his-PS14 1.325

[0309] 5.4 Determination of neutralizing titer

[0310] The mouse serum samples obtained in Example 5.2 were diluted by a certain multiple according to experience (for example, diluted 500-fold) and then mixed with an equal volume of pseudovirus 2019-nCoV PSV (from the National Institutes for Food and Drug Control). Samples without adding serum were used as positive controls, and samples without adding pseudovirus were used as negative controls. After incubating at 37°C for 1 hour, Vero E6 or 293FT / ACE2 cells (from Shenzhou Cell Engineering Co., Ltd.) were synchronously infected. After infection, the cells were cultured at 37°C under 5% CO2 for about 20 - 28 hours, and the RLU value was detected on a microplate luminometer. According to

[0311] Neutralization inhibition rate % = (lg(positive RLU) - lg(sample RLU)) / (lg(positive RLU) - lg(negative RLU)) x 100%, calculate the neutralization inhibition rate.

[0312] The neutralizing titer results of the sera of mice immunized with different immunocompositions are shown in Table 6 and Figure 3 .

[0313] Table 6 Neutralizing titers of immunocompositions (results on day 35, immunization dose 3 μg)

[0314]

[0315] Example 6. Polysaccharide enhances the immunogenicity of several viral antigens

[0316] Select several viral antigens, as shown in Table 7 (all from Beijing Sino Biological Inc.), and conjugate them with PS14 polysaccharide. The steps are as follows:

[0317] Streptococcus pneumoniae serotype 14 (PS14) was prepared according to Example 1, and activation was carried out according to the steps of Example 2.2, wherein the amount of sodium periodate added was adjusted according to Table 8. PS14 was conjugated with the carrier protein CRM197 according to the steps of Example 3, and the ratio of protein to polysaccharide during conjugation was as shown in Table 8. An aluminum adjuvant-containing immunocomposition was prepared according to Example 5.1.3. Mice were immunized according to Example 5.2, and the immunization dose was 3 μg of antigen per mouse.

[0318] The titer of the immune serum was detected, and the corresponding antigen was used for coating, without setting positive and negative controls. The operation steps were exactly the same as those in 5.3.1.

[0319] The titers of the immune sera of various antigen conjugates are shown in Table 8. It can be seen that the immunogenicity of most antigens is significantly improved after conjugation with polysaccharides.

[0320] Table 7 Names, Catalog Numbers and Corresponding Abbreviations of Several Virus Antigens

[0321]

[0322]

[0323] Table 8 Conjugation Conditions of Several Virus Antigens with Polysaccharides and Titers of Immune Sera of Conjugates

[0324]

[0325] The antigens used in Table 8 are all recorded in Table 7, and the His-tag is not shown.

[0326] According to the above data, the immunogenicity of protein antigens is significantly improved after conjugation with Streptococcus pneumoniae capsular polysaccharides. Under the condition of aluminum adjuvant, the antibody titer of the immune serum of the conjugate can reach up to 2.3 times that of the original. The neutralization activity of the conjugate is also greatly improved compared with that of the corresponding protein. Compared with aluminum adjuvant, MF59 adjuvant, MF59 and aluminum adjuvant mixed adjuvant, and MF59 adjuvant plus MPL adjuvant can all further improve the immune effect of the conjugate. The immune effects are similar when using Streptococcus pneumoniae serotype 14 capsular polysaccharide, Streptococcus pneumoniae serotype 7F capsular polysaccharide, and dextran as conjugating agents.

[0327]

[0328]

[0329]

[0330] SEQUENCE LISTING <110> Shenzhou Cell Engineering Co., Ltd. <120> A method for enhancing the immunogenicity of protein / peptide antigens <130> PCT69222SXB <160> 19 <170> PatentIn version 3.3 <210> 1 <211> 223 <212> PRT <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 1 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 <210> 2 <211> 234 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 2 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Ala 210 215 220 His His His His His His His His His His 225 230 <210> 3 <211> 457 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 3 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Ala 210 215 220 Asp Asp Asp Asp Lys Ala Val Pro Arg Asp Ser Gly Cys Lys Pro Cys 225 230 235 240 Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys 245 250 255 Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val 260 265 270 Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe 275 280 285 Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu 290 295 300 Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His 305 310 315 320 Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala 325 330 335 Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg 340 345 350 Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met 355 360 365 Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro 370 375 380 Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn 385 390 395 400 Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val 405 410 415 Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr 420 425 430 Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu 435 440 445 Lys Ser Leu Ser His Ser Pro Gly Lys 450 455 <210> 4 <211> 232 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 4 Asn His Lys Val Thr Ser Thr Thr Thr Ile Ile Gln Asp Ala Thr Ser 1 5 10 15 Gln Ile Lys Asn Thr Thr Pro Thr Tyr Leu Thr Gln Ser Pro Gln Leu 20 25 30 Gly Ile Ser Pro Ser Asn Pro Ser Glu Ile Thr Ser Gln Ile Thr Thr 35 40 45 Ile Leu Ala Ser Thr Thr Pro Gly Val Lys Ser Thr Leu Gln Ser Thr 50 55 60 Thr Val Gly Thr Lys Asn Thr Thr Thr Thr Gln Ala Gln Pro Ser Lys 65 70 75 80 Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro Pro Ser Lys Pro Asn Asn 85 90 95 Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys Ser 100 105 110 Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro Asn Lys Lys 115 120 125 Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Glu Glu Pro Thr Phe Lys 130 135 140 Thr Ala Lys Glu Asp Pro Lys Pro Gln Thr Thr Gly Ser Gly Glu Val 145 150 155 160 Pro Thr Thr Lys Pro Thr Gly Glu Pro Thr Ile Asn Thr Thr Lys Thr 165 170 175 Asn Ile Thr Thr Thr Leu Leu Thr Ser Asn Thr Thr Arg Asn Pro Glu 180 185 190 Leu Thr Ser Gln Met Glu Thr Phe His Ser Thr Ser Ser Glu Gly Asn 195 200 205 Pro Ser Pro Ser Gln Val Ser Ile Thr Ser Glu Tyr Leu Ser Gln Pro 210 215 220 Ser Ser Pro Pro Asn Thr Pro Arg 225 230 <210> 5 <211> 158 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 5 Ala Leu Glu Val Leu Phe Gln Gly Pro Tyr Glu Val Arg Asn Val Ser 1 5 10 15 Gly Ile Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr 20 25 30 Glu Ala Ala Asp Val Ile Met His Thr Pro Gly Cys Val Pro Cys Val 35 40 45 Arg Glu Gly Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr Leu 50 55 60 Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His Val 65 70 75 80 Asp Leu Leu Val Gly Thr Ala Ala Phe Cys Ser Ala Met Tyr Val Gly 85 90 95 Asp Leu Cys Gly Ser Ile Phe Leu Val Ser Gln Leu Phe Thr Phe Ser 100 105 110 Pro Arg Arg His Glu Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr Pro 115 120 125 Gly His Val Ser Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp 130 135 140 Ser Pro Thr Thr Ala Leu Val Val Ser Gln Leu Leu Arg Ile 145 150 155 <210> 6 <211> 278 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 6 Glu Thr His Thr Thr Gly Arg Val Ala Gly His Thr Thr Ser Gly Phe 1 5 10 15 Thr Ser Leu Phe Ser Ser Gly Ala Ser Gln Lys Ile Gln Leu Val Asn 20 25 30 Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp 35 40 45 Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe Tyr Ala His Lys Phe 50 55 60 Asn Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Ile Asp 65 70 75 80 Trp Phe Ala Gln Gly Trp Gly Pro Ile Thr Tyr Thr Lys Pro Asn Ser 85 90 95 Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly 100 105 110 Val Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro 115 120 125 Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Val Pro Thr Tyr 130 135 140 Ser Trp Gly Glu Asn Glu Thr Asp Met Met Leu Leu Asn Asn Thr Arg 145 150 155 160 Pro Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly 165 170 175 Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Val Gly 180 185 190 Asn Arg Thr Leu Ile Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu 195 200 205 Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys 210 215 220 Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Leu Asn 225 230 235 240 Phe Ser Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg 245 250 255 Leu Asn Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu Glu 260 265 270 Asp Arg Asp Arg Ser Glu 275 <210> 7 <211> 347 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 7 Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val 1 5 10 15 Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu 20 25 30 Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Glu 35 40 45 Thr Arg Gly Lys Leu Cys Pro Lys Cys Leu Asn Cys Thr Asp Leu Asp 50 55 60 Val Ala Leu Gly Arg Pro Lys Cys Thr Gly Lys Ile Pro Ser Ala Arg 65 70 75 80 Val Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro 85 90 95 Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg 100 105 110 Gly Tyr Glu His Ile Arg Leu Ser Thr His Asn Val Ile Asn Ala Glu 115 120 125 Asn Ala Pro Gly Gly Pro Tyr Lys Ile Gly Thr Ser Gly Ser Cys Pro 130 135 140 Asn Ile Thr Asn Gly Asn Gly Phe Phe Ala Thr Met Ala Trp Ala Val 145 150 155 160 Pro Lys Asn Asp Lys Asn Lys Thr Ala Thr Asn Pro Leu Thr Ile Glu 165 170 175 Val Pro Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly 180 185 190 Phe His Ser Asp Asn Glu Thr Gln Met Ala Lys Leu Tyr Gly Asp Ser 195 200 205 Lys Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr 210 215 220 Val Ser Gln Ile Gly Gly Phe Pro Asn Gln Thr Glu Asp Gly Gly Leu 225 230 235 240 Pro Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Ser Gly 245 250 255 Lys Thr Gly Thr Ile Thr Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys 260 265 270 Val Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro 275 280 285 Leu Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn 290 295 300 Lys Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn 305 310 315 320 Cys Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys 325 330 335 Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg 340 345 <210> 8 <211> 511 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 8 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 1 5 10 15 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 20 25 30 Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 35 40 45 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 50 55 60 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val 65 70 75 80 Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 85 90 95 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 100 105 110 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser 115 120 125 Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Thr Pro Ser Phe Phe 130 135 140 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile 145 150 155 160 Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Ile Leu Trp 165 170 175 Gly Ile His His Ser Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln 180 185 190 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg 195 200 205 Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 210 215 220 Arg Met Asp Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 225 230 235 240 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 245 250 255 Val Lys Lys Gly Asp Ser Ala Ile Val Lys Ser Glu Val Glu Tyr Gly 260 265 270 Asn Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser 275 280 285 Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 290 295 300 Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 305 310 315 320 Pro Leu Thr Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile 325 330 335 Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His 340 345 350 Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln 355 360 365 Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys 370 375 380 Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu 385 390 395 400 Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp 405 410 415 Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg 420 425 430 Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val 435 440 445 Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe 450 455 460 Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn 465 470 475 480 Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys Arg 485 490 495 Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gln 500 505 510 <210> 9 <211> 276 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 9 Ile Pro Leu Gly Val Val His Asn Asn Thr Leu Gln Val Ser Asp Ile 1 5 10 15 Asp Lys Leu Val Cys Arg Asp Lys Leu Ser Ser Thr Ser Gln Leu Lys 20 25 30 Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val Pro 35 40 45 Thr Ala Thr Lys Arg Trp Gly Phe Arg Ala Gly Val Pro Pro Lys Val 50 55 60 Val Asn Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Asp 65 70 75 80 Ile Lys Lys Ala Asp Gly Ser Glu Cys Leu Pro Glu Ala Pro Glu Gly 85 90 95 Val Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser Gly Thr 100 105 110 Gly Pro Cys Pro Glu Gly Tyr Ala Phe His Lys Glu Gly Ala Phe Phe 115 120 125 Leu Tyr Asp Arg Leu Ala Ser Thr Ile Ile Tyr Arg Ser Thr Thr Phe 130 135 140 Ser Glu Gly Val Val Ala Phe Leu Ile Leu Pro Glu Thr Lys Lys Asp 145 150 155 160 Phe Phe Gln Ser Pro Pro Leu His Glu Pro Ala Asn Met Thr Thr Asp 165 170 175 Pro Ser Ser Tyr Tyr His Thr Val Thr Leu Asn Tyr Val Ala Asp Asn 180 185 190 Phe Gly Thr Asn Met Thr Asn Phe Leu Phe Gln Val Asp His Leu Thr 195 200 205 Tyr Val Gln Leu Glu Pro Arg Phe Thr Pro Gln Phe Leu Val Gln Leu 210 215 220 Asn Glu Thr Ile Tyr Thr Asn Gly Arg Arg Ser Asn Thr Thr Gly Thr 225 230 235 240 Leu Ile Trp Lys Val Asn Pro Thr Val Asp Thr Gly Val Gly Glu Trp 245 250 255 Ala Phe Trp Glu Asn Lys Lys Asn Phe Thr Lys Thr Leu Ser Ser Glu 260 265 270 Glu Leu Ser Val 275 <210> 10 <211> 469 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 10 Met Pro Leu Gly Val Val Thr Asn Ser Thr Leu Glu Val Thr Glu Ile 1 5 10 15 Asp Gln Leu Val Cys Lys Asp His Leu Ala Ser Thr Asp Gln Leu Lys 20 25 30 Ser Val Gly Leu Asn Leu Glu Gly Ser Gly Val Ser Thr Asp Ile Pro 35 40 45 Ser Ala Thr Lys Arg Trp Gly Phe Arg Ser Gly Val Pro Pro Lys Val 50 55 60 Val Ser Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Glu 65 70 75 80 Ile Lys Lys Pro Asp Gly Ser Glu Cys Leu Pro Pro Pro Pro Asp Gly 85 90 95 Val Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys Ala Gln Gly Thr 100 105 110 Gly Pro Cys Pro Gly Asp Tyr Ala Phe His Lys Asp Gly Ala Phe Phe 115 120 125 Leu Tyr Asp Arg Leu Ala Ser Thr Val Ile Tyr Arg Gly Val Asn Phe 130 135 140 Ala Glu Gly Val Ile Ala Phe Leu Ile Leu Ala Lys Pro Lys Glu Thr 145 150 155 160 Phe Leu Gln Ser Pro Pro Ile Arg Glu Ala Val Asn Tyr Thr Glu Asn 165 170 175 Thr Ser Ser Tyr Tyr Ala Thr Ser Tyr Leu Glu Tyr Glu Ile Glu Asn 180 185 190 Phe Gly Ala Gln His Ser Thr Thr Leu Phe Lys Ile Asp Asn Asn Thr 195 200 205 Phe Val Arg Leu Asp Arg Pro His Thr Pro Gln Phe Leu Phe Gln Leu 210 215 220 Asn Asp Thr Ile His Leu His Gln Gln Leu Ser Asn Thr Thr Gly Arg 225 230 235 240 Leu Ile Trp Thr Leu Asp Ala Asn Ile Asn Ala Asp Ile Gly Glu Trp 245 250 255 Ala Phe Trp Glu Asn Lys Lys Asn Leu Ser Glu Gln Leu Arg Gly Glu 260 265 270 Glu Leu Ser Phe Glu Ala Leu Ser Leu Asn Glu Thr Glu Asp Asp Asp 275 280 285 Ala Ala Ser Ser Arg Ile Thr Lys Gly Arg Ile Ser Asp Arg Ala Thr 290 295 300 Arg Lys Tyr Ser Asp Leu Val Pro Lys Asn Ser Pro Gly Met Val Pro 305 310 315 320 Leu His Ile Pro Glu Gly Glu Thr Thr Leu Pro Ser Gln Asn Ser Thr 325 330 335 Glu Gly Arg Arg Val Gly Val Asn Thr Gln Glu Thr Ile Thr Glu Thr 340 345 350 Ala Ala Thr Ile Ile Gly Thr Asn Gly Asn His Met Gln Ile Ser Thr 355 360 365 Ile Gly Ile Arg Pro Ser Ser Ser Gln Ile Pro Ser Ser Ser Pro Thr 370 375 380 Thr Ala Pro Ser Pro Glu Ala Gln Thr Pro Thr Thr His Thr Ser Gly 385 390 395 400 Pro Ser Val Met Ala Thr Glu Glu Pro Thr Thr Pro Pro Gly Ser Ser 405 410 415 Pro Gly Pro Thr Thr Glu Ala Pro Thr Leu Thr Thr Pro Glu Asn Ile 420 425 430 Thr Thr Ala Val Lys Thr Val Leu Pro Gln Glu Ser Thr Ser Asn Gly 435 440 445 Leu Ile Thr Ser Thr Val Thr Gly Ile Leu Gly Ser Leu Gly Leu Arg 450 455 460 Lys Arg Ser Arg Arg 465 <210> 11 <211> 107 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 11 Val Ser Tyr Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro 1 5 10 15 Ala Glu Thr Leu His Gly Thr Val Thr Val Glu Val Gln Tyr Ala Gly 20 25 30 Thr Asp Gly Pro Cys Lys Val Pro Ala Gln Met Ala Val Asp Met Gln 35 40 45 Thr Leu Thr Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val Ile Thr 50 55 60 Glu Ser Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe 65 70 75 80 Gly Asp Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His 85 90 95 His Trp His Arg Ser Gly Ser Thr Ile Gly Lys 100 105 <210> 12 <211> 243 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 12 Asn His Lys Val Thr Ser Thr Thr Thr Ile Ile Gln Asp Ala Thr Ser 1 5 10 15 Gln Ile Lys Asn Thr Thr Pro Thr Tyr Leu Thr Gln Ser Pro Gln Leu 20 25 30 Gly Ile Ser Pro Ser Asn Pro Ser Glu Ile Thr Ser Gln Ile Thr Thr 35 40 45 Ile Leu Ala Ser Thr Thr Pro Gly Val Lys Ser Thr Leu Gln Ser Thr 50 55 60 Thr Val Gly Thr Lys Asn Thr Thr Thr Thr Gln Ala Gln Pro Ser Lys 65 70 75 80 Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro Pro Ser Lys Pro Asn Asn 85 90 95 Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys Ser Ile Cys Ser 100 105 110 Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro Asn Lys Lys 115 120 125 Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Glu Glu Pro Thr Phe Lys 130 135 140 Thr Ala Lys Glu Asp Pro Lys Pro Gln Thr Thr Gly Ser Gly Glu Val 145 150 155 160 Pro Thr Thr Lys Pro Thr Gly Glu Pro Thr Ile Asn Thr Thr Lys Thr 165 170 175 Asn Ile Thr Thr Thr Leu Leu Thr Ser Asn Thr Thr Arg Asn Pro Glu 180 185 190 Leu Thr Ser Gln Met Glu Thr Phe His Ser Thr Ser Ser Glu Gly Asn 195 200 205 Pro Ser Pro Ser Gln Val Ser Ile Thr Ser Glu Tyr Leu Ser Gln Pro 210 215 220 Ser Ser Pro Pro Asn Thr Pro Arg Ala His His His His His His His 225 230 235 240 His His His <210> 13 <211> 168 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 13 His His His His His His His His His His Ala Leu Glu Val Leu Phe 1 5 10 15 Gln Gly Pro Tyr Glu Val Arg Asn Val Ser Gly Ile Tyr His Val Thr 20 25 30 Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Val Ile 35 40 45 Met His Thr Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ser Ser 50 55 60 Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser 65 70 75 80 Val Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Thr 85 90 95 Ala Ala Phe Cys Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Ile 100 105 110 Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg His Glu Thr 115 120 125 Val Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His 130 135 140 Arg Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu 145 150 155 160 Val Val Ser Gln Leu Leu Arg Ile 165 <210> 14 <211> 289 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 14 Glu Thr His Thr Thr Gly Arg Val Ala Gly His Thr Thr Ser Gly Phe 1 5 10 15 Thr Ser Leu Phe Ser Ser Gly Ala Ser Gln Lys Ile Gln Leu Val Asn 20 25 30 Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp 35 40 45 Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe Tyr Ala His Lys Phe 50 55 60 Asn Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Ile Asp 65 70 75 80 Trp Phe Ala Gln Gly Trp Gly Pro Ile Thr Tyr Thr Lys Pro Asn Ser 85 90 95 Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly 100 105 110 Val Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro 115 120 125 Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Val Pro Thr Tyr 130 135 140 Ser Trp Gly Glu Asn Glu Thr Asp Met Met Leu Leu Asn Asn Thr Arg 145 150 155 160 Pro Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly 165 170 175 Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Val Gly 180 185 190 Asn Arg Thr Leu Ile Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu 195 200 205 Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys 210 215 220 Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Leu Asn 225 230 235 240 Phe Ser Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg 245 250 255 Leu Asn Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu Glu 260 265 270 Asp Arg Asp Arg Ser Glu Ala His His His His His His His His His 275 280 285 His <210> 15 <211> 358 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 15 Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val 1 5 10 15 Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu 20 25 30 Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Glu 35 40 45 Thr Arg Gly Lys Leu Cys Pro Lys Cys Leu Asn Cys Thr Asp Leu Asp 50 55 60 Val Ala Leu Gly Arg Pro Lys Cys Thr Gly Lys Ile Pro Ser Ala Arg 65 70 75 80 Val Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro 85 90 95 Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg 100 105 110 Gly Tyr Glu His Ile Arg Leu Ser Thr His Asn Val Ile Asn Ala Glu 115 120 125 Asn Ala Pro Gly Gly Pro Tyr Lys Ile Gly Thr Ser Gly Ser Cys Pro 130 135 140 Asn Ile Thr Asn Gly Asn Gly Phe Phe Ala Thr Met Ala Trp Ala Val 145 150 155 160 Pro Lys Asn Asp Lys Asn Lys Thr Ala Thr Asn Pro Leu Thr Ile Glu 165 170 175 Val Pro Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly 180 185 190 Phe His Ser Asp Asn Glu Thr Gln Met Ala Lys Leu Tyr Gly Asp Ser 195 200 205 Lys Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr 210 215 220 Val Ser Gln Ile Gly Gly Phe Pro Asn Gln Thr Glu Asp Gly Gly Leu 225 230 235 240 Pro Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Ser Gly 245 250 255 Lys Thr Gly Thr Ile Thr Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys 260 265 270 Val Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro 275 280 285 Leu Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn 290 295 300 Lys Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn 305 310 315 320 Cys Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys 325 330 335 Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg Ala His His His His 340 345 350 His His His His His His 355 <210> 16 <211> 522 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 16 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 1 5 10 15 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 20 25 30 Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 35 40 45 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 50 55 60 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val 65 70 75 80 Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 85 90 95 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 100 105 110 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser 115 120 125 Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Thr Pro Ser Phe Phe 130 135 140 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile 145 150 155 160 Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Ile Leu Trp 165 170 175 Gly Ile His His Ser Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln 180 185 190 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg 195 200 205 Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 210 215 220 Arg Met Asp Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 225 230 235 240 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 245 250 255 Val Lys Lys Gly Asp Ser Ala Ile Val Lys Ser Glu Val Glu Tyr Gly 260 265 270 Asn Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser 275 280 285 Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 290 295 300 Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 305 310 315 320 Pro Leu Thr Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile 325 330 335 Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His 340 345 350 Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln 355 360 365 Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys 370 375 380 Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu 385 390 395 400 Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp 405 410 415 Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg 420 425 430 Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val 435 440 445 Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe 450 455 460 Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn 465 470 475 480 Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys Arg 485 490 495 Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr Tyr Gln Ala 500 505 510 His His His His His His His His His His 515 520 <210> 17 <211> 514 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 17 Ile Pro Leu Gly Val Val His Asn Asn Thr Leu Gln Val Ser Asp Ile 1 5 10 15 Asp Lys Leu Val Cys Arg Asp Lys Leu Ser Ser Thr Ser Gln Leu Lys 20 25 30 Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val Pro 35 40 45 Thr Ala Thr Lys Arg Trp Gly Phe Arg Ala Gly Val Pro Pro Lys Val 50 55 60 Val Asn Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Asp 65 70 75 80 Ile Lys Lys Ala Asp Gly Ser Glu Cys Leu Pro Glu Ala Pro Glu Gly 85 90 95 Val Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser Gly Thr 100 105 110 Gly Pro Cys Pro Glu Gly Tyr Ala Phe His Lys Glu Gly Ala Phe Phe 115 120 125 Leu Tyr Asp Arg Leu Ala Ser Thr Ile Ile Tyr Arg Ser Thr Thr Phe 130 135 140 Ser Glu Gly Val Val Ala Phe Leu Ile Leu Pro Glu Thr Lys Lys Asp 145 150 155 160 Phe Phe Gln Ser Pro Pro Leu His Glu Pro Ala Asn Met Thr Thr Asp 165 170 175 Pro Ser Ser Tyr Tyr His Thr Val Thr Leu Asn Tyr Val Ala Asp Asn 180 185 190 Phe Gly Thr Asn Met Thr Asn Phe Leu Phe Gln Val Asp His Leu Thr 195 200 205 Tyr Val Gln Leu Glu Pro Arg Phe Thr Pro Gln Phe Leu Val Gln Leu 210 215 220 Asn Glu Thr Ile Tyr Thr Asn Gly Arg Arg Ser Asn Thr Thr Gly Thr 225 230 235 240 Leu Ile Trp Lys Val Asn Pro Thr Val Asp Thr Gly Val Gly Glu Trp 245 250 255 Ala Phe Trp Glu Asn Lys Lys Asn Phe Thr Lys Thr Leu Ser Ser Glu 260 265 270 Glu Leu Ser Val Ala Asp Asp Asp Asp Lys Glu Pro Lys Ser Ser Asp 275 280 285 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 290 295 300 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 305 310 315 320 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 325 330 335 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 340 345 350 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 355 360 365 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 370 375 380 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 385 390 395 400 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 405 410 415 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 420 425 430 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 435 440 445 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 450 455 460 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 465 470 475 480 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 485 490 495 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 500 505 510 Gly Lys <210> 18 <211> 480 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 18 Met Pro Leu Gly Val Val Thr Asn Ser Thr Leu Glu Val Thr Glu Ile 1 5 10 15 Asp Gln Leu Val Cys Lys Asp His Leu Ala Ser Thr Asp Gln Leu Lys 20 25 30 Ser Val Gly Leu Asn Leu Glu Gly Ser Gly Val Ser Thr Asp Ile Pro 35 40 45 Ser Ala Thr Lys Arg Trp Gly Phe Arg Ser Gly Val Pro Pro Lys Val 50 55 60 Val Ser Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Glu 65 70 75 80 Ile Lys Lys Pro Asp Gly Ser Glu Cys Leu Pro Pro Pro Pro Asp Gly 85 90 95 Val Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys Ala Gln Gly Thr 100 105 110 Gly Pro Cys Pro Gly Asp Tyr Ala Phe His Lys Asp Gly Ala Phe Phe 115 120 125 Leu Tyr Asp Arg Leu Ala Ser Thr Val Ile Tyr Arg Gly Val Asn Phe 130 135 140 Ala Glu Gly Val Ile Ala Phe Leu Ile Leu Ala Lys Pro Lys Glu Thr 145 150 155 160 Phe Leu Gln Ser Pro Pro Ile Arg Glu Ala Val Asn Tyr Thr Glu Asn 165 170 175 Thr Ser Ser Tyr Tyr Ala Thr Ser Tyr Leu Glu Tyr Glu Ile Glu Asn 180 185 190 Phe Gly Ala Gln His Ser Thr Thr Leu Phe Lys Ile Asp Asn Asn Thr 195 200 205 Phe Val Arg Leu Asp Arg Pro His Thr Pro Gln Phe Leu Phe Gln Leu 210 215 220 Asn Asp Thr Ile His Leu His Gln Gln Leu Ser Asn Thr Thr Gly Arg 225 230 235 240 Leu Ile Trp Thr Leu Asp Ala Asn Ile Asn Ala Asp Ile Gly Glu Trp 245 250 255 Ala Phe Trp Glu Asn Lys Lys Asn Leu Ser Glu Gln Leu Arg Gly Glu 260 265 270 Glu Leu Ser Phe Glu Ala Leu Ser Leu Asn Glu Thr Glu Asp Asp Asp 275 280 285 Ala Ala Ser Ser Arg Ile Thr Lys Gly Arg Ile Ser Asp Arg Ala Thr 290 295 300 Arg Lys Tyr Ser Asp Leu Val Pro Lys Asn Ser Pro Gly Met Val Pro 305 310 315 320 Leu His Ile Pro Glu Gly Glu Thr Thr Leu Pro Ser Gln Asn Ser Thr 325 330 335 Glu Gly Arg Arg Val Gly Val Asn Thr Gln Glu Thr Ile Thr Glu Thr 340 345 350 Ala Ala Thr Ile Ile Gly Thr Asn Gly Asn His Met Gln Ile Ser Thr 355 360 365 Ile Gly Ile Arg Pro Ser Ser Ser Gln Ile Pro Ser Ser Ser Pro Thr 370 375 380 Thr Ala Pro Ser Pro Glu Ala Gln Thr Pro Thr Thr His Thr Ser Gly 385 390 395 400 Pro Ser Val Met Ala Thr Glu Glu Pro Thr Thr Pro Pro Gly Ser Ser 405 410 415 Pro Gly Pro Thr Thr Glu Ala Pro Thr Leu Thr Thr Pro Glu Asn Ile 420 425 430 Thr Thr Ala Val Lys Thr Val Leu Pro Gln Glu Ser Thr Ser Asn Gly 435 440 445 Leu Ile Thr Ser Thr Val Thr Gly Ile Leu Gly Ser Leu Gly Leu Arg 450 455 460 Lys Arg Ser Arg Arg Ala His His His His His His His His His His 465 470 475 480 <210> 19 <211> 345 <212> PRT <213> Artificial <220> <223> The sequence is artificially synthesized. <400> 19 Val Ser Tyr Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro 1 5 10 15 Ala Glu Thr Leu His Gly Thr Val Thr Val Glu Val Gln Tyr Ala Gly 20 25 30 Thr Asp Gly Pro Cys Lys Val Pro Ala Gln Met Ala Val Asp Met Gln 35 40 45 Thr Leu Thr Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val Ile Thr 50 55 60 Glu Ser Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe 65 70 75 80 Gly Asp Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His 85 90 95 His Trp His Arg Ser Gly Ser Thr Ile Gly Lys Ala Asp Asp Asp Asp 100 105 110 Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 115 120 125 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 130 135 140 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 145 150 155 160 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 165 170 175 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 180 185 190 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 195 200 205 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 210 215 220 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 225 230 235 240 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 245 250 255 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 260 265 270 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 275 280 285 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 290 295 300 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 305 310 315 320 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 325 330 335 Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345

Claims

1. A method for enhancing the immunogenicity of a protein / peptide antigen, the method comprising conjugating a protein / peptide antigen with a sugar to form a sugar-protein / peptide antigen conjugate, wherein the sugar is Streptococcus pneumoniae capsular polysaccharide; wherein the protein / peptide antigen is selected from: SARS-CoV-2 RBD-mFc, wherein the Streptococcus pneumoniae capsular polysaccharide is selected from: Streptococcus pneumoniae serotype 14 capsular polysaccharide; or wherein the protein / peptide antigen is selected from: SARS-CoV-2 RBD-his, wherein the Streptococcus pneumoniae capsular polysaccharide is selected from: Streptococcus pneumoniae serotype 14 capsular polysaccharide or Streptococcus pneumoniae serotype 7F capsular polysaccharide; or wherein the protein / peptide antigen is selected from: MERS-COV RBD-his, wherein the Streptococcus pneumoniae capsular polysaccharide is selected from: Streptococcus pneumoniae serotype 14 capsular polysaccharide; or wherein the protein / peptide antigen is selected from: RSV-gpG-his, wherein the Streptococcus pneumoniae capsular polysaccharide is selected from: Streptococcus pneumoniae serotype 14 capsular polysaccharide; or wherein the protein / peptide antigen is selected from: Flu-B-HA1-his, wherein the Streptococcus pneumoniae capsular polysaccharide is selected from: Streptococcus pneumoniae serotype 14 capsular polysaccharide; or wherein the protein / peptide antigen is selected from: H5N1-HA-his, wherein the Streptococcus pneumoniae capsular polysaccharide is selected from: Streptococcus pneumoniae serotype 14 capsular polysaccharide; or wherein the protein / peptide antigen is selected from: Ebola-GP-Fc, wherein the Streptococcus pneumoniae capsular polysaccharide is selected from: Streptococcus pneumoniae serotype 14 capsular polysaccharide; or wherein the protein / peptide antigen is selected from: Ebola-GP1-his, wherein the Streptococcus pneumoniae capsular polysaccharide is selected from: Streptococcus pneumoniae serotype 14 capsular polysaccharide; or wherein the protein / peptide antigen is selected from: ZIKV-E-Fc, wherein the Streptococcus pneumoniae capsular polysaccharide is selected from: Streptococcus pneumoniae serotype 14 capsular polysaccharide; or wherein the protein / peptide antigen is selected from: HCV-E2-his, wherein the Streptococcus pneumoniae capsular polysaccharide is selected from: Streptococcus pneumoniae serotype 14 capsular polysaccharide; or wherein the protein / peptide antigen is selected from: HCV-E1-his; wherein the Streptococcus pneumoniae capsular polysaccharide is selected from: Streptococcus pneumoniae serotype 14 capsular polysaccharide.

2. The method according to claim 1, wherein the protein / peptide antigen comprises a sequence shown in any one of SEQ ID NO: 1 - SEQ ID NO:

19.

3. The method according to claim 1 or 2, wherein the molecular weight of the conjugate is 400 - 14000 KDa.

4. The method according to claim 1 or 2, wherein the sugar-protein / peptide antigen is further conjugated with a protein carrier, and the protein carrier is CRM197.

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