Method for improving the immunogenicity of protein / peptide antigens

Abstract

A method for improving the immunogenicity of a protein / peptide antigen is disclosed, comprising the step of conjugating the protein / peptide antigen with a sugar to form a glycoprotein / peptide antigen conjugate having improved immunogenicity compared to an unconjugated protein / peptide antigen. In particular, the method comprises the step of conjugating a pathogen, e.g., a viral surface protein antigen or a fragment thereof, with a polysaccharide, particularly a capsule-type polysaccharide of Streptococcus pneumoniae. The immunogenic conjugate may be used to prevent or treat diseases caused by pathogens, particularly diseases caused by coronaviruses.

Description

Technology Field

[0001] Citation of related applications

[0002] This application claims the benefit of Chinese patent application 202010369100.7 filed on May 1, 2020. The full text of the aforementioned application is incorporated herein by reference.

[0003] Technology field

[0004] The present invention relates to the field of immunogenic compositions, and more specifically, to a method for improving the immunogenicity of a protein / peptide antigen by conjugating the protein / peptide antigen with a saccharide to produce a glycoprotein / peptide antigen conjugate having increased immunogenicity compared to an unconjugated protein / peptide antigen. More specifically, the present invention relates to a method comprising a pathogen, such as a viral surface protein antigen or a fragment thereof, and a saccharide, particularly Streptococcus pneumoniae It involves conjugation with a capsular polysaccharide. The immunogenic conjugate can be used to prevent or treat diseases caused by pathogens, particularly diseases caused by coronaviruses. Background Technology

[0005] When a vertebrate individual is immunized with a vaccine in which an infectious microorganism, toxin, virus, or a subunit thereof is used as an antigen component, the aforementioned antigen component, which is an exogenous substance to the individual, will induce or stimulate a memory immune response to the exogenous molecule in the individual, thereby protecting the individual from damage caused by a secondary immune response when the individual is exposed to the exogenous molecule again.

[0006] The term "antigen" refers to an exogenous substance recognized (specifically bound) by antibodies or T-cell receptors, but it does not necessarily elicit an immune response. Exogenous substances that can be recognized (specifically bound) by antibodies or T-cell receptors and thus induce specific immunity are called "immunogenic antigens" or "immunogens."

[0007] Vaccines that use infectious microorganisms, toxins, or viral subunits—that is, cellular structures (bacteria or fungi) or parts of viruses—as antigens are inanimate vaccines and are widely used due to their safety. However, the ability of these subunits to induce a specific immune response is weak; in other words, the antigens have low immunogenicity.

[0008] The traditional method for enhancing immunogenicity is the addition of adjuvants. New methods for enhancing the immune response are continuously being investigated in ongoing research. One important tool is exogenous carrier macromolecules that have been successfully used for decades for low-immunogenic antigens, such as commonly seen encephalitis vaccines, Type B Haemophilus influenzae b The immunogenicity of the aforementioned low-immunogenic antigen is enhanced by conjugating it with a vaccine and a pneumonia vaccine; in this case, to prepare a more effective immunogenic composition, a purified encapsulated polysaccharide (encapsulated polymer) was mixed with a carrier protein (Schneerson et al. (1984) Infect. Immun. 45: 582-591). Commonly used carrier proteins include, for example, tetanus toxoid, tetanus toxoid fragment C, tetanus toxoid nonvirulent mutants, diphtheria toxoid, CRM197, and other nonvirulent mutants of diphtheria toxoid [e.g., CRM176, CRM197, CRM228, CRM45 (Uchida et al. Human J. Biol. ChemExamples include [218; 3838-3844, 1973); CRM9, CRM45, CRM02, CRM103, CRM107, and other mutants]. Since these polysaccharide antigens are thymocyte-independent antigens that do not induce a cellular immune response, they do not generate immune memory and cannot form protective antibodies in children or immunocompromised individuals. After the polysaccharide antigen is conjugated to a protein carrier containing a T cell epitope, the conjugate is endocytosed and processed by antigen-presenting cells or B cells, and a peptide fragment of the carrier protein appears on the surface of the corresponding cell, activating helper T cells and inducing multiple immune responses to generate protective antibodies and create immune memory.

[0009] However, the effect of bacterial polysaccharides on the immunogenicity of protein / peptide antigens has rarely been reported. US5192540A discloses a vaccine comprising an immunogenic conjugate of an oxidized polyribose-ribitol-phosphate polysaccharide fragment of Haemophilus influenzae type B, comprising a 38,000-dalton or 40,000-dalton outer membrane protein of Haemophilus influenzae type B, which can be used for immunization against diseases caused by Haemophilus influenzae type B. However, "the conjugate vaccine of the present invention exhibits very high immunogenicity in animal models. Their antibody response to PRP was much greater than previously reported. Additionally, the conjugate vaccine also induces antibodies against the major protein (38k or 40k protein) of Haemophilus influenzae type B."

[0010] US 9296795B discloses the use of an immunogenic polysaccharide-protein conjugate having a polysaccharide antigen derived from a pathogenic agent (or its oligosaccharide fragments expressing one or more antigenic epitopes) in an immunogenic composition, wherein the polysaccharide is conjugated to a Staphylococcus surface-attached carrier protein to induce an antibody response to the polysaccharide antigen and the Staphylococcus surface-attached carrier protein. "The conjugate described in the present invention has the unique advantage of inducing the production of antibodies against the polysaccharide antigen and the surface-attached vector protein (both virulence factors) and conferring immunity against diseases caused by pathogenic agents." In other words, the surface-attached protein itself can confer immunity to the body rather than merely acting as a protein carrier for the polysaccharide antigens. The titers of surface adhesion protein-specific antibodies induced by the stapled surface adhesion protein were similar to those of the unconjugated surface adhesion protein (Figs. 17-20). This confirms that the antigenic epitope is not modified by the binding of the surface adhesion protein to CP.

[0011] In the two aforementioned studies, there were only reports that protein / peptide antigens conjugated to polysaccharides could induce antibody production, but no enhancement of their immunogenicity was reported.

[0012] The inventors' pioneering discovery includes enhancing the immunogenicity of a protein / peptide antigen by conjugating the protein / peptide antigen with a sugar to form a glycoprotein / peptide antigen conjugate.

[0013] The inventors hypothesize that the principle is that protein aggregates stimulate the body's immune response and generate antibodies more readily than protein monomers. Furthermore, most pattern recognition receptors on the surface of antigen-presenting cells in the animal immune system are associated with sugars, and sugars produced by bacteria are important signals that stimulate the immune system. However, the present invention is not bound by this theory.

[0014] One aspect of the present invention relates to a method for improving the immunogenicity of a protein / peptide antigen, comprising the step of conjugating the protein / peptide antigen with a sugar to form a glycoprotein / peptide antigen conjugate.

[0015] In a specific embodiment of the present invention, the sugars are selected from polysaccharides, oligosaccharides, or monosaccharides;

[0016] Preferably, Neisseria encephalitis encapsulated polysaccharides, Type B Haemophilus influenzae encapsulated polysaccharides, s Treptococcus pneumoniae Capsule-type polysaccharides of, Group B Staphilococcus aureus ( Staphylococcus aureus Selected from encapsulated polysaccharides, dextran, mannan, starch, inulin, pectin, carboxymethyl starch, chitosan, and derivatives thereof;

[0017] More preferably, Streptococcus pneumoniae It is a capsule-type polysaccharide of;

[0018] Most preferably, Streptococcus pneumoniae Capsular polysaccharides of serotype 14, Streptococcus pneumoniae Capsule-type polysaccharides of serotype 6B and Streptococcus pneumoniae Selected from the capsule-type polysaccharides of serotype 7F, and

[0019] The above protein / peptide antigen is selected from pathogen-related protein / peptide antigens or tumor-related protein / peptide antigens, and

[0020] Herein, the pathogen is a coronavirus, human immunodeficiency virus HIV-1, human herpes simplex virus, cytomegalovirus, rotavirus, EB virus, herpes zoster virus, hepatitis virus, respiratory syncytial virus, parainfluenza virus, measles virus, mumps virus, human papillomavirus, flavivirus, or influenza virus, Neisseria , Moraxella , Bordetella; Mycobacterium tuberculosis including Mycobacterium; Enterotoxicity Escherichia coli including Esherikia ; Salmonella , Listeria , Helicobacter; Staphilococcus aureus, Staphilococcus epidermidis including Staphylococcus ; Borrelia; Chlamydia trachomatis , Chlamydia pneumoniae including Chlamydia ; Plasmodium falciparum including Plasmodium ; Toxoplasma , Candida Selected from;

[0021] Preferably, it is a protein / peptide associated with pathogen invasion into a host;

[0022] More preferably, the pathogen is a virus;

[0023] More preferably, the virus is Coronavirus family , Paramyxoviridae , Orthomyxoviridae , Filovirus family or Flaviviridae Selected from the virus,

[0024] The above tumors are selected from diffuse large B-cell lymphoma, follicular lymphoma, other lymphomas, leukemia, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdomyomas, hepatocellular carcinoma, prostate cancer, breast cancer, cholangiocarcinoma and gallbladder cancer, bladder cancer; brain tumors including neuroblastoma, schwann tumor, 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.

[0025] In a specific embodiment of the present invention, the protein / peptide antigen is a protein / peptide comprising the following:

[0026] Coronavirus family viral antigens;

[0027] Preferably, coronavirus spike protein;

[0028] More preferably, coronavirus spike protein S1 subunit;

[0029] More preferably, the coronavirus spike protein receptor binding region RBD;

[0030] Or a fragment or variant having immunogenicity for all of the above protein / peptide antigens.

[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 an antigen as previously defined and another protein or peptide.

[0033] Preferably, the fusion protein is 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 rat IgG Fc fragment.

[0037] In a specific embodiment of the present invention, the protein / peptide antigen comprises a sequence described as any one 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 glycoprotein / peptide antigen conjugate is 400-14000 KDa.

[0039] In a specific embodiment of the present invention, the protein / peptide antigen is a protein / peptide comprising the following:

[0040] Paramyxoviridae viral antigens;

[0041] Preferably, a paramyxovirus glycoprotein receptor binding region;

[0042] Preferably paramyxovirus glycoprotein F, glycoprotein G;

[0043] Or a fragment or variant having immunogenicity for all of the above protein / peptide antigens.

[0044] In a specific embodiment of the present invention, Paramyxoviridae The virus is the human respiratory syncytial virus.

[0045] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of an antigen as previously defined and another protein or peptide; preferably, the fusion protein is RSV-gpG-his.

[0046] 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.

[0047] In a specific embodiment of the present invention, the protein / peptide antigen is a protein / peptide comprising the following:

[0048] Orthomyxoviridae viral antigens;

[0049] Preferably, an orthomyxovirus glycoprotein receptor binding region;

[0050] Preferably, hemagglutinin (HA) protein and / or neuraminidase (NA) protein;

[0051] Or a fragment or variant having immunogenicity for all of the above protein / peptide antigens.

[0052] In a specific embodiment of the present invention, the orthomyxovirus is a type B influenza virus and / or a type A influenza H5N1 virus.

[0053] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of an antigen as previously defined and another protein or peptide, and

[0054] Preferably, the fusion protein is Flu-B-HA1-his or H5N1-HA-his.

[0055] In a specific embodiment of the present invention, the protein / peptide antigen comprises a sequence described as any one of SEQ ID NO. 7 and / or SEQ ID NO. 15, SEQ ID NO. 8 and / or SEQ ID NO. 16.

[0056] In a specific embodiment of the present invention, the protein / peptide antigen is a protein / peptide comprising the following:

[0057] Filovirus family viral antigens ;

[0058] Preferably, a filovirus envelope glycoprotein receptor binding region;

[0059] Preferably, filovirus envelope glycoproteins GP1 and / or GP2;

[0060] More preferably, filovirus envelope glycoprotein GP1;

[0061] Or a fragment or variant having immunogenicity for all of the above protein / peptide antigens.

[0062] In a specific embodiment of the present invention, the filovirus is the Ebola virus.

[0063] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of an antigen as previously defined and another protein or peptide.

[0064] Preferably, the fusion protein is Ebola-GP-Fc or Ebola-GP1-his;

[0065] Here, Fc is preferably an IgG Fc fragment, more preferably a human or rat IgG Fc fragment.

[0066] In a specific embodiment of the present invention, the protein / peptide antigen comprises a sequence described as any one of SEQ ID NO. 9 and / or SEQ ID NO. 17, SEQ ID NO. 10 and / or SEQ ID NO. 18.

[0067] In a specific embodiment of the present invention, the protein / peptide antigen is a protein / peptide comprising the following:

[0068] Flaviviridae viral antigens;

[0069] Preferably, Flavivirus or Hepacivirus viral antigens;

[0070] Preferably, Flavivirus The outer protein receptor binding region;

[0071] Preferably, Flavivirus At least one of the structural domains EDI, EDII, and EDIII of the coat protein;

[0072] More preferably, Flavivirus Structural domain EDIII of the coat protein; or

[0073] Preferably, Hepasivirus The outer glycoprotein receptor binding region;

[0074] Preferably, Hepasivirus Coat glycoprotein E1 and / or E2;

[0075] Or a fragment or variant having immunogenicity for all of the above protein / peptide antigens.

[0076] In a specific embodiment of the present invention, Flavivirus The virus is preferably the Zika virus; Hepasivirus The virus is preferably the hepatitis C virus.

[0077] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of an antigen as previously defined and another protein or peptide, and

[0078] Preferably, the fusion protein is ZIKV-E-Fc; where

[0079] Fc is preferably an IgG Fc fragment, more preferably a human or rat IgG Fc fragment; or

[0080] Preferably, the fusion protein is HCV-E2-his and / or HCV-E1-his.

[0081] In a specific embodiment of the present invention, the protein / peptide antigen comprises a sequence described as 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.

[0082] In a specific embodiment of the present invention, a glycoprotein / peptide antigen is further conjugated to a protein carrier.

[0083] In a specific embodiment of the present invention, the protein carrier is tetanus toxoid, tetanus toxoid fragment C, tetanus toxoid non-toxic mutant, diphtheria toxoid, CRM197, and other non-toxic mutants of diphtheria toxoid, preferably CRM197.

[0084] A second aspect of the present invention relates to a glycoprotein / peptide antigen conjugate having increased immunogenicity compared to an unconjugated protein / peptide antigen.

[0085] In a specific embodiment of the present invention, the sugars are selected from polysaccharides, oligosaccharides, or monosaccharides;

[0086] Preferably, Neisseria encephalates encapsulated polysaccharides, Type B Haemophilus influenzae encapsulated polysaccharides, s Treptococcus pneumoniae Capsule-type polysaccharides of, Group B Staphilococcus aureus Selected from encapsulated polysaccharides, dextran, mannan, starch, inulin, pectin, carboxymethyl starch, chitosan, and derivatives thereof;

[0087] More preferably, Streptococcus pneumoniae It is a capsule-type polysaccharide of;

[0088] Most preferably, Streptococcus pneumoniae Capsular polysaccharides of serotype 14, Streptococcus pneumoniae Capsule-type polysaccharides of serotype 6B and Streptococcus pneumoniae Selected from the encapsulated polysaccharides of serotype 7F;

[0089] The above protein / peptide antigen is selected from pathogen-related protein / peptide antigens or tumor-related protein / peptide antigens, and

[0090] Here, the pathogen is a coronavirus, human immunodeficiency virus HIV-1, human herpes simplex virus, cytomegalovirus, rotavirus, EB virus, herpes zoster virus, hepatitis virus, respiratory syncytial virus, parainfluenza virus, measles virus, mumps virus, human papillomavirus, flavivirus, or influenza virus, Neisseria , Moraxella , Bordetella; Mycobacterium tuberculosis including Mycobacterium; Enterotoxicity Esherikia Kolai including Esherikia ; Salmonella , Listeria , Helicobacter; Staphilococcus aureus, Staphilococcus epidermidis including Staphylococcus ; Borrelia; Chlamydia trachomatis , Chlamydia pneumoniae including Chlamydia ; Plasmodium falsipalum including Plasmodium ; Toxoplasma , Candida Selected from;

[0091] Preferably, it is a protein / peptide associated with the invasion of a host pathogen;

[0092] More preferably, the pathogen is a virus;

[0093] More preferably, the virus is Coronavirus family , Paramyxoviridae , Orthomyxoviridae , Filovirus family or Flaviviridae Selected from the virus,

[0094] Here, the tumor is selected from diffuse large B-cell lymphoma, follicular lymphoma, other lymphomas, leukemia, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdomyomas, hepatocellular carcinoma, prostate cancer, breast cancer, cholangiocarcinoma and gallbladder cancer, bladder cancer; brain tumors including neuroblastoma, schwann tumor, 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.

[0095] In a specific embodiment of the present invention, the protein / peptide antigen is a protein / peptide comprising the following:

[0096] Coronavirus family viral antigens;

[0097] Preferably, coronavirus spike protein;

[0098] More preferably, coronavirus spike protein S1 subunit;

[0099] More preferably, the coronavirus spike protein receptor binding region RBD;

[0100] Or a fragment or variant having immunogenicity for all of the above protein / peptide antigens.

[0101] In a specific embodiment of the present invention, the coronavirus is SARS-CoV-2 or Middle East Respiratory Syndrome coronavirus.

[0102] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of an antigen as previously defined and another protein or peptide.

[0103] Preferably, the fusion protein is SARS-CoV-2 RBD-mFc; or

[0104] SARS-CoV-2 RBD-his; or

[0105] MERS-COV RBD-his and;

[0106] Fc is preferably an IgG Fc fragment, more preferably a human or rat IgG Fc fragment.

[0107] In a specific embodiment of the present invention, the protein / peptide antigen comprises a sequence described as any one of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3.

[0108] In a specific embodiment of the present invention, the protein / peptide antigen is a protein / peptide comprising the following:

[0109] Paramyxoviridae Antigen containing;

[0110] Preferably, a paramyxovirus glycoprotein receptor binding region;

[0111] Preferably paramyxovirus glycoprotein F, glycoprotein G;

[0112] Or a fragment or variant having immunogenicity for all of the above protein / peptide antigens.

[0113] In a specific embodiment of the present invention, the paramyxovirus is a human respiratory syncytial virus.

[0114] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of an antigen as previously defined and another protein or peptide, and

[0115] Preferably, the fusion protein is RSV-gpG-his.

[0116] 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.

[0117] In a specific embodiment of the present invention, the protein / peptide antigen comprises the following:

[0118] Orthomyxoviridae Antigen containing;

[0119] Preferably, an orthomyxovirus glycoprotein receptor binding region;

[0120] Preferably, hemagglutinin (HA) protein and / or neuraminidase (NA) protein;

[0121] Or a fragment or variant having immunogenicity for all of the above protein / peptide antigens.

[0122] In a specific embodiment of the present invention, the orthomyxovirus is a type B influenza virus and / or a type A influenza H5N1 virus.

[0123] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of an antigen as previously defined and another protein or peptide;

[0124] Preferably, the fusion protein is Flu-B-HA1-his or H5N1-HA-his.

[0125] In a specific embodiment of the present invention, the protein / peptide antigen comprises a sequence described as any one of SEQ ID NO. 7 and / or SEQ ID NO. 15, SEQ ID NO. 8 and / or SEQ ID NO. 16.

[0126] In a specific embodiment of the present invention, the protein / peptide antigen comprises the following:

[0127] Filovirus family Antigen containing;

[0128] Preferably, a filovirus envelope glycoprotein receptor binding region;

[0129] Preferably, filovirus envelope glycoproteins GP1 and / or GP2;

[0130] More preferably, filovirus envelope glycoprotein GP1;

[0131] Or a fragment or variant having immunogenicity for all of the above protein / peptide antigens.

[0132] In a specific embodiment of the present invention, the filovirus is the Ebola virus.

[0133] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of an antigen as previously defined and another protein or peptide, and

[0134] Preferably, the fusion protein is Ebola-GP-Fc or Ebola-GP1-his;

[0135] Here, Fc is preferably an IgG Fc fragment, more preferably a human or rat IgG Fc fragment.

[0136] In a specific embodiment of the present invention, the protein / peptide antigen comprises a sequence described as any one of SEQ ID NO. 9 and / or SEQ ID NO. 17, SEQ ID NO. 10 and / or SEQ ID NO. 18.

[0137] In a specific embodiment of the present invention, the protein / peptide antigen comprises the following:

[0138] Flaviviridae Antigen containing;

[0139] Preferably, a viral antigen of a flavivirus or hepacivirus;

[0140] Preferably, Flavivirus The outer protein receptor binding region;

[0141] Preferably, Flavivirus At least one of the structural domains EDI, EDII, and EDIII of the coat protein;

[0142] More preferably, Flavivirus Structural domain EDIII of the coat protein; or

[0143] Preferably, Hepasivirus The outer glycoprotein receptor binding region;

[0144] Preferably, Hepasivirus Coat glycoprotein E1 and / or E2;

[0145] Or a fragment or variant having immunogenicity for all of the above protein / peptide antigens.

[0146] In a specific embodiment of the present invention, Flavivirus is preferably the Zika virus; or

[0147] Hepasivirus It is preferably the hepatitis C virus.

[0148] In a specific embodiment of the present invention, the protein / peptide antigen is a fusion protein of an antigen as previously defined and another protein or peptide;

[0149] Preferably, the fusion protein is ZIKV-E-Fc; where

[0150] Fc is preferably an IgG Fc fragment, more preferably a human or rat IgG Fc fragment; or

[0151] Preferably, the fusion protein is HCV-E2-his and / or HCV-E1-his.

[0152] In a specific embodiment of the present invention, the protein / peptide antigen comprises a sequence described as 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.

[0153] In a specific embodiment of the present invention, the molecular weight of the glycoprotein / peptide antigen conjugate is 400-14000 KDa.

[0154] In a specific embodiment of the present invention, a glycoprotein / peptide antigen is further conjugated to a protein carrier.

[0155] In a specific embodiment of the present invention, the protein carrier is tetanus toxoid, tetanus toxoid fragment C, tetanus toxoid non-toxic mutant, diphtheria toxoid, CRM197, and other non-toxic mutants of diphtheria toxoid, preferably CRM197.

[0156] A third aspect of the present invention relates to an immunogenic composition comprising the aforementioned glycoprotein / peptide antigen conjugate, an adjuvant, and an excipient.

[0157] In a specific embodiment of the present invention, the auxiliary agent is selected from aluminum auxiliary agents, oil-in-water emulsion auxiliary agents, MF59, QS-21, and lipid monophosphate A.

[0158] A fourth aspect of the present invention relates to the use of a glycoprotein / peptide antigen conjugate or an immunogenic composition for the prevention or treatment of a disease caused by a pathogen-associated protein / peptide antigen or a tumor-associated protein / peptide as defined above, wherein, preferably, the pathogen

[0159] Coronavirus, more preferably SARS-CoV-2 and / or MERS-CoV;

[0160] Paramyxovirus, more preferably human respiratory syncytial virus;

[0161] Orthomyxovirus, more preferably influenza B virus and / or influenza A H5N1 virus;

[0162] Filovirus, more preferably Ebola virus;

[0163] It is a flavivirus, preferably a Zika virus; or a hepatitis C virus.

[0164] A fifth aspect of the present invention relates to the use of a glycoprotein / peptide antigen conjugate or an immunogenic composition in the manufacture of a vaccine or drug for the prophylactic treatment of a disease caused by a pathogen-associated protein / peptide antigen or a tumor-associated protein / peptide as defined above, wherein, preferably, the pathogen

[0165] Coronavirus, more preferably SARS-CoV-2 and / or MERS-CoV;

[0166] Paramyxovirus, more preferably human respiratory syncytial virus;

[0167] Orthomyxovirus, more preferably influenza B virus and / or influenza A H5N1 virus;

[0168] Filovirus, more preferably Ebola virus;

[0169] It is a flavivirus, more preferably a Zika virus; or a hepatitis C virus. Brief explanation of the drawing

[0170] Figure 1 shows the titers of anti-SARS-COV-2 RBD antibodies in mouse serum immunized with an immune combination using SARS-COV-2 RBD-mFc as the antigen. The values ​​shown are absorbances detected at a dilution of 8,000x of the corresponding serum. Figure 2 illustrates a comparison of different adjuvants against the antigen SARS-COV-2 RBD-his-PS14 conjugate at a serum dilution of 32,000x and an immunization dose of 3 µg per mouse. Figure 3 illustrates the comparison of the serum neutralization activity of the SARS-COV-2 RBD and CRM197 immunological combination conjugated with PS14 as an antigen in mice immunized with a serum dilution of 1500x and an immunization dose of 3 μg per mouse as a SARS-COV-2 RBD protein-PS14 polysaccharide conjugate. Figure 4 shows the immunological results of PS7F, PS14, and dextran as conjugates for the conjugate. Specific details for implementing the invention

[0171] definition

[0172] Unless otherwise noted, all scientific and technical terms used herein have the meanings generally understood by those skilled in the art to which this invention pertains. For the purposes of this invention, the following terms are additionally defined.

[0173] Where the singular forms “one,” “a / an,” “another,” and “the” are used in this specification or the appended claims, they also include the plural forms of the subject, unless the context clearly indicates otherwise.

[0174] The term “include” or “comprise” means to include specific components without excluding any other components. Phrases such as “essentially composed of” may include other components or steps that do not impair the novel or essential properties of the invention; that is, these terms exclude other components or steps that impair the novel or essential properties of the invention. The term “composed of” means to include specific components or groups of components and to exclude all other components. In this specification and the appended claims, the phrase “wherein the protein / peptide antigen is a protein / peptide comprising X” means that the amino acid sequence of the said protein / peptide antigen comprises the protein / peptide sequence of X.

[0175] The term "antigen" refers to an exogenous substance (specifically bound) recognized by antibodies or T-cell receptors, but it does not necessarily induce an immune response. Exogenous substances that induce specific immunity are called "immunogenic antigens" or "immunogens." An "anti-antigen" is an antigen that cannot induce an immune response on its own (however, a combination of several anti-antigen molecules or a combination of an anti-antigen and a macromolecular carrier can induce an immune response).

[0176] "Humoral immune response" is an antibody-mediated immune response involving the introduction and production of antibodies that recognize and bind to the antigen of the immunogenic composition of the present invention with a specific affinity. "Cell-mediated immune response" is an immune response mediated by T cells and / or other leukocytes, induced by providing antigen epitopes associated with class I or II molecules of major histocompatibility complex (MHC), CD1, or other atypical MHC-like molecules.

[0177] The term "saccharides" may be used to refer to polysaccharides, oligosaccharides, or monosaccharides. Polysaccharides can be isolated from organisms such as bacteria, or they may be naturally occurring polysaccharides that are optionally resized to a specific degree by microfluidic methods. Resizing polysaccharides reduces the viscosity of polysaccharide samples and / or improves the filterability of conjugated products. Oligosaccharides are hydrolyzed polysaccharides containing a small number of repeating units (typically 5 to 30 repeating units). Polysaccharides may also be chemically synthesized.

[0178] The term "conjugate" is used in this specification and the appended claims to refer to a protein / peptide covalently conjugated with a sugar. The glycoprotein / peptide conjugate of the present invention and the immunogenic composition containing the same may contain a certain amount of free sugar, protein / peptide.

[0179] In this document, the term "conjugation" refers to the process in which sugars, such as bacterial encapsulated polysaccharides, are covalently bonded to proteins / peptides.

[0180] The term "immunogenic composition" refers to any pharmaceutical composition containing an antigen, such as a microorganism or a component thereof, that can be used to induce an immune response in an individual.

[0181] The term "carrier" may be used to refer to a diluent, adjuvant, excipient, or medium administered together with a pharmaceutical composition. Water, saline solution, and aqueous solutions of dextrose and glycerol may be used as liquid carriers, particularly for injectable solutions.

[0182] In this document, the term "immunogenicity" means the ability of an antigen (or an epitope of an antigen), such as a coronavirus spike protein receptor binding region or a glycoconjugate thereof, or an immunogenic composition containing the same, to induce a humoral or cell-mediated immune response or both of the above responses in a host (e.g., mammal).

[0183] A "protective" immune response is a humoral or cell-mediated immune response that protects an individual from infection, or the ability of an immunogenic composition to induce both of these responses. The protection provided does not need to be absolute; that is, it is not necessary to completely prevent or eradicate the infection as long as there is a statistically significant improvement compared to a control group of the individual (e.g., an infected animal not provided with the relevant vaccine or immunogenic composition). Protection may be limited to mitigating the severity of infection symptoms or the rapidity of attack.

[0184] The terms “immuno-dose” and “immuno-effective dose” are used interchangeably herein and mean that the antigen or immunogenic composition is sufficient to induce an immune response (cellular (T-cell) or humoral (B-cell or antibody) response, or both) when measured by standard analytical methods known to those skilled in the art.

[0185] The efficacy of an antigen as an immunogen can be measured, for example, by proliferation assays, cell lysis assays, or measurement of B-cell activity levels.

[0186] Method of the present invention for improving the immunogenicity of protein / peptide antigens

[0187] The present invention is a pioneering invention in which the inventors discovered that the immunogenicity of a protein / peptide antigen is improved by including the step of conjugating a protein / peptide antigen with a sugar to form a glycoprotein / peptide antigen conjugate.

[0188] Prior to the present invention, no studies have reported that the immunogenicity of a protein / peptide antigen is increased in a glycoprotein / peptide antigen conjugate. On the contrary, as mentioned in the background art, previous studies have described the immunogenicity of the protein / peptide antigen in the conjugate as conserved immunogenicity (US 5192540A / US 9296795B), or described the protein / peptide antigen epitopes as not being modified by conjugation (US 9296795B). These teachings are contrary to the purpose of the present invention.

[0189] Sugar-conjugated protein / peptide antigen of the present invention

[0190] 1. As an antigen Coronavirus family virus

[0191] Coronavirus family In the series Orthocoronavidae and Letovirology Subfamily is included.

[0192] Orthocoronavidae subfamily BetacoronavirusThe genus includes the well-known Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome-associated Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). These three viruses determine viral organization or host affinity by mediating viral invasion primarily through the binding of spike proteins (S proteins) to host cell receptors. The host cell receptor protein for SARS-CoV-2 is angiotensin-converting enzyme 2 (ACE2). The spike protein (S protein) binds to the ACE2 receptor and is cleaved by host protease into an S1 polypeptide containing a receptor binding domain (SARS-CoV-2 RBD) and an S2 polypeptide responsible for invading the host by mediating the fusion of the virus with the cell membrane.

[0193] In one embodiment of the present invention, the coronavirus SARS-CoV-2 spike protein (SARS-CoV-2 S protein), its extracellular domain, S1 subunit, or receptor binding domain is used as an antigen.

[0194] In one embodiment of the present invention, Middle East Respiratory Syndrome coronavirus is selected as an antigen, for example, its extracellular region, S1 subunit, or receptor binding region is used as an antigen.

[0195] 2. As an antigen Paramyxoviridae virus

[0196] Paramyxoviridae The series Paramyxovirinae and Pneumoviruse It includes two subfamilies. Human respiratory syncytial virus (RSV) is Paramyxovirus subfamily of RespirovirusIt is one of the respiratory viruses of the genus RSV. RSV encodes two major transmembrane surface glycoproteins: glycoprotein G (adhesion protein) and glycoprotein F (fusion protein). Glycoprotein G mediates viral binding to cell receptors, while glycoprotein F facilitates viral fusion to the cell membrane, allowing viral ribonucleoproteins to invade the cytoplasm (Lopez et al. (1998) J. Virology 72:6922-6928).

[0197] In one embodiment of the present invention, an RSV envelope glycoprotein is selected as the antigen. Human RSV envelope glycoproteins, such as RSV glycoprotein F or glycoprotein G, may be used.

[0198] 3. As an antigen Orthomyxoviridae virus

[0199] Orthomyxoviridae The family includes influenza A virus, influenza B virus, influenza C virus, and other genera.

[0200] Infections by type A, B, and C influenza viruses rely on two envelope proteoglycans, hemagglutinin (HA) and neuraminidase (NA), which are primarily responsible for viral attachment and the entry of viral particles into cells. Influenza virus infection is initiated by the attachment of the hemagglutinin (HA) protein to sialic acid-containing cell receptors (glycoproteins and glycolipids) on the surface of the virion. The neuraminidase (NA) protein mediates the processing of sialic acid receptors, and viral entry into cells relies on HA-dependent receptor-mediated cytokinesis (CN103865892B).

[0201] In one embodiment of the present invention, the influenza A H5N1 hemagglutinin (HA) protein is used as an antigen, and the influenza B hemagglutinin protein (HA1 subunit) may also be used as an antigen.

[0202] 4. As an antigen Filovirus familyvirus

[0203] Filovirus family Representative examples include the Ebola virus of the genus Ebolavirus and the Marburg virus of the genus Marburgvirus.

[0204] The only protein present on the surface of the Ebola virus is the glycoprotein (GP). The GP1,2 trimer forming the virus's surface spike consists of two subunits, GP1 and GP2, connected by disulfide bonds (Volchkova, VA). et al., (1998), Virology 250:408-414; Falzarano, D. et al. (2006) Chembiochem 7: 1605-1611). GP1 is known to mediate viral attachment 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).

[0205] In one embodiment of the present invention, an Ebola virus glycoprotein (GP) is selected as the antigen. For example, a GP protein subunit (GP1 and / or GP2) which is the GP extracellular structural domain is used as the antigen.

[0206] 5. As an antigen Flaviviridae virus

[0207] Flaviviridae virus In the series, mainly Flavivirus , Pestivirus , Pegivirus and Hepacivirus The interior is included, and here the above Flaviviridae It includes Zika virus (ZIKV), dengue fever (DV), West Nile virus, Japanese encephalitis virus, and yellow fever virus. Hepasivirus It includes the hepatitis C virus (HCV).

[0208] Flavivirus envelope proteins play a crucial role in host cell viral infection by mediating the entry of the corresponding virus into host cells. The envelope protein consists of three distinct structural envelope domains I, II, and III (EDI, EDII, and EDIII). EDI is the central structural domain of the envelope protein that stabilizes the overall orientation of the protein, and the glycosylation site of EDI is associated with viral production, pH sensitivity, and neuroinvasiveness. EDII plays a critical role in membrane fusion due to the immunological advantages of its fusion loop epitope and envelope dimer epitope. Additionally, EDIII is a primary target for antibody neutralization (Xingcui Zhang). et al. , (2017) Viruses . 2017 Nov; 9(11): 338. Structures and Functions of the Envelope Glycoprotein in Flavivirus Infections).

[0209] The Zika virus envelope protein ("E" or "EP") consists of three different structural domains. E structural domain I (E-DI) is the central structural domain that constitutes the overall E protein structure. E structural domain II (E-DII) is formed by two extended loops protruding from E-DI and is located within the pockets of E-DI and E structural domain III (E-DIII). E-DIII is an immunoglobulin-like structural domain that forms small protrusions on the surface of smooth, spherical mature viral particles and is thought to interact with cell receptors on target cells (CN109996560A).

[0210] The HCV RNA genome encodes a single polymeric protein that is cleaved at or after translation into three structural proteins (core, glycoproteins E1 and E2) and seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The envelope proteins, glycoproteins E1 and E2, form a heterodimer to constitute the viral envelope protein, which plays a crucial role in mediating viral entry and morphogenesis when the virus enters host cells. Hepatitis C virus envelope proteoglycans bind to specific proteins on the surface of host liver cells to initiate the entry process. This process involves multiple host receptors / co-receptors. Here, E2 acts as the primary HCV envelope proteoglycan and interacts directly with the receptors / co-receptors. It has long been thought that E1 does not interact directly with host receptors in this process, but rather induces membrane fusion with E2 by maintaining the functional E2 conformation necessary for receptor binding (Yimin, Tong. Et al. , (2018) Front Immunol . 2018; 9: 1411. Role of Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly).

[0211] In one embodiment of the present invention, Zika virus envelope protein E-DIII is selected as the antigen.

[0212] In one embodiment of the present invention, hepatitis C virus envelope glycoprotein E1 and / or E2 is selected as the antigen.

[0213] The viral antigens described above in this chapter can be obtained by extraction from natural pathogens or by genetic recombination. Modified forms thereof, such as their immunogenic fragments or variants, as well as, for example, their purified tags or fusion proteins with antibody Fc fragments, may also be used in the present invention.

[0214] Polysaccharides are bacterial polysaccharides, for example Neisseria encephalates encapsulated polysaccharides, Type B Haemophilus influenzae encapsulated polysaccharides, s Treptococcus pneumoniae Capsule-type polysaccharides of, Group B Staphilococcus aureus It may be not only encapsulated polysaccharides but also dextran and mannan. The polysaccharides may be plant-derived polysaccharides such as starch, inulin, pectin, etc., or derivatives of chemically modified polysaccharides such as carboxymethyl starch. In addition, the polysaccharides may be animal-derived polysaccharides such as chitosan or derivatives thereof.

[0215] The process of polysaccharide-protein conjugation is as follows: the polysaccharide is made to carry a reactive group by chemical reaction. Then, the reactive groups are reacted with chemical reactive groups on the protein molecule, such as amino, carboxyl, sulfhydryl groups, the imidazole ring on histidine, the indole ring on tryptophan, the phenyl ring on tyrosine, the phenyl ring on phenylalanine, the hydroxyl group on serine, and glutamine and asparagine to form covalent bonds.

[0216] One method of conjugating polysaccharides to protein molecules is to oxidize the polysaccharides using sodium periodate to generate aldehyde groups, which react with the amino groups of the protein molecules to form Schiff bases, which are reduced to stable single bonds in the presence of a reducing agent. This allows the polysaccharides to form covalent bonds with the protein molecules. Reducing agents such as sodium borohydrogenate can be added to the reaction system.

[0217] Another method of conjugating polysaccharides to protein molecules is to react the polysaccharides with cyanogen bromide or 1-cyano-4-dimethylaminopyridine tetrafluoroborate to produce reactive cyanate esters. The cyanate groups react with amino groups on the surface of the protein to form covalent bonds. The activated polysaccharides may also first react with linker arms such as hexanediamine, hexanedihydrazide, etc. Then, the product is reacted with the protein in the presence of a condensation agent to form covalent linkers.

[0218] In addition, polysaccharides can be activated with other chemical reagents and then reacted with proteins to form conjugates. Epichlorohydrin, triazine, diazine, divinylsulfone, and other reagents well known in the industry are included here.

[0219] To improve the immunogenicity of the glycoprotein / peptide antigen conjugate of the present invention, a protein carrier may be additionally added to the reaction of the sugar and the protein / peptide antigen conjugate to form a glycoprotein / peptide antigen-protein carrier conjugate. The protein carrier may be tetanus toxoid, tetanus toxoid fragment C, a tetanus toxoid non-toxic mutant, diphtheria toxoid, CRM197, or other non-toxic mutants of diphtheria toxoid, preferably CRM197, which are commonly used in the vaccine industry.

[0220] 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 nonionic surfactant, 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 an immune response when administered together with an immunogen or antigen. The compositions used in the present invention may or may not contain vaccine adjuvants. Adjuvants that may be used in the compositions of the present invention include, but are not limited to, the following: oil emulsion compositions including squalene-water emulsions, such as MF59; complete Freund adjuvants (CFA) and incomplete Freund adjuvants (IFA); saponin preparations; unique particles called immunostimulatory complexes (ISCOM) formed by the combination of saponin and cholesterol; and virosomes and virus-like particles.

[0223] The adjuvant used will vary depending on the individual receiving the immunogenic composition, the prescribed route of administration, and the frequency.

[0224] The immunogenic composition may optionally include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include carriers of animals (including humans and non-human mammals) that are or will be listed in national pharmacopoeias. The term “carrier” may be used to refer to a diluent, adjuvant, excipient, or medium administered with the pharmaceutical composition. Aqueous solutions of water, saline, dextrose, and glycerol may be used as liquid carriers, particularly for injectable solutions.

[0225] The immunogenic composition of the present invention may include one or more additional immunomodulators that are substances capable of disturbing or modifying the immune system to cause upregulation or downregulation of humoral and / or cell-mediated immunity to be observed. In one embodiment, upregulation of the humoral and / or cell-mediated capabilities (arms) of the immune system is provided. This includes, for example, adjuvants or cytokines.

[0226] Administration route of the immunogenic composition of the present invention

[0227] The immunogenic composition of the present invention for therapeutic or prophylactic treatment may be administered intramuscularly, intraperitoneally, intradermally, or subcutaneously; or administered through the mucous membranes into the oral / esophagus, respiratory tract, or genitourinary tract. Intranasal administration of the vaccine is preferred for the treatment of certain diseases, e.g., preferably pneumonia or otitis media. The vaccine of the present invention may be provided as a single dose, but its components may be co-administered simultaneously or administered at different times. In addition to a single route of administration, two different routes of administration may also be used.

[0228] The optimal amount of a component for a specific immunogenic composition can be determined by standard studies involving the observation of the appropriate immune response of individuals. After the initial vaccination, individuals may receive one or several additional vaccinations at appropriate time intervals.

[0229] Uses of the immunogenic composition of the present invention

[0230] The protein / peptide antigen conjugate and immunogenic composition of the present invention can prevent or treat diseases caused by pathogens such as coronavirus, paramyxovirus, orthomyxovirus, filovirus and flavivirus, more specifically SARS-CoV-2 and / or MERS-CoV virus, human respiratory syncytial virus, influenza B virus, influenza A H5N1 virus, Ebola virus, Zika virus and / or hepatitis C virus.

[0231] Abbreviation of the present invention

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

[0233] SARS-CoV-2 RBD-his: SARS-CoV-2 coronavirus spike protein receptor binding region with 6-histidine tag;

[0234] MERS-COV RBD-his: Middle East Respiratory Syndrome coronavirus spike protein receptor binding region with 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;

[0238] flu-B-HA1: Influenza B hemagglutinin protein (HA1 subunit);

[0239] H5N1-HA: Influenza A H5N1 hemagglutinin;

[0240] Ebola-GP: Ebola virus glycoprotein (receptor binding domain);

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

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

[0243] PS14: Streptococcus pneumoniae Capsule polysaccharides of serotype 14

[0244] PS7F: Streptococcus pneumoniae Capsule polysaccharides of serotype 7F

[0245] Alum: Aluminum adjuvant; in this case, it is an aluminum phosphate adjuvant.

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

[0247] Serotype 14 Streptococcus pneumoniae The seed was ATCC 6314, and serotype 7F Streptococcus pneumoniae The seed was ATCC 10351.

[0248] Preserved with 0.5 mL of glycerol Streptococcus pneumoniaeSeeds were heated in a 150 rpm shaker at 37°C for 10–16 hours in 500 ml of Hoeprich medium (VM Goncalves, Optimization of medium and cultivation conditions for capsular polysaccharide production by Streptococcus pneumoniae serotype 23F, AllpMicrobiolBiotechnol Added to (2002) 59:713-717), and OD 600 If this exceeded 1.0, the culture was stopped. 0.6 g of sodium deoxycholate was added, mixed well, and left to stand for at least 2 hours to allow the bacteria to be completely dissolved. The mixture was centrifuged at 14,000 g for 30 minutes, the supernatant was removed, and the solution was ultrafiltered at 100 kDa to concentrate it to approximately 400 ml, which is one-tenth of the original volume. 36% acetic acid was gradually added to the concentrate to adjust the pH to 3.5. After standing for 2 hours, the mixture was centrifuged at 14,000 g for 30 minutes, 390 ml of the supernatant was removed, mixed with 130 ml of anhydrous ethanol, and left overnight. The next day, the supernatant was centrifuged at 14,000 g for 30 minutes, 780 ml of anhydrous ethanol was added to the supernatant, and left overnight. The next day, after centrifugation at 14,000 g for 30 minutes, the supernatant was discarded. 300 ml of 75% ethanol was added to the precipitate, and after suspending the precipitate, it was centrifuged again at 14,000 g for 30 minutes. The supernatant was discarded, and the precipitate was dissolved in 10 ml of water to ensure that the polysaccharide concentration in the solution was at least 10 mg / ml. The resulting solution was Streptococcus pneumoniae It is a capsule-type polysaccharide solution.

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

[0250] The polysaccharide prepared in Example 1 Streptococcus pneumoniaeSerum type 14 (PS14) and 7F (PS7F) were encapsulated polysaccharides or dextran (dextran, Sigma, 00894, hereinafter the same).

[0251] 2.1 Polysaccharide Hydrolysis.

[0252] 10 mL of capsule-type polysaccharide purified to 10 mg / ml was added to 0.86 mL of 36% acetic acid, and the final concentration of acetic acid in the solution was 500 mM. After 2 hours in a 90°C water bath, 1 M NaOH was added to neutralize the pH to 6-7 to obtain a hydrolyzed polysaccharide sample.

[0253] The molecular weight of PS14 polysaccharide was measured to be approximately 500 kDa by HPLC-MALS and approximately 300 kDa after hydrolysis.

[0254] The molecular weight of the PS7F capsule polysaccharide was approximately 700 kDa and it was not hydrolyzed.

[0255] Dextran was not hydrolyzed.

[0256] 2.2 High activation of polysaccharides.

[0257] 100 mg of sodium periodate was added to 10 mL of a 10 mg / ml polysaccharide solution, mixed well, and reacted in a dark room for 1 hour. A centrifugal chromatography column containing 5 ml of Sephadex G 25 packing was taken, and 10 ml of 50 mM Na2HPO4 buffer (pH=7.0) was added. The buffer was allowed to flow through the column under gravity. Then, the column was placed in a centrifuge and centrifuged at 1000 g for 2 minutes. Afterward, the existing collection tube was replaced with a new tube, 1 ml of the polysaccharide solution oxidized with sodium periodate was added to the centrifugal column, and it was centrifuged again at 1000 g for 2 minutes. The effluent collected from the column was a highly activated polysaccharide solution.

[0258] 2.3 Activation of low activity of polysaccharides.

[0259] 30 mg of sodium periodate was added to 10 mL of a 10 mg / ml polysaccharide solution and mixed thoroughly. A centrifugal chromatography column containing 5 ml of Sephadex G 25 packing was taken, and 10 ml of 50 mM Na2HPO4 buffer (pH=7.0) was added. Then, the column was placed in a centrifuge and centrifuged at 1000 g for 2 minutes. Afterward, the existing collection tube was replaced with a new tube, 1 ml of the polysaccharide solution oxidized with sodium periodate was added to the column, and the column was centrifuged again at 1000 g for 2 minutes. The effluent collected from the column was a polysaccharide solution activated to a low level of activity.

[0260] Example 3: Protein / peptide antigen conjugated with polysaccharide

[0261] The protein / peptide antigen used is the coronavirus spike protein receptor binding domain, and the polysaccharide is Streptococcus pneumoniae It is a capsule-type polysaccharide or dextran. Specific ingredients, amounts, and volumes are shown in Table 1.

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

[0263] 2. Coronavirus spike receptor protein and polysaccharide conjugation: 3 mg of coronavirus spike receptor protein activated according to Table 1 Streptococcus pneumoniaeThe solution was added to the encapsulated polysaccharide or dextran and supplemented with 50 mM Na2HPO4 buffer (pH 7.0) to the final volume shown in Table 1. A 10 mg / mL sodium borohydride solution was added to the reaction solution (0.15 ml to 0.6 ml reaction systems and 0.375 ml to 1.5 ml reaction systems) and reacted at room temperature for 2 hours. Subsequently, the conjugate was aseptically filtered through a 100,000 MW ultrafiltration tube, and the final ultrafiltration volume was reduced to less than 2 ml by 10-fold exchange of PBS buffer. The fixed samples were aseptically filtered using a 0.22 µm filter and stored at 4°C.

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

[0265]

[0266] Example 4. Protein / peptide antigen conjugated with polysaccharides and protein carrier

[0267] The protein / peptide antigen used was SARS-COV-2 RBD, the coronavirus spike protein receptor binding domain, the protein vector was CRM197, and the polysaccharide was Streptococcus pneumoniae It was a capsule-type polysaccharide. The specific composition, amount, and volume are shown in Table 2.

[0268] CRM197 is a variant of diphtheria toxin (Geert J. Schenk, Efficient CRM197-mediated drμg targeting to monocytes, Journal of Controlled Release 158 (2012) 139-147). The coronavirus spike protein receptor binding domain and CRM197 Streptococcus pneumoniaeIt is conjugated with the capsule polysaccharide of serotype 14. The activation of the polysaccharide was carried out as in Example 2.2. 1.5 mg of polysaccharide, 2.7 mg of coronavirus spike protein receptor binding domain protein, and 0.3 mg of CRM197 were taken and conjugated according to the same procedure as in Example 3. The reaction conditions of the conjugate and the molecular weight of the product are shown in Table 2.

[0269]

[0270] Example 5. Immunogenicity of Coronavirus Spike Protein Receptor Binding Domain-Polysaccharide Conjugate

[0271] The coronavirus spike protein receptor binding domain-polysaccharide conjugates used are shown in Tables 3-6.

[0272] 5.1 Preparation of Immunogenic Compositions

[0273] An immunogenic composition was prepared using the coronavirus spike protein receptor binding domain protein or the conjugate prepared in Example 3 or 4 as an antigen.

[0274] 5.1.1 Preparation of MF59 Adjuvants

[0275] 200 ml of a 10 mM sodium citrate solution (adjusted to pH 6.5 with HCl) was prepared, and 1 ml of Tween 80 (Nanjing Well Pharmaceutical Co., LTD) was added and mixed well to dissolve it. 1 ml of Span 85 (Zhaoqing Chaoneng Industrial Co., Ltd.) was added to 10 ml of squalene (Merck) and mixed well to dissolve it. The two previous solutions were mixed and homogenized three times using a high-pressure homogenizer (AH-PILOTATS) set to 800 bar to obtain a homogeneous emulsion with MF59 as an adjuvant.

[0276] 5.1.2 Preparation of MF59 adjuvants containing monophosphatidyl lipid A (MPL)

[0277] 10 mg of MPL (MERCK L6895) was dispersed in 10 ml of sodium citrate buffer (10 mM, pH 6.5). Another 4 ml of MF59 adjuvant was added to 1 ml of the MPL dispersion and mixed to obtain an MF59 adjuvant containing MPL.

[0278] 5.1.3 Preparation of Aluminum Adjuvant Immunogenic Composition

[0279] The antigen was diluted with PBS to 0.02 mg / ml or 0.06 mg / ml (peptide / protein equivalent, hereinafter), and the aluminum adjuvant (Beijing Nuoning Biotechnology Co., Ltd.) was diluted with PBS to 1 mg / ml. The diluted antigen and the aluminum adjuvant were mixed in equal volumes. In this immunogenic composition, the protein concentration of the antigen was 0.01 mg / ml or 0.03 mg / ml, respectively.

[0280] 5.1.4 Preparation of MF59 Adjuvant Immunogenic Composition

[0281] The antigen was diluted to 0.02 mg / ml or 0.06 mg / ml, respectively, using PBS, and the diluted antigen was mixed with an equal volume of MF59 adjuvant. The protein concentration of the antigen in this immunogenic composition was 0.01 mg / ml or 0.03 mg / ml, respectively.

[0282] 5.1.5 Preparation of MF59 adjuvant immunogenic composition containing MPL

[0283] The antigen was diluted to 0.02 mg / ml or 0.06 mg / ml, respectively, using PBS, and the diluted antigen was mixed with an equal volume of MF59 adjuvant containing MPL. The protein concentration of the antigen in this immunogenic composition was 0.01 mg / ml or 0.03 mg / ml, respectively.

[0284] 5.1.6 Preparation of an Immunogenic Adjuvant Composition Mixed with MF59 and Aluminum Adjuvant

[0285] 1.5 ml of aluminum adjuvant and 1.5 ml of MF59 adjuvant were mixed, and then 0.18 ml of antigen at a concentration of 1 mg / ml was added. The protein concentration of the antigen in this immunogenic composition was 0.03 mg / ml.

[0286] 5.2 Immunized mouse.

[0287] Mice were selected from 4-6 week old Balb / c mice and immunized on days 14 and 28, respectively, by intraperitoneal injection of 0.1 ml of an immune composition at a concentration of 0.01 mg / ml or 0.03 mg / ml as described in Example 5.1. Blood was collected from the orbit on days 7, 21, and 35 to measure serum antibody titers and neutralization titers.

[0288] 5.3 Serum Efficacy Analysis

[0289] 5.3.1 Serum efficacy analysis when the antigen is SARS-COV-2 RBD or its fusion protein

[0290] A 96-well plate was coated with 100 μl / well using SARS-COV-2 RBD-mFc protein (SinoCelltech Ltd., same as the professional label) at a concentration of 5 μg / mL for 2 hours at room temperature, and the plate was closed by washing it with 2% BSA at room temperature for 1 hour using CD155(D1)-mFc (SinoCelltech Ltd., same as the professional label) as an unrelated control. The serum to be tested (prepared in Example 5.2) was diluted to different dilutions using PBS containing 0.1% bovine serum albumin (BSA) (the exact dilution factors varied depending on the time set for immunological collection, e.g., 1000x, 8000x, 16000x, and 32000x dilutions), mouse serum immunized with SARS-COV-2 RBD-mFc was used as a positive control, and mouse serum of an unrelated immune target (anti-CD70 serum, Beijing Sino Biological, Inc.) was used as a negative control. The serum to be tested and the goat anti-mouse IgG F(ab)2 / HRP (Beijing Sino Biological, Inc.) detection secondary antibody were added simultaneously at different dilutions at a rate of 100 μl / well. OD at specific dilution levels 450 It indicates the antibody titer.

[0291] 5.3.2 Serum efficacy analysis when the antigen is MERS-COV RBD or its fusion protein

[0292] In the MERS-COV RBD-his immunoserological assay, plates are coated with MERS-COV RBD-his (Beijing Sino Biological, Inc., 40071-V08B1) without positive or negative controls. The other steps are the same as in 5.3.1.

[0293] 5.3.3 Serum Efficacy Analysis Results

[0294] The results of the serum efficacy analysis are shown in Table 3-5 and Figure 1-4.

[0295] The efficacy of the aluminum adjuvant immunization combination was shown in Table 3 for mice immunized in serum at an 8000x dilution for 35 days.

[0296]

[0297] The efficacy results of the SARS-COV-2 RBD-his-PS14 aluminum / MF59 / MF59-aluminum / MF59-MPL adjuvant immunogenic composition in serum at a dilution of 32,000x on day 35 are shown in Table 4.

[0298]

[0299] The serum efficacy of the MF59 adjuvant immune composition in mice immunized in serum at 8000x dilution on day 21 is shown in Table 5.

[0300]

[0301] 5.4 Analysis of Neutralizing Efficacy

[0302] The mouse serum sample obtained in Example 5.2 was experimentally diluted a certain number of times (e.g., 500x dilution) and mixed with an equal volume of Pseudovirus 2019-nCoV PSV (China Food and Drug Administration). The serum sample was not added as a positive control, nor was the pseudovirus added as a negative control. After incubation at 37°C for 1 hour, the samples were co-infected with Vero E6 or 293FT / ACE2 cells (SinoCelltech Ltd.). The cells were incubated at 37°C with 5% CO2 for approximately 20–28 hours after infection, and RLU values ​​were measured on a microplate luminescence detector. The neutralization inhibition rate (%) was calculated according to the following formula: Neutralization inhibition rate (%) = (lg (Positive RLU) - lg (Sample RLU)) / (lg (Positive RLU) - lg (Negative RLU)) × 100%.

[0303] The results of the neutralization efficacy on the serum of mice immunized with different immunogenic compositions are shown in Table 6 and Figure 3.

[0304]

[0305] Example 6. Enhancement of immunogenicity of various viral antigens by polysaccharides

[0306] Various viral antigens shown in Table 7 (all provided by Beijing Sino Biological, Inc.) were selected and conjugated with PS14 polysaccharide in the next step.

[0307] Streptococcus pneumoniae Serotype 14 (PS14) was prepared according to Example 1 and activated according to the steps of Example 2.2, with the amount of sodium periodate added adjusted according to Table 8. PS14 was conjugated to the carrier protein CRM197 according to the steps of Example 3, and the ratio of protein to polysaccharide for conjugation is shown in Table 8. An aluminum-containing adjuvant immunogenic composition was prepared according to Example 5.1.3. Mice were immunized according to Example 5.2 with an antigen dose of 3 µg per mouse.

[0308] Immunoserogenic efficacy was analyzed using the corresponding antigen coating without positive and negative controls, and the procedure was exactly the same as in 5.3.1.

[0309] The immunoserolytic efficacy of various antigen conjugates is shown in Table 8. For most antigens, it can be seen that immunogenicity significantly increased after conjugation with polysaccharides.

[0310]

[0311]

[0312] The antigens used in Table 8 are listed in Table 7, but His-tags are not shown here.

[0313] According to the above data, the immunogenicity of protein antigens is that they Streptococcus pneumoniae It was significantly increased when conjugated with encapsulated polysaccharides. Under aluminum adjuvant conditions, the antibody efficacy of the conjugate immune serum reached up to 2.3 times the original efficacy. The neutralizing activity of the conjugate was also significantly higher than the activity of the corresponding proteins. Compared to the aluminum adjuvant, the MF59 adjuvant, MF59 mixed with the aluminum adjuvant, and MF59 adjuvant containing the MPL adjuvant all further enhance the immune effect of the corresponding conjugate. Streptococcus pneumoniae Serum type 14 capsule-type polysaccharide, Streptococcus pneumoniae When serotype 7F capsule polysaccharide and dextran were used as a conjugate, the immunological effects were similar.

[0314] Sequence list

[0315]

[0316]

[0317]

[0318]

Claims

Claim 1 In vitro (composed of the step of conjugating a protein / peptide antigen with a saccharide to form a glycoprotein / peptide antigen conjugate, thereby improving the immunogenicity of a protein / peptide antigen) in vitro As a method, the glycoprotein / peptide antigen is further conjugated to a protein carrier, and the sugars Streptococcus pneumoniae A method in vitro wherein the encapsulated polysaccharide of serotype 14, the carrier is CRM197, and the protein / peptide antigen is a pathogen-associated protein / peptide antigen selected from human respiratory syncytial virus glycoprotein G (RSV-gpG), hepatitis C virus envelope glycoprotein E1 (HCV-E1), hepatitis C virus envelope E2 protein (HCV-E2), influenza B hemagglutinin protein (Flu-B-HA1), influenza A H5N1 hemagglutinin (H5N1-HA), Ebola virus glycoprotein (Ebola-GP), Ebola virus glycoprotein GP1 (Ebola-GP1), Zika virus envelope protein (ZIKV-E), and coronavirus spike protein. Claim 2 The in vitro method according to claim 1, wherein the protein / peptide antigen is a fusion protein that is SARS-CoV-2 RBD-mFc, SARS-CoV-2 RBD-his; or MERS-COV RBD-his; and the Fc fragment is a human or rat IgG Fc fragment. Claim 3 In paragraph 2, the in vitro method wherein the protein / peptide antigen comprises a sequence described as any one of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO.

3. Claim 4 A test tube method according to any one of claims 1 to 3, wherein the molecular weight of the conjugate is 400-14000 KDa. Claim 5 In the in vitro method according to claim 1, wherein the protein / peptide antigen is a fusion protein of RSV-gpG-his. Claim 6 A method in vitro according to claim 1, wherein the protein / peptide antigen comprises the sequence described in SEQ ID NO. 4 and / or SEQ ID NO.

12. Claim 7 A method in vitro according to claim 1, wherein the protein / peptide antigen is a fusion protein that is Flu-B-HA1-his or H5N1-HA-his. Claim 8 A method in vitro according to claim 1, wherein the protein / peptide antigen comprises a sequence described as any one of SEQ ID NO. 7 and / or SEQ ID NO. 15, SEQ ID NO. 8 and / or SEQ ID NO.

16. Claim 9 The in vitro method according to claim 1, wherein the protein / peptide antigen is a fusion protein that is Ebola-GP-Fc or Ebola-GP1-his; and the Fc is a human or rat IgG Fc fragment. Claim 10 A method in vitro according to claim 1, wherein the protein / peptide antigen comprises a sequence described as any one of SEQ ID NO. 9 and / or SEQ ID NO. 17, SEQ ID NO. 10 and / or SEQ ID NO.

18. Claim 11 The in vitro method of claim 1, wherein the protein / peptide antigen is a fusion protein that is ZIKV-E-Fc; the Fc is a human or rat IgG Fc fragment; or the fusion protein is HCV-E2-his and / or HCV-E1-his. Claim 12 The in vitro method according to claim 1, wherein the protein / peptide antigen comprises a sequence described as 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. Claim 13 A glycoprotein / peptide antigen conjugate having increased immunogenicity compared to an unconjugated protein / peptide antigen, wherein the glycoprotein / peptide antigen conjugate is formed by conjugating the protein / peptide antigen with a sugar, the glycoprotein / peptide antigen is additionally conjugated with a protein carrier, and the sugar is Streptococcus pneumoniae A conjugate comprising a capsule-type polysaccharide of serotype 14, wherein the carrier is CRM197, and the protein / peptide antigen is a pathogen-associated protein / peptide antigen selected from: human respiratory syncytial virus glycoprotein G (RSV-gpG), hepatitis C virus envelope glycoprotein E1 (HCV-E1), hepatitis C virus envelope E2 protein (HCV-E2), influenza B hemagglutinin protein (Flu-B-HA1), influenza A H5N1 hemagglutinin (H5N1-HA), Ebola virus glycoprotein (Ebola-GP), Ebola virus glycoprotein GP1 (Ebola-GP1), Zika virus envelope protein (ZIKV-E), and coronavirus spike protein. Claim 14 In paragraph 13, the protein / peptide antigen is a fusion protein that is SARS-CoV-2 RBD-mFc; SARS-CoV-2 RBD-his; or MERS-COV RBD-his; and the Fc fragment is a human or rat IgG Fc fragment, conjugate. Claim 15 In claim 13, the protein / peptide antigen comprises a sequence described as any one of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, in a conjugate. Claim 16 In paragraph 13, the above protein / peptide antigen is a conjugate in which the protein / peptide antigen is a fusion protein of RSV-gpG-his. Claim 17 In claim 13, the conjugate wherein the protein / peptide antigen comprises the sequence described in SEQ ID NO. 4 and / or SEQ ID NO.

12. Claim 18 In paragraph 13, the above protein / peptide antigen is a fusion protein that is Flu-B-HA1-his or H5N1-HA-his, a conjugate. Claim 19 In claim 13, the protein / peptide antigen comprises a sequence described as any one of SEQ ID NO. 7 and / or SEQ ID NO. 15, SEQ ID NO. 8 and / or SEQ ID NO. 16, in a conjugate. Claim 20 In paragraph 13, the protein / peptide antigen is a fusion protein that is Ebola-GP-Fc or Ebola-GP1-his; and the Fc is a conjugate in which the Fc is a human or rat IgG Fc fragment. Claim 21 In claim 13, the protein / peptide antigen comprises a sequence described as any one of SEQ ID NO. 9 and / or SEQ ID NO. 17, SEQ ID NO. 10 and / or SEQ ID NO. 18, in a conjugate. Claim 22 In paragraph 13, the protein / peptide antigen is a fusion protein that is ZIKV-E-Fc; the Fc is a human or rat IgG Fc fragment; or the fusion protein is a conjugate that is HCV-E2-his and / or HCV-E1-his. Claim 23 A conjugate according to claim 13, wherein the protein / peptide antigen comprises a sequence described as 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. Claim 24 In paragraph 13, the conjugate having a molecular weight of 400-14000 KDa. Claim 25 An immunogenic composition comprising the glycoprotein / peptide antigen conjugate of claim 13, an adjuvant, and an excipient, for use in the prevention or treatment of diseases caused by pathogen-associated protein / peptide antigens defined in claim 13. Claim 26 An immunogenic composition comprising the glycoprotein / peptide antigen conjugate of claim 13, an adjuvant, and an excipient, for use in the manufacture of a vaccine or drug for the prevention or treatment of a disease caused by a pathogen-associated protein / peptide antigen defined in claim 13. Claim 27 An immunogenic composition according to claim 25, wherein the adjuvant is selected from aluminum adjuvant, oil-in-water emulsion adjuvant, MF59, QS-21, and lipid monophosphate A. Claim 28 An immunogenic composition according to claim 26, wherein the adjuvant is selected from aluminum adjuvant, oil-in-water emulsion adjuvant, MF59, QS-21, and lipid monophosphate A. Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete

Images