Methods to enhance antigen immunogenicity by forming Fc fragment fusion protein glycoconjugates

Abstract

A method for enhancing the immunogenicity of a protein / peptide antigen is provided, wherein the protein / peptide antigen forms a fusion protein with an Fc fragment, preferably with receptor-binding / complement-binding enhanced Fc fragment, and further forms a fusion protein glycoconjugate with a glycosyl group. In the preferred embodiment, the Fc fragment, due to alterations in its amino acid sequence and / or glycosylation, has an enhanced binding capacity to Fc receptors and / or complement protein C1q compared to its native form. Such vaccines can maintain long-term humoral and cellular immune responses, exhibiting higher cellular immune responses and producing higher titers of neutralizing antibodies in immunized animals. Using the SARS-CoV-2 RBD region as the antigen in an immunizing composition, it is formed into a fusion protein with an Fc region that has undergone amino acid sequence and fucose alteration, and further forms a fusion protein glycoconjugate with pneumococcal polysaccharide, for the prevention of SARS-CoV-2 infection-related diseases.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202010415139.8, filed on May 15, 2020, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of immunology, specifically relating to a method for enhancing the immunogenicity of protein / peptide antigens. The protein / peptide antigen forms a fusion protein with an Fc fragment, preferably with receptor- and complement-enhanced binding, and further conjugates with glycosyl groups to form a fusion protein glycoconjugate. In the preferred embodiment, the Fc fragment, due to alterations in its amino acid sequence and / or glycosylation, exhibits enhanced binding ability to Fc receptors and / or complement protein C1q compared to its native form. Such vaccines can maintain long-term humoral and cellular immune responses, with higher cellular immune responses and higher titers of neutralizing antibodies produced in immunized animals. One example of this invention uses the SARS-CoV-2 RBD region as the antigen in an immune composition, forming a fusion protein with an Fc region that has undergone alterations in its amino acid sequence and fucose content, and further conjugating it with pneumococcal polysaccharide to form a fusion protein glycoconjugate for the prevention of SARS-CoV-2 infection-related diseases. Background Technology

[0004] Safety and immunogenicity are core concerns in vaccine development. In vivo vaccines, which use subunits of infectious microorganisms, toxins, viruses, and tumor cells as antigens, are widely adopted due to their safety profile. However, subunit antigens have poor immunogenicity and a weaker ability to induce specific immune responses.

[0005] Traditional methods for enhancing immunogenicity involve adding immune adjuvants. New methods for enhancing immune responses are still under continuous research and exploration.

[0006] One important approach is to conjugate poorly immunogenic antigens with exogenous macromolecules used as carriers; this method has been successfully applied for decades. Fc fusion proteins represent another emerging approach, involving the fusion of a biologically active functional protein with an Fc fragment to create novel recombinant proteins. These proteins not only retain the biological activity of the functional protein molecule but also possess some antibody properties, such as the binding and mediating of related biological functions by Fc receptors. These two approaches will be described in detail below.

[0007] 1. Conjugate vaccine

[0008] Conjugate vaccines in common use are meningitis vaccine, Haemophilus influenzae type b vaccine and pneumococcal vaccine, in which purified capsular polysaccharide is conjugated to carrier protein to produce more effective immunogenic composition. Commonly used carrier proteins are tetanus toxoid, tetanus toxoid fragment C, tetanus toxin non-toxic mutant, diphtheria toxoid, CRM197, other non-toxic mutants of diphtheria toxin such as CRM176, CRM197, CRM228, CRM45 (Uchida et al. J. Biol. Chem. 218; 3838-3844, 1973); CRM9, CRM45, CRM02, CRM103 and CRM107 and other mutants.

[0009] It is worth noting that the above conjugate vaccines use polysaccharide as antigenic substance. Such polysaccharide antigens are non-thymus cell dependent antigens, which cannot produce cellular immune response and form immune memory. In children or immunocompromised population, protective antibodies cannot be formed. Conjugation of polysaccharide antigen with protein carrier having T cell epitope, endocytosis and processing of the conjugate of sugar and protein in antigen presenting cells or B cells, followed by display of polypeptide fragments of carrier protein on cell surface, activation of helper T cells, and generation of protective antibodies and immune memory in series immune response.

[0010] However, there is no report on the improvement of immunogenicity of protein / peptide antigen after conjugation with sugar. US5192540A discloses a vaccine containing immunogenic conjugate of Haemophilus influenzae type B 38,000 Dalton or 40,000 Dalton outer membrane protein and Haemophilus influenzae type B oxidized polyribosyl-ribitol-phosphate polysaccharide fragment, which can be used to immunize against diseases caused by Haemophilus influenzae type B. However, "the conjugate vaccines of the invention are highly immunogenic in animal models. They elicit significantly higher antibody responses to PRP than previously reported. The conjugate vaccines also induce antibodies to the major protein of Haemophilus influenzae type B (38K or 40k protein)".

[0011] US9296795B discloses the use of an immunogenic polysaccharide-protein conjugate having a polysaccharide antigen (or an oligosaccharide fragment thereof, representing one or more antigenic epitopes) derived from a hospital pathogen in an immunogenic composition. The polysaccharide-protein conjugate conjugate is conjugated to a Staphylococcus aureus surface adhesin carrier protein to elicit an antibody response against the polysaccharide antigen and the Staphylococcus aureus surface adhesin carrier protein. The patent discloses that "the conjugate of the present invention has the unique advantage of inducing the production of antibodies against both the polysaccharide antigen and the surface adhesin carrier protein (both of which are virulence factors), and conferring immunity against diseases caused by hospital pathogens. That is, the surface adhesin protein itself can also confer immunity, rather than simply acting as a protein carrier for the polysaccharide antigen." "The titers of surface adhesin protein-specific antibodies induced by the conjugated surface adhesin protein are similar to those of the unconjugated surface adhesin protein. This confirms that the antigenic epitope is not altered by the binding of the surface adhesin protein and CP."

[0012] To the inventor's knowledge, only the two studies mentioned above have reported that when protein / peptide-polysaccharide conjugates are used as antigens, the protein portion does not act as a carrier protein to enhance the immunogenicity of the polysaccharide, but rather exists as an immunogenic substance. However, these two studies stop at the fact that the protein / peptide portion can also induce antibody production, but there is no report of enhanced immunogenicity. The inventor's invention patent application entitled "A Method for Enhancing the Immunogenicity of Protein-Peptide Antigens," application number PCT / CN2021 / 090809 (prior national application priority application number CN202010369100.7), filed on April 29, 2021, reports the applicant's groundbreaking invention: by conjugating protein / peptide antigens with sugars to form sugar-protein / peptide antigen conjugates, the immunogenicity of protein / peptide antigens is improved.

[0013] The inventor's invention patent application entitled "Method for Enhancing the Immunogenicity of Protein / Peptide Antigens by Forming Fusion Proteins with Modified Fc Fragments", application number PCT / CN2021 / 092013 (prior national application priority application number CN202010394463.6), filed on May 7, 2021, reports another groundbreaking invention by the applicant: by forming fusion proteins with protein / peptide antigens and Fc fragments, preferably with receptor binding / complement binding enhancing the Fc fragments, the immunogenicity of protein / peptide antigens is improved.

[0014] 2. Fc receptor fusion protein vaccine

[0015] B cell-mediated humoral immunity is one of the vaccine-mediated protective mechanisms. Studies have shown that the interaction between Fc cells and Fc receptors (FcRs) and complement receptors (CRs) can mediate better antigen capture and presentation, promote B cell affinity maturation, and generate high-affinity antibodies. 1-6Furthermore, after Fc binds to receptors on follicular dendritic cells (fDCs) (mediated by FcR or CR), it is displayed on the surface of fDCs. This antigen is extremely important for maintaining the long-term presence of antigens and the survival of antigen-specific B cells. 7 ].

[0016] Furthermore, cytotoxic T lymphocytes (CTLs) play a crucial role in resisting viral infection and clearing virus-infected cells. Antigen cross-presentation allows exogenous antigens to enter the cell's endogenous processing and presentation mechanisms, thereby displaying exogenous antigen peptides on MHC class I molecules for T cell recognition and initiation of a CTL cell response. Cross-presentation of exogenous antigens is significant for effectively activating CTLs and triggering an antiviral immune response; therefore, enhancing the cross-presentation of subunit vaccines is one of the effective strategies for improving vaccine efficacy. Dendritic cells (DCs) are currently known as the most potent professional antigen-presenting cells and are the primary cells responsible for cross-presentation. Exogenous antigens enter DCs mainly through three pathways: phagocytosis, pinocytosis, and receptor-mediated endocytosis. Currently known endocytosis receptors involved in cross-presentation of exogenous antigens include C-type lectin receptors (CLRs), Fc receptors (FcRs) that recognize immune complex IgG, scavenger receptors that recognize apoptotic cells, and chemokine receptors. These receptors mediate antigen endocytosis, allowing the antigen to enter specific endosomes and bind to MHC class I molecules, thereby activating CD8+. + T cells. Antigen-antibody complexes (immune complexes) can be recognized by FcRs of dendritic cells (DCs), and the resulting cross-linking can internalize antigens and cross-process and present them, specifically activating CTL responses. 8-10 This FcR-mediated antigen cross-presentation has been shown to induce strong CTL responses. 11 ].

[0017] The Fc receptors (FcRγ) that bind to IgG in the human body mainly include FcγRI (CD64), FcγRIIA (CD32a), FcγRIIB (CD32B), and FcγRIII (CD16), etc. 12 FcγRIIB is an inhibitory receptor, mainly expressed on B cells, macrophages, and mast cells. 13 It can be further divided into FcγRIIB-1 and FcγRIIB-2. FcγRIIB-1 is expressed only on B cells and controls the overactivation of B cells and the recognition of self-antigens during B cell development. It transmits apoptosis signals to B cells through the intracellular receptor tyrosine inhibitory motif (ITIM) to achieve the negative selection process of B cells and regulate the development process of B cells. FcγRIIB-2 is expressed on other immune cells except NK and T cells. Through receptor cross-linking, it can effectively induce the phagocytosis of antigen-antibody complexes.14 , 15 The remaining Fc receptors are all activating receptors. CD64 is mainly expressed on monocytes, macrophages, and dendritic cells (DCs); CD32a is mainly expressed on neutrophils, monocytes, macrophages, and DCs; and CD16a is mainly expressed on NK cells, monocytes, and macrophages. 15 After recognizing antigen-antibody complexes, the intracellular receptor tyrosine activation motif (ITAM) determines the antigen uptake initiated by antigen-presenting cells (APCs) and the antigen presentation function carried out by MHC molecules after uptake. 16 ].

[0018] Fc fusion proteins are novel recombinant proteins produced by fusing a bioactive functional protein with an Fc fragment using genetic engineering and other technologies. They not only retain the biological activity of the functional protein molecule but also possess some antibody properties, such as the binding and mediating of Fc receptors. Antigen-Fc fusion proteins can serve as antigen delivery vehicles, using the Fc fragment to target and bind to antigen-presenting cells, shortening the antigen's free time in plasma, increasing its half-life, and thus enhancing antigen presentation and cross-presentation reactions.

[0019] Currently, the number of Fc fusion protein vaccines undergoing clinical trials is relatively small. In a phase IIb clinical trial, chronic cHBV-infected patients treated with a YIC immunogenic complex (a mixture of yeast-expressed hepatitis B surface antigen HBsAg and HBsAg-immunized human serum neutralizing antibody HBIG in a specific ratio, with the addition of aluminum adjuvant) at 60 μg / 4 weeks showed a significantly higher serum hepatitis B virus e antigen (HBeAg) seroconversion rate (21.8% vs 9%), a significantly lower viral titer, and the production of anti-E antigen antibodies, compared to the control group receiving only aluminum adjuvant. 22 Meanwhile, under in vitro conditions, YIC can increase the maturation (high expression of CD83), antigen recognition and presentation (expression of HLA-II, CD86, CD80, and CD40 markers) of DC cells from cHBV-infected patients, and secrete more inflammatory factors (IL-12). The patient's DC-PBMC mixed cells, stimulated with YIC, produced more T lymphocyte cytokines (Th1 cells: IL-2, IFNγ) and (Th2 cells: IL-5, IL-10) than those stimulated with HBsAg antigen alone. 23 However, excessive stimulation by YIC may induce immune fatigue in the body, thereby reducing cellular immune responses. 24 Therefore, a suitable immunization regimen is particularly important in order to achieve better immunization results.

[0020] Other vaccines based on viral antigen-Fc fusion proteins (RSV, HBV, DENV, TB) have also been tested in animal models, achieving effective activation of the immune system and producing antibodies that reduce serum viral antigen levels. 25- 28 ].

[0021] The safety risks arising from the activation of the immune system by Fc fusion protein vaccines are also a topic worthy of consideration. In the chronic hepatitis B cHBV-infected patients mentioned above, a small number experienced a transient increase in alanine aminotransferase (ALT) levels for a period of time after YIC treatment. The proportion of patients experiencing ALT elevation was similar compared to the control group receiving only aluminum adjuvant. The increase in transaminase levels reflects liver damage to some extent, but subsequent observations showed that ALT levels could return to normal. 24 In its Phase IIa clinical trials, elevated ALT levels were observed in some patients who had achieved HBeAg seroconversion. 23 The adverse events (AEs) data from the Phase IIb clinical trial of YIC showed that the proportions of serious AEs were similar in the YIC 30μg dose group, the YIC 60μg dose group, and the aluminum adjuvant control group (3.6% vs 5% vs 5.1%). The most common AEs in the YIC group were injection site-related reactions, including inflammatory reactions such as rash, swelling, and itching. Other systemic AEs, such as fever, headache, and nausea, showed no significant difference compared to the control group. 22 Therefore, immune complexes containing Fc have a relatively good safety profile in clinical practice, with no serious adverse reactions caused by excessive activation of the immune system.

[0022] Modifying the Fc fragment to enhance FcRs binding can yield Fe4-Fc modified molecules, which can increase the binding of Fc to complement protein C1q and Fc receptors CD16, CD32, CD64, etc. 17-21 This could potentially further enhance Fc and its receptor-mediated antigen capture and presentation, improve B cell maturation and the production of high-affinity antibodies, maintain long-term humoral immunity, and enhance CTL immune responses mediated by antigen cross-presentation.

[0023] The inventor's invention patent application, entitled "A Method for Enhancing the Immunogenicity of Protein / Peptide Antigens by Forming a Fusion Protein with a Modified Fc Fragment," with application number CN202010394463.6 and application date of May 11, 2020, reports the applicant's groundbreaking invention: by forming a fusion protein with a modified antibody Fc fragment, the immunogenicity of the protein / peptide antigen is enhanced, wherein the Fc fragment, due to changes in its amino acid sequence and / or glycosylation, has an enhanced binding capacity to Fc receptors and / or complement protein C1q compared to its native form. Summary of the Invention

[0024] In one aspect, the present invention provides a method for enhancing the immunogenicity of a protein / peptide antigen, the method comprising:

[0025] The protein / peptide antigen is fused with the Fc fragment to form a fusion protein, and then...

[0026] The fusion protein is conjugated with sugar to form a fusion protein glycoconjugate, preferably

[0027] The Fc fragment is an enhanced Fc fragment that binds to the receptor / complement.

[0028] In one embodiment, the protein / peptide antigen in the method is a pathogen-associated protein / peptide antigen or a tumor-associated protein / peptide antigen.

[0029] In one embodiment, the pathogen in the method is selected from:

[0030] 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, Flavivir or Influenza Virus, Neisseria spp., Moraxella spp., Bordetella spp., Mycobacterium spp., including Mycobacterium tuberculosis; Escherichia spp., including Enterotoxigenic Escherichia coli; Salmonella spp., Listeria spp., Helicobacter spp., Staphylococcus spp., including Staphylococcus aureus, Staphylococcus epidermidis; Treponema spp., Chlamydia spp., including Chlamydia trachomatis, Chlamydia pneumoniae; Plasmodium spp., including Plasmodium falciparum; Toxoplasma gondii, Candida albicans;

[0031] The tumors were selected from:

[0032] Diffuse large B-cell lymphoma, follicular lymphoma, other lymphomas, leukemia, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdomyosarcoma, 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 carcinoma, ovarian cancer, pancreatic cancer, renal cell carcinoma, rectal cancer, thyroid cancer, parathyroid tumors, uterine tumors and soft tissue sarcoma.

[0033] In one embodiment, the protein / peptide antigen in the method is selected from secreted proteins or full-length membrane proteins, or their functional domains, mutant proteins, truncated proteins, or modified proteins composed of one or more antigenic polypeptide epitopes.

[0034] In one embodiment, the protein / peptide antigen in the method is selected from...

[0035] Coronavirus antigen;

[0036] Preferred coronavirus spike protein;

[0037] More preferably, fragments of the coronavirus spike protein;

[0038] More preferably, the ACE2 receptor-binding domain (RBD) of the coronavirus spike protein;

[0039] More preferably, the S1 subunit of the coronavirus spike protein;

[0040] Preferably, the coronavirus is selected from SARS-CoV-2.

[0041] In one embodiment, the Fc fragment in the method is derived from the heavy chain constant region of a human antibody, mouse antibody, rabbit antibody, or other animal antibody.

[0042] In one embodiment, the Fc fragment in the method is derived from an IgG, IgM, or IgA subtype antibody of a human antibody.

[0043] Preferably, the antibody is derived from IgG1, IgG2, IgG3 or IgG4 subtypes;

[0044] More preferably, it is an IgG1-modified Fc fragment with altered amino acid sequence and / or glycosylation form for the purpose of improving the binding function with Fc receptor / C1q complement.

[0045] In one embodiment, the Fc receptor of the method is selected from CD16, CD32a, CD32b or CD64.

[0046] In one implementation, the method

[0047] The modified antibody Fc fragment is an enhanced fragment for binding to Fc receptors CD32a, CD32b, and CD64 / an enhanced fragment for binding to complement C1q;

[0048] Its amino acid sequence is shown in SEQ ID NO: 3.

[0049] In one implementation, the method

[0050] The modified antibody Fc fragment is an enhanced fragment for binding to Fc receptors CD16a, CD32a, CD32b, and CD64 / an enhanced fragment for binding to complement C1q.

[0051] Its amino acid sequence is shown in SEQ ID NO: 4, and it is produced using fucose-knockout mammalian cells, preferably, the mammalian cells are HEK-293 cells with the fut8 gene knocked out.

[0052] In one embodiment, the antigen in the method is preferably conjugated to other macromolecules via a linker, preferably, the other macromolecules being polysaccharides, peptides / proteins.

[0053] In one embodiment, the other macromolecules in the method are selected from polysaccharides, oligosaccharides, or monosaccharides;

[0054] Preferred ingredients include Neisseria gonorrhoeae capsular polysaccharide, Haemophilus influenzae B capsular polysaccharide, Streptococcus pneumoniae capsular polysaccharide, Staphylococcus aureus group B capsular polysaccharide, dextran, mannan, starch, inulin, pectin, carboxymethyl starch, chitosan and their derivatives;

[0055] More preferably, it is a polysaccharide from the capsular region of Streptococcus pneumoniae;

[0056] More preferably, it is capsular polysaccharide of Streptococcus pneumoniae serotype 14, capsular polysaccharide of Streptococcus pneumoniae serotype 6B and capsular polysaccharide of Streptococcus pneumoniae serotype 7F;

[0057] The preferred option is capsular polysaccharide from Streptococcus pneumoniae serotype 14.

[0058] In one embodiment, the molecular weight of the conjugate in the method is 800-6000 kDa.

[0059] In one embodiment, the method combines a protein / peptide antigen in the form of a fusion protein glycoconjugate with an immune adjuvant.

[0060] In one embodiment, the adjuvant in the method is selected from aluminum adjuvants, MF59, or mixtures thereof.

[0061] In another aspect, the present invention provides an immunogenically enhanced protein / peptide antigen comprising fusing the protein / peptide antigen with an Fc fragment to form a fusion protein, and then...

[0062] Protein / peptide-Fc fragment fusion protein glycoconjugates formed by glycoconjugation; preferably

[0063] The Fc fragment is an enhanced Fc fragment that binds to the receptor / complement.

[0064] In one embodiment, the protein / peptide antigen is a pathogen-associated protein / peptide antigen or a tumor-associated protein / peptide antigen.

[0065] In one embodiment, the protein / peptide antigen,

[0066] The pathogens mentioned therein are selected from:

[0067] 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, Flavivir or Influenza Virus, Neisseria spp., Moraxella spp., Bordetella spp., Mycobacterium spp., including Mycobacterium tuberculosis; Escherichia spp., including Enterotoxigenic Escherichia coli; Salmonella spp., Listeria spp., Helicobacter spp., Staphylococcus spp., including Staphylococcus aureus, Staphylococcus epidermidis; Treponema spp., Chlamydia spp., including Chlamydia trachomatis, Chlamydia pneumoniae; Plasmodium spp., including Plasmodium falciparum; Toxoplasma gondii, Candida albicans;

[0068] The tumors were selected from:

[0069] Diffuse large B-cell lymphoma, follicular lymphoma, other lymphomas, leukemia, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdomyosarcoma, 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 carcinoma, ovarian cancer, pancreatic cancer, renal cell carcinoma, rectal cancer, thyroid cancer, parathyroid tumors, uterine tumors and soft tissue sarcoma.

[0070] In one embodiment, the protein / peptide antigen is selected from secretory proteins or full-length membrane proteins, or their functional domains, mutant proteins, truncated proteins, or modified proteins composed of one or more antigenic polypeptide epitopes.

[0071] In one embodiment, the protein / peptide antigen is selected from...

[0072] Coronavirus antigen;

[0073] Preferred coronavirus spike protein;

[0074] More preferably, fragments of the coronavirus spike protein;

[0075] More preferably, the ACE2 receptor-binding domain (RBD) of the coronavirus spike protein;

[0076] More preferably, the S1 subunit of the coronavirus spike protein;

[0077] Preferably, the coronavirus is selected from SARS-CoV-2.

[0078] In one embodiment, the Fc fragment in the protein / peptide antigen is derived from the heavy chain constant region of a human antibody, mouse antibody, rabbit antibody, or other animal antibody.

[0079] In one embodiment, the Fc fragment in the protein / peptide antigen is derived from a human antibody subtype IgG, IgM, or IgA.

[0080] Preferably, the antibody is derived from IgG1, IgG2, IgG3 or IgG4 subtypes;

[0081] More preferably, it is an IgG1-modified Fc fragment with altered amino acid sequence and / or glycosylation form for the purpose of improving its binding function with Fc receptor and C1q complement.

[0082] In one embodiment, the protein / peptide antigen, Fc receptor is selected from CD16, CD32a, CD32b, or CD64.

[0083] In one embodiment, the protein / peptide antigen

[0084] The modified antibody Fc fragment is an enhanced fragment for binding to Fc receptors CD32a, CD32b, and CD64 / an enhanced fragment for binding to complement C1q;

[0085] Its amino acid sequence is shown in SEQ ID NO: 3.

[0086] In one embodiment, the protein / peptide antigen

[0087] The modified antibody Fc fragment is an enhanced fragment for binding to Fc receptors CD16a, CD32a, CD32b, and CD64 / an enhanced fragment for binding to complement C1q.

[0088] Its amino acid sequence is shown in SEQ ID NO: 4, and it is produced using fucose-knockout mammalian cells, preferably, the mammalian cells are HEK-293 cells with the fut8 gene knocked out.

[0089] In one embodiment, the antigen in the protein / peptide antigen is preferably conjugated to other macromolecules via a linker, and preferably, the other macromolecules are polysaccharides, peptides / proteins.

[0090] In one embodiment, the other macromolecules in the protein / peptide antigen are selected from polysaccharides, oligosaccharides, or monosaccharides.

[0091] Preferred ingredients include Neisseria gonorrhoeae capsular polysaccharide, Haemophilus influenzae B capsular polysaccharide, Streptococcus pneumoniae capsular polysaccharide, Staphylococcus aureus group B capsular polysaccharide, dextran, mannan, starch, inulin, pectin, carboxymethyl starch, chitosan and their derivatives;

[0092] More preferably, it is a polysaccharide from the capsular region of Streptococcus pneumoniae;

[0093] More preferably, it is capsular polysaccharide of Streptococcus pneumoniae serotype 14, capsular polysaccharide of Streptococcus pneumoniae serotype 6B and capsular polysaccharide of Streptococcus pneumoniae serotype 7F;

[0094] The preferred option is capsular polysaccharide from Streptococcus pneumoniae serotype 14.

[0095] In one embodiment, the molecular weight of the conjugate in the protein / peptide antigen is 800-6000 kDa.

[0096] In one embodiment, the adjuvant in the protein / peptide antigen is selected from aluminum adjuvant, MF59, or a mixture thereof.

[0097] In another aspect, the present invention provides an immune composition comprising...

[0098] The protein / peptide antigen described in this invention;

[0099] adjuvants; and

[0100] Pharmaceutically acceptable carriers, excipients, or stabilizers, preferably

[0101] Pharmaceutically acceptable carriers, excipients, or stabilizers in lyophilized or aqueous solutions.

[0102] In one embodiment, the adjuvant in the immune composition may be selected from at least one of aluminum adjuvant and MF59.

[0103] In another aspect, the present invention provides the use of the protein / peptide antigen or the immune composition described herein for the prevention of diseases / tumors caused by pathogens, preferably coronaviruses, more preferably SARS-CoV-2.

[0104] In another aspect, the present invention provides the use of the protein / peptide antigen or the immune composition described herein in the preparation of a vaccine for preventing diseases / tumors caused by pathogens, preferably coronaviruses, more preferably SARS-CoV-2.

[0105] In another aspect, the present invention provides an immune combination comprising...

[0106] The protein / peptide antigen described in this invention, or the immune composition described in this invention; and

[0107] One or more other immunogenic agents.

[0108] In another aspect, the present invention provides a kit comprising...

[0109] The protein / peptide antigen or the immune composition described in this invention;

[0110] Preferably,

[0111] It further includes a device for administering the immune composition.

[0112] In another aspect, the present invention provides a method for preventing disease / tumor caused by pathogens, preferably coronaviruses, more preferably SARS-CoV-2, comprising administering to a subject the protein / peptide antigen described in the present invention, the immune composition described in the present invention, the immune combination described in the present invention, or the kit described in the present invention.

[0113] In another aspect, the present invention provides a method for immunizing an animal, comprising administering the animal the protein / peptide antigen described in the present invention, the immune composition described in the present invention, the immune combination described in the present invention, or the kit described in the present invention to generate neutralizing antibodies. Attached Figure Description

[0114] Figure 1 The results show the IgG2a / IgG1 ratios of mice immunized with RBD-Fc-Fe4 and RBD-Fc-Fe4-PS14 as antigens, aluminum adjuvant, MF59 adjuvant, and a mixture of MF59 and aluminum adjuvant. Each adjuvant was further administered at three doses: 1 μg, 3 μg, and 10 μg. Serum samples were collected after the second immunization to determine the IgG2a / IgG1 ratio.

[0115] Figure 2 The comparison of serum IgG2a / IgG1 ratios for RBD-PS14, RBD-Fc-PS14, RBD-Fc-Ce3-PS14, and RBD-Fc-Fe4-PS14 antigens is shown. Invention Details

[0117] Building upon the two groundbreaking inventions mentioned above, the inventors have developed a method to further enhance the immunogenicity of protein / peptide antigens: fusing the protein / peptide antigen with an Fc fragment, preferably a receptor-binding / complement-binding enhanced Fc fragment, to form a fusion protein; then, conjugating this fusion protein with a glycosylation compound. The immunogenicity of this modified protein / peptide antigen is enhanced, and the aforementioned Fc fragment, due to its altered amino acid sequence and / or glycosylation, exhibits improved binding ability to Fc receptors and / or complement protein C1q.

[0118] The inventors used this invention to prepare a vaccine for the novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2).

[0119] SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, namely angiotensin-converting enzyme 2 (ACE2)

[29] . After the viral trimeric S protein binds to the ACE2 receptor, it is cleaved by the host protease into an S1 polypeptide containing a receptor binding domain (RBD) and an S2 polypeptide responsible for mediating viral fusion with the cell membrane

[30] . The specific interaction between S1 and ACE2 triggers a conformational change in the S2 subunit, which leads to the fusion of the viral envelope with the cell membrane or lysosomal membrane and release of viral nucleic acid into the cytoplasm

[31] . Data show that COVID-19 patients, especially those with severe symptoms, have significantly reduced lymphocytes and significantly increased plasma pro-inflammatory factors, suggesting that the immune system plays an important role in the disease process[32-34]. Analysis of serum antibodies in 23 COVID-19 patients after the onset of symptoms showed that most patients developed an antibody response against the RBD protein 10 days after the onset of symptoms

[35] . In the early stages of the disease, the proportion of patients with positive RBD protein antibodies was higher than that of patients with positive N protein antibodies, indicating that the body may first produce neutralizing antibodies to inhibit viral invasion of cells via RBD. Analysis of cellular immunity showed that the specific T cells against different antigens in newly discharged patients differed significantly from those in uninfected individuals, with RBD-specific T cells being the most widely distributed. Cellular immunity levels in patients followed up two weeks after recovery were significantly reduced. RBD not only induces humoral immunity and the production of neutralizing antibodies but also induces T-cell immune responses; therefore, RBD protein is an effective target for SARS-CoV-2 vaccines.

[0120] definition

[0121] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. For the purposes of this invention, the following terms are further defined.

[0122] When used herein and in the appended claims, the singular forms “a,” “an,” “another,” and “the” include the plural referents unless the context clearly indicates otherwise.

[0123] The terms "comprising" or "including" mean that a specific ingredient is included without excluding any other ingredients. Phrases such as "consistently composed of" allow the inclusion of other ingredients or steps that do not impair the novelty or essential characteristics of the invention; that is, they exclude other unlisted ingredients or steps that would impair the novelty or essential characteristics of the invention. The term "composed of" means that a specific ingredient or group of ingredients is included and all other ingredients are excluded.

[0124] The term " / " includes both "and" and "or" cases.

[0125] The term "antigen" refers to a foreign substance that is recognized (specifically bound) by an antibody or T-cell receptor, but which cannot definitively induce an immune response. Foreign substances that induce specific immunity are called "immunogenic antigens" or "immunogens." A "hapten" is an antigen that cannot elicit an immune response on its own (although a combination of several hapten molecules, or a combination of a hapten and a large molecular carrier, can elicit an immune response).

[0126] The term "RBD receptor-binding domain" specifically refers to the "ACE2 receptor-binding domain of the coronavirus spike protein (SARS-CoV-2 RBD)" in this specification and the appended claims. These terms are used interchangeably. SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, angiotensin-converting enzyme 2 (ACE2). After the viral trimeric S protein binds to the ACE2 receptor, it is cleaved by host proteases into an S1 polypeptide containing the receptor-binding domain (RBD) and an S2 polypeptide responsible for mediating viral fusion with the cell membrane.

[0127] "Humoral immune response" is an antibody-mediated immune response and involves the introduction and generation of antibodies that recognize and bind to antigens in the immunogenic compositions of the present invention with a certain affinity. "Cell-mediated immune response" is an immune response mediated by T cells and / or other leukocytes. "Cell-mediated immune response" is induced by the provision of antigenic epitopes associated with class I or II molecules of the major histocompatibility complex (MHC), CD1, or other atypical MHC-like molecules.

[0128] The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein” encompass chains of amino acids of any length, with relatively short chains (e.g., shorter than 100 amino acids) often referred to as peptides. This chain can be linear or branched, and may contain modified amino acids and / or intercalated non-amino acids.

[0129] The term "antibody" refers to an immunoglobulin molecule, meaning any form of antibody that expresses desired biological activity. This includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), and even antibody fragments. Typically, a full-length antibody structure preferably comprises four polypeptide chains, typically two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. Each light chain contains a light chain variable region and a light chain constant region. Besides this typical full-length antibody structure, other derived forms are also possible.

[0130] Based on the amino acid sequence of their heavy chain constant region, complete antibodies can be classified into five classes: IgA, IgD, IgE, IgG, and IgM. IgG and IgA can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Correspondingly, the heavy chains of these five antibody classes are classified as α, δ, ε, γ, and μ chains, respectively. Based on the amino acid sequence of their light chain constant region, the light chains of antibodies can be classified as κ and λ.

[0131] The term "variable region" refers to the domain in the antibody heavy or light chain that is involved in antibody binding to the antigen.

[0132] The term "constant region" refers to certain amino acid sequences on the light and heavy chains of an antibody that do not directly participate in the binding of the antibody to the antigen, but exhibit a variety of effector functions, such as antibody-dependent cytotoxicity.

[0133] The term "Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as an amino acid residue extending from Cys226 or from Pro230 to its C-terminus. The residues in the Fc region are numbered as per the EU index in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The IgG Fc region typically has two constant regions, CH2 and CH3.

[0134] The term “Fc receptor” or “FcR” refers to a receptor that binds to the Fc region of an antibody. Preferred are human FcRs of natural sequence, and more preferably are receptors (γ receptors) that bind to IgG antibodies, including FcγRI, FcγRII, and FcγRIII subtypes, as well as variants of these receptors. Other FcRs are included in the term “FcR”. The term also includes the neonatal receptor (FcRn), which is responsible for transporting maternal IgG to the fetus (Guyer et al., Journal of Immunology 117:587 (1976) and Kim et al., Journal of Immunology 24:249 (1994)).

[0135] The term "neonatal Fc receptor," or simply "FcRn," refers to the receptor that binds to the Fc region of IgG antibodies. Neonatal Fc receptors (FcRn) play a crucial role in the metabolic fate of IgG antibodies in vivo. FcRn functions to rescue IgG from lysosomal degradation pathways, thereby reducing its clearance in serum and prolonging its half-life. Therefore, the in vitro FcRn binding properties / characteristics of IgG indicate its in vivo pharmacokinetic properties in the bloodstream.

[0136] The term "Fc fusion protein" refers to a novel recombinant protein produced by fusing a biologically active functional protein with an Fc fragment using techniques such as genetic engineering. It not only retains the biological activity of the functional protein molecule, but also has some antibody properties, such as the binding of FcRs and related biological functions.

[0137] The terms “modified Fc peptide”, “modified Fc region” and “modified Fc” are used interchangeably herein to refer to an Fc peptide or a portion thereof that contains at least one amino acid modification or a modified glycosylation modification thereof.

[0138] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary from antibody isotype to antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor (e.g., CD16, CD32, CD64) binding, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0139] The term "sugar" can refer to polysaccharides, oligosaccharides, or monosaccharides. Polysaccharides can be isolated from organisms, such as bacteria, and can be naturally occurring polysaccharides. Optionally, their size can be adjusted using microfluidic methods. Size adjustment of polysaccharides can reduce the viscosity of polysaccharide samples and / or improve the filterability of conjugated products. Oligosaccharides are hydrolyzed polysaccharides with a small number of repeating units (typically 5-30 repeating units). Polysaccharides can also be chemically synthesized.

[0140] The term "conjugate" refers to a protein / peptide covalently conjugated to a sugar. The fusion protein glycoconjugates and immunogenic compositions comprising them of the present invention may contain a certain amount of free sugar, protein / peptide.

[0141] As used in this article, “conjugation” refers to the process of covalently linking sugars, such as bacterial capsular polysaccharides, to proteins / peptides.

[0142] The term "immunogenic composition" refers to any pharmaceutical composition containing an antigen that can be used to induce an immune response in an individual.

[0143] As used herein, “immunogenicity” means the ability of an antigen (or an epitope of an antigen) such as the receptor-binding domain of the coronavirus spike protein (SARS-CoV-2 RBD) or a fusion protein glycoconjugate or immunogenic composition containing the antigen to induce a humoral or cellular immune response in a host (e.g., a mammal) or both.

[0144] A “protective” immune response refers to the ability of an immunogenic composition to induce a humoral or cellular immune response, or both, to protect 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 (e.g., infected animals that have not been administered the vaccine or the immunogenic composition). Protection may be limited to mitigating the severity or rapid onset of infection symptoms.

[0145] The terms “immunogenic amount” and “immunogenically effective amount” are used interchangeably herein and refer to the amount of an antigen or immunogenic composition sufficient to elicit an immune response (cellular (T cell) or humoral (B cell or antibody) response, or both, as measured by standard assays known to those skilled in the art).

[0146] The effectiveness of an antigen as an immunogen can be measured by proliferation assay, cell lysis assay, or by measuring B cell activity levels.

[0147] The method of the present invention for improving the immunogenicity of protein / peptide antigens

[0148] This invention is based on two groundbreaking inventions by the inventors. A protein / peptide antigen is fused with an Fc receptor-binding / complement-binding enhancing fragment to form a fusion protein, which is then conjugated with a glycoside to form a fusion protein glycoconjugate. Immunizing animals with these fusion protein glycoconjugates as antigens in an immunizing composition can maintain long-term humoral and cellular immune responses, exhibiting a higher cellular immune response and producing higher titers of neutralizing antibodies.

[0149] The fusion protein glycoconjugate of the present invention as a protein / peptide antigen

[0150] Coronaviruses primarily mediate viral invasion by binding to host cell receptors via their spike protein (S protein), which also determines the virus's tissue or host tropism. The host cell receptor protein for SARS-CoV-2 is angiotensin-converting enzyme 2 (ACE2). After the viral trimeric spike protein (S protein) binds to the ACE2 receptor, it is cleaved by host proteases into an S1 polypeptide containing a receptor-binding domain (SARS-CoV-2RBD) and an S2 polypeptide responsible for mediating viral fusion with the cell membrane, thereby allowing the virus to invade the body.

[0151] One embodiment of the present invention uses SARS-CoV-2RBD as the protein / peptide antigen. The antigen can be obtained by extracting from the natural pathogen or by genetic recombination. The SARS-CoV-2RBD enhances the binding affinity to the Fc receptor and / or complement protein C1q by forming a fusion protein through genetic engineering. In a particularly preferred embodiment, the Fc fragment is an Fc receptor CD32a, CD32b, and CD64 binding enhancement fragment / complement C1q binding enhancement fragment; its amino acid sequence is shown in SEQ ID NO: 3. In a most preferred embodiment, the modified antibody Fc fragment is an Fc receptor CD16a, CD32a, CD32b, and CD64 binding enhancement fragment / complement C1q binding enhancement fragment; its amino acid sequence is shown in SEQ ID NO: 4, and it is produced using fucose-knockout CHO cells. The fusion protein is then conjugated with a sugar, which can be a polysaccharide, oligosaccharide, or monosaccharide.

[0152] The polysaccharides can be bacterial polysaccharides, such as common Neisseria gonorrhoeae capsular polysaccharides, Haemophilus influenzae B capsular polysaccharides, Streptococcus pneumoniae capsular polysaccharides, Staphylococcus aureus group B capsular polysaccharides, as well as dextran, mannan, etc. Polysaccharides can also be plant-derived polysaccharides, such as starch, inulin, pectin, etc., or chemically modified polysaccharide derivatives, such as carboxymethyl starch. The polysaccharides can also be animal-derived polysaccharides, such as chitosan and its derivatives.

[0153] Polysaccharides and proteins bind together through chemical reactions. First, the polysaccharide is activated, meaning it contains reactive groups. These reactive groups then react with chemically reactive groups on the protein molecule, such as amino groups, carboxyl groups, thiol groups, the imidazole ring on histidine, the indole ring on tryptophan, the benzene ring on tyrosine, the benzene ring on phenylalanine, the hydroxyl group on serine, and groups that can undergo chemical reactions, such as glutamine and asparagine, to form covalent bonds.

[0154] One method for polysaccharide-protein molecule conjugation involves the oxidation of polysaccharides using sodium periodate. This oxidation produces aldehyde groups on the polysaccharide, which then react with amino groups on the protein molecule to form a Schiff base. In the presence of a reducing agent, the Schiff base is reduced to a stable single bond, thus forming a covalent bond between the polysaccharide and the protein molecule.

[0155] The immune composition of the present invention

[0156] 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 detergent, a free radical oxidation inhibitor, a diluent, or a carrier.

[0157] An adjuvant is a substance that enhances the immune response when administered together with an immunogen or antigen. The immunogenic compositions of 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, at least one of MF59, QS-21, or MPL.

[0158] In one embodiment, the adjuvant in the immunogenic composition of the present invention is an aluminum-based adjuvant. The adjuvant used will depend on the individual being administered the immunogenic composition, the prescribed route of injection, and the number of injections.

[0159] The immunogenic composition may optionally contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include those used in national pharmacopoeias for animals (including humans and non-human mammals). The term carrier can be used to refer to a diluent, adjuvant, excipient, or medium administered with the pharmaceutical composition. Water, saline solutions, and solutions containing dextrose and glycerol may be used as liquid carriers, particularly for injectable solutions.

[0160] The immunogenic compositions of the present invention may also contain one or more additional immunogenic agents.

[0161] Administration of the immunogenic composition of the present invention

[0162] The immunogenic compositions of the present invention for therapeutic or prophylactic treatment can be administered via intramuscular, intraperitoneal, intradermal, or subcutaneous injection; or via mucosal administration to the oral / esophagus, respiratory tract, or genitourinary 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 in a single dose, their components can also be administered simultaneously or at different times. In addition to a single route of administration, two different routes of administration can be used.

[0163] The optimal amount of a component used in a particular immunogenic composition can be determined through standard studies involving the observation of an appropriate immune response in an individual. Following the initial vaccination, an individual may receive one or more adequately spaced booster immunizations.

[0164] Use of the immunogenic composition of the present invention

[0165] The protein / peptide antigens and immune complexes of this invention can prevent or treat diseases caused by pathogens, especially coronaviruses, and more particularly diseases caused by SARS-CoV-2 virus. The protein / peptide antigens and immune complexes of this invention can also prevent or treat tumor diseases. They can also be used to immunize animals to produce neutralizing antibodies.

[0166] The abbreviation for this invention is RBD or SARS-CoV-2RBD: SARS-CoV-2 coronavirus spike protein receptor-binding domain; RBD-Fc: fusion protein of the SARS-CoV-2 coronavirus spike protein receptor-binding domain and the Fc fragment of human IgG.

[0167] RBD-Fc-Ce3: A fusion protein of the SARS-CoV-2 coronavirus spike protein receptor-binding domain and the Fc fragment of human IgG, wherein the Fc sequence has been modified, as shown in SEQ ID NO:3. RBD-Fc-Fe4: A fusion protein of the SARS-CoV-2 coronavirus spike protein receptor-binding domain and the Fc fragment of human IgG, wherein the Fc sequence has been modified, as shown in SEQ ID NO:4, and expressed by HEK-293 cells with the fut8 gene knocked out. PS14: Streptococcus pneumoniae serotype 14 capsular polysaccharide.

[0168] RBD-PS14: RBD and PS14 conjugate

[0169] RBD-Fc-PS14: RBD-Fc and PS14 conjugate

[0170] RBD-Fc-Fe4-PS14: RBD-Fc-Fe4 and PS14 conjugate

[0171] RBD-Fc-Ce3-PS14: RBD-Fc-Ce3 and PS14 conjugate

[0172] Alum: Aluminum adjuvant, specifically aluminum phosphate adjuvant in this article.

[0173] MF59: Oil-in-water microemulsion adjuvant, see examples for details. Detailed Implementation

[0174] Example

[0175] Example 1: Construction and protein production of expression vectors for SARS-CoV-2 RBD, RBD-Fc, RBD-Fc-Ce3 and RBD-Fc-Fe4 fusion proteins

[0176] 1.1 Construction of SARS-CoV-2 RBD expression vector and protein production

[0177] The SARS-CoV-2-Spike-RBD sequence (SEQ ID NO:7) was obtained by PCR amplification (PCR amplification template was obtained from Beijing Yiqiao Shenzhou Technology Co., Ltd., the same below), which contains the signal peptide sequence (SEQ ID NO:6) and the SARS-CoV-2-Spike-RBD sequence (SEQ ID NO:5). It was then inserted into the pSE vector (source: Shenzhou Cell Engineering Co., Ltd., the same below) digested with Hind III+Xba I (source: Fermentas, the same below) by in-fusion method to obtain the pSE-CoV-2-RBD expression vector (SEQ ID NO:7).

[0178] Amplification primers:

[0179] RBD-1GTCACCGTCCTGACACGAAGCTT GGTACC(SEQ ID NO:16)RBD-2

[0180] TATAGAATAGGGCCCTCTAGATTTAGAAGTTCACACACTTGTTCTTCA CC(SEQ ID NO:17)

[0181] pSE-CoV-2-RBD plasmid was extracted, transfected into HEK-293 cells (source: Invitrogen, the same below) and cultured for expression for 7 days. High-purity SARS-CoV-2RBD protein was obtained after purification.

[0182] 1.2 Construction of SARS-CoV-2 RBD-mFc expression vector and protein production

[0183] The SARS-CoV-2-Spike-RBD sequence was amplified by PCR and inserted into the pSE-mFc vector (source: Shenzhou Cell Engineering Co., Ltd.) containing the signal peptide (SEQ ID NO:6), linker (SEQ ID NO:8), and mouse IgG1 constant region sequence (SEQ ID NO:13) digested with Afe I enzyme and dephosphated by FastAP to obtain the pSE-CoV-2-RBD-mFc expression vector (SEQ ID NO:14).

[0184] Amplification primers:

[0185] RBD-21TTTGTCTCTCCAGCAGGGTGGTGCCATCTGGAGATGT(SEQ ID NO:18)

[0186] RBD-22GTCATCGTCATCAGCGAAGTTCACACACTGGTTCTTAA(SEQ ID NO:19)

[0187] pSE-CoV-2-RBD-mFc plasmid was extracted, transfected into HEK-293 cells, and cultured for expression for 7 days. High-purity RBD-mFc protein was obtained by purification using a protein A purification column.

[0188] 1.3 Construction of SARS-CoV-2 RBD-Fc expression vector and protein production

[0189] The SARS-CoV-2-Spike-RBD sequence was amplified by PCR and inserted into the pSTEP2-Fc vector (source: Shenzhou Cell Engineering Co., Ltd.) containing the signal peptide (SEQ ID NO:6), linker (SEQ ID NO:8), and human IgG1 constant region sequence (SEQ ID NO:9) after being digested with Afe I and dephosphated by FastAP, to obtain the pSE-CoV-2-RBD-Fc expression vector (SEQ ID NO:10).

[0190] Amplification primers:

[0191] RBD-21TTTGTCTCTCCAGCAGGGTGGTGCCATCTGGAGATGT(SEQ ID NO:20)

[0192] RBD-22GTCATCGTCATCAGCGAAGTTCACACACTGGTTCTTAA(SEQ ID NO:21)

[0193] pSE-CoV-2-RBD-Fc plasmid was extracted, transfected into HEK-293 cells, and cultured for expression for 7 days. High-purity RBD-Fc protein was obtained by purification using a protein A purification column.

[0194] 1.4 Construction of SARS-CoV-2RBD-Fc-Ce3 expression vector and protein production

[0195] To enhance the CDC function mediated by the antibody Fc fragment, nucleotide mutations were performed on the constant region of the IgG1 subtype according to the literature [36,37] to obtain the genetically engineered heavy chain IgG1 constant region nucleotide sequence (Fc-Ce3, SEQ ID NO:11).

[0196] The SARS-CoV-2-Spike-RBD-Ce3-Fc sequence (SEQ ID NO:12) was amplified by PCR and inserted into the pSE vector digested with Hind III+Xba I via in-fusion to obtain the pSE-nCoV-2-RBD-Fc-Ce3 expression vector (SEQ ID NO:12).

[0197] Amplification primers:

[0198] RBD-23GTCACCGTCCTGACACGAAGCTTGGTACC(SEQ ID NO:22)RBD-24

[0199] GGGAGAGGCTCTTCTGCGTGTAGTGGTTGTGCAGAGCGCCATGCATCACG GAGCAT(SEQ ID NO:23)

[0200] RBD-25

[0201] TATAGAATAGGGCCCTCTAGATTTATTTACCCGGAGACAGGGAGAGG CTCTTCTGCGTGT(SEQ IDNO:24)

[0202] pSE-nCoV-2-RBD-Fc-Ce3 plasmid was extracted, transfected into HEK-293 cells, and cultured for expression for 7 days. High-purity RBD-Fc-Ce3 was obtained by purification using a protein A purification column.

[0203] 1.5 Construction of SARS-CoV-2 RBD-Fc-Fe4 expression vector and protein production

[0204] To further enhance the immune function mediated by the antibody Fc fragment, the pSE-nCoV-2-RBD-Fc-Ce3 plasmid was extracted, transfected into HEK-293 cells with the fut8 gene knockout (source: Shenzhou Cell Engineering Co., Ltd.), and cultured for expression for 7 days. The high-purity RBD-Fc-Fe4 was obtained by purification using a protein A purification column.

[0205] Example 2: Preparation of Streptococcus pneumoniae serotype 14 capsular polysaccharide (PS14)

[0206] The seed for serotype 14 Streptococcus pneumoniae is ATCC 6314.

[0207] Take 0.5 ml of glycerol-preserved Streptococcus pneumoniae seed and inoculate it into 500 ml of Hoeprich's medium (VMGoncalves, Optimization of medium and cultivation conditions for capsular polysaccharide production by Streptococcus pneumoniae serotype).

[0208] In 23F, Allp Microbiol Biotechnol (2002) 59:713-717), cultured on a shaker at 37℃ and 150 rpm for 10-16 hours until OD was reached. 600 Stop culturing when the pH exceeds 1.0. Add 0.6g of sodium deoxycholate, mix well, and let stand for at least 2 hours to allow complete bacterial lysis. Centrifuge at 14000g for 30 minutes, collect the supernatant, and concentrate it to one-tenth of its original volume (approximately 400ml) using 100kDa ultrafiltration. Gradually add 36% acetic acid to the concentrate to adjust the pH to 3.5. Let stand for 2 hours, centrifuge at 14000g for 30 minutes, collect 390ml of the supernatant, add 130ml of anhydrous ethanol, mix well, and let stand overnight. The next day, centrifuge at 14000g for 30 minutes, collect the supernatant, add another 780ml of anhydrous ethanol, mix well, and let stand overnight. The next day, centrifuge at 14000g for 30 minutes, discard the supernatant, add 300ml of 75% ethanol solution to the precipitate to suspend it, and then centrifuge again at 14000g for 30 minutes. Discard the supernatant, dissolve the precipitate in 10ml of water, and control the polysaccharide concentration in the solution to be greater than 10mg / ml. The resulting solution is a Streptococcus pneumoniae capsular polysaccharide solution.

[0209] Example 3: Activation of capsular polysaccharide of Streptococcus pneumoniae serotype 14

[0210] Take 10 mL of the polysaccharide solution prepared in Example 2 with a concentration of 10 mg / mL, add 100 mg of sodium periodate, mix well, and let stand in the dark for 1 hour. Take a centrifugal chromatography column packed with 5 mL of Sephadex G 25 packing material, add 10 mL of 50 mM Na2HPO4 buffer (pH 7.0), and let the buffer flow through the column under gravity. Then place the column in a centrifuge and centrifuge at 1000 g for 2 min. After that, replace the collection tube, take 1 mL of the polysaccharide solution oxidized with sodium periodate, add it to the centrifugal chromatography column, and centrifuge again at 1000 g for 2 min. The collected effluent from the centrifugal column is the activated polysaccharide solution.

[0211] Example 4: Conjugation of protein / peptide antigens with polysaccharides

[0212] Four SARS-CoV-2 RBD proteins, or their fusion proteins, namely RBD, RBD-Fc, RBD-Fc-Fe4, and RBD-Fc-Ce3, were selected as protein / peptide antigens and conjugated to Streptococcus pneumoniae serotype 14 capsular polysaccharide, as follows:

[0213] 1. Change the buffer for SARS-CoV-2RBD or its fusion protein: Take 5 mg of SARS-CoV-2RBD or its fusion protein and change the buffer to 50 mM Na2HPO4 buffer with pH 7.0 using a 30,000 MW ultrafiltration tube. Finally concentrate the buffer to a volume of less than 0.5 mL, i.e., the final protein concentration is ≥10 mg / mL.

[0214] 2. Conjugation of SARS-CoV-2 RBD or its fusion protein with polysaccharides: Take 3 mg of SARS-CoV-2 RBD or its fusion protein, add 0.5 mg of activated Streptococcus pneumoniae capsular polysaccharide, and add 50 mM Na2HPO4 buffer (pH 7.0) to a final volume of 0.6 ml. Then add 3.6 μl of 5 M sodium cyanoborohydride solution and mix by rotation for 1 h at room temperature in the dark. Next, add 0.15 ml of 10 mg / mL sodium borohydride solution to the reaction solution and react at room temperature for 2 h. Then, use a 100,000 MW ultrafiltration tube, replacing the buffer 10 times with PBS buffer until the final ultrafiltration volume is less than 2 ml. After aseptic filtration through a 0.22 μm filter, store the ultrafiltered conjugate sample at 4 °C.

[0215] 3. The molecular weight of the conjugate was determined using HPLC-MALLS.

[0216] Conjugate Conjugate molecular weight kDa RBD-PS14 6011 / 1237 * ]]> RBD-FC-PS14 5944 / 1242 / 881.9 ** <!-- 14 -->]]> RBD-Fe4-FC-Ce3-PS14 5948 / 1441 / 1337 ** ]]> RBD-Fc-Fe4-PS14 7,850 / 1,886 * ]]

[0217] *Two peaks in molecular weight distribution

[0218] **Three peaks in molecular weight distribution

[0219] Example 5. Preparation of an immunogenic composition of SARS-CoV-2 RBD / its fusion protein glycoconjugate and determination of its immunogenicity.

[0220] Immunogenic compositions were prepared and their immunogenicity was determined using RBD, RBD-Fc, RBD-Fc-Fe4, RBD-Fc-Ce3 and their corresponding polysaccharide conjugates RBD-PS14, RBD-FC-PS14, RBD-Fe4-FC-Ce3-PS14, and RBD-Fc-Fe4-PS14 as antigens, respectively.

[0221] 5.1 Preparation of Immunoassay

[0222] An immune composition was prepared using SARS-CoV-2 RBD protein / its fusion protein or the conjugate prepared in Example 4 as the antigen.

[0223] 5.1.1 Preparation of MF59 adjuvant

[0224] Prepare 200 ml of 10 mM sodium citrate solution (adjust pH to 6.5 with HCl), add 1 ml of Tween 80 (Nanjing Well Pharmaceutical Co., Ltd.), and mix thoroughly to dissolve. Separately, take 10 ml of squalene (Merck) and add 1 ml of Span 85 (Zhaoqing Chaoneng Industrial Co., Ltd.), and mix thoroughly to dissolve. Combine the two solutions and homogenize three times using an AH-PILOTATS high-pressure homogenizer at 800 bar to obtain a homogeneous emulsion, which is the MF59 adjuvant.

[0225] 5.1.2 Preparation of aluminum adjuvant immunomodulatory compositions

[0226] The antigen was diluted with PBS to 0.02 mg / ml, 0.06 mg / ml, or 0.2 mg / ml (based on peptide / protein ratio, the same below), and the aluminum adjuvant (Beijing Nuoning Biotechnology Co., Ltd.) was diluted with PBS to 1 mg / ml. The diluted antigen and aluminum adjuvant were mixed in equal volumes. The protein concentrations of the antigen in this immunizing composition were 0.01 mg / ml, 0.03 mg / ml, or 0.1 mg / ml, respectively.

[0227] 5.1.3 Preparation of MF59 adjuvant immunomodulatory composition

[0228] The antigen was diluted with PBS to 0.02 mg / ml, 0.06 mg / ml, or 0.2 mg / ml, respectively. The diluted antigen was then mixed with an equal volume of MF59 adjuvant. The protein concentration of the antigen in the immunization composition was 0.01 mg / ml, 0.03 mg / ml, or 0.1 mg / ml, respectively.

[0229] 5.1.4 Preparation of MF59 and aluminum adjuvant mixed adjuvant immunomodulatory composition

[0230] Take three 1.5ml portions of aluminum adjuvant, add 1.5ml of MF59 adjuvant to each portion, mix, and then add 0.03ml, 0.09ml, and 0.3ml of antigen with a concentration of 1mg / ml, respectively. The protein concentration of the antigen in this immune composition is 0.01mg / ml, 0.03mg / ml, or 0.1mg / ml, respectively.

[0231] 5.2 Immunization of mice:

[0232] Balb / c mice aged 4-6 weeks were selected and intraperitoneally injected with 0.1 ml of an immunization composition with an antigen concentration of 0.01 mg / ml, 0.03 mg / ml, or 0.1 mg / ml as described in Example 5.1. A booster immunization was performed on day 14 (at the same dose as the initial immunization). Blood was collected from the orbital sinus on day 7 and day 21 to determine serum titer, neutralizing titer, and the IgG2a / IgG1 ratio.

[0233] 5.3 Determination of serum titer

[0234] 5.3.1 Determination of serum titer when the antigen is SARS-CoV-2 RBD or its fusion protein

[0235] SARS-CoV-2RBD-mFc protein (Shenzhou Cell Engineering Co., Ltd., same throughout) at a concentration of 5 μg / mL was coated onto 96-well plates, 100 μl / well. The plates were incubated at room temperature for 2 hours, washed, and then blocked with 2% BSA at room temperature for 1 hour. CD155(D1)-mFc (Shenzhou Cell Engineering Co., Ltd., same throughout) was used as an irrelevant control with the same label. The serum to be tested (prepared in Example 5.2) was diluted to 1:8000 with PBS containing 0.1% bovine serum albumin (BSA). 100 μl / well each of the serum to be tested and goat anti-mouse IgG F(ab)2 / HRP (Beijing Yiqiao Shenzhou Technology Co., Ltd.) detection secondary antibody were added to the coated 96-well plates. After incubation for 2 hours, the plates were washed 5 times, and the substrate chromogenic solution was added for color development. After termination, the OD was read using a microplate reader. 450 OD at a certain dilution factor 450 This indicates the antibody titer.

[0236] The titers of serum (1:8000 dilution) of mice immunized on day 21 with immunization compositions of 1, 3 and 10 μg as antigens (RBD-Fc-Fe4 and RBD-Fc-Fe4-PS14, Alum, MF59 and MF59+Alum as adjuvants) were measured. The results are shown in Table 1.

[0237] Table 1. Serum antibody titers of mice immunized with immunotherapies using RBD-Fc-Fe4 and RBD-Fc-Fe4-PS14 as antigens.

[0238]

[0239] The titers of serum (1:8000 dilution) of mice immunized with immunization compositions containing RBD, RBD-Fc, RBD-Fc-Ce3, RBD-PS14, RBD-Fc-PS14, and RBD-Fc-Ce3-PS14 as antigens and MF59+Alum mixed adjuvant at doses of 1, 3, and 10 μg were measured on day 21. The results are shown in Table 2.

[0240] Table 2. Serum antibody titers of mice immunized with immunotherapies using RBD-Fc-Ce3 and RBD-Fc-Ce3-PS14 as antigens.

[0241] Antigen / immunizing dose / serum titer of immunizing composition 1 μg / OD 450 ]] 3 μg / OD 450 ]] 10 μg / OD 450 ]] RBD 0.988 0.997 1.006 RBD-Fc 1.103 1.235 1.298 RBD-Fc-Ce3 1.023 1.275 1.906 RBD-PS14 1.061 1.272 1.257 RBD-Fc-PS14 0.427 0.648 0.827 RBD-Fc-Ce3-PS14 0.450 0.469 0.882

[0242] 5.4 Determination of neutralizing potency

[0243] The mouse serum sample obtained in Example 5.2 was diluted 500-fold and mixed with an equal volume of 800 TCID50 / ml pseudovirus 2019-nCoV PSV (China National Institutes for Food and Drug Control). After incubation at 37°C for 1 hour, 3 × 10⁻⁶ mice were simultaneously infected. 4 / well Vero E6 or 293FT / ACE2 cells (Shenzhou Cell Engineering Co., Ltd.). After infection, cells were cultured at 37℃ and 5% CO2 for approximately 20-28 hours, and RLU values ​​were measured using a microplate chemiluminescence analyzer. A positive control was prepared with pseudovirus but without serum, and a negative control was prepared without both pseudovirus and serum. The neutralization inhibition rate (%) was calculated as: (lg(positive RLU) - lg(sample RLU)) / (lg(positive RLU) - lg(negative RLU)) x 100%.

[0244] The pseudovirus neutralizing titer of the serum (1:500 dilution) of mice immunized with RBD-Fc-Fe4 and RBD-Fc-Fe4-PS14 as antigens, Alum, MF59, and MF59+Alum as adjuvants, and immunization doses of 1, 3, and 10 μg was measured on day 21. The results are shown in Table 3.

[0245] Table 3. Neutralizing titer of pseudovirus in serum from mice immunized with immunomodulatory compositions using RBD-Fc-Fe4 and RBD-Fc-Fe4-PS14 as antigens.

[0246]

[0247] The pseudovirus neutralizing titers of serum (1:500 dilution) of mice immunized with RBD, RBD-Fc, RBD-Fc-Ce3, RBD-PS14, RBD-Fc-PS14, and RBD-Fc-Ce3-PS14 as antigens and MF59+Alum mixed adjuvant at immunization doses of 1, 3, and 10 μg were measured on day 21. The results are shown in Table 4.

[0248] Table 4. Neutralizing titer of pseudovirus in serum of mice immunized with immunomodulatory compositions using RBD-Fc-Ce3 and RBD-Fc-Ce3-PS14 as antigens.

[0249] Antigen / immunizing dose of immunizing composition 1 μg % inhibition 3 μg % inhibition 10 μg % inhibition RBD 6.2 9.8 14.0 RBD-Fc 26.4 24.0 25.4 RBD-Fc-Ce3 29.6 53.6 71.0 RBD-PS14 44.2 93.2 81.2 RBD-Fc-PS14 36.0 42.8 75.4 RBD-Fc-Ce3-PS14 26.4 44.8 78.8

[0250] 5.5 Determine and calculate IgG2a / IgG1

[0251] 5.5.1 Determination of IgG1 antibody in mouse serum

[0252] IgG1 antibodies in mouse serum were detected using an enzyme-linked immunosorbent assay (ELISA). The coating protein RBD was diluted to a concentration of 2 μg / mL, and 100 μl of the plate was coated and incubated overnight at 4°C. The plate was washed, and then blocked with 100 μl of TBST buffer containing 2% BSA (bovine serum albumin) for 2 hours at room temperature. The mouse serum sample from Example 5.2 was diluted to 1:500,000 using TBST diluent containing 0.1% BSA. 100 μl of the diluted mouse serum sample was added to the blocked plate and incubated at room temperature for 1 hour. The plate was washed, and 100 μl of 1 μg / mL HRP-labeled anti-IgG1 antibody R-mIgG1-R020-H (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added to the plate and incubated at room temperature for 1 hour. Colorimetric reagent was added for development, and the reaction was terminated. The readings were taken at 450 nm using an ELISA reader. OD of serum at a dilution of 1:500,000 450 The value represents the amount of IgG1.

[0253] 5.5.2 Quantitative determination of IgG2a antibody in mouse serum

[0254] The content of IgG2a antibody in mouse serum was detected by enzyme-linked immunosorbent assay (ELISA). The coating protein RBD was diluted to a concentration of 2 μg / mL, and 100 μl was used to coat the ELISA plate, which was then incubated overnight at 4°C. After washing, the plate was blocked with 100 μl of TBST buffer containing 2% BSA for 2 hours at room temperature, followed by washing. The mouse serum sample was diluted to 1:5000 with TBST diluent containing 0.1% BSA. 100 μl of the diluted mouse serum sample was added to the blocked ELISA plate and incubated at room temperature for 1 hour, followed by washing. 100 μl of 1 μg / mL HRP-labeled anti-IgG2a antibody R-mIgG2a-R005-H (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added to the ELISA plate and incubated at room temperature for 1 hour. Colorimetric reagent was added for color development, and after stopping the reaction, the OD value was read at 450 nm using an ELISA reader. The OD value of the serum at a 1:5000 dilution was recorded. 450 The value represents the amount of IgG2a antibody in mouse serum.

[0255] IgG2a / IgG1 = (1:5000 serum) OD 450 Value / (1:500000 serum) OD 450 The values ​​and measurement results are shown in Tables 5-6 and 6-6. Figure 1-2 As shown.

[0256] The IgG2a / IgG1 ratio of the serum of mice immunized with RBD-Fc-Fe4 and RBD-Fc-Fe4-PS14 as antigens, and Alum, MF59, and MF59+Alum as adjuvants, at immunization doses of 1, 3, and 10 μg, was measured on day 21. The results are shown in Table 5.

[0257] Table 5. Serum IgG2a / IgG1 ratio of mice immunized with immunotherapies using RBD-Fc-Fe4 and RBD-Fc-Fe4-PS14 as antigens

[0258]

[0259] The IgG2a / IgG1 ratio of the serum of mice immunized with RBD, RBD-Fc, RBD-Fc-Ce3, RBD-PS14, RBD-Fc-PS14, and RBD-Fc-Ce3-PS14 as antigens and MF59+Alum mixed adjuvant as adjuvant at immunization doses of 1, 3, and 10 μg was measured on day 21 (IgG2a was measured with serum diluted 1:5000; IgG1 was measured with serum diluted 1:500000). The results are shown in Table 6.

[0260] Table 6. Serum IgG2a / IgG1 ratio of mice immunized with three proteins and their corresponding conjugates as antigens.

[0261]

[0262]

[0263] Figure 1 The results of immunizing mice with RBD-Fc-Fe4 and RBD-Fc-Fe4-PS14 antigens using aluminum adjuvant, MF59 adjuvant, and a mixture of MF59 and aluminum adjuvant are shown. Each adjuvant was further administered at three doses: 1 μg, 3 μg, and 10 μg. Serum IgG2a / IgG1 ratios were measured after the second immunization. The figure shows that the IgG2a / IgG1 ratio in the serum immunized with the polysaccharide-conjugated antigen was significantly increased when using the MF59 adjuvant. A high IgG2a / IgG1 ratio indicates a stronger cellular immune response, suggesting better protective efficacy of the vaccine.

[0264] Figure 2The comparison of serum IgG2a / IgG1 ratios for RBD-PS14, RBD-Fc-PS14, RBD-Fc-Ce3-PS14, and RBD-Fc-Fe4-PS14 antigens is shown, with MF59+Alum adjuvant as the adjuvant. As can be seen from the figure, RBD-Fc-Fe4-PS14 has a higher IgG2a / IgG1 ratio compared to the other proteoglycan conjugates.

[0265] Based on the above data, for RBD-Fc-Fe4, when used concurrently with aluminum adjuvant, the antibody titer of the protein-only immune serum was higher compared to that of the polysaccharide conjugate. However, when using MF59 adjuvant, the antibody titer and pseudovirus neutralization titer of RBD-Fc-Fe4 and its corresponding polysaccharide conjugate immune serum were not significantly different. However, the IgG2a / IgG1 ratio of RBD-Fc-Fe4 and its polysaccharide conjugate immune serum was significantly higher. The IgG2a / IgG1 ratio reflects cellular immune propensity; a high IgG2a / IgG1 ratio predicts a stronger cellular immune response, which in turn indicates better vaccine protection. Comparing the IgG2a / IgG1 ratios of several antigen proteins and their corresponding polysaccharide conjugates, the RBD-Fc-Fe4 polysaccharide conjugate had the highest ratio, making it the optimal vaccine candidate molecule.

[0266] Sequence Listing

[0267] SEQ ID NO:1

[0268] SARS-COV-2RBD amino acid sequence SRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0269] SEQ ID NO:2

[0270] SARS-COV-2 RBD-Fc amino acid sequence SRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFADDDDKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0271] SEQ ID NO:3

[0272] SARS-COV-2 RBD-Fc-Ce3 amino acid sequence: SRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFADDDDKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHGALHNHYTQKSLSLSPGK

[0273] SEQ ID NO:4

[0274] Amino acid sequence of SARS-COV-2 RBD-Fc-Fe4 (sample obtained from RBD-Fc-Ce3+ CHO Fut8 knockout cells): SRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFADDDDKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHGALHNHYTQKSLSLSPGK

[0275] SEQ ID NO:5

[0276] SARS-COV-2 RBD nucleotide sequence: AGCAGGGTCCAACCAACAGAGAGCATTGTGAGGTTTCCAAACATCACCAACCTGTGTCCATTTGGAGAGGTGTTCAATGCCACCAGGTTTGCCTCTGTCTATGCCTGGAACAGGAAGAGGATTAGCAACTGTGTGGCTGACTACTCTGTGCTCTACAACTCTGCCTCCTTCAGCACCTTCAAGTGTTATGGAGTGAGCCCAACCAAACTGAATGACCTGTGTTTCACCAATGTCTATGCTGACTCCTTTGTGATTAGGGGAGATGAGGTGAGACAGATTGCCCCTGGACAAACAGGCAAGATTGCTGACTACAACTACAAACTGCCTGATGACTTCACAGGCTGTGTGATTGCCTGGAACAGCAACAACCTGGACAGCAAGGTGGGAGGCAACTACAACTACCTCTACAGACTGTTCAGGAAGAGCAACCTGAAACCATTTGAGAGGGACATCAGCACAGAGATTTACCAGGCTGGCAGCACACCATGTAATGGAGTGGAGGGCTTCAACTGTTACTTTCCACTCCAATCCTATGGCTTCCAACCAACCAATGGAGTGGGCTACCAACCATACAGGGTGGTGGTGCTGTCCTTTGAACTGCTCCATGCCCCTGCCACAGTGTGTGGACCAAAGAAGAGCACCAACCTGGTGAAGAACAAGTGTGTGAACTTC

[0277] SEQ ID NO:6

[0278] Signal peptide nucleotide sequence: ATGGATGCCATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTGTTTGTCTCTCCC

[0279] SEQ ID NO:7

[0280] The nucleotide sequence of SARS-COV-2 RBD containing a signal peptide: ATGGATGCCATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTGTTTGTCTCTCCCAGCAGGGTCCAACCAACAGAGAGCATTGTGAGGTTTCCAAACATCACCAACCTGTGTCCATTTGGAGAGGTGTTCAATGCCACCAGGTTTGCCTCTGTCTATGCCTGGAACAGGAAGAGGATTAGCAACTGTGTGGCTGACTACTCTGTGCTCTACAACTCTGCCTCCTTCAGCACCTTCAAGTGTTATGGAGTGAGCCCAACCAAACTGAATGACCTGTGT TTCACCAATGTCTATGCTGACTCCTTTGTGATTAGGGGAGATGAGGTGAGACAGATTGCCCCTGGACAAACAGGCAAGATTGCTGACTACAACTACAAACTGCCTGATGACTTCACAGGCTGTGTGATTGCCTGGAACAGCAACAACCTGGACAGCAAGGTGGGAGGCAACTACAACTACCTCTACAGACTGTTCAGGAAGAGCAACCTGAAACCATTTGAGAGGGACATCAGCACAGAGATTTACCAGGCTGGCAGCACACCATGTAATGGAGTGGAGGGCTTCAACTGTTACTTTCCACTCCAATCCTATGGCTTCCAACCAACCAATGGAGTGGGCTACCAACCATACAGGGTGGTGGTGCTGTCCTTTGAACTGCTCCATGCCCCTGCCACAGTGTGTGGACCAAAGAAGAGCACCAACCTGGTGAAGAACAAGTGTGTGAACTTC

[0281] SEQ ID NO:8

[0282] The nucleotide sequence of Linker: GCTGATGACGATGACAAG[[ID=]7]

[0283] SEQ ID NO:9

[0284] The nucleotide sequence of the human IgG1 constant region Fc

[0285] GAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTC

[0286] CTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT

[0287] CCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGG

[0288] TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC

[0289] GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC

[0290] AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG

[0291] CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT

[0292] ACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCC

[0293] TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC

[0294] CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCT

[0295] CTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG

[0296] CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT

[0297] CCGGGTAAATGA

[0298] SEQ ID NO:10

[0299]

[0300] SEQ ID NO:11

[0301] SARS-CoV-2 RBD-Fc-Ce3 nucleotide sequence: GAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCT CTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGGCGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAA

[0302] SEQ ID NO:12

[0303]

[0304] SEQ ID NO:13

[0305] Nucleotide sequence of murine IgG1 constant region mFc

[0306] GCTGTGCCCAGGGATTCTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCAT

[0307] CTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAA

[0308] GGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTG

[0309] GTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTT

[0310] CAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAAT

[0311] GGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAA

[0312] ACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCT

[0313] CCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTC

[0314] TTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTAC

[0315] AAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTC

[0316] AATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACAT

[0317] GAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGASEQ ID NO:14

[0318]

[0319] SEQ ID NO:15

[0320] The amino acid sequence of SARS-CoV-2 RBD-mFc: SRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFADDDDKAVPRDSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

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Claims

1. A method of enhancing the immunogenicity of a protein / peptide antigen, the method comprising fusing the protein / peptide antigen with an Fc fragment, forming a fusion protein, and then conjugating the fusion protein with a saccharide, forming a fusion protein saccharide conjugate, wherein the protein / peptide antigen is selected from SARS-CoV-2; and wherein the Fc fragment is an IgGl engineered Fc fragment that has been subjected to amino acid sequence mutation and / or glycosylation pattern alteration for the purpose of enhancing the binding function to Fc receptor / C1q complement; and wherein the Fc receptor is selected from CD16, CD32a, CD32b or CD64; and wherein the saccharide is S. pneumoniae serotype 14 capsular polysaccharide; and (1) wherein the Fc fragment is an Fc receptor CD32a, CD32b and CD64 binding enhancing fragment / C1q complement binding enhancing fragment, and the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 3; or (2) wherein the Fc fragment is an Fc receptor CD16a, CD32a, CD32b and CD64 binding enhancing fragment / C1q complement binding enhancing fragment, and the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 4, and produced with fucose knock-out mammalian cells.

2. The method of claim 1, wherein the mammalian cells are fut8 gene knock-out HEK-293 cells. wherein 3. The method of claim 1, wherein the molecular weight of the conjugate is 800-6000 KDa.

4. The method of any one of claims 1-3, wherein the protein / peptide antigen in the form of fusion protein saccharide conjugate is combined with an immunoadjuvant.

5. The method of claim 4, wherein the adjuvant is selected from an aluminum adjuvant, MF59 or a mixture thereof.

6. An immunogenically enhanced protein / peptide antigen comprising fusing the protein / peptide antigen with an Fc fragment to form a fusion protein, and then conjugating the fusion protein with a saccharide to form a protein / peptide-Fc fragment fusion protein saccharide conjugate; wherein the protein / peptide antigen is selected from SARS-CoV-2; and wherein the Fc fragment is an IgGl engineered Fc fragment that has been subjected to amino acid sequence mutation and / or glycosylation pattern alteration for the purpose of enhancing the binding function to Fc receptor, C1q complement; and wherein the Fc receptor is selected from CD16, CD32a, CD32b or CD64; and wherein the saccharide is S. pneumoniae serotype 14 capsular polysaccharide; and (1) wherein the Fc fragment is an Fc receptor CD32a, CD32b and CD64 binding enhancing fragment / C1q complement binding enhancing fragment, and the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 3; or (2) wherein the Fc fragment is an Fc receptor CD16a, CD32a, CD32b and CD64 binding enhancing fragment / C1q complement binding enhancing fragment, and the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 4, and produced with fucose knock-out mammalian cells. ​ ​ ​ ​ ​ ​ wherein ​ ​ ​ ​ ​ ​ 7. The protein / peptide antigen of claim 6, wherein the mammalian cell is a fut8 gene knockout HEK-293 cell.

8. The protein / peptide antigen of claim 7, wherein the molecular weight of the conjugate is 800-6000 KDa.

9. An immunological composition comprising a) the protein / peptide antigen of any one of claims 6-8; b) an adjuvant; and c) a pharmaceutically acceptable carrier, excipient or stabilizer.

10. The immunological composition of claim 9, wherein the pharmaceutically acceptable carrier, excipient or stabilizer is in the form of a lyophilized formulation or an aqueous solution.

11. The immunological composition of claim 9 or 10, wherein the adjuvant is selected from at least one of an aluminum adjuvant, MF59.

12. Use of the protein / peptide antigen of any one of claims 6-8, or the immunological composition of any one of claims 9-11, for the manufacture of a medicament for the prevention of a disease caused by SARS-CoV-2.

13. Use of the protein / peptide antigen of any one of claims 6-8, or the immunological composition of any one of claims 9-11, for the manufacture of a vaccine for the prevention of a disease caused by SARS-CoV-2.

14. A kit comprising the protein / peptide antigen of any one of claims 6-8, or the immunological composition of any one of claims 9-11. ​ ​ ​

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