Methods for improving immunogenicity by saccharide-coronavirus rbd antigen conjugates

Abstract

A method for enhancing the immunogenicity of coronavirus RBD, a glyco-coronavirus RBD antigen conjugate, and an immunogenic complex containing the antigen conjugate are disclosed. Specifically, a glyco-coronavirus RBD protein conjugate is formed by combining a more stable truncated RBD protein with pneumonia polysaccharide. Immunization of animals with this conjugate as an immunogen can maintain a long-term humoral and cellular immune response. The immune complex formed by the glyco-coronavirus RBD protein conjugate and MF59 adjuvant can generate higher titers of neutralizing antibodies and cellular immune responses. This immunogenic complex can be used to prevent diseases related to coronavirus infection (e.g., SARS-CoV-2).

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202010963735.X, filed on September 14, 2020, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of immunology and relates to a method for enhancing the immunogenicity of coronavirus RBD, a glyco-coronavirus RBD antigen conjugate, and an immunogenic complex containing the antigen conjugate. Specifically, a glyco-coronavirus RBD protein conjugate is formed by combining a more stable truncated RBD protein with pneumonia polysaccharide. Using this as an immunogen to immunize animals can maintain a long-term humoral and cellular immune response. Background Technology

[0004] SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, namely angiotensin-converting enzyme 2 (ACE2)[1]. 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[2]. 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[3]. 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[4-6]. 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[7]. 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.

[0005] One way to enhance the immune response is to conjugate poorly immunogenic antigens with exogenous macromolecules used as carriers; this method has been successfully applied for decades.

[0006] The applicant's invention patent application, entitled "A Method for Enhancing the Immunogenicity of Protein / Peptide Antigens," with application numbers CN202010369100.7 and PCT / CN2021 / 090809, reports the inventor's groundbreaking invention: by conjugating protein / peptide antigens with sugars to form glyco-protein / peptide antigen conjugates, the immunogenicity of protein / peptide antigens is enhanced. This invention differs from conventional glyco-protein peptide conjugate vaccines such as encephalitis vaccines, Haemophilus influenzae type b vaccines, and pneumonia vaccines, which produce more effective immunogenic compositions by binding purified capsular polysaccharides to carrier proteins.

[0007] To enhance protein stability, this invention truncates the RBD protein and forms a glyco-coronavirus RBD protein conjugate with pneumonia polysaccharide. Immunizing animals with this conjugate as an immunogen can maintain long-term humoral and cellular immune responses, generating higher-titer neutralizing antibodies and cellular immune responses for the prevention of coronavirus-related diseases such as SARS-CoV-2 infection. Summary of the Invention

[0008] A first aspect of the present invention provides a method for enhancing the immunogenicity of coronavirus RBD antigen, the method comprising conjugating coronavirus RBD antigen with a sugar to form a sugar-coronavirus RBD antigen conjugate.

[0009] In one embodiment, the sugar in the method is selected from polysaccharides, oligosaccharides, or monosaccharides; preferably, it is capsular polysaccharide of Neisseria gonorrhoeae, capsular polysaccharide of Haemophilus influenzae b, capsular polysaccharide of Streptococcus pneumoniae, capsular polysaccharide of Staphylococcus aureus group B, dextran, mannan, starch, inulin, pectin, carboxymethyl starch, chitosan, and their derivatives; more preferably, it is capsular polysaccharide of Streptococcus pneumoniae, and most 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.

[0010] In one embodiment, the coronavirus RBD antigen in the method comprises the amino acid sequence described in SEQ ID NO: 2 or an RBD truncated form of different lengths thereof, an amino acid sequence having at least 90%, 92%, 95%, 98% or 100% sequence identity thereof, or an RBD truncated form of different lengths thereof.

[0011] In one embodiment, the coronavirus RBD antigen in the method is conjugated with Streptococcus pneumoniae serotype 14 capsular polysaccharide / Streptococcus pneumoniae serotype 6B capsular polysaccharide.

[0012] In one embodiment, the Streptococcus pneumoniae serotype 14 capsular polysaccharide in the method is derived from ATCC6314, and the Streptococcus pneumoniae serotype 6B capsular polysaccharide is derived from ATCC6326.

[0013] In one embodiment, the coronavirus RBD antigen in the method is further fused with other proteins or peptides.

[0014] In one embodiment, the coronavirus RBD antigen in the method is used in combination with an immune adjuvant, preferably ALUM / MF59.

[0015] In one implementation, the method enhances the Th1 immune response.

[0016] Another aspect of the present invention provides a C-terminal truncated SARS-CoV-2 RBD antigen comprising the Arg319 to Thr531 fragments of the S1 subunit of the SARS-CoV-2 spike protein, preferably comprising the amino acid sequence described in SEQ ID NO: 2 or a truncated RBD of different lengths thereof, an amino acid sequence having at least 90%, 92%, 95%, 98% or 100% sequence identity thereof or a truncated RBD of different lengths thereof.

[0017] Another aspect of the present invention provides a glycocoronavirus RBD antigen conjugate that has enhanced immunogenicity compared to the unconjugated coronavirus RBD antigen.

[0018] In one embodiment, the sugar in the glyco-coronavirus RBD antigen conjugate is selected from polysaccharides, oligosaccharides, or monosaccharides; preferably, it is capsular polysaccharide of Neisseria gonorrhoeae, capsular polysaccharide of Haemophilus influenzae b, capsular polysaccharide of Streptococcus pneumoniae, capsular polysaccharide of Staphylococcus aureus group B, dextran, mannan, starch, inulin, pectin, carboxymethyl starch, chitosan, and their derivatives; more preferably, it is capsular polysaccharide of Streptococcus pneumoniae, and most 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.

[0019] In one embodiment, the coronavirus RBD antigen in the RBD antigen conjugate is a C-terminal truncated SARS-CoV-2 RBD antigen comprising the Arg319 to Thr531 fragments of the S1 subunit of the SARS-CoV-2 spike protein, preferably comprising the amino acid sequence described in SEQ ID NO: 2 or an RBD truncated version thereof of different lengths, an amino acid sequence having at least 90%, 92%, 95%, 98% or 100% sequence identity with it or an RBD truncated version thereof of different lengths.

[0020] In one embodiment, the coronavirus RBD antigen in the RBD antigen conjugate is conjugated with Streptococcus pneumoniae serotype 14 capsular polysaccharide / Streptococcus pneumoniae serotype 6B capsular polysaccharide.

[0021] In one embodiment, the pneumococcal serotype 14 capsular polysaccharide in the RBD antigen conjugate is derived from ATCC 6314, and the pneumococcal serotype 6B capsular polysaccharide is derived from ATCC 6326.

[0022] In one embodiment, the coronavirus RBD antigen in the RBD antigen conjugate is further fused with other proteins or peptides.

[0023] In one embodiment, the RBD antigen conjugate is used in combination with an immune adjuvant, preferably ALUM / MF59.

[0024] In one embodiment, the RBD antigen conjugate enhances the Th1 immune response when used as an antigen.

[0025] Another aspect of the present invention provides an immune complex comprising the coronavirus RBD antigen described in this invention or an RBD antigen conjugate described in this invention and an immune adjuvant.

[0026] In one embodiment, the adjuvant in the immune complex is selected from ALUM / MF59.

[0027] Another aspect of the invention provides the use of coronavirus RBD antigens, RBD antigen conjugates, or immune complexes as described herein for the prevention or treatment of diseases caused by coronaviruses.

[0028] Another aspect of the invention provides the use of coronavirus RBD antigens, RBD antigen conjugates, or immune complexes as described herein in the preparation of vaccines / medications for the prevention or treatment of diseases caused by coronaviruses. Attached Figure Description

[0029] Figure 1 The SEC and SDS-PAGE maps of the RBD(T4) recombinant protein pair are described.

[0030] Figure 2 The effects of PS14(A) and PS6B(B) on serum antibody titers in RBD(T4) immunized mice were described.

[0031] Figure 3 The effects of PS14(A) and PS6B(B) on the serum neutralizing antibody titers of RBD(T4) immunized mice were described.

[0032] Figure 4 The ratio of mIgG2a to mIgG1 antibody titers in the serum of RBD(T4) immunized mice was described by PS14(A) and PS6B(B).

[0033] Figure 5The study describes how PS14 enhances the Th1 T cell response in RBD(T4) immunized mice.

[0034] Figure 6 The effects of MF59 adjuvant on serum antibody titers (A) and neutralizing antibody titers (B) in PS14-RBD(T4) immunized mice were described.

[0035] Figure 7 The study described how MF59 adjuvant increased the ratio of mIgG2a to mIgG1 antibody titers in the serum of PS14-RBD(T4) immunized mice.

[0036] Figure 8 The study describes how MF59 adjuvant enhances the Th1 T cell response in PS14-RBD(T4) immunized mice. Invention Details

[0038] definition

[0039] 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.

[0040] 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.

[0041] The terms "comprising" or "including" mean that a specific ingredient is included without excluding any other ingredient. Terms such as "consistent with substantially" 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.

[0042] 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).

[0043] The term "spike protein (S protein)" refers to a protein that exists as a trimer on the surface of the coronavirus membrane. It binds to host cell receptors, mediating viral invasion and determining the virus's tissue or host tropism. The host cell receptor protein for SARS-CoV-2 is angiotensin-converting enzyme 2 (ACE2). After binding to the ACE2 receptor, the viral trimer spike protein (S protein) is cleaved by host proteases into an S1 polypeptide containing a receptor-binding domain (SARS-CoV-2 RBD) and an S2 polypeptide responsible for mediating viral fusion with the cell membrane.

[0044] The “RBD (Receptor Binding Domain, SARS-COV-2 RBD)” spike protein binds to the ACE2 receptor and is cleaved by host proteases into S1 and S2 subunits. The S1 subunit contains the receptor binding domain (SARS-COV-2 RBD), for example, a certain version of the S1 subunit is Arg319-Phe541.

[0045] "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.

[0046] 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.

[0047] The term "conjugate" refers to a protein / peptide covalently conjugated to a sugar. The sugar RBD antigen conjugates and immunogenic compositions comprising them of the present invention may contain a certain amount of free sugar, protein / peptide.

[0048] As used in this article, “conjugation” refers to the process of covalently linking sugars, such as bacterial capsular polysaccharides, to proteins / peptides.

[0049] The term "immunogenic composition" refers to any pharmaceutical composition containing an antigen, such as a microorganism or a component thereof, which can be used to induce an immune response in an individual.

[0050] As used herein, “immunogenicity” means the ability of an antigen (or an epitope of an antigen), such as the receptor-binding region of a coronavirus spike protein or a glycoconjugate or immunogenic composition containing that antigen, to induce a humoral or cellular immune response in a host (e.g., a mammal) or both.

[0051] 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.

[0052] The terms “immunogenic amount” and “immunogenically effective amount” are used interchangeably herein and refer to an amount of 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.

[0053] The effectiveness of an antigen as an immunogen can be measured by proliferation assay, cell lysis assay, or by measuring B cell activity levels.

[0054] The method of the present invention for improving the immunogenicity of protein / peptide antigens

[0055] The method for improving the immunogenicity of protein / peptide antigens of the present invention is achieved through the glycoRBD antigen conjugate of the present invention and the immunogenic composition of the present invention.

[0056] The sugar RBD antigen conjugate of the present invention

[0057] Coronaviruses primarily mediate viral invasion by binding to host cell receptors via their spike protein (S protein), which 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 SARS-CoV-2 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.

[0058] One approach of this invention uses a truncated RBD recombinant protein from the C-terminus of a coronavirus as an antigen. The antigen can be obtained by extraction from the natural pathogen or through gene recombination. Infection with the novel coronavirus SARS-CoV-2 depends on its spike protein, which contains two subunits: S1 and S2. The receptor-binding domain (RBD, Arg319-Phe541) located in the S1 subunit binds to the human cell receptor angiotensin-converting enzyme 2 (ACE2) to mediate viral invasion. Studies have shown that the RBD domain can not only induce humoral immunity to produce neutralizing antibodies but also induce T-cell immune responses; therefore, the RBD protein is an effective target for SARS-CoV-2 vaccines.

[0059] To improve the expression level, purity, and stability of the RBD recombinant protein, this invention uses a C-terminally truncated RBD recombinant protein. In one embodiment, it is a recombinant protein comprising the amino acid sequence described in SEQ ID NO: 2 (corresponding to the spike protein sequence R319-T531) or its active variant, named RBD(T4). To maintain long-term humoral and cellular immune responses, this invention further conjugates the truncated RBD recombinant protein with a glycosyl group.

[0060] The polysaccharides mentioned are 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 mentioned can also be animal-derived polysaccharides, such as chitosan and its derivatives.

[0061] In one embodiment, the RBD(T4) recombinant protein antigen is conjugated with Streptococcus pneumoniae serotype 14 capsular polysaccharide / Streptococcus pneumoniae serotype 6B capsular polysaccharide.

[0062] In one embodiment, the Streptococcus pneumoniae serotype 14 capsular polysaccharide is derived from ATCC6314, and this sugar-RBD antigen conjugate is referred to as PS14-RBD(T4). In another embodiment, the Streptococcus pneumoniae serotype 6B capsular polysaccharide is derived from ATCC6326, and this sugar-RBD antigen conjugate is referred to as PS6B-RBD(T4).

[0063] Under the same adjuvant conditions, compared with RBD(T4) immunization, immunization with PS14-RBD(T4) and PS6B-RBD(T4) conjugates both showed higher total antibody titers, and both were significantly enhanced. This indicates that conjugation with pneumococcal capsular polysaccharides PS14 or PS6B can significantly increase the total antibody titer in mice immunized with RBD(T4).

[0064] Conjugation of RBD(T4) with pneumococcal capsular polysaccharide PS14 or PS6B can significantly increase the serum neutralizing antibody titer in RBD(T4)-immunized mice.

[0065] Both PS14-RBD(T4) and PS6B-RBD(T4) conjugates showed higher mIgG2a / mIgG1 antibody titer ratios, indicating that conjugation of RBD(T4) with pneumococcal capsular polysaccharides PS14 or PS6B can enhance Th1 immune responses.

[0066] The immunogenic composition of the present invention

[0067] In one embodiment, the immunogenic composition of the present invention further comprises at least one selected from adjuvant, buffer, cryoprotectant, salt, divalent cation, nonionic detergent, free radical oxidation inhibitor, diluent, or 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 selected from ALUM / MF59 oil-in-water adjuvant.

[0068] Adjuvants are substances that enhance the immune response when administered together with an immunogen or antigen.

[0069] Compared with RBD(T4)+MF59 immunization, PS14-RBD(T4)+MF59 immunization slightly increased the expression level of IFN-γ specific to mouse spleen lymphocytes, but the IFN-γ:IL-4 ratio was significantly increased, indicating that PS14 can effectively enhance the production of specific cellular immunity against RBD(T4) protein in mice.

[0070] Compared to PS14-RBD(T4), PS14-RBD(T4)+Alum exhibits a higher level of stimulation-specific IL-4 expression. Figure 8 A), while PS14-RBD(T4)+MF59 has a higher level of stimulation-specific IFN-γ expression ( Figure 8 B) and a higher IFN-γ:IL-4 ratio ( Figure 8 C). Therefore, MF59 adjuvant has a stronger ability to enhance cellular response after PS14-RBD(T4) immunization in mice.

[0071] The PS14-RBD(T4)+MF59 immunization significantly increased the mIgG2a / mIgG1 antibody titer ratio, indicating that the MF59 adjuvant has a better function in improving the Th1 / Th2 balance.

[0072] 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.

[0073] The immunogenic compositions of the present invention may further comprise one or more additional immunomodulators, which are substances that disrupt or alter the immune system to thereby observe upregulation or downregulation of humoral and / or cell-mediated immunity. 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.

[0074] Administration of the immunogenic composition of the present invention

[0075] 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.

[0076] 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.

[0077] Use of the immunogenic composition of the present invention

[0078] The protein / peptide antigen conjugates and immune complexes of the present invention can prevent or treat diseases caused by pathogens, especially coronaviruses, and even more so diseases caused by SARS-CoV-2 virus. Detailed Implementation

[0079] Example 1: Construction and production of RBD(T4) recombinant protein

[0080] 1.1 Gene construction and expression of recombinant RBD(T4) protein

[0081] Infection with the novel coronavirus SARS-CoV-2 depends on its spike protein, which contains two subunits: S1 and S2. The receptor-binding domain (RBD, Arg319-Phe541) located in the S1 subunit binds to the human cell receptor angiotensin-converting enzyme 2 (ACE2) to mediate viral invasion. Studies have shown that the RBD domain can not only induce humoral immunity to produce neutralizing antibodies but also induce T-cell immune responses; therefore, the RBD protein is an effective target for SARS-CoV-2 vaccines. To improve the expression level, purity, and stability of the recombinant RBD protein, this embodiment designed a C-terminated recombinant RBD protein: RBD(T4). The specific design scheme is shown in Table 1.

[0082] Table 1 Design scheme for RBD(T4) recombinant protein

[0083]

[0084] ① Arginine (single-letter abbreviation)

[0085] The original sequence of the novel coronavirus RBD gene was obtained from NCBI (GenBank: MN908947.3). This gene underwent codon optimization to increase the expression level of the target antigen. The recombinant RBD(T4) protein construct was prepared using the primers shown below:

[0086] RBD-1 (SEQ ID NO: 7) GTCACCGTCCTGACACGAAGCTTGCCGCCACCATGAAACACCTGTGGTTCTTCCT RBD-2 (SEQ ID NO: 8) TAGAATAGGGCCCTCTAGATTTAGGTGCTCTTCTTTGGTCCACAC

[0087] The full-length sequence of the recombinant RBD(T4) protein gene (SEQ ID NO: 5), including the signal peptide gene sequence (SEQ ID NO: 3) and the RBD(T4) protein gene sequence (SEQ ID NO: 1), was obtained by PCR amplification. This sequence was then inserted into a pSE vector (source: Shenzhou Cell Engineering Co., Ltd.) digested with Hind III + Xba I (source: Fermentas) via in-fusion. After confirming the recombinant expression vector was correctly sequenced, the plasmid was extracted, transiently transferred into HEK-293 cells, and cultured for 7 days. The supernatant was collected by centrifugation. The obtained cell supernatant was purified using hydrophobic chromatography and mixed anion exchange chromatography to obtain high-purity RBD(T4) recombinant protein, which was then transferred to the target buffer via ultrafiltration.

[0088] 1.2 Stability Analysis of RBD(T4) Recombinant Protein The purified RBD(T4) recombinant protein was analyzed for purity by size exclusion high-performance liquid chromatography (SEC-HPLC, TSK-G2000) and non-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the purity of SEC-HPLC was 99.5% and the purity of non-reduced SDS-PAGE was 98.6%, indicating that the RBD(T4) recombinant protein has high purity. (See attached figures). Figure 1 .

[0089] The recombinant RBD(T4) protein was replaced with a buffer solution containing 0.36 mg / mL citrate, 2.35 mg / mL sodium citrate, 14.61 mg / mL NaCl, 0.13 g / kg sodium hydroxide, and pH 6.0, to a final concentration of approximately 0.6 mg / mL. The samples were stored at 25°C for one week (25T1W), 37°C for one week (37T1W), 45°C for one week (45T1W), and then thawed at -80°C for 3 hours followed by thaw at 45°C for 1 hour (5FT). This freeze-thaw cycle was repeated five times. Changes in purity before and after acceleration were analyzed using SEC-HPLC, dynamic light scattering (DLS), and non-reducing SDS-PAGE.

[0090] The accelerated stability test results of RBD(T4) recombinant protein are shown in Table 2. The results indicate that under multiple accelerated conditions, the SEC purity of RBD(T4) recombinant protein remained high, the aggregate level increased only slightly, and the fragment level remained unchanged, demonstrating good accelerated stability.

[0091] Table 2. Results of accelerated stability testing of RBD(T4) recombinant protein

[0092]

[0093] Example 2: Preparation of RBD(T4) recombinant protein pneumococcal serotype 14 capsular polysaccharide (PS14) and serotype 6B capsular polysaccharide (PS6B) conjugates

[0094] 2.1 Preparation of capsular polysaccharides from Streptococcus pneumoniae serotypes 14 (PS14) and 6B (PS6B)

[0095] The seed for serotype 14 Streptococcus pneumoniae is ATCC 6314, and the seed for serotype 6B Streptococcus pneumoniae is ATCC 6326.

[0096] 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 23F, Allp Microbiol Biotechnol (2002) 59: 713-717). Incubate at 37℃ on a shaker at 150 rpm for 10-16 h, and wait for OD. 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 35. 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.

[0097] Take 10 mL of a 10 mg / mL polysaccharide solution, add 100 mg of sodium periodate, mix well, and let stand in the dark for 1 hour. Take a 5 mL Sephadex G25 packing column, add 10 mL of 50 mM, pH 7.0 Na2HPO4 buffer, and allow the buffer to flow through the column under gravity. Then, place the column in a centrifuge and centrifuge at 1000 g for 2 minutes. After that, replace the collection tube with a new one, take 1 mL of the sodium periodate-oxidized polysaccharide solution, add it to the centrifuge column, and centrifuge again at 1000 g for 2 minutes. The collected effluent from the centrifuge column is the activated polysaccharide solution.

[0098] 2.2 Preparation of RBD(T4) conjugates with PS14 or PS6B

[0099] Change the medium for the receptor-binding domain of the coronavirus spike protein: Take 5 mg of RBD(T4) protein and change the medium to 50 mM Na2HPO4 buffer at pH 7.0 using a 30,000 MW ultrafiltration tube. Finally concentrate to a volume of less than 0.25 mL, i.e., the final protein concentration is ≥20 mg / mL.

[0100] Conjugation of the coronavirus spike protein receptor-binding domain to polysaccharides: Take 5 mg of RBD (T4), add 3 mg of activated Streptococcus pneumoniae capsular polysaccharide, and add 50 mM Na2HPO4 buffer (pH 7.0) to a final volume of 0.5 mL. Then add 0.5 μL of 5 M sodium cyanoborohydride solution and rotate to mix for 16 h at room temperature in the dark. Next, add 0.3 mL of 10 mg / mL sodium borohydride solution to the reaction solution and react at room temperature for 1 h. Then, use a 100,000 MW ultrafiltration tube to filter the conjugated sample PS14-RBD (T4) or PS6B-RBD (T4), replacing the buffer 10 times with PBS buffer until the final ultrafiltration volume is less than 5 mL. After aseptic filtration through a 0.22 μm filter, store the ultrafiltered conjugated sample at 4 °C.

[0101] 2.3 Preparation of PS14-RBD(T4) or PS6B-RBD(T4) conjugate immune compositions Using RBD(T4) or the PS14-RBD(T4) or PS6B-RBD(T4) conjugate prepared in Example 2.2 as antigens, and MF59 or Alum as adjuvants, immune compositions were prepared.

[0102] 2.3.1 Preparation of MF59 adjuvant

[0103] 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.

[0104] 2.3.2 Preparation of MF59 adjuvant immunomodulatory composition

[0105] The RBD(T4), PS14-RBD(T4), or PS6B-RBD(T4) conjugate antigens were diluted with PBS to a concentration of 0.02 mg / mL. The diluted antigens were then mixed with an equal volume of MF59 adjuvant, resulting in an immunization composition with a protein concentration of 0.01 mg / mL.

[0106] 2.3.3 Preparation of Alum adjuvant immunomodulatory compositions

[0107] The PS14-RBD(T4) conjugate antigen was diluted with PBS to a concentration of 0.02 mg / mL, and the aluminum adjuvant (Beijing Nuoning Biotechnology Co., Ltd.) was diluted with PBS to a concentration of 1 mg / mL. The diluted antigen and aluminum adjuvant were mixed in equal volumes. The protein concentration of the antigen in this immunizing composition was 0.01 mg / mL.

[0108] Example 3: Immunogenicity Study of RBD(T4) Recombinant Protein Pneumococcal Capsular Polysaccharide Conjugate

[0109] 3.1 Immunization of Mice Balb / c mice, aged 4-6 weeks, were selected. They were intraperitoneally injected with 0.1 mL of the immunization composition at a concentration of 0.01 mg / mL as described in Example 2.3. The PS14-RBD(T4) immunization group received booster immunizations on days 14 and 28, respectively. Blood was collected from the tail vein on day 35 (7 days after 3 immunizations), and the spleen was collected on day 26 (12 days after 3 immunizations) or day 49 (21 days after 3 immunizations). The PS6B-RBD(T4) immunization group received a booster immunization on day 14, and blood was collected from the tail vein on day 35 (21 days after 2 immunizations). After blood collection, serum total antibody titer, neutralizing titer, and IgG2a and IgG1 subtype antibody titers were measured. Specific T-cell responses were measured using mouse spleens.

[0110] 3.2 Pneumococcal capsular polysaccharide enhances the immunogenicity of RBD(T4) recombinant protein (humoral + cellular).

[0111] 3.2.1 Increased Antibody and Neutralizing Antibody Titers in Pneumococcal Capsular Polysaccharide-Enhanced RBD (T4) Mice Serum. SARS-COV-2 RBD protein (Shenzhou Cell Engineering Co., Ltd., same throughout) at a concentration of 5 μg / mL was coated onto 96-well plates at 100 μL / well, and incubated overnight at 2-8℃. After washing, 2% BSA was added for blocking at room temperature for 1 h. The serum to be tested was diluted to different dilutions using TBST containing 0.1% bovine serum albumin (BSA) (the specific dilution factor varied depending on the immunization blood collection time, such as 1000×, 8000×, 16000×, 32000× dilutions). The experiment used SARS-COV-2 RBD-mFc-immunized mouse serum as a positive control, healthy mouse serum (Shenzhou Cell Engineering Co., Ltd., same throughout) as a negative control, and serum-free serum as a blank control, at 100 μL / well, and incubated at room temperature for 1-2 h. Wash the plate three times to remove unbound antibodies. Add 100 μL of goat anti-mouse IgG F(ab)2 / HRP (Jackson Immuno Research, same throughout) as the secondary antibody and incubate at room temperature for about 1 hour. Wash the plate five times, add the substrate chromogenic solution for color development, and read the OD value using a microplate reader after termination. 450 . with OD 450 Value greater than negative serum OD 450Multiply by the maximum dilution factor of 2.1 to obtain the antibody titer. The results are as follows: Figure 2 As shown, under the same adjuvant conditions, compared with RBD(T4) immunization, immunization with PS14-RBD(T4) and PS6B-RBD(T4) conjugates both exhibited higher total antibody titers, and both showed a significant increase. This indicates that conjugation with pneumococcal capsular polysaccharides PS14 or PS6B can significantly enhance the total antibody titer in mice immunized with RBD(T4).

[0112] This embodiment further examined the neutralization level of mouse serum after immunization. Mouse serum was inactivated in a 37°C water bath for 30 min. Then, 50 μL of inactivated mouse serum sample at different dilution gradients (the specific dilution factors varied depending on the immunization collection time, such as 500×, 1581×, 5000×, and 15810× dilutions) was added to each well, along with 100 TCID50. 50 An equal volume of pseudovirus PSV-Luc-Spike(M) (Shenzhou Cell Engineering Co., Ltd., same text throughout) was mixed and incubated at 37°C for 1 hour, then used to infect 293FT-ACE2 cells (Shenzhou Cell Engineering Co., Ltd., same text throughout). 3 × 10⁶ cells of 293FT-ACE2 were then added. 4 100 μL of cells per well were evenly seeded into 96-well plates containing serum sample and pseudovirus incubation. The cell dilution medium was DMEM + 10% FBS + 50 μg / mL gentamicin sulfate. A positive control group M (cells seeded, no sample added, containing pseudovirus) and a negative control group M′ (cells seeded, no sample added, no pseudovirus) were established. The 96-well plates were incubated at 37℃ and 5% CO2 for 20–24 h. 50 μL / well was added to 1×PLB and reacted for 10 min. 40 μL / well was then transferred to a 96-well all-white fluorescent analysis plate, and bioluminescence detection was performed using a microplate bioluminescence detector. GraphPad Prism software was used to plot the results, with the horizontal axis representing the sample name and the vertical axis representing the neutralization inhibition rate of the sample against SARS-CoV-2 pseudovirus infection. The neutralization inhibition rate (%) was calculated as (positive control group M (RLU) - sample group (RLU)) / (positive control group M (RLU) - negative control group M′ (RLU)) × 100%. The results are as follows Figure 3 As shown, similar to mouse serum titers, conjugation of RBD(T4) with pneumococcal capsular polysaccharide PS14 or PS6B can significantly increase the serum neutralizing antibody titers of RBD(T4)-immunized mice.

[0113] 3.2.2 Pneumococcal capsular polysaccharide enhances cellular response in RBD(T4)-immunized mice

[0114] The titer of mIgG1 or mIgG2a subtype antibodies against RBD in mouse serum was detected using enzyme-linked immunosorbent assay (ELISA). SARS-CoV-2 RBD protein at a concentration of 5 μg / mL was coated onto 96-well plates (100 μL / well) and incubated overnight at 2–8°C. After washing, 2% BSA was added for blocking at room temperature for 1 h. The serum to be tested was diluted to different dilutions using TBST containing 0.1% bovine serum albumin (BSA) (the specific dilution factor varied depending on the immunization time, such as 1000×, 8000×, 16000×, and 32000× dilutions). Healthy mouse serum served as a negative control, and serum-free serum served as a blank control (100 μL / well). The plates were incubated at room temperature for 1–2 h. Wash the plate three times to remove unbound antibodies. Add 100 μL of 0.15 μg / mL labeled HRP anti-IgG1 detection antibody R-mIgG1-R020-H (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., same throughout) or anti-IgG2a detection antibody R-mIgG2a-R005-H (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., same throughout) to the ELISA plate and incubate at room temperature for about 1 hour. Add chromogenic buffer for color development. After stopping the reaction, read the value at 450 nm using an ELISA reader. Titrate the antibody titer of mIgG1 or mIgG2a subtypes in serum using the maximum dilution factor of 2.1, where the OD450 value is greater than the OD450 value of negative serum. mIgG2a represents Th1 immune response (cellular immunity), and mIgG1 represents Th2 immune response (humoral immunity). Data analysis: mIgG2a / mIgG1 ratio = log(mIgG2a titer) / log(mIgG1 titer). Results are as follows. Figure 4 As shown, under the same adjuvant conditions, compared with RBD(T4) immunization, both PS14-RBD(T4) and PS6B-RBD(T4) conjugate immunization had higher mIgG2a / mIgG1 ratios, indicating that RBD(T4) conjugated with pneumococcal capsular polysaccharides PS14 or PS6B can enhance the Th1 immune response.

[0115] The expression levels of specific IFN-γ (cellular immunity) and IL-4 (humoral immunity) in mouse spleen lymphocytes 7 days after PS14-RBD(T4) immunization were detected using the ELISpot method. Mouse spleen cells were isolated, and 100 μL / well was seeded into pre-treated ELISpot plates (Mabtech, same throughout) at a cell seeding density of 2 × 10⁶ cells / well. 5Cells / well were added, and then 100 μL / well of RBD recombinant protein was added to a final concentration of 2 μg / mL. The plates were incubated at 37°C with 5% CO2 for approximately 40 h. After incubation, the cell supernatant was removed from the ELISpot plate, and the plates were washed 5 times with PBS. Then, 100 μL / well of diluted detection antibody was added, and the plates were incubated for 2 h, followed by 5 washes with PBS. Then, 100 μL / well of diluted Streptavidin-ALP (1:1000) was added, and the plates were incubated at room temperature for 1 h, followed by 5 washes with PBS. Then, 100 μL / well of BCIP / NBT-plus substrate filtered through a 0.45 μm membrane was added, and the plates were incubated at room temperature in the dark for 10-30 min until clear spots appeared. The incubation was terminated with tap water. The ELISpot plates were then placed in a cool, shaded place at room temperature to air dry naturally. Results were analyzed using an ELISA spectrophotometer. Cells were analyzed every 10 wells. 6 SFCs (Spot-forming cells) of mouse spleen cells represent the number of antigen-specific IFN-γ positive T cells. Data were analyzed using GrapPad Prism software. Results are as follows: Figure 5 As shown, compared with RBD(T4)+MF59 immunization, PS14-RBD(T4)+MF59 immunization slightly increased the expression level of IFN-γ specific to mouse spleen lymphocytes, but the IFN-γ:IL-4 ratio was significantly increased, indicating that PS14 can effectively enhance the production of specific cellular immunity against RBD(T4) protein in mice.

[0116] 3.3 MF59 adjuvant increases PS14-RBD(T4) immunogenicity (humoral + cellular)

[0117] 3.3.1 MF59 adjuvant has a stronger ability to enhance serum antibody titers and neutralizing antibody titers in PS14-RBD(T4) immunized mice.

[0118] Referring to Example 3.2.1, the total antibody titer and neutralizing antibody titer of serum from mice immunized with an immunocombination of PS14-RBD(T4) with MF59 or Alum adjuvant were detected. The results are as follows: Figure 6 As shown, compared to PS14-RBD(T4) immunization alone, Alum adjuvant slightly increased total antibody titer and neutralizing titer, while MF59 adjuvant significantly increased total antibody titer. Figure 6 A) and neutralizing antibody titer ( Figure 6 B), and significantly superior to Alum adjuvant.

[0119] 3.3.2 MF59 adjuvant significantly enhanced cellular response in PS14-RBD(T4) immunized mice.

[0120] Referring to Example 3.2.2, the titers of RBD-specific mIgG2a or mIgG1 subtype antibodies in the serum of mice immunized with the immunocomposite of PS14-RBD(T4) with MF59 or Alum adjuvant were detected, and the mIgG2a / mIgG1 ratio was calculated. The results are as follows: Figure 7 As shown, PS14-RBD(T4) and PS14-RBD(T4)+Alum have similar mIgG2a / mIgG1 ratios, while PS14-RBD(T4)+MF59 immunization can significantly increase the mIgG2a / mIgG1 ratio, indicating that MF59 adjuvant has a better ability to enhance cellular response.

[0121] Following Example 3.2.2, the ELISpot method was used to detect the secretion levels of specific IFN-γ (cellular immunity) and IL-4 (humoral immunity) in splenic lymphocytes of mice 12 days after immunization. The results are as follows: Figure 8 As shown, compared to PS14-RBD(T4), PS14-RBD(T4)+Alum exhibits a higher level of stimulation-specific IL-4 expression. Figure 8 A), while PS14-RBD(T4)+MF59 has a higher level of stimulation-specific IFN-γ expression ( Figure 8 B) and a higher IFN-γ:IL-4 ratio ( Figure 8 C). Therefore, MF59 adjuvant has a stronger ability to enhance cellular response after PS14-RBD(T4) immunization in mice.

[0122] The results in summary indicate that PS14 and MF59 adjuvants can effectively enhance the production of specific humoral and cellular immune responses against RBD(T4) in mice, and may be used to prevent SARS-CoV-2 infection-related diseases.

[0123] Sequence List:

[0124]

[0125]

[0126] References

[0127] 1. Zhou, P., et al., Discovery of a novel coronavirus associated with therecent pneumonia outbreak in humans and its potential bat origin. BioRxiv, 2020.

[0128] 2.Wan,Y.,et al.,Receptor recognition by novel coronavirus:An analysisbased on decade-long structural studies of SARS.Journal of virology,2020.

[0129] 3.Bosch,B.J.,et al.,Severe acute respiratory syndrome coronavirus(SARS-CoV)infection inhibition using spike protein heptad repeat-derivedpeptides.Proceedings of the National Academy of Sciences of the United Statesof America,2004.101(22):p.8455-8460.

[0130] 4.Huang,C.,et al.,Clinical features of patients infected with2019novel coronavirus.Lancet,2020.395(10223):p.497-506.

[0131] 5.Zhou,F.,et al.,Clinical course and risk factors for mortality ofadult inpatients with COVID-19:a retrospective cohort study.The Lancet,2020.

[0132] 6.Dong,C.,et al.,Characterization of anti-viral immunity in recoveredindividuals infected by SARS-CoV-2.medRxiv,2020.

[0133] 7.To, KK, et al., Temporal profiles of viral load in posteriororopharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study. Lancet Infect Dis, 2020. 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Claims

1. A method for enhancing the immunogenicity of coronavirus RBD antigen, the method comprising conjugating coronavirus RBD antigen with a sugar to form a sugar-coronavirus RBD antigen conjugate; wherein the coronavirus RBD antigen is mixed with an immune adjuvant, wherein the adjuvant is MF59; The amino acid sequence of the coronavirus RBD antigen is shown in SEQ ID NO: 2; The coronavirus RBD antigen is conjugated with Streptococcus pneumoniae serotype 14 capsular polysaccharide / Streptococcus pneumoniae serotype 6B capsular polysaccharide.

2. The method of claim 1, wherein the Streptococcus pneumoniae serotype 14 capsular polysaccharide is derived from ATCC6314 and the Streptococcus pneumoniae serotype 6B capsular polysaccharide is derived from ATCC6326.

3. The method of any one of claims 1-2, wherein the coronavirus RBD antigen is further fused with a signal peptide.

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