WO2022053016A1 - 通过糖-冠状病毒rbd抗原缀合物提高免疫原性的方法 - Google Patents

通过糖-冠状病毒rbd抗原缀合物提高免疫原性的方法 Download PDF

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WO2022053016A1
WO2022053016A1 PCT/CN2021/117680 CN2021117680W WO2022053016A1 WO 2022053016 A1 WO2022053016 A1 WO 2022053016A1 CN 2021117680 W CN2021117680 W CN 2021117680W WO 2022053016 A1 WO2022053016 A1 WO 2022053016A1
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rbd
capsular polysaccharide
coronavirus
rbd antigen
antigen
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PCT/CN2021/117680
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English (en)
French (fr)
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谢良志
张延静
李靖
孙春昀
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神州细胞工程有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

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  • the invention belongs to the field of immunology, and relates to a method for improving the immunogenicity of coronavirus RBD, a carbohydrate-coronavirus RBD antigen conjugate and an immunogenic complex containing the antigen conjugate.
  • a saccharide-coronavirus RBD protein conjugate was formed between the RBD truncated protein with better stability and pneumococcal polysaccharide, which can maintain long-term humoral and cellular immune responses after immunizing animals with this as an immunogen.
  • SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, angiotensin-converting enzyme 2 (ACE2) [1].
  • ACE2 angiotensin-converting enzyme 2
  • the trimeric S protein of the virus binds to the ACE2 receptor and is cleaved by the host protease into the S1 polypeptide containing the receptor binding domain (RBD) and the S2 polypeptide responsible for mediating the fusion of the virus with the cell membrane [2].
  • RBD receptor binding domain
  • S2 polypeptide responsible for mediating the fusion of the virus with the cell membrane [2].
  • the specific interaction between S1 and ACE2 triggers a conformational change of the S2 subunit, which leads to fusion of the viral envelope with the cellular or lysosomal membrane and release of viral nucleic acids into the cytoplasm [3].
  • One of the means to enhance the immune response is to conjugate poorly immunogenic antigens to exogenous macromolecules used as carriers, which has been used successfully for decades.
  • the applicant's invention is titled "A method for enhancing the immunogenicity of protein/peptide antigens", and the invention patent applications with application numbers CN202010369100.7 and PCT/CN2021/090809 report the inventor's pioneering invention: by combining The protein/peptide antigen is conjugated with sugar to form a sugar-protein/peptide antigen conjugate, which improves the immunogenicity of the protein/peptide antigen.
  • This invention is different from conventional glyco-protein peptide conjugate vaccines such as encephalitis vaccine, haemophilus influenza b vaccine and pneumonia vaccine, which combine their purified capsular polysaccharides with carrier proteins to produce more potent immunogens sexual composition.
  • the present invention truncated and expressed the RBD protein and formed a carbohydrate-coronavirus RBD protein conjugate with pneumonia polysaccharide.
  • the conjugate After immunizing animals with the conjugate as an immunogen, long-term humoral and cellular immune responses can be maintained, and higher titers of neutralizing antibodies and cellular immune responses can be generated to prevent coronaviruses, such as SARS-CoV-2 infection related diseases.
  • a first aspect of the present invention provides a method for improving the immunogenicity of a coronavirus RBD antigen, the method comprising forming a saccharide-coronavirus RBD antigen conjugate by conjugating the coronavirus RBD antigen with a saccharide.
  • the saccharide in the method is selected from polysaccharides, oligosaccharides or monosaccharides; preferably Neisseria encephalitis capsular polysaccharide, Haemophilus influenzae b capsular polysaccharide, Streptococcus pneumoniae capsular polysaccharide, Group B Staphylococcus aureus capsular polysaccharide, glucan, mannan, starch, inulin, pectin, carboxymethyl starch, chitosan and its derivatives; more preferably Streptococcus pneumoniae capsular polysaccharide, most preferably Preferred are S. pneumoniae serotype 14 capsular polysaccharide, S. pneumoniae serotype 6B capsular polysaccharide and S. pneumoniae serotype 7F capsular polysaccharide.
  • the coronavirus RBD antigen in the method comprises the amino acid sequence of SEQ ID NO: 2 or RBD truncations of different lengths thereof, having at least 90%, 92%, 95%, 98% or Amino acid sequences of 100% sequence identity or RBD truncations of different lengths.
  • the coronavirus RBD antigen in the method is conjugated to S. pneumoniae serotype 14 capsular polysaccharide/S. pneumoniae serotype 6B capsular polysaccharide.
  • the S. pneumoniae serotype 14 capsular polysaccharide in the method is derived from ATCC6314 and the S. pneumoniae serotype 6B capsular polysaccharide is derived from ATCC6326.
  • the coronavirus RBD antigen in the method is further fused to other proteins or peptides.
  • the coronavirus RBD antigen in the method is used in combination with an immune adjuvant, preferably, the adjuvant is ALUM/MF59.
  • the method enhances a Th1-type immune response.
  • Another aspect of the present invention provides a C-terminally truncated SARS-CoV-2 RBD antigen comprising the Arg319 to Thr531 fragment of the S1 subunit of the SARS-CoV-2 spike protein, preferably, comprising SEQ ID NO: 2.
  • Another aspect of the present invention provides a saccharide-coronavirus RBD antigen conjugate with improved immunogenicity compared to unconjugated coronavirus RBD antigen.
  • the saccharide in the saccharide-coronavirus RBD antigen conjugate is selected from polysaccharides, oligosaccharides or monosaccharides; preferably Neisseria encephalococcal capsular polysaccharide, Haemophilus influenzae b capsular polysaccharide , Streptococcus pneumoniae capsular polysaccharide, group B Staphylococcus aureus capsular polysaccharide, glucan, mannan, starch, inulin, pectin, carboxymethyl starch, chitosan and its derivatives; more preferably S. pneumoniae capsular polysaccharide, most preferably S. pneumoniae serotype 14 capsular polysaccharide, S. pneumoniae serotype 6B capsular polysaccharide and S. pneumoniae serotype 7F capsular polysaccharide.
  • the coronavirus RBD antigen in the RBD antigen conjugate is a C-terminally truncated SARS-CoV-2 RBD antigen comprising Arg319 of the S1 subunit of the SARS-CoV-2 spike protein Fragments to Thr531, preferably, comprising the amino acid sequence described in SEQ ID NO: 2 or RBD truncations of different lengths, amino acids having at least 90%, 92%, 95%, 98% or 100% sequence identity therewith sequence or RBD truncations of different lengths.
  • the coronavirus RBD antigen in the RBD antigen conjugate is conjugated to S. pneumoniae serotype 14 capsular polysaccharide/S. pneumoniae serotype 6B capsular polysaccharide.
  • the S. pneumoniae serotype 14 capsular polysaccharide in the RBD antigen conjugate is derived from ATCC 6314 and the S. pneumoniae serotype 6B capsular polysaccharide is derived from ATCC 6326.
  • the coronavirus RBD antigen in the RBD antigen conjugate is further fused with other proteins or peptides.
  • the RBD antigen conjugate is used in combination with an immune adjuvant, preferably, the adjuvant is ALUM/MF59.
  • the RBD antigen conjugate enhances a Th1-type immune response when used as an antigen.
  • Another aspect of the present invention provides an immune complex comprising the coronavirus RBD antigen of the present invention or the RBD antigen conjugate of the present invention and an immune adjuvant.
  • the adjuvant in the immune complex is selected from ALUM/MF59.
  • Another aspect of the present invention provides the use of the coronavirus RBD antigen, RBD antigen conjugate or immune complex as described in the present invention to prevent or treat diseases caused by coronavirus.
  • Another aspect of the present invention provides coronavirus RBD antigens, RBD antigen conjugates or immune complexes as described in the present invention in the preparation of vaccines/medicines for preventing or treating diseases caused by coronaviruses.
  • Figure 1 depicts the SEC and SDS-PAGE profiles of the RBD(T4) recombinant protein pair.
  • FIG. 2 depicts that PS14 (A) and PS6B (B) enhance serum antibody titers in RBD (T4) immunized mice.
  • FIG. 3 depicts that PS14 (A) and PS6B (B) enhance serum neutralizing antibody titers in RBD (T4) immunized mice.
  • Figure 4 depicts that PS14 (A) and PS6B (B) increase the ratio of mIgG2a/mIgG1 antibody titers in the serum of RBD (T4) immunized mice.
  • FIG. 5 depicts that PS14 enhances Th1-type T cell responses in RBD(T4) immunized mice.
  • Figure 6 depicts that MF59 adjuvant enhances serum antibody titers (A) and neutralizing antibody titers (B) in PS14-RBD (T4) immunized mice.
  • Figure 7 depicts that MF59 adjuvant increases the ratio of mIgG2a/mIgG1 antibody titers in the serum of PS14-RBD(T4) immunized mice.
  • Figure 8 depicts that MF59 adjuvant enhances Th1-type T cell responses in PS14-RBD(T4) immunized mice.
  • antigen refers to a foreign substance that is recognized (specifically bound) by an antibody or T cell receptor, but which does not deterministically induce an immune response.
  • Foreign substances that induce specific immunity are called “immune antigens” or “immunogens”.
  • Hapen refers to an antigen that does not elicit an immune response by itself (although a combination of several molecules of a hapten, or a combination of a hapten and a macromolecular carrier, can elicit an immune response).
  • S protein The protein referred to by the term "Spike protein (S protein)" is distributed on the surface of the coronavirus membrane in the form of trimers, which bind to host cell receptors to mediate the invasion of the virus and determine the organization or organization of the virus. Host tropism.
  • the host cell receptor protein of SARS-CoV-2 is angiotensin-converting enzyme 2 (ACE2).
  • ACE2 angiotensin-converting enzyme 2
  • S protein trimeric spike protein of the virus binds to the ACE2 receptor and is cleaved by the host protease into the S1 polypeptide containing the receptor binding domain (SARS-COV-2 RBD) and responsible for mediating the virus with the cell membrane. Fusion S2 polypeptide.
  • RBD receptor binding domain, Receptor binding domain, SARS-COV-2 RBD
  • S1 subunit contains receptor binding domain (Receptor binding domain, SARS-COV-2 RBD), eg, Arg319-Phe541 of a certain version of the S1 subunit.
  • a “humoral immune response” is an antibody-mediated immune response and involves the introduction and production of antibodies that recognize and bind with a certain affinity to the antigens in the immunogenic compositions of the present invention, and a “cell-mediated immune response” is produced by T cells and /or other leukocyte-mediated immune responses.
  • a "cell-mediated immune response” is elicited by presenting epitopes associated with class I or class II molecules of the major histocompatibility complex (MHC), CD1 or other atypical MHC-like molecules.
  • MHC major histocompatibility complex
  • sugar may be used to refer to polysaccharides, oligosaccharides or monosaccharides.
  • the polysaccharide can be isolated from an organism, such as a bacteria, and can be a natural polysaccharide, optionally sized to some extent using microfluidization methods. Sizing the polysaccharide can reduce the viscosity of the polysaccharide sample and/or improve the filterability of the conjugated product.
  • Oligosaccharides are hydrolyzed polysaccharides with a small number of repeating units (typically, 5-30 repeating units). Polysaccharides can also be chemically synthesized.
  • conjugates refers to a protein/peptide covalently conjugated to a sugar.
  • saccharide RBD antigen conjugates of the present invention and immunogenic compositions comprising the same may contain certain amounts of free saccharides, proteins/peptides.
  • conjugation refers to the process by which a saccharide, such as a bacterial capsular polysaccharide, is covalently linked to a protein/peptide.
  • immunogenic composition refers to any pharmaceutical composition containing an antigen, such as a microorganism or a component thereof, that can be used to elicit an immune response in an individual.
  • Immunogenic means that an antigen (or epitope of an antigen), such as the coronavirus spike protein receptor binding region or a glycoconjugate or immunogenic composition comprising the antigen, is animal) to elicit a humoral or cell-mediated immune response, or both.
  • a “protective” immune response refers to the ability of an immunogenic composition to induce a humoral or cell-mediated immune response, or both, to protect an individual from infection.
  • the protection afforded does not have to be absolute, i.e., it does not have to completely prevent or eradicate the infection, so long as there is a statistically significant improvement relative to a population of control individuals (eg, infected animals not administered the vaccine or immunogenic composition) . Protection may be limited to moderating the severity of infection symptoms or the rapidity of onset.
  • Immunogenic amount and “immunogenically effective amount” are used interchangeably herein to mean that an antigen or immunogenic composition is sufficient to elicit an immune response (cellular (T cells) or humoral (B cells or antibodies) or both Or, the amount as measured by standard assays known to those skilled in the art.
  • the effectiveness of an antigen as an immunogen can be measured by a proliferation assay, by a cytolysis assay, or by measuring the level of B cell activity.
  • the method of improving the immunogenicity of a protein/peptide antigen of the present invention is achieved by the carbohydrate RBD antigen conjugate of the present invention and the immunogenic composition of the present invention.
  • Coronaviruses mainly mediate virus invasion through the binding of spike protein (S protein) to host cell receptors and determine the tissue or host tropism of the virus.
  • the host cell receptor protein of the new coronavirus SARS-CoV-2 is angiotensin-converting enzyme 2 (ACE2).
  • ACE2 angiotensin-converting enzyme 2
  • the trimeric spike protein (S protein) of the new coronavirus SARS-CoV-2 binds to the ACE2 receptor and is cleaved by the host protease into the S1 polypeptide containing the receptor binding domain (SARS-CoV-2 RBD) and The S2 polypeptide responsible for mediating the fusion of the virus with the cell membrane, and then invading the body.
  • a scheme of the present invention selects the RBD recombinant protein truncated at the C-terminal of the coronavirus as the antigen.
  • Antigens can be obtained by extracting natural pathogens or by genetic recombination.
  • the infection of the new coronavirus SARS-CoV-2 depends on the spike protein (Spike) on its surface.
  • the spike protein contains two subunits: S1 and S2, in which the receptor binding domain (RBD, Arg319) of the S1 subunit is located.
  • -Phe541) binds to the human cellular receptor angiotensin-converting enzyme 2 (ACE2) to mediate viral invasion.
  • ACE2 angiotensin-converting enzyme 2
  • the present invention adopts C-terminal truncated RBD recombinant protein.
  • the recombinant protein of the amino acid sequence or its active variant was named RBD(T4).
  • the present invention further conjugates the truncated RBD recombinant protein with sugar.
  • polysaccharide is bacterial polysaccharide, such as common Neisseria encephalococcus capsular polysaccharide, Haemophilus influenzae b capsular polysaccharide, Streptococcus pneumoniae capsular polysaccharide, group B Staphylococcus aureus capsular polysaccharide and glucan, Mannan, etc.
  • the polysaccharide can also be a plant-derived polysaccharide, such as starch, inulin, pectin, etc., or a derivative of a chemically modified polysaccharide, such as carboxymethyl starch.
  • the polysaccharide may also be of animal origin, such as chitosan and its derivatives.
  • the RBD(T4) recombinant protein antigen is conjugated to S. pneumoniae serotype 14 capsular polysaccharide/S. pneumoniae serotype 6B capsular polysaccharide.
  • the S. pneumoniae serotype 14 capsular polysaccharide is derived from ATCC6314, and the saccharide-RBD antigen conjugate is referred to as PS14-RBD(T4).
  • the S. pneumoniae serotype 6B capsular polysaccharide is derived from ATCC6326, and the saccharide-RBD antigen conjugate is designated PS6B-RBD(T4).
  • PS14-RBD(T4) and PS6B-RBD(T4) conjugate immunization both had higher total antibody titers, and both had significant improvement. It shows that the total antibody titer of RBD(T4) immunized mice can be significantly increased after conjugation with pneumococcal capsular polysaccharide PS14 or PS6B.
  • PS14-RBD(T4) and PS6B-RBD(T4) conjugates have higher mIgG2a/mIgG1 antibody titer ratios, indicating that RBD(T4) can be improved after conjugation with pneumococcal capsular polysaccharide PS14 or PS6B. Th1-type immune response.
  • the immunogenic composition of the present invention further comprises at least one of adjuvants, buffers, cryoprotectants, salts, divalent cations, nonionic detergents, free radical oxidation inhibitors, diluents or carriers A sort of.
  • the adjuvant in the immunogenic composition of the present invention is an aluminum-based adjuvant.
  • the adjuvant is selected from ALUM/MF59 oil-in-water adjuvant.
  • Adjuvants are substances that enhance the immune response when administered with an immunogen or antigen.
  • PS14-RBD(T4)+MF59 immunization slightly increased the expression level of splenic lymphocyte-specific IFN- ⁇ in mice, but IFN- ⁇ :IL-4 was significantly increased. This indicates that PS14 can effectively enhance the specific cellular immunity against RBD(T4) protein in mice.
  • PS14-RBD(T4)+Alum had higher stimulation-specific IL-4 expression levels (Fig. 8A), while PS14-RBD(T4)+MF59 had higher stimulation-specific Levels of IFN- ⁇ expression (FIG. 8B) and higher IFN- ⁇ :IL-4 ratio (FIG. 8C). Therefore, MF59 adjuvant has a stronger ability to enhance the cellular response of PS14-RBD(T4) immunized mice.
  • PS14-RBD(T4)+MF59 immunization can significantly increase the mIgG2a/mIgG1 antibody titer ratio, indicating that MF59 adjuvant has a better function of improving Th1/Th2 balance.
  • the immunogenic composition may optionally contain a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carriers include those in the pharmacopoeia of various countries for animals (including humans and non-human mammals).
  • the term carrier may be used to refer to a diluent, adjuvant, excipient or vehicle with which the pharmaceutical composition is administered. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid carriers, especially for injectable solutions.
  • the immunogenic compositions of the invention may also comprise one or more additional immunomodulatory agents, which are substances that perturb or alter the immune system such that up- or down-regulation of humoral and/or cell-mediated immunity is observed.
  • additional immunomodulatory agents are substances that perturb or alter the immune system such that up- or down-regulation of humoral and/or cell-mediated immunity is observed.
  • upregulation of humoral and/or cell-mediated arms of the immune system is provided.
  • adjuvants or cytokines include, for example, adjuvants or cytokines.
  • the immunogenic compositions of the invention for therapeutic or prophylactic treatment can be administered by intramuscular, intraperitoneal, intradermal or subcutaneous injection; or via mucosal administration to the oral/esophagus, respiratory tract, genitourinary tract. Intranasal administration of vaccines is preferred for the treatment of certain diseases, such as pneumonia or otitis media.
  • the vaccines of the present invention may be administered in a single dose, the components thereof may also be co-administered at the same time or in divided doses. In addition to a single route of administration, two different routes of administration can be used.
  • Optimal amounts of components for a particular immunogenic composition can be determined by standard studies involving observation of appropriate immune responses in individuals. Following an initial vaccination, an individual may receive one or several well-spaced booster immunizations.
  • the protein/peptide antigen conjugates and immune complexes of the present invention can prevent or treat diseases caused by pathogens, especially diseases caused by coronaviruses, and more particularly diseases caused by SARS-CoV-2 virus.
  • the infection of the new coronavirus SARS-CoV-2 depends on the spike protein (Spike) on its surface.
  • the spike protein contains two subunits: S1 and S2, in which the receptor binding domain (RBD, Arg319) of the S1 subunit is located.
  • -Phe541) binds to the human cellular receptor angiotensin-converting enzyme 2 (ACE2) to mediate viral invasion.
  • ACE2 human cellular receptor angiotensin-converting enzyme 2
  • RBD(T4) was designed in this example. The specific design scheme is shown in Table 1.
  • Amplification primers :
  • RBD-1 (SEQ ID NO: 7) GTCACCGTCCTGACACGAAGCTTGCCGCCACCATGAAACACCTGTGGTTTCTTCCT RBD-2 (SEQ ID NO: 8) TAGAATAGGGCCCTCTAGATTTAGGTGCTCTTCTTTGGTCCACAC
  • the complete sequence of the RBD(T4) recombinant protein gene (SEQ ID NO:5) was obtained by PCR amplification, including the signal peptide gene sequence (SEQ ID NO:3) and the RBD(T4) protein gene sequence (SEQ ID NO:1), Inserted into pSE vector (source: Shenzhou Cell Engineering Co., Ltd.) digested by Hind III+Xba I (source: Fermentas) by in-fusion method.
  • pSE vector source: Shenzhou Cell Engineering Co., Ltd.
  • Hind III+Xba I source: Fermentas
  • the plasmid was extracted, transiently transferred to HEK-293 cells, and the supernatant was collected by centrifugation after 7 days of culture.
  • the obtained cell supernatant is purified by hydrophobic chromatography and mixed anion exchange chromatography to obtain a relatively high-purity RBD (T4) recombinant protein, which is exchanged into the target buffer by ultrafiltration.
  • the purified RBD(T4) recombinant protein was analyzed by size exclusion high performance liquid chromatography (SEC-HPLC, TSK-G2000) and non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively.
  • SEC-HPLC size exclusion high performance liquid chromatography
  • SDS-PAGE non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • RBD(T4) recombinant protein with 0.36mg/mL citric acid, 2.35mg/mL sodium citrate, 14.61mg/mL NaCl, 0.13g/kg sodium hydroxide, pH6.0 buffer, final concentration at 0.6 mg/mL or so.
  • the samples were stored at 25°C for 1 week (25T1W), 37°C for 1 week (37T1W), 45°C for 1 week (45T1W), stored at -80°C for 3 hours, and then transferred to 45°C for thawing.
  • 1 h (5FT), repeated freezing and thawing was performed five times in this way, and SEC-HPLC, dynamic light scattering (DLS), and non-reducing SDS-PAGE were used to analyze the change of the purity of the samples before and after the acceleration.
  • the accelerated stability test results of the RBD(T4) recombinant protein are shown in Table 2. The results showed that the SEC purity of RBD(T4) recombinant protein was still high under multiple accelerated conditions, the aggregate level increased less, and the fragment level did not change, showing good accelerated stability.
  • Example 2 Preparation of RBD (T4) recombinant protein pneumococcal serotype 14 capsular polysaccharide (PS14) and serotype 6B capsular polysaccharide (PS6B) conjugates
  • Serotype 14 S. pneumoniae seed is ATCC 6314 and serotype 6B S. pneumoniae seed is ATCC 6326.
  • Centrifuge at 14000g for 30min take the supernatant, and use 100kDa ultrafiltration to concentrate to one-tenth of the original volume, about 400mL.
  • the concentrate was gradually adjusted to pH 3.5 by adding 36% acetic acid. Let stand for 2 h, centrifuge at 14,000 g for 30 min, take 390 mL of supernatant, add 130 mL of absolute ethanol, mix well, and let stand overnight. The next day, centrifuge at 14000g for 30min, take the supernatant, add 780mL absolute ethanol, mix well, and let stand overnight.
  • Coronavirus spike protein receptor binding region exchange medium take 5 mg of RBD(T4) protein, use a 30,000MW ultrafiltration tube to exchange the medium to 50mM Na 2 HPO 4 with a pH of 7.0, buffer, and finally concentrate to a volume of less than 0.25 mL, that is, the final protein concentration is ⁇ 20 mg/mL.
  • the coronavirus spike protein receptor binding region is conjugated with polysaccharide: take 5 mg of RBD (T4), add 3 mg of activated Streptococcus pneumoniae capsular polysaccharide, and add 50 mM, pH 7.0 Na 2 HPO 4 buffer to the total The final volume was 0.5 mL, then 0.5 ⁇ L of 5M sodium cyanoborohydride solution was added, and the reaction was rotated and mixed for 16 h at room temperature in the dark. Then, 0.3 mL of 10 mg/mL sodium borohydride solution was added to the reaction solution, and the reaction was carried out at room temperature for 1 h.
  • conjugated sample PS14-RBD (T4) or PS6B-RBD (T4) was used in a 100,000 MW ultrafiltration tube, PBS buffer was exchanged 10 times, and the final ultrafiltration volume was less than 5 mL.
  • the ultrafiltered conjugate samples were sterile filtered through a 0.22 ⁇ m filter and stored at 4°C.
  • the RBD(T4), PS14-RBD(T4) or PS6B-RBD(T4) conjugate antigen was diluted with PBS to 0.02 mg/mL, and the diluted antigen was mixed with an equal volume of MF59 adjuvant.
  • the protein concentration was 0.01 mg/mL.
  • the PS14-RBD(T4) conjugate antigen was diluted with PBS to 0.02 mg/mL, 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 concentration of the antigen in the immunization composition was 0.01 mg/mL.
  • mice were selected from 4-6 week Balb/c mice, and 0.1 mL of the immune composition with a concentration of 0.01 mg/mL as described in Example 2.3 was intraperitoneally injected.
  • Booster immunization was performed on the 28th day, blood was collected from the tail vein on the 35th day (3 days for 7 days), and the spleen was collected on the 26th day (3 days for 12 days) or 49 days (3 days for 21 days).
  • the PS6B-RBD (T4) immunized group was boosted on the 14th day, and the tail vein blood was collected on the 35th day (21 days after immunization). After blood collection, the serum total antibody titer, neutralization titer, IgG2a subtype and IgG1 subtype antibody titer were determined, and the mouse spleen was taken to determine the specific T cell response.
  • the SARS-COV-2 RBD protein (Shenzhou Cell Engineering Co., Ltd., the same as the full text) at a concentration of 5 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L/well, overnight at 2-8 °C. After washing the plate, 2% BSA was added for blocking at room temperature for 1 h.
  • TBST containing 0.1% bovine serum albumin (BSA) to dilute the serum to be tested to different dilutions (the specific dilution times are different according to the time of immune blood collection, such as 1000 ⁇ , 8000 ⁇ , 16000 ⁇ , 32000 ⁇ dilution),
  • the experiment set SARS-COV-2 RBD-mFc immunized mouse serum as positive control, healthy mouse serum (Shenzhou Cell Engineering Co., Ltd., the same as the full text) as negative control, and no serum as blank control, 100 ⁇ L/well, incubated at room temperature 1-2h.
  • BSA bovine serum albumin
  • the neutralization level of the immunized mouse serum was further detected.
  • the specific dilution ratio is different according to the time of immunization blood collection, such as 500 ⁇ , 1581 ⁇ , 5000 ⁇ , 15810 ⁇ dilution).
  • a positive control group M inoculated with cells, without sample, containing pseudovirus
  • a negative control group M' inoculated with cells, without sample, without pseudovirus
  • the 96-well plate was incubated at 37°C and 5% CO 2 for 20-24 h, 50 ⁇ L/well was added with 1 ⁇ PLB to react for 10 min, and 40 ⁇ L/well was transferred to a 96-well all-white fluorescence assay plate, and then the microplate format was used.
  • the luminescence detector performs bioluminescence detection.
  • Enzyme-linked immunosorbent assay was used to detect the mIgG1 or mIgG2a subtype antibody titers against RBD in mouse serum.
  • the SARS-COV-2 RBD protein at a concentration of 5 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L/well, overnight at 2-8 °C. After washing the plate, 2% BSA was added for blocking at room temperature for 1 h.
  • TBST containing 0.1% bovine serum albumin (BSA) to dilute the serum to be tested to different dilutions (the specific dilution times are different according to the time of immune blood collection, such as 1000 ⁇ , 8000 ⁇ , 16000 ⁇ , 32000 ⁇ dilution), Serum from healthy mice was used as negative control, and serum-free was used as blank control, 100 ⁇ L/well, incubated at room temperature for 1-2 h.
  • BSA bovine serum albumin
  • Antibody titers of the mIgG1 or mIgG2a subtype in the serum were titrated at the maximum dilution with an OD450 value greater than the OD450 of the negative serum multiplied by 2.1.
  • mIgG2a characterizes Th1-type immune responses (cellular immunity)
  • mIgG1 characterizes Th2-type immune responses (humoral immunity).
  • IFN- ⁇ cellular immunity
  • IL-4 humoral immunity
  • the cell seeding density is 2 ⁇ 10 5 cells/well
  • 100 ⁇ L/well is added to the final concentration of 2 ⁇ g/mL of RBD recombinant protein was incubated in a 37°C, 5% CO 2 incubator for about 40h.
  • the cell supernatant from the ELISpot plate was removed, and the plate was washed 5 times with PBS, and then 100 ⁇ L/well was added to the diluted assay.
  • Antibody after 2h incubation, the plate was washed 5 times with PBS, 100 ⁇ L/well was added with diluted Streptavidin-ALP (1:1000), after 1h incubation at room temperature, the plate was washed 5 times with PBS, and then 100 ⁇ L/well was added and filtered with a 0.45 ⁇ m filter.
  • the BCIP/NBT-plus substrate was developed at room temperature for 10-30 minutes in the dark until clear spots appeared and terminated with tap water.
  • the ELISpot plate was placed in a cool place at room temperature and allowed to dry naturally, and the results were analyzed by an enzyme-linked spot analyzer.
  • the number of antigen-specific IFN- ⁇ positive T cells was expressed as SFC (Spot-forming cells) per 10 6 mouse spleen cells, and the data was counted by GrapPad Prism software.
  • the results are shown in Figure 5.
  • PS14-RBD(T4)+MF59 immunization slightly increased the expression level of splenic lymphocyte-specific IFN- ⁇ in mice, but IFN- ⁇ :IL- 4 has a significant increase, indicating that PS14 can effectively enhance the specific cellular immunity against RBD (T4) protein in mice.
  • 3.3.1 MF59 adjuvant has stronger ability to increase serum antibody titer and neutralizing antibody titer of PS14-RBD(T4) immunized mice
  • Example 3.2.1 the total antibody titer and neutralizing antibody titer of the sera of mice immunized with the immune composition of PS14-RBD (T4) and MF59 or Alum adjuvant were detected.
  • the results are shown in Figure 6.
  • Alum adjuvant can slightly increase the total antibody titer and neutralizing titer
  • MF59 adjuvant can greatly increase the total antibody titer (Figure 6A) and Neutralizing antibody titers ( Figure 6B), and significantly better than Alum adjuvant.
  • Example 3.2.2 to detect the RBD-specific mIgG2a or mIgG1 subtype antibody titers in the sera of mice immunized with the immune composition of PS14-RBD (T4) and MF59 or Alum adjuvant, and calculate the mIgG2a/mIgG1 ratio.
  • the results are shown in Figure 7.
  • 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 Adjuvants have better ability to enhance cellular response.
  • spleen lymphocyte-specific IFN- ⁇ (cellular immunity) and IL-4 (humoral immunity) secretion levels of mice were detected after 2 immunization for 12 days.
  • the results are shown in Fig. 8.
  • PS14-RBD(T4)+Alum has a higher level of stimulation-specific IL-4 expression (Fig. 8A)
  • PS14-RBD(T4)+ MF59 had higher levels of stimulation-specific IFN- ⁇ expression
  • FIG. 8C IFN- ⁇ :IL-4 ratio

Abstract

一种提高冠状病毒RBD免疫原性的方法、糖-冠状病毒RBD抗原缀合物及含有该抗原缀合物的免疫原性复合物。具体地，将稳定性更优的RBD截短蛋白与肺炎多糖形成糖-冠状病毒RBD蛋白缀合物，以此为免疫原免疫动物后可维持长时程的体液免疫和细胞免疫反应。糖-冠状病毒RBD蛋白缀合物同MF59佐剂形成的免疫复合物可产生更高效价的中和抗体和细胞免疫反应。该免疫原性复合物可用于预防冠状病毒(例如SARS-CoV-2)感染相关疾病。

Description

通过糖-冠状病毒RBD抗原缀合物提高免疫原性的方法
相关申请的交叉引用
本申请要求2020年09月14日提交的中国专利申请202010963735.X的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明属于免疫学领域，涉及一种提高冠状病毒RBD免疫原性的方法、糖-冠状病毒RBD抗原缀合物及含有该抗原缀合物的免疫原性复合物。具体地，将稳定性更优的RBD截短蛋白与肺炎多糖形成糖-冠状病毒RBD蛋白缀合物，以此为免疫原免疫动物后可维持长时程的体液免疫和细胞免疫反应。
背景技术
SARS-CoV-2和SARS-CoV具有共同的宿主细胞受体蛋白，即血管紧张素转化酶2(ACE2)[1]。病毒的三聚体S蛋白同ACE2受体结合后被宿主蛋白酶切割为包含受体结合域(Receptor binding domain,RBD)的S1多肽和负责介导病毒同细胞膜融合的S2多肽[2]。S1和ACE2之间的特异性相互作用会触发S2亚基的构象变化，从而导致病毒包膜和细胞膜或溶酶体膜融合并释放病毒核酸进入细胞质[3]。数据表明COVID-19患者，尤其是在重症患者中，伴随肺炎症状，淋巴细胞显著降低，血浆促炎因子显著增加，提示了免疫系统在疾病进程中发挥着重要的作用[4-6]。对23例COVID-19患者出现症状后血清抗体的分析表明，大多数患者在出现症状10天后发生针对RBD蛋白的抗体反应[7]。发病早期血清中RBD蛋白抗体阳性患者比例高于N蛋白抗体阳性患者比例，说明机体可能先产生具有中和作用的抗体，以抑制病毒通过RBD侵入细胞。对细胞免疫的分析表明，刚出院患者针对不同抗原的特异性T细胞与未感染者的T细胞有显著差异，其中RBD特异性T细胞分布最广。康复两周后的随访患者细胞免疫水平则明显降低。RBD不仅可引起体液免疫，产生中和抗体，而且还可诱导T细胞免疫应答，因此RBD蛋白是SARS-CoV-2疫苗的有效靶标。
增强免疫反应的手段之一是将免疫原性差的抗原与用作载体的外源大分子缀合，这一手段已成功应用了数十年。
申请人的发明名称为《一种增强蛋白/肽抗原免疫原性的方法》、申请号为 CN202010369100.7和PCT/CN2021/090809的发明专利申请中报道了发明人的开创性的发明：通过将蛋白/肽抗原与糖缀合，形成糖-蛋白/肽抗原缀合物，提高了蛋白/肽抗原的免疫原性。该发明与习见的糖-蛋白肽缀合物疫苗如脑炎疫苗、嗜血流感细菌b疫苗和肺炎疫苗不同，后者以其纯化的荚膜多糖与载体蛋白结合而产生更有效的免疫原性组合物。
为增强蛋白稳定性，本发明将RBD蛋白进行了截短表达并同肺炎多糖形成糖-冠状病毒RBD蛋白缀合物。以该缀合物作为免疫原免疫动物后可维持长时程的体液免疫和细胞免疫反应，产生更高效价的中和抗体和细胞免疫反应以用于预防冠状病毒，例如SARS-CoV-2感染相关疾病。
发明内容
本发明的第一方面提供了一种提高冠状病毒RBD抗原免疫原性的方法，该方法包括通过将冠状病毒RBD抗原与糖缀合，形成糖-冠状病毒RBD抗原缀合物。
在一个实施方案中，所述方法中的糖选自多糖、寡糖或单糖；优选为奈瑟氏脑炎球菌荚膜多糖、嗜血性流感杆菌b荚膜多糖、肺炎链球菌荚膜多糖、B群金黄色葡萄球菌荚膜多糖、葡聚糖、甘露聚糖、淀粉、菊糖、果胶、羧甲基淀粉、壳聚糖及其衍生物；更优选为肺炎链球菌荚膜多糖，最优选为肺炎链球菌血清型14荚膜多糖、肺炎链球菌血清型6B荚膜多糖和肺炎链球菌血清型7F荚膜多糖。
在一个实施方式中，所述方法中的冠状病毒RBD抗原包含SEQ ID NO:2所述氨基酸序列或其不同长度的RBD截短体、与其具有至少90％，92％，95％，98％或100％序列同一性的氨基酸序列或其不同长度的RBD截短体。
在一个实施方案中，所述方法中的冠状病毒RBD抗原与肺炎链球菌血清型14荚膜多糖/肺炎链球菌血清型6B荚膜多糖缀合。
在一个实施方案中，所述方法中的肺炎链球菌血清型14荚膜多糖衍生于ATCC6314，肺炎链球菌血清型6B荚膜多糖衍生于ATCC6326。
在一个实施方案中，所述方法中的冠状病毒RBD抗原进一步与其他蛋白或肽融合。
在一个实施方案中，所述方法中的冠状病毒RBD抗原与免疫佐剂合用，优选的，佐剂为ALUM/MF59。
在一个实施方案中，所述方法提升了Th1型免疫反应。
本发明的另一个方面提供了一种C端截短的SARS-CoV-2 RBD抗原，其包含SARS-CoV-2刺突蛋白S1亚基的Arg319至Thr531片段，优选的，包含SEQ ID NO：2所描述的氨基酸序列或其不同长度的RBD截短体、与其具有至少90％，92％，95％，98％或 100％序列同一性的氨基酸序列或其不同长度的RBD截短体。
本发明的另一个方面提供了一种糖-冠状病毒RBD抗原缀合物，与未经缀合的冠状病毒RBD抗原相比，其免疫原性提高。
在一个实施方案中，所述糖-冠状病毒RBD抗原缀合物中的糖选自多糖、寡糖或单糖；优选为奈瑟氏脑炎球菌荚膜多糖、嗜血性流感杆菌b荚膜多糖、肺炎链球菌荚膜多糖、B群金黄色葡萄球菌荚膜多糖、葡聚糖、甘露聚糖、淀粉、菊糖、果胶、羧甲基淀粉、壳聚糖及其衍生物；更优选为肺炎链球菌荚膜多糖，最优选为肺炎链球菌血清型14荚膜多糖、肺炎链球菌血清型6B荚膜多糖和肺炎链球菌血清型7F荚膜多糖。
在一个实施方案中，所述RBD抗原缀合物中的冠状病毒RBD抗原是一种C端截短的SARS-CoV-2 RBD抗原，其包含SARS-CoV-2刺突蛋白S1亚基的Arg319至Thr531片段，优选的，包含SEQ ID NO：2所描述的氨基酸序列或其不同长度的RBD截短体、与其具有至少90％，92％，95％，98％或100％序列同一性的氨基酸序列或其不同长度的RBD截短体。
在一个实施方案中，所述RBD抗原缀合物中的冠状病毒RBD抗原与肺炎链球菌血清型14荚膜多糖/肺炎链球菌血清型6B荚膜多糖缀合。
在一个实施方案中，所述RBD抗原缀合物中的肺炎链球菌血清型14荚膜多糖衍生于ATCC 6314，肺炎链球菌血清型6B荚膜多糖衍生于ATCC6326。
在一个实施方案中，所述RBD抗原缀合物中的冠状病毒RBD抗原进一步与其他蛋白或肽融合。
在一个实施方案中，所述RBD抗原缀合物与免疫佐剂合用，优选的，佐剂为ALUM/MF59。
在一个实施方案中，所述RBD抗原缀合物用作抗原时，提升了Th1型免疫反应。
本发明的另一个方面提供了一种免疫复合物，其包含本发明所述的冠状病毒RBD抗原或本发明所述的RBD抗原缀合物及免疫佐剂。
在一个实施方案中，所述免疫复合物中的佐剂选自ALUM/MF59。
本发明的另一个方面提供了如本发明所述的冠状病毒RBD抗原、RBD抗原缀合物或免疫复合物预防或治疗冠状病毒引起的疾病的用途。
本发明的另一个方面提供了如本发明所述的冠状病毒RBD抗原、RBD抗原缀合物或免疫复合物在制备预防或治疗冠状病毒引起的疾病的疫苗/药物用途。
附图说明
图1描述了RBD(T4)重组蛋白对的SEC及SDS-PAGE图谱。
图2描述了PS14(A)及PS6B(B)增强RBD(T4)免疫小鼠血清抗体效价。
图3描述了PS14(A)及PS6B(B)增强RBD(T4)免疫小鼠血清中和抗体效价。
图4描述了PS14(A)及PS6B(B)增加RBD(T4)免疫小鼠血清中mIgG2a/mIgG1抗体滴度的比值。
图5描述了PS14提升RBD(T4)免疫小鼠Th1型T细胞反应。
图6描述了MF59佐剂增强PS14-RBD(T4)免疫小鼠血清抗体效价(A)及中和抗体效价(B)。
图7描述了MF59佐剂增加PS14-RBD(T4)免疫小鼠血清中mIgG2a/mIgG1抗体滴度的比值。
图8描述了MF59佐剂提升PS14-RBD(T4)免疫小鼠Th1型T细胞反应。
发明详述
定义
除非另有说明，本文使用的所有技术和科学术语具有本发明所属的技术领域的普通技术人员通常理解的含义。为了本发明的目的，进一步定义以下术语。
当用于本文和所附权利要求书中时，单数形式“一”、“一种”、“另一”和“所述”包括复数指代对象，除非上下文明确地另有指示。
术语“包括”、“包含”是指包括具体成分而不排除任何其他的成分。诸如“基本上由……组成”允许包括不损害本发明的新颖或基本特征的其他成分或步骤，即，它们排除损害本发明的新颖或基本的特征的其他未列举的成分或步骤。
术语“抗原”是指一种由抗体或T细胞受体所识别(特异性结合)的外源物质，但是其不能确定性地诱导免疫应答。诱导特异性免疫的外源性物质称为“免疫性抗原”或“免疫原”。“半抗原”是指一种本身不能引发免疫应答(尽管几个分子半抗原的结合物，或半抗原与大分子载体的结合物可引发免疫应答)的抗原。
术语“棘突蛋白(Spike蛋白，S蛋白)”所指的蛋白以三聚体的形式分布在冠状病毒膜表面，其与宿主细胞受体结合从而介导病毒的入侵，并决定病毒的组织或宿主嗜性。SARS-CoV-2的宿主细胞受体蛋白为血管紧张素转化酶2(ACE2)。病毒的三聚体棘突蛋白(S蛋白)同ACE2受体结合后被宿主蛋白酶切割为包含受体结合域(Receptor binding domain,SARS-COV-2 RBD)的S1多肽和负责介导病毒同细胞膜融合的S2多肽。
“RBD(受体结合域、Receptor binding domain、SARS-COV-2 RBD)”系棘突蛋白同 ACE2受体结合后被宿主蛋白酶切割为S1亚单位和S2亚单位，S1亚单位包含受体结合域(Receptor binding domain,SARS-COV-2 RBD)，例如，某一种版本的S1亚基的Arg319-Phe541。
“体液免疫应答”是抗体介导的免疫应答并且涉及引入和生成以一定亲和力识别和结合本发明的免疫原性组合物中的抗原的抗体，“细胞介导的免疫应答”是由T细胞和/或其他白细胞介导的免疫应答。“细胞介导的免疫应答”是通过提供与主要组织相容性复合物(MHC)的I类或II类分子、CD1或其他非典型MHC样分子相关的抗原表位而诱发的。
术语“糖”可以用于指多糖、寡糖或单糖。多糖可以自生物体，如细菌分离，可以是天然的多糖，任选地，用微流化方法，将其大小调整至一定程度。将多糖进行大小调整，可降低多糖样品的粘度并且/或者提高缀合的产品的过滤性。寡糖是具有少量重复单元的水解多糖(典型地，5-30个重复单元)。多糖亦可以是化学合成的。
术语“缀合物”是指与糖共价缀合的蛋白/肽。本发明的糖RBD抗原缀合物和包含其的免疫原性组合物可以包含一定量的游离糖、蛋白/肽。
本文所使用的“缀合”是指借以使例如细菌荚膜多糖的糖与蛋白/肽共价连接的过程。
术语“免疫原性组合物”是指含有抗原如微生物或其组分的任何药物组合物，该组合物可用于在个体中诱发免疫应答。
如本文所使用的“免疫原性”意指抗原(或抗原的表位)例如冠状病毒棘突蛋白受体结合区或包含该抗原的糖缀合物或免疫原性组合物在宿主(例如哺乳动物)中诱发体液或细胞介导的免疫应答或二者的能力。
“保护性”免疫应答是指免疫原性组合物诱发用于保护个体免于感染的体液或细胞介导的免疫应答或两者的能力。所提供的保护不必是绝对的，即，不必完全阻止或根除感染，只要相对于对照个体群体(例如未给药疫苗或免疫原性组合物的受感染动物)存在统计学上显著的改进即可。保护可限于缓和感染症状的严重性或发作快速性。
“免疫原性量”和“免疫有效量”二者在本文可交换使用，是指抗原或免疫原性组合物足以引发免疫应答(细胞(T细胞)或体液(B细胞或抗体)应答或二者，如通过本领域技术人员已知的标准测定所测量的量。
抗原作为免疫原的有效性可通过增殖测定、通过细胞溶解测定、或通过测量B细胞活性水平来测量。
本发明的提高蛋白/肽抗原免疫原性的方法
本发明的提高蛋白/肽抗原免疫原性的方法通过本发明的糖RBD抗原缀合物以及本发明的免疫原性组合物得以实现。
本发明的糖RBD抗原缀合物
冠状病毒主要通过棘突蛋白(S蛋白)与宿主细胞受体结合来介导病毒的入侵，并决定病毒的组织或宿主嗜性。其中，新冠病毒SARS-CoV-2的宿主细胞受体蛋白为血管紧张素转化酶2(ACE2)。新冠病毒SARS-CoV-2的三聚体棘突蛋白(S蛋白)同ACE2受体结合后被宿主蛋白酶切割为包含受体结合域(Receptor binding domain,SARS-CoV-2 RBD)的S1多肽和负责介导病毒同细胞膜融合的S2多肽，进而侵入体内。
本发明的一个方案选用冠状病毒C末端截短的RBD重组蛋白作为抗原。抗原可以通过提取天然病原获得，也可以通过基因重组获得。新型冠状病毒SARS-CoV-2的感染依赖于其表面的刺突蛋白(Spike)，刺突蛋白含有两个亚基:S1和S2，其中位于S1亚基的受体结合结构域(RBD，Arg319-Phe541)结合人细胞受体血管紧张素-转化酶2(ACE2)来介导病毒入侵。研究表明，RBD结构域不仅可引起体液免疫产生中和抗体，而且还可诱导T细胞免疫应答,因此RBD蛋白是SARS-CoV-2疫苗的有效靶点。
为提升RBD重组蛋白的表达水平、纯度和稳定性，本发明采用C末端截短的RBD重组蛋白，在一个实施方案中，为包含SEQ ID NO:2(对应刺突蛋白序列R319-T531)所述氨基酸序列或其活性变体的重组蛋白，命名为RBD(T4)。为维持长时程的体液免疫和细胞免疫反应，本发明进一步将该截短的RBD重组蛋白与糖缀合。
所述多糖是细菌多糖，如常见的奈瑟氏脑炎球菌荚膜多糖、嗜血性流感杆菌b荚膜多糖、肺炎链球菌荚膜多糖、B群金黄色葡萄球菌荚膜多糖以及葡聚糖、甘露聚糖等。多糖也可以是植物来源多糖，如淀粉、菊糖、果胶等，也可以是经化学改性的多糖的衍生物，如羧甲基淀粉。所述多糖也可以是动物来源多糖，如壳聚糖及其衍生物。
在一个实施方案中，RBD(T4)重组蛋白抗原与肺炎链球菌血清型14荚膜多糖/肺炎链球菌血清型6B荚膜多糖缀合。
在一个实施方案中，肺炎链球菌血清型14荚膜多糖衍生于ATCC6314，该糖-RBD抗原缀合物被称为PS14-RBD(T4)。在另一个实施方案中，肺炎链球菌血清型6B荚膜多糖衍生于ATCC6326，该糖-RBD抗原缀合物被称PS6B-RBD(T4)。
在相同佐剂条件下，相比RBD(T4)免疫，PS14-RBD(T4)和PS6B-RBD(T4)缀合物免疫均具更高的总抗体效价，且均具有显著性提升。表明同肺炎球菌荚膜多糖PS14或PS6B缀合后均可显著提升RBD(T4)免疫小鼠的总抗体效价。
RBD(T4)同肺炎球菌荚膜多糖PS14或PS6B缀合后均可显著提升RBD(T4)免疫小鼠的血清中和抗体效价。
PS14-RBD(T4)和PS6B-RBD(T4)缀合物免疫均具更高的mIgG2a/mIgG1抗体滴度比值， 说明RBD(T4)同肺炎球菌荚膜多糖PS14或PS6B缀合后均可提升Th1型免疫反应。
本发明的免疫原性组合物
在一个实施方案中，本发明的免疫原性组合物还包含佐剂、缓冲剂、冷冻保护剂、盐、二价阳离子、非离子清洁剂、自由基氧化抑制剂、稀释剂或载体中的至少一种。在一个实施方案中，本发明的免疫原性组合物中的佐剂是铝系佐剂。在一个实施方案中，所述佐剂是选自ALUM/MF59水包油佐剂。
佐剂是当与免疫原或抗原一起给药时增强免疫应答的物质。
相比RBD(T4)+MF59免疫，PS14-RBD(T4)+MF59免疫微弱提升了小鼠脾淋巴细胞特异性IFN-γ的表达水平，但IFN-γ：IL-4具有显著性升高，说明PS14能够有效提升小鼠产生针对RBD(T4)蛋白的特异性细胞免疫。
相比PS14-RBD(T4)，PS14-RBD(T4)+Alum具有更高的刺激特异性IL-4表达的水平(图8A)，而PS14-RBD(T4)+MF59具有更高的刺激特异性IFN-γ表达的水平(图8B)和更高的IFN-γ：IL-4比值(图8C)。因此MF59佐剂具有更强的提升PS14-RBD(T4)免疫小鼠后细胞反应的能力。
而PS14-RBD(T4)+MF59免疫可显著提升mIgG2a/mIgG1抗体滴度比值，说明MF59佐剂具有更优的提升Th1/Th2平衡的功能。
所述免疫原性组合物可任选地包含药学上可接受的载体。所述药学上可接受的载体包括各国药典用于动物(包括人类以及非人类哺乳动物)的载体。术语载体可用于指与药物组合物一起给药的稀释剂、佐剂、赋形剂或媒介物。可采用水、盐水溶液以及含水的右旋糖和甘油溶液作为尤其用于注射溶液剂的液体载体。
本发明的免疫原性组合物还可包含一种或多种额外的免疫调节剂，其是扰乱或改变免疫系统从而观察到体液和/或细胞介导的免疫的上调或下调的物质。在一个实施方案中，提供了免疫系统的体液和/或细胞介导的能力(arms)的上调。包括例如佐剂或细胞因子。
本发明的免疫原性组合物的给药形式
用于治疗或预防性治疗的本发明免疫原性组合物可以通过肌内注射、腹膜内注射、皮内注射或皮下注射；或者经由粘膜给药至口腔/食道、呼吸道、泌尿生殖道。鼻内给予疫苗对于治疗某些疾病，例如肺炎或中耳炎是优选的。虽然本发明的疫苗可单剂量给予，但是其组分也可同时或分时共同给予。除了单一给药途径以外，可以使用两种不同的给药途径。
用于特定免疫原性组合物的组分的最佳量可通过涉及在个体中观察适当免疫应答的标准研究来确定。在进行初始疫苗接种后，个体可接受一次或若干次充分间隔的加强免疫。
本发明的免疫原性组合物的用途
本发明蛋白/肽抗原缀合物及免疫复合物可以预防或治疗病原体引起的疾病，尤其是冠状病毒，更尤其是SARS-CoV-2病毒引起的疾病。
具体实施方式
实施例1：RBD(T4)重组蛋白的构建及生产
1.1 RBD(T4)重组蛋白的基因构建和表达
新型冠状病毒SARS-CoV-2的感染依赖于其表面的刺突蛋白(Spike)，刺突蛋白含有两个亚基:S1和S2，其中位于S1亚基的受体结合结构域(RBD，Arg319-Phe541)结合人细胞受体血管紧张素-转化酶2(ACE2)来介导病毒入侵。研究表明，RBD结构域不仅可引起体液免疫产生中和抗体，而且还可诱导T细胞免疫应答,因此RBD蛋白是SARS-CoV-2疫苗的有效靶点。为提升RBD重组蛋白的表达水平、纯度和稳定性，本实施例设计了C末端截短的RBD重组蛋白：RBD(T4)。具体设计方案如表1。
表1 RBD(T4)重组蛋白设计方案
Figure PCTCN2021117680-appb-000001
①表示精氨酸的单字母缩写
[根据细则26改正28.09.2021]　
新型冠状病毒RBD基因的原始序列来源于NCBI(GenBank:MN908947.3)，该基因经过密码子优化，以提高目标抗原的表达量。利用如下所示的引物，制备RBD(T4)重组蛋白构建体：
扩增引物：
RBD-1(SEQ ID NO:7) GTCACCGTCCTGACACGAAGCTTGCCGCCACCATGAAACACCTGTGGTTCTTCCT
RBD-2(SEQ ID NO:8) TAGAATAGGGCCCTCTAGATTTAGGTGCTCTTCTTTGGTCCACAC
通过PCR扩增获得RBD(T4)重组蛋白基因全序列(SEQ ID NO:5)，包含信号肽基因序列(SEQ ID NO:3)和RBD(T4)蛋白基因序列(SEQ ID NO:1)，通过in-fusion方法插入到Hind III+Xba I(来源：Fermentas)酶切的pSE载体(来源：神州细胞工程有限公司)中。重组表达载体经测序正确后提取质粒，瞬转到HEK-293细胞，培养7天后离心收集上清。获得的细胞上清液利用疏水层析以及混合阴离子交换层析进行纯化，即可得到较高纯度的RBD(T4)重组蛋白，通过超滤换液到目标缓冲液中。
1.2 RBD(T4)重组蛋白的稳定性分析
经纯化获得的RBD(T4)重组蛋白分别通过分子排阻高效液相色谱(SEC-HPLC，TSK-G2000) 及非还原十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)方法分析纯度，结果显示SEC-HPLC纯度为99.5非还原SDS-PAGE纯度为98.6，表明RBD(T4)重组蛋白具有较高的纯度，结果参见图1。
将RBD(T4)重组蛋白置换到0.36mg/mL枸橼酸，2.35mg/mL枸橼酸钠，14.61mg/mL NaCL，0.13g/kg氢氧化钠，pH6.0缓冲液，终浓度在0.6mg/mL左右。将样品分别置于25℃中保存1周(25T1W)、37℃中保存1周(37T1W)、45℃中保存1周(45T1W)、置于-80℃条件保存3h后转移至45℃条件解冻1h(5FT)，如此进行五次反复冻融，应用SEC-HPLC、动态光散射(DLS)、非还原SDS-PAGE分析样品的加速前后的纯度变化。
RBD(T4)重组蛋白的加速稳定性检测结果见表2。结果表明RBD(T4)重组蛋白在多个加速条件下，SEC纯度仍较高，聚集体水平增加较少，片段水平无变化，表现出了良好的加速稳定性。
表2 RBD(T4)重组蛋白加速稳定性检测结果
Figure PCTCN2021117680-appb-000002
实施例2：RBD(T4)重组蛋白肺炎球菌血清型14荚膜多糖(PS14)及血清型6B荚膜多糖(PS6B)缀合物的制备
2.1肺炎链球菌血清型14(PS14)和6B(PS6B)荚膜多糖的制备
血清型14肺炎链球菌种子为ATCC 6314、血清型6B肺炎链球菌种子为ATCC 6326。
取0.5mL甘油保存的肺炎链球菌种子接入500mL Hoeprich's培养基(V.M.Goncalves,Optimization of medium and cultivation conditions for capsular polysaccharide productionby Streptococcus pneumoniae serotype 23F,AllpMicrobiolBiotechnol(2002)59:713-717)中，37℃摇床培养，转速150rpm，培养10-16h，待OD 600大于1.0时停止培养。加入0.6g脱氧胆酸钠，混匀，静置2h以上，使细菌完全裂解。转速14000g条件下离心30min，取上清， 用100kDa超滤浓缩至原十分之一体积,约400mL。浓缩液逐渐加入36％乙酸，调至pH 3.5。静置2h，转速14000g离心30min，取上清390mL加入130mL无水乙醇混匀，静置过夜。次日在14000g转速条件下离心30min后取上清，再加入780mL无水乙醇混匀，静置过夜。次日转速14000g离心30min后弃上清，在沉淀中加入300mL75％乙醇溶液，悬浮沉淀，之后再次14000g离心30min。弃上清，沉淀用10mL水溶解，控制溶液中多糖浓度大于10mg/mL。所得溶液即为肺炎链球菌荚膜多糖溶液。
取10mL浓度10mg/mL的多糖溶液，加入100mg高碘酸钠，混匀，避光静置反应1h。取装有5mL Sephadex G25填料的离心层析柱，加入10mL 50mM，pH＝7.0的Na 2HPO 4缓冲液，缓冲液在其重力作用下流过层析柱。然后将层析柱放入离心机，转速1000g条件下离心2min。之后更换新的收集管，取1mL经高碘酸钠氧化的多糖溶液放入离心层析柱，再次1000g离心2min。收集的离心柱流出液，即为活化的多糖溶液。
2.2 RBD(T4)与PS14或PS6B缀合物的制备
冠状病毒棘突蛋白受体结合区换液：取RBD(T4)蛋白5mg，用30,000MW超滤管换液至50mM，pH为7.0的Na 2HPO 4,缓冲液中，最终浓缩至体积小于0.25mL，既蛋白终浓度≥20mg/mL。
冠状病毒棘突蛋白受体结合区与多糖缀合：取5mg RBD(T4)，加入3mg活化后肺炎链球菌荚膜多糖，并补加50mM，pH为7.0的Na 2HPO 4缓冲液，至总终体积为0.5mL，然后加入5M氰基硼氢化钠溶液0.5μL，在室温避光条件下旋转混合反应16h。然后向反应液中加入10mg/mL的硼氢化钠溶液0.3mL，室温反应1h。然后将缀合后样品PS14-RBD(T4)或PS6B-RBD(T4)用100,000MW超滤管，PBS缓冲液换液10倍，最终超滤后体积小于5mL。0.22μm滤器无菌过滤超滤后的缀合物样品后于4℃保存。
2.3 PS14-RBD(T4)或PS6B-RBD(T4)缀合物免疫组合物的制备
以RBD(T4)或实施例2.2制备的PS14-RBD(T4)、PS6B-RBD(T4)缀合物作为抗原，以MF59或Alum为佐剂制备免疫组合物。
2.3.1 MF59佐剂的制备
配制200mL 10mM柠檬酸钠溶液(HCl调节pH6.5)，加入1mL Tween 80(南京威尔药业股份有限公司)混匀充分溶解。另取10mL角鲨烯(Merck)并加入1mL Span 85(肇庆市超能实业有限公司)混匀充分溶解。将前面两种溶液混合，用高压匀质机(AH-PILOTATS)设置800bar均质3次，得到均匀的乳液即为MF59佐剂。
2.3.2 MF59佐剂免疫组合物的制备
RBD(T4)、PS14-RBD(T4)或PS6B-RBD(T4)缀合物抗原用PBS稀释成0.02mg/mL，稀释 后的抗原与等体积的MF59佐剂混合，该免疫组合物中抗原的蛋白浓度为0.01mg/mL。
2.3.3 Alum佐剂免疫组合物的制备
PS14-RBD(T4)缀合物抗原用PBS稀释成0.02mg/mL，铝佐剂(北京诺宁生物科技有限公司)用PBS稀释成1mg/mL。稀释后的抗原和铝佐剂等体积混合。该免疫组合物中抗原的蛋白浓度为0.01mg/mL。
实施例3：RBD(T4)重组蛋白肺炎球菌荚膜多糖缀合物免疫原性研究
3.1免疫小鼠
小鼠选用4-6周Balb/c小鼠，腹腔注射0.1mL如实施例2.3所述的浓度为0.01mg/mL的免疫组合物，PS14-RBD(T4)免疫组分别于第14天和第28天加强免疫，第35天(3免7天)尾静脉取血，第26天(3免12天)或49天(3免21天)取脾。PS6B-RBD(T4)免疫组于第14天加强免疫，第35天(2免21天)尾静脉取血。采血后测定血清总抗体效价、中和效价以及IgG2a亚型，IgG1亚型抗体效价，取小鼠脾测定特异性T细胞反应。
3.2肺炎球菌荚膜多糖增加RBD(T4)重组蛋白的免疫原性(体液+细胞)
3.2.1肺炎球菌荚膜多糖增加RBD(T4)免疫小鼠血清抗体效价及中和抗体效价
将浓度为5μg/mL的SARS-COV-2 RBD蛋白(神州细胞工程有限公司，全文同)包被于96孔板，100μL/孔，2-8℃过夜包被。洗板后加入2％BSA室温封闭1h。使用含0.1％牛血清白蛋白(BSA)的TBST将待检测血清稀释到不同稀释度(具体稀释倍数根据免疫采血时间设定有不同，如1000×、8000×、16000×、32000×稀释)，实验设定SARS-COV-2 RBD-mFc免疫的小鼠血清为阳性对照，健康小鼠血清(神州细胞工程有限公司，全文同)为阴性对照，无血清为blank对照，100μL/孔，室温孵育1-2h。洗板3遍去除未结合抗体，加入山羊抗鼠IgG F(ab) 2/HRP(Jackson ImmunoResearch公司，全文同)检测二抗，100μL/孔，室温孵育约1h。洗板5遍，加入底物显色液进行显色，终止后酶标仪读取OD 450。以OD 450值大于阴性血清OD 450乘以2.1的最大稀释倍数为抗体效价。结果如图2所示，在相同佐剂条件下，相比RBD(T4)免疫，PS14-RBD(T4)和PS6B-RBD(T4)缀合物免疫均具更高的总抗体效价，且均具有显著性提升。表明同肺炎球菌荚膜多糖PS14或PS6B缀合后均可显著提升RBD(T4)免疫小鼠的总抗体效价。
本实施例进一步检测了免疫后小鼠血清的中和水平。将小鼠血清置于37℃水浴锅灭活30min后，每孔加入不同稀释梯度(具体稀释倍数根据免疫采血时间设定有不同，如500×、1581×、5000×、15810×稀释)的灭活后的小鼠血清样品50μL，与100TCID 50/孔的假病毒PSV-Luc-Spike(M)(神州细胞工程有限公司，全文同)等体积混合，37℃孵育1h后，侵染293FT-ACE2细胞(神州细胞工程有限公司，全文同)。将293FT-ACE2细胞3×10 4个 细胞/孔(100μL)均匀接种于含有血清样品与假病毒孵育物的96孔板中，细胞稀释液为DMEM+10％FBS+50μg/mL硫酸庆大霉素。设阳性对照组M(接种细胞，不加样品，含假病毒)和阴性对照组M'(接种细胞，不加样品，不含假病毒)。将96孔板在37℃、5％CO 2条件下培养20-24h，50μL/孔加入1×PLB反应10min，移取40μL/孔转移至96孔全白荧光分析板中，然后采用微孔板式发光检测仪进行生物发光检测。利用GraphPad Prism软件作图，横坐标为样品名称，纵坐标为样品对SARS-CoV-2假病毒侵染的中和抑制率，中和抑制率(％)＝(阳性对照组M(RLU)-样品组(RLU))/(阳性对照组M(RLU)-阴性对照组M'(RLU))×100％。结果如图3所示，同小鼠血清效价相似，RBD(T4)同肺炎球菌荚膜多糖PS14或PS6B缀合后均可显著提升RBD(T4)免疫小鼠的血清中和抗体效价。
3.2.2肺炎球菌荚膜多糖提升RBD(T4)免疫小鼠细胞反应
采用酶联免疫法(ELISA)检测小鼠血清中针对的RBD的mIgG1或mIgG2a亚型抗体效价。将浓度为5μg/mL的SARS-COV-2 RBD蛋白包被于96孔板，100μL/孔，2-8℃过夜包被。洗板后加入2％BSA室温封闭1h。使用含0.1％牛血清白蛋白(BSA)的TBST将待检测血清稀释到不同稀释度(具体稀释倍数根据免疫采血时间设定有不同，如1000×、8000×、16000×、32000×稀释)，健康小鼠血清为阴性对照，无血清为blank对照，100μL/孔，室温孵育1-2h。洗板3遍去除未结合抗体，将100μL浓度为0.15μg/mL的标记HRP抗IgG1检测抗体R-mIgG1-R020-H(北京义翘神州生物技术有限公司，全文同)或抗IgG2a检测抗体R-mIgG2a-R005-H(北京义翘神州生物技术有限公司，全文同)加入到酶标板中室温作用约1h。加入显色液显色，终止反应后使用酶标仪在450nm下进行读数。以OD450值大于阴性血清OD450乘以2.1的最大稀释倍数滴定血清中的mIgG1或mIgG2a亚型的抗体效价。mIgG2a表征Th1型免疫反应(细胞免疫)，mIgG1表征Th2型免疫反应(体液免疫)。数据分析mIgG2a/mIgG1比值＝log(mIgG2a效价)/log(mIgG1效价)。结果如图4所示，相同佐剂条件下，相比RBD(T4)免疫，PS14-RBD(T4)和PS6B-RBD(T4)缀合物免疫均具更高的mIgG2a/mIgG1比值，说明RBD(T4)同肺炎球菌荚膜多糖PS14或PS6B缀合后均可提升Th1型免疫反应。
采用ELISpot方法检测PS14-RBD(T4)3免7天后小鼠脾淋巴细胞特异性IFN-γ(细胞免疫)和IL-4(体液免疫)表达水平。分离小鼠脾细胞，将小鼠脾细胞100μL/孔接种于提前处理好的ELISpot孔板(Mabtech，全文同)，细胞接种密度为2×10 5cells/孔，然后100μL/孔加入终浓度为2μg/mL的RBD重组蛋白，置37℃、5％CO 2培养箱内孵育约40h，孵育结束后去掉ELISpot孔板细胞上清，用PBS洗板5次，随后100μL/孔加入稀释好的检测抗体，孵育2h后用PBS洗板5次，100μL/孔加入稀释好的Streptavidin-ALP(1:1000)，室温 孵育1h后用PBS洗板5次，随后100μL/孔加入用0.45μm滤膜过滤的BCIP/NBT-plus底物，室温避光10-30min显色至有清晰斑点出现并用自来水终止。将ELISpot孔板放置在室温阴凉处，待其自然晾干，采用酶联斑点分析仪进行结果分析。以每10 6小鼠脾细胞的SFC(Spot-formingcells)表示抗原特异性的IFN-γ阳性T细胞数，GrapPad Prism软件进行数据统计。结果如图5所示，相比RBD(T4)+MF59免疫，PS14-RBD(T4)+MF59免疫微弱提升了小鼠脾淋巴细胞特异性IFN-γ的表达水平，但IFN-γ：IL-4具有显著性升高，说明PS14能够有效提升小鼠产生针对RBD(T4)蛋白的特异性细胞免疫。
3.3 MF59佐剂增加PS14-RBD(T4)免疫原性(体液+细胞)
3.3.1 MF59佐剂具有更强的提升PS14-RBD(T4)免疫小鼠血清抗体效价及中和抗体效价能力
参照实施例3.2.1检测PS14-RBD(T4)同MF59或Alum佐剂的免疫组合物免疫小鼠血清的抗体总效价及中和抗体效价。结果如图6所示，相比PS14-RBD(T4)单独免疫，Alum佐剂可小幅提升总抗体效价及中和效价，而MF59佐剂可大幅提升总抗体效价(图6A)及中和抗体效价(图6B)，且显著优于Alum佐剂。
3.3.2 MF59佐剂显著提升PS14-RBD(T4)免疫小鼠的细胞反应
参照实施例3.2.2检测PS14-RBD(T4)同MF59或Alum佐剂的免疫组合物免疫小鼠血清的RBD特异性mIgG2a或mIgG1亚型抗体效价，并计算mIgG2a/mIgG1比值。结果如图7所示，PS14-RBD(T4)和PS14-RBD(T4)+Alum具有相近的mIgG2a/mIgG1比值，而PS14-RBD(T4)+MF59免疫可显著提升mIgG2a/mIgG1比值，说明MF59佐剂具有更优的提升细胞反应能力。
参照实施例3.2.2ELISpot方法检测2免12天后小鼠脾淋巴细胞特异性IFN-γ(细胞免疫)和IL-4(体液免疫)分泌水平。结果如图8所示，相比PS14-RBD(T4)，PS14-RBD(T4)+Alum具有更高的刺激特异性IL-4表达的水平(图8A)，而PS14-RBD(T4)+MF59具有更高的刺激特异性IFN-γ表达的水平(图8B)和更高的IFN-γ：IL-4比值(图8C)。因此MF59佐剂具有更强的提升PS14-RBD(T4)免疫小鼠后细胞反应的能力。
综上结果表明，PS14和MF59佐剂可有效提升小鼠产生针对RBD(T4)的特异性体液及细胞免疫反应，可用于预防SARS-CoV-2感染相关疾病。
序列表：
Figure PCTCN2021117680-appb-000003
Figure PCTCN2021117680-appb-000004
参考文献
1.Zhou,P.,et al.,Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin.BioRxiv,2020.
[根据细则26改正28.09.2021]　
2.Wan,Y.,et al.,Receptor recognition by novel coronavirus: An analysis based on decade-long structural studies of SARS.Journal of virology,2020.
3.Bosch,B.J.,et al.,Severe acute respiratory syndrome coronavirus(SARS-CoV)infection inhibition using spike protein heptad repeat-derived peptides.Proceedings of the National Academy of Sciences of the United States of America,2004.101(22):p.8455-8460.
4.Huang,C.,et al.,Clinical features of patients infected with 2019novel coronavirus in Wuhan,China.Lancet,2020.395(10223):p.497-506.
5.Zhou,F.,et al.,Clinical course and risk factors for mortality of adult inpatients with COVID-19in Wuhan,China:a retrospective cohort study.The Lancet,2020.
6.Dong,C.,et al.,Characterization of anti-viral immunity in recovered individuals infected by SARS-CoV-2.medRxiv,2020.
7.To,K.K.,et al.,Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2:an observational cohort study.Lancet Infect Dis,2020.

Claims (21)

  1. 一种提高冠状病毒RBD抗原免疫原性的方法，该方法包括通过将冠状病毒RBD抗原与糖缀合，形成糖-冠状病毒RBD抗原缀合物。
  2. 如权利要求1所述的方法，其中糖选自多糖、寡糖或单糖；
    优选为奈瑟氏脑炎球菌荚膜多糖、嗜血性流感杆菌b荚膜多糖、肺炎链球菌荚膜多糖、B群金黄色葡萄球菌荚膜多糖、葡聚糖、甘露聚糖、淀粉、菊糖、果胶、羧甲基淀粉、壳聚糖及其衍生物；
    更优选为肺炎链球菌荚膜多糖，
    最优选为肺炎链球菌血清型14荚膜多糖、肺炎链球菌血清型6B荚膜多糖和肺炎链球菌血清型7F荚膜多糖。
  3. 如权利要求1或2所述的方法，其中的冠状病毒RBD抗原包含SEQ ID NO:2所述氨基酸序列或其不同长度的RBD截短体、与其具有至少90％，92％，95％，98％或100％序列同一性的氨基酸序列或其不同长度的RBD截短体。
  4. 如权利要求3所述的方法，其中的冠状病毒RBD抗原与肺炎链球菌血清型14荚膜多糖/肺炎链球菌血清型6B荚膜多糖缀合。
  5. 如权利要求4所述的方法，其中肺炎链球菌血清型14荚膜多糖衍生于ATCC 6314，肺炎链球菌血清型6B荚膜多糖衍生于ATCC6326。
  6. 如权利要求1-5之任一所述的方法，其中冠状病毒RBD抗原进一步与其他蛋白或肽融合。
  7. 如权利要求1-6之任一所述的方法，其中冠状病毒RBD抗原与免疫佐剂合用，优选的，佐剂为ALUM/MF59。
  8. 如权利要求1-7之任一所述的方法，该方法提升了Th1型免疫反应。
  9. 一种C端截短的SARS-CoV-2 RBD抗原，其包含SARS-CoV-2刺突蛋白S1亚基的Arg319至Thr 531片段，优选的，包含SEQ ID NO：2所描述的氨基酸序列或其不同长度的RBD截短体、与其具有至少90％，92％，95％，98％或100％序列同一性的氨基酸序列或其不同长度的RBD截短体。
  10. 一种糖-冠状病毒RBD抗原缀合物，与未经缀合的冠状病毒RBD抗原相比，其免疫原性提高。
  11. 如权利要求10所述的糖-冠状病毒RBD抗原缀合物，其中糖选自多糖、寡糖或单糖；优选为奈瑟氏脑炎球菌荚膜多糖、嗜血性流感杆菌b荚膜多糖、肺炎链球菌荚膜多糖、B 群金黄色葡萄球菌荚膜多糖、葡聚糖、甘露聚糖、淀粉、菊糖、果胶、羧甲基淀粉、壳聚糖及其衍生物；
    更优选为肺炎链球菌荚膜多糖，
    最优选为肺炎链球菌血清型14荚膜多糖、肺炎链球菌血清型6B荚膜多糖和肺炎链球菌血清型7F荚膜多糖。
  12. 如权利要求10或11所述的RBD抗原缀合物，其中的冠状病毒RBD抗原如权利要求9定义。
  13. 如权利要求12所述的RBD抗原缀合物，其中的冠状病毒RBD抗原与肺炎链球菌血清型14荚膜多糖/肺炎链球菌血清型6B荚膜多糖缀合。
  14. 如权利要求13所述的RBD抗原缀合物，其中肺炎链球菌血清型14荚膜多糖衍生于ATCC 6314，肺炎链球菌血清型6B荚膜多糖衍生于ATCC6326。
  15. 如权利要求10-14之任一所述的RBD抗原缀合物，其中冠状病毒RBD抗原进一步与其他蛋白或肽融合。
  16. 如权利要求10-15之任一所述的RBD抗原缀合物，其中RBD抗原缀合物与免疫佐剂合用，优选的，佐剂为ALUM/MF59。
  17. 如权利要求10-16之任一所述的RBD抗原缀合物，该抗原缀合物用作抗原时，提升了Th1型免疫反应。
  18. 一种免疫复合物，包含权利要求9的冠状病毒RBD抗原或权利要求10-17之任一的RBD抗原缀合物及免疫佐剂。
  19. 如权利要求18所述的免疫复合物，佐剂选自ALUM/MF59。
  20. 权利要求9所述的冠状病毒RBD抗原、权利要求10-17之任一所述的RBD抗原缀合物或权利要求18或19所述的免疫复合物预防或治疗冠状病毒引起的疾病的用途。
  21. 权利要求9所述的冠状病毒RBD抗原、权利要求10-17之任一所述的RBD抗原缀合物或权利要求18或19所述的免疫复合物在制备预防或治疗冠状病毒引起的疾病的疫苗/药物用途。
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