A mutant of the receptor binding domain of S protein and its application

Abstract

The present application relates to the fields of virology and immunology. In particular, the present application relates to a receptor binding domain (RBD) mutant of an S protein and its application. In addition, the present application also relates to a pharmaceutical composition (e.g., a vaccine) comprising the mutant and a method for preparing the mutant. The present application provides a mutant of the RBD of an S protein, which can induce protective antibodies against different coronaviruses (e.g., SARS‑CoV‑2, SARS‑CoV‑1), and achieve protection against different subtypes of coronaviruses.

Description

Technical Field

[0001] The present application relates to the fields of virology and immunology. In particular, the present application relates to a mutant of a receptor binding domain (RBD) of an S protein and its application. In addition, the present application also relates to a pharmaceutical composition (e.g., a vaccine) comprising the mutant, a method for preparing the mutant, and a method for using the mutant to prevent and / or treat coronavirus infection and / or a disease caused by the infection (e.g., novel coronavirus pneumonia). Background Art

[0002] Coronavirus infection can cause respiratory diseases in humans. Mild coronavirus infection can cause flu-like symptoms, while severe infection can develop into severe viral pneumonia, threatening human life and health. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a coronavirus that can cause coronavirus disease pneumonia (COVID-19) when it infects its host.

[0003] The coronaviruses (HCoV) that infect humans include HCoV-OC43, HCoV-HKU1, HCoV-229E and HCoV-NL63, as well as the highly pathogenic MERS-CoV, SARS-CoV and SARS-CoV-2. In the phylogenetic tree analysis, SARS-CoV-2 is highly similar to the SARS-CoV sequence and belongs to the genus Betacoronavirus together with the highly pathogenic MERS-CoV and the endemic HCoV-OC43 and HCoV-HKU1. Currently, three HCoV infections (SARS-CoV, MERS-CoV and SARS-CoV-2) have occurred that have caused severe syndromes. In addition, multiple variants of SARS-CoV-2 have emerged worldwide. Although the D614G mutation in the S protein significantly promoted the infectivity of the corresponding variant in susceptible cells, this residue substitution failed to cause immune escape. In addition, many results showed that the main SARS-CoV-2 variant in South Africa, B.1.351, has many key mutations in RBD (including K417N, E484K and N501Y), which will reduce the therapeutic effect of neutralizing antibodies and even lead to a significant reduction in the ability of SARS-CoV-2 vaccines designed based on the original SARS-CoV-2 sequence to prevent SARS-CoV-2 variant infection.

[0004] SARS-CoV-2 is an enveloped, single-stranded, positive-sense RNA virus, and its genome is highly homologous to SARS-CoV and some bat coronaviruses. SARS-CoV-2 contains three membrane proteins, including surface spike protein (S), integral membrane protein (M), and membrane protein (E). The receptor of SARS-CoV-2, like SARS-CoV, specifically binds to angiotensin-converting enzyme 2 (ACE2) on host cells through the receptor binding domain (RBD) on the S protein, which leads to membrane fusion and cell entry of the virus, and plays a vital role in the process of viral infection of cells. Therefore, the virus infection can be neutralized by interfering with the binding of the S protein to ACE2. Therefore, the S protein, especially the RBD region, is the main source and recognition region of coronavirus neutralizing antibodies, and the RBD protein is an ideal antigen for the preparation of SARS-CoV-2 vaccines. Since SARS-CoV-2 RBD and SARS-CoV RBD show high amino acid sequence similarity, it is reasonable to speculate that there are conserved neutralizing epitopes between the two, which has been proven by many studies.

[0005] Therefore, the universal SARS-CoV-2 vaccine based on the conserved antigenic epitopes of SARS-CoV-2 and SARS-CoV can not only improve our ability to prevent the risk of possible coronavirus pandemics in the future, but also has the potential to prevent infection by highly pathogenic SARS-CoV-2 variants. Summary of the invention

[0006] After a lot of experiments and repeated explorations, the inventors of the present application have modified the wild-type RBD of the S protein to have an N-linked glycosyl chain. The obtained RBD mutant can enhance the ability of the RBD protein to induce a broad spectrum of protective antibodies. The induced protective antibodies can recognize a variety of coronaviruses (e.g., SARS-CoV-2, SARS-CoV) and have a broader spectrum of protective effects. Based on this, the inventors of the present application have developed an RBD containing an N-linked glycosylation motif (e.g., containing a characteristic sequence NX-(S or T)), which can induce a broad spectrum of anti-coronavirus protective antibodies in vivo and can provide a broad spectrum of anti-coronavirus protection in vivo.

[0007] Therefore, in a first aspect, the present application provides a mutant of the receptor binding domain (RBD) of the S protein, which has an N-glycosylation motif.

[0008] In certain embodiments, the mutant of the RBD of the S protein has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) N-glycosylation motifs (NX-(S or T)) compared to the RBD of the wild-type S protein; wherein N represents asparagine, X represents any amino acid except proline, S represents serine, and T represents threonine.

[0009] N-glycosylation is a post-translational modification of a polypeptide, which refers to the attachment of a glycosyl chain to a free -NH2 group on a specific asparagine residue in a polypeptide chain. The N-glycosylation process is usually carried out in the endoplasmic reticulum (ER) and the Golgi apparatus (GA). Methods for N-glycosylation of proteins / polypeptides are common in the art, and can be found, for example, in Coligan et al., (2000) Current Protocols in Protein Science, Vol. 1-2, John Wiley and Sons, Inc., NY.

[0010] In certain embodiments, the N-glycosylation motif of the RBD mutant is located at amino acid residue 140, amino acid residue 157, amino acid residue 99, amino acid residue 103, amino acid residue 137, amino acid residue 169, amino acid residue 171, amino acid residue 172 and / or amino acid residue 175 of the wild-type RBD.

[0011] In certain embodiments, the N-glycosylation motif possessed by the mutant of the RBD is introduced by insertion and / or substitution. In the mutant of the RBD, the amino acid undergoing N-linked glycosylation is usually asparagine (N) in the characteristic sequence NX-(S or T), wherein N represents asparagine, X represents any amino acid except proline, S represents serine, and T represents threonine. Therefore, in certain preferred embodiments, the difference between the mutant and the wild RBD is at least that the mutant contains the characteristic sequence NX-(S or T); wherein N represents asparagine, X represents any amino acid except proline, S represents serine, and T represents threonine.

[0012] In certain preferred embodiments, the mutant of the RBD has the following mutations: asparagine is inserted and / or replaces at least one of the following amino acid residues of the wild-type RBD: amino acid residue 140, amino acid residue 157, amino acid residue 99, amino acid residue 103, amino acid residue 137, amino acid residue 169, amino acid residue 171, amino acid residue 172 and / or amino acid residue 175.

[0013] In certain preferred embodiments, the mutant of the RBD has the following mutations: at least one of the following amino acid residues of the wild-type RBD is inserted and / or replaced with serine: amino acid residue 142, amino acid residue 159, amino acid residue 101, amino acid residue 105, amino acid residue 139, amino acid residue 171, amino acid residue 173, amino acid residue 174 or amino acid residue 177.

[0014] In certain preferred embodiments, the mutant of the RBD has the following mutations: at least one of the following amino acid residues of the wild-type RBD is inserted and / or replaced with threonine: amino acid residue 142, amino acid residue 159, amino acid residue 101, amino acid residue 105, amino acid residue 139, amino acid residue 171, amino acid residue 173, amino acid residue 174 or amino acid residue 177.

[0015] In certain preferred embodiments, the mutant of the RBD has the following mutation: at least one of the following amino acid residues of the wild-type RBD is inserted and / or replaced with an NXS glycosylation motif: amino acid residue 140, amino acid residue 157, amino acid residue 101, amino acid residue 105, amino acid residue 139, amino acid residue 171, amino acid residue 173, amino acid residue 174 or amino acid residue 177.

[0016] In certain preferred embodiments, the mutant of the RBD has the following mutation: at least one of the following amino acid residues of the wild-type RBD is inserted and / or replaced with the NXT glycosylation motif: amino acid residue 140, amino acid residue 157, amino acid residue 101, amino acid residue 105, amino acid residue 139, amino acid residue 171, amino acid residue 173, amino acid residue 174 or amino acid residue 177.

[0017] In this article, the reference to amino acid residue 140 refers to the amino acid residue 140 of the wild RBD, and similarly, the reference to amino acid residue 157 refers to the amino acid residue 157 of the wild RBD. Therefore, when the glycosylation motif is inserted into amino acid residue 140 and amino acid residue 157 at the same time, amino acid residue 140 and amino acid residue 157 of the wild RBD are still used as the insertion positions.

[0018] In certain preferred embodiments, the mutant of RBD has the following mutations: replacement of amino acid residue 140 of the wild-type RBD with asparagine and replacement of amino acid residue 142 of the wild-type RBD with threonine.

[0019] In certain preferred embodiments, the mutant of the RBD has the following mutations: replacing the amino acid residue at position 157 of the wild-type RBD with asparagine; optionally, replacing the amino acid residue at position 159 of the wild-type RBD with threonine.

[0020] In certain preferred embodiments, the mutant of RBD has the following mutations: replacement of amino acid residue 99 of the wild-type RBD with asparagine and replacement of amino acid residue 101 of the wild-type RBD with threonine.

[0021] In certain preferred embodiments, the mutant of RBD has the following mutations: replacement of amino acid residue 103 of the wild-type RBD with asparagine and replacement of amino acid residue 105 of the wild-type RBD with threonine.

[0022] In certain preferred embodiments, the mutant of RBD has the following mutations: replacement of amino acid residue 137 of the wild-type RBD with asparagine and replacement of amino acid residue 139 of the wild-type RBD with threonine.

[0023] In certain preferred embodiments, the RBD mutant has the following mutation: amino acid residue 171 of the wild-type RBD is replaced with threonine.

[0024] In certain preferred embodiments, the mutant of RBD has the following mutations: replacement of amino acid residue 171 of the wild-type RBD with asparagine and replacement of amino acid residue 173 of the wild-type RBD with threonine.

[0025] In certain preferred embodiments, the RBD mutant has the following mutations: replacement of amino acid residue 172 of the wild-type RBD with asparagine and replacement of amino acid residue 174 of the wild-type RBD with threonine.

[0026] In certain preferred embodiments, the mutant of RBD has the following mutations: replacement of amino acid residue 175 of the wild-type RBD with asparagine and replacement of amino acid residue 177 of the wild-type RBD with threonine.

[0027] In certain preferred embodiments, the mutant of the RBD has the following mutations: replacement of the amino acid residue 140 of the wild-type RBD with asparagine and replacement of the amino acid residue 142 of the wild-type RBD with threonine; and, replacement of the amino acid residue 157 of the wild-type RBD with asparagine; optionally, replacement of the amino acid residue 159 of the wild-type RBD with threonine.

[0028] In certain embodiments, the substitution is a conservative substitution. It is known to those skilled in the art that conservative substitutions can be made to proteins or polypeptides without significantly affecting or changing the functions and properties of the proteins or polypeptides.

[0029] The method of inserting or replacing a certain amino acid residue in a protein / polypeptide chain with other amino acid residues is well known to those skilled in the art. For example, any amino acid residue in a protein / polypeptide chain can be modified (e.g., inserted or replaced) by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0030] In certain preferred embodiments, X used for insertion and / or replacement of the NXT and NXS glycosylation motifs of wild-type RBD can be one or more amino acid residues selected from the group consisting of alanine, glycine, valine, leucine, isoleucine, phenylalanine, tryptophan, serine, tyrosine, cysteine, methionine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.

[0031] In certain embodiments, the wild RBD is the RBD of the S protein of wild SARS-CoV-2.

[0032] In certain embodiments, the wild-type RBD has an amino acid sequence as shown in SEQ ID NO:1.

[0033] In certain embodiments, the mutated RBD has an amino acid sequence as shown in SEQ ID NO:2, 3, 4, 5, 6, 7, 8 or 9.

[0034] In certain embodiments, the mutant RBD has one or more chemical modifications or additional mutations.

[0035] In certain embodiments, the RBD mutants described above have an increased molecular weight compared to wild-type RBD, and the increase in molecular weight can be determined by methods well known in the art, such as SDS-PAGE or mass spectrometry.

[0036] In certain embodiments, such molecular weight increase is due to utilization of N-glycosylation sites, such as addition of N-glycans to RBD. Addition and modification of N-glycans to protein / polypeptide chains can be achieved by using eukaryotic or modified prokaryotic expression systems.

[0037] In a second aspect, the present application provides a recombinant protein comprising a mutant of the RBD as described above, and an additional peptide segment, wherein the additional peptide segment is connected to the mutant.

[0038] In certain embodiments, the additional peptide segment is directly connected to the mutant, or is connected to the mutant via a linker. In certain preferred embodiments, the additional peptide segment is connected to the mutant via a linker. Suitable prior art linkers can consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). In addition, other linkers, such as those described in Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol., may also be used.

[0039] In certain embodiments, the additional peptide segment is linked to the N-terminus or C-terminus of the mutant.

[0040] In certain embodiments, the recombinant protein comprises at least 1, at least 2, at least 3, at least 5 or more additional peptide segments.

[0041] It is easy to understand that these peptide segments can be connected to any end (N-terminus or C-terminus) of the mutant independently in various ways. For example, in certain preferred embodiments, the recombinant protein of the present invention may include two additional peptide segments, wherein one additional peptide segment is connected to the N-terminus of the mutant through a joint or without a joint, and another additional peptide segment is connected to the C-terminus of the mutant through a joint or without a joint. In certain preferred embodiments, the recombinant protein of the present invention may include two or more additional peptide segments, wherein the two or more additional peptide segments are independently connected to the N-terminus or C-terminus of the mutant through a joint or without a joint. In certain preferred embodiments, when two or more additional peptide segments are connected to the N-terminus of the mutant, the two or more additional peptide segments can be connected in series in any order and then connected to the N-terminus of the mutant through a joint or without a joint. Similarly, in certain preferred embodiments, when two or more additional peptide segments are connected to the C-terminus of the mutant, the two or more additional peptide segments can be connected in series in any order and then connected to the C-terminus of the mutant through a joint or without a joint.

[0042] The additional peptide segment may be selected according to actual needs. For example, in certain preferred embodiments, the additional peptide segment may be a signal peptide (e.g., a signal peptide as shown in SEQ ID NO: 4). Without being bound by any theory, it is generally believed that the use of a signal peptide may promote the secretion of a recombinant protein, thereby facilitating the recovery of the recombinant protein. Typically, such a signal peptide may be connected to the N-terminus of the mutant. In addition, during or after secretion, the signal peptide may be removed to produce a desired mutant or recombinant protein.

[0043] In certain embodiments, the additional peptide segment is selected from a signal peptide, a tag peptide, a folding motif, a detectable label, and any combination thereof.

[0044] In certain embodiments, the signal peptide is linked to the N-terminus of the mutant.In certain embodiments, the signal peptide has an amino acid sequence as shown in SEQ ID NO:4.

[0045] In certain embodiments, the folding motif is connected to the C-terminus of the mutant.In certain embodiments, the folding motif has an amino acid sequence as shown in SEQ ID NO:14.

[0046] In another aspect, the present application relates to a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding a mutant of the present invention or a recombinant protein of the present invention. In certain preferred embodiments, the nucleic acid molecule of the present invention is isolated or purified.

[0047] In another aspect, the present application relates to a vector comprising a nucleic acid molecule as described above. The vector of the present invention can be a cloning vector, a transfer vector, or an expression vector. In a preferred embodiment, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, etc. In a preferred embodiment, the vector can express the mutant of the present invention or the recombinant protein of the present invention in a eukaryotic cell (e.g., an insect cell). In a preferred embodiment, the vector is a baculovirus transfer vector, which can be used together with baculovirus genomic DNA to achieve the expression of the mutant of the present invention or the recombinant protein of the present invention in insect cells.

[0048] In another aspect, the present invention also relates to a host cell comprising a nucleic acid molecule or vector as described above. In certain embodiments, the host cell is a cell capable of N-glycosylation. N-glycosylation is the addition of a sugar moiety to an asparagine residue in a NXS / T motif. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cell of the present invention can also be a cell line, such as 293T cells. Without being bound by any theory, it is generally believed that the use of eukaryotic cells helps to maintain the correct conformation of the protein and promotes the folding of the protein. Therefore, in certain preferred embodiments, the host cell of the present invention is a eukaryotic cell such as an insect cell. In certain preferred embodiments, the host cell of the present invention is an insect cell, which comprises a baculovirus transfer vector containing the above-mentioned nucleic acid molecule, and a baculovirus genomic DNA.

[0049] In another aspect, the present invention also relates to a virus (eg, baculovirus) comprising the nucleic acid molecule or vector as described above. In certain preferred embodiments, the virus is a baculovirus.

[0050] In another aspect, the present application relates to a multimer, which comprises or consists of a plurality of mutants of the present invention or a plurality of recombinant proteins of the present invention. In certain preferred embodiments, the multimer is a trimer.

[0051] In another aspect, the present application also relates to a composition comprising the mutant, or the recombinant protein, or the nucleic acid molecule, or the vector, or the host cell, or the virus, or the polymer. In certain preferred embodiments, the composition comprises a mutant or recombinant protein of the present invention. In certain preferred embodiments, the composition comprises a polymer of the present invention. In certain preferred embodiments, the pharmaceutical composition further comprises the RBD of a wild S protein. In certain preferred embodiments, the RBD of the wild S protein is the RBD of a wild SARS-CoV-2 S protein. In certain preferred embodiments, the RBD of the wild S protein has an amino acid sequence as shown in SEQ ID NO: 1.

[0052] In another aspect, the present invention also relates to a pharmaceutical composition (e.g., a vaccine), comprising a mutant or recombinant protein or polymer of the present invention, and optionally further comprising a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition (e.g., a vaccine) of the present invention can be used to prevent or treat coronavirus infection or diseases caused by coronavirus infection, such as novel coronavirus pneumonia, etc. In certain preferred embodiments, the pharmaceutical composition further comprises the RBD of a wild S protein. In certain preferred embodiments, the RBD of the wild S protein is the RBD of a wild SARS-CoV-2 S protein. In certain preferred embodiments, the RBD of the wild S protein has an amino acid sequence as shown in SEQ ID NO: 1.

[0053] In certain preferred embodiments, the mutant or recombinant protein or polymer of the present invention is present in an effective amount for preventing or treating coronavirus infection or a disease caused by coronavirus infection. In certain preferred embodiments, the pharmaceutical composition (e.g., vaccine) of the present invention further comprises another active ingredient. In certain preferred embodiments, the other active ingredient can prevent or treat coronavirus infection or a disease caused by coronavirus infection. In certain preferred embodiments, the pharmaceutical composition (e.g., vaccine) of the present invention further comprises an adjuvant, such as an aluminum adjuvant. In certain preferred embodiments, the method further comprises administering the RBD of the wild S protein to the subject together with the mutant as described above.

[0054] In some preferred embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable carrier, excipient, stabilizer or other agents that can provide favorable properties for the administration of the pharmaceutical composition (e.g., administration to human subjects). Suitable pharmaceutical carriers include, for example, sterile water, saline, glucose, condensation products of castor oil and ethylene oxide, liquid acid, lower alcohols (e.g., C1-4 alcohols), oils (e.g., corn oil, peanut oil, sesame oil; it optionally also includes an emulsifier such as a mono- or di-glyceride of a fatty acid or a phospholipid such as lecithin), ethylene glycol, polyalkylene glycol, sodium alginate, poly (vinyl pyrrolidone) and the like. The carrier optionally may also include an adjuvant, a preservative, a stabilizer, a wetting agent, an emulsifier, a penetration enhancer, etc. In some preferred embodiments, the pharmaceutical composition is sterile. In addition, the viscosity of the drug combination can be controlled and maintained by selecting a suitable solvent or excipient. In certain preferred embodiments, the pharmaceutical composition is formulated to have a pH of 4.5-9.0, 5.0-8.0, 6.5-7.5, or 6.5-7.0.

[0055] The pharmaceutical composition (e.g., vaccine) of the present invention can be administered by methods known in the art, such as, but not limited to, oral administration or injection. In certain preferred embodiments, the pharmaceutical composition (e.g., vaccine) of the present invention is administered in a unit dosage form.

[0056] The amount of the pharmaceutical composition of the present invention (e.g., vaccine) required to prevent or treat a particular condition will depend on the route of administration, the severity of the condition to be treated, the patient's sex, age, weight and overall health condition, etc., and can be reasonably determined by a physician based on actual circumstances.

[0057] In certain preferred embodiments, the pharmaceutical composition (e.g., vaccine) of the present invention further comprises a wild RBD. In certain preferred embodiments, the wild RBD is a wild SARS-CoV-2 RBD. In certain preferred embodiments, the wild RBD has an amino acid sequence as shown in SEQ ID NO: 1.

[0058] In another aspect, the present invention relates to a method for preventing or treating coronavirus infection or a disease caused by coronavirus infection in a subject, comprising administering a preventive or therapeutically effective amount of a mutant or recombinant protein or polymer according to the present invention or a pharmaceutical composition of the present invention to the subject. In certain preferred embodiments, the disease caused by coronavirus infection is novel coronavirus pneumonia. In certain preferred embodiments, the subject is a mammal, such as a mouse and a human.

[0059] In certain preferred embodiments, the coronavirus is selected from human coronavirus OC43 strain (HCoV-OC43), human coronavirus HKU1 strain (HCoV-HKU1), human coronavirus 229E strain (HCoV-229E), human coronavirus NL63 strain (HCoV-NL63), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0060] In certain preferred embodiments, the methods of the present invention can be used to prevent and / or treat infection by SARS-CoV-2 virus and SARS-CoV virus, as well as diseases associated therewith (e.g., novel coronavirus pneumonia).

[0061] In another aspect, the present invention also relates to the use of the mutant or recombinant protein or polymer of the present invention in the preparation of a pharmaceutical composition (e.g., a vaccine), which is used to prevent or treat coronavirus infection or a disease caused by coronavirus infection in a subject. In certain preferred embodiments, the coronavirus is selected from human coronavirus OC43 strain (HCoV-OC43), human coronavirus HKU1 strain (HCoV-HKU1), human coronavirus 229E strain (HCoV-229E), human coronavirus NL63 strain (HCoV-NL63), Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In certain preferred embodiments, the disease caused by coronavirus infection is novel coronavirus pneumonia. In certain preferred embodiments, the subject is a mammal, such as a mouse and a human.

[0062] In another aspect, the present invention also relates to the above-mentioned mutant or recombinant protein or polymer, which is used to prevent or treat coronavirus infection or a disease caused by coronavirus infection in a subject. In certain preferred embodiments, the disease caused by coronavirus infection is novel coronavirus pneumonia. In certain preferred embodiments, the subject is a mammal, such as a mouse and a human. In certain preferred embodiments, the mutant or recombinant protein or polymer is used to prevent and / or treat infection by SARS-CoV-2 virus and SARS-CoV virus and diseases associated therewith (such as novel coronavirus pneumonia).

[0063] In another aspect, the present invention relates to a method for preparing the above-mentioned mutant or recombinant protein, which comprises culturing the host cell or virus of the present invention under conditions allowing the expression of the mutant or recombinant protein; and recovering the expressed mutant or recombinant protein.

[0064] In certain preferred embodiments, the method comprises: introducing the vector (e.g., expression vector) of the present invention into a host cell (e.g., eukaryotic cell), thereby expressing the mutant or recombinant protein in the host cell; and, recovering the expressed mutant or recombinant protein. In certain preferred embodiments, the method comprises: introducing a baculovirus transfer vector containing the above-mentioned nucleic acid molecule and baculovirus genomic DNA into an insect cell, thereby expressing the mutant or recombinant protein in the insect cell; and, recovering the expressed mutant or recombinant protein.

[0065] In another aspect, the present invention also relates to a method for preparing a vaccine, comprising mixing the mutant or recombinant protein or polymer of the present invention with a pharmaceutically acceptable carrier and / or excipient, optionally also mixing an adjuvant such as an aluminum adjuvant, and / or another active ingredient, such as another active ingredient capable of preventing or treating coronavirus infection or a disease caused by coronavirus infection. In certain preferred embodiments, the method for preparing a vaccine comprises the steps of mixing the mutant or recombinant protein or polymer of the present invention with an adjuvant (e.g., an aluminum adjuvant).

[0066] As discussed above, the obtained vaccine can be used to prevent or treat coronavirus infection or diseases caused by coronavirus infection, such as new coronavirus pneumonia.

[0067] In another aspect, the present invention also relates to an N-glycosylated RBD mutant, which comprises a mutant as described above or a recombinant protein as described above, which has been glycosylated by one or more N-glycans, wherein the N-glycans have been linked to one or more N-glycosylation motifs in the human RBD mutant.

[0068] In another aspect, the present invention also relates to a method for preparing a mutant as described above, the method comprising, under conditions allowing expression of the mutant, transfecting a nucleic acid encoding the mutant as described above into a cell capable of N-glycosylation, and recovering the expressed mutant.

[0069] In another aspect, the present invention also relates to a method for preparing the pharmaceutical composition as described above, the method comprising the steps of:

[0070] (a) expressing the nucleic acid molecule as described above or the vector as described above in a host cell capable of N-glycosylation;

[0071] (b) Purification of N-glycosylated RBD mutants of S protein;

[0072] In certain embodiments, the method further comprises the steps of:

[0073] (c) expressing a nucleotide sequence encoding the RBD of a wild-type S protein in a host cell;

[0074] (d) purifying the RBD of the wild-type S protein;

[0075] (e) mixing the product of step (b) and the product of step (d);

[0076] Optionally, a pharmaceutically acceptable carrier and / or excipient is prepared and mixed with the product obtained in step (e).

[0077] Definition of terms

[0078] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0079] As used herein, the terms "natural RBD", "wild RBD" and "wild S protein RBD" refer to the biologically active, naturally occurring receptor binding domain (RBD) of the S protein, which have the same meaning and can be used interchangeably. The amino acid sequence of the RBD of the natural S protein or the RBD of the wild S protein can be easily obtained from various public databases (e.g., GenBank database).

[0080] As used herein, when referring to the amino acid sequence of wild RBD, it is described using the sequence shown in SEQ ID NO: 1. For example, the expression "amino acid residue 140 of wild RBD" refers to the amino acid residue 140 shown in SEQ ID NO: 1. However, those skilled in the art understand that wild RBD may have multiple versions, which have substantially the same primary structure (i.e., amino acid sequence) and higher-order structure (i.e., spatial structure), and substantially the same biological function, but there may still be slight differences in the amino acid sequence between them. Therefore, in the present application, wild RBD is not limited to the protein shown in SEQ ID NO: 1, but is intended to cover all known wild RBDs. Therefore, in the present application, the term "wild RBD" should include various naturally occurring RBDs with biological functions, including, for example, the wild RBD shown in SEQ ID NO: 1 and its naturally occurring variants. In addition, when describing the amino acid position of RBD, it includes not only the specific amino acid position in SEQ ID NO: 1, but also the amino acid position corresponding to the specific amino acid position in its natural variant. For example, the expression "amino acid residue 140 of wild-type RBD" includes amino acid residue 140 of SEQ ID NO: 1, and corresponding amino acid positions in natural variants thereof. According to the present application, the expression "corresponding amino acid position" refers to the amino acid position at the equivalent position in the compared sequences when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage identity. Similarly, the expression "the position corresponding to position 140 of SEQ ID NO: 1" refers to the amino acid position at the equivalent position to position 140 of SEQ ID NO: 1 in the compared sequence when a sequence is optimally aligned with SEQ ID NO: 1, i.e., when a sequence is aligned with SEQ ID NO: 1 to obtain the highest percentage identity.

[0081] In certain preferred embodiments, the wild RBD has an amino acid sequence as shown in SEQ ID NO: 1. In certain preferred embodiments, the wild RBD is a naturally occurring, biologically functional receptor binding domain, and its amino acid sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In certain preferred embodiments, the wild RBD is a naturally occurring, biologically functional receptor binding domain, and its amino acid sequence is compared to SEQ ID NO: 1, and has one or more (e.g., 1-10 or 1-5 or 1-3) amino acid differences (e.g., conservative amino acid substitutions).

[0082] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0083] As used herein, the term "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)", formerly known as "new coronavirus" or "2019-nCov", belongs to the genus Beta coronavirus and is an enveloped single-stranded positive-sense RNA virus. The genome sequence of SARS-CoV-2 is known to those skilled in the art, and can be found, for example, in GenBank: MN908947. SARS-CoV-2 contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). The receptor of SARS-CoV-2, like SARS-CoV, specifically binds to angiotensin-converting enzyme 2 (ACE2) on the host cell through the receptor binding domain (RBD) on the S protein, which is connected to the membrane fusion and cell entry of the virus, and plays a vital role in the process of viral infection of cells.

[0084] As used herein, the terms "surface spike protein", "S protein" and "Spike protein" have the same meaning and can be used interchangeably. It refers to a viral membrane fusion protein. The S protein of the coronavirus can be combined into a trimer containing approximately 1,300 amino acids. The S protein contains two subunits, S1 and S2. Among them, S1 mainly contains a receptor binding domain (RBD), which is responsible for recognizing the cell's receptors. S2 contains the basic elements required for the membrane fusion process. The genomic sequence of the S protein is known to those skilled in the art, and can be found in various public databases, such as the NCBI database.

[0085] As used herein, the terms "new coronavirus pneumonia" and "COVID-19" refer to pneumonia caused by infection with SARS-CoV-2. The two have the same meaning and can be used interchangeably.

[0086] As used herein, the term "severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1)", also known as SARS-CoV, belongs to the genus Betacoronavirus, which is an enveloped single-stranded positive-sense RNA virus. The genome sequence of SARS-CoV-1 is known to those skilled in the art, and can be found, for example, in GenBank: ABF65836.1. The pneumonia caused by SARS-CoV-1 is called SARS.

[0087] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the biological activity of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0088] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0089] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0090] As used herein, the term "epitope" refers to the site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also referred to as "antigenic determinant" in the art. An epitope or antigenic determinant is usually composed of chemically active surface groups of a molecule such as amino acids or carbohydrates or sugar side chains and usually has specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation, which can be "linear" or "conformational". See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all the points of interaction between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction exist across separate protein amino acid residues.

[0091] As used herein, the term "polymer" refers to a polymer composed of a polypeptide molecule (e.g., a mutant or recombinant protein of the present invention) as a monomer, which can generally contain at least 2 (e.g., 3, 4, 5 or more) polypeptide monomers (e.g., a mutant or recombinant protein of the present invention). In such polymers, monomer molecules are polymerized to form polymers through intermolecular interactions (e.g., hydrogen bonds, van der Waals forces, hydrophobic interactions). In certain embodiments of the present invention, the polymer is a trimer containing 3 monomers.

[0092] As used herein, the term "isolated" or "isolated" refers to that obtained from the natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0093] As used herein, the term "N-glycosylation motif" refers to a characteristic motif NX-(S or T) capable of N-linked glycosylation, wherein N represents asparagine, X represents any amino acid except proline, S represents serine, and T represents threonine.

[0094] As used herein, the term "protective antibody" refers to an antibody that has a protective effect against a virus. Protective antibodies include, but are not limited to, antibodies that can neutralize the toxicity of a virus, antibodies that can inhibit the virus from recognizing and binding to a host cell, and antibodies that can inhibit the fusion of a virus with a host cell.

[0095] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, and ionic strength enhancers. For example, pH regulators include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant); ionic strength enhancers include, but are not limited to, sodium chloride.

[0096] As used herein, the term "adjuvant" refers to a non-specific immunopotentiator, which, when delivered into the body together with an antigen or in advance, can enhance the body's immune response to the antigen or change the type of immune response. There are many kinds of adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium brevis, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is used more in clinical experiments. In the present invention, it is particularly preferred that the adjuvant is an aluminum adjuvant.

[0097] As used herein, the term "effective amount" refers to an amount that can effectively achieve the intended purpose. For example, an effective amount for preventing or treating a disease (e.g., coronavirus infection) refers to an amount that can effectively prevent, stop or delay the occurrence of a disease (e.g., coronavirus infection), or alleviate, reduce or treat the severity of an existing disease (e.g., a disease caused by coronavirus infection). It is within the capabilities of those skilled in the art to determine such an effective amount. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0098] As used herein, the term "immunogenicity" refers to the ability to stimulate the body to form specific antibodies or sensitized lymphocytes. It refers to the ability of an antigen to stimulate specific immune cells, activate, proliferate, and differentiate immune cells, and ultimately produce immune effector substances such as antibodies and sensitized lymphocytes. It also refers to the ability of the body's immune system to form antibodies or sensitized T lymphocytes after the antigen stimulates the body. The specific immune response. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce an immune response in the host depends on three factors: the nature of the antigen, the host's reactivity, and the mode of immunity.

[0099] As used herein, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0100] As used herein, "subject" refers to an animal, such as a vertebrate. In certain embodiments, the subject is a mammal, such as a human, bovine, equine, feline, canine, rodent, or primate. Particularly preferably, the subject is a human. In this article, the term can be used interchangeably with "patient".

[0101] As used herein, "N-glycosylated RBD mutant" refers to an RBD mutant having one or more covalently attached glycan moieties. As used herein, "glycan" or "glycan moiety" refers to a glycosidically linked monosaccharide. The glycan is typically attached to the mutant via a free NH2 group to asparagine. Methods for N-glycosylation of proteins are common in the art and can be found, for example, in Coligan et al., (2000) Current Protocols in Protein Science, Vol. 1-2, John Wiley and Sons, Inc., NY.

[0102] Advantageous Effects of the Invention

[0103] The present application provides a mutant of the RBD of an S protein, which can induce protective antibodies against different coronaviruses (e.g., SARS-CoV-2, SARS-CoV-1), achieve protective effects against different subtypes of coronaviruses, and can therefore be used as a broad-spectrum vaccine capable of resisting multiple (e.g., 2, 3 or more subtypes) of coronaviruses, for preventing and / or treating multiple (e.g., 2, 3 or more subtypes) of coronavirus infections and diseases associated with the infections (e.g., novel coronavirus pneumonia). Moreover, the mutant of the RBD of the S protein of the present application has a greatly increased probability of inducing protective antibodies against different coronaviruses (e.g., SARS-CoV-2, SARS-CoV-1) compared to the RBD of the wild-type S protein.

[0104] Therefore, the present application provides a broad-spectrum coronavirus vaccine, which can provide cross-protection against multiple coronaviruses (e.g., SARS-CoV-2, SARS-CoV-1), and has an ideal immune effect, and is not easily rapidly ineffective due to coronavirus mutations, thereby overcoming the shortcomings of existing coronavirus vaccines such as loss of immune efficacy and unsatisfactory immune effects due to frequent virus mutations. Therefore, the broad-spectrum coronavirus vaccine of the present application has particularly significant advantages over existing vaccines.

[0105] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 The wild-type SARS-CoV-2 RBD (RBD wt ) and mutated SARS-CoV-2 RBD (RBD Glycan458,475 ) structure diagram, where 458 is RBD Glycan458,475 The 140th amino acid residue is the glycosylation site, and 475 is the RBD Glycan458,475 The glycosylation modification site of the 157th amino acid residue on the glycosylation site.

[0107] Figure 2 The schematic diagram of the construction of two recombinant proteins is shown, among which the recombinant RBD wt Proteins include signal peptide, RBD wt , linker sequence (GSGS) and mouse IgG2a subtype antibody heavy chain CH2-3; recombinant RBD Glycan458,475 Proteins include signal peptide, RBD Glycan458,475, connecting sequence (GSGS) and mouse IgG2a subtype antibody heavy chain CH2-3.

[0108] Figure 3 Shows RBD wt and RBD Glycan458,475 The polyacrylamide gel electrophoresis results are shown in Figure 1, where the first lane is the marker and the second lane is the RBD wt , the third lane is RBD Glycan458,475 .

[0109] Figure 4 The seven SARS-CoV-2 antibodies were shown to bind to RBD wt and RBD Glycan458,475 The combination of .

[0110] Figure 5 Shows RBD wt and RBD Glycan458,475 The titer of antibodies produced in the serum of immunized mice, Figure 5 A is the titer of antibodies and the S protein of SARS-CoV and SARS-CoV-2. Figure 5 B is RBD wt and RBD Glycan458,475 The probability of cross-antibodies (i.e., antibodies that can bind to both SARS-CoV and SARS-CoV-2) produced after immunization of mice.

[0111] Sequence information

[0112] The information of the partial sequences involved in the present invention is provided in Table 1 below.

[0113] Table 1: Description of sequences

[0114]

[0115]

[0116]

[0117]

[0118] DETAILED DESCRIPTION

[0119] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0120] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)); and ANIMAL CELL CULTURE (Animal Cell Culture). CELL CULTURE) (RI Freshney, ed. (1987)).

[0121] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0122] Example 1. Preparation of glycosylated SARS-CoV-2 RBD protein

[0123] 1. Molecular cloning design

[0124] The amino acid sequence of SARS-CoV-2 Spike protein was obtained from the GeneBank database (GenBank ID: MN908947.3). The R319-L518 segment in the amino acid sequence is the amino acid sequence of SARS-CoV-2 RBD protein in this study (abbreviated as RBD wt ), RBD wtThe amino acid sequence of is shown in SEQ ID NO: 1. The characteristic amino acid sequence of glycosylation is NXS or T (wherein N is asparagine Asn, X represents any amino acid except proline, S is serine Ser, and T is threonine Thr). wt The proteins were mutated separately, and a total of 8 mutants were prepared, as follows:

[0125] (1) The amino acid K at position 140 of SEQ ID NO: 1 was mutated to N, the amino acid N at position 142 was mutated to T, the amino acid A at position 157 was mutated to N, and the amino acid S at position 159 was mutated to T. The mutated protein was named SARS-CoV-2RBD Glycan458,475 (RBD Glycan458,475 ), whose amino acid sequence is shown in SEQ ID NO: 2. Compared with RBD wt , RBD Glycan458,475 Glycosylation will be performed at asparagine 140 and 157, masking the corresponding key epitopes;

[0126] (2) The amino acid K at position 99 of SEQ ID NO:1 was mutated to N, and the amino acid A at position 101 was mutated to T. The mutated protein was named SARS-CoV-2RBD Glycan417 (RBD Glycan417 ), whose amino acid sequence is shown in SEQ ID NO: 3. Compared with RBD wt , RBD Glycan417 Glycosylation modification will be performed at asparagine 99, masking the corresponding key epitope;

[0127] (3) The amino acid Y at position 103 of SEQ ID NO: 1 was mutated to N, and the amino acid Y at position 105 was mutated to T. The mutated protein was named SARS-CoV-2RBD Glycan421 (RBD Glycan421 ), whose amino acid sequence is shown in SEQ ID NO: 4. Compared with RBD wt , RBD Glycan421 It will be glycosylated at asparagine 103, masking the corresponding key epitope;

[0128] (4) The amino acid L at position 137 of SEQ ID NO: 1 was mutated to N, and the amino acid R at position 139 was mutated to T. The mutated protein was named SARS-CoV-2RBD Glycan455 (RBD Glycan455 ), whose amino acid sequence is shown in SEQ ID NO: 5. Compared with RBD wt , RBD Glycan455It will be glycosylated at asparagine 137, masking the corresponding key epitope;

[0129] (5) The amino acid Y at position 171 of SEQ ID NO: 1 was mutated to T, and the mutated protein was named SARS-CoV-2RBD Glycan487 (RBD Glycan487 ), whose amino acid sequence is shown in SEQ ID NO: 6. Compared with RBD wt , RBD Glycan487 Glycosylation modification will be performed at asparagine 169, masking the corresponding key epitope;

[0130] (6) The amino acid Y at position 171 of SEQ ID NO: 1 was mutated to N, and the amino acid P at position 173 was mutated to T. The mutated protein was named SARS-CoV-2RBD Glycan489 (RBD Glycan489 ), whose amino acid sequence is shown in SEQ ID NO: 7. Compared with RBD wt , RBD Glycan489 It will be glycosylated at asparagine 171, masking the corresponding key epitope;

[0131] (7) The amino acid F at position 172 of SEQ ID NO: 1 was mutated to N, and the amino acid L at position 174 was mutated to T. The mutated protein was named SARS-CoV-2RBD Glycan490 (RBD Glycan490 ), whose amino acid sequence is shown in SEQ ID NO: 8. Compared with RBD wt , RBD Glycan490 It will be glycosylated at asparagine 172, masking the corresponding key epitope;

[0132] (8) The amino acid Q at position 175 of SEQ ID NO: 1 was mutated to N, and the amino acid Y at position 177 was mutated to T. The mutated protein was named SARS-CoV-2RBD Glycan493 (RBD Glycan493 ), whose amino acid sequence is shown in SEQ ID NO: 9. Compared with RBD wt , RBD Glycan493 Glycosylation will be performed at asparagine 175, masking the corresponding key epitope.

[0133] 2. Construction of protein expression clones

[0134] The nucleotide sequences encoding GSGS amino acids were used to encode nine RBD proteins (RBD wtand the above 8 mutants) were connected to the nucleotide sequence encoding the mouse IgG2a subtype antibody heavy chain CH2-3 (amino acid sequence as shown in SEQ ID NO: 10), and the nucleotide sequence encoding RBD wt A nucleotide sequence encoding a signal peptide (SEQ ID NO: 11) was introduced into the 5' end of the nucleotide sequence of the above 8 mutants to construct a fusion expression gene. Figure 2 Shows RBD wt and RBD Glycan458,475 Schematic diagram of the connection. The gene was synthesized into the multiple cloning site of the eukaryotic expression vector pTT5 through the technical service of General Biosystems (Anhui) Co., Ltd. The vector powder with the target gene was dissolved in 50μl of double distilled water, and 1μl was added to 100μl of TOP10 competent cells (Escherichia coli). The sample was incubated on ice for 30min, heat shocked at 42℃ for 30sec, and incubated on ice for 2min. The reacted Escherichia coli was then added to LB medium and spread on an agarose medium plate containing ampicillin resistance for culture. After the colonies grow, pick the monoclonal colonies and the plasmid is purified by GenElute TM The plasmid was extracted using a plasmid miniprep kit (manufacturer: Sigma; catalog number: PLN350-1KT).

[0135] 3. Protein Expression and Purification

[0136] Prepare ExpiCHO suspension cells in logarithmic growth phase TM The cells were placed in a cell shaker at 125 rpm, 37°C, and 8% CO2 for culture until the density reached 6 × 10 6 / mL, viable cell rate> 98%. Take 25mL of cells and place them in a new cell culture bottle as a transfection system. Tube A: 1mL ExpiCHO TM Expression Medium contains 25 μg plasmid, tube B: 1 mL ExpiCHO TM The Expresssion Medium contains 80 μL of the transfection reagent in the ExpiFectamine TM CHO Transfection Kit (Manufacturer: Thermo Scientific; Catalog Number: A29129). Mix tube A and tube B and let stand at room temperature for 2 minutes. After 2 minutes, pour the mixture into the 25 mL prepared transfection cell system. Place in a cell shaker at 125 rpm, 37°C, 8% CO2 for 18-22 hours. Add ExpiFectamine to each bottle. TM150 μL of enhancer and 4 mL of auxiliary material in the CHO Transfection Kit were placed in a cell shaker at 125 rpm, 32°C, and 5% CO2 for 8-15 days. After the culture was completed, centrifuged at 4°C, 4000 rpm for 10 minutes, and the cell supernatant was collected.

[0137] Filter the supernatant with a 0.22 μm filter. Turn on the AKTA instrument, rinse the A and B pipes with A solution (200 mM sodium hydrogen phosphate dodecahydrate) and B solution (100 mM citric acid monohydrate) respectively, and install the protein A column. Use A solution to balance the protein A column for more than 15 minutes at a flow rate of 8 mL / min, and proceed to the next step after the UV value, pH value and conductivity detected by the instrument are stable. Load the sample at a flow rate of 6-10 ml / min, then the UV value will rise, and this peak is the penetration peak. Continue to wash the column with A solution, and collect the penetration peak sample for detection. After the pH value no longer changes, use B solution at a flow rate of 6-10 ml / min, then the pH value drops, the UV value rises, and this peak is the elution peak. The antibody is mainly present in the elution peak. Collect the elution peak sample for detection. Use A solution to balance the column, and then fill the pipe and protein A column with 20% ethanol, remove the column, and store at 4°C. The samples of the penetration peak and elution peak were purified and identified by SDS-PAGE (the samples were boiled in water for 5 minutes to open the disulfide bonds between the heavy and light chains of the antibody, see the second edition of Molecular Cloning Experiment Guide). The purified monoclonal antibody was dialyzed overnight with 20mM PBS buffer, and the concentration was measured by UV spectrometry or BCA, and then dispensed into 1.5ml tubes and stored at -20℃ for later use.

[0138] Example 2. Polyacrylamide gel electrophoresis analysis

[0139] Polyacrylamide gel electrophoresis (SDS-PAGE) experiment was used to analyze the SARS-CoV-2 RBD in this patent wtand the molecular weights of the above 8 mutants. The upper gel was a 5% concentrated gel (3.4 ml water, 830 μl 30% acrylamide, 630 μl 1M Tris (pH 6.8), 50 μl 10% SDS, 50 μl 10% ammonium persulfate and 5 μl TEMED). The lower gel was a 12% separation gel (3.3 ml water, 4 ml 30% acrylamide, 2.5 ml 1M Tris (pH 8.8), 100 μl 10% SDS, 100 μl 10% ammonium persulfate and 10 μl TEMED). The sample was electrophoresed at 150 V for 2 hours. After electrophoresis, the SDS-PAGE gel was stained overnight with 0.25% Coomassie Brilliant Blue (Sigma). Afterwards, decolorization was performed with a decolorization buffer (300 ml of methanol, 100 ml of acetic acid and 600 ml of double distilled water).

[0140] Result analysis: 8 mutants after glycosylation modification (RBD Glycan458,475 、RBD Glycan417 、RBD Glycan421 、RBD Glycan455 、RBD Glycan487 、RBD Glycan489 、RBD Glycan490 and RBD Glycan493 ) have a molecular weight greater than that of RBD wt Protein. Among them, Figure 3 Shows RBD wt and RBD Glycan458,475 Polyacrylamide gel electrophoresis results.

[0141] Example 3. Epitope Characterization Analysis of Glycosylation-Modified Mutants

[0142] RBD wt The coating solution was prepared by diluting 8 mutants with 50mM CB buffer (NaHCO3 / Na2CO3 buffer, final concentration of 50mM, pH 9.6) at pH 9.6, with a final concentration of 0.5μg / mL. 100μL of coating solution was added to each well of the 96-well ELISA plate, coated at 4℃ for 16 hours, and then coated at 37℃ for 2 hours. Wash once with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20). Then 200μl of blocking solution (20mM Na2HPO4 / NaH2PO4 buffer solution with pH 7.4 containing 20% ​​calf serum and 1% casein) was added to each well, blocked at 37℃ for 2 hours, and the blocking solution was discarded. After drying, it was stored in an aluminum foil bag at 4℃ for later use.

[0143] Take the human SARS-CoV-2RBD-specific monoclonal antibodies 3C11, 5C4, 6G3, 6H7, 4D10, 6G9 and 5C6 that recognize different epitopes (wherein the VH and VL sequences of the 7 antibodies are shown in SEQ ID NO: 17 to SEQ ID NO: 30 in Table 1, respectively, the 7 antibodies have the same heavy chain constant region, the specific sequence is shown in SEQ ID NO: 31, and the 7 antibodies have the same light chain constant region, the specific sequence is shown in SEQ ID NO: 32). Use PBS solution to start 5-fold gradient dilution from 100 μg / ml as the starting concentration, and dilute 8 gradients in total. Take the coated ELISA plate, add 100 μl of the diluted antibody sample to each well, and place it in a 37°C incubator for reaction for 60 minutes. The ELISA plate was washed 5 times with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20), 100μl of horseradish peroxidase (HRP) labeled goat anti-human IgG reaction solution was added to each well, and the plate was placed in a 37°C incubator for reaction for 30 minutes. After the enzyme marker reaction step was completed, the ELISA plate was washed 5 times with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20), 50μl of TMB color developer (purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd.) was added to each well, and the plate was placed in a 37°C incubator for reaction for 15 minutes. After the color development reaction step was completed, 50μl of stop solution (purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd.) was added to each well of the ELISA plate after the reaction, and the OD450 / 630 value of each well was detected on an ELISA reader.

[0144] Analysis of results: The test results showed that the binding ability of the 8 glycosylated mutants to some monoclonal antibodies decreased, proving that the glycosylated sites in these mutants can effectively mask the SARS-CoV-2 specific epitopes recognized by some monoclonal antibodies.

[0145] Figure 4 Shown are the interactions of 7 SARS-CoV-2 antibodies with RBD wt and RBD Glycan458,475 Among them, the binding activity of 6G3, 4D10, 5C6, 6G9 and 6H7 was not significantly affected by glycosylation modification. However, their binding ability with 3C11 and 5C4 decreased significantly, less than 1%. This result shows that RBD wt Glycosylation modification of amino acid residues 140 and 150 of the protein can effectively mask the SARS-CoV-2 specific epitopes recognized by monoclonal antibodies 3C11 and 5C4.

[0146] Example 4. Analysis of the ability of glycosylation-modified mutants to induce cross-binding antibody responses

[0147] 1. Mouse Immunization Experiment

[0148] Six-week-old female Balb / C mice were provided by the Experimental Animal Center of Xiamen University, weighing approximately 20 g. wt The 8 mutants were mixed with aluminum adjuvant at a volume ratio of 1:1 for immunization of mice. Each group consisted of 3 mice, and the immunization method was intramuscular injection. The immunization dose was 20 μg protein / mouse. After one injection, the mouse serum was collected 14 days after immunization. The serum samples were inactivated at 56°C for 30 minutes and stored at -20°C for later use.

[0149] 2. Evaluation of SARS-CoV-2 and SARS-CoV S protein-specific antibody titers in serum

[0150] With reference to the sequences of SARS-CoV-2 and SARS-CoV (Genbank ID: MN908947.3 and ABF65836.1), the nucleotide sequence encoding the amino acids 15 to 1213 of the S protein was selected (the amino acid sequence of the extracellular segment of the SARS-CoV-2 S protein at positions 15-1213 is shown in SEQ ID NO: 12, and the amino acid sequence of the extracellular segment of the SARS-CoV S protein at positions 15-1213 is shown in SEQ ID NO: 13). Then, a peptide segment containing a thrombin folding sequence (amino acid sequence as shown in SEQ ID NO: 14), a T4 trimerization element (amino acid sequence as shown in SEQ ID NO: 15) and a His purification tag (amino acid sequence as shown in SEQ ID NO: 16) was added to the C-terminus of SEQ ID NO: 12 and SEQ ID NO: 13, respectively. In addition, the amino acids 682 to 685 of the SARS-CoV-2 S protein (GenBank ID: MN908947.3) were replaced with "AGAG", and the amino acids 986 to 987 were replaced with "PP". The nucleotide sequences encoding the above two proteins were cloned into the baculovirus insect expression vector pAcgp67B, and recombinant baculovirus was prepared. The signal peptide on the vector was used for secretory expression in insect cells, and the modified SARS-CoV-2 S protein and SARS-CoV S protein were obtained by affinity chromatography purification.

[0151] The modified SARS-CoV-2 S protein and SARS-CoV S protein were diluted with 50mM CB buffer (NaHCO3 / Na2CO3 buffer, final concentration of 50mM, pH 9.6) at pH 9.6 to a final concentration of 0.5μg / ml. 100μl of coating solution was added to each well of the 96-well ELISA plate, coated at 4°C for 16 hours, and then coated at 37°C for 2 hours. Wash once with PBST washing solution (20mMPB7.4, 150mM NaCl, 0.1% Tween20). Then add 200μl of blocking solution (20mM Na2HPO4 / NaH2PO4 buffer solution with a pH of 7.4 containing 20% ​​calf serum and 1% casein) to each well, put it at 37°C for blocking for 2 hours, and discard the blocking solution. After drying, put it into an aluminum foil bag and store it at 4°C for use.

[0152] The serum to be tested was diluted 2 times in a gradient, with a total of 12 gradients. Take the ELISA plate coated with SARS-CoV-2 S protein or SARS-CoV S protein, add 100 μl of diluted serum sample to each well, and place it in a 37°C incubator for 60 minutes. Wash the ELISA plate with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20) 5 times, add 100 μl of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG reaction solution to each well, and place it in a 37°C incubator for 30 minutes. After completing the enzyme marker reaction step, wash the ELISA plate with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20) 5 times, add 50 μl of TMB color developer (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) to each well, and place it in a 37°C incubator for 15 minutes. After the color development step was completed, 50 μl of stop solution (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) was added to each well of the ELISA plate after the reaction, and the OD450 / 630 value of each well was detected on an ELISA reader to calculate the serum SARS-CoV-2 S protein or SARS-CoV S protein-specific antibody titer.

[0153] Results analysis: RBD wt After immunization with the eight mutants, specific antibody responses could be induced in mice. Two weeks after immunization, serum SARS-CoV-2 S protein and SARS-CoV S protein-specific antibodies were positively converted. In addition, the eight mutants induced higher titers of serum SARS-CoV-2 S protein-specific antibodies, which was consistent with RBD. wt There was no significant difference, and the serum SARS-CoV S protein-specific antibody titer was also higher, which was consistent with RBD wtThere was no significant difference. According to the formula: (SARS-CoV S protein specific antibody titer / SARS-CoV-2 S protein specific antibody titer) × 100%, the proportion of serum cross-binding antibodies was analyzed. The analysis results showed that the probability of cross-binding antibodies in the specific antibodies induced by the eight mutants was much higher than that of RBD. wt 3.65%. This indicates that the mutants with glycosylation modification have a stronger ability to induce the production of broad-spectrum antibodies. It can be seen that these 8 mutants are particularly suitable for use as broad-spectrum vaccines to induce protective antibodies with broad-spectrum neutralizing activity in vivo.

[0154] Figure 5 A shows the RBD wt and RBD Glycan458,475 The titer of antibodies produced in the serum of immunized mice, RBD Glycan458,475 The induced serum SARS-CoV-2 S protein-specific antibody titers were high, which was consistent with RBD wt No significant difference, RBD Glycan458,475 The induced serum SARS-CoV S protein-specific antibody titer was also high, which was consistent with RBD wt There is no significant difference. SARS-CoV-2RBD was calculated by the formula Glycan458,475 33.33% of the induced specific antibodies were cross-binding antibodies, much higher than RBD wt 3.65%( Figure 5 B), indicating that the modified RBD has glycosylation modification Glycan458,475 The protein has a stronger ability to induce the production of broad-spectrum antibodies. Glycan458,475 It is particularly suitable for use as a broad-spectrum vaccine for inducing protective antibodies with broad-spectrum neutralizing activity in vivo.

[0155] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A mutant of a receptor binding domain (RBD) of an S protein, wherein the mutant of the RBD of the S protein has any of the following compared with the RBD of a wild-type S protein: (1) replacing the amino acid residue 140 of the wild-type RBD with asparagine, the amino acid residue 142 of the wild-type RBD with threonine, the amino acid residue 157 of the wild-type RBD with asparagine, and the amino acid residue 159 of the wild-type RBD with threonine; (2) replacing the amino acid residue 99 of the wild-type RBD with asparagine and the amino acid residue 101 of the wild-type RBD with threonine; (3) replacing the amino acid residue 103 of the wild-type RBD with asparagine and the amino acid residue 105 of the wild-type RBD with threonine; (4) replacing the amino acid residue 137 of the wild-type RBD with asparagine and the amino acid residue 139 of the wild-type RBD with threonine; (5) replacing the amino acid residue 171 of the wild-type RBD with threonine; (6) replacing the amino acid residue 171 of the wild-type RBD with asparagine and the amino acid residue 173 of the wild-type RBD with threonine; (7) replacing the amino acid residue 172 of the wild-type RBD with asparagine and the amino acid residue 174 of the wild-type RBD with threonine; or (8) The amino acid residue 175 of the wild-type RBD was replaced with asparagine and the amino acid residue 177 of the wild-type RBD was replaced with threonine.

2. The mutant according to claim 1, wherein the wild RBD is the RBD of the S protein of wild SARS-CoV-2.

3. The mutant of claim 1, wherein the wild-type RBD has an amino acid sequence as shown in SEQ ID NO:

1.

4. The mutant of claim 1, wherein the mutated RBD has an amino acid sequence as shown in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8 or 9.

5. The mutant of claim 1, wherein the mutated RBD has one or more chemical modifications or additional mutations.

6. A recombinant protein comprising a mutant of the RBD according to any one of claims 1 to 5, and one or more additional peptide segments, wherein the additional peptide segments are connected to the mutant.

7. The recombinant protein of claim 6, wherein the additional peptide segment is directly linked to the mutant or is linked to the mutant via a linker.

8. The recombinant protein of claim 6, wherein the additional peptide segment is linked to the N-terminus or C-terminus of the mutant.

9. The recombinant protein of claim 6, wherein the additional peptide segment is selected from the group consisting of a signal peptide, a tag peptide, a folding motif, a detectable label, and any combination thereof.

10. The recombinant protein of claim 9, wherein the signal peptide is linked to the N-terminus of the mutant. The recombinant protein of claim 10 , wherein the signal peptide has an amino acid sequence as shown in SEQ ID NO:

4. The recombinant protein of claim 9 , wherein the folding motif is linked to the C-terminus of the mutant.

13. The recombinant protein of claim 12, wherein the folding motif has an amino acid sequence as shown in SEQ ID NO:

14.

14. A nucleic acid molecule comprising or consisting of a nucleotide sequence encoding the mutant according to any one of claims 1 to 5 or the recombinant protein according to any one of claims 6 to 13. A vector comprising the nucleic acid molecule according to claim 14 .

16. A host cell or virus comprising the nucleic acid molecule of claim 14 or the vector of claim 15.

17. A multimer comprising or consisting of a plurality of mutants according to any one of claims 1 to 5 or recombinant proteins according to any one of claims 6 to 13. The multimer of claim 17 , which is a trimer.

19. A composition comprising the mutant according to any one of claims 1 to 5, or the recombinant protein according to any one of claims 6 to 13, or the nucleic acid molecule according to claim 14, or the vector according to claim 15, or the host cell or virus according to claim 16, or the multimer according to claim 17 or 18.

20. The composition of claim 19, wherein the RBD of the wild S protein is the RBD of the S protein of the wild SARS-CoV-2.

21. The composition of claim 19, wherein the RBD of the wild S protein has an amino acid sequence as shown in SEQ ID NO:

1.

22. A pharmaceutical composition comprising the mutant according to any one of claims 1 to 5, the recombinant protein according to any one of claims 6 to 13, or the multimer according to claim 17 or 18, and optionally further comprising a pharmaceutically acceptable carrier and / or excipient.

23. The pharmaceutical composition of claim 22, further comprising the RBD of a wild-type S protein.

24. The pharmaceutical composition of claim 23, wherein the RBD of the wild S protein is the RBD of the S protein of the wild SARS-CoV-2.

25. The pharmaceutical composition of claim 23, wherein the RBD of the wild S protein has an amino acid sequence as shown in SEQ ID NO:

1.

26. Use of the mutant according to any one of claims 1 to 5, the recombinant protein according to any one of claims 6 to 13, or the multimer according to claim 17 or 18 in the preparation of a pharmaceutical composition for preventing or treating coronavirus infection or a disease caused by coronavirus infection in a subject.

27. The use of claim 26, wherein the coronavirus is selected from human coronavirus OC43 strain (HCoV-OC43), human coronavirus HKU1 strain (HCoV-HKU1), human coronavirus 229E strain (HCoV-229E), human coronavirus NL63 strain (HCoV-NL63), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

28. The use according to claim 26, wherein the disease caused by coronavirus infection is pneumonia.

29. The use according to claim 26, wherein the disease caused by coronavirus infection is novel coronavirus pneumonia.

30. The use of claim 26, wherein the subject is a mammal.

31. The use according to claim 26, wherein the subject is a mouse or a human.

32. A method for preparing the mutant described in any one of claims 1-5 or the recombinant protein described in any one of claims 6-13, comprising culturing the host cell or virus of claim 16 under conditions that allow expression of the mutant or recombinant protein; and recovering the expressed mutant or recombinant protein.

33. A method for preparing a vaccine, comprising mixing the mutant according to any one of claims 1 to 5, the recombinant protein according to any one of claims 6 to 13, or the multimer according to claim 17 or 18 with a pharmaceutically acceptable carrier and / or excipient.

34. The method of claim 33, further comprising admixing an adjuvant and / or an additional active ingredient.

35. The method of claim 34, wherein the mixed adjuvant is an aluminum adjuvant.

36. The method of claim 34, wherein the additional active ingredient is capable of preventing or treating coronavirus infection or a disease caused by coronavirus infection.

37. An N-glycosylated RBD mutant comprising a mutant according to any one of claims 1 to 5 or a recombinant protein according to any one of claims 6 to 13, which has been glycosylated by one or more N-glycans, wherein: The N-glycans are already attached to one or more N-glycosylation motifs in the human RBD mutant.

38. A method for preparing the mutant of claim 37, the method comprising, under conditions allowing expression of the mutant, transfecting a nucleic acid encoding the mutant of claim 37 into a cell capable of N-glycosylation, and recovering the expressed mutant.

39. A method for preparing a pharmaceutical composition according to any one of claims 22 to 25, comprising the steps of: (a) expressing the nucleic acid molecule of claim 14 or the vector of claim 15 in a host cell capable of N-glycosylation; (b) Purification of N-glycosylated RBD mutants of S protein.

40. The method of claim 39, further comprising the steps of: (c) expressing a nucleotide sequence encoding the RBD of a wild-type S protein in a host cell; (d) purifying the RBD of the wild-type S protein; (e) mixing the product of step (b) and the product of step (d).

41. The method of claim 40, further comprising the steps of: A pharmaceutically acceptable carrier and / or excipient is prepared and mixed with the product obtained in step (e).

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