Antigens of β-coronaviruses, preparation methods and uses thereof

Abstract

The embodiments of the present disclosure relate to antigens of β-coronaviruses, preparation methods and uses thereof. The amino acid sequence of the antigen of the β-coronavirus includes an amino acid sequence arranged in a (A-B)-(A-B) pattern or an amino acid sequence arranged in a (A-B)-C-(A-B) pattern or an amino acid sequence arranged in a (A-B)-(A-B′) pattern or an amino acid sequence arranged in a (A-B)-C-(A-B′) pattern. The antigen of the β-coronavirus has a single-chain dimer structure. A single-chain dirtier expressed according to examples of the present disclosure is stable in content and has excellent immunogenicity as an antigen of a β-coronavirus, and a vaccine prepared by using the single-chain dimer as an antigen of a β-coronavirus can elicit high-titer neutralizing antibodies in mice.

Claims

1. An antigen of a β-coronavirus, its amino acid comprising an amino acid sequence arranged in a (A-B)-(A-B) pattern or an amino acid sequence arranged in a (A-B)-C -(A-B) pattern or an amino acid sequence arranged in a (A-B)-(A-B′) pattern or an amino acid sequence arranged in a (A-B)-C-(A-B′) pattern, wherein A-B represents a partial amino acid sequence or an entire amino acid sequence of a receptor binding domain of a surface spike protein of the β-coronavirus; C represents an amino acid linker sequence; A-B′ represents an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids in the amino acid sequence of A-B; a protein encoded by A-B′ has an identical immunogenicity as a protein encoded by A-B; and the antigen of the β-coronavirus has a single-chain dimer structure.

2. The antigen of the β-coronavirus according to claim 1, wherein the β-coronavirus is selected from a group consisting of severe respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and 2019 novel coronavirus.

3. The antigen of the β-coronavirus according to claim 1, wherein the amino acid linker sequence comprises a (GGS).sub.n linker sequence, wherein n represents the number of GGSs, n is an integer more than or equal to 1; preferably, n is an integer selected from 1 to 10, and further preferably, n is an integer selected from 1 to 5.

4. The antigen of the β-coronavirus according to claim 1, wherein the partial amino acid sequence of the receptor binding domain of the surface spike protein of the β-coronavirus is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the entire amino acid sequence of the receptor binding domain of the surface spike protein of the β-coronavirus.

5. The antigen of the β-coronavirus according to claim 1, wherein: when the β-coronavirus is the Middle East respiratory syndrome coronavirus, the partial or the entire amino acid sequence of the receptor binding domain of the surface spike protein thereof is any one selected from a group consisting of following amino acid sequences: (1) SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; (2) an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids in the amino acid sequence (1), wherein a protein encoded by the amino acid sequence has an identical immunogenicity as a protein encoded by the amino acid sequence (1); alternatively, the partial amino acid sequence of the receptor binding domain of the surface spike protein thereof comprises SEQ ID NO: 2; when the β-coronavirus is the 2019 novel coronavirus, the partial or the entire amino acid sequence of the receptor binding domain of the surface spike protein thereof is any one selected from a group consisting of following amino acid sequences: (3) SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; (4) an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids in the amino acid sequence (3), wherein a protein encoded by the amino acid sequence has an identical immunogenicity as a protein encoded by the amino acid sequence (3); alternatively, the partial amino acid sequence of the receptor binding domain of the surface spike protein comprises SEQ ID NO: 6; and when the β-coronavirus is the severe respiratory syndrome coronavirus, the partial or the entire amino acid sequence of the receptor binding domain of the surface spike protein thereof is any one selected from a group consisting of following amino acid sequences: (5) SEQ ID NO: 8; (6) an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids in the amino acid sequence (5), wherein a protein encoded by the amino acid sequence has an identical immunogenicity as a protein encoded by the amino acid sequence (5).

6. The antigen of the β-coronavirus according to claim 5, wherein: when the β-coronavirus is the Middle East respiratory syndrome coronavirus, the amino acid sequence of the antigen of the β-coronavirus comprises any one selected from a group consisting of following amino acid sequences: (1) two repeated amino acid sequences of SEQ ID NO: 1 linked in tandem by a GGSGGS linker sequence; (2) two repeated amino acid sequences of SEQ ID NO: 1 linked in tandem by a GGS linker sequence; (3) two repeated amino acid sequences of SEQ ID NO: 1 linked directly in tandem; (4) two repeated amino acid sequences of SEQ ID NO: 2 linked in tandem by a GGS linker sequence; (5) two repeated amino acid sequences of SEQ ID NO: 2 linked directly in tandem; (6) two repeated amino acid sequences of SEQ ID NO: 3 linked by a GGSGGSGGSGS linker sequence; (7) two repeated amino acid sequences of SEQ ID NO: 3 linked in tandem by a GGSGGSGGSGS linker sequence; (8) two repeated amino acid sequences of SEQ ID NO: 3 linked in tandem by a GGSGGSGGS linker sequence; (9) two repeated amino acid sequences of SEQ ID NO: 3 linked in tandem by a GGS linker sequence; and (10) two repeated amino acid sequences of SEQ ID NO: 3 linked directly in tandem; alternatively, the amino acid sequence of the antigen of the β-coronavirus comprises two repeated amino acid sequences of SEQ ID NO: 2 directly linked in tandem; when the β-coronavirus is the 2019 novel coronavirus, the amino acid sequence of the antigen of the β-coronavirus comprises any one selected from a group consisting of following amino acid sequences: (1) two repeated amino acid sequences of SEQ ID NO: 5 linked directly in tandem; (2) two repeated amino acid sequences of SEQ ID NO: 6 linked directly in tandem; and (3) two repeated amino acid sequences of SEQ ID NO: 7 linked directly in tandem; alternatively, the amino acid sequence of the antigen of the β-coronavirus comprises two repeated amino acid sequences of SEQ ID NO: 6 directly linked in tandem; and when the β-coronavirus is the severe respiratory syndrome coronavirus, the amino acid sequence of the antigen of the β-coronavirus comprises two repeated amino acid sequences of SEQ ID NO: 8 linked directly in tandem.

7. The antigen of the β-coronavirus according to claim 6, wherein: a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 1 linked in tandem by the GGSGGS linker sequence is shown as SEQ ID NO: 9; a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 1 linked in tandem by the GGS linker sequence is shown as SEQ ID NO: 10; a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 1 linked directly in tandem is shown as SEQ ID NO: 11; a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 2 linked in tandem by the GGS linker sequence is shown as SEQ ID NO: 12; a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 2 linked directly in tandem is shown as SEQ ID NO: 13; a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 3 linked in tandem by the GGSGGSGGSGGSGGS linker sequence is shown as SEQ ID NO: 14; a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 3 linked in tandem by the GGSGGSGGSGGS linker sequence is shown as SEQ ID NO: 15; a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 3 linked in tandem by the GGSGGSGGS linker sequence is shown as SEQ ID NO: 16; a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 3 linked in tandem by the GGS linker sequence is shown as SEQ ID NO: 17; a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 3 linked directly in tandem is shown as SEQ ID NO: 18; a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 5 linked directly in tandem is shown as SEQ ID NO: 19; a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 6 linked directly in tandem is shown as SEQ ID NO: 20; a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 7 linked directly in tandem is shown as SEQ ID NO: 21; and a nucleotide sequence encoding the two repeated amino acid sequences of SEQ ID NO: 8 linked directly in tandem is shown as SEQ ID NO: 23.

8. A method for preparing the antigen of the β-coronavirus according to claim 1, comprising following steps: adding a sequence encoding a signal peptide to a 5′-terminal of a nucleotide sequence encoding the antigen of the β-coronavirus, adding a terminator codon to a 3′-terminal for cloning and expression, screening a correct recombinant, transfecting an expression system cell for expression, collecting a cell supernatant after expression, and purifying to obtain the antigen of the β-coronavirus.

9. The method according to claim 8, wherein the expression system cell is selected from a group consisting of mammalian cell, insect cell, yeast cell, and bacterial cell; preferably, the mammalian cell is 293T cell or CHO cell, and the bacterial cell is Escherichia coli cell.

10. A nucleotide sequence encoding the antigen of the β-coronavirus according to claim 1.

11. A recombinant vector comprising the nucleotide sequence according to claim 10.

12. An expression system cell comprising the recombinant vector according to claim 11.

13. A method for preparing a vaccine against the β-coronavirus by using the antigen of the β-coronavirus according to claim 1.

14. A β-coronavirus vaccine, comprising the antigen of the β-coronavirus according to claim 1 and an adjuvant.

15. The β-coronavirus vaccine according to claim 14, wherein the adjuvant is selected from a group consisting of an aluminum adjuvant, an MF59 adjuvant, and an MF59-like adjuvant.

16. A β-coronavirus DNA vaccine, comprising a recombinant vector comprising a DNA sequence encoding the antigen of the β-coronavirus according to claim 1.

17. A β-coronavirus RNA vaccine, comprising a recombinant vector comprising an mRNA sequence encoding the antigen of the β-coronavirus according to claim 1.

18. A β-coronavirus viral vector vaccine comprising a recombinant viral vector comprising a nucleotide sequence encoding the antigen of the β-coronavirus according to claim 1, alternatively, the viral vector is one or more selected from a group consisting of an adenovirus vector, a poxvirus vector, an influenza virus vector, and an adeno-associated virus vector.

19. A method for preparing a vaccine against the β-coronavirus by using the nucleotide sequence according to claim 10.

20. A method for preparing a vaccine against the β-coronavirus by using the recombinant vector according to claim 11.

21. A method for preparing a vaccine against the β-coronavirus by using the expression system cell according to claim 12.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows an ultraviolet absorption profile for the RBD protein obtained by means of a constructed vector pFastBac-SP-MERS-RBD (E367-Y606) in Example 1 subjected to molecular sieve chromatography using a Superdex 200 Hiload 16/60 column (GE), and an electrophoretogram for the protein subjected to SDS-PAGE under reduced conditions (+DTT) or non-reduced conditions (−DTT) obtained by collecting Dimer peaks and Monomer peaks in the ultraviolet absorption profile.

(2) FIG. 2 is a schematic diagram of the immunization and MERS-CoV challenge strategies in Examples 2 to 7.

(3) FIG. 3 shows the results of Example 3, i.e., the titers of MERS-CoV RBD specific IgG antibody in sera collected from mice according to the immunization strategy in FIG. 2 after the third immunization in Example 2, where Dimer indicates that MERS-CoV RBD-dimer was used as the immunogen; RBD-monomer indicates MERS-CoV RBD-monomer was used as the immunogen; AddaVax indicates the use of AddaVax adjuvant; Alum indicates the use of aluminum adjuvant; no indication of adjuvant means no use of adjuvant; and 3 μg, 10 μg, and 30 μg, indicate the amounts of the immunogen used per immunization. Significant difference analysis: ns, P>0.05; *, P<0.05; **, P<0.01; ***, p<0.001; ****, P<0.0001.

(4) FIG. 4 shows the results of Example 5, i.e., 90% neutralization titers antibodies against MERS-CoV pseudovirus in sera collected from mice according to the immunization strategy in FIG. 2 after the third immunization in Example 2, where Dimer indicates that MERS-CoV RBD dimer was used as the immunogen; RBD monomer indicates that MERS-CoV RBD monomer was used as the immunogen; AddaVax indicates the use of AddaVax adjuvant; Alum indicates the use of aluminum adjuvant; no indication of adjuvant means no use of adjuvant; and 3 μg, 10 μg, and 30 μg indicate the amounts of the immunogen used per immunization. Significant difference analysis: ns, P>0.05; ***, p<0.001.

(5) FIG. 5 shows the results of Example 6, i.e., 50% neutralization titers of antibodies against MERS-CoV euvirus (EMC strain) in sera collected from mice according to the immunization strategy in FIG. 2 after the third immunization in Example 2, where Dimer indicates that MERS-CoV RBD dimer was used as the immunogen; AddaVax indicates the use of Addavax adjuvant; Alum indicates the use of aluminum adjuvant; no indication of adjuvant means no use of adjuvant; and 3 μg, 10 μg, and 30 μg indicate the amounts of the immunogen used per Significant difference analysis: ns, P>0.05; ***, p<0.001; ****, P<0.0001.

(6) FIG. 6 shows the results of Example 7 in which mice after the third immunization were subjected to intranasal infection with adenovirus expressing hCD26 (hDPP4) according to the immunization strategy in FIG. 2, and were challenged with MERS-COV 5 days later, followed by the detection of viral titers (TCID.sub.50) on the tissue homogenates prepared from the lungs of the mice removed 3 days later, Dimer indicates that MERS-CoV RBD dimer was used as the immunogen; AddaVax indicates the use of AddaVax adjuvant; Alum indicates the use of aluminum adjuvant; no indication of adjuvant means no use of adjuvant; and 3 μg, 10 μg, and 30 μg indicate the amounts of the immunogen used per immunization. Significant difference analysis: ns, P>0.05; *, P<0.05; **, P<0.01; ***, p<0.001; ****, P<0.0001.

(7) FIG. 7 shows the pathological results of the examination of the protective efficacy of the vaccine on the lung tissue of mice in Example 8 in which the lungs from the challenged mice in Example 7 after necroscopy were fixed in 4% paraformaldehyde, embedded in paraffin, stained with hematoxylin and eosin and sliced to obtain tissue sections for observation of pathological changes, where AddaVax indicates the use of AddaVax adjuvant; Alum indicates the use of aluminum adjuvant; and 3 μg, 10 μg, and 30 μg indicate the amounts of the immunogen used per immunization. Slight, Mild and Severe indicate different grades of lung tissue lesions, respectively.

(8) FIG. 8 shows the structure of MERS-CoV-RBD dimer (E367-Y606) analyzed in Example 9.

(9) FIGS. 9A, 9B, and 9C show single-chain RBD dimers designed based on the MERS-CoV RBD-dimer structure in Example 10.

(10) FIG. 10 shows the results of Western blot conducted on MERS-RBD-C1 to MERS-RBD-C10 single-chain dimers expressed in Example 10 under reduced conditions (+DTT) or non-reduced conditions (−DTT), where RBD Monomer is MERS-CoV RBD Monomer protein.

(11) FIG. 11 shows an ultraviolet absorption profile for MERS-RBD-C5 single-chain dirtier expressed in Example 11 subjected to molecular sieve chromatography using a Superdex 200 Hiload 16/60 column (GE), and the results of SDS-PAGE of the purified single-chain dimer under reduced conditions (+DTT) or non-reduced conditions (−DTT).

(12) FIG. 12 shows the titers of MERS-CoV-RBD specific IgG antibody elicited by immunization of mice in Example 12 with single-chain MERS-CoV-RBD dimer and disulfide-linked non-single-chain dimer proteins, where sc-dimer is a single-chain dimer, and Dimer is a disulfide-linked non-single-chain dimer. Significant difference analysis: ns, P>0.05; *, P<0.05; ***, p<0.001; ****, P<0.0001.

(13) FIG. 13 shows the 90% neutralization titers of antibodies against MERS-CoV pseudovirus elicited by immunization of mice with single-chain MERS-CoV-RBD dimer and disulfide-linked non-single-chain dimer proteins in Example 12, where sc-dimer is a single-chain dimer, and Dimer is a disulfide-linked non-single-chain dimer. Significant difference analysis: ns, P>0.05; *, P<0.05; ****, P<0.0001.

(14) FIGS. 14A and 14B are comparison diagrams of the receptor binding domains (RBDs) of β-coronaviruses in Example 13, where the sequences in the two figures were consecutive, and the following β-coronaviruses were aligned:

(15) MERS-CoV (AFS88936), SARS-CoV (AAS00003), SARS-CoV-2 (QHR63290), Bat-CoV_HKU5 (ABN10875), Rousettus_bat-CoV (AOG30822), Bat-CoV_BM48-31 (ADK66841), Bat-CoV_HKU9 (ABN10911), Bat_Hp-betaCoV (AIL94216), SARS-related-CoV (APO40579), BtRs-Beta-CoV (QDF43825), Bat-SARS-like-CoV (ATO98231), SARS-like-CoV_WIV16 (ALK02457), Bat-CoV (ARI44804), BtR1 -Beta-CoV (QDF43815), HCoV_HKU1 (AZS52618), MCoV_MHV1 (ACN89742), BetaCoV_HKU24 (AJA91217), HCoV_OC43 (AAR01015), and BetaCoV_Erinaceus (AGX27810).

(16) FIG. 15 is a mimic diagram showing the structure of SARS-CoV-RBD dimer or 2019-nCoV-RBD dimer in example 13 and the construct of the expression 2019-nCoV-RBD dimer, the 2019-nCoV-RBD monomer and the SARS-CoV-RBD dimer designed.

(17) FIG. 16 shows the results of Western blot under reduced conditions (+DTT) or non-reduced conditions (−DTT) for several single-chain dimers of SARS-CoV-RBD and 2019-nCoV- RBD expressed in Example 13.

(18) FIG. 17 shows the ultraviolet absorbance at 280 nm for 2019-nCoV-RBD-C2 antigen purified in Example 14, and the results of SDS-PAGE of the purified single-chain dirtier under reduced conditions (+DTT) or non-reduced conditions (−DTT).

(19) FIG. 18 shows the ultraviolet absorbance at 280 nm for SARS-CoV-RBD-C1 antigen purified in Example 14, and the results of SDS-PAGE of the purified single-chain dimer under reduced conditions (+DTT) or non-reduced conditions (−DTT).

(20) FIG. 19 shows the titers of 2019-nCoV-RBD specific IgG antibody in sera collected from mice after three immunizations (19 days after the first immunization, 14 days after the second immunization, and 14 days after the third immunization) in Example 15, respectively, where sc-dimer indicates that single-chain nCoV-RBD dimer was used as the immunogen, and Monomer indicates that nCoV-RBD-monomer was used as the immunogen. Significant difference analysis: ****, P<0.0001.

(21) FIG. 20 shows the 90% neutralization titers of antibodies against 2019-nCoV pseudovirus in sera collected from mice after three immunizations (19 days after the first immunization, 14 days after the second immunization, and 14 days after the third immunization) in Example 15, where sc-dimer indicates that single-chain nCoV-RBD dimer was used as the immunogen, and Monomer indicates that nCoV-RBD-monomer was used as the immunogen. Significant difference analysis: ns, P=0.05; **, P<0.01; ****, P<0.0001.

(22) FIG. 21 shows the 50% neutralization titers of antibodies against 2019-nCoV euvirus (2020XN4276 strain) in sera collected after the second immunization (14 days after the second immunization) of mice in Example 15, where sc-dimer indicates that single-chain nCoV-RBD dimer was used as the immunogen, and Monomer indicates that nCoV-RBD-monomer was used as the immunogen.

(23) FIG. 22 shows the titers of SARS-RBD-specific IgG antibody in sera collected after three immunizations of mice in Example 16 (19 days after the first immunization, 14 days after the second immunization, and 14 days after the third immunization), where sc-dimer indicates that single-chain SARS-CoV-RBD dimer was used as the immunogen, and Monomer indicates that SARS-CoV-RBD-monomer was used as the immunogen. Significant difference analysis: ns, P>0.05; *, P<0.05; **, P<0.01; ****, P<0.0001.

(24) FIG. 23 shows the 90% neutralization titers of antibodies against SARS-CoV pseudovirus in sera collected from mice after three immunizations (19 days after the first immunization, 14 days after the second immunization, and 14 days after the third immunization) in Example 16, where sc-dimer indicates that single-chain SARS-CoV-RBD dimer was used as the immunogen, and Monomer indicates that SARS-RBD-monomer was used as the immunogen. Significant difference analysis: ns, P>0.05; *, P<0.05; **, P<0.01; ***, p<0.001; ****, P<0.0001.

DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE

(25) In order to make the objects, technical solutions and advantages of the examples of the present disclosure clearer, the technical solutions in the examples of the present disclosure will be clearly and completely described below. Apparently, the described examples are some, but not all examples of the present disclosure. All other examples derived from the examples of the present disclosure by a person skilled in the art without creative work shall fall within the scope of protection of the present disclosure.

(26) Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present disclosure. It will be understood by those skilled in the art that the present disclosure may be practiced without some of these specific details. In some examples, materials, elements, methods, procedures, and the like that are well known to those of skill in the art have not been described in detail so as not to obscure the present disclosure.

(27) Throughout the specification and claims, unless expressly indicated otherwise, the terms “comprise” or “include”, or variations such as “comprises” or “comprising”, “includes” or “including” will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.

(28) Explanation of Terms

(29) Disulfide-linked non-single-chain RBD dimer and RBD monomer refer to those obtained by inserting a nucleotide sequence encoding RBD monomer into a vector, then transfecting cells of an expression system for expression, collecting cell supernatants after expression, and purifying, where two RBD monomers in a disulfide-linked non-single-chain RBD dimer are simply disulfide-bonded through cysteines therein. As used herein, disulfide-linked non-single-chain RBD dimer and non-single-chain RBD-dimer protein have the same meaning; and RBD monomer, monomeric RBD, and RBD-monomer protein all have the same meaning.

(30) Single-chain RBD dimer is a recombinant protein obtained by linking two nucleotide sequences encoding identical or substantially identical RBD monomers in tandem directly or via a linker fragment, adding a sequence encoding a signal peptide to the 5′-terminal of the nucleotide sequence and a terminator codon to the 3′-terminal of the nucleotide sequence for cloning and expression, screening correct recombinants, transfecting cells of an expression system cell for expression, collecting cell supernatants after expression, and purifying, where the protein contains two RBD monomers which are identical or substantially identical and can be directly linked together with peptide bonds or linked together through a linker sequence (such as GGS, GGSGGS and the like). As used herein, single-chain RBD-dimer, single-chain RBD dimer, single-chain dimer, sc-RBD dimer, single-chain RBD dimer and the like all have the same meaning.

EXAMPLE 1

Preparation of Recombinant Baculovirus Expressing MERS-CoV Antigen, and Expression and Purification of RBD Protein

(31) A nucleotide sequence (shown as SEQ ID NO: 24) encoding an amino acid RBD (E367-Y606) sequence (shown as SEQ ID NO: 1) in MERS-CoV S protein (having a sequence shown as GenBank: AFS88936.1) was cloned between EcoR I and Xho I restriction enzyme cutting sites of a pFastBac vector (pFastBac-SP, available from Invitrogen) containing gp67 signal peptide after the addition of a translation termination codon to the 3′-terminal thereof, so that the protein encoding region was subjected to fusion expression behind the signal peptide gp67 sequence for secretion of the protein of interest having 6 histidines at the C-terminal thereof, thereby obtaining a vector pFastBac-SP-MFRS-RBD (E367-Y606). The vector was then transfected into the cells of the expression system for expression, and after expression, cell supernatants were collected and purified.

(32) The obtained RBD protein was purified through molecular sieve chromatography using a Superdex 200 Hiload 16/60 column (GE), and a typical ultraviolet absorption profile for protein purification is shown in FIG. 1. There was one dimer peak and one monomer peak, SDS-PAGE was conducted on the elution peak of MERS-RBD protein in the vicinity of the elution volume of 78 mL. Under non-reduced conditions (without DTT), the size of the protein in the vicinity of the elution volume of 78 mL was approximately 60 Kd; whereas under reduced conditions (with DTT added), the size was approximately 30 Kd, which confirmed that the protein obtained in this peak was a dimer. SDS-PAGE was conducted on the elution peak in the vicinity of 90 mL of the elution volume, the size of the protein of interest was approximately 30 Kd under non-reduced conditions (without DTT) and reduced conditions, which confirmed that the peak was mainly RBD monomer. The dimer or monomer used in each of Examples 2 to 9 below was the disulfide-linked non-single-chain RBD dirtier or RBD monomer obtained in this Example.

EXAMPLE 2

Experiment for Immunization of Mice with MERS-RBD Protein

(33) MF59 (AddaVax used below was an MF59-like adjuvant) and aluminum adjuvant two commonly used adjuvants approved by SFDA, were used as vaccine components to provide more direct guidance for subsequent clinical trials. An in vitro neutralization experiment, as a classic method, was conducted to detect the protective efficacy of vaccines. Therefore, different doses of antigen were mixed with AddaVax adjuvant and Imject™ Alum adjuvant separately for immunization. The immunization groups, the types of RBD used in each group, the amount of RBD used in each immunization and the adjuvants are shown in Table 1 in which the blank space indicates “None”.

(34) MERS-RBD antigen (dimer or monomer) obtained in Example 1 was diluted with normal saline to a desired concentration and emulsified with adjuvants in groups. BALB/c mice aged 4-6 weeks (average weight 15-20 g, similarly hereinafter) were immunized in groups, with 6 mice in each group.

(35) TABLE-US-00001 TABLE 1 Group Immunogen Forms Dose Adjuvant  1 RBD Dimer  3 μg Alum  2 RBD Dimer 10 μg Alum  3 RBD Dimer 30 μg Alum  4 RBD Dimer  3 μg AddaVax  5 RBD Dimer 10 μg AddaVax  6 RBD Dimer 30 μg AddaVax  7 RBD Monomer  3 μg Alum  8 RBD Monomer 10 μg Alum  9 RBD Monomer 30 μg Alum 10 RBD Monomer  3 μg AddaVax 11 RBD Monomer 10 μg AddaVax 12 RBD Monomer 30 μg AddaVax 13 PBS — 14 PBS — Alum 15 PBS — Addavax

(36) The immunization was conducted according to a strategy as shown in FIG. 2. i.e., by means of intramuscular injection into the thigh, each mouse received three immunizations with vaccine at day 0, day 21 and day 42, respectively, each time in a vaccination volume of 100 μl. On the day 56 (namely, the 14th day after the third immunization), blood was collected from the tails of mice. Mouse sera were obtained by centrifugation at 3000 rpm for 10 minutes after standing, and stored at −20° C. in a refrigerator for specific antibody titer assay and pseudovirus neutralization assay.

EXAMPLE 3

ELISA Assay of Vaccine-Elicited Specific Antibody Titer

(37) (1) The RBD-monomer protein of MERS-CoV was diluted to 3 μg/ml with an ELISA coating solution (Solarbio, C1050), and 100 μl of the resulting solution was added to each well of a 96-well ELISA plate (Coring, 3590) and placed at 4° C. for 12 hours.

(38) (2) The coating solution was removed, and then PBS was added to wash once. 5% skim milk prepared with PBS was added to a 96-well plate in an amount of 100 μper well as a blocking solution for blocking and placed at room temperature for 1 hour. After the completion of blocking, the plate was washed once with PBS solution.

(39) (3) Mouse serum was diluted during blocking. Serum samples were also diluted with the blocking solution. Serum samples were diluted from 20-fold. Then 100 μl of serum was added to each well of the ELISA plate, while the blocking solution was added for the negative control, incubated at 37° C., for 2 hours, and then washed with PBST for 4 times.

(40) (4) Goat anti-mouse IgG-ITIRP antibody (Abcam, ab6789) diluted 1:2000 with the blocking solution was added and incubated at 37° C. for 1.5 hours, and then washed with PBST for 5-6 times. Plates were developed with TMB substrate, which was followed by stopping the reactions with 2 M hydrochloric acid for a proper time, and the absorbance was measured at 450 nm using a microplate reader. Antibody titer values were defined as the highest dilution of serum with a response value greater than 2.5 times the negative control value. The titer of a sample was defined as half of the lowest dilution (limit of detection) at which the response value was still less than 2.5-fold background value, namely, 1: 10.

(41) As shown in FIG. 3, significantly different levels of antibodies were elicited for the RBD dimer group and monomer group at doses of 3 μg and 10 μg with the AddaVax adjuvant, and significantly different levels of antibodies were elicited for the two groups at doses of 3 μg, 10 μg and 30 μg with the aluminum adjuvant, and the dimer group elicited higher levels of antibodies, indicating that the dimeric RBD antigen had a significantly higher ability to activate the antibody response in mice than the RBD monomer vaccine.

(42) The RBD-monomer protein of MERS-CoV was used as the coating protein in all ELISA assays in the examples of the present disclosure,

EXAMPLE 4

Preparation of MERS-CoV Pseudovirus

(43) PNI43-Lucii Pseudovirus Packaging

(44) (1) Cell plating: on the day before transfection, 293T cells grown in logarithmic phase were harvested by trypsinization, counted, reseeded and cultured overnight in a 10 cm petri dish, and transfected (without antibiotics) when the confluence of the cells reached 70-90% over 18-24 hours.

(45) (2) Plasmid co-transfection by a PEI method: a total of 20 82 g of plasmid (10 μg of HIV pNL4-3.Luc.RE (Invitrogen) and 10 μg of pCAGGS-MERS-S which was obtained by inserting a DNA sequence encoding MERS Spike protein (M1-H1352) into EcoRI and XhoI sites of pCAGGS vector) and 40 μL of PEI (2 mg/ml) were dissolved in normal saline or HBS separately, to a final volume of 500 μL, and mixed evenly. After standing for 5 minutes, the two solutions were mixed, followed by standing for 20 minutes. The mixture was then added dropwise to the cell culture dish, and 4-6 hours later, the cells were washed twice with PBS and provided with a fresh serum-free medium.

(46) (3) Virus harvesting: after transfection for 48 hours, cells and supernatants were harvested, centrifuged slow at 1000 rpm for 10 minutes to remove cell debris, packed, and single use aliquots were stored at −80° C. to avoid the decrease of virus titers caused by repeated freezing and thawing.

(47) (4) Infection: on the first day, the cells were seeded and cultured overnight, and the cells reached 80-100% over 18-24 hours;

(48) On the next day, the susceptible cells were washed with PBS to remove serum and infected with the collected viral supernatant, and the culture medium was changed to a serum-containing medium 4-6 hours later. According to the experimental requirements, Luciferase values could be measured at different time points, with reference to the Luciferase Assay System Protocol or the Dual Luciferase Reporter Assay System Protocol of Promega Company. The harvested virus solution was diluted 5-fold and added to Huh7 cells (human hepatoma cells) in a 96-well plate. After 4 hours of infection, the virus solution was discarded, and the cells were washed twice with PBS, and provided with DMEM complete medium containing 10% serum. The medium was discarded 48 hours later, and the cells were washed twice with PBS and added with a cell lysis solution. After freezing and thawing once at −80° C., 20 μl of cell culture from each well was assayed for luciferase activity using a GloMax 96 Microplate Luminometer (Promega). TCID.sub.50) was calculated by Reed-Muech method.

EXAMPLE 5

Pseudovirus Neutralization Assay of Immune Serum

(49) The serum obtained in Example 2 was diluted in multiple ratios, mixed with 100 TCIDD.sub.50 pseudovirus, and incubated for 30 minutes at 37° C. The mixture was then added to a 96-well plate completely covered with Huh7 cells. After incubation at 37° C. for 4 hours, the virus solution was discarded, and the cells were washed twice with PBS, and provided with a complete medium DMEM containing 10% serum. After 48 hours, the culture medium was discarded, and the cells were washed twice with PBS and added with a cell lysis solution to assay the luciferase activity. Pseudovirus having spike protein on the surface infected cells to release DNA and express rather than replicate luciferase. If the pseudovirus could not infect the cells in the presence of neutralizing antibodies, the luciferase was not expressed. The neutralization titers of the serum were examined in this way.

(50) The results of immunogenicity assays after the third immunization are shown in FIG. 4. The result shown that the RBD dimer (E367-Y606) elicited neutralizing antibodies after three immunizations, regardless of the adavax adjuvant group or the aluminum adjuvant group (indicated by +Alum). Particularly, the mean value of the neutralizing antibodies NT.sub.90 in the AddaVax adjuvant 10 μg group could reach more than 1:1000 (as shown in FIG. 4); whereas the RBD-monomer (E379-E589) did not elicit neutralizing antibodies after three immunizations except low neutralizing antibody production in 2 mice (as shown in FIG. 4). Pseudovirus neutralization assay demonstrated that the neutralizing antibodies induced by the RBD-dimer was much higher than that induced by the monomeric RBD.

(51) The RBD monomer (E379-E589) was obtained by the following method: a nucleic acid fragment (shown as SEQ ID NO: 25) encoding the amino acid (E379-E589) sequence (shown as SEQ ID NO: 4) in MERS-CoV S protein was inserted into EcoRI and XhoI restriction enzyme cutting sites of pFastBac-SP to allow fusion expression of the protein coding region behind the signal peptide gp67 sequence for secretion of the protein of interest having 6 histidines at the C-terminal thereof, thereby obtaining a vector pEastBac-SP-MERS-RBD (E379-E589).

EXAMPLE 6

Euvirus Neutralization of Immune Serum (EMC Strain)

(52) Neutralization assay was conducted with serum after three immunizations for MERS-CoV euvirus (EMC strain, disclosed in Yao Y, Bao W, et al. An animal model of MERS produced by infection of rhesus macaques with NIERS coronavirus. J Infect Dis. 2014,209(2):236-242. doi:10.1093/infdis/jit590, supplied by the institute of laboratory animals of Peking Union Medical College). The results are shown in FIG. 5. The results showed that both AddaVax adjuvant and aluminum adjuvant could elicit high neutralizing antibodies in mice. The highest group (Addavax adjuvant 10 μg and RBD dimer) achieved an IC50 greater than 1:600. This result demonstrated that the dimeric RBD could elicit a higher level of neutralizing antibodies in mice by MERS-CoV euvirus neutralization assay.

EXAMPLE 7

Challenge Protection Experiment

(53) Mice immunized three times in Example 2 were intranasally infected with adenovirus expressing hCD26 (hDPP4) on day 77, as shown in FIG. 2. This allowed transient expression of MERS-CoV receptor hCD26 in lung, making mice susceptible to MERS-CoV (see Chi H et al. DNA vaccine encoding Middle East responsive syndrome coronavirus S1 protein induces protective immune responses in mice [J]. Vaccine, 2017, 35 (16): 2069-2075). Five days later, experiments with MERS-CoV (EMC strain) challenge were conducted with a challenge dose of 5×10.sup.5 pfu. Three days later after challenge, the lungs of mice were harvested, and tissue homogenate prepared therefrom was used to detect virus titers (TCID.sub.50). The results are shown in FIG. 6. Compared with PBS control group, the viral load in lung tissue of mice in vaccine group decreased significantly. The viral load in the group with AddaVax adjuvant 3 μg and RBD dimer decreased by nearly 1000 times compared with that of the PBS group, showing a good protective efficacy. These results showed that the RBD dimer, as a vaccine, had a markedly significant protective efficacy against MERS-Co V challenge.

EXAMPLE 8

Validation of Vaccine Protection for The Lung Tissue of Mice

(54) The lung tissue of mice in the MERS-CoV challenge experiment in Example 7 was fixed in 4% paraformaldehyde, and then stained with hematoxylin and eosin, and tissue sections were used to observe the pathological changes of the lung, with results as shown in FIG. 7. Lung tissues of all control mice (namely, PBS group) exhibited severe interstitial pneumonia, pulmonary alveolitis, diffuse inflammatory cell infiltration, and necrosis of bronchial epithelial cells (as shown in FIG. 7). However, milder lesions were observed in the group of mice immunized with the RBD-dimer and the pulmonary alveolus was highly visible with lower infiltration of inflammatory cells because both AddaVax and Alum adjuvants could greatly alleviate the lung injury caused by virus challenge. The small histopathological changes in the lung likely resulted from a direct inoculation of high amount (5×10.sup.5 pfu) of virus intranasally. Therefore, the RBD-dimer could substantially reduce the lung injury caused by MERS-CoV infection.

EXAMPLE 9

Crystallization and Structure Determination of MERS-RBD-Dimer

(55) The RBD (E367-Y606) protein was expressed according to the method of Example 1, After purification, the dimer protein peaks were collected. The protein was concentrated to 10 mg/ml and mixed with the reservoir solution in a volume ratio of 1:1, and then protein crystal screening was carried out by mosquito® Protein Crystallization Screening Liquid Workstation (TTP LabTech). Diffraction-quality crystals of MERS-CoV RBD-dimer were obtained at 18° C. The crystals were collected at the Shanghai Synchrotron Radiation Facility (SSRF), and finally 2.8 Å diffraction data were obtained. The data were processed with HKL2000 software, and the structure was solved by the molecular replacement module, with the structure of MERS-CoV RBD (PDB: 4KQZ) as the search models. The results are shown in FIG. 8.

EXAMPLE 10

Structure Design of a Single-Chain RBD Dimer (Sc-RBD Dinner) Based on MERS-RBD Dinner

(56) Based on the MERS-RBD crystal structure of FIG. 8, the N-terminal (N′) and C-terminal (C′) of the two subunits of RBD were arranged in an end-to-end form. The N-terminal and the C-terminal each had an invisible flexible sequence (as shown in FIG. 9A), which inspired us to link two subunits as a tandem repeat single chain, namely, single-chain RBD dimer (sc-RBD) dimer).

(57) The first design (as shown in FIG. 9A) was as follows:

(58) (1) two GGS linker sequences were added between two repeated tandem (E367-Y606) sequences to obtain MERS-RBD-C1 (abbreviated as C1), where the nucleotide sequence encoding the amino acid sequence was SEQ ID NO: 9.

(59) (2) one GGS linker sequence was added between two repeated tandem (E367-Y606) sequences to obtain MERS-RBD-C2 (abbreviated as C2), where the nucleotide sequence encoding the amino acid sequence is SEQ ID NO: 10;

(60) (3) two repeated (E367-Y606) sequences were directly linked in tandem to obtain MERS-RBD-C3 (abbreviated as C3), where the nucleotide sequence encoding the amino acid sequence is SEQ ID NO: 11.

(61) The second design (as shown in FIG. 9B), in order to avoid the effects of cysteine residue (C603) at the position 603 of the C-terminal on expression, a truncated construct at C-terminal residue N602 was conducted, which was specifically as follows:

(62) (4) one GGS linker sequence was added between two repeated tandem (E367-N602) sequences to obtain MERS-RBD-C4 (abbreviated as C4), where the nucleotide sequence encoding the amino acid sequence was SEQ ID NO: 12;

(63) (5) two repeated (E367-N602) sequences were linked in tandem directly to obtain MERS-RBD-C5 (abbreviated as C5), where the nucleotide sequence encoding the amino acid sequence was SEQ ID NO: 13.

(64) The third design in which (as shown in FIG. 9C) structurally visible sequences were directly expressed and linked by linker sequences of different lengths was specifically as follows:

(65) (6) five GCS linker sequences were added between two repeated tandem (V381-L588) sequences to obtain MERS-RBD-C6 (abbreviated as C6), where the nucleotide sequence encoding the amino acid sequence was SEQ ID NO: 14;

(66) (7) four GCS linker sequences were added between two repeated tandem (V381-L588) sequences to obtain MERS-RBD-C7 (abbreviated as C7), where the nucleotide sequence encoding the amino acid sequence was SEQ ID NO: 15;

(67) (8) three GGS linker sequences were added between two repeated tandem (V381-L588) sequences to obtain MERS-RBD-C8 (abbreviated as C8), where the nucleotide sequence encoding the amino acid sequence was SEQ ID NO: 16;

(68) (9) one GGS linker sequence was added between two repeated tandem (V381-L588) sequences to obtain MERS-RBD-C9 (abbreviated as C9), where the nucleotide sequence encoding the amino acid sequence was SEQ ID NO: 17;

(69) (10) two repeated sequences (V381-L588) were directly linked in tandem to obtain MERS-RBD-C10 (abbreviated as C10), where the nucleotide sequence encoding the amino acid sequence was SEQ ID NO: 18.

(70) The 5′-terminal of a nucleotide sequence encoding the above VIERS-RBD-C1 to C10 was added with a nucleotide sequence encoding the MERS-S protein self-signal peptide (MIHSVFLLMFLLTPTES), while the 3′-terminal was added with a nucleotide sequence encoding six histidines. A terminator codon was then added to the 3′-terminal, and the obtained nucleotide sequence was inserted between the EcoRI and XhoI restriction enzyme cutting sites of a pCAGGS vector, and a Kozak sequence gccacc was contained upstream of an initiator codon. The above plasmid was transfected into 293T cells, and 48 hours later, the supernatant was collected, and the N-terminus of the protein of interest was provided with a signal peptide. Western blot method was used to detect the expression of the protein of interest, with the results as shown in FIG. 10. The results showed that all constructs were expressed except C2. Under both reduced (+DTT) and non-reduced (−DTT) conditions, the protein was about the size of the dimer (50-60 Kda). Among them, C4 and C5 were expressed at the highest levels. In view of that no any exogenous sequence was introduced and the sequence of the MERS-CoV itself was completely used, the C5 construct would be more advantageous and safer for clinical use. The efficacy of MERS-RBD-C5 as a vaccine would be further assessed.

EXAMPLE 11

Mammalian Expression of Single-Chain MERS-CoV RBD Dimer (Sc-RBD Dimer) and Protein Purification

(71) MERS-RBD-C5 was expressed using mammalian 293T cells, After transfection of the plasmid into 293T cells, expression was conducted and the supernatant was harvested. The cell supernatant was filtered through a 0.22 μm filtration membrane to remove cell debris. The supernatant of cell culture was purified by Ni affinity chromatography column (Histrap) overnight at 4° C. The resin was washed with buffer A (20 mM Tris, 150 mM NaCl, pH 8.0) to remove non-specific binding proteins. Finally, the protein of interest was eluted from the resin with buffer B (20 mM Tris, 150 mM NaCl, pH 8.0, 300 mM imidazole), and the eluent was concentrated to be within 5 ml with a concentration tube of 10K MWCO. The protein of interest was further purified by molecular sieve chromatography using a Superdex 200 Hiload 16/60 column (GE). The buffer for molecular sieve chromatography was 20 mM Tris and 150 mM NaCl, with pH 8.0. After the molecular sieve chromatography, there was only one main peak near the elution volume of 80 mL. Proteins were collected for SDS-PAGE analysis. As can be seen from the results of SDS-PAGE, MERS-RBD-C5 protein showed a distinct protein band between 55 and 72 kd, which was the size of RBD dimer. It was demonstrated that single-chain MERS-RBD dimer was obtained, as shown in FIG. 11. By using the method of Example 10, 293T cells were used to express and purify the non-single-chain MERS RBD dimer for comparison with the sc-RBD dimer.

EXAMPLE 12

Mice Immunized with Single-Chain MERS-CoV RBD Dimer Sc-RBD Dimer) Protein

(72) The single-chain MERS-RBD dimer antigen obtained in Example 11 was diluted in normal saline and emulsified with adjuvants in groups. Then BALB/c mice aged 4-6 weeks were immunized in groups, with 6 mice in each group. in addition, one group of mice was immunized with PBS as a negative control. A group of mice immunized with 293T cells expressed a non-single-chain form of the dimer. Each mouse received three immunizations of vaccine by intramuscular injection into the thigh, at day 0, day 21 and day 42, respectively, at a vaccination volume of 100 μl each time (containing 10 μg of immunogen). Orbital blood was collected from mice 19 days later after the first immunization, 14 days later after the second immunization and 14 days later after the third immunization. Mouse serum was obtained by centrifugation at 3000 rpm for 10 minutes after standing, and stored in a refrigerator at −20° C. for specific antibody detection and pseudovirus neutralization detection.

(73) The serum specific antibody titer of the mice was detected by ELISA assay, using the method as shown in Example 3, with the results shown in FIG. 12. The RBD-sc-dimer group mice and disulfide-linked non-single-chain RBD-dimer group (indicated by Dimer) mice could be induced to produce antibody response. The titer mean value of the sc-dimer group was higher than that of the Dimer group, and the two groups had a significant difference after three immunizations (*, P<0.05). The results showed that the sc-dimer had excellent immunogenicity as the disulfide-linked non-single-chain RBD-dimer.

(74) The pseudovirus neutralization experiment was carried out with reference to Example 5, with the results shown in FIG. 13. The sc-dimer group mice and disulfide-linked non-single-chain RBD-dimer group (indicated by Dimer) mice could be induced to produce antibody response. The titer mean value of the sc-dimer group was higher than that of the Dimer group, and there was a significant difference between the two groups after the first immunization and the second immunization (FIG. 13). The mean values of the pseudovirus neutralization titers of sc-dimer group mice after three immunizations were already greater than 1:1000. The results indicated that the vaccine developed by the sc-dimer had great clinical development potential.

EXAMPLE 13

Application of Single-Chain RBD Dimer Technology in Other Coronavirus Vaccines

(75) To verify that this concept can be applied to vaccine design of all other coronaviruses, we compared the Receptor Binding Domains (RBDs) of the 19 common βcoronaviruses, the result is shown in FIG. 14A and 14B. All β-coronavirus RBDs exhibited a conserved cysteine at position C603 of MERS-CoV, as shown in FIG. 14B. 2019-nCoV (hereinafter referred to as nCoV) and SARS-CoV were selected for verification. According to the structure of SARS-RBD (PDB: 3D0G), the crystal structure of SARS-RBD was molded into the crystal structure of MERS-RBD dimer at a resolution of 2.8 Å by using Pymol software. A simulated SARS-RBD dimer structure as shown in FIG. 15 was obtained. It was found that, like MERS-RBD dimer. SARS-RBD dimer also existed in the form of end-to-end (as shown in FIG. 15). Since the RBD region of 2019-nCoV shared more than 75% homology with SARS-CoV, it was expected that the RBD dimer of 2019-nCoV would form this end-to-end arrangement. Considering that dimers in MERS-CoV could induce neutralizing antibodies with higher titers than monomers, it was considered that single-chain dimers (sc-dimers) were stilled used to design vaccines. Firstly, based on the S protein sequence of the 2019-nCoV WH01 strain, the construct of three single-chain dimers (sc-dimer) was designed, as shown in FIG. 15: (1) two R319-S530 were linked in tandem and named nCoV-RBD-C1 (the nucleotide sequence encoding the amino acid sequence was SEQ ID NO: 19); (2) two R319-K537 were linked in tandem and named nCoV-RBD-C2 (the nucleotide sequence encoding the amino acid sequence was SEQ ID NO: 20); (3) two R319-F541 were linked in tandem and named nCoV-RBD-C3 (the nucleotide sequence encoding the amino acid sequence was SEQ ID NO: 21); and (4) a further monomer was constructed as R319-F541, named nCoV-RBD-C4 (the nucleotide sequence encoding the amino acid sequence was SEQ ID NO: 22). in addition, a single-chain dimer of SARS-CoV was constructed, and two R306-Q523 were linked in tandem, as shown in FIGS. 14A and 14B, named SARS-CoV-RBD-C1. (the nucleotide sequence encoding this amino acid sequence was SEQ ID NO: 23).

(76) A nucleotide sequence encoding the above nCoV-RBD-C1 to C4 and a nucleotide sequence encoding SARS-CoV-RBD-C1 (SEQ. ID NO: 23) were added with the nucleotide sequence encoding the MERS-S protein self-signal peptide (MIHSVFLLMFLLPTES). After the nucleotide sequence encoding six histidines was added to the 3′-terminal, a terminator codon was added to the 3′-terminal, and inserted into the pCAGGS vector EcoRI and XhoI restriction enzyme cutting sites, and the Kozak sequence gccacc was contained upstream of the initiator codon thereof. The above plasmid was transfected into 293T cells. The supernatant was harvested 48 hours later and the expression of the protein of interest was detected by Western blot. The expression results are shown in FIG. 16. The results showed that the expression of nCoV-RBD-C2 was the highest among several antigen designs of 2019-nCoV. The SARS-CoV-RBD-C1 construct also had a high protein expression.

(77) The above results indicated that in the design of a single-chain dimer of the β-coronavirus, the optimal construct is from the first amino acid of FIG. 14A (marked as Start) to an amino acid before the last cysteine of FIG. 14B (marked as Stop).

EXAMPLE 14

Expression and Purification of Single-Chain 2019-nCoV-RBD Dimer Antigen and Single-Chain SARS-CoV-RBD Dimer Antigen

(78) Mammalian 293T cells were used to express nCoV-RBD-C2. After the plasmid was transfected into 293T cells, the supernatant was harvested. The cell supernatant was filtered through a 0.22 μm filtration membrane to remove cell debris. The supernatant of cell culture was purified by Ni affinity chromatography column (Histrap) overnight at 4° C. The resin was washed with buffer A (20 mM Tris, 150 mM NaCl, pH 8.0) to remove non-specific binding proteins. Finally, the protein of interest was eluted from the resin with buffer B (20 mM Tris, 150 mM NaCl, pH 8.0, 300 mM imidazole), and the eluent was concentrated to be within 5 ml with a concentration tube of 10K MWCO. The protein of interest was further purified by molecular sieve chromatography with a Superdex 200 Hiload 16/60 column (GE). The buffer for molecular sieve chromatography was 20 mM Tris and 150 mM NaCl, with PH 8.0. After molecular sieve chromatography, there was only one main peak near the elution volume of 80 ml. Proteins were collected for SDS-PAGE analysis. As can be seen from the results of SDS-PAGE, nCoV-RBD-C2 protein showed a distinct protein band between 48-63 kd, which was the size of RBD-dimer. It was demonstrated that single-chain 2019-nCoV-RBD dimer was obtained, as shown in FIG. 17. The purity was more than 9.5%. The results showed that such construct could produce sufficient and high-purity single-chain 2019-nCoV dimer protein.

(79) The monomeric RBD protein of 2019-nCoV (obtained by expression of nCoV-RBD-C4 construct), the monomeric RBD protein of SARS-CoV (SARS-CoV RBD R306-F527, having an amino acid sequence and a nucleotide sequence encoding the amino acid sequence as shown as SEQ ID NO: 26 and SEQ ID NO: 27) and the single-chain dimer protein of SARS-CoV (obtained by expression of SARS-CoV-RBD-C1 construct) were expressed and purified in the same way.

(80) As shown in FIG. 18, the result of the single-chain dimer protein of SARS-CoV showed that after the molecular sieve chromatography, there was only one main peak near the elution volume of 80 mL. Proteins were collected for SDS-PAGE analysis. As can be seen from the results of SDS-PAGE, SARS-CoV-RBD-C1 protein of interest showed a distinct protein band between 55 and72 kd, which was the size of RBD-dimer. It was demonstrated that single-chain SARS-RBD dimer was obtained, as shown in FIG. 18, and with high purity.

EXAMPLE 15

Mice Immunized with Single-Chain 2019-nCoV-RBD Dimer Protein

(81) The single-chain 2019-nCoV-RBD dimer and 2019-nCoV-RBD monomer obtained in Example 14 was diluted in PBS solution and emulsified with AddaVax adjuvant in groups. Then BALB/c mice (average body weight 15-20g, the same applies below) aged 6-8 weeks were immunized in groups, with 8 mice in each group. Each mouse received three immunizations of vaccine by intramuscular injection into the thigh, at day 0, day 21 and day 42, respectively, at a vaccination volume of 100 μl each time (containing 10 μg of immunogen). Blood samples were collected from mice 19 days later after the first immunization, 14 days later after the second immunization and 14 days later after the third immunization. Mouse senior was obtained by centrifugation at 3000 rpm for 10 minutes after standing, and stored in a refrigerator at −20° C. for specific antibody detection and pseudovirus neutralization detection.

(82) The serum specific antibody titer of 2019-CoV RBD of the mice was detected by ELISA assay, using the method as shown in Example 3, with the results shown in FIG. 19. The single-chain dimeric RBD (indicated by sc-dimer) and monomeric RBD (indicated by Monomer) could induce mice to produce antibody response. The titer mean value of the single-chain dimetic RBD group was higher than that of the monomeric RBD group, and the two groups had a significant difference after three immunizations (FIG. 19). The single-chain dimeric RBD induced mice to produce antibodies at levels of up to approximately 1:10.sup.6 after three immunizations. The results showed that the immunogenicity of the single-chain dimeric RBD antigen was stronger than that of the single-chain dimeric RBD antigen, and it had great potential as a potential new coronavirus vaccine.

(83) 2019-nCoV pseudovirus neutralization assay was carry out with reference to Example 5, with results as shown in FIG. 20. Neutralizing antibodies were induced in only the single-chain dimeric RBD (indicated by sc-dimer) group after the first immunization. Neutralizing antibodies were not detected in both the monomeric RBD (indicated by Monomer) and PBS groups, and there was a significant difference of neutralizing antibody titers between the single-chain dimeric RBD group and the monomeric RBD group (FIG. 20). After the second and third immunizations, both single-chain dimeric RBD and monomeric RBD could induce mice to produce neutralizing antibodies. After each immunization, the mean value of neutralizing antibody titers of the single-chain dimeric RBD group was higher than that of the monomeric RBD group (10-100 times higher), and there was a significant difference between the two groups after each immunization (FIG. 20). The single-chain dimeric RBD induced mice to produce antibodies at levels of up to approximately 1:10.sup.4 after three immunizations. The results showed that the single-chain dimeric RBD antigen could induce mice to produce higher neutralizing antibody level than the monomeric RBD antigen, and the single-chain dimeric RBD antigen had high advantages in use.

(84) Neutralization assay was conducted with serum after the second immunization for 2019-nCoV euvirus (2020XN4276 strain, which was published in Lu J, du Plessis L, Liu Z, et al. Genomic Epidemiology of SARS-CoV-2 in Guangdong Province, China. Cell. 2020;181(5):997-1003.e9. doi:10.1016/j.cell.2020.04.023, provided by Guangdong Provincial Center for Disease Control and Prevention). The experimental results are shown in FIG. 21. The results showed that the RBD dimer could induce mice to produce high levels of neutralizing antibodies against the novel coronavirus. The highest neutralizing NT50 was greater than 4096, and the lowest NT50 was 512 in a mouse. However, the neutralizing antibodies against the novel coronavirus were: detected in only 2 of the 8 mice in the RBD monomer group, with lower NT50, which were 128 and 256, respectively. The results indicated that the dimeric RBD could induce mice to produce higher levels of neutralizing antibodies against the novel coronavirus.

EXAMPLE 16

Mice Immunized with Single-Chain SARS-RBD Dimer Protein

(85) The single-chain SARS-RBD dimer and SARS-RBD monomer obtained in Example 14 were diluted in PBS solution and emulsified with AddaVax adjuvant in groups. Then BALB/c mice aged 6-8 weeks were immunized in groups, with 6 mice in each group. Each mouse received three immunizations of vaccine by intramuscular injection into the thigh, at day 0, day 2 and day 42, respectively, at a vaccination volume of 100 μl each time (containing 10 μg of immunogen). Blood samples were collected from mice 19 days later after the first immunization, 14 days later after the second immunization and 14 days later after the third immunization. Mouse serum was obtained by centrifugation at 3000 rpm for 10 minutes after standing, and stored in a refrigerator at −20° C. for specific antibody detection and pseudovirus neutralization detection.

(86) The serum specific antibody titer of SARS-RBD of the mice was detected by ELISA assay, using the method shown in Example 3, with the results shown in FIG. 22. The single-chain dimeric RBD (indicated by sc-dimer) and monomeric RBD (indicated by Monomer) could induce mice to produce antibody response. The titer mean value of the single-chain dimeric RBD group was higher than that of the monomeric RBD group, and the two groups had a significant difference after the second and the third immunizations (FIG. 22). The dimeric RBD induced mice to produce antibodies at levels of up to approximately 1:10.sup.6 after three immunizations. The results showed that the immunogenicity of the dimeric RBD antigen was stronger than that of the monomeric RBD antigen.

(87) The neutralization assay was conducted for SARS-CoV pseudovirus with reference to Example 5, with the results shown in FIG. 23. After the first and second immunizations, both dimeric RBD (indicated by sc-dimer) group and monomeric RBD (indicated by Monomer) group could induce mice to produce neutralizing antibodies. The mean value of neutralizing antibody titers of the dimeric RBD group was higher, and there was a significant difference between the two groups (FIG. 23). After the third immunization, the mean value of neutralizing antibody titers of the dimeric RBD group was still higher than that of the monomeric RBD group, and there was a significant difference (FIG. 23). The levels of neutralizing antibodies induced by dimeric RBD in mice after three immunizations were higher than 1:10.sup.3. The results showed that the dimeric RBD antigen could induce mice to produce higher neutralizing antibody level than the monomeric RBD antigen, and the dimeric RBD antigen had high advantages in use.

(88) Finally, it should be noted that the above examples are only intended to illustrate rather than limit the technical solutions of the present disclosure. Although the present disclosure has been described in detail with reference to the foregoing examples, it will be understood by a person skilled in the art that the technical solutions described in the foregoing examples may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the examples of the present disclosure.

INDUSTRIAL PRACTICAL APPLICABILITY

(89) The examples of the present disclosure relate to antigens of β-coronaviruses, preparation methods and uses thereof. An antigen of a β-coronavirus, its amino acid sequence comprises an amino acid sequence arranged in a (A-B)-(A-B) pattern or an amino acid sequence arranged in a (A-B)-C-(A-B) pattern or an amino acid sequence arranged in a (A-B)-(A-B′) pattern or an amino acid sequence arranged in a (A-B)-C-(A-B′) pattern, where A-B represents a partial amino acid sequence or the entire amino acid sequence of a receptor binding domain of a surface spike protein of the β-coronavirus; C represents an amino acid linker sequence; A-B′ represents an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids in the amino acid sequence of A-B; a protein encoded by A-B′ has the identical or substantially identical immunogenicity as a protein encoded by A-B; and the antigen of the β-coronavirus has a single-chain dimer structure. The single-chain dimer expressed according to the examples of the present disclosure is stable in content and has excellent immunogenicity as an antigen of a β-coronavirus, and the vaccine prepared by using the single-chain dimer as an antigen of a β-coronavirus can elicit high-titer neutralizing antibodies in mice.