Expression vector, cell and vaccine of multivalent coronavirus recombinant protein and preparation method of multivalent coronavirus recombinant protein

Abstract

The invention discloses an expression vector, a cell and a vaccine of multivalent coronavirus recombinant protein and a preparation method of the multivalent coronavirus recombinant protein, and relates to the technical field of preparation of protein vaccines of coronavirus. A recombinant protein technical means is utilized to prepare a novel multivalent coronavirus recombinant protein, the novel multivalent coronavirus recombinant protein is prepared into a candidate vaccine, the candidate vaccine can induce an organism to generate immunoreactions aiming at different mutant strains of beta new coronavirus and alpha human coronavirus, and the generated immunoreactions are humoral immunity, cellular immunity and / or mucosal immunity. Therefore, the expression vector and the recombinant cell for expressing the novel multivalent coronavirus recombinant protein, provided by the invention, have a good application prospect. The vaccine containing the multivalent coronavirus recombinant protein has a wider coronavirus protection spectrum, the application range of the vaccine is expanded, and the vaccine has a good immunogenicity enhancing effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein vaccine preparation of human coronavirus, in particular to a multivalent coronavirus recombinant protein expression vector, cell, vaccine and preparation method. BACKGROUND

[0002] Coronaviruses NL63 (alpha species), 229E (alpha species), OC43 (beta species), HKU1 (beta species), SARS (beta species), MERS (beta species), SARS-CoV-2 (beta species) can infect humans, have high pathogenicity to humans, and have a lasting impact on human life and health, but most of them have no vaccine approved for clinical use. Although several vaccines for SARS-CoV-2 have been approved for marketing, SARS-CoV-2 has high variability, making the effectiveness of the marketed vaccines continue to be challenged. Therefore, there is an urgency and necessity for research on human coronavirus multivalent broad-spectrum vaccine technology.

[0003] Coronaviruses are single-stranded RNA viruses, and their spike proteins (S) are located on the surface of the viral membrane and are composed of N-terminal S1 subunits and C-terminal S2 subunits. The receptor binding domain (RBD) in the S1 subunit binds to specific receptors to mediate viral invasion when the virus infects host cells, and is the core target of vaccines. Existing coronavirus multivalent or broad-spectrum vaccines usually use strategies such as mixing multiple antigens, antigen concatenation, or conserved sequences. Some of these strategies for new coronaviruses have been clinically validated for effectiveness, but the effectiveness of multivalent / broad-spectrum vaccines for human coronaviruses remains to be revealed.

[0004] Existing vaccine technology targeting the RBD of the new coronavirus shows that two or three molecules of RBD repeat concatenation or heterogeneous concatenation have certain broad-spectrum in vivo protective effects; human Fc-fused new coronavirus heterogeneous concatenation RBD proteins can effectively induce the production of specific binding and neutralizing antibodies in mice (see patent application No. 202111444421.X). The above studies are only for the new coronavirus, and there are few reports on broad-spectrum vaccines for coronaviruses. Therefore, it is of great scientific significance and urgent market demand to develop broad-spectrum vaccines for coronaviruses.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The present application aims to provide a multivalent coronavirus recombinant protein expression vector, cell, vaccine and preparation method to solve the above technical problems.

[0007] The application utilizes recombinant protein technology to prepare a new type of multivalent coronavirus recombinant protein, prepare a candidate vaccine, and evaluate the immune effect.

[0008] The application is implemented as follows:

[0009] In a first aspect, the application provides an expression vector of a multivalent coronavirus recombinant protein, which comprises a promoter, a secretion signal peptide, a coding sequence of an RBD polypeptide of a spike protein of a coronavirus, a coding sequence of an Fc segment of human, mouse or monkey immunoglobulin IgG1, and a terminator, the coronavirus being at least two selected from SARS, MERS, a novel coronavirus prototype (PT), an Omicron variant, an Alpha (B.1.1.7) variant, a Beta (B.1.351) variant, a Gamma (P.1) variant, a Delta (B.1.617.2) variant, and human coronaviruses OC43, NL63, 229E, HKU1; and the expression vector has, from 5'-3' end, the promoter, the secretion signal peptide, the coding sequence of the RBD polypeptide of the spike protein of the at least one coronavirus, and the coding sequence of the Fc segment of the human, mouse or monkey immunoglobulin IgG1, the RBD polypeptide of the spike protein of the coronavirus being any one selected from an artificially designed RBD soluble antigen protein and an RBD prototype polypeptide of a spike protein of a coronavirus.

[0010] In a second aspect, the application further provides a recombinant cell comprising the expression vector of the multivalent coronavirus recombinant protein described above.

[0011] In a third aspect, the application further provides use of the recombinant cell in preparation of a vaccine for preventing or treating a coronavirus.

[0012] In a fourth aspect, the application further provides a multivalent coronavirus recombinant protein vaccine, which comprises a fusion protein of an RBD polypeptide of a spike protein of a coronavirus and an Fc segment of human, mouse or monkey immunoglobulin IgG1.

[0013] The coronavirus is at least two selected from SARS, MERS, a novel coronavirus prototype (PT), an Omicron variant, an Alpha (B.1.1.7) variant, a Beta (B.1.351) variant, a Gamma (P.1) variant, a Delta (B.1.617.2) variant, and human coronaviruses OC43, NL63, 229E, HKU1; and in the multivalent coronavirus recombinant protein vaccine, one molecule or two molecules of the Fc segment of the human, mouse or monkey immunoglobulin IgG1 are fused to the C-terminus of the RBD polypeptide of the spike protein of the coronavirus in series to form a multivalent coronavirus recombinant protein with a symmetric or asymmetric structure tetramer.

[0014] In a fifth aspect, the present application also provides a method for preparing the above-mentioned multivalent coronavirus recombinant protein, which comprises any one of the following ways:

[0015] (1) transfecting a host cell with the expression vector of the above-mentioned multivalent coronavirus recombinant protein;

[0016] (2) culturing the recombinant cell mentioned above.

[0017] The present application has the following beneficial effects:

[0018] The present application uses recombinant protein technology to prepare a new type of multivalent coronavirus recombinant protein, which is prepared into a candidate vaccine for immune effect evaluation. The evaluation results of the subjects show that it can induce the body to produce an immune response against different mutant strains of beta genus new coronavirus and alpha genus human coronavirus, and the immune response produced is humoral immunity, cellular immunity and / or mucosal immunity. Therefore, the expression vector and the recombinant cell for expressing the new type of multivalent coronavirus recombinant protein provided by the present application have good application prospects, and the expression vector and the recombinant cell can prepare a vaccine against multiple coronaviruses. The vaccine containing the multivalent coronavirus recombinant protein has a wider coronavirus protection spectrum, expands the application range of the vaccine, and has a good effect of enhancing immunogenicity.

[0019] In addition, the various expression vectors of the coronavirus recombinant protein provided by the present application are mixed to prepare a candidate vaccine, which is expected to prevent infection of all current human coronaviruses, and has broad application value. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0021] Figure 1 Structure diagram of the target antigen molecule;

[0022] Figure 2 Experimental results of Fc enhancing respiratory mucosal immune response (A is a structure diagram of the antigen molecule used in the immunogenicity evaluation test, B is the detection results of specific neutralizing antibodies in serum, C left is the detection results of specific IgG in serum, and C right is the detection results of specific IgA in lung lavage fluid (BALF), nasal lavage fluid (Nasal lavage) and serum (Serum));

[0023] Figure 3 Results of cytokine detection;

[0024] Figure 4 Results of specific antibody detection in serum, nasal lavage, and lung lavage using recombinant protein W3681 prepared in Example 2 with different adjuvants and different immunization routes;

[0025] Figure 5 Results of specific neutralizing antibody titer detection;

[0026] Figure 6 Results of mouse weight and survival rate detection;

[0027] Figure 7 Results of tissue virus titer detection;

[0028] Figure 8 Results of vaccine protection test using the vaccine prepared based on the multivalent coronavirus recombinant protein W3230 provided in Example 3;

[0029] Figure 9 Results of vaccine protection test using the vaccine prepared based on the multivalent coronavirus recombinant protein W3402 provided in Example 4;

[0030] Figure 10 Results of vaccine protection test using the vaccine prepared based on the multivalent coronavirus recombinant protein RB2NX1 provided in Example 9. DETAILED DESCRIPTION

[0031] Reference will now be made in detail to embodiments of the present application, one or more examples of which are illustrated in the drawings. Each example is provided by way of explanation of the present application, not limitation thereof. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present application without departing from the scope or spirit of the application. For example, features illustrated or described as part of one embodiment, can be used with another embodiment to yield a still further embodiment.

[0032] In a first aspect, the present application provides an expression vector of a multivalent coronavirus recombinant protein, which comprises a promoter, a secretion signal peptide, a coding sequence of a RBD polypeptide of a spike protein of a coronavirus, a coding sequence of an Fc fragment of a human, murine or monkey immunoglobulin IgG1 and a terminator, the coronavirus being selected from at least two of SARS, MERS, a novel coronavirus prototype (PT), an Omicron variant, an Alpha (B.1.1.7) variant, a Beta (B.1.351) variant, a Gamma (P.1) variant, a Delta (B.1.617.2) variant and human coronaviruses NL63, 229E, OC43 and HKU1; and the expression vector has, from 5'-3' end, the promoter, the secretion signal peptide, the coding sequence of the RBD polypeptide of the spike protein of the coronavirus, the coding sequence of the Fc fragment of the human, murine or monkey immunoglobulin IgG1. The RBD polypeptide of the spike protein of the coronavirus is selected from any one of an artificially designed RBD soluble antigen protein and a RBD prototype polypeptide of a spike protein of a coronavirus.

[0033] The present application provides a new recombinant protein expression strategy, which can meet the efficient expression of a plurality of different coronavirus RBD polypeptides, and provides strong support for preparing vaccines against a plurality of coronaviruses. The Fc fragment of human immunoglobulin IgG1 plays a role in enhancing mucosal immunity, cellular immunity and / or humoral immunity. Experiments have proved that, whether it is muscle injection immunization or nasal immunization, the vaccine with Fc can induce high titers of specific antibodies in serum, nasal lavage fluid and alveolar lavage fluid compared with the vaccine without Fc. In particular, the antibody titer produced by nasal immunization is much higher than that produced by muscle injection immunization. Compared with the vaccine without Fc, the vaccine with Fc can induce higher titers of specific neutralizing antibodies in serum and enhance Th1 and Th2 type cellular immune responses. Through nasal immunization experiments, the Fc fragment can enhance the humoral immune response of the candidate vaccine while enhancing its mucosal immune response.

[0034] The artificially designed RBD soluble antigen protein refers to that the entire sequence of the antigen protein is designed by artificial or artificial intelligence, so as to improve the solubility of the antigen protein for subsequent secretion, purification and the like, or to improve the immunogenicity and conservation of the antigen protein. The artificially designed RBD soluble antigen protein can be completely free of the amino acid sequence of the original RBD.

[0035] In a preferred embodiment of the present application, the 5' end of the coding sequence of the RBD polypeptide of the spike protein of the coronavirus further has a signal peptide coding sequence; the signal peptide is arranged to help improve the yield of the recombinant protein.

[0036] In a preferred embodiment of the application, a protease recognition site sequence is further provided between the coding sequence of the RBD polypeptide of the spike protein of the coronavirus and the coding sequence of the Fc fragment of human immunoglobulin IgG1; the arrangement of the protease recognition site can facilitate the separation of the RBD polypeptide (or the multimeric protein) from the Fc by protease treatment.

[0037] In a preferred embodiment of the application, the Omicron variant is selected from at least one of Omicron BA.2.75, Omicron BA.4 / BA.5, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, Omicron XBB.1.5, Omicron XBB.1.16 and Omicron EG.5 variant;

[0038] Preferably, the human coronavirus is selected from at least one of human coronavirus OC43 and human coronavirus NL63, human coronavirus 229E, human coronavirus HKU1;

[0039] Preferably, the RBD polypeptide sequences of the novel coronavirus prototype (PT), the Alpha (B.1.1.7) variant, the Beta (B.1.351) variant, the Gamma (P.1) variant, the Delta (B.1.617.2) variant, Omicron BA.2.75, Omicron BA.4 / BA.5, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, Omicron XBB.1.5, Omicron XBB.1.16 and Omicron EG.5, human coronavirus OC43, human coronavirus NL63, human coronavirus 229E and human coronavirus HKU1 are sequentially shown in SEQ ID NO: 1-17; the RBD polypeptide sequences of SARS and MERS are shown in SEQ ID NO: 31 and SEQ ID NO: 32, respectively.

[0040] The nucleotide sequence of the expression cassette for expressing the RBD polypeptide of the prototype strain of the novel coronavirus is shown as SEQ ID NO: 18, the nucleotide sequence of the expression cassette for expressing the RBD polypeptide of Omicron XBB.1.5 is shown as SEQ ID NO: 19, the nucleotide sequence of the expression cassette for expressing the RBD polypeptide of the prototype strain of the novel coronavirus and Omicron XBB.1.5 is shown as SEQ ID NO: 20, the nucleotide sequence of the expression cassette for expressing the RBD polypeptide of Omicron BA.2.75 and the prototype strain of the novel coronavirus is shown as SEQ ID NO: 21, the nucleotide sequence of the expression cassette for expressing the RBD polypeptide of Omicron BA.4 / BA.5 and Delta (B.1.617.2) variant is shown as SEQ ID NO: 22, the nucleotide sequence of the expression cassette for expressing the RBD polypeptide of Omicron BA.4 / BA.5 and the prototype strain of the novel coronavirus is shown as SEQ ID NO: 23, the nucleotide sequence of the expression cassette for expressing the RBD polypeptide of human coronavirus NL63 and human coronavirus OC43 is shown as SEQ ID NO: 24, the nucleotide sequence of the expression cassette for expressing the RBD polypeptide of human coronavirus OC43 and human coronavirus NL63 is shown as SEQ ID NO: 25, the nucleotide sequence of the expression cassette for expressing the RBD polypeptide of SARS and MERS is shown as SEQ ID NO: 26, the nucleotide sequence of the expression cassette for expressing the RBD polypeptide of human coronavirus 229E and human coronavirus HKU1 is shown as SEQ ID NO: 27, the nucleotide sequence of the expression cassette for expressing the RBD polypeptide of the prototype strain of the novel coronavirus and Omicron BA.2.75 is shown as SEQ ID NO: 28, the nucleotide sequence of the expression cassette for expressing the RBD polypeptide of human coronavirus NL63 and Omicron XBB.1.16 is shown as SEQ ID NO: 29, the nucleotide sequence of the expression cassette for expressing the RBD polypeptide of Omicron BA.2.75 and Omicron EG.5 is shown as SEQ ID NO: 30.

[0041] The promoter in the expression vector is selected from CMV, U6, H1, SV40, RSV, etc.

[0042] In a second aspect, the present application also provides a recombinant cell comprising the expression vector of the multivalent coronavirus recombinant protein as described above.

[0043] In preferred embodiments of the application, the recombinant cell is selected from a mammalian cell. The mammalian cell is selected from any one of 293 cell, 293T cell, 293FT cell, CHO cell, COS cell, mouse L cell, LNCaP cell, 633 cell, Vero, BHK cell, CV1 cell, Hela cell, MDCK cell, Hep-2 cell and Per6 cell. Among them, 293 series cells, Per6 cells and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins, which are well known to those skilled in the art.

[0044] In preferred embodiments of the application, the recombinant cell is used to form a multivalent coronavirus recombinant protein with a symmetric or asymmetric structure tetramer.

[0045] Preferably, the multivalent coronavirus recombinant protein is a monovalent, bivalent, trivalent or tetravalent coronavirus recombinant protein.

[0046] In a third aspect, the present application also provides a use of a recombinant cell in the preparation of a vaccine for preventing or treating coronavirus.

[0047] In preferred embodiments of the application, the vaccine is used to generate mucosal immunity, cellular immunity and / or humoral immunity in a subject. The vaccine provided by the present application can induce mucosal immune response, cellular immune response and / or humoral immune response in a subject, and especially after the addition of an adjuvant, the immune response of the body can be enhanced.

[0048] The subject includes, but is not limited to, mammals such as humans, monkeys, mice, etc.

[0049] In preferred embodiments of the application, the vaccine is used for at least one immunization mode selected from nasal drop immunization, oral immunization, gavage immunization, enema immunization and injection immunization.

[0050] The vaccine is further prepared as a DNA vaccine, an RNA vaccine, a protein vaccine or a recombinant virus vaccine.

[0051] In a fourth aspect, the present application also provides a multivalent coronavirus recombinant protein vaccine, which comprises: a fusion protein of a RBD polypeptide of a spike protein of a coronavirus and a Fc segment of a human, mouse or monkey immunoglobulin IgG1;

[0052] The coronavirus is selected from at least two of SARS, MERS, a novel coronavirus prototype (PT), an Omicron variant, an Alpha (B.1.1.7) variant, a Beta (B.1.351) variant, a Gamma (P.1) variant, a Delta (B.1.617.2) variant, and human coronaviruses OC43, NL63, 229E, HKU1; and in the multivalent coronavirus recombinant protein vaccine, one molecule of Fc segment of human, murine or monkey immunoglobulin IgG1 is fused to the C-terminus of one molecule or two molecules of RBD polypeptide of the spike protein of the coronavirus in series to form a multivalent coronavirus recombinant protein with a symmetric or asymmetric structure tetramer. The RBD polypeptide of the spike protein of the coronavirus is selected from any one of the following: an artificially designed RBD soluble antigen protein, an RBD prototype polypeptide of the spike protein of the coronavirus

[0053] The Fc segment plays a role in enhancing humoral immunity, cellular immunity and / or mucosal immunity.

[0054] In a preferred embodiment of the application, the N-terminus of the RBD of the spike protein of the coronavirus further comprises a signal peptide.

[0055] In a preferred embodiment of the application, the multivalent coronavirus recombinant protein vaccine further comprises an adjuvant.

[0056] In a preferred embodiment of the application, the multivalent coronavirus recombinant protein and the adjuvant are mixed in a mass ratio of 1:20;

[0057] In a preferred embodiment of the application, the adjuvant is selected from at least one of the following: a Toll-like receptor agonist, a RIG-I-like receptor agonist, a NOD-like receptor agonist, a C-type lectin receptor, a STING agonist, a saponin, a cytokine and other adjuvants; the other adjuvant is selected from at least one of the following: heat shock proteins, A151, MF59, MnJ, RIBI adjuvant, lipopolysaccharide, GTP-GDP, sodium fluoride, aluminum hydroxide adjuvant, alkyl polyacrylate polymer, dimethyl dioctadecyl ammonium bromide, complete Freund's adjuvant, incomplete Freund's adjuvant and AS03 adjuvant.

[0058] In a preferred embodiment of the application, the Toll-like receptor agonist is selected from at least one of the following: CpG-ODN, CpG 1018, peptidoglycan, lipoteichoic acid, MPLA, imiquimod, resiquimod, bacterial flagellin and Poly I:C;

[0059] The RIG-I-like receptor agonist is selected from at least one of the following: 3pRNA and short double-stranded RNA;

[0060] The NOD-like receptor agonist is selected from at least one of muramyl dipeptide and N-acetylglucosamine;

[0061] The C-type lectin receptor is selected from at least one of beta-glucan and fucoidan disulfate;

[0062] The STING agonist is selected from at least one of CF501, cGAMP, c-di-AMP and c-di-GMP;

[0063] The saponin is selected from at least one of QS21, tomatine and Quil-A;

[0064] The cytokine is selected from at least one of GM-CSF, IL-2, IL-12, IL-6, IFN-gamma, Flt-3 and lymphotactin;

[0065] In a preferred embodiment of the application, the STING agonist is selected from CF501 and the other adjuvant is selected from AS03.

[0066] The inventors found that when the adjuvant is selected from the above types, both intramuscular immunization and nasal immunization can induce strong humoral immune responses. Especially when the adjuvant is selected from CF501 or AS03 adjuvant, it has the effect of inducing stronger humoral immune response and mucosal immune response.

[0067] Compared with traditional aluminum adjuvant, the use of CF501 as adjuvant for nasal immunization has stronger IgA secretion at the respiratory mucosa site; the combination of the use of AS03 adjuvant for intramuscular immunization has the highest specific neutralizing antibody titer, that is, it has a stronger humoral immune response.

[0068] In a preferred embodiment of the application, the Omicron variant is selected from at least one of Omicron BA.2.75, Omicron BA.4 / BA.5, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, Omicron XBB.1.5, Omicron XBB.1.16 and Omicron EG.5 variant;

[0069] Preferably, the human coronavirus is selected from at least one of human coronavirus OC43 and human coronavirus NL63, human coronavirus 229E, human coronavirus HKU1.

[0070] In a preferred embodiment of the application, the multivalent coronavirus recombinant protein vaccine is selected from any one of the following:

[0071] (1) N-prototype strain of novel coronavirus RBD polypeptide- RBD polypeptide of at least one Omicron variant-human immunoglobulin IgG1 Fc segment-C;

[0072] (2) N- RBD polypeptide of at least one Omicron variant - RBD polypeptide of prototype strain of SARS-CoV-2 - Fc fragment of human immunoglobulin IgG1 - C;

[0073] (3) N- RBD polypeptide of prototype strain of SARS-CoV-2 - RBD polypeptide of Delta (B.1.617.2) variant - Fc fragment of human immunoglobulin IgG1 - C;

[0074] (4) N- RBD polypeptide of Delta (B.1.617.2) variant - RBD polypeptide of prototype strain of SARS-CoV-2 - Fc fragment of human immunoglobulin IgG1 - C;

[0075] (5) N- RBD polypeptide of human coronavirus OC43 - RBD polypeptide of human coronavirus NL63 - Fc fragment of human immunoglobulin IgG1 - C;

[0076] (6) N- RBD polypeptide of human coronavirus NL63 - RBD polypeptide of human coronavirus OC43 - Fc fragment of human immunoglobulin IgG1 - C;

[0077] (7) N- RBD polypeptide of SARS - RBD polypeptide of MERS - Fc fragment of human immunoglobulin IgG1 - C;

[0078] (8) N- RBD polypeptide of MERS - RBD polypeptide of SARS - Fc fragment of human immunoglobulin IgG1 - C;

[0079] (9) N- RBD polypeptide of human coronavirus NL63 - RBD polypeptide of human coronavirus OC43 - Fc fragment of human immunoglobulin IgG1 - C; and N- RBD polypeptide of at least one Omicron variant - RBD polypeptide of at least one Omicron variant - Fc fragment of human immunoglobulin IgG1 - C;

[0080] (10) N- RBD polypeptide of human coronavirus NL63 - RBD polypeptide of at least one Omicron variant - Fc fragment of human immunoglobulin IgG1 - C; and N- RBD polypeptide of prototype strain of SARS-CoV-2 - RBD polypeptide of at least one Omicron variant - Fc fragment of human immunoglobulin IgG1 - C;

[0081] (11) N- RBD polypeptide of human coronavirus 229E - RBD polypeptide of human coronavirus HKU1 - Fc fragment of human immunoglobulin IgG1 - C.

[0082] In addition, in other embodiments, the multivalent coronavirus recombinant protein vaccine can also be a fusion product of a tandem protein of RBD polypeptides of any two or more coronaviruses and an Fc fragment of human immunoglobulin IgG1.

[0083] In a preferred embodiment of the application, the multivalent coronavirus recombinant protein vaccine is selected from any one of the following:

[0084] (1) N-RBD polypeptide of human coronavirus NL63-RBD polypeptide of human coronavirus OC43-Fc segment-C of human immunoglobulin IgG1; and N-RBD polypeptide of BA.2.75 variant-RBD polypeptide of EG.5 variant-Fc segment-C of human immunoglobulin IgG1.

[0085] (2) N-RBD polypeptide of human coronavirus NL63-RBD polypeptide of XBB.1.16 variant-Fc segment-C of human immunoglobulin IgG1; and N-RBD polypeptide of prototype strain of novel coronavirus-RBD polypeptide of BA.2.75 variant-Fc segment-C of human immunoglobulin IgG1. This vaccine can effectively induce immune responses against four viruses of human coronavirus NL63, XBB.1.16 variant, prototype strain of novel coronavirus and BA.2.75 variant, and exert complete protection against infection of corresponding variants.

[0086] In a preferred embodiment of the application, the sequences of RBD polypeptides of prototype strain of novel coronavirus (PT), alpha (B.1.1.7) variant, beta (B.1.351) variant, gamma (P.1) variant, delta (B.1.617.2) variant, Omicron BA.2.75, Omicron BA.4 / BA.5, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, Omicron XBB.1.5, Omicron XBB.1.16 and Omicron EG.5, human coronavirus OC43, human coronavirus NL63, human coronavirus 229E and human coronavirus HKU1 are shown in SEQ ID NO: 1-17, respectively. The sequences of RBD polypeptides of SARS and MERS are shown in SEQ ID NO: 31 and SEQ ID NO: 32, respectively.

[0087] In a preferred embodiment of the application, the RBD polypeptide of the spike protein of the coronavirus and the Fc segment of human immunoglobulin IgG1 further have a protease enzyme cleavage site.

[0088] In a preferred embodiment of the application, the protease is selected from thrombin (enzyme cleavage site amino acid sequence LVPRGS), enterokinase, TEV protease or HRC-3C protease.

[0089] In a fifth aspect, the present application also provides a method for preparing the above-mentioned multivalent coronavirus recombinant protein, which comprises any one of the following:

[0090] (1) transfecting the host cell with the expression vector of the multivalent coronavirus recombinant protein described above;

[0091] (2) culturing the recombinant cell described above.

[0092] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If specific conditions are not indicated in the embodiments, the conditions are implemented according to conventional conditions or the conditions suggested by the manufacturers. If the manufacturers of the reagents or instruments are not indicated, the reagents or instruments are conventional products that can be purchased in the market.

[0093] The features and performances of the present application will be further described in detail below in combination with the embodiments.

[0094] The present application modularly splices, combines and replaces the receptor binding domain (RBD) of the coronavirus core antigen-spike protein (S), the Fc segment of human immunoglobulin IgG1, and the host cell-specific highly expressed secretory protein signal peptide to form a sequence-optimized nucleotide, which is transformed into Chinese hamster ovary (CHO) cells, can be large-scale expressed and obtain high-purity RBD-Fc fusion protein, and then combined with different vaccine adjuvants to design and screen a multivalent subunit vaccine against multiple coronavirus mutant strains.

[0095] Embodiment 1

[0096] The present embodiment provides a design method of a multivalent coronavirus recombinant protein and an expression process of the recombinant protein. The target protein expressed by the eukaryotic expression system forms a Y-shaped structure in the expression process. The target protein with the Fc segment can be obtained by affinity chromatography purification. The RBD multimeric protein without the Fc segment can be obtained by treating the target protein with the Fc segment with thrombin because the thrombin cleavage site is contained between the RBD and the Fc. The amino acid sequence of the enzyme cleavage site of thrombin is LVPRGS.

[0097] Figure 1 The A figure in the above table is a schematic diagram of the primary structure and the higher structure of different antigen molecules of proteins including the signal peptide (SP), the RBD polypeptide and the Fc segment.

[0098] Specifically, the DNA sequences of one or two key amino acids of RBD are connected, and then connected with the Fc segment of human IgG1, and a secretory signal peptide is added at the N terminus, and cloned into a eukaryotic expression plasmid (pcDNA3.1, ThermoFisher, V79020). Specifically, the expression vector of the multivalent coronavirus recombinant protein includes an expression frame, and the expression frame includes a promoter, a secretory signal peptide, a coding sequence of the RBD polypeptide of the spike protein of the coronavirus, a coding sequence of the Fc segment of the human immunoglobulin IgG1 and a terminator.

[0099] The DNA sequence of the expression frame is inserted between the HindIII (ThermoFisher, FD0504) and BamH I (ThermoFisher, FD0055) sites of pcDNA3.1, the enzyme reaction system is: 5uL of endonuclease buffer, 1uL of each endonuclease, 5ug of DNA, and the rest is supplemented with sterile water to 50uL, 37°C for 1 hour. After the enzyme digestion is completed, the enzyme digestion product is recovered using a DNA recovery kit (omega BIO-TEK, D2500-02). The enzyme digestion product is mixed according to the molar ratio of vector: fragment = 1:3, and T4 ligase (NEB, M0202S) is used for 16°C connection for 12 hours to complete the connection. Among them, the RBD can be derived from different mutant strains, and all nucleotides have been codon optimized for mammalian expression systems. Among them, the RBD sequence can be derived from SARS, MERS, new crown prototype strain (prototype, PT), Omicron variant BA.2.75, BA.4, BA.5, XBB.1.5, XBB.1.16, EG.5, human coronavirus OC43, NL63, HKU1, 229E, new coronavirus prototype strain (prototype, PT), Alpha (B.1.1.7) variant, Beta (B.1.351) variant, Gamma (P.1) variant, Delta (B.1.617.2) variant, Omicron BA.2.75, Omicron BA.4 / BA.5, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, Omicron XBB.1.5, Omicron XBB.1.16 and Omicron EG.5, human coronavirus OC43, human coronavirus NL63, human coronavirus 229E and human coronavirus HKU1 RBD polypeptide sequences are shown in SEQ ID NO: 1-17, respectively. Codon optimization, new coronavirus prototype strain (prototype, PT), Alpha (B.1.1.7) variant, Beta (B.1.351) variant, Gamma (P.1) variant, Delta (B.1.617.2) variant, Omicron BA.2.75, Omicron BA.4 / BA.5, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, Omicron XBB.1.5, XBB.1.16 and Omicron EG.5, human coronavirus OC43, human coronavirus NL63, human coronavirus 229E and human coronavirus HKU1 expression frame nucleotide sequences are shown in any one of SEQ ID NO: 18-27.

[0100] The successfully constructed plasmid is transfected into Chinese hamster ovary (CHO) cells (100mL culture volume), and the experimental scheme is as follows:

[0101] Cell culture: Cells were passaged one day before transfection, 90 ml of 0.85*10 6 cells / ml of suspension cells, to get 2*10 6 cells / ml of cell density for transfection the next day. Incubate overnight at appropriate temperature, rotation speed, CO2 (e.g. 37℃, 125 rpm, 5%).

[0102] Prepare FectoPRO solution and DNA mixture: Warm Fecto Transfection Reagent to room temperature and mix gently. Take 10 ml of serum-free medium opti PRO-SFM to a sterile tube. Add 50 μg of DNA (50 μl of 1 μg / μl concentration), mix gently. Add 100 μl of Fecto Transfection Reagent to a new sterile tube. Add the DNA mixture to the sterile tube containing the Fecto Transfection Reagent, mix well. Incubate at room temperature for 10 minutes to allow complex reformation.

[0103] Add transfection complex to cells in complete medium: Add the transfection reagent and DNA complex to the cells in culture. Place the flask on a shaker incubator (125 rpm amplitude 50 mm) at 37℃ in 5% CO2. 0-4 hours after transfection, add 75 μl of Fecto Booster. 18-24 hours after transfection, transfer the cells from 37℃ to 32℃ for cold culture. After 12 days of culture, collect the culture supernatant for the purpose of protein purification.

[0104] Example 2

[0105] This example provides a multivalent coronavirus recombinant protein, numbered W3681.

[0106] It is a symmetrical bivalent tetramer structure, as shown in B of Figure 1 . The recombinant protein includes: N-terminal-XBB.1.5 RBD-new coronavirus prototype (PT) RBD-Fc segment of human IgG1. The expression frame includes a promoter, a secretion signal peptide, a coding sequence of XBB.1.5 RBD-new coronavirus prototype (PT) RBD, thrombin, Fc segment of human immunoglobulin IgG1, and a terminator. The DNA sequence information of the expression frame is shown as SEQ ID NO: 20, which is connected to the basic vector pcDNA3.1 according to the method of Example 1.

[0107] The DNA sequence encoding the RBD polypeptide is connected to the coding sequence of the Fc segment of human IgG1, and a secretion signal peptide is added to the N terminus, and is cloned into the open reading frame of the eukaryotic expression plasmid pcDNA3.1.

[0108] The protein is expressed according to the method of Example 1. During the expression of the target protein, due to intermolecular interactions, disulfide bonds and other forces, a Y-shaped structure dimer is formed. Protein A and G affinity chromatography column and AKTA automatic purification instrument can be used to obtain the target protein. The purification steps are as follows: the culture solution containing the target protein is filtered through a 0.22 um filter membrane, mixed with 50 mM NaCl, 20 mM NaH2PO4, pH 8.0 PBS at a volume ratio of 1:5, then added to the Protein A and G preloaded affinity chromatography column, 10 times the column volume of PBS is added to wash away the unbound proteins, two times the column volume of 0.2 M, pH 3.0 elution buffer is added to elute the target protein, and 2 M, pH 9.0 neutralization buffer is added to adjust the pH value of the target protein to 7.0.

[0109] The target protein obtained by the above purification is further purified using an AKTA automatic purification instrument.

[0110] Example 3

[0111] This example provides a multivalent coronavirus recombinant protein, numbered W3230.

[0112] It is a symmetric bivalent tetramer structure, refer to Figure 1 The recombinant protein includes: N-terminal-BA.4 / BA.5 RBD-new coronavirus prototype (prototype, PT) RBD-human IgG1 Fc segment. The expression frame includes a promoter, a secretion signal peptide, a BA.4 / BA.5 RBD-new coronavirus prototype (prototype, PT) RBD, a thrombin, a human immunoglobulin IgG1 Fc segment coding sequence and a terminator. The DNA sequence information of the expression frame is shown as SEQ ID NO: 23, which is connected to the basic vector pcDNA3.1 according to the method of Example 1.

[0113] The protein is expressed and purified according to the method of Example 2.

[0114] Example 4

[0115] This example provides a multivalent coronavirus recombinant protein, numbered W3402.

[0116] It is a symmetric bivalent tetramer structure, refer to Figure 1The recombinant protein includes: N-terminal-BA.2.75 RBD-SARS-CoV-2 prototype RBD-human IgG1 Fc segment. The expression cassette includes a promoter, a secretion signal peptide, a coding sequence of BA.2.75 RBD-SARS-CoV-2 prototype RBD, thrombin, and a human immunoglobulin IgG1 Fc segment, and a terminator. The DNA sequence information of the expression cassette is shown as SEQ ID NO: 21, which is connected to the basic vector pcDNA3.1 according to the method of Example 1.

[0117] The expression and purification of the protein were performed according to the method of Example 2.

[0118] Example 5

[0119] This example provides a multivalent coronavirus recombinant protein, numbered W3234.

[0120] It is a symmetric bivalent tetramer structure, referring to Figure 1 The recombinant protein includes: N-terminal-BA.4 / BA.5 RBD-Delta RBD-human IgG1 Fc segment. The expression cassette includes a promoter, a secretion signal peptide, a coding sequence of BA.4 / BA.5 RBD-Delta RBD, thrombin, and a human immunoglobulin IgG1 Fc segment, and a terminator. The DNA sequence information of the expression cassette is shown as SEQ ID NO: 22, which is connected to the basic vector pcDNA3.1 according to the method of Example 1.

[0121] The expression and purification of the protein were performed according to the method of Example 2.

[0122] Example 6

[0123] This example provides a multivalent coronavirus recombinant protein, numbered W3959.

[0124] It is a symmetric bivalent tetramer structure, referring to Figure 1 The recombinant protein includes: N-terminal-OC43 RBD-NL63 RBD-human IgG1 Fc segment. The expression cassette includes a promoter, a secretion signal peptide, a coding sequence of OC43 RBD-NL63 RBD, thrombin, and a human immunoglobulin IgG1 Fc segment, and a terminator. The DNA sequence information of the expression cassette is shown as SEQ ID NO: 24, which is connected to the basic vector pcDNA3.1 according to the method of Example 1.

[0125] The expression and purification of the protein were performed according to the method of Example 2.

[0126] Example 7

[0127] This embodiment provides a multivalent coronavirus recombinant protein, numbered W4084.

[0128] It is a bivalent symmetric tetramer structure, refer to Figure 1 B in the figure. The recombinant protein includes: N-terminal-SARS RBD-MERS RBD-human IgG1 Fc segment. The expression frame includes a promoter, a secretion signal peptide, a coding sequence of SARS RBD-MERS RBD, thrombin, a human immunoglobulin IgG1 Fc segment, and a terminator. The DNA sequence information of the expression frame is shown as SEQ ID NO: 26, which is connected to the basic vector pcDNA3.1 according to the method of Example 1.

[0129] The expression and purification of the protein are carried out according to the method of Example 2.

[0130] Example 8

[0131] This embodiment provides a multivalent coronavirus recombinant protein, numbered W2024.

[0132] It is a bivalent symmetric tetramer structure, refer to Figure 1 B in the figure. The recombinant protein includes: N-terminal-229E RBD-HKU1 RBD-human IgG1 Fc segment. The expression frame includes a promoter, a secretion signal peptide, a coding sequence of 229E RBD-HKU1 RBD, thrombin, a human immunoglobulin IgG1 Fc segment, and a terminator. The DNA sequence information of the expression frame is shown as SEQ ID NO: 27, which is connected to the basic vector pcDNA3.1 according to the method of Example 1.

[0133] The expression and purification of the protein are carried out according to the method of Example 2.

[0134] Example 9

[0135] This embodiment provides a multivalent coronavirus recombinant protein, numbered B2E5NO.

[0136] It is a non-symmetric tetravalent tetramer structure, refer to Figure 1 B in the figure. The recombinant protein includes: N-terminal-NL63 RBD-OC43 RBD-human IgG1 Fc segment; N-terminal-BA.2.75 RBD-EG.5 RBD-human IgG1 Fc segment. Expression frame 1 includes a promoter, a secretion signal peptide, a coding sequence of NL63 RBD-OC43 RBD, thrombin, a human immunoglobulin IgG1 Fc segment, and a terminator. Expression frame 2 includes a promoter, a secretion signal peptide, a coding sequence of BA.2.75 RBD-EG.5 RBD, thrombin, a human immunoglobulin IgG1 Fc segment, and a terminator.

[0137] The DNA sequence information of expression frame 1 is shown as SEQ ID NO: 24, and the DNA sequence information of expression frame 2 is shown as SEQ ID NO: 30, which are connected to the base vector pcDNA3.1 according to the method of Example 1. The protein expression and purification are carried out according to the method of Example 2.

[0138] Example 10

[0139] This example provides a multivalent coronavirus recombinant protein numbered EB2NX1.

[0140] It is an asymmetric tetravalent tetramer structure, as shown in B of Figure 1 The recombinant protein includes: N-terminal - new coronavirus prototype (prototype, PT) RBD - BA.2.75 RBD - Fc segment of human IgG1; N-terminal - NL63 RBD - XBB.1.16 RBD - Fc segment of human IgG1; expression frame 1 includes a promoter, a secretion signal peptide, a coding sequence of new coronavirus prototype (prototype, PT) RBD - BA.2.75 RBD, thrombin, Fc segment of human immunoglobulin IgG1 and a terminator, and expression frame 2 includes a promoter, a secretion signal peptide, a coding sequence of NL63 RBD - XBB.1.16 RBD, thrombin, Fc segment of human immunoglobulin IgG1 and a terminator.

[0141] The DNA sequence information of expression frame 1 is shown as SEQ ID NO: 28, and the DNA sequence information of expression frame 2 is shown as SEQ ID NO: 29, which are connected to the base vector pcDNA3.1 according to the method of Example 1. The protein expression and purification are carried out according to the method of Example 2.

[0142] Comparative Example 1

[0143] This example provides a new coronavirus prototype recombinant protein numbered Prototype RBD (PT RBD), which does not contain an Fc segment, and the protein expression and purification method is the same as that of Example 2.

[0144] Comparative Example 2

[0145] This example provides a new coronavirus prototype recombinant protein numbered W3230 RBD, which is different from Example 3 in that the recombinant protein in this comparative example does not contain an Fc segment, and the protein expression and purification method is the same as that of Example 2.

[0146] Comparative Example 3

[0147] This example provides a new coronavirus prototype recombinant protein numbered Prototype (PT)-Fc, which contains an Fc segment, and the protein expression and purification method is the same as that of Example 2

[0148] Experimental Example 1

[0149] This experimental example explores the effect of the Fc segment on the immunogenicity of the candidate vaccine.

[0150] 1. Preparation of the vaccine

[0151] An appropriate amount of the target protein was mixed with the adjuvant at a mass ratio of 1:20, and 100 μL / animal was used for intramuscular injection immunization and 50 μL / animal was used for nasal cavity immunization. The mixture was diluted to the corresponding volume using PBS.

[0152] 2. Animal immunization

[0153] The vaccine prepared in step 3 was used to immunize the mice at 50 μL / animal. After the mice were anesthetized with isoflurane, the vaccine was slowly dropped into the respiratory tract through the nasal cavity or injected into the thigh muscle. The second dose was administered on day 21 after the first dose. The blood serum was collected by orbital bleeding on day 14 (day 14) and day 28 (day 28) after the first dose.

[0154] 3. Detection of the immunogenicity of the candidate vaccine

[0155] On day 31 after the first dose, the mice were sacrificed by cervical dislocation, the nasal cavity was washed with 200 μL of PBS to collect the nasal lavage fluid, the lungs were washed with 500 μL of PBS to collect the alveolar lavage fluid, and the spleen was ground into dispersed single cells using a screen for standby use.

[0156] ELISA detection of specific antibody titers in serum or lavage fluid: The antigen protein was diluted to 1 μg / mL with the coating solution to coat the ELISA plate, 100 μL of liquid was added to each well, and the plate was coated at 4°C for 12 hours. The coating solution was discarded, and each well was washed with 200 μL of PBST for three times at an interval of 10 min. The PBST was discarded, and 100 μL of 5% skimmed milk powder-containing PBST blocking solution was added to each well for blocking at 37°C for 1 hour. The blocking solution was discarded, and each well was washed with 200 μL of PBST for three times at an interval of 10 min, and then patted dry. The serum or lavage fluid to be tested was diluted to an appropriate dilution with 5% skimmed milk powder-containing PBST, and then added to the ELISA plate, 100 μL of liquid was added to each well, and the plate was incubated at 37°C for 1.5 hours. The liquid was discarded, and each well was washed with 200 μL of PBST for five times at an interval of 10 min, and then patted dry. The ELISA secondary antibody was diluted to an appropriate concentration with 5% skimmed milk powder-containing PBST, and then added to the plate, 100 μL of liquid was added to each well, and the plate was incubated at 37°C for 1 hour. The secondary antibody was discarded, and each well was washed with 200 μL of PBST for five times at an interval of 10 min, and then patted dry. The diluted double-component TMB color developing liquid was added to each well, 100 μL of liquid was added to each well, and the plate was incubated at room temperature for 15 min. After 50 μL of 2M sulfuric acid was added to each well to terminate the reaction, the absorbance value at 450 nm was detected using an enzyme marker.

[0157] The results of specific neutralizing antibody detection in serum of Comparative Example 1 and Comparative Example 3 and Vehicle (the control group of this experimental example refers to the control group without any protein and only containing solvent PBS) Figure 2 In the middle B, Figure 2 In the middle C, the left side is the result of specific IgG detection in serum, Figure 2 In the middle C, the right side is the result of specific IgA detection in lung lavage fluid (BALF), nasal lavage fluid (Nasal lavage) and serum (Serum).

[0158] From the results of specific antibody titer detection in serum (middle C), Figure 2 In the middle C), it can be concluded that whether it is intramuscular immunization (i.m.) or intranasal immunization (i.n.), the vaccine with Fc can induce high titer of specific IgG. From the results of specific antibody titer detection in serum, lavage fluid (Nasal lavage), it can be concluded that compared with the vaccine without Fc, the vaccine with Fc can induce higher titer of specific antibody, and the antibody titer produced by intranasal immunization is much higher than that of intramuscular immunization. From the results of specific neutralizing antibody titer detection in serum (middle B), it can be concluded that compared with the vaccine without Fc, the vaccine with Fc can induce higher titer of specific neutralizing antibody. Figure 2

[0159] In summary, after intranasal immunization, the Fc segment can enhance the humoral immune response of the candidate vaccine and enhance its mucosal immune response.

[0160] This experimental example further carries out cytokine detection, and the detection method uses a cell flow cytometer.

[0161] Cell flow cytometry: 1 mL of spleen cells (density 1 x 10 7 ​The cells were transferred from the 24-well plate to an EP tube and then centrifuged at 2000 RPM for 5 min, and the supernatant was discarded. 10 μL of Zombie Aqua dye was diluted in 10 mL of PBS. Then, 100 μL of the cells were resuspended, and incubated at room temperature for 30 min in the dark. 1 mL of Flow Cytometry Staining Buffer (hereinafter referred to as Buffer) was added, and centrifuged at 2000 RPM for 5 min, and the supernatant was discarded. 100 uL of Buffer was added for resuspension, and 2 μL of Purified Rat Anti-Mouse CD16 / CD32 (Mouse BD Fc Block) (2.4G2) was added, and incubated at 4°C for 15 min. The extracellular antibody Mix was added. After mixing by blowing, it was incubated at 4°C in the dark for 30 min. 1 mL of Buffer was added, and centrifuged at 2000 RPM for 5 min, and the supernatant was discarded. 500 μL of fixing solution (containing 4.2% paraformaldehyde) was added, and vortexed, and then fixed at 4°C in the dark for 30 min. 10x membrane-breaking solution was diluted to 1x with ddH2O, and then 1 mL of the membrane-breaking solution was directly added to the EP tube, and centrifuged at 2000 RPM for 5 min, and the supernatant was discarded. 100 μL of the membrane-breaking solution was added to resuspend the cells, and then the intracellular antibody Mix (2.5 uL of each antibody was added for each sample) was added, and vortexed, and then incubated at 4°C in the dark for 45 min. 1 mL of Buffer was added, and centrifuged at 2000 RPM for 5 min, and the supernatant was discarded. 300 uL of Buffer (or fixing solution) was added to resuspend the cells, and then detected on the machine.

[0162] The recombinant proteins of Comparative Example 1 and Comparative Example 3 were used as stimulants, respectively, and the detection results are shown in Figure 3 As shown, the vaccine with the Fc segment can induce stronger Th1 and Th2 type cellular immune responses.

[0163] Compared with the vaccine without the Fc segment, the vaccine with the Fc segment can produce higher total IgG;

[0164] Compared with the vaccine without the Fc segment, the vaccine with the Fc segment enhances the secretion of IgA on the respiratory mucosa;

[0165] Compared with the vaccine without the Fc segment, the vaccine with the Fc segment enhances the cellular immune response;

[0166] Compared with intramuscular immunization, intranasal immunization enhances the secretion of IgA on the respiratory mucosa.

[0167] Experimental Example 2

[0168] This experimental example investigates the effects of different vaccines, different adjuvants, and different immunization routes on the immune effect of the candidate vaccine. In this experimental example, the recombinant protein of Example 2 is used as an example, aluminum adjuvant, CF501, and AS03 are used as adjuvants, and intramuscular injection immunization and intranasal immunization are performed, respectively, to detect humoral immune response and mucosal immune response. The detection method is the same as the method of detecting specific antibody titer in serum or lavage fluid by ELISA in Experimental Example 2.

[0169] From the results of specific antibody detection in serum ( Figure 4 ), it can be seen that using aluminum adjuvant, CF501, and AS03 as adjuvants, intramuscular injection immunization (i.m.) and intranasal immunization (i.n.) can all induce strong humoral immune response, but intranasal immunization with CF501 adjuvant induces the strongest mucosal immune response among all combinations, followed by intramuscular injection immunization with CF501 adjuvant, and intramuscular injection immunization with AS03 adjuvant and aluminum adjuvant almost cannot induce mucosal immune response. Regardless of the combination, it can induce the production of systemic specific IgG antibodies against both the prototype strain and XBB.1.16.

[0170] Further detection of specific neutralizing antibody titer was performed as follows:

[0171] Serum neutralization titer detection:

[0172] Vero E6 cells were plated in 24-well plates on the first day, and the cell density reached more than 90% on the second day. The immune serum was diluted in 6 gradients with serum-free DMEM, the cell culture medium in the 24-well plate was discarded, 75 μL of diluted serum and 100 PFUs of new coronavirus were added to each well, two replicates for each gradient, and the cells were infected in a 37°C cell incubator for 1 hour. The grinding liquid was discarded, 1 mL of 1% methyl cellulose 2% FBS DMEM medium was added to each well, and the cells were cultured in a 37°C cell incubator for 72 hours. The culture medium in the 24-well plate was discarded, 1 mL of 10% formalin was added to each well, and the cells were fixed at room temperature for 1 hour. The formalin in the plate was discarded, and the plate was placed in a sealed box filled with 75% alcohol. The plate was taken to the biosafety level II laboratory, the alcohol was washed off, 500 μL of 5% crystal violet solution was added to each well, and the cells were stained at room temperature for 15 minutes. The crystal violet staining solution was discarded, the plate was washed, the number of plaques in each well was counted, and the neutralization titer of the serum was calculated.

[0173] The results of the detection of prototype (PT) and XBB.1.16 specific neutralizing antibody titers are shown in Table 2.Figure 5 As shown in the figure, from the detection results of specific neutralizing antibody titers in serum, it can be concluded that the group using AS03 adjuvant muscle immunization has the highest specific neutralizing antibody titer, indicating that it has the strongest humoral immune response. Therefore, compared with the traditional aluminum adjuvant, the use of CF501 as an adjuvant for nasal immunization has the strongest IgA secretion at the respiratory mucosa site.

[0174] Experimental Example 3

[0175] In this experimental example, the recombinant protein of Example 2 is used as an example, CF501 is used as an adjuvant, 1 μg of vaccine is prepared for nasal immunization, and the protective effect of the vaccine is detected.

[0176] 1. The experimental method of the protective effect of the vaccine is as follows: After immunization, the mice are transferred to a biosafety level three laboratory, and the new coronavirus is diluted to the required concentration with serum-free DMEM medium. After the mice are anesthetized with isoflurane, 50 μL of the diluted virus is slowly dropped into the nasal cavity for infection, which is recorded as day 0. After nasal instillation, the body weight and death of the mice are recorded at a fixed time every day until day 14. On day 14, the mice are sacrificed, and the brain, lung, and concha are ground for detection of virus titer.

[0177] The detection results of the body weight and survival rate of the mice are shown in the figure Figure 6 As shown in the figure, after nasal immunization, the mice can completely survive after infection with the virus, while the control group is all dead, indicating that the vaccine can exert complete protection under this condition. Figure 6 PT W3681 i.n. refers to infection with the prototype strain of the new coronavirus, and nasal immunization with the fusion protein vaccine W3681 of the prototype strain of the new coronavirus and the mutant strain, and detection of the body weight and survival rate of the mice; XBB.1.16 W3681 i.n. refers to infection with the XBB.1.16 strain, and nasal immunization with the fusion protein vaccine W3681 of the prototype strain of the new coronavirus and the mutant strain, and detection of the body weight and survival rate of the mice. The detection results of virus titer in tissues Figure 7 ) show that the viral load in the mice receiving vaccine immunization decreases significantly, and on the 14th day of observation, the virus titer has approached the lowest detection line of the experimental system.

[0178] From Experimental Examples 2 and 3, it can be known that no matter which new coronavirus RBD is used as an antigen of the candidate vaccine, effective humoral immunity and mucosal immunity can be induced; using the design strategy of the present application, the candidate vaccine can exert complete protection against different mutant strains of the new coronavirus.

[0179] Experimental Example 4

[0180] For the multivalent coronavirus recombinant protein W3230 provided in Example 3, CF501 is used as an adjuvant to prepare 1 μg of vaccine for nasal immunization, and the protective effect of the vaccine is detected, and the experimental method is the same as that of Experimental Example 3.

[0181] Results Figure 8 As shown, the vaccine W3230 can produce specific antibodies against the corresponding mutant strains after intranasal immunization; the candidate vaccine can induce immune responses against the corresponding mutant strains and exert complete protection against infection of the corresponding mutant strains.

[0182] Experimental Example 5

[0183] The multivalent coronavirus recombinant protein W3402 provided in Example 4 was used as an antigen, CF501 was used as an adjuvant, 1 μg of the vaccine was prepared for intranasal immunization, and the protective effect of the vaccine was detected, and the experimental method was the same as that in Experimental Example 3.

[0184] In this experimental example, BA.2.75 and BA.2 are closely related, and BA.2 is used for infection, and the fusion protein W3402 of BA.2.75+PT is used for immunization, and the vehicle refers to a control containing no protein but only a solvent and an adjuvant.

[0185] Results Figure 9 As shown, the vaccine W3402 can produce specific antibodies against the corresponding mutant strains after intranasal immunization; the candidate vaccine can induce immune responses against the corresponding mutant strains and exert complete protection against infection of the corresponding mutant strains.

[0186] From Experimental Examples 4 and 5, it can be seen that no matter which new coronavirus RBD is used as an antigen of a candidate vaccine, effective humoral immunity and mucosal immunity can be induced; using the design strategy of the application, the candidate vaccine can exert complete protection against different new coronavirus mutant strains.

[0187] Experimental Example 6

[0188] The multivalent coronavirus recombinant protein RB2NX1 (tetravalent tetramer) provided in Example 9 was used as an antigen, CF501 was used as an adjuvant, 1 μg of the vaccine was prepared for intranasal immunization, and the protective effect of the vaccine was detected, and the experimental method was the same as that in Experimental Example 3.

[0189] Results Figure 10 As shown, the vaccine RB2NX1 can effectively induce immune responses against four viruses after intranasal immunization.

[0190] In summary, the design strategy shown in the application can obtain a vaccine against multiple coronaviruses; the vaccine designed in the application can not only induce immune responses against different mutant strains of beta genus new coronaviruses, but also induce immune responses against alpha genus human coronaviruses, thereby expanding the application range of the vaccine.

[0191] The above merely provides the preferred embodiments of the present application, and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the principles and technical scope of the present application shall fall into the scope of the present application.

Claims

1. An expression vector for recombinant multivalent coronavirus proteins, characterized in that, It includes: An expression frame comprising a promoter, a secretory signal peptide, a coding sequence for an RBD polypeptide of a coronavirus spike protein, a coding sequence for the Fc segment of human, mouse, or monkey immunoglobulin IgG1, and a terminator, wherein the coronavirus is selected from at least two of SARS, MERS, the prototype strain (PT) of novel coronavirus, the Omega-Jon variant, the Alpha (B.1.1.7) variant, the Beta (B.1.351) variant, the Gamma (P.1) variant, the Delta (B.1.617.2) variant, and human coronaviruses OC43, NL63, 229E, and HKU1; and wherein the expression vector, from the 5'-3' end, sequentially comprises a promoter, a secretory signal peptide, a coding sequence for at least one RBD polypeptide of a coronavirus spike protein, and a coding sequence for the Fc segment of human, mouse, or monkey immunoglobulin IgG1; wherein the RBD polypeptide of the coronavirus spike protein is selected from any one of the following: an artificially designed RBD soluble antigen protein, or a prototype RBD polypeptide of the coronavirus spike protein.

2. The expression vector for the recombinant multivalent coronavirus protein according to claim 1, characterized in that, The 5' end of the coding sequence of the RBD polypeptide of the spike protein of the coronavirus also has a signal peptide coding sequence. Preferably, the coding sequence of the RBD polypeptide of the coronavirus spike protein also contains a protease recognition site sequence between the coding sequence of the Fc segment of human, mouse or monkey immunoglobulin IgG1. Preferably, the Omeprone mutant strain is selected from at least one of Omeprone BA.2.75, BA.4 / BA.5, Omeprone BA.1, Omeprone BA.2, Omeprone BA.2.12.1, Omeprone XBB.1.5, Omeprone XBB.1.16 and Omeprone EG.5 mutant strains; Preferably, the human coronavirus is selected from at least one of human coronavirus OC43, human coronavirus NL63, human coronavirus 229E, and human coronavirus HKU1; Preferably, the sequences of the RBD peptides of the novel coronavirus prototype (PT), alpha (B.1.1.7) variant, beta (B.1.351) variant, gamma (P.1) variant, delta (B.1.617.2) variant, omeprion BA.2.75, omeprion BA.4 / BA.5, omeprion BA.1, omeprion BA.2, omeprion BA.2.12.1, omeprion XBB.1.5, omeprion XBB.1.16 and omeprion EG.5, human coronavirus OC43, human coronavirus NL63, human coronavirus 229E and human coronavirus HKU1 are shown in SEQ ID NO:1-17 respectively; the sequences of the RBD peptides of SARS and MERS are shown in SEQ ID NO:31 and SEQ ID NO:32 respectively; Preferably, the nucleotide sequence of the expression cassette for expressing the RBD peptide of the novel coronavirus prototype strain is shown in SEQ ID NO:18; the nucleotide sequence of the expression cassette for expressing the RBD peptide of Omeprone XBB.1.5 is shown in SEQ ID NO:19; the nucleotide sequence of the expression cassette for expressing the RBD peptide of the novel coronavirus prototype strain and Omeprone XBB.1.5 is shown in SEQ ID NO:20; the nucleotide sequence of the expression cassette for expressing the RBD peptide of Omeprone BA.2.75 and the novel coronavirus prototype strain is shown in SEQ ID NO:21; the nucleotide sequence of the expression cassette for expressing the RBD peptide of Omeprone BA.4 / BA.5 and Delta (B.1.617.2) variant is shown in SEQ ID NO:22; the nucleotide sequence of the expression cassette for expressing the RBD peptide of Omeprone BA.4 / BA.5 and the novel coronavirus prototype strain is shown in SEQ ID NO:23; and the nucleotide sequence of the expression cassette for expressing the RBD peptide of human coronavirus NL63 and human coronavirus OC43 is shown in SEQ ID NO:

23. As shown in NO:24, the nucleotide sequences of the expression frames for the RBD peptides of the human coronavirus OC43 and human coronavirus NL63 are shown in SEQ ID NO:25; the nucleotide sequences of the expression frames for the RBD peptides of SARS and MERS are shown in SEQ ID NO:26; the nucleotide sequences of the expression frames for the RBD peptides of the human coronavirus 229E and human coronavirus HKU1 are shown in SEQ ID NO:27; the nucleotide sequences of the expression frames for the RBD peptides of the novel coronavirus prototype strain and Omeprone BA.2.75 are shown in SEQ ID NO:28; the nucleotide sequences of the expression frames for the RBD peptides of the human coronavirus NL63 and Omeprone XBB.1.16 are shown in SEQ ID NO:29; and the nucleotide sequences of the expression frames for the RBD peptides of Omeprone BA.2.75 and Omeprone EG.5 are shown in SEQ ID NO:

30.

3. A recombinant cell, characterized in that, It includes the expression vector of the multivalent coronavirus recombinant protein as described in any one of claims 1-2; Preferably, the recombinant cells are selected from mammalian cells; Preferably, the recombinant cells are used to form multivalent coronavirus recombinant proteins having symmetrical or asymmetrical tetramer structures; Preferably, the multivalent coronavirus recombinant protein is a monovalent, bivalent, trivalent, or quadrivalent coronavirus recombinant protein.

4. The use of the recombinant cells as described in claim 3 in the preparation of vaccines for the prevention or treatment of coronaviruses; Preferably, the vaccine is used to induce mucosal immunity, cellular immunity, and / or humoral immunity in test subjects; Preferably, the vaccine is used for at least one immunization method selected from nasal immunization, oral immunization, gavage immunization, enema immunization and injection immunization; Preferably, the vaccine is further prepared as a DNA vaccine, RNA vaccine, protein vaccine, or recombinant viral vaccine.

5. A multivalent coronavirus recombinant protein vaccine, characterized in that, It includes: A fusion protein of the RBD peptide of the coronavirus spike protein with the Fc fragment of human, mouse, or monkey immunoglobulin IgG1; The coronavirus is selected from at least two of SARS, MERS, the prototype strain (PT) of novel coronavirus, Omeprone variant, Alpha (B.1.1.7) variant, Beta (B.1.351) variant, Gamma (P.1) variant, Delta (B.1.617.2) variant, and human coronaviruses OC43, NL63, 229E, and HKU1; and in the multivalent coronavirus recombinant protein vaccine, the Fc segment of a unit molecule of human, mouse, or monkey immunoglobulin IgG1 is fused to the C-terminus of one or two molecules of the RBD polypeptide of the coronavirus spike protein in tandem to form a multivalent coronavirus recombinant protein with a symmetrical or asymmetrical tetramer structure; the RBD polypeptide of the coronavirus spike protein is selected from any one of the following: artificially designed RBD soluble antigen protein, and the RBD prototype polypeptide of the coronavirus spike protein.

6. The multivalent coronavirus recombinant protein vaccine according to claim 5, characterized in that, The N-terminus of the RBD of the spike protein of the coronavirus also includes a signal peptide.

7. The multivalent coronavirus recombinant protein vaccine according to claim 5, characterized in that, The multivalent coronavirus recombinant protein vaccine also includes an adjuvant; Preferably, the multivalent coronavirus recombinant protein and the adjuvant are mixed at a mass ratio of 1:2-20. Preferably, the adjuvant is selected from at least one of the following groups: Toll-like receptor agonists, RIG-I-like receptor agonists, NOD-like receptor agonists, C-type lectin receptors, STING agonists, saponins, cytokines, and other adjuvants; wherein the other adjuvant is selected from at least one of heat shock proteins, A151, MF59, MnJ, RIBI adjuvant, lipopolysaccharide, GTP-GDP, sodium fluoride, aluminum hydroxide adjuvant, alkyl polyacrylate polymers, dimethyl dioctadecyl quaternary ammonium bromide, complete Freund's adjuvant, incomplete Freund's adjuvant, and ASO3 adjuvant; Preferably, the Toll-like receptor agonist is selected from at least one of CpG-ODN, CpG 1018, peptidoglycan, lipoteichoic acid, MPLA, imiquimod, remiquimod, bacterial flagellin, and PolyI:C; The RIG-I-like receptor agonist is selected from at least one of 3pRNA and short double-stranded RNA; The NOD-like receptor agonist is selected from at least one of muramyl dipeptide and N-acetylglucosamine; The C-type lectin receptor is selected from at least one of β-glucan and trehalose diborate; The STING agonist is selected from at least one of cGAMP, c-di-AMP and c-di-GMP; The saponins are selected from at least one of QS21, tomatine and Quil-A; The cytokines are selected from at least one of GM-CSF, IL-2, IL-12, IL-6, IFN-γ, Flt-3, and lymphocyte chemokines; Preferably, the STING agonist is selected from CF501, and the other adjuvant is selected from AS03.

8. The multivalent coronavirus recombinant protein vaccine according to claim 5, characterized in that, The Omeprone mutant strain is selected from at least one of the following: Omeprone BA.2.75, Omeprone BA.4 / BA.5, Omeprone BA.1, Omeprone BA.2, Omeprone BA.2.12.1, Omeprone XBB.1.5, Omeprone XBB.1.16, and Omeprone EG.5 mutant strain; Preferably, the human coronavirus is selected from at least one of human coronavirus OC43, human coronavirus NL63, human coronavirus 229E, and human coronavirus HKU1; Preferably, the multivalent coronavirus recombinant protein vaccine is selected from any one of the following: (1) N-RBD polypeptide of the prototype strain of novel coronavirus - at least one RBD polypeptide of Omecron variant - Fc fragment of human immunoglobulin IgG1 - C. (2) N-at least one RBD polypeptide of Omecron variant - RBD polypeptide of novel coronavirus prototype strain - Fc fragment of human immunoglobulin IgG1 - C. (3) RBD polypeptide of N-novel coronavirus prototype strain - RBD polypeptide of Delta (B.1.617.2) mutant strain - Fc fragment of human immunoglobulin IgG1; (4) RBD peptide of N-delta (B.1.617.2) variant - RBD peptide of novel coronavirus prototype strain - Fc segment of human immunoglobulin IgG1; (5) RBD peptide of N-human coronavirus OC43-RBD peptide of human coronavirus NL63-Fc fragment of human immunoglobulin IgG1-C; (6) RBD peptide of N-human coronavirus NL63-RBD peptide of human coronavirus OC43-Fc fragment of human immunoglobulin IgG1-C; (7) RBD peptide of N-SARS-RBD peptide of MERS-Fc fragment of human immunoglobulin IgG1-C; (8) RBD peptide of N-MERS-RBD peptide of SARS-Fc fragment of human immunoglobulin IgG1-C; (9) N-RBD peptide of human coronavirus NL63-RBD peptide of human coronavirus OC43-Fc fragment of human immunoglobulin IgG1-C; and N-RBD peptide of at least one Omecron variant-RBD peptide of at least one Omecron variant-Fc fragment of human immunoglobulin IgG1-C. (10) The RBD peptide of N-human coronavirus NL63 - at least one RBD peptide of Omecron variant - Fc segment of human immunoglobulin IgG1; and the RBD peptide of N-novel coronavirus prototype strain - at least one RBD peptide of Omecron variant - Fc segment of human immunoglobulin IgG1. (11) RBD polypeptide of N-human coronavirus 229E-RBD polypeptide of human coronavirus HKU1-Fc fragment of human immunoglobulin IgG1-C; Preferably, the multivalent coronavirus recombinant protein vaccine is selected from any one of the following: (1) RBD peptide of N-human coronavirus NL63-RBD peptide of human coronavirus OC43-Fc fragment of human immunoglobulin IgG1-C; and RBD peptide of N-BA.2.75 variant-RBD peptide of EG.5 variant-Fc fragment of human immunoglobulin IgG1-C; (2) The RBD peptide of N-human coronavirus NL63-XBB.1.16 variant-human immunoglobulin IgG1 Fc segment-C; and the RBD peptide of N-novel coronavirus prototype strain-BA.2.75 variant-human immunoglobulin IgG1 Fc segment-C. Preferably, the sequences of the RBD peptides of the novel coronavirus prototype (PT), alpha (B.1.1.7) variant, beta (B.1.351) variant, gamma (P.1) variant, delta (B.1.617.2) variant, omeprion BA.2.75, omeprion BA.4 / BA.5, omeprion BA.1, omeprion BA.2, omeprion BA.2.12.1, omeprion XBB.1.5, omeprion XBB.1.16 and omeprion EG.5, human coronavirus OC43, human coronavirus NL63, human coronavirus 229E and human coronavirus HKU1 are shown in SEQ ID NO:1-17 respectively; the sequences of the RBD peptides of SARS and MERS are shown in SEQ ID NO:31 and SEQ ID NO:32 respectively.

9. The multivalent coronavirus recombinant protein vaccine according to claim 5, characterized in that, The spike protein of coronaviruses also has a protease cleavage site between its RBD polypeptide and the Fc fragment of human immunoglobulin IgG1. Preferably, the protease is selected from thrombin, enterokinase, TEV protease, or HRC-3C protease; Preferably, the amino acid sequence of the thrombin cleavage site is LVPRGS.

10. A method for preparing the multivalent coronavirus recombinant protein according to any one of claims 5-9, characterized in that, It includes any of the following methods: (1) Transfecting host cells with the expression vector of the multivalent coronavirus recombinant protein according to any one of claims 1-2; (2) Cultivate the recombinant cells as described in claim 3.

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