Vaccine composition containing conserved recombinant protein fragment of coronavirus and application thereof

Abstract

The present invention provides chimeric proteins and immunogenic compositions based on coronavirus S2 subunit and N protein useful for the prevention and / or treatment of coronavirus infections, and uses of such chimeric proteins and immunogenic compositions.

Description

Technical Field

[0001] The present invention relates to the fields of immunology and molecular virology, in particular to the prevention and treatment of coronaviruses. Specifically, the present invention relates to chimeric proteins and immunogenic compositions based on coronavirus S2 subunits and N proteins that can be used to prevent and / or treat coronavirus infections, as well as uses of such chimeric proteins and immunogenic compositions. Background Art

[0002] The USAID PREDICT Program 1 (2009-2019) found 113 emerging coronaviruses in humans and animals in areas with high incidence of intensive spillover. Based on the above facts and the emergence of new variants of SARS-CoV-2, the scientific community has proposed a plan to develop a new generation of vaccines with more broad-spectrum protection - a broad-spectrum coronavirus vaccine (Rubin, 2021)(Morens et al., 2022). For the development of the broad-spectrum vaccine, the current focus is on multiple immunogenic antigens / regions based on the spike (S) protein (multivalent) and proteins / designs based on the conserved regions of coronaviruses. Multivalent vaccines require multiple immunodominant regions, and the range is usually limited to variants in these regions (Cohen et al., 2021; Liang et al., 2021; Wang et al., 2022). Another plan based on conserved antigens is to achieve broad-spectrum protection based on the degree of conservation of the antigen and its presentation.

[0003] One of the most relevant viral regions contained in the coronavirus broad-spectrum vaccine is the S2 subunit. The S2 subunit is the most conserved region of the S protein, involved in the fusion process of the virus invading the human body, and contains T cell antigen epitopes. One of the T cell antigen epitopes is conserved in multiple coronaviruses and is associated with a rapid response after infection with SARS-CoV-2, thereby reducing the severity of human infection (Loyal et al., 2021).

[0004] Due to the important function of membrane fusion, the region of the S2 subunit involved in the membrane fusion process is underexposed. In order to induce a significant immune response, the S2 subunit region needs to be presented in a highly immunogenic environment with an appropriate conformation to expose key motifs. However, this is extremely challenging. Studies have shown that no anti-S2 subunit immune response was detected in animals vaccinated with SARS-CoV-2 S2 subunit fragments. Some studies have inserted the key conserved region of the S2 subunit (i.e., fusion peptide, FP) into Escherichia coli with a simplified genome and expressed it on the cell surface. Immunological evaluation of the vaccinated animals found that neither antibodies against the fusion peptide nor cell-mediated immune responses were induced. In addition, although there was a certain alleviation of symptoms after heterologous virus attack, there was no statistical difference in the viral load in the samples tested (Fabris Maeda et al., 2021). Another report indicated that when the HR1-HR2 fragment of the S2 subunit was presented in ferritin particles, significant neutralizing antibodies could be induced only when the HR1-HR2 fragment was presented in combination with the receptor binding domain (RBD) in the same ferritin particle (Ma et al., 2020). Another report was about the immunological evaluation of a recombinant protein of the S2 subunit, which demonstrated that the recombinant protein of the S2 subunit could not induce functional antibodies in mice. In the same study, when the S2 subunit gene was inserted into a DNA vector as a vaccine platform, cell-mediated immune responses and anti-S2 subunit neutralizing antibodies were induced, indicating that the native conformation of the S2 subunit is very important for inducing an appropriate immune response (Ng KW et al, 2021).

[0005] It is well known that the conformation of the pre-fusion form of the S2 subunit is different from the conformation adopted during the fusion process (Fan et al., 2020). The transient fiber conformation of the S2 subunit is immediately involved in the binding of the virus and host cell membranes after the release of the S1 subunit, and exposes key areas during the binding process. Therefore, the transient fiber conformation of the S2 subunit has broad prospects and can be explored as a potential candidate vaccine target. At present, the research difficulties of the fiber structure (fusion form) as a vaccine target have not been overcome.

[0006] Regarding the study of cross-neutralizing antibodies and cell-mediated cross-immune responses induced by candidate vaccines based on the S2 subunit, although the S2 subunit region is conserved, the available information is still very limited. Two recombinant proteins based on the stable form of the S2 subunit of the MERS-CoV S protein failed to induce cross-neutralizing antibodies against SARS-CoV-2, indicating that inducing cross-neutralizing antibodies through recombinant protein constructs is difficult and complicated (Hsieh et al, 2021).

[0007] Mucosal immunity is considered an important way to prevent viral transmission. Most vaccine antigens used to induce mucosal immunity are based on full-length S protein in different vaccine platforms and formulations. However, no relevant studies have demonstrated the ability of intranasal candidate vaccines based on the S2 subunit to induce mucosal immune responses.

[0008] The combination of two conserved regions or two conserved proteins in the same vaccine formulation has not been studied in depth. Such a combination may increase the scope of cross-immune reactions and become an attractive point for the development of vaccine formulations to prevent future zoonotic epidemics caused by new viruses. Summary of the invention

[0009] In view of the above-mentioned defects in the prior art, the present invention aims to provide an immunogenic composition based on a recombinant conserved protein fragment of a coronavirus S2 subunit and an N protein, wherein the immunogenic composition is capable of inducing a humoral immune response and / or a cell-mediated immune response against a beta coronavirus in mice (e.g., in the mucosal system and / or the systemic system).

[0010] Chimeric Protein

[0011] Therefore, in one aspect, the present application provides a chimeric protein comprising a first peptide segment and a second peptide segment, wherein the first peptide segment comprises the sequence of the S2 subunit of the coronavirus S protein or a fragment thereof; the second peptide segment comprises the coronavirus N protein or a fragment thereof;

[0012] Wherein, the S protein and the N protein are derived from the same or different coronaviruses.

[0013] In certain embodiments, the chimeric protein comprises a first peptide segment and a second peptide segment, wherein the first peptide segment comprises the sequence of the S2 subunit of the coronavirus S protein or a fragment thereof, or consists of it; and the second peptide segment comprises the coronavirus N protein or a fragment thereof, or consists of it.

[0014] In certain embodiments, the fragment of the S2 subunit has the immunological activity of the S2 subunit from which it is derived (e.g., it is capable of inducing a humoral immune response and / or a cell-mediated immune response against the coronavirus in a subject (in the mucosal system and / or the systemic system)). In certain embodiments, the fragment of the N protein has the immunological activity of the N protein from which it is derived (e.g., it is capable of inducing a humoral immune response and / or a cell-mediated immune response against the coronavirus in a subject (in the mucosal system and / or the systemic system)).

[0015] In certain embodiments, the first peptide segment comprises at least 100, at least 120, at least 150 or at least 170 consecutive amino acid residues of the amino acid residues at positions corresponding to positions 806-1000 of SEQ ID NO:10 in the S protein; and the second peptide segment comprises at least 50, at least 80 or at least 90 consecutive amino acid residues of the amino acid residues at positions corresponding to positions 255-365 of SEQ ID NO:3 in the N protein.

[0016] In certain embodiments, the first peptide segment comprises the extramembranous region of the S2 subunit or a fragment thereof.

[0017] In certain embodiments, the first peptide segment comprises the same sequence as positions 810-982 (or positions 810-986, or positions 810-1000, or positions 810-1018, or positions 810-1020, or positions 810-1030, or positions 810-1161, or positions 806-982, or positions 806-986, or positions 806-1000, or positions 806-1018, or positions 806-1020, or positions 806-1030) of the S protein as positions 810-982 (or positions 810-986, or positions 810-1000, or positions 810-1018, or positions 806-1020, or positions 806-1030) of SEQ ID NO: 10. 6-1030, or 806-1161, or 800-982, or 800-986, or 800-1000, or 800-1018, or 800-1020, or 800-1030, or 800-1161, or 798-982, or 798-986, or 798-1000, or 798-1018, or No. 798-1020, or No. 798-1030, or No. 798-1161, or No. 790-982, or No. 790-986, or No. 790-1000, or No. 790-1018, or No. 790-1020, or No. 790-1030, or No. 790-1161, or No. 686-982, or No. 686-986, or No. 686-1000 or consisting of the amino acid residues at the corresponding positions of positions 684-1018, or 684-1020, or 684-1030, or 684-1161, or 684-982, or 684-986, or 684-1000, or 684-1018, or 684-1020, or 684-1030, or 684-1161).

[0018] In certain embodiments, the first peptide segment comprises the same sequence as positions 810-982 (or positions 810-986, or positions 810-1000, or positions 810-1018, or positions 810-1020, or positions 810-1030, or positions 806-982, or positions 806-986, or positions 806-1000, or positions 806-1018, or positions 806-1020, or positions 806-1030, or positions 800-982, or positions 800-986, or positions 800-1000, or positions 800-1 018, or 800-1020, or 800-1030, or 798-982, or 798-986, or 798-1000, or 798-1018, or 798-1020, or 798-1030, or 790-982, or 790-986, or 790-1000, or 790-1018, or 790-1020, or 790-1030) or consist of the amino acid residues at the corresponding positions.

[0019] In certain embodiments, the first peptide segment comprises or consists of amino acid residues at positions corresponding to positions 806-1000, or 806-1018, or 798-1000, or 798-1018 of SEQ ID NO:10 in the S protein.

[0020] In certain embodiments, the S protein has: (a) an amino acid sequence as shown in SEQ ID NO: 10 or 13; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 10 or 13; or, (c) a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence shown in SEQ ID NO: 10 or 13.

[0021] In certain embodiments, the first peptide segment comprises an amino acid sequence as shown in SEQ ID NO: 11 or 12, or a sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 11 or 12, or a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions, or consists of the same.

[0022] In certain embodiments, the second peptide segment comprises the peptide sequence of SEQ ID NO: 3's 260-360 (or 260-365, or 260-371, or 260-380, or 255-360, or 255-365, or 255-371, or 255-380, or 250-360, or 250-365, or 250-371, or 250-380, or 248-360, or 248-365, or 248-371, or 248-380, or 240-360, or 240-365, or 240-371, or 240-380) amino acid residues at corresponding positions or consisting of them.

[0023] In certain embodiments, the second peptide segment comprises or consists of amino acid residues at positions corresponding to positions 255-365, or 255-371, or 248-365, or 248-371 of SEQ ID NO: 3 in the N protein.

[0024] In certain embodiments, the N protein has: (a) an amino acid sequence as shown in SEQ ID NO: 2 or 3; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 2 or 3; or, (c) a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0025] In certain embodiments, the second peptide segment comprises an amino acid sequence as shown in SEQ ID NO: 4 or 5, or a sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 4 or 5, or a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions, or consisting of the same.

[0026] In certain embodiments, the first peptide segment comprises the same sequence as positions 810-982 (or positions 810-986, or positions 810-1000, or positions 810-1018, or positions 810-1020, or positions 810-1030, or positions 810-1161, or positions 806-982, or positions 806-986, or positions 806-1000, or positions 806-1018, or positions 806-1020, or positions 806-1030) of the S protein as positions 810-982 (or positions 810-986, or positions 810-1000, or positions 806-1018, or positions 806-1020, or positions 806-1030) of SEQ ID NO: 10. 0, or 806-1161, or 800-982, or 800-986, or 800-1000, or 800-1018, or 800-1020, or 800-1030, or 800-1161, or 798-982, or 798-986, or 798-1000, or 798-1018, or 798-1020 , or 798-1030, or 798-1161, or 790-982, or 790-986, or 790-1000, or 790-1018, or 790-1020, or 790-1030, or 790-1161, or 686-982, or 686-986, or 686-1000, or 686-1018, or 686-1020, or 686-1030, or 686-1161, or 684-982, or 684-986, or 684-1000, or 684-1018, or 684-1020, or 684-1030, or 684-1161) or consists of the amino acid residues at the corresponding positions of SEQ or consisting of the amino acid residues at the corresponding positions of positions 260-360 (or 260-365, or 260-371, or 260-380, or 255-360, or 255-365, or 255-371, or 255-380, or 250-360, or 250-365, or 250-371, or 250-380, or 248-360, or 248-365, or 248-371, or 248-380, or 240-360, or 240-365, or 240-371, or 240-380) of ID NO: 3.

[0027] In certain embodiments, the first peptide segment comprises or consists of amino acid residues at positions corresponding to positions 806-1000, or 806-1018, or 798-1000, or 798-1018 of SEQ ID NO:10 in the S protein; and the second peptide segment comprises or consists of amino acid residues at positions corresponding to positions 255-365, or 255-371, or 248-365, or 248-371 of SEQ ID NO:3 in the N protein.

[0028] In certain embodiments, the S protein and the N protein are each independently derived from a coronavirus selected from the genus Beta.

[0029] In certain embodiments, the S protein and the N protein are each independently derived from a coronavirus selected from the genus Beta, the subgenus Sarbe and the subgenus Merbe.

[0030] In certain embodiments, the S protein and the N protein are each independently derived from SARS-CoV-2, SARS-CoV-1, or MERS-CoV.

[0031] In certain embodiments, the S protein and the N protein are each independently derived from the Delta strain, Wuhan Hu-1 strain, B.1 strain, B.1.1.7 strain, B.1.351 strain, P.1 strain, B.1.671.2 strain, BA.1 strain, BA.2 strain, BA.3 strain, BA.4 / 5 strain, BA.2.12.1 strain, XBB strain, XBB.1.5 strain, XBB.1.16 strain, CH.1.1 strain, XBB.1.9 strain, XBB.2.3 strain, and EG.5.1 strain of SARS-CoV-2.

[0032] In certain embodiments, the S protein and the N protein are derived from the Delta strain of SARS-CoV-2.

[0033] In certain embodiments, the chimeric protein has one or more of the following features:

[0034] (1) The S2 subunit has: (a) an amino acid sequence as shown in SEQ ID NO: 16 or 17; (b) an amino acid sequence having at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence as shown in SEQ ID NO: 16 or 17; or, (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence as shown in SEQ ID NO: 16 or 17;

[0035] (2) the first peptide segment and the second peptide segment are optionally connected via a linker (e.g., a peptide linker, for example, a peptide linker comprising one or more glycine and / or one or more serine); for example, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO: 18;

[0036] (3) The first peptide segment is connected to the N-terminus or C-terminus of the second peptide segment through the peptide linker; in certain embodiments, the first peptide segment is connected to the N-terminus of the second peptide segment through the peptide linker;

[0037] (4) The chimeric protein has: (a) the amino acid sequence as shown in SEQ ID NO:7; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence as shown in SEQ ID NO:7; or (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence as shown in SEQ ID NO:7;

[0038] (5) the conformation of the S2 subunit or its fragment in the chimeric protein is different from the conformation of the pre-fusogenic form; in certain embodiments, the S2 subunit or its fragment in the chimeric protein is in a fibrous conformation (e.g., a fibrous conformation of a fusogenic form or a post-fusion form);

[0039] (6) The chimeric protein exists in the form of a monomer, a dimer, or a multimer (eg, a trimer).

[0040] In certain embodiments, the S2 subunit or fragment thereof in the chimeric protein is in a fibrous conformation (eg, a fusogenic form or a post-fusion form of the fibrous conformation), and the fibrous conformation exists stably in the chimeric protein.

[0041] It is easy for those skilled in the art to understand that during the process of coronavirus infecting host cells, when the RBD located in the S1 subunit in the S protein specifically binds to the corresponding site of the cell surface receptor (ACE2), the S2 subunit is induced to undergo a conformational change, so that it changes from a pre-fusion (pre-fusogenic) conformation to a fusion (fusogenic) form or a post-fusion (post-fusion) form of a fiber conformation (see Huang, Y., Yang, C., Xu, Xf. et al. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacol Sin 41, 1141-1149 (2020). https: / / doi.org / 10.1038 / s41401-020-0485-4). After the S1 subunit is released, the S2 subunit in the fiber conformation directly mediates the fusion of the coronavirus envelope with the host cell membrane. This step is the key to coronavirus infection of host cells.

[0042] Therefore, the chimeric protein provided in the present application comprising the S2 subunit or its fragment in the fiber conformation (for example, the fiber conformation that exists stably) is significantly advantageous for inducing an immune response against coronavirus infection and has broad application prospects.

[0043] Composition

[0044] On the other hand, the present application provides a composition comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the sequence of the S2 subunit of the coronavirus S protein or a fragment thereof; the second polypeptide comprises the coronavirus N protein or a fragment thereof;

[0045] Wherein, the S protein and the N protein are derived from the same or different coronaviruses.

[0046] In certain embodiments, the first polypeptide comprises the sequence of the S2 subunit of the coronavirus S protein or a fragment thereof; and the second polypeptide comprises the coronavirus N protein or a fragment thereof.

[0047] In certain embodiments, the first polypeptide comprises at least 250, at least 300, at least 330 or at least 350 consecutive amino acid residues of the amino acid residues at positions corresponding to positions 712-1204 of SEQ ID NO: 13 in the S protein; and the second polypeptide comprises at least 200, at least 250, at least 300 or at least 320 consecutive amino acid residues of the amino acid residues at positions corresponding to positions 14-365 of SEQ ID NO: 3 in the N protein.

[0048] In certain embodiments, the first polypeptide comprises the extramembranous region of the S2 subunit or a fragment thereof.

[0049] In certain embodiments, the first polypeptide comprises the S protein corresponding to positions 812-1200 (or positions 812-1204, or positions 812-1206, or positions 812-1207, or positions 812-1210, or positions 812-1213, or positions 788-1200, or positions 788-1204, or positions 788-1206, or positions 788-1207, or positions 788-1210) of SEQ ID NO: 13. 1210, or 788-1213, or 720-1200, or 720-1204, or 720-1206, or 720-1207, or 720-1210, or 720-1213, or 712-1200, or 712-1204, or 712-1206, or 712- 1207, or 712-1210, or 712-1213, or 708-1200, or 708-1204, or 708-1206, or 708-1207, or 708-1210, or 708-1213, or 700-1200, or 700-1204, or 700 -1206, or 700-1207, or 700-1210, or 700-1213, or 686-1200, or 686-1204, or 686-1206, or 686-1207, or 686-1210, or 686-1213) or consists of the amino acid residues at the corresponding positions.

[0050] In certain embodiments, the first polypeptide comprises the S protein corresponding to positions 720-1200 (or positions 720-1204, or positions 720-1206, or positions 720-1207, or positions 720-1210, or positions 720-1213, or positions 712-1200, or positions 712-1204, or positions 712-1206, or positions 712-1207, or positions 712-1210, or positions 712-1213, or positions 708-1209) of SEQ ID NO: 13. 1200, or 708-1204, or 700-1206, or 708-1207, or 708-1210, or 708-1213, or 700-1200, or 700-1204, or 700-1206, or 700-1207, or 700-1210, or 700-1213) or consist of the amino acid residues at the corresponding positions.

[0051] In certain embodiments, the first polypeptide comprises or consists of amino acid residues at positions corresponding to positions 712-1204, or 712-1207, or 708-1204, or 708-1207 of the S protein in SEQ ID NO: 13.

[0052] In certain embodiments, the S protein has: (a) an amino acid sequence as shown in SEQ ID NO: 13 or 10; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 13 or 10; or, (c) a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence shown in SEQ ID NO: 13 or 10.

[0053] In certain embodiments, the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 14 or 15, or a sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 14 or 15, or a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions, or consisting thereof.

[0054] In certain embodiments, the second polypeptide comprises amino acid residues at positions corresponding to positions 20-360 (or 20-365, or 20-371, or 20-410, or 20-419, or 14-360, or 14-365, or 14-371, or 14-410, or 14-419, or 10-360, or 10-365, or 10-371, or 10-410, or 10-419, or 1-360, or 1-365, or 1-371, or 1-410) of the N protein of SEQ ID NO: 3, or the second polypeptide comprises or consists of the full-length N protein.

[0055] In certain embodiments, the second polypeptide comprises amino acid residues at positions corresponding to positions 14-365, or 14-419, or 1-365 of SEQ ID NO: 3 in the N protein, or the second polypeptide comprises or consists of the full-length N protein.

[0056] In certain embodiments, the N protein has: (a) an amino acid sequence as shown in SEQ ID NO: 2 or 3; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 2 or 3; or, (c) a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0057] In certain embodiments, the second polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 2-3, 6, or a sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in any one of SEQ ID NOs: 2-3, 6, or a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions.

[0058] In certain embodiments, the first polypeptide comprises the extramembrane region of the S2 subunit or a fragment thereof; and the second polypeptide comprises the amino acid residues at positions corresponding to positions 20-360 (or 20-365, or 20-371, or 20-410, or 20-419, or 14-360, or 14-365, or 14-371, or 14-410, or 14-419, or 10-360, or 10-365, or 10-371, or 10-410, or 10-419, or 1-360, or 1-365, or 1-371, or 1-410) of the N protein in SEQ ID NO:3, or the second polypeptide comprises or consists of the full-length N protein.

[0059] In certain embodiments, the first polypeptide comprises the S protein corresponding to positions 812-1200 (or positions 812-1204, or positions 812-1206, or positions 812-1207, or positions 812-1210, or positions 812-1213, or positions 788-1200, or positions 788-1204, or positions 788-1206, or positions 788-1207, or positions 788-1210) of SEQ ID NO: 13. , or 788-1213th, or 720-1200th, or 720-1204th, or 720-1206th, or 720-1207th, or 720-1210th, or 720-1213th, or 712-1200th, or 712-1204th, or 712-1206th, or 712-1207th, or 71 2-1210, or 712-1213, or 708-1200, or 708-1204, or 708-1206, or 708-1207, or 708-1210, or 708-1213, or 700-1200, or 700-1204, or 700-1206, or 700-1207 7, or 700-1210, or 700-1213, or 686-1200, or 686-1204, or 686-1206, or 686-1207, or 686-1210, or 686-1213) or consists of the amino acid residues at the corresponding positions of SEQ The invention relates to an amino acid residue at a position corresponding to positions 20-360 (or 20-365, or 20-371, or 20-410, or 20-419, or 14-360, or 14-365, or 14-371, or 14-410, or 14-419, or 10-360, or 10-365, or 10-371, or 10-410, or 10-419, or 1-360, or 1-365, or 1-371, or 1-410) of ID NO:3, or, the second polypeptide comprises or consists of the full-length N protein.

[0060] In certain embodiments, the first polypeptide comprises or consists of amino acid residues at positions corresponding to positions 712-1204, or 712-1207, or 708-1204, or 708-1207 of SEQ ID NO: 13 in the S protein; and the second polypeptide comprises amino acid residues at positions corresponding to positions 14-365, or 14-419, or 1-365 of SEQ ID NO: 3 in the N protein, or the second polypeptide comprises or consists of the full-length N protein.

[0061] In certain embodiments, the S protein and the N protein are each independently derived from a coronavirus selected from the genus Beta.

[0062] In certain embodiments, the S protein and the N protein are each independently derived from a coronavirus selected from the genus Beta, the subgenus Sarbe and the subgenus Merbe.

[0063] In certain embodiments, the S protein and the N protein are each independently derived from SARS-CoV-2, SARS-CoV-1, or MERS-CoV.

[0064] In certain embodiments, the S protein and the N protein are each independently derived from the Delta strain, Wuhan Hu-1 strain, B.1 strain, B.1.1.7 strain, B.1.351 strain, P.1 strain, B.1.671.2 strain, BA.1 strain, BA.2 strain, BA.3 strain, BA.4 / 5 strain, BA.2.12.1 strain, XBB strain, XBB.1.5 strain, XBB.1.16 strain, CH.1.1 strain, XBB.1.9 strain, XBB.2.3 strain, and EG.5.1 strain of SARS-CoV-2.

[0065] In certain embodiments, the S protein and the N protein are derived from the Wuhan Hu-1 strain of SARS-CoV-2.

[0066] In certain embodiments, the composition has one or more of the following features:

[0067] (1) The S2 subunit has: (a) an amino acid sequence as shown in SEQ ID NO: 16 or 17; (b) an amino acid sequence having at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence as shown in SEQ ID NO: 16 or 17; or, (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence as shown in SEQ ID NO: 16 or 17;

[0068] (2) the first polypeptide and the second polypeptide are respectively present in different peptide chains;

[0069] (3) The first polypeptide in the composition exists in the form of a monomer, a dimer or a multimer (eg, a trimer).

[0070] In certain embodiments, the first polypeptide and the second polypeptide are linked covalently or non-covalently.

[0071] In certain embodiments, no linkage is formed between the first polypeptide and the second polypeptide.

[0072] In another aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a chimeric protein as described above or a first polypeptide and a second polypeptide as described above.

[0073] In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a chimeric protein as described above.

[0074] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a first polypeptide as described above, and a second nucleotide sequence encoding a second polypeptide as described above.

[0075] In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are located on the same nucleic acid molecule.

[0076] In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are located at different nucleic acid molecules, respectively. In certain embodiments, the nucleic acid molecule of the isolated nucleic acid molecule comprises: (i) a first nucleic acid molecule comprising a first nucleotide sequence encoding a first polypeptide as described above, and, (ii) a second nucleic acid molecule comprising a second nucleotide sequence encoding a second polypeptide as described above.

[0077] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule as described above.

[0078] In another aspect, the present application provides a host cell comprising the isolated nucleic acid molecule or vector as described above.

[0079] On the other hand, the present application provides a method for preparing the chimeric protein as described above or the first polypeptide and the second polypeptide as described above, comprising culturing the host cell as described above under suitable conditions, and recovering the chimeric protein or the first polypeptide and the second polypeptide from the cell culture.

[0080] In another aspect, the present application provides an immunogenic composition comprising the chimeric protein as described above or the composition as described above, and optionally a pharmaceutically acceptable carrier and / or excipient (eg, adjuvant).

[0081] In certain embodiments, the adjuvant is selected from: alum adjuvant, CpG adjuvant (e.g., ODN-39M), aluminum salt adjuvant, zinc-aluminum mixed adjuvant, Freund's adjuvant, oil emulsion adjuvant, cytokine, TLR agonist, nucleic acid adjuvant, liposome, saponin adjuvant, AS01B adjuvant and any combination thereof.

[0082] In certain embodiments, the adjuvant is selected from: alum adjuvant, CpG adjuvant (eg, ODN-39M).

[0083] In certain embodiments, the adjuvant is ODN-39M (eg, ODN-39M as set forth in SEQ ID NO:9).

[0084] In certain embodiments, the immunogenic composition is a vaccine.

[0085] In certain embodiments, the immunogenic composition is administered parenterally (e.g., subcutaneously, intradermally, intramuscularly), mucosally (e.g., respiratory (e.g., intranasal), digestive (e.g., oral), ocular, rectal), or simultaneously parenterally (e.g., subcutaneously, intradermally, intramuscularly) and mucosally (e.g., respiratory (e.g., intranasal), digestive (e.g., oral), ocular, rectal).

[0086] In certain embodiments, the immunogenic composition contains a composition as described above, and a CpG adjuvant (eg, ODN-39M, eg, ODN-39M as set forth in SEQ ID NO:9).

[0087] In certain embodiments, the immunogenic composition contains a composition as described above, and a CpG adjuvant (e.g., ODN-39M, e.g., ODN-39M as shown in SEQ ID NO:9), and the immunogenic composition is administered via a mucosal route (e.g., respiratory route (e.g., intranasal route), digestive route (e.g., oral route), ocular route, rectal route).

[0088] In certain embodiments, the immunogenic composition contains a chimeric protein as described above and a CpG adjuvant (eg, ODN-39M, eg, ODN-39M as set forth in SEQ ID NO:9).

[0089] In certain embodiments, the immunogenic composition contains a chimeric protein as described above and a CpG adjuvant (e.g., ODN-39M, e.g., ODN-39M as shown in SEQ ID NO:9), and the immunogenic composition is administered via a mucosal route (e.g., respiratory route (e.g., intranasal route), digestive route (e.g., oral route), ocular route, rectal route).

[0090] In certain embodiments, the immunogenic composition contains a first immunogenic composition comprising a chimeric protein as described above and a CpG adjuvant (e.g., ODN-39M, e.g., ODN-39M as shown in SEQ ID NO:9), and a second immunogenic composition comprising a chimeric protein as described above and an alum adjuvant; and the first immunogenic composition and the second immunogenic composition are used for combined vaccination (e.g., simultaneous or sequential vaccination).

[0091] In certain embodiments, the immunogenic composition comprises a first immunogenic composition comprising a chimeric protein as described above and a CpG adjuvant (e.g., ODN-39M, e.g., ODN-39M as shown in SEQ ID NO:9), and a second immunogenic composition comprising a chimeric protein as described above and an alum adjuvant; and the first immunogenic composition and the second immunogenic composition are used for combined vaccination (e.g., simultaneous or sequential vaccination), wherein the first immunogenic composition is administered via a mucosal route (e.g., a respiratory route (e.g., intranasal route), a digestive route (e.g., oral route), an ocular route, a rectal route), and the second immunogenic composition is administered via a parenteral route (e.g., subcutaneous route, intradermal route, intramuscular route).

[0092] In certain embodiments, the immunogenic composition is capable of inducing a humoral immune response and / or a cell-mediated immune response against a betacoronavirus in a subject (eg, in the mucosal system and / or the systemic system).

[0093] In certain embodiments, the immunogenic composition has one or more characteristics selected from the group consisting of:

[0094] (1) inducing the production of antibodies (e.g., binding antibodies, cross-binding antibodies, neutralizing antibodies and / or cross-neutralizing antibodies) against coronavirus (e.g., beta coronavirus, such as Sarbe subgenus and / or Merbe subgenus coronavirus) S protein (e.g., S2 subunit) in the subject (e.g., in the mucosal system and / or systemic system);

[0095] (2) inducing a cell-mediated immune response and / or a cell-mediated cross-immune response against the S protein (e.g., S2 subunit) of a coronavirus (e.g., a beta coronavirus, such as a Sarbe subgenus and / or a Merbe subgenus coronavirus) in a subject (e.g., in the mucosal system and / or the systemic system);

[0096] (3) inducing the production of antibodies (e.g., binding antibodies and / or cross-binding antibodies) against the N protein of a coronavirus (e.g., a beta coronavirus, such as a Sarbe subgenus and / or a Merbe subgenus coronavirus) in a subject (e.g., in the mucosal system and / or the systemic system);

[0097] (4) inducing a cell-mediated immune response and / or a cell-mediated cross-immune response against the N protein of a coronavirus (e.g., a beta coronavirus, such as a Sarbe subgenus and / or a Merbe subgenus coronavirus) in a subject (e.g., in the mucosal system and / or the systemic system);

[0098] (5) Inducing a Th1-biased immune response in the subject (e.g., in the mucosal system and / or the systemic system); for example, the immune response is an immune response against the S protein (e.g., S2 subunit) and / or N protein of a coronavirus (e.g., a β coronavirus, such as a Sarbe and / or Merbe subgenus coronavirus).

[0099] On the other hand, the present application provides a method for preparing an immunogenic composition, which comprises mixing the chimeric protein as described above or the first polypeptide and the second polypeptide as described above with a pharmaceutically acceptable carrier and / or excipient; optionally, the method further comprises mixing an adjuvant, and / or additional active ingredients, such as additional active ingredients that can prevent or treat coronavirus infection or diseases caused by coronavirus infection.

[0100] In certain embodiments, the method comprises admixing a chimeric protein as described above with an adjuvant and a pharmaceutically acceptable carrier and / or excipient.

[0101] In certain embodiments, the method comprises mixing the first polypeptide and the second polypeptide as described above with an adjuvant and a pharmaceutically acceptable carrier and / or excipient.

[0102] In certain embodiments, the adjuvant is selected from: alum adjuvant, CpG adjuvant (e.g., ODN-39M), aluminum salt adjuvant, zinc-aluminum mixed adjuvant, Freund's adjuvant, oil emulsion adjuvant, cytokine, TLR agonist, nucleic acid adjuvant, liposome, saponin adjuvant, AS01B adjuvant and any combination thereof.

[0103] In certain embodiments, the adjuvant is selected from: alum adjuvant, CpG adjuvant (eg, ODN-39M).

[0104] In certain embodiments, the adjuvant is ODN-39M (eg, ODN-39M as set forth in SEQ ID NO:9).

[0105] In certain embodiments, when the adjuvant is a CpG adjuvant (eg, ODN-39M), the method comprises: mixing the second polypeptide as described above with the CpG adjuvant (eg, ODN-39M), and then mixing with the first polypeptide.

[0106] On the other hand, the present application provides the use of the chimeric protein, or composition, or isolated nucleic acid molecule, or vector, or host cell, or immunogenic composition as described above in the preparation of a medicament for inducing an immune response against coronavirus in a subject and / or for preventing and / or treating coronavirus infection or a disease associated with coronavirus infection in a subject.

[0107] In certain embodiments, the medicament is a vaccine.

[0108] In certain embodiments, the coronavirus is a beta coronavirus, such as a Sarbe and / or Merbe subgenus coronavirus.

[0109] In certain embodiments, the coronavirus is SARS-CoV-2, SARS-CoV-1, or MERS-CoV.

[0110] In certain embodiments, the disease associated with coronavirus infection is COVID-19.

[0111] In certain embodiments, the subject is a mammal, such as a human.

[0112] On the other hand, the present application provides a method for inducing an immune response against a coronavirus in a subject and / or for preventing and / or treating a coronavirus infection or a disease associated with a coronavirus infection in a subject, comprising: administering an effective amount of the chimeric protein, or composition, or isolated nucleic acid molecule, or vector, or host cell, or immunogenic composition as described above to a subject in need thereof.

[0113] In certain embodiments, the coronavirus is a beta coronavirus, such as a Sarbe and / or Merbe subgenus coronavirus.

[0114] In certain embodiments, the coronavirus is SARS-CoV-2, SARS-CoV-1, or MERS-CoV.

[0115] In certain embodiments, the disease associated with coronavirus infection is COVID-19.

[0116] In certain embodiments, the subject is a mammal, such as a human.

[0117] Definition of terms

[0118] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the virology, biochemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0119] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0120] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0121] As used herein, the term "coronavirus" has a meaning commonly understood by those skilled in the art, and generally refers to a member of the coronavirus subfamily (Coronavirinae) belonging to the Coronaviridae family (Coronaviridae). Non-limiting examples of the coronavirus include: SARS-related coronaviruses (e.g., SARS-Cov-1, SARS-Cov-2), MERS-related coronaviruses (MERS-Cov). As used herein, the term "S protein" refers to the coronavirus spike protein (Spikeprotein), which belongs to the type I transmembrane protein, composed of two parts, S1 and S2 protein subunits, mediating the binding of the virus to the host cell surface receptor and the fusion of the cell membrane.

[0122] Those skilled in the art understand that in the amino acid sequence of the S protein (including the sequences of the S1 subunit and the S2 subunit), mutations or variations (including but not limited to, substitutions, deletions and / or additions, such as S proteins of different strains of coronavirus) may be naturally generated or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "S protein" shall include all S proteins corresponding to such sequences, for example, including the S protein shown in SEQ ID NO: 10 or 13 and its natural or artificial variants. Furthermore, when describing a sequence fragment or amino acid position of the S protein, it includes not only the sequence fragment or amino acid position of SEQ ID NO: 10 or 13, but also the corresponding sequence fragment or amino acid position in its natural or artificial variant.

[0123] In the present invention, when referring to the sequence fragment of the S protein or its amino acid position, the description is made with reference to the sequence shown in SEQ ID NO: 10 or 13. For example, the expression "amino acid residues at positions corresponding to positions 806-1000 of SEQ ID NO: 10 in the S protein" means the amino acid sites / residues in the compared sequence that are at equivalent positions to the amino acid residues 806-1000 of SEQ ID NO: 10 when the sequence is optimally aligned with SEQ ID NO: 10, i.e. when the sequence is aligned with SEQ ID NO: 10 to obtain the highest percentage identity.

[0124] In the present invention, the term "S2 subunit" or "S2 protein" has the same meaning and can be used interchangeably, and generally refers to the S2 subunit at the carboxyl terminus (C terminus) of the S protein. The S2 subunit is inserted into the viral envelope in a handle-like shape and is responsible for mediating the membrane fusion of the virus and the host cell after the S protein S1 subunit (i.e., the subunit at the amino terminus (N terminus) of the S protein) binds to the host cell membrane surface receptor. The sequence of the S2 subunit is separated from the S1 subunit by the Furin cleavage site in the S protein.

[0125] The amino acid sequence of the S2 subunit is well known to those skilled in the art, and generally includes a fusion peptide (FP) sequence, two heptad repeats (HR), a central helix (CH), a connector domain (CD), a transmembrane domain (TD) and a cytoplasm domain (CD). In certain embodiments, the amino acid sequence of the S2 subunit corresponds to the sequence of amino acid residues at positions corresponding to positions 684-1271 (or 686-1271) of SEQ ID NO: 10 in the S protein. In certain embodiments, the amino acid sequence of the S2 subunit corresponds to the sequence of amino acid residues at positions corresponding to positions 686-1273 of SEQ ID NO: 13 in the S protein.

[0126] In this article, the term "S2 subunit extracellular region" or "S2 protein extracellular region", also known as the S2 subunit / S2 protein extracellular domain (Ectodomain), refers to a fragment of the S2 subunit that does not contain a cytoplasm region (Cytoplasm Domain) and a transmembrane domain (Transmembrane Domain). In certain embodiments, the amino acid sequence of the S2 subunit extracellular region corresponds to the sequence of amino acid residues at positions corresponding to positions 684-1204 (or 686-1204) of SEQ ID NO: 10 in the S protein. In certain embodiments, the amino acid sequence of the S2 subunit extracellular region corresponds to the sequence of amino acid residues at positions corresponding to positions 686-1206 (or 686-1207, or 686-1213) of SEQ ID NO: 13 in the S protein. It is easy for those skilled in the art to understand that based on the different algorithms, standards or methods for transmembrane region prediction / verification, the C-terminal residue range of the extracellular region of the S2 subunit reported in different studies may differ by one to several amino acid residues.

[0127] As used herein, the term "N protein" refers to the coronavirus nucleocapsid protein, which can recognize viral RNA and package it into a ribonucleoprotein (RNP) complex, and participate in multiple processes such as viral transcription, replication, and immune regulation by binding to viral or host proteins.

[0128] Those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and / or additions, such as N proteins of different strains of coronavirus) may be naturally or artificially introduced into the amino acid sequence of the N protein without affecting its biological function. Therefore, in the present invention, the term "N protein" shall include all N proteins corresponding to such sequences, for example, including N proteins as shown in SEQ ID NO: 2 or 3 and natural or artificial variants thereof. Furthermore, when describing a sequence fragment or amino acid position of the N protein, it includes not only the sequence fragment or amino acid position of SEQ ID NO: 2 or 3, but also the corresponding sequence fragment or amino acid position in its natural or artificial variant.

[0129] In the present invention, when referring to a sequence fragment of the N protein or its amino acid position, the description is made with reference to the sequence shown in SEQ ID NO: 3. For example, the expression "amino acid residues at positions corresponding to positions 255-365 of the N protein in SEQ ID NO: 3" means the amino acid sites / residues in the compared sequence that are at equivalent positions to amino acid residues 255-365 of SEQ ID NO: 3 when the sequence is optimally aligned with SEQ ID NO: 3, i.e., when the sequence is aligned with SEQ ID NO: 3 to obtain the highest percentage identity.

[0130] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0131] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0132] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0133] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0134] Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the desired expression level, etc. A vector can be introduced into a host cell to thereby produce transcripts, proteins, or peptides, including proteins, isolated nucleic acid molecules, etc. as described herein.

[0135] In the present invention, the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0136] In the present invention, unless otherwise specified herein or clearly contradicted by the context, the terms "chimeric protein" and "fusion protein" have the same meaning and can be used interchangeably.

[0137] Advantageous Effects of the Invention

[0138] Compared with existing marketed vaccines and vaccines under development, the vaccine composition provided by the present invention has the following advantages:

[0139] (1) It can quickly induce cell-mediated cross-immune responses against conserved antigens such as the N protein and S protein (e.g., S2 protein) of coronaviruses (e.g., beta coronaviruses, such as SARS-CoV-2), that is, it can induce a broad-spectrum cell-mediated immune response.

[0140] Cell-mediated immune responses can effectively resist coronavirus infections including SARS-CoV-2, and the results of studies on SARS-CoV-1 infection show that the protective effect of the cell-mediated immune response induced by it can last up to 17 years. Therefore, the cell-mediated cross-immune response against N and S2 proteins induced by the S2ND preparation supplemented with appropriate mucosal adjuvants and the N+S2 protein combination preparation provided in this application has potential protective ability, which can at least resist infection by β-coronaviruses.

[0141] (2) Induce cross-humoral immune responses against S protein (e.g., S2 protein) and N protein with broad-spectrum neutralizing ability.

[0142] Neutralizing antibodies against S2 protein can inhibit the fusion process, which promotes infection in cells. The neutralizing antibodies have a protective effect in animal models and are correlated with the asymptomatic stage after natural infection in humans. At the same time, studies have also shown that induced anti-N protein antibodies also have a protective effect in animal models. The antigen-specific humoral immune response (neutralizing reaction against S2 and N proteins) induced by the S2ND preparation and the N+S2 protein combination preparation provided in the present application further expands its broad spectrum of resistance to coronavirus (e.g., beta coronavirus) infection.

[0143] (3) In particular, when inoculated via a mucosal route (e.g., intranasal route), the S2ND preparation and N+S2 protein combination preparation provided in the present application are particularly effective in inducing an immune response (e.g., a cell-mediated immune response as described in (1) and / or a humoral immune response as described in (2)). Based on the important role of mucosal immune response in blocking viral transmission, it is crucial to induce the above-mentioned cross-immune response in the mucosal system, which is a key advantage of the vaccine composition of the present invention. In addition, the intranasal route is more feasible in actual operation and can avoid related problems caused by injection administration. Therefore, it is particularly suitable for large-scale vaccination.

[0144] (4) In addition, it should be emphasized that the S2ND preparation and the N+S2 protein combination preparation provided in the present application have the immunogenicity of both N protein and S2 protein. Therefore, as a single vaccine preparation (for example, without the need to be used directly in combination with other vaccines), it can induce the above-mentioned immune response against the N protein and S2 protein of coronavirus (for example, beta coronavirus).

[0145] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] Figure 1a : Analysis of expression and purification of cloned S2ND protein; samples were analyzed by 12% SDS-PAGE experiment under reducing conditions; 1) molecular weight standard reference (MWM), 2) BL21 (DE3) whole cells expressing S2ND protein, 3) BL21 (DE3) whole cells (negative control), 4) purified S2ND protein.

[0147] Figure 1b : The expression and purification of the cloned S2ND protein were analyzed; samples were analyzed by western blotting (WB) under reducing conditions using an anti-N protein polyclonal antibody (40588-T62, purchased from Sino Biological Inc., Beijing, China); 1) MWM, 2) BL21(DE3) whole cells expressing S2ND protein, 3) BL21(DE3) whole cells (negative control), 4) purified S2ND protein.

[0148] Figure 2a : Analysis of expression and purification of cloned S2 protein; samples were analyzed by 12% SDS-PAGE under reducing conditions; 1) MWM, 2) HEK-293T cells (negative control), 3) HEK-293T-S2 cells, 4) HEK-293T cell culture supernatant (negative control), 5) HEK-293T-S2 cell culture supernatant, 6) purified S2 protein.

[0149] Figure 2b : The expression and purification of the cloned S2 protein were analyzed; the samples were analyzed by WB under reducing conditions using an anti-S2 protein polyclonal antibody (40590-T62, purchased from Sino Biological Technology Co., Ltd., Beijing, China); 1) MWM, 2) HEK-293T cells (negative control), 3) HEK-293T-S2 cells, 4) HEK-293T cell culture supernatant (negative control), 5) HEK-293T-S2 cell culture supernatant, 6) purified S2 protein.

[0150] Figure 3a: Analysis of expression and purification of cloned N protein; samples were analyzed by 12% SDS-PAGE under reducing conditions; 1) MWM, 2) BL21(DE3) whole cells (negative control), 3) BL21(DE3) whole cells expressing N protein, 4) purified N protein.

[0151] Figure 3b : The expression and purification of the cloned N protein were analyzed; samples were analyzed by WB under reducing conditions using an anti-N protein polyclonal antibody (40588-T62, purchased from Sino Biological Inc., Beijing, China); 1) MWM, 2) BL21(DE3) whole cells (negative control), 3) BL21(DE3) whole cells expressing N protein, 4) purified N protein.

[0152] Figure 4 : Recognition of recombinant proteins (S2ND protein, S2 protein, and N protein) by anti-SARS-CoV-2 sera in recovered volunteers infected with SARS-CoV-2.

[0153] Figure 5a : Recognition of recombinant S2ND protein with or without ODN-39M by anti-SARS-CoV-2 sera from recovered volunteers infected with SARS-CoV-2.

[0154] Figure 5b : Recognition of recombinant N protein with or without ODN-39M by anti-SARS-CoV-2 sera from recovered volunteers infected with SARS-CoV-2.

[0155] Figure 6 : The results of the anti-S2ND protein IgG ELISA test on the serum of immunized Balb / C mice were used to detect the immunogenicity of the S2ND preparation; after logarithmic transformation of the titer, one-way analysis of variance (One Way Anova) and Tukeys multiple comparison method were used for statistical analysis.

[0156] Figure 7 : The immunogenicity of S2ND preparation was tested by anti-S2 protein IgG ELISA test on the serum of immunized Balb / C mice; after logarithmic transformation of titers, statistical analysis was performed using One Way Anova method and Tukeys multiple comparison method.

[0157] Figure 8: The immunogenicity of S2ND preparation was tested by anti-N protein IgG ELISA test on the serum of immunized Balb / C mice; after logarithmic transformation of titers, statistical analysis was performed using One Way Anova method and Tukeys multiple comparison method.

[0158] Figure 9a : The detection results of IgG subclass antibodies in the serum of immunized Balb / C mice were evaluated by anti-N protein IgG1 ELISA test; after logarithmic transformation of the titers, One Way Anova method and Tukeys multiple comparison method were used for statistical analysis.

[0159] Figure 9b : The detection results of IgG subclass antibodies in the serum of immunized Balb / C mice were evaluated by anti-N protein IgG2a ELISA test; after logarithmic transformation of the titers, one-way analysis of variance (One Way Anova) and Tukeys multiple comparison method were used for statistical analysis.

[0160] Fig.10 The results of the humoral immune response induced by S2ND preparation were evaluated by anti-N protein IgA ELISA test on bronchoalveolar lavage fluid (BALF) (undiluted) of immunized Balb / C mice, and the Kruskal-Wallis nonparametric test and Dunns multiple comparison test were used for statistical analysis.

[0161] Fig.11 :Through the conserved peptide N 351-365 The IFNγ-ELISPOT assay after stimulating spleen cells was used to detect the cell-mediated immune response induced by S2ND preparation in mice. The results were statistically analyzed using the Kruskal-Wallis nonparametric test and Dunns multiple comparison test.

[0162] Fig.12 : The anti-S2 protein IgG ELISA test was performed on the sera of immunized C-57 / BL-6 mice to detect the immunogenicity of S2ND preparations and NO+S2 preparations. After logarithmic transformation of the titers, statistical analysis was performed using the One Way Anova method and Tukeys multiple comparison method.

[0163] Fig.13a : The detection results of IgG subclass antibodies in the serum of immunized C-57 / BL-6 mice were evaluated by anti-S2 protein IgG1 ELISA test; after logarithmic transformation of the titers, One Way Anova method and Tukeys multiple comparison method were used for statistical analysis.

[0164] Fig.13b : The detection results of IgG subclass antibodies in the serum of immunized C-57 / BL-6 mice were evaluated by anti-S2 protein IgG2a ELISA test; after logarithmic transformation of the titers, One Way Anova method and Tukeys multiple comparison method were used for statistical analysis.

[0165] Fig.14 : The results of the humoral immune response induced by S2ND preparations and S2 preparations in the mucosal system were evaluated by performing anti-S2 protein IgA ELISA test on BALF (undiluted) of immunized C-57 / BL-6 mice; the Kruskal-Wallis nonparametric test and Dunns multiple comparison test were used for statistical analysis.

[0166] Fig.15 : The immunogenicity of S2ND and S2 preparations was evaluated by anti-N protein IgG ELISA test on the sera of immunized C-57 / BL-6 mice; after logarithmic transformation of the titers, statistical analysis was performed using the One Way Anova method and Tukeys multiple comparison method.

[0167] Fig.16a : The results of IgG subclass antibody detection in the serum of immunized C-57 / BL-6 mice were evaluated by anti-N protein IgG1 ELISA test; after logarithmic transformation of titers, One Way Anova method and Tukeys multiple comparison method were used for statistical analysis.

[0168] Fig.16b : The results of IgG subclass antibody detection in the serum of immunized C-57 / BL-6 mice were evaluated by anti-N protein IgG2a ELISA test; after logarithmic transformation of titers, One Way Anova method and Tukeys multiple comparison method were used for statistical analysis.

[0169] Fig.17 : The results of the humoral immune response induced by S2ND preparations and S2 preparations in the mucosal system were evaluated by performing anti-N protein IgA ELISA test on BALF (undiluted) of immunized C-57 / BL-6 mice; the Kruskal-Wallis nonparametric test and Dunns multiple comparison test were used for statistical analysis.

[0170] Fig.18a: The cell-mediated immune response induced by S2ND preparations and S2 preparations in C-57 / BL-6 mice was detected by IFNγ-ELISPOT assay after spleen cells were stimulated with S2 protein. The results were analyzed statistically using Kruskal-Wallis nonparametric test and Dunns multiple comparison test.

[0171] Fig.18b : The cell-mediated immune response induced by S2ND preparation and S2 preparation in C-57 / BL-6 mice was detected by IFNγ-ELISPOT assay after splenocytes were stimulated with N protein. The Kruskal-Wallis nonparametric test and Dunns multiple comparison test were used for statistical analysis.

[0172] Fig.19 : To evaluate the cross-reaction of IgG antibodies with SARS-CoV-2S2 protein, SARS-CoV-1S2 protein, and MERS-CoV S2 protein in the serum of group C inoculated with NO+S2 preparation via intranasal route; after logarithmic transformation of titers, OneWay Anova method and Tukeys multiple comparison method were used for statistical analysis.

[0173] Fig.20a : To evaluate the cross-reaction of IgG antibodies in the serum of group B that received intranasal administration of S2NDO preparation with SARS-CoV-2Delta variant N protein, SARS-CoV-2Omicron variant N protein, and SARS-CoV-1N protein; the Kruskal-Wallis nonparametric test and Dunns multiple comparison test were used for statistical analysis.

[0174] Fig.20b : The results of cross-reaction of IgG antibodies in the serum of group C that received NO+S2 preparation via intranasal route with SARS-CoV-2Delta variant N protein, SARS-CoV-2Omicron variant N protein, and SARS-CoV-1N protein were evaluated; the Kruskal-Wallis nonparametric test and Dunns multiple comparison test were used for statistical analysis.

[0175] Fig.21: The anti-S2 protein (SARS-CoV-2S2 protein, SARS-CoV-1S2 protein and MERS-CoV S2 protein) IgA ELISA test was performed on the BALF (undiluted) of group C that was inoculated with NO+S2 preparations via intranasal route to evaluate the detection results of humoral cross-immune response induced by intranasal inoculation of NO+S2 preparations in the mucosal system; the Kruskal-Wallis nonparametric test and Dunns multiple comparison test were used for statistical analysis.

[0176] Fig.22a : The anti-N protein (SARS-CoV-2Delta variant N protein, SARS-CoV-2Omicron variant N protein and SARS-CoV-1N protein) IgA ELISA test was performed on the BALF (undiluted) of group B that was inoculated with S2NDO preparations via intranasal route to evaluate the detection results of humoral cross-immune response induced by S2NDO preparations in the mucosal system; the Kruskal-Wallis non-parametric test and Dunns multiple comparison test were used for statistical analysis.

[0177] Figure 22b : The anti-N protein (SARS-CoV-2Delta variant N protein, SARS-CoV-2Omicron variant N protein and SARS-CoV-1N protein) IgA ELISA test was performed on the BALF (undiluted) of group C that was inoculated with NO+S2 preparations via intranasal route to evaluate the detection results of humoral cross-immune response induced by intranasal inoculation of NO+S2 preparations in the mucosal system; the Kruskal-Wallis nonparametric test and Dunns multiple comparison test were used for statistical analysis.

[0178] Fig.23a : The sera of Group B that were inoculated with S2NDO preparations via the intranasal route were tested using the vesicular stomatitis virus (VSV) pseudovirus system (SARS-CoV-2Delta variant S-VSV, SARS-CoV-2Omicron variant S-VSV, SARS-CoV-1S-VSV, and MERS-CoV S-VSV) to evaluate the results of the cross-neutralizing antibody response induced by intranasal inoculation of S2NDO preparations.

[0179] Figure 23b: The sera of Group C that were inoculated with NO+S2 preparation via intranasal route were tested using the VSV pseudovirus system (SARS-CoV-2Delta variant S-VSV, SARS-CoV-2Omicron variant S-VSV, SARS-CoV-1S-VSV and MERS-CoV S-VSV) to evaluate the detection results of cross-neutralizing antibody response induced by intranasal inoculation of NO+S2 preparation.

[0180] Fig.24a : The BALF of group B, which was inoculated with S2NDO preparations via intranasal route, was tested using the VSV pseudovirus system (SARS-CoV-2Delta variant S-VSV, SARS-CoV-2Omicron variant S-VSV, SARS-CoV-1S-VSV and MERS-CoV S-VSV) to evaluate the detection results of cross-neutralizing antibody responses induced by intranasal inoculation of S2NDO preparations.

[0181] Figure 24b : The BALF of group C, which was inoculated with NO+S2 preparation via intranasal route, was tested using the VSV pseudovirus system (SARS-CoV-2Delta variant S-VSV, SARS-CoV-2Omicron variant S-VSV, SARS-CoV-1S-VSV and MERS-CoV S-VSV) to evaluate the detection results of cross-neutralizing antibody response induced by intranasal inoculation with NO+S2 preparation.

[0182] Fig.25a : The results of cell-mediated cross-immune response induced by intranasal inoculation of S2NDO preparation combined with subcutaneous inoculation of S2ND+alum preparation in group C were detected by IFNγ-ELISPOT test after spleen cells were stimulated with SARS-CoV-2Delta variant S2 protein, SARS-CoV-2Omicron variant S2 protein, SARS-CoV-1S2 protein or MERS-CoV S2 protein. The Kruskal-Wallis nonparametric test and Dunns multiple comparison test were used for statistical analysis.

[0183] Fig.25b : The results of the cell-mediated cross-immune response induced by group C inoculated with NO+S2 preparations via intranasal route were detected by IFNγ-ELISPOT test after spleen cells were stimulated with SARS-CoV-2Delta variant S2 protein, SARS-CoV-2Omicron variant S2 protein, SARS-CoV-1S2 protein or MERS-CoV S2 protein, and the Kruskal-Wallis nonparametric test and Dunns multiple comparison test were used for statistical analysis.

[0184] Fig.26a :Through SARS-CoV-2Delta variant N protein, SARS-CoV-2Omicron variant N protein, SARS-CoV-1N protein or conserved peptide N 351-365 The results of the cell-mediated cross-immune response induced by group B inoculated with S2NDO preparations via intranasal route were detected by IFNγ-ELISPOT assay after stimulating splenocytes, and statistical analysis was performed using the Kruskal-Wallis nonparametric test and Dunns multiple comparison test.

[0185] Figure 26b : Through SARS-CoV-2Delta variant N protein, SARS-CoV-2Omicron variant N protein, SARS-CoV-1N protein or conserved peptide N 351-365 The results of the cell-mediated cross-immune response induced by group B inoculated with NO+S2 preparations via intranasal route were detected by IFNγ-ELISPOT assay after stimulating splenocytes, and the Kruskal-Wallis nonparametric test and Dunns multiple comparison test were used for statistical analysis. DETAILED DESCRIPTION

[0186] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0187] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Compiled Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995. It is understood by those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the invention claimed.

[0188] Example 1 Cloning, expression, purification and antigenic characterization of S2ND protein, S2 protein fragment and N protein

[0189] The nucleotide sequence encoding the amino acid sequence of the S2ND protein (SEQ ID NO:7) was cloned into the PET-28 vector for expression in E. coli. The S2ND protein is a S2ND chimeric protein, whose DNA sequence is SEQ ID NO:8, formed by fusion of the S protein fragment aa.798-1018 and the N protein fragment aa.248-371 of the SARS-CoV-2Delta variant (YP 009724397.2). The expressed protein accounts for about 7% of the total protein in E. coli ( Figure 1a The obtained protein was purified by ion exchange chromatography and gel filtration to a purity of more than 95%. Anti-N protein polyclonal antibody (40588-T62, purchased from Beijing Sino Biological Technology Co., Ltd.) and Western blotting (WB) were used for verification ( Figure 1b ).

[0190] The amino acid sequence (SEQ ID NO: 14) encoding the S2 subunit fragment 712-1204 of the spike protein of the SARS-CoV-2 Wuhan Hu-1 strain (NP_828851.1) was cloned into an optimized mammalian cell vector. HEK-293 cells were transfected with an expression plasmid carrying the S2 subunit fragment. After culturing the cells, cell samples and supernatants were collected and characterized by SDS-PAGE experiments and WB using an anti-S2 protein polyclonal antibody (40590-T62, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.). Figure 2a As shown, an S2 subunit fragment close to the theoretical molecular weight was detected in the supernatant and verified by WB using an anti-S2 protein polyclonal antibody ( Figure 2b ).like Figure 2a and 2b As shown, the purity of the purified recombinant protein reached more than 95%.

[0191] The amino acid sequence (SEQ ID NO: 2) encoding the N protein of the SARS-CoV-2 Delta variant (YP 009724397.2) was cloned into the PET-28 vector for expression in E. coli. The resulting protein accounted for about 5% of the total protein in E. coli ( Figure 3a The obtained protein was purified by ion exchange chromatography and gel filtration to a purity of more than 95%, and was verified by WB experiment using anti-N protein polyclonal antibody (40588-T62, purchased from Beijing Sino Biological Technology Co., Ltd.) ( Figure 3b ).

[0192] Example 2 Characterization of N protein, S2 protein, S2ND protein and their reaction with ODN-39M

[0193] For antigen characterization, serum from recovered volunteers infected with SARS-CoV-2 in Guangdong Province, China was used to identify the recombinant protein. The results showed that the serum was positive for the S2ND protein, S2 protein, and N protein, which reflects the correct folding of the protein and the exposure of the relevant antigenic epitopes ( Figure 4 ).

[0194] In order to enhance the induction of cell-mediated immune response, the S2ND protein and N protein were combined with ODN-39M (DNA sequence is SEQ ID NO: 9) to prepare preparations. The ODN-39M is an effective mucosal adjuvant phosphodiester backbone CpG oligodeoxynucleotide (Gil et al, 2015). The sera of the same group of recovered volunteers were used for antigen characterization of the obtained preparations. As shown in Figure 5, the recognition level of the S2ND protein and the N protein by the serum was similar compared with the preparations not combined with the ODN-39M. This indicates that the addition of the ODN-39M does not affect the reasonable conformation of the S2ND protein and the N protein.

[0195] Example 3 Design of immunization scheme for Balb / C mice and immunological evaluation of different preparations based on the S2ND protein

[0196] Balb / C mice were used to evaluate the S2ND protein in different formulations. The Balb / C mouse model is particularly suitable for detecting cell-mediated immune responses against N protein because it can reasonably present immunodominant antigenic epitopes.

[0197] According to the following design, seven groups of Balb / C mice (female, 6-8 weeks old) were immunized on days 0, 7, and 21, specifically 10 μg of the recombinant protein was inoculated via intranasal administration or subcutaneous injection. All immunogens were dissolved in phosphate buffered saline (PBS). The inoculum size for the intranasal route was 50 μL, and the inoculum size for the subcutaneous injection route was 100 uL. Aluminum hydroxide was added as an adjuvant to the subcutaneous injection preparation.

[0198] Group 1: S2ND formulation, intranasal administration

[0199] Group 2: S2ND+ODN-39M (S2NDO) formulation, intranasal administration

[0200] Group 3: S2ND + alum preparation, subcutaneous injection route

[0201] Group 4: S2ND+ODN-39M formulation, subcutaneous injection route

[0202] Group 5: S2ND+ODN-39M+alum preparation, subcutaneous injection route (100 μL)

[0203] Group 6: PBS + alum preparation (negative control), subcutaneous injection route (100 μL)

[0204] Group 7: PBS (negative control), inoculated by intranasal route;

[0205] Among them, the amino acid sequence of the S2ND protein is shown in SEQ ID NO:7.

[0206] Mice were sacrificed on days 19 and 26 after the last immunization and the induced humoral and cell-mediated immune responses were evaluated.

[0207] Example 4 Evaluation of humoral immune response

[0208] Humoral immune response against the S2ND protein and S2 protein

[0209] On the 19th day after the last dose of immunization in Example 3, serum was collected from the blood samples of mice and analyzed by anti-S2ND purified protein IgG ELISA test. All groups vaccinated with S2ND preparations induced high levels of anti-S2ND protein antibodies ( Figure 6 ).

[0210] Serum samples were analyzed by IgG ELISA test against the primary SARS-CoV-2 virus strain S2 recombinant protein (40590-V08H1, purchased from Sino Biological Technology Co., Ltd., Beijing, China). Figure 7 As shown, all groups obtained low-level anti-S2 protein responses. In the group vaccinated with S2ND + alum preparation, 2 out of 5 mice showed positive reactions, with anti-S2 protein titers >10 3 .

[0211] The S2 region within the S2ND protein presents a fusogenic form of fibrous structure, which is very different from the structure of the pre-fusogenic form of the S2 protein. The low antibody response level indicates that the S2 protein conformation contained in the S2ND protein structure is different from the pre-fusogenic form of the S2 protein coated on the ELISA plate. The S2 protein conformation contained in the S2ND protein structure is conducive to the construction of the S2ND protein. However, the results of a certain level of anti-S2 protein antibody detection show that there are common antigenic epitopes in both conformations.

[0212] Humoral immune response against SAR-CoV-2Delta variant N protein

[0213] On the 19th day after the last dose of immunization in Example 3, serum from the mouse blood samples was collected and analyzed by anti-N protein (SAR-CoV-2Delta variant N protein, 40588-V07E29, purchased from Sino Biological Technology Co., Ltd., Beijing, China) IgG ELISA test. Figure 8As shown in Figure 2, all groups vaccinated with S2ND formulations induced high levels of anti-N protein antibodies. When measuring IgG subclass antibodies, the groups vaccinated with S2NDO formulations via both intranasal and subcutaneous routes induced the highest levels of IgG2a antibody responses, which were statistically significantly different from the group vaccinated with S2ND+Alum formulation via subcutaneous injection ( Figure 9b In addition, the levels of IgG1 antibodies induced by the intranasal route were relatively low in both groups compared with those induced by the subcutaneous route ( Figure 9a ).

[0214] The results of the IgA antibody response test against the N protein of the SAR-CoV-2Delta variant in BALF samples of all groups showed that only the group that received the S2ND preparation via intranasal route (including the group that received the S2ND preparation via intranasal route and the group that received the S2NDO preparation via intranasal route) induced an IgA antibody response in the BALF ( Fig.10 ).

[0215] Example 5 Evaluation of cell-mediated immune responses in immunized Balb / C mice

[0216] Anti-conserved peptide N 351-365 Cell-mediated immune response

[0217] On the 19th day after the last dose of immunization in Example 3, the conserved peptide N 351-365 After in vitro stimulation, the frequency of IFNγ production by splenocytes derived from the immunized mice was detected using Mabtech antibodies and well plates. 351-365 (ILLNKHIDAYKTFPP) is synthesized by Zhejiang Paipeptide Biotechnology Co., Ltd. in China with a purity of over 97%.

[0218] like Fig.11 As shown, in the conserved peptide N 351-365 Under stimulation, positive responses of IFNγ-secreting cells were detected in both the intranasal inoculation of S2ND preparation and S2NDO preparation groups, while no IFNγ-secreting cells were detected in the spleen cells of mice in the subcutaneous inoculation of S2NDO preparation, S2ND+Alum preparation and S2NDO+Alum preparation groups. In addition, only one mouse in the subcutaneous inoculation of S2NDO preparation tested positive.

[0219] Combining all the anti-N protein immune responses detected, it was concluded that intranasal administration of S2ND preparation is beneficial to inducing Th1 pattern and cell-mediated immune responses against N protein antigen epitopes.

[0220] Example 6 Design of vaccination plan for C-57 mice and immunological evaluation of different preparations of S2 protein, N protein and S2ND protein combined vaccination

[0221] In order to evaluate the immunogenicity of the S2 recombinant fragment, it was combined with N protein and S2ND protein into two preparations, and inoculated into C57BL / 6 mice by intranasal and subcutaneous injection. The second group of mouse experiments was designed. Because the C57BL / 6 mouse model can reasonably present immunodominant antigen epitopes, it is particularly suitable for detecting cell-mediated immune responses in the S2 region.

[0222] Six groups of C57BL / 6 mice (female, 6-8 weeks old) were immunized on days 0, 7, and 21 according to the following design. The inoculum size of S2ND and S2 recombinant proteins was 10 μg, and the inoculum size of N protein was 5 μg. All immunogens were dissolved in PBS. The inoculum size for the intranasal route was 50 μL, and the inoculum size for the subcutaneous injection route was 100 uL. Aluminum hydroxide was added as an adjuvant to the subcutaneous injection formulation.

[0223] Group 1: N+ODN-39M+S2 (NO+S2) formulation, intranasal inoculation

[0224] Group 2: S2 formulation, intranasal administration

[0225] Group 3: Intranasal inoculation of S2ND+ODN-39M (S2NDO) combined with subcutaneous inoculation of S2ND+Alum

[0226] Group 4: N+ODN-39M+S2+Alum (NO+S2+Al) preparation, subcutaneous injection Group 5: S2+Alum preparation,

[0227] Group 6: intranasal inoculation of PBS preparation combined with subcutaneous inoculation of PBS+alum preparation (negative control);

[0228] Wherein, the sequences of the S2ND chimeric protein, S2 protein and N protein in the preparation are shown as SEQ ID NO: 7, SEQ ID NO: 14 and SEQ ID NO: 2, respectively.

[0229] Mice were sacrificed on days 18 and 26 after the last immunization and the induced humoral and cell-mediated immune responses were evaluated.

[0230] Example 7 Evaluation of humoral immune response

[0231] Humoral immune response against S2 protein of primary SARS-CoV-2 virus strain

[0232] On the 18th day after the last dose of immunization in Example 6, the mouse serum was analyzed by IgG ELISA test against the primary SARS-CoV-2 virus strain S2 recombinant protein (40590-V08H1, purchased from Sino Biological Technology Co., Ltd., Beijing, China). Fig.12 As shown, all groups vaccinated with S2 preparations induced high levels of anti-S2 protein antibody responses. The group vaccinated with S2 preparation alone by intranasal route induced the same level of antibody response as the group vaccinated with S2+Alum preparation by subcutaneous injection route. The group vaccinated with NO+S2 preparation by intranasal route obtained the highest level of anti-S2 protein antibody response.

[0233] At the same time, the embodiment detected the anti-S2 protein antibody response of the group (Group 3) vaccinated with S2NDO preparation by intranasal route combined with S2ND+Alum preparation by subcutaneous injection. Similar to the previous Balb / C experiment, the induced anti-S2 protein antibody level was lower than the level induced by the group vaccinated with S2 preparation. This may be related to the different conformations of the two proteins.

[0234] The examples measured IgG subclass antibody responses. Fig.13b As shown, only the group that received the NO+S2 formulation intranasally induced a high level of anti-S2 protein IgG2a antibody response. All tested groups induced similar levels of IgG1 antibodies, except for the group that received the S2NDO formulation intranasally combined with the S2ND+Alum formulation subcutaneously (Group 3). Fig.13a The results showed that intranasal administration of NO+S2 preparation could induce a Th1-biased response against the S2 protein region.

[0235] The embodiment detected the anti-S2 protein IgA antibody response level in the BALF of the intranasal vaccination group. Fig.14 As shown, IgA antibody responses were induced in BALF in both groups vaccinated with S2 formulation via intranasal route, and the IgA antibody levels induced in the group vaccinated with NO+S2 formulation tended to be significantly higher.

[0236] According to the inventors' knowledge, this application reports for the first time the immunogenicity of the S2 subunit via intranasal administration. The data in this application show that even the preparation of the S2 subunit prepared only based on PBS exhibits good immunogenicity, while other SARS-CoV-2 recombinant proteins such as RBD or N protein cannot induce immune responses in serum and BALF via intranasal administration.

[0237] Humoral immune response against SARS-CoV-2Delta variant N protein

[0238] On the 18th day after the last dose of immunization in Example 6, the mouse serum was analyzed by IgG ELISA test against SARS-CoV-2Delta variant N protein (40588-V07E29, purchased from Sino Biological Technology Co., Ltd., Beijing, China). Fig.15 As shown in Figure 2, both groups vaccinated with N combined with S2 or S2ND formulations induced high levels of anti-N protein antibody responses. When measuring IgG subclass antibodies, the group vaccinated with NO+S2 formulation intranasally induced the highest level of IgG2a antibody response, although there was no significant statistical difference compared with the group vaccinated with S2NDO formulation intranasally combined with S2ND+Alum formulation subcutaneously (Group 3) ( Fig.16b ). No statistical difference was detected between the groups for the detection of IgG1 subclass antibodies ( Fig.16a ).

[0239] The Example tested the IgA antibody response against the N protein of the Delta variant in the BALF of the same group of intranasally inoculated mice. Fig.17 The same level of anti-N protein IgA antibody response was induced in BALF in the group that received NO+S2 formulation intranasally and the group that received S2NDO formulation intranasally combined with S2ND+Alum formulation subcutaneously.

[0240] In the group that received NO+S2 preparation via intranasal route and the group that received S2NDO preparation via intranasal route combined with S2ND+Alum preparation via subcutaneous injection, the inoculated immunogens were more likely to induce a Th1-biased anti-N protein humoral immune response at the mucosal site.

[0241] Example 8 Assessment of cell-mediated immune response

[0242] Cell-mediated immune response against S2 and N proteins

[0243] On the 26th day after the last dose of immunization in Example 6, after in vitro stimulation with the S2 protein of the primary strain of SARS-CoV-2 (40590-V08H1, purchased from Sino Biological Technology Co., Ltd., Beijing, China), Mabtech antibodies and well plates were used to detect positive spleen cells producing IFNγ. The group vaccinated with NO+S2 preparations by the intranasal route induced a positive response of IFNγ-secreting cells, while no IFNγ-secreting cell response was detected in the spleen cells of mice in the group vaccinated with S2 preparations by the intranasal route. In addition, a positive response was also detected in the group vaccinated with S2NDO preparations by the intranasal route combined with S2ND+Alum preparations by subcutaneous injection, while the response was not detected in the group vaccinated by subcutaneous injection ( Fig.18a ).

[0244] At the same time, after in vitro stimulation with the SARS-CoV-2Delta variant N protein (40588-V07E29, purchased from Sino Biological Technology Co., Ltd., Beijing, China), positive splenocytes producing IFNγ were detected. Only the group vaccinated with the NO+S2 preparation intranasally induced a positive response ( Fig.18b ). Such a response was not detected in the group that received the S2NDO formulation by intranasal route in combination with the S2ND+Alum formulation by subcutaneous route, because the N protein region included in the S2ND construct does not contain an antigenic epitope that is immunodominant in C57BL / 6 mice.

[0245] Based on the anti-S2 protein and N protein immunogenicity data obtained in Balb / C and C57BL / 6 mice, it was concluded that the groups vaccinated with NO+S2 preparations intranasally and S2NDO preparations intranasally combined with S2ND+Alum preparations subcutaneously could induce the broadest immune response in both the mucosal system and the systemic system.

[0246] Example 9 Design of vaccination scheme to detect cross immune response induced by vaccination with N+ODN-39M+S2 formulation and S2ND+ODN-39M / S2ND+Alum formulation

[0247] The ability of each protein to induce cross-immune response was tested by using N+ODN-39M+S2 preparation and S2ND+ODN-39M / S2ND+Alum preparation

[0248] Three groups of Balb / C mice (female, 6-8 weeks old) were inoculated with the specified immunogens on days 0, 15, and 30, and three groups of C-57BL / 6 mice (female, 6-8 weeks old) were inoculated with the specified immunogens on days 0, 7, and 21. The inoculation amount of S2ND and S2 protein preparations was 10 μg, and the inoculation amount of N protein preparations was 5 μg. All immunogens inoculated by the intranasal route were dissolved in PBS (50 μL per mouse). Alum (Alhydrogel purchased from InvivoGen, USA) was added as an adjuvant to the S2ND preparation inoculated by subcutaneous injection (100 μL per mouse). The design scheme is as follows:

[0249] 1. Group B: S2ND+ODN-39M (S2NDO) formulation, intranasally inoculated into Balb / C mice

[0250] 2. Group B: N+ODN-39M+S2 (NO+S2) formulation, intranasally inoculated into Balb / C mice

[0251] 3. Group B: PBS preparation (negative control), intranasal inoculation of Balb / C mice

[0252] 4. Group C: C-57BL / 6 mice were inoculated with S2ND+ODN-39M (S2NDO) preparation via intranasal route and S2ND+alum preparation via subcutaneous injection.

[0253] 5. Group C: N+ODN-39M+S2 (NO+S2) formulation, intranasally inoculated into C-57BL / 6 mice

[0254] 6. Group C: intranasal route combined with subcutaneous injection of PBS preparation (negative control), C-57BL / 6 mice;

[0255] Wherein, the sequences of the S2ND chimeric protein, S2 protein and N protein in the preparation are shown as SEQ ID NO: 7, SEQ ID NO: 14 and SEQ ID NO: 2, respectively.

[0256] After the last immunization, the mice were divided into two groups and sacrificed on days 18 and 26 to evaluate the induced humoral and cell-mediated immune responses.

[0257] Example 10 Evaluation of humoral immune response

[0258] On the 18th day after the last dose of immunization in Example 9, the sera of mice in Group C that were intranasally inoculated with the NO+S2 preparation were analyzed by IgG ELISA test against the primary SARS-CoV-2 virus strain S2 protein (40590-V08H1), SARS-CoV-1 S2 protein (40150-V08B3) and MERS-CoV S2 protein (40070-V08B). The above virus strain S2 proteins were purchased from Beijing Sino Biological Technology Co., Ltd. Fig.19 As shown, similar antibody titers were obtained for all tested antigens, indicating that cross-humoral immune responses against S2 protein can be induced.

[0259] At the same time, the sera of mice in group B inoculated with S2NDO preparations and group C inoculated with NO+S2 preparations were analyzed by IgG ELISA test against SARS-CoV-2Delta variant N protein (40588-V07E29), SARS-CoV-2Omicron variant N protein (40588-V07E34) and SARS-CoV-1N protein (40143-V08B). The N proteins of the above virus strains were purchased from Beijing Sino Biological Technology Co., Ltd. Fig.20a and Fig.20b As shown, all sera obtained similar antibody response levels against the N protein of the above-mentioned virus strains, indicating that the sera have extensive cross-reactivity characteristics with coronaviruses of the Sarbe subgenus.

[0260] Regarding mucosal immune responses, BALF of mice in group C that were intranasally inoculated with the NO+S2 formulation were analyzed by IgA ELISA assays against SARS-CoV-2 S2 protein, SARS-CoV-1 S2 protein, and MERS-CoV S2 protein. Fig.21 As shown, similar to the response levels obtained in serum, the BALF of C57BL / 6 mice inoculated intranasally with the NO+S2 preparation group showed positive responses against the S2 proteins of the above virus strains.

[0261] At the same time, the BALF samples of group C, which received NO+S2 preparations intranasally, and group B, which received S2NDO preparations intranasally, were tested for their response levels against the N protein of SARS-CoV-2Delta variant (40588-V07E29), SARS-CoV-2Omicron variant (40588-V07E34), and SARS-CoV-1N protein (40143-V08B). The N proteins of the above-mentioned virus strains were purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. The results showed that both groups obtained extensive cross-reactivity against the N proteins of the above-mentioned virus strains ( Fig.22a and 22b ).

[0262] Neutralization test

[0263] Neutralization tests were performed using a vesicular stomatitis virus (VSV) pseudovirus production system. When pseudoviruses are incubated with samples containing neutralizing antibodies before cell infection, they block entry into cells or block fusion with cells. The amount of blocked virus can be determined by the results of a chemiluminescent reduction assay, which reflects the level of neutralizing antibodies or molecular inhibitors in the sample. Four pseudoviruses carrying the SARS-CoV-2 Delta variant S protein, the SARS-CoV-2 Omicron variant S protein, the SARS-CoV-1 S protein, and the MERS-CoV S protein were used to detect the level of neutralizing antibodies in serum and BALF samples from the S2ND and S2 groups (specifically, group B, which received the S2NDO formulation intranasally, and group C, which received the NO+S2 formulation intranasally). Fig.23a and 23b The neutralizing antibody levels against each pseudovirus in the sera of the representative test group. The results show that neutralizing antibody titers were detected in all test samples. The positive neutralizing response against the four pseudoviruses indicates that each group induced a cross-response of neutralizing antibodies. Fig.24a and 24b Represents the neutralizing antibody titer against each pseudovirus in BALF. Although the titers in BALF were lower than those detected in serum, neutralizing activity against the four pseudoviruses was detected in all panels.

[0264] Example 11 Evaluation of cell-mediated immune responses

[0265] Based on the immunodominance of SARS-CoV-2 antigenic epitopes in different mouse strains, C57BL / 6 mice were selected to detect the immune response against S2 protein, and Balb / C mice were selected to detect the cell-mediated immune response against N protein.

[0266] On the 26th day after the last vaccination in Example 9, spleen cells from Group C, which was inoculated with S2NDO preparations by intranasal route combined with S2ND+alum preparations by subcutaneous injection, and Group C, which was inoculated with NO+S2 preparations by intranasal route, were collected, and after in vitro stimulation with SARS-CoV-2 primary virus strain S2 protein (40590-V08H1), SARS-CoV-1 S2 protein (40143-V08B), and MERS-CoV S2 protein (40070-V08B), positive spleen cells producing IFNγ were detected. The results showed that both groups obtained positive cell-mediated immune responses against the S2 proteins of the above virus strains, indicating that the S2ND protein and S2 protein in the test preparations can induce cell-mediated cross-immune responses ( Fig.25a and Fig.25b ).

[0267] In order to evaluate the anti-N protein immune response, group B, which was inoculated with S2NDO preparations by intranasal route, and group B, which was inoculated with NO+S2 preparations by intranasal route, were selected to carry out the cell-mediated immune response test against N protein. On the 26th day after the last vaccination, spleen cells were isolated and the SARS-CoV-2Delta variant N protein (40588-V07E29), SARS-CoV-2Omicron variant N protein (40588-V07E34), SARS-CoV-1N protein (40143-V08B) and conserved peptide N were used to detect the N protein. 351-365 The results of in vitro stimulation of splenocytes were as follows Fig.26a and 26b All groups obtained positive responses against the N protein of the above-mentioned virus strains, indicating that a broad spectrum of cell-mediated immune responses can be induced.

[0268] Based on the similar humoral immunity and cell-mediated immune cross-responses obtained by the combined preparation of S2 protein and N protein, the S2 protein and N protein can become candidate vaccine targets for the development of a broad-spectrum coronavirus vaccine.

[0269]

[0270] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

[0271] Sequence information

[0272] SEQ ID NO:1 Amino acid sequence of S2 subunit fragment 708-1207

[0273] SNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGI

[0274] AVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADA

[0275] GFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAG

[0276] AALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDV

[0277] VNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQ

[0278] LIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPA

[0279] QEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVI

[0280] GIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAK

[0281] NLNESLIDLQELGKYE

[0282] Amino acid sequence of SEQ ID NO:2 N protein (SARS-CoV-2 Delta variant, with Met at the N-terminus) MGSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEGLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSMGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKAYETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA

[0283] Amino acid sequence of SEQ ID NO:3 N protein (SARS-CoV-2 Delta variant, without Met at the N-terminus) GSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEGLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSMGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKAYETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA

[0284] SEQ ID NO:4 Amino acid sequence of N protein (SARS-CoV-2 Delta variant) aa.255-365 SKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPP

[0285] SEQ ID NO:5N protein (SARS-CoV-2Delta variant) amino acid sequence aa.248-371

[0286] KKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKD

[0287] SEQ ID NO:6N protein (SARS-CoV-2Delta variant) amino acid sequence aa.14-365

[0288] RITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEGLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSR SRNSSRNSTPGSSMGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPP

[0289] SEQ ID NO: Amino acid sequence of 7S2ND chimeric protein

[0290] MFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNQLQNQLVNQNQLVNQTLV SVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRAGGSSGKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQ GNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDKDPNFKDQVILLNKHIDAYKTFPPTEPKKD

[0291] SEQ ID NO:8S2NDDNA Connection

[0292]

[0293] SEQ ID NO: 9 DNA sequence of ODN-39M

[0294] 5'-ATC GAC TCT CGA GCG TTC TCG GGG GAC GAT CGT CGG GGG-3

[0295] SEQ ID NO: 10 Amino acid sequence of full-length S protein (SARS-CoV-2Delta variant)

[0296]

[0297] SEQ ID NO:11 Amino acid sequence of S protein (SARS-CoV-2Delta variant) aa.806-1000

[0298] DPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDE

[0299] MIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQF

[0300] NSAIGKIQDSLSSTASALGKLQNVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKV

[0301] EAEVQIDRLITGRLQ

[0302] SEQ ID NO:12S protein (SARS-COV-2Delta variant) amino acid sequence aa.798-1018

[0303] FNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTV

[0304] LPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYEN

[0305] QKLIANQFNSAIGKIQDSLSSTASALGKLQNVVNQNAQALNTLVKQLSSNFGAISSVLNDI

[0306] LSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA

[0307] SEQ ID NO:13 Amino acid sequence of full-length S protein (SARS-CoV-2 Wuhan-Hu-1 strain)

[0308] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSN

[0309] VTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNN

[0310] ATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLE

[0311] GKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLL

[0312] ALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKC

[0313] TLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCV

[0314] ADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYN

[0315] YKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG

[0316] VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0317] NFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGT

[0318] NTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSY

[0319] ECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVT

[0320] TEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFA

[0321] QVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIA

[0322] ARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAY

[0323] RFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLV

[0324] KQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLA

[0325] ATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICH

[0326] DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQP

[0327] ELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG

[0328] KYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPV

[0329] LKGVKLHYT

[0330] Amino acid sequence of S protein (SARS-CoV-2 Wuhan-Hu-1 strain) aa.712 - 1204

[0331] IAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG

[0332] Amino acid sequence of S protein (SARS-CoV-2 Wuhan-Hu-1 strain) aa. 708 - 1207, SEQ ID NO:15

[0333] SNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYE

[0334] Amino acid sequence (aa. 686 - 1271) of the S2 subunit (SARS-CoV-2 Delta variant), SEQ ID NO:16

[0335] ASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0336] Amino acid sequence (aa. 686 - 1273) of the S2 subunit (SARS-CoV-2 Wuhan-Hu-1 strain), SEQ ID NO:17

[0337] SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0338] SEQ ID NO:18 LINKER Amino Acid Sequence

[0339] GGSSGG。

Claims

1. A chimeric protein comprising a first peptide segment and a second peptide segment, wherein: The first peptide segment comprises the sequence of the S2 subunit of the coronavirus S protein or a fragment thereof; the second peptide segment comprises the coronavirus N protein or a fragment thereof; Wherein, the S protein and the N protein are derived from the same or different coronaviruses.

2. The chimeric protein of claim 1, wherein The first peptide segment comprises at least 100, at least 120, at least 150 or at least 170 consecutive amino acid residues of the amino acid residues at positions corresponding to positions 806-1000 of SEQ ID NO:10 in the S protein; the second peptide segment comprises at least 50, at least 80 or at least 90 consecutive amino acid residues of the amino acid residues at positions corresponding to positions 255-365 of SEQ ID NO:3 in the N protein.

3. The chimeric protein of claim 1 or 2, wherein The first peptide segment comprises the extramembrane region of the S2 subunit or a fragment thereof; Preferably, the first peptide segment comprises the S protein corresponding to positions 810-982 (or positions 810-986, or positions 810-1000, or positions 810-1018, or positions 810-1020, or positions 810-1030, or positions 810-1161, or positions 806-982, or positions 806-986, or positions 806-1000, or positions 806-1018, or positions 806-1020, or positions 806-1030) in SEQ ID NO:

10. 6-1030, or 806-1161, or 800-982, or 800-986, or 800-1000, or 800-1018, or 800-1020, or 800-1030, or 800-1161, or 798-982, or 798-986, or 798-1000, or 798-1018, or No. 798-1020, or No. 798-1030, or No. 798-1161, or No. 790-982, or No. 790-986, or No. 790-1000, or No. 790-1018, or No. 790-1020, or No. 790-1030, or No. 790-1161, or No. 686-982, or No. 686-986, or No. 686-1000 , or 686-1018, or 686-1020, or 686-1030, or 686-1161, or 684-982, or 684-986, or 684-1000, or 684-1018, or 684-1020, or 684-1030, or 684-1161) or consisting of the amino acid residues at the corresponding positions; Preferably, the first peptide segment comprises the S protein corresponding to positions 810-982 (or positions 810-986, or positions 810-1000, or positions 810-1018, or positions 810-1020, or positions 810-1030, or positions 806-982, or positions 806-986, or positions 806-1000, or positions 806-1018, or positions 806-1020, or positions 806-1030, or positions 800-982, or positions 800-986, or positions 800-1000, or positions 800-1 018, or 800-1020, or 800-1030, or 798-982, or 798-986, or 798-1000, or 798-1018, or 798-1020, or 798-1030, or 790-982, or 790-986, or 790-1000, or 790-1018, or 790-1020, or 790-1030) or consisting of the amino acid residues at the corresponding positions; Preferably, the first peptide segment comprises or consists of amino acid residues at positions corresponding to positions 806-1000, or positions 806-1018, or positions 798-1000, or positions 798-1018 of SEQ ID NO: 10 in the S protein; Preferably, the S protein has: (a) an amino acid sequence as shown in SEQ ID NO: 10 or 13; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 10 or 13; or, (c) a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence shown in SEQ ID NO: 10 or 13; Preferably, the first peptide segment comprises an amino acid sequence as shown in SEQ ID NO: 11 or 12, or a sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 11 or 12, or a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions, or consists of the same.

4. The chimeric protein of any one of claims 1 to 3, wherein The second peptide segment comprises or consists of amino acid residues at positions corresponding to positions 260-360 (or 260-365, or 260-371, or 260-380, or 255-360, or 255-365, or 255-371, or 255-380, or 250-360, or 250-365, or 250-371, or 250-380, or 248-360, or 248-365, or 248-371, or 248-380, or 240-360, or 240-365, or 240-371, or 240-380) of the N protein; Preferably, the second peptide segment comprises or consists of amino acid residues at positions corresponding to positions 255-365, or 255-371, or 248-365, or 248-371 of SEQ ID NO: 3 in the N protein; Preferably, the N protein has: (a) an amino acid sequence as shown in SEQ ID NO: 2 or 3; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 2 or 3; or, (c) a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence shown in SEQ ID NO: 2 or 3; Preferably, the second peptide segment comprises an amino acid sequence as shown in SEQ ID NO: 4 or 5, or a sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 4 or 5, or a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions, or consisting of the same.

5. The chimeric protein of any one of claims 1 to 4, wherein The S protein and the N protein are each independently derived from a coronavirus selected from the genus β; Preferably, the S protein and the N protein are each independently derived from a coronavirus selected from the genus β, the subgenus Sarbe and the subgenus Merbe; Preferably, the S protein and the N protein are each independently derived from SARS-CoV-2, SARS-CoV-1 or MERS-CoV; Preferably, the S protein and the N protein are derived from the Delta strain of SARS-CoV-2.

6. The chimeric protein of any one of claims 1 to 5, which has one or more of the following characteristics: (1) The S2 subunit has: (a) an amino acid sequence as shown in SEQ ID NO: 16 or 17; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence as shown in SEQ ID NO: 16 or 17; or, (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence as shown in SEQ ID NO: 16 or 17; (2) the first peptide segment and the second peptide segment are optionally connected via a linker (e.g., a peptide linker, for example, a peptide linker comprising one or more glycine and / or one or more serine); preferably, the peptide linker comprises an amino acid sequence as shown in SEQ ID NO: 18; (3) The first peptide segment is connected to the N-terminus or C-terminus of the second peptide segment through the peptide linker; preferably, the first peptide segment is connected to the N-terminus of the second peptide segment through the peptide linker; (4) The chimeric protein has: (a) the amino acid sequence as shown in SEQ ID NO:7; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence as shown in SEQ ID NO:7; or (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence as shown in SEQ ID NO:7; (5) The conformation of the S2 subunit or its fragment in the chimeric protein is different from that of the pre-fusion form; preferably, the S2 subunit or its fragment in the chimeric protein is in a fibrous conformation (e.g., a fibrous conformation of the fusion form); (6) The chimeric protein exists in the form of a monomer, a dimer or a multimer (eg, a trimer).

7. A composition comprising a first polypeptide and a second polypeptide, wherein: The first polypeptide comprises the sequence of the S2 subunit of the coronavirus S protein or a fragment thereof; the second polypeptide comprises the coronavirus N protein or a fragment thereof; Wherein, the S protein and the N protein are derived from the same or different coronaviruses.

8. The composition of claim 7, wherein The first polypeptide comprises at least 250, at least 300, at least 330 or at least 350 consecutive amino acid residues of the amino acid residues at positions corresponding to positions 712-1204 of SEQ ID NO: 13 in the S protein; the second polypeptide comprises at least 200, at least 250, at least 300 or at least 320 consecutive amino acid residues of the amino acid residues at positions corresponding to positions 14-365 of SEQ ID NO: 3 in the N protein.

9. The composition of claim 7 or 8, wherein The first polypeptide comprises the extramembrane region of the S2 subunit or a fragment thereof; Preferably, the first polypeptide comprises the S protein corresponding to positions 812-1200 (or 812-1204, or 812-1206, or 812-1207, or 812-1210, or 812-1213, or 788-1200, or 788-1204, or 788-1206, or 788-1207, or 788-1210) of SEQ ID NO:

13. 1210, or 788-1213, or 720-1200, or 720-1204, or 720-1206, or 720-1207, or 720-1210, or 720-1213, or 712-1200, or 712-1204, or 712-1206, or 712- 1207, or 712-1210, or 712-1213, or 708-1200, or 708-1204, or 708-1206, or 708-1207, or 708-1210, or 708-1213, or 700-1200, or 700-1204, or 700 -1206, or 700-1207, or 700-1210, or 700-1213, or 686-1200, or 686-1204, or 686-1206, or 686-1207, or 686-1210, or 686-1213) or consisting of the amino acid residues at the corresponding positions; Preferably, the first polypeptide comprises the S protein corresponding to positions 720-1200 (or positions 720-1204, or positions 720-1206, or positions 720-1207, or positions 720-1210, or positions 720-1213, or positions 712-1200, or positions 712-1204, or positions 712-1206, or positions 712-1207, or positions 712-1210, or positions 712-1213, or positions 708-1209) of SEQ ID NO:

13. 1200, or 708-1204, or 708-1206, or 708-1207, or 708-1210, or 708-1213, or 700-1200, or 700-1204, or 700-1206, or 700-1207, or 700-1210, or 700-1213) or consisting of the amino acid residues at the corresponding positions of the amino acid residues; Preferably, the first polypeptide comprises or consists of amino acid residues at positions corresponding to positions 712-1204, or 712-1207, or 708-1204, or 708-1207 of the S protein in SEQ ID NO: 13; Preferably, the S protein has: (a) an amino acid sequence as shown in SEQ ID NO: 13 or 10; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 13 or 10; or, (c) a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence shown in SEQ ID NO: 13 or 10; Preferably, the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 14 or 15, or a sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 14 or 15, or a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions, or consists of the same.

10. The composition of any one of claims 7 to 9, wherein The second polypeptide comprises the amino acid residues at positions corresponding to positions 20-360 (or 20-365, or 20-371, or 20-410, or 20-419, or 14-360, or 14-365, or 14-371, or 14-410, or 14-419, or 10-360, or 10-365, or 10-371, or 10-410, or 10-419, or 1-360, or 1-365, or 1-371, or 1-410) of the N protein in SEQ ID NO: 3, or the second polypeptide comprises or consists of the full-length N protein; Preferably, the second polypeptide comprises the amino acid residues at positions corresponding to positions 14-365, or 14-419, or 1-365 of SEQ ID NO: 3 in the N protein, or the second polypeptide comprises or consists of the full-length N protein; Preferably, the N protein has: (a) an amino acid sequence as shown in SEQ ID NO: 2 or 3; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 2 or 3; or, (c) a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence shown in SEQ ID NO: 2 or 3; Preferably, the second polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 2-3, 6, or a sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in any one of SEQ ID NOs: 2-3, 6, or a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions.

11. The composition of any one of claims 7 to 10, wherein The S protein and the N protein are each independently derived from a coronavirus selected from the genus β; Preferably, the S protein and the N protein are each independently derived from a coronavirus selected from the genus β, the subgenus Sarbe and the subgenus Merbe; Preferably, the S protein and the N protein are each independently derived from SARS-CoV-2, SARS-CoV-1 or MERS-CoV; Preferably, the S protein and the N protein are derived from the Wuhan Hu-1 strain of SARS-CoV-2.

12. The composition of any one of claims 7 to 11, having one or more selected from the following features: (1) The S2 subunit has: (a) an amino acid sequence as shown in SEQ ID NO: 16 or 17; (b) an amino acid sequence having at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence as shown in SEQ ID NO: 16 or 17; or, (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions compared to the amino acid sequence as shown in SEQ ID NO: 16 or 17; (2) the first polypeptide and the second polypeptide are respectively present in different peptide chains; (3) The first polypeptide in the composition exists in the form of a monomer, a dimer or a multimer (eg, a trimer).

13. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric protein of any one of claims 1 to 6 or the first polypeptide and the second polypeptide as defined in any one of claims 7 to 12; Preferably, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the chimeric protein of any one of claims 1 to 6; Preferably, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a first polypeptide as defined in any one of claims 7-12, and a second nucleotide sequence encoding a second polypeptide as defined in any one of claims 7-12.

14. A vector comprising the isolated nucleic acid molecule of claim 13.

15. A host cell comprising the isolated nucleic acid molecule of claim 13 or the vector of claim 14.

16. A method for preparing the chimeric protein of any one of claims 1 to 6 or the first polypeptide and the second polypeptide as defined in any one of claims 7 to 12, comprising culturing the host cell of claim 15 under suitable conditions, and recovering the chimeric protein or the first polypeptide and the second polypeptide from the cell culture.

17. An immunogenic composition comprising the chimeric protein of any one of claims 1 to 6 or the composition of any one of claims 7 to 12, and optionally a pharmaceutically acceptable carrier and / or excipient (e.g., adjuvant); Preferably, the adjuvant is selected from the group consisting of: alum adjuvant, CpG adjuvant (e.g., ODN-39M), aluminum salt adjuvant, zinc-aluminum mixed adjuvant, Freund's adjuvant, oil emulsion adjuvant, cytokine, TLR agonist, nucleic acid adjuvant, liposome, saponin adjuvant, AS01B adjuvant and any combination thereof; Preferably, the adjuvant is selected from: alum adjuvant, CpG adjuvant (e.g., ODN-39M); Preferably, the adjuvant is ODN-39M (e.g., ODN-39M as shown in SEQ ID NO: 9); Preferably, the immunogenic composition is a vaccine.

18. The immunogenic composition of claim 17, wherein The immunogenic composition is administered parenterally (e.g., subcutaneously, intradermally, intramuscularly), mucosally (e.g., respiratory (e.g., intranasal), digestive (e.g., oral), ocular, rectal), or simultaneously parenterally (e.g., subcutaneously, intradermally, intramuscularly) and mucosally (e.g., respiratory (e.g., intranasal), digestive (e.g., oral), ocular, rectal); For example, the immunogenic composition contains the composition of any one of claims 7 to 12, and a CpG adjuvant (e.g., ODN-39M, e.g., ODN-39M as shown in SEQ ID NO: 9); preferably, the immunogenic composition is inoculated via a mucosal route (e.g., a respiratory route (e.g., an intranasal route), a digestive route (e.g., an oral route), an ocular route, a rectal route); For example, the immunogenic composition comprises the chimeric protein of any one of claims 1 to 6 and a CpG adjuvant (e.g., ODN-39M, e.g., ODN-39M as shown in SEQ ID NO: 9); preferably, the immunogenic composition is inoculated via a mucosal route (e.g., a respiratory route (e.g., an intranasal route), a digestive route (e.g., an oral route), an ocular route, a rectal route); For example, the immunogenic composition comprises a first immunogenic composition comprising a chimeric protein of any one of claims 1 to 6 and a CpG adjuvant (e.g., ODN-39M, e.g., ODN-39M as shown in SEQ ID NO: 9), and a second immunogenic composition comprising a chimeric protein of any one of claims 1 to 6 and an alum adjuvant; and the first immunogenic composition and the second immunogenic composition are used for combined vaccination (e.g., simultaneous or sequential vaccination); preferably, the first immunogenic composition is administered via a mucosal route (e.g., a respiratory route (e.g., intranasal route), a digestive route (e.g., oral route), an ocular route, a rectal route), and the second immunogenic composition is administered via a parenteral route (e.g., subcutaneous route, intradermal route, intramuscular route).

19. The immunogenic composition of claim 17 or 18, which is capable of inducing a humoral immune response and / or a cell-mediated immune response against a coronavirus (e.g., a beta coronavirus, such as a Sarbe subgenus and / or a Merbe subgenus coronavirus) in a subject (e.g., in the mucosal system and / or the systemic system); Preferably, the immunogenic composition has one or more characteristics selected from the following: (1) inducing the production of antibodies (e.g., binding antibodies, cross-binding antibodies, neutralizing antibodies and / or cross-neutralizing antibodies) against coronavirus (e.g., beta coronavirus, such as Sarbe subgenus and / or Merbe subgenus coronavirus) S protein (e.g., S2 subunit) in the subject (e.g., in the mucosal system and / or systemic system); (2) inducing a cell-mediated immune response and / or a cell-mediated cross-immune response against the S protein (e.g., S2 subunit) of a coronavirus (e.g., a beta coronavirus, such as a Sarbe subgenus and / or a Merbe subgenus coronavirus) in a subject (e.g., in the mucosal system and / or the systemic system); (3) inducing the production of antibodies (e.g., binding antibodies and / or cross-binding antibodies) against the N protein of a coronavirus (e.g., a beta coronavirus, such as a Sarbe subgenus and / or a Merbe subgenus coronavirus) in a subject (e.g., in the mucosal system and / or the systemic system); (4) inducing a cell-mediated immune response and / or a cell-mediated cross-immune response against the N protein of a coronavirus (e.g., a beta coronavirus, such as a Sarbe subgenus and / or a Merbe subgenus coronavirus) in a subject (e.g., in the mucosal system and / or the systemic system); (5) Inducing a Th1-biased immune response in a subject (e.g., in the mucosal system and / or systemic system).

20. A method for preparing an immunogenic composition, comprising mixing the chimeric protein of any one of claims 1 to 6 or the first polypeptide and the second polypeptide as defined in any one of claims 7 to 12 with a pharmaceutically acceptable carrier and / or excipient; optionally, the method further comprises mixing an adjuvant, and / or another active ingredient, such as another active ingredient capable of preventing or treating coronavirus infection or a disease caused by coronavirus infection; For example, the method comprises mixing the chimeric protein of any one of claims 1 to 6 with an adjuvant and a pharmaceutically acceptable carrier and / or excipient; For example, the method comprises mixing a first polypeptide and a second polypeptide as defined in any one of claims 7 to 12 with an adjuvant and a pharmaceutically acceptable carrier and / or excipient; Preferably, the adjuvant is selected from the group consisting of: alum adjuvant, CpG adjuvant (e.g., ODN-39M), aluminum salt adjuvant, zinc-aluminum mixed adjuvant, Freund's adjuvant, oil emulsion adjuvant, cytokine, TLR agonist, nucleic acid adjuvant, liposome, saponin adjuvant, AS01B adjuvant and any combination thereof; Preferably, the adjuvant is selected from: alum adjuvant, CpG adjuvant (e.g., ODN-39M); Preferably, the adjuvant is ODN-39M (eg, ODN-39M as shown in SEQ ID NO: 9).

21. Use of the chimeric protein of any one of claims 1 to 6, or the composition of any one of claims 7 to 12, or the isolated nucleic acid molecule of claim 13, or the vector of claim 14, or the host cell of claim 15, or the immunogenic composition of any one of claims 17 to 19 in the preparation of a medicament for inducing an immune response against a coronavirus in a subject and / or for preventing and / or treating a coronavirus infection or a disease associated with a coronavirus infection in a subject; Preferably, the drug is a vaccine; Preferably, the coronavirus is a β coronavirus, such as a Sarbe subgenus and / or a Merbe subgenus coronavirus; Preferably, the subject is a mammal, such as a human.

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