Vaccine composition containing conserved recombinant protein fragment of coronavirus, and use of thereof

Abstract

A chimeric protein and immunogenic composition based on coronavirus S2 subunit and N protein, which can be used in preventing and / or treating coronavirus infection, and a use of the chimeric protein and immunogenic composition.

Description

Vaccine composition comprising conservative recombinant protein fragments of coronavirus and its application

[0001] This application is based on the application with CN application number 202311506781.7 and application date November 13, 2023, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] 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 the coronavirus S2 subunit and N protein that can be used to prevent and / or treat coronavirus infection, as well as uses of such chimeric proteins and immunogenic compositions. Background Art

[0003] The USAID PREDICT Program 1 (2009-2019) identified 113 emerging coronaviruses in humans and animals in areas of high risk for intensive spillover. Considering these findings and the emergence of new SARS-CoV-2 variants, the scientific community has proposed developing a new generation of vaccines with broader protection—broad-spectrum coronavirus vaccines (Rubin, 2021) (Morens et al., 2022). Current research on broad-spectrum vaccines is focusing on multiple immunogenic antigens / regions based on the spike (S) protein (multivalent) and proteins / designs based on conserved regions of coronaviruses. Multivalent vaccines require multiple immunodominant regions and are typically limited to variants within these regions (Cohen et al., 2021; Liang et al., 2021; Wang et al., 2022). Another approach based on conserved antigens aims to achieve broad protection based on the degree of antigen conservation and presentation.

[0004] One of the most relevant viral regions included in a broad-spectrum coronavirus vaccine is the S2 subunit. The S2 subunit is the most conserved region of the S protein, involved in the fusion process during viral invasion and containing T cell epitopes. One of these T cell epitopes is conserved across multiple coronaviruses and is associated with a rapid response to SARS-CoV-2 infection, thereby reducing the severity of infection in humans (Loyal et al., 2021).

[0005] Due to the important function of membrane fusion, the region of the S2 subunit involved in the membrane fusion process is insufficiently exposed. 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, capable of exposing 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. One study 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 were 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 on the immunological evaluation of a recombinant S2 subunit protein demonstrated that the S2 subunit recombinant protein failed to induce functional antibodies in mice. In the same study, when the S2 subunit gene was inserted into a DNA vector used as a vaccine platform, it induced a cell-mediated immune response and anti-S2 subunit neutralizing antibodies, indicating that the native conformation of the S2 subunit is important for the induction of an appropriate immune response (Ng KW et al., 2021).

[0006] It is well known that the conformation of the pre-fusion form of the S2 subunit differs from the conformation adopted during fusion (Fan et al., 2020). The transient fibrous conformation of the S2 subunit immediately participates in the binding of the virus and host cell membranes after the release of the S1 subunit, exposing key regions during the binding process. Therefore, the transient fibrous conformation of the S2 subunit has broad prospects and can be explored as a potential candidate vaccine target. Currently, the research difficulties of this fibrous structure (fusion form) as a vaccine target have not been overcome.

[0007] Although the S2 subunit region is conserved, limited information is available regarding the induction of cross-neutralizing antibodies and cell-mediated cross-immune responses by candidate vaccines based on the S2 subunit. Two recombinant proteins based on a stabilized form of the MERS-CoV S protein S2 subunit failed to induce cross-neutralizing antibodies against SARS-CoV-2, suggesting that inducing cross-neutralizing antibodies using recombinant protein constructs is difficult and complex (Hsieh et al, 2021).

[0008] Mucosal immunity is considered an important pathway for preventing viral transmission. Most vaccine antigens used to induce mucosal immunity are based on the full-length S protein in different vaccine platforms and formulations. However, no relevant studies have demonstrated the ability of intranasal vaccine candidates based on the S2 subunit to induce mucosal immune responses.

[0009] The combination of two conserved regions or two conserved proteins in a single vaccine formulation has not been extensively studied. Such combinations may increase the scope of cross-immunity and become attractive for vaccine development to prevent future zoonotic outbreaks caused by novel viruses.

[0010] Summary of the Invention

[0011] In view of the above-mentioned defects in the prior art, the present invention aims to provide an immunogenic composition of recombinant conserved protein fragments based on the coronavirus S2 subunit and N protein, which can induce a humoral immune response and / or a cell-mediated immune response against β-coronavirus in mice (e.g., in the mucosal system and / or systemic system).

[0012] Chimeric protein

[0013] 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;

[0014] Wherein, the S protein and the N protein are derived from the same or different coronaviruses.

[0015] 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 the sequence thereof; and the second peptide segment comprises the coronavirus N protein or a fragment thereof, or consists of the sequence thereof.

[0016] In certain embodiments, the fragment of the S2 subunit has the immunological activity of the S2 subunit from which it is derived (for example, 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 (for example, 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)).

[0017] 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.

[0018] In certain embodiments, the first peptide segment comprises the extramembranous region of the S2 subunit or a fragment thereof.

[0019] In certain embodiments, the first peptide segment comprises the same sequence as that of positions 810-982 (or 810-986, or 810-1000, or 810-1018, or 810-1020, or 810-1030, or 810-1161, or 806-982, or 806-986, or 806-1000, or 806-1018, or 806-1020, or 806-1030) of the S protein 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 684-1018, 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).

[0020] In certain embodiments, the first peptide segment comprises the same sequence as that of positions 810-982 (or 810-986, or 810-1000, or 810-1018, or 810-1020, or 810-1030, or 806-982, or 806-986, or 806-1000, or 806-1018, or 806-1020, or 806-1030) of the S protein as that of SEQ ID NO: 10. 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 consists of the amino acid residues at the corresponding positions.

[0021] In certain embodiments, 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 the S protein in SEQ ID NO: 10.

[0022] 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.

[0023] 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 to 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.

[0024] In certain embodiments, the second peptide segment comprises the peptide sequence of the N protein corresponding to SEQ ID or consisting of the amino acid residues at 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 NO: 3.

[0025] 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.

[0026] 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.

[0027] 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 consists of the same.

[0028] In certain embodiments, the first peptide segment comprises the same sequence as that of positions 810-982 (or 810-986, or 810-1000, or 810-1018, or 810-1020, or 810-1030, or 810-1161, or 806-982, or 806-986, or 806-1000, or 806- 1018, or 806-1020, or 806-1030, 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 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 positions corresponding to the amino acid residues of SEQ ID NO: 1 in the N protein; and the second peptide segment comprises or consists of the amino acid residues at the positions corresponding to the amino acid residues of SEQ ID NO: 1 in the N protein or consisting of the amino acid residues at 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.

[0029] 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.

[0030] In certain embodiments, the S protein and the N protein are each independently derived from a coronavirus selected from the genus Beta.

[0031] In certain embodiments, the S protein and the N protein are each independently derived from a coronavirus selected from the genus Beta, subgenus Sarbe and subgenus Merbe.

[0032] In certain embodiments, the S protein and the N protein are each independently derived from SARS-CoV-2, SARS-CoV-1, or MERS-CoV.

[0033] 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.

[0034] In certain embodiments, the S protein and the N protein are derived from the Delta strain of SARS-CoV-2.

[0035] In certain embodiments, the chimeric protein has one or more of the following characteristics:

[0036] (1) The S2 subunit has: (a) the 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;

[0037] (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 the amino acid sequence shown in SEQ ID NO: 18;

[0038] (3) The first peptide segment is connected to the N-terminus or C-terminus of the second peptide segment via the peptide linker; in certain embodiments, the first peptide segment is connected to the N-terminus of the second peptide segment via the peptide linker;

[0039] (4) The chimeric protein has: (a) the amino acid sequence of SEQ ID NO: 7; (b) an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to the amino acid sequence of SEQ ID NO: 7; or (c) a sequence that has 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 of SEQ ID NO: 7;

[0040] (5) The conformation of the S2 subunit or fragment thereof in the chimeric protein is different from the conformation of the pre-fusogenic form; in certain embodiments, the S2 subunit or fragment thereof in the chimeric protein is in a fibrous conformation (e.g., a fibrous conformation of the fusogenic form or the post-fusion form);

[0041] (6) The chimeric protein exists in the form of a monomer, dimer, or multimer (e.g., trimer).

[0042] In certain embodiments, the S2 subunit or a fragment thereof in the chimeric protein is in a fibrous conformation (eg, a fusogenic form or a post-fusion form), and the fibrous conformation is stably present in the chimeric protein.

[0043] It is easy for those skilled in the art to understand that in the process of coronavirus infecting host cells, when the RBD located in the S1 subunit of the S protein specifically binds to the corresponding site of the cell surface receptor (ACE2), it induces the S2 subunit to undergo a conformational change, causing it to change 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 and the host cell membrane. This step is the key to coronavirus infection of host cells.

[0044] Therefore, the chimeric protein provided in the present application comprising the S2 subunit or its fragment in the fiber conformation (for example, the stably existing fiber conformation) is significantly advantageous for inducing an immune response against coronavirus infection and has broad application prospects.

[0045] Composition

[0046] In another aspect, 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;

[0047] Wherein, the S protein and the N protein are derived from the same or different coronaviruses.

[0048] 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.

[0049] 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.

[0050] In certain embodiments, the first polypeptide comprises the extramembranous region of the S2 subunit or a fragment thereof.

[0051] In certain embodiments, 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- 1207th, or 712th-1210th, or 712th-1213th, or 708th-1200th, or 708th-1204th, or 708th-1206th, or 708th-1207th, or 708th-1210th, or 708th-1213th, or 700th-1200th, or 700th-1204th, or 700th -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.

[0052] In certain embodiments, the first polypeptide comprises the S protein corresponding to positions 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 708-1209) 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 consists of the amino acid residues at the corresponding positions.

[0053] In certain embodiments, the first polypeptide comprises or consists of amino acid residues at positions corresponding to positions 712-1204, or positions 712-1207, or positions 708-1204, or positions 708-1207 of the S protein of SEQ ID NO: 13.

[0054] 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 that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to 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.

[0055] In certain embodiments, the first polypeptide comprises the amino acid sequence 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 to 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.

[0056] In certain embodiments, 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 of SEQ ID NO: 3, or the second polypeptide comprises or consists of the full-length N protein.

[0057] In certain embodiments, the second polypeptide comprises amino acid residues at positions corresponding to positions 14-365, or 14-419, or 1-365 of the N protein in SEQ ID NO: 3, or the second polypeptide comprises or consists of the full-length N protein.

[0058] 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.

[0059] 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 to 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.

[0060] In certain embodiments, the first polypeptide comprises the extramembranous 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 of SEQ ID NO: 3, or the second polypeptide comprises or consists of the full-length N protein.

[0061] In certain embodiments, the first polypeptide comprises the same sequence as that at 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 the S protein as that at positions 812-1200 (or 812-1204, or 812-1206, or 812-1207, or 812-1210) 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 700-120 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 positions corresponding to SEQ ID NO: 3, or the second polypeptide comprises or consists of the amino acid residues at 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.

[0062] 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; and the second polypeptide comprises amino acid residues at positions corresponding to positions 14-365, or 14-419, or 1-365 of the N protein in SEQ ID NO: 3, or the second polypeptide comprises or consists of the full-length N protein.

[0063] In certain embodiments, the S protein and the N protein are each independently derived from a coronavirus selected from the genus Beta.

[0064] In certain embodiments, the S protein and the N protein are each independently derived from a coronavirus selected from the genus Beta, subgenus Sarbe and subgenus Merbe.

[0065] In certain embodiments, the S protein and the N protein are each independently derived from SARS-CoV-2, SARS-CoV-1, or MERS-CoV.

[0066] 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.

[0067] In certain embodiments, the S protein and the N protein are derived from the Wuhan Hu-1 strain of SARS-CoV-2.

[0068] In certain embodiments, the composition has one or more of the following characteristics:

[0069] (1) The S2 subunit has: (a) the 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;

[0070] (2) the first polypeptide and the second polypeptide are each present in different peptide chains;

[0071] (3) The first polypeptide in the composition exists in the form of a monomer, a dimer or a multimer (e.g., a trimer).

[0072] In certain embodiments, the first polypeptide and the second polypeptide are linked covalently or non-covalently.

[0073] In certain embodiments, no linkage is formed between the first polypeptide and the second polypeptide.

[0074] 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.

[0075] In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a chimeric protein as described above.

[0076] 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.

[0077] In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are located on the same nucleic acid molecule.

[0078] In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are located in different nucleic acid molecules. 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.

[0079] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule as described above.

[0080] In another aspect, the present application provides a host cell comprising the isolated nucleic acid molecule or vector as described above.

[0081] In another aspect, 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.

[0082] 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).

[0083] In certain embodiments, 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.

[0084] In certain embodiments, the adjuvant is selected from the group consisting of: alum adjuvant, CpG adjuvant (eg, ODN-39M).

[0085] In certain embodiments, the adjuvant is ODN-39M (eg, ODN-39M as set forth in SEQ ID NO:9).

[0086] In certain embodiments, the immunogenic composition is a vaccine.

[0087] 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).

[0088] 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).

[0089] In certain embodiments, the immunogenic composition comprises 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).

[0090] 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).

[0091] In certain embodiments, the immunogenic composition comprises a chimeric protein as described above and a CpG adjuvant (e.g., ODN-39M, e.g., ODN-39M as set forth 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).

[0092] 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 set forth 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).

[0093] 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 set forth 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., respiratory route (e.g., intranasal route), digestive route (e.g., oral route), ocular route, rectal route), and the second immunogenic composition is administered via a parenteral route (e.g., subcutaneous route, intradermal route, intramuscular route).

[0094] 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 (e.g., in the mucosal system and / or systemic system).

[0095] In certain embodiments, the immunogenic composition has one or more characteristics selected from the group consisting of:

[0096] (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 a subject (e.g., in the mucosal system and / or systemic system);

[0097] (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 systemic system);

[0098] (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 the subject (e.g., in the mucosal system and / or systemic system);

[0099] (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 systemic system);

[0100] (5) Inducing a Th1-biased immune response in the subject (e.g., in the mucosal system and / or 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 subgenus and / or a Merbe subgenus coronavirus).

[0101] 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 capable of preventing or treating coronavirus infection or diseases caused by coronavirus infection.

[0102] In certain embodiments, the method comprises admixing a chimeric protein as described above with an adjuvant and a pharmaceutically acceptable carrier and / or excipient.

[0103] 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.

[0104] In certain embodiments, 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.

[0105] In certain embodiments, the adjuvant is selected from the group consisting of: alum adjuvant, CpG adjuvant (eg, ODN-39M).

[0106] In certain embodiments, the adjuvant is ODN-39M (eg, ODN-39M as set forth in SEQ ID NO:9).

[0107] 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.

[0108] 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.

[0109] In certain embodiments, the medicament is a vaccine.

[0110] In certain embodiments, the coronavirus is a beta coronavirus, such as a Sarbe and / or Merbe subgenus coronavirus.

[0111] In certain embodiments, the coronavirus is SARS-CoV-2, SARS-CoV-1, or MERS-CoV.

[0112] In certain embodiments, the disease associated with coronavirus infection includes but is not limited to COVID-19.

[0113] In certain embodiments, the subject is a mammal, such as a human.

[0114] On the other hand, the present application provides a method 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, 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.

[0115] In certain embodiments, the coronavirus is a beta coronavirus, such as a Sarbe and / or Merbe subgenus coronavirus.

[0116] In certain embodiments, the coronavirus is SARS-CoV-2, SARS-CoV-1, or MERS-CoV.

[0117] In certain embodiments, the disease associated with coronavirus infection includes but is not limited to COVID-19.

[0118] In certain embodiments, the subject is a mammal, such as a human.

[0119] Definition of terms

[0120] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0121] When the terms "for example," "such as," "including," "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."

[0122] 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) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0123] 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) of the Coronaviridae family. 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, which is a type I transmembrane protein composed of two parts, S1 and S2 protein subunits, that mediates the binding of the virus to host cell surface receptors and the fusion of cell membranes.

[0124] Those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and / or additions, such as S proteins of different strains of coronavirus) may occur naturally or be artificially introduced into the amino acid sequence of the S protein (including the sequences of the S1 subunit and the S2 subunit) without affecting its biological function. Therefore, in the present invention, the term "S protein" should 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 variants.

[0125] In the present invention, when referring to a fragment of an S protein sequence or its amino acid position, the description is made with reference to the sequence set forth in SEQ ID NO: 10 or 13. For example, the expression "amino acid residues at positions 806-1000 of the S protein corresponding to positions 806-1000 of SEQ ID NO: 10" refers to the amino acid sites / residues in the compared sequence that are located at equivalent positions to 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.

[0126] In the present invention, the terms "S2 subunit" or "S2 protein" have the same meaning and are used interchangeably. They generally refer 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 stalk-like manner and is responsible for mediating membrane fusion between 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 sequences of the S2 subunit and the S1 subunit are separated by the Furin cleavage site in the S protein.

[0127] 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 the S protein in SEQ ID NO: 10. 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 the S protein in SEQ ID NO: 13.

[0128] Herein, 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 include the cytoplasm region (Cytoplasm Domain) and the 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.

[0129] 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.

[0130] 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 occur naturally or be 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 the N protein shown in SEQ ID NO: 2 or 3 and its natural or artificial variants. 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 variants.

[0131] In the present invention, when referring to a sequence fragment of the N protein or its amino acid positions, the description is made with reference to the sequence set forth in SEQ ID NO: 3. For example, the expression "amino acid residues at positions 255-365 of the N protein corresponding to positions 255-365 of SEQ ID NO: 3" refers to 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.

[0132] As used herein, the term "identity" refers to the match 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 6 positions match). Typically, two sequences are compared when aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using 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.

[0133] 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 of residues physically or functionally similar to 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 the 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).

[0134] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell via 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 (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (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, a vector may also contain a replication initiation site.

[0135] 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.

[0136] 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 a transcript, protein, or peptide, including proteins, isolated nucleic acid molecules, etc., as described herein.

[0137] In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0138] 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 are used interchangeably.

[0139] Advantageous Effects of the Invention

[0140] Compared with existing marketed vaccines and vaccines under development, the vaccine composition provided by the present invention has the following advantages:

[0141] (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.

[0142] Cell-mediated immune responses can effectively protect against coronavirus infections, including SARS-CoV-2, and studies of SARS-CoV-1 infection have shown that the protective effects of the cell-mediated immune responses induced by them can last up to 17 years. Therefore, the cell-mediated cross-immune responses 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 have potential protective capabilities, at least against infection by beta coronaviruses.

[0143] (2) Induce cross-humoral immune responses against S protein (e.g., S2 protein) and N protein with broad-spectrum neutralizing ability.

[0144] Neutralizing antibodies against the 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.

[0145] (3) In particular, when administered via a mucosal route (e.g., intranasal route), the S2ND formulation provided herein and the N+S2 protein combination formulation 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 the related problems caused by injection administration. Therefore, it is particularly suitable for large-scale vaccination.

[0146] (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).

[0147] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the 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

[0148] Figure 1a: Analysis of expression and purification of cloned S2ND protein; samples were analyzed by 12% SDS-PAGE under reducing conditions; 1) molecular weight marker (MWM), 2) BL21(DE3) whole cells expressing S2ND protein, 3) BL21(DE3) whole cells (negative control), 4) purified S2ND protein.

[0149] Figure 1b: Analysis of expression and purification of cloned S2ND protein; samples were analyzed by western blotting (WB) under reducing conditions using an anti-N protein polyclonal antibody (40588-T62, purchased from Sino Biological Technology Co., Ltd., Beijing, China); 1) MWM, 2) BL21(DE3) whole cells expressing S2ND protein, 3) BL21(DE3) whole cells (negative control), 4) purified S2ND protein.

[0150] 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.

[0151] Figure 2b: Analysis of expression and purification of cloned S2 protein; samples were analyzed by Western blotting under reducing conditions using an anti-S2 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.

[0152] 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.

[0153] Figure 3b: Analysis of expression and purification of cloned N protein; samples were analyzed by Western blotting under reducing conditions using an anti-N protein polyclonal antibody (40588-T62, purchased from Sino Biological Technology Co., Ltd., Beijing, China); 1) MWM, 2) BL21(DE3) whole cells (negative control), 3) BL21(DE3) whole cells expressing N protein, 4) purified N protein.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] Figure 6: Anti-S2ND protein IgG ELISA test on the serum of immunized Balb / C mice to detect the immunogenicity of S2ND preparations; after logarithmic transformation of the titer, one-way ANOVA and Tukey's multiple comparison method were used for statistical analysis.

[0158] Figure 7: Anti-S2 IgG ELISA test on sera from immunized Balb / C mice to detect the immunogenicity of S2ND preparations; after logarithmic transformation of titers, statistical analysis was performed using the One Way Anova method and Tukeys' multiple comparison method.

[0159] Figure 8: Anti-N protein IgG ELISA test on the serum of immunized Balb / C mice to detect the immunogenicity of S2ND preparation; after logarithmic transformation, the titer was statistically analyzed using the One Way Anova method and Tukey's multiple comparison method.

[0160] Figure 9a: Detection results of IgG subclass antibodies in the serum of immunized Balb / C mice evaluated by anti-N protein IgG1 ELISA test; after logarithmic transformation, the titers were statistically analyzed using the one-way anova method and Tukeys' multiple comparison method.

[0161] Figure 9b: Detection results of IgG subclass antibodies in the serum of immunized Balb / C mice evaluated by anti-N protein IgG2a ELISA test; after logarithmic transformation of the titers, one-way analysis of variance (ANOVA) and Tukeys' multiple comparison method were used for statistical analysis.

[0162] Figure 10: Anti-N protein IgA ELISA test was performed on the bronchoalveolar lavage fluid (BALF) of immunized Balb / C mice (undiluted) to evaluate the humoral immune response induced by S2ND preparation. The Kruskal-Wallis nonparametric test and Dunns multiple comparison test were used for statistical analysis.

[0163] Figure 11: Through the conserved peptide N 351-365 The cell-mediated immune response induced by S2ND preparation in mice was detected by IFNγ-ELISPOT assay after stimulating splenocytes. The results were statistically analyzed using Kruskal-Wallis nonparametric test and Dunns multiple comparison test.

[0164] Figure 12: Anti-S2 protein IgG ELISA test on the serum of immunized C-57 / BL-6 mice to detect the immunogenicity of S2ND preparation and NO+S2 preparation. After logarithmic transformation, the titer was statistically analyzed using the one-way anova method and Tukeys' multiple comparison method.

[0165] Figure 13a: Detection results of IgG subclass antibodies in the serum of immunized C-57 / BL-6 mice evaluated by anti-S2 protein IgG1 ELISA test; after logarithmic transformation, the titers were statistically analyzed using the one-way anova method and Tukeys' multiple comparison method.

[0166] Figure 13b: Detection results of IgG subclass antibodies in the serum of immunized C-57 / BL-6 mice evaluated by anti-S2 protein IgG2a ELISA test; after logarithmic transformation, the titers were statistically analyzed using the One Way Anova method and Tukeys' multiple comparison method.

[0167] Figure 14: Anti-S2 protein IgA ELISA test was performed on BALF (undiluted) of immunized C-57 / BL-6 mice to evaluate the humoral immune response induced by S2ND preparations and S2 preparations in the mucosal system; statistical analysis was performed using the Kruskal-Wallis non-parametric test and Dunns' multiple comparison test.

[0168] Figure 15: Anti-N protein IgG ELISA test on the sera of immunized C-57 / BL-6 mice to evaluate the immunogenicity of S2ND and S2 formulations; after logarithmic transformation of the titer, statistical analysis was performed using the One Way Anova method and Tukeys' multiple comparison method.

[0169] Figure 16a: Detection of IgG subclass antibodies in the serum of immunized C-57 / BL-6 mice assessed by anti-N protein IgG1 ELISA; titers were log-transformed and statistically analyzed using the One Way Anova method with Tukeys' multiple comparisons.

[0170] Figure 16b: IgG subclass antibody detection results in the serum of immunized C-57 / BL-6 mice evaluated by anti-N protein IgG2a ELISA test; after logarithmic transformation, the titers were statistically analyzed using the One Way Anova method and Tukeys' multiple comparison method.

[0171] Figure 17: Anti-N protein IgA ELISA test was performed on BALF (undiluted) of immunized C-57 / BL-6 mice to evaluate the humoral immune response induced by S2ND preparation and S2 preparation in the mucosal system; statistical analysis was performed using the Kruskal-Wallis non-parametric test and Dunns' multiple comparison test.

[0172] Figure 18a: The results of the cell-mediated immune response induced by S2ND preparation and S2 preparation in C-57 / BL-6 mice were detected by IFNγ-ELISPOT assay after splenocytes were stimulated with S2 protein. The Kruskal-Wallis nonparametric test and Dunns' multiple comparison test were used for statistical analysis.

[0173] Figure 18b: The results of the cell-mediated immune response induced by S2ND preparation and S2 preparation in C-57 / BL-6 mice were 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.

[0174] Figure 19: Evaluation of the cross-reactivity of IgG antibodies with SARS-CoV-2 S2 protein, SARS-CoV-1 S2 protein, and MERS-CoV S2 protein in the serum of Group C, which received the NO+S2 formulation via the intranasal route; the titers were log-transformed and statistically analyzed using the One Way Anova method and Tukeys' multiple comparison method.

[0175] Figure 20a: Results of the test evaluating the cross-reactivity of IgG antibodies in the serum of Group B that received the S2NDO formulation via the intranasal route with the N protein of the SARS-CoV-2 Delta variant, the N protein of the SARS-CoV-2 Omicron variant, and the N protein of SARS-CoV-1; statistical analysis was performed using the Kruskal-Wallis nonparametric test and the Dunns' multiple comparison test.

[0176] Figure 20b: Evaluation of the cross-reactivity of IgG antibodies in the serum of Group C, which received the NO+S2 formulation via the intranasal route, with the N protein of the SARS-CoV-2 Delta variant, the N protein of the SARS-CoV-2 Omicron variant, and the N protein of SARS-CoV-1; statistical analysis was performed using the Kruskal-Wallis nonparametric test and the Dunns' multiple comparison test.

[0177] Figure 21: Anti-S2 protein (SARS-CoV-2 S2 protein, SARS-CoV-1 S2 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 the intranasal route to evaluate the humoral cross-immune response induced by intranasal inoculation of NO+S2 preparations in the mucosal system; the Kruskal-Wallis non-parametric test and Dunns' multiple comparison test were used for statistical analysis.

[0178] Figure 22a: Anti-N protein (SARS-CoV-2 Delta variant N protein, SARS-CoV-2 Omicron variant N protein, and SARS-CoV-1 N protein) IgA ELISA test was performed on the BALF of group B (undiluted) that was inoculated with the S2NDO preparation via the intranasal route to evaluate the humoral cross-immune response induced by the S2NDO preparation in the mucosal system; the Kruskal-Wallis non-parametric test and Dunns' multiple comparison test were used for statistical analysis.

[0179] Figure 22b: Anti-N protein (SARS-CoV-2 Delta variant N protein, SARS-CoV-2 Omicron variant N protein and SARS-CoV-1 N protein) IgA ELISA test was performed on the BALF of group C (undiluted) inoculated with NO+S2 preparation via the intranasal route to evaluate the humoral cross-immune response induced by intranasal administration of NO+S2 preparation in the mucosal system; statistical analysis was performed using the Kruskal-Wallis non-parametric test and Dunns' multiple comparison test.

[0180] Figure 23a: The results of the test on group B sera that were inoculated with S2NDO formulations via the intranasal route were evaluated by using the vesicular stomatitis virus (VSV) pseudovirus system (SARS-CoV-2 Delta variant S-VSV, SARS-CoV-2 Omicron variant S-VSV, SARS-CoV-1 S-VSV and MERS-CoV S-VSV) to evaluate the cross-neutralizing antibody response induced by intranasal administration of S2NDO formulations.

[0181] Figure 23b: The sera of Group C that were inoculated with NO+S2 preparations via the intranasal route were tested using the VSV pseudovirus system (SARS-CoV-2 Delta variant S-VSV, SARS-CoV-2 Omicron variant S-VSV, SARS-CoV-1 S-VSV and MERS-CoV S-VSV) to evaluate the cross-neutralizing antibody response induced by intranasal administration of NO+S2 preparations.

[0182] Figure 24a: The BALF of group B that was inoculated with S2NDO preparations via intranasal route was tested using the VSV pseudovirus system (SARS-CoV-2 Delta variant S-VSV, SARS-CoV-2 Omicron variant S-VSV, SARS-CoV-1 S-VSV and MERS-CoV S-VSV) to evaluate the cross-neutralizing antibody response induced by intranasal administration of S2NDO preparations.

[0183] Figure 24b: The BALF of group C, which was inoculated intranasally with NO+S2 preparation, was tested using the VSV pseudovirus system (SARS-CoV-2 Delta variant S-VSV, SARS-CoV-2 Omicron variant S-VSV, SARS-CoV-1 S-VSV and MERS-CoV S-VSV) to evaluate the cross-neutralizing antibody response induced by intranasal inoculation with NO+S2 preparation.

[0184] Figure 25a: The results of the cell-mediated cross-immune response induced by intranasal administration of the S2NDO preparation combined with subcutaneous administration of the S2ND + alum preparation in group C were detected by IFNγ-ELISPOT assay after spleen cells were stimulated with SARS-CoV-2 Delta variant S2 protein, SARS-CoV-2 Omicron variant S2 protein, SARS-CoV-1 S2 protein, or MERS-CoV S2 protein. Statistical analysis was performed using the Kruskal-Wallis nonparametric test and Dunns' multiple comparison test.

[0185] Figure 25b: The results of the cell-mediated cross-immune response induced by group C inoculated with NO+S2 preparation via intranasal administration were detected by IFNγ-ELISPOT assay after splenocytes were stimulated with SARS-CoV-2 Delta variant S2 protein, SARS-CoV-2 Omicron variant S2 protein, SARS-CoV-1 S2 protein or MERS-CoV S2 protein. The Kruskal-Wallis nonparametric test and Dunns' multiple comparison test were used for statistical analysis.

[0186] Figure 26a: Through SARS-CoV-2 Delta variant N protein, SARS-CoV-2 Omicron variant N protein, SARS-CoV-1 N protein or conserved peptide N 351-365 The results of the IFNγ-ELISPOT assay after stimulating splenocytes were used to detect the cell-mediated cross-immune response induced by group B inoculated with S2NDO preparations via intranasal route. The Kruskal-Wallis nonparametric test and Dunns' multiple comparison test were used for statistical analysis.

[0187] Figure 26b: Through SARS-CoV-2 Delta variant N protein, SARS-CoV-2 Omicron variant N protein, SARS-CoV-1 N protein or conserved peptide N 351-365 The results of IFNγ-ELISPOT assay after stimulating splenocytes were used to detect the cell-mediated cross-immune response induced by group B inoculated with NO+S2 preparation via intranasal route. The Kruskal-Wallis nonparametric test and Dunns' multiple comparison test were used for statistical analysis. DETAILED DESCRIPTION

[0188] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0189] 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., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0190] Example 1 Cloning, expression, purification and antigenic characterization of S2ND protein, S2 protein fragment and N protein

[0191] 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 Escherichia coli. The S2ND protein is an S2ND chimeric protein with a DNA sequence of SEQ ID NO: 8, which is formed by fusion of the S protein fragment aa.798-1018 and the N protein fragment aa.248-371 of the SARS-CoV-2 Delta variant (YP 009724397.2). The expressed protein accounts for approximately 7% of the total protein in Escherichia 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 Yiqiao Shenzhou Technology Co., Ltd.) and Western blotting (WB) were used for verification (Figure 1b).

[0192] The amino acid sequence (SEQ ID NO: 14) encoding the SARS-CoV-2 Wuhan Hu-1 strain (NP_828851.1) spike protein S2 subunit fragment 712-1204 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.). As shown in Figure 2a, an S2 subunit fragment close to the theoretical molecular weight was detected in the supernatant, and was verified by WB using an anti-S2 protein polyclonal antibody (Figure 2b). As shown in Figures 2a and 2b, the purity of the purified recombinant protein reached more than 95%.

[0193] The amino acid sequence encoding the N protein of the SARS-CoV-2 Delta variant (YP 009724397.2) (SEQ ID NO: 2) was cloned into the PET-28 vector for expression in E. coli. The resulting protein accounted for approximately 5% of the total protein in E. coli (Figure 3a). The resulting protein was purified by ion exchange chromatography and gel filtration to a purity of over 95%, and was verified by Western blotting using an anti-N protein polyclonal antibody (40588-T62, purchased from Beijing Sino Biological Technology Co., Ltd.) (Figure 3b).

[0194] Example 2 Characterization of N protein, S2 protein, and S2ND protein and their reaction with ODN-39M

[0195] For antigenic 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, reflecting the correct folding of the protein and the exposure of the relevant antigenic epitopes (Figure 4).

[0196] To enhance the induction of cell-mediated immune responses, the S2ND and N proteins were each conjugated with ODN-39M (DNA sequence: SEQ ID NO: 9) to prepare formulations. ODN-39M is a phosphodiester-based CpG oligodeoxynucleotide (ODN) adjuvant with an effective mucosal delivery system (Gil et al., 2015). The resulting formulations were antigenically characterized using sera from the same group of recovered volunteers. As shown in Figure 5, serum recognition of the S2ND and N proteins was similar compared to that of the formulation without ODN-39M. This suggests that the addition of ODN-39M does not affect the proper conformation of the S2ND and N proteins.

[0197] Example 3 Design of Balb / C mouse immunization scheme and immunological evaluation of different formulations based on the S2ND protein

[0198] Balb / C mice were used to evaluate the S2ND protein in various formulations. The Balb / C mouse model is particularly suitable for detecting cell-mediated immune responses against the N protein because it can reasonably represent immunodominant epitopes.

[0199] Seven groups of Balb / C mice (female, 6-8 weeks old) were immunized on days 0, 7, and 21 with 10 μg of the recombinant protein via intranasal or subcutaneous injection according to the following protocol. All immunogens were dissolved in phosphate-buffered saline (PBS). The inoculum size for the intranasal route was 50 μL, and the subcutaneous injection size was 100 μL. Aluminum hydroxide was used as an adjuvant for the subcutaneous injection formulation.

[0200] Group 1: S2ND formulation, intranasal administration

[0201] Group 2: S2ND+ODN-39M (S2NDO) formulation, intranasal inoculation

[0202] Group 3: S2ND+alum preparation, subcutaneous injection route

[0203] Group 4: S2ND+ODN-39M formulation, subcutaneous injection

[0204] Group 5: S2ND+ODN-39M+alum preparation, subcutaneous injection (100 μL)

[0205] Group 6: PBS + alum preparation (negative control), subcutaneous injection (100 μL)

[0206] Group 7: PBS (negative control), inoculated via intranasal route;

[0207] The amino acid sequence of the S2ND protein is shown in SEQ ID NO: 7.

[0208] Mice were sacrificed on days 19 and 26 after the last immunization, and the induced humoral and cell-mediated immune responses were evaluated.

[0209] Example 4 Assessment of humoral immune response

[0210] Humoral immune response against the S2ND protein and S2 protein

[0211] On day 19 after the last immunization of 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).

[0212] Serum samples were analyzed by IgG ELISA against the primary SARS-CoV-2 virus strain S2 recombinant protein (40590-V08H1, purchased from Sino Biological Technology Co., Ltd., Beijing, China). As shown in Figure 7, 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 .

[0213] The S2 region within the S2ND protein exhibits a fusogenic, fibrous structure, significantly different from the pre-fusogenic form of the S2 protein. The low antibody response suggests that the S2 protein conformation contained within the S2ND protein structure differs from the pre-fusogenic form of the S2 protein used to coat the ELISA plate. The S2 protein conformation contained within the S2ND protein structure facilitates the construction of the S2ND protein. However, the presence of common antigenic epitopes in both conformations is suggested by the presence of certain anti-S2 antibody antibodies.

[0214] Humoral immune response against the N protein of the SAR-CoV-2 Delta variant

[0215] 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-N protein (SAR-CoV-2 Delta variant N protein, 40588-V07E29, purchased from Sino-Bio Technologies Co., Ltd., Beijing, China) IgG ELISA test. As shown in Figure 8, all groups vaccinated with the S2ND formulation induced high levels of anti-N protein antibodies. When measuring IgG subclass antibodies, the groups vaccinated with the S2NDO formulation via the intranasal route and the subcutaneous injection route induced the highest levels of IgG2a antibody response, which was statistically significantly different from the group vaccinated with the S2ND + alum formulation via the subcutaneous injection route (Figure 9b). In addition, compared with the subcutaneous injection route, the two groups vaccinated via the intranasal route induced relatively lower levels of IgG1 antibodies (Figure 9a).

[0216] The results of the IgA antibody response test against the N protein of the SAR-CoV-2 Delta variant in BALF samples of all groups showed that only the group vaccinated with the S2ND preparation via intranasal route (including the group vaccinated with the S2ND preparation via intranasal route and the group vaccinated with the S2NDO preparation via intranasal route) induced IgA antibody response in BALF (Figure 10).

[0217] Example 5 Evaluation of cell-mediated immune responses in vaccinated Balb / C mice

[0218] Anti-conserved peptide N 351-365 Cell-mediated immune response

[0219] 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) was synthesized by Zhejiang Paipeptide Biological Co., Ltd. in China with a purity of over 97%.

[0220] As shown in FIG11 , in the conserved peptide N 351-365 Upon stimulation, IFNγ-secreting cells were detected in both the intranasal and S2NDO groups. However, no IFNγ-secreting cells were detected in the spleen cells of mice in the subcutaneous S2NDO, S2ND+Alum, and S2NDO+Alum groups. Furthermore, only one mouse in the subcutaneous S2NDO group tested positive.

[0221] Based on all the anti-N protein immune responses tested, it was concluded that intranasal vaccination with S2ND preparation is beneficial to inducing Th1 pattern and cell-mediated immune responses against N protein antigen epitopes.

[0222] Example 6 Design of an Immunization Plan for C-57 Mice and Immunological Evaluation of Different Formulations of Combined Vaccination with S2 Protein, N Protein, and S2ND Protein

[0223] To evaluate the immunogenicity of the recombinant S2 fragment, two formulations were combined with the N protein and the S2ND protein and administered intranasally and subcutaneously to C57BL / 6 mice. The C57BL / 6 mouse model is particularly well-suited for testing cell-mediated immune responses to the S2 region, as it allows for the proper representation of immunodominant epitopes.

[0224] Six groups of C57BL / 6 mice (female, 6-8 weeks old) were immunized on days 0, 7, and 21 according to the following protocol. The inoculum size was 10 μg for S2ND and S2 recombinant proteins, and 5 μg for N protein. All immunogens were dissolved in PBS. The inoculum size was 50 μL for the intranasal route and 100 μL for the subcutaneous route. Aluminum hydroxide was used as an adjuvant for the subcutaneous formulation.

[0225] Group 1: N+ODN-39M+S2 (NO+S2) formulation, intranasal inoculation

[0226] Group 2: S2 formulation, intranasal administration

[0227] Group 3: Intranasal inoculation of S2ND+ODN-39M (S2NDO) formulation combined with subcutaneous inoculation of S2ND+Alum formulation

[0228] Group 4: N+ODN-39M+S2+Alum (NO+S2+Al) formulation, subcutaneous injection

[0229] Group 5: S2+Alum preparation,

[0230] Group 6: intranasal inoculation of PBS preparation combined with subcutaneous inoculation of PBS+alum preparation (negative control);

[0231] The sequences of the S2ND chimeric protein, S2 protein, and N protein in the preparation are shown in SEQ ID NO: 7, SEQ ID NO: 14, and SEQ ID NO: 2, respectively.

[0232] Mice were sacrificed on days 18 and 26 after the last immunization, and the induced humoral and cell-mediated immune responses were evaluated.

[0233] Example 7 Assessment of humoral immune response

[0234] Humoral immune response against S2 protein of primary SARS-CoV-2 strain

[0235] Eighteen days after the last immunization in Example 6, mouse sera were analyzed by IgG ELISA against the primary SARS-CoV-2 strain S2 recombinant protein (40590-V08H1, purchased from Sino Biological Technologies, Inc., Beijing, China). As shown in Figure 12, all groups vaccinated with the S2 formulation induced high levels of anti-S2 antibody responses. The groups vaccinated with the S2 formulation alone via the intranasal route induced the same level of antibody responses as the groups vaccinated with the S2 formulation plus alum via subcutaneous injection. The group vaccinated with the NO formulation plus the S2 formulation via the intranasal route achieved the highest anti-S2 antibody responses.

[0236] The examples also examined anti-S2 antibody responses in a group (Group 3) that received both the intranasal S2NDO formulation and the subcutaneous S2ND+Alum formulation. Similar to the previous Balb / C experiments, the induced anti-S2 antibody levels were lower than those in the group receiving the S2 formulation. This may be due to the different conformations of the two proteins.

[0237] The Examples measured IgG subclass antibody responses. As shown in Figure 13b, only the group receiving the NO+S2 formulation intranasally induced a high level of anti-S2 protein IgG2a antibody response. All test groups, except for the group receiving the S2NDO formulation intranasally combined with the S2ND+Alum formulation subcutaneously (Group 3), induced similar IgG1 antibody levels (Figure 13a). These results suggest that intranasal administration of the NO+S2 formulation can induce a Th1-biased response targeting the S2 protein region.

[0238] The Example detected the level of IgA antibody response against S2 protein in BALF of the intranasal vaccination group. As shown in Figure 14, IgA antibody response was induced in BALF of both groups vaccinated with S2 formulation intranasally, and the IgA antibody level induced in the group vaccinated with NO+S2 formulation was significantly higher.

[0239] To the best of the inventors' knowledge, this application reports for the first time the immunogenicity of the S2 subunit via intranasal administration. Data from this application show that even a formulation of the S2 subunit prepared solely based on PBS exhibits good immunogenicity, whereas other SARS-CoV-2 recombinant proteins, such as RBD or N protein, fail to induce an immune response in serum or BALF via intranasal administration.

[0240] Humoral immune response against the N protein of the SARS-CoV-2 Delta variant

[0241] On day 18 after the last dose of immunization in Example 6, mouse sera were analyzed by IgG ELISA test against SARS-CoV-2 Delta variant N protein (40588-V07E29, purchased from Sino-Bio Technologies, Inc., Beijing, China). As shown in Figure 15, the groups vaccinated with N combined with S2 or S2ND formulations all 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 to the group vaccinated with S2NDO formulation intranasally combined with S2ND+Alum formulation subcutaneously (Group 3) (Figure 16b). No statistical difference was detected between the groups for IgG1 subclass antibodies (Figure 16a).

[0242] The Example examined IgA antibody responses against the Delta variant N protein in the BALF of the same groups vaccinated intranasally, and the results are shown in Figure 17. Similar levels of anti-N protein IgA antibody responses were induced in the BALF of the groups vaccinated intranasally with the NO+S2 formulation and the group vaccinated intranasally with the S2NDO formulation combined with the S2ND+Alum formulation subcutaneously.

[0243] 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 immunogens administered were more likely to induce a Th1-biased anti-N protein humoral immune response at the mucosal site.

[0244] Example 8 Assessment of cell-mediated immune response

[0245] Cell-mediated immune responses against S2 and N proteins

[0246] On the 26th day after the last dose of immunization in Example 6, after in vitro stimulation with the primary SARS-CoV-2 virus strain S2 protein (40590-V08H1, purchased from Sino Biological Technology Co., Ltd., Beijing, China), positive splenocytes producing IFNγ were detected using Mabtech antibodies and well plates. The group vaccinated intranasally with the NO+S2 formulation induced a positive response in IFNγ-secreting cells, while no IFNγ-secreting cell response was detected in the splenocytes of mice in the group vaccinated intranasally with the S2 formulation. In addition, a positive response was also detected in the group vaccinated intranasally with the S2NDO formulation combined with the S2ND+Alum formulation, while no such response was detected in the group vaccinated subcutaneously (Figure 18a).

[0247] At the same time, splenocytes positive for IFNγ production were detected after in vitro stimulation with the SARS-CoV-2 Delta variant N protein (40588-V07E29, purchased from Sino Biological Technology Co., Ltd., Beijing, China). A positive response was induced only in the group receiving the NO+S2 formulation via intranasal administration (Figure 18b). This response was not detected in the group receiving the S2NDO formulation via intranasal administration combined with the S2ND+Alum formulation via subcutaneous injection, as the N protein region contained in the S2ND construct does not contain an immunodominant epitope in C57BL / 6 mice.

[0248] 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 via intranasal route and the groups vaccinated with S2NDO preparations via intranasal route combined with S2ND+Alum preparations via subcutaneous injection can induce the broadest immune responses in both the mucosal system and the systemic system.

[0249] Example 9 Design of vaccination scheme and detection of cross immune response induced by vaccination with N+ODN-39M+S2 formulation and S2ND+ODN-39M / S2ND+Alum formulation

[0250] 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.

[0251] Three groups of Balb / C mice (female, 6-8 weeks old) and three groups of C-57BL / 6 mice (female, 6-8 weeks old) were vaccinated with the designated immunogens on days 0, 15, and 30, and on days 0, 7, and 21. The inoculation dose of S2ND and S2 protein preparations was 10 μg, and the inoculation dose of N protein preparation was 5 μg. All immunogens administered intranasally were dissolved in PBS (50 μL per mouse). Alum (Alhydrogel purchased from InvivoGen, USA) was added as an adjuvant for the S2ND preparation administered subcutaneously (100 μL per mouse). The design scheme is as follows:

[0252] 1. Group B: S2ND+ODN-39M (S2NDO) formulation, intranasally inoculated into Balb / C mice

[0253] 2. Group B: N+ODN-39M+S2 (NO+S2) formulation, intranasally inoculated into Balb / C mice

[0254] 3. Group B: PBS preparation (negative control), intranasal inoculation of Balb / C mice

[0255] 4. Group C: C-57BL / 6 mice were intranasally inoculated with S2ND+ODN-39M (S2NDO) and subcutaneously inoculated with S2ND+alum.

[0256] 5. Group C: N+ODN-39M+S2 (NO+S2) formulation, intranasally inoculated into C-57BL / 6 mice

[0257] 6. Group C: C-57BL / 6 mice inoculated with PBS preparation via intranasal route combined with subcutaneous injection (negative control);

[0258] The sequences of the S2ND chimeric protein, S2 protein, and N protein in the preparation are shown in SEQ ID NO: 7, SEQ ID NO: 14, and SEQ ID NO: 2, respectively.

[0259] After the last dose of 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.

[0260] Example 10 Assessment of humoral immune response

[0261] On day 18 after the last dose of vaccination in Example 9, sera from mice in Group C that had been vaccinated intranasally with the NO+S2 formulation were analyzed by IgG ELISA against the primary SARS-CoV-2 strain S2 protein (40590-V08H1), SARS-CoV-1 S2 protein (40150-V08B3), and MERS-CoV S2 protein (40070-V08B). The S2 proteins of the above-mentioned strains were purchased from Beijing Sino Biological Technology Co., Ltd. As shown in Figure 19, similar antibody titers were obtained for all tested antigens, indicating that cross-humoral immune responses against the S2 protein can be induced.

[0262] At the same time, sera from mice in group B, which were intranasally inoculated with the S2NDO formulation, and group C, which were intranasally inoculated with the NO+S2 formulation, were analyzed by IgG ELISA against the SARS-CoV-2 Delta variant N protein (40588-V07E29), the SARS-CoV-2 Omicron variant N protein (40588-V07E34), and the SARS-CoV-1 N protein (40143-V08B). The N proteins of the above-mentioned virus strains were purchased from Beijing Sino Biological Science and Technology Co., Ltd. As shown in Figures 20a and 20b, all sera obtained similar antibody response levels against the N proteins of the above-mentioned virus strains, indicating that the sera have a broad cross-reactivity profile with coronaviruses of the Sarbe subgenus.

[0263] For mucosal immune responses, BALF from mice in Group C, which had been intranasally inoculated with the NO+S2 formulation, were analyzed using IgA ELISA assays against the SARS-CoV-2 S2 protein, SARS-CoV-1 S2 protein, and MERS-CoV S2 protein. As shown in Figure 21, similar to the response levels observed in the serum, BALF from C57BL / 6 mice in the group intranasally inoculated with the NO+S2 formulation showed positive responses against the S2 proteins of these viral strains.

[0264] At the same time, BALF samples from group C, which received the NO+S2 formulation intranasally, and group B, which received the S2NDO formulation intranasally, were tested for responses to the SARS-CoV-2 Delta variant N protein (40588-V07E29), the SARS-CoV-2 Omicron variant N protein (40588-V07E34), and the SARS-CoV-1 N protein (40143-V08B). The N proteins of the above-mentioned virus strains were purchased from Beijing Sino Biological Science and Technology Co., Ltd. The results showed that both groups obtained broad cross-reactivity against the N proteins of the above-mentioned virus strains (Figures 22a and 22b).

[0265] Neutralization test

[0266] Neutralization assays were conducted using a vesicular stomatitis virus (VSV) pseudovirus production system. When pseudoviruses are incubated with samples containing neutralizing antibodies prior to cell infection, they block entry or fusion with cells. The amount of virus blocked can be determined using chemiluminescent reduction assays, which reflect 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 measure neutralizing antibody levels in serum and BALF samples from groups receiving the S2ND and S2 formulations (specifically, Group B, which received the S2NDO formulation intranasally, and Group C, which received the NO+S2 formulation intranasally). Figures 23a and 23b represent the neutralizing antibody levels against each pseudovirus in the serum of the test groups. The results show that neutralizing antibody titers were detected in all test samples. Positive neutralization responses against the four pseudoviruses indicate that each group induced a cross-neutralizing antibody response. Figures 24a and 24b represent the neutralizing antibody titers 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 groups.

[0267] Example 11 Assessment of cell-mediated immune responses

[0268] Based on the immunodominance of SARS-CoV-2 antigenic epitopes in different mouse strains, C57BL / 6 mice were used to detect the immune response against S2 protein, and Balb / C mice were used to detect the cell-mediated immune response against N protein.

[0269] On the 26th day after the last vaccination in Example 9, splenocytes were collected from Group C, which was inoculated with the S2NDO preparation intranasally and the S2ND+Alum preparation subcutaneously, and from Group C, which was inoculated with the NO+S2 preparation intranasally. After in vitro stimulation with the primary SARS-CoV-2 virus strain S2 protein (40590-V08H1), SARS-CoV-1 S2 protein (40143-V08B), and MERS-CoV S2 protein (40070-V08B), IFNγ-positive splenocytes were detected. The results showed that both groups obtained a positive cell-mediated immune response against the S2 protein of the above-mentioned virus strains, indicating that the S2ND protein and S2 protein in the test preparation can induce a cell-mediated cross-immune response (Figures 25a and 25b).

[0270] In order to evaluate the anti-N protein immune response, group B, which was vaccinated with S2NDO preparation by intranasal route, and group B, which was vaccinated with NO+S2 preparation 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-2 Delta variant N protein (40588-V07E29), SARS-CoV-2 Omicron variant N protein (40588-V07E34), SARS-CoV-1 N protein (40143-V08B) and conserved peptide N were used to detect the N protein. 351-365 Splenocytes were stimulated in vitro, and the results are shown in Figures 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.

[0271] Based on the similar humoral immunity and cell-mediated immune cross-responses obtained through 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.

[0272] Example 12 Results Summary

[0273] Table 1 summarizes exemplary characteristics of exemplary vaccine compositions of the invention.

[0274] The candidate vaccine provided in this application is based on two conserved protein fragments of SARS-CoV-2 (N protein and S2 protein fragments), which can induce cross-humoral immune responses and cell-mediated cross-immune responses against the N protein and S2 protein of the beta coronavirus. At the same time, it can also induce humoral immune responses in the mucosal system.

[0275] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

[0276] Sequence information

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 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% (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 the amino acid sequence 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 the amino acid sequence shown in SEQ ID NO: 14 or 15, or has at least 90% (e.g. at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or a sequence having, or consisting of, one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions.

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., respiratory route (e.g., intranasal route), digestive route (e.g., oral route), ocular route, 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.

22. 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 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 to a subject in need thereof; Preferably, the coronavirus is a β coronavirus, such as a Sarbe subgenus and / or a Merbe subgenus coronavirus; Preferably, the disease associated with coronavirus infection includes but is not limited to COVID-19; Preferably, the subject is a mammal, such as a human.

Images