Coronavirus multivalent vaccine and application thereof

Abstract

The invention provides a coronavirus multivalent vaccine and application thereof. The coronavirus multivalent vaccine comprises a mutant coronavirus Spike protein extracellular structural domain or a truncated fragment thereof or a fusion protein containing the mutant coronavirus Spike protein extracellular structural domain. The invention also provides application of the coronavirus multivalent vaccine in preparation of drugs for preventing or treating coronavirus infection.

Description

A coronavirus multivalent vaccine and its application Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a coronavirus polyvalent vaccine and applications thereof. Background Art

[0002] Coronaviruses are non-segmented, single-stranded, positive-sense RNA viruses. Based on serotype and genomic characteristics, the subfamily Coronavirinae is divided into four genera: α, β, γ, and δ. They are named for the protruding projections on the viral envelope, resembling a corolla. The novel coronavirus (SARS-CoV-2 or 2019-nCoV) belongs to the β genus. It has an envelope, and its particles are round or oval, often pleomorphic, with a diameter of 60-140 nm. Current research shows that SARS-CoV-2 shares a high degree of homology with SARS-CoV.

[0003] The novel coronavirus, COVID-19, is primarily transmitted through the respiratory tract, though it can also be spread through contact. The disease affects a broad range of individuals, with the elderly and those with underlying medical conditions experiencing more severe illness. Children and infants can also develop the disease. The primary clinical symptoms are fever, fatigue, and a dry cough, while upper respiratory symptoms such as nasal congestion and runny nose are less common. In the early stages of the disease, patients may have normal or decreased white blood cell counts or decreased lymphocyte counts. Some patients may experience elevated liver enzymes, myoglobin, and creatinine levels. Chest imaging reveals early signs of multiple small patchy shadows and interstitial changes, particularly in the outer lung zones. These changes progress to multiple ground-glass opacities and infiltrates in both lungs. Severe cases may develop pulmonary consolidation, followed by progressive respiratory distress, and eventually acute respiratory distress syndrome (ARDS), shock, and multiple tissue damage and dysfunction in the lungs, heart, and kidneys. While most patients with mild infection have a good prognosis, those with severe disease often present in critical condition or even death.

[0004] Recently, basic, clinical, and epidemiological studies on COVID-19 have been continuously published or announced, and there is an urgent need for an effective vaccine against coronavirus in this field.

[0005] Summary of the Invention

[0006] The present invention provides a multivalent coronavirus vaccine. In some embodiments, the multivalent coronavirus vaccine comprises a coronavirus spike protein extracellular domain containing a mutation or a truncated fragment thereof. In some embodiments, the multivalent coronavirus vaccine comprises a fusion protein comprising a coronavirus spike protein extracellular domain containing a mutation or a truncated fragment thereof. The multivalent coronavirus vaccine of the present invention can induce a stronger neutralizing antibody response against coronavirus.

[0007] The virus particle first binds to angiotensin-converting enzyme 2 (ACE2) on the surface of lung epithelial cells through the receptor binding domain (RBD) in the S1 subunit of the Spike protein (S protein or spike protein). After the RBD binds to the receptor and is hydrolyzed by proteases, the S2 subunit located at the C-terminus of the S protein is exposed and embedded in the plasma membrane or endosomal membrane. The heptad repeat sequence 1 (HR1) and the heptad repeat sequence 2 (HR2) in the S2 subunit interact with each other to form a six-helix bundle (6-HB) fusion core, causing the viral capsid to fuse with the cell membrane. SARS-CoV or SARS-CoV-2 enters the cell and uses the cell to synthesize new virus particles. These new virus particles are then released outside the cell and infect surrounding normal cells in the same way.

[0008] In some embodiments, the coronavirus Spike protein extracellular domain or a truncated fragment thereof contains mutations, wherein the mutations include: 1) RRAR is mutated to GSAS; 2) there is a mutation in the turning region between HR1 and the central helix region (CH) that prevents HR1 and CH from forming a straight helix during fusion.

[0009] In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P / V987P in the turn region between HR1 and CH.

[0010] In some embodiments, the amino acid numbering of the coronavirus Spike protein is based on the amino acid numbering of the cryo-EM model PDB ID 6VSB or GenBank accession number MN908947.3 as a reference.

[0011] In some embodiments, the truncated fragment of the coronavirus Spike protein extracellular domain containing the mutation has 5-80 amino acid residues truncated at the C-terminus compared to the full-length extracellular domain of the coronavirus Spike protein. In some embodiments, the truncated fragment of the coronavirus Spike protein extracellular domain containing the mutation has 20-76 amino acid residues truncated at the C-terminus compared to the full-length extracellular domain of the coronavirus Spike protein. In some embodiments, the truncated fragment of the coronavirus Spike protein extracellular domain containing the mutation has 70 amino acid residues truncated at the C-terminus compared to the full-length extracellular domain of the coronavirus Spike protein.

[0012] In some embodiments, the coronavirus Spike protein extracellular domain or a truncated fragment thereof is from SARS-CoV-2, SARS-CoV or MERS-CoV. In some embodiments, the coronavirus Spike protein extracellular domain or a truncated fragment thereof is from the SARS-CoV-2 original strain or a variant thereof. In some embodiments, the coronavirus Spike protein extracellular domain or a truncated fragment thereof is from the SARS-CoV-2 original strain, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant.

[0013] In some embodiments, the mutation-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs: 3, 4, 6, 7, 9-12, 32-35, 78-83, or an amino acid sequence that is at least 80% or at least 90% identical to the amino acid sequence shown in any one of SEQ ID NOs: 3, 4, 6, 7, 9-12, 32-35, 78-83, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 3, 4, 6, 7, 9-12, 32-35, 78-83.

[0014] In some embodiments, the fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the linker is a GS linker. In some embodiments, the linker is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS)2, (GGGGS)3, or any combination thereof. In some embodiments, the linker is (G m S) n , wherein each m is independently 1, 2, 3, 4 or 5, and n is 1, 2, 3, 4 or 5. In some embodiments, the sequence of the linker is (GGGGS) n, n is 1, 2, 3, 4 or 5. In some embodiments, the linker is GGGGS. In some embodiments, the linker is (GGGGS)2. In some embodiments, the linker is (GGGGS)3. In some embodiments, the linker is (GGGGS)4. In some embodiments, the linker is (GGGGS)5. In some embodiments, the monomeric subunit protein is a self-assembled monomeric subunit protein. In some embodiments, the monomeric subunit protein is a monomeric ferritin subunit. In some embodiments, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, algal ferritin, insect ferritin, fungal ferritin and mammalian ferritin. In some embodiments, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit. In some embodiments, there is an N19Q mutation in the amino acid sequence of the Helicobacter pylori non-heme monomeric ferritin subunit. In some embodiments, the monomeric ferritin subunit comprises the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 14.

[0015] In some embodiments, the fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker, and the mutation comprises: 1) RRAR is mutated to GSAS; 2) there is a mutation in the turn region between HR1 and CH that prevents the formation of a straight helix during the fusion process. In some embodiments, the fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof is a fusion protein in which the C-terminus of the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof is connected to the N-terminus of the monomeric subunit protein through a linker. In some embodiments, the fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof is a fusion protein in which the C-terminus of the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof is connected to the N-terminus of the monomeric ferritin subunit through a linker.

[0016] In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof further comprises an N-terminal signal peptide.

[0017] In some embodiments, the signal peptide is selected from the group consisting of CSP, mschito, MF-α, pho1, HBM, t-pA, and the signal peptide of IL-3.

[0018] In some embodiments, the N-terminal signal peptide comprises an amino acid sequence as shown in SEQ ID NO: 2 or 5, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2 or 5, or an amino acid sequence having one or more conservative amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 2 or 5.

[0019] In some embodiments, the fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of the SARS-CoV-2 original strain Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of the SARS-CoV-2 original strain Spike protein containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutation comprises: 1) RRAR is mutated to GSAS; 2) the double mutation K986P / V987P is present in the turn region between HR1 and CH.

[0020] In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 Alpha variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 Alpha variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutation comprises: 1) RRAR to GSAS mutation; 2) the presence of a double mutation K986P / V987P in the turn region between HR1 and CH.

[0021] In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 Beta variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 Beta variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutation comprises: 1) RRAR to GSAS mutation; 2) the presence of a double mutation K986P / V987P in the turn region between HR1 and CH.

[0022] In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 Gamma variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 Gamma variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutation comprises: 1) RRAR to GSAS mutation; 2) the presence of a double mutation K986P / V987P in the turn region between HR1 and CH.

[0023] In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 Delta variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 Delta variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutations comprise: 1) RRAR to GSAS mutation; 2) the presence of a double mutation K986P / V987P in the turn region between HR1 and CH.

[0024] In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 Kappa variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 Kappa variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutation comprises: 1) RRAR to GSAS mutation; 2) the presence of a double mutation K986P / V987P in the turn region between HR1 and CH.

[0025] In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 Epsilon variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 Epsilon variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutation comprises: 1) RRAR to GSAS mutation; 2) the presence of a double mutation K986P / V987P in the turn region between HR1 and CH.

[0026] In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 lambda variant spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a SARS-CoV-2 lambda variant spike protein containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutation comprises: 1) RRAR to GSAS mutation; 2) the presence of a double mutation K986P / V987P in the turn region between HR1 and CH.

[0027] In some embodiments, the fusion protein comprising a coronavirus Spike protein extracellular domain containing a mutation or a truncated fragment thereof comprises a SARS-CoV-2 Omicron variant Spike protein extracellular domain containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising a coronavirus Spike protein extracellular domain containing a mutation or a truncated fragment thereof comprises a SARS-CoV-2 Omicron variant Spike protein extracellular domain containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutation comprises: 1) RRAR to GSAS mutation; 2) the presence of a double mutation K986P / V987P in the turn region between HR1 and CH.

[0028] In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67.

[0029] In some embodiments, the coronavirus multivalent vaccine comprises at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67.

[0030] In some embodiments, the coronavirus multivalent vaccine comprises at least two fusion proteins comprising an amino acid sequence as shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67.

[0031] In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 22, 23 or 29 and a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 43, 44 or 67.

[0032] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 22, 23 or 29 and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 43, 44 or 67 is (1-5): (1-5). In some embodiments, the mass ratio of the above two fusion proteins is (1-3): (1-3). In some embodiments, the mass ratio of the above two fusion proteins is (1-2): (1-2). In some embodiments, the mass ratio of the above two fusion proteins is 1:1.

[0033] In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO:22 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO:43.

[0034] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO:22 to the fusion protein comprising the amino acid sequence as shown in SEQ ID NO:43 is (1-5):(1-5). In some embodiments, the mass ratio of the above two fusion proteins is (1-3):(1-3). In some embodiments, the mass ratio of the above two fusion proteins is (1-2):(1-2). In some embodiments, the mass ratio of the above two fusion proteins is 1:1.

[0035] In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO:29 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO:67.

[0036] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO:29 to the fusion protein comprising the amino acid sequence as shown in SEQ ID NO:67 is (1-5):(1-5). In some embodiments, the mass ratio of the above two fusion proteins is (1-3):(1-3). In some embodiments, the mass ratio of the above two fusion proteins is (1-2):(1-2). In some embodiments, the mass ratio of the above two fusion proteins is 1:1.

[0037] In some embodiments, the fusion protein comprising the extracellular domain of a coronavirus spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a coronavirus spike protein containing a mutation or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto. In some embodiments, the Fc fragment of the immunoglobulin is derived from IgG, IgM, IgA, IgE, or IgD. In some embodiments, the Fc fragment of the immunoglobulin is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc fragment of the immunoglobulin is an Fc fragment of IgG1. In some embodiments, the Fc fragment of the immunoglobulin is an Fc fragment of human IgG1. In some embodiments, the Fc fragment of the immunoglobulin comprises the amino acid sequence as shown in SEQ ID NO: 38, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence having one or more conservative amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 38.

[0038] In some embodiments, the fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof is obtained by connecting the C-terminus of the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof to the N-terminus of the Fc fragment of an immunoglobulin.

[0039] In some embodiments, the fusion protein comprising a coronavirus spike protein ectodomain containing a mutation or a truncated fragment thereof comprises a SARS-CoV-2 original strain spike protein ectodomain containing a mutation or a truncated fragment thereof and an immunoglobulin Fc fragment linked thereto. In some embodiments, the mutations comprise: 1) RRAR to GSAS; 2) the presence of the double mutation K986P / V987P in the turn region between HR1 and CH.

[0040] In some embodiments, the fusion protein comprising a coronavirus spike protein ectodomain containing a mutation or a truncated fragment thereof comprises a SARS-CoV-2 alpha variant spike protein ectodomain containing a mutation or a truncated fragment thereof and an immunoglobulin Fc fragment linked thereto. In some embodiments, the mutation comprises: 1) a RRAR to GSAS mutation; and 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0041] In some embodiments, the fusion protein comprising a coronavirus spike protein ectodomain containing a mutation or a truncated fragment thereof comprises a SARS-CoV-2 Beta variant spike protein ectodomain containing a mutation or a truncated fragment thereof and an immunoglobulin Fc fragment linked thereto. In some embodiments, the mutation comprises: 1) a RRAR to GSAS mutation; and 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0042] In some embodiments, the fusion protein comprising a coronavirus spike protein ectodomain containing a mutation or a truncated fragment thereof comprises a SARS-CoV-2 gamma variant spike protein ectodomain containing a mutation or a truncated fragment thereof and an immunoglobulin Fc fragment linked thereto. In some embodiments, the mutation comprises: 1) a RRAR to GSAS mutation; and 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0043] In some embodiments, the fusion protein comprising a coronavirus spike protein ectodomain containing a mutation or a truncated fragment thereof comprises a SARS-CoV-2 Delta variant spike protein ectodomain containing a mutation or a truncated fragment thereof and an immunoglobulin Fc fragment linked thereto. In some embodiments, the mutation comprises: 1) a RRAR to GSAS mutation; and 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0044] In some embodiments, the fusion protein comprising a coronavirus spike protein ectodomain containing a mutation or a truncated fragment thereof comprises a SARS-CoV-2 kappa variant spike protein ectodomain containing a mutation or a truncated fragment thereof, linked to an immunoglobulin Fc fragment. In some embodiments, the mutations comprise: 1) RRAR to GSAS; and 2) the presence of the double mutation K986P / V987P in the turn region between HR1 and CH.

[0045] In some embodiments, the fusion protein comprising a coronavirus spike protein ectodomain containing a mutation or a truncated fragment thereof comprises a SARS-CoV-2 Epsilon variant spike protein ectodomain containing a mutation or a truncated fragment thereof and an immunoglobulin Fc fragment linked thereto. In some embodiments, the mutation comprises: 1) a RRAR to GSAS mutation; and 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0046] In some embodiments, the fusion protein comprising a coronavirus spike protein ectodomain containing a mutation or a truncated fragment thereof comprises a SARS-CoV-2 lambda variant spike protein ectodomain containing a mutation or a truncated fragment thereof and an immunoglobulin Fc fragment linked thereto. In some embodiments, the mutation comprises: 1) a RRAR to GSAS mutation; and 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0047] In some embodiments, the fusion protein comprising a coronavirus spike protein ectodomain containing a mutation or a truncated fragment thereof comprises a SARS-CoV-2 Omicron variant spike protein ectodomain containing a mutation or a truncated fragment thereof and an immunoglobulin Fc fragment linked thereto. In some embodiments, the mutation comprises: 1) a RRAR to GSAS mutation; and 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0048] In some embodiments, the fusion protein comprising the mutant coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76.

[0049] In some embodiments, the coronavirus multivalent vaccine comprises at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76.

[0050] In some embodiments, the coronavirus multivalent vaccine comprises at least two fusion proteins comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76.

[0051] In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 53, 54 or 72 and a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 61, 62 or 76.

[0052] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 53, 54 or 72 and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 61, 62 or 76 is (1-5): (1-5). In some embodiments, the mass ratio of the above two fusion proteins is (1-3): (1-3). In some embodiments, the mass ratio of the above two fusion proteins is (1-2): (1-2). In some embodiments, the mass ratio of the above two fusion proteins is 1:1.

[0053] In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO:53 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO:61.

[0054] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO:53 and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO:61 is (1-5):(1-5). In some embodiments, the mass ratio of the above two fusion proteins is (1-3):(1-3). In some embodiments, the mass ratio of the above two fusion proteins is (1-2):(1-2). In some embodiments, the mass ratio of the above two fusion proteins is 1:1.

[0055] In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO:72 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO:76.

[0056] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO:72 to the fusion protein comprising the amino acid sequence as shown in SEQ ID NO:76 is (1-5):(1-5). In some embodiments, the mass ratio of the above two fusion proteins is (1-3):(1-3). In some embodiments, the mass ratio of the above two fusion proteins is (1-2):(1-2). In some embodiments, the mass ratio of the above two fusion proteins is 1:1.

[0057] In some embodiments, the coronavirus multivalent vaccine further comprises a conserved fragment of a coronavirus Spike protein or a fusion protein comprising the same. In some embodiments, the conserved fragment of the coronavirus Spike protein is from SARS-CoV-2, SARS-CoV or MERS-CoV. In some embodiments, the conserved fragment of the coronavirus Spike protein is from the original strain of SARS-CoV-2 or a variant thereof. In some embodiments, the conserved fragment of the coronavirus Spike protein is from the original strain of SARS-CoV-2, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant.

[0058] In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein further comprises an N-terminal signal peptide.

[0059] In some embodiments, the signal peptide is selected from the group consisting of CSP, mschito, MF-α, pho1, HBM, t-pA, and the signal peptide of IL-3.

[0060] In some embodiments, the N-terminal signal peptide comprises an amino acid sequence as shown in SEQ ID NO: 2 or 5, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2 or 5, or an amino acid sequence having one or more conservative amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 2 or 5.

[0061] In some embodiments, the fusion protein comprising the conserved fragment of the coronavirus Spike protein comprises a conserved fragment of the coronavirus Spike protein and a monomeric subunit protein connected by a linker. In some embodiments, the linker is a GS linker. In some embodiments, the linker is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS)2, (GGGGS)3, or any combination thereof. In some embodiments, the linker is (G m S) n , wherein each m is independently 1, 2, 3, 4 or 5, and n is 1, 2, 3, 4 or 5. In some embodiments, the sequence of the linker is (GGGGS) n, n is 1, 2, 3, 4 or 5. In some embodiments, the linker is GGGGS. In some embodiments, the linker is (GGGGS)2. In some embodiments, the linker is (GGGGS)3. In some embodiments, the linker is (GGGGS)4. In some embodiments, the linker is (GGGGS)5. In some embodiments, the monomeric subunit protein is a self-assembled monomeric subunit protein. In some embodiments, the monomeric subunit protein is a monomeric ferritin subunit. In some embodiments, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, algal ferritin, insect ferritin, fungal ferritin and mammalian ferritin. In some embodiments, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit. In some embodiments, there is an N19Q mutation in the amino acid sequence of the Helicobacter pylori non-heme monomeric ferritin subunit. In some embodiments, the monomeric ferritin subunit comprises the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 14.

[0062] In some embodiments, the fusion protein comprising the conserved fragment of the coronavirus Spike protein is obtained by connecting the C-terminus of the conserved fragment of the coronavirus Spike protein to the N-terminus of the monomeric subunit protein via a linker.

[0063] In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises a conserved fragment of the SARS-CoV-2 original strain Spike protein and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises a conserved fragment of the SARS-CoV-2 original strain Spike protein and a monomeric ferritin subunit connected by a linker.

[0064] In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Alpha variant Spike protein and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Alpha variant Spike protein and a monomeric ferritin subunit connected by a linker.

[0065] In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Beta variant Spike protein and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Beta variant Spike protein and a monomeric ferritin subunit connected by a linker.

[0066] In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Gamma variant Spike protein and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Gamma variant Spike protein and a monomeric ferritin subunit connected by a linker.

[0067] In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Delta variant Spike protein and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Delta variant Spike protein and a monomeric ferritin subunit connected by a linker.

[0068] In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Kappa variant Spike protein and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Kappa variant Spike protein and a monomeric ferritin subunit connected by a linker.

[0069] In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Epsilon variant Spike protein and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Epsilon variant Spike protein and a monomeric ferritin subunit connected by a linker.

[0070] In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Lambda variant Spike protein and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Lambda variant Spike protein and a monomeric ferritin subunit connected by a linker.

[0071] In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Omicron variant Spike protein and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises a conserved fragment of a SARS-CoV-2 Omicron variant Spike protein and a monomeric ferritin subunit connected by a linker.

[0072] In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 45-46, 68, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in any one of SEQ ID NOs: 45-46, 68, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 45-46, 68.

[0073] In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises a conserved fragment of the coronavirus Spike protein and an Fc fragment of an immunoglobulin linked thereto. In some embodiments, the Fc fragment of the immunoglobulin is from IgG, IgM, IgA, IgE, or IgD. In some embodiments, the Fc fragment of the immunoglobulin is from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc fragment of the immunoglobulin is an Fc fragment of IgG1. In some embodiments, the Fc fragment of the immunoglobulin is an Fc fragment of human IgG1. In some embodiments, the Fc fragment of the immunoglobulin comprises the amino acid sequence as shown in SEQ ID NO: 38, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 38.

[0074] In some embodiments, the fusion protein comprising the conserved fragment of the coronavirus Spike protein is obtained by linking the C-terminus of the conserved fragment of the coronavirus Spike protein to the N-terminus of the Fc fragment of an immunoglobulin.

[0075] In some embodiments, the fusion protein comprising the conserved fragment of the coronavirus Spike protein comprises the conserved fragment of the Spike protein of the original strain of SARS-CoV-2 and the Fc fragment of the immunoglobulin linked thereto.

[0076] In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises a conserved fragment of the SARS-CoV-2 Alpha variant Spike protein and an Fc fragment of an immunoglobulin linked thereto.

[0077] In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises a conserved fragment of the SARS-CoV-2 Beta variant Spike protein and an Fc fragment of an immunoglobulin linked thereto.

[0078] In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises a conserved fragment of the SARS-CoV-2 Gamma variant Spike protein and an Fc fragment of an immunoglobulin linked thereto.

[0079] In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises a conserved fragment of the SARS-CoV-2 Delta variant Spike protein and an Fc fragment of an immunoglobulin linked thereto.

[0080] In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises a conserved fragment of the SARS-CoV-2 Kappa variant Spike protein and an Fc fragment of an immunoglobulin linked thereto.

[0081] In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises a conserved fragment of the SARS-CoV-2 Epsilon variant Spike protein and an Fc fragment of an immunoglobulin linked thereto.

[0082] In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises a conserved fragment of the SARS-CoV-2 Lambda variant Spike protein and an Fc fragment of an immunoglobulin linked thereto.

[0083] In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises a conserved fragment of the SARS-CoV-2 Omicron variant Spike protein and an Fc fragment of an immunoglobulin linked thereto.

[0084] In some embodiments, the fusion protein comprising a conserved fragment of a coronavirus Spike protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 63, 64, or 77, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in any one of SEQ ID NOs: 63, 64, or 77, or an amino acid sequence having one or more conservative amino acid substitutions with the amino acid sequence shown in any one of SEQ ID NOs: 63, 64, or 77.

[0085] In some embodiments, the coronavirus multivalent vaccine comprises at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67, and further comprises a conserved fragment of the coronavirus Spike protein or a fusion protein comprising the same.

[0086] In some embodiments, the coronavirus multivalent vaccine comprises: (1) at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67, and (2) a fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 45-46, 63, 64, 68, and 77.

[0087] In some embodiments, the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO:22, 23 or 29, (2) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO:43, 44 or 67, and (3) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO:63, 64 or 77.

[0088] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 22, 23 or 29, the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 43, 44 or 67, and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 63, 64 or 77 is (1-5): (1-5): (1-5). In some embodiments, the mass ratio of the above three fusion proteins is (1-3): (1-3): (1-3). In some embodiments, the mass ratio of the above three fusion proteins is (1-2): (1-2): (1-2). In some embodiments, the mass ratio of the above three fusion proteins is 1:1:1.

[0089] In some embodiments, the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:22, (2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:43, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:63.

[0090] In some embodiments, the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:29, (2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:67, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:77.

[0091] In some embodiments, the coronavirus multivalent vaccine comprises at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76, and further comprises a conserved fragment of the coronavirus Spike protein or a fusion protein comprising the same.

[0092] In some embodiments, the coronavirus multivalent vaccine comprises: (1) at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76, and (2) a fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 45-46, 63, 64, 68 and 77.

[0093] In some embodiments, the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 53, 54 or 72, (2) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 61, 62 or 76, and (3) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 63, 64 or 77.

[0094] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 53, 54 or 72, the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 61, 62 or 76, and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 63, 64 or 77 is (1-5): (1-5): (1-5). In some embodiments, the mass ratio of the above three fusion proteins is (1-3): (1-3): (1-3). In some embodiments, the mass ratio of the above three fusion proteins is (1-2): (1-2): (1-2). In some embodiments, the mass ratio of the above three fusion proteins is 1:1:1.

[0095] In some embodiments, the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:53, (2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:61, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:63.

[0096] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 53, the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 61, and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 63 is (1-5): (1-5): (1-5). In some embodiments, the mass ratio of the above three fusion proteins is (1-3): (1-3): (1-3). In some embodiments, the mass ratio of the above three fusion proteins is (1-2): (1-2): (1-2). In some embodiments, the mass ratio of the above three fusion proteins is 1:1:1.

[0097] In some embodiments, the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:72, (2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:76, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:77.

[0098] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO:72, the fusion protein comprising the amino acid sequence as shown in SEQ ID NO:76, and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO:77 is (1-5):(1-5):(1-5). In some embodiments, the mass ratio of the above three fusion proteins is (1-3):(1-3):(1-3). In some embodiments, the mass ratio of the above three fusion proteins is (1-2):(1-2):(1-2). In some embodiments, the mass ratio of the above three fusion proteins is 1:1:1.

[0099] In some embodiments, "at least 80% identity" is at least about 80% identity, at least about 81% identity, at least about 83% identity, at least about 84% identity, at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or a range between any two of these values ​​(including the endpoints), or any value therein.

[0100] In some embodiments, "at least 90% identity" is at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or a range between any two of these values ​​(including the endpoints), or any value therein.

[0101] In some embodiments, as a result of the antigen (e.g., a coronavirus Spike protein extracellular domain containing a mutation or a truncated fragment thereof or a conserved fragment of a coronavirus Spike protein) being linked to a self-assembled monomeric subunit protein (e.g., a monomeric ferritin subunit), nanoparticles displaying the antigen on their surface are produced.

[0102] In some embodiments, the coronavirus multivalent vaccine further includes a pharmaceutically acceptable carrier and / or adjuvant.

[0103] The present invention also provides a polynucleotide encoding the extracellular domain of the coronavirus Spike protein containing the mutation described herein or a truncated fragment thereof, a conserved fragment of the coronavirus Spike protein, or a fusion protein comprising the same.

[0104] The present invention also provides an expression vector comprising a polynucleotide encoding the extracellular domain of the coronavirus Spike protein containing the mutation described herein or a truncated fragment thereof, a conserved fragment of the coronavirus Spike protein, or a fusion protein comprising the same.

[0105] The present invention also provides a host cell comprising the polynucleotide or expression vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is a CHO cell, HEK293 cell, Cos1 cell, Cos7 cell, CV1 cell or mouse L cell.

[0106] The present invention also provides the use of the coronavirus multivalent vaccine described herein in the preparation of a medicament for preventing or treating coronavirus infection.

[0107] The present invention also provides the use of the coronavirus multivalent vaccine described herein in preventing or treating coronavirus infection. In some embodiments, the use of the coronavirus multivalent vaccine described herein in preventing or treating SARS or COVID-19 is provided.

[0108] The present invention also provides a method for preventing or treating coronavirus infection, comprising administering an effective amount of the coronavirus multivalent vaccine described herein to a patient in need.

[0109] In some embodiments, the coronavirus infection is an infection with the original strain of SARS-CoV-2 or a variant thereof. In some embodiments, the coronavirus infection is an infection with the original strain of SARS-CoV-2, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant, or SARS-CoV-2 Omicron variant. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 is the binding curve of the fusion protein and human ACE2; Figure 1a is the binding curve of the fusion protein D and human ACE2, and Figure 1b is the binding curve of the fusion protein G and human ACE2.

[0111] Figure 2 shows the serum anti-Spike protein IgG titers, with the bars representing the geometric mean (GMT) of the titers. Figures 2a, 2c, and 2e show the titers 14 days after the first dose (Day 14), and Figures 2b, 2d, and 2f show the titers 14 days after the second dose (Day 35).

[0112] Figure 3 shows the pseudovirus inhibition titer of the serum of vaccine-immunized mice, and the bars represent the geometric mean (GMT) of the titer; in the figure, wildtype represents SARS-CoV-2 Spike pseudovirus, Delta represents SARS-CoV-2 Spike (B.1.617.2) pseudovirus, BA.1 represents SARS-CoV-2 Spike (B.1.1.529) pseudovirus; BA.2.12.1 represents SARS-CoV-2 Spike (BA.2.12.1) pseudovirus; BA.3 represents SARS-CoV-2 Spike (BA.3) pseudovirus; BA.4 / 5 represents SARS-CoV-2 Spike (BA.4 / 5) pseudovirus.

[0113] Figure 4 shows the pseudovirus inhibition titer of serum from mice immunized with the bivalent vaccine, and the bars represent the geometric mean (GMT) of the titer; Figure 4a shows the comparison of neutralizing antibody titers in serum from different doses of antigen; Figure 4b shows the comparison of neutralizing antibody titers from different variants of the same dose of antigen (5 μg bivalent vaccine); in the figure, wildtype and WT represent SARS-CoV-2 Spike pseudovirus, Delta represents SARS-CoV-2 Spike (B.1.617.2) pseudovirus, BA.1 represents SARS-CoV-2 Spike (B.1.1.529) pseudovirus; BA.2.12.1 represents SARS-CoV-2 Spike (BA.2.12.1) pseudovirus; BA.3 represents SARS-CoV-2 Spike (BA.3) pseudovirus; BA.4 / 5 represents SARS-CoV-2 Spike (BA.4 / 5) pseudovirus; Alpha represents SARS-CoV-2 Spike (B.1.1.7 / VUI-202012 / 01,del145Y) pseudovirus; Gamma represents SARS-CoV-2 Spike (B.1.351 / 501Y.V2) pseudovirus; Beta represents SARS-CoV-2 Spike (P.1501Y.V3) pseudovirus.

[0114] Figure 5 shows the long-term anti-COVID-19 Spike protein antibody titers in mouse serum after immunization with the bivalent vaccine; the bars represent the geometric mean (GMT) of the titers (values ​​are shown in the box); in the figure, 2W is the serum 2 weeks after the second immunization, and 30W is the serum 30 weeks after the second immunization; Wildtype, Delta, and BA.1 are WT-Spike-His, Delta-Spike-His, and Omicron-Spike-His, respectively.

[0115] Figure 6 shows the serum anti-Spike protein IgG titers, with bars representing the geometric mean (GMT) of the titers; Figures 6a, 6c, and 6e show the titers 14 days after the first dose (Day 14), and Figures 6b, 6d, and 6f show the titers 14 days after the second dose (Day 35).

[0116] Figure 7 shows the antibody titers inhibiting the binding of hACE2 to Spike protein in the serum of mice immunized with the same dose of bivalent vaccine plus different doses of adjuvant; the Spike protein in Figure 7a is WT-Spike-His, the Spike protein in Figure 7b is Delta-Spike-His, and the Spike protein in Figure 7c is Omicron-Spike-His; the bar graph represents IC 50 The geometric means of the data are shown in the bar graphs, with error symbols indicating 95% confidence intervals.

[0117] Figure 8 shows the anti-Spike protein IgG titer of immune serum from K18-hACE2 transgenic mice, and the bars represent the geometric mean (GMT) of the titer. In the figure, low, medium, and high represent the low-dose group, medium-dose group, and high-dose group, respectively. Wildtype, Delta, and Omicron represent WT-Spike-His, Delta-Spike-His, and Omicron-Spike-His, respectively.

[0118] FIG9 shows the viable virus titer in the lungs of challenged mice.

[0119] Figure 10 shows the inhibition rate (%) of serum against SARS-CoV-2 Omicron BA.1.1.

[0120] the term

[0121] Unless otherwise stated, each of the following terms shall have the meaning set forth below.

[0122] definition

[0123] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0124] It should be noted that, as used herein and in the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. Thus, the terms "a," "an," "one or more," and "at least one" can be used interchangeably. Similarly, the terms "comprising," "including," and "having" can be used interchangeably and should generally be understood to be open-ended and non-restrictive, e.g., not excluding other unrecited elements or steps.

[0125] The term "amino acid" refers to an organic compound containing both an amino group and a carboxyl group, such as an α-amino acid, which can be encoded by a nucleic acid directly or in the form of a precursor. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code." Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0126] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue that has a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions are unlikely to substantially alter the functional properties of a protein. Examples of amino acid classes with chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; and 6) acidic side chains: aspartic acid and glutamic acid.

[0127] The term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids and does not refer to the specific length of the product. Thus, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other terms used to refer to two or more amino acid chains, and the term "polypeptide" may be used in place of or interchangeably with any of the above terms. The term "polypeptide" is also intended to refer to products of polypeptides that have been modified after expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but it need not be translated from a specified nucleic acid sequence and may be produced by any means, including chemical synthesis.

[0128] Unless otherwise indicated, fusion protein is a recombinant protein comprising the amino acid sequence from at least two unrelated proteins, and the at least two unrelated proteins have been linked together to form a single protein by a peptide bond. The amino acid sequences of unrelated proteins can be directly connected to each other, or a joint can be used to connect. As used herein, if the amino acid sequence of protein is usually not linked together via a peptide bond in its natural environment (for example, in a cell), then they are unrelated. For example, usually the amino acid sequence of bacterial enzymes such as bacillus stearothermophilus dihydrolipoyl transacetylase (E2p) and the amino acid sequence of coronavirus Spike protein are not linked together by a peptide bond.

[0129] The terms "homology," "identity," or "similarity" refer to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing alignable positions in each sequence. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between the sequences is a function of the number of matching or homologous positions shared by the sequences.

[0130] The term "encoding" when applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide and, in its native state or when manipulated by methods well known to those skilled in the art, can produce the polypeptide and / or its fragments via transcription and / or translation.

[0131] A polynucleotide is composed of a specific sequence of four bases: adenine (A), cytosine (C), guanine (G), and thymine (T), or, in the case of RNA, thymine replaced by uracil (U). A "polynucleotide sequence" can be represented by an alphabetic representation of the polynucleotide molecule. This alphabetic representation can be entered into a database on a computer with a central processing unit and used in bioinformatics applications, such as functional genomics and homology searches.

[0132] The terms "polynucleotide," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analogs. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If such modifications are present, structural modifications to the nucleotides can be made before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, for example by conjugation with a labeling component. This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any polynucleotide embodiment of the present disclosure includes a double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0133] A nucleic acid or polynucleotide sequence (or polypeptide or protein sequence) having a certain percentage (e.g., 90%, 95%, 98% or 99%) of "identity" or "sequence identity" to another sequence means that when the sequences are aligned, that percentage of bases (or amino acids) in the two sequences being compared are the same. The percent identity or sequence identity can be determined visually or using software programs known in the art, such as those described in Ausubel et al., eds. (2007), in Current Protocols in Molecular Biology. Preferably, the alignment is performed using the default parameters. One such alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expectation=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that have the above specified percentage identities and encode polypeptides having the same or similar biological activity.

[0134] As used herein with respect to cells, nucleic acids, polypeptides, antibodies, and the like, the term "isolated" refers to a molecule that has been separated from one or more of the other components of a cell's natural environment, such as DNA or RNA. The term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that do not exist in their natural state and do not exist in their natural state. The term "isolated" is also used herein to refer to cells or polypeptides that have been separated from other cellular proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, and the like are typically prepared by at least one purification step. In some embodiments, the purity of an isolated nucleic acid, polypeptide, antibody, and the like is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range (including the endpoints) between any two of these values, or any value therein.

[0135] The term "recombinant" in reference to a polypeptide or polynucleotide refers to a form of the polypeptide or polynucleotide that does not occur in nature, and by way of non-limiting example, can be produced by combination with a polynucleotide or polypeptide that does not normally exist.

[0136] "Antibody" and "antigen-binding fragment" refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody, any antigen-binding fragment thereof, or a single chain thereof. The term "antibody" therefore includes any protein or peptide that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen.

[0137] As used herein, the terms "antigen" or "immunogen" are used interchangeably and refer to a substance, typically a protein, that is capable of inducing an immune response in a subject. The term also refers to a protein that is immunologically active, i.e., capable of eliciting a humoral and / or cellular immune response against the protein upon administration to a subject (directly or by administering a nucleotide sequence or vector encoding the protein to the subject). Unless otherwise indicated, the term "vaccine antigen" is used interchangeably with "protein antigen" or "antigenic polypeptide."

[0138] "Neutralizing antibodies" are antibodies that reduce the infectivity of an infectious agent by binding to specific antigens on the agent. In some embodiments, the infectious agent is a virus. "Broadly neutralizing antibodies" are antibodies that bind to and inhibit the function of related antigens, such as antigens that have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the antigenic surface of the antigen. For antigens from pathogens, such as viruses, the antibody may bind to and inhibit the function of more than one class and / or subclass of antigens from the pathogen.

[0139] "cDNA" refers to DNA that is complementary or identical to mRNA and can be in single-stranded or double-stranded form.

[0140] "Epitope" refers to an antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic so that they elicit a specific immune response. For example, an epitope is a region of an antigen to which B and / or T cells respond. An epitope can be formed by contiguous amino acids or by non-contiguous amino acids juxtaposed by the tertiary folding of a protein.

[0141] Vaccine refers to a biological product that causes a preventive or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, vaccines cause antigen-specific immune responses to pathogens such as viral pathogens or cells composed of cells associated with pathological conditions. Vaccines can include polynucleotides (e.g., nucleic acids encoding known antigens), peptides or polypeptides (e.g., disclosed antigens), viruses, cells or one or more cell compositions. In some embodiments, vaccines or vaccine antigens or vaccine compositions are expressed from fusion protein expression vectors and self-assembled into nanoparticles that display antigen polypeptides or proteins on the surface.

[0142] The term "multivalent vaccine" will be recognized and understood by those of ordinary skill in the art, for example, to mean comprising antigens from different pathogens (e.g., Spike proteins from different SARS-CoV-2 coronavirus sources) or comprising a fusion protein or its nucleic acid sequence or its construct, or comprising different antigens from the same pathogen or comprising a fusion protein or its nucleic acid sequence or its construct. In the context of the present invention, a coronavirus multivalent vaccine may be comprising antigens from two or more different SARS-CoV-2 coronaviruses (e.g., Spike proteins from different SARS-CoV-2 coronavirus sources) or comprising a fusion protein or its nucleic acid sequence or its construct, or comprising different antigens from the same SARS-CoV-2 coronavirus or comprising a fusion protein or its nucleic acid sequence or its construct.

[0143] An effective amount of a vaccine or other agent refers to an amount sufficient to produce a desired response, such as to cause an immune response, prevent, alleviate or eliminate the signs or symptoms of a condition or disease (such as pneumonia). For example, this can be the amount necessary to inhibit viral replication or measurably change the outward symptoms of a viral infection. Typically, this amount will be sufficient to measurably inhibit the replication or infectivity of a virus (such as SARS-CoV-2). When administered to a subject, a dosage that reaches a target tissue concentration will typically be used, which has been shown to achieve in vitro inhibition of viral replication. In some embodiments, an "effective amount" is an amount for treating (including preventing) one or more symptoms and / or potential causes (such as treating coronavirus infection) of a condition or disease. In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount to prevent the development of one or more symptoms or signs (such as one or more symptoms or signs associated with coronavirus infection) of a specific disease or condition.

[0144] Nanoparticle refers to a spherical protein shell with a diameter of tens of nanometers and a well-defined surface geometry. The spherical protein shell is formed by identical copies of non-viral proteins that can automatically assemble into nanoparticles with an outward appearance similar to virus-like particles (VLPs). Examples include ferritin (FR), which is conserved between multiple species and forms 24 aggressiveness (24-mer), Bacillus stearothermophilus dihydrolipoyl transacetylase (E2P), hyperthermophilic dioxotetrahydropteridine synthase (LS) and Thermotoga maritima encapsulin, which all form 60 aggressiveness (60-mer). Self-assembling nanoparticles can spontaneously form after recombinant expression of proteins in appropriate expression systems. The production, detection and characterization of nanoparticles can use the same technology developed for VLPs.

[0145] Virus-like particles (VLP) refer to non-replicating viral capsids, which derive from any one of a variety of viruses. VLP generally includes one or more viral proteins, such as but not limited to the protein called capsid, coat protein, globular wall protein, those proteins of surface protein and / or envelope protein, or the polypeptide of the formation particle derived from these proteins. In a suitable expression system, after recombinant expression protein, VLP can form spontaneously. The method of producing specific VLP is known in the art. Conventional techniques known in the art (such as by electron microscope, biophysical characterization etc.) can be used to detect the existence of the VLP that follows recombinant expression viral protein. For example, VLP can be separated by density gradient centrifugation and / or identified by characteristic density bands. Alternatively, the vitrified water sample of the VLP goods in question can be subjected to cryo-electron microscopy, and image is recorded under suitable exposure conditions.

[0146] The terms "about" and "approximately" are used interchangeably and refer to the conventional error range of the corresponding numerical value that is readily known to those skilled in the relevant art. In some embodiments, "about" mentioned herein refers to the described numerical value and its ±10%, ±5% or ±1% range.

[0147] "ECMO" refers to extracorporeal membrane oxygenation (ECMO), which is a medical emergency technology device mainly used to provide continuous extracorporeal respiration and circulation to patients with severe heart and lung failure to maintain the patient's life.

[0148] "ICU" refers to the Intensive Care Unit, where treatment, nursing, and rehabilitation can be carried out simultaneously. It provides isolation facilities and equipment for critically ill or comatose patients, and offers the best care, comprehensive treatment, integrated medical care and nursing, as well as early postoperative rehabilitation, joint care and exercise therapy, and other services.

[0149] "IMV" stands for intermittent mandatory ventilation, which implements periodic volume or pressure ventilation based on a pre-set time interval, i.e., time trigger. This allows the patient to breathe spontaneously at any set base pressure level during mandatory ventilation. During spontaneous breathing, the patient can breathe spontaneously with continuous airflow support, or the machine will open the on-demand valve to allow spontaneous breathing. Most ventilators can provide pressure support during spontaneous breathing.

[0150] The term "subject" refers to any animal classified as a mammal, such as humans and non-human mammals. Examples of non-human animals include dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, mice, etc. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein. Preferably, the subject is a human.

[0151] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent, slow, ameliorate, or halt an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following results, whether detectable or undetectable, such as alleviation of symptoms, reduction in disease severity, stabilization of the disease state (i.e., non-worsening), delay or slowing of disease progression, improvement, palliation, alleviation, or elimination (whether partial or total) of the disease state, and prolongation of life expectancy relative to that expected in the absence of treatment. Patients in need of treatment include those already suffering from a condition or disorder, those susceptible to a condition or disorder, or those in whom such condition or disorder is to be prevented, and those who may or may be expected to benefit from the administration of the Spike protein nanoparticles or pharmaceutical compositions disclosed herein for treatment.

[0152] Overview

[0153] For SARS-CoV, MERS-CoV and SARS-CoV-2, the viral genome encodes spike (S), envelope (E), membrane (M) and nucleocapsid (N) structural proteins, among which the S glycoprotein (Spike protein) is responsible for binding to the host receptor through the receptor binding domain (RBD) in its S1 subunit, as well as the subsequent membrane fusion and viral entry driven by its S2 subunit. Receptor binding can help keep the RBD in a "standing" state, which facilitates the dissociation of the S1 subunit from the S2 subunit. When the S1 subunit dissociates from the S2 subunit, the second S2' cleavage releases the fusion peptide. The connecting region, HR1 and CH form a very long helical piece to insert the fusion peptide into the host cell membrane. Finally, HR1 and HR2 form a helical structure and assemble into a six-helix bundle to fuse the viral membrane and the host membrane.

[0154] The RBD consists of a core subdomain and a receptor binding motif (RBM). Although the core subdomains of the three coronaviruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, are highly similar, their RBMs are significantly different, resulting in different receptor specificities: SARS-CoV and SARS-CoV-2 recognize angiotensin-converting enzyme 2 (ACE2), while MERS-CoV binds dipeptidyl peptidase 4 (DPP4). Because the S glycoprotein is surface-exposed and mediates entry into host cells, it is the primary target of neutralizing antibodies (NAbs) after infection and a focus of vaccine design. The Spike trimer is extensively modified with N-linked glycans, which are important for proper folding and regulating accessibility to NAbs.

[0155] The present invention stabilizes the Spike trimer in its pre-fusion conformation with the host cell membrane by 1) inactivating mutations at the S1 / S2 cleavage site and 2) preventing the HR1 and CH from forming a straight helix during fusion in the turn region between HR1 and CH. In some embodiments, the coronavirus Spike protein extracellular domain containing the mutations, or a truncated fragment thereof, can be displayed on nanoparticles.

[0156] According to the research and exemplary designs described herein, the present invention provides a coronavirus vaccine comprising a coronavirus Spike protein ectodomain containing a mutation or a truncated fragment thereof or a fusion protein thereof. The present invention also provides a coronavirus multivalent vaccine comprising a coronavirus Spike protein ectodomain containing a mutation or a truncated fragment thereof or a fusion protein thereof. The present invention also provides related polynucleotides, expression vectors and pharmaceutical compositions. In some embodiments, the stable Spike trimer and RBD protein in the form of protein or nucleic acid (DNA / mRNA) carried by the viral vector can be used as a coronavirus vaccine. In addition, the stable Spike trimer and RBD presented by nanoparticles can also be used as a coronavirus vaccine.

[0157] The antigens and vaccines based on coronavirus Spike proteins of the present invention have many advantageous properties. The Spike trimer design described herein presents conserved neutralizing epitopes with its natural-like structure, making the Spike trimer useful as an antigen vaccine or for multivalent display on nanoparticles. The nanoparticle vaccine of the present invention allows Spike trimers derived from different coronaviruses to be displayed on known nanoparticles, such as ferritin, E2p, and I3-01, with a size range of 12.2 to 25.0 nm. Nanoparticles presenting all trimers can be produced in high yield in HEK293 cells, ExpiCHO cells, and CHO cells. The produced Spike protein nanoparticles can be purified by antibodies and size exclusion chromatography (SEC).

[0158] Unless otherwise stated herein, the mutant coronavirus Spike protein extracellular domain or its truncated fragment, coronavirus Spike protein S1 subunit, coronavirus Spike protein conserved fragment, fusion protein, Spike protein nanoparticle, coronavirus multivalent vaccine, encoding polynucleotide, expression vector and host cell of the present invention and related therapeutic applications can be produced or performed according to the methods exemplified herein or conventional methods well known in the art.

[0159] Unless otherwise indicated, the order of steps or the order in which certain operations are performed is not important as long as the invention remains operable. Furthermore, two or more steps or operations may be performed simultaneously.

[0160] Unless otherwise specified, any and all examples used herein, or exemplary language used herein (e.g., "such as" or "including"), are intended only to better illustrate the invention and do not limit the scope of the invention. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0161] Coronavirus Spike protein extracellular domain or its truncated fragment containing mutation

[0162] The present invention provides a coronavirus Spike protein ectodomain or a truncated fragment thereof containing a mutation that can be used to produce a vaccine. By introducing mutations into the coronavirus Spike protein ectodomain or a truncated fragment thereof, the mutated Spike trimer is stabilized. Some specific Spike proteins of specific SARS-CoV-2 strains or isolates are exemplified herein, such as SEQ ID NOs: 1, 8, and 31. Due to the functional similarity and sequence homology between different isolates or strains of a given coronavirus, mutated Spike proteins or truncated fragments thereof derived from orthologous sequences of other known coronavirus Spike proteins can also be generated according to the mutation strategy described herein. Many known coronavirus Spike protein sequences have been described in the literature. See, e.g., James et al., J. Mol. Biol. 432:3309-25, 2020; Andersen et al., Nat. Med. 26:450-452, 2020; Walls et al., Cell 180:281–292, 2020; Zhang et al., J. Proteome Res. 19:1351-1360, 2020; Du et al., Expert Opin. Ther. Targets 21:131-143.; 2017; Yang et al., Viral Immunol. 27:543-550, 2014; Wang et al., Antiviral Res. 133:165-177, 2016; Bosch et al., J. Virol. 77:8801-8811, 2003; Lio et al., TRENDS Microbiol. 12: 106-111, 2004; Chakraborti et al., Virol. J. 2: 73, 2005; and Li, Ann. Rev. Virol. 3: 237-261, 2016.

[0163] As described herein, some mutant Spike proteins or truncated fragments thereof of the present invention contain mutations that enhance the structural stability of the Spike protein or truncated fragment prior to fusion with the cell membrane. These mutations include mutations that inactivate the S1 / S2 cleavage site and mutations in the turn region between HR1 and CH that remove any strain in the turn region between HR1 and CH, thereby preventing the formation of a straight helix.

[0164] Some mutant coronavirus Spike protein extracellular domains or truncated fragments thereof (as shown in SEQ ID NO: 3, 4, 6, 7, 9-12, 32-35, 78-83) are derived from the SARS-CoV-2 virus that causes COVID-19. These polypeptides contain mutations that inactivate the S1 / S2 cleavage site and mutations in the turn region between HR1 and CH. As an example, the amino acid sequence of the SARS-CoV-2 original strain Spike protein used for mutation is shown in SEQ ID NO: 1 or as shown in residues 14-1213 of SEQ ID NO: 1 or as shown in residues 15-1213 of SEQ ID NO: 1. In some embodiments, the Spike protein used for mutation can be SEQ ID NO: 1, 8 or 31 or a variant thereof, such as a variant substantially identical thereto or a conservatively modified variant. Using the amino acid numbering based on the cryo-EM model PDB ID 6VSB or GenBank accession number MN908947.3 as a reference, the S1 / S2 cleavage site 682 RRAR 685 Inactivation of the S1 / S2 cleavage site can be achieved by many sequence changes (e.g., deletions or substitutions) within or around the site. As exemplified herein, one mutation that inactivates the S1 / S2 cleavage site without affecting protein structure is to replace the S1 / S2 cleavage site with 682 RRAR 685 Mutation 682 GSAS 685. In addition to inactivating the S1 / S2 cleavage site, a double mutation can be made in the turn region between HR1 and CH. This double mutation eliminates the strain in the turn region (between HR1 and CH motifs) during fusion by preventing the formation of a straight helix. In some embodiments, this double mutation can be K986G / V987G, K986P / V987P, K986G / V987P or K986P / V987G. In addition to the above-mentioned mutations that stabilize the structure of the pre-fusion Spike protein or its truncated fragments, some SARS-CoV-2 Spike proteins or their truncated fragments of the present invention may contain a deletion of most or all of the HR2 domain. Using the exemplary SARS-CoV-2 Spike protein sequence SEQ ID NO: 1 for illustration, such a deletion can include the deletion of residues 1144-1213 of SEQ ID NO: 1. In some embodiments, the deletion can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 76, 80 or more residues from the C-terminus of the ectodomain of a truncated Spike protein (e.g., SEQ ID NO: 1, 3, 4, 8-10, 31-33, 78, 80, or 82), or a range (inclusive of any two of these values) or any value therein. In some embodiments, the C-terminally truncated Spike protein can extend beyond the HR2 domain. In some embodiments, the Spike protein sequence can include an N-terminal signal peptide as set forth in SEQ ID NO: 2 or 5.

[0165] Exemplary coronavirus Spike protein extracellular domains or truncated fragments or variants thereof are as follows:

[0166] The full-length extracellular domain (ECD) of the original SARS-CoV-2 Spike protein, whose amino acid sequence is shown in SEQ ID NO: 1, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) is marked in italics, and the S1 / S2 cleavage site 682 RRAR 685 Use underlining, bolding, and italics.

[0167] The amino acid sequence of the full-length extracellular domain a1 of the mutated SARS-CoV-2 original strain Spike protein is shown in SEQ ID NO: 3. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is italicized, and the S1 / S2 cleavage site is 682 RRAR 685 Mutation 682GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0168] The amino acid sequence of the full-length extracellular domain a2 of the mutated SARS-CoV-2 original strain Spike protein is shown in SEQ ID NO: 4. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics, and the S1 / S2 cleavage site is shown in the following figure: 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0169] The amino acid sequence of the full-length extracellular domain a3 of the mutated SARS-CoV-2 original strain Spike protein is shown in SEQ ID NO: 78. The sequence does not contain a signal peptide or S1 / S2 cleavage site. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0170] The amino acid sequence of the C-terminal truncated fragment b1 of the extracellular domain of the mutated SARS-CoV-2 original strain Spike protein is shown in SEQ ID NO: 6. In the sequence, 70 amino acid residues are truncated at the C-terminus, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is marked in italics, and the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0171] The amino acid sequence of the C-terminal truncated fragment b2 of the extracellular domain of the mutated SARS-CoV-2 original strain Spike protein is shown in SEQ ID NO: 7. In the sequence, the C-terminus is truncated by 70 amino acid residues, and the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics, and the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0172] The amino acid sequence of the C-terminal truncated fragment b3 of the extracellular domain of the mutated SARS-CoV-2 original strain Spike protein is shown in SEQ ID NO: 79. In the sequence, the C-terminus is truncated by 70 amino acid residues, does not contain a signal peptide, and has an S1 / S2 cleavage site. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0173] The full-length extracellular domain (ECD) of the SARS-CoV-2 Delta variant Spike protein, whose amino acid sequence is shown in SEQ ID NO:8, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1 / S2 cleavage site 682 RRAR 685 Use underlining, bolding, and italics.

[0174] The amino acid sequence of the full-length extracellular domain c1 of the mutated SARS-CoV-2 Delta variant Spike protein is shown in SEQ ID NO: 9. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is italicized, and the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0175] The amino acid sequence of the full-length extracellular domain c2 of the mutated SARS-CoV-2 Delta variant Spike protein is shown in SEQ ID NO: 10. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics, and the S1 / S2 cleavage site is shown in 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0176] The amino acid sequence of the full-length extracellular domain c3 of the mutated SARS-CoV-2 Delta variant Spike protein is shown in SEQ ID NO: 80. The sequence does not contain a signal peptide or S1 / S2 cleavage site. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0177] The amino acid sequence of the C-terminal truncated fragment d1 of the extracellular domain of the mutated SARS-CoV-2 Delta variant Spike protein is shown in SEQ ID NO: 11. In the sequence, 70 amino acid residues are truncated at the C-terminus, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is italicized, and the S1 / S2 cleavage site is shown in SEQ ID NO: 11. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0178] The amino acid sequence of the C-terminal truncated fragment d2 of the extracellular domain of the mutated SARS-CoV-2 Delta variant Spike protein is shown in SEQ ID NO: 12. In the sequence, 70 amino acid residues are truncated at the C-terminus, and the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics, and the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0179] The amino acid sequence of the C-terminal truncated fragment d3 of the extracellular domain of the mutated SARS-CoV-2 Delta variant Spike protein is shown in SEQ ID NO: 81. In the sequence, the C-terminus is truncated by 70 amino acid residues, does not contain a signal peptide, and has an S1 / S2 cleavage site. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0180] The amino acid sequence of the S1 subunit of the Spike protein of the SARS-CoV-2 Delta variant is shown in SEQ ID NO: 13, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) is italicized.

[0181] The full-length extracellular domain (ECD) of the SARS-CoV-2 Omicron variant Spike protein, whose amino acid sequence is shown in SEQ ID NO:31, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1 / S2 cleavage site 682 RRAR 685 Use underlining, bolding, and italics.

[0182] The amino acid sequence of the full-length extracellular domain f1 of the mutated SARS-CoV-2 Omicron variant Spike protein is shown in SEQ ID NO: 32. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is italicized, and the S1 / S2 cleavage site is 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0183] The amino acid sequence of the full-length extracellular domain f2 of the mutated SARS-CoV-2 Omicron variant Spike protein is shown in SEQ ID NO: 33. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics, and the S1 / S2 cleavage site is shown in 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0184] The amino acid sequence of the full-length extracellular domain f3 of the mutated SARS-CoV-2 Omicron variant Spike protein is shown in SEQ ID NO: 82. The sequence does not contain a signal peptide or an S1 / S2 cleavage site. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0185] The amino acid sequence of the C-terminal truncated fragment g1 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant Spike protein is shown in SEQ ID NO: 34. In the sequence, 70 amino acid residues are truncated at the C-terminus, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is italicized, and the S1 / S2 cleavage site is shown in SEQ ID NO: 34. 682 RRAR 685 Mutation682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0186] The amino acid sequence of the C-terminal truncated fragment g2 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant Spike protein is shown in SEQ ID NO: 35. In the sequence, the C-terminus is truncated by 70 amino acid residues, and the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics, and the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0187] The amino acid sequence of the C-terminal truncated fragment g3 of the extracellular domain of the Spike protein of the mutant SARS-CoV-2 Omicron variant is shown in SEQ ID NO: 83. In the sequence, the C-terminus is truncated by 70 amino acid residues, does not contain a signal peptide, and has an S1 / S2 cleavage site. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0188] The amino acid sequence of the Spike protein S1 subunit of the SARS-CoV-2 Omicron variant is shown in SEQ ID NO:36, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is italicized.

[0189] The amino acid sequence of the conserved fragment O330 of the Spike protein of the SARS-CoV-2 Omicron variant is shown in SEQ ID NO:37.

[0190] Fusion protein

[0191] The present invention provides a fusion protein comprising a heterologous scaffold, wherein the heterologous scaffold displays at least one antigenic polypeptide or trimeric protein derived from a coronavirus Spike protein. In some embodiments, the coronavirus antigen used is a coronavirus Spike protein extracellular domain or a truncated fragment thereof containing the various stabilizing mutations described above. In some embodiments, the coronavirus antigen used comprises or is derived from the RBD domain of the coronavirus Spike protein. In some embodiments, the coronavirus antigen used comprises or is derived from the S1 subunit of the coronavirus Spike protein. In some embodiments, the coronavirus antigen used comprises or is derived from a conserved fragment of the coronavirus Spike protein. In an exemplary embodiment, the Spike protein sequence used comprises the sequence shown in any one of SEQ ID NOs: 1, 3, 4, 6-13, 31-37, 78-83, or a variant substantially identical thereto or conservatively modified therewith. After the expression vector expressing the fusion protein is transfected into the host cell, a nanoparticle vaccine displaying the antigen (e.g., Spike protein) on the surface will be produced due to the connection between the antigen (e.g., Spike protein) and the self-assembling protein (e.g., monomeric ferritin subunit).

[0192] Any heterologous scaffold can be used to present antigens in the construction of the vaccine of the present invention. This includes virus-like particles (VLPs), such as nanoparticles. Various nanoparticles can be used to produce the vaccine of the present invention. Typically, the nanoparticles used in the present invention need to be formed by multiple replicas of a single subunit. The nanoparticles are typically spherical and / or have rotational symmetry (e.g., having a 3-fold axis and a 5-fold axis), such as the icosahedral structure exemplified herein. Additionally or alternatively, the amino termini of the nanoparticle subunits must be exposed and in close proximity to the 3-fold axis, and the spacing of the three amino termini must closely match the spacing of the carboxyl termini of the displayed trimer-stabilized Spike protein.

[0193] In some embodiments, the self-assembled nanoparticles employed are about 25 nm in diameter or less (typically assembled from 12, 24, or 60 subunits) and have a 3-fold axis on the particle surface. Such nanoparticles provide suitable particles for producing multivalent vaccines. In some preferred embodiments, coronavirus antigens can be presented on self-assembled nanoparticles, for example, on self-assembled nanoparticles derived from ferritin (FR), as exemplified herein. Ferritin is a globular protein found in animals, bacteria, and plants whose primary function is to control the rate and location of multinuclear Fe(III)2O3 formation by transporting hydrated iron ions and protons to or from a mineralized core. The globular form of ferritin consists of a monomeric subunit protein (also referred to as a monomeric ferritin subunit), which is a polypeptide having a molecular weight of approximately 17-20 kDa. The sequences of the subunits of these proteins are known in the art. In some embodiments, nanoparticle vaccines of the invention may utilize any of these known nanoparticles, as well as conservatively modified variants thereof or variants having substantially identical (e.g., at least 90%, 95%, or 99% identical) sequences thereto.

[0194] In some exemplary embodiments, the fusion protein of the present invention comprises an Fc fragment (e.g., a human IgG Fc fragment). Typically, the C-terminus of a coronavirus Spike protein conserved sequence or a coronavirus Spike protein S1 subunit or a mutation-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof is fused to the N-terminus of the Fc fragment.

[0195] The amino acid sequence of human IgG Fc is as follows:

[0196] In some exemplary embodiments, the fusion protein of the present invention comprises a nanoparticle subunit sequence (e.g., a Helicobacter pylori non-heme monomeric ferritin subunit, whose amino acid sequence is shown in SEQ ID NO: 14), or a conservatively modified variant thereof or a sequence substantially identical thereto. Typically, the C-terminus of the coronavirus Spike protein conserved sequence or the coronavirus Spike protein S1 subunit or the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof is fused to the N-terminus of the self-assembling nanoparticle (NP) subunit. In some embodiments, the C-terminus of the coronavirus Spike protein conserved fragment or the coronavirus Spike protein S1 subunit or the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof is connected to the N-terminus of the nanoparticle subunit via a GS linker, such as GGGGS or GGGGSGGGGS.

[0197] The amino acid sequence of the non-heme monomeric ferritin subunit of Helicobacter pylori is as follows:

[0198] Nanoparticles displaying the conserved sequence of the coronavirus Spike protein described herein or the coronavirus Spike protein S1 subunit or any stable coronavirus Spike protein ectodomain containing mutations or their truncated fragments can be constructed by fusing the subunits of the antigen polypeptide or multimeric antigen protein (e.g., trimeric antigen) to the subunits of the nanoparticle (e.g., ferritin subunit) and other optional or alternative components described herein. In order to construct the fusion protein of the present invention, one or more linkers can be used to connect and maintain the overall activity of different functional proteins unchanged. Typically, the linker comprises a short peptide sequence, such as a GS-rich peptide. In some embodiments, the linker or linker motif can be any flexible peptide that connects two protein domains or motifs without interfering with their function. For example, the linker used can be a G4S linker or a (G4S)2 linker as shown herein to connect the spike protein and the nanoparticle scaffold sequence. The recombinant production of the fusion protein of the present invention can be based on the scheme described herein and / or other methods known in the art.

[0199] Exemplary fusion protein sequences are as follows:

[0200] Fusion protein A1: The C-terminus of the full-length extracellular domain a1 of the mutated SARS-CoV-2 original strain Spike protein (as shown in SEQ ID NO: 3) was connected to the N-terminus of the Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO: 14) via the linker GGGGS (as shown in SEQ ID NO: 15) to obtain fusion protein A1, the amino acid sequence of which is shown in SEQ ID NO: 16. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 16. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0201] Fusion protein A2: The C-terminus of the full-length extracellular domain a2 of the mutated SARS-CoV-2 original strain Spike protein (as shown in SEQ ID NO: 4) was connected to the N-terminus of the Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO: 14) via the linker GGGGS (as shown in SEQ ID NO: 15) to obtain fusion protein A2, the amino acid sequence of which is shown in SEQ ID NO: 17. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) was replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics and the S1 / S2 cleavage site is shown in SEQ ID NO: 17. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0202] Fusion protein B1: The C-terminus of the truncated fragment b1 of the extracellular domain of the mutated SARS-CoV-2 original strain Spike protein (as shown in SEQ ID NO: 6) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 14) via the linker GGGGS (as shown in SEQ ID NO: 15) to obtain fusion protein B1, whose amino acid sequence is shown in SEQ ID NO: 18. In the sequence, the C-terminus of the extracellular domain of the SARS-CoV-2 original strain Spike protein is truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 18. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0203] Fusion protein B2: The C-terminus of the truncated fragment b2 of the extracellular domain of the mutated SARS-CoV-2 original strain Spike protein (as shown in SEQ ID NO: 7) was connected to the N-terminus of the Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO: 14) via the linker GGGGS (as shown in SEQ ID NO: 15) to obtain fusion protein B2, the amino acid sequence of which is shown in SEQ ID NO: 19. In the sequence, the C-terminus of the extracellular domain of the SARS-CoV-2 original strain Spike protein was truncated by 70 amino acid residues, and the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) was replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 19. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0204] Fusion protein C1: The C-terminus of the mutant SARS-CoV-2 Delta variant Spike protein full-length extracellular domain c1 (as shown in SEQ ID NO: 9) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 14) through the linker GGGGS (as shown in SEQ ID NO: 15) to obtain fusion protein C1, whose amino acid sequence is shown in SEQ ID NO: 20. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 20. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0205] Fusion protein C2: The C-terminus of the mutant SARS-CoV-2 Delta variant Spike protein full-length extracellular domain c2 (as shown in SEQ ID NO: 10) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 14) through the linker GGGGS (as shown in SEQ ID NO: 15) to obtain the fusion protein C2, whose amino acid sequence is shown in SEQ ID NO: 21. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) was replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics and the S1 / S2 cleavage site is shown in SEQ ID NO: 21. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0206] Fusion protein D1: The C-terminus of the truncated fragment d1 of the extracellular domain of the mutant SARS-CoV-2 Delta variant Spike protein (as shown in SEQ ID NO: 11) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 14) via the linker GGGGS (as shown in SEQ ID NO: 15) to obtain the fusion protein D1, whose amino acid sequence is shown in SEQ ID NO: 22. In the sequence, the C-terminus of the extracellular domain of the SARS-CoV-2 Delta variant Spike protein is truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 22. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0207] Fusion protein D2: The C-terminus of the truncated fragment d2 of the extracellular domain of the mutant SARS-CoV-2 Delta variant Spike protein (as shown in SEQ ID NO: 12) was connected to the N-terminus of the Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO: 14) via the linker GGGGS (as shown in SEQ ID NO: 15) to obtain the fusion protein D2, whose amino acid sequence is shown in SEQ ID NO: 23. In the sequence, the C-terminus of the extracellular domain of the SARS-CoV-2 Delta variant Spike protein was truncated by 70 amino acid residues, and the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) was replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 23. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0208] Fusion protein E1: The C-terminus of the SARS-CoV-2 Delta variant Spike protein S1 subunit (shown in SEQ ID NO: 13) was linked to the N-terminus of the Helicobacter pylori non-heme monomeric ferritin subunit (shown in SEQ ID NO: 14) via the linker GGGGS (shown in SEQ ID NO: 15) to obtain fusion protein E1, whose amino acid sequence is shown in SEQ ID NO: 24. In the sequence, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) is italicized, and the linker is italicized and bolded.

[0209] Fusion protein E2: The C-terminus of the SARS-CoV-2 Delta variant Spike protein S1 subunit (as shown in SEQ ID NO:13) was linked to the N-terminus of the Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO:14) via the linker GGGGS (as shown in SEQ ID NO:15). The original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO:5), resulting in fusion protein E2, the amino acid sequence of which is shown in SEQ ID NO:25. In the sequence, the N-terminal signal peptide is italicized, and the linker is italicized and bolded.

[0210] Fusion protein E3: The C-terminus of the SARS-CoV-2 Omicron variant Spike protein S1 subunit (shown in SEQ ID NO:36) was linked to the N-terminus of the Helicobacter pylori non-heme monomeric ferritin subunit (shown in SEQ ID NO:14) via the linker GGGGS (shown in SEQ ID NO:15) to obtain fusion protein E3, the amino acid sequence of which is shown in SEQ ID NO:39. In the sequence, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is italicized, and the linker is italicized and bolded.

[0211] Fusion protein E4: The C-terminus of the SARS-CoV-2 Omicron variant Spike protein S1 subunit (shown in SEQ ID NO:36) was linked to the N-terminus of the Helicobacter pylori non-heme monomeric ferritin subunit (shown in SEQ ID NO:14) via the linker GGGGS (shown in SEQ ID NO:15). The original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5) to obtain fusion protein E4, the amino acid sequence of which is shown in SEQ ID NO:40. In the sequence, the N-terminal signal peptide is italicized, and the linker is italicized and bolded.

[0212] Fusion protein F1: The C-terminus of the mutant SARS-CoV-2 Omicron variant Spike protein full-length extracellular domain f1 (as shown in SEQ ID NO:32) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO:14) through the linker GGGGS (as shown in SEQ ID NO:15) to obtain fusion protein F1, whose amino acid sequence is shown in SEQ ID NO:41. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO:41. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0213] Fusion protein F2: The C-terminus of the mutant SARS-CoV-2 Omicron variant Spike protein full-length extracellular domain f2 (as shown in SEQ ID NO: 33) was connected to the N-terminus of the Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO: 14) through the linker GGGGS (as shown in SEQ ID NO: 15) to obtain the fusion protein F2, whose amino acid sequence is shown in SEQ ID NO: 42. In the sequence, the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaced the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2), the signal peptide is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 42. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0214] Fusion protein G1: The C-terminus of the truncated fragment g1 of the extracellular domain of the mutant SARS-CoV-2 Omicron variant Spike protein (as shown in SEQ ID NO: 34) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 14) through the linker GGGGS (as shown in SEQ ID NO: 15) to obtain the fusion protein G1, whose amino acid sequence is shown in SEQ ID NO: 43. In the sequence, the C-terminus of the extracellular domain of the SARS-CoV-2 Omicron variant Spike protein is truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 43. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0215] Fusion protein G2: The C-terminus of the truncated fragment g2 of the extracellular domain of the mutant SARS-CoV-2 Omicron variant Spike protein (as shown in SEQ ID NO: 35) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 14) through the linker GGGGS (as shown in SEQ ID NO: 15) to obtain the fusion protein G2, whose amino acid sequence is shown in SEQ ID NO: 44. In the sequence, the C-terminus of the extracellular domain of the SARS-CoV-2 Omicron variant Spike protein was truncated by 70 amino acid residues, and the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) was replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics and the S1 / S2 cleavage site is shown in SEQ ID NO: 44. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0216] Fusion protein H1: The original signal peptide, MFVFLVLLPLVSS (shown in SEQ ID NO:2), was added to the N-terminus of the O330 fragment (shown in SEQ ID NO:37). The C-terminus of the O330 fragment was then ligated to the N-terminus of the H. pylori non-heme monomeric ferritin subunit (shown in SEQ ID NO:14) via the linker GGGGS (shown in SEQ ID NO:15) to obtain fusion protein H1, the amino acid sequence of which is shown in SEQ ID NO:45. The original signal peptide is italicized, and the linker is italicized and bolded.

[0217] Fusion protein H2: The signal peptide MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5) was added to the N-terminus of the O330 fragment (shown in SEQ ID NO:37). The C-terminus of the O330 fragment was then ligated to the N-terminus of the H. pylori non-heme monomeric ferritin subunit (shown in SEQ ID NO:14) via the linker GGGGS (shown in SEQ ID NO:15) to obtain fusion protein H2, the amino acid sequence of which is shown in SEQ ID NO:46. The signal peptide is italicized, and the linker is italicized and bolded.

[0218] Fusion protein A1-1: The C-terminus of the full-length extracellular domain a1 of the mutated SARS-CoV-2 original strain Spike protein (as shown in SEQ ID NO: 3) was linked to the N-terminus of human IgG Fc (as shown in SEQ ID NO: 38) to obtain fusion protein A1-1, whose amino acid sequence is shown in SEQ ID NO: 47. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is italicized, and the S1 / S2 cleavage site is 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0219] Fusion protein A2-1: The C-terminus of the full-length extracellular domain a2 of the mutated SARS-CoV-2 original strain Spike protein (as shown in SEQ ID NO: 4) was connected to the N-terminus of human IgG Fc (as shown in SEQ ID NO: 38) to obtain fusion protein A2-1, whose amino acid sequence is shown in SEQ ID NO: 48. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) was replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 48. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0220] Fusion protein B1-1: The C-terminus of the truncated fragment b1 (as shown in SEQ ID NO: 6) of the extracellular domain of the mutated SARS-CoV-2 original strain Spike protein was connected to the N-terminus of human IgG Fc (as shown in SEQ ID NO: 38) to obtain fusion protein B1-1, whose amino acid sequence is shown in SEQ ID NO: 49. In the sequence, the C-terminus of the extracellular domain of the SARS-CoV-2 original strain Spike protein was truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 49. 682 RRAR 685 Mutation 682 GSAS685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0221] Fusion protein B2-1: The C-terminus of the truncated fragment b2 (as shown in SEQ ID NO: 7) of the extracellular domain of the mutated SARS-CoV-2 original strain Spike protein was connected to the N-terminus of human IgG Fc (as shown in SEQ ID NO: 38) to obtain fusion protein B2-1, whose amino acid sequence is shown in SEQ ID NO: 50. In the sequence, the C-terminus of the extracellular domain of the SARS-CoV-2 original strain Spike protein was truncated by 70 amino acid residues, and the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) was replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 50. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0222] Fusion protein C1-1: The C-terminus of the full-length extracellular domain c1 of the mutant SARS-CoV-2 Delta variant Spike protein (as shown in SEQ ID NO: 9) was linked to the N-terminus of human IgG Fc (as shown in SEQ ID NO: 38) to obtain fusion protein C1-1, whose amino acid sequence is shown in SEQ ID NO: 51. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is italicized, and the S1 / S2 cleavage site is 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0223] Fusion protein C2-1: The C-terminus of the full-length extracellular domain c2 of the mutant SARS-CoV-2 Delta variant Spike protein (as shown in SEQ ID NO: 10) was connected to the N-terminus of human IgG Fc (as shown in SEQ ID NO: 38) to obtain fusion protein C2-1, whose amino acid sequence is shown in SEQ ID NO: 52. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) was replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 52. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0224] Fusion protein D1-1: The C-terminus of the truncated fragment d1 (as shown in SEQ ID NO: 11) of the extracellular domain of the mutant SARS-CoV-2 Delta variant Spike protein was connected to the N-terminus of human IgG Fc (as shown in SEQ ID NO: 38) to obtain fusion protein D1-1, whose amino acid sequence is shown in SEQ ID NO: 53. In the sequence, the C-terminus of the extracellular domain of the SARS-CoV-2 Delta variant Spike protein is truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 53. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0225] Fusion protein D2-1: The C-terminus of the truncated fragment d2 of the extracellular domain of the mutant SARS-CoV-2 Delta variant Spike protein (as shown in SEQ ID NO: 12) was connected to the N-terminus of human IgG Fc (as shown in SEQ ID NO: 38) to obtain fusion protein D2-1, whose amino acid sequence is shown in SEQ ID NO: 54. In the sequence, the C-terminus of the extracellular domain of the SARS-CoV-2 Delta variant Spike protein was truncated by 70 amino acid residues, and the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) was replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 54. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0226] Fusion protein E1-1: The C-terminus of the SARS-CoV-2 Delta variant Spike protein S1 subunit (as shown in SEQ ID NO:13) was ligated to the N-terminus of human IgG Fc (as shown in SEQ ID NO:38) to obtain fusion protein E1-1, the amino acid sequence of which is shown in SEQ ID NO:55. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is italicized.

[0227] Fusion protein E2-1: The C-terminus of the SARS-CoV-2 Delta variant Spike protein S1 subunit (as shown in SEQ ID NO:13) was linked to the N-terminus of human IgG Fc (as shown in SEQ ID NO:38), and the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO:5) to obtain fusion protein E2-1, the amino acid sequence of which is shown in SEQ ID NO:56. In the sequence, the N-terminal signal peptide is italicized.

[0228] Fusion protein E3-1: The C-terminus of the SARS-CoV-2 Omicron variant Spike protein S1 subunit (shown in SEQ ID NO:36) was ligated to the N-terminus of human IgG Fc (shown in SEQ ID NO:38) to obtain fusion protein E3-1, the amino acid sequence of which is shown in SEQ ID NO:57. In the sequence, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is italicized.

[0229] Fusion protein E4-1: The C-terminus of the SARS-CoV-2 Omicron variant Spike protein S1 subunit (as shown in SEQ ID NO:36) was linked to the N-terminus of human IgG Fc (as shown in SEQ ID NO:38), and the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO:5) to obtain fusion protein E4-1, the amino acid sequence of which is shown in SEQ ID NO:58. In the sequence, the N-terminal signal peptide is italicized.

[0230] Fusion protein F1-1: The C-terminus of the full-length extracellular domain f1 of the mutant SARS-CoV-2 Omicron variant Spike protein (as shown in SEQ ID NO: 32) was linked to the N-terminus of human IgG Fc (as shown in SEQ ID NO: 38) to obtain fusion protein F1-1, the amino acid sequence of which is shown in SEQ ID NO: 59. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is italicized, and the S1 / S2 cleavage site is shown in SEQ ID NO: 59. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0231] Fusion protein F2-1: The C-terminus of the full-length extracellular domain f2 of the mutant SARS-CoV-2 Omicron variant Spike protein (as shown in SEQ ID NO: 33) was connected to the N-terminus of human IgG Fc (as shown in SEQ ID NO: 38) to obtain the fusion protein F2-1, whose amino acid sequence is shown in SEQ ID NO: 60. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) was replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics and the S1 / S2 cleavage site is shown in SEQ ID NO: 60. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0232] Fusion protein G1-1: The C-terminus of the truncated fragment g1 (as shown in SEQ ID NO: 34) of the extracellular domain of the mutant SARS-CoV-2 Omicron variant Spike protein was connected to the N-terminus of human IgG Fc (as shown in SEQ ID NO: 38) to obtain the fusion protein G1-1, whose amino acid sequence is shown in SEQ ID NO: 61. In the sequence, the C-terminus of the extracellular domain of the SARS-CoV-2 Omicron variant Spike protein is truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 61. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0233] Fusion protein G2-1: The C-terminus of the truncated fragment g2 (as shown in SEQ ID NO: 35) of the extracellular domain of the mutant SARS-CoV-2 Omicron variant Spike protein was connected to the N-terminus of human IgG Fc (as shown in SEQ ID NO: 38) to obtain the fusion protein G2-1, whose amino acid sequence is shown in SEQ ID NO: 62. In the sequence, the C-terminus of the extracellular domain of the SARS-CoV-2 Omicron variant Spike protein was truncated by 70 amino acid residues, and the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) was replaced by the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5). The signal peptide is marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO: 62. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0234] Fusion protein H1-1: The original signal peptide, MFVFLVLLPLVSS (shown in SEQ ID NO:2), was added to the N-terminus of the O330 fragment (shown in SEQ ID NO:37). The C-terminus of the O330 fragment was then ligated to the N-terminus of human IgG Fc (shown in SEQ ID NO:38) to obtain fusion protein H1-1, the amino acid sequence of which is shown in SEQ ID NO:63. The original signal peptide is indicated in italics.

[0235] Fusion protein H2-1: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO: 5) was added to the N-terminus of the O330 fragment (shown in SEQ ID NO: 37), and the C-terminus of the O330 fragment was then ligated to the N-terminus of human IgG Fc (shown in SEQ ID NO: 38) to obtain fusion protein H2-1, the amino acid sequence of which is shown in SEQ ID NO: 64. The signal peptide is indicated in italics.

[0236] Mature fusion protein A: Compared with fusion proteins A1 and A2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 26. In the sequence, the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685, which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0237] Mature fusion protein B: Compared with fusion proteins B1 and B2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 27. In the sequence, the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0238] Mature fusion protein C: Compared with fusion proteins C1 and C2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 28. In the sequence, the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0239] Mature fusion protein D: Compared with fusion proteins D1 and D2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 29. In the sequence, the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0240] Mature fusion protein E-1: Compared with fusion proteins E1 and E2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 30. In the sequence, the linker is marked in italics and bold.

[0241] Mature fusion protein E-2: Compared with fusion proteins E3 and E4, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 65. In the sequence, the linker is marked in italics and bold.

[0242] Mature fusion protein F: Compared with fusion proteins F1 and F2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 66. In the sequence, the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0243] Mature fusion protein G: Compared with fusion proteins G1 and G2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 67. In the sequence, the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0244] Mature fusion protein H: Compared with fusion proteins H1 and H2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 68. In the sequence, the linker is marked in italics and bold.

[0245] Mature fusion protein A-1: ​​Compared with fusion proteins A1-1 and A2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 69. In the sequence, the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0246] Mature fusion protein B-1: Compared with fusion proteins B1-1 and B2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 70. In the sequence, the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685, which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0247] Mature fusion protein C-1: Compared with fusion proteins C1-1 and C2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 71. In the sequence, the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0248] Mature fusion protein D-1: Compared with fusion proteins D1-1 and D2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 72. In the sequence, the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0249] Mature fusion protein E-3: Compared with fusion proteins E1-1 and E2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 73.

[0250] Mature fusion protein E-4: Compared with fusion proteins E3-1 and E4-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 74.

[0251] Mature fusion protein F-1: Compared with fusion proteins F1-1 and F2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 75. In the sequence, the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0252] Mature fusion protein G-1: Compared with fusion proteins G1-1 and G2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 76. In the sequence, the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0253] Mature fusion protein H-1: Compared with fusion proteins H1-1 and H2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 77.

[0254] SEQ ID NO: 26-30, 65-77 are mature fusion protein sequences with the N-terminal signal peptide (SEQ ID NO: 2 or 5) removed. In addition to these specific exemplified fusion proteins, the present invention also encompasses nanoparticle vaccines comprising a subunit sequence substantially identical to any of these exemplified nanoparticle vaccine sequences or a subunit sequence of a conservatively modified variant thereof.

[0255] Coronavirus polyvalent vaccine

[0256] In some embodiments, the coronavirus multivalent vaccine of the present invention comprises antigens from two or more viruses (e.g., Spike proteins from different SARS-CoV-2 coronavirus sources) or fusion proteins thereof, or comprises different antigens from the same SARS-CoV-2 coronavirus or fusion proteins thereof. In some embodiments, the coronavirus multivalent vaccine is a coronavirus bivalent vaccine. In some embodiments, the coronavirus multivalent vaccine is a coronavirus trivalent vaccine.

[0257] In some embodiments, the coronavirus multivalent vaccine comprises at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67.

[0258] In some embodiments, the coronavirus multivalent vaccine comprises at least two fusion proteins comprising an amino acid sequence as shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67.

[0259] In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 22, 23 or 29 and a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 43, 44 or 67.

[0260] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 22, 23 or 29 and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 43, 44 or 67 is (1-5): (1-5); or the mass ratio is 1:1.

[0261] In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO:22 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO:43.

[0262] In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO:29 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO:67.

[0263] In some embodiments, the coronavirus multivalent vaccine comprises at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76.

[0264] In some embodiments, the coronavirus multivalent vaccine comprises at least two fusion proteins comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76.

[0265] In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 53, 54 or 72 and a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 61, 62 or 76.

[0266] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 53, 54 or 72 and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 61, 62 or 76 is (1-5): (1-5); or the mass ratio is 1:1.

[0267] In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO:53 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO:61.

[0268] In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO:72 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO:76.

[0269] In some embodiments, the coronavirus multivalent vaccine further comprises a conserved fragment of the coronavirus Spike protein or a fusion protein comprising the same.

[0270] In some embodiments, the coronavirus multivalent vaccine comprises: (1) at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67, and (2) a fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 45-46, 63, 64, 68, and 77.

[0271] In some embodiments, the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO:22, 23 or 29, (2) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO:43, 44 or 67, and (3) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO:63, 64 or 77.

[0272] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 22, 23 or 29, the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 43, 44 or 67, and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 63, 64 or 77 is (1-5): (1-5): (1-5); or the mass ratio is 1:1:1.

[0273] In some embodiments, the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:22, (2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:43, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:63.

[0274] In some embodiments, the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:29, (2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:67, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:77.

[0275] In some embodiments, the coronavirus multivalent vaccine comprises: (1) at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76, and (2) a fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 45-46, 63, 64, 68 and 77.

[0276] In some embodiments, the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 53, 54 or 72, (2) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 61, 62 or 76, and (3) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 63, 64 or 77.

[0277] In some embodiments, the mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 53, 54 or 72, the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 61, 62 or 76, and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 63, 64 or 77 is (1-5):(1-5):(1-5); or the mass ratio is 1:1:1.

[0278] In some embodiments, the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:53, (2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:61, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:63.

[0279] In some embodiments, the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:72, (2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:76, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:77.

[0280] In some embodiments, the coronavirus multivalent vaccine is prepared by mixing two or more antigens or fusion proteins containing them in a certain proportion.

[0281] Polynucleotides and expression vectors

[0282] The mutation-containing coronavirus Spike protein extracellular domain or its truncated fragment, coronavirus Spike protein S1 subunit, coronavirus Spike protein conserved fragment, fusion protein or Spike protein nanoparticle of the present invention is generally produced by an expression vector, which comprises the coding sequence of the mutation-containing coronavirus Spike protein extracellular domain or its truncated fragment, coronavirus Spike protein S1 subunit, coronavirus Spike protein conserved fragment, fusion protein or Spike protein nanoparticle described herein. Therefore, in some related aspects, the present invention provides polynucleotides (DNA or RNA) encoding the mutation-containing coronavirus Spike protein extracellular domain or its truncated fragment, coronavirus Spike protein S1 subunit, coronavirus Spike protein conserved fragment, fusion protein or Spike protein nanoparticle described herein. Some polynucleotides of the present invention encode one of the mutation-containing coronavirus Spike protein extracellular domain or its truncated fragment described herein, for example, a truncated fragment of the SARS-CoV-2 Spike protein extracellular domain shown in SEQ ID NO: 12. Some polynucleotides of the present invention encode a subunit sequence of one of the nanoparticle vaccines described herein, for example, a fusion protein sequence shown in SEQ ID NO: 23. The fusion protein expressed by the present invention may not contain an N-terminal signal peptide, or some polynucleotide sequences may additionally encode an N-terminal signal peptide. For example, a polynucleotide encoding a fusion protein (e.g., SEQ ID NOs: 26-30) may further comprise a sequence encoding an N-terminal signal peptide as set forth in SEQ ID NOs: 2 or 5, or a sequence substantially identical thereto or a conservatively modified variant thereof.

[0283] The present invention also provides expression vectors having such polynucleotides and host cells (e.g., prokaryotic or eukaryotic cells, such as HEK293, CHO, ExpiCHO, and CHO-S cell lines) for producing a coronavirus Spike protein ectodomain or a truncated fragment thereof, a coronavirus Spike protein S1 subunit, a coronavirus Spike protein conserved fragment, or a fusion protein containing a mutation. Fusion proteins encoded by the polynucleotides or expressed by the vectors are also included in the present invention. As described herein, the nanoparticle subunit-fused Spike protein ectodomain or a truncated fragment thereof, Spike protein S1 subunit, or a Spike protein conserved fragment will self-assemble into a nanoparticle vaccine that displays the Spike protein or its truncated fragment, Spike protein S1 subunit, or a Spike protein conserved fragment on its surface.

[0284] Polynucleotides and related vectors can be produced by standard molecular biology techniques or the protocols exemplified herein. For example, general protocols for cloning, transfection, transient gene expression, and obtaining stably transfected cell lines are described in the art, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, (3rd edition, 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou edition, 2003). Mutations can also be introduced into polynucleotide sequences by known methods, such as PCR.

[0285] The selection of specific vector depends on the intended use of protein. For example, no matter the cell type is prokaryotic or eukaryotic, the vector selected must be able to drive the expression of protein in the desired cell type. Many vectors contain sequences that allow prokaryotic vectors to replicate and the eukaryotic expression of operably connected gene sequences. The vector that can be used for the present invention can replicate autonomously, that is, the vector exists outside the chromosome, and its replication need not be directly connected to the replication of the host cell genome. Alternatively, the replication of the vector can be connected to the replication of the host chromosome DNA, for example, the vector can be integrated into the chromosome of the host cell, which is achieved by retroviral vectors and in a stable transfected cell line. Viral-based expression vectors and non-viral-based expression vectors can be used to produce antigens in mammalian host cells. Non-viral vectors and systems include plasmids, additional vectors (usually having an expression cassette for expressing protein or RNA) and human artificial chromosomes. Alternative viral vectors include lentiviral or other retroviral based vectors, adenovirus, adeno-associated virus, cytomegalovirus, herpes virus, SV40 based vectors, papillomavirus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV).

[0286] Depending on the specific vector used to express the protein, various known cells or cell lines can be used in the practice of the present invention. The host cell can be any cell carrying the recombinant vector of the protein of the present invention, wherein the vector is allowed to drive the protein expression for the present invention. It can be prokaryotic, such as any of many bacterial strains, or can be eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells, including, for example, rodent, monkey or human cells. The cell expressing the protein of the present invention can be a primary culture cell or can be an established cell line. Therefore, in addition to the cell line (such as HEK293 cell) illustrated herein, many other host cell lines well known in the art can also be used in the practice of the present invention. These include, for example, a variety of Cos cell lines, CHO cells, HeLa cells, Sf9 cells, AtT20, BV2 and N18 cells, myeloma cell lines, transformed B cells and hybridomas.

[0287] The vector expressing the protein can be introduced into the selected host cell by any of a number of suitable methods known to those skilled in the art. To introduce the protein-encoding vector into mammalian cells, the method used will depend on the form of the vector. For plasmid vectors, DNA encoding the protein sequence can be introduced by any of a number of transfection methods, including, for example, liposome-mediated transfection ("lipofection"), DEAE-dextran-mediated transfection, electroporation, or calcium phosphate precipitation. These methods are described in detail, for example, in Brent et al. above. Among them, lipofection is widely accepted because it is simple to operate and does not require special equipment. For example, Lipofectamine (Life Technologies) or LipoTAXI (Stratagene) kits can be used for transfection. Other companies that provide lipofection reagents and methods include Bio-Rad Laboratories, CLONTECH, Glen Research, Life Technologies, JBL Scientific, MBI Fermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

[0288] In order to produce recombinant proteins in high yield over a long period of time, stable expression is preferred. Instead of using an expression vector comprising a viral origin of replication, a protein coding sequence and an optional marker controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) can be used to transform the host cell. The selective marker in the recombinant vector is resistant to selection and allows the cell to stably integrate the vector into its chromosome. Commonly used selective markers include neomycin (neo), which is resistant to the aminoglycoside G-418, and hygromycin (hygro), which is resistant to hygromycin.

[0289] In some embodiments, the recombinant expression vector comprises at least one promoter element, a protein coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on either side of the inserted sequence. Efficient transcription can be achieved using the early and late promoters of SV40, the early promoters of long terminal repeats from retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus. Other cellular promoters, such as the actin promoter, can also be used. Suitable expression vectors include pIRES1neo, pRetro-Off, pRetro-On, pLXSN, pLNCX, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), pSVL, pMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2. Commonly used mammalian cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells.

[0290] In some embodiments, the inserted gene fragment needs to contain a selection marker. Common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, and other selection markers to facilitate the screening and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking these genes. After culture in a selective medium, the successfully transfected cells grow in large numbers and produce the desired target protein.

[0291] In addition, standard techniques well known to those skilled in the art can be used to introduce mutations in the nucleotide sequences encoding the present invention, including but not limited to site-directed mutagenesis and PCR-mediated mutations that cause amino acid replacements. Variants (including derivatives) encode less than 50 amino acid replacements, less than 40 amino acid replacements, less than 30 amino acid replacements, less than 25 amino acid replacements, less than 20 amino acid replacements, less than 15 amino acid replacements, less than 10 amino acid replacements, less than 5 amino acid replacements, less than 4 amino acid replacements, less than 3 amino acid replacements, or less than 2 amino acid replacements relative to the original protein. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example, by saturation mutation, and the biological activity of the resulting mutants can be screened to identify mutants that retain activity.

[0292] In some embodiments, the substitutions described herein are conservative amino acid substitutions.

[0293] Pharmaceutical compositions and methods of treatment

[0294] The present invention also provides a pharmaceutical composition and related treatment methods. The pharmaceutical composition comprises an effective dose of fusion protein or Spike protein nanoparticles or coronavirus multivalent vaccine and a pharmaceutically acceptable carrier.

[0295] The term "pharmaceutically acceptable" refers to substances approved by a government regulatory agency or listed in other recognized pharmacopoeias for use in animals (particularly for humans). In addition, a "pharmaceutically acceptable carrier" generally refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary agent, etc.

[0296] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle that can be administered to a patient together with the active ingredient. Such carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the pharmaceutical composition can also contain a small amount of wetting agent, emulsifier, or pH buffer such as acetate, citrate or phosphate. Antimicrobial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and agents for regulating tonicity such as sodium chloride or dextrose are also contemplated. These pharmaceutical compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The pharmaceutical compositions can be formulated as suppositories using conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a clinically effective dose of the fusion protein or spike protein nanoparticles, preferably in purified form, together with an appropriate amount of carrier to provide a dosage form suitable for the patient. The formulation should be suitable for the mode of administration. The formulation can be packaged in ampoules, disposable syringes, or multidose vials made of glass or plastic.

[0297] In some embodiments, the pharmaceutical composition may comprise a fusion protein or a Spike protein nanoparticle or a coronavirus multivalent vaccine, and a polynucleotide or vector encoding the fusion protein described herein. In some embodiments, the viral (e.g., SARS-CoV-2) Spike protein extracellular domain or a trimer of a truncated fragment thereof can be used to prevent and treat corresponding viral infections. In some embodiments, nanoparticle vaccines comprising the fusion protein described herein can be used to prevent or treat corresponding diseases, such as infections caused by various coronaviruses. Some embodiments of the present invention relate to the use of SARS-CoV-2 antigens or vaccines in preventing or treating SARS-CoV-2 infection in human subjects. Some embodiments of the present invention relate to the use of SARS-CoV antigens or vaccines in preventing or treating SARS-CoV infection.

[0298] In the practice of some therapeutic methods of the present invention, the corresponding Spike protein nanoparticles or fusion proteins or coronavirus multivalent vaccines, or polynucleotides encoding fusion proteins as described herein, are administered to subjects in need of prevention or treatment of a disease (e.g., SARS-CoV-2 infection). Typically, the Spike protein nanoparticles, fusion proteins, coronavirus multivalent vaccines, or polynucleotides encoding fusion proteins disclosed herein are contained in pharmaceutical compositions. The pharmaceutical composition can be a therapeutic preparation or a preventive preparation. Typically, the pharmaceutical composition can additionally contain one or more pharmaceutically acceptable carriers, and optionally other therapeutic ingredients (e.g., antiviral drugs). Various pharmaceutically acceptable additives can also be used in the pharmaceutical composition.

[0299] Some pharmaceutical compositions of the present invention are vaccine compositions. For vaccine compositions, suitable adjuvants may be additionally included. Suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MF59, SEPIVAC SWE TM , MPL and IL-12. In some embodiments, the vaccine compositions described herein (e.g., SARS-CoV-2 vaccines) can be formulated as controlled-release or timed-release formulations. This can be achieved in compositions comprising sustained-release polymers or by microencapsulated delivery systems or bioadhesive gels. Various pharmaceutical compositions can be prepared according to standard procedures well known in the art. See, for example, U.S. Patents 4,652,441 and 4,917,893; U.S. Patents 4,677,191 and 4,728,721; and U.S. Patent 4,675,189.

[0300] The pharmaceutical composition of the present invention can be used in a variety of therapeutic or preventive applications, for example, for treating SARS-CoV-2 infection in a subject or for causing an immune response to SARS-CoV-2 in a subject. As an example, a coronavirus multivalent vaccine can be administered to a subject to induce an immune response to SARS-CoV-2, for example, to induce the production of broad-spectrum neutralizing antibodies against the virus. For subjects at risk of infection with SARS-CoV-2, the vaccine composition of the present invention can be administered to provide preventive protection against viral infection. The therapeutic and preventive applications of vaccines derived from other antigens described herein can be similarly performed. Depending on the specific subject and disease, the pharmaceutical composition of the present invention can be administered to a subject by a variety of administration methods known to those of ordinary skill in the art, for example, by parenteral routes such as intramuscular, subcutaneous, intravenous, intra-arterial, joint, and intraperitoneal routes. In some embodiments, the treatment methods of the present invention relate to methods for blocking coronaviruses (e.g., SARS-CoV or SARS-CoV-2) from entering host cells (e.g., human host cells), methods for preventing coronavirus Spike proteins from binding to host receptors, and methods for treating acute respiratory diseases associated with coronavirus infection. In some embodiments, the treatment methods and pharmaceutical compositions described herein can be used in combination with other known therapeutic agents and / or methods for treating or preventing coronavirus infection. Known therapeutic agents and / or methods include, for example, nuclease analogs or protease inhibitors (e.g., remdesivir), monoclonal antibodies against one or more coronaviruses, immunosuppressants or anti-inflammatory drugs (e.g., sarilumab or tocilizumab), ACE inhibitors, vasodilators, or any combination thereof.

[0301] For therapeutic applications, the pharmaceutical composition should contain a therapeutically effective amount of the fusion protein, Spike protein nanoparticles, or coronavirus multivalent vaccine described herein. For preventive applications, the pharmaceutical composition should contain a preventive effective amount of the fusion protein, Spike protein nanoparticles, or coronavirus multivalent vaccine described herein. The appropriate amount of antigen can be determined based on the specific disease or condition to be treated or prevented, the severity, age, and other personal attributes of the specific subject (e.g., the overall state of the subject's health). The determination of the effective dose is also guided by animal model studies, followed by human clinical trials, and by a dosing regimen that can significantly reduce the occurrence or severity of the subject's target disease condition or symptoms.

[0302] For preventive applications, before any symptoms, for example, before infection, a pharmaceutical composition is provided. The prophylactic administration of the pharmaceutical composition is used to prevent or improve any subsequent infection. Therefore, in some embodiments, the subject to be treated is, for example, a subject who has been infected (for example, SARS-CoV-2 infects) or a subject who is at risk of infection (for example, SARS-CoV-2 infects) due to exposure or possible exposure to a virus (for example, SARS-CoV-2). After administering a therapeutically effective amount of the disclosed pharmaceutical composition, the subject's infection (for example, SARS-CoV-2 infection) or the symptoms associated with infection (for example, SARS-CoV-2 infection) can be monitored.

[0303] For therapeutic applications, the pharmaceutical composition is provided at or after the onset of symptoms of a disease or infection, for example, after the onset of symptoms of an infection (e.g., SARS-CoV-2 infection) or after diagnosis of infection. Thus, the pharmaceutical composition can be provided before expected exposure to the virus so as to reduce the expected severity, duration, or extent of the infection and / or related disease condition after exposure or suspected exposure to the virus or after the initial stage of actual infection. The pharmaceutical compositions of the present invention can be combined with other agents known in the art for treating or preventing infection with related pathogens (e.g., SARS-CoV-2 infection).

[0304] Vaccine compositions (e.g., SARS-CoV-2 vaccines) or pharmaceutical compositions comprising the fusion proteins, spike protein nanoparticles, or multivalent coronavirus vaccines of the present invention can be provided as components of a kit. Optionally, such a kit includes additional components, including packaging, instructions for use, and various other reagents, such as buffers, substrates, antibodies or ligands (e.g., control antibodies or ligands), and detection reagents.

[0305] Various known delivery systems can be used to administer the fusion protein, Spike protein nanoparticles or coronavirus multivalent vaccine or derivative of the present invention or its encoding polynucleotide or expression vector, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the fusion protein or Spike protein nanoparticles, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432), construction of nucleic acid as part of a retrovirus or other vector, etc. DETAILED DESCRIPTION

[0306] The following is a detailed description of the technical solution of the present invention, which does not limit the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0307] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0308] Example 1: Preparation of fusion protein

[0309] Based on the sequence of the fusion protein described herein, the fusion protein can be prepared by the following method or other known methods: The DNA sequence encoding the fusion protein (such as SEQ ID NO: 16-30, 39-77) is cloned into an expression vector, and then electroporated into CHO-K1 cells, cultured, and purified to obtain the fusion protein.

[0310] The three-dimensional structures of fusion protein D and fusion protein G were analyzed using cryo-electron microscopy (Cryo-EM). Connecting the extracellular domain of SARS-CoV-2 Spike protein or its truncated fragment to the N-terminus of the monomeric ferritin subunit did not interfere with the self-assembly of ferritin. Nanoparticles were well formed and spikes appeared on the surface.

[0311] Example 2: Binding ability test of fusion protein to hACE2 protein

[0312] 1.1 Fusion protein binding ability test for hACE2 protein (ELISA)

[0313] This experiment uses ELISA to detect the binding ability of fusion protein D and fusion protein G to human ACE2 protein (hACE2), thereby evaluating whether the Spike protein-ferritin fusion protein of the present invention can well display the key antigenic epitopes of Spike protein. The method is briefly described as follows: 100 μL of 2 μg / mL antigen (WT-Spike-His, Delta-Spike-His, Omicron-Spike-His, fusion protein D, or fusion protein G) solution was coated overnight at 4°C; washed twice with PBST (PBS buffer containing 0.05% Tween-20); blocking solution (PBST containing 3% BSA) was added to each reaction well and incubated at 37°C for 2 hours; after blocking, the wells were washed three times with PBST; a serial dilution of human ACE2-his-biotin (Sino Biological, Cat. No.: 10108-H27B-B) was added (starting at 2.5 μg / mL and diluted 3-fold for a total of 10 serial dilutions) at 100 μL per well and incubated at 37°C for 1.5 hours; washed five times with PBST; and streptavidin-labeled catalase (Jackson Immunofluorescence ... Research, catalog number: 016-030-084; 1:10000), incubated at 37°C for 1 hour; washed 8 times with PBST; added TMB solution to the reaction wells at 100 μL / well and incubated at 37°C in the dark for 5-15 minutes; added 50 μL of stop solution (0.1 M H2SO4) to each reaction well to terminate the enzymatic color development reaction; set the detection wavelength to 450 nm for reading.

[0314] WT-Spike-His was constructed by adding 6×His (HHHHHH) to the C-terminus of the C-terminal truncated fragment b1 of the extracellular domain of the mutated SARS-CoV-2 original strain Spike protein (as shown in SEQ ID NO:6). Delta-Spike-His was constructed by adding 6×His (HHHHHH) to the C-terminus of the C-terminal truncated fragment d1 of the extracellular domain of the mutated SARS-CoV-2 Delta variant Spike protein (as shown in SEQ ID NO:11). Omicron-Spike-His was constructed by adding 6×His (HHHHHH) to the C-terminus of the C-terminal truncated fragment g1 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant Spike protein (as shown in SEQ ID NO:34).

[0315] The results are shown in Figure 1. hACE2 binds to fusion protein D, Delta, and the Spike protein of the original strain with similar affinity.50 The values ​​were 9.2, 8.1, and 5.7 ng / mL, respectively (Figure 1a); hACE2 binds to fusion protein G and Omicron Spike protein with similar affinity, EC 50 The values ​​were 9.3 and 8.2 ng / mL, respectively ( Figure 1b ).

[0316] 1.2 Binding ability test of fusion protein to hACE2 protein (BLI)

[0317] The affinity constants of fusion proteins D and G for hACE2 binding were determined using biomembrane interferometry (BLI) on a PALL Fortebio Octet RED&QK system. Multichannel parallel quantitative analysis was performed using WT-Spike-His (same as in Example 2, step 1.1), Delta-Spike-His (same as in Example 2, step 1.1), Omicron-Spike-His (same as in Example 2, step 1.1), fusion protein D, and fusion protein G. The concentration gradient was set at 50, 100, 200, and 400 nM. hACE2-Biotin (Acro biosystems, Cat. No. AC2-H5257) was coupled to an SA Biosensor (Octet, Cat. No. 2107002811).

[0318] The results are shown in Table 1. The results indicate that the binding affinities of fusion proteins D and G for hACE2 are significantly stronger than those of WT-Spike-His, Delta-Spike-His, and Omicron-Spike-His. This indicates that the Spike protein-ferritin fusion protein of the present invention can effectively display the key antigenic epitopes of the Spike protein.

[0319] Table 1 Binding kinetics of SARS-CoV-2 Spike protein to hACE2 receptor

[0320] Example 3: Immunogenicity of the vaccine in mice

[0321] To evaluate the immunogenicity of monovalent vaccines (fusion protein D, fusion protein G) and bivalent vaccines (i.e., fusion protein D and fusion protein G at a mass ratio of 1:1), the effects of different antigen doses on immunogenicity were explored using BALB / c mice.

[0322] 1.1 Immunization of mice

[0323] Balb / c mice (n=10 per group) were injected intramuscularly with different doses of vaccine on day 0 and day 21, respectively. Control mice were only given SEPIVAC SWE TMAdjuvant, SEPIVAC SWE per mouse TM The volume of adjuvant (SEPPIC SA, product number 80748J, batch number 210721010001) was fixed at 50 μL, and the total volume of each administration was 100 μL per animal. The group dosing schedule is shown in Table 2. Blood was collected on the 14th day, 35th day (2 weeks after the second immunization), and 30 weeks after the second immunization for ELISA detection of serum anti-Spike protein [original strain, Delta, and Omicron (BA.1)] IgG titers and SARS-CoV-2 Spike pseudovirus neutralizing antibody experiments.

[0324] Table 2 Grouped dosing regimen

[0325] 1.2 Detection of serum anti-Spike protein IgG titer by ELISA

[0326] WT-Spike-His (same as in step 1.1 of Example 2), Delta-Spike-His (same as in step 1.1 of Example 2), and Omicron-Spike-His (same as in step 1.1 of Example 2) were diluted to 1 μg / mL with PBS and added to a 96-well ELISA plate (Costar, Cat. No. 9018) at 100 μL / well and incubated at 4°C overnight; the plates were washed with PBST (0.05% by volume Tween-20 in 1× PBS); Blocking solution (PBST containing 3% BSA) was added and incubated at 37°C for 2 hours; washed with PBST; the mouse serum obtained in step 1.1 of Example 3 was added with a gradient dilution (the serum on day 14 was diluted 100-fold as the starting concentration, the serum on day 35 was diluted 1000-fold as the starting concentration, and then 11 3-fold gradient dilutions were performed), 100 μL per well, incubated at 37°C for 1.5 hours; washed with PBST; Peroxidase-AffiniPure 1:10000 diluted was added. Goat Anti-Mouse IgG (Jackson, Catalog No.: 115-035-003), 100 μL / well, incubate at 37°C for 1 hour; wash with PBST; add 100 μL / well TMB solution and incubate at 37°C, then terminate the reaction with 50 μL / well 0.1 M sulfuric acid; set the detection wavelength to 450 nm for reading, and fit the obtained OD values ​​with a nonlinear four-parameter equation curve model. The serum dilution corresponding to 2.0 times the mean OD450 value of the blank control was used as the antibody titer, and then the geometric mean titer (GMT) of each group was calculated.

[0327] The results are shown in Figures 2a to 2f. In all dose groups tested, no anti-Spike protein IgG titers were detected in mice given adjuvant alone. As shown in Figures 2a, 2c, and 2e, all mice produced IgG antibodies against WT-Spike-His, Delta-Spike-His, and Omicron-Spike-His after the initial immunization, and the antibody titers were dose-dependent. Fusion protein D showed a clear dose-response relationship for the geometric mean antibody titer (GMT) against Omicron-Spike-His (Figure 2e), but the dose-response relationship for WT-Spike-His and Delta-Spike-His was not significant (Figures 2a and 2c). Fusion protein G showed a clear dose-response relationship for the GMTs against WT-Spike-His and Delta-Spike-His (Figures 2a and 2c), but the dose-response relationship for the GMTs against Omicron-Spike-His was not significant (Figure 2e). It is worth noting that in the bivalent vaccine group, the dose ranged from 0.2μg to 10μg, and the GMT of IgG antibodies against WT-Spike-His, Delta-Spike-His and Omicron-Spike-His showed a clear dose-dependency.

[0328] After the second booster immunization, anti-Spike protein IgG titers in all mice increased 20-100-fold compared to those after the initial immunization, with no significant dose-response relationship. Compared with mice receiving the same dose of fusion protein D, mice receiving fusion protein G had significantly lower antibody titers against WT-Spike-His and Delta-Spike-His (Figures 2b, 2d); however, the antibody titer against Omicron-Spike-His in the fusion protein G group was significantly higher than that in the fusion protein D group (Figure 2f). Compared with fusion protein D and fusion protein G, the bivalent vaccine induced higher antibody titers against the original strain, Delta, and Omicron anti-Spike protein IgG.

[0329] The above results show that the bivalent vaccine has better broad spectrum than the monovalent vaccine fusion protein D or fusion protein G, and the antibody titers against different mutants are better than or equal to those of the monovalent vaccine.

[0330] 1.3 SARS-CoV-2 Spike pseudovirus neutralizing antibody experiment

[0331] The SARS-CoV-2 Spike pseudovirus was diluted 50-fold with DMEM medium containing 10% FBS, and the pseudovirus dilution was added to a 96-well white plate (Costar, Cat. No. 3917) at 25 μL / well. The mouse serum collected on the 35th day of Example 3, step 1.1, was diluted 40-fold with DMEM medium containing 10% FBS, and then a 3-fold gradient dilution was performed. The dilution gradient was 7 times and added to the 96-well plate containing the pseudovirus at 50 μL / well. After thorough shaking and mixing, the plate was placed in an incubator at 37°C for 1 hour. ACE2-293 cell suspension (2×10 4 cells / well), and placed the 96-well plate in an incubator for culture; after culturing for 48 hours, the 96-well plate was taken out and allowed to return to room temperature, and 50 μL Bio-Lite was added to each well. TM Luciferase Assay System (Vazyme, Catalog No. DD1201) solution was reacted at room temperature in the dark for 2 minutes, and the luminescence signal was read using the luminescence detection module of a microplate reader.

[0332] The ACE2-293 cell construction method is as follows: HEK293 cells were cultured in DMEM complete medium containing 10% FBS and transfected with an ACE2 expression plasmid (Sino Biological, HG10108-M) using lipofectamine 2000 transfection reagent (Thermo Fisher, 11668019). Subsequently, the cells were screened by hygromycin (200 μg / ml) pressure and flow cytometry (using 10 μg / ml anti-ACE2 and PE-conjugated anti-human IgG-Fc). The cells were further expanded and single clones with a PE positive rate >90% were selected for the next expansion step to screen for ACE2-expressing HEK293 cells, namely ACE2-293 cells.

[0333] Among them, the SARS-CoV-2 Spike pseudoviruses are: SARS-CoV-2 Spike pseudovirus (Jiman Bio, GM-0220PV07); SARS-CoV-2 Spike (B.1.617.2) pseudovirus (Jiman Bio, GM-0220PV45); SARS-CoV-2 Spike (B.1.1.7 / VUI-202012 / 01,del145Y) pseudovirus (Jiman Bio, GM-0220PV33); SARS-CoV-2 Spike (B.1.351 / 501Y.V2) pseudovirus (Jiman Bio, GM-0220PV32); SARS-CoV-2 Spike (P.1501Y.V3) pseudovirus (Jiman Bio, GM-0220PV47); SARS-CoV-2 Spike (B.1.1.529) pseudovirus (Jiman Biotechnology, GM-0220PV84); SARS-CoV-2 Spike (BA.2.12.1) pseudovirus (Jiman Biotechnology, GM-0220PV93); SARS-CoV-2 Spike (BA.3) pseudovirus (Jiman Biotechnology, GM-0220PV92); SARS-CoV-2 Spike (BA.4 / 5) pseudovirus (Jiman Biotechnology, GM-0220PV89).

[0334] The results in Figure 3 show that the bivalent vaccine has a better broad spectrum than the monovalent vaccine fusion protein D or fusion protein G, and the neutralizing antibody titers against different variants are better than or equal to those of the monovalent vaccine. The titers of fusion protein G against SARS-CoV-2 Spike pseudovirus and SARS-CoV-2 Spike (B.1.617.2) pseudovirus were significantly lower than those of fusion protein D and the bivalent vaccine; the titers of fusion protein D against SARS-CoV-2 Spike (B.1.1.529) pseudovirus and SARS-CoV-2 Spike (BA.3) pseudovirus were significantly lower than those of fusion protein G and the bivalent vaccine; while the bivalent vaccine maintained a very high titer against all tested pseudoviruses.

[0335] The results in Figure 4a show that the serum of mice immunized twice with different doses of the bivalent vaccine had high neutralizing antibody titers against all tested strains. The highest IC50 GMT values ​​for SARS-CoV-2 Spike (B.1.1.529) and SARS-CoV-2 Spike (BA.4 / 5) pseudoviruses were 10198 and 1018, respectively. The GMT values ​​for other strains were all >3,000, with the highest GMT value exceeding 10,000, showing a certain dose-effect relationship. The titer of the 0.2μg group was lower than that of the 1μg group, but the difference was not statistically significant. The results in Figure 4b show that the serum of mice immunized with the bivalent vaccine had high neutralizing antibody titers against all 9 strains tested.

[0336] 1.4 Durability of vaccine-induced immune responses

[0337] The long-term antibody titer after bivalent vaccination was evaluated. The ELISA method was used to detect the specific antibody titer of the serum against WT-Spike-His (same as step 1.1 of Example 2), Delta-Spike-His (same as step 1.1 of Example 2), and Omicron-Spike-His (same as step 1.1 of Example 2). The detection steps were the same as step 1.2 of Example 3, wherein the serum was the serum collected 2 weeks after the second immunization of Group 8 in Example 3 (diluted 1000 times as the starting concentration, then 3-fold gradient dilution), and the serum collected 30 weeks after the second immunization (diluted 1000 times as the starting concentration, then 3-fold gradient dilution). The results showed (Figure 5) that the serum antibody titer at 30 weeks after the second immunization was not significantly decreased compared with the serum at 2 weeks after the second immunization. The GMTs of the serum against WT-Spike-His, Delta-Spike-His, and Omicron-Spike-His at 2 weeks and 30 weeks after the second immunization were 9.1×10 6 and 9.6×10 6 , 7.2×10 6 and 6.0×10 6 , 6.3×10 6 and 5.6×10 6 , indicating that the bivalent vaccine can provide long-term protection.

[0338] Example 4: Effect of adjuvants on vaccine immunogenicity

[0339] 1.1 Immunization of mice

[0340] BALB / c mice (8 weeks old) received two intramuscular injections (day 0 and day 21) of different doses of SEPIVAC SWE. TMAdjuvant (SEPPIC SA, Catalog No. 80748J, Batch No. 210721010001) and bivalent vaccine (i.e., fusion protein D and fusion protein G at a mass ratio of 1:1) were administered in a total volume of 100 μL per animal. The group dosing schedule is shown in Table 3. Serum was collected on days 14 and 35.

[0341] Table 3 Grouped dosing regimen

[0342] 1.2 Detection of serum anti-Spike protein IgG titer by ELISA

[0343] The detection method is as described in step 1.2 of Example 3, and the results are shown in Figures 6a to 6f. Regardless of whether it is a single immunization or a secondary immunization, adding different doses of adjuvant to the same dose of antigen (5 μg) can significantly increase the antibody titer.

[0344] After the first immunization, compared with the 5 μg antigen unadjuvanted group, GMTs of IgG antibodies against WT-Spike-His, Delta-Spike-His, and Omicron-Spike-His increased by 29-, 22-, and 31-fold, respectively (Figures 6a, 6c, and 6e). After the second immunization, GMTs of IgG antibodies against WT-Spike-His, Delta-Spike-His, and Omicron-Spike-His increased by 60-, 37-, and 66-fold, respectively (Figures 6b, 6d, and 6f). Although anti-Spike protein antibody titers showed some adjuvant dose dependence after the first immunization, after the second immunization, no significant dose-dependence was observed when the adjuvant dose was 10, 15, 25, or 50 μL (Figures 6b, 6d, and 6f). Antibody titers increased significantly after the second immunization, increasing by 20- to 100-fold compared with the first immunization.

[0345] After the first immunization, the GMT of IgG antibodies against all Spike proteins (original strain, Delta type, and Omicron type) showed a dose-dependent relationship with the adjuvant / antigen ratio, with higher adjuvant dosages associated with higher GMTs. However, after the second immunization, when the adjuvant doses were 10, 15, 25, and 50 μL, no significant dose-dependent relationship was observed between the GMT of IgG antibodies against Spike proteins and the adjuvant / antigen ratio. This suggests that under the current experimental conditions, an adjuvant dose of 10-50 μL combined with a 5 μg dose of the bivalent vaccine can adequately elicit a humoral immune response in mice.

[0346] 1.3 hACE2 blocking experiment

[0347] To evaluate the effect of adjuvants on the immunogenicity of the bivalent vaccine, competitive ELISA was used to detect the inhibitory ability of serum on the binding of hACE2 to the Spike protein of three different variants.

[0348] Three recombinant protein antigens, WT-Spike-His (same as in step 1.1 of Example 2), Delta-Spike-His (same as in step 1.1 of Example 2), and Omicron-Spike-His (same as in step 1.1 of Example 2), were diluted in 1× PBS and added to a 96-well microtiter plate at 2 μg / mL, 100 μL / well, and incubated overnight at 2-8°C. The next day, the plates were washed twice with PBST (PBS buffer containing 0.05% Tween-20) and blocked with blocking solution (PBST containing 3% BSA) at 37°C for 2 hours. The cells were then washed three times with PBST solution, and serially diluted immune mouse serum collected on day 35 of step 1.1 of Example 4 was added (mouse serum was diluted 100-fold as the starting concentration, followed by serial dilutions of 3-fold). The cells were incubated at 37°C for 1 hour. 25 ng / mL biotinylated hACE2 (Acro, Catalog No. AC2-H82E7) was then added at 100 μL / well, and the cells were incubated at 37°C for 1 hour. After washing five times with PBST, a 1:10,000 dilution of Peroxidase se-conjugated Streptavidin enzyme-labeled secondary antibody (Jackson Immuno Research, Catalog No. 016-030-084) was added and incubated at 37°C for 0.5 hour. After washing eight times with PBST, 100 μL of TMB colorimetric solution was added for development at room temperature for 10 minutes, and the reaction was terminated by adding 50 μL of stop solution. The detection wavelength was set to 450 nm for reading, and the obtained OD value was fitted with a nonlinear four-parameter equation curve using SoftMax pro 7.02 software to calculate the antibody inhibition titer IC. 50 Value, for IC 50 For samples with values ​​below the detection limit, the IC 50 The value is counted as 100.

[0349] The results are shown in Figure 7a-c. Compared with the group without adjuvant, the addition of 5 μL adjuvant can increase the IC 50 GMTs increased significantly, from 111 to 803, 101 to 688, and 199 to 934, corresponding to the inhibition of hACE2 binding to WT-Spike-His, Delta-Spike-His, and Omicron-Spike-His, respectively. Furthermore, when the adjuvant dose was greater than or equal to 10 μL, there was no significant difference in GMTs between the dose groups, indicating that the adjuvant dose-effect relationship was not significant within this range and at 5 μg of antigen. However, all GMTs were significantly higher than those in the 5 μL adjuvant dose group.

[0350] Example 5: Protective effect of vaccine in K18-hACE2 transgenic mouse model

[0351] Mice were challenged with SARS-CoV-2 to assess the protective effect of vaccines against viral infection and to evaluate whether there were vaccine-related exacerbations. In this study, the K18-hACE2 transgenic mouse (C57BL / 6J) model was used. This model has been validated and widely used to study SARS-CoV-2 infection. It is highly susceptible to SARS-CoV-2 infection. K18-hACE2 mice infected with SARS-CoV-2 developed dose-dependent lung disease with features similar to human COVID-19, including diffuse alveolar damage, inflammatory cell infiltration, tissue damage, pulmonary vascular damage, significant weight loss, and death.

[0352] 1.1 Immunization of mice and intranasal challenge

[0353] Different doses of bivalent vaccine (i.e., fusion protein D and fusion protein G at a mass ratio of 1:1) and fixed dose of SEPIVAC SWE TM The adjuvant was injected intramuscularly twice into the mice (on days 0 and 21, respectively), with a total volume of 100 μL per mouse each time. The group dosing schedule is shown in Table 4. Blood was collected on day 35, and mice were infected with the novel coronavirus Omicron BA.1.1 strain via intranasal drops on day 42 at a viral intranasal dose of 5000 TCID50 per mouse.

[0354] Table 4 Grouped dosing regimen

[0355] 1.2 Detection of serum anti-Spike protein IgG titer by ELISA

[0356] To evaluate the immunogenicity of the bivalent vaccine (i.e., fusion protein D and fusion protein G in a mass ratio of 1:1) in K18-hACE2 transgenic mice, the IgG titers against the Spike proteins of the original strain, Delta, and Omicron variants in the mouse serum after the second immunization were tested by ELISA. The test method is shown in step 1.2 of Example 3. The mouse serum was collected on the 35th day of step 1.1 of Example 5. The serum was diluted 1000 times as the starting concentration, and then diluted 3-fold in a gradient, for a total of 11 gradients.

[0357] The results are shown in Figure 8. All mice vaccinated with the bivalent vaccine elicited robust anti-Spike protein immune responses. Anti-Spike protein IgG titers against the Delta and original strains were higher than those against the Omicron strain, and antibody titers were comparable between the groups receiving 0.25 μg and 2.5 μg of antigen, or between the groups receiving 2.5 μg and 10 μg of antigen. For all three Spike proteins, no anti-Spike protein IgG titers were detected in the blank control group.

[0358] 1.3 Mouse weight measurement

[0359] From the day of infection (recorded as Day 0) until the 10th day after infection (Day 10), the mice were weighed and their weights were recorded daily. The mice in the low, medium, and high dose groups did not show weight loss, and their weight changes were similar to those of the blank control mice that were not inoculated with the virus. In contrast, the mice in the model control group lost a significant amount of weight on the 5th day after virus infection, and all mice lost weight on the 6th day after infection, reaching the euthanasia standard (when the mouse weight loss exceeded 25%).

[0360] 1.4 Detection of active virus titer in lungs of mice after infection

[0361] The method for detecting live virus in the lungs is the Focus Forming Assay (FFA), which is briefly described as follows: 2 days after infection (Day 2), some mice in each group were euthanized and their lungs were collected and ground; the mouse lung tissue homogenate was centrifuged and the supernatant was taken, first diluted 1:3 and then diluted 1:10; the lung homogenate stock solution and the dilution were added to the pre-prepared Vero-E6 cell plate, 50 μL / well, and incubated at 37°C for 1 hour; the culture supernatant was discarded and 100 μL was added 1.6% sodium carboxymethylcellulose culture medium (Sigma, catalog number: C4888-500G), cultured at 37°C, 5% CO2 for 24 hours; the culture supernatant was discarded and 4% paraformaldehyde (biosharp, catalog number: BL539A) was added for fixation; after fixation, the cells were treated with 0.1% Triton-X100 (Sigma, catalog number: T8787-100mL) to perforate the membrane, and then blocked with blocking solution (PBST containing 3% BSA) for 2 hours; rabbit anti-new coronavirus nucleoprotein polyclonal antibody (Sino Biological, catalog number: 40143-T62) was used as the primary antibody, and HRP goat-anti rabbit IgG (abcam, catalog number: ab6721) was used as the secondary antibody, incubated in sequence, and then developed with True-blue colorimetric solution (KPL, catalog number: 50-78-02); images were acquired using an Immuno ELISPOT instrument, and image analysis was performed to obtain the number of spots.

[0362] The results are shown in Figure 9. The average titer of live virus in the lung tissue of mice in the model control group was 4.49×10 4 The lung virus titers of all mice receiving the bivalent vaccine were below the detection limit, indicating that the virus replication in the mouse lungs was completely inhibited.

[0363] 1.5 Neutralization titer of mouse serum against coronavirus Omicron BA.1.1 true virus

[0364] The neutralization titer of the immune serum against the 2019-nCoV Omicron BA.1.1 real virus was tested using the focus reduction neutralization test (FRNT). The method is briefly described as follows: the serum stock solution collected on the 35th day of Example 5, step 1.1, was diluted 1:8 with DMEM medium, and then diluted 2-fold with DMEM medium for a total of 6 dilutions; the diluted serum was mixed with an equal volume of 300-400 PFU of 2019-nCoV Omicron BA.1.1 real virus. The BA.1.1 solution was mixed (the final serum dilutions were 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512, respectively) and incubated at 37°C for 1 hour. The incubation mixture was then transferred to a pre-prepared Vero-E6 cell plate, 100 μL per well, and incubated for another 1 hour at 37°C, 5% CO2. The culture supernatant was discarded and 100 μL of 1.6% CMC culture medium (Sigma, Cat. No. C4888-500G) was added. , 37 ° C, 5% CO2 for 24 h; 4% paraformaldehyde (biosharp, catalog number: BL539A) was added to inactivate and fix the cells; after fixation, the cells were treated with 0.1% Triton-X100 (Sigma, catalog number: T8787-100 mL) to perforate the membrane, and then blocked with blocking solution (PBST containing 3% BSA) for 2 h; rabbit anti-new coronavirus nucleoprotein polyclonal antibody (Sino Biological, catalog number: 40143-T62) was used as the primary antibody, HRP Goat-anti rabbit IgG (abcam, Catalog No. ab6721) was used as a secondary antibody for sequential incubation, followed by development with True-blue colorimetric solution (KPL, Catalog No. 50-78-02). Images were acquired using an ImmunoELISPOT instrument, and image analysis was performed to determine the number of spots. The inhibition rate was calculated using the formula: 100 × (1 - number of spots in sample wells / number of spots in positive control wells). Positive control wells were serum-free (approximately 300-400 spots per well), and negative control wells were virus-free (no spots).

[0365] The results are shown in Figure 10. When the serum dilution was 1:512, the geometric mean inhibition rates of the low, medium and high dose groups were 81.5%, 88.4% and 93.8%, respectively, which were far higher than the inhibition rate of 50%. Therefore, the IC50GMT were all greater than 512.

Claims

1. A coronavirus multivalent vaccine, characterized in that: The coronavirus multivalent vaccine comprises a coronavirus Spike protein extracellular domain containing a mutation or a truncated fragment thereof or a fusion protein comprising the same; the mutation comprises: 1) RRAR is mutated to GSAS; 2) there is a mutation in the turning region between HR1 and CH that prevents HR1 and CH from forming a straight helix during the fusion process.

2. The coronavirus multivalent vaccine according to claim 1, wherein The mutations include: 1) RRAR is mutated to GSAS; 2) there is a double mutation K986P / V987P in the turning region between HR1 and CH.

3. The coronavirus multivalent vaccine according to claim 1 or 2, characterized in that The truncated fragment of the coronavirus Spike protein extracellular domain containing the mutation has 5-80 amino acid residues truncated at the C-terminus compared with the full-length extracellular domain of the coronavirus Spike protein; or, 20-76 amino acid residues truncated at the C-terminus; or, 70 amino acid residues truncated at the C-terminus.

4. The coronavirus multivalent vaccine according to any one of claims 1 to 3, characterized in that The coronavirus Spike protein extracellular domain or a truncated fragment thereof is from SARS-CoV-2, SARS-CoV or MERS-CoV; or, the coronavirus Spike protein extracellular domain or a truncated fragment thereof is from the SARS-CoV-2 original strain or a variant thereof; or, the coronavirus Spike protein extracellular domain or a truncated fragment thereof is from the SARS-CoV-2 original strain, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant; or, the mutation-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs: 3, 4, 6, 7, 9-12, 32-35, 78-83, or is identical to SEQ ID NOs: An amino acid sequence that is at least 80% or at least 90% identical to the amino acid sequence shown in any one of SEQ ID NOs: 3, 4, 6, 7, 9-12, 32-35, 78-83, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 3, 4, 6, 7, 9-12, 32-35, 78-83.

5. The coronavirus multivalent vaccine according to any one of claims 1 to 4, characterized in that The fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker; or, the linker is a GS linker; or, the linker is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS)2, (GGGGS)3, or any combination thereof; or, the linker is (G m S) n , wherein each m is independently 1, 2, 3, 4 or 5, and n is 1, 2, 3, 4 or 5; or, the monomeric subunit protein is a self-assembled monomeric subunit protein; or, the monomeric subunit protein is a monomeric ferritin subunit; or, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, algal ferritin, insect ferritin, fungal ferritin and mammalian ferritin; or, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit; or, the monomeric ferritin subunit comprises the amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having one or more conservative amino acid substitutions with the amino acid sequence as shown in SEQ ID NO:

14.

6. The coronavirus multivalent vaccine according to claim 5, characterized in that The fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof is obtained by connecting the C-terminus of the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof to the N-terminus of the monomeric subunit protein through a linker.

7. The coronavirus multivalent vaccine according to any one of claims 1 to 6, characterized in that The fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67.

8. The coronavirus multivalent vaccine according to any one of claims 1 to 7, characterized in that The coronavirus multivalent vaccine comprises at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67.

9. The coronavirus multivalent vaccine according to claims 1-8, characterized in that The coronavirus multivalent vaccine comprises at least two fusion proteins comprising an amino acid sequence as shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67.

10. The multivalent coronavirus vaccine according to any one of claims 1 to 9, characterized in that The coronavirus multivalent vaccine comprises a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 22, 23 or 29 and a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 43, 44 or 67.

11. The coronavirus multivalent vaccine according to claim 10, wherein The mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 22, 23 or 29 and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 43, 44 or 67 is (1-5): (1-5); or the mass ratio is 1:

1.

12. The multivalent coronavirus vaccine according to any one of claims 1 to 11, characterized in that The coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 22 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO:

43.

13. The multivalent coronavirus vaccine according to any one of claims 1 to 11, characterized in that The coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 29 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO:

67.

14. The coronavirus multivalent vaccine according to any one of claims 1 to 4, characterized in that The fusion protein comprising the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of a coronavirus Spike protein containing a mutation or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto; or, the Fc fragment of the immunoglobulin is from IgG, IgM, IgA, IgE or IgD; or, the Fc fragment of the immunoglobulin is from IgG1, IgG2, IgG3 or IgG4; or, the Fc fragment of the immunoglobulin is an Fc fragment of IgG1; or, the Fc fragment of the immunoglobulin is an Fc fragment of human IgG1; or, the Fc fragment of the immunoglobulin comprises the amino acid sequence as shown in SEQ ID NO: 38, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:

38.

15. The coronavirus multivalent vaccine according to claim 14, wherein The fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof is obtained by connecting the C-terminus of the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof to the N-terminus of the Fc fragment of the immunoglobulin.

16. The multivalent coronavirus vaccine according to any one of claims 1 to 4, 14 to 15, characterized in that The fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76.

17. The multivalent coronavirus vaccine according to any one of claims 1 to 4 and 14 to 16, characterized in that The coronavirus multivalent vaccine comprises at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76.

18. The multivalent coronavirus vaccine according to any one of claims 1 to 4 and 14 to 17, characterized in that The coronavirus multivalent vaccine comprises at least two fusion proteins comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76.

19. The multivalent coronavirus vaccine according to any one of claims 1 to 4 and 14 to 18, characterized in that The coronavirus multivalent vaccine comprises a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 53, 54 or 72 and a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 61, 62 or 76.

20. The coronavirus multivalent vaccine according to claim 19, wherein The mass ratio of the fusion protein comprising the amino acid sequence shown in SEQ ID NO: 53, 54 or 72 and the fusion protein comprising the amino acid sequence shown in SEQ ID NO: 61, 62 or 76 is (1-5):(1-5); or the mass ratio is 1:

1.

21. The multivalent coronavirus vaccine according to any one of claims 1 to 4 and 14 to 20, characterized in that The coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 53 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO:

61.

22. The multivalent coronavirus vaccine according to any one of claims 1 to 4 and 14 to 20, characterized in that The coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO:72 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO:

76.

23. The multivalent coronavirus vaccine according to any one of claims 1 to 4, characterized in that The coronavirus multivalent vaccine further comprises a conserved fragment of the coronavirus Spike protein or a fusion protein comprising the same; alternatively, the conserved fragment of the coronavirus Spike protein is from SARS-CoV-2, SARS-CoV or MERS-CoV; alternatively, the conserved fragment of the coronavirus Spike protein is from the original strain of SARS-CoV-2 or its variant; alternatively, the conserved fragment of the coronavirus Spike protein is from the original strain of SARS-CoV-2, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant.

24. The coronavirus multivalent vaccine according to claim 23, wherein The fusion protein comprising the conserved fragment of coronavirus Spike protein comprises the conserved fragment of coronavirus Spike protein and monomeric subunit protein connected by a linker; or, the linker is a GS linker; or, the linker is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS)2, (GGGGS)3, or any combination thereof; or, the linker is (G m S) n , wherein each m is independently 1, 2, 3, 4 or 5, and n is 1, 2, 3, 4 or 5; or, the monomeric subunit protein is a self-assembled monomeric subunit protein; or, the monomeric subunit protein is a monomeric ferritin subunit; or, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, algal ferritin, insect ferritin, fungal ferritin and mammalian ferritin; or, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit; or, the monomeric ferritin subunit comprises the amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having one or more conservative amino acid substitutions with the amino acid sequence as shown in SEQ ID NO:

14.

25. The coronavirus multivalent vaccine according to claim 23 or 24, wherein The fusion protein comprising the conserved fragment of the coronavirus Spike protein is obtained by connecting the C-terminus of the conserved fragment of the coronavirus Spike protein to the N-terminus of the monomeric subunit protein via a linker.

26. The multivalent coronavirus vaccine according to any one of claims 23 to 25, wherein The fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 45-46, 68, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 45-46, 68, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 45-46, 68.

27. The coronavirus multivalent vaccine according to claim 23, wherein The fusion protein comprising the conserved fragment of the coronavirus Spike protein comprises the conserved fragment of the coronavirus Spike protein and the Fc fragment of an immunoglobulin linked thereto; or, the Fc fragment of the immunoglobulin is from IgG, IgM, IgA, IgE or IgD; or, the Fc fragment of the immunoglobulin is from IgG1, IgG2, IgG3 or IgG4; or, the Fc fragment of the immunoglobulin is the Fc fragment of IgG1; or, the Fc fragment of the immunoglobulin is the Fc fragment of human IgG1; or, the Fc fragment of the immunoglobulin comprises the amino acid sequence as shown in SEQ ID NO: 38, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence having one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO:

38.

28. The coronavirus multivalent vaccine according to claim 27, wherein The fusion protein comprising the conserved fragment of coronavirus Spike protein is obtained by connecting the C-terminus of the conserved fragment of coronavirus Spike protein to the N-terminus of the Fc fragment of immunoglobulin.

29. The multivalent coronavirus vaccine according to claim 23, 27 or 28, wherein The fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 63, 64, and 77, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in any one of SEQ ID NOs: 63, 64, and 77, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 63, 64, and 77.

30. The multivalent coronavirus vaccine according to any one of claims 23 to 29, wherein The coronavirus multivalent vaccine comprises at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67.

31. The coronavirus multivalent vaccine according to claim 30, wherein The coronavirus multivalent vaccine comprises: (1) at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 16-23, 26-29, 41-44, 66, and 67, and (2) a fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 45-46, 63, 64, 68, and 77.

32. The coronavirus multivalent vaccine according to claim 31, wherein The coronavirus multivalent vaccine comprises: (1) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 22, 23 or 29, (2) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 43, 44 or 67, and (3) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 63, 64 or 77.

33. The coronavirus multivalent vaccine according to claim 32, wherein The mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 22, 23 or 29, the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 43, 44 or 67, and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 63, 64 or 77 is (1-5): (1-5): (1-5); or the mass ratio is 1:1:

1.

34. The coronavirus multivalent vaccine according to claim 32 or 33, wherein The coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 22, (2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 43, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:

63.

35. The coronavirus multivalent vaccine according to claim 32 or 33, wherein The coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 29, (2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 67, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:

77.

36. The multivalent coronavirus vaccine according to any one of claims 23 to 29, wherein The coronavirus multivalent vaccine comprises at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76.

37. The coronavirus multivalent vaccine according to claim 36, wherein The coronavirus multivalent vaccine comprises: (1) at least one fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76, and (2) a fusion protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 45-46, 63, 64, 68 and 77.

38. The coronavirus multivalent vaccine according to claim 37, wherein The coronavirus multivalent vaccine comprises: (1) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 53, 54 or 72, (2) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 61, 62 or 76, and (3) a fusion protein comprising an amino acid sequence as shown in SEQ ID NO: 63, 64 or 77.

39. The coronavirus multivalent vaccine according to claim 38, wherein The mass ratio of the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 53, 54 or 72, the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 61, 62 or 76, and the fusion protein comprising the amino acid sequence as shown in SEQ ID NO: 63, 64 or 77 is (1-5):(1-5):(1-5); or the mass ratio is 1:1:

1.

40. The coronavirus multivalent vaccine according to claim 38 or 39, wherein The coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 53, (2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 61, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:

63.

41. The coronavirus multivalent vaccine according to claim 38 or 39, wherein The coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:72, (2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:76, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:

77.

42. The multivalent coronavirus vaccine according to any one of claims 1 to 41, wherein The coronavirus multivalent vaccine also includes a pharmaceutically acceptable carrier and / or adjuvant.

43. Use of the coronavirus multivalent vaccine according to any one of claims 1 to 42 in the preparation of a medicament for preventing or treating coronavirus infection or in the prevention or treatment of coronavirus infection; or, the coronavirus infection is SARS-CoV-2, SARS-CoV or MERS-CoV infection; or, the coronavirus infection is infection with the original strain of SARS-CoV-2 or a variant thereof; or, the coronavirus infection is infection with the original strain of SARS-CoV-2, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant.

44. A method for preventing or treating coronavirus infection, characterized in that The invention comprises administering an effective amount of the coronavirus multivalent vaccine according to any one of claims 1 to 42 to a patient in need thereof; or, the coronavirus infection is infection with the original strain of SARS-CoV-2 or a variant thereof; or, the coronavirus infection is infection with the original strain of SARS-CoV-2, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant.

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