Fusion protein for preventing or treating coronavirus infection, Spike protein nanoparticles and application of Spike protein nanoparticles

Abstract

Fusion proteins, Spike protein nanoparticles and formulations thereof are provided for the prevention or treatment of coronavirus infections, the fusion proteins comprising a mutation-containing coronavirus Spike protein extracellular domain or truncated fragment thereof and a monomeric subunit protein linked by a linker.

Description

Fusion protein, Spike protein nanoparticles and applications thereof for preventing or treating coronavirus infection Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to fusion proteins and Spike protein nanoparticles for preventing or treating coronavirus infection and applications thereof. Background Art

[0002] Coronaviruses are non-segmented, single-stranded, positive-sense RNA viruses. Based on serotype and genomic characteristics, they are divided into four genera within the subfamily Coronavirinae: α, β, γ, and δ. They are named for the protruding projections on their viral envelope, resembling a corolla. The novel coronavirus (SARS-CoV-2 or 2019-nCoV), discovered in 2019, 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] COVID-19, the novel coronavirus pneumonia, 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. Based on current epidemiological studies, the incubation period for the novel coronavirus is generally 1-14 days, with most cases occurring within 3-7 days. 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. 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 and, gradually, dyspnea. In severe cases, acute respiratory distress syndrome (ARDS), shock, and multiple tissue damage and dysfunction in the lungs, heart, and kidneys may develop. While most patients with mild infection have a good prognosis, those with severe disease often present in critical condition or even death.

[0004] With the recent surge in basic, clinical, and epidemiological research on COVID-19, there is an urgent need for effective vaccines against coronaviruses. Furthermore, many protein vaccines are unstable to heat or long-term storage at 4°C or freeze-thaw cycles, resulting in loss of efficacy during storage. Improving thermal stability can address cold chain challenges, such as those faced in remote and impoverished areas, and extend the shelf life of vaccines. Therefore, there is a need to develop stable vaccine compositions with enhanced thermal stability and / or shelf life.

[0005] Summary of the Invention

[0006] The present invention provides a coronavirus spike protein extracellular domain or a truncated fragment thereof containing a mutation that stabilizes the protein structure, and a fusion protein containing the coronavirus spike protein extracellular domain or a truncated fragment thereof. The present invention also provides a coronavirus vaccine comprising nanoparticles formed by fusion of the coronavirus spike protein extracellular domain or a truncated fragment containing the mutation with a monomeric ferritin subunit, which can induce a stronger neutralizing antibody response against coronaviruses.

[0007] The virus particle first binds to an 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) on its surface. 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 endosome 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 shell to fuse with the cell membrane. SARS-CoV or SARS-CoV-2 enters the cell and uses the cell to synthesize new virus particles for it; the new virus particles are released outside the cell and then infect surrounding normal cells in the same way. The fusion protein, nanoparticles and vaccine of the present invention can induce a stronger neutralizing antibody response to coronavirus.

[0008] In some embodiments, a coronavirus Spike protein extracellular domain or a truncated fragment thereof containing a mutation is provided, wherein the mutation comprises: 1) RRAR is mutated to GSAS; 2) there is a mutation in the turning region between HR1 and the central helical 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 is SARS-CoV-2, SARS-CoV, or MERS-CoV.

[0013] In some embodiments, the coronavirus is the original strain of SARS-CoV-2 or a variant thereof.

[0014] In some embodiments, the coronavirus is the SARS-CoV-2 original strain, the SARS-CoV-2 Alpha variant, the SARS-CoV-2 Beta variant, the SARS-CoV-2 Gamma variant, the SARS-CoV-2 Delta variant, the SARS-CoV-2 Kappa variant, the SARS-CoV-2 Epsilon variant, the SARS-CoV-2 Lambda variant, or the SARS-CoV-2 Omicron variant.

[0015] In some embodiments, the coronavirus is a SARS-CoV-2 Omicron variant BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7, XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1, or XBB.1.16.

[0016] 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, 6-9, 19-24, 26-31, 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: 3, 4, 6-9, 19-24, 26-31, 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: 3, 4, 6-9, 19-24, 26-31.

[0017] In some embodiments, a fusion protein is provided comprising the extracellular domain of the coronavirus Spike protein containing the mutation described herein or a truncated fragment thereof.

[0018] Some embodiments provide a fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation described herein or a truncated fragment thereof and a monomeric subunit protein connected by a linker. 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 fusion protein is a C-terminus of the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof connected to the N-terminus of the monomeric subunit protein through a linker. In some embodiments, the fusion protein is a C-terminus of the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof connected to the N-terminus of the monomeric ferritin subunit through a linker.

[0019] 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 , wherein 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.

[0020] In some embodiments, the fusion protein further comprises an N-terminal signal peptide. 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. In some embodiments, the N-terminal signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 2 or 5, or an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 2 or 5, 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: 2 or 5.

[0021] In some embodiments, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, algal ferritin, insect ferritin, fungal ferritin, or mammalian ferritin. In some embodiments, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit. In some embodiments, the Helicobacter pylori non-heme monomeric ferritin subunit has an N19Q mutation in its amino acid sequence. In some embodiments, the monomeric ferritin subunit comprises the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 10, 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: 10.

[0022] In some embodiments, a fusion protein is provided, comprising a SARS-CoV-2 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 comprises a SARS-CoV-2 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) a RRAR to GSAS mutation; 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0023] In some embodiments, the SARS-CoV-2 is the original strain or a variant thereof.

[0024] In some embodiments, the SARS-CoV-2 is 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.

[0025] In some embodiments, the coronavirus is a SARS-CoV-2 Omicron variant BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7, XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1, or XBB.1.16.

[0026] In some embodiments, the coronavirus is SARS-CoV-2 Omicron variant BA.1, BA.2, BA.3, BA.4, BA.5.

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

[0028] In some embodiments, a fusion protein is provided, comprising a SARS-CoV-2 Omicron variant BA.1 Spike protein extracellular domain or a truncated fragment thereof containing a mutation and a monomeric subunit protein, connected by a linker. In some embodiments, the fusion protein comprises a SARS-CoV-2 Omicron variant BA.1 Spike protein extracellular domain or a truncated fragment thereof containing a mutation and a monomeric ferritin subunit, connected by a linker. In some embodiments, the mutation comprises: 1) a mutation of RRAR to GSAS; 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0029] In some embodiments, a fusion protein is provided, comprising a SARS-CoV-2 Omicron variant BA.2 Spike protein extracellular domain or a truncated fragment thereof containing a mutation and a monomeric subunit protein, connected by a linker. In some embodiments, the fusion protein comprises a SARS-CoV-2 Omicron variant BA.2 Spike protein extracellular domain or a truncated fragment thereof containing a mutation and a monomeric ferritin subunit, connected by a linker. In some embodiments, the mutation comprises: 1) a RRAR to GSAS mutation; 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0030] In some embodiments, a fusion protein is provided, comprising a SARS-CoV-2 Omicron variant BA.3 Spike protein extracellular domain or a truncated fragment thereof containing a mutation and a monomeric subunit protein, connected by a linker. In some embodiments, the fusion protein comprises a SARS-CoV-2 Omicron variant BA.3 Spike protein extracellular domain or a truncated fragment thereof containing a mutation and a monomeric ferritin subunit, connected by a linker. In some embodiments, the mutation comprises: 1) a RRAR to GSAS mutation; 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0031] In some embodiments, a fusion protein is provided, comprising a SARS-CoV-2 Omicron variant BA.4 Spike protein extracellular domain or a truncated fragment thereof containing a mutation and a monomeric subunit protein, connected by a linker. In some embodiments, the fusion protein comprises a SARS-CoV-2 Omicron variant BA.4 Spike protein extracellular domain or a truncated fragment thereof containing a mutation and a monomeric ferritin subunit, connected by a linker. In some embodiments, the mutation comprises: 1) a RRAR to GSAS mutation; 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0032] In some embodiments, a fusion protein is provided, comprising a SARS-CoV-2 Omicron variant BA.5 Spike protein extracellular domain or a truncated fragment thereof containing a mutation and a monomeric subunit protein, connected by a linker. In some embodiments, the fusion protein comprises a SARS-CoV-2 Omicron variant BA.5 Spike protein extracellular domain or a truncated fragment thereof containing a mutation and a monomeric ferritin subunit, connected by a linker. In some embodiments, the mutation comprises: 1) a RRAR to GSAS mutation; 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0033] In some embodiments, the fusion protein comprises a coronavirus Spike protein extracellular domain containing a mutation and a monomeric ferritin subunit connected by a linker, wherein the coronavirus Spike protein extracellular domain containing a mutation comprises the amino acid sequence shown in any one of SEQ ID NOs: 3, 4, 6, 19-21, 26-28, and the monomeric ferritin subunit comprises the amino acid sequence shown in SEQ ID NO: 10; the coronavirus Spike protein extracellular domain containing a mutation is connected to the monomeric ferritin subunit via a linker shown in SEQ ID NO: 11.

[0034] In some embodiments, the fusion protein comprises a truncated fragment of the extracellular domain of a coronavirus Spike protein containing a mutation and a monomeric ferritin subunit connected by a linker, wherein the truncated fragment of the extracellular domain of a coronavirus Spike protein containing a mutation comprises the amino acid sequence shown in any one of SEQ ID NOs: 7-9, 22-24, and 29-31, and the monomeric ferritin subunit comprises the amino acid sequence shown in SEQ ID NO: 10; the truncated fragment of the extracellular domain of a coronavirus Spike protein containing a mutation is connected to the monomeric ferritin subunit via a linker shown in SEQ ID NO: 11.

[0035] In some embodiments, the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 12-17, 32-43, 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: 12-17, 32-43, 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: 12-17, 32-43.

[0036] 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 93% identity, at least about 94% identity, at least about 95% 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.

[0037] 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.

[0038] In some embodiments, a polynucleotide encoding the extracellular domain of the coronavirus Spike protein containing the mutation described herein or a truncated fragment thereof, or a fusion protein is provided.

[0039] In some embodiments, an expression vector comprising a polynucleotide encoding the extracellular domain of the coronavirus Spike protein containing a mutation described herein, or a truncated fragment or fusion protein thereof is provided.

[0040] In some embodiments, a cell is provided that can express a coronavirus spike protein extracellular domain containing a mutation described herein or a truncated fragment thereof. In some embodiments, the cell comprises one or more polynucleotides encoding a fusion protein described herein or an expression vector comprising a polynucleotide encoding a fusion protein described herein. In some embodiments, the cell is an isolated cell. In some embodiments, the cell is a CHO cell, HEK293 cell, Cos1 cell, Cos7 cell, CV1 cell, or mouse L cell.

[0041] In some embodiments, the fusion protein comprises a coronavirus spike protein extracellular domain containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker, and the fusion protein includes the following features:

[0042] The mutations include: 1) a mutation that inactivates the S1 / S2 cleavage site; 2) a mutation in the turning region between HR1 and CH that prevents HR1 and CH from forming a straight helix during fusion; and / or

[0043] The C-terminus of the mutant coronavirus Spike protein extracellular domain or its truncated fragment is connected to the monomeric ferritin subunit via a linker; and / or

[0044] 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; and / or

[0045] The monomeric ferritin subunit is a Helicobacter pylori monomeric ferritin subunit, comprising the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 10.

[0046] In some embodiments, provided are Spike protein nanoparticles comprising the fusion proteins described herein.

[0047] In some embodiments, the use of the fusion protein or Spike protein nanoparticles described herein in the preparation of a vaccine for preventing or treating coronavirus infection is provided. In some embodiments, the coronavirus infection is SARS-CoV-2, SARS-CoV, or MERS-CoV infection. In some embodiments, the coronavirus infection is infection with the original strain of SARS-CoV-2 or a variant thereof. In some embodiments, 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. In some embodiments, the coronavirus infection is infection with SARS-CoV-2 Omicron variant BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7, XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1, or XBB.1.16.

[0048] In some embodiments, a coronavirus vaccine is provided, comprising a fusion protein as described herein and / or a Spike protein nanoparticle comprising the fusion protein. In some embodiments, the coronavirus vaccine further comprises a pharmaceutically acceptable carrier and / or adjuvant. In some embodiments, the coronavirus vaccine comprises a fusion protein as described herein and a pharmaceutically acceptable carrier and / or adjuvant. In some embodiments, the coronavirus vaccine comprises a Spike protein nanoparticle as described herein and a pharmaceutically acceptable carrier and / or adjuvant.

[0049] In some embodiments, a coronavirus vaccine formulation is provided, which comprises a fusion protein and one or more of a buffer, a stabilizer, an alkali metal or an alkali metal salt, and a surfactant, wherein the fusion protein is the fusion protein described herein and / or a Spike protein nanoparticle comprising the fusion protein.

[0050] In some embodiments, the fusion protein comprises a coronavirus 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) a RRAR to GSAS mutation; and 2) a double mutation K986P / V987P in the turn region between HR1 and CH. In some embodiments, the coronavirus is the original strain of SARS-CoV-2, the SARS-CoV-2 Alpha variant, the SARS-CoV-2 Beta variant, the SARS-CoV-2 Gamma variant, the SARS-CoV-2 Delta variant, the SARS-CoV-2 Kappa variant, the SARS-CoV-2 Epsilon variant, the SARS-CoV-2 Lambda variant, or the SARS-CoV-2 Omicron variant. In some embodiments, the coronavirus is a SARS-CoV-2 Omicron variant BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7, XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1, or XBB.1.16.

[0051] In some embodiments, the coronavirus vaccine formulation comprises a fusion protein and further comprises one or more of a buffer, a stabilizer, an alkali metal or an alkali metal salt, and a surfactant; the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0052] In some embodiments, the concentration of the fusion protein is 0.05-5 mg / mL. In some embodiments, the concentration of the fusion protein is 0.05-1 mg / mL. In some embodiments, the concentration of the fusion protein is 0.05-0.5 mg / mL. In some embodiments, the concentration of the fusion protein is about 0.05, 0.1, about 0.45, about 0.5, about 1, about 2, about 3, about 4, or about 5 mg / mL.

[0053] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, 10-30 mM buffer, a stabilizer, an alkali metal or an alkali metal salt, and a surfactant.

[0054] In some embodiments, the buffer is selected from histidine buffer, citric acid buffer, phosphate buffer or a combination thereof. In some embodiments, the buffer is a histidine buffer. In some embodiments, the buffer is a combination of histidine buffer and citric acid buffer. In some embodiments, the concentration of the buffer is 1-50mM or 1-40mM or 1-30mM or 5-25mM or 10-30mM or 15-25mM or 5-15mM. In some embodiments, the concentration of the buffer is about 1mM, about 5mM, about 8mM, about 10mM, 13mM, about 15mM, about 18mM, about 20mM, about 22mM, about 25mM, about 27mM, about 30mM, about 35mM, about 40mM, about 50mM, or the range (including endpoints) between any two values ​​in these numerical values ​​or any value therein. In some embodiments, the buffer is a 5-25 mM histidine buffer. In some embodiments, the buffer is a 10-30 mM histidine buffer. In some embodiments, the buffer is a 5-15 mM histidine buffer. In some embodiments, the buffer is a 15-25 mM histidine buffer. In some embodiments, the buffer is about 20 mM histidine buffer. In some embodiments, the buffer is about 10 mM histidine buffer. In some embodiments, the buffer is a combination of 5-15 mM histidine buffer and 2-10 mM citric acid buffer. In some embodiments, the buffer is a combination of about 10 mM histidine buffer and about 5 mM citric acid buffer. In some embodiments, the pH is 5.0-7.0. In some embodiments, the pH is 5.5-6.5. In some embodiments, the pH is 5.7-6.3. In some embodiments, the pH of the antibody formulation is about 5.0, about 5.3, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.8, about 7.0, or a range (including endpoints) between any two of these values ​​or any value therein. In some embodiments, the pH is about 6.1.

[0055] In some embodiments, the stabilizer is selected from polyols, sugars, amino acids or combinations thereof. In some embodiments, the stabilizer is selected from one or more of arginine or its salts, polyethylene glycol, sorbitol, mannitol, glycerol, monosaccharides, oligosaccharides, polysaccharides, cyclodextrins or their derivatives. In some embodiments, the stabilizer is selected from one or more of cyclodextrins or their derivatives, sucrose and trehalose. In some embodiments, the cyclodextrins or their derivatives are selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, their hydroxypropylated derivatives (e.g., hydroxypropyl beta-cyclodextrin), hydroxyethylated derivatives, ethylated derivatives and methylated derivatives, sulfobutyl ether beta-cyclodextrin, branched cyclodextrin, cyclodextrin polymers, or combinations thereof. In some embodiments, the stabilizer is selected from hydroxypropyl beta-cyclodextrin, sucrose and trehalose or combinations thereof. In some embodiments, the stabilizer is a combination of hydroxypropyl beta-cyclodextrin and trehalose. In the solid state, trehalose usually exists as trehalose dihydrate, so in some embodiments, trehalose dihydrate can be used when formulating the preparation, or other forms of trehalose (such as anhydrous trehalose) can be used. In some embodiments, the stabilizer concentration is 0.5-200 mg / mL. In some embodiments, the stabilizer concentration is 0.5-170 mg / mL. In some embodiments, the stabilizer concentration is 10-170 mg / mL. In some embodiments, the stabilizer concentration is 80-170 mg / mL. In some embodiments, the stabilizer concentration is 60-100 mg / mL. In some embodiments, the stabilizer concentration is 0.5-80 mg / mL. In some embodiments, the stabilizer concentration is 0.5-30 mg / mL. In some embodiments, the stabilizer concentration is 0.5-50 mg / mL. In some embodiments, the stabilizer is about 0.5 mg / mL, about 4 mg / mL, about 8 mg / mL, about 10 mg / mL, about 20 mg / mL, about 21 mg / mL, about 25 mg / mL, about 30 mg / mL, about 50 mg / mL, about 84 mg / mL, about 85 mg / mL, about 100 mg / mL, about 150 mg / mL, about 170 mg / mL, or a range between any two of these values ​​(including the endpoints), or any value therein.

[0056] In some embodiments, the stabilizer is 0.5-170 mg / mL trehalose dihydrate or sucrose. In some embodiments, the stabilizer is 10-170 mg / mL trehalose dihydrate or sucrose. In some embodiments, the stabilizer is 80-170 mg / mL trehalose dihydrate or sucrose. In some embodiments, the stabilizer is 60-100 mg / mL trehalose dihydrate or sucrose. In some embodiments, the stabilizer is 0-450 mM trehalose or sucrose. In some embodiments, the stabilizer is 14-450 mM trehalose or sucrose. In some embodiments, the stabilizer is 210-450 mM trehalose or sucrose. In some embodiments, the stabilizer is 150-270 mM trehalose or sucrose. In some embodiments, the stabilizer is about 222 mM trehalose (about 84 mg / mL trehalose dihydrate). In some embodiments, the stabilizer is about 224.7mM trehalose (about 85mg / mL trehalose dihydrate). In some embodiments, the stabilizer is about 55.5mM trehalose (about 21mg / mL trehalose dihydrate). In some embodiments, the stabilizer is 0.5-80mg / mL hydroxypropyl beta-cyclodextrin. In some embodiments, the stabilizer is 0.5-50mg / mL hydroxypropyl beta-cyclodextrin. In some embodiments, the stabilizer is 0.5-20mg / mL hydroxypropyl beta-cyclodextrin. In some embodiments, the stabilizer is about 8mg / mL hydroxypropyl beta-cyclodextrin. In some embodiments, the stabilizer is about 4mg / mL hydroxypropyl beta-cyclodextrin.

[0057] In some embodiments, the alkali metal or alkali metal salt is selected from magnesium chloride, potassium chloride, sodium chloride or its combination. In some embodiments, the alkali metal or alkali metal salt concentration is 0-22 mg / mL. In some embodiments, the alkali metal or alkali metal salt concentration is 8-20 mg / mL. In some embodiments, the alkali metal or alkali metal salt concentration is 8-17 mg / mL. In some embodiments, the alkali metal or alkali metal salt concentration is about 0.1, about 1, about 2, about 4, about 8, about 8.25, about 8.3, about 10, about 13, about 16, about 16.5, about 18, about 20, about 22 mg / mL, or the range (including endpoints) between any two values ​​in these numerical values ​​or any value therein. In some embodiments, the alkali metal or alkali metal salt is 0.1-22 mg / mL sodium chloride. In some embodiments, the alkali metal or alkali metal salt is 8-17 mg / mL sodium chloride.

[0058] In some embodiments, the surfactant is selected from poloxamer, polysorbate or its combination. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is poloxamer 188, polysorbate 20 or polysorbate 80. In some embodiments, the surfactant concentration is 0.1-10mg / mL. In some embodiments, the surfactant is 0.01-5mg / mL. In some embodiments, the surfactant is 0.01-2mg / mL. In some embodiments, the surfactant is about 0.01, about 0.1, about 0.2, about 0.4, about 0.5, about 0.8, about 1, about 1.2, about 1.5, about 1.8, about 2, about 5, about 8, about 10mg / mL, or the range (including endpoints) between any two values ​​in these numerical values ​​or any value therein. In some embodiments, the surfactant is 0.2-1mg / mL. In some embodiments, the surfactant is 0.01-2mg / mL polysorbate 80. In some embodiments, the surfactant is 0.1-2 mg / mL polysorbate 80.

[0059] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL of a fusion protein, 10-30 mM of a buffer, a stabilizer, an alkali metal or alkali metal salt, or one or more of a surfactant, wherein the fusion protein is a fusion protein described herein. In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, or 61.

[0060] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, 5-25 mM phosphate buffer, 14-450 mM trehalose, 0-22 mg / mL alkali metal or alkali metal salt, 0.01-2 mg / mL polysorbate 80, and a pH of about 6.0-8.0, wherein the fusion protein is a fusion protein described herein. In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0061] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, 5-25 mM phosphate buffer, 10-170 mg / mL trehalose dihydrate, 0-22 mg / mL alkali metal or alkali metal salt, 0.01-2 mg / mL polysorbate 80, and a pH of about 6.0-8.0, wherein the fusion protein is a fusion protein described herein. In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0062] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM phosphate buffer, about 26.5 mM trehalose (about 10 mg / mL trehalose dihydrate), about 0.4 mg / mL polysorbate 80, and a pH of about 7.0, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:56.

[0063] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM phosphate buffer, about 450 mM trehalose (about 170 mg / mL trehalose dihydrate), about 0.4 mg / mL polysorbate 80, and a pH of about 7.0, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:56.

[0064] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM phosphate buffer, about 224.7 mM trehalose (about 85 mg / mL trehalose dihydrate), about 0.4 mg / mL polysorbate 80, about 8 mg / mL sodium chloride, and a pH of about 7.0, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:56.

[0065] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, 5-25 mM histidine buffer, about 200-450 mM trehalose, 0.01-2 mg / mL polysorbate 80, and a pH of 5.0-7.0, and the fusion protein is a fusion protein described in the present invention. In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, 5-25 mM histidine buffer, about 80-170 mg / mL trehalose dihydrate, 0.01-2 mg / mL polysorbate 80, and a pH of 5.0-7.0, and the fusion protein is a fusion protein described in the present invention. In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0066] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, about 450 mM trehalose (about 170 mg / mL trehalose dihydrate), about 0.4 mg / mL polysorbate 80, and a pH of 5.6-6.3, wherein the fusion protein is a fusion protein described herein. In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0067] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, about 450 mM trehalose (about 170 mg / mL trehalose dihydrate), about 0.4 mg / mL polysorbate 80, and a pH of 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:56.

[0068] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 15-25 mM histidine buffer, about 150-300 mM trehalose, about 0.01-2 mg / mL polysorbate 80, about 8-17 mg / mL sodium chloride, and a pH of 5.0-7.0, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0069] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 15-25 mM histidine buffer, about 60-100 mg / mL trehalose dihydrate, about 0.01-2 mg / mL polysorbate 80, about 8-17 mg / mL sodium chloride, and a pH of 5.0-7.0, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0070] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, about 224.7 mM trehalose (about 85 mg / mL trehalose dihydrate), about 0.4 mg / mL polysorbate 80, about 8 mg / mL sodium chloride, and a pH of 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:56.

[0071] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, about 222 mM trehalose (about 84 mg / mL trehalose dihydrate), about 0.4 mg / mL polysorbate 80, about 8-17 mg / mL sodium chloride, and a pH of 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0072] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, about 222 mM trehalose (about 84 mg / mL trehalose dihydrate), about 0.4 mg / mL polysorbate 80, about 8-17 mg / mL sodium chloride, and a pH of 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:56.

[0073] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, about 222 mM trehalose (about 84 mg / mL trehalose dihydrate), about 0.4 mg / mL polysorbate 80, about 8 mg / mL sodium chloride, and a pH of 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:56.

[0074] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, about 222 mM trehalose (about 84 mg / mL trehalose dihydrate), about 0.4 mg / mL polysorbate 80, about 16 mg / mL sodium chloride, and a pH of 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 56 or 61.

[0075] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, 15-25 mM histidine buffer, 0.5-50 mg / mL hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL polysorbate 80, 8-33 mg / mL sodium chloride, and a pH of 5.0-7.0, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0076] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, 8-20 mg / mL hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL polysorbate 80, 8-17 mg / mL sodium chloride, and a pH of about 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0077] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, 0.5-50 mg / mL hydroxypropyl beta-cyclodextrin, about 0.4 mg / mL polysorbate 80, 8-33 mg / mL sodium chloride, and a pH of 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0078] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, 0.5-20 mg / mL hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL polysorbate 80, 8-33 mg / mL sodium chloride, and a pH of 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 56 or 61.

[0079] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, about 8 mg / mL hydroxypropyl beta-cyclodextrin, about 0.4 mg / mL polysorbate 80, about 16.5 mg / mL sodium chloride, and a pH of about 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NO: 17, 56 or 61.

[0080] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, 5-15 mM histidine buffer and 2-10 mM citric acid buffer, 0.5-50 mg / mL hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL polysorbate 80, 8-17 mg / mL sodium chloride, and a pH of approximately 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0081] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 10 mM histidine buffer and about 5 mM citric acid buffer, 0.5-50 mg / mL hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL polysorbate 80, 8-17 mg / mL sodium chloride, and a pH of about 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0082] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 10 mM histidine buffer and about 5 mM citric acid buffer, 0.5-20 mg / mL hydroxypropyl beta-cyclodextrin, about 0.4 mg / mL polysorbate 80, 8-17 mg / mL sodium chloride, and a pH of about 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 56 or 61.

[0083] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 10 mM histidine buffer and about 5 mM citric acid buffer, about 55.5 mM trehalose (about 21 mg / mL trehalose dihydrate), about 0.4 mg / mL polysorbate 80, about 2 mg / mL sodium chloride, and a pH of about 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0084] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, 5-25 mM histidine buffer, 60-100 mg / mL sucrose, 0.01-2 mg / mL polysorbate 80, 8-17 mg / mL sodium chloride, and a pH of approximately 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0085] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, about 80 mg / mL sucrose, about 0.4 mg / mL polysorbate 80, 8-17 mg / mL sodium chloride, and a pH of about 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

[0086] In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, about 80 mg / mL sucrose, about 0.4 mg / mL polysorbate 80, about 8 mg / mL sodium chloride, and a pH of about 5.6-6.3, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:17.

[0087] In some embodiments, the coronavirus vaccine formulation further comprises an adjuvant. In some embodiments, the adjuvant is an aluminum adjuvant, a SWE adjuvant, or an MF59 adjuvant. In some embodiments, the adjuvant is added in an amount of 1 / 10 to 5 / 10 of the total volume of the coronavirus vaccine formulation. In some embodiments, the adjuvant is added in an amount of 3 / 10 of the total volume of the coronavirus vaccine formulation.

[0088] In some embodiments, the fusion protein in the coronavirus vaccine formulation comprises at least two fusion proteins described herein. In some embodiments, the fusion protein in the coronavirus vaccine formulation comprises two fusion proteins described herein. In some embodiments, the fusion protein in the coronavirus vaccine formulation comprises three fusion proteins described herein.

[0089] In some embodiments, a multivalent coronavirus vaccine is provided, comprising at least two fusion proteins described herein. In some embodiments, the multivalent coronavirus vaccine comprises two fusion proteins described herein. In some embodiments, the multivalent coronavirus vaccine comprises three fusion proteins described herein.

[0090] In some embodiments, a multivalent coronavirus vaccine is provided, comprising a first fusion protein and a second fusion protein, wherein the first fusion protein is the fusion protein described above, and the second fusion protein comprises a SARS-CoV-2 Delta 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 multivalent coronavirus vaccine further comprises a pharmaceutically acceptable carrier and / or adjuvant.

[0091] In some embodiments, a multivalent coronavirus vaccine formulation is provided, comprising a first fusion protein and a second fusion protein, and further comprising one or more of a buffer, a stabilizer, an alkali metal or an alkali metal salt, and a surfactant, wherein the first fusion protein comprises the extracellular domain of the SARS-CoV-2 Omicron variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker, and the second fusion protein comprises the extracellular domain of the SARS-CoV-2 Delta variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker.

[0092] In some embodiments, the mutation-containing SARS-CoV-2 Delta variant Spike protein extracellular domain or a truncated fragment thereof comprises: 1) a mutation from RRAR to GSAS; 2) a mutation in the turn region between HR1 and CH that prevents the formation of a straight helix during fusion. In some embodiments, the mutation comprises: 1) a mutation from RRAR to GSAS; 2) a double mutation K986P / V987P in the turn region between HR1 and CH.

[0093] In some embodiments, the truncated fragment of the extracellular domain of the SARS-CoV-2 Delta variant Spike protein containing the mutation has 5-80 amino acid residues truncated at the C-terminus compared to the full-length extracellular domain of the SARS-CoV-2 Delta variant Spike protein; or, the C-terminus has 20-76 amino acid residues truncated; or, the C-terminus has 70 amino acid residues truncated.

[0094] In some embodiments, the mutation-containing SARS-CoV-2 Delta variant Spike protein extracellular domain or a truncated fragment thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs: 45-50, 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: 45-50, or an amino acid sequence having one or more conservative amino acid substitutions compared with the amino acid sequence shown in any one of SEQ ID NOs: 45-50.

[0095] In some embodiments, the second fusion protein is a protein obtained by connecting the C-terminus of the extracellular domain of the SARS-CoV-2 Delta variant Spike protein or a truncated fragment thereof containing a mutation to the N-terminus of the monomeric subunit protein via a linker.

[0096] In some embodiments, the linker of the second fusion protein is a GS linker. In some embodiments, the linker of the second fusion protein is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS)2, (GGGGS)3, or any combination thereof. In some embodiments, the linker of the second fusion protein 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.

[0097] In some embodiments, the monomeric subunit protein of the second fusion protein is a self-assembled monomeric subunit protein. In some embodiments, the monomeric subunit protein of the second fusion 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 or mammalian ferritin. In some embodiments, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit. In some embodiments, the monomeric ferritin subunit comprises the amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 10.

[0098] In some embodiments, the second fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 51-56, 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: 51-56, 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: 51-56.

[0099] In some embodiments, the first fusion protein comprises the sequence shown in SEQ ID NO: 14, 15 or 17, and the second fusion protein comprises the sequence shown in SEQ ID NO: 53, 54 or 56.

[0100] In some embodiments, the first fusion protein comprises the sequence shown in SEQ ID NO: 17, and the second fusion protein comprises the sequence shown in SEQ ID NO: 56.

[0101] In some embodiments, the mass ratio of the first fusion protein to the second fusion protein is (1-5):(1-5), or the mass ratio is (1-3):(1-3), or the mass ratio is (1-2):(1-2), or the mass ratio is 1:(1-2), or the mass ratio is (1-2):1, or the mass ratio is 1:1.

[0102] In some embodiments, the coronavirus multivalent vaccine comprises 0.01-2 mg / mL of the first fusion protein and 0.01-2 mg / mL of the second fusion protein.

[0103] In some embodiments, the first fusion protein comprises the sequence shown in SEQ ID NO: 17, the second fusion protein comprises the sequence shown in SEQ ID NO: 56, and the mass ratio of the first fusion protein to the second fusion protein is 1:1.

[0104] In some embodiments, the first fusion protein comprises the sequence shown in SEQ ID NO: 61, the second fusion protein comprises the sequence shown in SEQ ID NO: 56, and the mass ratio of the first fusion protein to the second fusion protein is 1:1.

[0105] In some embodiments, the coronavirus multivalent vaccine formulation comprises a first fusion protein and a second fusion protein, and further comprises one or more of a buffer, a stabilizer, an alkali metal or an alkali metal salt, and a surfactant, wherein the first fusion protein comprises the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises the sequence shown in SEQ ID NO: 56.

[0106] In some embodiments, the coronavirus multivalent vaccine formulation comprises 0.01-2 mg / mL of a first fusion protein and 0.01-2 mg / mL of a second fusion protein, and further comprises 5-15 mM histidine buffer and 2-10 mM citric acid buffer, 0.5-80 mg / mL of hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL of polysorbate 80, 8-17 mg / mL of sodium chloride, and a pH of 5.0-7.0, wherein the first fusion protein comprises the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises the sequence shown in SEQ ID NO: 56.

[0107] In some embodiments, the coronavirus multivalent vaccine formulation comprises 0.01-0.2 mg / mL of a first fusion protein and 0.01-0.2 mg / mL of a second fusion protein, and further comprises approximately 10 mM histidine buffer and approximately 5 mM citric acid buffer, 0.5-20 mg / mL of hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL of polysorbate 80, 8-17 mg / mL of sodium chloride, and a pH of 5.6-6.3, wherein the first fusion protein comprises the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises the sequence shown in SEQ ID NO: 56.

[0108] In some embodiments, the coronavirus multivalent vaccine formulation comprises 0.01-0.2 mg / mL of a first fusion protein and 0.01-0.2 mg / mL of a second fusion protein, and further comprises about 10 mM histidine buffer and about 5 mM citric acid buffer, about 4 mg / mL of hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL of polysorbate 80, and about 8.26 mg / mL of sodium chloride, wherein the first fusion protein comprises the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises the sequence shown in SEQ ID NO: 56.

[0109] In some embodiments, the coronavirus multivalent vaccine formulation comprises 0.01-2 mg / mL of a first fusion protein and 0.01-2 mg / mL of a second fusion protein, and further comprises 5-15 mM histidine buffer, 0.5-80 mg / mL of hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL of polysorbate 80, 8-17 mg / mL of sodium chloride, with a pH of 5.0-7.0, wherein the first fusion protein comprises the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises the sequence shown in SEQ ID NO: 56.

[0110] In some embodiments, the coronavirus multivalent vaccine formulation comprises 0.01-0.2 mg / mL of a first fusion protein and 0.01-0.2 mg / mL of a second fusion protein, and further comprises approximately 10 mM histidine buffer, 0.5-20 mg / mL of hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL of polysorbate 80, 8-17 mg / mL of sodium chloride, with a pH of 5.6-6.3, wherein the first fusion protein comprises the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises the sequence shown in SEQ ID NO: 56.

[0111] In some embodiments, the coronavirus multivalent vaccine formulation comprises 0.01-0.2 mg / mL of a first fusion protein and 0.01-0.2 mg / mL of a second fusion protein, and further comprises approximately 10 mM histidine buffer, approximately 4 mg / mL of hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL of polysorbate 80, and approximately 8.26 mg / mL of sodium chloride, wherein the first fusion protein comprises the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises the sequence shown in SEQ ID NO: 56.

[0112] In some embodiments, the coronavirus multivalent vaccine formulation further comprises an adjuvant. In some embodiments, the adjuvant is an aluminum adjuvant, a SWE adjuvant, or an MF59 adjuvant. In some embodiments, the adjuvant is added in an amount of 1 / 10 to 5 / 10 of the total volume of the formulation. In some embodiments, the adjuvant is added in an amount of approximately 3 / 10 of the total volume of the formulation. In some embodiments, the adjuvant is a SWE adjuvant.

[0113] In some embodiments, the coronavirus multivalent vaccine formulation comprises approximately 0.08 mg / mL of a first fusion protein and approximately 0.08 mg / mL of a second fusion protein, and further comprises approximately 10 mM histidine buffer, approximately 4 mg / mL of hydroxypropyl beta-cyclodextrin, approximately 0.2 mg / mL of polysorbate 80, approximately 8.26 mg / mL of sodium chloride and SWE adjuvant, with a pH of 5.6-6.3, wherein the first fusion protein comprises the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises the sequence shown in SEQ ID NO: 56.

[0114] In some embodiments, the coronavirus multivalent vaccine formulation comprises about 0.04 mg / mL of a first fusion protein and about 0.04 mg / mL of a second fusion protein, and further comprises about 10 mM histidine buffer, about 4 mg / mL of hydroxypropyl beta-cyclodextrin, about 0.2 mg / mL of polysorbate 80, about 8.26 mg / mL of sodium chloride and SWE adjuvant, with a pH of 5.6-6.3, wherein the first fusion protein comprises the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises the sequence shown in SEQ ID NO: 56.

[0115] In some embodiments, the coronavirus multivalent vaccine formulation has a specification of 0.5 mL, containing about 40 μg of the first fusion protein and about 40 μg of the second fusion protein, and also contains about 0.39 mg of histidine, about 0.53 mg of histidine hydrochloride, about 2 mg of hydroxypropyl beta-cyclodextrin, about 0.1 mg of polysorbate 80, about 4.13 mg of sodium chloride and 0.15 mL of SWE adjuvant, with a pH of 5.6-6.3, the first fusion protein comprises the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises the sequence shown in SEQ ID NO: 56.

[0116] In some embodiments, the coronavirus multivalent vaccine formulation has a specification of 0.5 mL, comprising a coronavirus multivalent vaccine comprising approximately 20 μg of a first fusion protein and approximately 20 μg of a second fusion protein, and further comprising approximately 0.39 mg of histidine, approximately 0.53 mg of histidine hydrochloride, approximately 2 mg of hydroxypropyl beta-cyclodextrin, approximately 0.1 mg of polysorbate 80, approximately 4.13 mg of sodium chloride and 0.15 mL of SWE adjuvant, wherein the first fusion protein comprises the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises the sequence shown in SEQ ID NO: 56.

[0117] The present invention also provides methods and uses for prevention or treatment. In some embodiments, the present invention provides methods for preventing or treating coronavirus infection, comprising administering an effective amount of the fusion protein, Spike protein nanoparticles, or coronavirus vaccine described herein to a patient in need. In some embodiments, the use of the fusion protein, Spike protein nanoparticles, or coronavirus vaccine described herein in preventing or treating SARS or COVID-19 is provided. In some embodiments, the use of the fusion protein or Spike protein nanoparticles described herein in the preparation of a vaccine for preventing or treating SARS-CoV-2 infection is provided. In some embodiments, the coronavirus infection is SARS-CoV-2, SARS-CoV, or MERS-CoV infection. In some embodiments, the coronavirus infection is 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, a SARS-CoV-2 Alpha variant, a SARS-CoV-2 Beta variant, a SARS-CoV-2 Gamma variant, a SARS-CoV-2 Delta variant, a SARS-CoV-2 Kappa variant, a SARS-CoV-2 Epsilon variant, a SARS-CoV-2 Lambda variant, or a SARS-CoV-2 Omicron variant. In some embodiments, the coronavirus infection is an infection with the SARS-CoV-2 Omicron variant BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7, XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1, or XBB.1.16. In some embodiments, the coronavirus infection is an infection with the SARS-CoV-2 Omicron variant BA.5. In some embodiments, the coronavirus infection is an infection with the SARS-CoV-2 Omicron variant XBB.1.5. In some embodiments, the coronavirus infection is SARS-CoV-2 Omicron variant XBB.1.16 infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 is the binding curve of fusion protein D and human ACE2.

[0119] Figure 2 is the binding curve of fusion protein 2-1 and human ACE2.

[0120] Figure 3 shows the Spike protein-specific IgG titers in the serum of mice immunized with the bivalent vaccine with or without adjuvant; WT represents WT-Spike protein, Delta represents Delta-Spike protein, BA.1 represents BA.1-Spike protein, and BA.5 represents BA.5-Spike protein.

[0121] Figure 4 shows the anti-Spike protein IgG antibody titer in mouse serum; Figures 4a, 4c, 4e, and 4g show the anti-Spike protein IgG antibody titer in mouse serum after the primary immunization, and Figures 4b, 4d, 4f, and 4h show the anti-Spike protein IgG antibody titer in mouse serum after the second booster immunization; wherein, the bars represent the geometric mean (GMT) of the titer (values ​​are shown within the bars), and the error bars represent the 95% confidence interval (CI).

[0122] Figure 5 shows the pseudovirus inhibition titer of serum after mice were immunized with fusion protein D, fusion protein 2-1 and bivalent vaccine; wherein, WT represents the SARS-CoV-2 original strain pseudovirus, Delta represents the SARS-CoV-2 Delta pseudovirus, BA.5 represents the SARS-CoV-2 BA.5 pseudovirus, BQ.1.1 represents the SARS-CoV-2 BQ.1.1 pseudovirus, XBB represents the SARS-CoV-2 XBB pseudovirus, and XBB.1.5 represents the SARS-CoV-2 XBB.1.5 pseudovirus; the bars represent the geometric mean (GMT) of the titers (the values ​​are shown within the bars); the error bars represent the 95% CI.

[0123] Figure 6 shows the inhibitory titer of mouse serum against the real new coronavirus after the second immunization with the bivalent vaccine; the bars represent the geometric mean (GMT) of the titers (the values ​​are shown within the bars); the error bars represent the 95% CI; and ULOD represents the upper limit of detection of the test.

[0124] FIG7 shows the anti-Spike protein IgG antibody titers in the serum of mice sequentially immunized with the bivalent vaccine; wherein, the bars represent the geometric mean (GMT) of the titers (the values ​​are shown within the bars); and the error bars represent the 95% CI.

[0125] Figure 8 shows the pseudovirus inhibition titer of serum of mice sequentially immunized with the bivalent vaccine; wherein, WT represents the SARS-CoV-2 original strain pseudovirus, Delta represents the SARS-CoV-2 Delta pseudovirus, BF.7 represents the SARS-CoV-2 BF.7 pseudovirus, and XBB.1 represents the SARS-CoV-2 XBB.1 pseudovirus; the bars represent the geometric mean (GMT) of the titers (the values ​​are shown within the bars); the error bars represent the 95% CI.

[0126] Figure 9 is the ELISpot of spleen cells of vaccine-immunized mice; among them, WT represents the Spike protein peptide pool of the original strain of the new coronavirus, Delta represents the Spike protein peptide pool of the new coronavirus variant Delta, and BA.5 represents the Spike protein peptide pool of the new coronavirus variant BA.5; the error bars represent the geometric mean ± 95% CI.

[0127] Figure 10 shows the anti-Spike protein IgG antibody titer in rat serum; WT represents WT-Spike protein, Delta represents Delta-Spike protein, and BA.5 represents BA.5-Spike protein; the bars represent the geometric mean (GMT) of the titer (values ​​are shown within the bars), and the error bars represent the 95% CI.

[0128] the term

[0129] Unless otherwise stated, each of the following terms shall have the meaning set forth below.

[0130] definition

[0131] 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.

[0132] 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.

[0133] 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 (three-letter code: Ala, one-letter code: 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).

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] "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.

[0145] 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."

[0146] "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.

[0147] "cDNA" refers to DNA that is complementary or identical to mRNA and can be in single-stranded or double-stranded form.

[0148] "Epitope" refers to an antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic, such 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.

[0149] 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.

[0150] 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.

[0151] 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 replicas 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 (24mer), 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 recombinantly expressing proteins in appropriate expression systems. The production, detection, and characterization of nanoparticles can use the same technology developed for VLPs.

[0152] 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.

[0153] 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 range of ±10%, ±5% or ±1%.

[0154] "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.

[0155] "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, integration of medical care and nursing, as well as early postoperative rehabilitation, joint care and exercise therapy, and other services.

[0156] "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.

[0157] 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.

[0158] "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.

[0159] Overview

[0160] 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.

[0161] 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.

[0162] 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.

[0163] Based on the research and exemplary designs described herein, the present invention provides fusion proteins, Spike protein nanoparticles, and vaccine compositions. The present invention also provides related polynucleotides, expression vectors, and pharmaceutical compositions. In some embodiments, stable Spike trimers and RBD proteins in the form of proteins or nucleic acids (DNA / mRNA) carried by viral vectors can be used as coronavirus vaccines. In addition, stable Spike trimers and RBDs presented by nanoparticles can also be used as coronavirus vaccines.

[0164] 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 and CHO cells. The produced Spike protein nanoparticles can be purified by antibodies and size exclusion chromatography (SEC).

[0165] Unless otherwise indicated herein, the mutant coronavirus Spike protein extracellular domain or its truncated fragment, fusion protein, Spike protein nanoparticle, 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.

[0166] 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.

[0167] 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.

[0168] Coronavirus Spike protein extracellular domain or its truncated fragment containing mutation

[0169] 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 NO: 1. 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.

[0170] 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.

[0171] Some mutant coronavirus Spike protein extracellular domains or truncated fragments thereof (as shown in SEQ ID NO: 3-4, 6-9, 19-24, 26-31, 45-50, 62) 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. In some embodiments, the Spike protein used for mutation can be SEQ ID NO: 1, 18, 25 or 44 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 a deletion of residues 1139-1208 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), or a range (inclusive) between 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.

[0172] Exemplary coronavirus Spike protein extracellular domains or truncated fragments or variants thereof are as follows:

[0173] The full-length extracellular domain (ECD) of the SARS-CoV-2 Omicron variant BA.5 Spike protein, the amino acid sequence of which is shown in SEQ ID NO: 1, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is italicized, 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 I-1 of the mutated SARS-CoV-2 Omicron variant BA.5 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 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 I-2 of the mutated SARS-CoV-2 Omicron variant BA.5 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 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 I-3 of the mutated SARS-CoV-2 Omicron variant BA.5 Spike protein is shown in SEQ ID NO: 6. The sequence does not contain a signal peptide or S1 / S2 cleavage site. 682 RRAR 685 Mutation682 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 I-4 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.5 Spike protein is shown in SEQ ID NO: 7. 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 is shown in SEQ ID NO: 7. 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 I-5 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.5 Spike protein is shown in SEQ ID NO: 8. 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 I-6 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.5 Spike protein is shown in SEQ ID NO: 9. 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 full-length extracellular domain (ECD) of the SARS-CoV-2 Omicron variant BA.2 Spike protein, whose amino acid sequence is shown in SEQ ID NO: 18, 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.

[0181] The amino acid sequence of the full-length extracellular domain I-6-1 of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein is shown in SEQ ID NO: 19. 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.

[0182] The amino acid sequence of the full-length extracellular domain I-7 of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein is shown in SEQ ID NO: 20. 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.

[0183] The amino acid sequence of the full-length extracellular domain I-8 of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein is shown in SEQ ID NO: 21. 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.

[0184] The amino acid sequence of the C-terminal truncated fragment I-9 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein is shown in SEQ ID NO: 22. 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 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.

[0185] The amino acid sequence of the C-terminal truncated fragment I-10 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein is shown in SEQ ID NO: 23. 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.

[0186] The amino acid sequence of the C-terminal truncated fragment I-11 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein is shown in SEQ ID NO: 24. 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.

[0187] The full-length extracellular domain (ECD) of the SARS-CoV-2 Omicron variant BA.3 Spike protein, the amino acid sequence of which is shown in SEQ ID NO: 25, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is italicized, and the S1 / S2 cleavage site 682 RRAR 685 Use underlining, bolding, and italics.

[0188] The amino acid sequence of the full-length extracellular domain I-12 of the mutated SARS-CoV-2 Omicron variant BA.3 Spike protein is shown in SEQ ID NO: 26. 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.

[0189] The amino acid sequence of the full-length extracellular domain I-13 of the mutated SARS-CoV-2 Omicron variant BA.3 Spike protein is shown in SEQ ID NO: 27. 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.

[0190] The amino acid sequence of the full-length extracellular domain I-14 of the mutated SARS-CoV-2 Omicron variant BA.3 Spike protein is shown in SEQ ID NO: 28. 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.

[0191] The amino acid sequence of the C-terminal truncated fragment I-15 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.3 Spike protein is shown in SEQ ID NO: 29. 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: 3. 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0192] The amino acid sequence of the C-terminal truncated fragment I-16 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.3 Spike protein is shown in SEQ ID NO: 30. 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.

[0193] The amino acid sequence of the C-terminal truncated fragment I-17 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.3 Spike protein is shown in SEQ ID NO: 31. 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.

[0194] The amino acid sequence of the C-terminal truncated fragment g1 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.1 Spike protein is shown in SEQ ID NO: 62. 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: 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.

[0195] The full-length extracellular domain (ECD) of the SARS-CoV-2 Delta variant Spike protein, the amino acid sequence of which is shown in SEQ ID NO:44, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is italicized, and the S1 / S2 cleavage site 682 RRAR 685 Use underlining, bolding, and italics.

[0196] 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: 45. 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.

[0197] 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: 46. 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.

[0198] 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: 47. 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.

[0199] 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: 48. 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 682 RRAR 685 Mutation 682 GSAS 685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized.

[0200] 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: 49. 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.

[0201] 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: 50. 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.

[0202] Fusion protein

[0203] 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 above-mentioned various stable mutations. In an exemplary embodiment, the Spike protein sequence used comprises the sequence shown in any one of SEQ ID NO: 1, 3-4, 6-9, 18-31, 44-50, and 62, 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 an 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).

[0204] 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.

[0205] 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.

[0206] 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: 10), or a conservatively modified variant thereof, or a sequence substantially identical thereto. Typically, the C-terminus of 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 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 linker, such as GGGGS or GGGGSGGGGS.

[0207] The amino acid sequence of the non-heme monomeric ferritin subunit of Helicobacter pylori is as follows:

[0208] Nanoparticles displaying any stable mutant-containing coronavirus Spike protein extracellular domain or its truncated fragment described herein can be constructed by fusing subunits of antigenic polypeptides or multimeric antigenic proteins (e.g., trimeric antigens) to subunits of nanoparticles (e.g., ferritin subunits) 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 described in the art.

[0209] Exemplary fusion protein sequences are as follows:

[0210] Fusion protein 1: The C-terminus of the mutant SARS-CoV-2 Omicron variant BA.5 Spike protein full-length extracellular domain I-1 (as shown in SEQ ID NO: 3) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain fusion protein 1, whose amino acid sequence is shown in SEQ ID NO: 12. 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: 12. 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.

[0211] Fusion protein 2: The C-terminus of the mutant SARS-CoV-2 Omicron variant BA.5Spike protein full-length extracellular domain I-2 (as shown in SEQ ID NO: 4) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain fusion protein 2, whose amino acid sequence is shown in SEQ ID NO: 13. 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: 13. 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.

[0212] Fusion protein 3: The C-terminus of the C-terminal truncated fragment I-4 of the extracellular domain of the mutant SARS-CoV-2 Omicron variant BA.5 Spike protein (as shown in SEQ ID NO: 7) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain fusion protein 3, the amino acid sequence of which is shown in SEQ ID NO: 14. In the sequence, the C-terminus 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 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 4: The C-terminus of the C-terminal truncated fragment I-5 of the extracellular domain of the mutant SARS-CoV-2 Omicron variant BA.5Spike protein (as shown in SEQ ID NO: 8) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain fusion protein 4, whose amino acid sequence is shown in SEQ ID NO: 15. In the sequence, 70 amino acid residues were truncated at the C-terminus, 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 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 5: The C-terminus of the mutant SARS-CoV-2 Omicron variant BA.2 Spike protein full-length extracellular domain I-6-1 (as shown in SEQ ID NO: 19) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain fusion protein 5, whose amino acid sequence is shown in SEQ ID NO: 32. 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: 32. 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 6: The C-terminus of the mutant SARS-CoV-2 Omicron variant BA.2 Spike protein full-length extracellular domain I-7 (as shown in SEQ ID NO: 20) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain fusion protein 6, whose amino acid sequence is shown in SEQ ID NO: 33. 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: 33. 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 7: The C-terminus of the C-terminal truncated fragment I-9 of the extracellular domain of the mutant SARS-CoV-2 Omicron variant BA.2 Spike protein (as shown in SEQ ID NO: 22) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) via the linker GGGGS (as shown in SEQ ID NO: 11) to obtain fusion protein 7, the amino acid sequence of which is shown in SEQ ID NO: 34. In the sequence, the C-terminus 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: 34. 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.

[0217] Fusion protein 8: The C-terminus of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein extracellular domain C-terminal truncated fragment I-10 (as shown in SEQ ID NO: 23) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain fusion protein 8, whose amino acid sequence is shown in SEQ ID NO: 35. In the sequence, 70 amino acid residues were truncated at the C-terminus, 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 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.

[0218] Fusion protein 9: The C-terminus of the mutant SARS-CoV-2 Omicron variant BA.3 Spike protein full-length extracellular domain I-12 (as shown in SEQ ID NO: 26) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain fusion protein 9, whose amino acid sequence is shown in SEQ ID NO: 36. 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: 36. 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.

[0219] Fusion protein 10: The C-terminus of the mutant SARS-CoV-2 Omicron variant BA.3 Spike protein full-length extracellular domain I-13 (as shown in SEQ ID NO: 27) was connected to the N-terminus of the Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain fusion protein 10, whose amino acid sequence is shown in SEQ ID NO: 37. 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: 37. 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.

[0220] Fusion protein 11: The C-terminus of the C-terminal truncated fragment I-15 of the extracellular domain of the mutant SARS-CoV-2 Omicron variant BA.3 Spike protein (as shown in SEQ ID NO: 29) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) via the linker GGGGS (as shown in SEQ ID NO: 11) to obtain fusion protein 11, the amino acid sequence of which is shown in SEQ ID NO: 38. In the sequence, the C-terminus 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: 38. 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.

[0221] Show).

[0222] Fusion protein 12: The C-terminus of the C-terminal truncated fragment I-16 of the extracellular domain of the mutant SARS-CoV-2 Omicron variant BA.3 Spike protein (as shown in SEQ ID NO: 30) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain fusion protein 12, the amino acid sequence of which is shown in SEQ ID NO: 39. In the sequence, the C-terminus 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 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.

[0223] 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:45) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO:10) through the linker GGGGS (as shown in SEQ ID NO:11) to obtain fusion protein C1, 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 marked in italics, and the S1 / S2 cleavage site is shown in SEQ ID NO:51. 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.

[0224] 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: 46) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain the fusion protein C2, 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, and the junction is italicized and bolded.

[0225] Fusion protein D1: The C-terminus of the C-terminal truncated fragment d1 of the extracellular domain of the mutant SARS-CoV-2 Delta variant Spike protein (as shown in SEQ ID NO: 48) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain fusion protein D1, whose amino acid sequence is shown in SEQ ID NO: 53. In the sequence, the C-terminus 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, and the junction is italicized and bolded.

[0226] Fusion protein D2: The C-terminus of the C-terminal truncated fragment d2 of the extracellular domain of the mutant SARS-CoV-2 Delta variant Spike protein (as shown in SEQ ID NO: 49) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain the fusion protein D2, whose amino acid sequence is shown in SEQ ID NO: 54. In the sequence, the C-terminus 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, and the junction is italicized and bolded.

[0227] Mature fusion protein G: The C-terminus of the C-terminal truncated fragment g1 of the extracellular domain of the mutant SARS-CoV-2 Omicron variant BA.1 Spike protein (as shown in SEQ ID NO: 62) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 10) through the linker GGGGS (as shown in SEQ ID NO: 11) to obtain the fusion protein G1. In the sequence of the fusion protein G1, the C-terminus was truncated by 70 amino acid residues, and the original signal peptide was: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2). The amino acid sequence of the fusion protein G is shown in SEQ ID NO: 61. Compared with the fusion protein G1, the N-terminal signal peptide was removed. 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.

[0228] Mature fusion protein 1-1: Compared with fusion proteins 1 and 2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 16. In the sequence, the S1 / S2 cleavage site 682 RRAR 685 Mutation 682 GSAS685 , which is underlined and bold, and contains the double mutation K986P / V987P, which is underlined and italicized, and the junction is italicized and bolded.

[0229] Mature fusion protein 2-1: Compared with fusion proteins 3 and 4, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 17. 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.

[0230] Mature fusion protein 3-1: Compared with fusion proteins 5 and 6, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 40. 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.

[0231] Mature fusion protein 4-1: Compared with fusion proteins 7 and 8, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 41. 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.

[0232] Mature fusion protein 5-1: Compared with fusion proteins 9 and 10, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 42. 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.

[0233] Mature fusion protein 6-1: Compared with fusion proteins 11 and 12, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 43. 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.

[0234] 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: 55. 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.

[0235] 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: 56. 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.

[0236] SEQ ID NO: 16, 17, 40-43, 55, 56, 61 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.

[0237] Polynucleotides and expression vectors

[0238] The mutated coronavirus Spike protein extracellular domain or its truncated fragment, fusion protein or Spike protein nanoparticle of the present invention is generally produced by an expression vector, which contains the coding sequence of the mutated coronavirus Spike protein extracellular domain or its truncated fragment, fusion protein or Spike protein nanoparticle described herein. Therefore, in some related aspects, the present invention provides polynucleotides (DNA or RNA) encoding the mutated coronavirus Spike protein extracellular domain or its truncated fragment, fusion protein or Spike protein nanoparticle described herein. Some polynucleotides of the present invention encode one of the mutated 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:7. Some polynucleotides of the present invention encode a subunit sequence of one of the nanoparticle vaccines described herein, such as the fusion protein sequence shown in SEQ ID NO:12. The fusion protein expressed by the present invention may not include an N-terminal signal peptide, or some polynucleotide sequences additionally encode an N-terminal signal peptide. For example, the polynucleotide encoding the fusion protein (eg, SEQ ID NO: 16 or 17) may further comprise a sequence encoding the N-terminal signal peptide shown in SEQ ID NO: 2 or 5, or a sequence substantially identical thereto or a conservatively modified variant thereof.

[0239] 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 containing a mutation or a truncated fragment thereof or a fusion protein. Fusion proteins encoded by the polynucleotides or expressed by the vectors are also included in the present invention. As described herein, the Spike protein ectodomain or a truncated fragment thereof fused to the nanoparticle subunits will self-assemble into a nanoparticle vaccine that displays the Spike protein or a truncated fragment thereof on its surface.

[0240] 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. Mutations can also be introduced into polynucleotide sequences by known methods, such as PCR.

[0241] 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).

[0242] 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.

[0243] The vector expressing the protein can be introduced into the selected host cell by any of many suitable methods known to those skilled in the art. In order to introduce the vector encoding the protein into mammalian cells, the method used will depend on the form of the vector. For plasmid vectors, the DNA encoding the protein sequence can be introduced by any of many transfection methods, including, for example, liposome-mediated transfection ("lipofection"), DEAE-dextran-mediated transfection, electroporation or calcium phosphate precipitation. Among them, lipofection is widely accepted because it is simple to operate and does not require special instruments and equipment. For example, Lipofectamine (Life Technologies) or LipoTAXI (Stratagene) kits can be used for transfection.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] In some embodiments, the substitutions described herein are conservative amino acid substitutions.

[0249] Pharmaceutical compositions and methods of treatment

[0250] 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 and a pharmaceutically acceptable carrier.

[0251] 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.

[0252] 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. Antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and agents for regulating tension such as sodium chloride or dextrose are also foreseeable. These pharmaceutical compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The pharmaceutical compositions can be formulated into suppositories using traditional 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, etc. 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.

[0253] It should be noted that, in the present invention, the "%" related to the formulation components refers to the weight volume (w / v) percentage. For example, a formulation containing 1% stabilizer means that 100 mL of the formulation contains 1 g of stabilizer, or the stabilizer content is 0.01 g / mL.

[0254] "About" or "approximately" refers 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" or "approximately" mentioned herein refers to the described numerical value and its ±10%, ±5% or ±1% range.

[0255] "Comprising" or "including" means that compositions and methods, etc. include the listed elements (such as components in a composition, steps in a method, etc.), but do not exclude other elements. When "consisting essentially of..." is used to define compositions and methods, it means excluding other elements that have a fundamental effect on the combination for the intended use, but does not exclude elements that do not substantially affect the characteristics of the composition or method. "Consisting of..." means excluding elements that are not specifically listed. Embodiments defined by each of these transition terms are within the scope of the present invention. For example, when a composition is described as including ingredients A, B, and C, a composition consisting essentially of A, B, and C and a composition consisting of A, B, and C are independently within the scope of the present invention.

[0256] The term "buffer," also referred to as a buffer system, includes, but is not limited to, organic acids and their salts, such as succinic acid, acetic acid, citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, or phthalic acid, and their salts; Tris, or inorganic acids and their salts, such as phosphate buffers. Furthermore, amino acids can also be used as buffers. Such amino acids include, but are not limited to, glycine, histidine, arginine, lysine, ornithine, isoleucine, leucine, alanine, glutamic acid, or aspartic acid, and their salts.

[0257] The amount of a buffer herein refers to the total amount of the buffer pair in the buffer system comprising the buffer. In some embodiments, the amount of a buffer is expressed in molar concentration, where the value refers to the molar concentration of the buffer pair in the buffer system comprising the buffer. For example, in the case of a histidine buffer composed of histidine and histidine hydrochloride, a given concentration of histidine buffer (e.g., 20 mM) refers to the combined concentration of histidine and histidine hydrochloride.

[0258] The preparation of the present invention can be prepared with the excipients or their hydrates. For example, histidine hydrochloride, also known as histidine hydrochloride, can be anhydrous histidine hydrochloride or a histidine hydrochloride hydrate, such as histidine hydrochloride monohydrate.

[0259] The term "surfactant" includes, but is not limited to, polysorbates (Tween, such as polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, myristyl betaine, or cetyl betaine. ; Lauramidopropyl betaine, cocoamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine or isostearamidopropyl betaine (e.g., lauramidopropyl); myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.); etc. In some embodiments, the surfactant is polysorbate 80, also known as PS80, Tween 80 or Tween 80.

[0260] As used herein, "salt" refers to acidic salts formed with inorganic and / or organic acids, and basic salts formed with inorganic and / or organic bases. Although other salts may also be used, pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred. Exemplary basic salts include ammonium salts, alkali metal salts (such as sodium, lithium and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts), zinc salts, salts formed with organic bases (e.g., organic amines) (such as N-Me-D-glucosamine, choline, trimethylamine, dicyclohexylamine, tert-butylamine), and salts formed with amino acids (such as arginine, lysine), etc.

[0261] As used herein, "stability" and "stable" refer to a formulation comprising an antibody in which the antibody (including its antibody fragment) does not, or only minimally, aggregate, degrade, or fragment under given production, preparation, transportation, and / or storage conditions. A "stable" formulation maintains biological activity under given production, preparation, transportation, and / or storage conditions. The stability of the antibody can be assessed by measuring the degree of aggregation, degradation, or fragmentation of the formulation, for example, by SEC-HPLC, IEC-HPLC, CE-SDS (NR), optical inspection, turbidity, insoluble particles, and particle size detection using DLS.

[0262] In some embodiments, the pharmaceutical composition may comprise a fusion protein or Spike protein nanoparticle, 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, the nanoparticle vaccines 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.

[0263] In the practice of some therapeutic methods of the present invention, the corresponding Spike protein nanoparticles or fusion proteins, or the polynucleotides encoding the fusion proteins 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, or polynucleotides encoding the fusion proteins disclosed herein are contained in a pharmaceutical composition. 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.

[0264] 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 adjuvants such as 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 slow-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. In some embodiments, the vaccines of the present invention contain a suitable adjuvant, which is SEPIVAC SWE TM , a squalene-based oil-in-water emulsion.

[0265] The pharmaceutical compositions 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 eliciting an immune response to SARS-CoV-2 in a subject. As an example, nanoparticle vaccines 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 compositions 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 methods of treatment and pharmaceutical compositions described herein can be used in combination with other known therapeutic agents and / or modalities for treating or preventing coronavirus infection. Known therapeutic agents and / or modalities 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.

[0266] For therapeutic applications, the pharmaceutical composition should contain a therapeutically effective amount of the fusion proteins or Spike protein nanoparticles described herein. For prophylactic applications, the pharmaceutical composition should contain a prophylactically effective amount of the fusion proteins or Spike protein nanoparticles described herein. The appropriate amount of antigen can be determined based on the specific disease or condition to be treated or prevented, the severity of the subject, the 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 significantly reduces the occurrence or severity of the subject's target disease condition or symptoms.

[0267] 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.

[0268] 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).

[0269] Vaccine compositions (e.g., SARS-CoV-2 vaccines) or pharmaceutical compositions comprising the fusion proteins or spike protein nanoparticles 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.

[0270] Various known delivery systems can be used to administer the fusion proteins, Spike protein nanoparticles or derivatives of the present invention, or their encoding polynucleotides or expression vectors, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the fusion protein or Spike protein nanoparticles, receptor-mediated endocytosis, construction of nucleic acids as part of retroviruses or other vectors, etc. DETAILED DESCRIPTION

[0271] 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.

[0272] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0273] Example 1: Preparation of fusion protein

[0274] 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: 12-17, 32-43, 51-56, 61) is cloned into an expression vector, and then electroporated into CHO-K1 cells, cultured, and purified to obtain the fusion protein.

[0275] The three-dimensional structure of the fusion protein was analyzed using cryo-electron microscopy (Cryo-EM). Connecting the extracellular domain of the 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.

[0276] Example 2: Binding ability test of fusion protein to hACE2 protein

[0277] 1.1 Fusion protein binding ability test for hACE2 protein (ELISA)

[0278] This experiment uses ELISA to detect the binding ability of the fusion protein 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 the Spike protein. The method is briefly described as follows: 100 μL of 2 μg / mL antigen (WT-Spike protein (as shown in SEQ ID NO: 57), Delta-Spike protein (as shown in SEQ ID NO: 58), BA.5-Spike protein (as shown in SEQ ID NO: 59), fusion protein D, fusion protein 2-1) solution is added to each reaction well of a 96-well ELISA plate (Costar, Catalog No.: 9018), coated overnight at 4°C; washed twice with PBST (PBS buffer containing 0.05% Tween-20); blocking solution (PBST containing 3% BSA) is added to each reaction well and incubated in a 37°C incubator for 2 hours; after blocking, wash three times with PBST; add serial dilutions of human ACE2-his-biotin (Sino Biologics, catalog number: 10108-H27B-B) was added to each well at a starting concentration of 2.5 μg / mL and diluted 3-fold for a total of 10 serial dilutions. 100 μL was added to each well and incubated at 37°C for 1.5 h. The cells were washed 5 times with PBST. Streptavidin-labeled catalase (Jackson Immuno Research, catalog number: 016-030-084; 1:10,000 dilution) was added to the reaction wells at 100 μL / well and incubated at 37°C for 1 h. The cells were washed 8 times with PBST. TMB solution (Huzhou Yingchuang Biotechnology Co., Ltd., TMB-S-001) was added to the reaction wells at 100 μL / well and incubated at 37°C in the dark for 5-15 min. 50 μL of stop solution (0.1 M H2SO4) was added to each reaction well to terminate the enzymatic colorimetric reaction. The analysis software SoftMax pro was used. 7.02 software, set the detection wavelength to 450nm for reading, and fit the obtained readings with a four-parameter equation curve model. The equation is: Where A is the lower limit of absorbance, B represents the slope of the curve, and C is the antibody concentration corresponding to half the maximum response value (EC 50 ), D is the upper limit of absorbance.

[0279] Among them, the method for constructing a Spike protein (such as WT-Spike protein, Delta-Spike protein, or BA.5-Spike protein) is as follows: the DNA sequence encoding the target protein is cloned into an expression vector, then electroporated into CHO-K1 cells. After MSX pressure screening, mother clones with high expression levels are selected for culture and purification to obtain the target protein.

[0280] WT-Spike protein amino acid sequence:

[0281] Delta-Spike protein amino acid sequence:

[0282] BA.5-Spike protein amino acid sequence:

[0283] The results are shown in Figures 1 and 2. hACE2 binds to fusion protein D, WT-Spike protein, and Delta-Spike protein with similar affinity. 50 The values ​​were 9.2, 5.8, and 8.1 ng / mL, respectively (Figure 1); hACE2 also had similar affinity for binding to fusion protein 2-1 and BA.5-Spike protein, EC 50 The values ​​were 24.0 and 81.5 ng / mL, respectively (Figure 2). Both fusion protein 2-1 and fusion protein D can bind well to human ACE2, indicating that the nanoparticle structures presented by fusion protein 2-1 and fusion protein D can well display antigen epitopes.

[0284] 1.2 Binding ability test of fusion protein to hACE2 protein (BLI)

[0285] The binding ability of the fusion protein to hACE2 was assessed using biomolecular interferometry (BLI) on a PALL Biomolecular Interaction Analyzer (Fortebio Octet QKe) to evaluate the fusion protein's ability to effectively display the antigenic epitope. Multichannel parallel quantitative analysis of spike proteins (WT-Spike protein (see Example 2, Step 1.1), Delta-Spike protein (see Example 2, Step 1.1), BA.5-Spike protein (see Example 2, Step 1.1), and fusion proteins (Fusion Protein D, Fusion Protein 2-1) was performed using a concentration gradient of 0, 50, 100, 200, and 400 nM. hACE2-Biotin (Acro Biosystems, Catalog No. AC2-H5257) was immobilized on SA Biosensors (Octet, Catalog No. 2107002811). The results are shown in Table 1.

[0286] Table 1 Kinetic parameters of SARS-CoV-2 Spike protein binding to hACE2 receptor determined by biolayer interferometry

[0287] As shown in Table 1, the affinity of fusion protein D for hACE2 is significantly stronger than that of WT-Spike protein and Delta-Spike protein for hACE2. Fusion protein 2-1 and BA.5-Spike protein both exhibited extremely high binding affinities for hACE2, exceeding the instrument's detection range (KD < 1.0E-12). Similarly, the affinity of fusion protein 2-1 for hACE2 was significantly stronger than that of WT-Spike protein for hACE2. Both fusion protein D and fusion protein 2-1 have extremely strong affinities for hACE2, demonstrating that the nanoparticle structures presented by fusion protein 2-1 and fusion protein D are capable of effectively displaying antigenic epitopes.

[0288] Example 3: Effect of adjuvants on vaccine immunogenicity

[0289] 1.1 Immunization of mice

[0290] Female BALB / c mice (8 weeks old) received intramuscular injections of either a SWE adjuvanted (SEPPIC SA, Catalog No. 80748J, Lot No. 210721010001) or unadjuvanted bivalent vaccine (fusion protein D and fusion protein 2-1 at a 1:1 mass ratio). The experimental design is shown in Table 2. Blood was collected on day 14. Serum IgG antibody titers against Spike proteins (original strain, Delta, and Omicron variants BA.1 and BA.5) were measured by ELISA.

[0291] Table 2 Experimental design

[0292] 1.2 ELISA for detection of serum anti-Spike protein IgG antibody titer

[0293] WT-Spike protein (see step 1.1 of Example 2), Delta-Spike protein (see step 1.1 of Example 2), BA.1-Spike protein (see SEQ ID NO: 60, the construction method is shown in Example 2 step 1.1), BA.5-Spike protein (see Example 2 step 1.1) to a final concentration of 2 μg / mL, added to a 96-well ELISA plate (Costar, 9018) at 100 μL / well, and incubated at 4 ° C overnight; washed twice with PBST (PBS buffer containing 0.05% Tween-20), added blocking solution (PBST containing 3% BSA), and incubated in a 37 ° C incubator for 2 hours; washed twice with PBST, and added the mouse serum obtained in Example 3 step 1.1 with gradient dilution (the serum was diluted 100 times, and then 11 gradients of 3-fold gradient dilution) at 100 μL / well, and incubated at 37 ° C for 1.5 hours; washed three times with PBST, and added Peroxidase-AffiniPure Goat Anti-Mouse IgG (Jackson, catalog number: 115-035-003), 100 μL / well, incubate at 37°C for 1 hour; wash 8 times with PBST, add TMB solution (Huzhou Yingchuang Biotechnology Co., Ltd., TMB-S-001) at 100 μL / well, incubate at 37°C in the dark for 15-25 minutes; add 50 μL stop solution (0.1 M H2SO4) to each reaction well to terminate the enzymatic color development reaction.

[0294] Plate reading: SoftMax pro 7.02 software, which comes with the microplate reader, was used to set the detection wavelength to 450 nm for reading. The obtained readings were fitted with a nonlinear four-parameter equation curve model. The equation is: Where A is the lower limit of absorbance, B represents the slope of the curve, and C is the antibody concentration corresponding to half the maximum response value (EC 50 ), D is the upper limit of absorbance.

[0295] Data processing: blank control OD 450 The serum dilution corresponding to 2 times the mean was used as the antibody titer, and the geometric mean titer (GMT) of each group was then calculated.

[0296] BA.1-Spike protein amino acid sequence:

[0297] The results are shown in Figure 3. After a single immunization, the geometric mean titer (GMT) of anti-Spike protein IgG against WT-Spike protein, Delta-Spike protein, BA.1-Spike protein, and BA.5-Spike protein in the adjuvant group was increased by 54 times, 55 times, 37 times, and 65 times, respectively, compared with the non-adjuvant group, indicating that the adjuvant significantly enhanced the humoral immune response.

[0298] Example 4: Immunogenicity of the vaccine in mice

[0299] 1.1 Immunization of mice

[0300] Balb / c mice were injected intramuscularly with different doses of fusion protein D, fusion protein 2-1, and a bivalent vaccine (1:1 mass ratio of fusion protein D and fusion protein 2-1) plus a fixed dose of SWE adjuvant (SEPPIC SA, Cat. No. 80748J, Batch No. 210721010001) on days 0 (D0) and 21 (D21). Control mice received SWE adjuvant alone. Blood samples were collected on days 14 (D14) and 35 (D35) after immunization. The experimental design is shown in Table 3. Serum anti-Spike protein IgG titers were measured by ELISA and cross-neutralizing antibody titers against multiple pseudoviruses were measured by SARS-CoV-2 spike pseudovirus neutralization assay.

[0301] Table 3 Experimental design

[0302] 1.2 Detection of serum anti-Spike protein IgG titer by ELISA

[0303] WT-Spike protein (see step 1.1 of Example 2), Delta-Spike protein (see step 1.1 of Example 2), BA.1-Spike protein (see step 1.2 of Example 3), and BA.5-Spike protein (see step 1.1 of Example 2) were diluted with 1×PBS to a final concentration of 2 μg / mL, and 100 μL / well were added to a 96-well ELISA plate (Costar, catalog number: 9018) and incubated at 4°C overnight. The cells were washed twice with 0.05% Tween-20 (1 mL / well); blocking solution (PBST containing 3% BSA) was added and the cells were incubated overnight at 4°C; the cells were washed twice with PBST; the mouse serum obtained in step 1.1 of Example 4 was diluted 1000-fold and then diluted 3-fold in 11 steps, 100 μL per well, and incubated at 37°C for 1.5 hours; the cells were washed three times with PBST; 100 μL / well of Peroxidase-AffiniPure Goat Anti-Mouse IgG (Jackson, Catalog No. 115-035-003) was diluted 1:10,000 and incubated at 37°C for 1 hour; the cells were washed eight times with PBST; 100 μL / well of TMB solution (Huzhou Yingchuang Biotechnology Co., Ltd., TMB-S-001) was added and the cells were incubated at 37°C in the dark for 15-25 minutes; the enzymatic color development reaction was terminated with 50 μL / well of 0.1 M sulfuric acid.

[0304] Plate reading: SoftMax pro 7.02 software, which comes with the microplate reader, was used to set the detection wavelength to 450 nm for reading. The OD values ​​obtained were fitted with a nonlinear four-parameter equation curve model. The equation is: Where A is the lower limit of absorbance, B represents the slope of the curve, and C is the antibody concentration corresponding to half the maximum response value (EC 50 ), D is the upper limit of absorbance.

[0305] Data processing: blank control OD 450 The serum dilution corresponding to 2 times the mean was used as the antibody titer, and the geometric mean titer (GMT) of each group was then calculated.

[0306] In all tested dose groups, no anti-Spike protein IgG titers were detected in mice given only the adjuvant. As shown in Figures 4a, 4c, 4e, and 4g, all mice produced IgG antibodies against WT-Spike protein, Delta-Spike protein, BA.1-Spike protein, and BA.5-Spike protein on day 14 after the primary immunization, and the geometric mean antibody titer (GMT) showed a clear dose-response relationship.

[0307] On day 35 after the second booster immunization, anti-Spike protein IgG titers in all mice were significantly elevated compared to those after the primary immunization. Compared with mice receiving the same dose of fusion protein D, mice receiving fusion protein 2-1 had significantly lower antibody titers against WT-Spike protein and Delta-Spike protein (Figures 4b, 4d). However, the antibody titer against BA.5-Spike protein in the fusion protein 2-1 group was significantly higher than that in the fusion protein D group (Figure 4h). Compared with the monovalent vaccines fusion protein D and fusion protein 2-1, the bivalent vaccine induced higher IgG antibody titers against WT-Spike protein, Delta-Spike protein, BA.1-Spike protein, and BA.5-Spike protein.

[0308] The results of anti-Spike protein IgG antibody titers showed that the bivalent vaccine had better immunogenicity than the monovalent vaccine fusion protein D or fusion protein 2-1, and the titer of two injections was significantly better than that of one injection. The antibody titers against different mutant strains remained better than or equal to those of the monovalent vaccine.

[0309] 1.3 SARS-CoV-2 Spike pseudovirus neutralizing antibody experiment

[0310] In order to evaluate the broad spectrum of the bivalent vaccine (mass ratio of fusion protein D and fusion protein 2-1 is 1:1), the inhibitory ability of the immune serum against the original strain of SARS-CoV-2, the Delta variant, and the variants BA.5, BQ.1.1, XBB and XBB.1.5 was tested.

[0311] The pseudovirus was slowly dissolved on ice and diluted 50 times with DMEM complete medium. The pseudovirus dilution was added to a 96-well white plate. The experimental group, cell control group (CC group) and virus control group (VC group) were set up. Except for the CC group, the rest were 25 μL / well. The mouse serum collected from group 3, group 6 and group 9 on the 35th day of step 1.1 of Example 4 was incubated at 56 ° C for 30 min to inactivate complement, diluted 40 times with DMEM complete medium, and then diluted 2 times with 12 gradients. The experimental group was added to the 96-well white plate to which the pseudovirus had been added at 50 μL / well. The CC group was added with 75 μL / well of DMEM complete medium, and the VC group was added with 50 μL / well of DMEM complete medium. The 96-well white plate was fully shaken and mixed and placed in an incubator at 37 ° C for 1 hour. After incubation for 1 hour, the 96-well white plate was taken out and ACE2-293 cell suspension (2×10 4cells / well), gently blow evenly with a pipette, and place the 96-well white plate in an incubator for incubation. After 48 hours of incubation, remove the 96-well white plate, wait for it to return to room temperature, discard the culture medium, and add 100 μL of Bio-LiteTM Luciferase Assay System solution (Vazyme, Cat. No. DD1201) to each well. After reacting at room temperature in the dark for 2 minutes, the luminescence signal was read using the luminescence detection module of a microplate reader. The % inhibition was analyzed using a nonlinear four-parameter curve fitting model and the IC was calculated. 50 .

[0312] 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 subjected to pressure selection with hygromycin (200 μg / ml) and flow cytometry sorting (using 10 μg / ml anti-ACE2 and PE-conjugated anti-human IgG-Fc). The cells were further expanded and single clones with a PE positivity rate >90% were selected for further expansion, thereby screening HEK293 cells expressing ACE2, namely ACE2-293 cells.

[0313] Among them, the pseudoviruses used in this experiment are: SARS-CoV-2 original strain pseudovirus (Vazyme, product number DD1702-03), SARS-CoV-2 Delta pseudovirus (Vazyme, product number DD1754-03), SARS-CoV-2 BA.5 pseudovirus (Vazyme, DD1776-03), SARS-CoV-2 BQ.1.1 pseudovirus (Vazyme, product number DD1792-03), SARS-CoV-2 XBB pseudovirus (Vazyme, DD1794-03), and SARS-CoV-2 XBB.1.5 pseudovirus (Vazyme, DD1797-03).

[0314] The results in Figure 5 demonstrate that, consistent with the anti-Spike protein antibody titer results described above, the bivalent vaccine exhibits superior immunogenicity compared to the monovalent vaccines, fusion protein D or fusion protein 2-1, maintaining neutralizing antibody titers superior to or equal to those of the monovalent vaccines against various variants. Fusion protein 2-1 exhibited significantly lower titers against the original SARS-CoV-2 pseudovirus and SARS-CoV-2 Delta pseudovirus than did fusion protein D and the bivalent vaccine. Fusion protein D exhibited significantly lower titers against SARS-CoV-2 BA.5, SARS-CoV-2 BQ.1.1, SARS-CoV-2 XBB, and SARS-CoV-2 XBB.1.5 pseudoviruses than did fusion protein 2-1 and the bivalent vaccine. The bivalent vaccine, however, maintained high titers against all tested pseudoviruses. This demonstrates the bivalent vaccine's broad spectrum of activity, with high neutralizing antibody titers against a wide range of strains.

[0315] 1.4 Serum neutralizing antibody titer against SARS-CoV-2

[0316] The inhibitory titer of the mouse serum after the second immunization of the bivalent vaccine against the new coronavirus variants BA.5, BQ.1 and XBB true virus was detected. The microplate method was used to evaluate the neutralization titer (IC99 Titer) of the mouse serum collected on the 35th day of step 1.1 of group 9 in Example 4 against the new coronavirus variants BA.5, BQ.1 and XBB true virus. The principle is: after the virus infects sensitive target cells (VERO-E6), it causes cell morphological changes and the occurrence of cytopathic effect (CPE). After the specific neutralizing antibodies in the serum bind to the virus, the virus particles lose their infectivity and inhibit the occurrence of CPE.

[0317] The test results showed (Figure 6) that the serum of mice after the second immunization with the bivalent vaccine had a strong inhibitory ability against the new coronavirus variants BA.5, BQ.1 and XBB true viruses. Among them, the inhibition titer of 6 mice in the BA.5 group had reached the upper limit of the test, the inhibition titer of 2 mice in the BQ.1 group had reached the upper limit of the test, and the inhibition titer of 3 mice in the XBB group had reached the upper limit of the test; this shows that the bivalent vaccine has extremely high neutralizing antibody titers against the currently popular new coronavirus variants BA.5, BQ.1 and XBB true viruses.

[0318] Example 5: Immunogenicity of the bivalent vaccine as a booster in mice vaccinated with two doses of inactivated vaccine

[0319] 1.1 Immunization of mice

[0320] Two groups of Balb / c mice were injected intramuscularly with 100 μL of inactivated vaccine (Beijing Sinovac Biotech Co., Ltd., 202112187N) on days 0 (D0) and 21 (D21). After three months of recovery, they were vaccinated with either the inactivated vaccine (Beijing Sinovac Biotech Co., Ltd., 202112187N) or the bivalent vaccine (fusion protein D and fusion protein 2-1 at a 1:1 mass ratio) on day 111 (D111). Blood was collected one day before the third injection (D110) and two weeks after vaccination (D125). The experimental design is shown in Table 4. Serum anti-Spike protein (original strain, Delta, Omicron BA.1, and BA.5) IgG titers were measured by ELISA, and cross-neutralizing antibody titers against multiple pseudoviruses were measured using a pseudovirus neutralization assay.

[0321] Table 4 Experimental design

[0322] 1.2 Detection of serum anti-Spike protein IgG titer by ELISA

[0323] WT-Spike protein (preparation method see step 1.1 of Example 2), Delta-Spike protein (preparation method see step 1.1 of Example 2), BA.1-Spike protein (preparation method see step 1.2 of Example 3), and BA.5-Spike protein (preparation method see step 1.1 of Example 2) were diluted with 1×PBS to a final concentration of 2 μg / mL, and 100 μL / well were added to a 96-well ELISA plate (Costar, product number: 9018) and incubated at 4°C overnight; The cells were washed twice with 1× PBS supplemented with 0.05% Tween-20; blocking solution (PBST containing 3% BSA) was added and the cells were incubated overnight at 4°C; the cells were washed twice with PBST; 100 μL of the mouse serum obtained in step 1.1 of Example 5 (serum was diluted 1000-fold and then 3-fold in 11 steps) was added to each well and the cells were incubated at 37°C for 1.5 hours; the cells were washed three times with PBST; a 1:10,000 dilution of Peroxidase-AffiniPure Goat Anti-Mouse IgG (Jackson, catalog number: 115-035-003), 100 μL / well, incubate at 37°C for 1 hour; wash 8 times with PBST; add 100 μL / well TMB solution (Huzhou Yingchuang Biotechnology Co., Ltd., TMB-S-001) and incubate at 37°C in the dark for 15-25 minutes; add 50 μL stop solution (0.1 M H2SO4) to each reaction well to terminate the enzymatic color reaction.

[0324] Plate reading: SoftMax pro 7.02 software, which comes with the microplate reader, was used to set the detection wavelength to 450 nm for reading. The obtained readings were fitted with a nonlinear four-parameter equation curve model. The equation is: Where A is the lower limit of absorbance, B represents the slope of the curve, and C is the antibody concentration corresponding to half the maximum response value (EC 50 ), D is the upper limit of absorbance.

[0325] Data processing: blank control OD 450 The serum dilution corresponding to 2 times the mean was used as the antibody titer, and the geometric mean titer (GMT) of each group was then calculated.

[0326] As shown in Figures 7a-d, before the sequential booster injection, the serum anti-Spike protein IgG titers of both groups of mice were at a low level. Among them, the antibody titers against BA.1-Spike protein and BA.5-Spike protein were significantly lower than those against WT-Spike protein and Delta-Spike protein. Two weeks after the sequential booster injection, the anti-Spike protein IgG titers of mice increased significantly regardless of whether they were vaccinated with inactivated or bivalent vaccines. The anti-Spike protein IgG titers of the bivalent vaccine group (Group 2) against Wildtype, Delta, BA.1 and BA.5 strains were much higher than those in the inactivated vaccine group (Group 1).

[0327] 1.3 SARS-CoV-2 Spike pseudovirus neutralization experiment

[0328] The inhibitory ability of immune serum against the original strain, Delta variant, and variants BF.7 and XBB.1 of SARS-CoV-2 was tested. The pseudovirus was diluted 50-fold with DMEM complete medium, and the pseudovirus dilution was added to a 96-well white plate. The experimental group, cell control group (CC group), and virus control group (VC group) were set up, with 25 μL / well of each except the CC group. The mouse serum collected in step 1.1 of Example 5 was incubated at 56°C for 30 minutes to inactivate complement, diluted 40-fold with DMEM complete medium, and then diluted 2-fold in a gradient, with 12 gradients. The experimental group was added to the 96-well white plate with pseudovirus at 50 μL / well, the CC group was added with 75 μL / well of DMEM complete medium, and the VC group was added with 50 μL / well of DMEM complete medium. The 96-well white plate was thoroughly shaken and mixed, and then placed in an incubator at 37°C for 1 hour. After incubation for 1 hour, the 96-well white plate was taken out and ACE2-293 cell suspension (2×10 4cells / well), gently blow evenly with a pipette, and place the 96-well white plate in an incubator at 37°C. After 48 hours, remove the 96-well white plate, wait for it to return to room temperature, discard the culture medium, and add 100 μL of Bio-LiteTM Luciferase Assay System solution (Vazyme, Cat. No. DD1201) to each well. After reacting at room temperature in the dark for 2 minutes, the luminescence signal is read using the luminescence detection module of a microplate reader. The % inhibition is analyzed using a nonlinear four-parameter curve fitting model and the IC is calculated. 50 .

[0329] Among them, the pseudoviruses used in this experiment are: SARS-CoV-2 original strain pseudovirus (Vazyme, catalog number DD1702-03), SARS-CoV-2 Delta pseudovirus (Vazyme, catalog number DD1754-03), SARS-CoV-2 BF.7 pseudovirus (Jiman Biotechnology, catalog number: GM-0220PV100-96T), and SARS-CoV-2 XBB.1 pseudovirus (Jiman Biotechnology, catalog number: GM-0220PV104-96T).

[0330] The results are shown in Figures 8a-d. Consistent with the above-mentioned results on anti-Spike protein antibody titers, the bivalent vaccine had superior neutralizing antibody titers against different variants than the inactivated vaccine. Before sequential immunization, both groups of mice had almost no detectable neutralizing antibody titers against SARS-CoV-2 BF.7 and SARS-CoV-2 XBB.1 pseudoviruses. After sequential immunization, some mice in the inactivated vaccine group (Group 1) produced extremely low neutralizing antibody titers against SARS-CoV-2 BF.7 pseudovirus (Figure 8c), and only one mouse produced trace amounts of neutralizing antibodies against SARS-CoV-2 XBB.1 pseudovirus (Figure 8d). In contrast, the bivalent vaccine group (Group 2) produced relatively significant neutralizing antibody titers against both SARS-CoV-2 BF.7 and SARS-CoV-2 XBB.1 pseudoviruses.

[0331] Example 6: Bivalent vaccine cellular immunity enzyme linked immunospot assay (ELISpot) detection

[0332] 1.1 Immunization of mice

[0333] Six-week-old Balb / C female mice were divided into two groups and immunized by intramuscular injection according to the dose in Table 5 on day 0 (D0) and day 21 (D21), respectively. The mice were euthanized 2 weeks after the second immunization, and the spleens were harvested for ELIspot assay.

[0334] Table 5 Experimental design Note: The bivalent vaccine is a 1:1 mass ratio of fusion protein D and fusion protein 2-1; the adjuvant is SWE adjuvant

[0335] 1.2 ELIspot test

[0336] In order to evaluate the cellular immunity level of mice immunized with bivalent vaccine against different variants of the new coronavirus, the Spike protein peptide pool of the original strain (wildtype, WT) and variant strains (Delta, BA.5) of the new coronavirus was used as a stimulator to stimulate the spleen lymphocytes of immunized mice. Phorbol 12-myristate 13-acetate (PMA) (MedChemExpress, product number 16561-29-8) was used as a positive control, and blank culture medium was used as a negative control. The ELISpot method was used to detect the secretion of IFN-γ, IL-2, IL-4 and other cytokines by the cells.

[0337] ELIspot assays were performed using the ELISpot Plus Mouse IL-2 Kit (Mabtech, Cat. No. 3441-4HPW), the ELISpot Plus Mouse IL-4 Kit (Mabtech, Cat. No. 3311-4HPW), and the ELISpot Plus Mouse IFN-γ Kit (Mabtech, Cat. No. 3321-4HPT). The steps are briefly described as follows:

[0338] Wash the pre-coated IL-2, IL-4, and IFN-γ ELISpot plates with sterile PBS and add 200 μL / well of sterile DMEM (Gibco, Cat. No. C11995500BT) supplemented with 10% FBS (ExCell Bio, Cat. No. FSP500). Incubate at room temperature overnight. Dilute PMA to 100 ng / mL in sterile DMEM supplemented with 10% FBS. Separately, dilute the spike protein peptide pools of the original SARS-CoV-2 strain (Qiangyao Bio, Cat. No. 04010065348), the spike protein peptide pools of the SARS-CoV-2 variant Delta (Qiangyao Bio, Cat. No. 04010068345), and the spike protein peptide pools of the SARS-CoV-2 variant BA.5 (Qiangyao Bio, Cat. No. 04010069259) to 2 μg / mL. Discard the medium from the ELISpot plates and add 50 μL / well of PMA and the three peptide pools. DMEM medium was used as the negative control well. The spleen obtained in step 1.1 of Example 6 was ground, and the resulting cell suspension was passed through a 70 μm mesh and washed once with DMEM medium. 2 mL of Ack lysing buffer (Gibco, A10492-01) was added to the cells of each mouse and incubated on ice for 10 min. 20 mL of DMEM medium was added to terminate the lysis, centrifuged, and the supernatant was discarded. The cells were resuspended in DMEM medium to obtain mouse spleen lymphocytes. The spleen lymphocytes of each mouse were divided into 2 × 10 5 Add 50 μL of cells / well to the ELISpot plate in a suspended state. Incubate the ELISpot plate at 37°C, 5% CO2 for 48 hours. Discard the cell suspension and wash the plate with sterile PBS. Dilute the detection antibodies corresponding to the three ELISpot plates to 1 μg / mL in PBS containing 0.5% FBS, filter through a 0.22 μm filter, and add 100 μL / well to the corresponding ELISpot plate. Incubate at room temperature for 2 hours. Wash the plate with sterile PBS. Dilute Streptavidin-HRP (Jackson; Cat. No. 016-030-084) 1000-fold in PBS containing 0.5% FBS, filter through a 0.22 μm filter, and add 100 μL / well to the ELISpot plate. Incubate at room temperature for 1 hour. Wash the plate with sterile PBS. Add 100 μL / well of sterile TMB substrate solution for color development until distinct spots appear in the wells. Discard the liquid in the wells, rinse with deionized water to stop color development, remove the backing sheet, and repeatedly rinse both sides of the membrane. Let it dry and count the spots in the wells.

[0339] The IFN-γ ELISpot results are shown in Figure 9a. Compared with the adjuvant-only immune adjuvant group (Group 4), the Spike protein peptide pool of the original strain of the new coronavirus, the Spike protein peptide pool of the new coronavirus variant Delta, and the Spike protein peptide pool of the new coronavirus variant BA.5 after vaccination with the monovalent or bivalent vaccine can stimulate the activation of IFN-γ-secreting T cells. Among them, the T cell response to the Spike protein peptide pool of the new coronavirus variant Delta and the Spike protein peptide pool of the new coronavirus variant BA.5 is higher than that to the Spike protein peptide pool of the original strain of the new coronavirus.

[0340] The IL-2 and IL-4 ELISpot results are shown in Figures 9b and 9c, respectively. Similar to the IFN-γ ELISpot results, after vaccination, the Spike protein peptide pool of the original strain of the new coronavirus, the Spike protein peptide pool of the new coronavirus variant Delta, and the Spike protein peptide pool of the new coronavirus variant BA.5 can all stimulate the activation of IL-2-secreting cells or IL-4-secreting cells, and the Spike protein peptide pool of the new coronavirus variant Delta and the Spike protein peptide pool of the new coronavirus variant BA.5 have more significant stimulating effects.

[0341] In addition, after stimulation with the Spike protein peptide pool of the new coronavirus variant Delta and the Spike protein peptide pool of the new coronavirus variant BA.5, the number of IL-2-secreting cells was significantly higher than that of IL-4-secreting cells. As shown in Figure 9c, after stimulation with the Spike protein peptide pool of the original strain of the new coronavirus, the Spike protein peptide pool of the new coronavirus variant Delta, and the Spike protein peptide pool of the new coronavirus variant BA.5, only a very small number of IL-4-secreting cells were activated. From this point of view, fusion protein D, fusion protein 2-1, and bivalent vaccine immunization can induce cellular immune responses, and are mainly Th1 type.

[0342] Example 7: Immunogenicity of the vaccine in rats

[0343] 1.1 Rat immunization

[0344] The study design is shown in Table 6. Healthy Sprague-Dawley rats (SPF), half male and half female, received a single intramuscular injection of 0 or 80 μg of the bivalent vaccine (fusion protein D and fusion protein 2-1 at a 1:1 mass ratio) on day 1 (D1) in a 0.5 mL injection volume. Blood was collected on day 15 (D15). Serum anti-Spike protein (original strain, Delta, BA.1, and BA.5) IgG titers were measured by ELISA.

[0345] Table 6 Experimental design Note: Adjuvant is SWE adjuvant

[0346] 1.2 Detection of serum anti-Spike protein IgG titer by ELISA

[0347] WT-Spike protein (see step 1.1 of Example 2), Delta-Spike protein (see step 1.1 of Example 2), and BA.5-Spike protein (see step 1.1 of Example 2) were diluted with 1×PBS to a final concentration of 2 μg / mL, and 100 μL / well were added to a 96-well ELISA plate (Costar, Cat. No. 9018) and incubated at 4°C overnight; washed twice with PBST (0.05% volume of Tween-20 added to 1×PBS); added blocking solution (PBST containing 3% BSA) and incubated in a 37°C incubator for 2 hours; washed twice with PBST; added gradient dilutions of mouse serum obtained in step 1.1 of Example 7 (diluted the serum 100-fold, then diluted 3-fold in 11 steps), 100 μL per well, and incubated at 37°C for 1.5 hours; washed three times with PBST; added 1:5000 diluted Goat anti-Rat IgG secondary antibody (HRP) (Sino The cells were incubated at 37°C for 1 hour at 100 μL / well with PBST (Huzhou Yingchuang Biotechnology Co., Ltd., TMB-S-001) at 100 μL / well and incubated in the dark at 37°C for 15-25 minutes. The enzymatic color development reaction was terminated by adding 50 μL of stop solution (0.1 M H2SO4) to each reaction well.

[0348] Plate reading: SoftMax pro 7.02 software, which comes with the microplate reader, was used to set the detection wavelength to 450 nm for reading. The obtained readings were fitted with a nonlinear four-parameter equation curve model. The equation is: Where A is the lower limit of absorbance, B represents the slope of the curve, and C is the antibody concentration corresponding to half the maximum response value (EC 50 ), D is the upper limit of absorbance.

[0349] Data processing: blank control OD 450 The serum dilution corresponding to 2 times the mean was used as the antibody titer, and the geometric mean titer (GMT) of each group was then calculated.

[0350] The results are shown in Figure 10. On day 14 (D15) after a single immunization, few rats in the adjuvant-only group (Group 2) had detectable anti-Spike protein IgG titers, or only a few rats had very low titers. In contrast, all rats in the bivalent vaccine group (Group 1) produced IgG antibodies against WT-Spike protein, Delta-Spike protein, and BA.5-Spike protein. These results demonstrate that the bivalent vaccine, at a dose of 80 μg per rat, elicited a robust humoral immune response in rats.

[0351] Example 8: Animal protection effect of vaccine

[0352] The SARS-CoV-2-infected hamster pneumonia model was used to evaluate the animal protection effect of the bivalent vaccine.

[0353] 1.1 Experimental methods

[0354] Male golden hamsters (18 weeks old) were divided into 6 groups, 3 of which were challenge groups and 3 were satellite groups. The challenge group and satellite group were set up in the same way, with 2 dose groups (1μg group and 5μg group) and 1 model group. The vaccine was injected intramuscularly on day 0 and day 21, respectively. In addition, the challenge group golden hamsters were injected with SARS-CoV-2 Omicron variant BA.5 at 10 μg on day 42. 5 TCID 50 The challenge was performed by intranasal dripping of an infectious dose of 100 mg / kg. The experimental design is shown in Table 7.

[0355] Table 7 Experimental design Note: The bivalent vaccine is a 1:1 mass ratio of fusion protein D and fusion protein 2-1; the adjuvant is SWE adjuvant

[0356] 1.2 Observation indicators

[0357] For the challenged hamsters, body weight changes were recorded continuously until the end of the experiment. All hamsters were sacrificed 5 days after challenge, and lung tissue viral load and lung tissue pathology were measured.

[0358] 35 days after the satellite group was immunized, blood was collected to test for neutralizing antibodies.

[0359] 1.3 Antiviral effect of drugs

[0360] 1.3.1 Animal weight

[0361] Hamsters in the challenge group and model group experienced weight loss after challenge, with the highest average percentage loss being 5.91%. Hamsters in the challenge group and bivalent vaccine 1 μg group experienced continued weight loss after challenge, with the highest average percentage loss being 4.16%, which was not significantly different from the challenge group and model group (p>0.05). Hamsters in the challenge group and bivalent vaccine 5 μg group experienced a slight weight loss after challenge, with an average percentage loss of 1.33% 5 days after challenge, which was significantly lower than that in the challenge group and model group (p<0.01). See Table 8.

[0362] Table 8 Body weight changes of hamsters in each group after challenge (mean ± SD, unit g) Note: Compared with the model group, *p<0.05 indicates significant difference, **p<0.01 indicates very significant difference

[0363] 1.3.2 Viral load

[0364] The average viral load of lung tissue in the model group was 10 5.48 The average viral load in lung tissue of hamsters in the challenge group with 1 μg of bivalent vaccine was 10 copies / mg 5 days after challenge. 4.25 The average viral load in lung tissue of hamsters in the challenge group with bivalent vaccine 5μg was 10 copies / mg after 5 days of challenge. 3.59 copies / mg, which was significantly lower than that in the challenge group and model group (p<0.01), with a decrease of 1.89lg value.

[0365] 1.3.3 Neutralizing Antibodies

[0366] Thirty-five days after the first immunization, the neutralizing antibody GMT in hamsters in the satellite bivalent vaccine 1 μg group was 349.05. The neutralizing antibody GMT in hamsters in the satellite bivalent vaccine 5 μg group was 269.67. The neutralizing antibody GMT in hamsters in the satellite model group was ≤10.

[0367] 1.3.4 Pathological results

[0368] In the challenge group, the bivalent vaccine 1 μg group: 4 cases (4 / 6) developed moderate pneumonia in the lungs, and 2 cases (2 / 6) developed mild pneumonia in the lungs. In the challenge group, the bivalent vaccine 5 μg group: 1 case (1 / 6) developed moderate pneumonia in the lungs, and 5 cases (5 / 6) developed mild pneumonia in the lungs. In the challenge group model group: 6 cases (6 / 6) developed moderate pneumonia in the lungs.

[0369] Example 9: Fusion Protein D Preparation

[0370] The fusion protein D preparation shown in Table 9 was prepared. The protein concentration of fusion protein D was 0.5 mg / mL, and the buffer was 20 mM phosphate buffer at pH 7.0.

[0371] Table 9 Fusion protein D preparation composition

[0372] Dynamic light scattering (DLS) detection experiments found that the addition of arginine, magnesium chloride, potassium chloride or glycerol to the formulation would produce protein particles with a particle size of about 10 nm, indicating that arginine, magnesium chloride, potassium chloride or glycerol would cause 24mer disaggregation.

[0373] SEC-HPLC analysis was performed on formulations A1, A4, A8, and A9 after 14 days at 40°C. As shown in Table 10, the SEC-HPLC results show that formulation A1, which contains trehalose and polysorbate 80, exhibited the highest 24mer content after 14 days at high temperature, indicating relatively stable 24mer content.

[0374] Table 10 SEC-HPLC test results

[0375] The excipient concentrations and buffers of the fusion protein D formulation were changed, and the formulation design was shown in Table 11, wherein the protein concentration of fusion protein D was 0.5 mg / mL; 20 mM PB7.0 was a 20 mM phosphate buffer solution comprising 10.26 mM disodium hydrogen phosphate and 9.74 mM sodium dihydrogen phosphate, with a pH of 7.0; and 20 mM His5.66 was a 20 mM histidine buffer solution comprising 6 mM histidine and 14 mM histidine hydrochloride, with a pH of 5.66.

[0376] Table 11 Fusion protein D preparation composition

[0377] The preparation was placed at 40°C for 6 days and then analyzed by SEC-HPLC. The results are shown in Table 12. It can be seen that fusion protein D produced fewer HMW aggregates at 20 mM His5.66 than at 20 mM PB7.0, and was more stable at 20 mM His5.66.

[0378] Table 12 SEC-HPLC test results

[0379] The fusion protein D preparation shown in Table 13 was further prepared, wherein the protein concentration of the fusion protein D was 0.2 mg / mL, 20 mM His5.66 was a 20 mM histidine buffer, pH 5.66; 20 mM His6.0 was a 20 mM histidine buffer, pH 6.0; 20 mM PB6.0 was a 20 mM phosphate buffer, pH 6.0; 20 mM PB7.0 was a 20 mM phosphate buffer, pH 7.0; 20 mM PB8.0 was a 20 mM phosphate buffer, pH 8.0; and 20 mM CB6.0 was a 20 mM citric acid buffer, pH 6.0.

[0380] Table 13 Fusion protein D preparation composition

[0381] Each preparation sample was placed at 50°C and samples were taken for SEC-HPLC detection on the 7th, 11th, 23rd, 50th and 93rd day (D); 50°C-4D+4°C-89D indicates that the preparation sample was first placed at 50°C for 4 days (4D), then the sample was taken out and placed at 4°C for 89 days, and then sampled for SEC-HPLC detection.

[0382] The SEC-HPLC test results are shown in Table 14. From the SEC results, it can be seen that preparation C7 has better results than preparation C8; the samples prepared with pH 5.66 histidine buffer and the samples prepared with pH 6.0 histidine buffer both have good stability, and the preparations with the addition of sodium chloride, polysorbate 80 and trehalose are more stable.

[0383] Table 14 SEC-HPLC test results of samples (%)

[0384] Example 10: Fusion Protein G Preparation

[0385] A fusion protein G preparation as shown in Table 15 was prepared, wherein the protein concentration of fusion protein G was 0.2 mg / mL, the buffer was 20 mM His buffer, and the pH was 6.0.

[0386] Table 15 Composition of Fusion Protein G Preparation

[0387] Stability tests were conducted on each formulation sample at 4°C and 50°C for 4 days (50°C-4D) and three freeze-thaw cycles (-65°C-25°C) (DR3). Lamp inspection revealed that formulation D2 exhibited opalescence and diffused matter after 4 days at 50°C; all other formulations tested normal. SEC-HPLC analysis results, shown in Table 16, demonstrate that formulation D3, containing hydroxypropyl-β-cyclodextrin, was more stable at 50°C, with the highest 24mer content as measured by SEC. After three freeze-thaw cycles, formulation D2 outperformed formulation D1, indicating that formulations containing polysorbate 80, trehalose, and sodium chloride offer superior freeze-thaw protection compared to formulations containing sodium chloride alone.

[0388] Table 16 SEC-HPLC test results of fusion protein G preparation

[0389] The fusion protein G preparation shown in Table 17 was further prepared, wherein the protein concentration of the fusion protein G was 0.2 mg / mL, the buffer was 20 mM His buffer, and the pH was 6.0.

[0390] Table 17 Composition of Fusion Protein G Preparation

[0391] Samples of each formulation were stored at 25°C, 40°C, and 50°C for 7 days before being sampled for optical inspection. The results are shown in Table 18. The addition of sodium chloride significantly improved the opalescence of the solution, while opalescence would still occur without sodium chloride. The addition of polysorbate 80 reduced precipitation.

[0392] Samples of each formulation were collected and analyzed by SEC-HPLC after being stored at 50°C for 7, 14, 27, and 50 days (D), at 40°C for 50 days (D), and at 27°C for 14, 27, and 50 days (D). The results are shown in Tables 19 and 20. Sodium chloride significantly affects the stability of the fusion protein G formulation, and the lower the polysorbate 80 concentration, the better the stability. The concentration of hydroxypropyl beta-cyclodextrin has a limited effect on stability. There is no significant difference in stability between the formulation containing 8 mg / mL hydroxypropyl beta-cyclodextrin and the formulation containing 16 mg / mL hydroxypropyl beta-cyclodextrin, but the stability is better than that of the formulation E16 containing trehalose.

[0393] Table 19 SEC-HPLC test results under high temperature conditions

[0394] Table 20 SEC-HPLC test results at 25°C

[0395] Example 11: Bivalent vaccine (fusion protein D + fusion protein G) preparation

[0396] The formulations shown in Table 21 were prepared, wherein F1-F11 were fusion protein D formulations, F12-F22 were fusion protein G formulations, and F23-F33 were bivalent vaccine formulations comprising fusion protein D and fusion protein G (mass ratio 1:1), wherein the protein concentration was the concentration of each fusion protein, for example, formulation F23 contained 0.2 mg / mL fusion protein D and 0.2 mg / mL fusion protein G. The adjuvant was SWE adjuvant (SEPPIC SA, Catalog No. 80748J), an aluminum-free adjuvant, and the added amount was 3 / 10 of the total volume of the formulation. Among them, 20mM His6.0 is a 20mM histidine buffer with a pH of about 6.0; 10mM His6.0 is a 10mM histidine buffer with a pH of about 6.0; 10mM His+5mM CB is a mixture of 20mM histidine buffer with a pH of 6.0 and 10mM citric acid buffer with a pH of 6.6 in a volume ratio of 1:1, with a pH of about 6.0.

[0397] Table 21 Composition of monovalent / bivalent vaccine formulations Note: HP-CD is hydroxypropyl beta-cyclodextrin.

[0398] The formulation samples were subjected to optical inspection at 4°C, 25°C, 40°C, and 50°C, under illumination (4500 lx ± 500 lx, 25°C) for 7 days, followed by five freeze-thaw cycles (-60°C to 25°C), and shaken at room temperature (200 rpm) for 48 hours. Formulations F1-F15, F17, F19-F21, and F23-F32 all showed normal optical inspection results under these conditions. Formulations F16 and F18 showed slight opalescence and diffuse matter after seven days at 4°C. Formulation F22 showed slight opalescence and diffuse matter after five freeze-thaw cycles. Formulation F33 showed slight opalescence and diffuse matter after seven days at 4°C, and slight diffuse matter after seven days at 25°C and 50°C.

[0399] Among the bivalent vaccine formulations, formulations F23, F27, F29, and F31 were further analyzed by SEC-HPLC. The results are shown in Table 22. Freeze-thaw conditions significantly affected the 24mer in the unadjuvanted bivalent vaccine formulations, while elevated temperatures had a minimal effect, essentially similar to that observed under 4°C conditions. Formulations F23, F27, F29, and F31 all exhibited good stability after storage at 50°C for 7 and 23 days. Formulation F27 was superior to formulation F23.

[0400] Table 22 SEC-HPLC test results Note: Illumination (4500lx±500lx, 25°C), freeze-thaw (-60°C~25°C), oscillation (room temperature, 200rpm).

[0401] Fusion protein G preparation F12, a sample diluted 50-fold with SWE adjuvant CB6.0 buffer (10 mM citric acid buffer, pH 6.0), a sample diluted 100-fold with SWE adjuvant CB6.0 buffer (10 mM citric acid buffer, pH 6.0), and a sample of preparation F17 in Example 11 stored at 25°C for 27 days were named DLS-1, DLS-2, DLS-3, and DLS-4, respectively, to examine the effects of adjuvants on the DLS of the samples. The DLS measurement results are shown in Table 23. It can be seen that the DLS of the samples after the addition of adjuvants did not change much. The average particle size of the protein particles of fusion protein G preparation F12 was 42.5 nm. The DLS measurement results of preparation F26 in Example 11 stored at 50°C for 7 days were similar to those of preparation F17.

[0402] Table 23 DLS measurement results

[0403] Example 12: Fusion Protein 2-1 Preparation

[0404] Prepare the formulation shown in Table 24, where the fusion protein 2-1 concentration is 0.45 mg / mL and the buffer is 20 mM His buffer, pH 6.0. Prepare a sample of the fusion protein 2-1 formulation by ultrafiltration using 20 mM His 6.0 buffer. After 8 volume changes, dilute the protein concentration to 0.9 mg / mL. Mix the sample with 2× the stock solution of each excipient at a 1:1 volume ratio. Filter each sample using a sterile filter.

[0405] Table 24 Fusion protein 2-1 formulation composition

[0406] Each formulation sample was packaged and subjected to optical inspection after 7 and 14 days at 4°C. Formulation H3 produced white foreign matter after 14 days at 4°C, indicating possible protein precipitation. Formulations containing sodium chloride and arginine exhibited poor stability. Formulation H4 showed precipitation during optical inspection, while formulations H1 and H2 showed normal results, indicating that protein aggregation and precipitation can occur when formulations do not contain polysorbate 80.

[0407] SEC-HPLC analysis was performed after 1 month at 25°C, 7 days at 40°C, and 21 days at 40°C. The results are shown in Table 25. Formulations H1 and H2 were both relatively stable at 40°C. The SEC-HPLC results of the formulation sample stored at 25°C for 1 month showed that the 24mer in Formulation H1 decreased less with increasing storage time.

[0408] Table 25 SEC-HPLC test results of fusion protein 2-1 preparation

[0409] Example 13: Stability Study of Bivalent Vaccine (Fusion Protein D + Fusion Protein 2-1) Formulation

[0410] According to the aforementioned studies, the excipient composition of the bivalent vaccine formulation involving fusion protein D + fusion protein 2-1 is: 10mM histidine salt buffer + 8.26mg / mL sodium chloride + 4mg / mL hydroxypropyl beta-cyclodextrin + 0.2mg / mL polysorbate 80 + SWE adjuvant (the added amount is 3 / 10 of the total volume of the formulation). The formulation specifications and composition are shown in Table 26, where the protein concentrations of fusion protein D and fusion protein 2-1 in the 80μg / 0.5mL formulation are both 0.08mg / mL; the protein concentrations of fusion protein D and fusion protein 2-1 in the 40μg / 0.5mL formulation are both 0.04mg / mL.

[0411] Table 26 Specifications and composition of bivalent vaccine preparations

[0412] 1. Long-term stability test:

[0413] Long-term stability testing was conducted on formulation samples stored at 5±3°C in the dark. Samples were taken regularly for testing and the results were compared to those at time 0. The results are shown in Tables 27 and 28. Samples 41A and 41B showed no significant change in any of the test items compared to time 0 after 3 months at 5±3°C, indicating that the product's key quality attributes remained stable. There were no significant changes in any of the physical, chemical, and biological properties of the bivalent vaccine formulation samples across all batches, and the finished product maintained stable quality attributes after 4 months of long-term storage.

[0414] Table 27 Stability data of finished bivalent vaccine formulation (41A) batch at 5±3°C

[0415] Table 28 Stability data of finished bivalent vaccine formulation (41B) batch at 5±3°C Note: “NA” means no testing was scheduled.

[0416] 2. Accelerated stability test:

[0417] The formulation samples were placed at 25±2°C and 40±2°C in the dark for accelerated testing, and samples were taken regularly for testing, with the test results compared to time 0. The results are shown in Tables 29-31. For samples 41A and 41B, after being stored at 25±2°C for 3 months, there was no significant change in any of the test items compared to time 0, and the key quality attributes of the products remained stable and within the qualified range. The results for the other two batches were similar, both within the qualified range, and the product quality was stable. For sample 41B, after being stored at 40±2°C for 3 months, there was no significant change in any of the test items compared to time 0, and the key quality attributes of the products remained stable and within the qualified range. The results for batch 41A were similar.

[0418] Table 29 Stability data of finished formulation (41A) batch at 25±2°C Note: “NA” means no testing was scheduled.

[0419] Table 30 Stability data of finished formulation (41B) batch at 25±2°C

[0420] Table 31 Stability data of finished formulation (41B) batch at 40±2°C Note: “NA” means no testing was scheduled.

[0421] 3. Influencing factors test:

[0422] Samples from batches 41A and 41B were selected for tests on influencing factors such as high temperature (50±2°C), strong light (4500±500lux), and oscillation (200rpm, room temperature).

[0423] Tables 32 and 33 show the test results for the bivalent vaccine formulation under high-temperature conditions. After exposure to 50°C for 21 days, the S protein antibody titer of the finished bivalent vaccine formulation slowly decreased over time, exceeding the acceptance criteria for batch 41A at 7 days and for batch 41B at 3 days. The results of other test items showed no significant trend compared to the time 0. High temperatures can significantly alter the critical quality attributes of this product.

[0424] Table 32 High temperature test data (50±2℃) of finished preparation (41A) Note: “NA” means no testing was scheduled.

[0425] Table 33 High temperature test data (50±2℃) of finished preparation (41B) Note: “NA” means no testing was scheduled.

[0426] The test results of the bivalent vaccine formulation under strong light conditions are shown in Tables 34 and 35. After 21 days of exposure to strong light, the pH, osmotic pressure, squalene content, and S protein antibody titer of the finished formulation were all within the specified ranges compared to time 0, indicating that product quality remained stable. This indicates that exposure to strong light for 21 days does not affect the product's critical quality attributes.

[0427] Table 34: Light test data of finished preparation (41A) (4500±500 lux) Note: “NA” means no testing was scheduled.

[0428] Table 35: Light test data of finished preparation (41B) (4500±500 lux) Note: “NA” means no testing was scheduled.

[0429] The test results of the bivalent vaccine formulation under oscillation conditions (200 rpm, room temperature) are shown in Tables 36 and 37. When the finished formulation was vibrated at 200 rpm for 48 hours at room temperature, no significant trend in any test item was observed compared to the results at time 0. This indicates that 200 rpm oscillation during transportation at room temperature for 48 hours does not affect the quality of the finished bivalent vaccine formulation.

[0430] Table 36: Oscillation test data of finished formulation (41A) (200 rpm, room temperature) Note: “NA” means no testing was scheduled.

[0431] Table 37: Oscillation test data of finished formulation (41B) (200 rpm, room temperature) Note: “NA” means no testing was scheduled.

Claims

1. A coronavirus Spike protein extracellular domain containing a mutation or a truncated fragment thereof, characterized in that: The mutations include: 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; alternatively, the coronavirus is a SARS-CoV-2 Omicron variant; alternatively, the coronavirus is a SARS-CoV-2 Omicron variant BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7, XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.

16.

2. The coronavirus Spike protein extracellular domain or a truncated fragment thereof containing a mutation 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 Spike protein extracellular domain or a truncated fragment thereof containing a mutation 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 Spike protein extracellular domain or a truncated fragment thereof containing a mutation according to any one of claims 1 to 3, characterized in that 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, 6-9, 19-24, 26-31, 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: 3, 4, 6-9, 19-24, 26-31, 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: 3, 4, 6-9, 19-24, 26-31.

5. A fusion protein, characterized in that The invention relates to a fusion protein comprising a coronavirus Spike protein extracellular domain containing a mutation or a truncated fragment thereof and a monomeric subunit protein according to any one of claims 1 to 4, connected by a linker; or, the fusion protein comprises a coronavirus Spike protein extracellular domain containing a mutation or a truncated fragment thereof, the C-terminus of which is connected to the N-terminus of the monomeric subunit protein via a linker.

6. The fusion protein according to claim 5, characterized in that The connector is a GS connector; or, the connector 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 linker is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS)2, (GGGGS)3, or any combination thereof.

7. The fusion protein according to claim 5 or 6, characterized in that The monomeric subunit protein is a self-assembled monomeric subunit protein; alternatively, the monomeric subunit protein is a monomeric ferritin subunit; alternatively, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, algal ferritin, insect ferritin, fungal ferritin or mammalian ferritin; alternatively, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit; alternatively, the monomeric ferritin subunit comprises the amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:

10.

8. The fusion protein according to any one of claims 5 to 7, wherein The fusion protein further comprises an N-terminal signal peptide; alternatively, the N-terminal signal peptide is selected from the group consisting of CSP, mschito, MF-α, pho1, HBM, t-pA, and IL-3 signal peptides; alternatively, 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.

9. The fusion protein according to any one of claims 5 to 8, wherein The fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 12-17, 32-43, 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: 12-17, 32-43, 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: 12-17, 32-43.

10. A biomaterial comprising (1) A polynucleotide characterized in that: The polynucleotide encodes the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof according to any one of claims 1 to 4, or the fusion protein according to any one of claims 5 to 9; or (2) An expression vector, characterized in that the expression vector comprises a polynucleotide encoding the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof according to any one of claims 1 to 4, or the fusion protein according to any one of claims 5 to 9; or (3) A cell, characterized in that the cell contains a polynucleotide encoding the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof as described in any one of claims 1 to 4, or the fusion protein as described in any one of claims 5 to 9, or an expression vector containing a polynucleotide encoding the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof as described in any one of claims 1 to 4, or the fusion protein as described in any one of claims 5 to 9.

11. A Spike protein nanoparticle comprising the fusion protein according to any one of claims 5 to 9.

12. A coronavirus vaccine, characterized in that The coronavirus vaccine comprises the fusion protein according to any one of claims 5 to 9 and / or the Spike protein nanoparticles according to claim 11; or, further comprises a pharmaceutically acceptable carrier and / or adjuvant.

13. A coronavirus multivalent vaccine, characterized in that The coronavirus multivalent vaccine comprises a first fusion protein and a second fusion protein, wherein the first fusion protein is the fusion protein according to any one of claims 5 to 9, and the second fusion protein comprises the extracellular domain of the SARS-CoV-2 Delta variant Spike protein containing mutations or a truncated fragment thereof and a monomeric subunit protein connected by a linker; or, further comprises a pharmaceutically acceptable carrier and / or adjuvant.

14. The coronavirus multivalent vaccine according to claim 13, wherein The SARS-CoV-2 Delta variant Spike protein extracellular domain or a truncated fragment thereof containing a mutation comprises: 1) RRAR is mutated to GSAS; 2) a mutation exists in the turning region between HR1 and CH that prevents the formation of a straight helix during fusion; or, the mutation comprises: 1) RRAR is mutated to GSAS; 2) a double mutation K986P / V987P exists in the turning region between HR1 and CH; or, the truncated fragment of the SARS-CoV-2 Delta variant Spike protein extracellular domain containing a mutation, compared with the full-length extracellular domain of the SARS-CoV-2 Delta variant Spike protein, has a C-terminal truncation of 5-80 amino acid residues; or, a C-terminal truncation of 20-76 amino acid residues; or, a C-terminal truncation of 70 amino acid residues; or, the SARS-CoV-2 Delta variant Spike protein extracellular domain or a truncated fragment thereof containing a mutation comprises an amino acid sequence as shown in any one of SEQ ID NOs: 45-50, or a sequence similar to SEQ ID NOs: 46-48. 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: 45-50, 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: 45-50.

15. The coronavirus multivalent vaccine according to claim 13 or 14, characterized in that The second fusion protein is formed by connecting the C-terminus of the extracellular domain of the SARS-CoV-2 Delta variant Spike protein or a truncated fragment thereof containing the mutation to the N-terminus of the monomeric subunit protein through a linker.

16. The multivalent coronavirus vaccine according to any one of claims 13 to 15, characterized in that The linker of the second fusion protein 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 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.

17. The multivalent coronavirus vaccine according to any one of claims 13 to 16, wherein The monomeric subunit protein of the second fusion protein is a self-assembled monomeric subunit protein; alternatively, the monomeric subunit protein is a monomeric ferritin subunit; alternatively, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, algal ferritin, insect ferritin, fungal ferritin or mammalian ferritin; alternatively, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit; alternatively, the monomeric ferritin subunit comprises the amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:

10.

18. The coronavirus multivalent vaccine according to claim 13, wherein The second fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 51-56, 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: 51-56, 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: 51-56.

19. The coronavirus multivalent vaccine according to claim 13, wherein The first fusion protein comprises the sequence shown in SEQ ID NO: 14, 15 or 17, and the second fusion protein comprises the sequence shown in SEQ ID NO: 53, 54 or 56.

20. The coronavirus multivalent vaccine according to claim 13, wherein The first fusion protein comprises the sequence shown in SEQ ID NO: 17, and the second fusion protein comprises the sequence shown in SEQ ID NO:

56.

21. The multivalent coronavirus vaccine according to any one of claims 13 to 20, wherein The mass ratio of the first fusion protein to the second fusion protein is (1-5):(1-5), or the mass ratio is (1-3):(1-3), or the mass ratio is (1-2):(1-2), or the mass ratio is 1:(1-2), or the mass ratio is (1-2):1, or the mass ratio is 1:

1.

22. Use of the fusion protein according to any one of claims 5 to 9 or the Spike protein nanoparticles according to claim 11 in the preparation of a vaccine for preventing or treating 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; or, the coronavirus infection is infection with SARS-CoV-2 Infection with Omicron variants BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7, XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1, or XBB.1.

16.

23. A coronavirus vaccine formulation comprising a fusion protein and one or more of a buffer, a stabilizer, an alkali metal or an alkali metal salt, and a surfactant; The fusion protein comprises a coronavirus spike protein extracellular domain or a truncated fragment thereof containing a mutation and a monomeric ferritin subunit connected by a linker, wherein the mutation comprises: 1) RRAR is mutated to GSAS; 2) a double mutation K986P / V987P exists in the turning region between HR1 and CH, and the coronavirus is 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 coronavirus is SARS-CoV-2 Omicron variant BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7, XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.

16.

24. The coronavirus vaccine formulation of claim 23, wherein the fusion protein concentration is 0.05-5 mg / mL; or, the fusion protein concentration is 0.05-1 mg / mL.

25. The coronavirus vaccine formulation of claim 23 or 24, comprising 0.05-1 mg / mL fusion protein, 10-30 mM buffer, stabilizer, alkali metal or alkali metal salt, and surfactant.

26. The coronavirus vaccine formulation of any one of claims 23-25, wherein the buffer is selected from the group consisting of: a histidine buffer, a citric acid buffer, a phosphate buffer, or a combination thereof; or, the buffer is a histidine buffer; or, the buffer is a combination of histidine and citric acid buffer.

27. The coronavirus vaccine formulation of any one of claims 23-26, wherein the stabilizer is selected from hydroxypropyl beta-cyclodextrin, sucrose, trehalose, or a combination thereof.

28. The coronavirus vaccine formulation of any one of claims 23-27, wherein the alkali metal or alkali metal salt is selected from magnesium chloride, potassium chloride, sodium chloride, or a combination thereof; or, the alkali metal or alkali metal salt is sodium chloride.

29. The coronavirus vaccine formulation of any one of claims 23-28, wherein the surfactant is selected from poloxamer, polysorbate, or a combination thereof; or, the surfactant is selected from poloxamer 188, polysorbate 20, polysorbate 80, or a combination thereof.

30. The coronavirus vaccine formulation according to any one of claims 23 to 29, further comprising an adjuvant; or, the adjuvant is an aluminum adjuvant, a SWE adjuvant or a MF59 adjuvant; or, the amount of the adjuvant added is 1 / 10-5 / 10 of the total volume of the coronavirus vaccine formulation, or, the amount of the adjuvant added is 3 / 10 of the total volume of the coronavirus vaccine formulation.

31. The coronavirus vaccine formulation of any one of claims 23-30, wherein the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

32. A coronavirus vaccine preparation comprising (1) 0.05-1 mg / mL fusion protein, 5-25 mM phosphate buffer, 10-170 mg / mL trehalose dihydrate compound, 0-22 mg / mL alkali metal or alkali metal salt, 0.01-2 mg / mL polysorbate 80, pH about 6.0-8.0; or, (2) 0.05-1 mg / mL fusion protein, 5-25 mM histidine buffer, 14-450 mM trehalose, 0.01-2 mg / mL polysorbate 80, pH 5.0-7.0; or (3) 0.05-1 mg / mL fusion protein, 15-25 mM histidine buffer, 150-270 mM trehalose, 0.01-2 mg / mL polysorbate 80, 8-17 mg / mL sodium chloride, pH 5.0-7.0; or (4) 0.05-1 mg / mL fusion protein, 15-25 mM histidine buffer, 0.5-80 mg / mL hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL polysorbate 80, 8-33 mg / mL sodium chloride, pH 5.0-7.0; or (5) 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, about 222 mM trehalose, about 0.4 mg / mL polysorbate 80, 8-17 mg / mL sodium chloride, pH 5.6-6.3; or, (6) 0.05-1 mg / mL fusion protein, about 20 mM histidine buffer, 0.5-50 mg / mL hydroxypropyl beta-cyclodextrin, about 0.4 mg / mL polysorbate 80, 8-33 mg / mL sodium chloride, pH 5.6-6.3; or (7) 0.05-1 mg / mL fusion protein, 5-15 mM histidine buffer and 2-10 mM citric acid buffer, 0.5-50 mg / mL hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL polysorbate 80, 8-17 mg / mL sodium chloride, pH approximately 5.6-6.3; or (8) 0.05-1 mg / mL fusion protein, 10 mM histidine buffer and 5 mM citric acid buffer, 0.5-50 mg / mL hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL polysorbate 80, 8-17 mg / mL sodium chloride, pH about 5.6-6.3; or, (9) 0.05-1 mg / mL fusion protein, 5-25 mM histidine buffer, 60-100 mg / mL sucrose, 0.01-2 mg / mL polysorbate 80, 8-17 mg / mL sodium chloride, pH approximately 5.6-6.3; or, (10) 0.05-1 mg / mL fusion protein, 20 mM histidine buffer, 80 mg / mL sucrose, 0.4 mg / mL polysorbate 80, 8-17 mg / mL sodium chloride, pH approximately 5.6-6.3; in, The fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.

33. A coronavirus multivalent vaccine formulation, comprising: a first fusion protein and a second fusion protein, and further comprising one or more of a buffer, a stabilizer, an alkali metal or an alkali metal salt, and a surfactant, wherein the first fusion protein comprises the extracellular domain of the SARS-CoV-2 Omicron variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker, and the second fusion protein comprises the extracellular domain of the SARS-CoV-2 Delta variant Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker.

34. A coronavirus multivalent vaccine formulation as described in claim 33, wherein the mass ratio of the first fusion protein to the second fusion protein is (1-3):(1-3), or the mass ratio is (1-2):(1-2), or the mass ratio is 1:(1-2), or the mass ratio is (1-2):1, or 1:1; or, the coronavirus multivalent vaccine formulation comprises 0.01-2 mg / mL of the first fusion protein and 0.01-2 mg / mL of the second fusion protein.

35. The coronavirus multivalent vaccine formulation of claim 33 or 34, wherein the buffer is selected from: a histidine buffer, a citric acid buffer, or a combination thereof; or, the buffer is a histidine buffer, or a combination of histidine and citric acid buffer.

36. The coronavirus multivalent vaccine formulation of any one of claims 33-35, wherein the stabilizer is selected from hydroxypropyl beta-cyclodextrin, sucrose, trehalose, or a combination thereof.

37. The coronavirus multivalent vaccine formulation of any one of claims 33 to 36, wherein the alkali metal or alkali metal salt is selected from magnesium chloride, potassium chloride, sodium chloride, or a combination thereof; or the alkali metal or alkali metal salt is sodium chloride.

38. According to any one of claims 33-37, the coronavirus multivalent vaccine formulation, wherein the surfactant is selected from poloxamer, polysorbate or a combination thereof; or, the surfactant is selected from poloxamer 188, polysorbate 20, polysorbate 80 or a combination thereof.

39. A coronavirus multivalent vaccine formulation as described in any one of claims 33 to 38, comprising (1) 0.01-2 mg / mL of the first fusion protein and 0.01-2 mg / mL of the second fusion protein, further comprising 5-15 mM histidine buffer and 2-10 mM citric acid buffer, 0.5-80 mg / mL of hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL of polysorbate 80, and 8-17 mg / mL of sodium chloride, with a pH of 5.0-7.0, or (2) 0.01-0.2 mg / mL of the first fusion protein and 0.01-0.2 mg / mL of the second fusion protein, further comprising about 10 mM histidine buffer and about 5 mM citric acid buffer, 0.5-20 mg / mL of hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL of polysorbate 80, and 8-17 mg / mL of sodium chloride, with a pH of 5.6-6.3; or, (3) 0.01-0.2 mg / mL of the first fusion protein and 0.01-0.2 mg / mL of the second fusion protein, further comprising about 10 mM histidine buffer and about 5 mM citric acid buffer, about 4 mg / mL of hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL of polysorbate 80, about 8.26 mg / mL of sodium chloride, with a pH of 5.6-6.3; or, (4) 0.01-2 mg / mL of the first fusion protein and 0.01-2 mg / mL of the second fusion protein, further comprising 5-15 mM histidine buffer, 0.5-80 mg / mL of hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL of polysorbate 80, and 8-17 mg / mL of sodium chloride, with a pH of 5.0-7.0; or (5) 0.01-0.2 mg / mL of the first fusion protein and 0.01-0.2 mg / mL of the second fusion protein, further comprising about 10 mM histidine buffer, 0.5-20 mg / mL of hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL of polysorbate 80, and 8-17 mg / mL of sodium chloride, with a pH of 5.6-6.3; or (6) 0.01-0.2 mg / mL of the first fusion protein and 0.01-0.2 mg / mL of the second fusion protein, further comprising about 10 mM histidine buffer, about 4 mg / mL of hydroxypropyl beta-cyclodextrin, 0.01-2 mg / mL of polysorbate 80, and about 8.26 mg / mL of sodium chloride, with a pH of 5.6-6.3; in, The first fusion protein comprises the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises the sequence shown in SEQ ID NO:

56.

40. The coronavirus multivalent vaccine formulation according to any one of claims 33 to 39, further comprising an adjuvant; or, the adjuvant is an aluminum adjuvant, a SWE adjuvant or a MF59 adjuvant; or, the amount of the adjuvant added is 1 / 10-5 / 10 of the total volume of the coronavirus multivalent vaccine formulation, or, the amount of the adjuvant added is about 3 / 10 of the total volume of the coronavirus multivalent vaccine formulation.

41. A coronavirus multivalent vaccine comprising About 0.08 mg / mL of the first fusion protein and about 0.08 mg / mL of the second fusion protein, further comprising about 10 mM histidine buffer, about 4 mg / mL of hydroxypropyl beta-cyclodextrin, about 0.2 mg / mL of polysorbate 80, about 8.26 mg / mL of sodium chloride, and an adjuvant, with a pH of 5.6-6.3; or, About 0.04 mg / mL of the first fusion protein and about 0.04 mg / mL of the second fusion protein, further comprising about 10 mM histidine buffer, about 4 mg / mL of hydroxypropyl beta-cyclodextrin, about 0.2 mg / mL of polysorbate 80, about 8.26 mg / mL of sodium chloride, and an adjuvant, with a pH of 5.6-6.3, in, The first fusion protein comprises the sequence shown in SEQ ID NO: 17, and the second fusion protein comprises the sequence shown in SEQ ID NO: 56 or 61; The adjuvant is an aluminum adjuvant, a SWE adjuvant or a MF59 adjuvant; or, the amount of the adjuvant added is 1 / 10-5 / 10 of the total volume of the coronavirus multivalent vaccine, or, the amount of the adjuvant added is 3 / 10 of the total volume of the coronavirus multivalent vaccine.

42. A method for preventing or treating coronavirus infection, characterized in that: The invention comprises administering to a patient in need thereof an effective amount of the fusion protein according to any one of claims 5 to 9, the Spike protein nanoparticle according to claim 11, the coronavirus vaccine according to claim 12, the coronavirus multivalent vaccine according to any one of claims 13 to 21, the coronavirus vaccine preparation according to any one of claims 23 to 32, or the coronavirus multivalent vaccine preparation according to any one of claims 33 to 41; or, the coronavirus infection is SARS-CoV-2, SARS-CoV or MERS-CoV infection; or, the coronavirus infection is SARS-CoV-2 original strain or variant thereof; or, the coronavirus infection is 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 infection; or, the coronavirus infection is SARS-CoV-2Omicron variant BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7, XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.16 infection.

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