WO2024061188A1 - Coronavirus multivalent vaccine and use thereof - Google Patents

Abstract

The present invention provides a coronavirus multivalent vaccine and use thereof. The coronavirus multivalent vaccine comprises a coronavirus Spike protein extracellular domain containing a mutation, a truncated fragment thereof, or a fusion protein comprising same. The present invention further provides use of the coronavirus multivalent vaccine in the preparation of a drug for preventing or treating coronavirus infection.

Description

A coronavirus multivalent vaccine and its application Technical field

The invention belongs to the field of biotechnology, and particularly relates to coronavirus multivalent vaccines and their applications.

Background technique

Coronavirus is a non-segmented single-stranded positive-strand RNA virus. According to the serotype and genome characteristics, the coronavirus subfamily is divided into four genera: α, β, γ and δ. Because the virus envelope has ridges that extend to all sides. It is named after its protrusions and shape like a corolla. The new coronavirus (SARS-CoV-2 or 2019-nCoV) belongs to the genus β and is enveloped. The particles are round or oval, often pleomorphic, and have a diameter of 60-140nm. Current research shows that SARS-CoV-2 and SARS-CoV are highly homologous.

The novel coronavirus infection COVID-19 is mainly transmitted through the respiratory tract, and it may also be transmitted through contact. The population is generally susceptible, and the elderly and those with underlying diseases will become more seriously ill after infection. Children and infants are also affected. The main clinical symptoms of infected people are fever, fatigue, and dry cough, while upper respiratory tract symptoms such as nasal congestion and runny nose are rare. In the early stage of the disease, the total number of white blood cells in patients is normal or reduced, or the number of lymphocytes is reduced. Some patients have increased liver enzymes, muscle enzymes and myoglobin. Chest imaging showed that the patient showed multiple small patchy shadows and interstitial changes in the early stage, which were obvious in the outer lungs; and then developed into multiple ground-glass shadows and infiltrates in both lungs. In severe cases, lung consolidation may occur, and dyspnea gradually develops. In severe cases, Patients develop acute respiratory distress syndrome (ARDS), shock, and various tissue injuries and dysfunctions in lung tissue, heart, and kidneys. Most patients with mild infections have a good prognosis, but patients with severe infections often become critically ill or even die.

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

Contents of the invention

The invention provides a coronavirus multivalent vaccine. In some embodiments, the coronavirus multivalent vaccine comprises a mutant coronavirus Spike protein extracellular domain or a truncated fragment thereof. In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising a mutated extracellular domain of the coronavirus Spike protein or a truncated fragment thereof. The coronavirus multivalent vaccine of the present invention can induce a stronger neutralizing antibody response to coronavirus.

Viral particles first interact with 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. combine. When 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 serosa or endosome membrane. Heptapeptide repeat sequence 1 (HR1) and heptapeptide repeat sequence 2 (HR2) in the S2 subunit interact with each other to form a six-helix bundle (6-HB) fusion core, leading to the fusion of the viral shell and the cell membrane, SARS-CoV or SARS- CoV-2 enters cells and uses cells to synthesize new virus particles; the new virus particles are released outside the cells and then use the same method to infect surrounding normal cells.

In some embodiments, the coronavirus Spike protein extracellular domain containing mutations or a truncated fragment thereof, the mutations comprise: 1) mutating RRAR to GSAS; 2) between HR1 and the central helical region (CH) There are mutations in the turning region between HR1 and CH that prevent HR1 and CH from forming a straight helix during fusion.

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

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

In some embodiments, the truncated fragment of the extracellular domain of the coronavirus Spike protein containing mutations has a C-terminal truncation of 5-80 amino acid residues compared to the full-length extracellular domain of the coronavirus Spike protein. base. In some embodiments, the truncated fragment of the extracellular domain of the coronavirus Spike protein containing mutations has a C-terminal truncation of 20-76 amino acid residues compared to the full-length extracellular domain of the coronavirus Spike protein. base. In some embodiments, the truncated fragment of the extracellular domain of the coronavirus Spike protein containing mutations has a C-terminal truncation of 70 amino acid residues compared to the full-length extracellular domain of the coronavirus Spike protein.

In some embodiments, the coronavirus Spike protein extracellular domain or a truncated fragment thereof is from SARS-CoV-2, SARS-CoV or MERS-CoV. In some embodiments, the extracellular domain of the coronavirus Spike protein or a truncated fragment thereof is from an original strain of SARS-CoV-2 or a mutant strain thereof. In some embodiments, the coronavirus Spike protein extracellular domain or a truncated fragment thereof is from an original strain of SARS-CoV-2, a SARS-CoV-2 Alpha variant, a SARS-CoV-2 Beta variant, or a 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 strain.

In some embodiments, the mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises any of SEQ ID NOs: 3, 4, 6, 7, 9-12, 32-35, 78-83. The amino acid sequence shown in one item, or compared with the amino acid sequence shown in any one of SEQ ID NO: 3, 4, 6, 7, 9-12, 32-35, 78-83, it has at least 80% or at least 90% % identity of the amino acid sequence, or one or more conservative amino acid substitutions compared to the amino acid sequence shown in any of SEQ ID NO: 3, 4, 6, 7, 9-12, 32-35, 78-83 amino acid sequence.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated coronavirus Spike protein extracellular domain connected through a linker or Its truncated fragments and monomeric subunit proteins. 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 ( _GmS ) _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 linker has the sequence (GGGGS) _n and n is 1, 2, 3, 4, or 5. In some embodiments, the linker is GGGGS. In some embodiments, the linker is (GGGGS) ₂ . In some embodiments, the linker is (GGGGS) ₃ . In some embodiments, the linker is (GGGGS) ₄ . In some embodiments, the linker is (GGGGS) ₅ . In some embodiments, the monomeric subunit protein is a self-assembled monomeric subunit protein. In some embodiments, the monomeric subunit protein is a monomeric ferritin subunit. In some embodiments, the monomeric ferritin subunit is selected from the group consisting of bacterial ferritin, plant ferritin, phycoferritin, insect ferritin, fungal ferritin, and mammalian ferritin. In some embodiments, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit. In some embodiments, the N19Q mutation is present in the amino acid sequence of the H. pylori non-heme monomeric ferritin subunit. In some embodiments, the monomeric ferritin subunit comprises the amino acid sequence set forth in SEQ ID NO: 14, or is at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14. A conservative amino acid sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 14.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof connected through a linker and Monomeric ferritin subunit, the mutations include: 1) mutating RRAR to GSAS; 2) having a mutation in the turning region between HR1 and CH that prevents the formation of a straight helix during fusion. In some embodiments, the fusion protein comprising a mutated extracellular domain of the coronavirus Spike protein or a truncated fragment thereof is a C-terminus of the mutated extracellular domain of the coronavirus Spike protein or a truncated fragment thereof. The linker is attached to the N-terminus of the monomeric subunit protein. In some embodiments, the fusion protein comprising a mutated extracellular domain of the coronavirus Spike protein or a truncated fragment thereof is a C-terminus of the mutated extracellular domain of the coronavirus Spike protein or a truncated fragment thereof. The linker is attached to the N-terminus of the monomeric ferritin subunit.

In some embodiments, the fusion protein comprising a mutated extracellular domain of the coronavirus Spike protein or a truncated fragment thereof 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 has at least 80% or at least 90% difference compared to the amino acid sequence set forth in SEQ ID NO: 2 or 5. % identical amino acid sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 2 or 5.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated SARS-CoV-2 original strain Spike protein extracellular domain connected through a linker. or its truncated fragments and monomeric subunit proteins. In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises a mutated SARS-CoV-2 original strain Spike protein extracellular domain connected through a linker or Its truncated fragments and monomeric ferritin subunits. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated extracellular domain of the coronavirus Spike protein or a truncated fragment thereof comprises a mutated extracellular structure of the SARS-CoV-2 Alpha variant Spike protein connected through a linker. domains or truncated fragments thereof and monomeric subunit proteins. In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises a mutated SARS-CoV-2 Alpha variant Spike protein extracellular domain connected through a linker. or truncated fragments and monomeric ferritin subunits thereof. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated extracellular domain of the coronavirus Spike protein or a truncated fragment thereof includes a mutated extracellular structure of the SARS-CoV-2 Beta variant Spike protein connected through a linker. domains or truncated fragments thereof and monomeric subunit proteins. In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises a mutated SARS-CoV-2 Beta variant Spike protein extracellular domain connected through a linker. or truncated fragments and monomeric ferritin subunits thereof. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated SARS-CoV-2 Gamma variant Spike protein extracellular structure connected through a linker. domains or truncated fragments thereof and monomeric subunit proteins. In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises a mutated SARS-CoV-2 Gamma variant Spike protein extracellular domain connected through a linker. or truncated fragments and monomeric ferritin subunits thereof. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated extracellular domain of the coronavirus Spike protein or a truncated fragment thereof comprises a mutated extracellular structure of the SARS-CoV-2 Delta variant Spike protein connected through a linker. domains or truncated fragments thereof and monomeric subunit proteins. In some embodiments, the fusion protein comprising a mutated extracellular domain of the coronavirus Spike protein or a truncated fragment thereof comprises a mutated extracellular domain of the SARS-CoV-2 Delta variant Spike protein connected through a linker. or truncated fragments and monomeric ferritin subunits thereof. exist In some embodiments, the mutation includes: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turning region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated SARS-CoV-2 Kappa variant Spike protein extracellular structure connected through a linker. domains or truncated fragments thereof and monomeric subunit proteins. In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises a mutated SARS-CoV-2 Kappa variant Spike protein extracellular domain connected through a linker. or truncated fragments and monomeric ferritin subunits thereof. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated extracellular domain of the coronavirus Spike protein or a truncated fragment thereof comprises a mutated extracellular structure of the SARS-CoV-2 Epsilon variant Spike protein connected through a linker. domains or truncated fragments thereof and monomeric subunit proteins. In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises a mutated SARS-CoV-2 Epsilon variant Spike protein extracellular domain connected through a linker. or truncated fragments and monomeric ferritin subunits thereof. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated extracellular domain of the coronavirus Spike protein or a truncated fragment thereof comprises a mutated extracellular structure of the SARS-CoV-2 Lambda variant Spike protein connected through a linker. domains or truncated fragments thereof and monomeric subunit proteins. In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises a mutated SARS-CoV-2 Lambda variant Spike protein extracellular domain connected through a linker. or truncated fragments and monomeric ferritin subunits thereof. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated SARS-CoV-2 Omicron variant Spike protein extracellular structure connected through a linker. domains or truncated fragments thereof and monomeric subunit proteins. In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises a mutated SARS-CoV-2 Omicron variant Spike protein extracellular domain connected through a linker. or truncated fragments and monomeric ferritin subunits thereof. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises any of SEQ ID NOs: 16-23, 26-29, 41-44, 66, 67. The amino acid sequence shown in any one of SEQ ID NO: 16-23, 26-29, 41-44, 66, and 67. Compared with an amino acid sequence having at least 80% or at least 90% identity, or having one or more amino acid sequences compared to the amino acid sequence shown in any one of SEQ ID NO: 16-23, 26-29, 41-44, 66, 67 An amino acid sequence with conservative amino acid substitutions.

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

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

In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 22, 23 or 29 and a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 43, 44 or 67 fusion protein.

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

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

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

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

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

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof and connected thereto. Fc fragment of an immunoglobulin. In some embodiments, the Fc fragment of an immunoglobulin is from IgG, IgM, IgA, IgE, or IgD. In some embodiments, the Fc fragment of an immunoglobulin is from IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc fragment of an immunoglobulin is an Fc fragment of IgGl. In some embodiments, the Fc fragment of the immunoglobulin is an Fc fragment of human IgG1. In some embodiments, the Fc fragment of the immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO:38, or is at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO:38. A conservative amino acid sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 38.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof is obtained by combining the mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof. The C-terminus is connected to the N-terminus of the Fc fragment of the immunoglobulin.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated SARS-CoV-2 original strain Spike protein extracellular domain or a truncated fragment thereof. segment and the Fc fragment of the immunoglobulin linked thereto. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated SARS-CoV-2 Alpha variant Spike protein extracellular domain or a truncated fragment thereof. The short fragment and the Fc fragment of the immunoglobulin linked to it. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated SARS-CoV-2 Beta variant Spike protein extracellular domain or a truncated fragment thereof. The short fragment and the Fc fragment of the immunoglobulin linked to it. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated SARS-CoV-2 Gamma variant Spike protein extracellular domain or a truncated fragment thereof. The short fragment and the Fc fragment of the immunoglobulin linked to it. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated SARS-CoV-2 Delta variant Spike protein extracellular domain or a truncated fragment thereof. The short fragment and the Fc fragment of the immunoglobulin linked to it. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated SARS-CoV-2 Kappa variant Spike protein extracellular domain or a truncated fragment thereof. The short fragment and the Fc fragment of the immunoglobulin linked to it. In some embodiments, the mutations comprise: 1) Mute RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turning region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated SARS-CoV-2 Epsilon variant Spike protein extracellular domain or a truncated fragment thereof. The short fragment and the Fc fragment of the immunoglobulin linked to it. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutant coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises a mutant SARS-CoV-2 Lambda variant Spike protein extracellular domain or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto. In some embodiments, the mutation comprises: 1) RRAR is mutated to GSAS; 2) a double mutation K986P/V987P exists in the turning region between HR1 and CH.

In some embodiments, the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof includes a mutated SARS-CoV-2 Omicron variant Spike protein extracellular domain or a truncated fragment thereof. The short fragment and the Fc fragment of the immunoglobulin linked to it. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.

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

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

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

In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 53, 54 or 72 and a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 61, 62 or 76 fusion protein.

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

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

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

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

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

In some embodiments, the coronavirus multivalent vaccine further comprises a conserved fragment of the coronavirus Spike protein or a fusion protein comprising the same. In some embodiments, the conserved fragment of the coronavirus Spike protein is from SARS-CoV-2, SARS-CoV or MERS-CoV. In some embodiments, the conserved fragment of the coronavirus Spike protein is from the original strain of SARS-CoV-2 or a variant thereof. In some embodiments, the conserved fragment of the coronavirus Spike protein is from the original strain of SARS-CoV-2, SARS-CoV-2 Alpha variant, SARS-CoV-2Beta 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 fusion protein comprising a conserved fragment of the coronavirus Spike 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 has at least 80% or at least 90% difference compared to the amino acid sequence set forth in SEQ ID NO: 2 or 5. % identity of the amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 2 or 5.

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein includes a conserved fragment of the coronavirus Spike protein and a monomeric subunit protein connected through a linker. In some embodiments, The connector is a GS connector. 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 ( _GmS ) _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 linker has the sequence (GGGGS) _n and n is 1, 2, 3, 4, or 5. In some embodiments, the linker is GGGGS. In some embodiments, the linker is (GGGGS) ₂ . In some embodiments, the linker is (GGGGS) ₃ . In some embodiments, the linker is (GGGGS) ₄ . In some embodiments, the linker is (GGGGS) ₅ . In some embodiments, the monomeric subunit protein is a self-assembled monomeric subunit protein. In some embodiments, the monomeric subunit protein is a monomeric ferritin subunit. In some embodiments, the monomeric ferritin subunit is selected from the group consisting of bacterial ferritin, plant ferritin, phycoferritin, insect ferritin, fungal ferritin, and mammalian ferritin. In some embodiments, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit. In some embodiments, the N19Q mutation is present in the amino acid sequence of the H. pylori non-heme monomeric ferritin subunit. In some embodiments, the monomeric ferritin subunit comprises the amino acid sequence set forth in SEQ ID NO: 14, or is at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14. A conservative amino acid sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 14.

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein is a fusion protein in which the C-terminus of the conserved fragment of the coronavirus Spike protein is connected to the N-terminus of the monomeric subunit protein through a linker.

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

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

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

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein includes a conserved fragment of the SARS-CoV-2 Gamma variant Spike protein and a monomeric subunit protein connected through a linker. In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein includes a conserved fragment of the SARS-CoV-2 Gamma variant Spike protein and a monomeric ferritin subunit connected through a linker.

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

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

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

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

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

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 45-46 and 68, or is identical to any one of SEQ ID NOs: 45-46 and 68. The amino acid sequence shown in one item has at least 80% or at least 90% identity compared to the amino acid sequence, or has one or more conservations compared to the amino acid sequence shown in any one of SEQ ID NO: 45-46 and 68. Amino acid sequence of amino acid substitutions.

In some embodiments, the fusion protein comprising a conserved fragment of coronavirus Spike protein includes a conserved fragment of coronavirus Spike protein and an Fc fragment of an immunoglobulin connected thereto. In some embodiments, the Fc fragment of an immunoglobulin is from IgG, IgM, IgA, IgE, or IgD. In some embodiments, the Fc fragment of an immunoglobulin is from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc fragment of an immunoglobulin is an Fc fragment of IgGl. In some embodiments, the Fc fragment of the immunoglobulin is an Fc fragment of human IgG1. In some embodiments, the Fc fragment of the immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO:38, or has an amino acid sequence set forth in SEQ ID NO:38 that is at least 80% Or an amino acid sequence that is at least 90% identical, or has one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 38.

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein is a fusion protein that connects the C-terminus of the conserved fragment of the coronavirus Spike protein to the N-terminus of the Fc fragment of an immunoglobulin.

In some embodiments, the fusion protein comprising a conserved fragment of coronavirus Spike protein includes a conserved fragment of SARS-CoV-2 original strain Spike protein and an Fc fragment of an immunoglobulin connected thereto.

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein includes a conserved fragment of the SARS-CoV-2 Alpha variant Spike protein and an Fc fragment of an immunoglobulin connected thereto.

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein includes a conserved fragment of the SARS-CoV-2 Beta variant Spike protein and an Fc fragment of an immunoglobulin connected thereto.

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein includes a conserved fragment of the SARS-CoV-2 Gamma variant Spike protein and an Fc fragment of an immunoglobulin connected thereto.

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein includes a conserved fragment of the SARS-CoV-2 Delta variant Spike protein and an Fc fragment of an immunoglobulin connected thereto.

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein includes a conserved fragment of the SARS-CoV-2 Kappa variant Spike protein and an Fc fragment of an immunoglobulin connected thereto.

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein includes a conserved fragment of the SARS-CoV-2 Epsilon variant Spike protein and an Fc fragment of an immunoglobulin connected thereto.

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein includes a conserved fragment of the SARS-CoV-2 Lambda variant Spike protein and an Fc fragment of an immunoglobulin connected thereto.

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein includes a conserved fragment of the SARS-CoV-2 Omicron variant Spike protein and an Fc fragment of an immunoglobulin connected thereto.

In some embodiments, the fusion protein comprising a conserved fragment of the coronavirus Spike protein includes an amino acid sequence as shown in any one of SEQ ID NO: 63, 64, and 77, or is identical to any of SEQ ID NO: 63, 64, and 77. The amino acid sequence shown in one item has at least 80% or at least 90% identity compared to the amino acid sequence, or has one or more conservations compared to the amino acid sequence shown in any one of SEQ ID NO: 63, 64, and 77. Amino acid sequence of amino acid substitutions.

In some embodiments, the coronavirus multivalent vaccine comprises at least one fusion protein comprising the amino acid sequence shown in any one of SEQ ID NO: 16-23, 26-29, 41-44, 66, 67, It also contains a conserved fragment of the coronavirus Spike protein or a fusion protein containing it.

In some embodiments, the coronavirus multivalent vaccine comprises: (1) at least one amino acid sequence comprising any one of SEQ ID NO: 16-23, 26-29, 41-44, 66, 67 A fusion protein, and (2) a fusion protein comprising the amino acid sequence shown in any one of SEQ ID NOs: 45-46, 63, 64, 68 and 77.

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

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

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

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

In some embodiments, the coronavirus multivalent vaccine comprises at least one fusion protein comprising the amino acid sequence shown in any one of SEQ ID NOs: 47-54, 59-62, 69-72, 75-76, It also contains a conserved fragment of the coronavirus Spike protein or a fusion protein containing it.

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

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

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

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

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

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

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

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% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, 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 these values The range between (inclusive) any two values in , or any value within it.

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, inclusive of the endpoints ) or any value therein.

In some embodiments, due to the interaction between the antigen (e.g., a mutant coronavirus Spike protein extracellular domain or a truncated fragment thereof or a conserved fragment of the coronavirus Spike protein) and a self-assembled monomeric subunit protein (e.g., monomeric iron protein subunits), will produce nanoparticles displaying antigens on their surface.

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

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

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

The invention also provides host cells comprising the polynucleotide or expression vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is a CHO cell, HEK293 cell, Cos1 cell, Cos7 cell, CV1 cell, or murine L cell.

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

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

The invention also provides a method of preventing or treating coronavirus infection, comprising administering to a patient in need thereof an effective amount of a coronavirus multivalent vaccine described herein.

In some embodiments, the coronavirus infection is an infection with the original strain of SARS-CoV-2 or a variant thereof. In some embodiments, the coronavirus infection is an original strain of SARS-CoV-2, a SARS-CoV-2 Alpha variant, a SARS-CoV-2 Beta variant, a SARS-CoV-2 Gamma variant, or a 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.

Description of the drawings

Figure 1 shows the binding curve of fusion protein and human ACE2; Figure 1a shows the binding curve of fusion protein D and human ACE2; Figure 1b shows the binding curve of fusion protein G and human ACE2.

Figure 2 shows the serum anti-Spike protein IgG titer. The bars represent the geometric mean (GMT) of the titer. Figure 2a, Figure 2c and Figure 2e show the titer 14 days after the first dose (day 14). , Figure 2b, Figure 2d and Figure 2f show the titer 14 days after the second dose (day 35).

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

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

Figure 5 shows the long-term anti-COVID-19 Spike protein antibody titer in mouse serum after immunization with the bivalent vaccine; the bar represents the geometric mean (GMT, the value is displayed in the box) of the titer; in the figure, 2W is after the second immunization The serum at the 2nd week, 30W is the serum at the 30th week after the second immunization; Wildtype, Delta, and BA.1 are WT-Spike-His, Delta-Spike-His, and Omicron-Spike-His respectively.

Figure 6 shows the serum anti-Spike protein IgG titer, and the bars represent the geometric mean (GMT) of the titer; Figure 6a, Figure 6c and Figure 6e show the titer 14 days after the first dose (day 14), Figure 6b, Figure 6d and Figure 6f show the titers 14 days after the second dose (day 35).

Figure 7 shows the antibody titer of serum inhibiting the binding of hACE2 to Spike protein after immunizing mice with the same dose of bivalent vaccine plus different doses of adjuvant; among them, the Spike protein in Figure 7a is WT-Spike-His, and the Spike protein in Figure 7b is Delta-Spike-His, and the Spike protein of Figure 7c is Omicron-Spike-His; the bar graph represents the geometric mean of IC ₅₀ (values are shown within the bar graph), and the error symbols represent the 95% confidence interval.

Figure 8 shows the anti-Spike protein IgG titer of the immune serum of K18-hACE2 transgenic mice. The bar represents the geometric mean (GMT) of the titer. In the figure, low, medium and high are the low-dose group, the medium-dose group and the high-dose group respectively. Dosage groups, wildtype, Delta, and Omicron are WT-Spike-His, Delta-Spike-His, and Omicron-Spike-His respectively.

Figure 9 shows the lung viable virus titers of challenge mice.

Figure 10 shows the inhibition rate (%) of serum against the new coronavirus Omicron BA.1.1.

the term

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

definition

Unless otherwise defined, all technical and scientific terms used herein have common meaning in the art to which this invention belongs. The same meaning as commonly understood by technical personnel.

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, nucleic acid molecule refers to one or more nucleic acid molecules. Accordingly, the terms "a", "an", "one or more" and "at least one" may be used interchangeably. Similarly, the terms "comprising", "including" and "having" may be used interchangeably and should generally be understood to be open-ended and non-limiting, e.g. not excluding other unrecited elements or steps.

The term "amino acid" refers to organic compounds containing both amino and carboxyl groups, such as alpha-amino acids, which may be encoded by nucleic acids directly or in the form of precursors. 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 the "degeneracy of the genetic code." Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), Glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine Acid (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

A "conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue containing a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). Generally speaking, conservative amino acid substitutions are unlikely to materially alter the functional properties of the protein. Examples of amino acid classes containing chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine. amino acid; 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) Basic side chains: lysine, Arginine and histidine; 6) Acidic side chains: aspartic acid and glutamic acid.

The term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptide" 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 chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain" or any other term used to refer to two or more amino acid chains, and the term "polypeptide" may Used instead of or interchangeably with any of the above terms. The term "polypeptide" is also intended to refer to the product of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-natural Amino acid modifications that occur. A polypeptide may be derived from natural biological sources or produced by recombinant techniques, but it does not have to be translated from a specified nucleic acid sequence and may be produced by any means including chemical synthesis.

Unless otherwise stated, a fusion protein is a recombinant protein that contains amino acid sequences from at least two unrelated proteins that have been linked together by peptide bonds to form a single protein. unrelated protein The amino acid sequences may be directly linked to each other, or they may be linked using a linker. As used herein, proteins are not related if their amino acid sequences are not normally linked together via peptide bonds in their natural environment (eg, within a cell). For example, the amino acid sequence of a common bacterial enzyme such as Bacillus stearothermophilus dihydrolipoate transacetylase (E2p) and the amino acid sequence of the coronavirus Spike protein are not linked together by peptide bonds.

The terms "homology," "identity" or "similarity" refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions within each sequence that can be aligned. 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 sequences is a function of the number of matches or homologous positions shared by the sequences.

The term "encoding" when applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide, which, 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.

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

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. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are examples of unrestricted 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. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If the modification is present, the structural modification of the nucleotides can be performed before or after assembling the polynucleotides. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotides 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.

A nucleic acid or polynucleotide sequence (or a polypeptide or protein sequence) is "identical" or "sequence identical" to another sequence by a certain percentage (eg, 90%, 95%, 98% or 99%). When sequences are aligned, this percentage of bases (or amino acids) in the two sequences being compared are identical. The alignment percent identity or sequence identity can be determined using visual inspection or software programs known in the art, such as those described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. It is preferred to use the default parameters for comparison. One of the comparison programs is BLAST using default parameters, such as BLASTN and BLASTP, which use the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions =50sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that share the percentage identity specified above and encode a polypeptide with the same or similar biological activity.

The term "isolated" used in the present invention with respect to cells, nucleic acids, polypeptides, antibodies, etc., such as "isolated" DNA, RNA, polypeptides, and antibodies, refers to other components in the natural environment of cells, such as DNA or RNA. one or more separated molecules. The term "isolated" as used herein also refers to nucleic acids or peptides that are substantially free of cellular material, viral material or cell culture media 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 native state and do not exist in their native state. The term "isolated" is also used herein to refer to cells or polypeptides separated from other cellular proteins or tissues. Isolated polypeptide is intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc. are generally prepared by at least one purification step. In some embodiments, the purity of the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or any of these values. The range between any two values (inclusive) or any value within them.

The term "recombinant" refers to a polypeptide or polynucleotide and means a form of the polypeptide or polynucleotide that does not occur in nature, and non-limiting examples may be combined to produce polynucleotides that do not normally exist or Peptides.

"Antibody" and "antigen-binding fragment" refer to polypeptides or polypeptide complexes that specifically recognize and bind to antigens. Antibodies can be complete antibodies, any antigen-binding fragments thereof, or single chains thereof. The term "antibody" thus includes any protein or peptide whose molecule contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen.

As used herein, the terms "antigen" or "immunogen" are used interchangeably and refer to a substance, typically a protein, 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 responses to the body fluids and/or Cell type immune response. Unless otherwise stated, the term "vaccine antigen" is used interchangeably with "protein antigen" or "antigenic polypeptide."

"Neutralizing antibodies" refer to antibodies that reduce the infectious titer of an infectious agent by binding to a specific antigen on that agent. In some embodiments, the infectious agent is a virus. A "broadly neutralizing antibody" is an antibody that binds to and inhibits the function of a related antigen, e.g., at least 85%, 90%, 95%, 96%, 97%, 98% identical to the antigenic surface of the antigen % or 99% identity to the antigen. For antigens from pathogens, such as viruses, the antibodies can bind to and inhibit the function of more than one class and/or subclass of antigens from the pathogen.

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

"Epitope" refers to an antigenic determinant. These are specific chemical groups or peptide sequences on molecules that are antigenic such that they elicit a specific immune response, for example, an epitope is an antigenic region to which B and/or T cells respond. Epitopes can be formed from contiguous amino acids, or from non-contiguous amino acids juxtaposed by the tertiary folding of the protein.

Vaccine refers to a biological product that induces a preventive or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, vaccines elicit an antigen-specific immune response against the antigens of pathogens, such as viral pathogens, or cellular components associated with pathological conditions. Vaccines may include polynucleotides (eg, nucleic acids encoding known antigens), peptides or polypeptides (eg, disclosed antigens), viruses, cells, or one or more cellular components. In some embodiments, a vaccine or vaccine antigen or vaccine composition is expressed from a fusion protein expression vector and self-assembles into nanoparticles displaying the antigenic polypeptide or protein on the surface.

The term "multivalent vaccine" will be recognized and understood by those of ordinary skill in the art, and for example means a fusion protein containing antigens from different pathogens (such as Spike proteins from different SARS-CoV-2 coronavirus sources) or a fusion protein thereof. A nucleic acid sequence or a construct thereof, or a fusion protein containing different antigens from the same pathogen or a nucleic acid sequence thereof or a construct thereof. In the context of the present invention, a coronavirus multivalent vaccine may be one that contains antigens from more than two different SARS-CoV-2 coronaviruses (such as Spike proteins from different SARS-CoV-2 coronavirus sources) or a fusion thereof. Proteins or nucleic acid sequences or constructs thereof, or fusion proteins containing different antigens from the same SARS-CoV-2 coronavirus or nucleic acid sequences or constructs thereof.

An effective amount of a vaccine or other agent is one sufficient to produce a desired response, such as eliciting an immune response, preventing, alleviating, or eliminating signs or symptoms of a condition or disease (e.g., pneumonia). For example, this may be an amount necessary to inhibit viral replication or measurably alter the outward symptoms of a viral infection. Typically, this amount will be sufficient to measurably inhibit the replication or infectivity of the virus (eg, SARS-CoV-2). When administered to a subject, a dose that achieves target tissue concentrations that has been shown to achieve inhibition of viral replication in vitro will generally be used. In some embodiments, an "effective amount" is an amount that treats (including prevents) one or more symptoms and/or underlying causes of a condition or disease (eg, treating a coronavirus infection). In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount that prevents the development of one or more symptoms or signs of a particular disease or condition (eg, one or more symptoms or signs associated with a coronavirus infection).

Nanoparticles refer to spherical protein shells that are tens of nanometers in diameter and have well-defined surface geometry. The spherical protein shell is formed from identical copies of non-viral proteins that self-assemble into nanoparticles with a similar appearance to virus-like particles (VLPs). Examples include ferritin (FR), which is conserved across multiple species and forms a 24-mer, Bacillus stearothermophilus dihydrolipoic acid transacetylase (E2P), hyperthermophile dioxygenase Tetrahydropterin synthase (LS) and Thermotoga maritima encapsulin, all of which form a 60-mer. Self-assembling nanoparticles can form spontaneously after recombinantly expressing proteins in an appropriate expression system. Methods for the production, detection and characterization of nanoparticles can use the same techniques developed for VLPs.

Virus-like particles (VLPs) refer to non-replicating viral shells derived from any of a variety of viruses. VLP Typically includes one or more viral proteins, such as, but not limited to, those proteins known as capsid proteins, coat proteins, globin proteins, surface proteins and/or envelope proteins, or particles derived from these proteins. Peptides. In an appropriate expression system, VLPs can form spontaneously after recombinantly expressed proteins. Methods for producing specific VLPs are known in the art. The presence of VLPs following recombinantly expressed viral proteins can be detected using conventional techniques known in the art (eg, by electron microscopy, biophysical characterization, etc.). For example, VLPs can be separated by density gradient centrifugation and/or identified by characteristic density bands. Alternatively, cryo-electron microscopy can be performed on vitrified water samples of the VLP preparation in question and images recorded under appropriate exposure conditions.

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 technical field. In some embodiments, reference herein to "about" refers to the recited value as well as the range of ±10%, ±5%, or ±1% thereof.

"ECMO" refers to Extracorporeal Membrane Oxygenation (ECMO), which is a medical emergency technical equipment mainly used to provide continuous extracorporeal breathing and circulation for patients with severe cardiopulmonary failure to maintain the patient's life.

"ICU" refers to the intensive care unit (Intensive Care Unit). Treatment, nursing, and rehabilitation can all be carried out simultaneously. It provides isolation places and equipment for critically ill or comatose patients, and provides the best care, comprehensive treatment, combination of medical and nursing care, and surgery. Early rehabilitation, joint care, sports therapy and other services.

"IMV" refers to intermittent mandatory ventilation, which implements periodic volume or pressure ventilation based on preset time intervals, that is, time triggers. This period allows the patient to breathe spontaneously at any set basal pressure level during mandatory ventilation. While breathing spontaneously, the patient can breathe on his own with continuous airflow support, or the machine will open the on-demand valve to allow for spontaneous breathing. Most ventilators can provide pressure support while breathing spontaneously.

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 stated, the terms "patient" or "subject" are used interchangeably herein. Preferably, the subject is human.

"Treatment" means therapeutic treatment and prophylactic or preventative measures designed to prevent, slow down, ameliorate or halt adverse physiological changes or disorders, such as the progression of a disease, including but not limited to the following whether detectable or undetectable The results include alleviation of symptoms, reduction in disease severity, stabilization of disease status (i.e. no worsening), delay or slowdown of disease progression, improvement, alleviation, reduction or disappearance of disease status (whether partial or complete), prolongation and Expected survival without treatment, etc. Patients in need of treatment include patients who already have a condition or disorder, are susceptible to a condition or disorder, or are in need of prevention of a condition or disorder that may or are expected to result from administration of the Spike protein nanoparticles or pharmaceutical compositions disclosed herein. For patients who benefit from treatment.

Overview

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, S glycoprotein ( Spike protein) is responsible for binding to host receptors via the receptor binding domain (RBD) in its S1 subunit, and subsequent membrane fusion and viral entry driven by its S2 subunit. Receptor binding can help keep the RBD in the "standing" state, which facilitates the dissociation of the S1 subunit from the S2 subunit. When the S1 subunit dissociates from the S2 subunit, a second S2' cleavage releases the fusion peptide. The linker region, HR1, and CH form a very long helix 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.

RBD contains a core subdomain and a receptor binding motif (RBM). Although the core subdomains between 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 recognizes 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 for neutralizing antibodies (NAbs) following infection and is the focus of vaccine design. Spike trimers are extensively modified with N-linked glycans, which are important for correct folding and regulating accessibility to NAbs.

The present invention stabilizes the Spike trimer by 1) mutations that inactivate the S1/S2 cleavage site and 2) the presence of mutations in the turning region between HR1 and CH that prevent HR1 and CH from forming a straight helix during fusion. In a pre-fusion configuration with the host cell membrane. In some embodiments, mutant-containing coronavirus Spike protein extracellular domains or truncated fragments thereof can be displayed on nanoparticles.

According to the research and exemplary designs described herein, the present invention provides a coronavirus vaccine comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof or a fusion protein comprising the same. The present invention also provides a coronavirus multivalent vaccine comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof or a fusion protein comprising the same. The invention also provides related polynucleotides, expression vectors and pharmaceutical compositions. In some embodiments, stable Spike trimers and RBD proteins in protein or nucleic acid (DNA/mRNA) forms carried by viral vectors can be used as coronavirus vaccines. Additionally, nanoparticle-presented stable Spike trimers and RBDs can also be used as coronavirus vaccines.

The coronavirus Spike protein-based antigens and vaccines of the present invention have many advantageous properties. The Spike trimer design described herein presents conserved neutralizing epitopes in its native-like conformation, allowing the Spike trimer to be used as an antigen vaccine or for multivalent display on nanoparticles. The nanoparticle vaccine of the present invention allows the display of Spike trimers derived from different coronaviruses on well-known nanoparticles, such as ferritin, E2p and I3-01, with sizes ranging from 12.2 to 25.0 nm. All trimer-presenting nanoparticles can be produced in HEK293 cells, ExpiCHO cells, and CHO cells with high yields. The produced Spike protein nanoparticles can be purified by antibody and size exclusion chromatography (SEC).

Unless otherwise stated herein, the mutant coronavirus Spike protein extracellular domain or its truncated fragments, coronavirus Spike protein S1 subunit, coronavirus Spike protein conserved fragment, fusion protein, Spike protein nanoparticles, Coronavirus multivalent vaccines, encoded polynucleotides, expression vectors and host cells, and related therapeutic applications can all be produced or performed according to the methods exemplified herein or conventional methods well known in the art.

Unless otherwise stated, the order of steps or the order in which certain operations are performed is not important so long as the invention remains operable. Furthermore, two or more steps or operations can be performed simultaneously.

Unless otherwise stated, any and all examples used herein, or exemplary language (such as "such as" or "including") used herein, are intended merely to better illuminate the invention and do not pose a limitation on the scope of the invention. . No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Containing mutated extracellular domain of coronavirus Spike protein or truncated fragments thereof

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

As described herein, some mutant Spike proteins or truncated fragments thereof of the present invention contain mutations that can enhance the stability of the structure of the Spike protein or truncated fragment thereof before fusion with the cell membrane. These mutations include mutations that inactivate the S1/S2 cleavage site, and mutations in the turning region between HR1 and CH, which remove any strain in the turning region between HR1 and CH, i.e., prevent the formation of a straight helix.

Some mutant coronavirus Spike protein extracellular domains or truncated fragments thereof (as shown in SEQ ID NO: 3, 4, 6, 7, 9-12, 32-35, 78-83) are derived from the SARS-CoV-2 virus that causes COVID-19. These polypeptides contain mutations that inactivate the S1/S2 cleavage site and mutations in the turn region between HR1 and CH. As an example, the amino acid sequence of the mutated SARS-CoV-2 original strain Spike protein is shown in SEQ ID NO: 1 or as shown in residues 14-1213 of SEQ ID NO: 1 or as shown in residues 15-1213 of SEQ ID NO: 1. In some embodiments, the Spike protein used for mutation can be SEQ ID NO: 1, 8 or 31 or a variant thereof, such as a variant substantially identical thereto or a conservatively modified variant. Using the amino acid numbering based on the cryo-EM model PDB ID 6VSB or GenBank accession number MN908947.3 as a reference, the inactivation of the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ can be achieved by a number of sequence changes (e.g., deletions or substitutions) within or around the site. As exemplified herein, a mutation that inactivates the S1/S2 cleavage site without affecting the protein structure is to mutate the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ to ⁶⁸² GSAS ^685. In addition to inactivating the S1/S2 cleavage site, a double mutation can be performed in the turning region between HR1 and CH, which eliminates the strain in the turning region (between HR1 and CH motifs) during the fusion process by preventing the formation of a straight helix. In some embodiments, the double mutation may 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 fragment, 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 to illustrate, such a deletion may include a deletion of residues 1144-1213 of SEQ ID NO: 1. In some embodiments, the deletion can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 76, 80 or more residues at the C-terminus of the truncated Spike protein extracellular domain (e.g., SEQ ID NO: 1, 3, 4, 8-10, 31-33, 78, 80 or 82), or a range (including endpoints) 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 shown in SEQ ID NO: 2 or 5.

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

The full-length extracellular domain (ECD) of the original strain Spike protein of SARS-CoV-2, its amino acid sequence is shown in SEQ ID NO: 1, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) is marked in italics , S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is underlined, bolded and italicized.

The full-length extracellular domain a1 of the mutant SARS-CoV-2 original strain Spike protein has an amino acid sequence as shown in SEQ ID NO: 3. 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 ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and contains double Mutation K986P/V987P, underlined and italicized.

The full-length extracellular domain a2 of the mutant SARS-CoV-2 original strain Spike protein has an amino acid sequence as shown in SEQ ID NO: 4. In the sequence, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide is marked in italics, and the S1/S2 cleavage site ^{The 682} RRAR ⁶⁸⁵ mutation is ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.

The full-length extracellular domain a3 of the mutant SARS-CoV-2 original strain Spike protein has an amino acid sequence as shown in SEQ ID NO: 78. In the sequence, there is no signal peptide, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.

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

The C-terminal truncated fragment b2 of the extracellular domain of the spike protein of the original strain of SARS-CoV-2 is mutated, and its amino acid sequence is shown in SEQ ID NO: 7. In the sequence, 70 amino acid residues were truncated at the C terminus, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide Italicized, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.

The C-terminal truncated fragment b3 of the extracellular domain of the spike protein of the original strain of SARS-CoV-2 is mutated, and its amino acid sequence is shown in SEQ ID NO: 79. In the sequence, 70 amino acid residues are truncated at the C terminus, without the signal peptide, and the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and contains the double mutation K986P/V987P. , underlined and italicized.

The full-length extracellular domain (ECD) of SARS-CoV-2 Delta variant Spike protein, its amino acid sequence is shown in SEQ ID NO:8, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is in italics Marked, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is underlined, bolded and italicized.

The full-length extracellular domain c1 of the mutated SARS-CoV-2 Delta variant Spike protein, its amino acid sequence is shown in SEQ ID NO: 9. 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 ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and contains double Mutation K986P/V987P, underlined and italicized.

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

The full-length extracellular domain c3 of the mutated SARS-CoV-2 Delta variant Spike protein has an amino acid sequence as shown in SEQ ID NO: 80. In the sequence, there is no signal peptide, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.

The mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d1, its amino acid sequence is shown in SEQ ID NO: 11. In the sequence, 70 amino acid residues are truncated at the C terminus. The original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics. The S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ . It is underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.

The mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d2, its amino acid sequence is shown in SEQ ID NO: 12. In the sequence, 70 amino acid residues were truncated at the C terminus, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide Italicized, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.

The mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d3, its amino acid sequence is shown in SEQ ID NO: 81. In the sequence, 70 amino acid residues are truncated at the C terminus, without the signal peptide, and the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and contains the double mutation K986P/V987P. , underlined and italicized.

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

The full-length extracellular domain (ECD) of the Spike protein of the SARS-CoV-2 Omicron variant, its amino acid sequence is shown in SEQ ID NO: 31, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) is marked in italics Out, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is underlined, bolded and italicized.

The full-length extracellular domain f1 of the mutated SARS-CoV-2 Omicron variant Spike protein, its 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 ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and contains double Mutation K986P/V987P, underlined and italicized.

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

The full-length extracellular domain f3 of the mutated SARS-CoV-2 Omicron variant Spike protein has an amino acid sequence as shown in SEQ ID NO: 82. In the sequence, there is no signal peptide, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.

The mutated SARS-CoV-2 Omicron variant Spike protein extracellular domain C-terminal truncated fragment g1, its amino acid sequence is shown in SEQ ID NO: 34. In the sequence, 70 amino acid residues are truncated at the C terminus. The original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics. The S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ . It is underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.

The mutated SARS-CoV-2 Omicron variant Spike protein extracellular domain C-terminal truncated fragment g2, its 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 (shown in SEQ ID NO: 2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide Italicized, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.

The mutated SARS-CoV-2 Omicron variant Spike protein extracellular domain C-terminal truncated fragment g3, its amino acid sequence is shown in SEQ ID NO: 83. In the sequence, 70 amino acid residues are truncated at the C terminus, without the signal peptide, and the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and contains the double mutation K986P/V987P. , underlined and italicized.

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

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

fusion protein

The present invention provides fusion proteins comprising a heterologous scaffold displaying at least one antigenic polypeptide or trimeric protein derived from the coronavirus Spike protein. In some embodiments, the coronavirus antigen used is the extracellular domain of the coronavirus Spike protein containing various stable mutations described above or truncated fragments thereof. In some embodiments, the coronavirus antigen employed comprises or is derived from the RBD domain of the coronavirus Spike protein. In some embodiments, the coronavirus antigen employed comprises or is derived from the S1 subunit of the coronavirus Spike protein. In some embodiments, the coronavirus antigen employed comprises or is derived from a conserved fragment of the coronavirus Spike protein. In an exemplary embodiment, the Spike protein sequence used includes the sequence shown in any one of SEQ ID NO: 1, 3, 4, 6-13, 31-37, 78-83, or is substantially identical or conserved therewith. Modified variants. table After the expression vector of the fusion protein is transfected into the host cell, since the antigen (such as Spike protein) is connected to the self-assembly protein (such as monomeric ferritin subunit), a nanoparticle vaccine showing the antigen (such as Spike protein) on the surface will be produced.

Any heterologous scaffold can be used to present antigens in the construction of the vaccines of the invention. This includes virus-like particles (VLPs) such as nanoparticles. A variety of nanoparticles can be used to produce the vaccines of the invention. Typically, nanoparticles for use in the present invention need to be formed from multiple replicas of a single subunit. Nanoparticles are typically spherical, and/or have rotational symmetry (eg, having 3-fold and 5-fold axes), such as having an 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 trimer-stabilized Spike protein shown.

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

In some exemplary embodiments, fusion proteins of the invention comprise an Fc fragment (e.g., a human IgG Fc fragment). Usually, the C-terminus of the conserved sequence of the coronavirus Spike protein or the S1 subunit of the coronavirus Spike protein or the extracellular domain of the coronavirus Spike protein containing mutations or a truncated fragment thereof is fused to the N-terminus of the Fc fragment.

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

In some exemplary embodiments, the fusion protein of the invention comprises a nanoparticle subunit sequence (for example, Helicobacter pylori non-heme monomeric ferritin subunit, the amino acid sequence of which is shown in SEQ ID NO: 14), or its conserved Modified variants or sequences substantially identical thereto. Usually, the coronavirus Spike protein conserved sequence or coronavirus The C-terminus of the S1 subunit of the coronavirus Spike protein or the extracellular domain of the mutated coronavirus Spike protein or its truncated fragment is fused to the N-terminus of the self-assembled nanoparticle (NP) subunit. In some embodiments, the C-terminus of the conserved fragment of the coronavirus Spike protein or the S1 subunit of the coronavirus Spike protein or the extracellular domain of the coronavirus Spike protein containing mutations or a truncated fragment thereof is connected to the nanoparticle subunit via a GS linker. At the N-terminus, the linker is, for example, GGGGS or GGGSGGGGS.

The amino acid sequence of the non-heme monomeric ferritin subunit (Ferritin) of Helicobacter pylori is as follows:

By fusing subunits of the antigenic polypeptide or multimeric antigenic protein (e.g., trimeric antigen) to subunits of the nanoparticle (e.g., ferritin subunits) and other optional or alternative components described herein, To construct nanoparticles showing the conserved sequence of the coronavirus Spike protein described herein or the S1 subunit of the coronavirus Spike protein or any stable mutant-containing extracellular domain of the coronavirus Spike protein or a truncated fragment thereof. In order to construct the fusion protein of the present invention, one or more linkers can be used to connect and maintain the overall activities of different functional proteins unchanged. Typically, linkers contain short peptide sequences, such as GS-rich peptides. In some embodiments, a 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 employed may be a _G4S linker or a ( _G4S ) ₂ linker as shown herein to connect the spike protein and the nanoparticle scaffold sequence. Recombinant production of fusion proteins of the invention can be based on the protocols described herein and/or other methods known in the art.

Exemplary fusion protein sequences are as follows:

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

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

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

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

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

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

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

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

Fusion protein E1: The C-terminus of the SARS-CoV-2 Delta variant Spike protein S1 subunit (as shown in SEQ ID NO:13) is combined with Helicobacter pylori non-blood red through the linker GGGGS (as shown in SEQ ID NO:15) The N-terminus of ferritin subunit (shown in SEQ ID NO: 14) is connected to obtain the fusion protein E1, the amino acid sequence of which is shown in SEQ ID NO: 24. In the sequence, the original signal peptide: MFVFLVLLPLLVSS (shown in SEQ ID NO:2) is marked in italics, and the linker is marked in italics and bold.

Fusion protein E2: The C-terminus of the SARS-CoV-2 Delta variant Spike protein S1 subunit (as shown in SEQ ID NO:13) is combined with Helicobacter pylori non-blood red through the linker GGGGS (as shown in SEQ ID NO:15) The N-terminus of ferritin subunit (shown in SEQ ID NO:14) is connected with a signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5) replaced the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) to obtain fusion protein E2, whose amino acid sequence is shown in SEQ ID NO:25. In the sequence, the N-terminal signal peptide is in italics and the linker is in italics and bold.

Fusion protein E3: The C-terminus of the SARS-CoV-2 Omicron variant Spike protein S1 subunit (as shown in SEQ ID NO:36) is combined with Helicobacter pylori non-blood red through the linker GGGGS (as shown in SEQ ID NO:15) The N-terminus of ferritin subunit (shown in SEQ ID NO:14) is connected to obtain the fusion protein E3, the amino acid sequence of which is shown in SEQ ID NO:39. In the sequence, the original signal peptide: MFVFLVLLPLLVSS (shown in SEQ ID NO:2) is marked in italics, and the linker is marked in italics and bold.

Fusion protein E4: The C-terminus of the SARS-CoV-2 Omicron variant Spike protein S1 subunit (as shown in SEQ ID NO:36) is combined with Helicobacter pylori non-blood red through the linker GGGGS (as shown in SEQ ID NO:15) Connect the N-terminus of ferritin subunit (as shown in SEQ ID NO:14), and replace the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) with the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO:5) shown), the fusion protein E4 was obtained, and its amino acid sequence is shown in SEQ ID NO: 40. In the sequence, the N-terminal signal peptide is in italics and the linker is in italics and bold.

Fusion protein F1: The C-terminus of the full-length extracellular domain f1 of the mutated SARS-CoV-2 Omicron variant Spike protein (as shown in SEQ ID NO:32) is passed through the linker GGGGS (as shown in SEQ ID NO:15) Connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:14) to obtain fusion protein F1, the amino acid sequence of which is shown in SEQ ID NO:41. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and contains double Mutation K986P/V987P, use underscore Lines and italics are indicated, and joints are italicized and bolded.

Fusion protein F2: The C-terminus of the mutant SARS-CoV-2 Omicron variant Spike protein full-length extracellular domain f2 (as shown in SEQ ID NO: 33) was connected to the N-terminus of the Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 14) through the linker GGGGS (as shown in SEQ ID NO: 15) to obtain the fusion protein F2, whose amino acid sequence is shown in SEQ ID NO: 42. 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 was marked in italics, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ was mutated to ⁶⁸² GSAS ⁶⁸⁵ , marked in underline and bold, and the double mutation K986P/V987P was also included, marked in underline and italics, and the linker was marked in italics and bold.

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

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

Fusion protein H1: Add the original signal peptide: MFVFLVLLPLLVSS (as shown in SEQ ID NO:2) to the N-terminus of the O330 fragment (as shown in SEQ ID NO:37), and then pass the C-terminus of the O330 fragment through the adapter GGGGS (as shown in SEQ ID NO:15) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:14) to obtain the fusion protein H1, whose amino acid sequence is shown in SEQ ID NO:45 . The original signal peptide is in italics and the linker is in italics and bold.

Fusion protein H2: Add the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO:5) to the N-terminus of the O330 fragment (as shown in SEQ ID NO:37), and then pass the C-terminus of the O330 fragment through The linker GGGGS (shown in SEQ ID NO:15) is connected to the N-terminus of the non-heme monomer ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:14) to obtain fusion protein H2, whose amino acid sequence is as shown in SEQ ID Shown in NO:46. The signal peptide is italicized and the linker is italicized and bold.

Fusion protein A1-1: The C-terminus of the full-length extracellular domain a1 of the mutant SARS-CoV-2 original strain Spike protein (as shown in SEQ ID NO:3) and human IgG Fc (as shown in SEQ ID NO:38 (shown) was connected to the N-terminus to obtain fusion protein A1-1, the amino acid sequence of which is shown in SEQ ID NO: 47. 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 ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and contains double Mutation K986P/V987P, underlined and italicized.

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

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

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

Fusion protein C1-1: The C-terminus of the full-length extracellular domain c1 of the mutated SARS-CoV-2 Delta variant Spike protein (as shown in SEQ ID NO:9) and human IgG Fc (as shown in SEQ ID NO:38 shown), the fusion protein C1-1 was obtained, and its 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 ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and contains double Mutation K986P/V987P, underlined and italicized.

Fusion protein C2-1: The C-terminus of the full-length extracellular domain c2 of the mutated SARS-CoV-2 Delta variant Spike protein (as shown in SEQ ID NO:10) and human IgG Fc (as shown in SEQ ID NO:38 shown), the fusion protein C2-1 was obtained, and its amino acid sequence is shown in SEQ ID NO: 52. In the sequence, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide is marked in italics, and the S1/S2 cleavage site ^{The 682} RRAR ⁶⁸⁵ mutation is ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.

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

Fusion protein D2-1: The C-terminus of the mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d2 (as shown in SEQ ID NO:12) and human IgG Fc (as shown in SEQ ID NO:38) was connected to obtain the fusion protein D2-1, the amino acid sequence of which is shown in SEQ ID NO:54. In the sequence, The C-terminus of the extracellular domain of the SARS-CoV-2 Delta variant Spike protein was truncated by 70 amino acid residues, and the original signal peptide: MFVFLVLLPLVSS (such as SEQ ID NO:2), the signal peptide is marked in italics, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , is underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized Mark out.

Fusion protein E1-1: combine the C-terminus of the S1 subunit of SARS-CoV-2 Delta variant Spike protein (as shown in SEQ ID NO:13) and the N-terminus of human IgG Fc (as shown in SEQ ID NO:38) The fusion protein E1-1 was obtained by ligation, and its amino acid sequence is shown in SEQ ID NO: 55. In the sequence, the original signal peptide: MFVFLVLLPLLVSS (shown in SEQ ID NO:2) is marked in italics.

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

Fusion protein E3-1: The C-terminus of the S1 subunit of the SARS-CoV-2 Omicron variant Spike protein (as shown in SEQ ID NO:36) and the N-terminus of human IgG Fc (as shown in SEQ ID NO:38) The fusion protein E3-1 was obtained by ligation, and its amino acid sequence is shown in SEQ ID NO: 57. In the sequence, the original signal peptide: MFVFLVLLPLLVSS (shown in SEQ ID NO:2) is marked in italics.

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

Fusion protein F1-1: The C-terminus of the full-length extracellular domain f1 of the mutated SARS-CoV-2 Omicron variant Spike protein (as shown in SEQ ID NO:32) and human IgG Fc (as shown in SEQ ID NO:38 shown), the fusion protein F1-1 was obtained, and its amino acid sequence is shown in SEQ ID NO: 59. 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 ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and contains double Mutation K986P/V987P, underlined and italicized.

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

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

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

Fusion protein H1-1: Add the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) to the N-terminus of the O330 fragment (as shown in SEQ ID NO:37), and then combine the C-terminus of the O330 fragment with human IgG Fc (shown in SEQ ID NO:38) to obtain the fusion protein H1-1, the amino acid sequence of which is shown in SEQ ID NO:63. The original signal peptide is in italics.

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

Mature fusion protein A: Compared with fusion proteins A1 and A2, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 26. In the sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .

Mature fusion protein B: Compared with fusion proteins B1 and B2, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 27. In the sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .

Mature fusion protein C: Compared with fusion proteins C1 and C2, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 28. In the sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .

Mature fusion protein D: Compared with fusion proteins D1 and D2, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 29. In the sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .

Mature fusion protein E-1: Compared with fusion proteins E1 and E2, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 30. In the sequence, linkers are italicized and bolded.

Mature fusion protein E-2: Compared with fusion proteins E3 and E4, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 65. In the sequence, linkers are italicized and bolded.

Mature fusion protein F: Compared with fusion proteins F1 and F2, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 66. In the sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .

Mature fusion protein G: Compared with fusion proteins G1 and G2, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 67. In the sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .

Mature fusion protein H: Compared with fusion proteins H1 and H2, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 68. In the sequence, linkers are italicized and bolded.

Mature fusion protein A-1: Compared with fusion proteins A1-1 and A2-1, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 69. In the sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ is mutated to ⁶⁸² GSAS ⁶⁸⁵ , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.

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

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

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

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

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

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

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

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

SEQ ID NO:26-30 and 65-77 are mature fusion protein sequences with the N-terminal signal peptide (SEQ ID NO:2 or 5) removed. In addition to these specifically exemplified fusion proteins, the invention also encompasses nanoparticle vaccines containing subunits that are substantially identical to any of these exemplified nanoparticle vaccine sequences, or conservatively modified variants thereof sequence.

Coronavirus multivalent vaccine

In some embodiments, the coronavirus multivalent vaccine of the present invention contains antigens from more than two viruses (such as Spike proteins from different SARS-CoV-2 coronavirus sources) or fusion proteins thereof, or contains the same SARS-CoV -2 Different antigens of coronavirus or fusion proteins containing them. In some embodiments, the coronavirus multivalent vaccine is a coronavirus bivalent vaccine. In some embodiments, the coronavirus multivalent vaccine is a coronavirus trivalent vaccine.

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

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

In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 22, 23 or 29 and a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 43, 44 or 67 fusion protein.

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

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

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

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

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

In some embodiments, the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 53, 54 or 72 and a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 61, 62 or 76 fusion protein.

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

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

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

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

In some embodiments, the coronavirus multivalent vaccine comprises: (1) at least one amino acid sequence comprising any one of SEQ ID NO: 16-23, 26-29, 41-44, 66, 67 A fusion protein, and (2) a fusion protein comprising the amino acid sequence shown in any one of SEQ ID NOs: 45-46, 63, 64, 68 and 77.

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

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

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

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

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

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

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

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

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

In some embodiments, the coronavirus multivalent vaccine is made by mixing two or more antigens or fusion proteins containing them in a certain ratio.

Polynucleotides and expression vectors

The coronavirus Spike protein extracellular domain containing mutations or its truncated fragments, coronavirus Spike protein S1 subunit, coronavirus Spike protein conservative fragments, fusion proteins or Spike protein nanoparticles of the present invention are usually produced by expression vectors, and the expression vectors contain the coding sequence of the coronavirus Spike protein extracellular domain containing mutations or its truncated fragments, coronavirus Spike protein S1 subunit, coronavirus Spike protein conservative fragments, fusion proteins or Spike protein nanoparticles described herein. Therefore, in some related aspects, the present invention provides polynucleotides (DNA or RNA) encoding the coronavirus Spike protein extracellular domain containing mutations or its truncated fragments, coronavirus Spike protein S1 subunit, coronavirus Spike protein conservative fragments, fusion proteins or Spike protein nanoparticles described herein. Some polynucleotides of the present invention encode one of the coronavirus Spike protein extracellular domain containing mutations or its truncated fragments described herein, for example, a truncated fragment of the SARS-CoV-2 Spike protein extracellular domain shown in SEQ ID NO: 12. Some polynucleotides of the present invention encode a subunit sequence of one of the nanoparticle vaccines described herein, such as the fusion protein sequence shown in SEQ ID NO: 23. The fusion protein expressed by the present invention may not contain an N-terminal signal peptide, or some polynucleotide sequences may additionally encode an N-terminal signal peptide. For example, a polynucleotide encoding a fusion protein (e.g., SEQ ID NO: 26-30) may also contain a sequence encoding an N-terminal signal peptide as shown in SEQ ID NO: 2 or 5, or a sequence substantially identical thereto or a conservatively modified variant thereof.

The present invention also provides expression vectors with such polynucleotides and used to produce coronavirus Spike protein extracellular domain containing mutations or truncated fragments thereof, coronavirus Spike protein S1 subunits, coronavirus Spike protein conserved fragments or Host cells for the fusion protein (e.g., prokaryotic or eukaryotic cells, such as HEK293, CHO, ExpiCHO and CHO-S cell lines). Fusion proteins encoded by polynucleotides or expressed from vectors are also included in the present invention. As described herein, the nanoparticle subunit fused Spike protein extracellular domain or truncated fragments thereof, the Spike protein S1 subunit or the Spike protein conserved fragment will self-assemble into a nanoparticle vaccine that displays on its surface Spike protein or its truncated fragment, Spike protein S1 subunit or Spike protein conserved fragment.

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 stable transfected cell lines have been described in the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, ( Third Edition, 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou Edition, 2003). You can also use known methods PCR introduces mutations into polynucleotide sequences.

The choice of specific carrier depends on the intended use of the protein. For example, regardless of whether the cell type is prokaryotic or eukaryotic, the vector chosen must be able to drive protein expression in the desired cell type. Many vectors contain sequences that permit replication of the prokaryotic vector and eukaryotic expression of the operably linked gene sequence. Vectors useful in the present invention can replicate autonomously, that is, the vector exists extrachromosomally, and its replication need not be directly linked to replication of the host cell genome. Alternatively, replication of the vector can be linked to replication of the host chromosomal DNA, for example, the vector can be integrated into the chromosome of the host cell, via a retroviral vector and in a stably transfected cell line. Both viral-based and non-viral-based expression vectors can be used to produce antigens in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (usually with expression cassettes for expressing proteins or RNA) and human artificial chromosomes. Alternative viral vectors include lentiviral or other retrovirus-based vectors, adenovirus, adeno-associated virus, cytomegalovirus, herpesvirus, SV40-based vectors, papillomavirus, HBP, Epstein Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV).

Depending on the particular vector used to express the protein, a variety of known cells or cell lines may be used in the practice of the invention. A host cell can be any cell carrying a recombinant vector for a protein of the invention, allowing the vector to drive expression of the protein for the invention. It may be prokaryotic, such as any of many bacterial strains, or eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells, including, for example, rodent, simian, or human cells . Cells expressing proteins of the invention may be primary cultured cells or may be established cell lines. Accordingly, in addition to the cell lines exemplified herein (eg, HEK293 cells), many other host cell lines well known in the art may be used in the practice of the present invention. These include, for example, various Cos cell lines, CHO cells, HeLa cells, Sf9 cells, AtT20, BV2 and N18 cells, myeloma cell lines, transformed B cells and hybridomas.

Vectors expressing the protein can be introduced into the host cell of choice by any of a number of suitable methods known to those skilled in the art. To introduce a protein-encoding vector into a mammalian cell, 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 a number of transfection methods, including, for example, liposome-mediated transfection ("lipofectamine"), DEAE-dextran-mediated guided transfection, electroporation or calcium phosphate precipitation. These methods are described in detail, for example, in Brent et al., supra. Among them, lipofectamine transfection is widely accepted because it is simple to operate and does not require special equipment. For example, transfection can be performed using Lipofectamine (Life Technologies) or LipoTAXI (Stratagene) kits. Other companies providing lipofection reagents and methods include Bio-Rad Laboratories, CLONTECH, Glen Research, Life Technologies, JBL Scientific, MBI Fermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. Instead of using expression vectors containing viral origins of replication, one can use expression vectors containing appropriate expression control elements (e.g. promoters, enhancers, sequences, transcription termination (e.g., polyadenylation site, etc.) to control the protein coding sequence and selectable markers to transform host cells. The selectable marker in the recombinant vector confers resistance to selection and allows the cell to stably integrate the vector into its chromosomes. Commonly used selectable markers include: neomycin (neo), which is resistant to the aminoglycoside G-418, and hygromycin (hygro), which is resistant to hygromycin.

In some embodiments, a recombinant expression vector includes 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 flanking the inserted sequence. Efficient transcription can be obtained through the early and late promoters of SV40, the long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, and other cellular promoters such as muscle can also be used. Kinesin promoter. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, pLXSN, pLNCX, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), pSVL , pMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc. Commonly used mammalian cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells.

In some embodiments, the inserted gene fragment needs to contain selection markers. Common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes to facilitate transfection. Screening isolation of successful cells. The constructed plasmid is transfected into host cells without the above genes, and then cultured in a selective medium. The successfully transfected cells grow in large quantities and produce the desired target protein.

In addition, standard techniques 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 substitutions. Variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original protein. Or mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutations, and the biological activity of the resulting mutants can be screened to identify mutants that retain activity.

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

Pharmaceutical compositions and methods of treatment

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

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

The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the active ingredient can be administered to a patient. Such carriers may be sterile liquids such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When pharmaceutical compositions are administered intravenously, water is the preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions may 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, glycerin, Propylene, ethylene glycol, water, ethanol, etc. If desired, the pharmaceutical compositions may also contain small amounts of wetting agents, emulsifying agents, or pH buffering agents such as acetates, citrates, or phosphates. Antimicrobial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity-adjusting agents such as sodium chloride or dextrose are also contemplated. These pharmaceutical compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, and the like. The pharmaceutical composition may be formulated as a suppository using traditional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin, which is hereby incorporated by reference. Such compositions will contain a clinically effective dose of the fusion protein or Spike protein nanoparticles, preferably in a 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 preparation may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

In some embodiments, a pharmaceutical composition may comprise a fusion protein or Spike protein nanoparticle or coronavirus multivalent vaccine, and a polynucleotide or vector encoding a fusion protein described herein. In some embodiments, viral (eg, SARS-CoV-2) Spike protein extracellular domains or trimers of truncated fragments thereof can be used to prevent and treat corresponding viral infections. In some embodiments, nanoparticle vaccines containing fusion proteins described herein can be used to prevent or treat corresponding diseases, such as infections caused by various coronaviruses. Some embodiments of the invention relate to the use of SARS-CoV-2 antigens or vaccines to prevent or treat SARS-CoV-2 infection in human subjects. Some embodiments of the invention relate to the use of SARS-CoV antigens or vaccines to prevent or treat SARS-CoV infection.

In the practice of some treatment methods of the present invention, the corresponding Spike protein nanoparticles or fusion proteins or coronavirus multivalent vaccines, or the coronavirus multivalent vaccines described herein, are administered to subjects in need of prevention or treatment of diseases (such as SARS-CoV-2 infection). The polynucleotide encoding the fusion protein. Typically, the Spike protein nanoparticles, fusion proteins, coronavirus multivalent vaccines or polynucleotides encoding fusion proteins disclosed herein are included in pharmaceutical compositions. Pharmaceutical compositions may be therapeutic or prophylactic formulations. Typically, the pharmaceutical composition may additionally comprise one or more pharmaceutically acceptable carriers, and optionally other therapeutic ingredients (eg, antiviral agents). Various pharmaceutically acceptable additives may also be used in the pharmaceutical compositions.

Some pharmaceutical compositions of the present invention are vaccine compositions. For vaccine compositions, suitable adjuvants may be additionally included. Suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MF59, SEPIVAC SWE ^™ , 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 a composition comprising a sustained release polymer or by a microcapsule delivery system or a bioadhesive gel. 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.

The pharmaceutical compositions of the present invention can be used in a variety of therapeutic or prophylactic applications, such as 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, a coronavirus multivalent vaccine can be administered to a subject to induce an immune response to SARS-CoV-2, e.g., inducing the production of broadly neutralizing antibodies against the virus. For subjects at risk of infection with SARS-CoV-2, the vaccine compositions of the invention can be administered to provide prophylactic protection against viral infection. Therapeutic and prophylactic applications of vaccines derived from other antigens described herein can be similarly pursued. Depending on the specific subject and disease, the pharmaceutical composition of the present invention can be administered to the subject by a variety of administration methods known to those of ordinary skill in the art, for example, by the intramuscular route, the subcutaneous route, the intravenous route, Parenteral routes such as intraarterial route, articular route, intraperitoneal route, etc. In some embodiments, the therapeutic methods of the invention involve methods of blocking the entry of a coronavirus (e.g., SARS-CoV or SARS-CoV-2) into a host cell (e.g., a human host cell), preventing the coronavirus Spike protein from binding to the host receptor methods, and methods to treat acute respiratory illness associated with coronavirus infection. In some embodiments, the treatment methods and pharmaceutical compositions described herein can be used in combination with other known therapeutic agents and/or modalities for treating or preventing coronavirus infections. 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.

For therapeutic applications, the pharmaceutical composition should contain a therapeutically effective amount of the fusion protein, Spike protein nanoparticles or coronavirus multivalent vaccine described herein. For prophylactic applications, the pharmaceutical composition should contain a prophylactically effective amount of the fusion protein, Spike protein nanoparticles or coronavirus multivalent vaccine described herein. The appropriate amount of antigen can be determined based on the particular disease or condition to be treated or prevented, the subject's severity, age, and other personal attributes of the particular subject (e.g., the overall state of the subject's health). Determination of effective doses is also guided by studies in animal models and subsequently by clinical trials in humans, and by dosing regimens that significantly reduce the occurrence or severity of the target disease condition or symptoms in subjects.

For prophylactic applications, the pharmaceutical composition is provided before any symptoms, such as infection. Prophylactic administration of pharmaceutical compositions serves to prevent or ameliorate any subsequent infection. Thus, in some embodiments, a subject to be treated is a subject who has become infected (e.g., SARS-CoV-2 infection) due to or may be exposed to a virus (e.g., SARS-CoV-2) or is in Subjects at risk for infection (e.g., SARS-CoV-2 infection). After administration of a therapeutically effective amount of a disclosed pharmaceutical composition, the subject can be monitored for infection (eg, SARS-CoV-2 infection), symptoms associated with the infection (eg, SARS-CoV-2 infection).

For therapeutic use, the pharmaceutical composition is provided at or after the onset of symptoms of a disease or infection, such as after the onset of symptoms of an infection (eg, SARS-CoV-2 infection) or after the infection is diagnosed. Accordingly, pharmaceutical compositions may be provided prior to anticipated exposure to the virus in order to attenuate the expected severity, duration or extent of infection and/or associated disease conditions following exposure or suspected exposure to the virus or after the initial onset of actual infection. The pharmaceutical compositions of the present invention may be combined with other agents known in the art for the treatment or prevention of infection by relevant pathogens, such as SARS-CoV-2 infection.

The vaccine composition (e.g., SARS-CoV-2 vaccine) or pharmaceutical composition comprising the fusion protein, Spike protein nanoparticles or coronavirus multivalent vaccine of the present invention can be provided as a component 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.

Various known delivery systems can be used to administer the fusion protein, Spike protein nanoparticles or coronavirus multivalent vaccines or derivatives of the present invention or their encoding polynucleotides or expression vectors, such as encapsulated in liposomes, microparticles, microcapsules, Recombinant cells capable of expressing the fusion protein or Spike protein nanoparticles, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), as retroviruses, or Construction of nucleic acids that are part of other vectors, etc.

Detailed ways

The technical solutions of the present invention will be further described below through specific examples, which do not limit the scope of protection of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still belong to the protection scope of the present invention.

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

Example 1: Preparation of fusion protein

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

Cryo-electron microscopy (Cryo-EM) was used to analyze the three-dimensional structures of fusion protein D and fusion protein G. The extracellular domain of the SARS-CoV-2 Spike protein or its truncated fragment was connected to the N-terminus of the monomeric ferritin subunit. Interfering with the self-assembly of ferritin, the nanoparticles formed well and showed spikes on the surface.

Example 2: Test of binding ability of fusion protein and hACE2 protein

1.1 Fusion protein and hACE2 protein binding ability test (ELISA)

This test detects the activity of fusion protein D, fusion protein G and human ACE2 protein (hACE2) through ELISA. Binding ability, thereby evaluating whether the Spike protein-ferritin fusion protein of the present invention can well display the key antigenic epitopes of Spike protein. The method is briefly described as follows: Add 100 μL of 2 μg/mL antigen (WT-Spike-His, Delta-Spike-His, Omicron-Spike-His, fusion protein) to each reaction well of a 96-well microplate (Costar, Cat. No.: 9018). D or fusion protein G) solution, coated overnight at 4°C; washed twice with PBST (PBS buffer containing 0.05% Tween-20); add blocking solution (PBST containing 3% BSA) to each reaction well and set aside Incubate in a 37°C incubator for 2 hours; wash 3 times with PBST after blocking; add gradient dilution of humanACE2-his-biotin (Yiqiao Shenzhou, Cat. No.: 10108-H27B-B), with a starting concentration of 2.5 μg/mL, and a 3-fold gradient. Dilute (10 serial dilutions in total), 100 μL per well, and incubate in a 37°C incubator for 1.5 h; wash 5 times with PBST; add streptavidin-labeled hydrogen peroxide to the reaction well at 100 μL/well. Enzyme (Jackson Immuno Research, Cat. No.: 016-030-084; 1:10000), incubate at 37°C for 1 hour; wash with PBST 8 times; add TMB solution at 100 μL/well to the reaction well, and incubate at 37°C in the dark for 5 ~15min; add 50μL of stop solution (0.1M H ₂ SO ₄ ) to each reaction well to terminate the enzymatic color reaction; set the detection wavelength to 450nm for reading.

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

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

1.2 Fusion protein and hACE2 protein binding ability test (BLI)

Biofilm interference technology (BLI) was used to measure the affinity constants of fusion protein D and fusion protein G binding to hACE2. The instrument was the Fortebio Octet RED&QK system of PALL Company. Multi-channel parallel quantitative analysis of WT-Spike-His (same as step 1.1 in Example 2), Delta-Spike-His (same as step 1.1 in Example 2), Omicron-Spike-His (same as step 1.1 in Example 2), and fusion protein D , Fusion protein G, the concentration gradient is set to: 50, 100, 200 and 400nM, hACE2-Biotin (Acro biosystems, Cat. No. AC2-H5257) coupled to SA Biosensors sensor (Octet, Cat. No. 2107002811).

The results are shown in Table 1. The results show that the binding affinity of fusion protein D and fusion protein G to hACE2 is significantly stronger than the affinity of WT-Spike-His, Delta-Spike-His and Omicron-Spike-His to hACE2. It shows that the Spike protein-ferritin fusion protein of the present invention can well display the key antigenic epitope of Spike protein.

Table 1 Binding kinetics of SARS-CoV-2 Spike protein and hACE2 receptor

Example 3: Immunogenicity of the vaccine in mice

In order to evaluate the immunogenicity of monovalent vaccines (fusion protein D, fusion protein G) and bivalent vaccines (ie, fusion protein D and fusion protein G with a mass ratio of 1:1), BALB/c mice were used to explore different antigen doses. Effect on immunogenicity.

1.1 Mouse immunization

Balb/c mice (n=10 per group) were injected with different doses of vaccine by intramuscular injection on days 0 and 21 respectively. The control mice were only given SEPIVAC SWE ^TM adjuvant. Each mouse was given SEPIVAC SWE ^TM adjuvant. The volume of the dose (SEPPIC SA, product number 80748J, batch number 210721010001) is fixed at 50 μL, and the total volume of each dose is 100 μL/animal. The grouped dosing plan is shown in Table 2. On the 14th and 35th days (after the second immunization 2 weeks) and 30 weeks after the second immunization, blood was collected for ELISA to detect serum anti-Spike protein [original strain, Delta and Omicron (BA.1)] IgG titers and SARS-CoV-2 Spike pseudovirus neutralization Antibody experiments.

Table 2 Grouped dosing regimen

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

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

The results are shown in Figure 2a to Figure 2f. No anti-Spike protein IgG titers were detected in mice given adjuvant alone in all dose groups tested. As shown in Figures 2a, 2c, and 2e, after the initial immunization, all mice produced IgG antibodies against WT-Spike-His, Delta-Spike-His, and Omicron-Spike-His, and the antibody titers were dose-dependent. sex. The fusion protein D group showed an obvious dose-effect relationship on the geometric mean antibody titer (GMT) of Omicron-Spike-His (Figure 2e), but the dose-effect relationship on the GMT of WT-Spike-His and Delta-Spike-His was not significant. (Figure 2a, 2c); Fusion protein G showed an obvious dose-effect relationship on the GMT of WT-Spike-His and Delta-Spike-His (Figure 2a, 2c), but fusion protein G showed an obvious dose-effect relationship on the antibody titer of Omicron-Spike-His. The dose-effect relationship of GMT was not significant (Figure 2e). It is worth noting that in the bivalent vaccine group, the dosage ranged from 0.2 μg to 10 μg, the GMT of WT-Spike-His, Delta-Spike-His and Omicron-Spike-His IgG antibodies was significantly dose-dependent.

After the second boosted immunization, the anti-Spike protein IgG titers of all mice increased 20-100 times compared with those after the first immunization, with no significant dose-effect relationship. Compared with those receiving the same dose of fusion protein D, the antibody titers of mice receiving fusion protein G against WT-Spike-His and Delta-Spike-His were significantly lower than those of the former (Figure 2b, 2d); however, the fusion protein G group had significantly lower antibody titers against Omicron-Spike-His antibody titers were significantly higher than fusion protein D (Figure 2f); compared with fusion protein D and fusion protein G, the bivalent vaccine induced high antibody titers against the original strain, Delta and Omicron anti-Spike protein IgG Spend.

The above results show that the bivalent vaccine has a better broad spectrum than the monovalent vaccine fusion protein D or fusion protein G, and the antibody titers against different mutant strains remain better than or equal to the monovalent vaccine.

1.3 SARS-CoV-2 Spike pseudovirus neutralizing antibody experiment

Dilute the SARS-CoV-2 Spike pseudovirus 50 times with DMEM medium containing 10% FBS. Add the pseudovirus dilution solution to a 96-well white plate (Costar, Cat. No. 3917), 25 μL/well; use DMEM containing 10% FBS. In the culture medium, dilute the mouse serum collected on the 35th day of step 1.1 of Example 3 40 times, then perform 3-fold gradient dilution, dilute 7 gradients, and add 50 μL/well to the 96-well plate with pseudovirus added; shake thoroughly Mix well and place in culture Incubate for 1 hour at 37°C in the incubator; add ACE2-293 cell suspension (2×10 ⁴ cells/well) at 50 μL/well, and place the 96-well plate in the incubator for culture; take out the 96-well plate after 48 hours of culture and wait for it to recover to room temperature, add 50 μL of Bio-Lite ^TM Luciferase Assay System (Vazyme, Cat. No. DD1201) solution to each well, react for 2 minutes at room temperature in the dark, and then read the luminescence signal using the Luminescence detection module of a microplate reader.

The construction method of ACE2-293 cells is as follows: culture HEK293 cells in DMEM complete medium containing 10% FBS, and use lipofectamine 2000 transfection reagent (Thermo Fisher, 11668019) to transform the ACE2 expression plasmid (Yiqiao Shenzhou, HG10108-M). stained, and then through pressure screening and flow sorting with hygromycin (200 μg/ml) (using 10 μg/ml anti-ACE2 and PE-conjugated Anti-Human IgG-Fc), the cells continued to amplify and select PE-positive cells. Single clones with a rate of >90% were amplified in the next step, and HEK293 cells expressing ACE2, namely ACE2-293 cells, were selected.

Among them, the SARS-CoV-2 Spike pseudovirus is: SARS-CoV-2 Spike pseudovirus (Yoshiman Biotechnology, GM-0220PV07); SARS-CoV-2 Spike (B.1.617.2) pseudovirus (Yoshiman Biotechnology, GM-0220PV07) GM-0220PV45); SARS-CoV-2 Spike (B.1.1.7/VUI-202012/01, del145Y) pseudovirus (Yoshiman Bio, GM-0220PV33); SARS-CoV-2 Spike (B.1.351/501Y .V2) Pseudovirus (Jiman Bio, GM-0220PV32); SARS-CoV-2 Spike (P.1501Y.V3) Pseudovirus (Jiman Bio, GM-0220PV47); SARS-CoV-2 Spike (B.1.1 .529) Pseudovirus (Jiman Bio, GM-0220PV84); SARS-CoV-2 Spike (BA.2.12.1) Pseudovirus (Jiman Bio, GM-0220PV93); SARS-CoV-2 Spike (BA.3 ) pseudovirus (Yoshiman Bio, GM-0220PV92); SARS-CoV-2 Spike (BA.4/5) pseudovirus (Yoshiman Bio, GM-0220PV89).

The results in Figure 3 show that the bivalent vaccine has a better broad spectrum than the monovalent vaccine fusion protein D or fusion protein G, and the neutralizing antibody titers against different mutant strains remain better than or equal to the monovalent vaccine. The titer of fusion protein G against SARS-CoV-2 Spike pseudovirus and SARS-CoV-2 Spike (B.1.617.2) pseudovirus is significantly lower than that of fusion protein D and bivalent vaccine; the titer of fusion protein D against SARS-CoV- 2 The titers of Spike (B.1.1.529) pseudovirus and SARS-CoV-2 Spike (BA.3) pseudovirus were significantly lower than those of fusion protein G and bivalent vaccines; while the bivalent vaccine was effective against all strains tested. The virus maintains high titers.

The results in Figure 4a show that the mouse sera after being immunized twice with different doses of the bivalent vaccine had high neutralizing antibody titers against all tested strains, and were very effective against SARS-CoV-2 Spike (B.1.1.529 ) The highest GMT values of IC50 of pseudovirus and SARS-CoV-2 Spike (BA.4/5) pseudovirus are 10198 and 1018 respectively; the GMT values of other strains are all >3,000, and the highest GMT value reaches more than 10,000, and is presented There was a certain dose-effect relationship, and the titer of the 0.2 μg group was lower than that of the 1 μg group, but the difference was not statistically significant. The results in Figure 4b show that the serum of mice immunized with the bivalent vaccine had high neutralizing antibody titers against all nine strains tested.

1.4 Durability of vaccine-induced immune responses

Long-term antibody titers following bivalent vaccination were assessed. ELISA method to detect serum response to WT-Spike-His (same as The specific antibody titers of Example 2 step 1.1), Delta-Spike-His (same as Example 2 step 1.1), and Omicron-Spike-His (same as Example 2 step 1.1), and the detection steps are the same as Example 3 step 1.2. The serum is the serum collected in the 2nd week after the second immunization in Group 8 of Example 3 (diluted 1000 times as the starting concentration, and then 3 times gradient diluted), and the serum collected in the 30th week after the second immunization (diluted 1000 times as starting concentration, followed by 3-fold serial dilutions). The results showed (Figure 5) that the serum antibody titer at the 30th week after the second immunization did not decrease significantly compared with the serum at the 2nd week after the second immunization. The GMT of serum against WT-Spike-His, Delta-Spike-His and Omicron-Spike-His were 9.1×10 ⁶ and 9.6×10 ⁶ , 7.2×10 ⁶ and 6.0×10 ⁶ , 6.3×10 ⁶ and 5.6 respectively. ×10 ⁶ , indicating that the bivalent vaccine can provide long-term protection.

Example 4: Effect of adjuvants on vaccine immunogenicity

1.1 Mouse immunization

BALB/c mice (8 weeks old) received two intramuscular injections (day 0 and day 21) of different doses of SEPIVAC SWE ^TM adjuvant (SEPPIC SA, Cat. No. 80748J, Lot No. 210721010001) and bivalent vaccine (i.e. Fusion protein D and fusion protein G with a mass ratio of 1:1), the total volume of each dose is 100 μL/animal, and the grouped dosing plan is shown in Table 3. Serum was collected on days 14 and 35.

Table 3 Group dosing regimen

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

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

After the first immunization, compared with the 5 μg antigen without adjuvant group, the GMT of IgG antibodies against WT-Spike-His, Delta-Spike-His, and Omicron-Spike-His could be increased by 29, 22, and 31 times, respectively (Figure 6a , 6c, 6e); after the second immunization, the GMT of IgG antibodies against WT-Spike-His, Delta-Spike-His, and Omicron-Spike-His could be increased by 60, 37, and 66 times respectively (Figures 6b, 6d, and 6f) . Although the antibody titer against Spike protein showed a certain adjuvant dose dependence after the first immunization, after the second immunization, the antibody titer was lower when the adjuvant dose was 10, 15, 25, or 50 μL. There was no obvious dose dependence (Fig. 6b, 6d, 6f). The antibody titer increased significantly after the second immunization, and was 20-100 times higher than the first immunization.

After the first immunization, the GMT of all Spike protein (original strain, Delta type, Omicron type) IgG antibodies and the adjuvant/antigen ratio showed a dose-dependent relationship, that is, the higher the adjuvant dosage, the higher the GMT. However, after the second immunization, when the adjuvant doses were 10, 15, 25, and 50 μL, there was no obvious dose-dependent relationship between Spike protein IgG antibody GMT and the adjuvant/antigen ratio. It shows that under the current experimental conditions, the ratio of 10-50 μL adjuvant dose and 5 μg bivalent vaccine can fully induce the humoral immune response in mice.

1.3 hACE2 blocking experiment

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

Dilute three recombinant proteins: WT-Spike-His (same as step 1.1 in Example 2), Delta-Spike-His (same as step 1.1 in Example 2), and Omicron-Spike-His (same as step 1.1 in Example 2) with 1×PBS. Antigen was added to the 96-well enzyme plate at 2 μg/mL, 100 μL/well, and incubated overnight at 2-8°C. The next day, wash twice with PBST (PBS buffer containing 0.05% Tween-20), add blocking solution (PBST containing 3% BSA), and block at 37°C for 2 hours. Then wash it 3 times with PBST solution, add the immune mouse serum collected on the 35th day in step 1.1 of Example 4 with gradient dilution (the mouse serum is diluted 100 times as the starting concentration, and then 3 times gradient dilution), and incubate at 37°C for 1 hours; then add 25ng/mL biotin-labeled hACE2 (Acro, Cat. No. AC2-H82E7), 100μL/well, and incubate at 37°C for 1 hour. After washing 5 times with PBST, add 1:10000 diluted Peroxida se-conjugated Streptavidin enzyme-labeled secondary antibody (Jackson Immuno Research, Cat. No. 016-030-084), and incubate at 37°C for 0.5 hours. After washing 8 times with PBST, add 100 μL TMB chromogenic solution to develop color at room temperature for 10 minutes, and then add 50 μL stop solution to terminate the reaction. Set the detection wavelength to 450nm for reading, use SoftMax pro 7.02 software to fit the OD value of the reading with a nonlinear four-parameter equation curve and calculate the antibody inhibitory titer IC ₅₀ value. For samples whose IC ₅₀ value is lower than the lower limit of detection, , its IC ₅₀ value is calculated as 100.

The results are shown in the figure (Figure 7a-c). Compared with the group without adjuvant, adding 5 μL of adjuvant can significantly increase the IC ₅₀ GMT from 111 to 803, 101 to 688, and 199 to 934, respectively, corresponding to the inhibition of hACE2 and WT-Spike-His, Delta-Spike-His and Omicron-Spike-His combination. In addition, when the adjuvant dose is greater than or equal to 10 μL, there is no significant difference in GMT between each dose group, indicating that when the adjuvant is within this range and the antigen is 5 μg, the adjuvant dose effect relationship is not obvious, but it is significantly higher than that of 5 μL adjuvant. dose group.

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

SARS-CoV-2 challenge experiments were conducted on mice to evaluate the protective effect of the vaccine against viral infection and to assess whether there are vaccine-related exacerbations. In this study, the K18-hACE2 transgenic mouse (C57BL/6J) model was used. This model has been validated and widely used to study SARS-CoV-2 virus infection. It is highly susceptible to SARS-CoV-2 infection. K18-hACE2 mice infected with SARS-CoV-2 develop dose-dependent lung disease, which is characterized by Similar to human COVID-19, including diffuse alveolar damage, inflammatory cell infiltration, tissue damage, pulmonary vascular damage, Significant weight loss and death.

1.1 Mouse immunization and intranasal challenge

Mice were immunized twice by intramuscular injection with different doses of bivalent vaccine (i.e., fusion protein D and fusion protein G with a mass ratio of 1:1) and a fixed dose of SEPIVAC SWE ^TM adjuvant (on days 0 and 21, respectively). day), the total volume of each dose is 100 μL/animal, and the group dosing plan is shown in Table 4. Blood was collected on the 35th day, and the new coronavirus Omicron BA.1.1 strain was infected through intranasal instillation on the 42nd day. The intranasal dose of the virus was 5000TCID50/animal.

Table 4 Grouped dosing regimen

1.2 Use ELISA method to detect serum anti-Spike protein IgG titer

In order to evaluate the immunogenicity of the bivalent vaccine (i.e., fusion protein D and fusion protein G with a mass ratio of 1:1) in K18-hACE2 transgenic mice, the mouse sera after the second immunization were tested by ELISA against The IgG titer of Spike protein of the original strain, Delta and Omicron mutant strains. For the test method, please refer to step 1.2 of Example 3. The mouse serum is the serum collected on the 35th day of step 1.1 of Example 5. The serum was diluted 1000 times as the starting concentration. Then 3-fold gradient dilution, a total of 11 gradients.

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

1.3 Mouse weight detection

From the day of challenge (recorded as Day 0) until the 10th day after challenge (Day 10), the body weight of the mice was regularly weighed and recorded every day. Mice in the low-, medium-, and high-dose groups did not show weight loss, and their weight changes were similar to those of the blank control mice that had not been vaccinated with the virus. In contrast, the mice in the model control group significantly lost weight on the 5th day after virus infection, and the weight loss of all mice reached the euthanasia standard on the 6th day after virus challenge (when the mouse body weight dropped by more than 25%).

1.4 Detection of live virus titers in the lungs of mice after challenge

The method for detecting live virus in the lungs is the Focus Forming Assay (FFA). The experiment was as follows: 2 days after the challenge (Day 2), some mice in each group were euthanized, and their lungs were collected and ground; the mouse lung tissue homogenate was centrifuged to obtain the supernatant, which was first diluted 1:3 and then 1:10; the lung homogenate stock solution and the dilution were added to the pre-prepared Vero-E6 cell plate, 50 μL/well, and incubated at 37°C for 1 hour; the culture supernatant was discarded, and 100 μL of 1.6% sodium carboxymethyl cellulose culture medium (Sigma, catalog number: C4888-500G) was added, and the culture was incubated at 37°C and 5% CO ₂ Culture for 24 hours; discard the culture supernatant and add 4% paraformaldehyde (biosharp, catalog number: BL539A) for fixation; after fixation, treat the cells with 0.1% Triton-X100 (Sigma, catalog number: T8787-100mL) to perforate the membrane, and then block with blocking solution (PBST containing 3% BSA) for 2 hours; use rabbit anti-new coronavirus nucleoprotein polyclonal antibody (Sino Biological, catalog number: 40143-T62) as the primary antibody and HRP goat-anti rabbit IgG (abcam, catalog number: ab6721) as the secondary antibody, incubate in sequence, and then use True-blue color developing solution (KPL, catalog number: 50-78-02) for color development; use Immuno ELISPOT instrument to acquire images, perform image analysis, and obtain the number of spots.

The results are shown in Figure 9. The average titer of live virus in the lung tissue of mice in the model control group was 4.49×10 ⁴ spot forming units (FFU)/g; while the lung virus titers of all mice receiving the bivalent vaccine were lower than detection limit, indicating that viral replication in mouse lungs was completely inhibited.

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

The neutralization titer of the immune serum against the new coronavirus Omicron BA.1.1 true virus was tested using focus reduction neutralization test (FRNT). The method is briefly described as follows: use DMEM culture medium to dilute the serum stock solution collected on the 35th day of step 1.1 of Example 5 1:8 times, and then use DMEM culture medium to perform 2-fold gradient dilution, a total of 6 dilutions; Mix the serum with an equal volume of Omicron BA.1.1 solution containing 300-400PFU of the new coronavirus (the final dilutions of the serum are: 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512) , incubate at 37°C for 1 hour; then transfer the incubation mixture to the previously prepared Vero-E6 cell plate, 100 μL per well, and incubate for another 1 hour at 37°C, 5% _CO2 ; discard the culture supernatant, and add 100 μL of 1.6 % CMC culture medium (Sigma, Cat. No.: C4888-500G), cultured at 37°C and 5% _CO2 for 24 hours; add 4% paraformaldehyde (biosharp, Cat. No.: BL539A) to inactivate and fix the cells; after fixation, use 0.1% Triton -X100 (Sigma, Cat. No.: T8787-100mL) was used to treat the cells to break the membrane and punch holes, and then blocked with blocking solution (PBST containing 3% BSA) for 2 hours; rabbit anti-COVID-19 nucleoprotein polyclonal antibody (Yiqiao Shenzhou, Cat. No.: 40143 -T62) as the primary antibody, HRP goat-anti rabbit IgG (abcam, Cat. No.: ab6721) as the secondary antibody, incubate in sequence, and then use True-blue chromogenic solution (KPL, Cat. No.: 50-78-02) for color development; Use the Immuno ELISPOT instrument to obtain images, perform picture analysis, obtain the number of spots, and calculate the inhibition rate. The inhibition rate calculation formula is: 100×(1-number of spots in the sample well/number of spots in the positive control well). No serum is added to the positive control hole (each There are about 300-400 spots in the well), and no virus is added to the negative control well (no spots).

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

Claims (44)

A coronavirus multivalent vaccine, characterized in that the coronavirus multivalent vaccine contains the coronavirus Spike protein extracellular domain containing mutations or a truncated fragment thereof or a fusion protein containing the same; the mutations include: 1) Mutation of RRAR to GSAS; 2) There is a mutation in the turning region between HR1 and CH that prevents HR1 and CH from forming a straight helix during the fusion process. The coronavirus multivalent vaccine according to claim 1, wherein the mutation includes: 1) mutating RRAR to GSAS; 2) there is a double mutation K986P/V987P in the turning region between HR1 and CH. The coronavirus multivalent vaccine according to claim 1 or 2, wherein the truncated fragment of the extracellular domain of the coronavirus Spike protein containing mutations is similar to the full-length extracellular domain of the coronavirus Spike protein. Than, the C-terminus is truncated by 5-80 amino acid residues; or, the C-terminus is truncated by 20-76 amino acid residues; or, the C-terminus is truncated by 70 amino acid residues. The coronavirus multivalent vaccine according to any one of claims 1 to 3, wherein the coronavirus Spike protein extracellular domain or its truncated fragment is from SARS-CoV-2, SARS-CoV or MERS- CoV; alternatively, the extracellular domain of the coronavirus Spike protein or a truncated fragment thereof is from the original strain of SARS-CoV-2 or a mutant strain thereof; alternatively, the extracellular domain of the coronavirus Spike protein or a truncated fragment thereof is from 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 strain, SARS-CoV-2 Epsilon variant strain, SARS-CoV-2 Lambda variant strain or SARS-CoV-2 Omicron variant strain; or, the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof Contains the amino acid sequence shown in any one of SEQ ID NO: 3, 4, 6, 7, 9-12, 32-35, 78-83, or the same as SEQ ID NO: 3, 4, 6, 7, 9- 12. An amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in any one of 12, 32-35, 78-83, or with SEQ ID NO: 3, 4, 6, 7, 9-12 , 32-35, 78-83 any one of the amino acid sequence compared to the amino acid sequence having one or more conservative amino acid substitutions. The coronavirus multivalent vaccine according to any one of claims 1 to 4, wherein the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof is connected through a linker. The mutation-containing coronavirus Spike protein extracellular domain or its truncated fragment and monomeric subunit protein; 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; alternatively, the linker is (G _m S) _n , where each m is independently 1, 2, 3, 4 or 5, n is 1, 2, 3, 4 or 5; or, the monomeric subunit protein is a self-assembled monomeric subunit protein; or, the monomeric subunit protein is a monomeric ferritin subunit; or, the The monomeric ferritin subunit is selected from the group consisting of bacterial ferritin, plant ferritin, phycoferritin, insect ferritin, fungal ferritin and lactate. Animal ferritin; alternatively, the monomeric ferritin subunit is a non-heme monomeric ferritin subunit of Helicobacter pylori; alternatively, the monomeric ferritin subunit comprises the amino acid sequence shown in SEQ ID NO: 14 , 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: 14, or having one or more conserved amino acids compared to the amino acid sequence shown in SEQ ID NO: 14 Substituted amino acid sequence. The coronavirus multivalent vaccine according to claim 5, wherein the fusion protein comprising the extracellular domain of the coronavirus Spike protein with mutations or a truncated fragment thereof is obtained by combining the coronavirus with mutations. The C-terminus of the extracellular domain of Spike protein or its truncated fragment is connected to the N-terminus of the monomeric subunit protein through a linker. The coronavirus multivalent vaccine according to any one of claims 1 to 6, wherein the fusion protein comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises SEQ ID NO: The amino acid sequence shown in any one of 16-23, 26-29, 41-44, 66, and 67, or the amino acid sequence shown in any one of SEQ ID NO: 16-23, 26-29, 41-44, 66, and 67 The amino acid sequence has at least 80% or at least 90% identity compared to the amino acid sequence, or compared to the amino acid sequence shown in any one of SEQ ID NO: 16-23, 26-29, 41-44, 66, 67 An amino acid sequence with one or more conservative amino acid substitutions. The coronavirus multivalent vaccine according to any one of claims 1-7, characterized in that the coronavirus multivalent vaccine contains at least one component such as SEQ ID NO: 16-23, 26-29, 41-44 , a fusion protein of the amino acid sequence shown in any one of 66 and 67. The coronavirus multivalent vaccine according to claims 1-8, characterized in that the coronavirus multivalent vaccine contains at least two species including SEQ ID NO: 16-23, 26-29, 41-44, 66, Fusion protein of the amino acid sequence shown in any one of 67. The coronavirus multivalent vaccine according to any one of claims 1 to 9, wherein the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 22, 23 or 29 and Fusion proteins containing the amino acid sequence shown in SEQ ID NO: 43, 44 or 67. The coronavirus multivalent vaccine of claim 10, wherein the fusion protein comprising the amino acid sequence shown in SEQ ID NO: 22, 23 or 29 and the fusion protein comprising the amino acid sequence shown in SEQ ID NO: 43, 44 or 67 The mass ratio of the fusion protein of the amino acid sequence shown is (1-5):(1-5); or the mass ratio is 1:1. The coronavirus multivalent vaccine according to any one of claims 1 to 11, wherein the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 22 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 22 Fusion protein of the amino acid sequence shown in NO:43. The coronavirus multivalent vaccine according to any one of claims 1 to 11, wherein the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 29 and a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 29 Fusion protein of the amino acid sequence shown in NO:67. The coronavirus multivalent vaccine as described in any one of claims 1 to 4, characterized in that the fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof comprises the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof and the Fc fragment of an immunoglobulin connected thereto; or, the Fc fragment of the immunoglobulin is from IgG, IgM, IgA, IgE or IgD; or, the Fc fragment of the immunoglobulin is from IgG1, IgG2, IgG3 or IgG4; or, the Fc fragment of the immunoglobulin is the Fc fragment of IgG1; or, the Fc fragment of the immunoglobulin is the Fc fragment of human IgG1; or, the Fc fragment of the immunoglobulin comprises the amino acid sequence as shown in SEQ ID NO:38, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in SEQ ID NO:38, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:38. The coronavirus multivalent vaccine according to claim 14, characterized in that the fusion protein comprising the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof is obtained by connecting the C-terminus of the extracellular domain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof to the N-terminus of the Fc fragment of the immunoglobulin. The coronavirus multivalent vaccine according to any one of claims 1-4 and 14-15, wherein the fusion protein comprising the mutated extracellular domain of the coronavirus Spike protein or a truncated fragment thereof comprises: The amino acid sequence shown in any one of SEQ ID NO:47-54, 59-62, 69-72, 75-76, or any of the amino acid sequences shown in SEQ ID NO:47-54, 59-62, 69-72, 75-76 An amino acid sequence that has at least 80% or at least 90% identity compared to the amino acid sequence shown in one item, or an amino acid sequence shown in any one of SEQ ID NO: 47-54, 59-62, 69-72, 75-76 Amino acid sequences are compared to amino acid sequences with one or more conservative amino acid substitutions. The coronavirus multivalent vaccine according to any one of claims 1-4 and 14-16, characterized in that the coronavirus multivalent vaccine contains at least one component such as SEQ ID NO: 47-54, 59-62 , a fusion protein of the amino acid sequence shown in any one of 69-72 and 75-76. The coronavirus multivalent vaccine according to any one of claims 1-4 and 14-17, characterized in that the coronavirus multivalent vaccine contains at least two species including SEQ ID NO: 47-54, 59-62 , a fusion protein of the amino acid sequence shown in any one of 69-72 and 75-76. The coronavirus multivalent vaccine according to any one of claims 1-4 and 14-18, wherein the coronavirus multivalent vaccine comprises an amino acid sequence as shown in SEQ ID NO: 53, 54 or 72 The fusion protein and the fusion protein comprising the amino acid sequence shown in SEQ ID NO: 61, 62 or 76. The coronavirus multivalent vaccine of claim 19, wherein the fusion protein comprising the amino acid sequence shown in SEQ ID NO: 53, 54 or 72 and the fusion protein comprising the amino acid sequence shown in SEQ ID NO: 61, 62 or 76 The mass ratio of the fusion protein of the amino acid sequence shown is (1-5):(1-5); or the mass ratio is 1:1. The coronavirus multivalent vaccine according to any one of claims 1-4 and 14-20, wherein the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 53 and A fusion protein containing the amino acid sequence shown in SEQ ID NO:61. The coronavirus multivalent vaccine according to any one of claims 1-4 and 14-20, wherein the coronavirus multivalent vaccine comprises a fusion protein comprising the amino acid sequence shown in SEQ ID NO:72 and A fusion protein containing the amino acid sequence shown in SEQ ID NO:76. The coronavirus multivalent vaccine according to any one of claims 1 to 4, characterized in that the coronavirus multivalent vaccine also contains a conserved fragment of the coronavirus Spike protein or a fusion protein comprising the same; or, the coronavirus multivalent vaccine The conserved fragment of the Spike protein is from SARS-CoV-2, SARS-CoV or MERS-CoV; or, the conserved fragment of the coronavirus Spike protein is from the original strain of SARS-CoV-2 or its mutant strain; or, the coronavirus Spike protein The conserved fragments come from the original SARS-CoV-2 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. The coronavirus multivalent vaccine according to claim 23, characterized in that the fusion protein comprising the conservative fragment of the coronavirus Spike protein comprises a conservative fragment of the coronavirus Spike protein and a monomeric subunit protein connected by a linker; or, the linker is a GS linker; or, the linker is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS) ₂ , (GGGGS) ₃ , or any combination thereof; or, the linker is (G _m S) _n , wherein each m is independently 1, 2, 3, 4 or 5, and n is 1, 2, 3, 4 or 5; or, the monomeric subunit protein is a self-assembled monomeric subunit protein; or, the monomeric subunit protein is a monomeric ferritin subunit; or, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, algae ferritin, insect ferritin, fungal ferritin and mammalian ferritin; or, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit; or, the monomeric ferritin subunit comprises the amino acid sequence shown in SEQ ID NO: 14, or with SEQ ID An amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence shown in SEQ ID NO:14, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:14. The coronavirus multivalent vaccine according to claim 23 or 24, wherein the fusion protein comprising a conserved fragment of the coronavirus Spike protein is a C-terminus of the conserved fragment of the coronavirus Spike protein. Connected to the N-terminus of the monomeric subunit protein through a linker. The coronavirus multivalent vaccine according to any one of claims 23-25, wherein the fusion protein comprising a conserved fragment of the coronavirus Spike protein comprises as shown in any one of SEQ ID NO: 45-46, 68 An amino acid sequence, 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 NO:45-46, 68, or an amino acid sequence with SEQ ID NO:45-46, 68 The amino acid sequence shown in any item is compared to the amino acid sequence having one or more conservative amino acid substitutions. The coronavirus multivalent vaccine according to claim 23, wherein the fusion protein comprising a conserved fragment of the coronavirus Spike protein includes a conserved fragment of the coronavirus Spike protein and an Fc fragment of an immunoglobulin connected thereto; or , the Fc fragment of the immunoglobulin is from IgG, IgM, IgA, IgE or IgD; or, the Fc fragment of the immunoglobulin is from IgG1, IgG2, IgG3 or IgG4; or, the Fc fragment of the immunoglobulin is The Fc fragment of IgG1; or the Fc fragment of the immunoglobulin is the Fc fragment of human IgG1; or the Fc fragment of the immunoglobulin includes the amino acid sequence shown in SEQ ID NO:38, or is the same as SEQ ID NO: An amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 38. The coronavirus multivalent vaccine as described in claim 27 is characterized in that the fusion protein comprising the conservative fragment of the coronavirus Spike protein is obtained by connecting the C-terminus of the conservative fragment of the coronavirus Spike protein to the N-terminus of the Fc fragment of the immunoglobulin. The coronavirus multivalent vaccine as described in claim 23, 27 or 28 is characterized in that the fusion protein comprising the conservative fragment of the coronavirus Spike protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 63, 64, 77, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in any one of SEQ ID NO: 63, 64, 77, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 63, 64, 77. The coronavirus multivalent vaccine according to any one of claims 23-29, characterized in that, the coronavirus multivalent vaccine includes at least one component such as SEQ ID NO: 16-23, 26-29, 41-44 , a fusion protein of the amino acid sequence shown in any one of 66 and 67. The coronavirus multivalent vaccine according to claim 30, wherein the coronavirus multivalent vaccine comprises: (1) at least one compound comprising SEQ ID NO: 16-23, 26-29, 41-44, A fusion protein of the amino acid sequence shown in any one of SEQ ID NO: 45-46, 63, 64, 68 and 77. The coronavirus multivalent vaccine according to claim 31, wherein the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 22, 23 or 29, ( 2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 43, 44 or 67, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 63, 64 or 77. The coronavirus multivalent vaccine according to claim 32, wherein the fusion protein comprising the amino acid sequence shown in SEQ ID NO: 22, 23 or 29, or the fusion protein comprising the amino acid sequence shown in SEQ ID NO: 43, 44 or 67 The mass ratio of the fusion protein of the amino acid sequence shown and the fusion protein containing the amino acid sequence shown in SEQ ID NO: 63, 64 or 77 is (1-5): (1-5): (1-5); Or the mass ratio is 1:1:1. The coronavirus multivalent vaccine according to claim 32 or 33, wherein the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 22, (2) A fusion protein comprising the amino acid sequence shown in SEQ ID NO: 43, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 63. The coronavirus multivalent vaccine according to claim 32 or 33, wherein the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 29, (2) A fusion protein comprising the amino acid sequence shown in SEQ ID NO: 67, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 77. The coronavirus multivalent vaccine according to any one of claims 23-29, characterized in that the coronavirus multivalent vaccine includes at least one component such as SEQ ID NO: 47-54, 59-62, 69-72 , a fusion protein of the amino acid sequence shown in any one of 75-76. The coronavirus multivalent vaccine according to claim 36, wherein the coronavirus multivalent vaccine comprises: (1) at least one compound comprising SEQ ID NO: 47-54, 59-62, 69-72, A fusion protein of the amino acid sequence shown in any one of SEQ ID NO: 45-46, 63, 64, 68 and 77. The coronavirus multivalent vaccine according to claim 37, wherein the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 53, 54 or 72, ( 2) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 61, 62 or 76, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 63, 64 or 77. The coronavirus multivalent vaccine of claim 38, wherein the fusion protein comprising the amino acid sequence shown in SEQ ID NO: 53, 54 or 72, or the fusion protein comprising the amino acid sequence shown in SEQ ID NO: 61, 62 or 76 shown The mass ratio of the fusion protein of the amino acid sequence and the fusion protein comprising the amino acid sequence shown in SEQ ID NO: 63, 64 or 77 is (1-5): (1-5): (1-5); or the mass ratio The ratio is 1:1:1. The coronavirus multivalent vaccine according to claim 38 or 39, wherein the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 53, (2) A fusion protein comprising the amino acid sequence shown in SEQ ID NO: 61, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 63. The coronavirus multivalent vaccine according to claim 38 or 39, wherein the coronavirus multivalent vaccine comprises: (1) a fusion protein comprising the amino acid sequence shown in SEQ ID NO: 72, (2) A fusion protein comprising the amino acid sequence shown in SEQ ID NO:76, and (3) a fusion protein comprising the amino acid sequence shown in SEQ ID NO:77. The coronavirus multivalent vaccine according to any one of claims 1 to 41, characterized in that the coronavirus multivalent vaccine further includes a pharmaceutically acceptable carrier and/or adjuvant. The use of the coronavirus multivalent vaccine according to any one of claims 1 to 42 in the preparation of drugs for preventing or treating coronavirus infection or in the prevention or treatment of coronavirus infection; or, the coronavirus infection is SARS-CoV- 2. SARS-CoV or MERS-CoV infection; or, the coronavirus infection is an infection by the original strain of SARS-CoV-2 or its mutant strain; or, the coronavirus infection is an infection by 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 strain, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant. A method for preventing or treating coronavirus infection, characterized in that it comprises administering an effective amount of the coronavirus multivalent vaccine according to any one of claims 1 to 42 to a patient in need; 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.