CN115785232A

CN115785232A - Fusion protein for preventing or treating coronavirus infection, spike protein nanoparticle and application thereof

Info

Publication number: CN115785232A
Application number: CN202211001505.0A
Authority: CN
Inventors: 苏华飞; 郑丹丹; 冯旭; 黄贤明; 李胜峰
Original assignee: Bio Thera Solutions Ltd
Current assignee: Bio Thera Solutions Ltd
Priority date: 2021-08-20
Filing date: 2022-08-19
Publication date: 2023-03-14
Also published as: WO2023020623A1

Abstract

The invention provides an extracellular domain containing mutation of coronavirus Spike protein or a truncated fragment thereof, a fusion protein, spike protein nanoparticles and application thereof for preventing or treating coronavirus infection.

Description

Fusion protein for preventing or treating coronavirus infection, spike protein nanoparticle and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to fusion protein and Spike protein nanoparticles for preventing or treating coronavirus infection and application thereof.

Background

Coronaviruses are nonsegmented single-stranded positive-strand RNA viruses, which are classified into four genera, alpha, beta, gamma and delta, according to the serotype and the genomic characteristics, and are named because the virus envelope has protrusions extending around, and is shaped like a corolla. The novel coronavirus (SARS-CoV-2 or 2019-nCoV) discovered in 2019 belongs to a novel coronavirus of beta genus, and has an envelope, a round or oval particle, usually polymorphism and a diameter of 60-140nm. Current studies show that SARS-CoV-2 has a high homology with SARS-CoV.

The novel coronavirus pneumonia COVID-19 is mainly transmitted through respiratory tract, and may also be transmitted by contact. The population is common and susceptible, the disease of the old and the people with basic diseases is serious after infection, and children and infants also have diseases. Based on current epidemiological investigations, the latency of the new coronaviruses is generally 1-14 days, mostly 3-7 days. The main clinical symptoms of the infected patients are fever, hypodynamia and dry cough, and the symptoms of upper respiratory tract such as nasal obstruction, watery nasal discharge and the like are rare. In the early stage of onset, the total white blood cell count of a patient is normal or reduced, or the number of lymphocytes is reduced, and liver enzyme, myozyme and myoglobin are increased in part of the patients. The chest image shows that the patient presents multiple small spot images and interstitial changes in early stage, and the extrapulmonary zone is obvious; further, the lung is developed into double-lung multiple-wear vitreous shadows and infiltrative shadows, the serious patients can have lung consolidation and breathing difficulty, and Acute Respiratory Distress Syndrome (ARDS), shock and various tissue injuries and dysfunctions of lung tissues, heart and kidney occur. The prognosis of most patients with mild infection is good, and severe patients are often critically ill or even die.

Recently, fundamental, clinical and epidemiological studies on COVID-19 have been published or published, and there is a strong need in the art for effective vaccines against coronaviruses.

Disclosure of Invention

The present invention provides mutant coronavirus Spike (Spike) protein extracellular domains or truncated fragments thereof comprising a stabilizable protein structure, and fusion proteins comprising mutant coronavirus Spike protein extracellular domains or truncated fragments thereof. The invention also provides a coronavirus vaccine containing the nanoparticle formed by fusing and self-assembling the mutant coronavirus Spike protein extracellular domain or the truncated fragment thereof and the monomeric ferritin subunit, and the coronavirus vaccine can induce stronger neutralizing antibody response to coronavirus.

The virus particles first bind to a protein called angiotensin converting enzyme 2 (ACE 2) on the surface of lung epithelial cells via the Receptor Binding Domain (RBD) in the S1 subunit of the Spike protein (S protein or Spike protein) on their surface. After the RBD binds to the receptor and is hydrolyzed by proteases, the S2 subunit at the C-terminus of the S protein is exposed and embedded in the serous or endocytic membrane. The heptad repetitive sequence 1 (HR 1) and the heptad repetitive sequence 2 (HR 2) in the S2 subunit interact with each other to form a six-helix bundle (6-HB) fusion core, so that the virus shell is fused with the cell membrane, and SARS-CoV or SARS-CoV-2 enters into the cell and is synthesized into a new virus particle by the cell; the new viral particles are released outside the cell and infect the surrounding normal cells in the same way. The fusion protein, the nanoparticle and the vaccine of the invention can induce stronger neutralizing antibody response to coronavirus.

In some embodiments, there is provided a coronavirus Spike protein extracellular domain or a truncated fragment thereof comprising a mutation comprising: 1) Mutating RRAR to GSAS; 2) There is a discontinuity in the turn region between HR1 and the central helical region (CH) that prevents HR1 and CH from forming a straight helix during fusion.

In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turn-around region between HR1 and CH.

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

In some embodiments, the truncated fragment of the extracellular domain of the mutant-containing coronavirus Spike protein is C-terminally truncated by 5-80 amino acid residues as compared to the full-length extracellular domain of the coronavirus Spike protein. In some embodiments, the truncated fragment of the extracellular domain of the mutant-containing coronavirus Spike protein is C-terminally truncated by 20-76 amino acid residues as compared to the full-length extracellular domain of the coronavirus Spike protein. In some embodiments, the truncated fragment of the extracellular domain of the mutant-containing coronavirus Spike protein is C-terminally truncated by 70 amino acid residues as compared to the full-length extracellular domain of the coronavirus Spike protein.

In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, or MERS-CoV.

In some embodiments, the coronavirus is wild-type SARS-CoV-2 or a variant thereof.

In some embodiments, the coronavirus is a wild-type SARS-CoV-2, SARS-CoV-2Alpha variant, SARS-CoV-2Beta variant, SARS-CoV-2Gamma variant, SARS-CoV-2Delta variant, SARS-CoV-2Kappa variant, SARS-CoV-2Epsilon variant, SARS-CoV-2Lambda variant, or SARS-CoV-2Omicron variant.

In some embodiments, the mutant-containing coronavirus Spike protein extracellular domain or truncated fragment thereof comprises an amino acid sequence set forth in any one of SEQ ID NOs 3-4, 6-7, 9-12, 32-35, 78-83, or an amino acid sequence having at least 80% or at least 90% identity to an amino acid sequence set forth in any one of SEQ ID NOs 3-4, 6-7, 9-12, 32-35, 78-83, or an amino acid sequence having one or more conservative amino acid substitutions to an amino acid sequence set forth in any one of SEQ ID NOs 3-4, 6-7, 9-12, 32-35, 78-83.

Also provided in some embodiments is a fusion protein comprising an extracellular domain of a mutant-containing coronavirus Spike protein described herein or a truncated fragment thereof.

Some embodiments provide a fusion protein comprising an extracellular domain of a mutation-containing coronavirus Spike protein or a truncated fragment thereof and a monomeric subunit protein as described herein linked by a linker. In some embodiments, the monomeric subunit protein is a self-assembled monomeric subunit protein. In some embodiments, the monomeric subunit protein is a monomeric ferritin subunit. In some embodiments, the fusion protein is a polypeptide comprising a mutant coronavirus Spike protein extracellular domain or a truncated fragment thereof linked at its C-terminus to the N-terminus of a monomeric subunit protein via a linker. In some embodiments, the fusion protein is formed by linking the C-terminus of the extracellular domain of the mutant-containing coronavirus Spike protein or a truncated fragment thereof to the N-terminus of the monomeric ferritin subunit via a linker.

In some embodiments, the linker is a GS linker. In some embodiments, the linker is selected from the group consisting of GS, GGS, GGGS, GGGGS, SGGGS, gggggg, GGSs, (ggggggs) ₂ ，(GGGGS) ₃ Or any combination thereof. In some embodiments, the linker is (G) _m S) _n Wherein each m is independently 1,2, 3, 4, or 5,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 fusion protein further comprises an N-terminal signal peptide. In some embodiments, the signal peptide is selected from the group consisting of CSP, mschito, MF- α, pho1, HBM, t-pA, and the signal peptide of IL-3. In some embodiments, the N-terminal signal peptide comprises an amino acid sequence as set forth in SEQ ID No. 2 or 5, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence set forth in SEQ ID No. 2 or 5, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID No. 2 or 5.

In some embodiments, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, algal ferritin, insect ferritin, fungal ferritin, or mammalian ferritin. In some embodiments, the monomeric ferritin subunit is a helicobacter pylori non-heme monomeric ferritin subunit. In some embodiments, the 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 shown as SEQ ID No. 14, or an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence shown as SEQ ID No. 14, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown as SEQ ID No. 14.

In some embodiments, a fusion protein is provided, comprising an extracellular domain of a mutant-containing wild-type SARS-CoV-2 Spike protein or a truncated fragment thereof and a monomeric subunit protein joined by a linker. In some embodiments, the fusion protein comprises an extracellular domain of a wild-type SARS-CoV-2 Spike protein containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit joined by a linker. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) The double mutation K986P/V987P is present in the turn-around region between HR1 and CH.

In some embodiments, a fusion protein is provided comprising an extracellular domain of a mutant SARS-CoV-2Alpha variant Spike protein, or a truncated fragment thereof, and a monomeric subunit protein, joined by a linker. In some embodiments, the fusion protein comprises an extracellular domain of a mutant SARS-CoV-2Alpha variant Spike protein, or a truncated fragment thereof, and a monomeric ferritin subunit, joined by a linker. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turn-around region between HR1 and CH.

In some embodiments, a fusion protein is provided comprising an extracellular domain of a mutant SARS-CoV-2Beta variant Spike protein, or a truncated fragment thereof, and a monomeric subunit protein, joined by a linker. In some embodiments, the fusion protein comprises an extracellular domain of a mutant SARS-CoV-2Beta variant Spike protein or a truncated fragment thereof and a monomeric ferritin subunit joined by a linker. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turn-around region between HR1 and CH.

In some embodiments, a fusion protein is provided comprising an extracellular domain of a mutant SARS-CoV-2Gamma variant Spike protein, or a truncated fragment thereof, and a monomeric subunit protein, linked by a linker. In some embodiments, the fusion protein comprises an extracellular domain of a mutant SARS-CoV-2Gamma variant Spike protein, or a truncated fragment thereof, and a monomeric ferritin subunit joined by a linker. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turn-around region between HR1 and CH.

In some embodiments, a fusion protein is provided comprising an extracellular domain of a mutant SARS-CoV-2Delta variant Spike protein, or a truncated fragment thereof, and a monomeric subunit protein, joined by a linker. In some embodiments, the fusion protein comprises an extracellular domain of a SARS-CoV-2Delta variant Spike protein containing a mutation, or a truncated fragment thereof, and a monomeric ferritin subunit joined by a linker. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) The double mutation K986P/V987P is present in the turn-around region between HR1 and CH.

In some embodiments, a fusion protein is provided comprising an extracellular domain of a mutant SARS-CoV-2Kappa variant Spike protein or a truncated fragment thereof and a monomeric subunit protein linked by a linker. In some embodiments, the fusion protein comprises an extracellular domain of a mutant SARS-CoV-2Kappa variant Spike protein or a truncated fragment thereof and a monomeric ferritin subunit joined by a linker. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turn-around region between HR1 and CH.

In some embodiments, a fusion protein is provided comprising an extracellular domain of a mutant SARS-CoV-2Epsilon variant Spike protein, or a truncated fragment thereof, and a monomeric subunit protein, joined by a linker. In some embodiments, the fusion protein comprises an extracellular domain of a mutant SARS-CoV-2Epsilon variant Spike protein, or a truncated fragment thereof, and a monomeric ferritin subunit joined by a linker. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turn-around region between HR1 and CH.

In some embodiments, a fusion protein is provided comprising an extracellular domain of a mutant SARS-CoV-2Lambda variant Spike protein, or a truncated fragment thereof, and a monomeric subunit protein, joined by a linker. In some embodiments, the fusion protein comprises an extracellular domain of a mutant SARS-CoV-2Lambda variant Spike protein, or a truncated fragment thereof, and a monomeric ferritin subunit joined by a linker. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turn-around region between HR1 and CH.

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

In some embodiments, the fusion protein comprises a mutant-containing coronavirus Spike protein extracellular domain and a monomeric ferritin subunit connected by a linker, the mutant-containing coronavirus Spike protein extracellular domain comprising an amino acid sequence set forth in any one of SEQ ID NOs 3-4, 9-10, 32-33, 78, 80, 82, and the monomeric ferritin subunit comprising an amino acid sequence set forth in SEQ ID No. 14; the mutant coronavirus Spike protein extracellular domain is connected with the monomer ferritin subunit through a linker as shown in SEQ ID NO. 15.

In some embodiments, the fusion protein comprises a truncated fragment comprising the extracellular domain of a mutant coronavirus Spike protein comprising an amino acid sequence as set forth in any one of SEQ ID NOs 6-7, 11-12, 34-35, 79, 81, 83 and a monomeric ferritin subunit comprising an amino acid sequence as set forth in SEQ ID No. 14, connected by a linker; the truncated fragment containing the mutant coronavirus Spike protein extracellular domain is connected with the monomer ferritin subunit through a linker as shown in SEQ ID NO. 15.

In some embodiments, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs 16-23, 26-29, 41-44, 66-67, or an amino acid sequence having at least 80% or at least 90% identity as compared to an amino acid sequence set forth in any one of SEQ ID NOs 16-23, 26-29, 41-44, 66-67, or an amino acid sequence having one or more conservative amino acid substitutions as compared to an amino acid sequence set forth in any one of SEQ ID NOs 16-23, 26-29, 41-44, 66-67.

In some embodiments, a fusion protein is provided comprising a coronavirus Spike protein S1 subunit and a monomeric subunit protein joined by a linker. In some embodiments, the monomeric subunit protein is a self-assembled monomeric subunit protein. In some embodiments, the monomeric subunit protein is a monomeric ferritin subunit. In some embodiments, the fusion protein is a coronavirus Spike protein S1 subunit linked at its C-terminus to the N-terminus of a monomeric subunit protein by a linker.

In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, or MERS-Cov. In some embodiments, the coronavirus is wild-type SARS-CoV-2 or a variant thereof. In some embodiments, the coronavirus is a wild-type SARS-CoV-2, SARS-CoV-2Alpha variant, SARS-CoV-2Beta variant, SARS-CoV-2Gamma variant, SARS-CoV-2Delta variant, SARS-CoV-2Kappa variant, SARS-CoV-2Epsilon variant, SARS-CoV-2Lambda variant, or SARS-CoV-2Omicron variant.

In some embodiments, a fusion protein is provided comprising a wild-type SARS-CoV-2 Spike protein S1 subunit and a monomeric ferritin subunit joined by a linker. In some embodiments, the fusion protein is a linkage of the C-terminus of the S1 subunit of the wild-type SARS-CoV-2 Spike protein to the N-terminus of the monomeric ferritin subunit via a linker.

In some embodiments, a fusion protein is provided, comprising a SARS-CoV-2Alpha variant Spike protein S1 subunit and a monomeric ferritin subunit joined by a linker. In some embodiments, the fusion protein is formed by linking the C-terminus of the S1 subunit of the SARS-CoV-2Alpha variant Spike protein to the N-terminus of the monomeric ferritin subunit via a linker.

In some embodiments, a fusion protein is provided comprising a SARS-CoV-2Beta variant Spike protein S1 subunit and a monomeric ferritin subunit joined by a linker. In some embodiments, the fusion protein is formed by linking the C-terminus of the S1 subunit of the Spike protein of the SARS-CoV-2Beta variant to the N-terminus of the monomeric ferritin subunit via a linker.

In some embodiments, a fusion protein is provided comprising a SARS-CoV-2Gamma variant Spike protein S1 subunit and a monomeric ferritin subunit joined by a linker. In some embodiments, the fusion protein is formed by linking the C-terminus of the S1 subunit of the Spike protein of the SARS-CoV-2Gamma variant to the N-terminus of the monomeric ferritin subunit via a linker.

In some embodiments, a fusion protein is provided comprising a SARS-CoV-2Delta variant Spike protein S1 subunit and a monomeric ferritin subunit joined by a linker. In some embodiments, the fusion protein is formed by linking the C-terminus of the S1 subunit of the Spike protein of the SARS-CoV-2Delta variant to the N-terminus of the monomeric ferritin subunit via a linker.

In some embodiments, a fusion protein is provided comprising a SARS-CoV-2Kappa variant Spike protein S1 subunit and a monomeric ferritin subunit joined by a linker. In some embodiments, the fusion protein is formed by linking the C-terminus of the S1 subunit of the Spike protein of the SARS-CoV-2Kappa variant to the N-terminus of the monomeric ferritin subunit via a linker.

In some embodiments, a fusion protein is provided comprising a SARS-CoV-2Epsilon variant Spike protein S1 subunit and a monomeric ferritin subunit joined by a linker. In some embodiments, the fusion protein is a SARS-CoV-2Epsilon variant Spike protein S1 subunit C-terminus linked to the N-terminus of the monomeric ferritin subunit through a linker.

In some embodiments, a fusion protein is provided comprising a SARS-CoV-2Lambda variant Spike protein S1 subunit and a monomeric ferritin subunit joined by a linker. In some embodiments, the fusion protein is formed by linking the C-terminus of the S1 subunit of the SARS-CoV-2Lambda variant Spike protein to the N-terminus of the monomeric ferritin subunit via a linker.

In some embodiments, a fusion protein is provided comprising a SARS-CoV-2Omicron variant Spike protein S1 subunit and a monomeric ferritin subunit joined by a linker. In some embodiments, the fusion protein is a SARS-CoV-2Omicron variant Spike protein S1 subunit C-terminus linked to the N-terminus of the monomeric ferritin subunit by a linker.

In some embodiments, the fusion protein comprises a coronavirus Spike protein S1 subunit and a monomeric ferritin subunit connected by a linker, the coronavirus Spike protein S1 subunit comprising an amino acid sequence set forth as SEQ ID NO 13 or 36, and the monomeric ferritin subunit comprising an amino acid sequence set forth as SEQ ID NO 14; the coronavirus Spike protein S1 subunit is connected with the monomer ferritin subunit through a joint shown as SEQ ID NO. 15.

In some embodiments, the fusion protein is a fusion protein of the C-terminus of the S1 subunit of the coronavirus Spike protein shown in SEQ ID NO 13 or 36 linked to the N-terminus of the H.pylori non-heme monomeric ferritin subunit shown in SEQ ID NO 14 via a linker GGGGS, while simultaneously binding the C-terminus of the S1 subunit of the coronavirus Spike protein to the N-terminus of the H.pylori non-heme monomeric ferritin subunit shown in SEQ ID NO 14 with a signal peptide: MEFGLSLVFLVLILKGVQC replaces the original signal peptide: MFVFLVLLPLVSSQ.

In some embodiments, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs 24-25, 30, 39-40, 65, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence set forth in any one of SEQ ID NOs 24-25, 30, 39-40, 65, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in any one of SEQ ID NOs 24-25, 30, 39-40, 65.

In some embodiments, a fusion protein is provided comprising a conserved fragment of a coronavirus Spike protein and a monomeric subunit protein linked by a linker. In some embodiments, the monomeric subunit protein is a self-assembled monomeric subunit protein. In some embodiments, the monomeric subunit protein is a monomeric ferritin subunit. In some embodiments, the fusion protein is a conserved fragment of a coronavirus Spike protein linked at the C-terminus to the N-terminus of the monomeric subunit protein by a linker.

In some embodiments, the linker is a GS linker. In some embodiments, the linker is selected from the group consisting of GS, GGS, GGGS, GGGGS, SGGGS, gggggg, GGSs, (ggggggs) ₂ ，(GGGGS) ₃ Or any combination thereof. In some embodiments, the linker is (G) _m S) _n Wherein each m is independently 1,2, 3, 4, or 5,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 . At one endIn some embodiments, the linker is (GGGGS) ₅ 。

In some embodiments, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, algal ferritin, insect ferritin, fungal ferritin, or mammalian ferritin. In some embodiments, the monomeric ferritin subunit is a helicobacter pylori non-heme monomeric ferritin subunit. In some embodiments, the 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 an amino acid sequence shown as SEQ ID No. 14, or an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence shown as SEQ ID No. 14, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the amino acid sequence shown as SEQ ID No. 14.

In some embodiments, a fusion protein is provided comprising a conserved fragment of wild-type SARS-CoV-2 Spike protein and a monomeric subunit protein linked by a linker. In some embodiments, the fusion protein comprises a conserved fragment of wild-type SARS-CoV-2 Spike protein and a monomeric ferritin subunit joined by a linker.

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

In some embodiments, a fusion protein is provided comprising a conserved fragment of the SARS-CoV-2Beta variant Spike protein and a monomeric subunit protein linked by a linker. In some embodiments, the fusion protein comprises a conserved fragment of the SARS-CoV-2Beta variant Spike protein and a monomeric ferritin subunit joined by a linker.

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

In some embodiments, a fusion protein is provided comprising a conserved fragment of the SARS-CoV-2Delta variant Spike protein and a monomeric subunit protein linked by a linker. In some embodiments, the fusion protein comprises a conserved fragment of the SARS-CoV-2Delta variant Spike protein and a monomeric ferritin subunit joined by a linker.

In some embodiments, a fusion protein is provided comprising a conserved fragment of the Spike protein of a SARS-CoV-2Kappa variant strain and a monomeric subunit protein linked by a linker. In some embodiments, the fusion protein comprises a conserved fragment of the SARS-CoV-2Kappa variant Spike protein and a monomeric ferritin subunit joined by a linker.

In some embodiments, a fusion protein is provided comprising a conserved fragment of the SARS-CoV-2Epsilon variant Spike protein and a monomeric subunit protein linked by a linker. In some embodiments, the fusion protein comprises a conserved fragment of the SARS-CoV-2Epsilon variant Spike protein and a monomeric ferritin subunit joined by a linker.

In some embodiments, a fusion protein is provided comprising a conserved fragment of SARS-CoV-2Lambda variant Spike protein and a monomeric subunit protein joined by a linker. In some embodiments, the fusion protein comprises a conserved fragment of SARS-CoV-2Lambda variant Spike protein and a monomeric ferritin subunit joined by a linker.

In some embodiments, a fusion protein is provided comprising a conserved fragment of the SARS-CoV-2Omicron variant Spike protein and a monomeric subunit protein linked by a linker. In some embodiments, the fusion protein comprises a conserved fragment of the SARS-CoV-2Omicron variant Spike protein and a monomeric ferritin subunit joined by a linker.

In some embodiments, the fusion protein comprises a conserved fragment of a coronavirus Spike protein comprising an amino acid sequence as set forth in SEQ ID No. 37 and a monomeric ferritin subunit comprising an amino acid sequence as set forth in SEQ ID No. 14, connected by a linker; the conserved fragment of the coronavirus Spike protein is connected with the monomeric ferritin subunit through a joint shown as SEQ ID NO. 15.

In some embodiments, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs 45-46, 68, or an amino acid sequence having at least 80% or at least 90% identity compared to an amino acid sequence set forth in any one of SEQ ID NOs 45-46, 68, or an amino acid sequence having one or more conservative amino acid substitutions compared to an amino acid sequence set forth in any one of SEQ ID NOs 45-46, 68.

In some embodiments, a fusion protein is provided comprising an extracellular domain of a mutation-containing coronavirus Spike protein described herein or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto. In some embodiments, the fusion protein is a C-terminus of the extracellular domain of a mutation-containing coronavirus Spike protein described herein or a truncated fragment thereof linked to the N-terminus of an Fc fragment of an immunoglobulin.

In some embodiments, the Fc fragment of the immunoglobulin is from an IgG, igM, igA, igE, or IgD. In some embodiments, the Fc fragment of the immunoglobulin is from IgG1, igG2, igG3, or IgG4. In some embodiments, the Fc fragment of the immunoglobulin is an Fc fragment of IgG 1. In some embodiments, the Fc fragment of the immunoglobulin is an Fc fragment of human IgG 1. In some embodiments, the Fc fragment of the immunoglobulin comprises an amino acid sequence as set forth in SEQ ID No. 38, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence set forth in SEQ ID No. 38, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID No. 38.

In some embodiments, a fusion protein is provided comprising an extracellular domain of a wild-type SARS-CoV-2 Spike protein containing a mutation or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turn-around region between HR1 and CH.

In some embodiments, a fusion protein is provided comprising an extracellular domain of a Spike protein of a SARS-CoV-2Alpha variant containing a mutation or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turn-around region between HR1 and CH.

In some embodiments, a fusion protein is provided comprising an extracellular domain of a Spike protein of a SARS-CoV-2Beta variant containing a mutation or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) The double mutation K986P/V987P is present in the turn-around region between HR1 and CH.

In some embodiments, a fusion protein is provided, comprising an extracellular domain of Spike protein of a SARS-CoV-2Gamma variant containing a mutation or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turn-around region between HR1 and CH.

In some embodiments, a fusion protein is provided, comprising an extracellular domain of a SARS-CoV-2Delta variant Spike protein containing a mutation or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turn-around region between HR1 and CH.

In some embodiments, a fusion protein is provided comprising an extracellular domain of Spike protein of a SARS-CoV-2Kappa variant strain containing a mutation or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turn-around region between HR1 and CH.

In some embodiments, a fusion protein is provided comprising an extracellular domain of a SARS-CoV-2Epsilon variant Spike protein containing a mutation or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) The double mutation K986P/V987P is present in the turn-around region between HR1 and CH.

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

In some embodiments, a fusion protein comprising an extracellular domain of Spike protein of a SARS-CoV-2Omicron variant containing mutation or a truncated fragment thereof and an Fc fragment of immunoglobulin linked thereto is provided. In some embodiments, the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a double mutation K986P/V987P in the turn-around region between HR1 and CH.

In some embodiments, the fusion protein comprises an amino acid sequence as set forth 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 as compared to an amino acid sequence set forth 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 as compared to an amino acid sequence set forth in any one of SEQ ID NOs 47-54, 59-62, 69-72, 75-76.

In some embodiments, a fusion protein is provided comprising a coronavirus Spike protein S1 subunit and an Fc fragment of an immunoglobulin linked thereto. In some embodiments, the fusion protein is a coronavirus Spike protein S1 subunit linked at its C-terminus to the N-terminus of an Fc fragment of an immunoglobulin.

In some embodiments, a fusion protein is provided comprising the S1 subunit of a wild-type SARS-CoV-2 Spike protein and an Fc fragment of an immunoglobulin linked thereto.

In some embodiments, a fusion protein is provided comprising the S1 subunit of the Spike protein of a SARS-CoV-2Alpha variant and an Fc fragment of an immunoglobulin linked thereto.

In some embodiments, a fusion protein is provided, comprising SARS-CoV-2Beta variant Spike protein S1 subunit and an Fc fragment of an immunoglobulin linked thereto.

In some embodiments, a fusion protein is provided comprising the S1 subunit of the Spike protein of a SARS-CoV-2Gamma variant strain and an Fc fragment of an immunoglobulin linked thereto.

In some embodiments, a fusion protein is provided comprising the S1 subunit of the Spike protein of a SARS-CoV-2Delta variant and an Fc fragment of an immunoglobulin linked thereto.

In some embodiments, a fusion protein is provided comprising the S1 subunit of the Spike protein of a SARS-CoV-2Kappa variant and an Fc fragment of an immunoglobulin linked thereto.

In some embodiments, a fusion protein is provided comprising the S1 subunit of the Spike protein of a SARS-CoV-2Epsilon variant and an Fc fragment of an immunoglobulin linked thereto.

In some embodiments, a fusion protein is provided comprising the S1 subunit of the Spike protein of a SARS-CoV-2Lambda variant and an Fc fragment of an immunoglobulin linked thereto.

In some embodiments, a fusion protein is provided comprising the S1 subunit of the Spike protein of a SARS-CoV-2Omicron variant and an Fc fragment of an immunoglobulin linked thereto.

In some embodiments, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs 55-58, 73-74, or an amino acid sequence having at least 80% or at least 90% identity compared to an amino acid sequence set forth in any one of SEQ ID NOs 55-58, 73-74, or an amino acid sequence having one or more conservative amino acid substitutions compared to an amino acid sequence set forth in any one of SEQ ID NOs 55-58, 73-74.

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

In some embodiments, the Fc fragment of the immunoglobulin is from IgG, igM, igA, igE, or IgD. In some embodiments, the Fc fragment of the immunoglobulin is from an IgG1, an IgG2, an IgG3, or an IgG4. In some embodiments, the Fc fragment of the immunoglobulin is an Fc fragment of IgG 1. In some embodiments, the Fc fragment of the immunoglobulin is an Fc fragment of human IgG 1. In some embodiments, the Fc fragment of the immunoglobulin comprises an amino acid sequence as set forth in SEQ ID No. 38, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence set forth in SEQ ID No. 38, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID No. 38.

In some embodiments, a fusion protein is provided comprising a conserved fragment of wild-type SARS-CoV-2 Spike protein and an Fc fragment of an immunoglobulin linked thereto.

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

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

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

In some embodiments, a fusion protein is provided, comprising a conserved fragment of SARS-CoV-2Delta variant Spike protein and an Fc fragment of an immunoglobulin linked thereto.

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

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

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

In some embodiments, a fusion protein is provided comprising a conserved fragment of Spike protein of a SARS-CoV-2Omicron variant and an Fc fragment of an immunoglobulin linked thereto.

In some embodiments, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs 63-64, 77, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence set forth in any one of SEQ ID NOs 63-64, 77, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in any one of SEQ ID NOs 63-64, 77.

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

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

In some embodiments, the one or more conservative amino acid substitutions is about 1, about 2, about 3, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 13, about 14, about 15, about 17, about 18, about 19, about 20, about 22, about 24, about 25, about 27, about 30, about 32, about 33, about 36 conservative amino acid substitutions, or a range between any two of these values (including the endpoints) or any value therein.

In some embodiments, a polynucleotide is provided that encodes the extracellular domain of a mutant-containing coronavirus Spike protein or a truncated fragment thereof, or a fusion protein as described herein.

In some embodiments, an expression vector is provided comprising a polynucleotide encoding an extracellular domain of a mutant-containing coronavirus Spike protein or a truncated fragment or fusion protein thereof as described herein.

In some embodiments, a cell is provided that can express the mutant-containing coronavirus Spike protein extracellular domain described herein or a truncated fragment thereof. In some embodiments, the cell comprises one or more polynucleotides encoding a fusion protein described herein or an expression vector comprising a polynucleotide encoding a fusion protein described herein. In some embodiments, the cell is an isolated cell. In some embodiments, the cell is a CHO cell, HEK293 cell, cos1 cell, cos7 cell, CV1 cell, or murine L cell.

In some embodiments, the fusion protein comprises a mutant-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker, the fusion protein comprising the following features:

the mutation comprises: 1) A mutation which inactivates the S1/S2 cleavage site; 2) There is a mutation in the turn region between HR1 and CH that prevents HR1 and CH from forming a straight helix during fusion; and/or

The C-terminal of the mutant-containing coronavirus Spike protein extracellular domain or the truncated fragment thereof is connected with the monomer ferritin subunit through a linker; and/or

The joint is (G) _m S) _n Wherein each m is independently 1,2, 3, 4, or 5,n is 1,2, 3, 4, or 5; and/or

The monomeric ferritin subunit is helicobacter pylori monomeric ferritin subunit, and comprises an amino acid sequence shown as SEQ ID NO. 14, or an amino acid sequence with at least 80% or at least 90% of identity compared with the amino acid sequence shown as SEQ ID NO. 14, or an amino acid sequence with one or more conservative amino acid substitutions compared with the amino acid sequence shown as SEQ ID NO. 14.

In some embodiments, there is provided a Spike protein nanoparticle comprising a fusion protein described herein.

In some embodiments, a coronavirus vaccine is provided comprising a fusion protein described herein and/or a Spike protein nanoparticle comprising the fusion protein. In some embodiments, the coronavirus vaccine further comprises a pharmaceutically acceptable carrier and/or adjuvant. The invention also provides a coronavirus vaccine. In some embodiments, the coronavirus vaccine comprises a fusion protein described herein and a pharmaceutically acceptable carrier and/or adjuvant. In some embodiments, the coronavirus vaccine comprises a Spike protein nanoparticle described herein and a pharmaceutically acceptable carrier and/or adjuvant.

The invention also provides a method of prevention or treatment and uses thereof. In some embodiments, the invention provides methods for preventing or treating a coronavirus infection, the methods comprising administering to a patient in need thereof an effective amount of a fusion protein, a Spike protein nanoparticle, or a coronavirus vaccine as described herein. In some embodiments, there is provided the use of the fusion proteins, spike protein nanoparticles, or coronavirus vaccines described herein for the prevention or treatment of SARS or COVID-19. In some embodiments, there is provided the use of the fusion protein or Spike protein nanoparticles described herein in the preparation of a vaccine for the prevention or treatment of SARS-CoV-2 infection. In some embodiments, the coronavirus infection is a SARS-CoV-2, SARS-CoV, or MERS-Cov infection. In some embodiments, the coronavirus infection is a wild-type SARS-CoV-2 infection or a variant thereof. In some embodiments, the coronavirus infection is infection by a wild-type SARS-CoV-2, SARS-CoV-2Alpha variant, SARS-CoV-2Beta variant, SARS-CoV-2Gamma variant, SARS-CoV-2Delta variant, SARS-CoV-2Kappa variant, SARS-CoV-2Epsilon variant, SARS-CoV-2Lambda variant, or SARS-CoV-2Omicron variant.

Drawings

FIG. 1 is a binding curve of a fusion protein with human ACE 2; FIG. 1a is the binding curve of fusion protein D and human ACE2, and FIG. 1b is the binding curve of fusion protein G and human ACE 2.

FIG. 2 is serum anti-Spike protein IgG titers, the bar graph represents the Geometric Mean (GMT) of the titers; in the figure, wildtype represents WT-Spike-His, delta represents Delta-Spike-His, and Omicron represents Omicron-Spike-His.

FIG. 3 shows anti-pseudovirus neutralization titers (IC) ₅₀ ) (ii) a In the figure, wildtype represents SARS-CoV-2 Spike pseudovirus, delta represents SARS-COV-2 Spike (B.1.617.2) pseudovirus, and Omicron represents SARS-COV-2 Spike (B.1.1.529) pseudovirus.

FIG. 4 is serum anti-Spike protein IgG titers, bar graph representing Geometric Mean of Titers (GMT); wherein, fig. 4a, fig. 4c and fig. 4e are titers 14 days (day 14) after the first administration, and fig. 4b, fig. 4d and fig. 4f are titers 14 days (day 35) after the second administration.

FIG. 5 shows anti-pseudovirus neutralization titers (IC) ₅₀ ) (ii) a In the figure, wildtype represents SARS-CoV-2 Spike pseudovirus, delta represents SARS-COV-2 Spike (B.1.617.2) pseudovirus, and Omicron represents SARS-COV-2 Spike (B.1.1.529) pseudovirus.

Term(s) for

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

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide the skilled artisan with a general definition of many of the terms used in the present invention: scientific Press Science and Technology (Academic Press Dictionary of Science and Technology), morris (ed., academic Press (1 st edition, 1992); oxford Biochemical and Molecular Biology Dictionary (Oxford Dictionary of Biochemistry and Molecular Biology), smith et al (ed.), oxford University Press, revised 2000; chemical encyclopedia Dictionary (encyclopedia Dictionary of Chemistry), kumar (ed.), anmol Publications Pvt Ltd (2002); dictionary of microorganisms and Molecular Biology (Dictionary of Microbiology and Molecular Biology), singleton et al (ed.), john Wiley and Sons (3 rd edition, 2002); chemical Dictionary (Dictionary of Chemistry), hunt (ed), routridge (1 st edition, 1999); the Dictionary of Pharmaceutical Medicine (Dictionary of Pharmaceutical Medicine), nahler (ed.), springer-Verlag Telos (1994); organic Chemistry Dictionary (Dictionary of Organic Chemistry), kumar and Anndand (ed.), anmol Publications Pvt. Co., ltd. (2002); and a biological Dictionary (A Dictionary of Biology) (Oxford Paperback Reference), martin and Hine (ed.), oxford University Press (Oxford University Press) (4 th edition 2000).

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

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

"conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue having a side chain (R group) of similar chemical nature (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Examples of classes of amino acids containing chemically similar side chains include: 1) Aliphatic side chain: glycine, alanine, valine, leucine, and isoleucine; 2) Aliphatic hydroxyl side chain: serine and threonine; 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) Basic side chain: lysine, arginine and histidine; 6) Acidic side chain: aspartic acid and glutamic acid.

The term "polypeptide" is intended to encompass both the singular "polypeptide" and the plural "polypeptide" and refers to a molecule composed of monomers of amino acids linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single chain or multiple chains of two or more amino acids and does not refer to a particular length of the product. Thus, included within the definition of "polypeptide" are peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to two or more amino acid chains, and the term "polypeptide" may be used in place of, or in alternation with, any of the above terms. The term "polypeptide" is also intended to refer to the product of post-expression modification of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modification. The polypeptide may be derived from a natural biological source or produced by recombinant techniques, but it need not be translated from a specified nucleic acid sequence, and it may be produced in any manner, including chemical synthesis.

Unless otherwise indicated, a fusion protein is a recombinant protein comprising amino acid sequences from at least two unrelated proteins that have been joined together by peptide bonds to form a single protein. The amino acid sequences of unrelated proteins may be directly linked to each other, or may be linked using a linker. As used herein, the amino acid sequences of proteins are not related if they are not normally linked together via peptide bonds in their natural environment (e.g., within a cell). For example, typically the amino acid sequence of a bacterial enzyme such as Bacillus stearothermophilus dihydrolipoic acid transacetylase (E2 p) and the amino acid sequence of a coronavirus Spike protein are not linked together by a peptide bond.

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 in each sequence that can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

The term "encoding" as applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide and/or fragments thereof that can be produced by transcription and/or translation in its native state or when manipulated by methods well known to those skilled in the art.

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

The terms "polynucleotide", "polynucleotide" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof. The polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. The following are examples of non-limiting polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), an exon, an intron, a messenger RNA (mRNA), a transfer RNA, ribosomal RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, structural modifications to the nucleotide can be made before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may 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, embodiments of any polynucleotide of the present disclosure include a double-stranded form and each of two complementary single-stranded forms known or predicted to comprise the double-stranded form.

A nucleic acid or polynucleotide sequence (or polypeptide or protein sequence) has a certain percentage (e.g., 90%,95%, 98%, or 99%) of "identity" or "sequence identity" with another sequence, meaning that the percentage of bases (or amino acids) in the two sequences being compared are the same when the sequences are aligned. The percent identity or sequence identity of the alignment can be determined using visual inspection or software programs known in the art, such as the software program described in Current Protocols in Molecular Biology, ausubel et al. Preferably, the alignment is performed using default parameters. One alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both using the following default parameters: geneticcode = standard; filter = none; strand = booth; cutoff =60; expect =10; matrix = BLOSUM62; descriptions =50sequences; sortby = HIGHSCORE; databases = non-redundant; genBank + EMBL + DDBJ + PDB + GenBank CDStranslations + Swi ssProtein + Spupdate + PIR. A biologically equivalent polynucleotide is a polynucleotide having the above specified percentage of identity and encoding a polypeptide having the same or similar biological activity.

The term "isolated" as used herein with respect to a cell, nucleic acid, polypeptide, antibody, etc., e.g., "isolated" DNA, RNA, polypeptide, antibody, refers to a molecule that is separated from one or more other components, e.g., DNA or RNA, respectively, in the natural environment of the cell. 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. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that do not occur in nature, and which do not occur in nature. The term "isolated" is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptides are intended to include both purified and recombinant polypeptides. Isolated polypeptides, antibodies, and the like are typically prepared by at least one purification step. In some embodiments, an isolated nucleic acid, polypeptide, antibody, etc., is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% pure, or ranges between any two of these values (including the endpoints) or any value therein.

The term "recombinant" refers to a polypeptide or polynucleotide, and means a form of a polypeptide or polynucleotide that does not occur in nature, and non-limiting examples may include combinations that produce polynucleotides or polypeptides that do not normally occur.

"antibody," "antigen-binding fragment," refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. The antibody may be a whole antibody and any antigen binding fragment thereof or a single chain thereof. The term "antibody" thus includes any protein or peptide in a molecule that contains at least a portion of an immunoglobulin molecule having biological activity that binds 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 an immune response to a humoral and/or cellular type of the protein upon administration to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein). Unless otherwise indicated, the term "vaccine antigen" is used interchangeably with "protein antigen" or "antigenic polypeptide".

By "neutralizing antibody" is meant an antibody that reduces the infectious titer of an infectious agent by binding to a specific antigen on the infectious 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 relevant antigen, e.g., an antigen that is at least 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to the antigenic surface of the antigen. For antigens from a pathogen, such as a virus, 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 to or identical to mRNA and may be in single-stranded or double-stranded form.

"epitope" refers to an antigenic determinant. These are specific chemical groups or peptide sequences on the molecule that are antigenic such that they elicit a specific immune response, e.g., epitopes are antigenic regions of B and/or T cell responses. Epitopes can be formed of contiguous amino acids or of non-contiguous amino acids juxtaposed by tertiary folding of the protein.

A vaccine refers to a biological product that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, vaccines elicit antigen-specific immune responses against antigens of pathogens, such as viral pathogens, or cellular components associated with pathological conditions. A vaccine can include a polynucleotide (e.g., a nucleic acid encoding a known antigen), a peptide or polypeptide (e.g., a disclosed antigen), a virus, a cell, or one or more cellular components. In some embodiments, the vaccine or vaccine antigen or vaccine composition is expressed from a fusion protein expression vector and self-assembles into nanoparticles that display the antigen polypeptide or protein on the surface.

An effective amount of a vaccine or other agent refers to an amount sufficient to produce a desired response, e.g., elicit an immune response, prevent, reduce, or eliminate signs or symptoms of a disorder or disease (e.g., pneumonia). For example, this may be the amount necessary to inhibit viral replication or measurably alter the external symptoms of viral infection. Typically, this amount will be sufficient to measurably inhibit replication or infectivity of the virus (e.g., SARS-CoV-2). When administered to a subject, a dose will generally be used that achieves a target tissue concentration, which has been shown to achieve inhibition of viral replication in vitro. In some embodiments, an "effective amount" is an amount that treats (including prevents) one or more symptoms and/or underlying causes of a disorder or disease (e.g., treats 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 disorder (e.g., one or more symptoms or signs associated with a coronavirus infection).

Nanoparticles refer to spherical protein shells with diameters of tens of nanometers and well-defined surface geometries. The globular protein shell is formed from identical replicas of non-viral proteins that are capable of self-assembling into nanoparticles having a similar appearance to virus-like particles (VLPs). Examples include Ferritin (FR), which is conserved across species and forms a 24-mer, bacillus stearothermophilus dihydrolipoate transacetylase (E2P), hyperthermophilus dioxytetrahydropteridine synthase (LS) and Thermotoga maritima encapsuin, all of which form a 60-mer. Self-assembled nanoparticles can form spontaneously upon recombinant expression of a protein in a suitable expression system. The methods of nanoparticle production, detection and characterization may use the same techniques developed for VLPs.

Virus-like particles (VLPs) refer to non-replicating viral shells, which are derived from any of a variety of viruses. VLPs typically include one or more viral proteins, such as, but not limited to, those proteins known as capsid, coat, wall, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. In a suitable expression system, VLPs may form spontaneously upon recombinant expression of the protein. Methods of producing particular VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art (e.g., by electron microscopy, biophysical characterization, etc.). See, for example, baker et al (1991) Biophys.J.60:1445-1456 and Hagensee et al (1994) J.Virol.68:4503-4505. For example, VLPs may be identified by density gradient centrifugation and/or by characteristic density bands. Alternatively, a vitrified water sample of the VLP preparation in question may be subjected to cryoelectron microscopy and images recorded under appropriate exposure conditions.

The terms "about" and "approximately" are used interchangeably and refer to the conventional range of error for corresponding numerical values as would be readily understood by a worker skilled in the relevant art. In some embodiments, reference herein to "about" refers to the numerical values recited and ranges of ± 10%, ± 5%, or ± 1% thereof.

"ECMO" refers to the Extracorporeal Membrane Oxygenation (ECMO), which is a medical emergency technology device mainly used to provide continuous Extracorporeal respiration and circulation to patients with severe cardiopulmonary failure to maintain the life of the patients.

The ICU refers to Intensive Care Unit (Intensive Care Unit), and can be used for synchronously carrying out treatment, nursing and rehabilitation, providing isolation places and equipment for patients with severe or coma, providing optimal nursing, comprehensive treatment, medical and nursing combination, early rehabilitation after operation, joint nursing exercise treatment and other services.

"IMV" refers to intermittent commanded ventilation (intermittent ventilation), which is the implementation of periodic volume or pressure ventilation based on a preset time interval, i.e., time trigger. During which the patient is allowed to breathe spontaneously at any set basal pressure level during the commanded ventilation. In spontaneous breathing, the patient may breathe spontaneously with continuous airflow support, or the machine will open an on-demand valve to allow spontaneous breathing. Most ventilators can provide pressure support during spontaneous breathing.

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, and the like. The terms "patient" or "subject" are used interchangeably herein, unless otherwise indicated. Preferably, the subject is a human.

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures, with the object of preventing, slowing, ameliorating, or halting undesirable physiological changes or disorders, such as the progression of a disease, including, but not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration, palliation, alleviation or abolition (whether partial or total) of disease state, extending the expected life span when not treated, and the like, whether detectable or undetectable. Patients in need of treatment include patients already having a condition or disorder, patients susceptible to a condition or disorder, or patients in need of prevention of the condition or disorder, patients who may or are expected to benefit from administration of the Spike protein nanoparticles or pharmaceutical compositions disclosed herein for treatment.

SUMMARY

For SARS-CoV, MERS-CoV and SARS-CoV-2, the viral genome encodes Spike (S), envelope (E), membrane (M) and nucleocapsid (N) structural proteins, where the 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 maintain the RBD in a "standing" state, which facilitates 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 junction 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 helix structure and assemble into a six-helix bundle to fuse the viral and host membranes.

The RBD comprises a core subdomain and a Receptor Binding Motif (RBM). Although the core subdomain is highly similar between the three coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2, their RBMs are significantly different, leading to different receptor specificities: SARS-CoV and SARS-CoV-2 recognize angiotensin converting enzyme 2 (ACE 2), while MERS-CoV binds to dipeptidyl peptidase 4 (DPP 4). Since the S glycoprotein is surface exposed and mediates entry into host cells, it is a major target of post-infection neutralizing antibodies (nabs) and is also the focus of vaccine design. Spike trimers are extensively modified with N-linked glycans, which are important for proper folding and regulation of accessibility to nabs.

The present invention stabilizes the Spike trimer in the pre-fusion conformation with the host cell membrane by 1) a mutation that inactivates the S1/S2 cleavage site and 2) the presence of a mutation in the turn region between HR1 and CH that prevents the formation of a straight helix in HR1 and CH during fusion. In some embodiments, the mutant-containing coronavirus Spike protein extracellular domain or truncated fragment thereof may be displayed on a nanoparticle.

According to the studies and exemplary designs described herein, the present invention provides fusion proteins, spike protein nanoparticles, and vaccine compositions. 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) form carried by viral vectors are useful as coronavirus vaccines. In addition, stable Spike trimers and RBDs presented by nanoparticles can also be used as coronavirus vaccines.

The antigens and vaccines based on coronavirus Spike proteins of the invention have a number of advantageous properties. The Spike trimers described herein are designed to present conserved neutralizing epitopes in their native-like conformation, making Spike trimers useful as antigen vaccines or multivalent display on nanoparticles. The nanoparticle vaccines of the present invention allow Spike trimers derived from different coronaviruses to be displayed on well-known nanoparticles, such as ferritin, E2p and I3-01, in the size range of 12.2 to 25.0nm. All trimer-presenting nanoparticles can be produced with high yield in HEK293 cells, expichho cells, CHO cells. The produced Spike protein nanoparticles can be purified by antibody and Size Exclusion Chromatography (SEC).

Unless otherwise indicated herein, the mutant-containing extracellular domain of a coronavirus Spike protein or a truncated fragment thereof, the coronavirus Spike protein S1 subunit, a conserved fragment of a coronavirus Spike protein, a fusion protein, a Spike protein nanoparticle, an encoded polynucleotide, an expression vector and a host cell of the present invention, and related therapeutic applications can be produced or performed according to the methods exemplified herein or by conventional methods well known in the art.

The order of steps or order for performing certain operations is immaterial unless otherwise indicated, so long as the invention remains operable. Also, two or more steps or operations may be performed simultaneously.

The use of any and all examples, or exemplary language (e.g., "such as" or "including") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Mutant-containing coronavirus Spike protein extracellular domain or truncated fragment thereof

The invention provides mutant-containing extracellular domains of coronavirus Spike proteins or truncated fragments thereof that are useful for the generation of vaccines. The mutated Spike trimers are stabilized by introducing mutations into the extracellular domain of the coronavirus Spike protein or a truncated fragment thereof. Some specific Spike proteins, such as SEQ ID NOS: 1, 8 and 31, of a specific SARS-CoV-2 strain or isolate are exemplified herein. Due to the functional similarity and sequence homology between different isolates or strains of a given coronavirus, mutated Spike proteins or truncated fragments thereof derived from orthologous sequences of other known coronavirus Spike proteins may also be generated according to the mutation strategies described herein. Many known coronavirus Spike protein sequences have been described in the literature. See, e.g., james et al, J.mol.biol.432:3309-25,2020; andersen et al, nat. Med.26:450-452,2020; walls et al, cell 180; zhang et al, J.Proteome Res.19:1351-1360,2020; du et al, expert opin. Ther. Targets 21; 2017; yang et al, viral Immunol.27:543-550,2014; wang et al, anti 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 of the mutated Spike proteins or truncated fragments thereof of the present invention comprise mutations that can enhance the stability of the structure of the Spike protein or truncated fragment thereof prior to fusion with a cell membrane. These mutations include those that inactivate the S1/S2 cleavage site, as well as mutations in the turn around region between HR1 and CH that remove any strain in the turn around region between HR1 and CH, i.e., prevent the formation of a straight helix.

Some mutant-containing coronavirus Spike protein extracellular domains or truncated fragments thereof (shown in SEQ ID NOS: 3-4, 6-7, 9-12, 32-35, 78-83) are derived from SARS-CoV-2 virus that causes COVID-19. These polypeptides contain mutations in the S1/S2 cleavage site which are inactive, as well as mutations in the turn region between HR1 and CH. As an example, the amino acid sequence of the wild-type SARS-CoV-2 Spike protein used for mutation is shown as SEQ ID NO. 1 or as shown as residues 15 to 1213 of SEQ ID NO. 1. In some embodiments, the Spike protein used for mutation may be SEQ ID NOs 1, 8, or 31 or variants thereof, e.g., variants substantially identical thereto or conservatively modified variants. Using amino acid numbering based on the cryo-EM model PDB ID 6VSB or GenBank accession number MN908947.3 as a reference, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Can be achieved by a number of sequence alterations (e.g., deletions or substitutions) within or around the site. As exemplified herein, one mutation that inactivates the S1/S2 cleavage site without affecting the protein structure is to inactivate the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ . In addition to inactivating the S1/S2 cleavage site, a double mutation in the turn region between HR1 and CH may be made that eliminates the strain in the turn region (between HR1 and CH motifs) during fusion by preventing the formation of a straight helix. In some embodiments, the double mutation can be K986G/V987G, K986P/V987P, K986G/V987P or K986P/V987G. In addition to the above mutations in the structure of the stable prefusion Spike protein or truncated fragment thereof, some SARS-CoV-2 Spike proteins or truncated fragments thereof of the invention may contain deletions of most or all of the HR2 domain. Using the exemplary SARS-CoV-2 Spike protein sequence SEQ ID NO 1 for illustrationIt is understood that such deletions may include deletions of residues 1144-1213 as shown in 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 C-terminal of the truncated Spike protein extracellular domain (e.g., SEQ ID NOs: 1, 3-4, 8-10, 31-33, 78, 80, or 82), or a range between any two of these values (including the endpoints), or any value therein. In some embodiments, the C-terminally truncated Spike protein may extend beyond the HR2 domain. In some embodiments, the Spike protein sequence may include an N-terminal signal peptide as set forth in SEQ ID NO 2 or 5.

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

the amino acid sequence of the full-length extracellular domain (ECD) of the wild SARS-CoV-2 Spike protein is shown in SEQ ID NO:1, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Underlined, bolded and italicized.

The amino acid sequence of the full-length extracellular domain a1 of the mutant wild SARS-CoV-2 Spike protein is shown in SEQ ID NO. 3. In sequence, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The amino acid sequence of the full-length extracellular domain a2 of the mutant wild SARS-CoV-2 Spike protein is shown in SEQ ID NO. 4. In sequence, with the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The amino acid sequence of the full-length extracellular domain a3 of the mutant wild SARS-CoV-2 Spike protein is shown in SEQ ID NO. 78. In the sequence, without signal peptide, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The amino acid sequence of the C-end truncated fragment b1 of the extracellular domain of the mutant wild SARS-CoV-2 Spike protein is shown as SEQ ID NO. 6. In the sequence, the C-terminus is truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The amino acid sequence of the C-end truncated fragment b2 of the extracellular domain of the mutant wild SARS-CoV-2 Spike protein is shown as SEQ ID NO. 7. In the sequence, the C-terminus is truncated by 70 amino acid residues, and the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The amino acid sequence of the C-end truncated fragment b3 of the extracellular domain of the mutant wild SARS-CoV-2 Spike protein is shown as SEQ ID NO. 79. In the sequence, the C terminal is truncated by 70 amino acid residues, no signal peptide is contained, and the S1/S2 cutting site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

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

The full-length extracellular domain c1 of the mutant SARS-CoV-2Delta variant Spike protein has the amino acid sequence shown in SEQ ID NO. 9. In sequence, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The full-length extracellular domain c2 of the mutant SARS-CoV-2Delta variant Spike protein has the amino acid sequence shown in SEQ ID NO. 10. In sequence, with the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The full-length extracellular domain c3 of the mutant SARS-CoV-2Delta variant Spike protein has the amino acid sequence shown in SEQ ID NO. 80. In sequence, without signal peptide, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains double mutationsK986P/V987P, underlined and italicized.

The amino acid sequence of the C-terminal truncated segment d1 of the extracellular domain of the mutant SARS-CoV-2Delta variant Spike protein is shown in SEQ ID NO. 11. In the sequence, the C-terminus is truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The amino acid sequence of the C-terminal truncated segment d2 of the extracellular domain of the mutant SARS-CoV-2Delta variant Spike protein is shown in SEQ ID NO. 12. In the sequence, the C-terminus is truncated by 70 amino acid residues, and the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The amino acid sequence of the C-terminal truncated segment d3 of the extracellular domain of the mutant SARS-CoV-2Delta variant Spike protein is shown as SEQ ID NO. 81. In the sequence, the C terminal is truncated by 70 amino acid residues, no signal peptide is contained, and the S1/S2 cutting site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

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

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

The full-length extracellular domain f1 of the Spike protein of the mutant SARS-CoV-2Omicron variant strain has the amino acid sequence shown in SEQ ID NO. 32. In sequence, the original signal peptide: MFVFLVLLPLVSSQ (Shown as SEQ ID NO: 2) in italics, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The full-length extracellular domain f2 of the Spike protein of the mutant SARS-CoV-2Omicron variant strain has the amino acid sequence shown in SEQ ID NO. 33. In sequence, with the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The full-length extracellular domain f3 of the Spike protein of the mutant SARS-CoV-2Omicron variant strain has the amino acid sequence shown in SEQ ID NO. 82. In the sequence, without signal peptide, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The C-terminal truncated fragment g1 of the extracellular domain of the mutant SARS-CoV-2Omicron variant Spike protein has the amino acid sequence shown in SEQ ID NO. 34. In the sequenceC-terminal is truncated by 70 amino acid residues, original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The C-terminal truncated fragment g2 of the extracellular domain of the mutant SARS-CoV-2Omicron variant Spike protein has the amino acid sequence shown in SEQ ID NO. 35. In the sequence, the C-terminus is truncated by 70 amino acid residues, and the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

The C-terminal truncated fragment g3 of the extracellular domain of the mutant SARS-CoV-2Omicron variant Spike protein has the amino acid sequence shown in SEQ ID NO. 83. In the sequence, the C terminal is truncated by 70 amino acid residues, no signal peptide is contained, and the S1/S2 cutting site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

SARS-CoV-2Omicron variant Spike protein S1 subunit, its amino acid sequence is shown in SEQ ID NO:36, original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) are italicized.

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

PNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFF (as shown in SEQ ID NO: 37).

Fusion proteins

The invention provides fusion proteins comprising a heterologous scaffold displaying at least one antigenic polypeptide or trimeric protein derived from a coronavirus Spike protein. In some embodiments, the coronavirus antigen used is an extracellular domain of a coronavirus Spike protein or a truncated fragment thereof containing the various stabilizing mutations described above. In some embodiments, the coronavirus antigen employed comprises or is derived from the RBD domain of a coronavirus Spike protein. In some embodiments, the coronavirus antigen employed comprises or is derived from the S1 subunit of a coronavirus Spike protein. In some embodiments, the coronavirus antigen employed comprises or is derived from a conserved fragment of a coronavirus Spike protein. In exemplary embodiments, the Spike protein sequence employed comprises the sequence set forth in any one of SEQ ID NOs 1, 3-4, 6-13, 31-37, 78-83, or a variant substantially identical or conservatively modified thereto. After transfection of the host cells with an expression vector expressing a fusion protein, nanoparticle vaccines will be generated that display the antigen (e.g., spike protein) on their surface as a result of the ligation of the antigen (e.g., spike protein) to a self-assembling protein (e.g., monomeric ferritin subunit).

Any heterologous scaffold can be used to present antigens in the construction of the vaccines of the present invention. This includes virus-like particles (VLPs), such as nanoparticles. Various nanoparticles can be used to produce the vaccines of the present invention. In general, nanoparticles for use in the present invention need to be formed from multiple copies of a single subunit. The nanoparticles are generally spherical and/or have rotational symmetry (e.g., having 3-fold and 5-fold axes), such as the icosahedral structures exemplified herein. Additionally or alternatively, the amino terminus of the nanoparticle subunit must be exposed and immediately adjacent to the 3-fold axis, and the spacing of the three amino termini must closely match the spacing of the carboxy termini of the displayed trimer-stable Spike protein.

In some embodiments, the self-assembled nanoparticles employed are about 25nm 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 for the production of multivalent vaccines. In some preferred embodiments, the coronavirus antigen may be presented on a self-assembled nanoparticle, for example on a self-assembled nanoparticle derived from Ferritin (FR) as exemplified herein. Ferritin is a globular protein found in animals, bacteria and plants, and its main role is to control polynuclear Fe (III) by transporting hydrated iron ions and protons to or from the mineralized core ₂ O ₃ The rate and location of formation. The globular form of ferritin is composed of monomeric subunit proteins (also known as monomeric ferritin subunits) which are polypeptides with molecular weights of about 17-20 kDa. The sequences of subunits of these proteins are known in the art. In some embodiments, the nanoparticle vaccines of the present invention can use any of these known nanoparticles, as well as conservatively modified variants thereof or variants having substantially the same (e.g., at least 90%,95%, or 99% identity) sequence thereto.

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

The amino acid sequence of human IgGFc is as follows:

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (as shown in SEQ ID NO: 38).

In some exemplary embodiments, the fusion proteins of the invention comprise a nanoparticle subunit sequence (e.g., the H.pylori non-heme monomeric ferritin subunit, amino acid sequence of which is shown in SEQ ID NO: 14), or conservatively modified variants thereof, or sequences substantially identical thereto. Typically, 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 the mutation or a truncated fragment thereof is fused to the N-terminus of the self-assembled Nanoparticle (NP) subunit. In some embodiments, the C-terminus of the conserved sequence of the coronavirus Spike protein or the coronavirus Spike protein S1 subunit or the extracellular domain of the coronavirus Spike protein containing the mutation, or a truncated fragment thereof, is linked to the N-terminus of the nanoparticle subunit via a GS linker, such as GGGGS or GGGGSGGGGS.

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

DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (as shown in SEQ ID NO: 14).

A construct showing conserved sequences of coronavirus Spike proteins or coronavirus Spike proteins described herein can be constructed by fusing subunits of antigenic polypeptides or multimeric antigenic proteins (e.g., trimeric antigens) to subunits of nanoparticles (e.g., ferritin subunits) and other optional or alternative components described hereinA white S1 subunit or any stable nanoparticle containing the mutant coronavirus Spike protein extracellular domain or a truncated fragment thereof. To construct the fusion proteins of the present invention, one or more linkers may be used to link and maintain the overall activity of the different functional proteins. Typically, the linker comprises a short peptide sequence, such as a GS-rich peptide. In some embodiments, a linker or linker motif may be any flexible peptide that links two protein domains or motifs without interfering with their function. For example, the linker employed may be G as shown herein ₄ S terminal or (G) ₄ S) ₂ A linker to connect the spike protein and the nanoparticle scaffold sequence. Recombinant production of the fusion proteins of the invention can be based on the protocols described herein and/or other methods already described in the art.

Exemplary fusion protein sequences are as follows:

fusion protein A1: the C-terminal of the full-length extracellular domain A1 (shown as SEQ ID NO: 3) of the mutant wild type SARS-CoV-2 Spike protein is connected with the N-terminal of the helicobacter pylori non-heme monomer ferritin subunit (shown as SEQ ID NO: 14) through a joint GGGGS (shown as SEQ ID NO: 15) to obtain the fusion protein A1, and the amino acid sequence of the fusion protein A1 is shown as SEQ ID NO: 16. In sequence, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Fusion protein A2: the C end of the full-length extracellular domain a2 (shown as SEQ ID NO: 4) of the mutant wild SARS-CoV-2 Spike protein is connected with the non-helicobacter pylori through a joint GGGGS (shown as SEQ ID NO: 15)The N-terminal of the heme monomer ferritin subunit (shown as SEQ ID NO: 14) is connected to obtain a fusion protein A2, and the amino acid sequence of the fusion protein A2 is shown as SEQ ID NO: 17. In sequence, with the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Fusion protein B1: the C-terminal of the truncated fragment B1 (shown as SEQ ID NO: 6) at the C-terminal of the extracellular domain of the mutant wild-type SARS-CoV-2 Spike protein is connected with the N-terminal of the helicobacter pylori non-heme monomer ferritin subunit (shown as SEQ ID NO: 14) through a joint GGGGS (shown as SEQ ID NO: 15) to obtain a fusion protein B1, and the amino acid sequence of the fusion protein B1 is shown as SEQ ID NO: 18. In the sequence, the C-terminus is truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while including the double mutation K986P/V987P, underlined and italicized, and linkers italicized and bolded.

Fusion protein B2: to be mutatedThe fusion protein B2 is obtained by connecting the C end of the truncated fragment B2 (shown as SEQ ID NO: 7) at the C end of the extracellular domain of the wild SARS-CoV-2 Spike protein with the N end of the helicobacter pylori non-heme monomer ferritin subunit (shown as SEQ ID NO: 14) through a joint GGGGS (shown as SEQ ID NO: 15), and the amino acid sequence of the fusion protein B2 is shown as SEQ ID NO: 19. In the sequence, the C-terminus is truncated by 70 amino acid residues, and the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Fusion protein C1: the C end of the full-length extracellular domain C1 (shown as SEQ ID NO: 9) of the mutant SARS-CoV-2Delta variant Spike protein is connected with the N end of the helicobacter pylori non-heme monomer ferritin subunit (shown as SEQ ID NO: 14) through a joint GGGGS (shown as SEQ ID NO: 15) to obtain the fusion protein C1, and the amino acid sequence of the fusion protein C1 is shown as SEQ ID NO: 20. In sequence, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Fusion protein C2: the C end of the full-length extracellular domain C2 (shown as SEQ ID NO: 10) of the mutant SARS-CoV-2Delta variant Spike protein is connected with the N end of the helicobacter pylori non-heme monomer ferritin subunit (shown as SEQ ID NO: 14) through a joint GGGGS (shown as SEQ ID NO: 15) to obtain a fusion protein C2, and the amino acid sequence of the fusion protein C2 is shown as SEQ ID NO: 21. In sequence, with the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Fusion protein D1: the C-terminal of the truncated fragment D1 (shown as SEQ ID NO: 11) at the C-terminal of the extracellular domain of the mutant SARS-CoV-2Delta variant Spike protein is connected with the N-terminal of the helicobacter pylori non-heme monomer ferritin subunit (shown as SEQ ID NO: 14) through a joint GGGGS (shown as SEQ ID NO: 15) to obtain the fusion protein D1, and the amino acid sequence of the fusion protein D1 is shown as SEQ ID NO: 22. In the sequence, the C-terminus is truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Fusion protein D2: the C-terminal of the truncated fragment D2 (shown as SEQ ID NO: 12) at the C-terminal of the extracellular domain of the mutant SARS-CoV-2Delta variant Spike protein is connected with the N-terminal of the helicobacter pylori non-heme monomer ferritin subunit (shown as SEQ ID NO: 14) through a joint GGGGS (shown as SEQ ID NO: 15) to obtain the fusion protein D2, and the amino acid sequence of the fusion protein D2 is shown as SEQ ID NO: 23. In the sequence, the C-terminus is truncated by 70 amino acid residues, and the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Fusion protein E1: the C-terminal of the S1 subunit of the Spike protein of the SARS-CoV-2Delta variant (shown as SEQ ID NO: 13) is connected with the N-terminal of the H.pylori non-heme monomer ferritin subunit (shown as SEQ ID NO: 14) through a joint GGGGS (shown as SEQ ID NO: 15) to obtain a fusion protein E1, and the amino acid sequence of the fusion protein E1 is shown as SEQ ID NO: 24. In sequence, the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) are in italics and the linker is in italics and bold.

Fusion protein E2: the C-terminal of the S1 subunit of the Spike protein of the SARS-CoV-2Delta variant (shown as SEQ ID NO: 13) is connected with the N-terminal of the helicobacter pylori non-heme monomer ferritin subunit (shown as SEQ ID NO: 14) through a joint GGGGS (shown as SEQ ID NO: 15), and the protein sequence is expressed by a signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown as SEQ ID NO: 2), fusion protein E2 is obtained, and the amino acid sequence is shown as SEQ ID NO: 25. In the sequence, the N-terminal signal peptide is italicized and the linker is italicized and bolded.

Fusion protein E3: the C-terminal of the S1 subunit of the Spike protein of the SARS-CoV-2Omicron variant strain (shown as SEQ ID NO: 36) is connected with the N-terminal of the H.pylori non-heme monomer ferritin subunit (shown as SEQ ID NO: 14) through a joint GGGGS (shown as SEQ ID NO: 15) to obtain a fusion protein E3, and the amino acid sequence of the fusion protein E3 is shown as SEQ ID NO: 39. In sequence, the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) are in italics and the linker is in italics and bold.

Fusion protein E4: the C-terminal of the S1 subunit of the SARS-CoV-2Omicron variant Spike protein (shown as SEQ ID NO: 36) is connected with the N-terminal of the helicobacter pylori non-heme monomeric ferritin subunit (shown as SEQ ID NO: 14) through a joint GGGGS (shown as SEQ ID NO: 15), and the content of the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), fusion protein E4 is obtained, and the amino acid sequence thereof is shown in SEQ ID NO: 40. In the sequence, the N-terminal signal peptide is italicized and the linker is italicized and bolded.

Fusion protein F1: the C-terminus of the full-length extracellular domain f1 (shown as SEQ ID NO: 32) of the mutant SARS-CoV-2Omicron variant Spike protein was passed throughThe linker GGGGS (shown as SEQ ID NO: 15) is connected with the N end of the helicobacter pylori non-heme monomer ferritin subunit (shown as SEQ ID NO: 14) to obtain the fusion protein F1, and the amino acid sequence of the fusion protein F1 is shown as SEQ ID NO: 41. In sequence, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Fusion protein F2: the C-terminal of the full-length extracellular domain F2 (shown as SEQ ID NO: 33) of the mutant SARS-CoV-2Omicron variant Spike protein is connected with the N-terminal of the helicobacter pylori non-heme monomeric ferritin subunit (shown as SEQ ID NO: 14) through a joint GGGGS (shown as SEQ ID NO: 15) to obtain a fusion protein F2, and the amino acid sequence of the fusion protein is shown as SEQ ID NO: 42. In sequence, with the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while including the double mutation K986P/V987P, underlined and italicized, and linkers italicized and bolded.

Fusion proteinsG1: the C-terminal of the truncated fragment G1 (shown as SEQ ID NO: 34) at the C-terminal of the extracellular domain of the mutant SARS-CoV-2Omicron variant Spike protein is connected with the N-terminal of the helicobacter pylori non-heme monomeric ferritin subunit (shown as SEQ ID NO: 14) through a linker GGGGS (shown as SEQ ID NO: 15) to obtain the fusion protein G1, and the amino acid sequence of the fusion protein G1 is shown as SEQ ID NO: 43. In the sequence, the C-terminus is truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Fusion protein G2: the C-terminal of the truncated fragment G2 (shown as SEQ ID NO: 35) at the C-terminal of the extracellular domain of the mutant SARS-CoV-2Omicron variant Spike protein is connected with the N-terminal of the helicobacter pylori non-heme monomeric ferritin subunit (shown as SEQ ID NO: 14) through a linker GGGGS (shown as SEQ ID NO: 15) to obtain the fusion protein G2, and the amino acid sequence of the fusion protein G2 is shown as SEQ ID NO: 44. In the sequence, the C-terminus is truncated by 70 amino acid residues, and the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Fusion protein H1: the original signal peptide was added to the N-terminus of the O330 fragment (as shown in SEQ ID NO: 37): MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), then the C-terminal of O330 fragment is connected with the N-terminal of helicobacter pylori non-heme monomer ferritin subunit (shown in SEQ ID NO: 14) through a linker GGGGS (shown in SEQ ID NO: 15) to obtain fusion protein H1, the amino acid sequence of which is shown in SEQ ID NO: 45. The original signal peptide is italicized and the linker is italicized and bolded.

Fusion protein H2: adding a signal peptide at the N terminal of an O330 fragment (shown as SEQ ID NO: 37): MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO: 5), then the C-terminal of O330 fragment is connected with the N-terminal of helicobacter pylori non-heme monomeric ferritin subunit (shown in SEQ ID NO: 14) through a joint GGGGS (shown in SEQ ID NO: 15) to obtain fusion protein H2, and the amino acid sequence of which is shown in SEQ ID NO: 46. The signal peptide is italicized and the linker is italicized and bolded.

Fusion protein A1-1: the C-terminal of the full-length extracellular domain A1 (shown as SEQ ID NO: 3) of the mutant wild type SARS-CoV-2 Spike protein is connected with the N-terminal of the human IgGFc (shown as SEQ ID NO: 38) to obtain the fusion protein A1-1, and the amino acid sequence of the fusion protein A1-1 is shown as SEQ ID NO: 47. In sequence, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

Fusion protein A2-1: the C-terminal of the full-length extracellular domain A2 (shown as SEQ ID NO: 4) of the mutant wild type SARS-CoV-2 Spike protein is connected with the N-terminal of the human IgGFc (shown as SEQ ID NO: 38) to obtain the fusion protein A2-1, and the amino acid sequence of the fusion protein A2-1 is shown as SEQ ID NO: 48. In sequence, with the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

Fusion protein B1-1: the C-terminal of the truncated fragment B1 (shown as SEQ ID NO: 6) at the C-terminal of the extracellular domain of the mutant wild type SARS-CoV-2 Spike protein is connected with the N-terminal of the human IgGFc (shown as SEQ ID NO: 38) to obtain the fusion protein B1-1, and the amino acid sequence of the fusion protein B1-1 is shown as SEQ ID NO: 49. In the sequence, the C-terminus is truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

Fusion protein B2-1: the mutant wild type SARS-CoV-2 Spike protein extracellular domain C end truncated fragment b2 (shown as SEQ ID NO: 7)The C-terminal is connected with the N-terminal of human IgGFc (shown as SEQ ID NO: 38) to obtain a fusion protein B2-1, and the amino acid sequence of the fusion protein is shown as SEQ ID NO: 50. In the sequence, the C-terminus is truncated by 70 amino acid residues, and the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

Fusion protein C1-1: the C end of the full-length extracellular domain C1 (shown as SEQ ID NO: 9) of the mutant SARS-CoV-2Delta variant Spike protein is connected with the N end of the human IgGFc (shown as SEQ ID NO: 38) to obtain the fusion protein C1-1, and the amino acid sequence of the fusion protein C1-1 is shown as SEQ ID NO: 51. In sequence, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

Fusion protein C2-1: the C end of the full-length extracellular domain C2 (shown as SEQ ID NO: 10) of the mutant SARS-CoV-2Delta variant Spike protein is connected with the N end of the human IgGFc (shown as SEQ ID NO: 38) to obtain the fusion protein C2-1,the amino acid sequence is shown in SEQ ID NO. 52. In sequence, with the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

Fusion protein D1-1: the C-terminal of the truncated fragment D1 (shown as SEQ ID NO: 11) at the C-terminal of the extracellular domain of the mutant SARS-CoV-2Delta variant Spike protein is connected with the N-terminal of the human IgGFc (shown as SEQ ID NO: 38) to obtain the fusion protein D1-1, and the amino acid sequence of the fusion protein D1-1 is shown as SEQ ID NO: 53. In the sequence, the C-terminus is truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

Fusion protein D2-1: the C-terminal of the truncated fragment D2 (shown as SEQ ID NO: 12) at the C-terminal of the extracellular domain of the mutant SARS-CoV-2Delta variant Spike protein is connected with the N-terminal of the human IgGFc (shown as SEQ ID NO: 38) to obtain the fusion protein D2-1, and the amino acid sequence of the fusion protein D2-1 is shown as SEQ ID NO: 54. In the sequence of the first and second,the C-terminal is truncated by 70 amino acid residues, and the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

Fusion protein E1-1: the C-terminal of the S1 subunit of the Spike protein of the SARS-CoV-2Delta variant (shown as SEQ ID NO: 13) is connected with the N-terminal of the human IgGFc (shown as SEQ ID NO: 38) to obtain the fusion protein E1-1, and the amino acid sequence of the fusion protein E1-1 is shown as SEQ ID NO: 55. In sequence, the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) are italicized.

Fusion protein E2-1: the C-terminal of the S1 subunit of the Spike protein of the SARS-CoV-2Delta variant (shown as SEQ ID NO: 13) was linked to the N-terminal of human IgGFc (shown as SEQ ID NO: 38), and the sequence was determined using a signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown as SEQ ID NO: 2), fusion protein E2-1 is obtained, and the amino acid sequence is shown as SEQ ID NO: 56. In the sequence, the N-terminal signal peptide is italicized.

Fusion protein E3-1: the C-terminal of the S1 subunit of the SARS-CoV-2Omicron variant Spike protein (shown as SEQ ID NO: 36) is connected with the N-terminal of the human IgGFc (shown as SEQ ID NO: 38) to obtain the fusion protein E3-1, and the amino acid sequence of the fusion protein E3-1 is shown as SEQ ID NO: 57. In sequence, the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) are italicized.

Fusion protein E4-1: the C-terminus of the S1 subunit of the Spike protein of the SARS-CoV-2Omicron variant strain (shown in SEQ ID NO: 36) was linked to the N-terminus of human IgGFc (shown in SEQ ID NO: 38), and the sequence was determined using a signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown as SEQ ID NO: 2), fusion protein E4-1 is obtained, and the amino acid sequence is shown as SEQ ID NO: 58. In the sequence, the N-terminal signal peptide is italicized.

Fusion protein F1-1: the C-terminal of the full-length extracellular domain F1 (shown as SEQ ID NO: 32) of the mutant SARS-CoV-2Omicron variant Spike protein was linked to the N-terminal of human IgGFc (shown as SEQ ID NO: 38) to obtain a fusion protein F1-1, the amino acid sequence of which is shown as SEQ ID NO: 59. In sequence, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

Fusion protein F2-1: the full-length extracellular domain f2 (shown as SEQ ID NO: 33) of the mutant SARS-CoV-2Omicron variant Spike proteinShown in the specification) and the N end of human IgGFc (shown in SEQ ID NO: 38) to obtain a fusion protein F2-1, and the amino acid sequence of the fusion protein is shown in SEQ ID NO: 60. In sequence, with the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

Fusion protein G1-1: the C-terminal of the truncated fragment G1 (shown as SEQ ID NO: 34) at the C-terminal of the extracellular domain of the mutant SARS-CoV-2Omicron variant Spike protein was ligated to the N-terminal of human IgGFc (shown as SEQ ID NO: 38) to obtain a fusion protein G1-1, the amino acid sequence of which is shown as SEQ ID NO: 61. In the sequence, the C-terminus is truncated by 70 amino acid residues, the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is italicized, with the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

Fusion protein G2-1: the C-terminal of the truncated fragment G2 (shown as SEQ ID NO: 35) at the C-terminal of the extracellular domain of the mutant SARS-CoV-2Omicron variant Spike protein was ligated to the N-terminal of human IgGFc (shown as SEQ ID NO: 38) to obtain fusion protein G2-1, the amino acid sequence of which is shown as SEQ ID NO: 62. In the sequence, the C-terminus is truncated by 70 amino acid residues, and the signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO: 5) replaces the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide is italicized, S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ By underliningAnd bold and contain the double mutation K986P/V987P, underlined and italicized.

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

Fusion protein H2-1: MEFGLSLVFLVLILKGVQC (shown as SEQ ID NO: 5) is added at the N-terminal of the O330 fragment (shown as SEQ ID NO: 37), and then the C-terminal of the O330 fragment is connected with the N-terminal of human IgG Fc (shown as SEQ ID NO: 38) to obtain the fusion protein H2-1, wherein the amino acid sequence of the fusion protein H2-1 is shown as SEQ ID NO: 64. The signal peptide is italicized.

Mature fusion protein a: compared with the fusion proteins A1 and A2, the N-terminal signal peptide is removed, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 26. In sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Mature fusion protein B: compared with the fusion proteins B1 and B2, the N-terminal signal peptide is removed, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 27. In sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Mature fusion protein C: compared with the fusion proteins C1 and C2, the N-terminal signal peptide is removed, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 28. In sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Mature fusion protein D: compared with the fusion proteins D1 and D2, the fusion protein has the amino acid sequence shown in SEQ ID NO. 29, and the N-terminal signal peptide is removed. In sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Mature fusion protein E-1: compared with the fusion proteins E1 and E2, the N-terminal signal peptide is removed, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 30. In the sequence, the adapters are indicated in italics and bold.

Mature fusion protein E-2: compared with the fusion proteins E3 and E4, the N-terminal signal peptide is removed, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 65. In the sequence, the adapters are indicated in italics and bold.

Mature fusion protein F: compared with the fusion proteins F1 and F2, the N-terminal signal peptide is removed, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 66. In sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Mature fusion protein G: compared with the fusion proteins G1 and G2, the N-terminal signal peptide, the amino acid thereof, is removedThe sequence is shown as SEQ ID NO. 67. In sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, while containing the double mutation K986P/V987P, underlined and italicized, and adaptor italicized and bolded.

Mature fusion protein H: compared with fusion proteins H1 and H2, the fusion protein has the N-terminal signal peptide removed, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 68. In the sequence, the adapters are indicated in italics and bold.

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

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

Mature fusion protein C-1: compared with the fusion proteins C1-1 and C2-1, the N-terminal signal peptide is removed, and the amino acid sequence of the fusion protein is shown as SEQ ID NO: 71. In sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

Mature fusion protein D-1: compared with the fusion proteins D1-1 and D2-1, the N-terminal signal peptide is removed, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 72. In sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

Mature fusion protein E-3: compared with the fusion proteins E1-1 and E2-1, the N-terminal signal peptide is removed, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 73.

CVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (as shown in SEQ ID NO: 73).

Mature fusion protein E-4: compared with the fusion proteins E3-1 and E4-1, the N-terminal signal peptide is removed, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 74.

CVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (as 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 is removed, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 75. In sequence, the S1/S2 cleavage site ⁶⁸² RRAR ⁶⁸⁵ Is mutated to ⁶⁸² GSAS ⁶⁸⁵ Underlined and bolded, and contains the double mutation K986P/V987P, underlined and italicized.

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

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

PNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (as shown in SEQ ID NO: 77).

SEQ ID NOS: 26-30 and 65-77 are mature fusion protein sequences with the N-terminal signal peptide (SEQ ID NOS: 2 or 5) removed. In addition to these specifically exemplified fusion proteins, the invention also encompasses nanoparticle vaccines that contain subunit sequences that are substantially identical to any of these exemplified nanoparticle vaccine sequences or conservatively modified variants thereof.

Polynucleotides and expression vectorsBody

The mutant-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof, coronavirus Spike protein S1 subunit, coronavirus Spike protein conserved fragment, fusion protein, or Spike protein nanoparticle of the invention is typically produced by an expression vector comprising a coding sequence for the mutant-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof, coronavirus Spike protein S1 subunit, coronavirus Spike protein conserved fragment, fusion protein, or Spike protein nanoparticle described herein. Thus, in some related aspects, the invention provides polynucleotides (DNA or RNA) encoding the mutant-containing extracellular domain of a coronavirus Spike protein or a truncated fragment thereof, the coronavirus Spike protein S1 subunit, a conserved fragment of a coronavirus Spike protein, a fusion protein, or a Spike protein nanoparticle described herein. Some polynucleotides of the invention encode one of the mutant-containing extracellular domains of coronavirus Spike proteins described herein or a truncated fragment thereof, e.g., a truncated fragment of the extracellular domain of SARS-CoV-2 Spike protein shown in SEQ ID NO: 12. Some polynucleotides of the 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 comprise an N-terminal signal peptide, or some polynucleotide sequences additionally encode an N-terminal signal peptide. For example, a polynucleotide encoding a fusion protein (e.g., SEQ ID NOS: 26-30) can further comprise a sequence encoding an N-terminal signal peptide shown in SEQ ID NOS: 2 or 5, or a sequence substantially identical thereto or a conservatively modified variant thereof.

The invention also provides expression vectors having such polynucleotides and host cells (e.g., prokaryotic or eukaryotic cells, such as HEK293, CHO, expichho and CHO-S cell lines) for producing mutant-containing extracellular domains of coronavirus Spike protein or truncated fragments thereof, coronavirus Spike protein S1 subunits, conserved fragments of coronavirus Spike protein, or fusion proteins. Fusion proteins encoded by the polynucleotides or expressed by the vectors are also encompassed by the present invention. As described herein, the nanoparticle subunit fused Spike protein extracellular domain or truncated fragment thereof, spike protein S1 subunit, or a Spike protein conserved fragment will self-assemble into a nanoparticle vaccine that displays on its surface a Spike protein or truncated fragment thereof, a Spike protein S1 subunit, or a Spike protein conserved fragment.

Polynucleotides and related vectors can be produced by standard molecular biology techniques or protocols illustrated herein. For example, general protocols for Cloning, transfection, transient gene expression and obtaining stably transfected cell lines have been described in the art, e.g., sambrook et al, molecular Cloning: A Laboratory Manual, cold Spring Harbor Press, N.Y. (third edition, 2000); and Brent et al, current Protocols in Molecular Biology, john Wiley & Sons, inc. (ringbou edition, 2003). Mutations can also be introduced into a polynucleotide sequence by PCR by known methods.

The choice of a particular carrier depends on the intended use of the protein. For example, whether the cell type is prokaryotic or eukaryotic, the vector chosen must be capable of driving expression of the protein in the desired cell type. Many vectors contain sequences that allow for the replication of prokaryotic vectors and eukaryotic expression of operably linked gene sequences. The vectors useful in the present invention may replicate autonomously, i.e., the vector exists extrachromosomally, and their replication does not have to be linked directly to replication of the host cell genome. Alternatively, replication of the vector may be linked to replication of the host chromosomal DNA, e.g., the vector may be integrated into the chromosome of the host cell by 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 expression of proteins or RNA) and human artificial chromosomes. Alternative viral vectors include lentiviral or other retroviral based vectors, adenovirus, adeno-associated virus, cytomegalovirus, herpes virus, SV40 based vectors, papilloma virus, 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 present invention. The host cell may be any cell which carries a recombinant vector for a protein of the invention, wherein the vector is allowed to drive the expression of the protein for use in the invention. It may be prokaryotic, such as any of a number of 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. The cells expressing the proteins of the invention may be primary culture cells or may be established cell lines. Thus, in addition to the cell lines exemplified herein (e.g., HEK293 cells), many other host cell lines well known in the art may also 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.

The vector expressing the protein may be introduced into the host cell of choice by any of a number of suitable methods known to those skilled in the art. For introducing a vector encoding a protein into mammalian cells, the method used will depend on the form of the vector. For plasmid vectors, the DNA encoding the protein sequence can be introduced by any of a number of transfection methods, including, for example, liposome-mediated transfection ("lipofection"), DEAE-dextran-mediated transfection, electroporation, or calcium phosphate precipitation. These methods are described in detail, for example, in Brent et al, supra. Among them, lipofection is widely accepted because of its simple operation and no need of special instruments and equipment. For example, transfection may be performed using Lipofectamine (Life technologies) or LipotaXI (Stratagene) kits. Other companies that provide lipofection reagents and methods include Bio-Rad laboratories, CLONTECH, glen Research, life Technologies, JBL Scientific, MBI Fermentas, panVera, promega, quantum Biotechnologies, sigma-Aldrich, and Wako Chemicals USA.

For producing recombinant proteins in high yield over a long period of time, stable expression is preferred. Instead of using an expression vector comprising a viral origin of replication, a host cell may be transformed with a protein coding sequence and optionally a marker controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). The selectable marker in the recombinant vector is resistant to selection and allows the cell to stably integrate the vector into its chromosome. Common selection markers include: neomycin (neo) resistant to the aminoglycoside G-418, and hygromycin (hygro) resistant to hygromycin.

In some embodiments, the 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 on both sides of the insert. Efficient transcription can be obtained by the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI and the early promoters of cytomegalovirus, and other cellular promoters such as actin can also be used. 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, among others. Commonly used mammalian cells include HEK293 cells, cos1 cells, cos7 cells, CV1 cells, murine L cells, CHO cells, and the like.

In some embodiments, the inserted gene fragment contains a selection marker, and common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, and the like, so as to facilitate the selection and isolation of cells that are transfected successfully. The constructed plasmid is transfected to host cells without the genes, and the cells successfully transfected grow in large quantities through selective culture medium culture to produce the target protein to be obtained.

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 mutations resulting in amino acid substitutions and PCR-mediated mutations. Variants (including derivatives) encode substitutions of less than 50 amino acids, less than 40 amino acids, less than 30 amino acids, less than 25 amino acids, less than 20 amino acids, less than 15 amino acids, less than 10 amino acids, less than 5 amino acids, less than 4 amino acids, less than 3 amino acids, or less than 2 amino acids relative to the original protein. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity 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 invention also provides pharmaceutical compositions and related methods of treatment. The pharmaceutical composition comprises an effective dose of fusion protein or Spike protein nanoparticles and a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" refers to substances that are approved by a governmental regulatory agency or other generally recognized pharmacopoeia for use in animals, particularly in humans. Furthermore, "pharmaceutically acceptable carrier" generally refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid, and the like.

The term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which the active ingredient may be administered to a patient. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. If desired, the pharmaceutical compositions may also contain minor amounts of wetting, emulsifying, or pH buffering agents, such as acetates, citrates or phosphates. Antimicrobial agents such as benzyl alcohol or methylparaben, 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 formulations and the like. The pharmaceutical compositions may be formulated as suppositories with conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable Pharmaceutical carriers are described in Remington's Pharmaceutical Sciences of e.w. martin, which is incorporated herein by reference. Such compositions will contain a clinically effective dose of the fusion protein or Spike protein nanoparticles, preferably in purified form, together with an appropriate amount of carrier to provide a form of administration suitable for the patient. The formulation should be suitable for the mode of administration. The formulations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In some embodiments, the pharmaceutical composition may comprise a fusion protein or a Spike protein nanoparticle, and a polynucleotide or vector encoding the fusion protein described herein. In some embodiments, trimers of the extracellular domain of the Spike protein of a virus (e.g., SARS-CoV-2) or truncated fragments thereof can be used to prevent and treat corresponding viral infections. In some embodiments, vaccines comprising nanoparticles described herein can be used to prevent or treat the corresponding diseases, such as infections caused by various coronaviruses. Some embodiments of the invention relate to the use of a SARS-CoV-2 antigen or vaccine in the prevention or treatment of SARS-CoV-2 infection in a human subject. Some embodiments of the invention relate to the use of a SARS-CoV antigen or vaccine in the prevention or treatment of SARS-CoV infection.

In the practice of some methods of treatment of the present invention, a subject in need of prevention or treatment of a disease (e.g., SARS-CoV-2 infection) is administered a corresponding Spike protein nanoparticle or fusion protein, or a polynucleotide encoding a fusion protein as described herein. Typically, the Spike protein nanoparticles, fusion proteins, or polynucleotides encoding fusion proteins disclosed herein are included in a pharmaceutical composition. The pharmaceutical composition may be a therapeutic or prophylactic formulation. Typically, the pharmaceutical composition may additionally comprise one or more pharmaceutically acceptable carriers, and optionally other therapeutic ingredients (e.g. antiviral agents). Various pharmaceutically acceptable additives may also be used in the pharmaceutical composition.

Some of the pharmaceutical compositions of the present invention are vaccine compositions. For vaccine compositions, suitable adjuvants may additionally be included. Suitable adjuvants include, for example, aluminum hydroxide, lecithin, freund's adjuvant, MF59, SEPIVAC SWE ^TM MPL and IL-12. In some embodiments, the vaccine compositions described herein (e.g., SARS-CoV-2 vaccine) can be formulated as a controlled-release or timed-release formulation. This can be achieved in a composition comprising a slow release polymer or by a microencapsulated delivery system or bioadhesive gel. Various pharmaceutical compositions can be prepared according to standard procedures well known in the art. See, for example, U.S. Pat. nos. 4,652,441 and 4,917,893; US patents US4,677,191 and US4,728,721; and U.S. Pat. No. 4,675,189.

The pharmaceutical compositions of the invention can be used in a variety of therapeutic or prophylactic applications, for example, for treating a SARS-CoV-2 infection in a subject or for eliciting an immune response to SARS-CoV-2 in a subject. As an example, a nanoparticle vaccine can be administered to a subject to induce an immune response against SARS-CoV-2, e.g., to induce 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 performed similarly. Depending on the particular subject and disease, the pharmaceutical composition of the present invention may be administered to the subject by a variety of administration means known to those of ordinary skill in the art, for example, by parenteral routes such as intramuscular route, subcutaneous route, intravenous route, intra-arterial route, articular route, intraperitoneal route, and the like. In some embodiments, the therapeutic methods of the invention relate to methods of blocking entry of a coronavirus (e.g., SARS-CoV or SARS-CoV-2) into a host cell (e.g., a human host cell), methods of preventing binding of a coronavirus Spike protein to a host receptor, and methods of treating acute respiratory disease associated with coronavirus infection. In some embodiments, the therapeutic 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., reidesavir), monoclonal antibodies against one or more coronaviruses, immunosuppressive or anti-inflammatory agents (e.g., sarilumab or Tocilizumab), ACE inhibitors, vasodilators, or any combination thereof.

For therapeutic applications, the pharmaceutical composition should comprise a therapeutically effective amount of the fusion protein, spike protein nanoparticles described herein. For prophylactic applications, the pharmaceutical composition should comprise a prophylactically effective amount of the fusion protein, spike protein nanoparticles described herein. The appropriate amount of antigen can be determined based on the particular disease or disorder to be treated or prevented, the severity of the subject, age, and other personal attributes of the particular subject (e.g., the overall status of the subject's health). Determination of an effective dose is also guided by animal model studies, followed by human clinical trials, and by dosing regimens that can significantly reduce the occurrence or severity of a disease condition or symptom of interest in a subject.

For prophylactic use, the pharmaceutical composition is provided prior to any symptoms, e.g. prior to infection. Prophylactic administration of the pharmaceutical composition is used to prevent or ameliorate any subsequent infection. Thus, in some embodiments, the subject to be treated is a subject that has been infected (e.g., a SARS-CoV-2 infection), or is at risk of infection (e.g., a SARS-CoV-2 infection), e.g., due to exposure or possible exposure to a virus (e.g., SARS-CoV-2). Following administration of a therapeutically effective amount of the disclosed pharmaceutical compositions, the subject can be monitored for infection (e.g., SARS-CoV-2 infection), symptoms associated with infection (e.g., SARS-CoV-2 infection).

For therapeutic applications, the pharmaceutical composition is provided at or after the onset of symptoms of a disease or infection, such as after symptoms of infection (e.g., SARS-CoV-2 infection) have occurred or after diagnosis of the infection. Thus, the pharmaceutical composition may be provided prior to anticipated exposure to the virus, so as to attenuate the anticipated severity, duration or extent of the infection and/or associated disease condition following exposure or suspected exposure to the virus or following the actual initial stage of infection. The pharmaceutical compositions of the present invention may be combined with other agents known in the art for treating or preventing infection by the relevant pathogen (e.g., SARS-CoV-2 infection).

Vaccine compositions (e.g., SARS-CoV-2 vaccine) or pharmaceutical compositions comprising the fusion protein, spike protein nanoparticles of the invention may be provided as components of a kit. Optionally, such kits include 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 may be used to administer the fusion proteins, spike protein nanoparticles or derivatives of the invention or polynucleotides or expression vectors encoding them, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the fusion proteins or Spike protein nanoparticles, receptor-mediated endocytosis (see, e.g., wu and Wu,1987, j.biol.chem.262, 4429-4432), construction of nucleic acids as part of a retrovirus or other vector, and the like.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others based on the teachings of the present invention are within the scope of the invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1: preparation of fusion proteins

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

The three-dimensional structures of the fusion protein D and the fusion protein G are analyzed by using a Cryo-electron microscope (Cryo-EM), the connection of the extracellular domain of the SARS-CoV-2 Spike protein or the truncated fragment thereof at the N end of the monomeric ferritin subunit does not interfere with the self-assembly of the ferritin, the nanoparticles are well formed, and the surface shows the spikes.

Example 2: test of binding ability of fusion protein to hACE2 protein

1.1 test of the binding Capacity of the fusion protein to hACE2 protein (ELISA)

The test detects the binding capacity of the fusion protein D and the fusion protein G with human ACE2 protein (hACE 2) by ELISA, so as to evaluate whether the Spike protein-ferritin fusion protein can well display the key epitope of the Spike protein. The method is briefly described as follows: adding 100 μ L of 2 μ G/mL antigen (WT-Spike-His, delta-Spike-His, omicron-Spike-His, fusion protein D or fusion protein G) solution to each reaction well of a 96-well enzyme-labeled plate (Costar, cat. No.: 9018), and coating overnight at 4 ℃; washing with PBST (0.05% Tween-20 in PBS buffer) 2 times; adding blocking solution (PBST containing 3% BSA) to each reaction well, incubating in an incubator at 37 ℃ for 2h; after blocking, washing with PBST for 3 times; adding gradient diluted human ACE2-his-biotin (product number: 10108-H27B-B, yi Qiao Shen) with initial concentration of 2.5 μ g/mL, gradient diluting 3 times, diluting 10 serial dilutions with concentration of 100 μ L per well, placing in 37 deg.C incubator, and incubating for 1.5H; wash 5 times with PBST; streptavidin-labeled catalase (Jackson Immuno Research, cat No. 016-030-084 1; washed 8 times with PBST; adding TMB solution into the reaction hole at a rate of 100 mu L/hole, and incubating for 5-15 min at 37 ℃ in a dark place; to each reaction well was added a stop solution (0.1M H) ₂ SO ₄ ) 50 μ L of the enzyme-catalyzed color reaction was terminated; the reading was performed with the detection wavelength set at 450 nm.

Wherein, the WT-Spike-His is constructed by adding 6 × His (HHHHHHHHHH) to the C-terminal of the truncated fragment b1 (shown as SEQ ID NO: 6) at the extracellular domain of the mutant wild type SARS-CoV-2 Spike protein. Delta-Spike-His is constructed by adding 6 × His (HHHHHHHHHH) to the C-terminal of the C-terminal truncated segment d1 (shown in SEQ ID NO: 11) of the extracellular domain of the mutant SARS-CoV-2Delta variant Spike protein. The Omicron-Spike-His was constructed by adding 6 × His (HHHHHHHH) to the C-terminus of a truncated fragment g1 (shown in SEQ ID NO: 34) of the extracellular domain C-terminus of the mutant SARS-CoV-2Omicron variant Spike protein.

The results are shown in FIG. 1, where hACE2 binds to fusion protein D and Delta, wild-type Spike protein with similar affinity, EC ₅₀ Values of 9.2, 8.1, 5.7ng/mL, respectively (FIG. 1 a); hACE2 binds to fusion protein G and the Spike protein of Omicron with similar affinity, EC ₅₀ The values were 9.3 and 8.2ng/mL, respectively (FIG. 1 b).

1.2 binding Capacity test (BLI) of fusion protein to hACE2 protein

The affinity constant of the combination of the fusion protein D and the fusion protein G with hACE2 is determined by adopting a biomembrane interference technology (BLI), and the apparatus is a Fortebio Octet RED & QK system of PALL company. Multichannel parallel quantitative analysis WT-Spike-His (same as example 2 step 1.1), delta-Spike-His (same as example 2 step 1.1), omicron-Spike-His (same as example 2 step 1.1), fusion protein D, fusion protein G, concentration gradient set as: 50. 100, 200 and 400nM, hACCE 2-Biotin (Acro biosystems, cat # AC 2-H5257) coupled with SA Biosensors sensors (Octet, cat # 2107002811).

The results are shown in Table 1 and indicate that fusion protein D, fusion protein G bind hACE2 with significantly greater affinity than WT-Spike-His, delta-Spike-His and Omicron-Spike-His to hACE 2. The Spike protein-ferritin fusion protein of the invention can well display the key antigen epitope of the Spike protein.

TABLE 1 binding kinetics of SARS-CoV-2 Spike protein to hACE2 receptor

Antigens	KD(M)	kon(1/Ms)	kdis(1/s)
				WT-Spike-His	3.28E-08	8.90E+04	2.92E-03
Delta-Spike-His	3.43E-08	9.85E+04	3.38E-03
				Omicron-Spike-His	8.37E-09	6.44E+04	5.39E-04
Fusion protein D	5.49E-10	4.02E+04	2.21E-05
				Fusion protein G	<1.0E-12	4.67E+04	<1.0E-07

Example 3: immunogenicity of vaccines in mice (subcutaneous injection)

1.1 immunization of mice

To study monovalent vaccines (fusional)Synprotein D and fusion protein G) were studied for immunogenicity in mice. Mixing and emulsifying fusion protein D and fusion protein G with immunologic adjuvant, and injecting subcutaneously Balb/c mice (6-8 weeks old) on day 0 and day 21 respectively, each mouse SEPIVAC SWE ^TM Adjuvant (SEPPIC s.a., cat No. 80748J, lot No. 210721010001) was fixed in volume of 50 μ l, total volume of 100 μ l/individual per administration, and the group dosing schedule is shown in table 2. Blood was collected from the orbit at day 35 after immunization, left to stand until serum was separated out, and centrifuged to obtain mouse serum for ELISA detection of serum antibodies [ wild type, delta and Omicron (BA.1) ]]IgG titer and SARS-CoV-2 Spike pseudovirus neutralizing antibody assay.

TABLE 2 group dosing regimen

Group of	Medicine	Dosage (mu g/body)	Immunization regimen
				1	Fusion protein D	5	s.c.
2	Fusion protein G	5	s.c.

1.2 detection of serum IgG titer against Spike protein by ELISA

WT-Spike-His (same as 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) antigens were diluted to 2. Mu.g/mL, added to a 96-well plate (Corning, 9018) at 100. Mu.L per well, and coated overnight at 4 ℃; the 96-well plate was washed 3 times in a washing buffer PBST (PBS buffer containing 0.05% Tween-20), a blocking solution (washing buffer PBST containing 3% BSA) was added, and incubated at 37 ℃ for 2 hours; washing a 96-well plate with a washing buffer PBST for 3 times, adding 100 mu L of mouse serum (the serum is diluted 1000 times to be used as an initial concentration and then diluted 3 times in a gradient way) obtained in the step 1.1 of the example 3 into each well, and incubating for 1.5h at 37 ℃; the 96-well plate was washed 5 times with washing buffer PBST, 100. Mu.L of Peroxidase-Affinipure Goat Anti-Mouse IgG (Jackson, cat # 115-035-003) diluted to 1 10000 was added, and incubated at 37 ℃ for 1 hour; the 96-well plate was washed 8 times with washing buffer PBST, and 100. Mu.L of TMB (Biopanda, cat. TMB-S-001) substrate was added thereto for color development; after developing for 10-15min, 50 μ L of 0.1M H is added ₂ SO ₄ The reaction was terminated, the absorbance value (OD value) was measured at a wavelength of 450nm, and the resulting read OD values were fitted with a nonlinear four-parameter equation curve model with 2.0 times the average of the negative well readings in each 96-well plate as a final value, thereby determining the titer of each serum sample.

The results are shown in FIG. 2 and indicate that both fusion protein D and fusion protein G induced strong IgG antibody titers against the wild type, delta and Omicron against Spike proteins, which were all able to elicit relevant antigen-specific antibody responses.

1.3 SARS-CoV-2 Spike pseudovirus neutralizing antibody assay

The mouse serum obtained in step 1.1 of example 3 was subjected to gradient dilution with 10% FBS-containing DMEM medium, and transferred to a 96-well plate at 50. Mu.L per well for use; diluting different SARS-CoV-2 Spike pseudoviruses with a 10% FBS-containing DMEM medium, transferring the diluted SARS-CoV-2 Spike pseudoviruses to the above-mentioned 96-well plate containing mouse serum at 25. Mu.L per well, mixing them, and incubating at 37 ℃ for 1 hour; digesting ACE2-293 cells with 0.25% trypsin-EDTA (Gibco, 25200-072), counting, adjusting cell densityTo 4X 10 ⁵ cells/mL, 50. Mu.L per well of the cells were added to the above 96-well plate, and the CO was reduced at 37 ℃ 5% ₂ Incubating for 48h in an incubator; mu.L of Bio-Lite Luciferase Assay System (Devovemedium, DD 1201-03) Assay reagent was added to each well, and after standing for 3 minutes, readings were taken, and the neutralization inhibition rate of mouse serum was calculated from the readings and the titer was calculated: neutralization inhibition rate = [1- (sample group-blank control group)/(negative control group-blank control group)]X is 100%; wherein, the sample group is added with SARS-CoV-2 Spike pseudovirus and mouse serum, the negative control group is added with SARS-CoV-2 Spike pseudovirus without mouse serum, and the blank control group is not added with SARS-CoV-2 Spike pseudovirus and without mouse serum.

The construction method of the ACE2-293 cell comprises the following steps: HEK293 cells were cultured in DMEM complete medium containing 10% FBS, ACE2 expression plasmid (HG 10108-M) was transfected using lipofectamine 2000 transfection reagent (Thermo Fisher, 11668019), and then monoclonals with PE positive rate >90% were selected for further amplification by pressure screening and flow sorting of hygromycin (200. Mu.g/ml) (using 10. Mu.g/ml Anti-ACE2 and PE coupled Anti-HumanIgG-Fc), and HEK293 cells expressing ACE2, namely ACE2-293 cells, were selected.

Wherein, the SARS-CoV-2 Spike pseudovirus is: SARS-CoV-2 Spike pseudovirus (Gimbago sp., GM-0220PV 07); SARS-COV-2 Spike (B.1.617.2) pseudovirus (Gimban organism, GM-0220PV 45); SARS-COV-2 Spike (B.1.1.529) pseudovirus (Gimbap. Gigantea, GM-0220PV 84).

The results are shown in FIG. 3, which shows that fusion protein D and fusion protein G all have inhibitory effects on SARS-CoV-2 Spike pseudovirus, SARS-COV-2 Spike (B.1.617.2) pseudovirus and SARS-COV-2 Spike (B.1.1.529) pseudovirus, and that fusion protein G has a better inhibitory effect on SARS-COV-2 Spike (B.1.1.529) pseudovirus.

Example 4: immunogenicity of vaccines in mice (intramuscular injection)

To assess the immunogenicity of monovalent vaccines (fusion protein D and fusion protein G), BALB/c mice were used to explore the effect of different antigen doses on immunogenicity.

1.1 immunization of mice

Balb/c mice (n =10 per group) were injected intramuscularly with different doses of vaccine on day 0 and 21, respectively, and control mice were given SEPIVAC SWE alone ^TM Adjuvant, SEPIVAC SWE per mouse ^TM Adjuvant (SEPPIC s.a., cat No. 80748J, lot No. 210721010001) was fixed in volume at 50 μ L, total volume at 100 μ L/stick per administration, and the group dosing schedule is shown in table 3. Blood was collected at day 14 and day 35 post immunization for ELISA detection of serum antibodies [ wild type, delta and Omicron (BA.1)]IgG titer and SARS-CoV-2 Spike pseudovirus neutralizing antibody assay.

TABLE 3 group dosing regimen

Group of	Medicine	Dosage (mu g/body)	Immunization regimen
				1	Fusion protein D	0.2	i.m.
2	Fusion protein D	1	i.m.
				3	Fusion protein D	5	i.m.
4	Fusion protein G	0.2	i.m.
				5	Fusion protein G	1	i.m.
6	Fusion protein G	5	i.m.

1.2 detection of serum IgG titer against Spike protein by ELISA

WT-Spike-His (same as 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) were diluted to 1. Mu.g/mL with PBS, and added to a 96-well microplate (Costar, cat # 9018) at 100. Mu.L/well overnight at 4 ℃; washing 2 times with PBST (0.05% volume Tween-20 in 1 XPBS); add blocking solution (PBST containing 3% BSA) and incubate 2 hours at 37 ℃; washing 3 times with PBST; a gradient of mouse serum obtained in step 1.1 of example 4 (serum was diluted 100-fold at day 14 as the starting concentration, 1000-fold at day 35 as the starting concentration, and then diluted 11 gradients in 3-fold gradient) was added at 100. Mu.L per well, and incubated at 37 ℃ for 1.5 hours; wash 5 times with PBST; adding 100 mu L/well of Peroxidase-Affinipure Goat Anti-Mouse IgG (Jackson, cat number: 115-035-003) diluted by 1; PBST wash 8 times; adding 100 mu L/hole TMB solution and incubating for 15-25 minutes at 37 ℃; the reaction was stopped with 50. Mu.L/well of 0.1M sulfuric acid; the reading was performed by setting the detection wavelength to 450nm, and the obtained reading OD value was fitted with a nonlinear four-parameter equation curve model, with 2.0 times the average value of the readings of the negative wells in each 96-well plate as a final value, to thereby determine the titer of each serum sample.

The results are shown in FIGS. 4a to 4f. On day 14 after the primary immunization, no anti-Spike protein IgG titers were detected in the adjuvant-only mice in all dose groups tested. All mice in the other groups had elevated IgG titers against Spike protein, and all mice had further significant elevated IgG titers after the second booster immunization. Furthermore, anti-Spike protein IgG titers increased dose-dependently over the dose range after a single immunization, but there was no significant difference between the 1 μ g and 5 μ g antigen groups after the second booster immunization. Furthermore, the mice receiving the fusion protein G had lower antibody titers against the wild-type and Delta anti-Spike proteins than those receiving the same dose of the fusion protein D, but the fusion protein G induced antibody titer against the Omicron anti-Spike protein was significantly higher than that of the fusion protein D. Fusion protein D induced strong anti-Spike protein IgG antibody titers to wild-type and Delta, but the antibody titer to Omicron decreased significantly.

These studies indicate that both test vaccines elicit dose-related antigen-specific antibody responses.

1.3 SARS-CoV-2 Spike pseudovirus neutralizing antibody assay

Gradient-diluting the mouse serum collected on day 35 of step 1.1 of example 4 with DMEM medium containing 10% FBS, and adding 50. Mu.L/well to a 96-well cell culture whiteboard; then, SARS-CoV-2 pseudovirus was diluted with 10% FBS-containing DMEM medium, and the pseudovirus dilution (25. Mu.L/well) was added to the 96-well plate to which the mouse serum dilution was added. The 96-well plate was incubated for 1h at 37 ℃ in an incubator. The 96-well plate after incubation for 1h was removed and ACE2-293 cell suspension (2X 10) was added at 50. Mu.L/well ⁴ cells/well), the 96-well plate was placed in an incubator for culture. After 48h incubation, the 96-well plates were removed and allowed to return to room temperature, 50. Mu.L Bio-Lite was added to each well ^TM The Luciferase Assay System (Vazyme, cat # DD 1201) solution was reacted for 2min at room temperature in the dark, and then the Luminescence signal was read by a microplate reader Luminescence detection module.

Wherein, the SARS-CoV-2 Spike pseudovirus is: SARS-CoV-2 Spike pseudovirus (Gimbago sp., GM-0220PV 07); SARS-COV-2 Spike (B.1.617.2) pseudovirus (Gimbala, GM-0220PV 45); SARS-COV-2 Spike (B.1.1.529) pseudovirus (Gimbap. Gigantea, GM-0220PV 84).

As shown in FIG. 5, the results showed that the fusion protein D and the fusion protein G all inhibited SARS-CoV-2 Spike pseudovirus, SARS-COV-2 Spike (B.1.617.2) pseudovirus and SARS-COV-2 Spike (B.1.1.529) pseudovirus, and that the fusion protein G had a good inhibitory effect on SARS-COV-2 Spike (B.1.1.529) pseudovirus.

Claims

1. A coronavirus Spike protein extracellular domain comprising a mutation or a truncated fragment thereof, wherein the mutation comprises: 1) Mutating RRAR to GSAS; 2) There is a sudden change in the turn region between HR1 and CH that prevents the formation of a straight helix during fusion; alternatively, the mutation comprises: 1) Mutating RRAR to GSAS; 2) The double mutation K986P/V987P exists in the diversion region between HR1 and CH; or, the truncated fragment of the extracellular domain of the coronavirus Spike protein containing the mutation is truncated by 5-80 amino acid residues at the C terminal compared with the full-length extracellular domain of the coronavirus Spike protein; or, the C-terminus is truncated by 20-76 amino acid residues; or, the C-terminus is truncated by 70 amino acid residues; alternatively, the coronavirus is SARS-CoV-2, SARS-CoV or MERS-CoV; or, the coronavirus is wild type SARS-CoV-2 or variant thereof; or, the coronavirus is a wild-type SARS-CoV-2, SARS-CoV-2Alpha variant, SARS-CoV-2Beta variant, SARS-CoV-2Gamma variant, SARS-CoV-2Delta variant, SARS-CoV-2Kappa variant, SARS-CoV-2Epsilon variant, SARS-CoV-2Lambda variant or SARS-CoV-2Omicron variant; alternatively, the mutant-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises an amino acid sequence as set forth in any one of SEQ ID NOs 3-4, 6-7, 9-12, 32-35, 78-83, or an amino acid sequence having at least 80% or at least 90% identity with an amino acid sequence as set forth in any one of SEQ ID NOs 3-4, 6-7, 9-12, 32-35, 78-83, or an amino acid sequence having one or more conservative amino acid substitutions with an amino acid sequence as set forth in any one of SEQ ID NOs 3-4, 6-7, 9-12, 32-35, 78-83.

2. A fusion protein comprising the mutant-containing coronavirus Spike protein extracellular domain or truncated fragment thereof of claim 1 and a monomer subunit protein linked by a linker; alternatively, the monomeric subunit protein is a self-assembled monomeric subunit protein; alternatively, the monomeric subunit protein is a monomeric ferritin subunit; or, the fusion protein is formed by connecting the C end of the mutant-containing coronavirus Spike protein extracellular domain or the truncated fragment thereof with the N end of the monomer subunit protein through a linker; or, the fusion protein is formed by connecting the C end of the mutant-containing coronavirus Spike protein extracellular domain or the truncated fragment thereof with the N end of the monomer ferritin subunit through a joint.

3. A fusion protein, comprising a coronavirus Spike protein S1 subunit and a monomeric subunit protein connected by a linker; alternatively, the monomeric subunit protein is a self-assembled monomeric subunit protein; alternatively, the monomeric subunit protein is a monomeric ferritin subunit; or, the fusion protein is formed by connecting the C end of the S1 subunit of the coronavirus Spike protein with the N end of the monomer subunit protein through a linker.

4. A fusion protein comprising a conserved fragment of a coronavirus Spike protein and a monomeric subunit protein linked by a linker; alternatively, the monomeric subunit protein is a self-assembled monomeric subunit protein; alternatively, the monomeric subunit protein is a monomeric ferritin subunit; or, the fusion protein is formed by connecting the C end of the conserved fragment of the coronavirus Spike protein with the N end of the monomer subunit protein through a linker.

5. The fusion protein of any one of claims 2-4, wherein the linker is a GS linker; alternatively, the linker is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGGG, GGSS, (GGGGS) ₂ ，(GGGGS) ₃ Or any combination thereof; or, the linker is (G) _m S) _n Wherein each m is independentIs 1,2, 3, 4 or 5,n is 1,2, 3, 4 or 5.

6. The fusion protein of any one of claims 2-5, wherein the monomeric ferritin subunit is selected from the group consisting of bacterial ferritin, plant ferritin, algal ferritin, insect ferritin, fungal ferritin, and mammalian ferritin; alternatively, the monomeric ferritin subunit is a helicobacter pylori non-heme monomeric ferritin subunit; alternatively, the monomeric ferritin subunit comprises an amino acid sequence as set forth 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 set forth in SEQ ID NO. 14, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO. 14.

7. A fusion protein comprising the extracellular domain of the mutation-containing coronavirus Spike protein of claim 1 or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto; alternatively, the C-terminus of the mutant-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof is linked to the N-terminus of the Fc fragment of an immunoglobulin.

8. A fusion protein comprising the S1 subunit of the coronavirus Spike protein and an Fc fragment of an immunoglobulin linked thereto; alternatively, the fusion protein is formed by connecting the C terminal of the S1 subunit of the coronavirus Spike protein with the N terminal of the Fc fragment of the immunoglobulin.

9. A fusion protein comprising a conserved fragment of the coronavirus Spike protein and an Fc fragment of an immunoglobulin linked thereto; alternatively, the fusion protein is formed by connecting the C end of the conserved fragment of the coronavirus Spike protein with the N end of the Fc fragment of the immunoglobulin.

10. The fusion protein of any one of claims 7-9, wherein the Fc fragment of the immunoglobulin is from an IgG, igM, igA, igE, or IgD; alternatively, 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 IgG 1; or, the Fc fragment of the immunoglobulin is the Fc fragment of human IgG 1; alternatively, the Fc fragment of the immunoglobulin comprises an amino acid sequence as shown in SEQ ID NO. 38, 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. 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.

11. The fusion protein of any one of claims 2-10, further comprising an N-terminal signal peptide; alternatively, the N-terminal signal peptide is selected from the group consisting of signal peptides of CSP, mschito, MF- α, pho1, HBM, t-pA, and IL-3; alternatively, the N-terminal signal peptide comprises an amino acid sequence as set forth in SEQ ID NO 2 or 5, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence set forth in SEQ ID NO 2 or 5, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO 2 or 5.

12. The fusion protein of any one of claims 2-11, wherein the coronavirus is SARS-CoV-2, SARS-CoV, or MERS-CoV; or, the coronavirus is wild type SARS-CoV-2 or variant thereof; alternatively, the coronavirus is a wild-type SARS-CoV-2, SARS-CoV-2Alpha variant, SARS-CoV-2Beta variant, SARS-CoV-2Gamma variant, SARS-CoV-2Delta variant, SARS-CoV-2Kappa variant, SARS-CoV-2Epsilon variant, SARS-CoV-2Lambda variant or SARS-CoV-2Omicron variant.

13. The fusion protein of any one of claims 2, 5 to 6, 11 to 12, comprising an amino acid sequence as set forth in any one of SEQ ID NOs 16 to 23, 26 to 29, 41 to 44, 66 to 67, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence set forth in any one of SEQ ID NOs 16 to 23, 26 to 29, 41 to 44, 66 to 67, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in any one of SEQ ID NOs 16 to 23, 26 to 29, 41 to 44, 66 to 67.

14. The fusion protein of any one of claims 3, 5 to 6, 11 to 12, comprising an amino acid sequence as set forth in any one of SEQ ID NOs 24 to 25, 30, 39 to 40, 65, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence set forth in any one of SEQ ID NOs 24 to 25, 30, 39 to 40, 65, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in any one of SEQ ID NOs 24 to 25, 30, 39 to 40, 65.

15. The fusion protein of any one of claims 4-6, 11-12, comprising an amino acid sequence as set forth in any one of SEQ ID NOs 45-46, 68, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence set forth in any one of SEQ ID NOs 45-46, 68, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in any one of SEQ ID NOs 45-46, 68.

16. The fusion protein of any one of claims 7 and 10-12, comprising an amino acid sequence as set forth 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 to an amino acid sequence as set forth 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 as compared to an amino acid sequence set forth in any one of SEQ ID NOs 47-54, 59-62, 69-72, 75-76.

17. The fusion protein of any one of claims 8 and 10 to 12, comprising an amino acid sequence as set forth in any one of SEQ ID NOs 55 to 58, 73 to 74, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence set forth in any one of SEQ ID NOs 55 to 58, 73 to 74, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in any one of SEQ ID NOs 55 to 58, 73 to 74.

18. The fusion protein of any one of claims 9 to 12, comprising an amino acid sequence as set forth in any one of SEQ ID NOs 63 to 64, 77, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence set forth in any one of SEQ ID NOs 63 to 64, 77, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in any one of SEQ ID NOs 63 to 64, 77.

19. A biomaterial is prepared from

(1) A polynucleotide encoding the extracellular domain of the mutation-containing coronavirus Spike protein of claim 1 or a truncated fragment thereof or the fusion protein of any one of claims 2-18; or the like, or, alternatively,

(2) An expression vector comprising a polynucleotide encoding the mutant-containing coronavirus Spike protein extracellular domain of claim 1 or a truncated fragment thereof or the fusion protein of any one of claims 2-18; or the like, or, alternatively,

(3) A cell comprising an expression vector encoding the mutant-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof according to claim 1, the fusion protein according to any one of claims 2 to 18, or a polynucleotide encoding the mutant-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof according to claim 1, the fusion protein according to any one of claims 2 to 18.

20. A Spike protein nanoparticle comprising the fusion protein of any one of claims 2-6, 11-15.

21. A coronavirus vaccine, comprising the fusion protein of any one of claims 2-18 and/or the Spike protein nanoparticle of claim 20; alternatively, a pharmaceutically acceptable carrier and/or adjuvant is also included.

22. Use of a fusion protein according to any one of claims 2 to 18 or a nanoparticle of Spike protein according to claim 20 for the preparation of a vaccine for the prevention or treatment of a coronavirus infection; alternatively, the coronavirus infection is a SARS-CoV-2, SARS-CoV or MERS-CoV infection; alternatively, the coronavirus infection is wild-type SARS-CoV-2 or variant thereof infection; alternatively, the coronavirus infection is infection with a wild-type SARS-CoV-2, SARS-CoV-2Alpha variant, SARS-CoV-2Beta variant, SARS-CoV-2Gamma variant, SARS-CoV-2Delta variant, SARS-CoV-2Kappa variant, SARS-CoV-2Epsilon variant, SARS-CoV-2Lambda variant or SARS-CoV-2Omicron variant.

23. A method of preventing or treating a coronavirus infection, comprising administering to a patient in need thereof an effective amount of the fusion protein of any one of claims 2-18 or the Spike protein nanoparticles of claim 20 or the coronavirus vaccine of claim 21; alternatively, the coronavirus infection is a SARS-CoV-2, SARS-CoV or MERS-CoV infection; alternatively, the coronavirus infection is wild-type SARS-CoV-2 or variant thereof infection; alternatively, the coronavirus infection is an infection with a wild-type SARS-CoV-2, SARS-CoV-2Alpha variant, SARS-CoV-2Beta variant, SARS-CoV-2Gamma variant, SARS-CoV-2Delta variant, SARS-CoV-2Kappa variant, SARS-CoV-2Epsilon variant, SARS-CoV-2Lambda variant or SARS-CoV-2Omicron variant.