WO2023020623A1 - 用于预防或治疗冠状病毒感染的融合蛋白、Spike蛋白纳米颗粒及其应用 - Google Patents

用于预防或治疗冠状病毒感染的融合蛋白、Spike蛋白纳米颗粒及其应用 Download PDF

Info

Publication number
WO2023020623A1
WO2023020623A1 PCT/CN2022/113747 CN2022113747W WO2023020623A1 WO 2023020623 A1 WO2023020623 A1 WO 2023020623A1 CN 2022113747 W CN2022113747 W CN 2022113747W WO 2023020623 A1 WO2023020623 A1 WO 2023020623A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
cov
sars
fusion protein
seq
Prior art date
Application number
PCT/CN2022/113747
Other languages
English (en)
French (fr)
Inventor
苏华飞
郑丹丹
冯旭
黄贤明
李胜峰
Original Assignee
百奥泰生物制药股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 百奥泰生物制药股份有限公司 filed Critical 百奥泰生物制药股份有限公司
Publication of WO2023020623A1 publication Critical patent/WO2023020623A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a fusion protein for preventing or treating coronavirus infection, Spike protein nanoparticles and applications thereof.
  • Coronavirus is a single-stranded positive-sense RNA virus that is not segmented. According to the serotype and genomic characteristics, the subfamily Coronaviridae is divided into four genera: ⁇ , ⁇ , ⁇ , and ⁇ . Protruding, shaped like a corolla and named after it.
  • the new coronavirus (SARS-CoV-2 or 2019-nCoV) discovered in 2019 belongs to the new coronavirus of the genus ⁇ , with an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 60-140nm. Current research shows that SARS-CoV-2 is highly homologous to SARS-CoV.
  • the novel coronavirus pneumonia COVID-19 is mainly transmitted through the respiratory tract, and it may also be transmitted through contact.
  • the crowd is generally susceptible, and the elderly and those with underlying diseases are more seriously ill after infection, and children and infants also have the disease.
  • the incubation period of the new coronavirus is generally 1-14 days, most of which are 3-7 days.
  • the main clinical symptoms of infected people are fever, fatigue, and dry cough, while upper respiratory symptoms such as nasal congestion and runny nose are rare.
  • the total number of white blood cells in patients is normal or decreased, or the number of lymphocytes is decreased, and some patients have increased liver enzymes, muscle enzymes and myoglobin.
  • Chest imaging showed multiple small patchy shadows and interstitial changes in the early stage, especially in the extrapulmonary zone; then it developed into multiple ground-glass shadows and infiltration shadows in both lungs, and in severe cases, lung consolidation and dyspnea gradually appeared.
  • Acute Respiratory Distress Syndrome (ARDS) shock, and various tissue damage and dysfunction of lung tissue, heart, and kidney occurred in patients. Most patients with mild infection have a good prognosis, while those with severe infection are often in critical condition and even die.
  • the present invention provides a coronavirus Spike (spike) protein ectodomain or a truncated fragment thereof comprising a mutation that can stabilize the protein structure, and a fusion comprising a mutated coronavirus Spike protein ectodomain or a truncated fragment thereof protein.
  • the present invention also provides a coronavirus vaccine comprising the mutated coronavirus Spike protein ectodomain or a truncated fragment thereof fused with a monomeric ferritin subunit and self-assembled to form a coronavirus vaccine capable of inducing a stronger response to the coronavirus Strong neutralizing antibody response.
  • Virus particles first communicate with angiotensin-converting enzyme 2 (ACE2) on the surface of lung epithelial cells through the receptor binding domain (RBD) in the S1 subunit of the Spike protein (S protein or spike protein) on its surface.
  • ACE2 angiotensin-converting enzyme 2
  • RBD receptor binding domain
  • S protein or spike protein Spike protein
  • the heptapeptide repeat sequence 1 (HR1) and the heptapeptide repeat sequence 2 (HR2) in the S2 subunit interact with each other to form a six-helix bundle (6-HB) fusion core, resulting in the fusion of the viral shell and the cell membrane, SARS-CoV or SARS-CoV CoV-2 enters the cell and uses the cell to synthesize new virus particles for it; the new virus particles are released outside the cell and then use the same method to infect surrounding normal cells.
  • the fusion protein, nanoparticle and vaccine of the present invention can induce a stronger neutralizing antibody response to coronavirus.
  • a coronavirus Spike protein ectodomain or a truncated fragment thereof containing a mutation comprising: 1) RRAR is mutated to GSAS; 2) in HR1 and the central helical region (CH)
  • the steering region in between contains mutations that prevent HR1 and CH from forming a straight helix during fusion.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • the amino acid numbering of the coronavirus Spike protein is based on the amino acid numbering of cryo-EM model PDB ID 6VSB or GenBank accession number MN908947.3 as a reference.
  • the truncated fragment of the ectodomain of the mutated coronavirus Spike protein has 5-80 amino acid residues truncated at the C-terminus compared with the full-length extracellular domain of the coronavirus Spike protein base. In some embodiments, the truncated fragment of the ectodomain of the coronavirus Spike protein containing mutations, compared with the full-length extracellular domain of the coronavirus Spike protein, the C-terminus is truncated by 20-76 amino acid residues base.
  • the truncated fragment of the ectodomain of the mutated coronavirus Spike protein has 70 amino acid residues truncated at the C-terminus compared with the full-length extracellular domain of the coronavirus Spike protein.
  • the coronavirus is SARS-CoV-2, SARS-CoV or MERS-Cov.
  • the coronavirus is wild-type SARS-CoV-2 or a variant thereof.
  • the coronavirus is wild-type SARS-CoV-2, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant.
  • the mutated coronavirus Spike protein ectodomain or its truncated fragment comprises any of SEQ ID NO: 3-4, 6-7, 9-12, 32-35, 78-83
  • Some embodiments also provide a fusion protein comprising the extracellular domain of the mutated coronavirus Spike protein described herein or a truncated fragment thereof.
  • a fusion protein comprising the ectodomain of the mutated coronavirus Spike protein described herein or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the monomeric subunit protein is a self-assembled monomeric subunit protein.
  • the monomeric subunit protein is a monomeric ferritin subunit.
  • the fusion protein is to connect the C-terminus of the mutated coronavirus Spike protein extracellular domain or its truncated fragment to the N-terminus of the monomeric subunit protein through a linker.
  • the fusion protein is to connect the C-terminus of the mutated coronavirus Spike protein extracellular domain or its truncated fragment with the N-terminus of the monomeric ferritin subunit through a linker.
  • the linker is a GS linker. In some embodiments, the linker is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGGG, GGSS, (GGGGS) 2 , (GGGGS) 3 , or any combination thereof. In some embodiments, the linker is ( GmS ) n , wherein each m is independently 1, 2, 3, 4, or 5 and n is 1, 2, 3, 4, or 5. In some embodiments, the sequence of the linker is (GGGGS) n , and the 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 .
  • the fusion protein further comprises an N-terminal signal peptide.
  • the signal peptide is selected from signal peptides of CSP, mschito, MF- ⁇ , pho1, HBM, t-pA, and IL-3.
  • the N-terminal signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2 or 5, or has at least 80% or at least 90% of the amino acid sequence shown in SEQ ID NO: 2 or 5 % identity amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 2 or 5.
  • the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, phycoferritin, insect ferritin, fungal ferritin, or mammalian ferritin.
  • the monomeric ferritin subunit is a H. pylori non-heme monomeric ferritin subunit.
  • the N19Q mutation is present in the H. pylori non-heme monomeric ferritin subunit amino acid sequence.
  • the monomeric ferritin subunit comprises the amino acid sequence set forth in SEQ ID NO: 14, or is at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14 A non-specific amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 14.
  • a fusion protein comprising a mutated wild-type SARS-CoV-2 Spike protein ectodomain or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutated wild-type SARS-CoV-2 Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutant SARS-CoV-2 Alpha variant Spike protein ectodomain or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutated SARS-CoV-2 Alpha variant Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutant SARS-CoV-2 Beta variant Spike protein ectodomain or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutant SARS-CoV-2 Beta variant Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutant SARS-CoV-2 Gamma variant Spike protein ectodomain or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutated SARS-CoV-2 Gamma variant Spike protein ectodomain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutant SARS-CoV-2 Delta variant Spike protein ectodomain or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutant SARS-CoV-2 Delta variant Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutant SARS-CoV-2 Kappa variant Spike protein ectodomain or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutant SARS-CoV-2 Kappa variant Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutant SARS-CoV-2 Epsilon variant Spike protein ectodomain or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutated SARS-CoV-2 Epsilon variant Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutant SARS-CoV-2 Lambda variant Spike protein extracellular domain or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutant SARS-CoV-2 Lambda mutant Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutant SARS-CoV-2 Omicron mutant Spike protein ectodomain or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutant SARS-CoV-2 Omicron variant Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • the fusion protein comprises a mutant coronavirus Spike protein ectodomain and a monomer ferritin subunit connected by a linker
  • the mutated coronavirus Spike protein ectodomain comprises such as SEQ
  • the amino acid sequence shown in any one of ID NO:3-4, 9-10, 32-33, 78, 80, 82, described monomer ferritin subunit comprises the amino acid sequence shown in SEQ ID NO:14;
  • the ectodomain of the coronavirus Spike protein containing the mutation is connected to the monomeric ferritin subunit by a linker as shown in SEQ ID NO:15.
  • the fusion protein comprises a truncated fragment containing a mutated coronavirus Spike protein extracellular domain and a monomeric ferritin subunit connected by a linker, and the mutated coronavirus Spike protein extracellular structure
  • the truncated fragment of the domain comprises the amino acid sequence shown in any one of SEQ ID NO:6-7, 11-12, 34-35, 79, 81, 83, and the monomer ferritin subunit comprises such as SEQ ID NO Amino acid sequence shown in: 14;
  • the truncated fragment containing the mutated coronavirus Spike protein extracellular domain is connected with monomer ferritin subunit by linker shown in SEQ ID NO: 15.
  • the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 16-23, 26-29, 41-44, 66-67, or with SEQ ID NO: 16-23, An amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence shown in any one of 26-29, 41-44, 66-67, or with SEQ ID NO: 16-23, 26-29, 41 - an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of 44, 66-67.
  • a fusion protein comprising a coronavirus Spike protein S1 subunit and a monomeric subunit protein connected by a linker.
  • the monomeric subunit protein is a self-assembled monomeric subunit protein.
  • the monomeric subunit protein is a monomeric ferritin subunit.
  • the fusion protein connects the C-terminal of the S1 subunit of the coronavirus Spike protein to the N-terminal of the monomeric subunit protein through a linker.
  • 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 wild-type SARS-CoV-2, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant.
  • the linker is a GS linker. In some embodiments, the linker is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGGG, GGSS, (GGGGS) 2 , (GGGGS) 3 , or any combination thereof. In some embodiments, the linker is ( GmS ) n , wherein each m is independently 1, 2, 3, 4, or 5 and n is 1, 2, 3, 4, or 5. In some embodiments, the sequence of the linker is (GGGGS) n , and the 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 .
  • the fusion protein further comprises an N-terminal signal peptide.
  • the signal peptide is selected from signal peptides of CSP, mschito, MF- ⁇ , pho1, HBM, t-pA, and IL-3.
  • the N-terminal signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2 or 5, or has at least 80% or at least 90% of the amino acid sequence shown in SEQ ID NO: 2 or 5 % identity amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 2 or 5.
  • the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, phycoferritin, insect ferritin, fungal ferritin, or mammalian ferritin.
  • the monomeric ferritin subunit is a H. pylori non-heme monomeric ferritin subunit.
  • the N19Q mutation is present in the H. pylori non-heme monomeric ferritin subunit amino acid sequence.
  • the monomeric ferritin subunit comprises the amino acid sequence set forth in SEQ ID NO: 14, or is at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14 A non-specific amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 14.
  • a fusion protein comprising a wild-type SARS-CoV-2 Spike protein S1 subunit and a monomeric ferritin subunit linked by a linker.
  • the fusion protein connects the C-terminal of the S1 subunit of the wild-type SARS-CoV-2 Spike protein to the N-terminal of the monomeric ferritin subunit through a linker.
  • a fusion protein comprising a SARS-CoV-2 Alpha variant Spike protein S1 subunit and a monomeric ferritin subunit connected by a linker.
  • the fusion protein is to connect the C-terminus of the S1 subunit of the Spike protein of the SARS-CoV-2 Alpha variant to the N-terminus of the monomeric ferritin subunit through a linker.
  • a fusion protein comprising a SARS-CoV-2 Beta variant Spike protein S1 subunit and a monomeric ferritin subunit linked by a linker.
  • the fusion protein is to connect the C-terminal of the S1 subunit of the Spike protein of the SARS-CoV-2 Beta variant strain to the N-terminal of the monomeric ferritin subunit through a linker.
  • a fusion protein comprising a SARS-CoV-2 Gamma variant Spike protein S1 subunit and a monomeric ferritin subunit linked by a linker.
  • the fusion protein is to connect the C-terminal of the S1 subunit of the Spike protein of the SARS-CoV-2 Gamma variant strain to the N-terminal of the monomeric ferritin subunit through a linker.
  • a fusion protein comprising a SARS-CoV-2 Delta variant Spike protein S1 subunit and a monomeric ferritin subunit linked by a linker.
  • the fusion protein is to connect the C-terminal of the S1 subunit of the Spike protein of the SARS-CoV-2 Delta variant strain to the N-terminal of the monomeric ferritin subunit through a linker.
  • a fusion protein comprising a SARS-CoV-2 Kappa variant Spike protein S1 subunit and a monomeric ferritin subunit linked by a linker.
  • the fusion protein is to connect the C-terminus of the S1 subunit of the Spike protein of the SARS-CoV-2 Kappa variant strain to the N-terminus of the monomeric ferritin subunit through a linker.
  • a fusion protein comprising a SARS-CoV-2 Epsilon variant Spike protein S1 subunit and a monomeric ferritin subunit linked by a linker.
  • the fusion protein is to connect the C-terminal of the S1 subunit of the Spike protein of the SARS-CoV-2 Epsilon mutant strain to the N-terminal of the monomeric ferritin subunit through a linker.
  • a fusion protein comprising a SARS-CoV-2 Lambda variant Spike protein S1 subunit and a monomeric ferritin subunit connected by a linker.
  • the fusion protein is to connect the C-terminal of the S1 subunit of the Spike protein of the SARS-CoV-2 Lambda mutant strain to the N-terminal of the monomeric ferritin subunit through a linker.
  • a fusion protein comprising the S1 subunit of the SARS-CoV-2 Omicron variant Spike protein and a monomeric ferritin subunit linked by a linker.
  • the fusion protein is to connect the C-terminus of the S1 subunit of the Spike protein of the SARS-CoV-2 Omicron variant through a linker to the N-terminus of the monomeric ferritin subunit.
  • the fusion protein comprises a coronavirus Spike protein S1 subunit connected by a linker and a monomeric ferritin subunit
  • the coronavirus Spike protein S1 subunit comprises a protein as shown in SEQ ID NO: 13 or 36.
  • the amino acid sequence of the monomeric ferritin subunit comprises the amino acid sequence shown in SEQ ID NO:14;
  • the coronavirus Spike protein S1 subunit is connected with the monomeric ferritin subunit by a linker shown in SEQ ID NO:15 connect.
  • the fusion protein is the C-terminus of the coronavirus Spike protein S1 subunit as shown in SEQ ID NO: 13 or 36 and the Helicobacter pylori as shown in SEQ ID NO: 14 through the linker GGGGS
  • the N-terminal linkage of the ferritin subunit of the heme monomer was obtained while replacing the original signal peptide: MFVFLVLLPLVSSQ with the signal peptide: MEFGLSLVFLVLILKGVQC.
  • the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 24-25, 30, 39-40, 65, or with SEQ ID NO: 24-25, 30, 39- An amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence shown in any one of 40, 65, or with any one of SEQ ID NO: 24-25, 30, 39-40, 65 An amino acid sequence is compared to an amino acid sequence having one or more conservative amino acid substitutions.
  • a fusion protein comprising a conserved fragment of a coronavirus Spike protein and a monomeric subunit protein connected by a linker.
  • the monomeric subunit protein is a self-assembled monomeric subunit protein.
  • the monomeric subunit protein is a monomeric ferritin subunit.
  • the fusion protein connects the C-terminus of the conserved fragment of the coronavirus Spike protein to the N-terminus of the monomeric subunit protein through a linker.
  • 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 wild-type SARS-CoV-2, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant.
  • the linker is a GS linker. In some embodiments, the linker is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGGG, GGSS, (GGGGS) 2 , (GGGGS) 3 , or any combination thereof. In some embodiments, the linker is ( GmS ) n , wherein each m is independently 1, 2, 3, 4, or 5 and n is 1, 2, 3, 4, or 5. In some embodiments, the sequence of the linker is (GGGGS) n , and the 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 .
  • the fusion protein further comprises an N-terminal signal peptide.
  • the signal peptide is selected from signal peptides of CSP, mschito, MF- ⁇ , pho1, HBM, t-pA, and IL-3.
  • the N-terminal signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2 or 5, or has at least 80% or at least 90% of the amino acid sequence shown in SEQ ID NO: 2 or 5 % identity amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 2 or 5.
  • the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, phycoferritin, insect ferritin, fungal ferritin, or mammalian ferritin.
  • the monomeric ferritin subunit is a H. pylori non-heme monomeric ferritin subunit.
  • the N19Q mutation is present in the H. pylori non-heme monomeric ferritin subunit amino acid sequence.
  • the monomeric ferritin subunit comprises the amino acid sequence set forth in SEQ ID NO: 14, or is at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14 A non-specific amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 14.
  • a fusion protein comprising a conserved fragment of the wild-type SARS-CoV-2 Spike protein and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a conserved fragment of the wild-type SARS-CoV-2 Spike protein and a monomeric ferritin subunit connected by a linker.
  • a fusion protein comprising a conserved fragment of Spike protein and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a conserved fragment of Spike protein and a monomeric ferritin subunit connected by a linker.
  • a fusion protein comprising a conserved fragment of Spike protein and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a conserved fragment of Spike protein and a monomeric ferritin subunit connected by a linker.
  • a fusion protein comprising a conserved fragment of Spike protein and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a conserved fragment of the Spike protein of the SARS-CoV-2 Gamma variant strain and a monomeric ferritin subunit connected by a linker.
  • a fusion protein comprising a conserved fragment of Spike protein and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a conserved fragment of Spike protein and a monomeric ferritin subunit connected by a linker.
  • a fusion protein comprising a conserved fragment of Spike protein and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a conserved fragment of the Spike protein of the SARS-CoV-2 Kappa variant strain connected by a linker and a monomeric ferritin subunit.
  • a fusion protein comprising a conserved fragment of Spike protein and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a conserved fragment of Spike protein and a monomeric ferritin subunit connected by a linker.
  • a fusion protein comprising a conserved fragment of Spike protein and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a conserved fragment of Spike protein and a monomeric ferritin subunit connected by a linker.
  • a fusion protein comprising a conserved fragment of Spike protein and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a conserved fragment of Spike protein and a monomeric ferritin subunit connected by a linker.
  • the fusion protein comprises a conserved fragment of the coronavirus Spike protein and a monomeric ferritin subunit connected by a linker, and the conserved fragment of the coronavirus Spike protein comprises the amino acid sequence shown in SEQ ID NO: 37,
  • the monomeric ferritin subunit comprises the amino acid sequence shown in SEQ ID NO: 14;
  • the conserved fragment of the coronavirus Spike protein is connected to the monomeric ferritin subunit through a linker shown in SEQ ID NO: 15.
  • the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 45-46, 68, or an amino acid sequence similar to that shown in any one of SEQ ID NO: 45-46, 68 Compared with an amino acid sequence having at least 80% or at least 90% identity, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 45-46, 68.
  • a fusion protein comprising the ectodomain of the mutated coronavirus Spike protein described herein or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto.
  • the fusion protein is to link the C-terminus of the ectodomain of the coronavirus Spike protein containing the mutation described herein or a truncated fragment thereof with the N-terminus of the Fc fragment of the immunoglobulin.
  • the fusion protein further comprises an N-terminal signal peptide.
  • the signal peptide is selected from signal peptides of CSP, mschito, MF- ⁇ , pho1, HBM, t-pA, and IL-3.
  • the N-terminal signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2 or 5, or has at least 80% or at least 90% of the amino acid sequence shown in SEQ ID NO: 2 or 5 % identity amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 2 or 5.
  • 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 IgGl, IgG2, IgG3 or IgG4. In some embodiments, the Fc fragment of the immunoglobulin is an IgG1 Fc fragment. In some embodiments, the Fc fragment of the immunoglobulin is the Fc fragment of human IgG1.
  • the Fc fragment of the immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO: 38, or is at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 38 A non-specific amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:38.
  • a fusion protein comprising a mutated wild-type SARS-CoV-2 Spike protein extracellular domain or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutated SARS-CoV-2 Alpha variant Spike protein extracellular domain or a truncated fragment thereof and an Fc fragment of an immunoglobulin connected thereto.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutated SARS-CoV-2 Beta variant Spike protein extracellular domain or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutated SARS-CoV-2 Gamma variant Spike protein extracellular domain or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutated SARS-CoV-2 Delta variant Spike protein extracellular domain or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutated SARS-CoV-2 Kappa variant Spike protein extracellular domain or a truncated fragment thereof and an Fc fragment of an immunoglobulin linked thereto.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutated SARS-CoV-2 Epsilon variant Spike protein extracellular domain or a truncated fragment thereof and an Fc fragment of an immunoglobulin connected thereto.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutated SARS-CoV-2 Lambda variant Spike protein extracellular domain or a truncated fragment thereof and an Fc fragment of an immunoglobulin connected thereto.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutated SARS-CoV-2 Omicron variant Spike protein extracellular domain or a truncated fragment thereof and an Fc fragment of an immunoglobulin connected thereto.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) double mutation K986P/V987P in the turn region between HR1 and CH.
  • the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO:47-54, 59-62, 69-72, 75-76, or with SEQ ID NO:47-54, An amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence shown in any one of 59-62, 69-72, 75-76, or with SEQ ID NO: 47-54, 59-62, 69 - An amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of 72, 75-76.
  • a fusion protein comprising a coronavirus Spike protein S1 subunit and an Fc fragment of an immunoglobulin linked thereto.
  • the fusion protein connects the C-terminal of the S1 subunit of the coronavirus Spike protein to the N-terminal of the Fc fragment of the immunoglobulin.
  • 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 wild-type SARS-CoV-2, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant.
  • the fusion protein further comprises an N-terminal signal peptide.
  • the signal peptide is selected from signal peptides of CSP, mschito, MF- ⁇ , pho1, HBM, t-pA, and IL-3.
  • the N-terminal signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2 or 5, or has at least 80% or at least 90% of the amino acid sequence shown in SEQ ID NO: 2 or 5 % identity amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 2 or 5.
  • 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 IgGl, IgG2, IgG3 or IgG4. In some embodiments, the Fc fragment of the immunoglobulin is an IgG1 Fc fragment. In some embodiments, the Fc fragment of the immunoglobulin is the Fc fragment of human IgG1.
  • the Fc fragment of the immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO: 38, or is at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 38 A non-specific amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:38.
  • a fusion protein comprising the S1 subunit of the wild-type SARS-CoV-2 Spike protein and the Fc fragment of an immunoglobulin linked thereto.
  • a fusion protein comprising the S1 subunit of the Spike protein of the SARS-CoV-2 Alpha variant and the Fc fragment of an immunoglobulin linked thereto.
  • a fusion protein comprising the S1 subunit of the Spike protein of the SARS-CoV-2 Beta variant and the Fc fragment of an immunoglobulin linked thereto.
  • a fusion protein comprising the S1 subunit of the Spike protein of the SARS-CoV-2 Gamma variant strain and the Fc fragment of an immunoglobulin linked thereto.
  • a fusion protein comprising the S1 subunit of the Spike protein of the SARS-CoV-2 Delta variant and the Fc fragment of an immunoglobulin linked thereto.
  • a fusion protein comprising the S1 subunit of Spike protein of SARS-CoV-2 Kappa variant strain and the Fc fragment of immunoglobulin linked thereto.
  • a fusion protein comprising the S1 subunit of the Spike protein of the SARS-CoV-2 Epsilon variant strain and the Fc fragment of an immunoglobulin linked thereto.
  • a fusion protein comprising the S1 subunit of Spike protein of SARS-CoV-2 Lambda mutant strain and the Fc fragment of immunoglobulin connected thereto.
  • a fusion protein comprising the S1 subunit of Spike protein of SARS-CoV-2 Omicron variant strain and the Fc fragment of immunoglobulin linked thereto.
  • the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO:55-58, 73-74, or with any one of SEQ ID NO:55-58, 73-74 An amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence of , or having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO:55-58, 73-74 amino acid sequence.
  • a fusion protein comprising a conserved fragment of a coronavirus Spike protein and an Fc fragment of an immunoglobulin linked thereto.
  • the fusion protein connects the C-terminus of the conserved fragment of the coronavirus Spike protein to the N-terminus of the Fc fragment of the immunoglobulin.
  • 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 wild-type SARS-CoV-2, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant.
  • the fusion protein further comprises an N-terminal signal peptide.
  • the signal peptide is selected from signal peptides of CSP, mschito, MF- ⁇ , pho1, HBM, t-pA, and IL-3.
  • the N-terminal signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2 or 5, or has at least 80% or at least 90% of the amino acid sequence shown in SEQ ID NO: 2 or 5 % identity amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 2 or 5.
  • 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 IgGl, IgG2, IgG3 or IgG4. In some embodiments, the Fc fragment of the immunoglobulin is an IgG1 Fc fragment. In some embodiments, the Fc fragment of the immunoglobulin is the Fc fragment of human IgG1.
  • the Fc fragment of the immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO: 38, or is at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 38 A non-specific amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:38.
  • a fusion protein comprising a conserved fragment of the wild-type SARS-CoV-2 Spike protein and an Fc fragment of an immunoglobulin linked thereto.
  • a fusion protein comprising a conserved fragment of the Spike protein of the SARS-CoV-2 Alpha variant strain and an Fc fragment of an immunoglobulin connected thereto.
  • a fusion protein comprising a conserved fragment of the Spike protein of the SARS-CoV-2 Beta variant strain and an Fc fragment of an immunoglobulin connected thereto.
  • a fusion protein comprising a conserved fragment of the Spike protein of the SARS-CoV-2 Gamma variant strain and an Fc fragment of an immunoglobulin connected thereto.
  • a fusion protein comprising a conserved fragment of the Spike protein of the SARS-CoV-2 Delta variant strain and an Fc fragment of an immunoglobulin connected thereto.
  • a fusion protein comprising a conserved fragment of the Spike protein of the SARS-CoV-2 Kappa variant strain and an Fc fragment of an immunoglobulin connected thereto.
  • a fusion protein comprising a conserved fragment of the Spike protein of the SARS-CoV-2 Epsilon mutant strain and an Fc fragment of an immunoglobulin connected thereto.
  • a fusion protein comprising a conserved fragment of the Spike protein of the SARS-CoV-2 Lambda variant strain and an Fc fragment of an immunoglobulin connected thereto.
  • a fusion protein comprising a conserved fragment of the Spike protein of the SARS-CoV-2 Omicron variant strain and an Fc fragment of an immunoglobulin connected thereto.
  • the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 63-64, 77, or an amino acid sequence similar to that shown in any one of SEQ ID NO: 63-64, 77 An amino acid sequence having at least 80% or at least 90% identity to it, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 63-64, 77.
  • 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 (inclusive) or where any value.
  • 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 % identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or a range between any two of these values, inclusive, or any of these values.
  • the one or more conservative amino acid substitutions are 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 (inclusive), or any value therein.
  • Some embodiments provide a polynucleotide encoding the ectodomain of the coronavirus Spike protein containing mutations described herein or a truncated fragment thereof, or a fusion protein.
  • an expression vector comprising a polynucleotide encoding the ectodomain of the mutated coronavirus Spike protein described herein or a truncated fragment or fusion protein thereof.
  • a cell expressing the ectodomain of the mutated coronavirus Spike protein described herein or a truncated fragment thereof comprises one or more polynucleotides encoding a fusion protein described herein or an expression vector comprising a polynucleotide encoding a fusion protein described herein.
  • the cells are isolated cells.
  • the cells are CHO cells, HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, or murine L cells.
  • the fusion protein comprises a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof and a monomer ferritin subunit connected by a linker, and the fusion protein includes the following characteristics:
  • the mutations comprise: 1) a mutation that inactivates the S1/S2 cleavage site; 2) a mutation in the steering region between HR1 and CH that prevents HR1 and CH from forming a straight helix during fusion; and/or
  • the C-terminus of the mutated coronavirus Spike protein ectodomain or its truncated fragment is connected to the monomeric ferritin subunit through a linker;
  • the linker is (G m S) n , wherein each m is independently 1, 2, 3, 4 or 5, and n is 1, 2, 3, 4 or 5; and/or
  • the monomeric ferritin subunit is a Helicobacter pylori monomeric ferritin subunit, comprising the amino acid sequence shown in SEQ ID NO: 14, or having at least 80% of the amino acid sequence shown in SEQ ID NO: 14 Or an amino acid sequence that is at least 90% identical, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 14.
  • Spike protein nanoparticles comprising the fusion proteins described herein.
  • a coronavirus vaccine comprising a fusion protein described herein and/or a Spike protein nanoparticle comprising a fusion protein.
  • the coronavirus vaccine further includes a pharmaceutically acceptable carrier and/or adjuvant.
  • the invention also provides a coronavirus vaccine.
  • the coronavirus vaccine comprises a fusion protein described herein and a pharmaceutically acceptable carrier and/or adjuvant.
  • the coronavirus vaccine comprises the Spike protein nanoparticles described herein and a pharmaceutically acceptable carrier and/or adjuvant.
  • the present invention also provides prevention or treatment methods and uses.
  • the present invention provides methods for preventing or treating coronavirus infection, the methods comprising administering an effective amount of the fusion protein, Spike protein nanoparticle or coronavirus vaccine described herein to a patient in need.
  • the use of the fusion protein, Spike protein nanoparticle or coronavirus vaccine described herein in the prevention or treatment of SARS or COVID-19 is provided.
  • the use of the fusion protein or Spike protein nanoparticles described herein in the preparation of a vaccine for preventing or treating SARS-CoV-2 infection is provided.
  • the coronavirus infection is a SARS-CoV-2, SARS-CoV or MERS-Cov infection.
  • the coronavirus infection is wild-type SARS-CoV-2 or a variant infection thereof.
  • the coronavirus infection is wild-type SARS-CoV-2, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV -2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant infection.
  • Figure 1 is the binding curve of fusion protein and human ACE2;
  • Figure 1a is the binding curve of fusion protein D and human ACE2, and
  • Figure 1b is the binding curve of fusion protein G and human ACE2.
  • Figure 2 is the serum anti-Spike protein IgG titer, and the bar graph represents the geometric mean (GMT) of the titer; in the figure, wildtype represents WT-Spike-His, Delta represents Delta-Spike-His, and Omicron represents Omicron-Spike-His .
  • Figure 3 is the anti-pseudovirus neutralization titer (IC 50 ); in the figure wildtype represents SARS-CoV-2 Spike pseudovirus, Delta represents SARS-COV-2 Spike (B.1.617.2) pseudovirus, Omicron represents SARS- COV-2 Spike (B.1.1.529) pseudovirus.
  • Fig. 4 is serum anti-Spike protein IgG titer, and bar graph represents the geometric mean (GMT) of titer;
  • Fig. 4a, Fig. 4c and Fig. 4e are 14 days (the 14th day) titer after administration for the first time
  • Figure 4b Figure 4d and Figure 4f are the titers 14 days after the second administration (the 35th day).
  • Figure 5 is the anti-pseudovirus neutralization titer (IC 50 ); in the figure wildtype represents SARS-CoV-2 Spike pseudovirus, Delta represents SARS-COV-2 Spike (B.1.617.2) pseudovirus, Omicron represents SARS- COV-2 Spike (B.1.1.529) pseudovirus.
  • nucleic acid molecule refers to one or more nucleic acid molecules. Accordingly, the terms “a”, “an”, “one or more” and “at least one” may be used interchangeably. Similarly, the terms “comprising”, “comprising” and “having” can be used interchangeably, and generally should be understood as open-ended and non-limiting, eg, other unrecited elements or steps are not excluded.
  • amino acid refers to an organic compound containing both amino and carboxyl groups, such as an ⁇ -amino acid, which can be encoded by a nucleic acid directly or in the form of a precursor.
  • a single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called “degeneracy of the genetic code”.
  • Amino acids include natural amino acids and unnatural amino acids.
  • Natural amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine amino acid (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).
  • a “conservative amino acid substitution” refers to the replacement of one amino acid residue with another amino acid residue containing a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions are unlikely to substantially alter the functional properties of a protein.
  • classes of amino acids that contain chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, Arginine and histidine; 6) acidic side chains: aspartic acid and glutamic acid.
  • polypeptide is intended to encompass the singular as well as the plural “polypeptides” and refers to a molecule composed of amino acid monomers linked linearly by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • the definition of “polypeptide” includes peptide, dipeptide, tripeptide, oligopeptide, "protein”, “amino acid chain” or any other term used to refer to a chain of two or more amino acids, and the term “polypeptide” may Used in place of, or interchangeably with, any of the above terms.
  • polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-natural Amino acid modifications that occur.
  • a polypeptide may be derived from natural biological sources or produced by recombinant techniques, but it need not be translated from a specified nucleic acid sequence, it may be produced by any means including chemical synthesis.
  • a fusion protein is a recombinant protein comprising amino acid sequences from at least two unrelated proteins that have been linked together by peptide bonds to form a single protein.
  • Amino acid sequences of unrelated proteins can be linked directly to each other, or can be linked using a linker.
  • proteins are not related if their amino acid sequences are not normally linked together by peptide bonds in their natural environment (eg, within a cell).
  • a bacterial enzyme such as Bacillus stearothermophilus dihydrolipoic acid transacetylase (E2p) and the coronavirus Spike protein are not linked together by a peptide bond.
  • homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the alignable positions in each sequence. When a position in the sequences being compared 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.
  • encoding when applied to a polynucleotide refers to a polynucleotide which is said to "encode” a polypeptide which, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce The polypeptide and/or fragments thereof.
  • a polynucleotide is composed of a specific sequence of four bases: adenine (A), cytosine (C), guanine (G), thymine (T), or when the polynucleotide is RNA Thymine was replaced with uracil (U).
  • a "polynucleotide sequence” may be denoted by the letters of the polynucleotide molecule. This letter designation can be entered into a database in a computer with a central processing unit and used in bioinformatics applications such as for functional genomics and homology searches.
  • polynucleotide polynucleotide
  • oligonucleotide oligonucleotide
  • a polynucleotide can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribose Somatic RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • nucleotides can be made before or after assembly of the polynucleotide.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • Polynucleotides may be further modified after polymerization, for example by conjugation with labeling components.
  • the term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of a polynucleotide of the present disclosure includes the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.
  • Identity or “sequence identity” of a nucleic acid or polynucleotide sequence (or polypeptide or protein sequence) with another sequence by a certain percentage means When sequences are aligned, the percentage of bases (or amino acids) in the two sequences being compared are the same. This alignment percent identity or sequence identity can be determined using visual inspection or software programs known in the art, such as those described by Ausubel et al.eds. (2007) in Current Protocols in Molecular Biology. It is preferred to use the default parameters for the alignment.
  • Biologically equivalent polynucleotides are polynucleotides that share the above indicated percentages of identity and encode a polypeptide having the same or similar biological activity.
  • isolated used in the present invention with respect to cells, nucleic acids, polypeptides, antibodies, etc., for example, "isolated" DNA, RNA, polypeptides, antibodies refers to the isolated components of the cell's natural environment, such as DNA or RNA. One or more of the isolated molecules.
  • isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is intended to include fragments of nucleic acid that do not occur in nature, and do not exist in nature.
  • isolated is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues.
  • Isolated polypeptide is intended to include purified and recombinant polypeptides.
  • Isolated polypeptides, antibodies, etc. will usually be prepared by at least one purification step.
  • the purity of the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or some of these values The range (inclusive) between any two values of , or any value therein.
  • polypeptides or polynucleotides refers to polypeptides or polynucleotides, meaning forms of polypeptides or polynucleotides that do not occur in nature, non-limiting examples may be produced by combination of polynucleotides or polynucleotides that do not normally exist or peptide.
  • Antibody and antigen-binding fragment refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen.
  • Antibodies can be whole antibodies and any antigen-binding fragments thereof or single chains thereof.
  • the term “antibody” thus includes any protein or peptide whose molecule contains at least a portion of an immunoglobulin molecule that has the biological activity to bind an antigen.
  • the terms "antigen” or “immunogen” are used interchangeably and refer to a substance, usually 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 humoral and/or Cell Type Immune Response.
  • vaccine antigen is used interchangeably with “protein antigen” or “antigenic polypeptide”.
  • Neutralizing antibody refers to an antibody that reduces the infectious titer of an infectious agent by binding to a specific antigen on the infectious agent.
  • 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., at least 85%, 90%, 95%, 96%, 97%, 98%, Antigens with % or 99% identity.
  • the antibodies may bind to and inhibit the function of antigens from more than one class and/or subclass of the pathogen.
  • cDNA refers to DNA that is complementary or identical to mRNA, and may be in single- or double-stranded form.
  • Epitope refers to an antigenic determinant. These are specific chemical groups or peptide sequences on molecules that are antigenic such that they elicit a specific immune response, eg, epitopes are regions of an antigen to which B and/or T cells respond. Epitopes can be formed from contiguous amino acids, or from non-contiguous amino acids juxtaposed by the tertiary folding of the protein.
  • Vaccines are biological products that elicit a prophylactic or therapeutic immune response in a subject.
  • the immune response is a protective immune response.
  • vaccines elicit an antigen-specific immune response against antigens of pathogens, such as viral pathogens, or cellular components associated with the pathological condition.
  • a vaccine may comprise a polynucleotide (eg, a nucleic acid encoding a known antigen), a peptide or polypeptide (eg, a disclosed antigen), a virus, a cell, or one or more cellular components.
  • the vaccine or vaccine antigen or vaccine composition is expressed from a fusion protein expression vector and self-assembles into nanoparticles displaying the antigenic polypeptide or protein on its surface.
  • an effective amount of a vaccine or other agent is sufficient to produce a desired response, eg, elicit an immune response, prevent, alleviate or eliminate signs or symptoms of a disorder or disease such as pneumonia.
  • a desired response eg, elicit an immune response
  • this may be the amount necessary to inhibit viral replication or measurably alter the outward symptoms of viral infection.
  • this amount will be sufficient to measurably inhibit replication or infectivity of a virus, such as SARS-CoV-2.
  • doses that have been shown to achieve inhibition of viral replication in vitro to achieve target tissue concentrations will generally be used.
  • an "effective amount" is an amount that treats (including prevents) one or more symptoms and/or underlying causes of a disorder or disease (eg, treats a coronavirus infection).
  • 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 (eg, 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. This spherical protein shell is formed from identical copies of non-viral proteins that are able to self-assemble into nanoparticles with a similar appearance to virus-like particles (VLPs). Examples include ferritin (FR), which is conserved across species and forms 24-mers (24-mer), Bacillus stearothermophilus dihydrolipoic acid transacetylase (E2P), hyperthermophilus dioxygenase Tetrahydropteridine synthase (LS) and Thermotoga maritima encapsulin, all of which form 60-mers. Self-assembling nanoparticles can form spontaneously after recombinantly expressing proteins in an appropriate expression system. Methods of production, detection and characterization of nanoparticles can use the same techniques developed for VLPs.
  • FR ferritin
  • E2P Bacillus stearothermophilus dihydrolip
  • VLPs refer to non-replicating viral capsids derived from any of a variety of viruses.
  • VLPs typically include one or more viral proteins such as, but not limited to, those known as capsid proteins, coat proteins, globular wall proteins, surface proteins and/or envelope proteins, or formed particles derived from these proteins of polypeptides.
  • VLPs can form spontaneously following recombinant expression of the protein. Methods of producing specific VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using routine techniques known in the art (eg, by electron microscopy, biophysical characterization, etc.). See, eg, Baker et al. (1991) Biophys.
  • VLPs can be separated by density gradient centrifugation and/or identified by characteristic density bands.
  • cryo-electron microscopy can be performed on vitrified water samples of the VLP preparation in question and images recorded under appropriate exposure conditions.
  • ECMO refers to extracorporeal membrane oxygenation (Extracorporeal Membrane Oxygenation, ECMO), which is a medical emergency technology equipment, mainly used to provide continuous extracorporeal respiration and circulation for patients with severe cardiopulmonary failure to maintain their lives.
  • ICU refers to the intensive care unit (Intensive Care Unit), where treatment, nursing, and rehabilitation can be carried out simultaneously, providing isolation places and equipment for severe or comatose patients, providing the best nursing care, comprehensive treatment, combination of medical care and nursing care, and surgery Postoperative early rehabilitation, joint care and sports therapy and other services.
  • Intensive Care Unit intensive care unit
  • Treatment, nursing, and rehabilitation can be carried out simultaneously, providing isolation places and equipment for severe or comatose patients, providing the best nursing care, comprehensive treatment, combination of medical care and nursing care, and surgery Postoperative early rehabilitation, joint care and sports therapy and other services.
  • IMV intermittent mandatory ventilation
  • intermittent mandatory ventilation implements periodic volume or pressure ventilation according to a preset time interval, that is, time trigger. This period allows the patient to breathe spontaneously at any set basal pressure level during mandatory ventilation.
  • spontaneous breathing the patient can breathe spontaneously with continuous airflow support, or the machine will open the valve on demand to allow spontaneous breathing. According to most ventilators can provide pressure support during spontaneous breathing.
  • 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. Unless stated otherwise, the terms “patient” or “subject” are used interchangeably herein. Preferably, the subject is a human.
  • Treatment means therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent, slow down, ameliorate or stop an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following whether detectable or undetectable Relief of symptoms, reduction of disease extent, stabilization of disease state (i.e. not worsening), delay or slowing of disease progression, amelioration, remission, alleviation or disappearance of disease state (whether partial or total), prolongation and Expected survival without treatment, etc.
  • Patients in need of treatment include those already suffering from a condition or disorder, those prone to suffer from a condition or disorder, or those in need of prevention of the condition or disorder, who can or are expected to benefit from the administration of the Spike protein nanoparticles or pharmaceutical compositions disclosed herein For patients who benefit from treatment.
  • the viral genome encodes the 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.
  • S glycoprotein Spike protein
  • RBD receptor-binding domain
  • Receptor binding can help keep the RBD in a "standing" state, which facilitates the dissociation of the S1 subunit from the S2 subunit.
  • a second S2' cleavage releases the fusion peptide.
  • HR1 and CH form a very long helical piece to insert the fusion peptide into the host cell membrane.
  • HR1 and HR2 form a helical structure and assemble into a six-helix bundle to fuse the viral and host membranes.
  • the RBD contains a core subdomain and a receptor binding motif (RBM).
  • RBM receptor binding motif
  • SARS-CoV and MERS-CoV-2 recognizes angiotensin-converting enzyme 2 (ACE2)
  • ACE2 angiotensin-converting enzyme 2
  • DPP4 dipeptidyl peptidase 4
  • S glycoprotein is surface-exposed and mediates entry into host cells, it is the primary target of neutralizing antibodies (NAbs) after infection and a focus of vaccine design.
  • Spike trimers are extensively modified with N-linked glycans that are important for proper folding and regulation of accessibility to NAbs.
  • the present invention stabilizes the Spike trimer by 1) a mutation inactivating the S1/S2 cleavage site and 2) the presence of a mutation in the steering region between HR1 and CH that prevents HR1 and CH from forming a straight helix during fusion In the conformation before fusion with the host cell membrane.
  • a mutated extracellular domain of a coronavirus Spike protein or a truncated fragment thereof can be displayed on a nanoparticle.
  • the present invention provides fusion proteins, Spike protein nanoparticles, and vaccine compositions.
  • the invention also provides related polynucleotides, expression vectors and pharmaceutical compositions.
  • the stabilized Spike trimer and RBD protein in the form of protein or nucleic acid (DNA/mRNA) carried by the viral vector can be used as a coronavirus vaccine.
  • the stabilized Spike trimer and RBD presented by nanoparticles can also be used as a coronavirus vaccine.
  • the coronavirus Spike protein-based antigens and vaccines of the present invention have a number of advantageous properties.
  • the design of Spike trimers described here presents conserved neutralizing epitopes in their native-like conformation, making Spike trimers useful as antigenic vaccines or multivalently displayed on nanoparticles.
  • the nanoparticle vaccine of the present invention allows the display of Spike trimers derived from different coronaviruses on well-known nanoparticles, such as ferritin, E2p and I3-01, with sizes ranging from 12.2 to 25.0 nm. All trimer-presenting nanoparticles could be produced in high yield in HEK293 cells, ExpiCHO cells, CHO cells.
  • the produced Spike protein nanoparticles can be purified by antibody and size exclusion chromatography (SEC).
  • the encoded polynucleotides, expression vectors and host cells and related therapeutic applications can be produced or performed according to the methods exemplified herein or conventional methods well known in the art.
  • the present invention provides the mutated extracellular domain of coronavirus Spike protein or its truncated fragments which can be used to produce vaccines.
  • the mutated Spike trimer is 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 NO: 1, 8 and 31, are exemplified herein for specific SARS-CoV-2 strains or isolates. Due to the functional similarity and sequence homology between different isolates or strains of a given coronavirus, it is also possible to generate orthologous sequences of Spike proteins derived from other known coronaviruses according to the mutation strategy described herein.
  • some mutated Spike proteins or truncated fragments thereof of the present invention contain mutations that enhance the stability of the structure of the Spike protein or truncated fragments thereof prior to fusion with the cell membrane. These mutations include mutations that inactivate the S1/S2 cleavage site, and mutations in the turning region between HR1 and CH that remove any strain in the turning region between HR1 and CH, i.e. prevent straight helix formation .
  • Some mutated coronavirus Spike protein ectodomains or truncated fragments thereof are derived from the virus that causes COVID- 19 of the SARS-CoV-2 virus. These peptides contain mutations that inactivate the S1/S2 cleavage sites as well as mutations in the steering region between HR1 and CH.
  • the amino acid sequence of the wild-type SARS-CoV-2 Spike protein used for mutation is shown in SEQ ID NO: 1 or the amino acid sequence shown in residues 15-1213 of SEQ ID NO: 1.
  • the Spike protein used for mutation may be SEQ ID NO: 1, 8 or 31 or a variant thereof, such as a variant substantially identical thereto or a conservatively modified variant.
  • inactivation of S1/S2 cleavage site 682 RRAR 685 can be altered by a number of sequences within or around the site (e.g., missing or replaced) to achieve.
  • one mutation that inactivates the S1/S2 cleavage site without affecting protein structure is to mutate the S1/S2 cleavage site 682 RRAR 685 to 682 GSAS 685 .
  • a double mutation in the turn region between HR1 and CH abolishes the turn region during fusion by preventing the formation of a straight helix (HR1 and CH motifs between) strains.
  • the double mutation may be K986G/V987G, K986P/V987P, K986G/V987P, or K986P/V987G.
  • some SARS-CoV-2 Spike proteins or their truncated fragments of the present invention may contain a deletion of most or the entire HR2 domain. Illustrated using the exemplary SARS-CoV-2 Spike protein sequence SEQ ID NO:1, such deletions may include deletions of residues 1144-1213 of SEQ ID NO:1. In some embodiments, the deletion can be 5, 10 of the C-terminus of the truncated Spike protein extracellular domain (eg, SEQ ID NO: 1, 3-4, 8-10, 31-33, 78, 80 or 82).
  • the C-terminally truncated Spike protein can extend beyond the HR2 domain.
  • the Spike protein sequence may include the N-terminal signal peptide shown in SEQ ID NO:2 or 5.
  • coronavirus Spike protein extracellular domain or its truncated fragment or its variant as follows:
  • ECD extracellular domain
  • SEQ ID NO: 1 The full-length extracellular domain (ECD) of the wild-type SARS-CoV-2 Spike protein, its amino acid sequence is shown in SEQ ID NO: 1, and the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is marked in italics , S1/S2 cleavage sites 682 RRAR 685 are underlined, bolded and italicized.
  • the amino acid sequence of the full-length extracellular domain a1 of the mutated wild-type SARS-CoV-2 Spike protein is shown in SEQ ID NO:3.
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO:2) is marked in italics
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , marked with underline and bold, and contains double Mutation K986P/V987P, underlined and italicized.
  • the amino acid sequence of the full-length extracellular domain a2 of the mutated wild-type SARS-CoV-2 Spike protein is shown in SEQ ID NO:4.
  • the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO:2) is replaced with signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5), the signal peptide is marked in italics, S1/S2 cleavage site
  • the mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is also included, which is underlined and italicized.
  • the amino acid sequence of the full-length extracellular domain a3 of the mutated wild-type SARS-CoV-2 Spike protein is shown in SEQ ID NO:78.
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which are underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the amino acid sequence of the C-terminal truncated fragment b1 of the ectodomain of the wild-type SARS-CoV-2 Spike protein is shown in SEQ ID NO:6.
  • the C-terminus is truncated by 70 amino acid residues
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) is marked in italics
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , It is underlined and bolded, while the double mutation K986P/V987P is included, it is underlined and italicized.
  • the amino acid sequence of the C-terminal truncated fragment b2 of the ectodomain of the wild-type SARS-CoV-2 Spike protein is shown in SEQ ID NO:7.
  • 70 amino acid residues were truncated at the C-terminus, and the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO: 5) was used to replace Marked in italics, the S1/S2 cleavage site 682 RRAR 685 mutation to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is also included, which is underlined and italicized.
  • the amino acid sequence of the C-terminal truncated fragment b3 of the extracellular domain of the wild-type SARS-CoV-2 Spike protein is shown in SEQ ID NO:79.
  • the C-terminus is truncated by 70 amino acid residues, does not contain a signal peptide, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which are underlined and bolded, and contain double mutations K986P/V987P , are underlined and italicized.
  • ECD extracellular domain
  • SEQ ID NO: 8 The full-length extracellular domain (ECD) of the Spike protein of the SARS-CoV-2 Delta mutant strain, its amino acid sequence is shown in SEQ ID NO: 8, and the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is marked in italics Out, S1/S2 cleavage sites 682 RRAR 685 are underlined, bolded and italicized.
  • the amino acid sequence of the full-length extracellular domain c1 of the mutated SARS-CoV-2 Delta variant Spike protein is shown in SEQ ID NO:9.
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO:2) is marked in italics
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , marked with underline and bold, and contains double Mutation K986P/V987P, underlined and italicized.
  • the amino acid sequence of the full-length extracellular domain c2 of the mutated SARS-CoV-2 Delta variant Spike protein is shown in SEQ ID NO:10.
  • the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO:2) is replaced with signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5), the signal peptide is marked in italics, S1/S2 cleavage site
  • the mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is also included, which is underlined and italicized.
  • the amino acid sequence of the full-length extracellular domain c3 of the mutated SARS-CoV-2 Delta variant Spike protein is shown in SEQ ID NO:80.
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which are underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the amino acid sequence of the C-terminal truncated fragment d1 of the extracellular domain of Spike protein of the mutated SARS-CoV-2 Delta variant strain is shown in SEQ ID NO:11.
  • the C-terminus is truncated by 70 amino acid residues
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) is marked in italics
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , It is underlined and bolded, while the double mutation K986P/V987P is included, it is underlined and italicized.
  • the amino acid sequence of the C-terminal truncated fragment d2 of the extracellular domain of Spike protein in the mutated SARS-CoV-2 Delta variant strain is shown in SEQ ID NO:12.
  • 70 amino acid residues were truncated at the C-terminus, and the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO: 5) was used to replace Marked in italics, the S1/S2 cleavage site 682 RRAR 685 mutation to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is also included, which is underlined and italicized.
  • the amino acid sequence of the C-terminal truncated fragment d3 of the extracellular domain of the Spike protein of the mutated SARS-CoV-2 Delta variant strain is shown in SEQ ID NO:81.
  • the C-terminus is truncated by 70 amino acid residues, does not contain a signal peptide, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which are underlined and bolded, and contain double mutations K986P/V987P , are underlined and italicized.
  • amino acid sequence of the S1 subunit of the Spike protein of the SARS-CoV-2 Delta mutant strain is shown in SEQ ID NO: 13, and the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2) is marked in italics.
  • ECD extracellular domain
  • SEQ ID NO:31 The full-length extracellular domain (ECD) of the Spike protein of SARS-CoV-2 Omicron mutant strain, its amino acid sequence is shown in SEQ ID NO:31, and the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO:2) is marked in italics Out, S1/S2 cleavage sites 682 RRAR 685 are underlined, bolded and italicized.
  • the full-length ectodomain f1 of the mutated SARS-CoV-2 Omicron variant Spike protein the amino acid sequence of which is shown in SEQ ID NO:32.
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO:2) is marked in italics
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , marked with underline and bold, and contains double Mutation K986P/V987P, underlined and italicized.
  • the full-length ectodomain f2 of the mutated SARS-CoV-2 Omicron variant Spike protein the amino acid sequence of which is shown in SEQ ID NO:33.
  • the original signal peptide: MFVFLVLLPLVSSQ shown in SEQ ID NO:2
  • signal peptide: MEFGLSLVFLVLILKGVQC shown in SEQ ID NO:5
  • the signal peptide is marked in italics
  • S1/S2 cleavage site The mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is also included, which is underlined and italicized.
  • the full-length ectodomain f3 of the mutated SARS-CoV-2 Omicron variant Spike protein the amino acid sequence of which is shown in SEQ ID NO:82.
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which are underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the amino acid sequence of the C-terminal truncated fragment g1 of the extracellular domain of the Spike protein of the mutated SARS-CoV-2 Omicron variant strain is shown in SEQ ID NO:34.
  • the C-terminus is truncated by 70 amino acid residues
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) is marked in italics
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , It is underlined and bolded, while the double mutation K986P/V987P is included, it is underlined and italicized.
  • the amino acid sequence of the C-terminal truncated fragment g2 of the extracellular domain of the Spike protein of the mutated SARS-CoV-2 Omicron variant strain is shown in SEQ ID NO:35.
  • 70 amino acid residues were truncated at the C-terminus, and the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2), signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO: 5) was used to replace Marked in italics, the S1/S2 cleavage site 682 RRAR 685 mutation to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is also included, which is underlined and italicized.
  • the amino acid sequence of the C-terminal truncated fragment g3 of the extracellular domain of the Spike protein of the mutated SARS-CoV-2 Omicron variant strain is shown in SEQ ID NO:83.
  • the C-terminus is truncated by 70 amino acid residues, does not contain a signal peptide, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which are underlined and bolded, and contain double mutations K986P/V987P , are underlined and italicized.
  • the amino acid sequence of the Spike protein S1 subunit of the SARS-CoV-2 Omicron mutant strain is shown in SEQ ID NO:36, and the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO:2) is marked in italics.
  • amino acid sequence of the conserved fragment O330 of the Spike protein of the SARS-CoV-2 Omicron variant is shown in SEQ ID NO:37.
  • the present invention provides a fusion protein comprising a heterologous scaffold exhibiting at least one antigenic polypeptide or trimeric protein derived from a coronavirus Spike protein.
  • the coronavirus antigen used is the extracellular domain of the coronavirus Spike protein or a truncated fragment thereof containing various stable mutations as described above.
  • the coronavirus antigen employed comprises or is derived from the RBD domain of the coronavirus Spike protein.
  • the coronavirus antigen employed comprises or is derived from the S1 subunit of the coronavirus Spike protein.
  • the coronavirus antigen employed comprises or is derived from a conserved fragment of the coronavirus Spike protein.
  • the Spike protein sequence employed comprises the sequence shown in any one of SEQ ID NO: 1, 3-4, 6-13, 31-37, 78-83, or is substantially identical or conserved therewith modified variant.
  • a nanoparticle vaccine displaying the antigen (e.g. Spike protein) on the surface will be produced due to the linkage of the antigen (e.g. Spike protein) to the self-assembling protein (e.g. monomeric ferritin subunit) .
  • Any heterologous scaffold can be used to present antigens in the construction of vaccines of the invention.
  • This includes virus-like particles (VLPs), such as nanoparticles.
  • VLPs virus-like particles
  • a variety of nanoparticles can be used to produce the vaccines of the invention.
  • nanoparticles useful in the present invention need to be formed from multiple copies of a single subunit. Nanoparticles are typically spherical, and/or have rotational symmetry (eg, with 3- and 5-fold axes), such as having an icosahedral structure as exemplified herein.
  • the amino-termini of the nanoparticle subunits must be exposed and in close proximity to the 3-fold axis, and the spacing of the three amino-termini must closely match the spacing of the carboxyl-termini of the trimer-stabilized Spike proteins shown.
  • the self-assembled nanoparticles employed have a diameter of about 25 nm or less (typically assembled from 12, 24, or 60 subunits) and have a 3-fold axis on the particle surface.
  • Such nanoparticles provide suitable particles for the production of multivalent vaccines.
  • coronavirus antigens may be presented on self-assembled nanoparticles, eg, self-assembled nanoparticles derived from ferritin (FR) as exemplified herein.
  • Ferritins are globular proteins found in animals, bacteria, and plants whose main role is to control multinucleation by transporting hydrated iron ions and protons to or from mineralized cores. Rate and location of Fe(III) 2O3 formation .
  • the globular form of ferritin is composed of monomeric subunit proteins (also called monomeric ferritin subunits), which are polypeptides with a molecular weight of approximately 17-20 kDa.
  • monomeric subunit proteins also called monomeric ferritin subunits
  • the sequences of the subunits of these proteins are known in the art.
  • the nanoparticle vaccines of the invention may employ any of these known nanoparticles, as well as their conservatively modified variants or be substantially identical (e.g., at least 90%, 95% or 99% identical) thereto variants of the sequence.
  • fusion proteins of the invention comprise an Fc fragment (eg, a human IgG Fc fragment).
  • Fc fragment eg, a human IgG Fc fragment.
  • the C-terminus of the conserved sequence of the coronavirus Spike protein or the S1 subunit of the coronavirus Spike protein or the ectodomain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof is fused to the N-terminus of the Fc fragment.
  • amino acid sequence of human IgG Fc is as follows:
  • the fusion protein of the present invention comprises a nanoparticle subunit sequence (such as Helicobacter pylori non-heme monomer ferritin subunit, its amino acid sequence is shown in SEQ ID NO: 14), or its conserved A modified variant or a sequence substantially identical thereto.
  • a nanoparticle subunit sequence such as Helicobacter pylori non-heme monomer ferritin subunit, its amino acid sequence is shown in SEQ ID NO: 14
  • the C-terminus of the coronavirus Spike protein conservative sequence or the coronavirus Spike protein S1 subunit or the mutated coronavirus Spike protein extracellular domain or its truncated fragment is fused to the N of the self-assembling nanoparticle (NP) subunit. end.
  • the C-terminus of the conserved sequence of the coronavirus Spike protein or the S1 subunit of the coronavirus Spike protein or the ectodomain of the coronavirus Spike protein containing a mutation or a truncated fragment thereof is connected to the nanoparticle subunit via a GS linker.
  • the linker is for example GGGGS or GGGGSGGGGS.
  • the amino acid sequence of the non-heme monomer ferritin subunit (Ferritin) of Helicobacter pylori is as follows:
  • linkers can be used to connect and maintain the overall activity of different functional proteins.
  • linkers comprise short peptide sequences, such as GS-rich peptides.
  • a linker or linker motif may be any flexible peptide that connects two protein domains or motifs without interfering with their function.
  • the linker employed may be a G4S linker or a ( G4S ) 2 linker as shown herein to connect the spike protein and nanoparticle scaffold sequence. Recombinant production of fusion proteins of the invention can be based on the protocols described herein and/or other methods already described in the art.
  • An exemplary fusion protein sequence is as follows:
  • Fusion protein A1 the C-terminus of the full-length ectodomain a1 (as shown in SEQ ID NO:3) of the mutant wild-type SARS-CoV-2 Spike protein is combined with the linker GGGGS (as shown in SEQ ID NO:15) N-terminal connection of Helicobacter pylori non-heme monomer ferritin subunits (as shown in SEQ ID NO:14) to obtain fusion protein A1, the amino acid sequence of which is shown in SEQ ID NO:16.
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO:2) is marked in italics, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , marked with underline and bold, and contains double
  • the mutation K986P/V987P is underlined and italicized, and the linker is italicized and bolded.
  • Fusion protein A2 the C-terminus of the full-length ectodomain a2 (as shown in SEQ ID NO: 4) of the mutant wild-type SARS-CoV-2 Spike protein is combined with the linker GGGGS (as shown in SEQ ID NO: 15) N-terminal connection of Helicobacter pylori non-heme monomeric ferritin subunits (as shown in SEQ ID NO:14) to obtain fusion protein A2, the amino acid sequence of which is shown in SEQ ID NO:17.
  • the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO:2) is replaced with signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5), the signal peptide is marked in italics, S1/S2 cleavage site
  • the mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is also underlined and italicized, and the linker is italicized and bolded.
  • Fusion protein B1 the C-terminus of the C-terminal truncated fragment b1 (as shown in SEQ ID NO: 6) of the mutant wild-type SARS-CoV-2 Spike protein extracellular domain is passed through the linker GGGGS (as shown in SEQ ID NO: 15) shown) and the N-terminus of Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO: 14) to obtain fusion protein B1, the amino acid sequence of which is shown in SEQ ID NO: 18.
  • the C-terminus is truncated by 70 amino acid residues
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) is marked in italics
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , It is underlined and bolded
  • the double mutation K986P/V987P is included, which is underlined and italicized, and the linker is italicized and bolded.
  • Fusion protein B2 The C-terminus of the C-terminal truncated fragment b2 (as shown in SEQ ID NO: 7) of the mutant wild-type SARS-CoV-2 Spike protein extracellular domain is passed through the linker GGGGS (as shown in SEQ ID NO: 15) shown) and the N-terminus of Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO: 14) to obtain fusion protein B2, the amino acid sequence of which is shown in SEQ ID NO: 19.
  • Fusion protein C1 the C-terminus of the full-length ectodomain c1 (as shown in SEQ ID NO: 9) of the mutated SARS-CoV-2 Delta variant Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO: 15) Ligated with the N-terminus of Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO:14) to obtain fusion protein C1, the amino acid sequence of which is shown in SEQ ID NO:20.
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO:2) is marked in italics, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , marked with underline and bold, and contains double
  • the mutation K986P/V987P is underlined and italicized, and the linker is italicized and bolded.
  • Fusion protein C2 the C-terminus of the full-length ectodomain c2 (as shown in SEQ ID NO: 10) of the mutated SARS-CoV-2 Delta variant Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO: 15) Ligated with the N-terminus of Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO: 14) to obtain fusion protein C2, the amino acid sequence of which is shown in SEQ ID NO: 21.
  • the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO:2) is replaced with signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5), the signal peptide is marked in italics, S1/S2 cleavage site
  • the mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is also underlined and italicized, and the linker is italicized and bolded.
  • Fusion protein D1 The C-terminus of the C-terminal truncated fragment d1 (as shown in SEQ ID NO: 11) of the extracellular domain of the mutated SARS-CoV-2 Delta variant Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO: 15 shown) and the N-terminus of Helicobacter pylori non-heme monomer ferritin subunit (shown in SEQ ID NO:14) to obtain fusion protein D1, the amino acid sequence of which is shown in SEQ ID NO:22.
  • the C-terminus is truncated by 70 amino acid residues
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) is marked in italics
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , It is underlined and bolded
  • the double mutation K986P/V987P is included, which is underlined and italicized, and the linker is italicized and bolded.
  • Fusion protein D2 The C-terminus of the C-terminal truncated fragment d2 (as shown in SEQ ID NO: 12) of the extracellular domain of the mutated SARS-CoV-2 Delta variant Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO: 15 shown) and the N-terminus of Helicobacter pylori non-heme monomer ferritin subunit (shown in SEQ ID NO:14) to obtain fusion protein D2, the amino acid sequence of which is shown in SEQ ID NO:23.
  • Fusion protein E1 The C-terminus of the Spike protein S1 subunit (as shown in SEQ ID NO: 13) of the SARS-CoV-2 Delta variant strain is combined with the non-hemoglobin Helicobacter pylori through the linker GGGGS (as shown in SEQ ID NO: 15) The N-terminus connection of the monomeric ferritin subunit (as shown in SEQ ID NO:14) obtains fusion protein E1, and its amino acid sequence is as shown in SEQ ID NO:24. In the sequence, the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) is marked in italics, and the linker is marked in italics and bold.
  • Fusion protein E2 The C-terminus of the Spike protein S1 subunit (as shown in SEQ ID NO: 13) of the SARS-CoV-2 Delta variant strain is combined with the non-hemoglobin Helicobacter pylori through the linker GGGGS (as shown in SEQ ID NO: 15)
  • the N-terminus connection of ferritin subunit (as shown in SEQ ID NO:14) of element monomer replaces original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) to obtain fusion protein E2, the amino acid sequence of which is shown in SEQ ID NO:25. In the sequence, the N-terminal signal peptide is italicized and the linker is italicized and bolded.
  • Fusion protein E3 The C-terminus of the Spike protein S1 subunit (as shown in SEQ ID NO:36) of the SARS-CoV-2 Omicron variant strain is combined with the non-hemoglobin Helicobacter pylori through the linker GGGGS (as shown in SEQ ID NO:15) The N-terminal connection of the monomeric ferritin subunit (as shown in SEQ ID NO:14) is connected to obtain fusion protein E3, and its amino acid sequence is as shown in SEQ ID NO:39. In the sequence, the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) is marked in italics, and the linker is marked in italics and bold.
  • Fusion protein E4 The C-terminus of the Spike protein S1 subunit (as shown in SEQ ID NO:36) of the SARS-CoV-2 Omicron variant strain is combined with the non-haemoglobin of Helicobacter pylori through the linker GGGGS (as shown in SEQ ID NO:15)
  • the N-terminal connection of ferritin subunit (as shown in SEQ ID NO:14) of element monomer replaces original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO:2) with signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO:5) Shown), obtain fusion protein E4, its amino acid sequence 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 (as shown in SEQ ID NO:32) of the mutated SARS-CoV-2 Omicron variant Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO:15) Ligated with the N-terminus of Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO:14) to obtain fusion protein F1, the amino acid sequence of which is shown in SEQ ID NO:41.
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO:2) is marked in italics, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , marked with underline and bold, and contains double
  • the mutation K986P/V987P is underlined and italicized, and the linker is italicized and bolded.
  • Fusion protein F2 the C-terminus of the full-length ectodomain f2 (as shown in SEQ ID NO:33) of the mutated SARS-CoV-2 Omicron variant Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO:15) Ligated with the N-terminus of Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 14) to obtain fusion protein F2, the amino acid sequence of which is shown in SEQ ID NO: 42.
  • Fusion protein G1 The C-terminus of the C-terminal truncated fragment g1 (as shown in SEQ ID NO: 34) of the extracellular domain of the mutated SARS-CoV-2 Omicron mutant Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO: 15 shown) and the N-terminus of Helicobacter pylori non-heme monomeric ferritin subunit (shown in SEQ ID NO:14) to obtain fusion protein G1, the amino acid sequence of which is shown in SEQ ID NO:43.
  • the C-terminus is truncated by 70 amino acid residues
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) is marked in italics
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , It is underlined and bolded
  • the double mutation K986P/V987P is included, which is underlined and italicized, and the linker is italicized and bolded.
  • Fusion protein G2 The C-terminus of the C-terminal truncated fragment g2 (as shown in SEQ ID NO: 35) of the extracellular domain of the mutated SARS-CoV-2 Omicron variant Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO: 15 shown) and the N-terminus of Helicobacter pylori non-heme monomer ferritin subunit (shown in SEQ ID NO:14) to obtain fusion protein G2, the amino acid sequence of which is shown in SEQ ID NO:44.
  • Fusion protein H1 Add the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO:2) to the N-terminus of the O330 fragment (shown in SEQ ID NO: 2), and then pass the C-terminus of the O330 fragment through the linker GGGGS (shown in SEQ ID NO:37) ID NO: 15) is connected to the N-terminus of Helicobacter pylori non-heme monomer ferritin subunit (as shown in SEQ ID NO: 14) to obtain fusion protein H1, and its amino acid sequence is shown in SEQ ID NO: 45 .
  • the original signal peptide is italicized and the linker is italicized and bolded.
  • Fusion protein H2 Add a signal peptide at the N-terminus of the O330 fragment (as shown in SEQ ID NO:37): MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO:5), and then pass the C-terminus of the O330 fragment through the linker GGGGS (as shown in SEQ ID Shown in NO:15) is connected with the N-terminus of Helicobacter pylori non-heme monomer ferritin subunit (shown in SEQ ID NO:14) to obtain fusion protein H2, and its amino acid sequence is shown in SEQ ID NO:46. Signal peptides are italicized and linkers are italicized and bolded.
  • Fusion protein A1-1 the C-terminus of the full-length ectodomain a1 (as shown in SEQ ID NO: 3) of the mutant wild-type SARS-CoV-2 Spike protein is combined with human IgG Fc (as shown in SEQ ID NO: 38) Shown) N-terminal connection to obtain fusion protein A1-1, its amino acid sequence is shown in SEQ ID NO:47.
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO:2) is marked in italics, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , marked with underline and bold, and contains double Mutation K986P/V987P, underlined and italicized.
  • Fusion protein A2-1 combine the C-terminus of the full-length ectodomain a2 (as shown in SEQ ID NO: 4) of the mutant wild-type SARS-CoV-2 Spike protein with human IgG Fc (as shown in SEQ ID NO: 38 Shown) N-terminal connection to obtain fusion protein A2-1, its amino acid sequence is shown in SEQ ID NO:48.
  • the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO:2) is replaced with signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5), the signal peptide is marked in italics, S1/S2 cleavage site
  • the mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is also included, which is underlined and italicized.
  • Fusion protein B1-1 combine the C-terminus of the C-terminal truncated fragment b1 (as shown in SEQ ID NO: 6) of the ectodomain of the mutant wild-type SARS-CoV-2 Spike protein with human IgG Fc (as shown in SEQ ID NO: 6) Shown in: 38) N-terminal connection obtains fusion protein B1-1, and its amino acid sequence is shown in SEQ ID NO: 49.
  • the C-terminus is truncated by 70 amino acid residues
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) is marked in italics
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , It is underlined and bolded, while the double mutation K986P/V987P is included, it is underlined and italicized.
  • Fusion protein B2-1 combine the C-terminus of the C-terminal truncated fragment b2 (as shown in SEQ ID NO: 7) of the ectodomain of the mutant wild-type SARS-CoV-2 Spike protein with human IgG Fc (as shown in SEQ ID NO: 7) : 38) N-terminal connection to obtain fusion protein B2-1, its amino acid sequence is shown in SEQ ID NO: 50.
  • Fusion protein C1-1 the C-terminus of the full-length extracellular domain c1 (as shown in SEQ ID NO: 9) of the mutated SARS-CoV-2 Delta variant Spike protein is combined with human IgG Fc (as shown in SEQ ID NO: 38 Shown) N-terminal connection to obtain fusion protein C1-1, its amino acid sequence is shown in SEQ ID NO:51.
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO:2) is marked in italics, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , marked with underline and bold, and contains double Mutation K986P/V987P, underlined and italicized.
  • Fusion protein C2-1 the C-terminus of the full-length ectodomain c2 (as shown in SEQ ID NO: 10) of the mutant SARS-CoV-2 Delta variant strain Spike protein is combined with human IgG Fc (as shown in SEQ ID NO: 38 shown) to obtain the fusion protein C2-1, the amino acid sequence of which is shown in SEQ ID NO:52.
  • the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO:2) is replaced with signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5), the signal peptide is marked in italics, S1/S2 cleavage site
  • the mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is also included, which is underlined and italicized.
  • Fusion protein D1-1 combine the C-terminus of the C-terminal truncated fragment d1 (as shown in SEQ ID NO: 11) of the extracellular domain of the mutant SARS-CoV-2 Delta strain Spike protein with human IgG Fc (as shown in SEQ ID shown in NO:38) to obtain the fusion protein D1-1, the amino acid sequence of which is shown in SEQ ID NO:53.
  • the C-terminus is truncated by 70 amino acid residues
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) is marked in italics
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , It is underlined and bolded, while the double mutation K986P/V987P is included, it is underlined and italicized.
  • Fusion protein D2-1 combine the C-terminus of the C-terminal truncated fragment d2 (as shown in SEQ ID NO: 12) of the extracellular domain of the mutated SARS-CoV-2 Delta variant Spike protein with human IgG Fc (as shown in SEQ ID NO: 12) shown in NO:38) to obtain the fusion protein D2-1, the amino acid sequence of which is shown in SEQ ID NO:54.
  • Fusion protein E1-1 the C-terminal of the Spike protein S1 subunit (as shown in SEQ ID NO: 13) of the SARS-CoV-2 Delta mutant strain and the N-terminal of human IgG Fc (as shown in SEQ ID NO: 38)
  • the connection obtains the fusion protein E1-1, the amino acid sequence of which is shown in SEQ ID NO:55.
  • the original signal peptide MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) is marked in italics.
  • Fusion protein E2-1 combine the C-terminus of the Spike protein S1 subunit (as shown in SEQ ID NO: 13) of the SARS-CoV-2 Delta mutant strain with the N-terminus of human IgG Fc (as shown in SEQ ID NO: 38) Connect, replace the original signal peptide with signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO:5): MFVFLVLLPLVSSQ (as shown in SEQ ID NO:2), obtain fusion protein E2-1, its amino acid sequence is as shown in SEQ ID NO:56 shown. In the sequence, the N-terminal signal peptide is italicized.
  • Fusion protein E3-1 the C-terminus of the Spike protein S1 subunit (as shown in SEQ ID NO:36) of the SARS-CoV-2 Omicron mutant strain and the N-terminus of human IgG Fc (as shown in SEQ ID NO:38) Connection obtains fusion protein E3-1, and its amino acid sequence is as shown in SEQ ID NO:57. In the sequence, the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) is marked in italics.
  • Fusion protein E4-1 the C-terminus of the Spike protein S1 subunit (as shown in SEQ ID NO:36) of the SARS-CoV-2 Omicron variant strain and the N-terminus of human IgG Fc (as shown in SEQ ID NO:38) Connect, replace the original signal peptide with signal peptide: MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO:5): MFVFLVLLPLVSSQ (as shown in SEQ ID NO:2), obtain fusion protein E4-1, its amino acid sequence is as shown in SEQ ID NO:58 shown. In the sequence, the N-terminal signal peptide is italicized.
  • Fusion protein F1-1 the C-terminus of the full-length ectodomain f1 (as shown in SEQ ID NO: 32) of the mutated SARS-CoV-2 Omicron variant strain Spike protein is combined with human IgG Fc (as shown in SEQ ID NO: 38 shown) to obtain fusion protein F1-1, the amino acid sequence of which is shown in SEQ ID NO:59.
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO:2) is marked in italics, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , marked with underline and bold, and contains double Mutation K986P/V987P, underlined and italicized.
  • Fusion protein F2-1 the C-terminus of the full-length ectodomain f2 (as shown in SEQ ID NO: 33) of the mutated SARS-CoV-2 Omicron variant strain Spike protein is combined with human IgG Fc (as shown in SEQ ID NO: 38 shown) to obtain the fusion protein F2-1, the amino acid sequence of which is shown in SEQ ID NO:60.
  • the original signal peptide: MFVFLVLLPLVSSQ (shown in SEQ ID NO:2) is replaced with signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5), the signal peptide is marked in italics, S1/S2 cleavage site
  • the mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is also included, which is underlined and italicized.
  • Fusion protein G1-1 the C-terminus of the C-terminal truncated fragment g1 (as shown in SEQ ID NO: 34) of the extracellular domain of the mutated SARS-CoV-2 Omicron mutant Spike protein and the human IgG Fc (as shown in SEQ ID NO: 34) shown in NO:38) to obtain the fusion protein G1-1, the amino acid sequence of which is shown in SEQ ID NO:61.
  • the C-terminus is truncated by 70 amino acid residues
  • the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO: 2) is marked in italics
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , It is underlined and bolded, while the double mutation K986P/V987P is included, it is underlined and italicized.
  • Fusion protein G2-1 the C-terminus of the C-terminal truncated fragment g2 (as shown in SEQ ID NO: 35) of the extracellular domain of the mutated SARS-CoV-2 Omicron variant Spike protein and the human IgG Fc (as shown in SEQ ID NO: 35) shown in NO:38) to obtain the fusion protein G2-1, the amino acid sequence of which is shown in SEQ ID NO:62.
  • Fusion protein H1-1 Add the original signal peptide: MFVFLVLLPLVSSQ (as shown in SEQ ID NO:2) to the N-terminus of the O330 fragment (as shown in SEQ ID NO: 37), and then combine the C-terminus of the O330 fragment with human IgG Fc (As shown in SEQ ID NO:38) the N-terminal connection obtains fusion protein H1-1, and its amino acid sequence is as shown in SEQ ID NO:63.
  • the original signal peptide is italicized.
  • Fusion protein H2-1 Add MEFGLSLVFLVLILKGVQC (as shown in SEQ ID NO:5) to the N-terminal of the O330 fragment (as shown in SEQ ID NO:37), and then combine the C-terminal of the O330 fragment with human IgG Fc (as shown in SEQ ID Shown in NO:38), the N-terminal connection obtains fusion protein H2-1, and its amino acid sequence is shown in SEQ ID NO:64. Signal peptides are italicized.
  • Mature fusion protein A Compared with fusion proteins A1 and A2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:26. In the sequence, the mutation of S1/S2 cleavage site 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and double mutation K986P/V987P is included, which is underlined and italicized, and the linker is italicized and bolded .
  • Mature fusion protein B Compared with fusion proteins B1 and B2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:27. In the sequence, the mutation of S1/S2 cleavage site 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and double mutation K986P/V987P is included, which is underlined and italicized, and the linker is italicized and bolded .
  • Mature fusion protein C Compared with fusion proteins C1 and C2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:28. In the sequence, the mutation of S1/S2 cleavage site 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and double mutation K986P/V987P is included, which is underlined and italicized, and the linker is italicized and bolded .
  • Mature fusion protein D Compared with fusion proteins D1 and D2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:29. In the sequence, the mutation of S1/S2 cleavage site 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and double mutation K986P/V987P is included, which is underlined and italicized, and the linker is italicized and bolded .
  • Mature fusion protein E-1 Compared with fusion proteins E1 and E2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:30. In sequences, linkers are italicized and bolded.
  • Mature fusion protein E-2 Compared with fusion proteins E3 and E4, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:65. In sequences, linkers are italicized and bolded.
  • Mature fusion protein F Compared with fusion proteins F1 and F2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:66. In the sequence, the mutation of S1/S2 cleavage site 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and double mutation K986P/V987P is included, which is underlined and italicized, and the linker is italicized and bolded .
  • Mature fusion protein G Compared with fusion proteins G1 and G2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:67. In the sequence, the mutation of S1/S2 cleavage site 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and double mutation K986P/V987P is included, which is underlined and italicized, and the linker is italicized and bolded .
  • Mature fusion protein H Compared with fusion proteins H1 and H2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:68. In sequences, linkers are italicized and bolded.
  • Mature fusion protein A-1 Compared with fusion proteins A1-1 and A2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:69. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which are underlined and bolded, and double mutations K986P/V987P are included, which are underlined and italicized.
  • Mature fusion protein B-1 Compared with fusion proteins B1-1 and B2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:70. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which are underlined and bolded, and double mutations K986P/V987P are included, which are underlined and italicized.
  • Mature fusion protein C-1 Compared with fusion proteins C1-1 and C2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:71. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which are underlined and bolded, and double mutations K986P/V987P are included, which are underlined and italicized.
  • Mature fusion protein D-1 Compared with fusion proteins D1-1 and D2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:72. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which are underlined and bolded, and double mutations K986P/V987P are included, which are underlined and italicized.
  • Mature fusion protein E-3 Compared with fusion proteins E1-1 and E2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:73.
  • Mature fusion protein E-4 Compared with fusion proteins E3-1 and E4-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:74.
  • Mature fusion protein F-1 Compared with fusion proteins F1-1 and F2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:75. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which are underlined and bolded, and double mutations K986P/V987P are included, which are underlined and italicized.
  • Mature fusion protein G-1 Compared with fusion proteins G1-1 and G2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:76. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which are underlined and bolded, and double mutations K986P/V987P are included, which are underlined and italicized.
  • Mature fusion protein H-1 Compared with fusion proteins H1-1 and H2-1, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO:77.
  • SEQ ID NO:26-30, 65-77 is the mature fusion protein sequence that removes the N-terminal signal peptide (SEQ ID NO:2 or 5).
  • the invention also encompasses nanoparticle vaccines comprising subunits of subunit sequences substantially identical to any of these exemplified nanoparticle vaccine sequences, or conservatively modified variants thereof sequence.
  • the mutated coronavirus Spike protein ectodomain or its truncated fragment, coronavirus Spike protein S1 subunit, coronavirus Spike protein conservative fragment, fusion protein or Spike protein nanoparticles of the present invention are usually produced by expression vectors, so Said expression vector comprises the coding sequence of the mutated coronavirus Spike protein ectodomain or its truncated fragment, coronavirus Spike protein S1 subunit, coronavirus Spike protein conservative fragment, fusion protein or Spike protein nanoparticle described herein .
  • the present invention provides an extracellular domain or a truncated fragment thereof, a coronavirus Spike protein S1 subunit, a conserved fragment of a coronavirus Spike protein, a fusion protein encoding a mutant coronavirus Spike protein described herein. or polynucleotides (DNA or RNA) of Spike protein nanoparticles.
  • Some polynucleotides of the present invention encode one of the mutated coronavirus Spike protein ectodomains or truncated fragments thereof described herein, for example, the SARS-CoV-2 Spike protein shown in SEQ ID NO: 12 Truncated fragment of the extracellular domain.
  • polynucleotides of the present invention encode a subunit sequence of one of the nanoparticle vaccines described herein, such as the fusion protein sequence shown in SEQ ID NO:23.
  • the fusion protein expressed in the present invention may not contain an N-terminal signal peptide, or some polynucleotide sequences additionally encode an N-terminal signal peptide.
  • a polynucleotide encoding a fusion protein e.g., SEQ ID NO: 26-30
  • the present invention also provides expression vectors with such polynucleotides and methods for producing mutated coronavirus Spike protein extracellular domains or truncated fragments thereof, coronavirus Spike protein S1 subunits, coronavirus Spike protein conservative fragments or Host cells for fusion proteins (eg, prokaryotic or eukaryotic cells, such as HEK293, CHO, ExpiCHO and CHO-S cell lines). Fusion proteins encoded by polynucleotides or expressed by vectors are also included in the present invention.
  • fusion proteins eg, prokaryotic or eukaryotic cells, such as HEK293, CHO, ExpiCHO and CHO-S cell lines.
  • the extracellular domain of the Spike protein or its truncated fragments, the S1 subunit of the Spike protein, or the conserved fragment of the Spike protein fused to nanoparticle subunits will self-assemble into a nanoparticle vaccine that displays on its surface Spike protein or its truncated fragment, Spike protein S1 subunit or Spike protein conserved fragment.
  • Polynucleotides and related vectors can be produced by standard molecular biology techniques or the protocols exemplified herein. For example, general protocols for cloning, transfection, transient gene expression, and obtaining 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., (3rd ed., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003). Mutations can also be introduced into polynucleotide sequences by known methods PCR.
  • vectors useful in the present invention are autonomously replicating, ie, the vector exists extrachromosomally and its replication is not necessarily directly linked to replication of the host cell genome.
  • replication of the vector can be linked to replication of the host chromosomal DNA, for example, integration of the vector into the chromosome of the host cell can be achieved by retroviral vectors and in stably transfected cell lines.
  • Non-viral vectors and systems include plasmids, episomal vectors (often with expression cassettes for expressing proteins or RNAs) and human artificial chromosomes.
  • Alternative viral vectors include lentivirus or other retrovirus-based vectors, adenovirus, adeno-associated virus, cytomegalovirus, herpesvirus, SV40-based vectors, papillomavirus, HBP Epstein Barr virus, vaccinia virus vector, and Semliki Forest virus (SFV).
  • a host cell can be any cell carrying a recombinant vector of a protein of the invention, wherein the vector is allowed to drive expression of the protein used in the invention. It can be prokaryotic, such as any of a number of bacterial strains, or it can be eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells, including, for example, rodent, simian, or human cells . Cells expressing proteins of the invention may be primary culture cells or may be established cell lines.
  • cell lines exemplified herein eg, HEK293 cells
  • 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.
  • a vector expressing a protein can be introduced into a host cell of choice by any of a number of suitable methods known to those skilled in the art.
  • the method used will depend on the form of the vector.
  • DNA encoding protein sequences can be introduced by any of a number of transfection methods including, for example, liposome-mediated transfection ("lipofection"), DEAE-dextran-mediated induced transfection, electroporation or calcium phosphate precipitation. These methods are described in detail, eg, in Brent et al., supra. Among them, lipofection is widely accepted because of its simple operation and no need for special equipment.
  • Lipofectamine Life Technologies
  • LipoTAXI LipoTAXI kits
  • Other companies offering liposome transfection 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.
  • protein coding sequences and optional markers controlled by appropriate expression control elements can be used Transform host cells.
  • the selectable marker in the recombinant vector confers resistance to selection and allows the cell to stably integrate the vector into its chromosome.
  • Commonly used selection markers include: neomycin (neo), which is resistant to aminoglycoside G-418, and hygromycin, which is resistant to hygromycin.
  • a recombinant expression vector includes at least one promoter element, a protein coding sequence, a transcription termination signal, and a polyA tail.
  • Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the inserted sequence.
  • High-efficiency transcription can be obtained through the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and early promoters of cytomegalovirus, and other cellular promoters such as muscle Kinetin promoter.
  • Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, Plncx, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), PSVL , PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc.
  • Commonly used mammalian cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells, etc.
  • the inserted gene fragment needs to contain selection markers, common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes, so as to facilitate transfection Screening of successful cell isolation.
  • selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes, so as to facilitate transfection Screening of successful cell isolation.
  • the constructed plasmid is transfected into host cells without the above-mentioned genes, and cultured in a selective medium, the successfully transfected cells grow in large numbers and produce the desired target protein.
  • mutations may be introduced in the nucleotide sequences encoding the present invention using standard techniques known to those skilled in the art, including but not limited to site-directed mutagenesis and PCR-mediated mutations resulting in amino acid substitutions.
  • Variants include derivatives
  • 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.
  • substitutions described herein are conservative amino acid substitutions.
  • compositions and methods of treatment are provided.
  • the invention also provides pharmaceutical compositions and related treatment methods.
  • the pharmaceutical composition comprises an effective dose of fusion protein or Spike protein nanoparticles and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to a substance approved by a governmental regulatory agency or listed in other recognized pharmacopoeias for use in animals, especially in humans.
  • pharmaceutically acceptable carrier generally refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary, etc.
  • carrier refers to a diluent, adjuvant, excipient or carrier with which the active ingredient can be administered to a patient.
  • Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable 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 can 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, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, Propylene, ethylene glycol, water, ethanol, etc.
  • the pharmaceutical composition if desired, can also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial 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 composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • compositions will contain a clinically effective dose of the fusion protein or Spike protein nanoparticles, preferably in a purified form, together with an appropriate amount of carrier to provide a dosage form suitable for the patient.
  • the formulation should be suitable for the mode of administration.
  • the preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • a pharmaceutical composition may comprise a fusion protein or Spike protein nanoparticle, and a polynucleotide or vector encoding a fusion protein described herein.
  • the virus such as SARS-CoV-2
  • the virus can be used to prevent and treat corresponding virus infection.
  • vaccines comprising nanoparticles described herein can be used to prevent or treat corresponding diseases, such as infections caused by various coronaviruses.
  • Some embodiments of the invention relate to the use of a SARS-CoV-2 antigen or vaccine for preventing or treating a SARS-CoV-2 infection in a human subject.
  • Some embodiments of the invention relate to the use of SARS-CoV antigens or vaccines in the prevention or treatment of SARS-CoV infection.
  • the corresponding Spike protein nanoparticles or fusion proteins, or the fusion proteins described herein are administered to subjects in need of prevention or treatment of diseases (such as SARS-CoV-2 infection).
  • diseases such as SARS-CoV-2 infection.
  • polynucleotide Typically, the Spike protein nanoparticles, fusion proteins or polynucleotides encoding fusion proteins disclosed herein are contained in pharmaceutical compositions.
  • a pharmaceutical composition may be a therapeutic formulation or a prophylactic formulation.
  • the pharmaceutical composition may additionally comprise one or more pharmaceutically acceptable carriers, and optionally other therapeutic ingredients (eg, antiviral drugs).
  • Various pharmaceutically acceptable additives can also be used in the pharmaceutical composition.
  • compositions of the invention are 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.
  • the vaccine compositions described herein eg, SARS-CoV-2 vaccines
  • Various pharmaceutical compositions can be prepared according to standard procedures well known in the art. See, eg, US Patents 4,652,441 and 4,917,893; US Patents 4,677,191 and 4,728,721; and US Patent 4,675,189.
  • the pharmaceutical composition 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 .
  • a nanoparticle vaccine can be administered to a subject to induce an immune response to SARS-CoV-2, e.g., to induce broadly neutralizing antibodies against the virus.
  • 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.
  • the pharmaceutical composition of the present invention can be administered to the subject by various administration methods known to those of ordinary skill in the art, for example, by intramuscular route, subcutaneous route, intravenous route, Intra-arterial route, joint route, intraperitoneal route and other parenteral routes.
  • the therapeutic methods of the present invention relate to methods of blocking entry of a coronavirus (such as SARS-CoV or SARS-CoV-2) into a host cell (such as a human host cell), preventing the binding of the coronavirus Spike protein to the host receptor A method, and a method for treating acute respiratory diseases associated with coronavirus infection.
  • a coronavirus such as SARS-CoV or SARS-CoV-2
  • a host cell such as a human host cell
  • the treatment methods and pharmaceutical compositions described herein may be used in combination with other known therapeutic agents and/or modalities for treating or preventing coronavirus infection.
  • known therapeutic agents and/or modalities include, for example, nuclease analogs or protease inhibitors (e.g., remdesivir), monoclonal antibodies against one or more coronaviruses, immunosuppressants or anti-inflammatory drugs (eg, sarilumab or tocilizumab), ACE inhibitors, vasodilators, or any combination thereof.
  • the pharmaceutical composition should contain a therapeutically effective amount of the fusion protein, Spike protein nanoparticles described herein.
  • the pharmaceutical composition should contain 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 condition to be treated or prevented, the severity, age of the subject, and other personal attributes of the particular subject (eg, the general state of the subject's health). Determination of effective dosages is also guided by studies in animal models, followed by human clinical trials, and by dosing regimens that significantly reduce the occurrence or severity of the target disease condition or symptom in a subject.
  • the pharmaceutical composition is provided prior to any symptoms, eg prior to infection.
  • Prophylactic administration of the pharmaceutical composition serves to prevent or ameliorate any subsequent infection.
  • the subject to be treated is a subject who has been infected (e.g., SARS-CoV-2 infection) or is in a state of Subjects at risk of infection (eg, SARS-CoV-2 infection).
  • the subject can be monitored for infection (eg, SARS-CoV-2 infection), symptoms associated with infection (eg, SARS-CoV-2 infection).
  • the pharmaceutical composition is provided at or after the onset of symptoms of a disease or infection, eg, after the onset of symptoms of an infection (eg, SARS-CoV-2 infection) or after diagnosis of an infection.
  • the pharmaceutical composition may be provided prior to anticipated exposure to the virus to attenuate the expected severity, duration or extent of the infection and/or associated disease condition following exposure or suspected exposure to the virus or following the initial stages of actual infection.
  • the pharmaceutical compositions of the present invention can be combined with other agents known in the art for the treatment or prevention of infection by related pathogens, such as SARS-CoV-2 infection.
  • a vaccine composition (such as a SARS-CoV-2 vaccine) or a pharmaceutical composition comprising the fusion protein of the present invention, Spike protein nanoparticles can be provided as a component of a kit.
  • 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 reagent.
  • fusion protein of the present invention Spike protein nanoparticles or derivatives or their encoding polynucleotides or expression vectors, for example encapsulated in liposomes, microparticles, microcapsules, capable of expressing the fusion protein or Spike protein nanoparticles recombinant cells, receptor-mediated endocytosis (see for example Wu and Wu, 1987, J.Biol.Chem.262:4429-4432), nucleic acid as part of retrovirus or other vectors build etc.
  • Embodiment 1 the preparation of fusion protein
  • the sequence of the fusion protein described herein can be prepared by the following method or other known methods.
  • the DNA sequence encoding the fusion protein (as shown in SEQ ID NO: 16-30, 39-77) was cloned into an expression vector, then electroporated into CHO-K1 cells, cultured and purified to obtain the fusion protein.
  • cryo-EM cryo-electron microscopy
  • Embodiment 2 fusion protein and hACE2 protein binding ability test
  • the method is briefly described as follows: Add 100 ⁇ L of 2 ⁇ g/mL antigen (WT-Spike-His, Delta-Spike-His, Omicron-Spike-His, fusion protein D or fusion protein G) solution, coated overnight at 4°C; washed twice with PBST (PBS buffer containing 0.05% Tween-20); adding blocking solution (PBST containing 3% BSA) to each reaction well Incubate in an incubator at 37°C for 2 hours; wash with PBST 3 times after blocking; add humanACE2-his-biotin (Shenzhou Yiqiao, product number: 10108-H27B-B) in serial dilution, the initial concentration is 2.5 ⁇ g/mL, 3-fold gradient Dilution, a total of 10 serial dilutions, 100 ⁇ L per well, incubated in a 37°C incubator for 1.5 h; washed 5 times with PBST; added streptavidin-labeled catalase to the reaction well
  • WT-Spike-His is to add 6 ⁇ His (HHHHHH) to the C-terminus of the C-terminal truncated fragment b1 (as shown in SEQ ID NO: 6) of the extracellular domain of the mutant wild-type SARS-CoV-2 Spike protein ) constructed from.
  • Delta-Spike-His is the addition of 6 ⁇ His (HHHHHH) to the C-terminus of the C-terminal truncated fragment d1 (as shown in SEQ ID NO: 11) of the extracellular domain of the mutated SARS-CoV-2 Delta variant Spike protein built.
  • Omicron-Spike-His is the addition of 6 ⁇ His (HHHHHH) to the C-terminus of the C-terminal truncated fragment g1 (as shown in SEQ ID NO: 34) of the extracellular domain of the mutant SARS-CoV-2 Omicron Omicron Spike protein built.
  • hACE2 has similar affinities to fusion protein D, Delta, and wild-type Spike protein, with EC 50 values of 9.2, 8.1, and 5.7 ng/mL, respectively ( Figure 1a); hACE2 binds to fusion protein G and Omicron Spike protein binding with similar affinity, EC50 values were 9.3, 8.2ng/mL ( Figure 1b).
  • Biomembrane interferometry was used to measure the affinity constants of fusion protein D and fusion protein G binding to hACE2, and the instrument was the Fortebio Octet RED&QK system of PALL Company.
  • Multi-channel parallel quantitative analysis of 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, the concentration gradient is set to: 50, 100, 200 and 400nM, hACE2-Biotin (Acro biosystems, product number AC2-H5257) coupled with SABiosensors sensor (Octet, product number 2107002811).
  • mice To investigate the immunogenicity of the monovalent vaccines (fusion protein D and fusion protein G), immunogenicity studies were performed in mice. Mix and emulsify fusion protein D and fusion protein G with immune adjuvant respectively, and immunize Balb/c mice (6-8 weeks old) by subcutaneous injection on day 0 and day 21 respectively, each mouse is adjuvanted with SEPIVAC SWE TM The volume of the dose (SEPPIC SA, product number 80748J, batch number 210721010001) was fixed at 50 ⁇ l, and the total volume of each administration was 100 ⁇ l/rat. See Table 2 for the grouping dosage regimen.
  • the blood was taken from the orbit, left to stand until the serum was precipitated, and the mouse serum was obtained by centrifugation, which was used to detect serum anti-[wild type, Delta and Omicron (BA.1)] IgG titer and SARS-CoV-2 by ELISA Spike pseudovirus neutralizing antibody experiment.
  • 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) antigens were diluted to 2 ⁇ g/mL respectively, Add 100 ⁇ L per well to a 96-well plate (Corning, 9018), and coat overnight at 4°C; wash the 96-well plate three times in washing buffer PBST (PBS buffer containing 0.05% Tween-20), and add blocking solution (washing buffer PBST containing 3% BSA), incubated at 37°C for 2h; the 96-well plate was washed 3 times with washing buffer PBST, and the mouse serum obtained in Step 1.1 of Example 3 of step dilution was added to each well (serum Dilute 1000 times as the initial concentration, then 3 times serial dilution) 100 ⁇ L, incubate at 37°C for 1.5h; wash the 96-well plate 5 times
  • the mouse serum obtained in step 1.1 of Example 3 was serially diluted with DMEM medium containing 10% FBS, and transferred to a 96-well plate at 50 ⁇ L per well for use; different SARS-CoV-2 Spike pseudoviruses were respectively Dilute with DMEM medium containing 10% FBS, transfer the diluted SARS-CoV-2 Spike pseudovirus at 25 ⁇ L per well to the above-mentioned 96-well plate containing mouse serum, mix well and incubate at 37°C for 1 hour ; ACE2-293 cells were digested with 0.25% Trypsin-EDTA (Gibco, 25200-072) and counted, the cell density was adjusted to 4 ⁇ 10 5 cells/mL, and the cells were added to the above-mentioned 96-well plate at 50 ⁇ L per well, Incubate in a 37°C, 5% CO 2 incubator for 48 hours; add 50 ⁇ L of Bio-Lite Luciferase Assay System (Novizan, DD1201-03)
  • Neutralization inhibition rate [1-(sample group-blank control group)/(negative control group-blank control group)] ⁇ 100%; Wherein, sample group adds SARS-CoV -2 Spike pseudovirus and mouse serum, negative control group added SARS-CoV-2 Spike pseudovirus without mouse serum, blank control group did not add SARS-CoV-2 Spike pseudovirus and did not add mouse serum.
  • ACE2-293 cells were cultured with DMEM complete medium containing 10% FBS, and lipofectamine 2000 transfection reagent (Thermo Fisher, 11668019) was used to transfect ACE2 expression plasmid (Shenzhou, HG10108-M).
  • the SARS-CoV-2 Spike pseudovirus is: SARS-CoV-2 Spike pseudovirus (Jimman Bio, GM-0220PV07); SARS-COV-2 Spike (B.1.617.2) pseudovirus (Jiman Bio, GM-0220PV45); SARS-COV-2 Spike (B.1.1.529) pseudovirus (Jiman Bio, GM-0220PV84).
  • the volume of the dose (SEPPIC SA, product number 80748J, batch number 210721010001) was fixed at 50 ⁇ L, and the total volume of each administration was 100 ⁇ L/rat.
  • Blood was collected on the 14th and 35th days after immunization, and used for ELISA detection of serum anti-[wild type, Delta and Omicron (BA.1)] IgG titers and SARS-CoV-2 Spike pseudovirus neutralizing antibody experiments.
  • mice serum obtained in step 1.1 of Example 4 was added in a serial dilution (100-fold dilution of the serum on the 14th day was used as the initial concentration, and on the 35th day Serum was diluted 1000 times as the initial concentration, then 3 times serially diluted (11 gradients), 100 ⁇ L per well, incubated at 37°C for 1.5 hours; washed 5 times with PBST; added 1:10000 diluted Peroxidase-AffiniPure Goat Anti-Mouse IgG (Jackson, catalog number: 115-035-003), 100 ⁇ L/well, incubate at 37°C for 1 hour; wash 8 times with PBST; add 100 ⁇ L/well TMB solution and incubate at 37°C for 15-25 minutes; terminate with 50 ⁇ L/well of 0.1M sulfuric acid Reaction; set the detection wavelength to 450nm for reading, and the obtained reading OD value is fitted with a nonlinear four-
  • mice receiving fusion protein G had lower antibody titers against wild-type and Delta anti-Spike proteins, but fusion protein G-induced antibodies against Omicron anti-Spike proteins The antibody titer was significantly higher than fusion protein D. Fusion protein D induced robust anti-Spike protein IgG antibody titers against wild-type and Delta, but significantly decreased antibody titers against Omicron.
  • the mouse serum collected on the 35th day of step 1.1 of Example 4 was serially diluted with DMEM medium containing 10% FBS, and 50 ⁇ L/well was added to a 96-well cell culture white plate; then the SARS-CoV-2 pseudovirus was mixed with 10 Dilute in DMEM medium with %FBS, add pseudovirus dilution to the 96-well plate with mouse serum dilution, 25 ⁇ L/well.
  • the 96-well plate was incubated in an incubator at 37°C for 1 h.
  • the 96-well plate after incubation for 1 h was taken out, ACE2-293 cell suspension (2 ⁇ 10 4 cells/well) was added at 50 ⁇ L/well, and the 96-well plate was placed in an incubator for culture.
  • the SARS-CoV-2 Spike pseudovirus is: SARS-CoV-2 Spike pseudovirus (Jimman Bio, GM-0220PV07); SARS-COV-2 Spike (B.1.617.2) pseudovirus (Jiman Bio, GM-0220PV45); SARS-COV-2 Spike (B.1.1.529) pseudovirus (Jiman Bio, GM-0220PV84).

Abstract

提供了用于预防或治疗冠状病毒感染的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段、融合蛋白、Spike蛋白纳米颗粒及其应用。

Description

用于预防或治疗冠状病毒感染的融合蛋白、Spike蛋白纳米颗粒及其应用 技术领域
本发明属于生物技术领域，尤其涉及用于预防或治疗冠状病毒感染的融合蛋白、Spike蛋白纳米颗粒及其应用。
背景技术
冠状病毒为不分节段的单股正链RNA病毒，根据血清型和基因组特点冠状病毒亚科被分为α、β、γ和δ四个属，由于病毒包膜上有向四周伸出的突起，形如花冠而得名。2019年发现的新型冠状病毒(SARS-CoV-2或2019-nCoV)属于β属的新型冠状病毒，有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60-140nm。目前研究显示，SARS-CoV-2与SARS-CoV具有高度同源性。
新型冠状病毒肺炎COVID-19主要通过呼吸道传染，其也可能通过接触传播。人群普遍易感，老年人及有基础疾病者感染后病情较重，儿童及婴幼儿也有发病。基于目前的流行病学调查，新型冠状病毒的潜伏期一般为1-14天，大多数在3-7天。感染者的主要临床症状是发热、乏力、干咳，而鼻塞、流涕等上呼吸道症状少见。在发病早期，患者的白细胞总数正常或降低，或淋巴细胞数目减少，部分患者出现肝酶、肌酶和肌红蛋白增高的现象。胸部影像显示患者早期呈现多发小斑片影及间质改变，以肺外带明显；进而发展为双肺多发磨玻璃影、浸润影，严重者可出现肺实变，并逐渐出现呼吸困难，严重者发生急性呼吸窘迫综合征(ARDS)、休克以及肺组织、心脏、肾脏多种组织损伤和功能障碍。多数轻度感染患者预后良好，重度患者病情常常危重，甚至死亡。
近期，有关COVID-19的基础、临床及流行病学研究不断发表或者公布，在本领域中迫切需要有效的针对冠状病毒的疫苗。
发明内容
本发明提供了包含可稳定蛋白结构的突变的冠状病毒Spike(刺突)蛋白胞外结构域或其截短片段，及包含含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的融合蛋白。本发明还提供了包含所述含突变的冠状病毒Spike蛋白胞外结构域或其截短片段与单体铁蛋白亚基融合并自组装形成的纳米颗粒的冠状病毒疫苗，能诱导对冠状病毒更强的中和抗体反应。
病毒颗粒首先通过其表面的Spike蛋白(S蛋白或棘突蛋白)的S1亚基中的受体结合域(RBD)与肺上皮细胞表面的一种称为血管紧张素转化酶2(ACE2)进行结合。当RBD与受体结合并被蛋白酶水解之后，位于S蛋白C端的S2亚基暴露，并嵌入浆膜或者内吞体膜中。S2亚基中的七肽重复序列1(HR1)与七肽重复序列2(HR2)彼此相互作用形成六螺旋束(6-HB)融合核心，导致病毒外壳与细胞膜融合，SARS-CoV或SARS-CoV-2进入细胞内，并利用细胞为其合成新的病毒颗粒；新的病毒颗粒释放到细胞外，再利用同样的方式侵染周围正常的细胞。本发明的融合蛋白、纳米颗粒及疫苗能诱导对冠状病毒更强的中和抗体反应。
在一些实施方案中提供了一种含突变的冠状病毒Spike蛋白胞外结构域或其截短片段，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和中央螺旋区(CH)之间的转向区域存在防止HR1和CH在融合过程中形成直螺旋的突变。
在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中，冠状病毒Spike蛋白的氨基酸编号是基于cryo-EM模型PDB ID 6VSB或GenBank登录号MN908947.3的氨基酸编号作为参考。
在一些实施方案中，所述含突变的冠状病毒Spike蛋白胞外结构域的截短片段，其与冠状病毒Spike蛋白全长胞外结构域相比，C端截短了5-80个氨基酸残基。在一些实施方案中，所述含突变的冠状病毒Spike蛋白胞外结构域的截短片段，其与冠状病毒Spike蛋白全长胞外结构域相比，C端截短了20-76个氨基酸残基。在一些实施方案中，所述含突变的冠状病毒Spike蛋白胞外结构域的截短片段，其与冠状病毒Spike蛋白全长胞外结构域相比，C端截短了70个氨基酸残基。
在一些实施方案中，所述冠状病毒为SARS-CoV-2、SARS-CoV或MERS-Cov。
在一些实施方案中，所述冠状病毒为野生型SARS-CoV-2或其变异株。
在一些实施方案中，所述冠状病毒为野生型SARS-CoV-2、SARS-CoV-2 Alpha变异株、SARS-CoV-2 Beta变异株、SARS-CoV-2 Gamma变异株、SARS-CoV-2 Delta变异株、SARS-CoV-2 Kappa变异株、SARS-CoV-2 Epsilon变异株、SARS-CoV-2 Lambda变异株或SARS-CoV-2 Omicron变异株。
在一些实施方案中，所述含突变的冠状病毒Spike蛋白胞外结构域或其截短片段包含如SEQ ID NO:3-4、6-7、9-12、32-35、78-83任一项所示的氨基酸序列，或与SEQ ID NO:3-4、6-7、9-12、32-35、78-83任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:3-4、6-7、9-12、32-35、78-83任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
一些实施方案中还提供了一种包含本文所述含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的融合蛋白。
一些实施方案提供了一种融合蛋白，所述融合蛋白包含通过接头连接的本文所述含突变的冠状病毒Spike蛋白胞外结构域或其截短片段和单体亚基蛋白。在一些实施方案中，所述单体亚基蛋白为自组装的单体亚基蛋白。在一些实施方案中，所述单体亚基蛋白为单体铁蛋白亚基。在一些实施方案中，所述融合蛋白是将含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的C端通过接头与单体亚基蛋白的N端连接。在一些实施方案中，所述融合蛋白是将含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的C端通过接头与单体铁蛋白亚基的N端连接。
在一些实施方案中，所述接头为GS接头。在一些实施方案中，所述接头选自GS，GGS，GGGS，GGGGS，SGGGS，GGGG，GGSS，(GGGGS) 2，(GGGGS) 3，或其任意组合。在一些实施方案中，所述接头为(G mS) n，其中每个m独立为1、2、3、4或5，n为1、2、3、4或5。在一些实施方案中，所述接头的序列为(GGGGS) n，所述n为1、2、3、4或5。在一些实施方案中，所述接头为GGGGS。在一些实施方案中，所述接头为(GGGGS) 2。在一些实施方案中，所述接头为(GGGGS) 3。在一些实施方案中，所述接头为(GGGGS) 4。在一些实施方案中，所述接头为(GGGGS) 5。
在一些实施方案中，所述融合蛋白还包含N端信号肽。在一些实施方案中，所述信号肽选自CSP，mschito，MF-α，pho1，HBM，t-pA，以及IL-3的信号肽。在一些实施方案中，所述N端信号肽包含如SEQ ID NO:2或5所示的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，单体铁蛋白亚基选自细菌铁蛋白、植物铁蛋白、藻铁蛋白、昆虫铁蛋白、真菌铁蛋白或哺乳动物铁蛋白。在一些实施方案中，所述单体铁蛋白亚基是幽门螺杆菌非血红素单体铁蛋白亚基。在一些实施方案中，幽门螺杆菌非血红素单体铁蛋白亚基氨基酸序列中存在N19Q突变。在一些实施方案中，所述单体铁蛋白亚基包含如SEQ ID NO:14所示的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的含突变的野生型SARS-CoV-2 Spike蛋白胞外结构域或其截短片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的含突变的野生型SARS-CoV-2 Spike蛋白胞外结构域或其截短片段和单体铁蛋白亚基。在一些实施方案中，所述突变包含：1)将RRAR 突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的含突变的SARS-CoV-2 Alpha变异株Spike蛋白胞外结构域或其截短片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的含突变的SARS-CoV-2 Alpha变异株Spike蛋白胞外结构域或其截短片段和单体铁蛋白亚基。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的含突变的SARS-CoV-2 Beta变异株Spike蛋白胞外结构域或其截短片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的含突变的SARS-CoV-2 Beta变异株Spike蛋白胞外结构域或其截短片段和单体铁蛋白亚基。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的含突变的SARS-CoV-2 Gamma变异株Spike蛋白胞外结构域或其截短片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的含突变的SARS-CoV-2 Gamma变异株Spike蛋白胞外结构域或其截短片段和单体铁蛋白亚基。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的含突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域或其截短片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的含突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域或其截短片段和单体铁蛋白亚基。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的含突变的SARS-CoV-2 Kappa变异株Spike蛋白胞外结构域或其截短片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的含突变的SARS-CoV-2 Kappa变异株Spike蛋白胞外结构域或其截短片段和单体铁蛋白亚基。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的含突变的 SARS-CoV-2 Epsilon变异株Spike蛋白胞外结构域或其截短片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的含突变的SARS-CoV-2 Epsilon变异株Spike蛋白胞外结构域或其截短片段和单体铁蛋白亚基。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的含突变的SARS-CoV-2 Lambda变异株Spike蛋白胞外结构域或其截短片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的含突变的SARS-CoV-2 Lambda变异株Spike蛋白胞外结构域或其截短片段和单体铁蛋白亚基。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的含突变的SARS-CoV-2 Omicron变异株Spike蛋白胞外结构域或其截短片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的含突变的SARS-CoV-2 Omicron变异株Spike蛋白胞外结构域或其截短片段和单体铁蛋白亚基。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中，所述融合蛋白包括通过接头连接的含突变的冠状病毒Spike蛋白胞外结构域和单体铁蛋白亚基，所述含突变的冠状病毒Spike蛋白胞外结构域包含如SEQ ID NO:3-4、9-10、32-33、78、80、82任一项所示的氨基酸序列，所述单体铁蛋白亚基包含如SEQ ID NO:14所示的氨基酸序列；含突变的冠状病毒Spike蛋白胞外结构域通过如SEQ ID NO:15所示的接头与单体铁蛋白亚基连接。
在一些实施方案中，所述融合蛋白包括通过接头连接的含突变的冠状病毒Spike蛋白胞外结构域的截短片段和单体铁蛋白亚基，所述含突变的冠状病毒Spike蛋白胞外结构域的截短片段包含如SEQ ID NO:6-7、11-12、34-35、79、81、83任一项所示的氨基酸序列，所述单体铁蛋白亚基包含如SEQ ID NO:14所示的氨基酸序列；含突变的冠状病毒Spike蛋白胞外结构域的截短片段通过如SEQ ID NO:15所示的接头与单体铁蛋白亚基连接。
在一些实施方案中，所述融合蛋白包含如SEQ ID NO:16-23、26-29、41-44、66-67任一项所示的氨基酸序列，或与SEQ ID NO:16-23、26-29、41-44、66-67任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:16-23、26-29、41-44、66-67任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案提供一种融合蛋白，包含通过接头连接的冠状病毒Spike蛋白S1亚基和单体亚基蛋白。在一些实施方案中，所述单体亚基蛋白为自组装的单体亚基蛋白。在一些实施方案中，所述单体亚基蛋白为单体铁蛋白亚基。在一些实施方案中，所述融合蛋白是将冠状病毒Spike蛋白S1亚基的C端通过接头与单体亚基蛋白的N端连接。
在一些实施方案中，所述冠状病毒为SARS-CoV-2、SARS-CoV或MERS-Cov。在一些实施方案中，所述冠状病毒为野生型SARS-CoV-2或其变异株。在一些实施方案中，所述冠状病毒为野生型SARS-CoV-2、SARS-CoV-2 Alpha变异株、SARS-CoV-2 Beta变异株、SARS-CoV-2 Gamma变异株、SARS-CoV-2 Delta变异株、SARS-CoV-2 Kappa变异株、SARS-CoV-2 Epsilon变异株、SARS-CoV-2 Lambda变异株或SARS-CoV-2 Omicron变异株。
在一些实施方案中，所述接头为GS接头。在一些实施方案中，所述接头选自GS，GGS，GGGS，GGGGS，SGGGS，GGGG，GGSS，(GGGGS) 2，(GGGGS) 3，或其任意组合。在一些实施方案中，所述接头为(G mS) n，其中每个m独立为1、2、3、4或5，n为1、2、3、4或5。在一些实施方案中，所述接头的序列为(GGGGS) n，所述n为1、2、3、4或5。在一些实施方案中，所述接头为GGGGS。在一些实施方案中，所述接头为(GGGGS) 2。在一些实施方案中，所述接头为(GGGGS) 3。在一些实施方案中，所述接头为(GGGGS) 4。在一些实施方案中，所述接头为(GGGGS) 5。
在一些实施方案中，所述融合蛋白还包含N端信号肽。在一些实施方案中，所述信号肽选自CSP，mschito，MF-α，pho1，HBM，t-pA，以及IL-3的信号肽。在一些实施方案中，所述N端信号肽包含如SEQ ID NO:2或5所示的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，单体铁蛋白亚基选自细菌铁蛋白、植物铁蛋白、藻铁蛋白、昆虫铁蛋白、真菌铁蛋白或哺乳动物铁蛋白。在一些实施方案中，所述单体铁蛋白亚基是幽门螺杆菌非血红素单体铁蛋白亚基。在一些实施方案中，幽门螺杆菌非血红素单体铁蛋白亚基氨基酸序列中存在N19Q突变。在一些实施方案中，所述单体铁蛋白亚基包含如SEQ ID NO:14所示的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的野生型SARS-CoV-2 Spike蛋白S1亚基和单体铁蛋白亚基。在一些实施方案中，所述融合蛋白是将野生型 SARS-CoV-2 Spike蛋白S1亚基的C端通过接头与单体铁蛋白亚基的N端连接。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Alpha变异株Spike蛋白S1亚基和单体铁蛋白亚基。在一些实施方案中，所述融合蛋白是将SARS-CoV-2 Alpha变异株Spike蛋白S1亚基的C端通过接头与单体铁蛋白亚基的N端连接。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Beta变异株Spike蛋白S1亚基和单体铁蛋白亚基。在一些实施方案中，所述融合蛋白是将SARS-CoV-2 Beta变异株Spike蛋白S1亚基的C端通过接头与单体铁蛋白亚基的N端连接。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Gamma变异株Spike蛋白S1亚基和单体铁蛋白亚基。在一些实施方案中，所述融合蛋白是将SARS-CoV-2 Gamma变异株Spike蛋白S1亚基的C端通过接头与单体铁蛋白亚基的N端连接。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Delta变异株Spike蛋白S1亚基和单体铁蛋白亚基。在一些实施方案中，所述融合蛋白是将SARS-CoV-2 Delta变异株Spike蛋白S1亚基的C端通过接头与单体铁蛋白亚基的N端连接。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Kappa变异株Spike蛋白S1亚基和单体铁蛋白亚基。在一些实施方案中，所述融合蛋白是将SARS-CoV-2 Kappa变异株Spike蛋白S1亚基的C端通过接头与单体铁蛋白亚基的N端连接。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Epsilon变异株Spike蛋白S1亚基和单体铁蛋白亚基。在一些实施方案中，所述融合蛋白是将SARS-CoV-2 Epsilon变异株Spike蛋白S1亚基的C端通过接头与单体铁蛋白亚基的N端连接。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Lambda变异株Spike蛋白S1亚基和单体铁蛋白亚基。在一些实施方案中，所述融合蛋白是将SARS-CoV-2 Lambda变异株Spike蛋白S1亚基的C端通过接头与单体铁蛋白亚基的N端连接。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Omicron变异株Spike蛋白S1亚基和单体铁蛋白亚基。在一些实施方案中，所述融合蛋白是将SARS-CoV-2 Omicron变异株Spike蛋白S1亚基的C端通过接头与单体铁蛋 白亚基的N端连接。
在一些实施方案中，所述融合蛋白包含通过接头连接的冠状病毒Spike蛋白S1亚基和单体铁蛋白亚基，所述冠状病毒Spike蛋白S1亚基包含如SEQ ID NO:13或36所示的氨基酸序列，所述单体铁蛋白亚基包含如SEQ ID NO:14所示的氨基酸序列；冠状病毒Spike蛋白S1亚基通过如SEQ ID NO:15所示的接头与单体铁蛋白亚基连接。
在一些实施方案中，所述融合蛋白是将如SEQ ID NO:13或36所示的冠状病毒Spike蛋白S1亚基的C端通过接头GGGGS与如SEQ ID NO:14所示的幽门螺旋杆菌非血红素单体铁蛋白亚基的N端连接，同时用信号肽：MEFGLSLVFLVLILKGVQC替换原始信号肽：MFVFLVLLPLVSSQ获得。
在一些实施方案中，所述融合蛋白包含如SEQ ID NO:24-25、30、39-40、65任一项所示的氨基酸序列，或与SEQ ID NO:24-25、30、39-40、65任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:24-25、30、39-40、65任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案提供一种融合蛋白，包含通过接头连接的冠状病毒Spike蛋白保守片段和单体亚基蛋白。在一些实施方案中，所述单体亚基蛋白为自组装的单体亚基蛋白。在一些实施方案中，所述单体亚基蛋白为单体铁蛋白亚基。在一些实施方案中，所述融合蛋白是将冠状病毒Spike蛋白保守片段的C端通过接头与单体亚基蛋白的N端连接。
在一些实施方案中，所述冠状病毒为SARS-CoV-2、SARS-CoV或MERS-Cov。在一些实施方案中，所述冠状病毒为野生型SARS-CoV-2或其变异株。在一些实施方案中，所述冠状病毒为野生型SARS-CoV-2、SARS-CoV-2 Alpha变异株、SARS-CoV-2 Beta变异株、SARS-CoV-2 Gamma变异株、SARS-CoV-2 Delta变异株、SARS-CoV-2 Kappa变异株、SARS-CoV-2 Epsilon变异株、SARS-CoV-2 Lambda变异株或SARS-CoV-2 Omicron变异株。
在一些实施方案中，所述接头为GS接头。在一些实施方案中，所述接头选自GS，GGS，GGGS，GGGGS，SGGGS，GGGG，GGSS，(GGGGS) 2，(GGGGS) 3，或其任意组合。在一些实施方案中，所述接头为(G mS) n，其中每个m独立为1、2、3、4或5，n为1、2、3、4或5。在一些实施方案中，所述接头的序列为(GGGGS) n，所述n为1、2、3、4或5。在一些实施方案中，所述接头为GGGGS。在一些实施方案中，所述接头为(GGGGS) 2。在一些实施方案中，所述接头为(GGGGS) 3。在一些实施方案中，所述接头为(GGGGS) 4。在一些实施方案中，所述接头为(GGGGS) 5。
在一些实施方案中，所述融合蛋白还包含N端信号肽。在一些实施方案中，所述信号肽选自CSP，mschito，MF-α，pho1，HBM，t-pA，以及IL-3的信号肽。在一些实施方案中，所述N端信号肽包含如SEQ ID NO:2或5所示的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，单体铁蛋白亚基选自细菌铁蛋白、植物铁蛋白、藻铁蛋白、昆虫铁蛋白、真菌铁蛋白或哺乳动物铁蛋白。在一些实施方案中，所述单体铁蛋白亚基是幽门螺杆菌非血红素单体铁蛋白亚基。在一些实施方案中，幽门螺杆菌非血红素单体铁蛋白亚基氨基酸序列中存在N19Q突变。在一些实施方案中，所述单体铁蛋白亚基包含如SEQ ID NO:14所示的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的野生型SARS-CoV-2 Spike蛋白保守片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的野生型SARS-CoV-2 Spike蛋白保守片段和单体铁蛋白亚基。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Alpha变异株Spike蛋白保守片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的SARS-CoV-2 Alpha变异株Spike蛋白保守片段和单体铁蛋白亚基。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Beta变异株Spike蛋白保守片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的SARS-CoV-2 Beta变异株Spike蛋白保守片段和单体铁蛋白亚基。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Gamma变异株Spike蛋白保守片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的SARS-CoV-2 Gamma变异株Spike蛋白保守片段和单体铁蛋白亚基。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Delta变异株Spike蛋白保守片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的SARS-CoV-2 Delta变异株Spike蛋白保守片段和单体铁蛋白亚基。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Kappa变异株Spike蛋白保守片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的SARS-CoV-2 Kappa变异株Spike蛋白保守片段和单体铁蛋白亚基。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Epsilon变异株Spike蛋白保守片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的SARS-CoV-2 Epsilon变异株Spike蛋白保守片段和单体铁蛋白亚基。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Lambda变异株Spike蛋白保守片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的SARS-CoV-2 Lambda变异株Spike蛋白保守片段和单体铁蛋白亚基。
在一些实施方案中提供了一种融合蛋白，包含通过接头连接的SARS-CoV-2 Omicron变异株Spike蛋白保守片段和单体亚基蛋白。在一些实施方案中，所述融合蛋白包含通过接头连接的SARS-CoV-2 Omicron变异株Spike蛋白保守片段和单体铁蛋白亚基。
在一些实施方案中，所述融合蛋白包含通过接头连接的冠状病毒Spike蛋白保守片段和单体铁蛋白亚基，所述冠状病毒Spike蛋白保守片段包含如SEQ ID NO:37所示的氨基酸序列，所述单体铁蛋白亚基包含如SEQ ID NO:14所示的氨基酸序列；冠状病毒Spike蛋白保守片段通过如SEQ ID NO:15所示的接头与单体铁蛋白亚基连接。
在一些实施方案中，所述融合蛋白包含如SEQ ID NO:45-46、68任一项所示的氨基酸序列，或与SEQ ID NO:45-46、68任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:45-46、68任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案提供一种融合蛋白，包含本文所述含突变的冠状病毒Spike蛋白胞外结构域或其截短片段和与其连接的免疫球蛋白的Fc片段。在一些实施方案中，所述融合蛋白是将本文所述含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的C端与免疫球蛋白的Fc片段的N端连接。
在一些实施方案中，所述融合蛋白还包含N端信号肽。在一些实施方案中，所述信号肽选自CSP，mschito，MF-α，pho1，HBM，t-pA，以及IL-3的信号肽。在一些实施方案中，所述N端信号肽包含如SEQ ID NO:2或5所示的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述免疫球蛋白的Fc片段来自IgG、IgM、IgA、IgE或IgD。在一些实施方案中，所述免疫球蛋白的Fc片段来自IgG1、IgG2、IgG3或IgG4。在一 些实施方案中，所述免疫球蛋白的Fc片段为IgG1的Fc片段。在一些实施方案中，所述免疫球蛋白的Fc片段为人IgG1的Fc片段。在一些实施方案中，所述免疫球蛋白的Fc片段包含如SEQ ID NO:38所示的氨基酸序列，或与SEQ ID NO:38所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:38所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中提供了一种融合蛋白，包含含突变的野生型SARS-CoV-2 Spike蛋白胞外结构域或其截短片段和与其连接的免疫球蛋白的Fc片段。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含含突变的SARS-CoV-2 Alpha变异株Spike蛋白胞外结构域或其截短片段和与其连接的免疫球蛋白的Fc片段。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含含突变的SARS-CoV-2 Beta变异株Spike蛋白胞外结构域或其截短片段和与其连接的免疫球蛋白的Fc片段。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含含突变的SARS-CoV-2 Gamma变异株Spike蛋白胞外结构域或其截短片段和与其连接的免疫球蛋白的Fc片段。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含含突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域或其截短片段和与其连接的免疫球蛋白的Fc片段。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含含突变的SARS-CoV-2 Kappa变异株Spike蛋白胞外结构域或其截短片段和与其连接的免疫球蛋白的Fc片段。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含含突变的SARS-CoV-2 Epsilon变异株Spike蛋白胞外结构域或其截短片段和与其连接的免疫球蛋白的Fc片段。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向 区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含含突变的SARS-CoV-2 Lambda变异株Spike蛋白胞外结构域或其截短片段和与其连接的免疫球蛋白的Fc片段。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中提供了一种融合蛋白，包含含突变的SARS-CoV-2 Omicron变异株Spike蛋白胞外结构域或其截短片段和与其连接的免疫球蛋白的Fc片段。在一些实施方案中，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
在一些实施方案中，所述融合蛋白包含如SEQ ID NO:47-54、59-62、69-72、75-76任一项所示的氨基酸序列，或与SEQ ID NO:47-54、59-62、69-72、75-76任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:47-54、59-62、69-72、75-76任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案提供一种融合蛋白，包含冠状病毒Spike蛋白S1亚基和与其连接的免疫球蛋白的Fc片段。在一些实施方案中，所述融合蛋白是将冠状病毒Spike蛋白S1亚基的C端与免疫球蛋白的Fc片段的N端连接。
在一些实施方案中，所述冠状病毒为SARS-CoV-2、SARS-CoV或MERS-Cov。在一些实施方案中，所述冠状病毒为野生型SARS-CoV-2或其变异株。在一些实施方案中，所述冠状病毒为野生型SARS-CoV-2、SARS-CoV-2 Alpha变异株、SARS-CoV-2 Beta变异株、SARS-CoV-2 Gamma变异株、SARS-CoV-2 Delta变异株、SARS-CoV-2 Kappa变异株、SARS-CoV-2 Epsilon变异株、SARS-CoV-2 Lambda变异株或SARS-CoV-2 Omicron变异株。
在一些实施方案中，所述融合蛋白还包含N端信号肽。在一些实施方案中，所述信号肽选自CSP，mschito，MF-α，pho1，HBM，t-pA，以及IL-3的信号肽。在一些实施方案中，所述N端信号肽包含如SEQ ID NO:2或5所示的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述免疫球蛋白的Fc片段来自IgG、IgM、IgA、IgE或IgD。在一些实施方案中，所述免疫球蛋白的Fc片段来自IgG1、IgG2、IgG3或IgG4。在一些实施方案中，所述免疫球蛋白的Fc片段为IgG1的Fc片段。在一些实施方案中，所 述免疫球蛋白的Fc片段为人IgG1的Fc片段。在一些实施方案中，所述免疫球蛋白的Fc片段包含如SEQ ID NO:38所示的氨基酸序列，或与SEQ ID NO:38所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:38所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中提供了一种融合蛋白，包含野生型SARS-CoV-2 Spike蛋白S1亚基和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Alpha变异株Spike蛋白S1亚基和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Beta变异株Spike蛋白S1亚基和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Gamma变异株Spike蛋白S1亚基和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Delta变异株Spike蛋白S1亚基和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Kappa变异株Spike蛋白S1亚基和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Epsilon变异株Spike蛋白S1亚基和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Lambda变异株Spike蛋白S1亚基和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Omicron变异株Spike蛋白S1亚基和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中，所述融合蛋白包含如SEQ ID NO:55-58、73-74任一项所示的氨基酸序列，或与SEQ ID NO:55-58、73-74任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:55-58、73-74任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案提供一种融合蛋白，包含冠状病毒Spike蛋白保守片段和与其连接的免疫球蛋白的Fc片段。在一些实施方案中，所述融合蛋白是将冠状病毒Spike蛋白保守片段的C端与免疫球蛋白的Fc片段的N端连接。
在一些实施方案中，所述冠状病毒为SARS-CoV-2、SARS-CoV或MERS-Cov。 在一些实施方案中，所述冠状病毒为野生型SARS-CoV-2或其变异株。在一些实施方案中，所述冠状病毒为野生型SARS-CoV-2、SARS-CoV-2 Alpha变异株、SARS-CoV-2 Beta变异株、SARS-CoV-2 Gamma变异株、SARS-CoV-2 Delta变异株、SARS-CoV-2 Kappa变异株、SARS-CoV-2 Epsilon变异株、SARS-CoV-2 Lambda变异株或SARS-CoV-2 Omicron变异株。
在一些实施方案中，所述融合蛋白还包含N端信号肽。在一些实施方案中，所述信号肽选自CSP，mschito，MF-α，pho1，HBM，t-pA，以及IL-3的信号肽。在一些实施方案中，所述N端信号肽包含如SEQ ID NO:2或5所示的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述免疫球蛋白的Fc片段来自IgG、IgM、IgA、IgE或IgD。在一些实施方案中，所述免疫球蛋白的Fc片段来自IgG1、IgG2、IgG3或IgG4。在一些实施方案中，所述免疫球蛋白的Fc片段为IgG1的Fc片段。在一些实施方案中，所述免疫球蛋白的Fc片段为人IgG1的Fc片段。在一些实施方案中，所述免疫球蛋白的Fc片段包含如SEQ ID NO:38所示的氨基酸序列，或与SEQ ID NO:38所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:38所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中提供了一种融合蛋白，包含野生型SARS-CoV-2 Spike蛋白保守片段和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Alpha变异株Spike蛋白保守片段和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Beta变异株Spike蛋白保守片段和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Gamma变异株Spike蛋白保守片段和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Delta变异株Spike蛋白保守片段和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Kappa变异株Spike蛋白保守片段和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Epsilon变异株Spike 蛋白保守片段和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Lambda变异株Spike蛋白保守片段和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中提供了一种融合蛋白，包含SARS-CoV-2 Omicron变异株Spike蛋白保守片段和与其连接的免疫球蛋白的Fc片段。
在一些实施方案中，所述融合蛋白包含如SEQ ID NO:63-64、77任一项所示的氨基酸序列，或与SEQ ID NO:63-64、77任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:63-64、77任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，至少80％同一性为至少约80％同一性、至少约81％同一性、至少约83％同一性、至少约84％同一性、至少约85％同一性、至少约86％同一性、至少约87％同一性、至少约88％同一性、至少约89％同一性、至少约90％同一性、至少约91％同一性、至少约93％同一性、至少约94％同一性、至少约95％同一性、至少约97％同一性、至少约98％同一性、至少约99％同一性，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
在一些实施方案中，至少90％同一性为至少约90％同一性、至少约91％同一性、至少约92％同一性、至少约93％同一性、至少约94％同一性、至少约95％同一性、至少约96％同一性、至少约97％同一性、至少约98％同一性、至少约99％同一性，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
在一些实施方案中，一个或多个保守氨基酸取代为约1个、约2个、约3个、约5个、约6个、约7个、约8个、约9个、约10个、约12个、约13个、约14个、约15个、约17个、约18个、约19个、约20个、约22个、约24个、约25个、约27个、约30个、约32个、约33个、约36个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
一些实施方案中提供了一种编码本文所述含突变的冠状病毒Spike蛋白胞外结构域或其截短片段，或融合蛋白的多聚核苷酸。
在一些实施方案中提供了一种包含编码本文所述含突变的冠状病毒Spike蛋白胞外结构域或其截短片段或融合蛋白的多聚核苷酸的表达载体。
在一些实施方案中提供了一种可以表达本文所述含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的细胞。在一些实施方案中，所述细胞包含编码本文所述融合蛋白的一种或多种多聚核苷酸或包含编码本文所述融合蛋白的多聚核苷酸的表达载 体。在一些实施方案中，所述细胞为分离的细胞。在一些实施方案中，所述细胞为CHO细胞、HEK293细胞、Cos1细胞、Cos7细胞、CV1细胞或鼠L细胞。
在一些实施方案中，所述融合蛋白包含通过接头连接的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段和单体铁蛋白亚基，所述融合蛋白包括以下特征：
所述突变包含：1)使S1/S2切割位点失活的突变；2)在HR1和CH之间的转向区域存在防止HR1和CH在融合过程中形成直螺旋的突变；和/或
所述含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的C-末端通过接头与单体铁蛋白亚基进行连接；和/或
所述接头为(G mS) n，其中每个m独立为1、2、3、4或5，n为1、2、3、4或5；和/或
所述单体铁蛋白亚基为幽门螺杆菌单体铁蛋白亚基，包含如SEQ ID NO:14所示的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中提供了包含本文所述融合蛋白的Spike蛋白纳米颗粒。
在一些实施方案中提供了一种冠状病毒疫苗，所述冠状病毒疫苗包含本文所述融合蛋白和/或包含融合蛋白的Spike蛋白纳米颗粒。在一些实施方案中，所述冠状病毒疫苗还包括药学上可接受的载体和/或佐剂。本发明还提供了一种冠状病毒疫苗。在一些实施方案中，所述冠状病毒疫苗包含本文所述融合蛋白以及药学上可接受的载体和/或佐剂。在一些实施方案中，所述冠状病毒疫苗包含本文所述Spike蛋白纳米颗粒以及药学上可接受的载体和/或佐剂。
本发明还提供了预防或治疗方法和用途。在一些实施方案中，本发明提供了用于预防或治疗冠状病毒感染的方法，这些方法包括向有需要的患者施用有效量的本文所述融合蛋白、Spike蛋白纳米颗粒或冠状病毒疫苗。在一些实施方案中，提供了本文所述融合蛋白、Spike蛋白纳米颗粒或冠状病毒疫苗在预防或治疗SARS或COVID-19中的应用。在一些实施方案中，提供了本文所述融合蛋白或Spike蛋白纳米颗粒在制备预防或治疗SARS-CoV-2感染的疫苗中的应用。在一些实施方案中，所述冠状病毒感染为SARS-CoV-2、SARS-CoV或MERS-Cov感染。在一些实施方案中，所述冠状病毒感染为野生型SARS-CoV-2或其变异株感染。在一些实施方案中，所述冠状病毒感染为野生型SARS-CoV-2、SARS-CoV-2 Alpha变异株、SARS-CoV-2 Beta变异株、SARS-CoV-2 Gamma变异株、SARS-CoV-2 Delta变异株、SARS-CoV-2 Kappa变异株、SARS-CoV-2 Epsilon变异株、SARS-CoV-2 Lambda变异株或SARS-CoV-2 Omicron变 异株感染。
附图说明
图1为融合蛋白与人ACE2结合曲线；图1a为融合蛋白D与人ACE2结合曲线，图1b为融合蛋白G与人ACE2结合曲线。
图2为血清抗Spike蛋白IgG滴度，条形图表示滴度的几何平均值(GMT)；图中wildtype代表WT-Spike-His，Delta代表Delta-Spike-His，Omicron代表Omicron-Spike-His。
图3为抗假病毒中和滴度(IC 50)；图中wildtype代表SARS-CoV-2 Spike假病毒，Delta代表SARS-COV-2 Spike(B.1.617.2)假病毒，Omicron代表SARS-COV-2 Spike(B.1.1.529)假病毒。
图4为血清抗Spike蛋白IgG滴度，条形图表示滴度的几何平均值(GMT)；其中，图4a、图4c和图4e为第一次给药后14天(第14天)滴度，图4b、图4d和图4f为第二次给药后14天(第35天)滴度。
图5为抗假病毒中和滴度(IC 50)；图中wildtype代表SARS-CoV-2 Spike假病毒，Delta代表SARS-COV-2 Spike(B.1.617.2)假病毒，Omicron代表SARS-COV-2 Spike(B.1.1.529)假病毒。
术语
除非另作说明，否则下列的每一个术语应当具有下文所述的含义。
定义
除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的相同含义。以下参考文献为技术人员提供了本发明中使用的许多术语的一般定义：科学出版社科学与技术(Academic Press Dictionary of Science and Technology)，Morris(编辑)，学术出版社(Academic Press)(第1版，1992年)；牛津生物化学和分子生物学词典(Oxford Dictionary of Biochemistry and Molecular Biology)，Smith等(编辑)，牛津大学出版社(Oxford University Press)，2000年修订；化学百科全书字典(Encyclopaedic Dictionary of Chemistry)，Kumar(编辑)，Anmol Publications Pvt有限公司(2002)；微生物和分子生物学词典(Dictionary of Microbiology and Molecular Biology)，Singleton等(编辑)，John Wiley和Sons(第3版，2002)；化学词典(Dictionary of Chemistry)，Hunt(编辑)，Routledge(第1版，1999)；药物医学词典(Dictionary of Pharmaceutical Medicine)，Nahler(编辑)，Springer-Verlag Telos(1994)；有机化学词典(Dictionary of Organic Chemistry)，Kumar和Anandand(编辑)，Anmol Publications Pvt.有限公司(2002)；以及生物词典(A Dictionary of Biology)(牛津平装参考，Oxford  Paperback Reference)，Martin和Hine(编辑)，牛津大学出版社(Oxford University Press)(2000年第4版)。
应当注意的是，如本文中及权利要求书中使用的，单数形式“一个”、“一种”和“该/所述”包括复数提及物，除非上下文另有明确规定。例如，核酸分子指一种或多种核酸分子。因此，术语“一个”、“一种”、“一个/种或多个/种”和“至少一个/种”可以互换使用。类似地，术语“包含”、“包括”和“具有”可以互换使用，通常应当理解为开放式且非限制性的，例如，不排除其他未列举的要素或步骤。
术语“氨基酸”是指既含氨基又含羧基的有机化合物，比如α-氨基酸，其可直接或以前体的形式由核酸编码。单个氨基酸由三个核苷酸(所谓的密码子或碱基三联体)组成的核酸编码。每一个氨基酸由至少一个密码子编码。相同氨基酸由不同密码子编码称为“遗传密码的简并性”。氨基酸包括天然氨基酸和非天然氨基酸。天然氨基酸包括丙氨酸(三字母代码：Ala，一字母代码：A)、精氨酸(Arg，R)、天冬酰胺(Asn，N)、天冬氨酸(Asp，D)、半胱氨酸(Cys，C)、谷氨酰胺(Gln，Q)、谷氨酸(Glu，E)、甘氨酸(Gly，G)、组氨酸(His，H)、异亮氨酸(Ile，I)、亮氨酸(Leu，L)、赖氨酸(Lys，K)、甲硫氨酸(Met，M)、苯丙氨酸(Phe，F)、脯氨酸(Pro，P)、丝氨酸(Ser，S)、苏氨酸(Thr，T)、色氨酸(Trp，W)、酪氨酸(Tyr，Y)和缬氨酸(Val，V)。
“保守氨基酸取代”是指一个氨基酸残基被另一个含有化学性质(例如电荷或疏水性)相似的侧链(R基团)的氨基酸残基所取代。一般而言，保守氨基酸取代不大会在实质上改变蛋白质的功能性质。含有化学性质相似侧链的氨基酸类别的实例包括：1)脂族侧链：甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸；2)脂族羟基侧链：丝氨酸和苏氨酸；3)含酰胺的侧链：天冬酰胺和谷氨酰胺；4)芳族侧链：苯丙氨酸、酪氨酸和色氨酸；5)碱性侧链：赖氨酸、精氨酸和组氨酸；6)酸性侧链：天冬氨酸和谷氨酸。
术语“多肽”旨在涵盖单数的“多肽”以及复数的“多肽”，并且是指由通过酰胺键(也称为肽键)线性连接的氨基酸单体组成的分子。术语“多肽”是指两个或更多个氨基酸的任何单条链或多条链，并且不涉及产物的特定长度。因此，“多肽”的定义中包括肽、二肽、三肽、寡肽、“蛋白质”、“氨基酸链”或用于指两个或多个氨基酸链的任何其他术语，并且术语“多肽”可以用来代替上述任何一个术语，或者与上述任何一个术语交替使用。术语“多肽”也意在指多肽表达后修饰的产物，包括但不限于糖基化、乙酰化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割或非天然发生的氨基酸修饰。多肽可以源自天然生物来源或通过重组技术产生，但其不必从指定的核酸序列翻译所得，它可能以包括化学合成的任何方式产生。
除非另有说明，融合蛋白是包含来自至少两个不相关蛋白的氨基酸序列的重组蛋 白，所述至少两个不相关蛋白已经通过肽键连接在一起以形成单个蛋白。不相关蛋白的氨基酸序列可以彼此直接连接，或者可以使用接头连接。如本文所用，如果蛋白的氨基酸序列通常在其天然环境中(例如，在细胞内)通常不经由肽键连接在一起，则它们是不相关的。例如，通常细菌酶例如嗜热脂肪芽孢杆菌二氢硫辛酸转乙酰基酶(E2p)的氨基酸序列和冠状病毒Spike蛋白的氨基酸序列不通过肽键连接在一起。
术语“同源性”、“同一性”或“相似性”是指两个肽之间或两个核酸分子之间的序列相似性。可以通过比较每个序列中可以比对的位置来确定同源性。当被比较的序列中的位置被相同的碱基或氨基酸占据时，则分子在该位置是同源的。序列之间的同源程度是由序列共有的匹配或同源位置的数目组成的一个函数。
术语“编码”应用于多核苷酸时，是指被称为“编码”多肽的多核苷酸，在其天然状态或当通过本领域技术人员公知的方法操作时，经转录和/或翻译可以产生该多肽和/或其片段。
多聚核苷酸是由四种碱基的特定序列组成：腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)、胸腺嘧啶(T)，或当多聚核苷酸是RNA时胸腺嘧啶换为尿嘧啶(U)。“多聚核苷酸序列”可以以多聚核苷酸分子的字母表示。该字母表示可以被输入到具有中央处理单元的计算机中的数据库中，并用于生物信息学应用，例如用于功能基因组学和同源性搜索。
术语“多核苷酸”、“多聚核苷酸”和“寡核苷酸”可互换使用，是指任何长度的核苷酸的聚合形式，无论是脱氧核糖核苷酸还是核糖核苷酸或其类似物。多聚核苷酸可以具有任何三维结构并且可以执行已知或未知的任何功能。以下是不受限制的多聚核苷酸的实施例：基因或基因片段(例如探针、引物、EST或SAGE标签)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、核糖酶、cDNA、dsRNA、siRNA、miRNA、重组多聚核苷酸、分支的多聚核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针和引物。多聚核苷酸可以包含修饰的核苷酸，例如甲基化的核苷酸和核苷酸类似物。如果存在该修饰，则对核苷酸的结构修饰可以在组装多聚核苷酸之前或之后进行。核苷酸的序列可以被非核苷酸组分中断。聚合后可以进一步修饰多聚核苷酸，例如通过与标记组分缀合。这个术语也指双链和单链分子。除另有说明或要求外，本公开的任何多聚核苷酸的实施例包括双链形式和已知或预测构成双链形式的两种可互补单链形式中的每一种。
核酸或多聚核苷酸序列(或多肽或蛋白序列)与另一序列有具有一定百分比(例如90％、95％、98％或者99％)的“同一性”或“序列同一性”是指当序列比对时，所比较的两个序列中该百分比的碱基(或氨基酸)相同。可以使用目测或本领域已知的软件程序来确定该比对同一性百分比或序列同一性，比如Ausubel et al.eds.(2007)在 Current Protocols in Molecular Biology中所述的软件程序。优选使用默认参数进行比对。其中一种比对程序是使用默认参数的BLAST，例如BLASTN和BLASTP，两者使用下列默认参数：Geneticcode＝standard；filter＝none；strand＝both；cutoff＝60；expect＝10；Matrix＝BLOSUM62；Descriptions＝50sequences；sortby＝HIGHSCORE；Databases＝non-redundant；GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR。生物学上等同的多聚核苷酸是具有上述指定百分比的同一性并编码具有相同或相似生物学活性的多肽的多聚核苷酸。
本发明中关于细胞、核酸、多肽、抗体等所使用的术语“分离的”，例如“分离的”DNA、RNA、多肽、抗体是指分别于细胞天然环境中的其它组分如DNA或RNA中的一种或多种所分离的分子。本发明使用的术语“分离的”还指当通过重组DNA技术产生时基本上不含细胞材料、病毒材料或细胞培养基的核酸或肽，或化学合成时的化学前体或其他化学品。此外，“分离的核酸”意在包括不以天然状态存在的核酸片段，并且不会以天然状态存在。术语“分离的”在本发明中也用于指从其他细胞蛋白质或组织分离的细胞或多肽。分离的多肽意在包括纯化的和重组的多肽。分离的多肽、抗体等通常通过至少一个纯化步骤制备。在一些实施方案中，分离的核酸、多肽、抗体等的纯度至少为约50％、约60％、约70％、约80％、约90％、约95％、约99％，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
术语“重组”涉及多肽或多聚核苷酸，意指天然不存在的多肽或多聚核苷酸的形式，不受限制的实施例可以通过组合产生通常并不存在的多聚核苷酸或多肽。
“抗体”、“抗原结合片段”是指特异性识别和结合抗原的多肽或多肽复合物。抗体可以是完整的抗体及其任何抗原结合片段或其单链。因此术语“抗体”包括分子中含有具有与抗原结合的生物学活性的免疫球蛋白分子的至少一部分的任何蛋白质或肽。
如本文所用，术语“抗原”或“免疫原”可互换使用，其指能够在受试者中诱导免疫响应的物质，通常是蛋白质。该术语还指具有免疫活性的蛋白质，即一旦向受试者给药(直接或通过向受试者给药编码该蛋白质的核苷酸序列或载体)就能够引起针对该蛋白质的体液和/或细胞类型的免疫响应。除非另有说明，术语“疫苗抗原”与“蛋白质抗原”或“抗原多肽”可互换使用。
“中和抗体”是指通过与传染原上的特定抗原结合来降低所述传染原的感染滴度的抗体。在一些实施方案中，传染原是病毒。“广谱中和抗体”是与相关抗原结合并抑制其功能的抗体，所述相关抗原例如与所述抗原的抗原性表面具有至少85％、90％、95％、96％、97％、98％或99％同一性的抗原。对于来自病原体的抗原例如病毒，所述抗体可与来自所述病原体的多于一种类和/或亚类的抗原结合并抑制其功能。
“cDNA”是指与mRNA互补或相同的DNA，可以是单链或双链形式。
“表位”是指抗原决定簇。这些是具有抗原性的分子上的特定化学基团或肽序列，以至于它们引发特异性的免疫响应，例如，表位是B和/或T细胞响应的抗原区域。表位可以由连续氨基酸形成，或者由蛋白质的三级折叠而并列的非连续氨基酸形成。
疫苗是指在受试者体内引起预防性或治疗性免疫响应的生物制品。在某些情况下，免疫响应是保护性免疫响应。通常，疫苗引起针对病原体例如病毒病原体的抗原或与病理状况相关的细胞组成的抗原特异性免疫响应。疫苗可包括多核苷酸(例如，编码已知抗原的核酸)，肽或多肽(例如公开的抗原)，病毒，细胞或一种或多种细胞组成。在一些实施方式中，疫苗或疫苗抗原或疫苗组合物从融合蛋白表达载体表达并自组装成在表面上显示了抗原多肽或蛋白质的纳米颗粒。
有效量的疫苗或其他试剂，指的是足以产生所需的响应，例如引起免疫响应、预防、减轻或消除病症或疾病(如肺炎)的体征或症状。例如，这可以是抑制病毒复制或可测量地改变病毒感染的外在症状所必需的量。通常，该量将足以可测量地抑制病毒(例如SARS-CoV-2)的复制或传染性。当施用于受试者时，通常将使用达到目标组织浓度的剂量，该剂量已显示出实现了体外抑制病毒复制。在一些实施方式中，“有效量”是治疗(包括预防)病症或疾病的一种或多种症状和/或潜在原因(例如治疗冠状病毒感染)的量。在一些实施方式中，有效量是治疗有效量。在一些实施方式中，有效量是防止特定疾病或病症的一种或多种症状或体征(例如与冠状病毒感染相关的一种或多种症状或体征)发展的量。
纳米颗粒是指球形蛋白质壳，其直径为数十纳米并且具有明确定义的表面几何形状。该球形蛋白质壳由非病毒蛋白质的相同复制品形成，该非病毒蛋白质能够自动组装成具有与病毒样颗粒(VLP)类似外观的纳米颗粒。实例包括铁蛋白(FR)，其在多物种间是保守的并形成24聚体(24-mer)，嗜热脂肪芽孢杆菌二氢硫辛酸转乙酰基酶(E2P)，超嗜热菌二氧四氢喋啶合酶(LS)和海栖热袍菌encapsulin，其中全部形成60聚体(60-mer)。自组装纳米颗粒可以在适当的表达系统中重组表达蛋白质后自发形成。纳米颗粒的生产、检测和表征的方法可以使用开发用于VLP的相同技术。
病毒样颗粒(VLP)是指非复制的病毒壳，其来源于多种病毒中的任何一种。VLP通常包括一种或多种病毒蛋白，例如但不限于被称为衣壳的蛋白，外壳蛋白，球壁蛋白，表面蛋白和/或包膜蛋白的那些蛋白，或衍生自这些蛋白的形成颗粒的多肽。在适当的表达系统中，在重组表达蛋白质后，VLP可以自发形成。生产特定VLP的方法是本领域已知的。可以使用本领域已知的常规技术(例如通过电子显微镜，生物物理表征等)来检测遵循重组表达病毒蛋白的VLP的存在。参见，例如，Baker等(1991)Biophys.J.60:1445-1456以及Hagensee等(1994)J.Virol.68:4503-4505。例如，VLP 可以通过密度梯度离心分离和/或通过特征密度带来识别。可选地，可以对所讨论的VLP制品的玻璃化水样进行冷冻电子显微镜检查，并在适当的曝光条件下记录图像。
术语“约”和“大约”可以互换使用，是指相关技术领域技术人员容易知道的相应数值的常规误差范围。在一些实施方式中，本文中提到“约”指所描述的数值以及其±10％、±5％或±1％的范围。
“ECMO”即指体外膜肺氧合(Extracorporeal Membrane Oxygenation，ECMO)，其是一种医疗急救技术设备，主要用于对重症心肺功能衰竭患者提供持续的体外呼吸与循环，以维持患者生命。
“ICU”是指重症加强护理病房(Intensive Care Unit)，治疗、护理、康复均可同步进行，为重症或昏迷患者提供隔离场所和设备，提供最佳护理、综合治疗、医养结合，以及术后早期康复、关节护理运动治疗等服务。
“IMV”即指间歇性指令通气(intermittent mandatory ventilation)，其是根据预先设置的时间间隔即时间触发，来实施周期性的容量或压力通气。这期间允许患者在指令通气期间以任何设定的基础压力水平进行自主呼吸。在自主呼吸时，患者可以在持续气流支持下自主呼吸，或者机器将按需阀门打开以允许自主呼吸。据大多数呼吸机都可以在自主呼吸时提供压力支持。
术语“受试者”是指被分类为哺乳动物的任何动物，例如人类和非人类哺乳动物。非人类动物的例子包括狗，猫，牛，马，绵羊，猪，山羊，兔子、大鼠、小鼠等。除非另有说明，否则术语“患者”或“受试者”在本文中可互换使用。优选地，受试者是人类。
“治疗”是指治疗性治疗和预防性或防治性措施，其目的是预防、减缓、改善或停止不良的生理改变或紊乱，例如疾病的进程，包括但不限于以下无论是可检测还是不可检测的结果，症状的缓解、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减缓、疾病状态的改善、缓和、减轻或消失(无论是部分还是全部)、延长与不接受治疗时预期的生存期限等。需要治疗的患者包括已经患有病症或紊乱的患者，容易患有病症或紊乱的患者，或者需要预防该病症或紊乱的患者，可以或预期从施用本发明公开的Spike蛋白纳米颗粒或药物组合物用于治疗中受益的患者。
概述
对于SARS-CoV、MERS-CoV和SARS-CoV-2，病毒基因组编码刺突(S)、包膜(E)、膜(M)和核衣壳(N)结构蛋白，其中，S糖蛋白(Spike蛋白)负责通过其S1亚单位中的受体结合结构域(RBD)结合宿主受体，以及由其S2亚单位驱动的随后的膜融合和病毒的进入。受体结合可以帮助将RBD保持在“站立”状态，这有助于S1亚单位与S2 亚单位的解离。当S1亚单位与S2亚单位解离时，第二S2′切割可释放融合肽。连接区域、HR1和CH形成一个非常长的螺旋件以将融合肽插入宿主细胞膜。最后，HR1和HR2形成螺旋结构，并组装成六螺旋束以融合病毒膜和宿主膜。
RBD包含一个核心子域和一个受体结合基序(RBM)。尽管SARS-CoV、MERS-CoV和SARS-CoV-2三种冠状病毒之间的核心子域高度相似，但它们的RBM明显不同，从而导致不同的受体特异性：SARS-CoV和SARS-CoV-2识别血管紧张素转换酶2(ACE2)，而MERS-CoV结合二肽基肽酶4(DPP4)。由于S糖蛋白是表面暴露的并介导进入宿主细胞，因此它是感染后中和抗体(NAb)的主要目标，也是疫苗设计的重点。Spike三聚体广泛地用N-连接的聚糖修饰，N-连接的聚糖对于正确折叠和调节对NAb的可及性很重要。
本发明通过1)使S1/S2切割位点失活的突变和2)在HR1和CH之间的转向区域存在防止HR1和CH在融合过程中形成直螺旋的突变，从而使Spike三聚体稳定在与宿主细胞膜融合前构造中。在一些实施方案中，可以将含突变的冠状病毒Spike蛋白胞外结构域或其截短片段显示在纳米颗粒上。
根据本文所述的研究和示例性设计，本发明提供了融合蛋白、Spike蛋白纳米颗粒和疫苗组合物。本发明还提供了相关的多核苷酸、表达载体和药物组合物。在一些实施方案中，病毒载体携带的呈蛋白质或核酸(DNA/mRNA)形式的稳定的Spike三聚体和RBD蛋白可用作冠状病毒疫苗。另外，纳米颗粒呈递的稳定的Spike三聚体和RBD也可以用作冠状病毒疫苗。
本发明的基于冠状病毒Spike蛋白的抗原和疫苗具有许多有利的特性。本文所述的Spike三聚体设计以其天然样构造呈现保守的中和表位，使Spike三聚体可用作抗原疫苗或在纳米颗粒上多价显示。本发明的纳米颗粒疫苗允许将源自不同冠状病毒的Spike三聚体显示在公知的纳米颗粒上，例如铁蛋白、E2p和I3-01，其尺寸范围为12.2至25.0nm。可以在HEK293细胞、ExpiCHO细胞、CHO细胞中高产地生产所有呈递三聚体的纳米颗粒。生产的Spike蛋白纳米颗粒可通过抗体和分子排阻色谱(SEC)纯化。
除非本文另有说明，否则本发明的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段、冠状病毒Spike蛋白S1亚基、冠状病毒Spike蛋白保守片段、融合蛋白、Spike蛋白纳米颗粒、编码的多核苷酸、表达载体和宿主细胞以及相关的治疗应用都可以根据本文举例的方法或本领域熟知的常规方法来产生或进行。
除非另有说明，步骤的顺序或执行某些操作的顺序并不重要，只要本发明保持可操作性即可。而且，可以同时进行两个或更多个步骤或操作。
除非另有说明，本文中使用的任何和所有示例，或本文所使用的示例性语言(例如“诸如”或“包括”)仅旨在更好地说明本发明，而不对本发明的范围构成限制。说明书中的任何语言都不应解释为任何未要求保护的要素对于实施本发明是必不可少的。
含突变的冠状病毒Spike蛋白胞外结构域或其截短片段
本发明提供了可用于产生疫苗的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段。通过将突变引入冠状病毒Spike蛋白胞外结构域或其截短片段中，使突变后的Spike三聚体稳定。本文举例说明了特定SARS-CoV-2毒株或分离物的一些特定Spike蛋白，例如SEQ ID NO:1、8和31。由于给定冠状病毒的不同分离物或毒株之间的功能相似性和序列同源性，因此也可以根据本文所述的突变策略来产生衍生自其他已知冠状病毒Spike蛋白直系同源序列的突变的Spike蛋白或其截短片段。在文献中已经描述了许多已知的冠状病毒Spike蛋白序列。参见，例如，James等,J.Mol.Biol.432:3309-25,2020；Andersen等,Nat.Med.26:450-452,2020；Walls等,Cell 180:281–292,2020；Zhang等,J.Proteome Res.19:1351-1360,2020；Du等,Expert Opin.Ther.Targets 21:131-143.；2017；Yang等,Viral Immunol.27:543-550,2014；Wang等,Antiviral Res.133:165-177,2016；Bosch等,J.Virol.77:8801-8811,2003；Lio等,TRENDS Microbiol.12:106-111,2004；Chakraborti等,Virol.J.2:73,2005；以及Li,Ann.Rev.Virol.3:237-261,2016.。
如本文所述，本发明的一些突变的Spike蛋白或其截短片段包含可以增强与细胞膜融合前Spike蛋白或其截短片段结构的稳定性的突变。这些突变包括使S1/S2切割位点失活的突变，以及在HR1和CH之间的转向区域的突变，该突变去除了HR1和CH之间的转向区域中的任何应变，即防止形成直螺旋。
一些含突变的冠状病毒Spike蛋白胞外结构域或其截短片段(如SEQ ID NO:3-4、6-7、9-12、32-35、78-83所示)来源于引起COVID-19的SARS-CoV-2病毒。这些多肽中含有S1/S2切割位点失活的突变以及在HR1和CH之间的转向区域的突变。作为示例，用于突变的野生型SARS-CoV-2 Spike蛋白的氨基酸序列如SEQ ID NO:1所示或如SEQ ID NO:1的第15-1213残基所示的氨基酸序列。在一些实施方案中，用于突变的Spike蛋白可以是SEQ ID NO:1、8或31或其变体，例如与其基本相同的变体或保守修饰的变体。使用基于cryo-EM模型PDB ID 6VSB或GenBank登录号MN908947.3的氨基酸编号作为参考，S1/S2切割位点 682RRAR 685的失活可以通过位点内或位点周围的许多序列改变(例如，缺失或替代)来实现。如本文所示例的，使S1/S2切割位点失活而不影响蛋白质结构的一种突变是将S1/S2切割位点 682RRAR 685突变为 682GSAS 685。除了使S1/S2切割位点失活外，还可在HR1和CH之间的转向区域进行双重突变，该双重突变通过防止直螺旋的形成而消除了融合过程中转向区域(HR1和CH基序之间) 的应变。在一些实施方案中，这种双重突变可以是K986G/V987G、K986P/V987P、K986G/V987P或K986P/V987G。除了上述稳定融合前Spike蛋白或其截短片段结构的突变以外，本发明的一些SARS-CoV-2 Spike蛋白或其截短片段可含有大部分或整个HR2结构域的缺失。使用示例性的SARS-CoV-2 Spike蛋白序列SEQ ID NO:1来说明，这种缺失可以包括如SEQ ID NO:1的第1144-1213残基的缺失。在一些实施方案中，缺失可以是截短Spike蛋白胞外结构域(例如SEQ ID NO:1、3-4、8-10、31-33、78、80或82)的C端5个、10个、15个、20个、25个、30个、35个、40个、45个、50个、55个、60个、65个、70个、75个、76个、80个或更多个残基，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，C端截短的Spike蛋白可以延伸超过HR2结构域。在一些实施方案中，Spike蛋白序列可包括SEQ ID NO:2或5所示的N端信号肽。
示例性的冠状病毒Spike蛋白胞外结构域或其截短片段或其变体如下：
野生型SARS-CoV-2 Spike蛋白全长胞外结构域(ECD)，其氨基酸序列如SEQ ID NO:1所示，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685用下划线、加粗和斜体标出。
Figure PCTCN2022113747-appb-000001
Figure PCTCN2022113747-appb-000002
突变的野生型SARS-CoV-2 Spike蛋白全长胞外结构域a1，其氨基酸序列如SEQ ID NO:3所示。在序列中，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000003
突变的野生型SARS-CoV-2 Spike蛋白全长胞外结构域a2，其氨基酸序列如SEQ ID NO:4所示。在序列中，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000004
突变的野生型SARS-CoV-2 Spike蛋白全长胞外结构域a3，其氨基酸序列如SEQ ID NO:78所示。在序列中，不含信号肽，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000005
Figure PCTCN2022113747-appb-000006
突变的野生型SARS-CoV-2 Spike蛋白胞外结构域C端截短片段b1，其氨基酸序列如SEQ ID NO:6所示。在序列中，C端截短了70个氨基酸残基，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000007
Figure PCTCN2022113747-appb-000008
突变的野生型SARS-CoV-2 Spike蛋白胞外结构域C端截短片段b2，其氨基酸序列如SEQ ID NO:7所示。在序列中，C端截短了70个氨基酸残基，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000009
突变的野生型SARS-CoV-2 Spike蛋白胞外结构域C端截短片段b3，其氨基酸序列如SEQ ID NO:79所示。在序列中，C端截短了70个氨基酸残基，不含信号肽，S1/S2 切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000010
SARS-CoV-2 Delta变异株Spike蛋白全长胞外结构域(ECD)，其氨基酸序列如SEQ ID NO:8所示，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685用下划线、加粗和斜体标出。
Figure PCTCN2022113747-appb-000011
Figure PCTCN2022113747-appb-000012
突变的SARS-CoV-2 Delta变异株Spike蛋白全长胞外结构域c1，其氨基酸序列如SEQ ID NO:9所示。在序列中，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000013
Figure PCTCN2022113747-appb-000014
突变的SARS-CoV-2 Delta变异株Spike蛋白全长胞外结构域c2，其氨基酸序列如SEQ ID NO:10所示。在序列中，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000015
突变的SARS-CoV-2 Delta变异株Spike蛋白全长胞外结构域c3，其氨基酸序列如 SEQ ID NO:80所示。在序列中，不含信号肽，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000016
突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域C端截短片段d1，其氨基酸序列如SEQ ID NO:11所示。在序列中，C端截短了70个氨基酸残基，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000017
Figure PCTCN2022113747-appb-000018
突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域C端截短片段d2，其氨基酸序列如SEQ ID NO:12所示。在序列中，C端截短了70个氨基酸残基，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000019
Figure PCTCN2022113747-appb-000020
突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域C端截短片段d3，其氨基酸序列如SEQ ID NO:81所示。在序列中，C端截短了70个氨基酸残基，不含信号肽，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000021
Figure PCTCN2022113747-appb-000022
SARS-CoV-2 Delta变异株Spike蛋白S1亚基，其氨基酸序列如SEQ ID NO:13所示，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出。
Figure PCTCN2022113747-appb-000023
SARS-CoV-2 Omicron变异株Spike蛋白全长胞外结构域(ECD)，其氨基酸序列如SEQ ID NO:31所示，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685用下划线、加粗和斜体标出。
Figure PCTCN2022113747-appb-000024
Figure PCTCN2022113747-appb-000025
突变的SARS-CoV-2 Omicron变异株Spike蛋白全长胞外结构域f1，其氨基酸序列如SEQ ID NO:32所示。在序列中，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000026
突变的SARS-CoV-2 Omicron变异株Spike蛋白全长胞外结构域f2，其氨基酸序列如SEQ ID NO:33所示。在序列中，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000027
突变的SARS-CoV-2 Omicron变异株Spike蛋白全长胞外结构域f3，其氨基酸序列如SEQ ID NO:82所示。在序列中，不含信号肽，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000028
Figure PCTCN2022113747-appb-000029
突变的SARS-CoV-2 Omicron变异株Spike蛋白胞外结构域C端截短片段g1，其氨基酸序列如SEQ ID NO:34所示。在序列中，C端截短了70个氨基酸残基，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000030
Figure PCTCN2022113747-appb-000031
突变的SARS-CoV-2 Omicron变异株Spike蛋白胞外结构域C端截短片段g2，其氨基酸序列如SEQ ID NO:35所示。在序列中，C端截短了70个氨基酸残基，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000032
Figure PCTCN2022113747-appb-000033
突变的SARS-CoV-2 Omicron变异株Spike蛋白胞外结构域C端截短片段g3，其氨基酸序列如SEQ ID NO:83所示。在序列中，C端截短了70个氨基酸残基，不含信号肽，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000034
SARS-CoV-2 Omicron变异株Spike蛋白S1亚基，其氨基酸序列如SEQ ID NO:36所示，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出。
Figure PCTCN2022113747-appb-000035
Figure PCTCN2022113747-appb-000036
SARS-CoV-2 Omicron变异株Spike蛋白保守片段O330的氨基酸序列如SEQ ID NO:37所示。
PNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFF(如SEQ ID NO:37所示)。
融合蛋白
本发明提供了包含异源支架的融合蛋白，所述异源支架显示了至少一种源自冠状病毒Spike蛋白的抗原多肽或三聚体蛋白。在一些实施方案中，所使用的冠状病毒抗原是含有上述各种稳定突变的冠状病毒Spike蛋白胞外结构域或其截短片段。在一些实施方案中，所采用的冠状病毒抗原包含或衍生自冠状病毒Spike蛋白的RBD结构域。在一些实施方案中，所采用的冠状病毒抗原包含或衍生自冠状病毒Spike蛋白的S1亚基。在一些实施方案中，所采用的冠状病毒抗原包含或衍生自冠状病毒Spike蛋白的保守片段。在示例性的实施方案中，所采用的Spike蛋白序列包含SEQ ID NO:1、3-4、6-13、31-37、78-83任一所示的序列，或与其基本上相同或保守修饰的变体。将表达融合蛋白的表达载体转染宿主细胞后，由于抗原(例如Spike蛋白)与自组装蛋白(例如单体铁蛋白亚基)连接，将产生表面上显示抗原(例如Spike蛋白)的纳米颗粒疫苗。
任何异源支架可用于在本发明疫苗的构建中呈递抗原。这包括病毒样颗粒(VLP)，例如纳米颗粒。各种纳米颗粒可用于产生本发明的疫苗。通常，用于本发明的纳米颗粒需要由单个亚单位的多个复制品形成。纳米颗粒通常是球形的，和/或具有旋转对称性(例如，具有3重轴和5重轴)，例如具有本文示例的二十面体结构。另外地或可替代地，纳米颗粒亚单位的氨基末端必须暴露并紧邻3重轴，并且三个氨基末端的间隔必须紧密匹配显示的三聚体稳定的Spike蛋白的羧基末端的间隔。
在一些实施方案中，所采用的自组装纳米颗粒的直径为约25nm或更小(通常由12、24或60个亚基组装而成)，并且在粒子表面上具有3重轴。这种纳米颗粒提供了合适的颗粒以生产多价疫苗。在一些优选的实施方案中，冠状病毒抗原可以呈递在自组装纳米颗粒上，例如呈递在衍生自本文举例说明的铁蛋白(FR)的自组装纳米颗粒上。铁蛋白是在动物、细菌和植物中发现的球状蛋白，其主要作用是通过将水合的铁离子和质子运输到矿化核心或通过将水合的铁离子和质子从矿化核心运输出来以控制多核Fe(III) 2O 3形成的速率和位置。铁蛋白的球状形式由单体亚单位蛋白(也称为单体铁蛋白亚基)组成，该单体亚单位蛋白是分子量约为17-20kDa的多肽。这些蛋白质的亚单位的序列是本领域已知的。在一些实施方案中，本发明的纳米颗粒疫苗可以使用任何这些已知的纳米颗粒，以及它们的保守修饰的变体或与其具有基本相同(例如，至少90％，95％或99％同一性)序列的变体。
在一些示例性实施方案中，本发明的融合蛋白包含Fc片段(例如人IgG Fc片段)。通常，将冠状病毒Spike蛋白保守序列或冠状病毒Spike蛋白S1亚基或含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的C端融合到Fc片段的N端。
人IgG Fc的氨基酸序列如下：
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(如SEQ ID NO:38所示)。
在一些示例性实施方案中，本发明的融合蛋白包含纳米颗粒亚单位序列(例如幽门螺旋杆菌非血红素单体铁蛋白亚基，其氨基酸序列如SEQ ID NO：14所示)，或其保守修饰的变体或与其基本相同的序列。通常，将冠状病毒Spike蛋白保守序列或冠状病毒Spike蛋白S1亚基或含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的C端融合到自组装纳米颗粒(NP)亚单位的N端。在一些实施方案中，冠状病毒Spike蛋白保守序列或冠状病毒Spike蛋白S1亚基或含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的C端经GS接头连接至纳米颗粒亚单位的N端，接头例如为GGGGS或GGGGSGGGGS。
幽门螺旋杆菌非血红素单体铁蛋白亚基(Ferritin)的氨基酸序列如下：
DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS(如SEQ  ID NO:14所示)。
可通过将抗原多肽或多聚抗原蛋白(例如，三聚体抗原)的亚单位融合至纳米颗粒的亚单位(例如，铁蛋白亚单位)以及本文所述的其他任选或替代的组分，来构建显示本文所述的冠状病毒Spike蛋白保守序列或冠状病毒Spike蛋白S1亚基或任何稳定的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的纳米颗粒。为了构建本发明的融合蛋白，可以采用一个或多个接头来连接并维持不同功能蛋白的整体活性不变。通常，接头包含短肽序列，例如富含GS的肽。在一些实施方案中，接头或接头基序可以是连接两个蛋白质结构域或基序而不干扰其功能的任何柔性肽。例如，所采用的接头可以是如本文所示的G 4S接头或(G 4S) 2接头以连接刺突蛋白和纳米颗粒支架序列。本发明的融合蛋白的重组生产可以基于本文所述的方案和/或本领域中已经描述的其他方法。
示例性的融合蛋白序列如下：
融合蛋白A1：将突变的野生型SARS-CoV-2 Spike蛋白全长胞外结构域a1(如SEQ ID NO:3所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白A1，其氨基酸序列如SEQ ID NO:16所示。在序列中，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000037
Figure PCTCN2022113747-appb-000038
融合蛋白A2：将突变的野生型SARS-CoV-2 Spike蛋白全长胞外结构域a2(如SEQ ID NO:4所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白A2，其氨基酸序列如SEQ ID NO:17所示。在序列中，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000039
Figure PCTCN2022113747-appb-000040
融合蛋白B1：将突变的野生型SARS-CoV-2 Spike蛋白胞外结构域C端截短片段b1(如SEQ ID NO:6所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白B1，其氨基酸序列如SEQ ID NO:18所示。在序列中，C端截短了70个氨基酸残基，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000041
Figure PCTCN2022113747-appb-000042
融合蛋白B2：将突变的野生型SARS-CoV-2 Spike蛋白胞外结构域C端截短片段b2(如SEQ ID NO:7所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白B2，其氨基酸序列如SEQ ID NO:19所示。在序列中，C端截短了70个氨基酸残基，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000043
Figure PCTCN2022113747-appb-000044
融合蛋白C1：将突变的SARS-CoV-2 Delta变异株Spike蛋白全长胞外结构域c1(如SEQ ID NO:9所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白C1，其氨基酸序列如SEQ ID NO:20所示。在序列中，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000045
Figure PCTCN2022113747-appb-000046
融合蛋白C2：将突变的SARS-CoV-2 Delta变异株Spike蛋白全长胞外结构域c2(如SEQ ID NO:10所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白C2，其氨基酸序列如SEQ ID NO:21所示。在序列中，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000047
Figure PCTCN2022113747-appb-000048
融合蛋白D1：将突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域C端截短片段d1(如SEQ ID NO:11所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白D1，其氨基酸序列如SEQ ID NO:22所示。在序列中，C端截短了70个氨基酸残基，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000049
融合蛋白D2：将突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域C端截 短片段d2(如SEQ ID NO:12所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白D2，其氨基酸序列如SEQ ID NO:23所示。在序列中，C端截短了70个氨基酸残基，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000050
融合蛋白E1：将SARS-CoV-2 Delta变异株Spike蛋白S1亚基(如SEQ ID NO:13所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白E1，其氨基酸序列 如SEQ ID NO:24所示。在序列中，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000051
融合蛋白E2：将SARS-CoV-2 Delta变异株Spike蛋白S1亚基(如SEQ ID NO:13所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，获得融合蛋白E2，其氨基酸序列如SEQ ID NO:25所示。在序列中，N端信号肽用斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000052
Figure PCTCN2022113747-appb-000053
融合蛋白E3：将SARS-CoV-2 Omicron变异株Spike蛋白S1亚基(如SEQ ID NO:36所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白E3，其氨基酸序列如SEQ ID NO:39所示。在序列中，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000054
融合蛋白E4：将SARS-CoV-2 Omicron变异株Spike蛋白S1亚基(如SEQ ID NO:36所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，获得融合蛋白E4，其氨基酸序列如SEQ ID NO:40所示。在序列中，N端信号肽用斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000055
融合蛋白F1：将突变的SARS-CoV-2 Omicron变异株Spike蛋白全长胞外结构域f1(如SEQ ID NO:32所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白F1，其氨基酸序列如SEQ ID NO:41所示。在序列中，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000056
Figure PCTCN2022113747-appb-000057
融合蛋白F2：将突变的SARS-CoV-2 Omicron变异株Spike蛋白全长胞外结构域f2(如SEQ ID NO:33所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白F2，其氨基酸序列如SEQ ID NO:42所示。在序列中，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000058
Figure PCTCN2022113747-appb-000059
融合蛋白G1：将突变的SARS-CoV-2 Omicron变异株Spike蛋白胞外结构域C端截短片段g1(如SEQ ID NO:34所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白G1，其氨基酸序列如SEQ ID NO:43所示。在序列中，C端截短了70个氨基酸残基，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000060
Figure PCTCN2022113747-appb-000061
融合蛋白G2：将突变的SARS-CoV-2 Omicron变异株Spike蛋白胞外结构域C端截短片段g2(如SEQ ID NO:35所示)的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白G2，其氨基酸序列如SEQ ID NO:44所示。在序列中，C端截短了70个氨基酸残基，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000062
Figure PCTCN2022113747-appb-000063
融合蛋白H1：在O330片段(如SEQ ID NO:37所示)的N端添加原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，然后将O330片段的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白H1，其氨基酸序列如SEQ ID NO:45所示。原始信号肽用斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000064
融合蛋白H2：在O330片段(如SEQ ID NO:37所示)的N端添加信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)，然后将O330片段的C端通过接头GGGGS(如SEQ ID NO:15所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:14所示)的N端连接获得融合蛋白H2，其氨基酸序列如SEQ ID NO:46所示。信号肽用斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000065
融合蛋白A1-1：将突变的野生型SARS-CoV-2 Spike蛋白全长胞外结构域a1(如SEQ ID NO:3所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白A1-1，其氨基酸序列如SEQ ID NO:47所示。在序列中，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划 线和斜体标出。
Figure PCTCN2022113747-appb-000066
融合蛋白A2-1：将突变的野生型SARS-CoV-2 Spike蛋白全长胞外结构域a2(如SEQ ID NO:4所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白A2-1，其氨基酸序列如SEQ ID NO:48所示。在序列中，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000067
融合蛋白B1-1：将突变的野生型SARS-CoV-2 Spike蛋白胞外结构域C端截短片段b1(如SEQ ID NO:6所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白B1-1，其氨基酸序列如SEQ ID NO:49所示。在序列中，C端截短了70个氨基酸残基，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000068
Figure PCTCN2022113747-appb-000069
融合蛋白B2-1：将突变的野生型SARS-CoV-2 Spike蛋白胞外结构域C端截短片段b2(如SEQ ID NO:7所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白B2-1，其氨基酸序列如SEQ ID NO:50所示。在序列中，C端截短了70个氨基酸残基，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000070
Figure PCTCN2022113747-appb-000071
融合蛋白C1-1：将突变的SARS-CoV-2 Delta变异株Spike蛋白全长胞外结构域c1(如SEQ ID NO:9所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白C1-1，其氨基酸序列如SEQ ID NO:51所示。在序列中，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000072
Figure PCTCN2022113747-appb-000073
融合蛋白C2-1：将突变的SARS-CoV-2 Delta变异株Spike蛋白全长胞外结构域c2(如SEQ ID NO:10所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白C2-1，其氨基酸序列如SEQ ID NO:52所示。在序列中，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000074
Figure PCTCN2022113747-appb-000075
融合蛋白D1-1：将突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域C端截短片段d1(如SEQ ID NO:11所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白D1-1，其氨基酸序列如SEQ ID NO:53所示。在序列中，C端截短了70个氨基酸残基，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000076
Figure PCTCN2022113747-appb-000077
融合蛋白D2-1：将突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域C端截短片段d2(如SEQ ID NO:12所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白D2-1，其氨基酸序列如SEQ ID NO:54所示。在序列中，C端截短了70个氨基酸残基，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000078
Figure PCTCN2022113747-appb-000079
融合蛋白E1-1：将SARS-CoV-2 Delta变异株Spike蛋白S1亚基(如SEQ ID NO:13所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白E1-1，其氨基酸序列如SEQ ID NO:55所示。在序列中，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出。
Figure PCTCN2022113747-appb-000080
融合蛋白E2-1：将SARS-CoV-2 Delta变异株Spike蛋白S1亚基(如SEQ ID NO:13所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，获得融合蛋白E2-1，其氨基酸序列如SEQ ID NO:56所示。在序列中，N端信号肽用斜体标出。
Figure PCTCN2022113747-appb-000081
融合蛋白E3-1：将SARS-CoV-2 Omicron变异株Spike蛋白S1亚基(如SEQ ID NO:36所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白E3-1，其氨基酸序列如SEQ ID NO:57所示。在序列中，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出。
Figure PCTCN2022113747-appb-000082
Figure PCTCN2022113747-appb-000083
融合蛋白E4-1：将SARS-CoV-2 Omicron变异株Spike蛋白S1亚基(如SEQ ID NO:36所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，获得融合蛋白E4-1，其氨基酸序列如SEQ ID NO:58所示。在序列中，N端信号肽用斜体标出。
Figure PCTCN2022113747-appb-000084
融合蛋白F1-1：将突变的SARS-CoV-2 Omicron变异株Spike蛋白全长胞外结构域f1(如SEQ ID NO:32所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白F1-1，其氨基酸序列如SEQ ID NO:59所示。在序列中，原始信号 肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000085
融合蛋白F2-1：将突变的SARS-CoV-2 Omicron变异株Spike蛋白全长胞外结构域f2(如SEQ ID NO:33所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白F2-1，其氨基酸序列如SEQ ID NO:60所示。在序列中，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000086
融合蛋白G1-1：将突变的SARS-CoV-2 Omicron变异株Spike蛋白胞外结构域C端截短片段g1(如SEQ ID NO:34所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白G1-1，其氨基酸序列如SEQ ID NO:61所示。在序列中，C端截短了70个氨基酸残基，原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000087
融合蛋白G2-1：将突变的SARS-CoV-2 Omicron变异株Spike蛋白胞外结构域C端截短片段g2(如SEQ ID NO:35所示)的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白G2-1，其氨基酸序列如SEQ ID NO:62所示。在序列中，C端截短了70个氨基酸残基，用信号肽：MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)替换了原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000088
Figure PCTCN2022113747-appb-000089
融合蛋白H1-1：在O330片段(如SEQ ID NO:37所示)的N端添加原始信号肽：MFVFLVLLPLVSSQ(如SEQ ID NO:2所示)，然后将O330片段的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白H1-1，其氨基酸序列如SEQ ID NO:63所示。原始信号肽用斜体标出。
Figure PCTCN2022113747-appb-000090
融合蛋白H2-1：在O330片段(如SEQ ID NO:37所示)的N端添加 MEFGLSLVFLVLILKGVQC(如SEQ ID NO:5所示)，然后将O330片段的C端与人IgG Fc(如SEQ ID NO:38所示)的N端连接获得融合蛋白H2-1，其氨基酸序列如SEQ ID NO:64所示。信号肽用斜体标出。
Figure PCTCN2022113747-appb-000091
成熟的融合蛋白A：与融合蛋白A1和A2相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:26所示。在序列中，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000092
Figure PCTCN2022113747-appb-000093
成熟的融合蛋白B：与融合蛋白B1和B2相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:27所示。在序列中，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000094
成熟的融合蛋白C：与融合蛋白C1和C2相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:28所示。在序列中，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头 用斜体和加粗标出。
Figure PCTCN2022113747-appb-000095
成熟的融合蛋白D：与融合蛋白D1和D2相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:29所示。在序列中，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000096
Figure PCTCN2022113747-appb-000097
成熟的融合蛋白E-1：与融合蛋白E1和E2相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:30所示。在序列中，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000098
Figure PCTCN2022113747-appb-000099
成熟的融合蛋白E-2：与融合蛋白E3和E4相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:65所示。在序列中，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000100
成熟的融合蛋白F：与融合蛋白F1和F2相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:66所示。在序列中，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000101
Figure PCTCN2022113747-appb-000102
成熟的融合蛋白G：与融合蛋白G1和G2相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:67所示。在序列中，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000103
Figure PCTCN2022113747-appb-000104
成熟的融合蛋白H：与融合蛋白H1和H2相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:68所示。在序列中，接头用斜体和加粗标出。
Figure PCTCN2022113747-appb-000105
成熟的融合蛋白A-1：与融合蛋白A1-1和A2-1相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:69所示。在序列中，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000106
Figure PCTCN2022113747-appb-000107
成熟的融合蛋白B-1：与融合蛋白B1-1和B2-1相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:70所示。在序列中，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000108
Figure PCTCN2022113747-appb-000109
成熟的融合蛋白C-1：与融合蛋白C1-1和C2-1相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:71所示。在序列中，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000110
Figure PCTCN2022113747-appb-000111
成熟的融合蛋白D-1：与融合蛋白D1-1和D2-1相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:72所示。在序列中，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000112
成熟的融合蛋白E-3：与融合蛋白E1-1和E2-1相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:73所示。
CVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(如SEQ ID NO:73所示)。
成熟的融合蛋白E-4：与融合蛋白E3-1和E4-1相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:74所示。
CVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(如SEQ ID NO:74所示)。
成熟的融合蛋白F-1：与融合蛋白F1-1和F2-1相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:75所示。在序列中，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体标出。
Figure PCTCN2022113747-appb-000113
成熟的融合蛋白G-1：与融合蛋白G1-1和G2-1相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:76所示。在序列中，S1/S2切割位点 682RRAR 685突变为 682GSAS 685，用下划线和加粗标出，同时包含双重突变K986P/V987P，用下划线和斜体 标出。
Figure PCTCN2022113747-appb-000114
成熟的融合蛋白H-1：与融合蛋白H1-1和H2-1相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:77所示。
PNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(如SEQ ID NO:77所示)。
SEQ ID NO:26-30、65-77是去除N端信号肽(SEQ ID NO:2或5)的成熟的融合蛋白序列。除了这些具体示例的融合蛋白之外，本发明还涵盖纳米颗粒疫苗，该疫苗含有与任何这些示例的纳米颗粒疫苗序列中的任一个基本相同的亚单位序列或其保守修饰的变体的亚单位序列。
多核苷酸和表达载体
本发明的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段、冠状病毒Spike蛋白S1亚基、冠状病毒Spike蛋白保守片段、融合蛋白或Spike蛋白纳米颗粒通常通过表达载体来生产，所述表达载体包含本文所述的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段、冠状病毒Spike蛋白S1亚基、冠状病毒Spike蛋白保守片段、融合蛋白或Spike蛋白纳米颗粒的编码序列。因此，在一些相关方面，本发明提供了编码本文所述的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段、冠状病毒Spike蛋白S1亚基、冠状病毒Spike蛋白保守片段、融合蛋白或Spike蛋白纳米颗粒的多核苷酸(DNA或RNA)。本发明的一些多核苷酸编码本文所述的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段中的一种，例如，SEQ ID NO:12所示的SARS-CoV-2 Spike蛋白胞外结构域的截短片段。本发明的一些多核苷酸编码本文所述的纳米颗粒疫苗中的一种的亚单位序列，例如SEQ ID NO:23所示的融合蛋白序列。本发明表达的融合蛋白可以不包含N端信号肽，或者一些多核苷酸序列额外地编码N端信号肽。例如，编码融合蛋白(例如，SEQ ID NO:26-30)的多核苷酸还可以包含编码SEQ ID NO:2或5所示的N端信号肽、或与其基本上相同的序列或保守修饰的变体的序列。
本发明还提供了具有此类多核苷酸的表达载体和用于产生含突变的冠状病毒Spike蛋白胞外结构域或其截短片段、冠状病毒Spike蛋白S1亚基、冠状病毒Spike蛋白保守片段或融合蛋白的宿主细胞(例如，原核或真核细胞，如HEK293，CHO，ExpiCHO和CHO-S细胞系)。由多核苷酸编码或由载体表达的融合蛋白也包括在本发明中。如本文所述，纳米颗粒亚单位融合的Spike蛋白胞外结构域或其截短片段、Spike蛋白S1亚基或Spike蛋白保守片段将自组装成纳米颗粒疫苗，该纳米颗粒疫苗在其表面上显示了Spike蛋白或其截短片段、Spike蛋白S1亚基或Spike蛋白保守片段。
多核苷酸和相关载体可以通过标准分子生物学技术或本文举例说明的方案产生。例如，用于克隆，转染，瞬时基因表达和获得稳定转染的细胞系的通用方案在本领域中已有描述，例如，Sambrook等，Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Press,N.Y.,(第三版,2000年)；以及Brent等,Current Protocols in Molecular Biology,John Wiley&Sons股份有限公司(ringbou版,2003)。也可以通过已知方法PCR将突变引入多核苷酸序列中。
具体载体的选择取决于蛋白的预期用途。例如，无论该细胞类型是原核还是真核细胞，选择的载体必须能够驱动蛋白在所需细胞类型中的表达。许多载体含有允许原核载体复制和可操作地连接的基因序列的真核表达的序列。可用于本发明的载体可以自主复制，即，该载体在染色体外存在，并且其复制不必直接与宿主细胞基因组的复制相连。可选地，载体的复制可以与宿主染色体DNA的复制相连，例如，可以将载体整合到宿主细胞的染色体中，这通过逆转录病毒载体且在稳定转染的细胞系中实现。基于病毒的表达载体和基于非病毒的表达载体均可用于在哺乳动物宿主细胞中产生抗原。非病毒载体和系统包括质粒，附加型载体(通常具有用于表达蛋白质或RNA的表达盒)和人类人工染色体。可选的病毒载体包括基于慢病毒或其他逆转录病毒的载体，腺病毒，腺伴随病毒，巨细胞病毒，疱疹病毒，基于SV40的载体，乳头瘤病毒，HBP Epstein Barr病毒，牛痘病毒载体和Semliki Forest病毒(SFV)。
取决于用于表达蛋白的特定载体，在本发明的实践中可以使用各种已知的细胞或细胞系。宿主细胞可以是携带本发明蛋白的重组载体的任何细胞，其中允许载体驱动用于本发明的蛋白表达。它可以是原核的，例如许多细菌菌株中的任何一种，或者可以是真核的，例如酵母或其他真菌细胞，昆虫或两栖动物细胞，或哺乳动物细胞，包括例如啮齿动物，猿猴或人细胞。表达本发明蛋白的细胞可以是原代培养细胞或可以是已确立的细胞系。因此，除了本文举例说明的细胞系(例如HEK293细胞)外，本领域中熟知的许多其他宿主细胞系也可以用于本发明的实践中。这些包括，例如，多种Cos细胞系，CHO细胞，HeLa细胞，Sf9细胞，AtT20，BV2和N18细胞，骨髓瘤细胞系，转化的B细胞和杂交瘤。
通过本领域技术人员已知的许多合适方法中的任一种，可以将表达蛋白的载体引入选择的宿主细胞。为了将编码蛋白的载体引入哺乳动物细胞，所使用的方法将取决于载体的形式。对于质粒载体，可以通过许多转染方法中的任一种引入编码蛋白序列的DNA，这些方法包括例如脂质体介导的转染(“脂质体转染”)，DEAE-葡聚糖介导的转染，电穿孔或磷酸钙沉淀法。这些方法例如在上文的Brent等中有详细描述。其中，脂质体转染因操作简单且不需要特殊仪器设备而被广泛接受。例如，可以使用Lipofectamine(生命技术)或LipoTAXI(Stratagene)试剂盒转染。提供脂质体转染试剂和方法的其他公司包括Bio-Rad实验室，CLONTECH，Glen Research，Life Technologies，JBL Scientific，MBI Fermentas，PanVera，Promega，Quantum Biotechnologies，Sigma-Aldrich和Wako Chemicals USA。
为了长期高产量地生产重组蛋白，稳定表达是优选的。代替使用包含病毒复制起点的表达载体，可以用由适当的表达控制元件(例如启动子，增强子，序列，转录终止子，聚腺苷酸化位点等)控制的蛋白编码序列和可选的标记转化宿主细胞。重组载体中的选择标记对选择具有抗性，并使细胞将载体稳定整合到其染色体中。常用的选择标 记包括：对氨基糖苷G-418具有抗性的新霉素(neo)，以及对潮霉素具有抗性的潮霉素(hygro)。
在一些实施方案中，重组表达载体包括至少一个启动子元件，蛋白编码序列，转录终止信号和polyA尾巴。其他元件包括增强子，Kozak序列及插入序列两侧RNA剪接的供体和受体位点。可以通过SV40的前期和后期启动子，来自逆转录病毒的长末端重复序列如RSV、HTLV1、HIVI及巨细胞病毒的早期启动子来获得高效的转录，也可应用其它一些细胞的启动子如肌动蛋白启动子。合适的表达载体可包括pIRES1neo，pRetro-Off，pRetro-On，PLXSN，Plncx，pcDNA3.1(+/-)，pcDNA/Zeo(+/-)，pcDNA3.1/Hygro(+/-)，PSVL，PMSG，pRSVcat，pSV2dhfr，pBC12MI和pCS2等。常使用的哺乳动物细胞包括HEK293细胞，Cos1细胞，Cos7细胞，CV1细胞，鼠L细胞和CHO细胞等。
在一些实施方案中，插入基因片段需含有筛选标记，常见的筛选标记包括二氢叶酸还原酶，谷氨酰胺合成酶，新霉素抗性，潮霉素抗性等筛选基因，以便于转染成功的细胞的筛选分离。将构建好的质粒转染到无上述基因的宿主细胞，经过选择性培养基培养，转染成功的细胞大量生长，产生想要获得的目的蛋白。
此外，可以使用本领域技术人员已知的标准技术在编码本发明所述的核苷酸序列中引入突变，包括但不限于导致氨基酸取代的定点突变和PCR介导的突变。变体(包括衍生物)编码相对于原蛋白来说少于50个氨基酸的取代、少于40个氨基酸的替换、少于30个氨基酸的取代、少于25个氨基酸的取代、少于20个氨基酸的取代、少于15个氨基酸的取代、少于10个氨基酸的取代、少于5个氨基酸的取代、少于4个氨基酸的取代、少于3个氨基酸的取代或少于2个氨基酸的取代。或者可以沿着全部或部分编码序列时随机引入突变，例如通过饱和突变，以及可以筛选所得突变体的生物活性以鉴定保留活性的突变体。
在一些实施方案中，本文所述取代为保守氨基酸取代。
药物组合物和治疗方法
本发明还提供了药物组合物和相关的治疗方法。所述药物组合物包含有效剂量的融合蛋白或Spike蛋白纳米颗粒以及药学上可接受的载体。
术语“药学上可接受的”是指由政府的监管机构批准的或其他公认的药典中列出的用于动物(特别是用于人类)的物质。此外，“药学上可接受的载体”通常是指任何类型的无毒固体、半固体或液体填充剂、稀释剂、包封材料或制剂助剂等。
术语“载体”是指可以与活性成分一起施用于患者的稀释剂、佐剂、赋形剂或载体。此类载体可以是无菌液体，如水和油，包括石油、动植物或合成来源的油，如花生油、 大豆油、矿物油、芝麻油等。当药物组合物静脉内给药时，水是优选的载体。盐水溶液和葡萄糖水溶液和甘油溶液也可用作液体载体，特别是用于注射溶液。合适的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙烯、乙二醇、水、乙醇等。如有需要，药物组合物还可以含有少量的润湿剂、乳化剂，或pH缓冲剂如乙酸盐、柠檬酸盐或磷酸盐。抗菌剂如苯甲醇或对羟基苯甲酸甲酯、抗氧化剂如抗坏血酸或亚硫酸氢钠、螯合剂如乙二胺四乙酸，以及调节张力的试剂如氯化钠或右旋葡萄糖也是可以预见的。这些药物组合物可以采取溶液、悬液、乳剂、片剂、丸剂、胶囊、散剂、缓释制剂等形式。该药物组合物可以用传统的粘合剂和载体如甘油三酯配制成栓剂。口服制剂可以包括标准载体，例如药物等级的甘露糖醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。合适的药物载体的实例在E.W.Martin的Remington's Pharmaceutical Sciences中有描述，在此通过引用并入本发明。此类组合物将含有临床有效剂量的融合蛋白或Spike蛋白纳米颗粒，优选以纯化后的形式，连同合适数量的载体，以提供适合于患者的给药形式。该制剂应该适用于给药模式。制剂可以封装在安瓿瓶、一次性注射器或由玻璃或塑料制成的多剂量小瓶中。
在一些实施方案中，药物组合物可包含融合蛋白或Spike蛋白纳米颗粒，以及编码本文所述的融合蛋白的多核苷酸或载体。在一些实施方案中，病毒(例如SARS-CoV-2)Spike蛋白胞外结构域或其截短片段三聚体可以用于预防和治疗相应的病毒感染。在一些实施方案中，包含本文所述的纳米颗粒疫苗可用于预防或治疗相应疾病，例如各种冠状病毒引起的感染。本发明的一些实施方案涉及SARS-CoV-2抗原或疫苗在预防或治疗人类受试者的SARS-CoV-2感染中的用途。本发明的一些实施方案涉及SARS-CoV抗原或疫苗在预防或治疗SARS-CoV感染中的用途。
在本发明的一些治疗方法的实践中，对需要预防或治疗疾病(例如SARS-CoV-2感染)的受试者施用相应的Spike蛋白纳米颗粒或融合蛋白，或本文所述的编码融合蛋白的多核苷酸。通常，本文公开的Spike蛋白纳米颗粒、融合蛋白或编码融合蛋白的多核苷酸包含在药物组合物中。药物组合物可以是治疗制剂或预防制剂。通常，该药物组合物可以另外包含一种或多种药学上可接受的载体，以及任选地其他治疗成分(例如抗病毒药)。药物组合物中也可以使用各种药学上可接受的添加剂。
本发明的一些药物组合物是疫苗组合物。对于疫苗组合物，可以另外包括合适的佐剂。合适的佐剂包括例如氢氧化铝、卵磷脂、弗氏佐剂、MF59、SEPIVAC SWE TM、MPL和IL-12。在一些实施方案中，本文所述的疫苗组合物(例如SARS-CoV-2疫苗)可以配制为控释或定时释放制剂。这可以在包含缓释聚合物的组合物中或通过微囊递送系统或生物粘附凝胶来实现。各种药物组合物可以根据本领域众所周知的标准程序来制备。参见，例如美国专利US4,652,441和US4,917,893；美国专利US4,677,191 和US4,728,721；以及美国专利US4,675,189。
本发明的药物组合物可以用于多种治疗或预防应用中，例如用于治疗受试者体内的SARS-CoV-2感染或用于引起对受试者体内的SARS-CoV-2的免疫响应。作为示例，可以将纳米颗粒疫苗给药受试者以诱导对SARS-CoV-2的免疫响应，例如，诱导产生针对病毒的广谱中和抗体。对于有风险感染SARS-CoV-2的受试者而言，可以施用本发明的疫苗组合物以提供针对病毒感染的预防性保护。可以类似地进行衍生自本文所述的其他抗原的疫苗的治疗性和预防性应用。取决于具体的受试者和疾病，本发明的药物组合物可以通过本领域普通技术人员已知的多种给药方式给予受试者，例如，通过肌内途径、皮下途径、静脉内途径、动脉内途径、关节途径、腹膜内途径等肠胃外途径。在一些实施方案中，本发明的治疗方法涉及阻断冠状病毒(例如SARS-CoV或SARS-CoV-2)进入宿主细胞(例如人宿主细胞)的方法，预防冠状病毒Spike蛋白与宿主受体结合的方法，以及治疗与冠状病毒感染有关的急性呼吸道疾病的方法。在一些实施方案中，本文所述的治疗方法和药物组合物可以与用于治疗或预防冠状病毒感染的其他已知治疗剂和/或方式结合使用。已知的治疗剂和/或方式包括，例如，核酸酶类似物或蛋白酶抑制剂(例如，瑞德西韦)，针对一种或多种冠状病毒的单克隆抗体，免疫抑制剂或抗炎药(例如，sarilumab或Tocilizumab)，ACE抑制剂，血管扩张剂或其任意组合。
对于治疗应用，药物组合物应包含治疗有效量的本文所述的融合蛋白、Spike蛋白纳米颗粒。对于预防应用，药物组合物应包含预防有效量的本文所述的融合蛋白、Spike蛋白纳米颗粒。可以基于要治疗或预防的特定疾病或病症、受试者的严重程度、年龄以及特定受试者的其他个人属性(例如，受试者健康状况的总体状况)来确定适当的抗原量。有效剂量的确定还由动物模型研究指导，随后由人类临床试验指导，并由可显著减少受试者的目标疾病病症或症状的发生或严重程度的给药方案指导。
对于预防性应用，在任何症状之前，例如在感染之前，提供药物组合物。药物组合物的预防性给药用于预防或改善任何随后的感染。因此，在一些实施方案中，待治疗的受试者是例如由于暴露或可能暴露于病毒(例如SARS-CoV-2)而已经感染(例如，SARS-CoV-2感染)的受试者或处于感染(例如，SARS-CoV-2感染)风险中的受试者。在给予治疗有效量的所公开的药物组合物之后，可以监测受试者的感染(例如SARS-CoV-2感染)、与感染(例如SARS-CoV-2感染)相关的症状。
对于治疗应用，在疾病或感染的症状发作时或之后，例如在感染(例如SARS-CoV-2感染)症状发生后或诊断感染后，提供药物组合物。因此，可以在预期暴露于病毒之前提供药物组合物，以便在暴露于或怀疑暴露于病毒之后或在实际感染初期之后，减弱感染和/或相关疾病病症的预期严重性、持续时间或程度。本发明的药物组合物可以与 本领域已知的用于治疗或预防相关病原体的感染(例如SARS-CoV-2感染)的其他试剂组合。
包含本发明所述融合蛋白、Spike蛋白纳米颗粒的疫苗组合物(例如SARS-CoV-2疫苗)或药物组合物可以提供作为试剂盒的组分。任选地，这种试剂盒包括另外的组成，该另外的组成包括包装、使用说明书和各种其他试剂，例如为缓冲液、底物、抗体或配体(例如对照抗体或配体)以及检测试剂。
各种已知输送系统可用于施用本发明融合蛋白、Spike蛋白纳米颗粒或衍生物或其编码多核苷酸或表达载体，例如包封于脂质体、微粒、微胶囊、能够表达所述融合蛋白或Spike蛋白纳米颗粒的重组细胞、受体介导的内吞作用(参见例如Wu andWu,1987,J.Biol.Chem.262:4429-4432)、作为逆转录病毒或其它载体的一部分的核酸的构建等。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：融合蛋白的制备
根据本文所述融合蛋白的序列，可以通过以下方法或其他已知方法制备。将编码融合蛋白(如SEQ ID NO:16-30、39-77所示)的DNA序列克隆至表达载体，然后电转CHO-K1细胞，培养并纯化获得融合蛋白。
使用冷冻电镜(Cryo-EM)对融合蛋白D和融合蛋白G的三维结构进行解析，在单体铁蛋白亚基的N端连接SARS-CoV-2 Spike蛋白胞外结构域或其截短片段没有干扰铁蛋白的自组装，纳米颗粒形成良好，表面显示出刺突。
实施例2：融合蛋白与hACE2蛋白结合能力试验
1.1融合蛋白与hACE2蛋白结合能力试验(ELISA)
本试验通过ELISA检测融合蛋白D和融合蛋白G与人ACE2蛋白(hACE2)的结合能力，从而评价本发明的Spike蛋白-铁蛋白融合蛋白能否良好展示Spike蛋白关键的抗原表位。方法简述如下：向96孔酶标板(Costar，货号：9018)每个反应孔中加100μL 2μg/mL抗原(WT-Spike-His、Delta-Spike-His、Omicron-Spike-His、融合蛋白D或融合蛋白G)溶液，4℃包被过夜；用PBST(含0.05％Tween-20的PBS缓冲液)洗涤2次；向每个反应孔中加入封闭液(含3％BSA的PBST)置37℃培养箱孵育2h；封闭后用PBST洗涤3次；加入梯度稀释的humanACE2-his-biotin(义翘神州， 货号：10108-H27B-B)，起始浓度为2.5μg/mL，3倍梯度稀释，共10个系列稀释浓度，每孔100μL，置于37℃培养箱温育1.5h；用PBST洗涤5次；以100μL/孔向反应孔中加入链酶亲和素标记的过氧化氢酶(Jackson Immuno Research，货号：016-030-084；1:10000)，于37℃温育1h；用PBST洗涤8次；以100μL/孔向反应孔中加入TMB溶液，37℃避光孵育5～15min；向每个反应孔中加终止液(0.1M H 2SO 4)50μL终止酶促显色反应；设定检测波长为450nm进行读数。
其中，WT-Spike-His是在突变的野生型SARS-CoV-2 Spike蛋白胞外结构域C端截短片段b1(如SEQ ID NO:6所示)的C-末端添加6×His(HHHHHH)构建而成。Delta-Spike-His是在突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域C端截短片段d1(如SEQ ID NO:11所示)的C-末端添加6×His(HHHHHH)构建而成。Omicron-Spike-His是在突变的SARS-CoV-2 Omicron变异株Spike蛋白胞外结构域C端截短片段g1(如SEQ ID NO:34所示)的C-末端添加6×His(HHHHHH)构建而成。
结果如图1所示，hACE2与融合蛋白D及Delta、野生型的Spike蛋白结合具有类似亲和力，EC 50值分别为9.2、8.1、5.7ng/mL(图1a)；hACE2与融合蛋白G及Omicron的Spike蛋白结合具有类似的亲和力，EC 50值分别为9.3、8.2ng/mL(图1b)。
1.2融合蛋白与hACE2蛋白结合能力试验(BLI)
采用生物膜干涉技术(BLI)对融合蛋白D和融合蛋白G与hACE2结合的亲和力常数测定，仪器为PALL公司的Fortebio Octet RED&QK系统。多通道平行定量分析WT-Spike-His(同实施例2步骤1.1)、Delta-Spike-His(同实施例2步骤1.1)、Omicron-Spike-His(同实施例2步骤1.1)、融合蛋白D、融合蛋白G，浓度梯度设定为：50、100、200和400nM，hACE2-Biotin(Acro biosystems，货号AC2-H5257)偶联SABiosensors传感器(Octet，货号2107002811)。
结果如表1所示，结果表明融合蛋白D、融合蛋白G与hACE2结合的亲和力要显著强于WT-Spike-His、Delta-Spike-His和Omicron-Spike-His与hACE2的亲和力。表明本发明的Spike蛋白-铁蛋白融合蛋白可以很好地展示Spike蛋白的关键抗原表位。
表1 SARS-CoV-2 Spike蛋白与hACE2受体结合动力学
抗原 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
融合蛋白D 5.49E-10 4.02E+04 2.21E-05
融合蛋白G <1.0E-12 4.67E+04 <1.0E-07
实施例3：疫苗在小鼠体内的免疫原性(皮下注射)
1.1小鼠免疫
为了研究单价疫苗(融合蛋白D和融合蛋白G)的免疫原性，在小鼠体内进行了免疫原性研究。将融合蛋白D、融合蛋白G分别与免疫佐剂混合乳化，分别于第0天和第21天通过皮下注射免疫Balb/c小鼠(6-8周龄)，每只小鼠SEPIVAC SWE TM佐剂(SEPPIC S.A.，货号80748J，批号210721010001)体积固定为50μl，每次给药总体积为100μl/只，分组给药方案见表2。于免疫后第35天眼眶取血，静置待血清析出，离心获得小鼠血清，用于ELISA检测血清抗[野生型、Delta和Omicron(BA.1)]IgG滴度和SARS-CoV-2 Spike假病毒中和抗体实验。
表2分组给药方案
组别 药物 剂量(μg/只) 免疫方式
1 融合蛋白D 5 s.c.
2 融合蛋白G 5 s.c.
1.2采用ELISA法检测血清抗Spike蛋白IgG滴度
将WT-Spike-His(同实施例2步骤1.1)、Delta-Spike-His(同实施例2步骤1.1)、Omicron-Spike-His(同实施例2步骤1.1)抗原分别稀释至2μg/mL，按照每孔100μL加入96孔板(Corning，9018)中，4℃包被过夜；将96孔板在清洗缓冲液PBST(含0.05％Tween-20的PBS缓冲液)中清洗3次，加入封闭溶液(含3％BSA的清洗缓冲液PBST)，37℃孵育2h；将96孔板用清洗缓冲液PBST涤洗3次，每孔添加梯度稀释的实施例3步骤1.1获得的小鼠血清(将血清稀释1000倍作为起始浓度，然后3倍梯度稀释)100μL，37℃孵育1.5h后；将96孔板用清洗缓冲液PBST洗涤5次，加入100μL的1:10000稀释后的Peroxidase-AffiniPure Goat Anti-Mouse IgG(Jackson，货号：115-035-003)，37℃孵育1h；将96孔板用清洗缓冲液PBST洗涤8次，再添加100μL TMB(Biopanda，货号TMB-S-001)底物进行显色；显色10-15min后，加入50μL 0.1M H 2SO 4终止反应，在450nm波长下测量吸光度值(OD值)，对所得读数OD值以非线性四参数方程曲线模型进行拟合，以每个96孔板中的阴性孔读数平均值的2.0倍作为终点值，从而确定每个血清样品的滴度。
结果如图2所示，结果表明融合蛋白D和融合蛋白G都能诱导出强大的针对野生型、Delta和Omicron的抗Spike蛋白IgG抗体滴度，都能引起相关的抗原特异性抗体反应。
1.3 SARS-CoV-2 Spike假病毒中和抗体实验
将实施例3步骤1.1获得的小鼠血清用含有10％FBS的DMEM培养基进行梯度稀释，按照每孔50μL转移至96孔板中，待用；将不同的SARS-CoV-2 Spike假病毒分别用含有10％FBS的DMEM培养基进行稀释，将稀释后的SARS-CoV-2 Spike假病毒按照每孔25μL转移至上述含有小鼠血清的96孔板中，混匀后于37℃孵育1小时；将ACE2-293细胞用0.25％Trypsin-EDTA(Gibco，25200-072)消化后计数，将细胞密度调至4×10 5cells/mL，按照每孔50μL将细胞加入到上述96孔板中，在37℃、5％CO 2培养箱中孵育48h；每孔加入50μL Bio-Lite Luciferase Assay System(诺维赞，DD1201-03)检测试剂，静置3分钟后进行读数，并根据读数计算小鼠血清的中和抑制率并计算滴度：中和抑制率＝[1-(样品组-空白对照组)/(阴性对照组-空白对照组)]×100％；其中，样品组添加SARS-CoV-2 Spike假病毒和小鼠血清，阴性对照组添加SARS-CoV-2 Spike假病毒不添加小鼠血清，空白对照组不添加SARS-CoV-2 Spike假病毒和不添加小鼠血清。
ACE2-293细胞的构建方法为：将HEK293细胞用含10％FBS的DMEM完全培养基培养，采用lipofectamine 2000 transfection reagent(Thermo Fisher，11668019)进行ACE2表达质粒(义翘神州，HG10108-M)的转染，之后通过潮霉素(200μg/ml)的加压筛选和流式分选(采用10μg/ml anti-ACE2和PE偶联的Anti-Human IgG-Fc)，细胞继续扩增挑选出PE阳性率>90％的单克隆进行下一步扩增，筛选出表达ACE2的HEK293细胞，即ACE2-293细胞。
其中，SARS-CoV-2 Spike假病毒为：SARS-CoV-2 Spike假病毒(吉满生物，GM-0220PV07)；SARS-COV-2 Spike(B.1.617.2)假病毒(吉满生物，GM-0220PV45)；SARS-COV-2 Spike(B.1.1.529)假病毒(吉满生物，GM-0220PV84)。
结果见图3，结果显示融合蛋白D和融合蛋白G对SARS-CoV-2 Spike假病毒、SARS-COV-2 Spike(B.1.617.2)假病毒、SARS-COV-2 Spike(B.1.1.529)假病毒都有抑制作用，融合蛋白G对SARS-COV-2 Spike(B.1.1.529)假病毒的抑制效果较好。
实施例4：疫苗在小鼠体内的免疫原性(肌肉注射)
为了评估单价疫苗(融合蛋白D和融合蛋白G)的免疫原性，利用BALB/c小鼠探索了不同抗原剂量对免疫原性的影响。
1.1小鼠免疫
Balb/c小鼠(每组n＝10)分别在第0天和第21天以肌肉注射方式注射不同剂量的疫苗，对照小鼠仅给予SEPIVAC SWE TM佐剂，每只小鼠SEPIVAC SWE TM佐剂(SEPPIC S.A.，货号80748J，批号210721010001)体积固定为50μL，每次给药总体积为100μL/只，分组给药方案见表3。于免疫后第14天和第35天采血，用于ELISA检测血清抗[野生型、Delta和Omicron(BA.1)]IgG滴度和SARS-CoV-2 Spike假病毒中 和抗体实验。
表3分组给药方案
组别 药物 剂量(μg/只) 免疫方式
1 融合蛋白D 0.2 i.m.
2 融合蛋白D 1 i.m.
3 融合蛋白D 5 i.m.
4 融合蛋白G 0.2 i.m.
5 融合蛋白G 1 i.m.
6 融合蛋白G 5 i.m.
1.2采用ELISA法检测血清抗Spike蛋白IgG滴度
将WT-Spike-His(同实施例2步骤1.1)、Delta-Spike-His(同实施例2步骤1.1)、Omicron-Spike-His(同实施例2步骤1.1)分别用PBS稀释到1μg/mL，以100μL/孔加入到96孔酶标板(Costar，货号：9018)中，4℃过夜；用PBST(在1×PBS中加入0.05％体积的Tween-20)洗涤2次；加入封闭液(含3％BSA的PBST)37℃孵育2小时；用PBST洗涤3次；加入梯度稀释的实施例4步骤1.1获得的小鼠血清(将第14天血清稀释100倍作为起始浓度、第35天血清稀释1000倍作为起始浓度，然后3倍梯度稀释11个梯度)，每孔100μL，37℃孵育1.5小时；用PBST洗涤5次；加入1:10000稀释后的Peroxidase-AffiniPure Goat Anti-Mouse IgG(Jackson，货号：115-035-003)，100μL/孔，37℃孵育1小时；PBST洗涤8次；加入100μL/孔TMB溶液37℃孵育15～25分钟；用50μL/孔的0.1M硫酸终止反应；设定检测波长为450nm进行读数，对所得读数OD值以非线性四参数方程曲线模型进行拟合，以每个96孔板中的阴性孔读数平均值的2.0倍作为终点值，从而确定每个血清样品的滴度。
结果见图4a至图4f。在初次免疫后的第14天，在所有测试的剂量组中，仅给予佐剂的小鼠没有检测到抗Spike蛋白IgG滴度。其它组所有的小鼠抗Spike蛋白IgG滴度都有升高，在第二次增强免疫后所有小鼠的抗IgG滴度均进一步明显升高。此外，在单次免疫后，抗Spike蛋白IgG滴度在剂量范围内呈剂量依赖性增加关系，但在第二次增强免疫后，1μg和5μg抗原组之间无显著差异。此外，与接受相同剂量的融合蛋白D的小鼠相比，接受融合蛋白G的小鼠具有较低的针对野生型和Delta抗Spike蛋白抗体滴度，但融合蛋白G诱导的针对Omicron抗Spike蛋白抗体滴度则显著高于融合蛋白D。融合蛋白D诱导了强大的针对野生型和Delta的抗Spike蛋白IgG抗体滴度，但针对Omicron的抗体滴度显著下降。
这些研究表明，两种试验疫苗都引起了剂量相关的抗原特异性抗体反应。
1.3 SARS-CoV-2 Spike假病毒中和抗体实验
用含10％FBS的DMEM培养基将实施例4步骤1.1第35天采集的小鼠血清进行梯度稀释，50μL/孔加入96孔细胞培养白板中；然后将SARS-CoV-2假病毒用含10％FBS的DMEM培养基稀释，于加有小鼠血清稀释液的96孔板中加入假病毒稀释液，25μL/孔。将96孔板置于培养箱中37℃孵育1h。取出孵育1h后的96孔板，以50μL/孔加入ACE2-293细胞悬液(2×10 4cells/孔)，将96孔板置于培养箱培养。培养48h后取出96孔板，待其恢复至室温，每孔加入50μLBio-Lite TM Luciferase Assay System(Vazyme，货号DD1201)溶液，室温避光反应2min后，用酶标仪Luminescence检测模块读取发光信号。
其中，SARS-CoV-2 Spike假病毒为：SARS-CoV-2 Spike假病毒(吉满生物，GM-0220PV07)；SARS-COV-2 Spike(B.1.617.2)假病毒(吉满生物，GM-0220PV45)；SARS-COV-2 Spike(B.1.1.529)假病毒(吉满生物，GM-0220PV84)。
结果如图5所示，结果显示融合蛋白D和融合蛋白G对SARS-CoV-2 Spike假病毒、SARS-COV-2 Spike(B.1.617.2)假病毒、SARS-COV-2 Spike(B.1.1.529)假病毒都有抑制作用，融合蛋白G对SARS-COV-2 Spike(B.1.1.529)假病毒的抑制效果较好。

Claims (39)

  1. 一种含突变的冠状病毒Spike蛋白胞外结构域或其截短片段，其特征在于，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在防止融合过程中形成直螺旋的突变。
  2. 如权利要求1所述的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段，其特征在于，所述突变包含：1)将RRAR突变为GSAS；2)在HR1和CH之间的转向区域存在双重突变K986P/V987P。
  3. 如权利要求1或2所述的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段，其特征在于，所述含突变的冠状病毒Spike蛋白胞外结构域的截短片段，其与冠状病毒Spike蛋白全长胞外结构域相比，C端截短了5-80个氨基酸残基；或者，C端截短了20-76个氨基酸残基；或者，C端截短了70个氨基酸残基。
  4. 如权利要求1-3任一项所述的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段，其特征在于，所述冠状病毒为SARS-CoV-2、SARS-CoV或MERS-Cov；或者，所述冠状病毒为野生型SARS-CoV-2或其变异株；或者，所述冠状病毒为野生型SARS-CoV-2、SARS-CoV-2 Alpha变异株、SARS-CoV-2 Beta变异株、SARS-CoV-2 Gamma变异株、SARS-CoV-2 Delta变异株、SARS-CoV-2 Kappa变异株、SARS-CoV-2 Epsilon变异株、SARS-CoV-2 Lambda变异株或SARS-CoV-2 Omicron变异株。
  5. 如权利要求1-4任一项所述的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段，其特征在于，所述含突变的冠状病毒Spike蛋白胞外结构域或其截短片段包含如SEQ ID NO:3-4、6-7、9-12、32-35、78-83任一项所示的氨基酸序列，或与SEQ ID NO:3-4、6-7、9-12、32-35、78-83任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:3-4、6-7、9-12、32-35、78-83任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  6. 一种融合蛋白，其特征在于，包含通过接头连接的如权利要求1-5任一项所述的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段和单体亚基蛋白；或者，所述单体亚基蛋白为自组装的单体亚基蛋白；或者，所述单体亚基蛋白为单体铁蛋白亚基。
  7. 如权利要求6所述的融合蛋白，其特征在于，所述融合蛋白是将含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的C端通过接头与单体亚基蛋白的N端连接； 或者，所述融合蛋白是将含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的C端通过接头与单体铁蛋白亚基的N端连接。
  8. 一种融合蛋白，其特征在于，包含通过接头连接的冠状病毒Spike蛋白S1亚基和单体亚基蛋白；或者，所述单体亚基蛋白为自组装的单体亚基蛋白；或者，所述单体亚基蛋白为单体铁蛋白亚基。
  9. 如权利要求8所述的融合蛋白，其特征在于，所述融合蛋白是将冠状病毒Spike蛋白S1亚基的C端通过接头与单体亚基蛋白的N端连接。
  10. 一种融合蛋白，其特征在于，包含通过接头连接的冠状病毒Spike蛋白保守片段和单体亚基蛋白；或者，所述单体亚基蛋白为自组装的单体亚基蛋白；或者，所述单体亚基蛋白为单体铁蛋白亚基。
  11. 如权利要求10所述的融合蛋白，其特征在于，所述融合蛋白是将冠状病毒Spike蛋白保守片段的C端通过接头与单体亚基蛋白的N端连接。
  12. 如权利要求6-11任一项所述的融合蛋白，其特征在于，所述接头为GS接头。
  13. 如权利要求6-12任一项所述的融合蛋白，其特征在于，所述接头选自GS，GGS，GGGS，GGGGS，SGGGS，GGGG，GGSS，(GGGGS) 2，(GGGGS) 3，或其任意组合。
  14. 如权利要求6-12任一项所述的融合蛋白，其特征在于，所述接头为(G mS) n，其中，每个m独立为1、2、3、4或5，n为1、2、3、4或5。
  15. 如权利要求6-14任一项所述的融合蛋白，其特征在于，所述单体铁蛋白亚基选自细菌铁蛋白、植物铁蛋白、藻铁蛋白、昆虫铁蛋白、真菌铁蛋白或哺乳动物铁蛋白；或者，所述单体铁蛋白亚基是幽门螺杆菌非血红素单体铁蛋白亚基。
  16. 如权利要求6-15任一项所述的融合蛋白，其特征在于，所述单体铁蛋白亚基包含如SEQ ID NO:14所示的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:14所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  17. 一种融合蛋白，其特征在于，包含如权利要求1-5任一项所述的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段和与其连接的免疫球蛋白的Fc片段。
  18. 如权利要求17所述的融合蛋白，其特征在于，所述含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的C端与免疫球蛋白的Fc片段的N端连接。
  19. 一种融合蛋白，其特征在于，包含冠状病毒Spike蛋白S1亚基和与其连接的免疫球蛋白的Fc片段。
  20. 如权利要求19所述的融合蛋白，其特征在于，所述融合蛋白是将冠状病毒Spike蛋白S1亚基的C端与免疫球蛋白的Fc片段的N端连接。
  21. 一种融合蛋白，其特征在于，包含冠状病毒Spike蛋白保守片段和与其连接的免疫球蛋白的Fc片段。
  22. 如权利要求21所述的融合蛋白，其特征在于，所述融合蛋白是将冠状病毒Spike蛋白保守片段的C端与免疫球蛋白的Fc片段的N端连接。
  23. 如权利要求17-22任一项所述的融合蛋白，其特征在于，所述免疫球蛋白的Fc片段来自IgG、IgM、IgA、IgE或IgD；或者，所述免疫球蛋白的Fc片段来自IgG1、IgG2、IgG3或IgG4；或者，所述免疫球蛋白的Fc片段为IgG1的Fc片段；或者，所述免疫球蛋白的Fc片段为人IgG1的Fc片段。
  24. 如权利要求17-23任一项所述的融合蛋白，其特征在于，所述免疫球蛋白的Fc片段包含如SEQ ID NO:38所示的氨基酸序列，或与SEQ ID NO:38所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:38所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  25. 如权利要求6-24任一项所述的融合蛋白，其特征在于，所述融合蛋白还包含N端信号肽。
  26. 如权利要求25所述的融合蛋白，其特征在于，所述N端信号肽选自CSP，mschito，MF-α，pho1，HBM，t-pA，以及IL-3的信号肽。
  27. 如权利要求25所述的融合蛋白，其特征在于，所述N端信号肽包含如SEQ ID NO:2或5所示的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:2或5所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  28. 如权利要求6-27任一项所述的融合蛋白，其特征在于，所述冠状病毒为 SARS-CoV-2、SARS-CoV或MERS-Cov；或者，所述冠状病毒为野生型SARS-CoV-2或其变异株；或者，所述冠状病毒为野生型SARS-CoV-2、SARS-CoV-2 Alpha变异株、SARS-CoV-2 Beta变异株、SARS-CoV-2 Gamma变异株、SARS-CoV-2 Delta变异株、SARS-CoV-2 Kappa变异株、SARS-CoV-2 Epsilon变异株、SARS-CoV-2 Lambda变异株或SARS-CoV-2 Omicron变异株。
  29. 如权利要求6-7、12-16、25-28任一项所述的融合蛋白，其特征在于，所述融合蛋白包含如SEQ ID NO:16-23、26-29、41-44、66-67任一项所示的氨基酸序列，或与SEQ ID NO:16-23、26-29、41-44、66-67任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:16-23、26-29、41-44、66-67任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  30. 如权利要求8-9、12-16、25-28任一项所述的融合蛋白，其特征在于，所述融合蛋白包含如SEQ ID NO:24-25、30、39-40、65任一项所示的氨基酸序列，或与SEQ ID NO:24-25、30、39-40、65任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:24-25、30、39-40、65任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  31. 如权利要求10-16、25-28任一项所述的融合蛋白，其特征在于，所述融合蛋白包含如SEQ ID NO:45-46、68任一项所示的氨基酸序列，或与SEQ ID NO:45-46、68任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:45-46、68任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  32. 如权利要求17-18、23-28任一项所述的融合蛋白，其特征在于，所述融合蛋白包含如SEQ ID NO:47-54、59-62、69-72、75-76任一项所示的氨基酸序列，或与SEQ ID NO:47-54、59-62、69-72、75-76任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:47-54、59-62、69-72、75-76任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  33. 如权利要求19-20、23-28任一项所述的融合蛋白，其特征在于，所述融合蛋白包含如SEQ ID NO:55-58、73-74任一项所示的氨基酸序列，或与SEQ ID NO:55-58、73-74任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:55-58、73-74任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  34. 如权利要求21-28任一项所述的融合蛋白，其特征在于，所述融合蛋白包含如SEQ ID NO:63-64、77任一项所示的氨基酸序列，或与SEQ ID NO:63-64、77任一项所示的氨基酸序列相比具有至少80％或至少90％同一性的氨基酸序列，或与SEQ ID NO:63-64、77任一项所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  35. 一种生物材料，为
    (1)一种多聚核苷酸，其特征在于，所述多聚核苷酸编码如权利要求1-5任一项所述的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段或如权利要求6-34任一项所述的融合蛋白；或，
    (2)一种表达载体，其特征在于，所述表达载体包含编码如权利要求1-5任一项所述的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段或如权利要求6-34任一项所述的融合蛋白的多聚核苷酸；或，
    (3)一种细胞，其特征在于，所述细胞包含编码如权利要求1-5任一项所述的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段、如权利要求6-34任一项所述的融合蛋白的多聚核苷酸或包含编码如权利要求1-5任一项所述的含突变的冠状病毒Spike蛋白胞外结构域或其截短片段、如权利要求6-34任一项所述的融合蛋白的多聚核苷酸的表达载体。
  36. 一种包含权利要求6-16、25-31任一项所述的融合蛋白的Spike蛋白纳米颗粒。
  37. 一种冠状病毒疫苗，其特征在于，所述冠状病毒疫苗包含如权利要求6-34任一项所述的融合蛋白和/或如权利要求36所述的Spike蛋白纳米颗粒；或者，还包括药学上可接受的载体和/或佐剂。
  38. 权利要求6-34任一项所述的融合蛋白或权利要求36所述的Spike蛋白纳米颗粒在制备预防或治疗冠状病毒感染的疫苗中的应用；或者，所述冠状病毒感染为SARS-CoV-2、SARS-CoV或MERS-Cov感染；或者，所述冠状病毒感染为野生型SARS-CoV-2或其变异株感染；或者，所述冠状病毒感染为野生型SARS-CoV-2、SARS-CoV-2 Alpha变异株、SARS-CoV-2 Beta变异株、SARS-CoV-2 Gamma变异株、SARS-CoV-2 Delta变异株、SARS-CoV-2 Kappa变异株、SARS-CoV-2 Epsilon变异株、SARS-CoV-2 Lambda变异株或SARS-CoV-2 Omicron变异株感染。
  39. 一种预防或治疗冠状病毒感染的方法，其特征在于，包括向有需要的患者施用有效量的权利要求6-34任一项所述的融合蛋白或权利要求36所述的Spike蛋白纳米颗粒或权利要求37所述的冠状病毒疫苗；或者，所述冠状病毒感染为SARS-CoV-2、SARS-CoV或MERS-Cov感染；或者，所述冠状病毒感染为野生型SARS-CoV-2或其变异株感染；或者，所述冠状病毒感染为野生型SARS-CoV-2、SARS-CoV-2 Alpha变异株、SARS-CoV-2 Beta变异株、SARS-CoV-2 Gamma变异株、SARS-CoV-2 Delta变异株、SARS-CoV-2 Kappa变异株、SARS-CoV-2 Epsilon变异株、SARS-CoV-2 Lambda变异株或SARS-CoV-2 Omicron变异株感染。
PCT/CN2022/113747 2021-08-20 2022-08-19 用于预防或治疗冠状病毒感染的融合蛋白、Spike蛋白纳米颗粒及其应用 WO2023020623A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202110961117.6 2021-08-20
CN202110961117 2021-08-20
CN202210253722.2 2022-03-15
CN202210253722 2022-03-15

Publications (1)

Publication Number Publication Date
WO2023020623A1 true WO2023020623A1 (zh) 2023-02-23

Family

ID=85239469

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/113747 WO2023020623A1 (zh) 2021-08-20 2022-08-19 用于预防或治疗冠状病毒感染的融合蛋白、Spike蛋白纳米颗粒及其应用

Country Status (2)

Country Link
CN (1) CN115785232A (zh)
WO (1) WO2023020623A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024061188A1 (zh) * 2022-09-19 2024-03-28 百奥泰生物制药股份有限公司 一种冠状病毒多价疫苗及其应用
CN117586425A (zh) * 2024-01-19 2024-02-23 北京安百胜生物科技有限公司 一种重组呼吸道合胞病毒颗粒抗原其制备方法和应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111533809A (zh) * 2020-04-21 2020-08-14 中国科学院武汉病毒研究所 针对新型冠状病毒的亚单位疫苗及应用
CN111560074A (zh) * 2020-03-20 2020-08-21 中山大学 一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒s蛋白单区域亚单位纳米疫苗
CN111607002A (zh) * 2020-02-24 2020-09-01 中山大学 一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗
CN112358533A (zh) * 2020-10-30 2021-02-12 上海泽润生物科技有限公司 重组刺突蛋白及其制备方法和用途
CN112538105A (zh) * 2020-06-29 2021-03-23 斯克里普斯研究院 稳定的冠状病毒刺突(s)蛋白抗原和相关疫苗
CN113185613A (zh) * 2021-04-13 2021-07-30 武汉大学 新型冠状病毒s蛋白及其亚单位疫苗
CN113234170A (zh) * 2021-04-13 2021-08-10 武汉大学 新型冠状病毒突变株s蛋白及其亚单位疫苗

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111607002A (zh) * 2020-02-24 2020-09-01 中山大学 一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗
CN111560074A (zh) * 2020-03-20 2020-08-21 中山大学 一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒s蛋白单区域亚单位纳米疫苗
CN111533809A (zh) * 2020-04-21 2020-08-14 中国科学院武汉病毒研究所 针对新型冠状病毒的亚单位疫苗及应用
CN112538105A (zh) * 2020-06-29 2021-03-23 斯克里普斯研究院 稳定的冠状病毒刺突(s)蛋白抗原和相关疫苗
US20210139543A1 (en) * 2020-06-29 2021-05-13 The Scripps Research Institute Stabilized Coronavirus Spike (S) Protein Immunogens and Related Vaccines
CN112358533A (zh) * 2020-10-30 2021-02-12 上海泽润生物科技有限公司 重组刺突蛋白及其制备方法和用途
CN113185613A (zh) * 2021-04-13 2021-07-30 武汉大学 新型冠状病毒s蛋白及其亚单位疫苗
CN113234170A (zh) * 2021-04-13 2021-08-10 武汉大学 新型冠状病毒突变株s蛋白及其亚单位疫苗

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
POLLET JEROEN, CHEN WEN-HSIANG, STRYCH ULRICH: "Recombinant protein vaccines, a proven approach against coronavirus pandemics", ADVANCED DRUG DELIVERY REVIEWS, ELSEVIER, AMSTERDAM , NL, vol. 170, 1 March 2021 (2021-03-01), Amsterdam , NL , pages 71 - 82, XP055824752, ISSN: 0169-409X, DOI: 10.1016/j.addr.2021.01.001 *

Also Published As

Publication number Publication date
CN115785232A (zh) 2023-03-14

Similar Documents

Publication Publication Date Title
CN111217918B (zh) 一种基于2,4-二氧四氢喋啶合酶的新型冠状病毒s蛋白双区域亚单位纳米疫苗
US20230295242A1 (en) Virus-like particles and methods of use
WO2023020623A1 (zh) 用于预防或治疗冠状病毒感染的融合蛋白、Spike蛋白纳米颗粒及其应用
CN105669838B (zh) 来自水痘-带状疱疹病毒gE蛋白的中和表位及针对其的抗体
JP2021527077A (ja) 新規構造成分を含有するナノ粒子ワクチン
JP6643981B2 (ja) インフルエンザウイルスワクチンおよびその使用
KR20170102905A (ko) 신규 다가 나노입자 기반 백신
JPH09501933A (ja) 植物、動物、およびヒトのワクチンとして並びに免疫療法に有用な抗体の免疫優性エピトープの弱化
SG191295A1 (en) Modified influenza hemagglutinin proteins and uses thereof
US10987417B2 (en) Engineered and multimerized human immunodeficiency virus envelope glycoproteins and uses thereof
JP2015521592A (ja) 安定化されたｇｐ１２０
WO2022096899A1 (en) Viral spike proteins and fusion thereof
US20230212267A1 (en) Dna antibody constructs for use against sars-cov-2
JP5290576B2 (ja) 修飾されたｈｉｖ−１エンベロープタンパク質
WO2023138333A1 (zh) 一种重组新型冠状病毒蛋白疫苗、其制备方法和应用
WO2024061188A1 (zh) 一种冠状病毒多价疫苗及其应用
JP2002522448A (ja) ウイルス疾患の予防および治療
JP2024502783A (ja) コロナウイルスワクチン
EP1301637B1 (en) Dna vaccines encoding hiv accessory proteins
US20060142219A1 (en) Pharmaceutical compositions comprising an hiv envelope protein and cd4
Mitchell et al. Inactivation of a common epitope responsible for the induction of antibody-dependent enhancement of HIV
WO2024061239A1 (zh) 用于预防或治疗冠状病毒感染的融合蛋白、Spike蛋白纳米颗粒及其应用
CN109851678A (zh) 一种改良的亚稳定态牛呼吸道合胞病毒融合前体f蛋白质及编码的dna分子和其应用
AU2001283493A1 (en) DNA vaccines encoding HIV accessory proteins
US11305004B2 (en) Compositions and methods related to ebolavirus vaccines

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22857932

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE