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WO2024061239A1 - Fusion protein for preventing or treating coronavirus infection, spike protein nanoparticle, and use thereof - Google Patents

Fusion protein for preventing or treating coronavirus infection, spike protein nanoparticle, and use thereof Download PDF

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Publication number
WO2024061239A1
WO2024061239A1 PCT/CN2023/119845 CN2023119845W WO2024061239A1 WO 2024061239 A1 WO2024061239 A1 WO 2024061239A1 CN 2023119845 W CN2023119845 W CN 2023119845W WO 2024061239 A1 WO2024061239 A1 WO 2024061239A1
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WIPO (PCT)
Prior art keywords
fusion protein
coronavirus
cov
amino acid
protein
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PCT/CN2023/119845
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French (fr)
Chinese (zh)
Inventor
苏华飞
郑丹丹
冯旭
黄俊杰
欧锦新
李颖欣
黄贤明
刘翠华
邹勇娟
邓秋娟
张卫孝
李胜峰
Original Assignee
百奥泰生物制药股份有限公司
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Publication of WO2024061239A1 publication Critical patent/WO2024061239A1/en

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    • 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

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to fusion proteins and Spike protein nanoparticles for preventing or treating coronavirus infection and their applications.
  • Coronavirus is a non-segmented single-stranded positive-strand RNA virus. According to the serotype and genome characteristics, the coronavirus subfamily is divided into four genera: ⁇ , ⁇ , ⁇ and ⁇ . Because the virus envelope has ridges that extend to all sides. It is named after its protrusions and shape like a corolla.
  • the new coronavirus (SARS-CoV-2 or 2019-nCoV) discovered in 2019 belongs to the beta genus and is enveloped. The particles are round or oval, often pleomorphic, and have a diameter of 60-140nm. Current research shows that SARS-CoV-2 and SARS-CoV are highly homologous.
  • COVID-19 is mainly transmitted through the respiratory tract, and it may also be transmitted through contact.
  • the general population is susceptible, and the elderly and those with underlying diseases are more seriously ill after infection. Children and infants also get sick.
  • the incubation period of the new coronavirus is generally 1-14 days, and most of them are 3-7 days.
  • the main clinical symptoms of the infected are fever, fatigue, and dry cough, while upper respiratory tract symptoms such as nasal congestion and runny nose are rare.
  • the total white blood cell count of the patient is normal or decreased, or the number of lymphocytes is decreased, and some patients have increased liver enzymes, myoenzymes and myoglobin.
  • Chest imaging shows that patients present with multiple small patchy shadows and interstitial changes in the early stage, which are obvious in the outer lungs; then develop into multiple ground-glass shadows and infiltration shadows in both lungs, severe cases may have pulmonary consolidation, and gradually develop dyspnea, severe cases develop acute respiratory distress syndrome (ARDS), shock, and multiple tissue damage and dysfunction of lung tissue, heart, and kidney. Most patients with mild infection have a good prognosis, while severe patients are often in critical condition or even die.
  • ARDS acute respiratory distress syndrome
  • the present invention provides a fusion containing a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof that can stabilize the protein structure, and a fusion comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof. protein.
  • the present invention also provides a coronavirus Spike protein extracellular domain containing mutations or a truncation thereof. Coronavirus vaccines in which fragments are fused to monomeric ferritin subunits and self-assemble into nanoparticles can induce stronger neutralizing antibody responses to coronaviruses.
  • Viral particles first interact with an angiotensin-converting enzyme 2 (ACE2) on the surface of lung epithelial cells through the receptor binding domain (RBD) in the S1 subunit of the Spike protein (S protein or spike protein) on its surface. combine.
  • ACE2 angiotensin-converting enzyme 2
  • RBD receptor binding domain
  • S protein or spike protein Spike protein
  • Heptapeptide repeat sequence 1 (HR1) and heptapeptide repeat sequence 2 (HR2) in the S2 subunit interact with each other to form a six-helix bundle (6-HB) fusion core, leading to the fusion of the viral shell and the cell membrane, SARS-CoV or SARS- CoV-2 enters cells and uses cells to synthesize new virus particles; the new virus particles are released outside the cells and then use the same method to infect surrounding normal cells.
  • the fusion protein, nanoparticles and vaccine of the present invention can induce stronger neutralizing antibody responses to coronavirus.
  • a coronavirus Spike protein extracellular domain or a truncated fragment thereof containing a mutation comprising: 1) mutating RRAR to GSAS; 2) in HR1 and the central helical region (CH) There are mutations in the turning region between HR1 and CH that prevent HR1 and CH from forming a straight helix during fusion.
  • the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
  • 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 extracellular domain of the coronavirus Spike protein containing mutations has a C-terminal truncation of 5-80 amino acid residues compared to the full-length extracellular domain of the coronavirus Spike protein. base. In some embodiments, the truncated fragment of the extracellular domain of the coronavirus Spike protein containing mutations has a C-terminal truncation of 20-76 amino acid residues compared to the full-length extracellular domain of the coronavirus Spike protein. base.
  • the truncated fragment of the extracellular domain of the coronavirus Spike protein containing mutations has a C-terminal truncation of 70 amino acid residues compared to the full-length extracellular domain of the coronavirus Spike protein.
  • the coronavirus is SARS-CoV-2, SARS-CoV, or MERS-CoV.
  • the coronavirus is an original strain of SARS-CoV-2 or a variant thereof.
  • the coronavirus is an original strain of SARS-CoV-2, a variant strain of SARS-CoV-2 Alpha, a variant strain of SARS-CoV-2 Beta, a variant strain of SARS-CoV-2 Gamma, a variant strain of 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 coronavirus is SARS-CoV-2 Omicron variant strain BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7 ,XBB,XBB.1,XBB.1.5,XBB.1.5.1,XBB.1.9.1 Or XBB.1.16.
  • the mutation-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises as shown in any one of SEQ ID NO: 3, 4, 6-9, 19-24, 26-31
  • Some embodiments also provide a fusion protein comprising the mutated extracellular domain of the coronavirus Spike protein described herein or a truncated fragment thereof.
  • a fusion protein comprising the mutation-containing extracellular domain of the coronavirus Spike protein described herein or a truncated fragment thereof and a monomeric subunit protein linked 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 a C-terminus containing a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof connected to the N-terminus of a monomeric subunit protein through a linker.
  • the fusion protein is a C-terminus containing a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof connected to the N-terminus of a 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, GGSS, (GGGGS) 2 , (GGGGS) 3 , or any combination thereof. In some embodiments, the linker is ( GmS ) n , wherein each m is independently 1, 2, 3, 4, or 5 and n is 1, 2, 3, 4, or 5. In some embodiments, the linker has the sequence (GGGGS) n and n is 1, 2, 3, 4, or 5. In some embodiments, the linker is GGGGS. In some embodiments, the linker is (GGGGS) 2 . In some embodiments, the linker is (GGGGS) 3 . In some embodiments, the linker is (GGGGS) 4 . In some embodiments, the linker is (GGGGS) 5 .
  • the fusion protein further comprises an N-terminal signal peptide.
  • the signal peptide is selected from the group consisting of CSP, mschito, MF- ⁇ , pho1, HBM, t-pA, and the signal peptide of IL-3.
  • the N-terminal signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 2 or 5, or has at least 80% or at least 90% difference compared to the amino acid sequence set forth in SEQ ID NO: 2 or 5. % identity of the amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 2 or 5.
  • 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 Helicobacter pylori non-heme monomeric ferritin subunit.
  • the N19Q mutation is present in the amino acid sequence of the H. pylori non-heme monomeric ferritin subunit.
  • the monomeric ferritin The subunit comprises an amino acid sequence as set forth in SEQ ID NO: 10, or an amino acid sequence having at least 80% or at least 90% identity as compared to the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 10
  • the amino acid sequences shown are compared to amino acid sequences with one or more conservative amino acid substitutions.
  • a fusion protein comprising a mutation-containing extracellular domain of the SARS-CoV-2 Spike protein or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutated 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) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
  • the SARS-CoV-2 is the original strain or a variant thereof.
  • the SARS-CoV-2 is an original strain of SARS-CoV-2, a variant strain of SARS-CoV-2 Alpha, a variant strain of SARS-CoV-2 Beta, a variant strain of SARS-CoV-2 Gamma, SARS-CoV-2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant.
  • the coronavirus is SARS-CoV-2 Omicron variant strain BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7 , XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.16.
  • the coronavirus is SARS-CoV-2 Omicron variant strain BA.1, BA.2, BA.3, BA.4, BA.5.
  • a fusion protein comprising a mutation-containing SARS-CoV-2 Omicron variant Spike protein extracellular domain or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutated 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) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising the mutation-containing SARS-CoV-2 Omicron variant BA.1 Spike protein extracellular domain or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutated SARS-CoV-2 Omicron variant BA.1 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) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising a mutation-containing SARS-CoV-2 Omicron variant BA.2 Spike protein extracellular domain or a truncated fragment thereof and a monomeric subunit protein connected by a linker. white.
  • the fusion protein comprises a mutated SARS-CoV-2 Omicron variant BA.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) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising the mutation-containing SARS-CoV-2 Omicron variant BA.3 Spike protein extracellular domain or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutated SARS-CoV-2 Omicron variant BA.3 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) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
  • a fusion protein comprising an extracellular domain of a SARS-CoV-2 Omicron variant BA.4 Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises an extracellular domain of a SARS-CoV-2 Omicron variant BA.4 Spike protein containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker.
  • the mutation comprises: 1) mutating RRAR to GSAS; 2) a double mutation K986P/V987P in the turning region between HR1 and CH.
  • a fusion protein comprising the mutation-containing SARS-CoV-2 Omicron variant BA.5 Spike protein extracellular domain or a truncated fragment thereof and a monomeric subunit protein connected by a linker.
  • the fusion protein comprises a mutated SARS-CoV-2 Omicron variant BA.5 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) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
  • the fusion protein includes a mutation-containing coronavirus Spike protein extracellular domain and a monomeric ferritin subunit connected by a linker, the mutation-containing coronavirus Spike protein extracellular domain comprising SEQ.
  • the amino acid sequence shown in any one of ID NO: 3, 4, 6, 19-21, 26-28, the monomeric ferritin subunit includes the amino acid sequence shown in SEQ ID NO: 10; containing a mutated crown
  • the extracellular domain of the viral Spike protein is connected to the monomeric ferritin subunit through the linker shown in SEQ ID NO:11.
  • the fusion protein includes a truncated fragment of the extracellular domain of the coronavirus Spike protein containing mutations and a monomeric ferritin subunit connected by a linker, the extracellular structure of the coronavirus Spike protein containing mutations
  • the truncated fragment of the domain includes the amino acid sequence shown in any one of SEQ ID NO:7-9, 22-24, 29-31, and the monomeric ferritin subunit includes the amino acid sequence shown in SEQ ID NO:10 Sequence; the truncated fragment containing the mutated extracellular domain of the coronavirus Spike protein is connected to the monomeric ferritin subunit through the linker shown in SEQ ID NO: 11.
  • the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 12-17, 32-43, or is identical to any one of SEQ ID NO: 12-17, 32-43
  • the amino acid sequence has at least 80% or at least 90% identity compared to the amino acid sequence, or has one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 12-17, 32-43 amino acid sequence.
  • "at least 80% identity” is at least about 80% identity, at least about 81% identity, at least about 83% identity, at least about 84% identity, at least about 85% identity, at least About 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 93% identical, 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 of the endpoints) or any value therein.
  • "at least 90% identity” is at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least About 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or a range between any two of these values, inclusive of the endpoints ) or any value therein.
  • Some embodiments provide a polynucleotide encoding a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof, or a fusion protein described herein.
  • an expression vector comprising a polynucleotide encoding a mutated coronavirus Spike protein extracellular domain as described herein, or a truncated fragment or fusion protein thereof.
  • a cell that can express a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof as described herein.
  • the cell comprises one or more polynucleotides encoding a fusion protein described herein or an expression vector comprising a polynucleotide encoding a fusion protein described herein.
  • 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 mutation-containing extracellular domain of the coronavirus Spike protein or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker, and the fusion protein includes the following features:
  • the mutations include: 1) a mutation that inactivates the S1/S2 cleavage site; 2) a mutation in the turning region between HR1 and CH that prevents HR1 and CH from forming a straight helix during the fusion process; and/or
  • the C-terminus of the mutated coronavirus Spike protein extracellular domain or its truncated fragment is connected to the monomeric ferritin subunit through a linker;
  • the linker is (G m S) n , where 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: 10, or having at least 80% of the amino acid sequence shown in SEQ ID NO: 10. Or an amino acid sequence that is at least 90% identical, or has one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 10.
  • Spike protein nanoparticles comprising a fusion protein described herein are provided.
  • the use of the fusion protein or Spike protein nanoparticles described herein in preparing a vaccine to prevent or treat coronavirus infection is provided.
  • the coronavirus infection is a SARS-CoV-2, SARS-CoV or MERS-CoV infection.
  • the coronavirus infection is an infection with the original strain of SARS-CoV-2 or a variant thereof.
  • the coronavirus infection is an original strain of SARS-CoV-2, a SARS-CoV-2 Alpha variant, a SARS-CoV-2 Beta variant, a SARS-CoV-2 Gamma variant, or a SARS-CoV -2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant.
  • the coronavirus infection is SARS-CoV-2 Omicron variant strain BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF. 7. XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.16 infection.
  • 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 coronavirus vaccine comprises a fusion protein described herein and a pharmaceutically acceptable carrier and/or adjuvant.
  • the coronavirus vaccine comprises Spike protein nanoparticles described herein and a pharmaceutically acceptable carrier and/or adjuvant.
  • a coronavirus vaccine preparation which includes a fusion protein and one or more of a buffer, a stabilizer, an alkali metal or alkali metal salt, and a surfactant, and the fusion protein is Fusion proteins and/or Spike protein nanoparticles comprising the fusion proteins described herein.
  • the fusion protein comprises a mutation-containing coronavirus 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) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
  • the coronavirus is an original strain of SARS-CoV-2, a variant strain of SARS-CoV-2 Alpha, a variant strain of SARS-CoV-2 Beta, a variant strain of SARS-CoV-2 Gamma, a variant strain of 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 coronavirus is SARS-CoV-2 Omicron variant strain BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7 , XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.16.
  • the coronavirus vaccine preparation includes a fusion protein and one or more of a buffer, a stabilizer, an alkali metal or alkali metal salt, and a surfactant;
  • the fusion protein includes SEQ ID The amino acid sequence shown in any one of NO: 12-17, 32-43, 51-56, and 61.
  • the concentration of the fusion protein is 0.05-5 mg/mL. In some embodiments, the concentration of the fusion protein is 0.05-1 mg/mL. In some embodiments, the concentration of the fusion protein is 0.05-0.5 mg/mL. In some embodiments, the concentration of the fusion protein is about 0.05, 0.1, about 0.45, about 0.5, about 1, about 2, about 3, about 4, about 5 mg/mL.
  • the coronavirus vaccine formulation contains 0.05-1 mg/mL fusion protein, 10-30 mM buffer, stabilizer, alkali metal or alkali metal salt, and surfactant.
  • the buffer is selected from histidine buffer, citric acid buffer, phosphate buffer or a combination thereof. In some embodiments, the buffer is a histidine buffer. In some embodiments, the buffer is a combination of a histidine buffer and a citric acid buffer. In some embodiments, the concentration of the buffer is 1-50mM, or 1-40mM, or 1-30mM, or 5-25mM, or 10-30mM, or 15-25mM, or 5-15mM.
  • the concentration of the buffer is about 1mM, about 5mM, about 8mM, about 10mM, 13mM, about 15mM, about 18mM, about 20mM, about 22mM, about 25mM, about 27mM, about 30mM, about 35mM, about 40mM, about 50mM, or any range (including endpoints) between any two values in these numerical values or any value therein.
  • the buffer is a 5-25mM histidine buffer. In some embodiments, the buffer is a 10-30mM histidine buffer. In some embodiments, the buffer is a 5-15mM histidine buffer. In some embodiments, the buffer is a 15-25mM histidine buffer.
  • the buffer is about 20mM histidine buffer. In some embodiments, the buffer is about 10mM histidine buffer. In some embodiments, the buffer is a combination of 5-15mM histidine buffer and 2-10mM citric acid buffer. In some embodiments, the buffer is a combination of about 10mM histidine buffer and about 5mM citric acid buffer. In some embodiments, the pH is 5.0-7.0. In some embodiments, the pH is 5.5-6.5. In some embodiments, the pH is 5.7-6.3.
  • the pH of the antibody formulation is about 5.0, about 5.3, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.8, about 7.0, or a range (including endpoints) between any two of these values or any value therein. In some embodiments, the pH is about 6.1.
  • the stabilizer is selected from polyols, sugars, amino acids, or combinations thereof. In some embodiments, the stabilizer is selected from one of arginine or a salt thereof, polyethylene glycol, sorbitol, mannitol, glycerol, monosaccharide, oligosaccharide, polysaccharide, cyclodextrin or derivatives thereof. Kind or variety. In some embodiments, the stabilizer is selected from one or more of cyclodextrin or its derivatives, sucrose, and trehalose.
  • the cyclohexan Essence or its derivatives are selected from ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin, its hydroxypropylated derivatives (such as hydroxypropyl beta cyclodextrin), hydroxyethylated derivatives , ethylated derivatives and methylated derivatives, sulfobutyl ether ⁇ -cyclodextrin, branched cyclodextrin, cyclodextrin polymers, or combinations thereof.
  • the stabilizer is selected from hydroxypropyl betacyclodextrin, sucrose, and trehalose, or combinations thereof.
  • the stabilizer is a combination of hydroxypropyl betacyclodextrin and trehalose.
  • trehalose usually exists as trehalose dihydrate, so in some embodiments, trehalose dihydrate can be used to prepare the preparation, or other forms of trehalose (such as anhydrous trehalose) can be used.
  • the stabilizer concentration is 0.5-200 mg/mL. In some embodiments, the stabilizer concentration is 0.5-170 mg/mL. In some embodiments, the stabilizer concentration is 10-170 mg/mL. In some embodiments, the stabilizer concentration is 80-170 mg/mL. In some embodiments, the stabilizer concentration is 60-100 mg/mL.
  • the stabilizer concentration is 0.5-80 mg/mL. In some embodiments, the stabilizer concentration is 0.5-30 mg/mL. In some embodiments, the stabilizer concentration is 0.5-50 mg/mL. In some embodiments, the stabilizer is about 0.5 mg/mL, about 4 mg/mL, about 8 mg/mL, about 10 mg/mL, about 20 mg/mL, about 21 mg/mL, about 25 mg/mL, about 30 mg/mL. mL, about 50 mg/mL, about 84 mg/mL, about 85 mg/mL, about 100 mg/mL, about 150 mg/mL, about 170 mg/mL, or a range between any two of these values (including endpoints) or therein any value.
  • the stabilizer is 0.5-170 mg/mL trehalose dihydrate or sucrose. In some embodiments, the stabilizer is 10-170 mg/mL trehalose dihydrate or sucrose. In some embodiments, the stabilizer is 80-170 mg/mL trehalose dihydrate or sucrose. In some embodiments, the stabilizer is 60-100 mg/mL trehalose dihydrate or sucrose. In some embodiments, the stabilizer is 0-450 mM trehalose or sucrose. In some embodiments, the stabilizer is 14-450 mM trehalose or sucrose. In some embodiments, the stabilizer is 210-450 mM trehalose or sucrose.
  • the stabilizer is 150-270 mM trehalose or sucrose. In some embodiments, the stabilizer is about 222 mM trehalose (about 84 mg/mL trehalose dihydrate). In some embodiments, the stabilizer is about 224.7 mM trehalose (about 85 mg/mL trehalose dihydrate). In some embodiments, the stabilizer is about 55.5 mM trehalose (about 21 mg/mL trehalose dihydrate). In some embodiments, the stabilizer is 0.5-80 mg/mL hydroxypropyl betacyclodextrin. In some embodiments, the stabilizer is 0.5-50 mg/mL hydroxypropyl betacyclodextrin.
  • the stabilizer is 0.5-20 mg/mL hydroxypropyl betacyclodextrin. In some embodiments, the stabilizer is about 8 mg/mL hydroxypropyl betacyclodextrin. In some embodiments, the stabilizer is about 4 mg/mL hydroxypropyl betacyclodextrin.
  • the alkali metal or alkali metal salt is selected from magnesium chloride, potassium chloride, sodium chloride, or combinations thereof. In some embodiments, the alkali metal or alkali metal salt concentration is 0-22 mg/mL. In some embodiments, the alkali metal or alkali metal salt concentration is 8-20 mg/mL. In some embodiments, the alkali metal or base The metal salt concentration is 8-17 mg/mL. In some embodiments, the alkali metal or alkali metal salt concentration is about 0.1, about 1, about 2, about 4, about 8, about 8.25, about 8.3, about 10, about 13, about 16, about 16.5, about 18. About 20, about 22 mg/mL, or a range between any two of these values (inclusive of endpoints) or any value therein. In some embodiments, the alkali metal or alkali metal salt is 0.1-22 mg/mL sodium chloride. In some embodiments, the alkali metal or alkali metal salt is 8-17 mg/mL sodium chloride.
  • the surfactant is selected from poloxamers, polysorbates, or combinations thereof. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is poloxamer 188, polysorbate 20, or polysorbate 80. In some embodiments, the surfactant concentration is 0.1-10 mg/mL. In some embodiments, the surfactant is 0.01-5 mg/mL. In some embodiments, the surfactant is 0.01-2 mg/mL.
  • the surfactant is about 0.01, about 0.1, about 0.2, about 0.4, about 0.5, about 0.8, about 1, about 1.2, about 1.5, about 1.8, about 2, about 5, about 8 , about 10 mg/mL, or a range between any two of these values (inclusive of the endpoints) or any value therein.
  • the surfactant is 0.2-1 mg/mL.
  • the surfactant is 0.01-2 mg/mL polysorbate 80. In some embodiments, the surfactant is 0.1-2 mg/mL polysorbate 80.
  • the coronavirus vaccine preparation contains one or more of 0.05-1 mg/mL fusion protein, 10-30 mM buffer, stabilizer, alkali metal or alkali metal salt, and surfactant,
  • the fusion protein is a fusion protein as described herein.
  • the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.
  • the coronavirus vaccine formulation contains 0.05-1 mg/mL fusion protein, 5-25mM phosphate buffer, 14-450mM trehalose, 0-22mg/mL alkali metal or alkali metal salt, 0.01-2mg/ mL polysorbate 80, pH is about 6.0-8.0, and the fusion protein is the fusion protein described herein.
  • the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.
  • the coronavirus vaccine formulation contains 0.05-1 mg/mL fusion protein, 5-25 mM phosphate buffer, 10-170 mg/mL trehalose dihydrate, 0-22 mg/mL alkali metal or alkali metal salt , 0.01-2mg/mL polysorbate 80, pH is about 6.0-8.0, and the fusion protein is the fusion protein described herein.
  • the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM phosphate buffer, about 26.5 mM trehalose (about 10 mg/mL trehalose dihydrate), about 0.4 mg/mL poly Sorbitate 80, pH is about 7.0, and the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM phosphate buffer, about 450 mM trehalose (about 170 mg/mL trehalose dihydrate), about 0.4 mg/mL polysorbate Ester 80, pH is about 7.0, and the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM phosphate buffer, about 224.7 mM trehalose (about 85 mg/mL trehalose dihydrate), about 0.4 mg/mL poly Sorbitate 80, about 8 mg/mL sodium chloride, pH about 7.0, the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, 5-25 mM histidine buffer, about 200-450 mM trehalose, 0.01-2 mg/mL polysorbate 80, pH 5.0 -7.0, the fusion protein is the fusion protein of the present invention.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, 5-25 mM histidine buffer, about 80-170 mg/mL trehalose dihydrate, 0.01-2 mg/mL polysorbate 80, pH is 5.0-7.0, and the fusion protein is the fusion protein of the present invention.
  • the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.
  • the coronavirus vaccine formulation comprises 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 450 mM trehalose (about 170 mg/mL trehalose dihydrate), about 0.4 mg/mL polysorbate 80, and a pH of 5.6-6.3, and the fusion protein is the fusion protein described in the present invention.
  • the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, and 61.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 450 mM trehalose (about 170 mg/mL trehalose dihydrate), about 0.4 mg/mL Polysorbate 80, pH is 5.6-6.3, and the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 15-25 mM histidine buffer, about 150-300 mM trehalose, about 0.01-2 mg/mL polysorbate 80, about 8-17 mg/mL sodium chloride, pH 5.0-7.0, the fusion protein includes the amino acid sequence shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, and 61.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 15-25 mM histidine buffer, about 60-100 mg/mL trehalose dihydrate, about 0.01-2 mg/mL poly Sorbitate 80, about 8-17 mg/mL sodium chloride, pH 5.0-7.0, the fusion protein includes the amino acids shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, 61 sequence.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20mM histidine buffer, about 224.7mM trehalose (about 85mg/mL trehalose dihydrate), about 0.4mg/ mL polysorbate 80, about 8 mg/mL sodium chloride, pH 5.6-6.3, and the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 222 mM trehalose (about 84 mg/mL trehalose dihydrate), about 0.4 mg/mL Polysorbate 80, about 8-17 mg/mL sodium chloride, pH 5.6-6.3, the fusion protein includes as shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, 61 Amino acid sequence.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 222 mM trehalose (about 84 mg/mL trehalose dihydrate), about 0.4 mg/mL Polysorbate 80, about 8-17 mg/mL sodium chloride, pH 5.6-6.3, the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 222 mM trehalose (about 84 mg/mL trehalose dihydrate), about 0.4 mg/mL Polysorbate 80, about 8 mg/mL sodium chloride, pH 5.6-6.3, the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 222 mM trehalose (about 84 mg/mL trehalose dihydrate), about 0.4 mg/mL Polysorbate 80, about 16 mg/mL sodium chloride, pH 5.6-6.3, the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56 or 61.
  • the coronavirus vaccine formulation comprises 0.05-1 mg/mL fusion protein, 15-25 mM histidine buffer, 0.5-50 mg/mL hydroxypropyl beta-cyclodextrin, 0.01-2 mg/mL polysorbate 80, 8-33 mg/mL sodium chloride, and a pH of 5.0-7.0
  • the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, and 61.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, 8-20 mg/mL hydroxypropyl betacyclodextrin, 0.01-2 mg/mL polysorbate Ester 80, 8-17mg/mL sodium chloride, pH is about 5.6-6.3, the fusion protein includes the amino acid sequence shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, 61 .
  • the coronavirus vaccine formulation comprises 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, 0.5-50 mg/mL hydroxypropyl-beta-cyclodextrin, about 0.4 mg/mL polysorbate 80, 8-33 mg/mL sodium chloride, pH 5.6-6.3
  • the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, 61.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, 0.5-20 mg/mL hydroxypropyl betacyclodextrin, 0.01-2 mg/mL polysorbate Ester 80, 8-33mg/mL sodium chloride, pH 5.6-6.3, the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56 or 61.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 8 mg/mL hydroxypropyl betacyclodextrin, about 0.4 mg/mL polysorbate 80. About 16.5 mg/mL sodium chloride, pH is about 5.6-6.3, and the fusion protein includes the amino acid sequence shown in any one of SEQ ID NO: 17, 56 or 61.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, 5-15 mM histidine buffer and 2-10 mM citrate buffer, 0.5-50 mg/mL hydroxypropyl betacycline paste Essence, 0.01-2mg/mL polysorbate 80, 8-17mg/mL sodium chloride, pH is about 5.6-6.3, the fusion protein includes SEQ ID NO: 12-17, 32-43, 51-56, The amino acid sequence shown in any one of 61.
  • the coronavirus vaccine formulation comprises 0.05-1 mg/mL fusion protein, about 10 mM histidine buffer and about 5 mM citric acid buffer, 0.5-50 mg/mL hydroxypropyl beta-cyclodextrin, 0.01-2 mg/mL polysorbate 80, 8-17 mg/mL sodium chloride, and a pH of about 5.6-6.3, and the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, 61.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 10 mM histidine buffer and about 5 mM citrate buffer, 0.5-20 mg/mL hydroxypropyl betacyclodextrin, about 0.4 mg/mL polysorbate 80, 8-17 mg/mL sodium chloride, pH approximately 5.6-6.3, the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56 or 61.
  • the coronavirus vaccine formulation includes 0.05-1mg/mL fusion protein, about 10mM histidine buffer and about 5mM citrate buffer, about 55.5mM trehalose (about 21mg/mL trehalose dihydrate ), about 0.4 mg/mL polysorbate 80, about 2 mg/mL sodium chloride, pH about 5.6-6.3, the fusion protein includes SEQ ID NO: 12-17, 32-43, 51-56, 61 The amino acid sequence shown in any one.
  • the coronavirus vaccine formulation contains 0.05-1 mg/mL fusion protein, 5-25 mM histidine buffer, 60-100 mg/mL sucrose, 0.01-2 mg/mL polysorbate 80, 8-17 mg /mL sodium chloride,
  • the pH is about 5.6-6.3
  • the fusion protein includes the amino acid sequence shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, and 61.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 80 mg/mL sucrose, about 0.4 mg/mL polysorbate 80, 8-17 mg/mL Sodium chloride, pH is about 5.6-6.3, and the fusion protein includes the amino acid sequence shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, and 61.
  • the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 80 mg/mL sucrose, about 0.4 mg/mL polysorbate 80, about 8 mg/mL chloride Sodium chloride, pH is about 5.6-6.3, and the fusion protein includes the amino acid sequence shown in SEQ ID NO: 17.
  • the coronavirus vaccine formulation further includes an adjuvant.
  • the adjuvant is aluminum adjuvant, SWE adjuvant, or MF59 adjuvant.
  • the adjuvant is added in an amount of 1/10-5/10 of the total volume of the coronavirus vaccine formulation. In some embodiments, the adjuvant is added in an amount of 3/10 of the total volume of the coronavirus vaccine formulation.
  • the fusion protein in the above-described coronavirus vaccine formulation includes at least two fusion proteins described herein. In some embodiments, the fusion protein in the above-described coronavirus vaccine formulation includes two fusion proteins described herein. In some embodiments, the fusion protein in the above-described coronavirus vaccine formulation includes three fusion proteins described herein.
  • a multivalent coronavirus vaccine comprising at least two fusion proteins described herein.
  • the coronavirus multivalent vaccine comprises two fusion proteins described herein.
  • the coronavirus multivalent vaccine includes three fusion proteins described herein.
  • a coronavirus multivalent vaccine comprising a first fusion protein and a second fusion protein, the first fusion protein being the fusion protein described above, and the third fusion protein being the fusion protein described above.
  • the dual fusion protein contains the mutated extracellular domain of the SARS-CoV-2 Delta variant Spike protein or its truncated fragment and the monomeric subunit protein connected through a linker.
  • the coronavirus multivalent vaccine further includes a pharmaceutically acceptable carrier and/or adjuvant.
  • a coronavirus multivalent vaccine preparation comprising a first fusion protein and a second fusion protein, and further comprising one or more of a buffer, a stabilizer, an alkali metal or an alkali metal salt, and a surfactant.
  • the first fusion protein includes the mutation-containing extracellular domain of the SARS-CoV-2 Omicron variant Spike protein or its truncated fragment and the monomeric subunit protein connected through a linker
  • the second fusion protein includes the mutation-containing extracellular domain of the Spike protein.
  • the mutation-containing SARS-CoV-2 Delta variant Spike protein extracellular domain or a truncated fragment thereof comprises: 1) mutation of RRAR to GSAS; 2) a mutation that prevents the formation of a straight helix during fusion exists in the turning region between HR1 and CH.
  • the mutation comprises: 1) mutation of RRAR to GSAS; 2) a double mutation K986P/V987P exists in the turning region between HR1 and CH.
  • the truncated fragment of the extracellular domain of the SARS-CoV-2 Delta variant Spike protein containing mutations is compared with the full-length extracellular domain of the SARS-CoV-2 Delta variant Spike protein.
  • 5-80 amino acid residues are truncated at the C terminus; or 20-76 amino acid residues are truncated at the C terminus; or 70 amino acid residues are truncated at the C terminus.
  • the mutation-containing SARS-CoV-2 Delta variant Spike protein extracellular domain or a truncated fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NO: 45-50, or is identical to An amino acid sequence that is at least 80% or at least 90% identical to the amino acid sequence shown in any one of SEQ ID NO: 45-50, or compared to the amino acid sequence shown in any one of SEQ ID NO: 45-50 An amino acid sequence with one or more conservative amino acid substitutions.
  • the second fusion protein is a C-terminus of the extracellular domain of the SARS-CoV-2 Delta variant Spike protein or a truncated fragment thereof through a linker and the N-terminus of the monomeric subunit protein. connect.
  • the linker of the second fusion protein is a GS linker. In some embodiments, the linker of the second fusion protein is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS) 2 , (GGGGS) 3 , or any combination thereof. In some embodiments, the linker of the second fusion protein is (G m S) n , wherein each m is independently 1, 2, 3, 4, or 5, and n is 1, 2, 3, 4, or 5 .
  • the monomeric subunit protein of the second fusion protein is a self-assembled monomeric subunit protein. In some embodiments, the monomeric subunit protein of the second fusion protein is a monomeric ferritin subunit. In some embodiments, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, phycoferritin, insect ferritin, fungal ferritin, or mammalian ferritin. In some embodiments, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit.
  • the monomeric ferritin subunit comprises the amino acid sequence set forth in SEQ ID NO: 10, or is at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 10.
  • the second fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 51-56, or has an amino acid sequence as compared to the amino acid sequence set forth in any one of SEQ ID NO: 51-56 An amino acid sequence that is at least 80% or at least 90% identical, or has an amino acid sequence compared to the amino acid sequence shown in any one of SEQ ID NO: 51-56 An amino acid sequence with one or more conservative amino acid substitutions.
  • the first fusion protein comprises a sequence as shown in SEQ ID NO:14, 15 or 17, and the second fusion protein comprises a sequence as shown in SEQ ID NO:53, 54 or 56.
  • the first fusion protein comprises the sequence set forth in SEQ ID NO: 17 and the second fusion protein comprises the sequence set forth in SEQ ID NO: 56.
  • the mass ratio of the first fusion protein to the second fusion protein is (1-5):(1-5), or the mass ratio is (1-3):(1-3), or the mass ratio is (1-2):(1-2), or the mass ratio is 1:(1-2), or the mass ratio is (1-2):1, or the mass ratio is 1:1.
  • the coronavirus multivalent vaccine comprises 0.01-2 mg/mL of a first fusion protein and 0.01-2 mg/mL of a second fusion protein.
  • the first fusion protein includes the sequence shown in SEQ ID NO: 17
  • the second fusion protein includes the sequence shown in SEQ ID NO: 56
  • the mass ratio of protein is 1:1.
  • the first fusion protein includes the sequence shown in SEQ ID NO: 61
  • the second fusion protein includes the sequence shown in SEQ ID NO: 56
  • the first fusion protein and the second fusion protein The mass ratio of protein is 1:1.
  • the coronavirus multivalent vaccine preparation includes a first fusion protein and a second fusion protein, and also includes one or more of a buffer, a stabilizer, an alkali metal or an alkali metal salt, and a surfactant.
  • the first fusion protein includes the sequence shown in SEQ ID NO:17 or 61
  • the second fusion protein includes the sequence shown in SEQ ID NO:56.
  • the coronavirus multivalent vaccine formulation contains 0.01-2 mg/mL first fusion protein and 0.01-2 mg/mL second fusion protein, and further contains 5-15mM histidine buffer and 2-10mM lemon Acid buffer, 0.5-80 mg/mL hydroxypropyl betacyclodextrin, 0.01-2 mg/mL polysorbate 80, 8-17 mg/mL sodium chloride, pH 5.0-7.0,
  • the first fusion protein includes
  • the second fusion protein contains the sequence shown in SEQ ID NO: 17 or 61
  • the second fusion protein contains the sequence shown in SEQ ID NO: 56.
  • the coronavirus multivalent vaccine formulation includes 0.01-0.2 mg/mL first fusion protein and 0.01-0.2 mg/mL second fusion protein, and further includes about 10 mM histidine buffer and about 5 mM citric acid Buffer, 0.5-20 mg/mL hydroxypropyl betacyclodextrin, 0.01-2 mg/mL polysorbate 80, 8-17 mg/mL sodium chloride, pH 5.6-6.3
  • the first fusion protein includes as follows The sequence shown in SEQ ID NO:17 or 61
  • the second fusion protein includes the sequence shown in SEQ ID NO:56.
  • the coronavirus multivalent vaccine formulation comprises 0.01-0.2 mg/mL first fusion protein Baihe 0.01-0.2mg/mL second fusion protein also contains about 10mM histidine buffer and about 5mM citrate buffer, about 4mg/mL hydroxypropyl betacyclodextrin, 0.01-2mg/mL polysorbate 80.
  • the coronavirus multivalent vaccine formulation includes 0.01-2 mg/mL first fusion protein and 0.01-2 mg/mL second fusion protein, and further includes 5-15 mM histidine buffer, 0.5-80 mg/ mL hydroxypropyl betacyclodextrin, 0.01-2mg/mL polysorbate 80, 8-17mg/mL sodium chloride, pH 5.0-7.0, the first fusion protein includes SEQ ID NO: 17 or 61 The sequence shown is that the second fusion protein contains the sequence shown in SEQ ID NO:56.
  • the coronavirus multivalent vaccine formulation includes 0.01-0.2 mg/mL first fusion protein and 0.01-0.2 mg/mL second fusion protein, and also includes about 10 mM histidine buffer, 0.5-20 mg /mL hydroxypropyl betacyclodextrin, 0.01-2mg/mL polysorbate 80, 8-17mg/mL sodium chloride, pH is 5.6-6.3, the first fusion protein includes SEQ ID NO: 17 or The sequence shown in SEQ ID NO: 56, the second fusion protein contains the sequence shown in SEQ ID NO: 56.
  • the coronavirus multivalent vaccine formulation includes 0.01-0.2 mg/mL first fusion protein and 0.01-0.2 mg/mL second fusion protein, and also includes about 10 mM histidine buffer, about 4 mg/mL mL hydroxypropyl betacyclodextrin, 0.01-2mg/mL polysorbate 80, about 8.26mg/mL sodium chloride, the first fusion protein includes the sequence shown in SEQ ID NO: 17 or 61, the first The two fusion proteins include the sequence shown in SEQ ID NO:56.
  • the coronavirus multivalent vaccine formulation further includes an adjuvant.
  • the adjuvant is aluminum adjuvant, SWE adjuvant, or MF59 adjuvant.
  • the adjuvant is added in an amount ranging from 1/10 to 5/10 of the total volume of the formulation. In some embodiments, the adjuvant is added in an amount of approximately 3/10 of the total volume of the formulation. In some embodiments, the adjuvant is a SWE adjuvant.
  • the coronavirus multivalent vaccine formulation includes about 0.08 mg/mL first fusion protein and about 0.08 mg/mL second fusion protein, and also includes about 10 mM histidine buffer, about 4 mg/mL hydroxypropanol Betacyclodextrin, about 0.2 mg/mL polysorbate 80, about 8.26 mg/mL sodium chloride and SWE adjuvant, pH 5.6-6.3, the first fusion protein comprising SEQ ID NO: 17 or The sequence shown in SEQ ID NO: 56, the second fusion protein contains the sequence shown in SEQ ID NO: 56.
  • the coronavirus multivalent vaccine formulation comprises about 0.04 mg/mL of a first fusion protein and about 0.04 mg/mL of a second fusion protein, and further comprises about 10 mM histidine buffer, about 4 mg/mL of hydroxypropyl beta-cyclodextrin, about 0.2 mg/mL of polysorbate 80, about 8.26 mg/mL of sodium chloride, and a SWE adjuvant, with a pH of 5.6-6.3, wherein the first fusion protein comprises a sequence as shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises Contains the sequence shown in SEQ ID NO:56.
  • the coronavirus multivalent vaccine formulation has a specification of 0.5 mL, comprising about 40 ⁇ g of a first fusion protein and about 40 ⁇ g of a second fusion protein, and also comprises about 0.39 mg of histidine, about 0.53 mg of histidine hydrochloride, about 2 mg of hydroxypropyl beta-cyclodextrin, about 0.1 mg of polysorbate 80, about 4.13 mg of sodium chloride and 0.15 mL of SWE adjuvant, with a pH of 5.6-6.3, wherein the first fusion protein comprises a sequence as shown in SEQ ID NO:17 or 61, and the second fusion protein comprises a sequence as shown in SEQ ID NO:56.
  • the coronavirus multivalent vaccine preparation has a specification of 0.5 mL, and the coronavirus multivalent vaccine contains about 20 ⁇ g of the first fusion protein and about 20 ⁇ g of the second fusion protein, and also contains about 0.39 mg of histidine, about 0.53 mg histidine hydrochloride, about 2 mg hydroxypropyl betacyclodextrin, about 0.1 mg polysorbate 80, about 4.13 mg sodium chloride and 0.15 mL SWE adjuvant, the first fusion protein comprising SEQ ID NO: 17 or 61, the second fusion protein contains the sequence shown in SEQ ID NO: 56.
  • the present invention also provides methods and uses for prevention or treatment.
  • the present invention provides methods for preventing or treating coronavirus infection comprising administering to a patient in need thereof an effective amount of a fusion protein, Spike protein nanoparticle or coronavirus vaccine described herein.
  • use of the fusion proteins, Spike protein nanoparticles, or coronavirus vaccines described herein in preventing or treating SARS or COVID-19 is provided.
  • the coronavirus infection is a SARS-CoV-2, SARS-CoV or MERS-CoV infection.
  • the coronavirus infection is an infection with the original strain of SARS-CoV-2 or a variant thereof.
  • the coronavirus infection is an original strain of SARS-CoV-2, a SARS-CoV-2 Alpha variant, a SARS-CoV-2 Beta variant, a SARS-CoV-2 Gamma variant, or a SARS-CoV -2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant.
  • the coronavirus infection is SARS-CoV-2 Omicron variant strain BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF. 7. XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.16 infection.
  • the coronavirus infection is a SARS-CoV-2 Omicron variant BA.5 infection.
  • the coronavirus infection is a SARS-CoV-2 Omicron variant XBB.1.5 infection.
  • the coronavirus infection is a SARS-CoV-2 Omicron variant XBB.1.16 infection.
  • Figure 1 shows the binding curve of fusion protein D and human ACE2.
  • Figure 2 shows the binding curve of fusion protein 2-1 and human ACE2.
  • Figure 3 shows the serum IgG titer specific to Spike protein after immunizing mice with the bivalent vaccine with or without adjuvant;
  • WT represents WT-Spike protein
  • Delta represents Delta-Spike protein
  • BA.1 represents BA.1-Spike protein
  • BA.5 represents BA.5-Spike protein.
  • Figure 4 shows the anti-Spike protein IgG antibody titers in mouse serum
  • Figures 4a, 4c, 4e, and 4g show the anti-Spike protein IgG antibody titers in mouse serum after the first immunization
  • Figures 4b, 4d, 4f, and 4h show the second enhancement Anti-Spike protein IgG antibody titer in mouse serum after immunization
  • the bar represents the geometric mean (GMT) of the titer (the value is displayed within the bar)
  • the error bar represents the 95% confidence interval (CI).
  • Figure 5 shows the inhibitory titer of serum against pseudovirus after immunizing mice with fusion protein D, fusion protein 2-1 and bivalent vaccine; among them, WT represents the original strain of SARS-CoV-2 pseudovirus, and Delta represents SARS-CoV-2 Delta.
  • BA.5 represents SARS-CoV-2 BA.5 pseudovirus
  • BQ.1.1 represents SARS-CoV-2 BQ.1.1 pseudovirus
  • XBB represents SARS-CoV-2 XBB pseudovirus
  • XBB.1.5 represents SARS- CoV-2 XBB.1.5 pseudovirus
  • bars represent the geometric mean (GMT) of titers (values are shown within the bars); error bars represent 95% CI.
  • Figure 6 shows the inhibitory titer of mouse serum against the true coronavirus after secondary immunization with the bivalent vaccine; among them, the bar represents the geometric mean (GMT) of the titer (the value is displayed within the bar); the error bar represents 95% CI; ULOD indicates the upper limit of detection of the test.
  • GTT geometric mean
  • ULOD indicates the upper limit of detection of the test.
  • Figure 7 shows the serum anti-Spike protein IgG antibody titers of mice sequentially immunized with the bivalent vaccine; the bars represent the geometric mean (GMT) of the titers (values are displayed within the bars); the error bars represent the 95% CI.
  • Figure 8 shows the inhibitory titers of serum pseudoviruses in mice sequentially immunized with the bivalent vaccine; among them, WT represents the original strain of SARS-CoV-2 pseudovirus, Delta represents the SARS-CoV-2 Delta pseudovirus, and BF.7 represents SARS-CoV. -2 BF.7 pseudovirus, XBB.1 represents SARS-CoV-2 XBB.1 pseudovirus; bars represent the geometric mean (GMT) of titers (values are shown within bars); error bars represent 95% CI .
  • WT represents the original strain of SARS-CoV-2 pseudovirus
  • Delta represents the SARS-CoV-2 Delta pseudovirus
  • BF.7 represents SARS-CoV. -2 BF.7 pseudovirus
  • XBB.1 represents SARS-CoV-2 XBB.1 pseudovirus
  • bars represent the geometric mean (GMT) of titers (values are shown within bars)
  • error bars represent 95% CI .
  • Figure 9 shows the ELISpot of spleen cells of vaccine-immunized mice; among them, WT represents the Spike protein peptide pool of the original strain of the new coronavirus, Delta represents the Spike protein peptide pool of the new coronavirus mutant strain Delta, and BA.5 represents the Spike protein peptide pool of the new coronavirus mutant strain BA.5. Spike protein peptide pool; error bars represent geometric mean ⁇ 95% CI.
  • Figure 10 shows the rat serum anti-Spike protein IgG antibody titer; where, WT represents WT-Spike protein, Delta represents Delta-Spike protein, and BA.5 represents BA.5-Spike protein; the bar represents the geometric mean of the titer. (GMT) (values shown within bars), error bars represent 95% CI.
  • nucleic acid molecule refers to one or more nucleic acid molecules. Accordingly, the terms “a”, “an”, “one or more” and “at least one” may be used interchangeably. Similarly, the terms “comprising”, “including” and “having” may be used interchangeably and should generally be understood to be open-ended and non-limiting, e.g. not excluding other unrecited elements or steps.
  • amino acid refers to organic compounds containing both amino and carboxyl groups, such as alpha-amino acids, which may be encoded by nucleic acids directly or in the form of precursors.
  • a single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called the "degeneracy of the genetic code.”
  • Amino acids include natural amino acids and unnatural amino acids.
  • Natural amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine 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) with similar chemical properties (eg, charge or hydrophobicity). Generally speaking, conservative amino acid substitutions are unlikely to materially alter the functional properties of the protein. Examples of amino acid classes containing chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine.
  • Amide-containing side chains asparagine and glutamine
  • Aromatic side chains phenylalanine, tyrosine and tryptophan
  • Basic side chains lysine, Arginine and histidine
  • Acidic side chains aspartic acid and glutamic acid.
  • polypeptide is intended to encompass the singular “polypeptide” as well as the plural “polypeptide” and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any single chain or chains of two or more amino acids and does not refer to a specific length of the product.
  • the definition of “polypeptide” includes peptide, dipeptide, tripeptide, oligopeptide, "protein,” “amino acid chain” or any other term used to refer to two or more amino acid chains, and the term “polypeptide” may Used instead of or interchangeably with any of the above terms.
  • polypeptide is also intended to refer to the product of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-natural Amino acid modifications that occur.
  • a polypeptide may be derived from natural biological sources or produced by recombinant techniques, but it does not have to be translated from a specified nucleic acid sequence and may be produced by any means including chemical synthesis.
  • a fusion protein is a recombinant protein that contains amino acid sequences from at least two unrelated proteins that have been linked together by peptide bonds to form a single protein.
  • Amino acid sequences of unrelated proteins can be linked directly to each other, or they can be linked using linkers.
  • linkers As used herein, if protein's Amino acid sequences are not related together if they are not normally linked together via peptide bonds in their natural environment (eg, within a cell).
  • the amino acid sequence of a common bacterial enzyme such as Bacillus stearothermophilus dihydrolipoate transacetylase (E2p) and the amino acid sequence of the coronavirus Spike protein are not linked together by peptide bonds.
  • homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions within each sequence that can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences.
  • encoding when applied to a polynucleotide refers to a polynucleotide that is said to "encode” a polypeptide that, in its native state or when manipulated by methods well known to those skilled in the art, may be transcribed and/or translated to produce the polypeptide and/or fragment thereof.
  • a polynucleotide is composed of a specific sequence of four bases: adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U) when the polynucleotide is RNA.
  • a "polynucleotide sequence" can be represented by the letters of the polynucleotide molecule. This letter representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, such as functional genomics and homology searches.
  • polynucleotide refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or the like.
  • Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown.
  • genes or gene fragments e.g.
  • polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If such modifications are present, structural modifications to the nucleotide may be made before or after assembly of the polynucleotide.
  • sequence of nucleotides can be interrupted by non-nucleotide components.
  • the polynucleotide can be further modified after polymerization, for example by conjugation with a labeling component.
  • This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, embodiments of any polynucleotide of the present disclosure include double-stranded forms and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.
  • a nucleic acid or polynucleotide sequence is "identical” or “sequence identical” to another sequence by a certain percentage (eg, 90%, 95%, 98% or 99%). When sequences are aligned, this percentage of bases (or amino acids) in the two sequences being compared are identical.
  • the alignment percent identity or sequence identity can be determined using visual inspection or software programs known in the art, such as those described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. It is preferred to use the default parameters for comparison.
  • Biologically equivalent polynucleotides are polynucleotides that share the percentage identity specified above and encode a polypeptide with the same or similar biological activity.
  • isolated used in the present invention with respect to cells, nucleic acids, polypeptides, antibodies, etc., such as “isolated” DNA, RNA, polypeptides, and antibodies, refers to other components in the natural environment of cells, such as DNA or RNA. one or more separated molecules.
  • isolated as used herein also refers to nucleic acids or peptides that are substantially free of cellular material, viral material or cell culture media when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is intended to include nucleic acid fragments that do not exist in their native state and do not exist in their native state.
  • isolated is also used herein to refer to cells or polypeptides separated from other cellular proteins or tissues.
  • Isolated polypeptide is intended to include purified and recombinant polypeptides.
  • Isolated polypeptides, antibodies, etc. are generally prepared by at least one purification step.
  • the purity of the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or any of these values. The range between any two values (inclusive) or any value within them.
  • recombinant refers to a polypeptide or polynucleotide and means a form of the polypeptide or polynucleotide that does not occur in nature, and non-limiting examples may be combined to produce polynucleotides that do not normally exist or Peptides.
  • Antibody and antigen-binding fragment refer to polypeptides or polypeptide complexes that specifically recognize and bind to antigens. Antibodies can be complete antibodies, any antigen-binding fragments thereof, or single chains thereof. The term “antibody” thus includes any protein or peptide whose molecule contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen.
  • the terms "antigen” or “immunogen” are used interchangeably and refer to a substance, typically a protein, capable of inducing an immune response in a subject.
  • the term also refers to a protein that is immunologically active, i.e., capable of eliciting responses to the body fluids and/or Cell type immune response.
  • vaccine antigen is used interchangeably with “protein antigen” or “antigenic polypeptide.”
  • Neutralizing antibodies refer to antibodies that reduce the infectious titer of an infectious agent by binding to a specific antigen on that agent.
  • the infectious agent is a virus.
  • a “broadly neutralizing antibody” is an antibody that binds to and inhibits the function of a related antigen, e.g., at least 85%, 90%, 95%, 96%, 97%, 98% identical to the antigenic surface of the antigen % or 99% identity to the antigen.
  • the antibodies can bind to and inhibit the function of more than one class and/or subclass of antigens from the pathogen.
  • cDNA refers to DNA that is complementary or identical to mRNA and may be in single- or double-stranded form.
  • Epitope refers to an antigenic determinant. These are specific chemical groups or peptide sequences on molecules that are antigenic, So much so that they elicit a specific immune response, for example, an epitope is an antigenic region to which B and/or T cells respond. Epitopes can be formed from contiguous amino acids, or from non-contiguous amino acids juxtaposed by the tertiary folding of the protein.
  • Vaccine refers to a biological product that induces a preventive or therapeutic immune response in a subject.
  • the immune response is a protective immune response.
  • vaccines elicit an antigen-specific immune response against the antigens of pathogens, such as viral pathogens, or cellular components associated with pathological conditions.
  • Vaccines may include polynucleotides (eg, nucleic acids encoding known antigens), peptides or polypeptides (eg, disclosed antigens), viruses, cells, or one or more cellular components.
  • a vaccine or vaccine antigen or vaccine composition is expressed from a fusion protein expression vector and self-assembles into nanoparticles displaying the antigenic polypeptide or protein on the surface.
  • An effective amount of a vaccine or other agent is sufficient to produce a desired response, such as eliciting an immune response, preventing, alleviating, or eliminating signs or symptoms of a condition or disease (e.g., pneumonia).
  • a desired response such as eliciting an immune response, preventing, alleviating, or eliminating signs or symptoms of a condition or disease (e.g., pneumonia).
  • this may be an amount necessary to inhibit viral replication or measurably alter the outward symptoms of a viral infection.
  • this amount will be sufficient to measurably inhibit the replication or infectivity of the virus (eg, SARS-CoV-2).
  • a dose that achieves target tissue concentrations and has been shown to achieve inhibition of viral replication in vitro will generally be used.
  • an "effective amount” is an amount that treats (including prevents) one or more symptoms and/or underlying causes of a condition or disease (eg, treating a coronavirus infection). In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount that prevents the development of one or more symptoms or signs of a particular disease or condition (eg, one or more symptoms or signs associated with a coronavirus infection).
  • Nanoparticles refer to spherical protein shells that are tens of nanometers in diameter and have well-defined surface geometry.
  • the spherical protein shell is formed from identical copies of non-viral proteins that self-assemble into nanoparticles with an appearance similar to virus-like particles (VLPs).
  • VLPs virus-like particles
  • examples include ferritin (FR), which is conserved across multiple species and forms a 24mer, Bacillus stearothermophilus dihydrolipoic acid transacetylase (E2P), Hyperthermophila dioxytetrahydrogen pteridine synthase (LS) and Thermotoga maritima encapsulin, all of which form a 60-mer.
  • Self-assembling nanoparticles can form spontaneously after recombinantly expressing proteins in an appropriate expression system. Methods for the production, detection and characterization of nanoparticles can use the same techniques developed for VLPs.
  • VLPs refer to non-replicating viral shells derived from any of a variety of viruses.
  • VLPs typically include one or more viral proteins, such as, but not limited to, those proteins known as capsid proteins, coat proteins, wall proteins, surface proteins and/or envelope proteins, or formed particles derived from these proteins of peptides.
  • viral proteins such as, but not limited to, those proteins known as capsid proteins, coat proteins, wall proteins, surface proteins and/or envelope proteins, or formed particles derived from these proteins of peptides.
  • VLPs can form spontaneously after recombinantly expressed proteins. Methods for producing specific VLPs are known in the art.
  • the presence of VLPs following recombinantly expressed viral proteins can be detected using conventional techniques known in the art (eg, by electron microscopy, biophysical characterization, etc.). For example, VLPs can be separated by density gradient centrifugation and/or identified by characteristic density bands. Alternatively, cryo-electron microscopy can be performed on vitrified water
  • ECMO Extracorporeal Membrane Oxygenation
  • ICU refers to the intensive care unit (Intensive Care Unit). Treatment, nursing, and rehabilitation can all be carried out simultaneously. It provides isolation places and equipment for critically ill or comatose patients, and provides the best care, comprehensive treatment, combination of medical and nursing care, and surgery. Early rehabilitation, joint care, sports therapy and other services.
  • IMV intermittent mandatory ventilation
  • This period allows the patient to breathe spontaneously at any set basal pressure level during mandatory ventilation. While breathing spontaneously, the patient can breathe on his own with continuous airflow support, or the machine will open the on-demand valve to allow for spontaneous breathing. Most ventilators can provide pressure support while breathing spontaneously.
  • subject refers to any animal classified as a mammal, such as humans and non-human mammals.
  • non-human animals include dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, mice, etc.
  • patient or subject are used interchangeably herein.
  • the subject is human.
  • Treatment means therapeutic treatment and prophylactic or preventative measures designed to prevent, slow down, ameliorate or halt adverse physiological changes or disorders, such as the progression of a disease, including but not limited to the following whether detectable or undetectable
  • the results include alleviation of symptoms, reduction in disease severity, stabilization of disease status (i.e. no worsening), delay or slowdown of disease progression, improvement, alleviation, reduction or disappearance of disease status (whether partial or complete), prolongation and Expected survival without treatment, etc.
  • Patients in need of treatment include patients who already have a condition or disorder, are susceptible to a condition or disorder, or are in need of prevention of a condition or disorder that may or are expected to result from administration of the Spike protein nanoparticles or pharmaceutical compositions disclosed herein. For patients who benefit from treatment.
  • S spike
  • E envelope
  • M membrane
  • N nucleocapsid
  • 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.
  • RBD receptor binding domain
  • Receptor binding can help keep the RBD in the "standing" state, which facilitates the dissociation of the S1 subunit from the S2 subunit.
  • a second S2' cleavage releases the fusion peptide.
  • the linker region, HR1, and CH form a very long helix to insert the fusion peptide into the host cell membrane.
  • HR1 and HR2 form a helical structure and assemble into a six-helix bundle to fuse the viral membrane and the host membrane.
  • RBD contains a core subdomain and a receptor binding motif (RBM).
  • RBM receptor binding motif
  • SARS-CoV and MERS-CoV-2 recognizes angiotensin-converting enzyme 2 (ACE2), while MERS-CoV binds dipeptidyl peptidase 4 (DPP4).
  • ACE2 angiotensin-converting enzyme 2
  • DPP4 dipeptidyl peptidase 4
  • the present invention stabilizes the Spike trimer by 1) mutations that inactivate the S1/S2 cleavage site and 2) the presence of mutations in the turning region between HR1 and CH that prevent HR1 and CH from forming a straight helix during fusion.
  • mutant-containing coronavirus Spike protein extracellular domains or truncated fragments thereof can be displayed on nanoparticles.
  • the present invention provides fusion proteins, Spike protein nanoparticles, and vaccine compositions.
  • the invention also provides related polynucleotides, expression vectors and pharmaceutical compositions.
  • stable Spike trimers and RBD proteins in protein or nucleic acid (DNA/mRNA) forms carried by viral vectors can be used as coronavirus vaccines.
  • nanoparticle-presented stable Spike trimers and RBDs can also be used as coronavirus vaccines.
  • the coronavirus Spike protein-based antigens and vaccines of the present invention have many advantageous properties.
  • the Spike trimer design described herein presents conserved neutralizing epitopes in its native-like conformation, allowing the Spike trimer to be used as an antigen vaccine or for multivalent display on nanoparticles.
  • the nanoparticle vaccine of the present invention allows the display of Spike trimers derived from different coronaviruses on well-known nanoparticles, such as ferritin, E2p and I3-01, with sizes ranging from 12.2 to 25.0 nm. All trimer-presenting nanoparticles can be produced in HEK293 cells and CHO cells with high yields.
  • the produced Spike protein nanoparticles can be purified by antibody and size exclusion chromatography (SEC).
  • mutant coronavirus Spike protein extracellular domain or truncated fragments thereof, fusion proteins, Spike protein nanoparticles, encoded polynucleotides, expression vectors and host cells of the present invention and related treatments Applications can be produced or performed according to the methods exemplified herein or conventional methods well known in the art.
  • the present invention provides a mutation-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof that can be used to produce a vaccine.
  • the mutated Spike trimer is stabilized by introducing mutations into the extracellular domain of the coronavirus Spike protein or its truncated fragments.
  • This article illustrates some specific Spike proteins of specific SARS-CoV-2 strains or isolates, such as SEQ ID NO:1. Due to the functional similarities and sequence homologies between different isolates or strains of a given coronavirus, it is also possible to generate spike proteins derived from orthologous sequences of other known coronavirus Spike proteins according to the mutation strategies described here. Mutated Spike protein or truncated fragments thereof. Many known coronavirus Spike protein sequences have been described in the literature.
  • some mutant Spike proteins or truncated fragments thereof of the invention comprise 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 a mutation that inactivates the S1/S2 cleavage site, and a mutation in the turn region between HR1 and CH, which removes any strain in the turn region between HR1 and CH, i.e. preventing the formation of a straight helix .
  • Some coronavirus Spike protein extracellular domains containing mutations or truncated fragments thereof are derived from COVID-19 SARS-CoV-2 virus. These polypeptides contain mutations that inactivate the S1/S2 cleavage site and mutations in the turning region between HR1 and CH.
  • the Spike protein used for mutation may be SEQ ID NO: 1, 18, 25, or 44, or a variant thereof, such as a variant that is substantially identical thereto or a conservatively modified variant thereof.
  • inactivation of the S1/S2 cleavage site 682 RRAR 685 can be achieved by a number of sequence changes within or around the site (e.g., missing or substituted) to achieve.
  • one mutation that inactivates the S1/S2 cleavage site without affecting the protein structure is to mutate the S1/S2 cleavage site 682 RRAR 685 to 682 GSAS 685 .
  • a double mutation can be made in the turn region between HR1 and CH, which eliminates the turn region during fusion by preventing the formation of straight helices (HR1 and CH motifs between).
  • this double mutation can be K986G/V987G, K986P/V987P, K986G/V987P or K986P/V987G.
  • some SARS-CoV-2 Spike proteins or truncated fragments thereof of the present invention may contain deletions of most or the entire HR2 domain.
  • deletions may include deletions such as residues 1139-1208 of SEQ ID NO: 1.
  • the deletion may be 5, 10, 15, 20, 25, 30, 35, 40 of the C-terminus of the extracellular domain of the Spike protein (eg, SEQ ID NO: 1). , 45, 50, 55, 60, 65, 70, 75, 76, 80 or more residues, or a range between any two of these values, including endpoint) or any value therein.
  • a 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 domains or truncated fragments thereof or variants thereof are as follows:
  • ECD extracellular domain
  • SARS-CoV-2 Omicron variant BA.5Spike protein The full-length extracellular domain (ECD) of SARS-CoV-2 Omicron variant BA.5Spike protein, its amino acid sequence is shown in SEQ ID NO: 1, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) is used Italicized, S1/S2 cleavage site 682 RRAR 685 underlined, bolded and italicized.
  • the full-length extracellular domain I-1 of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein has an amino acid sequence as shown in SEQ ID NO: 3.
  • the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains double Mutation K986P/V987P, underlined and italicized.
  • the full-length extracellular domain I-2 of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein has an amino acid sequence as shown in SEQ ID NO: 4.
  • the original signal peptide: MFVFLVLLPLVSS shown in SEQ ID NO:2
  • the signal peptide: MEFGLSLVFLVLILKGVQC shown in SEQ ID NO:5
  • the signal peptide is marked in italics, and the S1/S2 cleavage site
  • the 682 RRAR 685 mutation is 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the full-length extracellular domain I-3 of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein has an amino acid sequence as shown in SEQ ID NO: 6. In the sequence, there is no signal peptide, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the amino acid sequence of the C-terminal truncated fragment I-4 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein is shown in SEQ ID NO: 7.
  • the original signal peptide: MFVFLVLLPLVSS (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
  • contains the double mutation K986P/V987P marked with underline and italics.
  • the signal peptide in italics S1/S2 cleavage site
  • the 682 RRAR 685 mutation is 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • ECD extracellular domain
  • SARS-CoV-2 Omicron variant BA.2 Spike protein The full-length extracellular domain (ECD) of SARS-CoV-2 Omicron variant BA.2 Spike protein, its amino acid sequence is shown in SEQ ID NO:18, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) Italicized, S1/S2 cleavage site 682 RRAR 685 underlined, bolded and italicized.
  • the full-length extracellular domain I-6-1 of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein has an amino acid sequence as shown in SEQ ID NO: 19.
  • the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains double Mutation K986P/V987P, underlined and italicized.
  • the full-length extracellular domain I-7 of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein has an amino acid sequence as shown in SEQ ID NO: 20.
  • the original signal peptide: MFVFLVLLPLVSS shown in SEQ ID NO:2
  • the signal peptide: MEFGLSLVFLVLILKGVQC shown in SEQ ID NO:5
  • the signal peptide is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 mutates to 682 GSAS 685 , underlined and bold out, and also contains the double mutation K986P/V987P, which is underlined and italicized.
  • the full-length extracellular domain I-8 of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein has an amino acid sequence as shown in SEQ ID NO: 21. In the sequence, there is no signal peptide, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein extracellular domain C-terminal truncated fragment I-9, its amino acid sequence is shown in SEQ ID NO: 22. In the sequence, 70 amino acid residues are truncated at the C terminus.
  • the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics.
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 . It is underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.
  • the signal peptide Italicized the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.
  • the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein extracellular domain C-terminal truncated fragment I-11, its amino acid sequence is shown in SEQ ID NO: 24. In the sequence, 70 amino acid residues are truncated at the C terminus, without the signal peptide, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains the double mutation K986P/V987P. , underlined and italicized.
  • ECD extracellular domain
  • SARS-CoV-2 Omicron variant BA.3Spike protein The full-length extracellular domain (ECD) of SARS-CoV-2 Omicron variant BA.3Spike protein, its amino acid sequence is shown in SEQ ID NO:25, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is used Italicized, S1/S2 cleavage site 682 RRAR 685 underlined, bolded and italicized.
  • the full-length extracellular domain I-12 of the mutant SARS-CoV-2 Omicron variant BA.3 Spike protein whose amino acid sequence is shown in SEQ ID NO: 26.
  • the original signal peptide: MFVFLVLLPLVSS (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 , which is underlined and bolded, and contains the double mutation K986P/V987P, which is underlined and italicized.
  • the full-length extracellular domain I-13 of the mutated SARS-CoV-2 Omicron variant BA.3Spike protein has an amino acid sequence as shown in SEQ ID NO: 27.
  • the original signal peptide: MFVFLVLLPLVSS shown in SEQ ID NO:2
  • the signal peptide: MEFGLSLVFLVLILKGVQC shown in SEQ ID NO:5
  • the signal peptide is marked in italics, and the S1/S2 cleavage site
  • the 682 RRAR 685 mutation is 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the full-length extracellular domain I-14 of the mutated SARS-CoV-2 Omicron variant BA.3Spike protein has an amino acid sequence as shown in SEQ ID NO: 28. In the sequence, there is no signal peptide, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the mutated SARS-CoV-2 Omicron variant BA.3 Spike protein extracellular domain C-terminal truncated fragment I-15, its amino acid sequence is shown in SEQ ID NO: 29. In the sequence, 70 amino acid residues are truncated at the C terminus.
  • the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics.
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 . It is underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.
  • the signal peptide Italicized the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.
  • the amino acid sequence of the C-terminal truncated fragment g1 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.1 Spike protein is shown in SEQ ID NO: 62.
  • 70 amino acid residues are truncated at the C-terminus
  • the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is marked in italics
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , marked with underline and bold
  • contains the double mutation K986P/V987P marked with underline and italics.
  • ECD extracellular domain
  • SEQ ID NO:44 The full-length extracellular domain (ECD) of SARS-CoV-2 Delta variant Spike protein, its amino acid sequence is shown in SEQ ID NO:44, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics Out, S1/S2 cleavage site 682 RRAR 685 is underlined, bolded and italicized.
  • the full-length extracellular domain c1 of the mutant SARS-CoV-2 Delta variant Spike protein whose amino acid sequence is shown in SEQ ID NO: 45.
  • the original signal peptide: MFVFLVLLPLVSS (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 , which is underlined and bolded, and contains the double mutation K986P/V987P, which is underlined and italicized.
  • the full-length extracellular domain c2 of the mutated SARS-CoV-2 Delta variant Spike protein has an amino acid sequence as shown in SEQ ID NO: 46.
  • the original signal peptide: MFVFLVLLPLVSS shown in SEQ ID NO:2
  • the signal peptide: MEFGLSLVFLVLILKGVQC shown in SEQ ID NO:5
  • the signal peptide is marked in italics, and the S1/S2 cleavage site
  • the 682 RRAR 685 mutation is 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the full-length extracellular domain c3 of the mutated SARS-CoV-2 Delta variant Spike protein has an amino acid sequence as shown in SEQ ID NO: 47. In the sequence, there is no signal peptide, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d1 is shown in SEQ ID NO: 48.
  • 70 amino acid residues are truncated at the C terminus, and the original signal peptide is: MFVFLVLLPLVSS (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 , is underlined and bolded, and the double mutation K986P/V987P is included, underlined and Italicized.
  • the mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d2, its amino acid sequence is shown in SEQ ID NO: 49. In the sequence, 70 amino acid residues were truncated at the C terminus, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5).
  • the signal peptide Italicized the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.
  • the mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d3, its amino acid sequence is shown in SEQ ID NO: 50.
  • 70 amino acid residues are truncated at the C terminus, without the signal peptide, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains the double mutation K986P/V987P. , underlined and italicized.
  • the present invention provides fusion proteins comprising a heterologous scaffold displaying at least one antigenic polypeptide or trimeric protein derived from the coronavirus Spike protein.
  • the coronavirus antigen used is the extracellular domain of the coronavirus Spike protein containing various stable mutations described above or truncated fragments thereof.
  • the Spike protein sequence used includes the sequence shown in any one of SEQ ID NO: 1, 3-4, 6-9, 18-31, 44-50, 62, or is substantially identical thereto. or conservatively modified variants.
  • the expression vector expressing the fusion protein is transfected into the host cell, since the antigen (such as Spike protein) is connected to the self-assembly protein (such as monomeric ferritin subunit), a nanoparticle vaccine showing the antigen (such as Spike protein) on the surface will be produced. .
  • the antigen such as Spike protein
  • the self-assembly protein such as monomeric ferritin subunit
  • Any heterologous scaffold can be used to present antigens in the construction of the vaccines of the invention.
  • This includes virus-like particles (VLPs) such as nanoparticles.
  • VLPs virus-like particles
  • a variety of nanoparticles can be used to produce the vaccines of the invention.
  • nanoparticles for use in the present invention need to be formed from multiple replicas of a single subunit. Nanoparticles are typically spherical, and/or have rotational symmetry (eg, having 3-fold and 5-fold axes), such as having an icosahedral structure exemplified herein.
  • the amino termini of the nanoparticle subunits must be exposed and in close proximity to the 3-fold axis, and the spacing of the three amino termini must closely match the spacing of the carboxyl termini of the trimer-stabilized Spike protein shown.
  • self-assembled nanoparticles are employed that are about 25 nm or less in diameter (typically assembled from 12, 24, or 60 subunits) and have a 3-fold axis on the particle surface. Such nanoparticles provide suitable particles to produce multivalent vaccines.
  • coronavirus antigens may be presented on self-assembling nanoparticles, such as self-assembling nanoparticles derived from ferritin (FR) as exemplified herein.
  • Ferritin is a globular protein found in animals, bacteria, and plants whose primary role is to control multinucleation by transporting hydrated iron ions and protons to and from the mineralized core Rate and location of Fe(III) 2 O 3 formation.
  • the globular form of ferritin consists of a monomeric subunit protein (also called a monomeric ferritin subunit), which is a polypeptide with a molecular weight of approximately 17-20 kDa.
  • the sequences of the subunits of these proteins are known in the art.
  • the nanoparticle vaccines of the invention may use any of these known nanoparticles, as well as conservatively modified variants thereof or that are substantially identical (e.g., at least 90%, 95% or 99% identical) Sequence variants.
  • the fusion protein of the invention comprises a nanoparticle subunit sequence (for example, Helicobacter pylori non-heme monomeric ferritin subunit, the amino acid sequence of which is shown in SEQ ID NO: 10), or its conserved Modified variants or sequences substantially identical thereto.
  • a nanoparticle subunit sequence for example, Helicobacter pylori non-heme monomeric ferritin subunit, the amino acid sequence of which is shown in SEQ ID NO: 10
  • the C-terminus of the extracellular domain of the coronavirus Spike protein or its truncated fragment containing mutations is fused to the N-terminus of the self-assembled nanoparticle (NP) subunit.
  • the C-terminus of the extracellular domain of the coronavirus Spike protein containing mutations or a truncated fragment thereof is connected to the N-terminus of the nanoparticle subunit via a linker, such as GGGGS or GGGGSGGGGS.
  • one or more linkers can be used to connect and maintain the overall activities of different functional proteins unchanged.
  • linkers typically contain short peptide sequences, such as GS-rich peptides.
  • a linker or linker motif can 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 the nanoparticle scaffold sequence. Recombinant production of fusion proteins of the invention can be based on the protocols described herein and/or other methods that have been described in the art.
  • Exemplary fusion protein sequences are as follows:
  • Fusion protein 1 The C-terminus of the full-length extracellular domain I-1 (as shown in SEQ ID NO: 3) of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO: 11) and the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO: 10) to obtain fusion protein 1, the amino acid sequence of which is shown in SEQ ID NO: 12.
  • the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains double
  • the mutation K986P/V987P is underlined and italicized, and the linker is italicized and bolded.
  • Fusion protein 2 The C-terminus of the full-length extracellular domain I-2 of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein (as shown in SEQ ID NO: 4) is passed through the linker GGGGS (as shown in SEQ ID NO: 11) and the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO: 10) to obtain fusion protein 2, the amino acid sequence of which is shown in SEQ ID NO: 13. In the sequence, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5).
  • the signal peptide is marked in italics, and the S1/S2 cleavage site
  • the mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the linker is italicized and bolded.
  • Fusion protein 3 The C-terminus of the C-terminal truncated fragment I-4 (as shown in SEQ ID NO: 7) of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 3, whose amino acid sequence is shown in SEQ ID NO:14 . In the sequence, 70 amino acid residues are truncated at the C terminus.
  • the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics.
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 . It is underlined and bolded.
  • the double mutation K986P/V987P is also underlined and italicized.
  • the linker is italicized and bolded.
  • Fusion protein 4 The C-terminus of the C-terminal truncated fragment I-5 (as shown in SEQ ID NO: 8) of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 4, whose amino acid sequence is shown in SEQ ID NO:15 .
  • Fusion protein 5 The C-terminus of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein full-length extracellular domain I-6-1 (as shown in SEQ ID NO:19) is passed through the linker GGGGS (as shown in SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 5, whose amino acid sequence is shown in SEQ ID NO:32 .
  • the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains double
  • the mutation K986P/V987P is underlined and italicized, and the linker is italicized and bolded.
  • Fusion protein 6 The C-terminus of the full-length extracellular domain I-7 of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein (as shown in SEQ ID NO:20) is passed through the linker GGGGS (as shown in SEQ ID NO :11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 6, the amino acid sequence of which is shown in SEQ ID NO:33.
  • the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5).
  • the signal peptide is marked in italics, and the S1/S2 cleavage site
  • the mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the linker is italicized and bolded.
  • Fusion protein 7 The C-terminus of the C-terminal truncated fragment I-9 (shown in SEQ ID NO: 22) of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein is passed through the linker GGGGS (such as SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 7, whose amino acid sequence is as shown in SEQ ID NO:34 Show. In the sequence, 70 amino acid residues are truncated at the C terminus.
  • the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics.
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 . It is underlined and bolded.
  • the double mutation K986P/V987P is also underlined and italicized.
  • the linker is italicized and bolded.
  • Fusion protein 8 The C-terminus of the C-terminal truncated fragment I-10 (shown in SEQ ID NO: 23) of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein is passed through the linker GGGGS (such as SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 8, whose amino acid sequence is as shown in SEQ ID NO:35 Show.
  • Fusion protein 9 The C-terminus of the full-length extracellular domain I-12 (as shown in SEQ ID NO: 26) of the mutated SARS-CoV-2 Omicron variant BA.3Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO: 11) and the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO: 10) to obtain fusion protein 9, the amino acid sequence of which is shown in SEQ ID NO: 36.
  • the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains double
  • the mutation K986P/V987P is underlined and italicized, and the linker is italicized and bolded.
  • Fusion protein 10 The C-terminus of the full-length extracellular domain I-13 (as shown in SEQ ID NO: 27) of the mutated SARS-CoV-2 Omicron variant BA.3Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO: 11) and the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO: 10) to obtain fusion protein 10, the amino acid sequence of which is shown in SEQ ID NO: 37.
  • the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5).
  • the signal peptide is marked in italics, and the S1/S2 cleavage site
  • the mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the linker is italicized and bolded.
  • Fusion protein 11 The C-terminus of the C-terminal truncated fragment I-15 (as shown in SEQ ID NO: 29) of the extracellular domain of the mutant SARS-CoV-2 Omicron variant BA.3Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 11, whose amino acid sequence is shown in SEQ ID NO:38 . In the sequence, 70 amino acid residues are truncated at the C terminus.
  • the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics.
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 . It is underlined and bolded.
  • the double mutation K986P/V987P is also underlined and italicized.
  • the linker is italicized and bolded.
  • Fusion protein 12 The C-terminus of the C-terminal truncated fragment I-16 (as shown in SEQ ID NO: 30) of the extracellular domain of the mutant SARS-CoV-2 Omicron variant BA.3Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 12, whose amino acid sequence is shown in SEQ ID NO:39 .
  • Fusion protein C1 the full-length extracellular domain c1 of the mutated SARS-CoV-2 Delta variant Spike protein (As shown in SEQ ID NO: 45)
  • the C-terminus of Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO: 10) is connected through the linker GGGGS (as shown in SEQ ID NO: 11) N-terminal ligation obtains fusion protein C1, whose amino acid sequence is shown in SEQ ID NO: 51.
  • the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains double
  • the mutation K986P/V987P is underlined and italicized, and the linker is italicized and bolded.
  • Fusion protein C2 The C-terminus of the full-length extracellular domain c2 of the mutated SARS-CoV-2 Delta variant Spike protein (as shown in SEQ ID NO:46) is passed through the linker GGGGS (as shown in SEQ ID NO:11) The fusion protein is obtained by ligating the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (as shown in SEQ ID NO:10) White C2, its amino acid sequence is shown in SEQ ID NO:52.
  • the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5).
  • the signal peptide is marked in italics, and the S1/S2 cleavage site
  • the mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
  • the linker is italicized and bolded.
  • Fusion protein D1 The C-terminus of the mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d1 (as shown in SEQ ID NO:48) is passed through the linker GGGGS (as shown in SEQ ID NO:11 (shown in SEQ ID NO: 10) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO: 10) to obtain the fusion protein D1, the amino acid sequence of which is shown in SEQ ID NO: 53.
  • Fusion protein D2 The C-terminus of the mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d2 (as shown in SEQ ID NO:49) is passed through the linker GGGGS (as shown in SEQ ID NO:11 (shown in SEQ ID NO: 10) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO: 10) to obtain fusion protein D2, the amino acid sequence of which is shown in SEQ ID NO: 54.
  • Mature fusion protein G The C-terminus of the C-terminal truncated fragment g1 (as shown in SEQ ID NO: 62) of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.1 Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (as shown in SEQ ID NO:10) to obtain fusion protein G1. In the sequence of fusion protein G1, the C-terminus is truncated.
  • MFVFLVLLPLVSSQ shown in SEQ ID NO: 2
  • the amino acid sequence of fusion protein G is shown in SEQ ID NO: 61.
  • the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized.
  • the linker is italicized and bolded.
  • Mature fusion protein 1-1 Compared with fusion proteins 1 and 2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 16. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .
  • Mature fusion protein 2-1 Compared with fusion proteins 3 and 4, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 17. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .
  • Mature fusion protein 3-1 Compared with fusion proteins 5 and 6, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 40. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .
  • Mature fusion protein 4-1 Compared with fusion proteins 7 and 8, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 41. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .
  • Mature fusion protein 5-1 Compared with fusion proteins 9 and 10, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 42. In the sequence, the S1/S2 cleavage site 682 RRAR 685 was mutated to 682 GSAS 685 , It is underlined and bolded. The double mutation K986P/V987P is also underlined and italicized. The linker is italicized and bolded.
  • Mature fusion protein 6-1 Compared with fusion proteins 11 and 12, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 43. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .
  • Mature fusion protein C Compared with fusion proteins C1 and C2, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 55. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .
  • Mature fusion protein D Compared with fusion proteins D1 and D2, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 56. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .
  • SEQ ID NO:16, 17, 40-43, 55, 56, and 61 are mature fusion protein sequences with the N-terminal signal peptide (SEQ ID NO: 2 or 5) removed.
  • the invention also encompasses nanoparticle vaccines containing subunits that are substantially identical to any of these exemplified nanoparticle vaccine sequences, or conservatively modified variants thereof sequence.
  • the mutation-containing coronavirus Spike protein extracellular domain or its truncated fragment, fusion protein or Spike protein nanoparticle of the present invention is usually produced by an expression vector, the expression vector contains the mutation-containing coronavirus Spike protein described herein
  • the coding sequence of the extracellular domain or its truncated fragment, fusion protein or Spike protein nanoparticle is usually produced by an expression vector, the expression vector contains the mutation-containing coronavirus Spike protein described herein. Therefore, in some related aspects, the present invention provides polynucleotides (DNA or RNA) encoding the mutated coronavirus Spike protein extracellular domain or truncated fragments thereof, fusion proteins or Spike protein nanoparticles described herein.
  • Some polynucleotides of the present invention encode one of the mutated coronavirus Spike protein extracellular domains described herein or truncated fragments thereof, for example, the SARS-CoV-2 Spike protein shown in SEQ ID NO:7 Truncated fragments of the extracellular domain.
  • Some polynucleotides of the present invention encode the subunit sequence of one of the nanoparticle vaccines described herein, such as the fusion protein sequence shown in SEQ ID NO: 12.
  • the fusion protein expressed in the present invention may not contain an N-terminal signal peptide, or some polynucleotide sequences may additionally encode an N-terminal signal peptide.
  • a polynucleotide encoding a fusion protein may also include an N-terminal signal peptide encoding SEQ ID NO: 2 or 5, or a sequence substantially identical or conservatively modified thereto. Variant sequence.
  • the invention also provides expression vectors having such polynucleotides and host cells (e.g., prokaryotic or eukaryotic cells, such as HEK293, CHO, ExpiCHO and CHO-S cell lines). Fusion proteins encoded by polynucleotides or expressed from vectors are also included in the present invention. As described herein, nanoparticle subunit fused extracellular domains of Spike protein or truncated fragments thereof will self-assemble into nanoparticle vaccines displaying Spike protein or truncated fragments thereof on their surface.
  • host cells e.g., prokaryotic or eukaryotic cells, such as HEK293, CHO, ExpiCHO and CHO-S cell lines.
  • Polynucleotides and related vectors can be produced by standard molecular biology techniques or the protocols exemplified herein. For example, general protocols for cloning, transfection, transient gene expression, and obtaining stable transfected cell lines are well known in the art. has been described in . Mutations can also be introduced into the polynucleotide sequence by PCR, a known method.
  • vectors useful in the present invention can replicate autonomously, that is, the vector exists extrachromosomally, and its replication need not be directly linked to replication of the host cell genome.
  • replication of the vector can be linked to replication of the host chromosomal DNA, for example, the vector can be integrated into the chromosome of the host cell, via a retroviral vector and in a stably transfected cell line.
  • Non-viral vectors and systems include plasmids, episomal vectors (usually with expression cassettes for expressing proteins or RNA) and human artificial chromosomes.
  • Alternative viral vectors include lentiviral or other retrovirus-based vectors, adenovirus, adeno-associated virus, cytomegalovirus, herpesvirus, SV40-based vectors, papillomavirus, HBP, Epstein Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV).
  • a host cell can be any cell carrying a recombinant vector for a protein of the invention, allowing the vector to drive expression of the protein for the invention. It may be prokaryotic, such as any of many bacterial strains, or eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells, including, for example, rodent, simian, or human cells . Cells expressing proteins of the invention may be primary cultured cells or may be established cell lines.
  • cell lines exemplified herein eg, HEK293 cells
  • host cell lines well known in the art may be used in the practice of the present invention. These include, for example, various Cos cell lines, CHO cells, HeLa cells, Sf9 cells, AtT20, BV2 and N18 cells, myeloma cell lines, transformed B cells and hybridomas.
  • Vectors expressing the protein can be introduced into the host cell of choice by any of a number of suitable methods known to those skilled in the art.
  • the method used will depend on the form of the vector.
  • the DNA encoding the protein sequence can be introduced by any of a number of transfection methods, including, for example, liposome-mediated transfection ("lipofectamine"), DEAE-dextran-mediated guided transfection, electroporation or calcium phosphate precipitation.
  • lipofectamine transfection is widely accepted because it is simple to operate and does not require special equipment.
  • transfection can be performed using Lipofectamine (Life Technologies) or LipoTAXI (Stratagene) kits.
  • protein-coding sequences controlled by appropriate expression control elements e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.
  • selectable markers 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 chromosomes.
  • Commonly used selectable markers include neomycin (neo), which is resistant to the aminoglycoside G-418, and hygromycin, which is resistant to hygromycin. (hygro).
  • a recombinant expression vector includes at least one promoter element, a protein coding sequence, a transcription termination signal, and a polyA tail.
  • Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the inserted sequence. Efficient transcription can be obtained through the early and late promoters of SV40, the long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus. Promoters from other cells such as muscle can also be used. Kinesin promoter.
  • Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, pLXSN, pLNCX, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), pSVL , pMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc.
  • Commonly used mammalian cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells.
  • the inserted gene fragment needs to contain a selection marker.
  • selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes to facilitate transfection. Screening isolation of successful cells. The constructed plasmid is transfected into host cells without the above genes, and then cultured in a selective medium. The successfully transfected cells grow in large quantities and produce the desired target protein.
  • mutations can be introduced into the nucleotide sequences encoding the invention using standard techniques known to those skilled in the art, including, but not limited to, site-directed mutagenesis resulting in amino acid substitutions and PCR-mediated mutagenesis.
  • 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 present invention also provides a pharmaceutical composition and a related treatment method.
  • the pharmaceutical composition comprises an effective dose of fusion protein or Spike protein nanoparticles and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to substances approved by a governmental regulatory agency or listed in other recognized pharmacopoeias for use in animals, particularly 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 vehicle with which the active ingredient can be administered to a patient.
  • Such carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.
  • water is a preferred carrier.
  • Saline solutions Liquid and glucose aqueous solution and glycerol solution can also be used as liquid carrier, particularly for injection solution.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc.
  • the pharmaceutical composition can also contain a small amount of wetting agent, emulsifier, or pH buffer such as acetate, citrate or phosphate.
  • Antibacterial agents such as benzyl alcohol or methyl parahydroxybenzoate, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tension regulating agents such as sodium chloride or dextrose are also foreseeable.
  • These pharmaceutical compositions can take the form of solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release preparation, etc.
  • the pharmaceutical composition can be formulated into suppositories with traditional adhesives and carriers such as triglycerides.
  • Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.
  • compositions will contain a clinically effective dose of 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 preparation should be suitable for the mode of administration.
  • the preparation can be packaged in an ampoule, a disposable syringe, or a multi-dose vial made of glass or plastic.
  • % related to the preparation components refers to the weight volume (w/v) percentage.
  • 1% stabilizer in the preparation means that 100 mL of preparation contains 1 g of stabilizer, or the stabilizer content is 0.01g/mL.
  • references herein to "about” or “approximately” refer to the recited value and its range of ⁇ 10%, ⁇ 5%, or ⁇ 1%.
  • compositions, methods, etc. include the listed elements (such as components in the composition, steps in the method, etc.), but do not exclude other elements.
  • Consisting essentially of is used to define compositions and methods, it is meant to exclude other elements that materially affect the composition for its intended use, but does not exclude elements that do not materially affect the characteristics of the composition or method. .
  • Consisting of means excluding elements not specifically enumerated. Embodiments defined by each of these transitional terms are within the scope of the invention. For example, when a composition is described as including ingredients A, B, and C, compositions consisting essentially of A, B, and C and compositions consisting of A, B, and C are independently within the scope of the invention .
  • buffer is also known as a buffer system, which includes but is not limited to organic acids and their salts, such as succinic acid, acetic acid, citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid or phthalic acid and their salts; Tris , or inorganic acids and their salts, such as phosphate buffers.
  • amino acids can also be used as buffering agents. Such amino acids include, but are not limited to, glycine, histidine, arginine, lysine, ornithine, isoleucine, leucine, alanine, glutamic acid or aspartic acid and salts thereof.
  • the amount of buffering agent in the present invention refers to the total amount of buffering pairs in the buffering system that constitutes the buffering agent.
  • molar concentration is used as the unit for the amount of buffer, and its numerical value refers to the molar concentration of the buffer pair in the buffer system of the buffer.
  • a histidine buffer consists of histidine and histidine hydrochloride
  • a given concentration of histidine The buffer (eg 20mM) is a combined concentration of histidine and histidine hydrochloride.
  • histidine hydrochloride also known as histidine hydrochloride
  • histidine hydrochloride can be anhydrous histidine hydrochloride or histidine hydrochloride hydrate, such as histidine hydrochloride monohydrate.
  • surfactant includes, but is not limited to, polysorbate (Tween, such as polysorbate 20 and polysorbate 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS) ); sodium lauryl sulfate; octyl glycoside sodium salt; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine or stearyl sulfobetaine; lauryl sulfobetaine Acid, myristyl sarcosine, linoleyl sarcosine or stearyl sarcosine; linoleyl betaine, myristyl betaine or cetyl betaine; laurolamidopropyl betaine , cocamidopropyl betaine, linoleamidopropyl betaine, myristamido
  • lauramidopropyl betaine propyl myristamidopropyldimethylamine, palmitamidopropyldimethylamine or isostearamidopropyldimethylamine; sodium methylcocoyltaurate or methyloleyltaurate Disodium acid acid; polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (such as Pluronics, PF68, etc.); etc.
  • the surfactant is polysorbate 80, also known as PS80, Tween 80 or Tween 80.
  • salts refer to acidic salts formed with inorganic and/or organic acids, and basic salts formed with inorganic and/or organic bases. Although other salts may be used, pharmaceutically acceptable (ie, nontoxic, physiologically acceptable) salts are preferred.
  • Exemplary basic salts include ammonium salts, alkali metal salts (e.g., sodium, lithium, and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), zinc salts, and organic bases (e.g., organic amines) (e.g., N-Me -Salts formed by -D-glucosamine, choline, trimethylamine, dicyclohexylamine, tert-butylamine) and salts formed with amino acids (such as arginine, lysine), etc.
  • alkali metal salts e.g., sodium, lithium, and potassium salts
  • alkaline earth metal salts e.g., calcium and magnesium salts
  • zinc salts e.g., zinc salts
  • organic bases e.g., organic amines
  • organic amines e.g., N-Me -Salts formed by -D-glucosamine, choline, trimethylamine,
  • Stability and “stable” herein refer to the fact that in preparations containing antibodies, the antibodies (including antibody fragments thereof) do not, or only rarely, develop under given production, preparation, transportation and/or storage conditions. Aggregation, degradation or fragmentation. A “stable” formulation retains biological activity under given conditions of production, preparation, transportation and/or storage. The degree of aggregation, degradation or fragmentation of the preparation can be measured by techniques such as SEC-HPLC, IEC-HPLC, CE-SDS (NR), light inspection and turbidity, insoluble particles, DLS detection of particle size, etc., thereby The antibodies were assessed for stability.
  • a pharmaceutical composition can comprise a fusion protein or Spike protein nanoparticle, and a polynucleotide or vector encoding a fusion protein described herein.
  • viral eg, SARS-CoV-2
  • Spike protein extracellular domains or trimers of truncated fragments thereof can be used to prevent and treat corresponding viral infections.
  • vaccines containing 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 SARS-CoV-2 antigens or vaccines to prevent or treat SARS-CoV-2 infection in human subjects.
  • Some embodiments of the invention relate to the use of SARS-CoV antigens or vaccines to prevent or treat SARS-CoV infection.
  • the corresponding Spike protein nanoparticles or fusion proteins, or the fusion protein encoding fusion proteins described herein are administered to a subject in need of preventing or treating a disease (e.g., SARS-CoV-2 infection).
  • a disease e.g., SARS-CoV-2 infection.
  • the Spike protein nanoparticles, fusion proteins, or polynucleotides encoding fusion proteins disclosed herein are included in pharmaceutical compositions.
  • Pharmaceutical compositions may be therapeutic or prophylactic formulations.
  • the pharmaceutical composition may additionally comprise one or more pharmaceutically acceptable carriers, and optionally other therapeutic ingredients (eg, antiviral agents).
  • Various pharmaceutically acceptable additives may also be used in the pharmaceutical compositions.
  • compositions of the invention are vaccine compositions.
  • suitable adjuvants may additionally be included.
  • suitable adjuvants include, for example, aluminum adjuvants such as 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.
  • a suitable adjuvant for the vaccine of the invention is SEPIVAC SWE TM , a squalene-based oil-in-water emulsion.
  • compositions of the present invention can be used in a variety of therapeutic or preventive applications, such as for treating SARS-CoV-2 infection in a subject or for causing 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, for example, to induce the production of broad-spectrum neutralizing antibodies against the virus.
  • the vaccine composition of the present invention can be administered to provide preventive protection against viral infection.
  • the therapeutic and preventive applications of vaccines derived from other antigens described herein can be similarly performed.
  • the pharmaceutical composition of the present invention can be administered to a subject by a variety of administration methods known to those of ordinary skill in the art, for example, by parenteral routes such as intramuscular routes, subcutaneous routes, intravenous routes, intra-arterial routes, joint routes, and intraperitoneal routes.
  • parenteral routes such as intramuscular routes, subcutaneous routes, intravenous routes, intra-arterial routes, joint routes, and intraperitoneal routes.
  • the treatment methods of the present invention relate to methods for blocking coronaviruses (such as SARS-CoV or SARS-CoV-2) from entering host cells (such as human host cells), methods for preventing coronavirus Spike proteins from binding to host receptors, and methods for treating acute respiratory diseases associated with coronavirus infection.
  • the methods of treatment and pharmaceutical compositions described herein can be used in combination with other known therapeutic agents and/or methods for treating or preventing coronavirus infection.
  • known therapeutic agents and/or methods include, for example, nuclease analogs or protease inhibitors (e.g., remdesivir), monoclonal antibodies against one or more coronaviruses, immunosuppressants or anti-inflammatory drugs (e.g., sarilumab or tocilizumab), ACE inhibitors, vasodilators, or any combination thereof.
  • 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 and Spike protein nanoparticles described herein.
  • Appropriate antibiotics may be determined based on the specific disease or condition to be treated or prevented, the subject's severity, age, and other personal attributes of the particular subject (e.g., the overall state of the subject's health). original amount. Determination of effective doses is also guided by studies in animal models and subsequently by clinical trials in humans, and by dosing regimens that significantly reduce the occurrence or severity of the target disease condition or symptoms in subjects.
  • a pharmaceutical composition for preventive applications, before any symptoms, such as before infection, a pharmaceutical composition is provided.
  • the preventive administration of the pharmaceutical composition is used to prevent or improve any subsequent infection.
  • the subject to be treated is, for example, a subject who has been infected (e.g., SARS-CoV-2 infection) or a subject at risk of infection (e.g., SARS-CoV-2 infection) due to exposure or possible exposure to a virus (e.g., SARS-CoV-2).
  • the subject's infection e.g., SARS-CoV-2 infection
  • symptoms associated with infection e.g., SARS-CoV-2 infection
  • the pharmaceutical composition is provided at or after the onset of symptoms of a disease or infection, such as after the onset of symptoms of an infection (eg, SARS-CoV-2 infection) or after the infection is diagnosed.
  • pharmaceutical compositions may be provided prior to anticipated exposure to the virus in order to attenuate the expected severity, duration or extent of infection and/or associated disease conditions following exposure or suspected exposure to the virus or after the initial onset of actual infection.
  • the pharmaceutical compositions of the present invention may be combined with other agents known in the art for the treatment or prevention of infection by relevant pathogens, such as SARS-CoV-2 infection.
  • kits include additional components including packaging, instructions for use, and various other reagents, such as buffers, substrates, antibodies or ligands (e.g., control antibodies or ligands), and detection Reagents.
  • additional components including packaging, instructions for use, and various other reagents, such as buffers, substrates, antibodies or ligands (e.g., control antibodies or ligands), and detection Reagents.
  • 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 recombinant cells of Spike protein nanoparticles, receptor-mediated endocytosis, construction of nucleic acids as part of retroviruses or other vectors, etc.
  • 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: 12-17, 32-43, 51-56, and 61) is cloned into an expression vector, then electroporated into CHO-K1 cells, cultured and purified to obtain the fusion protein.
  • the three-dimensional structure of the fusion protein was analyzed using cryo-electron microscopy (Cryo-EM).
  • the extracellular domain of the SARS-CoV-2 Spike protein or its truncated fragment was connected to the N-terminus of the monomeric ferritin subunit without interfering with the self-regulation of ferritin. Assembled, the nanoparticles formed well and showed spikes on the surface.
  • Example 2 Test of the binding ability of fusion protein to hACE2 protein
  • This test detects the binding ability of the fusion protein to human ACE2 protein (hACE2) through ELISA, thereby evaluating whether the Spike protein-ferritin fusion protein of the present invention can well display the key antigenic epitopes of the Spike protein.
  • the method is briefly described as follows: Add 100 ⁇ L of 2 ⁇ g/mL antigen (WT-Spike protein (shown in SEQ ID NO: 57), Delta-Spike protein ( As shown in SEQ ID NO:58), BA.5-Spike protein (as shown in SEQ ID NO:59), fusion protein D, fusion protein 2-1) solution, coated at 4°C overnight; use PBST (containing 0.05 % Tween-20 PBS buffer); add blocking solution (PBST containing 3% BSA) to each reaction well and incubate in a 37°C incubator for 2 hours; wash 3 times with PBST after blocking; add gradient dilutions of human ACE2-his-biotin (Yiqiao Shenzhou, product number: 10108-H27B-B), starting concentration is 2.5 ⁇ g/mL, 3-fold gradient dilution, a total of 10 serial dilution concentrations, 100 ⁇ L per well, cultured at 37°C Incubate for 1.5h in
  • Spike protein such as WT-Spike protein, Delta-Spike protein or BA.5-Spike protein
  • clone the DNA sequence encoding the target protein into an expression vector then electroporate CHO-K1 cells, and pressurize with MSX After screening, mother clones with high expression levels are selected for culture and purification to obtain the target protein.
  • hACE2 binds to fusion protein D, WT-Spike protein and Delta-Spike protein with similar affinities, and the EC 50 values are 9.2, 5.8 and 8.1ng/mL respectively ( Figure 1); hACE2 and Fusion protein 2-1 and BA.5-Spike protein binding also have similar affinities, with EC 50 values of 24.0 and 81.5ng/mL respectively ( Figure 2). Both fusion protein 2-1 and fusion protein D can bind well to human ACE2, indicating that the nanoparticle structures presented by fusion protein 2-1 and fusion protein D can well display antigen epitopes.
  • the biofilm interference technology was used to bind the fusion protein to hACE2, and the instrument was PALL's Biomolecular Interaction Analyzer (Fortebio Octet QKe) to evaluate whether the fusion protein could display the antigenic epitope well.
  • Multi-channel parallel quantitative analysis of Spike protein WT-Spike protein (see step 1.1 of Example 2), Delta-Spike protein (see step 1.1 of Example 2), BA.5-Spike protein (see step 1.1 of Example 2) and fusion Protein (Fusion Protein D, Fusion Protein 2-1), the concentration gradient was set as: 0, 50, 100, 200 and 400nM, hACE2-Biotin (Acro biosystems, Cat. No. AC2-H5257) was fixed on SA Biosensors (Octet, Cat. No. 2107002811 ). The results are shown in Table 1.
  • the binding affinity of fusion protein D to hACE2 is significantly stronger than the affinity of WT-Spike protein and Delta-Spike protein to hACE2.
  • Fusion protein 2-1 and hACE2 and BA.5-Spike protein and hACE2 both show extremely high affinity, which is beyond the detection range of the instrument (KD ⁇ 1.0E-12).
  • the same affinity of fusion protein 2-1 and hACE2 It is also significantly stronger than WT-Spike protein and hACE2.
  • Both fusion protein D and fusion protein 2-1 have extremely strong affinity with hACE2, indicating that the nanoparticle structure presented by fusion protein 2-1 and fusion protein D can well display antigen epitopes.
  • mice Female BALB/c mice (8 weeks old) received intramuscular injection of bivalent vaccine (fusion protein D and fusion protein with a mass ratio of 1:1) with SWE adjuvant (SEPPIC S.A., Catalog No. 80748J, Lot No. 210721010001) or without adjuvant. Protein 2-1), the experimental design is shown in Table 2, and blood was collected to collect serum on the 14th day. The ELISA method was used to detect the IgG antibody titer of serum against Spike protein (original strain, Delta, and Omicron variant strains BA.1 and BA.5).
  • Dilute WT-Spike protein see step 1.1 of Example 2
  • Delta-Spike protein see step 1.1 of Example 2
  • BA.1-Spike protein shown in SEQ ID NO: 60
  • construction method See step 1.1 of Example 2
  • BA.5-Spike protein see step 1.1 of Example 2
  • a final concentration of 2 ⁇ g/mL and add it to a 96-well enzyme plate (Costar, 9018) at 100 ⁇ L/well at 4°C.
  • PBST PBS buffer containing 0.05% Tween-20
  • blocking solution PBST containing 3% BSA
  • incubate in a 37°C incubator for 2 hours wash twice with PBST, add gradient dilution
  • the mouse serum obtained in step 1.1 of Example 3 (dilute the serum 100 times, and then dilute it 3 times to 11 gradients), 100 ⁇ L/well, incubate at 37°C for 1.5h; wash 3 times with PBST, add 1:10000 dilution Peroxidase-AffiniPure Goat Anti-Mouse IgG (Jackson, Cat.
  • Plate reading Use the analysis software SoftMax pro 7.02 software that comes with the microplate reader, set the detection wavelength to 450nm for reading, and fit the obtained readings with a nonlinear four-parameter equation curve model.
  • the equation is: Among them, A is the lower limit of absorbance, B represents the slope of the curve, and C is the antibody concentration corresponding to half of the maximum response value (EC 50 ). D is the upper limit of absorbance.
  • fusion protein D fusion protein D
  • fusion protein 2-1 bivalent vaccine
  • SWE adjuvant SEPPIC SA, Cat. No. 80748J, Lot No. 210721010001
  • control mice were only given SWE adjuvant.
  • Blood was collected on the 14th day (D14) and the 35th day (D35) after immunization.
  • Table 3 Used to detect serum anti-Spike protein IgG titer and SARS-CoV-2 Spike pseudovirus by ELISA Neutralization experiments detect cross-neutralizing antibody titers against multiple pseudoviruses.
  • Dilute WT-Spike protein see step 1.1 of Example 2), Delta-Spike protein (see step 1.1 of Example 2), BA.1-Spike protein (see step 1.2 of Example 3), and BA.5 respectively with 1 ⁇ PBS.
  • -Spike protein (see step 1.1 in Example 2) to a final concentration of 2 ⁇ g/mL, added to a 96-well enzyme plate (Costar, Cat.
  • Plate reading Use the analysis software SoftMax pro 7.02 software that comes with the microplate reader, set the detection wavelength to 450nm for reading, and fit the obtained reading OD value with a nonlinear four-parameter equation curve model.
  • the equation is: Among them, A is the lower limit of absorbance, B represents the slope of the curve, C is the antibody concentration (EC 50 ) corresponding to half of the maximum response value, and D is the upper limit of absorbance.
  • mice given only adjuvant In all the tested dose groups, no anti-Spike protein IgG titers were detected in mice given only adjuvant. As shown in Figures 4a, 4c, 4e, and 4g, all mice produced IgG antibodies against WT-Spike protein, Delta-Spike protein, BA.1-Spike protein, and BA.5-Spike protein on the 14th day after the primary immunization, and the geometric mean antibody titer (GMT) showed a clear dose-effect relationship.
  • GTT geometric mean antibody titer
  • Anti-Spike protein IgG titers in all mice on day 35 after the second booster immunization compared with those after the primary immunization were significantly elevated.
  • mice receiving fusion protein 2-1 had significantly lower antibody titers against WT-Spike protein and Delta-Spike protein ( Figure 4b, 4d); but fusion protein 2-1
  • the antibody titer against BA.5-Spike protein in the group was significantly higher than that against fusion protein D ( Figure 4h); compared with the monovalent vaccine fusion protein D and fusion protein 2-1, the bivalent vaccine induced antibodies against WT-Spike protein and Delta- High antibody titers of Spike protein, BA.1-Spike protein and BA.5-Spike protein IgG.
  • the results of the anti-Spike protein IgG antibody titer showed that the bivalent vaccine has better immunogenicity than the monovalent vaccine fusion protein D or fusion protein 2-1, and the titer of two doses of vaccination is significantly better than that of one dose of vaccination , the antibody titers against different mutant strains remained better than or equal to the monovalent vaccine.
  • CC group cell control group
  • VC group virus control group
  • CC group 25 ⁇ L/well for each other; put the mouse serum collected on the 35th day of step 1.1 of Example 4, group 3, group 6, and group 9, incubate at 56°C for 30 minutes to inactivate complement, and dilute it 40 times with DMEM complete culture medium , then perform 2-fold gradient dilution, dilute 12 gradients, and add 50 ⁇ L/well of the test group to the 96-well white plate where the pseudovirus has been added.
  • the CC group adds 75 ⁇ L/well of DMEM complete medium, and the VC group adds DMEM complete medium. 50 ⁇ L/well; shake the 96-well white plate thoroughly and mix well, then place it in an incubator and incubate at 37°C for 1 hour. Take out the 96-well white plate after incubation for 1 hour, add ACE2-293 cell suspension (2 ⁇ 10 4 cells/well) at 50 ⁇ L/well, gently blow evenly with a pipette, and place the 96-well white plate in the incubator for culture.
  • ACE2-293 cells The construction method of ACE2-293 cells is as follows: culture HEK293 cells in DMEM complete medium containing 10% FBS, and use lipofectamine 2000 transfection reagent (Thermo Fisher, 11668019) to transfect ACE2 expression plasmid (Yiqiao Shenzhou, HG10108-M) Transfection, followed by pressure selection with hygromycin (200 ⁇ g/ml) and flow sorting (using 10 ⁇ g/ml anti-ACE2 and PE-conjugated Anti-Human IgG-Fc), cells continued to amplify to select out PE Single clones with a positive rate of >90% were amplified in the next step, and HEK293 cells expressing ACE2, namely ACE2-293 cells, were screened out.
  • lipofectamine 2000 transfection reagent Thermo Fisher, 11668019
  • ACE2 expression plasmid Yiqiao Shenzhou, HG10108-M
  • the pseudoviruses used in this experiment SARS-CoV-2 original strain pseudovirus (Vazyme, product number DD1702-03), SARS-CoV-2 Delta pseudovirus (Vazyme, product number DD1754-03), SARS-CoV-2 BA .5 Pseudovirus (Vazyme, DD1776-03), SARS-CoV-2 BQ.1.1 Pseudovirus (Vazyme, Cat. No. DD1792-03), SARS-CoV-2 XBB Pseudovirus (Vazyme, DD1794-03), SARS-CoV -2 XBB.1.5 Pseudovirus (Vazyme, DD1797-03).
  • the titer of fusion protein 2-1 against SARS-CoV-2 original strain pseudovirus and SARS-CoV-2 Delta pseudovirus is significantly lower than that of fusion protein D and bivalent vaccine; the titer of fusion protein D against SARS-CoV-2 BA.5
  • the titers of pseudovirus, SARS-CoV-2 BQ.1.1 pseudovirus, SARS-CoV-2 XBB pseudovirus and SARS-CoV-2 XBB.1.5 pseudovirus were significantly lower than those of fusion protein 2-1 and bivalent vaccine; while The bivalent vaccine maintained high titers against all pseudovirus strains tested. This shows that the bivalent vaccine is broad-spectrum and has high neutralizing antibody titers against a variety of strains.
  • the inhibitory titers of mouse serum after secondary immunization with the bivalent vaccine against the new coronavirus mutant strains BA.5, BQ.1 and XBB true virus were tested.
  • the microplate method was used to evaluate the neutralizing titer (IC99 Titer) of the mouse serum collected on the 35th day of step 1.1 of Group 9 of Example 4 against the new coronavirus mutant strains BA.5, BQ.1 and XBB true virus.
  • the principle is: after the virus infects sensitive target cells (VERO-E6), it causes morphological changes in the cells and the occurrence of cytopathic effect (CPE).
  • CPE cytopathic effect
  • Example 5 Immunogenicity of bivalent vaccine as booster shot in mice that have received 2 doses of inactivated vaccine
  • the ELISA method was used to detect the serum anti-Spike protein (original strain, Delta, Omicron BA.1 and BA.5) IgG titers, and the pseudovirus neutralization experiment was used to detect the cross-neutralizing antibody titers against multiple pseudoviruses.
  • Dilute WT-Spike protein (see step 1.1 of Example 2 for the preparation method), Delta-Spike protein (see step 1.1 of Example 2 for the preparation method), and BA.1-Spike protein (see step 1.1 of Example 2 for the preparation method) with 1 ⁇ PBS respectively. 1.2), BA.5-Spike protein (for the preparation method, see step 1.1 of Example 2) to a final concentration of 2 ⁇ g/mL, add 100 ⁇ L/well into a 96-well enzyme plate (Costar, Cat.
  • Plate reading Use the analysis software SoftMax pro 7.02 software that comes with the microplate reader, set the detection wavelength to 450nm for reading, and fit the obtained readings with a nonlinear four-parameter equation curve model.
  • the equation is: Among them, A is the lower limit of absorbance, B represents the slope of the curve, C is the antibody concentration (EC 50 ) corresponding to half of the maximum response value, and D is the upper limit of absorbance.
  • the serum anti-Spike protein IgG titers of both groups of mice were at low levels, among which, against BA.1-Spike protein and BA.5-Spike The antibody titer of the protein was significantly lower than that of WT-Spike protein and Delta-Spike protein.
  • the anti-Spike protein IgG titers of mice increased significantly regardless of whether they were vaccinated with inactivated vaccine or bivalent vaccine.
  • the anti-Spike protein IgG titers of the bivalent vaccine group (Group 2) against Wildtype, Delta, BA.1 and BA.5 strains were much higher than those of the inactivated vaccine group (Group 1).
  • the inhibitory ability of immune serum against the original SARS-CoV-2 strain, Delta variant strain, and variant strains BF.7, XBB.1, etc. was tested. Dilute the pseudovirus 50 times with DMEM complete culture medium, add the pseudovirus dilution solution to the 96-well white plate, and set up a test group, a cell control group (CC group) and a virus control group (VC group), except for the CC group The remaining 25 ⁇ L/well; the mouse serum collected in step 1.1 of Example 5 was placed at 56°C for 30 minutes to inactivate complement, diluted 40 times with DMEM complete culture medium, and then 2 times gradient diluted, diluted 12 gradients, test group Add 50 ⁇ L/well to the 96-well white plate with pseudovirus in sequence.
  • CC group cell control group
  • VC group virus control group
  • CC group For the CC group, add 75 ⁇ L/well of DMEM complete medium.
  • VC group For the VC group, add 50 ⁇ L/well of DMEM complete medium. Shake and mix the 96-well white plate thoroughly and place it on the Incubate in the incubator at 37°C for 1 hour. Take out the 96-well white plate after incubation for 1 hour, add ACE2-293 cell suspension (2 ⁇ 10 4 cells/well) at 50 ⁇ L/well, gently blow evenly with a pipette, and place the 96-well white plate in the incubator at 37°C. .
  • the pseudoviruses used in this experiment SARS-CoV-2 original strain pseudovirus (Vazyme, product number DD1702-03), SARS-CoV-2 Delta pseudovirus (Vazyme, product number DD1754-03), SARS-CoV-2 BF .7 fake virus (Jiman Biotechnology, product number: GM-0220PV100-96T), SARS-CoV-2 XBB.1 fake virus (Jiman Biotechnology, product number: GM-0220PV104-96T).
  • mice in the inactivated vaccine group (Group 1) produced extremely low neutralizing antibody titers against SARS-CoV-2 BF.7 pseudovirus ( Figure 8c), and only 1 mouse was resistant to SARS-CoV-2 XBB
  • the .1 pseudovirus produced trace amounts of neutralizing antibodies ( Figure 8d); while the bivalent vaccine group (Group 2) produced both SARS-CoV-2 BF.7 pseudovirus and SARS-CoV-2 XBB.1 pseudovirus. Relatively significant neutralizing antibody titers.
  • mice Six-week-old Balb/C female mice were divided into two groups and immunized by intramuscular injection according to the doses in Table 5 on day 0 (D0) and day 21 (D21), respectively. The mice were euthanized 2 weeks after the second immunization, and the spleens were removed for ELIspot assay.
  • the bivalent vaccine is fusion protein D and fusion protein 2-1 with a mass ratio of 1:1; the adjuvant is SWE adjuvant.
  • the Spike protein peptide pool of the original strain (wildtype, WT) and variants (Delta, BA.5) of the new coronavirus was used as a stimulant to stimulate the spleen lymphocytes of immunized mice.
  • Phorbol 12-myristate 13-acetate (PMA) (MedChemExpress, Cat. No. 16561-29-8) was used as a positive control, and blank culture medium was used as a negative control.
  • the ELISpot method was used to detect the secretion of IFN- ⁇ , IL-2, IL-4 and other cytokines by the cells.
  • Ack lysing buffer Gibco, A10492-01
  • ELISpot plate Incubate the ELISpot plate at 37°C, 5% CO2 for 48 hours. Discard the cell fluid in the plate and wash the plate with sterile PBS. Dilute the detection antibodies corresponding to the three ELISpot plates to 1 ⁇ g/mL with PBS solution containing 0.5% FBS. After filtering with a 0.22 ⁇ m filter, add 100 ⁇ L/well to the corresponding ELISpot plate and incubate at room temperature for 2 hours. Wash the plate with sterile PBS. Streptavidin-HRP (Jackson; Cat. No.: 016-030-084) was diluted 1000 times with PBS solution containing 0.5% FBS.
  • the IFN- ⁇ ELISpot results are shown in Figure 9a.
  • the Spike protein peptide pool of the original strain of the new coronavirus and the Spike protein peptide of the new coronavirus mutant strain Delta were used. Both the pool and the Spike protein peptide pool of the new coronavirus mutant strain BA.5 can stimulate the activation of IFN- ⁇ -secreting T cells. Among them, T cells respond to the Spike protein peptide pool of the new coronavirus mutant strain Delta and the new coronavirus mutant strain BA.5. Sp The response of the ike protein peptide pool is higher than that of the Spike protein peptide pool of the original strain of the new coronavirus.
  • the IL-2 and IL-4 ELISpot results are shown in Figures 9b and 9c respectively. They are the same as the IFN- ⁇ ELISpot results.
  • the Spike protein peptide pool of the virus variant BA.5 can stimulate the activation of IL-2 secreting cells or IL-4 secreting cells, and the Spike protein peptide pool of the new coronavirus variant Delta and the new coronavirus variant BA.5 The stimulating effect of Spike protein peptide pool is more significant.
  • the research design is shown in Table 6. Healthy Sprague-Dawley rats (SPF grade), half male and half female, were given a single intramuscular injection of 0 or 80 ⁇ g/rat bivalent vaccine (fusion protein D with a mass ratio of 1:1) on day 1 (D1). and fusion protein 2-1), the injection volume was 0.5mL/animal, and blood was collected on the 15th day (D15).
  • the ELISA method was used to detect serum anti-Spike protein (original strain, Delta, BA.1 and BA.5) IgG titers.
  • the adjuvant is SWE adjuvant
  • WT-Spike protein (see step 1.1 of Example 2), Delta-Spike protein (see step 1.1 of Example 2), and BA.5-Spike protein (see step 1.1 of Example 2) were diluted with 1 ⁇ PBS to a final concentration of 2 ⁇ g/mL, and 100 ⁇ L/well were added to a 96-well ELISA plate (Costar, catalog number: 9018) and incubated at 4°C overnight; washed twice with PBST (0.05% volume of Tween-20 was added to 1 ⁇ PBS); blocking solution (PBST containing 3% BSA) was added and incubated in a 37°C incubator for 2 hours; washed twice with PBST; the mouse serum obtained in step 1.1 of Example 7 was added with gradient dilution (the serum was diluted 100 times, and then 11 gradients were diluted 3 times), 100 ⁇ L per well, and incubated at 37°C for 1.5 hours; washed three times with PBST; Goat anti-Rat IgG secondary antibody (H
  • Plate reading Use the analysis software SoftMax pro 7.02 software that comes with the microplate reader, set the detection wavelength to 450nm for reading, and fit the obtained readings with a nonlinear four-parameter equation curve model.
  • the equation is: Among them, A is the lower limit of absorbance, B represents the slope of the curve, C is the antibody concentration (EC 50 ) corresponding to half of the maximum response value, and D is the upper limit of absorbance.
  • the SARS-CoV-2 infected hamster pneumonia model was used to evaluate the animal protection effect of the bivalent vaccine.
  • the bivalent vaccine is fusion protein D and fusion protein 2-1 with a mass ratio of 1:1; the adjuvant is SWE adjuvant.
  • the weight changes of the hamsters in the challenge group were recorded continuously until the end of the experiment. All hamsters were killed 5 days after the challenge, and the viral load and lung tissue pathology were detected.
  • the hamsters in the model group of the challenge group showed weight loss after being challenged with the virus, with the highest average decrease percentage being 5.91%.
  • the body weight of the hamsters in the 1 ⁇ g bivalent vaccine group continued to decrease after challenge, with the highest average decrease percentage being 4.16%, which was not significantly different from the challenge model group (p>0.05).
  • the body weight of the hamsters in the 5 ⁇ g bivalent vaccine group decreased slightly after challenge.
  • the average decrease percentage 5 days after challenge was 1.33%, which was significantly lower than the challenge model group (p ⁇ 0.01). See Table 8.
  • the average viral load in the lung tissue of hamsters in the model group of the challenge group was 10 5.48 copies/mg 5 days after challenge.
  • the average viral load in the lung tissue of hamsters in the 1 ⁇ g bivalent vaccine group was 10 4.25 copies/mg 5 days after challenge, which was significantly lower than the challenge model group (p ⁇ 0.01), with a decrease of 1.23 lg.
  • the average viral load in the lung tissue of hamsters in the 5 ⁇ g bivalent vaccine group was 10 3.59 copies/mg 5 days after challenge, which was significantly lower than the challenge model group (p ⁇ 0.01), with a decrease of 1.89 lg.
  • the neutralizing antibody GMT of the hamsters in the 1 ⁇ g bivalent vaccine group of the satellite group was 349.05.
  • the neutralizing antibody GMT of hamsters in the 5 ⁇ g bivalent vaccine group of the satellite group was 269.67.
  • the neutralizing antibody GMT of hamsters in the satellite group model group was ⁇ 10.
  • the protein concentration of fusion protein D is 0.5mg/mL, and the buffer is 20mM phosphate buffer with pH 7.0.
  • the preparation design is shown in Table 11.
  • the protein concentration of fusion protein D is 0.5mg/mL
  • 20mM PB7.0 is 20mM phosphate buffer, including 10.26mM disodium hydrogen phosphate and 9.74mM sodium dihydrogen phosphate, pH 7.0
  • 20mM His5.66 is a 20mM histidine buffer, including 6mM histidine and 14mM histidine hydrochloride, pH 5.66.
  • fusion protein D produces less HMW aggregates under the condition of 20mM His5.66 than under the condition of 20mM PB7.0, and is more stable under the condition of 20mM His5.66.
  • fusion protein D preparation shown in Table 13, in which the protein concentration of fusion protein D is 0.2mg/mL, 20mM His5.66 is 20mM histidine buffer, and the pH is 5.66; 20mM His6.0 is 20mM histidine.
  • Each preparation sample was placed at 50°C, and samples were taken for SEC-HPLC detection on days 7, 11, 23, 50, and 93 (D); 50°C-4D+4°C-89D indicates that the preparation sample was placed at 50°C first. 4 days (4D), and then the samples were taken out and kept at 4°C for 89 days, and then samples were taken for SEC-HPLC detection.
  • the SEC-HPLC test results are shown in Table 14. It can be seen from the SEC results that preparation C7 has better results than preparation C8; the samples prepared with pH5.66 histidine buffer and the samples prepared with pH6.0 histidine buffer are both Have better The stability of the preparation with sodium chloride, polysorbate 80 and trehalose added at the same time is more stable.
  • fusion protein G preparation as shown in Table 15, in which the protein concentration of fusion protein G is 0.2mg/mL, the buffer is 20mM His buffer, and the pH is 6.0.
  • preparation D2 was better than preparation D1, indicating that the protective effect of preparations containing polysorbate 80, trehalose and sodium chloride on freezing and thawing was better than that of preparations containing only sodium chloride.
  • fusion protein G preparation as shown in Table 17, in which the protein concentration of fusion protein G is 0.2mg/mL, the buffer is 20mM His buffer, and the pH is 6.0.
  • Each preparation sample was sampled for SEC-HPLC at 50°C for 7, 14, 27, and 50 days (D), 40°C for 50 days (D), and 27°C for 14, 27, and 50 days (D). Test, the results are shown in Table 19 and Table 20.
  • Sodium chloride has a significant impact on the stability of fusion protein G preparations. The lower the concentration of polysorbate 80, the better the stability. The concentration of hydroxypropyl betacyclodextrin has limited impact on the stability.
  • F1-F11 are fusion protein D preparations
  • F12-F22 are fusion protein G preparations
  • F23-F33 are bivalent preparations containing fusion protein D and fusion protein G (mass ratio 1:1).
  • Vaccine formulations in which the protein concentration is the concentration of each fusion protein for example, formulation F23 contains 0.2 mg/mL fusion protein D and 0.2 mg/mL fusion protein G.
  • the adjuvant is SWE adjuvant (SEPPIC S.A., Cat. No. 80748J), which is an aluminum-free adjuvant. The added amount is 3/10 of the total volume of the preparation.
  • 20mM His6.0 is a 20mM histidine buffer with a pH of about 6.0
  • 10mM His6.0 is a 10mM histidine buffer with a pH of about 6.0
  • 10mM His+5mM CB is 20mM histidine with a pH of 6.0.
  • the buffer solution is mixed with 10mM citrate buffer solution with a pH of 6.6 at a volume ratio of 1:1, and the pH is approximately 6.0.
  • HP-CD hydroxypropyl betacyclodextrin
  • the preparation samples were placed at 4°C, 25°C, 40°C, 50°C, light (4500lx ⁇ 500lx, 25°C) for 7 days, frozen and thawed (-60°C ⁇ 25°C) 5 times, and shaken at room temperature (200rpm) Light inspections were conducted separately under 48-hour conditions. Among them, the light inspection results of preparations F1-F15, F17, F19-F21, and F23-F32 under the above conditions were all normal. The light examination results of preparations F16 and F18 when placed at 4°C for 7 days showed slight opalescence and dispersion. The light inspection results of preparation F22 under the condition of freezing and thawing 5 times showed slight opalescence and dispersion. Formulation F33 at 4°C The light inspection results under the condition of being placed under the condition of 7 days at 25°C and 50°C showed slight opalescence and dispersion.
  • preparations F23, F27, F29 and F31 were selected for further SEC-HPLC testing, and the results are shown in Table 22. Freezing and thawing conditions have a greater impact on the 24mer of the bivalent vaccine preparation without adjuvant, while high temperature has a smaller impact on the preparation, which is basically similar to the 4°C condition. Preparations F23, F27, F29, and F31 all have good stability when stored at 50°C for 7 days and 23 days. Formulation F27 was superior to formulation F23.
  • Fusion protein G preparation F12 SWE adjuvant CB6.0 buffer (10mM citrate buffer, pH6.0) diluted 50-fold sample, SWE adjuvant CB6.0 buffer (10mM citrate buffer, pH6 .0)
  • SWE adjuvant CB6.0 buffer 10mM citrate buffer, pH6 .0
  • the samples diluted 100 times and the preparation F17 in Example 11 placed at 25°C for 27 days were named DLS-1, DLS-2, DLS-3, and DLS-4 respectively to examine the effect of the adjuvant on The influence of sample DLS.
  • the DLS measurement results are shown in Table 23. It can be seen that the DLS of the sample after adding the adjuvant does not change much.
  • the average particle size of the protein particles of the fusion protein G preparation F12 is 42.5nm.
  • the DLS measurement results of formulation F26 in Example 11 after being placed at 50°C for 7 days were similar to those of formulation F17.
  • the protein concentration of fusion protein 2-1 is 0.45mg/mL
  • the buffer is 20mM His buffer
  • the pH is 6.0.
  • Prepare the fusion protein 2-1 preparation sample use 20mM His6.0 buffer to ultrafiltrate and change the liquid. After 8 times the volume of the liquid change, dilute the protein concentration to 0.9mg/mL; then mix it with each 2 ⁇ excipient stock solution according to the volume ratio of 1 :1 Mix. Filter samples individually using a sterile filter.
  • Preparation H3 produced white foreign matter when placed at 4°C for 14 days, and protein may precipitate.
  • the stability of the preparation containing sodium chloride and arginine was poor.
  • Preparation H4's light test results showed precipitation, and the light test results of preparations H1 and H2 The test results are all normal, indicating that when polysorbate 80 is not included in the preparation, protein aggregation and precipitation will occur.
  • SEC-HPLC detection was performed after being placed at 25°C for 1 month, 40°C for 7 days, and 21 days. The results are shown in Table 25. Under the condition of 40°C, formulations H1 and H2 are relatively stable. The SEC-HPLC results of the preparation samples stored at 25°C for 1 month show that the 24mer of preparation H1 decreases less with the extension of storage time.
  • Example 13 Stability study of bivalent vaccine (fusion protein D + fusion protein 2-1) preparation
  • the auxiliary material composition of the bivalent vaccine preparation involving fusion protein D + fusion protein 2-1 is: 10mM histidine salt buffer + 8.26mg/mL sodium chloride + 4mg/mL hydroxypropyl betacycline paste Essence + 0.2 mg/mL polysorbate 80 + SWE adjuvant (the amount added is 3/10 of the total volume of the preparation).
  • the specifications and composition of the preparation are shown in Table 26.
  • the protein concentrations of fusion protein D and fusion protein 2-1 in the 80 ⁇ g/0.5 mL preparation are both 0.08 mg/mL; the protein concentrations of fusion protein D and fusion protein in the 40 ⁇ g/0.5 mL preparation are both The protein concentration of 2-1 is 0.04mg/mL.
  • Samples 41A and 41B were batched at 5 ⁇ 3 When stored under °C conditions for 3 months, there is no obvious change trend in each inspection item compared with 0, and the key quality attributes of the product remain stable. There were no significant changes in the physical, chemical and biological properties of each batch of bivalent vaccine preparation samples. The quality attributes of the finished product were stable after being stored under long-term conditions for 4 months.
  • Samples 41A and 41B were placed at 25 ⁇ 2°C for 3 months, and there was no obvious change trend in each test item compared with time 0.
  • the key quality attributes of the product remained stable and were all within the qualified range; the results of the other two batches were similar, all within the qualified range, and the product quality was stable.
  • Sample 41B was placed at 40 ⁇ 2°C for 3 months, and there was no obvious change trend in each test item compared with time 0.
  • the key quality attributes of the product remained stable and were all within the qualified range.
  • the results of batch 41A were also similar.
  • Batch 41A and 41B samples were selected for testing on influencing factors such as high temperature (50 ⁇ 2°C), strong light (4500 ⁇ 500lux), and vibration (200rpm, room temperature).
  • the test results of the bivalent vaccine preparation under high temperature conditions are shown in Tables 32 and 33.
  • the finished product of the bivalent vaccine preparation was placed at a high temperature of 50°C for 21 days.
  • the S protein antibody titer value slowly decreased with time.
  • Batch 41A exceeded the acceptance standard at 7 days
  • batch 41B exceeded the acceptance standard at 3 days; other inspection results were relatively There is no obvious trend at 0. High temperatures will cause significant changes in the critical quality attributes of this product.
  • the test results of the bivalent vaccine preparation under strong light conditions are shown in Tables 34 and 35.
  • the finished preparation was placed under strong light conditions for 21 days. Compared with the time at 0, the pH, osmotic pressure, squalene content and S protein antibody titer values were all within the scope of the quality standards, and the product quality remained stable; indicating that under strong light conditions Leaving it for 21 days will not affect the product's critical quality attributes.
  • test results of the bivalent vaccine preparation under shaking (200 rpm, room temperature) conditions are shown in Tables 36 and 37.
  • the finished product was vibrated at room temperature at 200 rpm for 48 hours. Compared with time 0, there was no obvious change trend in the results of each inspection item. It shows that 200rpm vibration during transportation at room temperature within 48 hours will not affect the quality of the finished bivalent vaccine preparation.

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Abstract

Provided are a fusion protein for preventing or treating coronavirus infection, a Spike protein nanoparticle, and a preparation thereof. The fusion protein comprises a mutation-containing coronavirus Spike protein extracellular domain which is linked by means of a linker, or a truncated fragment and monomeric subunit protein thereof.

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Fusion proteins and Spike protein nanoparticles for preventing or treating coronavirus infection and their applications 技术领域Technical field
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。The invention belongs to the field of biotechnology, and in particular relates to fusion proteins and Spike protein nanoparticles for preventing or treating coronavirus infection and their applications.
背景技术Background technique
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。Coronavirus is a non-segmented single-stranded positive-strand RNA virus. According to the serotype and genome characteristics, the coronavirus subfamily is divided into four genera: α, β, γ and δ. Because the virus envelope has ridges that extend to all sides. It is named after its protrusions and shape like a corolla. The new coronavirus (SARS-CoV-2 or 2019-nCoV) discovered in 2019 belongs to the beta genus and is enveloped. The particles are round or oval, often pleomorphic, and have a diameter of 60-140nm. Current research shows that SARS-CoV-2 and SARS-CoV are highly homologous.
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。COVID-19 is mainly transmitted through the respiratory tract, and it may also be transmitted through contact. The general population is susceptible, and the elderly and those with underlying diseases are more seriously ill after infection. Children and infants also get sick. Based on current epidemiological surveys, the incubation period of the new coronavirus is generally 1-14 days, and most of them are 3-7 days. The main clinical symptoms of the infected are fever, fatigue, and dry cough, while upper respiratory tract symptoms such as nasal congestion and runny nose are rare. In the early stage of the disease, the total white blood cell count of the patient is normal or decreased, or the number of lymphocytes is decreased, and some patients have increased liver enzymes, myoenzymes and myoglobin. Chest imaging shows that patients present with multiple small patchy shadows and interstitial changes in the early stage, which are obvious in the outer lungs; then develop into multiple ground-glass shadows and infiltration shadows in both lungs, severe cases may have pulmonary consolidation, and gradually develop dyspnea, severe cases develop acute respiratory distress syndrome (ARDS), shock, and multiple tissue damage and dysfunction of lung tissue, heart, and kidney. Most patients with mild infection have a good prognosis, while severe patients are often in critical condition or even die.
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。同时，由于许多蛋白疫苗对热不稳定，或在4℃下长期储存或冻融不稳定，因此它们在储存时会失去效力。热稳定性的提高可以解决例如在偏远和贫困地区可能面临的冷链问题，并且延长疫苗的保质期，因此需要开发具有增加的热稳定性和/或增加的保质期的稳定疫苗组合物制剂。Recently, basic, clinical and epidemiological studies on COVID-19 have been continuously published or released, and there is an urgent need for effective vaccines against coronavirus in this field. At the same time, because many protein vaccines are unstable to heat, or are unstable to long-term storage at 4°C or freeze-thaw, they will lose effectiveness during storage. Improved thermal stability can solve cold chain problems that may be faced in remote and poor areas, for example, and extend the shelf life of vaccines, so there is a need to develop stable vaccine composition formulations with increased thermal stability and/or increased shelf life.
发明内容Summary of the invention
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。The present invention provides a fusion containing a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof that can stabilize the protein structure, and a fusion comprising a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof. protein. The present invention also provides a coronavirus Spike protein extracellular domain containing mutations or a truncation thereof. Coronavirus vaccines in which fragments are fused to monomeric ferritin subunits and self-assemble into nanoparticles can induce stronger neutralizing antibody responses to coronaviruses.
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。Viral particles first interact with an angiotensin-converting enzyme 2 (ACE2) on the surface of lung epithelial cells through the receptor binding domain (RBD) in the S1 subunit of the Spike protein (S protein or spike protein) on its surface. combine. When RBD binds to the receptor and is hydrolyzed by proteases, the S2 subunit located at the C-terminus of the S protein is exposed and embedded in the serosa or endosome membrane. Heptapeptide repeat sequence 1 (HR1) and heptapeptide repeat sequence 2 (HR2) in the S2 subunit interact with each other to form a six-helix bundle (6-HB) fusion core, leading to the fusion of the viral shell and the cell membrane, SARS-CoV or SARS- CoV-2 enters cells and uses cells to synthesize new virus particles; the new virus particles are released outside the cells and then use the same method to infect surrounding normal cells. The fusion protein, nanoparticles and vaccine of the present invention can induce stronger neutralizing antibody responses to coronavirus.
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。In some embodiments, a coronavirus Spike protein extracellular domain or a truncated fragment thereof containing a mutation is provided, the mutation comprising: 1) mutating RRAR to GSAS; 2) in HR1 and the central helical region (CH) There are mutations in the turning region between HR1 and CH that prevent HR1 and CH from forming a straight helix during fusion.
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。In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
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。In some embodiments, the amino acid numbering of the coronavirus Spike protein is based on the amino acid numbering of cryo-EM model PDB ID 6VSB or GenBank accession number MN908947.3 as a reference.
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。In some embodiments, the truncated fragment of the extracellular domain of the coronavirus Spike protein containing mutations has a C-terminal truncation of 5-80 amino acid residues compared to the full-length extracellular domain of the coronavirus Spike protein. base. In some embodiments, the truncated fragment of the extracellular domain of the coronavirus Spike protein containing mutations has a C-terminal truncation of 20-76 amino acid residues compared to the full-length extracellular domain of the coronavirus Spike protein. base. In some embodiments, the truncated fragment of the extracellular domain of the coronavirus Spike protein containing mutations has a C-terminal truncation of 70 amino acid residues compared to the full-length extracellular domain of the coronavirus Spike protein.
在一些实施方案中，所述冠状病毒为SARS-CoV-2、SARS-CoV或MERS-CoV。In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, or MERS-CoV.
在一些实施方案中，所述冠状病毒为SARS-CoV-2原始株或其变异株。In some embodiments, the coronavirus is an original strain of SARS-CoV-2 or a variant thereof.
在一些实施方案中，所述冠状病毒为SARS-CoV-2原始株、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。In some embodiments, the coronavirus is an original strain of SARS-CoV-2, a variant strain of SARS-CoV-2 Alpha, a variant strain of SARS-CoV-2 Beta, a variant strain of SARS-CoV-2 Gamma, a variant strain of 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.
在一些实施方案中，所述冠状病毒为SARS-CoV-2 Omicron变异株BA.1、BA.2、BA.3、BA.4、BA.5、BQ.1、BQ.1.1、BF.7、XBB、XBB.1、XBB.1.5、XBB.1.5.1、XBB.1.9.1 或XBB.1.16。In some embodiments, the coronavirus is SARS-CoV-2 Omicron variant strain BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7 ,XBB,XBB.1,XBB.1.5,XBB.1.5.1,XBB.1.9.1 Or XBB.1.16.
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、6-9、19-24、26-31任一项所示的氨基酸序列，或与SEQ ID NO:3、4、6-9、19-24、26-31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、6-9、19-24、26-31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。In some embodiments, the mutation-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof comprises as shown in any one of SEQ ID NO: 3, 4, 6-9, 19-24, 26-31 An amino acid sequence, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence shown in any one of SEQ ID NO: 3, 4, 6-9, 19-24, 26-31, or An amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 3, 4, 6-9, 19-24, 26-31.
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。Some embodiments also provide a fusion protein comprising the mutated extracellular domain of the coronavirus Spike protein described herein or a truncated fragment thereof.
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。Some embodiments provide a fusion protein comprising the mutation-containing extracellular domain of the coronavirus Spike protein described herein or a truncated fragment thereof and a monomeric subunit protein linked by a linker. In some embodiments, the monomeric subunit protein is a self-assembled monomeric subunit protein. In some embodiments, the monomeric subunit protein is a monomeric ferritin subunit. In some embodiments, the fusion protein is a C-terminus containing a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof connected to the N-terminus of a monomeric subunit protein through a linker. In some embodiments, the fusion protein is a C-terminus containing a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof connected to the N-terminus of a monomeric ferritin subunit through a linker.
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，GGSS，(GGGGS)2，(GGGGS)3，或其任意组合。在一些实施方案中，所述接头为(GmS)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。In some embodiments, the linker is a GS linker. In some embodiments, the linker is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS) 2 , (GGGGS) 3 , or any combination thereof. In some embodiments, the linker is ( GmS ) n , wherein each m is independently 1, 2, 3, 4, or 5 and n is 1, 2, 3, 4, or 5. In some embodiments, the linker has the sequence (GGGGS) n and n is 1, 2, 3, 4, or 5. In some embodiments, the linker is GGGGS. In some embodiments, the linker is (GGGGS) 2 . In some embodiments, the linker is (GGGGS) 3 . In some embodiments, the linker is (GGGGS) 4 . In some embodiments, the linker is (GGGGS) 5 .
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。In some embodiments, the fusion protein further comprises an N-terminal signal peptide. In some embodiments, the signal peptide is selected from the group consisting of CSP, mschito, MF-α, pho1, HBM, t-pA, and the signal peptide of IL-3. In some embodiments, the N-terminal signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 2 or 5, or has at least 80% or at least 90% difference compared to the amino acid sequence set forth in SEQ ID NO: 2 or 5. % identity of the amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 2 or 5.
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:10所示的氨基酸序列，或与SEQ ID NO:10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:10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。In some embodiments, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, phycoferritin, insect ferritin, fungal ferritin, or mammalian ferritin. In some embodiments, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit. In some embodiments, the N19Q mutation is present in the amino acid sequence of the H. pylori non-heme monomeric ferritin subunit. In some embodiments, the monomeric ferritin The subunit comprises an amino acid sequence as set forth in SEQ ID NO: 10, or an amino acid sequence having at least 80% or at least 90% identity as compared to the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 10 The amino acid sequences shown are compared to amino acid sequences with one or more conservative amino acid substitutions.
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 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 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。In some embodiments, a fusion protein is provided, comprising a mutation-containing extracellular domain of the SARS-CoV-2 Spike protein or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprises a mutated SARS-CoV-2 Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
在一些实施方案中，所述SARS-CoV-2为原始株或其变异株。In some embodiments, the SARS-CoV-2 is the original strain or a variant thereof.
在一些实施方案中，所述为SARS-CoV-2为SARS-CoV-2原始株、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。In some embodiments, the SARS-CoV-2 is an original strain of SARS-CoV-2, a variant strain of SARS-CoV-2 Alpha, a variant strain of SARS-CoV-2 Beta, a variant strain of SARS-CoV-2 Gamma, 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.
在一些实施方案中，所述冠状病毒为SARS-CoV-2 Omicron变异株BA.1、BA.2、BA.3、BA.4、BA.5、BQ.1、BQ.1.1、BF.7、XBB、XBB.1、XBB.1.5、XBB.1.5.1、XBB.1.9.1或XBB.1.16。In some embodiments, the coronavirus is SARS-CoV-2 Omicron variant strain BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7 , XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.16.
在一些实施方案中，所述冠状病毒为SARS-CoV-2 Omicron变异株BA.1、BA.2、BA.3、BA.4、BA.5。In some embodiments, the coronavirus is SARS-CoV-2 Omicron variant strain BA.1, BA.2, BA.3, BA.4, BA.5.
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。In some embodiments, a fusion protein is provided, comprising a mutation-containing SARS-CoV-2 Omicron variant Spike protein extracellular domain or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprises a mutated SARS-CoV-2 Omicron variant Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
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BA.1Spike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BA.1Spike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。In some embodiments, a fusion protein is provided, comprising the mutation-containing SARS-CoV-2 Omicron variant BA.1 Spike protein extracellular domain or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprises a mutated SARS-CoV-2 Omicron variant BA.1 Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
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BA.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SARS-CoV-2 Omicron变异株BA.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。In some embodiments, a fusion protein is provided, comprising a mutation-containing SARS-CoV-2 Omicron variant BA.2 Spike protein extracellular domain or a truncated fragment thereof and a monomeric subunit protein connected by a linker. white. In some embodiments, the fusion protein comprises a mutated SARS-CoV-2 Omicron variant BA.2 Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
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BA.3Spike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BA.3Spike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。In some embodiments, a fusion protein is provided, comprising the mutation-containing SARS-CoV-2 Omicron variant BA.3 Spike protein extracellular domain or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprises a mutated SARS-CoV-2 Omicron variant BA.3 Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
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BA.4Spike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BA.4Spike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。In some embodiments, a fusion protein is provided, comprising an extracellular domain of a SARS-CoV-2 Omicron variant BA.4 Spike protein containing a mutation or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprises an extracellular domain of a SARS-CoV-2 Omicron variant BA.4 Spike protein containing a mutation or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutation comprises: 1) mutating RRAR to GSAS; 2) a double mutation K986P/V987P in the turning region between HR1 and CH.
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BA.5Spike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BA.5Spike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。In some embodiments, a fusion protein is provided, comprising the mutation-containing SARS-CoV-2 Omicron variant BA.5 Spike protein extracellular domain or a truncated fragment thereof and a monomeric subunit protein connected by a linker. In some embodiments, the fusion protein comprises a mutated SARS-CoV-2 Omicron variant BA.5 Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH.
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、6、19-21、26-28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:10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:11所示的接头与单体铁蛋白亚基连接。In some embodiments, the fusion protein includes a mutation-containing coronavirus Spike protein extracellular domain and a monomeric ferritin subunit connected by a linker, the mutation-containing coronavirus Spike protein extracellular domain comprising SEQ. The amino acid sequence shown in any one of ID NO: 3, 4, 6, 19-21, 26-28, the monomeric ferritin subunit includes the amino acid sequence shown in SEQ ID NO: 10; containing a mutated crown The extracellular domain of the viral Spike protein is connected to the monomeric ferritin subunit through the linker shown in SEQ ID NO:11.
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:7-9、22-24、29-31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:10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:11所示的接头与单体铁蛋白亚基连接。 In some embodiments, the fusion protein includes a truncated fragment of the extracellular domain of the coronavirus Spike protein containing mutations and a monomeric ferritin subunit connected by a linker, the extracellular structure of the coronavirus Spike protein containing mutations The truncated fragment of the domain includes the amino acid sequence shown in any one of SEQ ID NO:7-9, 22-24, 29-31, and the monomeric ferritin subunit includes the amino acid sequence shown in SEQ ID NO:10 Sequence; the truncated fragment containing the mutated extracellular domain of the coronavirus Spike protein is connected to the monomeric ferritin subunit through the linker shown in SEQ ID NO: 11.
在一些实施方案中，所述融合蛋白包含如SEQ ID NO:12-17、32-43任一项所示的氨基酸序列，或与SEQ ID NO:12-17、32-43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:12-17、32-43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。In some embodiments, the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 12-17, 32-43, or is identical to any one of SEQ ID NO: 12-17, 32-43 The amino acid sequence has at least 80% or at least 90% identity compared to the amino acid sequence, or has one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 12-17, 32-43 amino acid sequence.
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。In some embodiments, "at least 80% identity" is at least about 80% identity, at least about 81% identity, at least about 83% identity, at least about 84% identity, at least about 85% identity, at least About 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 93% identical, 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 of the endpoints) or any value therein.
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。In some embodiments, "at least 90% identity" is at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least About 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or a range between any two of these values, inclusive of the endpoints ) or any value therein.
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。Some embodiments provide a polynucleotide encoding a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof, or a fusion protein described herein.
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。In some embodiments, an expression vector is provided comprising a polynucleotide encoding a mutated coronavirus Spike protein extracellular domain as described herein, or a truncated fragment or fusion protein thereof.
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。In some embodiments, a cell is provided that can express a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof as described herein. In some embodiments, the cell comprises one or more polynucleotides encoding a fusion protein described herein or an expression vector comprising a polynucleotide encoding a fusion protein described herein. In some embodiments, the cells are isolated cells. In some embodiments, the cells are CHO cells, HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, or murine L cells.
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：In some embodiments, the fusion protein comprises a mutation-containing extracellular domain of the coronavirus Spike protein or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker, and the fusion protein includes the following features:
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The mutations include: 1) a mutation that inactivates the S1/S2 cleavage site; 2) a mutation in the turning region between HR1 and CH that prevents HR1 and CH from forming a straight helix during the fusion process; and/or
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The C-terminus of the mutated coronavirus Spike protein extracellular domain or its truncated fragment is connected to the monomeric ferritin subunit through a linker; and/or
所述接头为(GmS)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 The linker is (G m S) n , where each m is independently 1, 2, 3, 4 or 5 and n is 1, 2, 3, 4 or 5; and/or
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:10所示的氨基酸序列，或与SEQ ID NO:10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:10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。The monomeric ferritin subunit is a Helicobacter pylori monomeric ferritin subunit, comprising the amino acid sequence shown in SEQ ID NO: 10, or having at least 80% of the amino acid sequence shown in SEQ ID NO: 10. Or an amino acid sequence that is at least 90% identical, or has one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 10.
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。In some embodiments, Spike protein nanoparticles comprising a fusion protein described herein are provided.
在一些实施方案中，提供本文所述的融合蛋白或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。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SARS-CoV-2原始株或其变异株感染。在一些实施方案中，所述冠状病毒感染为SARS-CoV-2原始株、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。在一些实施方案中，所述冠状病毒感染为SARS-CoV-2 Omicron变异株BA.1、BA.2、BA.3、BA.4、BA.5、BQ.1、BQ.1.1、BF.7、XBB、XBB.1、XBB.1.5、XBB.1.5.1、XBB.1.9.1或XBB.1.16感染。In some embodiments, the use of the fusion protein or Spike protein nanoparticles described herein in preparing a vaccine to prevent or treat coronavirus infection is provided. In some embodiments, the coronavirus infection is a SARS-CoV-2, SARS-CoV or MERS-CoV infection. In some embodiments, the coronavirus infection is an infection with the original strain of SARS-CoV-2 or a variant thereof. In some embodiments, the coronavirus infection is an original strain of SARS-CoV-2, a SARS-CoV-2 Alpha variant, a SARS-CoV-2 Beta variant, a SARS-CoV-2 Gamma variant, or a SARS-CoV -2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant. In some embodiments, the coronavirus infection is SARS-CoV-2 Omicron variant strain BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF. 7. XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.16 infection.
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。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。In some embodiments there is provided a coronavirus vaccine comprising a fusion protein described herein and/or a Spike protein nanoparticle comprising a fusion protein. In some embodiments, the coronavirus vaccine further includes a pharmaceutically acceptable carrier and/or adjuvant. In some embodiments, the coronavirus vaccine comprises a fusion protein described herein and a pharmaceutically acceptable carrier and/or adjuvant. In some embodiments, the coronavirus vaccine comprises Spike protein nanoparticles described herein and a pharmaceutically acceptable carrier and/or adjuvant.
在一些实施方案中提供了一种冠状病毒疫苗制剂，其包含融合蛋白，还包含缓冲剂、稳定剂、碱金属或碱金属盐、表面活性剂中的一种或多种，所述融合蛋白为本文所述融合蛋白和/或包含融合蛋白的Spike蛋白纳米颗粒。In some embodiments, a coronavirus vaccine preparation is provided, which includes a fusion protein and one or more of a buffer, a stabilizer, an alkali metal or alkali metal salt, and a surfactant, and the fusion protein is Fusion proteins and/or Spike protein nanoparticles comprising the fusion proteins described herein.
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。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，所述冠状病毒为SARS-CoV-2原始株、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，所述冠状病毒为SARS-CoV-2 Omicron变异株BA.1、BA.2、BA.3、BA.4、BA.5、BQ.1、BQ.1.1、BF.7、XBB、XBB.1、XBB.1.5、XBB.1.5.1、XBB.1.9.1或XBB.1.16。 In some embodiments, the fusion protein comprises a mutation-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof and a monomeric ferritin subunit connected by a linker. In some embodiments, the mutations comprise: 1) mutation of RRAR to GSAS; 2) presence of a double mutation K986P/V987P in the turn region between HR1 and CH. In some embodiments, the coronavirus is an original strain of SARS-CoV-2, a variant strain of SARS-CoV-2 Alpha, a variant strain of SARS-CoV-2 Beta, a variant strain of SARS-CoV-2 Gamma, a variant strain of 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. In some embodiments, the coronavirus is SARS-CoV-2 Omicron variant strain BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7 , XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.16.
在一些实施方案中，所述冠状病毒疫苗制剂包含融合蛋白，还包含缓冲剂、稳定剂、碱金属或碱金属盐、表面活性剂中的一种或多种；所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine preparation includes a fusion protein and one or more of a buffer, a stabilizer, an alkali metal or alkali metal salt, and a surfactant; the fusion protein includes SEQ ID The amino acid sequence shown in any one of NO: 12-17, 32-43, 51-56, and 61.
在一些实施方案中，所述融合蛋白的浓度为0.05-5mg/mL。在一些实施方案中，所述融合蛋白的浓度为0.05-1mg/mL。在一些实施方案中，所述融合蛋白的浓度为0.05-0.5mg/mL。在一些实施方案中，所述融合蛋白的浓度为约0.05、0.1、约0.45、约0.5、约1、约2、约3、约4、约5mg/mL。In some embodiments, the concentration of the fusion protein is 0.05-5 mg/mL. In some embodiments, the concentration of the fusion protein is 0.05-1 mg/mL. In some embodiments, the concentration of the fusion protein is 0.05-0.5 mg/mL. In some embodiments, the concentration of the fusion protein is about 0.05, 0.1, about 0.45, about 0.5, about 1, about 2, about 3, about 4, about 5 mg/mL.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、10-30mM缓冲剂、稳定剂、碱金属或碱金属盐和表面活性剂。In some embodiments, the coronavirus vaccine formulation contains 0.05-1 mg/mL fusion protein, 10-30 mM buffer, stabilizer, alkali metal or alkali metal salt, and surfactant.
在一些实施方案中，所述缓冲剂选自组氨酸缓冲剂、柠檬酸缓冲剂、磷酸缓冲剂或其组合。在一些实施方案中，所述缓冲剂为组氨酸缓冲剂。在一些实施方案中，所述缓冲剂为组氨酸缓冲剂和柠檬酸缓冲剂的组合。在一些实施方案中，所述缓冲剂的浓度为1-50mM、或1-40mM、或1-30mM、或5-25mM、或10-30mM、或15-25mM、或5-15mM。在一些实施方案中，所述缓冲剂的浓度为约1mM、约5mM、约8mM、约10mM、13mM、约15mM、约18mM、约20mM、约22mM、约25mM、约27mM、约30mM、约35mM、约40mM、约50mM，或这些数值中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，所述缓冲剂为5-25mM的组氨酸缓冲剂。在一些实施方案中，所述缓冲剂为10-30mM的组氨酸缓冲剂。在一些实施方案中，所述缓冲剂为5-15mM的组氨酸缓冲剂。在一些实施方案中，所述缓冲剂为15-25mM的组氨酸缓冲剂。在一些实施方案中，所述缓冲剂为约20mM的组氨酸缓冲剂。在一些实施方案中，所述缓冲剂为约10mM的组氨酸缓冲剂。在一些实施方案中，所述缓冲剂为5-15mM组氨酸缓冲剂和2-10mM柠檬酸缓冲剂的组合。在一些实施方案中，所述缓冲剂为约10mM组氨酸缓冲剂和约5mM柠檬酸缓冲剂的组合。在一些实施方案中，所述pH为5.0-7.0。在一些实施方案中，所述pH为5.5-6.5。在一些实施方案中，所述pH为5.7-6.3。在一些实施方案中，所述抗体制剂的pH为约5.0、约5.3、约5.6、约5.7、约5.8、约5.9、约6.0、约6.1、约6.2、约6.3、约6.4、约6.5、约6.8、约7.0，或这些数值中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，所述pH为约6.1。In some embodiments, the buffer is selected from histidine buffer, citric acid buffer, phosphate buffer or a combination thereof. In some embodiments, the buffer is a histidine buffer. In some embodiments, the buffer is a combination of a histidine buffer and a citric acid buffer. In some embodiments, the concentration of the buffer is 1-50mM, or 1-40mM, or 1-30mM, or 5-25mM, or 10-30mM, or 15-25mM, or 5-15mM. In some embodiments, the concentration of the buffer is about 1mM, about 5mM, about 8mM, about 10mM, 13mM, about 15mM, about 18mM, about 20mM, about 22mM, about 25mM, about 27mM, about 30mM, about 35mM, about 40mM, about 50mM, or any range (including endpoints) between any two values in these numerical values or any value therein. In some embodiments, the buffer is a 5-25mM histidine buffer. In some embodiments, the buffer is a 10-30mM histidine buffer. In some embodiments, the buffer is a 5-15mM histidine buffer. In some embodiments, the buffer is a 15-25mM histidine buffer. In some embodiments, the buffer is about 20mM histidine buffer. In some embodiments, the buffer is about 10mM histidine buffer. In some embodiments, the buffer is a combination of 5-15mM histidine buffer and 2-10mM citric acid buffer. In some embodiments, the buffer is a combination of about 10mM histidine buffer and about 5mM citric acid buffer. In some embodiments, the pH is 5.0-7.0. In some embodiments, the pH is 5.5-6.5. In some embodiments, the pH is 5.7-6.3. In some embodiments, the pH of the antibody formulation is about 5.0, about 5.3, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.8, about 7.0, or a range (including endpoints) between any two of these values or any value therein. In some embodiments, the pH is about 6.1.
在一些实施方案中，所述稳定剂选自多元醇、糖类、氨基酸或其组合。在一些实施方案中，所述稳定剂选自精氨酸或其盐、聚乙二醇、山梨醇、甘露醇、甘油、单糖、寡糖、多糖、环糊精或其衍生物中的一种或多种。在一些实施方案中，所述稳定剂选自环糊精或其衍生物、蔗糖和海藻糖中的一种或多种。在一些实施方案中，所述环糊 精或其衍生物选自α-环糊精、β-环糊精、γ-环糊精、其羟丙基化衍生物(例如羟丙基倍他环糊精)、羟乙基化衍生物、乙基化衍生物和甲基化衍生物、磺丁基醚β-环糊精、支链环糊精、环糊精聚合物，或它们的组合。在一些实施方案中，所述稳定剂选自羟丙基倍他环糊精、蔗糖和海藻糖或其组合。在一些实施方案中，所述稳定剂为羟丙基倍他环糊精和海藻糖的组合。固体状态下，海藻糖通常以海藻糖二水合物存在，所以在一些实施方案中，配制制剂时可以采用海藻糖二水合物，也可以采用其他形式的海藻糖(例如无水海藻糖)配制。在一些实施方案中，所述稳定剂浓度为0.5～200mg/mL。在一些实施方案中，所述稳定剂浓度为0.5-170mg/mL。在一些实施方案中，所述稳定剂浓度为10-170mg/mL。在一些实施方案中，所述稳定剂浓度为80-170mg/mL。在一些实施方案中，所述稳定剂浓度为60-100mg/mL。在一些实施方案中，所述稳定剂浓度为0.5-80mg/mL。在一些实施方案中，所述稳定剂浓度为0.5-30mg/mL。在一些实施方案中，所述稳定剂浓度为0.5-50mg/mL。在一些实施方案中，所述稳定剂为约0.5mg/mL、约4mg/mL、约8mg/mL、约10mg/mL、约20mg/mL、约21mg/mL、约25mg/mL、约30mg/mL、约50mg/mL、约84mg/mL、约85mg/mL、约100mg/mL、约150mg/mL、约170mg/mL，或这些数值中任何两个值之间的范围(包括端点)或其中任何值。In some embodiments, the stabilizer is selected from polyols, sugars, amino acids, or combinations thereof. In some embodiments, the stabilizer is selected from one of arginine or a salt thereof, polyethylene glycol, sorbitol, mannitol, glycerol, monosaccharide, oligosaccharide, polysaccharide, cyclodextrin or derivatives thereof. Kind or variety. In some embodiments, the stabilizer is selected from one or more of cyclodextrin or its derivatives, sucrose, and trehalose. In some embodiments, the cyclohexan Essence or its derivatives are selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, its hydroxypropylated derivatives (such as hydroxypropyl beta cyclodextrin), hydroxyethylated derivatives , ethylated derivatives and methylated derivatives, sulfobutyl ether β-cyclodextrin, branched cyclodextrin, cyclodextrin polymers, or combinations thereof. In some embodiments, the stabilizer is selected from hydroxypropyl betacyclodextrin, sucrose, and trehalose, or combinations thereof. In some embodiments, the stabilizer is a combination of hydroxypropyl betacyclodextrin and trehalose. In the solid state, trehalose usually exists as trehalose dihydrate, so in some embodiments, trehalose dihydrate can be used to prepare the preparation, or other forms of trehalose (such as anhydrous trehalose) can be used. In some embodiments, the stabilizer concentration is 0.5-200 mg/mL. In some embodiments, the stabilizer concentration is 0.5-170 mg/mL. In some embodiments, the stabilizer concentration is 10-170 mg/mL. In some embodiments, the stabilizer concentration is 80-170 mg/mL. In some embodiments, the stabilizer concentration is 60-100 mg/mL. In some embodiments, the stabilizer concentration is 0.5-80 mg/mL. In some embodiments, the stabilizer concentration is 0.5-30 mg/mL. In some embodiments, the stabilizer concentration is 0.5-50 mg/mL. In some embodiments, the stabilizer is about 0.5 mg/mL, about 4 mg/mL, about 8 mg/mL, about 10 mg/mL, about 20 mg/mL, about 21 mg/mL, about 25 mg/mL, about 30 mg/mL. mL, about 50 mg/mL, about 84 mg/mL, about 85 mg/mL, about 100 mg/mL, about 150 mg/mL, about 170 mg/mL, or a range between any two of these values (including endpoints) or therein any value.
在一些实施方案中，所述稳定剂为0.5-170mg/mL海藻糖二水合物或蔗糖。在一些实施方案中，所述稳定剂为10-170mg/mL海藻糖二水合物或蔗糖。在一些实施方案中，所述稳定剂为80-170mg/mL海藻糖二水合物或蔗糖。在一些实施方案中，所述稳定剂为60-100mg/mL海藻糖二水合物或蔗糖。在一些实施方案中，所述稳定剂为0-450mM海藻糖或蔗糖。在一些实施方案中，所述稳定剂为14-450mM海藻糖或蔗糖。在一些实施方案中，所述稳定剂为210-450mM海藻糖或蔗糖。在一些实施方案中，所述稳定剂为150-270mM海藻糖或蔗糖。在一些实施方案中，所述稳定剂为约222mM海藻糖(约84mg/mL海藻糖二水合物)。在一些实施方案中，所述稳定剂为约224.7mM海藻糖(约85mg/mL海藻糖二水合物)。在一些实施方案中，所述稳定剂为约55.5mM海藻糖(约21mg/mL海藻糖二水合物)。在一些实施方案中，所述稳定剂为0.5-80mg/mL羟丙基倍他环糊精。在一些实施方案中，所述稳定剂为0.5-50mg/mL羟丙基倍他环糊精。在一些实施方案中，所述稳定剂为0.5-20mg/mL羟丙基倍他环糊精。在一些实施方案中，所述稳定剂为约8mg/mL羟丙基倍他环糊精。在一些实施方案中，所述稳定剂为约4mg/mL羟丙基倍他环糊精。In some embodiments, the stabilizer is 0.5-170 mg/mL trehalose dihydrate or sucrose. In some embodiments, the stabilizer is 10-170 mg/mL trehalose dihydrate or sucrose. In some embodiments, the stabilizer is 80-170 mg/mL trehalose dihydrate or sucrose. In some embodiments, the stabilizer is 60-100 mg/mL trehalose dihydrate or sucrose. In some embodiments, the stabilizer is 0-450 mM trehalose or sucrose. In some embodiments, the stabilizer is 14-450 mM trehalose or sucrose. In some embodiments, the stabilizer is 210-450 mM trehalose or sucrose. In some embodiments, the stabilizer is 150-270 mM trehalose or sucrose. In some embodiments, the stabilizer is about 222 mM trehalose (about 84 mg/mL trehalose dihydrate). In some embodiments, the stabilizer is about 224.7 mM trehalose (about 85 mg/mL trehalose dihydrate). In some embodiments, the stabilizer is about 55.5 mM trehalose (about 21 mg/mL trehalose dihydrate). In some embodiments, the stabilizer is 0.5-80 mg/mL hydroxypropyl betacyclodextrin. In some embodiments, the stabilizer is 0.5-50 mg/mL hydroxypropyl betacyclodextrin. In some embodiments, the stabilizer is 0.5-20 mg/mL hydroxypropyl betacyclodextrin. In some embodiments, the stabilizer is about 8 mg/mL hydroxypropyl betacyclodextrin. In some embodiments, the stabilizer is about 4 mg/mL hydroxypropyl betacyclodextrin.
在一些实施方案中，所述碱金属或碱金属盐选自氯化镁、氯化钾、氯化钠或其组合。在一些实施方案中，所述碱金属或碱金属盐浓度为0-22mg/mL。在一些实施方案中，所述碱金属或碱金属盐浓度为8-20mg/mL。在一些实施方案中，所述碱金属或碱 金属盐浓度为8-17mg/mL。在一些实施方案中，所述碱金属或碱金属盐浓度为约0.1、约1、约2、约4、约8、约8.25、约8.3、约10、约13、约16、约16.5、约18、约20、约22mg/mL，或这些数值中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，所述碱金属或碱金属盐为0.1-22mg/mL氯化钠。在一些实施方案中，所述碱金属或碱金属盐为8-17mg/mL氯化钠。In some embodiments, the alkali metal or alkali metal salt is selected from magnesium chloride, potassium chloride, sodium chloride, or combinations thereof. In some embodiments, the alkali metal or alkali metal salt concentration is 0-22 mg/mL. In some embodiments, the alkali metal or alkali metal salt concentration is 8-20 mg/mL. In some embodiments, the alkali metal or base The metal salt concentration is 8-17 mg/mL. In some embodiments, the alkali metal or alkali metal salt concentration is about 0.1, about 1, about 2, about 4, about 8, about 8.25, about 8.3, about 10, about 13, about 16, about 16.5, about 18. About 20, about 22 mg/mL, or a range between any two of these values (inclusive of endpoints) or any value therein. In some embodiments, the alkali metal or alkali metal salt is 0.1-22 mg/mL sodium chloride. In some embodiments, the alkali metal or alkali metal salt is 8-17 mg/mL sodium chloride.
在一些实施方案中，所述表面活性剂选自泊洛沙姆、聚山梨酯或其组合。在一些实施方案中，所述表面活性剂为聚山梨酯。在一些实施方案中，所述表面活性剂为泊洛沙姆188、聚山梨酯20或聚山梨酯80。在一些实施方案中，所述表面活性剂浓度为0.1-10mg/mL。在一些实施方案中，所述表面活性剂为0.01-5mg/mL。在一些实施方案中，所述表面活性剂为0.01-2mg/mL。在一些实施方案中，所述表面活性剂为约0.01、约0.1、约0.2、约0.4、约0.5、约0.8、约1、约1.2、约1.5、约1.8、约2、约5、约8、约10mg/mL，或这些数值中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，所述表面活性剂为0.2-1mg/mL。在一些实施方案中，所述表面活性剂为0.01-2mg/mL聚山梨酯80。在一些实施方案中，所述表面活性剂为0.1-2mg/mL聚山梨酯80。In some embodiments, the surfactant is selected from poloxamers, polysorbates, or combinations thereof. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is poloxamer 188, polysorbate 20, or polysorbate 80. In some embodiments, the surfactant concentration is 0.1-10 mg/mL. In some embodiments, the surfactant is 0.01-5 mg/mL. In some embodiments, the surfactant is 0.01-2 mg/mL. In some embodiments, the surfactant is about 0.01, about 0.1, about 0.2, about 0.4, about 0.5, about 0.8, about 1, about 1.2, about 1.5, about 1.8, about 2, about 5, about 8 , about 10 mg/mL, or a range between any two of these values (inclusive of the endpoints) or any value therein. In some embodiments, the surfactant is 0.2-1 mg/mL. In some embodiments, the surfactant is 0.01-2 mg/mL polysorbate 80. In some embodiments, the surfactant is 0.1-2 mg/mL polysorbate 80.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、10-30mM缓冲剂、稳定剂、碱金属或碱金属盐、表面活性剂中的一种或多种，所述融合蛋白为本文所述的融合蛋白。在一些实施方案中，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine preparation contains one or more of 0.05-1 mg/mL fusion protein, 10-30 mM buffer, stabilizer, alkali metal or alkali metal salt, and surfactant, The fusion protein is a fusion protein as described herein. In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、5-25mM磷酸缓冲剂、14-450mM海藻糖、0-22mg/mL碱金属或碱金属盐、0.01-2mg/mL聚山梨酯80，pH约为6.0-8.0，所述融合蛋白为本文所述的融合蛋白。在一些实施方案中，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation contains 0.05-1 mg/mL fusion protein, 5-25mM phosphate buffer, 14-450mM trehalose, 0-22mg/mL alkali metal or alkali metal salt, 0.01-2mg/ mL polysorbate 80, pH is about 6.0-8.0, and the fusion protein is the fusion protein described herein. In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、5-25mM磷酸缓冲剂、10-170mg/mL海藻糖二水合物、0-22mg/mL碱金属或碱金属盐、0.01-2mg/mL聚山梨酯80，pH约为6.0-8.0，所述融合蛋白为本文所述的融合蛋白。在一些实施方案中，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation contains 0.05-1 mg/mL fusion protein, 5-25 mM phosphate buffer, 10-170 mg/mL trehalose dihydrate, 0-22 mg/mL alkali metal or alkali metal salt , 0.01-2mg/mL polysorbate 80, pH is about 6.0-8.0, and the fusion protein is the fusion protein described herein. In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM磷酸缓冲剂、约26.5mM海藻糖(约10mg/mL海藻糖二水合物)、约0.4mg/mL聚山梨酯80，pH约为7.0，所述融合蛋白包含如SEQ ID NO:56所示的氨基酸序列。 In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM phosphate buffer, about 26.5 mM trehalose (about 10 mg/mL trehalose dihydrate), about 0.4 mg/mL poly Sorbitate 80, pH is about 7.0, and the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM磷酸缓冲剂、约450mM海藻糖(约170mg/mL海藻糖二水合物)、约0.4mg/mL聚山梨酯80，pH约为7.0，所述融合蛋白包含如SEQ ID NO:56所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM phosphate buffer, about 450 mM trehalose (about 170 mg/mL trehalose dihydrate), about 0.4 mg/mL polysorbate Ester 80, pH is about 7.0, and the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM磷酸缓冲剂、约224.7mM海藻糖(约85mg/mL海藻糖二水合物)、约0.4mg/mL聚山梨酯80、约8mg/mL氯化钠，pH约为7.0，所述融合蛋白包含如SEQ ID NO:56所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM phosphate buffer, about 224.7 mM trehalose (about 85 mg/mL trehalose dihydrate), about 0.4 mg/mL poly Sorbitate 80, about 8 mg/mL sodium chloride, pH about 7.0, the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、5-25mM组氨酸缓冲剂、约200-450mM海藻糖、0.01-2mg/mL聚山梨酯80，pH为5.0-7.0，所述融合蛋白为本发明所述的融合蛋白。在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、5-25mM组氨酸缓冲剂、约80-170mg/mL海藻糖二水合物、0.01-2mg/mL聚山梨酯80，pH为5.0-7.0，所述融合蛋白为本发明所述的融合蛋白。在一些实施方案中，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, 5-25 mM histidine buffer, about 200-450 mM trehalose, 0.01-2 mg/mL polysorbate 80, pH 5.0 -7.0, the fusion protein is the fusion protein of the present invention. In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, 5-25 mM histidine buffer, about 80-170 mg/mL trehalose dihydrate, 0.01-2 mg/mL polysorbate 80, pH is 5.0-7.0, and the fusion protein is the fusion protein of the present invention. In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, and 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、约450mM海藻糖(约170mg/mL海藻糖二水合物)、约0.4mg/mL聚山梨酯80，pH为5.6-6.3，所述融合蛋白为本发明所述的融合蛋白。在一些实施方案中，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 450 mM trehalose (about 170 mg/mL trehalose dihydrate), about 0.4 mg/mL polysorbate 80, and a pH of 5.6-6.3, and the fusion protein is the fusion protein described in the present invention. In some embodiments, the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, and 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、约450mM海藻糖(约170mg/mL海藻糖二水合物)、约0.4mg/mL聚山梨酯80，pH为5.6-6.3，所述融合蛋白包含如SEQ ID NO:56所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 450 mM trehalose (about 170 mg/mL trehalose dihydrate), about 0.4 mg/mL Polysorbate 80, pH is 5.6-6.3, and the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约15-25mM组氨酸缓冲剂、约150-300mM海藻糖、约0.01-2mg/mL聚山梨酯80、约8-17mg/mL氯化钠，pH为5.0-7.0，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 15-25 mM histidine buffer, about 150-300 mM trehalose, about 0.01-2 mg/mL polysorbate 80, about 8-17 mg/mL sodium chloride, pH 5.0-7.0, the fusion protein includes the amino acid sequence shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, and 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约15-25mM组氨酸缓冲剂、约60-100mg/mL海藻糖二水合物、约0.01-2mg/mL聚山梨酯80、约8-17mg/mL氯化钠，pH为5.0-7.0，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。 In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 15-25 mM histidine buffer, about 60-100 mg/mL trehalose dihydrate, about 0.01-2 mg/mL poly Sorbitate 80, about 8-17 mg/mL sodium chloride, pH 5.0-7.0, the fusion protein includes the amino acids shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, 61 sequence.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、约224.7mM海藻糖(约85mg/mL海藻糖二水合物)、约0.4mg/mL聚山梨酯80、约8mg/mL氯化钠，pH为5.6-6.3，所述融合蛋白包含如SEQ ID NO:56所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20mM histidine buffer, about 224.7mM trehalose (about 85mg/mL trehalose dihydrate), about 0.4mg/ mL polysorbate 80, about 8 mg/mL sodium chloride, pH 5.6-6.3, and the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、约222mM海藻糖(约84mg/mL海藻糖二水合物)、约0.4mg/mL聚山梨酯80、约8-17mg/mL氯化钠，pH为5.6-6.3，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 222 mM trehalose (about 84 mg/mL trehalose dihydrate), about 0.4 mg/mL Polysorbate 80, about 8-17 mg/mL sodium chloride, pH 5.6-6.3, the fusion protein includes as shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, 61 Amino acid sequence.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、约222mM海藻糖(约84mg/mL海藻糖二水合物)、约0.4mg/mL聚山梨酯80、约8-17mg/mL氯化钠，pH为5.6-6.3，所述融合蛋白包含如SEQ ID NO:56所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 222 mM trehalose (about 84 mg/mL trehalose dihydrate), about 0.4 mg/mL Polysorbate 80, about 8-17 mg/mL sodium chloride, pH 5.6-6.3, the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、约222mM海藻糖(约84mg/mL海藻糖二水合物)、约0.4mg/mL聚山梨酯80、约8mg/mL氯化钠，pH为5.6-6.3，所述融合蛋白包含如SEQ ID NO:56所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 222 mM trehalose (about 84 mg/mL trehalose dihydrate), about 0.4 mg/mL Polysorbate 80, about 8 mg/mL sodium chloride, pH 5.6-6.3, the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、约222mM海藻糖(约84mg/mL海藻糖二水合物)、约0.4mg/mL聚山梨酯80、约16mg/mL氯化钠，pH为5.6-6.3，所述融合蛋白包含如SEQ ID NO:56或61所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 222 mM trehalose (about 84 mg/mL trehalose dihydrate), about 0.4 mg/mL Polysorbate 80, about 16 mg/mL sodium chloride, pH 5.6-6.3, the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56 or 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、15-25mM组氨酸缓冲剂、0.5-50mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-33mg/mL氯化钠，pH为5.0-7.0，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg/mL fusion protein, 15-25 mM histidine buffer, 0.5-50 mg/mL hydroxypropyl beta-cyclodextrin, 0.01-2 mg/mL polysorbate 80, 8-33 mg/mL sodium chloride, and a pH of 5.0-7.0, and the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, and 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、8-20mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH约为5.6-6.3，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, 8-20 mg/mL hydroxypropyl betacyclodextrin, 0.01-2 mg/mL polysorbate Ester 80, 8-17mg/mL sodium chloride, pH is about 5.6-6.3, the fusion protein includes the amino acid sequence shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, 61 .
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20 mM组氨酸缓冲剂、0.5-50mg/mL羟丙基倍他环糊精、约0.4mg/mL聚山梨酯80、8-33mg/mL氯化钠，pH为5.6-6.3，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, 0.5-50 mg/mL hydroxypropyl-beta-cyclodextrin, about 0.4 mg/mL polysorbate 80, 8-33 mg/mL sodium chloride, pH 5.6-6.3, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-17, 32-43, 51-56, 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、0.5-20mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-33mg/mL氯化钠，pH为5.6-6.3，所述融合蛋白包含如SEQ ID NO:56或61所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, 0.5-20 mg/mL hydroxypropyl betacyclodextrin, 0.01-2 mg/mL polysorbate Ester 80, 8-33mg/mL sodium chloride, pH 5.6-6.3, the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56 or 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、约8mg/mL羟丙基倍他环糊精、约0.4mg/mL聚山梨酯80、约16.5mg/mL氯化钠，pH约为5.6-6.3，所述融合蛋白包含如SEQ ID NO:17、56或61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 8 mg/mL hydroxypropyl betacyclodextrin, about 0.4 mg/mL polysorbate 80. About 16.5 mg/mL sodium chloride, pH is about 5.6-6.3, and the fusion protein includes the amino acid sequence shown in any one of SEQ ID NO: 17, 56 or 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、5-15mM组氨酸缓冲剂和2-10mM柠檬酸缓冲剂、0.5-50mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH约为5.6-6.3，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, 5-15 mM histidine buffer and 2-10 mM citrate buffer, 0.5-50 mg/mL hydroxypropyl betacycline paste Essence, 0.01-2mg/mL polysorbate 80, 8-17mg/mL sodium chloride, pH is about 5.6-6.3, the fusion protein includes SEQ ID NO: 12-17, 32-43, 51-56, The amino acid sequence shown in any one of 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约10mM组氨酸缓冲剂和约5mM柠檬酸缓冲剂、0.5-50mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH约为5.6-6.3，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation comprises 0.05-1 mg/mL fusion protein, about 10 mM histidine buffer and about 5 mM citric acid buffer, 0.5-50 mg/mL hydroxypropyl beta-cyclodextrin, 0.01-2 mg/mL polysorbate 80, 8-17 mg/mL sodium chloride, and a pH of about 5.6-6.3, and the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约10mM组氨酸缓冲剂和约5mM柠檬酸缓冲剂、0.5-20mg/mL羟丙基倍他环糊精、约0.4mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH约为5.6-6.3，所述融合蛋白包含如SEQ ID NO:56或61所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 10 mM histidine buffer and about 5 mM citrate buffer, 0.5-20 mg/mL hydroxypropyl betacyclodextrin, about 0.4 mg/mL polysorbate 80, 8-17 mg/mL sodium chloride, pH approximately 5.6-6.3, the fusion protein includes the amino acid sequence shown in SEQ ID NO: 56 or 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约10mM组氨酸缓冲剂和约5mM柠檬酸缓冲剂、约55.5mM海藻糖(约21mg/mL海藻糖二水合物)、约0.4mg/mL聚山梨酯80、约2mg/mL氯化钠，pH约为5.6-6.3，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1mg/mL fusion protein, about 10mM histidine buffer and about 5mM citrate buffer, about 55.5mM trehalose (about 21mg/mL trehalose dihydrate ), about 0.4 mg/mL polysorbate 80, about 2 mg/mL sodium chloride, pH about 5.6-6.3, the fusion protein includes SEQ ID NO: 12-17, 32-43, 51-56, 61 The amino acid sequence shown in any one.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、5-25mM组氨酸缓冲剂、60-100mg/mL蔗糖、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠， pH约为5.6-6.3，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation contains 0.05-1 mg/mL fusion protein, 5-25 mM histidine buffer, 60-100 mg/mL sucrose, 0.01-2 mg/mL polysorbate 80, 8-17 mg /mL sodium chloride, The pH is about 5.6-6.3, and the fusion protein includes the amino acid sequence shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, and 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、约80mg/mL蔗糖、约0.4mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH约为5.6-6.3，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 80 mg/mL sucrose, about 0.4 mg/mL polysorbate 80, 8-17 mg/mL Sodium chloride, pH is about 5.6-6.3, and the fusion protein includes the amino acid sequence shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, and 61.
在一些实施方案中，所述冠状病毒疫苗制剂包含0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、约80mg/mL蔗糖、约0.4mg/mL聚山梨酯80、约8mg/mL氯化钠，pH约为5.6-6.3，所述融合蛋白包含如SEQ ID NO:17所示的氨基酸序列。In some embodiments, the coronavirus vaccine formulation includes 0.05-1 mg/mL fusion protein, about 20 mM histidine buffer, about 80 mg/mL sucrose, about 0.4 mg/mL polysorbate 80, about 8 mg/mL chloride Sodium chloride, pH is about 5.6-6.3, and the fusion protein includes the amino acid sequence shown in SEQ ID NO: 17.
在一些实施方案中，所述冠状病毒疫苗制剂还包含佐剂。在一些实施方案中，所述佐剂为铝佐剂、SWE佐剂或MF59佐剂。在一些实施方案中，所述佐剂的添加量为所述冠状病毒疫苗制剂总体积的1/10-5/10。在一些实施方案中，所述佐剂的添加量为所述冠状病毒疫苗制剂总体积的3/10。In some embodiments, the coronavirus vaccine formulation further includes an adjuvant. In some embodiments, the adjuvant is aluminum adjuvant, SWE adjuvant, or MF59 adjuvant. In some embodiments, the adjuvant is added in an amount of 1/10-5/10 of the total volume of the coronavirus vaccine formulation. In some embodiments, the adjuvant is added in an amount of 3/10 of the total volume of the coronavirus vaccine formulation.
在一些实施方案中，上述冠状病毒疫苗制剂中融合蛋白包含至少二种本文所述融合蛋白。在一些实施方案中，上述冠状病毒疫苗制剂中融合蛋白包含二种本文所述融合蛋白。在一些实施方案中，上述冠状病毒疫苗制剂中融合蛋白包含三种本文所述融合蛋白。In some embodiments, the fusion protein in the above-described coronavirus vaccine formulation includes at least two fusion proteins described herein. In some embodiments, the fusion protein in the above-described coronavirus vaccine formulation includes two fusion proteins described herein. In some embodiments, the fusion protein in the above-described coronavirus vaccine formulation includes three fusion proteins described herein.
在一些实施方案中提供一种冠状病毒多价疫苗，所述冠状病毒多价疫苗包含至少二种本文所述融合蛋白。在一些实施方案中，所述冠状病毒多价疫苗包含二种本文所述融合蛋白。在一些实施方案中，所述冠状病毒多价疫苗包含三种本文所述融合蛋白。In some embodiments there is provided a multivalent coronavirus vaccine comprising at least two fusion proteins described herein. In some embodiments, the coronavirus multivalent vaccine comprises two fusion proteins described herein. In some embodiments, the coronavirus multivalent vaccine includes three fusion proteins described herein.
在一些实施方案中提供一种冠状病毒多价疫苗，所述冠状病毒多价疫苗包含第一融合蛋白和第二融合蛋白，所述第一融合蛋白为上文所述的融合蛋白，所述第二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，所述冠状病毒多价疫苗还包括药学上可接受的载体和/或佐剂。In some embodiments, a coronavirus multivalent vaccine is provided, the coronavirus multivalent vaccine comprising a first fusion protein and a second fusion protein, the first fusion protein being the fusion protein described above, and the third fusion protein being the fusion protein described above. The dual fusion protein contains the mutated extracellular domain of the SARS-CoV-2 Delta variant Spike protein or its truncated fragment and the monomeric subunit protein connected through a linker. In some embodiments, the coronavirus multivalent vaccine further includes a pharmaceutically acceptable carrier and/or adjuvant.
在一些实施方案中提供一种冠状病毒多价疫苗制剂，包含第一融合蛋白和第二融合蛋白，还包含缓冲剂、稳定剂、碱金属或碱金属盐、表面活性剂中的一种或多种，所述第一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，所述第二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。In some embodiments, a coronavirus multivalent vaccine preparation is provided, comprising a first fusion protein and a second fusion protein, and further comprising one or more of a buffer, a stabilizer, an alkali metal or an alkali metal salt, and a surfactant. species, the first fusion protein includes the mutation-containing extracellular domain of the SARS-CoV-2 Omicron variant Spike protein or its truncated fragment and the monomeric subunit protein connected through a linker, and the second fusion protein includes the mutation-containing extracellular domain of the Spike protein. Adapter-linked extracellular domain of spike protein of SARS-CoV-2 Delta variant or its truncated fragments and monomeric subunits protein.
在一些实施方案中，所述含突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域或其截短片段，其突变包含：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。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。In some embodiments, the mutation-containing SARS-CoV-2 Delta variant Spike protein extracellular domain or a truncated fragment thereof comprises: 1) mutation of RRAR to GSAS; 2) a mutation that prevents the formation of a straight helix during fusion exists in the turning region between HR1 and CH. In some embodiments, the mutation comprises: 1) mutation of RRAR to GSAS; 2) a double mutation K986P/V987P exists in the turning region between HR1 and CH.
在一些实施方案中，所述含突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域的截短片段，其与SARS-CoV-2 Delta变异株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。In some embodiments, the truncated fragment of the extracellular domain of the SARS-CoV-2 Delta variant Spike protein containing mutations is compared with the full-length extracellular domain of the SARS-CoV-2 Delta variant Spike protein. , 5-80 amino acid residues are truncated at the C terminus; or 20-76 amino acid residues are truncated at the C terminus; or 70 amino acid residues are truncated at the C terminus.
在一些实施方案中，所述含突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域或其截短片段包含如SEQ ID NO:45-50任一项所示的氨基酸序列，或与SEQ ID NO:45-50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:45-50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。In some embodiments, the mutation-containing SARS-CoV-2 Delta variant Spike protein extracellular domain or a truncated fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NO: 45-50, or is identical to An amino acid sequence that is at least 80% or at least 90% identical to the amino acid sequence shown in any one of SEQ ID NO: 45-50, or compared to the amino acid sequence shown in any one of SEQ ID NO: 45-50 An amino acid sequence with one or more conservative amino acid substitutions.
在一些实施方案中，所述第二融合蛋白是将含突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域或其截短片段的C端通过接头与单体亚基蛋白的N端连接。In some embodiments, the second fusion protein is a C-terminus of the extracellular domain of the SARS-CoV-2 Delta variant Spike protein or a truncated fragment thereof through a linker and the N-terminus of the monomeric subunit protein. connect.
在一些实施方案中，所述第二融合蛋白的接头为GS接头。在一些实施方案中，所述第二融合蛋白的接头选自GS，GGS，GGGS，GGGGS，SGGGS，GGSS，(GGGGS)2，(GGGGS)3，或其任意组合。在一些实施方案中，所述第二融合蛋白的接头为(GmS)n，其中，每个m独立为1、2、3、4或5，n为1、2、3、4或5。In some embodiments, the linker of the second fusion protein is a GS linker. In some embodiments, the linker of the second fusion protein is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS) 2 , (GGGGS) 3 , or any combination thereof. In some embodiments, the linker of the second fusion protein is (G m S) n , wherein each m is independently 1, 2, 3, 4, or 5, and n is 1, 2, 3, 4, or 5 .
在一些实施方案中，所述第二融合蛋白的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，所述第二融合蛋白的单体亚基蛋白为单体铁蛋白亚基。在一些实施方案中，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。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，所述单体铁蛋白亚基包含如SEQ ID NO:10所示的氨基酸序列，或与SEQ ID NO:10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:10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。In some embodiments, the monomeric subunit protein of the second fusion protein is a self-assembled monomeric subunit protein. In some embodiments, the monomeric subunit protein of the second fusion protein is a monomeric ferritin subunit. In some embodiments, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, phycoferritin, insect ferritin, fungal ferritin, or mammalian ferritin. In some embodiments, the monomeric ferritin subunit is a Helicobacter pylori non-heme monomeric ferritin subunit. In some embodiments, the monomeric ferritin subunit comprises the amino acid sequence set forth in SEQ ID NO: 10, or is at least 80% or at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 10. A unique 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:10.
在一些实施方案中，所述第二融合蛋白包含如SEQ ID NO:51-56任一项所示的氨基酸序列，或与SEQ ID NO:51-56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:51-56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。In some embodiments, the second fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 51-56, or has an amino acid sequence as compared to the amino acid sequence set forth in any one of SEQ ID NO: 51-56 An amino acid sequence that is at least 80% or at least 90% identical, or has an amino acid sequence compared to the amino acid sequence shown in any one of SEQ ID NO: 51-56 An amino acid sequence with one or more conservative amino acid substitutions.
在一些实施方案中，所述第一融合蛋白包含如SEQ ID NO:14、15或17所示的序列，第二融合蛋白包含如SEQ ID NO:53、54或56所示的序列。In some embodiments, the first fusion protein comprises a sequence as shown in SEQ ID NO:14, 15 or 17, and the second fusion protein comprises a sequence as shown in SEQ ID NO:53, 54 or 56.
在一些实施方案中，所述第一融合蛋白包含如SEQ ID NO:17所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列。In some embodiments, the first fusion protein comprises the sequence set forth in SEQ ID NO: 17 and the second fusion protein comprises the sequence set forth in SEQ ID NO: 56.
在一些实施方案中，所述第一融合蛋白和第二融合蛋白的质量比为(1-5):(1-5)，或质量比为(1-3):(1-3)，或质量比为(1-2):(1-2)，或质量比为1:(1-2)，或质量比为(1-2):1，或质量比为1:1。In some embodiments, the mass ratio of the first fusion protein to the second fusion protein is (1-5):(1-5), or the mass ratio is (1-3):(1-3), or the mass ratio is (1-2):(1-2), or the mass ratio is 1:(1-2), or the mass ratio is (1-2):1, or the mass ratio is 1:1.
在一些实施方案中，所述冠状病毒多价疫苗包含0.01-2mg/mL第一融合蛋白和0.01-2mg/mL第二融合蛋白。In some embodiments, the coronavirus multivalent vaccine comprises 0.01-2 mg/mL of a first fusion protein and 0.01-2 mg/mL of a second fusion protein.
在一些实施方案中，所述第一融合蛋白包含如SEQ ID NO:17所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列，所述第一融合蛋白和第二融合蛋白的质量比为1:1。In some embodiments, the first fusion protein includes the sequence shown in SEQ ID NO: 17, the second fusion protein includes the sequence shown in SEQ ID NO: 56, the first fusion protein and the second fusion protein The mass ratio of protein is 1:1.
在一些实施方案中，所述第一融合蛋白包含如SEQ ID NO:61所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列，所述第一融合蛋白和第二融合蛋白的质量比为1:1。In some embodiments, the first fusion protein includes the sequence shown in SEQ ID NO: 61, the second fusion protein includes the sequence shown in SEQ ID NO: 56, the first fusion protein and the second fusion protein The mass ratio of protein is 1:1.
在一些实施方案中，所述冠状病毒多价疫苗制剂包含第一融合蛋白和第二融合蛋白，还包含缓冲剂、稳定剂、碱金属或碱金属盐、表面活性剂中的一种或多种，所述第一融合蛋白包含如SEQ ID NO:17或61所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列。In some embodiments, the coronavirus multivalent vaccine preparation includes a first fusion protein and a second fusion protein, and also includes one or more of a buffer, a stabilizer, an alkali metal or an alkali metal salt, and a surfactant. , the first fusion protein includes the sequence shown in SEQ ID NO:17 or 61, and the second fusion protein includes the sequence shown in SEQ ID NO:56.
在一些实施方案中，所述冠状病毒多价疫苗制剂包含0.01-2mg/mL第一融合蛋白和0.01-2mg/mL第二融合蛋白，还包含5-15mM组氨酸缓冲剂和2-10mM柠檬酸缓冲剂、0.5-80mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH为5.0-7.0，所述第一融合蛋白包含如SEQ ID NO:17或61所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列。In some embodiments, the coronavirus multivalent vaccine formulation contains 0.01-2 mg/mL first fusion protein and 0.01-2 mg/mL second fusion protein, and further contains 5-15mM histidine buffer and 2-10mM lemon Acid buffer, 0.5-80 mg/mL hydroxypropyl betacyclodextrin, 0.01-2 mg/mL polysorbate 80, 8-17 mg/mL sodium chloride, pH 5.0-7.0, the first fusion protein includes The second fusion protein contains the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein contains the sequence shown in SEQ ID NO: 56.
在一些实施方案中，所述冠状病毒多价疫苗制剂包含0.01-0.2mg/mL第一融合蛋白和0.01-0.2mg/mL第二融合蛋白，还包含约10mM组氨酸缓冲剂和约5mM柠檬酸缓冲剂、0.5-20mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH为5.6-6.3，所述第一融合蛋白包含如SEQ ID NO:17或61所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列。In some embodiments, the coronavirus multivalent vaccine formulation includes 0.01-0.2 mg/mL first fusion protein and 0.01-0.2 mg/mL second fusion protein, and further includes about 10 mM histidine buffer and about 5 mM citric acid Buffer, 0.5-20 mg/mL hydroxypropyl betacyclodextrin, 0.01-2 mg/mL polysorbate 80, 8-17 mg/mL sodium chloride, pH 5.6-6.3, the first fusion protein includes as follows The sequence shown in SEQ ID NO:17 or 61, and the second fusion protein includes the sequence shown in SEQ ID NO:56.
在一些实施方案中，所述冠状病毒多价疫苗制剂包含0.01-0.2mg/mL第一融合蛋 白和0.01-0.2mg/mL第二融合蛋白，还包含约10mM组氨酸缓冲剂和约5mM柠檬酸缓冲剂、约4mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、约8.26mg/mL氯化钠，所述第一融合蛋白包含如SEQ ID NO:17或61所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列。In some embodiments, the coronavirus multivalent vaccine formulation comprises 0.01-0.2 mg/mL first fusion protein Baihe 0.01-0.2mg/mL second fusion protein also contains about 10mM histidine buffer and about 5mM citrate buffer, about 4mg/mL hydroxypropyl betacyclodextrin, 0.01-2mg/mL polysorbate 80. About 8.26 mg/mL sodium chloride, the first fusion protein includes the sequence shown in SEQ ID NO: 17 or 61, and the second fusion protein includes the sequence shown in SEQ ID NO: 56.
在一些实施方案中，所述冠状病毒多价疫苗制剂包含0.01-2mg/mL第一融合蛋白和0.01-2mg/mL第二融合蛋白，还包含5-15mM组氨酸缓冲剂、0.5-80mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH为5.0-7.0，所述第一融合蛋白包含如SEQ ID NO:17或61所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列。In some embodiments, the coronavirus multivalent vaccine formulation includes 0.01-2 mg/mL first fusion protein and 0.01-2 mg/mL second fusion protein, and further includes 5-15 mM histidine buffer, 0.5-80 mg/ mL hydroxypropyl betacyclodextrin, 0.01-2mg/mL polysorbate 80, 8-17mg/mL sodium chloride, pH 5.0-7.0, the first fusion protein includes SEQ ID NO: 17 or 61 The sequence shown is that the second fusion protein contains the sequence shown in SEQ ID NO:56.
在一些实施方案中，所述冠状病毒多价疫苗制剂包含0.01-0.2mg/mL第一融合蛋白和0.01-0.2mg/mL第二融合蛋白，还包含约10mM组氨酸缓冲剂、0.5-20mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH为5.6-6.3，所述第一融合蛋白包含如SEQ ID NO:17或61所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列。In some embodiments, the coronavirus multivalent vaccine formulation includes 0.01-0.2 mg/mL first fusion protein and 0.01-0.2 mg/mL second fusion protein, and also includes about 10 mM histidine buffer, 0.5-20 mg /mL hydroxypropyl betacyclodextrin, 0.01-2mg/mL polysorbate 80, 8-17mg/mL sodium chloride, pH is 5.6-6.3, the first fusion protein includes SEQ ID NO: 17 or The sequence shown in SEQ ID NO: 56, the second fusion protein contains the sequence shown in SEQ ID NO: 56.
在一些实施方案中，所述冠状病毒多价疫苗制剂包含0.01-0.2mg/mL第一融合蛋白和0.01-0.2mg/mL第二融合蛋白，还包含约10mM组氨酸缓冲剂、约4mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、约8.26mg/mL氯化钠，所述第一融合蛋白包含如SEQ ID NO:17或61所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列。In some embodiments, the coronavirus multivalent vaccine formulation includes 0.01-0.2 mg/mL first fusion protein and 0.01-0.2 mg/mL second fusion protein, and also includes about 10 mM histidine buffer, about 4 mg/mL mL hydroxypropyl betacyclodextrin, 0.01-2mg/mL polysorbate 80, about 8.26mg/mL sodium chloride, the first fusion protein includes the sequence shown in SEQ ID NO: 17 or 61, the first The two fusion proteins include the sequence shown in SEQ ID NO:56.
在一些实施方案中，所述冠状病毒多价疫苗制剂还包含佐剂。在一些实施方案中，所述佐剂为铝佐剂、SWE佐剂或MF59佐剂。在一些实施方案中，所述佐剂的添加量为制剂总体积的1/10-5/10。在一些实施方案中，所述佐剂的添加量约为制剂总体积的3/10。在一些实施方案中，所述佐剂为SWE佐剂。In some embodiments, the coronavirus multivalent vaccine formulation further includes an adjuvant. In some embodiments, the adjuvant is aluminum adjuvant, SWE adjuvant, or MF59 adjuvant. In some embodiments, the adjuvant is added in an amount ranging from 1/10 to 5/10 of the total volume of the formulation. In some embodiments, the adjuvant is added in an amount of approximately 3/10 of the total volume of the formulation. In some embodiments, the adjuvant is a SWE adjuvant.
在一些实施方案中，所述冠状病毒多价疫苗制剂包含约0.08mg/mL第一融合蛋白和约0.08mg/mL第二融合蛋白，还包含约10mM组氨酸缓冲剂、约4mg/mL羟丙基倍他环糊精、约0.2mg/mL聚山梨酯80、约8.26mg/mL氯化钠和SWE佐剂，pH为5.6-6.3，所述第一融合蛋白包含如SEQ ID NO:17或61所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列。In some embodiments, the coronavirus multivalent vaccine formulation includes about 0.08 mg/mL first fusion protein and about 0.08 mg/mL second fusion protein, and also includes about 10 mM histidine buffer, about 4 mg/mL hydroxypropanol Betacyclodextrin, about 0.2 mg/mL polysorbate 80, about 8.26 mg/mL sodium chloride and SWE adjuvant, pH 5.6-6.3, the first fusion protein comprising SEQ ID NO: 17 or The sequence shown in SEQ ID NO: 56, the second fusion protein contains the sequence shown in SEQ ID NO: 56.
在一些实施方案中，所述冠状病毒多价疫苗制剂包含约0.04mg/mL第一融合蛋白和约0.04mg/mL第二融合蛋白，还包含约10mM组氨酸缓冲剂、约4mg/mL羟丙基倍他环糊精、约0.2mg/mL聚山梨酯80、约8.26mg/mL氯化钠和SWE佐剂，pH为5.6-6.3，所述第一融合蛋白包含如SEQ ID NO:17或61所示的序列，第二融合蛋白包 含如SEQ ID NO:56所示的序列。In some embodiments, the coronavirus multivalent vaccine formulation comprises about 0.04 mg/mL of a first fusion protein and about 0.04 mg/mL of a second fusion protein, and further comprises about 10 mM histidine buffer, about 4 mg/mL of hydroxypropyl beta-cyclodextrin, about 0.2 mg/mL of polysorbate 80, about 8.26 mg/mL of sodium chloride, and a SWE adjuvant, with a pH of 5.6-6.3, wherein the first fusion protein comprises a sequence as shown in SEQ ID NO: 17 or 61, and the second fusion protein comprises Contains the sequence shown in SEQ ID NO:56.
在一些实施方案中，所述冠状病毒多价疫苗制剂规格为0.5mL，包含约40μg第一融合蛋白和约40μg第二融合蛋白，还包含约0.39mg组氨酸、约0.53mg盐酸组氨酸、约2mg羟丙基倍他环糊精、约0.1mg聚山梨酯80、约4.13mg氯化钠和0.15mL SWE佐剂，pH为5.6-6.3，所述第一融合蛋白包含如SEQ ID NO:17或61所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列。In some embodiments, the coronavirus multivalent vaccine formulation has a specification of 0.5 mL, comprising about 40 μg of a first fusion protein and about 40 μg of a second fusion protein, and also comprises about 0.39 mg of histidine, about 0.53 mg of histidine hydrochloride, about 2 mg of hydroxypropyl beta-cyclodextrin, about 0.1 mg of polysorbate 80, about 4.13 mg of sodium chloride and 0.15 mL of SWE adjuvant, with a pH of 5.6-6.3, wherein the first fusion protein comprises a sequence as shown in SEQ ID NO:17 or 61, and the second fusion protein comprises a sequence as shown in SEQ ID NO:56.
在一些实施方案中，所述冠状病毒多价疫苗制剂规格为0.5mL，包含冠状病毒多价疫苗包含约20μg第一融合蛋白和约20μg第二融合蛋白，还包含约0.39mg组氨酸、约0.53mg盐酸组氨酸、约2mg羟丙基倍他环糊精、约0.1mg聚山梨酯80、约4.13mg氯化钠和0.15mL SWE佐剂，所述第一融合蛋白包含如SEQ ID NO:17或61所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列。In some embodiments, the coronavirus multivalent vaccine preparation has a specification of 0.5 mL, and the coronavirus multivalent vaccine contains about 20 μg of the first fusion protein and about 20 μg of the second fusion protein, and also contains about 0.39 mg of histidine, about 0.53 mg histidine hydrochloride, about 2 mg hydroxypropyl betacyclodextrin, about 0.1 mg polysorbate 80, about 4.13 mg sodium chloride and 0.15 mL SWE adjuvant, the first fusion protein comprising SEQ ID NO: 17 or 61, the second fusion protein contains the sequence shown in SEQ ID NO: 56.
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SARS-CoV-2原始株或其变异株感染。在一些实施方案中，所述冠状病毒感染为SARS-CoV-2原始株、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。在一些实施方案中，所述冠状病毒感染为SARS-CoV-2 Omicron变异株BA.1、BA.2、BA.3、BA.4、BA.5、BQ.1、BQ.1.1、BF.7、XBB、XBB.1、XBB.1.5、XBB.1.5.1、XBB.1.9.1或XBB.1.16感染。在一些实施方案中，所述冠状病毒感染为SARS-CoV-2 Omicron变异株BA.5感染。在一些实施方案中，所述冠状病毒感染为SARS-CoV-2 Omicron变异株XBB.1.5感染。在一些实施方案中，所述冠状病毒感染为SARS-CoV-2 Omicron变异株XBB.1.16感染。The present invention also provides methods and uses for prevention or treatment. In some embodiments, the present invention provides methods for preventing or treating coronavirus infection comprising administering to a patient in need thereof an effective amount of a fusion protein, Spike protein nanoparticle or coronavirus vaccine described herein. In some embodiments, use of the fusion proteins, Spike protein nanoparticles, or coronavirus vaccines described herein in preventing or treating SARS or COVID-19 is provided. In some embodiments, there is provided the use of the fusion protein or Spike protein nanoparticles described herein in the preparation of a vaccine to prevent or treat SARS-CoV-2 infection. In some embodiments, the coronavirus infection is a SARS-CoV-2, SARS-CoV or MERS-CoV infection. In some embodiments, the coronavirus infection is an infection with the original strain of SARS-CoV-2 or a variant thereof. In some embodiments, the coronavirus infection is an original strain of SARS-CoV-2, a SARS-CoV-2 Alpha variant, a SARS-CoV-2 Beta variant, a SARS-CoV-2 Gamma variant, or a SARS-CoV -2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant. In some embodiments, the coronavirus infection is SARS-CoV-2 Omicron variant strain BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF. 7. XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.16 infection. In some embodiments, the coronavirus infection is a SARS-CoV-2 Omicron variant BA.5 infection. In some embodiments, the coronavirus infection is a SARS-CoV-2 Omicron variant XBB.1.5 infection. In some embodiments, the coronavirus infection is a SARS-CoV-2 Omicron variant XBB.1.16 infection.
附图说明Description of the drawings
图1为融合蛋白D与人ACE2结合曲线。Figure 1 shows the binding curve of fusion protein D and human ACE2.
图2为融合蛋白2-1与人ACE2结合曲线。Figure 2 shows the binding curve of fusion protein 2-1 and human ACE2.
图3为双价疫苗加或不加佐剂免疫小鼠后的血清对Spike蛋白特异性IgG滴度； 其中，WT代表WT-Spike蛋白，Delta代表Delta-Spike蛋白、BA.1代表BA.1-Spike蛋白、BA.5代表BA.5-Spike蛋白。Figure 3 shows the serum IgG titer specific to Spike protein after immunizing mice with the bivalent vaccine with or without adjuvant; Among them, WT represents WT-Spike protein, Delta represents Delta-Spike protein, BA.1 represents BA.1-Spike protein, and BA.5 represents BA.5-Spike protein.
图4为小鼠血清抗Spike蛋白IgG抗体滴度；图4a、4c、4e、4g为初次免疫后小鼠血清抗Spike蛋白IgG抗体滴度，图4b、4d、4f、4h为第二次增强免疫后小鼠血清抗Spike蛋白IgG抗体滴度；其中，条形表示滴度的几何平均值(GMT)(值显示在条形内)，误差线条表示95％置信区间(confidence interval，CI)。Figure 4 shows the anti-Spike protein IgG antibody titers in mouse serum; Figures 4a, 4c, 4e, and 4g show the anti-Spike protein IgG antibody titers in mouse serum after the first immunization, and Figures 4b, 4d, 4f, and 4h show the second enhancement Anti-Spike protein IgG antibody titer in mouse serum after immunization; among them, the bar represents the geometric mean (GMT) of the titer (the value is displayed within the bar), and the error bar represents the 95% confidence interval (CI).
图5为融合蛋白D、融合蛋白2-1及双价疫苗免疫小鼠后血清对假病毒抑制滴度；其中，WT代表SARS-CoV-2原始株假病毒，Delta代表SARS-CoV-2 Delta假病毒，BA.5代表SARS-CoV-2 BA.5假病毒，BQ.1.1代表SARS-CoV-2 BQ.1.1假病毒，XBB代表SARS-CoV-2 XBB假病毒，XBB.1.5代表SARS-CoV-2 XBB.1.5假病毒；条形表示滴度的几何平均值(GMT)(值显示在条形内)；误差线条表示95％CI。Figure 5 shows the inhibitory titer of serum against pseudovirus after immunizing mice with fusion protein D, fusion protein 2-1 and bivalent vaccine; among them, WT represents the original strain of SARS-CoV-2 pseudovirus, and Delta represents SARS-CoV-2 Delta. Pseudovirus, BA.5 represents SARS-CoV-2 BA.5 pseudovirus, BQ.1.1 represents SARS-CoV-2 BQ.1.1 pseudovirus, XBB represents SARS-CoV-2 XBB pseudovirus, XBB.1.5 represents SARS- CoV-2 XBB.1.5 pseudovirus; bars represent the geometric mean (GMT) of titers (values are shown within the bars); error bars represent 95% CI.
图6为双价疫苗二次免疫后小鼠血清对新冠病毒真病毒抑制滴度；其中，条形表示滴度的几何平均值(GMT)(值显示在条形内)；误差线条表示95％CI；ULOD表示该试验的检测上限。Figure 6 shows the inhibitory titer of mouse serum against the true coronavirus after secondary immunization with the bivalent vaccine; among them, the bar represents the geometric mean (GMT) of the titer (the value is displayed within the bar); the error bar represents 95% CI; ULOD indicates the upper limit of detection of the test.
图7为双价疫苗序贯免疫小鼠血清抗Spike蛋白IgG抗体滴度；其中，条形表示滴度的几何平均值(GMT)(值显示在条形内)；误差线条表示95％CI。Figure 7 shows the serum anti-Spike protein IgG antibody titers of mice sequentially immunized with the bivalent vaccine; the bars represent the geometric mean (GMT) of the titers (values are displayed within the bars); the error bars represent the 95% CI.
图 8为双价疫苗序贯免疫小鼠血清假病毒抑制滴度；其中，WT代表SARS-CoV-2原始株假病毒，Delta代表SARS-CoV-2 Delta假病毒，BF.7代表SARS-CoV-2 BF.7假病毒，XBB.1代表SARS-CoV-2 XBB.1假病毒；条形表示滴度的几何平均值(GMT)(值显示在条形内)；误差线条表示95％CI。Figure 8 shows the inhibitory titers of serum pseudoviruses in mice sequentially immunized with the bivalent vaccine; among them, WT represents the original strain of SARS-CoV-2 pseudovirus, Delta represents the SARS-CoV-2 Delta pseudovirus, and BF.7 represents SARS-CoV. -2 BF.7 pseudovirus, XBB.1 represents SARS-CoV-2 XBB.1 pseudovirus; bars represent the geometric mean (GMT) of titers (values are shown within bars); error bars represent 95% CI .
图9为疫苗免疫小鼠脾细胞ELISpot；其中，WT代表新冠病毒原始株的Spike蛋白肽池，Delta代表新冠病毒变异株Delta的Spike蛋白肽池，BA.5代表新冠病毒变异株BA.5的Spike蛋白肽池；误差线表示几何平均值±95％CI。Figure 9 shows the ELISpot of spleen cells of vaccine-immunized mice; among them, WT represents the Spike protein peptide pool of the original strain of the new coronavirus, Delta represents the Spike protein peptide pool of the new coronavirus mutant strain Delta, and BA.5 represents the Spike protein peptide pool of the new coronavirus mutant strain BA.5. Spike protein peptide pool; error bars represent geometric mean ±95% CI.
图10为大鼠血清抗Spike蛋白IgG抗体滴度；其中，WT代表WT-Spike蛋白，Delta代表Delta-Spike蛋白，BA.5代表BA.5-Spike蛋白；条形表示滴度的几何平均值(GMT)(值显示在条形内)，误差线条表示95％CI。Figure 10 shows the rat serum anti-Spike protein IgG antibody titer; where, WT represents WT-Spike protein, Delta represents Delta-Spike protein, and BA.5 represents BA.5-Spike protein; the bar represents the geometric mean of the titer. (GMT) (values shown within bars), error bars represent 95% CI.
术语the term
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。Unless otherwise stated, each of the following terms shall have the meaning set forth below.
定义definition
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。 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
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。It should be noted that, as used herein and in the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, nucleic acid molecule refers to one or more nucleic acid molecules. Accordingly, the terms "a", "an", "one or more" and "at least one" may be used interchangeably. Similarly, the terms "comprising", "including" and "having" may be used interchangeably and should generally be understood to be open-ended and non-limiting, e.g. not excluding other unrecited elements or steps.
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)。The term "amino acid" refers to organic compounds containing both amino and carboxyl groups, such as alpha-amino acids, which may be encoded by nucleic acids directly or in the form of precursors. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called the "degeneracy of the genetic code." Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine 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).
“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。A "conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue containing a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). Generally speaking, conservative amino acid substitutions are unlikely to materially alter the functional properties of the protein. Examples of amino acid classes containing chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine. amino acid; 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) Basic side chains: lysine, Arginine and histidine; 6) Acidic side chains: aspartic acid and glutamic acid.
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。The term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptide" and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain" or any other term used to refer to two or more amino acid chains, and the term "polypeptide" may Used instead of or interchangeably with any of the above terms. The term "polypeptide" is also intended to refer to the product of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-natural Amino acid modifications that occur. A polypeptide may be derived from natural biological sources or produced by recombinant techniques, but it does not have to be translated from a specified nucleic acid sequence and may be produced by any means including chemical synthesis.
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。Unless otherwise stated, a fusion protein is a recombinant protein that contains amino acid sequences from at least two unrelated proteins that have been linked together by peptide bonds to form a single protein. Amino acid sequences of unrelated proteins can be linked directly to each other, or they can be linked using linkers. As used herein, if protein's Amino acid sequences are not related together if they are not normally linked together via peptide bonds in their natural environment (eg, within a cell). For example, the amino acid sequence of a common bacterial enzyme such as Bacillus stearothermophilus dihydrolipoate transacetylase (E2p) and the amino acid sequence of the coronavirus Spike protein are not linked together by peptide bonds.
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。The terms "homology," "identity" or "similarity" refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions within each sequence that can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences.
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。The term "encoding" when applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide that, in its native state or when manipulated by methods well known to those skilled in the art, may be transcribed and/or translated to produce the polypeptide and/or fragment thereof.
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。A polynucleotide is composed of a specific sequence of four bases: adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U) when the polynucleotide is RNA. A "polynucleotide sequence" can be represented by the letters of the polynucleotide molecule. This letter representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, such as functional genomics and homology searches.
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。The terms "polynucleotide," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or the like. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are examples of non-limiting 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 polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If such modifications are present, structural modifications to the nucleotide may be made before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, for example by conjugation with a labeling component. This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, embodiments of any polynucleotide of the present disclosure include double-stranded forms and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.
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。A nucleic acid or polynucleotide sequence (or a polypeptide or protein sequence) is "identical" or "sequence identical" to another sequence by a certain percentage (eg, 90%, 95%, 98% or 99%). When sequences are aligned, this percentage of bases (or amino acids) in the two sequences being compared are identical. The alignment percent identity or sequence identity can be determined using visual inspection or software programs known in the art, such as those described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. It is preferred to use the default parameters for comparison. One of the alignment programs is BLAST using default parameters, such as BLASTN and BLASTP, both of which use The following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+ GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that share the percentage identity specified above and encode a polypeptide with the same or similar biological activity.
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。The term "isolated" used in the present invention with respect to cells, nucleic acids, polypeptides, antibodies, etc., such as "isolated" DNA, RNA, polypeptides, and antibodies, refers to other components in the natural environment of cells, such as DNA or RNA. one or more separated molecules. The term "isolated" as used herein also refers to nucleic acids or peptides that are substantially free of cellular material, viral material or cell culture media when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that do not exist in their native state and do not exist in their native state. The term "isolated" is also used herein to refer to cells or polypeptides separated from other cellular proteins or tissues. Isolated polypeptide is intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc. are generally prepared by at least one purification step. In some embodiments, the purity of the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or any of these values. The range between any two values (inclusive) or any value within them.
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。The term "recombinant" refers to a polypeptide or polynucleotide and means a form of the polypeptide or polynucleotide that does not occur in nature, and non-limiting examples may be combined to produce polynucleotides that do not normally exist or Peptides.
“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。"Antibody" and "antigen-binding fragment" refer to polypeptides or polypeptide complexes that specifically recognize and bind to antigens. Antibodies can be complete antibodies, any antigen-binding fragments thereof, or single chains thereof. The term "antibody" thus includes any protein or peptide whose molecule contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen.
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。As used herein, the terms "antigen" or "immunogen" are used interchangeably and refer to a substance, typically a protein, capable of inducing an immune response in a subject. The term also refers to a protein that is immunologically active, i.e., capable of eliciting responses to the body fluids and/or Cell type immune response. Unless otherwise stated, the term "vaccine antigen" is used interchangeably with "protein antigen" or "antigenic polypeptide."
“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。"Neutralizing antibodies" refer to antibodies that reduce the infectious titer of an infectious agent by binding to a specific antigen on that agent. In some embodiments, the infectious agent is a virus. A "broadly neutralizing antibody" is an antibody that binds to and inhibits the function of a related antigen, e.g., at least 85%, 90%, 95%, 96%, 97%, 98% identical to the antigenic surface of the antigen % or 99% identity to the antigen. For antigens from pathogens, such as viruses, the antibodies can bind to and inhibit the function of more than one class and/or subclass of antigens from the pathogen.
“cDNA”是指与mRNA互补或相同的DNA，可以是单链或双链形式。"cDNA" refers to DNA that is complementary or identical to mRNA and may be in single- or double-stranded form.
“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。"Epitope" refers to an antigenic determinant. These are specific chemical groups or peptide sequences on molecules that are antigenic, So much so that they elicit a specific immune response, for example, an epitope is an antigenic region to which B and/or T cells respond. Epitopes can be formed from contiguous amino acids, or from non-contiguous amino acids juxtaposed by the tertiary folding of the protein.
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。Vaccine refers to a biological product that induces a preventive or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, vaccines elicit an antigen-specific immune response against the antigens of pathogens, such as viral pathogens, or cellular components associated with pathological conditions. Vaccines may include polynucleotides (eg, nucleic acids encoding known antigens), peptides or polypeptides (eg, disclosed antigens), viruses, cells, or one or more cellular components. In some embodiments, a vaccine or vaccine antigen or vaccine composition is expressed from a fusion protein expression vector and self-assembles into nanoparticles displaying the antigenic polypeptide or protein on the surface.
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。An effective amount of a vaccine or other agent is sufficient to produce a desired response, such as eliciting an immune response, preventing, alleviating, or eliminating signs or symptoms of a condition or disease (e.g., pneumonia). For example, this may be an amount necessary to inhibit viral replication or measurably alter the outward symptoms of a viral infection. Typically, this amount will be sufficient to measurably inhibit the replication or infectivity of the virus (eg, SARS-CoV-2). When administered to a subject, a dose that achieves target tissue concentrations and has been shown to achieve inhibition of viral replication in vitro will generally be used. In some embodiments, an "effective amount" is an amount that treats (including prevents) one or more symptoms and/or underlying causes of a condition or disease (eg, treating a coronavirus infection). In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount that prevents the development of one or more symptoms or signs of a particular disease or condition (eg, one or more symptoms or signs associated with a coronavirus infection).
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(24mer)，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。Nanoparticles refer to spherical protein shells that are tens of nanometers in diameter and have well-defined surface geometry. The spherical protein shell is formed from identical copies of non-viral proteins that self-assemble into nanoparticles with an appearance similar to virus-like particles (VLPs). Examples include ferritin (FR), which is conserved across multiple species and forms a 24mer, Bacillus stearothermophilus dihydrolipoic acid transacetylase (E2P), Hyperthermophila dioxytetrahydrogen pteridine synthase (LS) and Thermotoga maritima encapsulin, all of which form a 60-mer. Self-assembling nanoparticles can form spontaneously after recombinantly expressing proteins in an appropriate expression system. Methods for the production, detection and characterization of nanoparticles can use the same techniques developed for VLPs.
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。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。Virus-like particles (VLPs) refer to non-replicating viral shells derived from any of a variety of viruses. VLPs typically include one or more viral proteins, such as, but not limited to, those proteins known as capsid proteins, coat proteins, wall proteins, surface proteins and/or envelope proteins, or formed particles derived from these proteins of peptides. In an appropriate expression system, VLPs can form spontaneously after recombinantly expressed proteins. Methods for producing specific VLPs are known in the art. The presence of VLPs following recombinantly expressed viral proteins can be detected using conventional techniques known in the art (eg, by electron microscopy, biophysical characterization, etc.). For example, VLPs can be separated by density gradient centrifugation and/or identified by characteristic density bands. Alternatively, cryo-electron microscopy can be performed on vitrified water samples of the VLP preparation in question and images recorded under appropriate exposure conditions.
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。The terms "about" and "approximately" are used interchangeably and refer to corresponding numbers that are readily known to those skilled in the relevant technical fields. The normal error range for the value. In some embodiments, reference herein to "about" refers to the recited value as well as the range of ±10%, ±5%, or ±1% thereof.
“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。"ECMO" refers to Extracorporeal Membrane Oxygenation (ECMO), which is a medical emergency technical equipment mainly used to provide continuous extracorporeal breathing and circulation for patients with severe cardiopulmonary failure to maintain the patient's life.
“ICU”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。"ICU" refers to the intensive care unit (Intensive Care Unit). Treatment, nursing, and rehabilitation can all be carried out simultaneously. It provides isolation places and equipment for critically ill or comatose patients, and provides the best care, comprehensive treatment, combination of medical and nursing care, and surgery. Early rehabilitation, joint care, sports therapy and other services.
“IMV”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。"IMV" refers to intermittent mandatory ventilation, which implements periodic volume or pressure ventilation based on preset time intervals, that is, time triggers. This period allows the patient to breathe spontaneously at any set basal pressure level during mandatory ventilation. While breathing spontaneously, the patient can breathe on his own with continuous airflow support, or the machine will open the on-demand valve to allow for spontaneous breathing. Most ventilators can provide pressure support while breathing spontaneously.
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。The term "subject" refers to any animal classified as a mammal, such as humans and non-human mammals. Examples of non-human animals include dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, mice, etc. Unless otherwise stated, the terms "patient" or "subject" are used interchangeably herein. Preferably, the subject is human.
“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。"Treatment" means therapeutic treatment and prophylactic or preventative measures designed to prevent, slow down, ameliorate or halt adverse physiological changes or disorders, such as the progression of a disease, including but not limited to the following whether detectable or undetectable The results include alleviation of symptoms, reduction in disease severity, stabilization of disease status (i.e. no worsening), delay or slowdown of disease progression, improvement, alleviation, reduction or disappearance of disease status (whether partial or complete), prolongation and Expected survival without treatment, etc. Patients in need of treatment include patients who already have a condition or disorder, are susceptible to a condition or disorder, or are in need of prevention of a condition or disorder that may or are expected to result from administration of the Spike protein nanoparticles or pharmaceutical compositions disclosed herein. For patients who benefit from treatment.
概述Overview
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。 For SARS-CoV, MERS-CoV and SARS-CoV-2, the viral genome encodes spike (S), envelope (E), membrane (M) and nucleocapsid (N) structural proteins, among which, S glycoprotein ( Spike protein) is responsible for binding to host receptors via the receptor binding domain (RBD) in its S1 subunit, and subsequent membrane fusion and viral entry driven by its S2 subunit. Receptor binding can help keep the RBD in the "standing" state, which facilitates the dissociation of the S1 subunit from the S2 subunit. When the S1 subunit dissociates from the S2 subunit, a second S2' cleavage releases the fusion peptide. The linker region, HR1, and CH form a very long helix to insert the fusion peptide into the host cell membrane. Finally, HR1 and HR2 form a helical structure and assemble into a six-helix bundle to fuse the viral membrane and the host membrane.
RBD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。RBD contains a core subdomain and a receptor binding motif (RBM). Although the core subdomains are highly similar between the three coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2, their RBMs are significantly different, resulting in different receptor specificities: SARS-CoV and SARS-CoV -2 recognizes angiotensin-converting enzyme 2 (ACE2), while MERS-CoV binds dipeptidyl peptidase 4 (DPP4). Because the S glycoprotein is surface-exposed and mediates entry into host cells, it is the primary target for neutralizing antibodies (NAbs) following infection and is the focus of vaccine design. Spike trimers are extensively modified with N-linked glycans, which are important for correct folding and regulating accessibility to NAbs.
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。The present invention stabilizes the Spike trimer by 1) mutations that inactivate the S1/S2 cleavage site and 2) the presence of mutations in the turning region between HR1 and CH that prevent HR1 and CH from forming a straight helix during fusion. In a pre-fusion configuration with the host cell membrane. In some embodiments, mutant-containing coronavirus Spike protein extracellular domains or truncated fragments thereof can be displayed on nanoparticles.
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。Based on the studies and exemplary designs described herein, the present invention provides fusion proteins, Spike protein nanoparticles, and vaccine compositions. The invention also provides related polynucleotides, expression vectors and pharmaceutical compositions. In some embodiments, stable Spike trimers and RBD proteins in protein or nucleic acid (DNA/mRNA) forms carried by viral vectors can be used as coronavirus vaccines. Additionally, nanoparticle-presented stable Spike trimers and RBDs can also be used as coronavirus vaccines.
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、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。The coronavirus Spike protein-based antigens and vaccines of the present invention have many advantageous properties. The Spike trimer design described herein presents conserved neutralizing epitopes in its native-like conformation, allowing the Spike trimer to be used as an antigen vaccine or for multivalent display on nanoparticles. The nanoparticle vaccine of the present invention allows the display of Spike trimers derived from different coronaviruses on well-known nanoparticles, such as ferritin, E2p and I3-01, with sizes ranging from 12.2 to 25.0 nm. All trimer-presenting nanoparticles can be produced in HEK293 cells and CHO cells with high yields. The produced Spike protein nanoparticles can be purified by antibody and size exclusion chromatography (SEC).
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、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。Unless otherwise stated herein, the mutant coronavirus Spike protein extracellular domain or truncated fragments thereof, fusion proteins, Spike protein nanoparticles, encoded polynucleotides, expression vectors and host cells of the present invention and related treatments Applications can be produced or performed according to the methods exemplified herein or conventional methods well known in the art.
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。Unless otherwise stated, the order of steps or the order in which certain operations are performed is not important so long as the invention remains operable. Furthermore, two or more steps or operations can be performed simultaneously.
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。 Unless otherwise stated, any and all examples used herein, or exemplary language (such as "such as" or "including") used herein, are intended merely to better illuminate the invention and do not pose a limitation on the scope of the invention. . No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
含突变的冠状病毒Spike蛋白胞外结构域或其截短片段Containing mutated extracellular domain of coronavirus Spike protein or truncated fragments thereof
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。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。The present invention provides a mutation-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof that can be used to produce a vaccine. The mutated Spike trimer is stabilized by introducing mutations into the extracellular domain of the coronavirus Spike protein or its truncated fragments. This article illustrates some specific Spike proteins of specific SARS-CoV-2 strains or isolates, such as SEQ ID NO:1. Due to the functional similarities and sequence homologies between different isolates or strains of a given coronavirus, it is also possible to generate spike proteins derived from orthologous sequences of other known coronavirus Spike proteins according to the mutation strategies described here. Mutated Spike protein or truncated fragments thereof. Many known coronavirus Spike protein sequences have been described in the literature.
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。As described herein, some mutant Spike proteins or truncated fragments thereof of the invention comprise 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 a mutation that inactivates the S1/S2 cleavage site, and a mutation in the turn region between HR1 and CH, which removes any strain in the turn region between HR1 and CH, i.e. preventing the formation of a straight helix .
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、6-9、19-24、26-31、45-50、62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。在一些实施方案中，用于突变的Spike蛋白可以是SEQ ID NO:1、18、25或44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682RRAR685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682RRAR685突变为682GSAS685。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1139-1208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)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。 Some coronavirus Spike protein extracellular domains containing mutations or truncated fragments thereof (as shown in SEQ ID NO: 3-4, 6-9, 19-24, 26-31, 45-50, 62) are derived from COVID-19 SARS-CoV-2 virus. These polypeptides contain mutations that inactivate the S1/S2 cleavage site and mutations in the turning region between HR1 and CH. In some embodiments, the Spike protein used for mutation may be SEQ ID NO: 1, 18, 25, or 44, or a variant thereof, such as a variant that is substantially identical thereto or a conservatively modified variant thereof. Using amino acid numbering based on the cryo-EM model PDB ID 6VSB or GenBank accession number MN908947.3 as a reference, inactivation of the S1/S2 cleavage site 682 RRAR 685 can be achieved by a number of sequence changes within or around the site (e.g., missing or substituted) to achieve. As exemplified herein, one mutation that inactivates the S1/S2 cleavage site without affecting the protein structure is to mutate the S1/S2 cleavage site 682 RRAR 685 to 682 GSAS 685 . In addition to inactivating the S1/S2 cleavage site, a double mutation can be made in the turn region between HR1 and CH, which eliminates the turn region during fusion by preventing the formation of straight helices (HR1 and CH motifs between). In some embodiments, this double mutation can be K986G/V987G, K986P/V987P, K986G/V987P or K986P/V987G. In addition to the above-mentioned mutations that stabilize the structure of the pre-fusion Spike protein or truncated fragments thereof, some SARS-CoV-2 Spike proteins or truncated fragments thereof of the present invention may contain deletions of most or the entire HR2 domain. Using the exemplary SARS-CoV-2 Spike protein sequence SEQ ID NO: 1 to illustrate, such deletions may include deletions such as residues 1139-1208 of SEQ ID NO: 1. In some embodiments, the deletion may be 5, 10, 15, 20, 25, 30, 35, 40 of the C-terminus of the extracellular domain of the Spike protein (eg, SEQ ID NO: 1). , 45, 50, 55, 60, 65, 70, 75, 76, 80 or more residues, or a range between any two of these values, including endpoint) or any value therein. In some embodiments, a C-terminally truncated Spike protein can extend beyond the HR2 domain. In some embodiments, the Spike protein sequence may include the N-terminal signal peptide shown in SEQ ID NO: 2 or 5.
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：Exemplary coronavirus Spike protein extracellular domains or truncated fragments thereof or variants thereof are as follows:
SARS-CoV-2 Omicron变异株BA.5Spike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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685用下划线、加粗和斜体标出。
The full-length extracellular domain (ECD) of SARS-CoV-2 Omicron variant BA.5Spike protein, its amino acid sequence is shown in SEQ ID NO: 1, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) is used Italicized, S1/S2 cleavage site 682 RRAR 685 underlined, bolded and italicized.
突变的SARS-CoV-2 Omicron变异株BA.5Spike蛋白全长胞外结构域I-1，其氨基酸序列如SEQ ID NO:3所示。在序列中，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

The full-length extracellular domain I-1 of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein has an amino acid sequence as shown in SEQ ID NO: 3. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains double Mutation K986P/V987P, underlined and italicized.

突变的SARS-CoV-2 Omicron变异株BA.5Spike蛋白全长胞外结构域I-2，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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

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

突变的SARS-CoV-2 Omicron变异株BA.5Spike蛋白全长胞外结构域I-3，其氨基酸序列如SEQ ID NO:6所示。在序列中，不含信号肽，S1/S2切割位点682RRAR685突变为682GSAS685，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。

The full-length extracellular domain I-3 of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein has an amino acid sequence as shown in SEQ ID NO: 6. In the sequence, there is no signal peptide, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.

突变的SARS-CoV-2 Omicron变异株BA.5Spike蛋白胞外结构域C端截短片段I-4，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，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。
The amino acid sequence of the C-terminal truncated fragment I-4 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein is shown in SEQ ID NO: 7. In the sequence, 70 amino acid residues are truncated at the C-terminus, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is marked in italics, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , marked with underline and bold, and contains the double mutation K986P/V987P, marked with underline and italics.
突变的SARS-CoV-2 Omicron变异株BA.5Spike蛋白胞外结构域C端截短片段I-5，其氨基酸序列如SEQ ID NO:8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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点 682RRAR685突变为682GSAS685，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。
The mutated SARS-CoV-2 Omicron variant BA.5Spike protein extracellular domain C-terminal truncated fragment I-5, its amino acid sequence is shown in SEQ ID NO: 8. In the sequence, 70 amino acid residues were truncated at the C terminus, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide In italics, S1/S2 cleavage site The 682 RRAR 685 mutation is 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
突变的SARS-CoV-2 Omicron变异株BA.5Spike蛋白胞外结构域C端截短片段I-6，其氨基酸序列如SEQ ID NO:9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682RRAR685突变为682GSAS685，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。

The mutated SARS-CoV-2 Omicron variant BA.5 Spike protein extracellular domain C-terminal truncated fragment I-6, its amino acid sequence is shown in SEQ ID NO: 9. In the sequence, 70 amino acid residues are truncated at the C terminus, without the signal peptide, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains the double mutation K986P/V987P. , underlined and italicized.

SARS-CoV-2 Omicron变异株BA.2 Spike蛋白全长胞外结构域(ECD)，其氨基酸序列如SEQ ID NO:18所示，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685用下划线、加粗和斜体标出。

The full-length extracellular domain (ECD) of SARS-CoV-2 Omicron variant BA.2 Spike protein, its amino acid sequence is shown in SEQ ID NO:18, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) Italicized, S1/S2 cleavage site 682 RRAR 685 underlined, bolded and italicized.

突变的SARS-CoV-2 Omicron变异株BA.2 Spike蛋白全长胞外结构域I-6-1，其氨基酸序列如SEQ ID NO:19所示。在序列中，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。
The full-length extracellular domain I-6-1 of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein has an amino acid sequence as shown in SEQ ID NO: 19. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains double Mutation K986P/V987P, underlined and italicized.
突变的SARS-CoV-2 Omicron变异株BA.2 Spike蛋白全长胞外结构域I-7，其氨基酸序列如SEQ ID NO:20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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。
The full-length extracellular domain I-7 of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein has an amino acid sequence as shown in SEQ ID NO: 20. In the sequence, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 mutates to 682 GSAS 685 , underlined and bold out, and also contains the double mutation K986P/V987P, which is underlined and italicized.
突变的SARS-CoV-2 Omicron变异株BA.2 Spike蛋白全长胞外结构域I-8，其氨基酸序列如SEQ ID NO:21所示。在序列中，不含信号肽，S1/S2切割位点682RRAR685突变为682GSAS685，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。

The full-length extracellular domain I-8 of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein has an amino acid sequence as shown in SEQ ID NO: 21. In the sequence, there is no signal peptide, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.

突变的SARS-CoV-2 Omicron变异株BA.2 Spike蛋白胞外结构域C端截短片段I-9，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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

The mutated SARS-CoV-2 Omicron variant BA.2 Spike protein extracellular domain C-terminal truncated fragment I-9, its amino acid sequence is shown in SEQ ID NO: 22. In the sequence, 70 amino acid residues are truncated at the C terminus. The original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics. The S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 . It is underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.

突变的SARS-CoV-2 Omicron变异株BA.2 Spike蛋白胞外结构域C端截短片段I-10，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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。
The mutated SARS-CoV-2 Omicron variant BA.2 Spike protein extracellular domain C-terminal truncated fragment I-10, its amino acid sequence is shown in SEQ ID NO: 23. In the sequence, 70 amino acid residues were truncated at the C terminus, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide Italicized, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.
突变的SARS-CoV-2 Omicron变异株BA.2 Spike蛋白胞外结构域C端截短片段I-11，其氨基酸序列如SEQ ID NO:24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682RRAR685突变为682GSAS685，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。
The mutated SARS-CoV-2 Omicron variant BA.2 Spike protein extracellular domain C-terminal truncated fragment I-11, its amino acid sequence is shown in SEQ ID NO: 24. In the sequence, 70 amino acid residues are truncated at the C terminus, without the signal peptide, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains the double mutation K986P/V987P. , underlined and italicized.
SARS-CoV-2 Omicron变异株BA.3Spike蛋白全长胞外结构域(ECD)，其氨基酸序列如SEQ ID NO:25所示，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685用下划线、加粗和斜体标出。

The full-length extracellular domain (ECD) of SARS-CoV-2 Omicron variant BA.3Spike protein, its amino acid sequence is shown in SEQ ID NO:25, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is used Italicized, S1/S2 cleavage site 682 RRAR 685 underlined, bolded and italicized.

突变的SARS-CoV-2 Omicron变异株BA.3Spike蛋白全长胞外结构域I-12，其氨基酸序列如SEQ ID NO:26所示。在序列中，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

The full-length extracellular domain I-12 of the mutant SARS-CoV-2 Omicron variant BA.3 Spike protein, whose amino acid sequence is shown in SEQ ID NO: 26. In the sequence, the original signal peptide: MFVFLVLLPLVSS (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 , which is underlined and bolded, and contains the double mutation K986P/V987P, which is underlined and italicized.

突变的SARS-CoV-2 Omicron变异株BA.3Spike蛋白全长胞外结构域I-13，其氨基酸序列如SEQ ID NO:27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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

The full-length extracellular domain I-13 of the mutated SARS-CoV-2 Omicron variant BA.3Spike protein has an amino acid sequence as shown in SEQ ID NO: 27. In the sequence, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide is marked in italics, and the S1/S2 cleavage site The 682 RRAR 685 mutation is 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.

突变的SARS-CoV-2 Omicron变异株BA.3Spike蛋白全长胞外结构域I-14，其氨基酸序列如SEQ ID NO:28所示。在序列中，不含信号肽，S1/S2切割位点682RRAR685突变为682GSAS685，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。
The full-length extracellular domain I-14 of the mutated SARS-CoV-2 Omicron variant BA.3Spike protein has an amino acid sequence as shown in SEQ ID NO: 28. In the sequence, there is no signal peptide, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
突变的SARS-CoV-2 Omicron变异株BA.3Spike蛋白胞外结构域C端截短片段I-15，其氨基酸序列如SEQ ID NO:29所示。在序列中，C端截短了70个氨基酸残基，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

The mutated SARS-CoV-2 Omicron variant BA.3 Spike protein extracellular domain C-terminal truncated fragment I-15, its amino acid sequence is shown in SEQ ID NO: 29. In the sequence, 70 amino acid residues are truncated at the C terminus. The original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics. The S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 . It is underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.

突变的SARS-CoV-2 Omicron变异株BA.3Spike蛋白胞外结构域C端截短片段I-16，其氨基酸序列如SEQ ID NO:30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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

The mutated SARS-CoV-2 Omicron variant BA.3 Spike protein extracellular domain C-terminal truncated fragment I-16, its amino acid sequence is shown in SEQ ID NO: 30. In the sequence, 70 amino acid residues were truncated at the C terminus, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide Italicized, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.

突变的SARS-CoV-2 Omicron变异株BA.3Spike蛋白胞外结构域C端截短片段I-17，其氨基酸序列如SEQ ID NO:31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682RRAR685突变为682GSAS685，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。

The mutated SARS-CoV-2 Omicron variant BA.3 Spike protein extracellular domain C-terminal truncated fragment I-17, its amino acid sequence is shown in SEQ ID NO: 31. In the sequence, 70 amino acid residues are truncated at the C terminus, without the signal peptide, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains the double mutation K986P/V987P. , underlined and italicized.

突变的SARS-CoV-2 Omicron变异株BA.1Spike蛋白胞外结构域C端截短片段g1，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，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。
The amino acid sequence of the C-terminal truncated fragment g1 of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.1 Spike protein is shown in SEQ ID NO: 62. In the sequence, 70 amino acid residues are truncated at the C-terminus, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO: 2) is marked in italics, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , marked with underline and bold, and contains the double mutation K986P/V987P, marked with underline and italics.
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:44所示，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685用下划线、加粗和斜体标出。

The full-length extracellular domain (ECD) of SARS-CoV-2 Delta variant Spike protein, its amino acid sequence is shown in SEQ ID NO:44, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics Out, S1/S2 cleavage site 682 RRAR 685 is underlined, bolded and italicized.

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:45所示。在序列中，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

The full-length extracellular domain c1 of the mutant SARS-CoV-2 Delta variant Spike protein, whose amino acid sequence is shown in SEQ ID NO: 45. In the sequence, the original signal peptide: MFVFLVLLPLVSS (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 , which is underlined and bolded, and contains the double mutation K986P/V987P, which is underlined and italicized.

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:46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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

The full-length extracellular domain c2 of the mutated SARS-CoV-2 Delta variant Spike protein has an amino acid sequence as shown in SEQ ID NO: 46. In the sequence, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide is marked in italics, and the S1/S2 cleavage site The 682 RRAR 685 mutation is 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.

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:47所示。在序列中，不含信号肽，S1/S2切割位点682RRAR685突变为682GSAS685，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。
The full-length extracellular domain c3 of the mutated SARS-CoV-2 Delta variant Spike protein has an amino acid sequence as shown in SEQ ID NO: 47. In the sequence, there is no signal peptide, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized.
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:48所示。在序列中，C端截短了70个氨基酸残基，原始信号肽： MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。
The mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d1, its amino acid sequence is shown in SEQ ID NO: 48. In the sequence, 70 amino acid residues are truncated at the C terminus, and the original signal peptide is: MFVFLVLLPLVSS (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 , is underlined and bolded, and the double mutation K986P/V987P is included, underlined and Italicized.
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:49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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

The mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d2, its amino acid sequence is shown in SEQ ID NO: 49. In the sequence, 70 amino acid residues were truncated at the C terminus, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide Italicized, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized.

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:50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682RRAR685突变为682GSAS685，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。

The mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d3, its amino acid sequence is shown in SEQ ID NO: 50. In the sequence, 70 amino acid residues are truncated at the C terminus, without the signal peptide, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains the double mutation K986P/V987P. , underlined and italicized.

融合蛋白fusion protein
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、6-9、18-31、44-50、62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。The present invention provides fusion proteins comprising a heterologous scaffold displaying at least one antigenic polypeptide or trimeric protein derived from the coronavirus Spike protein. In some embodiments, the coronavirus antigen used is the extracellular domain of the coronavirus Spike protein containing various stable mutations described above or truncated fragments thereof. In an exemplary embodiment, the Spike protein sequence used includes the sequence shown in any one of SEQ ID NO: 1, 3-4, 6-9, 18-31, 44-50, 62, or is substantially identical thereto. or conservatively modified variants. After the expression vector expressing the fusion protein is transfected into the host cell, since the antigen (such as Spike protein) is connected to the self-assembly protein (such as monomeric ferritin subunit), a nanoparticle vaccine showing the antigen (such as Spike protein) on the surface will be produced. .
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。Any heterologous scaffold can be used to present antigens in the construction of the vaccines of the invention. This includes virus-like particles (VLPs) such as nanoparticles. A variety of nanoparticles can be used to produce the vaccines of the invention. Typically, nanoparticles for use in the present invention need to be formed from multiple replicas of a single subunit. Nanoparticles are typically spherical, and/or have rotational symmetry (eg, having 3-fold and 5-fold axes), such as having an icosahedral structure exemplified herein. Additionally or alternatively, the amino termini of the nanoparticle subunits must be exposed and in close proximity to the 3-fold axis, and the spacing of the three amino termini must closely match the spacing of the carboxyl termini of the trimer-stabilized Spike protein shown.
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)2O3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。 In some embodiments, self-assembled nanoparticles are employed that are about 25 nm or less in diameter (typically assembled from 12, 24, or 60 subunits) and have a 3-fold axis on the particle surface. Such nanoparticles provide suitable particles to produce multivalent vaccines. In some preferred embodiments, coronavirus antigens may be presented on self-assembling nanoparticles, such as self-assembling nanoparticles derived from ferritin (FR) as exemplified herein. Ferritin is a globular protein found in animals, bacteria, and plants whose primary role is to control multinucleation by transporting hydrated iron ions and protons to and from the mineralized core Rate and location of Fe(III) 2 O 3 formation. The globular form of ferritin consists of a monomeric subunit protein (also called a monomeric ferritin subunit), which is a polypeptide with a molecular weight of approximately 17-20 kDa. The sequences of the subunits of these proteins are known in the art. In some embodiments, the nanoparticle vaccines of the invention may use any of these known nanoparticles, as well as conservatively modified variants thereof or that are substantially identical (e.g., at least 90%, 95% or 99% identical) Sequence variants.
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：10所示)，或其保守修饰的变体或与其基本相同的序列。通常，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，含突变的冠状病毒Spike蛋白胞外结构域或其截短片段的C端经接头连接至纳米颗粒亚单位的N端，接头例如为GGGGS或GGGGSGGGGS。In some exemplary embodiments, the fusion protein of the invention comprises a nanoparticle subunit sequence (for example, Helicobacter pylori non-heme monomeric ferritin subunit, the amino acid sequence of which is shown in SEQ ID NO: 10), or its conserved Modified variants or sequences substantially identical thereto. Usually, the C-terminus of the extracellular domain of the coronavirus Spike protein or its truncated fragment containing mutations is fused to the N-terminus of the self-assembled nanoparticle (NP) subunit. In some embodiments, the C-terminus of the extracellular domain of the coronavirus Spike protein containing mutations or a truncated fragment thereof is connected to the N-terminus of the nanoparticle subunit via a linker, such as GGGGS or 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：
The amino acid sequence of Helicobacter pylori non-heme monomer ferritin subunit (Ferritin) is as follows:
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G4S接头或(G4S)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。By fusing subunits of the antigenic polypeptide or multimeric antigenic protein (e.g., trimeric antigen) to subunits of the nanoparticle (e.g., ferritin subunits) and other optional or alternative components described herein, To construct nanoparticles displaying any stable mutant coronavirus Spike protein extracellular domain or truncated fragments thereof described herein. In order to construct the fusion protein of the present invention, one or more linkers can be used to connect and maintain the overall activities of different functional proteins unchanged. Typically, linkers contain short peptide sequences, such as GS-rich peptides. In some embodiments, a linker or linker motif can be any flexible peptide that connects two protein domains or motifs without interfering with their function. For example, the linker employed may be a G4S linker or a ( G4S ) 2 linker as shown herein to connect the spike protein and the nanoparticle scaffold sequence. Recombinant production of fusion proteins of the invention can be based on the protocols described herein and/or other methods that have been described in the art.
示例性的融合蛋白序列如下：Exemplary fusion protein sequences are as follows:
融合蛋白1：将突变的SARS-CoV-2 Omicron变异株BA.5Spike蛋白全长胞外结构域I-1(如SEQ ID NO:3所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白1，其氨基酸序列如SEQ ID NO:12所示。在序列中，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Fusion protein 1: The C-terminus of the full-length extracellular domain I-1 (as shown in SEQ ID NO: 3) of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO: 11) and the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO: 10) to obtain fusion protein 1, the amino acid sequence of which is shown in SEQ ID NO: 12. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains double The mutation K986P/V987P is underlined and italicized, and the linker is italicized and bolded.

融合蛋白2：将突变的SARS-CoV-2 Omicron变异株BA.5Spike蛋白全长胞外结构域I-2(如SEQ ID NO:4所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白2，其氨基酸序列如SEQ ID NO:13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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Fusion protein 2: The C-terminus of the full-length extracellular domain I-2 of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein (as shown in SEQ ID NO: 4) is passed through the linker GGGGS (as shown in SEQ ID NO: 11) and the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO: 10) to obtain fusion protein 2, the amino acid sequence of which is shown in SEQ ID NO: 13. In the sequence, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide is marked in italics, and the S1/S2 cleavage site The mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized. The linker is italicized and bolded.

融合蛋白3：将突变的SARS-CoV-2 Omicron变异株BA.5Spike蛋白胞外结构域C端截短片段I-4(如SEQ ID NO:7所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白3，其氨基酸序列如SEQ ID NO:14所示。在序列中，C端截短了70个氨基酸残基，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Fusion protein 3: The C-terminus of the C-terminal truncated fragment I-4 (as shown in SEQ ID NO: 7) of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 3, whose amino acid sequence is shown in SEQ ID NO:14 . In the sequence, 70 amino acid residues are truncated at the C terminus. The original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics. The S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 . It is underlined and bolded. The double mutation K986P/V987P is also underlined and italicized. The linker is italicized and bolded.

融合蛋白4：将突变的SARS-CoV-2 Omicron变异株BA.5Spike蛋白胞外结构域C端截短片段I-5(如SEQ ID NO:8所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白4，其氨基酸序列如SEQ ID NO:15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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Fusion protein 4: The C-terminus of the C-terminal truncated fragment I-5 (as shown in SEQ ID NO: 8) of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.5Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 4, whose amino acid sequence is shown in SEQ ID NO:15 . In the sequence, 70 amino acid residues were truncated at the C terminus, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide Italicized, the S1/S2 cleavage site 682 RRAR 685 mutation to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized. The linker is italicized and bolded. out.

融合蛋白5：将突变的SARS-CoV-2 Omicron变异株BA.2 Spike蛋白全长胞外结构域I-6-1(如SEQ ID NO:19所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白5，其氨基酸序列如SEQ ID NO:32所示。在序列中，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Fusion protein 5: The C-terminus of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein full-length extracellular domain I-6-1 (as shown in SEQ ID NO:19) is passed through the linker GGGGS (as shown in SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 5, whose amino acid sequence is shown in SEQ ID NO:32 . In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains double The mutation K986P/V987P is underlined and italicized, and the linker is italicized and bolded.

融合蛋白6：将突变的SARS-CoV-2 Omicron变异株BA.2 Spike蛋白全长胞外结构域I-7(如SEQ ID NO:20所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白6，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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Fusion protein 6: The C-terminus of the full-length extracellular domain I-7 of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein (as shown in SEQ ID NO:20) is passed through the linker GGGGS (as shown in SEQ ID NO :11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 6, the amino acid sequence of which is shown in SEQ ID NO:33. In the sequence, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide is marked in italics, and the S1/S2 cleavage site The mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized. The linker is italicized and bolded.

融合蛋白7：将突变的SARS-CoV-2 Omicron变异株BA.2 Spike蛋白胞外结构域C端截短片段I-9(如SEQ ID NO:22所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白7，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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Fusion protein 7: The C-terminus of the C-terminal truncated fragment I-9 (shown in SEQ ID NO: 22) of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein is passed through the linker GGGGS (such as SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 7, whose amino acid sequence is as shown in SEQ ID NO:34 Show. In the sequence, 70 amino acid residues are truncated at the C terminus. The original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics. The S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 . It is underlined and bolded. The double mutation K986P/V987P is also underlined and italicized. The linker is italicized and bolded.

融合蛋白8：将突变的SARS-CoV-2 Omicron变异株BA.2 Spike蛋白胞外结构域C端截短片段I-10(如SEQ ID NO:23所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白8，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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Fusion protein 8: The C-terminus of the C-terminal truncated fragment I-10 (shown in SEQ ID NO: 23) of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.2 Spike protein is passed through the linker GGGGS (such as SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 8, whose amino acid sequence is as shown in SEQ ID NO:35 Show. In the sequence, 70 amino acid residues were truncated at the C terminus, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide Italicized, the S1/S2 cleavage site 682 RRAR 685 mutation to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized. The linker is italicized and bolded. out.

融合蛋白9：将突变的SARS-CoV-2 Omicron变异株BA.3Spike蛋白全长胞外结构域I-12(如SEQ ID NO:26所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白9，其氨基酸序列如SEQ ID NO:36所示。在序列中，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Fusion protein 9: The C-terminus of the full-length extracellular domain I-12 (as shown in SEQ ID NO: 26) of the mutated SARS-CoV-2 Omicron variant BA.3Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO: 11) and the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO: 10) to obtain fusion protein 9, the amino acid sequence of which is shown in SEQ ID NO: 36. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains double The mutation K986P/V987P is underlined and italicized, and the linker is italicized and bolded.

融合蛋白10：将突变的SARS-CoV-2 Omicron变异株BA.3Spike蛋白全长胞外结构域I-13(如SEQ ID NO:27所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白10，其氨基酸序列如SEQ ID NO:37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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Fusion protein 10: The C-terminus of the full-length extracellular domain I-13 (as shown in SEQ ID NO: 27) of the mutated SARS-CoV-2 Omicron variant BA.3Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO: 11) and the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO: 10) to obtain fusion protein 10, the amino acid sequence of which is shown in SEQ ID NO: 37. In the sequence, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide is marked in italics, and the S1/S2 cleavage site The mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized. The linker is italicized and bolded.

融合蛋白11：将突变的SARS-CoV-2 Omicron变异株BA.3Spike蛋白胞外结构域C端截短片段I-15(如SEQ ID NO:29所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白11，其氨基酸序列如SEQ ID NO:38所示。在序列中，C端截短了70个氨基酸残基，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。
Fusion protein 11: The C-terminus of the C-terminal truncated fragment I-15 (as shown in SEQ ID NO: 29) of the extracellular domain of the mutant SARS-CoV-2 Omicron variant BA.3Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 11, whose amino acid sequence is shown in SEQ ID NO:38 . In the sequence, 70 amino acid residues are truncated at the C terminus. The original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is marked in italics. The S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 . It is underlined and bolded. The double mutation K986P/V987P is also underlined and italicized. The linker is italicized and bolded.
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融合蛋白12：将突变的SARS-CoV-2 Omicron变异株BA.3Spike蛋白胞外结构域C端截短片段I-16(如SEQ ID NO:30所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白12，其氨基酸序列如SEQ ID NO:39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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。
Fusion protein 12: The C-terminus of the C-terminal truncated fragment I-16 (as shown in SEQ ID NO: 30) of the extracellular domain of the mutant SARS-CoV-2 Omicron variant BA.3Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO:10) to obtain fusion protein 12, whose amino acid sequence is shown in SEQ ID NO:39 . In the sequence, 70 amino acid residues were truncated at the C terminus, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide Italicized, the S1/S2 cleavage site 682 RRAR 685 mutation to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is included, underlined and italicized. The linker is italicized and bolded. out.
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:45所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白C1，其氨基酸序列如SEQ ID NO:51所示。在序列中，原始信号肽：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。
Fusion protein C1: the full-length extracellular domain c1 of the mutated SARS-CoV-2 Delta variant Spike protein (As shown in SEQ ID NO: 45) The C-terminus of Helicobacter pylori non-heme monomeric ferritin subunit (as shown in SEQ ID NO: 10) is connected through the linker GGGGS (as shown in SEQ ID NO: 11) N-terminal ligation obtains fusion protein C1, whose amino acid sequence is shown in SEQ ID NO: 51. In the sequence, the original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) is marked in italics, and the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded, and contains double The mutation K986P/V987P is underlined and italicized, and the linker is italicized and bolded.
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:46所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋 白C2，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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。
Fusion protein C2: The C-terminus of the full-length extracellular domain c2 of the mutated SARS-CoV-2 Delta variant Spike protein (as shown in SEQ ID NO:46) is passed through the linker GGGGS (as shown in SEQ ID NO:11) The fusion protein is obtained by ligating the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (as shown in SEQ ID NO:10) White C2, its amino acid sequence is shown in SEQ ID NO:52. In the sequence, the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO:2) is replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide is marked in italics, and the S1/S2 cleavage site The mutation of 682 RRAR 685 to 682 GSAS 685 is underlined and bolded, and the double mutation K986P/V987P is included, which is underlined and italicized. The linker is italicized and bolded.
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:48所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白D1，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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。
Fusion protein D1: The C-terminus of the mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d1 (as shown in SEQ ID NO:48) is passed through the linker GGGGS (as shown in SEQ ID NO:11 (shown in SEQ ID NO: 10) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO: 10) to obtain the fusion protein D1, the amino acid sequence of which is shown in SEQ ID NO: 53. In the sequence, 70 amino acids are truncated at the C-terminus Acid residues, original signal peptide: MFVFLVLLPLVSS (as shown in SEQ ID NO:2) are marked in italics, S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , is underlined and bolded, and contains double The mutation K986P/V987P is underlined and italicized, and the linker is italicized and bolded.
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:49所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白D2，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：MFVFLVLLPLVSS(如SEQ ID NO:2所示)，信号肽用斜体标出，S1/S2切割位点682RRAR685突变为682GSAS685，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。
Fusion protein D2: The C-terminus of the mutated SARS-CoV-2 Delta variant Spike protein extracellular domain C-terminal truncated fragment d2 (as shown in SEQ ID NO:49) is passed through the linker GGGGS (as shown in SEQ ID NO:11 (shown in SEQ ID NO: 10) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (shown in SEQ ID NO: 10) to obtain fusion protein D2, the amino acid sequence of which is shown in SEQ ID NO: 54. In the sequence, 70 amino acid residues were truncated at the C terminus, and the original signal peptide: MFVFLVLLPLVSS (shown in SEQ ID NO: 2) was replaced with the signal peptide: MEFGLSLVFLVLILKGVQC (shown in SEQ ID NO:5). The signal peptide Italicized, S1/S2 cleavage site 682 RRAR 685 mutated to 682 GSAS 685 , underlined and bold, including double mutations K986P/V987P, underlined and italicized, connectors italicized and bolded.
成熟的融合蛋白G：将突变的SARS-CoV-2 Omicron变异株BA.1Spike蛋白胞外结构域C端截短片段g1(如SEQ ID NO:62所示)的C端通过接头GGGGS(如SEQ ID NO:11所示)与幽门螺旋杆菌非血红素单体铁蛋白亚基(如SEQ ID NO:10所示)的N端连接获得融合蛋白G1，在融合蛋白G1的序列中，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)。融合蛋白G的氨基酸序列如SEQ ID NO:61所示。与融合蛋白G1相比，去除了N端信号肽。S1/S2切割位点682RRAR685突变为682GSAS685，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。

Mature fusion protein G: The C-terminus of the C-terminal truncated fragment g1 (as shown in SEQ ID NO: 62) of the extracellular domain of the mutated SARS-CoV-2 Omicron variant BA.1 Spike protein is passed through the linker GGGGS (as shown in SEQ ID NO:11) is connected to the N-terminus of the non-heme monomeric ferritin subunit of Helicobacter pylori (as shown in SEQ ID NO:10) to obtain fusion protein G1. In the sequence of fusion protein G1, the C-terminus is truncated. 70 amino acid residues are shortened, and the original signal peptide is: MFVFLVLLPLVSSQ (shown in SEQ ID NO: 2). The amino acid sequence of fusion protein G is shown in SEQ ID NO: 61. Compared with fusion protein G1, the N-terminal signal peptide is removed. The S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded.

成熟的融合蛋白1-1：与融合蛋白1和2相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:16所示。在序列中，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Mature fusion protein 1-1: Compared with fusion proteins 1 and 2, the N-terminal signal peptide is removed, and its amino acid sequence is shown in SEQ ID NO: 16. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .

成熟的融合蛋白2-1：与融合蛋白3和4相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:17所示。在序列中，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Mature fusion protein 2-1: Compared with fusion proteins 3 and 4, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 17. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .

成熟的融合蛋白3-1：与融合蛋白5和6相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:40所示。在序列中，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Mature fusion protein 3-1: Compared with fusion proteins 5 and 6, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 40. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .

成熟的融合蛋白4-1：与融合蛋白7和8相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:41所示。在序列中，S1/S2切割位点682RRAR685突变为682GSAS685，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。
Mature fusion protein 4-1: Compared with fusion proteins 7 and 8, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 41. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .
成熟的融合蛋白5-1：与融合蛋白9和10相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:42所示。在序列中，S1/S2切割位点682RRAR685突变为682GSAS685， 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。
Mature fusion protein 5-1: Compared with fusion proteins 9 and 10, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 42. In the sequence, the S1/S2 cleavage site 682 RRAR 685 was mutated to 682 GSAS 685 , It is underlined and bolded. The double mutation K986P/V987P is also underlined and italicized. The linker is italicized and bolded.
成熟的融合蛋白6-1：与融合蛋白11和12相比，去除了N端信号肽，其氨基酸序列如SEQ ID NO:43所示。在序列中，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Mature fusion protein 6-1: Compared with fusion proteins 11 and 12, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 43. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .

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:55所示。在序列中，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Mature fusion protein C: Compared with fusion proteins C1 and C2, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 55. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .

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:56所示。在序列中，S1/S2切割位点682RRAR685突变为682GSAS685，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。

Mature fusion protein D: Compared with fusion proteins D1 and D2, the N-terminal signal peptide has been removed, and its amino acid sequence is shown in SEQ ID NO: 56. In the sequence, the S1/S2 cleavage site 682 RRAR 685 is mutated to 682 GSAS 685 , which is underlined and bolded. It also contains the double mutation K986P/V987P, which is underlined and italicized. The linker is italicized and bolded. .

SEQ ID NO:16、17、40-43、55、56、61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。SEQ ID NO:16, 17, 40-43, 55, 56, and 61 are mature fusion protein sequences with the N-terminal signal peptide (SEQ ID NO: 2 or 5) removed. In addition to these specifically exemplified fusion proteins, the invention also encompasses nanoparticle vaccines containing subunits that are substantially identical to any of these exemplified nanoparticle vaccine sequences, or conservatively modified variants thereof sequence.
多核苷酸和表达载体Polynucleotides and expression vectors
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、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、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、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:7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:12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:16或17)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。The mutation-containing coronavirus Spike protein extracellular domain or its truncated fragment, fusion protein or Spike protein nanoparticle of the present invention is usually produced by an expression vector, the expression vector contains the mutation-containing coronavirus Spike protein described herein The coding sequence of the extracellular domain or its truncated fragment, fusion protein or Spike protein nanoparticle. Therefore, in some related aspects, the present invention provides polynucleotides (DNA or RNA) encoding the mutated coronavirus Spike protein extracellular domain or truncated fragments thereof, fusion proteins or Spike protein nanoparticles described herein. Some polynucleotides of the present invention encode one of the mutated coronavirus Spike protein extracellular domains described herein or truncated fragments thereof, for example, the SARS-CoV-2 Spike protein shown in SEQ ID NO:7 Truncated fragments of the extracellular domain. Some polynucleotides of the present invention encode the subunit sequence of one of the nanoparticle vaccines described herein, such as the fusion protein sequence shown in SEQ ID NO: 12. The fusion protein expressed in the present invention may not contain an N-terminal signal peptide, or some polynucleotide sequences may additionally encode an N-terminal signal peptide. For example, a polynucleotide encoding a fusion protein (e.g., SEQ ID NO: 16 or 17) may also include an N-terminal signal peptide encoding SEQ ID NO: 2 or 5, or a sequence substantially identical or conservatively modified thereto. Variant sequence.
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。The invention also provides expression vectors having such polynucleotides and host cells (e.g., prokaryotic or eukaryotic cells, such as HEK293, CHO, ExpiCHO and CHO-S cell lines). Fusion proteins encoded by polynucleotides or expressed from vectors are also included in the present invention. As described herein, nanoparticle subunit fused extracellular domains of Spike protein or truncated fragments thereof will self-assemble into nanoparticle vaccines displaying Spike protein or truncated fragments thereof on their surface.
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。也可以通过已知方法PCR将突变引入多核苷酸序列中。Polynucleotides and related vectors can be produced by standard molecular biology techniques or the protocols exemplified herein. For example, general protocols for cloning, transfection, transient gene expression, and obtaining stable transfected cell lines are well known in the art. has been described in . Mutations can also be introduced into the polynucleotide sequence by PCR, a known method.
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)。The choice of specific carrier depends on the intended use of the protein. For example, regardless of whether the cell type is prokaryotic or eukaryotic, the vector chosen must be able to drive protein expression in the desired cell type. Many vectors contain sequences that permit replication of the prokaryotic vector and eukaryotic expression of the operably linked gene sequence. Vectors useful in the present invention can replicate autonomously, that is, the vector exists extrachromosomally, and its replication need not be directly linked to replication of the host cell genome. Alternatively, replication of the vector can be linked to replication of the host chromosomal DNA, for example, the vector can be integrated into the chromosome of the host cell, via a retroviral vector and in a stably transfected cell line. Both viral-based and non-viral-based expression vectors can be used to produce antigens in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (usually with expression cassettes for expressing proteins or RNA) and human artificial chromosomes. Alternative viral vectors include lentiviral or other retrovirus-based vectors, adenovirus, adeno-associated virus, cytomegalovirus, herpesvirus, SV40-based vectors, papillomavirus, HBP, Epstein Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV).
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。Depending on the particular vector used to express the protein, a variety of known cells or cell lines may be used in the practice of the invention. A host cell can be any cell carrying a recombinant vector for a protein of the invention, allowing the vector to drive expression of the protein for the invention. It may be prokaryotic, such as any of many bacterial strains, or eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells, including, for example, rodent, simian, or human cells . Cells expressing proteins of the invention may be primary cultured cells or may be established cell lines. Accordingly, in addition to the cell lines exemplified herein (eg, HEK293 cells), many other host cell lines well known in the art may be used in the practice of the present invention. These include, for example, various Cos cell lines, CHO cells, HeLa cells, Sf9 cells, AtT20, BV2 and N18 cells, myeloma cell lines, transformed B cells and hybridomas.
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。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。Vectors expressing the protein can be introduced into the host cell of choice by any of a number of suitable methods known to those skilled in the art. To introduce a protein-encoding vector into a mammalian cell, the method used will depend on the form of the vector. For plasmid vectors, the DNA encoding the protein sequence can be introduced by any of a number of transfection methods, including, for example, liposome-mediated transfection ("lipofectamine"), DEAE-dextran-mediated guided transfection, electroporation or calcium phosphate precipitation. Among them, lipofectamine transfection is widely accepted because it is simple to operate and does not require special equipment. For example, transfection can be performed using Lipofectamine (Life Technologies) or LipoTAXI (Stratagene) kits.
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)。For long-term, high-yield production of recombinant proteins, stable expression is preferred. Instead of using expression vectors containing viral origins of replication, protein-coding sequences controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers 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 chromosomes. Commonly used selectable markers include neomycin (neo), which is resistant to the aminoglycoside G-418, and hygromycin, which is resistant to hygromycin. (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。In some embodiments, a recombinant expression vector includes at least one promoter element, a protein coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the inserted sequence. Efficient transcription can be obtained through the early and late promoters of SV40, the long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus. Promoters from other cells such as muscle can also be used. Kinesin promoter. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, pLXSN, pLNCX, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), pSVL , pMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc. Commonly used mammalian cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells.
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。In some embodiments, the inserted gene fragment needs to contain a selection marker. Common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes to facilitate transfection. Screening isolation of successful cells. The constructed plasmid is transfected into host cells without the above genes, and then cultured in a selective medium. The successfully transfected cells grow in large quantities and produce the desired target protein.
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个氨基酸的取代、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。Furthermore, mutations can be introduced into the nucleotide sequences encoding the invention using standard techniques known to those skilled in the art, including, but not limited to, site-directed mutagenesis resulting in amino acid substitutions and PCR-mediated mutagenesis. Variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions relative to the original protein Substitution of amino acids, substitution of less than 15 amino acids, substitution of less than 10 amino acids, substitution of less than 5 amino acids, substitution of less than 4 amino acids, substitution of less than 3 amino acids or substitution of less than 2 amino acids replace. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.
在一些实施方案中，本文所述取代为保守氨基酸取代。In some embodiments, the substitutions described herein are conservative amino acid substitutions.
药物组合物和治疗方法Pharmaceutical compositions and methods of treatment
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。The present invention also provides a pharmaceutical composition and a related treatment method. The pharmaceutical composition comprises an effective dose of fusion protein or Spike protein nanoparticles and a pharmaceutically acceptable carrier.
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。The term "pharmaceutically acceptable" refers to substances approved by a governmental regulatory agency or listed in other recognized pharmacopoeias for use in animals, particularly in humans. In addition, "pharmaceutically acceptable carrier" generally refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary, etc.
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。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。The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the active ingredient can be administered to a patient. Such carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions Liquid and glucose aqueous solution and glycerol solution can also be used as liquid carrier, particularly for injection solution. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the pharmaceutical composition can also contain a small amount of wetting agent, emulsifier, or pH buffer such as acetate, citrate or phosphate. Antibacterial agents such as benzyl alcohol or methyl parahydroxybenzoate, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tension regulating agents such as sodium chloride or dextrose are also foreseeable. These pharmaceutical compositions can take the form of solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release preparation, etc. The pharmaceutical composition can be formulated into suppositories with traditional adhesives and carriers such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions will contain a clinically effective dose of 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 preparation should be suitable for the mode of administration. The preparation can be packaged in an ampoule, a disposable syringe, or a multi-dose vial made of glass or plastic.
需要说明的是，本发明中，涉及到制剂组分的“％”指重量体积(w/v)百分比，比如制剂中含1％稳定剂表示100mL制剂中含有1g稳定剂，或稳定剂含量为0.01g/mL。It should be noted that in the present invention, the "%" related to the preparation components refers to the weight volume (w/v) percentage. For example, 1% stabilizer in the preparation means that 100 mL of preparation contains 1 g of stabilizer, or the stabilizer content is 0.01g/mL.
“约”或“大约”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”或“大约”指所描述的数值以及其±10％、±5％或±1％的范围。"About" or "approximately" refers to the conventional error range of the corresponding numerical value that is easily known to those skilled in the relevant technical fields. In some embodiments, references herein to "about" or "approximately" refer to the recited value and its range of ±10%, ±5%, or ±1%.
“包括”或“包含”指组合物和方法等等包括所列举的元素(如组合物中的组分，方法中的步骤等)，但不排除其它元素。当“基本上由……组成”用于定义组合物和方法时，指排除对用于预期用途的组合有根本影响的其它元素，但不排除不会本质上影响组合物或方法的特征的元素。“由……组成”指排除未特别列举的元素。由这些过渡术语中的每一者定义的实施方案均在本发明的范围内。举例来说，当组合物被描述为包括成分A、B以及C时，基本上由A、B以及C组成的组合物和由A、B以及C组成的组合物独立地在本发明的范围内。"Including" or "comprising" means that compositions, methods, etc. include the listed elements (such as components in the composition, steps in the method, etc.), but do not exclude other elements. When “consisting essentially of” is used to define compositions and methods, it is meant to exclude other elements that materially affect the composition for its intended use, but does not exclude elements that do not materially affect the characteristics of the composition or method. . “Consisting of” means excluding elements not specifically enumerated. Embodiments defined by each of these transitional terms are within the scope of the invention. For example, when a composition is described as including ingredients A, B, and C, compositions consisting essentially of A, B, and C and compositions consisting of A, B, and C are independently within the scope of the invention .
术语“缓冲剂”也被称为缓冲体系，其包括但不限于有机酸和其盐，如琥珀酸、醋酸、柠檬酸、抗坏血酸、葡糖酸、碳酸、酒石酸或苯二甲酸及其盐；Tris，或无机酸和其盐，如磷酸盐缓冲剂。此外，氨基酸也可以用作缓冲剂。这样的氨基酸包括但不限于甘氨酸、组氨酸、精氨酸、赖氨酸、鸟氨酸、异亮氨酸、亮氨酸、丙氨酸、谷氨酸或天冬氨酸和其盐。The term "buffer" is also known as a buffer system, which includes but is not limited to organic acids and their salts, such as succinic acid, acetic acid, citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid or phthalic acid and their salts; Tris , or inorganic acids and their salts, such as phosphate buffers. In addition, amino acids can also be used as buffering agents. Such amino acids include, but are not limited to, glycine, histidine, arginine, lysine, ornithine, isoleucine, leucine, alanine, glutamic acid or aspartic acid and salts thereof.
本发明中缓冲剂的量，是指组成缓冲剂的缓冲体系中缓冲对的总量。在一些实施方式中，采用摩尔浓度作为缓冲剂的量的单位，其数值指缓冲剂的缓冲体系中缓冲对的摩尔浓度。如，由组氨酸和盐酸组氨酸组成的组氨酸缓冲剂时，给定浓度的组氨酸 缓冲剂(如20mM)是组氨酸和盐酸组氨酸的组合浓度。The amount of buffering agent in the present invention refers to the total amount of buffering pairs in the buffering system that constitutes the buffering agent. In some embodiments, molar concentration is used as the unit for the amount of buffer, and its numerical value refers to the molar concentration of the buffer pair in the buffer system of the buffer. For example, when a histidine buffer consists of histidine and histidine hydrochloride, a given concentration of histidine The buffer (eg 20mM) is a combined concentration of histidine and histidine hydrochloride.
本发明所述的制剂可以用所述辅料或其水合物配制。比如组氨酸盐酸盐，又称盐酸组氨酸，可以是无水组氨酸盐酸盐，也可以是组氨酸盐酸盐水合物，如组氨酸盐酸盐一水合物。The preparations of the present invention can be formulated with the excipients or hydrates thereof. For example, histidine hydrochloride, also known as histidine hydrochloride, can be anhydrous histidine hydrochloride or histidine hydrochloride hydrate, such as histidine hydrochloride monohydrate.
术语“表面活性剂”包括但不限于聚山梨酯(Tween，如聚山梨酯20和聚山梨酯80)；泊洛沙姆(例如泊洛沙姆188)；Triton；十二烷硫酸钠(SDS)；月桂硫酸钠；辛基配糖物钠盐；月桂基磺基甜菜碱、肉豆蔻基磺基甜菜碱、亚油烯基磺基甜菜碱或硬脂基磺基甜菜碱；月桂基肌氨酸、肉豆蔻基肌氨酸、亚油烯基肌氨酸或硬脂基肌氨酸；亚油烯基甜菜碱、肉豆蔻基甜菜碱或鲸蜡基甜菜碱；月桂酰胺基丙基甜菜碱、椰油酰胺基丙基甜菜碱、亚油酰胺基丙基甜菜碱、肉豆蔻酰胺基丙基甜菜碱、棕榈酰胺基丙基甜菜碱或异硬脂酰胺基丙基甜菜碱(例如月桂酰胺基丙基)；肉豆蔻酰胺基丙基二甲胺、棕榈酰胺基丙基二甲胺或者异硬脂酰胺基丙基二甲胺；甲基椰油酰基牛磺酸钠或甲基油基牛磺酸二钠；聚乙二醇，聚丙二醇，和乙烯与丙烯二醇的共聚物(例如Pluronics，PF68等)；等等。在一些实施方式中，表面活性剂为聚山梨酯80，又名PS80，吐温80或Tween 80。The term "surfactant" includes, but is not limited to, polysorbate (Tween, such as polysorbate 20 and polysorbate 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS) ); sodium lauryl sulfate; octyl glycoside sodium salt; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine or stearyl sulfobetaine; lauryl sulfobetaine Acid, myristyl sarcosine, linoleyl sarcosine or stearyl sarcosine; linoleyl betaine, myristyl betaine or cetyl betaine; laurolamidopropyl betaine , cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitoamidopropyl betaine or isostearamidopropyl betaine (e.g. lauramidopropyl betaine propyl); myristamidopropyldimethylamine, palmitamidopropyldimethylamine or isostearamidopropyldimethylamine; sodium methylcocoyltaurate or methyloleyltaurate Disodium acid acid; polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (such as Pluronics, PF68, etc.); etc. In some embodiments, the surfactant is polysorbate 80, also known as PS80, Tween 80 or Tween 80.
如在本文中所使用的，“盐”指与无机和/或有机酸形成的酸性盐，以及与无机和/或有机碱形成的碱性盐。尽管也可以使用其他盐，但是药学上可接受的(即，无毒性、生理上可接受的)盐是优选的。示例性碱性盐包括铵盐，碱金属盐(如钠、锂和钾盐)，碱土金属盐(如钙和镁盐)，锌盐，与有机碱(例如，有机胺)(如N-Me-D-葡糖胺、胆碱、三甲胺、二环己胺、叔丁胺)形成的盐以及与氨基酸(如精氨酸、赖氨酸)形成的盐等。As used herein, "salts" refer to acidic salts formed with inorganic and/or organic acids, and basic salts formed with inorganic and/or organic bases. Although other salts may be used, pharmaceutically acceptable (ie, nontoxic, physiologically acceptable) salts are preferred. Exemplary basic salts include ammonium salts, alkali metal salts (e.g., sodium, lithium, and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), zinc salts, and organic bases (e.g., organic amines) (e.g., N-Me -Salts formed by -D-glucosamine, choline, trimethylamine, dicyclohexylamine, tert-butylamine) and salts formed with amino acids (such as arginine, lysine), etc.
本文的“稳定性”、“稳定”，是指包含抗体的制剂中，抗体(包括其抗体片段)在给定的生产、制备、运输和/或贮存条件下不发生、或仅极少地发生聚集、降解或片段化。“稳定”制剂在给定的生产、制备、运输和/或贮存条件下保持生物学活性。可通过例如SEC-HPLC、IEC-HPLC、CE-SDS(NR)、灯检及浑浊度、不溶性颗粒、DLS检测粒子粒径等技术测量的所述制剂的聚集、降解或片段化程度等，从而评估所述抗体的稳定性。"Stability" and "stable" herein refer to the fact that in preparations containing antibodies, the antibodies (including antibody fragments thereof) do not, or only rarely, develop under given production, preparation, transportation and/or storage conditions. Aggregation, degradation or fragmentation. A "stable" formulation retains biological activity under given conditions of production, preparation, transportation and/or storage. The degree of aggregation, degradation or fragmentation of the preparation can be measured by techniques such as SEC-HPLC, IEC-HPLC, CE-SDS (NR), light inspection and turbidity, insoluble particles, DLS detection of particle size, etc., thereby The antibodies were assessed for stability.
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。 In some embodiments, a pharmaceutical composition can comprise a fusion protein or Spike protein nanoparticle, and a polynucleotide or vector encoding a fusion protein described herein. In some embodiments, viral (eg, SARS-CoV-2) Spike protein extracellular domains or trimers of truncated fragments thereof can be used to prevent and treat corresponding viral infections. In some embodiments, vaccines containing 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 SARS-CoV-2 antigens or vaccines to prevent or treat SARS-CoV-2 infection in human subjects. Some embodiments of the invention relate to the use of SARS-CoV antigens or vaccines to prevent or treat SARS-CoV infection.
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。In the practice of some therapeutic methods of the present invention, the corresponding Spike protein nanoparticles or fusion proteins, or the fusion protein encoding fusion proteins described herein, are administered to a subject in need of preventing or treating a disease (e.g., SARS-CoV-2 infection). Polynucleotides. Typically, the Spike protein nanoparticles, fusion proteins, or polynucleotides encoding fusion proteins disclosed herein are included in pharmaceutical compositions. Pharmaceutical compositions may be therapeutic or prophylactic formulations. Typically, the pharmaceutical composition may additionally comprise one or more pharmaceutically acceptable carriers, and optionally other therapeutic ingredients (eg, antiviral agents). Various pharmaceutically acceptable additives may also be used in the pharmaceutical compositions.
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铝佐剂如氢氧化铝、卵磷脂、弗氏佐剂、MF59、SEPIVAC SWETM、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。在一些实施方案中，本发明的疫苗包含合适的佐剂为SEPIVAC SWETM，为一种基于角鲨烯的水包油乳液。Some pharmaceutical compositions of the invention are vaccine compositions. For vaccine compositions, suitable adjuvants may additionally be included. Suitable adjuvants include, for example, aluminum adjuvants such as aluminum hydroxide, lecithin, Freund's adjuvant, MF59, SEPIVAC SWE ™ , MPL and IL-12. In some embodiments, the vaccine compositions described herein (eg, SARS-CoV-2 vaccines) can be formulated as a controlled release or timed release formulation. This can be achieved in compositions containing slow-release polymers or via microencapsulated delivery systems or bioadhesive gels. Various pharmaceutical compositions can be prepared according to standard procedures well known in the art. See, for example, US Patents 4,652,441 and 4,917,893; US Patents 4,677,191 and 4,728,721; and US Patent 4,675,189. In some embodiments, a suitable adjuvant for the vaccine of the invention is SEPIVAC SWE ™ , a squalene-based oil-in-water emulsion.
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。The pharmaceutical compositions of the present invention can be used in a variety of therapeutic or preventive applications, such as for treating SARS-CoV-2 infection in a subject or for causing an immune response to SARS-CoV-2 in a subject. As an example, a nanoparticle vaccine can be administered to a subject to induce an immune response to SARS-CoV-2, for example, to induce the production of broad-spectrum neutralizing antibodies against the virus. For subjects at risk of infection with SARS-CoV-2, the vaccine composition of the present invention can be administered to provide preventive protection against viral infection. The therapeutic and preventive applications of vaccines derived from other antigens described herein can be similarly performed. Depending on the specific subject and disease, the pharmaceutical composition of the present invention can be administered to a subject by a variety of administration methods known to those of ordinary skill in the art, for example, by parenteral routes such as intramuscular routes, subcutaneous routes, intravenous routes, intra-arterial routes, joint routes, and intraperitoneal routes. In some embodiments, the treatment methods of the present invention relate to methods for blocking coronaviruses (such as SARS-CoV or SARS-CoV-2) from entering host cells (such as human host cells), methods for preventing coronavirus Spike proteins from binding to host receptors, and methods for treating acute respiratory diseases associated with coronavirus infection. In some embodiments, the methods of treatment and pharmaceutical compositions described herein can be used in combination with other known therapeutic agents and/or methods for treating or preventing coronavirus infection. Known therapeutic agents and/or methods include, for example, nuclease analogs or protease inhibitors (e.g., remdesivir), monoclonal antibodies against one or more coronaviruses, immunosuppressants or anti-inflammatory drugs (e.g., sarilumab or tocilizumab), ACE inhibitors, vasodilators, or any combination thereof.
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。For therapeutic applications, the pharmaceutical composition should contain a therapeutically effective amount of the fusion protein, Spike protein nanoparticles described herein. For prophylactic applications, the pharmaceutical composition should contain a prophylactically effective amount of the fusion protein and Spike protein nanoparticles described herein. Appropriate antibiotics may be determined based on the specific disease or condition to be treated or prevented, the subject's severity, age, and other personal attributes of the particular subject (e.g., the overall state of the subject's health). original amount. Determination of effective doses is also guided by studies in animal models and subsequently by clinical trials in humans, and by dosing regimens that significantly reduce the occurrence or severity of the target disease condition or symptoms in subjects.
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。For preventive applications, before any symptoms, such as before infection, a pharmaceutical composition is provided. The preventive administration of the pharmaceutical composition is used to prevent or improve any subsequent infection. Therefore, in some embodiments, the subject to be treated is, for example, a subject who has been infected (e.g., SARS-CoV-2 infection) or a subject at risk of infection (e.g., SARS-CoV-2 infection) due to exposure or possible exposure to a virus (e.g., SARS-CoV-2). After administering a therapeutically effective amount of the disclosed pharmaceutical composition, the subject's infection (e.g., SARS-CoV-2 infection) and symptoms associated with infection (e.g., SARS-CoV-2 infection) can be monitored.
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。For therapeutic use, the pharmaceutical composition is provided at or after the onset of symptoms of a disease or infection, such as after the onset of symptoms of an infection (eg, SARS-CoV-2 infection) or after the infection is diagnosed. Accordingly, pharmaceutical compositions may be provided prior to anticipated exposure to the virus in order to attenuate the expected severity, duration or extent of infection and/or associated disease conditions following exposure or suspected exposure to the virus or after the initial onset of actual infection. The pharmaceutical compositions of the present invention may be combined with other agents known in the art for the treatment or prevention of infection by relevant pathogens, such as SARS-CoV-2 infection.
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。Vaccine compositions (such as SARS-CoV-2 vaccines) or pharmaceutical compositions containing the fusion proteins, Spike protein nanoparticles of the present invention can be provided as components of the kit. Optionally, such kits include additional components including packaging, instructions for use, and various other reagents, such as buffers, substrates, antibodies or ligands (e.g., control antibodies or ligands), and detection Reagents.
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、作为逆转录病毒或其它载体的一部分的核酸的构建等。Various known delivery systems can be used to administer the 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 recombinant cells of Spike protein nanoparticles, receptor-mediated endocytosis, construction of nucleic acids as part of retroviruses or other vectors, etc.
具体实施方式Detailed ways
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。The technical solutions of the present invention will be further described below through specific examples, which do not limit the scope of protection of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still belong to the protection scope of the present invention.
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。Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.
实施例1：融合蛋白的制备Example 1: Preparation of fusion protein
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:12-17、32-43、51-56、61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。 According to the sequence of the fusion protein described herein, it can be prepared by the following method or other known methods. The DNA sequence encoding the fusion protein (as shown in SEQ ID NO: 12-17, 32-43, 51-56, and 61) is cloned into an expression vector, then electroporated into CHO-K1 cells, cultured and purified to obtain the fusion protein.
使用冷冻电镜(Cryo-EM)对融合蛋白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。The three-dimensional structure of the fusion protein was analyzed using cryo-electron microscopy (Cryo-EM). The extracellular domain of the SARS-CoV-2 Spike protein or its truncated fragment was connected to the N-terminus of the monomeric ferritin subunit without interfering with the self-regulation of ferritin. Assembled, the nanoparticles formed well and showed spikes on the surface.
实施例2：融合蛋白与hACE2蛋白结合能力试验Example 2: Test of the binding ability of fusion protein to hACE2 protein
1.1融合蛋白与hACE2蛋白结合能力试验(ELISA)1.1 Fusion protein and hACE2 protein binding ability test (ELISA)
本试验通过ELISA检测融合蛋白与人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蛋白(如SEQ ID NO:57所示)、Delta-Spike蛋白(如SEQ ID NO:58所示)、BA.5-Spike蛋白(如SEQ ID NO:59所示)、融合蛋白D、融合蛋白2-1)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human ACE2-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稀释)，于37℃温育1h；用PBST洗涤8次；以100μL/孔向反应孔中加入TMB溶液(湖州英创生物科技有限公司，TMB-S-001)，37℃避光孵育5～15min；向每个反应孔中加终止液(0.1M H2SO4)50μL终止酶促显色反应；用酶标仪自带分析软件SoftMax pro 7.02软件，设定检测波长为450nm进行读数，对所得读数以四参数方程曲线模型进行拟合，方程为其中A为吸光度下限，B代表曲线斜率，C为最大反应值一半对应的抗体浓度(EC50)，D为吸光度上限。This test detects the binding ability of the fusion protein to human ACE2 protein (hACE2) through ELISA, thereby evaluating whether the Spike protein-ferritin fusion protein of the present invention can well display the key antigenic epitopes of the Spike protein. The method is briefly described as follows: Add 100 μL of 2 μg/mL antigen (WT-Spike protein (shown in SEQ ID NO: 57), Delta-Spike protein ( As shown in SEQ ID NO:58), BA.5-Spike protein (as shown in SEQ ID NO:59), fusion protein D, fusion protein 2-1) solution, coated at 4°C overnight; use PBST (containing 0.05 % Tween-20 PBS buffer); add blocking solution (PBST containing 3% BSA) to each reaction well and incubate in a 37°C incubator for 2 hours; wash 3 times with PBST after blocking; add gradient dilutions of human ACE2-his-biotin (Yiqiao Shenzhou, product number: 10108-H27B-B), starting concentration is 2.5 μg/mL, 3-fold gradient dilution, a total of 10 serial dilution concentrations, 100 μL per well, cultured at 37°C Incubate for 1.5h in a box; wash 5 times with PBST; add streptavidin-labeled catalase (Jackson Immuno Research, Cat. No.: 016-030-084; 1:10000 dilution) to the reaction well at 100 μL/well. , incubate at 37°C for 1 hour; wash 8 times with PBST; add TMB solution (Huzhou Yingchuang Biotechnology Co., Ltd., TMB-S-001) to the reaction well at 100 μL/well, and incubate at 37°C in the dark for 5 to 15 minutes; Add 50 μL of stop solution (0.1M H 2 SO 4 ) to each reaction well to stop the enzymatic color reaction; use the analysis software SoftMax pro 7.02 software that comes with the microplate reader, set the detection wavelength to 450 nm for reading, and calculate the reading as The four-parameter equation curve model is fitted, and the equation is Among them, A is the lower limit of absorbance, B represents the slope of the curve, C is the antibody concentration (EC 50 ) corresponding to half of the maximum response value, and D is the upper limit of absorbance.
其中，Spike蛋白(例如WT-Spike蛋白、Delta-Spike蛋白或BA.5-Spike蛋白)的构建方法：将编码目的蛋白的DNA序列克隆至表达载体，然后电转CHO-K1细胞，经MSX加压筛选后，挑选表达量高的母克隆进行培养并纯化，获得目的蛋白。Among them, the construction method of Spike protein (such as WT-Spike protein, Delta-Spike protein or BA.5-Spike protein): clone the DNA sequence encoding the target protein into an expression vector, then electroporate CHO-K1 cells, and pressurize with MSX After screening, mother clones with high expression levels are selected for culture and purification to obtain the target protein.
WT-Spike蛋白氨基酸序列：

WT-Spike protein amino acid sequence:

Delta-Spike蛋白氨基酸序列：
Delta-Spike protein amino acid sequence:
BA.5-Spike蛋白氨基酸序列：

BA.5-Spike protein amino acid sequence:

结果如图1和图2所示，hACE2与融合蛋白D、WT-Spike蛋白及Delta-Spike蛋白结合具有类似亲和力，EC50值分别为9.2、5.8、8.1ng/mL(图1)；hACE2与融合蛋白2-1及BA.5-Spike蛋白结合也具有类似的亲和力，EC50值分别为24.0、81.5ng/mL(图2)。融合蛋白2-1和融合蛋白D均可以与人ACE2很好的结合，说明融合蛋白2-1和融合蛋白D呈现的纳米颗粒结构均能良好的展示抗原表位。The results are shown in Figures 1 and 2. hACE2 binds to fusion protein D, WT-Spike protein and Delta-Spike protein with similar affinities, and the EC 50 values are 9.2, 5.8 and 8.1ng/mL respectively (Figure 1); hACE2 and Fusion protein 2-1 and BA.5-Spike protein binding also have similar affinities, with EC 50 values of 24.0 and 81.5ng/mL respectively (Figure 2). Both fusion protein 2-1 and fusion protein D can bind well to human ACE2, indicating that the nanoparticle structures presented by fusion protein 2-1 and fusion protein D can well display antigen epitopes.
1.2融合蛋白与hACE2蛋白结合能力试验(BLI)1.2 Binding ability test of fusion protein and hACE2 protein (BLI)
采用生物膜干涉技术(BLI)融合蛋白与hACE2的结合能力，仪器为PALL公司的生物分子相互作用分析仪(Fortebio Octet QKe)，从而评估融合蛋白能否良好的展示抗原表位。多通道平行定量分析Spike蛋白(WT-Spike蛋白(见实施例2步骤1.1)、Delta-Spike蛋白(见实施例2步骤1.1)、BA.5-Spike蛋白(见实施例2步骤1.1)和融合蛋白(融合蛋白D、融合蛋白2-1)，浓度梯度设定为：0、50、100、200和400nM，hACE2-Biotin(Acro biosystems，货号AC2-H5257)固定在SA Biosensors(Octet，货号2107002811)。结果见表1。The biofilm interference technology (BLI) was used to bind the fusion protein to hACE2, and the instrument was PALL's Biomolecular Interaction Analyzer (Fortebio Octet QKe) to evaluate whether the fusion protein could display the antigenic epitope well. Multi-channel parallel quantitative analysis of Spike protein (WT-Spike protein (see step 1.1 of Example 2), Delta-Spike protein (see step 1.1 of Example 2), BA.5-Spike protein (see step 1.1 of Example 2) and fusion Protein (Fusion Protein D, Fusion Protein 2-1), the concentration gradient was set as: 0, 50, 100, 200 and 400nM, hACE2-Biotin (Acro biosystems, Cat. No. AC2-H5257) was fixed on SA Biosensors (Octet, Cat. No. 2107002811 ). The results are shown in Table 1.
表1生物层干涉法测定SARS-CoV-2 Spike蛋白与hACE2受体结合动力学参数
Table 1 Determination of binding kinetic parameters between SARS-CoV-2 Spike protein and hACE2 receptor by biolayer interference method
从表1可以看出，融合蛋白D与hACE2结合的亲和力要显著强于WT-Spike蛋白、Delta-Spike蛋白与hACE2的亲和力。融合蛋白2-1与hACE2及BA.5-Spike蛋白与hACE2均显示出极高的亲和力，且超出仪器的检测范围(KD<1.0E-12)，同样的融合蛋白2-1与hACE2的亲和力也显著强于WT-Spike蛋白与hACE2。融合蛋白D和融合蛋白2-1均与hACE2具有极强的亲和力，表明了融合蛋白2-1和融合蛋白D呈现的纳米颗粒结构可以良好的展示抗原表位。As can be seen from Table 1, the binding affinity of fusion protein D to hACE2 is significantly stronger than the affinity of WT-Spike protein and Delta-Spike protein to hACE2. Fusion protein 2-1 and hACE2 and BA.5-Spike protein and hACE2 both show extremely high affinity, which is beyond the detection range of the instrument (KD<1.0E-12). The same affinity of fusion protein 2-1 and hACE2 It is also significantly stronger than WT-Spike protein and hACE2. Both fusion protein D and fusion protein 2-1 have extremely strong affinity with hACE2, indicating that the nanoparticle structure presented by fusion protein 2-1 and fusion protein D can well display antigen epitopes.
实施例3：佐剂对疫苗免疫原性的影响Example 3: Effect of adjuvants on vaccine immunogenicity
1.1小鼠免疫1.1 Mouse immunization
雌性BALB/c小鼠(8周龄)分别接受肌肉注射有SWE佐剂(SEPPIC S.A.，货号80748J，批号210721010001)或无佐剂的二价疫苗(质量比为1:1的融合蛋白D和融合蛋白2-1)，实验设计见表2，第14天分别采血收集血清。用ELISA法检测血清抗Spike蛋白(原始株、Delta、及Omicron变异株BA.1和BA.5)的IgG抗体滴度。Female BALB/c mice (8 weeks old) received intramuscular injection of bivalent vaccine (fusion protein D and fusion protein with a mass ratio of 1:1) with SWE adjuvant (SEPPIC S.A., Catalog No. 80748J, Lot No. 210721010001) or without adjuvant. Protein 2-1), the experimental design is shown in Table 2, and blood was collected to collect serum on the 14th day. The ELISA method was used to detect the IgG antibody titer of serum against Spike protein (original strain, Delta, and Omicron variant strains BA.1 and BA.5).
表2实验设计
Table 2 Experimental design
1.2 ELISA法检测血清抗Spike蛋白的IgG抗体滴度1.2 ELISA method to detect serum anti-Spike protein IgG antibody titer
用1×PBS分别稀释WT-Spike蛋白(见实施例2步骤1.1)、Delta-Spike蛋白(见实施例2步骤1.1)、BA.1-Spike蛋白(如SEQ ID NO:60所示，构建方法见实施例2步骤1.1)、BA.5-Spike蛋白(见实施例2步骤1.1)至终浓度为2μg/mL，以100μL/孔加入到96孔酶标板(Costar，9018)中，4℃过夜；用PBST(含0.05％Tween-20的PBS缓冲液)洗涤2次，加入封闭溶液(含3％BSA的PBST)，置37℃培养箱孵育2h；用PBST涤洗2次，加入梯度稀释的实施例3步骤1.1获得的小鼠血清(将血清稀释100倍，然后3倍梯度稀释11个梯度)，100μL/孔，37℃孵育1.5h；用PBST洗涤3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1h；用PBST洗涤8次，按100μL/孔加入TMB溶液(湖州英创生物科技有限公司，TMB-S-001)，37℃避光孵育15～25min；向每个反应孔中加50μL终止液(0.1M H2SO4)终止酶促显色反应。Dilute WT-Spike protein (see step 1.1 of Example 2), Delta-Spike protein (see step 1.1 of Example 2), and BA.1-Spike protein (shown in SEQ ID NO: 60) with 1×PBS respectively, construction method (See step 1.1 of Example 2), BA.5-Spike protein (see step 1.1 of Example 2) to a final concentration of 2 μg/mL, and add it to a 96-well enzyme plate (Costar, 9018) at 100 μL/well at 4°C. Overnight; wash twice with PBST (PBS buffer containing 0.05% Tween-20), add blocking solution (PBST containing 3% BSA), and incubate in a 37°C incubator for 2 hours; wash twice with PBST, add gradient dilution The mouse serum obtained in step 1.1 of Example 3 (dilute the serum 100 times, and then dilute it 3 times to 11 gradients), 100 μL/well, incubate at 37°C for 1.5h; wash 3 times with PBST, add 1:10000 dilution Peroxidase-AffiniPure Goat Anti-Mouse IgG (Jackson, Cat. No.: 115-035-003), 100 μL/well, incubate at 37°C for 1 hour; wash 8 times with PBST, add TMB solution (Huzhou Yingchuang Biotechnology Co., Ltd. at 100 μL/well Company, TMB-S-001), incubate at 37°C in the dark for 15-25 minutes; add 50 μL stop solution (0.1M H 2 SO 4 ) to each reaction well to terminate the enzymatic color reaction.
读板：用酶标仪自带分析软件SoftMax pro 7.02软件，设定检测波长为450nm进行读数，对所得读数以非线性四参数方程曲线模型进行拟合，方程为其中A为吸光度下限，B代表曲线斜率，C为最大反应值一半对应的抗体浓度(EC50)， D为吸光度上限。Plate reading: Use the analysis software SoftMax pro 7.02 software that comes with the microplate reader, set the detection wavelength to 450nm for reading, and fit the obtained readings with a nonlinear four-parameter equation curve model. The equation is: Among them, A is the lower limit of absorbance, B represents the slope of the curve, and C is the antibody concentration corresponding to half of the maximum response value (EC 50 ). D is the upper limit of absorbance.
数据处理：以空白对照OD450均值的2倍值对应的血清稀释度作为抗体滴度。然后再计算每组的几何平均滴度(GMT)。Data processing: The serum dilution corresponding to 2 times the mean value of OD 450 of the blank control was used as the antibody titer. The geometric mean titer (GMT) of each group was then calculated.
BA.1-Spike蛋白氨基酸序列：
BA.1-Spike protein amino acid sequence:
结果如图3所示，单次免疫后，与不加佐剂组相比，加佐剂组针对WT-Spike蛋白、Delta-Spike蛋白、BA.1-Spike蛋白、BA.5-Spike蛋白的抗Spike蛋白IgG几何平均滴度(GMT)可分别提高54倍、55倍、37倍、65倍，表明佐剂显著增强了体液免疫应答。The results are shown in Figure 3. After a single immunization, compared with the group without adjuvant, the anti-Spike protein in the adjuvant group was stronger against WT-Spike protein, Delta-Spike protein, BA.1-Spike protein, and BA.5-Spike protein. The geometric mean titer (GMT) of protein IgG can be increased by 54 times, 55 times, 37 times and 65 times respectively, indicating that the adjuvant significantly enhances the humoral immune response.
实施例4：疫苗在小鼠体内的免疫原性Example 4: Immunogenicity of vaccine in mice
1.1小鼠免疫1.1 Immunization of mice
Balb/c小鼠分别在第0天(D0)和第21天(D21)以肌肉注射方式注射不同剂量的融合蛋白D、融合蛋白2-1和双价疫苗(质量比为1:1的融合蛋白D和融合蛋白2-1)抗原加固定剂量的SWE佐剂(SEPPIC S.A.，货号80748J，批号210721010001)，对照小鼠仅给予SWE佐剂。于免疫后第14天(D14)和第35天(D35)采血，实验设计见表3。用于ELISA检测血清抗Spike蛋白IgG滴度和SARS-CoV-2 Spike假病毒 中和实验检测针对多种假病毒的交叉中和抗体滴度。Balb/c mice were injected intramuscularly with different doses of fusion protein D, fusion protein 2-1 and bivalent vaccine (fusion protein with a mass ratio of 1:1) on day 0 (D0) and day 21 (D21). Protein D and fusion protein 2-1) antigen plus a fixed dose of SWE adjuvant (SEPPIC SA, Cat. No. 80748J, Lot No. 210721010001), and control mice were only given SWE adjuvant. Blood was collected on the 14th day (D14) and the 35th day (D35) after immunization. The experimental design is shown in Table 3. Used to detect serum anti-Spike protein IgG titer and SARS-CoV-2 Spike pseudovirus by ELISA Neutralization experiments detect cross-neutralizing antibody titers against multiple pseudoviruses.
表3试验设计
Table 3 Experimental design
1.2采用ELISA法检测血清抗Spike蛋白IgG滴度1.2 Use ELISA method to detect serum anti-Spike protein IgG titer
用1×PBS分别稀释WT-Spike蛋白(见实施例2步骤1.1)、Delta-Spike蛋白(见实施例2步骤1.1)、BA.1-Spike蛋白(见实施例3步骤1.2)、BA.5-Spike蛋白(见实施例2步骤1.1)至终浓度为2μg/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)置4℃培养箱孵育过夜；用PBST洗涤2次；加入梯度稀释的实施例4步骤1.1获得的小鼠血清(将血清稀释1000倍，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3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(湖州英创生物科技有限公司，TMB-S-001)，37℃避光孵育15～25分钟；用50μL/孔的0.1M硫酸终止酶促显色反应。Dilute WT-Spike protein (see step 1.1 of Example 2), Delta-Spike protein (see step 1.1 of Example 2), BA.1-Spike protein (see step 1.2 of Example 3), and BA.5 respectively with 1×PBS. -Spike protein (see step 1.1 in Example 2) to a final concentration of 2 μg/mL, added to a 96-well enzyme plate (Costar, Cat. No.: 9018) at 100 μL/well, overnight at 4°C; incubate with PBST (in 1×PBS Add 0.05% volume of Tween-20) and wash twice; add blocking solution (PBST containing 3% BSA) and incubate in a 4°C incubator overnight; wash twice with PBST; add gradient dilutions obtained in step 1.1 of Example 4. Mouse serum (dilute the serum 1000 times, and then dilute it 3 times to 11 gradients), 100 μL per well, incubate at 37°C for 1.5 hours; wash 3 times with PBST; add 1:10000 diluted Peroxidase-AffiniPure Goat Anti-Mouse IgG (Jackson, Cat. No.: 115-035-003), 100 μL/well, incubate at 37°C for 1 hour; wash 8 times with PBST; add 100 μL/well TMB solution (Huzhou Yingchuang Biotechnology Co., Ltd., TMB-S-001), Incubate at 37°C in the dark for 15 to 25 minutes; use 50 μL/well of 0.1M sulfuric acid to terminate the enzymatic color reaction.
读板：用酶标仪自带分析软件SoftMax pro 7.02软件，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，方程为其中A为吸光度下限，B代表曲线斜率，C为最大反应值一半对应的抗体浓度(EC50)，D为吸光度上限。Plate reading: Use the analysis software SoftMax pro 7.02 software that comes with the microplate reader, set the detection wavelength to 450nm for reading, and fit the obtained reading OD value with a nonlinear four-parameter equation curve model. The equation is: Among them, A is the lower limit of absorbance, B represents the slope of the curve, C is the antibody concentration (EC 50 ) corresponding to half of the maximum response value, and D is the upper limit of absorbance.
数据处理：以空白对照OD450均值的2倍值对应的血清稀释度作为抗体滴度。然后再计算每组的几何平均滴度(GMT)。Data processing: The serum dilution corresponding to 2 times the mean value of OD 450 of the blank control was used as the antibody titer. The geometric mean titer (GMT) of each group was then calculated.
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。如图4a、4c、4e、4g所示，在初次免疫后的第14天所有的小鼠产生了针对WT-Spike蛋白、Delta-Spike蛋白、BA.1-Spike蛋白和BA.5-Spike蛋白IgG抗体，抗体几何平均抗体滴度(GMT)呈明显的剂量效应关系。In all the tested dose groups, no anti-Spike protein IgG titers were detected in mice given only adjuvant. As shown in Figures 4a, 4c, 4e, and 4g, all mice produced IgG antibodies against WT-Spike protein, Delta-Spike protein, BA.1-Spike protein, and BA.5-Spike protein on the 14th day after the primary immunization, and the geometric mean antibody titer (GMT) showed a clear dose-effect relationship.
在第二次增强免疫后第35天所有小鼠的抗Spike蛋白IgG滴度与初次免疫后相比 均显著升高。与接受相同剂量的融合蛋白D相比，接受融合蛋白2-1的小鼠针对WT-Spike蛋白和Delta-Spike蛋白抗体滴度显著低于前者(图4b、4d)；但融合蛋白2-1组针对BA.5-Spike蛋白抗体滴度显著高于融合蛋白D(图4h)；与单价疫苗融合蛋白D、融合蛋白2-1相比，双价疫苗诱导了针对WT-Spike蛋白、Delta-Spike蛋白、BA.1-Spike蛋白和BA.5-Spike蛋白IgG的高抗体滴度。Anti-Spike protein IgG titers in all mice on day 35 after the second booster immunization compared with those after the primary immunization were significantly elevated. Compared with those receiving the same dose of fusion protein D, mice receiving fusion protein 2-1 had significantly lower antibody titers against WT-Spike protein and Delta-Spike protein (Figure 4b, 4d); but fusion protein 2-1 The antibody titer against BA.5-Spike protein in the group was significantly higher than that against fusion protein D (Figure 4h); compared with the monovalent vaccine fusion protein D and fusion protein 2-1, the bivalent vaccine induced antibodies against WT-Spike protein and Delta- High antibody titers of Spike protein, BA.1-Spike protein and BA.5-Spike protein IgG.
抗Spike蛋白IgG抗体滴度的结果表明，双价疫苗比单价疫苗融合蛋白D或融合蛋白2-1具有更好的免疫原性，而且接种两针的滴度明显优于接种一针的滴度，对不同变异株的抗体滴度均保持优于或等于单价疫苗。The results of the anti-Spike protein IgG antibody titer showed that the bivalent vaccine has better immunogenicity than the monovalent vaccine fusion protein D or fusion protein 2-1, and the titer of two doses of vaccination is significantly better than that of one dose of vaccination , the antibody titers against different mutant strains remained better than or equal to the monovalent vaccine.
1.3 SARS-CoV-2 Spike假病毒中和抗体实验1.3 SARS-CoV-2 Spike pseudovirus neutralizing antibody experiment
为了评估双价疫苗(融合蛋白D和融合蛋白2-1的质量比1:1)的广谱性，检测了免疫血清对SARS-CoV-2原始株、Delta变异株以及变异株BA.5、BQ.1.1、XBB及XBB.1.5等的抑制能力。In order to evaluate the broad spectrum of the bivalent vaccine (the mass ratio of fusion protein D and fusion protein 2-1 is 1:1), immune serum was tested against the original strain of SARS-CoV-2, the Delta variant strain, and the variant strain BA.5, Suppression capabilities of BQ.1.1, XBB and XBB.1.5, etc.
将假病毒置于冰上缓慢溶解，使用DMEM完全培养基稀释50倍，于96孔白板中加入假病毒稀释液，设置试验组、细胞对照组(CC组)和病毒对照组(VC组)，除CC组外其余各25μL/孔；将实施例4步骤1.1组3、组6、组9第35天采集的小鼠血清置于56℃孵育30min灭活补体，用DMEM完全培养基稀释40倍，再进行2倍梯度稀释，稀释12个梯度，试验组按照50μL/孔依次加到已加入假病毒的96孔白板中，CC组加入DMEM完全培养基75μL/孔，VC组加入DMEM完全培养基50μL/孔；将96孔白板充分震荡混匀后置于培养箱中37℃孵育1h。取出孵育1h后的96孔白板，以50μL/孔加入ACE2-293细胞悬液(2×104cells/孔)，用移液枪轻轻吹匀，将96孔白板置于培养箱培养。培养48h后取出96孔白板，待其恢复至室温，弃去培养基，每孔加入100μL Bio-LiteTM Luciferase Assay System溶液(Vazyme，货号DD1201)，室温避光反应2min后，用酶标仪Luminescence检测模块读取发光信号。通过非线性四参数曲线拟合模型分析％抑制并计算IC50。Slowly dissolve the pseudovirus on ice, dilute it 50 times with DMEM complete culture medium, add the pseudovirus diluent to the 96-well white plate, and set up a test group, a cell control group (CC group) and a virus control group (VC group). Except for the CC group, 25 μL/well for each other; put the mouse serum collected on the 35th day of step 1.1 of Example 4, group 3, group 6, and group 9, incubate at 56°C for 30 minutes to inactivate complement, and dilute it 40 times with DMEM complete culture medium , then perform 2-fold gradient dilution, dilute 12 gradients, and add 50 μL/well of the test group to the 96-well white plate where the pseudovirus has been added. The CC group adds 75 μL/well of DMEM complete medium, and the VC group adds DMEM complete medium. 50 μL/well; shake the 96-well white plate thoroughly and mix well, then place it in an incubator and incubate at 37°C for 1 hour. Take out the 96-well white plate after incubation for 1 hour, add ACE2-293 cell suspension (2×10 4 cells/well) at 50 μL/well, gently blow evenly with a pipette, and place the 96-well white plate in the incubator for culture. After culturing for 48 hours, take out the 96-well white plate, wait until it returns to room temperature, discard the culture medium, add 100 μL Bio-LiteTM Luciferase Assay System solution (Vazyme, Cat. No. DD1201) to each well, react for 2 minutes at room temperature in the dark, and use a microplate reader for Luminescence detection. The module reads the luminous signal. % inhibition was analyzed by a nonlinear four-parameter curve fitting model and IC50 was calculated.
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转染试剂(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。The construction method of ACE2-293 cells is as follows: culture HEK293 cells in DMEM complete medium containing 10% FBS, and use lipofectamine 2000 transfection reagent (Thermo Fisher, 11668019) to transfect ACE2 expression plasmid (Yiqiao Shenzhou, HG10108-M) Transfection, followed by pressure selection with hygromycin (200μg/ml) and flow sorting (using 10μg/ml anti-ACE2 and PE-conjugated Anti-Human IgG-Fc), cells continued to amplify to select out PE Single clones with a positive rate of >90% were amplified in the next step, and HEK293 cells expressing ACE2, namely ACE2-293 cells, were screened out.
其中，本试验所用的假病毒：SARS-CoV-2原始株假病毒(Vazyme，货号DD1702-03)，SARS-CoV-2 Delta假病毒(Vazyme，货号DD1754-03)，SARS-CoV-2 BA.5假病毒(Vazyme，DD1776-03)，SARS-CoV-2 BQ.1.1假病毒(Vazyme，货号DD1792-03)，SARS-CoV-2 XBB假病毒(Vazyme，DD1794-03)，SARS-CoV-2 XBB.1.5 假病毒(Vazyme，DD1797-03)。Among them, the pseudoviruses used in this experiment: SARS-CoV-2 original strain pseudovirus (Vazyme, product number DD1702-03), SARS-CoV-2 Delta pseudovirus (Vazyme, product number DD1754-03), SARS-CoV-2 BA .5 Pseudovirus (Vazyme, DD1776-03), SARS-CoV-2 BQ.1.1 Pseudovirus (Vazyme, Cat. No. DD1792-03), SARS-CoV-2 XBB Pseudovirus (Vazyme, DD1794-03), SARS-CoV -2 XBB.1.5 Pseudovirus (Vazyme, DD1797-03).
图5结果表明，与上述抗Spike蛋白抗体滴度的结果一致，双价疫苗比单价疫苗融合蛋白D或融合蛋白2-1具有更好的免疫原性，对不同变异株的中和抗体滴度均保持优于或等于单价疫苗。融合蛋白2-1对SARS-CoV-2原始株假病毒及SARS-CoV-2 Delta假病毒的滴度显著低于融合蛋白D及双价疫苗；融合蛋白D对SARS-CoV-2 BA.5假病毒、SARS-CoV-2 BQ.1.1假病毒、SARS-CoV-2 XBB假病毒及SARS-CoV-2 XBB.1.5假病毒的滴度显著低于融合蛋白2-1及双价疫苗；而双价疫苗对所有检测的毒株假病毒保持很高的滴度。说明双价疫苗具有广谱性，对多种毒株均具有较高的中和抗体滴度。The results in Figure 5 show that, consistent with the above results on anti-Spike protein antibody titers, the bivalent vaccine has better immunogenicity than the monovalent vaccine fusion protein D or fusion protein 2-1. Neutralizing antibody titers against different mutant strains All remain better than or equal to monovalent vaccines. The titer of fusion protein 2-1 against SARS-CoV-2 original strain pseudovirus and SARS-CoV-2 Delta pseudovirus is significantly lower than that of fusion protein D and bivalent vaccine; the titer of fusion protein D against SARS-CoV-2 BA.5 The titers of pseudovirus, SARS-CoV-2 BQ.1.1 pseudovirus, SARS-CoV-2 XBB pseudovirus and SARS-CoV-2 XBB.1.5 pseudovirus were significantly lower than those of fusion protein 2-1 and bivalent vaccine; while The bivalent vaccine maintained high titers against all pseudovirus strains tested. This shows that the bivalent vaccine is broad-spectrum and has high neutralizing antibody titers against a variety of strains.
1.4血清对新冠真病毒中和抗体滴度1.4 Serum neutralizing antibody titer against true coronavirus
检测了双价疫苗二次免疫后的小鼠血清对新冠病毒变异株BA.5、BQ.1和XBB真病毒的抑制滴度。采用微量板法，评估实施例4步骤1.1组9第35天采集的小鼠血清对新冠病毒变异株BA.5、BQ.1和XBB真病毒的中和滴度(IC99 Titer)。原理为：病毒感染敏感靶细胞(VERO-E6)后，引起细胞形态学变化，出现细胞病变效应(CPE)，而血清中的特异性中和抗体与病毒结合后，可使病毒颗粒失去感染性，抑制CPE的出现。The inhibitory titers of mouse serum after secondary immunization with the bivalent vaccine against the new coronavirus mutant strains BA.5, BQ.1 and XBB true virus were tested. The microplate method was used to evaluate the neutralizing titer (IC99 Titer) of the mouse serum collected on the 35th day of step 1.1 of Group 9 of Example 4 against the new coronavirus mutant strains BA.5, BQ.1 and XBB true virus. The principle is: after the virus infects sensitive target cells (VERO-E6), it causes morphological changes in the cells and the occurrence of cytopathic effect (CPE). The specific neutralizing antibodies in the serum combine with the virus to make the virus particles lose their infectivity. , inhibit the emergence of CPE.
试验结果表明(图6)，双价疫苗二次免疫后小鼠血清对新冠病毒变异株BA.5、BQ.1和XBB真病毒具有强烈的抑制能力，其中BA.5组有6只小鼠抑制滴度已达到试验上限、BQ.1组有2只小鼠抑制滴度已达到试验上限、XBB组有3只小鼠抑制滴度已达到试验上限；说明双价疫苗对当前流行的新冠病毒变异株BA.5、BQ.1和XBB真病毒具有极高的中和抗体滴度。The experimental results showed (Figure 6) that the mouse serum after the second immunization with the bivalent vaccine had a strong inhibitory ability against the new coronavirus variants BA.5, BQ.1 and XBB true viruses. Among them, the inhibition titer of 6 mice in the BA.5 group had reached the upper limit of the test, the inhibition titer of 2 mice in the BQ.1 group had reached the upper limit of the test, and the inhibition titer of 3 mice in the XBB group had reached the upper limit of the test; this indicates that the bivalent vaccine has extremely high neutralizing antibody titers against the currently popular new coronavirus variants BA.5, BQ.1 and XBB true viruses.
实施例5：双价疫苗作为加强针在已接种2剂灭活苗的小鼠体内的免疫原性Example 5: Immunogenicity of bivalent vaccine as booster shot in mice that have received 2 doses of inactivated vaccine
1.1小鼠免疫1.1 Mouse immunization
两组Balb/c小鼠分别在第0天(D0)和第21天(D21)以肌肉注射方式注射100μL灭活苗(北京科兴中维生物技术有限公司，202112187N)，恢复3个月后，在第111天(D111)分别接种灭活苗(北京科兴中维生物技术有限公司，202112187N)或双价疫苗(质量比为1:1的融合蛋白D和融合蛋白2-1)，并在第三针接种前一天(D110)与接种后两周(D125)采血，试验设计见表4。采用ELISA法检测血清抗Spike蛋白(原始株、Delta、Omicron BA.1和BA.5)IgG滴度，利用假病毒中和实验检测针对多种假病毒的交叉中和抗体滴度。Two groups of Balb/c mice were injected intramuscularly with 100 μL of inactivated vaccine (Beijing Sinovac Zhongwei Biotechnology Co., Ltd., 202112187N) on day 0 (D0) and day 21 (D21) respectively, and recovered after 3 months. , receive inactivated vaccine (Beijing Sinovac Zhongwei Biotechnology Co., Ltd., 202112187N) or bivalent vaccine (fusion protein D and fusion protein 2-1 with a mass ratio of 1:1) on day 111 (D111), and Blood was collected one day before the third dose of vaccination (D110) and two weeks after vaccination (D125). The experimental design is shown in Table 4. The ELISA method was used to detect the serum anti-Spike protein (original strain, Delta, Omicron BA.1 and BA.5) IgG titers, and the pseudovirus neutralization experiment was used to detect the cross-neutralizing antibody titers against multiple pseudoviruses.
表4试验设计

Table 4 Experimental design

1.2采用ELISA法检测血清抗Spike蛋白IgG滴度1.2 Use ELISA method to detect serum anti-Spike protein IgG titer
用1×PBS分别稀释WT-Spike蛋白(制备方法见实施例2步骤1.1)、Delta-Spike蛋白(制备方法见实施例2步骤1.1)、BA.1-Spike蛋白(制备方法见实施例3步骤1.2)、BA.5-Spike蛋白(制备方法见实施例2步骤1.1)至终浓度为2μg/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)，置4℃培养箱孵育过夜；用PBST洗涤2次；加入梯度稀释的实施例5步骤1.1获得的小鼠血清(将血清稀释1000倍，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3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(湖州英创生物科技有限公司，TMB-S-001)，37℃避光孵育15～25分钟；向每个反应孔中加50μL终止液(0.1M H2SO4)终止酶促显色反应。Dilute WT-Spike protein (see step 1.1 of Example 2 for the preparation method), Delta-Spike protein (see step 1.1 of Example 2 for the preparation method), and BA.1-Spike protein (see step 1.1 of Example 2 for the preparation method) with 1×PBS respectively. 1.2), BA.5-Spike protein (for the preparation method, see step 1.1 of Example 2) to a final concentration of 2 μg/mL, add 100 μL/well into a 96-well enzyme plate (Costar, Cat. No.: 9018), overnight at 4°C ; Wash twice with PBST (0.05% volume of Tween-20 in 1×PBS); add blocking solution (PBST containing 3% BSA) and incubate in a 4°C incubator overnight; wash twice with PBST; add gradient Dilute the mouse serum obtained in step 1.1 of Example 5 (dilute the serum 1000 times, and then dilute it 3 times to 11 gradients), 100 μL per well, incubate at 37°C for 1.5 hours; wash 3 times with PBST; add 1:10000 dilution The final Peroxidase-AffiniPure Goat Anti-Mouse IgG (Jackson, Cat. No.: 115-035-003), 100 μL/well, incubated at 37°C for 1 hour; washed 8 times with PBST; added 100 μL/well TMB solution (Huzhou Yingchuang Biotechnology Co., Ltd. Company, TMB-S-001), incubate at 37°C in the dark for 15 to 25 minutes; add 50 μL stop solution (0.1M H 2 SO 4 ) to each reaction well to terminate the enzymatic color reaction.
读板：用酶标仪自带分析软件SoftMax pro 7.02软件，设定检测波长为450nm进行读数，对所得读数以非线性四参数方程曲线模型进行拟合，方程为其中A为吸光度下限，B代表曲线斜率，C为最大反应值一半对应的抗体浓度(EC50)，D为吸光度上限。Plate reading: Use the analysis software SoftMax pro 7.02 software that comes with the microplate reader, set the detection wavelength to 450nm for reading, and fit the obtained readings with a nonlinear four-parameter equation curve model. The equation is: Among them, A is the lower limit of absorbance, B represents the slope of the curve, C is the antibody concentration (EC 50 ) corresponding to half of the maximum response value, and D is the upper limit of absorbance.
数据处理：以空白对照OD450均值的2倍值对应的血清稀释度作为抗体滴度。然后再计算每组的几何平均滴度(GMT)。Data processing: The serum dilution corresponding to 2 times the mean value of OD 450 of the blank control was used as the antibody titer. The geometric mean titer (GMT) of each group was then calculated.
如图7a-d所示，在接种序贯免疫加强针前，两组小鼠的血清抗Spike蛋白IgG滴度都处于较低水平，其中，针对BA.1-Spike蛋白和BA.5-Spike蛋白的抗体滴度显著低于WT-Spike蛋白和Delta-Spike蛋白。在序贯加强免疫后2周，无论接种的是灭活苗还是双价疫苗，小鼠的抗Spike蛋白IgG滴度均显著升高。双价疫苗组(组2)对Wildtype、Delta、BA.1和BA.5等毒株的抗Spike蛋白IgG滴度都远远高于灭活苗组(组1)。As shown in Figure 7a-d, before vaccination with sequential immune booster shots, the serum anti-Spike protein IgG titers of both groups of mice were at low levels, among which, against BA.1-Spike protein and BA.5-Spike The antibody titer of the protein was significantly lower than that of WT-Spike protein and Delta-Spike protein. Two weeks after the sequential booster immunization, the anti-Spike protein IgG titers of mice increased significantly regardless of whether they were vaccinated with inactivated vaccine or bivalent vaccine. The anti-Spike protein IgG titers of the bivalent vaccine group (Group 2) against Wildtype, Delta, BA.1 and BA.5 strains were much higher than those of the inactivated vaccine group (Group 1).
1.3 SARS-CoV-2 Spike假病毒中和实验1.3 SARS-CoV-2 Spike pseudovirus neutralization experiment
检测了免疫血清对的SARS-CoV-2原始株、Delta变异株以及变异株BF.7、XBB.1等的抑制能力。将假病毒用DMEM完全培养基稀释50倍，于96孔白板中加入假病毒稀释液，设置试验组、细胞对照组(CC组)和病毒对照组(VC组)，除CC组外 其余各25μL/孔；将实施例5步骤1.1采集的小鼠血清置于56℃孵育30min灭活补体，用DMEM完全培养基稀释40倍，再进行2倍梯度稀释，稀释12个梯度，试验组按照50μL/孔依次加到已加入假病毒的96孔白板中，CC组加入DMEM完全培养基75μL/孔，VC组加入DMEM完全培养基50μL/孔；将96孔白板充分震荡混匀后置于培养箱中37℃孵育1h。取出孵育1h后的96孔白板，以50μL/孔加入ACE2-293细胞悬液(2×104cells/孔)，用移液枪轻轻吹匀，将96孔白板置于培养箱37℃培养。48h后取出96孔白板，待其恢复至室温，弃去培养基，每孔加入100μL Bio-LiteTM Luciferase Assay System溶液(Vazyme，货号DD1201)，室温避光反应2min后，用酶标仪Luminescence检测模块读取发光信号。通过非线性四参数曲线拟合模型分析％抑制并计算IC50。The inhibitory ability of immune serum against the original SARS-CoV-2 strain, Delta variant strain, and variant strains BF.7, XBB.1, etc. was tested. Dilute the pseudovirus 50 times with DMEM complete culture medium, add the pseudovirus dilution solution to the 96-well white plate, and set up a test group, a cell control group (CC group) and a virus control group (VC group), except for the CC group The remaining 25 μL/well; the mouse serum collected in step 1.1 of Example 5 was placed at 56°C for 30 minutes to inactivate complement, diluted 40 times with DMEM complete culture medium, and then 2 times gradient diluted, diluted 12 gradients, test group Add 50 μL/well to the 96-well white plate with pseudovirus in sequence. For the CC group, add 75 μL/well of DMEM complete medium. For the VC group, add 50 μL/well of DMEM complete medium. Shake and mix the 96-well white plate thoroughly and place it on the Incubate in the incubator at 37°C for 1 hour. Take out the 96-well white plate after incubation for 1 hour, add ACE2-293 cell suspension (2×10 4 cells/well) at 50 μL/well, gently blow evenly with a pipette, and place the 96-well white plate in the incubator at 37°C. . After 48 hours, take out the 96-well white plate, wait until it returns to room temperature, discard the culture medium, add 100 μL Bio-LiteTM Luciferase Assay System solution (Vazyme, Cat. No. DD1201) to each well, react for 2 minutes at room temperature in the dark, and use the Luminescence detection module of a microplate reader Read the luminescence signal. % inhibition was analyzed by a nonlinear four-parameter curve fitting model and IC50 was calculated.
其中，本试验所用的假病毒：SARS-CoV-2原始株假病毒(Vazyme，货号DD1702-03)，SARS-CoV-2 Delta假病毒(Vazyme，货号DD1754-03)，SARS-CoV-2 BF.7假病毒(吉满生物科技，货号：GM-0220PV100-96T)，SARS-CoV-2 XBB.1假病毒(吉满生物科技，货号：GM-0220PV104-96T)。Among them, the pseudoviruses used in this experiment: SARS-CoV-2 original strain pseudovirus (Vazyme, product number DD1702-03), SARS-CoV-2 Delta pseudovirus (Vazyme, product number DD1754-03), SARS-CoV-2 BF .7 fake virus (Jiman Biotechnology, product number: GM-0220PV100-96T), SARS-CoV-2 XBB.1 fake virus (Jiman Biotechnology, product number: GM-0220PV104-96T).
结果如图8a-d所示，与上述抗Spike蛋白抗体滴度的结果一致，双价疫苗对不同变异株的中和抗体滴度均优于灭活苗。在序贯免疫前，两组小鼠对SARS-CoV-2 BF.7假病毒和SARS-CoV-2 XBB.1假病毒几乎都没有可检测到的中和抗体滴度，序贯免疫后，灭活苗组(组1)部分小鼠对SARS-CoV-2 BF.7假病毒产生了极低的中和抗体滴度(图8c)，仅有1只小鼠对SARS-CoV-2 XBB.1假病毒产生了微量的中和抗体(图8d)；而双价疫苗组(组2)对SARS-CoV-2 BF.7假病毒和SARS-CoV-2 XBB.1假病毒都产生了较为显著的中和抗体滴度。The results are shown in Figure 8a-d. They are consistent with the above-mentioned anti-Spike protein antibody titer results. The neutralizing antibody titers of the bivalent vaccine against different mutant strains are better than those of the inactivated vaccine. Before sequential immunization, both groups of mice had almost no detectable neutralizing antibody titers against SARS-CoV-2 BF.7 pseudovirus and SARS-CoV-2 XBB.1 pseudovirus. After sequential immunization, Some mice in the inactivated vaccine group (Group 1) produced extremely low neutralizing antibody titers against SARS-CoV-2 BF.7 pseudovirus (Figure 8c), and only 1 mouse was resistant to SARS-CoV-2 XBB The .1 pseudovirus produced trace amounts of neutralizing antibodies (Figure 8d); while the bivalent vaccine group (Group 2) produced both SARS-CoV-2 BF.7 pseudovirus and SARS-CoV-2 XBB.1 pseudovirus. Relatively significant neutralizing antibody titers.
实施例6：双价疫苗细胞免疫酶联免疫斑点试验(enzyme linked immunospot assay，ELISpot)检测Example 6: Detection of bivalent vaccine cellular immunity enzyme-linked immunospot assay (ELISpot)
1.1小鼠免疫1.1 Mouse immunization
6周龄的Balb/C雌性小鼠分2组，分别在第0天(D0)和第21天(D21)通过肌肉注射按表5剂量进行免疫接种，在第二次免疫后2周对小鼠实施安乐死，取脾脏用于ELIspot试验。Six-week-old Balb/C female mice were divided into two groups and immunized by intramuscular injection according to the doses in Table 5 on day 0 (D0) and day 21 (D21), respectively. The mice were euthanized 2 weeks after the second immunization, and the spleens were removed for ELIspot assay.
表5试验设计

注：双价疫苗为质量比为1:1的融合蛋白D和融合蛋白2-1；佐剂为SWE佐剂Table 5 Experimental design

Note: The bivalent vaccine is fusion protein D and fusion protein 2-1 with a mass ratio of 1:1; the adjuvant is SWE adjuvant.
1.2 ELIspot试验1.2 ELIspot test
为了评估双价疫苗免疫小鼠对不同新冠病毒变异株的细胞免疫水平，利用新冠病毒原始株(wildtype，WT)及变异株(Delta、BA.5)的Spike蛋白肽池作为刺激物，刺激免疫后的小鼠脾淋巴细胞，以Phorbol 12-myristate 13-acetate(PMA)(MedChemExpress，货号16561-29-8)作为阳性对照，以空白培养基作为阴性对照，通过ELISpot方法检测细胞分泌的IFN-γ、IL-2、IL-4等细胞因子。In order to evaluate the cellular immunity level of mice immunized with bivalent vaccine against different variants of the new coronavirus, the Spike protein peptide pool of the original strain (wildtype, WT) and variants (Delta, BA.5) of the new coronavirus was used as a stimulant to stimulate the spleen lymphocytes of immunized mice. Phorbol 12-myristate 13-acetate (PMA) (MedChemExpress, Cat. No. 16561-29-8) was used as a positive control, and blank culture medium was used as a negative control. The ELISpot method was used to detect the secretion of IFN-γ, IL-2, IL-4 and other cytokines by the cells.
使用ELISpot Plus Mouse IL-2试剂盒(Mabtech，货号3441-4HPW)，ELISpot Plus Mouse IL-4试剂盒(Mabtech，货号3311-4HPW)，ELISpot Plus Mouse IFN-γ试剂盒(Mabtech，货号3321-4HPT)进行ELIspot试验，步骤简述如下：Use ELISpot Plus Mouse IL-2 Kit (Mabtech, Catalog No. 3441-4HPW), ELISpot Plus Mouse IL-4 Kit (Mabtech, Catalog No. 3311-4HPW), ELISpot Plus Mouse IFN-γ Kit (Mabtech, Catalog No. 3321-4HPT ) to perform the ELIspot test. The steps are briefly described as follows:
将预包被的IL-2、IL-4、IFN-γELISpot板用无菌PBS清洗，加入200μL/孔无菌的含有10％FBS(ExCell Bio，货号FSP500)的DMEM培养基(Gibco，货号C11995500BT)，于室温孵育过夜。用无菌的含有10％FBS的DMEM培养基将PMA稀释至100ng/mL，另将新冠病毒原始株的Spike蛋白肽池(强耀生物，货号04010065348)、新冠病毒变异株Delta的Spike蛋白肽池(强耀生物，货号04010068345)、新冠病毒变异株BA.5的Spike蛋白肽池(强耀生物，货号04010069259)稀释至2μg/mL。弃掉ELISpot板内的培养基，分别加入PMA及三种肽池，50μL/孔。以DMEM培养基作为阴性对照孔。取实施例6步骤1.1获得的脾脏研磨，将得到的细胞悬液通过70μm筛网，用DMEM培养基清洗一遍；向每只小鼠的细胞内加入2mL Ack lysing buffer(Gibco，A10492-01)冰上孵育10min，加入20mL DMEM培养基终止裂解作用，离心，弃上清，用DMEM培养基重悬细胞，得到小鼠的脾淋巴细胞。将各小鼠的脾淋巴细胞按照2×105细胞/50μL/孔悬空滴加进ELISpot板。将ELISpot板置于37℃，5％CO2孵育48小时。弃去板内细胞液，用无菌的PBS清洗平板。分别将三种ELISpot板对应的检测抗体用含0.5％FBS的PBS溶液稀释至1μg/mL，用0.22μm滤膜过滤后，100μL/孔加入对应ELISpot板，室温孵育2小时。用无菌的PBS清洗平板。将Streptavidin-HRP(Jackson；货号：016-030-084)用含0.5％FBS的PBS溶液稀释1000倍，用0.22μm滤膜过滤后，100μL/孔加入ELISpot板，室温孵育1小时。用无菌的PBS清洗平板。加入100μL/孔无菌的TMB底物溶液显色，直至孔内有明显的斑点出现。弃掉孔内液体，用去离子水冲洗终止显色，撕去背面的胶板，反复冲洗膜两侧。晾干，读取孔内斑点数。Wash the pre-coated IL-2, IL-4, and IFN-γ ELISpot plates with sterile PBS, and add 200 μL/well of sterile DMEM medium containing 10% FBS (ExCell Bio, Cat. No. FSP500) (Gibco, Cat. No. C11995500BT). ) and incubate at room temperature overnight. Use sterile DMEM medium containing 10% FBS to dilute PMA to 100ng/mL, and add the Spike protein peptide pool of the original strain of the new coronavirus (Qiangyao Biotechnology, Cat. No. 04010065348) and the Spike protein peptide pool of the new coronavirus mutant strain Delta. (Qiangyao Biotechnology, Catalog No. 04010068345), and the Spike protein peptide pool of the new coronavirus mutant strain BA.5 (Qiangyao Biotechnology, Catalog No. 04010069259) were diluted to 2 μg/mL. Discard the culture medium in the ELISpot plate, and add PMA and three peptide pools respectively, 50 μL/well. DMEM medium was used as a negative control well. Grind the spleen obtained in step 1.1 of Example 6, pass the obtained cell suspension through a 70 μm mesh, and wash it once with DMEM culture medium; add 2 mL of Ack lysing buffer (Gibco, A10492-01) to the cells of each mouse on ice Incubate for 10 minutes, add 20 mL of DMEM medium to terminate the lysis, centrifuge, discard the supernatant, and resuspend the cells in DMEM medium to obtain mouse spleen lymphocytes. The spleen lymphocytes of each mouse were suspended and dropped into the ELISpot plate at a rate of 2×10 5 cells/50 μL/well. Incubate the ELISpot plate at 37°C, 5% CO2 for 48 hours. Discard the cell fluid in the plate and wash the plate with sterile PBS. Dilute the detection antibodies corresponding to the three ELISpot plates to 1 μg/mL with PBS solution containing 0.5% FBS. After filtering with a 0.22 μm filter, add 100 μL/well to the corresponding ELISpot plate and incubate at room temperature for 2 hours. Wash the plate with sterile PBS. Streptavidin-HRP (Jackson; Cat. No.: 016-030-084) was diluted 1000 times with PBS solution containing 0.5% FBS. After filtering with a 0.22 μm filter, 100 μL/well was added to the ELISpot plate and incubated at room temperature for 1 hour. Wash the plate with sterile PBS. Add 100 μL/well of sterile TMB substrate solution to develop color until obvious spots appear in the wells. Discard the liquid in the well, rinse with deionized water to stop color development, peel off the rubber plate on the back, and rinse both sides of the membrane repeatedly. Let dry and read the number of spots in the hole.
IFN-γELISpot结果如图9a所示，与只免疫佐剂组(组4)相比，单价或双价疫苗接种后用新冠病毒原始株的Spike蛋白肽池、新冠病毒变异株Delta的Spike蛋白肽池、新冠病毒变异株BA.5的Spike蛋白肽池都可以刺激IFN-γ分泌型T细胞的激活，其中，T细胞对新冠病毒变异株Delta的Spike蛋白肽池和新冠病毒变异株BA.5的Sp ike蛋白肽池的响应要高于新冠病毒原始株的Spike蛋白肽池。The IFN-γ ELISpot results are shown in Figure 9a. Compared with the adjuvant-only group (group 4), after monovalent or bivalent vaccination, the Spike protein peptide pool of the original strain of the new coronavirus and the Spike protein peptide of the new coronavirus mutant strain Delta were used. Both the pool and the Spike protein peptide pool of the new coronavirus mutant strain BA.5 can stimulate the activation of IFN-γ-secreting T cells. Among them, T cells respond to the Spike protein peptide pool of the new coronavirus mutant strain Delta and the new coronavirus mutant strain BA.5. Sp The response of the ike protein peptide pool is higher than that of the Spike protein peptide pool of the original strain of the new coronavirus.
IL-2和IL-4ELISpot结果分别如图9b和9c所示，与IFN-γELISpot结果相同，疫苗接种后用新冠病毒原始株的Spike蛋白肽池、新冠病毒变异株Delta的Spike蛋白肽池、新冠病毒变异株BA.5的Spike蛋白肽池都可以刺激IL-2分泌型细胞或IL-4分泌型细胞的激活，且新冠病毒变异株Delta的Spike蛋白肽池和新冠病毒变异株BA.5的Spike蛋白肽池的刺激效果更显著。The IL-2 and IL-4 ELISpot results are shown in Figures 9b and 9c respectively. They are the same as the IFN-γ ELISpot results. After vaccination, the Spike protein peptide pool of the original strain of the new coronavirus, the Spike protein peptide pool of the new coronavirus mutant strain Delta, and the new coronavirus were used. The Spike protein peptide pool of the virus variant BA.5 can stimulate the activation of IL-2 secreting cells or IL-4 secreting cells, and the Spike protein peptide pool of the new coronavirus variant Delta and the new coronavirus variant BA.5 The stimulating effect of Spike protein peptide pool is more significant.
另外，在新冠病毒变异株Delta的Spike蛋白肽池和新冠病毒变异株BA.5的Spike蛋白肽池刺激后，IL-2分泌型细胞数量要显著高于IL-4分泌型细胞。如图9c所示，在新冠病毒原始株的Spike蛋白肽池、新冠病毒变异株Delta的Spike蛋白肽池和新冠病毒变异株BA.5的Spike蛋白肽池刺激后，仅有极少量的IL-4分泌型细胞激活。由此来看，融合蛋白D、融合蛋白2-1和双价疫苗免疫后都可以诱导细胞免疫应答，且以Th1型为主。In addition, after stimulation by the Spike protein peptide pool of the new coronavirus variant Delta and the Spike protein peptide pool of the new coronavirus variant BA.5, the number of IL-2 secreting cells was significantly higher than that of IL-4 secreting cells. As shown in Figure 9c, after stimulation by the Spike protein peptide pool of the original strain of the new coronavirus, the Spike protein peptide pool of the new coronavirus variant Delta, and the Spike protein peptide pool of the new coronavirus variant BA.5, only a very small amount of IL- 4 Secretory cell activation. From this point of view, fusion protein D, fusion protein 2-1 and bivalent vaccine can all induce cellular immune responses after immunization, and the Th1 type is dominant.
实施例7：疫苗在大鼠体内的免疫原性Example 7: Immunogenicity of the vaccine in rats
1.1大鼠免疫1.1 Rat immunization
本研究设计如表6所示。健康Sprague-Dawley大鼠(SPF级)，雌雄各半，在第1天(D1)以肌肉注射方式单次给药0或80μg/只的双价疫苗(质量比为1:1的融合蛋白D和融合蛋白2-1)，注射体积为0.5mL/只，于第15天(D15)采血。采用ELISA法检测血清抗Spike蛋白(原始株、Delta、BA.1和BA.5)IgG滴度。The research design is shown in Table 6. Healthy Sprague-Dawley rats (SPF grade), half male and half female, were given a single intramuscular injection of 0 or 80 μg/rat bivalent vaccine (fusion protein D with a mass ratio of 1:1) on day 1 (D1). and fusion protein 2-1), the injection volume was 0.5mL/animal, and blood was collected on the 15th day (D15). The ELISA method was used to detect serum anti-Spike protein (original strain, Delta, BA.1 and BA.5) IgG titers.
表6试验设计

注：佐剂为SWE佐剂Table 6 Experimental design

Note: The adjuvant is SWE adjuvant
1.2采用ELISA法检测血清抗Spike蛋白IgG滴度1.2 Use ELISA method to detect serum anti-Spike protein IgG titer
用1×PBS分别稀释WT-Spike蛋白(见实施例2步骤1.1)、Delta-Spike蛋白(见实施例2步骤1.1)、BA.5-Spike蛋白(见实施例2步骤1.1)至终浓度为2μg/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)，置37℃培养箱孵育2小时；用PBST洗涤2次；加入梯度稀释的实施例7步骤1.1获得的小鼠血清(将血清稀释100倍，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3次；加入1:5000稀释后的Goat anti-Rat IgG secondary antibody(HRP)(Sino Biological，货号SSA005)，100μL/孔，37℃孵育1小 时；PBST洗涤8次；加入100μL/孔TMB溶液(湖州英创生物科技有限公司，TMB-S-001)，37℃避光孵育15～25分钟；向每个反应孔中加50μL终止液(0.1M H2SO4)终止酶促显色反应。WT-Spike protein (see step 1.1 of Example 2), Delta-Spike protein (see step 1.1 of Example 2), and BA.5-Spike protein (see step 1.1 of Example 2) were diluted with 1×PBS to a final concentration of 2 μg/mL, and 100 μL/well were added to a 96-well ELISA plate (Costar, catalog number: 9018) and incubated at 4°C overnight; washed twice with PBST (0.05% volume of Tween-20 was added to 1×PBS); blocking solution (PBST containing 3% BSA) was added and incubated in a 37°C incubator for 2 hours; washed twice with PBST; the mouse serum obtained in step 1.1 of Example 7 was added with gradient dilution (the serum was diluted 100 times, and then 11 gradients were diluted 3 times), 100 μL per well, and incubated at 37°C for 1.5 hours; washed three times with PBST; Goat anti-Rat IgG secondary antibody (HRP) (Sino Biological, catalog number SSA005), 100 μL/well, incubate at 37°C for 1 hour ; Wash 8 times with PBST; Add 100 μL/well TMB solution (Huzhou Yingchuang Biotechnology Co., Ltd., TMB-S-001) and incubate at 37°C in the dark for 15-25 minutes; Add 50 μL stop solution (0.1M H 2 SO 4 ) to each reaction well to terminate the enzymatic color development reaction.
读板：用酶标仪自带分析软件SoftMax pro 7.02软件，设定检测波长为450nm进行读数，对所得读数以非线性四参数方程曲线模型进行拟合，方程为其中A为吸光度下限，B代表曲线斜率，C为最大反应值一半对应的抗体浓度(EC50)，D为吸光度上限。Plate reading: Use the analysis software SoftMax pro 7.02 software that comes with the microplate reader, set the detection wavelength to 450nm for reading, and fit the obtained readings with a nonlinear four-parameter equation curve model. The equation is: Among them, A is the lower limit of absorbance, B represents the slope of the curve, C is the antibody concentration (EC 50 ) corresponding to half of the maximum response value, and D is the upper limit of absorbance.
数据处理：以空白对照OD450均值的2倍值对应的血清稀释度作为抗体滴度。然后再计算每组的几何平均滴度(GMT)。Data processing: The serum dilution corresponding to 2 times the mean value of OD 450 of the blank control was used as the antibody titer. The geometric mean titer (GMT) of each group was then calculated.
结果如图10所示，在单次免疫后的第14天(D15)，仅给予佐剂组(组2)几乎没有或仅有个别大鼠检测到极低的抗Spike蛋白IgG滴度，而双价疫苗组(组1)所有大鼠产生了针对WT-Spike蛋白、Delta-Spike蛋白和BA.5-Spike蛋白IgG抗体。结果表明，双价疫苗在80μg/只的剂量下可以引起大鼠极强的体液免疫反应。The results are shown in Figure 10. On the 14th day (D15) after a single immunization, almost no or very low anti-Spike protein IgG titers were detected in individual rats in the adjuvant-only group (Group 2), while All rats in the bivalent vaccine group (Group 1) produced IgG antibodies against WT-Spike protein, Delta-Spike protein and BA.5-Spike protein. The results showed that the bivalent vaccine could induce a strong humoral immune response in rats at a dose of 80 μg/animal.
实施例8：疫苗的动物保护效果Example 8: Animal Protection Effect of Vaccine
应用SARS-CoV-2感染地鼠肺炎模型，评价双价疫苗的动物保护效果。The SARS-CoV-2 infected hamster pneumonia model was used to evaluate the animal protection effect of the bivalent vaccine.
1.1实验方法1.1 Experimental methods
将雄性金黄地鼠(18周龄)分为6组，其中3组为攻毒组，3组为卫星组。攻毒组和卫星组设置相同，设置2个剂量组(1μg组和5μg组)和1个模型组。分别在第0天和第21天以肌肉注射方式注射疫苗。另外，攻毒组黄金地鼠在第42天用SARS-CoV-2 Omicron变异株BA.5以105TCID50/只的感染剂量滴鼻进行攻毒。试验设计见表7。Male golden hamsters (18 weeks old) were divided into 6 groups, of which 3 groups were challenge groups and 3 groups were satellite groups. The challenge group and satellite group have the same settings, with 2 dose groups (1 μg group and 5 μg group) and 1 model group. The vaccine was administered intramuscularly on days 0 and 21, respectively. In addition, the golden hamsters in the challenge group were challenged intranasally with the SARS-CoV-2 Omicron variant BA.5 at an infectious dose of 10 5 TCID 50 /mouse on the 42nd day. The experimental design is shown in Table 7.
表7试验设计

注：双价疫苗为质量比为1:1的融合蛋白D和融合蛋白2-1；佐剂为SWE佐剂Table 7 Experimental design

Note: The bivalent vaccine is fusion protein D and fusion protein 2-1 with a mass ratio of 1:1; the adjuvant is SWE adjuvant.
1.2观察指标 1.2 Observation indicators
攻毒组地鼠，攻毒后连续记录体重变化至实验结束。攻毒后5天处死全部地鼠，检测肺组织病毒载量和肺组织病理。The weight changes of the hamsters in the challenge group were recorded continuously until the end of the experiment. All hamsters were killed 5 days after the challenge, and the viral load and lung tissue pathology were detected.
卫星组免疫35天后，采集血液检测中和抗体。Thirty-five days after the satellite group was immunized, blood was collected to detect neutralizing antibodies.
1.3药物抗病毒效果1.3 Antiviral effect of drugs
1.3.1动物体重1.3.1 Animal weight
攻毒组模型组地鼠攻毒后出现体重下降，平均下降百分比最高为5.91％。攻毒组双价疫苗1μg组地鼠攻毒后体重持续下降，平均下降百分比最高为4.16％，与攻毒组模型组无显著差异(p＞0.05)。攻毒组双价疫苗5μg组地鼠攻毒后体重略有下降，攻毒后5天平均下降百分比为1.33％，显著低于攻毒组模型组(p＜0.01)。见表8。The hamsters in the model group of the challenge group showed weight loss after being challenged with the virus, with the highest average decrease percentage being 5.91%. The body weight of the hamsters in the 1 μg bivalent vaccine group continued to decrease after challenge, with the highest average decrease percentage being 4.16%, which was not significantly different from the challenge model group (p>0.05). The body weight of the hamsters in the 5 μg bivalent vaccine group decreased slightly after challenge. The average decrease percentage 5 days after challenge was 1.33%, which was significantly lower than the challenge model group (p<0.01). See Table 8.
表8各组地鼠攻毒后体重变化(平均值±SD，单位g)

注：与模型组比较，*p<0.05有显著性差异，**p<0.01有非常显著性差异Table 8 Body weight changes of hamsters in each group after challenge (mean ± SD, unit g)

Note: Compared with the model group, *p<0.05 has significant difference, **p<0.01 has very significant difference
1.3.2病毒载量1.3.2 Viral load
攻毒组模型组地鼠攻毒后5天肺组织平均病毒载量检测结果为105.48copies/mg。攻毒组双价疫苗1μg组地鼠攻毒后5天肺组织平均病毒载量为104.25copies/mg，显著低于攻毒组模型组(p＜0.01)，下降1.23lg值。攻毒组双价疫苗5μg组地鼠攻毒后5天肺组织平均病毒载量为103.59copies/mg，显著低于攻毒组模型组(p＜0.01)，下降1.89lg值。The average viral load in the lung tissue of hamsters in the model group of the challenge group was 10 5.48 copies/mg 5 days after challenge. The average viral load in the lung tissue of hamsters in the 1 μg bivalent vaccine group was 10 4.25 copies/mg 5 days after challenge, which was significantly lower than the challenge model group (p<0.01), with a decrease of 1.23 lg. The average viral load in the lung tissue of hamsters in the 5 μg bivalent vaccine group was 10 3.59 copies/mg 5 days after challenge, which was significantly lower than the challenge model group (p<0.01), with a decrease of 1.89 lg.
1.3.3中和抗体1.3.3 Neutralizing antibodies
第1次免疫后35天卫星组双价疫苗1μg组地鼠中和抗体GMT＝349.05。卫星组双价疫苗5μg组地鼠中和抗体GMT＝269.67。卫星组模型组地鼠中和抗体GMT≤10。35 days after the first immunization, the neutralizing antibody GMT of the hamsters in the 1 μg bivalent vaccine group of the satellite group was 349.05. The neutralizing antibody GMT of hamsters in the 5 μg bivalent vaccine group of the satellite group was 269.67. The neutralizing antibody GMT of hamsters in the satellite group model group was ≤10.
1.3.4病理结果1.3.4 Pathological results
攻毒组双价疫苗1μg组：4例(4/6)肺脏呈中度肺炎，2例(2/6)肺脏呈轻度肺炎。攻毒组双价疫苗5μg组：1例(1/6)肺脏呈中度肺炎，5例(5/6)肺脏呈轻度肺炎。攻毒组模型组：6例(6/6)肺脏呈中度肺炎。Challenge group: Bivalent vaccine 1 μg group: 4 cases (4/6) showed moderate pneumonia in the lungs, and 2 cases (2/6) showed mild pneumonia in the lungs. Challenge group Bivalent vaccine 5 μg group: 1 case (1/6) developed moderate pneumonia in the lungs, and 5 cases (5/6) developed mild pneumonia in the lungs. Challenge group and model group: 6 cases (6/6) showed moderate pneumonia in the lungs.
实施例9：融合蛋白D制剂Example 9: Fusion Protein D Preparation
配制表9所示的融合蛋白D制剂，融合蛋白D的蛋白浓度为0.5mg/mL，缓冲液为pH 7.0的20mM磷酸缓冲液。 Prepare the fusion protein D preparation shown in Table 9. The protein concentration of fusion protein D is 0.5mg/mL, and the buffer is 20mM phosphate buffer with pH 7.0.
表9融合蛋白D制剂组成
Table 9 Fusion protein D preparation composition
通过动态光散射(DLS)检测实验发现加入精氨酸、氯化镁、氯化钾或甘油的处方会产生粒径为10nm左右的蛋白颗粒，说明精氨酸、氯化镁、氯化钾或甘油会导致24mer解聚。Through dynamic light scattering (DLS) detection experiments, it was found that adding arginine, magnesium chloride, potassium chloride or glycerol to the recipe will produce protein particles with a particle size of about 10nm, indicating that arginine, magnesium chloride, potassium chloride or glycerol can cause 24mer Depolymerization.
对制剂A1、A4、A8、和A9在40℃高温下放置14d时进行SEC-HPLC检测。如表10所示，从SEC-HPLC检测结果来看，加入海藻糖、聚山梨酯80的处方A1在高温放置14d后，24mer含量最高，比较稳定。SEC-HPLC detection was performed on preparations A1, A4, A8, and A9 when they were placed at a high temperature of 40°C for 14 days. As shown in Table 10, from the SEC-HPLC test results, the recipe A1 containing trehalose and polysorbate 80 has the highest 24mer content after being left at high temperature for 14 days, which is relatively stable.
表10 SEC-HPLC检测结果
Table 10 SEC-HPLC detection results
改变融合蛋白D制剂辅料浓度和缓冲剂，制剂设计如表11所示，其中融合蛋白D的蛋白浓度为0.5mg/mL；20mM PB7.0为20mM磷酸缓冲液，包括10.26mM磷酸氢二钠和9.74mM磷酸二氢钠，pH为7.0；20mM His5.66为20mM组氨酸缓冲液，包括6mM组氨酸和14mM组氨酸盐酸盐，pH为5.66。Change the fusion protein D preparation excipient concentration and buffer. The preparation design is shown in Table 11. The protein concentration of fusion protein D is 0.5mg/mL; 20mM PB7.0 is 20mM phosphate buffer, including 10.26mM disodium hydrogen phosphate and 9.74mM sodium dihydrogen phosphate, pH 7.0; 20mM His5.66 is a 20mM histidine buffer, including 6mM histidine and 14mM histidine hydrochloride, pH 5.66.
表11融合蛋白D制剂组成

Table 11 Composition of Fusion Protein D Preparation

将制剂放置在40℃高温下考察6天，进行SEC-HPLC检测。结果如表12所示，可以看出融合蛋白D在20mM His5.66条件下比20mM PB7.0条件下产生的HMW聚集体要少，在20mM His5.66条件下更稳定。The preparation was placed at a high temperature of 40°C for 6 days and subjected to SEC-HPLC detection. The results are shown in Table 12. It can be seen that fusion protein D produces less HMW aggregates under the condition of 20mM His5.66 than under the condition of 20mM PB7.0, and is more stable under the condition of 20mM His5.66.
表12 SEC-HPLC检测结果
Table 12 SEC-HPLC detection results
进一步配制表13所示的融合蛋白D制剂，其中融合蛋白D的蛋白浓度为0.2mg/mL，20mM His5.66为20mM组氨酸缓冲液，pH为5.66；20mM His6.0为20mM组氨酸缓冲液，pH为6.0；20mM PB6.0为20mM磷酸缓冲液，pH为6.0；20mM PB7.0为20mM磷酸缓冲液，pH为7.0；20mM PB8.0为20mM磷酸缓冲液，pH为8.0；20mM CB6.0为20mM柠檬酸缓冲液，pH为6.0。Further prepare the fusion protein D preparation shown in Table 13, in which the protein concentration of fusion protein D is 0.2mg/mL, 20mM His5.66 is 20mM histidine buffer, and the pH is 5.66; 20mM His6.0 is 20mM histidine. Buffer, pH 6.0; 20mM PB6.0 is 20mM phosphate buffer, pH 6.0; 20mM PB7.0 is 20mM phosphate buffer, pH 7.0; 20mM PB8.0 is 20mM phosphate buffer, pH 8.0; 20mM CB6.0 is 20mM citrate buffer, pH 6.0.
表13融合蛋白D制剂组成
Table 13 Composition of Fusion Protein D Preparation
各制剂样品在放置50℃条件下，在第7、11、23、50、93天(D)取样进行SEC-HPLC检测；50℃-4D+4℃-89D表明制剂样品在50℃先放置了4天(4D)，然后取出样品在4℃放置了89天，然后取样进行SEC-HPLC检测。Each preparation sample was placed at 50°C, and samples were taken for SEC-HPLC detection on days 7, 11, 23, 50, and 93 (D); 50°C-4D+4°C-89D indicates that the preparation sample was placed at 50°C first. 4 days (4D), and then the samples were taken out and kept at 4°C for 89 days, and then samples were taken for SEC-HPLC detection.
SEC-HPLC检测结果如表14所示，从SEC结果可以看出制剂C7比制剂C8结果较好；pH5.66组氨酸缓冲液配制的样品和pH6.0组氨酸缓冲液配制的样品均具有较好 的稳定性，同时加入氯化钠、聚山梨酯80和海藻糖的制剂更稳定。The SEC-HPLC test results are shown in Table 14. It can be seen from the SEC results that preparation C7 has better results than preparation C8; the samples prepared with pH5.66 histidine buffer and the samples prepared with pH6.0 histidine buffer are both Have better The stability of the preparation with sodium chloride, polysorbate 80 and trehalose added at the same time is more stable.
表14样品的SEC-HPLC检测结果(％)
Table 14 SEC-HPLC test results of samples (%)
实施例10：融合蛋白G制剂Example 10: Fusion Protein G Preparation
配制如表15所示的融合蛋白G制剂，其中融合蛋白G的蛋白浓度为0.2mg/mL，缓冲液为20mM His缓冲液，pH为6.0。Prepare the fusion protein G preparation as shown in Table 15, in which the protein concentration of fusion protein G is 0.2mg/mL, the buffer is 20mM His buffer, and the pH is 6.0.
表15融合蛋白G制剂的组成
Table 15 Composition of Fusion Protein G Preparations
各制剂样品分别在4℃、50℃下放置4天(50℃-4D)和冻融(-65℃-25℃)3次(DR3)的条件下对进行稳定性考察。灯检结果显示制剂D2在50℃下放置4天后灯检呈乳光，弥散物；其他制剂的灯检均为正常。SEC-HPLC检测结果如表16所示，结果表明，加入羟丙基倍他环糊精的制剂D3在50℃高温条件下更稳定，SEC结果中24mer含量最高。冻融3次后，制剂D2优于制剂D1，表明加入聚山梨酯80、海藻糖和氯化钠的制剂对冻融的保护作用优于只含氯化钠的制剂。Each preparation sample was placed at 4°C and 50°C for 4 days (50°C-4D) and freeze-thawed (-65°C-25°C) three times (DR3) to examine the stability. The light inspection results showed that preparation D2 showed opalescence and dispersion after being placed at 50°C for 4 days; the light inspection of other preparations was normal. The SEC-HPLC test results are shown in Table 16. The results show that the preparation D3 with hydroxypropyl betacyclodextrin added is more stable under high temperature conditions of 50°C, and the 24mer content is the highest in the SEC results. After freezing and thawing three times, preparation D2 was better than preparation D1, indicating that the protective effect of preparations containing polysorbate 80, trehalose and sodium chloride on freezing and thawing was better than that of preparations containing only sodium chloride.
表16融合蛋白G制剂的SEC-HPLC检测结果
Table 16 SEC-HPLC detection results of fusion protein G preparations
进一步配制如表17所示的融合蛋白G制剂，其中融合蛋白G的蛋白浓度为0.2mg/mL，缓冲液为20mM His缓冲液，pH为6.0。Further prepare the fusion protein G preparation as shown in Table 17, in which the protein concentration of fusion protein G is 0.2mg/mL, the buffer is 20mM His buffer, and the pH is 6.0.
表17融合蛋白G制剂的组成
Table 17 Composition of Fusion Protein G Preparation
各制剂样品在25℃、40℃、50℃条件下放置7天时取样对进行灯检考察。灯检结 果如表18所示，氯化钠的加入对溶液的乳光现象有明显的改善，不加氯化钠会有乳光现象产生，聚山梨酯80的加入可以减少溶液的析出现象。Samples of each preparation were taken for light inspection after being placed at 25°C, 40°C, and 50°C for 7 days. Light inspection junction As shown in Table 18, the addition of sodium chloride can significantly improve the opalescence phenomenon of the solution. Without the addition of sodium chloride, opalescence will occur. The addition of polysorbate 80 can reduce the precipitation of the solution.
各制剂样品在50℃放置7、14、27、50天(D)、40℃下放置50天(D)、27℃下放置14、27、50天(D)的条件下取样进行SEC-HPLC检测，结果如表19和表20所示。氯化钠对融合蛋白G制剂的稳定性有明显影响，聚山梨酯80浓度越低稳定性越好；羟丙基倍他环糊精的浓度对稳定性的影响有限，含8mg/mL羟丙基倍他环糊精的制剂和16mg/mL羟丙基倍他环糊精的制剂的稳定性之间没有明显区别，但比加入海藻糖的制剂E16稳定性更好。Each preparation sample was sampled for SEC-HPLC at 50°C for 7, 14, 27, and 50 days (D), 40°C for 50 days (D), and 27°C for 14, 27, and 50 days (D). Test, the results are shown in Table 19 and Table 20. Sodium chloride has a significant impact on the stability of fusion protein G preparations. The lower the concentration of polysorbate 80, the better the stability. The concentration of hydroxypropyl betacyclodextrin has limited impact on the stability. Containing 8mg/mL hydroxypropyl betacyclodextrin There is no obvious difference in the stability between the formulation of hydroxypropyl betacyclodextrin and the formulation of 16mg/mL hydroxypropylbetacyclodextrin, but it is more stable than the formulation E16 with trehalose added.
表19高温条件下的SEC-HPLC检测结果

Table 19 SEC-HPLC detection results under high temperature conditions

表20 25℃条件下的SEC-HPLC检测结果
Table 20 SEC-HPLC detection results at 25°C
实施例11：双价疫苗(融合蛋白D+融合蛋白G)制剂Example 11: Bivalent vaccine (fusion protein D + fusion protein G) preparation
配制如表21所示的制剂，其中F1-F11为融合蛋白D制剂，F12-F22为融合蛋白G制剂，F23-F33为包含融合蛋白D和融合蛋白G(质量比1:1)的双价疫苗制剂，其中蛋白浓度为每种融合蛋白的浓度，例如，制剂F23包含0.2mg/mL融合蛋白D和0.2mg/mL融合蛋白G。佐剂为SWE佐剂(SEPPIC S.A.，货号80748J)，是一种无铝的佐剂，添加量为制剂总体积的3/10。其中，20mM His6.0为20mM组氨酸缓冲液，pH约为6.0；10mM His6.0为10mM组氨酸缓冲液，pH约为6.0；10mM His+5mM CB为pH为6.0的20mM组氨酸缓冲液和pH为6.6的10mM柠檬酸缓冲液体积比1:1混合得到，pH约为6.0。Prepare preparations as shown in Table 21, wherein F1-F11 are fusion protein D preparations, F12-F22 are fusion protein G preparations, and F23-F33 are bivalent preparations containing fusion protein D and fusion protein G (mass ratio 1:1). Vaccine formulations in which the protein concentration is the concentration of each fusion protein, for example, formulation F23 contains 0.2 mg/mL fusion protein D and 0.2 mg/mL fusion protein G. The adjuvant is SWE adjuvant (SEPPIC S.A., Cat. No. 80748J), which is an aluminum-free adjuvant. The added amount is 3/10 of the total volume of the preparation. Among them, 20mM His6.0 is a 20mM histidine buffer with a pH of about 6.0; 10mM His6.0 is a 10mM histidine buffer with a pH of about 6.0; 10mM His+5mM CB is 20mM histidine with a pH of 6.0. The buffer solution is mixed with 10mM citrate buffer solution with a pH of 6.6 at a volume ratio of 1:1, and the pH is approximately 6.0.
表21单价/双价疫苗制剂的组成


注：HP-CD为羟丙基倍他环糊精。Table 21 Composition of monovalent/bivalent vaccine preparations


Note: HP-CD is hydroxypropyl betacyclodextrin.
将制剂样品分别在4℃、25℃、40℃、50℃、光照(4500lx±500lx,25℃)下放置7天、冻融(-60℃～25℃)5次、室温下振荡(200rpm)48小时的条件下分别进行灯检检测。其中，制剂F1-F15、F17、F19-F21、F23-F32在上述条件下的灯检检测结果均为正常。制剂F16和F18在4℃下放置7天的条件下的灯检结果为轻微乳光，弥散物。制剂F22在冻融5次的条件下的灯检结果为轻微乳光，弥散物。制剂F33在4℃ 下放置7天的条件下的灯检结果为轻微乳光，弥散物；在25℃、50℃下放置7天的条件下的灯检结果为轻微弥散物。The preparation samples were placed at 4℃, 25℃, 40℃, 50℃, light (4500lx±500lx, 25℃) for 7 days, frozen and thawed (-60℃～25℃) 5 times, and shaken at room temperature (200rpm) Light inspections were conducted separately under 48-hour conditions. Among them, the light inspection results of preparations F1-F15, F17, F19-F21, and F23-F32 under the above conditions were all normal. The light examination results of preparations F16 and F18 when placed at 4°C for 7 days showed slight opalescence and dispersion. The light inspection results of preparation F22 under the condition of freezing and thawing 5 times showed slight opalescence and dispersion. Formulation F33 at 4°C The light inspection results under the condition of being placed under the condition of 7 days at 25℃ and 50℃ showed slight opalescence and dispersion.
在双价疫苗制剂中选择制剂F23、F27、F29和F31进行进一步的SEC-HPLC检测，结果如表22所示。冻融条件对未加佐剂的双价疫苗制剂的24mer影响比较大，而高温对制剂的影响较小，基本和4℃条件相近。制剂F23、F27、F29、F31在50℃放置7天和23天的条件下均具有良好的稳定性。制剂F27优于制剂F23。Among the bivalent vaccine preparations, preparations F23, F27, F29 and F31 were selected for further SEC-HPLC testing, and the results are shown in Table 22. Freezing and thawing conditions have a greater impact on the 24mer of the bivalent vaccine preparation without adjuvant, while high temperature has a smaller impact on the preparation, which is basically similar to the 4°C condition. Preparations F23, F27, F29, and F31 all have good stability when stored at 50°C for 7 days and 23 days. Formulation F27 was superior to formulation F23.
表22 SEC-HPLC检测结果

注：光照(4500lx±500lx,25℃)、冻融(-60℃～25℃)、振荡(室温，200rpm)。Table 22 SEC-HPLC detection results

Note: Illumination (4500lx±500lx, 25℃), freezing and thawing (-60℃～25℃), shaking (room temperature, 200rpm).
将融合蛋白G制剂F12、SWE佐剂用CB6.0缓冲液(10mM柠檬酸缓冲液，pH6.0)稀释50倍的样品、SWE佐剂用CB6.0缓冲液(10mM柠檬酸缓冲液，pH6.0)稀释100倍的样品、实施例11中的制剂F17在25℃条件下放置27天的样品分别命名为DLS-1、DLS-2、DLS-3、DLS-4，以考察佐剂对样品DLS的影响，DLS测定结果如表23所示，可见加入佐剂后的样品的DLS变化不大，融合蛋白G制剂F12的蛋白颗粒平均粒径为42.5nm。实施例11中的制剂F26在50℃条件下放置7天的DLS测定结果与制剂F17类似。Fusion protein G preparation F12, SWE adjuvant CB6.0 buffer (10mM citrate buffer, pH6.0) diluted 50-fold sample, SWE adjuvant CB6.0 buffer (10mM citrate buffer, pH6 .0) The samples diluted 100 times and the preparation F17 in Example 11 placed at 25°C for 27 days were named DLS-1, DLS-2, DLS-3, and DLS-4 respectively to examine the effect of the adjuvant on The influence of sample DLS. The DLS measurement results are shown in Table 23. It can be seen that the DLS of the sample after adding the adjuvant does not change much. The average particle size of the protein particles of the fusion protein G preparation F12 is 42.5nm. The DLS measurement results of formulation F26 in Example 11 after being placed at 50°C for 7 days were similar to those of formulation F17.
表23 DLS测定结果

Table 23 DLS measurement results

实施例12：融合蛋白2-1制剂Example 12: Fusion protein 2-1 preparation
配制如表24所示的制剂，其中融合蛋白2-1的蛋白浓度为0.45mg/mL，缓冲液为20mM His缓冲液，pH为6.0。配制融合蛋白2-1制剂样品，使用20mM His6.0缓冲液超滤换液，8倍体积换液后，将蛋白浓度稀释至0.9mg/mL；再与各个2×辅料母液，按照体积比1:1混合。使用无菌过滤器分别过滤样品。Prepare the preparation shown in Table 24, in which the protein concentration of fusion protein 2-1 is 0.45mg/mL, the buffer is 20mM His buffer, and the pH is 6.0. Prepare the fusion protein 2-1 preparation sample, use 20mM His6.0 buffer to ultrafiltrate and change the liquid. After 8 times the volume of the liquid change, dilute the protein concentration to 0.9mg/mL; then mix it with each 2× excipient stock solution according to the volume ratio of 1 :1 Mix. Filter samples individually using a sterile filter.
表24融合蛋白2-1制剂组成
Table 24 Fusion protein 2-1 preparation composition
将各制剂样品分装，在4℃条件下放置7天和14天时进行灯检。制剂H3在4℃条件下放置14天时产生白色异物，可能有蛋白析出，制剂组成包含氯化钠以及精氨酸时稳定性较差，制剂H4的灯检结果有析出，制剂H1和H2的灯检结果均为正常，说明制剂中不含聚山梨酯80时，会产生蛋白聚集析出的现象。Pack each preparation sample into aliquots and place them at 4°C for 7 and 14 days for light inspection. Preparation H3 produced white foreign matter when placed at 4°C for 14 days, and protein may precipitate. The stability of the preparation containing sodium chloride and arginine was poor. Preparation H4's light test results showed precipitation, and the light test results of preparations H1 and H2 The test results are all normal, indicating that when polysorbate 80 is not included in the preparation, protein aggregation and precipitation will occur.
在25℃条件下放置1月、40℃条件下放置7天和21天时分别进行SEC-HPLC检测，结果如表25所示。在40℃的条件下，制剂H1、H2都较为稳定。在25℃条件下放置1个月的制剂样品的SEC-HPLC的结果可以看出，制剂H1的24mer随放置时间的延长下降幅度更小。SEC-HPLC detection was performed after being placed at 25°C for 1 month, 40°C for 7 days, and 21 days. The results are shown in Table 25. Under the condition of 40℃, formulations H1 and H2 are relatively stable. The SEC-HPLC results of the preparation samples stored at 25°C for 1 month show that the 24mer of preparation H1 decreases less with the extension of storage time.
表25融合蛋白2-1制剂的SEC-HPLC检测结果
Table 25 SEC-HPLC detection results of fusion protein 2-1 preparation
实施例13：双价疫苗(融合蛋白D+融合蛋白2-1)制剂稳定性研究Example 13: Stability study of bivalent vaccine (fusion protein D + fusion protein 2-1) preparation
根据前述的研究，涉及融合蛋白D+融合蛋白2-1的双价疫苗制剂的辅料组成为：10mM组氨酸盐缓冲液+8.26mg/mL氯化钠+4mg/mL羟丙基倍他环糊精+0.2mg/mL聚山梨酯80+SWE佐剂(添加量为制剂总体积的3/10)。制剂规格和组成如表26所示，其中80μg/0.5mL的制剂中融合蛋白D和融合蛋白2-1的蛋白浓度均为0.08mg/mL；40μg/0.5mL的制剂中融合蛋白D和融合蛋白2-1的蛋白浓度均为0.04mg/mL。According to the aforementioned research, the auxiliary material composition of the bivalent vaccine preparation involving fusion protein D + fusion protein 2-1 is: 10mM histidine salt buffer + 8.26mg/mL sodium chloride + 4mg/mL hydroxypropyl betacycline paste Essence + 0.2 mg/mL polysorbate 80 + SWE adjuvant (the amount added is 3/10 of the total volume of the preparation). The specifications and composition of the preparation are shown in Table 26. The protein concentrations of fusion protein D and fusion protein 2-1 in the 80 μg/0.5 mL preparation are both 0.08 mg/mL; the protein concentrations of fusion protein D and fusion protein in the 40 μg/0.5 mL preparation are both The protein concentration of 2-1 is 0.04mg/mL.
表26双价疫苗制剂规格和组成
Table 26 Bivalent vaccine preparation specifications and composition
1.长期稳定性试验：1. Long-term stability test:
将制剂样品放置在5±3℃避光条件下进行长期稳定性试验，并定期取样检测，检测结果跟0时进行比较，结果如表27和28所示，样品41A和41B批在5±3℃条件下放置3个月各检项较0时均无明显变化趋势，产品关键质量属性保持稳定。各批次双价疫苗制剂样品的理化和生物性质各检项无明显变化，成品在长期条件下存放4个月产品质量属性稳定。Place the preparation samples under light-proof conditions at 5±3°C for long-term stability testing, and take samples for testing regularly. The test results are compared with those at 0:00. The results are shown in Tables 27 and 28. Samples 41A and 41B were batched at 5±3 When stored under ℃ conditions for 3 months, there is no obvious change trend in each inspection item compared with 0, and the key quality attributes of the product remain stable. There were no significant changes in the physical, chemical and biological properties of each batch of bivalent vaccine preparation samples. The quality attributes of the finished product were stable after being stored under long-term conditions for 4 months.
表27双价疫苗制剂成品(41A)批在5±3℃条件下稳定性数据
Table 27 Stability data of batch of finished bivalent vaccine preparation (41A) at 5±3°C
表28双价疫苗制剂成品(41B)批在5±3℃条件下稳定性数据

注：“NA”表示未安排检测。Table 28 Stability data of batch of finished bivalent vaccine preparation (41B) at 5±3°C

Note: "NA" means no testing is scheduled.
2.加速稳定性试验：2. Accelerated stability test:
将制剂样品放置于25±2℃和40±2℃避光条件下进行加速试验，并定期取样检测，检测结果跟0时进行比较。结果如表29-31所示，样品41A和41B批在25±2℃条件下放置3个月各检项较0时均无明显变化趋势，产品关键质量属性保持稳定，均在合格范围内；其他两个批次的结果也类似，均在合格范围内，产品质量稳定。样品41B在40±2℃条件下放置3个月各检项较0时均无明显变化趋势，产品关键质量属性保持稳定，均在合格范围内，41A批的结果也类似。The preparation samples were placed at 25±2℃ and 40±2℃ in the dark for accelerated testing, and samples were taken regularly for testing, and the test results were compared with those at time 0. The results are shown in Tables 29-31. Samples 41A and 41B were placed at 25±2℃ for 3 months, and there was no obvious change trend in each test item compared with time 0. The key quality attributes of the product remained stable and were all within the qualified range; the results of the other two batches were similar, all within the qualified range, and the product quality was stable. Sample 41B was placed at 40±2℃ for 3 months, and there was no obvious change trend in each test item compared with time 0. The key quality attributes of the product remained stable and were all within the qualified range. The results of batch 41A were also similar.
表29制剂成品(41A)批在25±2℃条件下稳定性数据

注：“NA”表示未安排检测。 Table 29 Stability data of batch of finished preparation (41A) at 25±2°C

Note: "NA" means no testing is scheduled.
表30制剂成品(41B)批在25±2℃条件下稳定性数据
Table 30 Stability data of batch of finished preparation (41B) at 25±2°C
表31制剂成品(41B)批在40±2℃条件下稳定性数据

注：“NA”表示未安排检测。Table 31 Stability data of batch of finished preparation (41B) at 40±2°C

Note: "NA" means no testing is scheduled.
3.影响因素试验：3. Test of influencing factors:
选择41A和41B批样品进行高温(50±2℃)、强光照(4500±500lux)以及振荡(200rpm，室温)等影响因素试验。Batch 41A and 41B samples were selected for testing on influencing factors such as high temperature (50±2°C), strong light (4500±500lux), and vibration (200rpm, room temperature).
双价疫苗制剂在高温条件下的试验结果如表32和33所示。双价疫苗制剂成品在50℃高温条件下放置21天，S蛋白抗体滴度值随时间增加缓慢下降，41A批在7天时超出接受标准，41B批在3天时超出接受标准；其他检项结果较0时无明显变化趋势。高温会使本品关键质量属性发生显著变化。 The test results of the bivalent vaccine preparation under high temperature conditions are shown in Tables 32 and 33. The finished product of the bivalent vaccine preparation was placed at a high temperature of 50°C for 21 days. The S protein antibody titer value slowly decreased with time. Batch 41A exceeded the acceptance standard at 7 days, and batch 41B exceeded the acceptance standard at 3 days; other inspection results were relatively There is no obvious trend at 0. High temperatures will cause significant changes in the critical quality attributes of this product.
表32制剂成品(41A)高温试验数据(50±2℃)

注：“NA”表示未安排检测。Table 32 High temperature test data of finished preparation (41A) (50±2℃)

Note: "NA" means no testing is scheduled.
表33制剂成品(41B)高温试验数据(50±2℃)

注：“NA”表示未安排检测。Table 33 High temperature test data of finished preparation (41B) (50±2℃)

Note: "NA" means no testing is scheduled.
双价疫苗制剂在强光照条件下的试验结果如表34和35所示。制剂成品在强光照条件下放置21天，同0时比较，pH、渗透压、角鲨烯含量和S蛋白抗体滴度值均在质量标准规定范围内，产品质量保持稳定；说明强光照条件下放置21天不会影响产品的关键质量属性。The test results of the bivalent vaccine preparation under strong light conditions are shown in Tables 34 and 35. The finished preparation was placed under strong light conditions for 21 days. Compared with the time at 0, the pH, osmotic pressure, squalene content and S protein antibody titer values were all within the scope of the quality standards, and the product quality remained stable; indicating that under strong light conditions Leaving it for 21 days will not affect the product's critical quality attributes.
表34制剂成品(41A)光照试验数据(4500±500lux)

注：“NA”表示未安排检测。Table 34 Light test data of finished preparation (41A) (4500±500lux)

Note: "NA" means no testing is scheduled.
表35制剂成品(41B)光照试验数据(4500±500lux)

注：“NA”表示未安排检测。Table 35 Light test data of finished preparation (41B) (4500±500lux)

Note: "NA" means no testing is scheduled.
双价疫苗制剂在振荡(200rpm，室温)条件下的试验结果如表36和37所示。制剂成品在室温条件下以转速为200rpm条件下振动48小时，同0时比较，各检项结果均无明显变化趋势。说明48小时内室温环境下运输过程200rpm振荡对双价疫苗制剂成品的质量不会产生影响。The test results of the bivalent vaccine preparation under shaking (200 rpm, room temperature) conditions are shown in Tables 36 and 37. The finished product was vibrated at room temperature at 200 rpm for 48 hours. Compared with time 0, there was no obvious change trend in the results of each inspection item. It shows that 200rpm vibration during transportation at room temperature within 48 hours will not affect the quality of the finished bivalent vaccine preparation.
表36制剂成品(41A)振荡试验数据(200rpm，室温)


注：“NA”表示未安排检测。Table 36 Oscillation test data of finished preparation (41A) (200rpm, room temperature)


Note: "NA" means no testing is scheduled.
表37制剂成品(41B)振荡试验数据(200rpm，室温)

注：“NA”表示未安排检测。 Table 37 Oscillation test data of finished preparation (41B) (200rpm, room temperature)

Note: "NA" means no testing is scheduled.

Claims (42)

  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；或者，所述冠状病毒为SARS-CoV-2 Omicron变异株；或者，所述冠状病毒为SARS-CoV-2 Omicron变异株BA.1、BA.2、BA.3、BA.4、BA.5、BQ.1、BQ.1.1、BF.7、XBB、XBB.1、XBB.1.5、XBB.1.5.1、XBB.1.9.1或XBB.1.16。A mutation-containing coronavirus Spike protein extracellular domain or a truncated fragment thereof, characterized in that the mutation includes: 1) mutating RRAR to GSAS; 2) the presence of a turning region between HR1 and CH to prevent fusion A mutation that forms a straight helix in the process; or the coronavirus is a SARS-CoV-2 Omicron variant; or the coronavirus is a SARS-CoV-2 Omicron variant BA.1, BA.2, or BA.3 , BA.4, BA.5, BQ.1, BQ.1.1, BF.7, XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.16.
  2. 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。The coronavirus Spike protein extracellular domain containing mutations or a truncated fragment thereof according to claim 1, characterized in that the mutation includes: 1) mutating RRAR to GSAS; 2) between HR1 and CH There is a double mutation K986P/V987P in the turning region.
  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。The mutation-containing extracellular domain of coronavirus Spike protein or a truncated fragment thereof according to claim 1 or 2, characterized in that the truncated fragment of the mutation-containing extracellular domain of coronavirus Spike protein is identical to Compared with the full-length extracellular domain of the coronavirus Spike protein, 5-80 amino acid residues are truncated at the C-terminus; or 20-76 amino acid residues are truncated at the C-terminus; or 70 amino acid residues are truncated at the C-terminus Amino acid residues.
  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、6-9、19-24、26-31任一项所示的氨基酸序列，或与SEQ ID NO:3、4、6-9、19-24、26-31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、6-9、19-24、26-31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。The coronavirus Spike protein extracellular domain containing mutations or a truncated fragment thereof according to any one of claims 1 to 3, characterized in that the coronavirus Spike protein extracellular domain containing mutations or a truncated fragment thereof The segment contains the amino acid sequence shown in any one of SEQ ID NO: 3, 4, 6-9, 19-24, 26-31, or is identical to SEQ ID NO: 3, 4, 6-9, 19-24, 26 An amino acid sequence with at least 80% or at least 90% identity compared to the amino acid sequence shown in any one of -31, or with any one of SEQ ID NO: 3, 4, 6-9, 19-24, 26-31 The amino acid sequences shown are compared to amino acid sequences with one or more conservative amino acid substitutions.
  5. 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1-4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和单体亚基蛋白；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端通过接头与单体亚基蛋白的N端连接。A fusion protein, characterized by comprising the mutation-containing coronavirus Spike protein extracellular domain or its truncated fragment and monomeric subunit protein as described in any one of claims 1-4, connected through a linker; or , the fusion protein is a C-terminal containing a mutated coronavirus Spike protein extracellular domain or a truncated fragment thereof connected to the N-terminal of a monomeric subunit protein through a linker.
  6. 如权利要求5所述的融合蛋白，其特征在于，所述接头为GS接头；或者，所述接头为(GmS)n，其中，每个m独立为1、2、3、4或5，n为1、2、3、4或5；或者，所述接头选自GS，GGS，GGGS，GGGGS，SGGGS，GGSS，(GGGGS)2，(GGGGS)3，或其任意组合。The fusion protein of claim 5, wherein the linker is a GS linker; or, the linker is ( GmS ) n , wherein each m is independently 1, 2, 3, 4 or 5 , n is 1, 2, 3, 4 or 5; alternatively, the linker is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS) 2 , (GGGGS) 3 , or any combination thereof.
  7. 如权利要求5或6所述的融合蛋白，其特征在于，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；或者，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；或者，所述单体铁蛋白亚基包含如SEQ ID NO:10所示的氨基酸序列，或与SEQ ID NO:10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:10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。The fusion protein of claim 5 or 6, wherein the monomeric subunit protein is a self-assembled monomeric subunit protein; or, the monomeric subunit protein is a monomeric ferritin subunit; Alternatively, the monomeric iron egg The white subunit is selected from bacterial ferritin, plant ferritin, phycoferritin, insect ferritin, fungal ferritin or mammalian ferritin; or, the monomeric ferritin subunit is a non-heme monomeric ferritin subunit of Helicobacter pylori base; alternatively, the monomeric ferritin subunit comprises the amino acid sequence shown in SEQ ID NO: 10, or has at least 80% or at least 90% identity compared to the amino acid sequence shown in SEQ ID NO: 10 Amino acid sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 10.
  8. 如权利要求5-7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；或者，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；或者，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。The fusion protein according to any one of claims 5 to 7, characterized in that the fusion protein also includes an N-terminal signal peptide; or, the N-terminal signal peptide is selected from CSP, mschito, MF-α, pho1, HBM, t-pA, and the signal peptide of IL-3; alternatively, the N-terminal signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2 or 5, or is identical to the amino acid sequence shown in SEQ ID NO: 2 or 5 An amino acid sequence that has at least 80% or at least 90% identity compared to an amino acid sequence, 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: 2 or 5.
  9. 如权利要求5-8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:12-17、32-43任一项所示的氨基酸序列，或与SEQ ID NO:12-17、32-43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:12-17、32-43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。The fusion protein according to any one of claims 5-8, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NO: 12-17, 32-43, or is the same as SEQ ID NO : An amino acid sequence that has at least 80% or at least 90% identity compared to the amino acid sequence shown in any one of 12-17 and 32-43, or an amino acid sequence shown in any one of SEQ ID NO: 12-17 and 32-43 The amino acid sequence is compared to the amino acid sequence having one or more conservative amino acid substitutions.
  10. 一种生物材料，为a biological material for
    (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1-4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或如权利要求5-9任一项所述的融合蛋白；或，(1) A polynucleotide, characterized in that the polynucleotide encodes the mutation-containing extracellular domain of the coronavirus Spike protein according to any one of claims 1-4 or a truncated fragment thereof segment or the fusion protein according to any one of claims 5-9; or,
    (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1-4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或如权利要求5-9任一项所述的融合蛋白的多聚核苷酸；或，(2) An expression vector, characterized in that the expression vector contains the extracellular domain encoding the mutation-containing coronavirus Spike protein according to any one of claims 1 to 4 or a truncated fragment thereof or as claimed in claim 1 The polynucleotide of the fusion protein according to any one of 5-9; or,
    (3)一种细胞，其特征在于，所述细胞包含编码如权利要求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、如权利要求5-9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1-4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、如权利要求5-9任一项所述的融合蛋白的多聚核苷酸的表达载体。(3) A cell, characterized in that the cell contains an extracellular domain encoding the mutation-containing coronavirus Spike protein according to any one of claims 1-4 or a truncated fragment thereof, as claimed in claim 5- 9 The polynucleotide of any one of the fusion proteins or the polynucleotide encoding the mutation-containing coronavirus Spike protein extracellular domain or its truncated fragment as claimed in any one of claims 1-4, as claimed in Expression vector for the polynucleotide of the fusion protein described in any one of 5-9.
  11. 一种包含权利要求5-9任一项所述的融合蛋白的Spike蛋白纳米颗粒。 A Spike protein nanoparticle comprising the fusion protein according to any one of claims 5-9.
  12. 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5-9任一项所述的融合蛋白和/或如权利要求11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。A coronavirus vaccine, characterized in that the coronavirus vaccine includes the fusion protein according to any one of claims 5-9 and/or the Spike protein nanoparticles according to claim 11; or, it also includes pharmaceutical acceptable carriers and/or adjuvants.
  13. 一种冠状病毒多价疫苗，其特征在于，所述冠状病毒多价疫苗包含第一融合蛋白和第二融合蛋白，所述第一融合蛋白为权利要求5-9任一项所述的融合蛋白，所述第二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；或者，还包括药学上可接受的载体和/或佐剂。A multivalent coronavirus vaccine, characterized in that the coronavirus multivalent vaccine includes a first fusion protein and a second fusion protein, and the first fusion protein is the fusion protein of any one of claims 5-9 , the second fusion protein includes the mutated SARS-CoV-2 Delta variant Spike protein extracellular domain or a truncated fragment thereof and a monomeric subunit protein connected through a linker; or, it also includes a pharmaceutically acceptable Carriers and/or adjuvants.
  14. 如权利要求13所述的冠状病毒多价疫苗，其特征在于，所述含突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域或其截短片段，其突变包含：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；或者，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；或者，所述含突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域的截短片段，其与SARS-CoV-2 Delta变异株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；或者，所述含突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域或其截短片段包含如SEQ ID NO:45-50任一项所示的氨基酸序列，或与SEQ ID NO:45-50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:45-50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。The coronavirus multivalent vaccine according to claim 13, wherein the mutation-containing SARS-CoV-2 Delta variant Spike protein extracellular domain or its truncated fragment, the mutation includes: 1) RRAR Mutation to GSAS; 2) There is a mutation in the turn region between HR1 and CH that prevents the formation of a straight helix during fusion; alternatively, the mutation includes: 1) Mutating RRAR to GSAS; 2) Between HR1 and CH There is a double mutation K986P/V987P in the turning region; or, the truncated fragment of the extracellular domain of the SARS-CoV-2 Delta variant Spike protein containing the mutation is the same as the full-length SARS-CoV-2 Delta variant Spike protein. Compared with the external domain, 5-80 amino acid residues are truncated at the C terminus; or 20-76 amino acid residues are truncated at the C terminus; or 70 amino acid residues are truncated at the C terminus; or, all The extracellular domain of the SARS-CoV-2 Delta variant Spike protein containing mutations or its truncated fragment contains the amino acid sequence shown in any one of SEQ ID NO:45-50, or is identical to SEQ ID NO:45-50 An amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence shown in any one of them, or one or more conserved amino acids compared to the amino acid sequence shown in any one of SEQ ID NO: 45-50 Substituted amino acid sequence.
  15. 如权利要求13或14所述的冠状病毒多价疫苗，其特征在于，所述第二融合蛋白是将含突变的SARS-CoV-2 Delta变异株Spike蛋白胞外结构域或其截短片段的C端通过接头与单体亚基蛋白的N端连接。The coronavirus multivalent vaccine according to claim 13 or 14, wherein the second fusion protein is a protein containing the extracellular domain of the mutated SARS-CoV-2 Delta variant Spike protein or a truncated fragment thereof. The C-terminus is connected to the N-terminus of the monomeric subunit protein through a linker.
  16. 如权利要求13-15任一项所述的冠状病毒多价疫苗，其特征在于，所述第二融合蛋白的接头为(GmS)n，其中，每个m独立为1、2、3、4或5，n为1、2、3、4或5；或者，所述接头为GS接头；或者，所述接头选自GS，GGS，GGGS，GGGGS，SGGGS，GGSS，(GGGGS)2，(GGGGS)3，或其任意组合。The coronavirus multivalent vaccine according to any one of claims 13 to 15, wherein the linker of the second fusion protein is (G m S) n , wherein each m is independently 1, 2, or 3 , 4 or 5, n is 1, 2, 3, 4 or 5; or, the linker is a GS linker; or, the linker is selected from GS, GGS, GGGS, GGGGS, SGGGS, GGSS, (GGGGS) 2 , (GGGGS) 3 , or any combination thereof.
  17. 如权利要求13-16任一项所述的冠状病毒多价疫苗，其特征在于，所述第二融合蛋白的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；或者，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；或者，所述单体铁蛋白亚基包含如SEQ ID NO:10所示的 氨基酸序列，或与SEQ ID NO:10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:10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。The coronavirus multivalent vaccine according to any one of claims 13 to 16, wherein the monomer subunit protein of the second fusion protein is a self-assembled monomer subunit protein; or, the monomer The subunit protein is a monomeric ferritin subunit; or, the monomeric ferritin subunit is selected from bacterial ferritin, plant ferritin, phycoferritin, insect ferritin, fungal ferritin or mammalian ferritin; or, The monomeric ferritin subunit is a non-heme monomeric ferritin subunit of Helicobacter pylori; alternatively, the monomeric ferritin subunit comprises as shown in SEQ ID NO: 10 Amino acid sequence, or an amino acid sequence having at least 80% or at least 90% identity compared to the amino acid sequence shown in SEQ ID NO: 10, or having one or more amino acid sequences compared to the amino acid sequence shown in SEQ ID NO: 10 Amino acid sequence with conservative amino acid substitutions.
  18. 如权利要求13所述的冠状病毒多价疫苗，其特征在于，所述第二融合蛋白包含如SEQ ID NO:51-56任一项所示的氨基酸序列，或与SEQ ID NO:51-56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:51-56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。The coronavirus multivalent vaccine as described in claim 13 is characterized in that the second fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 51-56, or an amino acid sequence having at least 80% or at least 90% identity with the amino acid sequence shown in any one of SEQ ID NOs: 51-56, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 51-56.
  19. 如权利要求13所述的冠状病毒多价疫苗，其特征在于，所述第一融合蛋白包含如SEQ ID NO:14、15或17所示的序列，第二融合蛋白包含如SEQ ID NO:53、54或56所示的序列。The coronavirus multivalent vaccine of claim 13, wherein the first fusion protein includes the sequence shown in SEQ ID NO: 14, 15 or 17, and the second fusion protein includes the sequence shown in SEQ ID NO: 53 , 54 or 56.
  20. 如权利要求13所述的冠状病毒多价疫苗，其特征在于，所述第一融合蛋白包含如SEQ ID NO:17所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列。The coronavirus multivalent vaccine according to claim 13, wherein the first fusion protein includes the sequence shown in SEQ ID NO:17, and the second fusion protein includes the sequence shown in SEQ ID NO:56 .
  21. 如权利要求13-20任一项所述的冠状病毒多价疫苗，其特征在于，所述第一融合蛋白和第二融合蛋白的质量比为(1-5):(1-5)，或质量比为(1-3):(1-3)，或质量比为(1-2):(1-2)，或质量比为1:(1-2)，或质量比为(1-2):1，或质量比为1:1。The coronavirus multivalent vaccine according to any one of claims 13 to 20, wherein the mass ratio of the first fusion protein and the second fusion protein is (1-5): (1-5), or The mass ratio is (1-3):(1-3), or the mass ratio is (1-2):(1-2), or the mass ratio is 1:(1-2), or the mass ratio is (1- 2):1, or the mass ratio is 1:1.
  22. 权利要求5-9任一项所述的融合蛋白或权利要求11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SARS-CoV-2原始株或其变异株感染；或者，所述冠状病毒感染为SARS-CoV-2原始株、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；或者，所述冠状病毒感染为SARS-CoV-2 Omicron变异株BA.1、BA.2、BA.3、BA.4、BA.5、BQ.1、BQ.1.1、BF.7、XBB、XBB.1、XBB.1.5、XBB.1.5.1、XBB.1.9.1或XBB.1.16感染。Application of the fusion protein according to any one of claims 5 to 9 or the Spike protein nanoparticles according to claim 11 in the preparation of vaccines for preventing or treating coronavirus infection; or, the coronavirus infection is SARS-CoV- 2. SARS-CoV or MERS-CoV infection; or, the coronavirus infection is an infection by the original strain of SARS-CoV-2 or its mutant strain; or, the coronavirus infection is an infection by the original strain of SARS-CoV-2, SARS- CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant strain, SARS-CoV-2 Lambda variant strain or SARS-CoV-2 Omicron variant strain; or, the coronavirus infection is SARS-CoV-2 Omicron variant strain BA.1, BA.2, BA.3, BA .4, BA.5, BQ.1, BQ.1.1, BF.7, XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.16 infection.
  23. 一种冠状病毒疫苗制剂，其包含融合蛋白，还包含缓冲剂、稳定剂、碱金属或碱金属盐、表面活性剂中的一种或多种；A coronavirus vaccine preparation, which contains a fusion protein and one or more of a buffer, a stabilizer, an alkali metal or an alkali metal salt, and a surfactant;
    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，其中所述突变包含：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，所述冠状病毒为SARS-CoV-2 原始株、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；或者，所述冠状病毒为SARS-CoV-2 Omicron变异株BA.1、BA.2、BA.3、BA.4、BA.5、BQ.1、BQ.1.1、BF.7、XBB、XBB.1、XBB.1.5、XBB.1.5.1、XBB.1.9.1或XBB.1.16。The fusion protein includes the mutation-containing coronavirus Spike protein extracellular domain or its truncated fragment and the monomeric ferritin subunit connected through a linker, wherein the mutation includes: 1) mutating RRAR to GSAS; 2) in There is a double mutation K986P/V987P in the turning region between HR1 and CH, and the coronavirus is SARS-CoV-2 Original strain, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 Delta variant, SARS-CoV-2 Kappa variant, SARS- CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant; alternatively, the coronavirus is SARS-CoV-2 Omicron variant BA.1, BA.2, BA. 3. BA.4, BA.5, BQ.1, BQ.1.1, BF.7, XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.16.
  24. 如权利要求23所述的冠状病毒疫苗制剂，所述融合蛋白浓度为0.05-5mg/mL；或者，所述融合蛋白浓度为0.05-1mg/mL。The coronavirus vaccine preparation of claim 23, wherein the fusion protein concentration is 0.05-5 mg/mL; or, the fusion protein concentration is 0.05-1 mg/mL.
  25. 如权利要求23或24所述的冠状病毒疫苗制剂，其包含0.05-1mg/mL融合蛋白、10-30mM缓冲剂、稳定剂、碱金属或碱金属盐和表面活性剂。The coronavirus vaccine preparation according to claim 23 or 24, which contains 0.05-1mg/mL fusion protein, 10-30mM buffer, stabilizer, alkali metal or alkali metal salt and surfactant.
  26. 如权利要求23-25任一项所述的冠状病毒疫苗制剂，所述缓冲剂选自：组氨酸缓冲剂、柠檬酸缓冲剂、磷酸缓冲剂或其组合；或者，所述缓冲剂为组氨酸缓冲剂；或者，所述缓冲剂为组氨酸和柠檬酸缓冲剂的组合。The coronavirus vaccine preparation according to any one of claims 23 to 25, the buffer is selected from: histidine buffer, citric acid buffer, phosphate buffer or a combination thereof; or, the buffer is a group consisting of: Acid buffer; alternatively, the buffer is a combination of histidine and citric acid buffers.
  27. 如权利要求23-26任一项所述的冠状病毒疫苗制剂，所述稳定剂选自羟丙基倍他环糊精、蔗糖和海藻糖或其组合。The coronavirus vaccine formulation according to any one of claims 23 to 26, wherein the stabilizer is selected from hydroxypropyl-beta-cyclodextrin, sucrose and trehalose or a combination thereof.
  28. 如权利要求23-27任一项所述的冠状病毒疫苗制剂，所述碱金属或碱金属盐选自氯化镁、氯化钾、氯化钠或其组合；或者，所述碱金属或碱金属盐为氯化钠。The coronavirus vaccine preparation according to any one of claims 23 to 27, the alkali metal or alkali metal salt is selected from magnesium chloride, potassium chloride, sodium chloride or a combination thereof; or, the alkali metal or alkali metal salt is sodium chloride.
  29. 如权利要求23-28任一项所述的冠状病毒疫苗制剂，所述表面活性剂选自泊洛沙姆、聚山梨酯或其组合；或者，所述表面活性剂选自泊洛沙姆188、聚山梨酯20、聚山梨酯80或其组合。The coronavirus vaccine preparation according to any one of claims 23 to 28, the surfactant is selected from poloxamer, polysorbate or a combination thereof; or, the surfactant is selected from poloxamer 188 , polysorbate 20, polysorbate 80 or combinations thereof.
  30. 如权利要求23-29任一项所述的冠状病毒疫苗制剂，还包含佐剂；或者，所述佐剂为铝佐剂、SWE佐剂或MF59佐剂；或者，所述佐剂的添加量为所述冠状病毒疫苗制剂总体积的1/10-5/10，或者，所述佐剂的添加量为所述冠状病毒疫苗制剂总体积的3/10。The coronavirus vaccine preparation according to any one of claims 23 to 29, further comprising an adjuvant; or, the adjuvant is aluminum adjuvant, SWE adjuvant or MF59 adjuvant; or, the added amount of the adjuvant It is 1/10-5/10 of the total volume of the coronavirus vaccine preparation, or the added amount of the adjuvant is 3/10 of the total volume of the coronavirus vaccine preparation.
  31. 如权利要求23-30任一项所述的冠状病毒疫苗制剂，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。The coronavirus vaccine preparation according to any one of claims 23-30, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, and 61.
  32. 一种冠状病毒疫苗制剂，其包含A coronavirus vaccine preparation comprising
    (1)0.05-1mg/mL融合蛋白、5-25mM磷酸缓冲剂、10-170mg/mL海藻糖二水 合物、0-22mg/mL碱金属或碱金属盐、0.01-2mg/mL聚山梨酯80，pH约为6.0-8.0；或者，(1)0.05-1mg/mL fusion protein, 5-25mM phosphate buffer, 10-170mg/mL trehalose dihydrate Compounds, 0-22 mg/mL alkali metal or alkali metal salts, 0.01-2 mg/mL polysorbate 80, pH approximately 6.0-8.0; or,
    (2)0.05-1mg/mL融合蛋白、5-25mM组氨酸缓冲剂、14-450mM海藻糖、0.01-2mg/mL聚山梨酯80，pH为5.0-7.0；或者，(2) 0.05-1mg/mL fusion protein, 5-25mM histidine buffer, 14-450mM trehalose, 0.01-2mg/mL polysorbate 80, pH 5.0-7.0; or,
    (3)0.05-1mg/mL融合蛋白、15-25mM组氨酸缓冲剂、150-270mM海藻糖、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH为5.0-7.0；或者，(3)0.05-1mg/mL fusion protein, 15-25mM histidine buffer, 150-270mM trehalose, 0.01-2mg/mL polysorbate 80, 8-17mg/mL sodium chloride, pH 5.0-7.0 ;or,
    (4)0.05-1mg/mL融合蛋白、15-25mM组氨酸缓冲剂、0.5-80mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-33mg/mL氯化钠，pH为5.0-7.0；或者，(4)0.05-1mg/mL fusion protein, 15-25mM histidine buffer, 0.5-80mg/mL hydroxypropyl betacyclodextrin, 0.01-2mg/mL polysorbate 80, 8-33mg/mL chloride sodium chloride, pH 5.0-7.0; or,
    (5)0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、约222mM海藻糖、约0.4mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH为5.6-6.3；或者，(5) 0.05-1mg/mL fusion protein, approximately 20mM histidine buffer, approximately 222mM trehalose, approximately 0.4mg/mL polysorbate 80, 8-17mg/mL sodium chloride, pH 5.6-6.3; or ,
    (6)0.05-1mg/mL融合蛋白、约20mM组氨酸缓冲剂、0.5-50mg/mL羟丙基倍他环糊精、约0.4mg/mL聚山梨酯80、8-33mg/mL氯化钠，pH为5.6-6.3；或者，(6)0.05-1mg/mL fusion protein, approximately 20mM histidine buffer, 0.5-50mg/mL hydroxypropyl betacyclodextrin, approximately 0.4mg/mL polysorbate 80, 8-33mg/mL chloride Sodium, pH 5.6-6.3; or,
    (7)0.05-1mg/mL融合蛋白、5-15mM组氨酸缓冲剂和2-10mM柠檬酸缓冲剂、0.5-50mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH约为5.6-6.3；或者(7)0.05-1mg/mL fusion protein, 5-15mM histidine buffer and 2-10mM citric acid buffer, 0.5-50mg/mL hydroxypropyl betacyclodextrin, 0.01-2mg/mL polysorbate 80. 8-17mg/mL sodium chloride, pH approximately 5.6-6.3; or
    (8)0.05-1mg/mL融合蛋白、10mM组氨酸缓冲剂和5mM柠檬酸缓冲剂、0.5-50mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH约为5.6-6.3；或者，(8)0.05-1mg/mL fusion protein, 10mM histidine buffer and 5mM citrate buffer, 0.5-50mg/mL hydroxypropyl betacyclodextrin, 0.01-2mg/mL polysorbate 80, 8- 17mg/mL sodium chloride, pH approximately 5.6-6.3; or,
    (9)0.05-1mg/mL融合蛋白、5-25mM组氨酸缓冲剂、60-100mg/mL蔗糖、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH约为5.6-6.3；或者，(9)0.05-1mg/mL fusion protein, 5-25mM histidine buffer, 60-100mg/mL sucrose, 0.01-2mg/mL polysorbate 80, 8-17mg/mL sodium chloride, pH approximately 5.6 -6.3; or,
    (10)0.05-1mg/mL融合蛋白、20mM组氨酸缓冲剂、80mg/mL蔗糖、0.4mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH约为5.6-6.3；(10) 0.05-1mg/mL fusion protein, 20mM histidine buffer, 80mg/mL sucrose, 0.4mg/mL polysorbate 80, 8-17mg/mL sodium chloride, pH approximately 5.6-6.3;
    其中，所述融合蛋白包含如SEQ ID NO:12-17、32-43、51-56、61任一项所示的氨基酸序列。Wherein, the fusion protein includes the amino acid sequence shown in any one of SEQ ID NO: 12-17, 32-43, 51-56, and 61.
  33. 一种冠状病毒多价疫苗制剂，其包含：第一融合蛋白和第二融合蛋白，还包含缓冲剂、稳定剂、碱金属或碱金属盐、表面活性剂中的一种或多种，所述第一融合 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，所述第二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。A coronavirus multivalent vaccine preparation, which contains: a first fusion protein and a second fusion protein, and also contains one or more of a buffer, a stabilizer, an alkali metal or an alkali metal salt, and a surfactant, said first fusion The protein includes the extracellular domain of the SARS-CoV-2 Omicron variant Spike protein containing mutations or its truncated fragment and the monomeric subunit protein connected through a linker, and the second fusion protein includes the SARS containing mutations connected through a linker. -CoV-2 Delta variant Spike protein extracellular domain or its truncated fragments and monomeric subunit proteins.
  34. 如权利要求33所述的冠状病毒多价疫苗制剂，所述第一融合蛋白和第二融合蛋白的质量比为(1-3):(1-3)，或质量比为(1-2):(1-2)，或质量比为1:(1-2)，或质量比为(1-2):1，或为1:1；或者，所述冠状病毒多价疫苗制剂包含0.01-2mg/mL第一融合蛋白和0.01-2mg/mL第二融合蛋白。The coronavirus multivalent vaccine preparation according to claim 33, the mass ratio of the first fusion protein and the second fusion protein is (1-3): (1-3), or the mass ratio is (1-2) :(1-2), or the mass ratio is 1:(1-2), or the mass ratio is (1-2):1, or 1:1; or the coronavirus multivalent vaccine preparation contains 0.01- 2mg/mL first fusion protein and 0.01-2mg/mL second fusion protein.
  35. 如权利要求33或34所述的冠状病毒多价疫苗制剂，所述缓冲剂选自：组氨酸缓冲剂、柠檬酸缓冲剂或其组合；或者，所述缓冲剂为组氨酸缓冲剂、组氨酸和柠檬酸缓冲剂的组合。In the coronavirus multivalent vaccine formulation as described in claim 33 or 34, the buffer is selected from: a histidine buffer, a citric acid buffer or a combination thereof; or, the buffer is a combination of a histidine buffer, a histidine and a citric acid buffer.
  36. 如权利要求33-35任一项所述的冠状病毒多价疫苗制剂，所述稳定剂选自羟丙基倍他环糊精、蔗糖和海藻糖或其组合。The coronavirus multivalent vaccine preparation according to any one of claims 33 to 35, wherein the stabilizer is selected from hydroxypropyl betacyclodextrin, sucrose and trehalose or a combination thereof.
  37. 如权利要求33-36任一项所述的冠状病毒多价疫苗制剂，所述碱金属或碱金属盐选自氯化镁、氯化钾、氯化钠或其组合；或者所述碱金属或碱金属盐为氯化钠。The coronavirus multivalent vaccine preparation according to any one of claims 33 to 36, the alkali metal or alkali metal salt is selected from magnesium chloride, potassium chloride, sodium chloride or a combination thereof; or the alkali metal or alkali metal salt Salt is sodium chloride.
  38. 如权利要求33-37任一项所述的冠状病毒多价疫苗制剂，所述表面活性剂选自泊洛沙姆、聚山梨酯或其组合；或者，所述表面活性剂选自泊洛沙姆188、聚山梨酯20、聚山梨酯80或其组合。The coronavirus multivalent vaccine preparation according to any one of claims 33 to 37, the surfactant is selected from poloxamer, polysorbate or a combination thereof; or, the surfactant is selected from poloxamer Polysorbate 188, polysorbate 20, polysorbate 80 or combinations thereof.
  39. 如权利要求33-38任一项所述的冠状病毒多价疫苗制剂，其包含The coronavirus multivalent vaccine preparation according to any one of claims 33-38, comprising
    (1)0.01-2mg/mL第一融合蛋白和0.01-2mg/mL第二融合蛋白，还包含5-15mM组氨酸缓冲剂和2-10mM柠檬酸缓冲剂、0.5-80mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH为5.0-7.0，或者，(1) 0.01-2mg/mL first fusion protein and 0.01-2mg/mL second fusion protein, also containing 5-15mM histidine buffer, 2-10mM citrate buffer, 0.5-80mg/mL hydroxypropyl Betacyclodextrin, 0.01-2mg/mL polysorbate 80, 8-17mg/mL sodium chloride, pH 5.0-7.0, or,
    (2)0.01-0.2mg/mL第一融合蛋白和0.01-0.2mg/mL第二融合蛋白，还包含约10mM组氨酸缓冲剂和约5mM柠檬酸缓冲剂、0.5-20mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH为5.6-6.3；或者，(2) 0.01-0.2mg/mL first fusion protein and 0.01-0.2mg/mL second fusion protein, also containing about 10mM histidine buffer, about 5mM citrate buffer, 0.5-20mg/mL hydroxypropyl cyclodextrin, 0.01-2 mg/mL polysorbate 80, 8-17 mg/mL sodium chloride, pH 5.6-6.3; or
    (3)0.01-0.2mg/mL第一融合蛋白和0.01-0.2mg/mL第二融合蛋白，还包含约10mM组氨酸缓冲剂和约5mM柠檬酸缓冲剂、约4mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、约8.26mg/mL氯化钠，pH为5.6-6.3；或者， (3) 0.01-0.2mg/mL first fusion protein and 0.01-0.2mg/mL second fusion protein, also containing about 10mM histidine buffer, about 5mM citrate buffer, and about 4mg/mL hydroxypropyl beta Cyclodextrin, 0.01-2mg/mL polysorbate 80, approximately 8.26mg/mL sodium chloride, pH 5.6-6.3; or,
    (4)0.01-2mg/mL第一融合蛋白和0.01-2mg/mL第二融合蛋白，还包含5-15mM组氨酸缓冲剂、0.5-80mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH为5.0-7.0；或者，(4) 0.01-2mg/mL first fusion protein and 0.01-2mg/mL second fusion protein, also containing 5-15mM histidine buffer, 0.5-80mg/mL hydroxypropyl betacyclodextrin, 0.01- 2mg/mL polysorbate 80, 8-17mg/mL sodium chloride, pH 5.0-7.0; or,
    (5)0.01-0.2mg/mL第一融合蛋白和0.01-0.2mg/mL第二融合蛋白，还包含约10mM组氨酸缓冲剂、0.5-20mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、8-17mg/mL氯化钠，pH为5.6-6.3；或者，(5) 0.01-0.2mg/mL first fusion protein and 0.01-0.2mg/mL second fusion protein, also containing about 10mM histidine buffer, 0.5-20mg/mL hydroxypropyl betacyclodextrin, 0.01 -2mg/mL polysorbate 80, 8-17mg/mL sodium chloride, pH 5.6-6.3; or,
    (6)0.01-0.2mg/mL第一融合蛋白和0.01-0.2mg/mL第二融合蛋白，还包含约10mM组氨酸缓冲剂、约4mg/mL羟丙基倍他环糊精、0.01-2mg/mL聚山梨酯80、约8.26mg/mL氯化钠，pH为5.6-6.3；；(6) 0.01-0.2mg/mL first fusion protein and 0.01-0.2mg/mL second fusion protein, also containing about 10mM histidine buffer, about 4mg/mL hydroxypropyl betacyclodextrin, 0.01- 2mg/mL polysorbate 80, approximately 8.26mg/mL sodium chloride, pH 5.6-6.3;;
    其中，所述第一融合蛋白包含如SEQ ID NO:17或61所示的序列，第二融合蛋白包含如SEQ ID NO:56所示的序列。Wherein, the first fusion protein includes the sequence shown in SEQ ID NO:17 or 61, and the second fusion protein includes the sequence shown in SEQ ID NO:56.
  40. 如权利要求33-39任一项所述的冠状病毒多价疫苗制剂，还包含佐剂；或者，所述佐剂为铝佐剂、SWE佐剂或MF59佐剂；或者，所述佐剂的添加量为所述冠状病毒多价疫苗制剂总体积的1/10-5/10，或者，所述佐剂的添加量约为所述冠状病毒多价疫苗制剂总体积的3/10。The coronavirus multivalent vaccine preparation according to any one of claims 33 to 39, further comprising an adjuvant; or, the adjuvant is aluminum adjuvant, SWE adjuvant or MF59 adjuvant; or, the adjuvant is The added amount is 1/10-5/10 of the total volume of the coronavirus multivalent vaccine preparation, or the added amount of the adjuvant is about 3/10 of the total volume of the coronavirus multivalent vaccine preparation.
  41. 一种冠状病毒多价疫苗，其包含A multivalent coronavirus vaccine containing
    约0.08mg/mL第一融合蛋白和约0.08mg/mL第二融合蛋白，还包含约10mM组氨酸缓冲剂、约4mg/mL羟丙基倍他环糊精、约0.2mg/mL聚山梨酯80、约8.26mg/mL氯化钠，和佐剂，pH为5.6-6.3；或者，About 0.08mg/mL first fusion protein and about 0.08mg/mL second fusion protein, also containing about 10mM histidine buffer, about 4mg/mL hydroxypropyl betacyclodextrin, about 0.2mg/mL polysorbate 80. About 8.26 mg/mL sodium chloride, and adjuvant, pH 5.6-6.3; or,
    约0.04mg/mL第一融合蛋白和约0.04mg/mL第二融合蛋白，还包含约10mM组氨酸缓冲剂、约4mg/mL羟丙基倍他环糊精、约0.2mg/mL聚山梨酯80、约8.26mg/mL氯化钠，和佐剂，pH为5.6-6.3，About 0.04mg/mL first fusion protein and about 0.04mg/mL second fusion protein, also containing about 10mM histidine buffer, about 4mg/mL hydroxypropyl betacyclodextrin, about 0.2mg/mL polysorbate 80. About 8.26mg/mL sodium chloride, and adjuvant, pH 5.6-6.3,
    其中，所述第一融合蛋白包含如SEQ ID NO:17所示的序列，第二融合蛋白包含如SEQ ID NO:56或61所示的序列；Wherein, the first fusion protein includes the sequence shown in SEQ ID NO:17, and the second fusion protein includes the sequence shown in SEQ ID NO:56 or 61;
    所述佐剂为铝佐剂、SWE佐剂或MF59佐剂；或者，所述佐剂的添加量为所述冠状病毒多价疫苗总体积的1/10-5/10，或者，所述佐剂的添加量为所述冠状病毒多价疫苗总体积的3/10。 The adjuvant is aluminum adjuvant, SWE adjuvant or MF59 adjuvant; or, the added amount of the adjuvant is 1/10-5/10 of the total volume of the coronavirus multivalent vaccine, or the adjuvant The amount added is 3/10 of the total volume of the coronavirus multivalent vaccine.
  42. 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5-9任一项所述的融合蛋白或权利要求11所述的Spike蛋白纳米颗粒或权利要求12所述的冠状病毒疫苗或权利要求13-21任一项所述的冠状病毒多价疫苗或权利要求23-32任一项所述的冠状病毒疫苗制剂或权利要求33-41任一项所述的冠状病毒多价疫苗制剂；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SARS-CoV-2原始株或其变异株感染；或者，所述冠状病毒感染为SARS-CoV-2原始株、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；或者，所述冠状病毒感染为SARS-CoV-2Omicron变异株BA.1、BA.2、BA.3、BA.4、BA.5、BQ.1、BQ.1.1、BF.7、XBB、XBB.1、XBB.1.5、XBB.1.5.1、XBB.1.9.1或XBB.1.16感染。 A method for preventing or treating coronavirus infection, characterized in that it comprises administering to a patient in need thereof an effective amount of the fusion protein of any one of claims 5 to 9, the Spike protein nanoparticle of claim 11, the coronavirus vaccine of claim 12, the coronavirus multivalent vaccine of any one of claims 13 to 21, the coronavirus vaccine preparation of any one of claims 23 to 32, or the coronavirus multivalent vaccine preparation of any one of claims 33 to 41; or, the coronavirus infection is SARS-CoV-2, SARS-CoV or MERS-CoV infection; or, the coronavirus infection is SARS-CoV-2 original strain or its variant infection; or, the coronavirus infection is SARS-CoV-2 original strain, SARS-CoV-2 Alpha variant, SARS-CoV-2 Beta variant, SARS-CoV-2 Gamma variant, SARS-CoV-2 Delta variant, SARS-CoV-2 Kappa variant, SARS-CoV-2 Epsilon variant, SARS-CoV-2 Lambda variant or SARS-CoV-2 Omicron variant infection; or, the coronavirus infection is SARS-CoV-2 Omicron variant BA.1, BA.2, BA.3, BA.4, BA.5, BQ.1, BQ.1.1, BF.7, XBB, XBB.1, XBB.1.5, XBB.1.5.1, XBB.1.9.1 or XBB.1.16 infection.
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