WO2024082681A1 - 截短的呼吸道合胞病毒f蛋白及其用途 - Google Patents

截短的呼吸道合胞病毒f蛋白及其用途 Download PDF

Info

Publication number
WO2024082681A1
WO2024082681A1 PCT/CN2023/102342 CN2023102342W WO2024082681A1 WO 2024082681 A1 WO2024082681 A1 WO 2024082681A1 CN 2023102342 W CN2023102342 W CN 2023102342W WO 2024082681 A1 WO2024082681 A1 WO 2024082681A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
seq
protein
amino acid
sequence
Prior art date
Application number
PCT/CN2023/102342
Other languages
English (en)
French (fr)
Inventor
郑子峥
林敏�
尹一凡
汪晨
赵小蒙
陈莉
张军
夏宁邵
Original Assignee
厦门大学
厦门万泰沧海生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 厦门大学, 厦门万泰沧海生物技术有限公司 filed Critical 厦门大学
Publication of WO2024082681A1 publication Critical patent/WO2024082681A1/zh

Links

Classifications

    • 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
    • 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
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18533—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
    • G01N2333/135—Respiratory syncytial virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to the field of biomedicine, and specifically, the present application relates to a fusion protein, and a nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein.
  • the present application also relates to a vaccine comprising the fusion protein or the nucleic acid molecule.
  • the present invention also relates to a method for preventing and/or treating RSV infection or a disease and/or symptom caused by RSV infection using these fusion proteins, nucleic acid molecules and vaccines.
  • Respiratory syncytial virus is one of the most important pathogens causing lower respiratory tract infections in infants and young children worldwide. According to statistics, 33 million children under the age of 5 are infected with RSV each year, causing nearly 160,000 deaths. RSV infection cannot provide sustained immunity and can cause repeated infections. More than 99% of children under the age of 2 have been infected with RSV at least once, and 70% of them have been hospitalized for infection and complicated by bronchiolitis, pneumonia and asthma. In addition to infants and children, elderly people with low immunity and immunosuppression are also at high risk of RSV infection. The elderly often develop obstructive pulmonary disease with cardiopulmonary complications. Despite the serious disease and economic burden caused to the world, there is still no safe and effective vaccine for RSV.
  • RSV belongs to the genus Pneumovirus and the family Pneumoviridae. It is an enveloped single-stranded negative-sense RNA virus. The genome is about 15.2kb long, transcribes ten genes, and encodes a total of eleven proteins. Among them, the adhesion glycoprotein G and the membrane fusion protein F are located on the surface of the viral membrane. Compared with the G protein, the F protein is more conservative and is therefore considered to be the most important protective antigen of RSV.
  • the F protein of RSV belongs to type I integral membrane protein. Its precursor protein F0 is composed of 574 amino acids. Three F0s form a trimer through hydrophobic interaction. When passing through the Golgi apparatus, it is cleaved by the host's furin protease between amino acids 109 and 110 and between amino acids 136 and 137. After cleavage, the F protein releases a short peptide P27 containing 27 amino acids. The remaining two segments F2 and F1 are connected by two disulfide bonds to form a mature F protein, which is displayed on the cell membrane or the surface of the virion by budding. At this time, the F protein is in a high-energy metastable state and is very unstable, called pre-F.
  • F1 At the N-terminus of F1 is a highly hydrophobic fusion peptide FP, which is located in the hydrophobic cavity of the trimer. Under the triggering of currently unknown factors, a series of drastic structural changes occur at the N-terminus of F1. This process allows FP to be inserted into the cell membrane surface to help the virus complete membrane fusion and thus infect cells, and also causes the pre-F structure to become a stable low-energy state post-F conformation.
  • the RSV pre-F protein can be divided into two parts: the "handle" region near the membrane, including domain I and domain II, which is mainly composed of ⁇ -chain structure and terminates in the C-terminal helix that enters the membrane; and a membrane-distal part, the RSV F "head” region, including domain III, which is mainly ⁇ -helix.
  • the RSV F head contains at least two epitopes associated with neutralizing antibodies.
  • Site I and site V are located at the top of the pre-F trimer and can be recognized by strong neutralizing antibodies including D25, AM22, 5C4 and hRSV90.
  • Site II is located in the middle of the pre-F trimer and is the binding site of the commercial antibody palivizumab.
  • Site I and site IV which are also recognized by low-efficiency antibodies, are located in the stalk region of the pre-F protein.
  • the stalk region of the pre-F protein accounts for 55% of the total surface area of the trimer, which may be related to the high neutralizing epitope in the head region. or site V competition, resulting in a weakened antibody response against high- and low-density epitopes.
  • the main purpose of RSV vaccine research with F protein as the main protective antigen is to produce high- to medium-neutralizing antibody titers. Improving the immunogenicity of RSV vaccines with F protein as the protective antigen is of great significance to RSV vaccine development.
  • the present application provides a fusion protein comprising a first truncation, a second truncation, and a connector connecting the first truncation and the second truncation;
  • the first truncation is compared to the F2 protein of wild-type respiratory syncytial virus (RSV), wherein the N-terminus of the F2 protein of wild-type RSV is truncated by 5 to 25 amino acids (e.g., 5-8, 9-12, 13-16, 17-20, 21-25), and the C-terminus is truncated by 3-5 amino acids (e.g., 3, 4, 5);
  • RSV respiratory syncytial virus
  • the second truncation has 7-9 amino acids (e.g., 7, 8, 9) truncated at the N-terminus of the F1 protein of wild-type RSV and 238 to 268 amino acids (e.g., 238-241, 242-245, 246-249, 250-253, 254-257, 258-261, 262-265, 266-268) truncated at the C-terminus.
  • the truncation length of the C-terminus of the second truncation has a certain range (i.e., truncation of 238 to 268 amino acids), and the truncation length of the N-terminus of the first truncation has a certain range (i.e., truncation of 5 to 25 amino acids). This is because within these ranges, the first truncation and the second truncation are still able to keep intact several protein secondary structures that maintain conformational stability, such as ⁇ 2/ ⁇ 7, ⁇ 1 and ⁇ 4, etc.
  • the first truncation is truncated by 5 or 25 amino acids at the N-terminus and 4 amino acids at the C-terminus of the F2 protein of wild RSV compared to the F2 protein of wild RSV.
  • the F2 protein of wild-type RSV has an amino acid sequence as shown in SEQ ID NO:13.
  • the first truncation has an amino acid sequence as shown in SEQ ID NO:16 or 17.
  • the second truncation is truncated by 8 or 9 amino acids at the N-terminus and 252 or 268 amino acids at the C-terminus of the F1 protein of wild-type RSV compared to the F1 protein of wild-type RSV.
  • the F1 protein of wild RSV has an amino acid sequence as shown in SEQ ID NO:12.
  • the second truncation has an amino acid sequence as shown in SEQ ID NO:14 or 15.
  • the linker comprises at least 1 (eg, 1, 2) proline.
  • the linker has 3-20 amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20). In certain embodiments, the linker has 5-8 amino acids.
  • the linker comprises a plurality (eg, 2, 3, 4, 5, 6, 7) of glycines and at least 1 (eg, 1, 2) of proline.
  • the linker has a sequence as shown in SEQ ID NO:2.
  • the first truncation is located at the N-terminus of the linker.
  • the second truncation is located at the C-terminus of the linker.
  • the fusion protein comprises, from N-terminus to C-terminus, a first truncation, a connector, and a second truncation.
  • the fusion protein has an amino acid sequence as shown in SEQ ID NO:3 or 4.
  • the fusion protein further comprises one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions). loss or addition).
  • amino acid substitutions, deletions or additions e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions. loss or addition).
  • the first truncation and/or the second truncation of the fusion protein comprises one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions).
  • the sequence of the fusion protein has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 3-7;
  • the substitutions are conservative substitutions.
  • the sequence of the fusion protein has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 4.
  • amino acid substitutions, deletions or additions e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions
  • the sequence of the fusion protein has an amino acid substitution in the 29th position corresponding to SEQ ID NO: 4, and the sequence of the fusion protein also has an amino acid substitution at any one of the positions 47 to 214 (for example, position 47, position 212, position 214, position 88, position 94, position 114, position 101) corresponding to SEQ ID NO: 4.
  • the sequence of the fusion protein has an amino acid substitution at the 114th position corresponding to SEQ ID NO: 4, and the sequence of the fusion protein also has an amino acid substitution at any one of the positions 29 to 94 corresponding to SEQ ID NO: 4.
  • the sequence of the fusion protein has one or several amino acid substitutions in the following positions corresponding to SEQ ID NO: 4: at position 29, position 47, position 212, position 214, position 88, position 94, position 114 and position 101.
  • the sequence of the fusion protein has amino acid substitutions in positions 29 and 212 corresponding to SEQ ID NO: 4, compared to the sequence shown in SEQ ID NO: 4.
  • the sequence of the fusion protein has amino acid substitutions in positions 29 and 214 corresponding to SEQ ID NO: 4, compared to the sequence shown in SEQ ID NO: 4.
  • the sequence of the fusion protein has amino acid substitutions in the positions corresponding to positions 29 and 114 of SEQ ID NO:4.
  • the sequence of the fusion protein has amino acid substitutions at positions 29, 47, 101 and 214 corresponding to SEQ ID NO: 4, compared to the sequence shown in SEQ ID NO: 4.
  • the sequence of the fusion protein has amino acid substitutions at positions 88, 101, 114 and 214 corresponding to SEQ ID NO: 4, compared to the sequence shown in SEQ ID NO: 4.
  • the sequence of the fusion protein has an amino acid substitution in the 94th and 114th positions corresponding to SEQ ID NO: 4.
  • the substitution at position 29 is from isoleucine I to leucine L.
  • the substitution at position 212 is from valine V to methionine M.
  • the substitution at position 214 is from alanine A to leucine L.
  • the substitution at position 94 is from valine V to isoleucine I.
  • the substitution at position 47 is from methionine M to alanine A.
  • the substitution at position 114 is from tyrosine Y to isoleucine I.
  • the substitution at position 101 is from valine V to isoleucine I.
  • the substitution at position 214 is from alanine A to leucine L.
  • the substitution at position 88 is from amino acid leucine L to alanine A.
  • the fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 28-34.
  • the fusion protein further comprises: a signal peptide, a multimerization motif (eg, a trimerization motif) and/or a membrane anchoring region.
  • a signal peptide e.g, a signal peptide
  • a multimerization motif e.g, a trimerization motif
  • the membrane anchoring region has an amino acid sequence as shown in SEQ ID NO:8.
  • the signal peptide has an amino acid sequence as shown in SEQ ID NO:9.
  • the multimerization motif has an amino acid sequence as shown in SEQ ID NO:21 or 22.
  • the signal peptide is located at the N-terminus of the first truncation. In certain embodiments, the signal peptide is connected to the first truncation via a first connecting peptide.
  • the membrane anchoring region is located at the C-terminus of the second truncation.
  • the membrane anchoring region is connected to the second truncation through a second connecting peptide.
  • the multimerization motif is located at the C-terminus of the second truncation. In certain embodiments, the multimerization motif is connected to the second truncation via a second connecting peptide.
  • the first connecting peptide and the second connecting peptide each independently comprise one or more (e.g., 1, 2, or 3) sequences as shown in (GmS)n, wherein m is selected from an integer of 1-6, and n is selected from an integer of 1-6. In certain embodiments, m is 3, 4, or 5. In certain embodiments, n is 1 or 2. In certain embodiments, the first connecting peptide and the second connecting peptide have the sequence shown in SEQ ID NO: 18.
  • the fusion protein comprises, from N-terminus to C-terminus, a signal peptide, a first connecting peptide, a first truncation, a connector, a second truncation, a second connecting peptide, and a membrane anchoring region.
  • the fusion protein has the sequence shown in SEQ ID NO: 19, 20, 45 or 46.
  • the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein as described above.
  • the nucleotide sequence is codon-optimized or non-optimized according to the codon preference of the host cell.
  • the present application provides a vector comprising the nucleic acid molecule as described above.
  • the vector is a viral vector.
  • the viral vector is selected from: an influenza virus vector, an enterovirus vector, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a poxvirus vector, a baculovirus vector, a papillomavirus vector, or a papillomasovavirus vector.
  • the present application provides a host cell comprising the fusion protein as described above, the nucleic acid molecule as described above, or the vector as described above.
  • the host cell is selected from a prokaryotic cell (eg, an E. coli cell), a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell, such as a mouse cell or a human cell.
  • the fusion protein comprises a membrane anchoring region and is displayed on the surface of the cell membrane of the host cell.
  • the present application provides a method for expressing or producing the fusion protein as described above, the method comprising culturing the host cell as described above under conditions allowing protein expression, and optionally, recovering or purifying the expressed fusion protein.
  • the present application provides a kit comprising an antigen component and a carrier component capable of displaying the antigen component; wherein,
  • the antigen component comprises (i) the fusion protein as described above, (ii) the nucleic acid molecule as described above, and/or (iii) the mRNA product transcribed from the nucleic acid molecule as described above;
  • the carrier component is selected from: nanomaterials (e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers and biomimetic nanoparticles), bacterial outer membrane vesicles (OMVs), polymerized bases, virus-like particles (VLPs), or any combination thereof.
  • nanomaterials e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers and biomimetic nanoparticles
  • OMVs bacterial outer membrane vesicles
  • VLPs virus-like particles
  • the antigen component and the carrier component in the kit are provided separately or in the form of a complex.
  • the antigenic component is a monomer or a multimer (eg, a dimer, a trimer, a tetramer, a pentamer).
  • the multimerization base and/or VLP in the kit is provided in the form of a protein or a nucleic acid molecule comprising a nucleotide sequence encoding the protein.
  • the polymerizing base has an amino acid sequence as shown in SEQ ID NO:21 or 22.
  • the VLPs are assembled from proteins obtained from RSV, hepatitis E virus (HEV), hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).
  • HSV hepatitis E virus
  • HBV hepatitis B virus
  • HPV human papillomavirus
  • HIV human immunodeficiency virus
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and/or excipient, and any one or more selected from the following (1) to (4):
  • the present application provides methods for producing vaccines against respiratory syncytial virus (RSV), which methods include providing a kit or pharmaceutical composition as described above and formulating it into a pharmaceutically acceptable vaccine.
  • RSV respiratory syncytial virus
  • the present application provides a vaccine comprising the fusion protein as described above, or the nucleic acid molecule as described above, or the mRNA product transcribed from the nucleic acid molecule as described above, or the vector as described above.
  • the vaccine is prepared by the kit or pharmaceutical composition as described above.
  • the vaccine further comprises an adjuvant.
  • the above-mentioned vaccine can be administered to a subject, such as a human subject.
  • the total dose of the fusion protein in the vaccine for separate administration can be, for example, about 0.01 ⁇ g to about 10 mg, such as 1 ⁇ g-1 mg, such as 10 ⁇ g-100 ⁇ g. Determining the dose to be administered can be determined experimentally, and determining the dose is routine for those skilled in the art.
  • the present application provides a method for inducing antibodies against RSV, which comprises administering (e.g., injecting) an effective amount of the fusion protein as described above, or the nucleic acid molecule as described above, or the vector as described above, or the host cell as described above, or the pharmaceutical composition as described above, or the vaccine as described above in cells in vitro or in a subject.
  • the antibody is a neutralizing antibody.
  • administration includes parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, or mucosal administration, e.g., intranasal, oral, etc.
  • parenteral administration such as intradermal, intramuscular, subcutaneous, transdermal, or mucosal administration, e.g., intranasal, oral, etc.
  • the composition is administered by intramuscular injection.
  • the protein of the present invention can be used as a diagnostic tool, for example, to test the immune status of a subject by determining whether there are antibodies capable of binding to the fusion protein of the present application in the serum of the subject.
  • the present application provides a method for detecting whether a subject is infected with RSV in vitro, the method comprising: contacting a biological sample obtained from the subject with the fusion protein as described above; and detecting whether a complex formed by the fusion protein and the antibody is present.
  • the subject is a mammal, eg, a mouse, a human.
  • the biological sample is selected from whole blood, serum, plasma, or any combination thereof.
  • the present application provides a method for screening candidate drugs capable of inhibiting RSV infection of cells, the method comprising: adding the fusion protein as described above, or the vector as described above, or the pharmaceutical composition as described above, or Before, simultaneously with or after contacting the host cells with the vaccine as described above, the host cells are contacted with the candidate drug.
  • the present application provides the use of the fusion protein as described above, or the nucleic acid molecule as described above, or the vector as described above, or the host cell as described above, or the pharmaceutical composition as described above, or the vaccine as described above in the preparation of a kit for inducing an immune response to RSV in a subject.
  • the immune response includes inducing the production of antibodies against RSV in the subject.
  • the antibody is a neutralizing antibody.
  • the subject is a mammal, eg, a mouse, a human.
  • the present application provides the use of the fusion protein as described above, or the nucleic acid molecule as described above, or the vector as described above, or the host cell as described above, or the pharmaceutical composition as described above, or the vaccine as described above in the preparation of a kit for preventing and/or treating RSV infection or diseases and/or symptoms caused by RSV infection.
  • the subject is a mammal, for example, a mouse, a human.
  • the diseases and symptoms caused by RSV infection are selected from bronchiolitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.
  • RSV respiratory syncytial virus
  • the RSV genome is about 15Kb in length, contains 10 genes, and encodes 11 proteins, including 8 structural proteins (F, G, M2-1, M2-2, SH, N, P, L) and 3 non-structural proteins (NS1, NS2, NS3).
  • F fusion protein
  • adhesion protein attachment protein, G
  • F protein is a type I glycoprotein.
  • the sequence of F protein can be obtained from public databases (e.g., GenBank database).
  • the amino acid sequence of wild F protein is shown in SEQ ID NO:1.
  • wild or wild are used interchangeably. When these terms are used to describe a nucleic acid molecule, polypeptide, or protein, it means that the nucleic acid molecule, polypeptide, or protein exists in nature, is found in nature, and has not been modified or processed by humans.
  • wild respiratory syncytial virus The F1/F2 protein of (RSV) refers to a naturally occurring, biologically active F1/F2 protein.
  • GenBank database e.g., GenBank database.
  • the amino acid sequence of the F2 protein of wild RSV can be shown as SEQ ID NO: 13.
  • the amino acid sequence of the F1 protein of wild RSV can be shown as SEQ ID NO: 12.
  • C-terminal truncated by X amino acids means that the most consecutive X amino acids at the C-terminus are truncated.
  • N-terminal truncated by X amino acids means that the most consecutive X amino acids at the N-terminus are truncated.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell.
  • Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc.
  • plasmids such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1
  • bacteriophages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses include adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes.
  • the vector may also contain a replication initiation site.
  • the term "host cell” refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • codons have degeneracy. That is, in the translation process of protein, each amino acid may correspond to one or more codons, for example, up to 6 codons. Different species have great differences in the use of degenerate codons encoding a certain amino acid, and have different preferences. This preference phenomenon is called "codon preference”. Therefore, as used herein, the term "codon preference" refers to the situation where a species prefers to use certain specific codons to encode amino acids. It is particularly advantageous in some cases to optimize the sequence of nucleic acid molecules according to codon preference, for example, it may help to improve the expression level of the protein encoded by the nucleic acid molecule.
  • Escherichia coli or human cells
  • virus-like particle is a polymeric particle whose structure is similar to that of natural
  • VLP is a natural virus particle.
  • VLP is a virus-like particle assembled from proteins. It has been confirmed that some viruses (e.g., RSV, HBV, HEV, HPV) proteins (e.g., capsid proteins, surface proteins, envelope proteins) can spontaneously form VLPs after recombinant expression in an appropriate expression system.
  • viruses e.g., RSV, HBV, HEV, HPV
  • capsid proteins e.g., capsid proteins, surface proteins, envelope proteins
  • the term “pharmaceutically acceptable” means that it is recognized in the pharmaceutical field that it can be used in animals, especially in humans.
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed.
  • pH adjusters including but not limited to phosphate buffers
  • surfactants including but not limited to cationic, anionic or non-ionic surfactants, such as Tween-80
  • adjuvants including but not limited to sodium chloride
  • ionic strength enhancers including but not limited to sodium chloride
  • pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including oils derived from petroleum, animals, plants or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.
  • physiological saline is a preferred carrier.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.
  • pharmaceutically acceptable excipients may include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc.
  • the pharmaceutical composition may also include a wetting agent, or an emulsifier such as sodium hyaluronate, or a pH buffer.
  • the pharmaceutical composition may be in the form of a solution, a suspension, an emulsion, a tablet, a pill, a capsule, a powder, a sustained-release formulation, etc.
  • the term "subject” refers to mammals, including but not limited to humans, rodents (mice, rats, guinea pigs), dogs, horses, cows, cats, pigs, monkeys, chimpanzees, etc.
  • the subject is a human.
  • the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • a disease prevention effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease
  • a disease treatment effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.
  • conservative substitution refers to a substitution that does not adversely affect or alter the amino acid sequence comprising the residue.
  • Amino acid substitutions that alter the expected properties of a protein/polypeptide can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions that replace amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the fusion protein of the present application contains RSV F1 truncation and F2 truncation in a unique truncation manner.
  • the ability of the truncated fusion protein to induce specific antibodies is comparable to that of the full-length pre-F protein, and can also induce neutralizing antibody titers significantly higher than those of the full-length pre-F protein.
  • the connector contained in the fusion protein also plays a role in improving the stability of the pre-F specific epitope site ⁇ . Furthermore, after obtaining the above-mentioned fusion protein, it was mutated, and the mutated fusion protein has a stronger affinity with the antibody, higher thermal stability, and stronger immunogenicity.
  • the fusion protein of the present application shows good protection and safety, and is suitable for various forms of vaccine platforms, such as nucleic acid vaccines, recombinant protein vaccines, viral vector vaccines, and granulated vaccines, etc. Therefore, the fusion protein of the present application has great potential in inducing immune responses to RSV in subjects, and preventing and/or treating RSV infection or diseases and/or symptoms caused by RSV infection.
  • Figure 1 shows the high neutralizing antibody epitope site II and The expression result.
  • FIG2 shows a schematic diagram of the structures of the wild-type F protein F wt and the F protein truncated form LC2.
  • Figure 3 shows a schematic diagram of the structure of the F protein truncated LC2 predicted by AlphaFold 2.
  • Figure 4 shows the expression of F protein truncations detected by Western blot.
  • the first lane is the Marker
  • the second lane is the pre-F protein SC-TM
  • the third lane is the F protein truncations LC2.
  • Figure 5 shows the immunofluorescence detection of F protein truncated LC2 and site II specific antibody mota (A in Figure 5) and Binding of specific antibodies D25 and AM22 (B and C in FIG. 5 ).
  • Figure 6 shows the serum binding antibody titer and serum antibody typing produced by mice induced by the mRNA vaccine encoding the F protein truncation.
  • Figure 7 is a schematic diagram of the neutralizing antibody titer results induced in mice by the mRNA vaccine encoding the F protein truncation.
  • Figure 8 shows a schematic diagram of the construction of the wild-type F protein Fwt and the mutant F protein truncate LC2A, wherein the wild-type F protein Fwt includes a signal peptide, F2, F1, a membrane anchor region and a cytoplasmic region; the mutant F protein truncate LC2A includes a signal peptide, a first connecting peptide, a mutant first truncate, a connecting body, a mutant second truncate, a second connecting peptide, and a multimerization motif.
  • FIG9 shows the polyacrylamide gel electrophoresis results of the control protein SC-TM and the mutant F protein truncation, wherein the first lane is a marker, the second lane is LC2A, and the third lane is SC-TM.
  • FIG10 shows the binding of SC-TM and seven mutated F protein truncations to three F protein antibodies.
  • FIG11 shows the affinity results of SC-TM and seven mutated F protein truncations to two F protein antibodies detected by Biacore, the protein thermal stability results detected by DSF, and the epitope thermal stability results detected by ELISA.
  • Figure 12 shows the antibody titers in the serum of mice immunized with SC-TM and seven mutant F protein truncations, wherein Figure 12A shows the antibody titers binding to Pre-F, and Figure 12B shows the antibody titers binding to Post-F.
  • the pcDNA3.1 eukaryotic expression vector was connected to construct a recombinant plasmid of F protein containing a flexible connecting peptide, which were named LA, LB, LC, LD, LE and LF, respectively. Immunofluorescence was used to detect the high neutralizing antibody epitope site II and expression.
  • Alexa Fluor 568 (1:2000 dilution) (Manufacturer: Thermo; Catalog Number: A-11013) fluorescent secondary antibody to each well and incubate at room temperature for 1 hour.
  • Figure 1 shows the high neutralizing antibody epitope site II and The results showed that the F protein containing the flexible linker peptides LA and LC retained the highest level of expression.
  • the amino acid sequence of RSV A2Fusion protein was obtained from the GenBank database (GenBank ID: FJ614814.1), and the amino acid sequence of F wt is shown in SEQ ID NO: 1.
  • the amino acid sequence characteristics of the F protein truncate are that the N-terminus and C-terminus of the F2 peptide chain and the F1 peptide chain are truncated respectively, and the truncated F2 and F1 are connected by a flexible connecting peptide containing proline to help stabilize the pre-F conformation.
  • the C-terminus of the F1 peptide chain is connected to different display bases through a flexible connecting peptide.
  • a total of 10 mutants were prepared, as follows:
  • the amino acids 31 to 105 of SEQ ID NO:1 were connected to the amino acids 145 to 322 through a flexible linker peptide containing proline (SEQ ID NO:2).
  • the truncated F protein had a total length of 314 amino acids and was named LC1. Its amino acid sequence is shown in SEQ ID NO:3.
  • LC1 retains Site V, site III and site II epitopes.
  • the amino acids 56 to 105 of SEQ ID NO:1 were connected to the amino acids 145 to 306 through a flexible linker peptide containing proline (SEQ ID NO:2).
  • the truncated F protein had a total length of 280 amino acids and was named LC3. Its amino acid sequence is shown in SEQ ID NO:5. LC3 retains and siteII epitope.
  • the amino acids 51 to 105 of SEQ ID NO:1 were connected to the amino acids 145 to 297 through a flexible linker peptide containing proline (SEQ ID NO:2).
  • the truncated F protein had a total length of 276 amino acids and was named LC4. Its amino acid sequence is shown in SEQ ID NO:6. LC4 retains and siteII epitope.
  • SEQ ID NO:1 The amino acids 56 to 105 of SEQ ID NO:1 were linked to the amino acids 145 to 297 through a flexible proline-containing linker peptide (SEQ ID NO:2).
  • SEQ ID NO:2 The truncated F protein had a total length of 271 amino acids and was named LC5. Its amino acid sequence is shown in SEQ ID NO:7. LC5 retained the site II epitope.
  • the nucleotide sequences encoding the five F protein truncations were connected to the nucleotide sequence encoding the F protein cytoplasmic segment (amino acid sequence as shown in SEQ ID NO: 8) through the nucleotide sequence encoding the flexible connecting peptide.
  • the nucleotide sequence encoding the signal peptide (amino acid sequence as shown in SEQ ID NO: 9) was introduced into the 5' end of the nucleotide sequence of the truncation.
  • the nucleotide sequence of the F protein truncation was submitted to Shanghai Shenggong Biotechnology Co., Ltd.
  • FIG. 2 shows a schematic diagram of the connection of the F protein truncation LC2.
  • Figure 3 shows a schematic diagram of the structure of the F protein truncation predicted by AlphaFold 2.
  • the full-length F protein SC-TM recombinant plasmid in the pre-fusion structure was constructed according to the above method as a control (the sequence of the SC-TM protein was obtained from GenBank: 5C6B_F) and handed over to Shanghai Shenggong for synthesis.
  • the plasmids that were successfully sequenced were subjected to xBaI single enzyme digestion after plasmid extraction, and the single enzyme digestion results were identified by capillary electrophoresis.
  • the plasmids that were correctly identified were detected by spectrophotometer for OD260 and OD280, and their DNA concentration and purity were determined.
  • the plasmids were stored at -20°C.
  • the sample loading amount for electrophoresis is 20 ⁇ g/well. ⁇ -mercaptoethanol and loading buffer are added to the sample and heat denatured at 100°C for 10 minutes.
  • Alexa Fluor 568 (1:2000 dilution) (Manufacturer: Thermo; Catalog Number: A-11013) fluorescent secondary antibody to each well and incubate at room temperature for 1 hour.
  • RNA transcription-related elements were constructed in an in vitro transcription plasmid containing F protein truncated LC2 and full-length pre-F protein SC-TM.
  • the plasmid extraction and identification were consistent with those described in Example 1.
  • the transcription template was digested with DNase I.
  • the mRNA was purified by precipitation using lithium chloride (manufacturer: Invitrogen; catalog number: AM9480).
  • mice The feeding and experimental operations of experimental animals were carried out in accordance with the procedures established by the Experimental Animal Use and Management Committee of Xiamen University.
  • the experimental animals were BALB/c female mice, 6-8 weeks old, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and housed in the Experimental Animal Center of Xiamen University.
  • mice were randomly divided into groups, with 4 mice in each group. 5 ⁇ g of mRNA-LNP was injected into the quadriceps femoris of one side of the thigh, and the control group was PBS without mRNA-LNP.
  • a second booster immunization was performed 2 weeks after the initial immunization, for a total of two immunizations.
  • pre-F protein and post-F protein for coating is as follows: download the sequences of pre-F and post-F proteins from GenBank (GenBank ID: LY628284.1, GenBank ID: 6APB_A), clone the nucleotide sequences encoding the two proteins into the pcDNA3.1 eukaryotic expression vector, express them using the CHO eukaryotic expression system, and obtain pre-F protein and post-F protein by affinity chromatography purification.
  • the endpoint titer is determined by 3 times the reading of the negative well.
  • Figure 5 A shows that the F protein truncated LC2 of the present application induced a high level of pre-F binding antibody titer, which is comparable to SC-TM (ie, full-length pre-F protein).
  • Figure 6 B shows that when immunized in the form of mRNA vaccine, a higher level of IgG2a antibody was induced, indicating that it exhibited Th-1 bias.
  • the F protein truncated form described in the embodiment of the present invention induces a higher level of pre-F specific antibodies and a significantly higher neutralizing antibody titer than the full-length F protein, showing good protection and safety, and is suitable for various forms of vaccine platforms, such as nucleic acid vaccines, recombinant protein vaccines, viral vector vaccines, and granulated vaccines.
  • This example is based on the F protein truncated form LC2 (SEQ ID NO: 4) obtained in the above example, and further mutations are performed on it to prepare a total of 7 mutants, as follows:
  • the amino acid I at position 29 of SEQ ID NO: 4 was mutated to L, and the amino acid V at position 212 was mutated to M.
  • the mutated protein was named LC2A, and its amino acid sequence is shown in SEQ ID NO: 28.
  • the 47th amino acid M of SEQ ID NO: 4 was mutated to A, and the 114th amino acid Y was mutated to I.
  • the mutated protein was named LC2D, and its amino acid sequence is shown in SEQ ID NO: 31.
  • the amino acid I at position 29 of SEQ ID NO: 4 was mutated to L
  • the amino acid M at position 47 was mutated to A
  • the amino acid V at position 101 was mutated to I
  • the amino acid A at position 214 was mutated to L.
  • the mutated protein was named LC2E, and its amino acid sequence is shown in SEQ ID NO: 32.
  • the 88th amino acid L of SEQ ID NO: 4 was mutated to A, the 101st amino acid V was mutated to I, the 114th amino acid Y was mutated to I, and the 214th amino acid A was mutated to L.
  • the mutated protein was named LC2F, and its amino acid sequence is shown in SEQ ID NO: 33.
  • Example 11 Construction of a recombinant plasmid expressing a truncated F protein
  • the nucleotide sequences encoding the 7 mutant F protein truncations were connected to the nucleotide sequence encoding the trimerization motif foldon (amino acid sequence as shown in SEQ ID NO: 21) through the nucleotide sequence encoding the GSGS amino acid, and the nucleotide sequence encoding the signal peptide (SEQ ID NO: 9) was introduced at the 5' end of the nucleotide sequence encoding the 7 mutant F protein truncations to construct a fusion expression gene.
  • the nucleotide sequence of the mutant F protein truncations was submitted to Shanghai Shenggong Biotechnology Co., Ltd.
  • Tube A 1 mL ExpiCHOTM Expresssion Medium contains 25 ⁇ g of plasmid
  • tube B 1 mL ExpiCHOTM Expresssion Medium contains 80 ⁇ L of the transfection reagent in the ExpiFectamineTM CHO Transfection Kit (Manufacturer: Thermo Scientific; Catalog No.: A29129).
  • Opti-MEM culture medium Take another 125 ⁇ L of opti-MEM culture medium into another 1.5 mL centrifuge tube, add 4 ⁇ L of Lipofectamine3000 (manufacturer: Invitrogen; catalog number: L3000015), vortex and mix, and mark it as B. Add tube A to tube B, vortex and mix, and let it stand for 15 minutes. Then add the solution to the 293t six-well plate, shake it gently, and place it in a cell culture incubator for culture. On the third day, discard the culture medium and add pre-cooled PBS to wash the cells twice. Add 150 ⁇ L of RIPA lysis buffer containing protease inhibitors and lyse the cells on ice for 30 minutes.
  • SC-TM and 7 mutants were diluted with PBS buffer to prepare coating solution with a final concentration of 1 ⁇ g/mL. 100 ⁇ L of the diluted coating solution was added to a 96-well ELISA plate and coated overnight at 4°C. Wash once with PBST (20mM PB7.4, 150mM NaCl, 0.1% Tween20) washing solution and spin dry. Then add 200 ⁇ L of blocking solution (containing 20% calf serum and 1% casein, 20mM Na2HPO4/NaH2PO4 buffer solution with a pH value of 7.4) to each well, block at 37°C for 2 hours, and discard the blocking solution. After drying, put it into an aluminum foil bag and store it at 4°C for later use.
  • PBST 20mM PB7.4, 150mM NaCl, 0.1% Tween20
  • the binding affinity of the mutant F protein truncate to the F protein-specific antibody D25 and hRSV90 was determined by surface plasmon resonance analysis (SPR).
  • SPR surface plasmon resonance analysis
  • the mutant F protein truncate with histidine tag was immobilized on an NTA sensor chip (Cytiva) at a concentration of about 100 nM using Biacore-8K (Cytiva).
  • Antibodies were added after gradient dilution (concentration range of 200 to 0.78 nM).
  • the RU data were fitted to a 1:1 binding model using Biacore TM Insight software.
  • DSF Differential Scanning Fluorimetry
  • the feeding and experimental operations of experimental animals were carried out in accordance with the procedures established by the Experimental Animal Use and Management Committee of Xiamen University.
  • the experimental animals were BALB/c female mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., 6-8 weeks old, and were raised in the Experimental Animal Center of Xiamen University.
  • the immunization scheme was to mix SC-TM and 7 mutant F protein truncations with aluminum adjuvant in a volume ratio of 1:1 for immunization of mice.
  • Each group consisted of 5 mice, and the immunization method was unilateral thigh muscle injection, and the immunization dose was 20 ⁇ g protein/mouse.
  • the mouse orbital blood was collected on the 7th day after immunization, and the serum was separated. The serum samples were inactivated at 56°C for 30 minutes and placed at 4°C for use.
  • the Pre-F protein used for coating was SC-TM, and the sequence of the Post-F protein was downloaded from GenBank (GenBank ID: 6APB_A, SEQ ID NO: 44).
  • GenBank GenBank ID: 6APB_A, SEQ ID NO: 44.
  • the nucleotide sequence encoding the Post-F protein was cloned into the pcDNA3.1 eukaryotic expression vector and expressed using the ExpiCHO TM system.
  • the expression steps were the same as those described in Example 3.
  • the protein coating and blocking steps were the same as those described in Example 4.
  • the serum was diluted 200 times in the first well, 5 times in the gradient, and a total of 8 gradients were diluted.
  • the diluted serum was added to the blocked 96-well plate at a volume of 100 ⁇ L/well and incubated at 37°C for 1 hour.
  • the serum was discarded, washed 5 times with PBST, and dried.
  • a 1:5000 dilution of horseradish peroxidase-labeled goat anti-mouse IgG antibody (manufacturer: Abcam; catalog number: ab97265) was added at a volume of 100 ⁇ L/well and incubated at 37°C for 1 hour.
  • the secondary antibody was discarded and washed five times with PBST.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Wood Science & Technology (AREA)
  • Public Health (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Mycology (AREA)
  • Toxicology (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Pulmonology (AREA)

Abstract

提供一种融合蛋白，以及包含编码所述融合蛋白的核苷酸序列的核酸分子。还涉及包含所述融合蛋白或所述核酸分子的疫苗。进一步的，还涉及这些融合蛋白、核酸分子和疫苗用于预防和/或治疗RSV感染或由RSV感染所引起的疾病和/或症状的方法。

Description

截短的呼吸道合胞病毒F蛋白及其用途 技术领域
本申请涉及生物医药领域，具体而言，本申请涉及一种融合蛋白，以及包含编码所述融合蛋白的核苷酸序列的核酸分子。本申请还涉及包含所述融合蛋白或所述核酸分子的疫苗。进一步的，本发明还涉及这些融合蛋白、核酸分子和疫苗用于预防和/或治疗RSV感染或由RSV感染所引起的疾病和/或症状的方法。
背景技术
呼吸道合胞病毒(respiratory syncytial virus,RSV)是世界范围内引起婴幼儿下呼吸道感染最主要的病原体之一。据统计，全球每年5岁以下儿童感染RSV人数达3300万，造成近16万人的死亡。RSV感染后无法获得持续免疫，可出现反复感染，超过99％的2岁以下儿童至少感染过一次RSV，且其中70％患者发生住院感染并发细支气管炎、肺炎及哮喘。除婴幼儿及儿童外，免疫力低下的老年人及免疫抑制人群也是RSV感染的高危人群，老人常导致阻塞性肺病且伴有心肺并发症。尽管给全球造成严重的疾病和经济负担，目前针对RSV仍然没有安全有效的疫苗。
RSV属于肺炎病毒属、肺病毒科，是一种有包膜的单股负链RNA病毒。基因组全长约15.2kb，转录十个基因，共编码十一种蛋白。其中黏附糖蛋白G及膜融合蛋白F位于病毒膜表面，相比于G蛋白，F蛋白在保守性上更具优势，因此被认为是RSV的最重要的保护性抗原。
RSV的F蛋白属于Ⅰ型整合膜蛋白，其前体蛋白F0由574个氨基酸组成，三个F0之间通过疏水作用形成三聚体，在通过高尔基体时被宿主的弗林蛋白酶在第109、110位氨基酸之间及136、137位氨基酸之间进行切割。切割后的F蛋白释放出含有27个氨基酸的短肽P27，其余两段F2及F1通过两个二硫键连接形成成熟的F蛋白，通过出芽的方式展示到细胞膜或病毒体表面。此时的F蛋白处于高能级亚稳态，十分不稳定，被称为pre-F。在F1的N末端是一段高度疏水的融合肽FP，位于三聚体的疏水空腔内，在目前未知因素的触发下，F1的N末端发生一系列剧烈的结构变化，这个过程使得FP可以插到细胞膜表面帮助病毒完成膜融合从而感染细胞，也导致pre-F结构变成稳定的低能态的post-F构象。
2013年，McLellan等人首次通过点突变的方式获得了稳固的pre-F蛋白并命名为 DS-Cav1，随后又有几个pre-F蛋白被相继报道，包括DS-Cav1-Cys-zipper、SC-DM以及SC-TM。相比于post-F蛋白，这些pre-F蛋白被证实可以激发显著更高水平的中和抗体滴度。RSV的pre-F蛋白可分为两部分：近膜端的“柄”区域，包括结构域I和结构域II，主要由β链结构组成，终止于进入膜的c端螺旋；和一个膜远端部分，RSV F“头部”区域，包括结构域III，主要是α-螺旋。RSV F头部含有至少两个与中和抗体相关的表位。及site V位于pre-F三聚体的顶端，可分别被包括D25、AM22、5C4及hRSV90在内的强中和抗体识别。Site II位于pre-F三聚体的中部，是商品化抗体palivizumab的结合位点。同样被低效抗体识别的site I和site IV位于pre-F蛋白的柄区。Pre-F蛋白的柄区占三聚体总表面积的55％，可能与头部区的高中和表位或site V竞争，导致针对高中和表位的抗体应答减弱。
以F蛋白为主要保护性抗原的RSV疫苗研究的主要目的在于产生高中和滴度的抗体，提高以F蛋白作为保护性抗原的RSV疫苗的免疫原性对RSV疫苗研发具有重要意义。
发明内容
包含截短体的融合蛋白
在第一方面，本申请提供了一种融合蛋白，其包含第一截短体，第二截短体，以及连接所述第一截短体和第二截短体的连接体；其中，
第一截短体与野生的呼吸道合胞病毒(RSV)的F2蛋白相比，在野生的RSV的F2蛋白的N端截短了5个至25个氨基酸(例如，5个-8个，9个-12个，13个-16个，17个-20个，21个-25个)，以及C端截短了3-5个氨基酸(例如，3个，4个，5个)；
第二截短体与野生的RSV的F1蛋白相比，在野生的RSV的F1蛋白的N端截短了7-9个氨基酸(例如，7个，8个，9个)，以及C端截短了238个至268个氨基酸(例如，238个-241个，242个-245个，246个-249个，250个-253个，254个-257个，258个-261个，262个-265，266个-268个)。
在某些实施方案中，第二截短体的C端的截短长度具有一定的范围(即，截短238个至268个氨基酸)，第一截短体的N端的截短长度具有一定的范围(即，截短5个至25个氨基酸)，这是由于在这些范围内，第一截短体和第二截短体仍然能够使维持构象稳定性的几种蛋白质二级结构保持完整，例如β2/β7、α1和α4等。
在某些实施方案中，所述第一截短体与野生的RSV的F2蛋白相比，在野生的RSV的F2蛋白的N端截短了5个或25个氨基酸，以及C端截短了4个氨基酸。
在某些实施方案中，野生的RSV的F2蛋白具有如SEQ ID NO:13所示的氨基酸序列。
在某些实施方案中，所述第一截短体具有如SEQ ID NO:16或17所示的氨基酸序列。
在某些实施方案中，所述第二截短体与野生的RSV的F1蛋白相比，在野生的RSV的F1蛋白的N端截短了8个或9个氨基酸，以及C端截短了252个或268个氨基酸。
在某些实施方案中，野生的RSV的F1蛋白具有如SEQ ID NO:12所示的氨基酸序列。
在某些实施方案中，所述第二截短体具有如SEQ ID NO:14或15所示的氨基酸序列。
在某些实施方案中，所述连接体包含至少1个(例如，1个，2个)脯氨酸。
在某些实施方案中，所述连接体具有3-20个氨基酸(例如，3个，4个，5个，6个，7个，8个，9个，10个，15个，20个)。在某些实施方案中，所述连接体具有5-8个氨基酸。
在某些实施方案中，所述连接体包含多个(例如，2个，3个，4个，5个，6个，7个)甘氨酸以及至少1个(例如，1个，2个)脯氨酸。
在某些实施方案中，所述连接体具有如SEQ ID NO:2所示的序列。
在某些实施方案中，所述第一截短体位于所述连接体的N端。
在某些实施方案中，所述第二截短体位于所述连接体的C端。
在某些实施方案中，所述融合蛋白从N端至C端依次包含：第一截短体，连接体，第二截短体。
在某些实施方案中，所述融合蛋白具有如SEQ ID NO:3或4所示的氨基酸序列。
包含截短的突变体的融合蛋白
在某些实施方案中，所述融合蛋白还包含1个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺 失或添加)。
在某些实施方案中，在所述融合蛋白的第一截短体和/或第二截短体上包含1个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)。
在某些实施方案中，所述融合蛋白的序列与SEQ ID NO：3-7任一项所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)；
在某些实施方案中，所述置换为保守置换。
在某些实施方案中，所述融合蛋白的序列与SEQ ID NO：4所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)。
在某些实施方案中，与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第29位位点中具有氨基酸的置换，并且，所述融合蛋白的序列在对应于SEQ ID NO：4的第47位-第214位(例如，第47位，第212位，214位，第88位，第94位，第114位，第101位)位点中的任意一个位点也具有氨基酸的置换。
在某些实施方案中，与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第114位位点中具有氨基酸的置换，并且，所述融合蛋白的序列在对应于SEQ ID NO：4的第29位-第94位位点中的任意一个位点也具有氨基酸的置换。
在某些实施方案中，与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的下述位点中具有一个或几个氨基酸的置换：在第29位，第47位，第212位，214位，第88位，第94位，第114位和第101位。
在某些实施方案中，与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第29位和第212位位点中具有氨基酸的置换。
在某些实施方案中，与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第29位和第214位位点中具有氨基酸的置换。
在某些实施方案中，与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第29位和第114位位点中具有氨基酸的置换。
在某些实施方案中，与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第29位，第47位，第101位和第214位位点中具有氨基酸的置换。
在某些实施方案中，与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第88位，第101位，第114位和第214位位点中具有氨基酸的置换。
在某些实施方案中，与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第94位，第114位位点中具有氨基酸的置换。
在某些实施方案中，所述第29位位点的置换是由异亮氨酸I置换为亮氨酸L。
在某些实施方案中，所述第212位位点的置换是由缬氨酸V置换为甲硫氨酸M。
在某些实施方案中，所述第214位位点的置换是由丙氨酸A置换为亮氨酸L。
在某些实施方案中，所述第94位位点的置换是由缬氨酸V置换为异亮氨酸I。
在某些实施方案中，所述第47位位点的置换是由甲硫氨酸M置换为丙氨酸A。
在某些实施方案中，所述第114位位点的置换是由酪氨酸Y置换为异亮氨酸I。
在某些实施方案中，所述第101位位点的置换是由缬氨酸V置换为异亮氨酸I。
在某些实施方案中，所述第214位位点的置换是由丙氨酸A置换为亮氨酸L。
在某些实施方案中，所述第88位位点的置换是由氨基酸亮氨酸L置换为丙氨酸A。
在某些实施方案中，所述融合蛋白具有如SEQ ID NO：28-34任一项所示的氨基酸序列。
在某些实施方案中，所述融合蛋白还包含：信号肽，多聚化基序(例如，三聚化基序)和/或膜锚定区。
在某些实施方案中，所述膜锚定区具有如SEQ ID NO:8所示的氨基酸序列。
在某些实施方案中，所述信号肽具有如SEQ ID NO:9所示的氨基酸序列。
在某些实施方案中，所述多聚化基序具有如SEQ ID NO:21或22所示的氨基酸序列。
在某些实施方案中，所述信号肽位于第一截短体的N端。在某些实施方案中，所述信号肽通过第一连接肽与第一截短体连接。
在某些实施方案中，所述膜锚定区位于第二截短体的C端。在某些实施方案中，所 述膜锚定区通过第二连接肽与第二截短体连接。
在某些实施方案中，所述多聚化基序位于第二截短体的C端。在某些实施方案中，所述多聚化基序通过第二连接肽与第二截短体连接。
在某些实施方案中，所述第一连接肽和第二连接肽各自独立地包含一个或几个(例如1个、2个或3个)如(GmS)n所示的序列，其中m选自1-6的整数，n选自1-6的整数。在某些实施方案中，m为3、4、或5。在某些实施方案中，n为1或2。在某些实施方案中，所述第一连接肽和第二连接肽具有SEQ ID NO：18所示的序列。
在某些实施方案中，所述融合蛋白从N端至C端依次包含：信号肽，第一连接肽，第一截短体，连接体，第二截短体，第二连接肽，膜锚定区。
在某些实施方案中，所述融合蛋白具有SEQ ID NO：19、20、45或46所示的序列。
在另一方面，本申请提供了一种核酸分子，其包含编码如前所述的融合蛋白的核苷酸序列。
在某些实施方案中，所述核苷酸序列根据宿主细胞的密码子偏好性进行了密码子优化或未进行优化。
在另一方面，本申请提供了一种载体，其包含如前所述的核酸分子。
在某些实施方案中，所述载体是病毒载体。
在某些实施方案中，所述病毒载体选自：流感病毒载体，肠道病毒载体，逆转录酶病毒载体，腺病毒载体，腺相关病毒载体，疱疹病毒载体，痘病毒载体，杆状病毒载体，乳头瘤病毒载体，或乳头多瘤空泡病毒载体。
在另一方面，本申请提供了一种宿主细胞，其包含如前所述的融合蛋白或如前所述的核酸分子或如前所述的载体。
在某些实施方案中，所述宿主细胞选自原核细胞(例如大肠杆菌细胞)，真核细胞。
在某些实施方案中，所述真核细胞是哺乳动物细胞，例如小鼠细胞、人细胞。
在某些实施方案中，所述融合蛋白包含膜锚定区，并展示在所述宿主细胞的细胞膜的表面。
在另一方面，本申请提供了一种表达或产生如前所述的融合蛋白的方法，所述方法包括，在允许蛋白质表达的条件下，培养如前所述的宿主细胞，以及任选地，回收或纯化所表达的融合蛋白。
在另一方面，本申请提供了一种试剂盒，其包含抗原组分，以及能够展示所述抗原组分的载体组分；其中，
所述抗原组分包含(i)如前所述的融合蛋白，(ii)如前所述的核酸分子，和/或(iii)由如前所述的核酸分子转录的mRNA产物；
所述载体组分选自：纳米材料(例如，脂质纳米颗粒、蛋白质纳米颗粒、聚合物纳米颗粒、无机纳米载体和仿生纳米颗粒)，细菌的外膜囊泡(OMVs)，多聚化基座，病毒样颗粒(VLP)，或其任意组合。
在某些实施方案中，所述试剂盒中的抗原组分和载体组分单独提供，或形成复合物提供。
在某些实施方案中，所述抗原组分是单体或多聚体(例如，二聚体，三聚体，四聚体，五聚体)。
在某些实施方案中，所述试剂盒中的多聚化基座和/或VLP以蛋白或包含编码所述蛋白的核苷酸序列的核酸分子的形式提供。
在某些实施方案中，所述多聚化基座具有如SEQ ID NO:21或22所示的氨基酸序列。
在某些实施方案中，所述VLP是由获自RSV，戊型肝炎病毒(HEV)，乙型肝炎病毒(HBV)，人乳头瘤病毒(HPV)，或人类免疫缺陷病毒(HIV)的蛋白组装而成。
在另一方面，本申请提供了一种药物组合物，其包含药学上可接受的载体和/或赋性剂，以及选自下列(1)至(4)的任意一项或多项：
(1)如前所述的融合蛋白；
(2)如前所述的核酸分子；
(3)如前所述的载体；
(4)如前所述的宿主细胞。
在另一方面，本申请提供了用于产生对抗呼吸道合胞病毒(RSV)的疫苗的方法，这些方法包括提供如前所述的试剂盒或药物组合物，并将其配制成药学上可接受的疫苗。
在另一方面，本申请提供了一种疫苗，其包含如前所述的融合蛋白，或如前所述的核酸分子，或由如前所述的核酸分子转录的mRNA产物，或如前所述的载体。
在某些实施方案中，所述疫苗由如前所述的试剂盒或药物组合物制备而成。
在某些实施方案中，所述疫苗还包含佐剂。
在某些实施方案中，可以将上述疫苗给予受试者，例如人类受试者。用于单独给予的疫苗中的融合蛋白的总剂量可以是例如约0.01μg至约10mg，例如1μg-1mg，例如10μg-100μg。确定所给与的剂量可以通过实验确定，确定剂量对于本领域技术人员来说是常规的。
在另一方面，本申请提供了一种诱导针对RSV的抗体的方法，所述方法包括在体外的细胞中或在受试者体内施用(例如，注射)有效量的如前所述的融合蛋白，或如前所述的核酸分子，或如前所述的载体，或权如前所述的宿主细胞，或如前所述的药物组合物，或如前所述的疫苗。
在某些实施方案中，所述抗体为中和抗体。
在某些实施方案中，施用包括胃肠外给予，如皮内、肌内、皮下、经皮、或粘膜给予，例如鼻内、经口等。在某些实施方案中，通过肌内注射来给予组合物。
此外，本发明的蛋白可以用作诊断工具，例如通过确定受试者的血清中是否存在能够结合本申请的融合蛋白的抗体来测试受试者的免疫状态。
因此，在另一方面，本申请提供了一种用于在体外检测受试者是否存在RSV感染的方法，所述方法包括：使获得自所述受试者的生物样品与如前所述的融合蛋白接触；以及，检测是否存在由所述融合蛋白和抗体形成的复合物。
在某些实施方案中，所述受试者为哺乳动物，例如，小鼠，人。
在某些实施方案中，所述生物样品选自全血，血清，血浆，或其任何组合。
在另一方面，本申请提供了筛选能够抑制RSV感染细胞的候选药物的方法，所述方法包括，在将如前所述的融合蛋白，或如前所述的载体，或如前所述的药物组合物，或 如前所述的疫苗与宿主细胞接触之前、同时或之后，将所述宿主细胞与所述候选药物接触。
在另一方面，本申请提供了如前所述的融合蛋白，或如前所述的核酸分子，或如前所述的载体，或如前所述的宿主细胞，或如前所述的药物组合物，或如前所述的疫苗在制备试剂盒中的用途，所述试剂盒用于诱导受试者对RSV的免疫应答。
在某些实施方案中，所述免疫应答包括诱导受试者产生针对RSV的抗体。
在某些实施方案中，所述抗体为中和抗体。
在某些实施方案中，所述受试者为哺乳动物，例如，小鼠，人。
在另一方面，本申请提供了如前所述的融合蛋白，或如前所述的核酸分子，或如前所述的载体，或如前所述的宿主细胞，或如前所述的药物组合物，或如前所述的疫苗在制备试剂盒中的用途，所述试剂盒用于预防和/或治疗RSV感染或由RSV感染所引起的疾病和/或症状。
在某些实施方案中，所述受试者为哺乳动物，例如，小鼠，人。在某些实施方案中，由RSV感染所引起的疾病和症状选自细支气管炎，肺炎，哮喘，阻塞性肺病，心肺并发症。
术语定义
在本文中，术语“呼吸道合胞病毒”和“RSV”具有相同的含义，其是属于肺炎病毒科，肺病毒属的一种病毒。RSV基因组全长约15Kb，包含10个基因，编码11种蛋白，包括8种结构蛋白(F、G、M2-1、M2-2、SH、N、P、L)和3种非结构蛋白(NS1、NS2、NS3)。其中融合蛋白(fusion protein,F)和粘附蛋白(attachment protein,G)是两个主要的包膜糖蛋白，F蛋白为I型糖蛋白，经细胞蛋白酶裂解为F1和F2后具有生物学活性，能使病毒包膜与宿主细胞膜融合形成多核巨细胞。F蛋白的序列可从公共数据库中获得(例如，GenBank数据库)。在某些实施方案中，野生的F蛋白的氨基酸序列如SEQ ID NO:1所示。
如本文中所使用的，术语“野生的”或“天然的”可互换地使用。当这些术语用于描述核酸分子、多肽或蛋白时，其表示该核酸分子、多肽或蛋白在自然界中存在，发现于自然界，并且未经过人工的任何修饰或加工。如本文中所使用的，野生的呼吸道合胞病毒 (RSV)的F1/F2蛋白是指天然存在的，具有生物学活性的F1/F2蛋白。本领域技术人员可以方便地从各种公共数据库(例如GenBank数据库)获得F1和F2蛋白的氨基酸序列。例如，野生的RSV的F2蛋白的氨基酸序列可如SEQID NO：13所示。野生的RSV的F1蛋白的氨基酸序列可如SEQID NO：12所示。
如本文中所使用的，术语“C端截短X个氨基酸”是指，C端最末端连续的X个氨基酸被截短。同理，术语“N端截短X个氨基酸”是指，N端最末端连续的X个氨基酸被截短。
如本文中所使用的，术语“载体”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本领域技术人员所知晓的，密码子存在简并性。即，在蛋白质的翻译过程中，每个氨基酸可对应1种或多种密码子，例如可对应多达6种密码子。不同的物种在使用编码某一氨基酸的简并密码子时存在着很大的差异，有着不同的偏好。这种偏好现象即被称为“密码子偏好性”。因此，如本文中所使用的，术语“密码子偏好性”是指某一物种偏爱使用某些特定的密码子来编码氨基酸的情况。根据密码子偏好性来优化核酸分子的序列在某些情况下是特别有利的，例如，可能有助于提高核酸分子所编码的蛋白质的表达水平。例如，当使用大肠杆菌(或人细胞)来表达蛋白或其片段时，针对大肠杆菌(或人细胞)的密码子偏好性来优化编码蛋白或其片段的核酸序列将是潜在有利的。
如本文中所使用的，术语“病毒样颗粒(VLP)”是一种多聚体颗粒，其结构与天然 的病毒颗粒相似或不相似。在某些实施方案中，VLP是天然的病毒颗粒。在某些实施方案中，VLP是由蛋白组装成的病毒样颗粒。经证实，一些病毒(例如，RSV，HBV，HEV，HPV)的蛋白(例如，衣壳蛋白，表面蛋白，包膜蛋白)在适当的表达系统中重组表达之后，可以自发形成VLP。
如本文中所使用的，术语“药学上可接受的”意指，制药领域公认的可用于动物，特别是可用于人的。如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂(包括但不限于磷酸盐缓冲液)，表面活性剂(包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80)，佐剂，离子强度增强剂(包括但不限于氯化钠)，稀释剂，赋形剂，用于容纳或施用治疗剂的介质，以及其任何组合。
如本文中所使用的，药学上可接受的载体可以是无菌液体，诸如水和油，包括源自石油、动物、植物的或合成的油，诸如花生油、大豆油、矿物油、芝麻油等等。当静脉内施用药用组合物时，生理盐水是优选的载体。盐水溶液以及水性右旋糖和甘油溶液也可用作液态载体，特别是用于可注射溶液。
如本文中所使用的，药学上可接受的赋形剂可包括淀粉、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、滑石、氯化钠、奶粉、甘油、丙烯、乙二醇、水、乙醇等等。如果需要，药物组合物还可以包含润湿剂，或乳化剂例如透明质酸钠，或pH缓冲剂。药物组合物可以采取溶液、悬浮液、乳状液、片剂、丸剂、胶囊、粉剂、缓释配方等形式。
如本文中所使用的，术语“受试者”是指哺乳动物，包括但不限于，人，啮齿类动物(小鼠，大鼠，豚鼠)，狗，马，牛，猫，猪，猴，黑猩猩等。优选地，受试者是人。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病有效量是指，足以预防，阻止，或延迟疾病的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的 蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
发明的有益效果
本申请的融合蛋白包含独特截短方式的RSV F1截短体和F2截短体，截短的融合蛋白诱导特异性抗体的能力与全长的pre-F蛋白相当，还能够诱导显著高于全长pre-F蛋白的中和抗体滴度。在这其中，融合蛋白所包含的连接体也起到了提高pre-F特异性表位siteΦ的稳定性的作用。进一步的，在获得了上述融合蛋白后，对其进行了突变，突变后的融合蛋白与抗体具有更强的亲和力，还具有更高的热稳定性，和更强的免疫原性。
综上，本申请的融合蛋白表现出了良好的保护性及安全性，适用于各种形式的疫苗平台，例如核酸疫苗、重组蛋白疫苗、病毒载体疫苗及颗粒化疫苗等。因此，本申请的融合蛋白在诱导受试者对RSV的免疫应答，以及预防和/或治疗RSV感染或由RSV感染所引起的疾病和/或症状中具有较大的潜力。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了包含不同连接肽的F蛋白转染293t细胞后，高中和抗体表位site II及的表达结果。
图2显示了野生型F蛋白Fwt及F蛋白截短体LC2的结构示意图。
图3显示了AlphaFold 2预测的F蛋白截短体LC2的结构示意图。
图4为免疫印迹法(Western blot)检测F蛋白截短体的表达，第一泳道为Marker，第二泳道为pre-F蛋白SC-TM，第三泳道为F蛋白截短体LC2。
图5为免疫荧光(Immunofluorescence)检测F蛋白截短体LC2与site II特异性抗体mota(图5中的A)和特异性抗体D25、AM22(图5中的B、C)的结合。
图6为编码F蛋白截短体的mRNA疫苗诱导小鼠产生的血清结合抗体滴度及血清抗体分型。
图7为编码F蛋白截短体的mRNA疫苗诱导小鼠产生的中和抗体滴度结果示意图。
图8显示了野生型F蛋白Fwt，及突变的F蛋白截短体LC2A的构建示意图。其中，野生型F蛋白Fwt包括信号肽，F2，F1，膜锚定区和胞质区；突变的F蛋白截短体LC2A包括信号肽，第一连接肽，突变的第一截短体，连接体，突变的第二截短体，第二连接肽，多聚化基序。
图9显示了对照蛋白SC-TM及突变的F蛋白截短体的聚丙烯酰胺凝胶电泳结果图，其中，第一泳道为marker，第二泳道为LC2A，第三泳道为SC-TM。
图10显示了SC-TM及7种突变的F蛋白截短体与三种F蛋白抗体的结合情况。
图11显示了通过Biacore检测SC-TM和7种突变的F蛋白截短体与两种F蛋白抗体的亲和力结果，通过DSF检测的蛋白质热稳定性结果以及通过ELISA检测的表位热稳定性结果。
图12显示了SC-TM及7种突变的F蛋白截短体免疫小鼠后血清中的抗体滴度。其中，图12A为与Pre-F的结合抗体滴度，图12B为与Post-F的结合抗体滴度。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述






具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。例如，本发明中所使用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等常规技术，可参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR 2：实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1.F蛋白截短体的柔性连接肽的筛选实验
RSV F蛋白在变构过程中，疏水空腔内融合肽的释放是一个非常关键的触发事件。为了帮助融合肽稳定在疏水空腔内以稳固pre-F构象，在F2(SEQ ID NO:12)的C端及F1(SEQ ID NO:11)的N端引入不同长度的柔性连接肽。共设计了6种柔性连接肽，氨基酸序列分别如SEQ ID NO:23，2，24，25，26和27所示。上述柔性连接肽修饰的F蛋白核苷酸序列提交上海生工生物工程公司进行人源化密码子优化，在5’端及3’端分别引入HindIII及xBaI酶切位点，接入pcDNA3.1真核表达载体，构建含柔性连接肽的F蛋白重组质粒，分别命名为LA，LB，LC，LD，LE和LF。使用免疫荧光检测柔性连接肽修饰的F蛋白高中和抗体表位site II及的表达。
1)第一天晚上接种3×104个/孔293T细胞到96孔板中。
2)第二天，当细胞汇合度达到85％时进行质粒转染。取5μL的opti-MEM培养基于96孔U底板中，加入0.2μg的质粒，0.3μL的P3000，涡旋震荡混匀，标记为A。再取5μL的opti-MEM培养基于96孔U底板中，加入0.3μL的Lipofectamine3000，涡旋震荡混匀标记为B。将A管加入到B管，涡旋震荡混匀，静置15分钟。之后将溶液加到铺好的293t细胞板中，轻轻摇匀后置于细胞培养箱中培养。
3)转染24小时后，加入每孔加入100μL的4％甲醛溶液，室温固定30分钟。
4)弃掉细胞培养液及甲醛溶液，用PBS清洗3次，每次5分钟。
5)每孔加入100μL含2％脱脂奶粉的PBS，室温封闭2小时。
6)PBS清洗一次后，分别加入识别不同表位的抗体(site II：motavizumab， AM22、D25)，室温孵育1小时。
7)用PBS清洗3次。
8)每孔加入100μL Alexa Fluor568(1：2000稀释)(厂家：Thermo；目录号：A-11013)荧光二抗，室温孵育1小时。
9)用PBS清洗3次。
10)每孔加入100μL细胞核染料DAPI(厂家：Invitrogen；目录号：D1306)，染色10分钟。
11)用PBS清洗3次。
12)于高内涵成像系统拍照及定量分析。
图1显示了包含不同连接肽的F蛋白转染293t细胞后，高中和抗体表位site II及 的表达结果。结果显示，包含柔性连接肽LA、LC的F蛋白保留了最高水平的 的表达。
实施例2.F蛋白截短体的设计方案
自GenBank数据库获得RSV A2Fusion蛋白氨基酸序列(GenBank ID:FJ614814.1)，Fwt的氨基酸序列如SEQ ID NO:1所示。F蛋白截短体的氨基酸序列特征为，F2肽链及F1肽链的N端及C端分别进行截短，并且通过含脯氨酸的柔性连接肽将截短后的F2与F1进行连接，帮助稳固pre-F构象。F1肽链的C末端通过柔性连接肽与不同的展示基座连接。共制备了种突变体，具体如下：
(1)将SEQ ID NO:1的第31位到105位氨基酸与145位到322位氨基酸通过含脯氨酸的柔性连接肽(SEQ ID NO:2)连接，截短后的F蛋白全长314个氨基酸，命名为LC1，其氨基酸序列如SEQ ID NO:3所示。LC1保留了site V、site III及siteII表位。
(2)将SEQ ID NO:1的第51位到105位氨基酸与145位到306位氨基酸通过含脯氨酸的柔性连接肽(SEQ ID NO:2)连接，截短后的F蛋白全长284个氨基酸，命名为LC2，其氨基酸序列如SEQ ID NO:4所示。LC2保留了site V及siteII表位。
(3)将SEQ ID NO:1的第56位到105位氨基酸与145位到306位氨基酸通过含脯氨酸的柔性连接肽(SEQ ID NO:2)连接，截短后的F蛋白全长280个氨基酸，命名为LC3，其氨基酸序列如SEQ ID NO:5所示。LC3保留了及siteII表位。
(4)将SEQ ID NO:1的第51位到105位氨基酸与145位到297位氨基酸通过含脯氨酸的柔性连接肽(SEQ ID NO:2)连接，截短后的F蛋白全长276个氨基酸，命名为LC4，其氨基酸序列如SEQ ID NO:6所示。LC4保留了及siteII表位。
(5)将SEQ ID NO:1的第56位到105位氨基酸与145位到297位氨基酸通过含脯氨酸的柔性连接肽(SEQ ID NO:2)连接，截短后的F蛋白全长271个氨基酸，命名为LC5，其氨基酸序列如SEQ ID NO:7所示。LC5保留了siteII表位。
实施例3.表达F蛋白截短体的重组质粒的构建
通过编码柔性连接肽的核苷酸序列分别将编码5种F蛋白截短体的核苷酸序列与编码F蛋白胞质段(氨基酸序列如SEQ ID NO:8所示)的核苷酸序列进行连接。并且在上 述截短体的核苷酸序列的5’端引入编码信号肽(氨基酸序列如SEQ ID NO:9所示)的核苷酸序列。将F蛋白截短体的核苷酸序列提交上海生工生物工程公司进行人源化密码子优化，在5’端及3’端分别引入HindIII及xBaI酶切位点，接入pcDNA3.1真核表达载体，构建含F蛋白截短体目的基因的重组质粒。图2显示了F蛋白截短体LC2的连接示意图。图3显示了AlphaFold 2预测的F蛋白截短体的结构示意图。另外按照前述方法构建了处于融合前结构的全长F蛋白SC-TM重组质粒作为对照(SC-TM蛋白的序列获自GenBank：5C6B_F)，交由上海生工合成。测序成功的质粒经质粒大提后进行xBaI单酶切，利用毛细管电泳鉴定单酶切结果，鉴定正确后的质粒通过分光光度计检测OD260及OD280，测定其DNA浓度及纯度，-20℃保存质粒。
实施例4：免疫印迹(Western Blot)法检测重组质粒的表达
1)第一天晚上接种8×105个/孔293T细胞到6孔板中。
2)第二天，当细胞汇合度达到85％时进行质粒转染。取125μL的opti-MEM(厂家：Gibco；目录号：31985-070)培养基于1.5mL离心管中，加入2μg的质粒，4μL的P3000，涡旋震荡混匀，标记为A。再取125μL的opti-MEM培养基于另一个1.5mL离心管中，加入4μL的Lipofectamine3000(厂家：Invitrogen；目录号：L3000015)，涡旋震荡混匀标记为B。将A管加入到B管，涡旋震荡混匀，静置15分钟。之后将溶液加到铺好的293t六孔板中，轻轻摇匀后置于细胞培养箱中培养。
3)第三天，弃掉培养液，加入预冷的PBS清洗细胞两遍。
4)加入150μL的含蛋白酶抑制剂的RIPA裂解液，冰上裂解细胞30分钟。
5)12000rpm，4℃离心20分钟后收集上清。
6)使用BCA定量试剂(厂家：PIERCE；目录号：23225)对上清的蛋白浓度进行测定。
7)电泳样品上样量为20μg/孔，样品中加入β巯基乙醇及Loading buffer，100℃热变性10分钟。
8)加入商品化的12％预制胶，180V的电压，电泳40分钟。
9)电泳完成后，用湿转的方法将蛋白转印到NC膜上。
10)用含5％脱脂奶粉的PBS溶液封闭2小时。
11)PBST清洗一遍。
12)人源单克隆抗体Motavizumab用2％脱脂奶粉1：2000稀释，将NC膜转移到稀 释好的抗体中，室温摇床孵育2小时。
13)用PBST清洗三次，每次5分钟。
14)用含2％脱脂奶粉的PBS稀释GAH-HRP，将NC膜转移到稀释好的二抗中，室温摇床孵育1h。
15)用PBST清洗三次，每次五分钟。
16)加入底物显色，于WB成像系统检测并拍照。
17)如图4所示，Western Blot分析F蛋白截短体在293t细胞上的表达水平。检测抗体为Motavizumab。图中最左侧通道为180kD的蛋白Marker。SC-TM为对照的融合前F蛋白，LC2为本发明所述的F蛋白截短体。
实施例5：免疫荧光(Immunofluorescence)检测F蛋白截短体的表位完整性
1)第一天晚上接种3×104个/孔293T细胞到96孔板中。
2)第二天，当细胞汇合度达到85％时进行质粒转染。取5μL的opti-MEM培养基于96孔U底板中，加入0.2μg的质粒，0.3μL的P3000，涡旋震荡混匀，标记为A。再取5μL的opti-MEM培养基于96孔U底板中，加入0.3μL的Lipofectamine3000，涡旋震荡混匀标记为B。将A管加入到B管，涡旋震荡混匀，静置15分钟。之后将溶液加到铺好的293t细胞板中，轻轻摇匀后置于细胞培养箱中培养。
3)转染24小时后，加入每孔加入100μL的4％甲醛溶液，室温固定30分钟。
4)弃掉细胞培养液及甲醛溶液，用PBS清洗3次，每次5min。
5)每孔加入100μL含2％脱脂奶粉的PBS，室温封闭2小时。
6)PBS清洗一次后，分别加入识别不同表位的抗体(site II：motavizumab， AM22、D25)，室温孵育1小时。
7)用PBS清洗3次。
8)每孔加入100μL Alexa Fluor568(1：2000稀释)(厂家：Thermo；目录号：A-11013)荧光二抗，室温孵育1小时。
9)用PBS清洗3次。
10)每孔加入100μL细胞核染料DAPI(厂家：Invitrogen；目录号：D1306)，染色10分钟。
11)用PBS清洗3次。
12)于高内涵成像系统拍照及定量分析。
如图5所示，免疫荧光展示了全长pre-F蛋白SC-TM，F蛋白截短体LC2的重组质粒转染293t细胞后，高中和抗体表位site II及的表达。
实施例6.F蛋白截短体mRNA疫苗的构建
按照Andrew J Bett等人发表的文献(PMID:32128257)中记载的方法将RNA转录相关元件构建于含F蛋白截短体LC2及全长pre-F蛋白SC-TM的体外转录质粒，质粒抽提及鉴定如实施例1所述一致。质粒大提后用限制性内切酶BspQ1线性化。用T7体外转录试剂盒(厂家：南京诺唯赞生物科技有限公司；目录号：DD4202)进行转录获得加帽的mRNA。使用DNase I消化转录模板。使用氯化锂(厂家：Invitrogen；目录号：AM9480)沉淀纯化mRNA。
将纯化后的mRNA溶解于酸性柠檬酸钠缓冲液中待用。脂纳米颗粒LNP的制备：将阳离子脂质：DSPC：胆固醇：DMG-PEG2000按摩尔比为50：10：38.5：1.5在乙醇中溶解、混合。将mRNA与脂质混合溶液分别按照流速比3：1包封。包封完成后，将其透析到PBS溶液中,获得mRNA疫苗。
实施例7.F蛋白截短体mRNA疫苗的免疫
实验用动物的饲养及实验操作均按照厦门大学实验动物使用和管理委员会制定的程序进行。实验动物为购自北京维通利华实验动物技术有限公司的BALB/c雌性小鼠，6-8周龄，饲养于厦门大学实验动物中心。
1)购买的小鼠随机分组，每组4只。按5μg/只单侧大腿股四头肌注射mRNA-LNP，对照组为不含mRNA-LNP的PBS。
2)初次免疫2周后进行二次加强免疫，共免疫两次。
3)加强免疫后14天眼眶采血，分离血清以备分析。
实施例8.血清抗体滴度及分型检测
1)包被用的pre-F蛋白及post-F蛋白的构建方法如下：从GenBank下载pre-F及post-F蛋白的序列(GenBank ID:LY628284.1,GenBank ID:6APB_A)，将编码两种蛋白的核苷酸序列克隆到pcDNA3.1真核表达载体上，利用CHO真核表达体系进行表达，亲和层析纯化获得pre-F蛋白及post-F蛋白。
2)第一天晚上将pre-F蛋白(SC-TM)及post-F蛋白分别用PBS稀释，以100ng/ 孔的用量包被96孔板，4℃过夜。
3)弃掉包被蛋白液，用PBST洗涤一次。
4)以200μL/孔的用量加入含5％胎牛血清的封闭液，37℃放置2小时。
5)弃掉封闭液，用PBST洗涤一次。
6)以100μL/孔的用量加入梯度稀释的小鼠血清(首孔200倍，5倍梯度)，37℃放置1小时。
7)弃掉血清稀释液，用PBST洗涤五次。
8)以100μL/孔的用量加入1：5000稀释的辣根过氧化物酶标记的山羊抗小鼠的IgG抗体、IgG1抗体及IgG2a抗体(厂家：Abcam；目录号：ab97265)，37℃孵育1小时。
9)弃掉二抗，用PBST洗涤五次。
10)以100μL/孔的用量加入显色液，室温避光显色10分钟。
11)以50μL/孔的用量加入终止液，酶标仪检测450nm条件时的吸光度。
12)终点滴度的判断标准为：阴性孔读值的3倍。
实验结果如图5所示。图5中的A显示了本申请的F蛋白截短体LC2诱导了高水平的pre-F结合抗体滴度，与SC-TM(即，全长pre-F蛋白)相当。图6中的B显示了以mRNA疫苗形式免疫时，诱导了更高水平的IgG2a抗体，表明呈现Th-1偏向性。
实施例9.中和抗体滴度的检测
1)眼眶采血后分离的血清于56℃孵育30分钟灭活补体。2)梯度稀释血清：首孔10倍，4倍梯度共稀释10个梯度。3)加入等体积的rRSV-mkatushka2(MOI＝0.1)，混合后于37℃孵育1小时。4)将血清病毒混合液以100μL/孔的体积转移到铺有单层Hela细胞的96孔板中，37℃孵育。5)培养24小时后，使用SpectraMax Paradigm Multi-Mode Microplate Reader读板。
实验结果如图7所示。实验结果显示，与SC-TM(即，全长pre-F蛋白)相比，本申请的F蛋白截短体LC2诱导了显著高于SC-TM的中和抗体滴度。
综上所述，本发明实施例所述的F蛋白截短体，相比于全长的F蛋白，诱导了更高水平的pre-F特异性抗体，且诱导了显著更高的中和抗体滴度。表现出了良好的保护性及安全性，适用于各种形式的疫苗平台，例如核酸疫苗、重组蛋白疫苗、病毒载体疫苗及颗粒化疫苗等。
实施例10.突变的F蛋白截短体的设计方案
本实施例以上述实施例中获得的F蛋白截短体—LC2(SEQ ID NO：4)为基础，对其进行进一步突变，共制备了7种突变体，具体如下：
(1)将SEQ ID NO：4的第29位氨基酸I突变为L，第212位氨基酸V突变为M，突变后的蛋白命名为LC2A，其氨基酸序列如SEQ ID NO：28所示。
(2)将SEQ ID NO：4的第29位氨基酸I突变为L，第214位氨基酸A突变为L，突变后的蛋白命名为LC2B，其氨基酸序列如SEQ ID NO：29所示。
(3)将SEQ ID NO：4的第29位氨基酸I突变为L，第94位氨基酸V突变为I，突变后的蛋白命名为LC2C，其氨基酸序列如SEQ ID NO：30所示。
(4)将SEQ ID NO：4的第47位氨基酸M突变为A，第114位氨基酸Y突变为I，突变后的蛋白命名为LC2D，其氨基酸序列如SEQ ID NO：31所示。
(5)将SEQ ID NO：4的第29位氨基酸I突变为L，第47位氨基酸M突变为A，第101位氨基酸V突变为I，第214位氨基酸A突变为L，突变后的蛋白命名为LC2E，其氨基酸序列如SEQ ID NO：32所示。
(6)将SEQ ID NO：4的第88位氨基酸L突变为A，第101位氨基酸V突变为I，第114位氨基酸Y突变为I，第214位氨基酸A突变为L，突变后的蛋白命名为LC2F，其氨基酸序列如SEQ ID NO：33所示。
(7)将SEQ ID NO：4的第94位氨基酸V突变为I，第114位氨基酸Y突变为I突变后的蛋白命名为LC2G，其氨基酸序列如SEQ ID NO：34所示。
实施例11.表达F蛋白截短体的重组质粒的构建
通过编码GSGS氨基酸的核苷酸序列分别将编码7种突变的F蛋白截短体的核苷酸序列与编码三聚化基序foldon(氨基酸序列如SEQ ID NO:21所示)的核苷酸序列进行连接，并且，分别在编码7种突变的F蛋白截短体的核苷酸序列的5’端引入编码信号肽(SEQ ID NO:9)的核苷酸序列，以构建融合表达基因。将突变的F蛋白截短体的核苷酸序列提交上海生工生物工程公司进行人源化密码子优化，在5’端及3’端分别引入HindIII及xBaI酶切位点，接入pcDNA3.1真核表达载体，构建含F蛋白截短体目的基因的重组质粒。另外按照前述方法构建了处于融合前结构的全长F蛋白SC-TM重组质粒作为对照(SC-TM蛋白的序列获自GenBank：5C6B_F，SEQ ID NO:35)，交由上海 生工合成。图8显示了野生型F蛋白Fwt，对照F蛋白SC-TM以及突变的F蛋白截短体LC2A的连接示意图。
将带有目的基因的质粒干粉用10μL双蒸水溶解，取1μL加入100μL DH5α感受态细胞中(大肠杆菌)。样品冰上孵育30min，42℃热击90sec，冰上孵育2min。然后把反应后的大肠杆菌加入LB培养基中，并铺到含有氨苄抗性的琼脂糖培养基平板中进行培养。待菌落长出后，挑取单克隆菌落，质粒用无内毒素质粒大提试剂盒(厂家：天根；目录号：DP117)提取备用。
实施例12.蛋白的表达和纯化
准备处于对数生长期的悬浮细胞ExpiCHOTM，将其置于125rpm，37℃，8％CO2的细胞摇床进行培养，至密度为6×106个/mL，活细胞率>98％。取25mL细胞置于新的细胞培养瓶中，作为一个转染体系。A管：1mL ExpiCHOTM Expresssion Medium中包含25μg质粒，B管：1mL ExpiCHOTM Expresssion Medium中包含80μL ExpiFectamine TM CHO Transfection Kit(厂家：Thermo Scientific；目录号：A29129)中的转染试剂。将A管与B管混合，室温静置2min，2min后将混合液倒入25mL准备好的转染细胞体系中。置于125rpm，37℃，8％CO2细胞摇床进行培养18-22h。每瓶加入ExpiFectamineTM CHO Transfection Kit中的150μL增强剂以及4mL辅料，置于125rpm，32℃，5％CO2的细胞摇床培养8-15d。培养完成后，4℃，4000rpm离心10min，收集细胞上清。
用0.22μm滤器过滤上述上清。打开AKTA仪器，先分别用A液(200mM十二水合磷酸氢二钠)和B液(100mM一水柠檬酸)冲洗A管道和B管道，装上protein A柱子。用A液以8mL/min的流速平衡protein A柱子15min以上，待仪器检测的UV值、pH值和电导率稳定后进行下一步。以6-10ml/min流速上样，随后UV值会上升，该峰为穿透峰，并继续用A液洗柱，同时收集穿透峰样品待检测。待pH值不再变化后用B液以6-10ml/min流速进液，随后pH值下降，UV值上升，该峰为洗脱峰，抗体主要存在于洗脱峰中。收取洗脱峰样品待检测。用A液平衡柱子，再用20％乙醇充满管道和protein A柱子，取下柱子，4℃保存。将穿透峰和洗脱峰的样品进行纯化，并进行SDS–PAGE鉴定(对样品进行沸水煮5min，可以使抗体重链和轻链间的二硫键打开，见《分子克隆实验指南》第二版)。纯化的单克隆抗体用20mM PBS缓冲液透析过夜，并采用紫外分光或者BCA测取浓度，分装至1.5ml管中，存放于-20℃备用。
实施例13.聚丙烯酰胺凝胶电泳
第一天晚上接种8×105个/孔293T细胞到6孔板中。第二天，当细胞汇合度达到85％时进行质粒转染。取125μL的opti-MEM(厂家：Gibco；目录号：31985-070)培养基于1.5mL离心管中，加入2μg的质粒，4μL的P3000，涡旋震荡混匀，标记为A。再取125μL的opti-MEM培养基于另一个1.5mL离心管中，加入4μL的Lipofectamine3000(厂家：Invitrogen；目录号：L3000015)，涡旋震荡混匀标记为B。将A管加入到B管，涡旋震荡混匀，静置15分钟。之后将溶液加到铺好的293t六孔板中，轻轻摇匀后置于细胞培养箱中培养。第三天，弃掉培养液，加入预冷的PBS清洗细胞两遍。加入150μL的含蛋白酶抑制剂的RIPA裂解液，冰上裂解细胞30分钟。12000rpm，4℃离心20分钟后收集上清。使用BCA定量试剂(厂家：PIERCE；目录号：23225)对上清的蛋白浓度进行测定。电泳样品上样量为20μg/孔，样品中加入β巯基乙醇及Loading buffer，100℃热变性10分钟。加入商品化的12％预制胶，180V的电压，电泳40分钟。电泳后，SDS-PAGE凝胶用0.25％的考马斯亮蓝(Sigma)染色过夜。之后，用脱色缓冲液进行脱色(300ml甲醇，100ml乙酸和600ml双蒸水)。
结果分析：对照蛋白SC-TM分子量在60kDa附近，突变的F蛋白截短体分子量较小，约30kDa。图9显示了SC-TM和LC2A的聚丙烯酰胺凝胶电泳结果。
实施例14.突变的F蛋白截短体的表位特征分析
将SC-TM和7种突变体用PBS缓冲液稀释制备包被液，终浓度为1μg/mL。在96孔酶标板中加入100μL稀释好的包被液，4℃包被过夜。用PBST(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤液洗涤一次，甩干。然后每孔加入200μL的封闭液(含有20％小牛血清及1％酪蛋白，pH值为7.4的20mM Na2HPO4/NaH2PO4缓冲液溶液)，37℃条件下封闭2小时，弃去封闭液。干燥后装入铝箔袋4℃保存备用。取识别不同表位的F蛋白特异的单克隆抗体MOTA，D25，hRSV90(3种抗体的VH和VL序列分别如表1中的SEQ ID NO：36至SEQ ID NO：41所示，3种抗体具有相同的重链恒定区，具体序列如SEQ ID NO：42所示，3种抗体具有相同的轻链恒定区，具体序列如SEQ ID NO：43所示)。以PBS溶液从100μg/ml为起始浓度开始5倍梯度稀释，共稀释8个梯度。取已包被的酶标板，每孔加入100μl已稀释的抗体样品，置于37℃温箱反应60分钟。将酶标板用PBST洗液(20mM PB7.4，150mM NaCl，0.1％Tween20) 洗涤5遍，每孔加入100μl辣根过氧化物酶(HRP)标记的羊抗人IgG反应液，置于37℃温箱反应30分钟。完成酶标记物反应步骤后，将酶标板用PBST洗液(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤5遍，每孔加入50μl TMB显色剂(购自北京万泰生物药业股份有限公司)，置于37℃温箱反应15分钟。完成显色反应步骤后，在反应完的酶标板中每孔加入50μl终止液(购自北京万泰生物药业股份有限公司)，并于酶标仪上检测各孔的OD450/630值。
结果分析：结果显示，几种突变的F蛋白截短体与检测抗体的结合能力均优于SC-TM，尤其是其中的LC2A，LC2B和LC2E，与高中和抗体D25及hRSV90的结合显著增强(图10)。
实施例15.突变的F蛋白截短体与抗体的亲和力分析
通过表面等离子共振分析(SPR)技术测定突变的F蛋白截短体与F蛋白特异性抗体D25及hRSV90的结合亲和力。用Biacore-8K(Cytiva)将带有组氨酸标签的突变F蛋白截短体以约100nM的浓度固定到NTA传感器芯片(Cytiva)上。加入梯度稀释后的抗体(浓度范围为200至0.78nM)。使用BiacoreTM Insight软件将RU数据拟合至1：1结合模型。
结果分析：结果显示，几种突变的F蛋白截短体与检测抗体的亲和力与SC-TM相当，其中LC2A与D25的亲和力相较于SC-TM提高3.85倍，LC2B与D25的亲和力相较于SC-TM提高1.25倍，LC2B与hRSV90的亲和力相较于SC-TM提高3.85倍(图11)。
实施例16.突变的F蛋白截短体的热稳定性分析
(1)使用差示扫描荧光法(Differential Scanning Fluorimetry,DSF)分析SC-TM及7种突变的F蛋白截短体的热稳定性。先将待测蛋白样品用无菌PBS溶液稀释到浓度为1mg/mL，2倍梯度稀释，共做8个梯度，确定合适的样品浓度，以空白PBS作为对照。用去离子水稀释染料SYPRO Orange(厂家：SIGMA-ALDRICH；目录号：S5692-500UL)，使其终浓度为50X。取45μL稀释好的待测蛋白样品加入伯乐PCR板中，随后加入5μL 50X的SYPRO Orange染料。将上述配置好的体系放入荧光定量PCR仪中(BIO-RAD)，激发光和发射波长分别调至荧光染料相应波长，升温速率设为0.5℃/10s，温度变化范围从0℃到100℃。使用BIO-RAD manager处理数据，绘制荧光 强度随温度的变化曲线，通过拟合波尔兹曼(Boltzmann transport equation，BTE)方程，计算出蛋白样品的Tm值。
结果分析：结果显示，与对照蛋白Pre-F相比，7种突变的F蛋白截短体的热稳定性没有显著差异，其中，LC2A的Tm值略高于SC-TM(59.3℃vs.58.4℃)(图11)。
(2)使用酶联免疫吸附实验(ELISA)分析SC-TM与7种突变的F蛋白截短体的表位热稳定性。使用PBS溶液将待测蛋白稀释至10μg/mL，分别放置在25℃、50℃、70℃和90℃温度下处理1小时，随后立刻至于冰上冷却。包板及检测步骤同实施例4所述一致。
结果分析：结果显示，对照蛋白SC-TM与7种突变的F蛋白截短体在70℃和90℃时仍然可检测到与D25的结合，且7种突变体的结合均优于SC-TM(图11)。
实施例17.突变的F蛋白截短体的免疫原性分析
(1)小鼠免疫实验
实验用动物的饲养及实验操作均按照厦门大学实验动物使用和管理委员会制定的程序进行。实验动物为购自北京维通利华实验动物技术有限公司的BALB/c雌性小鼠，6-8周龄，饲养于厦门大学实验动物中心。免疫方案为将SC-TM和7种突变的F蛋白截短体分别与铝佐剂按1：1的体积比混合，用于免疫小鼠。每组为5只小鼠，免疫方式为单侧大腿肌肉注射，免疫剂量为20μg蛋白/只小鼠。两针免疫，于加免后第7天采集小鼠眼眶血，分离血清，血清样本在56℃下灭活30分钟，置于4℃备用。
(2)血清中Pre-F和Post-F蛋白特异性抗体滴度评估
包被用的Pre-F蛋白为SC-TM，Post-F蛋白的序列从GenBank上下载(GenBank ID:6APB_A，SEQ ID NO：44)，将编码Post-F蛋白的核苷酸序列克隆到pcDNA3.1真核表达载体上，利用ExpiCHOTM体系表达，表达步骤同实施例3所述一致。蛋白包被及封闭步骤同实施例4所述一致。梯度稀释血清，首孔200倍，5倍梯度，共稀释8个梯度，以100μL/孔的量将稀释好的血清加入封闭好的96孔板中，37℃孵育1小时。弃掉血清，用PBST洗涤5次，甩干。以100μL/孔的用量加入1：5000稀释的辣根过氧化物酶标记的山羊抗小鼠的IgG抗体(厂家：Abcam；目录号：ab97265)，37℃孵育1小时。弃掉二抗，用PBST洗涤五次。以100μL/孔的用量加入显色液，室温避光显色10分钟。以50μL/孔的用量加入终止液，酶标仪检测450nm条件时的吸光度。终点滴度的判断标准为：阴性孔读值的3倍。
结果分析：结果显示7种突变的F蛋白截短体均可以诱导Pre-F偏向性的抗体应答，其中LC2A，LC2B，LC2C，LC2D和LC2F的Pre-F结合抗体滴度与全长Pre-F蛋白SC-TM相当(图12)。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (20)

  1. 一种融合蛋白，其包含第一截短体，第二截短体，以及连接所述第一截短体和第二截短体的连接体；其中，
    第一截短体与野生的呼吸道合胞病毒(RSV)的F2蛋白相比，在野生的RSV的F2蛋白的N端截短了5个至25个氨基酸(例如，5个-8个，9个-12个，13个-16个，17个-20个，21个-25个)，以及C端截短了3个，4个或5个氨基酸；
    第二截短体与野生的RSV的F1蛋白相比，在野生的RSV的F1蛋白的N端截短了7个，8个或9个氨基酸，以及C端截短了238个至268个氨基酸(例如，238个-241个，242个-245个，246个-249个，250个-253个，254个-257个，258个-261个，262个-265，266个-268个)。
  2. 权利要求1所述的融合蛋白，其中，第一截短体与野生的RSV的F2蛋白相比，在野生的RSV的F2蛋白的N端截短了5个或25个氨基酸，以及C端截短了4个氨基酸；
    优选地，野生的RSV的F2蛋白具有如SEQ ID NO:13所示的氨基酸序列；
    优选地，所述第一截短体具有如SEQ ID NO:16或17所示的氨基酸序列。
  3. 权利要求1或2所述的融合蛋白，其中，第二截短体与野生的RSV的F1蛋白相比，在野生的RSV的F1蛋白的N端截短了8个或9个氨基酸，以及C端截短了252个或268个氨基酸；
    优选地，野生的RSV的F1蛋白具有如SEQ ID NO:12所示的氨基酸序列；
    优选地，所述第二截短体具有如SEQ ID NO:14或15所示的氨基酸序列。
  4. 权利要求1-3任一项所述的融合蛋白，其中，所述连接体包含至少1个(例如，1个，2个)脯氨酸；
    优选地，所述连接体具有3-20个氨基酸(例如，3个，4个，5个，6个，7个，8个，9个，10个，15个，20个)；更优选地，所述连接体具有5-8个氨基酸；
    优选地，所述连接体包含多个(例如，2个，3个，4个，5个，6个，7个)甘氨酸以及至少1个(例如，1个，2个，3个)脯氨酸；
    优选地，所述连接体具有如SEQ ID NO:2所示的序列；
    优选地，所述第一截短体位于所述连接体的N端；
    优选地，所述第二截短体位于所述连接体的C端；
    优选地，所述融合蛋白从N端至C端依次包含：第一截短体，连接体，第二截短体；
    优选地，所述融合蛋白具有如SEQ ID NO:3或4所示的氨基酸序列。
  5. 权利要求1-4任一项所述的融合蛋白，其中，所述融合蛋白还包含1个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)；
    优选地，在所述融合蛋白的第一截短体和/或第二截短体上包含1个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)；
    优选地，所述融合蛋白的序列与SEQ ID NO：3-7任一项所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)；
    优选地，所述置换为保守置换。
  6. 权利要求5所述的融合蛋白，其中，所述融合蛋白的序列与SEQ ID NO：4所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)；
    优选地，与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第29位位点中具有氨基酸的置换，并且，所述融合蛋白的序列在对应于SEQ ID NO：4的第47位-第214位位点中的任意一个位点也具有氨基酸的置换；
    优选地，与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第114位位点中具有氨基酸的置换，并且，所述融合蛋白的序列在对应于SEQ ID NO：4的第29位-第94位位点中的任意一个位点也具有氨基酸的置换；
    优选地，与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的下述位点中具有一个或几个氨基酸的置换：在第29位，第47位，第212位，214位，第88位，第94位，第114位和第101位；
    优选地，所述融合蛋白具有选自下列的一项或多项特征：
    (1)与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第29位和第212位位点中具有氨基酸的置换；
    (2)与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第29位和第214位位点中具有氨基酸的置换；
    (3)与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第29位和第114位位点中具有氨基酸的置换；
    (4)与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第29位，第47位，第101位和第214位位点中具有氨基酸的置换；
    (5)与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第88位，第101位，第114位和第214位位点中具有氨基酸的置换；
    (6)与SEQ ID NO：4所示的序列相比，所述融合蛋白的序列在对应于SEQ ID NO：4的第94位，第114位位点中具有氨基酸的置换。
  7. 权利要求6所述的融合蛋白，其中，所述融合蛋白具有选自下列的一项或多项特征：
    (1)所述第29位位点的置换是由异亮氨酸I置换为亮氨酸L；
    (2)所述第212位位点的置换是由酪氨酸V置换为甲硫氨酸M；
    (3)所述第214位位点的置换是由丙氨酸A置换为亮氨酸L；
    (4)所述第94位位点的置换是由氨基酸V置换为异亮氨酸I；
    (5)所述第47位位点的置换是由甲硫氨酸M置换为丙氨酸A；
    (6)所述第114位位点的置换是由酪氨酸Y置换为异亮氨酸I；
    (7)所述第101位位点的置换是由缬氨酸V置换为异亮氨酸I；
    (8)所述第214位位点的置换是由丙氨酸A置换为亮氨酸L；
    (9)所述第88位位点的置换是由氨基酸亮氨酸L置换为丙氨酸A；
    优选地，所述融合蛋白具有如SEQ ID NO：28-34任一项所示的氨基酸序列。
  8. 权利要求1-7任一项所述的融合蛋白，其中，所述融合蛋白还包含：信号肽，多聚化基序(例如，三聚化基序)和/或膜锚定区；
    优选地，所述膜锚定序列具有如SEQ ID NO:8所示的氨基酸序列；
    优选地，所述信号肽具有如SEQ ID NO:9所示的氨基酸序列；
    优选地，所述多聚化基序具有如SEQ ID NO:21或22所示的氨基酸序列；
    优选地，所述信号肽位于第一截短体的N端；优选地，所述信号肽通过第一连接肽与第一截短体连接；
    优选地，所述膜锚定区位于第二截短体的C端；优选地，所述膜锚定区通过第二连接肽与第二截短体连接；
    优选地，所述多聚化基序位于第二截短体的C端；优选地，所述多聚化基序通过第二连接肽与第二截短体连接；
    优选地，所述第一连接肽和第二连接肽各自独立地包含一个或几个(例如1个、2个或3个)如(GmS)n所示的序列，其中m选自1-6的整数，n选自1-6的整数；优选地，m为3、4、或5；优选地，n为1或2；优选地，所述第一连接肽和第二连接肽具有SEQ ID NO：18所示的序列；
    优选地，所述融合蛋白从N端至C端依次包含：信号肽，第一连接肽，第一截短体，连接体，第二截短体，第二连接肽，膜锚定区；
    优选地，所述融合蛋白具有SEQ ID NO：19、20、45或46所示的序列。
  9. 一种核酸分子，其包含编码权利要求1-8任一项所述的融合蛋白的核苷酸序列；
    优选地，所述核苷酸序列根据宿主细胞的密码子偏好性进行了密码子优化或未进行优化。
  10. 一种载体，其包含权利要求9所述的核酸分子；
    优选地，所述载体是病毒载体；
    优选地，所述病毒载体选自：流感病毒载体，肠道病毒载体，逆转录酶病毒载体，腺病毒载体，腺相关病毒载体，疱疹病毒载体，痘病毒载体，杆状病毒载体，乳头瘤病毒载体，或乳头多瘤空泡病毒载体。
  11. 一种宿主细胞，其包含权利要求1-8任一项所述的融合蛋白或权利要求9所述的核酸分子或权利要求10所述的载体；
    优选地，所述宿主细胞选自原核细胞(例如大肠杆菌细胞)，真核细胞；
    优选地，所述真核细胞是哺乳动物细胞，例如小鼠细胞、人细胞；
    优选地，所述融合蛋白展示在所述宿主细胞的细胞膜的表面。
  12. 一种表达或产生权利要求1-8任一项所述的融合蛋白的方法，所述方法包括，在允许蛋白质表达的条件下，培养权利要求所述11的宿主细胞，以及任选地，回收或纯化所表达的融合蛋白。
  13. 一种试剂盒，其包含抗原组分，以及能够展示所述抗原组分的载体组分；其中，
    所述抗原组分包含(i)权利要求1-8任一项所述的融合蛋白，(ii)权利要求9所述的核酸分子，和/或(iii)由权利要求9所述的核酸分子转录的mRNA产物；
    所述载体组分选自：纳米材料(例如，脂质纳米颗粒、蛋白质纳米颗粒、聚合物纳米颗粒、无机纳米载体和仿生纳米颗粒)，细菌的外膜囊泡(OMVs)，多聚化基座，病毒样颗粒(VLP)，或其任意组合；
    优选地，所述试剂盒中的抗原组分和载体组分单独提供，或形成复合物提供；
    优选地，所述抗原组分是单体或多聚体(例如，二聚体，三聚体，四聚体，五聚体)；
    优选地，所述试剂盒中的多聚化基座和/或VLP以蛋白或核酸的形式提供；
    优选地，所述多聚化基座具有如SEQ ID NO:21或22所示的氨基酸序列；
    优选地，所述VLP是由获自RSV，戊型肝炎病毒(HEV)，乙型肝炎病毒(HBV)，人乳头瘤病毒(HPV)，或人类免疫缺陷病毒(HIV)的蛋白组装而成。
  14. 一种药物组合物，其包含药学上可接受的载体和/或赋性剂，以及选自下列(1)至(4)的任意一项或多项：
    (1)权利要求1-8任一项所述的融合蛋白；
    (2)权利要求9所述的核酸分子；
    (3)权利要求10所述的载体；
    (4)权利要求11所述的宿主细胞。
  15. 一种疫苗，其包含权利要求1-8任一项所述的融合蛋白，或权利要求9所述的核酸分子，或由权利要求9所述的核酸分子转录的mRNA产物，或权利要求10所述的载体；
    优选地，所述疫苗由权利要求13所述的试剂盒制备而成；
    优选地，所述疫苗还包含佐剂。
  16. 一种诱导针对RSV的抗体的方法，所述方法包括在体外的细胞中或在受试者体内施用(例如，注射)有效量的权利要求1-8任一项所述的融合蛋白，或权利要求9所述的核酸分子，或权利要求10所述的载体，或权利要求11所述的宿主细胞，或权利要求14所述的药物组合物，或权利要求15所述的疫苗；
    优选地，所述抗体为中和抗体。
  17. 一种用于在体外检测受试者是否存在RSV感染的方法，所述方法包括：使获得自所述受试者的生物样品与权利要求1-8任一项所述的融合蛋白接触；以及，检测是否存在由所述融合蛋白和抗体形成的复合物；
    优选地，所述受试者为哺乳动物，例如，小鼠，人；
    优选地，所述生物样品选自全血，血清，血浆，或其任何组合。
  18. 一种筛选能够抑制RSV感染细胞的候选药物的方法，所述方法包括，在将权利要求1-8任一项所述的融合蛋白，或权利要求10所述的载体，或权利要求14所述的药物组合物，或权利要求15所述的疫苗与宿主细胞接触之前、同时或之后，将所述宿主细胞与所述候选药物接触。
  19. 权利要求1-8任一项所述的融合蛋白，或权利要求9所述的核酸分子，或权利要求10所述的载体，或权利要求11所述的宿主细胞，或权利要求14所述的药物组合物，或权利要求15所述的疫苗在制备试剂盒中的用途，所述试剂盒用于诱导受试者对RSV的免疫应答；
    优选地，所述免疫应答包括诱导受试者产生针对RSV的抗体；
    优选地，所述抗体为中和抗体；
    优选地，所述受试者为哺乳动物，例如，小鼠，人。
  20. 权利要求1-8任一项所述的融合蛋白，或权利要求9所述的核酸分子，或权利要求10所述的载体，或权利要求11所述的宿主细胞，或权利要求14所述的药物组合物， 或权利要求15所述的疫苗在制备试剂盒中的用途，所述试剂盒用于预防和/或治疗RSV感染或由RSV感染所引起的疾病和/或症状；
    优选地，所述受试者为哺乳动物，例如，小鼠，人；
    优选地，由RSV感染所引起的疾病和症状选自细支气管炎，肺炎，哮喘，阻塞性肺病，心肺并发症。
PCT/CN2023/102342 2022-10-17 2023-06-26 截短的呼吸道合胞病毒f蛋白及其用途 WO2024082681A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211267043.7A CN116478296B (zh) 2022-10-17 2022-10-17 截短的呼吸道合胞病毒f蛋白及其用途
CN202211267043.7 2022-10-17

Publications (1)

Publication Number Publication Date
WO2024082681A1 true WO2024082681A1 (zh) 2024-04-25

Family

ID=87210704

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/102342 WO2024082681A1 (zh) 2022-10-17 2023-06-26 截短的呼吸道合胞病毒f蛋白及其用途

Country Status (2)

Country Link
CN (1) CN116478296B (zh)
WO (1) WO2024082681A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117567652B (zh) * 2024-01-19 2024-05-14 北京安百胜生物科技有限公司 一种重组呼吸道合胞病毒颗粒抗原

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140271699A1 (en) * 2013-03-13 2014-09-18 The United States Of America, As Represented By The Secretary, Department Of Health And Human Serv Prefusion rsv f proteins and their use
CN105473604A (zh) * 2013-03-13 2016-04-06 美国政府(由卫生和人类服务部的部长所代表) 融合前rsv f蛋白和其用途
CN109069611A (zh) * 2016-03-29 2018-12-21 美国政府(由卫生和人类服务部的部长所代表) 取代修饰的融合前rsv f蛋白及其用途
US20190330277A1 (en) * 2016-12-16 2019-10-31 Institute For Research In Biomedicine Novel Recombinant Prefusion RSV F Proteins And Uses Thereof
CN111405907A (zh) * 2017-08-07 2020-07-10 考尔德生物科技有限公司 构象稳定的rsv预融合f蛋白
CN111655715A (zh) * 2018-01-29 2020-09-11 默沙东公司 稳定化的rsv f蛋白及其用途
CN113811549A (zh) * 2019-02-21 2021-12-17 Xencor股份有限公司 非靶向和靶向性il-10 fc融合蛋白

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2014231357B2 (en) * 2013-03-15 2018-06-14 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Epitope of RSV fusion protein and antibody identifying same
CN103204943B (zh) * 2013-03-20 2014-07-23 中国科学院武汉病毒研究所 呼吸道合胞病毒F蛋白与Fc的融合蛋白及其用途
RS58436B1 (sr) * 2013-04-25 2019-04-30 Janssen Vaccines & Prevention Bv Stabilizovani rastvorljivi pre-fuzioni rsv f polipeptidi
WO2018130072A1 (zh) * 2017-01-12 2018-07-19 厦门大学 稳定呼吸道合胞病毒融合蛋白的方法
CN111808187B (zh) * 2019-08-02 2022-05-17 苏州高泓利康生物科技有限公司 针对呼吸道合胞病毒的蛋白结合分子

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140271699A1 (en) * 2013-03-13 2014-09-18 The United States Of America, As Represented By The Secretary, Department Of Health And Human Serv Prefusion rsv f proteins and their use
CN105473604A (zh) * 2013-03-13 2016-04-06 美国政府(由卫生和人类服务部的部长所代表) 融合前rsv f蛋白和其用途
CN109069611A (zh) * 2016-03-29 2018-12-21 美国政府(由卫生和人类服务部的部长所代表) 取代修饰的融合前rsv f蛋白及其用途
US20190330277A1 (en) * 2016-12-16 2019-10-31 Institute For Research In Biomedicine Novel Recombinant Prefusion RSV F Proteins And Uses Thereof
CN111405907A (zh) * 2017-08-07 2020-07-10 考尔德生物科技有限公司 构象稳定的rsv预融合f蛋白
CN111655715A (zh) * 2018-01-29 2020-09-11 默沙东公司 稳定化的rsv f蛋白及其用途
CN113811549A (zh) * 2019-02-21 2021-12-17 Xencor股份有限公司 非靶向和靶向性il-10 fc融合蛋白

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JEFFREY C. BOYINGTON, JOYCE, SASTRY MALLIKA, STEWART-JONES GUILLAUME B. E., CHEN MAN, KONG WING-PUI, NGWUTA JOAN, THOMAS PAUL, TSY: "Structure-Based Design of Head-Only Fusion Glycoprotein Immunogens for Respiratory Syncytial Virus", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, vol. 11, no. 7, 27 June 2016 (2016-06-27), pages e0159709, XP055383885, DOI: 10.1371/journal.pone.0159709 *
KRARUP, A. ET AL.: "A Highly Stable Prefusion RSV F Vaccine Derived from Structural Analysis of the Fusion Mechanism", NATURE COMMUNICATIONS, vol. 6, 3 September 2015 (2015-09-03), XP002755858, DOI: 10.1038/ncomms9143 *
RUIZ-ARGUELLO, B.M. ET AL.: "Thermostability of the Human Respiratory Syncytial Virus Fusion Protein before and after Activation: Implications for the Membrane-Fusion Mechanism", JOURNAL OF GENERAL VIROLOGY, vol. 85, 31 December 2004 (2004-12-31), XP007917291, DOI: 10.1099/vir.0.80318-0 *

Also Published As

Publication number Publication date
CN116478296B (zh) 2024-02-23
CN116478296A (zh) 2023-07-25

Similar Documents

Publication Publication Date Title
US20230265127A1 (en) Stabilized soluble pre-fusion rsv f polypeptides
WO2022262142A1 (zh) 一种产生广谱交叉中和活性的重组新型冠状病毒rbd三聚体蛋白疫苗、其制备方法和应用
JP6679475B2 (ja) 安定化された可溶性融合前ｒｓｖ ｆポリペプチド
US11786591B2 (en) Recombinant metapneumovirus F proteins and their use
Melero et al. Structural, antigenic and immunogenic features of respiratory syncytial virus glycoproteins relevant for vaccine development
CN112469661A (zh) 具有新结构组分的纳米粒疫苗
JP6975709B2 (ja) 安定化したウイルスクラスｉ融合タンパク質
JP2016527253A (ja) 立体構造的に安定化されたｒｓｖ融合前ｆタンパク質
BR112020022393A2 (pt) composição, vacina compreendendo uma composição, método para produzir uma composição, molécula de ácido nucleico, e, vetor, célula hospedeira, kit compreendendo uma composição, e, vacinas para uso na profilaxia e/ou tratamento de infecção por citomegalovírus e por vírus sincicial respiratório.
WO2024082681A1 (zh) 截短的呼吸道合胞病毒f蛋白及其用途
CN115246874A (zh) 一种重组新型冠状病毒s-rbd三聚体蛋白、其制备方法和应用
WO2023138334A1 (zh) 一种重组新型冠状病毒蛋白疫苗、其制备方法和应用
US9896484B2 (en) Influenza virus recombinant proteins
WO2019076218A1 (zh) H3n2亚型流感病毒血凝素蛋白的突变体及其应用
CN113227123A (zh) 重组呼吸道合胞病毒f蛋白以及包含该蛋白的疫苗组合物
KR20180038557A (ko) 타겟 폴리펩티드를 제시하기 위한 폴리펩티드 캐리어 및 이의 용도
JP2023523423A (ja) ＳＡＲＳ－ＣｏＶ－２に対するワクチン及びその調製物
KR102332725B1 (ko) 오량체 기반 재조합 단백질 백신 플랫폼 및 이를 발현하는 시스템
US11872279B2 (en) SARS-CoV-2 antigens and uses thereof
WO2018175518A1 (en) Mini-protein immunogens displayng neutralization epitopes for respiratory syncytial virus (rsv)
US11723968B2 (en) Stabilized recombinant hantaviral spike proteins comprising mutations in Gc
WO2018175560A1 (en) Nanoparticle immunogens to elicit responses against the influenza receptor binding site on the hemagglutinin head domain
CN118108812A (zh) Rsv f蛋白的突变体
CN113801206A (zh) 利用受体识别域诱导抗新冠病毒中和抗体的方法
CN116964104A (zh) 免疫原性冠状病毒融合蛋白及相关方法

Legal Events

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

Ref document number: 23878678

Country of ref document: EP

Kind code of ref document: A1